Rev. bras. farmacogn. vol.26 número2

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Metanolic

extract

of

Malpighia

emarginata

bagasse:

phenolic

compounds

and

inhibitory

potential

on

digestive

enzymes

Tamara

R.

Marques

,

Aline

A.

Caetano,

Anderson

A.

Simão,

Flávia

Cíntia

de

O.

Castro,

Vinicius

de

Oliveira

Ramos,

Angelita

D.

Corrêa

LaboratóriodeBioquímica,DepartamentodeQuímica,UniversidadeFederaldeLavras,Lavras,MG,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16May2015 Accepted18August2015 Availableonline29November2015

Keywords: Malpighiaemarginata

␣-Amylase

␣-Glucosidase Lipase Trypsin Inhibitor

a

b

s

t

r

a

c

t

Addingvaluetofruitresiduesisofgreatinterest,sincetheycanbepresentedasaviablesolution insearchofnewdrugsforthetreatmentofobesityandrelateddiseases,duetobioactivesubstances, especiallyphenoliccompounds.Thus,theobjectiveofthisstudywastopreparethemethanolextractof acerolabagasseflour,inordertoevaluateitspotentialasasourceofinhibitorsoftheenzymes␣-amylase, ␣-glucosidase,lipaseandtrypsin,anddeterminethecontentofphenoliccompoundsbyhighperformance liquidchromatography.Enzymaticinhibitionassayswereconductedinthepresenceorabsenceof sim-ulatedgastricfluid.Inthemethanolextractofacerolabagasseflour,thefollowingphenoliccompounds wereidentified:gallicacid,syringicandp-coumaricacid,catechin,epigallocatechingallate,epicatechin andquercetin;epicatechinwasthemajorcompound.Intheabsenceofgastricfluid,simulatedenzymes hadavariableinhibitionoftheacerolabagasseflourextract,exceptforlipase,whichwasnotinhibited. Inthepresenceofsimulatedgastricfluid,therewasaninhibitionof170.08IEU(InhibitedEnzymeUnit in␮molmin−1_g−1_{)for␣-amylaseand69.29IEUfor␣-glucosidase,indicatingthatthisextractshows} potentialasanadjuvantinthetreatmentofobesityandotherdyslipidemia.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Obesityisadiseaseresultingfromtheexcessiveaccumulation ofbodyfat,andbringsmultipleoutcomesforhealth,suchasthe prevalenceandprogressionofcardiovasculardiseases(especially heartdiseasesandstroke),whichwerethemajorcausesofdeath in2012;Sometypesofcancer(endometrium,breastandcolon); skeletalmuscledisturbs(speciallyosteoarthritis–ahighly inca-pacitatingdegenerativedisease);hypertensionandtype2diabetes mellitus(WanderleyandFerreira,2010;WHO,2015).

Between1980and2014,theworld’sobesityprevalency dou-bled.DatafromtheWorldHealthOrganizationreportthat,in2014, morethan1.9billionadultswereoverweightand,amongthem, morethan600millionwereobese(WHO,2015).

One way to fight this epidemic disease is drug treatment. Medicinetofightweightgain,whichhastheobjectivetorestrict energy absorption and cause weight loss, is widely available (Boniglia et al., 2008). However,these drugs cause side effects andareprohibitedbyAnvisasince2011(Abeso, 2014).Another

∗ Correspondingauthor.

E-mail:angelita@dqi.ufla.br(T.R.Marques).

alternativebroadlyemployedistheuseofplantextracts.Overthe lastyears,therewasasubstantialincreaseinitsuse,bythefact thatthepopulationbelievesitsintakeisharmless,withalowcost, andmayinhibitdigestiveenzymes,leadingtobeneficialchangesin metabolism(Simãoetal.,2012).Howevernotallnaturalproducts arebeneficialandfurtherstudiesarenecessarytoevaluatetheir effectsontheorganism.

Enzymes like ␣-amylase and ␣-glycosidase, responsible for processingdietarycarbohydrates,actonstarchbreakdown, result-inginmonosaccharideabsorptionbyenterocytes.Therefore,their inhibitionoffersapromisingstrategyforthepreventionofobesity, aswellastype2diabetesassociatedtohyperglycemia,by inhibi-tingstarchbreakdownandglucoseabsorptioninthesmallintestine (Kwonetal.,2008;Balasubramaniametal.,2013).

Lipase,involvedinfatmetabolism,isalsoanimportanttarget forinhibitors,sinceitsinhibitionlimitstriacylglycerolabsorption, leadingtoadecreaseincaloricyieldandweightloss.Ontheother hand,trypsininhibition,involvedinproteindigestion,hasamalefic effect,onceitimpairsthecompleteaminoacidabsorptioninfood, essentialfortheorganism.

Research has been carried out for evaluating the effects of naturalproductsonthetreatmentofobesityandassociated comor-bidities, reinforcing theneed for the search of new sources of

http://dx.doi.org/10.1016/j.bjp.2015.08.015

amylase, glycosidase and lipase inhibitors (Souza et al., 2011; Pereira et al., 2011a; Simão et al., 2012). Therefore, digestive inhibitorswhoassistinreducingfatandcarbohydrateabsorption inthesmallintestinemaybeusefulhelpersinthetreatmentof obesity.

Naturalproductshavebeengainingspaceandimportancein thepharmaceuticalindustry,sincetheyhavebioactivesubstances capableofinspiringnewphytomedicinesandphytotherapic prod-ucts. Phenolic compounds are among those substances. These compoundspresentchemicalstructureswithhydroxylsand aro-maticrings,whichcanbesimplestructuresorpolymers,originated from plant secondary metabolism and largely found in fruits (AngeloandJorge,2007).Manystudiesreportthebenefitsof phe-noliccompoundsasanadjunctinthetreatmentofobesity(Klaus etal.,2005;Henetal.,2006;Alterioetal.,2007;Santiago-Mora etal.,2011;Vogeletal.,2015;Zhangetal.,2015).

Alterioetal. (2007)andKlaus etal. (2005)reportthat phe-nolic compoundsact in theprevention of obesitydue totheir thermogeniceffects,abilitytooxidizebodyfatandbydecreasing intestinalabsorptionoffatsandcarbohydratescausedbythe inhi-bition of digestive enzymes, resulting in weight loss. Phenolic compounds,suchastannins,havetheabilityofcombiningwith digestiveenzymes,proteinsandotherpolymers(suchas carbo-hydrates),formingstable complexes, impairingabsorption and, therefore,makingthempossibleinhibitorsofsomeofthese diges-tiveenzymes(Wonetal.,2007;Gholamhoseinianetal.,2010).

In this context, the useof agro industrial residues of fruits ispromising fortheextractionofactive principlesthatmaybe employedasanalternativetothetreatmentofobesityand corre-lateddiseases.Bydiscardingtheseresidues,secondarymetabolites ofgreataggregatedvaluewithpossibleapplicationsin pharma-ceutical and food industries, are also eliminated. For example, theacerolabagasseoriginatedinjuiceprocessingis,accordingto

Marquesetal.(2013),richin phenoliccompounds,withrecord contentsof10.82g100g−1 _{drymatter;however,thesephenolic}

compoundswerenotyetidentified.

Given the above,the objective of thepresent study wasto preparethemethanolextractofacerolabagasseflour(ABF), eval-uateitspotentialasasourceof␣-amylase,␣-glycosidase,lipase andtrypsininhibitors,anddeterminethephenoliccompoundsby highperformanceliquidchromatography(HPLC),aimingtouseit asanauxiliaryinthetreatmentofobesityandcorrelateddiseases, aggregatingvaluetothisresidue.

Materialandmethods

Preparationofacerolabagasseflour

AcerolaMalpighiaemarginataDC.,Malpighiaceae(BRS238 Fru-tacor)bagassewasobtainedfromplantsgrowninthemunicipality ofPerdões,MG,Brazil(21◦₀₅′₂₇′′_S;45◦₀₅′₂₇′′_{W,848maltitude);}

thelocalclimateaccordingtotheKöppensystemisclassifiedas Cwa:mildandrainysummerswithmoderatetemperatures,annual averagetemperaturebelow21◦_{C,averageannualprecipitationof}

1529.7mm,andrelativehumidityof76%(Emater,2002).Acerola fruitswereusedforpulpextraction,andtheresidualbagassewas provided in threebatches by a fruitpulp plantfirm located in Perdões,MG,Brazil.

Acerolabagasse(4kg)wasfrozenat−18◦_{Candlyophilizedin}

glasscontainersprotectedfromlight for7 daystoobtain450g drybagasse.Afterlyophilization,acerolabagassewashomogenized usingmortarandpestle,waspassedinsievesandmostflour parti-cleswereretainedonsievessized40mesh(0.425mm)to80mesh (0.180mm),thus,classifiedasfineandthenplacedinahermetically sealedflask,protectedfromlightinarefrigeratorat4◦_C.

Obtentionoftheextract

Toobtainthemethanolextractofacerolabagasseflour(ABF),1g ofacerolabagasselyophilizedpowderwastransferredtoa250ml erlenmeyerandthenadded50mlof50%methanolsolutioninthree repetitions.Afterwards,itcoveredwithagroundglassjointand putonahotplateat80◦_{C.Afterboilingfor15min,theextractwas}

filteredinfilterpaperandcollectedtoa250mlbecker.Theresidue wasonceagainputonanerlenmeyerandthisprocessrepeatedfor twomoretimes.Afterthethirdfiltration,thebeckerwastakento thehotplatetoevaporatethemethanoluntilthevolumereaches 16ml(AOAC,2012),andthensubmittedtoenzymaticinhibition analysis.

Forthechromatographyprocess,thebeckerwastakentothehot platetoevaporatethemethanol,posteriorlyfrozenandlyophilized (FreeZone®_{2.5literFreezeDrySustems).Lyophilyzedextract(1g)}

wassolubilizedin16mlultrapurewaterobtainedfromaMilli-Q system(EMDMillipore,Billerica,MA,USA).

Identificationandquantificationofphenoliccompounds

HPLCwasperformedusingaShimadzuUHPLCchromatograph (ShimadzuCorporation,Kyoto,Japan)equippedwithtwoLC-20AT high-pressurepumps,anSPD-M20AUV–visdetector,aCTO-20AC oven,aCBM-20Ainterface,andanautomaticinjectorwithan SIL-20Aautosampler.SeparationswereperformedusingaShim-pack VP-ODS-C18 (250mm×4.6mm)column, connected toa

Shim-pack Column Holder (10mm×4.6mm)pre-column (Shimadzu,

Japan).

Themobilephaseconsistedofthefollowingsolutions:2%acetic acidinwater(A)andmethanol:water:aceticacid(70:28:2,v/v/v) (B).Analyseswereperformedforatotaltimeof65minat40◦_C,flux

of1mlmin−1_{,wavelengthof280nm,andinjectionvolumeof20␮l}

inagradient-typesystem(100%solventAfrom0.01to5min;70% solventAfrom5to25min;60%solventAfrom25to43min;55% solventAfrom43to50min;and0%solventAfor10min)untilthe endoftherun.SolventAwasincreasedto100%,seekingto main-taina balancedcolumn.Aceticacidandmethanol(HPLCgrade; Sigma–Aldrich,USA)wereusedinthepreparationofthemobile phase.

Addition of standards to the extracts was also used as an identificationparameter.Thephenolicstandardsusedwere gal-lic acid, catechin, epigallocatechin gallate, epicatechin, syringic acid,p-coumaric acid,ferulicacid,salicylicacid,resveratroland quercetinallobtainedfromSigma–Aldrich(St.Louis,MO,USA).The stockstandardsolutionswerepreparedinmethanol(HPLCgrade; Sigma–Aldrich,USA).

TheABFextractandthestandardswerefilteredthrougha

0.45-␮mnylonmembrane(EMDMillipore,USA)anddirectlyinjected intothechromatographicsystem,inthreereplicates.The pheno-liccompoundsintheextractwereidentifiedbycomparisonwith retention times of standards. Quantification wasperformed by theconstructionofanalyticalcurvesobtainedbylinearregression usingOrigin6.1computersoftware(OriginLab,Northampton,MA, USA)andconsideringthecoefficientofdetermination(R2)equalto 0.99.

Enzymeobtention

O

HO

HO

HO

HO HO

HO _HO

OH

OH 500 000

400 000

300 000

200 000

Intensity (mV)

Time (min)

100 000

–100 000

–200 000 0

0 5 10 15 20 25 30 35 40 45 50 55 60 65

1 2

3 4

5

6 7

OH OH

OH

OH

OH OH

OH OH

OH OH

OH OH

OH OH

OH OH OH OH

OH

H3C

CH3

OH OH

OH

O

O

O O

O

O

O

O

O O

O

1

2

5

6

7

3

4

Fig.1.Chromatogramofacerolabagasseflourextract,withpeakidentification:(1)Gallicacid(time=6.541min);(2)catechin(time=10.419min);(3)epigallocatechingallate (time=11.987min);(4)epicatechin(time=13.139min);(5)syringicacid(time=14.988min);(6)p-coumaricacid(time=19.892min)and(7)quercetin(time=51.185min).

Activityof˛-amylase,˛-glucosidase,lipaseandtrypsin

The␣-amylaseactivitywasdeterminedusingthemethodology proposedbyNoeltingandBernfeld(1948).Thus,theextractand␣ -amylaseenzymewerepre-incubatedfor20min,inawaterbathat 37◦_{C.Thesubstratewasthe1%starchpreparedinTris0.05moll}−1_,

pH7.0bufferwith38mmoll−1_{NaCland0.1mmoll}−1_CaCl2.After

additionof100␮lofthesubstrate,themixturewasincubatedfor fourperiodsoftime.Thereactionwasinterruptedadding200␮lof 3.5dinitrosalicylicacidandtheproductreadinspectrophotometer at540nm.

The␣-glucosidaseactivitywasdeterminedaccordingtoKwon etal.(2008),using5mmoll−1_{p-nitrophenyl-␣-}d-glucopyranoside

ina0.1moll−1_{pH7.0citrate–phosphatebufferassubstrate.Inthe}

assay,extractand␣-glucosidaseenzymewereincubatedinawater bath,at37◦_{C,forfourperiodsoftime,afteradditionofthe}

sub-strate.Thereactionwasinterruptedadding1.000␮lof0.05moll−1

NaOHandtheproductwasreadinaspectrophotometerat410nm. Thelipase activitywasdetermined accordingtoSouzaetal. (2011), using 8mmoll−1 _{p-nitrophenylpalmitate in Tris–HCl}

0.05mmoll−1_{,pH8.0buffercontaining0.5%Triton-X100as}

sub-strate.Intheassay,extractandlipaseenzymewasincubatedina waterbath,at37◦_{C,forfourperiodsoftime,afteradditionofthe}

substrate.Thereactionwasstopped,transferringthetubestoan icebathandaddingTris–HCl0.05mmoll−1 _{pH8.0buffer.Thep}

-nitrophenol,ofyellowcoloration, aproductof thelipase action onp-nitrophenylpalmitate,wasread in aspectrophotometer at 410nm.

Thetrypsinactivitywasdeterminedaccordingtothe method-ologyproposedbyErlangeretal.(1961).Thus,extractandtrypsin enzymewereincubatedinawaterbath,at37◦_{C,forfourperiods}

oftime,afteradditionofp-benzoyl-dl-arginine-p-nitroanilide

sub-strate(BApNA),preparedinTris0.05moll−1_{,pH8.2.Thereaction}

wasinterruptedadding200␮lof30%aceticacidandtheproduct

readinaspectrophotometerat410nm.

Foreachassayofenzymaticactivity,thevolumeofextractwas differentand its dilution rangedso that theenzyme inhibition rangedfrom50to80%,accordingtothemethodology.

Theinhibitionoftheenzymeswereobtainedfromthe deter-minationoftheslopesofthestraightlines(absorbance×time)of

thecontrolenzyme(withoutextract)andenzymes+inhibitor(with extract)activityassays.Theslopeofthestraightlineisduetothe speedofproductformationperminuteofreactionandthepresence oftheinhibitorcausesadecreaseinthatinclination.Fromthat incli-nation,theabsorbancevalueswereconvertedintomicromolesof productthroughastandardglucosecurvefortheamylaseandof

p-nitrophenolforglycosidaseandlipase,while,forthetrypsin,the ofBApNAmolarextinctioncoefficientdeterminedbyErlangeretal. (1961)wasused.

Preparationofsimulatedgastricfluid

Withtheobjectiveofsimulatingthedigestionprocessinthe stomachinvitro,enzymaticactivityassaysinthepresenceofa sim-ulatedgastricfluidwerealsocarriedout.Forsuch,theextractwas incubatedwiththesimulatedgastricfluidpreparedaccordingto

TheUnitedStatesandPharmacopeia,(2005),for1hinawaterbath at37◦_{C.Subsequently,wasneutralizedwithsodiumbicarbonate}

salttopH7.2andonlythenrealizedtheactivityassays.

Resultsanddiscussion

Each100gABFyielded48goflyophilizedextract(48%yield). The following phenolic compounds were identified in the ABF extract,inmgl−1_{:gallicacid(3.32±0.23),catechin(11.33±0.33),}

epigallocatechin gallate (9.13±0.89), epicatechin (91.86±1.49),

syringic acid (37.16±0.12), p-coumaric acid (2.41±0.13) and

quercetin(0.29±0.02)(Fig.1);gallicacidisahydrolyzabletannin

monomer,andepigallocatechingallate,catechinandepicatechin arecondensedtanninmonomers.Itwaspossibletoobservethat epicatechinhadthehighestcontent,followedbysyringicacid.The compoundsepicatechin,ferulicacid,salicylicacidandresveratrol werenotidentifiedintheABFextract.

LinandLin-Shiau(2006),Alterioetal.(2007),Choetal.(2010)

andRainsetal.(2011)reportedthatphenoliccompoundssuchas caffeicandchlorogenicacid,catechin,epigallocatechingallateand quercetinhavethermogeniceffect,abilitytooxidizefats,control appetite,regulatelevelsofhormonesrelatedtoobesityandinhibit digestiveenzymesinvolvedintheabsorptionofcarbohydratesand lipids.Thus,thisstudyshowsthattheacerolabagasseextracthas bioactivesubstancesandcanbeexploitedbythepharmaceutical industryinsearchofdrugstocontrolobesityandrelateddiseases. Theresultsforenzymatic inhibitionof ABFbeforethe expo-sure togastric fluids are shown in Table 1. The ABFmethanol extract inhibited theactivity of ␣-amylase, presenting an inhi-bition potential of 238.96␮molmin−1_g−1 _{dry matter – DM.}

Thispotential exceeds theone foundby Pereira et al.(2011b), who analyzed the white bean crude extract and detected an inhibition of 54.1␮molmin−1_g−1_{. Simão et al. (2012),}

study-ingaqueousextractsofmedicinalplants,observedaninhibition of2907.13␮molmin−1_g−1 _{DMforTournefortiapaniculataCham.}

Table1

Inhibitionofdigestiveenzymesbyacerolabagassepowderbeforeandafterthe exposuretosimulatedgastricfluid.

Enzyme Inhibition(IEUb₎a

Beforeexposure Afterexposure

␣-Amylase 238.96±1.64 170.08±1.06

␣-Glycosidase 78.51±1.78 69.29±0.28

Lipase ndc _ndc

Trypsin 227.52±3.59 84.73±5.41

Datafromthreerepetitions,withmean±standarddeviation.

a_{TheABFextractmeasuredforeachoftheenzymeswasdilutedtoprovidean}

inhibitionbetween50%and80%,inordertoensureresultreliability.

b _{IEU=InhibitedEnzymeUnitin␮molmin}−1_g−1_{drymatter–DM.} c _{nd=inhibitionnotdetected.}

relatedwiththedigestionofcarbohydratesand,consequently,with theelevationinglycemiclevelsafterameal.Highglycemiclevels leadtoserioushealthproblemsinthepopulation,suchastype2 diabetes.Theintakefoodrichin␣-amylaseposesasan interest-ingstrategyinthepreventionandtreatmentofhyperglycemia,by slowingpostprandialglucoselevelsinbloodaftertheingestionof carbohydrates(Vadiveletal.,2011).

Theinhibitionof␣-glucosidasebytheABFextractwasabout 78.51␮molmin−1_g−1_{DM.TheinhibitorypotentialofABPfound}

inthispapersurpasses theonesverifiedbySimão etal.(2012)

who,studyingaqueousextractsofmedicinalplantslikeAloevera

(L.) Burm.(Aloe), Simaba ferruginea St. Hil.(calunga), Baccharis trimera(Less.)DC (carqueja),Garcinia cambogiaDesr.(garcinia),

T.paniculataCham.(marmelinho),foundinhibitionsof0.58 and 35.46␮molmin−1_g−1 _{DM, as well as those from Pereira et al.} (2011a),who analyzedcommercialsamplesofHoodia gordonni, usedasanauxiliaryinthetreatmentofobesity,andfound inhi-bitionsof10.40e16.70␮molmin−1_g−1_DM.

Theinhibitionof␣-glucosidaseextendsgastricemptying,leads

tosatietyandweightloss,effectswhichcanbeusefulinthe treat-mentofobesity(Chenetal.,2008).

Therefore, theinhibition of ␣-amylase and ␣-glycosidaseby naturalproductscanprovideanalternativeforthetreatmentof obesityinsubstitutiontosyntheticdrugsnowavailableonthe mar-ket,besidescontrollingglucoselevelsinbloodintype2diabetes patients(McDougalletal.,2005a).

The ABF extract was not able to inhibit lipase, an enzyme involvedinlipidmetabolism,neitherbeforenoraftertheexposure tosimulatedgastricfluid. However,for trypsin,aninhibition of 227.52␮molmin−1_g−1_{DMwasobserved.Whentrypsininhibitors}

arepresentinthediet,thesemayleadtoareductioningrowthrate inanimals,followedbyadecreaseinproteindigestibility,leadingto weightlossandendogenousproteincatabolism(McDougalletal., 2005a).Therefore,thetrypsininhibitorisconsideredan antinutri-tionalfactor.

ThepassageoftheABFextractthroughthegastrointestinal cav-itymayleadtostructuralmodificationsontheinhibitorsbecause ofthepHofthegastricacid.Consideringtheexpressiveinhibition of␣-amylase,␣-glycosidaseandtrypsininthepresenceoftheABF extract,thisextractwassubmittedtoagastricfluidassay(Table1). Inthepresenceof simulatedgastricfluid, theABFmethanol extractstillmaintained71%ofitsinhibitoryactivityover␣-amylase and88%ofinhibitoryactivityover␣-glycosidase.Therefore,the extractdidnotshowaconsiderableinhibitoryactivityoverthese twoenzymesaftertheyweresubmittedtosimulatedgastricfluid. TheABFextractdecreasedtheinhibitionoftrypsinby63%in thepresenceofsimulatedgastricfluid.Thisreductionintrypsin inhibitionisconsideredpositivesince,wheninhibitionoccurs,it isconsideredantinutritional,impairingproteindigestion,which isthemainsourceofessentialaminoacids.However,aresidual inhibitoryactivityof37%wasstillobserved.Itisnotedthatthe

Table2

Contentofphenoliccompoundsinthemethanolextractofacerolabagasseflour, usedineachenzymaticassay.

Phenoliccompound(␮g) Enzymaticassays

␣-Amylasea _{␣-Glycosidase}b _Lipasec _Trypsinc

Gallicacid 0.02 0.06 0.17 0.66 Catechin 0.07 0.19 0.57 2.26 Epigallocatechin-gallate 0.05 0.15 0.46 1.83 Epicatechin 0.54 1.53 4.60 18.4 Siringicacid 0.22 0.62 1.86 7.44

p-Cumaricacid 0.01 0.04 0.12 0.48 Quercetin 1.71×10−3 _4.83

×10−3 _{0.01 0.06}

Phenoliccompounds 0.91 2.59 7.79 31.13

a_{Extractdilution1:7.5.} b_{Extractdilution1:2.} c _{Crudeextract.}

resistanceoftheinhibitortopassthroughthesimulatedgastric fluidisastrongindicativethattheseresultswillrepeatininvivo

assays.

Inthisstudy,theinhibitionofdigestiveenzymescanprobablybe explainedbythepresenceofphenoliccompoundsinthemethanol ABFextract,whoselevelsweredifferentforeachenzymaticassay assessed(Table2).␣-Amylasewastheone thathad the small-estcontentofphenoliccompounds,whichledtoaninhibitionof 0.91␮g.Ontheotherhand,thecontentofphenoliccompoundswas higher(31.13␮g)forthetrypsinassay,thatis,34timessuperior tothatfoundfor␣-amylase.Therefore,thissuggeststhatsmaller contentsofphenoliccompoundsmaynotleadtotrypsininhibition, whichwouldbebeneficial,sinceitcouldreducetheabsorptionof carbohydratesandallowproteindigestion.

Gallicacidisconsideredahydrolyzabletannin,whenfoundin theformofgallicacidesters,whilecatechin,epicatechingallateand epicatechin,whenfoundintheformofflavonoids,areconsidered condensed tannins. These compounds have strong interactions with metal ions and macromolecules such as polysaccharides, besidestheability toformsoluble complexeswithseveral pro-teins,asdigestivesenzymes(Wonetal.,2007;Gholamhoseinian etal.,2010).Thisabilitythattanninsexhibittointeractwith pro-teinsmakesthis classofsubstances powerfuldigestiveenzyme inhibitors.

McDougalletal.(2005b)reportthatredfruitextractsinphenolic compoundsinhibittwomainenzymesinvolvedinstarch diges-tion,␣-amylaseand␣-glycosidase,invitro.Inasimilarway,recent studieswithredfruitsreportedinhibitionof ␣-amylase and␣ -glycosidase,andmentionedthattanninswerethemosteffective compoundsininhibitingtheseenzymes(Boathetal.,2012).Kam etal.(2013),studyingtheeffectsofextractsfromdifferentparts ofpomegranate(pulp,peels,seedsandflower)overthedigestive enzymes␣-amylaseand␣-glycosidase,showedthatthemethanol extractfromthepomegranateflower,wherethephenolic com-pounds gallic acidandellagic acidarefound, exhibitsa potent inhibitoreffectontheseenzymes.

Klausetal.(2005)reportedthatratsfedepigallocatechingallate, purifiedfromgreentea,hadanobesitydecreaseinducedbythe diet,duetoareductioninenergyabsorptionandanincreasein lipidoxidation.Ontheotherhand,Bryansetal.(2007)reported thatblackteaisefficientinreducingpostprandialbloodglucose levelsandrelatedthisfacttothepresenceofphenoliccompounds suchasepigallocatechin,epigallocatechingallate,epicatechinand epicatechingallate.

Flavonoids like quercetin comprise a heterogeneous class of phenolic compounds present in plants, which can also act in the organism, inhibiting digestive enzymes. Wenzel (2013)

carbohydratedigestionandcontrollingpostprandialglucoselevels inblood,thusconfirmingtheresultobtainedbyTaderaetal.(2006), whoreportedtheinhibitoryactivityofquercetininthepresenceof

␣-amylase.

LinandLin-Shiau(2006),reportthatflavonoidshavetheability toactonthesympatheticnervoussystemthroughthemodulation ofnoradrenaline,thusincreasingthermogenesisandfatoxidation. Italsopreventstheincreaseinthesizeandnumberofadipocytes, thereforepreventingthedepositionoffatinthebodyand regulat-ingbodyweight.

Phenolicextractsoflentilscontainingp-hydroxybenzoicacid, syringicacid,trans-p-coumaricacid,epicatechingallate,quercetin andkaempferolwereshowntobegoodinhibitorsoflipaseand␣ -glycosidase,contributingtocontrolglucoselevelsinblood,aswell asobesity(Zhangetal.,2015).

Most phenols previously mentioned were foundin the ABF extract,which couldhaveledtoa complexationwithdigestive enzymesand,probably,contributedtoitsinhibition.Theinhibition ofdigestiveenzymesisapromisingalternativeforthetreatment ofobesityandtype2diabetes,especiallybythefacttheyactin thesmallintestine,withoutactinginthecentralnervoussystem, whereprescribedanorexigenicdrugsusuallyact.

Conclusion

The ABF methanol extract that contains the phenolic com-pounds gallic acid, catechin, epicatechin gallate, epicatechin, siringic acid, p-cumaricacidand quercetin, wasable toinhibit

in vitro digestive enzymes ␣-amylase and ␣-glucosidase. This shows that the ABF extract may represent a good source of inhibitors,andcanbeusedasanauxiliaryinthetreatmentof obe-sity,associatedcomorbiditiesandinthecontroloftype2diabetes.

Authors’contributions

TRM contributed in running the laboratory work, analysis of thedata and draftedthe paper.AAC contributed in running thelaboratorywork.AASparticipatedofenzymeactivityassays. FCOCcontributedtochromatographicanalysis.VORparticipatedof enzymeactivityassays.ADCdesignedthestudy,supervisedthe lab-oratoryworkandcontributedtocriticalreadingofthemanuscript. Alltheauthorshavereadthefinalmanuscriptandapprovedthe submission.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorswouldliketothankFundac¸ãodeAmparoàPesquisa doEstadodeMinasGerais,CAPESandCNPqforthegrantsprovided.

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