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PLENARYLECTURES


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PLANTSANDPEOPLE:ALONGHISTORYOFHUMAN-ENVIRONMENTINTERACTIONTHROUGHARCHAEOBOTANICALRESEARCH

Peña-ChocarroL.

EscuelaEspañoladeHistoriayArqueologíaenRoma(EEHAR),ConsejoSuperiordeInvestigacionesCientíficas(CSIC)Madrid,Spain

email:[email protected] history of the human species is intrinsically related to that of plants. Their role in nutrition but also the myriad ofdifferentpurposes(medicines,drinks,dyes,shelter,fodder,fuel,crafts,cosmetics,rituals,building,etc.)forwhichplantsareused demonstrate their central role in societies. In the past, this relationship pervaded every aspect of human life(subsistence, health, clothing, heating…) and thus, the study of archaeological plant remains provides an extraordinaryopportunity for exploring human-environment interactions throughout time. Consequently, archaeobotany has become amostvaluabletoolforapproachingthesubjectbyencompassingboththearchaeologicalandscientificrecords.My lecture aims at showing the power of archaeobotanical research for exploring plant-human interactions in the pastthrough examples from different research projects in the Mediterranean region. From prehistoric hunter-gatherers toMedievalfarmers,acrossthewesternMediterranean,Iwillsurveyplantencountersprovidingevidenceoftheimportantroleofplantsforbothprehistoricforagersandagriculturalists.Althoughhumanshavebeenhuntergatherersforamuchlongerperiodthanfarmers,asignificantpartofarchaeobotanicalresearchhasfocusedonagriculturalcommunitiesso,thewayplantfoodandresourceswereusedbyprehistoricpre-agrariangroupsisstilllargelyunknown.Inmypresentation,Iwilldiscussnotonlyexamplesfromfarmingcommunitiesbutalsolatestresearchonpre-agrarianplantexploitation.Particularemphasiswillalsobegiventoethnographicapproachesasatoolforbetterunderstandingtheintertwiningrelationshipbetweenplantsandpeopleinthepast.


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THEIMPACTOFDIET,PRE-ANDPROBIOTICSTRATEGIES

ONGUTMICROBIOTACOMPOSITIONANDHEALTH

DuncanSH. andFlintHJ.MicrobiologyGroup,GutHealthProgramme,RowettInstituteofNutritionandHealth,Foresterhill,Aberdeen,Scotland

email:[email protected]

Arichbalanceofhumangutmicrobialspeciesisnecessarytosustainhealthymetabolismandimmunefunction.Disturbanceinthecolonicmicrobialbalancecanhavenegativeconsequencesforhealthresultinginelevatedinflammationandinfectionwhicharealsocontributoryfactorsindiabetes,boweldiseaseandcancer.Changesinthespeciescompositionofthehumanfaecalmicrobiotacanbedetectedwithinseveraldaysoftransferringtoanewdiet.Thereisagrowingawarenessthatthemicrobial balance in the colonmaybecome increasinglyperturbedwith aging and thereforehastens theonsetof certaindiseases.Dietandagearethereforemajorfactorsdrivingmicrobialcommunitycompositionbothintheshortandlongterm.Significantprogresshasbeenmadeindefiningsomeofthedominantmembersofthemicrobialcommunity inthehealthylargeintestineandinidentifyingtheirrolesinmetabolism.Forexample,certainbacterialphylotypesidentifiedfrom16SrRNAgene sequences respond to increases in particular dietary non-digestible carbohydrates across many individuals. This isdespite considerable inter-individual variation in microbiota composition. Furthermore, controlled dietary interventionstudieshaveshownthatonlycertainspecificgroupsofFirmicutesarestimulatedby resistantstarchor inulin,whileotherrepresentativesofthissamephylumarestimulatedbycomplexnon-starchpolysaccharide(NSP)fibressuchaswheatbranorpectin.FunctionalanalysisofculturedstrainssuggeststhattheseFirmicutesbacteriaarelikelytoplayakeyroleininitiatingthe degradation of these insoluble substrates in the colon. The relationship between dietary intake of fermentablecarbohydrates and metabolic outputs of the gut microbiota, including the potentially beneficial short chain fatty acids,propionate and butyrate is of particular interest in terms of human health. In additionwheat bran products have a highcontentofpotentiallybioactivephenolicacids,especiallyferulicacid,thatarelargelyboundwithintheplantcellwall.Thesephenolic acids are released only upon degradation by microorganisms in the large intestinewhich can also have healthconsequences.Todeterminehealtheffectsrequiresanexcellentunderstandingofthebacterialgroupsinvolvedinsubstratebreakdown, thedistributionofmetabolicpathwaysandcross-feeding interactions involved inproduct formationandhowthisisdrivenbythegutenvironment.Currently,thereisavitalneedforbetterawarenessoftheimpactofdiet,prebioticandprobiotic strategies in driving human colonic microbial composition for maintaining healthy gut function and wellbeingacrossallagegroups.


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PLANTPATTERNSFORETHNOBOTANICALFOODANDNUTRACEUTICALUSES:THECASESTUDYFROM

TRADITIONALVILLAGESOFBALI(INDONESIA)

CanevaG.1 ,TraversettiL.1,SujarwoW.2,ZuccarelloV.3¹DepartmentofScience,UniversityRomaTre,Rome-Italy;2BaliBotanicalGardens,IndonesianInstituteofSciences(LIPI),CandikuningBaturitiTabanan,Bali-Indonesia;3DeptofSciencesandBiologicalandEnvironmentalTechnology,SalentoUniversity,Lecce-Monteroni,Lecce-Italy

email:giulia.caneva@uniroma3.itThemedicinalpropertiesofplantshaveincreasinglybeingstudiedinthelastdecadesduetotheimportanceofextractactiveprinciplesfornutraceuticalapplications.TraditionalEthnobotanicalKnowledge(TEK)representsoneofthemostimportantways toachieve thegoalof increasingandpromoting such information.Due to increasingurbanizationand technologicalinnovation, fewer and fewer people belonging toWestern culturemaintain this kind of knowledge whilst still there arevillagesinlessdevelopedareasoftheWorld,wherethisknowledgeisstillwidelyusedandhandeddownfromgenerations.TEK is especially threatened in tropical areas, where single indigenous individuals and entire communities are rapidlychangingtheircultureundertheinfluenceofWesternmodels(Brosietal.2007;QuinlanandQuinlan2007;Voeks2010).In this study,we analyse ethnobotanical information for the Indonesian islandofBali, as a relevant example in theAsiancontextconservingitstraditionsandculture,inordertoidentifyandanalyzepatternsofplantsethnobotanicallyusedonalocal scale. Due to the abundance of native flora, this study should contribute also to increase the knowledge on theapplicationofwildplantsinthetreatmentorpreventionofdiseasesthroughtraditionalmedicine.Ethnobotanicaldatawerecollectedusingdifferentinterviewmethods(semi-structuredinterviews,keyinformantinterviews,individual discussions, and focus group discussions) (Alexiades and Sheldon 1996; Sujarwo et al. 2015). Interviews werecarriedoutin13Traditionalvillages(BaliAga)involving50informants,mainlybelongingtomalegender,sincefemalewereless confident in giving information. In order to assess similarities in plant pattern within villages, we used the Jaccardsimilarity index. The similarity matrix was then used as reference matrix for cluster analysis with the complete linkagemethod.Threeprincipalcoordinatesanalyses(PCoA)wererunbyusingthesimilaritymatrix,withspeciesgroupedasfollow:i) according togroupsobtained fromcluster analysis, ii) according to thebiological form, iii) according to theusehumanmadeofeachplant.The113-recordedspeciesbelongto50familiesand91genera.Thedominantlifeformsaretrees(39.29%),followedbyshrubs(21.43%),perennialherbs(19.64%),climbers(18.75%),andwoodyclimbers(0.89%).Themostfrequentlyusedpartsareleaves,fruits,tuberousroots,andseeds.Theclustertreeclearlyshowedaseparationbetweenspeciesinto12groups.Fewspecieswere completely separated from the others, while half of the species are grouped inside a pattern. That shows certainhomogeneityamongplantusedinthevillages,asconfirmedfromthethreeprincipalcoordinatesanalysis.Our findings confirmed thecentral roleofTEKas sourceof information topreserveand toenhance thecultureofnativepeople.ThewideuseoftraditionalspeciestheymadeunderlinethemedicinalpropertiesofBaliplants,bothforfoodusesandtoextractactiveprinciplesfornutraceuticalapplications.1. Alexiades,M.N.andJ.W.Sheldon.Selectedguidelinesforethnobotanicalresearch:Afieldmanual.TheNewYorkBotanicalGardenPress,NewYork.2. Brosi,B.etal.2007.Culturalerosionandbiodiversity:Canoe-makingknowledgeinPohnpei,Micronesia.ConservationBiology1996,21:875–879.3. Quinlan, M. and Quinlan R.Modernization and medicinal plant knowledge in a Caribbean horticultural village. Medical Anthropology Quarterly 2007,

21:169–192.4. Sujarwo,W.,etal.EthnobotanicalstudyofLoloh:TraditionalherbaldrinksfromBali(Indonesia).JournalofEthnopharmacology2015,169:34-48.5. Voeks,R.A.Ecotourismandethnobotanicalerosion:ApossiblerescueeffectinBrazil’sChapadaDiamantina.In:Recentdevelopmentandcasestudiesin

ethnobotany,eds.U.P.AlbuquerqueandN.Hanazaki,2010,pp.228–245.SociedadeBrasileiradeEtnobiologiaeEtnoecologia.


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HELICHRYSUMITALICUM:WAKINGUPTHESLEEPINGGIANTOFMEDITERRANEANHERBALMEDICINE

AppendinoG.

DipartimentodiScienzedelFarmaco,UniversitàdelPiemonteOrientale,Novara-Italyemail:[email protected]

Helichrysumitalicum(Roth.)Don.(Asteraceae),aniconicplantoftheMediterraneanarea,hasnowbecomealsoaniconofluxurybecauseof itsuse inglamorousperfumesandpersonalcareproducts[1].H. italicum is,however,alsoan importantmedicinal plant, and recent studies have provided the basis for a veritable Helichrysum Renaissance, rationalizing thefascinating ethnopharmacology of the plant in the light of molecular investigations on its constituents and theirpharmacologicaltargets[2].ExtractsfromH.Italicumhavethepotentialtobedevelopedasanovelingredientformedicinalandhealthfoodproductsjustlikeitsessentialoilhasbeeninperfumeryandaromatherapy,butawakeningthissleepinggiantof the Mediterranean herbal medicine will required a multidisciplinary joint effort. One of the major challenges to beaddressedistheestablishmentareliablesupplychaintoovercometheplightofspontaneousharvestthatisthreateningthewildpopulationoftheplantinSardiniaandCorsica,wherethemostvaluablechemotypeoftheplantgrows.Asecondissuethat needs to be addressed is the high variability of the phytochemical profile of the plant, where the concentration ofheterodimericpyrones,themajorbioactiveconstituentsoftheplant,canrangefromundetectabletoalmost1%.1.AppendinoG.etal.,Helichrysumitalicum:ThesleepinggiantofMediterraneanherbalmedicine.Herbalgram2015;105:34-45.2.Taglialatela-ScafatiO.etal.AntimicrobialphenolicsandunusualglyceridesfromHelichrysumitalicumsubsp.microphyllum.JNatProd2013;76:346−353


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THEROLEOFTHEFOODCHEMISTINTHEENHANCEMENTOFTHEFOODFARMINGCHAIN

DugoG.mo

UniversityofMessina,B.I.O.M.O.R.FDepartment,Messina-Italyemail:[email protected]

ThefoodsectorplaysastrategicroleinbothItalianandworldeconomy,aswellasinthesocialdevelopment.The food industry is facing lots of changes compared to the past and in this scenario the physical-chemical andmicrobiologicalanalysisofrawmaterialsandoffinalproductsareessential.Todatetheconsumersafetyisthemainrequirement,andinthisfiledthefoodchemistisoneofthemainfiguressuitableforthe whole food production chain control, from the origin of raw materials and processing methods , until their properdistribution.Themicro andmacro constituent analyses allows us to determinate not only the traceability of the product, but also itshealthaspects.Themonitoringoforganic,inorganicandmicrobiologicalcontaminationisthemostimportanttoolforfoodsafetydetermination.Thefoodchemistfigureisimportantalsointhemonitoringofrowproductcultivationandmakingprocesses;infact,itplaysafundamentalroleinsectoroperatorsorentirecompaniestraining.Byusinginnovativeandnewgreentechnologies,severalbenefitsderivebyitsworksuchas:improvementofproduction;innovationofproductiontechnologies;legal,economicandtaxonstart-upconsulting;qualityand safety of the environment and the workplace ;assessment of risks due to the presence of contaminants from theproductionchain;consultingonthestudyofeffectsonhumanhealthoffoodprepared.The scientific progress allows us to get high performance targets and to reduce the wastes processes by using lowenvironmentalimpacttechnologies.


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BIOCATALYSIS:THEGREENSIDEOFCHEMISTRY

RivaS.

IstitutodiChimicadelRiconoscimentoMolecolare,C.N.R.,Milan-Italyemail:[email protected]

Sustainabilityhasbecomeoneofthekeywordsofourmodernsociety.Whenappliedtochemistryithasbeencodifiedintheso-called“greenchemistry”principles:1theinventionandtheexploitationofchemicalprocesseswhichdonotuseatallorlimittheuseofsubstancesthatmightbedangeroustotheenvironmentandtohumanhealth.Andwhatismoreintrinsicallygreenthannatureitself,withitsproductsanditscatalysts?Natureisaninexhaustiblesourceofbioactivemolecules. The structural complexity and the "fragility" of these natural productsmakes them an ideal traininggroundfornewsyntheticstrategies.Morespecifically,biocatalysis,exploitingthepeculiarpropertiesofenzymesintermsofselectivityandefficiencyundermildreactionconditions,offersinterestingsolutions.Thispresentationwilldiscusssomeoftheresultsobtainedbyourresearchgroup inthisarea,movingfromtheregio-andstereoselectiveoxidoreductionofpolyhydroxylatedsteroids,2totheregioselectiveacylationofnaturalglycosidescatalyzedbylipasesandproteasesinorganicsolvents.3Someexampleswillbealsorelatedtothesyntheticexploitationoflaccases,agroupofoxidoreductasesbelongingtotheso-called blue-copper oxidases.4 These enzymes proved to be efficient catalysts for the oxidization of a broad range ofmolecules,includingalkaloids5-6andphenols,7directlyorinthepresenceoflow-molecular-weightredoxmediators.Reactiveradical intermediates, generated by biocatalyzed oxidation at the expense of molecular oxygen, further undergo self-coupling reactions leading to the formationofC-OandC-Cdimersandoligomers, are trappedby reactivecosubstrates indominoreactions,or,inmorerareexamples,originatehydroxylatedproducts.1. AnastasP,andWarnerJC.GreenChemistry:TheoryandPractise,OxfordUniversityPress,20082. MontiD,etal.,One-potmultienzymaticsynthesisof12-ketoursodeoxycholicacid:subtlecofactorspecificitiesrulethereactionequilibriaoffivebiocatalysts

workinginarow.Adv.Synth.Catal.2009,351,1303–1311andreferencestherein3. RivaS.Enzymaticmodificationofthesugarmoietiesofnaturalglycosides.J.Mol.Cat.B-Enzymatic2002,19-20,43-54andreferencestherein4. RivaS.Laccases:blueenzymesforgreenchemistry.TrendsBiotechnol.,2006,24,219-2265. ChirivìC.etal.TheQuestforNewMildandSelectiveModificationsofNaturalStructures:Laccase-CatalysedOxidationofErgotAlkaloidsLeadstoUnexpected

StereoselectiveC-4HydroxylationChem.Eur.J.2012,18,10355-103616. SaguiF.etal.,Laccase-catalyzedcouplingofcatharanthineandvindoline:anefficientapproachtothebisindolealkaloidanhydrovinblastineTetrahedron,

2009,65,312–3177. MontiD.etal.Redoxreactionscatalyzedbyisolatedenzymes.Chem.Rev.,2011,111,4111-4140andreferencestherein


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EFFICIENTANALYTICALSTRATEGIESFORANEARLYIDENTIFICATIONOFBIOACTIVENATURAL

PRODUCTSATTHEHERBARIUMSAMPLESCALE

WolfenderJL.SchoolofPharmaceuticalSciences,UniversityofGeneva,UniversityofLausanne,Geneva-Switzerland

email:jean-luc.wolfender@unige.chWiththerecentprogressesmadeinmetaboliteprofilingmethodsandminiaturizationofbioassays,aquestionthatarisesis:dowestillneedtoperformconventionallargescalebioactivity-guidedisolationofnaturalproducts(NPs)?High resolution mass spectrometry (HRMS) and data dependent MS/MS analyses provide very valuable information onsecondarymetabolitesforin-depthmetabolomeannotationstudies[1].Therecentdevelopmentofmolecularnetwork(MN)approachesfortheminingofsuchdatagivesthepossibilitytoestablishrelationshipsbetweenmetabolites,thussignificantlyimprovingtheefficiencyofdereplication,particularlywhencombinedwithhighqualitychemotaxonomicdata[2].Suchtypesofinformationcanbegeneratedwithafewmgofextractonlyandarereadilyapplicabletoherbariumscalesamples.ForcompletedenovoidentificationofnewcompoundsMS-targetedmicro-isolationcanbeperformed,andsensitive1Dand2D microNMR data are acquired with microgram amounts of purified metabolites. For bioactivity determination, manybioassaysalsofittothisscale.Usinganidealcombinationofsuchmethods,itisthusvirtuallypossibletofullyidentifyanybioactiveprinciplesinthisway.Integrationofotherfilterstothisapproach,suchaspermeationstudiesonextracts,additionallyprovidekeyinformationonthe possible bioavailability of NPs prior to their isolation [3]. Furthermore the link of a given bioactivity result to thosepreviously reported for compounds similar to those identified can be rationalised through in silico chemical spaceapproaches.Ideally, a combinationall these state-of-the-artmethods shouldenable the identificationofvaluableNPsefficientlyat theanalyticalscale.InsuchawaylargescaleMS-targetedisolationofsuchselectedNPsonlycanbecomeaveryrationalwaytoconductinvestigations.Various examples will illustrate the power, but also the bottlenecks, of such tools. Discussion on the potential of suchapproachesandthechangesofparadigmthattheymightinduceinnaturalproductresearchwillbemade.1.WolfenderJL,etal.Currentapproachesandchallengesforthemetaboliteprofilingofcomplexnaturalextracts.JChromatogrA2015;1382:136-1642. Allard PM, et al. Integration of Molecular Networking and In-Silico MS/MS Fragmentation for Natural Products Dereplication. Anal Chem 2016;

10.1021/acs.analchem.5b048043.PetitC,etal.High-throughputpredictionofpassiveintestinalabsorptionofnaturalproductsandplantextractswiththePAMPAassay.PlantaMed2015;81:

SL1A_03


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UNTARGETEDMETABOLOMICSTOIDENTIFYBIOACTIVECOMPOUNDSANDSYNERGISTSFROM

BOTANICALMEDICINES

CechNB.DepartmentofChemistryandBiochemistry,TheUniversityofNorthCarolinaGreensboro,Greensboro(NC)-USA

email:[email protected] central challenge in botanical medicine research is distilling complex mixtures down to bioactive compounds. Toaccomplish this goal, the gold standard approach is bioassay-guided fractionation, in which the mixture is subjected tosuccessive rounds of purification andbioassays until an active compound is identified. Bioassay guided fractionation hashistoricallyplayedacriticalroleindrugdiscovery,butis,nonetheless,fraughtwithchallenges.Theprocessisbiasedtowardsthemostabundantandeasilyisolatablemixturecomponents,whichmaynotbethemostbiologicallyactive.Furthermore,ifmultiplecompoundscontributeeitheradditively,antagonistically,orsynergisticallytotheobservedbiologicalactivityofthemixture,activitymaybelostuponisolation.Asacomplementarystrategytobioassay-guidedfractionation,ourresearchgrouphasdevelopeduntargetedmetabolomicsstrategiestoaidintheidentificationofbioactivemixturecomponents.Thesestrategiesinvolveprofilingbotanicalmixturesusing ultraperformance chromatography coupled to high resolving power mass spectrometry on an Orbitrap massspectrometer.Theresultingchemicaldataisthenintegratedwithbiologicalassaydatausingbiochemometricdataanalysisstrategies. Several case studieswill be presented illustrating how this approach can be applied to identify antimicrobialcompoundsfromfungiandbotanicalextracts.Acknowledgments:Thisworkisacollectiveeffortofmanyoutstandingscientists.TheseincludeDr.NicholasOberlies,Dr.HuzefaRaja,Dr.DanielTodd,andDr.JoshuaKelloggattheUniversityNorthCarolinaGreensboro,Dr.OlavKvalheimattheUniversityofBergeninNorway,andDr.AlexanderHorswillattheUniversityofIowa.Countlessundergraduateandgraduatestudentshavealsomadecriticalcontributions.FundingwasprovidedbytheNationalCenterforComplementaryandIntegrativeHealth,acomponentoftheNationalInstitutesofHealth(grantnumberR01AT006860).


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EVALUATIONOFTHEBIOLOGICALACTIVITYOFPHYTOCHEMICALSUSINGINVIVOMODELORGANISMS

Santos-BuelgaC. ,Ayuda-DuránB.,González-ManzanoS.,González-ParamásA.M.

GrupodeInvestigaciónenPolifenoles(GIP-USAL),UniversityofSalamanca,FacultyofPharmacy,Salamanca-Spainemail:[email protected]

Phytochemicals,andamongthemphenoliccompounds,havebeenrelatedwithavarietyofbeneficialeffectsonhuman health. However, the actual mechanisms subjacent to their in vivo biological activity are not fullyunderstood.Classically,theyhavebeenassociatedtotheirantioxidantandradicalscavengingabilities.Thatviewhas mostly originated from in vitro assays performed with the compounds as they are present in the plant,whereas invivo studies that take intoaccount thecomplex interactions thatoccur inacompleteorganismarelimited. It is known that most phenolic compounds are little bioavailable and extensively metabolized in thehumanorganism,sothatthecompoundsthatmayreachthebiologicaltargetsaredifferentfromthosepresentinplant tissues. Currently, it is thought that the mechanisms involved in the biological activity of phenoliccompoundsaremorecomplexthan initiallysupposedandcould involvemetabolites interactionswithdifferentsignalingandtranscriptionfactors,andmodulationoftheactivityofenzymesimpliedincelldefensemechanisms.Differentexvivoand invivoapproaches,suchastheuseofcellculturesoranimal(e.g.,murine)modelscanbeemployed to elucidate themechanisms involved in the biological effects of phytochemicals. However, animalstudiesareexpensiveandhavebioethical limitations,andassays incell linesarenotalwayseasytoextrapolateandwouldrequiretheuseofthecompoundsthatcanbefoundatthecellularlevel,whicharenotalwaysknownand/or commercially available. In this respect, the assays in simple model organisms, such as the nematodeCaenorhabditiselegans,representanattractivealternativetoperforminvivostudieswithphytochemicalsbeforetheirassessmentinhigheranimalsandhumans.C.elegansoffersseveraladvantagesforbiochemicalandgeneticstudies.ItpossessesashortlifespanandallowsaneasyandinexpensivestorageandculturingonPetridishesorliquidmedia.Italsoshowsastrongconservationinmolecularandcellularpathwaysinrelationtomammals,witharelevantnumberofhumandiseasegenesanddisease pathways present in theworm. Furthermore, its robust genetic foundation that includes a completelysequenced genome facilitates the application of techniques such as transformation and RNAi for geneticmanipulation. Finally, as an invertebrate, its use does not pose ethical concerns. For these reasons, it isincreasinglyusedasamodel tostudybiologicaleffectsofbeneficialandtoxicsubstancesaswellasto identifynewpharmacologicaltargets.Theseaspectswillbereviewedinthepresenttalk,whereexamplesontheapplicationofthisinvivomodelfortheassessmentofthebiologicaleffectsandmechanismsofactivityofphenoliccompoundswillbepresented.Acknowledgments.TheGIP-USALisfinanciallysupportedbytheSpanishGovernmentthroughthegrantsBFU2012-35228andAGL2015-64522-C2-2-R.


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ANTIOXIDANTTHERAPIESTHROUGHSIGNALTRANSDUCTIONANDGENE-REGULATORYPATHWAYS

NúñezSellésAJ.

NationalEvangelicUniversity,SantoDomingo,DominicanRepublicemail:[email protected]

Oxidative stress caused by the body imbalance of oxygen and nitrogen reactive species (ROS/RNS) may induce gene-regulatoryeffectsandinfluencesignaltransductionpathways.Ourpreviousfindingsontheeffectofamangobarkextract(MangiferaindicaL.)showedadose-dependentinhibitionoftumornecrosisfactor(TNFα)andnuclearfactorκB(NfκB)bothinvitroand invivo indicatingthat itsantioxidanteffectwasdown-regulatedbythesefactors1.Othertranscriptionalfactors(JAK1/2,STAT3,AKT,MAPK,IKK)andtheVEGFhavebeenalsoinhibitedbymangiferin,themajorcomponentofthemangoextract2. It has been described recently that the Antioxidant Responsive Element (ARE) acts as specific enhancer ofantioxidantenzymesregulationthroughthetranscriptionalfactorNfr23. ThisfactormayexertanautoregulatoryeffectonmafGandBSV1transcriptionalregulation4,5. Theseefectsareunderlyingthehumanantioxidantdefensepathwaysandmayexplainthepotentialbenefitsofantioxidanttherapiesforchronicdegenerativediseases.Acknowledgments:worksupportedbyFONDOCYTProject2012-2013-2A1-58.1. NuñezSellesAJ,etal.MangoPolyphenolsandItsProtectiveEffectsonDiseasesAssociatedtoOxidativeStress.CurrPharmacBiotechnol2015,16,272-280.2. RajendranP,etal.Mangiferinincancerchemopreventionandtreatment:pharmacokineticsandmoleculartargets.JRecepSignTrands2015,35,76-843. KimYC,etal.Hemin-inducedActivationoftheThioredoxinGenebyNrf2:adifferentialregulationoftheantioxidantresponsiveelementbyaswitchofits

bindingfactors.JBiolChem2001,276,18399-406.4. KatsuokaF,etal.Nrf2transcriptionallyactivatesthemafGgenethroughanantioxidantresponseelement.JBiolChem2005,280,4483-4490.5. Jyrkkanen HK, et al. Novel insights into the regulation of antioxidant-response-element-mediated gene expression by electrophiles: induction of the

transcriptionalrepressorBACH1byNrf2.BiochemJ2011,440,167-174.


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ORALCOMMUNICATIONS


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MATERIALANDIMMATERIALCONSTITUTIVEELEMENTSOFHEALINGSITES-

THEMAYANTUYACUCASESTUDYINTHEPERUVIANAMAZON.

ReT.UnescoChairAnthropologyofHealth,Biosphereandsystemsofcare,UniversityofGenoa,Genova-Italy

email:[email protected],[email protected] times,healingsiteshavebeen identified forpeculiargeomorphologicalassets,hydrological structuresandaspecificbiosphere.Astheplace isconsideredalreadypartofthetherapy,theprocessofhealingbecomes linkedtomanydifferentelements.Theemergenceofmodernmedicineandtheindifferentlocalizationofthesiteslinkedtotheplanningofcaretherapiesandthe undifferentiated urbanization has in fact neglected - apart from a few isolated cases - this aspect of the cultures ofhealth.Westernmedicineandethnomedicinearetodaybridginggapstowardsanewapproachbyexchangingknowledgeandexperiences.Theresearchprojectinvolvesamultidisciplinaryteamofanthropologists,physicists,chemists,ethnobotanical,architectsandmedicaldoctorsfromdifferentinstitutions.Itsprimaryobjectiveistheanalysisofaspecificplaceandthedifferentprocessesgeneratinghealing.ThecasestudyisMayantuyacu,ahealingcenterofashaninkamedicinelocatedinthePeruvianAmazon.The aim of the process is to identify the combination of geomorphology, hydrology, biosphere, medical plants andtraditionalmythology,music,landscapeandarchitectureinvolvedintheactionofhealingandcaring.The research on field, laboratory and academic is articulated in several disciplines and the first results are planned to bepublishedbytheUniversityPressofApusGraphincoeditionwiththeUnescoChairoftheUniversityofGenoa.The research analyzes the following elements: water, music (icaros), plant teachers, ceremonial architectures (malocas)involvedduringtherapiesandhealingactivitiesandtheirimpactondifferentspecies(plants,animalsandhumans).Thecrossreference of the results of the different research field activities generates an independent and objective set of empiricalevidence.1.Architecture: the studyof theethnographic relevanceof sacred spacesand theirperiodic re-construction in ceremonialeventsconstitutesthecentraleventofthetransmissionofacosmovisionandwithinthedynamicsofa living landscape.ATechnomorphologyofthesiteandadigital3Drecordofthecosmologyofthemainmalocaisunderdevelopment.2.Water:thestudyoftheelectricalconductivitymeasurementsondifferentsamplesisindicatingthedifferentstructuresofthewatersandtheireffectsinthepreparationofmedicines.3.Music:thestudyoftheheartmechanicalvibrationswith laserDopplervibrometrycomparestherecordedsignalsforanindividualindifferentconditions.Ananalysisofthemusicusesthedecompositionmethodintimedomain,inordertostudyself-similarstructuresofthelistenedmusic.4. Ethnomedicine: The study of the different properties of the ashanika plant teachers is presented through clinical andbotanicalcases,inordertotranslatethehealingprocessintoascientificmethodology.1.ReTandVenturaC.TransculturalPerspectiveonConsciousness:abridgebetweenAnthropology,MedicineandPhysicsin:“CosmosandHistory:TheJournal

ofNaturalandSocialPhilosophy”2015vol.11,2:228-241


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THEROLEOFFAMILIARGARDENSINPRESERVINGTHEETHNOBOTANICALKNOWLEDGEOFA

PERUVIANNATIVECOMMUNITY

PaduanoC.1,CornaraL.1 ,GiordaniP.2 ,MariottiMG.1 ¹UniversityofGenova,DISTAV,Genova-Italy;2UniversityofGenova,DIFAR,Genova-Italy

email:[email protected];[email protected];m.mariotti@unige.itThestudywasconductedinthenativecommunityofMaime(Pasco,Peru),belongingtotheYaneshaethnicgroup.ThemaineconomicactivityoftheinhabitantsisthecultivationofcoffeetosellatthenearcityofVillaRica.Duringtheyearsthesocialand cultural influence of this city has become stronger and stronger and led to a profound transformation of the habit,lifestyle, cosmovision of native people. The home gardens have been much studied in the tropics, where are generallycharacterizedbyawidevarietyofplantsusefulfordifferentpurposes,soastoresemblethesurroundingecosystem.Homegardenshostedible,decorative,medicalnativespecies;whichtraditionallyhaveasubsistencefunctionandaretheresultofaculturalheritage.Inthiswork,weaimedtotestwhetherhomegardensstillhadanactualroleinYaneshafamiliesforthesupplyoftraditionalherbalmedicinalresources,withspecialreferencetothoseconsideredasmostimportant.Afloristicinventorywithnativeguideswascarriedoutacrossthewholeterritoryofthecommunitycoveringboththewildvegetationandhomegardens,inordertodeterminethecurrentuseofnaturalhealthremediesandthewayoftransmittingtraditionalskills.Wevisited23familygardens,accompaniedbythenatives,forevaluatingthefrequencyofmedicinalplants,wildorcultivatedinthevicinityofthehouses.Threesamplesforeachspecieswerecollected,withpermission,tobedriedandidentifiedattheuniversityherbariumUNMSMinLima.Wealsoevaluatedtherelationshipbetweentheimportanceofaspeciesforthecommunity(IV),thefrequencyofuseanditsfrequencyinthegardens.ThiswasdonebycalculatingthevalueoftheSpearmancorrelationcoefficientbetweenthevariables.Eighty-fourmedicinal taxahavebeen inventoried, including 28wild and56 cultivated species.Among themain cultivatedplantsthemostcommonwereColocasiaesculenta,whichoccurredin55%ofvisitedgardens,Mangiferaindica(36%),Musa×paradisiaca andNicotiana tabacum (both in 32% of cases), Citrus limon, Persea americana, Piper umbellatum and Solanumquitoense (27%). Themost frequent spontaneous includeAcmella alba,Plantagomajor andVerbena litoralis (46%). SixteenspecieswithahighIVwerepreservedinfamilygardens.Sixofthese(incl.Bixaorellana,Minthostachysmollis,Crassulasp.and3 species of the genus Cyperus) were exclusively found in home gardens. Particularly, Cyperus species, called "epe" , inYanesha,or"piripiri" , inSpanish,arethoughttohavedifferentproperties,whichsometimesfall intothemagicalsphere(Valadeau,2010;Luziatelli,2010).Overall,25%ofmedicinalplantsfoundduringthefloristicinventorywereextremelyrareinnaturalhabitats.Amongthem,only 10%wascultivated inat least3homegardens,whilemorethanhalfdidnotoccurred.Apparentlyhomegardensdonotplayeithertheancientsubsistencefunctionoramorecurrentroleintheprotectionofthemost relevant species for the community. Substitutability of medicinal plants , a lack of awareness of the resourcedegradationissuesandaccessibilityofalternativeremedies,suchasmodernmedicines,arefactorshavingabearingonthistrend.1. Luziatelli,G.,etal.Asháninkamedicinalplants:acasestudyfromthenativecommunityofBajoQuimiriki,Junín,Peru.J.EthnobiologyEthnomedicine2010,

6:212. Valadeau,C.,etal.Therainbowhurtsmyskin:MedicinalconceptsandplantusesamongtheYanesha(Amuesha),anAmazonianPeruvianethnicgroup.J.

Ethnopharmacology2010,127:175-192.


15

BIODIVERSITYUSE:PRODUCTIVECHAINANDETHNOKNOWLEDGE

(ITACOATIARA,AMAZONAS-BRASIL)

CostaRC. ,PereiraUA.NationalInstituteofAmazonianResearch-INPA,SocialStudiesLaboratory,Manaus(AM)-Brazil.

email:[email protected] severalpotentialities amongwhicharemedicinalplants. Theseareused in the traditionalway,supportedinethnoknowledgewhichisculturallyconstructedforgenerations.ThisstudywasconductedinItacoatiara-AM,wheretheseplantsareindicatedforthetreatmentofvariousdiseases.Therearenopublicpoliciesfortheimprovementandstrengtheningofproductivechainsdirectedtomedicinalplants,althoughtheimportanceofmedicinalplantsasasocialnecessity,economicandplantbiodiversityusepolicyisanelementofsocialjustice.ForthisworkhasbeendonefieldworkinItacoatiara(Amazonas,Brazil)inJanuary2016andabibliographicresearch,aimingtheuseofbiodiversityformedicinalpurposesandthedeterminationofthestructuresthatcomprisetheproductionchain.InItacoatiara,peasantscultivatemedicinalplantsintheirsmallfarms/yards.Theseareusedforfamilyuseandpartoftheproductiongoestothecountyfair.Medicinalplantsarepartofaproductionchain,whereproductionstartswiththepeasantwork,which,fromitsethnicknowledgerelatingtomedicinalplants,destinatepartoftheirproductiontomarket,whichstartto have exchange value, and in a domestic case, a use value. In the distribution and circulation there is themiddleman(atravessador),which goes to theproducers to buy their production and take it to sell in the county fairs. In the caseofItacoatiara,oftenthemiddlemanfunctionisexercisedbythepeasanthimself,whotakeshisproductiontothefairwhereheownsasaleplace.AtthefairoftheruralproducerofItacoatiara-AM,itwasidentifiedthemarketingofcat'sclaw,whichisindicated for inflammation; embauba for the treatment of diabetes, high bloodpressure and hemorrhage; grass smellingused to control the heart racing. There are also potions (garrafadas) being produced, which are composed by severalmedicinalplants,forexample,jatoba,jucáandsaratudo,wheninfieldworktheywerepackedintwo-literbottles,pricedatR$10,00(~U$2.5).Inthiscontexttheethnoknowledgeisofhighimportanceinthecommoditytradingprocess(medicinalplant); there is thediversityofmedicinalAmazonianplantsa functionalityasakey factor tomarketing in the fairof ruralproducer in themunicipality of Itacoatiara. The productive chain ofmedicinal plants strengthens and is strengthened byethnoknowledgeandtheneedsofitsuse.Acknowledgments:ToPPBio/CNPq,CT-Agro/CNPq,CAPES(Pro-Amazônia)andCENBAM/CNPq/MCTIforthefinancialsupport.1. CostaR.C.andNunezC.V.2011.Ethno-knowledgeandbioproductsmarketinManaus-AM.EmiratesJ.FoodandAgriculture2011,23:237-242.2. CostaR.C.etal.AlgumasdimensõesdomercadodabiodiversidadeemManaus-AM.AteliêGeográfico(UFG).2011,5:103-117.3. Costa R.C. andNunez C.V.Processos de Cadeia Produtiva para oMercado da Biodiversidade. In: Costa, R. C. et al. (orgs.)Mercado e Biodiversidade.

Manaus.INPA.2015.


16

BIODIVERSITYOFMELIPONINIBEESINECUADOR:

ANINTANGIBLEHERITAGETOPROTECTGENUINEHONEY

VitP.1,2 ,PedroS.R.M.3,FrisoneV.4¹UniversidaddeLosAndes,FacultyofPharmacy,FoodScienceDepartment,Mérida-Venezuela;2TheUniversityofSydney,DisciplineofBiomedicalScience,CumberlandCampus,Lidcombe(Sydney)-Australia;3UniversidadedeSãoPaulo,Phylosophy,ScienceandLiterature

Department,RibeirãoPreto(SãoPaulo)-Brazil;4MuseodiArcheologiaeScienzeNaturali‘G.Zannato’,MontecchioMaggiore(Vicenza)-Italyemail:[email protected]

Megabiodiversityresultsfromeventsofspeciationandextinctioninnaturalhistory.BeesproducinghoneyincerumenpotsbelongtothetribeMeliponiniwithsome400speciesintheNeotropics(CamargoandPedro,2007).Theyareknownbytheethnic name “stingless bees”. A fossil record of the extinct Cretotrigona prisca, from the late Cretaceous amber in NewJersey,USA,c.a.65-96Ma(MichenerandGrimaldi 1988) is theoldestbeepresence inourplanet.Cultural traditionsareasource to know stingless species richness in Argentina (Zalmudio and Hilghert, 2012). The drop of meliponiculture, thereducedmanagementandthelossofcoloniesduetotheincreaseofdisturbedhabitat,hadmoreimpacttodeclineMeliponabeecheiithancompetitionwithAfricanizedhoneybeesinMexico(Villanuevaetal.,2005).ThebiodiversityofstinglessbeesinEcuadorisrecordedfromdiversecontexts.Thedriverofthisworkwastheentomologicaloriginofpot-honeyneededfortheproposalofqualitystandardsintheEcuadorianhoneynorm(NTEINEN1572).TheparadoxofagreatbiodiversityofMeliponinipot-honeyandthepresenceoffakehoneys.Honeyswerecollectedinstinglessbeehivesorpurchasedinthelocalmarkets.Stinglessbeespecimenswerepreparedfortaxonomic identificationsbyDr. Pedro SRM. The ethnic nameswere retrieved as knowledgeof ethnospeciesbesides theusesofbeeproductsandthetypeofbeemanagement.ThebiodiversityofEcuadorianstinglessbeesobservedduringthehoneyqualityresearchcontributedtoincreasethenumberoftaxa identified inpreviousstudies(Coloma,1986;Rasmussen,2004;CamargoandPedro,2007)upto132.TheancestralknowledgesessioninthePlanBuenVivir“SumakKawsay”,hasnobudgettosupportmeliponicultureinEcuador.Bothpot-honeyhuntingandstinglessbeekeepingareindividualinitiatives.ThereisnocensusofstinglessbeekeepersinEcuador,andwe contributed to that database. One of the artificial honeys uncovered with an authenticity test (Vit, 1998), andcharacterizedbyphysicochemicalindicators(Vitetal.,2016)wasdetectedbytwoKichwafemaleassessorsfromtheAmazoninPastazaprovinceduringaFree-ChoiceProfilesensorystudy(VitandDeliza,2015).TheProject“RouteofLivingMuseumsofMeliponiniBees intheWorld” lodgesintangibleancestralknowledgeof indigenousandruralmeliponicultorstoprotecttheenvironmentwheretheythrive.Itisalsoanactionforpreservingtheirculturalheritage.Acknowledgements:ToConsejodeDesarrolloCientífico,Humanístico,TecnológicoyArtístico,UniversidaddeLosAndesCDCHTA-ULAforcontinuoussupportto the Research Group APIBA ADG-FA-97-08. To the Scholarship Prometeo-SENESCYT-UTMACH 2014 – 2015 to P. Vit. To Instituto Ecuatoriano deNormalizaciónINENtoponderpot-honey.TostinglessbeekeepersofEcuadorforpreservingtheirancientart.}1. CamargoJMF.andPedroSRM.MeliponiniLepeletier,1836.In:JSMoure,DUrbanandGARMelo,editors.CatalogueofBees(Hymenoptera,Apoidea)inthe

NeotropicalRegion.SociedadeBrasileiradeEntomologia,Curitiba,Brazil,2007,pp272-578.2. Coloma LA.Contribución para el conocimiento de las abejas sin aguijón (Meliponinae:Apidae:Hymenoptera) de Ecuador. Tesis para obtener el título de

LicenciadoenCienciasBiológicas.PontificiaUniversidadCatólicadeEcuador,Quito,Ecuador,1986,ppi-xiv,1-146.3. MichenerCD.andGrimaldiDA.Theoldestfossilbee:apoidhistory,evolutionarystasis,andantiquityofsocialbehavior.ProceedingsNationalAcademyof

Science1988,85:6424-6426.4. NTEINEN.NormaTécnicaEcuatorianaObligatoria.InstitutoEcuatorianodeNormalización.MieldeAbejas,Requisitos.NTEINEN1572.INEN,Quito,Ecuador,1988,pp1-4.5. RasmussenC.BeesfromSouthernEcuador.Lyonia2004,7:29-35.6. VillanuevaGR.etal.ExtinctionofMeliponabeecheiiandtraditionalbeekeepingintheYucatánpeninsula.BeeWorld2005,86:35-41.7. VitP.Atesttodetectcanesugarhoney.ALAN1998,48:62-64.8. VitP.andDelizaR.SensorydiscriminationofgenuineandfalsehoneysbynativeKichwaassesorsfromRioChico,Pastazaprovince,Ecuador.11thPangborn

SensoryScienceSymposium,Gothenborg,Sweden,23-27August2015;CrossCulturalSensoryandConsumerResearchSection.9. VitP.etal..UncoveringartificialhoneysfromEcuadorianmarkets.SILAE2016,Modena,Italy,11-15September2016,PP1_1910. ZamudioF.andHilgertNI.Cómolosconocimientos localesaportaninformaciónsobre lariquezadeespeciesdeabejassinaguijón(Apidae:Meliponini)del

nortedeMisiones,Argentina?Interciencia2012,37:36–43.


17

POLLENASINDICATOROFHUMANBEHAVIOUR

MercuriAM.

UniversitàdiModenaeReggioEmilia,DipartimentoScienzeVita,LaboratoriodiPalinologiaePaleobotanica,Modena-Italyemail:[email protected]

Archaeobotany is an extraordinary tool to investigate and comprehend the ethnobotanical evidence on a long timeperspective. Palynology applied to archaeological sciences puts particular emphasis on the pollen produced by plantshandled during human activities. In fact, the palynology of archaeological sites (on-site spectra) helps to recognizeanthropogenicpollenindicators(API;Mercurietal.,2013a),andtoinvestigatethehumanroleinexploitingorchangingtheplantcovernearthesite.Aspollenismainlytransportedintothesitebyhumans,andingeneralonlyaminorpartarrivesbyairorwater,highpercentagesorconcentrationofpolleninacertaincontextareindicativeoftransportofhighamountoforganicmatterinthelayer,includingbothcollectionsofplantsandfaecescontainingpollenfromeatenplants(Faegrietal.,1989;Mercuri,2008a).Pollensampleshavebeencollectedfromarchaeologicallayersofmorethanahundredsitesselectedfromprehistoricalandhistoricalsites located in Italyand inthecentralSahara(Mercuri,2008b;Mercurietal.,2015).Thesampleswerepreparedusing tetra-Na-pyrophosphate, HCl 10%, acetolysis, separation with Na-metatungstate hydrate, HF 40% and ethanol(Florenzano et al., 2012). Pollen slidesweremounted on glycerol jelly. Lycopodium tabletswere added for calculation ofconcentrations(expressedaspollenpergram-p/g).Pollenwasidentifiedat1000xmagnifications,withthehelpofatlasesandthereferencecollectionofthelaboratoryofModena.Pollen analyses focused on selected anthropogenic pollen indicators (cultivated plants, weeds, ruderal flora) that wererecurrentinspectrafromarchaeologicalsitesoftheMediterraneanarea(Mercurietal.,2013a,b).Themajorityofpollenfromarchaeologicalsites isaclearmarkerofhumanenvironmentsshapedbyfoodproduction(e.g.cereals, legumes) and plant exploitation by people and domestic animals (developing chicory or plantain vegetation).Similaritieswithcurrentusesofplants inpopularmedicineandtraditionsareeasily foundbyobservingthehuman/naturerelationshipsover a long time scale. Plant accumulation/transport in archaeological sites is evident fromhigh amountsofpollen(andmacroremains)thatmayrepresentaselectionofparticularspeciesforfood(e.g.Triticum),medicine(Artemisia)fodder(grasses),fuel(oaks,andothertrees),textiles(hemp),constructionorotheruses.Moreover,theconstantrecoveryofspecieswithhighnutritionalvalues,suchascereals,testifiesthefoodhistoryandtheagrarianvocationofthemodern-dayterritories. Pollen data show unambiguous evidence of past human handling of useful species which commonly weremultipurposeplants(e.g.TyphaandCannabis)throughtheselectionandtransportofspecies,confirmingdeepknowledgeonthe‘greenworld’byancientsocieties/humangroups.1. FaegriK,KalandPE,KrzywinskiK(eds.),1989.Textbookofpollenanalysis.4thedn.,JohnWileyandSonsLtd,Chichester,UK.2. FlorenzanoA,etal.ThesignificanceofintestinalparasiteremainsinpollensamplesfromMediaevalpitsinthePiazzaGaribaldiofParma,EmiliaRomagna,

NorthernItaly.Geoarchaeology2012,27:34-47.3. Mercuri AM. Plant exploitation and ethnopalynological evidence from theWadi Teshuinat (Tadrart Acacus, Libyan Sahara). Journal of Archaeological

Science2008a,35:1619-1642.4. MercuriAM. 2008b.Human influence, plant landscape, evolution and climate inferences from the archaeobotanical records of theWadi Teshuinat area

(LibyanSahara).J.AridEnvironments2008b,72,pp.1950-1967.5. MercuriAMetal.AnthropogenicPollenIndicators(API)fromarchaeologicalsitesaslocalevidenceofhuman-inducedenvironmentsintheItalianpeninsula.

AnnalidiBotanica2013a,3:pp.143-153.6. MercuriAM,etal.Olea,JuglansandCastanea:theOJCgroupaspollenevidenceofthedevelopmentofhuman-inducedenvironmentsintheItalianpeninsula.

QuaternaryInternational2013b:303,pp.24-42.7. MercuriA.M.,etal.PollenandmacroremainsfromHolocenearchaeologicalsites:adatasetfortheunderstandingofthebio-culturaldiversityoftheItalian

landscape.ReviewofPalaeobotanyandPalynology2015,218,pp.250-266.


18

APRELIMINARYSTUDYOFMETHODOLOGIESFORANINVESTIGATIONABOUTICHTHYOLOGYANDGASTRONOMYINCLASSICALSOURCES

CrescentiC.,AlfaM.,DugoG.mo

DipartimentoBIOMORF,UniversitàdegliStudidiMessina,Messina-Italyemail:[email protected]

Thiswork proposes an interdisciplinary approach to ichthyology and fishing in theMediterranean sea area in the Greek-Romanworld.Themainpurpose is toanswer toonebasicquestionand tootherquestions strictly related to the former:which fish specieswidelywell-known in theancient timesare still appreciated, investigatedandcooked inmodern times?Whichdifferencesor analogies couldwenotice?Which fish species, instead,wemay say, fell inobsolescence?Moreover,whichmethodologiesshouldbeusedinsimilarcases?Thedenominationoffishspecies(orplantspecies), indeed,changesthrough the centuries, making the exact identification of each rather difficult. The classical literature is full of scientifictreatisesandreferencesaboutseaproducts,cooking,medicine,pharmacologyanddidacticpoetry,intheformofcompleteworks or, more often, of fragments. The works of Hippocrates the physician, Aristotle’s History of animals, the Parts ofanimalsandGenerationofanimals,TheophrastusofEresus,AristophanesofByzantium,theOvid’streatiseonfishing,PlinytheElder,Apicius,Oppian’sHalieutica,ClaudiusAelianusthenaturalist,Polluxandseveralmorearetheprimarysourcesofthis investigation. Furthermore, the Greek comedy abounds in references to food and, in particular, to fish. The ancientcomedyisarealisticpictureoflifeinGreeceandRome.Forexample,Antiphanes(fr.130K-A):“So,wehavethestewedSting-ray(Dasyatispastinaca),thebackoftheRay(Rajabrachyura),theMullet(Mugilcephalus),theSeaBass(Dicentrarchuslabrax),theSnapper(Lutjanuscampechanus),thestomachoftheFishingFrog,theheadoftheConger(Congerconger),thehipoftheTunny (Thunnus thynnus), theRedMullet (Mullusbarbatus), theneckofGlaucus (Glaucus atlanticus)”. Lotsof informationaboutseaproductsmaybededucedbytheabovementionedOppian.Forexample,(Hal.,1.98):“Nowfishesdifferinbreedandhabitandintheirpathinthesea,andnotallfisheshavelikerange.Forsomekeepbythelowshores,feedingonsandand whatever things grow in the sand; to wit, the Sea-horse (Hippocampus sp.), the swift Cuckoo-fish (Synodontismultipunctatus),theyellowErythinus(Strombuserythrinus),theCitharusandtheRedMullet(Mullusbarbatus)andthefeebleMelanurus(Halichoeresmelanurus),theshoalsoftheTrachurus(Trachurusmediterraneus)andtheSole(Soleasolea)andthePlatyurus (Thamnocephalus platyurus), the weak Ribbon-fish (Desmodema polystictum) and the Mormyrus (Mormyruscaschive)ofvariedhueandtheMackerel(Scomberscombrus)andtheCarp(Cyprinuscarpio)andallthatlovetheshores”Itwould be interesting to understandwhich species exactly the Citharus belongs to. In the Athenaeus’Deipnosophists theCitharusiscalled‘acharnus’anditismentionedtooinCallia’sCiclops:“Aroastedharp-fish,andaray,andtheheadofawell-fedtunny”.LSJ1describes itas:“akindofflatfish,sacredtoApollo”.PliniustheElderwithderogativewordssays:“it’stheworsttypeofrhombus”.TheCitharusisprobablyaspeciesofflatfish,belongingtothePleuronectiformes.Theaimoftheworkwastocomparemodernandancientknowledgeaboutfishspecies.Thesourceswereanalyzedtobetterunderstandthetechnicallexiconofichthyologyandfishing,thephilologicalaspectsandtheancientgastronomy.1. Athenaeus,TheDeipnosophists,ed.diBurtonGulickC.,19272. GarnseyP.,FoodandSocietyinClassicalAntiquity,19993. GarcíaSolerMJ.,Elpescadoenlacomediagriega,in“Sociedad,PolíticayLiteratura:Comediagriegaantigua”,1996,pp.279-2854. Liddell-Scott-JonesJSJ.AGreek-EnglishLexicon,18195. MeinekeA.,Fragmentacomicorumgraecorum,18396. Oppian,Colluthus,Tryphiodorus,ed.diMairA.W.,1928


19

THEPALEONUTRITIONFROM500BCTO500AD:

CASESTUDIESBETWEENTHEADDAANDTICINORIVER(NORTHERNITALY)

MotellaDeCarloS.1,BrunelloV.2,CortiC.2,RampazziL.2,Castelletti,L.1-3

¹LaboratoryofArchaeobiology,CivicMuseums,Como-Italy;²UniversityofInsubria,DepartmentofScienceandHighTechnology,Como-Italy;3CatholicUniversity,Milan-Italyemail:[email protected]

Inthe lastdecades, remainsofarchaeologicalvegetal foodshavebeengiven increasingattentionbythedomainscientificliterature.Theseconsistoffindswithrecognizablemorphology,oradheringtovessels,orfragmentsofbiogenicmaterial.AstheanalysisofthispaleobotanicalmaterialthroughopticalorSEMmicroscopyishightimeconsuming,newfindsareoftenonlyreportedwithnoadditionalmicromorphologicalorchemicaldocumentation.The University of Insubria and the Laboratory of Archaeobiology of the Como CivicMuseums have just launched a newresearchinitiativeonthemicroscopicalanalysisofarchaeologicalremainsofcharredfood.Preliminaryresultshavealreadybeenpublishedonthe"bread"oftheBronzeAgeintheGardaLakearea,northernItaly(Castellettietal.,2015;Castellettietal.,inpress).The analysis focusesonmicromorphology andassesses thedegreeof conservationof structuresbyopticalmicroscopicalinspection(inparticularreflectedlightmicroscopy),scanningelectronmicroscope(SEM),combinedwithchemicalsurveybyelectronmicroprobe(EDAX),andIRspectroscopy.This work concerns charred food remains of proto-historic age, classical and early Middle Ages discovered in severalarchaeological sites in the area between the Adda and the Ticino river, northern Italy. The remains are related to theGolaseccaCulture,RomanImperialAgeandtheearlystagesoftheMiddleAges,anddatebacktoaperiodrangingfrom500BCand500AD,approximately.The findsderive from funeralpyresor firesofbuildings.Remainsofphytoliths, andotheranatomicalstructuresrelated,for instance,tothecaryopsesofcerealsandtotheseedsof legumes,arestill recognizable.Tracesoffungalattacks,mainlyintheformofhyphaeandspores,arealsoobservable.Thesedatamakeitpossibletoidentifytheingredients,theproceduresforthepreparationoffood,thetechniquesofrawmaterialsprocessingandthenutritionalvalueofrecognizablecomponents.1. CastellettiL,etal.LasituazionedellericerchetraarcheobotanicaealimentazioneinPiemontenelquadrodelleattivitàdelLaboratoriodiArcheobiologia,

AttidellaSocietàdeiNaturalistieMatematicidiModena2006,137:pp.275-290,Modena2. CastellettiL,etal.L’alimentazionenellaPreistoriadell’ItaliaSettentrionale:casidistudioemetodidiindagine,50ariunioneIIPP,Roma(Italy),2015.3. CastellettiL,etal.inpress.Unpaninodi3800annifaaCavriana(Mantova).4. MotellaDeCarloS.Iroghifunerari:unachiavediletturaperilpaesaggiovegetaleeperilritualefunebre.InGambariFMandCerriR.(acuradi)L’albadella

città.LeprimenecropolidelcentroprotourbanodiCastellettoTicino,2011,pp.209-224,Novara.


20

NOTONLYFORBEAUTY,THREERITUALPLANTSINCLASSICALTIMES

BosiG. ,RisoFM.,RinaldiR.,MazzantiM.

Lab.PalinologyandPaleobotany,Dep.LifeSciences,UniversityofModenaandReggioEmilia,Modena-Italyemail:[email protected]

Theplantshavealwaysbeenimportantinhumanhistory,notonlybecausetheyprovidefood,medicines,fuelandmaterials,butalsofortheirsocialuses,includingreligiousones,asthefuneralrites.InMediterraneanthetraditionslinkedtoplantscanhaveveryancientroots,whichhoweverarecodifiedfromtheArchaicGreecetotheRomanWorld.During a PhD research the archaeobotanical remains (seeds/fruits and charcoals), recovered in graves and funerarystructures(1st–5thcent.AD,RomanAge)ofModena(EmiliaRomagna,NorthernItaly),havebeeninvestigatedandarestillunder study.Up tonow,about35 contexts from3differentnecropoliswereanalyzed. (Rinaldiet al., inpress;Risoet al.,2016).Inthelaboratory,moreover,wearestudyingtheplanticonographyofhundredstombstones(30graves,end5th–3rdcent.BC) fromPaestum(Ferrarietal., in litteris).Thesestudiesalsopermitacomparisonof thearcheobotanicaldataandthat from the Greek-Roman written sources regarding the role of plants in the classical societies (e.g. Baumann, 1993;Bernhardt, 2008; Caneva, 2010; Caneva and Bohuny, 2003; Castoldi, 2014; Ducourthial, 2003; Forster, 1936; Frazer, 2012;Giesecke,2014;Raven,2000;Repici,2000;SenaChiesaandPontrandolfo,2015).InModenagraveshavesofarbeen identifiedaround50carpological taxa, inaccordancewiththeresultsobtainedfromareviewofabout300RomancremationsofNorthernItaly(RottoliandCastiglioni,2011).IntheinvestigationofthePaestum’stombstones,wehaveidentified10plantspecieswithcertainty.Wenoticedthatthreewoodyplantsarepresentbothinthepictures of Paestum and, with their "fruits", among Roman funerary offerings inModena and they also appear inmanyclassical sources: olive (Olea europaea), date palm (Phoenix dactylifera) and grapevine (Vitis vinifera), are plants thatrepresentacommonthreadbetweenGreekandRomanworld,fortheirbeautyandthesymbolicmeaningsascribedtothem.Theseplants,amongtheoldestfruitcropsknownintheMediterraneanarea,havepropertieswhichmadethemessentialfornutraceuticandmedicinefields,fromancienttimesuntiltoday.Olives,datepalmandgrapevineare,amongthevegetables,theperfectexampleofformrelatedtotheusefulnessandsymbolicaspectsconnectedwiththeusesineverydaylife.1. BaumannH,1993.TheGreekPlantWorldinMyth,ArtandLiterature.TimberPress,Portland.°2. BernhardtP,2008.GodsandGoddessesintheGarden.RutgersUniversityPress,NewBrunswick.°3. CanevaG,2010.IlCodiceBotanicodiAugusto.Roma–AraPacis.ParlarealpopoloattraversoleimmaginidellaNatura.Gangemi,Roma.°4. CanevaG,BohunyL,2003.BotanicalanalysisontheLivia’sVillapaintedflora(PrimaPorta,Roma).ScienceTechCultHerit4:149-155.°5. CastoldiM,2014.AlberidiBronzo.Pianteinbronzoeinmetallipreziosinell'anticaGrecia.EDIPUGLIA,Bari.°6. DucourthialG,2003.Floremagiqueetastrologiquedel'antiquité.Belin,Paris.°7. ForsterES,1936.TreesandPlantsinHomer.TheClassicalReview,50(3):97-104.°8. FrazerJF,2012.Ilramod'oro.BollatiBoringhieri,Torino.°9. GieseckeA,2014.TheMythologyofPlants-BotanicalLorefromAncientGreeceandRome.Getty,LosAngeles.°10. RavenJE,2000.PlantsandPlantLoreinAncientGreece.Leopard'sHead,Oxford.°11. RepiciL,2000.Uominicapovolti-LepiantenelpensierodeiGreci.Laterza,Bari.°12. RottoliM,CastiglioniE,2011.PlantofferingsfromRomancremationsinnorthernItaly:areview.VegHistArchaeobot20:495-506.13. Rinaldi R, Bosi G, Mazzanti M, in press.Offerte vegetali in contesti funerari. In: Malnati L (ed.) Archeologia a Novi Park. Ricerche interdisciplinari di

archeologiaurbananelsuburbiodiModenaromanaemedievale(IXsec.a.C.-XVIIsec.d.C.).All'InsegnadelGiglio,Firenze.14. RisoFM,BosiG,RinaldiR,LabateD,2016.AnalisiarcheobotanicheaconfrontotranecropolisuburbanadiMutina(scavoexParcoNoviSad)enecropoli

predialenell'agrocenturiatomutinense(scavicavaCorpusDomini–Marzaglia).In:MarchettiMC(ed.)AbstractTRAC26/RAC12,Quasar,Roma,p.109.15. SenaChiesaG,PontrandolfoA(eds.),2015.MitoeNatura.DallaGreciaaPompei.Electa,Milano


21

PERSICARIALAPATHIFOLIA(POLYGONUMLAPATHIFOLIUM,PALESMARTWEED)SEEDSASFOODDURINGTHEIRONAGESOUTHOFTHEALPS?NEWDATAFROMTHESITEOFPADOVA-S.EUFEMIA(NE-ITALY)

MartinelliE.1,2 ,MotellaS.1,2,BehreKE.3,AndreisC.4,CastellettiL.2,5

¹UniversitàdegliStudidell’Insubria,Como-Italy;²LaboratoriodiArcheobiologiadeiMuseiCivicidiComo,Como-Italy;3NiedersächsischesInstitutfürhistorischeKüstenforschung,Wilhelmshaven-Germany;4UniversitàdegliStudidiMilano,Milano-

Italy;5UniversitàCattolicadiMilano,Milano-Italyemail:[email protected]

IthasbeendemonstratedthatwildherbspecieswereintentionallygatheredforhumanconsumptionasanadditiontodietNorthoftheAlpsduringtheIronAge;inparticular,highquantitiesofPersicarialapathifolia(palesmartweed)oftenoccurredin German and Denmark Iron Age bog bodies, cadavers of sacrificed or executed people 1. In this study we will discusswhetherpalesmartweedandotherPolygonaceaewereusedas foodduring the IronAgealsoSouthof theAlps.WealsodiscussthehypothesisthattheuseofpalesmartweedasfoodarrivedinN-ItalyfromtheCarpathiansarea(SE-Europe).Wepresent newpalaeocarpological data from the siteof S.Eufemia, located in the city of Padova (NE-Italy),mainly usedduringtheIronAgebetweenVIIIandVcent.BCforritualpurposes2.WeexaminedthemacrofossilrecordsofcoevalsitesinN-ItalyandinSE-Europetoverifytheoccurrenceofpalesmartweed.WefinallycheckeditsuseasfoodduringtheXIXandXXcenturies in Europe. Pale smartweed is oneof the threemost represented carpological taxa in the site 3,4 (ca.14%of thetotal),particularly inwoodcharcoal layers found inwidepits (VII-VI cent.BC).These structureswereexcavated to setonlarge ritual fires and for the deposition of human and animal remains ceremonially sacrificed. In coeval N-Italy sites thisspeciesiswellrepresented(>10%)onlyinahouseinBressanone(NE-Italy)5.Palesmartweedisatherophytewhichhaveapaleotemperatedistributionranging,intheOldWorld,fromN-AfricatothewholeEuropeasfarasSiberiaandsub-SiberianAsia.InNE-Italyitiswidelypresentinareas,evenverylarge,alongwatercoursesonsilty-sandysubstratesrichinnutrients.IntheCarpathiansarea(e.g.Bulgaria)6;Popova2009)thisspecieswasusedasaparallelsourceoffoodbetweenVandIIIcent.BC. A large and pure assembly of seeds of this taxon, clearly purposefully collected, dating back to the XII cent. wasrecovered in Germany 1. Pale smartweed leaves and seeds represented a supplementary food source forced by poverty,faminesandwarsduringtheXIXcent.7andarealsonowadayseatenraworcooked(e.g.inPolish)8,9.SomePoligonaceaearefoodplants incertaincountries inAsia 10: rootstocksareconsumedraworroastedandusedtoobtainstarch inMongolia,China11andSiberia7.Persicariamaculosaisnowadaysoneofthe56wildherbsconstitutingadishtypicalinNE-Italy11.Wecanconcludethatpalesmartweedwasprobablyusedasfoodduringthe IronAgealsoSouthoftheAlps,even if thispracticeseemsrestrictedtoNE-Italy:probablyitcamefromtheCarpathiansareaanddidn’tdiffuseduringthatperiodtowardsNW-Italy,whereitwasconsideredassupplementaryfoodonlylaterduringtheXIXcent.7.TheuseofPoligonaceaewasforcedbypoverty and famines, as attested for Italy and NE-Europe during the XIX cent., and also by adverse climate conditions,nowadays encountered in certainAsiatic regions. InNE-Italy the useof Poligonaceaehas remaineduntil now in the foodtradition.Itisnottobeexcludedasymbolicvalueofpalesmartweed,sinceitwasinvolvedinritualcelebrationinS.Eufemiaandwaspartofthelastmealsofbogbodies.1. BehreK.E.Collectedseedsandseedsfromherbsasprehistoricfood.Veget.Hist.Archaeobot.2008,17:65–73.2. RutaSerafiniA.andMicheliniP.Offerteesacrifici‘allimite’dell’anticaPadova.InRaviolaF.(ed.)Hesperìa,2013,30:1199-1223.3. MartinelliE.Analisiantracologicheepaleocarpologicheperlaricostruzionedelpaesaggiovegetaledurantel’OloceneinPianuraPadana:nuovidatidalsitodi

S.EufemiaaPadova.Master’sdegreethesisinAnalysisandmanagementofNaturalEnvironments,UniversitàdegliStudidiMilano,2009.4. MartinelliE.etal.Ambienteeritualitànell’areapadovanafraetàdelFerroedetàromana: irestimacroscopicivegetalidiPadovaS.Eufemia,Archeologia

classicaepost-classicatraItaliaeMediterraneo.ScrittiinricordodiMariaPiaRossignani,inpress5. TecchiatiU.andNegriP.Analisideimacroresticarpologiciprovenientidaunedificiodell’EtàdelFerro(Vsec.a.C.)scavatoaBressanone,ViaElvas12e16

(BZ).AttiAcc.Rov.Agiati2009,259,seriesVIII:167-194.6. PopovaT.PalaeobotaniccatalogueofsitesandstudiedplantsremainsintheterritoryofBulgaria(1980-2008).Interdiscip.Stud.2009,20–21:71–165.7. MattiroloO.etal.Phytoalimurgiapedemontana.Comealimentarsiconlepiante.Torino,BluEdizioni,Naturaeambiente,2011.8. KunkelG.PlantsforHumanConsumption.Koenigatein,KoeltzScientificBooks,19849. ŁuczajŁ.-SzymańskiW.M.WildvascularplantsgatheredforconsumptioninthePolishcountryside:areview.J.EthnobiologyEthnomedicine2007,3:17-39.10. MitichL.W.IntriguingWorldofWeeds.PaleSmartweed(PolygonumlapathifoliumL.)andotherPolygonums.WeedTechnology201,12:560-562.11. DreonA.L.PisticedaltreerbeselvaticheinFriuli.Natural2010,1:30-37.


22

WAYUUTRADITIONALMEDICINEANDTHEUSEOFMEDICINALPLANTSINTHECONTROLANDPREVENTIONOFHYPERGLYCEMIA

MorenoMI. ,RosadoJR.

UniversityofLaGuajira,Riohacha,LaGuajira-Colombiaemail:[email protected],[email protected]

The Wayuu Indians, who live in the department of La Guajira (Colombia), have traditionally used dry forest plants formedicinal purposes to prevent, cure or control the various diseases that afflict them. Diabetes has become one of thediseases that affectingmillionsof peopleworldwide, ofwhich theWayuuarenoexception. The aimof the studywas toobtain information from practicants of Wayuu traditional medicine (Chaman, Outsüü) about the role played by somemedicinalplantsinthecontrolandpreventionofhyperglycemia.Itwasrealizedaquantitativeresearch,throughinterviewswithshamanssettledinthree(3)municipalitiesinthedepartmentofLaGuajira(Maicao,UribiaandManaure)wherethereisthelargestindigenouspopulation.Theinformationobtainedwascomparedwiththeexistingliteratureonascientificlevel.The outcomes showed that theWayuu using 16 plants for the treatment of hyperglycemia, overhanging by its effectiveBauhiniaglabra (bejucocadena),Curatellaamericana (peralejo),Prosopis juliflora (trupillo),Eugenia jambolana (uvitamorá),Parquinsoniaaculeata(sauceguajiro),Cissussicyoides(uvitae’culebra),Jacquiniapungens(mancamulo).Themostcommonlyusedmethodswerecooking(50%)andtheinfusion(25%);thepartsoftheplantsmostfrequentlyusedwerethewholeplant(37.5%), stems, leavesandbark (18.75%each)and roots (12.5%).Currently, theplants reportedare subjectofexperimentalresearch and according to the scientific literature there are significant advances in the control and prevention ofhyperglycemiaworldwide.1. BaileyC,andDayC.Traditionalplantmedicinesastreatmentsfordiabetes.DiabetesCare,1989,12:553-564.2. ChaturvediA,etal.AntidiabeticandantiulcereffectsofextractofEugenia jambolanaseed inmilddiabeticrats:studyongastricmucosaloffensiveacid-

pepsinsecretion.IndianJPhysiolPharmacol.,2009,53:137-146.3. García A and Rocha R. Efecto de Mangifera indica sobre la hiperglicemia aguda en ratas normoglicémicas, 2005. Available at:

http://www.repositorio.uchile.cl/handle/2250/110632.Accesson:15Feb2016.4. Hundekari GI, et al. Antidiabetic activity of aqueous extract of Parkinsonia aculeata in alloxan induced diabetic rats, with emphasis on diabetic

complications.IntJPharmaBioSci.,2013,4:533-541.5. OspinaL,etal.ComprobacióndelaactividadhipoglicemianteycaptadoraderadicaleslibresoxigenadosdelosprincipiosactivosdeCuratellaamericanaL.

RevistaColombianadeCienciasQuímico-Farmacéuticas,1995,24:6-11.6. VianaGS.etal.HypoglycemicandantilipemiceffectsoftheaqueousextractfromCissussicyoides.BMCPharmacol.,2004,8:4-9.


23

ETHNOPHARMACOLOGICALSTUDYOFSOMEPLANTSTRADITIONALLYUSED

ASANTIDIABETICINTHECOLOMBIANPACIFICREGION

LévuokMenaKP. ,PrietoJA. PontificiaUniversidadJaveriana,BogotáD.C.-Colombia

email:[email protected],[email protected](DM2)isametabolicandmultifactorialdiseasewhichnowadaysissufferedbymorethan382millionpeople1.Oneofthemostimportantcomplicationsinthetreatmentofdiabetesisthatitcanmanifestwithobesity,presentedinabout60to90%ofpatientswithDM22.Theintrinsicrelationshipbetweenthetwodiseasescanbeacceleratedbyoxidativestressthat isgeneratedfromthenon-oxidativemetabolismof lipidsandcarbohydrates3.Thismetabolicprofile indiabeticpatients requires an integrated treatment of the disease. The worldwide increase in the mortality of this disease haspromotedmultiple researches fornewandbetter therapeuticalternatives 4. Thus, thewidevarietyofunexplorednaturalsubstancesoffersanalternativetreatmentofthisdisease.TheknowledgedevelopedbythetraditionalmedicineinElChocooffers valuable information about a variety of plants used effectively as treatment against diabetes and/or associatedsymptoms.However,theancientpracticesoftheafrocommunitiesintheColombianPacificbehindthepreparationofthesenatural medicines have not been enough studied experimentally. The objective of this research is to provide a scientificsupporttothetraditionaluseofsomemedicineplantsrecognizedasnaturalantidiabeticsintheColombianPacific.Thestudiedspecieswereselectedviaethnobotanicalstudy,takingintoaccountthosespecieswithusingvaluegreaterthan60%. Fromdried and powderedmaterial ofArtocarpus altilis,Bauhinia picta,Momordica balsamina,Neurolaena lobata andVismiamacrophylla,hydroalcoholicextractswereobtainedbymaceration.Theseextractswerecharacterizedchemicallybyapreliminar phytochemical analysis on TLC and by quantification of total phenols5. They also were submitted tospectrophotometric and autographic assays to identify substances capable to inhibit the enzymes α-glycosidase andpancreaticlipase6;finallydecolorationproofsofthefreeradicalDPPH•7.Theextractsofthefivespecieswerecharacterizedbythepresenceofphenoliccompounds,steroidsand/ortriterpenoids,beingthemostrepresentativethephenoliccompounds,withestimatedvaluesbetween5.04±0.78to34.00±1.11mgEAG/gofsample,withgreaterpresenceintheextractsofV.macrophylla.ThehydroalcoholicextractsfromtheleavesandbarkofV.macrophyllapossessthegreatercapacityto inhibittheenzymaticactivityoftheα-glucosidasewith IC50valuesbetween18and 104 times lower than the IC50 values of the acarbose (678±2.90 µg/mL), respectively. The leaves extract ofN. lobatacaused the greatest inhibitionof pancreatic lipasewith an IC50 = 17.00±0.87 µg/mL, showing a slightly lower activity thanorlistat (4.44±1.07 µg/mL). Besides, in the autographic assay with pancreatic lipase, around 37 inhibitory zones wereidentifiedbetweenthesixextracts,relatedtothepresenceofflavonoids,saponinesandsteroids,especiallyinN.lobata,B.pictayM.balsamina.All theextracts,excepttheM.balsamina, showanexcellentbehavioras inhibitorsof thefreeradicalDPPH•withvaluesIC50between10.63±1.08and152.90±0.67µg/mL.Byrelatingthetotalphenoliccontentofthesixextractswith the results of the three activiades evaluated, it was possible to establish that the three biological activities have acorrelationabove75%withthetotalphenoliccontent,indicatingthatthesesubstancesarelargelyresponsiblefortheeffectsofextractsonenzymesandDPPH•.Inconclusion,thespeciesV.macrophyllaandN. lobataarethemostpromisingasrawmaterialforthesearchofnewintegraltreatmentsfordiabetestype2.1. NamHC,etal.ExecutiveSummary-IDFDiabetesAtlas.6thed.DiabetesAtlas.InternationalDiabetesFederation,2013,p.162. JeonWS,andPark,CY,Antiobesitypharmacotherapyforpatientswithtype2diabetes:focusonlong-termmanagement.EndocrinolMetab2014,29,410–

417.3. RaghebR,etal,Freefattyacid-inducedmuscleinsulinresistanceandglucoseuptakedysfunction:evidenceforPKCactivationandoxidativestress-activated

signalingpathways.BiochemBiophysResCommun.2009,389,211–216.4. LebovitzHE,Type2diabetesmellitus--currenttherapiesandtheemergenceofsurgicaloptions.NatRevEndocrinol.2011,7,408–419.5. BounatirouS,etal,Chemicalcomposition,antioxidantandantibacterialactivitiesoftheessentialoilsisolatedfromTunisianThymuscapitatusHoff.etLink.

FoodChem.2007,105,146–155.6. LuyenBTT,etal,.AnewphenylpropanoidandanalkylglycosidefromPiperretrofractumleaveswiththeirantioxidantandα-glucosidaseinhibitoryactivity

BioorgMedChemLett.2014,24,4120–4124.7. CuendetM,etal.IridoidglucosideswithfreeradicalscavengingpropertiesfromFagraeablumeiHelvChimActa.1997,80,1144–1152.


24

ALKALOIDSFROMHIPPEASTRUMCANASTRENSEJ.DUTILH&R.S.OLIVEIRA(AMARYLLIDACEAE)

VicenteAR.1,AndradeJP.1,FronzaM.2,BastidaJ.3,Borges,WS.1

¹DQUI/CCE/UFES,Vitória(ES)-Brazil;2DepartmentofPharmaceuticalSciences/UVV,VilaVelha(ES)-Brazil;3DepartmentofBiology,HealthandEnvironment/UB,Barcelona-Spain.

email:warley.borges@ufes.brAmaryllidaceaeisawell-knownfamilyofmonocotyledonouswhicharedistributedwidelyinBrazilianterritory,beingfoundinthe biomes Amazon, Cerrado, Caatinga, Pampa, Pantanal and Atlantic Forest. This plant family is characterized by thepresenceofanexclusivegroupofisoquinolinealkaloids,whichhavebeenisolatedfromplantsofallthegenusofthisfamily.1Severalbiologicalactivitieshavealreadybeenreportedforthesealkaloids includingacetylcholinesterase(AChE) inhibition,antimalarial,anticancer,antiviral,amongothers.1TheaimofthisworkwastocarryoutachemicalinvestigationofthealkaloidspresentintheBrazilianspeciesH.canastrense.Seventeen compounds were purified including typical Amaryllidaceae alkaloids and some are new homolycorine-typederivatives.ThespeciesHippeastrumcanastrenseJ.DutilhR.S.Oliveirawerecollectedanddividedinfreshbulbs(900g)andaerialparts(720g),crushedseparatelyandextractedwithmethanol.ThecombinedmaceratewasevaporatedtodrynessandthecrudeextractwasacidifiedtopH2with2%H2SO4,theneutralmaterialremovedusingEt2O(3x200ml)andEtOAc(3x200ml).Theaqueousphasewas thenbasifiedup topH 11withNH4OH (25%, v/v) andextractedwithn-Hex (10 x 200ml), followedbyEtOAc (10 x 200ml) and finally EtOAc:MeOH (3:1, v/v, 3 x 200ml). These enriched-alkaloidal fractionswere submitted toextensivechromatographictechniquesforalkaloidpurificationandwerealsotestedagainstmousehepatomacells(H1C1C7)and the fraction EtOAc from aerial parts along with the alkaloid candimine were tested against human breastadenocarcinomacellline(MCF-7).The Amaryllidaceae alkaloids sanguinine, hippeastrine, candimine, 7-methoxy-O-methyllycorenine, homolycorine,albomaculine, 2α-methoxyhomolycorine, 2α,7-dimethoxyhomolycorine, 2α-hydorxyhomolycorine, 2α-hydroxyalbomaculine,8-O-demethylhomolycorine, lycorine and norpluviine were isolated and identified in H. canastrense. Furthermore, thecompletecharacterizationoffournewhomolycorine-typealkaloidsiscurrentlyunderstudy.Concerningthecytotoxicassay,apercentagerangeofcell-deathbetween70%to80%wereobservedfortheextractEtOAcfrombulbsandaerialpartsandfortheextractn-Hexfrombulbs.Therangeofconcentrationofallextractsvariedbetween4-500mg.ml-1.ThefractionEtOAcfromaerialpartsdisplayedthebestcytotoxicactivityagainsthepatomacellline(cell-deathpercentageof73%in4mg.ml-1ofextract)andwasalsotestedforthehumanbreastadenocarcinomacellline(MCF-7),alongwithcandimine.TheIC500.58and28.7mg.ml-1wereobservedforEtOAcextractandcandimine,respectively.Inthisattempt,theextractshowedaremarkablecytotoxicpotentialforthesetumorcellline.Insummary,theindigenousAmaryllidaceaeH.canastrenseshowedtobeabletosynthesizeagreatnumberofhomolycorine-typederivatives,thisfeature istypicallyobserved intheHippeastrumgenus.Thealkaloid-richfractionsfromH.canastrensedisplayed an important cytotoxic activity against mouse hepatoma and human breast adenocarcinoma cell lines. In thisattempt,cytotoxicassaysfortheallisolatedcompoundsarestillinprogress.Acknowledgements:theauthorsthankCAPES,FAPESandCNPq

1. Bastida,J.etal..ChemicalandbiologicalaspectsofAmaryllidaceaealkaloids.In:RecentAdvancesinPharmaceuticalSciences,Muñoz-Torrero,D.,ed.;TransworldResearchNetwork:Kerala,2011,pp.65-100.


25

PHYTOCHEMICALINVESTIGATIONOFETHANOLICEXTRACTS

FROMKIGELIAAFRICANA(LAM.)BENTH.FRUITS

CostaR.1 ,AlbergamoA.2,PellizzeriV.2D’AngeloV.1,MicheliV.1,OcchiutoF.1,FerracaneA.1,DugoG.1,2¹UniversityofMessina,Messina-Italy;²Science4Life,aspin-offoftheUniversityofMessina,Messina-Italy


Kigeliaafricana(Lam.)Benth.isatreeendemictotheAfricancontinent(tropicalzone).Alltheplantparts,includingleaves,fruits,stembark,androots,arewidelyusedinthelocalmedicine.Theplantiscommonlyknownas“sausagetree”,becauseofthesingularshapeofthebigfruits.Thelatterconstitutethemostuseddrugobtainedfromthetree,andareconsideredavalidremedytotreatseveralailments.Beyondconventionaluses,suchastheantimicrobial,thefruitsarealsoemployedinreligiousritualstopromotefertilityandprosperity.Powdered fruits of K. africana collected in Mali (Africa) were subjected to solvent (ethanol) extraction. The extractunderwent a preliminary TLC screening with UV spectroscopy detection. Successively, the extract was further purifiedthroughSPEcartridges,inordertobeanalyzedbyHPLC-PDAandHPLC-ESI-MS.TLC analysis evidenced the presence in the ethanolic extract of polyphenols, iridoids, and phytosterols. A deeperinvestigationcarriedoutthroughLCtechniqueswithbothPDAandmassspectrometricdetection,allowedto identifyandquantifysixphenolicacids,threeiridoids,andtwounassignedcomponents,presumablybeingtwoextrairidoids.


26

NOVELSECONDARYMETABOLITESEXTRACTEDFROMTHEECUADORIANPLANT

MACROCARPAEALENAEJ.R.GRANT

MalagónAvilésO.1 ,GilardoniG.1,HerreraSarangoC.1,HerreraToroM.1,ArmijosRiofrioC.1,VidariG.2¹UniversidadTécnicaParticulardeLoja,Loja-Ecuador;2UniversitádegliStudidiPavia,DipartimentodiChimica,Pavia-Italy

email:[email protected] lenae J.R. Grant is a plant belonging to the family Gentianaceae, growing in Colombia, Ecuador and Perubetween 1000-3500ma.s.l.,mainlyon theeasternAndeanslopes.ThegenusMacrocarpaea counts75 recognizedspecies,morethan30ofwhicharepresentinEcuador.ThespeciesMacrocarpaealenaeJ.R.Grantwasclassifiedforthefirsttimein2003[1],afterbeingcollectedintheParqueNacionalPodocarpus, intheZamora-Chinchipeprovince;howeverthesamplesemployedinthisstudieswererecollectednearthetownofSaraguro,intheprovinceofLoja.InfactthisplantisemployedinthetraditionalmedicineoftheSaraguropeoplewiththenameofTabacodeCerro,wereitisappliedtothetreatmentofcoldandofsomesupernaturaldiseases.This is the firstchemical studyon this species;however thegenusMacrocarpaea countsononepublication [2],about theextractionofxanthonesfromMacrocarpaeaglabra.Dry leaves were exhaustively extracted four times with ethyl acetate and methanol. The methanol extract was thensubjected to acidic extraction with 2% sulfuric acid, followed by alkalinization to pH 11 and repeated extraction withchloroform,affordingthealkaloidfractionoftheplant.Theextractsweresubjectedtochromatographicfractionation,bothonsilicagelandC-18reversedphase,untiltheobtainmentofpuremetabolites.

In a previous edition of this congress (Marsala, 2014), some spectroscopicevidence about the presence of the alkaloid gentianine was shown. Afterthat, foursecondarymetaboliteswere isolatedasalmostpurecompounds.Twoofthesemoleculesresultedtobeunknowninscientificliterature(1-2),while theother twoare knownashirsutanineA (3) and sweroside (4). Thestructure of the new molecules was elucidated with spectroscopictechniques, through 1H, 13C, DEPT, COSY, HSQC, HMBC and NOESY NMRexperiments and by mass spectrometry. In this moment, some intent todetectabiologicalactivityisinprogress.

Acknowledgements: Part of this studywas conducted under the financial support of the PROMETEO PROJECT to GG, patronized by Secretaría de EducaciónSuperior,Ciencia,TecnologíaeInnovaciònofEcuadorin2014-2015.1. GrantJ.R.,StruweL.DeMacrocarpaeaeGrisebach(ExGentianaceis)SpeciebusNovisIii:SixNewSpeciesOfMoon-Gentians(Macrocarpaea,Gentianaceae:

Helieae)FromParqueNacionalPodocarpus,Ecuado.rHarvardPapersInBotany,2003,8:61–812. StoutG.H.etal.TheXanthonesofMacrocarpaeaGlabraPhytochemistry,1969,8:1969,2417-2419

(1) (2)

(3) (4)


27

CHEMICALCOMPOSITIONANDBIOLOGICALACTIVITIESOFHELIANTHEMUMSESSILIFLORUM

BenabdelazizI.1,HabaH.1 ,LavaudC.2,MarcourtL.3,BenkhaledM.1,WolfenderJL.3

¹Batna-1University,ChemistryDepartment,LaboratoryofChemistryandEnvirnmentalChemistryLCCE,Batna-Algeria;2ReimsUniversity,InstitutdeChimieMoléculairedeReims,CNRSUMR6229,Reims-France;3UniversityofGeneva,PhytochemistryandBioactiveNaturalProducts,School

ofPharmaceuticalScience,Geneva-Switzerlandemail:[email protected]

Helianthemumisagenusofplantsincludingaround110species,belongstotheCistaceaefamilyalsoknownasrockrose.Thisgenus is growing inAmerica, Europe andNorthernAfrica.However, theMediterranean region is considered its center ofdiversity (Mabberly, 1997). Helianthemum sessiliflorum Pers. is one of species of this genus that showed previously anti-inflammatoryandanalgesicactivities.Inaddition,theaerialpartsofthisplantarerecommendedinfolkmedicineincaseofcutaneous lesion.Thephytochemical investigationof theAcOEtandn-BuOHextractsof theaerialpartsofH. sessiliflorumresultedinthecharacterizationofonenewlignannamed1-O-acetylprinsepiol1,togetherwithtwentysevenknownbioactivecompounds2-28(Benabdelazizetal.,2015).Furthermore,theantioxidantandantibacterialactivitiesofdifferentextractsofH.sessiliflorumweredetermined.Thestructuresofalltheisolatedcompounds1-28weredeterminedbyspectralmethodsincluding1D(1Hand13CNMR)and2DNMR(COSY,HSQC,TOCSY,HMBCandNOESY),HR-ESI-MS,UV,IR,valuesofopticalrotationandchemicalcorrelationswithknowncompoundsthathavebeendescribedintheliterature.Driedaerialparts(1Kg)oftheplantmaterialweremaceratedwith70%EtOH(3× 10L)at roomtemperature.TheEtOHextractwasconcentratedthendilutedwithH2Oandpartitionedsuccessivelywithcyclohexane(3×150mL),AcOEt(3×150mL)andn-buthanol(3×150mL).TheAcOEtextract(3.2g)wasfractionatedonVLC(SiO2)withthesolventsystemcyclohexane/AcOEt(100:0to0:100)thenAcOEt/MeOH(100:0to0:100)togive four fractions (F1-F4). The purification of these fractions afforded twenty secondarymetabolites 1-20. Then-butanolextract (8 g) was chromatographed on a polyamide column eluted with H2O/MeOH (100:0 to 0:100) to give five mainfractions(F1-F5).Thepurificationoftheobtainedfractionsgaveeightpolyphenolics21-28.Repeatedcolumnchromatographyover silicagel (SiO2), reversed-phase (RP-18),polyamideSC-6andsephadexLH-20,andHPLC(RP-18)oftheAcOEtextractaffordedtwentycompounds,thenewfurofuranlignan;1-O-acetylprinsepiol1,alongwithnineteenknownonesnamed1α-hydroxypinoresinol2, (+)-cycloolivil3, ( ̶ )˗pinellicacid4,benzoicacid5,p-hydroxybenzoicacid 6, protocatechuic acid 7, vanillic acid 8, gallic acid 9, ( ̶ )˗epicatechin 10, ( ̶ )˗catechin 11, ( ̶ )˗epigallocatechin 12, ( ̶)˗gallocatechin13,astragalin14,tiliroside15,quercetrin16, isoquercetrin17,myricitrin18,β-sitosterol19anddaucosterol20.The chemical investigation of n-butanol extract using also several chromatographic methods, afforded eight knownpolyphenoliccompoundscalledisolaricireinol9'-O-β-D-glucopyranoside21,nicotiflorin22,kaempferol3-O-vicianoside23,rutin24,neoisorutin25,vicenin-226andtwoauronolsasenantiomericpairs;hovetrichosideC27and28.Theantioxidantactivitywas performed by DPPH radical scavengingmethodwhich indicated that ethyl acetate extract had the best antioxidantpotential (IC50 = 32.75 ± 2.07 µg/ml). The significant linear correlation was done between the values of the totalphenolic/flavonoid contents and antioxidant activity of plant extracts. The antibacterial activity of different extracts wasassessedagainstsomebacterial strains.Theethylacetateandn-butanolextractsshowedmoderateactivity.Thiscouldbeexplainingthetraditionaluseofthisplantagainstskininfections.1. MabberlyDJ.Theplantbook.Cambridgeuniversitypress,Cambridge,19972. BenabdelazizI.etal.LignansandotherconstituentsfromHelianthemumsessiliflorum.RecordsofNaturalProducts2015,9:342-348.


28

PROANTHOCYANIDINA2FROMDIPLOTROPISPURPUREALEAVES

SilvaJN.1,CursinoLMC.1,NunezCV.2

¹INPA-LABB,Manaus(Amazonas)-Brazil;2NationalInstituteofAmazonianResearch–INPA,BioprospectionandBiotechnologyLaboratory–LABB,Manaus(Amazonas)-Brazil

email:[email protected](Kleinhoonte)Amshoff,whichisknownassucupira,sucupira-pretaandothers(StasiandHiruma-Lima 2002) has been chosen as part of our bioprospection research line. It belongs to Fabaceae family andPapilionoideaesub-family.Chemicalstudyonthisspecieshasbeendescribedasflavonoids,triterpeneandsteroidsisolatedfromtrunkbarks(Braz-Filhoetal.1973).Inordertoidentifyothercompounds,thisworkdescribesthephytochemicalstudyofmethanolicextractfromleavesofDiplotropispurpurea.Leaves of Diplotropis purpurea were collected in June 2012 at the Reserva Particular do Patrimônio Natural (RPPN) inPresidenteFigueiredo,AM.Theplantmaterialwasextractedwithhexane,methanolandwaterbyusingultrasoundbathfor20minforeachsolventrepeated3times.Themethanolicextract(4g)waspartitionedtoobtain3phases:CH2Cl2,EtOAcandMeOH/H2O.TheEtOAcphasewassubmittedtoopenchromatographiccolumnonFlorisilusing thesolventsCH2Cl2,EtOAcandMeOHasgradient elution system. 32 fractionswereobtained and analyzedby TLC. Combined fractions 15-17 (21mg)wereseparatedbypreparativeTLCusingCH2Cl2/MeOH8:2aselutionsystemyieldingcompound1(16.6mg).Thephytochemicalworkyieldedonebiflavonoidasthemajorcompoundofthefraction15-17. The fractionwasanalyzedbyTLC,MSandNMR.The fraction showeda red spotwhenrevealed with anisaldehyde sulfuric reagent. The HR-ESI-MS spectrum revealed themolecularionatm/z577[M+H]+,indicatingC30H24O12asthemolecularformulae.The1H-NMRspectra analysis showed signals at δH 4.07 (d, J = 3.27, H-3) and δH 4.41 (d, J = 3.27, H-4)belongingtotheC-ringofflavanol.HMBCspectracorrelationsatδH4.41withδC107.21and152.42confirmedadoublylinkedinterflavanylbondsofDringofthesecondflavanolmoietydescribed inthe literature(Nametal.,2015).Othersignalsat4.93(s),4.24(d,J=3.73Hz),2.76(dd,J=17.0and5.2Hz),2.90(dd,J=17.0and2.2Hz)allowedtheidentificationofFring.All these data in comparison with the literature allowed the biflavonoid identification asproanthocyanidin A2. As far as we know, this is the first report of this biflavonoid in thegenusDiplotropis.Additionalstudieswillbeperformedtoevaluatetheantibacterialactivity.Acknowledgments:CT-Agro/CNPq,Pro-Amazônia/CAPES,ProgramaCiênciasemFronteiras/CNPq,PPBio/CNPq,INCT/CENBAM/CNPq,FAPEAM.1. Braz-FilhoR.etal.FlavonoidsfromAmazonianLeguminosae.Phytochemistry,1973,12:1184-1186.2. NamJ.etal.SubtleChemicalShiftsExplaintheNMRFingerprintsofOligomericProanthocyanidinswithHighDentinBiomodificationPotency.J.Org.Chem.,

2015,80:7495-7507.3. StasiLC.andHiruma-LimaCA.PlantasMedicinaisnaAmazôniaenaMataAtlântica.Unesp,SãoPaulo,SP,Brazil,2002,pp300.

O

OH

OH O

O OH

OH

OH

OH

OH

OH

OH


29

CYCLOARTANEGLYCOSIDESFROMASTRAGALUSGOMBO

MaamriaL.1,BenkhaledM.1 ,LongC.2,HabaH.1,LavaudC.3,Cannac,A.2

¹Batna-1University,ChemistryDepartment,LaboratoryofChemistryandEnvironmentalChemistryLCCE,Batna-Algeria;²USR3388CNRS-PierreFabre,3AvenueHubertCurien,Toulouse-France;3ReimsUniversity,InstitutdeChimieMoléculairedeReims,CNRSUMR6229,Reims-France

email:[email protected] is represented inAlgeriabyabout40species, includingAstragalusgomboCoss.&Dur.ThisendemicperennialplantgrowsinsandyaridanddesertpasturesofAlgeria(QuezelandSanta,1963).SeveralAstragalusspeciesareused in traditionalmedicine as antiperspirants, diuretics, tonics, in treatment of nephritis, diabetes, leukemia and uterinecancer (Avunduk et al., 2008). Previous phytochemical studies on Astragalus saponins have shown the importance ofcycloartane-type triterpene glycosides as major compounds (Barbić et al., 2010). They are known for their interestingbiological properties, including immunostimulating, antiviral, anti-inflammatory and cytotoxic activities. The chemicalinvestigationofthen-butanolextractoftheaerialpartsofAstragalusgomboresultedintheisolationofsixnewcycloartane-typetriterpeneglycosides1–6,inadditiontoanotherknowncycloartanetriterpeneglycoside7(Maamriaetal.,2015).Thestructuresoftheisolatedcompoundshavebeendeterminedonthebasisof1Dand2Dhomo-andheteronuclearNMRandmass spectrometry, aswell asby comparisonwith reported literaturedata.Partof theair-driedandpowderedplantmaterial ofA. gombo (aerial parts; 1 Kg)wasmacerated two times (10 l x 2, each 48 h) with EtOH-H2O (70:30) at roomtemperature.TheEtOHextractwasconcentratedandpartitionedwithpetroleumether,EtOAcandn-butanol(eachsolvent,500mLx3).Then-BuOHextract(14g)wasseparatedoveraVLCchromatography(vacuumliquidchromatography)usingreverse-phasematerial.ElutionwasperformedwithH2O-MeOH(100:0to0:100)togivefourteenfractions(F1-F14).FractionF10(679mg)wassubjectedtosilicagelcolumnchromatographywiththesolventsystemCH2Cl2-MeOH(100:0to60:40)togive8sub-fractions(F10-1toF10-8).Thesub-fractionF10-1(166mg)waschromatographedbysemi-preparativeHPLCusinganelutionH2O-acetonitrile(50:50,50:50,0:100,0:100)asmobilephasetoyieldcompounds3(Rt=9.521min,80.7mg)and4(Rt= 12.346 min, 32.4 mg). Sub-fraction F10-3 (95 mg) was purified by semi-preparative HPLC using a gradient of H2O-acetonitrile(75:25,44:56,0:100)toyieldcompounds1(Rt=5.851min,2mg),2(Rt=6.678min,2.5mg),5(Rt=7.743min,2.9mg) and6 (Rt= 8.728min, 5.9mg). Fraction F11 (300mg)was subjected to CC on Sephadex LH-20 in CHCl3giving 5 sub-fractions (F11-1 to F11-5). Preparative TLC of sub-fraction F11-2 (50 mg), developed with CHCl3-MeOH (90:10), affordedcompound7(15mg).Columnchromatographyoversilicagel(SiO2),preparativeTLC,sephadexLH-20andHPLC(RP-18)affordedsevencycloartaneglycosides 1-7, from then-BuOHextract of the aerial parts ofA. gombo. This result is in agreementwithprevious studiesperformed on Astragalus species. The phytochemical investigation of A. gombo confirms the occurrence of cycloartanetriterpeneglycosidesintheAstragalusgenus.Infact,thischemicalstudyallowedtheisolationandcharacterizationofsevencycloartane glycosides of which six compounds 1-6 called gombosides A-F are described for the first time. The knowncompound7wasidentifiedastomentosideII.1. QuezelPandSantaS.NouvelleFloredel’Algérieetdesrégionsdésertiquesméridionales,V.1–2.CNRS,Paris,19632. AvundukSetal.TriterpeneglycosidesfromtherootsofAstragalusflavescent..J.Nat.Prod.2008,71,pp141-145.3. BarbićMetal.CycloastragenolglycosidesfromAstragalusillyricus.BiochememicalSystematicsandEcology2010,38,pp460-462.4. MaamriaLetal.NewcycloartaneglycosidesfromAstragalusgombo.PhytochemistryLetters2015,11,pp286-291.


30

NATURALANDSEMISYNTHETICCOMPOUNDSFROMARGENTINIANPOLYGONUMACUMINATUM

AGAINSTHUMANPATHOGENICYEASTS

DiLibertoMG.²,ZacchinoSA.2,DeritaMG.1,2 ¹CONICET,UniversidadNacionaldelLitoral/FacultaddeCienciasAgrarias,CátedradeCultivosIntensivos,SantaFe-Argentina;²CONICET,UniversidadNacionaldeRosario/FacultaddeCienciasBioquímicasyFarmacéuticas,CátedradeFarmacognosia,SantaFe-Argentina

email:[email protected] the last years, fungi havebecome themajor causeof human infections, especially among immunocompromisedhosts,having an enormous impact on morbidity and mortality. Although there are many available drugs for the treatment ofsystemic and superficial mycoses, they are not completely effective for their eradication. In addition, they all possess acertaindegreeoftoxicityandquicklydevelopresistanceduetoinappropriateandabusiveuse.Therefore,thereisanurgentneedforthedevelopmentofnewantifungalchemicalstructuresalternativestotheexistingones.1Inthiscontext,exploringnatural and semisynthetic products, turn out to be of great importance. Previously, we reported antifungal activities ofpolygodialisolatedfromPolygonumacuminatumKunthandsomemoleculesderivativesfromthisnaturalproduct,againstapanel of human pathogenic fungi.2 In this work, we expanded the list of yeasts using clinical isolates of Candida andCryptococcusstrains.P.acuminatumwascollectedduringthefloweringseason(March2014)inSantaFeprovince(Argentina)anddepositedattheHerbarium FCA-UNL (Arturo Ragonese) with identifying data SF # MD97. Natural products polygodial (1) and p-methoxycinamoylpolygodial(2),aswellassemisyntheticcompoundsconfertifolin(3),drimenal(4)andisodrimenol(5)wereobtainedaccordingtopreviousreports2andtestedagainstclinically isolatedfungifromCentrodeReferenciaenMicología(CEREMIC) andMalbrán Institute. They included9 strainsofCandida spp. (5ofC. albicans and4ofC.non-albicans) and6strains of Cryptococcus neoformans. Inocula of yeasts suspensions were obtained according to reported procedures andadjustedto1x105ColonyFormingUnits(CFU)/mL.3MIC50wasdefinedasthelowestconcentrationofeachcompoundthatshowed50%growthreductionrespect to thecontrolandwasdeterminedusingVERSAMaxmicroplate reader (MolecularDevices,Sunnyvale,CA,USA).AmphotericinBwasusedaspositivecontrolandtestswereperformedbyduplicate.

Table1.MIC50(μg(mL)meanconcentrationsofcompounds(1-5)inducing50%growthreductioncomparedtocontrol.

CCC=CentrodeReferenciaenMicologia(Rosario,Argentina),IM=MalbranInstitute(BuenosAires,Argentina),I=inactive>250μg/mL.

ResultsareshowninTable1.AllCandidaandCryptocuccusstrainsweremoresensitivetonaturalproductspolygodial(1)(MICs50 between 1.9 and 15.6 µg/mL) andp-methoxycinamoylpolygodial (2) (MICs50 between 3.9 and 250 µg/mL) than tosemisynthetic derivatives confertifolin (3), drimenal (4) and isodrimenol (5). Among these, compound 4 showed higheractivity than 3 and 5, since it inhibited the growth of all evaluated strains with MICs50 between 15.6 and 125 µg/mL.Compounds3 and5were active against allCryptococcus strains evaluated (MICs50 between 15.6 and 250 µg/mL) but lessactive against Candida strains and in some cases they resulted inactive.With these resultswe conclude that compoundsisolatedfromthenaturalsourceP.acuminatumarehighlymorepowerfullasantifungalthansemisyntheticones.Acknowledgments:CONICETforMDdoctoralfellowshipandPIP11220150100524CO,UNRprojectBIO4111. KouznetsovV.etal.SynthesisandantifungalactivityofdiverseC-2pyridinylandpyridinylvinylsubstitutedquinolones.Bioorg.Med.Chem.2012,20:6506-12.2. Derita,M.,etal.Structuralrequirementsfortheantifungalactivitiesofnaturaldrimanesesquiterpenesandanalogues,supportedbyconformationaland

electronicstudies.Molecules2013,18:2029-2051.3. ClinicalandLaboratoryStandardsInstitute(CLSI).ReferencemethodforbrothdilutionantifungalsusceptibilitytestingforfilamentousfungiM-27A2.2002,

Vol.28,(Wayne,L.ed).


31

COMPARATIVEPHYTOCHEMICALPROFILESOFLIPPIAORIGANOIDESGROWNWILDANDINVITRO

CastilhoCVV.1 ,SilvaVD.1,LeitãoGG.2,daSilvaNCB.1,LeitãoSG.1,BoylanF.3

¹UniversidadeFederaldoRiodeJaneiro,FaculdadeFarmácia,CidadeUniversitária,RiodeJaneiro-Brazil;2InstitutodePesquisasdeProdutosNaturais,RiodeJaneiro-Brazil;3TrinityCollegeDublin,SchoolofPharmacyandPharmaceuticalSciences,Facultyof

HealthSciences,PanozInstitute,Dublin-Ireland.email:[email protected]

Lippiaoriganoides (Verbenaceae) is commonlyknownas“sálva-de-marajó”,generallyuseddue to itsmedicinalproperties(carminative,repellentandforthetreatmentof infectiousdiseases,genitourinaryandgastrointestinaldisorders)andfood(condiment)[1].ItoccursinNorthernSouthAmerica,mainlyinBrazil,ColombiaandVenezuela[2].Invitroculturetechniquesareanalternativetosustainableandlarge-scaleproductionofplantsformedicinaluse.Themaingoalofthisstudywastoevaluate the effects of different plant growth regulators on L. origanoides in vitrodevelopment and to achieve the bestchromatographicprofileinordertoenableagoodseparationofthechemicalconstituentsofthisethnomedicinalplant.AerialpartsofL.origanoidescollectedfromAmazonregion(Manaus-Brazil)wereextractedbypercolationusingcommercialethanoltoobtainthehydroalcoholiccrudeextract(LONM).Amother-plantkeptinagreenhouselocatedatRiodeJaneiroMountainRegionwasusedasdonortoinvitrocultureestablishmentandanhydroalcoholiccrudeextractwasalsopreparedfromit(LONP). InthisstudyculturesofLONPmaintained invitro inbasicmedium[3]withoutgrowthregulators(MS0)orsupplementedwith2.5µMofnaphthaleneaceticacid(NAA)or0.5µMofjasmonicacid(JA)or100µMsalicylicacid(SA)wereprepared.After60d,plantletswereevaluated fornumberofshootsandnodal segments, shoot length, rootedplants (%),callus development and fresh/dry biomass. The datawere submitted to ANOVA and themean valueswere compared byTukey’s test. Crude hydroalcoholic extracts for the treated plants were prepared by maceration and the results werecompared to LONM and LONP. High-Speed Countercurrent Chromatography (HSCCC) and High Performance LiquidChromatography (HPLC) coupledwithultraviolet (UV)were the techniquesused for the comparison among theextracts.HSCCCresultsweremonitoredbyThinLayerChromatography(TLC)andHPLC-UVresultswerecomparedwithaprofileof6flavonoidstandardsanalysedinthesamechromatographicconditions.Hormonal treatmentofLippiaoriganoides by tissue culture influencedqualitatively andquantitatively the chemicalprofilewhencomparedtonaturalplant(LONP).Thehighestnumberofshoots/plantwasobtainedwithMS+2.5µMNAA.Ageneralsolvent system using hexane-ethanol-water (4:3:1, v/v) was employed to achieve a good separation by polarity of theconstituentsoftheextractsbyHSCCC.Thechemicalprofilingshowedanincreaseofthenumberofsteroidsandterpenesfortheinvitrocultures.MS0;MS+0.5µmJAandMS+100µMSAtreatmentsshowedayellowfluorescenceontheTLCplatethatwas indicative of the presence of flavonoids. Through analysis by HPLC-UV using isocratic mobile phase composed ofmethanol-acetone-aceticacid-phosphoricacid-water(200:100:10:10:200,v/v),thepresenceofnaringeninwasverifiedinwildplants,MS+0.5µmJAandMS+100µMSA.Acknowledgments:Oliveira,D.R.forsupplyingtheplantmaterial.1. Oliveira,D.R.etal.EthnopharmacologicalstudiesofLippiaoriganoides.BrazilianJournalofPharmacognosy,2014,24,pp206-214.2. Pascual,M.E.etal.Lippia:traditionaluses,chemistryandpharmacology:areview.JournalofEthnopharmacology,2001,76,pp201–214.3. Murashige,T.andSkoog,F.Arevisedmediumforrapidgrowthandbioassayswithtabacotissuecultures.PhysiologiaPlantarum,1962,15,pp473-497.


32

IDENTIFICATIONOFPHENOLICCOMPOUNDSFROMCARDUNCELLUSHELENIOIDES

BYHPLC-ESI-MS(TOF)

MeratateF.1,DemiratasI.2,AkkalS.3

1DepartmentofChemistry,UniversityofM’sila,M’sila-Algeria;2UniversityofÇankırıKaratekin,FacultyofScience,DepartmentofChemistry,BallicaCampus,Çankırı-Turkey;3Vvarenbimol,ChemistryDepartment,UniversityofConstantine,Constantine-Algérie

email:[email protected] large familyAsteraceae or Compositae formedmany flowering plants: it includes nearly 24,000 species in over 1,500genera,thegenusCarthamusinclude18speciesofwhichC.tinctoriusL.istheonlycultivatedspecies.The aim of this studywas to characterize the polyphenolic composition of Carduncellus helenioides and to evaluate theirantioxidantandantimicrobialactivities.Byusing theHPLC-ESI-TOF-MSmethod,a totalof 12compoundscorresponding tophenolic acids and the flavonoid familywere identified fromalmond skin in only 11min. Thephenolic profile showed thepresenceof fourphenolicacidderivatives (Gallicacid, Gentisicacid,Chlorogenic acid,Caffeicacid,p-Coumaricacid),andthree flavonoidglycosides (rutin,hesperidin,apigenin-7-O-glucoside).Theextractswerescreenedforantioxidantactivitiesusing two in vitro assaymodels: DPPH radical scavenging assay. The antimicrobial activitywas determined using the discdiffusionmethod.


33

PROFILECHARACTERIZATIONOFVOLATILECOMPOUNDSONWILDAND“INVITRO”PLANTSOF

CLINOPODIUMODORUM(LAMIACEAE)USINGHS-SPME/GC-MSANALYSIS

VázquezAM.1,AimarML.2,GoleniowskiME.3 1LaboratoryofChemicalTechnology,CollegeofChemistry,Cordoba-Argentine;2DepartmentofChemistry,CollegeofExactPhysicandNaturalSciences,NationalCordobaUniversity,Cordoba-Argentine;3Ceprocor,ScienceandTechnologyMinistry,Córdoba-

Argentine.email:[email protected]

ClinopodionOdorumisusedasaflavouringagentinfood,andhasgainedconsiderableimportanceinthetraditionalmedicinebythelocalinhabitantsasananticatarrhal,antispasmodic,stringent,carminative,diuretic,laxative,stomachic,anti-oxidant,antiacid,stimulant,soporific,vermifuge,menstrualsuppressor,or in thetreatmentof flatulence,colic,altitudesicknesses,headachesandstomach-aches,aswellasananti-spasmodicandtohelpinparturition.Asitsproductionoccurentirelyfromwildcollection,thisspeciesissubjecttoahighextractionpressureresultingfromitsuseasanherbalcomponentofyerbamatecomposite(anArgentinetraditionalinfusion),folkmedicineandinthepreparationofliquorcompanies.TheaimofthisworkwastochooseamonginvitroClinopodiumodorumplants,thosethatgrewinthebestmediaconditionatdifferentconcentrationsandcombinationsof6-Benzylaminopurine(BAP)and2-Naphthaleneaceticacid(NAA)(mg/l-1)orwithout these growth regulators (PGRs). All the plantlets grew at different concentrations and combinations of growthregulatorsanddifferentmediawithoutthesehormoneswerefoundoutasuitablestartingplantmaterialforvolatileorganiccompoundsyield.Free-hormone,WPmediumshowedanefficientsurvivalpercentageofgrowingplantlets.ComparativeanalysiswascarriedoutonwildplantincomparisonwiththatC.odorumtissueculture.Consideringthecompositionofeachvolatilecompoundhadbeenshowedsimilaritiesanddifferences,sotheresultshaveawidevariability.(-)Menthoneisthepredominantmonoterpeneproducedinthemostofplantmediastudiedeithercontrolor with the addition of growth regulators. Pulegone showed a higher sensitivity to the nutrient composition media, itbiosynthesisisfavoredonlowsaltconcentrationsmedia.ThiscomponentisaccumulatedinhigherconcentrationonMSandB5athalf-strengthsaltmedia(MS1⁄2andB51⁄2).Theaddition to culturemediaof somenutrients asnitrate, calcium,phosphate, sulfur amongother independentofPGRsshowedapositiveeffectongrowthandvolatilecompoundsproduction.Acknowledgments:toFONCYT(NationalArgentineScientificFounds)forsupportingthisresearch:Startup:PICT-2013-0458-:Argentine.1. Diazetal.InVitroPropagationofMuña-Muña(Clinopodiumodorum(Griseb.)Harley).Biotechnologyresearchinternational2012,2012(4):196583.


34

CAPSICUMANNUMSEEDASSOURCEOFFOOD:PHYSICOCHEMICALANDMICROBIOLOGICAL

CHARACTERIZATION

Colín-CruzMA.1 ,Bautista-CorralE.2,Guadarrama-LezamaA.1,Martínez-HernándezV.1¹UniversidadAutónomadelEstadodeMéxico,FacultaddeQuímica,Toluca-México;²FoodScience,UniversidadAutónomadel

EstadodeMéxico,FacultaddeQuímica,Toluca,México. email:[email protected],chilli(Capsicumannum)seedhasbeenconsumedsincepre-Hispanictimes.Currently,thedryseedisgroundandusedforpreparingdiversedishes.Productionofchilli(inMexico)isabout2.2milliontonsperyear(SAGARPA,2015)ofwhich40%iscommercializedasdrychilli.Indryform,itcontains15-20%ofseedthatrepresentssomethousandtonsoffood.Theseedhasbeenextensivelystudiedfromanagronomicpointofview(asseedforsowing)(Morenoetal.,2011;Reyesetal.,2006),butitsphysicochemical(ornutritious)characteristicsarescarcelyknown(Carrilloetal.,2009).TheaimofthisresearchwastostudythephysicochemicalandmicrobiologicalcharacteristicsoftheseedoffourvarietiesofCapsicumannuminordertoknowitsvalueasfoodandtopromoteitsconsumption.TheseedsoffourvarietiesofdryCapsicumannumwerestudied:“mulato”,“ancho”“pasilla”and“guajillo”.Theconstituentsof the chilliwere quantified: pericarp, peduncle,membranes and seeds. Chemical compositionwas determined:moisture(atmospheric oven), protein (by Kjeldhal), fat (by solvent extraction), ashes (by calcination) fiber (by acid and alkalineextraction), minerals (by atomic absorption), fatty acid profile (by gas chromatography). Molds, yeasts and mesophilicmicroorganismswerealsodetermined.Thestudywascarriedoutintriplicate.Results aregivenas average for all four typesof chilli/seeds,withexception forpercentageof seedsandmicroflora. Themajorconstituentoftheanalyzedchilieswasthepericarp70.81%,thepedunclerepresents5.74%andthemembranes2.57%.Seedcontentwas21.24%, 18.74%, 18.17%and14.16%for“pasilla”,“mulato”,“guajillo”and“ancho”chilies, respectively.Thenumberofseeds(insideonechilli)was294andtheweightofaseedwas8.7mg.Thechemicalcompositionofseedswasasfollows:moisture7.37%,protein19%,fat16.14%,fiber37.8%andashes3.5%.Theseedscontainthefollowingminerals(ppm):Na (20),K (263),Mg (30),Ca (1.2), Zn (0.7), Fe (4.9).Amongheavymetals, the leadwaspresentat8ppm.Populationofmesophilic bacteria was 104 CFU/g, 103 CFU/g, 102 CFU/g and 0 CFU/g for “guajillo”, “ancho” “mulato” and “pasilla”,respectively.Thepresenceofmoldsoryeastsinseedswasnodetected.Withrespecttootherthree,the“guajillo”seedsaresmaller,clearerandbrighter,andthechillicontainsthelowestnumberofseeds.Thereweresignificantdifferencesinchemicalcompositionaswellasinmineralscontentamongvarietiesofseeds.Leadidentifiedinseedscouldcomefromirrigationwater.Eventhoughtheseedsareenclosedinthepericarp,theycontainmesophilicmicroorganisms.Thestudy revealed that thechilli seedsanalyzedhaveabalancedchemical composition fromanutritiouspointofview:ahighcontentofprotein,fatandfiber.1. CarrilloEP.etal.Calidaddelasemillaencolectasdechiledeagua(CapsicumannuumL.)delosvallescentralesdeOaxaca,México.AgriculturaTécnicaen

México,2009,35(3),257-266.2. MorenoPérezE.D.etal.,DiversidadMorfológicaenColectasdeChileGuajillo(CapsicumannuumL.)delCentro-NortedeMéxico.RevistaChapingo.Serie

Horticultura,2011,17(1),23-30.3. ReyesRivasE.etal.RentabilidaddelChileSecoenZacatecas,México.RevistaFitotecniaMexicana,2006,29(002),137-144.4. SAGARPA (Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación) (2015). Producción de chile mexicano. Available at http://

www.gob.mx/sagarpa.Acceson:14Apr2016.


35

THEPEPTIDEPROFILEOFPARMIGIANOREGGIANOCHEESE:EVOLUTIONTHROUGHTHEGASTROINTESTINALTRANSITANDUTILIZATIONBYBIFIDOBACTERIAANDLACTOBACILLI

AmarettiA.1 ,BottariB.2,QuartieriA.1,PrandiB.2,RaimondiS.1,

RossiM.1,UlriciA.1,GattiM.2,SforzaS.2,NocettiM.31Dept.ofLifeScineces,UniversityofModenaandReggioEmilia,Modena/ReggioEmilia-Italy;2Dept.ofFoodSciences,UniversityofParma,

Parma-Italy;3ConsorziodelFormaggioParmigianoReggiano,ReggioEmila-Italy.email:[email protected]

Parmigiano Reggiano cheese (PR) is a raw-milk, hard cooked, long-ripened cheese of high quality and nutritional value,produced ina restrictedarea innorthern Italy.Longripeningtimesallowforextensiveproteolysisofmilkproteinsby therennet and the enzymes of starter and non-starter bacteria [1]. Proteolysis contributes to digestibility and flavor, reduceallergenicity,andyieldspotentiallybioactivepeptides(e.g.antihypertensiveorimmunomodulatory)[2].Thisstudyaimedtoidentify the fateofPRpeptidesduringgastrointestinal transit and toevaluate if theycouldmodulate thegutmicrobiota,supportingthegrowthofbeneficialbacteriasuchasbifidobacteriaandlactobacilli.Differently aged PR samples (16, 24, and 36 months) were subjected to in vitro digestion, simulating oral, gastric, andduodenaltransit.Thedigestsweregivento27strainsofBifidobacteriumand30ofLactobacillusasthesolenitrogensourcetoevaluatetheirabilitytoutilizethePRpeptides.CulturesofhumangutmicrobiotawerecarriedouttoevaluateifPRdigestscouldaffect theabundanceofbifidobacteriaand lactobacilliamong intestinalbacteria.Thepeptides inPRsamples, in thedigests,andinculturalbrothswerecharacterizedbyHPLC/ESI-MS/MSandquantifiedbyUPLC/ESI/MS.Peptideutilizationbybacteriawasanalyzedbyprincipalcomponentanalysis(PCA)Beforedigestion,acommonpeptideprofilewasdetectedinalltheripenedPRsamples,regardlessoftheirripeningtime.Atotalof63peptideswereidentified,witha length2to103residuesandamolecularweightspreadoverawiderange.Thedigestioncaused thedisappearanceof the serumproteinsandmostof theoriginalpeptides,while71newpeptideswerefound,withanincreaseofshorterones.ThepeptidesofPRdigestscanbeutilizedasnitrogensourceforthegrowthofpureculturesofBifidobacteriumandLactobacillus,belongingtospecieswhicharenaturalcolonizersofthehumanhindgutand/orwhich could findapplicationasprobiotics.All the strainsgrew inPR-basedmedia, inmanycaseswithhighbiomass yield.Bifidobacteria generally consumed a greater amount of peptides than lactobacilli, in terms of both the mean peptideconsumptionandthenumberofpeptidesconsumed.PCArevealedstrongstrainspecificityintheabilitytoutilizeeachsinglepeptide,butdidnotrevealanygroupingbasedonthemaincharacteristicofthepeptides(e.g.thelengthortheabundanceof hydrophobic, polar, andpositively/negatively charged aminoacids). Inmicrobiota cultures, thePRdigest promoted thegrowth of bifidobacteria, even though rapid and effective consumption of peptides by proteolytic colonic bacteriamadeimpossibleany relationshipbetweengrowthofbifidobacteriaandpeptidepreferences.These resultsconfirmthepositiveroleofPRongrowthofbeneficialbacteriaandpotentialmodulationoftheintestinalmicrobiota,asafurthermechanismofhealthpromotion.Theincreaseofbifidobacteriaisuniversallyregardedasahealthpromotingeffect,albeitthecomplexityofthecommunityinhabitingthecolonthatcantakeadvantageofPRpeptides.Acknowledgement:ThestudywassupportedbyFondazioneCassadiRisparmiodiModena,grantno.SIME2013.06271. McSweeneyP.L.H.,Biochemistryofcheeseripening.Int.J.DairyTech.2004,57,127–144.2. TidonaF.etal.,Bioactivepeptidesindairyproducts.Ital.J.Anim.Sci.2009,8,315-340.


36

EVALUATIONOFTHEANTIOXIDANTCAPACITYANDCONTENTOFPOLYPHENOLSOBTAINEDFROMTEA(CamelliaSinensis)OFFOURBRANDSSOLDINCOLOMBIABYEXTRACTIONATROOM

TEMPERATURE

Ramiréz-AristizabalLS.1 ,OrtizA.

2,Restrepo-AristizabalMF.

1,Salinas-VilladaJF.

1

1UniversidadTecnológicadePereira,Carrera27#10-02BarrioAlamosRisaralda-Colombia;2CentroNacionaldeInvestigacionesdeCafé–

CenicaféSedePlanalto,Manizales(Caldas)-Colombiaemail:[email protected]

Herbal medicines have long been used to treat chronic diseases such as cancer, neurodegeneration and diabetes,usually inthe formofherbal teas,alsocalled tisanes[1].Greenteaiscatchingmoreinterestthanotherbeverageduetoitshealty benefical effects and has become the most consumed beverage all over the word, after water [2], moreovercontainingcatechinswichhaveanti-oxidative,anti-carcinogenic,anti-microbial,anti-viral,anti-nflammatory,andanti-diabeticproperties [3]. Theconsumptionofgreen tea inColombia is a recent trendandthemarket is continuouslygrowing, thenthemost common commercially available types of green teaweretested in this study;Oriental, Lipton,Hindúand Jaibel.Themainobjectiveof this studywas todetermineandcompare the amount of polyphenols present in tea samplesconsidering extraction in water at room temperature and stirring every 30 seconds for 5 minutes. The antioxidantcapacity was determined by the 1,1- diphenyl-2-picrylhydrazyl (DPPH) free-radical scavenging assay and the oxygenradicalabsorbance.Capacity(ORAC)assay.Concentrationsrangingfrom22.36±0.98to41.29±0.86mgTroloxequivalents /gdrysampleforDPPHassayand22.95±1.31 to46.25±2.05mgTroloxequivalent / gdry simpleweredetermined forORACassay. Itwasalsofoundthattheamountoftotalphenolsinteasamplesrangingfrom2.53 ±0.25 to 14.63 ±0.53mg gallic acid equivalent (GAE) / g dry sample and total flavonoid content concentrationwasobtainedfrom2.67±0.20to7.08±0.38mgcatechinequivalent/gdrysimple.Theantioxidantactivityandtotalflavonoid

contentwere highly correlated for bothDPPH (r2= 0.9911) asORAC(r2 =0.9968). Teaextracts from theOriental brandhad the highest polyphenol content showing greater biological activity, contrary to Jaibel tea brandwhich recorded thelowestconcentrationsinallanalyzes.Todate,thereappeartobenothoroughstudiesonhowantioxidantactivityofteasmaybeaffectedbyhotorcoldwatersteeping and how this may be related to their polyphenol content. The results obtained contribute to gaining furtherknowledgeonhowthepotentialhealthbenefitsofthispopularbeveragemaybemaximizedbythedifferentmethodsofpreparation.1. BüyükbalciA.andSedefNE.Determinationof InVitroAntidiabeticEffects,AntioxidantActivitiesandPhenolContentsofSomeHerbalTea.PlantFoods

HumNutr.2008,63:27-332. Pasrija,D.andC.Anandharamakrishnan.TechniquesforExtractionofGreenTeaPolyphenols:AReview.FoodandBioprocessTechnology2015,8:935–50.3. AnaningsihVK.etal.GreenTeaCatechinsduringFoodProcessingandStorage:AReviewonStabilityandDetection.FoodResearchInternational2013,50:

469–79.


37

CHEMICALCHARACTERIZATIONOFJATOBÁ:OIL,FRUITANDFLOUR

GonçalvesRodriguesdasDoresR.1 ,CiceroN.3,4,PellizzeriV.3,FingerFL.2,SalvoA.3,MicalizziG.3,SignorinoV.3,GiuffridaD.3,DugoG.mo3,4

1UniversidadeFederaldeOuroPreto.CampusUniversitario,OuroPreto(MG)-Brazil;2UniversidadeFederaldeViçosa,Viçosa(MinasGerais)-Brazil;3DipartimentodiScienzebiomediche,odontoiatricheedelleimmaginimorfologicheefunzionali,Università

diMessina,Messina-Italy;4Science4lifes.r.l.,SpinOffCompanyUniversitàdiMessina,Messina-Italyemail:[email protected]

BraziliantropicalfruitsoccurredinCerradotypicalbiome,thatoriginally,coveredabout2,039.386km2,justunder24%oftheBrazilian territory. Because high incidence of endemic life forms, has placed the biome among the world's "hot spots"(ALHO,2005).ThenativefruitstreesoccupyaprominentplaceinCerradoecosystem.HymenaeacoubarilL.var.stilbocarpa(Hayne) Lee et Lang. (Caesalpinaceae), or Jatobá tree produces fruitswhich contain a flour pulpwhich is extracted andconsumed by native population for diet helps, to reduce the risk of gastrointestinal and coronary illnesses (LORENZI &MATOS, 2008). The aimof thiswordwas study thebiometry, characterized fresh anddrymass, polyphenols contents offruits,identifymajoritariancontentofbarkoil,andmineralcharacteristicallyofflourfruits.The fruitswerecollected inBrazil, transportedandmeasured innatura(integers,barkandseedout, flour). Thebiometryparameters was does to digital pachymetry and data measured in mm. The fruits were measured (mg and mm) andsegregatedinbark,seedsandflour.OilwasobtainedforhydrodistillationbyEuropeanPharmacopeia8th(2014)where100gdebarkwereextracted forwaterdistillation inClevengerapparatus for4h.Theoilobtainedwas reunitedandperceptualyieldwasestimated.TheCGanalyseswasexecutedShimadzuGCMS-QP2010system,andthevolumeinjectionwas0.4µL.The software used was GCMS solution (Shimadzu). Polyphenols analyzed to HPLC, compered con 25 standards andexpressedinppm.ThemineralanalyzedwasdoforIPC-MSandresultsexpressedinmgkg-1Themedianof length,width andweightof fruits, bark, seeds, flourwere 114.8613 ± 5.9089mm to41.0563 ± 2.0701mm,51.4589±4.1760g in fruits;wherebarkweightwas35.3274± 2.5618g, in flourwith seedsandonly flour were 16.0731 ±2.1006 g and 6.5886 ± 0.7437g, and seeds 8.9898 ± 3.0260 g by fruit. From to 100 g of Jatobá bark were obtained forhydrodistillation inClevengerapparatusabout2.0976±0.5260%ofessentialoil.Themajoritariancompoundsof jatobáoilwere Germacreno D, Alpha-copaene Cadineno-delta, Muurolene Gamma, Biciclogermacrene, Cadineno-gamma. Thepolyphenols identifywere tirosole (522ppm)andt-cinnamicacid (230ppm).Thenutritionalcompositionof flourwereNa4.954,Mg604.636,K1507.245,Ca917.821,Mn147.098,Fe45.579,Cu28.516mgKg-1(ppm),Cr0.287,Ni0.551,As0.071,Se0.380, Cd 0.125, Pb 0.181 ppm. The importance of this study was to obtain quality biometric parameter settings(organography)dependingonthefruitmaturationstage(maturefruits),toobtainessentialoilandflour.Ingeneral,greenfruit has a higher oil content as a function of the defense mechanisms against herbivores. Germacreno D (19.56%), thechemicalmarker, ispotentialanti-microbial,bactericideandantioxidant, justifyingtheethnomedicinaluse.Theantioxidantactivityinflourispresent,andinfunctionofpolyphenolswithcharacteristicsactbyantioxidantincellularmembranes.Thenutritional composition suggest theuseof this flour inpreparationsof functional foods, asbreads, cakesor foods rich incalciumusadjuvantalimentary.Acknowledgements:ToCNPq,FAPEMIGandUNIME.


38

POLYPHENOLS,MICROBIOTA,ANDBIFIDOBACTERIA:RECENTINSIGHTS

RossiM.1 ,García-VillalbaR.2,AmarettiA.1,RaimondiS.1,LeonardiA.1,QuartieriA.1,Tomàs-BarberànF.2

1Dept.ofLifeScineces,UniversityofModenaandReggioEmilia,Modena-Italy;2Dept.ofFoodScienceandTechnology,CEBAS-CSIC,Murcia-Spain

email:maddalena.rossi@unimore.itInformationfrommetagenomeanalysissuggestthatconsiderabledifferences ingutmicrobiotacompositionoccuramongsubjects,wherethisdiversityisresponsibleofdifferentmetabolicactivities.Consistently,studiesdealingwithbioavailabilityofphytochemicalmetabolitesdemonstratedthatdifferenttransformationsare largelyattributabletothediversebacterialpopulations.Objectsofthisstudywerechlorogenicacid(C-QA)andsecoisolariciresinoldiglucoside(SDG),amajorphenolicacidandoneof themost common lignans in thewestern diet, respectively. C-QA exerts several health effects, due to the antioxidantproperties. The dietary intake of lignans is recognized to exert several positive effects, due tometabolites produced bybacterial transformationsoccurring in thecolon.The residentmicrobiota releases theaglyconesecoisolariciresinol (SECO)and transforms it into theenterolignansenterodiol (ED)andenterolactone (EL) thatpresent improvedbioavailability andpotentbiologicalactivity,especiallyasphytoestrogens.Interindividual diversity in C-QA and SDG metabolism were explored. C-QA biotransformation were performed with 9different human colonic microbiota, through resting cells experiments. The diverse bacterial communities were able tohydrogenate, dehydroxylate C-QA, or break the quinic acid moiety also before hydrolyzing its ester bond. All thetransformation pathways converged on 3-(3-hydroxyphenyl)-propanoic acid, the finalmetabolite inmost of the samples,with some novel compoud identified for the first time in the transformation carried out with the microbiota of a solevolunteer.ToevaluatewhetherthefateofC-QAmaybemodifiedbythepresenceofapotentiallyprobioticstrainabletohydrolyzeC-QAtoCA,continuousmicrobiotaculturesweresupplementedwithB.animalissubsp.lactisWC0432,astrainableto hydrolyze C-QA into CA. The transformationpathways of C-QA in presenceofB. animalis subsp. lactisWC0432 did notchange,eventhoughthekineticswerediverse.Transformations carried out by colonic microbiota on SDG were performed to identify and quantify the metabolitesproduced in vitro by the human gut microbiota of five healthy subjects, to evaluate in vitro the efficacy of a potentialprobiotic strainofB.pseudocatenulatum able tohydrolyzeSDG, and to identify in vivo the lignanmetabolitesdetected invitroandtheirpossibleconjugatesthroughtheanalysisofurineandfecalsamplesofthesamevolunteersafteringestionofflaxseed.ThetwoglucosemoietiesofSDGweresuccessivelyremovedtoyieldSMG,andthenSECO.Deglycosylationwasnotthelimitingstepofthebiotransformationpathway,sincethemicrobiotaofallthesubjectswasefficientinreleasingSECO.Consistently, the utilization of the probiotic strain potentially increasing the release of the aglycone did not improve thebioavailability of enterolignans. SECO underwent successive dehydroxylations and demethylations yielding ED (4–18%conversion)andEL (0.2–6%)after24h.Novel intermediates related toSECO,matairesinolandanhydrosecoisolariciresinol,were identified infecalcultures.Thesemetaboliteswerealsofoundafterflaxseedconsumption infecesandurine intheirnativeformand/ormodifiedbyphaseIIhumanenzymes(glucuronide,sulfateandsulfoglucuronideconjugates).


39

CHARACTERIZATIONOFFOSANDINULINSBYHPAEC-PEDANALYSES

CorradiniC. ,BignardiC.,CavazzaA.,LaganàC.,SalvadeoP.

DepartmentofChemistry,UniversityofParma,Parma-Italyemail:[email protected]

Theanalysisofcarbohydratesinfoodhasextremenutritionalimportancebecausetheyareaprimarysourceofenergyandhave healthy beneficial effects resulting mainly from dietary fibers and other unavailable carbohydrates resistant todigestion.High-Performance Anion-Exchange Chromatography (HPAEC) coupled with pulsed electrochemical detection (PED) is apowerfulanalyticaltoolincarbohydrateseparationdueitsabilitytoseparateallclassesofalditols,aminosugars,mono,oligoand polisaccharides, according to structural features such as size, composition, anomericity and linkage isomerism.Moreover, this technique has been a major impact on the analysis of oligo- and polysaccharides. The compatibility ofelectrochemicaldetectionwithgradientelutioncoupledwith thehighselectivityof theanion-exchangestationaryphasesallowsmixturesofsimplesugars,aswellasoligo-andpolysaccharidessuchasfrurcooligosaccharides(FOS)andinulintobeseparatedwithhighresolutioninasinglerun.FOSandinulinareeffectiveprebiotics,whichbeneficiallyaffectthehostbyselectivelystimulatingthegrowthand/oractivityofthemicrobialgroups.Theyoccurnaturally inmanyfoodsofvegetableorigin,suchasonions,Jerusalemartichokes,asparagus,leeks,garlicandchicory.Carbohydrate analyses were performedwith a Dionex apparatus consisted of a GP50 low-pressure quaternary gradientpumpequippedwithanautosamplerAS50andapulsedelectrochemicaldetector (ED50)consistingofanamperometricflow through cell and twoelectrodes: a silver-silver chloride referenceelectrode andagoldworkingelectode. TheED50detectordeliveredtotheelectrochemicalcell thefollowingpotentialwaveform:E1=0,1V(t1=0.00-0.40s), integratingfrom0.20and0.40s,E2=-2.0V(t2=0.41-0.42s);E3=0.6V(t3=0.43s);E4=-0.1V(t4=0.44-0.50s).Elutions of FOS and inulin at different degree of polymerization were performed at room temperature using DionexCarbopaccolumnsandsuitableelutiongradientprograms.MALDI-MSmeasurementswereperformedusingaMALDI-LRTOFmassspectrometer(Micromass,Manchester,UK)operatinginthepositivelinearionmode.IonsformedbyapulsedUVlaserlamp(ʎ=337nm),wereacceleratedat15keV.Laserstrengthwasvariedfromsampletosampletoobtainthebestsignal.Forallsampledifferentmatricesweretested.FOSandinulinhavingdifferentdegreeofpolymerizationwerecharacterizedbyHPAEC-PEDmethodsspeciallyoptimizedandvalidated.TheassignmentofchromatographicpeakswasbasedonthegenerallyacceptedassumptionsthattheretentiontimeofahomologousseriesofcarbohydratesincreasedastheDPincreased.IntheinvestigatedshortchainFOSproducts,astudyonthedetectorresponse in relationshipwiththedegreeofpolymerizationwasalsocarriedout.Toverify thechainlenghdistributionoftheanalyzedFOSandinulionbyHPAEC-PED,thesameproductswereanalyzedbyMALDI-TOFMS.TheproposedHPAEC-PEDmethodsallow thedeterminationof FOSand inulnwhichareusually employedby food industry asfunctionalingredientseitherfortheirprebioticpropertiesorasafatreplacergivingafat-likemouthfeelandtexture.FOS and inulin at different degree of polymerization can be charactirize by optimizedHPAEC-PEDmethos. The proposedmethodsmethodmayalso contribute to clarify the real importance, theprebiotic effectivenessand thebiological roleofthesecarbohydrates.


40

MACRONUTRIENTCOMPOSITIONOFGIANTWATERBUG(LETHOSERUSSP.)EDIBLEINSECT

Melo-RuizV. ,Quirino-BarredaT.,Sánchez-HerreraK.,Márquez-CruzB.,Díaz-GarcíaR.

UniversidadAutónomaMetropolitana-X.Calz.delHueso,MéxicoCity-Méxicoemail:[email protected].

Macronutrientmalnutritionischaracterizedasadiseaseofpovertyinthemidstofplenty.Itmeansthatwhilemacronutrientsrichsourcesarereadilyavailabletheyarenotconsumeornotintakeonadequateamountsbyvulnerablegroupsthroughalack of awareness. In Mexico and Thailand there are a large reproduction of an under-utilized edible insect of theBelestomidae family, giant water bug, Lethoserus americanus, from Mexico and Lethoserus indicus from Thailand, bothinhabitantsof freshwater riversandstreams that canbeeasily collected either in theairbornewhenattracted tobrightlightsorwithnets fromthewater.Theaimof this studywasassessmacronutrient compositionofGiantWaterbug fromMexico and from Thailand to investigate if there is a difference in chemical composition of insects from two differentgeographicallocationswhereinsectsareconsumed.Insect sampleswere obtain in summer time 2014, at the ChangMai nightmarket in Thailand and conventional samplingcollected at Xochimilco canals of Mexico City, and macronutrients assess as proteins, lipids, minerals, fiber and solublecarbohydratesofadultsindrybasis,wasprovidedaccordingAOAC,1995techniques.DataobtainedforinsectsfromMexico1),andThailand2)were:1)proteins60.12%,2)53.11%;lipids1)5.72%,2)8.15%;minerals1)5.46%, 2) 6.75%; fiber 10.95%, 2) 12.23%; soluble carbohydrates 1)17.75%, 2) 19.74%. Themacronutrient composition of giantwaterbug,showadifferenceinmacronutrients,duetotheenvironmentfloraandfaunaofthewaterconditions,wheretheyacquire the nutrients necessary for growth and reproduction. Proteins are higher in insects fromMexico, but the rest ofmacronutrientsarelower.Giantwaterbugsareavailableallyearroundandareagoodsourceofproteinsnecessaryforthehumanhealth.


41

PROFILINGOFMAJORANDTRACEELEMENTSOFTHEMEDITERRANEANSEPIAINKBYIDUCTIVELY

COUPLED-MASSSPECTROMETRY

BuaDG.1,AnnuarioG.1,AlbergamoA.2 ,DonatoD.2,ZammutoV.3,CiceroN.1.2,CostaR.1,DugoG.mo1,21DepartmentofBiomedicalSciences,DentalandMorphologicalandFunctionalImages,UniversityofMessina,Messina-Italy;

2Science4LifeS.r.l.,aSpin-offoftheUniversityofMessina,Messina-Italy;3DepartmentofChemical,BiologicalandPharmaceuticalSciences,UniversityofMessina,Messina-Italy

email:[email protected], such as cuttlefish, contain very fewnon-edible portions and constitute a valid alternative to theover exploited fish and lean meat sources, due to a typical high protein/fat ratio,which makes them highlyvaluableforhumandiet.AlthoughtheyrepresentaminorfisherysourceinEurope,specificenvironmentalfactorsof theMediterranean area encourage their biodiversity and abundance (Keller et al., 2016), thus contributingpositivelytotheexpansionoffisheriesandtradingofcephalopodsandderivedproducts.Manystudiesalreadyfocusedonthenutritionalcompositionofcuttlefish’sediblepartscomingfromtheMediterraneanarea(Özyurtetal.,2006;Villanuevaetal.,2004),butnostudyhasbeenstillperformedontheassessmentofthenutritionalvalueoftheMediterraneancuttlefishink,increasinglyappreciatedintheMediterraneanculinaryculture.Theaimof thepresentworkwasto investigatethenutritionalcompositionof theSepiaofficinalis ink in termsofmajor(Na,Mg,K, Ca andP) and trace (Cu, Zn, Cr, Fe,Mo, Co,V,Ni,Mn,As, Cd, Pb andHg) elements, bymeansofinductivelycoupledplasmamass-spectrometry(ICP-MS).Black inks of Sepia officinalis specimens sampled in six different sampling sites, located along the north- andsouth-easternSiciliancoasts,wereconsidered.Eachinksamplewaspre-treatedbyamicrowavedigestionsystemand the major and trace element profile was subsequently characterized through a fully validated ICP-MSmethod.A Principal ComponentAnalysis (PCA)was also conducted to reducedatadimensionality and clustermajorandtracemetalcontentsdependingontheconsideredvariables(e.g. samplingsites).Furthermore,datawere statistically interpreted by chemometrics and a benefit/risk analysis was conducted to assess thenutrient/contaminantintakederivedfromtheconsumptionofSepiaink.Sepiainkrevealedtobebothareservoirof“healthy”elements,andasinkofpotentialtoxicmetals.Mg (6246.4±452.1μgg-1)andNa (6426±531.7μgg-1) showedtobe themostabundantmajorelements.Amongtraceelements,FeandCuwerefoundinconsiderablelevels(35.95±5.53μgg-1and11.09±1.28μgg-1,respectively),whiletrascurableamountsofCr,ZnandMowererevealedinallthesamples(<0.60μgg-1).HeavymetalssuchasAs,Cd,PbandHgshowedtoberelatedtotheenvironmentalconditionsofthesamplingarea.Indeed,inksamples from Capo Milazzo showed the highest levels of all considered elements (Pb 1.90±0.24 μg g-1, Hg0.90±0.08μgg-1,As0.90±0.04μgg-1andCd0.09±0.006μgg-1),duetothestrongpollutionofsuchindustrializedarea. The benefit/risk analysiswas performed considering a typical portion of Sepia ink (~3g). It highlighted agood contribution of major (K, Mg and Na) and essential trace metals (Cu and Fe) to the established RDAs.AlthoughparticularlyhighlevelswerefoundintheinksamplesfromCapoMilazzo,theassumptionofpotentiallytoxicelementsviaSepiainkdidnotrepresentariskforhumanconsumers.1. KellerS.etal.Large-scalespatio-temporalpatternsofMediterraneancephalopoddiversity.PloSone2016,11:e0146469.2. ÖzyurtG.etal.SeasonalchangesoffattyacidsofcuttlefishSepiaofficinalis(Mollusca:Cephalopoda)inthenortheasternMediterranean

sea.Foodchemistry2006,95,382-385.3. TayelF.T.etal.Assessmentofheavymetalsandnonessentialcontentofsomeedibleandsofttissues.EgyptianJ.AquaticRes.2007,33:

85-97


42

EFFECTSOFJASMONICACIDANDINDOLE-3-ACETICACIDINTHEVOLATILEPROFILEOFAnemia

TomentosaINVITROPLANTS,ANAROMATICFERN

NetoJF.1 ,BarretoCS.2,PintoSC.3,LeitãoSG.4,SilvaNCB.2 ¹UniversidadeFederaldoRiodeJaneiro(UFRJ),PósgraduaçãoemBiotecnologiaVegetal,CidadeUniversitária,RiodeJaneiro-Brazil;2UFRJ,

FaculdadeFarmácia,CidadeUniversitária,RiodeJaneiro-Brazil;3UFRJ,CampusMacaé,FaculdadedeFarmácia,Macaé-Brazil.email:[email protected];[email protected]

Ferns are the second largest group of plants. However, research related to their in vitro development aimed at theproductionofindividualsfortheornamentalplantsmarket,leavingthechemicalresearchbeside.TheessentialoilfromwildplantsofAnemiatomentosavar.anthriscifoliahasbeenstudiedandshowedthepresenceoftriquinane-typesesquiterpenesinwildplantsandinvitroplants(1)(2),whichareresponsibleforitswoodyscent.In thiswork,we tried tooptimize theproductionof the trichinane sesquiterpenes in in vitro culturesofA. tomentosa byaddingdifferentconcentrationsof theplantgrowthregulators jasmonicacid(0,1; 1; 10µmol)and indole-3-aceticacid(2.8;5.7;11.4µmol)totheMSmediumculture.Plant’sleafs(±5g)weresubjectedtoSDEextraction(SimultaneousDistillationandExtraction).ThesampleswereanalyzedbyGC/FIDandGC/MS inAgilent6890NandAgilent5973Nsystems,bothwithHP-5MS fused silica capillary columns Hydrogen was used as carrier gas for GC/FID and helium for GC/MS. The percentagecomposition was obtained by normalization from FID. Volatile compounds were identified by comparison of both massspectraandlinearretentionindiceswithspectrallibraryandliterature(3)Cultureswerealsoanalyzedfortheirfreshbiomassproduction(FB),drybiomass(DB)andnumberoffullyexpandedleaves.ThemajorcomponentsfoundinSDEextractswerethemonoterpenestrans-pinocarveol(24.23%),pinocarvone(19.73%)andα-pinene(17.04%).Themajoraveragesinfreshanddrybiomassproductionwereobservedintreatmentswithjasmonicacid(FB:0.8g;0.4g;0.25gDB:0.78g;0.35g;0.24grespectivelytotreatments0.1;1;10µmol),andtheloweraverageswerefoundinthetreatmentwith indole-3-aceticacid(FB:0.30g;0.17g;0.12gDB:0.22g;0.12g;0.08g).Also,thenumberoffullyexpandedleaveswas greater in the treatmentswith jasmonic acid (12, 10, 8 respectively to treatments 0.1;1;10 µmol). The chemicalprofileofthevolatilecomponentspresentedapredominanceofmonoterpenesoversesquiterpenesforthe invitroplants(bothtreatments)similarly topreviousworkwithouttheuseofgrowthregulators (2).Besidesan increase in thebiomassproduction,jasmonicacidincreasedthediversityofvolatiles,whereasindole-3-aceticacidappearstosuppressthisdiversity.1. Pinto SC. Chemical composition and antimycobacterial activity of the essential oil from Anemia tomentosa var. anthriscifolia. Natural Product

Communications,2009,4:1675-1678.2. PintoSC.etal.ChemicalcompositionofthevolatilefractionsfromwildandinvitroplantsofAnemiatomentosavar.anthriscifolia(Pteridophyta).Journalof

EssentialOilResearch,2013,25:198-202.1- 3AdamsR.P.IdentificationofEssentialOilComponentsbyGasChromatography/MassSpectrometry.4thedn.AlluredPubl.Corp.,CarolStream,2007.


43

TRANSFORMATIONOFFOODAGRICULTUREWASTEINTOVALUEADDEDPRODUCTS:

BIOETHANOLANDSCP

TropeaA.1 ,DiBellaG.1,PotortìAG.1,BuaG.D.1,GervasiT.1,DugoG.mo1,2¹UniversityofMessina,BIOMORFDept.,Messina-Italy;2Science4Lifesrl,Spin-OffCompany,UniversityofMessina,Sant’Agata

(Messina)-Italyemail:[email protected]

Currentlythereisincreasedinterestintheproductionofbioethanolandsinglecellprotein(SCP)fromrenewableresourcesandthere ismuchemphasisonexploitinglignocellulosesources,representingthemostabundantandlowcostmaterial intheworld(Germecetal.,2016).Somewastestreams,containbothcellulosicandnon-cellulosicsugars.Pineapplewastesarerichinintracellularsugarsandplantcellwallswhicharecomposedmainlyofcellulose,pecticsubstancesandhemicellulosessuitableforethanolproduction(Tropea,etal.2014).ThegoalofthisstudywastoproduceethanolandSCPfrompineapplewaste after plant cell walls enzymatic saccharification and co-fermentation carried out by two different strains,SaccharomycescerevisiaeATTC4126andPichiastipitisATCC58785.Fermentation testwas carriedout ina 5 Lbatch fermenter (C.BraunFermenter, Sartorius). The fermenterwasequippedwithonefour-bladedrushtonturbine,andthecommoncontrolsystems:temperatureandpHprobes,CO2detectorandgasmass flow meter. Pineapple wastes, enclosing fruit skin and core, were homogenized in a fruit blender. The resultinghomogenate,withadrymattercontentof14%(w/w),wasdilutedwithwatertoa9%drymatter,inaworkingvolumeof3.5Land immediately treated at 100˚C for 10 min under continuous mixing to inactivate endogenous enzymes and reducemicrobial spoilage. No further sterilization procedure was adopted. Before fermentation starting the media wassupplemented by digestive enzymes, urea phosphate, Ca-panthothenate and biotin (Tropea et al., 2015). Fermentationparameterswere:25°C,pH5.5andconstantstirringat200rpm.CO2evolutionwasmeasuredduringthefermentationandrepresentative samples were taken at regular intervals for ethanol, soluble and insoluble sugars determination by HRGC(Blakeney et al., 1983) methods and moreover protein determination by Kjeldahl method according to AOAC methods(2006).Substrateinitialfibersandsolublesugarswere26and44%respectively.Initialproteincontentwas4%.By 24 h soluble sugars and fiber utilization by the yeasts, as well as ethanol production stopped. The highest ethanolproductionwas 8%, reaching a 97% of the Theoretical Yield (TY) (Hossain et al., 2010). Though the alcohol concentrationobtainedappearsratherlow,dueofcoursetothelowsugarcontent,thisstudycouldbeattractivebecauseTY,calculatedondrymatterloss,wasreallyhighmakingthesewastesanexcellentrawmaterialforethanolproductionbyS.cerevisiaeandP.stipitis.Moreoversubstrateresultingfromthefermentationprocesswasenrichedinprotein,risinguptothe25.6%,makingthisbiomasssuitableforbothanimalfeedandenergyproduction.1. GermecM.etal.Ethanolproduction fromricehullusingPichiastipitisandoptimizationofacidpretreatmentanddetoxificationprocess.Biotechnology

progress.2016.DOI10.1002/btpr.2275.2. TropeaA.etal.BioethanolProductionFromPineappleWastes.JournalofFoodResearch.2014,3:60-70.3. TropeaA.etal.DevelopmentofminimalfermentationmediasupplementationforethanolproductionusingtwoSaccharomycescerevisiaestrains.Natural

ProductRes.2015,30:1009-16.4. BlakeneyAB.etal.Asimpleandrapidpreparationofalditolacetatesformonosaccharideanalysis.CarbohydrateResearch,1983,113:291-299.5. AssociationofOfficialAnalyticalChemist.2006.MethodsofficialofAnalysis.AOAC,Washington,DC.6. HossainA.,etal.Creationofalternativeenergybybio-ethanolproductionfrompineapplewasteandtheusageofitspropertiesforengine.AfricanJournal

ofMicrobiologyResearch,2010,4:813-819.


44

BIOCONVERSIONOFALKALINEHYDROLYSATESOBTAINEDFROMAGROINDUSTRIALWASTETO

PRODUCEFLAVORINGAGENTS

NumpaqueMA.,DurangoDL. ,GarcíaCM.,GilJH. UniversidadNacionaldeColombia,Medellín,Antioquia,Colombia

email:[email protected],[email protected]

Agro-industrialwaste is an important supply of useful renewable resources in the production of raw industrialmaterials;however,thepotentialusehasbeenlittleexplored.Ferulicacid(FA),fromagro-industrialwaste,constitutesaviablesourceofantioxidantsandprecursorsofflavoringagentsthatareof interest inthefood,cosmeticandpharmaceutical industries.Thisproductcanbeusedasasubstrate for thebiologicalsynthesisof"naturaladditives",offeringmultipleeconomicandenvironmentaladvantagessuchas lowcosts,highavailabilityof startingmaterialsandefficientuseofundervaluedwastethatcausesenvironmentalproblems.Abiotechnologicalprocesswasevaluatedtoobtainnatural flavoringagentsthroughthemicrobial conversion of FA (whichwas obtained through base hydrolysis of lignin rich agricultural byproducts) usingphytopathogenicfungi,suchasBotryodiplodiatheobromaeandColletotrichumacutatum.The FA content in the alkaline hydrolyzate, obtained from agro-industrial waste, such as white and yellow corn bran,corncobs,ricehusks,bagasse,bananapseudostems,coconuthusksandcoffeepulp,wasquantified.Threeliquidmedia(Czapeck-Dox,Sabouraudandhydrolyzed)wereformulatedtoevaluatethemycelialgrowthofthefungiB. theobromae and C. acutatum. Themediawere adjustedwith agar (2%) and the corncobswere hydrolyzated to a finalconcentrationof1000or1400mgFA/mL.Theradialgrowthofthefungiwasmeasuredandtheinhibitionpercentagewascalculated. The biotransformations of FA and the hydrolyzation were performed in a Czapeck-Dox medium in 250 mLErlenmeyer flasks (stirred shaker). The progress of the biotransformation was followed with the chromatographictechniquescoupledtoamassspectrometry(GC-MS).Afterperformingalkalinehydrolysisunderstandardconditions, thehighestFAcontent(1385mg/100g)wasobtainedfromthecorncobs.ThishydrolyzatewasusedasthesourceofAFinthebiotransformationprocesswiththefungi.Thecompounds4-ethylguaiacol,4-vinylguaiacol,vanillin,homovanillicalcoholandacetovanillinwereidentifiedasbiotransformationprocessmetabolites from FA by the fungi C. acutatum and B. theobromae. Taking into account the metabolites found in thebiotransformationprocesses,ametabolicpathwayforthebiotransformationofFAbyC.acutatumandB.theobromaewasproposed.For both fungi, it is observed that the hydrolyzate substantially limited the growth ofmicroorganisms, with amaximuminhibition of ~ 90% (C. acutatum) and ~ 88% (B. theobromae). Possibly, the hydrolyzate contained hydroxycinnamic andbenzoic acids,which inhibit the growth ofmicroorganisms, and lacked the nutrients needed for the development of thefungi.Tocarryoutthebiotransformationprocessusingthehydrolyzateasasubstrate,itwasnecessarytosupplementtheculturemediumwithnutrients;theCzapeck-Doxmediumwasappropriateforobtainingflavoringagentssuchas4-ethylguaiacol,4-vinylguaiacolandvanillin,amongothers.1. DiGioia,D.,Luziatelli,F.,Negroni,A.,Ficca,A.G.,Fava,F.,yRuzzi,M.(2011).MetabolicengineeringofPseudomonasfluorescensfortheproductionof

vanillinfromferulicacid.JournalofBiotechnology,156(4),309–316..2. Kumar,N.,PruthiV.(2014).Potentialapplicationsofferulicacidfromnaturalsources.BiotechnologyReports,4,86–93.


45

THERESCUEOFCOFFEEFRUIT(Coffeaarábica)ASAFUNCTIONALFOOD

GarcíaR.1 ,PastorN.2

1FacultaddeQuímicoFarmacobiología,UMSNH,Morelia(Mich.)-México;2FacultadIngenieriaCivil,UMSNH,CiudadUniversitaria,Morelia(Mich.)-México


The food industry releases large amounts of byproducts, which are a source of serious pollution and environmentalproblems, posing a threat to the environment by contaminating water and soil. Coffee is one of the most tradedcommoditiesintheworld,butgeneratesalotofwasteproductsduringprocessingfromthecoffeefruittothedrink1.Peels,pulpandskincomprisealmost45%ofthecoffeefruit,andarethemainproductsoffood industry.Despiterecentfindingsdemonstratinghighlevelsofantioxidantcompoundsandotherphytonutrients,littleprogresshasbeenmadeinitsuseasanadditiveorasafoodperse2.Aimofthisworkwastoobtainafunctionalfood,bydevelopingasnackmadeoutofcoffeepulp(Coffea arábica) dehydrated with a solar dehydrator, roasted and finally covered with chocolate. For this purposes, twochocolate substitutes were used, the SILKO SUMMER AND CEBES LSX-80which were provided by the Company AarhusKarlshamnMexico,S.AdeC.V.andwerecomparedwithatrademarkMoctezuma®Chocolate.PulpCoffea arábica cherrywas used already in themature state, and cropswere located in areas surrounding the city ofMorelia,Michoacán.Coffeecherrypulpwaswashedandseparatedmanuallyforsubsequentdehydrationandanalyses.Aftersolar drying, the pulp was dried at 100 0C for 10 min in order to obtain a crispy texture. Dehydrated and roasted pulpunderwent total polyphenols quantitation by Folin Ciocalteu methodology. The roasted pulp was then covered withchocolatesubstitutes:SILKOSUMMERANDCEBESLSX-80andresultscomparedwiththoseobtainedusingacoverofthetrademarkMoctezuma®Chocolate.Sensory studieswereconductedusing testingpanels for choosing thebest coverage.Finally, the finished product underwent additional analyses to quantify major components and to determine the energycontentofthisnewsnack.After solardehydration, themoisturecontent in thepulpwas 12.72%.Resultsofproximateanalysis showedthatpulpwascharacerizedbymoisture(13.72%),ash(6.16%),totalfat(8.85%),protein(3.85%),dietaryfiber(29.26%)andtotalcarbohydrates(38.16%).Highmethoxylpectinwas 1.35% inhulls and4.58% inpulp. The caffeine content in thepulpwas0.42%. The totalpolyphenol resulted in a concentration of 2.09 mg/L. Sensory results concerning the cover (substitutes vs commercialchocolate) indicate a greater acceptance of the formulation using the commercial chocolate. Proximate analysis of thefinishedproductrevealedcarbohydrates(32.0%),fiber(29.9%),proteins(3.8%)andlipids(22.8%).Finally,asnackportionof100gwillprovide378.3Kcal.1. RamirezCoronelM.et al. Estudiosdel contenidodeácidoshidroxicinamicosde lapulpadel cafèpara subiotransformacion. XII CongresoNacional de

BiotecnologiayBioingenieria,20112.CastilloS,etal.Algodiferenteenelmundodelossnacks:Chipsdevegetales.2009


46

METABOLITEFINGERPRINTINGOFPUNICAGRANATUML.(POMEGRANATE)POLYPHENOLSBYMEANS

OFHPLC-UV/DAD,ESI-MSANDMS2

PellatiF.1 ,BrighentiV.1,GroothuisSF.1,2,PrencipeFP.1,AmirR.3,4,BenvenutiS.1¹DepartmentofLifeSciences,UniversityofModenaandReggioEmilia,Modena-Italy;²HanzeUniversityofAppliedSciences,InstituteofLife

ScienceandTechnology,Groningen-TheNetherlands;3LaboratoryofPlantScience,MigalGalileeTechnologyCenter,KiryatShmona-Israel;4Tel-HaiCollege,UpperGalilee-Israel

email:[email protected].,commonlyknownaspomegranate,isanancientfruitwidelyconsumedallovertheworldasfreshfruitorjuice.Inaddition,itisextensivelyusedintherapeuticformulas,cosmeticsandfoodseasonings.Theincreasinginterestinits health-promoting properties is fully justified by themost recent findings, according towhich the fruit can be a usefulagentfortreatingawiderangeofhumandisordersanddiseases.Pomegranatejuiceisusedincosmeticsandfoodforitswell-knownantioxidantactivity.Otherpropertiesaredocumentedforpomegranatemesocarp and exocarp extracts, such as anti-inflammatory, antimicrobial and anticancer; these extracts arealsoabletoexertacardiovascularprotectiverole.Thewiderangeofhealth-promotingbiologicalactivitiesdescribedabovearerelatedtothesynergisticpresenceofauniqueandcomplexphytochemicalcompositionthatencompassesanthocyanins,phenolicacidsandhydrolyzabletannins.However, despite the large number of studies on pomegranate chemical composition, there is no validated analyticalmethod that includes the simultaneous determination of all pomegranate constituents. In the light of all the above, thedevelopment and validation of a new highly efficient analyticalmethod for the completemetabolite fingerprinting of allpomegranatepolyphenolsbymeansHPLC-UV/DAD,ESI-MSandMS2wasperformedinthisstudyforthefirsttime.While pomegranate juice was directly analysed after simple centrifugation, different extraction techniques, includingmaceration, heat reflux extraction, ultrasound-assisted extraction and microwave-assisted extraction, were compared inordertoobtainahighyieldofthetargetanalytesfrompomegranatepeels.Dynamicmacerationwithamixtureofwaterandethanol 80:20 (v/v)with 0.1% hydrochloric acid as the extraction solvent provided the best result in terms of recovery ofpomegranatesecondarymetabolites.Thequali-andquantitativeanalysisofpomegranatesecondarymetaboliteswasperformedbyHPLC-UV/DAD,HPLC-ESI-MSandMS2analyses.Theapplicationofthefused-corecolumntechnologyallowedustoobtainan improvementoftheHPLCperformance incomparisonwiththatofconventionalparticulatestationaryphases,thusenablingagoodseparationofallconstituentsinashortertimeandwithlowsolventusage.TheanalyticalmethodwascompletelyvalidatedtoshowcompliancewithICHguidelinesandthenitwassuccessfullyappliedto the chemical characterization of commercial and experimental pomegranate cultivars, thus demonstrating to be anefficient tool for the fingerprinting of this plant material. The quantitative data collected were submitted to principalcomponent analysis (PCA), in order to highlight the possible presence of pomegranate varieties with high nutraceuticalvalue.Fromthestatisticalanalysis,fourexperimentalvarietiesshowedaremarkablecontentofbioactivecompoundsinthepeels,whilecommercialvarietiesstillrepresentthebestsourceofhealthyjuice.The ability to fully analyze different parts of pomegranate cultivars conform to the ICH guidelines could be useful in thechemical analysis of nutraceutical and food products containing this plant extract. This research showed also the greatpotential that pomegranate peels have in relation to hydrolysable tannins and could turn the current waste stream ofpomegranatepeelsintoanewsourceofhealthyanimalfoodorherbalproducts.


47

TYPIFICATIONOFAVOCADOFRUIT(PerseaamericanaMill.)CULTIVATEDINCOLOMBIABY

MULTIVARIATESTATISTICALANALYSISWITHAVIEWTOWARDINDUSTRIALIZATION

RobayoMAT. ,DelgadoAWA.,CucaSLE.UniversidadNacionaldeColombia,DepartamentodeQuímica,Bogotá-Colombia

email:atrobayom@unal.edu.coInColombiaavocadoproductionisgrowingandstandsitasthethirdworldproducerafterMexicoandDominicanRepublic(FAO Statistical; 2013); commercial crops of avocados var. guatemalensis, var. americana (known as ‘criollos’) and hybridsbetween them are distributed throughout Colombian territory (Perea, M; 2010), therefore avocado fruits with differentphenotypic characteristics such as shape, size, epicarp color as well as taste and texture can be found. In this studyseventeenavocadosamplescultivated invariousregionsofColombiawere includedmainlyforfurther informationonthisfruit.Moreover,wewonder if theypresentvariationonfatcontentandfattyacidprofile toselect thebestones for foodindustryandtoindustrialoilextractionpurposes.The fruits were weighed and epicarp, mesocarp and endocarp were separated from each other. Mesocarp was vacuumdryingandfatcontentwasdeterminatedbySoxhletextraction.FattyextractswerederivatizedwiththecomplexBF3/MeOH14%(AOCS;1973)andthefattyacidmethylesterswereanalyzedbyGC/MS.Dataobtainedwereprocessedusingmultivariatestatisticalanalysis.Avocadofruitsincludedinthisstudyhaveweightsbetween60.1and606.8g.Epicarpproportionisvaryingfrom6.3to18.6%;mesocarpproportionfrom42.8to80.5%andendocarpfrom7.8to39.2%.Crudefatcontent isbetween5.4and28.4%w.b.whichcorrespondtoacalorificvaluefrom72.9to274.0kcal/100gfreshpulp.CultivarsGuatemalteco,HassandFuertehadthehighestfatcontent,expressedaspercentoffreshpulp(mean±standarddeviation):27.9%±0.5,25.5%±0.1and24.8±0.1,respectively. This feature show themas cultivars interesting for industrialoil extractionpurposes.Monounsaturated fattyacids-MUFA’s-were themajor constituents in avocadooil regardlessof cultivar (56.8-83.3%);oleic acidwas theprincipalMUFAwithvaluesbetween36.6and73.2%, followedbypalmitoleicacid (4.2- 15.0%).Palmitic (13.2-32.8%)andstearic (0.5-1.2%)acidswerethesaturatedfattyacidsfoundinavocadooil.Polyunsaturatedfattyacids-PUFA’s-aslinoleicacid(7.7-16.5%)and linolenicacid (0.4-1.6%)were found.Avocadosvar.guatemalensishad themajorconcentrationofMUFA’s (78.3-82.8%)andminorcontentofsaturatedfattyacids (13.8-17.5%).Avocadosvar.americanahadhigherproportionsofsaturatedfattyacids (28.5- 33.7%) and less content of MUFA’s (56.8-59.7%). A principal component analysis including the morphologicalvariablesandfattyacidprofileforeachcultivar,showsthattheseventeensamplescanbetypifiedintofourgroups.Thefirstgroupiscomposedofcultivarswithcharacteristicsofavocadosvar.guatemalensis.Asecondgroupisintegratedbycultivarswith featuresofavocadosvar. americana.The thirdcluster is locatedbetween theabove twogroupsandcontainshybridcultivars of avocado. Cultivar grown in Santa Bárbara- Santander forms the last cluster and presents a particular aniseedsmellthatischaracteristicofavocadosvar.drymifolia.Inconclusion,thereisagreatvariabilitybetweendifferentsamplesincludedinthisstudybasedontheirphenotypicfeaturesandcontentofmetabolites,whichmayberesultfromgeneticdiversity;thereforefourclustersthatobeythethreeavocadoracesandhybridsofthesewereformed.Thisstatisticalmodelallowsapproachesongeneticdiversityofcultivarswhichtheirgenotypearenotknown;nevertheless,thisinformationmustbeconfirmedbyanalysisofgeneticmaterial.Acknowledgments:TheauthorsacknowledgethefinancialsupportprovidedbyFondoNacionaldeFinanciamientoparalaCiencia,laTecnologíaylaInnovación,FranciscoJosédeCaldas(ContractNo.0459-2013,RIFRUTBIO)andtheDepartmentofChemistryatUniversidadNacionaldeColombia-SedeBogotá.1. AOCS.1973.MethodCe2-66.2. FAO.2013.Availableat:http://faostat3.fao.org/compare/E.Accesson:18Mar2016.3. Perea,M.et.al.2010.Biotecnologíaaplicadaalmejoramientodeloscultivosdefrutastropicales.Ed.UNALColombia.p.233-259.


48

VALIDATIONOFANALYTICALMETHODBYHPLC-DADFORIDENTIFICATIONANDQUANTIFICATIONOF

CAFFEOYLQUINICACIDDERIVATIVESINCYNARASCOLYMUSL.TABLETSANDCAPSULES

CarneiroAA.,SimeoniLA.,MagalhãesPO.,SilveiraD.,Fonseca-BazzoYM. FacultyofHealthScience,UniversityofBrasilia,Brasília-Brazil.

email:[email protected]. leaves,popularlyknownasartichoke,havecholagogueandcholereticspropertiesandareusedforthetreatmentofdigestivedisorders. Itseffectsareattributedtocaffeoylquinicacidderivatives.ThisstudyaimedatvalidatingtheanalyticalmethodfordeterminationofcaffeoylquinicacidderivativesinartichoketabletsandcapsulesbyHPLC-DAD.ItwasusedaC18column(250x4.6mm,5um)withtemperaturemaintainedat40°C,flowrate1.2mL/min,injectionvolumeof25µLanddetectionat330nm.Themobilephasewascomposedbyagradientofphosphoricacid0.5%andphosphoricacid0.5%inacetonitrile.Sampleswerepreparedattheconcentrationof4mg/mLinamixtureofmethanolandwater(3:7),usingnot less than 20 tablets and accuratelyweighing thequantity equivalent to the labeled amountof artichoke extract. Themethod was adapted from European Pharmacopoeia - 7th edition to the finished product and inclusion of markerscryptochlorogenic, neochlorogenic, isochlorogenic acids and cynarin. Method selectivity evaluation was carried outcomparingthreesamples:onewithouthydrolysis induction,onewithacidichydrolysis,andtheotherwithbasichydrolysis.Forprecisionanalysis,sixsamplesat100%concentrationwereanalyzed inthesameday.Linearityofstandardcompoundswasperformedbythreeanalyticcurveswithsixconcentrationsforeachcompound.Fromthelinearitycurveitwaspossibletocalculatethequantificationanddetectionlimits.Theaccuracyevaluationwascarriedoutthroughthestandardadditionmethodatconcentrationsof80, 100and120%withthreereplicates.Finally,3commercialproductsofartichoketabletsorcapsuleswereevaluatedtodeterminethecaffeoylquinicacidderivativescontents.Themethodproved to be selective, precise and accurate. Furthermore, the standard compounds showed linearity in theconcentrationrangefrom1to50µg/mL.Detectionandquantificationlimitsofstandardscompoundsranged0.20to1.05and0.68to3.48µg/mL,respectively.CommercialproductsA,BandChaddifferentlabeledamountofartichokeextract(200,335and300mg,respectively)anddifferentconcentrationsofstandardcompoundspermgofextract.BrandBwastheonlyonethatpresentedcynarinon itsprofileandgotsignificantlyhigherconcentrations (p<0.005atone-wayANOVAfollowedbytukey)ofneochlorogenic,chlorogenicandisochlorogenicAandCacidsinrelationtoothercommercialproducts.Moreover,brandBhadthehighesttotalcontentofcaffeoylquinicacidderivatives(26.73µg/mg)followedbyC(9.32µg/mg)andA(8.47µg/mg).Suchdifferencesmaybringasignificantimpactontherapeuticoutcome.Inconclusion,themethodwassuitablefortheidentificationandquantificationofcaffeoylquinicacidderivativesintabletsandcapsules,whichcouldbeusedinqualitycontrol,ensuringthesafetyandefficacyofherbalmedicine.Acknowledgement:FAPDF,CAPES,CNPq,FINATECandDPP-UniversityofBrasilia.1. EMA.EuropeanMedicinesAgency,2011.AssessmentreportonCynarascolymusL.,folium.Availableat:

http://www.ema.europa.eu/docs/en_GB/document_library/Herbal_HMPC_assessment_report/2011/12/WC500119940.pdf.Accesson:10may2016.2. EMA.EuropeanMedicinesAgency,2011.CommunityherbalmonographonCynarascolymusL.folium.Availableat:

http://www.ema.europa.eu/docs/en_GB/document_library/Herbal__Community_herbal_monograph/2011/12/WC500119942.pdf.Accesson:10may2016.3. EuropeanPharmacopoeia.7thedition.Strasbourg:CouncilofEurope,2010.


49

ESSENTIALOILS:PHYTOCHEMICALCOMPOSITION,CYTOTOXICITYANDANTIMICROBIALACTIVITY

TardugnoR. ,PellatiF.,BenvenutiS.

UniversityofModenaandReggioEmilia,DepartmentofLifeSciences,Modena-Italyemail:[email protected]

Essential oils (EOs) are very complexmixtures composedof natural volatile constituents. They are producedby aromaticplant specializedglands asdefensive secondarymetabolites [1, 2]. EOs arewell known from theMiddleAges and canbeobtainedinmostcasesbysteamorhydro-distillation[1,2].Nowadays,theuseofthesephytocomplexesisincreasingbothinethnomedicine and in companies involved in the pharmaceutical, agronomic, food and cosmetic fields. Due to the EOcomplexity,deepqualitativeandquantitative investigationsarenecessarytoexploretheircomposition,toguaranteetheirgenuinenessandtoallowarationalinvestigationabouttheirpotentialbiologicalactivities.Inthelightofalltheabove,thisresearchprojectwas focusedon thedevelopmentofhighlyefficientmethods tostudy thephytochemical compositionofseveralEOs.All the EOs were analysed both on Agilent gaschromatograph coupled with mass spectrometer (GC-MS) and Agilentgaschromatograph coupled with flame ionization detector (GC-FID), for qualitative and semi-quantitative purposes,respectively.CompoundswereseparatedonanAgilentTechnologiesHP-5cross-linked5%diphenyl-95%dimethylpolysiloxane(30mx0.32mmi.d.,0.25mmfilmthickness)capillarycolumn.Theelutionwasobtainedunderprogrammed-temperature.Heliumwasusedasthecarriergas.Compoundswereidentifiedbycomparisonoftheirlinearretentionindices(LRI)relativetoC8-C40n-alkanesobtainedontheHP-5columnwiththoseprovidedintheliterature[3].Furthermore,theidentificationofthe several constituents has been carried out by comparison of their mass spectra with those recorded in the NationalInstituteofStandardsandTechnology(NISTversion2.0d,2005)and,whenitwasnecessary,the identificationwascarriedoutbyco-injectionofavailablereferencecompounds.Relativepercentageamountofindividualcomponentswasexpressedas the percent peak area relative to the total composition of the essential oil obtained by the GC-FID analysis. Semi-quantitativedatawereacquiredasthemeanoftriplicateanalysesforeachsample.The studywas focused on EOs obtained from plants cultivated in Italy or in other countries,with particular attention toLavandulaangustifoliaP.Miller,LavandulaxintermediaEmericexLoisel.,ThymusvulgarisL.andCedrelopsisgreveiH.Baillon.The chemical characterization developed in this work had a key role in classification, selection and explanation of thepossiblebiologicalactivityoftheeachEO,whichwasevaluatedincollaborationwithotherinstitutions,oncelllinesincludingA549,Caco-2,Hep-2andWKD.Moreover,EOsweretestedonseveralmicroorganismssuchas,Candidaalbicans,Escherichiacoli,Listeriamonocytogenes,Pseudomonas aeruginosa,Salmonella enterica, Staphylococcus aureus,Streptococcus agalactiaeandStreptococcusmutans.ThestudyofEOscompositionandthebiologicalpropertieshighlightedinterestingcorrelationsbetweentheanalytesandthebioactivity.Asdemonstratedby the resultsobtained, thedevelopmentandapplicationofanalyticalmethods tostudy theEOscompositionishighlyrecommendedtoensuretheirgenuinenessandtherapeuticefficacy.Finally,theachievedresultsmightbeavaluablecontributionintheselectionofeffectivephytocomplexesandmoleculestofightmicrobialinfections,ashighlightedbyWHO.1. BakkaliF.etal.Biologicaleffectsofessentialoils–areview.Food.Chem.Toxicol.2008,46:446-475.2. ShaabanaHAE.etal.,Bioactivityofessentialoilsandtheirvolatilearomacomponents:Review.J.Essent.OilRes.2012,24:203-212.3. AdamsRP.Identificationofessentialoilsbygaschromatography/massspectrometry.CarolStream:AlluredPublishingCorporation(2007).


50

DETERMINATIONOFSQUALENEINE.V.O.O.BYDIFFERENTANALYTICALTECHNIQUES

SalvoA.1,2 ,RotondoA.1,LaTorreGL.1 ,ManganoV.1,CasaleKE.1,CiceroN.1,2 ,PellizzeriV.1,Saija

E.1,DugoG.mo1,21DepartmentofBiomedicalandDentalScienceandMorphofunctionalImaging,Messina-Italy;2Science4Lifesrl,SpinOffCompany,

UniversityofMessina,Messina-Italyemail:[email protected];[email protected];[email protected]

Squalene(SQ)isanisoprenoidcompoundwith30carbonatoms,containingsixdoublebondsanditispresentmainlyintheoil of cod liver. For the first time it was isolated from the oil liver shark, but is widely distributed in nature in both thevegetableandanimaltissues(Tsujimoto,1916).Inhumans,about60%ofSQisabsorbedfromfoodandmainsourceisoliveoil,particularlyextravirginoliveoil (E.V.O.O.)whichcontainsabout0.2-0.7% (Smitth,2000).Manystudies reportdifferentbeneficialpropertiesofSQsuchas:a)antioxidanteffect;b)protectiveeffectagainstskinphoto-oxidation,c)abilitytoboosttheimmunesystem,d)aptitudetoadjusttheamountofcarrierproteinsofLDLfat(Kelly,1999;Nicolaidesetal.,1968).OneofthefirststudiesrelatedtotheSQdeterminationinvolvedacolorimetricmethod(Rothblatetal.,1962);generallySQinfoods, oils and fats was determined by titrimetric (AOAC, 1999) or chromatographic procedures (HPLC, GC, hypenatedchromatographic techniques) (Popa et al, 2015). The purpose of this study was to compare three different analyticaltechniques(UPLC-DAD,GC-MSandNMR)fortheSQquantificationinEVOOs.A fast UPLC-DAD method was used for SQ determination in 33 biological EVOO samples. Analytical sample treatmentincludedtheclean-uppurificationbyusingDiscoverySPEDSC-SiSiliceTubes.TheSQanalysiswascarriedoutusinganAcquityUPLC®WatersBEHC18columnof1.7mm(2.1x50mm).TheGC-MS analysiswere carried out on samples previously subjected to chromatographic purificationon columnpackedwithglasswoolanddeactivatedsilica.ThemassspectrometerwasusedinfullscanandinSIMmodefortheidentificationofthecharacteristicfragments.1HNMRanalysisrequiredthesimpledilutionof125mlofsamplein475µldiCDCl3.Standard1Hspectrawererunat500MHzwith 70° pulses repeated for 64 transients, 2 sec as acquisition, and 2 sec of relaxation time (about 10 minutes ofexperimentaltime).TheUPLCmethodhasprovedusefulandversatile,thesamplepreparationwasfoundtoberelativelyfast(about30min)andthechromatographicruntookonlyabout3min.TheGCmethodshowedagoodsensitivityandreproducibility,howeverthistechniqueprovidedforlongsamplepreparationtimeandlongtimeofanalysis.TheNMR,ontheotherhand,showedthat inabout15min, includingsampling, integrationandquantification,turnsouttohaveagoodprecisionandaccuracy.StatisticanalysisofthedatashowedthatallEVOOsaregroupedbycountryoforiginandthattheSicilianshavemoreSQthanthesamplesofothercountries.1. Tsujimoto,M.A.,1916..HighlyUnsaturatedHydrocarboninSharkLiverOil.JournalofChemical&Engineering,8,889-896.2. Smith,T.J.,2000.Squalene:potentialchemopreventiveagent.Expertopinionofinvestigationaldrugs,9,1841-1848.3. Kelly,G.S.(1999).Squaleneanditspotentialclinicaluses.Journalofclinicaltherapeutic,4,29-36.4. Nicolaides,N.etal.,1968.Theskinsurfacelipidsonmancomparedwiththoseofeighteenspeciesofanimals.RiceJournalofinvestigativeDermatology,

51,83-89.5. Rothblat,G.H.etal.,1962.Aquantitativecolorimetricassayforsqualene.AnalyticalBiochemistry,4,52–56.6. Association of Official Analytical Chemists (AOAC). Officialmethod of analysis (OMA). 1999. Squalene in Oils and Fats. TitrimetricMethod. Official

MethodsofAnalysisofAOACInternational,16thedn.,AOACInternational,Arlington,AOACOfficialMethod41.1.40.7. Popa,O.etal.,2015.Methodsforobtaininganddeterminationofsqualenefromnaturalsources.BioMedResearch International,Article ID367202,16

pages,http://dx.doi.org/10.1155/2015/367202.


51

CHEMICALSTUDIESONPSIDIUMFRIEDRICHSTHALIANUMNIED.FRUITFLAVOURANDTHEIR

PRECURSORS

CuadradoCT.1,DelPozo-BayónM.2,OsorioC.1 1DepartamentodeQuímica,UniversidadNacionaldeColombia-SedeBogotá,Bogotá-Colombia;2InstitutodeInvestigaciónen

CienciasdelaAlimentación(CIAL)(CSIC-UAM),CampusdeCantoblanco,Madrid-Spainemail:[email protected]

TheMyrtaceae family compriseswoody trees and shrubs containing a variety of oilswhich release characteristic odours.Centresofdiversity for thefamilyarethewest tropics (mainlySouthAmerica),Australia,andtropicalAsia.Thereare fourgeneraofinterestwhichproduceediblefruit:Psidium,Eugenia,Syzygium,andFeijoa.Amongthem,“guayabaagria”orsourguava (Psidium friedrichsthalianum Nied.) grows naturally from southernMexico to northern South America. The fruit isconsumed fresh (pulp)or it isused toprepared juicesandsorbets,andexhibitsapleasantand intensearoma.Untilnow,thereisonlyonestudythatreportsthevolatilecompositionofthisfruit[1],howevernoanysensoryanalysiswasperformedbeforetoidentifytheodour-activevolatiles.Theodour-activevolatilesofwholefruitswereisolatedbySolventAssistedFlavourEvaporation(SAFE)ofdichloromethaneextract. GC-O andGCMS analyses allow identifying all volatile organic compounds relevant to the flavour of this fruit, bycomparisonwith standards.A thiol-rich fractionwasobtainedby affinity chromatographyonmercurated agarosegel [2].Theglycosidic-richfractionwasisolatedbyselectiveadsorptiononAmberliteXAD-4glasscolumnandmethanolelution[3].Glycosidically bound volatiles were released by enzymatic hydrolysis with a b-glucosidase (emulsin, Sigma-Aldrich) andanalysedalsobyGCMS.Basedon thepresenceof sulphurvolatilecompounds, thecysteine-S-conjugateswere isolatedbyusing a Dowex 50WX8 resin and eluting with ammonia [4]. The identity of glucosides and cysteine-S-conjugates wereelucidatedfromtheiranalysesbyHPLC-ESI/MS.GC-OandGC-MSanalysesofP. friedrichsthalianum fruit SAFEextract allow identifyingethylbutanoate, (Z)-3-hexenal, andethyl hexanoate as key aroma compounds of this fruit. Other relevant odorants to the overall aroma were the sulphurcompounds: dimethyl disulfide, 2-methyl 1,3-dithiolane, methional, 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, andbis(methylthio)methane,aswellas,d-dodecalactone.Sulphurcompoundswerereportedhereforfirsttimeasodour-activevolatiles in this fruit and based on sensory experiments, they could be considered the responsible for the characteristicflavour of this fruit. Hexyl b-D-glucopyranoside was identified as aroma precursor after HPLC-ESI/MS analyses of theglycosidic mixture and GCMS analyses of enzymatically released volatiles with a glucosidase. The role of hexyl-b-D-glucopyranoside and 3-(1-hexanol)-L-cysteine as precursors of hexanol, and 3-sulfanyl hexanol, respectively, wasdemonstratedbyenzymatichydrolysisofcorrespondingprecursormixtures.Inconclusion,therelevanceofaliphaticesters,C6-compounds, and sulphur compounds as odour-active volatiles of P. friedrichsthalianum fruit was demonstrated in thisstudythroughthecorrelationofanalyticaldataandsensoryanalyses.Acknowledgments:AuthorsgreatlyappreciatethefinancialsupportprovidedbyFondoNacionaldeFinanciamientoparalaCiencia,laTecnologíaylaInnovación,FranciscoJosédeCaldas,contractNo.0459–2013,RedNacionalparalaBioprospeccióndeFrutasTropicales-RIFRUTBIO.1. PinoJ.A.etal.CharacterizationofvolatilesinCostaRicanguava[Psidiumfriedrichsthalianum(Berg)Niedenzu]fruit.J.AgricFood.Chem.2002,50:

6023−6026.2. SteinhausM.etal.Comparisonofthemostodour-activevolatilesindifferenthopvarietiesbyapplicationofacomparativearomaextractdilutionanalysis.

Eur.FoodRes.Tech.2007,226:45–55.3. MayorgaH.etal.Hydroxyesterdisaccharidesfromfruitsofcapegooseberry(Physalisperuviana).Phytochemistry2002,59:439-445.4. StarkenmannC.andNiclass,Y.Newcysteine-S-conjugateprecursorsofvolatilesulfurcompoundsinbellpeppers(CapsicumannuumL.cultivar).J.Agric.

FoodChem.2011,59:3358–3365


52

FLAVONOIDSASASCAFFOLDFORTHEDEVELOPMENTOFANTITRYPANOSOMATIDICAGENTS

BorsariC.1,LincianoP.1,Cordeiro-da-SilvaA.2,GulS.3,AlundaJM.4,ClosJ.5,TaitA.1,CostantinoL.1,PellatiF.1,Wade,R.C.6;Mangani,S.7;Ferrari,S.1;Costi,M.P.1

1DipartimentodiScienzedellaVita,UniversitàdegliStudidiModenaeReggioEmilia,Modena-Italy;2IBMC-InstitutodeBiologiaMoleculareCelular,UniversidadedoPorto,Oporto-Portogual;3FraunhoferInstituteforMolecularBiologyandAppliedEcologyScreeningPort(Fraunhofer-IMESP),Hamburg-Germany;4ComplutenseUniversityofMadrid,CiudadUniversitaria,Madrid-Spain;5Bernhard-Nocht-InstitutfuerTropenmedizin(Bnitm)Hamburg-Germany;6HeidelbergInstituteforTheoreticalStudies(HITS)

GmbH,Heidelberg-Germany;7DipartimentodiBiotecnologia,ChimicaeFarmacia,UniverisitàdiSiena,Siena-Italy.email:[email protected]

Parasites of the family of Trypanosomatidae are agents of serious human diseases, including African sleeping sickness,ChagasdiseaseandLeishmaniasis.Sincedrugscurrently inusehave limitations intermsofsafety,efficacyanddurationoftreatment, there is an urgent requirement for new effective drugs1. The folate pathway is a successful target for thetreatmentofbacterialinfectionsandsomeparasiticdiseases(e.g.malaria).However,theclassicalinhibitorsofdihydrofolatereductase (DHFR) are ineffective against Leishmania and Trypanosoma because Pteridin Reductase 1 (PTR1), atrypanosomatidicenzyme,overlapsDHFRactivity2-3.Therefore,PTR1isapromisingtargetforthedevelopmentofimprovedtherapies.Computational studieswere performed to screen a library of 98 natural compounds fromplants of different origins.Wecombined target-based screening on pteridine reductase 1 with a phenotypic screening on Trypanosoma brucei, for hitidentification.FlavonoidsturnedouttobeaninterestingclasstobeexploredasPTR1inhibitors.Eventhoughflavonoidshavebeenwidelyexploredandoftenpleiotropicpropertiescanbeobservedleadingtopromiscuousinhibition,theyrepresentaninteresting starting point for drug discovery in trypanosomatidic infections. Aiming to explore the flavonol scaffold, 16compoundswere synthesizedandbiologicallyevaluated.FourX-ray crystallographic structureswere solvedand, togetherwithdockinganalysis, allowedSARelucidation.Twelvecompounds showedEC50valuesagainstT.bruceibelow 10µM.Awide panel of in vitro toxicity propertieswere evaluated to guide compounds selection. Compound2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA orcyclodextrins resulted in lower toxicity when compared to the free compound. Combination studies with methotrexaterevealedthatcompound13(3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one)hasthehighestsynergisticeffectandadosereduction indexattheEC50valueof14.6.Ourresultssuggestthat,usinganappropriatechemicalanddeliverystrategy,flavonoidsmaybepromisingleadsfordrugdevelopmentagainsttrypanosomatidicdiseases.Acknowledgments:ThisprojecthasreceivedfundingfromtheEuropeanUnion’sSeventhFrameworkProgrammeforresearch,technologicaldevelopmentanddemonstrationundergrantagreementn°603240(NMTrypI-NewMedicineforTrypanosomatidicInfections).1. CastilloE.Thekinetoplastidchemotherapyrevisited:currentdrugs,recentadvancesandfutureperspectives.CurrMedChem.2010,17,4027-4051.2. GilbertI.H.InhibitorsofdihydrofolatereductaseinLeishmaniaandtrypanosomes.BiochimBiophysActa.2002.1587,249-257.3. DawsonA.StructureandreactivityofTrypanosomabruceipteridinereductase:inhibitionbythearchetypalantifolatemethotrexate.Mol.Microbiol.2006,

61,1457-1468.


53

SYNTHESISANDEVALUATIONLEISHMANICIDALOFANALOGUESENT-BEYER-15-EN-18-OLAND

ISOESTEVIOL.

EscobarG.1 ,QuiñonesW.1,EcheverriLF.1,TorresLF.1,ArchboldR.1,MurilloJ.2,RodríguezG.2,AranaLM.2,RobledoSM.3

¹UniversityofAntioquia,InstituteofChemistry,QOPN,Medellin-Colombia;2UniversityofAntioquia,Medellin-Colombia;3UniversityofAntioquia,SchoolofMedicine,PECET,Medellin-Colombia

email:[email protected](orleishmaniasis)isasetofzoonoticandanthroponoticdiseasescausedbyprotozoaofthegenusLeishmania.Itoccursmainlyinthreeforms:leishmaniasisvisceral(LV),cutaneous(LC)andmucocutaneous(LM).Itisestimatedthateachyearabout1.3millioninfectedpeople,beingdeadlytonear25,000[1].Inthesearchfornewcompoundsagainsttheaforementionedparasite,twoisomersatC-4ofent-Beyer-15-en-18-olFigure1)isolatedfromtheplantBaccharistola[2]showedhighactivity,invitro,againstamastigotesofLeishmaniapanamensis(LC),with80%inhibitionat4.6ug/ml.Thesecompoundsarepresentinverylowpercentagesintheplant,withthedisadvantageofnotbeingavailableinColombia.Steviosideandrebaudiosidesarewidelyusedasnon-caloricsweeteners[3],whichareidealforobtainingent-Beyer-15-en-18-ol in importantquantities(Fig.1),allowingteststoanimalscaleandfacilitatesthegenerationofgoodamountofsyntheticderivatives[4,5].

Figure1.Synthesisofent-Beyer-15-en-18-olfromstevioside.

Variousderivativesshowedincreasedactivitythanthestartingcompound.especiallythosewhoretainedthehydroxylatC-1Acknowledgments:Rodriguez,G.wassponsoredbyColciencias-COLOMBIA(projectNo111,565,843,059,contract618-2014)1. Worldhealthorganization.2015.Availableat:http://www.who.int/mediacentre/factsheets/fs375/en/.2. SanMartinA.etal.DiterpenoidsfromBaccharistola.Phytochemistry,1983,22:146-1463.3. GeunsJ.MoleculesofInterestStevioside.Phytochemistry,2003,64:913–921.4. LohoelterC.etal.(–)-IsosteviolasaVersatileEx-Chiral-PoolBuldingBlockforOrganicChemistry.Eur.J.Org.Chem.2013,5539–5554.5. HuangT.J.etal.Synthesisandantiviraleffectsofisosteviol-derivedanaloguesagainstthehepatitisBvirus.Phytochemistry,2014,99,107-114.


54

GANGLIOSIDESN-ACETYLANDN-GLICOLYLGM3.

STRATEGYTOSUPPORTTHEVSSPPLATFORMINVACCINESAGAINSTCANCER.

RodríguezMonteroMC.,GonzálezLavautJA. ,LópezLópezM.,RuizGarcíaJA.,ArteagaGLR.,MorenoMR.,LoraJ.,GonzálezD.,BencomoVV.,GutiérrezJM.,GuillénB.,GonzálezL.,RodríguezM.,

FernándezLE.,CarrA.CenterofChemicalBiomolecular(CQB)andCenterofMolecularImmunology(CIM),BIOCUBAFARMA,Havana,Cuba.


ForseveralyearswhentheproductionofthegangliosidesbeganintheCQBstartingfromerythrocytes,itwasrevealedthattheyieldsobtainedbythisprocedurewereverylowtobeabletosatisfythedemandsrequiredinmedicalpracticeduetothelowconcentrationofgangliosides inerythrocytes.AstrategywasreportedfortheproductionofthegangliosidesN-acetylandN-glicolyl GM3 in order to guarantee the VSSP platform in the development of vaccines against cancer to be used inclinicaltrials.Furthermore,thesocioeconomicimportancethatthesevaccineshaveforthecountrydemandshighcapacitytoproduce thesegangliosides.Although itwaspossible to scale up to 50L the extractionprocess starting frommammalianerythrocytes, the obtained average yield of N-AcGM3 and N-GcGM3 would not be able to satisfy the possible demand,especiallyifthetechnicalcapabilitiesoftheinfrastructuresandthedelaysthatmayoccurduringtheprocessofproductiveextractive-purificationhavetobetakenintoconsideration.Additionally,biginternationalregulatoryrestrictionsexistontheuse of components of animal origin, thus having a strong impact on medicinal drug policy making. The different stepsstarting from the extractionprocedure to the chemical synthesiswere described aswell as the characterizationphysical-chemicalcharacterizationandthechemical-pharmacieuticalevaluationallowingtoestablishqualityspecifications.Moreover,a physico-chemical comparability of natural vs synthetic compounds produced, the limitations that the proposed processmay have to become a scalable procedure, the scientific state of the art and the technological solutions were alsoinvestigated. In 2009 the procedure of the laboratory scale upwas proposed and the development of theN-AcGM3wasprioritized in 2010 due to the expectations that in Europe vaccines containing these components were going to beregistered.TheproductionofthesyntheticN-AcGM3beganin2011andsubsequentlyin2012aprocesswasdevelopedabletotriplicateprodutyield.Sincethen,dozensoflotsofthesyntheticN-AcGM3wereproducedallowingtoperformcomparabilitystudiesandtocarryoutpharmaceuticaldevelopments,biologicalevaluationsandclinicalevaluations.


55

EFFECTOFSECONDARYMETABOLITESOFCANELOBARKFRACTIONS

(DRIMYSWINTERIFORST)ONTHEVIABILITYOFHELICOBACTERPYLORI.

NiloCM.1,EspinozaSJ.1 ,ArriagadaChavarriaC,2;KennedyNB.3,ToledoH.4.1ColegioLosAlercesdeTalagante,R.M,Chile;2ColegioLosAlercesdeTalagante,R.M,Chile;3UniversidaddeChile,Santiago,R.M,

Chile;4UniversidaddeChile,Santiago,R.M,Chile.email:[email protected],[email protected],[email protected]

In Chile the seroprevalence ofHelicobacter pylori is bigger than 75% in children under 5 years and reaches 50% in lowersocioeconomicclassand15%inthemiddleclass.(Figueroaetal.,1993)Thishighinfectionatinfantilelevel,perhapsexplainswhythiscountryhasoneofthehighestrateofgastriccancerintheworld.(FONTECNo.94-0356,August,1996.)H.pylorieradication requires the use of at least two antibiotics, bismuth or associated to an antisecretor, a treatment that ismassively used in almost all patients, but showing discomfortwhen swallowing, liver damage andother effects (MedicalJournalofChile,2000).ForthisreasontheeffectsofanaturalextractofCanelowereinvestigated(DrimyswinteriForst)ontheviabilityofthepathogeninvitro.Anextractof183grs.WaspreparedofdriedandgroundbarkofCaneloandoneliterof96%ethanol.Itrested48hours,obtaining3.34grs.Ofafoamymaterialandsemicristallizedinarotaryevaporator.Solublefractionswerethenremovedinasilicagelchromatographycolumn,withmixturesof increasingpolarityhexaneetilo.Chromatographywasmadeinathinlayer of resulting 150 fractions, which were revealed with iodine and analyzed with ultraviolet light. Some initial,intermediate and final fractions for biological testing and analysis byNMRprotons. For selectedbiological tests,HelicobacterpyloriwasculturedinagarTSAsupplementedwithhorseserum,supplementDentandVitox,bothlaboratoryusedOxoidLimited. 6.51x109bac/ml inspecialconditions ofpressure, temperature andhumidity, inagrowthchamber for 48 hours and subsequently measured and tabulated results. It was used 10 µl of compounds dissolved inDMSO,differentconcentrationonplanting.The fractions of a higher biological activity were those intermediate fractions whose NMR spectrum contained apredominanceofsignalscorrespondingtoaliphaticchainsasodoriferoussubstancessuchasPolygodial, IsotadeonalandstructuresderivedfromDrimano.Otherintermediatefractionsshowedthepresenceofflavonoids,withclearsignsofsimilarspectrumSafroleandpresenceofα-glycoside.Allthesefractionsconcentratedin80µg/mlinvitrobiologicaltestsshowedhalos of inhibition of bacterial growth from 2.2 cm. The final fractions gave as result aliphatic chains compatible withSesquiterpenesprobably derivativated fromDrimano withoutdoublebondsandwithoutoxygen functions,which in thebiologicaltestsinconcentrationof80µg/ml.presentedgreatandimmeasurablegrowthinhibitionbacterialhales.ItcouldbeconcludedthatallintermediateandfinalfractionsobtainedcontainingflavonoidsandDrimanos,hadbactericidaleffectonthepathogeninvitro,showinghalosofinhibitionofbacterialgrowth,higherthanthoseobtainedwithGentamicinusedasabactericidecontrol.ItcanbeconcludedthatCanelobarkcontainsFlavonoidsandDrimanosthatinvitrobactericidal,actasphytodrugonHelicobacterpylori,microorganismthatisresponsibleofduodenalulcersandgastriccancerinhumans.1. BustinzaFandCaballeroA.EffectofFlavonolsonClostridiumbotulinum.1947WesternRegionalResearchlaboratory,Science;105:125-31California,USA2. Figueroaetal.TestforDiagnosisofHelicobacterPylori.1996,Bios-Chile.FONTECProjectN°94-0356.3. Gonzalez-SegoviaR.etal.EffectoftheFlavonoidquercetinoninflammationandperoxidationinducedbyHelicobacterpylori ingastricmucosaofguinea

pig.J.Gastroenterology2008,43:441-7.4. Harris P. et al. Usefulness of serologic diagnosis of Helicobacter pylori infection in children. Revista Chilena de Pediatría/ Chilean Paediatrics

Magazine,Santiago,Chile.2005,76;241-2515. TratamientodeerradicacióndeHelicobacterPylorienelpacienteconúlcerapéptica.Guíaclínica/ClinicalGuide.MINSAL.(2013)6. MuñozO.etal.UseofmedicinalplantsinChile.EditorialUniversitariaSantiago,Chile,2001,pp101-104.


56

AROEIRA’S(MYRACRODRUONURUNDEUVA)EXTRACTBACTERICIDALACTIVITY

ANDFNC1,FNC2,FNE1,FNE2NANOPARTICULESINSTREPTOCOCCUSAUREUSANDESTAFILOCOCCUSCOLISTRAINS.

MachadoAC.1,3 ,LopesMMR.2,DurangoLGC.3,ForimMR.3,DamanteCA.4,PeresSHCS1.

1FOB-FaculdadedeOdontologiadeBauru,DepartamentodeSaúdeColetiva,Bauru(SP)-Brazil;2FOB-FaculdadedeOdontologiadeBauru,CentroIntegradodePesquisas,Bauru(SP)-Brazil;3UFSCAR-UniversidadeFederaldeSãoCarlos,

DepartamentodeQuimica,SãoCarlos(SP)-Brazil;4FOB-FaculdadedeOdontologiadeBauru,DepartamentodePeriodontia,Bauru(SP)-Brazil

email:[email protected] plants act in cure and disease prevention. Aroeira (M. urundeuva) is the importance actually because yourleaves present antinflammatory and antimicrobial activity beyond other therapeutic property. The plants and thenanoparticules’sinclusionhavethebeneficitandabilityindiscoverspecifictargetstotissueandorgans.Thenanoparticulesbearsandreleasedrugsandvaccinethroughbeing100to200timeslowerthanthehumancells.Outstandingthemedicinalplants knowledge and your performance near the nanoparticule. The objective study available the aroeira’s extract andaroeira’s extract (M. urundeuva) nanoparticules 2 nanocapsules and 2 nanospheres and your dilution in relation to yourbactericidalandbacteriostaticactivityinsS.aureusandE.colistrains.Theconcentrationextractandaroeira’snanoparticuleswere 1, 1:10, 1:100, 1:1000 e 1:10000mg/mL. For the bacteriostatico test it standardized bacterial suspensions since a 24hculture and concentration 1x106 bacterium/ml. The trial was realized in 96 well’s plate (quintuplicate) with BHI means,extract’sdilutionsandaroeira’snanoparticules,bacteria,negativeandpositivecontrolandblank.After24hinstove,the96wellplateswerereadin590nmspectrophotometer.Nextthebactericidaltestwasrealized,sinceextractseriatedilutionandthe4nanoparticulesample,andwereplatedintriplicateinBHIagarandincubatedinbacteriologicalstoveovernighttolatercoloniesformerunitscount(CFU).Thevariancehomogeneityanalysiswasobteinedbythenormalitytest,ANOVArepeatedmeasurenext by the Tukey’s test (normal distribution) or Kruskal-Wallis following Tukey’s test (not normal), adopting 5%significance level. Aroeira’s extract, control and crude extract didn’t present bactericidal activity. In E.coli strains the 1:10(48,36%death)dilutionfollowthe1:10000(33,60%)dilutionwasthatpresentbactericidalactivitymajor.TheS.aureusstrainsin rude’s extract 100%) contact and 1:1000 (86,78%) dilution didn’t present growing. In bactericidal activity concentrationnanoparticuleFNC1andFNC21:1000wasthatmoreliquidateS.aureus(73,78%),(77,40%)andE.coli.(60,27%)(45,87%)strainsin relation to the control and others concentrations.In FNE1, the 1:10000 (52,35%) concentration was that more presentbactericidalactivity inS.aureusstrainsnext 1:1000(42,94%)concentration. InE.coli strainsthe1mgnanoparticuleobtained25,92%bacterialdeath,next1:10000(11,11%).InFNE2,1:10000obtainedmorebactericidalactivityasS.aureus(94,27%)strainsasE.coli(57,77%)strains.Concludethatevenmoredilutiontheextractandnanoparticule,moreefficacy.


57

ESTERSOFFATTYACIDSASALTERNATIVESORADJUVANTSOFANTIBIOTICSINANIMALFEED

DeLeoR.1 ,SberveglieriV.2,QuartieriA.1,PulvirentiA.1,2

¹DepartmentofLifeScience,UniversityofModenaandReggioEmilia,ReggioEmilia-Italy;²CNR-INOSENSORlab,Brescia-Italyemail:[email protected]

Thewidespreaduseofantibiotics in theanimal feeding industry iscausinggreatconcern for the increasingoccurrenceofantibiotic resistance.Natural substancessuchasessentialoils,organicacidsandshortchain fattyacidsmayconstituteanalternativetoantibiotics,sincetheycanexertantimicrobialandantioxidantactivities(DesboisandSmith,2010).Inthisstudy,theantimicrobialactivityofacommercialblendofshortchainfattyacidsandtheiresters(BL),wasevaluated.BLcompositionwasinvestigatedbyGC-MS.ItsantimicrobialactivityagainstpathogenicstrainsofEscherichiacoli,Salmonellaenterica subsp.entericaTyphimurium,Staphylococcusaureus,Streptococcusagalactiae andStreptococcusuberiswas testedby Kirby-Bauer method (Fekete et al., 1994) and was compared with 4 antibiotics commonly added to animal feeds:doxycyclinehyclate(DH), lincomycinhydrochloride(LH),chlortetracyclinewithsulfadimethoxine(CS),andamoxicillinwithcolistinsulphate(ACS).Furthermore,thesynergiceffectofBLandACS indifferentamountswasevaluatedbyKirby-Bauertest.ThecompositionofBL,analizedbyGC-MS,wasamixtureofmono-glyceridesofbutyricacid(C4),lauricacid(C12);caprylicacid(C8)andcapricacid(C10).TheactivityofBLwasgloballycomparablewiththeoneofantibiotics,withtheonlyexceptionofACS,thatshowedahigherinhibitoryefficacy.DifferentmixturesofBLandACSshowedhigherantimicrobialactivitythanACSitself,suggestingasynergybetweentraditionalantibioticsanddifferentantimicrobialcompounds.TheutilizationofBLorsimilar formulationscouldconstituteavalidalternativetoantibioticsandaddressadoublegoalofprevention and therapy: its administration in animal feed could reduce the possibility of bacterial infection in livestockovercoming the concerns about thedevelopmentof antibiotic resistance, anda synergicmixwith appropriate antibioticsusedintherapycouldenhancetheeffectivenessofthetreatment.1. DesboisAPandSmithVJ.Antibacterialfreefattyacids:activities,mechanismsofactionandbiotechnologicalpotential.ApplMicrobiolBiotechnol.2010;

85:1629-42.2. FeketeT,etal.Acomparisonofserialplateagardilution,Bauer-Kirbydiskdiffusion,andtheVitekAutoMicrobicsystemforthedeterminationof

susceptibilitiesofKlebsiellaspp.,Enterobacterspp.,andPseudomonasaeruginosatotenantimicrobialagents.DiagnMicrobiolInfectDis.1994,18:251-8.


58

EFFECTOFAMIXTUREOFALLERGENSASORALIMMUNOTHERAPYONAMURINEMODELOF

ALLERGENINDUCEDASTHMA

Aguirre-LópezAE.1,Mena-LópezJA.1,Ríos-GarcíaE.1,Ramírez-SánchezI.1,Ceballos-ReyesGM.1,Trejo-GómezOrozcoL.1,Trejo-TapiaD.1 ,Hernández-OrtegaM.2

¹InstitutoPolitécnicoNacional,EscuelaSuperiordeMedicina,CiudaddeMexico-México;2UniversidadAnáhuacNorte,Facultadde

CienciasdelaSalud,NaucalpandeJuárez-Méxicoemail:[email protected];[email protected]

Worldwide235millionpeoplesufferfromasthmaandaconstantincreaseintheglobalprevalence,morbidity,mortality,andeconomicburdenassociatedwithasthmaisbeingobserved1,2.Asthmaisclinicallycharacterizedbybronchialhyperreactivityand symptoms of airway obstruction 3. The global burden of allergic diseases demands effective disease preventionstrategiesand in recent years,promising studieshaveemerged regardingoral immunotherapy (OIT).Anovel formulationobtained from a mixture of four allergens: Candida albicans, Staphylococcus aureus, StaphylococcusepidermisandDermatophagoidespteronyssinusatfixedconcentrationhasbeendeveloped4.InordertoexploreitsefficacytheaimofthepresentworkwastoevaluatetheeffectoftheoralconsumptionofthisallergenmixtureonMDAandproteincarbonylcontent,andhistologychangesonlungtissueinamurinemodelofallergeninducedasthma.BALB/cfemalemice(7-8weeksold)weresensitized)intraperitoneallyonday0and7with20μgofovalbuminemulsifiedin0.2mlofsterilephosphatebufferedsalinecontaining2mgofaluminumhydroxide.Ondays14,16,18,20and22afterinitialsensitization,micewereairchallengedbyexposurefor20mintonebulized1%dustmite(Dermatophagoidespteronyssinus)or 0.1% Californian Pepper tree pollen (Schinusmolle) delivered via a nebulizer. The allergenmixturewas administered at1mg/kgdosefromday23to28.Anotherairchallengewasdonefromday29to375.Onday38animalsweredividedintwogroups:Onegroupwaseuthanizedwithxylazine/ketaminemixtureanlungwasextractedforMDAformationandproteincarbonyl content. The other group was anesthetized with xylazine and ketamine (50 and 10 mg/kg, respectively) andtranscardialperfusionfixationwasperformedforhistopathologyanalysis.Allergen mixture treated mice showed a decrease in MDA formation and protein carbonyl content compared with theuntreatedmice.Histopathologyindicateaprotectiveeffectofallergenmixtureininflammationandmucushyper-secretionduetogobletcellhyperplasia.Ourstudyshowssignificantprotectiveefficacyof allergenmixtureagainstoxidativestressandinflammationduringasthmaandprovidesevidencethatuseoftheallergenmixturecanhelpinbettermanagementofinflammatorylungdisorders.Acknowledgments:ThisworkwassupportedbytheInstitutoPolitécnicoNacional,COFAAandCONACyT.1. WHO,2011.FactSheetNo.307.Availableat:http://www.who.int/mediacentre/factsheets/fs307/en/Acesson:10April20162. BramanS.S.Theglobalburdenofasthma.Chest2006,130(1Suppl.),4S–12S.3. TemelkovskiJ.,etal.Improvedmurinemodelofasthma:selectiveairwayinflammation,epitheliallesionsandincreasedmethacholineresponsiveness

followingchronicexposuretoaerosolisedallergen.Thorax1998;53:849–8564. Trejo-TapiaD.Anti-allergicvaccinefortreatingrecurrentinflammationoftherespiratorytract.WO/2009/136780.12/11/2009.5. Fraga-IrisoR.etal.DevelopmentofaMurineModelofAirwayInflammationandRemodelinginExperimentalAsthma.ArchBronconeumol.2009,45:422–

428.


59

HYPOGLYCEMICACTIVITYOFinvitroPLANTSOFAzadirachtaindica

Méndez-SánchezLI.1,Cruz-CruzDG1,Sanchez-OcampoPM.2 ,CapatazTafurJ.3

¹DivisióndeEstudiosdePosgrado,UniversidaddelPapaloapan,Tuxtepec(Oaxaca)-México;²CONACyT-UNPA,UniversidaddelPapaloapan,Tuxtepec(Oaxaca)-México;3LaboratoriodeCultivodeCélulasVegetales,InstitutodeBiotecnología,Universidaddel

Papaloapan,Tuxtepec(Oaxaca)-Méxicoemail:[email protected],[email protected]

Azadirachta indica ismultipurpose plant that has been used in traditionalmedicine, because of its high content of activeprinciplesmainly tetranortriterpenoids, which are known for their therapeutic potential were screened for pancreatic α-amylaseinhibition,aknownanti-diabetictarget(Ponnusamyetal.,2015).InMéxicoitsusedtotreatdiabetesmellitus(DM),thesecondcauseofdeathaccordingtotheNationalInstituteofStatisticsandGeography2014.TheaimofthepresentworkwastoassessfortheirhypoglycemicactivityextractsofdifferentpolarityfrominvitroandwildplantofA.indica.Biologicalactivityarerelatedtothechemicalprofileoftheextracts.Forevaluatethehypoglycemicactivityofinvitroandwildplant’sextracts(hexane,ethylacetate,methanolandaqueous)ofA. indica, we used normoglycemic and diabetic (glucose levels >120mg/dl) CD-1 mice. The DM type II was inducedintraperitoneally with STZ (40 mg/kg) after intraperitoneal injection of nicotinamide (68 mg/kg) (Masiello et al., 1998).ExtractsfromA.indicaandglybenclamidewereadministeredatdoseof200mg/kgand600µg/kg,respectively(Akinolaetal., 2010) administrationwas orally every 24 hours for seven days. The extractswere further subjected to phytochemicalstudies(Chaovanalikit&Wrolstad,2004;Dewantoetal.,2002;Capatazetal.,2007;Daietal1999)TheoraladministrationofA. indicaextractsatdoseof200mg/kgforsevendaysproducedahypoglycemiceffect.Hexaneextract of in vitro plant waswhich showed greater decrease in blood glucose (44.19%), decreasing by 20.72%more thanglybenclamidewhileaqueousandmethanolextractfromwildplantwereexertedthehighesteffect(33.6%and26.8%).ThechemicalprofilebyTLCofhexaneextractof in vitro plant showed thepresenceof twodiferentsbands (maybe salannin,nimbidin)incomparisontoTLCoftheother invitroextract;thoughitstotalsecondarymetabolitescontentlesscomparedwith the other extracts. Extracts in vitro plantA. indica were showed greater decrease in blood glucose that wild plantextracts.InvitropropagatedplantsofA.indicaareanalternativesourceofhypoglycemiccompoundtowildgrowingplants.Thisactivitywereassociateswithitstraditionalusefordiabetestreatment.Acknowledgments:FinancialsupportfromCONACyT(grant18395).Méndez-SánchezisindebtedtoCONACyTforfellowshipawarded(N°22133).1. AkinolaOB. et al.Chronic treatmentwith ethanolic extract of the leaves ofAzadirachta indica ameliorates lesions of pancreatic islets in streptozotocin

diabetes.InternationalJournalofMorphology,2010,28:291-302.2. CapatazTafurJ.etal.EfectoantialimentariodelosextractosdesuspensionescelularesdeAzadirachtaindicasobreSpodopterafrugiperdaJ.E.Smithen

condicionesdelaboratorio.FacultadNacionaldeAgronomíaMedellín.2007,60:3703-3715.3. ChaovanalikitA.andWrolstadR.E.Totalanthocyaninsandtotalphenolicsoffreshandprocessedcherriesandtheirantioxidantproperties.FoodChemistry

andToxicology,2004,69:67–72.4. DaiJ.etal.Extractionandcolorimetricdeterminationofazadirachtin-relatedlimonoidsinneemseedkernel.J.AgriculturalFoodChem,1999,47,3738-3742.5. DewantoV.etal.Thermalprocessingenhancesthenutritionalvalueoftomatoesbyincreasingtotalantioxidantactivity.J.AgricultureFood,2002,50:3010–

3014.6. MasielloP.etal.ExperimentalNIDDM:developmentofanewmodelinadultratsadministeredstreptozotocinandnicotinamide.Diabetes,1998,47:224-229.7. Ponnusamy S. et al.Gedunin and Azadiradione: Human Pancreatic Alpha-Amylase Inhibiting Limonoids fromNeem (Azadirachta indica) as Anti-Diabetic

Agents.PLOSone2015,10:1-19.


60

BIOLOGICALASSESSMENTOFPISTACIALENTISCUSEXTRACTS:PHYTOPHARMACOLOGICAL

PROPERTIES

GilMV.1,PeralD.2,Delgado-AdámezJ.3,Fernández-BolañosJG.4,Martín-VertedorD.3,LópezÓ.4 ,LagunesI.5,PadrónJ.M.5

1Dpto.deQuímicaOrgánicaeInorgánica,FacultaddeCiencias,UniversidaddeExtremadura,Badajoz-España;2Dpto.deTerapeúticaMédico-Quirúrgica,FacultaddeMedicina,UniversidaddeExtremadura,Badajoz-España;3InstitutoTecnológicoAgroalimentario(CICYTEX-INTAEX),Badajoz-Spain;4Dpto.deQuímicaOrgánica,FacultaddeQuímica,UniversidaddeSevilla,Sevilla-España.;5BioLab,InstitutoUniversitariodeBio-Orgánica“AntonioGonzález”(IUBO-AG),CentrodeInvestigaciones

BiomédicasdeCanarias(CIBICAN),UniversidaddeLaLaguna,LaLaguna-Españaemail:[email protected]

Pistacia lentiscus is an evergreen shrub belonging to the Anacardiaceae family, and particularly abundant in theMediterraneanarea, includingtheIberianPeninsula,Greece,NorthAfricaandtheNearEast.Both,theaerialpartsandtheresin of this shrub have beenwidely used in ethnomedicine for the treatment of cough, eczema, stomach-ache, icterus,diabetes, or respiratory diseases, among others [1,2]. Numerous studies indicate, for instance, that the resin exhibitsimportantantioxidant,anti-inflammatory,hypolipidemicandanticanceractivities[3].In this communication we present the obtention of aqueous and organic extracts from Pistacia lentiscus leaves via alixiviation process and their biological assessment. In particular, their antioxidant profile, their capacity for inhibitingglycosidasesandacetylcholinesterase,aswellastheinvitroantiproliferativeactivityagainstaseriesofhumansolidtumorshavebeenanalyzed.Theaqueousextractturnedouttobeapotent inhibitorofα-glucosidase,andacetylcholinesterase,soitcouldbeuseful inthetreatmentoftype2diabetesandAlzheimer’sdisease,respectively.Moreover,both,theaqueousandorganicextractsexhibitedagoodantiproliferativeactivityagainstthetestedcelllines.Acknowledgments:WethanktheDirecciónGeneraldeInvestigación(CTQ2011-28417-C02-01,CEI10/00018),JuntadeAndalucía(FQM134,P11-CVI-7427),InstitutodeSaludCarlosIII(PI11/00840),FEDERfunds,7thIEUFrameworkProgramme(FP7-REGPOT-2012-CT2012-31637-IMBRAIN)forthefinancialsupport.1. Vuorinen A. et al. Pistacia lentiscus oleoresin: Virtual screening and identification of masticadienonic and isomasticadienonic acids as inhibitors of 11β-

hydroxysteroiddehydrogenase1.PlantaMedica2015,81,525.2. LjubuncicP.etal.TheeffectsofaqueousextractspreparedfromtheleavesofPistacialentiscusinexperimentalliverdisease.J.Ethnopharmacol.2005,100,

198.3. Georgiadis I.etal.BeneficialhealtheffectsofChiosgummasticandperoxisomeproliferator-activated receptors: indicationsof commonmechanisms.J.

Med.Food2015,18,1.


61

DOYOUREALLYTHINKANTIOXIDANTSAREEFFECTIVESANDSAFE?

EcheverriF. ,FernandoTorresF.,QuiñonesW.

UniversityofAntioquia,InstituteofChemistry,Medellín-Colombiaemail:[email protected]

Inthelastdecadeagreatnutritionalrevolutionhasbeencarriedoutintheworld,notonlybyeffortstoreducecaloricfoodbutalsobytheaddedadvantagetopreventorreliefseveraldiseases,throughingestionofsecondarymetabolites.Oneofthemost important groupsof this classof compounds are theAntioxidants (Fanget al, 2002). Cardiovascular sickness inaddition to neurodegenerative, gastrointestinal, immunologic diseases and of course sexual difficulties and aging aresusceptibletobetreatedwiththesecompounds(Pelusoetal.,2015).Ontheotherhands,severalcommercialantioxidantsareconsideredassafeanddisplaystructuralanalogiestotheexperimentalandnaturalantioxidants,althoughpeoplethinkthatthesesubstancesarecarcinogenicandtheirconsumptionhasalreadybeenbanned(Carochoetal.,2013).AconcisesearchofAntioxidantEffectsinthelastfiveyearswasconductedinagreatdiversityofscientificjournals,includingthoseofChemistry,Biology,Biochemistry,Pharmacy,NutritionandMedicine.Lateron,dataobtainedandexperimentswereorganizedinseveralgroups(seebelow)Results and Experimental sections of each paper were compared accordingly to principles of biology, chemistry andbiochemistry;severalmajorproblemsweredetected,asfollows(Bauretal.,2006,Lewandowskaetal.,2013;Schaichetal.,2015):• Alowbioavailabilitywithafasthepaticmetabolism• Anunknownandspecifictarget• Absenceofarationaltherapeuticscheme• Thelackofaconfidentandreliablemechanismofaction• Alotofchemicalassays,noteverextrapolated• Ignoranceofchemicalreactioncoursewitharadical,ametal,ananionoracationRecently more evidences have indicated an additional risk from antioxidants including cancer, since many moleculesoriginally classified as antioxidants possess a high prooxidant capability (Nimse et al., 2015, Perera et al., 2011) Then theinappropriateuseofantioxidantcouldbedangerousforhumanhealth,sinceseveralbiochemicalreactionsinvolvearadicalmechanism,e.g.prostanoidbiosynthesis,oxygentransportanderectionmechanism,amongothers.Finallyandaftermorethan 20 years of antioxidant expectative, at the endof the conclusion section there are frequent additional explanationsaboutdoubtsoftheresultsandtheneedformorestudiesaswellaspreciseinvivoinvestigationstoclarifytherealeffecttopreventorcuresomehumandiseases.1. FangYZetal.Freeradicals,antioxidants,andnutrition.Nutrition.2002,18:872-9.2. PelusoIandPalmeryM.Flavonoidsatthepharma-nutritioninterface:Isatherapeuticindexindemand?BiomedPharmacother.2015,71:102-7.3. CarochoMandFerreiraICFR.Areviewonantioxidants,prooxidantsandrelatedcontroversy:naturalandsyntheticcompounds,screeningandanalysis

methodologiesandfutureperspectives.FoodChemToxicol.2013,51:15–254. BaurJAandSinclairDA.Therapeuticpotentialofresveratrol:theinvivoevidence.NatRevDrugDiscov.2006,5:493-506.5. LewandowskaUetal.Overviewofmetabolismandbioavailabilityenhancementofpolyphenols.J.Agric.FoodChem.2013,6,12183–12199.6. SchaichKMet al.Reprint of “Hurdles and pitfalls inmeasuring antioxidant efficacy: A critical evaluation of ABTS, DPPH, andORAC assays. J. Function.

Foods.2015,doi:10.1016/j.jff.2015.05.0247. NimseS.B.andPalD.Freeradicals,naturalantioxidants,andtheirreactionmechanisms.RSCAdv.2015,5:27986-280068. PereraRMandBardeesyN.Cancer:whenantioxidantsarebad.Nature.2011,475:43-4.


62

CYTOTOXICALKENYLPHENOLSFROMPipereriopodon(PIPERACAE)

MuñozDR.1,2 ,CucaLE.1 ,SandovalAG.1,ArboledaG.1

¹UniversidadNacionaldeColombia,Bogotá-Colombia;2UniversidaddeCienciasAplicadasyAmbientalesU.D.C.A,Bogotá-Colombia

email:[email protected],[email protected],cancerdiseaseisoneoftheleadingcausesofdeathworldwideandin2030,thenumber of people with cancer will reach 26.4 millions in the whole world. Natural products have been the mostrepresentativesourcesofsmallmoleculesforcancertherapyandprovidedenormouscontributionstonewdrugdiscoveryandmechanismsofaction.Moreover,manydevelopingcountriesarestillusingtraditionalmedicinesduetotheir lowcostandlimitedaccesstopharmaceuticaldrugs.Theethnobotanicalknowledgerepresentsthreforethebasisforthediscoveryofnewactiveprinciples.Infact,theinterestonsimplephenolicstructureshasbeenrenewedbecauseoftheantioxidant,anti-inflammatory,anti-estrogenic,anti-mutagenicandanti-carcinogeniceffectsofthesecompoundsbothinvitroandinvivo.InColombiamanyspeciesaretraditionallyusedbylocalhealersanditisnecessarytolinkchemicalcompositionstobiologicaleffects.PhytochemicalreviewsonthegenusPiperhaveshownthatitcontainshighamountofphenoliccompoundssuchasflavonoids, lignansandsimplephenols thatwehaverecentlydemonstratedtoexerthighcytotoxicactivityagainstcancercells. The present study aims to investigate the cytotoxic activity of two new compounds isolated from the leaves ofP.eriopodon, againstPC3 (prostate) andA549 (lung) cancer cell lines, by the colorimetric assayofMTT (methyl tetrazoliumtiazole).Theirstructureswereelucidatedonthebasisofspectroscopicanalysisof1Dand2DRMN.The investigated compounds were obtained using conventional chromatographic techniques and NMR spectra weremeasuredonBrukerAvancespectrometerat400MHzfor1HNMRand100MHzfor13CNMR.ThehumancelllinesA549(lungcarcinoma)andPC3(prostatecarcinoma)wereculturedinDulbecco'sModifiedEagle'sMedium(DMEM)supplementedwithinactivatedfetalbovineserum(FBS,10%),penicillin(100µg/ml)andstreptomycin(100µg/ml)inahumidifiedatmosphereat37°Cin5%CO2.ThesolventusedinthecolorimetricassaywasDMSO(0.2%).Cellswereharvested,countedandplated(1x104cell/well)ina96wellmicrotiterplate.After24h,cellswereexposedtovariousconcentrationsofthetwocompounds(100,50, 10, 5, 1, 0.1 and0.5 µg/mL). Plateswere incubated for 48 h at 37 °C in humidified airwith 5% CO2. After the indicatedincubation times, 10 µL ofMTT solutionwas added to eachwell (0.45mg/mL final concentration) and incubated for 4 h.Finallythemediumwasremovedand100µLofsaline lysisbufferwasadded.CellularviabilitywasdeterminedbytheMTTreductionassayandtheopticaldensitywasmeasuredat595nm.ThetwonewalkenylphenolsexhibitedconsiderableantiproliferativeactivityagainstA549andPC3humancancercell lineswithIC50valuesbetween2.5and30.0µg/mL.ThevaluesoftheIC50ofthesecompoundsarecomparabletothoseofreportedtoembelin,aquinonethatboundselectivitytoXIAPprotein,inducingapoptosisincancercells.Acknowledgments: Authors acknowledge the financial support provided by the Administrative Department of Science, Technology and Innovation fromColombiaandtheDepartmentofChemistryatUniversidadNacionaldeColombia-SedeBogotá.1. Cragg G and Newman D. Beneficial health effects of Chios gum mastic and peroxisome proliferator-activated receptors: indications of common

mechanisms.BiochimicaetBiophysicaActa.2013,1830,3670-3695.2. Wangetal.AnticancerprinciplesfrommedicinalPiper(胡椒HúJiāo)plants.J.TraditionalComplementaryMed.2014,4:8-16.3. Freitas,etal.Cytotoxicnon-aromaticB-ringflavanonesfromPipercarniconnectivumC.DC.Phytochemistry2014,97:81-87.4. LopesAPetal.etal.AntioxidantandCytotoxicEffectsofCrudeExtract,Fractionsand4-NerolidylcathecolfromAerialPartsofPothomorpheumbellataL.

(Piperaceae).BioMedResearchInternational2013,ArticleID20658


63

CHEMICALCHARACTERIZATIONANDCYTOTOXICEFFECTOFLEAFESSENCIALOILFROMLippiagracilis

ANDLippiasidoides

ScherR. ,AlmeidaPOS.,FrançaMNF.,ReisES.,BlankAF.,CorreaCB.FederalUniversityofSergipe,SaoCristovao,Sergipe-Brazil

email:scher@ufs.brEssentialoilsarecomplexmixtureofdifferentcompoundsthatconferstothemawiderangeofbiologicalproperties.Severalstudieshaveshown invitroand invivoantitumoractivityofmanyessentialoilsobtainedfromplantsandthisactivityhasbeenrelatedtothepresenceofmonoterpenesintheircomposition(Ferrazetal.,2013;Nikolicetal.,2014).Lungcanceristhedeadliesttypeofcancerforbothmenandwomen,andthenumberofdeathseachyearisincreasing.Theavailabletherapiesarenotcompletelyefficientandtheyshowserioussideeffects.So,developmentofnewtherapies isnecessaryinordertoovercomethesebarriers.Inthisscenario,theessentialoilsfromplantsofLippiaspeciescanbefeatureasapotentialsourceoftherapeuticagentsforthedevelopmentofalternativedrugsagainstlungcancer.Thus,wehaveperformedthechemicalcharacterizationofessentialoilfromdifferentchemotypesofLippiasidoides(Cham.)andLippiagracilis(Schauer).Moreover,theircytotoxiceffectonlungcarcinomacellsA549wasassessed.TheessentialoilfromleafofbothspecieswasextractedbyhydrodistillationusingClevengerequipment(Ehlertetal.,2006).Chemical composition analysis was performed in a gas chromatograph coupled to a mass spectrometer (GC–MS) andquantitative analysis of the chemical constituents was performed by flame ionization gas chromatography (FID). Thecytotoxic effect of increasing concentrations of essential oils (from 20 to 160 μg/mL)was evaluated byMTT colorimetricassay.Toevaluatealterationsinmorphology,aftertreatment,cellsgrownovercircularcoverslipswere,fixedwithmethanol,stainedwithhematoxylin–eosinandexaminedbylightmicroscopy.GC-MSanalysishasshownthatbothessentialoil fromL.gracilischemotype110(LGEO110)andfromL.sidoideschemotype104 (LSEO104) have carvacrol as themain compound. On the other hand, thymol was themost abundantmonoterpenefound inthecompositionofL.gracilisessentialoilchemotype102(LGEO102)and inL.sidoidesessentialoilchemotype106(LSEO106). Even though all chemotypes evaluated have shown cytotoxic effect onA459 cells, the effect of those rich inthymol,wasmorepronouncedthanthatrichincarvacrol.TheCC50sobtainedforLGEO106andLSEO102were,respectively,72.42 and 97.93 μg/mL. For LGEO110 and LSEO104, the CC50swere 158.60 and 150.70 μg/mL, respectively. In fact, itwasdemonstratedbyMeloetal.(2014)thatessentialoilsrichinthymolandthethymolisolatedaremoreeffectivecomparedtocarvacrol. Morphological changes, including cytoplasm and chromatin condensation and nuclear fragmentation wereobserved inall chemotypesevaluated in thiswork.Oncemore, theeffectof thymol-richessentialoilswasstrongest thancarvacrol-richones.About95%ofA459cell treatedwith75.0μg/mLofLGEO106haveshownsomemorphological changewhereastheproportionofchangesinducedbythesameconcentrationofLGEO110didnotdifferfromthenegativecontrol.The same profile of action was observed between LSEO102 and LSEO104. Thus, we can conclude that the differencesobservedinthecytotoxiceffectofessentialoilsfromdifferentchemotypesofL.gracilisandL.sidoidesmightbedue,atleastinpart,tothepresenceofthymolorcarvacrolintheircomposition.Finally,themorphologicalchangesdetectedsuggestthatthecytotoxicactivitycanbeduetoessentialoilabilityintriggeringcelldeathprocessesbyapoptosis.1. EhlertPADetal.Tempodehidrodestilaçcãonaextraçãodeóleoessencialdeseteespéciesdeplantasmedicinais.Rev.Bras.Pl.Med.2006,8:79–80.2. FerrazRPC.etal.CytotoxiceffectofleafessentialoilofLippiagracilisSchauer(Verbenaceae).Phytomedicine.2013,20:615–621.3. Melo JO. et al. Cytotoxic effects of essential oils from three Lippia gracilis Schauer genotypes on HeLa, B16, and MCF-7 cells and normal human

fibroblasts.Gen.Mol.Res.2014,13:2691-2697.4. NikolicM.etal.Chemicalcomposition,antimicrobial,antioxidantandantitumoractivityofThymusserpyllumL.,ThymusalgeriensisBoiss.andReutand

ThymusvulgarisL.essentialoils.Ind.Crop.Prod.2014,52:183–190.


64

COMPARATIVEASSESSMENTOFTHEAPOPTOTICPOTENTIALOFCUBANANDBRAZILIANPROPOLISIN

HUMANLARYNGEALEPIDERMOIDCARCINOMACELLS

Frión-HerreraY.1 ,Díaz-GarcíaA.2,Ruiz-FuentesJ.3,Rodríguez-SánchezH.3,SforcinJM.41UNESP,SaoPaulo-Brazil/HavanaUniversity,Havana-Cuba;2LaboratoriesofBiofarmaceuticalsandChemistriesProductions

(LABIOFAM),Havana-Cuba;3TropicalMedicineInstitutePedroKouri,Havana-Cuba;4UNESP,SaoPaulo-Brazilemail:[email protected]

Propolishasbeenusedinfolkmedicineforcenturiesbecauseofitsbeneficialeffectsincludinganticancerproperties.Duetosignificantdifferencesinthechemicalcompositionofsamplesoriginatingfromdifferentgeographicandclimaticzones,theevaluation of antitumor effects of each type of propolis is crucial. The aim of this study was to compare the cytotoxicmechanismofCubanredpropolis (CP)andBraziliangreenpropolis (BP)onhuman laryngealcarcinoma(Hep-2),basedontheirapoptoticpotential.Theapoptoticefficiencyofbothsamplesofpropoliswasconfirmedusingaseriesofassays,includingcellviability,leakageoflactatedehydrogenase,mitochondrialmembranepotential,fluorescencestainingandtheexpressionofapoptosis-relatedgenes(p53,caspase-3,bax,bcl-2,bcl-xL,andp21).Cellviabilityandcytotoxicassayssuggestedadose-dependenttoxiceffectofCPandBPextractswithpossibleassociationofintracellular reactive oxygen species production. Apoptotic potencial was confirmed by decrease of mitochondrialmembranepotentialandfluorescencestaining,revealingthatbothsamplesinducedcellularapoptosisviaactivationofp53,caspase-3, bax, p21 signaling, and downregulation of bcl-2 and bcl-xL. Particularly, CP extract showed a higher apoptoticpotentialthanBPextract.Ourfindingsopenperspectivestostudythemaincomponentsofeachpropolis,whichmayleadtothedevelopmentofasuitabledrugforthetreatmentoflaryngealcancer1 RochaABetal.Naturalproductsinanticancertherapy.CurrOpinPharmacol,2001,1:364-9.2 Silva-CarvalhoRetal.Propolis:AComplexNaturalProductwithaPlethoraofBiologicalActivitiesThatCanBeExploredforDrugDevelopment.EvidBased

ComplementAlternatMed,2015:,2064393 ToretiVCetal.Recentprogressofpropolis for itsbiologicalandchemical compositionsand itsbotanicalorigin.EvidBasedComplementAlternatMed,

2013,697390.4 Frión-Herrera Y et al.Brazilian green propolis induced apoptosis in human lung cancer A549 cells throughmitochondrial-mediated pathway. Journal of

PharmacyandPharmacology,2015;67:1448-1456.5 DemirSetal.AntiproliferativeandproapoptoticactivityofTurkishpropolisonhumanlungcancercellline.NutrCancer,2016,65:165-1726 deMendonca,ICetal.Brazilianredpropolis:phytochemicalscreening,antioxidantactivityandeffectagainstcancercells.BMCComplementAlternMed,

2015,15:357.7 TsaiYetal.InductionofoxidativeDNAdamagebyflavonoidsofpropolis:itsmechanismandimplicationaboutantioxidantcapacity.ChemicalResearchin

Toxicology,2012,25:191-6.8 ChenCNetal..ApoptosisofhumanmelanomacellsinducedbythenovelcompoundspropolinAandpropolinBfromTaiwenesepropolis.CancerLett,2007,

245:218-31.9 Chen MJ et al. Caffeic acid phenethyl ester induces apoptosis of human pancreatic cancer cells involving caspase and mitochondrial dysfunction.

Pancreatology,2008,8:566-76.10 BegniniKRetal.Brazilianredpropolisinducesapoptosis-likecelldeathanddecreasesmigrationpotentialinbladdercancercells.EvidBasedComplement

AlternatMed,2014:639856


65

MASTICADIENONICAND3a-HYDROXYMASTICADIENONICACIDSINHIBITPROLIFERATIONPROSTATE

CANCERCELLSINVIVOANDINVITROBYINDUCINGAPOPTOSIS.

Martínez-VázquezM. ,Sánchez-MonroyB.,Martínez-VázquezA.UniversidadNacionalAutónomadeMéxico,InstitutodeQuímica,Coyoacán-México

email:marvaz@unam.mxPreviousstudiesindicatetheimportanceoftriterpenoidsasanticanceragentsinvivo,althoughtheircytotoxicitytohumancancer lines is small. Therefore,one formtoevaluate some triterpenesasanticanceragents is toassess invivo theiranti-tumoreffect(Yadav,etal.,2010).Studiesbyourgroupshowedthatmasticadienonic(AMD)and3αhydroxylmasticadienonic(3αAMD)acids,triterpenesisolatedfromA.adstrigens,aretheresponsiblefortheanti-inflammatoryandcytotoxicactivitiesofthisspecies(Oviedo,etal.,2004).Taking intoaccountabovewedecidedtoevaluate invivo theantitumorpropertiesofthese triterpenes using nudemice. The cell cycle arrest and induction of apoptosis by AMD y 3αAMD in vitro alsoweredetermined.Xenograftswereinducedbyinoculationto60nu/numalemiceasuspensionofPC-3cells.Threeweekslaterthemicewererandomly distributed into ten experimental groups with n = 6 each. Two groups were administered with vehicle, thefollowingtwowithcisplatin4mg/kgandtheotherwithAMDand3αAMD(60,125,250mg/kg)respectively.Attheendofthexenograftexperiment,thetumorsweredissectedandfixedin10%formaldehyde,dehydratedandembeddedinparaffintodeterminedPCNAandKi67expressionbyimmunohistochemistryassay.Apoptoticcellsweredetectedintumortissuesusingdetection kit Promega® kit (G7131) in situ. To determine a possible mechanism of AMD and 3αAMD tumor action,determination of inhibition of proliferation by crystal violet, cell cycle arrest and induction of apoptosis in vitro wereperformed. The expressionof p-Aktwasdeterminedbywesternblot using the supernatantof lysedPC-3 cells previouslytreatedwithAMDand3αAMD.TheantitumoractivityofAMDand3aAMDwasdemonstratedbythereductionoftumorgrowthsimilartocis-platinum(CP)inducedby inoculationofPC-3 cells innudemice. TheKi67protein is associatedwith cell proliferationandhas importantclinicalimplicationsincancer.Ki67analysisoftissuesshowedthatthegroupstreatedwithAMD,3aAMDandCPhavefewerproliferating cells and intensity of Ki67 compared with the control group. PCNA protein facilitates and controls DNAreplication.Detecting PCNA in tumor tissues showedgreatest number of positive cells and intensity brand in the controlgroupcomparedtothegroupstreatedwithAMDand3α-AMD.OurresultsshowedthatAMDand3aAMDinduceapoptosisinbothtumortissuesandPC-3cellaswellastheyarrestedthePC-3cyclecell.AlsoourresultsshowedthatAMDand3aAMDinhibittheactivationofAktinPC-3cells.ThelethaldosewasdeterminedinCD-1miceresulting400mg/kgforAMDand250mg/kgforthe3α-AMD.1. YadavV.Ret al.Boswellic acid inhibits growthandmetastasis of human colorectal cancer inorthotopicmousemodelbydownregulating inflammatory,

proliferative,invasiveandangiogenicbiomarkers.Int.J.Cancer,2012,130:2176–2184.2. Oviedo-ChávezI.etal.PrinciplesofthebarkofAmphipterygiumadstringens(Julianaceae)withanti-inflammatory.Phytomedicine2004,11:436-445


66

THEUSEFULNESSOFOREGANO’SPHYTOCOMPLEXCRUDEEXTRACT:NOVELANTI-PROLIFERATIVE

EFFECTSINADRENOCORTICALTUMORCELLS

PezzaniR.1 ,RubinR.1,MonticelliH.1,BertazzaL.1,RedaelliM.2,ZorzanM.3,SensiF.1,CeccatoF.1,ScaroniC.1,RomanoD.4,MianC.1,ArmaniniD.1,BertoliniC.4,BarolloS.1,BoscaroM.1

1EndocrinologyUnit,DepartmentofMedicine,UniversityofPadova,Padova-Italy;2DepartmentofAnalyticalChemistry,3ALaboratorisrl.,Padova-Italy;3DepartmentofMolecularMedicine,UniversityofPadova,Padova-Italy;4DepartmentofFood,

EnvironmentalandNutritionalSciences(DeFENS),SectionofFoodmicrobiologyandBioprocesses,UniversityofMilan,Milan-Italyemail:[email protected]

Oregano (Origanum vulgare L.) is a common aromatic plant broadly used in theMediterranean and Asian Regions. Sinceancient times, it has been consumed in the diet as flavor enhancer and used in folk medicine as curative herb treatingrespiratory diseases, painful menstruation, rheumatoid arthritis, dyspepsia, etc. Recently its role as an anticancerphytocomplex has been suggested in human preclinical models, although a crude extract of oregano has been neverevaluated into adrenocortical tumor cell models. This study analyzed the anti-proliferative effects of crude extract oforeganointo2tumorcelllines,SW13andH295R.Main components of oregano were studied by GC/MS analysis. Different procedures evaluated the effects of oregano,testing its properties on cell viability, survival and morphology. Moreover cell death processes and cell cycle wereinvestigatedaswellasmainintracellularpathwaysmodulation.Our findingsdemonstrated that thecrudeextractoforeganomaydecreasecell viability, vitalityandsurvivalofSW13andH295Rcells.Inaddition,oreganoinducedcelldeaththroughnecroticprocessandalteredcellcycle,producingadecreaseinG0/G1phase and an increase in subG0/G1phase.Westernblot revealed an impact onMAPKandPI3K/Akt pathways,withspecificreactivitydecrease,affectingp-Erk1/2andp-Akt.Thisworkunderlinestheanti-proliferativeactivityoforeganoasacrudeextractintumorcellmodels(SW13andH295Rcells),butmoredataareneededtosubstantiateitspotentialuseinthetreatmentofadrenocorticalneoplasiaorasanintegrativedietphytocomplex.


67

ANTITUMOREFFECTOFAQUEOUSEXTRACTOFJatrophadioicaSesséexCervLYMPHOMAL5178YIN

CD1/CMICE.

Montejano-RodríguezJR. ,Hernández-CeruelosA.,Muñoz-JuárezS.,DelaO-ArciniegaM.,Hernández-RomeroJ.,Almaguer-VargasG.

UniversityHidalgoState,PharmacyAcademicArea,SanAgustínTlaxiaca,Pachuca(Hidalgo)-México.email:[email protected]

Cancerisagroupofdiseaseswithhighincidencenationallyandworldwide1.In2008,themortalityrateinMexicowasinthethirdplace.Mostofthetime,treatmentsareveryexpensiveandsometimestoxicandinefficient,moreoveritisnecessarytosearch for more antitumor options. Natural alternatives, including medicinal plants, may represent an option for thetreatmentofthesepathologiccondition.Oneexample istheplantcalled"SangredeDrago"(Jatropha), that isusedasanantitumoral.TheaimofthisstudywastoevaluatetheantitumoreffectofaqueousextractofJatrophadioica inamodeloflymphomaL5178Y,inBalb/cmouse.Jatropadoicawas collected inTezontepecdeAldama,Hidalgo,Mexico. Theaqueousextractwaspreparedasan infusionfromtherootofthedriedplantthatwasprocessedandtrituratedaccordingtothePharmacopoeiaHerbolariaMexicana2

(2001).Thedecoctionwastested inthreedifferentdoses(0.92mg/Kg,2.40mg/kgand4.8mg/kg)equivalenttoone,threeandsixcupsper70kgofbodyweight.Forantitumortest,animalsweredividedintofivegroupsofsevenmaleBalb/cmice30g and subjected to an adaptation period of seven days. Into each mouse the tumor L5178Y lymphoma was inoculatedintraperitoneally. Survival time and tumor growth (weight) in mice were evaluated using the statistical test analysis ofvariance(ANOVA).ResultsshowthatmediumandhighdosesoftheaqueousextractofJ.dioicainhibitedtumorL5178Ygrowthinmicebalb/candmice survivedmore days comparedwith the control and the vincristine group of animals. The antitumor effect canattributedtothepresenceofterpenesandcyclicpeptidesandtothepresenceofaproteincalledcurcinthathasbeentestedinvitroshowingeffectsontheactivityofN-glycosidase3.Similarly,anantitumoreffectwasalsoobservedinamodelofP-388lymphocytic leukemia using a lactam Jatrophamacrorhiza 4. The group treatedwith J. dioica appeared healthier than thevincristinegroup.1.ANÓNIMO.2008.OrganizaciónMundialdelaSalud(OMS),centrodeprensa.Datosycifras.NotadescriptivaN°297.2.COMISIÓNPERMANENTEDELAFEUM.2001.Anexo5.Preparacióntisanas,pp.169.YGeneralidades,requisitosgenerales,tamañodelcortepág.20In:

FarmacopeaHerbolariadelosEstadosUnidosMexicanos.SecretariadeSaludEditor.D.F.,México.3.WiedhopfR.Metal.AntitumoragentsfromJatrophamacrorhiza(euphorbiaceae)I:Isolationandcharacterizationofjatropham.J.PharmaceuticalSciences

1973,62:1206–12074.CarlaW.etal.Medicinalproperty,phytochemistryandpharmacologyofseveralJatrophaspecies(Euphorbiaceae):Areview.Phytochemistry2013,85:7–29.


68

HERBALDRUGINTERACTIONSANDREMEDIES:ACHALLENGINGPROBLEMINTHERAPY

ShrivastavaN.1 ,JhaV.2

1DepartmentofPharmacology,SchoolofPharmacy,ITMUniversityGwalior-India;2DepartmentofPharmaceuticalChemistry,SchoolofPharmacy,ITMUniversityGwalior,Gwalior-India

email:[email protected],vibhujha16@gmail.comAlthoughherbalmedicinesarebyfarthelargestcomponentofnon-orthodoxremediestheydonothaveexclusiveclaims;itmustbemadeclearthattherearevarioustypesofotheralternativetreatmentsrangingfrompreparationsofanimalorigin,tominerals,vitaminsandaminoacids.Itmustbeappreciatedthatthequalitycontrolovermostherbalremedies,whicharenotofficiallyregisteredasmedicines,isoftenpoorandmorelikelytobenon-existent,andthatmostherbalpreparationsarenotstandardizedforpotencyinbiologicaltestsystems.Asaconsequencetheirpotencymayvaryconsiderablyfromsampleto sample. Furthermore they may be contaminated or adulterated with pesticides, toxic metals, botanicals, animalsubstancesand/ororthodoxdrugswhichmayleadtoadditionalandunexpectedadversedrugreactionsandinteractions.WereviewedvariousResearchesandreviewarticlesforthisworkandthefindingsareincludesinthisarticle.Generallytoolittle isknownabouttheconsequencesofsuchcombinationsalthoughtheclinicalreportsof interactionsthat infrequentlyappearinthemedicalandpharmaceuticalpresssuggestthatmanymoreinteractionsmaybeoccurringthatarenotrealizedassuchandarenotreportedintheliterature.Reducingtheriskofdruginteractionsisachallengethatembracesanumberofconsiderations.Thefollowingareguidelinestoreduceandmanagedruginteractions.Factorssuchasage,thenatureofthepatient’smedicalproblem(e.g.,impairedrenalfunction),dietaryhabits,smoking,andproblems such as alcoholism influence the effect of certain drugs and should be considered during the initial patientinterview.Acknowledgments:AuthorsarethankfultoMr.RamashankarSingh(Chancellor,ITMUniversity),Dr.D.S.Chauhan(ManagingDirector,ITMUniversity),Prof.(Dr.)VandanaKushwah(Vice-Chancellor,ITMUniversity)andDr.RichaKothari(Head,SchoolofPharmacyITMUniversity)forprovidingfinancialsupportandnecessaryfacilitiesforthiswork.WearealsothankfultoallsupportivestaffofSchoolofPharmacy,ITMUniversitywhoweredirectlyorindirectlyinvolvedinthiswork.1. CapobiancoDJetaLProximal-muscleweaknessinducedbyherbs.NEnglJMed1993,329,1430.2. D'ArcyPF.Adversereactionsandinteractionswithherbalmedicines.Part2--Druginteractions.AdverseDrugReactToxicolRev1993,12,147.3. DeSmetPAGM.RitualenemasandsnuffsintheAmericas,Ferris,Dordrecht.19854. DeSmetPAGM,KellerK,HanselR,ChandlerRF(eds)AdverseEffectsofHerbalDrug,SpringerVerlag(Berlin),19925. DeahlM.Betelnut-inducedextrapyramidalsyndrome:Anunusualdruginteraction.MovDiscord1989,4:330.6. GoldmanJA,andMyersonG.Chineseherbalmedicine:camouflagedprescriptionantiinflammatorydrugs,corticosteroids,andlead.ArthritisRheum1991,

34.1207.7. JosephAMetaLStealthsteroids.NEnglJMed1991,324.62.8. Khin-Maung-Uetal.Clinicaltrialofberberineinacutewaterydiarrhoea.BrMedJ.1985,291.1601.9. OttJ.Ayahuascaanalogues.Pangaeanentheogens.NaturalProducts,Kennewick.1994


69

ECHINACEAANGUSTIFOLIADC.EXTRACTQUALI-QUANTITATIVEANALYSISANDFIBROBLASTCELL

GROWTHEVALUATION

MilellaL.1 ,BartoloM.1,FaraoneI.1,BenedettoC.1,ValentaoP.2,AndradeP.2,CarmosinoM.1,deTommasiN.3

1DipartimentodiScienze,UniversitàdegliStudidellaBasilicata,Potenza-Italy;2RededeQuímicaeTecnologia/LaboratórioAssociadoparaaQuímicaVerde(REQUIMTE/LAQV),LaboratóriodeFarmacognosia,DepartamentodeQuímica,Faculdadede

Farmácia,UniversidadedoPorto,Porto-Portugal;3DipartimentodiFarmacia,UniversitàdegliStudidiSalerno,Fisciano(SA)-Italyemail:[email protected]

EchinaceaangustifoliaDC.,nativefromNorthAmerica, isoneofthethreeEchinaceaspeciesthatarewidelyusedasherbalremedy, mainly for their immunostimulant properties. Chemical analysis of plants in the genus Echinacea has identifiedseveralgroupsofmedically importantcomponents includingpolysaccharides, flavonoids,caffeicacidderivatives,essentialoils,polyacetylenes,alkylamides(1).Theaimofthisstudywasthequali-quantitativeanalysisofsecondarymetabolitesinextractsobtainedbydifferentextractivetechniques,theevaluationofpolysaccharidescontentandinvitrocicatrizingactivityonfibroblastcelllines.The extraction of bioactive compounds from dried roots of E. angustifolia was performed by different techniques asmaceration, ultrasound, microwave and enzymatic-assisted extraction. Temperature and pH were monitored duringextractionprocedures (2 hours).Quali-quantitative analysiswasperformedbyHPLC-DAD system (2),whereas the solublepolysaccharidescontentwasevaluatedbyphenol-sulphuricacidmethod(3).Thestimulationof fibroblastcellgrowthwascarriedoutbywoundhealingassay.Solventextractionconsistsofliberatingplantcompoundsintoarecipientsolvent.Thechoiceofthesolventtypedependsonthesolubilityofthesoluteinthesolventandthecombinationwithanextractionprocesscouldensurethemaximumyield.Extracts of E. angustifolia roots showed similar qualitative phytochemical profile reporting the presence of caftaric acid,chlorogenicacid,caffeicacid, ferulicacid,chicoricacid,echinacoside,verbascoside,3,4and3,5di-O-caffeoylquinicacid.Ontheotherhandextractsreportedsignificantquantitativedifferencesandtheenzymatic-assistedextractionwasfoundtobemore efficient to recover high amount of secondary metabolites than other extraction techniques in aqueous solvent.Compoundsas3,4and3,5di-O-caffeoylquinicacidwerefoundintracesinalmostallextracts(4).Echinacoside,instead,wasthe main phenolic component in this species. Polysaccharides in Echinacea root were quantified and the extracts alsoshowedquantitativedifferences,alsoevidentwhenstimulationoffibroblastcellbyinvitroassaywasperformed.RootsofEchinaceaangustifoliaareanimportantnaturalsourceforchemical-pharmaceuticalandfoodsupplementfieldandenzymaticextractioncouldbeusefultoincreasethesecondarymetaboliteyield.1. CozzolinoRP.etal.StructuralanalysisofthepolysaccharidesfromEchinaceaangustifoliaradix.CarbohydPolym.2006,65,263–272.2. RussoDP.Etal.EvaluationofAntioxidant,AntidiabeticandAnticholinesteraseActivitiesofSmallanthussonchifoliusLandracesandCorrelationwithTheir

PhytochemicalProfiles.IntJMolSci.2015,31,17696-718.3. GlavačNK.etal.OptimizationanduseofaspectrophotometricmethodfordeterminingpolysaccharidesinEchinaceapurpurea.CentEurJBiol.2012,7,126-

131.4. RussoD.etal.NutraceuticalpropertiesofCitrusclementinajuices.Pharmacologyonline2012,1(SPL.1)84-93.


70

EFFECTOFSPIRULINAPLATENSISINPATIENTSWITHMETABOLICSYNDROME

CasamayorLaimeZ.LABIOFAM,Habana-Cuba

email:zclaime@infomed.sld.cuThemetabolicsyndromeisanimportantcauseofmorbidityandmortality,consideredariskfactorforcardiovasculardisease.Carbohydratedisorders,obesity,dyslipidemiaandhypertensionassociatedcharacterize it.Spirulina for itshighcontentofcarotenoidsandotherantioxidantscanbenefitpreventionandimprovement,decreasingthebloodlipidconcentrationandinterveneinstoppingtheoxidativeprocessesresponsibleforthedeteriorationofthewallsofveinsandarteries,improvingbloodpressure.AimofthestudywastoevaluatetheeffectofSpirulinaplatensisinpatientswithmetabolicsyndrome.A longitudinal, prospective study of 45 adults with a diagnosis of metabolic syndrome, who attended the MedicalExaminationControl,wasconducted for theperiod2010-2013.They randomlyassigned twogroups:acontrolwithdietaryadviceandonestudywithdietaryadviceplus6tabletsdailySpirel®100%naturalfor6months.ChangesinlipidprofileandIPS,wereevaluatedfromtestWilcoxonsignedranksaftertreatmentineachgroup,withalpha=0.05.Thedecreaseincholesterolwassignificantinthestudygroup(p=0.001).Whencomparedwiththecontrolgroup,thet-testshowednostatisticallysignificantdifference(p=0.297).Triglyceridesdecreasedsignificantly inthestudygroup(p=0.001).Bothgroupsexperiencedsignificantchanges in the IPS, in thecontrolgroupvaluesdecreased (p=0.000)and in thestudygroupwas significantly increased (p=0.000), resulted in improvedperipheral vascular insufficiency.Noadverse reactionswerereported.Consumption of Spirel®100% natural for 6months is effective and safe in improvingmetabolic syndrome and peripheralvascularinsufficiency.


71

TURNERADIFFUSA(DAMIANA)ASSOURCEOFHEPATOPROTECTIVEANDHYPOGLUCEMIC

COMPOUNDS

WaksmanN.1 ,DelgadoC.1,ParraA.1,BarriosJ.1,CorderoP.1,SalazarR.1,AcevedoJ.21UniversidadAutónomadeNuevoLeón,FacultaddeMedicina,MaderoyAguirrePequeño,Monterrey(N.L.)-México;2Universidad

AutónomadelEstadodeMorelos,FacultaddeMedicina,Cuernavaca(Morelos)-Méxicoemail:[email protected]

Liverdiseasesanddiabetesareserioushealthproblemsworldwide.Mexico isacountrywithagreatbiodiversityofplantsandahistoricaltraditionofusingthem.Turneradiffusa(damiana)isashrubnativefromMéxicowithdifferenttraditionallyuses1. Previously we reported the hepatoprotective and hypoglycemic effect of a methanolic extract obtained from thisplant2,aswellasitsgreatantioxidantpotential3.Therefore,basedonabioassay-guidedfractionationofamethanolicextractobtained from the aerial part ofT. diffusa, we present in the present contribution the isolation and identification of thecompoundsresponsible forthepharmacologicalaction,aswellas theeffortstowardstheelaborationofaphytomedicinewiththisplant.The extracts were obtained by maceration with MeOH at room temperature and where further purified by solid phaseextraction, silica vacuum liquid chromatography, reverse-phase and countercurrent chromatography, to obtain thecompoundsresponsibleforthepharmacologicalaction.PuritywasdeterminedbyHPLCandtlc.StructureswereestablishedbyNMR,MSandiontrapHPLC/MS.CytotoxicityandhepatoprotectionwereassessedinaninvitromodelusingHepG2cellline and in an ex vivo model by means of precision-cut hepatic tissue slices. Silibinine was used as positive control.HypoglycemicactivitywasevaluatedinvivowithnormoglycemicCD1mices.Insulinwasusedaspositivecontrol.ThefractionationprocedureyieldedaC-glycosideofluteolinewhichexhibitedafourtimesgreaterhepatoprotectiveeffectthanthewidelyusedhepatoprotectiveagentsilibininagainstcarbontetrachloridedamageinaninvitromodelusingHepG2cells. The activitywas confirmed using an ex vivomodelwith precision-cut hepatic tissue slices. From another fraction aterpenewas isolatedand identified,which resultedresponsible for thehypoglycemicactionof thisplant.All these resultsvalidatethetraditionallyusesoftheplant,andopeninsighttofurtherinvestigationsAlthoughthesefindingsarepromising,the recuperationof thecompounds is lowandcostsassociated forpurificationsarehigh; thereforeweare tryingnowtoobtainanstandardizedextractofthisplantthatcouldfulfilltherequirementsofaphytomedicineproposal.Acknowlegment:FundingSEP-CONACYT2012grantNo.180977andPRODEP:funding103-5/15/141561. Szewczyk, K et al. Ethnobotany, phytochemistry, and bioactivity of the genus Turnera (Passifloraceae) with a focus on damiana—Turnera diffusa J.

Ethnopharmacol.2014,152,424–4432. Torres-Gonzálezetal,ProtectiveeffectoffourMexicanplantsagainstCCl₄-induceddamageontheHuh7humanhepatomacellline.Ann.Hepatol.2011,10,

73–79.3. Pérez-Meseguer, J et al. Development and validation of an HPLC-DAD analytical procedure for quality control of damiana (Turnera diffusa), using an

antioxidantmarkerisolatedfromtheplant.J.AOACInt.2010,93,1161–1168.


72

ROSSOPURO™:AREDYEASTRICEEXTRACTFORTHEMANAGEMENTOFHYPERCHOLESTEROLEMIA

RussoR.1 ;ColelliF.2,SignorinoG.2,Pisano,C.2,TerruzziC.1

¹GiellepiS.p.A.HealthScience,Lissone(MB)-Italy;²Biogem,ResearchInstitute,ArianoIrpino(AV)-Italy

email:[email protected] yeast rice extract is obtained following rice fermentation byMonascus purpureus. It has been used for centuries intraditionalChinesemedicineas remedyofdifferentdiseases related tocirculationanddigestion.Evidenceexistsabout itsefficacyinthecontrolofbloodcholesterollevels.Infact,themainproductoffermentationismonacolinK,whichinhibitsthesynthesis of cholesterol by inhibition of hydroxymethylglutaryl-CoA (HMG-CoA) reductase, the key enzyme of cholesterolsynthesisinhumanbody(1,2).TheaimofthecurrentstudywastoevaluatetheefficacyofROSSOPURO™,ourredyeastriceextractrichinactivecompounds,inimprovinglipidprofileinananimalmodelofdiet-inducedhyperlipidemia.C57BL/6J mice were used for experimental procedures. Animals were divided into 4 groups according to treatment (14animals/group). 3groupswere feddailywithahigh-fatdiet for4 consecutiveweeks.After thisperiod, serum lipidswereanalyzed (total cholesterol and triglycerides) and the animals,maintainedwith high-fat diet, were treated by gavage forfurther 4 weeks according to the following scheme: group 1: administered with ROSSOPURO™; group 2: treated withtraditional redyeast rice(reference item);group3: treatedwithvehicle.Conversely, thenegativecontrolgroup(group4)werefedwitharodentstandarddietandreceivedthevehiclebygavage.Atboththepre-dose(week4)andtheendofthetreatment,bloodsampleswerecollectedfromthemandibularveinofalltheanimalsunderalightanesthesia,afterafastingperiod.Thebloodsamplesweredividedintodifferenttest-tubeswithoutanyanticoagulantandanalyzedbythemeansoftheautomaticanalyzerVitroVet(SCIL),inordertoassessserumlipidprofile(total cholesterol, HDL, LDL, triglycerides), glucose, lipase, lactate dehydrogenase, creatine kinase, CRP. The atherogenicindex (AI)were calculatedbyHDL-cholesterol/total cholesterol andLDL-cholesterol/HDL-cholesterol.Duringall the in vivostudy,theanimalsweremonitoredforclinicalsignsandmortality,bodyweight,foodandwaterconsumptions.Resultsof thecurrent study showed thatROSSOPURO™ improved lipidprofileofanimalsbetter than the reference item.These results suggest that our technological approach lead to a bio-enhanced ingredient of dietary supplements andcandidate it as a valid alternative or complementary therapy to conventional treatment for the hypercholesterolemiamanagement,andthushavearoleincardiovascularpreventionstrategies.1. BarbagalloCM.etal.RoleofNutraceuticalsinHypolipidemicTherapy.FrontCardiovascMed.2015,2:22.2. LiY.etal.Ameta-analysisofredyeastrice:aneffectiveandrelativelysafealternativeapproachfordyslipidemia.PlosOne.2014,9:e98611.


73

EFFECTSOFPERALEJO(CuratellaamericanaL.)INTHEEXPULSIONOFKIDNEYSTONES,

USINGTRADITIONALMETHODS

RosadoJR.UniversityofLaGuajira,Riohacha,LaGuajira-Colombia

email:[email protected],[email protected] the experimental level, peralejo bark (Curatella americana L.) has been used to demonstrate their antihyperglycemicactivity in mice, but there are no scientific reports of its use in dissolving of kidney stones. At the level of wayuupharmacopoeiaasinglecaseofuseinsuchactivityisreported.Theaimoftheresearchwasdemonstratethroughtraditionalmethodstheeffectivenessofthebarkintheexpulsionofkidneystones.It was used bark branches with blackish gray color, 5 to 8 cmwide, washedwith cleanwater and dried 2 days indirectsunlight.Thebarkwascrumbledmanuallyandchipsobtainedwereplacedinaglassbottlecontainingpotablealcohol60°atarateof330gofbarkper1 literofalcohol,thiswaskeptinadarkplace,andstirredfor1minutedailyby14days.Then, itproceededtofilterthroughacleanclothandthefilteredliquidwasplacedin20mlplasticbottle.Becauseofdifficulties inachieving alcohol, this was replaced by artisanal rum of sugar cane (chirrinche) with grades similar to potable alcohol.Through the Health Center and community in generalwere selected approximately 50 patientswith problems of clinicaldiagnosis of stones in ureters, urethra and kidney chalices level. Clinic patients were also included for treatment afterconsultationwith the urologist. Each patient was given a bottle with the plant dye, prescribing a dose of 20 drops in atablespooninthemorningandbeforebedtimeuntilfinish20mlofthebottlecontents.Theoutcomesdemonstrate theeffectivenessofperalejobarkat all levelsof renal structure,noting that small stonesaredissolvedat 5daysof treatment, andpatients reporturinationwith sandstone,whilepatientswith larger stonesdelayedeven2monthsfromthestartoftreatmentandthenexpelthem.Allpatientsreported,almost immediately,paindecreasecausedinthelumbarpartbythepresenceofkidneystones,andaftertreatmentscansshowtheabsenceofthem.1. AriasHCetal.Plantasmedicinales.6taed.Bogotá,Colombia.19632. RosadoJR.FarmacopeaGuajira.2nded.EdicionesUniversidaddeLaGuajira,Ed.GenteNueva,Bogotá,Colombia.2009,p.4603. GómezHA.etal.Actividadantiinflamatoriadeproductosnaturales.BoletínLatinoamericanoydelCaribedePlantasMedicinalesyAromáticas2011,10.

Availableat:http://www.redalyc.org/articulo.oa?id=85618379003.Accesson:5Apr2016.


74

NRF2-MEDIATEDANTIOXIDANTACTIVITYOFT.ROSEA(BERTOL)DCANDTCHRYSANTHA(JACQ)G.

NICHOLSONEXTRACTS.

GarzónSC1,2,JiménezFJ.1,LoperaIA.1,SillerFR.2,VelozaL.A.1,Sepúlveda-AriasJC.1 1UniversidadTecnológicadePereira,Pereira(Risaralda)-Colombia;2FundaciónUniversitariaAutónomadelasAméricas,Pereira

(Risaralda)-Colombia.email:[email protected]

Plants from the genus Tabebuia have been used in traditional medicine as anti-inflammatory, anti-carcinogenic and anti-microbial agents, in rural areas of South America. Also, several ethnobotanical and ethnopharmacological studies haveshown the therapeutic potential of these plants for the treatment of several human diseases. These studies suggest apotential use of plant biodiversity in order to find new molecules with potential importance in pharmacology andtherapeutics (Jiménez-González., et al. 2013). However, most of the studies have been done on Tabebuia impetiginosa(FrancoOspina.,etal.2013).Theaimofthisstudywastoevaluate invitrotheantioxidantactivityoftheextractsfromtheinnerbarkofTabebuia rosea andTabebuia chrysanthamediatedby thenuclear factorE2-related factor 2 (Nrf2), using celllinesHEK-293(Humankidney),HepG2(humanhepatocellularcarcinoma)andHeLa(humancervicaladenocarcinoma).Theantioxidantactivityofextracts (n-hexane,chloroform,ethylacetate,n-butanolandaqueous)obtained fromthe innerbarkofTroseaandT.chrysanthawasevaluatedusingtheORACtechnique.TheeffectofextractsontheviabilityofHEK-293,HepG2 andHeLa cellswas determined using theMTTmethod. To evaluate the protective effect of extracts to theH2O2-inducedoxidativestresswedidusetheMTTmethod.TheeffectofextractsonthetranslocationofNrf2tothenucleuswasevaluatedwiththeNrf2transcriptionfactorassaykit (CaymanChemicalCo),accordingtothemanufacturer's instructions.The induction of the antioxidant response genes (NQO1, HO-1 and MT1A) was evaluated using real-time PCR with theTaqMan®RNA-to-CTTM1-stepkit(AppliedByosistems).The ethyl acetate extract obtained from the inner bark frombothT. rosea andT. chrysantha displayed thehighestORACactivity(12523and6325mmolesEqTrolox/gextract,respectively).TheevaluationoftheexpressionofantioxidantresponsegenessuchasNQO1andHO-1showedthattheseextractshavetheabilitytoinducetheexpressionofbothgenesinHeLaandHEK-293 cell lines, after 6 and 12 hours of exposure to the extracts, whereas in the HepG2 cell line only theNQO1 geneexpressionwas induced.Theextracts alsohad theability toactivateand translocateNrf2 to thenucleusafter4hoursofexposureofHepG2andHeLacellstotheextracts.TheseresultssuggestthattheethylacetateextractsfromtheinnerbarkofT.chrysanthaandT.roseahaveanimportantantioxidantactivityandmaybeusedasanewsourceofnaturalantioxidantsAcknowledgmentsto:PatrimonioAutónomoFondoNacionaldeFinanciamientoparalaCiencia,laTecnologíaylaInnovación,FranciscoJosédeCaldas,ContratoRC-0572-2012-Bio-Red-Co-CENIVAM, Vicerrectoría de Investigaciones Innovación y Extensión Universidad Tecnológica de Pereira and Fundación UniversitariaAutónomadelasAméricasforfinancialsupport.1. Jiménez-GonzálezFJ.etal.,Anti-infectiousactivityinplantsofthegenusTabebuia.UniversitasScientiarum,2013.18:257-267.2. Franco Ospina L.A., et al., Actividad antiinflamatoria, antioxidante y antibacteriana de dos especies del género Tabebuia. Revista Cubana de Plantas

Medicinales,2013,18:34-46


75

ANTIOXIDANTACTIVITYANDAPOPTOSISINDUCTIONASSESSEDBYFLOWCYTOMETRYOFABACTRIS

GUINEENSISPOLYPHENOLEXTRACT

QuesadaS.1 ,QuesadaMS2,AzofeifaG.1,PérezAM.31UniversidaddeCostaRica,DepartamentodeBioquímica,EscueladeMedicina,SanJosé-CostaRica;2UniversidaddeCostaRica,

FacultaddeFarmacia/InstitutodeInvestigacionesFarmaceúticas,SanJosé-CostaRica;3UniversidaddeCostaRica,CentroNacionalenCienciayTecnologíadeAlimentos,SanJosé-CostaRica


Bactrisguineensis (Aracaceae),alsoknown inCostaRicaasgüiscoyoloruvita,corozo inHonduras,Panamá,ColombiaandVenezuela, is a wild palm that grows in the plains. Its fruit has a high polyphenol content (824.4± 77.5 mg GAE/100 g),comparedwithanotherfruitslikeblueberryandplums.TheantioxidantactivityofBactrisguineensiswasreportedinliteraturewiththemethodsDPPHandORAC(Osorioetal.2011andRojanoetal.2012),howeveritisimportanttocorrelateitwithanotherassaysclosertoinvivosystems,liketheeffectsoncellculture.FullripefruitswerecollectedintheprovinceofGuanacaste,CostaRica.Thefruitwasextractedwithacetone/waterandthensubjected to polyphenol purification using anAmberlite XAD-7 column (150mmx 20mm). The columnwaswashedwithwater, the phenolic compoundswere elutedwithmethanol:water (80:20), concentrate under vacuum (40ºC) and freezedried.TheextractwasanalyzedbyUPLC-MS.AntioxidantactivitywasassessedbyusingtheDCFDAfluorescentdye.TheintracellularROSproductionwasinducedontheVero cell linewith tert-butyl-hydroperoxide (TBHP) in presence and absence of the polyphenol extract. The fluorescencepositivecellsweredeterminedbyflowcytometryandtheanalysisperformedbyCellQuestsoftware.InordertoanalyzeiftheobservedcytotoxicactivityofthepolyphenolextractonthecoloncarcinomacelllineSW620(IC50:16.6±1.3mg/mL)was because of an activation of apoptoticmechanisms, apoptotic cellswere quantified using Annexin V-FITC/PIassayandanalyzedbyflowcytometry.ThepolyphenolsidentifiedbyUPLC-MSintheextractwerecyanidin3-rutinoside,procyanidinB1andprocyanidin3-rutinoside.Theantioxidanteffectsofthepolyphenolsinsideofcells,usingtheDCFDAassay,showsthattheextractreducedtheinducedintracellularROS in adose-dependentmanner,withan IC50: 151.7mg/mL.Also theantioxidant activitywasdeterminedbyDPPH(IC50:3.3±0.2µg/mL)andORAC(979.1µmolTE/gextract).ThecytotoxiceffectobservedovertheSW620celllineseemstobehappenbyapoptosis.Weobserved50.3%ofcellsinearlyapoptosisanda17.7%ofcellsinlateapoptosis,afterthepolyphenolextracttreatment.However,furtherinvestigationwillbeneededinordertoexplaintheapoptoticmechanismsinvolvedinitsactivity.Inconclusion,withitspolyphenolcontent,highantioxidantactivityanditscytotoxiceffectoncoloncarcinomacells,Bactrisguineensisseemstobeafruitwithahighpotentialasfunctionalfood.Acknowledgments:financialsupportbyVicerrectoríadeInvestigación,UniversidaddeCostaRica(ProjectNo.735-B2-659)1. RojanoB.et al.Estabilidadde antocianinas y valoresde capacidadde absorbanciade radicalesoxígeno (ORAC)de extractos acuososde corozo (Bactris

guineensis).RevistaCubanadePlantasMedicinales2012,17:244-255.2. OsorioC.etal.Antioxidantactivityofcorozo(Bactrisguineensis)fruitbyelectronparamagneticresonance(EPR)spectroscopy.EuropeanFoodResearch

Technology2011:233:103-108.


76

InVitroANTIOXIDANTANDANTICHOLINESTERASEACTIVITIESOFCOLOMBIANPLANTSASPOTENTIALNEUROPROTECTIVEAGENTS

PlazasEA. ,CucaLE. ,AvilaMC.,PatiñoOJ.,DelgadoWA.

NationalUniversityofColombia,Bogotá-Colombiaemail:[email protected],[email protected]

Neurodegenerative diseases comprise a complex and heterogeneous group of central nervous system (CNS) disorders,characterizedby theprogressive and irreversible loss of neuronal cell populations from specific regions of thebrain. TheprototypicalneurodegenerativedisordersincludeAlzheimer'sdisease(AD),Parkinson'sdisease(PD)andamyotrophiclateralsclerosis(ALS).Acommonfeatureofthesediseasesistheextensiveevidenceofoxidativestress,whichmightberesponsibleforthedysfunctionordeathofneuronalcellsthatcontributestodiseasepathogenesis(Barnhametal,2004).ADisthemostprevalent neurodegenerative disorder worldwide and is the major cause of dementia associated with aging. It is amultifactorialdisorderwithacomplexpathogenesis.Thelargenumberofbiochemicalpathwaysaredistinguishbyabnormalaccumulations of extracellular (β-amyloid plaques) and intracellular (tau) proteins, mitochondrial abnormalities andneuroinflammatoryprocesses;triggeringaprogressivedegenerationofmemoryandcognitivefunction.Althoughthecausesand mechanisms of neuronal death in AD are unknown some factors to play an important role, such as the cholinergichypothesis and oxidative stress. The inhibition of AChE is an important target strategy not only for AD, also for seniledementia,ataxiaandPD.Actually,cholinesterase inhibitorsaretheonlyapproveddrugsfortreatingofADasGalantaminaand Rivastigmina; however, these only improve cognitive ability and have considerable side effects. Consequently, thedevelopmentfornewtherapeuticallyagentsmoreeffectiveandsafetyisrequired.Inrecentyears,theresearchhavefocusedon the search formultitarget agents like a promising option (Rosini et al, 2008). In thisworkwas performed an in vitroscreening for antioxidant and acetylcholinesterase inhibitory activity of Colombian Lauraceae, Piperaceae, Rutaceae andMyristicaceaespecies.Thepreliminaryactivitywasdeterminateusingan invitroTLC-bioautographicassayusingsilicagelasstationaryphaseandtwodifferentmobilephases,92ethanolicextractsof49Colombianspecieswereevaluated.Theantioxidantpotentialwasassessed by DPPH• scavenging activity by TLC-bioautographic, the active extracts were evaluated the total antioxidantcapacityandtheIC50weredeterminedandexpressedasVCEAC(mg/L) incomparisonwithascorbicacid(Dengetal,2011).AChEinhibitoryactivitywascarriedoutbybioautographicassaydescribedpreviously(Marstonetal.,2002).TheenzymaticactivityofextractswasmeasuredusinganadaptationofthecolorimetricEllmanmethod(Ingkaninanetal.,2003).InthepreliminaryTLCassaywereevaluated18Myristicaeae,32Lauraceae,30Piperaceaeand12Rutaceaeextracts.InDPPH•assay38extracts(300µg)ofthetotaltestedshowedcompoundswithhighcapacityofradicalscavenging,themarkedactiveextracts are mainly of the families Lauraceae and Myristicaceae. In most cases these compounds has medium to highpolarities, however some Piperaceae extracts exhibit nonpolar compounds with high radical scavenging capacity. In theAChEassay,15extracts(300µg)of7species(LauraceasandRutaceas)showedcompoundswithhighinhibitoryactivity.Thespecies of greatest activity againstAChEbelong to thegenus Zanthoxylum andOcoteaand they are characterizedby thepresenceofalkaloids;thesecompoundsarewidelyrecognizedtobeAChEinhibitors(Mukherjeeetal,2007).Bycomparison,to the antioxidant and anticholinesterase activities, it was possible to identify least nine extracts (5 species)which haveactivity in the both assays, therefore they can be considered promising for rational isolation of potential multitargettherapeuticagentsinthetreatmentofneurodegenerativediseases.1. Barnham,K.etal,Neurodegenerativediseasesandoxidativestress.Naturereviews2004,3:205-214.2. Deng,J.etal.Anovelantioxidantactivityindex(AAU)fornaturalproductsusingtheDPPHassay.FoodChemistry.2011,125:1430-1435.3. IngkaninanK.etal.ScreeningforacetylcholinesteraseinhibitoryactivityinplantsusedinThaitraditionalrejuvenatingandneurotonicremedies.Journalof

Ethnopharmacology.2003,89:261–264.4. Marston,A.etal.ArapidTLCbioautographicmethodforthedetectionofacetylcholinesteraseandbutyrylcholinesterase inhibitors inplantsPhytochem.

Anal.2002,13:51-54.5. Mukherjee,P.etal.Acetylcholinesteraseinhibitorsfromplants.Phytomedicine.2007,4:289–300.6. Rosini,M.etalInhibitionofAcetylcholinesterase,β-AmyloidAggregation,andNMDAReceptorsinAlzheimer’sDisease:APromisingDirectionfortheMulti-

target-DirectedLigandsGoldRush.J.Med.Chem.2008,51:4381–4384.


77

ANTIOXIDANTANDANTIPROLIFERATIVEACTIVITYOFHaematoxylombrasilettoKarst

Bello-MartínezJ.1 ,VenalonzoMM.2,Robles-ZepedaR.3,VelázquezCC.3,VidalGM.3,TorresGF.4,

Rosas-AcevedoJL.1¹UniversidadAutónomadeGuerrero,UnidaddeCienciasdeDesarrolloRegional,Acapulco(Guerrero)-México;²UniversidadAutónomadeGuerrero,UnidadAcadémicadeCienciasQuímicoBiológicas,LaboratoriodeQuímicadeProductosNaturales,Chilpancingo(Guerrero),México;3UniversidaddeSonora,DepartamentodeCienciasQuímicoBiológicas,CentroHermosillo

(Sonora)-México;4UniversidadAutónomadeGuerrero,UnidaddeCienciasNaturales,Petaquillas(Guerrero)-México.email:[email protected]

Traditionalmedicineincludestheuseofmedicinalplantsthathavebeenusedforthousandsofyearsbyruralandindigenouscommunities.Manyplantscontainantioxidantcompoundswhichprotectcellsfromtheadverseeffectsoffreeradicalsandoxidativestress,acauseofcancer(3).Haematoxylumbrasilettowoodyplant, isusedintraditionalmedicine inthestateofGuerrero,Méxicoforthetreatmentof:kidneyinfections,kidneystonesandanti-inflammatoryagent.(1,2)InthisstudyitwasevaluatetheantioxidantactivityofextractsandfractionsindifferentconcentrationsbyDPPHmethod0.1mM.ThepowderofheartwoodofH.brasiletto(1kg)wasextractedwith96%EtOHforthreetimestogivecrudeextract(50g), theextractwas then suspended in amixtureofwater andMetOH3:2 andpartitionedwithhexane,CH2Cl2 andEtOAcsuccessivelytogivefractionsHBM-2A(500mg),HBM-2B(2g)andHBM-2C(10g).Itwasfoundthattheethanolicextract(IC50=152,mg/mL±0.47)andtheorganicfractionHBM-2A(IC50=80,14mg/mL±0.25),HBM-2B(IC50=40,17mg/mL±0.15)andHBM-2C (IC50= 40,05 mg/mL ± 0.25) showed an antioxidant capacity significantly higher among extracts and fractionsanalyzed (p<0.001,a=0.05).TheTroloxwas takenasapositivecontrol forantioxidantactivitywithahighpercentageofinhibitionof(IC50=43,9mg/mL±0.41)comparedwiththeresultsobtainedforextractsandfractionsofH.brasiletto.Additionally,weevaluatedtheantiproliferativeactivityofthosefractionsbyMTTassayonRAW264.7andA549cancercelllines,usingL-929asanotcancerouscellline.HBM-2BshowedasignificantinhibitoryeffectonA549(22.57±0.86µg/mL),andRAW264.7(23.87±0.84µg/mL)cells.ThisfractionexhibitedactivityagainstL-929notcancerouscell(30.89±0.84µg/mL).Itcan be concluded that extracts ofHaematoxylum brasiletto, are important sources of antioxidants with anti-proliferativepotential.1. ArguetaA..Atlasdelasplantasdelamedicinatradicionalmexicana,vol.1.InstitutoNacionalIndigenista,MéxicoCity,México,20042. Li-GenL.et.al.NaturallyocurringHomoisoflavonoidsFuctionsaspotentproteinTyrosinekinaseInhibitorsbyc-Src-basedhigh-throughputscreening.J.

MedicinalChemistry,2008,51:No15.3. MamedovN.MedicinalPlantsStudies:History,ChallengesandProspective.MedAromatPlants.2012,1:1000-e133.


78

ANTIOXIDANT,HYPOGLYCEMICANDANTIMICROBIALACTIVITYOFTWOALGERIANTANNINSORGHUMSCRUDEXTRACTSCORRELATEDWITHPOLYPHENOLSANDTANNINCONTENTS

KadriF.1,2 ,BoudjenibaM.2,WahteletB.3

1Départ.deBiologieetPhysiologieCellulaire,FacultédesSciencesdelaNatureetdelaVie,UniversitéSaadDahlab-Blida,Blida-Algeria;2Laboratoired’EthnobotaniqueetSubstanceNaturelle,EcoleNormaleSupérieure,Kouba-Alger-Algeria;3Industrial

biologicalChemistryUnit,AgroBiotechfacultyofGembloux,LiegeUniversity,Liege-Belgium.email:[email protected]

Highlevelsofphenolicacids,procyanidinsandanthocyaninshavebeenreportedinsorghums.Polyphenolsincludingtanninshavebeenshowntohaveantioxydantandfreeradicalscaveringeffects.Antioxidantactivityof70%acetoneextractsof twoAlgerian tanninsorghumseeds,browntannin sorghum(BTS) and redtannin sorghum (RTS),was examined in relation to its contents in total polyphenols and tannins. Total phenols contentsestimated by Folin-Ciocalteu method and tannins contents estimated y HCl- Vanillin method. antioxidant activity.investigatedbyFreeradical,2,2-diphenyl-1-picrylhydrazyl(DPPH*)scaveringessay.Hypoglycemicactivityofmethanolandacetoneextractsofredtaninsorghumwasexaminedonratessubmittedtoalloxanetreatmentandtheantibacterialactivitywastestedusingdiscdiffusionmethod.Total phenol varied between 35.20 ± 2 in BTS extract and 10.34 ± 3mg g -1 in RTS extract s.while Tannins contentswerebetween61.36±1.3mgg-1and25.34±5mgg-1.ThehighestradicalscavengingeffectwasobservedinBTSwithIC50=5µgml–1crud extract. The potency of radical scavenging effect of this sorghumextractwas about 5 times greater than syntheticantioxidantButylatedHydroxy-Toluene(BHT)andtwotimesmorethanButylatedHydroxy-Anisole(BHA). Inaddition,theRTSshowedanIC50valueof15µgml–1whichstillmoreeffectivethanBHT.Glycemiavaluesof ratestreatedwithacetoneextractweresignificantly lowerthanthoseofpositivecontrolandthoseofrates treated with methanol extract. This, probably due to the difference in tannins composition between the twogenotypes.SorghumextractsshowedahighinhibitoryeffectonS.epedermidisandS.aureusgrowthwhilealowereffectwasobservedonE.coligrowth.The results obtained in the present study indicate that polyphenol and tannin content correlated strongly with theirbiologicalactivitiesandthattanninsorghumseedsareapotentialsourceofnaturalbioactivecompounds.1. DlaminiN.R.etal.TheeffectofsorghumtypeandprocessingontheantioxidantpropertiesofAfricansorghum-basedfoods.FoodChemistry2007,105:

1412–14192. DykesL.andRooneyL.W.Sorghumandmilletphenolsandantioxidants.J.CerealScience,2006,44,236–251.3. DykesL.etal.Phenoliccompoundsandantioxidantactivityofsorghumgrainsofvaryinggenotypes.J.AgriculturalandFoodChemistry,2005,53:6813–

6818.4. ChungIM.etal.AntidiabeticeffectsofthreeKoreansorghumphenolicextractsinnormalandstreptozotocin-induceddiabeticrats.FoodResearch

International,2011,44:127-132.


79

POSTERS


80

NUTRACEUTICEFFECTOFCUETLASArsenuraarmidaCEDIBLEINSECTLOCALFOOD

ATIXCAQUIXTLA,MEXICO

Melo-RuízV. ,Quirino-BarredaT.,Macin-CabreraS.,Sánchez-HerreraK.,Díaz-GarcíaR.UniversidadAutónomaMetropolitana,MexicoCity-Mexico.

email:[email protected], insect consumption is a cultural tradition inMexico since prehispanic time, practice that provides balancenutrientstoseveralethnicgroupsinMexico.Selectedspeciesfrominsectsplayanimportantroleatfestivitiesandcommoninsectsaresourceoftherequestednutrientsforadailydietofpopulation.Ixcaquixtla,semiruralvillagelocatedSouthEastPuebla,Stateatanaridzonewithalowfoodproductionhasseveralspeciesofedibleinsectsthatlocalpeopleintakebutnotasa regularbasis, since theyhavenot information regarding thenutraceutic functions thesesmallanimalscanprovide tohumanhealth.Nutraceuticaltermisdefinedasfoodorfoodcomponentsthatprovidehealthormedicalbenefits,includingpreventionortreatmentofdeceaseinhumans.Cuetlascommonnameofthelarvaestageofabutterflyisoneoftheinsectsconsumed by local people of Ixcaquixtla. The aim of this research is to chemical analyzemacronutrient composition andinvestigatethenutraceuticbenefitsinhealthandinformtolocalpeopleofthem.ConventionsamplingwastakeplaceatAugust2014,nearbythevillageandrawdry insectswereanalyzedproteins, lipids,minerals,fiberandsolublecarbohydratesindrybasisaccordingAOAC(19995)techniques.Dataobtainedwas:proteins56:93%;lipids14.76%;minerals10.07%;fiber2.13%;solublecarbohydrates16.11%.Cuetlascontentagoodamountofproteins,essentialmacromoleculesforhumanlife,essentialfattyacidsimportantsourcesoffuelforbraincellsandinparticularforhearthandskeletalmuscle,minerals,notdeterminedindividually,formetabolismprocesses,fiber,andsolublecarbohydratesforenergy.CuetlasedibleinsectsforIxcaquixtlacommunityprovidenutraceuticnutrientsthatbenefitandimprovelocalpeoplehealth.


81

ARGENTINIANMEDICINALPLANTSAGAINSTPOSTHARVESTPHYTOPATHOGENICSFUNGIISOLATED

FROMORANGES,STRAWBERRIESANDPEACHES

DiLibertoMG.1,SvetazLA.1;DeritaMG.1,2 1CONICET,UniversidadNacionaldeRosario,FacultaddeCienciasBioquímicasyFarmacéuticas,CátedradeFarmacognosia,Rosario(SantaFe)-Argentina;2CONICET,UniversidadNacionaldelLitoral,FacultaddeCienciasAgrarias,CátedradeCultivosIntensivos,

Esperanza(SantaFe),Argentina.email:[email protected]

Continuous and excessive use of synthetics compounds in order to prevent fungal infection in postharvested fruits hascauseddangerousconsequencesnotonly totheenvironmentbutalsotohumanhealth. In thiscontext,exploringnaturalsources based on native plant extracts or compounds isolated from them, turns out to be of great economic and socialimportance.1Inthiswork,threeArgentineannativespecieswithpreviouslyreportedhumanantifungalproperties(SolidagochilensisMeyen2,PolygonumacuminatumKunth3,4andDrimyswinteriiForst5),wereevaluatedagainstthreepathogenicfungi(Penicilium digitatum, Botrytis cinerea andMonilinia fructicola) which strongly affect oranges, strawberries and peachesrespectively.S. chilensis,P. acuminatum andD.winterii were collected during the flowering season (March 2014) in Santa Fe province(Argentina)anddepositedattheHerbariumFCA-UNL(ArturoRagonese)withidentifyingdataSF#MD16,MD97andMD108respectively.Air-driedaerial partsof each species (100g)werepowderedand successivelymacerated (3×24heach)withpetroleum ether (H), ethyl acetate (EtOAc) and methanol (MeOH) with mechanical stirring to obtain the correspondingextracts,afterfiltrationandevaporation.Fortheantifungalevaluation,standardizedstrainsfromNationalInstituteofAgriculturalInvestigation(INIA)Salto(Uruguay)andNational InstituteofAgricultural Technology (INTA) SanPedro (Argentina)wereused.P. digitatum INIA-S22 (P.d.),B.cinerea INIA-S26 (B.c.) andM. fructicola INTA-SP345 (M.f.) strainswere grown on Potato-Dextrose-Agar (PDA) using Petridishes for 48 h up to 6 days at 15-18 °C (as needed for each fungi growth). Inocula of spore suspensionswere obtainedaccording to reported procedures (CLSI)6 and adjusted to 1 x104 Colony Forming Units (CFU)/mL.1 Minimum InhibitoryConcentrations (MICs) of each plant extract were determined by using broth microdilution techniques according to theguidelinesoftheCLSIforfilamentousfungi(M38A2).6Imazalilandcarbendazimwereusedaspositivecontrols.Endpointsweredefinedasthe lowestconcentrationofextractresulting intotal inhibition(MIC100)ofvisualgrowthcomparedtothegrowthinthecontrolwellscontainingnoantifungal.

Results are shown in Table 1. Regarding S. chilensis, MeOHextractwas themost active one, withMICs between 250-1000µg/mL and showing the highest potency against P.d., H andEtOAcextractsweremoreactiveagainstM.f.(MICs=125µg/mL)buthardlyactiveagainstB.c.(MICs=1000µg/mL).P.acuminatumH and EtOAc extracts weremoderately active against P.d. andB.c. (MICs between 250-500 µg/mL) but showed importantinhibition for M.f. (MICs= 31.2 µg/mL), while MeOH extractresulted inactive.D. winteriiH and EtOAc extracts were hardlyactiveagainstP.d.andB.c.(MICsbetween500-1000µg/mL)butthey exhibited activity against M.f. (MICs= 125 µg/mL), MeOHextractalsoturnedoutinactive.Acknowledgement:CONICETforMDdoctoralfellowshipandPIP11220150100524CO,UNRprojectBIO411.

1. Singh,H.;Batish,D.;Kohlil,R.2003.CriticalReviewinPlantSciences22:239-311.2. Vila,R.;MundinaM.;Tomi,F.;Furlán,R.;Zacchino,S.;Casanova,J.;Cañigueral,S.2002.PlantaMedica68:164-167.3. Derita,M.;Leiva,M.;Zacchino,S.2009.JournalofEthnopharmacology124:377-383.4. Derita,M.;Zacchino,S.2011.NaturalProductCommunications6(7):931-933.5. Malheiros,A.;EtAl.2005.JournalofPharmacyandPharmaceuticalSciences8:335-339.6. ClinicalandLaboratoryStandardsInstitute(CLSI).2002.ReferencemethodforbrothdilutionantifungalsusceptibilitytestingforfilamentousfungiM-38

A2.Vol.28,p.1-35.(Wayne,L.ed).

Table1:MinimumInhibitoryConcentrations(MICs)(μg/mL)ofeach plant extract evaluated. I= inactive (MIC > 1000 µg/mL).P.d: Penicillium digitatum, B.c: Botrytis cinerea, M.f: Moniliniafructicola

PlantMaterial Extract MICs(µg/mL)P.d B.c M.f

S.chilensis

(aerealparts)

H I 1000 125AcOEt I 1000 125MeOH 250 500 1000

P.acuminatum(aerealparts)

H 250 250 31.2AcOEt 500 500 31.2MeOH I I I

D.winterii(bark)

H 500 500 125AcOEt 1000 1000 125MeOH I I I

Imazalil 7.8 0.9 0.12Carbendazim 15.6 7.8 0.49


82

CACAOCO-PRODUCTS:ANALTERNATIVETOOBESITYCONTROL

Hidalgo-PérezTejadaEI.1,Aguirre-LópezAE.1,Mena-LópezJA.1,Plazola-JacintoCP.2,Valadez-CarmonaL.2,Ortiz-MorenoA.2,Hernández-NavarroMD.3,Ceballos-ReyesGM.1 ,Hernández-OrtegaM.3

¹InstitutoPolitécnicoNacional,EscuelaSuperiordeMedicina,CiudaddeMéxico-México;²InstitutoPolitécnicoNacional,EscuelaNacionaldeCinciasBiológicas,CiudaddeMéxico-México;3UniversidadAutónomadelEstadodeMéxico,FacultaddeQuímica,

TolucadeLerdo(EstadodeMéxico)-Méxicoemail:[email protected],[email protected]

Overweightandobesityhavebeenhighlyincreasinginthelastyears,becomingaseriouspublichealthproblem;amongthestrategies to control the problem are functional foods therefore different strategies are being sought to control thisepidemic, including functional foods.Cacao is rich in flavonoidsmainly (-)-epicatechinand fiber,whichhavebeen showedtherapeutic effects thus, the use of cacao co-products represents an attractive alternative as an adjuvant for handlingoverweightandobesity. Therefore theaimof thepresentworkwas toevaluate theeffectof cacaoco-productsonbodyweight,lipidprofile,bloodpressureandhepaticenzymesinarodentmodelwithobesityinducedbydiet1,2.Inthisstudy,Wistarmaleratswerefedwithanobesogenicdiet(21%offat)and20%fructosesolutionasconsumptionwaterduring 5weeks. After 5weeks of feedingwith the high fat diet, ratswere distributed into 4 groups: Control, I, II and III(obesogenicdietplus141.54,23.55and11.751mgofequivalentsofGallicacidrespectivelyobtainedfromcacaoco-products).After5weeksoftreatmentbloodsampleswereobtainedviaretro-orbitalpunctures;bloodwasallowedtoclotfor30min,centrifuged at 3500 r.p.m. for 15 min, and then the serum was collected and analyzed. Glucose and triglycerides weredeterminedusingcommerciallyavailablecolorimetrickits(RandoxS.A.s,Mexico).Systolicbloodpressurewasmeasuredbyanon-invasivetail-cuffmethodbeforestartingthedietaryinductionperiod,atweek5andatstudycompletion3.Thegroupthatpresentedanimprovementinthecardiometabolicprofilewastheonethatreceivedatotaldoseof23.55mgofcacaoco-productsphenoliccontentshowingasignificantdecreased(p<0.05) incholesterol (36%), triglycerides(63%),c-LDL(37%)concentrations,andalsointherelationTG/c-HDL(64%)comparedtothedataobtainedintheobesogenicgroup.Nosignificantchanges(p>0.05)wereobservedinthec-HDLconcentration.Bloodpressuredecreasedsignificantly(p<0.05)ina27%andalsothehepaticenzymesdecreasedtheirconcentration(p<0.05).Theresultssuggestthatphenolsofcacaoco-productsimprovethelipidprofileofobeseratsindicatingthattheuseofco-productsasrawmaterialfortheproductionoffunctionalfoodscanbeusedasanadjuvantinthemanagementofoverweightandobesity.Acknowledgments:ThisworkwassupportedbytheInstitutoPolitécnicoNacionalandCONACyT.1. Martínez R, Torres P,MenesesMA, Figueroa JG, Pérez AJ, ViudaMM. (2012). "Chemical, technological and in vitro antioxidant properties of cocoa

(TheobromacacaoL.)co-products".FoodResearchInternational.49:39–45.2. GutierrezG.,Ortiz P., Vacaseydel V.C., Garduno L., ChamorroG.,Meaney E., Villafana S., Villarreal F., CeballosG. y Ramirez I. (2014). "Effects of (-)-

epicatechinonadiet-inducedratmodelofcardiometabolicriskfactors."EuropeanJournalofPharmacology.728:24-30.3. Kubota, Y,Umegaki, K, Kagota, S, Tanaka,N,Nakamura, K, Kunitomo,M, et al., 2006. Evaluation of blood pressuremeasured by tail-cuffmethods

(withoutheating)inspontaneouslyhypertensiverats.Biol.Pharm.Bull.29(8),1756–1758.


83

BOTRICIDEACTIVITYOFDRIMENOLANDDERIVATIVES

CarrascoH.1 ,OleaA.1,SilvaE.2 ,Robles-KellyC2,MartínezR.3,SedanA.3,ThomasM.3

¹InstitutodeQuímicaAplicada,FacultaddeIngenieríaUniversidadAutónomadeChile,Santiago-Chile;²InstitutodeCienciasBiomédicas,FacultaddeMedicinaUniversidadAutónomadeChile,Santiago-Chile;3InstitutodeQuímica,UniversidadAndrés

Bello,Valparaíso-Chile.email:[email protected];[email protected]

Botrytis cinerea, also known as “graymould fungus”, causes serious pre- and post-harvest diseases in at least 200 plantspecies,includingagronomicallyimportantcropsandharvestedcommodities.Nevertheless,themagnitudeofthefungicidaltreatments against this fungus has induced the appearance of resistant isolates, necessitating the identification anddevelopmentofnewmolecules.1The“canelo” isamedicinal treenativeformChilethathasbeentraditionallyusedbytheMapuchepeople,phytochemicalstudies have demonstrated that this plant containsmainly sesquiterpenes of the drimane-type and flavonoids, being themajoritydrimenolandpolygodial.Severalstudieshavereportedbiologicalactivitybothessentialsoilof“canelo”andtheirmetabolites.2,3Fungitoxicityofdrimenolandtheirderivativesandthecommercial fungicideBC1000wasassessedusingtheradialgrowth

testonmalt-yeastextractagar.ThecompoundsexcepttheBC-1000,weredissolved indichloromethaneatdifferent final concentrations (20,40,80,160 ppm). The medium with or without Drimenol, their derivatives andBC1000waspoured into6 cmdiameterPetri dishes.After evaporationofthe solvent, the Petri disheswere inoculatedwith 0.5 cm agar discswiththinmycelium ofB. cinerea. Cultureswere incubated in the dark at 22 ºCduring several days. Mycelial growth diameters were measured daily.Results were also expressed as EC50 (the concentration that reducedmycelialgrowthby50%).Eachexperimentwasdoneatleastintriplicate.Inthisworktheeffectofdrimenolanditsderivatives,Ac-Epox-drimenol(1)and Drimenone (2), on B. cinerea have been studied. Drimenol, asesquiterpeneofdrimane,wasobtainedfromD.winteri'sbarkaccordingtostandarprocedure.4Table 1showedtheeffectonBotrytiscinereamycelial

growthatdifferentconcentrations.Results showedwhat derivatives (1) and (2)were able to inhibit the hyphal growth at the same concentrations used fordrimenol.Nevertheless,theinhibitioneffectof(1)waslowerthandrimenoland(2).However,allcompoundstestedinthisworkshowedamuchlowerantifungalactivitythanthecommercialfungicide,Bc1000(Table1).Acknowledgments:TheauthorsthankstoFondecytforGrants1130742and11140194.1. LerouxP.,D.Debieu, C.Albertini,A.Arnold, J. Bach, F. Chapeland, E. Fournier,R. Fritz,M.Gredt, T.Giraud,M.Hugon,C. Lanen,C.Malosse andG.

Thebaud,2002.(H-W.Dehne,U.Gisi,K.H.Kuck,P.E.Russel,H.Lyr,ed.),AgroConceptGmbH,Bonn,Germany29–40.EDIT2010.Institute_ABC.2. Derita, M.; Montenegro, I.; Garibotto, F.; Enriz, R.; Cuellar, M.; Zacchino, S. Structural requirementsfor the antifungal activities of natural drimane

sesquiterpenesandanalogues,supportedbyconformationalandelectronicstudies.Molecules2013,18,2029–2051.3. Montenegro,I.,Tomasoni,G.,Bosio,C.,Quiñones,N.,Madrid,A.,Carrasco,H.,Olea,A.,Martinez,R.,Cuellar,M.andVillena,J.StudyontheCytotoxic

ActivityofDrimaneSesquiterpenesandNordrimaneCompoundsagainstCancerCellLines.Molecules2014,19,18993-19006.4. CechinelFilho,V.andYunes,R.A.Estratégiasparaaobtençãodecompostosfarmacologicamenteativosapartirdeplantasmedicinais,conceitossobre

modificaçãoestruturalparaaotimizaçãodaatividade.Quim.Nova,1998,21,99-105.

Table1. Effectofdrimenolandbyproductsonin vitro mycelial growth of B. cinerea anestimationofmedianeffectivedoses (ED50)wasbasedoncolonydiametermeasurementsafter4daysofincubation.


84

ANEWIRIDOIDANDOTHERCONSTITUENTSFROMPterocephalusnestorianusNab.

AbdullahFO.1 ,HussainFHS1,VitaFinziP2,VidariG.2

1DepartmentofChemistry,CollegeofScience,UniversitySalahaddin-Erbil,Erbil-Iraq;2DipartimentodiChimica,UniversitàdiPavia,Pavia-Italy

email:fuadchem@yahoo.comTheKurdistanRegionofIraqhasacentennialtraditionintheuseofmedicinalplants;however,themajorityofthemarestilluninvestigated from a phytochemically and pharmacologically point of view. Therefore, recently, we decided to start anambitiousprojectontheisolationandcharacterizationofmainbioactivecompoundsfromKurdishmedicinalplants.Withinthisprogram,wereport,forthefirsttime,themainconstituentsisolatedfromPterocephalusnestorianusNab.(Dipsacaceae),known in Kurdistan with the name of Lawa. This plant grows in sunny, dry, rock crevices, mostly inWestern Asia (Iran,Turkey), and it is used inKurdistan for treatingoral diseases and inflammation.Pterocephalus species are, indeed,widelyusedasornamentalplantsandinseveralfolkloricmedicinesallaroundtheworld.DryflowersandrootsofP.nestorianus(100g)wereseparatelyexhaustivelyextractedbymacerationatroomtemperatureinsolventsofincreasingpolarity:hexane,EtOAc,MeOH,MeOH-H2O,70:30.Subsequently,chlorophyllswereremovedfromtheMeOHextractsbySPE technique.Thechlorophyll freeextractswere then separately fractionatedby repartitionbetweensolvents and repetitive MPLC on successive C-18 reversed phase columns. The structures of isolated compounds wereestablishedby2DNMRspectroscopicmethodsand,incaseofknowncompounds,bycomparisonwithliterature.Themajorbioactiveisolatedproductswerekaempferolglycosidesandthreeiridoids: loganicacid(1), loganin(2) andthenewdimer,secologanylester(3).Theseiridoidsshowacharacteristichighanti-inflammatoryactivity,whichthuscorroboratethetraditionalusesoftheplant.Acknowledgments:ThisresearchhasbeenfinancedbytheUniversityofSalahaddin-Erbil/IraqandtheUniversityofPavia-Italy,intheframeworkofthesplit-sideprogramforthesupporttothePhDstudiesofFuadO.Abdullah.1. KawtherAS.SystematicStudyoftheGenusPterocephalusVaillEXAdans.(Dipsacaceae)inKurdistanofIraq.M.Sc.thesis,SalahaddinUniversity-Erbil.

Iraq.2007.2. GhahremanA,1995.FloraofIran.Researchinstituteofforestandrangelands.TehranUniversityPress,Tehran.3. Vahedi H, Nasrabadi M.; Lari J; Halimi M, 2011. Volatile constituents and antimicrobial activities of Pterocephalus canus. Journal of Medicinal Plants

Research,5,5646-5648.


85

BOTRICIDEACTIVITYOFEUGENOLANDDERIVATIVES

CarrascoH.1 ,OleaA.1,SilvaE.2 ,BarrazaD.2,Robles-KellyC.2,MartínezR.3,SedanA.3,ThomasM.3¹InstitutodeQuímicaAplicada,FacultaddeIngenieríaUniversidadAutónomadeChile,Santiago-Chile;²InstitutodeCienciasBiomédicas,FacultaddeMedicinaUniversidadAutónomadeChile,Santiago-Chile;3InstitutodeQuímica,UniversidadAndrés

Bello,Valparaíso-Chile.email:[email protected];[email protected]

Eugenol is amajor component of essential oil isolated from the Eugenia caryoplyllata,which has beenwidely used as anherbaldrug.Itisaremarkablyversatilemoleculeincorporatedasafunctionalingredientinnumerousproductsandhasfoundapplicationinit’savastrangeofpharmacologicalactivitieshasbeenwell-researchedandincludesantimicrobial,anti-oxidantandanticanceractivities,amongstothers.1,2Botrytis cinerea, also known as “gray mould fungus”, cause serious pre- and post-harvest diseases in at least 200 plantspecies, including agronomically important crops and harvested commodities, such as grapevine, tomato, strawberry,cucumber,bulbflower,cutflowerandornamentalplants(Jarvis,1980).3Fungitoxicityofeugenoland theirderivativesand thecommercial fungicideBC1000wasassessedusing the radialgrowthtestonmalt-yeast extract agar. The compoundsexcept theBC-1000,weredissolved indichloromethaneatdifferent finalconcentrations(20,40,80,160ppm).Themediumwithorwithouteugenol,theirderivativesandBC1000waspouredinto6cmdiameterPetridishes.Afterevaporationofthesolvent,thePetridisheswereinoculatedwith0.5cmagardiscswiththinmyceliumofB. cinerea. Cultureswere incubated in thedarkat 22 ºCduring severaldays.Mycelial growthdiametersweremeasureddaily.In this work the effect of eugenol, eugenol acetate, isoeugenol, isoeugenol acetate and 4-allyl-6-nitro-2-methoxyphenol(nitroeugenol),onB.cinereahavebeenstudied.Figure1showedtheeffectonBotrytiscinereamycelialgrowthatdifferentconcentrations.

Figure1.EffectofeugenolandderivativesonmycelialgrowthofB.cinereainsolidmedia.Percentageofgrowthinhibitionwasdeterminedafter48hoursofincubation.Everybarrepresentstheaverageofatleastthreeindependentexperiments±standarddeviation.Resultsshowedwhateugenolacetate,isoeugenolandisoeugenolacetatewereabletoinhibitthehyphalgrowthwithvaluesofEC50of112.77,56.55and67.84ppmrespectively.However,allcompoundstestedinthisworkshowedalowerantifungalactivitythanthecommercialfungicide,Bc1000.Acknowledgement:TheauthorsthankstoFondecytforGrants1130742and11140194.1. CarrascoAltamirano,H.;Espinoza,L.;Gallardo,C.;Cardona,W.;Catalán,K.;RussoA.;Cardile,V.; Lombardo,L.;Cuellar,M.Eugenoland its synthetic

analoguesinhibitcellgrowthofhumancancercells(PartI).J.Braz.Chem.Soc.2008,19,543–548.2. Héctor Carrasco, Marcela Raimondi, Laura Svetaz, Melina Di Liberto, María V. Rodriguez, Luis Espinoza, Alejandro Madrid and Susana Zacchino.

AntifungalActivityofEugenolAnalogues.InfluenceofDifferentSubstituentsandStudiesonMechanismofAction.Molecules2012,17,1002-10243. JarvisWREpidemiology.In:Coley-SmithJR,VerhoeffKandJarvisWR(eds)TheBiologyofBotrytis.AcademicPress,London,UK.1980.219-250


86

THELONGHISTORYOFPASTORALISMINSOUTHERNITALYASSHOWNBYARCHAEOPALYNOLOGY

FlorenzanoA.

UniversitàdiModenaeReggioEmilia,DipartimentoScienzeVita,LaboratoriodiPalinologiaePaleobotanica,Modena,Italyemail:[email protected]

PastoralismisoneofthemaineconomicactivitiesofthepeoplelivinginSouthernItaly.Animalbreedingandtranshumance(seasonalmigrationoflivestockandherdersbetweenmountainsandvalleysorcoasts)havealongtraditioninthisterritoryandcertainlyplayedanimportantroleinshapingthelandscape.Thankstothelong-timescalearchaeobotanicalresearch,itispossible to find the roots of this traditional practice in regions where vegetation and landforms have been significantlymodifiedbysheeptracksandpastures.The study of pasture indicators of biological origin from archaeological sites of southern Italy is particularly useful indiscriminatinglandusesandpastoral/breedingactivities,allowingtheidentificationofpastoralsitesandactivitieswhentheyarevirtuallyimpossibletoidentifyinthearchaeologicalrecordalone(Florenzano2015).ThisresearchhasbeencarriedoutonsamplesfromthemainarchaeologicalsitesofBasilicataregion located inatransectfromtheApennines(1site)tothecoastalongtheBradanoriver(7sites).The8siteshavebeenstudiedincollaborationwiththeUniversityofBasilicataandtheUniversityofTexasatAustin(Florenzano2013).A set of 121 pollen sampleswere taken from archaeological layers – small trenches, rooms or floors of houses, and spotsamples.Accordingtothechronologyofthesearchaeologicalsites,thetemporaltransectspansabout20centuries -fromtheArchaictoMedievalperiods.Ingeneral,thelocalevidenceofhuman-inducedenvironmentsismarkedbyhighvaluesofAnthropogenicPollenIndicators(API), plant taxa directly associatedwith human activities, such as crop plants, floras of arableweeds, and ruderal floras(Mercuri etal. 2013). Inpollendiagrams, theclearest signal forpastoralism isgivenby theabundanceofplants reflectinganimal breeding and grazing areas, such as daisy-family (Cichorieae and Asteroideae; Florenzano et al. 2015). The highpresenceofotherpollentaxamaybeindicativethoughindirectevidenceofpasturelands:e.g.plantain-Plantagoiscommonintrampledareas,andnettle-Urtica innitrophilousenvironments.Inadditiontothesepollenpastureindicators,non-pollenpalynomorphs-NPPscanbeusedtoassessthepresenceofpastfauna,inparticularherbivores.Ascosporesofdung-relatedfungi Sordaria, Sporormiella, Podospora and Cercophora are the most reliable indicators associated with local grazingactivities(vanGeeletal.2003).Datapointtoanopenlandscapedominatedbypasturesandcerealfields.Importantevidenceofpastoral farming rises from the joint recordofpollengrazing indicators and sporesof coprophilous fungi. ThisdatasethighlightthepressureofpastoralisminthepastandsupporttheideaoftheimportanceoftheancientpasturefarmingasamajoragentoflandscapetransformationinthisMediterraneanregion.1. FlorenzanoA,2013.Evoluzionediunpaesaggiomediterraneonellaricostruzionearcheoambientaledisitilucani.PhDThesis,UNIMORE,Italy.2. FlorenzanoA,2015.Lapastorizianell’economiaenelmodellamentodelpaesaggiomediterraneo.EsempidasitiarcheologicidelsudItalia.In:CambiF.,

DeVenutoG.,GoffredoR.(Eds.),StoriaeArcheologiaGlobale2.Ipascoli,icampi,ilmare.Edipuglia,Bari,Italy,pp.245-252.3. FlorenzanoA,MarignaniM,Rosati L, Fascetti S,MercuriAM,2015.AreCichorieaean indicatorofopenhabitatsandpastoralism in currentandpast

vegetationstudies?PlantBiosystems149,pp.154-165.4. MercuriAM,BandiniMazzantiM,FlorenzanoA,MontecchiMC,RattighieriE,TorriP,2013.AnthropogenicPollenIndicators(API)fromarchaeological

sitesaslocalevidenceofhuman-inducedenvironmentsintheItalianpeninsula.AnnalidiBotanica3,pp.143-153.5. vanGeelB,BuurmanJ,BrinkkemperO,SchelvisJ,AptrootA,vanReenenG,HakbijlT,2003.EnvironmentalreconstructionofaRomanPeriodsettlement

siteinUitgeest(TheNetherlands),withspecialreferencetocoprophilousfungi.JournalofArchaeologicalScience30,pp.873-883.


87

MULTIDISCIPLINARYINVESTIGATIONONEARLY-MIDHOLOCENEWILDCEREALSFOUNDAT

TAKARKORI(CENTRALSAHARA)

FornaciariR.1,2 ,ArruL.3,MercuriAM.1,diLerniaS.4,51UniversitàdiModenaeReggioEmilia,DipartimentoScienzeVita,LaboratoriodiPalinologiaePaleobotanica,Modena,Italy;

2CentroInterdipartimentaleBiogest-Siteia,ReggioEmilia-Italy;3UniversitàdiModenaeReggioEmilia,DipartimentoScienzeVita,ReggioEmilia-Italy;4UniversitàLaSapienza,DipartimentodiScienzedell’Antichità,Roma-Italy;5SchoolofGeography,

ArchaeologyandEnvironmentalStudies,UniversityoftheWitwatersrand,Johannesburg-SouthAfrica.email:[email protected]

Plantmacroremains from rock sheltersof central Saharagive information about the environmental conditionsduring theHolocene, and the adaptive strategies of human groups living in the area. Takarkori was excavated by the Italian-LibyanArchaeologicalMissionintheAcacusandMessak(directedbyS.diLernia,SapienzaUniversityofRome)anditschronologyrangesfromca.10,200toca.4,600calyrBP(Cremaschietal.2014).For the morphometrical analysis, fertile spikelets of Panicum laetum, Echinochloa colona and Sorghum bicolor subsp.verticilliflorumwereselectedasrepresentativeofdifferentculturalcontest.1,600desiccatedspecimens,foundas18“seedaccumulations”inthesite,weremeasuredusingimageanalysistechniquesanddatawereelaboratedbymeansofPrincipalComponentAnalysis (PCA). For themolecular analysis, aDNAwasextracted from the spikelets testingdifferentprotocolsand thenwas studied bymeans of theDNAbarcoding approach, using four chloroplastmarkers (rbcL,matK, trnH-psbA,trnL).AneighbourjoiningclusteringwasperformedonthecombineddatasetofthebarcodesequencesusingthestatisticalpackagePAST–PAleontologicalStatistics.Becauseof thepresent-day importanceofwildcereals in theSaharanandsub-Saharanregions,ethnobotanicalinformationwereinvestigatedfromliterature.Thearchaeobotanicalrecordshowshomogeneoustypologyanduniformsizeofthespikelets.Moreover,somerelationsarefounded inthecomparisonwiththemeasuresoftherelevantmodernspecies.ThiscouldbeassociatedwiththeactionofcollectionofthosecerealsbythehumangroupswholivedatTakarkori(Fornaciarietal.submitted).BioinformaticanalysisoftheaDNAsequencesallowstoinspectthephylogeneticrelationshipsbetweenthearchaeobotanicalrecordsandthemodernspeciesofAfricanwildcereals(Fornaciarietal.submitted).NextGenerationSequence(NGS)analysisisinprogress,andwillallowadeeperstudyofthedomesticationlevel,theoriginanddevelopingofminorcerealsinAfrica,eventuallyhelpingthearchaeologists in understanding changes in cultural trajectories. The specimens found at Takarkori, selected by type, areevidenceofadeepknowledgeoftheplantsdistributed intheregionandofthepresenceofasmallgroupofwildcerealscollectedcontinuouslyintheareaforaverylongtime.GrainsofthiswildspeciesarestillnowadaysessentialinthehumandietinAfricaandareharvestedbynomadicpeopleforfood,fodderandmanyotherpurposes(Mercuri2008).1. Cremaschi M, Zerboni A, Mercuri AM, Olmi L, Biagetti S, di Lernia S, 2014. Takarkori rock shelter (SW Lybia): an archive of Holocene climate and

environmentalchangesinthecentralSahara.QuaternaryScienceReview,101,pp36–60.2. FornaciariR,ArruL,TerenzianiR,MercuriAM,diLerniaS.MorphometryofwildcerealsfromHolocenearchaeologicalrecordsofcentralSahara(SW

Libya).Submittedto“PLANTSANDPEOPLEINTHEAFRICANPAST:ProgressinAfricanArchaeobotany”.3. FornaciariR,FornaciariS,FranciaE,MercuriAM,ArruL.PanicumspikeletsfromEarlyHoloceneTakarkorirockshelter(S-WLibya):archaeo-molecular

and-botanicalinvestigations.SubmittedtoPlantBiosystems.4. Mercuri AM, 2008. Plant exploitation and ethnopalynological evidence from the Wadi Teshuinat area (Tadrart Acacus, Libyan Sahara). Journal of

ArchaeologicalScience,35/6,pp1619–1642.


88

MULTIMEDIA,PREVENTIONANDMITIGATIONOFRISKINCARIBBEANCOUNTRIESAFFECTEDBY

NATURALDISASTERS

CalaCalaM1 ,FerreiroCantilloO1,CarballoDS2,PerezTrejoJM2,PeñaAriasY.1ProvincialDelegationoftheMinistryofScienceTechnologyandEnvironment;2CenterforResearchandTechnology

Management,CITMA,Guantánamo-Cuba.email:[email protected]

Among themain factors influencing the imbalance and health of plants, animals and human beings, there are poor diet,hereditary factors,andrepressivedrugs, lackofexercise,destructiveemotionsandenvironmentalpollution.Hence, ithasdeveloped a multimedia product, under the agreement Food Sovereignty and Local Development, aimed at vulnerablegroups affectedbynatural disasters inCaribbean countries, project fundedbyNGOsSpanish, toworkonprevention andmitigationnaturalandhealthdisasters.Thedevelopmentofvaluessuchassolidarity,cooperation,exchange,growth,socialcommunication;whilerespectingthecharacteristicsandpeculiaritiesofeachprovince,municipalityandcommunitywhereintervenedevidencedinthiswork.Inthemultimediausersorcustomerscanaccessmorethan1500documents(manuals,books,series,regulatoryframework,morethan900termsforabetterunderstandingofarticlesandmorethan250internationalenvironmentalephemeris,etc.)informatspdf,ppt,docandvideos;wherebasicandspecificinformationonnaturaldisasters(hydrometeorologicalfloods,hurricanes, storms, sea flooding, drought, earthquakes, forest fires) are shown, technological and health (epidemics,zoonosesandepidemics)thatisexposedtoanyterritoryorcountry.


89

BIOACCESSSIBILITYANDBIOACTIVITYOFPOLYPHENOLSEXTRACTEDFROMSIXCHERRYCULTIVARS

MartiniS. ,TagliazucchiD.,ConteA.

UniversityofModenaandReggioEmilia,DepartmentofLifeSciences,ReggioEmilia-Italyemail:[email protected]

Thereisapossibleassociationbetweenconsumptionoffruitandvegetablesandreducedriskofcancer,particularlycancerof thedigestive tract. This anti-cancer activity hasbeen attributed inpart topolyphenolspresent in foods [1]. Cherries inparticulararearichsourceofpolyphenols,especiallyanthocyaninsandhydroxycinnamicacids[2].Cherriesfromsixdifferentcultivars(DuronedellaMarca,Celeste,DuroneNeroI,Bigarreau,LapinsandMoretta)wereinvitrodigestedwith a procedure thatmimicked thephysiochemical conditionsof thegastro-intestinal tract [3]. After digestion,polyphenol-rich extracts were obtained by preparative chromatography on C18 column. Polyphenols were also extractedfromun-digestedcherrieswithmethanol-water-formicacidsolution(70:28:2v/v)andchromatographedonC18column.Totalpolyphenols and anthocyanins concentration were determined by Folin-Ciocalteau assay and pH differential method,respectively[3].Polyphenolswereidentifiedusingliquidchromatographymassspectrometry.Thedigestedandun-digestedpreparationswereassessedfortheircytotoxicandanti-proliferativeactivitiesusingtwohumancolonadenocarcinomacelllines(Caco-2andSW480)andIC50valuesofanti-proliferativeactivityweredetermined.Thetotalpolyphenolconcentrationintheun-digestedsamplesvariedfrom70.2mg/100gofcherryinthecultivarDuronedellaMarcato280.6mg/100gofcherriesinthecultivarDuroneNeroI.Thetotalanthocyanincontentvariedfrom0.12mg/100gofcherryinthecultivarDuronedellaMarcatoabout17mg/100gofcherryinthetwodarkercultivars(LapinsandMoretta). Invitrodigestionresultedinadecreaseinbothtotalpolyphenolsandanthocyanins.Totalpolyphenolsdecreaserangedfrom56to80%,whereastotalanthocyanindecreasewasmorethan90%inallthetestedcultivars.Massspectrometryshowedthatthe gastro-intestinal digestion decreased the content of hydroxycinnamic acids and anthocyanins. Other classes ofpolyphenolsaremorestabletodigestion.Breakdownproductsofpolyphenolsarealsopresent.Digestedandun-digestedpolyphenol-rich extracts showed no cytotoxic activity. Un-digested polyphenol-rich extract from the cultivar Bigarreaushowedthehighestanti-proliferativeeffect (IC50of7.1± 1.1µgpolyphenols/mL)ontheSW480cells.The invitrodigestionincreased the anti-proliferative activityof cherry extracts.Polyphenol-richextracts fromdigestedDuronedellaMarca andCelestewere themost activewith IC50 values of 1.7 ± 0.8 and 3.5 ± 1.2 µgpolyphenols/mL, respectively. All of the testedextractsshowednoanti-proliferativeactivityonCaco-2cells.Our results showed that cherry polyphenols were effective anti-proliferative agents on the more undifferentiated colonadenocarcinoma cells SW480 and that their digestion increased the effect. The increasemay be due to the formation ofpolyphenolmetabolites.Agreatdifferencewasobservedbetweenthecultivarsused.Furtherresearchisrequiredtoidentifythecompoundsresponsiblefortheobservedanti-proliferativeeffect.1. DelRio et al. (2013)Dietary (poly)phenolics in humanhealth: structures, bioavailability, and evidenceof protective effects against chronic diseases.

AntioxidRedoxSign18:1818–18922. Ferretietal.(2010)Cherryantioxidants:fromfarmtotable.Molecules15:6993–70053. Tagliazucchietal.(2010)Invitrobio-accessibilityandantioxidantactivityofgrapepolyphenols.FoodChem120:599-606


90

INFLUENCEOFTHEGELATOBASEFORMULATIONONTHESURVIVALOFLactobacilluscaseiSTRAINS

DURINGGELATOPRODUCTIONANDSTUDYOFTHEIRIMPACTONTHERHEOLOGICALPROFILE

ChiariP. ,BelmonteAM.,PulvirentiA. ,GiudiciP.Dept.ofLifeScience,UniversityofModenaandReggioEmilia,ReggioEmilia-Italy

email:[email protected];[email protected] research is focused on the production of Italian artisanal gelato enriched with probiotics. It has been used twopotentiallyprobioticstrainsdepositedinUNIMOREMicrobialCultureCollection(UMCC)isolatedfromParmigianoReggiano:LactobacilluscaseiPRA041andLactobacilluscasei225PRA.Thestudywascarriedoutevaluatingdifferentformulationsofgelatobasesandproceedingwithdirectinoculationofthestrainintothegelatomix,or, instead,byinoculatingthecellsinmilk, effecting fermentation and adding the fermented milk into gelato mix. We studied the influence of formulationsdifferentforfatcontentsandwithorwithoutaprebioticfiber(inulin)onthesurvivalofthestrainsandthechangesintherheologicalpropertiesofgelato.LactobacilluscaseiPra041andLactobacilluscaseiPra205stockcultureswerekeptat-80°CincryogenicvialscontainingMRSBroth(OxoidLtd,Basingstoke,Hampshire,England)andglycerol.Itwaspreparedabiomass.Thecellsarebeenseparatedbycentrifugationat5000gfor10’at4°C.Twobasesfortraditionalgelatohaveformulatedabletobepreparedeitherusingthehotprocess,eitherthecoldprocess.Thebasesdonotcontainintheformulationthepowderedfat.Oneofthebases,calledBASEAwastoincludea10%ofaprebioticfiber.TheprebioticfiberinulinOraftiGR(Beneo-OraftiAandorenstrat1,B-3300TienenBelgium)hasbeenused.Thesecondbase,calledBASEB,however,hasnottoincludeanyfiber.Bothbases(BaseAandBaseB)wereaddedwithpasteurizedcreamtoobtainmixtureto2.7%,4%and6%fat.Afirstseriesoftestswascarriedout adding to each formulation the sweet inoculum (19.2%). The second series of tests was carried out adding the acidinoculum(19.2%).Withthesetests, ithasbeentriedtofindtheformulathatguaranteedgreaterprotectionduringfreezingphase.Thisformulawasthentestedwithboththestrains(L.caseiPRA041andL.caseiPRA205)andalsousingboththecoldprocess, and thehotprocess.GelatohasbeenperformedbyCarpigianiCompactagelatomachine and stored intoaventilatedshowcaseISAequippedwithasystemofautodefrostandsetatatemperatureof-14°C(Tair)thatcorrespondedtoaTofthegelatoinpanof-11/-12°CContrary towhat reported in literature, the fat content or the presenceof inulin , does not substantially influence thesurvival of themicrobial strains to the freezingphase. Toobtain agood survival of the strains inoculatedneed tohave aformulation respectful of the correct parameters of balancing and a good stabilization. The speed, then,withwhich thegelatomachine isable to freezethemixture, is theothercrucialaspect.During freezingphasethere isa reductionof theinitialchargeequalto1log,itisessentialstartingfromalacticinitialcharge>108ufc/g,toobtainachargeingelatoofatleast107ufc/g. This value is in accordancewith the recommendations asdaily intake/g forprobiotics. Inorder toevaluate anychangestothestructureofthegelato,ithasbeenused,insteadofthespreadabilityofgelato,parameterverysubjective,itshardness.Itisusedforthispurposeadynamometeronwhichwasinstalledthepenetrometerprobe.Throughtheanalysisofthedatasomedifferenceswerefoundinstructurebetweenthestandardgelatoandthegelatoaddedofbacteria,mainlywhenithasbeenaddedanacidinoculum.


91

HERBALREMEDIESINTHENORTHOFALGERIA:

POTENTIALADVERSEINTERACTIONSWITHANTICANCERAGENTS

ZitouniH.,BengueddaFZB.,BenhamidaW.,BenallaL.,BairaM.,ToumiH.UniversityofOran,Oran-Algeria

email:[email protected] in the use of herbal products has grown in the world. Recent estimates suggest an overall prevalencefor herbal preparation use of 13% to 63% among cancer patients. With the narrow therapeutic range associatedwith most anticancer drugs, there is an increasing need for understanding possible adverse drug interactions inmedicaloncology.In this presentation, a literature overview is provided of known or suspected interactions of the most used herbsinOran,aNorthWesttowninAlgeria,withconventionalallopathictherapiesforcancer.Herbs with the potential to significantly modulate the activity of drug-metabolizing enzymes (notablycytochrome P450 isozymes) and/or the drug transporter P-glycoprotein include garlic (Allium sativum), peachleaves (Prunus persica), Fenugreek (Trigonella foenum graecum), saltbush (Atriplex halimus), Barberry (Berberisvulgaris),andmanyotherplants.Alloftheseproductsparticipateinpotentialinteractionswithanticancerdrugs.It is suggested that health care professionals and consumers should be aware of the potential for adverseinteractions with these herbs, question their patients on their use of them, especially among patients whosedisease is not responding to treatments as expected, and urge patients to avoid herbs that could confound theircancercare.


92

PORTULACAOLERACEAL.INTHEERAOFGLOBALISATION:

ASPECIESOFGREATNUTRACEUTICALVALUE

MantovaniV.1 ,BuldriniF.²,BosiG.²,VolpiN.1¹Dept.ofLifeSciences,UniversityofModenaandReggioEmilia,Modena-Italy;²Lab.ofPalinologyandPalaeobotany-Dept.ofLife

Sciences,UniversityofModenaandReggioEmilia,Modena-Italyemail:[email protected]

Portulaca oleracea L. is a common ruderal, synanthropic, cosmopolitan taxon, highly polymorphic, typical of warm sites(DaninandRaus,2012;Daninetal.,2014;Daninetal.,submitted).InItalyitsstatusasanativespeciesisdoubtful(Pignatti,1982).Itiswellknownsincetheantiquityforitsmedicinalandnutrientqualities(Bosietal.,2009):allpartsoftheplanthavetherapeutic properties (Gastaldo, 1987). It has been used for a long time as an analgesic, anti-inflammatory, antipyretic,diuretic, emollient, lenitive and anaphrodisiac. Many of such properties have been recently confirmed; furthermore, P.oleraceaisveryrichinomega-3polyunsaturatedfattyacids(Ezekweetal.,1999),sothatitsuseisrecommendedtocontrastthe excess of fatty acids assumedby fast foods (Picchi andPieroni, 2005) and its seeds are good to counteract diabetesmellitus(El-Sayed,2011).InthisworkwetestedthecontentoffattyacidsinseedsfromRomanperiodtopresent,asacomparisontothecontentoffatty acidsof the aerial partsof theplant: 12 freshplant and seeds samples, collected in Summer 2015, 19 seeds samplescomingfromItalianherbaria,datingfrom1920to2011,and20seedssamplescomingfromarchaeologicalexcavations,datedfrom1stto15thcent.AD.TotallipidswereextractedaccordingtoamodifiedmethodofFolch(1957)andmethylesterswereanalyzedbygaschromatography(GC)-FID.We found a high content of polyunsaturated fatty acids in fresh plants and seeds.Moreover, a quite similar profilewithcomparablesaturated,monounsaturatedandpolyunsaturatedfattyacidswasobservedinbothseedsandaerialpartsoftheplant, even if significant differences were measured in singular fatty acid percentages. Furthermore, comparing seedssamplesfromdifferenthistoricalperiods,wedeterminedanevidentdecrease inpolyunsaturatedfattyacidsdependingonincreasing time, from~69%of fresh seedsup to~11% in archaeological sampleswith theconsequent increase in saturatedfattyacids.This isprobablyduetoadegradationprocess,causedbyatmosphericoxygenexpositionandunsaturatedfattyacidsoxidationduringtime.Theappliedreportedmethodologywouldbeusefulforthedeterminationoffattyacidscontentandprofilealsoinancientsamplesofseedsandpartsofplants.1. BosiG,etal.Ethnobotanyofpurslane(PortulacaoleraceaL.) in Italyandmorfo-biometricanalysesofseeds fromarchaeological sitesofEmiliaRomagna

(Northern Italy). In: Morel JP, Mercuri AM. (eds.) Plants and Culture: seeds of the cultural heritage of Europe, EdiPuglia, Bari, 2009, pp. 129-139.http://www.plants-culture.unimore.it/book/11%20Bosi%20et%20alii.pdf

2. Danin A, and Raus T. A key to 19 microspecies of the Portulaca oleracea aggregate. In: Timonin AK, Sukhorukov AP, Harper GH, Nilova MV (eds.)ProceedingsoftheSymposiumCaryophyllales.LomonosovStateUniversity,Moscow2012,pp.70-83.

3. DaninA,etal.ThehistoryofthePortulacaoleraceaaggregateintheEmilia-RomagnaPoPlain(Italy)fromtheRomanagetothepresent.PlantBiosys2014,148:622-634.

4. DaninA,.etal.DiversificationofPortulacaoleraceaL.complexintheItalianpeninsulaandadjacentislands.ActaBotGallica,submitted5. El-Sayed MIK. Effects of Portulaca oleracea L. seeds in treatment of type-2 diabetes mellitus patients as adjunctive and alternative therapy. J

Ethnopharmacol137:643-651,20116. EzekweMO,etal.Nutritivecharacterizationofpurslaneaccessionsasinfluencedbyplantingdate.PlFoodsHumNutr1999,54:183-191.7. FolchJ,etal.Asimplemethodfortheisolationandpurificationoftotallipidsfromanimaltissues.JBiolChem1957,6:497-509.8. GastaldoP.CompendiodellaFloraOfficinaleItaliana.PiccinNuovaLibraria,Padova,1987.9. PicchiG,andPieroniA.Atlantedeiprodottitipici.LeErbe.INSOR/RAI-AGRA,Roma,2005,pag.317.10. PignattiS.Florad’Italia.Edagricole,Bologna,1982.


93

ANTIOXIDANTACTIVITYANDAPOPTOSISINDUCTIONASSESSEDBYFLOWCYTOMETRYOFABACTRIS

GUINEENSISPOLYPHENOLEXTRACT

QuesadaS.1 ,QuesadaMS.2,AzofeifaG.1,PérezAM.31UniversidaddeCostaRica,DepartamentodeBioquímica,EscueladeMedicina,SanJosé-CostaRica;2UniversidaddeCosta

Rica/FacultaddeFarmacia/InstitutodeInvestigacionesFarmaceúticas,SanJosé-CostaRica;3UniversidaddeCostaRica,CentroNacionalenCienciayTecnologíadeAlimentos,SanJosé-CostaRica


Bactrisguineensis (Aracaceae),alsoknown inCostaRicaasgüiscoyoloruvita,corozo inHonduras,Panamá,ColombiaandVenezuela, is a wild palm that grows in the plains. Its fruit has a high polyphenol content (824.4± 77.5 mg GAE/100 g),comparedwithanotherfruitslikeblueberryandplums.TheantioxidantactivityofBactrisguineensiswasreportedinliteraturewiththemethodsDPPHandORAC(Osorioetal.2011andRojanoetal.2012),howeveritisimportanttocorrelateitwithanotherassaysclosertoinvivosystems,liketheeffectsoncellculture.FullripefruitswerecollectedintheprovinceofGuanacaste,CostaRica.Thefruitwasextractedwithacetone/waterandthensubjected to polyphenol purification using anAmberlite XAD-7 column (150mmx 20mm). The columnwaswashedwithwater, the phenolic compoundswere elutedwithmethanol:water (80:20), concentrate under vacuum (40ºC) and freezedried.TheextractwasanalyzedbyUPLC-MS.AntioxidantactivitywasassessedbyusingtheDCFDAfluorescentdye.TheintracellularROSproductionwasinducedontheVero cell linewith tert-butyl-hydroperoxide (TBHP) in presence and absence of the polyphenol extract. The fluorescencepositivecellsweredeterminedbyflowcytometryandtheanalysisperformedbyCellQuestsoftware.InordertoanalyzeiftheobservedcytotoxicactivityofthepolyphenolextractonthecoloncarcinomacelllineSW620(IC50:16.6±1.3mg/mL)was because of an activation of apoptoticmechanisms, apoptotic cellswere quantified using Annexin V-FITC/PIassayandanalyzedbyflowcytometry.ThepolyphenolsidentifiedbyUPLC-MSintheextractwerecyanidin3-rutinoside,procyanidinB1andprocyanidin3-rutinoside.Theantioxidanteffectsofthepolyphenolsinsideofcells,usingtheDCFDAassay,showsthattheextractreducedtheinducedintracellularROS in adose-dependentmanner,withan IC50: 151.7mg/mL.Also theantioxidantactivitywasdeterminedbyDPPH(IC50:3.3±0.2µg/mL)andORAC(979.1µmolTE/gextract).ThecytotoxiceffectobservedovertheSW620celllineseemstobehappenbyapoptosis.Weobserved50.3%ofcellsinearlyapoptosisanda17.7%ofcellsinlateapoptosis,afterthepolyphenolextracttreatment.However,furtherinvestigationwillbeneededinordertoexplaintheapoptoticmechanismsinvolvedinitsactivity.Inconclusion,withitspolyphenolcontent,highantioxidantactivityanditscytotoxiceffectoncoloncarcinomacells,Bactrisguineensisseemstobeafruitwithahighpotentialasfunctionalfood.Acknowledgement:financialsupportbyVicerrectoríadeInvestigación,UniversidaddeCostaRica(ProjectNo.735-B2-659)1. Rojano,B.,Zapata,IC.&Cortes,B.2012.RevistaCubanadePlantasMedicinales17(3):244-255.2. Osorio,C.,Carriazo,JG.&Almaza,O.2011.EuropeanFoodResearchTechnology233:103-108.


94

ASSESSMENTOFTHEHEAVYMETALCONTENTINAROMATICSPICESBYINDUCTIVELYCOUPLED

PLASMA-MASSSPECTROMETRY

BuaDG.1,AnnuarioG.1,AlbergamoA.2 ,BartolomeoG.2,CiceroN1,2,DugoG.mo1,21DepartmentofBiomedicalSciences,DentalandMorphologicalandFunctionalImages,UniversityofMessina,Messina-Italy;

2Science4LifeS.r.l.,aSpin-offoftheUniversityofMessina,Messina-Italyemail:[email protected]

AimofthestudywastodeterminethecontentofCd,Hg,AsandPbinspicescommonlyavailableintheItalianmarket,sincesuch heavymetals pose amajor health concern and are considered among themost hazardous chemicals (WHO, 2007).ResultswerecomparedwiththemaximumpermissiblelimitssetbythenationalLegislativeDecree(L.D.)n.107implementingtheCouncilDirective88/388/EEC,andbyinternationalorganizations,suchasFoodandAgricultureOrganization(FAO)andWorld Health Organization (WHO). The risk derived from the intake of heavy metals via the assumption of spices wasassessed,consideringtheTolerableWeeklyIntake(TWI)andtheProvisionalTolerableWeeklyintake(PTWI)respectivelyforCdandHg,andthe95%lowerconfidencelimitofthebenchmarkdoseof1%extrarisk(BMDL01)forAsandPb.Seven samples of 3 types of spices produced in different countries (namely, cinnamon from Indonesia,Madagascar andVietnam;curcumafromIndiaandSriLanka;gingerfromIndiaandJapan)wereconsidered.Eachsamplewaspre-treatedbyamicrowavedigestionsystem;then,theheavymetalcontentwasdeterminedthroughavalidatedICP-MSmethod.One-wayanalysisofvariance (ANOVA)andpost-hocTukey’shonest significantdifference testwereapplied tocheck for significantdifferences(p<0,05)amongthecontentofeachheavymetalinthesamples.Allthetestedspicesrevealedtocontainheavymetalsandtobeapotentialcontaminationrouteforhumanconsumers.Cdwasdetectedinallthesamples:itslowestandhighestcontentwererespectivelymeasuredintheIndianginger(0,023±0,011)andintheSriLankancurcuma(0,288±0,004mgKg-1).CdshowedtoaccumulatebelowthelimitsetbytheLDn.107(1mgKg-

1)andFAO/WHO(0,3mgKg-1).ConsideringHg,thecurcumafromSriLankashowedthehighestHgcontent,representingthe28%ofthetotaltoxicantsinvestigatedinsuchspice.Nevertheless,allthesamplesmetthenationalandinternationalsafetylimitof1mgKg-1

.ThecinnamonfromMadagascarandtheJapanesegingerwerecharacterizedbythelowestandhighestAscontent(0,006±0,003and0,697±0,003mgKg-1,respectively).However,AslevelswerewithinthesafetylimitsetbytheLDn.107(3mgKg-1)andFAO/WHO(1mgKg-1).Pbmadeforthe65%ofthetotalinvestigatedmetals:thelowestandthehighestPbcontentwererespectivelyfoundintheIndiancurcumaandinthecinnamonfromVietnam(0,171±0,005and2,224±0,708mgKg-1,respectively).Pbcontentwasfoundtobebelowthepermissiblenationalandinternational limit(10mgKg-1) inallthe spices. Assessing the heavy metal intake through the assumption of spices, it resulted that in the cinnamon fromVietnam, the contribution of Cd and Pb respectively exceeded the TWI (151%) and BMDL01(431%); while in the Japaneseginger,thecontributionofAs(BMDL01=663-24,8%)andPb(BMDL01=219%)werewellabovethesafetylimits.Finally,theSriLankancurcumacontributedmosttotheintakeofhazardouselements,sinceeachmetalinvestigatedsignificantlyexceededthesafetylimitssetbyEFSAandJEFCA.1. LegislativeDecreeno.107.1992.Attuazionedelledirettive88/388/CEEe91/71/CEErelativeagliaromidestinatiadessereimpiegatineiprodottialimentari

edaimaterialidibaseperlaloropreparazione.Gazz.Uff.n.392. European Commission. 1988. Council Directive 88/388/EEC on the approximation of the laws of theMember States relating to flavorings for use in

foodstuffsandtosourcematerialsfortheirproduction.OfficialJournalL.1843. FAO/WHO - FoodandAgricultureOrganizationandWorldHealthOrganization Joint Food standardprogram. 1984.CodexAlimentarius.Commission

contaminationCAC/Vol.XVII4. WHO - World Health Organization. 2007. Safety evaluation of certain food additives and contaminants. Available from:

http://re.indiaenvironmentportal.org.in/files/9789241660594_eng.pdf


95

ANTI-INFLAMMATORYEFFECTANDPHYTOCHEMICALSCREENINGOFFICUSPERTUSA

MedinaFD.,OrejonGomezD.,HerreraHernándezN.

FacultaddeCienciasFarmacéuticasyBioquímica/UniversidadIncaGarcilasodelaVega,PuebloLibre,Lima-Perú.email:[email protected]

Ficuspertusa (Moraceae)grows in regionsasLoreto,MadredeDiosandPasco (Perú) [1]; ithasbeen traditionallyused totreat to rheumatism andwomen's ailments [2,3]. In studies chemical previous have reported the presence of flavonoids,alkaloids,tannins,saponinsandanthraquinones[4].Themainobjetivewastocontributetothephytochemicalstudyofthestembarkofthisspecieandassesstheiranti-inflammatoryeffect.PlantmaterialwascollectedinLoretoandidentifiedintheNHM(UNMSM)collectionundernumber3796.Thephytochemicalscreeningwas in crude ethanolic extract of dry stembark. The anti-inflammatory activitywas evaluated on carrageenan-inducedpawedemainrats[5]andexperimentalgroupsofratsweretreatedwithvehicle,diclofenacgel1%,ethanolextractsgel(EEG)0.5%,1%and10%.The phytochemical screening revealed presence of phenolics compounds, quinones, flavonoids, condensated tannins,terpenes,lactonesα,β-unsaturatedandalkaloids.FlavonoidsandstilbenescanbeconsideredmarkersquimiotaxonomicosoftheMoraceae [6]. Researches shows terpenes, terpenoids, lactones and alkaloids in the genus Ficus [7-10]. For the anti-inflammatorytest,theEEG10%usedinourstudyinhibitedtheedemainducedbycarrageenanat15%ascompareddiclofenac1%gelin2hours.Ourinvestigationrevealedanti-inflammatoryeffect,invitroofourgelextractethanolic.Acknowledgments:ThisworkhasbeengrantedbyUIGV1. CaceresBelloP,ReyenelRodriguezC.LosarbolesdeFicusdelvalledechanchamayodpdejuninPerú.Lima:Universidadnacionalagrarialamolina;2013.

79p2. Sanz-Biset J, Cañigueral S. Plant use in the medicinal practices known as “strict diets” in Chazuta valley (Peruvian Amazon). J Ethnopharmacol.

2011;137:271–883. KvistLP,AndersenMK,StagegaardJ,HesselsoeM,LlapapascaC.ExtractionfromwoodyforestplantsinfloodplaincommunitiesinAmazonianPeru:

Use,choice,evaluationandconservationstatusofresources.ForEcolManage.2001;150:147–74.4. FabiánMedinaDR,DiazReyJP,ArroyoAcevedoJ,BonillaRiveraPE,HerreraHernandezNG.Thewoundhelingeffectandpossible flavonesofFicus

paraensis.XXIVSILAECongressInternationalCongressonIntegrativeMedicine.PuntaCana;2015.ReportNo.:ps072.5. MandalSC,MaityTK,DasJ,SabaBP,PalM.Anti-inflammatoryevaluationofFicusracemosaLinn.leafextract.JEthnopharmacol.2000;72:87–926. RoweJW,ConnerAH.ExtractivesinEasternHardwoods:AReview.wisconsin:USDAForestServiceGeneralTechnicalReportFPL18.ForestProducts

Laboratory;1979.72p.7. SubramaniamG,AngKKH,NgS,BussAD,ButlerMS.AbenzopyrroloisoquinolinealkaloidfromFicusfistulosa.PhytochemLett[Internet].2009;2:88–90.

Availablefrom:http://linkinghub.elsevier.com/retrieve/pii/S18743900090000208. Yap VA, Loong BJ, Ting KN, Loh SHS, Yong KT, Low YY, et al. Hispidacine, an unusual 8,4’-oxyneolignan-alkaloid with vasorelaxant activity, and

hispiloscine,anantiproliferativephenanthroindolizidinealkaloid,fromFicushispidaLinn.Phytochemistry.2015;109:96–1029. YapVA,QazzazME,RajaVJ,BradshawTD,LohHS,SimKS,etal.FistulopsinesAandBantiproliferativesepticine-typealkaloids fromFicus fistulosa.

PhytochemLett[Internet].PhytochemicalSocietyofEurope;2016;15:136–41.Availablefrom:http://dx.doi.org/10.1016/j.phytol.2015.12.00710. YessoufouK,ElansaryHO,MahmoudEa.,Skalicka-WoźniakK.Antifungal,antibacterialandanticanceractivitiesofFicusdrupaceaL.stembarkextract

and biologically active isolated compounds. Ind Crops Prod [Internet]. 2015;74:752–8. Available from:http://linkinghub.elsevier.com/retrieve/pii/S0926669015301710


96

VISCERALADIPOSETISSUEREDUCTIONTHROUGHCACAOCO-PRODUCTSCONSUMPTION

Hidalgo-PérezTejadaEI.1,Valadez-CarmonaL.1,Plazola-JacintoCP.1,Hernández-OrtegaM.2,Ceballos-

ReyesGM.3,Ortiz-MorenoA.1 ¹InstitutoPolitécnicoNacional,EscuelaNacionaldeCienciasBiológicas,CiudaddeMéxico-México;2UniversidadAutónomadelEstadodeMéxico,FacultaddeQuímica,TolucadeLerdo(EstadodeMéxico)-México;3InstitutoPolitécnicoNacional,Escuela

SuperiordeMedicina,CiudaddeMéxico-México.email:[email protected]

The consumption of diets with high levels of fat and carbohydrates has an impact on the weight leading to developoverweight,obesity,cardiovasculardiseasesanddyslipidemiaamongothers1;theuseoffunctionalfoodsrichinpolyphenolsis analternative for the treatmentof such conditions. Therefore theobjectiveof this studywas toevaluate theeffectofcacaoco-productsontheweightofvisceraladiposetissueinarodentmodelwithobesityinducedbydiet2,3.Inthisstudy,Wistarmaleratswerefedwithanobesogenicdiet(21%offat)and20%fructosesolutionasconsumptionwaterduring5weeks.After5weeksoffeedingwiththehighfatdiet,ratsweredistributedinto5groups:Control,Obesogenic,I,IIand III (obesogenic diet plus 141.54, 23.55 and 11.751mgof equivalents of Gallic acid respectively obtained from cacao co-products).After5weeksoftreatmentratsweresacrificedandthevisceraladiposetissuewasweighted.Thegroupthatconsumedtheobesogenicdiethadvisceraladiposetissueaccumulationwhichwasreflectedinitsweight;theincrementwas8foldcomparedwithnormaldietgroup.Thetreatedgroupsreducethevisceraladiposetissuein12%and20%ingroupsIIandIII,whilegroupIdidnotshowsignificantdifference(p>0.05)withobesogenicgroup.Theresultssuggestthatthedecreaseinvisceraladiposetissuecouldbeattributedtophenolspresentsincacaoco-productsmakingthemagoodmaterial for the production of functional foods that can be used as an adjuvant in themanagement of overweight andobesity.Acknowledgments:ThisworkwassupportedbytheInstitutoPolitécnicoNacionalandCONACyT..1. GonzalesC.,LvalleG.,RíosG.2009.Síndromemetabólicoyenfermedadcardiovascular.3ªed.Intersistemas,Mexico.335-338.2. Martínez R, Torres P,MenesesMA, Figueroa JG, Pérez AJ, ViudaMM. (2012). "Chemical, technological and in vitro antioxidant properties of cocoa

(TheobromacacaoL.)co-products".FoodResearchInternational.49:39–45.3. GutierrezG.,Ortiz P., Vacaseydel V.C., Garduno L., ChamorroG.,Meaney E., Villafana S., Villarreal F., CeballosG. y Ramirez I. (2014). "Effects of (-)-

epicatechinonadiet-inducedratmodelofcardiometabolicriskfactors."EuropeanJournalofPharmacology.728:24-30.


97

ANTIMALARIALACTIVITYOFXANTHONESISOLATEDFROMGARCINIAMANGOSTANA

QuiñonesW.1 ,EcheverriLF.1,RobledoSM.2,TorresLF.1,GilJF.1,EscobarGA.1,ArchboldR.1

1UniversityofAntioquia,InstituteofChemistry,QOPN,Medellín-Colombia;2UniversityofAntioquia,SchoolofMedicine,PECET,Medellín-Colombia

email:[email protected] isaparasiticendemicdiseasethat iswidelydistributedworldwideandaffectsa largeeconomicallydisadvantagedpopulationlivinginruralareasoftropicalandsubtropicalcountries.1,2Conventionaltreatmentsincludecompoundssuchasaminoquinolines, antifolates, Atovaquone® a hydroxy-naphthoquinone, and artemisinin derivatives. The use of thesecompounds in affected populations presents disadvantages related to their relatively high cost and the development ofresistantparasites.3,4Therefore,thereisanurgentneedtodiscoveranddevelopnewandbetterdrugs.Unfortunately,thedrugdiscoveryanddevelopmentprocessesarenotonlyexpensivebutalsohighlytime-consumingandcomplicated.Oneofthemostimportantbottlenecksinthediscoveryanddevelopmentofnewdrugsfromnaturalproductsistheavailabilityofsufficientplantmaterialtoobtainpureactivemoleculesformoreadvancedevaluation.Based on the previously reported in vitro antiplasmodial activity of several xanthones from Garcinia mangostana, twoxanthones, α-mangostin and δ-mangostin, were isolated from mangosteen husk, and the in vitro antiplasmodial andcytotoxiceffectsweredetermined.α-MangostinwasmoreactiveagainsttheresistantPlasmodiumfalciparumchloroquine-resistant(FCR3)strain(IC50=0.2±0.01μM)thanδ-mangostin(IC50=121.2±1.0μM).Furthermore,thetherapeuticresponseaccording to the administration route was evaluated in a Plasmodium berghei malarial murine model. The greatesttherapeutic response was obtained with intraperitoneal administration; these xanthones reduced parasitemia byapproximately80%withadailydoseof100mg/kgadministeredtwiceadayfor7daysoftreatmentAcknowledgements:ThisworkwassponsoredbyColciencias-COLOMBIA(grant111571249866),CIDEPRO(CIIEs,UniversidaddeAntioquia)1. WorldMalariaReport2013.http://www.who.int/malaria/publications/world_malaria_report/2013/en/2. MurrayCJ,RosenfeldLC,LimSS,AndrewsKG,ForemanKJ,HaringD,FNancy,NaghaviM,LozanoR,LopezAD.Globalmalariamortalitybetween1980

and2010:asystematicanalysis.Lancet2012,379:413-431.3. Worldhealthstatistics2013.http://www.who.int/gho/publications/world_health_statistics/2013/en/4. Toshihiro M, Kazuyuki T. Evolution of Plasmodium falciparum drug resistance: implications for the development and containment of artemisinin

resistance.JpnJInfectDis2012,65:465-475.


98

UNCOVERINGARTIFICIALHONEYSFROMECUADORIANMARKETS

VitP.1 ,GonzálezI.2,MazaF.3

¹UniversidaddeLosAndes,FacultyofPharmacy,FoodScienceDepartment,Mérida-Venezuela;²UniversidaddeLosAndes,FacultyofPharmacy,FoodScienceDepartment,Mérida-Venezuela;3UniversidadTécnicadeMachala,FacultyofAgriculturaland

LivestockSciences,Machala(ElOro)[email protected]

Honeys are imitated using less expensive sweeteners to earn more profits on this sugary bee product. Economicallymotivated adulteration (EMA) is the term used for fraudulent and intentional additions or substitutions of substances toincreaseeconomicearnings(EasterStrayeretal.,2014).Honeystandardsaretheofficial referenceforauthenticitytestingbutbesidesspecializedtechniques,theseanalysisarenotalwaysavailableindevelopingcountries.Thesuitabilityofasimpleauthenticitytest(Vit,1995;Vit,1998)iscomparedherewithphysico-chemicalhoneyqualitystandardstoassessthepresenceofartificialhoneysintheEcuadorianmarket.Artificial honeyswell identified by sensory observationswere collected in 12 Ecuadorian provinces. The ash, free acidity,qualitative diastase activity, qualitative hydroxymethylfurfural (HMF), moisture, reducing sugars, and apparent sucrosecontentsweremeasuredfollowingCOVENIN2136-84(1984a)methods.The21artificialhoneysandonegenuinehoneyfromaPichinchaprovince apiarywere tested for authenticity after 1:1 honey:distilledwater, and vigorous shakingwith 1:1 honeydilution:diethylether(Vit,1998).Venezuelan(COVENIN2191-84)andEcuadorian(NTEINEN1572)standardsfromNationalHoneyNormswerecomparedwiththephysico-chemicalcompositionofEcuadorianfakehoneys.QualitativediastaseactivityandHMFoftheEcuadorianfakehoneysanalyzeddidnotsatisfythehoneystandards,as inastudywith500commercialhoneysfromVenezuela(Vitetal.,1994).HeatedandoldgenuinehoneysalsomayhavelowdiastaseactivityandincreasedHMFasdefects.Ashandmoisturearenotaproblem formanufacturedhoneys that tend tobemoreviscousandwith lowermoisture thangenuine tropicalhoneys; thereforethesetwoparametersarenothandytopursuedetectionof fakehoneys.Theremaininggroupofthreehoneyqualityfactorsdiscriminatedonlyfewfakehoneys.Higherfreeacidity(upto102.0meq/kg),higherapparentsucrose(upto23.89g/100g)andlowerreducingsugars(51.80g/100g)thanthemaximum40meq/kg,maximum5g/100g,minimum65g/100gcontentsrespectivelypermittedinthehoneystandards(COVENIN,1984b;NTEINEN,1988)werefound.Thetestbasedonthenumberofphasesofhoneydilutionsshakedwithdiethylether,wasreliabletouncoverallthe21fakehoneysfromEcuadorianmarkets.Acknowledgments:ToConsejodeDesarrolloCientífico,HumanísticoyTecnológico,UniversidaddeLosAndesCDCHT-ULAforcontinuoussupporttotheResearchGroupAPIBAADG-FA-97-08.TotheScholarshipPrometeo-SENESCYT-UTMACHjune2014–june2015toP.Vit.ToInstitutoEcuatorianodeNormalizaciónINENtoextendinvitationstojointheTechnicalCommitteetoreviewtheNTEINEN1572HoneyNorm.1. COVENIN. Comisión Venezolana de Normas Industriales, 1984a. Miel de Abejas. Métodos de Ensayo. COVENIN 2136-84. Fondonorma, Caracas,

Venezuela,pp1-36.2. COVENIN.ComisiónVenezolanadeNormasIndustriales,1984b.MieldeAbejas.COVENIN2194-84.Fondonorma,Caracas,Venezuela,pp1-5.3. EasterStrayerS,EverstineK,KennedyS,2014.EconomicallyMotivatedAdulterationofHoney:QualityControlVulnerabilitiesintheInternationalHoney

Market.FoodProt.Trends,34:8-14.4. NTEINEN.NormaTécnicaEcuatorianaObligatoria,1988.InstitutoEcuatorianodeNormalización.MieldeAbejas,Requisitos.NTEINEN1572.INEN,Quito,

Ecuador,pp1-4.5. VitP, 1995.Patent: Tubopistónvolumétricopara análisis (diseño industrial).Resolución 1286.BoletínServicioAutónomode laPropiedad Industrial.

Caracas,05deMayode1995.184°y136°.Venezuela.6. VitP,1998.Atesttodetectcanesugarhoney.ALAN48:62-64.7. VitP,GonzálezdeMartorelliI,López-PalaciosS,1994.Clasificacióndemielescomercialesvenezolanas.ALAN44:47-56.


99

ANTI-INFLAMMATORYSCREENINGOFPLANTEXTRACTSUSEDINFOLKMEDICINEOFCOLOMBIAN

CARIBBEANCOAST

CastroJP.1,OcampoYC.1,MejiaNM.1,BolívarS.2,DíazF.2,FrancoLA.1 ¹UniversityofCartagena,BiologicalEvaluationofPromisingSubstancesGroup,Cartagena-Colombia;2UniversityofCartagena,

LaboratoryofPhytochemicalandPharmacologicalResearches(LIFFUC),Cartagena-Colombiaemail:[email protected]

Responding to the need to find new, more effective and safe therapeutic alternatives to treat the clinical conditionsassociatedwithinflammatoryprocessesand attemptingtoexploittheethnobotanicalrichnessoftheColombianCaribbeanCoast,weevaluatedtheanti-inflammatorypotentialofseventotalethanolicextractsofbotanicalspecieswidelyusedinthefolkmedicineofthisregion. The total extracts of: Ambrosia cumanensis, Bauhinia guianensis, Capparis odoratissima, Cecropia peltata, Hyptis capitata,MammeaAmericanaandMurrayaexótica;wereobtainedbymacerationwithethanol.Presenceofdifferentmetaboliteswasdeterminedqualitatively,whileflavonoidsandphenoliccompoundsweredeterminedquantitatively.Theanti-inflammatorypotential of the extractswere assessed determining their activity on the production of nitric oxide (NO•), in RAW264.7macrophagescell line,quantifiedspectrophotometricallybytheaccumulationofnitrite (NO2

−),usingtheGriess reaction(1).Additionally, NO• scavenging effect was determined, using sodium nitroprusside as a radical donor. The extracts weretested at non-toxic concentrations and below 100 µg/mL in all cases, which were previously determined evaluating cellviabilitythroughtheMTTreductioncolorimetricassay.Ultimately,weevaluatedpossibleantioxidanteffectsoftheextracts,using the free radical scavenging DPPH! (2,2-diphenyl-1-picrylhydrazyl) and ABTS+! (2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonicacid))spectrophotometricmethods(2,3).The total ethanolic extracts of Ambrosia cumanensis, Cecropia peltata, Hyptis capitata, Mammea americana and Murrayaexótica showedapotent inhibitory activityof theNO•production (70-100%),without significant scavengingeffectof theNO•freeradical.TheseextractsdidnotshowsignificantscavengingeffectsofDPPH!andABTS+!freeradicals,atthesameconcentrations used in tests with cell cultures, suggesting that its potential anti-inflammatory activity is unrelated toneutralizationof oxidative stress associatedwith inflammatory processes. These five plant species should continue to bestudied,inordertoisolateandidentifycompoundsresponsiblefortheactivity,whichcouldbecomeastartingpointforthediscoveryofnewanti-inflammatoryagents.Acknowledgments:ThisstudywassupportedbytheUniversityofCartagena(Grant054-2013).JennyCastroisdeeplygratefultoColcienciasandtheUniversityofCartagenaforherPhDfellowshipthroughtheNationalProgramforDoctoralFormation(Grant647-2014).1. Ding, A. H., C. F. Nathan, et al. (1988). Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal

macrophages.Comparisonofactivatingcytokinesandevidenceforindependentproduction.JImmunol141(7):2407-2412.2. Re,R.,N.Pellegrini,etal.(1999).AntioxidantactivityapplyinganimprovedABTSradicalcationdecolorizationassay.FreeRadicBiolMed26(9-10):1231-

1237.3. Silva,B.M.,P.B.Andrade,etal. (2004).Quince(CydoniaoblongaMiller)fruit(pulp,peel,andseed)andJam:antioxidantactivity.JAgricFoodChem

52(15):4705-4712.


100

BIOGUIDEDFRACTIONATIONOFTrichiliahirtaSEEDSEXTRACTWITHCYTOTOXICACTIVITYAGAINSTABREASTCANCERCELLLINE

CaroD.1,OcampoY.1,BolivarS.2,DiazF.2,AristizábalFA.3,FrancoLA.1

¹UniversityofCartagena,BiologicalEvaluationofPromisingSubstancesGroup,Consulado,Cartagena-Colombia;2UniversityofCartagena,LaboratoryofPhytochemicalandPharmacologicalResearches(LIFFUC),Cartagena-Colombia;3NationalUniversityof

Colombia,CancerPharmacogeneticsResearchGroup,Bogotá-Colombiaemail:[email protected]

CaribbeanColombianflorahaslongbeenusedtotreatdiseases,Trichiliahirtaisaspeciesconventionallyusedbypatientstotreatmentthecancer(1).Inthiswork,abioguidedfractionationoftheethanolicextractofT.hirtaseedswasperformedbasedonitscytotoxicactivityontheMDA-MB-231breastcancercellline,aswellasthecancercellselectivityoftheextractsanditsactivefractionson3T3-L1fibroblasts.SeedsofT.hirtawerewashed,driedatroomtemperature,groundtoapowderandextractedwithethanoluntilexhaustion.The dry extract was fractionated using chromatographic techniques under a bio-guide scheme based on the cytotoxicactivityagainstMDA-MB-231cells,employingthemethyl-tetrazoliumbromidecolorimetricmethod(MTT)(2).Theextractandfractionswereclassifiedintothreecategoriesas:active(LC50≤20μg/ml),moderatelyactive(20<LC50<100μg/ml)andinactive(IC50≥100 μg/ml)(3). In order to determine the selectivity index (SI), we evaluated the cytotoxic effect of the extract andfractionsagainst3T3-L1fibroblastsasnormalcellsandcalculateditasaratioofLC50Fibroblast/LC50CancerCell.ChromatographicseparationofT.hirtatotalextract(19,9g),ledtoobtainfourmajorfractionsidentifiedasTH-F1(12,46g-62,6%),TH-F2(2,55g-10%),TH-F3(0,75g-3,7%)andTH-F4(1,02g-5,1%),whichwereevaluatedfortheircytotoxicactivity.ThetotalextractwasactiveonMDA-MB-231cells(LC50=7.71μg/mL-SI=1,86).Thisbiologicalactivitywaskeptonthreefractions,TH-F1 (LC50=15.35μg/mL), TH-F2 (LC50=14.13μg/mL), TH-F3 (LC50=58.75μg/mL). Interestingly, the cytotoxicitywashigher infractionselutedwithdichloromethane,indicatingthatthemainactivecompoundshavelowpolarity.Thisstudyprovidesanimportant basis to further researchdirected to the isolation and identificationof active constituents potentially useful tocancertreatment,usingColombianCaribbeanplantsasavaluablesource.Acknowledgements:ThisstudywassupportedbyColcienciasandtheUniversityofCartagena(Grant110756933390).DaneivaCaroisdeeplygratefultoColcienciasandtheUniversityofCartagenaforherPhDfellowshipthroughtheNationalProgramforDoctoralFormation(Grant647-2014).1. Hernández, E.,Mora,N.,et al. (2013).Actividad citotóxicadeextractos acuososdehojasdeTrichilia hirta sobre células tumoraleshumanas.Revista

CubanadeInvestigacionesBiomédicas,32(1),93-101.2. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of

immunologicalmethods,65(1-2),55-63.3. Manosroi, A., Akazawa, H., et al. (2015). In vitro anti-proliferative activity on colon cancer cell line (HT-29) of Thai medicinal plants selected from

Thai/Lannamedicinalplantrecipedatabase“MANOSROIIII”.Journalofethnopharmacology,161,11-17.


101

CAPTURINGTHEDIVERSITYOFFUNGALPOPULATIONINHUMANCOLONICMICROBIOTATHROUGH

CULTURE-DEPENDENTANDINDEPENDENTAPPROACHES

GozzoliC.1,RaimondiS.1,SomoneM.1,OrsiCF.2,PeppoloniS.2,BlasiE.2,CavalieriD.3,RossiM.1 1DepartmentofLifeSciences,UniversityofModenaandReggioEmilia,Modena-Italy;2DepartmentofDiagnosticMedicine,ClinicalandPublicHealth,UniversityofModenaandReggioEmilia,Modena-Italy;3DepartmentofBiology,UniversityofFlorence,Sesto

Fiorentino-Italyemail:[email protected]

Asmallfractionofgutmicrobiota isrepresentedbyfungi,thatremainrelativelyunexplored.oneshaveneverbeendeeplyinvestigated.Thisstudywantedto investigatethe inter-individualand intra-individualdifferencesof fungalpopulation in3fecalsamplesfrom8healthysubjects,withinthetime-spanofayear.Thecompositionofthefungalpopulation,intermsofconcentrationandtaxonomicassignment,wasexploredwithbothculturedependentandindependentmethodsatthestartof the study, after 3 months, and after a year. Strategies of interaction with the host of the isolates were investigateddeterminingthecapabilitytoadhereandtoformbiofilms,andtheinductionofHumanβ-defensin2secretionbyCaco-2celllines(HBD-2).IsolationoffungifromfecalsampleswascarriedoutonPDAsupplementedwithantibiotics. Theisolateswereclusterizedinto different biotypes through RAPD-PCR, then amplification and sequencing of rDNA genes and ITS spacer lead totaxonomic identification. Further characterization of theC. albicans strainswas done through SSCP andMLP. Each singlebiotype was tested for biofilm production and cellular adhesion. HBD-2 levels were determined in Caco-2 epithelial cellsstimulatedwithfungalisolates.Compositionofthefungalmicrobiotawasmonitoredinperiodicallycollectedfecalsamplesby454-pyrosequencingoftheinternaltranscribedspacerregionsof1and2rRNAgenes.Cultivablefungiwerequantifiedatthreetimepointsinthefecesof8healthyvolunteers.Thecountsrangedfrom1.4x102to4.5x 105 cfu/g, andonly for5outof 24 samples thechargeswereunder the limitofdetection.Awidequantitative intra-voluntary variability over the time was detected. The majority of the biotypes belonged to the genera Candida, and inparticular to the species C. albicans and C. zeylanoides, and to Galactomyces. All but one C. albicans strains were able toproducelargeamountsofbiofilmafter48hofincubationincompletemedium,whereasboththenon-albicansCandida(NAC)strains and all but one of the other fungi did not produce biofilms. All C. albicans strains adhered very efficiently to theepithelial cells. Appreciable, but lower levels of adhesionwere alsoobservedwith theNAC strains, but not for theotherfungi.The intestinalCaco-2epithelialcellsreleasedhigh levelsofHBD-2whenchallengedwithC.albicans isolates,whereasthe concentration of HBD-2 was markedly lower when Caco-2 cells were stimulated with NAC strains and other fungi.Metagenomeanalysisoffungalpopulationsucceededonlyfor13outof24fecalsamples.95diverseOTUswereidentifiedinthe13samples, resulting indetectionof59generaand36additional lineagesnotclassifiedatgenus level.Onaverage,24OTUs/sampleweredetected.Thesamplepresentingthewidestbiodiversityhostedfungiascribedto45diverseOTUs,theleastdiverse,8.Ascomycotadominatedthefungalpopulation(89.2%oftotalreads),withonly6.8and4.0%ofZygomycotaandBasidiomycota,respectively.MostofthereadsbelongedtothegeneraPenicilliumandAspergillus.Largedifferences infungal population determined by cultural methods and metagenome were detected. However, isolation of fungi wasnecessarytostudyinvitrotheinteractionoftheisolateswiththehost.


102

SUBDOMINANTBUTCONSTANTLACTOBACILLUSPOPULATION

INTHEHUMANGUTMICROBIOTAASREVEALEDBYWHOLEGENOMESEQUENCING

RossiM.1 ,Martinez-MartinezD.2,AmarettiA.1,UlriciA.3,RaimondiS.1,Moya,A.21DepartmentofLifeSciences,UniversityofModenaandReggioEmilia,Modena-Italy;2FoundationforthePromotionofHealthandBiomedicalResearchintheValencianCommunity(FISABIO),Valencia-Spain;3DepartmentofLifeSciences,Universityof

ModenaandReggioEmilia,ReggioEmilia-Italy.email:[email protected]

The genus Lactobacillus includes 217 species that colonize plants, foods, sewage, and the gastrointestinal tract (GIT) ofhumansandanimals.InthehumanGIT,Lactobacilluspopulationismadeupbybothresidentandtransientmembers,theselatterbeingingestedwithfermentedorspoiledfoodandbeverages,andwithprobiotics.Thisstudylongitudinallysurveyedthe Lactobacillus species and strains in the GIT of a healthy subject over a period of almost 2 years, throughmining themetagenomicwholegenomesequence(WGS).Metagenomic whole genome shotgun sequences were analyzed with LMAT (Livermore Metagenomics Analysis Toolkit)softwarepackage(Amesetal.,2013)(version1.2.4,withFeb2015releaseofdatabaseLMAT-Grand).The“Grand”database,releaseFeb’15wasused.WeconvertedrawLMAToutput intoa listofspeciesandstrainsof interest,filteringthembytheAverageReadScore(values>0)..Lactobacillusspeciesandstrainswereselectedfromthefiltereddatatodothesubsequentcalculations about persistence. The number of bacteria per gram of faeces belonging to each species and strain wasextrapolatedfromtherespectiverelativeamount(%),assumingthatthethreefecalsampleshavethesameconcentrationofmicroorganisms(1x1012cellsg-1).ThedatawereexpressedasLog(cellsg-1).Inthreetime-points(0,670and700days),58differentspecieswereidentified,16ofthembeingretrievedforthefirsttimeinhuman feces.L. rhamnosus,L. ruminis,L. delbrueckii,L. plantarum,L. casei, andL. acidophiluswere themost represented,beingestimatedat themagnitudesof6-8Log(cells/g).Other speciesweredetectedat themagnitudeof4-5 Log(cells/g).Many of them have never been associated to human feces (e.g. L. composti, L. farciminis, L. farraginis, L. harbinensis, L.namurensis,L.parabrevis,L.sanfranciscensis,L.shenzhenensis,L.suebicus,L.versmoldensis,L.zeae),thusmodifyingthevisionofLactobacillusecology.WGSalsoenabledthe identificationof86Lactobacillusstrains,belongingto52species.43strainslikelyoccupied theGITas true residents,being recurrently foundovera time-spanof 23monthswith limitedquantitativefluctuations.Asawhole,astablecommunityoflactobacilliwasdisclosed,withwideandunderstudiedbiodiversity.Amajorchallenge is still determining the specific GIT district where this plethora of lactobacilli replicates and grows, in order todiscriminate if theyare indigenousresidentofthecolon,orwhethertheyreach itsheddingfromupstreamsitesthattheycolonize.Thiscanmakethedifferenceintermsofmicrobe-immunesystemrelationship.1. Ames,S.K.,Hysom,D.A.,Gardner,S.N.,Lloyd,G.S.,Gokhale,M.B.,andAllen,J.E.(2013)Scalablemetagenomictaxonomyclassificationusingareference

genomedatabase.Bioinformatics29:2253-2260.


103

PRELIMINARYSTUDYOFPESTICIDERESIDUESINPETITGRAINDISTILLATEDFRACTIONS

BartolomeoG.1 ,RandoR.1,TropeaA.1,CiceroN.1,2,RecuperoS.3,DugoG.mo1,2

¹UniversityofMessina,BIOMORFDept.,Messina-Italy;2Science4Lifesrl,Spin-OffCompany,UniversityofMessina,Sant’Agata(Messina)-Italy;3EssenzeFlorealiAgrumidiRecuperoA.&C.S.N.C.,BarcellonaPozzodiGotto(Messina)-Italy

email:gbartolomeo@unime.itCitruspetitgrainessentialoilsareobtainedbysteamdistillationoftheleaves,budsandsmallbranchesofthedifferentcitrusspeciesandareused in soap-manufacturing,perfumeryandcosmetics.Among these,bitterorangepetitgrain is themostappreciated and is produced mainly in the Mediterranean countries and Paraguay (Dugo G et al., 2002). To guaranteegenuineness and quality of products it is necessary to evaluate some organic contaminants, like organophosphorus andorganochlorinepesticides,evenifamaximumlimitofresidueshasbeenestablishedbylegislationonlyforcitrusfruits.Mostscientificstudieshavefocusedonthedeterminationoftheseorganiccontaminantsresiduesincitrusessentialoils,butnotinpetitgrainones(DiBellaetal.,2010;DugoG.moetal.,2010).Inthisstudy,citruspetitgrainessentialoilsandtheirdistillatedfractions collected at predetermined time intervals during a full cycle of distillationwere analyzed to try to evaluate thecontamination.Organophosphorus and organochlorine pesticides were investigated respectively by Dani Master, HRGC-FPD, and TSQQuantumXLSThermoScientific,HRGC-MS/MS,inlemonpetitgrainoilssamples3fromconventionalagricultureand3frombiologicalagricultureand3bitterorangeoilssamples.Furthermore,duringthesteamdistillationcycleofeachsample,fourfractionswerecollectedevery25minuteeach.Allsamples,producedinMessina(Sicily,Italy)intheseason2014/2015,andallfractionswereaddedtointernalstandardsolutionanddirectlyanalyzed,withoutanyclean-upprocedure.Residuesoforganochlorinepesticideswerealways<LOQbothinpetitgrainoils,astheyareandintheirfractions.Amongtheorganophosphorus,methyl (meanvalueof0.026mg/L)andethylchlorpyriphos(1.920mg/L)andmethylpirimiphos(0.027mg/L) were found in 100% of bitter orange petitgrain oils. During the distillation cycle the pesticides content increasedsignificantlyinthethirdandfourthfractions:infactitwasobservedanaverageincreaseof50%and100%respectively.In lemon samples obtained from conventional and biological agriculture ethyl chlorpyriphos was the only determinedpesticidewithameanconcentrationof0.990and2.320mg/Lrespectively.Intheirfractionsitwasfoundthesametrendasobservedforbitterorangesamples.Since the final fractionsshowedahigherconcentrationofpesticidescomparedto the initialones, it couldbeassumedtoreducetheextractionprocess,whichhadayieldverylowpercentage.Obviouslythesedatawillbeimplementedwiththestudyofthevolatilefractionofeachdistillatedfractions.1. Dugo,etal.,2002.Citrus:thegenusCitrus.Taylor&Francis,London,UK.2. DiBella,etal.,2010.Pesticideandplasticizerresiduesincirusessentialoilsfromdifferentcountries.NaturalProductCommunications,4:1-6.3. Dugo,etal.,2010.Citrusoils:composition,advancedanalyticaltechniques,contaminants,andbiologicalactivity.Taylor&FrancisGroup,Abingdon,UK.


104

PRODUCTIONOFSINGLECELLPROTEINBYSACCHAROMYCESCEREVISIAEFROMFOODWASTE

GervasiT.1 ,CiceroN.1,2,RussoE1,NaccariC1,DugoG.mo1,2

¹UniversityofMessina,BIOMORFDept.,Messina-Italy;2Science4Lifesrl,Spin-OffCompany,UniversityofMessina,Sant’Agata(Messina)-Italy.

email:[email protected] intheworld iswaste,particularly foodwaste,both inthedevelopingandthedevelopedcountries(Huangetal.2015).Large-scaleretaildistributionwastesespeciallyfruitandvegetableswastecontainconsiderableamounts of fermentable and non fermentable sugars that could be utilized to produce value-added products by usingmicroorganisms suchas single cell protein (SCP). Foodwastehavebeenusedas animal feed (Theodoroset.al., 2013), foroxidative andhydrolytic enzymeproduction (Zilly, et.al 2012), cellulases production (Oberoiet. al 2010), enzymatic extractproduction(Silvestreetal2012),andasgrowthmediaforstarterculturessuchaskefirforbreadmakingandalcoholicanddairyfermentations.Theaimofthisworkwastostudythepromotionaleffectofwholediscardedfood,deriving fromthelarge-scale retaildistribution,onSCPproductionbySaccharomycescerevisiaeyeastusingsubstratesconsistingof fruitandvegetablemixturesthroughabatchfermentationprocesses(Plessas,etal.2008).Expired foodwaste, previously collected from large-scale retail distribution in the period between January and July 2015,wereused.Expiredvegetablessuchastomato, lettuce,potato,aubergine,pumpkin,pepperandcauliflowertogetherwithexpired fruit such as apple, strawberry, pinapple and pear were characterized and used as growth media. Dry mattercontent,ashcontentandproteinconcentrationweredeterminedaccordingtotheAssociationofOfficialAnalyticalChemistsmethod(AOAC,1984).S.cerevisieATCC4126wasused.A15ltotalvolumeautomaticBiostatEreactor(B.BraunMelsungenAG,Germany),runinbatchmodeandequippedwiththecommoncontrolswasused:pH:5.05withoutanyfurthercorrection;mixing:300;airflow:4l/min;airsterilization:filtrationthrough0.2mmSartofluorfilterandpressure:200mbar.Attheendofthebatchcultivationsubstratewastestedfortotalcellcount(CFU/mL),biomassconcentration(g/L)andproteincontent(%).ThepercentageofproteinintheSCPfromaninitialvalueof15.3%reachesafinalvalueof39.8%.Thesupernatanttotalnitrogenvaluesremainalmostconstantduringtheentirefermentationprocess.Thesupernatantdrymatter passes from a 18.9 g/100ml value at time 0, to 12.7g/100ml after four days of fermentation, and then present anincreasingtrenduptotheendofthefermentationprocess,thisbehaviorisprobablyduetothedecreaseofthesugar.Thegreat amount of food waste daily produced by the large-scale retail distribution makes their elimination necessaries.Unprocessedfoodwastecouldbewidelyusedfortheformulationofaddvalueproducts.1. Huang et al., 2015. Butanol production from food waste: a novel process for producing sustainable energy and reducing environmental pollution.

BiotechnolBiofuels.8:147.2. Theodoroset al., 2013.DiscardedOrangesandBrewer’s SpentGrains asPromoting Ingredients forMicrobialGrowthbySubmergedandSolidState

FermentationofAgro-industrialWasteMixturesApplBiochemBiotechnol170:1885–1895.3. Zilly,etal.2012.Solid-statebioconversionofpassionfruitwastebywhite-rotfungiforproductionofoxidativeandhydrolyticenzymes.FoodBioprocess

Technology,5:1573–1580.4. Oberoi, etal., 2010.Productionof cellulases throughsolid state fermentationusingkinnowpulpasamajor substrate. FoodBioprocessTechnology,

3:528–536.5. Silvestre,etal.,2012.EffectofpHandtemperatureontheactivityofenzymaticextractsfrompineapplepeel.FoodBioprocessTechnology,5:1824–1831.6. Plessasetal.2008.Upgradingofdiscardedorangesthroughfermentationusingkefirinfoodindustry.FoodChemistry,106:40–49.


105

DEVELOPMENTOFANEWMULTIRESIDEMETHODTOANALYZE67CONTAMINANTSINFOODWASTE

BYGC-MS/MS

FedeMR.1 ,DiBellaG.1,PotortìAG.1,LoTurcoV.1,MotteseA.1,DugoG.mo1,2¹UniversityofMessina,BIOMORFDept.,Messina-Italy;2Science4Lifesrl,Spin-OffCompany,UniversityofMessina,Sant’Agata

(Messina)-Italyemail:[email protected]

Thecontaminants found in foodcanbederived fromtheenvironment, fromthecultivationandorproductionprocesses.Theirpresencemayimpairthefoodqualityandalsoinvolveanhealthrisk.Duetothechemicalstabilityofthesesubstances,theycancontaminatealsovegetablesandfruits,limitingthepossibilityofreusingtheirwastesuchmatricesassubstratesforcompostoranimalfeed.Theaimofthisworkwastosetanewanalyticalmethodtoanalyzesimultaneously67contaminantsbelonging to different classes (PCBs, PAH, organochloride, organophosphorus and other types of pesticides) based onQuEChERStechniqueandtoanalyze70samplesoffoodwaste.31samplesoffruitwaste(pears,strawberries,peaches,bananas,pineapplesandmango)and39samplesofvegetablewaste(stripped and white zucchini, fennels, eggplants, tomatoes, lettuces, sweet peppers, cauliflowers and cucumber) wereanalyzed. The two testedextractionmethods consists in a liquid-liquidextractionwithhexaneandethyl acetateand twotypologiesofQuEChERS,onecomposedofMgSO4andNaClandthesecondcomposedofMgSO4andCH3COONa.Thechoiceof the best preparationmethodwas done based on the recovery values obtained. In particular, the first allow to obtainrecoveries in therangeof21,44% -97,76% insteadwiththesecondtherecoverieswerebetween78,84%and134,11%.Thesedata permit to choose the second method. Thus all the samples were analyzed by GC-MS/MS (GC/QqQ tandem massspectrometry, Thermo Scientific) with SRM modality selecting two MS/MS transitions for each compound, the first toquantifyandthesecondfortheconfirmation.Themethodvalidationalloweddetectionatlevelsrangedfrom0,2µg/Land10µg/L,insteadLOQswerebetween0,6and30µg/LandR2wasalwaysupto0,9806.Analysisofvegetablesandfruitsshowedthatonlythe24,28%(17samplesofwhich13fruitsand4vegetables)ofthewastesampleshadresiduesuptothespecificLODs.Alltheresidues,disciplinatedbytheECRegulationn°396/2005andsubsequentamendmentsandadditions,wereunderthespecificMRLs.Theseresultsconsenttoadfirmthatthiskindoffoodwastecanbeusedinsafetytoobtainnewproductswithhighaddedvalue.1. Gamón,etal.,2001.MultiresidueDeterminationofPesticides inFruitandVegetablesbyGasChromatography/TandemMassSpectrometry. Journalof

AOACInternational84,4.2. Chamkasem, et al., 2013. Analysis of 136 Pesticides in Avocado Using a Modified QuEChERS Method with LC-MS/MS and GC-MS/MS. Journal of

AgriculturalandFoodChemistry,61,2315−2329.3. Kaewsuya, et al., 2013. Automated QuEChERS Tips for Analysis of Pesticide Residues in Fruits and Vegetables by GC-MS. J. Agric. Food Chem., 61,

2299−2314.


106

MOLECULARCHARACTERIZATIONOFHYBRIDSTRAINSOFPLEUROTUSBYISSR

AguilarDL.1 ,ValenciaTG.1 ,ZarateSPB.1,VillanuevaAR.1,GuadarramaMPC.2,SánchezHA.2,Garín

AME.31UPIBI-InstitutoPolitécnicoNacional,Ticomán-México;²InstitutodeAgroindistria,UniversidadaTecnologicadelaMixteca,

Oaxaca-Mexico;3FacultaddeEstudiosSuperioresIztacala-UNAM,Tlalnepantla-Méxicoemail:[email protected],[email protected]

Mushrooms of Pleurotus genus are commercially cultivated in worldwide by their nutritional and medicinal properties(Huerta et al., 2010). The germplasm obtaining from native strains is very important for genetic improvement of ediblemushroom, allowing the production of hybrids with attractive attributes. In recent years, many researches have beenreportedforhybridproductionthroughneohaplontsmating,previously recoveredbychemicaldedikaryotization(ValenciaandLeal,2002,Guadarrama,etal.,2014)andisconvenienttorealizethegeneticanalysisofbothmonokarioticcomponentsand hybrid strains produced. The use of genetic markers inter simple sequence repeats (ISSR) for determination ofpolymorphismsinbasidiomyceteshasbeenamethodwithhighreproducibilityandhighlyinformative(Wangetal2012;Rainaet al 2001). The objective of present study was the genetic characterization of parental strains, reconstituted strains,neohaplontsandhybridsstrainsofPleurotusbyISSRmarkers.TwowildstrainsofthegenusPleurotuscollectedinHuajuapandeLeon,Oaxaca,Mexicowereused,alsofourhybridstrainsand two strains reconstituted, all generated by compatible pairings of neohaplonts previously produced were used(Guadarrama,etal.,2014).ForgDNAextractionofeachstrainwasusedtheChargeSwitchgDNAPlantKit(Invitrogen,USA)and for identification of genetics profiles six ISSR markers were used: UBC807 (AGAGAGAGAGAGAGAGT), UBC811(GAGAGAGAGAGAGAGAC), ISR02 (CAGCAGCAGCAGCAG), ISR11 (CACCACCACGC), ISR12 (GAGGAGGAGGC), ISR15(GCAGCAGCACT). PCR reaction for ISSR were in 25 µL reaction mixture containing 30 ng template DNA, 2 mMoligonucleotide,23µLPCRSuperMix(Invitrogen).TheISSRamplificationconditionwas:5mininitialdenaturationat94°C;40cyclesconsistingof1mindenaturationat94°C,1minprimerannealingrangesfrom53.2to63.8°Cand3minextensionat72°Candafinalextensionfor10minat72°C.AmplifiedISSRfragmentswereseparatedon1.5%agarose.84fragmentsreproduciblewith96%ofpolymorphismweredetected.Thesixoligonucleotidesamplifiedfragmentsinthe14strainsstudied,withthenumberofamplifiedfragmentsrangingfromeleven(ISSR11)toseventeen(ISSR12)andranginginweight to 200-2200 bp.Mallick and Sikdar (2014) uses these oligonucleotides in strains ofPleurotus florida and Lentinulaedodesandget13bandsforoligonucleotideISSR11300-3000bp,oligonucleotideISSR12andget12bandsfrom300to2500bp.WiththeanalysisusingmarkersISSRpatternsreproduciblebandingwereobtained,thusachievingcharacterizestrainsofPleurotus,parental,reconstituted,neohaplontesandhybridallowingtodetectgeneticvariationbetweenstrainsofPleurotushybridregardingparentalwhichtheyarose.Acknowledgnents:FinancialSupport:IPN-SIPProject:20160240,ICyTDF200/2012(PICSO12-096).1. Guadarrama,M.P.C.,Valencia, T.G.,Ramírez,C.R.,Robles,M. F., Yáñez, F. J.,Garín,A.M.E.,Hernández,C.G., BravoV.G. 2014.Morphologyand

mycelialgrowthrateofPleurotusspp.strainsfromtheMexicanmixtecregion.BrazilianJournalofMicrobiology45,3,861-8722. Huerta,G.,MartinezC.D,Sánchez,J.E.,Lara,L.H,Vilgalys,R.2010.GeneticrelationshipsbetweenmexicanspeciesofPleurotusanalyzingtheITS-region

fromrDNA.MicologiaAplicadaInternational.22:15-25.3. Mallick,P., Sikdar,R.S. 2014.Productionandmolecular characterizationof somatichybridsbetweenPleurotus florida andLentinulaedodes.World J

MicrobiolBiotechnol.30(8):2283-93.4. Raina,S.N.,Rani,V.,Kojima,T.,Ogihara,Y.,Singh,K.P,Devarumath,R.M.2001.RAPDandISSRfingerprintsasusefulgeneticmarkersforanalysisof

geneticdiversity,varietalidentification,andphylogeneticrelationshipsinpeanut(Arachishypogaea)cultivarsandwildspecies.Genome:44:763–772.5. Valencia,T.G.,Leal,L.H.2002.FriutbodycolorinPleurotusspp-hybridstrainsobtainedbymatingsofcompatibleneohaplonts.MushroomBiologyand

MushroomProducts.UAEM.ISBN968-878-105-3.151-159.6. Wang, S., Yin, Y., Liu, Y., Xu, F. 2012. Evaluationof genetic diversity amongChinesePleurotus eryngii cultivars by combinedRAPD/ISSRmarker. Curr

Microbiol.65:424–431.


107

SERJANICACIDOBTAINEDFROMCECROPIATELENITIDAREGULATESBLOODLIPIDLEVELSAND

REDUCESTHEEXPRESSIONOFPRO-INFLAMMATORYCYTOKINES

BalcazarN.1 ,GuillenA.2,MuñozDL.2,Ramirez-PinedaJR.3,MontoyaG.4,AcínS.1 ¹GrupoGENMOL,SededeInvestigaciónUniversitaria,UniversidaddeAntioquia,Medellín-Colombia;2GrupoNatura,Facultadde

CienciasNaturales,UniversidadICESI,Cali-Colombia;3DepartamentodeFisiologíayBioquímica,FacultaddeMedicina,UniversidaddeAntioquia,Medellin-Colombia;4GrupodeInmunomodulación,SededeInvestigaciónUniversitaria,UniversidaddeAntioquia,

Medellín-Colombiaemail:[email protected],[email protected]

Cecropia telenitida is a species of tropical tree located in the Andean region of Venezuela, Colombia, Peru and Ecuador(Frannco-Roselli,P.etal.,1997).Inthisarea,C.telenitidaisusedasatraditionalmedicineforthetreatmentofinflammatorydiseases and diabetesmellitus type 2. (Schinella G et al., 2008; AragaoDM., et al 2010). Serjanic acid is one of themostabundantpentacyclictriterpenespresentintherootsofC.telenitida(MontoyaPeláez,etal2013).Inapreliminaryworkwehave shown thismoleculehashypoglycemicactivity and reduces thegeneticexpressionofpro-inflammatory cytokines inadipose tissueof amousemodel of diet-inducedobesity and insulin resistance (BalcazarN. et al., 2014). In this studyweevaluatedtheeffectofserjanicacidinplasmalipidandadipokinelevelsusingthesamemousemodel.Wealsoanalyzedthegeneticexpressionofpro-inflammatorycytokinesinahumanmacrophagecelllinemodelafterserjanicacidtreatment.Ourobjective is to evaluate the effect of serjanic acid in themolecularmechanisms involved in the development of systemicinsulinresistance.Anethylacetateextractofair-dried rootsofC. telenitidawaspartitionedsuccessivelywithhexaneandethanol.Theethylacetatewasconcentrated,diluted inmethanolandpurifiedbySephadexLH-20.The fractionscontaining triterpeneswerepooledandusedforanalyticalandbiologicalassays.ConventionalisolationsaspreparativeHPLCwereusedtopurifyserjanicacid.Weevaluatedtheeffectofserjanicacidonblood lipidandadipokine levels inC57BL/6Jmicefedwithahigh-fat (HF)diet.After 10 serjanic acid oral doses, animals were sacrificed. Blood samples were taken to evaluate total cholesterol,triglycerides,LDLcholesterol,HDLcholesterol,leptin,adiponectinandresistinlevels.DifferentiatedhumanmonocyticTHP-1cellline,activatedwithlipopolysaccharide(LPS)andINF-γ,wasusedtoevaluateacidserjanicanti-inflammatoryaction.GeneexpressionlevelsofTNF-α,IL-6,IL-1βandMCP-1weredetectedbyreal-timeRT-PCR.Data showed no change in triglycerides and LDL cholesterol levels. However, serjanic acid administration reduced totalcholesterolinmicefedwithaHFdiet.Inthisgroup,HDLcholesterolbloodlevelswereincreasedaftertheadministrationofserjanicacid.Theadministrationofthismoleculealsoreducedsignificantlyplasma leptin levelsandweobservedaslightlyincreaseofadiponectinandresistinlevels.WecorroboratedthatserjanicacidreducesmRNAexpressionofpro-inflammatorycytokines MCP-1, IL-1β, IL-6 and TNF-α in human activated macrophages. Conclusions: This study demonstrates that theadministration of serjanic acid couldmodulate blood lipid and adipokine levels in an obese and insulin resistancemousemodel.Theseresultsalsoconfirmtheanti-inflammatoryactionofserjanicacidinahumancellmodel.1. Franco-RoselliP,BergC.Distributionalpatternsofcecropia(cecropiaceae):apanbiogeographicanalysis.Caldasia.1997;192. SchinellaG,AquilaS,DadeM,GinerR,delCarmenRecioM,SpegazziniE,etal.Anti-InflammatoryandApoptoticActivitiesofPomolicAcidIsolatedfrom

Cecropiapachystachya.PlantaMed.2008;74:215,20.3. AragãoDM,GuarizeL,LaniniJ,daCostaJC,GarciaRM,ScioE.HypoglycemiceffectsofCecropiapachystachyainnormalandalloxan-induceddiabetic

rats.JEthnopharmacol.2010;128:629-33.4. Guillermo L. Montoya Peláeza, Jelver A. Sierra, Fernando Alzate, Ulrike Holzgrabee, José R. Ramirez-Pineda. Pentacyclic triterpenes from Cecropia

telenitidawithimmunomodulatoryactivityondendriticcells.Rev.bras.farmacogn.2013vol.23no.5;754–7615. Balcazar, N1; Tabares-Guevara,Jorge;Gonzalez,P;Ramirez-Pineda,Jose;Montoya,G Hypoglycemic activity of Serjanic acid obtained from Cecropia

telenitida:preliminarydata.PO78AbstractsBookof23°SILAECongress2014pag.183.


108

PRELIMINARYSTUDYONTHEPHENOLYCPROFILEOFBOLETUSAEREUS

PellizzeriV.1 ,LoTurcoV.1,DiBellaG.1,SaittaM.1,GervasiT.1,DugoG.mo1,2

1UniversityofMessina,BIOMORFDept.,Messina-Italy;2Science4Lifesrl,Spin-OffCompany,UniversityofMessina,Sant’Agata(Messina)-Italy

email:[email protected] aereus and related species are among the most common and popular edible mushroom due to their particularsensoryquality.Thechemicalcomposition,aswellasthephenoliccomposition,ofediblemushroomswasevaluatedbymanyauthors,butaverylowreferenceisavailableonB.aereus.Inthepresentwork,itisgivenamethodtoinvestigate24phenoliccompoundsinB.aereusbyUHPLCsystem.Several samplesofBoletus aereuswere collected inNovember 2015 at thewoodsof PeloritaniMountains,Messina, Italy.Aftercollectionhalfofthoseweremaintainedat-20°Candtheotherhalfweredriedonfilterpaperatroomtemperaturefortendaysandtheninovenat45°cfor20min,tosimulateanindustrialprocessofpreservation.Aknownamountoffreshmushroomswasextractedwithamixtureofmethanol/water80:20inultrasonicsystemandthenanalyzedwithaWathersAcquityUHPLCsystem,equippedwithUVdetectorat280nm.Allthetestsforthemethodvalidationwerecarriedout:recoveryvaluesisabout80-90%obtainedbyspikedsamplebeforeextraction;thecalibrationcurvesof24standardofphenoliccompoundwerecarriedoutinthelinearrangeof0,025–0,2µg,withLOD=0,00043µgandLOQ=0,0014µgforHydroxytyrosol.Thenanotheramountofthedriedmushroomwasanalyzedbythesameprocedure.Hydroxytyrosolandt-Cinnamicacidandwere identifiedandquantified inall freshsamplesofBoletusaereusreachingupameanconcentrationof101mg/kgand0,94mg/kgrespectively.WhereasinthedriedsampleonlyHydroxytytrosolwasfound,havingameanconcentrationof299mg/kg.Noothercompoundamongthoseinvestigatedwasdetected.Protocatechuicacid,p-Hydroxybenzoicacidandp-Cumaricacid,weredetectedinpreviousstudyonediblemushrooms,butthey have not made a comparison between fresh and dried matter, furthermore there are no references aboutHydroxytyrosolandt-CinnamicacidinBoletusspp.Currently, thedataobtained in thepresent study seem tobe very interesting,especially if compared to thepreservationmethods.1. Dundar,V.Okumus,S.Ozdemir,K.S.Celik,M.Boga,E.Ozcagli,G.Ozhan,A.Yildiz.Antioxidant,antimicrobial,cytotoxicandanticholinesteraseactivites

ofsevenmuchroomspecieswiththeirphenolicacidcomposition.J.horticulture2015,2:42. HelenoSA.,L.Barros,M.J.Sousa,A.Martins,C.Santos-Buelga,I.C.F.R.Ferreira.TargetedmetabolitesanalysisinwildBoletusSpecies.LWT-foodScience

andTechnology44(2011)1343-1348


109

ETHNOBOTANICALINVESTIGATIONINTHEITALIANCENTRALALPS:HEALTHBENEFITSFROMEDIBLE

PLANTSPECIES

VitaliniS.1 ,PuricelliC.2,IritiM.1¹MilanStateUniversity,DepartmentofAgriculturalandEnvironmentalSciences,Milan-Italy;²MilanStateUniversity,Brera

BotanicalGarden,Milan-Italyemail:[email protected]

Inpopulartradition,plantresourcesareoftenemployedmulti-contextually,forexample,bothasfoodandmedicine(Pieroniet al., 2002).Considering thisdual feature,wedecide to carriedoutanethnobotanical survey inValtellina, a valleyof theLombardyregion,borderingSwitzerland,wherethetraditionaluseoftheplantsisstillalive.Morethan400peoplewereinterviewedfromninemunicipalitiesusingsemi-structuredquestionnaires.Datawereanalysedby quantitative parameters such as ethnobotanicity (EI) and ethnophytonomic (EPI) indices, factor informant consensus(FIC).Abandonedandcurrentuseswerecompared.EachreportedspecieswereexsiccatedandthesamplesdepositedattheHerbarium of the Brera Botanical Garden, Milan State University. Their scientific names were identified based onInternationalPlantNameIndex(IPNI).Inhabitantsquoted56outof220collectedplantspeciesasmedicinalfoodsbelongingto26families(Asteraceae,Rosaceae,Gentianaceae, Brassicaceae and Lamiaceae,mainly). Forty-seven specieswerewild, twelve cultivated andonepurchased.About 16%was reported for its exclusive use in the considered category. The local people still resorted to 94.6% of theirknownuses.Theplantpartsmostcommonlyusedwere leavesand fruits (20.6% respectively), flowers (19.1%), aerialparts(14.7%)androots(11.8%).Almostallplantswereusedinthepreparationofherbaltea,soups,salads,omeletsandliqueurs.The preference ranking identified Achillea moschata Wulfen at first place, followed by Taraxacum officinale F.H.Wigg.,ArtemisiagenipiStechm.,SambucusnigraL.andChenopodiumbonus-henricusL..Thestudyparticipantsconsideredthemedicinalcuisineanintegralpartofhealthypractices,withbeneficialeffectsmainlyonthedigestive(46.4%),respiratory(10.9%),nutritional(10.9%)andgenitourinary(9.4%)disorders.Ourdatatestifythatsomefunctionalfoodsarerootedinthetraditionalcultureofthestudyarea.1. PieroniA,NebelS,QuaveC,MunzH,Heinrich,M,2002.Ethnopharmacologyofliakra:traditionalweedyvegetablesoftheArberesheoftheVulturearea

inSouthernItaly.J.Ethnopharmacology81,165–185.2. IPNI-InternationalPlantNameIndex.Online:www.ipni.org


110

EFFECTOFCOMBINEDADMINISTRATIONOFARTHROSPIRA(SPIRULINA)MAXIMAANDWASTEOFCOCOSNUCIFERAL.ONLIPIDPROFILEINMALEMICEFEDWITHHYPERCHOLESTEROLEMICDIET

GarcíaGE.,MartínezE.,ChamorroG,OrtízA.,GarduñoL.

InstitutoPolitécnicoNacional,EscuelaNacionaldeCienciasBiológicas,UnidadProfesionalAdolfoLópezMateos,CiudaddeMéxico-México


MetabolicSyndrome (SM), is a seriousdiseasewithmany risk factors suchas insulin resistance,dyslipidemia,decreaseofhighdensitylipoproteins(HDL),abdominalobesityandhypertension;whichisassociatedwiththeincreasedindevelopmentofmetabolicandcardiovasculardiseases.ForcounteracttheSMtheyhavebeenincludedfunctionalfoodsindietthatmaycontentsantioxidantaspolyphenols,dietaryfiber,andpolyunsaturatedfattyacidΩ3y6;oneofthesefoodsisaunicellularcyanobacteriumArthrospira(Spirulina)maxima,hasbeendemonstratedtohaveantiatherogenicactivity,suggestingbenefitseffectsonhyperglycemiaandhypercholesterolemia.Otherfunctional foodwithhighbeneficialpotential iscoconut(CocosnuciferaL.),whichhavepointedantioxidants,antiviral,antifungal,antidiabetic,immune,hepatoprotective,cardioprotective,antibacterialeffects.Therefore,theobjectiveofthepresentstudywastoevaluatetheeffectofcombinedadministrationofArthrospira(Spirulina)maximaandwasteofCocosnuciferaL.onlipidprofileinmalemicefedahypercholesterolemicdiet.MalesICRmice(28-30gbodyweight)wereacclimatedfor7days,with12hourslight/darkperiods,temperatures22°C,withwater and food ad libitum; the animals were randomly assigned in 8 groups (n=10), 1) normal diet (DN), 2)hypercholesterolemicdiet(DH),3)normaldiet+spirulina(DN+S),4)normaldiet+coconutwaste(DN+C),5)normaldiet+spirulinacombinationcoconutwaste(DN+SC),6)hypercholesterolemicdiet+spirulina(DH+S),7)hypercholesterolemicdiet + coco waste (DH + C), 8) hypercholesterolemic diet + spirulina combination coco waste (DH + SC); the dosesadministeredwereof400mg/kgofSpirulina(S)and350mg/kgofcoconutwaste(C)andthetreatmentswereadministrateddailyandintragastricallyfor6days.Attheendofstudy,abloodsamplewasobtained,thenbycentrifugationgettingserum,miceweresacrificedfollowingtherecommendationsoftheNOM-ZOO-062.Fromtheserumobtainedweredeterminedtotalcholesterol, triglycerides, HDL-C and glucose byWienner Lab Selectrawith RANDOXKits, LDL-Cwas calculated using theFriedewaldformula.ResultsshownasignificantdecreaseintriglyceridesconcentrationbycombiningspirulinaandcoconutwastecomparingtotheDHgroup,thisisapositiveeffectbecausesuggestinganimprovingontransportationsoftriglycerideswithlipoproteinsduetoantioxidantcapacitythathaveSpirulinatocapturingfreeradicalsanddietaryfiberfromCoconutwaste,fermentableinthelargeintestineandproducesshortchainfattyacids.Asfortheeffectsonlipidprofileintotalcholesterolconcentration,HDL-C, LDL-C therewasno significantdifferencewith any treatments. In conclusion, in this present study for 6dayswasobserveapositivetherapeuticeffecttodecreasethetriglyceridesconcentrationinmouseserum,probablywouldhavemoreeffectmakinganewstudychangingdosesandtreatmenttime.1. DeLaraAndradeR,cols,2005,LaimportanciadeSpirulinaenlaalimentaciónacuícola.ContactoS57,pp13-16.2. Deb Mandal M, cols, 2001, Coconut (Cocos nucifera L.: Arecaceae): In health promotion and disease prevention, Asian Pacific Journal of Tropical

Medicine,pp241-247.3. EstradaJ,cols,2001,AntioxidantactivityofdifferentfractionsofSpirulinaplatensisproteanextract,Farmaco,56,pp497-500.4. Monro,J.A.,cols,1985,DietaryfiberofcoconutfromaPacificatoll:Solubleandinsolublecomponentsinrelationtomaturity,JournaloftheScienceof

FoodandAgriculture,36,1013−1018.


111

BIOLOGICALACTIVITIESOFLAVANDULAANTINEAEMAIRE.ENDEMICSPECIESINALGERIA

KrimatS.1 ,DobT.1,LamariL.2,KsouriA.1,MetidjiH.1,NouasriA.1

1Laboratoryofbioactiveproductsandbiomassvalorizationresearch,ENS,Kouba-Algeria;2Departmentofnaturalsciences,ENS,Kouba-Algeria


LavandulaantineaeMaire(Lamiaceae)isanendemicmedicinalplantofAlgeriawhichistraditionallyusedforthetreatmentofChills, Bruises, oedema and rheumatism (1). However, no antioxidant or antimicrobial activities of its extracts have beenpreviouslypublished.TheobjectiveofthisstudyistoevaluateantioxidantandantimicrobialactivityofLavandulaantineae.The hydromethanolic crude extract and its fractions (chloroform, diethyl ether, ethyl acetate and n-butanol) wereinvestigatedfortheirantioxidantactivitiesusing2,2-diphenyl-1-picrylhydrazyl(DPPH),reducingpowerandβ-carotene–linoleicacidtests.Totalphenoliccontentsandflavonoidcontentswerealsomeasured.Antimicrobialactivitywasexaminedagainst10microbialstrainsbydiscdiffusionandagardilutionmethods.Totalphenolicandflavonoidcontentsoftheextractsvariedbetween12.49-262.35mgGAE/gextractand1.35-4.03mgQE/gextract,respectively.Thehighestradicalscavengingactivitywasdetectedinethylacetateanddiethyletherfractions.Ethylacetatefractionhadalsothehighestreducingcapacity.Themosteffectivefractionsonlipidperoxidationarediethyletherandethylacetatefractions.Incaseofantimicrobialscreening,crudeextractanditsfractionsshowedmoderateantimicrobialactivityagainsttestedmicroorganisms.Amongthetestedsamples,thecrudehydromethanolicextracthavebeenthemostactive.The findings of the present study suggest that hydromethanolic extract and its fractions of Lavandula antineae possessantioxidantandantimicrobialpropertiesandhencecanbeapotentialnaturalsourceinhealthandmedicine.1.HammicheV&MaizaK..,2006.TraditionalmedicineinCentralSahara:PharmacopoeiaofTassiliN’ajjer.JEthnopharmacol,105,358-367.


112

PRIMARYSCREENINGOFBIOLOGICALACTIVITIESINEXTRACTSOBTAINEDFROMAROMATICAND

MEDICINALPLANTSGROWNINCOLOMBIA

FlechasMC.,BarriosSX.,CuadrosAJ.,StashenkoEE.,FuentesJL.,OcazionezRE. UniversidadIndustrialdeSantander,Bucaramanga(Santander)-Colombia.

email:relocaz@uis.edu.coHerbaltherapycouldbebeneficialinattenuatingseverityofhumandiseases.ColombiahasarichherbalrepertoireandtheVademecumonMedicinalPlantsprescribesmedicinesforavarietyofdiseases,butthenumberofplantsstudiedislimited.ThecurrentworkwasundertakentoidentifyplantspeciesfromColombiawhoseextract(EXT)couldbeselectedasprimarysource for discovering natural medicines to alleviate symptoms of cancer, dengue, and infectious caused byMethicillin-ResistantStaphylococcusaureus(MRSA).Supercritical fluid(CO2)extractionwascarriedouttoobtainplantEXTs,whichwerecollectedat 40(fraction1)and/or80(fraction2)bar.Fiftysamplesfrom27plantspecieswereprofiledforcytotoxicityandantiproliferativeactivitiesusingMTTscreening, in six human and animal cell types derived from targets of xenobiotic toxicity (liver, kidney, skin), and fromcancerousorgans(liver,cervix,breast).HierarchicalclusteringofcytotoxicprofileswasperformedusingCC50values,andthesumof individualsweightedhierarchyvalues(∑iWH)wasadoptedassimilarity indicator. Inhibitoryeffectondenguevirus(DENV)wasevaluatedbyusingbothareafractionoutputmethod(withImageJfreeware)andvirus-antigenreductiontest.AmodifiedmicrodilutionmethodwasusedfortestinginhibitionontheMRSAgrowth.SomeEXTswerenon-cytotoxic(n=12;CC50>200µg/mL;∑iWH>7.0)orcytotoxic(n=14;CC50<200µg/mL;∑iWH<3.2)toallcell types,whereas others exhibited cell type–specific cytotoxicity (n = 24; CC50: 500 – 77.8 200 µg/mL;∑iWH: 6.3 – 3.3).FractionsofEXTs fromthesameplant frequentlyshoweddifferentpatternsofcytotoxicity.EXTs fromBursera tomentosaand Eriope crassipes seem to be active (IC50 < 50 µg/mL; selectivity index > 6.4) against cells (HeLa) from cancer cervix,though none of EXTs presented relevant activity on other cancerous cell. Five EXTs reduced (66.8 – 31.4%) cellular deathcausedbyinfectionat leastofoneDENV-serotype,anddifferentpatternofantiviralactivityrespecttofractionofEXTwasobserved.Hyptissuaveolensfraction1,butnotfraction2,wastheonlyEXTactiveagainstallfourDENVserotypes(IC50:16.6–78.7μg/ml;SI>5.4).RelevantactivityagainstMRSA(MIC:0.029-0.059mg/mL)wasfoundinEXTsfromPipersubflavumvar.Espejuelanum,Pipereriopodon,andHyptissuaveolens.Discussion and Conclusion: Analysis of biological activities of products derived from plants is an option for identifyingstartingsamplesfordiscoveringofherbalmedicines.ThisstudyresultedintheidentificationofEXTsfromColombianplantswithpromisingactivitiesagainstcervixcancer,DENVandMRSA.Protectiveefficacystudiesinananimalmodelarerequiredtoconfirmactivities.This research was carried out thanks to financial support from the Colombian Institute of Science and Technology(Colciencias),GrantRC-245-2011(PatrimonioAutónomodelFondoNacionaldeFinanciamientoparalaCiencia,TecnologíaeInnovación,FranciscoJosédeCaldas).1. Bernal HY, et al. (2011). Pautas para el conocimiento, conservación y uso sostenible de las plantas medicinales nativas en Colombia. Instituto de

InvestigacióndeRecursosBiológicosAlexandervonHumbold.EdiprintLtda.,Bogotá,Colombia.2. EfferthT.Biopiracyofnaturalproductsandgoodbioprospectingpractice.Phytomedicine.201615;23(2):166-73.3. DutraRC,etal.MedicinalplantsinBrazil:Pharmacologicalstudies,drugdiscovery,challengesandperspectives.PharmacolRes.2016Jan23.pii:S1043-

6618(16)00023-2.doi:10.1016/j.phrs.2016.01.021.


113

ETHNOBOTANICALSURVEYONTHEUSEOFTRADITIONALMEDICINEFORTHETREATMENTOF

OSTEOARTHRITISINORAN,ALGERIA

LardjamA1,2,3,4 ,MazidR.1,3,SadaouiA.3,BensahailaS.3,Khalfa3.KhitriW.3,4,AzaizA.3,DjebliN.4,ToumiH.1,2,3

1PharmacovigilanceService-EHUd’Oran;2PharmaceuticalDevelopmentResearchLaboratory;3FacultyofMedicineofOran;4PharmacognosyApi-Phytotherapylaboratory,UniversityofOran,Oran-Algeria

email:lardmhadjer@outlook.frOsteoarthritisisaverycommonrheumaticdiseasethatisassociatedwithacoupleofdisabilitiesandchronicpains.Though,curativetreatmentarenotavailable.Recently,moreandmorepatientsareusingcomplementarymedicinesandparticularlyphytotherapy.The aim of the present study is to determine the frequency of phytotherapyin patients with osteoarthritis. Twoethnobotanical surveys were conducted in patients and herbalists, all data were processed and analysed by using SPSS(StatisticalPackagefortheSocialSciences,IBMCorporation;Chicago,IL)andExcelsoftwareprogram.The first surveywas carried out on 314 osteoarthritis,at the level of the Rheumatology department “EPSP Akid II, Oran-Algeria-”,andshowedthat38%ofthestudiedpopulationusemedicinalplants,about54speciesand43recipeshavebeenrecorded, belonging to 27 botanical families. However, themost used part is the fruit and themajority of remedies arepreparedasoil.Thesecondsurveywasconductedon52herbalistsintheregionofOran,52plantspecies(plantsalone,15recipes)havebeenidentified belonging to 27 botanical families and 37 commercial products. The most used part is the leaf and the mostrecommendedmodeofpreparationisthepowderform.Phytotherapyisstillapracticeusedbyosteoarthritispatients,bothsurveyshavehelpedidentifyimportantmedicinalplantsused by the local population and recommended by herbalists in order to treat osteoarthritis.These plants can provide aconvenient solutionto the problems of osteoarthritis and being a therapeutic prospect in order to improve the diseasemanagement.


114

GREENSYNTHESISOFNEWLIGANDSSTARTINGFROMCHIRALp-HALOGENATEDAMINES

HernándezTéllezMG. ,MorenoMoralesGE.,GutiérrezArgüellesD.,PortilloMorenoÓ.,Gutiérrez

PérezR.BeneméritaUniversidadAutónomadePuebla,FacultaddeCienciasQuímicas,LaboratorioSíntesisdeComplejos,Puebla-México.

email:guadalupe.hernandez@correo.buap.mxThisresearchwascarriedoutwithanewandmoreefficientmethodofsynthesis,achievingtheprinciplesofGreenChemistry.Thisnewtechniquereducestheuseofdangerouschemicals inthedesignanddevelopmentofthesynthesis.Ontheotherhand, iminesareversatile ligands,whichare synthesized from thecondensationofa carboxylic compoundandanamine.These compounds and theirmetallic complexes are very important as catalysts in different biological systems, polymers,colorantsandinthemedicalandpharmaceuticalfields.We report the synthesis of three new chiral imines derived from 2-thiophencarboxaldehyde and optically active primaryaromatic amines bearing halogen atoms in para- position: (R)-(+)-1-(4-fluorophenyl)ethylamine, (S)-(-)-1-(4-chlorophenyl)ethylamineand(S)-(-)-1-(4-bromophenyl)ethylamine,byusingatechniquefromGreenChemistry:“Solventfreereactions”,whichgaveexcellentyieldsoftheimines.InSolventfreereactions,theprocessesoccurinmanycasesinamoreefficientandselectivewaythaninreactionsinsolution,sinceinthesolidstatethemoleculesinthecrystalhadarrangedinaregularandcompactway.Solventfreereactionshavealsomanyadvantages:lowpollution,lowcostandeasywork-up.Thereactionof2-thiophenecarboxaldehydeand(R)-(+)-1-(4-fluorophenyl)ethylamineina1/1ratioleadstothecorrespondingimine(R)-(-)-[1-(4-fluorophenyl)-N-(tiophene-2-yl)methylidene]ethylamine,asawhitesolid.Mp=69-70°C,yield=97%,[𝛼]!!"°=-153.7º (c = 1, CHCl3). In the sameway, the reaction of 2-thiophenecarboxaldehyde and (S)-(-)-1-(4-chlorophenyl)ethylaminegives(S)-(+)-[1-(4-chlorophenyl)-N-(thiophene-2-yl)methylidene]ethylamineasawhitesolid.Mp=41-42°C,yield=98%,[𝛼]!!"° =+174.3º(c=1,CHCl3).Finally,thereactionof2-thiophenecarboxaldehydewith(S)-(-)-1-(4-bromophenyl)ethylamineproduces(S)-(+)-[1-(4-bromofenil)-N-(tiofen-2-il)metilideno]etilaminaasawhitesolidalso.Mp=58-59°C,yield=95%,[𝛼]!!"°=+198.7º(c=1,CHCl3).TheproductswerefullycharacterizedbyFT-IR,NMR1Hand13C,EI-MSandthestructuresofthenewligandswasconfirmedbyX–raydiffractionstudies.1. TanakaK.,(2003).Solvent-freeOrganicSynthesis.Wiley-VCHGmbH&CoKGaA,p.294.2. GetinetTamiruTigineh,Yuh-ShengWen,Ling-KangLiu.(2015).Solvent-freemechanochemicalconversionof3-ethoxysalicylaldehydeandprimary

aromaticaminestocorrespondingSchiff-Bases.Tetrahedron.71,170-175.3. Zhang,J.,Zhao,F.,Zhu,X.,Wong,W.K.,.(2012)Mater.Chem.22,16448-16457.


115

GREENSYNTHESISOFANEWCHIRALLIGANDHALOGENATEDANDITSPd(II)COMPLEX

HernándezTéllezMG. ,MorenoMoralesGE,GutiérrezArgüellesD.,PortilloMorenoÓ.,GutiérrezPérezR.

BeneméritaUniversidadAutónomadePuebla,FacultaddeCienciasQuímicas,LaboratorioSíntesisdeComplejos,Puebla-México.email:[email protected]

Nowadays,manygroupsworldwidecarryoutnumerousresearchesabouttheoriginandtreatmentofcarcinogenicdiseases,wherethedesignandsynthesisofnewdrugsareconstantlyprovedwiththegoalofeliminatingorcontrollingsuchdiseases.Thisreportisaboutsulfur-containingmoleculesintheirstructuretoformachiralligandwithboththeC=NgroupandaSulfuratom.ThesefeatureshavespecialinterestintheformationofmetalliccomplexessincemetalscanalsocoordinatewiththeSulfuratom.Asantecedent,ourresearchgrouphasalreadysynthesizedPd(II)complexeswithanticanceractivity.Duetotheimportanceofthesecomplexesinthemedicalfield,oneofourmainobjectivesisthesynthesisofmetalliccomplexesusingchiralligands.We report the synthesis of a newchiral ligandderived from2-(methylthio)-benzaldehyde and theoptically activeprimaryaromatic amine: (S)-(-)-1-(4-chlorophenyl)ethylamine, using a technique from Green Chemistry: “Solvent free reactions”,whichgaveexcellentyieldsoftheimineandthePd(II)complex.InSolventfreereactions,theprocessesoccurinmanycasesinamoreefficientand selectiveway than in reactions in solution, since in the solid state themolecules in thecrystal arearrangedinaregularandcompactway.Solventfreereactionshavealsomanyadvantages:lowpollution,lowcostandeasywork-up.The productswere characterizedby using spectroscopic techniques: FT-IR,NMRH andC, EI-MS and the structure of thePd(II)complexwasfullyconfirmedbyX-raydiffraction.ThecrystallinestructureofthecomplexshowedthatthePalladiumatomcoordinateswiththeSulfuratomandtheNitrogenatomoftheiminegroup,asexpected.1. GutiérrezD.,BernèsS.,HernándezG.,PortilloO.,MorenoG.E.,SharmaM.,SharmaP.,GutiérrezR.,(2015).NewChirala-ketoimine-Pd(II)complexesand

theiranticanceractivity.JournalofCoordinationChemistry21,3805-3813.2. GarayL.A.,PichonA.,JamesS.L.,(2007)Solvent-freesynthesisofmetalcomplexes.Chem.Soc.Rev.,36,846-855.3. TanakaK.,(2003).Solvent-freeOrganicSynthesis.Wiley-VCHGmbH&CoKGaA,p.294.


116

EFFECTIVENESSANDSAFETYOFNUTRISOLASNUTRITIONALSUPPLEMENTINTHETREATMENTOF

ADOLESCENTSWITHNUTRITIONALDEFICIENCY.

GonzálezS.1 ,DomínguezMI.1,CasamayorZ.1,HernándezB.1,CondeE.1,LealA.1,FilgueiraI.1,AgramonteG.1,CasasMJ.2,PoveaE.2,HeviaD.2,DíazM.2,ÁlvarezB.2,PérezIM.2,EspluganA.2,Bejerano

CJ.2,AlbertineM.21GrupoEmpresarialLABIOFAM,LaHabana-Cuba;2HospitalUniversitarioPediátricoWilliamSoler,Boyeros,Habana-Cuba


Thenutritionalrequirementsdependonthespendingneededtokeepthegrowthrhythm,changesinbodycompositionandintheenergyconsumption,hencetheneedforanadequatenutritionforadolescentstodeveloptheirbiologicalpotentialtothemaximum.To evaluate the effectiveness and safety of Nutrisol as a nutritional supplement in the treatment of adolescents withnutritionaldeficiency.A prospective, longitudinal and analytic study at the University Pediatric Hospital William Soler was performed. 40adolescents,20ofeachsex,withdiagnosisofnutritionaldeficiencyandpercentilevaluesequalto10orlesswereincluded.Theteenagersweretreatedforaperiodofsixmonths.GroupIreceivedtheusualtreatment(vitaminC,Polivitanddietaryassessment)andgroupIIwastreatedwithNutrisolanddietaryassessment.Anthropometricmeasurements,laboratorydataandclinicalparameterswereevaluated.Theevolutionof the responsevariableswasperformedusing thenon-parametricFriedmantest.AdolescentswhoconsumedNutrisolshowedgreatervariabilityinthestudiedanthropometricvariablesthanthosereceivingtheusualtreatment(p=0.001).TheexploringofhaematologicalvariablesshowedaslightincreaseofnormalvaluesforbothsexesafterconsumptionofNutrisol.Teenageboyshadbetterresponsetotreatmentthanfemales.Therewasnochangeintheparametersforsafetyvariablesassessedduringthestudy.Nauseaandlossofappetiteweretheadverseeventsreportedduringtheinvestigation.Noseriousadverseeventswerereported.TheuseofNutrisoliseffectiveandsafeintreatingadolescentswithnutritionaldeficiencies.


117

ANTI-STAPHYLOCOCCALACTIVITYINVITROANDINVIVOOFZINNIAPERUVIANA.HISTOLOGICALSTUDY.

EcheniqueDR.1,MattanaCM.1,LaciarAL.1,AguileraMerloC.2,CruceñoAM.2,SatorresSE.1

MicrobiologyArea1,MorphologyArea2,FacultyofChemistry,BiochemistryandPharmacyNationalUniversityofSanLuis,SanLuis.Argentina.

email:[email protected] aureus is apathogenanda frequent colonizerofhumanmucosaand skin. It cancauseavarietyof infectionsbeingresponsibleformostinfectionsofskinandsofttissue.ZinniaperuvianaL.(Asteraceae)isaherbaceousplantusedinfolkmedicinewhichextendsinthecentralregionofArgentina.TheaimofthisstudywastoevaluateantibacterialactivityinvitroofZ. peruviana extract, the effect of this extract in skin infection of BALB/cmice bymethicillin-resistant S. aureus (MRSA) andperformhistologicalstudiesofinjuries.Theextractwaspreparedusingsolventsethylacetate (AcOEt)andmixturesofn-hexaneandAcOEtof increasingpolarityonflashchromatography.Theminimalinhibitoryconcentration(MIC)andminimalbactericidalconcentration(MBC)weretestedina concentration ranging from 8000 to 100µg/ml. The first day, fourmice BALB/c were inoculated subcutaneously with a 1:1mixturev/vof1x108CFU/mlofMRSAstrainATCC43300and15:85%ethylacetate:n-hexane(AcOEt:HEX)Z.peruvianaextractinaconcentrationof8000µg/ml.OthergroupsoffourmicewereinfectedwithanequalconcentrationofMRSA(controlwithoutextract).Onthesecondday,theabscessopenedandallmiceweresacrificed.Histologicalstudiesoflesionswereperformedandthelesionsandinternalorgans(kidneyandspleen)wereremovedandhomogenizedforbacterialquantification.TheextractshowedantibacterialactivityagainstMRSAwithMIC8000µg/ml.TheMBCvaluewasidenticaltotheMICvalue.Nodifferencewasobservedintheevolutionandsizeoflesionsinbothgroupsofmice.However,asignificantdecreaseinbacterialcount of lesion inmice infectedwith themixtureMRSA and organic extractwas found (p=0.0076).No bacterial countwasobservedinkidneyandspleen.Therearefewreportsontheantimicrobialactivityofthisplant.SomeauthorsdemostratedthataqueousandalcoholicextractsfromZ.peruvianashoweddifferentdegreesofantibacterialactivityagainstGram-positiveandGram-negativemicroorganisms(Amanietal.,1998).Toourknowledge,therearefewreportsavailableintheliteratureonthephytochemicalscreeningofZ.peruviana,NamboCamachodetectedcompoundssuchasterpenesandsesquiterpenelactonesfromZ.peruviana(NamboCamacho,2015).Ontheotherhand,othersauthorsdetectedcompoundssuchassaponins,steroid,flavonoids, phenols and glycosides from Zinnia elegans (Mohamed et al., 2015), an interesting group of biologically activephytochemicals. So, this leads us to think that if some of these compounds are present in Z. peruviana, they could beresponsible, partly or completely, of antimicrobial activity observed in this study. Adipocytes have been suggested to beimmunologically active, but their role in host defense againstmicrobial infection is unclear (Zhang et al., 2015). Histologicalexamination of the mice natural infection with S. aureus showed the presence of rapid proliferation of adipocytes andexpansionof thedermal fat layer. Inmice treatedwithS. aureus+Z.peruviana,histologyshowed lessexpansionofadiposetissueandreductionofthepresenceofdermaladipocytes.Howeveraninfiltrateofleukocytesandformationoflargefolliclespreferablylocatedinthedeepdermiswasobserved.AmostprofoundstudyoftheactionofZinniaextractonskintissueshouldbeperformed.1. AmaniS.M., IslaM.I,VattuoneM.A.,PochM.J.,CudmaniN.GandSampietroA.R.. 1998Antimicrobialactivities in someArgentinemedicinalplants.Acta

Horticulturae.501:115-122.2. Tesis Digitales. Universidad Autónoma de México. 2015 Antonio Amado Nambo Camacho Available at: 132.248.9.195/ptd2015/abril/

304008605/304008605.pdf1. Mohamed A.H., Ahmed F.A., Ahmed O.K. 2015. Hepatoprotective and Antioxidant Activity of Zinnia Elegans Leaves Ethanolic Extract. International

JournalofScientificandEngineeringResearch.6:154-161.2. ZhangL.J.,Guerrero-JuarezC.F.,HataT.,BapatS.P.,MaksimR.R,PlikusV.,Gallo,R.L.2015.DermaladipocytesprotectagainstinvasiveStaphylococcus

aureusskininfection.Science.347:67-71


118

AZORELLATRIFURCATAANDZINNIAPERUVIANAEXTRACTS:

ACTIVITYAGAINSTCLINICALISOLATESSTAPHYLOCOCCUSAUREUS

EcheniqueDR.,MattanaCM.,LaciarAL.,AlcarázLE.,SatorresSE. MicrobiologyArea.FacultyofChemistry,BiochemistryandPharmacyNationalUniversityofSanLuis,SanLuis-Argentina

email:[email protected] causedbyStaphylococcus aureus andmainlyby strains resistant to antibiotics are a seriousproblem in all fieldsofmedicine,this,hascausedatrendtosearchfordrugsofnaturalorigin.Methicillin-resistantS.aureus(MRSA)aremicroorganismswhich lead to serious epidemiological and therapeutic problems. The natural products derived from medicinal plants haveproven to be an abundant source of compoundswith antibacterial activity.Azorella trifurcata (Gaertn.) Pers (Apiaceae) andZinniaperuviana(L.)(Asteraceae)arenativeplantsdistributedinArgentinaandChileusedinfolkmedicineforthetreatmentofdifferentdiseases(Barbozaetal.,2009).In this study theantibacterialactivityofZ.peruviana 100%methanolandA. trifurcata 100%n-hexaneextractswereevaluatedagainstfourMRSAstrains,twowereisolatedfromurinecultures,onefrombloodculturesandoneisolatedfromfootulcer.Ofthethreemethicillin-sensitiveS.aureus (MSSA)strains,onewas isolatedfromurineculturesandtwofrombloodcultures.AllMRSAstrainspresentedassociatedresistancetootherantibiotics.S.aureusATCC43300strainandS.aureusATCC25923wereusedascontrolsstrains.Theextractswerepreparedusingflashchromatography.Theantibacterialactivitywasassayedusingmicroplatemethodintripticasesoyabrothsupplementedwithtriphenyltetrazoliumchlorideusedasvisualindicatorofbacterialgrowth.Theinoculumofeachstrainwasadjustedtoconcentrationof106CFU/ml.Organicextractsweretestedfrom8000to100µg/ml.Theantibacterialactivity(MIC)wasdefinedasthelowestconcentrationoftheextractinthemediuminwhichtherenovisiblegrown.Extractsweresubmittedtoasubcultureontripticasesoyaagarplates toevaluate theminimalbactericidalconcentration(MBC).Extract of A. trifurcata100% n-hexane showed the highest antibacterial activity. All clinical isolates of MRSA assayed wereinhibitedwithMICsof 4000µg/ml and this extractwas active againstS. aureus ATCC43300at8000µg/ml. Clinical isolatesMSSA strains and S. aureus ATCC 25923 were sensitive to extract of A. trifurcata to concentration of 8000 µg/ml. The Z.peruviana100%methanolextractshowedactivityagainstatthreeMRSAstrainswithMICsof4000µg/ml.AllotherstrainsofS.aureus assayed were inhibited at a concentration of 8000 µg/ml. In general, MIC values were bacteriostatic. Higherconcentrationsofextractwereneeded tohavebactericidaleffect.Plants from thegenusAzorella haveyieldedanumberofditerpenoidswithdiverseinterestingskeletonssuchasmulinane,yaretane,azorellaneandmadreporane.Also,diterpeneshavebeenisolatedfromtheaerialpartsofA.trifurcata(Arecheetal.,2014).Ontheotherhandsomeauthorsdetectedcompoundssuch as saponins, steroid, flavonoids, phenols and glycosides fromZinnia elegans (Mohamed et al., 2015), and terpenes andsesquiterpene lactones were isolated from Z. peruviana, compounds which exhibit variety of biological activities such ascytotoxic,analgesic,antimicrobialamongothers(NamboCamacho,2015).Theantimicrobialpotencyoftheseplantsisprobablyduetooneorseveralof theseactivecompounds.Thehighprevalenceofmultidrug-resistantbacteria increases thebacterialinfections spectrum difficult to treat. The study of the antibacterial properties of plant extracts and its active compounds,remainsanimportantwaytonewtherapeuticoptions.1. Barboza,G.E.,CanteroJ.J.,NúñezC.,PacciaroniA.,EspinarL.A.2009.Medicinalplants:Ageneralreviewandaphytochemicalandethnopharmacological

screeningofthenativeArgentineFlora.Kurtziana.34:7-365.2. ArecheC.,SepulvedaB.,SanMartinA.,Garcia-BeltránO.,SimirgiotisM.,CañeteA.2014.AnunusualmulinanediterpenoidfromtheChileanplantAzorella

trifurcata(Gaertn)Pers.OrganicandBiomolecularChemistry0:1–83. MohamedA.H.,AhmedF.A.,AhmedO.K.2015.HepatoprotectiveandAntioxidantActivityofZinniaElegansLeavesEthanolicExtract.InternationalJournal

ofScientificandEngineeringResearch.6:154-161.4. Tesis Digitales. Universidad Autónoma de México. 2015 Antonio Amado Nambo Camacho Available at: 132.248.9.195/ptd2015/abril/304008605/

304008605.pdf


119

ANTIPROLIFERATIVEACTIVITYONHUMANPROSTATECELLSOFESSENTIALOILSOFTHREELEBANESE

SALVIASPECIES

RussoA.1 ,CardileV.2,GrazianoACE.2,FormisanoC.3,AvolaR2,ArnoldNA.4,SenatoreF.3,RiganoD.31DepartmentofDrugSciences,BiochemistrySection,UniversityofCatania,Catania-Italy;2DepartmentofBio-medical

SciencesandBiotechnologies,UniversityofCatania,Catania-Italy;3DepartmentofPharmacy,UniversityofNaplesFedericoII,Naples-Italy;4FacultédesSciencesAgronomiques,UniversitéSaintEsprit,Kaslik(Beyrouth)-Lebanon


Prostatecancer is the leadingcauseofdeathfromcancer inoldermenandthemostcommonlydiagnosedcancer inmenoverall.Manypatients are diagnosedwhen the cancer has already advanced tometastasis.Most patientswith advanceddisease respond initially to androgen-ablative therapies, but frequently in this stage, prostate cancer growth anddevelopmentbecomeindependentofandrogenandrendersandrogenablationtherapyineffective[1].Atpresentavailabletreatments for advanced, androgen-independent prostate cancer are only marginally effective [1]. In the Lebanese folkmedicine,Salviaspeciesareusedbymanypeopleinvariousvillagesandtownsforthetherapeuticvalueoftheiressentialoilsandwaterextracts[2].Inthisstudywereportthepotentialanticancereffectinandrogen-insensitive(DU-145)andandrogen-sensitive (LNCaP) human prostate cancer cells of essential oils from three Salvia species well known in Lebanon ascomponent inseveralmultiherbproductsusedforthetreatmentofcancerandotherdiseases:S.aureaL.,S. judaicaBoissandS.viscosaJacq.AerialpartsfromSalviaaureaS.judaicaandS.viscosawerecollectedatthefullfloweringstagefromplantswildgrowingatEl Kfour, Lebanon, in August 2012. The air-dried samples were ground in a Waring blender and then subjected tohydrodistillation for 3 h using n-hexane as a solvent according to the standard procedure described in EuropeanPharmacopoeia(2008).Analyticalgaschromatographywascarriedoutaspreviouslyreported[2].Thebiologicalactivityoftheessentialoils,wasinvestigatedagainsthumanprostatecancercells,testingseveralbiochemicalparameters[2,3],suchascellvitality(MTTassay),cellmembraneintegrity(lactatedehydrogenaserelease)andcaspase-3activity.Inaddition,theexpressionofBcl-2andBaxproteinswasevaluated.Theresultsrevealedthatallthetest essentialoilswereabletoinhibit,after72hoftreatment,thegrowthofcancercells.Ourdataalsodemonstratethatthesenaturalproductsinduceapoptoticcelldeaththatcouldberelatedtoanoverallactionofthesesquiterpenecompounds,particularlycaryophylleneoxide,foundincomparableconcentrationinallthreesamples.Inconclusion,theexperimentalevidencespresentedhere,couldprovideafurther invitro scientificsupportfortheuseofthesespeciesintraditionalherbalpreparationsknowninLebanonandsurroundingcountriesforthetreatmentofcancer.Inaddition,theselectivityoftheessentialoilsS.aurea,S. judaicaandS.viscosa intargetingcancercells,sparingnormalcells,suggests that thesenatural products for their active components couldbe candidate for further analysiswith the aim toreducethetoxicsideeffectsofchemotherapeuticsinprostatecancerpatients.1. FerlayJ,etal.,2015.Cancerincidenceandmortalityworldwide:sources,methodsandmajorpatternsinGLOBOCAN2012.Int.J.Cancer136,E359–3861. RussoA,etal., inpress.Comparativephytochemicalprofileandantiproliferativeactivityonhumanmelanomacellsofessentialoilsofthreelebanese

Salviaspecies.IndCropProd.2. Madrid,etal.,2015.Psoraleaglandulosaasapotentialsourceofanticanceragentsformelanomatreatment.Int.J.Mol.Sci.16,7944-7959.


120

ANTIMICROBIALPROPERTIESOFGARLIC(AlliumsativumL.)ANDONION

(AlliumcepaL.)EXTRACTONCOMMONCARP(Cyprinuscarpio)RESTRUCTUREDMEATDURINGSTORAGEIN4°C

Dublán-GarcíaO.1 ,Rodríguez-QuirozML.1,Gómez-OlivánLM.1,López-MartínezLX.2,Díaz-BanderaD.1,

Cira-ChávezLA.3,Hernández-NavarroMD.1,Jáuregui-RodríguezB.1¹UniversidadAutónomadelEstadodeMéxico,FacultaddeQuímica,CerrilloPiedrasBlancas,Toluca-México;2Centrode

InvestigaciónenAlimentaciónyDesarrollo,A.C.UnidadCuliacán,Culiacán(Sinaloa)-México;3InstitutoTecnológicodeSonora,CiudadObregón(Sonora)-México.

email:[email protected];[email protected] is an activity that has been the basis for numerous investigations that have been reflected in importanttechnologicaladvances(Montañoetal.,2006).Commoncarp(Cyprinuscarpio) isaspeciesthathasgreatadaptabilityandreproductive capacity; additionally it is a source of high quality protein. However, the high water content, the properbacterial floraoffisheryproductsandhighenzymeactivity,cause it tobeverysusceptibletodeterioration(Medinaetal.,2008) in order to counteract thesedamages havebeenused antimicrobials natural origin such as garlic andonions, theycontainphenolic compoundsand sulfurwhichexert anantioxidantandantimicrobial effect. Theaimof this studywas toevaluatetheeffectofanaqueousextractofgarlic(AlliumsativumL.)andonion(AlliumcepaL.)onmicrobialcharacteristicsduringtheshelflifeofrestructuredmeatofcommoncarp(Cyprinuscarpio).Amixtureof aqueous extractsof garlic andonionwas conducted inorder to assess themixturewithgreater amountofphenoliccompoundsandantimicrobialactivity.Forthedeterminationoftotalphenoliccompounds(TFC),wasaccordingtoLópez et al. (2014). For antimicrobial activity, microbial challenge was performed with microorganisms of importance inpublichealth suchasE coli, S. aureusandP. aeruginosa (NOM-113-SSA1-1994).A restructuredmeatwasdesignedbasedoncommoncarpatwhichthemixtureofextractsofgarlicandonionwasappliedanditsantimicrobialeffectwasevaluatedfor21daysunderrefrigeration(4°C)alsocarriedoutanacceptancetestwith200judgesuntrained.Allstudieswereconductedintriplicate,analyzedbyANOVAandTukeymeansdifference(p<0.05).It was found that a 50:50 mixture of the aqueous extracts of garlic and onion had the best TFC content (p <0.05) andantimicrobialeffect(p<0.05)inhibitinggrowthofS.aureus,E.coli,andP.aeruginosa.Theaqueousextractwasappliedtotherestructuredmeatobservedthataftercooking,17%oftheTFCisretainedwithrespecttotheamountobtainedintheinitialmixturewasalsoin42%oftheantioxidantactivity.Nomicrobialgrowthwasobservedduringthe21daysofstorage,notsoforthecontrolthathadgrowthat5daysofstorage.Acceptancetestingresultedinsignificantlyfinalproductacceptancebyconsumers.TheseresultsmightsuggestthattheuseofcarpmeatincombinationwithTFCgarlicandonionareanalternativeconsumption of this species of economic importance, being a product sensorially accepted also can be amethod usefulconservationandlowcost.1. Montaño,K.,VillalpandoyVargas,“AmplifiedFragmentLengthPolymorphismysuaplicaciónenacuicultura”.Interciencia,Vol.3,1p.563-5692006.2. Medina,I.,González,M.,Lois,S.,Iglesias,J.Maestre,R.,yPazosM.(2008)“AntioxidantMechanisminvolvedintheactivityofnaturalprocyanidinsinsea

foodproducts”.EJEAFChe.España.Vol.7p.p.3315-3319.3. López-Martínez, L.X., Aguilar- Cisneros, L.M., Dublán-García O. (2014) “Actividad antioxidante e inhibidora de α-glucosidasa y α-amilasa de tres

variedadesdecebolla(AlliumcepaL.)”.RevistaElectrónicaNovaScientia.12:6(2)p.p.234-247.4. NOM-113-SSA1-1994.Availableinwww.salud.gob.mx/unidades/cdi/nom/113ssa14.html.


121

EFFECTOFCHEMICALCOMPOUNDSAGAINSTBACTERIALBIOFILMSUNDERMONOSPECIESANDDUAL-

SPECIESCONDITIONS

MohamedAM.1,SatorresSE.1,NovoaRE.2,CifuentesDA.2,MattanaCM.1 1ÁreaMicrobiología,2ÁreaQuímicaOrgánica,FacultaddeQuímica,BioquímicayFarmacia.UniversidadNacionaldeSanLuis,San

Luis-Argentina.email:[email protected]

ListeriamonocytogenesandStaphylococcusaureusarepathogensofconcern inthefood industryfor itspotentialabilitytoproducebiofilms.Thisleadstoserioushealthproblemsandeconomiclosses.Thebiofilmservesasareservoirforcontinuousreleaseofbacteriaprocessedfoodsincontactwiththosesurfaces,asitisfoundthatadherentbacteriaaremoreresistanttothe processes of cleaning and sanitizing allowing survival for long periods of storage and difficult to eradicate. In thiscontext,invitrostudieswereaimed,onproductionofbacterialfilmsformedinmonocultures(abacterialspecie)andmixedcultures (interactionof twobacterial species), anderadicationofbiofilmsbynewcompoundswithbiological activities (1)obtainedfromcommercialketonesasanalternative,comparedtotheactionofchemicaldisinfectantscommonlyused.Bacterialinoculumswereadjustedtoaturbiditycorrespondingtothetube0.5MacFarlandscale(108CFU/mL).Ineachwell,150μLoftryptonesoybroth(TSB),supplementedwith1%glucoseand50μLofmonocultures(L.monocytogenes,S.aureus)andmixedcultures(ListeriaandStaphylococcus)wereplacedandthen,incubatedat37°Cfor48h.Attheendofincubationtime,thecontentofthewellswasdiscardedand100μLofsyntheticcompoundscalledsemicarbazoneof(E)-chalcone(DC-RN-3), 1-indanone semicarbazone (DC-RN-5), p-fluoroacetophenone semicarbazone (DC-RN-8) and strongly oxidizingdisinfectantssuchassodiumhypochlorite(NaClO)andhydrogenperoxide(H2O2)wereaddedandincubated24hat37°C.Thesynthetic compounds were evaluated at 10 mg/mL and 1 mg/mL. In turn, NaClO and H2O2 were tested at differentconcentrations:1/20,1/40,1/100,1/1,000,1/10,000,1/100,000.Thecontentofthewellswasdiscardedandwashed3timeswithphosphatebuffersolution(PBS)toremovenonadherentbacterialcells(planktoniccells)andfixedwithmethanoladherentcells(sessilecells).Subsequently,thesessilecellswerestainedwithcrystalvioletsolution1%for15min,airdryfor2handthebiofilmwasmeasuredat492nm inELISAreader.Control strainsproducingandnon-producingbiofilmwere included.Theexperimentswereperformedintriplicateandwererepeatedtwiceusingindependentbacterialcultures.Astatisticalanalysisoftheresultswithone-wayANOVAnonparametrictestwasconductedatsignificant levelofp<0.05.Regardingtheeffectofsyntheticcompounds,onlyDC-RN3at10mg/mLactedonstrainsofListeria,Staphylococcusandtheircombinationinhibitingbiofilmformation,liketheremainingcompoundsDC-RN5andDC-RN8wereactiveat1mg/mLonlytoeradicate biofilms of Listeria. Moreover, the disinfectants tested showed strong anti-biofilm power to all concentrationstested, with statistically significant differences about controls. Both species reached similar ability forming biofilm andinterspeciesinteractionsdidnothaveanysignificanteffecteitheronthebiofilmformingorontheantimicrobialresistanceofeachindividualspecies.1. BeraldoH,GambinoD.2004.TheWidePharmacologicalVersatilityofSemicarbazones,ThiosemicarbazonesandTheirMetalComplexes.Mini-Reviewsin

MedicinalChemistry.4:31-39.


122

EVALUATIONOFGENOTOXICACTIVITYOFTHREEAQUEOUSEXTRACTSOFNATIVEPLANTSFROMARGENTINA

MohamedAM.1,CangianoMA.2,AlcarázLE.1,SatorresSE.1,LaciarAL.1,MattanaCM.1

1ÁreaMicrobiología.2ÁreaBiología.FacultaddeQuímica,BioquímicayFarmacia.UniversidadNacionaldeSanLuis,SanLuis-Argentina.

email:cmmattan@unsl.edu.arTheArgentineanflorahasagreatdiversityofspeciesusedformedicinalpurposesfora longtime.AmongthemareAcaciacavenvar. cavenDC (commonly known as “espinillo”), Acacia furcatispina(known as “garabato negro”) and ProsopistorquataCav. Ex. Lag DC (known as “tintitaco”). These plants are used as alternative medicine for wound healing, asantisepticsand inthetreatmentofgastrointestinaldisorders.However,therearefewstudiestoknowthesafetyoftheseplantsandensuretheyarenotharmfultohealth. Inthiscontext,theaimofthisworkwastoevaluatethegenotoxicityofthreeextractsobtainedofplants,bycometassay,single-cellgelelectrophoresis.Humanbloodpoolwasobtainedbyvenouspuncturefromhealthy,adult,youngandnon-smokingvolunteers.Then,50µLofheparinisedwholebloodwasmixedwithRPMI-1640,centrifugedat2000gfor5minatroomtemperatureandincubatedfor2 h a 37ºC. Cellular viabilitywasdeterminedby exclusionmethodwith TrypanBlue. Isolated lymphocyteswere incubatedwiththeaqueousextractsofespinillo,garabatoandtintitaco,eachatthreeconcentrations:0.1,10and20mg/mLforespinilloandgarabato;and0.2,2and20mg/mLfortintitacoat37ºCduring2h.Negativeandpositivecontrolswereincluded.Thecellsuspensionswereembeddedin100µLof1%lowmeltingpointagarose(LMPA)andtheywerespreadonaslidepre-coatedwithafilmof1%normalmeltingpointagarose.Twoslideswerepreparedforeachsampleinwhichagarosecellsuspensionswereallowedtosolidifyat4ºC.Aftertheslidesweretransferredtolysissolution,pH10at4ºCfor1h.Slideswereplacedinanelectrophoresischamberexposedtoalkalifor20minbyunwindingofDNA.Later,electrophoresiswasperformedfor20minat25V/300mAandthen,theslideswereneutralizedandstainedwithgelred(Biotium).Celldamagewasdeterminedbythemethodofvisualcount,whichconsistsinobservationandclassificationthelevelofdamageinfivecategories,accordingtotheappearanceofthecomet:from0(cellswithoutmigration)to4(mostoftheDNAinthetail).Thedamageindex(DI)wascalculated.ResultswereanalyzedusingnoparametricKruskalWallistest.TherewasnodamageDNAbytheA.cavensandA.furcatispinaextracts at 0.1 and 10 mg/mL but at 20 mg/mL induced significant differences DNA primary damage in human bloodperipheral cells (p< 0.05) showing moderate level damage (category 2). On the other hand, P. torquata was stronglygenotoxicatallconcentrationstestedshowingdifferentlevelsofgenotoxicitywithincreasingconcentration.At20mg/mLtheDIwas greater even than thepositive control (DI=310±1). So it could not be used in concentrations tested. Previousstudiesfromourresearchgroup(1)haveshownthataqueousextractofA.cavens,A.furcatispinaandP.torquatahavegoodantibacterialactivitybutwasnotfoundscientificinformationonsafeuseoftheseextracts.Inthissense,ourstudyprovidesdataonthepresenceorabsenceofgenotoxicpotentialriskofproductsderivedfromtheseplants.1. MartínezM,MattanaC,SatorresS,SosaA,FuscoM,LaciarA,AlcarázL.2014.ScreeningphytochemicalandantibacterialactivityofthreeSanLuisnative

speciesbelongingattheFabaceaefamily.PharmacologyOnLine30(3):1-6


123

MEDICINALPLANTSOFCOLOMBIAWITHPOTENTIALFORTREATMENTINFECTIOUSDISEASES

Hernández-RodríguezP.1,3,PabónLC.1,3 ,RodríguezMF.2,31DepartmentofBasicSciencs,UniversidaddeLaSalle,Bogotá-Colombia;2FacultyofHealthSciences,UniversidaddeLaSalle,

Bogotá-Colombia;3BIOMIGEN,UniversidaddeLaSalle,Bogotá-Colombia.mail:[email protected]

Infectiousdiseaseshavegeneratedproblemsassociatedwithmorbidity,aftermathandmortality, inhumansand inanimalspecies, affectingpublichealthand livestockproduction.Currently, about 25%ofdeathsworldwidearedirectly related toinfectiousdisease,occurringmainlyintheregionoftheAmericaswith44%.Additionally, ithasbeenreportedthat57.8%ofthe events of emergingdiseases areof bacterial origin, beingmostly of newpathogens (40%) and antimicrobial resistantmicroorganisms(31%).Inthissense,theuseofplantsfortreatmentofcommoninfectiousdiseaseshasbeenknownformanyyears and therefore plant species have been used as a source of drugs for modern medicine, either by providing purecompounds or as prototypes modified to synthesis of new drugs. To work with natural products based on traditionalknowledge, ithasbeenproposedasaneffectivemethod for the identificationofmedicinalplants, extractsandbioactiveprinciples, reducing empiricism and improving the chances of success in drug development. 2,404 medicinal species forColombia,where1,656arenativeto theNeotropicsandonly 12.5%havereferencesthatshowtheir traditional therapeuticuseareknown;allowingestablishthatthecountryhas littleresearchonthesubjectandhasnotbeenratedthisbotanicalheritageasahealthalternative.Therefore, the objective of this work was to increase the phytochemical knowledge of some medicinal plants of theColombian biodiversity. Eleven plants were selected with the greatest factor consensus for the treatment of infectiousdiseases among herbalists subsequently extracts of these plants were subjected to evaluation of antimicrobial activityagainstPseudomonas aeruginosa,Escherichia coli,Staphylococcus aureus andStreptococcuspneumoniae themethodofHT-spoti. During the study it found that 80% of the plants showed antimicrobial activity against at least one of the selectedbacteria,Kohleriaspicatabeingtheonlyplantthatpresentactivityagainstfourmicroorganisms.Inaddition,thisspecieswithArnica montana, Equisetum giganteum and Levisticum officinale were those with the greatest potential to possessantimicrobial MIC values below 4 mg/mL. This project is presented as the first contribution to chemical and biologicalknowledgeofsomemedicinalspeciesofColombiandiversity,contributingtotheassessmentofthebotanicalheritageasaresourceforthedevelopmentofnewinnovativetherapeuticagents.1. BernalH,MartínezH,LondoñoC,MolanoM,QuevedoG,Vasquez,C.(2011).Pautasparaelconocimiento,conservaciónyusosostenibledelasplantas

medicinalesnativasenColombia.EstrategiaNacionalparalaConservacióndePlantas.Bogotá:EdiprintLtda.,primeraedición.1-2272. CarlsonT.(2002).Medicalethnobotanicalresearchasamethodtoidentifybioactiveplantstotreatinfectiousdiseases.AdvancesinPhytomedicine,1:

45-53.3. EdelsteinM,HeymannD,GieseckeJ,WeinbergJ.(2012)ValidityofInternationalHealthRegulationsinReportingEmergingInfectiousDiseases.Emerg

InfecDis.18,7:1115-20.4. JeevanA,BhakshuL,VenkataR. (2004) Invitroantimicrobial activityof certainmedicinalplants fromEasternGhats, India,used for skindiseases. J

Ethnopharmacol.90:353-7.5. LouwC,RegnierT,KorstenL. (2002).MedicinalbulbousplantsofSouthAfricaandtheir traditional relevance in thecontrolof infectiousdiseases. J

Ethnopharmacol.82:147-54.6. Mgole S, Ngassapa O, Pieters L, Vlietinck A. (2007). Ethnopharmacological survey of the Bunda district, Tanzania: Plants used to treat infectious

diseases.JEthnopharmacol,113(3):457-70.


124

INHIBITORYCAPACITYOFAQUEUSOREGANOEXTRACT(ORIGANUMVULGAREL.)ON

MICROORGANISMS(S.AUREUS,E.COLIANDSALMONELLATYPHIMURIUM)ANDIT´SEFFECTONTHESHELFLIFEOFAGEL-TYPEMADEOFPROTEINFROMGIANTSQUID(DOSIDICUSGIGAS).

Dublán-GarcíaO.1 ,López-MedinaFA.1,Gómez-OlivánLM.1,López-MartínezLX.2,Díaz-BanderaD.1,

Cira-ChávezLA.3,Hernández-NavarroMD.1,Jáuregui-RodríguezB.1¹UniversidadAutónomadelEstadodeMéxico,FacultaddeQuímica,CerrilloPiedrasBlancas,Toluca-México;2Centrode

InvestigaciónenAlimentaciónyDesarrollo,A.C.UnidadCuliacán,Culiacán(Sinaloa)-México;3InstitutoTecnológicodeSonora,CiudadObregón(Sonora)-México.

email:[email protected];odublang@uaemex.mxTheuseofnaturalextractsinfoodapplicationsandespeciallyinhighlyperishableproductsisbasedoncertaincharacteristicssuch as availability, functional attributes, antimicrobial properties, color contribution and improvement of flavor. Studieshaveshown,itcanbeusedasadditives,becauseofhisantioxidantsandantimicrobialeffects.Organicextractsfromoregano(Origanum vulgare L.) had shown antimicrobial effects againstE. coli, S. aureus andSalmonella typhimurium, the principalcomponentsofthisextractsarenon-polarandhasinteractionswiththecellwall.Theinformationfromaqueusextractsfromthissourcerecentlyhadgrownandit´sbeenprovedondifferentmicroorganismsanddifferentproducts,butnotspecificallygiantsquidproducts.Ourgoalistheuseofaqueusextractsformoreganointheproductionofagel-typeproductmadeofproteinextractedfromgiantsquid(Dosidicusgigas)asanalternativetoextendtheshelflifeofthisproduct.Extractionofthecomponentsfromoreganowasmadeusingabufferofphosphates(100mMpH6.0,withNaCl0.6M)astheextraction medium, using a ratio 1:10 (oregano: solution) and 30 minutes of extraction in continuous agitation. TheantimicrobialeffectagainstmicroorganismswasdeterminedbytheBauer-Kirbymethod.Addedconcentrationofextracttothegel-typewasdeterminedbyflavorandcolorcontribution,withamaximumof5%(v/w).Gel-typeproductswereevaluatedinthreecharacteristics:sensoryattributes(flavorandcolor),structureattributes(hardness,elasticityandcontinuousaspect)andmicrobiologyattributes(totalcoliformsbyMPNmethod).The oregano extract obtained shown antimicrobial characteristics againstS. aureusATCC 25922 (17mm) andE. coliATCC25923(12mm),butnotagainstSalmonellatyphimurium(fromclinicalsource).Fourconcentrationswereevaluated,0%,1%,3%and 5%; as the concentrationwas higher, the sensorial, structural andmicrobiological characteristicsweremaintained, asshownintable.1Concentration(v/w)

0% 1% 3% 5% Table. 1- Percentage of extract added to the gel-type product and the days ofstoragethatcharacteristicsweremaintained

Daysofstorage 7 9 13 17Microbiological analysis until the 20th daydidn´t showngrowthofmicroorganisms that use lactose and canproduceCO2,determinedinthefirststepofthedeterminationoftotalcoliformsbyMPNmethod.Itcanbeduetotheasepticelaborationprocessandthemanufacturingsteps.The increase in days of storage and maintenance of characteristics can be attributed to the growth inhibition ofmicroorganisms related todetrimentaleffects,and the interactionsbetweenextract componentsandproteinsof thegel-type.1. RodríguezSauceda,ElviaNereyda,USODEAGENTESANTIMICROBIANOSNATURALESENLACONSERVACIÓNDEFRUTASYHORTALIZASR.Ximhai[en

linea]2011,7(Enero-Abril):[Fechadeconsulta:7dejuniode2016]Disponibleen:<http://www.redalyc.org/articulo.oa?id=46116742014>ISSN1665-0441.2. Gómez-Estaca,Montero,Fernández-Martín,AlemányGómez-Guillén.Physicalandchemicalpropertiesoftuna-skinandbovine-hidegelatinfilmswith

addedaqueousoreganoandrosemaryextracts.JournalofFoodHydrocolloids,2009,23:1334-1341.


125

ERYSIMUMCRASSIPESASNEGLECTEDMEDICINALPLANT:STORAGEATEARLYBRONZEAGEKÜLLÜOBA/TURKEY

ÇizerO. ,GuseJ.

Eberhard-KarlsUniversität,InstitutefürNaturwissenschaftlicheArchäologie,Tübingen-Germanyemail:[email protected]

Atpresent,inmodernmedicine,numerousspeciesofErysimumarewellknownascontainingchemicalcompoundsinformofthesteroidglycosidesusedagainstcardiacdiseases,andessentialoilswithexpectorant,laxativeanddiureticeffects(Baueretal.1960,Maslennikovaetal.1967,Umarovaetal.1977,Makarevichetal.1993,Leietal.2000,Jawlaetal.2010).Essentialoils likedillapiolearedetectedrecently inothermedicinalErysimumspeciesasanti-inflammatorycompounds (AkhgarandRoknabadi 2015). As a part of Iranio-Turanian vegetation, species of genus Erysimum, E. crassipes is widely distributed inAnatolia and in the Near East (Davis 1965-1988). Despite the extended use of numerous Erysimum species for medicinalpurposes,ethno-medicinaluseofE.crassipesismentionedsolelyfromtheHighlandsofNorthernJordan(OranandEl-Aisawi2014).Archaeologically,atanEarlyBronzeAgesiteinTurkey,Erysimumcrassipesfoundinasmallpotwithca.2,5millionseedcounts,constitutesthefirstrecordedfindintheWorld(Cizer2015).EssentialoilandglycosideprofileofmodernE.crassipesseedscanbeobtainedusingdifferentchromatographicandspectrometricmethods(Es-Safietal.2005,Marchetal.2006).The seeds are collected by a plant taxonomist from well documented accession in Turkey. Identification and definitionchemical compoundsofErysimumcrassipes and their effectsonhumanbodymayenlighten thepossiblemedicinaluseofErysimumcrassipes.Suchkindofstudywillnotonlybecruciallyimportantfortheplant’sarchaeologicalmeaning,butalsoforthemodernbiochemistryandmedicine.1. Akhgar,M.R.andRoknabadi,S.(2015)CompositionoftheessentialofErysimumcrassicaule.ChemistryofNaturalCompounds51(3):575-576.2. Bauer, S.,OrszághŠ., Bauerová,O.,Mokrý, J.,Masler, L. Šikl,D. andTomko, J. (1960) Isolierung vonErysimosidundHevetikosid ausgraublätrigem

Hederich(ErysimumcanascensRoth.)durchGegenstromverteilung.PlantaMed:8(2):145-151.3. Cizer,Ö. (2015)Archaeobotanical investigations of plant cultivation and husbandry practices at the Early BronzeAge settlementKüllüoba inWest-

CentralTurkey:Considerationsonenvironment,climateandeconomy.BARInternationalSeries2766,Oxford.4. Davis,P.H.(eds.)1965-1988.FloraofTurkeyandtheeastAegeanIslands.(Vol1.Crucifereae)EdinburgUniversityPress.5. Es-SafiN.-E.,Kerhoas,L.,Einhorn,J.,Paul-Henri,D.(2005)ApplicationofESI/MS,CID/MSandtandemMS/MStothefragmentationstudyoferiodictyol7-

O-glucosyl-(→2)-glucosideandluteolin7-O-glucosyl-(1→2)-glucoside.InternationalJournalofMassSpectrometry247:93–100.6. Jawla,S.,Mogla,O.P.andKumar,Y.(2010)HerbalRemediesforAsthma:AnOverviewJournalofChemicalandPharmaceuticalResearch2(1):267-2727. Lei,H-Z.,Yahara,S.,Nohara,T.,Tai,B.-S.,Xiong,J.-Z.,andMa,Y.-L.(2000)CardiacGlycosidesfromErysimumcheiranthoides.Chemical&pharmaceutical

bulletin48(2):290-292.8. Makarevich, I. F., Zhernoklev, K. V., Slyusarskaya, T. V. andYarmolenko,G.N. (1993)CardiacGlycosides ofErysimum contractumHI. Glucocanescein

ChemistryofNaturalCompounds29(5):488-489.9. March,R.E.,Lewars,E.G.,Stadey,C.J.,Miao,X.-S.,Zhao,X.andMetcalfe,C.D.(2006)Acomparisonofflavonoidglycosidesbyelectrospraytandemmass

spectrometry.InternationalJournalofMassSpectrometry248(61–85).10. Maslennikova,V.A.,Genkina,G.L.,Umarova,R.U.,Navruzova,A.M.andAbubakirov,N.K.(1967)GlycosidesofErysimumVI.CardenolidesofE.altaicum,

E.cuspidatum,E.diffusum,E.marschallianum,E.nuratensae,E.violascens.KhimiyaPrirodnykhSoedinenii(ChemistryofNaturalCompounds)3(2):173-178.11. Oran, S. and Al-Eisawi, D. (2014) Medicinal plants in the high mountains of northern Jordan. International Journal of Biodiversity and Conservation:

6(6):436-443.12. Umarova,R.U.,Maslennikova,V.A.andAbubakirov,N.K.(1977)steroidcompoundsofErysimumXI.sterolsofErysimumdiffusumandE.euspidatum

ChemistryofNaturalCompounds13(6):692-693.


126

LEVELSOFHEAVYMETALSINOCTOPUS(OCTOPUSVULGARIS)FROMSOUTHTYRRHENIAN:

PRELIMINARYRESULTS

ArianoA.1,D’AmbolaM.1,VassalloA.2,SmaldoneG.1,VelottoS.1,SeverinoL.1

1UniversityofNaplesFedericoII,DepartmentofVeterinaryMedicineandAnimalProduction,DivisionofToxicology,Naples-Italy;2UniversityofBasilicata,DepartmentofScience,Potenza-Italy.

email:[email protected](Octopusvulgaris)isavoraciouspredatornormallydistributedonrocky,sandyandmuddybottoms.Itis characterizedby fastgrowth rates anda short lifespanandknown for its ability to accumulatehigh levelsof elementsessentialtometabolicfunctionsandnonessentialelements.Ingeneral,feedingisconsideredtobetheprimarypathwayfortraceelementbioaccumulationincephalopodsandsecondseawater.AlthoughO.vulgarisisbenthic,livingindirectcontactwith the substratum, which represents another possible pathway for trace element accumulation. The objectives of thepresentstudyweretoevaluateCd,PbandHglevelsinthemuscleofoctopus(Octopusvulgaris)collectedfromthesouthernTyrrhenianSeainItalyandtoassessthehealthriskrelatedtohumanconsumption.Samples (n.12) ofOctopus vulgariswere caught along the coast of the southern Tyrrhenian Sea between September andDecember 2015. Organisms were stored in individual plastic bags and immediately frozen onboard in order to minimizemobilizationofmetalsbetweenorgans/tissues.Sampleswerekeptat-25°Cforamaximumof2weeksbeforedissection.Inthelaboratory,armandmantlewasdissectedexcludingtheskin.Weight,mantlelengthandsexweredeterminedforeachindividualandwassubsequentlyhomogenizedbymeansofa laboratorymixer formetalanalysis.Aliquotsofeachsamplewere digested in ultrapure 65% HNO3 and H2O2 in a microwave digestion system. Pb, Cd and Hg concentrations weredeterminedbyatomicabsorptionspectrometer(GF-AAS).Cd and Hg concentrations were found very low in all samples of octopus muscles (Cd: 0.011±0.018 mg/kg w.w.; Hg:0.017±0.040mg/kgw.w.)andwerebelowthelegallimitforhumanconsumption.TheconcentrationofPbwasgenerallyhighinsamplesofmuscle(0.851±0.662mg/kgw.w.)andalltestedsampleswereabovethemaximumconcentrationlevel(RegCE1881/2006)excludingthisproducttohumanconsumption.Toestablishpossiblehumanhealthimplicationsrelatedtoconsumptionofoctopus,thePbestimateweeklyintakes(ISMEA,2010)weresubsequentlycomparedwiththeprovisionaltolerableweekly intake(PTWI)of25µg/kgofbodyweight(EFSA,2010).Considering the level of Pb, the consumption of octopus muscle may increase Pb intake, but it would not contributesignificantly to thePTWI. Incontrast,mayreachhighEWIvalues in theheavyconsumerofoctopus,whentheothermaincontributorstodietaryPbintakewereincludedintheexposureassessment.The preliminary results obtained in the current study showed the presence of Pb in all samples analyzed, underlying itspresenceintheenvironment.MonitoringstudiesonheavymetalsinOctopusvulgarisandotherspeciesofthemarinefoodchaininagreaternumberwillprovidemoredetailedinformationofthehumanexposuretoPbinthisarea.1. Bustamante,P.,Cosson,R.P.,Gallien, I., Caurant, F.,Miramand,P., 2002.Cadmiumdetoxificationprocesses in thedigestiveglandof cephalopods in

relationtoaccumulatedcadmiumconcentrations.Mar.Environ.Res.53,227–242. Miramand,P.,Bentley,D., 1992.Concentrationanddistributionofheavymetals in tissuesof twocephalopods,Eledone cirrhosa andSepiaofficinalis,

fromtheFrenchCoastoftheEnglishChannel.Mar.Biol.114,407–414.3. Storelli,M.,Marcotrigiano,G.,1999.CadmiumandtotalmercuryinsomecephalopodsfromtheSouthAdriaticSea(Italy).FoodAddit.Contam.16,261–

265.


127

PHYSICOCHEMICALEVALUATIONOFTHEOLEORESINFROMCopaiferapauperaCOLLECTED

SEASONALLYINACRE,BRAZIL

NakamuraMJ.1,VaucherAS.2,SianiAC.1 ,MazzeiJL.1,GuarinoESG.3,FreitasO.4,RamosMFS.2 1DepartamentodeProdutosNaturais,InstitutodeTecnologiaemFármacos,FundaçãoOswaldoCruz,RiodeJaneiro-Brazl;

2DepartamentodeFármacoseMedicamentos,FaculdadedeFarmácia,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro-Brazil;3CentrodePesquisaAgroflorestaldoAcre,Acre-Brazil;4DepartamentodeCiênciasFarmacêuticas,FaculdadedeCiências

FarmacêuticasdeRibeirãoPreto,SãoPaulo-Brazi.lemail:[email protected],[email protected]

Thecopaibaoleoresinhasbeenincreasinglycommercializedworldwideasavaluablenon-timberforestproductfromtherainforests.Effortshavebeendriventowardassessingchemicalstandardstoestablishthequalityandaggregatevaluetothisasrawmaterial.InAcreState,Brazil,theoleoresincollectorscategorizethecopaibatreesaswhite,yellow,redandblacktypes,according to thebark color, regardless thebotanical species.Our goalwas to assess the terpene composition, refractiveindex,densityandacidic indexvariations in theoleoresin,according toaseasonalcollectionofCopaiferapauperaHerzog,whichembracesallthetypesabove.Oleoresins collected from forty-eightGPS-marked individualsofC.paupera in theEmbrapaExperimental Fieldarea, atRioBranco-Porto Velho road, Acre State, Brazil. Samples and color type were duly registered. Samples (15-50 ml) werecentrifuged (2100 rcf, 30 min, 10 °C) to eliminate debris. Densities were measured in a pre-calibrated Mettler digitaldensimeter. Refractive indices (RI) were determined using an Abbe refractometer. Acidic indices (AI) were obtainedaccording by Pharmacopeia titration method. Chemical compositionss were assessed by GC-MS using a HP 6890NchromatographequippedwithaHP-5MScolumn(30m×0.32mm×0.25μm);heliumflow0.5ml/min; 1:20split ratiomode;oventemperaturefrom70˚C(held5minutes)to290˚Cat4˚C/min(held10minutes);ionsourceat250˚C;EI70eV.Samplealiquots(1μLfrom5mg/mlCH2Cl2solution)werepreviouslymethylatedwithdiazomethane.ConstituentswereidentifiedbycomparingmassspectrastoredinWiley59943Bdatabasisandretentionindex.RIvariedfrom1.45-1.54anddensityfrom0.956a1.056g/ml(statisticallynon-significant,p>0.05)foralltheoleoresinsamples.Acidicindexrangedfrom30-100mgKOH/g.Allsampleswereconstitutedbymixturesofsesquiterpeneandditerpeneacids.Mostfrequentcompounds,β-caryophyllene(20%avg,maximumproducedinApril)appearedin77%andcopalicacid(3%avg)in84%overallsamples.Caryophyllane/humulane-typesesquiterpeneswerepredominant inthered-,black-andyellow-typecopaiba,whilst elemane/cadinane-typewere predominant in thewhite copaiba oleoresin, as corroborated by Permanovastatistical analysis. These two tendencies reflects competitive biosynthetic routes in the plant. Danielic acid was foundabundantandexclusivelyinthewhite-typecopaiba.Thislatteroleoresinistheleastpreferredbybuyers,afactthatmayberelated to lowerβ-caryophyllenecontents.Valuesobtained forRI,densityandAIwere tentatively related to thechemicalcompositionandcomparedwiththoseobtainedtoediblefatsoils.SomepatternstodetectadulterationinoleoresinsfromC.pauperadestinedtocommercializationwerepreliminarilyinferred.Acknowledgments:Finanacialsupport:EmbrapaAcre(Macroprogram3)1. AdamsRP.Identificationofessentialoilcomponentsbygaschromatography–massspectrometry.4thed.Illinois:AlluredPubl.Corp.,2007.2. BarbosaP.etal.Influenceofabioticfactorsonthechemicalcompositionofcopaibaoil(CopaiferamultijugaHayne):soilcomposition,seasonalityand

diameteratbreastheight.JBrazChemSoc23,1823-1833(2012).3. FarmacopeiaBrasileira,1988-1996.4aed.,SãoPaulo:Atheneu.


128

ACETICACIDBACTERIAANDCELLULOSEPRODUCTION:STRAINSELECTIONANDPOLYMERCHARACTERIZATION

GulloM.1 ,ZanichelliG.1,SolaA.2,MontorsiM.3,MessoriM.2,GiudiciP.1

1DepartmentofLifeScience,UniversityofModenaandReggioEmilia,ReggioEmilia-Italy;²DepartmentofEngineering“EnzoFerrari”,UniversityofModenaandReggioEmilia,Modena-Italy;3DepartmentofEngineeringSciencesandMethods,Universityof

ModenaandReggioEmilia,ReggioEmilia-Italy.email:[email protected]

Bacterial cellulose (BC) is an insoluble, extracellular polysaccharide that is producedby different bacteria species. It is anattractivebiomaterialhavingpotentialuseinmanyfieldsasnaturalpolymerorincompositematerials.AmongBCproducingaceticacidbacteria(AAB),Komagataeibacterxylinusistherepresentativespeciesofcellulosesynthesis.Inthisstudy50AABisolatedfromKombuchatea,abeverageobtainedbythefermentationofteathroughacultureofyeastsandAAB,werescreenedfortheirabilitytoproduceBC.Candidatestrainswerepre-selectedonthebasisofqualitativetraitssuchas rapidgrowthonbasic culturemedia containingglucose andability toproduceBCas a layerordeposit in testingtubes. Quantitative assays to select the best candidate for optimized BC production in different culture conditions andculturemedia(standardGYmediumandtheoptimizedmedium(ET))wereconductedbymicroscaletests.ProcessscalabilityandoptimizationofBCproductionweretestedinbatchusingthestandardGYmediumandtheoptimizedone(ET).Then,theselectedBCmembranewascharacterizedfromamicrostructuralandthermo-mechanicalpointofview.SelectedstrainsshowedhighvariabilityinBCproduction.StrainK2G30achievedthehighestBCyieldinalltestedconditionsandthereforeitwasfullycharacterizedandidentified.DifferencesinBCyieldduetocarbonsourceswerestated.Thepoolofstrains collected and typed are amicrobial resource to studymechanisms of cellulose synthesis and to develop tailoredcelluloseprocesses;theyalsorepresentastartingpointforafurtheroptimizationinordertoincreasethecelluloseyield.TheSEMobservationoftheBCmembraneproducedbyK2G30highlightedahighdegreeofporosityduetotheultrafine3Dnetwork of BC nanofibres. Moreover, it did not show any difference between the two sides of the membrane, whichpresented an average thickness of about 20μm. TheXRD analysis estimated a degreeof crystallinity (Segal equation) ofabout80%.Thewaterabsorptionrate(WAR)testsprovedtheexcellentabilityofthepolymertoabsorbwater,sincetheBCproducedbyK2G30achievedaWARofabout400%in48hours.TheBCmembraneproducedbyK2G30,onaccountof itsmicrostructure,promising thermo-mechanicalbehaviorandhighwater-absorptionability, isan idealcandidatefortheproductionofbiomedicaldevices,especiallyforskinrepair,andfoodadditives.1. MamloukD,GulloM,2013.Aceticacidbacteria:physiologyandcarbonsourcesoxidation,Indianjournalofmicrobiology53(4),377-3842. X.Feng,N.Ullah,X.Wang,X.Sun,C.Li,Y.Bai,L.Chen,Z.Li,CharacterizationofbacterialcellulosebyGluconacetobacterhanseniiCGMCC3917,Journalof

FoodScience80(2015)E2217–E2227


129

AMARANTHGRAIN(AMARANTHUSHYPOCHONDRIACUS)ASSUSTAINABLEALTERNATIVEPROTEIN

SOURCESTOMEAT

Vilchis-PerezA. ,Melo-RuízV. ,García-SerraldeL.,Gazga-UriosteC.,Diaz-GarcíaR.MetropolitanAutonomousUniversity,DelegaciónCoyoacán,MéxicoCity-México


Amaranthgrain,nativewildplantfromLatinAmericawascultivatedbytheAztecssinceancienttimesandconsumedaspartoftheirdailydietandalsousedinreligiousceremonies.AmaranthcorpswerebannedbySpaniardsupontheirconquestofMexico, however the plant has continued to grow as weed since that time, and it´s genetic base has beenmaintained.NowadaysseveralspeciesofAmaranthuscaudatus,AmaranthuscrudentsandAmaranthushypocondriacusarestillcultivatedin some states of Mexico. Currently the main protein source for most Mexican diets is meat, which due to it´s cost ofproduction,meatisnotavailabletoallsocialgroups.ThereforeothersustainablesourcesofproteinsuchasAmaranthplantshouldbeinvestigated.Theaimofthisstudyistoevaluatenutritionalvalueofmacronutrientsthatcanbeuseassustainablealternativeproteinsourcesfocusonpartialreplacementofmeatinahealthybalanceddiets.MacronutrientsofAmaranthushypocondriacusgrainswereassessedindrybasis,accordingtoAOAC1995methods.Sampleswereobtainedearlysummer2015atMexicoState.Thesamplesweredriedat60°Cfor24handmilledtoafinepowdertoanalyze moisture, proteins, lipids, minerals, fibre and soluble carbohydrates. Proteins were calculated from the nitrogencontent by the Kjeldahlmethod using the conversion factor 6.25. Lipidswere determined by an extraction processwithpetroleumetherat120°Cfor6husingaGoldfishapparatus,mineralswereanalyzedafterincineratingthesampleat600°Cinamuffle furance for 6h.Crude Fibreby acid hydrolysis, followedby alkalinehydrolysis and soluble carbohydratesby [100-(protein+lipids+minerals+crudefibre)].Data obtained were: moisture 3.8%; proteins 14.80%; lipids 6.5%; minerals 2.22%; crude fibre 3.15%; soluble carbohydrates73.33%.Dataobtainedmightvaryduetobioticandabioticenvironmentconditions.ConsumptionofAmaranthusgrainisnotinaregulardietduetothelackof informationaboutthebenefitsthattheyprovidetohealth.However,Amaranthusplantofferanalternativesourceofproteintomeat,additionallygrainscontainrichsourcesoffattyacidsandmineralsrecognizedfortheirpotentialhealthbenefits.1. AOAC,1995.OfficialMethodsofAnalysis.15thed,AssociationofOfficialAnalyticalchemist,Washington,DC.2. ArqueologíaMexicana,2002.CocinaPrehispánicaRecetario.No.12,México,p.101.3. Castelló-Yturbide,T,1986.PresenciadelaComidaPrehispánica.FomentoCulturalBanamexA.C,México,p.193.4. Sahagún,F.B.,1980.CódiceFlorentino.LibroIV,f.72v.México.5. Suzanne-Nielsen,S,2010.FoodAnalysisLaboratoryManual.Springer,Secondedition,USA,p177.


130

AGRONOMICANDPHYTOCHEMICALSTUDYOFSICILIANSALVIASCLAREAL.

TuttolomondoT.1,NapoliE.2,RubertoG.2,LetoC.1,VirgaG.1,GennaroMC.1,LicataM.1,LaBellaS.1 ¹UniversitàdegliStudidiPalermo,DepartmentofAgriculturalandForestSciences,Palermo-Italy;²IstitutodelCNRdiChimica

Biomolecolare,Catania-Italy.email:[email protected]

SalviasclareaL. isabiennialxerophytebelongingtotheLamiaceaefamily,oneofthemostabundantplant familiesoftheMediterraneanflora.ManyoftheMediterranean’smedicinalandaromaticplantsbelongtothisfamily(Kintziosetal.,2000;Licataetal.,2015).;).ItistypicallyfoundintheMediterraneanBasinofEuropeandAfricaasfarastheAtlanticOcean,andiscultivatedforitsessentialoil(EO)inFrance,Bulgaria,Russia,USA,andChina(Lawrence,1992).TheEOishighlyvaluedinthecosmeticsandfoodindustries(Jibaoetal.,2005,Yaseenetal2015).Itwastraditionallyusedformedicinalpurposesandhasshownclearantimicrobial, anti-inflammatory,antioxidant,anti-depressiveand insecticideproperties. (Azikhanietal., 2016;Yanaetal.,2013;Mounaetal.,2012;Geunetal.,2010).BibliographicalstudiesandexploratorytripstoareasofSicilyledtotheidentificationof3siteswherewildpopulationsofS.sclareawerefoundgrowing:AlcaraLiFusi(ME),S.StefanoQuisquina(AG)andPrizzi(PA).InAugust2008,seedwascollectedfromeachofthepopulations.SeedplantingwascarriedoutinMarch2009andtheplantlingsweremovedtothefieldinthesecondhalfofMay.Layoutdensitywas0.75plants/m2.Harvestingwaseffectuatedduringthe“balsamicphase”(periodofhighestconcentrationofactiveingredients),andthemainbiometricandyieldparameterswereobserved.GC-MSwascarriedoutontheinflorescenceEO.Thebiotypesshowedexcellentvegetativedevelopment,reachingfull-floweringstagewithnospecificcropcare.Averagedrybiomassproductionwas412.8±167.6gin2009and193.1±24.6gin2010.SimilarresultswerefoundforEOyieldsfromtheinflorescences(1.20–2.25%in2009and1.5–2.6%in2010).TheAlcaraLiFusiecotypeswerethemostproductive.GCanalysisevidenced76compounds,representingapprox.98%ofthechemicalprofile(Adams,2007).Oxygenatedmonoterpenesweremostabundant(67-79%),andofthose, linalylacetateand linalool,detectedatarateof2:1,representedalmost60%ofthetotal;α-Terpineolwasthethirdmostabundantcomponent.AlsoworthnotingistheconspicuouspresenceofgermacreneDfromamongstthesesquiterpenes.Theabovesubdivisionwasfoundtobesimilarforallofthesamplesanalysed;thissamesimilarity was found between the components present in minor quantities. The biotypes analysed belong to the linalylacetate/linalool chemotype. S. sclarea essential oil provides both an ecological function, connected to the attraction of anumberofpollinators,andarangeofbiologicalactivities,suchasantimicrobialactionagainstnumerousstrainsofbacteria(Azikhanietal.,2016;Yaseenetal.,2015).1. Adams,R.,2007.IdentificationofEssentialOilComponentsbyGasChromatography/MassSpectroscopy.CarolStream,AlluredPubl.Co,Illinois.2. Azikhani,M., Tooryan, F., Azikhani,M., 2016. Inhibitory potential of Salvia sclarea andOcimum basilicum against chemical andmicrobial spoilage in

cheese.JournalofFoodSafety,36(1),109-119.3. Geun,H.S.,2010.Antidepressant-likeeffectofSalviasclareaisexplainedbymodulationofdopamineactivitiesinrats.JournalofEthnopharmacology130,

pp187-190.4. Jibao,C.,2005.Comparativeanalysisofclarysage(S.sclareaL.)oilvolatilesbyGC–FTIRandGC–MS.FoodChemistry,pp401-407.5. Kintzios,S.E.,2000.Sage-ThegenusSalvia.HarwoodAmsterdam,Netherland.6. Lawrence,B.M.,1992.Advancesinlabiatescience,R.M.Harley&T.Reynolds,Kew,UK:RoyalBotanicalGardens,pp.399–401.7. Licata,M.,Tuttolomondo,T.,Dugo,G.,Ruberto,G., Leto,C.,Napoli, E.M.,Rando,R.,RitaFede,M.,Virga,G., Leone,R.,LaBella,S. (2015).Studyof

quantitativeandqualitativevariationsinessentialoilsofSicilianoreganobiotypes.JournalofEssentialOilResearch27,293-306.8. Mouna,B.T.,2012.Fattyacids,phenolicchangesandantioxidantactivityofclarysage(SalviasclareaL.)rosette leavesgrownundersalineconditions.

IndustrialCropsandProducts38,pp58-63.9. Yana,H.,2013.ChemicalcompositionandantifungalactivityofessentialoilofSalviasclareaL.fromBulgariaagainstclinicalisolatesofCandidaspecies.

JournalofBioScienceandBiotechnology2(1),pp39-44.10. Yaseen,M.,Kumar,B.,Ram,D.,Singh,M.,Anand,S.,Yadav,H.K.,Samad,A.,2015.Agromorphological,chemicalandgeneticvariabilitystudiesforyield

assessmentinclarysage(SalviasclareaL.).IndustrialCropsandProducts77,pp640-647.


131

EVALUATIONOFTHEHYPOLIPEMIANTANDHYPOGLUCEMIANTEFFECTOFABASEPOWDERTOELABORATEFUNCTIONALFOODOBTAINEDFROMTOMATOPEEL(Physalisixocarpa)INMICE

ZambranoE.,PérezG.,PérezPasténR.

InstitutoPolitécnicoNacional,EscuelaNacionaldeCienciasBiológicas,UnidadProfesionalAdolfoLópezMateos,CiudaddeMéxico-México.

email:[email protected] is characterized by abnormalities in blood lipid levels, such as cholesterol, triglycerides, HDL high densitylipoproteinsandlowdensitylipoproteinsLDL.Havingnocontrolinthisconditionmayoccurdiabetes,whichistheincreasedbloodglucose,whichresultsinthedevelopmentofvariouscomplications,consideringoneoftheleadingcausesofmortalityin Mexico; so the search for alternative treatments to control these conditions is necessary. In this research thehypoglycemicand lipid-loweringactivityofabasepowderwasevaluatedtodevelopfunctional foodderivedfromtomatopeel;bythepresenceofsubstancesinitspeel,asacilsacarosas,itiscreditedwithlipid-loweringandhypoglycemiceffect.Male ICR mice were used (25-35 g body weight) and were acclimated for 7 days, with 12 hours light/dark periods,temperatures22°C,withwaterandfoodadlibitum; theanimalswererandomlyassignedin6groups(n=6),1)normaldiet(N),2)hypercholesterolemicdiet(H),3)normaldiet+tomatopeel500mg/kg(N+CT),4)hypercholesterolemicdiet+tomatopeel125mg/kg(H+CT),5)hypercholesterolemicdiet+tomatopeel250mg/kg(H+CT),6)hypercholesterolemicdiet+tomatopeel500mg/kg(H+CT),thetreatmentswereadministrateddailyand intragastricallyfor7days. .Attheendofthestudy,ablood sample was obtained, and then by centrifugation serum was obtained, then mice were sacrificed following therecommendationsof theNOM-ZOO-062.Fromtheserumobtained weredeterminedserumglucoseand lipidicprofile,byusingvitaLabSelectraII,LDL-CwascalculatedusingtheFriedewaldformula.Theresultsobtained,shown inrelationtotheglucoseconcentrationasignificantdifferencebetweenthegroupH+CT500mg / kg compared to theH group; in relation to cholesterol, therewas a significant difference between the groupH+CT250mg/kgcomparedtotheHgroup;inrelationtotriglyceridestherewasasignificantdifferencebetweenH+CT500mg/kggroup compared to theHgroup; in relation to LDL-C therewas a significantdifferencebetweengroupH+CT 500mg / kgcompared to theH group. It is concluded that the powder obtained from the tomato peel has a hypoglycemic and lipidloweringactivity,withthedoseof500mg/kgthemosteffective,suggestingthatsuchactivityisbecausethetomatopeelinhibitstheabsorptionofcholesterolinthebody.1. Armstrong E.J. and CohenDE. Pharmacology of cholesterol and lipoprotein metabolism. In. “Priciples of Pharmacology” Golan DE, Tashjian AH.,

ArmstrongEJ,ArmstrongAW.(eds),Lippincott,2005,417-438.2. RamosI.,etal.Diabetes,estrésoxidativoyantioxidantes.Investigaciónensalud,volVIII,núm001.3. IllnaitP.J.Ladislipidemiaenelpacientediabético.ParteI:Bioquímicapatológica.RevCubanaMedGenIntegr1997;13(4):372-377.4. Meléndez E., et al. Efectohipoglucemiantedel extractode la cáscarade tomate (physalis philadelphica lam) en ratones tratados con aloxana:Rev.

México.Cien.Farm.31(2),2000,10-12.


132

BIOACTIVECOMPOUNDSANDANTIOXIDANTACTIVITY

OFARTOCARPUSHETEROPHYLLUSLAM

Figueroa-IbarrraLM.1 ,Hernández-NavarroMD.1 ,Chamorro-CevallosGA.2,Hernández-OrtegaMM.1,Gil-EscamillaJO.1,Jáuregui-RodríguezB.1,Gómez-OlivánLM.1,Dublán-GarcíaO.1,Islas-FloresH.1¹DepartmentofPharmacy,FacultyofChemistry,AutonomousUniversityofStateMexico,Toluca(StateofMexico)-Mexico;

²Laboratoryofpreclinicaltoxicology,NationalSchoolofBiologicalSciences.MexicoD.F.-Mexicoemail:[email protected],[email protected]

ArtocarpusheterophyllusisanexoticfruitnativeofAsia,commonlyknownasjak,whichhasbeenusedintraditionalmedicalasatreatmentfordiabetesmellitus,anti-inflammatory,anthelmintic,antibacterialandhypoglycemic.InMexicoithasbeencultivated and commercialize formany years ago, as juice, jamor fresh fruit.However, in last years it hasbeenobjectofstudiesbecauseitsbiologicalproperties.Theaimofthepresentstudywastoquantifiedtheamountofbioactivecompoundssuchastotalphenolicandtotalflavonoidcontentsofleaves,exocarp,mesocarp,andseeds,aswelltoevaluateditsradicalscavengercapacitybyDPPHassay.Fruitand leaveswereobtainedofJackFruitQuiñonesCompany located inNayarit,Mexico.Thefruitwas identifiedbytheHerbarium of the UNAM´s Botanical garden in Mexico City. A voucher specimen that identifies the plant as Artocarpusheterophyllus hasbeendeposited at theDepartmentof Pharmacy (FS9157). The extracts of leaves, shell, and seedswereperformedfollowingthemethodAsongalemetal. (2004)withafewmodification.Thepulpwasmaceratedandsoaked in80%EtOH(Umeshetal,2010).Hydroalcoholicextractofleaves,shell,pulpandseedwereanalyzedwiththephytochemicalpreliminaryevaluation.ThecontentoftotalflavonoidcontentwasperformedfollowingtheprocedureofOsorioetal.(2011)andthetotalphenoliccompoundsbythemethodologyofAkroutetal.(2011).TheantioxidantactivitywasmeasuredbytheDPPHassay.Finally,theacutetoxicityofeachofthehydroalcoholicextractswasmeasuredbythelethaldose50(DL50)methodinCD1strainmice(OECD423,2001).Preliminary phytochemical assay revealed that A. heterophyllus contained tannins, glycosides, flavonoids and phenoliccompounds.Thetotalphenoliccontentoftheleaveswas75.44±0.14mggalicacideq./100gd.b.,shellhave72.24±0.33mggalicacideq./100gd.b,pulp8.79±0.09mggalicacideq./100gd.b.andseedshave11.04±0.16mggalicacideq./100gd.bThetotalflavonoidcontentoftheleaveswas38.88±0.66mgcatechineq./100g,d.b.,shellhave27.57±0.10mgcatechineq./100g,d.b,pulp6.10±0.36mgcatechineq./100g,d.bandseedshave7.50±0.14mgeq.catechin/100g.TheantioxidantactivityofthehydroalcoholicextractpresentedthehigherinhibitionpercentageofDPHHradical(50%ofinhibition).Thehydroalcoholicextractsdidnotcausemortality,morbiditysymptoms,ordeleteriouseffects.LD50valuewasgreaterthan2000mg/kg,whichindicatesthatitisnottoxic,soitcanbeusedforlaterbiologicalstudies.Thepresentstudyshowedthatthehydroalcoholicextracthaveastrongantioxidantactivity.1. Akrout,A.Alarcon,L.ElJani,H.Campra,P,(2011).AntioxidantandantitumoractivitiesofArtemisiacampestrisandThymelaeahirsutefromsouthern

Tunisia.FoodandChemicalToxicology.49(2):342-347.2. AsongalemE.A., Foyet,H.S., Ekobo, S.,Dimo, T., Kamtchouing, P., (2004). Antiinflammatory, lack of central analgesia and antipyretic properties of

Acanthusmontanus(Ness)T.Anderson.JournalofEthnopharmacology.95(1):63–68.3. López,L.X.Aguilar,M.L.Dublán,O.,(2014).Actividadantioxidanteeinhibidoradeα-glucosidasayα-amilasadetresvariedadesdecebolla(Alliumcepa

L).NovaScienta.6(2):234-247.4. Osorio,O.Moreno,A.ÁlvarezV.ÁlvarezL.GiustiM.,(2011).Phenolic,betacyaninsandantioxidantactivityinOpuntiajoconostlefruits.FoodResearch

International.44(7).2160-2168.5. OECD,“Acuteoraltoxicity—acutetoxicclassmethod,”Tech.Rep.423,OECDPublishing,2001.6. Umesh,B.,Shrimant,N.,Bapat,V.(2010).EvaluationofantioxidantCapacityandphenolcontentinJackfruit(ArtocarpusheterophyllusLam)fruitpulp.

PlantsFoodfortheHumanNutrition.65:99-104.


133

ANTIOXIDANTACTIVITYOFPORIFERANSFROMTHECOLOMBIANCARIBBEANROCKYCOASTBYDPPH

ANDABTSMETHOD

TovarBGP.1,RamírezALS.2,Valle-MolinaresRH.1 ,ValenciaJA.3 ,ArboledaVJW.1 1UniversidaddelAtlántico,FacultaddeCienciasBásicas,Barranquilla(Atlántico)-Colombia;2UniversidadTecnológicade

Pereira,EscueladeQuímica,BarrioAlamos(Risaralda)-Colombia;3DepartamentodeProducciónAgropecuaria,FacultaddeCienciasAgropecuarias,UniversidaddeCaldas,Manizales-Colombia

email:[email protected],[email protected],[email protected],[email protected]

Marineinvertebrates,especiallythespeciesbelongingtothePhylumPoríferarepresentsarichsourceofnewbiologicalandchemical compoundsof great interest.Manyof thesebioactivemolecules havebeen considered tohavegreat potential,suchasantibacterial, anticancer, anti-inflammatoryandantioxidants, amongotherpotentialbiological activities.Themaingoal of this studywas to evaluate the antioxidant activityof aqueous extracts from theColombianCaribbeanPoriferans:Suberites aurantiacus, Desmapsamma anchorata and Ircinia campana, using the in vitro models DPPH and ABTS; and theresultswerecomparedagainsttheresultsobtainedwiththeantioxidantcommercial,hydroquinone.IntheABTSassay,theaqueousextractsandfractionscontainingprecipitatedproteinsexhibitedhigh inhibitoryactivityof radicalcationshowingmorethan77%ofinhibitionincomparisonwiththehydroquinone,whilethefreeradicalscaptationbyDPPHintheI.campanawas146,73%whencomparedtothepositivecontrol(91,01%).TheresultssuggestthatthemarinespongesoftherockycoastoftheColombianCaribbeanarepromisingsourcesofantioxidantsandothersmoleculeswithusefulpotentialtobeusedinbiotechnologicalprocesses.1. Dhanalakshmi,J.,B.Kipkirui&S.Selvi.2012.Aninvitroantimicrobialactivityandbioactivitiesofproteinisolatedfrommarinesponge-Callyspongiasp.

Researchinpharmacy,2(1):36-41.2. Koopmans,M.,D.Martens&R.Wijffels.2009.Towardscommercialproductionofspongemedicines.Marinedrugs,7(4):787-802.3. Liu,Y.,H.Ji,J.Dong,S.Zhang,K.Lee,&S.Matthew.2008.AntioxidantalkaloidfromtheSouthChinaseamarinespongeIotrochotasp.Zeitschriftfür

naturforschungC,63(9-10):636-638.4. Seradj, H.,M.Moein,M. Eskandari & F.Maaref. 2012. Antioxidant activity of sixmarine sponges collected from the Persian Gulf.Iranian Journal of

PharmaceuticalSciences,8(4):249-255.


134

SESQUITERPENESANDMONOTERPENESFROMAMMOIDESATLANTICA(COSS.ETDUR.)WOLF

D’AmbolaM.1,BoudermineS.2,VassalloA.3 ,BenayacheS.2,SeverinoL.1,

DeTommasiN.4,MalafronteN.41UniversityofNaplesFedericoII,DepartmentofVeterinaryMedicineandAnimalProductions,Naples-Italy;2UniversityofConstantine1,DepartmentofChemistry,ResearchUnit,DevelopmentofNaturalResources,BioactiveMoleculesand

PhysiochemicalandBiologicalAnalysis,Constantine-Algeria;3UniversityofBasilicata,DepartmentofScience,Potenza-Italy;4UniversityofSalerno,DepartmentofPharmacy,Fisciano(SA)-Italy.

email:[email protected] genusAmmoides (Apiaceae) tribe of Ammineae includes two species in Algeria, one ofwhich is endemic:Ammoidesatlantica (Coss. etDur.)Wolf; theotherone,Ammoides pusilla (Brot.) Breistr, iswidespread in theMediterranean region.These species are used in folk medicine as antibacterial, anti-diarrhoea, anti-fever, antiinfluenza and to treat vitiligo inadditionoftheiruseascondiments(BellakhdarJ,1997).Ammoidesatlanticaisbiennalorperennialplantwiththickstemfittedwitharosetteofbasalleaves.Slightlybranchedstems.LeavesUmbels3-6rays.Fruit2-2.5mm,itisfoundinlawnsmountainsabove1000m(QuezelandSanta.,1962).In Algerian traditional medicine, the arial parts of Ammoides atlantica are reported to have a wide range of biologicalactivitiessuchasantibacterial,antioxidant,anthidiarrheicanddiabeticactivies(Ababsaetal.,2013;Laoueretal.,2003).Ammoidesatlanticaarialpartsweresubjectedtoextractionwithsolventspolarity increased,n-hexane,CHCl3,CHCl3-MeOH(9:1) andMeOH. The studyof the extractswas carriedout usingdifferent chromatographic techniques such as Silica gel,SephadexLH-20,MPLC,andrp-HPLC.Partofchloroformextract(4.5g)wassubjectedtocolumnchromatographyusingsilicagelandelutingwithCHCl3followedbyincreasingconcentrationsofMeOHinCHCl3(between1%and100%).Fractionsof50mlwerecollectedandanalyzedbyTLC.PreliminarystudyofCHCl3extractledtoisolationofsomepurecompoundswhosestructureswereelucidatedby1D-and2D-NMRSpectroscopy(1H,13C,13CDEPT,DQF-COSY,HSQC,HMBC,ROESY)andconfirmedbymassspectrometry.SevencompoundsfromCHCl3extractwereisolated:onemonoterpenechrysanthenone,twoglicosidesmonoterpenesasl-borneolO-β-D-glucopyranoside (Asano et al., 1993) and (-)-cis-chrysanthenol-β-D-glucopyranoside (Miyakado et al., 1974),three guaianolides as 9α-Acetoxyartecanin, 3α-Chloro-9α-acetoxy-4β,10α-dihydroxy-1β,2β-epoxy-5α,7αH-guai-11(13)-en-12,6α-olideandapressin(Trifunovic´etal.,2006)andanewbisabolenesesquiterpenederivative1,4-dihydroxybisabol-2,11-diene-10-one.1. AbabsaZEA,IhoualS,KaraAliW,LouaarS,LaouerH,AkkalS,MedjroubiK,2013.Antioxidantactivityofthespecies:Ammoidesatlantica(Coss.EtDur.)

Wolf.offamilyApiaceae.InternationalJournalofMedicinalandAromaticPlants(IJMAP).3(3):pp.401-403.2. Asano T,Murayama T,Hirai Y, Shoji J, 1993. Comparative studies on the constituents of ophiopogonis tuber and its congeners. VIII. Studies on the

glycosidesofthesubterraneanpartofOphiopogonjaponicusKer-Gawlercv.Nanus.(2)Chemical&PharmaceuticalBulletin41(3):pp566-570.3. BellakhdarJ,1997.Lapharmacopéemarocainetraditionnelle.Ibisspress,Paris,France.4. LaouerH,ZerrougMM,sahliF,ChakerAN,ValentiniG,FerrettiG,GrandeMandAnayaJ,2003.CompositionandantimicrobialactivityofAmmoides

pusilla(Brot.)Breistr.Essentialoil.JournalofEssentialOilResearch15(2):pp135-138.5. MiyakadoM, Ohno N, Hirai H, Yoshioka H,Mabry TJ, 1974. (-)-cis-ChrysanthenolO-β-D-glucopyranoside, a newmonoterpene glucoside fromDicoria

canescens.Phytochemistry(Elsevier)13(12),pp2881-2882.6. QuezelP,SantaS,1962.Nouvellefloredel’Algèrieetdesregionsdèsertiques,TomeII,EditionCNRS,Paris,France,p671.7. Trifunović S, Vajs V, Juranić Z, Zizak Z, Tesević V, Macura S, Milosavljević S, 2006. Cytotoxic constituents of Achillea clavennae fromMontenegro.

Phytochemistry,67(9):887-93.


135

PLANTCELLCULTUREASEXPERIMENTALMODELAPPLIEDTOTHEBIOSYNTHESISPATHWAYSTUDY

OFCOUMARINANDCHLOROGENICACIDINMIKANIAGLOMERATA(ASTERACEAE)

AranhaNettoL. ,AndreazzaNL.,SawayaACHF.,SalvadorMJ. DepartmentofPlantBiology,InstituteofBiology,UniversityofCampinas-UNICAMP,Campinas-Brazil


Mikaniaglomerata,popularlyknownasguaco,arecommonlyusedastreatmentforrespiratorysystemdiseasesduetotheirbronchodilatoreffect,activityassignedtocoumarinasactivecompound.However,ithasbeenobservedanaccumulationofchlorogenicacidandlowlevelsorevenabsenceofcoumarininM.glomerata,raisingthequestionwhetherthecoumarincouldbeusedaschemicalmarkerinthequalitycontrolofthemedicineswhichcontainsM.glomerata.Therefore,thereisaneed to investigate thebiosyntheticpathwayof these secondarymetabolites in this species.Plant cell culturesareverypromisingfortheproductionofsecondarymetabolites invitro,sincetheyallowarapidcellproliferation.Moreover,theyallowstudiesof regulationofaparticularsecondarymetaboliteundercontrolledconditionsandareeasy tomanipulate.Thisresearchaimwastoinvestigatetheuseofplantcellandtissueculturesasexperimentalmodelsappliedtothestudyofbiosyntheticpathwaysofcoumarinandchlorogenicacidinthisspecie.TheexplantsobtainedfromleavesofM.glomeratawerecollectedfromplantskeptinthegreenhouseoftheDepartmentofPlantBiologyoftheUnicampBiologyInstitute.Thentheywerewashedandsterilizedtopreventthegrowthoffungiandbacteriayhaymightbeassociatedwiththeplant.ThecalliweremaintainedonMSmedium1supplementedwith30.0g.L-1sucrose.Todeterminetheoptimalconcentrationsofthehormonesandensurethebestgrowingconditions,severaltestswere held with explants in media with different proportions of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzilaminopurina (BAP). Cell suspensionswereestablishedby inoculating cells from friable calli into liquidMSmediumwiththesamesupplements;howeveritwasobservedthatincreasingthesucroseconcentrationofthemediumto50.0g.L-1promotes the growth of these cells. Analysis for identification and quantification of the phenylpropanoid biosyntheticpathwayproductsweredonebyUPLC-MS,usingmethodologydescribedbyMelo2.Mediacontaining 4.52mMof2,4-Dand26uMBAPwereestablishedas thebestconditions forcallus induction for thisspecies since the calli development was fast and there was little root formation. M. Glomerata cell cultures havedemonstrated viable as a biological model for application in studies of the biosynthetic pathway of coumarin andchlorogenicacid.Preliminaryresultsfromtests inwhichchlorogenicacidwasinoculatedinM.glomeratacellsuspensionssuggeststhatthereisapositivemetabolismmodulation,whichdivertsthebiosynthesispathwaytocoumarinproduction.On the other hand, these tests showed that chlorogenic acid content in the cells was negatively correlated with anincreasingchlorogenicacidconcentrationaddedtothesuspensions.Despitethepositiveresults,furtherinvestigationsarenecessary to confirm the potential of the use of plant cell and tissue cultures ofM. glomerata as experimentalmodelsappliedtothestudyofbiosyntheticpathwaysofcoumarinandchlorogenicacidinthisspecies.Acknowledgments:CAPES,CNPq,FAEPEX-UnicampandFAPESP.1. Murashige,T.,Skoog,F.(1962).Arevisedmediumforrapidgrownandbioassaywithtabaccotissuecultures.PhysiologiaPlantarum.15:473-497.2. DeMelo,L.V.,Sawaya,A.C.H.F. (2015)UHPLC–MSquantificationofcoumarinandchlorogenicacid inextractsof themedicinalplantsknownas

guaco(MikaniaglomerataandMikanialaevigata).RevistaBrasileiradeFarmacognosia,25(2):105–110.


136

BIOACTIVITYANDCHEMICALCOMPOSITIONOFTHEESSENTIALOILFROMTHELEAVESOFANNONA

PARVIFLORA(A.ST.-HIL.)H.RAINER(ANNONACEAE)

SiqueiraCAT.1,AranhaNettoLA.1 ,LourençoCC.1,SerainAF.1,MesquitaJT.2,TemponeAG.2,Salvador

MJ.1 1DepartmentofPlantBiology,InstituteofBiology,UniversityofCampinas—UNICAMP,Campinas(SP)-Brazil;2AdolfoLutz

Institute,CenterforParasitologyandMycology,UniversityofSãoPaulo,SaoPaulo(SP)-Brazil.email:[email protected];[email protected]

Annonaceaefamilyarewidelydistributedaroundtheworldandisthelargestfamily inMagnolialeswithapproximately135generae2500species,that isconcentrated inthetropicswithfewspeciesfound intemperateregionswithtrees,shrubs,andwoodyclimbers (Siqueiraetal., 2015).Manyspeciesarevaluable for their largepulpy fruits, someareuseful for theirtimber,andothersareprizedasornamentals.ThegenusAnnona isoneof33genusofAnnonaceaeoccurring intheBrazilwithaboutof250speciesisdescribedinthecountry.Amongthemainchemicalgroupsofmetabolitespresentinthistaxon,wemention:alkaloids,acetogenins,volatileoil,flavonoids,amongothers(Siqueiraetal,2011).TheAnnonaparvifloraspeciesis knownas "pinha-brava"and so farhavenotbeendescribed scientific studiesof this species, so that encouragedus tostudy the essential oil. This species as part of a research project focusing on phytochemical investigation of Brazilianmedicinal plants searching for new bioactivity of natural products, in this study, we report the chemical analysis of theessentialoilfromtheleavesofAnnonaparviflorabyGC/MSandevaluationoftheirantimicrobialactivity.TheleavesofA.parviflora,werecollectedintheAgronomicInstituteofCampinas(IAC),thecoordinates(S23°06'89")and(W46°56'12"),Jundíai,SãoPauloState,Brazil.Thebotanical identificationperformedbyProf.Dr.JorgeYoshioTamashiro(IB-UNICAMP), and a voucher specimenof thematerialwasdeposited in theherbariumof IB-UNICAMP (register numberUEC-84100).TheessentialoilwasobtainedbyhydrodistillationfromfreshleavesofA.parvifloraandanalyzedbygascapillarychromatography (GC/MS). Antimicrobial and antileishmanial activities were evaluated in vitro according to Siqueira et al,(2015).ThisisthefirstreportontheanalysisofthevolatileconstituentsfromthefleshleavesofA.parvifloraandevaluationoftheirantibacterial and antileishmanial activity. Fifty-six compoundswere identified in the essential oil, representing 94.74% thetotaloil.Theessentialoilswerecharacterizedmainlyduetoincreasedpresenceofsesqueterpenes(60.07%),monoterpenes(34.67)andotherconstituents(4.81%),andthemaincompoundsidentifiedwerebicyclogermacrene(12.56%),germacreneD(10.32%),β-linalool(8.92%),(E)-caryophyllene(8.74%)andα-pinene(7.43%).Thisresultshowedtobe inaccordancewiththeclassesofvolatileconstituentsfoundintheAnnonagenusandintheAnnonaceaefamily(Siqueiraetal.,2007).TheessentialoilfromtheleavesofA.parviflorashowedsignificantly invitrobioactivityagainstmicroorganisms(bacteriaandprotozoa).AntimicrobialactivityoftheoilwasverifiedagainstEscherichiacoliATCC-10799,EnterobacteraerogenesandStaphylococcusaureus ATCC-14458 with minimum inhibitory concentration between 1.0 and 0.5 mg/mL. Moreover, the oil also showedpromisingantileishmanialactivityagainstL.(L.)chagasiwithIC50of44.2µg/mL.Thesignificantbiologicalactivitiespresentedbytheessentialoilssuggestthatthisspecieisarichsourceofbiologicallyactivecompounds.However,furtherinvestigationsareneededtoconfirmitstherapeuticpotential.Acknowledgments:CAPES,CNPq,FAEPEX-UnicampandFAPESP.1. SiqueiraCAT.etal.Bioactivityandchemicalcompositionoftheessentialoil fromthe leavesofGuatteriaaustralisA.St.-Hil.Naturalproductresearch,

2015,29:1966-1969.2. Siqueira CAT. et al. Chemical constituents of the volatile oil from leaves ofAnnona coriacea Mart., Annonaceae, and in vitro antiprotozoal activity.

BrazilianJournalofPharmacognosy,2011,21:33-40.


137

ANTIMICROBIALANDANTIOXIDANTACTIVITYOFSEVERALAGRO-INDUSTRIALWASTESFROMCOFFEEANDCOCOAPRODUCTION

OrtizA.1 ,BetancurPJF.2,Valle-MolinaresRH.3,ValenciaJA.4 ,ArboledaVJW.3

1CentroNacionaldeInvestigacionesdeCafé,Fisiologíavegetal,Chinchiná-Colombia;2UniversidaddeManizales,FacultaddeCienciasContables,EconómicasyAdministrativas,CIMAD,Manizales-Colombia;3UniversidaddelAtlántico,Facultadde

CienciasBásicas,Barranquilla(Atlántico)-Colombia,4UniversidaddeCaldas,FacultaddeCienciasAgropecuarias,DepartamentodeProducciónAgropecuaria,Manizales-Colombia.

email:[email protected],[email protected],[email protected] inthepursuitofhumanwelfareofferingnewalternativeresourcesforbioprospectingandsearchingofnewmolecules.Plantsareoneof these resources, since theyare consideredasnatural laboratorieswherealargenumberofchemicalcompoundsarebiosynthesized,especiallyproductsfromthesecondarymetabolism.Compoundspresentincocoaandcoffeeplayanimportantroleinthequalityofderivedfoods.Further,thesecompoundsexhibitawiderange of biological effects that make them very useful in the prevention of coronary diseases and neurodegenerativedisorders[1].Exploringplantsasapotentialsourceofantimicrobialpropertiesandantioxidantscouldaddadditionalvaluetoagro-productsderivedfromcocoaandcoffee, thusreducingeffectsgeneratedbypollution. Thecorrespondingbiologicalmaterial of coffee was collected in the Experimental Station CENICAFE-Naranjal (04º59'Norte, 75º39'Oeste) and cocoasampleswereprovidedby the FarmLuker (05º04'Norte, 75º41'Oeste) at departmentof Caldas, Colombia.Bioassayswereconducted in order to determine the antimicrobial and antioxidant activity from coffee and cocoa extracts.Bioassays ofantibacterialactivitywerecarriedoutagainstthebacteriaEscherichiacoli(ATCC25922),Klebsiellapneumoniae(ATCC70503),Staphylococcusaureus(ATCC25923)andMethicillin-resistantStaphylococcusaureus(MRSA)(ATCC43300).Antimicrobialactivity.Resultsshowpromisingantimicrobialactivityfromacetoneextractsofcoffee,againstallbacteriathatweretested.K.pneumoniae(9%)wasthebacteriathatshowedlesssusceptibilitytotheextracts,whileS.aureusdidshowthehighestpercentageofinhibition(52%).MethanolicextractsfromcocoadidnotshowinhibitoryactivityagainstK.pneumoniaewhereasforE.colistrain,showedlowerpercentagesofinhibition.Ingeneral,theextractshaveahigherantimicrobialactivityagainstgram-positivebacteriawhencomparedwithgram-negativebacteria,becausegram-negativebacteriahaveanoutermembranesurroundingthecellwallthatrestrictsthediffusionofhydrophobiccompoundsthroughthelipopolysaccharidethatcoverit.S.aureusandmethicillin-resistantS.aureusstrainsweresusceptibletoextractsfromcocoapodandfermentedroastedcocoabeanshowingan inhibition rangingbetween29.58and33.08%.Theactivityof theseextractsonbacteria isprobablyduetothepossibilitytheycangeneratecomplexeswithextracellularproteinsandsolubleproteins.Antioxidant capacity,DPPHmethod.Valueswithgreaterbiological activitywerepresented in coffee leavesextractedwithacetoneandmethanol solvent respectively (393.94±23.13mgTrolox /g sampleand271.34± 11.61mgTrolox /g sample).TheseresultsagreewiththoseresultsobtainedbyDudonnéetal.2009,whichstatethatinthecaseofgreencoffeevaluesarehigher formethanolextracts(60%ofDPPH),eventhoughthereviewedstudyusedaqueousextraction(41%ofDPPH).Theantioxidantcapacityofsamplesofgreencacaoandroastedcacaodifferbetweenthem,aswellasbetweenfermentedandunfermentedcocoa.Antioxidantcapacity,ORACmethod.Theresultsprovethatperformingmethanolicextractions;greenunfermentedcocoaandgreencoffeeshowsthehighestvaluesofantioxidantcapacitymeasuredasORACwith1694.323and698.794mmol/gsamplerespectively,whilethemucilageofcoffeehadthelowestcontentwith9,955μmol/gsample.Thehigheractivityvalueswerefound incoffee leavesandcoffeepulp (530.368and348.368mmol/gsample respectively),whensampleswereextractedusingacetone,while samplesof cocoahusksandcoffeemucilagepresented the lowest activities (5,775and9,070μmol/gsample respectively). It hasbeen shown that coffeeand cocoabeansandproductsmade from themcontain a varietyofantioxidantssuchassolublephenoliccompoundsandinsolublephenolicpolymers[2].Inrecentyears,thebacterialresistancetodrugsandthedevelopmentofcardiovasculardiseaseshaveincreasedsignificantlyworldwide, so the search and the discovery of new antimicrobial and antioxidant plant compounds represent abiotechnologicalalternativeandaneconomicallyinterestingfieldtoimprovesectorssuchasmedicine,agroandcosmetics.1. BoudetAM.(2007).Evolutionandcurrentstatusofresearchinphenoliccompounds.Phytochemistry.68:2722-35.2. ScalbertA.andWilliamsonG.(2000).Dietaryintakeandbioavailabilityofpolyphenols.J.Nutr.,130:2073–2085.


138

EVALUATIONOFANTIOXIDANTPROPERTIESANDACUTETOXICITYOFCYANOBACTERIAPIGMENTSANDMETHANOLICEXTRACTS

GaletovićA.1,2 ,NeiraI.3,VegaM.5,OchoaP.5,GallardoV.6,NúñezJ.4,SeuraF.6,TapiaC.4,ValdiviaC.4,

Gómez-SilvaB.1,2¹BiochemistryLab,BiomedicalDepartment,UniversityofAntofagasta,Antofagasta-Chile;2CentreforBiotechnologyandBioengineeringCeBiB,UniversityofAntofagasta,Antofagasta-Chile;3TechnologyDepartment,UniversityofAntofagasta,

Antofagasta-Chile;5NutritionandDieteticsDepartment,UniversityofAntofagasta,Antofagasta-Chile.email:[email protected]

Cyanobacteria are an interesting and innovative source of various biomolecules and some compounds have anti-inflammatory and antioxidants properties. Thesemicroorganisms synthesize and accumulate colored proteins (pigments)knownasphycobiliproteins(phycoerythrin,phycocyaninandallophycocyanin),participatingasanantennapigments inthephotosyntheticapparatus.Differentstudieshaveshowntheantioxidantcapacityofphycoerythrinandphycocyanin(Romayet al., 2003). The antioxidant phycocyanin pigment is related to its ability to sequester hydroxyl ions that promote theprocessof lipo-peroxidation(Romayetal.,2001).Consequently,cyanobacteria fromtheAtacamaDesertarean innovativesourceofnaturalantioxidantswithpotentialprotective roleagainstoxidativestress.Thegoalof thiswork is tostudytheantioxidantpropertiesandtoxicityofthepigmentsofcyanobacteriaforsubsequentapplicationinthefood,pharmaceuticalsandcosmeticsindustry.Cyanobacteria cells in the exponential growth phase were suspended in 75% methanol. The cells were disrupted byultrasound,thesuspensionwascentrifugedandthesupernatant(75%methanolextract)wasrecoveredbyandfiltered.Thepigments phycoerythrin and phycocyaninwere purified from cyanobacteria strains fromAtacamaDesert by precipitationwithammoniumsulfatesaturation(0-35%and35-60%)respectively.Thephycobiliproteinsweredializedandfreeze-driedandusedinantioxidantassays.Themethanolextractandfreezed-diedpigmentswereusedinABTSantioxidantassaybasedonprotocoldescribedbyReetal.(1999).ThevaluesobtainedwereexpressedasµmolesTroloxequivalentpergramfreshmassormilligramofpigment.Acute toxicityassaywasperformedusingage-synchronizedC.elegansN2 (wild-type).Nematodesplusaddedpigments(0.5-0.005mg/mL)were incubatedat20°Cfor24h.Later,nematodeswerecountedandclassifiedasactive(alive)orinactive(dead).TheABTSassaydonewithmethanolextractsfromcyanobacteriastrainsLLA-10,CAQ-15andLLC-10,showedvaluesrangingfrom1.95±0.38to7.17±0.61µmolesTE/gfreshmass.Theassayswithpigmentsphycocyanin(LLA-FC)andphycoerythrin(CAQ-FE)renderedvaluesrangingfrom1.98±0.45to3.12±0.56µmolesTE/mgpigment.Thesepreliminaryresultssuggestthatbothpigmentsandmethanolextractsfromcyanobacteriahaveantioxidantpowerthatcomparestofruitsasmulberry,pineappleor passion-fruit (Kuskoski et al., 2005). However, it is necessary to determinate the antioxidant power with additionalantioxidantsmethods.WealsohaveobservedthatthepigmentsusedhereshowednotoxiceffectsonC.elegans.Finally,thisis the first study on the antioxidant activity of purified pigmented proteins and methanol extracts from Atacamacyanobacteria. These results may contribute to future biotechnological applications on the pharmaceutical, cosmeticindustriesandonfunctionalfooddevelopments.Acknowledgements:CONICYT-Chile(CeBiBFB-0001)andUniversidaddeAntofagasta(SI-5305;CODEI-5364).1. KuskoskiE.M,.AsueroA.G,AnaM.TroncosoA.M.,Mancini-Filho,J.,Fett,R.(2005).Aplicacióndediversosmétodosquímicosparadeterminaractividad

antioxidanteenpulpadefrutos.Ciênc.Tecnol.Aliment.,Campinas,25(4):726-732.2. Re,R.,Pellegrini,N.,Proteggente,A.,Pannala,A.,Yang,M.andRice-Evans,C.(1999).AntioxidantActivityApplyinganImprovedABTSRadicalCation

DecolorizationAssay.FreeRadicalBiology&Medicine,Vol.26,Nos.9/10,pp.1231–1237.3. RomayCH.,RemirezD.,GonzálezR.(2001).ActividadAntioxidantedelaficocianinafrentearadicalesperoxílicosylaperoxidaciónmicrosomal.Rev.

CubanaInvestBiomed20(1):38-41.4. RomayCH.,GonzálezR.,LedónN.,RemirezD.andRimbauV.C-Phycocyanin:Abiliproteinwithantioxidant,anti-Inflammatoryandneuroprotective

effects(2003).CurrentProteinandPeptideScience4:207-216.


139

ADVANCESINKNOWLEDGEOFCHILIOTRICHUMDIFFUSUM(ASTERACEAE),ANATIVESPECIESFROM

PATAGONIAUSEDINTRADITIONALMEDICINEOFTHEONAS

AlcaldeBahamondeSM.1,Weisntein--OppenheimerC.2,FloresML.3,CórdobaOL.4 ¹FarmacologíaI,FacultaddeCienciasNaturales,UniversidadNacionaldelaPatagoniaSanJuanBosco,Chubut-Argentina;

²EscueladeQuímicayFarmacia,FacultaddeFarmacia,UniversidaddeValparaíso,Valparaíso-Chile;3Farmacognosia,FacultaddeCienciasNaturales,UniversidadNacionaldelaPatagoniaSanJuanBosco,Chubut-Argentina;4QuímicaBiológicaII,GQBMRNP-CRIDECIT,FacultaddeCienciasNaturales,UniversidadNacionaldelaPatagoniaSanJuanBosco,Chubut-

Argentina.email:[email protected]

Chiliotrichumdiffusumisashrubthat inhabitsthesouthwestofPatagoniaArgentina,popularlyknownas"matanegra"or"kóor"(inShelknamorOna´slanguage).TheOnatraditionallyusedflowersformedicinalpurposesto“clarifytheview”andaerialpartswereusedinrituals(tattoos),toimprovememory,treatheadaches,crampsandvarices[1,2].Inpreviousstudieswe showed that, aswithotherAsteraceae,C.diffusumbiosynthesized flavonoidsandphenolic acids related toanalgesic,anti-inflammatory [3] and vasodilator activities, and its traditional uses. In thisworkwepresent advances in the studyofbiologicalactivityandphytochemicalcharacterization.TheaerialpartsandflowersChiliotrichumdiffusum(G.Forst.)Kuntze(Asteraceae)werecollected insummerof2003and2006, in the villageof 28 deNoviembre (Santa Cruz,Argentina). Thesewere dried at room temperature, protected fromlight, ground and sieved. The aerial parts were exhaustively extracted with ethanol 96° at room temperature; then theextractwasfractionatedwithsolventsofincreasingpolarity.Extractionofflowerswasperformedwithwaterbydecoction[3, 4]. The extracts were analyzed by RP-HPLC-DAD. Cytotoxicity was assessed with the Artemia salina test (BST) andinhibition of root elongation of wheat test. Antiproliferative activity on tumor cell line SH-SY5Y (ATCC® CRL-2266™)wasmeasured using the resazurin method, which is based on the ability of viable cells to reduce this molecule to form afluorescentproductwithanexcitationwavelengthat544nmandanemissionat590nm;fibroblastwasusedaslinecontrol.AntioxidantactivitywasmeasuredbyinhibitiontestofDPPH(1,1-diphenylradical-picrylhydrazyl-2).Ethanolicextractfractionswereanalyzedatconcentrationsof10,100and500µg/ml,showingacytotoxicitywithaLC50<40µg/ml against Artemia salina test. Decocto from flowers showed inhibition of root elongation of wheat of 56 and 69 %(dilutions0.05and0.5%respectively)withaIC50of58.1µg/ml.DecoctoalsoshowedantiproliferativeactivityonthecelllineSH-SY5Y(IC500.62±0.1mg/ml)andonfibroblast(IC501.35±0.84mg/ml,dosesince1.25mg/ml).Consequently,theselectivityindexoftheantiproliferativeactivitywas2.1±1.6.Inbothextracts,flavonoidsandphenolicacidswerethemajormetabolites,standingoutintheethanol,vitexin,kaempferol-O-glc,apigenin-7-O-glcandquercetin-3-O-glc(isoquercitrin).Decoctoshowedcaffeicandchlorogenicacid,quercetin-3-O-gal(hyperoside),isoquercitrin,quercetin-3-O-rham(quercitrin),quercetinandkaempferol-3-O-rham(afzelin).Thesemetaboliteshavebeendescribedasantioxidants,anti-tumor,anti-inflammatory.Inthestudiesofpotentialanti-tumorsubstances,extractswhichhaveaLC50orIC50<200µg/mlinBSTorrootinhibitionassayare of interest. In this study, the results obtained with the decocto of flowers were important, and related to thephytochemicalprofile.Theseresultsandthestrongantioxidantactivity,supportingthetraditionalusesofthisspecies.1 VeraJC,1993-94.AnalesdelInstitutodelaPatagonia.UniversidaddeMagallanes.SerieCienciasHumanas,22,155-168.2 Catastro de Flora nativa usada con finesmedicinales por la Agrupación en la Reserva NacionalMagallanes en la Región deMagallanes y Antártica

Chilena;FondodeProteccióndeAmbiental–FPA.MinisteriodelMedioAmbiente,AsociaciónIndígenaWilliLafkendePuntaArenas,Chile,2010.3 AlcaldeBahamondeSM,FloresML,CórdobaOL,TairaCA,GorzalczanyS,2013.Antinociceptiveandanti-inflammatoryactivitiesofChiliotrichumdiffusum

(Asteraceae)aqueousextract.Braz.J.ofPharmacognosy,23(4),699-705.4 FarmacopeaArgentina,2003.FA-ANMAT,7°Ed.,Vol.1,Argentina.


140

ESI-MSANDUHPLC-MSANALYSISOFRESIDUESOFGREENPROPOLISANDEVALUATIONOFTHEIR

ANTIOXIDANTANDANTIMICROBIALACTIVITIES

CorrêaWR.1,2,RuedaRYR.1,PolancoIM.1,Hernandez-TascoAJ.1,MarinhoJVN.1,LópezBGC.1,PradoSC.2,CunhaIBS.1,3,SawayaACHF.1,SalvadorMJ.1

1InstituteofBiology,Dept.PlantBiology,CampinasStateUniversity-UNICAMP,Campinas-Brazil;2FederalInstituteofEducation,ScienceandTechnology,Inconfidentes(MinasGerais)-Brazil,3ThomsonMassSpectrometryLab.,IQ-UNICAMP,Campinas-Brazil.

email:[email protected] is a bee product produced by Apis mellifera, mainly composed of plant resins. It presents complex chemicalcomposition and its main bioactive components are flavonoids and a wide variety of phenolic compounds. Propolis hasseveraltherapeuticalactivities, includingantitumour,antioxidant,anti-inflammatory,hepatoprotective, immunomodulatoryand antimicrobial effects that makes propolis a product widely used in Brazil. Therefore, this study aimed to verify thepharmacological potential of a draff of greenpropolis and to identify andquantify certain ionsmarkers, characteristic ofgreenpropolis, certainlypresent indraffofpropolis. For this reason,weproceeded toevaluate theantioxidantcapacity,antimicrobialactivity,andthechemicalprofilebyESI-MS,aswellastoquantifytheconcentrationsoftheionmarkersintheresidueofgreenpropolisbyUHPLC-MS,comparedtothepropoliscontrol.Inthisstudy,weevaluatedthecompositionoftheresiduesofsubsequentextractionsofrawpropoliscomparedtothatofthefirstextraction(EEP)inordertodeterminetheappropriatenumberofextractionsthatcouldbeused.Threesamplesofgreen propolis, two of them from small apiaries and another from a larger apiary (all with similar composition) wereanalyzed.AllthreesampleswerecollectedinthestateofMinasGeraisintheSoutheasternregionofBrazil,wherepropolisispredominantlyderivedfromBaccharisdracunculifoliaplant.Thechemicalcompositionoftheresiduesandthecrudepropoliswas evaluatedbydirect insertionelectrospray ionizationmass spectrometry in thenegative ionmode (ESI(-)-MS)using aTQDAcquitymassspectrometer.AcomparisonoftheESI(-)-MSfingerprintsshowedthatbioactivesubstancesstillremaininthe residues after one or two extractions. The chromatographic were performed on a UPLC Acquity chromatographercoupledwithaTQDAcquitymassspectrometerwithanESIsource.AC18BEHWatersAcquitycolumn(2.1mmx50mmx1.7µmparticlesize)wasused.SolventAwasmili-Qpurifiedwaterwith0.1%formicacidandsolventBwasacetonitrile.Theflowratewas0.2mL/minand5µLofsampleswereinjected;withalineargradientstartingat5%acetonitrileandincreasingtoup100% methanol in nine minutes, held until 12 minutes and then returning to the initial conditions. The compounds wereidentifiedbycomparisonoftheirESI-MS/MSfragmentationspectrawithliteraturedata(Sawayaetal.,2011).TheantioxidantactivitywasassessedbyDPPHandORACassaysandtheantimicrobialactivitywasstudiedaswellinaccordancetoSalvadoretal.,(2011)andSalvadoretal,(2002),respectively.Toourknowledgethisisthefirstreportofchemicalcompositionandbiologicalactivityoftheresiduesoftheextractionsofgreenpropolis.ArtepellinCwasfoundtobepresentingreenpropolisextract(EEP)andalsointheresidues,howeveritwaspresent in lower concentrations.Nevertheless, another important substance, dicaffeoylquinic acid,was only found in thepropolis extract and in the first two residues. In theDPPH andORAC assays a sequential loss of antioxidant activitywasobserved. The antimicrobial activity of EEP and extract of the first two residues was investigated in vitro against gram-positive and gram-negative bacteria and EEP and the first extract presented inhibition values of (1 mg ml-1) or less. Weconclude that two sequential extractions of raw green propolis are appropriate to avoid the loss of important chemicalcomponentsintheresidue.Furtherinvestigationsarenecessarytoconfirmthepotentialofthesenaturalproductsandtheirconstituentsasusefulagentsforbiotecnologicalapplications.Propolisresidueobtainedafterthethirdextractionshowsnobiologicalactivity,howeveritmightbeusefulforotherpurposesotherthanforitsantimicrobialandantioxidantactivityandfurtherstudiesofthismaterialwouldbeappropriate.Acknowledgments:toCAPES,CNPq,FAPESPandFAEPEX-Unicampforthefinancialsupport..A.C.H.F.SawayawouldliketothankMrAntonioCarlosPresident-ApisFlora-forthesupplyofpropolis.CunhawouldlikethanktoMrEdilsonAEmidio(PROVMEL)forthesupplyofpropolisfromhisapiary.1. SalvadorMJ.etal.IsolationandHPLCquantitativeanalysisofantioxidantflavonoidsfromAlternantheratenellaColla.ZeitschriftfurNaturforschungC-J.

Biosciences,2006,61:19-25.2. SalvadorM.J.etal.,BioactivityofcrudeextractsandsomeconstituentsofBlutaparonportulacoides(Amaranthaceae).Phytomedicine,2002,9:566-571.3. SalvadorMJ.etal.,AntioxidantcapacityandphenoliccontentoffourMyrtaceaeplantsofthesouthofBrazil.Nat.ProductCommun.2011,6:977-982.4. SawayaA.Etal.AnalyticalmethodsappliedtodiversetypesofBrazilianpropolis.ChemistryCentralJournal,2011,5:27-30


141

DEVELOPMENTANDCHARACTERIZATIONOFACTIVEALGINATE-BASEDEDIBLEFILMSFORFOOD

PACKAGINGAPPLICATIONS

BignardiC.1,2,CaràD.1,CavazzaA.1,2,CorradiniC.1,2 ,GrimaldiM.1,SalvadeoP.1,21DepartmentofChemistry,UniversityofParma,ParcoAreadelleScienze,Parma-Italy;2HighMaterialsInnovationSpin-off

UniversitàdegliStudidiParma,Parma-Italye-mail:[email protected]

Inrecentyears, thedemandforsafe,highquality, freshnessandextendshelf lifeof foodhave ledtothedevelopmentofinnovativeapproachesinfoodpackagingnamed“activepackaging”whichcanbeclassifiedintotomainkinds:i- Nonmigratoryactivepackagingactingwithoutintenctionalmigrationii- Activereleasingpackagingallowingacontrolledmigrationofcompoundsintheatmospheresurroundingthefoodoron

foodsurfaceincontactwithpackaging.Recentlyantimicrobialpackagingsystemsobtainedbyahybridorganic-inorganiccoatingwithantimicrobialpropertieswerepreparedinourlaboratoryandthereleasebehaviorinwater,otherfoodsimulants,aswellasselectedfoodwasevaluated(1,2).In thispresentation results regarding thedevelopmentandcharacterizationofalginate-besededible“active” filmswillbepresented.Theproposedactivefilmsrepresentaninnovativematerialcharacterizedbybeingbiodegradable,andknowntobesuitablefor edible food packaging. Their also show good mechanical properties and high resistance, and has been successfullyemployedforactivefoodpackagingcontainingnaturalantioxidantagents.Resultswillbepresentedanddiscussed.1. CorradiniC.AlfieriI.,CavazzaA.,LantanoC.,LorenziA.,ZucchettoN.,MonteneroA.(2013)Antimicrobialfilmscontaininglysozymeforactivepackaging

obtainedbysol-geltechnique.JFoodEngineering,119,580-587.2. LantanoC.,AlfieriI.,CavazzaA.,CorradiniC.,LorenziA.,ZucchettoN.,MonteneroA.(2014)Natamycinbasedsol-gelantimicrobialcoatingsonpolylactic

acidfilmsforfoodpackaging.FoodChem.,165,342-347.


142

ANTIMICROBIALACTIVITYOFTWONEWTETRAHYDROQUINOLINES

Valle-MolinaresRH.1,3,Marsiglia-LansMA.1,ArboledaVJW.2,DelOlmo-FernandezE.1,

Melendez-GómezCM.2 ¹UniversityofAtlántico,Barranquilla(Atlántico)-Colombia;2DepartmentofPharmaceuticalChemistry,FacultyofPharmacy

(IBSAL-CIETUS),UniversityofSalamanca,Salamanca-Spain;3UniversityofCartagena,Cartagena(Bolivar)-Colombiaemail:[email protected]

Oneofthemostimportantpathologiescontributingtoincreasemorbidity,disabilityandmortalityworldwideareinfectiousdiseases causedby bacteria [1]. In addition, the excessive and inadequate use of antibiotics has led to the emergenceofpathogensresistanttodrugscurrentlyavailable,asinthecaseofEscherichiacoli,KlebsiellapneumoniaeandStaphylococcusaureus that have developed different resistance mechanisms [2]. Currently, in the search for new molecules withantibacterialactivitytwoapproachesarefollowedinordertodesignalternativesforthetreatmentofinfectionscausedbyresistant bacteria: (I) search for new molecules obtained from natural sources and (II) diversification of molecules bysynthesis from different natural sources, with known biological activity. Tetrahydroquinolines (THQ) have ubiquitousdistributionamongnaturalproductsandmedicinalagents.Forthisreason,theyhavebeenlsrgelystudiedandrepresentanimportant basis for synthetic chemicals [3]. In this work, two newly synthesized THQ-based (CM725 and CM728) wereobtainedbyusingtheimineDiels-Aldermethodology(DA)andtandemreactionAza-Michael-Idadihydropyrrole;afterwards,theirantibacterialactivitywasevaluatedonthe followingstrains:MethicillinresistantStaphylococcusaureus (SAMr)ATCC43300, Klebsiella pneumoniae ATCC 1705, Staphylococcus aureus wild type ATCC 25923, Escherichia coli ATCC 25922,Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 27853. Minimal Inhibitory Concentration (MIC) wascalculated following the recommendations of the CLSI-2012 for microdilution (IC50) by using the Graphpad Prism 5.0statisticalpackage.AserialdilutionsetofeachmoleculewaspreparedusingDMSOassolvent(75,35,20,10and5μg/mL).Thefinalvolumewas100μLandthefinalconcentrationofDMSOintheassaydidnotexceed1%.Eachtreatmentwascarriedout by triplicate and the cultures were incubating at 37°C and the growth was monitoring at OD600 until reaching thestationary phase. Gentamicin (20μg/mL) was used as positive control. The inhibition percentage was calculated fromequationusedbyValle-Molinaresetal.[4].

Fig1.CM728MoleculeStructure Fig2.CM725MoleculeStructure

Results show that compound CM728had the lowestMIC (0,06917μg /mL) for Enterococcus faecalis, whereas the highestvaluewas37,01μg/mLforS.aureus.However,compoundCM725showsbroadspectrumwithMICvaluesof7,417μg/mLand 14,84μg /mL toKlebsiella pneumoniae andEnterococcus faecalis, respectively. Thedifferencebetween theactivityofthesemoleculescouldbeattributedtothetypeofsubstituentspresentinthequinolinering.Otherstudieshavepreviouslyreportedthataliphaticsubstituentscontributetoenhancethebiologicalactivity[5].Incomparisonwiththeantibioticusedaspositivecontrol, it is importanttopointoutthatthesemoleculesshowedbetter inhibitionpercentageson thebacteriatested,particularlyagainstresistantbacteria(SAMr).Likewise,MICvaluesobtainedforthesemoleculesarelowerthanMICof positive control (20μg /mL). In conclusion, the screeningof natural and syntheticmolecules represents an interestingstrategyfordevelopingnewtoolstocontrolinfectionscausedbyresistantbacteria.Thediversityofquinolineringallowstoincreasethepotentialofthesemoleculesbyusingsubstituentsasanimportantcharacteristictodesignnewdrugs.1. WorldHealthOrganization,Antimicrobialresistance:GlobalReportonSurveillance,Geneva,20142. MshanaSE.etal.Antimicrobial resistance inhumanandanimalpathogens inZambia,DemocraticRepublicofCongo,MozambiqueandTanzania:an

urgentneedofasustainablesurveillancesystem,Ann.Clin.Microbiol.Antimicrob.12(2013)28.3. NgemenyaMN.etal.,Invitroscreeningof1-aryl-6-hydroxy-1,2,3,4-tetrahydroisoquinolines:Structurerelatedactivityagainstpathogenicbacteria,Asian

Pac.J.Trop.Biomed.5(2015)472–477.4. Valle Molinares RH. et al., Antimicrobial activity of metallo tetra (4-Carboxyphenil) Phthalocyanine useful in photodynamic therapy,

PharmacologyOnline.2(2015)131–1375. Li Q. et al. Novel triazolyl-functionalized chitosan derivatives with different chain lengths of aliphatic alcohol substituent: Design, synthesis, and

antifungalactivity,Carbohydr.Res.418(2015)44–49.


143

PHENOLICPRODUCTIONANDANTIRADICALARACTIVITIESOFINVIVOPLANTS

ANDINVITROCULTURESOFSEVERALHYPTISSPECIESCOLLECTEDINCERRADOAREA(GOIÁS,BRAZIL)

RiosRM.1,SilvaFG.2,PereiraPS.2,DiasAP.1 ¹UniversidadedoMinho,BiologyDepartament,CampusGualtar,Braga-Portugal;²InstitutoFederalGoiano,BiologyDepartament,

CampusRioVerde,RioVerde,Goiás-Brasil.email:[email protected]

Hyptis is a Lamiaceae genus, consisting of various medicinal and aromatic species widely distributed in South America,particularlyinBrazil.Amongthesespecies,H.suaveolens(SH),H.marrubioides(HM)andH.pectinata(HP)arewidelyusedinfolkmedicine in the regionofGoiás (Midwest,Brazil).Theyareusedas infusionsorethanol/aqueousextracts for treatingdiseasessuchasnasopharyngitis,sinuscongestion,skindisorders,headache,stomachproblems,fever,dentalpain,bacterialandfungalinfections,cancer,etc.Thispracticebecomesamajorconcerninthescientificcommunitybecauselittleisknownaboutthereliabilityandsafetyofuseoftheseplants.Inordertobetterunderstandandvalidatesomeofthetraditionalusesoftheseplantsbiomassoftheseplantswerestudiedconcerningphenoliccompositionandantiradicalaractivities.HS,HMandHPplantswerecollected invivo intheregionofRioVerde(GioásBrasil).Additionaly, invitroculturesofthesespecieswereestablished.Differenthormonecombinationsofauxin2,4-D/NAAandcytokininBAP/Kinwereusedfor invitrocultures.Thebestcombinationswereselectedforcalliand/orshootculturesproduction.Bothbiomassofinvivoplantsandin vitro cultures were taken and lyophilized. The dried biomass was ground and aliquots were taken for preparation ofethanolicextracts(80%).Theseextractswerefilteredandsubsequentlyanalyzedforphenoliccontent(HPLCanalysis)andantiradicalactivity(DPPHmethod).The in vivo plants produced mainly quercetin (Q) and luteolin (L) derivatives, and rosmarinic acid (RA) derivatives. Thephenolicprofilesof in vitro cultureswere similar to the in vivo counterparts, butwithdifferences in composition; in vitroculturesproducedmorecompoundsofRAtype.Theextractsofthethreespeciesshowngoodantiradicalarpotential,withthebestEC50obtainedforHP(57µg/ml).Invitroextractsofculturesshowedsimilaranti-radicalproperties;nosignificantdifferences were observed for EC50s of in vivo and in vitro biomass. Phenolic compounds may be responsible for theantiradicalaractivitiesofthesespeciesandthereforetheirpositiveeffectagainstseveralpathologicalprocesses.


144

APOPTOTICEFFECTOFCARDENOLIDEGLYCOSIDESFROMAsclepiassubulata

Robles-ZepedaRE.1 ,VelázquezC.1,Garibay-EscobarA.1,VilegasW.2,Medina-JuárezLA.3,Gámez-Meza

N.3,Rascón-ValenzuelaLA.11DepartamentodeCienciasQuímicoBiológicas,DivisióndeCienciasBiológicasydelaSalud,UniversidaddeSonora.EncinasyRosalesHermosillo(Sonora)-México;2UNESP-SãoPauloStateUniversity–InstituteofBiosciences,CoastalCampusofSãoVicente,SaoVicente(SP)--Brasil;3DepartamentodeInvestigacionesCientíficasyTecnológicasdelaUniversidaddeSonora,

DivisióndeCienciasBiológicasydelaSalud,Hermosillo(Sonora)-México.email:[email protected]

Aimof the studywas todetermine the cell deathpathways that the cardenolideglycosideswith antiproliferative activityfoundinthemethanolextractofA.subulataareabletoactivate.TheeffectofcardenolideglycosidesisolatedofA.subulataoninductionofapoptosisincancercellswasevaluatedthroughthemeasuringofseveralkeyeventsofapoptosis.A549cellsweretreatedfor12hwithdosesof3.0,0.2,3.0and1.0µMof12,16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, respectively. Apoptotic andnecroticcell levelsweremeasuredbydoublestainingwithannexinV-FITC/PI.Mitochondrialmembranedepolarizationwasexamined through JC-1 staining. Apoptosis cell death and the apoptosis pathways activated by cardenolide glycosidesisolatedofA.subulatawerefurthercharacterizedbythemeasurementofcaspase-3,caspase-8andcaspase-9activity.ApoptoticassaysshowedthatthefourcardenolideglycosidesisolatedofA.subulatainducedapoptosisinA549cells,whichwasevidencingbyphosphatidylserineexternalization in 18.2, 17.0,23.9and22.0% for 12, 16-dihydroxicalotropin,calotropin,corotoxigenin3-O-glucopyranosideanddesglucouzarin,respectively,comparedwith4.6%ofcontrolcells.Celldeathwasalsoassociatedwithadecreaseinmitochondrialmembranepotential,whichwasmorethan75%inthetreatedculturesrespecttocontrol.Theactivationofcaspase-3wasobserved inall cardenolideglycosides-treatedcancercells indicating thecaspase-dependent apoptosis of A549 cells. Extrinsic and intrinsic apoptosis pathways were activated by cardenolide glycosidestreatmentatthedosestested.In this study we have found that cardenolide glycosides, 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranosideanddesglucouzarin,isolatedfromA.subulatainducedthecelldeathtroughcaspase-dependentapoptosis,whichwasactivated,preferably,byextrinsicpathway.


145

ANTIPSORIATICACTIVITYOFLATINAMERICANNATIVEPLANTS

PintoNCC.1,MendesRF.1,SilvaJB.1,DuqueAPN.1,CastañonMCMN.2,ScioE.1

¹LaboratoryofBioactiveNaturalProducts,DepartmentofBiochemistry,FederalUniversityofJuizdeFora,JuizdeFora(MG)-Brazil;²DepartmentofMorphology,FederalUniversityofJuizdeFora,JuizdeFora(MG)-Brazil


Preliminarystudiesperformedbyourgrouphaveshownthemarkedanti-inflammatorypotentialoftheLatinAmericannativeplantsPereskiaaculeata,XylopiasericeaandVernoniacondensata,traditionallyusedtotreatinflammatorydisorders(Lorenzi.2009; Da Silva et al., 2011; Pinto et al., 2015). Psoriasis is an important chronic inflammatory and autoimmune-mediateddermatoseswhich affects 1-3% of theworld Caucasian population. So, the presentwork aimed to investigate the in vivoantipsoriaticactivityofthosespecies.The antipsoriatic activity was evaluated by the mouse tail test (Bosman et al., 1992). Different topical pharmaceuticalformulations(20mg)containing12%ofhexanefractionofP.aculeataleaves(HF),5%ofmethanolextractofX.sericeafruits(XE),5%ofethylacetatefractionofV.condensata leaves(EF),vehicleorPsorex®weretopicallyapplieddailyforfivedaysaweekduring twoweekson the tail of Swissmice (n=5). Thenaivegroupdidnot receive any treatment (vehicle control).Next,theanimalswereeuthanizedandthetailsweresubmittedtohistopathologicalanalysisusinghematoxylinandeosinstaining.TheslideswerehistometricallyanalyzedusingthesoftwareImageProPlus6.0®asfollows:(1)thetotal lengthofthescaleregion;(2)thelengthofthegranularlayerofthescaleregion;(3)theorthokeratosisdegree,whichwascalculatedbytheratioof(2)/(1);(4)therelativeactivityofeachtreatmentcalculatedbytheequation RA=(OKT–OKN)x100/(100–OKN),whereOKTmeanstheorthokeratosisdegreeof thetreatedgroupandOKNmeanstheorthokeratosisdegreeof thenaivegroup.ANOVAfollowedbytheNewman-Keulstestwasusedforstatisticalanalysis(COBEA–Protocolnº028/2014).Themousetailtestiswidelyappliedasitiseasilyaccomplished,providesreproducibleresultsandpresentshighcorrelationwith theactivityofantipsoriaticdrugscurrentlyused forhumanskin. In this test, theorthokeratosisdegree indicates thetherapeuticpotentialofatesteddrug.Thenaivegroupandthetopicalpharmaceuticalformulationscontainingvehicle,HF,XE,EForPsorex®showed,respectively,49.62±3.1,52.25±4.5,78.14±3.1***,72.16±2,8***,62.75±3.1*and86.83±2.5***oforthokeratosisdegree(*p<0,05;***p<0.001vsnaive).Therelativeactivitieswere0(naive),5.79(vehicle),56.88(HF),44.47(XE),26.29(EF)and74.06(Psorex®).Ourfindingsstronglysuggestedtheseplantspresentcompoundsendowedwithantipsoriaticpotential.Acknowledgements:ThisworkwassupportedbythegrantsfromFAPEMIG,CAPESandCNPq.1. BosmanB,MatthiesenT,HessV, Friderichs E, 1992.Aquantitativemethod formeasuring antipsoriatic activityof drugsby themouse tail test. Skin

Pharmacology,5:41-48.2. PintoNCC,MachadoDC,DaSilvaJM,ConegundesJL,GualbertoAC,GameiroJ,ChedierLM,CastañonMC,ScioE,2015.PereskiaaculeataMillerleaves

presentinvivotopicalanti-inflammatoryactivityinmodelsofacuteandchronicdermatitis.JournalofEthnopharmacology,173:330-337.3. DaSilva,JB,TemponiVS,FernandesFV,AlvesGAD,MatosDM,GasparettoCM,RibeiroA,PinhoJJRG,AlvesMS,SousaOV,2011.Newapproachesto

clarifyantinociceptiveandanti-inflammatoryeffectsoftheethanolextractfromVernoniacondensataleaves.Internationaljournalofmolecularsciences,12:8993-9008

4. LorenziH,2009.ÁrvoresBrasileiras:manualdeidentificaçãoecultivodeplantasarbóreasdoBrasil.NovaOdessa,InstitutoPlantarum.


146

STUDYOFHEAVYMETALS,TAA,POLYPHENOLSINTRITICUMDURUMWHEATSAMPLESGROUNDWITH

ANCIENTMILLSTONE

OliveriF.1,BrancatoP.1,GottusoV.1,CosenzaM.1,PittiR.1,BuaDG.2,AnnuarioG.2,RealeS1,3,CiceroN.2.4 ,DugoG.mo1,2,4

1ConsorziodiRicercasulRischioBiologicoinAgricoltura(Co.Ri.Bi.A.),Palermo-Italy;2DepartmentofBiomedicalSciences,DentalandMorphologicalandFunctionalImages,UniversityofMessina,Messina-Italy;3IstitutoZooprofilatticodiPalermo,

DipartimentodiTecnologieDiagnosticheInnovative,Palermo-Italy;4Science4LifeS.r.l.,aSpin-offoftheUniversityofMessina,Messina-Italy.


Inthiswork,samplesofTriticumdurumwheatandpastafromanancientsicilianmillstone(AnticoMulinoLazzara,Messina)were studied. Heavy metals, antioxidant properties and total polyphenols content were investigated. All samples wereanalizedbymolecularmarkers,preliminarydata.Allsampleswerepre-treatedbyamicrowavedigestionsystemandthecontentofCd,Hg,PbandAswasdetectedbyICP-MS(inductivelycoupledplasm-massspectrometry).Thetotalantioxidantactivity(mMolteac/100g)onthesamesampleswasdeterminedby themethodof theDPPH (2,2-diphenyl-1-picrylhydrazyl) and the contentof totalpolyphenols (expressed inmg/kgofequivalentsofacidgallic-GAE)bythemethodofFolinCiocalteau.All samples studiedhave shown thepresenceofheavymetals in traces. The concentrationof lead,mercury , arsenic andcadmium is lower than the legal limits. In fact, the regulation 1881/2006establishes forPb andCdamaximum allowableamountof0.20ppb(Reg.1881/2006),andtheobtainedresultsarebelowthethresholdlimit.TheaverageconcentrationofPbinpastais0,050ppband0,032ppbinflours,whilethecontentofCdislessthan0,015ppbinallsamples.AnalysisaboutHgshowsmeanvalueof0,020ppbinpastasamplesand0.018ppbinfloursamples,andaboutasinpastathemediumvalueis0,020ppbwhileintheflourthemediumvalueis0,015ppb(thereisnoregulationsconcerningcontentincereals).Analyticaldata obtained indicate that wheat and flour samples of Tumminia variety have higher antioxidant capacity than othervarieties(SimetoandSen.Cappelli),whichbasicallyarethesame.MakingacomparisonamongvaluesobtainedintheSimetochain, itwasobserved that theamountofantioxidantactivity resultshigher in the integral flour (66.30mMol teac/100g)thanpasta, probably this dependson transformationprocesses towhich it is subjected.Major concentrationof TAAwasfounded inbran sample (123.09mMol teac/100g). The contentofpolyphenols, in SenatoreCappelli flour sample ishigher(331.00 mg/kg), decreases in Tumminia, in Simeto wholemeal flour and in Simeto flour (247,12 mg/kg). Very high is thecontentinthebran(944mg/kg).Forthegeneticprofile,allsampleswereanalizedusing10ssr(simplesequencerepeats):8Xgwm (GaterslebenWheatMicrosatellite) and 2 Xwmc (WheatMicrosatellite Consortium) appropriately selected from apublic database (http://wheat.pw.usda.gov). Only four ssr are selected: Xwmc 415, Xwmc 597, Xwmc 544 and 368 Xwmcmarkers.1. REGOLAMENTO (CE) N. 1881/2006 DELLA COMMISSIONE del 19 dicembre 2006 che definisce i tenori massimi di alcuni contaminanti nei prodotti

alimentari.2. http://wheat.pw.usda.gov3. MillerHE,RigelhofF,MarquartL,PrakashA,KanterM.Antioxidantcontentofwholegrainbreakfastcereals,fruitsandvegetables.JAmCollNutr.2000,

19(3Suppl):312S-319S4. ManginiG., Taranto F.,Giove S.L.,GadaletaA. andBlancoA. 2010. IdentificationofDurumWheatCultivarsby aMinimumNumberofMicrosatellite

MarkersCerealResearchCommunications2010,38:155–162


147

ETNOPHARMACOLOGICALINVESTIGATIONOFPLANTSUSEDBYTHERIVERINEPOPULATIONONTHE

MICROREGIONOFNORTHARAGUAIA,MATOGROSSO,BRAZIL.

RibeiroRV.1,2,MartinsDTO.1 1FederalUniversityofMatoGrosso,FacultyofMedicine,AreaofPharmacology,Cuiabá(MatoGrosso)-Brazil;2Federal

InstituteofEducationScienceandTechnologyofMatoGrosso,LucasdoRioVerde(MatoGrosso)-Brazilemail:[email protected]

ThemicroregionofNorthAraguaiainMatoGrossocomprises14municipalitiescoveredundertheAmazonandtheBrazilianSavannah, bathed mainly by the Araguaia and Xingu rivers, and holds an enormous ethnocultural diversity. To survey,identifyandrecordmedicinalplantspeciesusedbyriverinepopulationsthatinhabitthemicroregionofNorthAraguaiaandselectnativeplantsforfurtherchemicalandpharmacologicalstudiesthroughconsultationinspecializesliterature.This was a cross-sectional ethnopharmacological study by non-probabilistic sampling (n=60), employing the snowballmethodfortheselectionofriverinedwellers locallyknownasspecialists intheuseofmedicinalplantsthroughoutthe14municipalities of the microregion. The data was obtained on the period between July and November in 2013 throughinterviews utilizing a semi-structured form. The relative importance (RI) and the informant consensus factor (ICF) weredetermined, and comparisonbetween taxonomic groups andbiological systemswas carriedout by principal componentanalysis (PCA)andChi-square (X²). The researchwasonly initiatedafter compliancewithall ethical aspects regarding theinformantsandfederalorganizations.Informantscited309plantspeciesbelongingto85botanicalfamilies,ofwhich73%werenative.ThemostcitedspecieswereCopaifera langsdorffii Desf. (133) Lafoensia pacari A. St.-Hil. (131) and Cecropia pachystachya Trécul. (126), and the plantsreportedinthisresearchwereindicatedfor18of22diseaseclassificationsinICD-10,withhighernumbersoninfectiousandparasiticdiseases(A00-B99,ChapterI),showing16,6%ofusagecitations.Amongtheplantscitedforthisdiseasecategory(A00-B99),ChenopodiumambrosioidesL.wasthespecieswithhighestusagecitations(50).TheRIvaluesrangedfrom0.1to1.9,withhighestvaluestoC.langsdorffii,C.pachystachyaandL.pacari(1.9;1.8and1.7,respectively).AsforICF,thehighestvalueswererelatedtoA00-B99(0.79),J00-J99(0.79)andK00-K93(0.78),andthemostcitednativeplantsindicatedforthetreatment of these disease categories were Bidens pilosa L., Vernonia ferruginea Less and L. pacari, respectively. PCAsshowed thatplants in theFabaceae familywere themostused for themost recurrent categories in the study (A00-B99,N00-N99andK00-K93).WealsoverifiedthatthebiologicalsystemsR00-R99,K00-K93,S00-T98,A00-B99,N00-N99,M00-M99, J00-J99andF00-F99 show strong correlation tooneanother and thus tend tobe treatedby the sameplants. Chi-squareanalysissuggestedthatthediseasecategorieswereinsomewaydifferentfromoneanotherregardingthespeciesofthefamiliesusedintheirtreatment.Outofthe9investigatednativeplants,C.langsdorffiiandBrosimumgaudichaudiistoodout, seeing as their phytochemical and pharmacological properties have been studied, they have also been sold aspharmaceuticalproductsandhaveapatentdeposit.LocalriverinespecialistsofthemicroregionofNorthAraguaiamakeuseofabroadvarietyofmedicinalplantsinhealthself-care, predominantly those used in the treatment of A00-B99, and some of these plants already have their therapeuticpotential scientifically validated. However, there are other whose pharmacological effect and safety have not yet beenproperlyinvestigated.Itwasalsoobservedthattheinformantsdonotuseplantsatrandom,butfollowingwellestablishedcriteriafortheselectionofthespeciesusedformedicinalpurposes.Thus,thepresentstudy,inadditiontoservingasabasisforfuturestudiesofchemical,pharmacologicalandagriculturalbioprospecting,mayalsocontributetothedevelopmentofmanagement,conservationandsustainableuseofmedicinalfloraofthemicroregion.Acknowledgments:FinancialsupportfromCAPES/Pró-Amazônia,CNPq/Bionorte.


148

SCREENINGOFANTIBIOTICPRODUCINGACTINOMYCETESFROMTHESEDIMENTSOFUNDISTURBED

FORESTAREASOFASELLA,ETHIOPIAANDITSHYPERACTIVITYAFTERMUTATION

KumarAP. ,KarpagamP.DepartmentofMicrobiology,SokotoStateUniversity,Sokoto-Nigeria

email:drpashokkumar@gmail.comWideanduncontrolledusageofantibioticshasmadethepathogenstobecomeresistanttocurrentlyusedantibiotics.Thereisanurgentneedfordevelopmentofanewdrugorahighlyactivemoleculeforcontrollingantibioticresistantstrains.Inthisstudy32strainsofActinomyceteswereisolatedandsubjectedtoprimaryscreeningbygiantcolonymethodagainstBacillus subtilis, Escherichia coli, Pseudomonas aeruginosaand Aeromonashydrophilia. The secondary screeningwas carriedoutbyfermentationprocess.Antibacterialactivitywasevaluatedbywellplatemethod.Theextractofisolateswassubjectedtowellplatemethodagainstpathogenicbacteria.B.subtilisandE.coliwerehighlyinhibitedbyAK1isolate.Otherisolatesshowedlimitedinhibitionofbacteria.TheAK1isolatewasmutatedbyUVirradiation.Themutantsdifferedfromthewildparentinreducedgrowthrates,changesintheshapeandsizeofthecolony,sporulationlevel,antibioticactivityandvariationinthecolorofthemycelium.ThezoneofinhibitionwashigherwithActinomycetesAK1 inoculatedplates.ThesecondarymetaboliteproductionwasenhancedbyUVmutagenesiswhencomparedtowildtype.Actinomycetesmayproducedifferentmoleculethatcaninhibitdifferenttypesofpathogens,however efforts like strain development can be done to produce new bioactive components againstmultidrug resistantbacteria.1. Freire-Moran Laura, BoAronsson, VhrisManz, Inge C.Gyssens, Anthony D. So, Dominique L.Monnet andOtto Cars. (2011). Critical shortage of new

antibioticsindevelopmentagainstmultidrug-resistantbacteria—Timetoreactisnow.DrugResistUpdat:14:118–124.2. JavedS,MerajM,BukhariSA,IrfanR,MahmoodS.(2013).Hyper-productionofAlkalineProteasebyMutagenicTreatmentofBacillussubtilisM-9using

AgroindustrialWastesinSubmergedFermentation.JMicrobBiochemTechnol5:074-0803. KumarKS, Anuradha S, SarmaGR, Venkateshwaralu Y, Kishan V. (2012). Screening, Isolation, Taxonomy and fermentation of an antibiotic producer

Streptomycesxinghaiensisfromsoilcapableofactingagainstlinezolidresistantstrains.IndianJournalofExperimentalBiology.50:718-728.


149

CHARACTERIZATIONANDEVALUATIONOFSAPONINSFROMYUCCABACCATAAGAINSTGIARDIA

INTESTINALISINVITRO

León-TrujilloRC.1 ,Astiazarán-GarcíaH.2,Robles-ZepedaR.3,Hernández-MartínezJ.4,Quihui-CotaL.11DepartamentodeNutriciónPúblicaySalud,CoordinacióndeNutrición,CentrodeInvestigaciónenAlimentaciónyDesarrollo,Hermosillo(SON)-México;2DepartamentodeNutriciónyMetabolismo,CoordinacióndeNutrición,CentrodeInvestigaciónenAlimentaciónyDesarrollo,Hermosillo(SON)-México;3DepartamentodeCienciasQuímicoBiológicas,UniversidaddeSonora,

Hermosillo(SON)-México;4UnidaddeServiciosdeApoyoenResoluciónAnalítica,UniversidadVeracruzana,Xalapa(Ver)-México.email:[email protected]

Humangiardiasisisaglobalpublichealthproblem,includingMexico.Ithasbeenestimatedthatabout200millionpeopleareinfectedwithgiardiasisannually. Itsprevalence inSonora ishigh (68%),although there isanationaldewormingcampaignsince 1993. Treatments of choice are expensive and may be accompanied by side effects, especially in children. In thiscontext, research in natural products chemistry is a viable option for bioactive compoundswith antiparasitic alternative.Recently,hasbeenreportedthatextractsofnativeplantYuccaschidigerafromsouthwesternUnitedStateshasanti-Giardiaactivity.On theother hand, SonoranDesert has an endemic flora researched.Yuccabaccata is distributed in the SonoranDesert and present similar Yucca schidigera activity. Previous studies in our laboratory have indicated that Y. baccatabutanolicextractsareactiveagainstG.intestinalisininfectedgerbils.TheaimofthisworkwastoisolateandcharacterizeY.baccatastemsaponinsanddeterminetheiranti-giardiainvitroactivity.Theresultsofprotonmagneticresonance(NMR1H)ofbutanolicextractsobtainedfrom2.0kgY.baccatastem(EEYB)flour,indicatethepresenceofsaponinsandactivityagainstGiardiaintestinalis.TheEEYBwassubjectedtocolumnchromatographyusingsilicagel60,230-400meshanda AcOEt-MeOH-H2Omobilephase, isolatingasaponinspiroestansteroidaltype(F8),whichwascharacterizedbyNMR1H,13C,andtwodimensionsHSQCexperiments,HMBC.Ontheotherhand,an inoculumof105trophozoitesofG. intestinalisGS/M-83-H7 inTYI-S-33growthmedium,wasused.Tothis, he added different concentrations of saponin (F8) from (0.1-50 µg / mL). It past 24 hours exposure viability wasobservedusinglightmicroscopy.AnalysisbyHR-ESI-MSshowedanionwithm/z739.4232forC39H63O13.SaponinF8showedanIC50of3.125ug/mLagainstG. intestinalis(P<0.001),whiletheIC50ofMetronidazolewas1.5ug/mL.Inconclusion,theanti-giardiaF8saponinactivitysuggestspotentialasadewormer.Itisimportanttocontinuestudyingtheanti-giardiaF8saponinofY.baccatatoelucidateitsmechanismofactionandtoxicity.1. Quihui-Cota,L..etal.,MarkedAntigiardialActivityofYuccabaccataExtracts:APotentialNaturalAlternativeforTreatingProtozoanInfections.BiomedRes

Int2014,2014:823492.


150

CHARACTERIZATIONOFANTHOCYANINSIN

BILBERRY(VACCINIUMMYRTILLUSL.)FOODDERIVATIVES

BenvenutiS.1 ,BrighentiV.1,RanieriC.1,PellatiF.1,BonaciniC.21DepartmentofLifeSciences,UniversityofModenaandReggioEmilia,Modena-Italy;2EdizioniArtestampa,Modena-Italy

email:stefania.benvenuti@unimore.itVacciniummyrtillusL.isaspontaneousplantofmountainareasofNorthernandCentralEurope,widelydiffusedintheItalianAlpsandApennines.Thefruitsofthisspecies,commonlynamed“bilberry”,havealargecommercialimportanceinthefood(asfreshfruits,jam,juice,liquor),dietary(asdriedbilberryfruitsorextract)andpharmaceuticalindustries(asstandardizedextractintabletorcapsules).Thebilberrycanbeconsideredasoneofthefewmedicinalplants, ifnottheonly,whichisnotconvenienttocultivate;forthis reason it is importantandurgenttodedicateattentiontotheprotectionandenhancementof thissmall fruitandtheethnobotany,nutraceuticalandgastronomicheritageconnectedtoitsapplication.Extensive studies on bilberries have been conducted over several years by researchers of the University ofModena andReggioEmilia and the recognitionof thevirtueshealth-accreditedby these studiesand the importanceofprotectingandenhancingV.myrtillushavesuggestedmorerecently,withintheChamberofCommerceofModena,tointroducethis"smallfruit" inthe"TraditionandflavorsofModena"basketwiththeaimtopromotethequalityoftheApenninebilberry intheprovinceofModena,untilconfinedtotheprovincesofTuscany.Thephytotherapeutic quality ofV.myrtillus is substantially attributable to the high content in active principles and to thechemicalspecificitywhichcharacterizesthem.Amongthesesurelyanthocyaninsarethecomponentswithhealth-protectingattributes such as antioxidant properties, which result in capillaroprotective activities, anti-edema, anti-inflammatory andanti-aging.The common uses of this precious treasure plant range from use in traditional cuisine to the industrial exploitation forpharmaceuticalpurposes.Freshisanexcellentfunctionalfoodthat, inadditiontowell-knownorganolepticproperties,hasbeneficialvirtues.Asconcentratedjuiceisa“nutraceuticalproduct"or“foodsupplement”,andforpharmaceuticalindustryis a real Phytomedicine, "Bilberry dry extract", titrated and standardized in anthocyanins, described in the majorPharmacopoeia,comesinsupplementsandinvariousdrugsusedinophthalmologyforthetreatmentofretinaldiseasesandinthetreatmentofotherdisordersofthemicrocirculation.WiththeaimtodescribethephytochemicalprofileofVacciniummyrtilluswehavecarriedoutmanystudiesandthispresentresearchdescribes the chemical characteristics ofmany samplesof juice, jamand liquorprepared from local company tocontrolthequalityandthegenuinenessoftheseproducts.Thesederivatives,withexcellentandtypicalflavor,aresubjectedtopreparationprocesseswhichcanaffectthephytochemicalandbiologicalprofileofbilberry.23samplesof jam,14of juiceand14of liquor“Mirtillino”wereanalyzedwiththeEuropeanPharmacopeiamethodforthedeterminationoftheanthocyaninscontentand,todescribethedifferentanthocyanins inthesepreparations, anHPLC-UVmethod according to a validated analytical technique was applied. In parallel, a group of expert tasters evaluated theorganolepticcharacteristicsofthesamplesandtheresultsofchemicalandtastingtestshaveledtoarankingofthe“Bestproductaward”forbothcontentanthocyaninsandfortaste.1. C.C.I.A.A.diModena,http://www.tradizionesaporimodena.it2. MelegariM,A.Albasini, (1988) Caratteristichequalitative dei prodotti dei vaccinietimodenesi conparticolare riferimento almirtillo nero (Vaccinium

myrtillusL.)”,AttiSoc.Nat.Mat.Modena,119,31-413. Melegari M, S.Benvenuti, (1998) Caratterizzazione fitochimica, proprietà biologiche e nuove prospettive di utilizzo di ribes, lamponi, mirtilli”,

Frutticoltura,12,334. VampaG.,etal.,HPTLCDeterminationofascorbicacidinfruitsofthegeneraRibes,RubusandVaccinium.PlantaMedica1992,58Suppl.7,A6755. PrencipeFP.Etal.,Metaboliteprofilingofpolyphenols inVacciniumberriesanddeterminationof theirchemopreventiveproperties, JPharmBiomed

Anal.2014,89,257-676. http://www.artestampaweb.it/news


151

POLIACETILENSEXTRACTIONINDAUCUSCAROTA

SperaD.M. ,NapolitanoTR.,VenettacciN.,DiGiammatteoV.ConsortiumofResearchAppliedtoBiotechnology-CRAB,Avezzano(AQ)-Italy

email:[email protected]"polyacetylenes"describesawholerangeofnaturalproductsthatarenotpolymers,infactmanyprecursorsandmanymetabolitescontainonlyasingleacetylenicbond.Thepolyacetylenesintheplantkingdomaresecondarymetabolites;they are therefore metabolics products but does not perform essential vital functions to the plant itself instead as theprimarymetabolites.Althoughtheyarenotessential,theystillplayanimportantroleinthegrowthanddevelopmentoftheplantdefensesystem.Chemicallythepolyacetylenesarecharacterizedbylongchainsofcarbonatomsandbythepresenceof some triple C-C bonds, this makes the molecules particularly reactive. The polyacetylenes, in fact, are unstable andsensitivecompoundstowardsthechemicalandenzymaticoxidativedegradation.Carrotsareconsideredthemainfoodsourceofpolyacetylenes.IncarrotsmajorpolyacetylenesarethreeandbelongtothefamilyofaliphaticC17polyacetylenesoffalcarinol.Thestructureisshowninfigure1.

Figure1:StructureoftheC17aliphaticpolyacetylenesfalcarinolfamily.

Theattentionof thescientificworldhascenteredon falcarinolbecause it isabioactivemolecule thathasbeenshowntohaveapronouncedcytotoxicactivityagainstvarioustumorcells.Theextractionstep is representedbyasolid - liquidextractionusingdicloromentanoasanextractionsolvent. Inorder toestimatetheconcentrationoffalcarinolinoilymatricesextractedfromcarrotsitwasnecessarytousefalcarinolstandardfortheconstructionofacalibration line.Thefalcarinolstandardhasbeenfoundat theShechemGmbHcompany(SiriusFineChemicals ,Fahrenheitstraβe,1,Bremen,Germany).Theproduct,ofanamountequalto20m, itappearedasa liquidoil isstoredinanambervial.1H-NMRspectrashowedtheproductisidentifiedandspecifieda≥95%purity.Fortheidentificationphase of the extracts we used the UPLC / ESI-MS system: mass spectrometry coupled to an ultra high performancechromatographicsystem.Thisprecisetechnique,accurateandsensitiveoffershighselectivityoverotherHPLCtechniques.The technique used could be applied for the determination of polyacetylenes also at other plants. The ESI (electrosprayionisationsource),used inpositivemodeconnectedtothemassspectrometershowedthepresenceofclusters inthegasphaseoffalcarinol.Thetrendistoformadductswiththesolventusedintheelutionstep(especiallywithacetonitrile).1. KreutzmannS.,ChristensenL.P.,EdelenbosM,2007.Theinfluenceoffrozenstorageonpolyacetylenesincarrots(DaucuscarotaL.).RecentHighlights

inFlavorChemistry&Biology.InProceedingsoftheWartburgSymposiumonFlavourChemistryandBiology,Eisenach,Germany,pp284-287.2. Czepa, A., and Hofmann, T. (2004). Quantitative studies and sensory analysis on the influence of cultivar, spatial tissue distribution and industrial

processingonthebitteroff-tasteofCarrots(DaucuscarotaL.)andCarrotproducts.JournalofAgriculturalandFoodChemistry.52,4508-45143. Kobæk-Larsen,M.,Christensen,L.P.,Vach,W.,Ritskes-Hoitinga,J.,&Brandt,K.(2005).Inhibitoryeffectsoffeedingwithcarrotsor(-)-falcarinol

ondevelopmentofazoxymethane-inducedpreneoplasticlesionsintheratcolon.JournalofAgriculturalandFoodChemistry,53,1823–18274. Christensen, L. P., Kreutzmann, S. (2007). Determination of polyacetylenes in carrot roots (Daucus carota L.) by high-performance liquid

chromatographycoupledwithdiodearraydetection.J.SeparationScience.30,483-490


152

NUTRACEUTICALPROPERTIESOFPRUNUSDULCISEXTRACTSOBTAINEDBYSUPERCRITICALFLUID

EXTRACTION

CarlucciG.1,SperaD.M.2 ,AnselmiA.2,CarlucciM.3,FerroneV.11DipartimentodiFarmacia,UniversitàdegliStudi“G.d’Annunzio”ChietiPescara,Chieti-Italy;2CRAB,Avezzano(AQ),Italy;3DipartimentodiScienzeMediche,OralieBiotecnologiche,UniversitàdegliStudi“G.d’Annunzio”ChietiPescara,Chieti-Italy

email:[email protected] thealmondseedsstemsmainly fromtheirhigh lipidcontent,whichconstitutesan importantsource of calories, but does not contribute to the formation of cholesterol in humans, because of their high level ofunsaturatedfattyacids,mainlyfattyacidsmonounsaturated(MUFA),whichareinverselycorrelatedwithserumcholesterollevels.Furthermore,thealmondoilhasahighcontentoftocopherolsthathashighfunctionalpropertiesandnutraceutical.The α-tocopherol (Fig.1) is chemically (2R)-3,4-Dihydro-2,5,5,8-tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-2H-1-benzopyran-6-ol, a derivative of the hydroxy chromane, formed in turn by a condensed benzene nucleus with a pyrannucleus,andacarrierisoacetilicchaininposition2.

From the same vegetable matrix, subjected to different extraction processes, you are obtained extracts that can differgreatlyevenforappearance,chemicalcompositionandbiologicalactivity[1].Thisworkhashelpedtohighlightthefeaturesandcurrentusagefieldsofalmondanddemonstratehowthealmondanditsderivativesintheformofextractsorscrapsoftraditionalfoodprocessingcouldbecomethefinestrawmaterialsforthefoodindustryandallowingfarmacosmetictoopennewmarketsfordifferent industrial fields. It’swas investigatedtheuseofsupercritical fluidsassolvents intheseparationand extraction techniques. This technique represents an important resource at both the scientific and industrial. Thenumerousareasof interest includingfoodproduction,pharmaceutical,petrochemicalandcosmetics.Theunitoperationofsupercriticalextractionhasbeenplacedincomparisonwiththeconventionalextractiontechniques.Thestudyoftheprocessparameters, such as temperature, pressure and flow ratehave allowed toobtain thebestoperating conditions. Thebestoperatingconditionsfortheextractionofoilandtocopherolswerefoundtobe:atemperatureof50±2degrees,apressureof420±20barandaflowrateof25±5kgh-1usingpulverizedalmondseeds[2].Thereareobviousdifferencesinfattyacidcompositionbetweenthetwomethodsofextraction.Fromtheanalysis,themainalmond oil components results are the following (g kg-1): Oleic acid (700), linoleic acid (200) and palmitic acid (70). Thecompositionofmonoandpolyunsaturatedfattyacidsrepresentabout700and200gkg-1oftotalfattyacids[3].TheuseofSFE, offers the advantage of offering of almond oil fractions enriched in tocopherols as noted by several authors. Thedeterminationsoftochopherolandfattyacidinalmondoilareverysimpleandrapidandgivesaccurateandpreciseresults.1. KodadOetal.,Variability intocopherolconcentration inalmondoiland itsuseasaselectioncriterion inalmondbredding,J.Horticult.lSci.Biotechnol..

2006,81:501-507.2.LeoL.etal.Supercriticalcarbondioxideextractionofoilandα-tocopherolfromalmondseeds,J.SciFoodAgric,2005,85:2167-2174.3.YildirimAetal,TotalOilContentandFattyAcidprofileofsomealmond(AmygdaluscommunisL.)Cultivars,J.FoodNutr.Sci.2016,66:inpress


153

INVITROPOTENTIALOFTWOLICHENSFROMDEPARTMENTTOLIMA-COLOMBIAASANTIOXIDANTS

ANDINHIBITORSOFDIGESTIVEENZYMES

OrtizLT. ,PrietoJA. PontificiaUniversidadJaveriana,Bogotá-Colombia

email:[email protected],juliet.prieto@javeriana.edu.coDiseasesgeneratedbymetabolicdisordersareaseriouspublichealthproblem,beingobesitythemostchronicandsystemicdiseasewithworldwideepidemicproportions.Itsfundamentalcauseisanenergyimbalancebetweencaloriesconsumedandcalories expended, and is considered a major risk factor for other diseases such as: diabetes, multifactorial metabolicdeficiency, cardiovasculardiseases (mainlyheartdiseaseand stroke),musculoskeletal disorders (especiallyosteoarthritis),and somecancers (includingendometrial, breast and colon) (WHO, 2014). In turn,oxidative stresshasbeen linked to theetiology of many degenerative diseases: atherosclerosis, cancer, inflammatory processes, diabetes, hypertension,neurodegenerativediseasesandevennaturalprocessessuchasaging(Vidal,etal.,2001).Moreover, thestudyofenzymemechanismshaveprovidedaninterfacebetweenorganicchemistry,drugdiscovery(Khosla,2000);consideringenzymesasmarkersofpharmacologicalimportanceinthedevelopmentofnewtherapeuticagents(Komatsuetal.,2013).Inthesearchfornewsourcesofbioactivecompoundscouldbeconsideredlichens;asymbioticassociationwithnutricional,cosmeticandfarmacological potential. Among the biological activities exhibited by the lichen it can be mentioned: antimicrobial,antioxidant, allelopathic, antiviral, antiproliferative, antiinflammatory, antipyretic, analgesic, among other activities. Theobjectiveofthisresearchistoestablishtheantioxidantcapacityandinhibitoryactivityofdigestiveenzymes,todeterminethetherapeuticpotentialoflichensdepartmentofTolima-Colombia.The lichenic material of dried and ground Usnea angulata y Parmotrema robustum from the upper basin of the riverCombeima (04º19’30’’ y 04º39’57’’ north latitude y los 75º10’11’’ y 75º23’23’’ west longitude) in the department of Tolima-Colombia, was extracted by maceration with a hydro-alcoholic mixture. Each extract was evaluated with respect to itsantioxidantcapacityinvitro,usingthefollowingcolorimetricmethods:DPPHscavenging,ABTSradicalcationdecolorization,totalantioxidantcapacity(TAC)andferricreducing-antioxidantpower(FRAP)(Perez&Saura,2007).Inaddition,theinvitroinhibitoryactivityoftheextractswasestablishedagainstα-glucosidase,lipasepancreaticandtheα-amylaseenzymes(Luyenetal,2014).TheantioxidantactivityresultsshowedpromisingactivityofP.robustumagainstDPPHradical(IC50=184,7±0,63μg/mL)andTAC(134,2±1,02mg/EAA),andofU.angulataagainstABTSradical(IC50=154.9±0.47μg/mL)withareductivecapacityFRAP(555,7±1,55μmFe+2). Inmostcases, theresultswerebetterthanthecontrolsused(BHT,Clorogenicacid).Concerningtheeffectofenzymaticinhibition,theresultsshowthatbothextractshaveamoderate-strongactivityagainsttwoofthethreeenzymestested,P.robustumbeingthemostpromisingforpancreaticlipase(IC50=338,3±2,56μg/mL)andU.angulataforα-amylase (IC50= 611,5±0.62 μg/mL). In the α-glucosidase assay it was determined that both extracts have greater capacityinhibition, with inhibitory concentrations between 3 and 11 times lower than acarbose control (345 ± 11,05 μg/mL). Inconclusion, based on the antiradical and antioxidant potential, the ability of enzyme inhibition revealed by species doforeseesapromisingfutureforU.angulataandP.robustumasasourceofnewbioactivecompounds,usefulastherapeuticsindiseasesgeneratedbymetabolicdisorders,suchasobesityanddiabetes.1. WorldHealthOrganization:WHO,2014.Recuperadode:http://www.who.int/topics/obesity/en/2. Vidal,etal.,2001.EndocrinolMetab.29,410–417.3. Ragheb,R,etal,2009.BiochemBiophysResCommun.389,211–216.4. Khosla,2000.TheJournalofOrganicChemistry.65,8127-8133.5. Komatsuetal.,2013.JournaloftheAmericanChemicalSociety.135,6002-6005.6. Perez&Saura,2007.Vcongresoiberoamericanodetecnologíapostcosechayagroexportaciones,1150–1160.7. Luyen,BTT,etal,2014.BioorgMedChemLett.24,4120–4124.


154

HEAVY-METALBIOACCUMULATIONCAPACITYOFWILDHERBACEOUSPLANTSINTHEPALERMO

URBANAREA

TuttolomondoT.1,LetoC.1,VirgaG.1 ,GennaroMC.1,LicataM.1,LaBellaS.1,GentileF².,DugoG.mo²¹UniversitàdegliStudidiPalermo,DepartmentofAgriculturalandForestSciences,Palermo-Italy;²UniversityofMessina,

DepartmentofBiomedicalandDentalScienceandMorphofunctionalImaging,Messina-Italyemail:[email protected]

In recent years, pollution from heavymetals has gained attention in terms of risk to the environment and public health.Urbanareasareknowntobethemainsourceofcontaminantsduetohighpollutantemissionsfromvarioushumanactivitiesand vehicle traffic (Wiseman et al, 2013; Zereini et al., 2007). Of particular concern in built-up areas is an increase in airpollutionduetoanumberofheavymetals,suchasCo,Ni,Cu,Zn,As,Mo,Cd,PbandHg.Thecapacityofplantstoabsorbtheseelementsintheirtissuescanbeusedasamethodofmonitoringheavy-metallevelsinurbansoils(Maliziaetal.,2012;Yanqunetal.,2005;Elekesetal.,2010).However,theefficacyofsuchamethodreliesonthesensitivityofanygivenspeciestoapollutant.Thisstudyseekstoexaminethecapacityofwildherbaceousplants locatedintheurbanareaofPalermotobio-accumulateheavymetals.Research was carried out in one of the main roads running through the centre of Palermo, Viale della Regione Siciliana,subjecttoextremelyhightrafficintensity.ThespeciesmostcommonlyfoundinallsamplingstationsalongthearterialroadwerePlantagolanceolata,Sorghumhalepense,VerbascumsinuatumandDaucuscarota.Soilandplantsamplesweretakenofthe 4 species in the study from all of the stations for chemical analyses. Plantmaterialwas collected both in spring andsummer.Heavy-metalconcentrationsfoundinthesampleswerecomparedtothosepublishedinanEUstudy(Soilpollutionbyheavymetals),whereasresultsofthesoilanalyseswerecomparedtolegalthresholds.Soilanalysesshowedhigh levelsofZn, followedbyPb,CuandNi. Otherelementswerefoundtobebelow10mgkg-1. Inspring,high-levelaccumulationofZnwasfoundinplanttissues,atarateofalmost40mgkg1,followedbyCu.Tracelevelswerefoundofalltheotherelements(Co,Ni,As,Mo,Cd,Pb,andHg).Concentrationlevelsofheavymetalsfoundinthesoilandplanttissuescanbeconsideredwithinthenormalrange,exceptforHglevelsinP.lanceolata,whichwerecritical.Duringthesummermonths,onceagainresultsshowedhigh-levelaccumulationofZninplanttissues,followedbyPb,NiandCu.Forallthespeciesconcerned,levelsofCo,Ni,As,Mo,Cd,PbandHgwerefoundtobegreaterthanthosefoundinspring,evenupto20timesgreater.Ni,HgandPblevelsinthetissueofV.sinuatumexceededthresholds.SimilarresultswerefoundforHg levels inD. carota andS.halepense. Thewildherbaceousplants in the studywere found, ingeneral, tobemoderatelytoleranttohighlevelsofheavymetalsinthesoil.Variationsinselectiveabsorptionofheavymetalswerefoundamongstthespecies.V.sinuatum,inparticular,showedgoodaccumulationcapacityofPb,NiandHg.AsimilarcapacitywasfoundforD.carotaandS.halepenseonlyforHg.1. MaliziaD.,GiulianoA.,OratggiG.,MasottiA.,2012.Commonplantsasalternativeanalyticaltoolstomonitorheavymetalsinsoil.Chem.Cent.J.6(Suppl

2),56.2. YanqunZ,YuanL,JianjunC,HaiyanC,LiQ,SchvartzC.HyperaccumulationofPb,ZnandCdinherbaceousgrownonlead-zincminingareainYunnan,

China.EnvironInt.2005;31(5):755–62.3. Elekes CC, Dumitriu I, Busuioc G, IliescuNS. The appreciation ofmineral element accumulation level in some herbaceous plants species by ICP-AES

method.EnvironSciPollutResInt.2010;17(6):1230–6.4. WisemanC.L.S.,ZereiniF.,PuttmannW.,2013.Traffic-relatedtraceelementfateanduptakebyplantscultivatedinroadsidesoilsinToronto,Canada.Sci.

TotalEnviron.442,86–95.5. ZereiniF.,WisemanC.L.S.,PuttmannW.,2007.Changesinpalladium,platinum,andrhodiumconcentrations,andtheirspatialdistributioninsoilsalonga

majorhighwayinGermanyfrom1994to2004.Environ.Sci.Technol.41,451–456.6. SoilpollutionbyheavymetalsPE-SO89.5a,Strasbourg24April1989.


155

CAROTENOIDSEXTRACTEDFROMLYCIUMSPPPROMOTEDTHEACTIVATIONOFARYLHYDROCARBON

RECEPTOR

MontesanoD.1 ,CossignaniL.2,ScalisiG.3,TurcoA.3,SimonettiMS.2,BlasiF.1¹DepartmentofPharmaceuticalSciences,UniversityofPerugia,Perugia-Italy;2DepartmentofPharmaceuticalSciences,University

ofPerugia,Perugia-Italy;3DepartmentofExperimentalMedicine,UniversityofPerugia,Perugia-Italyemail:[email protected]

LyciumplantsareSolanaceousdefoliatedshrubsthatprimarilygrowinnorthwestChinaandotherpartsofAsia.Theirfruits(gojiberries)arereportedtohavevariousbiologicalactivitiesandhealth-promotingpropertiesandhavebeenusedinAsiancountriesasatraditionalherbalmedicineandfunctionalfood(Amagase&Farnsworth,2011;Amagase,Sun,&Borek,2009).GenusLyciumpresentsahighcontentofcarotenoidsandthisprovidesahighprovitaminAvalueandantioxidativecapacity.Zeaxanthindipalmitaterepresentsthemajorcarotenoidofthefruitsand it is thefocusofcurrentresearchforpreventionandtreatmentofatrophicage-relatedmaculardegeneration(AMD).Inthispaper,asimpleextractionmethodandaHPLC-DAD-ESIanalysisfortheseparationandquantificationofzeaxanthindipalmitateandtheothercarotenoidsestersingojiberriesisdescribed.Carotenoidtotalcontentinthecrudesampleswasalso determined by usingUV-VIS spectrophotometric assay. The total amount of carotenoids and zeaxanthin dipalmitatewerecomparedamongfruitsofdifferentorigin.Thetotalcarotenoidextract, the isolatezeaxanthindipalmitateandpurestandard zeaxanthin were tested for their capability to act as agonist ligands for Aryl hydrocarbon Receptor (AhR), areceptorinvolvedinxenobioticdetoxificationandimmunoregulation.Themain componentsof the carotenoid fraction in all the considered sampleswas the zeaxanthindipalmitate andotherdifferentcarotenoidesters.ItwasobservedthatthezeaxanthindipalmitatepromotedAhRactivationtoasimilardegreeofwellknownAhRligands.Thesameresultshavenotbeenobtainedwithstandardzeaxanthin.Forthefirsttimethereportedresultssuggestthatzeaxanthininthedipalmitateformmaymediateanti-inflammatoryfunctionsthroughttheinvolvementofAhR.Acknowledgments:TheauthorsaregratefultotheSudRienergys.r.l.farm(CoriglianoCalabro,Italy)forthesupplyofgojiberry1. Amagase, H., & Farnsworth, N. R. (2011). A review of botanical characteristics, phytochemistry, clinical relevance in efficacy and safety of Lycium

barbarumfruit(goji).FoodResearchInternational,44,1702-1717.2. Amagase,H., Sun,B.,&Borek,C. (2009).Lyciumbarbarum (goji) juice improves invivoantioxidantbiomarkers in serumofhealthyadults.Nutrition

Research,29,19-25.3. StockingerB.,DiMeglioP.,GialitakisM.,&Duarte,J.H.(2014).Thearylhydrocarbonreceptor:multitaskingintheimmunesystem.AnnualReviewof

Immunology32,403-432.


156

ANTIOXIDANTCAPACITYANDPHENOLPROFILESOFSOMEBEEHIVEPRODUCTS

FROMUMBRIA(ITALY)

BlasiF.1 ,CossignaniL.2,SimonettiMS.2,MontesanoD.1¹DepartmentofPharmaceuticalSciences,UniversityofPerugia,Perugia-Italy;2DepartmentofPharmaceuticalSciences,University

ofPerugia,Perugia-Italyemail:[email protected]

Beehiveproductsareeasilyaccessiblenaturalproductswhicharebecomingincreasinglypopularduetotheirpotentialroleincontributing to humanhealth, thanks to their antioxidant, antimicrobial, and anti-inflammatoryproperties (da Silva et al.,2016). Total phenol content, antioxidant capacity and phenol profiles of some honey, propolis, and royal jelly samples,harvestedinUmbria(centralItaly),wereinvestigated.Totalphenolcontentwasdeterminedspectrophotometrically followingthemethodrecommendedbySingletonandRossi(1965)basedontheuseofFolin-Ciocalteureagent.Theanalyseswerecarriedoutonfiltered1%aqueoussolutionsofhoneyandroyaljellysamples,whilepollensampleswereextractedwith60%ethanolandthenfiltered(Carpesetal.,2009).The qualitative and quantitative analysis of beehive product phenols was performed by using high-performance liquid-chromatography with diode array detector (HPLC-DAD) and, in order to identify the phenol compounds, a massspectrometer(MS)detectorwasalsoused.Beforeanalysis,thesamplesweresuitablypurified.Twodifferentinvitroassays,DPPHandABTS,wereusedtoobtaindataabouttheantioxidantcapacityofsamples.The DPPH free radical-scavenging activity of beehive products was determined according to the method described byAssimopoulouetal.(2005),whiletheABTSassaywascarriedoutfollowingtheprocedurereportedbyTawahaetal.(2007).Allanalyseswerecarriedoutinduplicate.Thehighestvalueoftotalphenolcontentwasobservedforapollensample,andamonghoneysthemostinterestingvaluewasfoundforonionhoney.Thesamesampleswerecharacterizedbyhigherantiradicalcapacity,determinedbothbyDPPHandABTS assays. Thephenol compounds, identified andquantifiedbyHPLC-DAD-MS,were essentially phenolic acids andflavonoids.Acknowledgments:ThisworkwasfundedbyPSRUmbria2007–2013Mis.1.2.4,EuropeanFundforRuralDevelopment,‘‘Drinkenergizzantenaturalediorigineumbra’’project.1. AssimopoulouANetal.2005.PhytotherapyResearch19,997-1000.2. CarpesSTetal.2009.BrazilianJournalofFoodTechnology12,220-229.3. daSilvaPMetal.2016.FoodChemistry196,309-323.4. SingletonVLandRossiJA,1965.AmericanJournalofEnologyandViticulture16,144-158.5. TawahaKetal.2007.FoodChemistry104,1372-1378.


157

SECONDARYMETABOLITESFROMCHILEANPLANTSANDTHEIRBIOLOGICALACTIVITY

AlarcónJE. ,CespedesCL.,MuñozE.,QuirozS.

UniversityofBio-Bio,Chillan-Chileemail:[email protected],[email protected]

Plants of the Celastraceae and Rhamnaceae that growth in Chile have been studies. These plants have very interestingmetaboliteswithepoxyeudesmane(agarofurane),benzylisoquinolinic,aporphinicandcyclopeptideskeleton.Westudythebiologicalactivityassociateatthesecompounds.Air dried and powdered material was exhaustively extracted with MeOH. The resulting MeOH extract was filtered andconcentrated in vacuum. The residue was suspended in H2O and the sequentially extracted with solvent of increasingpolarity. The resulting fraction were concentrated and chromatographed over silica gel column. Column fraction wereanalyzed by TLC (silica gel 60 F254), and fraction with similar TLC patterns were combined to give pure compounds. AhyphenatedprocedurecombiningHPLC-MS-NMRallowsthedeterminationofchemicalstructuresoftheisolates.Fromtheextracts,fractionsandpurecompoundsforinhibitorycapacityofacetylcholinoesteraseenzymeanditsinsecticidalactivityagainstCydiapomonella,Tenebriomolitorandotherinsectpestswereevaluated.The results show that the extracts, fractions and pure compounds obtained fromChilean plants of the Celastraceae andRhamnaceaefamilypresentaninterestinginsecticidalactivityandinhibitoryactivityofacetylcholinoesteraseenzyme.Acknowledgment:ToFondecytprogramgrant# 1130463.Theauthorswishto thankto internalgrant fromDireccionde InvestigacionUniversidaddelBio-BioDIUBB#083009-2R,#122509and#132209GI/C,UniversidaddelBioBio,Chillan,Chile.1. Alarcón J, Cespedes CL, Muñoz E, Balbontin C, Valdes F, Gutierrez M, Astudillo L, and Seigler DS. 2015. Dihydroagarofuranoid Sesquiterpenes as

AcetylcholinesteraseInhibitorsfromCelastraceaePlants:MaytenusdistichaandEuonymusjaponicas.JAgricFoodChem63,10250-10256.2. AlarcónJ.,CespedesCL.2015.ChemicalconstituentsandbiologicalactivitiesofSouthAmericanRhamnaceae.PhytochemRev14:389–401.


158

DETECTIONOFPANCREATICLIPASEANDACETYLCHOLINESTERASEINHIBITORSINEXTRACTSFROM

Pipercf.asperiusculumANDPiperpertomentellumBYTLC-AUTOGRAPHYMETHODS.

PatiñoOJ.1 ,PrietoJA.2 ,PatiñoWR.3 ¹UniversidadNacionaldeColombia,BogotáD.C.-Colombia;²PontificiaUniversidadJaveriana,BogotáD.C.-Colombia;3Universidad

NacionaldeColombia,BogotáD.C.-Colombiaemail:[email protected],[email protected],[email protected]

Naturalproductshavebeenusedforthousandsofyearsforthebenefitofhumans.Inmedicine,plantshaveprovidedvariousactive principleswhich have inspired the development and synthesis ofmore active compounds (Fonnegra and Jimenez,2007).Neurodegenerativediseasesandobesityarepathologiesthatcurrentlyattracttheattentionofresearchersduetothenegative impacts that they haveon the quality of life of people (González andRodríguez, 2011; Rodríguez andGutierrez,2014).Thus, thewidevarietyofunexplorednaturalsubstancesoffersanalternative treatmentof thesediseases.PlantsofgenusPiper(Piperaceae)arespeciesofgreatimportanceduetoitsuseintraditionalmedicine,becauseareassociatingwithpharmacological activities as astringent, anesthetic, antipyretic, antimalarial, antifungal and insecticide (Dallas, 2004).Despitethebiologicalpotentialofthespeciesofpipergenus,therearestillmanyspeciesthatlackofchemicalandbiologicalactivity studies, as in the case of P. asperiusculum and P. pertomentellum. The objective of this research is CharacterizechemicallyandbiologicallytheethanolextractsfromleavesandinflorescencesofP.cf.asperiusculumvar.glabricauleandP.pertomentellum by a preliminary phytochemical study on TLC and evaluation of enzymatic inhibition of lipase andacetylcholinesteraseusingTLC-autograpyassays.Thepreliminary chemical compositionof ethanolic extractsof leaves and inflorescenceswasdeterminedby apreliminaryphytochemicalanalysisemployingchromatoplatesofsilicagelandcoloringreagents(Merk,2000).TheenzymaticinhibitiononpancreaticlipaseandacetylcholinesterasewereevaluatedbyTLC-autographymethods,takingadvantagethecapacityofthe enzymes to convert α-naphtyl acetate into naphthol which reacts with Fast Blue B salt to make purple-coloredbackgroundonTLCplates,producingwhitespotsonthebackgroundwheretheenzymaticinhibitorsarepresent(Hassam,2012;Marstonetal.,2002).TodetectacetylcholinesteraseinhibitorsalsowasusedtheEllman'smethodonTLCplates(Yangetal.,2012).Preliminaryphytochemicalanalysisallowedtodeterminethepresenceofflavonoids,phenols,terpenesand/orsteroids inextractsofthespeciesstudied.Thetwomethodsusedtodetectacetylcholinesteraseinhibitorsprovidedthesameresults,butthemethodthatusesFastBluesalt isamoreeconomicalmethod,making itamoreappropriatedmethodfordevelopbio-directedstudies.TheextractinwhichareobservedthelargestnumberofzonesofinhibitionofacetylcholinesteraseandpancreaticlipasecorrespondstoinflorescencesofP.pertomentellum.Theobtainedresultsshowthatinthefourevaluatedextracts there are presentmore thanone typeof compoundhaving inhibitory activity on acetylcholinesterase, being themost of inhibition areas related with the spots that revealed the presence of flavonoids and phenolic compounds inpreliminary phytochemical analysis. In inhibiting pancreatic lipase, it is observed that this effect possibly ismainly due totriterpenesand/orsteroids.ThisworkmakescontributionstotheresearchofthespeciesP.cf.var.glabricauleasperiuculumandP.pertomentellumbeing the first reportofchemicalcharacterizationandbiologicalactivity,makingevident thegreatpotentialofthesespeciestoinhibitenzymessuchaspancreaticlipaseandacetylcholinesterase,beingimportantsourcesofstudytofindalternativetherapiesforthetreatmentofobesityandneurodegenerativediseaseslikeAlzheimer.1. DallasLC.2004.Toxicol.Lett.150:85-96.2. FonnegraR.yJimenezS.2007.EditorialUniversidaddeAntioquia.2°edición.XI-XV.3. GonzálezCM,RodríguezCA.2011.Pharmacol.Res.64:438-455.4. HassanAM.2012.PhytochemAnal.23(4):405-407.5. MartsonA,KisslingJ,HostettmannK.2002.PhytochemAnal.13(1):51-54.6. MerckE.Reactivosdecoloraciónparacromatografíaencapafinayenpapel.Darmstadt.R.F.Alemania:Merck.2000.7. RodríguezJLl,GutiérrezHR.2014.Rev.Cub.SaludPública.40:378–387.8. YangZ,etal.2012.Med.Chem.Res.21(6):734-738.


159

CHEMICAL-FUNCTIONALCHARACTERIZATIONOFCOUROUPITAGUIANENSISBARK

RizzoD.1 ,ReT.2 ,VenturaC.1,FanizziFP.3,MiceliA.3

¹SchoolofMedicine,UniversityofBologna,Bologna-Italy;²UnescoChair“HealthAnthropology,BiosphereandHealingsystems”UniversityofGenoa,Genoa-Italy;3DiSTeBA,UniversityofSalento,Lecce-Italy.


Medicinalplantshavebeenusedinhealthcareandpreventionofmostoftheillnesses,neverthelesstheirusesastherapeuticapproachisoftenunderestimated.Then,consideringtheirusesforthousandyearsthatpersisttilltoday,itremainsnecessaryto thoroughly investigate the intrinsic potential of the plant’s Biodiversity as a source of molecules, or betterphytocomplexes,abletoprevent,alleviateortreatmanydiseaseswhichwearestillexposed.CouroupitaguianensisisamedicinalplantofthefamilyoftheLecythidaceaeandithasawideuseamongAmazonianNative.Itwasexperimentallyshownmanyactivitiesaboutthisplant,like:(ascelta).Theaimof the study is to characterize theprofileof secondarymetabolites and themineral contentof the aqueous andethanolicextractsofC.guianensisbark,using:qualitativecolorimetrictests,quantitativetests(Folin-Ciocalteau,FastBlueBB,Aluminiumchloride),InductiveCouplingPlasma(ICP)Test.Asecondsetofanalysisincluding2,2-diphenyl-1-picrylhydrazyl(DPPH)assay,MTTassayandMTTassayininducedoxidativestressonPlacentalMesenchymalStemCellstotesttheantioxidantactivityandtheabsenceofcytotoxicityonthiscellline.IthasbeendemonstratedthepresenceofTriterpenoids,ReducingSugars,Alkaloids,Saponins,PhenolsandAnthroquinonesintheextractsofC.guianensis.FlavonoidcontentwasevaluatedbyAluminiumchloridemethodandresultedof0,173-0,223mg/mlQuercitinequivalents.PhenoliccontentwasevaluatedwithFBBBMethodandFolin-CiocalteauMethod.FortheFBBBMethodresultedtobe4,774-2,790mg/mlGallicacidequivalent.UsingFolin-CiocalteauMethodwasfound1,780-1,010mg/mlGallicacidequivalent.18severalmetalswasquantifiedusingICP.Antioxidantactivitywasevaluatedusing2,2-diphenyl-1-picrylhydrazyl (DPPH)assaythatshowedvaluesof300-437μg/ml intermsofIC50.CitotoxicactivitywasmeasuredbyMTTassayat24-48-72hfromtheadministrationoftheextracts,atseveralconcentration,onPlacentalMesenchymalStemCells. Itwasdemonstratedthat0,1%concentration is themostactive.ThesameresultswasobtainedusingMTTassayinconditionofinducedoxidativestress.


160

COMPARISONOFTWOEXTRACTIONTECHNIQUESFORTHECOMPREHENSIVECHARACTERIZATIONOF

BIOACTIVEPHENOLICCOMPOUNDSINONIONWATREUSINGBYUHPLC-DAD-ESI-HRMS

CamponeL.1,CelanoR.1,PiccinelliAL.2,IbañezE.3,CarabettaS.,diSanzoR.,RastrelliL.2 ,RussoM1 .1DipartimentodiAgraria,UniversitàMediterraneadiReggioCalabria,loc.FeodiVito,ReggioCalabria-Italy;2Dipartimentodi

Farmacia,UniversitàdiSalerno,Fisciano(SA)-Italy;3DepartamentodeCaracterizacióndeAlimentos,InstitutodeFermentacionesIndustriales(CSIC),Madrid-Spain.

email:[email protected];mariateresa.russo@unirc.itFoodindustryproducesalargeamountofonionwastes,makingitnecessarytosearchforpossiblewaysfortheirutilization.Onewaycouldbetousetheseonionwastesasanaturalsourceofhigh-valuefunctionalingredients,sinceonionarerichinseveralgroupsofcompounds,suchasQuercetinanditsglycosylatedformandotherflavonols[1].Thesecompoundsarewellknowntobepotentfreeradicalscavengersandantioxidants,andhaveperceivedbenefitstohumanhealth[1].Theobjectiveof thiswork is carried out an optimization and a comparative study between two environmentally friendly and selectiveextractiontechniques,suchassupercriticalfluidextraction(SFE)andpressurized liquidextraction(PLE)havebeencarriedoutfocusinginthebioactivephenoliccompoundspresentinonionwaste.FortheanalysisoftheSFEandPLEextracts,anewmethodology for qualitative characterization has been developed, based on the use of ultra-high-performance liquidchromatography (UHPLC), coupled with two different detection systems photodiode array detector (DAD) and of highresolutionmass spectrometry (HRMS-MS) detector with an electrospray ionization interface (H-ESI). The two developedextractionmethodandwasausefultoolsforthedeterminationandcharacterizationofmanyphenoliccompoundspresentinOnionwastethatcouldbeusedasagoodsourceofsourceofphenoliccompounds.Acknowledgments: thisworkwas supportedby project PON03 PE_00090_3 “Modelli sostenibili e nuove tecnologie per la valorizzazione delle filiere vegetalimediterranee”[1]Rice-EvansC.etal.Antioxidantpropertiesofphenoliccompounds.TrendsPlantScience2.4(1997):152-159.[2]Jin,EunYoung,etal.Optimizationofvariousextractionmethodsforquercetinfromonionskinusingresponsesurfacemethodology.FoodScienceand

Biotechnology20.6(2011):1727-1733.


161

AFULLYAUTOMATEDMETHODFORDETERMINATIONOFOCHRATOXINAINWINEANDBEERBY

ULTRAHIGHLIQUIDCHROMATOGRAPHYTANDEMMASSSPECTROMETRY.

CamponeL.1,CelanoR.1,RastrelliL.2 ,RussoM.1,PiccinelliAL.21DipartimentodiAgraria,UniversitàMediterraneadiReggioCalabria,ReggioCalabria-Italy;2DipartimentodiFarmacia,Università

diSalerno,Fisciano(SA)-Italy.email:[email protected]

Ochratoxin A (OTA) is natural foodstuff contaminant belonging to the class of mycotoxins mainly produced by fungi ofgeneraAspergillus,Penicillium.Thismycotoxincanbefoundonawiderangeofagriculturalcommoditiessuchaswine,beer,cereals,driedfruits,nuts,coffee[1].The aim of this work was developed a rapid and accurate method based on automated solid-phase extraction-liquidchromatography/electrospray-tandemmassspectrometry(SPE-LC/ESI-MS-MS)fordeterminationofOchratoxinAinwineandbeer.On-lineSPEwasperformedbyloading100μLofwineorbeeratpH8throughanoasisMAXcartridge(anionexchange).After a washing step to removing interfering compounds, Ochratoxin A was eluted with an isocratic mobile phase(MeOH/Water 8:2 0.5% formic) and refocused on column in order to get high chromatography efficiency and good peakshape.Chromatographicseparationwasachievedinlessthan10minusingakinetexC-18reversedphaseanalyticalcolumn.For unequivocal identification and confirmation of analyte, two multiple reaction monitoring (MRM) transitions wereacquiredforOTAinthepositiveelectrosprayionizationmode(ESI+).ThedevelopedmethodwasvalidatedaccordingtoEU’srequirements[2]andaccuracyexpressedasrecoveryrangedfrom80to116%withmethodlimitsofquantification(LODs)inwine and beer were well below the maximum residue limits (MRLs) set by the European Union. Finally, the developedmethodwasappliedtotheanalysisoffiftyrealsamplesofwineandbeer.Thisnewdevelopedmethodoffershighsensitivityandaccuracywithaminimumsamplepre-treatment,offeringahighthroughputanalysis,withareductionofanalysistimeandsolventconsumption1. Rice-EvansC.etal.Antioxidantpropertiesofphenoliccompounds.Trendsinplantscience2(1997):152-159.2. Jin EY. et al. Optimization of various extraction methods for quercetin from onion skin using response surface methodology. Food Science and

Biotechnology20(2011):1727-1733


162

INVITROEFFECTOFBRAZILIANPROPOLISONTHEEXPRESSIONLEVELSOF

MIR-27A-3PANDMIR-19A-3P

DagliaM.1 ,CurtiV.1,ZaccariaV.2,DiLorenzoA.1,MeneghiniS1,FachiniA.1¹DepartmentofDrugSciences.UniversityofPavia,Pavia-Italy;²NaturalResearchandDevelopmentUnit,Corbetta(Milano)-Italy


Propolis is a non-toxic resinous product, collected from plants buds and exudates by Apis mellifera bees. Its chemicalcompositiondependsonbotanicalorigin,geographicalandclimaticzone,andbeespecies.Flavonoidareoneof themainchemicalclasses,contributinggreatly to itsbiologicalproperties. In fact,propolishasbeenusedsinceancienttimesforitsphysiologicalandpharmacologicalproperties,includingantimicrobial(antibacterial,antiviraland antifungal activities), antioxidant, and anti-inflammatory activities (Salim et al.,2015 - Zhang at al., 2016). Growingevidence supports the wide applications of propolis in modern medicine. Nevertheless, in the scientific literature themechanismsofactionatthebasisofpropolispropertieshasnotbeencompletelyelucidated.Thisstudyaimstoinvestigatethe ability of high quality Green Brazilian propolis extract tomodulate the expression levels ofmiRNAs connectedwithoxidativestressandinflammationanditsepigeneticeffects.The expression levels of two miRNAs, miR-27a-3p and miR-19a-3p, connected with oxidative stress and inflammation,respectively,wasevaluatedinHaCatcells(humankeratinocytes)thatweretreatedwithsub-toxicincreasingconcentrationsof green Brazilian propolis extract (prepared according to a patented technology). Moreover, the expression levels ofmRNAscodingforNFE2L2andTNF-α(validatedtargetofmiR-27a-3pandmiR-19a-3p,respectively)werealsodetermined.The study showed that the expression levels of miRNAs and target mRNAs were influenced by green Brazilian propolistreatment. Thus, this study demonstrates that propolis exerts anti-oxidant and anti-inflammatory effects through anepigeneticmechanism.1. SalimEI.etal.AntitumoralandAntioxidantPotentialofEgyptianPropolisAgainstthePC3ProstateCancerCellLine.AsianPacJCancerPrev,2015,16:

7641-7651.2. ZhangJ,etal.AntioxidantactivitiesandmolecularmechanismsoftheethanolextractsofBaccharispropolisandEucalyptuspropolisinRAW64.7cells.

PharmBiol,2016,6:1-16.


163

DESIGNFORFOOD

MartuscielloS.

DepartmentofArchitectureandIndustrialDesignSecondUniversityofNaples,Napoli-Italyemail:[email protected]

The SOCIAL DESIGN COLLECTION FOR FOOD [Diaeta Mediterranea: LANDesign/ali-ment-azione]® is characterized fromcontainers,tools,supportsandfittingsonali-ment-azioneforfoodconsideredtobe"thethingsthatpeopleoranimalseat,particular type of food, the things that plants need in order to grow" in the respect of environment andwelfare of thepeople, for the presentation and storage of food, with particular attention to the tools and procedures of production,preparation, distribution, food origins, traditions and market (FOODesign) and design of games, too large for about 40participants on the educational theme of food, to be used in the spaces of the schools or in public green areas withtemporaryurbandesignfunction(GIOCODesign).The SOCIAL DESIGN COLLECTION FOR FOOD was born from the Applied Research Project [Diaeta Mediterranea:LANDesign/ali-ment-azione]®, scientific directors Prof. Sabina Martusciello / Prof. Maria Dolores Morelli Department ofArchitectureandIndustrialDesignSecondUniversityofNapleswithDepartmentofPharmacyUNISAscientificdirectorProf.LucaRastrelli andBENECONScarl Regional CompetenceCentre for Cultural Ecology EconomySUN,Departmentof SocialPolicticsandCommunicationUNISA,EuropeandForeignDepartmentNationalCouncilof Architects, incollaborationwiththeCampaniaRegionandtheGeneralDirectorateMinistryofEducationCampania-highcultural,scientificandhumanitarianprofile-hasestablishedthevirtuouschain[University+School+Family+Institutes+Companies].The Applied Research Project [DiaetaMediterranea: LANDesign/ali-ment-azione]® is a focus about the relations betweenmanandenvironment,foodandhealth,landanddesign,contentandcontainer.Thegoalistodevelopservicesandproductoffoodproductionchaininnutraceutical,gastronomic,fashion,design,tourismsectorsaswellastothesettingupofnewagriculturalandgreeneconomyenterprises;TheAppliedResearchProject[DiaetaMediterranea:LANDesign/ali-ment-azione]® isbasedonamultidisciplinaryapproach:[ali-ment-azione]“ali”wingssuchascreativeingredient+“mente”mindsuchasscientificingredient+“azione”actionsuchasproductiveingredient;The Applied Research Project [Diaeta Mediterranea: LANDesign/ali-ment-azione]® is a testimonial of “The University forEXPO 2015” and “School Project EXPO 2015”, selected for the ADI COMPASSO D’ORO INTERNATIONAL AWARDS 2015“DESIGN FOR FOOD AND NUTRITION”, winner of the “Special Participatory Planning Award” and the “On-line Award -Sectionrealisedworks”oftheXContestIQU2015(InnovationandUrbanQuality)promotedbytheGruppoMaggioli;secondplaceattheInternationalDesignContestpromotedbyPOLI.DesignthePolitecnicodiMilano“The5seasons2015”IIIAwardat"Ars.Artthatcreatessocialemployment”withspecialmentionoftheAccentureFoundationMIBACTMinistryofCulturalHeritageandTourismin2013;“OSCARGREEN”AwardColdirettiin2011.


164

DESIGNFORNUTRITION

MorelliMD.

DepartmentofArchitectureandIndustrialDesign,SecondUniversityofNaples,Napoli-Italyemail:[email protected]

TheSOCIALDESIGNCOLLECTIONFORNUTRITION[DiaetaMediterranea:LANDesign/ali-ment-azione]®ischaracterizedfromaccessories,clothes,containers,supportsandfurniturefornutritionconsideredas"somenthingthatkeepsyouhealthy,thescienceoffoodanditseffectonhealthandgrowth"thatusefoodanditswaste,designedandprototypedinrespectingtheenvironmentandthewelfareofthepeople,aimedatthecosmo-ethics,actionsandprocessesrelatedbenefitstopeopleandtheenvironment(ACTIVEMODesign)anddesignofcontainers,supportsorfurnishingsandlogosforthenutraceutical:food,cosmetics,medicinesandsupplements,andstrategiesofdrugmarketinglanguages,sanitaryspaceswhichplayanimportanttherapeuticactiononhumanhealthandonthegrowthoraimedatpeoplewithdisabilities(PHARMAFOODesign).The SOCIAL DESIGN COLLECTION FOR NUTRITION was born from the Applied Research Project [Diaeta Mediterranea:LANDesign/ali-ment-azione]®, scientific directors Prof. Sabina Martusciello / Prof. Maria Dolores Morelli Department ofArchitectureandIndustrialDesignSecondUniversityofNapleswithDepartmentofPharmacyUNISAscientificdirectorProf.LucaRastrelli andBENECONScarl Regional CompetenceCentre for Cultural Ecology EconomySUN,Departmentof SocialPolicticsandCommunicationUNISA,EuropeandForeignDepartmentNationalCouncilof Architects, incollaborationwiththeCampaniaRegionandtheGeneralDirectorateMinistryofEducationCampania-highcultural,scientificandhumanitarianprofile-hasestablishedthevirtuouschain[University+School+Family+Institutes+Companies].The Applied Research Project [DiaetaMediterranea: LANDesign/ali-ment-azione]® is a focus about the relations betweenmanandenvironment,foodandhealth,landanddesign,contentandcontainer.Thegoalistodevelopservicesandproductoffoodproductionchaininnutraceutical,gastronomic,fashion,design,tourismsectorsaswellastothesettingupofnewagriculturalandgreeneconomyenterprises;TheAppliedResearchProject[DiaetaMediterranea:LANDesign/ali-ment-azione]® isbasedonamultidisciplinaryapproach:[ali-ment-azione]“ali”wingssuchascreativeingredient+“mente”mindsuchasscientificingredient+“azione”actionsuchasproductiveingredient;TheAppliedResearchProject[DiaetaMediterranea:LANDesign/ali-ment-azione]®isatestimonialof“TheUniversityforEXPO2015”and“SchoolProjectEXPO2015”,selectedfortheADICOMPASSOD’OROINTERNATIONALAWARDS2015“DESIGNFORFOODANDNUTRITION”,winner of the “Special Participatory PlanningAward” and the “On-lineAward - Section realisedworks”of theXContest IQU2015 (InnovationandUrbanQuality)promotedby theGruppoMaggioli; secondplaceat theInternationalDesignContestpromotedbyPOLI.DesignthePolitecnicodiMilano“The5seasons2015”IIIAwardat"Ars.Artthatcreatessocialemployment”withspecialmentionoftheAccentureFoundationMIBACTMinistryofCulturalHeritageandTourismin2013;“OSCARGREEN”AwardColdirettiin2011.


165

AUTHENTICATIONOFHONEYSBOTANICALORIGINBYUHPLC-MS/MSAMINOACIDSDETERMINATION.

APRELIMINARYSTUDY

CarabettaS.1,CamponeL.1,DiSanzoR.1,CefalyV.1,RastrelliL.2,RussoM.1

1DepartmentofAgriculture,LaboratoryofFoodChemistry,UniversityofReggioCalabria,ReggioCalabria-Italy;2DepartmentofPharmacy,UniversityofSalerno,Fisciano(SA)-Italy

email:[email protected](EU)hasadoptedaproposaltoamendtheCouncilDirective74/409/EECconcerninghoney[1].ThisDirectivelayingdowncommonrulesandqualityparameters,suchashumidity,diastaticindex,HMFcontentandpesticideslevels,butsomeoftheseparametershavenorelationshiptogeographicalorbotanicaloriginofhoney.Theaminoacidprofile couldbeuseful tools to identify thebotanicalorgeographicaloriginofhoneysamples.Theaimof thisworkwasthedevelopmentofanalyticalmethodfordeterminationoffreeaminoacids inhoneys.ThemethodisbasedonSPEcleanupofdilutedhoneybyacationexchangecartridge(MCX).ThepurifiedsamplewasdirectlyanalysedbyHydrophilicinteraction chromatography-tandem mass spectrometry (UHPLC–MS/MS) for determination of 20 amino acids. TheparametersofmassspectrometrysourceandMRMtransitionwerecarefullyoptimized.Moreover, inordertoachievethebestchromatographyseparationandagoodpeakresolution,differentcolumnsandmobilephasesweretested.BEHAmidestationary phase using a gradientwith acetonitrile andwater containing formic acid, ammonium acetate and ammoniumformate,hasbeenusedtoobtain,injust15minutes,thecompleteseparationof20α-aminoacidsinvestigated.TheproposedmethodwasappliedtoCalabrianhoneysofdifferentbotanicalorigins(Citrus,Chestnut,Robinia,EucaliptusandSulla).Theobtained results suggested that thedevelopedmethodology is robust and reliable for the analysis of free amino acids inhoney.Acknowledgments:Thisworkwassupportedbyproject“ARAC–Valorizzazionedeiprodottidell’apicolturacalabreseetecnicheinnovativeedecocompatibiliperilrisanamentodeiprodottiapisticicontaminati”RegioneCalabriaAPQAzione3.1. EEC (1974) CouncilDirectiveof 22 July 1974on theharmonizationof the lawsof theMember States relating to honey,74/409/EEC.OfficialJournaloftheEuropeanCommunitiesNo.L221/14.


166

SECHIUMEDULEALKALOIDEXTRACTAPOPTOSISINDUCEDONHELACELLSCERVICALCANCER

RiveraPIA.1,FigueroaAP.2,LunaTAL.3,SilvaTR.1

1EscuelaNacionaldeCienciasBiológicasdelI.P.N.,Mexico;2EscuelaSuperiordeMedicinadelI.P.N.,México;3EscuelaSuperiordeMedicinayHomeopatíadelI.P.N.,México.email:[email protected]

Cervical cancer is considered the secondmost common cancer in theworld, if half amillion new cases are reported andabout2333.400deathsannually. InMexico isconsideredthesecondcauseofdeath.Becausethe importanceofpathologychemotherapeutictreatmentscurrentlyexistfocusedoneliminationoftumorcells.Derivedfromtheextensiveknowledgeof ethnobotany, chayote (S. edule)which is endemic in TraditionalMexicanMedicine as a treatment for various ailmentsincludingconsideringthepossibleanti-tumoreffect.Weproposetodemonstratethelikelyabilityofthealkaloidsobtainedfromchayote(Sechiumedule)theeffectof inducingapoptosisofacelllineoftumororigin,derivedfromcervicalcancer(HeLa).Evaluatetheeffectofapoptosiswithinductorand on HeLa cancer cell line and get the crude chayote (S. edule) as well as fractions of the same extract forchromatographicseparationinwhichapoptosiscontainingalkaloids.ThecellwasdeterminedbyAnnexintincipoonV-FITC/propidiumiodidebothquantifiedbyflowcytometryinassessingeachcases.ApplyingalkaloidsofSechiumeduletocellsculturedHeLacervicalcancercausedagreaterreductioninpercentageofviablecellsatallconcentrationstested(2.5,5.0,7.5and10.0.mu.L).A2.5uLvolumes,5.0uLand7.5uLareductionof10,8and10%of cell viability versus the negative controls, respectively, effect that was greater than that achieved by actinomycin D(positivecontrol)wasachieved.Themaximaleffectisachievedwithavolumeof10.0uLalkaloids,achievinga15%reductionincellviability.Therefore,itisdemonstratedthatalkaloidsS.edulereducecellviability,possiblybyinducingapoptosisincellsculturedHeLacervicalcancer.1. DelCampoJM,PratA,Gil-MorenoA,(2008).Updateonnoveltherapeuticagentsforcervicalcancer.GynecolOncol.110:S72-S76.2. JayshreeRS,SreenivasA,TessyM,KrishnaS.(2009)Cellintrinsic&extrinsicfactorsincervicalcarcinogenesis.JMedRes.13::286-295.


167

ANTITUMOREFFECTANDTOXICITYACUTEETHANOLEXTRACTOFGanodermacurtisii(Berk.)Murrill

LYMPHOMAL5178YINBALB/CMICE.

Montejano-RodríguezJR.1 ,Ríos-RamírezMI.2,Romero-BautistaL.2,Islas-SantillánMA.1,Bautista-AvilaM.1,Almaguer-VargasG.1

1UniversityHidalgoState,PharmacyAcademicArea,SanAgustínTlaxiaca,Pachuca(Hidalgo)-México;2UniversityHidalgoState,BiologyAcademicArea,MineraldelaReforma,Pachuca(Hidalgo)-México


Canceristheuncontrolledproliferationofabnormalcells,thatis,lossofnormalpatternsofcellbehavior1.Theseillnessesareamajorcauseofdeath intheworld2.Malignanttumors inMéxicoranksthird inmortality3andfortheHidalgoStaterankssecond4. Is necessary searching one natural alternative for the treatment of these diseases, one natural choice cheap,accessible,andsafe.Ganodermaisagenusofmushroomusedover4000yearsagoineasternculturesbecauseofmedicinalproperties. In China, your name is Reishi and is called "themushroom of the 10,000 years"; and Ling Zhi "mushroom ofimmortality".MannetakeandshitakeinJapaneseandKorean,YoungZhi5.ThepopulationusesGanodermafortreatmentofcancer6.Theaimoftheresearch istoevaluatetheantitumoreffectofethanolextractofGanodermacurtisii in lymphomaL5178Y(ATCC.CRL-9518)mousemodelinvivoandacutetoxicityinBalb/cmice.ThecollectionwasmadeinthemunicipalityofAcaxochitlánHgo.,inforestQuercus-Pinusandtaxonomicidentification.Theethanolextractwasobtainedbyrotaryevaporation(40 °C).Forantitumortest,7groupsof6maleBalb/cmice30gwereformed, subjected to adjustment period for seven days, weighed and marked. L5178Y lymphoma was inoculatedintraperitoneallyintoeachmouse.ThetoxicitytestwasconductedbasedontheLorkemethod;ninemalemicewereusedtoformthreebatcheswiththreemiceeach,indicatinglowdose1600mg/kg,2900mgaveragedose/kgandhighdose5000mg/kg.DayssurvivalofmiceafterinoculationL5178YlymphomaandtreatmentwithlowdoseofGcurtisii.(0.0128g/kg)wereonaverageof11.22days;andformediumdose(0.128g/kg)were11.8days,butthehighdose(0.64g/kg)causedimmediatedeathofthemice.Accordingtostatisticalanalysis(ANOVA),nosignificantstatisticaldifferencebetweenthelowdoseandtheaveragedosecomparedwiththecontrolgroup.Noanti-tumoreffectwasfound inthedosesusedcomparedwiththecontrolgroup.Results indicate thatG.curtisiididnotshowantitumoreffecton lymphomaL5178Y.G.curtisiiwasvery toxicfrom1600mg/kg.1. NationalCancerInstitute,2011.2. WorldHealthOrganization.2015.3. NationalInstituteofStatisticsandInformationGeographic,2013.4. HealthSecretaryHidalgo,2012.5. RusellRandM.Paterson.Ganoderma:Atherapeuticfungalbiofactory.J.Phytochemystry2006,67:1985-20016. HobbsC.MedicinalMushrooms.InterweavePress,1996,p.98


168

SYNTHESISANDEVALUATIONOFTHEANTI-INFLAMMATORYACTIVITYOFOPTICALLYACTIVE

TRIAZINES

ArteagaDV.1,AboníaR.1 ,Sepúlveda-AriasJC2.,VelozaLA.3 ¹UniversidadDelValle,DepartmentofChemistry,Cali-Colombia;2UniversidadTecnológicadePereira,Pereira-Colombia;

4UniversidadTecnológicadePereira,Pereira-Colombia.email:[email protected],[email protected]

It is known that most of the potential chiral synthetic drugs are generally obtained as racemates. This fact could be adrawback because it is documented that in several cases one of such enantiomers reduces the activity of the other orproduces undesirable side effects. For that, the searching for enantioselective synthetic approaches in drug discoveryprograms continuebeing an issueof highpriority.1 Thepresent researchwork is directed toward the development of anenantioselectivesyntheticapproachofnovelpyrrolidinetriazineswithpotentialanti-inflammatoryactivity,mediatedbytheuseofL-prolineasthekeystartingmaterial.Thesynthesisofourtargetmolecules4wasplannedasdepictedinthefollowingScheme.

Figure1.Proposedsyntheticsequenceforthenovelchiralpyrrolidinetriazines4

TheL-prolinemethylester2wasobtainedbytreatmentofL-proline1withSOCl2inMeOHatreflux,byfollowingapreviousreport.2Then,ester2wassubjectedtorefluxinMeOHwithphenylhydrazineaffordingthehydrazide3(R1=H).Finally,thetreatmentofhydrazide3withbenzaldehydesinEtOHatrefluxinthepresenceofAcOHascatalyst,affordedtheexpectedchiralproducts4ingoodyields.Threenewproducts4(derivedfrom4-ClC6H4CHO,4-MeOC6H4CHOand3,4-Cl2C6H3CHO)havebeenobtainedthroughthissimplemethodology.Lookingforawiderscope,thesynthesisoffurtherproducts4iscurrentlyinprogressstartingfromdiversephenylhydrazineandbenzaldehydederivatives.Inaddition,experimentsareinprogressinorder to evaluate de anti-inflammatory activity of 4 and its derivatives, usingmurinemacrophages (RAW 264.7 cell line)stimulatedwithbacteriallipopolysaccharide(LPS)atdifferenttimepoints.AcknowledgmentstoUniversidaddelValleandUniversidadTecnológicadePereira(Grant9-16-3)forfinancialsupport.1. LaccourreyeO.etal.,Eur.Ann.Otorhinolaryngol.HeadNeckDis.2015,132,31–34;(b)Johannsen,M.J.Chromatogr.2001,937,135–1382. KlegerisA.etal.,J.Neuroimmunol.2004,152,73–77.3. XinZ-Q.etal.,Tetrahedron:Asymmetry2002,13,1937-1940.


169

SYNTHESISANDSTUDYOFPHYSICO-CHEMICALPROPERTYOFDITHIOL-ONE'SDERIVATIVESFROMDITHIOL-THIONE

LaifaEA.

UniversityofConstantine1,Constantine-Algeriaemail:[email protected]

1,2- dithiole- 3-ones and 1,2-D ithiole-3-thiones are an important class of oxygen and sulfur containingheterocycles with antioxidant, chemopreventive and radioprotective actvities, the biological action of which has beenattributed to their redox reactions1. One of the main metabolites of the cancer chemopreventive agent, 4-methyl-5-(2-pyrazinyl)-3H-1,2-dithiole-3-thione(Oltipraz),isaresultofareductivemethylationprocess.ThismetabolitewassynthesizedalsobychemicalreductionofOltiprazwithsodiumsulfidefollowedbyalkylationwithmethyliodide2.Therefore, the reduction of 1,2-dithiole-3-thiones was intensively studied and has been shown that both biochemical3,electrochemical4 and reduction with sodium sulfide affords a dianionic intermediate, which can be alkylated to givedithioesterderivativesorundergoesanintramolecularcyclizationasincaseofOltiprazthisproductinhibitsHIV-1(AIDS)5.Anothergroupdescribedthesynthesisof4-fluoro-5-polyfluroalkyl-1,2-dithiole-3-thiones(R1=CF3,HCF2CF2,R2=F)andstudiedtheir cycloaddition properties as dienophiles and as 1,3-dipoles6. Also The natural product leinamycin, an antitumor,antibiotic isolated from Streptomyces sp, displays potent antitumor and cytotoxic activities and an interesting activityagainstGram-positivebacteria.We therefore undertook a study in order to develop a method to synthesize, identification and study biological andpharmaceutical interest of various derivatives of 1,2-dithiolan-3-one 1-oxides. The intramolecular cyclization of2,2’dithiodibenzoic acid and they derivatives offered 3H-1,2-benzodithiol-3-thione which, by oxidation gave 3H-1,2-benzodihiol-3-thione1-oxidewhichwereportinthiscommunication.1. KwakMK,P.A.Egner,P.M.Dolan,M.Ramos-Gomez,J.D.Groopman,K.Itoh,M.Yamamoto,T.W.Kensler,Mutat.Res.480–481(2001)305–315.2. LargeronM,D.Fleury,M.B.Fleury,Tetrahedron42(1986)409–415.3. LevronB,G.Burgot,J.-L.Burgot,Arch.Biochem.Biophys.382(2000)189–1944. DarchenA,P.Berthelot,C.Vaccher,M.NazarethViana,M.Debaert,J.-L.Burgot,J.Heterocycl.Chem.23(1986)1603–1607.5. AbazidM,J.L.Burgot,A.Darchen,M.Saidi,PhosphorusSulfurSiliconRelat.Elem.53(1990)425–427.6. TimoshenkoVM,J.-P.Bouillon,Yu.G.Shermolovich,C.Portella,TetrahedronLett.43(2002)5809–581


170

COLON-AVAILABLEDIETARYMELANOIDINSEXHIBITCYTOTOXICACTIVITYONHUMANCOLONADENOCARCINOMACELLLINES

TagliazucchiD. ,MartiniS.,ConteA.

¹UniversityofModenaandReggioEmilia,DepartmentofLifeSciences,ReggioEmilia-Italyemail:[email protected]

Emergingevidencefrom invitroand invivostudiessuggestedthatthegastro-intestinaltractmightbethekeysiteforthebiologicalactionof foodmelanoidins[1].Recently,Vitaglioneetal. [2] reviewedthepossiblemechanismsbywhichcoffeemelanoidins may influence the risk of colorectal cancer development. In this work, we tested the water-soluble highmolecularweightmelanoidins(HMWM)obtainedfromdifferentfoodsourcesfortheircytotoxicactivityagainstcoloncancercelllines.HMWMwereobtainedbyultrafiltration(cut-off10kDa)frominstantcoffee,cocoabrew,darkbeerandinstantbarleycoffeebeverages [2]. HMWM have been chemically characterized for their content in total polyphenols, protein, andpolysaccharidesaswell as their spectroscopicproperties.ThevalueofKmix420nm (definedas theabsorptionat420nmofasolution of HMWM at a concentration of 1 g/L) provides information on the relative amount of melanoidins (Kmix 420nm)respecttoothercompoundsfoundintheHMWMofbeverages.Cytotoxicactivityagainsttwocoloncancercelllines(Caco-2andSW480)wasassayedbyMTTassay.HMWMobtainedfromtheabovebeveragessubmittedto invitrogastro-intestinaldigestion[3],weresubjectedtothesamechemicalanalysisandassayedfortheircytotoxicactivity.Barley coffee had the highestHMWMcontent (83.3g/100g of drymatter) followed by coffee (34.4g/100g of drymatter),cocoa(28.1g/100gofdrymatter)anddarkbeer(20.0g/100gofdrymatter).TheHMWMofcoffeehadthehighestKmix420nmvalue,suggestingthatthisfractionisrelativelyrichinmelanoidins.Onthecontrary,despitethehighestcontentinHMWM,barleycoffeewasthesamplewiththelowestmelanoidinscontent.ProteincontentofHMWMrangedfrom6.1%(darkbeer)to10.6%(coffee)whereaspolysaccharidescontentrangedfrom42%(darkbeer)to61%(cocoa).CocoaandcoffeeHMWMhadthehighestphenoliccontent(6and12%,respectively).TheinvitrodigestioncausedageneraldecreaseinthepolysaccharideandproteincontentofalltheanalysedHMWMbecauseoftheactionofamylolyticandproteolyticenzymes.Theanalysisofthecontentofpolyphenolsshowedadecreaseinthesecomponentsasaresultofthedigestion.Onthecontrary,theinvitrodigestion caused an increase in the Kmix420nm of all the HMWM investigated suggesting that this process resulted in anenrichmentinmelanoidins.TheeffectofHMWMonthehumancolonadenocarcinomaCaco-2andSW480cellcultureswasinvestigated. Un-digested HMWM had no significant cytotoxic effect against both the cell lines. However, the in vitrodigestiongreatlyincreasedthecytotoxicactivityofHMWMagainstboththecelllines.CoffeeHMWMwerethemostactiveagainstCaco-2cellswhereascocoaHMWMwerefoundtobemostactiveagainstSW480cells.Consideringthat thecolonaccumulatesitscontentoveratleast24hinamaximumvolumeof2l,itispossibletoestimatetheconcentrationsofHMWMreachingdailythecolonwithdietaccordingtothedifferentfoodmatrices[1].Datashowedthat,theconsumptionofcoffee,cocoaandbarleycoffeebeveragesmayresultinintestinalconcentrationsofHMWM,whicharecytotoxicagainstCaco-2andSW480celllines.1. Tagliazucchietal.Thegastro-intestinaltractasthemajorsiteofbiologicalactionofdietarymelanoidins.AminoAcids2015,47:1077-10892. Vitaglioneetal.Coffee,colonfunction,andcolorectalcancer.FoodFunct2012,3:916–9223. Tagliazucchietal.Effectofdietarymelanoidinsonlipidperoxidationduringsimulatedgastricdigestion:theirpossibleroleinthepreventionofoxidative

damage.JAgricFoodChem2010,58:2413-2519


171

AFACILESYNTHESISOF6-AZASTEROIDS

MartínezPascualR.1,MontielSmithS.1 ,VegaBaezJL.1

BeneméritaUniversidadAutónomadePuebla,FacultaddeCienciasQuímicas,Puebla-México.email:[email protected]

Azasteroidshavebeendescribedtopossessrelevantbiologicalactivitiessuchas5α-reductaseinhibitors[1].Inthiscontext,some 6-azaesteroids have been described to exhibit equivalent inhibitory activity against 5α-reductase compared tofinasteride (4-azasteroid), which is the first drug accepted by US to treat benign prostatic hyperplasia [1]. A recurringmethodologytoprepare6-azasteroidsfrom3-hydroxy-Δ5steroidsconsistsofarouteofatleastsixsteps.Theseproceduresusually employ chromium-based oxidants or ozone to afford a 6-carboxy-secosteroid, which is the key precursor in thisprocedure[2].Herein,weintroduceanovelmethodologytosynthetizetwo6-azaesteroidsusingcholesterolanddiosgeninacetateasstartingmaterialswithouttheuseofpollutingoxidants.Inordertoinsertanitrogen-containingmotifattheC-6positionofcholesterolordiosgeninacetates,weplannedtoobtainthe oximes 2a or 2b as key precursors, which in turn could be transformed into the 5,6-seconitrile derivatives 3a or 3b.Subsequentreactionsonthenitrilefunctioncouldaffordthetarget6-azasteroids4aand4b(Figure1).

Figure1:Generalmethodforthesynthesisof6-azasteroids.Keyprecursors2aor2bwereobtainedfollowingatwo-stepsequence:1)TreatmentofcholesterolordiosgeninacetatewithNaNO2,BF3

.OEt2inAcOHafforded3β,5α-diacetoxy-6-nitroiminederivatives[4].2)ThenitroiminesmoietiesweretransformedintothecorrespondingoximeswithNaOAcandNH2OH.HClinrefluxingethanol.CleavageofBringwasachievedthroughasecondorderBeckmann rearrangementof theoximesaffordingseconitrilederivatives3aor3b.Thenitrileswerepartiallyhydrolyzed to the corresponding amides [5]. Finally, one pot Hoffmann rearrangement/cyclocondensation reaction wastestedtoobtainthe6-azasteroids.Insummary,wehavedevelopedanovelproceduretoobtain6-azaesteroidsavoidingtheuseofpollutingoxidantsorozone.Acknowledgments:WethankCONACYTforthescholarshiptoR.M.P.andforthefinancialsupportviatotheproject240329.1 AggarwalS.etal.Anoverviewon5α-reductaseinhibitors.Steroids,2010,75,109-153.2 FryeS.V.etal.6-Azasteroids:Structure-ActivityRelationshipsforInhibitionofType1and2Human5.alpha.-ReductaseandHumanAdrenal3.beta.-Hydroxy-

.DELTA.5-steroidDehydrogenase/3-Keto-.DELTA.5-steroidIsomerase.J.MedChem.1994,22,2352-2360.3 Ibrahim-OualiaM.andRocheblaveL.Recentadvancesinazasteroidschemistry.Steroids2008,73,375-407.4 LópezY.etal.MechanisticinsightsandnewproductsofthereactionofsteroidsapogeninswithNaNO2andBF3·Et2Oinaceticacid.Steroids2008,73,657-

668.5 LeeJ.etal.Anhydroushydrationofnitrilestoamidesusingaldoximesasthewatersource.Org.Lett.2009,11,5598-5601.


172

REACTIVITYOFCEMBRENESANDVERTICILLENESFROMBURSERACEAEUNDERACIDTREATMENTS

Hernández-HernándezJD.1 ,delRío-ChávezAA.1,EscobarFloresKD.1,Román-MarínLU.1,García-

GutiérrezHA.1,Cerda-García-RojasCM.2,Joseph-NathanP.21InstitutodeInvestigacionesQuímico-Biológicas,UniversidadMichoacanadeSanNicolásdeHidalgo,Morelia(Michoacán)-México;

2DepartamentodeQuímica,CentrodeInvestigaciónydeEstudiosAvanzadosdelInstitutoPolitécnicoNacional,CiudaddeMéxico-México.

email:jdiego@umich.mxPaclitaxelasanantitumoragenthasledtotheopeningofaveryextensivefieldresearchincludingstudiesontricyclictaxanediterpenes,verticillenebicyclicderivativesandmonocycliccembrenoids,ofwhichareallofthembiogeneticallyconnected[1].Giventhepotentialtherapeuticofferingallthesestructures,itisimportanttoprepareandstudynovelderivativeswhichcanbeobtainedbychemical transformationofnaturalproducts.Theverticilleneandcembrenediterpenoidswasfound indiferent sources including several species of the family Burseraceae. Of the hexane extracts from the stems of Burserakerberi [2], Bursera suntui and Bursera multijuga [3] was isolated several verticillene and cembrene derivatives; thiscompoundswere treatedwithm-chloroperbenzoic acid toprepare epoxidizedderivatives selectively varying the reactionconditions such as time and concentration, also were treated first with Et2O:BF3 and other side HClO4 generating

dehidrationandrearrengementproductsalsovaryingthereactionconditionssuchastime,temperatureandconcentration.FinallysomeofepoxidizedderivativesweretreatedwithHClO4generatingnewketonedrivativesasopeningproductsof

epoxide.ThestructuresofthenewcompoundsweredeterminedbyNMRinoneandtwodimensions.1. Hosoyama,H.;Inubushi,A.;Katsui,T.;Shigemori,H.;Kobayashi,J.Tetrahedron,1996,52,13145.2. Hernández-Hernández,J.D.;Román-Marín,L.U.;Cerda-García-Rojas,C.M.;Joseph-Nathan,P.J.Nat.Prod.2005,68,1598.3. JuanD.Hernández-Hernández,HugoGarcía-Gutiérrez;LuisaUraniaRomán-Marín;YunuénI.Torres-Blanco;CarlosM.Cerda-García-Rojas;PedroJoseph-

Nathan,NaturalProductCommunications,2014,9,1249-1252


173

ANTI-INFLAMMATORYANDANALGESICEVALUATIONOFFLOWERSSEDUMPRAEALTUMDC

Márquez-FloresYK.,OrtegaFLF.,Silva-TorresTR.,Meléndez-CamargoME.

EscuelaNacionaldeCienciasBiológicas,InstitutoPolitécnicoNacional,UnidadProfesionalAdolfoLópezMateos,GustavoA.Madero,CiudaddeMéxico-Méxicoemail:[email protected]

SedumpraealtumDC(Crassulaceae)isaplantwidelyusedintheMexicantraditionalmedicinetotreatbucalulcers,gumsandeyesirritationandithasbeneficialeffectinteethpainandburns.Inexperimentalmodels,extractofstemsandleaves,andthelyophilizedfromthisplanthavedemonstratedahighanti-inflammatoryactivityincottonpelletgranulomamodelinratsandthisresponsewasdose-dependent.Also,ithasbeenreportedthatmaincomponentsofextractarealkaloids,flavonoidsand terpines (Meléndez et al., 2002; Castro-Mussot et al., 2011). Inflammation is widely relatedwith pain, because theseprocessarepathophysiologicalresponsesofthebodytoharmfulstimuliandsomesignalingpathwaysarewellcorrelated.Basedontheabove,theobjectiveofthepresentwasevaluatetheanti-inflammatoryandanalgesiceffectsofflowersethanolextractofS.praealtuminmurinemodels.Femalemice30±5gandWistarrats200±20gofbodyweight(b.w.)wereused.FlowersethanolS.praealtumextract(SEE)waspreparedtoadministrate100and200mg/kgdoses(SEE100andSEE200groups).Preliminaryphytochemicalanalysiswasrealized.Animalsweregroupedandresultswerecomparedwithshamgroup.Aceticacidinducedwrithingresponseandhot plate models were perform to evaluate analgesic activity. Cotton pellet-induced granuloma formation were used toevaluateanti-inflammatoryactivity.Indomethacin5mg/kgofb.w.(IND)andacetylsalicylicacid100mg/kgofb.w.(AAS)wereusedasanti-inflammatoryreferencedrugs.Pain isacomplexunpleasantphenomenoncomposedofsensoryexperiences that includetime,space, intensity,emotion,cognition and motivation originating from damage tissue or abnormal physiological condition. In acetic acid inducedwrithing,theanimalreactswithacharacteristicstretchingbehaviordueto irritationofserousmembraneswhilehotplatemodelproducestwobehavioralcomponentsthancanbemeasuredintermsoftheirreactiontimes,namelypawlickingandjumping.Bothare considered tobe supra-spinally integrated responses (MilindandMonu, 2013).Abdominal constrictionsinduced by intraperitoneal injection of 0.6 % acetic acid were reduced with SEE 100 and 200 (P < 0.05 and P < 0.001,respectively).Similareffectwasobservedinhotplatemodel,inwhichSEE100wascapabletodecreaselatencytimeat2h(P<0.001)withthehighest inhibitionpercentage(68.5%)comparedwithallgroups).SEE200 inducedadecreaseof latencytimeat1,2and3h(P<0.001,P<0.001andP<0.01,respectively).TheseresultsdemonstratedthatSEEiscapabletodown-regulatedthenociceptionprocessatperipheralandcentrallevel,sincethemechanisminvolvedinthefirstmodelisrelatedwithreleaseofarachidonicacidmetabolitesviacyclooxygenase(COX)andprostaglandinbiosynthesis(Franzottietal.,2000)andthesecondwithcentralresponsetothermalstimuli.For theotherway,weevaluatedSEE 100 incottonpellet-inducedgranulomaformationmodel,observing that theextractdecreasethewet,dryandfinalgranulomaweight(P<0.05,P<0.001andP<0.01,respectively),similarlytoIND.UnlikeIND,SEE did not produce stomach or duodenal injury. This results revealed that SEE is decrease transudative, exudative andproliferativephasesof the response toa subcutaneously implantedcottonpellet (SwingleandShideman, 1972).With theabove, we can conclude that flowers ethanol S. praealtum extract possesses anti-inflammatory and analgesic in murinemodels.1. MeléndezCME,BuendíaRM,RamosZD,CardonaCP,VillarrealMME,2002.Studyoftheanti-inflammatoryeffectofSedumpraealtum(Siempreviva)in

therat:dose-dependentresponse.ProcWestPharmacolSoc,45:129-130.2. Castro-Mussot ME, Silva-Torres R, Meléndez-Camargo, 2011. Anti-inflammatory and anti-phagocytic activities in the extract of Sedum praealtum

(Siempreviva).In:AwaadAS,KaushikG,GovilJN.Mechanismandactionofphytoconstituents.RecentProgressinMedicinalPlants.Vol31.pp1-8.3. MilindP,MonuY,2013.Laboratorymodelsforscreeninganalgesics.IRPJ,4(1):15-19.4. FranzottiEM,SantosCVF,RodriguesHMSL,MourãoRHV,AndradeMR,AntoniolliAR,2000.Anti-inflammatory,analgesicactivityandacutetoxicityof

SidacordifoliaL.(Malva-branca).JEthopharmacol,72:273-278.


174

MorusnigraL.LEAVESEXTRACTSSTANDARDIZEDINCHLOROGENICACID,RUTINANDISOQUERCITRIN:

TYROSINASEINHIBITION

FreitasMM.,FontesP.,Homem-de-MelloM.,SouzaPM.,SilveiraD.,Fonseca-BazzoY.,SimeoniLA.,MagalhãesPO.

UniversityofBrasilia,HealthScienceSchool,LaboratoryofNaturalProducts,Brasília(DF)-Brazilemail:[email protected];[email protected]

Melanogenesis isaprocess responsible formelaninproduction,which isstored inmelanocytescontainingtyrosinase.Thisenzymeisresponsibleforskinhyperpigmentationduetotheoverproductionofmelanin.Inhibitionofthisenzymeisatargetinthecosmeticsindustry,sinceitcontrolsundesirableskinconditions.PlantspeciesoftheMorusgenusareknownforthebeneficialusesoffered indifferentpartsof theplant, includingtyrosinase inhibition.Thus, thisprojectaimedtostudy theinhibitoryactivityoftyrosinasebyextractsfromMorusnigra leaves,aswellasthecharacterizationofthechromatographicprofileofitsextractandviabilityofincorporationincosmeticsfromtheircytotoxicity,inordertobecomeanewtherapeuticoptionfromanaturalsource.M.nigra leaveswerecollected,pulverized,equallydivided into fivebatchesandthestandardizedextractwasobtainedbypassivemaceration.Thevoucherspecimenwasdeposited in theHerbariumof theUniversityofBrasília,numberFaggCW2302. Inorder toassessextractionreproducibility, thetotalsolidscontent,yieldandmoisturecontentweremeasuredforeachbatch.TyrosinaseenzymaticactivitywasperformedforeachbatchaccordingtoKhatibetal.(2005)withmodifications(1), providing the percentage of enzyme inhibition and IC50 values, which were obtained by constructing dose-responsecurvesandcomparedwithkojicacid,awell-knowntyrosinaseinhibitor.AHighPerformanceLiquidChromatography(HPLC)wasusedtodeterminechlorogenicacid, rutinand isoquercitrin levels inM.nigraethanolextract.ADionexUltiMate3000liquidchromatographysystemequippedwithaDiodeArrayDetectorDAD-3000operatingin280,330and354nmwasused.ThestandardizedM.nigraleavesextractshowednosignificantdifferencebetweenbatchesfortotalsolidscontent,yieldandmoisturecontent,whichshowsgoodreproducibilityoftheextractionprocess,alsodemonstratedbyTLCchromatographicprofile.High inhibitionoftyrosinaseactivitywasobserved(above90%at 15,625µg/mL)and IC50valuesrangingfrom5.00µg/mL ± 0.23 to 8.49 µg/mL ± 0.59, comparable to kojic acid (3.37 µg/mL ± 0.65). According to ICH guidelines, thechromatographicmethodemployedusingHighProfileLiquidChromatography(HPLC)wasvalidated.HPLCanalysisrevealedthepresenceof isoquercitrinas itsmajorcompound,followedbyrutinandchlorogenicacid,withsimilarchromatographicprofiles on all batches. Thus, M. nigra leaf extract was standardized using these polyphenols as markers, in whichisoquercitrinconcentrationis,onaverage,2.34timesgreaterthanchlorogenicacidand,onaverage,1.54timesgreaterthanrutin in all batches. This study demonstrated the potential ofM. nigra extract as a promisingwhitening agent of naturalsourceagainstskinhyperpigmentation.Acknowledgments: Authors are grateful to National Council of Technological and Scientific Development (CNPq), Support Research of the Federal DistrictFoundation (FAPDF), Higher Education Personnel Improvement Coordination (CAPES), Scientific and Technological Enterprise Foundation (FINATEC) andUniversityofBrasilia(UnB)forprovidingfundstoperformthepresentresearch.1.KhatibS.etal.Chalconesaspotenttyrosinaseinhibitors:theimportanceofa2,4-substitutedresorcinolmoiety.BioorgMedChem.2005,13:433-41.


175

ANGIOTENSIN-CONVERTINGENZYME(ACE)INHIBITORYACTIVITYOFSALVIALERIIFOLIABENTH.

EXTRACTS

Loizzo,M.R.1 ,RastrelliL.2,FalcoT.3,TundisR.1

¹UniversityofCalabria,Pharmacy,HealthandNutritionalSciences,UniversityofCalabria,ArcavacatadiRende(CS)-Italy;2DepartmentofPharmacy,UniversityofSalerno,Fisciano(SA)-Italy;3DepartmentofEnvironmentalandChemicalEngineering,

UniversityofCalabria,Rende(CS)-Italy.email:[email protected]

Salviaspecieshavebeenusedsinceancienttimesinfolkmedicineinthetreatmentofcardiovasculardiseases.Severalpre-clinical studiespoint towardspromising effects ofSalvia speciesononeof themost important cardiovascular risk factor:hypertension.AngiotensinConvertingEnzyme(ACE)inhibitorsarewidelyuseforthetreatmentofhypertension(Shlipaketal. 2001). As a part of our research for a natural therapeutic approach to the treatment of high blood pressure, in vitroscreeninghasbeenconductedtoevaluatetheACEinhibitoryactivityofS.leriifoliaextracts.S.leriifoliaaerialpartswereextractedbymacerationwithmethanolandsuccessivelypartitionedwithdichloromethane,ethylacetateandn-butanol.TheACEinhibitoryactivitywasmeasuredusingthemethoddescribedbyHansenetal.(1995).Folin-Ciocalteaumethodwasusedtodeterminingthetotalphenolscontent.Themethanol extract inhibited ACE enzymewith an IC50 value of 199.7 µg/mL. After fractionation, ethyl acetate fractiondemonstrated thehighestACE inhibitoryactivity,withan IC50valueof 104.3µg/mL, followedbydichloromethane fraction(IC50 value of 158.7 µg/mL). The ethyl acetate fraction a high total phenols content. TheACE inhibitory activity of severalSalvia specieswas previously demonstrated. S. acetabulosa methanol extract inhibited ACEwith IC50 value of 52.7 µg/mL(Loizzoetal.2008).Morerecently,Jiménez-Ferreretal.(2010)demonstratedtheinvivoACEinhibitoryactivityofS.eleganshydroalcoholicextract (50.27%).Thisextractat thedoseof0.75mg/kgsignificantlydecreasedsystolicbloodpressureandevenhadanantihypertensiveeffectthatwasgreaterthanthattreatmentwithlosartan.In conclusion, our result confirm the potential use of Salvia extract for the treatment of hypertension. Further study arenecessarytoidentifyS.leriifoliabioactiveconstituents.Acknowledgments:AuthorsthankFarsadNadjafi,MedicinalPlantsandDrugsResearchInstitute,ShahidBeheshtiUniversity,GC,Tehran,Iran,forsupplyingtheplantmaterial.1. HansenK.etal.Invitroscreeningoftraditionalmedicinesforanti-hypertensiveeffectbasedoninhibitionoftheangiotensinconvertingenzyme(ACE).J

Ethnopharmacol1995,48:43-51.2. Jiménez-FerrerE.etal.,AntihypertensiveactivityofSalviaelegansVahl.(Lamiaceae):ACEinhibitionandangiotensinIIantagonism.JEthnopharmacol.

2010,130:340-6.3. LoizzoMR.etal.InvitroinhibitoryactivitiesofplantsusedinLebanontraditionalmedicineagainstangiotensinconvertingenzyme(ACE)anddigestive

enzymesrelatedtodiabetes.JEthnopharmacol.2008,119:109-16.4. ShlipakMGetal.Comparisonoftheeffectsofangiotensinconverting-enzymeinhibitorsandbetablockersonsurvivalinelderlypatientswithreduced

leftventricularfunctionaftermyocardialinfarction.AmJMed.2001,110:425-433.


176

CHEMICALCOMPOSITIONANDENHANCEMENTOFTECHNOLOGICALPROPERTIESOFHALIMIUM

HALIMIFOLIUMEXTRACTWITHANTIOXIDANTACTIVITYKerbabK.1,MekhalfiT.1,ZaiterL.1,BenayacheS.1,BenayacheF.1,PicernoP.2,MencheriniT.2,Sansone

F.2,AquinoRP.2,RastrelliL.2 ,SmithAB.²,BrownABC.1,TaylorA.1,EvansAB.2¹UnitéderechercheValorisationdesRessourcesNaturelles,MoléculesBioactivesetAnalysesPhysicochimiquesetBiologiques

(VARENBIOMOL),DépartementdeChimie,FacultédesSciencesExactes,UniversitéConstantine1,Constantine-Algérie;²DipartimentodiFarmacia,UniversitàdegliStudidiSalerno,Fisciano(Salerno)-Italy

email:[email protected] isanendemicplant inAlgeriausedforthetreatmentofdigestivediseasesTodate,afewmolecularconstituentshavebeenisolatedfromthisplantandchemicallycharacterized,buttheirbiologicalactivitieshaveneverbeenreported. Inourcontinuingsearch forbioactivecompounds fromAlgerian medicinalplants [1],wereportedthechemicalcompositionandenhancementoftechnologicalpropertiesofHalimiumhalimifoliumextractwithantioxidantactivity.ABrukerDRX-600NMRspectrometer,operatingat599.19MHzfor1Hand150.858MHzfor13C,usingtheWINXNMRsoftwarepackage,wasused forNMRexperiments inCD3OD.Chemical shifts areexpressed inδ (partspermillion) referring to thesolventpeaksδH3.31andδC49.05forCD3OD,withcouplingconstants,J,inHertz.1H-13CHSQC,andHMBCexperimentswereobtainedusingconventionalpulsesequences.SemipreparativeHPLC,with isocraticelution,wasperformedwithaWaters590 seriespumping systemequippedwithaWatersR401 refractive indexdetector, a LunaC-18 column (250× 10.00mm,particlesize10mm;Phenomenex,Inc.),andaRheodyneinjector(100μLloop).QuantitativeHPLCanalysiswascarriedoutinanAgilent1260seriessystem.Phytochemical investigationofthisspecieshasledtotheisolationof14constituents.Allthestructureswereelucidatedbyspectroscopic methods including 1D and 2D NMR experiments, as well as ESIMS analysis. Quantitative détermination ofphenolic constituent from H. halimifolium water infusion was performed by HPLC-MS /MS. H. halimifolium exhibitedsignificant free-radical scavengingactivity,evaluatedbyDPPH-test.However,H.halimifolium has showna lowsolubility inwaterandinbiologicalfluids,whichcouldlimititsbioavailabilityaswellastherapeuticeffectafteroraladministration.Inthisresearch, a systemwereproducedby a spray-dryingprocess using a combinationof cellulose acetate phthalate (CAP) ascoating gastroresistant polymer and swelling or surfactant agents as enhancers of dissolution rate [2]. Themicroencapsulationhavegivenlong-lastingstoragestabilityandhavepreservedtheantioxidantproperties.1. Kerbab,K.,Mekhelfi,T.,Zaiter,L.,Benayache,S.,Benayache,F.,Picerno,P.,Mencherini,T.,Sansone,S.,Aquino,R.P.,&Rastrelli,L.(2015).Chemical

compositionandantioxidantactivityofapolarextractofThymelaeamicrophyllaCoss.etDur.NaturalProductResearch,29,671-675.2. Sansone,F.,Mencherini,T.,Picerno,P.,Esposito,T.,DelGaudio,P.,Russo,P.,Pepe,G.,Lauro,M.R.&Aquino,R.P.(2014).Microencapsulationbyspray

dryingofLanneamicrocarpaextract:technologicalcharacteristicsandantioxidantactivity.JournalofPharmacyandPharmacologyResearch,2,100-109.


177

SYNTHESISOF(E)-1-(FURAN-2-YLMETHYLENE)-2-PHENYLHYDRAZINE

Cabrera-VivasBM.1 ,RamírezJuanC.1,AguirreCabreraC.1,Flores-CastilloZA.1,ToledanoMY.2,García-

RamosJC.31FacultaddeCienciasQuímicas,BeneméritaUniversidadAutónomadePuebla,Puebla-México;2UniversidadNacionalAutónomadeMéxico,InstitutodeInvestigacionesBiomédicas,DepartamentodeInmunología,MexicoCity-Mexico;3UniversidadNacional

AutónomadeMéxico,FacultaddeQuímica,DepartamentodeQuímicaInorgánicayNuclear,MexicoCity-Mexico.email:[email protected]

Cancerisamajorburdenofdiseaseworldwide1.Eachyear,10millionsofpeoplearediagnosewithcanceraroundtheworld,andmorethanhalfofthepatientseventuallydiefromit.Inmanycountries,cancerranksthesecondmostcommoncauseofdeath following cardiovascular diseaseas.2 With significant improvement in treatment and prevention of cardiovasculardiseases, cancer has or will soon become the number one killer inmany parts of theworld. As elderly people aremostsusceptibletocancerandpopulationagingcontinuesinmanycountries,cancerwillremainamajorhealthproblemaroundtheglobe.

Phenylhydrazine (3.12 mmol, 577 mg) was dissolved in ethanol, a chemical equivalent (300 mg) of aldehyde which waspreviouslydissolvedinthesamesolventanditwasaddeddropbydropstirringconstantly.TheactionmixturewaskeptatroomtemperatureandwasmonitoredbyTLC,andthenvacuumfiltered.Thehydrazoneswererecrystallizedbyacontinuousand controlled process until light yellow crystals with adequate size and purity were developed in order to obtain X-raystudiesandcharacterizationwasperformedbym.p.,U.V,I.R.1HNMR,13CNMR.3(E)-1,1’-Diphenyl-2-(thiophen-2-ylmethylene)hydrazine.LightYellow crystals; yield: 75% at 25 oC,mp. 117–119 oC.Anal.calc. for C17H14N2S (MW= 278gmol-1) C, 73.3;H, 5.1;N, 10.1.Found: C, 73.2; H, 5.0; N, 10.15%. UV lmax = 412 nm. FT. IR(film): (cm-1): 3097 n(C–H), 1667–1920 n(Ph), 1585 n(C=N),1490n(C=C),695n(C–S).1HNMR400MHz,(CD3)2CO:(d/ppm,J/Hz):7.44(m,4H,C3’),7.37(d,1H,C5),7.36(s,1H,C=N),7.21(m,2H,C4’), 7.17 (dd, 4H, C2’) 7.02 (dd, 1H, C3), 6.98 (dd, 1H,C4). 13CNMR (300MHz, (CD3)2CO): (d/ppm): 143.39 (C2), 141.67(C1’),130.52(C5),129.89(C3’),127.32(C4),127.01(C3),125.61(C=N),124.64(C4’),122.24(C2’).MS-EI:m/z=278.37M+.

Ourinvestigationworkgrouphasmadeanexperimentalapproachwiththeevaluationofsynthetizedhydrazonesas((E)-1,1’-Diphenyl-2-(thiophen-2-ylmethylene)hydrazinepresented IC50of0.98mM, that represents thebestpotencyofcellgrowthinhibitionwithrespecttometronidazole(IC50=6.8mM).5Moreover,ourgroup isworkingon thesynthesisofmetallic complex inorderof intensify thepharmacologicalactivity, inparticulartheamoebicidalactivity.1. http://www.who.int/mediacentre/factsheets/fs297/en/index.html(accessed03.06.14), http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx (accessed

03.06.14).2. XiaomeiMaandHerbertYu*,YaleJ.Biol.Med.2006Dec;79(3-4):85–94.GlobalBurdenofCancer.3. Flores-Álamo,M.;Cabrera-Vivas,B.M.;Meléndrez-Luévano,R.;Hernández,P.J.M.;Ruiz-Azuara,L.1-{(E)-[5-(2-Nitro-phenyl)furan-2-yl]methyl-idene}-2,2-

diphenyl-hydrazine.ActaCrystallogr.E.2013,69,doi:10.1107/S1600536812050246.4. Y.Toledano,R.MeléndrezLuévano,M.Navarro-Olivarria, J.C.García-Ramos,M.Flores-Alamo,L.Ortiz-Frade,L.Ruiz-Azuara,B.M.Cabrera-Vivas*, J.

Med.Chem.Comm,2014,5,7,989-996.


178

SYNTHESISOF(E)-1,1-DIPHENYL-2-(THIOPHEN-2-YLMETHYLENE)HYDRAZINEANDCELLEVALUATION

AGAINSTAMIBIASIS

Meléndez-BalbuenaL.1 ,Cabrera-VivasBM.1,AguirreCabreraC.1,RamírezJuanC.1,García-RamosJC.2,Juárez-PosadasJR.3

1FacultaddeCienciasQuímicas,BeneméritaUniversidadAutónomadePuebla,Puebla,-México;2UniversidadNacionalAutónomadeMéxico,FacultaddeQuímica,DepartamentodeQuímicaInorgánicayNuclear,MexicoCity-Mexico;3ChemistryCenter,Science

Institute,UniversidadAutónomadePuebla,Puebla-México.email:[email protected]

TheChagasdiseaseisapublichealthproblem,beinginthefifthplacewithinthetoptenmostimportantparasiticdiseasesworldwide and takes the second place only aftermalaria in the American continent that can be transmitted by insects.1Hydrazonesandderivativesaremost frequentlyusedasdrugsbecausetheyarenitroheterocycliccompundsasnitrofuran(nifurtimox)andnitroimidazolederivative(benznidazole),whichareusedinthetreatmentoptionforT.cruziinfections2.OurinvestigationgroupdesignedsomecompoundsthathavestructuralsimilaritieswithNifurtimox,which isoneof twomostuseddrugs for treatmentofparasiticTrypanosomacruzi.3 On theotherhand,Nitroimidazolor itsderivativesas commondrugs, like Metronidazole, are well recognized for their amoebicidal properties that can heal bacterial infections of thevagina, stomach, skin, jointsand respiratory tract.Nitroimidazolederivativeshavebeenassayedasamoebicidesand theiractivities are associated with the redox biotransformation of the nitro group that generates a short-lived nitroso freeradical.4493mg(2.67mmol)diphenylhydrazineweredissolved inethanolandaceticacid(0.5mL)andwereslowlyadded intothissolutionwhilestirring;300mg(2.67mmol)ofthiophene-2-carbaldehydewereaddeddropbydropintotheabovesolutionwith strongly stirring and the resultingmixturewas kept at room temperature until it became a light yellow transparentsolution.Afteroneandahalfhourstheorangesolutionprecipitated.ThereactionwasmonitoredbyTLC,aluminumAlugramSilG/UV254.Themixturewasseparatedbyfiltrationinvacuosystemandtheprecipitatewaswashedthreetimeswithcoldmethanol. Recrystallization was performed three times with ethanol to obtain orange crystals for X-ray analysis andcharacterizationwasperformedbym.p.,U.V,I.R.1HNMR,13CNMR.LightYellowcrystals; yield: 75%at 25 oC,mp. 117–119 oC.Anal. calc. forC17H14N2S (MW=278gmol-1)C, 73.3;H,5.1;N, 10.1.Found: C, 73.2; H, 5.0; N, 10.15%. UV lmax = 412 nm. FT. IR (film): (cm-1): 3097 n(C–H), 1667–1920 n(Ph), 1585n(C=N), 1490n(C=C),695n(C–S).1HNMR400MHz,(CD3)2CO:(d/ppm,J/Hz):7.44(m,4H,C3´),7.37(d,1H,C5),7.36(s,1H,C=N),7.21(m,2H,C4´),7.17(dd,4H,C2´)7.02(dd, 1H,C3),6.98(dd, 1H,C4). 13CNMR(300MHz,(CD3)2CO): (d/ppm):143.39(C2), 141.67(C1´),130.52(C5),129.89(C3´),127.32(C4),127.01(C3),125.61(C=N),124.64(C4´),122.24(C2´).MS-EI:m/z=278.37M+.Our work group has made an experimental approach with the evaluation of synthetized hydrazones as (E)-2-(2-nitrobenzylidene)-1,1-diphenylhydrazinepresented IC50of 7.6mM, thatowns the samepotencyof cell growth inhibitionasmetronidazole(IC50=6.8mM).31. http://www.cdc.gov/parasites/chagas/2. Pintos-DiasJ.1984.Mem.Inst.OswaldoCruz79,suppl:85-91.3. TekwaniBLandR.K.Mehlotra,MicrobesInfect.,1999,1,385.4. Toledano-MagañaY.etal.,Med.Chem.Commun.,2014,5,989.5. Cabrera-VivasBM.etal.,ActaCrystallographica,2014.VolumeE70,o80.

PP2_14

Nifurtimox


179

SYNTHESISOF(E)-2-((5-(2-NITROPHENYL)FURAN-2-YL)METHYLENE)-1,1-DIPHENYLHYDRAZINEAND

ANTICANCERCELLEVALUATION

Cabrera-VivasBM.1 ,AguirreCabreraC.1,García-DíazIM.1,Ariza-RamírezJ.1,Ramírez-GarcíaJC.1,Orea-FloresML.2

1FacultaddeCienciasQuímicas,BeneméritaUniversidadAutónomadePuebla,Puebla-México;2InstitutodeQuímica,BeneméritaUniversidadAutónomadePuebla,Puebla-México.

email:[email protected] cancer is the most common type of cancer affecting more than 1 million women and causing high mortalityworldwide.Itisestimatedthattherehavebeen232,620newcasesofbreastcancerand39,970deathsoccurringintheUSAin 2011.1 A large number of breast cancers express estrogen receptor (ER) as well as progesterone receptor (PR) 2 andrespondtohormonaltherapyoraromataseinhibitors.However,thereisagroupofpatients(12–17%)whodonotrespondtosuch treatment because of the absence of these two receptors as well as the receptor HER-2/neu (ErbB2); this grouprepresentsahighlyaggressivebreastcancersubtype,knownastriple-negativebreastcancers(TNBC),3thataredifficulttotreat. In recent years, TNBC has gained attention due to its aggressive behavior, poor prognosis and lack of targetedtherapies.

Diphenylhydrazine(1.38mmol,254mg)wasdissolved inasolvent,achemicalequivalent(300mg)ofaldehydewhichwaspreviouslydissolved inthesamesolventand itwasaddeddropbydropstirringconstantlyandwhichwasdevelopedwithgreenerprocedure.TheactionmixturewaskeptatroomtemperatureandwasmonitoredbyTLC,andthenvacuumfiltered.ThehydrazoneswererecrystallizedbyacontinuousandcontrolledprocessuntilwinecrystalswithadequatesizeandpurityweredevelopedinordertoobtainX-raystudiesandcharacterizationwasperformedbym.p.,U.V,I.R.1HNMR,13CNMR.4

Winecrystals;yield:90.6%at25oC,mp.393-395K.Anal.calc.forC17H14N2S(MW=278gmol-1)C,73.3;H,5.1;N,10.1.Found:C,73.2;H,5.0;N,10.15%.UV lmax = 412 nm. FT. IR (film): (cm-1): 3097 n(C–H), 1667–1920 n(Ph),1585n(C=N), 1490 n(C=C), 695 n(C–S). 1H NMR (400 MHz, (CD3)2CO: (δ/p.p.m.,J/Hz):7.77(dd,H-3,J=7.94H3–H4J=1.20couplingWH3–H5);7.64(dd,H-6,J=8.10H6–H5andJ=1.16couplingWH6–H4);7.54(td,H-5, couplingH-4H-6 J=7.74 and couplingWH-3, J=1.20); 7.42 (t,H-3);7.36(td,H-4couplingH-3H-5,J=7.74,couplingWH-6,J=1.36);7.20(m,4H,H-2,H2,H-4);6.99(s,H-i);6.69(d,H-4couplingH-3,J=3.60);6.58(d,H-3 coupling H-4, J = 3.60). 13C NMR (100MHz, (CD3)2CO): (δ/ p.p.m.):153.0 (C2), 147.56 (C5), 147.17 (C2), 142.92 (C1), 131.77 (C5), 129.84 (C3),128.50(C3),127.97(C4),124.87(C2),124.79(iminic-C),123.81(C1),123.69(C6),122.40(C4),111.91(C4),110.10(C3).

Ourwork grouphasmade an experimental approachwith the cell evaluation in order tomeasure the anticancer activityagainstHTB-38,HTB-177,Molt-4.31. Siegel,R.etal.,CancerJ.Clin.2011,61,212.2. Deroo,B.J.andKorach,K.S.J.Clin.Invest.2006,116,561.3. Irvin,W.J.,Jr.andCarey,L.A.Eur.J.Cancer2008,44,2799.4. Flores-Alamo,M.etal.,ActaCrystallogr.E.2013,69,doi:10.1107/S1600536812050246.

PP2_15

020406080100120140

1µM 0.1µM

0.01µM

1µM 0.1µM

0.01µM

1µM 0.1µM

HTB-38 HTB-177 MOLT-4

Colchicina MTX A37


180

INFLUENCEOFGRANULARPREPARATIONMETHODANDTABLETHARDNESSINTHEDISSOLUTIONOF

APHYTOPHARMACEUTICALPRODUCT

ArchboldR.1 ,QuiñonesW.1,EcheverriLF.1,CaballeroC.2,TorresLF.1,EscobarGA.11UniversityofAntioquia,InstituteofChemistry,QOPN,Medellin-Colombia;2UniversityofBurgos,Burgos-España


Thegrowingdemandforherbalmedicineproductsobservedinthelastdecade,sustainedtheneedtolookfortechnological-pharmaceutical improvements in the preparation of such products. In the present study, we have analyzed andsimultaneouslyverifiedchangesthattablethardness(fractureresistance)andgranulatepreparationmethodmayexertoninproductdisintegrationanddissolution.Itisinfactimportanttounderlinethatthequalityoftheseproductsisbasedonthecharacteristicsthatdefineanddeterminetheirsuitability,allowingthemtobeclassifiedastherapeuticallyuseful.TabletsusedinthepresentstudywerepreparedaccordingtoformulasinTable1.Granulesproductionwasobtainedusingfourmethods:processof"simplemixture" (F1),processof"doublecompression"(F2),processof"wetgranulation" (F3)and theprocessof "mixedmethod" (F4). Mechanical, physical and technological propertiesof tabletswereevaluated inordertocomparetheinfluenceoftheseprocessesontablethardness(resistancetofracture),disintegrationandsolubility.

As illustrated in graphs of the dissolution profiles, changes in thehardness of tablets exert an increase in the disintegration time and adecreaseintheaverageconcentrationofcomponentsfromthedissolvedphytodrug. Results suggest that changes in these parameters maycriticallyinfluencetheefficiencyofherbalmedicinalproducts.

1. Lozano,M.C.,etal.2012.ManualdeTecnología Farmacéutica. Ed. ElsevierEspaña,S.L.Barcelona,España.2. Alvarez, C. N. S. et al. 2012.Tecnología Farmacéutica. Edita: EditorialClubUniversitario.SanVicente(Alicante),España.3. Nash, R. et al 2003. Pharmaceuticalprocessvalidation.3rded.MarcelDekker,NewYork.4. Levin, M. 2005. Pharmaceuticalprocess scaled-up.Marcel Dekker Taylor&Francis.NewYork.5. World Health Organization. 2007.Qualityassuranceofpharmaceutical.2nd.India.6. McCormick, K. andWylie Mcvay, Jr.(2012). Pharmaceutical process, designand management. Gower publishinglimited.England.7. Hermosilla E. R. et al. 2013. RevistaCubana de Plantas Medicinales. Estudiofito-químico y control de calidad deextractos de hojas de Rheedia aristataGriseb.18(3),pp.361-367


181

INVITROVALIDATIONOFANTI-INFLAMMATORY,HEALINGANDANTI-LEISHMANIALACTIVITYOF

Tabebuiachrysantha,TabebuiaroseaCalendulaoficinalisMatricariachamomilla,ThymusvulgarisandOreganumvulgare

RobledoSM.1 ,RiosYK.2 ,JuradoDS.1 ,EcheverriF.3

¹InstitutoInvestigacionesMédicas,UniversidaddeAntioquia,Medellín-Colombia;2UniversidaddeSantander,Cúcuta-Colombia;3QuímicaOrgánicadeProductosNaturales,Medellín-Colombia

email:[email protected];[email protected];[email protected];[email protected]

Cutaneousleishmaniasis(CL)isaworldwidedisease,whichmainlyoccursinthepoorestcountries.Thediseasesaffectmillionpeople causing high morbidity. Despite CL does not cause death per se, it is highly associated with social stigma anddiscrimination.Unfortunately,lackoftherapeuticalternatives,decreasedefficacy,sideeffectsandhighcosts,emphasizetheneed foradvance in thedevelopmentofnewandbetterdrugs.Naturalproducts remainasan important sourceofnovelchemical structures and therapeutic medicines. However, bio-guided studies to develop innovative structures withtherapeuticpotential are required.Given thatCL is a chronic inflammatorydisease that allowsparasitepersistence in thehost and facilitates the progression of lesions, an useful strategy is the combination of anti-inflammatory and healingpropertiesofthedrugcandidateswiththecapabilitytokillLeishmania.Theabilityofregulatingtheinflammatoryresponseandstimulatingthehealingactivity,couldacceleratetheresolutionofinflammationandscarring,whichinturnwouldleadtoatreatmentwithbetterbenefitseitherbytheshorterschemeorimprovementofthescarappearance.Thus,thisstudywasaimed to determine the possible effect of various natural products and synthetic derivatives with activity against L. (V)panamensisontheinflammatoryandhealingprocesses.Ethanol extracts from flowers of Tabebuia chrysantha, T. rosea and Calendula oficinalis and essential oils of Matricariachamomilla,ThymusvulgarisandOreganumvulgareweretestedforcytotoxicityinU937,BHK-21andDetroit551celllinesandprimary cultures of monocyte-derived macrophages (MDM) using the MTT method. The anti-leishmanial activity wasevaluated invitro againstL. (V)panamensis amastigotesby flowcytometry.Theeffectof theseproductson inflammationandhealingprocesseswasdeterminedquantifyingtheproductionofIFNg,TGFb,MCP-1,COX-2andvariousdermalgrowthfactors in culture supernatantsofhumanmonocyte-derived fibrocytes (FDM)byELISA. Lastly, the scarring capabilitywastestedinvitroaccordingtotheeffectofthesecompoundsonthefibrocytemigrationusingthecytoselectTMsystem.The anti-leishmanial activity but also the production of immunological factors associatedwith inflammation and scarringvariedamongextractsandessentialoils. TheethanolextractofC.oficinalis and theessentialoilofM. charantiawere themost effective compounds which meet all the characteristics pursued in the main objective of this work and could bepostulatedas“hit”tocontinuethedrugdevelopmentprocess.AcknowledgementtoUniversityofAntioquia(CIIEs–CIDEPRO).1. NikmehrB,GhaznaviH,RahbarA,SadrS,MehrzadiS.Invitroanti-leishmanialactivityofmethanolicextractsofCalendulaofficinalisflowers,Datura

stramoniumseeds,andSalviaofficinalisleaves.ChinJNatMed.2014;12(6):423-7.2. Jiménez-GonzálezF,VelozaL,Sepúlveda-AriasJ.Anti-infectiousactivityinplantsofthegenusTabebuia.Univ.Sci.2013;18(3):257-267


182

ANTI-INFLAMMATORYACTIVITYOFAPhysalisangulataL.DICHLOROMETHANEFRACTIONINDSS-

INDUCEDCOLITIS

RiveraDE.1,OcampoYC.1,FrancoLA.1 ¹UniversityofCartagena,BiologicalEvaluationofPromisingSubstancesGroup,Cartagena-Colombia


Physalis angulata is a plant commonly used in folk medicine of Latin American countries to treat inflammation relatedpathologies[1]. Inthiswork,weevaluatedtheanti-inflammatorypotentialofanenrichedfractionobtainedfromitscalycesusing a Dextran Sulfate Sodium (DSS)-induced colitis model in mice and Lipopolysaccharide (LPS)-stimulated RAW 264.7macrophagesinvitro.The total ethanolic extract of the P. angulata calyces was fractioned using liquid-liquid partition with petroleum ether,dichloromethane andmethanol. The dichloromethane fraction proved to have the highest anti-inflammatory potential invitro,andwasthereforeselectedforthenextexperiments. IntheDSSmodel,thefractionwasadministeredtomicefor7days, after twoDSS/Water cycles of 7/10 days, respectively.Weight changes, stool consistency and disease activity indexweremonitoredthroughouttheexperiment,whilecolonandmesentericlymphaticnodes(MLN)wereextractedattheendto assess the level of local and systemic inflammation. In vitro, the fractionwas evaluated on LPS-stimulated RAW 264.7macrophages,toascertainitsinfluenceontheexpressionlevelsofpro-inflammatorygeneslikeIL-1β,NOS2,COX2andTNF-α,aswell as genes that promote an anti-inflammatory, tissue repairing environment like ARG1, IL-10 and TGF-β1, using RealTime-PolymeraseChainReaction(RT-PCR)[2].The dichloromethane P. angulata fraction reduced the disease activity index and colon shortening induced by DSS. Thefractionshowedan importanteffectontheexpressionof theevaluatedgenes,downregulatingthepro-inflammatoryandupregulating the anti-inflammatory genes. This work confirms the therapeutic potential of P. angulata metabolites tomodulateinflammatoryprocesses.Acknowledgments:ThisstudywassupportedbyColciencias(grant110772553569)andtheUniversityofCartagena(grant003-2015).1. Kffuri,C.W., Lopes,M.A.,Ming,L.C.,Odonne,G.,&Kinupp,V. F. (2016).Antimalarialplantsusedby indigenouspeopleof theUpperRioNegro in

Amazonas,Brazil.Journalofethnopharmacology,178,188-198.2. Van Stijn, C. M., Kim, J., Lusis, A. J., Barish, G. D., & Tangirala, R. K. (2015). Macrophage polarization phenotype regulates adiponectin receptor

expressionandadiponectinanti-inflammatoryresponse.TheFASEBJournal,29(2),636-649.


183

CYTOTOXICEFFECTOFETHANOLICEXTRACTANDFRACTIONSFROMCrotonmalamboBARKAGAINSTMDA-MB-231CELLLINE.

CaroD.1,OcampoY.1,BolivarS.2,DiazF.2,SalasRD.1,AristizábalFA.3,FrancoLA.1

¹UniversityofCartagena,BiologicalEvaluationofPromisingSubstancesGroup,Cartagena-Colombia;2UniversityofCartagena,LaboratoryofPhytochemicalandPharmacologicalResearches(LIFFUC),Cartagena-Colombia;3NationalUniversityofColombia,

CancerPharmacogeneticsResearchGroup,Bogotá-Colombia.email:[email protected]

Breast cancer is themostcommon inwomenworldwide, is a seriouspublichealth issuewithnearly 1.7millionnewcasesdiagnosed in2012(1); thereforeaneffectivemanagementandtreatmentofthisdisease isundoubtedlycrucial. Inthissensethe ethnopharmacology research of botanical species has proved to be a useful approach to find potential anti-canceragents.Inthiswork,weevaluatedthecytotoxiceffectofethanolicextractofCrotonmalambobarkanditsfractionsontheMDA-MB-231breastcancercellline,usingtheMTTmethod.TheethanolicextractobtainedoftheC.malambobarkwasloadedonacolumnchromatographyoversilicagel,andelutedwith solvents in crescentorderofpolarity,underabio-guide schemebasedon thecytotoxicactivityagainstMDA-MB-231cells,employingthemethyl-tetrazoliumbromidecolorimetricmethod(MTT)(2).Theextractandfractionswereclassifiedintothreecategoriesas:active(LC50≤20μg/ml),moderatelyactive(20<LC50<100μg/ml)andinactive(IC50≥100μg/ml)(3).ChromatographicfractionationofC.malambototalextract,ledtoobtainsixmajorfractionsidentifiedasCM-F1,CM-F2,CM-F3,CM-F4,CM-F5andCM-F6,whichwereevaluatedinthebiologicalmodel.ThetotalextractwasmoderatelyactiveonMDA-MB-231cells (LC50=63.68μg/mL), thisactivitywasmaintained in twoofsix fractionwithabettercytotoxiceffect (CM-F5 -LC50=43.45 μg/mL and CM-F6 - LC50=43.35 μg/mL), which were eluted with ethyl acetate. These fractions represent apromising source of cytotoxic compounds that should be further explored, aiming to obtain new active compounds thatimprovethetherapeuticarsenalavailableforbreastcancertreatment.Acknowledgments:ThisstudywassupportedbyColcienciasandtheUniversityofCartagena(Grant110756933390).DaneivaCaroisdeeplygratefultoColcienciasandtheUniversityofCartagenaforherPhDfellowshipthroughtheNationalProgramforDoctoralFormation(Grant647-2014).1. Ferlay J., et al. (2012). Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11. GLOBOCAN 2012 1.1. Available from:

http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx,accessedon07/03/20162. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of

immunologicalmethods,65(1-2),55-63.3. Manosroi, A., Akazawa, H., et al. (2015). In vitro anti-proliferative activity on colon cancer cell line (HT-29) of Thai medicinal plants selected from

Thai/Lannamedicinalplantrecipedatabase“MANOSROIIII”.Journalofethnopharmacology,161,11-17.


184

ANTICONVULSANTEFFECTOFTANACETUMPARTHENIUM(L.)SCHULTZ-BIPONPENTYLENETETRAZOLE-

INDUCEDSEIZURESINMICE

Reyes-PérezV.I. ,Hernández-NavarroMD.UniversidadAutonomadelEstadodeMexico,Toluca-Mexico

email:[email protected],Tanacetumparthenium isusedtotreatepilepsyandseizures(Jägeretal.,2006).Basedonthisbackground,therehavebeenseveralstudiesinvitrooftheextractsofthisplantoritscomponents,suggestingananticonvulsanteffectthroughinteractionwiththeGABAergicsystemintheCNS(Violaetal.,1995;Jägeretal.,2006;Jägeret al., 2009). However, it is not known with certainty the components responsible for the anticonvulsant activity ormechanismthroughwhichtheyoperate.Themainobjectiveofthisstudywasconducting invivotestsallowingtocharacterizetheanticonvulsanteffectofaqueousand aqueous-alcoholic extract of T. parthenium and explore its possible mechanism of action, using a model of acuteadministrationofpentylenetetrazole(PTZ).Chemical characterization of aqueous and hydroalcoholic extracts of T. parthenium, respectively was performed. Thepresence of total polyphenols and flavonoids were quantified, and identified the main compounds by HPLC-DAD-ESI-MSanalysis.AcutetoxicitytestsfordeterminingtheLD50wereperformed.TheacutemodelofconvulsiveactivityinducedwithPTZinmicewasusedfortheevaluationofanticonvulsanteffect.ThehydroalcoholicextractofT.partheniumproducedthebesteffect(p<0.01)atdosesof100mgkgreducingthenumberanddurationoftonic-clonicseizuresinducedbyPTZ.Theaqueousextractatdoseof100mg/kg,significantlydecreasedthenumberofmyoclonicseizures(p<0.001)comparedwiththecontrolgroupandagainstthereferencedrug(Valproicacid)(p<0.05),andalsoincreasedthelatencytotonic-clonicseizures,althoughthisincreasewasnotstatisticallysignificant.Forthechemicalcharacterizationoftheextractstwomajorcompoundswereisolated:santinandsantamarin.ThelatterwastestedinthemodelofPTZ,producinghisbestanticonvulsanteffectatthedoseof1mg/kgbyreducingthenumber(p<0.05)andduration(p<0.01)oftonic-clonicseizuresandnumberofmyoclonicseizures(p<0.01)comparedwiththecontrolgroup,andanLD50greaterthan10mg/kg.TheLD50fortheaqueoushydroalcoholicextractwasgreaterthan5000mg/kg.The aqueous and hydroalcoholic extracts of T. parthenium and the sesquiterpene lactone santamarin exercised theiranticonvulsant effect inmice treatedwith PTZ. These results suggest that the anticonvulsant effect of these products ismediatedatleastinpartbytheGABAergicsystem,inparticularforGABAAreceptorsintheCNSfromapossiblesynergisticactivityofitscomponents(SucheryCarles,2015).Sothat,thepotentialofTanacetumpartheniumisproposedforitsuseasatherapeuticalternativeaccessible,andlowtoxicity,forthetreatmentofepilepsy.1. Jäger AK, Gauguin B, Andersen A, Gudiksen, L., 2006. Screening of plants used in Danish folk medicine to treat epilepsy and convulsions. J

Ethnopharmacol,105,294-300.2. JägerAK,KrydsfeldtK,RasmussenHB.,2009.Bioassay-guidedIsolationofApigeninwithGABA-benzodiazepineActivityfromTanecetumparthenium.

PhytotherRes,23,1642-1644.3. Sucher NJ, Carles MC., 2015. A pharmacological basis of herbal medicines for epilepsy. Epilepsy Behav.Available at:

http://dx.doi.org/10.1016/j.yebeh.2015.05.0124. ViolaH,Wasowski C, Levi de SteinM., 1995.Apigenin, a componentofMatricaria recutita flowers, is a central benzodiazepine receptor-ligandwith

anxiolyticeffects.PlantaMed,61,213-216.


185

LC/MSANALYSISANDHPLCQUANTIFICATIONOFMAINCOMPOUNDSOFFIVEHYPERICUMSPECIES

INDIGENOUSINTHEPELOPONNESE

ZeliouK.1 ,KoulakiotisNS.2,VogiatzoglouA.1,IatrouG.3,TsarbopoulosA.2,4,LamariFN.11LaboratoryofPharmacognosyandChemistryofNaturalProducts,DepartmentofPharmacy,UniversityofPatras,Patras-Greece;

2GAIAResearchCenter,GoulandrisNaturalHistoryMuseum,Athens-Greece;3DivisionofPlantBiology,DepartmentofBiology,UniversityofPatras,Patras-Greece;4DepartmentofPharmacology,SchoolofMedicine,UniversityofAthens,Athens-Greece.

email:[email protected](Hypericaceae) iswidelyknownbecauseofHypericumperforatumL.,aspecieswithnumeroustraditionaluses especially treating mild depression; action mainly attributed to hyperforin, a prenylated phloroglucinol.NaphthodianthronesarefoundexclusivelyinaerialpartsofHypericumgenusalongwithflavonolsandflavonolsglycosides,biflavonoids, and other phenolic compounds (as organic acids)[1]. In Greece except H. perforatum L., other 40 taxa ofHypericumgenusare indigenous,moreoverone thirdof themendemic [2].However theirphytochemical compositionhasnotbeeninvestigatedsufficiently.In this study we focused on the chemodiversity of five species of Hypericum growing wild in Peloponesse (Greece), H.perforatum L.,H. perfoliatum subsp. veronense (Schrank) A. Fröhl,H. triquetrifolium Turra,H. empetrifoliumWilld. subsp.empetrifoliumandH.vesiculosumGriseb.Aerialpartsofthespecieswerecollectedduringthefloweringperiodanddriedatroomtemperature.Thephytochemicalcompositionofthedrymethanolicextractswasstudiedusing“UltraPerformance”Liquid Chromatography (UPLC) coupled toQ-TOF (quadrupole - time of flight)Mass Spectrometry. Several organic acids,flavonoids, naphthodianthrones and phloroglucinols were identified by LC-MS. Total flavonoids, naphthodianthrones andprenylatedphloroglucinolderivativesaswellaschlorogenicacid,rutin,isoquercitrin,hyperoside,quercetin,II8-I3-biapigenin,pseudohypericinandhypericinwerequantifiedinallextractsbyHighPerformanceLiquidChromatography(HPLC-DAD),onaC-18column,withamethodpreviouslydevelopedandvalidatedinourlaboratory[3].Themajorityofthemaincompoundswerecommonamongtheexaminedspecies;howeversomedifferencesweredetectedespecially as regards the phloroglucinol derivatives.H. perforatum had the highest quantity of compounds permgof dryextract,doublethantheotherspecies,andthehighest%concentrationoftotalflavonoidsandphloroglucinolderivatives.H.vesiculosum had the highest content of napthodianthrones, while H. empetrifollium, had the lowest content ofnapthodianthrones and flavonoids, and the highest content of phenolic acids. The results of this study shed a light onHypericumchemodiversityandmayevenrevealothertaxawhichcouldbevaluableassourcesofthemedicinalpreparations.1. ErnstE.Hypericum:TheGenusHypericum.Taylor&FrancisLtd.UnitedKingdom,2003.2. PylaraA.,KaratzaE.IatrouG.(InGreek).ThegenusHypericumL.inGreece.στηνΕλλάδα.UniversityofPatras3. ZeliouK.,KontaxisN.I.,MargianniE.,PetrouC.,andLamariF.N.,2016.Submittedin“PlantaMedica”.


186

OPTIMIZATIONANDVALIDATIONOFTWOMETHODSOFENZYMEINHIBITIONRELATEDTOANTI-

HYPERGLYCEMICACTIVITY

Granados-GuzmánG.,Castro-RíosR.,WaksmandeTorresN.,Salazar-ArandaR. FacultaddeMedicina,UniversidadAutónomadeNuevoLeón,MadreoyGonzalitos,Monterrey(N.L.)-México

email:[email protected],simple,inexpensiveandreducereagents,residuesandtheconsumptionofexperimentalanimalsintheinitialscreeningofbiologicalactivity.However,theyhavelowreproducibilityandlowcorrelationwithinvivomodels,possiblybychangesmadeineachlaboratorythatcanaffectaccuracy.Inthisworkwereoptimizedandvalidatedinvitromicroscaleassaysrelatedwithantihyperglycemicactivity.Optimizationwascarriedoutusingafractionalfactorialdesignandabasicsequentialsimplexmethod,takingintoaccountthe best inhibition percentage and the absorbances of the controls. With the optimized experimental conditions themethodswerevalidated.Theoptimized conditionsofα-glucosidasemethodwere: enzyme concentrationof 0.55U/mL, substrate concentrationof111.5 uM, 17.5min incubation at 37 ° C. In the validation of thismethod, a linear range between 100 and 310.2 ug/mL foracarbose with a value of R2 0995, was established. The percentage coefficient of variation were less than 2% and errorpercentageswerebelow3%,indicatinggoodprecisionandaccuracy.TheZfactorwasgreaterthan0.96.Optimalconditionsfor the α-amylase method were: enzyme concentration of 0.5 U/mL, substrate concentration of 1.11 mM, 7.2 minpreincubation,incubationof5.5minand15.6mininwaterbath.Thelinearrangewasestablishedbetween0.5and12ug/mLofacarbose.The%CVwerebelow5%anderror%werelower6%.AZ-factorgreaterthan0.94wascalculated.Inconclusioninvitromicroscalemethods,preciseandaccurateabletodeterminethepotentialantihyperglycemicactivityinnaturalextractsmethodsweredeveloped.


187

THEORETICALSTUDYOFTHECONVERSION(E)-a,b-UNSATURATEDOXAZOLIDINONES

Ramírez-GarcíaJC. ,Cabrera-VivasBM. ,AguirreC.,PinedaFP.,MárquezCG.

BeneméritaUniversidadAutónomadePuebla,FacultaddeCienciasQuímicas,Puebla-México.email:[email protected],[email protected]

Stereochemistryhasbeenstudiedintheconversionof(E)-a,b-unsaturatedoxazolidinestotheb,g-unsaturated.TheZ/Eratiosof the resultingb,g-unsaturated compounds vary according tog-substituensof the (E )-a,b-unsaturatedoxazolidines. It isassumedthatthisreactionoccursbyinteractionofs->p*orvia6p-electronaromaticity,whichiscalledthesyneffect.ThereactionilustratedinFigure1consitingintheconversionof(E)-a,b-unsaturatedoxazolidinonestob,g-unsaturatedwasstudied using computational methods. Gaussian09 hasbee used to perform the electronic structure calculation, whereasmolecular orbital calculations were done using polarized and diffuse basis set. Molecular orbitals were displayed usingMOLDEN.Intrinsicreactioncoordinateswerealsomeasured.The stereochemistry of the conversion of (E )-a,b-unsaturated oxazolidines to the corresponding b,g-unsaturated wassuggestiveforasyn-effectanditcanbeproposedthattherelativedegreeontheg-substituentsconsistsoftheinteractionbetweenorbitalss->p.

Figure1.Conversionof(E)-a,b-unsaturatedoxazolidinestothecorrespondingZ/Eb,g-unsaturated,Looking at MOLDEN-generated graphics it could be suggested that molecular orbitals of the three molecules exhibitsignificantdifferences.ItwasalsoobservedthattheMOsarebiggerinthemoleculesb,g-unsaturated.Fora,b-unsaturatedoxazolidines, the g-substituents gavedifferent ratios according to the substituent. The IRCgavealso some insight of themomentofconversion.Acknowledgments:ThankstotheVIEP/BUAPforsupportingtheproject.1. Nakano,Soeta,Endo,Inomata,Ukaji.J.Org.Chem.2013,78,12654.2. EijiTayama,YukaToma.Tetrahedron2015,71,554.3. JaquelineJiménez,JuanCarlosRamírez,GabrielaHuelgas,RuthMeléndrez,BlancaM.Cabrera-Vivas,Estibaliz,Sansinenea,AurelioOrtiz.Tetrahedron

2015,71,4590


188

COMPUTATIONALANALYSISONVITAMINDRECEPTOR

Ramírez-GarcíaJC.1 ,Cabrera-VivasBM.2 ,AguirreC.2,PinedaFP.2,MárquezCG.2

BeneméritaUniversidadAutónomadePuebla,FacultaddeCienciasQuímicas,Puebla-Méxicoemail:[email protected],[email protected]

TheVitaminDReceptor(VDR)isamemberofnuclearreceptorsuperfamily,whichmediatesthebiologicalactionsofvitaminD, the hormone 1a, 25-dihydroxivitaminD3[1, 25(OH)2D3]. Also a receptor for Lithocholic acid (LCA), is a potential entericcarcinogenic,whichinducesitsmetabolismtroughVDRinteraction.AreexaminedtheoreticallytheinteractionofLCAandD3onVDR.Ithasbeenused4Q0Ay 1DB1 structures fromProteinDataBank to compare theelectronic interactions, andgeometricalcomparisonsbetween1α,25-dihydroxyvitaminD3andLithocholicacid (LCA),whichbothstimulate theVitaminDReceptor.ThistheoreticalstudywascarriedonusingSpartanandVMDsuiteofprograms.The purpose is to identifywhat kind of structural, electronic or electrostatic differences are present between these twomolecules.Themoleculesstudiedarepresentedinfigure1.Themolecule1a,25-(OH)2D3interactswithVDRnuclearreceptorwhenisinthenuclear receptor, ina spaceof26.508Angstrom large, 10.553highand 16.618wide.TwomoleculesofLithocholicAcidinteractwithVDR,oneof themamongnuclear receptorandotherbyoutside.Thedistancebetweenthe twoLCAare forbothC=Ois11.784andforCH319.432Angstrom.Electronicandenergeticpropertiesaredifferentforthemetabolites.Acknowledgments:thisprojectwassupportedbyVIEP/BUAP

Figure1.a)1a,25-(OH)2D3,b)VitaminDandVDR,c)LithocholicAcid.

1. Darius Wlochowitz, Martin Haubrock, Jetcy Arackal, Annalen Bleckmann, Alexander Wolff, Tim Beißbarth, Edgar Wingender, Mehmet Gültas.ComputationalIdentificationofKeyRegulatorsinTwoDifferentColorectalCancerCellLines.FrontGenet.2016;7:42.

2. RocioOtero,SamuelSeoane,RitaSigüeiro,AnnaY.Belorusova,MiguelA.Maestro,RomanPérez-Fernández,NatachaRochel andAntonioMouriño.Carborane-baseddesignofapotentvitaminDreceptoragonist.Chem.Sci.,2016,7,1033-1037.

3. AnnaY.Belorusova,JérômeEberhardt,NoëllePotier,RolandH.Stote,AnnickDejaegere,andNatachaRochel,.J.Med.Chem.,2014,57(11),4710–4719.4. KellyA. Teske, JonathonW.Bogart, LuisM. Sanchez,OliviaB. Yu, JoshuaV. Preston, JamesM.Cook,NicholasR. Silvaggi,DanielD.Bikle, LeggyA.

Arnold.EuropeanJournalofMedicinalChemistry109(2016)238e246


189

MEDICALAPPLICATIONSOFOZONIZEDOIL.ANUP-DATE

Martínez-Sánchez,G.1 ,Magistrelli,D.2

¹MedicalCenterBeautyBenefit,SanBiagiodiOsimo,Osimo(Ancona)-Italy;2DepartmentofAgriculturalandEnvironmentalScience-UniversityofMilan,Milan-Italy.

email:[email protected]:damiano.magistrelli@guest.unimi.itEarlyevidenceontheclinicaluseofozonatedoilsfirstappearinscientificliteraturein1859.Thislecturereviewsthegeneralandmainclinicalapplicationsofozonatedoilsthathaveappearedinscientificliteraturebetween1859-2016.Anin-depthscreeningofprimarysourcesofinformationonline-viaScopusdatabasesaswellasEmbase,PubMed,andtheCochraneDatabaseofSystemicReviews-wasperformed.Themostsignificantpapersofthelast25yearsarepresentedandtheirproposalscriticallyevaluated.Themain applicationsof theseagents are for infectiousdiseases. Theoxidationproductsgeneratedafter the reactionofozonewithfattyacidsandothersubstratescanactsasagermicide,immunestimulantandtissuerestorationagent.Someofthe advantages of ozonated oils are: low cost, similar or superior effects compared to traditional antibiotics, a broadantimicrobial spectrum and a low rate of adverse events. The stability of the ozonated oils allows the development ofstandardformulationsthatdeliverthebenefitsofozone,inaproductthatcanbeconventionalstoredforfutureclinicaluse.Thisisincontrasttothetherapeuticapplicationoftheozonegas,wherethegasmustbeappliedimmediatelyfollowingitsproduction.Themainclinicalstudiesthatsupporttheuseofozonizedoilsapplyozonizedsunfloweroilorozonizedoliveoilin different clinical conditions. The applications are essentially for external use, however there is evidence of immune-stimulatingandrepaireffectswhenusedorally.Adeepstudyofthebiologicaleffectsofvariousformulationsalreadyonthemarketwillsupportthescientificbasisoftheuseofozonizedoilindifferentpathologies.1. Martínez-Sánchez G., Re L., Perez-Davison G, Horwat Delaporte R, 2012. Las aplicaciones médicas de los aceites ozonizados, actualización. Revista

EspañoladeOzonoterapia.Vol.2,nº1,pp.121-139.http://www.revistaespañoladeozonoterapia.es/index.php/reo/article/view/23/23


190

BIOSAFETYASPECTSINTHEREGULATIONOFMODERNBIOTECHNOLOGYINPLANTS

BorgesBJP.1 ,AbreuPMV.1,CarminatiLS.1,ArantesOMN.2,FernandesAAR.1,FernandesPMB.1

¹FederalUniversityofEspíritoSanto,Vitória(EspíritoSanto)-Brazil;2StateUniversityofLondrina,Londrina(Paraná)-Brazilemail:[email protected]

The RNA interference (RNAi) technology, involving small non-coding RNAs (sRNA) is currently one of the main tools ofmodern biotechnology to develop resistant plants, especially against virus, and with desired characteristics (Zhou et al.,2013).microRNAs(miRNA),atypeofsRNA,hadbeenusedinanewstrategyfordevelopingplantforfood(Sherman,etal.2015).Ofextremeinterestofthisnewtechnologyisthefactthatitenablesgeneticmodification(GM)oftheplantwithnoproductionoftransgenicprotein.Nevertheless,theseadvancesshouldnotignorethepotentialriskstohuman,animal,andtotheenvironment(Cascubertaetal.,2013).Inthissense,itisnecessarytodiscussmiRNA-GMplantsriskassessmentintheregulatoryframework.ThisworkaimstopromoteananalysisontheBiosafetyregulationoftheGMtechniquesusingmiRNA.The question “Is there a specific biosafety regulation formiRNA technique or strategy for plants?”was pursued for thedescriptiveandcomparativeanalysisofthefoodbiosafetyregulationaboutthemiRNAtechniqueinthelegalframeworksofBrazil (http://ctnbio.mcti.gov.br), European Union (EU) (http://eur-lex.europa.eu/homepage.html) and United States (US)(https://www.loc.gov).IntheUS,therearefiveGMplantsdevelopedwiththeRNAitechnologyapprovedtomarket,namely,apple,potato,tomato,tobaccoandsoybean.TheriskassessmentoftheseGMplantsissimilartothatusedforGMplantsthatproducetransgenicproteins. The environmental impact is determined empirically. Nevertheless, the US are reviewing the legal frameworkprocess on this subject since 2015. In the EU, which adopts the precautionary principle, the list of genetic engineeringtechnologiesdoesnotmention anyof theRNAi-based strategies.Hence adebatehas started in theEUwithparticipantsfrom academy, industry, government and non-governmental organizations. Although, neither a consensus about whichcriteriashouldbeemployednorawaytoregulatesuchplantshasbeenreachedatthemoment,twoGMplantsusingRNAiwere approved in the EU, potato and soybean, but potato is no longer marketed. In Brazil, which also adopts theprecautionary principle, the regulation establishes a case-by-case analysis and does not describes specific criteria for GMplantsusingRNAi-basedstrategy.AGMplantusingthisstrategy,abeanresistanttotheGoldenMosaicVirus,wasrecentlyapprovedinthecountryanditwassubjectedtoriskassessmentwithoutapplicationofdifferentialcriteria.ThedialogueonriskassessmentandupdatingtheregulatoryGMplantsusingnewtechnologieshavealreadystartedinalllegalframeworksstudied;theregulationintheUSandBrazilremaindefiningtheapplicationofthesamebiosafetycriteriaforbothGMplantsusing RNAi-based strategy and other GM plants produced by other techniques of modern biotechnology. These resultssuggestapoliticalandconservativepositioninsteadofscience-based.Thecurrentscientificevidenceindicatesadegreeofrisk differentiation (or safety) between the RNAi techniques or strategies and those with other techniques of modernbiotechnology.Weconcludethatthisevidencecouldsupporttheapplicationofdifferential(specificorsimplified)criteriaintheriskassessmentthathasnotbeenappliedinthelegalframeworksstudied.Acknowledgments:wethankCAPES,CNPqandFAPESforfinancialsupport.1. Casacuberta,etal.(2015).BiotechnologicalusesofRNAiinplants:riskassessmentconsiderations.TrendsBiotechnol33,145-147.2. Sherman,etal.(2015).RNAitechnologiesinagriculturalbiotechnology:TheToxicologyForum40thAnnualSummerMeeting.RegulToxicolPharmacol

73,671-680.3. Zhou,etal.(2013).MicroRNA-mediatedgeneregulation:potentialapplicationsforplantgeneticengineering.PlantMolBiol83,59-75.


191

ANTIOXIDANTANDANTIMICROBIALACTIVITIESOFLEPIDIUMSATIVUMEXTRACTS

BouamraD.1,BakiCA.1,TouabetL.1,MelianiS.1,BouchebourA.1,DahamnaS.1,HarzallahD.2

1LaboratoryofPhytotherapyAppliedtoChronicDiseases,DepartmentofAnimalBiologyandPhysiology,FacultyofNaturalandLifeSciences,UniversityFerhatAbbas,Setif-Algeria;2LaboratoryofAppliedMicrobiology,DepartmentofMicrobiology,Facultyof

NaturalandLifeSciences,UniversityFerhatAbbas,Sétif-Algeria.email:[email protected]

Lepidium sativum, commonly known as Habarachad, is an edible grass with fast growth potential belonging to theBrassicaceae family. The seeds of this medicinal plant are diuretic and tonic; they are used to treat bacterial and fungalinfections.Themainaimofthisstudyistoevaluatethephenoliccomposition,theantioxidantandantimicrobialactivitiesofextractspreparedfromL.sativumseeds.Theyieldoftheextractionwas12.09%and0.87%forMethanol(MeOH)andAqueous(Aq.)Extractsrespectively.TheAq.extractpresentedthehighestcontentoftotalpolyphenols(39.38±0.001mg.GallicAcidequivalent /g.ofextract), followedbytheMeOHextract (31.38±0.024mgG.A.E. /g.ofextract),whereasthecontentsofflavonoidswere 14.12 ± 3.69 and 7.10 ± 7.86mg.Rutin equivalent /g. dry extract forMeOHandAq. extracts respectively.Moreover,theantioxidantactivitywasevaluatedbyusingDPPHtest[1],whereastheantimicrobialactivitywasassessedviadisc-diffusionmethod[2].TheresultsrevealedthatthetwoextractsshowedsignificanteffectsontheradicalDPPH;withanIC50fortheAq.Extract(0.104±0.006mg/ml)thentheMeOHextract(0.166±0.007mg/ml).Furthermore,thestudyoftheantimicrobialactivityofthetwoextractsshowedsomeeffectsagainstAspergillusflavus(28.33mm.indiameterfortheAq.extract) andAspergillus niger (21mm. in diameter for the same extract). However, no effect on the bacterial strainswasrecordedforbothextracts.Acknowledgments:TheauthorswouldliketothanktheDGRSDT,MinistryofHigherEducationandScientificResearchofAlgeriaforfinancialassistance.1. RapisardaPetal.(1999)JournalofAgriculturalandFoodChemistry,47(11):4718-4723.2. ZaidanMRetal.(2005)TropBiomed,22(2):165-70.


192

ANTIMICROBIALACTIVITYOFESSENTIALOILSAGAINSTSALMONELLAANDSTAPHYLOCOCCUSAUREUS

SerraccaL. ,RossiniI.,BattistiniR.,ImberciadoriM.,CorsiM.,TerarolliA.,TomeiL.,DeMontisG.,

ErcoliniC.¹IstitutoZooprofilatticoSperimentaleDelPiemonteLiguriaeValled'Aosta,LaSpezia-Italy

email:[email protected] the last decades, antibiotic resistance in many clinical pathogens increased significantly (Kassim et al., 2016). Diskdiffusionisoneoftheoldestapproachestoantimicrobialsusceptibilitytesting(AST)andremainoneofthemostwidelyusedASTmethodsinroutineclinicalmicrobiologylaboratories(Matuscheketal.,2013).Recently,theessentialoils(EO)havebeenevaluated for their antimicrobial activities (Mondello et al., 2009). EO and other plant extracts possess antibacterial,antifungal, and antiviral properties and have been screened worldwide as potential sources of novel antimicrobialcompounds(Maidaetal.,2014).ThusEOandtheirconstituentscanhopefullybeconsidered inthefutureformoreclinicalevaluationsandpossibleapplicationstocurrentmedications(Edris2007;Maidaetal.,2014).Salmonella (a strain isolated froma sausage sample) andStaphylococcus aureus (ATCC33862)were thebacteria chose totest.Eachbacteria strainwasgrownat37°C in liquidmedium(BPW).TheEO testedagainst thebacteriawere: red thyme(Thymus vulgaris), white thyme (Thymus serpyllum), linalool thyme (Thymus vulgaris ct linalool), thuyanol thyme (Thymusvulgaris ct thuyanol), true lavander (Lavandula officinalis), hybrid lavender (Lavandula hybrida), eucalyptus (Eucalyptusglobulus), tea tree (Melaleuca alternifolia), carnation (Syzygium aromaticum), lemon (Citrus limon), cajeput (Melaleucaleucadendronvar.cajeput).Sterilefilterpaperdisksweresoakedwith20µlofEOand40µlofTween80.PaperdiskswereplacedonthesurfaceofMullerHiltondisheswherebacterialcultureswerepreviouslystreakedwithsterilecottonswabs.Theplateswereincubatedat37°Cfor24haerobically.Afterincubation,thediameteroftheinhibitionzoneswasmeasuredinmillimeters, including thediameterofdisk.The sensitivity toEOswasclassifiesby thediameterof the inhibitionzonesasfollow:Sensitivefortotaldiameter20≥mm,Intermediateforatotaldiameter15-19mm,andResistantforatotaldiameyter≤14mm(CLSI).Thesensitivityof Salmonella toEOs revealed:36,4% (4/11)Sensistive for red thyme (Thymusvulgaris),white thyme (Thymusserpyllum), tea tree (Melaleuca alternifolia) and carnation (Syzygium aromaticum), 18,2% (2/11) Intermediate for eucalyptus(Eucalyptusglobulus),lemon(Citruslimon),45,4%(5/11)Resistantfortheremaining.ThesensitivityofStaphylococcusaureustoEOsrevealed:72,7%(8/11)Sensistiveforredthyme(Thymusvulgaris),whitethyme(Thymusserpyllum),linaloolthyme(Thymusvulgaris ct linalool), true lavander (Lavandula officinalis), hybrid lavender (Lavandula hybrida), eucalyptus (Eucalyptusglobulus), tea tree (Melaleuca alternifolia), carnation (Syzygium aromaticum), 18,2% (2/11) Intermediate for thuyanol thyme(Thymusvulgarisctthuyanol),cajeput(Melaleucaleucadendronvar.cajeput),9,1%(1/11)Resistantfortheremaining.1. CLSIPerformanceStandardsforAntimicrobialSiskabdDiffusionSusceptibilityTestsforBacteria IsolatedFromAnimals;ApprovedStandards-Fourfth

Edition.CLSIdocumentVET01-A4.Wayne,PA:ClinicalandLaboratoryStandardsInstitute;2013.2. EdrisAE.2007.Pharmaceuticalandtherapeuticpotentialsofessentialoilsandtheir individualvolatileconstituents:areview.PhytotherapyResearch,

pp308-323.3. Kassim A, Omuse G, Premji Z, Ravathi G. 2016. Comparison of Clinical Laboratory Standards Institute and European Committee on Antimicorbial

SusceptibilityTestingguidelines for the interpretationofantibiotic susceptibilityataUniversity teachinghospital inNairobi,Kenya:across-sectionalstudy.AnnalsofClinicalMicrobiologyandAntimicrobials,pp1-7.

4. MaidaI,LoNostroA,PesaventoG,BarnabeiM,CalonicoC,PerrinE,ChielliniC,FondiM,MengoniA,MagginiV,VannacciA,GalloE,BiliaAR,FlaminiG,Gori L, Firenzuoli F, Fani R. 2014. Exploring the anti-Burkholderia cepacia Complex activity of Essential Oils: A preliminary Analysis. Evidence-BasedComplementaryandAlternativeMedicine,pp1-8.

5. MondelloF,GirolamoA,ScaturoM,RicciML.2009.DeterminitionofLegionellapneumonihilasuscptibilitytoMelaleucaalternifoliaCheel(teatree)oilbyanimprovedbrothmicro-dilutionmethodundervapourcontrolledconditions.JournalofMicrobiologicalMethods,pp243-248.


193

TRITERPENES-ENRICHEDFRACTIONSOFEUCALYPTUSTERETICORNISMODULATEGENEEXPRESSION

ONHUMANADIPOSETISSUECELLLINESMODELS

BalcazarN.1,2 ,GuillenA.1,CastañoA.2,EcheverriLF.3,AcínS.1 ¹GENMOLGroup.SededeInvestigaciónUniversitaria,UniversidaddeAntioquia,Medellín-Colombia;2DepartmentofPhysiologyandBiochemistry,SchoolofMedicine,UniversidaddeAntioquia,Medellin-Colombia;3QOPNGroup-SchoolofNaturalandExacts

Sciences.SededeInvestigaciónUniversitaria,UniversidaddeAntioquia,Medellín-Colombiaemail:[email protected],[email protected]

Obesity is associated with adipose tissue inflammation that eventually results in insulin resistance and it is linked withcardiovascular disease (CVD) and type 2 diabetes mellitus (T2DM) (Khan, SE. et al., 2006; Esser N. et al. 2014). Previousstudies established the beneficial effects of enriched-triterpenes fractions from Eucalyptus tereticornis (Eu) on insulinresistance, glucose intolerance and hyperglycaemia in a diabetic mouse model (Guillén A., et al. 2015). In addition, theyshowedareductioninlevelsofpro-inflammatorycytokinesinadiposetissue(GuillénA.,etal.2015).Ouraimistoevaluatetheeffectoftriterpenes-enrichedfractionsfromEuonhumanadipocyteandmacrophagecelllinesmodels.A crudemethanolic extract (OBE100) from leaves of Euwas partitionedwith ethyl acetate, concentrated andpurified bySephadexLH-20.Thefractionscontainingtriterpeneswerepooled(OBE104),andusedforbiologicalassays.Adiposetissue-derived cellswere obtained by enzymatic digestion of adipose tissue from healthy patients. These cellswere induced todifferentiateand treated for7dayswithOBE100andOBE 104.Fattyacidcontentwasevaluatedbyoil red staining.Geneexpression levels of PPAR-γ, C/EBPα, FABP4, Glut4, adiponectin and leptin were analyzed by real-time RT-PCR. Humanmacrophage cell line U937, differentiated and activated with lipopolysaccharide (LPS) and INF-γ, was used to evaluateOBE100andOBE104anti-inflammatoryaction.GeneexpressionlevelsofIL-6,IL-1βandMCP-1weredetectedbyreal-timeRT-PCR.BothcelllinesviabilitywasdeterminedusingMTTassay.Both fractions of Eu (OBE100-OBE104) presented no toxicity on macrophage or adipocyte cells. We observed a dose-dependentreductionofthe intracellularfattyacidcontent inadipocytestreatedfor7dayswithEufractions.OBE100andOBE104increasedthegeneticexpressionofPPAR-γinadipocytes.ItwasalsoobservedasignificantreductionofleptinandGlut4 mRNA levels when we treated these cells with the extracts. However, the expression of C/EBPα, FABP4 andadiponectingeneswasregulateddifferentlybyOBE100andOBE104.OBE100increasedtheexpressionofthesegenesandOBE 104 decreased their expression, this effect was significantly observed when we treated the adipocytes with a highconcentrationoftheextract.BothfractionsreducedmRNAexpressionofpro-inflammatorycytokineIL-6inhumanactivatedmacrophages,withnomodificationoftheexpressionofMCP-1andIL-1βgenes.Conclusions: This study demonstrates that the treatment of differentiated human adipocytes with triterpenes-enrichedfractionsofEucalyptustereticornis reducestheirfattyacidcontent.Theseplantextractsalsoaltertheexpressionofgenesinvolvedinadipocytedifferentiationandmacrophageinflammatoryresponse.WehaveconfirmedinahumancellularmodeltheeffectsofEucalyptustereticornisobservedpreviouslyinmicemodels.Acknowledgements:ThisresearchwassupportedbytheColcienciasgrant1115657406561. KahnSE,HullRL,UtzschneiderKM.Mechanismslinkingobesitytoinsulinresistanceandtype2diabetes.Nature.2006Dec14;444(7121):840-6.2. EsserN,Legrand-PoelsS,PietteJ,ScheenAJ,PaquotN.Inflammationasalinkbetweenobesity,metabolicsyndromeandtype2diabetes.DiabetesRes

ClinPract.2014Aug;105(2):141-50.3. GuillénA,GranadosS,RivasKE,EstradaO,EcheverriLF,BalcázarN.AntihyperglycemicActivityofEucalyptustereticornisinInsulin-ResistantCellsanda

NutritionalModelofDiabeticMice.AdvPharmacolSci.2015;2015:418673.


194

ACTIVITYAGAINSTTHEFUNGUSColletotrichumlindemuthianumOFPHASEOLLINALONEORIN

COMBINATIONWITHSTRUCTURALLYRELATEDCOMPOUNDS,PHENYLPROPANOIDSANDAROMATICMONOTERPENES

DurangoD.1 ,QuiñonesW.2EscobarG.2

¹UniversidadNacionaldeColombia-SedeMedellín,EscueladeQuímica,GrupodeQuímicadelosProductosNaturalesylosAlimentos,Medellín(Antioquia)-Colombia;²UniversidaddeAntioquia,InstitutodeQuímica,GrupodeQuímicaOrgánicade

ProductosNaturales,Medellín(Antioquia)-Colombiaemail:[email protected]

Thecommonbean(Phaseolusvulgaris L.) is themost importantedible legume in theworld; it represents50%of thegrainlegumesconsumedworldwide.Unfortunately,thiscropisseriouslyaffectedbynumerousfungalpathogensthatcancausesevereyieldlosses.InColombia,themostwidespreadanddestructivediseaseincommonbeanistheanthracnose(causedbyColletotrichumlindemuthianumSacc.&Magnus;Scribner).Traditionally,thediseasehasbeenacceptablycontrolledbytheapplicationofconventionalsynthetic fungicides,someofwhichcanpresentpotentialharmfuleffectsontheenvironmentandhumanhealth(Tripathi&Dubey,2004).Nowadays,oneofthealternativesthatarebeingintenselyexploredtocontrolphytopathogenic fungus is based in the natural protectivemechanisms of plants,which include the accumulation of lowmolecularweightantimicrobialcompoundscalledphytoalexins. Incommonbean, themainphytoalexincorrespondtothepterocarpan phaseollin. In the present work, the inhibitory effect against C. lindemuthianum of phaseollin, alone or incombinationwithgenistein,daidzein,chromone,4-thiochromanone,4-chromanone,homopterocarpin,eudesmin,carvacrol,thymol,eugenol,andanetholewasevaluated.The fungus was isolated from diseased P. vulgaris pods and characterized by morphological analysis. The toxicity ofcompounds against C. lindemuthianum was evaluated through the poisoned food technique (Grover & Moore, 1962).EvaluationsweremadeinsterilePetridishesof70mmdiametertowhichwasadded7mLofpotatodextroseagar(PDA).Thecompoundswereaddedtoaconcentrationof200μg/mL;ethanolwasusedassolvent(<2μL/L).Petridisheswithandwithout ethanol were used as controls. The Petri dishes were incubated at room temperature andmycelial growthwasmeasuredeach24h(during96h).Therelativegrowthinhibitionofthetreatmentscomparedtothecontrolswascalculatedaspercentage,usingtheformula:Inhibition(%)={1–[radialgrowthoftreatment(mm)/radialgrowthofcontrol(mm)]}x100.Additionally, fungitoxicity of combinations of thymol, carvacrol, homopterocarpin, eudesmin, anethole, eugenol, 4-chromanone,chromone,4-thiochromanonewithphaseollin,inrelation5.5:1.5,3.5:3.5,and1.5:3.5,v/v)wasevaluated.Theresultsshowthateudesmin,carvacrol,and4-thiochromanoneexhibitedthehighestfungistaticeffect(>97%).Phaseollindisplayedarelativelyhighinhibitoryeffectagainstthefungus(between87and69%).However,theinhibitionwasincreased(additive interaction) by combining thephytoalexinwith eugenol, carvacrol and eudesmin. This effectwasdependentonspecificstructuralrequirementsinthecompoundsevaluated.Ontheotherhand,asubtractiveinteractionwasexhibitedformixtures of phaseollin and anethole, thymol, 4-thiochromanone, 4-chromanone, genistein, daidzein, and chromone. Theresultspresentedcanprovideusefulinformationforthedevelopmentofantifungalagentsmoreselectiveandfriendlytotheenvironment,tocontrolbeananthracnose.1. Tripathi P, DubeyNK. 2004. Exploitation of natural products as alternative strategy to control post-harvest fungal rotting of fruits and vegetables.

PostharvestBiologyandTechnology32(3):235-245.2. GroverRK,MooreJD.1962.ToxicometricstudiesoffungicidesagainstthebrowningorganismsSclerotiniafructicolaandS.lava.Phytopathology52:876-

880.


195

CORDIACURASSAVICA:PRIMARYSOURCEFORDISCOVERINGANATURALMEDICINEFORDENGUE

TREATMENT

VelandiaSA.1,LoperaIA2,Sepúlveda-AriasJC2,VelozaLA2,StashenkoEE1,OcazionezRE1 ¹UniversidadIndustrialdeSantander,Bucaramanga(Santander)-Colombia;2UniversidadTecnológicadePereira,Risaralda-

Colombiaemail:[email protected]

Dengueposes a substantialeconomicand diseaseburdenin endemic countries around the world. Severedenguehassignificanthospitalcoststhatincreaseduringepidemics.Thereiscurrentlynospecificantiviraltherapyavailablefordenguetreatment.Herbal therapy for reductionof virus load and theproductionof inflammatorymediatorsmaybebeneficial inattenuating severityofdengue.Colombiahasa richherbal repertoireand theVademecumonMedicinalPlantsprescribesmedicinesforavarietyofdiseases,butdengueassuchisnotincluded.The current work was carried out in order to identify anti-dengue activities in essential oils (EOs) from plants grown inColombia.Nineplantsspecieswereselected.EOsextractionwascarriedoutbyusingmicrowave-assistedhydrodistillation.Asystematicbioassay-guided screening approach was used to identify samples with relevant inhibitory effects on virus infection andproductionofinflammatorymediators.AnEO,whichshowedanIC50value≤50μg/ml,SI(selectivityindex)≥4.0andreducedinflammatory mediators by more than 25%, was designated to be active as an inhibitor of dengue virus (DENV) cellularinfection.An MTT assay showed that EOs obtained from Cordia curassavica, Baccharis decussata, Lippia origanoides, Piper holtonii,Psidiumsartorianum,Calycolpusmoritzianus,WedeliacalycinaandZorniabrasiliensishadnodiscerniblecytotoxicity(CC50>80μg/ml)onfourcelllinesderivedfromhuman(HepG-2andHEK-293)andanimal(Vero,andB16F10)organs.EOfromTagetescaracasana resulted toxic (CC50 between60 and 26μg/ml) on animal cells.All EOs (30µg/mL)were screened for antiviralactivities in DENV-1-infected Vero-cells by using an area-fraction output method with ImageJ freeware. Only Cordiacurassavica reduced (46.6%) cellulardeath causedby virus infection, and inhibitedDENV-2 (IC50: 38.2μg/ml; SI: 5),DENV-4(IC50: 10.0 μg/ml; SI: 19) andDENV-1 (IC50: 69.7 μg/ml; SI: 2.7) in a virus protein (NS1) reduction assay. A reduction in theproductionofprostaglandinE2(42.4%)andtheIL-6cytokine(37.6%),butnot(<20%)ininflammatorymediatorssuchusNO,TNF-αandIL-1β,wasobservedinmurinemacrophagecells(Raw264.7)treatedwithC.cursavvica.Analysis of biological properties of indigenous species is an option for identifying starting material for discovering bothherbal medicines and synthetic antivirals for dengue treatment. This study has resulted in the identification of Cordiacurassavica EO,whichpresents inhibitoryeffectoncell infectioncausedbyDENVserotypes.TheEOcouldbeselected forprotectiveefficacystudiesinananimalmodelinordertoconfirmantiviral,immunomodulationandtoxicityactivities.Acknowledgments:ThisresearchwascarriedoutthankstofinancialsupportfromtheColombianInstituteofScienceandTechnology(Colciencias),GrantRC-245-2011(PatrimonioAutónomodelFondoNacionaldeFinanciamientoparalaCiencia,TecnologíaeInnovación,FranciscoJosédeCaldas).1. YacoubS.etal.Thepathogenesisofdengue.CurrOpinInfectDis2013,26:284-289.2. KadirSLA.etal.Potentialanti-denguemedicinalplants:Areview.JNat.Med.2013,67:677–689.3. CastroNP.etal.Potentialinteractionofcomponentsfromessentialoilswithdenguevirusproteins.Blacpma2015,14:141-155.


196

ESSENTIALOILSANDTHEIRCOMMONMETABOLITEΒ-CARYOPHYLLENEASPRIMARYSOURCEFOR

DISCOVERINGANANTIVIRALAGENTAGAINSTDENGUEVIRUSES

VelandiaSA.,FlechasMC.,StashenkoEE.,OcazionezRE.

UniversidadIndustrialdeSantander,Bucaramanga(Santander)-Colombia.email:[email protected]

Dengue Virus (DENV) causes millions of infections in tropical countries around the world. There is currently no specificantiviraltherapyavailablefordenguetreatment.Analysisofbiologicalpropertiesofproductsderivedfrommedicinalplantsisanoptionforidentifyingstartingsamplesfordiscoveringofherbalmedicinesandsyntheticantivirals.Colombiahasarichherbalrepertoire,butknowledgeaboutanti-DENVactivitiesislimited.Thecurrentworkwasundertaken to investigateanti-DENVactivitiesofessentialoils (EOs) fromplantscommonlyused intraditionalfolkmedicineinColombia,andtoassessfurthertheanti-DENVactivityoftheirmostcommonmetabolite.Seven plants species were selected for EOs extraction by using microwave-assisted hydrodistillation. Chromatographic(GC/FDI, GC/MS) and statistical analyseswere applied for identification of EOmetabolites. A systematic bioassay-guidedscreeningapproachinhumanliver(HepG-2)andanimalkidney(Vero)cellswasfollowedforscreeningofantiviralactivitiesbyusinganarea-fractionoutputmethod(withImageJfreeware),andvirus-antigenreductiontests.EOsfromCordiacurassavica,Pipermarginatum,Pipermedium,Baccharisdecussata,Lippiaoriganoides,Hyptisbrachiate,andZorniabrasiliensis hadnodiscerniblecytotoxicityonfourcell linesfromhumanandanimalorgans(CC50>80μg/ml forallcells)inaMTTassay.OnlyEOsfromCordiacurassavicaandPipermarginatumreduced(46.3–60.2%)cellulardeathcausedbyvirusinfection,andreducedcellularinfectionofallfourDENVserotypes(IC50between10.0and86.5µg/mL;SIbetween30and 1.6) in virus-antigen reduction assays. Twenty-oneEOs constituentswere identified, andβ-caryophyllenewas theonlymetabolitecommontoalloils.β-CaryophyllenewasactiveagainstallfourDENVserotypes(IC50between8.4and15.6µMwithSIbetween22.2and41.3),andtime-of-drug-additionassaysrevealedthatitinhibitedatveryearlystagesofvirus-infection(0–4haftervirusadsorption).Productsderived fromplantsused in local traditional folkmedicineare sourceof compounds fordiscoveringofmedicinedrugs.ThisstudyidentifiedEOsandtheirmetaboliteβ-caryophyllenewithpromisinganti-DENVactivities,whichcouldbeusedasstartingpointsforconsiderationindesigningeffectiveherbalmedicinesand/orsyntheticantiviralsfordenguetreatment.Studiesonprotectiveefficacyinananimalmodelarenecessaryinordertoconfirmantiviralandtoxicityactivities.Acknowledgments:thisresearchwascarriedoutthankstofinancialsupportfromtheColombianInstituteofScienceandTechnology(Colciencias),GrantRC-245-2011(PatrimonioAutónomodelFondoNacionaldeFinanciamientoparalaCiencia,TecnologíaeInnovación,FranciscoJosédeCaldas).1 YacoubS.etal..Thepathogenesisofdengue.CurrOpinInfectDis2013,26:284–289.2 KadirSLA.etal.Potentialanti-denguemedicinalplants:Areview.JNatMed2013,67:677–689.3 BehnamMA.etal.TheMedicinalChemistryofDengueVirus.JMedChem2016,59:5622–5649


197

PHARMACOLOGICALINVESTIGATIONSONKIGELIAAFRICANA(LAM)BENTH.,FRUITSEXTRACT

VeronicaMicheliV.1,RokiaSanogoR.2,ValeriaD’AngeloV.3,OcchiutoF.3

1FundationA.Imbesi,UniversityofMessina,Messina-Italy;2DepartmentofMedicineTraditional,UniversityofBamako,Bamako-Mali;3DepartmentofChemical,Biological,PharmaceuticalandEnvironmentalSciences,UniversityofMessina,Messina-Italy.

email:[email protected] africana (Lam) Benth., syn.K. pinnata (Bignoniaceae) is abundant in the tropics and is widely used in Southern,CentralandWestAfricaasaherbalremedyforvariousailmentssuchasmalaria,eczema,rheumatism.Thefruitsinparticularhasawidereputationinfolkmedicineforthetreatmentofinfertility,sexualimpotence,postpartumhemorrhages,butthereis no scientific evidence thatKigelia is effective in these case [1, 2, 3]. Chemical constituents of K. africana include somenaftochinons,iridoids,phytosterols,isocumarin,lignan,flavonoids,phenolicacids,alkaloids,saponinsandtannins[4,5,6,7].Pharmacological studies of different Kigelia extracts have shown antibacterial activity of the fruits [8] and antiossidantactivityofthefruitandleaves[9].InthepresentstudytheKigeliahydroethanolicfruitsextractwasstudiedforitspossibleuterotonicactivity,onspontaneousandagonists-inducedcontractionsinisolatednon-pregnanthumanmyometriuminordertovalidateitstraditionaluseinthetreatmentofpost-partumbleeding.AuthenticatedfruitsofK.Africana,collectedinLimakoleandSibyvillages(Mali),wereextractedinSoxhletwithethanol60%.Myometrialstrips,takenduringthefollicularstageofthemenstrualcycle,wereobtainedfromhysterectomyspecimensofpremenopausalwomen. The longitudinalmuscle stripsof 1.5 cmweredissected andmountedvertically in a 20ml tissuebaths under physiological conditions to record their isometric contractions by a force transducer. The responses ofcumulative concentrations of plant extract on spontaneous contractions in the presence and absence of α-adrenoceptorblocker (prazosin), muscarinic receptor blocker (atropine) or cyclooxygenase inhibitor (indomethacin), and on agonist-inducedcontractions,wereinvestigated.TheeffectsofextractonmyometriummaybecomparedwiththoseprovokedbyPGE2,usedaspositivecontrol,inthesameexperimentalconditions.ionoftheactivity.Kigeliaextractincreased,inaconcentration-dependentmanner,muscularbasictonusandamplitudeofspontaneousuterinecontractionsatconcentrations ranging from50-400µg/ml.Pretreatmentwith prazosin,atropineor indomethacindidnotaffect theuterine responses toKigelia extract. Furthermore,Kigelia extract increased thecontractile response inducedbyvasopressinandacetylcholine.These investigations indicating thatplantextractexertuterotoniceffectsofmuscolotropictype on human myometrial muscles and his justifies the traditional use of this plant in the treatment of post-partumhaemorrhage.1. KokwaroJO.MedicinalPlantsofEastAfrica.3rded.UniversityofNairobiPress,Nairobi,Kenya,2009.2. NeuwingerHD.AfricanEthnobotanyPoisonsandDrugs.ChemistryPharmacologyToxicology.Chapman&Hall,19963. NeuwingerHD.AfricantraditionalMedicine.AdictionaryofPlantsUseandApplications.Medpharm.Sci.Publishers,Stuttgart,2000.4. GoudaG.Y.,etal.IridoidsfromKigeliapinnataDC.Fruits.Phytochemisrty2003,63:887-892.5. GoudaY.G.,etal.PhenylpropanoidsandphenylethanoidderivatesfromKigeliapinnataDC.Fruits.NatProdRes.2006,20:935-939.6. SidjuiL.S.etal.TriterpenesandLignansfromKigeliaafricana.J.AppliedPharmaceuticalScience2015,5:001-006.7. El-SayyadS.M.FlavonoidsoftheleavesandfruitsofKigeliapinnata.Fitoterapia1981,4:189-918. GraceOM.etal.AntibacterialactivityandisolationofactivecompoundsfromfruitofthetraditionalAfricanmedicinaltreeKigeliaafricana.SouthAfrJBot.

2002,68:220-222.9. Solomon I.S. Phytochemical screening and free radical scavenging activities of the fruit and leaves of Kigelia africana (Bignoniaceae). ARPN J. Science

Technology2014:4:45-51.


198

ANTILEISHMANIALACTIVITYANDINVIVOTOXICITYOFFOURCHEMOTYPESOFLIPPIA(FAM.

VERBENACEAE)ESSENTIALOILS

NeiraLF.1,HernandezJC.2,EscobarP.1 ,StashenkoEE.3

1CentrodeInvestigacióndeEnfermedadesTropicales(CINTROP),UniversidadIndustrialdeSantander,Bucaramanga-Colombia;2PAT-UIS,EscueladeMedicina,DepartamentodePatología,UniversidadIndustrialdeSantander,Bucaramanga-Colombia;3Centro

deInvestigaciónenBiomoléculas(CIBIMOL,CENIVAM),UniversidadIndustrialdeSantander,Bucaramanga-Colombiaemail:[email protected]

Essential oils (EOs) from plants distributed in Colombia and belonging to the genus Lippia are interesting candidates forcutaneous leishmaniasispharmaceuticsystems.TheEO-chemotypesofLippiaalbaandL.origanoidescharacterizedbytheirmajorcompoundsnamelycitral(EO1),thymol(EO2),carvacrol(EO3),andphellandrene(EO4)weretestedagainstLeishmaniaandtheirtoxicologyprofilewasdeterminedinBALB/cmice.Essential oils were obtained by microwave radiation-assisted hydrodistillation and characterized by gas chromatographycoupled tomassspectrometry 1.ActivitiesagainstLeishmania (Viannia)panamensis, L. (V.)braziliensis andcell toxicitiesonTHP-1cellsweredeterminedbystandardmethodologiesandIC50andCC50werecalculated2.Skinirritationwasstudiedaftertopicaltreatmentwith10,50and100%ofEO(w/winoliveoil)andacutetoxicityafteroraladministrationof2000mg/kg.Thecontacthypersensitivitywasdeterminedbypre-treatment/challengerwithEOsand theEO-inducedgenotoxicityafteroraladministration of 100 mg/kg/14 days by comet and micronucleolus (MN) assay. Cyclophosphamide and ethylmethanesulfonatewereusedascontrols3.All EOswere active against Leishmaniapromastigotes (IC50 0.39-11.18 µg/mL) and EO1 and EO3were active against L. (V.)panamensis(IC5042.66and63.87µg/mL)andL.(V.)braziliensis(IC509.19and15.43µg/mL)amastigotes.TheEO-celltoxicitiesonTHP-1cellswereintherangeofCC5026.27-52.55µg/mL.EO2 and EO3 used at 100% (pure) induced oedema, severe erythema and crusting with histopathology features ofhyperkeratosis, parakeratosis, spongiosis, and exocytosis of inflammatory cells. No irritation signs were observed usinglower EO-concentration.No changesofweight, food intake and serum levels of urea (34.16 to 70.0mg/dL) andAST/GOT(69.55to80.10U/L)wereobservedafteroraltreatment.IncreasedlevelsofalkalinephosphatasewereobservedafterEO2andEO3 treatment (82.32and559.80U/Lversus23.95-55.46U/L) suggestingapossible transient-liverdamage.Comparedwiththepositivecontrol(1-fluoro-2,4-dinitrobenzene),neitherincreaseinearsthickness(0.15versus1.0mm)norhistologicalinflammatory changeswere induced in EOs-sensitized animals after EOs-challenger. No DNA damagewas observed aftertreatmentwithserialdosesofEOs.The%ofDNAdamagewas22-28.75%(Control:74.38%)andforMNwas0.5-3.17(Control:175-200MN)inpolychromaticerythrocytes.AllEOschemotypesshowed invitroanti-parasiticactivity.Atthehigherconcentrationusedofthymol(EO2)andcarvacrol(EO3)chemotypes,theyshowedirritationandsomesignsofacutetoxicity.LowerEO-concentrationsthanthetestedinthisstudycouldbeusedsafetyincaseofpharmacologicalformulations.Acknowledgement:Thisstudywasconductedthankstothe“PatrimonioAutónomo,FondoNacionaldeFinanciamientoparalaCiencia,FranciscoJosedeCaldas,”contractRC-0572-2012-Bio-Red-CENIVAMandtotheUniversidadIndustrialdeSantander.1. StashenkoE.E.etal.LippiaoriganoideschemotypedifferentiationbasedonessentialoilGC-MSandprincipal componentanalysis. JournalofSeparation

Science,2010,33:93-1032. Escobar P. et al. Chemical composition and antiprotozoal activities of Colombian Lippia spp essential oils and their major components. Memórias do

InstitutoOswaldoCruz,2010,105:184–1903. VasquezM.Z.Combining the in vivo cometandmicronucleus assays: apractical approach togenotoxicity testinganddata interpretation.Mutagenesis,

2010,25:187–99


199

OXIDATIVESTRESSINDIFFERENTCLINICALCONDITIONS,USINGREDOXINDEXESOFDIAGNOSTIC

VALUE.

LizetteGildelValleLG.1,GuevaraM.2 1InstitutodeMedicinaTropicalPedroKourí,IPKAutopistaNoviadelMediodí,LaHabana,Cuba;2LABIOFAM,LaHabana,Cuba

email:marielaguevara@infomed.sld.cuTheredoxstatusimbalancecouldberelatedtodiverseclinicalentities.Recognizedanalyticmethodologiesquantifyingboth:oxidative biomolecular damage and antioxidant activity were used for the characterization of redox balance in humansamples(urine,lymphocytes,plasmaandserum)inrelationwithprogressionmarkersofdiverseclinicalconditions.Case and control studies of Human immunodeficiency virus (HIV), Dengue, Diabetes mellitus (DM) type 1, Human-Tlymphotropicvirustype-1patientsandapparentlyhealthyindividuals(18-84years)werecarriedout.Theevaluationwasalsoappliedininterventiondesigns.Theresultsevidencedoxidativealterationsandantioxidantcapacitydecreasedsignificantly(p<0.05)inpatientscomparedtohealthy individuals related inageandgender.Studiesofnutritional interventionandantioxidant supplementationwithVimang®in40and81HIVCubanpatientsrespectivelyshowedsignificantbeneficialchanges(p<0.05) in56%and43.9%ofcases respectively. An observational study in 56 HIV Cuban patients was carried out involving two combinations ofantiretroviral therapy. The study showed evidences of oxidative modifications significant (p < 0.05) in 87% of the cases,finding differences among combinations. An observational study involving 40 Cuban DM type 1 patients with change ofneutralprotamineHagedorninsulinfrompigtohumanrecombinant,showedasignificantbeneficialchange(p<0.05)in81%ofthecasesafterthechange.Theintegralcharacterizationcouldbeusefulforfollowupandindividuals’managementbutalsocontributetotheknowledgeofthemolecularmechanismsunderlyingintheseillnesses.1. HalliwellB,andWhitemanM.Measuringreactivespeciesandoxidativedamage invivoand incellculture:howshouldyoudo itandwhatdotheresults

mean?BrJPharmacol.2004,142:231-55.2. SantiniSA.etal.,DefectiveplasmaantioxidantdefensesandenhancedsusceptibilitytolipidperoxidationinuncomplicatedIDDM.Diabetes.1997,46:1853-8.3. AtalayM.etal.Treatmentswithsodiumselenateordoxycyclineoffsetdiabetesinducedperturbationsofthioredoxin-1levelsandantioxidantcapacity.Mol

CellBiochem.2011,351:125-31.4. Pérez-MatuteP.etal.Reactivespeciesanddiabetes:counteractingoxidativestresstoimprovehealth.CurrOpinPharmacol.2009,9:771-9.


200

ANTIOXIDANTANDANTIMICROBIALACTIVITIESOFLEPIDIUMSATIVUMEXTRACTS

BouamraD.1 ,BakiCA.1,TouabetL.1,MelianiS.1,BouchebourA.1,DahamnaS.1,HarzallahD.2

¹LaboratoryofPhytotherapyAppliedtoChronicDiseases,DepartmentofAnimalBiologyandPhysiology,FacultyofNaturalandLifeSciences,UniversityFerhatAbbas,Setif-Algeria,2LaboratoryofAppliedMicrobiology,DepartmentofMicrobiology,Facultyof

NaturalandLifeSciences,UniversityFerhatAbbas,Setif-Algeria.email:[email protected]

Nowadays,theuseofofficinalplantsisstillattopofmedicalpracticesinmanycountries,itisestimatedthat80%oftheworldpopulation use a wide variety of herbs and regional plants to treat illnesses with a great sureness of their remedialeffectiveness (Sandhya et al., 2006). Therefore, this study aims to evaluate the antioxidant and antimicrobial activities ofLepidiumsativum,aworldwideknownspeciesandlargelyutilizedinMediterraneanfolksmedicine.Lepidiumsativumseedswererinsed,driedandstockedinaroomtemperatureconditions.TheaqueousextractofLepidiumsativumseedswasobtainedfollowingtheproceduredescribedbyDivekaretal.(2010),andthemethanolicextractwasgotfollowing the procedure of Markham (1982). The phenolic content of the extracts was measured by the Folin-Ciocalteumethod (Li et al., 2007), the Flavonoid contentwasmeasured following the AlCl3method (Bahorun et al., 1996); for theantioxidantactivitytheDPPHtestwascarriedoutasdescribedbyRapisardaetal.(1999),andfortheantimicrobialactivitythedisc-diffusiontestwasperformedasdescribedbyZaidanetal.(2005).The phytochemical study shows that theMeOH extract of Lepidium sativum seeds got better yield and was richer withflavonoids, whereas the aqueous extract had better content of total phenolic compounds, which claim the therapeuticimportanceoftheplantanditsroleasasourceofbioactivecompounds.Moreover,theresultsoftheDPPHassayshowthattheaqueousextractofLepidiumsativumseedshadabetterscavengereffectthantheMeOHextract,butbothextractgotweakantioxidantactivityregardingthestandards,whichmaybeduetotheweakcontentoftheactivecompoundswithintheextracts.Furthermore,theresultsofthediscdiffusiontestrevealthatbothextractshavenoantibacterialeffectagainstthetestedstrains.However,bothextractsofLepidiumsativumseedsshowedagoodantifungalactivityagainstAspergillusflavusandAspergillusniger,andthismaybeduetothepropertiesofpolyphenolsfoundintheextractsoftheseeds,notablytheaqueousextract.ResultsfromthepresentstudyencourageformoreresearchonthenatureofthecompoundsfoundintheseedsofLepidiumsativumaswellasthetoxicologicalaspectoftheplant.1. Bahorun,T.,Gressier,B.,Trotin,F.,Brunet,C.,Dine,T.,Luyckx,M.,Vasseur,J.,Cazin,M.,Cazin,J.C.&Pinkas,M.(1996)Oxygenspeciesscavenging

activityofphenolicextractsfromhawthornfreshplantorgansandpharmaceuticalpreparations.Arzneim.-Forsch.46:1086-1089.2. DivekarVB,KalaskarMG,ChougulePD,RedasaniVK,BahetiDG,2010,isolationandcharacterizationofmucilagefromlepidiumsativumlinn.Seeds.Int.J.

Pharm.Res.Dev.,2(1),1-5.3. Li H.B., Cheng K.W., Fan K.W. and Jiang Y., 2007. Evaluation of antioxidant capacity and total phenolic content of different fraction of selected

microalgae.FoodChem.,102(3),pp771-776.4. MarkhamK.R.,1982.TechniqueofflavonoidsidentificationAcademicpress,LondonChap.Land.2,pp1-113.5. RapisardaP.,TomainoA.,LoCascioR.,BoninaF.,DePasqualeA.,andSaijaA.,1999.Antioxidanteffectivenessasinfluencedbyphenoliccontentoffresh

orangejuices.J.Agr.FoodChem.,47(11),pp4718-4723.6. SandhyaB.,ThomasS., IsabelW.andShenbagarathaiR.,2006.EthnomedicinalplantsusedbytheValaiyancommunityofPairanmalaihills (Reserved

Forest),TamilNadu,India-Apilotstudy.AfricanJTraditComple.3(1),pp101-114.7. ZaidanM.R.,NoorRainA.,BadrulA.R.,AdlinA.,NorazahA.andZakiahI.,2005.Invitroscreeningoffivelocalmedicinalplantsforantibacterialactivity

usingdiscdiffusionmethod.TropicalBiomedicine,22(2),pp165-170.


201

QUANTITATIVEHPLC-UV/DADANALYZESOFTHEMAINC-GLYCOSIDEFLAVONOIDSINTHREEDIFFERENTMETHANOLICEXTRACTSOFALTERNANTHERATENELLACOLLAAERIALPARTS

MarinhoJVN.1 ,SomaR.1,Hernández-TascoAJ.1,SalvadorMJ.2

¹DepartmentofPlantBiology,InstituteofBiology,UniversityofCampinas-UNICAMP,Campinas-Brazil;²DepartmentofPharmaceuticalSciences,FacultyofPharmacy,UNICAMP,Campinas-Brazil


AlternantheratenellaColla,belongingtotheAmaranthaceaefamily,isabushwidelydistributedinLatinAmerica,andusedinBraziliantraditionalmedicineforthetreatmentofinfectionsandinflammatorydiseases(Ferreiraetal.,2003;Salvadoretal.,2004).Severalmethodsofextractioncanbeemployedtoobtainplantextractsandtheirqualitydependsontheprocedureused.Thisworkcompares threedifferentextractingmethods:ultrasound,packed-bed reactorandmaceration inorder toprepareA.tenellaCollastandardizedextracts.TheC-glycosideflavonoids(2"-O-α-L-rhamnopyranosylvitexinandvitexin)wereanalyzedbymeansofhigh-performanceliquidchromatographycoupledwithdiodearraydetection(HPLC–UV/DAD).AlternantheratenellaCollawascollectedinRibeirãoPreto(SP,Brazil)andavoucherspecimenwaskeptintheFFCLRP/USPHerbarium (reference SPFR02968). The aerial part of theplantwaspowdered, air-dried and submitted to a packed-bedreactor(PR)andultrasound-assisted(US)40kHz,for30and60min.,30°Candmaceration(MAC)for720and1440min.,30°C.Methanolwasusedasextractingsolvents (1:20,m/v).TheHPLC-UV-DADanalysiswerecarriedout inaLC-20-ATShimadzuProminenceLiquidChromatographusingaC-18(Kinetix-Phenomenex)column(5µm,150x4.6mmid)withaguardcolumnC 18 (Phenomenex, 5μm, 2.1 x4.6mm id). Themobilephasewas composedofdistilledwater: formic acid (99:1, v/v) andacetonitrile:THF(tetrahydrofuran)(95:5,v/v).Theseparationwasachievedbyusingagradientelutionas follows:0-15minfrom15%to22%B;15-22minfrom22%to20%B;22-25minfrom20%to40%B;25–35minfrom40%to15%B;whichwasheldfor5min.Theflow-ratewas0.65mL/min,injectionvolumewas20μL,at25°C.Chromatogramswereacquiredat350nm.Thevalidation of the HPLC-UV/DAD method was performed in agreement with the international guidelines for analyticaltechniques for thequalitycontrolofpharmaceuticals (ICHguidelines).Thestandardsusedwerepreviously isolated inoutlaboratoryfromthissameplantfamily.TheHPLC-UV/DADmethoddevelopedlinearitywas(r2>0.99)forthereferencestandards:2"-O-α-L-rhamnopyranosylvitexin,andvitexin.Thelimitofdetection:0.156µg/ml,limitofquantification:0.625µg/ml,repeatability<2.0%,accuracy94-102%.Thismethodwasapplied toquantify the standards. The concentrationof 2"-O-α-L-rhamnopyranosylvitexin, identified in theA.tenellametanolicextractfoundinµg/gwas:US30min:17.36±0.06;US60min:21.22±0.12;PR30min:25.36±0.07;PR60min:23.81±0.36; Mac 720 min: 27.50±0.19 and MAC 1440 min: 36.80±0.05.The concentration of vitexin was (µg/g): US 30:3.88±0.01;US60:4.48±0.03;PR30:4.70±0.01;PR60:4.07±0.15);Mac720:5.75±0.05andMAC1440:6.73±0.03.DeSantanaAquino et al. (2015) showed that 2″-O-α-L-rhamnopyranosylvitexin, significantly inhibited paw edema and reduced bothleukocytemigration and the leakage of protein into the pleural cavity. Themaceration extraction presented the highestamountofflavonoidscontent,howeverthepacked-bedreactorextractiontimewaslowerandshowedaround50-90%oftheflavonoid quantity found in maceration extracts, as well as exhibited higher content of the C-glycoside flavonoids incomparationwith the amount found in the extracts obtained from theultrasound technical. Thepacked-bed reactor andultrasound-assistedmethodsemployed inthisstudy improvedtheextractionprocessofA.tenella,showedagoodyieldofmasstransferinashortertime,whencomparedtotraditionalmacerationmethod.Acknowledgements:toCAPES,CNPq,FAPEAM,FAPESPandFAEPEX-UNICAMP1. DeSantanaAquino,D.F.,Piccinelli,A.C.,Soares,F.L.P.,Arena,A.C.,Salvador,M.J.,&Kassuya,C.A.L.,2015.Anti-HyperalgesicAndAnti-Inflammatory

ActivityOfAlternantheraMaritimaExtractAnd2″-O-Α-L-RhamnopyranosylvitexinInMice.Inflammation,38(6),2057-2066.2. Ferreira,E.A.,Procópio,S.O.,Silva,E.A.M.,Silva,A.A.,&Rufino,R.J.N,2003.EstudosAnatômicosDeFolhasDeEspéciesDePlantasDaninhasDe

GrandeOcorrênciaNoBrasil. Iv-AmaranthusDeflexus,AmaranthusSpinosus,AlternantheraTenellaEEuphorbiaHeterophylla.PlantaDaninha,21(2),Pp.263-271.

3. Salvador,M.J.;Ferreira,E.O.;Mertens-Talcott,S.U.;Castro,W.V.;Butterweck,V.;Derendorf,H.;Dias,D.A.,2006.IsolationAndHplcQuantitativeAnalysisOfAntioxidantFlavonoidsFromAlternantheraTenellaColla.Z.Naturforschung,61c.


202

SPECTROSCOPYCHARACTERIZATIONOF(E)-2-(2,6-DICHLOROBENZYLIDENE)-1,1-DIPHENYLHYDRAZINE

Cabrera-VivasB.M.1 ,Ariza-RamírezJ.1,AguirreCabreraC.1,RamírezJuanC.1,García-RamosJC.2,

Toledano-MagañaY.31FacultaddeCienciasQuímicas,BeneméritaUniversidadAutónomadePuebla,Puebla-México;2UniversidadNacionalAutónomadeMéxico,FacultaddeQuímica,DepartamentodeQuímicaInorgánicayNuclear,MexicoCity-Mexico;3UniversidadNacional

AutónomadeMéxico,InstitutodeInvestigacionesBiomédicas,DepartamentodeInmunología,MexicoCity-Mexicoemail:[email protected]

Thehydrazonesarean importantgroupwhichhasabiologicalactivity.Hydrazonesarevery importantgroupofanalyticalregentsforthedeterminationofvariousmetalionsbyusingvariousanalyticaltechniques.Besidesthisuseofhydrazonesarealsohavingbiologicalactivitiesalso.Lakshmietaldiscussedabout thechemicalnatureofhydrazonesand theirbiologicalactivitiesandfocusedontheanalyticalapplicationsspectrophotometricandspectrofluorimetricofhydrazones.1

MoreoverHydrazonesderivativesofpyridineareversatileclassofanimportantgroupoforganiccompoundsthatareusedasbactericides,fungicides,anticanceragents.2

Lightgreencrystals;yield:74%at25°C,mp.100-102ºC.UVʎmax=349nm.FT.IR(film):(cm-1):3134n(C-H),1465,n(C=N),1300n(C=N),1231d(arC-Hip),881n(C-Cl)746(τ)-CH2.1HNMR(300MHz,(CD3)2CO:(d/ppm,J/Hz):7.46(m,4H,C2’,2H,C4’),7.38(s,1H,C=N),7.29(s,1H,C3),7.26(m,6H,C4,5,3’).13CNMR(300MHz,(CD3)2CO):(d/ppm):143.43(C1’,C=N),134.12(C2),131.70(C1),130.55(C5),130.21(C3’),129.44(C4),129.37(C3),125.31(C4’),122.57(C2’).MS-EI:m/z=341.24M+.C19H14Cl2N2

Diphenylhydrazine (1.42mmol, 263.01mg)was dissolved in a solvent, a chemical equivalent (1.42mmol, 250mg) of 2,6-dichlorobenzaldehydewhichwaspreviouslydissolvedinthesamesolventanditwasaddeddropbydropstirringconstantlyandwhichwasdevelopedwithgreenerprocedure.TheactionmixturewaskeptatroomtemperatureandwasmonitoredbyTLC,andthenvacuumfiltered.ThehydrazoneswererecrystallizedbyacontinuousandcontrolledprocessuntilwinecrystalswithadequatesizeandpurityweredevelopedinordertoobtainX-raystudies.Ourworkgrouphasbeensynthetizingmanyinterestingstructuresofhydrazonesthathave been characterized by m. p., U.V, I.R. 1H NMR, 13C NMR.3 (E)-2-(2,6-dichlorobenzylidene)-1,1-diphenylhydrazine.41. Lakshmi Narayana Suvarapu, Young Kyo Seo, Sung Ok Baek,Varada Reddy Ammireddy, Review on

Analytical and Biological applications of Hydrazones and their Metal Complexes, E-Journal ofChemistry,2012,9(3),1288-1304.

2. B.K.Banik,F.F.Becker,Eur.J.Med.Chem.45(2010)4687-4691.3. Blanca M. Cabrera-Vivas, Marcos Flores-Alamo, Ruth Meléndrez-Luevano, Lidia Meléndez-Balbuena,

and Juan C. Ramírez. Acta Crystallographica, 2014. Volume E70, o80. Section E, Supplementarymaterials1-8,StructureReports,(E)-1,1-Diphenyl-2-(thiophen-2-ylmethylidene)hydrazine.ISSN1600-5368.EditorsW.T.A.Harrison,H.Stoeckli-Evans,E.R.T.TiekinkandM.Weil.Doi:10.1107/S1600536813033874.

4. Yanis Toledano-Magaña, RuthMeléndrez-Luévano,MarisolNavarro-Olivarria, Juan CarlosGarcía-Ramos,Marcos Flores-Alamo, LuisOrtiz-Frade, LenaRuiz-Azuara and Blanca M. Cabrera-Vivas, Synthesis, characterization and evaluation of the substituent effect on the amoebicide activity of newhydrazonederivativesMed.Chem.Commun.,2014,5,989.


203

ANTIBACTERIALACTIVITYOFTHEESSENTIALOILOFORIGANUMGLANDULOSUMONBACTERIAL

STRAINSOFHOSPITALORIGINMOSTIMPLICATEDINNOSOCOMIALINFECTIONS

MazidR.1,3 ,LardjamH.1,2,3,4,KhitriW.1,3,4,BoudgheneSY.3,IzaroukenA.3,DjebliN.4,ToumiH.1,2,31PharmacovigilanceService-EHUofOran;2PharmaceuticalDevelopmentResearchLaboratory,3FacultyofMedicineofOran;

4PharmacognosyApi-Phytotherapylaboratory,UniverisityofOran,Oran-Algeriaemail:[email protected]

Origanumglandulosumisanaromaticplant,commoninAlgeriaandwidelyusedbylocalpeopleforitsmedicinalproperties.Theessentialoilfromthisplant,whichgrowsinthewestofAlgeria,wasstudiedtoevaluateanddetermineitsantibacterialactivity.Theextractionoftheessentialoilwasperformedbywatersteamdistillation;theyieldobtainedfromtheaerialparts(1.78%)isinteresting,itschromatographicprofilerevealedbyTLCshowedthepresenceofphenoliccompoundsthymolandcarvacrol.TheevaluationoftheactivityoftheessentialoilofOriganumglandulosum,onbacterialstrainsofhospitalorigin,ATCC,MRB,andHRB,mostimplicatedinnosocomialinfections(StaphylococcusaureusATCC25923,StaphylococcusaureusATCC 43300, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853,Staphylococcusaureusresistanttometicillin,Enterococcusfaecium,VARandRTEC,Acinetobacterbaumanii,IMPRandRCAZ,Klebsiellapneumoniacarbapenemase-producing)bythemethodofaromatogrammeandmicroatmosphere,showsthatthe antibacterial potency of this oil is very high, expressed by significant inhibition diameters on all strains exceptPseudomonasaeruginosa,andlowMICsandischaracterizedbyabactericidalaction.


204

PHYTOTHERAPYINDIABETICPATIENTS:ETHNOBOTANICALSURVEY,PHYTOCHEMICALANALYSISAND

EVALUATIONOFTHEANTIOXIDANTACTIVITYOFFIVEMEDICINALPLANTS

LardjamH.1,2,3,4 ,MazidR.1,3,BoudgheneSY.3,IzaroukenA.3,MoussaouiN.1PharmacovigilanceService-EHUofOran;2PharmaceuticalDevelopmentResearchLaboratory,3FacultyofMedicineofOran;

4PharmacognosyApi-Phytotherapylaboratory,UniversityofOran,Oran-Algeriaemail:[email protected]

Diabetesisoneofnon-communicablediseasesandisconsideredasamajorpublichealthburden,despitetheexistenceofavariety of chemical synthesis ofmedicines used for the treatment of this disease. Currently,more andmore patients areusingherbalmedicine.Forthis,wehavesetthefollowingobjectives:-Determinethefrequencyoftype2diabeticpatientswhoareusingmedicinalplants-Undergoneanidentificationofthecompositionofsomeherbalteasreadytouse-Performaphytochemicalstudyoffiveplantsknownasanti-diabeticsAnethnobotanical surveywas conducted among 314 type 2 diabetic patients, data collectionwas done through a surveyform,thesedatawereenteredandanalysedusingEpiData3.1software.The identificationofthecompositionofsomeherbalteas,sold inourareafordiabeticpopulation,wasperformedusingastereoscope,magnifiersandacamera.Forthephytochemicalanalysis,wedeterminedtotalpolyphenols,flavonoidsandcondensedtanninsthenweevaluated,byspectrophotometry,theantioxidantactivityinthetopfiveplantsknownasanti-diabeticscitedinoursurvey.Oursurveyshowedthat35.40%ofthestudypopulationusemedicinalplants,56specieshavebeenidentifiedbelongingto28families.Theidentificationofthecompositionofherbalteashasallowedustorevealseveralforgeries,whichexplainspoorregulationoftheseproducts.Thephytochemicalanalysisandtheevaluationoftheantioxidantactivity,whichwereconductedonfiveplants:fenugreek,olive, sagebrush, berbéris, oregano, showed high levels of total polyphenols, which are consistent with significantantioxidantactivities.Phytotherapy is still practiced by diabetic patients in addition to conventional treatment. The ethnobotanical surveyperformedindiabeticpatients,hasallowedtoidentifythemainplantsusedbythispopulation.Theseplantscanprovideabroadresponsetothecomplexproblemsofdiabetesmellitusandtherapeuticprospectsforimprovedmanagement.


205

INVITROANDINVIVOANTILEISHMANIALACTIVITYOFArtemisiaannuaLEAVESPOWDERANDPOTENTIALUTILITYINTHETREATMENTOFUNCOMPLICATEDCUTANEOUSLEISHMANIASIS

MesaLA.1,VásquezDA.2,LutgenP.3,VélezID.2,RestrepoAM.2,OrtizIC.4,RobledoSM.1

1UniversidaddeAntioquia,Medellín-Colombia;2PECET-UniversidaddeAntioquia,Medellín-Colombia;3IFBV-BELHERB,Niederanven-Luxembourg;4UniversidadPontificaBolivariana,Medellín-Colombia

email:[email protected] is a tropical disease that affect about 12millionpeople aroundworld.Although still effective, the currentlyuseddrugstotreatthisdiseasehaveserioussideeffectsandtherefore,alternativesdrugsareurgentlyneeded.Because80%oftheworldpopulationusetraditionalmedicineandplantderivedmaterials,theWorldHealthOrganizationhasprioritizedthevalidationoftraditionalmedicineproducts.Recently,amixtureofArtemisiaannuapowdercapsuleswasdevelopedasanewgalenicformulationofdriedherbpowder(“totum”)fortheoraltreatmentofmalariaandotherparasitosis.TheantileishmanialactivityandcytotoxicityofA.annuapowdercapsuleswasdeterminedby invitroand invivostudies.Apreliminarevaluationofthetherapeuticalpotentialasantileishmanial treatment inhumanswasdone intwopatientswithuncomplicatedcutaneousleishmaniasis(CL).TheA.annuapowdercapsulesshowedmoderateinvitroactivityagainstamastigotesofL.panamensisandnocytotoxicityinU-937macrophagesnorgenotoxicity inhuman lymphocyteswasobserved.Fiveof6hamsters (83.3%) treatedwith theA.annua leavespowderatadoseof500mg/kg/dayduring30dayscuredat theendof thestudy. Indeed, twopatientswithuncomplicatedCLcausedbyL.panamensistreatedwithcapsulesofA.annualeavespowderat30gtotalshowedcompletecureafter45daysoftreatmentendedwithoutadversereactions.Bothpatientsremaincuredat22and20monthsafterhavefinishedthetreatment,respectively.Inconclusion,A.annuapowdercapsulesmayrepresentanoptionfortreatinguncomplicatedCLandtheevaluationoftheefficacyandsafetyofthisherbproductinCLbyrandomizedcontrolledtrialsarenecessary.Acknowledgments: Authors thank the patients for agreeing to participate in the study. This work was supported by COLCIENCIAS (CT-695-2014) and theUniversityofAntioquia(CIDEPRO-CIIEs-2009-2014).1. IslamuddinM.etal.ExtractsofArtemisiaannualeavesandseedsmediateprogrammedcelldeathinLeishmaniadonovani.JMedMicrobiol2012,61:1709-

1718.2. IslamuddinM.etal.LeishmanicidalactivitiesofArtemisiaannualeafessentialoilagainstVisceralLeishmaniasis.FrontMicrobiol2014,5:626.3. ElfawalMA.etal.DriedwholeplantArtemisiaannuaasanantimalarialtherapy.PLoSOne2012,7:e52746.


206

ANTILEPTOSPIRALPOTENTIALOFMEDICINALPLANTSCOMMONLYUSEDFORTHETREATMENTOFINFECTIOUSDISEASES

Hernández-RodríguezP.1,4 ,PabónLC.1,4,RodriguezMF.2,4,GómezA.3,4

1DepartmentofScienceBasics,UniversidaddeLaSalle,Bogotá-Colombia;2FacultyofHealthSciences,UniversidaddeLaSalle,Bogotá-Colombia;3FacultyofAgriculturalSciences,UniversidaddeLaSalle,Bogotá-Colombia;4ResearchGroup

BIOMIGEN,UniversidaddeLaSalle,Bogotá-Colombia.email:[email protected]

LeptospirosisisazoonosiscausedbyLeptospiraspp,whichistransmittedtohumansbydirectcontactwiththeurineofwildor domestic animals infectedwith the bacteria. The diseaseoccursworldwidebut ismost often in placeswhere sanitaryconditionsarepoor,havingan impactonhumanhealth, livestockandagriculture.Thisdiseasecanrepresentupto20%offebrilediseasesofunknownorigin,ithasanendemicoverallaverageannualincidenceof5per100,000inhabitants,about17%ofpeoplehospitalizedwithleptospirosissufferacutelunginjuryandofthese25%die;inaddition,inthecaseoftheAmericastheincidenceis12.5per100,000population,andinColombiaof3.05casesper100,000inhabitants.Additionally,thereisnodifferentialdiagnosisofleptospirosiswhichhasledtofailuresincontrollingthediseaseandtreatmentdecisionsfavoringthebacteriaresistancetoantimicrobialastrimethoprim/sulfa,neomycin,andfluoroquinolones.Thatiswhyitrequireslow-costtreatmentoptions,lesstoxicandmoreeffectivetocontrolthedisease;inthissense,anovelalternativeandlittlestudiedfortreatmentofleptospirosisistheuseofnaturalantibiotics.Therefore,theobjectiveofthisstudywastoidentifywhichofthemedicinalplantscommonlyusedtotreatinfectionsinColombia,canbeusedtocontrolleptospirosis.Forthisit11plantswereselected, with the greatest consensus of use for the treatment of infectious diseases, which were extracted by coldmacerationwith96%ethanoland itsantimicrobialeffectbythemicrodilution(MDT)wasevaluatedagainstLeptospira sppserovarCanicola andHardjo itwasused as revealer viability ofAlamarblue.As a result of study identified thatParietariaofficinalis, Achyrocline bogotensis, Equisetum giganteum and Pulmonaria officinalis presented antileptospiral potential to30mg/mL against the two serovars ofLeptospira sppwhilePolypodium calaguala only showed activity againstHardjo andPhytolacca bogotensi against Canicola. This type of study contributes to knowledge about the susceptibility of strainsLeptospira spp against natural antimicrobials, allowing evaluate the use ofmedicinal plants with potential for control ofLeptospirosis.Wellasstudiesaimedatobtainingbio-activeprincipleforthepreventionandcontrolofthiszoonosis.1. Chandan S, Umesha S, Balamurugan V. Antileptospiral, Antioxidant and DNA damaging properties of Eclipta alba and Phyllanthus amarus. (2012).

ScientificReports.1:231.doi:10.4172/scientificreports.2312. ChanderM,KumarK,ShriramA,VijayachariP. (2014)Anti-leptospiralactivitiesofanendemicplantGlyptopetalumcalocarpum (Kurz.)prainusedasa

medicinal plant by Nicobarese of Andaman and Nicobar Islands, Natural Product Research: Formerly Natural Product Letters DOI:10.1080/14786419.2014.985679

3. Grace D, Jones B. (2011). Zoonoses:Wildlife Domestic Animal Interactions. A report to DFID. Nairobi and London: International Livestock ResearchInstituteandRoyalVeterinaryCollege.

4. Lerg.(2011).ReportoftheSecondMeetingoftheLeptospirosisBurdenEpidemiologyReferenceGroup.who.Genova:WHO.5. MiragliaF,MatsuoM,MoraisZ,DellagostinaO,SeixasF,FreitasJ,HartskeeriR,MorenoL,CostaB,SouzaG,VasconcellosS,Moreno,A.(2013).

Molecualrcharacterization,serotypingandantibioticsusceptibilityprofileofLeptospirainterrogansserovarCopenhageniislatesfromBrazil.Diagnosticmicrobiologyandinfectiousdisease,77,195-199.

6. NimalaD,PeriyanayagamK,IsmialM.(2008)InvitroantileptospiralactivityofPlectranthusamboinicus(lour)spreng.Pharmacologyonline.2:95-987. PrabhuN,MeeraJ,AlwinR,NatarajaseenivasanK,UmaA.(2014).InvitroantileptospiralactivityofchloroformextractofPiperbetleL.WorldJPharm

Sci.2(8):711-715


207

HYPOGLYCEMICEFFECTOFETHANOLICEXTRACTOFPARMENTIERAACULEATA(KUNTH)SEEMFRUIT

ANDBARKINMALEWISTARRATS

Muñoz-MuñizOD.1 ,Alcántara-LópezMG.2,Vázquez-HernándezM.1,Domínguez-OrtizMA.3¹UnidadSARA-UniversidadVeracruzana,Luis,Xalapa(Veracruz)-México;2FacultaddeQuímicaFarmacéuticaBiológica-

UniversidadVeracruzana,Xalapa(Veracruz)-México;3InstitutodeCienciasBásicas-UniversidadVeracruzana,Xalapa(Veracruz)-México

email:[email protected] (DM) isasyndromecharacterizedbychronichyperglycemiaanddisturbances incarbohydrates, fatsandproteinsmetabolismassociatedwithabsoluteor relativedeficiency in insulinsecretionand/ortheiraction. It isestimatedthat366millionpeoplehadDMin2011andby2030thiswouldhaverisento552million(Olokobaetal,2012).Inthissense,manyplantsusedintraditionalmedicinesuchasCissussicyoides,Argemonemexicana,JuglansregiaL.areusingtotreatthissyndrome.Inthissense,Parmentieraaculeata(Kunth)isatreecommonlyknowninMexicoas“cuajilote”,itbelongstothefamilyofBignoniaceae.Fruit isusedfor thetreatmentofseveral illnesses in theMexicanfolklore likediabetes,headache,gallstones,deafnessanddiarrhea.Frombarkandfruithavebeenisolatedseveralsecondarymetabolitessuchasβ-sitosterolandstigmasterolregardedashypoglycemic.AlthoughinsomeplacesofMéxicoisusuallyusedforthetreatmentofdiabetes(Andrade-CettoandHeinrich,2005;Juárez-Vázquezetal.,2013),therearenotreportsaboutthebiologicalactivityofthebark.For these reasons, in the present work we decided to evaluate the ethanolic extract (EtOH-E) from bark and fruit ofParmentieraaculeatausinganoralglucosetolerance(OGTT)inordertocorroborateitsethnomedicaluse.BarkandfruitwerecollectedinMisantla,VeracruzState,Mexico.Plantmaterialwerecut,driedandextractedbyexhaustivesequentialmaceration(hexane,chloroformandethanol).Phytochemicalanalysisofextractswasundertakenusingstandardqualitativemethods.Glucosetolerancetestwasperformedin32maleWistarrats(weighing250-300g).Ineachgroupeightrandomlyselectedanimalswereused.Thedoseof200mg/kgbodyweightofEtOH-EbarkandEtOH-Efruitwasgivenp.o.involume 2 mL/Kg. Glibenclamide (Gli, 20 mg/Kg) was the pharmacological control and water with tween 80 (5 %) andpolyethylene glycol (5 %) as vehicle (Veh). All treatmentswere dissolved in vehicle solution. Animalswere 12 h overnightfasted and ad lib access to water only. At morning, a first blood sample by tail bleeding was taken at 0 min (t = 0).Immediatelythecorrespondingtreatmentswereadministered.After30minutesanoraldoseofglucosewasgiven(50%w/v,5mL/Kg).Blood sampleswere taken at 0.5, 1, 2, 3, 4 and 24h after glucose loading, andbloodglucose level (BGL)weremeasured.Phytochemical screening revealed the presence of phenolic compounds (flavonoids and coumarins) and sterols in bothextracts; terpenoidsonlyweredetected in theextractofbarkandquinonesonly in fruit.Noweredetectedpresencesofalkaloids in both extracts. Treatmentwith ethanolic extract of bark significantly decreased the BGL like Gli; however, nosignificantdifferenceswerefoundwhenethanolicextractoffruitwasusedwithrespecttothevehiclegroup(F=11.375,p≤0.0001).Statisticalanalysisrevealedsignificantdifferencesoninteractiontimepertreatment(F=2.227,p≤0.010).TheBGLofallgroupspriortotreatmentadministration(0hour)showednoapparentdifferencecomparedtoeachother.Allgroups,however, showed increase in BGL 30 min following glucose loading (one hour after oral treatment administration),confirmingtheinductionofhyperglycemia.EtOH-EbarkstatisticallydecreasedtheBGLonehouraftertheglucoseloading,and hypoglycemic effect was sustained up to 4 hours. However, the hyperglycemia with glucose challenge was notsignificantlybroughtdownwithEtOH-Efruit.Theseresultsareimportantconsideringthattraditionalmedicineisoftenusedmainlythefruitandleavesofthisplantasaninfusionforthetreatmentofdiabetes.1. Andrade-CettoA,HeinrichM.(2005).Mexicanplantswithhypoglycaemiceffectusedinthetreatmentofdiabetes.JEthnopharmacol,99(3):325-48.2. Juárez-VázquezMD,Carranza-ÁlvarezC,Alonso-CastroAJ,González-AlcarazVF,Bravo-AcevedoE,Chamarro-TinajeroFJ,SolanoE.(2013).Ethnobotany

ofmedicinalplantsusedinXalpatlahuac,Guerrero,México.JEthnopharmacol,pii:S0378-8741(13)327-9.3. OlokobaAB,ObateruOA,OlokobaLB.(2012).Type2diabetesmellitus:areviewofcurrenttrends.OmanMedJ,27(4):269-73.


208

DEVELOPMENTOFREPELLENTWITHESSENTIALOILSFROMMEDICINALPLANTS

Vázquez-HernándezM.1 ,Muñoz-MuñizOD.1,Alcántara-LópezMG.2,Pérez-GutiérrezRA.3

¹UnidadSARA-UniversidadVeracruzana,Xalapa(Veracruz)-México;2FacultaddeQuímicaFarmacéuticaBiológica-UniversidadVeracruzana,Xalapa(Veracruz)-México;3FacultaddeQuímicaFarmacéuticaBiológica-UniversidadVeracruzana,Xalapa

(Veracruz)-Méxicoemail:[email protected]

Essentialoilsextractedfromseveralnaturalresourcesareusuallyusedaseffectiverepelentagainstmosquitoesthatcouldbe a vector for tropical diseases such as: malaria, dengue, chikungunya and zika. Those natural products constitute anorganicalternativetosyntheticcompoundscomerciallyavailableforthispurpose,sincetheyaregenerallylessfriendlytotheenvironment and in recent years have generated controversy due to increased resistance developedbymosquitoes. Thistropicaldiseasessuchhaveahighmortalityrateinsomecountries,sotheuseofrepellentsisapointofvitalimportancefortheircontrol.Inthiswork,chemicalcompositionofsixteenessentialoilsfromplantscultivatedinMexicowereanalyzedinordertodevelopaneffectiverepellentagainstmosquitobasedonstatisticalanalysisbyPCA(PrincipalComponentAnalysis).Speciesstudiedwere:Teatree(Melaleucaalternifolia),orange(Citrusaurantium),cinnamon(Cinnamomumspp.),Citronella(Cymbopogon spp.), Cloves (Eugenia caryophyllata), eucalyptus (Eucalyptus spp.), “Hierba del burro” (Hyptis mutabilis),lavender(LavenderangustifoliaandLavenderspp.),lime(Citrusaurantifolia),lemon(Citrusmedica),mint(MentaarvensisandMentapiperita),rosemary(Rosmarinusofficinalis),thyme(Thymusvulgaris)andverbena(Verbenaofficinalis).AnalysisofessentialoilswerecarriedoutinagaschromatographAgilentTechnologies®model6890M,usingaDB-5columnphenylmethylpolysiloxane,5%,witha lengthof60m,0.25mm id,0.25μmfilmthickness, thisequipmentwascoupledtoamass spectrometer. Individual identification of componentswas performed by comparisonwith themass spectra of thedatabaseversion2.0NIST05MS.Sampleswereinjectedintriplicateandtheresultswereexpressedasmean±S.E.Moftheareaunder the curveof each component. Informationobtained from theGC-MSanalysiswasorderedand cataloged in amatrixthatallowedstatisticalanalysisbythePCAmethodwithStatistica2.1program(variationcoeficient0.95,0.7and0.4.)inordertoselecttheessentialoilswiththebestfeaturesofrepellency.Seventy five different compoundswere determined in analyzed essential oils and forty-two of them have reports aboutrepellentactivity.D-limonene,α-pinene,β-pinene,caryophyllene,eucaliptolandmyrcenewerethemostcommonterpenespresentinsamples.TheanalysisofvariablesinthisstudyusingPCAallowedtodefinethecomponentsthatexplainmostoftheobservedvariability.Thedistributionofdatafortheanalysiswascarriedoutin3stages,takingintoaccountthefollowingfactors:repellantproperties,presenceinthelargestnumberofsamplesandtheinteractionbetweenthesefactors,usingthecoefficientsofvariationobtainedbyPCAmodel.Thisschemeofanalysisresultedintwopossiblecombinationsofrepellentmixturesbasedonthesimilarityofitscomponents,eachofwhichbringstogether6essentialoils.ThecombinationAandBhave in common citronella oils, orange blossom, lime, eucalyptus and verbena but differ in one component,while A haslavenderessentialoilBcontainsPeppermint.Fromtheresultsobtainedweproceededtothepreparationoftworepellentssolutionswhich sensory acceptancewas assessed (odor) using ahedonic scaleof 5pointsbeing the solutionA themostacceptedwithout differences by age group or gender.We are currently working on the development of a standardizedformulationthatallowstheevaluationoftherepellentproperties,stabilityandviability.1. AburjaiT.,NatshehF.(2003).Plantsusedincosmetics.PhytotherRes17(9):987–1000.2. McAllisterJ.,AdamsM.,(2010)Modeofactionfornaturalproductsisoledfromessentialoilsoftwotreesisdifferentfromavailablemosquito

adulticides.JMedEntomol.47(6),1123-1126.3. OMS(2016).WorldMalariareport.Availableat:http://www.who.int/malaria/publications/world-malaria-report-2015/en/Accesson:June242016.


209

EFFECTOFCOMBINATIONOFCHIA(Salviahispanica)ANDSPIRULINA(Arthrospiramaxima)

ONDIABETES

Vargas-ChávezS.1,Martínez-GaleroE.1,Chamorro-CevallosGA.1,Quevedo-CoronaL.2,Ortega-NavaME.1,García-GutierrezGE.1,Garduño-SicilianoL.1

¹DepartamentodeFarmacia,,EscuelaNacionaldeCienciasBiológicasdelInstitutoPolitécnicoNacional,Zacatenco,MexicoCity-Mexico;2DepartamentodeFisiología,EscuelaNacionaldeCienciasBiológicasdelInstitutoPolitécnicoNacional,Zacatenco,

MexicoCity-Mexico.emai:[email protected]

Type2diabetes(T2D)isaprogressivemetabolicdisorderwithdiversepathologicalmanifestationsassociatedwithabnormallipidandcarbohydratemetabolism.T2Disapublichealthproblematworldwidelevelandasaresultofitshighprevalence,therearehighcostsandhighratesofmorbidityandmortality.Consideringthelimitationsofcurrenttreatments,itiscrucialto identifynewtherapies whith lowundesirableeffectsandhigherefficacythanconventionaltherapies.Naturalproductsareavalidalternative. Inthepresentstudy,twonaturalproducts,namelychia(Salviahispanica)andspirulina(Arthrospiramaxima),wereevaluatedfortheireffectonglucoseconcentrationandlipidprofileindiabeticmice.Diabeteswasinducedbyauniquealloxaninjection.Onthe3thday(72hlater),micewithbloodglucoseconcentrationabove200 mg/dl were used. Animals were separated into five groups: a) no diabetic, b) diabetic, c) diabetic-chia, d) diabetic-spirulinae)diabetic-combination.Glucoseconcentrationwasmeasuredweekly.After28daysoftreatment,bloodsamplesweretakenfromtheretro-orbitalsinusandcentrifugedat3,000gfor15mintoisolateserumfortotalcholesterol(TC),high-densitylipoproteincholesterol(HDL-C)andtriglyceride(TG)determination.Allmice had free access to food andwater andwere kept on a 12 h light, 12 h dark cycle. Animal care and experimentalprocedureswerecarriedoutaccording to theofficialmexicannormNOM-062-ZOO-1999entitled“Technical specificationsfortheproduction,careanduseoflaboratoryanimals”.After28days, thediabetic state (D)significantlymodifiedglucoseconcentrationandsomeserum lipidprofileparameterscompared to thenegativecontrol (ND)groupofmice. Inparticular, total cholesterol increased,whereas triglyceridesandHDL cholesterol decreased. Addition of chia to the drinking water (DCH) significantly decreased glucose concentration,increased triglycerides and HDL levels and decreased the amount of total cholesterol compared to untreated diabeticanimals(D).Similarly,administrationofthespirulin(DSP)significantlydecreasedglucoseconcentration,increasedHDLlevelsanddecreasedtotalcholesterolcomparedtodiabeticmice(D).Nosignificantchangeswereobervedontriglyceridesafterspirulin supplementation. Interestingly, combined administration of spirulina-chia to diabetic mice (DCHSP) produced nosignificantchangesonglucose,triglycerides,totalcholesterolorHDL.Results indicate that thecombinationof thesetwonaturalproductsdonotexert thesametherapeuticeffectofchiaandspirulinawhentheyareadministeredalone,thussuggestingthatthesecompounsmaycompetewitheachother.1. Andrade-CettoA.,HeinrichM.:Mexicanplantswithhypoglycaemiceffectusedinthetreatmentofdiabetes.JEthnopharmacol2005;99:325–3482. Lee,E.H.,Park, J.-E., Choi,Y.-J.,Huh,K.-B.,&Kim,W.-Y. (2008).A randomized study toestablish theeffectsof spirulina in type2diabetesmellitus

patients.NutritionResearchandPractice,2(4),295–300.3. Mazokopakis, E. E., Papadomanolaki,M. G., Fousteris, A. A., Kotsiris, D. A., Lampadakis, I.M., & Ganotakis, E. S. (2014). The hepatoprotective and

hypolipidemiceffectsofSpirulina (Arthrospiraplatensis) supplementation inaCretanpopulationwithnon-alcoholic fatty liverdisease:aprospectivepilotstudy.AnnalsofGastroenterology :QuarterlyPublicationoftheHellenicSocietyofGastroenterology,27(4),387–394.

4. VillaverdeG.C.,M.O.C.,BlancoG.M.D.,RamírezR.J.(2005).Metabolismo.InM.O.C.VillaverdeG.C.,BlancoG.M.D.,RamírezR.J.(Ed.),FundamentosdeBioquímicaMetabólica(Firteditioned.,pp.13-20):EditorialAlfaomega.

5. Vuksan,V.,Whitham,D.,Sievenpiper,J.L.,Jenkins,A.L.,Rogovik,A.L.,Bazinet,R.P.,yHanna,A.(2007).SupplementationofconventionaltherapywiththenovelgrainSalba(SalviahispanicaL.)improvesmajorandemergingcardiovascularriskfactorsintype2diabetes:resultsofarandomizedcontrolledtrial.DiabetesCare,30(11),2804-2810.

6. Vuksan, V., Jenkins, A. L., Dias, A. G., Lee, A. S., Jovanovski, E., Rogovik, A. L., yHanna, A. (2010). Reduction in postprandial glucose excursion andprolongationofsatiety:possibleexplanationofthelong-termeffectsofwholegrainSalba(SalviaHispanicaL.).EurJClinNutr,64(4),436-438.


210

ORALANTI-INFLAMMATORYACTIVITYOFDICHLOROMETHANEFRACTIONOFLacistemapubescens

MART.LEAVES

ConegundesLMC.,SilvaJM.,PintoNCC.,FernandesMF.,ScioE. LaboratoryofBioactiveNaturalProducts,DepartmentofBiochemistry,FederalUniversityofJuizdeFora,JuizdeFora-Brazil


LacistemapubescensMart.(Lacistemataceae)isaplantspeciesoriginallyfromBrazilandwidelydistributedinLatinAmerica.Theleavesaretraditionallyusedtotreatseveraldiseases,includingrheumatism,bodyachesandfever(Roumyetal.,2007).Preliminarystudiesperformedbyourgroupshowedamarkedanti-inflammatorypotentialofthedichloromethanefraction(DF)of the leavesobtained fromthemethanolcrudeextract,whichencouraged further invivoevaluationof itsoralanti-inflammatoryactivity.Thecrotonoil-inducedearedema testwasperformedasdescribedbySchiantarelli et al. (1982).Micewereorally treatedwithDF(100and300mg/kg),indomethacin(10mg/kg),usedaspositivecontrol,andvehicle(12%Tweeninsaline).Next,20µLofcrotonoil(2.5%inacetone)wereappliedontheinnersurfaceoftherightearofeachmouse.Theleftearwastreatedonlywithacetone.Theanimalswereeuthanized6hours later,andafragmentof6mmdiameterwasremovedfrombothearsusingametallicpunch.Theweightdifferencebetweenthefragmentsindicatedtheedema(inflammationreaction).Thedatawereexpressedasmean±SEM.ANOVAfollowedbyNewman-KeulstestwasusedforstatisticalanalysisusingthePrism5.0(GraphPadSoftwareInc.)statisticprogram.DF100,DF300andindomethacinwereabletosignificantlyreducetheedemaby54%,52%and44%,respectively.Crotonoilisan inflammatory agent that induces an intense cutaneous vasodilatation and erythema, followed by the increase of earthicknessasaresultofcellextravasation.ThisinflammatoryprocessinvolvestheactivationofproteinkinaseC,thevascularpermeability increasingandthesynthesisofarachidonicacid(AA)metabolites,COX-2,IL-1β,TNF-αandadhesionmolecules(Saraivaetal.,2010).Forthisreason,glucocorticoidsanddrugscapabletointerferewithAAmetabolism,asCOXinhibitorsand/orLOX,mayefficientlyrespondtothistest(Gábor,2000).Thus,thesefindingsstronglysuggestedthatDFisendowedwithnaturalcompoundswithanti-inflammatorypotential.Acknowledgments:ThisworkwassupportedbythegrantsfromFAPEMIG,CAPESandCNPq.1. GaborM.MouseEarInflammationModelsandtheirPharmacologicalApplications.Budapeste:AkadémiaiKiadó.2000,p.3362. Roumy V. et al. Amazonian plants from Peru used by Quechua and mestizo to treat malaria with evaluation of their activity. Journal of

Ethnopharmacology2007,112:482-489.3. SaraivaR.A.etal.Topicalanti-inflammatoryeffectofCaryocarcoriaceumWittm.(Caryocaraceae)fruitpulpfixedoilonmiceearedemainducedbydifferent

irritantagents.JournalofEthnopharmacology2010,136:504-510.4. SchiantarelliP.etal.Anti-inflammatoryactivityandbioavailabilityofpercutaneouspiroxican.Arzneimittel-Forschung/DrugResearch1982,32:230-235.


211

WOUNDHEALINGACTIVITYOFGELSCONTAININGANEXTRACTOFCecropiapachystachyaLEAVES

DuqueAPN.1,PintoNCC.1,MendesRF.1,AragãoDMO.1,CastañonMCMN.2,ScioE.1

¹LaboratoryofBioactiveNaturalProducts,DepartmentofBiochemistry,FederalUniversityofJuizdeFora,JuizdeFora-Brazil;²DepartmentofMorphology,FederalUniversityofJuizdeFora,JuizdeFora-Brazil


Wound is a rupture in the epithelial integrity of the skinwhich resulted fromviolenceor trauma andmaybe followedbydisruptionofthestructureandfunctionofunderlyingnormaltissue(Attamaetal.,2011).Effortsarebeingmadeallovertheworld to discover agents that can promote a good healing and thereby reduce the cost of hospitalization and save thepatientfromamputationorotherseverecomplications(Mahmudetal.,2010;Mallaetal.,2015).So,theaimsofthisstudywere toevaluate the in vivowoundhealingofgels containingextractofCecropiapachystachya (ECP)and toperform thechemicalfingerprintoftheextract.Thewoundhealingactivityof thegelswasevaluatedfor21days,usingtheexcisionmodel in rats.Afteranesthetizedandbeingplacedintheproneposition,theanimalswereshavedandacylindricalskinfragmentwasremovedfromthemediumlineofthedorsalregionwitha10mmdiameterbiopsypunch.Thedepthoftheskinwoundincludedtheepidermis,derme,hipodermandmuscularlayer,sothatthefasciasuperficialiswasexposed.Afterthesurgery,theskinwoundsweretopicallytreatedwith200μLofcarbopolgel1%(controlgroup),ECP2%orECP5%.Theanimalsweretreatedonceaday,duringpre-establishedperiodsof3,7,14and21daysforeachsubgroup.Attheendofeachtreatment,theanimalswereeuthanizedandthe elliptical excision encompassing the area of the healing process was removed from each animal. The material wassubjected to histological process and hematoxylin and eosin staining. The slideswere histometrically analyzed using thesoftwareImageProPlus6.0®.Toidentifythechemicalmarkers,aHPLCanalysiswasperformed.ThestudywasapprovedbytheBrazilianCollegeofAnimalExperimentation(COBEA)protocolnº050/2012.The group treatedwith ECP 5% presented edema and inflammatory infiltratewith less intensity than ECP 2% and controlgroups.Inaddition,neovascularizationwassignificantlylowerinECP5%group,indicatingthattheinflammatoryreactionwasnot prolonged and the process of tissue repair wasmore advanced in this group. Both ECP 2% and 5% gels showed lessneovascularization and cellularity, and better tissue repair when compared to the control, which showed a younger andhomogeneous tissue. The present study has demonstrated that the ECP gels promoted the acceleration of the healingprocess when compared to the control group. Wound contraction, angiogenesis, epithelialization and the collagendepositionsupportfurtherevaluationofC.pachystachyaleavesinthetopicaltreatmentandmanagementofskinwounds.Thecompoundsorientin, isoorientinandchlorogenicacid identified in theextracthadalreadybeenreportedwithhealingactivity(Aragãoetal.,2010).Acknowledgments:ThisworkwassupportedbythegrantsfromFAPEMIG,CAPESandCNPq.1. AragãoDMO,GuariziL,LaniniJ,daCostaJC,GarciaRM,ScioE.;2010.HypoglycemiceffectsofCecropiapachystachyainnormalalloxan-induceddiabetic

rats.JournalofEthnopharmacology,128:629-6332. AttamaAA,UzorPF,NnadiCO,OkaforCG.;2011.Evaluationof thewoundhealingactivityofgel formulationsof leafextractofAspilaafricanaFam,

Compositae.JournalofChemicalandPharmaceuticalResearch,3:718-724.3. MahmudS,ShareefH,AhmadM,GouharS,RizwaniGH.;2010.PharmacognosticstudiesonfreshmatureleavesofHolopteleaintegrifolia(roxb)planch.

PakistanJournalofBotany,42:3705-3708.4. MallaB,GauchanDP,ChhetriRB.;2015.AnethnobotanicalstudyofmedicinalplantesusedbyethnicpeopleinParbatdistrictofwesternNepal.Journal

ofEthnopharmacology,165:103-117.


212

ANTIOXIDANTACTIVITYOFCecropiapachystachyaTRÉCUL(URTICACEAE)

INNORMALANDDIABETICRATS

FernandesMF.,AragãoDM.deO.,AssisCM.,PiresPP.,ScioE. ¹LaboratoryofBioactiveNaturalProducts,DepartmentofBiochemistry,FederalUniversityofJuizdeFora,JuizdeFora,Brazil

email:elitascio887@gmail.comDiabetesmellitusiscausedbytheincreaseofglycemia.Inthelongterm,hyperglycemiaisassociatedwithincreasedoxidativestress, contributing to the development of diabetic complications (Tiwari and Rao, 2002).Many plant species have beenstudiedsincetheyhaveahighcontentofantioxidants(Chunetal.,2005).Thus,thisstudyaimedtoevaluatetheantioxidanteffectoftheethylacetateextractofCecropiapachystachyaTrécul(Urticaceae)(ECP)innormalanddiabeticrats.Wistarratsweredividedinto5groups(n=6):diabeticanimalsreceivingvehicle(negativecontrol),diabeticanimalsreceivingECP 80mg/kg, diabetic animals receiving glibenclamide solution 3mg/kg (positive control), normal animals receiving thevehicleandnormalanimalsreceivingECP80mg/kg.Diabeteswasinducedbystreptozotocin(40mg/kg)intravenously.Thetreatmentoccurreddailyat7hamforaperiodofsixmonths.Atthetimeofeuthanasia,liverwasremovedforanalysisoftheantioxidantactivityofECP.Theenzymescatalase,superoxidedismutase,andglutathionereductasewereevaluatedina96-wellplate.ANOVAfollowedbyTukeytestwasusedforstatisticalanalysis.Bothnormal anddiabetic animals treatedwith ECPpresented the levels of the antioxidant enzymes close tonormal. FordiabeticanimalstreatedwithECP,thelevelsofcatalase,superoxidedismutase,andglutathionereductasewere6.16±0.45;13.97±0.82and7.77±0.62U/mL,respectivelywhilenormalanimalsshowed5.95±0.89;11.76±0.40and7.64±0.90U/mL,respectively.FordiabeticanimalsnottreatedwithECP,thelevelofenzymesremainedmuchlower(0.28±0.06;0.57±0.20e0.68±0.21U/mL,respectively).Itisimportanttohighlightthatglibenclamide,usedaspositivecontrol,failedtoincreasethelevelofenzymes(0.73±0.23; 1.25±0.36;0.65±0.13U/mL, respectively).This fact reflectedthegreatstabilityand integrityofthe antioxidant system in animals treated with ECP. Those results can be partially attributed to the presence of theantioxidantcompoundschlorogenicacid,isorientinandorientin,previouslyidentifiedinECP(Aragãoetal.,2010).Acknowledgments:ThisworkwassupportedbythegrantsfromFAPEMIG,CAPESandCNPq.1. Chun,O.K.;Kim,D.;Smith,N.;Schroeder,D.;Han, J.T.;Lee,C.Y.2005.Dailyconsumptionofphenolicsandtotalantioxidantcapacity fromfruitsand

vegetablesintheAmericandiet.JournaloftheScienceofFoodandAgriculture,85:1715-1724.2. Tiwari,A.K.;Rao, J.M.2002.Diabetesmellitus andmultiple therapeuticapproachesofphytochemicals:Present statusand futureprospects.Current

Science,83(1):30-38.3. Aragão, D.M.O; Guarize, L.;Lanini, J.; Costa, J.C.; Garcia, R.M.G.; Scio, E. 2010. Hypoglycemic effects ofCecropia pachystachyain normal and alloxan-

induceddiabeticrats.JournalEthnopharmacology,128:629–633.


213

PREPARATIONOFPARVIFOLINEDERIVATIVESASTUBULININHIBITORS

Silva-GarcíaEM.1,Cerda-García-RojasCM.1 ,delRíoTorresRE.2,Joseph-NathanP.1

1DepartamentodeQuímicayProgramadePosgradoenFarmacología,CentrodeInvestigaciónydeEstudiosAvanzadosdelInstitutoPolitécnicoNacional,MexicoCity-Mexico;2InstitutodeInvestigacionesQuímicoBiológicas,Morelia(Michoacán)-Mexico.

email:[email protected],cancertreatmentshavemanypharmacologicaltargets.Oneofthemisthestructuralproteintubulinwhichformshighly organized polymeric structures namedmicrotubules. They are involved in cellular functions as signaling, vesiculartransport, migration, and proliferation.1 Microtubules maintain a dynamic equilibrium among shortening, growth, andstabilizationofthelength,dependingonspecificcellularconditions.Thisphenomenoncanbereplicatedandstudiedinvitro2to find new substanceswithmodulatory effect on this protein and its functions. Tubulin interacting agents have awidestructuraldiversity;althoughinmanyofthemthepresenceofaromaticringsandelectronegativeatomscanberecognized,asincolchicine,whichhasabenzenemoietyfusedtoaseven-memberedring,aswellassevenelectronegativeatoms.Inthiswork we prepared and studied several derivatives of parvifoline3 [(–)-(6Z,10R)-5,8,9,10-tetrahydro-3,6,10-trimethyl-2-benzocyclo-octenol]whichsharesomestructuralfeatureswithcolchicineandthereforecouldinteractwithtubulininhibitingitspolymerizationprocess.Parvifolinewas isolated from the rootsofAcourtiahumboltii. The reactions for thepreparationof itsderivatives includedepoxidationswithm-chloroperbenzoic acid, esterificationswith benzoyl chloride,methylationswith dimethyl sulfate, andtranspositionswithEt2O:BF3.Purificationswerecarriedoutusingcolumnchromatographyandthestructureelucidationsfornewcompoundsweredoneby1Dand2DNMRandmassspectrometry.InvitroanalyseswereperformedwithtubulinusingtheprotocoldescribedbyShelanskietal.,4butthewavelengthforreadingswaschangedfrom340to450nm.Colchicinewasemployedasthepositivecontrolforinhibitionofpolymerization.5EachmoleculewasmodeledinSpartan'04andoptimizedusingDensityFunctionalTheoryattheB3LYP/DGDZVPlevelinGaussian03.TheoptimizedstructureswereusedfordockingstudiesinAutoDock4.A series of 14 new parvifoline derivatives were prepared by reaction sequences that comprised combinations ofepoxidations, hydroxylations or incorporation of ethers or ketones into the eight-membered ring. In one group ofcompoundsthephenolichydroxylgroupofparvifolinewasleftunchanged,whileintwoothercollectionsthehydroxylgroupwas either esterified or methylated to afford three different sets of compounds. According to the docking analysis, theinhibitoryactivityofparvifolineandsomeofitsderivativesontubulinpolymerizationwasinagreementwiththehydrophobicinteractions found in the colchicine binding site located in α-tubulin, mainly with Phe 296, Tyr 312, Lys 341 and Ile 311.Parvifoline itself also displayed twohydrogenbonds, oneof thembetween thephenolic oxygen atomand thehydrogenatomofTyr312aminogroup,andtheotheronebetweenthephenolichydrogenandGln342amidicoxygenatom.ParvifolinebenzoateshowednonpolarinteractionswithLys338,Pro298,Pro307andpolarinteractionswithHis309andGln342,whileO-methylparvifolinesustainedhydrophobicinteractionswithPro298andGln342,aswellasapolarcontactwithGly310.Acknowledgement:ThisworkwassupportedbyConacyt-Mexico(grant241053).1. Jordan,M.A.;Wilson,L.Microtubulesasatargetforanticancerdrugs.NatureRev.Cancer2004,4,253-265.2. Nogales,E.;Wolf,S.;Downing,K.H.Structureoftheα-βtubulindimerbyelectroncrystallography.Nature1998,391,199-203.3. Joseph-Nathan,P.;Hernández,J.D.;Román,L.U.;GarcíaG.,E.;Mendoza,V.SesquiterpenesfromPereziacarpholepis.Phytochem.1982,21,669-672.4. Shelanski,M.L.;Gaskin,F.;Cantor,C.R.Microtubuleassemblyintheabsenceofaddednucleotides.Proc.Natl.Acad.Sci.1973,70,765-768.5. Andreu,J.M.;Timasheff,S.N.Tubulinboundtocolchicineformspolymersdifferentfrommicrotubules.Proc.Natl.Acad.Sci.1982,79,6753-6756.


214

GC/MSANALYSISANDBIOLOGICALACTIVITIESOFTHEESSENTIALOILFROMTHEDRIEDANDFLESH

LEAVESOFANNONACACANS(ANNONACEAE)

LourençoCC.1,MarinhoJVN.1 ,Aranha-NettoL.1,SiqueiraCAT.1,SerainAF.1,MesquitaJT.2,TemponeAG.2,SalvadorMJ.1

1DepartmentofPlantBiology,InstituteofBiology,UniversityofCampinas-UNICAMP,Campinas-Brazil;2AdolfoLutzInstitute,CenterforParasitologyandMycology,UniversityofSãoPaulo,SaoPaulo-Brazil.


SeveralAnnonaceaespeciesareusedinfolkmedicineandbiologicalpropertiesofessentialoilshavebeenreported,includingantioxidant, antimicrobial, anti-trypanosoma, larvicidal, anti-inflamatory and anti-tumoral effects. As parts of a researchproject focusing on phytochemical investigation of Brazilian medicinal plants searching for new bioactivity of naturalproducts, in this study,ouraimwas to report thechemical analysisof theessentialoil from thedriedand flesh leavesofAnnona cacans fromSãoPaulo,Brazil byGC/MSandevaluationof their antimicrobial activity againstbacteria, fungus andprotozoan.TheessentialoilwasobtainedbyhydrodistillationfromdriedandfreshleavesofAnnonacacansandanalyzedbygascapillary(GC/MS).AntimicrobialandantileishmanialactivitieswereevaluatedinvitroaccordingtoSIQUEIRAetal,(2015).Thirty-one compoundswere identified from fresh leaves and twenty-seven compoundswere identified fromdried leaves,representing60.78%and57.44%respectivelyofthetotaloftheoil.Themaincompoundsidentifiedintheessentialoilofthefleshleaveswerespathulenol(24.02%),β-(E)-ocimene(21.70%),germacreneB(13.26%)andlinalool(10.52%),andessentialoilfromdried leavesweregermacreneB (28.25%), spathulenol (20.83%)and linalool (10.29%).Antimicrobialactivityof theoilswere evaluated and good results were verified against Escherichia coli ATCC-10799, Enterobacter aerogenes andStaphylococcusaureusATCC-14458.Moreover,theoilsalsoshowedpromisingantileishmanialactivity,givingresultsagainstLeishmania(L.)chagasiwithIC50between25.0and40.0µg.mL-1fordriedandfreshleavesoils.ThisisthefirstreportontheanalysisofthevolatileconstituentsfromthedriedandfleshleavesofA.cacansandevaluationoftheirantimicrobialactivity.Bothessentialoils fromthedriedand flesh leavesofA. cacans exhibitedpromisingactivitiesagainstL. (L.)chagasi, werenegativeagainstfungistrainsevaluatedandshowntobesensibleagainstsomebacteria,eliminatingbetween80%and90%ofthetestedstrains.Thesignificantbiologicalactivitiespresentedbytheessentialoilssuggestthatthesespeciesarearichsourceofbiologicallyactivecompounds.Thisstudyalsoconfirmstheimportanceofchemicalandbiologicalinvestigationsofessential oils of Annonaceous species, in particular Annona spp. in the search for new and safer antibacterial andantileishmanialagents.Acknowledgments:CAPES,CNPq,FAPEAM,FAEPEX-UnicampandFAPESP.1. SiqueiraC.A.T.etal.BioactivityandchemicalcompositionoftheessentialoilfromtheleavesofGuatteriaaustralisA.St.-Hil.NaturalProductRes.2015,29:

1966-1969.


215

CHEMICALCOMPOSITIONANDANTIOXIDANTANDANTIMICROBIALACTIVITIESFROMPLATYMISCIUM

GRACILEBENTH.SAWDUST

CuéllarJE. ,MartínezJ.,GilJH.,GarcíaCM.,RojanoBA.,DurangoDL. UniversidadNacionaldeColombia,sedeMedellín,Medellín(Antioquia)-Colombia


Platymiscium gracile Benth. (Fabaceae), is a large tree from tropical rainforests. This ismainly distributed in the Amazonrainforest of Peru and Colombia (Herrera and Ferreira, 2006). In South Colombia it is widely used for the production ofhandicraftsand furnituredue to thehighqualityof itswoodand resistance to fungi and insects.During theprocessesofcutting,grinding,drilling,andsanding,alargeamountofsawdustisproducedasaby-productandusuallydiscarded.OtherPlatymiscium species have been reported to contain isoflavonoids,which have shown to have important antioxidant andantimicrobialproperties(Reyes-Chilpaetal.,1998).Aspartofoursearchprogramofbioactivenaturalproductsfromagro-industrialwaste,inthepresentstudythechemicalcompositionandantifungal,antibacterialandantioxidantactivitiesofP.gracileBenth.sawdust,wereanalyzed.Four extracts from sawdust ofP. gracilewere prepared by successive percolationwithn-Hexane, dichloromethane, ethylacetateandmethanol.Then,eightcompoundswereobtainedbychromatographictechniques,andtheirstructuresassignedbyspectroscopicmethods(NMR)correspondingtoscoparone,homopterocarpine,calycosin,medicarpin,coniferylalcohol,coniferyl aldehyde, isoliquiritigenin, and naringenin. The antifungal activitywas carried out by the poison food technique(Grover and Moore et al., 1962) using the fungiColletotrichum gloeosporioides(Penz.) Penz. & Sacc.andColletotrichumacutatumSimmonds.isolatedfrominfectedtamarillofruits(SolanumbetaceumCav.,Solanaceae).Theresultsareexpressedaspercentageofinhibitionofmycelialgrowthinrelationtoanegativecontrol.Allconcentrationsweretestedintriplicate.TheantibacterialactivitywasevaluatedagainstStaphylococcusaureusATCC25923,EscherichiacoliATCC25922,BacilluscereusATCC10876andEnterococcusfaecalisstrainsbythebrothdilutiontechnique.MICcalculatingwasmadebyastain with 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) (Balouiri et al., 2016). The antioxidant activity and free radicalinhibitory (FR)was performed by DPPH, FRAP,ORAC and ABTS assays by spectrophotometricmethods. The calculationswere made taking as reference Trolox (DPPH, ORAC, ABTS) and ascorbic acid (ORAC). Quantification of total phenoliccontent(TPC)ineachextractwasmadebytheFolin-Ciocalteumethodusinggallicacidasstandard(Preciadoetal.,2015).The n-hexane-soluble fraction exhibited the greatest inhibitory effect against both fungi. Homopterocarpin has a strongantifungalactivityagainstC.acutatumandC.gloeosporioideswithinhibitionpercentagesat24hrangingfrom40to70%and24to54%,respectively.TheTPCvaluesforfractionsrangedfrom3235±221(ethylacetate)and36350±3049mggallicacid/100gextract(n-hexane).Theevaluationoftheantioxidantactivity(ORAC)revealedthatthehighestvalueswere9601±613(isoliquiritigenin)and5928±430troloxµmol/100g(medicarpin).ThefractionsandisolatedcompoundsfromP.graciledidnotshowantibacterialactivityundertheconditionsused.TheresultsshowthatP.gracilesawdustisapromisingsourceofcompoundswithantifungalandantioxidantactivity.1. HerreraF,FerreyraRandVargasC,2006.EllibrorojodelasplantasendémicasdelPerú,RevistaPeruanadeBiología,Lima,Peru,pp334s2. ReyesR,GómezF,MorenoG,JiménezM,andQuirozV, 1998.FlavonoidsandIsoflavonoidswithAntifungalPropertiesfromPlatymisciumyucatanum

Heartwood.Holzforschung,MexicoCity,Mexico,pp459-461.3. GroverR,MooreJ,1962.Toximetricstudiesoffungicidesagainstbrownrotorganism.SclerotinafrutícolaandS.laxa,Phytopathology,pp876-880.4. BalouiriM,SadikiM,KoraichiS,2016.Methodsforinvitroevaluatingantimicrobialactivity:Areview.JournalofPharmaceuticalAnalysis,pp71-79.5. PreciadoM,EcheverriT,PereañezJ,ZapataKandRojanoB,2015.AntioxidantsfromthreeSwieteniaSpecies(Meliaceae),JournalofMedicinalPlants

Research,Medellin,Antioquia,Colombia,pp9-17.


216

EVALUATIONOFTOTALBETALAINSOFSTENOCEREUSSPPANDCHARACTERIZATIONOFPHENOLIC

COMPOUNDSANDANTIOXIDANTCAPACITY

GilEscamillaJO. ,Hernández-NavarroMD. ,HernándezLópezAV.,Figueroa-IbarraLM.,Gómez-OlivánLM.,Dublán-GarcíaO.,Islas-FloresH.,Hernández-OrtegaMM.

DepartmentofPharmacy,FacultyofChemistry,AutonomousUniversityofStateMexico,Toluca,StateofMexico,Mexicoemail:[email protected];[email protected]

The daily intake of fruits and vegetables is related to the decrease of radical species of oxygen and nitrogen that causeoxidative stress for its antioxidantproperties and its adjuvant action in variousdegenerativepathogeneses (Simirgiotis&Schemeda-Hirschman, 2011). In recent years, the content of bioactive compounds from natural products have beenincorporated in food formulations, dietary supplements, edible films and coatings considered as functional products.Betalains are red pigments present in some Mexican cactaceae (red tunas, xoconostle and pitaya) recognized for itsantioxidant,hypoglycemic,hypocholesterolemic,andantinociceptiveeffects(LiChenWu,et.al,2006);however,littleissaidaboutitstoxicity.Therefore,theaimofthisstudywastoevaluatetheacutetoxicityofanacetonicextractofbetalainsfromthe fruit of Pitaya in amousemodel and characterized the content of phenolics and total flavonoids in the exocarp andmesocarp,respectively.Exocarpandmesocarp(50geach)wereextractedseparatelywithacetone(1:1w/v)usingaBlender.TheslurrywasfilteredthroughaWhatmannº1filterpaperbyvacuumsuctionusingaBuchnerfunnel.Thesamplewasre-extractedwith70%(v/v)aqueous acetone twomore times. The filtrateswere combined and transferred to a separatory funnel,mixedwith 2 volchloroformandgentlymixed.Thesampleswerestoredinadarkroomuntilaclearseparationbetweenthetwophaseswasobtaneid. After partition the aqueous phase was transferred to a boiling flask and the residual acetone/chloroformwasremoved using a rotary evaporator at 40ºC under vacuum and proceeded to freeze drying. In order to obtain aqueousextracts the lyophilized was washing with acid citric solution 0.1% in relation 1:20 (w/v). The acute toxicity LD50 wasperformance according to Lorke method (1983). For characterization of the extracts of total phenols content it wasdeterminedindifferentpartsofthefruitbythemethoddescribedbyChenet.al.,2007.ThedeterminationoftotalflavonoidswasperformedaccordingtodescribedbyOsorioet.al.,2013.WhilethescavengingeffectoftheradicalofeachpieceoffruitwasevaluatedbytheDPPHmethodandcomparedwithcatechin.Micegiventheconcentratedextracttotalbetalainsshowednomacroscopicorhistopathologicaltoxicologicalsigns.TheLD50washigher than5000mg / kg, therefore theextract is consideredaccording to the classificationof toxicity aspracticallynontoxic.Thebetalainscontentwashighestintheaqueousextractlyophilizedpulp(386.26±42.75mgeq.betacyanin/100g),aswellasphenoliccompoundsandflavonoids(535.41±11.65mgeq.A.G/100gy139.50±7.29mgeq.cat./100g)comparedtocetonicpulpandpeelextracts(47.19±11.12mgeq.betacyanin/100g,0.203±0.01mgeq.betacyanin/100g)respectively.Despitethelow-betalainsinpericarpthispresentedahighantioxidantcapacity(88.28±0.46%inhibitionofDPPH)andacontentoftotalphenolsandflavonoids(38.78±0.44mg/100gand22.49±1.91mg/100g)comparedtofreshpulppresentapercentinhibition of 25.57 ± 10.27%, the content of flavonoids and phenolic compounds was (43.611±0.71 mg eq.A.G/100g y7.461±0.470mgeq. cat./100g). The results indicate that thepulp lyophilizedpitaya contains thehighest concentrationofcompoundsofbiological interestcomparedtosolventextraction,althoughnoneoftheextracts istoxic, lyophilization isasafemethodforobtainingthisfunctionalingredient.1. Chen,H.Y.,Lin,Y.C.,Hsieh,C.L., (2007).Evaluationofantioxidantactivityofaqueousextractof someselectednutraceuticalherbs.FoodChem. 104,

1418–1424.2. Li-chenWu,Hsiu-WenHsu,Yun-ChenChen,Chih-ChungChiu,Yu-InLin,Ja-anAnnieHo(2006),Antioxidantandantiproliferativeactivitiesofredpitaya,

FoodChem,95,319-327.3. Osorio,O.Moreno,A.ÁlvarezV.ÁlvarezL.GiustiM.,(2011).Phenolic,betacyaninsandantioxidantactivityinOpuntiajoconostlefruits,FoodResearch

International,Springer,MexicoCity,Mexico,pp2160-2168.4. Simirgiotis,M.J.,&Schmeda-Hirschman,G. (2010).Determinationofphenolic compositionandantioxidanteactivity in fruits, rhizomesand leavesof

white strawberry (Fragaria chiloensis spp.Chiloensis formchiloensis) usingHPLC-DAD-ESI-MSand free radical quencing techniques. Journalof FoodCompositionandAnalysis.23.545-55


217

ANTIOXIDANTEFFECTOFBETALAINSFROMSTENOCEREUSSPP.AGAINSTN-NITROSODIETHYLAMINE

INMICE

Hernández-LópezAV.1,Figueroa-IbarraLM.1,Hernández-NavarroMD.1 ,Juaregui-RodriguezB.1,Gil-EscamillaJO.1,Gallegos-OrtizMR.1,Osorio-EsquivelO.2,Gómez-OlivánLM.1,Dublán-GarcíaO.1,Islas-

FloresH.11DepartmentofPharmacy,FacultyofChemistry,AutonomousUniversityofStateMexico,Toluca,StateofMexico,Mexico;

2UniversityofChalcatongo.Oaxaca,México.email:[email protected]

Nitrosaminesarenitrogencompoundsfoundinnature,smokedfoods,meatproducts,alcoholicbeverages,somecosmeticsandcertaindrugs.Recentstudieshavepointedtoacorrelationbetweencancerandnitrosaminesexposure.Nitrosaminesrequire metabolic bioactivation by a β-hroxilation. This reaction is catalized by cyp450, producing alquilan agents thatdamageDNAduetothereactiveoxygenspeciesleadingtooxidativestress.Epidemiologicalstudieshaveshownthatintakeoffruitsandvegetablesprovidetothedietdiversephytochemicalsthatpossessantioxidantproperties.Recently,ithasbeendemonstrated that betalains from beetroot and sour prickly fruits amount other red- colored fruits are powerfulantioxidants. Thus, our goalwas to evaluate the antioxidant capacityof betalains extracted from redpitaya (Stenocereusspp.)ininvitroandinvivostudiesagainstn-nitrosodiethylamine.ThebetalainsextractionandidentificationwascarriedoutaccordingtoOsorioetal.,(2011).AntioxidantcapacityinvitrowasperformedbyABTS andDPPHmethods (Re, 1999;Osorio et al., 2011).Oxidative injurywas evaluated inmice.Micewereintragastric gavage 28 days with betalains extracts at 0, 20, 60 and 180 mg/kg/day. On the 27th day, the mice wereadministeredasingledoseofNDEA(150mg/kg).SeveralparameterssuchasLipidperoxidation,CarboniladesProteins,SuperOxideDdismutaseandCatalaseenzymeswereevalatedinbloodserum.Thebetalainscontent inmethanolicextractwasfoundtobe17.5±0.3mgbetaninequivalents/100gwetbasis.Antioxidantactivityinvitrowas10.95±0.53µmoltroloxequivalents/gand9.06±0.27mgGAEwithABTSandDPPHmethod,respectively.Weobserved antioxidant protective effect frombetalains in vivo. The betalains extract showed a significant decrease onoxidative effect produced by NDEA in groups treated with betalains extracts compare to control group. These resultssuggest that fruits from Stenocereus spp are a good source of phytocompounds which are capable to protect againstoxidativedamageproducedbyhighconsumptionoffoodrichinnitritesandnitrates.1 Krajka-Kuźniak,V.,H.Szaefer,E.Ignatowicz,T.Adamska&W.Baer-Dubowska(2012)BeetrootjuiceprotectsagainstN-nitrosodiethylamine-inducedliver

injuryinrats.FoodandChemicalToxicology.50(6):2027-332 Osorio-Esquivel,O.,M.AliciaOrtiz,V.B.Álvarez,L.Dorantes-Álvarez&M.M.Giusti (2011)Phenolics,betacyaninsandantioxidantactivity inOpuntia

joconostlefruits.FoodResearchInternational,44,2160-2168.3 Re, R., N. Pellegrini, A. Proteggente, A. Pannala, M. Yang & C. Rice-Evans (1999) Antioxidant activity applying an improved ABTS radical cation

decolorizationassay.FreeRadicalBiologyandMedicine,26,1231-1237.


218

CANNABISSATIVAL.:DEVELOPMENTANDVALIDATIONOFANEWHPLCMETHODWITHUV/DADAND

ESI-MSnDETECTIONFORTHEANALYSISOFNON-PSYCHOACTIVECANNABINOIDS

BrighentiV. ,MaranD.,PellatiF.,TardugnoR.,BenvenutiS.DepartmentofLifeSciences,UniversityofModenaandReggioEmilia,Modena-Italy

email:[email protected]. isgainingarenewed interest, thankstothebiomedical relevanceofthephyto-cannabinoids[1]. Ingeneral, themainphytocannabinoidspresentinfibertypeplantsaretheacids,mainlycannabidiolicacid(CBDA),followedbytheirneutralforms, mainly cannabidiol (CBD) [2]. Cannabinoids are usually analyzed by using gas-chromatographic techniques, whichinvolvehighoperating temperature, leading to thedecarboxylationof thenativeacidiccompounds to theirneutral formsandtoanalterationoftherealcannabinoidprofilingoftheplantmaterial[2,3].Theaimoftheworkwasthedevelopmentandvalidationofareliablemethodforthemetaboliteprofilingofthemainbio-activecompoundsinhempcultivarsofdifferentorigins,inordertoselectthosethatcouldbeappliedforthepreparationofextractswithhighpharmaceuticalvalue.Thestudywasalsoaddressedtothe isolationofphytocannabinoidsfromspecifichempvarieties,withparticularattentiontothebioactivecannabidiol(CBD).Themetabolite profiling of hemp non-psychoactive phyto-cannabinoidswas performed by developing a RP-HPLCmethodcoupledwithUV/DADandESI-MSndetectiontakingagainadvantageofthefused-corestationaryphase.The purification of non-psychoactive cannabinoids from hemp samples was carried out by means of normal phasepreparativeliquidchromatography(NP-LC)withsilicagelasthestationaryphaseandchloroformandmethanolasthemobilephase.ThestructureofthetwoisolatedcompoundswasconfirmedbytheuseofNMRspectroscopy.Theanalyticalmethodoptimized in this studywas completely validated for linearity, sensitivity, accuracyandprecision toshowcompliancewithinternationalrequirements(ICHguidelines)[4]andsuccessfullyappliedtoseveralhempvarietiesfortheselectionofthosewithahighercontentinthebioactivephytocannabinoids.TwooftheanalyzedsampleswerefoundtobehighlyrichinCBDandCBDAand,thus,theywereselectedfortheisolationoftheaforementionedcompounds.Twooftheanalyzed samples were found to be highly rich in CBD and CBDA and, thus, they were selected for the isolation of theaforementionedcompounds.Preparative LC allowed us to obtain the isolation of two fractionswith a high content in CBD (99.6%) and CBDA (92.5%),respectively,withameanyieldof1.2%w/wfromtherawmaterial.thetwopurifiedcompoundswereconfirmedtobeCBDandCBDAbymeansofNMRspectroscopy.1. DeBackerB.etal.,J.Chromatogr.B,2009,877,4115-4124.2. PeschelW.,M.Politi,Talanta,2015,140,150-1653. UNODC,Recommendedmethods for the identificationandtheanalysisofcannabisandcannabisproducts.Manual forusebynationaldruganalysis

laboratories.UnitedNations,2009,pp.25-474. InternationalConferenceonHarmonizationofTechnicalRequirementsfortheRegistrationofPharmaceuticalsforHumanUse(ICH),GuidelinesQ2(R1)-

ValidationofAnalyticalProcedures:TextandMethodology,ICHSecretariat,c/oIFPMA,Geneva,2005,pp.1


219

BRONCHODILATORANDANTITUSIVEEFFECTSOFBlepharocalixsalicifolius(“ANACAHUITA”)

Jimenez-HernandezJ.1,2,ConsoliniAE.1

1NationalUniversityofLaPlata(UNLP),SchoolofExactSciences,Pharmacology,LaPlata-Argentina;²UNLP,LaPlata-Argentinaemail:[email protected]

The tree of Blepharocalyx salicifolius Kunth O. Berg (Myrtaceae) grows near the costs of Rio de la Plata, it is known as“anacahuita”andtheleavesareusedtoalleviatecough,broncospasmanddiarrhea,inpartduetothepresenceof1,8-cineolintheessentialoil(EO-B.s)andtincture(T-B.s)(1,2).Nevertheless,therearenotpharmacologicalreportsabouttheseeffectsand the mechanism of action. In a previous communication we showed that both EO-B.s and T-B.s non-competitivelyinhibitedthecontractility inducedbycarbachol intheisolatedsmallbowel(3,4).Theaimofthisworkwastoevaluatethedirecteffectsoftheessentialoilandthoseof1,8-cineolontheisolatedrattrachea.Also,theantitussiveeffectwasassessedinthemodelofcoughinducedbyammoniachlorideinmice.LeavesofB.swerecollectedinthe2013-2014summeranddriedatair.Theessentialoilwasisolatedbyhydrodistillation.TheEOwasdilutedinDMSObeforedillutinginTyrodethedayoftheexperiment.Concentration-Relaxationcurves(CRC)oftheEO-B.sweredoneinratisolatedtrachea,overthecontractureobtainedbycarbacholandcomparedwithpapaverine(Pap).Contractilitywasmeasuredbyaforcetransducerandacquistedonacomputer.Forstudyingthemechanismonthesmoothmuscle,theEOwasalsoevaluatedonCRCofcalcium(Ca2+)inahigh[K+]mediainisolatedsmallbowel(4).Inmice,theEOwasinjectedintraperitoneally30minbeforeexposingthemousetonebulizationofammoniachlorideandsodiumhydoxidetoformvolatileammonia.Numberofcoughswerecountedduring5minutes.Apositivecontrolofcodeinephosphatewasused.Inratisolatedtrachea,theEOrelaxedthecontractureobtainedbycarbachol(IC50:1.5±0.2µg/mL,n=11),whichwaslower(thatis,8timesmorepotent)thantheCIofpapaverine(IC50:12±1.2µg/mL,n=5).AlsothemaximalbronchodilationofEO-B.s was about 140% of the maximal carbachol contraction while papaverine relaxed to about 70% of the carbacholcontracture.TheEO-B.salsoinhibitedinanon-competitivewaytheconcentration-responsecurves(CRC)ofcalcium(Ca2+)inahigh[K+]mediainisolatedsmallbowelfindingaIC50of1.77±0.34µg/mLfortheEOand23.7±5.5µg/mLfor1,8-cineol.Inmice,90mg/KgEO-B.sreducedthecoughfrequencytoabout25%(n=11),aswellascodeine30mg/kg(n=7).ResultssuggestthattheEOofBlepharocalyxsalicifoliusareeffectivefortreatingcoughandbronchospasms,becauseitisamore potent relaxant than papaverine, associated to inhibition of Ca influx to the smooth muscle. Also, the EO ofBlepharocalyxsalicifoliusisagoodcentralantitussive,aswellascodeine.Acknowledgements:grantUNLPX-642.1. Gameauetal.(2013).JournalofEssentialOilResearch,25(3),166-170.2. Lahitte,H.B.&J.A.Hurrell(1999)“Árbolesrioplatenses”.LOLAEditorial,BuenosAires.3. Jimenez-HernandezJ,ColaredaGA,ConsoliniAE.(2014)SILAEcongress,PhOL20144. Blancoetal.JournalofEthnopharmacology149:803-809,2013.


220

PHENOLICPROFILEANDCYTOTOXICPROPERTIESOFPOLAREXTRACTSFROMBASALLEAVESANDFLOWERSOFISATISTINCTORIAL.

TavianoMF.1 ,FilocamoA.2,1,RagusaS.3,DugoP.1,CacciolaF.4,GuzmanML.5,HsuHT.5,GallettiG.6,

MiceliN.11DepartmentofChemical,Biological,PharmaceuticalandEnvironmentalSciences,UniversityofMessina,Messina-Italy;

2Foundation“Prof.AntonioImbesi”,UniversityofMessina,Messina-Italy;3DepartmentofHealthSciences,University“MagnaGraecia”,Catanzaro-Italy;4DepartmentofBiomedicalandDentalSciencesandMorphofunctionalImaging,Messina-Italy;5DepartmentofPharmacologyinMedicine,WeillCornellMedicalCollegeNewYork-USA;6DepartmentofHematologyand

Oncology,WeillCornellMedicalCollegeNewYork-USAemail:[email protected]

IsatistinctoriaL.(Brassicaceae)isanancientEuropeandyeandmedicinalplant.Historically,I.tinctoriahasbeencultivatedfortheproductionofindigodye(bluecolour)inEurope,andtraditionallyusedforthetreatmentofwounds,ulcersandtumours,haemorrhoids,snakebitesandvarious inflammatoryailments(Hamburger,2002).Although I.tinctoria isnotconsideredasanediblevegetableworldwide,ruralpeoplelivingaroundVulcanEtna(Sicily,Italy)consumeboiledflowerbudsofthisplantas ingredients for salads and omelets (Galletti et al., 2008).Many authors have reported the anti-inflammatory and anti-cancer properties of lipophilic extracts and isolated compounds from I. tinctoria, whereas only a few studies have beencarried out to investigate the biological activities of polar constituents present in this species. In continuation of ourresearches,thisworkwasdesignedtocharacterizethephenolicprofileandtoevaluatethecytotoxicpropertiesofthepolarextractsfrombasalleaves(It-B)andflowers(It-F).ofI.tinctoriagrownwildaroundAcireale(Catania,Sicily,Italy).Plantmaterialwaslyophilizedandsequentiallyextractedwithdichloromethaneand70%methanolatatemperatureof50°C.Characterizationof thephenolicprofileof thehydroalcoholicextractswasattainedbyHPLC-PDA-ESI-MSanalysis.ArtemiasalinalethalitybioassaywascarriedouttoinvestigatethepotentialcytotoxicityofIt-BandIt-F(Micelietal.,2016).Totestinvitro theanti-proliferativeeffectsof theextracts, ahumanacutemonocytic leukemia (AML-M5a) cell line (MOLM-13)wasused.After24and48hoursof treatment,cellswerestainedwithAnnexinVand7-amino-actinomycinDand fluorescencewasevaluatedbyflowcytometry.ByHPLC-PDA-ESI-MSanalysis13and8compoundsweresuccessfullyseparatedandidentifiedforIt-BandIt-F,respectively.Itinctoriaextractshaveshownaprettysimilarphenolicfingerprint;thetotalamountoftheidentifiedphenolicswashigherinIt-F(19.95mg/gextract)thanIt-B(10.13mg/gextract).Flavonoidsrepresentedthemainclassofcompounds,withluteolin-glucuronide and stellarin-2 being present in the greatest amount in It-B and It-F, respectively. Cinnamic acidsweremoreabundantinIt-BthaninIt-F.In theA. salina lethality bioassay both extracts resulted non-toxic to brine shrimp larvae (LC50 > 1000 µg/ml). It-B extractdemonstratedgoodcytotoxiceffectagainstMOLM-13cellsatbothtimepoints;particularly,after48hexposure,areductionof viability close to 100%was observed at the highest tested concentration (1mg/ml). It-F extract showed lower activity,causingabout40%growthinhibition.Furtherinvestigationsareneededtobetterexplorethepromisingcytotoxicpropertiesofthepolarextractfrom I.tinctoriabasalleavesandtodetermineitsactivecomponents.Acknowledgments:Theauthorswishtothankthe“UniversityofMessina”withinthe“ResearchandMobility”Project.1. HamburgerM,2002.PhytochemistryReviews,1,333-344.2. GallettiS,RighettiL,BagattaM,2008.FirstEuropeanFoodCongress.Ljubljana(Slovenia).3-9November.3. MiceliN.,BuongiornoLP,CeliMG,etal.,2016.NaturalProductResearch,30(11),1229-1239.


221

ANTI-GLYCANTANDANTI-DIABETICACTIVITIESOFAROMATICGUANYLHYDRAZONEDERIVATIVES

SarmentoPA.1,Silva-JúniorEF.2,FrançaPHB.2,RibeiroEAN.1,BastosMLA.1,AragãoDMO.3,FontesES.3,Araújo-JúniorJ.X.1,2

¹FederalUniversityofAlagoas/NursingandPharmacySchool,Maceio(Alagoas)-Brazil;2FederalUniversityofAlagoas,ChemistryandBiotechnologyInstitute,Maceio(Alagoas)-Brazil;3FederalUniversityofJuizdeFora,DepartmentofBiochemistry,Juizde

Fora(MinasGerais)-Brazilemail:[email protected]

In 2015 were estimated 415 million people living with diabetes, remaining on the ranking of the most important globaldiseasestoday.Vascularcomplicationsthatleadtoheartattacks,blindnessandamputationsaregeneratedfrombiochemicaldisordersduetoAdvancedGlycationEndProducts(AGEs)accumulation.Somearomaticsystemswereintroducedataminoguanidinestructure,knownasananti-AGE,resultingin19derivatives.Toevaluate the anti-glycant activity, AGEs were produced, in vitro, from combination between glucose and bovine serumalbumin(BSA).Derivativeswithbestinvitroresponseweresubjectedtotestofacuteinvivotoxicityatconcentrationsof2mgkg-1,10mgkg-1and100mgkg-1,showingtheanti-glycantandanti-diabeticpotentialsindiabeticrats(Wistar),inducedbystreptozotocin.In in vitro anti-glycant assay, 13 of these derivatives showed more than 50% of inhibition in the AGE-formation atconcentrationof2mgml-1,whencomparedtoaminoguanidinemolecule. Inserialdosage,0.5to2.5mgml-1,checkingtheinhibitionpercentageeverysevendaysduring49days,theLQM13derivedreachedto94.6%inhibition,whileaminoguanidineinhibitedupto74%AGE-formation,atconcentrationof1.0mgml-1.TheinvivostudiesshowedthatLQM3,LQM13andLQM15werenot cytotoxic. In tests for anti-diabetic activity andanti-glycant, in vivo, these threederivativeswereable to reducebloodglucose levelsupto65%,whencomparedtoglimepiridedrug.Furthermore,the levelsoffructosamineandglycatedhemoglobinlevelsremainedlowerintreatedgroupswiththesecompounds.Starting from results,we conclude that the studiedderivatives demonstrate low cytotoxicity, showanti-glycant potential(AGEs-BSA) and are able to reduce blood glucose and fructosamine levels and glycated hemoglobin in diabetic rats.WeacknowledgetoFAPEAL,CNPq,CAPESandFINEPforfinancialsupport.1. Majd,A.A.etAl.AminoguanidinePartiallyPreventsTheReductionInLiverPyruvateKinaseActivityInDiabeticRats.AdvancedBiomedicalResearch,V.

3,N.260,2014.Doi:10.4103/2277-9175.1482332. Nowotny,K.etAl.AdvancedGlycationEndProductsAndOxidativeStressInType2DiabetesMellitus.Biomolecules,V.5,N.1,P.194-222,2015.Doi:

10.3390/Biom50101943. Sarmento,P.A.etAl.SynthesisAndPharmacologicalEvaluationOfGuanidineDerivativesWithPotentialHypoglycemicActivity.BmcProceedings,V.8,

P.1-2,2014.Doi:10.1186/1753-6561-8-S4-P10


222

ASTUDYOFGUANYLHYDRAZONEDERIVATIVESWITHVASORELAXANTACTIVITYUSINGDYNAMIC

SIMULATIONS,MOLECULARDOCKINGAND2D-QUANTITATIVESTRUCTURE-ACTIVITYRELATIONSHIPANALYSIS

Silva-JúniorEF.1,HerculanoEA.2,FrançaPHB.1,CostaCDE.2,RibeiroEAN.2,AquinoTM.1,Araújo-Júnior

JX.1 ¹FederalUniversityofAlagoas,ChemistryandBiotechnologyInstitute,Maceio(Alagoas)-Brazil;²FederalUniversityofAlagoas,

NursingandPharmacySchool,Maceio(Alagoas)-Brazil.email:[email protected]

Cardiovasculardiseasesrepresentamajorpublichealthproblem,whichstandouthypertension,responsiblebyone-thirdofall deaths worldwide.1 Actually, different chemical structures from guanidine-guanylhydrazones classes are used in theantihypertensive treatment, i.e. Clonidine, Brimonidine, Guanfacine, Guanabenz (GBZ), Guanethidine, Guanazodine andTizanidine.2DuethestructuralsimilarityofthesecompoundswiththeGBZdrug,nowwehaveinvestigatedthevasorelaxantpotential effects in spontaneously hypertensive rats (SHR), an experimentalmodel of genetic hypertension similar to thehumanformofincreasedbloodpressure.3Inaddition,wearereportingtheDynamicSimulationsandMolecularDockingand2D-QSARanalysis.All experimental procedures were carried out in accordance with the internationally accepted principles for laboratoryanimaluseandcareascontainedinEuropeanCommunityguidelines.4DynamicsimulationswereperformedonDESMOND®softwarebySchrödingerSuite.ThecoordinatesforthepossibletargetdeterminedbyX-raycrystallographywasappliedtoobtaintheinitialsystemfordynamicsimulationsstudies.5Thecrystalstructureofβ2-adrenergicGprotein-coupledreceptor(PDBentry:2HR1)waspreliminarilyanalyzedbyusingGOLD®software.Themodelinganalysis,calculations,andvisualizationsfor2D-QSARwereperformedbyusingtheVLifeMolecularDesignSuite4.4.Guanylhydrazone derivatives (LQM01-15)were investigated,where LQM07 (pD2= 5.5±0.09), LQM08 (pD2= 4.75±0.07) andLQM14 (pD2= 5.6±0.1) presented similar potency to GBZ (pD2= 6.49±0.06). The β2-receptor was considered as targetinvolved. Dynamic simulations were used as relaxation method, leading to RMSF satisfactory values. Molecular dockingdemonstrated these compounds are able to interactwith ASP113, VAL114, VAL117, PHE193, PHE289, PHE290, and ASN312.Finally, 2D-QSAR studies were performed to investigate the structural requirements of β2-receptor antagonists. Twostatistically significant models (r2= 0.99, q2= 0.99, pred_r2= 0.99) were selected for predictive analysis. The informationgenerated by 2D-QSARmodelsmay lead to a better understanding of structural requirements to aid the design of novelantihypertensiveagents.Compounds LQM07 and LQM14 showed thebest activity profile,with efficacies comparable toGBZ, althoughwith lowerpotencythanthelatter.Inaddition,moleculardynamicsanddockingstudiessuggestthatthischemotypemayinteractwithβ2-adrenergicreceptors,asprobablemechanismofaction,sinceinteractionswithkeyresiduesforcarazololbindingmaybeobserved for them. Lastly, the built 2D-QSAR models for that series of compounds showed good predictability forguanylhydrazonesusedanexternalvalidationset(testset1and2),withactivitynearlysimilartoexperimentaldata.Acknowledgements:toFAPEAL,CNPq,CAPESandFINEPforfinancialsupport.1. Manciaetal.,2013.ESH/ESCguidelinesforthemanagementofarterialhypertension:TheTaskForceforthemanagementofarterialhypertensionofthe

EuropeanSocietyofHypertension(ESH)andoftheEuropeanSocietyofCardiology(ESC).EurHeartJ.pp2159-2219.2. Mukaetal.,2016.Theroleofepigeneticmodificationsincardiovasculardisease:Asystematicreview.IntJCardiol.pp174-183.3. Yamorietal.,1973.Spontaneoushypertensioninratsversusessentialhypertensioninman.SingaporeMedJ.pp393–394.4. TheCounciloftheEuropeanCommunities;EU(Directive86/609/EEC)1986,NoL358,1.5. Ajloo et al., 2015. Synthesis, characterization, spectroscopy, cytotoxic activity and molecular dynamic study on the interaction of three palladium

complexesofphenanthrolineandglycinederivativeswithcalfthymusDNA.InorganicaChimActa.pp144-160.


223

Baccharistrimera(LESS.)DC(ASTERACEAE)EXTRACTSAREPROMISINGANTIBACTERIALAND

ANTIOXIDANTAGENTS

Feres-NettoC.1,PedrosaDM.1,FernandesLS.1,CostaYFG.1,PintoNCC.2,ScioE.2,Del-Vechio-VieiraG.3,SousaOV.4,AlvesMS.4

¹FacultyofPharmacy,UniversidadeFederaldeJuizdeFora,JuizdeFora-Brazil;2LaboratoryofBioactiveNaturalProducts,DepartmentofBiochemistry,InstituteofBiologicalSciences,UniversidadeFederaldeJuizdeFora,JuizdeFora-Brazil;

3LaboratoryofPharmacologyofNaturalProducts,DepartmentofPharmaceuticalSciences,UniversidadeFederaldeJuizdeFora,JuizdeFora-Brazil;4DepartmentofPharmaceuticalSciences,UniversidadeFederaldeJuizdeFora,JuizdeFora-Brazil

email:[email protected](Less.)DC(Asteraceae),popularlyknowninBrazilas"carqueja",isaSouthAmerica’snativemedicinalplant.Thisvegetalspecie istraditionallyusedashypoglycemic,hepatoprotective,digestive,anti-inflammatory,analgesicandalsoto treat skin wounds (Lorenzi; Matos, 2008). The current study aimed to characterize the chemical profile and theantioxidantandantibacterialactivitiesofhexane(HE),ethylacetate(EAE)andethanol(EE)extractsofB.trimeraaerialparts.Phytochemicalscreeningwascarriedoutbyidentifyingchemicalreactions(Matos,2007).TotalphenolicandflavonoidsweredeterminedbyFolin-Ciocalteuandaluminumchloridecolorimetricmethods,respectively(Singletonetal.,1999;Sobrinhoetal.,2008).ThechemicalprofilewasestablishedbyreversephaseHPLC-DAD.Theantioxidantactivitywasexploredby2,2-diphenyl-1-picrylhydrazyl (DPPH), Ferric Reducing Antioxidant Power (FRAP) and β-carotene Bleaching Test (BCBT) assays(Koleva et al., 2002;Mensor et al., 2001; Oyaizu, 1986). The antibacterial activity was assessed by theMinimal InhibitoryConcentration (MIC)usingmicrodilutionmethod followedby theMinimalBactericidal Concentration (MBC),which allowsclassificationof theantibacterialeffectasbacteriostaticorbactericidal (Andrews, 2001;CLSI, 2012).Staphylococcusaureussubsp. aureus (ATCC® 29213™), Escherichia coli (ATCC® 10536™), Salmonella enterica subsp. enterica serovar Typhimurium(ATCC®13311™)andPseudomonasaeruginosa(ATCC®9027™)weretested.Tannins,flavonoids,coumarins,terpenoidsandsteroidsweredetected.Totalphenolicandflavonoidcontentsrangedfrom0.22 (HE) to8.66 (EE)g/100gand0.00 (HE) to4.62 (EE)g/100g, respectively.UV spectraobtained fromchromatographicanalysis of HE, EAE and EE suggested the presence of chlorogenic acid and flavonoids, especially flavonols and flavones.DPPHandFRAPassaysrevealedEC50valuesof61.61(EE)to390.75(HE)μg/mLand218.95(EAE)to566.75(HE)μg/mL,inthisorder. According BCBT, I% varied from 28.64 (EE) to 41.27 (HE) %. Considering S. aureus (ATCC® 29213™), HE, EAE and EEexhibitedMICvaluesof5000,1250and5000µg/mL, inthisorder,withbacteriostaticeffect.EEalsorevealedMICvalueof5000µg/mLagainstE.coli(ATCC®10536™),withthesameeffect.TheseresultssuggestthatB.trimeraisasourceofbioactivesubstances,especiallyflavonoids,withantioxidantandantibacterialproperties.Acknowledgments:ThisinvestigationwassupportedbyUFJF,FAPEMIG(CDS-APQ-04680-10),CNPqandCAPES.1. AndrewsJ.M.Determinationofminimuminhibitoryconcentrations.JournalofAntimicrobialChemotheraphy,48:5-16,2001.2. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved

StandardNinthEdition(M07-A9).Wayne:CLSI,2012.3. Koleva,I.I.;VanBeek,T.A.;Linssen,J.P.H.;DeGroot,A.;Evstatieva,L.N.Screeningofplantextractsforantioxidantactivity:acomparativestudyon

threetestingmethods.PhytochemicalAnalysis,13:8-17,2002.4. Lorenzi,H.;Matos,F.J.A.PlantasmedicinaisnoBrasil,nativaseexóticas.InstitutoPlantarumdeestudosdaFloraLtda.,SãoPaulo,Brazil,2008.5. Matos,F.J.Introduçãoàfitoquímicaexperimental.EdiçõesUFC,Fortaleza,Brazil,2007.6. Mensor,L.L.;Menezes,F.S.;Leitão,G.G.;Reis,A.S.;DosSantos,T.C.;Coube,C.S.;Leitão,S.G.ScreeningofBrazilianplantextractsforantioxidant

activitybytheuseofDPPHfreeradicalmethod.PhytotherapyResearch,15:127-130,2001.7. Oyaizu,M.Studiesonproductofbrowningreaction -antioxidativeactivitiesofproductsofbrowningreactionpreparedfromglucosamine.Japanese

JournalofNutrition,44:307-315,1986.8. Singleton,V. L.;Orthofer,R.; Lamuela-Raventós,R.M.Analysisof total phenols andotheroxidation substrates andantioxidantsbymeansof Folin-

CiocalteuReagent.MethodsinEnzymology,299:152-178,1999.9. Sobrinho, T. J. S. P.; Silva, C. H. T. P.; Nascimento, J. E.; Monteiro, J. M.; Albuquerque, U. P.; Amorim, E. L. C. Validação de metodologia

espectrofotométricaparaquantificaçãodosflavonoidesdeBauhiniacheilantha(Bongard)Steudel.RevistaBrasileiradeCiênciasFarmacêuticas,44:683-689,2008.


224

MOLECULARDESCRIPTORSASPREDICTINGCRITERIAINTHESEARCHOFACETYLCHOLINESTERASE(AChE)ISOFLAVONE-LIKEINHIBITORS

Orduz-DiazLL. ,Coy-BarreraED.

UniversidadMilitarNuevaGranada,Cajicá-Colombiaemail:[email protected],[email protected]

Acetylcholinesterase(AChE) isanenzymethatcatalyzesthehydrolysisofacetylcholine,aneurotransmitter insynaptichalfprocesses. Acetylcholine deficiency impedes neuronal communication and the nerve impulses transmission which leadsneurodegenerativediseases,e.g.,Alzheimer(1).Thesediseasesarecharacterizedbecausetheyarenotcured,buttreatedtoimprovethequalityoflifeofpeopleaffectedby(2);withinthealternativetreatmentsistheuseofnaturalproductssuchasisoflavones,whichreportedasAChEinhibitors(3).Additionally,thesecompoundsaregoodantioxidantsandcouldpreventneuronalapoptosisandoxidativestress-inducedbraindamage(4).Agroupof isoflavones (reported in the Fabaceae family)were assessedat in silico-level throughmoleculardockingusingAutodock/Vina.ResultswereexpressedasVinaScores(affinityinkcal/mol).TheligandspossessingthebestdockingresultswerestudiedbymoleculardynamicssimulationswithintheactivesiteofAChEduring5000psusingGromacs.Thus,forthemost-stable isoflavone-enzyme complexes, a detailed structural interactions analysis and their relevance were alsodocumented. The affinity values were analyzed with molecular descriptors (CDK generated) for each ligand by principalcomponentanalysis(PCA)andpartiallesssquareregression(PLS)analysis.Dockingresults let tothe identificationof twohits forAChE inhibition.Particular interactions intodeactivesitewerealsoobservedtoexplainthecomplexstability. Inaddition,crucialresiduesfortheenzyme:ligandinteractionwere identifiedbymoleculardynamicsanditsstabilitywasfoundtobeconstantaccrosstime.PCAallowedfindingdifferencesandcorrelationsfrommoleculardescriptorsforthosestudiedisoflavones.Relationships(PLS)betweenaffinitiesforeachenzyme-isoflavonecomplex respect tosomedescriptorswere likewiseobserved.This factcouldbeusedaspredictors in thesearchofAChEinhibitorsasalternativesinthetreatmentofneurodegenerativedisorderslikeAlzheimer.Acknowledgments:ThepresentworkisaproductderivedbytheProjectINV-CIAS-2050financedbyVicerrectoríadeInvestigacionesatUMNG-Validity2016.1. Reitz,C.,&Mayeux,R.(2014).Alzheimerdisease:epidemiology,diagnosticcriteria,riskfactorsandbiomarkers.Biochemicalpharmacology,88(4),640-

651.2. Prince,M.,Albanes,E.,Guerchet,M.,PrinaM.(2014).WorldAlzheimerReport2014.DementiaandRiskReduction.Ananalysisofprotectiveand

modifiablefactors(ADI),London.3. Murray,A.P.,Faraoni,M.B.,Castro,M.J.,Alza,N.P.,&Cavallaro,V.(2013).NaturalAChEinhibitorsfromplantsandtheircontributiontoAlzheimer’s

diseasetherapy.Currentneuropharmacology,11(4),388-413.4. Zhao,B.(2009).NaturalantioxidantsprotectneuronsinAlzheimer’sdiseaseandParkinson’sdisease.Neurochemicalresearch,34(4),630-638.


225

IN-SILICOSTUDIESONPTEROCAPARNSASANALTERNATIVEINTREATMENTOFALZHEIMER’SDISEASE

Orduz-DiazLL. ,Coy-BarreraED.

UniversidadMilitarNuevaGranada,Cajicá-Colombiaemail:[email protected],[email protected]

Alzheimer's disease (AD) is a neurodegenerative disorder affecting 33.6 million people worldwide (1). There are severalhypothesesabouttheetiologyofADsuchasthecholinergichypothesis,accumulationofproteinβ-amyloidandtau,geneticfactors, lifestyleandenvironmentalfactors.OneofthemostusedADtreatments istheuseofacetylcholinesterase(AChE)inhibitors-based therapy; this enzyme catalyzes the acetylcholine hydrolysis, a neurotransmitter that mediates synapticnervous system activity (2). Among the compounds reported as AChE inhibitors are flavonoids-like compounds such aspterocarpans,whichareproducedasphytoalexinsinplantsoftheFabaceaefamily(3).Agroupofpterocarpans (reported in theFabaceae family)wasassessedat in silico-level throughmoleculardocking.Testcompounds were structurally optimized at DFT level in SPARTAN. Docking calculations in Autodock/Vina per eachAChE:ligandcomplexwereperformedtentimesandtheresultingdata(affinityinkcal/molandRMSDinÅ)wereanalyzedbymultivariateanalysis.Inaddition,betterinteractionswereanalyzedatmolecularlevelasselectingcriteria.Theaffinityvalueswerecorrelatedwithmoleculardescriptors(CDKgenerated)foreachligandbypartiallesssquareregression(PLS)analysis.Eachpterocarpan-typecompoundsexhibitedparticularinteractions(mainlypolarcontacts)withtheresiduesintotheAChEactive site.However, someof themwere found tohavehigheraffinityandgoodconvergence (RMSD<2Å).AffinityandRMSDvalueswerealsocorrelatedwithsomeofthemoleculardescriptorsthroughmultivariatestatisticalanalysis,indicatingthatthosedescriptoscouldbeusedaspredictorsforfindingalternativesbasedonAChEinhibitors inthetreatmentofAD.Acknowledgments:ThepresentworkwasperformedwithintheProjectINV-CIAS-2050grantedbyVicerrectoríadeInvestigacionesatUMNG-Validity2016.1. Prince,M.,Albanes,E.,Guerchet,M.,PrinaM.WorldAlzheimerReport2014.DementiaandRiskReduction.Ananalysisofprotectiveandmodifiablefactors

(ADI).London,20142. ReitzC.,andMayeuxR.Alzheimerdisease:epidemiology,diagnosticcriteria,riskfactorsandbiomarkers.Biochemicalpharmacology,2014,88:640-651.3. MurrayA.P.etal.NaturalAChEinhibitorsfromplantsandtheircontributiontoAlzheimer’sdiseasetherapy.Currentneuropharmacology,2013,11:388-413.


226

NEWMULTITARGETDRUGSDERIVEDFROMNATURALPOLYPHENOLS

BeginesP.1,RoldánJ.2,OlieteA.1,PlataGB.2,JoséM.PadrónJM.2,LópezÓ.1 ,MayaI.1,Fernández-

BolañosJG.11Dpto.deQuímicaOrgánica,FacultaddeQuímica,UniversidaddeSevilla,Sevilla-España;2BioLab,InstitutoUniversitariodeBio-

Orgánica“AntonioGonzález”(IUBO-AG),CentrodeInvestigacionesBiomédicasdeCanarias(CIBICAN),UniversidaddeLaLaguna,LaLaguna-España

email:[email protected];Polyphenolsaresecondarymetabolitesparticularlyabundantinthevegetalkingdom,andfeaturedwithrelevantbiologicalproperties; in this context, they have been reported to be strong antioxidants, anti-inflammatory, antimicrobial,neuroprotective, or antiproliferative agents [1]. Therefore, polyphenols constitute an attractive family of potential drugsagainstaseriesofseverepathologieslikechronicinflammatoryprocesses,neurodegenerativedisorders,orcancer.Moreover,thecomplexandmultifactorialetiologyofsuchdiseasesmadeitnecessarytochangetheclassicalpointofviewinMedicinal Chemistry; in this context, multi-target drugs appeared as a promising approach for targeting simultaneouslyseveralbiologicalreceptors[2].Concernedby the increasing ratesofAlzheimer’sdiseaseandcancer,wedesigned twonewfamiliesofmulti-targetdrugsbased on polyphenols. In both cases, dual molecules were prepared as two pharmacophores were incorporated: apolyphenolicmotif, for tacklingoxidative stress,onecommon featureof suchdiseases [3], andan specificmotif foreachpathology (a selenoureido group for cancer, and a tacrine residue (1,2,3,4-tetrahydroacridin-9-amine) for Alzheimer’sdisease).Allthederivativeswerefoundtobestrongantioxidantagents,capableofefficientlyscavengingReactiveOxygenSpecies,likefree radicals, or peroxides, responsible for deleterious oxidative stress. Moreover, the combination of polyphenols withselenoureidomotifsaffordedstrongantiproliferativeagentsinthelowmicromolarrange.On the other hand, in Alzheimer’s patients, a common feature is the particularly low levels of the neurotransmitteracetylcholine,leadingtoaprogressiveandseverdeclineincognitivefunctions;therefore,acetylcholinesteraseinhibitors,liketacrine, the first marketed drug in this context, are claimed to be potentially useful drugs against memory loss in thisneurodegenerativedisease[4].We have accomplished the synthesis ofN-alkyl tacrine derivatives decoratedwith a polyphenolicmotif; such compoundsturnedout tobesubmicromolar inhibitorsofacetylcholinesterase,withapotencyof thesameordercompared toparenttacrine,andalsocapableofreducingtheoxidativestressprovokedbytacrine,whichleadstohepatotoxicity[5].Acknowledgments:WethanktheDirecciónGeneraldeInvestigación(CTQ2011-28417-C02-01,CEI10/00018),JuntadeAndalucía(FQM134,P11-CVI-7427),InstitutodeSaludCarlosIII(PI11/00840),FEDERfunds,7thIEUFrameworkProgramme(FP7-REGPOT-2012-CT2012-31637-IMBRAIN)forthefinancialsupport.1. ChoiDY.,Y.-J.Lee,J.T.Hong,H.-J.Lee,BrainRes.Bull.2012,87,144–153.2. BansalY.,O.Silakari,Eur.J.Med.Chem.2014,76,31–42.3. MencalhaA.,V.J.Victorino,R.Cecchini,C.Panis,AnticancerRes.2014,34,1127–1140.4. ShaikhS.,A.Verma,S.Siddiqui,S.S.Ahmad,S,M.Rizvi,S,Shakil,D.Biswas,D,SinghD,M.H.Siddiqui,S.Shakil,S.Tabrez,M.A.Kamal,CNSNeurol.Disord.

DrugTargets,2014,13,391–401.5. ParkSM.etal.Biol.Pharm.Bull.2015,38,184-192.


227

EVALUATIONOFGELPRODUCTIONANDANTIRADICALARACTIVITYINSEVERALALOESPECIES

GrčićN.1,CapelaP.2,DiasACP.1

¹CITAB-CentrefortheResearchandTechnologyofAgro-EnvironmentalandBiologicalSciences,DepartmentofBiology,UniversityofMinho,Braga-Portugal;²Ing.Agr,ViveirosSubtropicais,SãoMamededeInfesta-Portugal

email:[email protected](FamilyLiliaceae)isworldwideknowngenuswith600speciesnaturallyfromAfrica.Aloeshavebeenlongusedintraditionalmedicine, specially thegelproduced,andnowadaysareused for severalpurposes– likecosmeticsanddietarysupplements.Nowadays somespecies,namelyAloebarbadensis, are cultivatedas raw-material foragro-food, cosmeticoreven pharmaceutical industries. Nevertheless, as indicated, many Aloe species exists that might be also used for thesepurposes.In this work our goals were i- evaluate several aloe species concerning gel production and antioxidant activity, and ii-compareeventualdifferencesbetweenspeciesgrowningreenhouseoropenfieldconditions.Atotalof19differentAloespecieswereevaluatedforgelproductioncapacity,inpercentageoffreshepidermalpartofleaf(skin,peel)andfreshparenchymatransparentmucilaginousgel(gel),andfortheirantioxidantcapacity(byABTSmethod).Thespecieswerecultivatedinequivalentconditionsinthesamefield,inSantarem(Portugal).Additionally,somespecies(10)were grown both outside (ext) and in a greenhouse (int) conditions. Samples (leaves) from different plants (3) of eachspeciesandcondition(intorext)werecollected inFebruaryandJuneof2015, inaperiodthatdoesnotrainforat leastaweek.Fresh leafswereweighed,and thegelandpeelcollectedseparatelyandweight.Gelandpeelwere lyophilizedandstoredat-20°C,untiltheprocessing.Inordertoevaluateantiradicalarcapacity,aliquotsoflyophilizedgelsweresolubilizedinDMSO(1mg/mL).ABTSassaywasusedforantiradicalaractivityevaluationandcomparisonamongspeciesandgrowthconditions.The lowest percentage of fresh gel (8.2% ± 0.6)wasmeasured inAloe zebrina (ext). The highest percentage of fresh gel(69.2%± 1.4)wasobtained inhybridofAloearborescensxAloebarbadensis (int). Theproductionsofgelwereaffectedbygrowingplantsoutsideandingreenhouse,butnocleartendencywasobserved.Aloe chabaudii, andAloe inyangensis, both at greenhouse conditions (int), showed thehighest antiradicalar activitieswith57.1%±0.4and39.9%±0.6,respectively.The lowestscavengingactivitywasassessed inAloezebrina (int):2.3%±1.7.Aloebarbadensis(ext)and(int)showedanactivityof16.5%±1.3and5.9%±0.1,respectively.AloearborescensxAloebarbadensishybrid (int) and (ext)was33.2%± 1.3and3.9%± 1.2. So thevariabilityamongAloe species, concerninggelproductionandantiradicalaractivity,ishighandAloebarbadensismightnotbethebestspeciesintermsofthesepropertiesforagriculturepractices.


228

ANTILEISHMANIALACTIVITYOFGuareaguidonia(MELIACEAE)

TorresSuarezE.,Coy-BarreraCA.,Delgado-MurciaLG.,Coy-BarreraED.

UniversidadMilitarNuevaGranada,Cajicá-Colombiaemail:[email protected]

Meliaceaefamilyischemicallyhighlightedduetoabundanceanddiversityofthoselimonoidstheirplantscanproduced[1].Theuseversatilityofthese limonoids(commonlycalledasmeliacins)hasmotivated itscontinuoussearchanddiscovering,since some plants of the Meliaceae family (which are well-recognized as a source of tetranortriterpenoids) have beentraditionally used for pest control and for medicinal purposes. For example, some plants of the Guarea genus haveethnomedicinalrecordsforthecontrolofLeishmaniasis[2].However,thechemistryandbiologicalactivityofplant-derivedmaterialsfromthisgenuscouldbeconsideredunexplored.Thus,aspartofourresearchonMeliaceaeplants,aLC-MS-basedchemicalstudyandanantileishmanialactivityevaluationwereperformedwithG.guidonia,aneotropicaltreepossessingfewchemicalstudies.Leaves, seeds, wood and bark of test plant were collected in Vaupés, Colombia. These plantmaterials were then dried,pulverized and subjected tomaceration processwith ethanol. Chemical profiles were recorded by LC-MS analysis of theresulting crude extracts. In order to explore the effectiveness and security of the extracts as parasiticide agents, crudeextracts were then tested against extracellular (promastigotes) and internalized (amastigotes) Leishmania panamensisparasites and two macrophages cell lines (murine J774 and human macrophages). Cell viability of promastigotes andmacrophages,undertreatmentswithseveraldosesofextractsincomparisontoacontrol,wasdeterminedbyAlamarBlueassay.TheactivityofextractsagainstinternalizedamastigoteswasevaluatedbyfluorescencemicroscopypreviousmarkingwithSYBRgreenunderexpositiontovariousdosesofextracts.ActivityresultswereexpressedasEC50(parasites)andLC50(macrophages)inμg/mL(threereplicates).Ethanol-soluble extractswere found to be very rich in limonoid-like compounds and other kind ofmetabolites by LC-MSanalysis.Thisfact indicatedthatthiskindofphytoconstituentscouldbepossiblyresponsiblefortheactivityofG.guidonia-derived extracts (promastigotes EC50 46-200 µg/mL range; amastigotes EC50 54-200 µg/mL range). Additionally, somematerials exhibited low cytotoxic effect against both macrophages cell lines (LC50 > 200 µg/mL) and showed selectivityindexes>2aswell.Acknowledgments: This work was supported by grants from the Fondo de Investigación en Salud, Francisco José de Caldas (Contract No. RC463-2012)administeredbyColciencias.1. DaSilvaM,GottliebO,DreyerD.1984.BiochemicalSystematicsandEcology,12(3),299–310.2. MesquitaML,DesrivotJulie,BoriesC,FournetA,PaulaJE,GrellierP,EspindolaLS.2005.MemóriasdoInstitutoOswaldoCruz,100(7),783-787.


229

UFLC-ESI-MS-BASEDPROFILING,CYTOTOXICITYANDANTILEISHMANIALACTIVITYOFEXTRACTSOF

AzadiracthaindicaSEEDS

Monroy-VelandiaD.1,TorresSuarezE.2,Coy-BarreraCA.1,Delgado-MurciaLG.2,Coy-BarreraED.1 ¹UniversidadMilitarNuevaGranada,Cajicá-Colombia;2UniversidadNacionaldeColombia,Bogotá-Colombia

email:[email protected] indica (also known as neem) is a tree typically grown in tropical and semi-tropical regions. This plant wasintroducedinColombiaduetotheexcellentpesticidepropertiesamongothers(1).TherearesomestudiesdirectedontheevaluationoftheparasiticideactivityofA.indicaseeds(e.g.,againstLeishmaniaamazonensisparasites)(2).However,thereisa lackof informationabouttheeffectofA. indicaonL.panamensis.Thus,aspartofourresearchonMeliaceaeplants,thepresentstudywasdirectedtothephytochemicalexplorationandtheevaluationofcytotoxicityandantileishmanialactivity(againstL.panamensisparasites)ofA.indicaseeds-derivedextracts.SeedsofA.indicawerecollectedinHuiladepartment,Colombia.Thisplantmaterialwasthendried,pulverizedandsubjectedto several extraction processes including S-L extraction (using 96% ethanol), Soxhlet extraction and extrusion. ResultingcrudeextractswerechemicallycharacterizedbyLC-ESI-MS-basedanalysesemployingavalidatedmethodforprofilingherbalextracts. Extracts were also tested against extracellular (promastigotes) and internalized (amastigotes) Leishmaniapanamensis parasites and two macrophages cell lines (murine J774 and human macrophages) in order to examine theeffectiveness and security of the extracts as parasiticide agents Cell viability of promastigotes and macrophages, undertreatmentswithseveraldosesofextractsincomparisontoacontrol,wasdeterminedbyAlamarBlueassay.Theactivityofextracts against internalized amastigotes was evaluated by fluorescence microscopy previous marking with SYBR greenunderexpositiontovariousdosesofextracts.ActivityresultswereexpressedasEC50(parasites)andLC50(macrophages)inμg/mL(threereplicates).Extracts were found to have limonoid-related metabolites. However, particular differences were observed in the LC-MSprofiles regarding the presence/absence of some metabolites. Extracts exhibited EC50 values for promastigotes andamastigotes in the77-200and 143-200µg/mL ranges, respectively. Ethanol-soluble crudeextract fromA. indica seedswasthen the most potent antileishmanial material. In addition, low cytotoxiciy against J774 and human macrophages wasexhibitedforallextracts(LC50>150µg/mL)revealingselectivityindexes>2.Thus,abioguidedfractionationleadingtoobtainthebioactive compounds is currently in course. Thepresent study is the first reporton the activityofA. indica againstL.panamensis.Acknowledgments: This work was supported by grants from the Fondo de Investigación en Salud, Francisco José de Caldas (Contract No. RC463-2012)administeredbyColciencias.1. BoekeSJ,BoersmaMG,AlinkGM,vanLoonJJA,vanHuisA,DickeM,RietjensIMCM.2004.JournalofEthnopharmacology,94(1),25-41.2. CarneiroSM,CarvalhoFA,SantanaLC,SousaAP,NetoJM,ChavesMH.2012.BiologicalResearch.45(2):111-6.


230

ANTI-INFLAMMATORYACTIVITYOFEXTRACSFROMANINVITROCULTUREOFSambucusnigraL.

Cruz-CruzDG.1,Méndez-SánchezLI.1,Herrera-RuizML.2,CapatazTafurJ.3 ,Sánchez-OcampoPM.4

¹UniversidaddelPapaloapan.,Tuxtepec(Oaxaca)-México;2PharmacyLaboratory,CentrodeInvestigaciónBiomedicadelSur,Xochitepec(Morelo)-México;3PlantCellCultureLaboratory,BiotechnologyInstitute,UniversidaddelPapaloapan,Tuxtepec

(Oaxaca)-México;4CONACYT-UniversidaddelPapaloapan,Tuxtepec(Oaxaca)-Méxicoemail:[email protected],[email protected]

Sambucus nigra L. (Acanthaceae) is a plant used in traditional Mexican medicine to treat a number of health disordersincluding cough, fever, flu, diarrhea, dysentery, stomach pain, swelling, and found accumulate active compounds asanthocyanins, flavonols,phenolicacidswithantioxidant,antiviralandantibacterialactivities.However, thisplantdoesnothavestudiesthatsupportthatinvitrocultureofS.nigraproducesthesamecompoundsaswildplantsandalsopreservetheirbiologicalactivity.Inaddition,duetothatiscollectedandsoldindiscriminatelycouldbereduceitspopulationandpossiblyendangerofextinction.Theaimofthisstudywasevaluatedtheanti-inflammatoryactivityofextractfrominvitrocultureofS.nigrapreviouslyestablishmentashomogeneousplantmaterialsourceforbioactivecompoundsproduction.The in vitro culture calluswere induced fromexplants (flowers, leaves, buds and nodes) onMSmedium (Murashige andSkoog),andgrowthregulatorscombination,NAA(naphthalene-1acid),BAP(6-benzylaminopurine),IBA(indolebutyricacid)and 2,4-D (dichlorophenoxyacetic acid), at different concentrations (Cruz et al., 2015). For anti-inflammatory activity of invitrocultureandwildplantextractsofSnigra(hexane,ethylacetate,methanolandaqueous,doses1.6mg/ear)weusedTPA-inducedmouseearoedemaassay(12-O-tetradecanoylphorbol-13-acetate;dose2.5µg/ear)(Payaetal.,1993).Dexamethasonewasusedasapositivecontrol(1.0mg/ear).Furthermore,wereevaluatedthedoses-responseofactiveS.nigraextractsfromwild plants at doses of 0.4, 0.8 and 1.6 mg/ear, both ethyl acetate and methanol extracts. The extracts were furthersubjectedtophytochemicalstudies(Chaovanalikit&Wrolstad,2004;Dewantoetal.,2002).The topical administrationofS. nigraextracts at doseof 1.6mg/earproducedanti-inflammatory effect. Ethyl acetate andmethanolextractfromwildplantsshowedinflammationinhibitionabout88.5%and57.1%respectivelywithED50=1.01mg/earforethylacetateextract.Theinvitrocultureextracts,alsoshowninflammationinhibition,themethanolextractwith93.5%ofinhibition similar to ethyl acetate extract from wild plants and to times more that dexamethasone, which is our drugreference. The chemical profile by TLCof ethyl acetate extracts fromwild plant showed the presenceof different bands(phenolic compounds and flavonoids) in comparison to TLC of the in vitro extract; and its total secondary metabolitescontentwas high comparedwith the other extracts. The in vitro culture extract of S. nigra showed greater decrease ofinflammationthanourreferencedrugdexamethasone,andareanalternativesourceofanti-inflammatorycompoundtowildgrowingplants.Thisactivitywasassociateswithitstraditionaluseforinflammationtreatment.Acknowledgements: Financial support from CONACyT (grant 183958). Proyecto 3212 Catedras CONACyT (No245640). Cruz-Cruz is indebted to CONACyT forfellowshipawarded(N°330824).1. CruzD.G.,EstablecimientodeCultivoinvitrodeSambucusnigraL.SegundoEncuentroNacionaldeBiotecnologíaUATx,Tlaxcala,México.2. PayáM,FerrándizML,SanzMJ,BustosG,BlascoR,RiosJLandAlcarazMJ. 1993.Studyoftheantioedemaactivityofsomeseaweedandsponge

extractsfromthemediterrâneacoastinmice.PhytotherRes7:159-162.3. Chaovanalikit,A.,&Wrolstad,R.E.(2004).Totalanthocyaninsandtotalphenolicsoffreshandprocessedcherriesandtheirantioxidantproperties.Food

ChemistryandToxicology,69,67–72.4. Dewanto,V.,Wu,X.,Adom,K.K.,&Liu,R.H. (2002).Thermalprocessingenhancesthenutritionalvalueof tomatoesby increasingtotalantioxidant

activity.JournalofAgricultureandFoodChemistry,50,3010–3014.


231

ANTI-INFLAMMATORYACTIVITYOFSambucusnigraEXTRACTSFROMCALLUSINVITROCULTURE

Cruz-CruzDG.1,Méndez-SánchezLI.1,Herrera-RuizML.2,CapatazTafurJ.3 ,Sánchez-OcampoPM.4 ¹UniversidaddelPapaloapan.,Tuxtepec(Oaxaca)-México;2PharmacyLaboratory,CentrodeInvestigaciónBiomedicadelSur,Xochitepec(Morelo)-México;3PlantCellCultureLaboratory,BiotechnologyInstitute,UniversidaddelPapaloapan,Tuxtepec

(Oaxaca)-México;4CONACYT-UniversidaddelPapaloapan,Tuxtepec(Oaxaca)-Méxicoemail:[email protected],[email protected]

Sambucus nigra L. (Acanthaceae) is a plant used in traditional Mexican medicine to treat a number of health disordersincluding cough, fever, flu, diarrhea, dysentery, stomach pain, swelling, and found accumulate active compounds asanthocyanins, flavonols,phenolicacidswithantioxidant,antiviralandantibacterialactivities.However, thisplantdoesnothavestudiesthatsupportthatinvitrocultureofS.nigraproducesthesamecompoundsaswildplantsandalsopreservetheirbiologicalactivity.Inaddition,duetothatiscollectedandsoldindiscriminatelycouldbereduceitspopulationandpossiblyendangerofextinction.Theaimofthisstudywasevaluatedtheanti-inflammatoryactivityofextractfrominvitrocultureofS.nigrapreviouslyestablishmentashomogeneousplantmaterialsourceforbioactivecompoundsproduction.The in vitro culture calluswere induced fromexplants (flowers, leaves, buds and nodes) onMSmedium (Murashige andSkoog),andgrowthregulatorscombination,NAA(naphthalene-1acid),BAP(6-benzylaminopurine),IBA(indolebutyricacid)and2,4-D (dichlorophenoxyacetic acid), atdifferent concentrations (Cruzet al., 2015). Foranti-inflammatoryactivityof invitrocultureandwildplantextractsofSnigra(hexane,ethylacetate,methanolandaqueous,doses1.6mg/ear)weusedTPA-inducedmouseearoedemaassay(12-O-tetradecanoylphorbol-13-acetate;dose2.5µg/ear)(Payaetal.,1993).Dexamethasonewasusedasapositivecontrol(1.0mg/ear).Furthermore,wereevaluatedthedoses-responseofactiveS.nigraextractsfromwild plants at doses of 0.4, 0.8 and 1.6 mg/ear, both ethyl acetate and methanol extracts. The extracts were furthersubjectedtophytochemicalstudies(Chaovanalikit&Wrolstad,2004;Dewantoetal.,2002).The topical administrationofS. nigraextracts at doseof 1.6mg/earproducedanti-inflammatory effect. Ethyl acetate andmethanolextractfromwildplantsshowedinflammationinhibitionabout88.5%and57.1%respectivelywithED50=1.01mg/earforethylacetateextract.Theinvitrocultureextracts,alsoshowninflammationinhibition,themethanolextractwith93.5%ofinhibition similar to ethyl acetate extract from wild plants and to times more that dexamethasone which is our drugreference. The chemical profile by TLCof ethyl acetate extracts fromwild plant showed the presenceof different bands(phenolic compounds and flavonoids) in comparison to TLC of the in vitro extract; and its total secondary metabolitescontentwas high comparedwith the other extracts. The in vitro culture extract of S. nigra showed greater decrease ofinflammationthanourreferencedrugdexamethasone,andareanalternativesourceofanti-inflammatorycompoundtowildgrowingplants.Thisactivitywasassociateswithitstraditionaluseforinflammationtreatment.Acknowledgments: Financial support from CONACyT (grant 183958). Proyecto 3212 Catedras CONACyT (No 245640). Cruz-Cruz is indebted to CONACyT forfellowshipawarded(N°330824).1. CruzD.G.,Mendez-SanchezL, I., Capataz-Tafur J., Sanchez-OcampoP.,M. (2015).Establecimientodecultivo invitrodeSambucusnigraL. Segundo

EncuentroNacionaldeBiotecnologíaUATx,Tlaxcala,México.2. PayáM,FerrándizML,SanzMJ,BustosG,BlascoR,RiosJLandAlcarazMJ. 1993.Studyoftheantioedemaactivityofsomeseaweedandsponge

extractsfromthemediterrâneacoastinmice.PhytotherRes7:159-162.3. Chaovanalikit,A.,&Wrolstad,R.E.(2004).Totalanthocyaninsandtotalphenolicsoffreshandprocessedcherriesandtheirantioxidantproperties.Food

ChemistryandToxicology,69,67–72.4. Dewanto,V.,Wu,X.,Adom,K.K.,&Liu,R.H. (2002).Thermalprocessingenhancesthenutritionalvalueof tomatoesby increasingtotalantioxidant

activity.JournalofAgricultureandFoodChemistry,50,3010–3014.


232

ACUTETOXICITY"INVIVO"OFMETHANOLICEXTRACTOFRhipsalisbaccifera

Echeverría-PérezGM.1,Castro-TorresIG.2,Castro-TorresVA.2,Martínez-VázquezM.2,

Naranjo-RodríguezEB.3 1DepartamentodeFarmacia,FacultaddeQuímica,UniversidadNacionalAutónomadeMéxico,CiudaddeMéxico-México;

2InstitutodeQuímica,UniversidadNacionalAutónomadeMéxico,CiudaddeMéxico-Méxicoemail:[email protected]

Rhipsalisbaccifera is anepiphytic cactusknownas“mistletoecactus”andgrows indifferentpartsofMexico,Central andSouthAmerica.ThisplantbelongstotheCactaceaefamilyandiscommonlyusedinthetreatmentoffractures,topromotehairgrowth,strengtheningtheintestinalflora,asantiparasiticandantidoteforsnakebite.InourLaboratory,weperformhypoglycemicstudieswiththemethanolicextractofRhipsalisbaccifera,soourobjectiveisinthisworkwastheevaluationofacuteoraltoxicityoftheplant,accordingtotheOECDGuidelines,2001.Micewereweighed,marked and separated into five experimental groups: Group 1, control; 2, vehicle (water); groups 3, 4 and 4 wereadministered with different doses of methanolic extract of the plant (1, 10 and 100 mg/kg). Administration wasintragastrically, themicewereobserved for 1, 12,24,48and72hpost-administration.WeperformedHPLCanalysisof theextract.Methanolicextractoftheplantupto100 mg/kgdidnotproduceanytoxiceffects inmice.Ourresultsofchromatographicprofile indicated that themethanolic extract contains awide varietyof polar secondarymetabolites;HPLCof the extractshows two abundant peaks, which theymay be associatedwith secondarymetabolites responsible of the hypoglycemiceffectoftheextract.Acknowledgments:SupportedProjectfortheProgramadeApoyoaInvestigaciónyPosgrado(PAIP).FacultaddeQuímica.UNAM.Clave:TechnicalsupportQ.F.B.LuisAntonioMontoyaSalas.UniversidaddeCoahuila.México.1. Arts,J.H.,Zwart,A.,Schoen,E.D.,Klokman-Houweling,J.M.Determinationofconcentration-time-mortalityrelationshipsversusLC50saccordingto

OECDguideline403.1989,ExpPathol,37:62-66.


233

HYDROALCOHOLICEXTRACTOFEryngiumcarlinaeFORTHETREATMENTOF

HYPERCHOLESTEROLEMIAINMICE

Castro-TorresIG.1 ,DelaO-ArciniegaM.2,Naranjo-RodríguezE.B.3,Martínez-VázquezM.1¹InstitutodeQuímica,UniversidadNacionalAutónomadeMéxico,CiudaddeMéxico-México;2InstitutodeCienciasdelaSalud,UniversidadAutónomadelEstadodeHidalgo,Tilcuautla(Hidalgo)-México;3DepartamentodeFarmacia,FacultaddeQuímica,

UniversidadNacionalAutónomadeMéxico,CiudaddeMéxico-Méxicoemail:[email protected]

Hypercholesterolemia is a problem of public health in Mexico. The main factors for the development of this metabolicdiseaseareobesityandafatrichdiet.Eryngiumcarlinae isaplantbelongingtotheUmbelliferaefamilyandhas importantethnobotanicalusesforthetreatmentoflipiddiseases.WeexaminedtheeffectofhydroalcoholicextractofEryngiumcarlinae inserumlipidsconcentrationonmaleCD1micefedwithahypercholesterolemicdiet(cholesterol2%,cholicacid0.5%).Theexperimentalgroups(n=5)consistedin:normaldiet,hypercholesterolemic diet, ezetimibe (10 mg/kg) and hydroalcoholic extract of the plant (100 mg/kg). Histopathologicalstudiesoftheliverwereperformedandconcentrationsoftotalcholesterol,HDLandnon-HDLweremeasured.After4weeks, thehypercholesterolemicdietsignificantly increasedplasmaticcholesterolconcentration inmice; it causedincreases in LDL cholesterol concentration; the levels of HDL cholesterol decreased significantly. In mice fed with acholesterol richdiet increased liverweight. Inaddition, therewasmoderate infiltrationofneutrophilicgranulocytes in theliver during the entire study period. After treatmentwith the plant extract (for 4weeks), therewas a decrease in totalcholesterolandnon-HDLcholesterolconcentrationandtherewasanincreaseinlevelsofHDL(p<0.05).Livertissueshowedvacuolardegeneration.TheplantextractincreasedtheexpressionoftheintestinalsteroltransporterAbcg8withoutalteringthe expression of Abcg5 in mice. The same results were observed in the treatment with ezetimibe at 10 mg/kg; ThehypocholesterolemiceffectofEryngiumcarlinaemaybeassociatedwiththeincreasedexpressionofthetransporterAbcg8,whichfacilitatesanincreaseinintestinalcholesterolefflux.1. Castro-Torres IG,Cárdenas-VázquezRJ,Velázquez-GonzálezC,Ventura-MartínezR,De laO-ArciniegaM,Naranjo-RodríguezEB,Martínez-VázquezM.

2015.Futuretherapeutictargetsforthetreatmentandpreventionofcholesterolgallstones.EurJPharmacol765,366-374.2. Noriega-CisnerosR,Ortiz-ÁvilaO,Esquivel-GutiérrezE,Clemente-GuerreroM,Manzo-AvalosS,Salgado-GarcigliaR,Cortés-RojoC,BoldoghI,Saavedra-

MolinaA.2012.HypolipidemicactivityofEryngiumcarlinaeonstreptozotocin-induceddiabeticrats.BiochemResInt2012,603501.


234

SYNTHESISANDBIOLOGICALACTIVITYOFALKOXYLATEDCHALCONEDP7ONIMPORTANTPLANT

PATHOGENS

RemškarA.1 ,BetancurPJF.2,TrillerasVJE.3,ValenciaJA.4 ,ArboledaVJW.1,2

1UniversityofLjubljana,BiotechnicalFaculty,Ljubljana-Slovenia;2UniversidaddeManizales,FacultaddeCienciasContables,EconómicasyAdministrativas,CentrodeInvestigacionesenMedioAmbienteyDesarrollo–CIMAD,FacultaddeCienciasdelaSalud,Manizales-Colombia;3UniversidaddelAtlántico,FacultaddeCienciasBásicas,Barranquilla(Atlántico)-Colombia;

4DepartamentodeProducciónAgropecuaria,FacultaddeCienciasAgropecuarias,UniversidaddeCaldas,Manizales-Colombiaemail:[email protected],[email protected],[email protected],

[email protected]

Chalcones arenatural compounds that arewidelydistributed in a varietyofplant species. Their diversebiological activity(includingantimicrobial,antifungalandanticanceractivity)andthesimplicityinthepreparationofsyntheticchalconeshaveattractedtheattentionofpharmacologists(Xiao-Guang,2014)aswellasphytopharmacologists.1,3-diphenyl-2-propen-1-oneis a central core for all chalcones and the efficiency of their bioactivity depends on side groups, substitutions andconcentrationoftheagent(Lahtchenetal.,2008).Inordertoevaluatethebiologicalpotentialofthiscompoundsonplantpathogens; the alkoxylated chalcone DP7 was synthetized and tested, by using different bioassays, on Colletrotichumgloesporioides andRalstonia solanacearum, bothphytopathogens that attacks important crops such as tomato andwheatcropsworldwide.The ((E)-1-alkoxyphenyl-3-arylprop-2-en-1-one derivatives were prepared with acceptable yields starting with 3,4-dimethoxyacetophenoneor4-phenoxyacetophenoneandaromaticaldehydes inthepresenceofNaOH(solid)andethanolunder ultrasonic irradiation at room temperature during 5–15 minutes. The yield of the reaction was estimated bystoichiometricanalysisofthestartingmaterialsandthefinalweightofthefinalproductwaspurifiedbyrecrystallizationwithethanol and then subject to complete drying (compared to the stoichiometric expected quantity). The structures of thesynthesizedcompoundDP7wasconfirmedbyIR,1H-NMR,13C-NMRandMS.Bioassayswereconductedinordertodeterminethe antimicrobial activity on the pathogens Colletrotichum gloesporioides and Ralstonia solanacearum by using routinelyantimicrobialtestincludingdiffusionandbrothmicrodilutionmethodology.Chalconesareflavonoidprecursorswithadiversebioactivitypresentinvariousplantspecies.Thesecompoundscouldbeanature-friendlier alternative as anti-phytopathogenic agents thus replacing pesticides now in use. Our results showedbioactivityoftestedchalcone(DP7)againstthespeciesthatwereusedintheexperiment.Fungistaticactivityoftheassayedmoleculewas increasing until the third day of incubation but dropped in the last day according to PIC analysis. Previousstudiesshowedthatchalconesareveryefficientatsuppressingnotonlythegrowthoffungusandbacteriabutalsocancercells.Additionally,theresultsshowmoderateinhibitionatdifferentconcentrationsonR.solanacearumgrowthincomparisonwiththenegativecontrol(DMSO).Inconclusion,theefficiencyofchalconesisrelative.Itsbiologicalactivitydependsonthecell wall density, concentration and susceptibility. However, this kind of screening is necessary in order to design newproductsthatcouldbeusedasalternativemethodtocontrolimportantplantpathogensundersustainablepractices.1. Xiao-GuangBai,Chang-LiangXu,Shuang-ShuangZhaoetal.:SynthesisandCytotoxicEvaluationofAlkoxylatedChalcones,molecules2014, 19: 17256-

17278.2. K.L.Lahtchev,D.I.Batovska,St.P.Parushevetal.:Antifungalactivityofchalcones:Amechanisticstudyusingvariousyeaststrains,EuropeanJournalof

MedicinalChemistry2008,43:2220-2228.3. T.M. Osório, F.D. Monache, L.D. Chiaradia et al: Antibacterial activity of chalcones, hydrazones and oxadiazoles against methicillin-resistant

Staphylococcusaureus,Bioorganic&MedicinalChemistryLetters2012,22,225–230.


235

KalanchoebrasiliensisCAMB.(CRASSULACEAE):ANINTERESTINGSOURCEOFBIOACTIVESUBSTANCES

TOTREATINFECTIOUSDISEASESCAUSEDBYSalmonellaSTRAINS

MayorgaOAS.1,FlorencioJR.1,Feres-NettoC.2,PedrosaDM.2,CostaYFG.2,PintoNCC.3,ScioE.3,SousaOV.4,AlvesMS.4

¹UniversidadeFederaldeJuizdeFora,JuizdeFora-Brazil;2FacultyofPharmacy,UniversidadeFederaldeJuizdeFora,JuizdeFora-Brazil;3LaboratoryofBioactiveNaturalProducts,DepartmentofBiochemistry,InstituteofBiologicalSciences,Universidade

FederaldeJuizdeFora,JuizdeFora-Brazil;4DepartmentofPharmaceuticalSciences,FacultyofPharmacy,UniversidadeFederaldeJuizdeFora,JuizdeFora-Brazilemail:[email protected]

KalanchoebrasiliensisCamb.(Crassulaceae)isaBrazilianvegetalspeciespopularlyknownas"saião"and"couramabranca"traditionallyusedtotreatinjuries,abscesses,enlargedgangliaandinflammatoryprocesses(Costaetal.,1994;Mourãoetal.,1999).However,therearefewreportsaboutthepharmacologicalactivitiesofthismedicinalplant,mainlyaboutitspossibleantibacterial activity. The current study was designed to explore the chemical composition and the antibacterial activityagainstSalmonellareferenceandroutinestrainsofhydroethanolicextracts(HEE)obtainedfromfreshleavesofK.brasiliensiscollectedbeforeblooming,usingthreedifferentconcentrationsofethanol[30%(HEE230),50%(HEE250)and70%(HEE270)v/v].Thechemical characterizationwasestablishedby reversephaseHPLC-DAD.Theantibacterial activitywasassessedby theMinimal Inhibitory Concentration (MIC) using microdilution method followed by the Minimal Bactericidal Concentration(MBC),whichallowstheclassificationoftheantibacterialeffectasbacteriostaticorbactericidal(Andrews,2001;CLSI,2012).Two reference strains Salmonella enterica subsp. enterica serovar Choleraesuis (ATCC® 10708™) and Salmonella entericasubsp. enterica serovar Typhimurium (ATCC® 13311™) and five routine strains Salmonella enterica subsp. enterica serovarEnteritidis1406591,Salmonellaentericasubsp.entericaserovarEnteritidis1418594,Salmonellaentericasubsp.entericaserovarEnteritidis 1628260, Salmonella spp. 1266695 and Salmonella spp. 1507788 isolated from clinical human specimens weretested.UVspectraobtainedfromchromatographicanalysissuggestedthepresenceofthesamesixflavonolsinHEE230,HEE250andHEE270.Consideringtheantibacterialactivity,HEE230andHEE250wereactiveagainstS.Choleraesuis(ATCC®10708™)andS.Typhimurium (ATCC® 13311™), with MIC values of 5000 µg/mL and bacteriostatic effect. However, HEE270 inhibited allSalmonella strains tested,withMIC values of 5000 µg/mL and bacteriostatic effect, being themost active extract amongthoseinvestigated.TheseresultsindicatethatK.brasiliensisisaninterestingsourceofbioactivesubstances,beingeffectiveagainstSalmonellastrains,includingantibiotic-resistantones.Acknowledgments:ThisinvestigationwassupportedbyUFJF,FAPEMIG(CDS-APQ-04680-10),CAPES,andPAECOEA-GCUB. 1. Costa,S.S.;Jossang,A.;Bodo,B.;Souza,M.L.;Moraes,V.L.PatuletinacetylrhamnosidesfromKalanchoebrasiliensisasinhibitorsofhumanlymphocyte

proliferativeactivity.JournalofNaturalProducts,57:1503-1510,1994.2. Mourão,R.H.;Santos,F.O.;Franzotti,E.M.;Moreno,M.P.;Antoniolli,A.R.Antiinflammatoryactivityandacutetoxicity(LD50)ofthejuiceofKalanchoe

brasiliensis(Camb.)leavespickedbeforeandduringblooming.PhytotherapyResearch,13:352-354,1999.3. AndrewsJ.M.Determinationofminimuminhibitoryconcentrations.JournalofAntimicrobialChemotheraphy,48:5-16,2001.4. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved

Standard-NinthEdition(M07-A9).Wayne:CLSI,2012.


236

INVITROANTIBACTERIALACTIVITYOFHYPOESTESFORSSKAOLIIVAHL.ROEM&SCHULT.

(ACANTHACEAE)AGAISTMETHICILLIN-RESISTANTSTAPHYLOCOCCUSAUREUS:PRELIMINARYRESULTS

D’AmbolaM.1,NoceraFP.1,VassalloA.2,FlorioS.1,SeverinoL.1 ,DeMartinoL.11UniversityofNaplesFedericoII,DepartmentofVeterinaryMedicineandAnimalProductions,DivisionofToxicology,Naples-Italy;

2UniversityofBasilicata,DepartmentofScience,Potenza-Italy,email:[email protected]

Hypoestes forsskaolii is a perennial herb belonging to Acanthaceae family, which shows several beneficial properties asantioxidant, antibacterial, anticancer, antifungal and antiparasitic activity (Mothana et al. 2009; Ojo-Amaize et al. 2007;Rasoamiaranjanaharyetal.2003).Plantsproducewidearrayofbioactivemolecules,mostofwhichprobablyevolvedaschemicaldefenseagainstpredationorinfection(Samieetal.2010).Amedicinalplantisanyplantwhich,inoneormoreofitsorgan,containssubstancethatcanbeusedfortherapeuticpurposeorwhichisaprecursorforsynthesisofusefuldrugs(Edwards2004).Inordertodevelopinnovativeantibacterialcompoundswithpotencialapplicationsinpreventingmicrobialproliferation, inthisstudyweevaluatedtheantibacterialactivityofH.forsskaoliiagainstmethicillin-resistantStaphylococcusaureus(MRSA)ATCC33591.The crude extracts ofH. forsskaolii were obtained through a polarity solventmaceration process (n-hexane, chloroform,chloroform:methanol(9:1v/v)andmethanolof326gofroots.Theextractconcentrationsusedforthisstudyrangedfrom50µg/mLto500µg/mL.TheantimicrobialactivitywasmeasuredbysuspendingMRSA(onesingleporousbead,acarriertosupportmicroorganismsinMicrobankvialcontainingcryopreservative)inBrainHeartInfusionBroth(Oxoid)for24hat37°C;thereafter,theOpticalDensityat600nm(OD600)of thebacterial suspensionwasadjustedto0.1withtheadditionofsterilebroth.Testswerecarriedout inmicrotiterplatewiththebacterialsuspensionbyaddingtheextractsatdifferentconcentrations(50,70,80,100,200,500µg/mL).TheMinimal InhibitoryConcentration(MIC)wasdefinedasthe lowestconcentrationoftheextractsabletopreventthemicrobialgrowth.Theanalyseswereperformedintriplicateonthreeindependentexperiments.Wedemonstrated thatonly thechloroformicextractwaseffective ingrowth inhibitionof the testedMRSAstrainusingaconcentrationupto100µg/mL.Theantimicrobialactivityreportedcouldbeduetotheterpenoidswhicharepresentinthechloroformicextract.However,furtherstudiesareneededtobetterdefinetheeffectivenessofinvitroactivityofH.forsskaoliichloroformicextract.1. Edwards,R.2004.Noremedyinsightforherbalransack.Newscience181:10-11.2. Mothana, R. A., U. Lindequist, R. Gruenert, and P. J. Bednarski. 2009. Studies of the in vitro anticancer, antimicrobial and antioxidant potentials of

selectedYemenimedicinalplantsfromtheislandSoqotra.BMCComplementAlternMed9:7.3. Ojo-Amaize,E.A.,E.J.Nchekwube,H.B.Cottam,O.A.Oyemade,A.A.Adesomoju,andJ.I.Okogun.2007.Plasmodiumberghei:antiparasiticeffectsof

orallyadministeredhypoestoxideinmice.ExpParasitol117(2):218-221.4. Rasoamiaranjanahary, L., D. Guilet, A. Marston, F. Randimbivololona, and K. Hostettmann. 2003. Antifungal isopimaranes from Hypoestes serpens.

Phytochemistry64(2):543-548.5. Samie,A.,T.Tambani,E.Harshfield,E.Green,J.N.Ramalivhana,andP.O.Bessong.2010.AntifungalactivitiesofselectedVendamedicinalplantsagainst

Candidaalbicans,CandidakruseiandCryptococcusneoformans isolatedfromSouthAfricanAIDSpatients.AfricanJournalofBiotechnology9(20):2965-2976.


237

SEASONALVARIATIONANDCHEMOTYPEIDENTIFICATIONINVOLATILEANDNON-VOLATILEFRACTIONSFROMSchinusmolleLEAVESBYCHROMATOGRAPHICPROFILINGCOUPLEDWITH

CHEMOMETRICS

MaciasYP.1,BernalFA.2 ,Coy-BarreraE.2 ¹UniversidadDistritalFranciscoJosédeCaldas,Bogotá-Colombia;2MilitaryUniversityNuevaGranada,Cundinamarca,Colombia;²


Schinusmolle isaplantcommonlydistributedaroundtheworld,which isusedforpurelyornamentalpurposes.Despite itswidedistributionanditsgreatadaptationabilitytogrowunderseveralenvironmentalconditions,studiesonthebiologicalpotential and chemical composition of this species is currently limited. In fact, only few reports about the chemicalcomposition from fruit essential oils have been published [1], whereas chemical components from other parts and non-volatilefractionsremainalmostunexplored.Therefore,aspartofourresearchonmetabolomicsfromplantspeciesgrowingin Colombia, variability on chemical composition of volatile and non-volatile fractions fromS.molle leaveswas evaluatedusingGC-MSandLC-DAD-ESI-MScoupledwithchemometrictechniques.Atotalof48specimensofS.mollewerecollectedaroundBogotá,Colombia.Eachsamplewassubmittedtoextractionofbothvolatileandnon-volatilecomponents.Thefirstonewasaccomplishedbysupercriticalfluidextractionwhilethelatteronewascarriedoutbyultrasound-assistedextraction.Subsequently,chromatographicprofilingbyGC-MSandLC-DAD-ESI-MS for volatile and non-volatile fractions, respectively, was performed. The corresponding profiles were independentlyaligned,normalizedandscaled,andafterwardsstatisticallycomparedunderunsupervisedandsupervisedmodels,includingPCA and OPLS-DA. Total phenolic and total flavonoid contents were also determined for non-volatile extracts byconventionalspectrophotometricmethods.Comparison of Total Phenolic Contents for the analyzed samples showed to be significantly different among them asindicated by ANOVA (p<0.05). For its part, Total Flavonoid Contents resulted to be less changing, although significantdifferencescouldbealsoobserved.PCAandHCAdemonstratedsamplescanbeclassifiedinfourdifferentgroupsaccordingto their LC-UV-profiles, whichmeans significant variations in composition along thewhole set of samples was thereforeevident.OPLS-DAusingcollectiondateassupervisionvariableaffordedcleardifferentiationofsamples, indicatingmarkedcompositional changes with the season, which could help out defining chemotypes in S. molle. On the other hand, thevariations on phenolic content were related to changes in chemical profiles as demonstrated by OPLS. In contrast, anyrelationshipbetweenflavonoidcontentsandchromatographicprofilescouldnotbedefine.Incaseofthevolatilefractions,besides they were characterized as sesquiterpene-rich oils, four different groups by HCA according their profiles wereobserved.Moreover,seasonalvariations in theirchemicalcompositionsweredemonstratedaswell, supportingtheabovementionedresultsfornon-volatilecomponents.Insummary,statisticalmodelsletdemonstratetheexistenceofchemotypesamongS.molle specimens growing in Bogotá,which can be attributed to seasonalmetabolic variations. Identification ofchemicalmarkersforeachchemotypearecurrentlyinprogress.Acknowledgments:ThepresentworkwasperformedwithintheProjectINV-CIAS-2050grantedbyVicerrectoríadeInvestigacionesatUMNG-Validity2016.1. BendaoudH,etal.,2010,ChemicalCompositionandAnticancerandAntioxidantActivitiesofSchinusmolleL.andSchinusterebinthifoliusraddiberries

EssentialOils.JFoodSci75,C466-C472.


238

PHOTODYNAMICANTIMICROBIALACTIVITYOFSYZYGIUMCUMINIEXTRACTSONKLEBSIELLA

PNEUMONIAE

QuiguaORM.,Valle-MolinaresRH.,VallejoLWA.,DiazUCE. ,ArboledaVJW. ¹UniversityofAtlántico,Barranquilla(Atlántico)-Colombia


The genus Klebsiella frequently causes human nosocomial infections [1]. The most important specie from this genus isKlebsiellapneumoniae, abacteria responsibleof infectionsaspneumoniaandsepticemias, that require intensehealthcare[2].Itischaracterizedbyacapsule(virulencedeterminant),siderophores andfilamentaryprojections(Pilus)presentonthebacterial surface which favour adhesion to host organisms. Because of their ability to spread rapidly in the hospitalenvironment,thesebacteriatendtocausenosocomialoutbreaks.Recently,newtypesofstrains,calledextended-spectrum-β-lactamase (ESBL) producers, represent a challenge in the searchof effective alternatives for themanagement of theseinfections[3].Reactiveoxygenspecies(ROS)aremolecules involved inavarietyofchemical,biologicalandenvironmentalprocesses[1-3].ROS generationispromisinginantimicrobialphotodynamictherapysincetheirpresence leadstodamagesofthemicrobialmembraneandtoproteindegradation .However,synthesisofthesecompoundsrequiresa varietyofreagentsexpensiveandsometimestoxictohumansandtothe environment.Forthisreason,withtheaimtoselectnewcompoundswith potential biological activity onK. Pneumoniae, extracts obtained from the plant Syzygium cumini were evaluated byusingphotodynamictherapyandbioassays. EthanolextractofSyzygiumcuminifruitwasobtainedaccordingtothefollowingprotocol:freshgrapeswerepicked,cleanedwith distilled water and heated at 45°C for 5 days until constant weight. Dry samples were then isolated from shell. Anamountofcrushedmaterialwasplaced intoapercolator;thesolventethanolwasaddedtocompletelycoverthematerial(380 mL). Percolation process was performed for 1 day, thereafter 350 mL of the mixture were collected. Solvent wasrecoveredbysimpledistillationandreturnedtopercolator.Thenaproportionalamountoftheextract-ethanolmixtureatthebottomwas collected and the percolation process performed until constantweight. The antimicrobial activity of ethanolextractofSyzygiumcuminifruitwasevaluatedonKlebsiellapneumoniaeATCC70503byusingbrothmicrodilutionprotocolin96wellmicroplateat37°C.Differentconcentrationsofirradiatedandnon-irradiatedextractswereusedastreatments(51.8,41.4, 25.9, 15.5and 10,4µg/µl). The final volumewas 100µLand the final concentrationofDMSO(negativecontrol) in theassaydidnotexceed1%.TheOD600wasmonitoredandthe inhibitionpercentandMinimalInhibitoryConcentration(MIC)was calculated following the recommendationsof theCLSI-2012 formicrodilution (IC50)byusing theGraphpadPrism5.0statisticalpackage.DatawerecomparedwiththoseusingGentamicin(20µg/mL)aspositivecontrol.Results show statistically significant differences between treatments and negative and positive controls. In addition,irradiationtreatment increasedthebiologicalactivitysustainingtheuseofphotodynamictherapy.Weobservedapositivecorrelation between extract concentration and bacterial growth inhibition, values ranging between 41,6% and 90,5% forirradiatedtreatments.Nonirradiatedtreatmentsshowedinhibitionrangingbetween0%and58,6%.TheMICvaluesincaseofirradiatedtreatmentswas7.25µg/µL.The incidence of ESBL-producing strains among clinicalKlebsiella spp. isolates has been steadily increasing over the pastyears. Limitations in therapeuticoptionsdemandnewmeasures tocounteracthospital infections. For this reason,naturalsensitizers represent practible alternatives for selecting new compoundswith antibacterial activity [4]. In addition, thesenaturalsensitizersarecheap,accessible,abundantandhavelowerenvironmentalimpactcomparedtosyntheticcompounds.Innature,fruit,flowerandleafofplantsshowdifferentcolorsfromredtopurpleduetothepresenceofnaturaldyes,whichcanbeusedindifferentapplication.ItcanbesuggestedthatnaturalpigmentsfromplantsasSyzygiumcuminiwillbeusedaspotentialantibioticsrepresentingavaluableopportunitytocounteractthiskindofmultidrug-resistantbacteria.1. LópezJandEcheverriL.K.pneumoniae:lanueva“superbacteria”?Patogenicidadepidemiologíaymecanismosderesistencia,Iatreia.2010:23,157–1652. GonzálezAC.etal.,CaracterizacióndecepasdeKlebsiellapneumoniaeproductoradeB-lactamasasdeespectroextendidoaisladasendosunidadesde

cuidadosintensivos,RevChil.Infetcol.2013,30:374–3803. PfallerMAandSegretiJ.,OverviewoftheEpidemiologicalProfileandLaboratoryDetectionofExtended-Spectrumb-Lactamases,Epidemiol.Detect.

ESBLs.2016,42:153–1634. PereiraRosaL.,AntimicrobialPhotodynamicTherapy:ANewTherapeuticOptiontoCombatInfections,J.Med.Microbiol.Diagnosis.03(2014).

doi:10.4172/2161-0703.100015


239

IDENTIFICATIONOFPHENYLPROPANOIDGLYCOSIDESFROMALOYSIAPOLYSTACHYA(GRISEB.ET

MOLDENKE)BYHPLC-MSANALYSIS

BertelliD. ,GraziosiR.,PlessiM.DepartmentofLifeSciences,UniversityofModenaandReggioEmilia,Modena-Italy

email:[email protected] on the chemical composition of Aloysia polystachya (Griseb. et Moldenke) are poor in the literature. Phenoliccompoundscharacterizationinplantmatrixcanbeverydifficult,owingtobondtodifferentsugarmoieties,orconjugationto form complex structures. Despite a great number of investigations, also the separation and the simultaneousdetermination of polyphenolics of different groups remain difficult. Among the different methods available, high-performanceliquidchromatography(HPLC) ispreferablefortheseparationandquantificationofpolyphenolics.TheHPLC-MS characterization of the phenylpropanoid glycosides fraction, purified by Solid Phase Extraction (SPE), in Aloysiapolystachya(Griseb.etMoldenke)extractwasperformed.TwodifferentMS techniqueshavebeencoupled toHPLC, to identify the compoundspresent in theextract, and supporttentative structure identification: ion trap mass spectrometry (Ion Trap LC/MS), and the quadrupole-time of flight highresolutionmassspectrometry(Q-TOFHRMS).Nine phenylpropanoid glycosideswere identified inmethanolic extract of the Aloysia polystachya (Griseb. etMoldenke)leaves.Amongthem,thecompoundsforsythosideA,plantainosideC,purpureasideD,martynosideanditstwoisomerswereidentifiedhereforthefirsttime.The results present here couldbehelpful in the futuredeveloping and validatingof an analyticalmethod toquantify theisolated compounds, and to assess the quality of Aloysia polystachya (Griseb. et Moldenke) leaves. Knowledge of thecontentofphenylpropanoidglycosidescouldfurthercontributetothechemotaxonomyofthegenus.1. PiccinelliAL,DeSimoneF,PassiS,RastrelliL.2004.JAgricFoodChem52:5863-5868.2. Quirantes-PinèR,Herranz-LopezM,FunesL,Borras-LinaresI,MicolV,Segura-CarreteroA,Fernandez-GutierrezA.2013.Phytomedicine20:1112-1118.


240

ESSENTIALOILSASPOTENTIALFUMIGANTAGENTSFORTHECONTROLOFSitophiluszeamais

PatiñoWR.1 ,PatiñoOJ.1 ,CucaLE.1,DelgadoWA.1,PrietoJA.2

1NationalUniversityofColombia,Bogotá,Colombia;2PontificiaUniversityJaveriana,Bogotá,Colombiaemail:[email protected],[email protected]

Grainslikerice,wheatandmaizeareconsideredfundamentalbytheWorldHealthOrganization(WHO)toensuretheglobalfoodsecurity.Duetoacceleratedworldpopulationgrowth,by2050isestimatedthatglobaldemandforstoredgrainswillincreasearound40%ofcurrentproduction,exceedingthe300milliontonsperyear.Despitethegreatimportanceofthesecereals, indevelopingcountries likeColombiatheproductionforownconsumption is insufficient, inthecaseofmaize4.5milliontonsareimportedannually.Themainfactorsaffectingmaizeproductionarethelowagriculturaltechnologyandcroplossescausedbyinsectsanddiseasesattack(FAO2016).Thepostharvestinsectsarethemostresponsibleforthedamageofthestoredproduct.ColeopteraofSitophilusgenusareacosmopolitanplaguethataffectsstoredgrainsandcereals,withinthem is S. zeamais known asmaizeweevil (Pingali and Pandey, 2001; García and Bergvinson, 2007). The control of theseinsectsisdonewithsyntheticandsystemicpesticides,whichareexpensive,highlytoxicandarebecominglesseffectiveasresult of the emergence of resistant species. Therefore the discovery and development of new effective and safetybiocontrol agents is necessary. The botanical volatile metabolites like essential oils are a potential source of newagrochemicals due to high volatility, rich chemical composition and high distribution (Rees D. 2004). In this work wasperformedascreeningoffumigantactivityofessentialoilsagainstSitophiluszeamais.Theplantmaterialof52nativeandintroducedspecieswascollectedoracquiredindifferentpartsoftheColombianAndeanregion. From the fresh material the essential oils were extracted by steam distillation technique. The chemicalcharacterizationofessentialoilswasperformedbyGC-MSonHP-5andCarbowaxcolumns,thecomponentswereidentifiedbycomparisonofretentiontimesandmassspectra.ThefumigantactivityofessentialoilswasdeterminedagainstS.zeamaisusingthevialintovialmethod.Filterpaperswereimpregnatedwithessentialoilatcalculateddosestogiveafinalfumigantconcentrationof500µL/Linair.Theimpregnatedfilterpapersweredisposedina1,5mlvial,thesewereplacedina22mlvial,whichcontained10adultsinsect.Todeterminethefumigantactivitytwovariationsoftheassaywereevaluated,inthefirstassaytheinsecthadthepossibilitytocomeincontactwiththeimpregnateddisc.Otherwise,inthesecondtestwasavoidedthecontactcoveringthetopofthesmallvialwhitasemipermeablemembrane(Pascualetal.,2004;PrietoJ.A.2012).In the preliminary screening of fumigant activity 24 essential oils of the 52 tested showed insecticidal activity against S.zeamais.Bycomparisontothemortalityratesobtainedbythetwomethodswerefoundvariationsintheactivityofsomeoils.Pimpinelaanisum,OcimumbasilicumyArtemisiavulgarisessentialoilspresentedactivitymainlybycontact,inthefirstassaytheyshowedmortalityratesof80%,63%and40%respectively,whileinthesecondtesttheyweretotallyinactive.Moreover,theLavandulastoechas,RosmariusofficinalisyLippiaalbaessentialoilsshowedfumigantactionpredominantlywithmortalityrateshigherthan85%inbothmethodsassays.Throughthescreeningofpreliminaryactivitywasdeterminedtheinsecticidepotential of 24 essential oils andaswell as themethodsbywhich conducted theeffectby fumigant abilityor as contactinsecticides.1. FAO2016,Ahorrarparacrecerenlapráctica.http://www.fao.org/3/a-i4009s.pdf[Consultado:11/06/2016]2. García,L.S.,Bergvinson,D.J.2007.AgriculturaTécnicaenMéxico.33(2)181-1893. Pingali,P.L.,Pandey,S.2001.CIMMYT1999–2000.4. ReesD.2004.CSIROPublishing.1-3,46-51,114-1195. Pascualetal.2004.Bol.San.Veg.Plagas,30.279-286.6. PrietoJ.A.,2012.TesisDoctoralUniversidadNacionaldeColombia.136-140.


241

SYNTHESISOFBIOACTIVECHROMENESINSPIREDONTHOSEISOLATEDFROMPIPERCF.CUMANENSE

KUNTH(PIPERACEAE).

ParraAJE.1 ,PatiñoL.OJ.2,AvilaMMC.2,CucaSLE.2,Gamba-SánchezD.31UniversidadNacionaldeColombia,DepartamentodeQuímica,Bogotá-Colombia;2UniversidadNacionaldeColombia,DepartamentodeQuímica,Bogotá-Colombia;3UniversidaddelosAndes,DepartamentodeQuímica,Bogotá-Colombia

email:[email protected],alsoknownasbenzopyrones,areorganiccompoundsofacyclicfusedstructurebetweenanaromaticringandapyrone.Suchcompoundsarewidelydistributedinnatureandexistastwotypesofisomers:2H-chromene(2H-1-benzopyran)and 4H-chromene (4H-1-benzopyran). Suchmetabolites are biologically interesting compounds that have shown antiviral,mutagenic,antimicrobial,antitumor,anti-HIV,andantifungalactivity.Furthermore,theyarealsoactiveinthecentralnervoussystem, among others (1). They have been isolated from various genuses belonging to the families Apiacae, Asteraceae,Fabaceae,RutaceaeandPiperaceae,mainly. In thePiperaceae family theyhavebeengenerally reported in speciesof thegenusPiperandPeperomia(2).ChromenesfoundinthegenusPiperarecharacterizedbydifferentalkylchainsatposition2,adoublebondbetween3and4,carboxylorestergroupatposition6andsometimessubstituentsatposition8hydroxyltype or aliphatic chains like prenyl, geranyl and farnesyl are also common. Recently, we have demonstrated that someisolatedchromenesfromPipercf.cumanenseKunth(Piperaceae)showpotentantifungalactivity(3,4).Inordertoestablishthestructure-activityrelationship,westartedthesynthesisofseveralanalogsofchromenesinspiredonthoseisolatedfromthegenusPiperandpreliminaryresultsaredescribed.The synthesis of the chromenes is based on protocols previously described for different precursors (5).We startedwithsubstitutedphenols,subjectedtoanacetylationreactiononthehydroxylgroup,followedbyaFriesrearrangementtoform2-acetophenones (with different substituents). Subsequently a condensation with different ketones was performed, andtherefore the generation of the benzopira-2-one ring was conducted. Finally the obtained products were subjected toreduction,dehydrationand/ortoprenylacionestogeneratethedifferentanalogs.Themethoddevelopedisefficient,yieldsare very good providing a new class of chomenes and analogs. Furthermore, the obtained core allows structuralmodificationsandtheintroductionofdifferentesubstituents.Applyingthisprocedure,15analogueswithdifferentelectron-donatingandelectron-whitdrawinggroupsanddifferentchainsandallylichalogensubstituentsweresynthesized.Resultscontributes to research development on chromenes mainly isolated from the genus Piper, thus providing the basis forstructure-activitystudiesaimingtofindsubstanceswithgreaterantifungalactivity.Acknowledgments: Authors greatfully acknowledge financial supports provided by Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y laInnovación,FranciscoJosédeCaldas,(ContractNo.0459–2013,RIFRUTBIO)andbyColciencias(1101-05-17783),UniversidadNacionaldeColombiaandUniversidaddelosAndes.1. OrjalaJ.,etal.,TwochromenesandaprenylatedbenzoicacidderivativefromPiperaduncum.Phytochemistry1993,34:813-818.2. Pratap,R.andV.J.Ram,NaturalandSyntheticChromenes,FusedChromenes,andVersatilityofDihydrobenzochromenes inOrganicSynthesis.Chemical

Reviews2014,114:10476-10526.3. ParraJE.etal.Cumanensicacid,anewchromeneisolatedfromPipercf.cumanenseKunth(Piperaceae).PhytochemistryLetters2011,4:280-282.4. ParraJE.etal.AnewbenzoicacidderivativeisolatedfromPipercf.cumanenseKunth(Piperaceae).PhytochemistryLetters2013,6:590-592.5. Tawfik HA. et al. ChemInform Abstract: Synthesis of Chromones and Their Applications During the Last Ten Years. ChemInform, 46: no.

doi:10.1002/chin.201513331


242

CYTOTOXICITYOFDQ-35ISMEDIATEDBYG2-ARRESTANDMICRONUCLEIFORMATIONWITHOUTNUCLEARDAMAGE

OcampoY.1,RiveraD.1,AcuñaJ.1,CaroD.1,CastroJ.1,OliveroJ.2,MotilvaV.3,MüllerK.4,FrancoL.1

¹UniversityofCartagena,BiologicalEvaluationGroup,Cartagena-Colombia;2UniversityofCartagena,EnvironmentalandChemistryGroup,Cartagena-Colombia;3UniversityofSeville,AppliedandMolecularPharmacologyGroup,SchoolofPharmacy,

Seville-Spain;4UniversityofMünster,InstituteofPharmaceuticalandMedicinalChemistry,Münster-Germanyemail:[email protected]

Quinones represent a diverse family of naturally occurring secondary metabolites. Interest in these substances hasintensified in recent years due to their pharmacological importance [1]. In particular,many clinically important antitumoragentspossessaplanarringsystemcontainingthequinonenucleus.Inapreviousstudy,weidentifiedQD-35,athiophene-naphthoquinone, as a candidate molecule to develop colorectal cancer treatment [2]. Here, we demonstrated that thecytotoxiceffectofthiscompoundismediatedthroughG2-arrestwithoutnucleardamage.QD-35 was obtained by chemical synthesis using the method described by Zuse et. al. [3]. Structure was confirmed byspectroscopicdata(FT-IR,MNRandMS).Colorectalcancercells,HT-29(ATCC®-HTB-38™),weretreatedwithQD-35(LC50≈1.8µM) for 48 h. Thereafter, genotoxic effect was evaluated by cytokinesis-block micronucleus (MN) assay with somemodifications[4],aswellascometassayinneutralconditionsasdescribedby[5].Inaddition,theeffectoftestcompoundoncellcycleprogressionwasassessedbyflowcytometryusingpropidiumiodidestaining.MN formation is a hallmark of property of drug-induced genotoxicity. Treatment of HT-29 cells with QD-35 significantlyincreased theMN frequency. In contrast, test compound did not induce comet formation.QD-35 treatment resulted in aremarkable increase in the proportion of 4N cell population, and significant decrease of 2N population, suggesting aninduction of G2/M arrest. Moreover, we found a remarkable increase of >4N population, which is consistent with theinductionofMN.Insummary,ourresultsindicatetheabilityofQD-35toinducechromosomaldamageandgenomeinstabilityinHT-29cellswithoutinducingDNAdamage.Acknowledgments:ThisstudywassupportedbyColcienciasandtheUniversityofCartagena(Grant110756933930).1. Reis, M.I.P., et al., A new and efficient procedure for the synthesis of hexahydropyrimidine-fused 1,4-naphthoquinones. Beilstein Journal of Organic

Chemistry,2015.11:p.1235-1240.2. Franco,L.,etal.,Invitroscreeningofanticanceractivityof38syntheticquinoidderivativesonHT-29colorectalcancercellline.DrugDiscovery&Therapy

WorldCongress2015,2015.PO-76:p.1.3. Zuse, A., et al., 9-Benzylidene-naphtho [2, 3-b] thiophen-4-ones as Novel Antimicrotubule Agents Synthesis, Antiproliferative Activity, and Inhibition of

TubulinPolymerization.Journalofmedicinalchemistry,2006.49(26):p.7816-7825.4. Fenech,M.,Cytokinesis-blockmicronucleuscytomeassay.Nat.Protocols,2007.2(5):p.1084-1104.5. Cabarcas-Montalvo,M., J. Olivero-Verbel, andH. Corrales-Aldana,Genotoxic effects in blood cells ofMusmusculus and Iguana iguana living near coal

miningareasinColombia.ScienceofTheTotalEnvironment,2012.416:p.208-214.


243

SUSCEPTIBILITYOFTWOSTAPHYLOCOCCUSAUREUSSTRAINSFROMTHECLINICALENVIRONMENT

TOANNONAPURPUREAFRACTIONS

MárquezVRL1 ,JiménezCAP.1TamaraRY.1,ParejoAMS.1,MuñozNAS1 ,DeLaRosaTCR2¹UniversidaddeSucre,Sincelejo(Sucre)-Colombia;2UniversidaddelAtlántico,Barranquilla(Atlántico)-Colombia

email:[email protected] diseases can be controlled by various chemotherapeutic agents, however microorganisms can change theirantigenic structure, thus developing resistance to, even highly active, commercial antibiotics. To overcome this seriousproblem,itwassuggestedthatnaturalresourcessuchasplantscanbeusedasphytotherapeutics.PlantsoftheAnnonaceaefamily are almost exclusively typical of tropics, being distributed in tropical and subtropical regions of both hemisferios.Antibacterial activity has been shown for leaves of Annona diversifolia and for seeds and leaves of A. purpurea and A.Rollinia5.AnnonapurpureaisaplantcommonlyknowninthedepartmentofSucreColombiaasMatimba.In this study, the survival of two strains of Staphylococcus aureuswere evaluated to assess the antimicrobial activity offractions of ethanol extract from the dried bark of Annona purpurea. Fractions a, b, c, d were obtained by liquid-liquidfractionation of the total bark extract of Annona purpurea and evaluated by the method of diffusion in well, atconcentrations of 50 mg/mL, 30 mg/mL and 10mg/mL. Treatments were performed in triplicate. The bacteriumStaphylococcus aureus isolated from the clinical environment appeared sensitive to three of the five fractions tested.Concentrationsof30and50mg/mLshowedthegreatestinhibition.


244

HISTOCHEMISTRYANDGASCHROMATOGRAPHICPROFILEOFTHEETHYLETHERFRACTION

EXTRACTEDFROMFRESHSHEETSOFCNIDOSCOLUSACONTIFOLIUS(EUPHORBIACEAE)

MárquezVRL1 ,MartínezPMA.,JiménezCAP.1,TamaraRY.1,ParejoAMS.1,EstradaRDJ.1,CarpioZCM.1,CastilloCOJ.1,DeLaRosaTCR.2

¹UniversidaddeSucre,GrupodeInvestigaciónenProductosNaturales,UniversidaddeSucre,Sincelejo(Sucre)-Colombia;2UniversidaddelAtlántico,GrupodeInvestigaciónFituquímica,Barranquilla(Atlántico)-Colombia.

email:rita.marquez@unisucre.edu.coHistochemistryperformedonCnidoscolussacontifoliusleaveshowedthepresenceofcrystalsofcalciumoxalate,flavonoids,lignans,saponins,tanninsandalcaloides1.TheethyletherfractionobtainedfromthetheaqueousextractofC.acontifoliusleavesexhibitlowtoxicityagainstArtemianaupliisalina2.Thegaschromatographicprofileofthefractionshowstwopeaks(8and13)withpercentagesofareasof7,711and30,25andretention times of 68,184 and 86,589 minutes, respectively. Peaks were analyzed with a mass selective detector (MS),operatinginfullscanmodetoperformacomparativeanalysisoffragmentationpatternsandmassspectradatabasesNISTandWileyAdams.Forpeak(8)thefollowingdatawererecorded:molecularionpeaksatm/z:429,3(2.38%)andatm/z355.1(4.76%)[M+-74.2],309,3(1.9%)[M+-120],281(24.76%)[M+-148.3], 239(2.38%)[M+-190], 207(55,23%)[M+-222.3], 178(1,90%)[M+-251.3],169(3.33%)[M+-260],141,2(6,66%)[M+-288.1],113,2(10%)[M+-316.1],85,2(56,19%)[M+-344.1],70,2(66,19%)[M+-359.1],57,2(100%) [M+-372.1]. The base peak of 57.2 was characteristic for isoquinoline alkaloids described in the Euphorbiaceaefamily. The molecular formula C27H27NO4 corresponds to a component derived from Hemiargyrine3. For peak (13) thefollowingdatawererecorded:molecularionpeaksatm/z429(2.38%)andatm/z408,4(11.02)[M+-20,6],355.2(9,48%)[M+-73.8], 325,2 (5,17%)[M+- 103,8], 281 (50,00%) [M+-148], 249 (12.06%)[M+-180], 207 (100%)[M+-222], 190 (95,68%)[M+-239],175(26,72%)[M+- 254] , 148(22,41%) [M+-281], 121(58,62%) [M+-308],81,2(60,34%)[M+-347,8], 53,1(9,48%) [M+-375,9].Thebasepeak of 207.1 is characteristic for benzophenanthridine alkaloids already described in the Euphorbiaceae family4,5. ThemolecularformulaC22H23NO8correspondstoacomponentderivedfromangoline6.1. LarsonR.1988.Phytochemistry27969-9782. Torrico,F.,etal.2003.RevistaFacultaddeFarmacia.6658-66.3. Salatinoetal.,2007.Journal.Braz.Chem.Soc.,18,No.1,11-334. Alves,etal.1999.TetrahedronLetters40(2):205-208.5. MurilloJ.2000.AcademiaColombianadeCienciasExactas,FísicasyNaturales24.359-370.6. FonzesL,etal.1968.Phytochemistry71889-1890.


245

ESSENTIALOILSFROMCITRUSSINENSIS,CYMBOPOGONCITRATUS,PINUSSYLVESTRISANDLIPPIA

ORIGANOIDESEXHIBITLARVICIDEACTIVITYAGAINSTRHIPICEPHALUSSANGUINEUS

BravoM.1,UzcáteguiJ.1,SanabriaM.2,GarcíaL.3,RojasL.4,GualtieriM.3

1UnidaddeInvestigaciónenParasitologíaVeterinaria.DecanatodeCienciasVeterinarias.UniversidadCentroccidental“LisandroAlvarado”.Barquisimeto.Venezuela;2DecanatodeAgronomía.PosgradodeFitopatología.UniversidadCentroccidental“LisandroAlvarado”. Barquisimeto. Venezuela; 3Laboratorio de Investigación de Medicamentos Orgánicos. Facultad de Farmacia yBioanalisis. Universidad de Los Andes. Merida. Venezuela. 4Instituto de Investigaciones. Facultad de Farmacia y Bioanalisis.UniversidaddeLosAndes.Mérida,Venezuela.

email:[email protected] sanguineus (Acari: Ixodidae)or "dog tick" is anhematophagousectoparasite arthropodactingas vectorofseveral pathogenswhich can parasitize non only domestic andwild animals, but also humans [1]. Essential oils (EO) are"bioactive" molecules obtained from a number of plant species, which exhibit various properties, such as: fungistatic,bactericidal,analgesic, contraceptive,antiaflatoxigénic, insecticide,antioxidantandcytotoxic [2].Citrus sinensisor"sweetorange"(Rutaceae)contains,initsfruitexocarp,essentialoilscharacterizedbythepresenceoflimonene,myrcene,α-pinenesabineno and [3,4]. Cymbopogon citratus or "lemongrass" (Poaceae) has aromatic leaves containg amont the maincomponents, geraniol and citronellol [5]. Lippia origanoides or "oregano" (Verbenaceae), whose essential oil is obtainedfromtheleaves,containsmainlycarvacrol,α-cymene,γ-terpineneandthymol[6,7].Pinussylvestris(Pinaceae)isatreethathas essential oils in their aerial parts, containing mainly cariofileno γ-terpinene, α-terpinene, camphene, terpinolene, β-pinene,limonene,borneolandcadinene[8].EOwereobtainedbyhydrodistillationusingaClevengertrap.Yield:Citrussinensis2.1%,Cymbopogoncitratus0.68%,Lippiaoriganoides 0.42%, Pinus Sylvestri 0.17%, were dried over anhydrous Na2SO4, stored at 4 ° C and analyzed by gaschromatography-massspectrometry(GC-MS)[9].Thelarvicidalactivitywasdeterminedbythelarvalimmersiontest(LIT)ofengorgedR.sanguineusadultfemalesforeggcollection.Larvae(100-120)wereplacedinPetridisheswith2mLofeachEOdissolved in acetone at concentrations of 5%, 2.5%, 2%, 1.25%, 1%, 0.5%, 0.25%, 0.12%, 0.625%, 0.312%, 0.156% 0.06%. The totalnumberofdeadlarvae[10]wasrecorded.ResultsrevealedamortalityR.sanguineus larvaefrom100%to82%attheconcentrationstestedofEOfromPinussylvestris,CymbopogoncitratusandLippiaoriganoides.Bycontrast,EOfromCitrussinensisyieldedresultsof100%and58.33%mortalityat concentrations of 5% and 2.5%, respectively. In conclusion, results show the larvicidal activity of these essential oils,representingpossiblealternativetocontrolthisectoparasite.1 LatreilleP.1806.ParisiisetArgentorati,apudAmandKoenig.Paris,302pp.2 StanojevićJ,L.Stanojević,D.Cvetković,B.Danilović.2015.Advancedtechnologies.4(2):19-25.3 AzarP,M.Nekoei,K.Larijani,S.Bahraminasab.2011.J.Serb.Chem.Soc.76(12):1627–1637.4 AraújoC,C.daCamara,I.Neves,N.Ribeiro,C.Gomes,M.deMoraes,P.Botelho.2010.NatProdCommun.5(3):471-6.5 WangX,Q.Hao,Y.Chen,S.Jiang,Q.Yang,Q.Li.2015.JInsectSci.7;15.116.6 HenaoJ,L.Muñoz,L.Padilla,E.Ríos.2010.Rev.Invest.Univ.Quindío(21):82-86.7 ArangoO,A.Hurtado,I.Toro.2012.ActaAgronómica.61(3):207-213.8 KupcinskieneE,A.Stikliene,A.Judzentiene.2008.EnvironmentalPollution.155(3):481–491.9 BicchiS.1987.Capillarygaschromatographyinessentialoilanalysis.Huethig,Heidelberg.10 ShawR.(1965).Entomol.Res.56:389-405.


246

SECONDARYMETABOLITESFROMCHILEANPLANTSANDTHEIRBIOLOGICALACTIVITY

AlarcónJE. ,CespedesCL,MuñozE,QuirozS.UniversityofBio-Bio,Chillan-Chile

emal:[email protected],[email protected]

Plants of the Celastraceae and Rhamnaceae that growth in Chile have been studies. These plants have very interestingmetaboliteswithepoxyeudesmane(agarofurane),benzylisoquinolinic,aporphinicandcyclopeptideskeleton.Westudythebiologicalactivityassociateatthesecompounds.Air dried and powdered material was exhaustively extracted with MeOH. The resulting MeOH extract was filtered andconcentrated in vacuum. The residue was suspended in H2O and the sequentially extracted with solvent of increasingpolarity. The resulting fraction were concentrated and chromatographed over silica gel column. Column fraction wereanalyzed by TLC (silica gel 60 F254), and fraction with similar TLC patterns were combined to give pure compounds. AhyphenatedprocedurecombiningHPLC-MS-NMRallowsthedeterminationofchemicalstructuresoftheisolates.Fromtheextracts,fractionsandpurecompoundsforinhibitorycapacityofacetylcholinoesteraseenzymeanditsinsecticidalactivityagainstCydiapomonella,Tenebriomolitorandotherinsectpestswereevaluated.Results show that the extracts, fractions and pure compounds obtained from Chilean plants of the Celastraceae andRhamnaceaefamilypresentaninterestinginsecticidalactivityandinhibitoryactivityofacetylcholinoesteraseenzyme.Acknowledgments:ToFondecytprogramgrant#1130463.Theauthorswishtothankto internalgrantfromDireccionde InvestigacionUniversidaddelBio-BioDIUBB#083009-2R,#122509and#132209GI/C,UniversidaddelBioBio,Chillan,Chile.1. Alarcón J,et al.DihydroagarofuranoidSesquiterpenesasAcetylcholinesterase Inhibitors fromCelastraceaePlants:MaytenusdistichaandEuonymus

japonicas.JAgricFoodChem2015,63:10250-10256.2. AlarcónJ.andCespedesCL.ChemicalconstituentsandbiologicalactivitiesofSouthAmericanRhamnaceae.PhytochemRev2015,14:389–401.


247

ETHANOLCRUDEEXTRACTFROMLEAVESOFSINNINGIASCHIFFNERI(GESNERIACEAE)ANDTHEIR

FRACTIONSASNATURALPHOTOSENSITIZERINPHOTODYNAMICCHEMOTHERAPY

SerainAF.1 ,AndreazzaNL.1,StefanelloMEA.2,SalvadorMJ.1 ¹UniversidadeEstadualdeCampinas(UNICAMP),InstitutodeBiologia,DepartamentodeBiologiaVegetal,PPGBTPB,Campinas

(SP)-Brasil;²UniversidadeFederaldoParaná(UFPR),DepartamentodeQuimicaOrgânica,Curitiba(PR)-Brasilemail:[email protected],[email protected]

ThePhotodynamicchemotherapy(PDT)hasbeenusedformanypurposes,suchasantitumor,performingasanon-invasivetherapyandmoderateorminimalsideeffects.Photosensitizersareagentsthat,underilluminationatacertainwavelengthand inthepresenceofoxygen,producereactiveoxygenspeciesthat interactwithbiologicalsystemsandarecytotoxictothe target cells (Brown et al., 2004; Andreazza et al., 2015). TheSinningia schiffneri belong to Gesneriaceae family whatcomprisesaround150generaand3500species,distributedintropicsandsubtropicsaroundtheworld.InBrazil,thefamilyisrepresentedby27generaand211species,beingSinningia themost importantgenus.PhytochemicalstudieswithSinningiareportedtheidentificationofphenolicglycosides,anthocyaninsandanthraquinoneswithbiologicalproperties(Scharfetal.,2016;Verdanetal.,2015). It isknownthatanthraquinoneashypericinarenaturalphotosensitizerswithapplication inPDT(Wainwright,2009).Thispromptedusto investigatethepotentialofextractsfromS.schiffneri foremploymentasnaturalphotosensitizerinPDTagainsthumanmelanomacellline. The leaves of S. schiffneri was dried at 40° C and the powder subjected to themaceration procedurewith ethanol, andpartitionedwith organic solvents in order of increasing polarity (hexane, dichloromethane, ethyl acetate and n-butanol).Then,wasdeterminedtheabsorptionprofileofthecrudeextractandofthefractions inthevisibleregionof interestPDT(400-850 nm). The production of singlet oxygen was determined using the method of the photodecomposition of 1,3-diphenylisobenzofuran, 1,3-DPBF (Andreazzaetal., 2015). Theantiproliferativeactivityof thecrudeextract againsthumanmelanomacelllines(UACC62)wasassessedcolorimetricallybythereductionofatetrazoliumsalt(MTT).Absorbanceswereexpressedaspercentagesrelativetountreatedcontrols.Ascontrol,wasusedthechemotherapeuticDoxorubicin(positivecontrol),thediluent(negativecontrol)andthemethyleneblue(positivecontrolofthephotosensitizer),andanon-irradiatedplate.Thebioassayswerecarriedoutintriplicate.Oneplateineachassaywasirradiatedwhiletheotherwasnotirradiated,theantiproliferativeeffectwasinvestigated,anddataanalyzedbytheTukeytest.ThechemicalcompositionoftheextractwasdeterminedbyESI-MS/MSandHPLC-MS/UV-DADtechniques.The crude ethanol extract from the leaves ofS. schiffineri and their hexane, dichloromethane and ethyl acetate fractionswere obtained and showed absorption from 600 to 700nm. Also, singlet molecular oxygen (1O2) production (type IIphotosensitizationreaction)wasexamined,andtheresultsshowthat1,3-DPBFphotodegradationwasgreatlyenhancedinthepresenceofethanolcrudeextractofS.schiffineriandtheirhexaneanddichloromethanefractionsindicatingproductionofsingletoxygen.Laserirradiationaloneat660nmusingdiodelaser,outputpowerof35mW,andenergyof28J/cm2,ornon-irradiated crude extracts in sub-inhibitory concentration did not reduce the cell viability significantly, whereas irradiatedethanolextractofS.schiffneri,insub-inhibitoryconcentrations,exhibitedantiproliferativeeffectagainstUACC62celllines.Itis suggested the presence of substances with photosensitizing potential in bioactive samples. Naphthoquinones,anthraquinones and phenolic compounds were identified in the bioactive extract by ESI-MS/MS and HPLC-MS/UV-DADanalysis.Despitethepositiveresults,furtherinvestigationsarenecessarytoconfirmthepotentialofthisnaturalproductasphotosensitizerinPDT.Acknowledgments:FinancialSupportfromFapesp,Faepex-Unicamp,CapesandCNPq1. BrownSB,etal.Thepresentandfutureroleofphotodynamictherapyincancertreatment.LancetOncol.,2004,5:497-508.2. WainwrightM.Photosensitisersinbiomedicine.GreatBritain,Chippenham,Wiltshire,UK:JohnWiley&SonsLtda.2009,p.283.3. ScharfDR.etal.NaphthochromenesandRelatedConstituentsfromtheTubersofSinningiaallagophylla.J.Nat.Prod.,2016,79:1-6.4. VerdanMH.etal.ANewNaphthoquinonefromSinningialeucotricha(Gesneriaceae).Nat.Prod.Communications,2015,10:625-626.5. AndreazzaNL.etal.Photodynamicantimicrobialeffectsofbis-indolealkaloidindigofromIndigoferatruxillensisKunth(Leguminosae).LasersMed.Sci.,

2015,30:1315-1324.


248

THESYNERGISTICEFFECTSOFANTIOXIDANTSINTHECONTROLOFREDOXBALANCEINCULTUREDFIBROBLASTSANDPOSSIBLEINFLUENCEONPATHOLOGICCALCIFICATION

BoraldiF. ,CostaS.,AnnoviG.

DepartmentofLifeSciences,UniversityofModenaandReggioEmilia,Modenaemail:[email protected]

Inthelastdecadeslifespanisprogressivelyincreased,but,atthesametime,higherincidenceofage-associatedpathologicconditions as cancer, cardiovascular diseases, diabetes and Alzheimer were also observed [1]. Both genetic andenvironmentalfactors,includingdietaryhabits,cancontributetomodulatethesusceptibilityofindividualstotheoccurrenceandseverityofage-relateddiseases[2].Reactiveoxygenspecies(ROS),includingthosephysiologicallyproducedbycellularmetabolism,areknowntoaccumulatewithaging,thuscontributingtodamagenucleicacids,lipidsandproteinsandtherebyalteringtheirfunctions[3-4].Thisobservationsuggestedthatantioxidantsupplementationcanrestoreredoxbalance,andtherefore great attention is paid to increase the antioxidant properties of dietary products eventually adding antioxidantpolyphenols, vitamins, long-chainunsaturated fatty acids and carotenoids [2].However, there is a largebodyof evidencedemonstratingthatROScanalsoactassignallingmolecules,thusposingthequestionwhethercellularhomeostasiscanbeefficiently improved by a combination of antioxidants either in physiologic or in pathologic conditions. Moreover, theobservation that ectopic calcification, a frequent complication of age-related disease (namely atherosclerosis, diabetes,uremia),isassociatedtoincreasedoxidativestress,suggestedthatantioxidantsmayinterferewiththecalcificationprocess[5-7].The present study investigates in human dermal fibroblasts cultured from healthy adult individuals if a combination ofantioxidantsatlowandhighconcentrations(Table)canmodulatetheexpressionofdifferentparametersofredoxbalance[hydrogen peroxide, anion superoxide, lipid peroxidation (LPO), proteincarbonylationandtotalantioxidantstatus(TAS)].Furthermore,fibroblastswerealsoculturedinvitroinacalcifyingmediumandalkalinephosphataseactivityaswellasmineraldepositionwereevaluated.Results indicate that: a) reduction of ROS was observed only with theantioxidantmixathighconcentration;b)LPOwasdecreasedbyantioxidantsatboth low and high concentrations; c) surprisingly, protein carbonylation appeared increased in cells cultured withantioxidants at high concentration, d) TAS was never increased by treatment; e) treatments determined an increase ofalkalinephosphataseactivityandofthenumberofcalcifiedareas.Tobenotedthathighdosesofantioxidantsforseveraldaysareassociatedtocelldeath,calcificationbeing,atleastinpart,theconsequenceofthenecroticprocess.Thesedataindicatethatacombinationofquercetin,kaempferolandvitaminsCandE,althoughcapabletomodifytheredoxbalance,arenotonly inefficient incounteractingectopiccalcification,butonthecontrary,seemtofavourthecalcificationprocess also at lowdoses in the absence of evident cytotoxic effects. Therefore, antioxidantmix exertsmany additionaleffects,besidethoseonredoxbalance,andthereforetheirusemayrequireacarefulevaluationespeciallywhenaregivenassupplements.Acknowledgments:worksupportedbyFondazioneCRMO(#2015-0306)1. KanasiEetal.,Theagingpopulation:demographicsandthebiologyofaging.Periodontol2000.2016,72:13-8.2. RiscutaG.NutrigenomicsattheInterfaceofAging,Lifespan,andCancerPrevention.JNutr.2016,inpress.3. KaushalK.etal.Stress,OxidativeInjuryandDisease.IndianJ.Clin.Biochem.2015,30:3–104. HouT.etal.Mitochondrialflashes:newinsightsintomitochondrialROSsignallingandbeyond.JPhysiol.2014,592:3703-13.5. Agharazii M. et al. Inflammatory cytokines and reactive oxygen species as mediators of chronic kidney disease-related vascular calcification. Am J

Hypertens.2015,28:746-55.6. YamadaS.etal.Theantioxidanttempolamelioratesarterialmedialcalcificationinuremicrats:importantroleofoxidativestressinthepathogenesisof

vascularcalcificationinchronickidneydisease.JBoneMinerRes.2012,27:474-85.7. Roman-GarciaP.etal.Naturalantioxidantsandvascularcalcification:apossiblebenefit.JNephrol.2011,24:669-72.


249

FLASHCHROMATOGRAPHYMS-TARGETEDISOLATIONOFNATURALPRODUCTSUNDERNORMALPHASECONDITIONS

RighiD. ,AntonioAzzolliniA.,EmersonF.QueirozEF.,WolfenderJL.

SchoolofPharmaceuticalSciences,EPGL,UniversityofGeneva,UniversityofLausanne,Geneva,Switzerlandemail:[email protected]

Reversedphaseliquidchromatography(RP-LC)iswidelyusedforthemetaboliteprofilingofcomplexnaturalextractsandisnowmoreandmoreusedfortargetedMSisolationofbiomarkers.Normalphasechromatography(NP-LC)iswellsuitedforthe purification of lipophilic secondary metabolites, also offering some advantages compared to RP, like low operatingpressures and cheaper stationary phases. The complementary usage of both of stationary phases andMS detection formetabolitepurificationat thepreparative scaleusinggeneric separationmethodshasbeen investigatedonan innovativeFlash chromatography system integrating MS and ELSD detection (PuriFlash®- MS). A mixture of representative naturalproduct standards was chosen and analysed under both normal phase/reverse phase conditions. All parameters werecarefully optimized for both separation and detection (gradient system, split rate, flow rate, temperature, inj. volume,column length, ionizationsourceparameters).Aspecialcarewas taken to findMS ionizationandsplittingconditions thatprovide good detection and preclude source contamination. The HPLC analytical gradient was transferred to flashchromatographyfollowingageometricgradienttransfermethodaftercalibrationofthechromatographicsystems.1MS, incomplementtoUVdetection,enabledthemonitoringofNPswithweakandstrongchromophoresandtheselectivityofMSwasofgreathelpforaprecisecollectionofpartiallyco-elutingcompounds.APCI-MSdetectionwithoptimizedsplittingandpost-columnelutionofappropriatesolventwasfoundrobustandwell-suitedforpurificationsinbothNPandRPmodes.Thisstrategy allows an efficient and rational targeted isolation of tens to hundreds mg of compounds for further structuralidentificationorbioactivitycharacterizationstudies.Acknowledgements:Interchim(Monluçon,France)forloanofinstrumentandtechnicalsupport

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