LC Fingerprinting for Assessment

LCFingerprintingforAssessmentoftheQualityoftheLipophilicComponentsofSalviamiltiorrhizaDong-FengYang1,2,Zong-SuoLiang1,2,&,Jian-LinLiu1,21CollegeofLifeScienceofNorthwestA&FUniversity,712100Yangling,China;E-Mail:[e-mail]2ShaanxiResearchCenterinTCMFingerprintandNPLibrary,712100Yangling,ChinaReceived:14June2008/Revised:22October2008/Accepted:23October2008Onlinepublication:11December2008AbstractWithanappropriatemethodoffingerprinting,andbyanalysisofthreemarkercompounds,thefactorsaffectingthelipophiliccomponentsinSalviamiltiorrhizawereinvestigatedinthiswork.Astandardfingerprintofthelipophiliccomponentsofrootperidermwasconstructedat270nmandalimitednumberofmarkercompoundswereproposedonthebasisoftenbatchesofS.miltiorrhizafromShangluoGAP(goodagriculturalpractice)base.TheabsoluteamountsofthesecomponentsinS.miltiorrhizawereseriouslyaffectedbyproductionarea,harvesttime,rootdisease,rootcolor,androotdiameter.Relativeamountswere,however,consistentunderdifferentconditions.AccumulationofsomecomponentspeakedinAugustandwassignificantlyacceleratedbydiseasessuchasrootrotandnematodiasis.Theredrootswerethebestquality,comparedwithsableandyellowishroots.Thequalityofrootsofdifferentdiameterwasvariable.Forexample,amongsevengroupsofsamplesitwasbestforrootsofdiameter0.2cmandbetween0.5and0.8cm,andworstforrootheadsandfibrousroots.Inconclusion,thequalityofthelipophiliccomponentsofS.miltiorrhizawascomprehensivelyassessed.KeywordsColumnliquidchromatographySalviamiltiorrhizaBungeLamiaceaeQualityassessmentLipophiliccomponentFingerprintingIntroductionSalviamiltiorrhizaisoneofthemostimportantandpopulartraditionalChinesemedicinalplants.Itsroot,calledDansheninChinese,iswidelyusedforpreventionandtreatmentofcoronaryheartdisease[1],chronicrenalfailure[2],atherosclerosis[3],myocardialinfarction[4],anginapectoris[5],myocardialischemia[6],andliverfibrosisandcirrhosis[7].LipophiliccompoundsarethemainbiologicallyactivecomponentsofS.miltiorrhiza.Manyofthesehavebeenidentified,forexampletanshinoneIIA,tanshinoneI[8],dihydrotanshinoneI[9],andcryptotanshinone[10].TanshinoneIIAisthemainlipophiliccompoundthatimprovesbloodcircula-tion[11],preventsmyocardialinfarction[1],andactsasanantioxidant[12].TanshinoneIisusuallyusedfortreat-mentofanginapectoris[13].Dihydro-tanshinoneIhasaninhibitoryeffectontheactionofDNAtopoisomeraseI[14]andinductionofapoptosis[15].Chemicalfingerprintinghasbeenwidelyusedforqualitycontroloftradi-tionalChinesemedicine(TCM)[16].AssessmentofthequalityofS.miltiorrhiza2009,69,555–560DOI:10.1365/s10337-008-0918-60009-5893/09/03
2008Vieweg+Teubner|GWVFachverlageGmbHFullShortCommunicationChromatographia2009,69,March(No.5/6)555byfingerprintingmethods[17–23]andbyquantitativeanalysisofmarkercom-pounds[24]hasbeenreported.Intheworkdiscussedinthispaper,achromatographicfingerprintingmethodandquantitativeanalysisofthreemarkercompoundshavebeenusedtoinvesti-gatefactorsaffectingthequalityofS.miltiorrhiza,includingproductionarea,harvesttime,rootdisease,rootdiameter,rootcolor,andthedifferentpartsoftheplant.MaterialsandMethodsChemicalsandMaterialsLC-grademethanolwaspurchasedfromFisherScientific(Fairlawn,NJ,USA).Analytical-gradeethanolwasfromAnteBiochemistry(Suzhou,China).LC-qual-itywaterwasobtainedfromaUPWultrapurewatersystempurchasedfromShanghaiUltrapureTechnology(Shanghai,China).SalviamiltiorrhizawassuppliedbyShaanxiTaslyPlantsPharmaceutical(Shangluo,China)andthemedicalplantsgardenofNorthwestA&FUniversity.TanshinoneIIA,tanshinoneI,anddihydrotanshinoneIwerepurchasedfromtheNationalInstitutefortheControlofPharmaceuticalandBiologicalProducts(Beijing,China).SamplePreparationSamplesofS.miltiorrhizaweredriedat60C,comminutedtopowder,andsievedthrougha0.45-mmscreen.Thepowder(0.25g)wasextractedultrasoni-callywith50mLethanolfor1handtheextractswerecentrifugedat12,000rpmfor15minandthenfilteredthrougha0.45-lmMilliporefilter.StocksolutionsoftanshinoneIIA,dihydrotanshinone,IandtanshinoneIwerepreparedinmethanolanddilutedtothedesiredconcentrations.ChromatographyLCwasperformedwithaWaters(Milford,MA,USA)binarypumpandphotodiode-arraydetector(DAD).ThecolumnwasaWatersSunFireC18(250mm94.6mm,5-lmparticle).DatawereacquiredandprocessedbyuseofEmpower2software.Separationwasachievedbyelutionwithalineargradientpreparedfrommethanolandwater.Thegradientwas:t=0min60%methanol;t=25min,70%methanol;t=35min,75%meth-anol;t=45min,90%methanol;t=50min,90%methanol;t=55min,100%methanol.Theflowratewas1mLmin-1,thecolumntemperature30C,andthesamplesize20lL.Theeffluentwasmonitoredbetween190and500nmbyuseofthePDA.ResultsandDiscussionMethodValidationTheextractofsample#1wasinjectedfivetimes.Peak8(tanshinoneIIA)wasusedasthereferencepeakandtherela-tiveretentiontimes(RRT)andrelativepeakareas(RPA)oftheothermainpeakswerecalculated.Therepeatabilityofthemethodwasassessedbyanalysisoffiveindependentlypreparedextractsofsample#1.Theprecisionoftheanalysiswasdeterminedbyreplicateanalysisofthesameextractofsample#1overaperiodof48h.Theresultsrevealedthemethodperformedwell.Detectorresponsewasalinearfunc-tionoftheconcentrationofthecom-poun