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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 14␮g/mL),nitricoxideradicalconcentration

(IC50 30.51␮g/mL)and hydrogenperoxidescavengingactivity (IC50 265.53␮g/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.

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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].

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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 (25,50,100,150,200and250␮g/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 50–500␮g/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, 25,50,75,100and150␮g/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

atgradedconcentrationsof50–500␮g/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 50–500␮g/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 150␮g/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

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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. Theascorbicacidcontentoftheextractwas8.74␮g/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

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Fig.2.ThereducingpoweractivityofthepolyphenolicextractofT.

chebulafruits.Dataarepresentedasthemeans±SEMoftriplicates.

***p<0.001fromthecontrol.

transformFe3+toFe2+atvariousconcentrations(25,50, 100,150,200and250␮g/mL).Theresultsrevealedthat thereducingactivitysignificantlyincreasedasthe con-centrationoftheextractwasincreased(r2=0.989),with amaximumincreaseat250␮g/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 50–500␮g/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 150␮g/mL, with an IC50 value of 14±0.05␮g/mL (r2=0.976). Ascorbic acid showed an IC50 value of 16±0.06␮g/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

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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 500␮g/mL, with an IC50 value of30.51␮g/mL, inaconcentration-dependentmanner (r2=0.977; Fig. 5). However, ascorbic acid exhibited maximum per cent inhibition of 189%, with an IC50 valueof42␮g/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 valueof265.53␮g/mL,whereastheIC50 value for ascorbic acid was 278␮g/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

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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.

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References

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