Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 591313 6 pageshttpdxdoiorg1011552013591313
Research ArticleAqueous Extract of Annona macroprophyllataA Potential 120572-Glucosidase Inhibitor
F Brindis1 M E Gonzaacutelez-Trujano2 M Gonzaacutelez-Andrade3
E Aguirre-Hernaacutendez4 and R Villalobos-Molina1
1 Unidad de Biomedicina Facultad de Estudios Superiores-Iztacala Universidad Nacional Autonoma de MexicoAvenida de los Barrios 1 Los Reyes Iztacala 54090 Tlalnepantla MEX Mexico
2 Laboratorio de Neurofarmacologıa de Productos Naturales de la Direccion de Investigaciones en NeurocienciasInstituto Nacional de Psiquiatrıa Ramon de la Fuente Muniz Calzada Mexico-Xochimilco 101 Colonia San Lorenzo Huipulco14370 Mexico City DF Mexico
3 Instituto Nacional de Medicina Genomica (INMEGEN) Periferico sur 4809 Arenal Tepepan Tlalpan Secretarıa de Salud14610 Mexico City DF Mexico
4Departamento de Ecologıa y Recursos Naturales Facultad de Ciencias Universidad Nacional Autonoma de MexicoCiudad Universitaria Coyoacan 04510 Mexico City DF Mexico
Correspondence should be addressed to F Brindis brindis77unammx
Received 29 April 2013 Accepted 25 July 2013
Academic Editor Ruth C R Meex
Copyright copy 2013 F Brindis et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Annona genus contains plants used in folkmedicine for the treatment of diabetes In the present study an aqueous extract preparedfrom Annona macroprophyllata (Annonaceae also known as A diversifolia) leaves was evaluated on both the activity of yeast120572-glucosidase (an in vitro assay) and sucrose tolerance in Wistar rats The results have shown that the aqueous extract from Amacroprophyllata inhibits the yeast 120572-glucosidase with an IC
50= 118mgmL in a competitive manner with a 119870
119894= 097mgmL
a similar value to that of acarbose (119870119894= 079mgmL) The inhibitory activity of A macroprophyllata was reinforced by its
antihyperglycemic effect at doses of 100 300 and 500mgkg in rats Chromatographic analysis identified the flavonoids rutinand isoquercitrin in the most polar fractions of A macroprophyllata crude extract suggesting that these flavonoids are part of theactive constituents in the plant Our results support the use ofAmacroprophyllata inMexican folkmedicine to control postprandialglycemia in people with diabetes mellitus involving active constituents of flavonoid nature
1 Introduction
Diabetes mellitus (DM) is a chronic disease associated withabnormal and constant high blood glucose (hyperglycemia)that results from defects in insulin secretion action orboth In type 2 diabetes mellitus (DM2) postprandial hyper-glycemia is important in the development of the disease itis associated with micro- and macrovascular complicationsand it has been proposed as an independent risk factorfor cardiovascular disease [1 2] The postprandial phase ischaracterized by a rapid and increased glycemia These post-prandial ldquohyperglycemic spikesrdquo may be relevant to thepathophysiological conditions of late diabetes [3 4]
One important factor that arises in postprandial hyper-glycemia is the fast uptake of glucose in the intestine inwhich 120572-glucosidases hydrolyze starch and oligosaccharides[5]The120572-glucosidases exoglycosidases found in the luminalsurface of enterocytes contain maltaseglucoamylase andsucroseisomaltase activities [6] It is believed that inhibitionof these enzymes can effectively control the postprandialelevation of blood glucose Therefore an important strategyfor managing postprandial hyperglycemia is to inhibit 120572-glucosidase activity [7]The 120572-glucosidase inhibitors such asacarbose have been used in the clinic to control blood glucoseincrease especially postprandial in DM2 [8] these chemicaldrugs have strong inhibitory activity against 120572-glucosidases
2 BioMed Research International
but have the disadvantage of undesirable side effects suchas abdominal distention flatulence meteorism and diar-rhea [9] Previous research indicates that side effects couldbe caused by excessive inhibition of pancreatic 120572-amylaseresulting in the abnormal bacterial fermentation of undi-gested carbohydrates in the colon Therefore more effectiveinhibitors for the enzymes should have strong inhibitoryeffect against 120572-glucosidase andmild inhibitory effect against120572-amylase which can be an effective therapy for managingpostprandial hyperglycemia with minimal side effects [10]
Annonaceae is a family of plants consisting of 2300 to2500 species included in more than 130 genera in fact it isthe largest family of the order Magnoliales Only four genera(Annona Rollinia Uvaria andAsimina) produce edible fruitssuch as annona [11ndash13] Annona macroprophyllata DonnSm is a species belonging to the Annona genus it is acommon tree in central Mexico known as ldquoilamardquo (Statesof Colima Guerrero and Mexico) whereas in the southeast(Tehuantepec region and Yucatan) it is called ldquopapaucerdquo orldquoanona blancardquo [14 15] The fruits of this plant are used asfood but its leaves are employed as anticonvulsant [16] aswell as analgesic and anti-inflammatory agents in traditionalMexicanmedicine [17] However so far there are no scientificreports supporting its probable antihyperglycemic proper-ties even though other species of the genus are known tohave those properties that isAnnona squamosa A muricataA glabra and A cherimola [18ndash20] then we have used achemotaxonomic criterion in order thatA macroprophyllatabesides its described medicinal properties [14ndash17] was inves-tigated to search for its likely antidiabetic properties
2 Materials and Methods
21 Plant Material and Extract Annona macroprophyllataDonn Sm specimens were collected in Tejupilco Guerreroin September 2010 Dr E Cedillo-Portugal botanist from theUniversidad Autonoma de Chapingo (UACH) certified theauthenticity of the plant and a voucher specimen (AN9702)was deposited in the herbarium ldquoHerbario de Plantas UtilesEfraım Hernandez Xrdquo at the UACH in the State of MexicoMexico for future reference Aqueous extract was obtainedby using 50 g of dried and powdered leaves in a process ofinfusion in 500mL of boiling water The aqueous extract wasseparated from the residues by gravity filtration samples werefrozen in liquid nitrogen and then lyophilized during 12 husing a Heto FD3 Lab lyophilizer to yield 111 g (222)
22 HPLC Quantification of Flavonoids An HPLC analysiswas performed using an Agilent Technologies Chromato-graph The separation was performed on an ODS HypersilC18 column (15mm times 4mm id and 5120583m particle size) Thesamples were injected through a 20 120583L loop The columnwas thermostatically controlled at 30∘C using a 1mLminflow rate The mobile phase consisted of 15 85 acetonitrile-trifluoroacetic acid solution at pH 25 The detection wasmonitored at 350 nm wavelength and the system was runfor 15min Calibration curves were done for the standards
kaempferol hesperidin naringenin naringin rutin iso-quercitrin and quercetin
23 120572-Glucosidase Assay In Vitro 120572-Glucosidase inhibitionwas measured at pH 70 and 30∘C using p-nitrophenyl-120572-D-glucopyranoside (pNPG) as substrate and 075 IUmLof yeast 120572-glucosidase in 01M sodium phosphate bufferAcarbose and aqueous extract were dissolved in phosphatebuffer and serial dilutions from 14 to 02mgmL werepreparedThe increments in absorption at 405 nm due to thehydrolysis of pNPG by the 120572-glucosidase were determinedon a microplate reader DTX 880 Multimode Detector fromBeckman Coulter Ten 120583L of acarbose or extract solution(in triplicate) were incubated during 5min with 20120583L ofenzyme stock After incubation 10 120583L of substrate wereadded and further incubated for 35min at 30∘C Finallythe reaction was stopped by adding 30 120583L of 5M Na
2CO3
The concentration required to inhibit the enzyme activity by50 (IC
50) was calculated by regression analysis using the
following equation
V =119860100
1 + (119868IC50)119904 (1)
where V is the percentage of inhibition A100
is the maximuminhibition I is the inhibitor concentration IC
50is the concen-
tration required to inhibit the enzyme activity by 50 and 119904is the cooperative degree
24 Enzyme Kinetics The mode of inhibition for 120572-glucosidase was determined by the Lineweaver-Burk plotsAll the results are expressed as the mean of at least threeexperiments plusmn SEM Kinetic parameters such as 119881
119898119870119898 and
119870119894were evaluated by using the nonlinear regression method
based on the following inhibition equation
V =Vmax119878
119870119898(1 + 119868119870
119894) + 119878 (1 + 119868119870
1015840
119894)
(2)
where V is the initial velocity in either the Presence or absenceof the inhibitor 119878 and 119868 are the concentrations of substrate andinhibitor respectively119881
119898is the maximum velocity119870
119898is the
Michaelis-Menten constant 119870119894is the competitive inhibition
constant and 1198701015840119894is the uncompetitive inhibition constant
The kinetic data were analyzed using a computer program fornonlinear regressions (Origin 80)
25 Experimental Animals MaleWistar normoglycemic ratsweighing 200ndash250 g were obtained from our animal facilitiesat Facultad de Estudios Superiores Iztacala UNAM Pro-cedures involving animals and their care fulfilled the Mex-ican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999) and were in compliance with internationalrules on care and use of laboratory animals Furthermoreclearance for conducting the studies was obtained from theEthics Committee for the Use of Animals in Pharmacologicaland Toxicological Testing of Facultad de Estudios SuperioresIztacala UNAM For the pharmacological studies groupsof four animals were used All doses are in mgkg of body
BioMed Research International 3
weightThe ratswere housed in groups of four under standardlaboratory conditions (12 h lightdark cycle at 22 plusmn 1∘C) andmaintained on a standard pellet diet and water ad libitum
26 Preparation of the Test Samples and Determinationof Glycemia All aqueous administrations were suspendedin 09 saline solution and given by intragastrical routeAcarbose (Sigma-Aldrich Co St Louis MO USA) wasused as antihyperglycemic drug Sucrose (Reasol ReactivosAnalıticosMexico) was used as carbohydrate to carry out thesucrose tolerance tests The control rats received only vehicle(saline solution) in the same volume (05mL of vehicle100 gof body weight) by the same route Blood samples werecollected from the caudal vein by performing a small incisionat the end of the tail Blood glucose (mgdL) was estimatedusing a commercial glucometer (Accu-Chek sensor RocheMannheim Germany) The variation of glycemia as percent-age for each group was calculated with respect to the initial(0 h) level according to the following equation
Variation of glycemia = [119866119905minus 119866119894
119866119894
] times 100 (3)
where 119866119894is the initial glycemia values and 119866
119905is the glycemia
value after treatments administration [21]
27 Oral Sucrose Tolerance Test (OSTT) Rats were fastedduring 12 h before the experiment having free access to waterThe crude extract of A macroprophyllata was tested at thedoses of 100 300 and 500mgkg Acarbose (5mgkg) wassuspended in the same vehicle Time 0min was set beforetreatmentwith the extract 30min later a sucrose load (3 gkg)was administered to the rats Blood samples were obtained 1530 60 90 and 120min after the carbohydrate load [21]
28 Statistical Analysis Data represent the mean plusmn SEM of119899 = 4 rats for the OSTT and 119899 = 3 assays for in vitroexperiments Differences were analyzed using Studentrsquos 119905-test (Sigma Stat version 30) and significant differences wereconsidered at 119875 lt 005
3 Results and Discussion
In Mexico it has been reported the use of A muricata Aglabra and A cherimola as antidiabetic species [18] whichsuggested to us that A macroprophyllata is an interestingspecies to study since it may have the same metabolic path-ways that synthesize the compounds as the other membersof the genus and thus may show their therapeutic propertiesThat is we tested a chemotaxonomic criterion for the study
Elution of A macroprophyllata leaves aqueous extractyielded rutin (288 120583gmg of crude extract RT 3973min)and isoquercitrin (071120583gmg of crude extract RT 6872) aswell as some traces of astragalinisoquercitrin (003 120583gmg ofcrude extract) (Figure 1)
Annona macroprophyllata ethanolic extract has beeninvestigated as antinociceptive anti-inflammatory andanticonvulsant agents [15ndash17] however the species has not
been tested for antidiabetic actions as their counterpartshave done [19 20 22ndash25] Our study found rutin as one of itscomponents as well as isoquercitrin these two flavonoidsalong with quercetin have been tested on diabetic rats thatis rutin decreased fasting plasma glucose increased insulinlevels and improved the antioxidant status by decreasinglipid peroxidative products and increasing enzymic andnonenzymic antioxidants [26] Rutin has been associatedwith marked decrease of hepatic and cardiac levels oftriglycerides and elevated glycogen which suggest thatrutin can improve hyperglycemia and dyslipidemia whileinhibiting the progression of liver and heart dysfunction inSTZ-induced diabetic rats [27] and rutin has hypoglycemiceffect in normal and diabetic rats [28ndash30] Rutin metabolitesare capable of inhibiting 120572-glucosidase activity both in vivoand in vitro [31] and the formation of advanced glycationend products formed via protein glycation which correlateswith aging and diabetes complications [32]
To our knowledge this is the first report that the aqueousextract from A macroprophyllata inhibited the activity ofyeast 120572-glucosidase with a low IC
50(118mgmL) very
similar to that of acarbose (027mgmL) This result agreeswith that reported for rutin as inhibitor of 120572-glucosidase(IC50= 0196mM) compared with acarbose (IC
500091mM)
[33] therefore rutin might participate in the aqueous extractof A macroprophyllata on the 120572-glucosidase inhibition andantihyperglycemic effects
The next step was to determine the nature of the inhibi-tion exerted by the aqueous extract of A macroprophyllatathen a kinetic analysis of the inhibition of enzyme activityusing different amounts of aqueous extract or acarbose wasconducted The results yielded a 119870
119894of 097mgmL and 119870
119894
of 079mgmL to aqueous extract and acarbose respectivelyThese values show that the extract inhibited the enzymeand could be used to control postprandial hyperglycemiaLineweaver-Burk plots for 120572-glucosidase from yeast in thepresence of acarbose or A macroprophyllata extract atdifferent concentrations (Figure 2) revealed typical curvesfor competitive inhibitors implying binding to the catalyticsite Then we suggest that disaccharidases are targets offlavonoids in the regulation of glucose release and conse-quently glucose absorption by the gut This type of inhibitorhas several advantages over others that is its inhibition isnot permanent and undesirable effects are easily attenuatedwith decreasing dose therefore the aqueous extract ofA macroprophyllata represents a good alternative to avoidhyperglycemia
On the other hand to confirmwhether our results in vitrocould be reproduced in a whole organism the response onglycemia after single oral sucrose ingestion was examinedfor A macroprophyllata As observed in Figure 3 the testextract significantly avoided the hyperglycemic responseat doses of 100 300 and 500mgkg compared to control(119875 lt 005) The postprandial blood glucose peak wasdiminished from 15min in all three doses (100 300 and500mgkg) however the highest activity was 300 gt 500gt 100mgkg throughout the time-curve postingestion ofsucrose The glycemic lowering effect of A macroprophyllataat doses of 100 300 and 500mgkg was not greater than that
4 BioMed Research International
O
O
O
Isoquercitrin
OHO
HO
HO
HO
HO
HO
O
OOH
OHOH
OH
OH
OHOH
OH
OH
OH
OH
OHOO
Rutin
CH3
CH2
Figure 1 Structure of flavonoids rutin and isoquercitrin
1V
(mM
min
)minus1
1[pNPG (mM)]
60
50
40
30
20
10
0
minus10
minus2 0 2 4 6 8 10 12 14 16 18
(a)
1V
(mM
min
)minus1
70
60
50
40
30
20
10
0
minus10
1[pNPG (mM)]minus2 0 2 4 6 8 10 12 14 16 18
(b)
Figure 2 Lineweaver-Burk plots of 120572-glucosidase inhibition at different concentrations of (a) acarbose and (b) extract aqueous from Amacroprophyllata 00mgmL (◼) 02mgmL (e) 04mgmL (998771) 06mgmL (998787) 08mgmL (998819) 10mgmL (998835) 12mgmL (⧫) 14mgmL(C)
with acarbose a therapeutic drug used as positive control(5mgkg) however maximum effect obtained at a dose of300mgkg was not statistically different with acarbose dosefrom 60 min on results in vivo that agree to the reportedfor other species of the genus Annona as is the case of Asquamosa A muricata and A cherimola which also showantioxidant hypoglycemic antihyperglycemic and hypolipi-demic properties [19 20 22ndash25] These data support the ideathat this species synthesizes similar secondary metabolitesthat act as antidiabetic directly by lowering glucose avail-ability in the gut or indirectly by decreasing oxidative stressandor lipids attenuating the effect of these two factors indisease Our results indicate that the control of postprandial
glycemia showed by A macroprophyllata involves an anti-hyperglycemic effect mediated by the regulation of glucoseuptake from the intestinal lumen through the inhibition ofcarbohydrate digestion putatively by inhibition of intestinal120572-glucosidases complex as observed in vitro for yeast 120572-glucosidase In addition the search for the active principleof the extract could generate a new 120572-glucosidase inhibitoruseful for the development of new antidiabetic or antiobesityagents
In conclusion our results indicate that A macroprophyl-lata aqueous extract has potent antihyperglycemic effectand according to the in vitro studies it acts as a competi-tive inhibitor of the 120572-glucosidase Last but not least this
BioMed Research International 5
70
60
50
40
30
20
10
0
minus10
0 20 40 60 80 100 120Minutes
Varia
tion
of g
lyce
mia
()
lowast lowast
lowastlowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowast
Figure 3 Effects of the crude extract of the leaves of A macro-prophyllata on blood glucose levels in rats on the OSTT Vehicle(◼) acarbose 5mgkg (998819) extract A macroprophyllata 100mgkg(e) extract A macroprophyllata 300mgkg (998771) and extract Amacroprophyllata 500mgkg (998787) Each value is the mean plusmn SEM for4 rats in each group lowast119875 lt 005 is significantly different t-test forcomparison with respect to negative control values at the same timeBaseline glycemia 77mgdL
study provides scientific support to use A macroprophyllataleaves in Mexican traditional medicine for the treatment ofDM2
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
This work was supported by grants from PAPIIT DGAPAUNAM (IN214812) and INMEGEN (062012l) F Brindisacknowledges postdoctoral fellowship awarded by DGAPA-UNAM
References
[1] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004
[2] W Kerner and J Bruckel ldquoDefinition classification and diag-nosis of diabetes mellitusrdquoDiabetologie und Stoffwechsel vol 4no 2 pp S115ndashS117 2009
[3] E Bonora and M Muggeo ldquoPostprandial blood glucose as arisk factor for cardiovascular disease in type II diabetes theepidemiological evidencerdquo Diabetologia vol 44 no 12 pp2107ndash2114 2001
[4] A Ceriello ldquoPostprandial hyperglycemia and diabetes compli-cations is it time to treatrdquoDiabetes vol 54 no 1 pp 1ndash7 2005
[5] R Quezada-Calvillo C C Robayo-Torres A R Opekun etal ldquoContribution of mucosal maltase-glucoamylase activitiesto mouse small intestinal starch 120572-glucogenesisrdquo Journal ofNutrition vol 137 no 7 pp 1725ndash1733 2007
[6] A J Krentz and C J Bailey ldquoOral antidiabetic agentsrdquo Drugsvol 65 no 3 pp 385ndash411 2005
[7] E Borges de Melo A da Silveira Gomes and I Carvalho ldquo120572-and 120573-glucosidase inhibitors chemical structure and biologicalactivityrdquo Tetrahedron vol 62 no 44 pp 10277ndash10302 2006
[8] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of the novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985
[9] A Horii M Emi N Tomita et al ldquoPrimary structure of humanpancreatic 120572-amylase gene its comparison with human salivary120572-amylase generdquo Gene vol 60 no 1 pp 57ndash64 1987
[10] Y-I Kwon E Apostolidis and K Shetty ldquoIn vitro studiesof eggplant (Solanum melongena) phenolics as inhibitors ofkey enzymes relevant for type 2 diabetes and hypertensionrdquoBioresource Technology vol 99 no 8 pp 2981ndash2988 2008
[11] Z J Canizares Las Frutas Anonaceas Ediciones Fruticus LaHabana Cuba 1966
[12] S E Ruiz and A L Morett Las Anonas en el Mexico Pre-hispanico Memorias Congreso Internacional de AnonaceasChapingo Estado de Mexico 169-186 1997
[13] W PopenoeManual of Tropical and Sub-Tropical Fruits HafnerPress New York NY USA 1920
[14] M E Gonzalez-Trujano A Navarrete B Reyes and E HongldquoSome pharmacological effects of the ethanol extract of leavesof Annona diversifolia on the central nervous system in micerdquoPhytotherapy Research vol 12 pp 600ndash602 1998
[15] M E Gonzalez-Trujano A Navarrete B Reyes E Cedillo-Portugal and E Hong ldquoAnticonvulsant properties and bio-guided isolation of palmitone from leaves of Annona diversifo-liardquo Planta Medica vol 67 no 2 pp 136ndash141 2001
[16] M E Gonzalez-Trujano E Tapia L Lopez-Meraz A Navar-rete A Reyes-Ramırez and AMartınez ldquoAnticonvulsant effectofAnnona diversifolia Saff and palmitone on penicillin-inducedconvulsive activity A behavioral and EEG study in ratsrdquoEpilepsia vol 47 no 11 pp 1810ndash1817 2006
[17] A I Carballo A L Martınez M E Gonzalez-Trujano etal ldquoAntinociceptive activity of Annona diversifolia Saff leafextracts and palmitone as a bioactive compoundrdquo Pharmacol-ogy Biochemistry and Behavior vol 95 no 1 pp 6ndash12 2010
[18] A Andrade-Cetto and M Heinrich ldquoMexican plants withhypoglycaemic effect used in the treatment of diabetesrdquo Journalof Ethnopharmacology vol 99 no 3 pp 325ndash348 2005
[19] A Shirwaikar K Rajendran C Dinesh Kumar and RBodla ldquoAntidiabetic activity of aqueous leaf extract of Annonasquamosa in streptozotocin-nicotinamide type 2 diabetic ratsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 171ndash175 2004
[20] D O Adeyemi O A Komolafe O S Adewole et alldquoAnti hyperglycemic activities of Annona muricata (Linn)rdquoAfrican Journal of Traditional Complementary and AlternativeMedicines vol 6 no 1 pp 62ndash69 2009
[21] S Cristians J A Guerrero-Analco A Perez-Vasquez et alldquoHypoglycemic activity of extracts and compounds from theleaves of Hintonia standleyana and H latiflora potential alter-natives to the use of the stem bark of these speciesrdquo Journal ofNatural Products vol 72 no 3 pp 408ndash413 2009
[22] R K Gupta A N Kesari P S Murthy R Chandra V Tandonand GWatal ldquoHypoglycemic and antidiabetic effect of ethano-lic extract of leaves of Annona squamosa L in experimentalanimalsrdquo Journal of Ethnopharmacology vol 99 no 1 pp 75ndash81 2005
6 BioMed Research International
[23] R K Gupta A N Kesari S Diwakar et al ldquoIn vivo evalu-ation of anti-oxidant and anti-lipidimic potential of Annonasquamosa aqueous extract in type 2 diabetic modelsrdquo Journalof Ethnopharmacology vol 118 no 1 pp 21ndash25 2008
[24] G Gupta-Elera A R Garrett A Martinez R A Robison andK L OrsquoNeill ldquoThe antioxidant properties of the cherimoya(Annona cherimola) fruitrdquo Food Research International vol 44no 7 pp 2205ndash2209 2011
[25] M R Loizzo R Tundis M Bonesi et al ldquoRadical scavengingantioxidant and metal chelating activities of Annona cherimolaMill (cherimoya) peel and pulp in relation to their total phe-nolic and total flavonoid contentsrdquo Journal of Food Compositionand Analysis vol 25 no 2 pp 179ndash184 2012
[26] N Kamalakkannan and P S Mainzen Prince ldquoRutin improvesthe antioxidant status in streptozotocin-induced diabetic rattissuesrdquo Molecular and Cellular Biochemistry vol 293 no 1-2pp 211ndash219 2006
[27] A A Henrique Fernandes E L Barbosa Novelli K Okoshi etal ldquoInfluence of rutin treatment on biochemical alterations inexperimental diabetesrdquo Biomedicine and Pharmacotherapy vol64 no 3 pp 214ndash219 2010
[28] O Hnatyszyn J Mino G Ferraro and C Acevedo ldquoThehypoglycemic effect of Phyllanthus sellowianus fractions instreptozotocin-induced diabetic micerdquo Phytomedicine vol 9no 6 pp 556ndash559 2002
[29] O M Ahmed A A Moneim I A Yazid and A MMahmoudldquoAntihyperglycemic antihyperlipidemic and antioxidant effectsand the probable mechanisms of action of Ruta graveolens infu-sion and rutin in nicotinamide-streptozotocin-induced diabeticratsrdquo Diabetologia Croatica vol 39 no 1 pp 15ndash35 2010
[30] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity potential targets forglucose homeostasisrdquo Nutrition vol 27 no 11-12 pp 1161ndash11672011
[31] S Adisakwattana S Yibchok-Anun P Charoenlertkul and NWongsasiripat ldquoCyanidin-3-rutinoside alleviates postprandialhyperglycemia and its synergism with acarbose by inhibitionof intestinal 120572-glucosidaserdquo Journal of Clinical Biochemistry andNutrition vol 49 no 1 pp 36ndash41 2011
[32] S Pashikanti D R de Alba G A Boissonneault and DCervantes-Laurean ldquoRutin metabolites novel inhibitors ofnonoxidative advanced glycation end productsrdquo Free RadicalBiology and Medicine vol 48 no 5 pp 656ndash663 2010
[33] Y Qin F Chao F Gao et al ldquoComparative evaluationof quercetin isoquercetin and rutin as inhibitors of 120572-glucosidaserdquo Journal of Agricultural and Food Chemistry vol 57no 24 pp 11463ndash11468 2009
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MEDIATORSINFLAMMATION
of
2 BioMed Research International
but have the disadvantage of undesirable side effects suchas abdominal distention flatulence meteorism and diar-rhea [9] Previous research indicates that side effects couldbe caused by excessive inhibition of pancreatic 120572-amylaseresulting in the abnormal bacterial fermentation of undi-gested carbohydrates in the colon Therefore more effectiveinhibitors for the enzymes should have strong inhibitoryeffect against 120572-glucosidase andmild inhibitory effect against120572-amylase which can be an effective therapy for managingpostprandial hyperglycemia with minimal side effects [10]
Annonaceae is a family of plants consisting of 2300 to2500 species included in more than 130 genera in fact it isthe largest family of the order Magnoliales Only four genera(Annona Rollinia Uvaria andAsimina) produce edible fruitssuch as annona [11ndash13] Annona macroprophyllata DonnSm is a species belonging to the Annona genus it is acommon tree in central Mexico known as ldquoilamardquo (Statesof Colima Guerrero and Mexico) whereas in the southeast(Tehuantepec region and Yucatan) it is called ldquopapaucerdquo orldquoanona blancardquo [14 15] The fruits of this plant are used asfood but its leaves are employed as anticonvulsant [16] aswell as analgesic and anti-inflammatory agents in traditionalMexicanmedicine [17] However so far there are no scientificreports supporting its probable antihyperglycemic proper-ties even though other species of the genus are known tohave those properties that isAnnona squamosa A muricataA glabra and A cherimola [18ndash20] then we have used achemotaxonomic criterion in order thatA macroprophyllatabesides its described medicinal properties [14ndash17] was inves-tigated to search for its likely antidiabetic properties
2 Materials and Methods
21 Plant Material and Extract Annona macroprophyllataDonn Sm specimens were collected in Tejupilco Guerreroin September 2010 Dr E Cedillo-Portugal botanist from theUniversidad Autonoma de Chapingo (UACH) certified theauthenticity of the plant and a voucher specimen (AN9702)was deposited in the herbarium ldquoHerbario de Plantas UtilesEfraım Hernandez Xrdquo at the UACH in the State of MexicoMexico for future reference Aqueous extract was obtainedby using 50 g of dried and powdered leaves in a process ofinfusion in 500mL of boiling water The aqueous extract wasseparated from the residues by gravity filtration samples werefrozen in liquid nitrogen and then lyophilized during 12 husing a Heto FD3 Lab lyophilizer to yield 111 g (222)
22 HPLC Quantification of Flavonoids An HPLC analysiswas performed using an Agilent Technologies Chromato-graph The separation was performed on an ODS HypersilC18 column (15mm times 4mm id and 5120583m particle size) Thesamples were injected through a 20 120583L loop The columnwas thermostatically controlled at 30∘C using a 1mLminflow rate The mobile phase consisted of 15 85 acetonitrile-trifluoroacetic acid solution at pH 25 The detection wasmonitored at 350 nm wavelength and the system was runfor 15min Calibration curves were done for the standards
kaempferol hesperidin naringenin naringin rutin iso-quercitrin and quercetin
23 120572-Glucosidase Assay In Vitro 120572-Glucosidase inhibitionwas measured at pH 70 and 30∘C using p-nitrophenyl-120572-D-glucopyranoside (pNPG) as substrate and 075 IUmLof yeast 120572-glucosidase in 01M sodium phosphate bufferAcarbose and aqueous extract were dissolved in phosphatebuffer and serial dilutions from 14 to 02mgmL werepreparedThe increments in absorption at 405 nm due to thehydrolysis of pNPG by the 120572-glucosidase were determinedon a microplate reader DTX 880 Multimode Detector fromBeckman Coulter Ten 120583L of acarbose or extract solution(in triplicate) were incubated during 5min with 20120583L ofenzyme stock After incubation 10 120583L of substrate wereadded and further incubated for 35min at 30∘C Finallythe reaction was stopped by adding 30 120583L of 5M Na
2CO3
The concentration required to inhibit the enzyme activity by50 (IC
50) was calculated by regression analysis using the
following equation
V =119860100
1 + (119868IC50)119904 (1)
where V is the percentage of inhibition A100
is the maximuminhibition I is the inhibitor concentration IC
50is the concen-
tration required to inhibit the enzyme activity by 50 and 119904is the cooperative degree
24 Enzyme Kinetics The mode of inhibition for 120572-glucosidase was determined by the Lineweaver-Burk plotsAll the results are expressed as the mean of at least threeexperiments plusmn SEM Kinetic parameters such as 119881
119898119870119898 and
119870119894were evaluated by using the nonlinear regression method
based on the following inhibition equation
V =Vmax119878
119870119898(1 + 119868119870
119894) + 119878 (1 + 119868119870
1015840
119894)
(2)
where V is the initial velocity in either the Presence or absenceof the inhibitor 119878 and 119868 are the concentrations of substrate andinhibitor respectively119881
119898is the maximum velocity119870
119898is the
Michaelis-Menten constant 119870119894is the competitive inhibition
constant and 1198701015840119894is the uncompetitive inhibition constant
The kinetic data were analyzed using a computer program fornonlinear regressions (Origin 80)
25 Experimental Animals MaleWistar normoglycemic ratsweighing 200ndash250 g were obtained from our animal facilitiesat Facultad de Estudios Superiores Iztacala UNAM Pro-cedures involving animals and their care fulfilled the Mex-ican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999) and were in compliance with internationalrules on care and use of laboratory animals Furthermoreclearance for conducting the studies was obtained from theEthics Committee for the Use of Animals in Pharmacologicaland Toxicological Testing of Facultad de Estudios SuperioresIztacala UNAM For the pharmacological studies groupsof four animals were used All doses are in mgkg of body
BioMed Research International 3
weightThe ratswere housed in groups of four under standardlaboratory conditions (12 h lightdark cycle at 22 plusmn 1∘C) andmaintained on a standard pellet diet and water ad libitum
26 Preparation of the Test Samples and Determinationof Glycemia All aqueous administrations were suspendedin 09 saline solution and given by intragastrical routeAcarbose (Sigma-Aldrich Co St Louis MO USA) wasused as antihyperglycemic drug Sucrose (Reasol ReactivosAnalıticosMexico) was used as carbohydrate to carry out thesucrose tolerance tests The control rats received only vehicle(saline solution) in the same volume (05mL of vehicle100 gof body weight) by the same route Blood samples werecollected from the caudal vein by performing a small incisionat the end of the tail Blood glucose (mgdL) was estimatedusing a commercial glucometer (Accu-Chek sensor RocheMannheim Germany) The variation of glycemia as percent-age for each group was calculated with respect to the initial(0 h) level according to the following equation
Variation of glycemia = [119866119905minus 119866119894
119866119894
] times 100 (3)
where 119866119894is the initial glycemia values and 119866
119905is the glycemia
value after treatments administration [21]
27 Oral Sucrose Tolerance Test (OSTT) Rats were fastedduring 12 h before the experiment having free access to waterThe crude extract of A macroprophyllata was tested at thedoses of 100 300 and 500mgkg Acarbose (5mgkg) wassuspended in the same vehicle Time 0min was set beforetreatmentwith the extract 30min later a sucrose load (3 gkg)was administered to the rats Blood samples were obtained 1530 60 90 and 120min after the carbohydrate load [21]
28 Statistical Analysis Data represent the mean plusmn SEM of119899 = 4 rats for the OSTT and 119899 = 3 assays for in vitroexperiments Differences were analyzed using Studentrsquos 119905-test (Sigma Stat version 30) and significant differences wereconsidered at 119875 lt 005
3 Results and Discussion
In Mexico it has been reported the use of A muricata Aglabra and A cherimola as antidiabetic species [18] whichsuggested to us that A macroprophyllata is an interestingspecies to study since it may have the same metabolic path-ways that synthesize the compounds as the other membersof the genus and thus may show their therapeutic propertiesThat is we tested a chemotaxonomic criterion for the study
Elution of A macroprophyllata leaves aqueous extractyielded rutin (288 120583gmg of crude extract RT 3973min)and isoquercitrin (071120583gmg of crude extract RT 6872) aswell as some traces of astragalinisoquercitrin (003 120583gmg ofcrude extract) (Figure 1)
Annona macroprophyllata ethanolic extract has beeninvestigated as antinociceptive anti-inflammatory andanticonvulsant agents [15ndash17] however the species has not
been tested for antidiabetic actions as their counterpartshave done [19 20 22ndash25] Our study found rutin as one of itscomponents as well as isoquercitrin these two flavonoidsalong with quercetin have been tested on diabetic rats thatis rutin decreased fasting plasma glucose increased insulinlevels and improved the antioxidant status by decreasinglipid peroxidative products and increasing enzymic andnonenzymic antioxidants [26] Rutin has been associatedwith marked decrease of hepatic and cardiac levels oftriglycerides and elevated glycogen which suggest thatrutin can improve hyperglycemia and dyslipidemia whileinhibiting the progression of liver and heart dysfunction inSTZ-induced diabetic rats [27] and rutin has hypoglycemiceffect in normal and diabetic rats [28ndash30] Rutin metabolitesare capable of inhibiting 120572-glucosidase activity both in vivoand in vitro [31] and the formation of advanced glycationend products formed via protein glycation which correlateswith aging and diabetes complications [32]
To our knowledge this is the first report that the aqueousextract from A macroprophyllata inhibited the activity ofyeast 120572-glucosidase with a low IC
50(118mgmL) very
similar to that of acarbose (027mgmL) This result agreeswith that reported for rutin as inhibitor of 120572-glucosidase(IC50= 0196mM) compared with acarbose (IC
500091mM)
[33] therefore rutin might participate in the aqueous extractof A macroprophyllata on the 120572-glucosidase inhibition andantihyperglycemic effects
The next step was to determine the nature of the inhibi-tion exerted by the aqueous extract of A macroprophyllatathen a kinetic analysis of the inhibition of enzyme activityusing different amounts of aqueous extract or acarbose wasconducted The results yielded a 119870
119894of 097mgmL and 119870
119894
of 079mgmL to aqueous extract and acarbose respectivelyThese values show that the extract inhibited the enzymeand could be used to control postprandial hyperglycemiaLineweaver-Burk plots for 120572-glucosidase from yeast in thepresence of acarbose or A macroprophyllata extract atdifferent concentrations (Figure 2) revealed typical curvesfor competitive inhibitors implying binding to the catalyticsite Then we suggest that disaccharidases are targets offlavonoids in the regulation of glucose release and conse-quently glucose absorption by the gut This type of inhibitorhas several advantages over others that is its inhibition isnot permanent and undesirable effects are easily attenuatedwith decreasing dose therefore the aqueous extract ofA macroprophyllata represents a good alternative to avoidhyperglycemia
On the other hand to confirmwhether our results in vitrocould be reproduced in a whole organism the response onglycemia after single oral sucrose ingestion was examinedfor A macroprophyllata As observed in Figure 3 the testextract significantly avoided the hyperglycemic responseat doses of 100 300 and 500mgkg compared to control(119875 lt 005) The postprandial blood glucose peak wasdiminished from 15min in all three doses (100 300 and500mgkg) however the highest activity was 300 gt 500gt 100mgkg throughout the time-curve postingestion ofsucrose The glycemic lowering effect of A macroprophyllataat doses of 100 300 and 500mgkg was not greater than that
4 BioMed Research International
O
O
O
Isoquercitrin
OHO
HO
HO
HO
HO
HO
O
OOH
OHOH
OH
OH
OHOH
OH
OH
OH
OH
OHOO
Rutin
CH3
CH2
Figure 1 Structure of flavonoids rutin and isoquercitrin
1V
(mM
min
)minus1
1[pNPG (mM)]
60
50
40
30
20
10
0
minus10
minus2 0 2 4 6 8 10 12 14 16 18
(a)
1V
(mM
min
)minus1
70
60
50
40
30
20
10
0
minus10
1[pNPG (mM)]minus2 0 2 4 6 8 10 12 14 16 18
(b)
Figure 2 Lineweaver-Burk plots of 120572-glucosidase inhibition at different concentrations of (a) acarbose and (b) extract aqueous from Amacroprophyllata 00mgmL (◼) 02mgmL (e) 04mgmL (998771) 06mgmL (998787) 08mgmL (998819) 10mgmL (998835) 12mgmL (⧫) 14mgmL(C)
with acarbose a therapeutic drug used as positive control(5mgkg) however maximum effect obtained at a dose of300mgkg was not statistically different with acarbose dosefrom 60 min on results in vivo that agree to the reportedfor other species of the genus Annona as is the case of Asquamosa A muricata and A cherimola which also showantioxidant hypoglycemic antihyperglycemic and hypolipi-demic properties [19 20 22ndash25] These data support the ideathat this species synthesizes similar secondary metabolitesthat act as antidiabetic directly by lowering glucose avail-ability in the gut or indirectly by decreasing oxidative stressandor lipids attenuating the effect of these two factors indisease Our results indicate that the control of postprandial
glycemia showed by A macroprophyllata involves an anti-hyperglycemic effect mediated by the regulation of glucoseuptake from the intestinal lumen through the inhibition ofcarbohydrate digestion putatively by inhibition of intestinal120572-glucosidases complex as observed in vitro for yeast 120572-glucosidase In addition the search for the active principleof the extract could generate a new 120572-glucosidase inhibitoruseful for the development of new antidiabetic or antiobesityagents
In conclusion our results indicate that A macroprophyl-lata aqueous extract has potent antihyperglycemic effectand according to the in vitro studies it acts as a competi-tive inhibitor of the 120572-glucosidase Last but not least this
BioMed Research International 5
70
60
50
40
30
20
10
0
minus10
0 20 40 60 80 100 120Minutes
Varia
tion
of g
lyce
mia
()
lowast lowast
lowastlowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowast
Figure 3 Effects of the crude extract of the leaves of A macro-prophyllata on blood glucose levels in rats on the OSTT Vehicle(◼) acarbose 5mgkg (998819) extract A macroprophyllata 100mgkg(e) extract A macroprophyllata 300mgkg (998771) and extract Amacroprophyllata 500mgkg (998787) Each value is the mean plusmn SEM for4 rats in each group lowast119875 lt 005 is significantly different t-test forcomparison with respect to negative control values at the same timeBaseline glycemia 77mgdL
study provides scientific support to use A macroprophyllataleaves in Mexican traditional medicine for the treatment ofDM2
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
This work was supported by grants from PAPIIT DGAPAUNAM (IN214812) and INMEGEN (062012l) F Brindisacknowledges postdoctoral fellowship awarded by DGAPA-UNAM
References
[1] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004
[2] W Kerner and J Bruckel ldquoDefinition classification and diag-nosis of diabetes mellitusrdquoDiabetologie und Stoffwechsel vol 4no 2 pp S115ndashS117 2009
[3] E Bonora and M Muggeo ldquoPostprandial blood glucose as arisk factor for cardiovascular disease in type II diabetes theepidemiological evidencerdquo Diabetologia vol 44 no 12 pp2107ndash2114 2001
[4] A Ceriello ldquoPostprandial hyperglycemia and diabetes compli-cations is it time to treatrdquoDiabetes vol 54 no 1 pp 1ndash7 2005
[5] R Quezada-Calvillo C C Robayo-Torres A R Opekun etal ldquoContribution of mucosal maltase-glucoamylase activitiesto mouse small intestinal starch 120572-glucogenesisrdquo Journal ofNutrition vol 137 no 7 pp 1725ndash1733 2007
[6] A J Krentz and C J Bailey ldquoOral antidiabetic agentsrdquo Drugsvol 65 no 3 pp 385ndash411 2005
[7] E Borges de Melo A da Silveira Gomes and I Carvalho ldquo120572-and 120573-glucosidase inhibitors chemical structure and biologicalactivityrdquo Tetrahedron vol 62 no 44 pp 10277ndash10302 2006
[8] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of the novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985
[9] A Horii M Emi N Tomita et al ldquoPrimary structure of humanpancreatic 120572-amylase gene its comparison with human salivary120572-amylase generdquo Gene vol 60 no 1 pp 57ndash64 1987
[10] Y-I Kwon E Apostolidis and K Shetty ldquoIn vitro studiesof eggplant (Solanum melongena) phenolics as inhibitors ofkey enzymes relevant for type 2 diabetes and hypertensionrdquoBioresource Technology vol 99 no 8 pp 2981ndash2988 2008
[11] Z J Canizares Las Frutas Anonaceas Ediciones Fruticus LaHabana Cuba 1966
[12] S E Ruiz and A L Morett Las Anonas en el Mexico Pre-hispanico Memorias Congreso Internacional de AnonaceasChapingo Estado de Mexico 169-186 1997
[13] W PopenoeManual of Tropical and Sub-Tropical Fruits HafnerPress New York NY USA 1920
[14] M E Gonzalez-Trujano A Navarrete B Reyes and E HongldquoSome pharmacological effects of the ethanol extract of leavesof Annona diversifolia on the central nervous system in micerdquoPhytotherapy Research vol 12 pp 600ndash602 1998
[15] M E Gonzalez-Trujano A Navarrete B Reyes E Cedillo-Portugal and E Hong ldquoAnticonvulsant properties and bio-guided isolation of palmitone from leaves of Annona diversifo-liardquo Planta Medica vol 67 no 2 pp 136ndash141 2001
[16] M E Gonzalez-Trujano E Tapia L Lopez-Meraz A Navar-rete A Reyes-Ramırez and AMartınez ldquoAnticonvulsant effectofAnnona diversifolia Saff and palmitone on penicillin-inducedconvulsive activity A behavioral and EEG study in ratsrdquoEpilepsia vol 47 no 11 pp 1810ndash1817 2006
[17] A I Carballo A L Martınez M E Gonzalez-Trujano etal ldquoAntinociceptive activity of Annona diversifolia Saff leafextracts and palmitone as a bioactive compoundrdquo Pharmacol-ogy Biochemistry and Behavior vol 95 no 1 pp 6ndash12 2010
[18] A Andrade-Cetto and M Heinrich ldquoMexican plants withhypoglycaemic effect used in the treatment of diabetesrdquo Journalof Ethnopharmacology vol 99 no 3 pp 325ndash348 2005
[19] A Shirwaikar K Rajendran C Dinesh Kumar and RBodla ldquoAntidiabetic activity of aqueous leaf extract of Annonasquamosa in streptozotocin-nicotinamide type 2 diabetic ratsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 171ndash175 2004
[20] D O Adeyemi O A Komolafe O S Adewole et alldquoAnti hyperglycemic activities of Annona muricata (Linn)rdquoAfrican Journal of Traditional Complementary and AlternativeMedicines vol 6 no 1 pp 62ndash69 2009
[21] S Cristians J A Guerrero-Analco A Perez-Vasquez et alldquoHypoglycemic activity of extracts and compounds from theleaves of Hintonia standleyana and H latiflora potential alter-natives to the use of the stem bark of these speciesrdquo Journal ofNatural Products vol 72 no 3 pp 408ndash413 2009
[22] R K Gupta A N Kesari P S Murthy R Chandra V Tandonand GWatal ldquoHypoglycemic and antidiabetic effect of ethano-lic extract of leaves of Annona squamosa L in experimentalanimalsrdquo Journal of Ethnopharmacology vol 99 no 1 pp 75ndash81 2005
6 BioMed Research International
[23] R K Gupta A N Kesari S Diwakar et al ldquoIn vivo evalu-ation of anti-oxidant and anti-lipidimic potential of Annonasquamosa aqueous extract in type 2 diabetic modelsrdquo Journalof Ethnopharmacology vol 118 no 1 pp 21ndash25 2008
[24] G Gupta-Elera A R Garrett A Martinez R A Robison andK L OrsquoNeill ldquoThe antioxidant properties of the cherimoya(Annona cherimola) fruitrdquo Food Research International vol 44no 7 pp 2205ndash2209 2011
[25] M R Loizzo R Tundis M Bonesi et al ldquoRadical scavengingantioxidant and metal chelating activities of Annona cherimolaMill (cherimoya) peel and pulp in relation to their total phe-nolic and total flavonoid contentsrdquo Journal of Food Compositionand Analysis vol 25 no 2 pp 179ndash184 2012
[26] N Kamalakkannan and P S Mainzen Prince ldquoRutin improvesthe antioxidant status in streptozotocin-induced diabetic rattissuesrdquo Molecular and Cellular Biochemistry vol 293 no 1-2pp 211ndash219 2006
[27] A A Henrique Fernandes E L Barbosa Novelli K Okoshi etal ldquoInfluence of rutin treatment on biochemical alterations inexperimental diabetesrdquo Biomedicine and Pharmacotherapy vol64 no 3 pp 214ndash219 2010
[28] O Hnatyszyn J Mino G Ferraro and C Acevedo ldquoThehypoglycemic effect of Phyllanthus sellowianus fractions instreptozotocin-induced diabetic micerdquo Phytomedicine vol 9no 6 pp 556ndash559 2002
[29] O M Ahmed A A Moneim I A Yazid and A MMahmoudldquoAntihyperglycemic antihyperlipidemic and antioxidant effectsand the probable mechanisms of action of Ruta graveolens infu-sion and rutin in nicotinamide-streptozotocin-induced diabeticratsrdquo Diabetologia Croatica vol 39 no 1 pp 15ndash35 2010
[30] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity potential targets forglucose homeostasisrdquo Nutrition vol 27 no 11-12 pp 1161ndash11672011
[31] S Adisakwattana S Yibchok-Anun P Charoenlertkul and NWongsasiripat ldquoCyanidin-3-rutinoside alleviates postprandialhyperglycemia and its synergism with acarbose by inhibitionof intestinal 120572-glucosidaserdquo Journal of Clinical Biochemistry andNutrition vol 49 no 1 pp 36ndash41 2011
[32] S Pashikanti D R de Alba G A Boissonneault and DCervantes-Laurean ldquoRutin metabolites novel inhibitors ofnonoxidative advanced glycation end productsrdquo Free RadicalBiology and Medicine vol 48 no 5 pp 656ndash663 2010
[33] Y Qin F Chao F Gao et al ldquoComparative evaluationof quercetin isoquercetin and rutin as inhibitors of 120572-glucosidaserdquo Journal of Agricultural and Food Chemistry vol 57no 24 pp 11463ndash11468 2009
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
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Pharmaceutics
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MEDIATORSINFLAMMATION
of
BioMed Research International 3
weightThe ratswere housed in groups of four under standardlaboratory conditions (12 h lightdark cycle at 22 plusmn 1∘C) andmaintained on a standard pellet diet and water ad libitum
26 Preparation of the Test Samples and Determinationof Glycemia All aqueous administrations were suspendedin 09 saline solution and given by intragastrical routeAcarbose (Sigma-Aldrich Co St Louis MO USA) wasused as antihyperglycemic drug Sucrose (Reasol ReactivosAnalıticosMexico) was used as carbohydrate to carry out thesucrose tolerance tests The control rats received only vehicle(saline solution) in the same volume (05mL of vehicle100 gof body weight) by the same route Blood samples werecollected from the caudal vein by performing a small incisionat the end of the tail Blood glucose (mgdL) was estimatedusing a commercial glucometer (Accu-Chek sensor RocheMannheim Germany) The variation of glycemia as percent-age for each group was calculated with respect to the initial(0 h) level according to the following equation
Variation of glycemia = [119866119905minus 119866119894
119866119894
] times 100 (3)
where 119866119894is the initial glycemia values and 119866
119905is the glycemia
value after treatments administration [21]
27 Oral Sucrose Tolerance Test (OSTT) Rats were fastedduring 12 h before the experiment having free access to waterThe crude extract of A macroprophyllata was tested at thedoses of 100 300 and 500mgkg Acarbose (5mgkg) wassuspended in the same vehicle Time 0min was set beforetreatmentwith the extract 30min later a sucrose load (3 gkg)was administered to the rats Blood samples were obtained 1530 60 90 and 120min after the carbohydrate load [21]
28 Statistical Analysis Data represent the mean plusmn SEM of119899 = 4 rats for the OSTT and 119899 = 3 assays for in vitroexperiments Differences were analyzed using Studentrsquos 119905-test (Sigma Stat version 30) and significant differences wereconsidered at 119875 lt 005
3 Results and Discussion
In Mexico it has been reported the use of A muricata Aglabra and A cherimola as antidiabetic species [18] whichsuggested to us that A macroprophyllata is an interestingspecies to study since it may have the same metabolic path-ways that synthesize the compounds as the other membersof the genus and thus may show their therapeutic propertiesThat is we tested a chemotaxonomic criterion for the study
Elution of A macroprophyllata leaves aqueous extractyielded rutin (288 120583gmg of crude extract RT 3973min)and isoquercitrin (071120583gmg of crude extract RT 6872) aswell as some traces of astragalinisoquercitrin (003 120583gmg ofcrude extract) (Figure 1)
Annona macroprophyllata ethanolic extract has beeninvestigated as antinociceptive anti-inflammatory andanticonvulsant agents [15ndash17] however the species has not
been tested for antidiabetic actions as their counterpartshave done [19 20 22ndash25] Our study found rutin as one of itscomponents as well as isoquercitrin these two flavonoidsalong with quercetin have been tested on diabetic rats thatis rutin decreased fasting plasma glucose increased insulinlevels and improved the antioxidant status by decreasinglipid peroxidative products and increasing enzymic andnonenzymic antioxidants [26] Rutin has been associatedwith marked decrease of hepatic and cardiac levels oftriglycerides and elevated glycogen which suggest thatrutin can improve hyperglycemia and dyslipidemia whileinhibiting the progression of liver and heart dysfunction inSTZ-induced diabetic rats [27] and rutin has hypoglycemiceffect in normal and diabetic rats [28ndash30] Rutin metabolitesare capable of inhibiting 120572-glucosidase activity both in vivoand in vitro [31] and the formation of advanced glycationend products formed via protein glycation which correlateswith aging and diabetes complications [32]
To our knowledge this is the first report that the aqueousextract from A macroprophyllata inhibited the activity ofyeast 120572-glucosidase with a low IC
50(118mgmL) very
similar to that of acarbose (027mgmL) This result agreeswith that reported for rutin as inhibitor of 120572-glucosidase(IC50= 0196mM) compared with acarbose (IC
500091mM)
[33] therefore rutin might participate in the aqueous extractof A macroprophyllata on the 120572-glucosidase inhibition andantihyperglycemic effects
The next step was to determine the nature of the inhibi-tion exerted by the aqueous extract of A macroprophyllatathen a kinetic analysis of the inhibition of enzyme activityusing different amounts of aqueous extract or acarbose wasconducted The results yielded a 119870
119894of 097mgmL and 119870
119894
of 079mgmL to aqueous extract and acarbose respectivelyThese values show that the extract inhibited the enzymeand could be used to control postprandial hyperglycemiaLineweaver-Burk plots for 120572-glucosidase from yeast in thepresence of acarbose or A macroprophyllata extract atdifferent concentrations (Figure 2) revealed typical curvesfor competitive inhibitors implying binding to the catalyticsite Then we suggest that disaccharidases are targets offlavonoids in the regulation of glucose release and conse-quently glucose absorption by the gut This type of inhibitorhas several advantages over others that is its inhibition isnot permanent and undesirable effects are easily attenuatedwith decreasing dose therefore the aqueous extract ofA macroprophyllata represents a good alternative to avoidhyperglycemia
On the other hand to confirmwhether our results in vitrocould be reproduced in a whole organism the response onglycemia after single oral sucrose ingestion was examinedfor A macroprophyllata As observed in Figure 3 the testextract significantly avoided the hyperglycemic responseat doses of 100 300 and 500mgkg compared to control(119875 lt 005) The postprandial blood glucose peak wasdiminished from 15min in all three doses (100 300 and500mgkg) however the highest activity was 300 gt 500gt 100mgkg throughout the time-curve postingestion ofsucrose The glycemic lowering effect of A macroprophyllataat doses of 100 300 and 500mgkg was not greater than that
4 BioMed Research International
O
O
O
Isoquercitrin
OHO
HO
HO
HO
HO
HO
O
OOH
OHOH
OH
OH
OHOH
OH
OH
OH
OH
OHOO
Rutin
CH3
CH2
Figure 1 Structure of flavonoids rutin and isoquercitrin
1V
(mM
min
)minus1
1[pNPG (mM)]
60
50
40
30
20
10
0
minus10
minus2 0 2 4 6 8 10 12 14 16 18
(a)
1V
(mM
min
)minus1
70
60
50
40
30
20
10
0
minus10
1[pNPG (mM)]minus2 0 2 4 6 8 10 12 14 16 18
(b)
Figure 2 Lineweaver-Burk plots of 120572-glucosidase inhibition at different concentrations of (a) acarbose and (b) extract aqueous from Amacroprophyllata 00mgmL (◼) 02mgmL (e) 04mgmL (998771) 06mgmL (998787) 08mgmL (998819) 10mgmL (998835) 12mgmL (⧫) 14mgmL(C)
with acarbose a therapeutic drug used as positive control(5mgkg) however maximum effect obtained at a dose of300mgkg was not statistically different with acarbose dosefrom 60 min on results in vivo that agree to the reportedfor other species of the genus Annona as is the case of Asquamosa A muricata and A cherimola which also showantioxidant hypoglycemic antihyperglycemic and hypolipi-demic properties [19 20 22ndash25] These data support the ideathat this species synthesizes similar secondary metabolitesthat act as antidiabetic directly by lowering glucose avail-ability in the gut or indirectly by decreasing oxidative stressandor lipids attenuating the effect of these two factors indisease Our results indicate that the control of postprandial
glycemia showed by A macroprophyllata involves an anti-hyperglycemic effect mediated by the regulation of glucoseuptake from the intestinal lumen through the inhibition ofcarbohydrate digestion putatively by inhibition of intestinal120572-glucosidases complex as observed in vitro for yeast 120572-glucosidase In addition the search for the active principleof the extract could generate a new 120572-glucosidase inhibitoruseful for the development of new antidiabetic or antiobesityagents
In conclusion our results indicate that A macroprophyl-lata aqueous extract has potent antihyperglycemic effectand according to the in vitro studies it acts as a competi-tive inhibitor of the 120572-glucosidase Last but not least this
BioMed Research International 5
70
60
50
40
30
20
10
0
minus10
0 20 40 60 80 100 120Minutes
Varia
tion
of g
lyce
mia
()
lowast lowast
lowastlowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowast
Figure 3 Effects of the crude extract of the leaves of A macro-prophyllata on blood glucose levels in rats on the OSTT Vehicle(◼) acarbose 5mgkg (998819) extract A macroprophyllata 100mgkg(e) extract A macroprophyllata 300mgkg (998771) and extract Amacroprophyllata 500mgkg (998787) Each value is the mean plusmn SEM for4 rats in each group lowast119875 lt 005 is significantly different t-test forcomparison with respect to negative control values at the same timeBaseline glycemia 77mgdL
study provides scientific support to use A macroprophyllataleaves in Mexican traditional medicine for the treatment ofDM2
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
This work was supported by grants from PAPIIT DGAPAUNAM (IN214812) and INMEGEN (062012l) F Brindisacknowledges postdoctoral fellowship awarded by DGAPA-UNAM
References
[1] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004
[2] W Kerner and J Bruckel ldquoDefinition classification and diag-nosis of diabetes mellitusrdquoDiabetologie und Stoffwechsel vol 4no 2 pp S115ndashS117 2009
[3] E Bonora and M Muggeo ldquoPostprandial blood glucose as arisk factor for cardiovascular disease in type II diabetes theepidemiological evidencerdquo Diabetologia vol 44 no 12 pp2107ndash2114 2001
[4] A Ceriello ldquoPostprandial hyperglycemia and diabetes compli-cations is it time to treatrdquoDiabetes vol 54 no 1 pp 1ndash7 2005
[5] R Quezada-Calvillo C C Robayo-Torres A R Opekun etal ldquoContribution of mucosal maltase-glucoamylase activitiesto mouse small intestinal starch 120572-glucogenesisrdquo Journal ofNutrition vol 137 no 7 pp 1725ndash1733 2007
[6] A J Krentz and C J Bailey ldquoOral antidiabetic agentsrdquo Drugsvol 65 no 3 pp 385ndash411 2005
[7] E Borges de Melo A da Silveira Gomes and I Carvalho ldquo120572-and 120573-glucosidase inhibitors chemical structure and biologicalactivityrdquo Tetrahedron vol 62 no 44 pp 10277ndash10302 2006
[8] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of the novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985
[9] A Horii M Emi N Tomita et al ldquoPrimary structure of humanpancreatic 120572-amylase gene its comparison with human salivary120572-amylase generdquo Gene vol 60 no 1 pp 57ndash64 1987
[10] Y-I Kwon E Apostolidis and K Shetty ldquoIn vitro studiesof eggplant (Solanum melongena) phenolics as inhibitors ofkey enzymes relevant for type 2 diabetes and hypertensionrdquoBioresource Technology vol 99 no 8 pp 2981ndash2988 2008
[11] Z J Canizares Las Frutas Anonaceas Ediciones Fruticus LaHabana Cuba 1966
[12] S E Ruiz and A L Morett Las Anonas en el Mexico Pre-hispanico Memorias Congreso Internacional de AnonaceasChapingo Estado de Mexico 169-186 1997
[13] W PopenoeManual of Tropical and Sub-Tropical Fruits HafnerPress New York NY USA 1920
[14] M E Gonzalez-Trujano A Navarrete B Reyes and E HongldquoSome pharmacological effects of the ethanol extract of leavesof Annona diversifolia on the central nervous system in micerdquoPhytotherapy Research vol 12 pp 600ndash602 1998
[15] M E Gonzalez-Trujano A Navarrete B Reyes E Cedillo-Portugal and E Hong ldquoAnticonvulsant properties and bio-guided isolation of palmitone from leaves of Annona diversifo-liardquo Planta Medica vol 67 no 2 pp 136ndash141 2001
[16] M E Gonzalez-Trujano E Tapia L Lopez-Meraz A Navar-rete A Reyes-Ramırez and AMartınez ldquoAnticonvulsant effectofAnnona diversifolia Saff and palmitone on penicillin-inducedconvulsive activity A behavioral and EEG study in ratsrdquoEpilepsia vol 47 no 11 pp 1810ndash1817 2006
[17] A I Carballo A L Martınez M E Gonzalez-Trujano etal ldquoAntinociceptive activity of Annona diversifolia Saff leafextracts and palmitone as a bioactive compoundrdquo Pharmacol-ogy Biochemistry and Behavior vol 95 no 1 pp 6ndash12 2010
[18] A Andrade-Cetto and M Heinrich ldquoMexican plants withhypoglycaemic effect used in the treatment of diabetesrdquo Journalof Ethnopharmacology vol 99 no 3 pp 325ndash348 2005
[19] A Shirwaikar K Rajendran C Dinesh Kumar and RBodla ldquoAntidiabetic activity of aqueous leaf extract of Annonasquamosa in streptozotocin-nicotinamide type 2 diabetic ratsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 171ndash175 2004
[20] D O Adeyemi O A Komolafe O S Adewole et alldquoAnti hyperglycemic activities of Annona muricata (Linn)rdquoAfrican Journal of Traditional Complementary and AlternativeMedicines vol 6 no 1 pp 62ndash69 2009
[21] S Cristians J A Guerrero-Analco A Perez-Vasquez et alldquoHypoglycemic activity of extracts and compounds from theleaves of Hintonia standleyana and H latiflora potential alter-natives to the use of the stem bark of these speciesrdquo Journal ofNatural Products vol 72 no 3 pp 408ndash413 2009
[22] R K Gupta A N Kesari P S Murthy R Chandra V Tandonand GWatal ldquoHypoglycemic and antidiabetic effect of ethano-lic extract of leaves of Annona squamosa L in experimentalanimalsrdquo Journal of Ethnopharmacology vol 99 no 1 pp 75ndash81 2005
6 BioMed Research International
[23] R K Gupta A N Kesari S Diwakar et al ldquoIn vivo evalu-ation of anti-oxidant and anti-lipidimic potential of Annonasquamosa aqueous extract in type 2 diabetic modelsrdquo Journalof Ethnopharmacology vol 118 no 1 pp 21ndash25 2008
[24] G Gupta-Elera A R Garrett A Martinez R A Robison andK L OrsquoNeill ldquoThe antioxidant properties of the cherimoya(Annona cherimola) fruitrdquo Food Research International vol 44no 7 pp 2205ndash2209 2011
[25] M R Loizzo R Tundis M Bonesi et al ldquoRadical scavengingantioxidant and metal chelating activities of Annona cherimolaMill (cherimoya) peel and pulp in relation to their total phe-nolic and total flavonoid contentsrdquo Journal of Food Compositionand Analysis vol 25 no 2 pp 179ndash184 2012
[26] N Kamalakkannan and P S Mainzen Prince ldquoRutin improvesthe antioxidant status in streptozotocin-induced diabetic rattissuesrdquo Molecular and Cellular Biochemistry vol 293 no 1-2pp 211ndash219 2006
[27] A A Henrique Fernandes E L Barbosa Novelli K Okoshi etal ldquoInfluence of rutin treatment on biochemical alterations inexperimental diabetesrdquo Biomedicine and Pharmacotherapy vol64 no 3 pp 214ndash219 2010
[28] O Hnatyszyn J Mino G Ferraro and C Acevedo ldquoThehypoglycemic effect of Phyllanthus sellowianus fractions instreptozotocin-induced diabetic micerdquo Phytomedicine vol 9no 6 pp 556ndash559 2002
[29] O M Ahmed A A Moneim I A Yazid and A MMahmoudldquoAntihyperglycemic antihyperlipidemic and antioxidant effectsand the probable mechanisms of action of Ruta graveolens infu-sion and rutin in nicotinamide-streptozotocin-induced diabeticratsrdquo Diabetologia Croatica vol 39 no 1 pp 15ndash35 2010
[30] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity potential targets forglucose homeostasisrdquo Nutrition vol 27 no 11-12 pp 1161ndash11672011
[31] S Adisakwattana S Yibchok-Anun P Charoenlertkul and NWongsasiripat ldquoCyanidin-3-rutinoside alleviates postprandialhyperglycemia and its synergism with acarbose by inhibitionof intestinal 120572-glucosidaserdquo Journal of Clinical Biochemistry andNutrition vol 49 no 1 pp 36ndash41 2011
[32] S Pashikanti D R de Alba G A Boissonneault and DCervantes-Laurean ldquoRutin metabolites novel inhibitors ofnonoxidative advanced glycation end productsrdquo Free RadicalBiology and Medicine vol 48 no 5 pp 656ndash663 2010
[33] Y Qin F Chao F Gao et al ldquoComparative evaluationof quercetin isoquercetin and rutin as inhibitors of 120572-glucosidaserdquo Journal of Agricultural and Food Chemistry vol 57no 24 pp 11463ndash11468 2009
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
4 BioMed Research International
O
O
O
Isoquercitrin
OHO
HO
HO
HO
HO
HO
O
OOH
OHOH
OH
OH
OHOH
OH
OH
OH
OH
OHOO
Rutin
CH3
CH2
Figure 1 Structure of flavonoids rutin and isoquercitrin
1V
(mM
min
)minus1
1[pNPG (mM)]
60
50
40
30
20
10
0
minus10
minus2 0 2 4 6 8 10 12 14 16 18
(a)
1V
(mM
min
)minus1
70
60
50
40
30
20
10
0
minus10
1[pNPG (mM)]minus2 0 2 4 6 8 10 12 14 16 18
(b)
Figure 2 Lineweaver-Burk plots of 120572-glucosidase inhibition at different concentrations of (a) acarbose and (b) extract aqueous from Amacroprophyllata 00mgmL (◼) 02mgmL (e) 04mgmL (998771) 06mgmL (998787) 08mgmL (998819) 10mgmL (998835) 12mgmL (⧫) 14mgmL(C)
with acarbose a therapeutic drug used as positive control(5mgkg) however maximum effect obtained at a dose of300mgkg was not statistically different with acarbose dosefrom 60 min on results in vivo that agree to the reportedfor other species of the genus Annona as is the case of Asquamosa A muricata and A cherimola which also showantioxidant hypoglycemic antihyperglycemic and hypolipi-demic properties [19 20 22ndash25] These data support the ideathat this species synthesizes similar secondary metabolitesthat act as antidiabetic directly by lowering glucose avail-ability in the gut or indirectly by decreasing oxidative stressandor lipids attenuating the effect of these two factors indisease Our results indicate that the control of postprandial
glycemia showed by A macroprophyllata involves an anti-hyperglycemic effect mediated by the regulation of glucoseuptake from the intestinal lumen through the inhibition ofcarbohydrate digestion putatively by inhibition of intestinal120572-glucosidases complex as observed in vitro for yeast 120572-glucosidase In addition the search for the active principleof the extract could generate a new 120572-glucosidase inhibitoruseful for the development of new antidiabetic or antiobesityagents
In conclusion our results indicate that A macroprophyl-lata aqueous extract has potent antihyperglycemic effectand according to the in vitro studies it acts as a competi-tive inhibitor of the 120572-glucosidase Last but not least this
BioMed Research International 5
70
60
50
40
30
20
10
0
minus10
0 20 40 60 80 100 120Minutes
Varia
tion
of g
lyce
mia
()
lowast lowast
lowastlowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowast
Figure 3 Effects of the crude extract of the leaves of A macro-prophyllata on blood glucose levels in rats on the OSTT Vehicle(◼) acarbose 5mgkg (998819) extract A macroprophyllata 100mgkg(e) extract A macroprophyllata 300mgkg (998771) and extract Amacroprophyllata 500mgkg (998787) Each value is the mean plusmn SEM for4 rats in each group lowast119875 lt 005 is significantly different t-test forcomparison with respect to negative control values at the same timeBaseline glycemia 77mgdL
study provides scientific support to use A macroprophyllataleaves in Mexican traditional medicine for the treatment ofDM2
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
This work was supported by grants from PAPIIT DGAPAUNAM (IN214812) and INMEGEN (062012l) F Brindisacknowledges postdoctoral fellowship awarded by DGAPA-UNAM
References
[1] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004
[2] W Kerner and J Bruckel ldquoDefinition classification and diag-nosis of diabetes mellitusrdquoDiabetologie und Stoffwechsel vol 4no 2 pp S115ndashS117 2009
[3] E Bonora and M Muggeo ldquoPostprandial blood glucose as arisk factor for cardiovascular disease in type II diabetes theepidemiological evidencerdquo Diabetologia vol 44 no 12 pp2107ndash2114 2001
[4] A Ceriello ldquoPostprandial hyperglycemia and diabetes compli-cations is it time to treatrdquoDiabetes vol 54 no 1 pp 1ndash7 2005
[5] R Quezada-Calvillo C C Robayo-Torres A R Opekun etal ldquoContribution of mucosal maltase-glucoamylase activitiesto mouse small intestinal starch 120572-glucogenesisrdquo Journal ofNutrition vol 137 no 7 pp 1725ndash1733 2007
[6] A J Krentz and C J Bailey ldquoOral antidiabetic agentsrdquo Drugsvol 65 no 3 pp 385ndash411 2005
[7] E Borges de Melo A da Silveira Gomes and I Carvalho ldquo120572-and 120573-glucosidase inhibitors chemical structure and biologicalactivityrdquo Tetrahedron vol 62 no 44 pp 10277ndash10302 2006
[8] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of the novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985
[9] A Horii M Emi N Tomita et al ldquoPrimary structure of humanpancreatic 120572-amylase gene its comparison with human salivary120572-amylase generdquo Gene vol 60 no 1 pp 57ndash64 1987
[10] Y-I Kwon E Apostolidis and K Shetty ldquoIn vitro studiesof eggplant (Solanum melongena) phenolics as inhibitors ofkey enzymes relevant for type 2 diabetes and hypertensionrdquoBioresource Technology vol 99 no 8 pp 2981ndash2988 2008
[11] Z J Canizares Las Frutas Anonaceas Ediciones Fruticus LaHabana Cuba 1966
[12] S E Ruiz and A L Morett Las Anonas en el Mexico Pre-hispanico Memorias Congreso Internacional de AnonaceasChapingo Estado de Mexico 169-186 1997
[13] W PopenoeManual of Tropical and Sub-Tropical Fruits HafnerPress New York NY USA 1920
[14] M E Gonzalez-Trujano A Navarrete B Reyes and E HongldquoSome pharmacological effects of the ethanol extract of leavesof Annona diversifolia on the central nervous system in micerdquoPhytotherapy Research vol 12 pp 600ndash602 1998
[15] M E Gonzalez-Trujano A Navarrete B Reyes E Cedillo-Portugal and E Hong ldquoAnticonvulsant properties and bio-guided isolation of palmitone from leaves of Annona diversifo-liardquo Planta Medica vol 67 no 2 pp 136ndash141 2001
[16] M E Gonzalez-Trujano E Tapia L Lopez-Meraz A Navar-rete A Reyes-Ramırez and AMartınez ldquoAnticonvulsant effectofAnnona diversifolia Saff and palmitone on penicillin-inducedconvulsive activity A behavioral and EEG study in ratsrdquoEpilepsia vol 47 no 11 pp 1810ndash1817 2006
[17] A I Carballo A L Martınez M E Gonzalez-Trujano etal ldquoAntinociceptive activity of Annona diversifolia Saff leafextracts and palmitone as a bioactive compoundrdquo Pharmacol-ogy Biochemistry and Behavior vol 95 no 1 pp 6ndash12 2010
[18] A Andrade-Cetto and M Heinrich ldquoMexican plants withhypoglycaemic effect used in the treatment of diabetesrdquo Journalof Ethnopharmacology vol 99 no 3 pp 325ndash348 2005
[19] A Shirwaikar K Rajendran C Dinesh Kumar and RBodla ldquoAntidiabetic activity of aqueous leaf extract of Annonasquamosa in streptozotocin-nicotinamide type 2 diabetic ratsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 171ndash175 2004
[20] D O Adeyemi O A Komolafe O S Adewole et alldquoAnti hyperglycemic activities of Annona muricata (Linn)rdquoAfrican Journal of Traditional Complementary and AlternativeMedicines vol 6 no 1 pp 62ndash69 2009
[21] S Cristians J A Guerrero-Analco A Perez-Vasquez et alldquoHypoglycemic activity of extracts and compounds from theleaves of Hintonia standleyana and H latiflora potential alter-natives to the use of the stem bark of these speciesrdquo Journal ofNatural Products vol 72 no 3 pp 408ndash413 2009
[22] R K Gupta A N Kesari P S Murthy R Chandra V Tandonand GWatal ldquoHypoglycemic and antidiabetic effect of ethano-lic extract of leaves of Annona squamosa L in experimentalanimalsrdquo Journal of Ethnopharmacology vol 99 no 1 pp 75ndash81 2005
6 BioMed Research International
[23] R K Gupta A N Kesari S Diwakar et al ldquoIn vivo evalu-ation of anti-oxidant and anti-lipidimic potential of Annonasquamosa aqueous extract in type 2 diabetic modelsrdquo Journalof Ethnopharmacology vol 118 no 1 pp 21ndash25 2008
[24] G Gupta-Elera A R Garrett A Martinez R A Robison andK L OrsquoNeill ldquoThe antioxidant properties of the cherimoya(Annona cherimola) fruitrdquo Food Research International vol 44no 7 pp 2205ndash2209 2011
[25] M R Loizzo R Tundis M Bonesi et al ldquoRadical scavengingantioxidant and metal chelating activities of Annona cherimolaMill (cherimoya) peel and pulp in relation to their total phe-nolic and total flavonoid contentsrdquo Journal of Food Compositionand Analysis vol 25 no 2 pp 179ndash184 2012
[26] N Kamalakkannan and P S Mainzen Prince ldquoRutin improvesthe antioxidant status in streptozotocin-induced diabetic rattissuesrdquo Molecular and Cellular Biochemistry vol 293 no 1-2pp 211ndash219 2006
[27] A A Henrique Fernandes E L Barbosa Novelli K Okoshi etal ldquoInfluence of rutin treatment on biochemical alterations inexperimental diabetesrdquo Biomedicine and Pharmacotherapy vol64 no 3 pp 214ndash219 2010
[28] O Hnatyszyn J Mino G Ferraro and C Acevedo ldquoThehypoglycemic effect of Phyllanthus sellowianus fractions instreptozotocin-induced diabetic micerdquo Phytomedicine vol 9no 6 pp 556ndash559 2002
[29] O M Ahmed A A Moneim I A Yazid and A MMahmoudldquoAntihyperglycemic antihyperlipidemic and antioxidant effectsand the probable mechanisms of action of Ruta graveolens infu-sion and rutin in nicotinamide-streptozotocin-induced diabeticratsrdquo Diabetologia Croatica vol 39 no 1 pp 15ndash35 2010
[30] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity potential targets forglucose homeostasisrdquo Nutrition vol 27 no 11-12 pp 1161ndash11672011
[31] S Adisakwattana S Yibchok-Anun P Charoenlertkul and NWongsasiripat ldquoCyanidin-3-rutinoside alleviates postprandialhyperglycemia and its synergism with acarbose by inhibitionof intestinal 120572-glucosidaserdquo Journal of Clinical Biochemistry andNutrition vol 49 no 1 pp 36ndash41 2011
[32] S Pashikanti D R de Alba G A Boissonneault and DCervantes-Laurean ldquoRutin metabolites novel inhibitors ofnonoxidative advanced glycation end productsrdquo Free RadicalBiology and Medicine vol 48 no 5 pp 656ndash663 2010
[33] Y Qin F Chao F Gao et al ldquoComparative evaluationof quercetin isoquercetin and rutin as inhibitors of 120572-glucosidaserdquo Journal of Agricultural and Food Chemistry vol 57no 24 pp 11463ndash11468 2009
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
BioMed Research International 5
70
60
50
40
30
20
10
0
minus10
0 20 40 60 80 100 120Minutes
Varia
tion
of g
lyce
mia
()
lowast lowast
lowastlowastlowast
lowastlowast
lowastlowastlowastlowast
lowastlowast
lowast
Figure 3 Effects of the crude extract of the leaves of A macro-prophyllata on blood glucose levels in rats on the OSTT Vehicle(◼) acarbose 5mgkg (998819) extract A macroprophyllata 100mgkg(e) extract A macroprophyllata 300mgkg (998771) and extract Amacroprophyllata 500mgkg (998787) Each value is the mean plusmn SEM for4 rats in each group lowast119875 lt 005 is significantly different t-test forcomparison with respect to negative control values at the same timeBaseline glycemia 77mgdL
study provides scientific support to use A macroprophyllataleaves in Mexican traditional medicine for the treatment ofDM2
Conflict of Interests
The authors declare no conflict of interests
Acknowledgments
This work was supported by grants from PAPIIT DGAPAUNAM (IN214812) and INMEGEN (062012l) F Brindisacknowledges postdoctoral fellowship awarded by DGAPA-UNAM
References
[1] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004
[2] W Kerner and J Bruckel ldquoDefinition classification and diag-nosis of diabetes mellitusrdquoDiabetologie und Stoffwechsel vol 4no 2 pp S115ndashS117 2009
[3] E Bonora and M Muggeo ldquoPostprandial blood glucose as arisk factor for cardiovascular disease in type II diabetes theepidemiological evidencerdquo Diabetologia vol 44 no 12 pp2107ndash2114 2001
[4] A Ceriello ldquoPostprandial hyperglycemia and diabetes compli-cations is it time to treatrdquoDiabetes vol 54 no 1 pp 1ndash7 2005
[5] R Quezada-Calvillo C C Robayo-Torres A R Opekun etal ldquoContribution of mucosal maltase-glucoamylase activitiesto mouse small intestinal starch 120572-glucogenesisrdquo Journal ofNutrition vol 137 no 7 pp 1725ndash1733 2007
[6] A J Krentz and C J Bailey ldquoOral antidiabetic agentsrdquo Drugsvol 65 no 3 pp 385ndash411 2005
[7] E Borges de Melo A da Silveira Gomes and I Carvalho ldquo120572-and 120573-glucosidase inhibitors chemical structure and biologicalactivityrdquo Tetrahedron vol 62 no 44 pp 10277ndash10302 2006
[8] H Bischoff W Puls H P Krause H Schutt and G ThomasldquoPharmacological properties of the novel glucosidase inhibitorsBAY m 1099 (miglitol) and BAY o 1248rdquo Diabetes Research andClinical Practice vol 1 pp 53ndash62 1985
[9] A Horii M Emi N Tomita et al ldquoPrimary structure of humanpancreatic 120572-amylase gene its comparison with human salivary120572-amylase generdquo Gene vol 60 no 1 pp 57ndash64 1987
[10] Y-I Kwon E Apostolidis and K Shetty ldquoIn vitro studiesof eggplant (Solanum melongena) phenolics as inhibitors ofkey enzymes relevant for type 2 diabetes and hypertensionrdquoBioresource Technology vol 99 no 8 pp 2981ndash2988 2008
[11] Z J Canizares Las Frutas Anonaceas Ediciones Fruticus LaHabana Cuba 1966
[12] S E Ruiz and A L Morett Las Anonas en el Mexico Pre-hispanico Memorias Congreso Internacional de AnonaceasChapingo Estado de Mexico 169-186 1997
[13] W PopenoeManual of Tropical and Sub-Tropical Fruits HafnerPress New York NY USA 1920
[14] M E Gonzalez-Trujano A Navarrete B Reyes and E HongldquoSome pharmacological effects of the ethanol extract of leavesof Annona diversifolia on the central nervous system in micerdquoPhytotherapy Research vol 12 pp 600ndash602 1998
[15] M E Gonzalez-Trujano A Navarrete B Reyes E Cedillo-Portugal and E Hong ldquoAnticonvulsant properties and bio-guided isolation of palmitone from leaves of Annona diversifo-liardquo Planta Medica vol 67 no 2 pp 136ndash141 2001
[16] M E Gonzalez-Trujano E Tapia L Lopez-Meraz A Navar-rete A Reyes-Ramırez and AMartınez ldquoAnticonvulsant effectofAnnona diversifolia Saff and palmitone on penicillin-inducedconvulsive activity A behavioral and EEG study in ratsrdquoEpilepsia vol 47 no 11 pp 1810ndash1817 2006
[17] A I Carballo A L Martınez M E Gonzalez-Trujano etal ldquoAntinociceptive activity of Annona diversifolia Saff leafextracts and palmitone as a bioactive compoundrdquo Pharmacol-ogy Biochemistry and Behavior vol 95 no 1 pp 6ndash12 2010
[18] A Andrade-Cetto and M Heinrich ldquoMexican plants withhypoglycaemic effect used in the treatment of diabetesrdquo Journalof Ethnopharmacology vol 99 no 3 pp 325ndash348 2005
[19] A Shirwaikar K Rajendran C Dinesh Kumar and RBodla ldquoAntidiabetic activity of aqueous leaf extract of Annonasquamosa in streptozotocin-nicotinamide type 2 diabetic ratsrdquoJournal of Ethnopharmacology vol 91 no 1 pp 171ndash175 2004
[20] D O Adeyemi O A Komolafe O S Adewole et alldquoAnti hyperglycemic activities of Annona muricata (Linn)rdquoAfrican Journal of Traditional Complementary and AlternativeMedicines vol 6 no 1 pp 62ndash69 2009
[21] S Cristians J A Guerrero-Analco A Perez-Vasquez et alldquoHypoglycemic activity of extracts and compounds from theleaves of Hintonia standleyana and H latiflora potential alter-natives to the use of the stem bark of these speciesrdquo Journal ofNatural Products vol 72 no 3 pp 408ndash413 2009
[22] R K Gupta A N Kesari P S Murthy R Chandra V Tandonand GWatal ldquoHypoglycemic and antidiabetic effect of ethano-lic extract of leaves of Annona squamosa L in experimentalanimalsrdquo Journal of Ethnopharmacology vol 99 no 1 pp 75ndash81 2005
6 BioMed Research International
[23] R K Gupta A N Kesari S Diwakar et al ldquoIn vivo evalu-ation of anti-oxidant and anti-lipidimic potential of Annonasquamosa aqueous extract in type 2 diabetic modelsrdquo Journalof Ethnopharmacology vol 118 no 1 pp 21ndash25 2008
[24] G Gupta-Elera A R Garrett A Martinez R A Robison andK L OrsquoNeill ldquoThe antioxidant properties of the cherimoya(Annona cherimola) fruitrdquo Food Research International vol 44no 7 pp 2205ndash2209 2011
[25] M R Loizzo R Tundis M Bonesi et al ldquoRadical scavengingantioxidant and metal chelating activities of Annona cherimolaMill (cherimoya) peel and pulp in relation to their total phe-nolic and total flavonoid contentsrdquo Journal of Food Compositionand Analysis vol 25 no 2 pp 179ndash184 2012
[26] N Kamalakkannan and P S Mainzen Prince ldquoRutin improvesthe antioxidant status in streptozotocin-induced diabetic rattissuesrdquo Molecular and Cellular Biochemistry vol 293 no 1-2pp 211ndash219 2006
[27] A A Henrique Fernandes E L Barbosa Novelli K Okoshi etal ldquoInfluence of rutin treatment on biochemical alterations inexperimental diabetesrdquo Biomedicine and Pharmacotherapy vol64 no 3 pp 214ndash219 2010
[28] O Hnatyszyn J Mino G Ferraro and C Acevedo ldquoThehypoglycemic effect of Phyllanthus sellowianus fractions instreptozotocin-induced diabetic micerdquo Phytomedicine vol 9no 6 pp 556ndash559 2002
[29] O M Ahmed A A Moneim I A Yazid and A MMahmoudldquoAntihyperglycemic antihyperlipidemic and antioxidant effectsand the probable mechanisms of action of Ruta graveolens infu-sion and rutin in nicotinamide-streptozotocin-induced diabeticratsrdquo Diabetologia Croatica vol 39 no 1 pp 15ndash35 2010
[30] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity potential targets forglucose homeostasisrdquo Nutrition vol 27 no 11-12 pp 1161ndash11672011
[31] S Adisakwattana S Yibchok-Anun P Charoenlertkul and NWongsasiripat ldquoCyanidin-3-rutinoside alleviates postprandialhyperglycemia and its synergism with acarbose by inhibitionof intestinal 120572-glucosidaserdquo Journal of Clinical Biochemistry andNutrition vol 49 no 1 pp 36ndash41 2011
[32] S Pashikanti D R de Alba G A Boissonneault and DCervantes-Laurean ldquoRutin metabolites novel inhibitors ofnonoxidative advanced glycation end productsrdquo Free RadicalBiology and Medicine vol 48 no 5 pp 656ndash663 2010
[33] Y Qin F Chao F Gao et al ldquoComparative evaluationof quercetin isoquercetin and rutin as inhibitors of 120572-glucosidaserdquo Journal of Agricultural and Food Chemistry vol 57no 24 pp 11463ndash11468 2009
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
6 BioMed Research International
[23] R K Gupta A N Kesari S Diwakar et al ldquoIn vivo evalu-ation of anti-oxidant and anti-lipidimic potential of Annonasquamosa aqueous extract in type 2 diabetic modelsrdquo Journalof Ethnopharmacology vol 118 no 1 pp 21ndash25 2008
[24] G Gupta-Elera A R Garrett A Martinez R A Robison andK L OrsquoNeill ldquoThe antioxidant properties of the cherimoya(Annona cherimola) fruitrdquo Food Research International vol 44no 7 pp 2205ndash2209 2011
[25] M R Loizzo R Tundis M Bonesi et al ldquoRadical scavengingantioxidant and metal chelating activities of Annona cherimolaMill (cherimoya) peel and pulp in relation to their total phe-nolic and total flavonoid contentsrdquo Journal of Food Compositionand Analysis vol 25 no 2 pp 179ndash184 2012
[26] N Kamalakkannan and P S Mainzen Prince ldquoRutin improvesthe antioxidant status in streptozotocin-induced diabetic rattissuesrdquo Molecular and Cellular Biochemistry vol 293 no 1-2pp 211ndash219 2006
[27] A A Henrique Fernandes E L Barbosa Novelli K Okoshi etal ldquoInfluence of rutin treatment on biochemical alterations inexperimental diabetesrdquo Biomedicine and Pharmacotherapy vol64 no 3 pp 214ndash219 2010
[28] O Hnatyszyn J Mino G Ferraro and C Acevedo ldquoThehypoglycemic effect of Phyllanthus sellowianus fractions instreptozotocin-induced diabetic micerdquo Phytomedicine vol 9no 6 pp 556ndash559 2002
[29] O M Ahmed A A Moneim I A Yazid and A MMahmoudldquoAntihyperglycemic antihyperlipidemic and antioxidant effectsand the probable mechanisms of action of Ruta graveolens infu-sion and rutin in nicotinamide-streptozotocin-induced diabeticratsrdquo Diabetologia Croatica vol 39 no 1 pp 15ndash35 2010
[30] D Fontana Pereira L H Cazarolli C Lavado et al ldquoEffectsof flavonoids on 120572-glucosidase activity potential targets forglucose homeostasisrdquo Nutrition vol 27 no 11-12 pp 1161ndash11672011
[31] S Adisakwattana S Yibchok-Anun P Charoenlertkul and NWongsasiripat ldquoCyanidin-3-rutinoside alleviates postprandialhyperglycemia and its synergism with acarbose by inhibitionof intestinal 120572-glucosidaserdquo Journal of Clinical Biochemistry andNutrition vol 49 no 1 pp 36ndash41 2011
[32] S Pashikanti D R de Alba G A Boissonneault and DCervantes-Laurean ldquoRutin metabolites novel inhibitors ofnonoxidative advanced glycation end productsrdquo Free RadicalBiology and Medicine vol 48 no 5 pp 656ndash663 2010
[33] Y Qin F Chao F Gao et al ldquoComparative evaluationof quercetin isoquercetin and rutin as inhibitors of 120572-glucosidaserdquo Journal of Agricultural and Food Chemistry vol 57no 24 pp 11463ndash11468 2009
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of