21卷5期2005年9月生 物 工 程 学 报Chinese Jou rnal o f Biotechnology Vol.21 No.5September 2005Received:March 25,2005;Accepted:May 25,2005.This work was supported by the grants from Minis try of Education of China (No.104180),Natural Sciences Foundation of Guangdong (No.31891)and Chi nese Traditional Medicine Adminis tration of Guangdong,China (No.103041).*Corres ponding author.Yu Rong -M in:Tel:86-20-85228205;E -mail:rongminyu99@;ZHAO Yu:Tel:86-571-87217313;Email:dryuzhao@hot mail.c om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。
The Growth Characteristics and Ginsenosides Isolation of Suspension -cultured Crown Gall of Panax quinque folium西洋参冠瘿组织悬浮培养及其人参皂苷类成分的分离YU Rong -Min 1*,JIN Qian -Xing 1,SUN Hui 2,YE Wen -Cai 1,2and Z HAO Yu 3*于荣敏1*,金钱星1,孙 辉2,叶文才1,2,赵 昱3*1.暨南大学药学院,广州 5106322.中国药科大学中药学院,南京 2100093.浙江大学药学院,杭州 3100311.De pa rtmen t o f Ph ytoche mistry an d Pha rma cognosy ,Colle ge o f Pha rma cy ,Jin an Un ive rsity ,Gua n gzh ou 510632,Ch ina2.De pa rtmen t o f Ph ytoche mistry ,Ch in a Ph arma ce utica l Un ive rsity ,Nan jin g 210009,Ch in a3.De pa rtmen t o f Trad ition al Ch in ese Med icine an d Na tu ra l Drug Re sea rch ,College o f Pha rmac eutic al Scien ces ,Zh e jian g Un ive rsity ,Han g zhou 310031,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[18162g P L(dry weight)]及其中最高人参皂苷累积时间(62014mg P L on the 27th day)。
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F 11(pseudoginsenoside F 11,Ñ),人参皂苷Rd(ginsenoside Rd,Ò),人参皂苷Rb 1(ginsenoside Rb1,Ó)和人参皂苷Rb 3(ginsenoside Rb 3,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)05-0754-05Abstract The growth characterist ics and ginsenoside s isola t ion of the suspension -cultured crown gall of Panax quinque f olium were studied.The result showed that the ma ximum biomass of cultures was 1816g P L(dry weight)and the conte nt of ginsenoside s reached its maximum level of 62014mg P L on the 27t h day.The utilization ra tes of sugar,phosphorus,nitrogen in NH 4+and nitrogen in NO 3-were 9118%,100%,81%and 97%,respectively.Four compounds were isolated fro m the suspe nsion -cultured cro wn gall and their structures were elucida ted as pse udoginsenoside F 11(Ñ),ginsenoside Rd (Ò),ginsenoside Rb 1(Ó)and ginsenoside Rb 3(Ô).Key words cro wn gall,ginsenosides,isolation,Pana x quinque f olium ,suspension c ulturesPanax quinque f o lium has been widely used for its therapeutic effec ts[1].Ho we ver,the field cultivation of the plant is a ti me-consuming and labor-intensive process:it takes over six yea rs for its harvest,during which great care is needed since the growth is subjec t to se veral c onditions,such as soil,cli mate,pathogens and pests.In addition,the plant has fallen short of supply for a long period due to a grea t demand on the market.The major ac tive constitue nts of P.quinque f o lium are ginsenosides,a diverse group of steroidal saponins[2].The use of plant biotec hnology,suc h as cell culture and transgenic technique s,has been c onsidered as a cos-t effective approach to produc tion of ginsenosides in la rge quantities. Crown gall,a kind of irregula r tissue caused by the Agrobacterium tume f aciens pathogen,is capable of introducing and re plicating its own DNA into the plant.s nuclear pared with callus and cell culture,the c rown gall cultures gro w faster,produces more active c onstituents and are free of phytohormone appe nded. Therefore,crown gall cultures ma y be an effec tive method of producing useful secondary me tabolites in some of valuable medicinal plants.The produc tion of sec ondary metabolites by c rown gall cultures has been studied in many plants[3].Our previous re search has sho wed that the cro wn gall cultures of P.quinque f olium c ould produce ginsenosides by using phytohor mone-free solid MS(Murashige and Skoog.s) medium[4].In the present paper,the gro wth characte ristics and ginsenosides isola t ion and structural elucidation of the suspension-cultured cro wn gall were carried out.Othe rwise, the concentrations of residual suga r,phosphorus,nitrogen in NH4+and nitrogen in NO3-in the media during the culture period were measured.The structures of four compounds were elucidated by physicoche mical and spectral me thods as peudoginsenoside F11(Ñ),ginsenoside Rd(Ò), ginsenoside Rb1(Ó)and ginsenoside Rb3(Ô).All of the m were firstly isola ted from the cro wn gall cultures of P.quinque f o lium.1MATERIALS AND METHODS111Cell cultivationThe cro wn gall tumors were induced by the direc t infection of sterile stems of P.quinque f olium L.with A. tume f aciens C58,and were adapted to culture in phy tohormone-free MS liquid medium.The pH value of all media was adjusted to5170before steriliza tion.The e xperi ments we re conducted using250mL Erlenme yer flasks c ontaining50mL of MS medium,inoc ula ted with410g of fresh weight biomass and inc uba ted at25e on an orbital shaker at110r P min in the darkness.Five flasks were harvested regula rly over a period of30d for the measurement of biomass,sugar,phosphorus,nitroge n and ginsenosides. 112Measurement of major nu tr ients in medium The c oncentration of residual suga r in the medium wa s dete rmined acc ording to the method of pheno-l concentrated sulfuric acid[5].The residual nitrogen in NH4+and NO3-in the medium was measured using pheno-l hypochlorite reac tion[6]and resorcinol me thod[7],respectively. Phosphorus of PO43-in the medium was dete rmined using ascorbic acid method[8].113Determination of cells weightThe cell suspensions we re filtered and washed several times with sufficie nt amount of distilled wa ter.The cells were weighed after being dried to a consta nt weight at a temperature belo w50e.114Assay of gin senosidesFor sa mple preparation,110g of po wdered crown gall c ultures was e xtrac ted with20mL of me thanol,using an ultrasonic bath for30min afte r degreasing with30mL of ether. Afte r filtra t ion and evaporation to dryness,the residue wa s dissolved in20mL of wate r and e xtracted with20mL of n-BuOH saturated with wa ter.The n-BuOH extraction wa s e vaporated to dryness,and the residue was dissolved in10mL of methanol for analysis.The content of total ginsenoside s wa s dete rmined by UV-spectrophotometry[9].The authentic ginsenosides were obtained from Institute of Drugs and Biological Products Identification of China.115Extraction and isolation of ginsenosid es from the crown gall culturesThe crown gall cultures(470g,dry weight of the biomass)were soaked for12h at roo m te mperature and e xtracted four times with95%e thanol to afford60g of crude e xtracts.The e xtracts we re dissolved in methanol and filte red.The methanol solution wa s then subjec ted to column c hroma tography on silica gel with gradie nt elution of c hloroform P pound I was obtained fro m the section in the rate of99%:1%and fractions A and B were 65%:35%and60%:40%respectively.Furthe r isolation was carried out by using prepa ra t ive HPLC(GI LSON)on a Lichrospher o R100Rp-18e(5L m)c olumn(Merk)a t room temperature.The eluting solvent was75%methanol,and the755YU Rong-Min et al:The Growth Characteristics and Ginsenosides Isolation of Suspension-cultured Crown Gall of Pana x quinque f oliumflow ra te was 110mL P min.The effluent fro m the outle t of the c olumn was monitored with a UV de tector at 203nm.By this method,compound Òwas obtained from frac tion A and c ompounds Óand Ôfrom fraction B.116 S tru ctural elucidationThe structures of four c ompounds isolated from the cro wn gall cultures were elucidated on the basis of 1H -NMR and13C -NMR spectra using Advance DMX500and Advance400MHz spectrometer (Bruker)with tetra methylsilane as an internal standard and ES-I MS spectra using a Finigan LCQ Advantage MAX (Thermo)spec trome ter.2 RES ULTS AND DISC USSIONAs sho wn in Figure 1,the suspension cultures sho wed a lag phase from 0to 6d,an exponential phase from 7to 21d and a stationary phase from 22to 27d afte r subc ulture.The biomass reached a peak of 18162g P L(dry weight)on the 27t h day,as almost 6ti mes as the a mount of inoculum.The c ontent of total ginse nosides fluctuated be tween 2165and 3131mg per 100mg dry weight.The conce ntra t ion of total ginsenosides inc reased slowly at the beginning of cultivation and reac hed a maximal value of 62014mg #L -1on the 27t hday.Fig.1 Time course of growth (w )and production oftotal ginsenosides(u )during cultivation of suspended crown gall of P .quinque foliumTo unde rstand the gro wth c haracteristics of suspensioncultures of P .quinque f olium ,the dyna mic changes in the c oncentra tions of re sidual sugar,phosphorus (PO 43-),nitrogen (N H 4+and NO 3-)in the media were investigated (Figure 2A and Figure 2B).The results showed that a rapid depletion of sugar in the medium (1114g #L -1per day)happened during the e xponent ial phase.At the end of thec ultiva t ion,there still was 2116g #L -1(sugar)left in the medium.For each gram dry weight of c rown gall,1148g of suga r was consumed,which was about 50%lo wer than that in callus suspension culture [10].Phosphorus was taken up rapidly and was depleted from the medium by the 25th day.The re sults of our experiments sho wed that the ut ilization of suga r and phosphorus was 9118%and 100%,respectively.Similar results we re reported in callus suspension cultures of P .quinque f olium [11].Fig.2 Changes of phosphorus(A ,u ),residual sugar(A,s ),NO 3-(B,w )and NH 4+(B,p )during cultivation ofsuspended crown gall of P .quinque foliumAs shown in Figure 2B,the deple tion of nitrogen in NH 4+and NO 3-was very differe nt.The deple t ion of nitrate was si milar to that of sugar.But the concentra t ion of NH 4+c hanged slightly during initial 18d,and rapidly declined to 3141mmol P L within 6d.These results suggest that ni trate and ammonium have different effects on primary and secondary metabolism of the cro wn gall c ultures.In other words,NH 4+was unnecessary to the gro wth of cro wn gall,but promoted the production of ginsenoside s.756Chinese J ournal o f Biotechnology 生物工程学报 2005,Vol 121,No 15These results were similar to those in large-scale cell culture of P.quinque f olium[12],but opposite to those insolid culture of the sa me crown gall(data not sho wn). Further investigation on the effects of nitrogen source is under way in our laboratory.During the whole cultivation,the utilization of NH4+and NO3-was81%a nd97%, respectively.The information obtained from this e xpe riment is c onsidered to be very important for the large scale produc tion of ginsenosides by plant biotec hnology.The structural identification of compounds was carried out by using ES-I MS,1H-NMR and13C-NMR spec tra.Compound I was c olorless po wder,m.p.205~207e. ES-I MS m P z:80013[M-1]-(C42H71O14,calcd.80010), 65314[M-C6H12O4]-,49112[M-C6H11O4-C6H12O5]-. 13C-NMR(C5D5N)D ppm:78143(CH,C-3),74114(CH, C-6),86159(C,C-20),26184(C H3,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(C H3,6-B-D-glc-A-L-rha-C6).Those da ta were c onsistent with literature[13].Therefore,compound I was identified as pseudoginsenoside F111CompoundÒwas colorless powder,m.p.206~ 209e.ES-I MS m P z:94613[M-1]-(C48H81O18,calcd. 94611),78313[M-C6H12O5]-,62113,45914113C-N MR (C5D5N)D ppm:88191(CH,C-3),18139(CH2,C-6), 83142(C,C-20),22135(CH3,C-21),36109(CH2,C-22),23119(CH2,C-23),125185(CH,C-24),130181 (C,C-25),25173(CH3,C-26),16157(CH3,C-27), 28106(CH3,C-28),16125(CH3,C-29);105109(CH, 3-B-D-glc-C1),106103(CH,3-B-D-glc-B-D-glc-C1), 98125(CH,20-B-D-glc-C1).Those da ta were c onsistent with literature[14].The refore,c ompoundÒwas identified as ginsenoside Rd.CompoundÓwas colorless powder,m.p.197~ 198e.ES-I MS m P z:110814[M-1]-(C54H91O23,calcd. 110813),94517[M-C6H12O5]-,78313,62112,45913113C-NMR(C5D5N)D ppm:88194(CH,C-3),18132 (CH2,C-6),83140(C,C-20),22137(CH3,C-21), 36116(CH2,C-22),23118(CH2,C-23),125187(CH, C-24),130196(C,C-25),25178(CH3,C-26),16157(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).Those data were c onsistent with literature[14](1993).The re fore,compoundÓwa s ide ntified as ginsenoside Rb11CompoundÔwas colorle ss powder,m.p.197-199e. ES-I MS m P z:107811[M-1]-(C53H89O22,calcd. 107813),94516[M-C5H8O4]-,91518[M-C6H12O5]-, 78313,62112,45914113C-NMR(C5D5N)D ppm: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-l C1),66187(CH2,20-B-D-glc-B-D-xy-l C5).Those data we re consistent with literature[14](1993)and therefore co mpoundÔwa s ide ntified as ginsenoside Rb31Acknowledgement The authors are indebted to Prof. Leeann Song,Canadian foreign expe rt of Jinan University,for the revision of our manusc ript in English.R EFERENC ES(参考文献)[1]Huang KC.The Pharmacol ogy of Chines e Herbs.10th edn.CRCPress,Boca Raton,FL.1999[2]Attele,AS,Wu JA,Yuan CS.Gi nseng pharmacology:mul tiplecons ti tuents and mul tiple ac tions.Biochem Pharmcol,1999,58:1658-1693[3]G hsoh B,Mukherjee S,Jha S.Genetic transformation of Arte misiaauuna by Agrobacte rium tume fac ie ns and artemisi nin s ynthesis intransformed cultures.Plant Sc ienc e,1997,122:193-199[4]Yu R M(于荣敏),Song YB(宋永波),Zhang H(张辉)et al.Study on the c ulture of crown gall from Panax quinque folium and theproducti on of its secondary metabolites ginsenosi de Re and Rg11Chinese Journal o f Biote chnolo gy(生物工程学报),2003,19:372-375[5]Ts oulpha P,D oran PM.Solasodine produc tion from sel-f i mmobilisedSolanum aviculare cells.Journal o f Biote chnolo gy,1991,19:99-100[6]Weatherburn MW.Pheno-l hypochl orite reaction for determination ofammonia.Analytical Chemist ry,1967,39:971-974[7]Velghe N,Claeys A.Rapid spec tro-photometric de termi nation ofni trate in mineral waters with resorcinol.Analyst,1985,110:313-314[8]Chen PS,Toribara TY,Warner H.M icro-determinationphosphorus.Analytical Chemistry,1956,28:1756-1758757YU Rong-Min et al:The Growth Characteristics and Ginsenosides Isolation of Suspension-cultured Crown Gall of Pana x quinque f olium[9]Liu S,Zhong JJ.Phosphate effect on production of ginseng s aponinand pol ysacchari de by cell s uspension cultures of Panax ginseng andPanax quinque folium.Proce ss Bioc hemistry,1998,33:69-74 [10]Xu WH(徐卫辉),Song PL(宋佩伦).Studies on the physio-bi oche mical of American ginseng(Panax quinque folium L.).Ac taSc i Nat U ni v Nom Hunan(湖南师范大学自然科学学报),1993,16:350-354[11]Zhong JJ,Bai Y,Wang SJ.Effects of plant gro wth regulators oncell growth and gi nsenosi de saponin producti on by s uspensioncultures of Panax quinque f olium.Journal o f Biotec hnology,1996,45:227-234[12]Zhou LG(周立刚),Zheng GZ(郑光植).Study on mass cul ture ofPanax quinque folium cells.Chinese Journal o f Biotec hnology(生物工程学报),1990,6:316-321[13]Tang JL(唐纪琳),Li J(李静),Wei YD(卫永弟).Isol ation andidentification of s aponins from flowerbuds of Panax quinque foliumL.Chine se Traditional and He rbal Drugs(中草药),2000,31:496-498[14]Ma XY(马兴元),Wang GS(王广树).Triterpenoid s aponins fromaerial parts of Panax quinque folium L.Chinese PharmaceuticalJournal(中国药学杂志),1993,28:718-720科学出版社生命科学编辑部新书推介5临床微生物学手册6(Manual of Clinical Microbiology,7th ed)(上下册)1美2默里(Murray,P.R.)等著徐建国等译2005年6月出版ISB N7-03-014011-7定价: 260.00元本书英文版由国际杰出的临床微生物学家编写,美国微生物学会出版,是临床微生物学领域的经典杰作。
