antioxidant, cytotoxic, and toxic activities of propolis

Research ArticleAntioxidant Cytotoxic and Toxic Activities ofPropolis from Two Native Bees in Brazil Scaptotrigona depilisandMelipona quadrifasciata anthidioides

Thaliny Bonamigo1 Jaqueline Ferreira Campos1

TamaehMonteiro Alfredo1 Joseacute Benedito Perrella Balestieri1

Claudia Andrea Lima Cardoso2 Edgar Julian Paredes-Gamero34

Kely de Picoli Souza1 and Edson Lucas dos Santos1

1School of Environmental and Biological Science Federal University of Grande Dourados Dourados MS Brazil2Course of Chemistry State University of Mato Grosso do Sul Dourados MS Brazil3Department of Biochemistry Federal University of Sao Paulo SP Brazil4Interdisciplinary Center of Biochemistry Investigation University of Mogi das Cruzes Mogi das Cruzes SP Brazil

Correspondence should be addressed to Edson Lucas dos Santos edsonsantosphdgmailcom

Received 10 November 2016 Revised 27 January 2017 Accepted 1 February 2017 Published 9 March 2017

Academic Editor Jasminka Giacometti

Copyright copy 2017 Thaliny Bonamigo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Propolis is a natural mixture of compounds produced by various bee species including stingless bees This compound has beenshown to exhibit antioxidant antiproliferative and antitumor activities The present study aimed to determine the chemicalconstituents as well as the antioxidant cytotoxic and toxic activities of ethanol extracts of propolis obtained from the stinglessbees Scaptotrigona depilis and Melipona quadrifasciata anthidioides which are found in Brazil Phytosterols terpenes phenoliccompounds and tocopherol were identified in the ethanol extracts of propolis (EEPs) in different concentrations The compoundsstigmasterol taraxasterol vanilic acid caffeic acid quercetin luteolin and apigenin were found only in EEP-M The EEPs wereable to scavenge the free radicals 22-diphenyl-1-picrylhydrazyl and 221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) andprotected human erythrocytes against lipid peroxidation with the latter effect being demonstrated by their antihemolytic activityand inhibition of malondialdehyde formation The EEPs showed cytotoxic activity against erythroleukemic cells and necrosis wasthe main mechanism of death observed In addition the concentrations at which the EEPs were cytotoxic were not toxic againstCaenorhabditis elegans In this context it is concluded that EEP-S and EEP-M show antioxidant and cytotoxic activities and arepromising bioactive mixtures for the control of diseases associated with oxidative stress and tumor cell proliferation

1 Introduction

Stingless bees also known as meliponini belong to the tribeMeliponini and are distributed across more than 32 genera[1] Most species in this group exhibit eusocial habits and arefound in tropical and subtropical regions and 244 specieshave been described in Brazil [2 3]

This group of bees plays an important ecological rolecontributing to the preservation of plant species throughpollination Moreover they produce pollen honey wax andpropolis which are used in the hive and are consumed by

humans as nutraceuticals [4ndash6] Among these compoundspropolis is obtained through the collection of exudates fromdifferent parts of plants and combined with salivary enzymesfrom bees resulting in a resinous material that is used torepair cracks and damage to the hive defend againstmicroor-ganisms and mummify the bodies of other insects [7 8]

Propolis is generally composed of 50 to 60 resins andbalsams 30 to 40 waxes 5 to 10 essential oils and5 pollen grains and micronutrients with small amounts ofvitamins B1 B2 B6 C and E [9] The color and chemicalcomposition of this resin vary depending on the plant species

HindawiOxidative Medicine and Cellular LongevityVolume 2017 Article ID 1038153 12 pageshttpsdoiorg10115520171038153

2 Oxidative Medicine and Cellular Longevity

from which bees collect the raw material and the bee speciesthat produces it [8]

Therefore studies on propolis collected from differentgeographical regions and bee species are of great importancebecause these elements affect the chemical composition andconsequently the biological properties of propolis

The therapeutic activity of propolis from stingless beeshas beenwidely investigated in recent decades including des-criptions of its antioxidant activity [10ndash12] antimicrobial acti-vity [5 13 14] anti-inflammatory activity [15 16] and anti-tumor activity [7 12 17]

Among bee species Scaptotrigona depilis popularlyknown as ldquomandaguarirdquo andMelipona quadrifasciata anthid-ioides known as ldquomandacaiardquo are stingless species foundin South American countries including Paraguay Argentinaand Brazil [1] and their genetic and behavioral characteristicshave been well described [18ndash21] However studies on thebiological activity of propolis in these species are scarce inthe literature particularly in view of the difficulty in findingcolonies in their natural environment where species are dis-appearing because of anthropogenic activity

The pharmacological properties of propolis in thesetwo species were evaluated by Velikova et al [13] whodescribed the antimicrobial activity of propolis extracts fromM q anthidioides and by Sawaya [10] who described theantioxidant activity of propolis extracts from S depilis In thiscontext the present study aimed to determine the chemicalconstituents as well as the antioxidant cytotoxic and toxicactivities of ethanol extracts of propolis from the stingless beespecies S depilis andM q anthidioides from the state ofMatoGrosso do Sul in Midwest Brazil

2 Materials and Methods

21 Research Ethics No specific permits were required for thedescribed field studies All field works to collect the propolissamples were conducted on private land and with ownerpermission The field studies did not involve endangered orprotected speciesTheprotocol to collect of humanperipheralblood was approved by the Research Ethics Committee(Comite de Etica em Pesquisa CEP) of the UniversityCenter of GrandeDourados (CentroUniversitario daGrandeDourados UNIGRAN) Brazil (CEP process number 12312)All subjects provided written informed consent for participa-tion

22 Preparation of the Ethanol Extract of Propolis (EEPs)Propolis samples from S depilis (8381 g) and M q anthid-ioides (3642 g) were collected from the state of Mato Grossodo Sul (22∘1310158401210158401015840Sndash54∘491015840210158401015840W) in the Midwest Region ofBrazil with a total of seven collections being performed foreach species The ethanol extract of propolis (EEPs) wasprepared using 45mL of 80 ethanol per 1 g of propolisThismixture was incubated in a water bath at 70∘C in a sealedcontainer until total dissolution and subsequently filtered infilter paper qualitative 80 gm2 (Prolab Sao Paulo Brazil) toobtain the EEPs of S depilis (EEP-S) and M q anthidioides(EEP-M) [22] After preparation of the extracts they werekept at a temperature of minus20∘C until analysis

23 Chemical Analysis

231 Preparation of the Samples The samples (1mg) wasfractionated with hexane and water in proportion 1 1 v vand fraction soluble in hexane was analyzed by GC-MS andfraction in water by HPLC

232 GC-MS Samples were injected and analyzed by gaschromatography-mass spectrometry (GC-MS) The GC-MSanalysis was performed on a gas chromatograph (GC-2010Plus Shimadzu Kyoto Japan) equipped with amass spectrom-eter detector (GC-MS Ultra 2010) using LM-5 (5 phenyldimethyl poly siloxane) capillary column (15m length times02mm id and 02 120583m film thickness) with initial oventemperature set at 150∘C and heating from 150∘C to 280∘C at15∘Cminminus1 and a hold at 280∘C for 15min The carrier gaswas helium (9999) supplied at a flow rate of 10mLminwith split ratio 1 20 1 120583L injection volumeThe injector tem-perature was 280∘C and the quadrupole detector temperaturewas 280∘C The MS scan parameters included an electron-impact ionization voltage of 70 eVmass range of 45ndash600119898119911and scan interval of 03 s The identifications were completedby comparing the mass spectra obtained in the NIST21and WILEY229 libraries In some cases the compound wasconfirmed by comparison of standards Standards of the stig-masterol120573-sitosterol120573-amyrin120572-amyrin120573-amyrin acetateand tocopherol (Sigma-Aldrich with purity ge97) were pre-pared in the concentration initial of 1000 120583gmLThe concen-trations of compoundswere determined by extern calibrationafter dilutions appropriated in the range of 01ndash50 120583gmLThequantification of taraxasterol was performed in relation tostigmasterol The procedure was performed in triplicate

233 HPLC The extracts were analyzed in an analyticalHPLC (LC-6AD Shimadzu Kyoto Japan) system with adiode array detector (DAD) monitored at 120582 = 200ndash600 nmTheHPLC column was a C-18 (25 cm times 46mm particle size5 120583m Luna Phenomenex Torrance CA USA) with a smallprecolumn (25 cm times 3mm) containing the same packingused to protect the analytical column In each analysis theflow rate and the injected volume were set as 10mLminminus1and 20 120583L respectively All chromatographic analyses wereperformed at 22∘C Elution was carried out using an binarymobile phase of water with 6 acetic acid and 2mM sodiumacetate (eluent A) and acetonitrile (eluent B) The followingapplied gradients are as follows 5B (0min) 15B (30min)50B (35min) and 100B (45min) Standards of the vanilicacid caffeic acid ferulic acid p-coumaric acid benzoic acidcinnamic acid quercetin luteolin apigenin and vanillin(Sigma-Aldrich ge97) were prepared in the concentrationinitial of 1000120583gmLThe concentrations of compoundsweredetermined by extern calibration after dilutions appropriatedin the range of 001ndash10 120583gmLThe procedure was performedin triplicate

24 Antioxidant Activity

241 DPPH Free Radical Scavenging Activity The freeradical-scavenger activity was determined by the DPPH

Oxidative Medicine and Cellular Longevity 3

(22-diphenyl-1-picrylhydrazyl) assay as described previouslyby D Gupta and R K Gupta [23] with some modificationsThe antiradical activity of extracts was evaluated using adilution series in order to obtain a large spectrum of sampleconcentrations This involved the mixing of 18mL of DPPHsolution (011mMDPPH in 80 ethanol) with 02mLof EEP-S or EEP-M (1ndash300 120583gmL) followed by homogenizationAfter 30min quantification of the remaining DPPH radicalswas recorded by using absorption set at 517 nm Ascorbicacid and butylated hydroxytoluene (BHT) were used asreference antioxidantsThe tests were performed in duplicatein 2 independent experiments DPPH solution without thetested sample was used as control The percentage inhibitionwas calculated from the control with the following equa-tion

Scavenging activity () = (1 minus Abs sampleAbs control

) times 100 (1)

242 ABTS Free Radical Scavenging Activity Free radicalscavenging capacity for EEP was studied as described byRe et al [24] through the evaluation of the free radicalscavenging effect on 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicalThe stock solutions included7mM ABTS solution and 140mM potassium persulfatesolution The ABTS∙+ radical was then prepared by mixingthe two stock solutions (5mL of ABTS solution and 88120583Lpotassium persulfate solution) and left for 12ndash16 h at roomtemperature in the dark The solution was then diluted bymixing 1mL ABTS∙+ radical with ethanol absolute to obtainan absorbance of 070 nm plusmn 005 units at 734 nm usinga spectrophotometer Then 20120583L of EEP-S or EEP-M (1ndash300 120583gmL) was mixed with 1980120583L of the ABTS∙+ radicaland the absorbance was taken at 734 nm after 6min usinga spectrophotometer Ascorbic acid and butylated hydroxy-toluene (BHT) were used as positive controlsThree indepen-dent experiments were performed in triplicate The percent-age of inhibition of theABTS radical was calculated accordingto the following equation where Abscontrol is the absorb-ance of ABTS∙+ radical without the tested sample

inhibition of ABTS

= ((Abscontrol minus Abssample)Abscontrol

) times 100 (2)

243 Antioxidant Assay Using the Human Erythrocyte Model

(1) Preparation of Erythrocyte Suspensions Following ap-proval by the Research Ethics Committee 20mL of periph-eral blood was collected from healthy donors into sodiumcitrate-containing tubes and was subsequently centrifuged at1500 rpm for 10min After centrifugation the blood plasmaand leukocyte layers were discarded and the erythrocyteswere washed 3 times with saline solution and centrifugedat 1500 rpm for 10min Finally 10 erythrocyte suspensions

were prepared in saline solution to obtain 25 after thetreatment

(2) Oxidative Hemolysis Inhibition AssayThe protective effectof the propolis extracts was evaluated according to themethod described by Campos et al [12] with minor modi-fications The assays were conducted with erythrocyte sus-pensions The erythrocytes were preincubated at 37∘C for30min in the presence of different concentrations of EEPor ascorbic acid (50ndash125 120583gmL) Then 50mM 221015840-azobis-(2-amidinopropane) dihydrochloride (AAPH) solution wasadded A sample of 1 ethanol was used as a negative controlTotal hemolysis was induced by incubating erythrocyteswith distilled water Basal hemolysis caused by EEP wasassessed by incubating erythrocytes with the extract withoutthe presence of AAPH and the control was assessed inerythrocytes incubated only with 09 NaCl This mixturewas incubated at 37∘C for 240 min with periodical stirringHemolysis was determined after every 120 180 and of 240minutes of incubation specifically sample were centrifugedat 1500 rpm for 10min and aliquots of there were transferredto tubes with saline after which the absorbance of the super-natant was read spectrophotometrically at 540 nm Thepercentage hemolysis was measured with the formula 119860119861 times100 where (119860) is the sample absorbance and (119861) is the totalhemolysis Five independent experiments were performed induplicate

(3) Inhibitory Efficiency against Lipid Peroxidation A 25erythrocyte suspension was used to assess the protectiveeffects of EEP against lipid peroxidation as described byCampos et al [12] with some modifications Erythrocyteswere preincubated at 37∘C for 30min with different concen-trations of EEPs or ascorbic acid (50ndash125120583gmL) A sampleof 1 ethanol was used as a negative control Next 50mMAAPHwas added to the erythrocyte solution whichwas thenincubated at 37∘C for 4 hours with periodical stirring At120 180 and 240 minutes of incubation the samples werecentrifuged at 1500 rpm for 10min and 500120583L aliquots of thesupernatant were transferred to tubes with 1mL of 10 nmolthiobarbituric acid (TBA) As a standard control 500120583L of20mMmalondialdehyde (MDA) solution was added to 1mLof TBA The samples were incubated at 96∘C for 45min Thesamples were then cooled 4mL of n-butyl alcohol was addedand the samples were centrifuged at 3000 rpm for 5min Theabsorbance of supernatants sample was read at 532 nmThreeindependent experiments were performed in duplicateMDAlevels in the samples were expressed in nmolmL obtainedwith the following formula

MDA = Abs sample times (20 times 22032Abs standard

) (3)

25 Cytotoxic Activity and Cell Death Profile K562 erythro-leukemia cells line was grown is suspension in RPMI 1640media (Cultilab Campinas Sao Paulo Brazil) supplementedwith 10 fetal bovine serum (FBS Cultilab) 100UmL ofpenicillin and 100 120583gmL of streptomycin in a humidifiedatmosphere at 37∘C in 5 CO

2 The cytotoxic activity and


cell death profile were evaluated according to the methoddescribed by Paredes-Gamero et al [25] with minor mod-ifications Cells were seeded into 96-well plates (2 times 104cellwell) and cultured in medium with 10 FBS in theabsence or presence of EEP-S or EEP-M (31ndash500120583gmL) for24 h As negative controls were used cells were incubatedwith02 ethanol (highest concentration of ethanol in extract)All effects of the EEPs were compared with negative controlsAfter this period the K562 cells were washed with PBSand resuspended in annexin-labeling buffer (001M HEPESpH 74 014M NaCl and 25mM CaCl

2) The suspensions

were stained with annexin-FITC and propidium iodide (PI)(Becton Dickinson Franklin Lakes NJ USA) according tothe manufacturersquos instructions The cells were incubated atroom temperature for 15min Three thousand events werecollected per sample and the analyses were performed ona FACSCalibur flow cytometer (Becton Dickinson) withCellQuest software (Becton Dickinson)

26 In Vivo Toxicity

261 Rearing and Maintenance of Caenorhabditis elegansTo perform the in vivo toxicity assay we used the wild-type N2 strain of the nematode Caenorhabditis elegans Thespecimens were incubated at 20∘C in Petri dishes contain-ing nematode growth medium (NGM) agar and fed withEscherichia coli strain OP50-1 The nematode culture wassynchronized through treatment of pregnant hermaphroditeswith 2 sodium hypochlorite and 5M sodium hydroxide

262 Assessment of Toxicity in C elegans A toxicity assayfor the EEPs was performed in C elegans [26] in 96-wellplates Each well contained 10 nematodes at the L4 stagewhich were incubated for 24 hours at 20∘C with EEP-S andEEP-M at different concentrations (250ndash1000120583gmL) in M9medium After this period nematode viability was evaluatedby repeatedly touching the worms with a microspatula Forthe manipulation and examination of nematodes a modelMotic SMZ-140 amp W10X23 (British Columbia Canada)stereomicroscope was used The data were calculated fromtwo independent experiments in duplicate

27 Statistical Analyses All data are shown as the mean plusmnstandard error of mean (SEM) and for statistical significantdifferences between the groups using the analysis of variance(ANOVA) and posttest Dunnett comparing the treatmentwith the control using the Prism 6 GraphPad software Theresults were considered significant when 119901 lt 0053 Results

31 Chemical Composition The compounds identified inEEP-S and EEP-M are shown in Tables 1 and 2 Phytosterolsterpenes phenolic compounds and tocopherol were identi-fied in the two extracts in different concentrations EEP-Spresented a higher content of amyrins (triterpenes) and 120573-sitosterol (phytosterols) whereas EEP-M exhibited a higherconcentrations of tocopherol amyrins and apigenin (flavo-noid)The compounds stigmasterol taraxasterol vanilic acid

caffeic acid quercetin luteolin and apigenin were found onlyin EEP-M


321 DPPHandABTS Free Radical Scavenging Activity EEP-S and EEP-M were observed to scavenge free radicals invitro In both of the evaluatedmethods EEP-M showed betterantioxidant activity compared with EEP-S In the DPPHassay EEP-M showed 50 inhibition of free radicals (IC

50)

at a concentration of 6091 plusmn 201 120583gmL The IC50

was notcalculated for EEP-S The maximum activity of EEP-M wasachieved at a concentration of 300 120583gmL (Table 3)

In the assay with the ABTS radical IC50

values ofthe EEPs were 8004 plusmn 031 120583gmL (EEP-S) and 1345 plusmn181 120583gmL (EEP-M) and they showed maximal activity atconcentrations of 200120583gmL and 100 120583gmL respectivelyThe antioxidant activity of EEP-M was similar to that of thesynthetic antioxidant BHT (Table 3)

322 Oxidative Hemolysis Inhibition Assay The standardantioxidant ascorbic acid and the EEPs showed concentra-tion- and time-dependent antihemolytic activity EEP-Sdecreased hemolysis for 120min with hemolysis inhibitionreaching 635 plusmn 107 at a concentration of 125 120583gmL whencompared with the AAPH sample At the same concen-tration ascorbic acid and EEP-M protected erythrocytesagainst hemolysis induced by the oxidant 221015840-azobis(2-aminopropane) hydrochloride (AAPH) for up to 240minwith hemolysis inhibition reaching 565 plusmn 128 and 377 plusmn104 at 240min respectively compared with erythrocytestreated with AAPH (Figure 1) At the various concentrationstested the basal hemolysis observed using ascorbic acid andEEPs without the AAPH inducer was similar to the controltreatments with saline and ethanol (data not shown)

323 Efficiency of EEPs in the Inhibition of AAPH-InducedLipid Peroxidation The effectiveness of EEPs in inhibitinglipid peroxidation induced by AAPH in human erythrocytescan be determined by measuring malondialdehyde (MDA)levels Ascorbic acid and EEPs decreased MDA levels in aconcentration- and time-dependentmanner EEP-S inhibitedlipid peroxidation for 180min The ascorbic acid controlsolution inhibited lipid peroxidation by 656 plusmn 89 whereasEEP-M inhibited peroxidation by 744 plusmn 61 for 240min at aconcentration of 125120583gmL when compared with the AAPHsample (Figure 2)

33 Cytotoxic Activity and Cell Death Profile The ethanolextracts of propolis showed concentration-dependent cyto-toxicity At the highest concentration tested (500120583gmL)the cell growth of erythroleukemic cells (K562) were 326 plusmn32 (EEP-S) and 212 plusmn 41 (EEP-M) (Figure 3) At thisconcentration after 24 h of treatment EEP-S promoted deathby necrosis in 529 plusmn 41 of cells and death by late apoptosisin 121 plusmn 06 of cells (Figures 4(a) and 4(b)) whereas EEP-M promoted death by necrosis in 575 plusmn 38 of cells anddeath by late apoptosis in 194 plusmn 16 of cells (Figures 5(a) and5(b))


Table 1 Compounds identified in unpolar fraction of the EEPs from Scaptotrigona depilis andMelipona quadrifasciata anthidioides by GC-MS

Peak Retention time (min) Compounds Molecular mass EEP-S (mgg) EEP-M (mgg)1 1702 Stigmasterollowast 412 mdash 48 plusmn 012 1772 120573-Sitosterollowast 414 96 plusmn 02 54 plusmn 023 1793 120573-Amyrinlowast 426 143 plusmn 03 116 plusmn 034 1809 Taraxasterol 426 mdash 30 plusmn 015 1845 120572-Amyrinlowast 426 105 plusmn 03 50 plusmn 016 1965 120573-Amyrin acetatelowast 468 215 plusmn 04 137 plusmn 047 2456 Tocopherollowast 430 36 plusmn 01 150 plusmn 05lowastCompound was confirmed by comparison of standardData are shown as media plusmn standard deviation

Table 2 Compounds identified in polar fraction of the EEPs from Scaptotrigona depilis andMelipona quadrifasciata anthidioides by HPLC

Peak Retention time (min) Compounds Molecular mass EEP-S (mgg) EEP-M (mgg)1 795 Vanilic acid 168 mdash 59 plusmn 012 864 Caffeic acid 180 mdash 61 plusmn 023 1044 Vanillin 152 55 plusmn 02 57 plusmn 014 1348 p-Coumaric acid 164 63 plusmn 02 61 plusmn 025 1728 Ferulic acid 194 54 plusmn 02 61 plusmn 026 1999 Benzoic acid 122 68 plusmn 02 69 plusmn 017 3533 Quercetin 302 mdash 99 plusmn 028 3668 Luteolin 286 mdash 13 plusmn 019 4001 Cinnamic acid 148 134 plusmn 04 132 plusmn 0310 4262 Apigenin 270 mdash 156 plusmn 04lowastCompound was confirmed by comparison of standardData are shown as media plusmn standard deviation

34 Toxicity in C elegans EEP-S and EEP-M were not toxicto the nematodes after 24 h of incubation at any of theconcentrations evaluated compared with the control group(Figure 6)

4 Discussion

Propolis is a bee product that is widely used in the cosmeticsand food industries and is considered a functional food(nutraceutical) able to prevent diseases when included infood products [5] The chemical constituents present inpropolis are responsible for its therapeutic properties [7 1127] including its antibacterial antifungal and antiviral activ-ities [5 14] as well as its anti-inflammatory and antitumoractivities [15 16 28 29]

The major compounds identified in the EEP-S were 120573-amyrin 120573-amyrin acetate and 120572-amyrin and in the EEP-M were tocopherol 120573-amyrin acetate and apigenin Bothextracts show similar amounts of 120573-amyrin vanillin p-coumaric acid ferulic acid cinnamic acid and benzoic acidhowever the EEP-S showed higher content of amyrins thanEEP-M By contrast EEP-M exhibited approximately fourtimes the amount of tocopherol found in EEP-S and othercompounds which were found exclusively on the EEP-MDespite presenting the same chemical constituents variationsin the concentrations of these compounds may influence thebiological activities of the extracts

The compounds phenolic and flavonoid are correlatedwith the antioxidant and antitumor activity of propolis [810ndash12 30] Additionally other compounds identified in thepropolis such as caffeic acid apigenin and triterpenes aredescript with important blockers of oncogenic kinase PAK1well known to be responsible for a variety of diseases such asinfectious diseases Alzheimerrsquos disease diseases inflamma-tory diabetes hypertension obesity and cancer [31]

Phenolic compounds and terpenes have been found inpropolis extracts of other species of stingless bees from thesame geographical region [12 29] which may be related tothe plant species from which the bees collect raw materialsfor propolis production

The terpenes and phenolic compounds found in EEPshave been described as compounds responsible for the anti-oxidant activities of various plant species [32ndash34] Antioxi-dants are compounds that when present at low concentra-tions retard or prevent the oxidation of substrates and arehighly beneficial to health due to protecting cells and macro-molecules from oxidizing agents [35]

The most common oxidants in the body include thesuperoxide (O

2

minus) hydroxyl (OH∙) peroxyl (ROO) alkoxyl(RO) and hydroperoxyl (HO

2) radicals which are collec-

tively known as reactive oxygen species (ROS) These freeradicals are produced via gradual reduction ofmolecular oxy-gen and generate unpaired electrons which cause oxidativestress when they are out of equilibrium [36]


Table 3 IC50and maximum DPPH and ABTS radical scavenging activity of standard antioxidants EEP-S and EEP-M

SampleDPPH ABTS

IC50(120583gmL) Maximum inhibition IC

50(120583gmL) Maximum inhibition

120583gmL 120583gmLAscorbic acid 332 plusmn 065 9675 plusmn 041 50 250 plusmn 048 9737 plusmn 155 10BHT 2284 plusmn 787 8936 plusmn 230 200 2046 plusmn 278 9536 plusmn 180 100EEP-S ND 1491 plusmn 173 300 8004 plusmn 031 7342 plusmn 347 200EEP-M 6091 plusmn 201 9747 plusmn 003 300 1345 plusmn 181 9931 plusmn 012 100Values are means plusmn SEM DPPH (119899 = 2) and ABTS (119899 = 3) ND not determined

Control 50 75 100 125 50 75 100 125 50 75 100 125

AAPHEEP-MEEP-SAscorbic acid

lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

0

10

20

30

40

50

Hem

olys

is (

)

(a)

0

10

20

30

40

50

Control 50 75 100 125 50 75 100 125 50 75 100 125


Hem

olys

is (

)

lowast

lowast

lowast

lowast

lowast

lowastlowast

(b)

0

10

20

30

40

50

Hem

olys

is (

)

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowast lowast

lowast

(c)

Figure 1 Protective effects of ascorbic acid (standard antioxidant) and ethanol extracts of propolis from S depilis and M q anthidioidesagainst AAPH-induced hemolysis determined using a human erythrocyte suspension at 120min (a) 180min (b) and 240min (c) Ethanolwas employed as a negative control The results are expressed as the mean plusmn SEM (standard error of the mean) 119899 = 5 lowastSignificantly different(119901 lt 005) compared with the AAPH group

Both EEPs stabilized the free radicals 22-diphenyl-1-picrylhydrazyl (DPPH) and 221015840-azino-bis(3-ethylbenzothia-zoline-6-sulfonic acid) (ABTS) However EEP-M showedhigher antioxidant activity than EEP-S which may be relatedto the different concentrations of tocopherol in the extractsSome studies have reported the importance of tocopherols asantioxidants [37 38]

In addition the amyrins may be associated with the anti-oxidant activity of the extracts Tocopherols and these triter-penes are fat-soluble antioxidants that scavenge ROS [34 3940] These compounds may have been responsible for theincreased antioxidant activity of the EEPs observed in theassay with the free radical ABTS as this method is appliedto hydrophilic and lipophilic antioxidant systems [41]

Therefore the higher solubility of these compounds in thesolvent used in this assay produced greater antioxidantactivity

These results corroborate those obtained in the assaysinvolving the inhibition of lipid peroxidation in whichthe EEPs presented antihemolytic activity and protectiveactivity against lipid peroxidation when incubated withhuman erythrocytes in the presence of an oxidizing agentEEPs may also inhibit the peroxyl radical (ROO) whichinduces peroxidation of lipids and proteins present in humanerythrocyte membranes [42]

Oxidative stress leads to lipid peroxidation and conse-quently cell damage due to the oxidation of essential cellularcompounds including lipids proteins and nucleic acids An


0

500

1000

1500

2000

2500

3000

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowast

lowast

lowastlowast

lowast

lowast lowast lowastlowast

MD

A (n

mol

ml)

(a)

0

500

1000

1500

2000

2500

3000

lowastlowastlowast

lowast

lowast lowastlowast

lowastlowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(b)

0

500

1000

1500

2000

2500

3000

lowastlowast

lowastlowast

lowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(c)

Figure 2 Protective effects of ascorbic acid (standard antioxidant) and ethanol extracts of propolis from S depilis and M q anthidioidesagainst the production of malondialdehyde (MDA)ndasha byproduct of lipid peroxidationndashin a human erythrocyte suspension at 120min (a)180min (b) and 240min (c) Ethanol was used as a negative control The results are expressed as the mean plusmn SEM (standard error of themean) 119899 = 3 lowastSignificantly different (119901 lt 005) compared with the AAPH group

0 31 62 125 250 500 31 62 125 250 500EEP-S EEP-M

0

20

40

60

80

100

Cel

l via

bilit

y (

)

lowast

lowast

lowast

lowastlowastlowastlowast

Figure 3 Cytotoxic activity of EEPs from S depilis (EEP-S) andMq anthidioides (EEP-M) against the K562 erythroleukemia cell linelowast119901 lt 005 for the treated group versus control viable cells

excess of these free radicals can promote cell aging and thedevelopment of various diseases including Alzheimerrsquos can-cer arthritis and diabetes and can increase the risk of cardio-vascular disease [36 43]

Therefore the evaluated EEPs contain important antioxi-dant compounds that can limit the spread of oxidative stress-related diseases The free radicals scavenging and antihemo-lytic ability demonstrated by the EEP-M were more efficientthan results observed for propolis from the stingless beeTetragonisca fiebrigi [29] and Melipona orbignyi [12] from

Midwest Region of Brazil and some extracts of Apis mellifera[44 45]

In the present study EEP-S and EEP-M exhibited cyto-toxic activity against K562 erythroleukemic cells In additionthe decrease in cell viability was greater in cells treated withEEP-M than in those treatedwith EEP-SHowever both EEPscaused necrosis in most of the cells at a concentration of500 120583gmLThe cytotoxic effect of propolis was also observedin other cell lines as human lung adenocarcinoma epithe-lial (A549) human cervical adenocarcinoma (HeLa) andhuman breast adenocarcinoma (MCF-7) but themechanismsinvolved in the death of these tumor cells were apoptosis [46ndash48]Therefore the use of EEP-S andEEP-Mmay constitute analternative treatment for chronic myeloid leukemia as K562cells are resistant to apoptosis induced by various agents [49]

Some compounds found in EEPs may play an impor-tant role in anticancer activity including tocopherol whichshows antitumor activity in esophageal cancer cells [50] andbreast cancer in vitro and in vivo [51] Furthermore caffeatederivatives are cytotoxic against human carcinoma cell lines[52]

Other phenolic compounds present in propolis exhibitantiproliferative and cytotoxic effects against various tumorcell lines including those obtained from renal cell carcinomas[53] and the colon [30] pancreas [54] skin [55] and lungs[56] Amyrins can be isolated from plants and is known asnatural potent anticancer its compounds induces tumor cell


Control 31 62

125 250 500Concentration (휇gmL)

104

103

102

101

100

FLH annexin10

410

310

210

110

0

FLH annexin FLH annexin

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

(a)

0 31 62 125 250 500

Viable cellsApoptosis

NecrosisLate apoptosis

+ +

++

+

lowastlowast


lowast

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

Concentration (휇gmL)

(b)

Figure 4 Cytotoxic action of EEP from S depilis against the K562 erythroleukemia cell line (a) Representative diagrams obtainedvia flow cytometry of cells stained with annexin V-FITCPI the lower left quadrant (PIminusAnminus) represents viable cells the lower rightquadrant (PIminusAn+) represents apoptotic cells the upper left quadrant (PI+Anminus) represents cells undergoing necrosis and the upper rightquadrant (PI+An+) represents cells in late apoptosis (b) Frequency of cell death obtained from the corresponding diagrams for the testedconcentrations lowast119901 lt 005 for the treated group versus control viable cells lowastlowast119901 lt 005 for the treated group versus control apoptosis 119901 lt 005for the treated group versus control necrosis +119901 lt 005 for the treated group versus control late apoptosis


Control 31 62

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100

FLH annexinFLH annexin FLH annexin

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

(a)

+ + ++

+

+

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

0 31 62 125 250 500



lowast

lowastlowastlowast


lowastlowast


(b)

Figure 5 Cytotoxic action of EEP fromM q anthidioides against the K562 erythroleukemia cell line (a) Representative diagrams obtainedvia flow cytometry of cells stainedwith annexinV-FITCPI the lower left quadrant (PIminusAnminus) represents viable cells the lower right quadrant(PIminusAn+) represents apoptotic cells the upper left quadrant (PI+Anminus) represents cells in necrosis and the upper right quadrant (PI+An+)represents cells in late apoptosis (b) Frequency of cell death obtained from the corresponding diagrams for the tested concentrations lowast119901 lt005 for the treated group versus control viable cells lowastlowast119901 lt 005 for the treated group versus control apoptosis 119901 lt 005 for the treated groupversus control necrosis +119901 lt 005 for the treated group versus control late apoptosis


0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)

Figure 6 Toxicity of EEPs from S depilis EEP-S and M qanthidioides (EEP-M) against C elegans lowast119901 lt 005 for the treatedgroup versus the control with untreated nematodes

death as human bladder carcinoma (NTUB1) [34 57] andleukemia cells (HL-60) [58]

Although the EEPs presented cytotoxic activity againstK562 cells no toxic or lethal effects were observed against thenematode C elegans

In vivo experimental models serve as a tool to understandeffects of natural products in whole organisms These resultssuggest that the evaluated propolis samples show specificityagainst leukemic cells considering that these nematodeswerenot affected This specificity may be important for the treat-ment of leukemia because drug toxicity and low specificity areamong the major difficulties in the treatment of this disease[37]

Corroborating with the toxicity data of the EEPs recentstudy showed that the crude extract of propolis presentedanticancer effects in human lung cancer cell and is antime-lanogenic in themelanoma cell line additionally it was able toprolong the life ofC elegans [48] In addition the caffeic acidthemajor constituent of propolis does no present toxic effectsand also was able to increase the survival of the nematodeC elegans after infection with the fungal pathogen [59] Theability of propolis or caffeic acid to extend lifespan in Celegans was associated with inactivation of oncogenic kinasePAK1 [48 59]

Previous studies have shown thatC elegans can be used asan experimental model for obtaining rapid results in toxicitystudies for pharmacological compounds [60 61] because it isa multicellular organism with a high reproduction rate andshort life cycle which makes it an excellent in vivo model forcomplementing cell culture-based systems [61]

Therefore we conclude that the tested EEPs exhibitantioxidant and cytotoxic activities attributed to their chem-ical composition which includes phytosterols terpenesphenolic compounds and tocopherol and possibly to thesynergy between different compounds present in propolisMoreover these EEPs show therapeutic potential for use inthe prevention and treatment of diseases associated withoxidative stress and the proliferation of tumor cells

Abbreviations

AAPH 221015840-Azobis-(2-amidinopropane)dihydrochloride

Abs AbsorbanceAn Annexin V-FITCBHT ButylhydroxytolueneDAD Diode array detectorDPPH 22-Diphenyl-1-picrylhydrazylCG-MS Gas chromatography-mass spectrometryHPLC High performance liquid chromatographyEEPs Ethanol extract of propolisEEP-S Ethanol extract of propolis of

Scaptotrigona depilisEEP-M Ethanol extract of propolis ofMelipona

quadrifasciata anthidioidesMDA MalondialdehydeNaCl Sodium chloridePI Propidium iodideSEM Standard error of the meanTBA Thiobarbituric acid

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors thankMarcelo Mori PhD for intellectual assis-tance This work was supported by grants from Fundacao deApoio ao Desenvolvimento do Ensino Ciencia e Tecnologiado Estado deMatoGrosso do Sul (FUNDECT) Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPES)andConselhoNacional deDesenvolvimentoCientıfico e Tec-nologico (CNPq) Edson Lucas dos Santos Claudia AndreaLima Cardoso and Edgar J Paredes-Gamero were recipientof fellowship from CNPq Brazil

References

[1] J M F Camargo and S R M Pedro ldquoMeliponini Lepeletierrdquoin Catalogue of Bees (Hymenoptera Apidae) in the NeotropicalRegion J S Moure D Urban and G A R Melo Eds 2016httpwwwmourecriaorgbrcataloguecontente101016

[2] M Cortopassi-Laurino V L Imperatriz-Fonseca DW Roubiket al ldquoGlobal meliponiculture challenges and opportunitiesrdquoApidologie vol 37 no 2 pp 275ndash292 2006

[3] S RM Pedro ldquoThe stingless bee fauna in Brazil (hymenopteraapidae)rdquo Sociobiology vol 61 no 4 pp 348ndash354 2014

[4] D W Roubik Ecology and Natural History of Tropical BeesCambridge University Press New York NY USA 1989

[5] M Viuda-Martos Y Ruiz-Navajas J Fernandez-Lopez andJ A Perez-Alvarez ldquoFunctional properties of honey propolisand royal jellyrdquo Journal of Food Science vol 73 no 9 pp R117ndashR124 2008

[6] AAjibola J P Chamunorwa andKH Erlwanger ldquoNutraceuti-cal values of natural honey and its contribution to human healthand wealthrdquo Nutrition and Metabolism vol 9 article 61 2012

[7] S Castaldo and F Capasso ldquoPropolis an old remedy used inmodern medicinerdquo Fitoterapia vol 73 no 1 pp S1ndashS6 2002


[8] V Bankova M Popova and B Trusheva ldquoPropolis volatilecompounds chemical diversity and biological activity areviewrdquo Chemistry Central Journal vol 8 article 28 pp 1ndash82014

[9] Y K Park SMAlencar andC L Aguiar ldquoBotanical origin andchemical composition of Brazilian propolisrdquo Journal of Agri-cultural and Food Chemistry vol 50 no 9 pp 2502ndash2506 2002

[10] A C H F Sawaya ldquoComposition and antioxidant activity ofpropolis from three species of Scaptotrigona stingless beesrdquoJournal of ApiProduct and ApiMedical Science vol 1 no 2 pp37ndash42 2009

[11] N S S Guimaraes J C Mello J S Paiva et al ldquoBaccharisdracunculifolia the main source of green propolis exhibitspotent antioxidant activity and prevents oxidative mitochon-drial damagerdquo Food and Chemical Toxicology vol 50 no 3-4pp 1091ndash1097 2012

[12] J F Campos U P dos Santos L F B Macorini et al ldquoAnti-microbial antioxidant and cytotoxic activities of propolis fromMelipona orbignyi (Hymenoptera Apidae)rdquoFood andChemicalToxicology vol 65 pp 374ndash380 2014

[13] M Velikova V Bankova I Tsvetkova A Kujumgiev and MCMarcucci ldquoAntibacterial ent-kaurene fromBrazilian propolisof native stingless beesrdquo Fitoterapia vol 71 no 6 pp 693ndash6962000

[14] L Barrientos C L Herrera G Montenegro et al ldquoChemicaland botanical characterization of chilean propolis and biolog-ical activity on cariogenic bacteria Streptococcus mutans andStreptococcus sobrinusrdquo Brazilian Journal of Microbiology vol44 no 2 pp 577ndash585 2013

[15] M Barbaric K Miskovic M Bojic et al ldquoChemical compo-sition of the ethanolic propolis extracts and its effect on HeLacellsrdquo Journal of Ethnopharmacology vol 135 no 3 pp 772ndash7782011

[16] R LimaCavendish J De Souza Santos R BeloNeto et al ldquoAnti-nociceptive and anti-inflammatory effects of Brazilian redpropolis extract and formononetin in rodentsrdquo Journal of Ethno-pharmacology vol 173 pp 127ndash133 2015

[17] K S Borges M S Brassesco C A Scrideli A E E Soaresand L G Tone ldquoAntiproliferative effects of Tubi-bee propolisin glioblastoma cell linesrdquo Genetics and Molecular Biology vol34 no 2 pp 310ndash314 2011

[18] H V van Tome G F Martins M A P Lima L A O Camposand R N C Guedes ldquoImidacloprid-induced impairment ofmushroom bodies and behavior of the native stingless beemelipona quadrifasciata anthidioidesrdquo PLOS ONE vol 7 no 6Article ID e38406 2012

[19] L B de Faria K P Aleixo C A Garofalo V L Imperatriz-Fonseca andC I da Silva ldquoForaging of Scaptotrigona aff depilis(Hymenoptera Apidae) in an urbanized area seasonality inresource availability and visited plantsrdquoPsyche vol 2012 ArticleID 630628 12 pages 2012

[20] M H Karcher C Menezes D A Alves O S Beveridge V-LImperatriz-Fonseca and F L W Ratnieks ldquoFactors influencingsurvival duration and choice of virgin queens in the stinglessbeeMelipona quadrifasciatardquo Naturwissenschaften vol 100 no6 pp 571ndash580 2013

[21] G Figueiredo-Mecca L R Bego and F S Nascimento ldquoFor-aging behavior of scaptotrigona depilis (hymenoptera apidaemeliponini) and its relationship with temporal and abioticfactorsrdquo Sociobiology vol 60 no 3 pp 277ndash282 2013

[22] S M Alencar T L C Oldoni M L Castro et al ldquoChemicalcomposition and biological activity of a new type of Brazilian

propolis red propolisrdquo Journal of Ethnopharmacology vol 113no 2 pp 278ndash283 2007

[23] D Gupta and R K Gupta ldquoBioprotective properties of Dragonrsquosblood resin in vitro evaluation of antioxidant activity andantimicrobial activityrdquo BMC Complementary and AlternativeMedicine vol 11 no 13 pp 1ndash9 2011

[24] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[25] E J Paredes-Gamero M N C Martins F A M CappabiancoJ S Ide and A Miranda ldquoCharacterization of dual effectsinduced by antimicrobial peptides Regulated cell death ormembrane disruptionrdquo Biochimica et Biophysica ActamdashGeneralSubjects vol 1820 no 7 pp 1062ndash1072 2012

[26] J A Lewis and J T Fleming ldquoBasic culturemethodsrdquo inCaenor-habditis Elegans Modern Biological Analysis of an Organism HF Epstein and D C Shakes Eds pp 3ndash29 Academic Press SanDiego Calif USA 1995

[27] V D Wagh ldquoPropolis a wonder bees product and its pharma-cological potentialsrdquo Advances in Pharmacological Sciences vol2013 Article ID 308249 11 pages 2013

[28] N Paulino S R Abreu Y Uto et al ldquoAnti-inflammatory effectsof a bioavailable compound Artepillin C in Brazilian propolisrdquoEuropean Journal of Pharmacology vol 587 no 1ndash3 pp 296ndash3012008

[29] J F Campos U P Santos P S da Rocha et al ldquoAntimicro-bial antioxidant anti-inflammatory and cytotoxic activities ofpropolis from the stingless bee Tetragonisca fiebrigi (Jataı)rdquoEvidence-Based Complementary and Alternative Medicine vol2015 Article ID 296186 11 pages 2015

[30] A H Banskota T Nagaoka L Y Sumioka et al ldquoAntiprolifera-tive activity of the Netherlands propolis and its active principlesin cancer cell linesrdquo Journal of Ethnopharmacology vol 80 no1 pp 67ndash73 2002

[31] H Maruta ldquoHerbal therapeutics that block the oncogenickinase PAK1 a practical approach towards PAK1-dependentdiseases and longevityrdquo Phytotherapy Research vol 28 no 5 pp656ndash672 2014

[32] A A Azlim Almey C Ahmed Jalal Khan I Syed Zahir KMustapha Suleiman M R Aisyah and K Kamarul RahimldquoTotal phenolic content and primary antioxidant activity ofmethanolic and ethanolic extracts of aromatic plantsrsquo leavesrdquoInternational Food Research Journal vol 17 no 4 pp 1077ndash10842010

[33] A S Awaad and N A Al-Jaber ldquoAntioxidant natural plantrdquo inEthnomedicine Source amp Mechanism I vol 27 pp 1ndash35 RPMP2015

[34] L H Vazquez J Palazon and A Navarro-Ocana ldquoThe pen-tacyclic triterpenes 120572 120573-amyrins a review of sources andbiological activitiesrdquo in PhytochemicalsmdashA Global Perspectiveof Their Role in Nutrition and Health p 538 InTech RijekaCroatia 2012

[35] B Halliwell R Aeschbach J Loliger and O I Aruoma ldquoThecharacterization of antioxidantsrdquo Food and Chemical Toxicol-ogy vol 33 no 7 pp 601ndash617 1995

[36] A Kurek-Gorecka A Rzepecka-Stojko M Gorecki J StojkoM Sosada and G Swierczek-Zieba ldquoStructure and antioxidantactivity of polyphenols derived from propolisrdquo Molecules vol19 no 1 pp 78ndash101 2014

[37] M L De Mesquita R M Araujo D P Bezerra et al ldquoCytotox-icity of 120575-tocotrienols from Kielmeyera coriacea against cancer


cell linesrdquo Bioorganic andMedicinal Chemistry vol 19 no 1 pp623ndash630 2011

[38] P X Chen Y Tang M F Marcone et al ldquoCharacterizationof free conjugated and bound phenolics and lipophilic antiox-idants in regular- and non-darkening cranberry beans (Phaseo-lus vulgaris L)rdquo Food Chemistry vol 185 pp 298ndash308 2015

[39] R Amarowicz and R B Pegg ldquoLegumes as a source of naturalantioxidantsrdquo European Journal of Lipid Science and Technologyvol 110 no 10 pp 865ndash878 2008

[40] Z Wang A M Joshi K Ohnaka et al ldquoDietary intakes ofretinol carotenes vitamin C and vitamin E and colorectalcancer risk the Fukuoka colorectal cancer studyrdquoNutrition andCancer vol 64 no 6 pp 798ndash805 2012

[41] A Floegel D-O Kim S-J Chung S I Koo and O K ChunldquoComparison of ABTSDPPH assays to measure antioxidantcapacity in popular antioxidant-rich US foodsrdquo Journal of FoodComposition and Analysis vol 24 no 7 pp 1043ndash1048 2011

[42] C G Zou N S Agar and G L Jones ldquoOxidative insult tohuman red blood cells induced by free radical initiator AAPHand its inhibition by a commercial antioxidant mixturerdquo LifeSciences vol 69 no 1 pp 75ndash86 2001

[43] M M Silva M R Santos G Caroco R Rocha G Justino andL Mira ldquoStructure-antioxidant activity relationships of flavo-noids a re-examinationrdquo Free Radical Research vol 36 no 11pp 1219ndash1227 2002

[44] H Shi H Yang X Zhang and L Yu ldquoIdentification and quan-tification of phytochemical composition and anti-inflammatoryand radical scavenging properties of methanolic extracts ofChinese propolisrdquo Journal of Agricultural and Food Chemistryvol 60 no 50 pp 12403ndash12410 2012

[45] N Mercan I Kivrak M E Duru et al ldquoChemical compositioneffects onto antimicrobial and antioxidant activities of propoliscollected from different regions of TurkeyrdquoAnnals of Microbiol-ogy vol 56 no 4 pp 373ndash378 2006

[46] S Khacha-Ananda K Tragoolpua P Chantawannakul andY Tragoolpua ldquoPropolis extracts from the northern regionof Thailand suppress cancer cell growth through induction ofapoptosis pathwaysrdquo Investigational New Drugs vol 34 no 6pp 707ndash722 2016

[47] M Tartik E Darendelioglu G Aykutoglu and G BaydasldquoTurkish propolis supresses MCF-7 cell death induced byhomocysteinerdquo Biomedicine and Pharmacotherapy vol 82 pp704ndash712 2016

[48] N Taira B C Q Nguyen P T Be Tu and S Tawata ldquoEffectof Okinawa propolis on PAK1 activity Caenorhabditis eleganslongevity melanogenesis and growth of cancer cellsrdquo Journal ofAgricultural and Food Chemistry vol 64 no 27 pp 5484ndash54892016

[49] C-D Kang S-D Yoo B-W Hwang et al ldquoThe inhibitionof ERKMAPK not the activation of JNKSAPK is primarilyrequired to induce apoptosis in chronic myelogenous leukemicK562 cellsrdquo Leukemia Research vol 24 no 6 pp 527ndash534 2000

[50] S Lim J Y Lee W H Jung et al ldquoAnticancer effects ofastaxanthin and 120572-tocopherol in esophageal cancer cell linesrdquoThe Korean Journal of Helicobacter and Upper GastrointestinalResearch vol 11 no 3 pp 170ndash175 2011

[51] W Yu L Jia S-K Park et al ldquoAnticancer actions of naturaland synthetic vitamin E forms RRR-120572-tocopherol blocks theanticancer actions of 120574-tocopherolrdquo Molecular Nutrition andFood Research vol 53 no 12 pp 1573ndash1581 2009

[52] Y-C Chen Y-H Kuo N-C Yang C-W Liu W-T Changand C-L Hsu ldquoCytotoxic and apoptotic effects of caffeate

derivatives on A549 human lung carcinoma cellsrdquo Journal of theChinese Medical Association vol 77 no 10 pp 535ndash543 2014

[53] M J Valente A F Baltazar R Henrique L Estevinho andM Carvalho ldquoBiological activities of Portuguese propolis pro-tection against free radical-induced erythrocyte damage andinhibition of human renal cancer cell growth in vitrordquo Food andChemical Toxicology vol 49 no 1 pp 86ndash92 2011

[54] F Li S Awale Y Tezuka H Esumi and S Kadota ldquoStudy onthe constituents of mexican propolis and their cytotoxic acti-vity against PANC-1 human pancreatic cancer cellsrdquo Journal ofNatural Products vol 73 no 4 pp 623ndash627 2010

[55] C Chen M Weng C Wu and J Lin ldquoComparison of radicalscavenging activity cytotoxic effects and apoptosis inductionin human melanoma cells by taiwanese propolis from differ-ent sourcesrdquo Evidence-Based Complementary and AlternativeMedicine vol 1 no 2 pp 175ndash185 2004

[56] F Li S Awale Y Tezuka and S Kadota ldquoCytotoxic constituentsfromBrazilian red propolis and their structure-activity relation-shiprdquo Bioorganic and Medicinal Chemistry vol 16 no 10 pp5434ndash5440 2008

[57] K-W Lin A-MHuang H-Y Tu et al ldquoXanthine oxidase inhi-bitory triterpenoid and phloroglucinol from guttiferaceousplants inhibit growth and induced apoptosis in human ntub1cells through a ROS-dependentmechanismrdquo Journal of Agricul-tural and Food Chemistry vol 59 no 1 pp 407ndash414 2011

[58] FWA Barros PN Bandeira D J B Lima et al ldquoAmyrin estersinduce cell death by apoptosis in HL-60 leukemia cellsrdquo Bio-organic and Medicinal Chemistry vol 19 no 3 pp 1268ndash12762011

[59] J J Coleman T Komura J Munro et al ldquoThe immunomodu-latory activity of caffeic acid phenethyl ester in Caenorhabditiselegans is mediated by the CED-10 (Rac-1)PAK1 pathwayrdquoFuture Medicinal Chemistry vol 8 no 17 pp 2033ndash2046 2016

[60] M Dengg and J C A van Meel ldquoCaenorhabditis elegans asmodel system for rapid toxicity assessment of pharmaceuti-cal compoundsrdquo Journal of Pharmacological and ToxicologicalMethods vol 50 no 3 pp 209ndash214 2004

[61] M C K Leung P L Williams A Benedetto et al ldquoCaenorhab-ditis elegans an emerging model in biomedical and environ-mental toxicologyrdquo Toxicological Sciences vol 106 no 1 pp 5ndash28 2008

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


from which bees collect the raw material and the bee speciesthat produces it [8]

Therefore studies on propolis collected from differentgeographical regions and bee species are of great importancebecause these elements affect the chemical composition andconsequently the biological properties of propolis

The therapeutic activity of propolis from stingless beeshas beenwidely investigated in recent decades including des-criptions of its antioxidant activity [10ndash12] antimicrobial acti-vity [5 13 14] anti-inflammatory activity [15 16] and anti-tumor activity [7 12 17]

Among bee species Scaptotrigona depilis popularlyknown as ldquomandaguarirdquo andMelipona quadrifasciata anthid-ioides known as ldquomandacaiardquo are stingless species foundin South American countries including Paraguay Argentinaand Brazil [1] and their genetic and behavioral characteristicshave been well described [18ndash21] However studies on thebiological activity of propolis in these species are scarce inthe literature particularly in view of the difficulty in findingcolonies in their natural environment where species are dis-appearing because of anthropogenic activity

The pharmacological properties of propolis in thesetwo species were evaluated by Velikova et al [13] whodescribed the antimicrobial activity of propolis extracts fromM q anthidioides and by Sawaya [10] who described theantioxidant activity of propolis extracts from S depilis In thiscontext the present study aimed to determine the chemicalconstituents as well as the antioxidant cytotoxic and toxicactivities of ethanol extracts of propolis from the stingless beespecies S depilis andM q anthidioides from the state ofMatoGrosso do Sul in Midwest Brazil

2 Materials and Methods

21 Research Ethics No specific permits were required for thedescribed field studies All field works to collect the propolissamples were conducted on private land and with ownerpermission The field studies did not involve endangered orprotected speciesTheprotocol to collect of humanperipheralblood was approved by the Research Ethics Committee(Comite de Etica em Pesquisa CEP) of the UniversityCenter of GrandeDourados (CentroUniversitario daGrandeDourados UNIGRAN) Brazil (CEP process number 12312)All subjects provided written informed consent for participa-tion

22 Preparation of the Ethanol Extract of Propolis (EEPs)Propolis samples from S depilis (8381 g) and M q anthid-ioides (3642 g) were collected from the state of Mato Grossodo Sul (22∘1310158401210158401015840Sndash54∘491015840210158401015840W) in the Midwest Region ofBrazil with a total of seven collections being performed foreach species The ethanol extract of propolis (EEPs) wasprepared using 45mL of 80 ethanol per 1 g of propolisThismixture was incubated in a water bath at 70∘C in a sealedcontainer until total dissolution and subsequently filtered infilter paper qualitative 80 gm2 (Prolab Sao Paulo Brazil) toobtain the EEPs of S depilis (EEP-S) and M q anthidioides(EEP-M) [22] After preparation of the extracts they werekept at a temperature of minus20∘C until analysis

23 Chemical Analysis

231 Preparation of the Samples The samples (1mg) wasfractionated with hexane and water in proportion 1 1 v vand fraction soluble in hexane was analyzed by GC-MS andfraction in water by HPLC

232 GC-MS Samples were injected and analyzed by gaschromatography-mass spectrometry (GC-MS) The GC-MSanalysis was performed on a gas chromatograph (GC-2010Plus Shimadzu Kyoto Japan) equipped with amass spectrom-eter detector (GC-MS Ultra 2010) using LM-5 (5 phenyldimethyl poly siloxane) capillary column (15m length times02mm id and 02 120583m film thickness) with initial oventemperature set at 150∘C and heating from 150∘C to 280∘C at15∘Cminminus1 and a hold at 280∘C for 15min The carrier gaswas helium (9999) supplied at a flow rate of 10mLminwith split ratio 1 20 1 120583L injection volumeThe injector tem-perature was 280∘C and the quadrupole detector temperaturewas 280∘C The MS scan parameters included an electron-impact ionization voltage of 70 eVmass range of 45ndash600119898119911and scan interval of 03 s The identifications were completedby comparing the mass spectra obtained in the NIST21and WILEY229 libraries In some cases the compound wasconfirmed by comparison of standards Standards of the stig-masterol120573-sitosterol120573-amyrin120572-amyrin120573-amyrin acetateand tocopherol (Sigma-Aldrich with purity ge97) were pre-pared in the concentration initial of 1000 120583gmLThe concen-trations of compoundswere determined by extern calibrationafter dilutions appropriated in the range of 01ndash50 120583gmLThequantification of taraxasterol was performed in relation tostigmasterol The procedure was performed in triplicate

233 HPLC The extracts were analyzed in an analyticalHPLC (LC-6AD Shimadzu Kyoto Japan) system with adiode array detector (DAD) monitored at 120582 = 200ndash600 nmTheHPLC column was a C-18 (25 cm times 46mm particle size5 120583m Luna Phenomenex Torrance CA USA) with a smallprecolumn (25 cm times 3mm) containing the same packingused to protect the analytical column In each analysis theflow rate and the injected volume were set as 10mLminminus1and 20 120583L respectively All chromatographic analyses wereperformed at 22∘C Elution was carried out using an binarymobile phase of water with 6 acetic acid and 2mM sodiumacetate (eluent A) and acetonitrile (eluent B) The followingapplied gradients are as follows 5B (0min) 15B (30min)50B (35min) and 100B (45min) Standards of the vanilicacid caffeic acid ferulic acid p-coumaric acid benzoic acidcinnamic acid quercetin luteolin apigenin and vanillin(Sigma-Aldrich ge97) were prepared in the concentrationinitial of 1000120583gmLThe concentrations of compoundsweredetermined by extern calibration after dilutions appropriatedin the range of 001ndash10 120583gmLThe procedure was performedin triplicate


241 DPPH Free Radical Scavenging Activity The freeradical-scavenger activity was determined by the DPPH




) times 100 (1)


inhibition of ABTS


) times 100 (2)







) (3)





2) The suspensions


26 In Vivo Toxicity








50)














4 Discussion







2






SampleDPPH ABTS




Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

0

10

20

30

40

50

Hem

olys

is (

)

(a)

0

10

20

30

40

50

Control 50 75 100 125 50 75 100 125 50 75 100 125


Hem

olys

is (

)

lowast

lowast

lowast

lowast

lowast

lowastlowast

(b)

0

10

20

30

40

50

Hem

olys

is (

)

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowast lowast

lowast

(c)








0

500

1000

1500

2000

2500

3000

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowast

lowast

lowastlowast

lowast


MD

A (n

mol

ml)

(a)

0

500

1000

1500

2000

2500

3000

lowastlowastlowast

lowast

lowast lowastlowast

lowastlowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(b)

0

500

1000

1500

2000

2500

3000

lowastlowast

lowastlowast

lowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(c)


0 31 62 125 250 500 31 62 125 250 500EEP-S EEP-M

0

20

40

60

80

100

Cel

l via

bilit

y (

)

lowast

lowast

lowast










Control 31 62


104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

(a)

0 31 62 125 250 500



+ +

++

+

lowastlowast


lowast

0

20

40

60

80

100

Cel

l pop

ulat

ion

()


(b)



Control 31 62

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

(a)

+ + ++

+

+

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

0 31 62 125 250 500



lowast

lowastlowastlowast


lowastlowast


(b)



0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)








Abbreviations





Competing Interests


Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















) times 100 (1)


inhibition of ABTS


) times 100 (2)







) (3)





2) The suspensions


26 In Vivo Toxicity








50)














4 Discussion







2






SampleDPPH ABTS




Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

0

10

20

30

40

50

Hem

olys

is (

)

(a)

0

10

20

30

40

50

Control 50 75 100 125 50 75 100 125 50 75 100 125


Hem

olys

is (

)

lowast

lowast

lowast

lowast

lowast

lowastlowast

(b)

0

10

20

30

40

50

Hem

olys

is (

)

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowast lowast

lowast

(c)








0

500

1000

1500

2000

2500

3000

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowast

lowast

lowastlowast

lowast


MD

A (n

mol

ml)

(a)

0

500

1000

1500

2000

2500

3000

lowastlowastlowast

lowast

lowast lowastlowast

lowastlowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(b)

0

500

1000

1500

2000

2500

3000

lowastlowast

lowastlowast

lowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(c)


0 31 62 125 250 500 31 62 125 250 500EEP-S EEP-M

0

20

40

60

80

100

Cel

l via

bilit

y (

)

lowast

lowast

lowast










Control 31 62


104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

(a)

0 31 62 125 250 500



+ +

++

+

lowastlowast


lowast

0

20

40

60

80

100

Cel

l pop

ulat

ion

()


(b)



Control 31 62

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

(a)

+ + ++

+

+

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

0 31 62 125 250 500



lowast

lowastlowastlowast


lowastlowast


(b)



0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)








Abbreviations





Competing Interests


Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2) The suspensions


26 In Vivo Toxicity








50)














4 Discussion







2






SampleDPPH ABTS




Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

0

10

20

30

40

50

Hem

olys

is (

)

(a)

0

10

20

30

40

50

Control 50 75 100 125 50 75 100 125 50 75 100 125


Hem

olys

is (

)

lowast

lowast

lowast

lowast

lowast

lowastlowast

(b)

0

10

20

30

40

50

Hem

olys

is (

)

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowast lowast

lowast

(c)








0

500

1000

1500

2000

2500

3000

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowast

lowast

lowastlowast

lowast


MD

A (n

mol

ml)

(a)

0

500

1000

1500

2000

2500

3000

lowastlowastlowast

lowast

lowast lowastlowast

lowastlowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(b)

0

500

1000

1500

2000

2500

3000

lowastlowast

lowastlowast

lowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(c)


0 31 62 125 250 500 31 62 125 250 500EEP-S EEP-M

0

20

40

60

80

100

Cel

l via

bilit

y (

)

lowast

lowast

lowast










Control 31 62


104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

(a)

0 31 62 125 250 500



+ +

++

+

lowastlowast


lowast

0

20

40

60

80

100

Cel

l pop

ulat

ion

()


(b)



Control 31 62

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

(a)

+ + ++

+

+

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

0 31 62 125 250 500



lowast

lowastlowastlowast


lowastlowast


(b)



0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)








Abbreviations





Competing Interests


Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















4 Discussion







2






SampleDPPH ABTS




Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

0

10

20

30

40

50

Hem

olys

is (

)

(a)

0

10

20

30

40

50

Control 50 75 100 125 50 75 100 125 50 75 100 125


Hem

olys

is (

)

lowast

lowast

lowast

lowast

lowast

lowastlowast

(b)

0

10

20

30

40

50

Hem

olys

is (

)

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowast lowast

lowast

(c)








0

500

1000

1500

2000

2500

3000

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowast

lowast

lowastlowast

lowast


MD

A (n

mol

ml)

(a)

0

500

1000

1500

2000

2500

3000

lowastlowastlowast

lowast

lowast lowastlowast

lowastlowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(b)

0

500

1000

1500

2000

2500

3000

lowastlowast

lowastlowast

lowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(c)


0 31 62 125 250 500 31 62 125 250 500EEP-S EEP-M

0

20

40

60

80

100

Cel

l via

bilit

y (

)

lowast

lowast

lowast










Control 31 62


104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

(a)

0 31 62 125 250 500



+ +

++

+

lowastlowast


lowast

0

20

40

60

80

100

Cel

l pop

ulat

ion

()


(b)



Control 31 62

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

(a)

+ + ++

+

+

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

0 31 62 125 250 500



lowast

lowastlowastlowast


lowastlowast


(b)



0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)








Abbreviations





Competing Interests


Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















SampleDPPH ABTS




Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowastlowast

lowast

lowastlowast

lowastlowast

lowast

0

10

20

30

40

50

Hem

olys

is (

)

(a)

0

10

20

30

40

50

Control 50 75 100 125 50 75 100 125 50 75 100 125


Hem

olys

is (

)

lowast

lowast

lowast

lowast

lowast

lowastlowast

(b)

0

10

20

30

40

50

Hem

olys

is (

)

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowast lowast

lowast

(c)








0

500

1000

1500

2000

2500

3000

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowast

lowast

lowastlowast

lowast


MD

A (n

mol

ml)

(a)

0

500

1000

1500

2000

2500

3000

lowastlowastlowast

lowast

lowast lowastlowast

lowastlowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(b)

0

500

1000

1500

2000

2500

3000

lowastlowast

lowastlowast

lowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(c)


0 31 62 125 250 500 31 62 125 250 500EEP-S EEP-M

0

20

40

60

80

100

Cel

l via

bilit

y (

)

lowast

lowast

lowast










Control 31 62


104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

(a)

0 31 62 125 250 500



+ +

++

+

lowastlowast


lowast

0

20

40

60

80

100

Cel

l pop

ulat

ion

()


(b)



Control 31 62

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

(a)

+ + ++

+

+

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

0 31 62 125 250 500



lowast

lowastlowastlowast


lowastlowast


(b)



0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)








Abbreviations





Competing Interests


Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

500

1000

1500

2000

2500

3000

Control 50 75 100 125 50 75 100 125 50 75 100 125


lowastlowastlowast

lowast

lowast

lowastlowast

lowast


MD

A (n

mol

ml)

(a)

0

500

1000

1500

2000

2500

3000

lowastlowastlowast

lowast

lowast lowastlowast

lowastlowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(b)

0

500

1000

1500

2000

2500

3000

lowastlowast

lowastlowast

lowast lowast

Control 50 75 100 125 50 75 100 125 50 75 100 125


MD

A (n

mol

ml)

(c)


0 31 62 125 250 500 31 62 125 250 500EEP-S EEP-M

0

20

40

60

80

100

Cel

l via

bilit

y (

)

lowast

lowast

lowast










Control 31 62


104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

(a)

0 31 62 125 250 500



+ +

++

+

lowastlowast


lowast

0

20

40

60

80

100

Cel

l pop

ulat

ion

()


(b)



Control 31 62

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

(a)

+ + ++

+

+

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

0 31 62 125 250 500



lowast

lowastlowastlowast


lowastlowast


(b)



0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)








Abbreviations





Competing Interests


Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control 31 62


104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

(a)

0 31 62 125 250 500



+ +

++

+

lowastlowast


lowast

0

20

40

60

80

100

Cel

l pop

ulat

ion

()


(b)



Control 31 62

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

(a)

+ + ++

+

+

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

0 31 62 125 250 500



lowast

lowastlowastlowast


lowastlowast


(b)



0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)








Abbreviations





Competing Interests


Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control 31 62

104

103

102

101

100

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH

PI

FLH annexin10

410

310

210

110

0


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100


104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

104

103

102

101

100

(a)

+ + ++

+

+

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

0 31 62 125 250 500



lowast

lowastlowastlowast


lowastlowast


(b)



0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)








Abbreviations





Competing Interests


Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 250 500 1000 250 500 1000EEP-S EEP-M

0

20

40

60

80

100

Viab

le w

orm

s (

)








Abbreviations





Competing Interests


Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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