Characterization and antioxidant activity of a polysaccharide extracted from Sarcandra glabra

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CarbohydratePolymers

journalhomepage:www.elsevier.com/locate/carbpol

CharacterizationandantioxidantactivityofapolysaccharideextractedfromSarcandraglabra

LeiJin1,XinGuan1,WeiLiu,XianZhang,WeiYan,WenbingYao∗,XiangdongGao∗∗

StateKeyLaboratoryofNaturalMedicines,SchoolofLifeScienceandTechnology,ChinaPharmaceuticalUniversity,Nanjing210009,PRChina

article

info

abstract

Articlehistory:

Received5April2012

Receivedinrevisedform15May2012Accepted19May2012

Available online 27 May 2012

Keywords:

SarcandraglabrapolysaccharideBox–Behnkendesign

MonosaccharidecompositionAntioxidantactivity

Responsesurfacemethodology(RSM)wasemployedtooptimizetheparametersforpolysaccharideextractedfromtheaerialpartsofSarcandraglabra(SGP).Theoptimumconditionswerepredictedasfollows,ratioofwatertorawmaterialat30,extractingtemperatureat85◦C,extractingdurationat3h,andyieldwasestimatedat4.55%.TheexperimentalyieldofSGPundertheoptimumconditionswas4.49±0.09%.Ahomogenouspolysaccharide(SGP-1)wasobtainedbypurificationusingDEAE-cellulose-52andSephacrylS-400columnchromatography.SGP-1showedasinglesymmetricalpeakinhighperformancesize-exclusionchromatography(HPSEC)andtheaveragemolecularweight(Mw)wasesti-matedtobe1.06×104Da.Itwascomposedofglucose,galactose,andmannoseinaratioof8.38:3.13:1determinedbygaschromatography(GC).TheinvitroantioxidanttestsshowedthatSGP-1hassignificantinhibitioneffectsonhydroxyl,superoxideanion,DPPH,ABTSradicalsinadose-dependentmanner.ThisstudyindicatedthatSGP-1couldbeusedasapotentialnaturalantioxidant.

© 2012 Elsevier Ltd. All rights reserved.

1.Introduction

Sarcandraglabra(Thunb.)Nakai(Chinesename:ZhongjiefengorCaoshanhu)isconsideredasanimportantherbintraditionalChi-nesemedicines(TCMs).Medicalcomponentspreparedfromthisherbareusedasantitumororanti-inflammatorydrugsinChina(Zheng,Wang,Chen,&Hu,2003).Commercialsliketoothpaste,healthtea,cosmetics,andchewinggummadefromthisplantarealsobestselling.S.glabraismainlydistributedinthesouthernofChinaandSoutheastofAsia.Itisestimatedthatabout100millionkilogramsmaterialsofS.glabraarerequiredin5yearstosatisfythehugemarket(Cai&Chen,2010).Untilnow,mostoftheresearcheswerefocusonisolationandactivityofflavonoids,coumarins,triter-penoids,sesquiterpenes,andotherphenoliccomponentsofthisplant(Fengetal.,2010;Heetal.,2011;Xiao,Guo,Deng,&Li,2009),butlittleattentionwasdevotedtotheextractionandfunctionofbiomacromolecules(especiallypolysaccharides)fromS.glabra.Polysaccharidesfromnaturalsourcesareconsideredtobeeffective,non-toxicsubstances.Manypolysaccharidescouldbeexploredintodrugsfortheirpharmacologicalactivities.Antioxidantsarehelpfultoalleviatestress-induceddiseasesincludinginflammatory,cardiacdisorders,diabetesmellitus,and

neurodegenerativediseases(Ananthietal.,2010).Recently,severalreportsindicatedsomepolysaccharidesextractedfrombotanicalsandfunguscouldbeexploredaspotentialantioxidants(Huaetal.,2012;Jin,2012).Hotwaterextractionisaclassicalpolysaccharideextractionmethod.Intheprocessofhotwaterextraction,thecrudepolysaccharideyieldwasdependedontheparametersasfollows,solid-to-liquidratio,extractiontemperature,duration,andextrac-tiontimes.OptimizationofextractionparametersisimportantforfurtherstudyofthepolysaccharidefromS.glabra.

Thepresentstudywasconductedfirstlytooptimizeextract-ingparametersusingresponsesurfacemethodology(RSM).Afterisolationandpurification,preliminarycharacterizationofthehomogenouspolysaccharide(SGP-1)wasperformed.Inaddition,theantioxidantactivityofSGP-1wasestimatedthroughaseriesoftests,includinghydroxyl,superoxideanion,DPPH,ABTSradicalscavenging,ferrousionchelating,andreducingpowerdetermina-tions.

2.Materialsandmethods

2.1.Materialsandchemicals

∗Correspondingauthor.Tel.:+862583271218;fax:+862583271218.∗∗Correspondingauthor.Tel.:+862583271298;fax:+862583271249.

E-mailaddresses:wbyao@cpu.edu.cn(W.Yao),xiangdonggao@yahoo.com.cn(X.Gao).1

Theseauthorscontributedequallytothiswork.

ThematerialsofS.glabrawerepurchasedfromNanchang,JiangxiProvince,China,inOctober2010.Thediethylaminoethyl-cellulose(DEAE-52),SephacrylS-400forchromatographyanddextranstandardsofT-70,T-40,T-20,T-10,andT-5wereobtainedfromPharmaciaCo.Ltd.(Uppsala,Sweden).ThirtypercenthydrogenperoxidewaspurchasedfromSinopharm

0144-8617/$–seefrontmatter© 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.carbpol.2012.05.074

L.Jinetal./CarbohydratePolymers90 (2012) 524–532

525

Table1

Box–Behnkenexperimentaldesignandresultsforextractionyield.

Run

Factor1A:watertosolid

Factor2

B:temperature

Factor3C:extractduration

Experimentalyield(%)

12345671011121314151617

25(0)20(−1)25(0)25(0)25(0)25(0)30(1)20(−1)30(1)25(0)25(0)25(0)20(−1)30(1)20(−1)30(1)25(0)

90(1)80(0)80(0)80(0)70(−1)70(−1)80(0)90(1)70(−1)90(1)80(0)80(0)70(−1)90(1)80(0)80(0)80(0)

3(1)3(1)2(0)2(0)1(−1)3(1)1(−1)2(0)2(0)1(−1)2(0)2(0)2(0)2(0)1(−1)3(1)2(0)

4.273.914.204.283.3.753.793.883.3.6.3.343.4.153.6.534.37

±±±±±±±±±±±±±±±±±

0.060.050.090.060.070.110.030.090.040.110.050.010.130.100.130.070.04

chemicalreagentCo.Ltd.d-Glucose(Glc),d-galactose(Gal),d-mannose(Man),l-rhamnose(Rha),d-xylose(Xyl),d-arabinose(Ara),Gallicacid,trichlorideferric(FeCl3),ferroussulfate(FeSO4),ethylenediaminetetra-aceticacid(EDTA),ferrozine,nitrobluetetrazolium(NBT),Tris–HClbuffer,thiobarbituricacid(TBA),2,2-diphenyl-2-picrylhydrazylhydrate(DPPH),2-2-azino-bis-(3-ethyl-benzthia-zoline-6-sulfonicacid)(ABTS),andascorbicacid(Vc)werepurchasedfromSigmaChemicalCo.(St.Louis,MO,USA).Trifluoroaceticacid(TFA)wasfromMerck(Germany).AllotherreagentsofanalyticalgradewerepurchasedfromShanghaiChem-icalCo.(Shanghai,China).

realvalueonthecenterpointand󰀁Xiwasthestepchangevalue.Therangeofindependentvariables,aBBDmatrixandtheresponsevaluescarriedoutfordevelopingthemodelwerelistedinTable1.Thewholedesignedexperimentconsistedof17trialpointsinarandomorder.Thesetrialsweredividedinto12factorialpoints,and5replicateswhichwereusedfortheestimationofapureerrorsumofsquaresatthecenterofthedesign.Theresponsevalueineachtrialwasanaverageofduplicates.BasedontheBBDexperi-mentaldata,regressionanalysiswascarriedoutandfittedintotheempiricalquadraticsecond-orderpolynomialmodel:

2.2.Extractionmethod

Y=

󰀁

A0+

3󰀁i=1

AiXi

3󰀁i=1

AiiXi2

+

32󰀁󰀁i=1j=i+1

AijXiXj

(3)

5kgaerialpartsofS.glabrawascrushedtopassthrough80

meshsieveandrefluxedwith90%ethanolat80◦Cinwaterbathfor3htoremovefats,pigments,andsomeoligosaccharides.Eachdriedpretreatedsample(50g)wasextractedwithhotwaterindesignedconditions.Theextractionsolutionswerecollectedbycentrifugation(4000r/min,5min).Subsequently,aqueousextractwasconcentratedto1/3oftheoriginalvolume,cooled,andprecip-itatedwithfourvolumesofabsoluteethanolforovernightat4◦C.Theprecipitatescollectedbycentrifugation(4000r/min,5min)werewashedthreetimeswithabsoluteethanolanddriedunderreducedpressure.Thus,thecrudepolysaccharidenamedSGPwasobtainedforfollow-upstudy.Theyields(%)ofcrudepolysaccharideintheextractionwerecalculatedbythefollowingequations:

whereYrepresentedtheresponsefunction,A0,Ai,Aii,Aijweretheregressioncoefficientsofvariablesforintercept,linear,quadratic,andinteractiontermsrespectively,andXi,Xjwerelevelsof

/j).Thecoefficientsofthesecondtheindependentvariables(i=

polynomialmodelandtheresponsesurfacesobtainedfromtheexperimentaldesignwerefittedtomultiplenonlinearregressionsusingsoftwareDesign-Expert7.1.3(Stat-Ease,Inc.,USA).Thefit-nessofthepolynomialmodelequationwasrepresentedbythecoefficientofdeterminationR2,thestatisticalsignificancewascheckedbyF-testataprobability(P-value)of0.01or0.05.Thesig-nificancesoftheregressioncoefficientswerealsotestedbyF-test.

Polysaccharidesyield%(w/w)

2.4.Purificationofpolysaccharide

=

driedcrudeextractionweight

×100%

powderweight(50g)

(1)

2.3.Experimentaldesign

Onthebasisofsingle-factorexperimenttest(datawerenotshown),aBox–Behnkendesign(BBD)withthreeindependentvari-ables(X1,ratioofwatertorawmaterial,X2,extractiontemperature,X3,extractionduration)atthreelevelswereperformed.Forstatisti-calcalculation,thevariableswerecodedbythefollowingequation:

X−X0

xi=i

󰀁Xi

(2)

wherexiwastheindependentvariablecodedvalue,Xiwastheindependentvariablerealvalue,X0wastheindependentvariable

ThecrudepolysaccharideextractedfromS.glabra(0.5g)wasdissolvedindeionizedwater,centrifuged,andthenthesuper-natantwasappliedtoaDEAE-cellulose-52column(2.6cm×30cm)equilibratedwithdeionizedwater.Elutionwascarriedoutwithdeionizedwater,subsequentlywithalineargradientof0–2MNaClaqueoussolutionsataflowrateof18mL/h.Eachfractionintesttube(3mL)wascollectedbyanautomatedstep-by-stepfrac-tioncollector,andmonitoredbythephenol–sulfuricacidmethodat490nm.ThedeionizedwaterfractionwasthenappliedtoaSephacrylS-400column(1.6cm×100cm)equilibratedandelutedwithdeionizedwateratroomtemperatureataflowrateof18mL/h.Fractionswerecollectedandanalyzedbyphenol–sulfuricacidmethodat490nm.Thehomogeneousfractionsweregathered,con-centrated,andlyophilizedtogiveawhitepowderypolysaccharidewhichwascodedasSGP-1.ThetotalcarbohydratecontentofSGP-1wasdeterminedbyphenol–sulfuricacidmethodwithd-glucose

526L.Jinetal./CarbohydratePolymers90 (2012) 524–532

asstandardat490nm(Dubois,Gilles,Hamilton,Rebers,&Smith,1956).

2.5.Molecularweightdetermination

TheaveragemolecularweightofSGP-1wasdeterminedbyhighperformancesize-exclusionchromatography(HPSEC),whichwasperformedonanAgilent1100HPLCsystemequippedwithaShodexSUGARKS-805column(8.0mmID×300mm)andaRIDdetec-tor.Themobilephasewasdeionizedwater,andtheflowratewas1.0mL/minat30◦C.1mgsamplewasdissolvedinthemobilephase(1.0mL),and20␮Lofsupernatantwasinjectedaftercentrifuged(8000rpm;3min).Themolecularmasswasestimatedbythecom-parisontoacalibrationcurvepreparedwiththeT-seriesdextranstandardsofknownmolecularmasses(T-70,T-40,T-20,T-10,andT-5).

2.6.Analysisofmonosaccharidecompositions

SGP(5mg)washydrolyzedin1mLof2MTFAat100◦Cfor8h.TheproductwasreducedwithNaBH4at65◦Cfor1h,acety-latedwithmixtureofpyridineandaceticanhydride(1:1,v/v)at100◦Cfor1h,andthenanalyzedbygaschromatography(GC)withaHP-5capillarycolumn(HP6820,Hewlett–Packard).Thetemper-atureofthecolumnwasheldat150◦Cfor2min,increasedto220◦Catarateof2◦C/min,andsequentiallyincreasedto280◦Catarateof30◦C/minwithN2asthecarriergasandinositolastheinternalstandard.Standardmonosaccharides(l-rhamnose,d-arabinose,d-xylose,d-mannose,d-glucose,andd-galactose)werealsoderivedandanalyzedunderthesameprocedureasreferences(Honda,Suzuki,Kakehi,Honda,&Takai,1981).

2.7.FT-IRspectroscopy

TheFT-IRspectrumofSGP-1wasrecordedwithaNicolet5700IRspectrometerwiththerangeof4000–400cm−1.ThesamplewasanalyzedasKBrpellets.

2.8.Totalphenolicsdetermination

TotalphenolicscontentofSGP-1wasdeterminedbythemethod(Khokhar&Magnusdottir,2002)usingFolin-Ciocalteureagent,usinggallicacidasthestandard.Thecontentoftotalphenolicswascalculatedonthebasisofthecalibrationcurveofgallicacidandexpressedasmg/100mgofthedryweightofSGP-1.

2.9.Antioxidantactivity

2.9.1.Hydroxylradicalassay

ThehydroxylradicalscavengingabilityofSGP-1wasmea-suredaccordingtothemethoddescribedpreviously(Halliwell,Gutteridge,&Aruoma,1987)withaminormodification.Reactionsolutionmixtureinatotalvolumeof1.0mLcontainedphosphatebuffer(pH7.4,20mM),EDTA(100␮M),deoxyribose(60mM),fer-rictrichloride(100␮M),H2O2(1mM),ascorbicacid(100␮M),anddifferentconcentrationsofSGP-1(0,0.0625,0.125,0.25,0.5,1.0,and2.0mg/mL).Solutionsofascorbicacidandferrictrichloridewerepreparedimmediatelybeforeuse.Thereactionsolutionswereincubatedfor30minatroomtemperature,andthen1mLof1%TBAand1mLof30%(v/v)HClwereaddedtothemixture.Themix-tureswereboiledfor10minandcooledwithicebath.Deionizedwaterandascorbicacidwereaddedasblankandpositivecon-trol,respectively.Themixturesweremeasuredat532nm,andthe

scavengingactivityofhydroxylradical(%)wascalculatedaccordingtothefollowingequation:

ScavengingA532(blank)−A532(sample)

effect(%)=

A532(blank)

×100

whereA532(blank)wastheabsorbanceofthecontrol(deionizedwater,insteadofsample)andA532(sample)wastheabsorbanceofthetestsamplemixedwithreactionsolution.

2.9.2.Superoxideradicalassay

ThesuperoxideradicalscavengingactivityofSGP-1wasdeter-minedaccordingtotheliteratureprocedure(Sun,Wang,Fang,Gao,&Tan,2004)withafewmodifications.Briefly,superoxideradicalswerecreatedin3.0mLof16mMTris–HClbuffer(pH8.0),contain-ing78mMreducednicotinamideadeninedinucleotide(NADH),50␮Mnitrobluetetrazolium(NBT),10␮Mphenazinmethosulfate(PMS),andsamplesatgivenconcentrations(0,0.0625,0.125,0.25,0.5,1.0,and2.0mg/mL).Thecolorablereactionofsuperoxiderad-icalswithNBTwasassessedat560nm.Thedeionizedwaterwasusedastheblankcontrolandascorbicacidwasusedaspositivecon-trol.Thescavengingeffectofsuperoxideradicals(%)wascalculatedaccordingtothefollowingequation:

ScavengingA560(blank)−A560(sample)

effect(%)=

A560(blank)

×100

whereA560(blank)wastheabsorbanceofthecontrol(deionizedwater,insteadofsample)andA560(sample)wastheabsorbanceofthetestsamplemixedwithreactionsolution.

2.9.3.ScavengingactivityofDPPHradical

Thescavengingactivityof1,1-dihpenyl-2-picrylhydrazyl(DPPH)radicalwasmeasuredaccordingtoaliteraturemethod(Bracaetal.,2001)withslightmodification.0.5mLofsamples(0,0.0625,0.125,0.25,0.5,1.0,and2.0mg/mL)wasaddedto3.0mLofa0.01%(v/v)ethanolsolutionofDPPH.Absorbanceat517nmwasmeasuredafter30min.ThescavengingactivityofDPPHradical(%)wascalculatedaccordingtothefollowingequation:

Scavengingeffect(%)A517(blank)−A517(sample)

=

A×100

517(blank)

whereA517(blank)wastheabsorbanceofthecontrol(deionizedwater,insteadofsample)andA517(sample)wastheabsorbanceofthetestsamplemixedwithreactionsolution.

2.9.4.Totalantioxidantactivity

Thetotalradicalscavengingcapacitywasassessedwithareportedprocedure(Katalinic,Milos,Kulisic,&Jukic,2006)withslightmodification.TheABTSradicalcation(ABTS+)wasgeneratedbymixinganABTS(7mM)solutionwithapotassiumpersulfate(2.45mM)aqueoussolutionandleavingthemixturesinadarkplaceatroomtemperaturefor16h.Then3.6mLoftheABTS+solu-tionwasaddedto0.4mLofvariousconcentrations(0,0.0625,0.125,0.25,0.5,1.0,and2.0mg/mL)oftheSGP-1solutions.Afterreactingfor20minatroomtemperature,theabsorbancewasmeasuredat734nm.ThescavengingactivityofABTS(%)wascalculatedaccord-ingtothefollowingequation:

ScavengingeffectA734(blank)−A734(sample)

(%)=

A734(blank)

×100

whereA734(blank)wastheabsorbanceofthecontrol(deionizedwater,insteadofsample)andA734(sample)wastheabsorbanceofthetestsamplemixedwithreactionsolution.

2.9.5.DeterminationofFe2+-chelatingability

ThechelatingactivityofSGP-1onFe2+wascarriedoutfol-lowingareportedprocedure(Dinis,Madeira,&Almeida,1994).

L.Jinetal./CarbohydratePolymers90 (2012) 524–532

527

Table2

Regressioncoefficientsofthepredictedquadraticpolynomialmodel.

Sources

Sumofsquares

Degreeoffreedom

Meansquare

Fvalue

Significancelevel

Modelx1x2x3x1x2x1x3x2x3x1x1x2x2x3x3

ResidueLackoffitPureerrorCortotal

1.720.110.270.420.0340.0580.0420.120.480.0120.0720.0530.0191.80

911111111173416

0.190.110.270.420.0340.0580.0420.120.480.120.0100.018

4.770E−003

18.5510.4726.5340.553.325.584.0711.66.1911.65

0.00040.01430.00130.00040.11140.05020.08340.01120.00030.0112

**a*b****

****

3.72

0.1186

R2=0.9597

AdjR2=0.9098

CV=2.55

ab

Significanceat0.01level.Significanceat0.05level.

DifferentconcentrationofSGP-1(0,0.0625,0.125,0.25,0.5,1.0,and2.0mg/mL)wasmixedwith3.7mLdeionizedwater,andthenreactedwithFeCl2(2mM,0.1mL).After0.2mLof5mMferrozinewasadded,thesolutionwasmixed,lefttostandfor10minatroomtemperature,andthentheabsorbanceofthemixturewasdeter-minedat562nm.ThedeionizedwaterandEDTAwasrespectivelymeasuredastheblankandpositivecontrol.Thechelatingactiv-ityofSGP-1onFe2+(%)wascalculatedaccordingtothefollowingequation:

AconsiderablevariationintheyieldsofSGP,whichdependedupontheextractionconditionsatdifferentexperimentalcombinationswereshowninTable1.Byemployingmultipleregressionanalysisontheexperimentaldata,thepredictedresponseYfortheyieldofpolysaccharidescanbeobtainedbythefollowingsecond-orderpolynomialequation:

Y=4.31+0.12x1+0.19x2+0.23x3+0.092x1x2+0.12x2x3

Chelatingability(%)=

A562(blank)−A562(sample)

×100

A562(blank)

222

+0.10x1x3−0.17x1−0.34x2−0.17x3

(4)

whereA562(blank)wastheabsorbanceofthecontrol(deionized

water,insteadofsample)andA562(sample)wastheabsorbanceofthetestsamplemixedwithreactionsolution.

2.9.6.Determinationofreducingpower

ThereducingpowerofSGP-1wasevaluatedaccordingtoapre-viousmethod(Oyaizu,1986)withslightmodification.Thereactionmixturescontained2.5mLphosphatebuffer(pH6.6,0.2M),2.5mLpotassiumferricyanide(1%,w/v)andSGP-1(0,0.0625,0.125,0.25,0.5,1.0,and2.0mg/mL).Afterincubatingat50◦Cfor20min,2.5mLoftrichloroaceticacid(10%,w/v)wasaddedtothemixtureforter-minatingthereaction,andthencentrifugedat1000×gfor10min.Analiquotof2.5mLsupernatantwascollectedandmixedwith2.5mLdeionizedwaterand0.5mLFeCl3(0.1%,w/v).Afterincubat-ingatroomtemperaturefor15min,theabsorbanceofthemixturewasmeasuredat700nm,usingascorbicacidasapositivecontrol.

2.10.Statisticalanalysis

Allthedatawerepresentedasmeans±standarddeviation(SD)fromtriplicates.StatisticalanalysisinvolveduseoftheSigmaplotsoftwarepackage12.0(SystatSoftwareInc.)andExcel2007(MicrosoftCorp.).DifferencewasconsideredsignificantwhenP<0.05.

3.Resultsanddiscussions

3.1.Optimizationoftheextractionprocess

Inthisstudy,RSMwasintroducedtoevaluatethemultipleparametersandtheirinteractionsinahotwaterextractionpro-cess.Becauseofsavingtime,spaceandrawmaterial,BoxBehnkendesign(BBD)ismoreadvantageousthanthetraditionalsingleparameteroptimization(Box&Behnken,1960;Qiaoetal.,2009).

Table2listedthefitstatisticsofpolysaccharideextractionyield(Y)forthedesignedpolynomialquadraticmodel.F-valueof18.55andP-valueof0.0004indicatesthemodelwassignificant.Thelackoffitevaluatesthemodel’sfailureonthepointsinthedesignedexperimentaldatadomainthatwerenotincludedintheregres-sion.Thelowvalueof3.72forF-valueand0.1186forP-valueoflackoffitelucidatedthemodelwasnotsignificantrelativetothepureerror.Theaccuracyofthemodelswasevaluatedbydeter-minationofcoefficientR2andadjustedcoefficientR2values.ThevalueofR2calculatedfromANOVAanalysiswas0.9597,indicat-ingthatonly4.03%ofthetotalvariationswerenotexplainedbythemodel.TheadjustedR2valueof0.9098alsoconfirmedthatthemodelwassignificant.ThedegreeofprecisionfromthecomparedexperimentswasexhibitedbyCoefficientofVariation(CV)value.Alowvalue2.55ofCVclearlyindicatedahighdegreeofprecisionandagoodreliabilityoftheexperimentalvalues.Thesignificanceofeachcoefficientandinteractionstrengthbetweeneachindepen-dentvariablewasestimatedusingP-values.DatashowinginTable2indicatedthatallthelinearcoefficients(x1,x2,x3)andquadratic

2,x2,x2)significantlyaffectedtheyieldofSGP,termcoefficient(x1

23

withP-valuessmallerthan0.05.Theothertermcoefficients(x1x2,x2x3,x1x3)werenotsignificant(P>0.05).

3Dresponsesurfaceand2Dcontourplotsbasedontheregres-sionequationcouldprovideaprocesstovisualizetherelationshipbetweenresponsesandtriallevelsofeachvariableandthespecificinteractionsbetweentwotestvariables.Whethertheinteractionsbetweenthevariablesaresignificantornotcouldbeindicatedbytheshapesofthecontourplots.Circularcontourplotexhibitsthattheinteractionsbetweenthecorrespondingvariablesareneg-ligible,whileellipticalcontourplotshowsthattheinteractionsbetweenthecorrespondingvariablesaresignificant(Muralidhar,Chirumamil,Marchant,&Nigam,2001).AsshowninFig.1,ahighvalueyieldofSGPoccuratahighratioofwatertorawmaterialandamoderatetemperature(Fig.1aandd),ahighratioofwatertorawmaterialandalongduration(Fig.1bande)andalongduration

528L.Jinetal./CarbohydratePolymers90 (2012) 524–532

Fig.1.Responsesurface(3D)andcontourplots(2D)showingtheeffectsofvariables(x1:ratioofwatertorawmaterial;x2:extractiontemperature,◦C;andx3:extractionduration,h)ontheresponseY.

andamoderatetemperature(Fig.1candf).Fig.1showedthattheresponsesurfaceofeachvariablewasindependentwitheachother.Theinteractionsofthreevariableswerenotsignificant,whicharealsoobservedfromtheP-valuesinTable2.TheoptimalconditionsforSGPextractionobtainedfromtheseplotswereratioofwatertorawmaterialat30,extractingtemperatureat85◦Candextract-ingdurationfor3h.ThemaximumpredictedyieldofSGPunderthisconditionwas4.55%.Threetestswerecarriedouttoevaluatethereliabilityofpredictedoptimumconditions.Comparedwiththeyieldof4.53±0.07%oftheRun16thinTable1,thevalueof4.49±0.09%wasconsiderednotsignificantdifference(P>0.05).

3.3.FT-IRanalysis

3.2.Homogeneity,molecularweight,carbohydratecontent,andmonosaccharidescompositionofSGP-1

TheFT-IRspectrumofSGP-1wasshowninFig.4,abroadbandaround3400cm−1exhibitedOHstretchvibrationandthepeakat2929cm−1wasassignedtoCHstretchvibration.Thepeakaround18cm−1wasacharacteristicabsorptionbandofthebondedwater(Maréchal,2004).Threestretchingpeaksat1026,1080,and1155cm−1indicatedthepresenceofCObondsandpyranoseringinthemonosaccharideinSGP-1.Themoderateintensebandsintheregionof1350–1450cm−1werecorrespondedtosymmetricaldeformationsofCH2andCOHgroups.Theabsorptionat2cm−1belongedtothe␤-anomericconfiguration.Thepeakat937cm−1wasbelongedtotheskeletalmodeofpyranosering(Yietal.,2012).ThosesignalsallindicatedthatSGP-1hadthetypicalsaccharidemoietyabsorptionpeaks.

Thehighperformancesize-exclusivechromatograph(HPSEC)wasemployedtodeterminethehomogeneityandrelativemolecu-larweightofSGP-1,usingstandardglucansasreferences.AsshowninFig.2,SGP-1waselutedassingleandsymmetricpeakfromGPCprofile,whichindicatedthatitwashomogeneous.TherelativemolecularweightofSGP-1wasestimatedtobe10.6kDa.ReactionwithCoomassieBrilliantG-250wasnegativeandnoabsorptionat260or280nmintheUVspectrumindicatingtheabsenceofpro-teinandnucleicacidinSGP-1.TotalcarbohydratecontentinSGP-1wasdeterminedtobe95.57%.GCispreferredtomonosaccharidesquantitativeandqualitativedeterminationforitssensitive.Com-paredtotheretentiontimeoftheacetatederivativesinGC,SGP-1consistedofthreedifferentmonosaccharides,includingglucose,galactose,mannose(Fig.3),andthemolarratioof8.38:3.13:1.00wasmeasuredbyinternalstandardmethod.

Fig.2.HPSECtraceofSGP-1detectedbyRID.

L.Jinetal./CarbohydratePolymers90 (2012) 524–532

529

Fig.3.(a)RelativeretentiontimesonGCofacetatederivativeofstandardmonosaccharides.(b)RelativeretentiontimesonGCofacetatederivativeofmonosaccharidesinSGP-1.

3.4.TotalphenoliccontentinSGP-1

Thecompoundsextractedfromnaturalresourceswithgoodantioxidantactivityexceptofpolysaccharidesisresponsiblephe-nolicsaswell.ThetotalphenolcontentofSGP-1wasabout0.124±0.011mg/100mg(n=5).ForthetotalphenolicswerealmostremovedbythepurificationusingDEAE-cellulose-52andSephacrylS-400columnchromatography,theantioxidantactivityofSGP-1mainlyresultedfrompolysaccharides.

ascorbicacid(77.26%).Whentheconcentrationsincreasedfrom1.0mg/mLto2.0mg/mL,bothascorbicacidandSGP-1didnotshowanyhigherscavengingeffects.ThemechanismofSGP-1onclean-ingofthehydroxylradicalcouldrelatetochelateironandinducetheminactiveinFentonreaction.

3.5.Antioxidantactivity

3.5.1.Scavengingabilityofhydroxylradicals

Thehydroxylradicalisgenerallyacceptedasahighlypotentoxidant.Aftercrossingintocell,hydroxylradicalreadilyreactswithdiversebiomolecules,suchasproteins,lipidsDNA,andcarbo-hydratesincells,whichaccordinglycausestissuedamageorcellapoptosis(Yangetal.,2011).Fig.5ashowedthehydroxylrad-icalscavengingabilityofSGP-1atdifferentconcentrationswithascorbicacidasthepositivecontrol.SGP-1exhibitedscavengingactivityonhydroxylradicalsinaconcentration-dependentman-neratthetestconcentrations.Attheconcentrationof1.0mg/mL,thehighesthydroxylradicalscavengingrate(59.03%)ofSGP-1wasachieved,whichwas18.23%lesscomparedwiththatof1.0mg/mL

3.5.2.Scavengingabilityofsuperoxideanion

Superoxideradicaliscell-injuringthroughcausingdamagetoDNAandmembranelipidofcell(MacDonald,Galley,&Webster,2003).SGP-1wascomparedwithascorbicacidforsuperoxideradicalscavengingactivity.AsshowninFig.5b,atthetestcon-centrations,SGP-1exhibitedscavengingactivityonsuperoxideradicalsinaconcentration-dependentmanner.Themaximumscavengingability(70.82%)wasobservedforSGP-1ataconcentra-tionof1.0mg/mL,whileascorbicacidcouldachievethemaximumscavengingactivityof90.46%at0.25mg/mL.Atlowconcentra-tions(0–0.5mg/mL),ascorbicacidexhibitmuchhigherscavengingactivitythanSGP-1.However,asconcentrationincreasedtoupto1.0mg/mL,SGP-1showedstrongscavengingability.Polysaccha-rideswithspecialconformations,hydrogeninOHbondscouldbeeasilyliberatedandthuscouldstabilizesuperoxideanion.ThemechanismofSGP-1onscavengingsuperoxideanionmaybeasso-ciatedwiththedissociationenergyofOHbond.

Fig.4.IRspectraofSGP-1.

3.5.3.DPPHradicalscavengingactivity

TheantioxidantmechanismofDPPHradicalscavengingisrelatedtotheacceptanceofhydrogenbytheDPPHradical.Withthehydrogendonatedbytheantioxidant,theDPPHwasconvertedintoDPPH-H,anon-radicalform.ThemodelofscavengingDPPHradicaliswellacknowledgedandwidelyappliedtoestimatethefreeradicalscavengingabilityofvariousantioxidants(Yuan,Zhang,Fan,&Yang,2008).Thehydrogen-donatingabilityoftheseantiox-idantsdecidestheirantioxidantcapacity.Fig.5cdepictedDPPHradicalscavengingactivityofSGP-1andcomparedwithascorbicacidaspositivecontrol.Undertheexperimentalconditions,thescavengingeffectofSGP-1wascorrelatedwellwiththeincreas-ingconcentrations.TheDPPHradicalscavengingrateofSGP-1increasedfrom10.87%to77.59%,attheconcentrationsfrom0.0625to2.0mg/mL.TheresultindicatedthatSGP-1hadnoticeableeffectonthescavengingoffreeDPPHradicals.However,thescavengeeffectofascorbicacidisstrongerthanthatofSGP-1ontheidenticalconcentration.

530L.Jinetal./CarbohydratePolymers90 (2012) 524–532

Fig.5.AntioxidantactivitiesofSGP-1.(a)Scavengingabilityofhydroxylradicals,(b)scavengingabilityofsuperoxideanion,(c)DPPHradicalscavengingactivity,(d)ABTSradicalscavengingactivity,(e)ferrousionchelatingability,and(f)reducingpower.Alltreatmentswereconductedintriplicate,andmeanvaluesarereported.Theverticalbarsrepresentthestandarddeviationofeachdatapoint.Valuesmarkedbythesamecodearesignificantlydifferentcomparedwiththeblanks(P<0.01).

3.5.4.ABTSradicalscavengingactivity

ABTSassayisanacceptedmethodinmeasuringthetotalantiox-idantpowerofapotentialantioxidant.ThescavengingabilityofSGP-1andascorbicacidonABTSfreeradicalsisshowninFig.5d.ThescavengingpowerofSGP-1correlatedwellwiththeincreas-ingconcentrations,butwasslightlylowerthanascorbicacidatthesameconcentration.Themaximumscavengingabilityofascorbicacidwasattheconcentrationof0.5mg/mL,whileSGP-1obtained

themaximumeffectattheconcentrationof2.0mg/mL.SGP-1exhibitedmorethan50%scavengingABTSradicalcapacitysincetheconcentrationreached0.5mg/mL.TheresultsindicatedthatSGP-1hadstrongscavengingpowerforABTSradicalandcouldbeexploredasapotentialantioxidant.Thisassaycanbeusedinbothorganicandaqueoussolventsystemsandcanalsobeusedasanindextoreflecttheantioxidantactivityofthetestsamples(Aparicio,Peinado,Escrig,&Rupérez,2010;Luoetal.,2010).

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3.5.5.Ferrousionchelatingability

Themetalchelatingabilitywasrecognizedasacorrelativeactiv-itytoantioxidant.Sometimessamples’antioxidantcapacitycouldalsobeincreasedbyenhancingtheirmetalchelatingactivity.TheferrousionchelatingactivityofSGP-1atdifferentconcentrationsisshowninFig.5e.ComparedwithEDTAintheequivalentcon-centration,SGP-1exhibitedamuchweakermetalchelatingability.ThechelatingrateforSGP-1wasonly31.73%evenat2.0mg/mL,whilethatofEDTAwas90.90%.Severalstudieshavedemonstratedthatthemetalionchelatingabilityofpolysaccharidescouldbeduetoformationofcross-bridgebetweencarboxylgroupinuronicacidanddivalention,anddifferenceinferrousionchelatingabil-ityamongvariousacidicpolysaccharideswasminor.However,theresultofmonosaccharidecompositiondidnotshowSGP-1containanycarboxylgroup(Fan,Li,Deng,&Ai,2012).TheroleofSGP-1intheprocessofferrousionchelatingneedstobefurtherexplored.

3.5.6.Reducingpower

Thereducingpower,whichservesasasignificantpotentialactivityindex,couldbeassessedbyaFe3+–Fe2+reductionreac-tion.ThepresenceofreductantinthereactioncausesthereductionofFe3+/ferricyanidecomplextotheferrousformandcanbemonitoredbytheformationofPerl’sPrussianblueat700nm.ThereducingpowerofSGP-1andascorbicacidwascomparedinFig.5f,higherabsorbancevalueindicatesstrongerreducingpower.ThemaximumreducingpowerofascorbicacidandSGP-1wereobtainedat0.25mg/mL(absorbance=1.749)and2.0mg/mL(absorbance=1.178),respectively.Therearevariousmechanismstoexplaintheactivitiesofantioxidants,suchasdecompositionofperoxide,preventionofcontinuedhydrogenabstraction,preven-tionofchaininitiation,andchelatingoftransitionmetalions.Sun,Liu,andKennedy(2010)reportedthatantioxidantpolysaccharidecouldbedefinedaselectrondonors,whichcouldreactwithfreeradicals.ThemoderatereducingpowerofSGP-1showedthatSGP-1mayhassomefreeelectronstoreactwithFe3+.However,thereducingpowerofSGP-1wasmuchweakercomparedwiththatofascorbicacid.

4.Conclusion

Responsesurfacemethodologywasaneffectivetoolforopti-mizingconditionsofSGPextractedfromS.glabra.Theoptimalexperimentalextractionyield4.49±0.09%wasobtainedwhentheextractionparametersasfollowingconditions,ratioofwatertorawmaterialat30,extractiontemperatureat85,andtheextractiondurationat3h.Undertheoptimumcondition,theexperimentalextractionyieldofSGPagreedcloselywiththepredictedyieldof4.55%.AfterpurifiedbyDEAE-cellulose-52andSephacrylS-400gelchromatography,ahomogenouspolysaccharideSGP-1wasobtained.TheaveragemolecularweightofSGP-1was10.6kDa,whichwasdetectedbytheHPSEC.Themonosaccharidecompo-sitionstudydemonstratedthatSGP-1isaheteropolysaccharideconsistingofglucose,galactose,andmannoseintheratioof8.38:3.13:1.Theresultofinvitroantioxidantmeasurementdemon-stratedthatSGP-1canscavengefreeradicals,whichmaycontributetotheabilityofS.glabratotreattheinflammationdiseasecausedbyoxidantdamage.

However,furtherinvestigationofstructuralidentificationsisrequiredtoelucidatetheantioxidantmechanism.

Acknowledgments

ThisstudywassupportedbytheNationalNaturalScienceFoundationofChina(Nos.30873201and81072570),theNationalScientificandTechnologicalMajorProjectforSignificantNew

DrugsCreation(2012ZX09502001-004),theProjectProgramofStateKeyLaboratoryofNaturalMedicines,ChinaPharmaceuticalUniversity(No.JKGP201103)andNaturalScienceFoundationofJiangsuProvinceofChina(No.BK2011621).

References

Ananthi,S.,Raghavendran,H.R.B.,Sunil,A.G.,Gayathri,V.,Ramakrishnan,G.,&Vas-anthi,H.R.(2010).Invitroantioxidantandinvivoanti-inflammatorypotentialofcrudepolysaccharidefromTurbinariaornata(MarineBrownAlga).FoodandChemicalToxicology,48,187–192.

Aparicio,I.M.,Peinado,C.M.,Escrig,A.J.,&Rupérez,P.(2010).Multifunctional

antioxidantactivityofpolysaccharidefractionsfromthesoybeanbyproductokara.CarbohydratePolymers,82,245–250.

Box,G.E.P.,&Behnken,D.W.(1960).Somenewthreeleveldesignsforthestudy

ofquantitativevariables.Technometrics:AJournalofStatisticsforthePhysical,Chemical,andEngineeringSciences,2,455–475.

Braca,A.,De-Tommasi,N.,Di-Bari,L.,Pizza,C.,Politi,M.,&Morelli,I.(2001).

AntioxidantprinciplesfromBauhiniatarapotensis.JournalofNaturalProducts,,2–5.

Cai,H.L.,&Chen,L.F.(2010).Feasibilityanalysisofcultivationandexploitationfor

SarcandraglabrainJiangleCounty.ModernAgriculturalSciencesandTechnology,20,158–159.

Dinis,T.C.P.,Madeira,V.M.C.,&Almeida,L.M.(1994).Actionofphenolicderivatives

(acetaminophen,salicylate,and5-aminosalicylate)asinhibitorsofmembranelipidperoxidationandasperoxyradicalscavengers.ArchivesofBiochemistryandBiophysics,315,161–169.

Dubois,M.,Gilles,K.A.,Hamilton,J.K.,Rebers,P.A.,&Smith,F.(1956).Colorimetric

methodfordeterminationofsugarsandrelatedsubstances.AnalyticalChemistry,28,350–356.

Fan,L.P.,Li,J.W.,Deng,K.Q.,&Ai,L.Zh.(2012).Effectsofdryingmethodson

theantioxidantactivitiesofpolysaccharidesextractedfromGanodermalucidum.CarbohydratePolymers,87,1849–18.

Feng,S.X.,Xu,L.X.,Wu,M.,Hao,J.,Qiu,S.X.,&Wei,X.Y.(2010).Anewcoumarin

fromSarcandraglabra.Fitoterapia,81,472–474.

Halliwell,B.,Gutteridge,J.M.C.,&Aruoma,O.I.(1987).Oxidativedamage,lipid

peroxidationandantioxidantprotectioninchloroplasts.ChemistryandPhysicsofLipids,44,327–340.

He,X.F.,Zhang,S.,Zhu,R.X.,Yang,S.P.,Yuan,T.,&Yue,J.M.(2011).Sarcanolides

AandB:TwosesquiterpenoiddimerswithanonacyclicscaffoldfromSarcandrahainanensis.Tetrahedron,67,3170–3174.

Honda,S.,Suzuki,S.,Kakehi,K.,Honda,A.,&Takai,T.(1981).Analysisofthemonosac-charidecompositionsoftotalnon-dialyzableurinaryglycoconjugatesbythedithioacetalmethod.JournalofChromatography,226,341–350.

Hua,Y.L.,Yang,B.,Tang,J.,Ma,Zh.H.,Gao,Q.,&Zhao,M.M.(2012).Structuralanaly-sisofwater-solublepolysaccharidesinthefruitingbodyofDictyophoraindusiataandtheirinvivoantioxidantactivities.CarbohydratePolymers,87,343–347.Jin,X.C.(2012).Bioactivitiesofwater-solublepolysaccharidesfromfruitshellof

CamelliaoleiferaAbel:Antitumorandantioxidantactivities.CarbohydratePoly-mers,87,2198–2201.

Katalinic,V.,Milos,M.,Kulisic,T.,&Jukic,M.(2006).Screeningof70medicinalplant

extractsforantioxidantcapacityandtotalphenols.FoodChemistry,94,550–557.Khokhar,S.,&Magnusdottir,S.G.M.(2002).Totalphenol,catechin,andcaffeinecon-tentsofteascommonlyconsumedintheUnitedKingdom.JournalofAgriculturalandFoodChemistry,50(3),565–570.

Luo,A.X.,He,X.J.,Zhou,S.D.,Fan,Y.J.,Luo,A.S.,&Chun,Z.(2010).Purifica-tion,compositionanalysisandantioxidantactivityofthepolysaccharidesfromDendrobiumnobileLindl.CarbohydratePolymers,79,1014–1019.

MacDonald,J.,Galley,H.F.,&Webster,N.R.(2003).Oxidativestressandgene

expressioninsepsis.BritishJournalofAnaesthesia,90,221–232.

Maréchal,Y.(2004).Observingthewatermoleculeinmacromoleculesusinginfrared

spectrometry:Structureofthehydrogenbondnetworkandhydrationmecha-nism.JournalofMolecularStructure,700,217–223.

Muralidhar,R.V.,Chirumamil,R.R.,Marchant,R.,&Nigam,P.(2001).Aresponse

surfaceapproachforthecomparisonoflipaseproductionbyCandidacylindraceausingtwodifferentcarbonsources.BiochemicalEngineeringJournal,9,17–23.Oyaizu,M.(1986).Studiesonproductsofbrowningreaction-antioxidativeactivi-tiesofproductsofbrowningreactionpreparedfromglucosamine.TheJapaneseJournalofNutrition,44,307–315.

Qiao,D.L.,Hu,B.,Gan,D.,Sun,Y.,Ye,H.,&Zeng,X.X.(2009).Extractionoptimizedby

usingresponsesurfacemethodology,purificationandpreliminarycharacteri-zationofpolysaccharidesfromHyriopsiscumingii.CarbohydratePolymers,76,422–429.

Sun,C.,Wang,J.W.,Fang,L.,Gao,X.D.,&Tan,R.X.(2004).Freeradicalscavenging

andantioxidantactivitiesofEPS2,anexopolysaccharideproducedbyamarinefilamentousfungusKeissleriellasp.YS4108.LifeSciences,75,1063–1073.

Sun,Y.X.,Liu,J.Ch.,&Kennedy,J.F.(2010).Purification,compositionanalysis

andantioxidantactivityofdifferentpolysaccharideconjugates(APPs)fromthefruitingbodiesofAuriculariapolytricha.CarbohydratePolymers,82,299–304.Xiao,X.H.,Guo,Z.N.,Deng,J.C.,&Li,G.K.(2009).Separationandpurification

ofisofraxidinfromSarcandraglabrabymicrowave-assistedextractioncoupledwithhigh-speedcounter-currentchromatography.SeparationandPurificationTechnology,68,250–2.

532L.Jinetal./CarbohydratePolymers90 (2012) 524–532

Yang,L.Q.,Zhao,T.,Wei,H.,Zhang,M.,Zou,Y.,Mao,G.H.,etal.(2011).

CarboxymethylationofpolysaccharidesfromAuriculariaauriculaandtheirantioxidantactivitiesinvitro.InternationalJournalofBiologicalMacromolecules,49,1124–1130.

Yi,Y.,Zhang,M.W.,Liao,S.T.,Zhang,R.F.,Deng,Y.Y.,Wei,Zh.Ch.,etal.(2012).Struc-turalfeaturesandimmunomodulatoryactivitiesofpolysaccharidesoflonganpulp.CarbohydratePolymers,87,636–3.

Yuan,J.F.,Zhang,Z.Q.,Fan,Z.C.,&Yang,J.X.(2008).Antioxidanteffectsand

cytotoxicityofthreepurifiedpolysaccharidesfromLigusticumchuanxiongHort.CarbohydratePolymers,74,822–827.

Zheng,W.J.,Wang,S.F.,Chen,X.G.,&Hu,Z.D.(2003).AnalysisofSarcandra

glabraanditsmedicinalpreparationsbycapillaryelectrophoresis.Talanta,60,955–960.