银杏愈伤组织培养及银杏内酯测定的研究_英文_

收稿日期:19982072011　通讯联系人银杏愈伤组织培养及银杏内酯测定的研究郑玉果　于荣敏1　姚新生　张　辉　邵　刚沈阳药科大学中药系,沈阳　110015摘　要　国内首次采用固体培养的方法对组织培养法生产银杏内酯进行了研究.考察了银杏不同外植体的愈伤组织的诱导情况;对得到的各种愈伤组织进行了继代驯化,并考察了光照、MS 培养基成分等多种理化因子及激素对愈伤组织的诱导及生长的影响;应用生物法(PAF )和HPLC 对愈伤组织中的银杏内酯A 和B 进行了测试.结果显示,愈伤组织中的银杏内酯A 和B 的含量在10×10-6～50×10-6之间.关键词　银杏;愈伤组织培养;银杏内脂;HPLC 分类号　Q9461　IntroductionGi nkgo biloba tree is the only living species of the order G inkgoales ,whose ancestry has beentraced to the J urassic period.Baiguo ,Semen G inkgo is the dry ripe seeds of the plant collected in the fall when the seeds are ripe.This crude drug is officially listed in the Chinese Pharmacopoeia and used in a traditional Chinese medicine as an antiasthmatic and against polyuria. G inkgolides (Fig.1)are obtained from the root ,bark ,and leaves of the G.biloba tree 〔1〕.G inkgolides are specific platelet 2aggregating factor (PAF )antagonists.PAF is a potent mediator of inflammation ,which is secreted by a variety of leucocytes and serves to increase vascular perme 2Fig.1　The chemical structure of ginkgolide A and Bability and elicit chemotaxis and aggregation.PAF has been implicated as a mediator in a num 2ber of clinical conditions ranging from asthma and various allergic disorders to transplant rejec 2tion ,shock states ,such as septicemia and renaldisease 〔2〕.G inkgolide B appears to be the mostpotent inhibitor of PAF. The cultivation of G.biloba cell culture wasfirst reported by Tulecke 〔3〕,but it had not been proved for the existence of ginkgolides in the cal 2lus or the cell cultures until 1991.Carrier 〔4〕de 2scribed the determination of ginkgolide A ,and traces of ginkgolide B were detected with signal第16卷　第1期沈　阳　药　科　大　学　学　报Vol 116 No 111999年1月Journal of Shenyang Pharmaceutical University　Jan 11999　p 110to noise ratio ,being too low for positive confirmation of this last product.In 1993,K im 〔5〕gave thefirst report of 3813×10-6of ginkgolide B in the callus culture.2　Establishment of C alli Culture211　Materials Explants ,which are induced from the root of seedling ,were used for the callus culture.G inkgolide standards ginkgolide A (G A )and ginkgolide B (G B )were purified from the leaves of G.biloba and identified by means of IR ,MS ,NMR and other related methods by our group.212　Selection of optimum media Five media were used in our experiments for the induction of callus of leaves ,embryo ,root ,stem and shoot under the light of 8hrs within 24hrs supplemented with 1mg/L NAA and 011mg/L KT.The period of cultivation lasted 30days.The illumination with two G eneral Fluorse 2cent Lamp (40W )placed 40cm from the cultures.The influence of media on growth of G.biloba explans were summarized in Table1.T ab.1　The influence of media on grow th of G.biloba calliMedia MS H B56,72V N6Leaves ++++++++++++Embryo +++-+++++++++Root +----Petiole ++++++++++++Stem++++++++++++ “-”—no callus ; “+”—the rate of induction is below 20%; “++”—the rate of induction is between 20～50%; “+++”—the rate of induction is between 50～80%; “++++”—the rate of induction is between 80～100% The optimum medium is different from above explants.MS is the most suitable medium for the induction of leaves and petiole ,stem is N6,and no inductive action was found for the root in all testing media except MS.The result also showed the more mineral substances were contained in media ,the greater the inductive rate appeared.213　The influence of plant grow th regulators Studies of plant growth regulators (P GR )on calli cultures were conducted to find out the con 2ditions of calli growth rate and how to increase its medicinal contents.Different growth regulators were tested to obtain a better maintenance medium using MS mineral salt formulations.The best calli inductive rate on MS supplemented with the growth regulator 210mg/L of NAA was ob 2served and the increase in the dry weight of calli was found to be 210mg/L of 2,42D.No pro 2moting effect appeared when using singly indole 232acetic acid (IAA )and gibberellin (G A 3)in the induction and growth of G.biloba calli.The result of the influence of plant growth regulators on dry weight (DW )and inductive rate (IR )was shown in Figure 2and Figure 3.214　Comparision of callus culture for different explantsThe following is an order of inductive rate for all kinds of explants from high to low :11　第1期郑玉果等:银杏愈伤组织培养及银杏内酯测定的研究Fig.2　The effect of PGR onDW Fig.3　The effect of PGR on IRStem>Embryo>Leaves>Petiole>Shoot>RootExplant from the field2grown tree>Explant from tube seedlingTab.2　is a demonstration of the inductive rate(IR)of different explants in four weeks.T ab.2　The induction rate(IR)of different explantsExplants Stem Embryo Leaves Petiole ShootIR%in4wks1009521009029870285402603　G inkgolide Detection Lobstein2Guth〔1〕developed a purification method involiving liquid2liquid exraction and preparative chromatography for the analysis of ginkgolides obtained from leaf and bark extracts They used HPLC with ultaviolet(UV)detection at220nm and the limit of detection was approx2 imately30μm.Van Beek〔6〕reduced he detection limit to1μm,using refractive index.This limit is similar to that obtained by Tallevi and Kurz〔7〕which were used for the analysis biomass pro2 duced by shake flask cultures(Carrier et al.1990),no ginkgolide was detected.K im〔5〕reported their detection method using GC2MS,in which the content of G B reached a higher level(9～38×10-6)in suspension culture cells.In this paper,we developed successfully two sensitive ad useful methods in callus culture.311　The biological quantitative method(platelet aggregation induced by PAF)of ginkgolides 31111　Pri nciplePharmacological studies on pure ginkgolides A,B and C specifically inhibited the binding of platelet2activating factor to its receptor in isolated rabbit platelet membranes and inhibited platelet2activating factor2dependent in vitro platelt aggregation.G inkgolide B was the most active ginkgolide with an IC50of ca.10-7M.After oral administration,ginkgolide B inhibited platelet2 activating actor2dependent platelet aggregation in rabbits and inhibited thrombus formation in2 duced by electric stimulation of the carotid artery in rats.G inkgolide B laso inhibited platelet2acti2 vating factor2dependent human polymorphonuclear leukocyte aggregation in vitro.G inkgolides did not significantly affect arachidonic acid metabdolism and was not bound to neuotransmitter recep2 tors.G inkgolides may have a clinical use and might also be of values as pharmacological tools for 21沈　阳　药　科　大　学　学　报第16卷studying biological activities of the platelet 2activating factor.Fig.4　The curve of IHR 2C of the root callus31112　Gi nkgoli de analysis3111211　The LC 50detection of authentic sam 2ple of G BThe standard solution of G B was made up and then the curve of platelet aggregation was mea 2sured.The aggregation rate (AR )and inhibition rate (IHR )were got ,using MeOH as the stan 2dard.The standard curve was shown in Fig.4and the result was described in Table 3.3111212　The IC 50detection of callus samplesT ab.3　Datasheet of IC 50for authentic sample of GBConc/mg ・mL -10105010250101250100830100750100625Control PA/%17.619.222.834.051.276.480.0IHR/%78.076.071.556.936.04.5IC 500.00792(17.5μM ) The root callus was used as the detective sample ,and the analysis method is this same with the above.The result was shown in Table 4.The concentraton of G B was described in equivalent concentration (EC ).T ab.4　The IC 50of root calusConc/mg ・mL -10120110.080.06control AR/%36414956100IR/%645951440.07g ・mL -1EC of G B113×10-6312　The quantitative detection of ginkgolide by HPLCThe biological quantitative method of ginkgolide has some advantages ,such as strong selec 2tion ,high sensitivity ,etc ,but the content of G B obtained is of equivalent concentration.HPLC will be a good choice for the determination of pure constituent of ginkgolides.31211　Chrom atographic onditionsCloumn :Inertsil ODS column ,416mm ×250mm ,5μm ,G L Sciences Inc.Eluent :Ethanol 2Water (45∶55);Flow rate :1ml/min ;Deteetor :Differential detector ;Sensitivity :015×10-4.31212　Q uantif icationThe G.biloba calli to be investigated were analysed three times after purification.The con 2centrations of ginkgolide A and B were calculated with be following equations.Concentration (×10-6)for G A =area (G )/area (I.S.)×Wi.s.×1130Concentration (×10-6)for G A =area (G )/area (I.S.)×Wi.s.×1136where area (G )is the peak area of the ginkgolide compound of interest ,area (I.S.)is the peak area of the internal standard (benzyl alcohol ),Wi.s.is the mass of internal standard ,it equals :31　第1期郑玉果等:银杏愈伤组织培养及银杏内酯测定的研究41沈　阳　药　科　大　学　学　报第16卷Volume of internal standard(mL)×2109mg/mL.1130and1136are the response factors of rela2 tive weight of G A,G B with the internal standard(IS).The result of the quantitative detection of HPLC is shown is Table5,Which is modified from Van Beek(1991)’s report,in which neither G A nor G B was detected from G.biloba culture.T ab.5　The content(×10-6)of G A and GB measured by HPLC in root callus/n=3Group123mean Content of GA48.947.749.548.6±3.6Content of G B13.212.812.512.8±1.54　The Choice of Optimun C alli Containing High Content of G inkgolides in Different Explants and the Influences of Cultural Condi2 tions on the Production of G inkgolides411　The choice of optimum calli containing high content of,ginkgolides in diferent explants The comparison of different calli obtained from roots,stems,leaves,petioles,embryos,buds and shoots was made for their ability of production of ginkgolides.Table6gave the results of mea2 surement of the biological quantitiative method(BQM)and HPLC.T ab.6　The content(×10-6)of ginkgolides obtained from different calli Calli from root stem leaves petiole embryo bud shootG B BQM113.037.795.424.779.266.056.6GA HP LC10251365G B HP LC17262-38412　The influences of cultural conditions on the production of ginkgolides41211　The ef f ect of plant grow th regulators on the calli f or thei r ability of produci ng gi nkgoli desStudies of plant growth regulator on callus culture were conducted to find out the conditions of callus growth rate and how to increase its medicinal contents.The results showed that,when used singly,both NAA and2,42D,were satisfactory(Table7),but IBA was found ineffective on the production of ginkgolides.T ab.7　The effect of PGR on the production of ginkgolides(U nit:×10-6)PGR/mg・mL-1content of GA content of G B PGR R Y of GA R Y of G B NAA1.07180.06870.48 1.24NAA2.0310.07230.220.07NAA4.03-0.08910.27-IBA1.0--0.0305--IBA2.02-0.04250.09-IBA4.0--0.0498--2,42D110-80.0402-0.322,42D210-250.0510- 1.282,42D410-330.0791- 2.61R GR〔1〕:relative growth rate, R GR =(1/T )×ln (W 2/W 1)where T is the period of culture ,W 2is the weight of callus then harvested ,and W 1is the weight of callus when inoculated.R Y 〔2〕:is means rate of yield , R Y =R GR ×Content of G inkgolied 41212　The ef f ect of ill um i nation on the production of gi nkgoli desThe illumination would affect the growth of callus and the production of ginkgolides.The result of examination was found in Table 8.T ab.8　The effect of light on the root calli for the procuction of ginkgolides(U nit :×10-6)Type of culture Content of GAContent of G BRGR R Y of GA R Y of G B light culture 10170.04670.470.79dark culture1-0.06360.06REFERENCES1　Lobstein 2Guth A ,Briancon 2Scheid F ,Anton R.Analysis of terpenes from G inkgo biloba L.by hight performance liquid chro 2matography.J Chromatography ,1983,267:431～4382　Braquet P ,Touqui L ,Shen Y T.Pharmacol Reviews ,1987,39(2):97～1453　Tulecke W R.Tissue derived form the Pollen of G inkgo biloba.Science ,1953,117:599～6004　Carrier J ,Cosentio G ,Ndeufeld R.Nutritional and hormonal requirements or G inkgo biloba embryoderived callus and suspension cell culture.Plant Cell Rep ,1990,8:635～6385　K im Y C ,Jeon M H ,Sung S H.Production of G inkgolides in cell culture.PCT Int Appl WO 930,204(CL.C12P17/02),1993-02-046　Vanbeek T A ,Scheeren H A ,Rantio T.Determination of G inkgolides and bilobalide in G inkgo biloba Leaves and phytopharma 2ceuticals.J Chro Matography ,1991,543:375～3877　AL 2Tel T H ,Abu Zarga M H ,Sabri S S.New antural colchicinoids :Indications of two possible catabolic routes for the colchininealkaloids.J Nat Prool ,1990,53:623～6291　Author to whom correspondence should be addressed.Studies on the C allus Cultures of Ginkgo biloba and the Identi 2f ication of G inkgolidesZheng Yugou ,Yu Rongmin 1,Yao Xinsheng ,Zhang Hui ,Shao GangDepart ment of T raditional Chi nese Medici nes ,S henyang Pharmaceutical U niversity ,S henyang 110015Abstract The production of ginkgolides in callus culture of Gi nkgo biloba was reported.The af 2fection of some physical factors and chemical substances on the induction and growth of calli was also investigated.A bilogically quantitative method (platelet aggregation induced by PAF )and HPLC were successfully used for the determination of ginkgolides A and B in al kinds of callus cultures.The result showed that the content of ginkgolides A and B in the cell cultures varies from 10×10-6to 50×10-6.K ey w ords Gi nkgo biloba ;callus culture ;ginkgolides ;HPLC51　第1期郑玉果等:银杏愈伤组织培养及银杏内酯测定的研究。