JournalofTaibahUniversityforScience10(2016)805–812
Availableonlineatwww.sciencedirect.com
ScienceDirect
Antioxidant
activity
of
polyphenolic
extract
of
Terminalia
chebula
Retzius
fruits
Sarmistha
Saha
∗,
Ramtej
J.
Verma
DepartmentofZoology,UniversitySchoolofSciences,GujaratUniversity,Ahmedabad380009,India
Availableonline25November2014
Abstract
TheobjectiveofthisstudywastoinvestigatetheinvitroantioxidantactivitiesofpolyphenolicextractofTerminaliachebula Retzius(Combretaceae)fruits.ThepolyphenolicextractofT.chebulafruitswasevaluatedforantioxidantactivitybydetermining thereducingpower,totalantioxidantcapacity,DPPHradicalconcentration(IC50 14g/mL),nitricoxideradicalconcentration
(IC50 30.51g/mL)and hydrogenperoxidescavengingactivity (IC50 265.53g/mL)underin vitroconditions. Moreover,the
phytochemicalcharacterisationoftheextractwasalsomeasuredbydeterminingthetotalphenolic,flavonoid,tanninandascorbic acidcontents.CharacterisationoftheextractwasalsoperformedbyHPLCprofilingwiththestandardgallicacid.Thepresentstudy demonstratedthattheextracthassignificantreducingcapacityandnitricoxidescavengingactivity.Italsoscavengeshydrogen peroxide-inducedradicals.Theactivityoftheextractmaybeduetothetotalpolyphenoliccontent.Theantioxidantactivityofthe extractissignificantlyhigherthanthestandardascorbicacid,anditsactivityisconcentration-dependent.Itisconcludedthata polyphenolic-richfractionofT.chebulafruitsisapotentialsourceofnaturalantioxidants.
©2014TheAuthors.ProductionandhostingbyElsevierB.V.onbehalfofTaibahUniversity.Thisisanopenaccessarticleunder theCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Keywords: Terminaliachebula;Ascorbicacid;Totalantioxidativecapacity;DPPHradicalscavengingactivity;Nitricoxideradicalscavenging
assay;Hydrogenperoxidescavengingassay
1. Introduction
It is currentlyhypothesised that many diseases are duetooxidative stress that resultsfrom an imbalance between the formation and detoxification of pro-oxidants.Oxidativestressisinitiatedbyreactiveoxygen species(ROS),whichareproducedas aby-productof electrontransportinmitochondria[1].Theincreasein
∗Correspondingauthor.Tel.:+918460619412.
E-mailaddress:sarmisthapharmacol@yahoo.com(S.Saha).
PeerreviewunderresponsibilityofTaibahUniversity.
Production and hosting by Elsevier
ELSEVIER
http://dx.doi.org/10.1016/j.jtusci.2014.09.003
1658-3655©2014TheAuthors.ProductionandhostingbyElsevierB.V.onbehalfofTaibahUniversity.Thisisanopenaccessarticleunderthe CCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).
ROSgenerationordecreasedantioxidantavailabilitycan resultinanetincreaseinintracellularROS.Normally, intracellularmoleculesincludingmitochondrial antiox-idantspreventcellulardamageproducedbyendogenous ROS.Inlivingsystems,freeradicalsandROSare con-stantlygeneratedandcauseextensivedamagetotissues andin various disease conditions, particularly degen-erative diseases, and also lead to extensive lysis [2]. Therefore,the mechanismofactionof manysynthetic drugsinvolvesfreeradicalscavenging, whichprotects againstoxidativedamagebuthasadversesideeffects[3]. Analternativeistheconsumptionofnaturalantioxidants fromvariousfoodsupplementsandtraditionalmedicines [4], and there is increased interest among phytother-apyresearcherstousemedicinalplantswithantioxidant activityforprotectionagainstoxidativestress.
Terminalia chebula Retzius (Combretaceae), com-monlyknownasharitaki,exhibitsanumberofmedicinal activitiesduetothepresenceofalargenumberof dif-ferentphyto-constituents.Theplanthasaroundcrown andspreadingbranches. The barkisdarkbrown with somelongitudinalcracks.Theleavesareovateand ellip-tical, with two large glands at the top of the petiole. Thefruitordrupeisapproximately1–2inchesinsize. It has five lines or five ribs on the outer skin. The fruitsofT.chebulaarereportedtohavehepatoprotective activityagainstCCl4 andtert-butylhydroperoxide[5]. The fruitsalso displaycytoprotective [6], antidiabetic [7,8],antioxidant[9],antibacterial[10,11],anti-arthritic [12],hypo-cholesterolaemic[13]andanti-inflammatory activities[14].
Antioxidant activities increase proportionally with thepolyphenolcontent,primarilybecauseoftheirredox properties [15]. Among the diverseroles of polyphe-nols,they protect cell constituents against destructive oxidative damage, thus limiting the risk of various degenerativediseasesassociatedwithoxidativestressby actingaspotentfreeradicalscavengers.Thepolyphenol antioxidantactivityisduetothechemicalstructureand abilitytodonate/accept electrons,therebydelocalising theunpairedelectronwithinthearomaticstructure[16]. Recentstudiesshowedtheantioxidantpropertiesof dif-ferentextractsofT.chebulafruits.Inanearlierreport,a 70%methanolextractofT.chebulafruitswasfoundto havegoodefficacyinradicalscavengingabilities[17]. Inanotherreport,chloroform,ethanolic,n-butanolicand organicaqueousextractswereinvestigatedforanti-lipid peroxidation,anti-superoxideradicalformationandfree radicalscavenging activities[18].The results showed thatalltestedextractsexhibited antioxidantactivity at differentmagnitudesofpotency[18].Casuarinin, chebu-lanin,chebulinicacidand1,6-di-O-galloyl-b-d-glucose, isolatedfromT.chebula,werealsotestedinthisstudy andshowedsignificantantioxidantactivity[18].Chang and Lin [19] reported antioxidant activities of water, methanoland95%ethanolextractsoftheair-driedfruit ofT.chebula.However,theantioxidantactivity ofthe polyphenolic-richextractofT.chebulafruithasnotbeen previously evaluated. Therefore, the objective of this studywastoassessthefreeradicalscavengingpotential ofthepolyphenolicextractofT.chebulafruits.
2. Materialsandmethods
2.1. Chemicals
Gallicacidstandard(BatchNo.90618)wasprocured from Himedia LaboratoriesPvt. Ltd., Mumbai, India.
AscorbicacidwasobtainedfromMerckSpecialtiesPvt. Ltd.,Mumbai,India.HPLC-grademethanoland acetoni-trile,as wellas aceticacidwereobtained fromMerck SpecialtiesPvt.Ltd.,Mumbai,India.Allofthe chemi-calsusedinthisstudywereofanalyticalreagentgrade, unlessspecified.
2.2. Plantmaterialandextractpreparation
The T. chebula fruits were collected from a local marketofAhmedabad,India,duringJanuary2012and were taxonomically identified and authenticated as T. chebulafruitsby Dr.YogeshT.Jasrai, Departmentof Botany,GujaratUniversity,India.Avoucherspecimen wasdepositedintheherbariumforfuturereference(Ref. No.GU/2013/53).
The T. chebula fruits were thoroughly cleaned, dried under the shade and coarsely powdered. The polyphenolic-rich extract wasprepared accordingtoa previously reportedmethod [20].The powderedplant material wasmixed with70% methanolandstoredat roomtemperaturefor5days.After5days,itwasfiltered and thesolvent was evaporated.The residue was dis-solvedinwater,andtheaqueouslayerwaswashedwith petroleumetherseveraltimesuntilaclearupperlayerof petroleumetherwasobtained.Thelowerlayerwasthen treatedwithethylacetatecontainingglacialaceticacid (10mL/L).Extractionofthepolyphenolswasperformed for 36hat roomtemperature,andthe combinedethyl acetate layer was thenconcentrated. The residue was lyophilised.Theextractobtainedwasdriedandstoredin anairtightcontainerat4◦C.Theyieldofthedry poly-phenolicextractwas30.5%(w/w).Thedriedextractwas dissolvedinMilli-Qwaterandusedforfurtherstudy.
2.3. Phytochemicalstudies
Qualitativeanalysisfordeterminingthepresenceof tannins, saponins, glycosides,flavonoids, steroids and alkaloids in the polyphenolic extract was performed usingstandardmethods[21].
2.3.1. Totalphenoliccontent(TPC)
Thetotalphenoliccontentofthepolyphenolicextract ofT.chebulawasestimatedbythereportedmethod[22]. Variousconcentrationsofgallicacidwereusedtoplotthe standardcurve.Thetotalphenoliccontentoftheextract isexpressedasmggallicacidequivalents/gdryweight ofextract.
2.3.2. Flavonoidcontent
ThetotalflavonoidcontentintheT.chebula polyphe-nolicextractwasestimatedbythestandardmethod[23].
Theflavonoidcontentoftheextractisexpressedasmg quercetinequivalents/gdryweightofextract.
2.3.3. Tannincontent
The tannin content of the T. chebula polyphenolic extractwasestimatedbythemethoddescribedby Hager-manandButler[24].Thetannincontentoftheextractis expressedasmgrutinequivalents/gdryweightofextract.
2.3.4. Ascorbicacidcontent
Ascorbic acid,alsocalled vitaminC,is oneof the mostabundantantioxidantsinT.chebulaextractandwas quantifiedaccordingtothepreviouslyreportedmethod [25].Theascorbiccontentoftheextractisexpressedas
g/gdryweightofextract.
2.4. Standardisationofextract
The polyphenolic extract of T. chebula fruits was standardised using gallic acid as a standard. For reverse-phaseseparation of gallic acidfrom the other constituents,aSpectra-PhysicsHPLC-UVsystem(San Jose, CA, USA) witha Spectra systemP1000 pump, a Spectra system UV1000 detector and a Rheodyne 7125 injector with a 20-L loop was used. Chro-matographicseparationwasachievedonaCOSMOSIL C18column(250mm×4.6mm,length×inner diame-terwith5-mparticlesize), maintained at35◦Cina columnoven.Themobilephaseconsistedofdeionised water:acetonitrile:aceticacid(88:10:2,v/v/v).Theflow rateofthemobilephasewasmaintainedat1.0mL/min, andthewavelengthofthedetectorwassetat280nm.
2.5. Reducingpowerassay
The reducing power was determined based on the ability of the antioxidant toform acoloured complex with potassium ferricyanide, TCA and FeCl3. It was measuredbyamodifiedmethod[26]inwhichtheextract of T. chebula fruits (1mL) at various concentrations (25,50,100,150,200and250g/mL)wasmixedwith potassium ferricyanideandphosphate buffer(pH 6.6) andincubated at50◦Cfor 20min.Then, TCA (10%) wasaddedandcentrifugedat3000rpmfor10min.The supernatantwasremovedandmixedwithFeCl3(0.1%). Theabsorbancewasthenmeasuredat700nm.Ahigher absorbance of the reaction mixture indicates a higher reducingpower.
2.6. Totalantioxidantcapacity
The total antioxidant capacity of the polyphe-nolic extract of T. chebula was measured using a
spectrophotometer method at concentrations of 50–500g/mL [27]. Ascorbic acid equivalents were calculatedusingastandardcurveofascorbicacid.The experimentwasconductedintriplicate,andthevalues are expressed as g equivalents of ascorbic acid per
g/mLofextract.
2.7. DPPHradicalscavengingassay
The ability of T. chebula polyphenolic extract/ascorbic acid to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured by the reportedmethod Shimada et al.[27].The addition of 0.1mMDPPH solution invariousconcentrations (10, 25,50,75,100and150g/mL)ofplantextract/ascorbic acidinthepresenceofTris–HClbuffer(50mM,pH7.4) resultedindecreasedabsorbance,whichwasmeasured at517nm. A mixture of methanol andextract served as the blank. The per cent inhibition was calculated by measuring the absorbance of extract/ascorbic acid treatedsamples againsttheblank.TheIC50 valuesfor thepolyphenolicextractwerecalculatedandcompared withthestandardreferencecompoundascorbicacid.
2.8. Nitricoxideradicalscavengingassay
TheabilityofthepolyphenolicextractofT.chebula
atgradedconcentrationsof50–500g/mLtoscavenge nitric oxide radical was determined by the modified method [28]. Various concentrations of polyphenolic extractwereaddedto10mMsodiumnitroprussideand incubated for 2.5h. After incubation, Griess reagent wasaddedtothetubesandtheabsorbanceofthe chro-mophoreformedwasreadat590nm.Theblanksolution containedamixtureof0.5mLPBSand0.5mLextract. TheIC50valuesandpercentinhibitionbyvarious con-centrationsofextractwerecalculatedbycomparingthe absorbancevaluesofthecontrolandtestcompounds.
2.9. Hydrogenperoxidescavengingassay
The hydrogen peroxide scavenging activity of T. chebula atconcentrations of 50–500g/mL was esti-mated using a hydrogen peroxide solution [29]. A solution of hydrogen peroxide (2mmol/L) was pre-pared inphosphatebuffer(pH 7.4). The polyphenolic extract/ascorbic acid (25, 50, 75, 100, 125 and 150g/mL)wasaddedtothehydrogenperoxide solu-tion(0.6mL). Amixture ofphosphatebuffer(3.3mL) andextract(0.5mL)servedastheblank.Theabsorbance of hydrogen peroxideat 230nm was determinedafter 10min against a blank solution containing phosphate
buffer without hydrogen peroxide and compared to ascorbicacid,whichwasthereferencecompound.
2.10. Statisticalanalysis
Theresultsareexpressedasthemean±standarderror of the mean (SEM). The statistical analysis and lin-earregressionanalysiswereperformedusingGraphPad Instat,software,version5.0.TheIC50 valueswere cal-culated and compared by paired t tests. p<0.05 was consideredsignificant.
3. Resultsanddiscussion
3.1. Phytochemicalscreening
Thequalitativephytochemicalscreeningofthe poly-phenolic extract revealed the presence of tannins, saponins,flavonoids andalkaloids.The totalphenolic contentoftheextractofT.chebulawas134.47mggallic acidequivalent/gdryweight.Theflavonoidcontentof theextractwas7.934mgofquercetinequivalent/gdry weight.Thetannincontentofthepolyphenolicextractof
T.chebulawas31.47mgrutinequivalent/gdryweight. Theascorbicacidcontentoftheextractwas8.74g/g dry weight of extract. Phytochemical analysis of T. chebulashowedthepresenceofgallicacid,ellagicacid, tannicacid,ethylgallate,chebulicacid,chebulagicacid, corilagin,mannitol,ascorbicacid(vitaminC),andother compounds[30].AnotherreportfoundT.chebulawith 32% tannin content[31].Polyphenols are widely dis-tributedinplants,andphenolicantioxidantsactasfree radicalscavengersandmetal chelators[32]. Recently, bioflavonoidsandpolyphenolsofplantoriginhavebeen usedextensivelyforfreeradicalscavengingandtoinhibit membranelipidperoxidation[33].
3.2. Standardisationofextract
ThepolyphenolicextractofT.chebulafruitsshowed thepresenceofgallicacidwitharetentionfactorof2.50 (Fig.1b),whichiscomparabletotheretentionfactorof 2.40of thepure standardgallic acid(Fig. 1a). More-over,thepolyphenolicextract ofT.chebulafruitsalso showedthepresenceofotherpolyphenoliccompounds, withretentionfactorsof1.40and1.80.Recently,many natural antioxidants havebeen isolated from different plant materials [34,35], and some types of polyphe-nols,suchaschebulanin,corilagin,neochebulinicacid, ellagicacid,gallicacid,chebulagicacid,andchebulinic acid,are presentinthe fruitsof T.chebula[36].Inan earlierstudy, Rangsriwonget al.[37]investigated the
Fig.1.(a)Chromatogramofthestandardgallicacidand(b) chro-matogramofthepolyphenolicextractofT.chebulafruits.
separation of polyphenoliccompounds, such as gallic acid and ellagic acid, from T. chebula fruits by sub-critical water extraction. The decoction of T. chebula
fruit contained 3,4,6-tri-O-galloyl-d-glucose, chebulic acid, -punicalagin, corilagin, ␣-punicalagin, chebu-lagicacid,gallicacid,1,3,4,6-tri-O-galloyl--d-glucose, chebulinic acid, 1,2,3,4,6-penta-O-galloyl-d-glucose, ellagic acid, and 1,6-di-O-galloyl-d-glucose [38]. Flavonoids also have significant antioxidant activity underbothinvivoandinvitroconditions[39].
3.3. Reducingpowerassay
The reducing capacity of the polyphenolic extract of theT. chebulafruitswasmeasuredby itsabilityto
Fig.2.ThereducingpoweractivityofthepolyphenolicextractofT.
chebulafruits.Dataarepresentedasthemeans±SEMoftriplicates.
***p<0.001fromthecontrol.
transformFe3+toFe2+atvariousconcentrations(25,50, 100,150,200and250g/mL).Theresultsrevealedthat thereducingactivitysignificantlyincreasedasthe con-centrationoftheextractwasincreased(r2=0.989),with amaximumincreaseat250g/mL(Fig.2).The reduc-tivecapacity of acompounddepends onthe presence ofreductones, whichexhibitantioxidativepotentialby breakingthefreeradicalchainanddonatingahydrogen atom.Therefore,reducingactivityleadstothe termina-tionoftheradicalchainreactionsthatmayotherwisebe verydamaging[40].Thepresenceofantioxidant reduc-tants in the polyphenolicextract of T.chebula causes the reduction of the Fe3+/ferricyanide complex to the ferrousform,indicatingthatthepolyphenolicextractof
T.chebulahassignificantreducingpower.Lietal.[41] reportedtheexistenceofasimilarlinearco-relationship betweenthereducingpowerandtotalphenoliccontent (TPC).
3.4. Totalantioxidantcapacity
The total antioxidant capacity of the polyphenolic extractofT.chebulafruitsrangedfrom0.140to0.387at graded concentrations of 50–500g/mL (r2=0.963; Fig.3).Gallicacid,apolyphenylpresentinT.chebula, was previously evaluated for free radical scavenging activityandhassignificantreducingpowerand antioxi-dantactivity[42].
3.5. DPPHradicalscavengingassay
The DPPH radicalscavengingactivity of the poly-phenolicextractofT.chebulaatdifferentconcentrations is shown in Fig. 4 and is compared to the refer-ence compoundascorbic acid.Theradicalscavenging
Fig.3.Total antioxidantcapacities of thepolyphenolic extract of
T. chebula fruits and ascorbic acid. Data are presented as the
means±SEMoftriplicates.*p<0.05;***p<0.001fromthecontrol.
Fig.4.DPPHradicalscavengingactivityoftheT.chebulaextractand ascorbicacid.Dataarepresentedasthemeans±SEMoftriplicates. ***p<0.001fromthecontrol.
activity of the extract was the highest at the max-imum dose of 150g/mL, with an IC50 value of 14±0.05g/mL (r2=0.976). Ascorbic acid showed an IC50 value of 16±0.06g/mL (r2=0.987). The effectwas concentration-dependent.DPPHisastable, nitrogen-centred free radical, which, upon accepting hydrogen from the antioxidants present in the poly-phenolicextract,isconvertedintoastablediamagnetic molecule,diphenyl-picrylhydrazine[43].Theobserved reductionofDPPHbytheextractwaseitherduetothe transferofahydrogenatomorthetransferofanelectron. Phenoliccompoundsarealsoeffectivehydrogendonors, whichmakesthemgoodantioxidants[44].
3.6. Nitricoxideradicalscavengingassay
Inthepresentstudy,thenitricoxideradical quench-ingactivityofthepolyphenolicextractwasdetectedand
Fig. 5.Nitric oxide radicalscavenging activity of the extract of
T. chebula fruits and ascorbic acid. Data are presented as the
means±SEMoftriplicates.***p<0.001fromthecontrol. comparedwiththe standardascorbicacid.Theextract exhibited the maximum per cent inhibition of 267% at a concentration of 500g/mL, with an IC50 value of30.51g/mL, inaconcentration-dependentmanner (r2=0.977; Fig. 5). However, ascorbic acid exhibited maximum per cent inhibition of 189%, with an IC50 valueof42g/mL(r2=0.955;Fig.5).Thescavenging activityoftheextractagainstnitricoxidewasdetected byitsabilitytoinhibittheformationof nitritethrough direct competition with oxygen and oxides of nitro-genin the reaction mixture [40].The decrease inthe concentrationofthenitricoxideradicalwasmore signif-icantthanascorbicacid,whichisduetotheantioxidant activity of the polyphenolic extract. Nitric oxide is a potentpleiotropicmediatorofphysiologicalprocesses, suchassmoothmusclerelaxation,neuronalsignalling, inhibitionofplateletaggregationandregulationof cell-mediatedtoxicity.Itisadiffusiblefreeradicalthatplays manyrolesasaneffectormoleculeindiversebiological systems,includingneuronalcommunication, vasodilata-tion and antimicrobial and antitumor activities [45]. Moreover,inpathologicalconditions,nitricoxidereacts withsuperoxideanionandformspotentiallycytotoxic molecules,suchasperoxynitrite.
3.7. Hydrogenperoxidescavengingactivity
The polyphenolic-rich extract of T. chebula
showed significant scavenging activity of H2O2 in a concentration-dependent manner (r2=0.990, Fig. 6), withanIC50 valueof265.53g/mL,whereastheIC50 value for ascorbic acid was 278g/mL (r2=0.993, Fig.6).Hydrogen peroxideis aweakoxidisingagent andcandirectlyinactivate afewenzymes, usuallyby oxidationofessentialthiolgroups.Hydrogenperoxide
Fig.6.Hydrogenperoxideradicalscavengingactivityofthe polyphe-nolicextractofT.chebulafruitsandascorbicacid.Dataarepresented asthemeans±SEMoftriplicates.**p<0.01;***p<0.001fromthe control.
cancrosscellmembranesrapidly,andonceinsidethe cell, H2O2 likelyreacts withFe2+,andpossibly Cu2+ ions, to form hydroxyl radicals, which then become powerfuloxidisingagents.Thismaycausemanytoxic effects.Thephenoliccompoundsmayactasfreeradical scavengers because of their hydrogen-donatingability andscavengingability[46].
3.8. Correlationbetweenthetotalphenoliccontent andantioxidantactivity
Thetotalphenoliccontentofthepolyphenolicextract of T. chebula is significantly correlated with its total antioxidant capacity (R=0.992, p<0.05), DPPH rad-ical scavenging activity (R=0.971, p<0.05), nitric oxide radical quenching activity (R=0.995, p<0.05) andhydrogenperoxide scavengingactivity(R=0.990,
p<0.05).Thisresultindicatesthatthephenoliccontents ofT.chebulaareresponsibleforitsantioxidantactivity.It hasbeenproposedthattheantioxidantactivityofplants may be due totheir phenolic compounds [47].Many plantsexhibitefficientantioxidantpropertiesbecauseof theirphenolicconstituents[48].
4. Conclusion
Polyphenols are valuable plantconstituents for the scavenging of free radicals because of their phenolic hydroxylgroups[49].This,togetherwiththeobtained results,suggeststhatastheamountofpolyphenolic com-poundsincreases,theantioxidantactivityalsoincreases. In conclusion, the presentstudy demonstratesthat the polyphenolicextractofT.chebulafruitscanprotectthe body from oxidative stress from ROS, whichmay be duetothephyto-chemicalsintheformofpolyphenols
thatoccurintheplant.Thesemaybeusedin nutraceu-ticalsandthefoodindustry.However,additionalstudies arenecessarytodevelopamethodforthefractionation andidentificationofpolyphenolsandtodeterminethe mostactiveantioxidantcompoundsinthepolyphenolic extract.
Acknowledgement
ThefinancialassistancetoSarmisthaSahafromLady TataMemorialTrust(LTMT/AD/Q2/2011-2013)inthe form of a Senior Research Fellowship is gratefully acknowledged.
References
[1]Y.Liu,G.Fiskum,D.Schubert,Generationofreactiveoxygen speciesbythemitochondrialelectrontransportchain,J. Neu-rochem.80(2002)780–787.
[2]B. Halliwell, J.M. Gutteridge, Free Radicals in Biology and Medicine,OxfordUniversityPress,Oxford,1998.
[3]R.Yazdanparast,A.Ardestani,Invitroantioxidantandfree rad-icalscavengingactivityofCyperusrotundus,J.Med.Food10 (2007)667–674.
[4]R.Yazdanparast,S.Bahramikias,A.Ardestani,Nasturtium ofic-inalereducesoxidativestressandenhancesantioxidantcapacity inhypercholesterolaemicrats,Chem.Biol.Interact.172(2008) 176–184.
[5]S.A.Tasaduq,K.Singh,S.Sethi,S.C.Sharma,K.L.Bedi,J. Singh,B.S.Jaggi,R.K.Johri,Hepatocurativeandantioxidant pro-fileofHP-1,apolyherbalphytomedicine,Hum.Exp.Toxicol.22 (2003)639–645.
[6]N.A. Minkyun,B.A.E.Wan,S.S.Kang,B.S.Min,J.K. Yoo, O.K.Yuk,S.Yu-Ichiro,Y.Sciichi,M.Nobuhiko,Cytoprotective effectonoxidativestressandinhibitoryeffectoncellularaging ofTerminialiachebulafruit,Phytother.Res.18(2004)737–741.
[7]G.P.Senthilkumar, S.P.Subramanian,Biochemical studieson theeffectofTerminaliachebulaonthelevelsofglycoproteins instreptozotocin-inducedexperimentaldiabetesinrats,J.Appl. Biomed.6(2008)105–115.
[8]V.R.Kannan, G.S.Rajasekar,P.Rajesh,V.Balasubramanian, N.Ramesh,E.K.Solomon,D.Nivas,S.Chandru,Anti-diabetic activityonethanolicextractsoffruitsofTerminaliachebulaRetz. alloxaninduceddiabeticrats,Am.J.DrugDiscov.Dev.2(2012) 135–142.
[9]H.S.Lee,S.H.Jung,B.S.Yun,K.W.Lee,Isolationofchebulic acidfromTerminaliachebulaRetz.anditsantioxidanteffectin isolatedrathepatocytes,Arch.Toxicol.81(2007)211–218.
[10]F. Malckzadeh, H. Ehsanifar, N. Shahamat, M. Levin, R.R. Colwell,Antibacterialactivityofblackmyrobalan(Terminalia chebulaRetz.)againstHelicobactorpyroli,Int.J.Antimicrobiol. Agents18(2001)85–88.
[11]D.Lee,K.Boo,J.Woo,F.Duan,K.Lee,T.Kwon,H.Y.Lee, K.Z.Riu,D.Lee,Anti-bacterialandanti-viralactivitiesofextracts fromTerminaliachebulabarks,J.KoreanSoc.Appl.Biol.Chem. 54(2011)295–298.
[12]V.Nair,S.Singh,Y.K.Gupta,Anti-arthriticanddisease modify-ingactivityofTerminaliachebulaRetz.inexperimentalmodels, J.Pharm.Pharmacol.62(2010)1801–1806.
[13]D.A.Israni,K.V.Patel,T.R.Gandhi,Anti-hyperlipidemicactivity ofaqueousextractofTerminaliachebulaandGaumutrainhigh cholesteroldietfedrats,Int.J.Pharm.Sci.1(2010)48–59.
[14]N.Pratibha,V.S.Saxena,A.Amit,P.D’Souza,M.Bagchi,D. Bagchi,Anti-inflammatoryactivitiesofAller-7,anovel poly-herbalformulationforallergicrhinitis,Int.J.TissueReact.26 (2004)43–51.
[15]G.K.Rasineni,D.Siddavattam,A.R.Reddy,Freeradical quench-ingactivityandpolyphenolsinthreespeciesofColeus,J.Med. PlantsRes.2(2008)285–291.
[16]J.A. Ross, C.M. Kasum, Dietary flavonoids: bioavailability, metaboliceffects,andsafety,Ann.Rev.Nutr.22(2002)19–34.
[17]B.Hazra,R.Sarkar,S.Biswas,N.Mandal,Comparativestudyof theantioxidantandreactiveoxygenspeciesscavengingproperties in theextracts of thefruits ofTerminalia chebula, Termina-liabelericaandEmblicaofficinalis,BMCComplement.Altern. Med.(10)(2010)20.
[18]H.Cheng,T.Lin,K.Yu,C.Yang,C.Lin,Antioxidantandfree radicalscavengingactivitiesofTerminaliachebula,Biol.Pharm. Bull.26(2003)1331–1335.
[19]C.L.Chang,C.S.Lin,Phytochemicalcomposition,antioxidant activity,andneuroprotectiveeffectofTerminaliachebulaRetzius extracts,Evid.BasedComplement.Altern.Med.2012(2012) 1–7.
[20]C.T.Kumarappan,E.Thilagam,S.C.Mandal,Antioxidant activ-ityofpolyphenolicextractsofIchnocarpusfrutescens,SaudiJ. Biol.Sci.19(2012)349–355.
[21]A. Sofowara, Medicinal Plants and Traditional Medicine in Africa,SpectrumBooksLtd.,Ibadan,Nigeria,2009,pp.289.
[22]V.L.Singleton,R.Orthofer,R.M.Lamuela-Raventos,Analysisof totalphenolsandotheroxidationsubstratesandantioxidantsby meansofFolin–Ciocalteureagent,MethodsEnzymol.299(1999) 152–178.
[23]A.Meda,C.E.Lamien,M.Romito,J.Millogo,O.G.Nacoulma, Determination of the total phenolic, flavonoid and proline contentsinBurkinaFasanhoney,aswellastheirradical scav-engingactivity,FoodChem.91(2005)571–577.
[24]A.E.Hagerman,L.G.Butler,Proteinprecipitationmethodforthe quantitativedeterminationoftannins,J.Agric.FoodChem.26 (1978)809–812.
[25]B.C.Adedayo,G.Oboh,A.A.Akindahunsi,Changesinthetotal phenolcontentandantioxidantpropertiesofpepperfruit( Dennet-tiatripetala)withripening,Afr.J.FoodSci.4(2010)403–409.
[26]G.K.Jayaprakash,R.P.Singh,K.K.Sakariah,Antioxidantactivity ofgrapeseedextractsonperoxidationmodelsinvitro,J.Agric. FoodChem.55(2001)1018.
[27]K.Shimada,K.Fujikawa,K.Yahara,T.Nakamura, Antioxida-tivepropertiesofxanthanontheanti-oxidationofsoybeanoilin cyclodextrinemulsion,J.Agric.FoodChem.40(1992)945–948.
[28]N.Sreejayan,M.N.A.Rao,Nitricoxidescavengingby curcumi-noids,J.Pharm.Pharmacol.49(1997)105–107.
[29]R.J.Ruch,S.J.Cheng,J.E.Klaunig,Preventionofcytotoxicity andinhibitionofintercellularcommunicationbyantioxidant cat-echinsisolatedfromChinesegreentea,Carcinogenesis10(1989) 1003–1008.
[30]I.S.Grover,S.Bala,AntimutagenicactivityofT.chebula (myrob-lan)inSalmonellatyphimurium,Ind.J.Exp.Biol.30(1992) 339–341.
[31]R.R.Chattopadhyay,S.K.Bhattacharyya,Plantreview Termina-liachebula:anupdate,Pharmacog.Rev.1(1)(2007)151–156.
[32]L.J.Juang,S.J.Sheu,T.C.Lin,Determinationofhydrolyzable tanninsinthefruitofTerminaliachebulabyhigh-performance
liquidchromatographyandcapillaryelectrophoresis,J.Sep.Sci. 27(2004)718–724.
[33]A.A.Newairy,H.M.Abdou,Protectiveroleofflaxlignansagainst leadacetate-inducedoxidativedamageandhyperlipidemiainrats, FoodChem.Toxicol.47(2009)813–818.
[34]L.Packer,A.S.H.Ong,BiologicalOxidantsandAntioxidants: MolecularMechanismsandHealthEffects,AOCSPress, Cham-paign,IL,1997.
[35]S.V.Jovanovic,M.G.Simic,Antioxidantsinnutrition,Ann.N.Y. Acad.Sci.899(2000)326–334.
[36]T.Bhaumik,P.C.Joshi,A.K.Dey,A.B.Kundu,Chemical inves-tigationofTerminaliachebulaRetz.,Bull.Med.Ethnobot.Res. 10(1989)190–192.
[37]P.Rangsriwong,N.Rangkadilok,J.Satayavivad,M.Goto,A. Shotipruk, Subcriticalwater extraction of polyphenolic com-poundsfromTerminaliachebulaRetz.fruits,Sep.Purif.Technol. 66(2009)51–56.
[38]F.Pellati,R.Bruni,D.Righi,A.Grandini,M.Tognolini,F.P.Pio, F.Poli,S.Benvenuti,D.D.Rio,D.Rossi,Metaboliteprofilingof polyphenolsinaTerminaliachebulaRetziusayurvedicdecoction andevaluationofitschemopreventiveactivity,J. Ethnopharma-col.147(2013)277–285.
[39]P.G.Pietta,Flavonoidsasantioxidants,J.Nat.Prod.63(2000) 1035–1042.
[40]C.Alasalvar,M.Karamac,R.Amarowicz,F.Shahidi,Antioxidant andantiradicalactivitiesinextractsofhazelnutkernel(Corylus avellanaL.)andhazelnutgreenleafycover,J.Agric.FoodChem. 54(2006)4826–4832.
[41]H.Li,Z.Hao,X.Wang,L.Huang,J.Li,Antioxidantactivitiesof extractsandfractionsfromLysimachiafoenum-graecumHance, Bioresour.Technol.100(2009)970–974.
[42]G.Yen,P.Duh,H.Tsai,Antioxidantandpro-oxidantproperties ofascorbicacidandgallicacid,FoodChem.79(2002)307–313.
[43]S.V.Knezevic,B.Blazekovic,M.B.Stefan,A.Alegro,T.Koszegi, J.Petrik,Antioxidantactivitiesandpolyphenoliccontentsofthree selectedMicromeriaspeciesfromCroatia,Molecules16(2011) 1454–1470.
[44]A.Michalak,Phenoliccompoundsandtheirantioxidantactivity inplantsgrowingunderheavymetalstress,PolishJ.Environ. Stud.15(2006)523–530.
[45]F. Shahidi,C.Alasalvar, C.M.Liyana-Pathirana, Antioxidant phytochemicals in hazelnutkernel(Corylusavellana L.) and hazelnutbyproduct,J.Agric.FoodChem.55(2007)1212–1220.
[46]O.Beyhan,M.Elmastas,F.Gedikli,Totalphenoliccompounds andantioxidantcapacityofleaf,dryfruitandfreshfruitof fei-joa(Accasellowiana,Myrtaceae),J.Med.PlantsRes.4(2010) 1065–1072.
[47]N.C.Cook,S.Samman,Flavonoids–chemistry,metabolism, car-dioprotectiveeffects,anddietarysources,J.Nutr.Biochem.7 (1996)66–76.
[48]R.A.Larson,Theantioxidantsofhigherplants,Phytochemistry 27(1988)969–978.
[49]T.Hatano,H.Kagawa,T.Yasuhara,T.Okuda,Twonewflavonoids andotherconstituentsinlicoriceroottheirrelativeastringency andradicalscavengingeffects,Chem.Pharm.Bull.36(1988) 2090–2097.