西洋参冠瘿组织悬浮培养及其人参皂苷类成分的分离_英文_于荣敏

21卷5期

2005年9月生 物 工 程 学 报ChineseJournalofBiotechnologyVol.21 No.5September 2005TheGrowthCharacteristicsandGinsenosidesIsolationof Suspension-culturedCrownGallofPanaxquinquefolium西洋参冠瘿组织悬浮培养及其人参皂苷类成分的分离

YURong-Min1*,JINQian-Xing1,SUNHui2,YEWen-Cai1,2andZHAOYu3*

于荣敏1*,金钱星,孙 辉,叶文才121,2,赵 昱3*

1.暨南大学药学院,广州 510632

2.中国药科大学中药学院,南京 210009

3.浙江大学药学院,杭州 310031

1.DepartmentofPhytochemistryandPharmacognosy,CollegeofPharmacy,JinanUniversity,Guangzhou510632,China

2.DepartmentofPhytochemistry,ChinaPharmaceuticalUniversity,Nanjing210009,China

3.DepartmentofTraditionalChineseMedicineandNaturalDrugResearch,CollegeofPharmaceuticalSciences,ZhejiangUniversity,Hangzhou310031,China摘 要 对西洋参冠瘿组织悬浮培养生长特征进行了考察,并对其悬浮培养物中的人参皂苷类成分进行了提取、分离和鉴定。研究得到了培养物最大生物量收获时间[18162gPL(dryweight)]及其中最高人参皂苷累积时间(62014mgPLonthe27thday)。培养基中碳源、磷、氨基氮、硝基氮的利用率分别为9118%,100%,81%和97%。利用现代分离纯化方法从培养物中分离得到了4种人参皂苷类成分,利用理化及谱学技术分别鉴定为假人参皂苷F11(pseudoginsenosideF11,Ñ),人参皂苷Rd(ginsenosideRd,Ò),人参皂苷Rb1(ginsenosideRb1,Ó)和人参皂苷Rb3(ginsenosideRb3,Ô)。

关键词 西洋参,冠瘿组织,悬浮培养,人参皂苷,分离

中图分类号 R915;S56715 文献标识码 A 文章编号1000-3061(2005)05-0754-05

Abstract Thegrowthcharacteristicsandginsenosidesisolationofthesuspension-culturedcrowngallofPanaxquinquefoliumwerestudied.Theresultshowedthatthemaximumbiomassofcultureswas1816gPL(dryweight)andthecontentofginsenosidesreacheditsmaximumlevelof62014mgPLonthe27thday.Theutilizationratesofsugar,phosphorus,nitrogeninNH4+andnitrogeninNO3-were9118%,100%,81%and97%,respectively.Fourcompoundswereisolatedfromthesuspension-culturedcrowngallandtheirstructureswereelucidatedaspseudoginsenosideF11(Ñ),ginsenosideRd(Ò),ginsenosideRb1(Ó)andginsenosideRb3(Ô).

Keywords crowngall,ginsenosides,isolation,Panaxquinquefolium,suspensioncultures

Received:March25,2005;Accepted:May25,2005.

ThisworkwassupportedbythegrantsfromMinistryofEducationofChina(No.104180),NaturalSciencesFoundationofGuangdong(No.31891)andChineseTraditionalMedicineAdministrationofGuangdong,China(No.103041).

*Correspondingauthor.YuRong-Min:Tel:86-20-85228205;E-mail:rongminyu99@http://wendang.chazidian.com;

ZHAOYu:Tel:86-571-87217313;Email:dryuzhao@http://wendang.chazidian.com

国家教育部科学技术研究重点项目基金(No.104180),广东省自然科学基金(No.31891),广东省中医药局科研计划项目基金(No.103041)资助。

YURong-Minetal:TheGrowthCharacteristicsandGinsenosidesIsolationofSuspension-culturedCrownGallofPanaxquinquefolium755

Panaxquinquefoliumhasbeenwidelyusedforitstherapeuticeffects

[1]

experimentswereconductedusing250mLErlenmeyerflaskscontaining50mLofMSmedium,inoculatedwith410goffreshweightbiomassandincubatedat25eonanorbitalshakerat110rPmininthedarkness.Fiveflaskswereharvestedregularlyoveraperiodof30dforthemeasurementofbiomass,sugar,phosphorus,nitrogenandginsenosides.112 Measurementofmajornutrientsinmedium

Theconcentrationofresidualsugarinthemediumwasdeterminedaccordingtothemethodofpheno-lconcentratedsulfuricacid[5].TheresidualnitrogeninNH4+andNO3-inthemediumwasreaction[6]

and

measuredresorcinol

[8]

.However,thefieldcultivationofthe

plantisatime-consumingandlabor-intensiveprocess:ittakesoversixyearsforitsharvest,duringwhichgreatcareisneededsincethegrowthissubjecttoseveralconditions,suchassoil,climate,pathogensandpests.Inaddition,theplanthasfallenshortofsupplyforalongperiodduetoagreatdemandonthemarket.

ThemajoractiveconstituentsofP.quinquefoliumareginsenosides,adiversegroupofsteroidalsaponins[2].Theuseofplantbiotechnology,suchascellcultureandtransgenictechniques,hasbeenconsideredasacos-teffectiveapproachtoproductionofginsenosidesinlargequantities.Crowngall,Agrobacterium

akindofirregulartissuecausedbythetumefaciens

pathogen,

is

capable

of

usingpheno-lhypochlorite

respectively.

method[7],

PhosphorusofPO43-inthemediumwasdeterminedusingascorbicacidmethod

.

113 Determinationofcellsweight

Thecellsuspensionswerefilteredandwashedseveraltimeswithsufficientamountofdistilledwater.Thecellswereweighedafterbeingdriedtoaconstantweightatatemperaturebelow50e.114 Assayofginsenosides

Forsamplepreparation,110gofpowderedcrowngallcultureswasextractedwith20mLofmethanol,usinganultrasonicbathfor30minafterdegreasingwith30mLofether.Afterfiltrationandevaporationtodryness,theresiduewasdissolvedin20mLofwaterandextractedwith20mLofn-BuOHsaturatedwithwater.Then-BuOHextractionwasevaporatedtodryness,andtheresiduewasdissolvedin10mLofmethanolforanalysis.ThecontentoftotalginsenosideswasdeterminedbyUV-spectrophotometry[9].BiologicalProductsIdentificationofChina.

115 Extractionandisolationofginsenosidesfromthecrowngallcultures

Thecrowngallcultures(470g,dryweightofthebiomass)weresoakedfor12hatroomtemperatureandextractedfourtimeswith95%http://wendang.chazidian.compoundIwasobtainedfromthesectionintherateof99%:1%andfractionsAandBwere65%:35%and60%:40%respectively.FurtherisolationwascarriedoutbyusingpreparativeHPLC(GILSON)onaLichrospher

Ro

http://wendang.chazidian.comparedwithcallusandcellculture,thecrowngallculturesgrowfaster,

producesmoreactive

constituentsandarefreeofphytohormoneappended.Therefore,crowngallculturesmaybeaneffectivemethodofproducingusefulsecondarymetabolitesinsomeofvaluablemedicinalplants.Theproductionofsecondarymetabolitesbycrowngallcultureshasbeenstudiedinmanyplants

[3]

.

OurpreviousresearchhasshowedthatthecrowngallculturesofP.quinquefoliumcouldproduceginsenosidesbyusingphytohormone-freesolidMS(MurashigeandSkoog.s)medium.Inthepresentpaper,thegrowthcharacteristicsandginsenosidesisolationandstructuralelucidationofthesuspension-culturedcrowngallwerecarriedout.Otherwise,theconcentrationsofresidualsugar,phosphorus,nitrogeninNH4

+

[4]

Theauthentic

ginsenosideswereobtainedfromInstituteofDrugsand

andnitrogeninNO3

-

inthemediaduringtheculture

periodweremeasured.ThestructuresoffourcompoundswereelucidatedbyphysicochemicalandspectralmethodsaspeudoginsenosideF11(Ñ),

ginsenosideRd(Ò),

ginsenosideRb1(Ó)andginsenosideRb3(Ô).AllofthemwerefirstlyisolatedfromthecrowngallculturesofP.quinquefolium.

1 MATERIALSANDMETHODS

111 Cellcultivation

ThecrowngalltumorswereinducedbythedirectinfectionofsterilestemsofP.quinquefoliumL.withA.tumefaciensC58,

andwereadaptedto

culturein

phytohormone-freeMSliquidmedium.ThepHvalueofall100Rp-18e(5Lm)column(Merk)atroom

756

ChineseJournalofBiotechnology 生物工程学报 2005,Vol121,No15

flowratewas110mLPmin.TheeffluentfromtheoutletofthecolumnwasmonitoredwithaUVdetectorat203nm.Bythismethod,compoundÒwasobtainedfromfractionAandcompoundsÓandÔfromfractionB.116 Structuralelucidation

Thestructuresoffourcompoundsisolatedfromthecrowngallcultureswereelucidatedonthebasisof1H-NMRand

13

cultivation,therestillwas2116g#L-1(sugar)leftinthemedium.Foreachgramdryweightofcrowngall,1148gofsugarwasconsumed,whichwasabout50%lowerthanthatincallussuspensionculture[10].Phosphoruswastakenuprapidlyandwasdepletedfromthemediumbythe25thday.Theresultsofourexperimentsshowedthattheutilizationofsugarandphosphoruswas9118%and100%,respectively.SimilarresultswerereportedincallussuspensionculturesofP.quinquefolium[11].

C-NMRspectrausingAdvanceDMX500andAdvance

400MHzspectrometer(Bruker)withtetramethylsilaneasaninternalstandardandES-IMSspectrausingaFiniganLCQAdvantageMAX(Thermo)spectrometer.

2 RESULTSANDDISCUSSION

AsshowninFigure1,thesuspensionculturesshowedalagphasefrom0to6d,anexponentialphasefrom7to21dandastationaryphasefrom22to27daftersubculture.Thebiomassreachedapeakof18162gPL(dryweight)onthe27thday,asalmost6timesastheamountofinoculum.Thecontentoftotalginsenosidesfluctuatedbetween2165and3131mgper100mgdryweight.Theconcentrationoftotalginsenosidesincreasedslowlyatthebeginningofcultivationandreachedamaximalvalueof62014mg#L-1onthe27th

day.

Fig.2 Changesofphosphorus(A,u),residualsugar(A,

NO3-(B,

Fig.1 Timecourseofgrowth(w)andproductionof

totalginsenosides(u)duringcultivationofsuspendedcrowngallofP.quinquefolium

w)

s),

andNH4+(B,p)duringcultivationof

suspendedcrowngallofP.quinquefolium

AsshowninFigure2B,thedepletionofnitrogeninNH4+andNO3-wasverydifferent.Thedepletionofnitratewassimilartothatofsugar.ButtheconcentrationofNH4+changedslightlyduringinitial18d,andrapidlydeclinedto3141mmolPLwithin6d.Theseresultssuggestthatnitrateandammoniumhavedifferenteffectsonprimaryandsecondarymetabolismofthecrowngallcultures.Inotherwords,NH4

+

Tounderstandthegrowthcharacteristicsofsuspension

culturesofP.quinquefolium,thedynamicchangesintheconcentrationsofresidualsugar,phosphorus(PO4

3-

),

nitrogen(NH4+andNO3-)inthemediawereinvestigated(Figure2AandFigure2B).Theresultsshowedthatarapiddepletionofsugarinthemedium(1114g#L-1perday)

wasunnecessarytothegrowthofcrowngall,butpromotedthe

YURong-Minetal:TheGrowthCharacteristicsandGinsenosidesIsolationofSuspension-culturedCrownGallofPanaxquinquefolium757

Theseresultsweresimilartothoseinlarge-scalecellcultureofP.quinquefolium

[12]

(CH3,C-27),28106(CH3,C-28),16124(CH3,C-29);105108(CH,3-B-D-glc-C1),106102(CH,3-B-D-glc-B-D-glc-C1),98105(CH,20-B-D-glc-C1),105135(CH,20-B-D-glc-B-D-glc-C1).Thosedatawereconsistentwithliterature[14](1993).Therefore,identifiedasginsenosideRb11

CompoundÔwascolorlesspowder,m.p.197-199e.ES-IMSmPz:

107811[M-1]-(C53H89O22,

-13

,butoppositetothosein

solidcultureofthesamecrowngall(datanotshown).Furtherinvestigationontheeffectsofnitrogensourceisunderwayinourlaboratory.Duringthewholecultivation,theutilizationofNH4+respectively.

Theinformationobtainedfromthisexperimentisconsideredtobeveryimportantforthelargescaleproductionofginsenosidesbyplantbiotechnology.

ThestructuralidentificationofcompoundswascarriedoutbyusingES-IMS,1H-NMRandES-IMSmPz:80013[M-1]

13

-13

compoundÓwas

andNO3-

was81%and97%,

calcd.

-

107813),94516[M-C5H8O4],91518[M-C6H12O5],78313,62112,459141

C-NMR(C5D5N)Dppm:88197

(CH,C-3),18139(CH2,C-6),83137(C,C-20),22124(CH3,C-21),36113(CH2,C-22),23112(CH2,C-23),125191(CH,C-24),130191(C,C-25),25178(CH3,C-26),16157(CH3,C-27),28106(CH3,C-28),16123(CH3,C-29),105108(CH,3-B-D-glc-C1),105196(CH,3-B-D-glc-B-D-glc-C1),98107(CH,20-B-D-glc-C1),105181(CH,20-B-D-glc-B-D-xy-lC1),66187(CH2,20-B-D-glc-B-D-xy-lC5).

Thosedatawereconsistentwith

literature[14](1993)andthereforecompoundÔwasidentifiedasginsenosideRb31

Acknowledgement TheauthorsareindebtedtoProf.LeeannSong,CanadianforeignexpertofJinanUniversity,fortherevisionofourmanuscriptinEnglish.

C-NMRspectra.

CompoundIwascolorlesspowder,m.p.205~207e.

(C42H71O14,calcd.80010),

65314[M-C6H12O4]-,49112[M-C6H11O4-C6H12O5]-.C-NMR(C5D5N)Dppm:78143(CH,C-3),74114(CH,C-6),86159(C,C-20),26184(CH3,C-21),32164(CH2,C-22),28166(CH2,C-23),85150(CH,C-24),70119(C,C-25),27106(CH3,C-26),27163(CH3,C-27),32132(CH3,C-28),16177(CH3,C-29),101168(CH,6-B-D-glc-C1),101186(CH,6-B-D-glc-A-L-rha-C1),18164(CH3,6-B-D-glc-A-L-rha-C6).Thosedatawereconsistentwithliterature[13].Therefore,compoundIwasidentifiedaspseudoginsenosideF111

CompoundÒwascolorlesspowder,m.p.206~209e.ES-IMSmPz:94613[M-1]94611),78313[M-C6H12O5]

--

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13

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