从香草茶,绞股蓝提取的免疫刺激多糖的分离和鉴定

J.Agric.FoodChem.2008,56,6905–6909IsolationandCharacterizationofImmunostimulatoryPolysaccharidefromanHerbTea,GynostemmapentaphyllumMakino6905XINGBINYANG,*,†YANZHAO,‡YINGYANG,†ANDYUNRUAN†KeyLaboratoryofMinistryofEducationforMedicinalPlantResourceandNaturalPharmaceuticalChemistry,CollegeofLifeSciences,ShaanxiNormalUniversity,Xi’an710062,andSchoolofPharmaceuticalSciences,FourthMilitaryMedicalUniversity,Xi’an710032,ChinaWater-solublepolysaccharidefromGynostemmapentaphyllumherbtea(PSGP)wasisolatedbyhot-waterextractionandethanolprecipitation.Thechemicalcomponentsandpreliminaryimmunomodu-latingactivityofPSGPwereinvestigatedbothinvitroandinvivo.CapillaryzoneelectrophoresisanalysisshowedthatPSGPwasatypicalnonstarchheteropolysaccharide,withglucosebeingthemaincomponentmonosaccharide(23.2%),followedbygalactose(18.9%),arabinose(10.5%),rhamnose(7.7%),galacturonicacid(4.7%),xylose(3.9%),mannose(3.1%),andglucuronicacid(1.2%).PSGPcouldsignificantlystimulateperitonealmacrophagestoreleasenitricoxide,reactiveoxygenspecies,andtumornecrosisfactor-Rinadose-dependentmanner.ThisimmunostimulatingactivityofPSGPwasfurtherdemonstratedbyitsinhibitionontheproliferationofhumancoloncarcinomaHT-29andSW-116cellsincubatedwiththesupernatantofPSGP-stimulatedmacrophageculture.ItisevidentthatPSGPisaveryimportantingredientresponsibleforatleastinparttheimmunomodulatingactivityofG.pentaphyllumherbtea.KEYWORDS:Gynostemmapentaphyllumherbtea;polysaccharide;capillaryelectrophoresis;immuno-stimulatingactivityINTRODUCTIONTheconsumptionofherbteaispopularinAsiancountries.GynostemmapentaphyllumMakino,named“Jiao-Gu-Lan”inChinese,isawell-knownherbteainAsia.Itisasaponin-richplantandcontainsabout90dammarane-typeglycosideswhicharecloselyrelatedtothecomponentsaponinsinexpensiveginseng,andhence,cheapG.pentaphyllumisregardedas“secondginseng”(1–7).Inrecentyears,G.pentaphyllumhasattractedgreatattentionowingtoitswidebioactivitiesforthetreatmentsofhepatitis,hypertension,chronicbronchitis,gastritis,cancer,andotherdiseases(1,7).Forthisreason,itisclaimedthatdrinkingherbteaofG.pentaphyllumcouldpromotehealthandalleviatetheseverityofmanydisorders.G.pentaphyllumherbteahasbeenproducedonalargescaleinChinaandhasalsobeensoldinEuropeasanherbteawithaclaimofbenefittohealthandbeauty(4,8).Traditionally,low-molecular-weightcompounds,suchasessentialoilandsaponins,havebeenconsideredastheactiveingredientsofthisplant(8–10).Recently,thewater-solublepolysaccharideshavealsobeendemonstratedtobepartiallyresponsibleforsomebioactivityofG.pentaphyllumherbtea(9,10).Inparticular,*Towhomcorrespondenceshouldbeaddressed.Phone:[phone].Fax:[phone].E-mail:[e-mail]greaterbioavailabilitybecausetheyareeasilysoakedoutintoteainfusioncomparedwithessentialoilandsaponins.Interestinantitumor,immunostimulatory,andantioxidantactivityofnaturalpolysaccharidesisgrowing(9–11).Tothebestofourknowledge,littleisknownabouttheimmunomodu-latoryactivityofpolysaccharidefromG.pentaphyllumherbtea.ThepresentworkwasthereforeundertakentoisolateandcharacterizethepolysaccharideinG.pentaphyllumherbteaandtostudyitsimmunomodulatoryactivitybothinvivoandinvitro.MATERIALSANDMETHODSMaterialsandReagents.TheherbteaofG.pentaphyllumwaspurchasedfromPingliCountryFiveleafGynostemmaInstitute,Shaanxiprovince,China.D-Mannose,L-rhamnose,D-glucuronicacid,D-galac-turonicacid,D-glucose,D-xylose,D-galactose,L-arabinose,D-fucose,and2,7-dichlorofluoresceindiacetate(DCF-DA)wereobtainedfromSigma(St.Louis,MO).CoomassiebrilliantblueG250andbovineserumalbumin(BSA)werepurchasedfromHuameiBiochemistryCo.(Shanghai,China).m-HydroxydiphenylwasfromSigmaCo.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT),dimeth-ylsulfoxide(DMSO),RPMI1640,phosphate-bufferedsaline(PBS),andfetalbovineserum(FBS)weretheproductsofGibcoBRL(Gaithersburg,MD).Trifluoroaceticacid(TFA)wasobtainedfromMerck(Darmstadt,Germany).1-Phenyl-3-methyl-5-pyrazolone(PMP)†‡ShaanxiNormalUniversity.FourthMilitaryMedicalUniversity.waspurchasedfromtheBeijingReagentPlant(Beijing,China).Allotherchemicalswereoftheanalyticalgradeavailable.10.1021/jf801101uCCC:$40.752008AmericanChemicalSocietyPublishedonWeb07/18/20086906J.Agric.FoodChem.,Vol.56,No.16,2008Yangetal.IsolationofPolysaccharidefromG.pentaphyllum(PSGP).Polysaccharidewasisolatedaswepreviouslydescribed(12).Briefly,theherbteaofG.pentaphyllum(stemandleaf)wasdriedat110Cfor2handmashedintoapowder.Thesample(500g)wasrefluxedat80Cfor3hinabsolutealcohol.Afterthemixturewasfiltered,theresiduesweredriedinairandthenextractedinhotwater(1:10,w/v)at80Cthreetimes,for1heachtime.Theextractedsolutionwasconcentratedto10%oftheoriginalvolumeinarotaryevaporatorunderreducedpressure,andthenthepolysaccharidewasprecipitatedbyadding4volumesof95%(v/v)ethanolat4Cfor24h.Thesedimentwascollectedbycentrifugation(3000g,10min).Thenthepolysac-charidesedimentwasfurtherrefinedbybeingrepeatedlywashedwithethanol,acetone,andetheralternately.Therefinedpolysaccharidewasdissolvedindistilledwaterandintensivelydialyzed(MW10000)againstdistille