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银杏愈伤组织培养及银杏内酯测定的研究
http://www.100md.com 《沈阳药科大学学报》 1999年第1期
     作者:郑玉果 于荣敏 姚新生 张 辉 邵 刚

    单位:郑玉果 于荣敏(通讯联系人) 姚新生 张辉 邵刚 沈阳药科大学中药系,沈阳 110015

    关键词:银杏;愈伤组织培养;银杏内脂;HPLC

    沈阳药科大学学报990103 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.

    分类号 Q946

, http://www.100md.com     Studies on the Callus Cultures of Ginkgo biloba and the Identification of Ginkgolides

    Zheng Yugou,Yu Rongmin1,Yao Xinsheng,Zhang Hui,Shao Gang

    Department of Traditional Chinese Medicines,Shenyang Pharmaceutical University,Shenyang 110015

    Abstract The production of ginkgolides in callus culture of Ginkgo biloba was reported.The affection 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 10×10-6 to 50×10-6.
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    Key words Ginkgo biloba;callus culture;ginkgolides;HPLC

    1 Introduction

    Ginkgo biloba tree is the only living species of the order Ginkgoales,whose ancestry has been traced to the Jurassic period.Baiguo,Semen Ginkgo 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.
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    Ginkgolides(Fig.1)are obtained from the root,bark,and leaves of the G.biloba tree1.Ginkgolides are specific platelet-aggregating factor(PAF)antagonists.PAF is a potent mediator of inflammation,which is secreted by a variety of leucocytes and serves to increase vascular permeability and elicit chemotaxis and aggregation.PAF has

    Fig.1 The chemical structure of ginkgolide A and B
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    been implicated as a mediator in a number of clinical conditions ranging from asthma and various allergic disorders to transplant rejection,shock states,such as septicemia and renal disease2.Ginkgolide B appears to be the most potent inhibitor of PAF.

    The cultivation of G.biloba cell culture was first reported by Tulecke3,but it had not been proved for the existence of ginkgolides in the callus or the cell cultures until 1991. Carrier4 described the determination of ginkgolide A,and traces of ginkgolide B were detected with signal to noise ratio,being too low for positive confirmation of this last product.In 1993,Kim5 gave the first report of 38.3×10-6 of ginkgolide B in the callus culture.
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    2 Establishment of Calli Culture

    2.1 Materials

    Explants,which are induced from the root of seedling,were used for the callus culture.Ginkgolide standards ginkgolide A(GA)and ginkgolide B(GB)were purified from the leaves of G.biloba and identified by means of IR,MS,NMR and other related methods by our group.

    2.2 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 8 hrs within 24 hrs supplemented with 1 mg/L NAA and 0.1 mg/L KT.The period of cultivation lasted 30 days.The illumination with two General Fluorsecent Lamp(40W) placed 40 cm from the cultures.The influence of media on growth of G.biloba explans were summarized in Table1.
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    Tab.1 The influence of media on growth of G.biloba calli Media

    MS

    H

    B5

    6,7-V

    N6

    Leaves

    ++++

    +

    +++

    ++

    ++
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    Embryo

    +++

    -

    ++++

    +++

    ++

    Root

    +

    -

    -

    -

    -

    Petiole

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    +

    +++

    ++

    ++

    Stem

    +++

    +

    ++

    ++

    ++++

    “-”—no callus; “+”—the rate of induction is below 20%; “++”—the rate of induction is between 20~50%;
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    “+++”—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.

    2.3 The influence of plant growth regulators
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    Studies of plant growth regulators(PGR)on calli cultures were conducted to find out the conditions 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 2.0 mg/L of NAA was observed and the increase in the dry weight of calli was found to be 2.0 mg/L of 2,4-D. No promoting effect appeared when using singly indole-3-acetic acid(IAA)and gibberellin(GA3)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 2 and Figure 3.
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    Fig.2 The effect of PGR on DW

    Fig.3 The effect of PGR on IR

    2.4 Comparision of callus culture for different explants

    The following is an order of inductive rate for all kinds of explants from high to low:

    Stem>Embryo>Leaves>Petiole>Shoot>Root

    Explant from the field-grown tree>Explant from tube seedling
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    Tab.2 is a demonstration of the inductive rate(IR)of different explants in four weeks.

    Tab.2 The induction rate(IR) of different explants Explants

    Stem

    Embryo

    Leaves

    Petiole

    Shoot

    IR%in 4 wks

    100
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    95-100

    90-98

    70-85

    40-60

    3 Ginkgolide Detection

    Lobstein-Guth1developed a purification method involiving liquid-liquid exraction and preparative chromatography for the analysis of ginkgolides obtained from leaf and bark extracts They used HPLC with ultaviolet(UV) detection at 220 nm and the limit of detection was approximately 30 μm.Van Beek6 reduced he detection limit to 1 μm,using refractive index.This limit is similar to that obtained by Tallevi and Kurz7 which were used for the analysis biomass produced by shake flask cultures(Carrier et al.1990),no ginkgolide was detected.Kim5reported their detection method using GC-MS,in which the content of GB 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.
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    3.1 The biological quantitative method(platelet aggregation induced by PAF)of ginkgolides

    3.1.1 Principle

    Pharmacological studies on pure ginkgolides A,B and C specifically inhibited the binding of platelet-activating factor to its receptor in isolated rabbit platelet membranes and inhibited platelet-activating factor-dependent in vitro platelt aggregation.Ginkgolide B was the most active ginkgolide with an IC50 of ca.10-7 M. After oral administration,ginkgolide B inhibited platelet-activating actor-dependent platelet aggregation in rabbits and inhibited thrombus formation induced by electric stimulation of the carotid artery in rats.Ginkgolide B laso inhibited platelet-activating factor-dependent human polymorphonuclear leukocyte aggregation in vitro.Ginkgolides did not significantly affect arachidonic acid metabdolism and was not bound to neuotransmitter receptors.Ginkgolides may have a clinical use and might also be of values as pharmacological tools for studying biological activities of the platelet-activating factor.
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    Fig.4 The curve of IHR-C of the root callus

    3.1.2 Ginkgolide analysis

    3.1.2.1 The LC50 detection of authentic sample of GB

    The standard solution of GB was made up and then the curve of platelet aggregation was measured.The aggregation rate(AR) and inhibition rate(IHR) were got,using MeOH as the standard.The standard curve was shown in Fig.4 and the result was described in Table 3.
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    3.1.2.2 The IC50 detection of callus samples

    Tab.3 Datasheet of IC50 for authentic sample of GB Conc/mg.mL-1

    0.05

    0.025

    0.012 5

    0.008 3

    0.007 5

    0.006 25

    Control
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    PA/%

    17.6

    19.2

    22.8

    34.0

    51.2

    76.4

    80.0

    IHR/%

    78.0

    76.0

    71.5

    56.9
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    36.0

    4.5

    IC50

    0.007 92

    (17.5 μM)

    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 GB was described in equivalent concentration(EC).Tab.4 The IC50 of root calus Conc/mg.mL-1
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    0.2

    0.1

    0.08

    0.06

    control

    AR/%

    36

    41

    49

    56

    100

    IR/%

    64
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    59

    51

    44

    0.07 g.mL-1

    EC of GB

    113×10-6

    3.2 The quantitative detection of ginkgolide by HPLC

    The biological quantitative method of ginkgolide has some advantages,such as strong selection,high sensitivity,etc,but the content of GB obtained is of equivalent concentration.HPLC will be a good choice for the determination of pure constituent of ginkgolides.
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    3.2.1 Chromatographic onditions

    Cloumn:Inertsil ODS column,4.6 mm×250 mm,5 μm,GL Sciences Inc.Eluent:Ethanol-Water(45∶55);Flow rate:1 ml/min;Deteetor:Differential detector;Sensitivity:0.5×10-4.

    3.2.2 Quantification

    The G.biloba calli to be investigated were analysed three times after purification.The concentrations of ginkgolide A and B were calculated with be following equations.
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    Concentration(×10-6)for GA=area(G)/area(I.S.)×Wi.s.×1.30

    Concentration(×10-6)for GA=area(G)/area(I.S.)×Wi.s.×1.36

    where 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:Volume of internal standard(mL)×2.09 mg/mL.1.30 and 1.36 are the response factors of relative weight of GA,GB with the internal standard(IS).
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    The result of the quantitative detection of HPLC is shown is Table 5,Which is modified from Van Beek(1991)'s report,in which neither GA nor GB was detected from G.biloba culture.Tab.5 The content(×10-6) of GA and GB measured by HPLC in root callus /n=3 Group

    1

    2

    3

    mean

    Content of GA
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    48.9

    47.7

    49.5

    48.6±3.6

    Content of GB

    13.2

    12.8

    12.5

    12.8±1.5

    4 The Choice of Optimun Calli Containing High Content of Ginkgolides in Different Explants and the Influences of Cultural Conditions on the Production of Ginkgolides
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    4.1 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.Table 6 gave the results of measurement of the biological quantitiative method(BQM) and HPLC.

    Tab.6 The content(×10-6) of ginkgolides obtained from different calli Calli from
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    root

    stem

    leaves

    petiole

    embryo

    bud

    shoot

    GBBQM

    113.0

    37.7

    95.4

    24.7

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    66.0

    56.6

    GAHPLC

    10

    2

    5

    1

    3

    6

    5

    GBHPLC

    17
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    2

    6

    2

    -

    3

    8

    4.2 The influences of cultural conditions on the production of ginkgolides

    4.2.1 The effect of plant growth regulators on the calli for their ability of producing ginkgolides

    Studies 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 and 2,4-D, were satisfactory(Table 7),but IBA was found ineffective on the production of ginkgolides.Tab.7 The effect of PGR on the production of ginkgolides(Unit:×10-6) PGR/mg.mL-1
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    content of GA

    content of GB

    PGR

    RYof GA

    RY of GB

    NAA1.0

    7

    18

    0.068 7

    0.48

    1.24

    NAA2.0
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    3

    1

    0.072 3

    0.22

    0.07

    NAA4.0

    3

    -

    0.089 1

    0.27

    -

    IBA1.0

    -
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    -

    0.030 5

    -

    -

    IBA2.02

    -

    0.042 5

    0.09

    -

    IBA4.0

    -

    -

    0.049 8
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    -

    -

    2,4-D1.0

    -

    8

    0.040 2

    -

    0.32

    2,4-D2.0

    -

    25

    0.051 0

    -
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    1.28

    2,4-D4.0

    -

    33

    0.079 1

    -

    2.61

    RGR1:relative growth rate,RGR=(1/T)×ln(W2/W1)

    where T is the period of culture,W2 is the weight of callus then harvested,and W1 is the weight of callus when inoculated.
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    RY〔2〕:is means rate of yield,RY=RGR×Content of Ginkgolied

    4.2.2 The effect of illumination on the production of ginkgolides

    The illumination would affect the growth of callus and the production of ginkgolides.The result of examination was found in Table 8.Tab.8 The effect of light on the root calli for the procuction of ginkgolides(Unit:×10-6) Type of culture

    Content of GA
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    Content of GB

    RGR

    RY of GA

    RY of GB

    light culture

    10

    17

    0.046 7

    0.47

    0.79

    dark culture

    1
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    -

    0.063 6

    0.06

    REFERENCES

    [1] Lobstein-Guth A,Briancon-Scheid F,Anton R.Analysis of terpenes from Ginkgo biloba L. by hight performance liquid chromatography.J Chromatography,1983,267:431~438

    [2] Braquet P,Touqui L,Shen Y T.Pharmacol Reviews,1987,39(2):97~145

    [3] Tulecke W R.Tissue derived form the Pollen of Ginkgo biloba.Science,1953,117:599~600
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    [4] Carrier J,Cosentio G,Ndeufeld R.Nutritional and hormonal requirements or Ginkgo biloba embryoderived callus and suspension cell culture.Plant Cell Rep,1990,8:635~638

    [5] Kim Y C,Jeon M H,Sung S H.Production of Ginkgolides in cell culture.PCT Int Appl WO 930,204 (CL.C12P17/02),1993-02-04

    [6] Vanbeek T A,Scheeren H A,Rantio T.Determination of Ginkgolides and bilobalide in Ginkgo biloba Leaves and phytopharmaceuticals.J Chro Matography,1991,543:375~387

    [7] AL-Tel T H,Abu Zarga M H,Sabri S S.New antural colchicinoids:Indications of two possible catabolic routes for the colchinine alkaloids.J Nat Prool,1990,53:623~629

    收稿日期:1998-07-01, http://www.100md.com