前景诱人的银杏叶制剂

银杏叶中的 多种化学成分具 有很好的药效作 用。从60年代 至今,对银杏叶 成分的提取、药 理和临床应用的研究不断深入,银杏叶的药用价值日益看好。银杏叶制剂在医学上应用日渐广泛。 一、含银杏叶提取物的药剂 从银杏叶中提取的含黄酮苷和内酯的银杏叶提取物(即浸膏,EGb)主要用于制备防治心脑血管疾病的药剂。通常把德国斯瓦贝(W.Schwabe)公司生产的EGb761作为银杏叶的标准提取物,这种提取物中含有24％的黄酮苷和6％的内酯(银杏内酯和白果内酯)。药理试验表明:银杏黄酮类化合物用于豚鼠离休     

银杏萜内酯的分布与矮壮素对其生物合成的调节

银杏萜内酯分为银杏内酯A、B、C、J、M(ginkgolide A、B、C、J、M)和白果内酯(bilobalide),主要存在于银杏叶与根内,近年的研究指出银杏萜内酯分别在银杏叶和根中生物合成[1],Cartayrade等人[2]通过叶片生根实验发现生根叶片的银杏萜内酯含量显著高于未生根叶,因而认为银杏萜内酯是在根部合成,然后运输到叶中积累,目前对此还缺乏进一步的研究报道.     

复合酶预处理法对银杏叶总萜内酯提取率的影响

为研究复合酶预处理法对银杏叶总萜内酯浸出率的影响。本文采用单因素实验对银杏叶酶解预处理过程中的酶种类、酶用量、酶解时间、酶解温度、p H值五个关键因素进行探索,酶解后用20%乙醇-水溶液对银杏叶酶解液进行回流提取,LX-5型大孔吸附树脂对提取液进行纯化,浓缩干燥得银杏叶提取物产品。高效液相色谱法检测其总萜内酯含量。结果表明,银杏叶酶解预处理法的最佳工艺为:复合酶(纤维素酶∶果胶酶=1∶1),用量为银杏叶的1/300、酶解时间4.0 h、酶解温度50℃、初始p H值4.0。在该工艺条件下获得的银杏叶总萜内酯提取率为0.55%,银杏叶提取物中总萜内酯含量为8.96%,高于银杏叶提取物国际商务标准(总内酯含量≥6%)。说明复合酶预处理条件下银杏叶总萜内酯提取率高,研究为工业化生产高质量银杏叶提取物提供了理论依据。     

银杏叶黄酮含量对银杏叶提取物大孔树脂纯化工艺条件的影响研究

为考察银杏叶中黄酮含量对银杏叶提取物质量、得率及黄酮提取率的影响,为银杏叶提取物的生产提供原料标准和对应的工艺参数,本文选取黄酮含量分别为1.0%、0.8%和0.6%的三批银杏叶,在保证提取物质量满足中国药典要求的前提下,探究银杏叶质量的变化对提取物大孔树脂纯化工艺条件的影响。采用统计回归方法分别建立了银杏叶提取物的黄酮含量、提取物得率及黄酮提取率与纯化乙醇浓度和体积之间的关系。研究发现银杏叶提取物中黄酮含量与乙醇浓度及洗脱液体积正相关;提取物得率与洗脱液体积负相关、与乙醇浓度的关系随银杏叶中黄酮含量而变化;低黄酮含量银杏叶的提取物质量对工艺参数最为敏感;提取物中内酯含量与银杏叶中黄酮含量呈正相关关系并与提取物中黄酮含量、洗脱液体积呈负相关关系。为保证提取物质量、工艺条件和得率的稳定,建议通过不同质量原料混合的方式使银杏叶中黄酮含量维持在1%,采用2倍量15%乙醇进行洗脱,可获得黄酮含量大于24%、内酯含量6%,得率2.6%、黄酮提取率大于66%的银杏叶提取物。     

不同采收期银杏叶总内酯及黄酮甙的含量测定

采用ＨＰＬＣ法对不同采收期银杏叶总内酯及黄酮甙含量进行测定,结果表明,八至九月采收的银杏叶总内酯及黄酮甙含量均较高。     

银杏黄酮苷和萜类内酯含量的季节变化

以银杏（Ginkgo biloba L.)2年生实生苗和大树为试材,分析根、茎和叶中银杏黄酮苷及萜类内酯含量的季节变化规律。银杏叶中萜类内酯含量从春季起逐渐增加,至夏末秋初达最高值,随后逐渐减少;根和茎中萜类内酯含量的季节变化与叶中相类似,但在冬季休眠期维持较高含量,进入春季伴随叶的萌发生长降低到全年的最低点。银杏茎中萜类内酯含量最低,相当于叶含量的1／3和根含量的1／2。叶中白果内酯含量在总萜类内酯中所占比例较高,而在根和茎中所占比例则较低。随着树龄增加,银杏叶萜类内酯含量下降,这可能与萜类内酯合成能力下降有关。银杏黄酮苷含量在春季幼叶中最高,夏季和秋季相对较低且变化不明显;长枝叶中槲皮素较多,而短枝叶中山柰黄素较多。对不同季节和不同部位的不同成分含量的相关机理进行了讨论。     

贵州传统发酵豆制品中水解银杏黄酮苷的微生物β-葡萄糖苷酶筛选

[目的]微生物β-葡萄糖苷酶法水解银杏黄酮苷具有重要意义,不过目前这方面的研究极少。因此,本文目的是筛选到水解银杏黄酮苷的酶活高的微生物β-葡萄糖苷酶,并分析其底物选择性机制。[方法]以银杏叶提取物作为唯一碳源富集培养,从贵州传统发酵豆豉中筛选产对银杏黄酮苷水解酶活高的β-葡萄糖苷酶的菌株,并对该菌株进行鉴定。然后比较此β-葡萄糖苷酶对不同底物的选择性,同时测定此酶水解银杏黄酮苷反应的米氏常数Km及最大反应速率Vmax。最后,对不同的底物进行分子对接,分析其底物特异性机制。[结果]结果表明,筛选到的菌株GUXN01所产β-葡萄糖苷酶水解银杏黄酮苷的酶活最高,被鉴定为枯草芽孢杆菌。此β-葡糖糖苷酶对β构型的糖类以及苷类等具有广泛的底物特异性和不同的选择性,尤其对银杏黄酮苷具有很好的亲和性。分子对接研究表明枯草芽孢杆菌β-葡萄糖苷酶对银杏黄酮苷和其他糖苷类具有不同亲和性和选择性的原因主要是酶结构和底物分子结构的相互作用力的差异导致的。[结论]这些发现为GUXN01所产的β-葡萄糖苷酶应用于水解银杏黄酮苷类生产相应苷元奠定了良好的基础。     

不同产地及株龄果用银杏叶中总银杏酸含量的比较

银杏(Ginkgo biloba L.)是中国特有的集叶用、果用、材用、防护和观赏于一体的多功能树种。银杏叶和果实具有独特的药理效应,广泛应用于食品、保健品、化妆品和药品中,已逐渐成为全球植物药制剂与保健食品的前列品种。银杏叶为银杏的干燥叶,其提取物制剂在临床上用于治疗冠心病[1]、高血压[2]、脑梗死[3]、痴呆[4-5]、哮喘[6]、乙型肝炎[7]和糖尿病[8]等疾病。银杏叶主要含黄酮类和萜内酯类成分,还含有银杏酸     

不同银杏无性系叶药用成分差异及聚类分析

运用HPLC技术分析了54个银杏(Ginkgo biloba)无性系叶总黄酮和萜内酯及其组分含量的差异,并进行了聚类。结果表明,银杏无性系间叶中总黄酮、萜内酯及其组分含量存在遗传变异,且萜内酯及其组分含量的变异系数明显高于总黄酮及其组分。总黄酮含量较高的无性系有18、42、32和50号,其槲皮素、异鼠李素、山奈酚含量均较高。萜内酯含量较高的无性系为13、42、33、51和65号,其银杏内酯A(GA)、银杏内酯B(GB)、银杏内酯C(GC)及白果内酯(BB)含量均较高。通过对总黄酮-萜内酯进行联合复选,显示叶中总黄酮和萜内酯含量均较高的无性系为13、65、33、51、18、32和42号。这些无性系可通过嫁接、扦插直接在银杏采叶园进行推广种植,或作为叶用银杏新品种的育种材料。     

鄂西南不同核用银杏品种叶中主要黄酮苷元及碳、氮元素含量比较研究

为了比较鄂西南不同核用银杏品种叶中主要黄酮苷元含量,并进一步探讨影响其含量的因素,采用RPHPLC法对样品中黄酮苷元进行定量分析,同时采用德国elementar vario MICRO CUBE元素分析仪测定样品中碳、氮等元素含量。结果表明:不同银杏品种叶片中黄酮含量存在差异,三种主要黄酮苷元组成比例在各品种间差异更加显著,恩银15号及恩银23号具有较好苷元比例,可以作为叶用银杏发展的优先考虑品种。进一步分析表明,叶片总黄酮含量与叶中氮含量之间显著正相关,提示合理施氮有助于改善三种苷元的比例关系,从而使其比例达到最佳。通过比较鄂西南不同银杏品种银杏叶中有效成分的含量,为提高银杏叶质量,优化银杏叶资源提供了理论依据。     

<Emphasis Type="Italic">In vitro</Emphasis> selection of salt tolerant cell lines in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Cell lines able to grow on media containing 50, 100, 150 or 200 mM NaCl were established from potato callus cultures by direct recurrent selection or gradual selection. In callus subjected to direct selection only small clusters of cells survived on medium with 150 or 200 mM NaCl, whereas on 100 mM small cell portions appear necrotic. When cell lines were obtained by successive subcultures on media with increased concentrations of NaCl, salt-tolerant calli were more compact and developed a greenish colour free from necrotic areas. The response of calli lines grown on media with NaCl was compared to control line. The NaCl-tolerant calli showed a decrease in relative growth rate and water content, with higher reductions in the 150 mM tolerant callus. Lipid peroxidation was increased in 50 mM and 100 mM NaCl-tolerant calli, while in 150 mM tolerant callus remained similar to 100 mM values. There was a significant increase in ascorbic acid content in 100 mM and 150 mM NaCl-tolerant calli as compared to the 50 mM, that was two-fold the value found in the control. Also, the contents of soluble and insoluble proteins increased in salt-tolerant lines. SDS-PAGE of soluble proteins showed the synthesis of specific polypeptides in the presence of NaCl in culture medium and the synthesis of a new polypeptide.     

Flavonol glycoside production in callus cultures of Epimedium diphyllum.

Callus cultures of Epimedium diphyllum produced a large amount of epimedoside A in addition to a small amount of diphylloside B, ikarisoside C, epimedoside E, diglycosides of des-O-methylanhydroicaritin (8-gamma, gamma-dimethylallylkaempfero). Icariin, epimedins A-C, which are glycosides of anhydroicaritin, were also produced in the callus cultures. Contents of the flavonol glycosides in callus tissue were higher than those of mother plants, but the composition of each flavonol glycoside mixture in the callus cultures was different from that of the original plants. The time-course experiments showed that an inverse relationship existed between cell growth and flavonol glycoside production. Effects of hormonal factors on cell growth and flavonol glycoside production indicated that 2,4-dichlorophenoxyacetic acid was needed for the production of flavonol glycosides.     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

Cell suspension as a tool to study the biosynthesis of pilocarpine in Jaborandi

Jaborandi (Pilocarpus microphyllus) is a species that naturally occurs in the North and Northeast of Brazil, whose leaves produce pilocarpine (an imidazole alkaloid that has been used to treat glaucoma and xerostomy), the biosynthesis of which is still uncertain. The aim of this work was to establish cell lineages and select them according to an alkaloid profile similar to the one from Jaborandi leaves. The induction of callus was done in different culture media and growth regulators. Calluses from primary cultures or those subcultured several times were used as explants for the obtainment of six cell lineages. Alkaloids content analyses and growth curves showed that lines obtained from primary cultures produced more alkaloids and a better development. Cell lines from 12 subcultures presented a decrease in pilocarpine and pilosine production. After 24 subcultures, the production of alkaloids remained constant. ESI-MS analysis showed that cell culture extracts have the same alkaloid composition as extracts made from leaves. The results indicate that cell suspensions can be used as a model to study the biosynthesis of the imidazole alkaloid in P. microphyllus.     

Tolerance to NaCl induces changes in plasma membrane lipid composition,fluidity and H+-ATPase activity of tomato calli

Two tomato ( Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 m M NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H⁺-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 m M NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 m M NaCl, growth of calli tolerant to 100 m M NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 m M NaCl. Growth in the presence of 100 m M NaCl provoked an increase of Na⁺ and Cl⁻ content, but no significant changes in K⁺ and Ca²⁺. As compared with NaCl-sensitive and 50 m M NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 m M NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 m M NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 m M NaCl showed lower ATP hydrolysis and ATP-dependent H⁺-pumping activities, as well as a lower passive permeability to H⁺ than plasma membrane from NaCl-sensitive and 50 m M NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H⁺-ATPase activity in salt tolerance of tomato calli is discussed.     

Effect of varying NaCl doses on flavonoid production in suspension cells of Ginkgo biloba: relationship to chlorophyll fluorescence,ion homeostasis,antioxidant system and ultrastructure

Ginkgo suspension cells were used to investigate the mechanism that governs the shift between primary and secondary metabolism under NaCl elicitation. The production of three flavonol glycosides, chlorophyll fluorescence, ion content, the antioxidant system, and the cellular ultrastructure in the presence of NaCl doses from 5 to 175 mM were examined. At low salt doses (5–50 mM), cell growth and flavonol glycosides accumulation were stimulated without damaging cell structure or inducing oxidative stress by maintaining high K⁺ and chlorophyll content. At moderate salt doses (75–125 mM), the cells could withstand the salt stress without an impact on survival by changing internal cellular structure, maintaining high levels of K⁺ and Ca²⁺ and increasing anti-oxidative enzyme activities rather than flavonol glycosides to counteract the inhibition of the photosystem II, the accumulation of Na⁺ and hydrogen peroxide (H₂O₂) in the cells. This allowed cells to divert their metabolism from growth to defense-related pathways and tolerate NaCl stress. At higher salinity (150–175 mM), the cellular structure was damaged, and the high Na⁺ and low K⁺ content led to osmotic stress, and therefore, the stimulation of peroxidase (POD) and catalase (CAT) was not enough to cope with high H₂O₂ accumulation. The high production of flavonol glycosides may be a response of elicitation stimulation to serious damage at 175 mM NaCl. In conclusion, the use of 175 mM NaCl may be desirable for the induction of flavonol glycoside production in Ginkgo suspension cells.     

The Stimulatory Effect of the Antibiotic Cefotaxime on Plant Regeneration in Maize Tissue Culture

In order to elucidate the effects of the antibiotic cefotaxime on callus growth and morphogenesis, we incubated embryogenic maize calli (Zea mays L.) of A188 and R91 lines and of their F1 hybrid with 50–500 mg/l cefotaxime throughout several subcultures. Cefotaxime did not affect the induction frequency and growth of the embryogenic callus but enhanced its morphogenesis. In both tested lines and a hybrid, the highest increase in the number of regenerated plants was observed at the antibiotic concentration of 150 mg/l. The degree of morphogenesis stimulation and the range of cefotaxime concentrations effective in stimulation of plant regeneration depended on the properties of calli obtained from tested genotypes.     

In vitro selection of salt tolerant variants following <Superscript>60</Superscript>Co gamma irradiation of long-term callus cultures of <Emphasis Type="Italic">Zoysia matrella</Emphasis> [L.] Merr.

To study growth in the presence of NaCl, in vitro plantlets regenerated from callus of manilagrass (Zoysia matrella [L.] Merr.) were cultured on regeneration medium supplemented with or without 0.3 M NaCl. The results indicated that growth was significantly inhibited by NaCl, with the leaves becoming relatively shorter and thicker. The differences of in vitro plantlets grown under NaCl stress provided specific criteria for the selection of salt tolerant variants. The 6-year maintained calli were treated with different doses (0, 5, 10, 20, 40, 80, 100, 150, 200, 250, and 300 Gy) of ₆₀Co γ rays. Regeneration rate and regeneration capacity of the calli were highest after treatment with 20 Gy ⁶⁰Co γ rays, 27.08 and 91.67% respectively. When the irradiation dose was increased to 100 Gy, 10.42% of the calli developed shoots, but at 150 Gy, both regeneration capacity and regeneration rate declined significantly, and no shoot was observed after 6 weeks of regeneration. Therefore, 100–150 Gy is the most appropriate irradiation span for inducing somaclonal variation. The irradiated calli were selected in vitro for NaCl tolerance. Five NaCl tolerant variant lines, Ze1, Fv1, Te1, Tw1, Fr1, were selected on subculture medium supplemented with 0.35 M NaCl, then transferred to regeneration medium containing 0.25 M NaCl, and grown in a greenhouse. The dark green colour index (DGCI) was used to identify the amount of injury caused by NaCl treatment. This was significantly higher in four of the lines, Ze1, Fv1, Te1, Fr1 (30.88, 31.17, 30.45 and 37.70%, respectively) compared to the control line (CK), which was regenerated from calli subcultured monthly (27.39%), indicating that watering with NaCl caused less injury in these four lines. These lines had lower proline contents than CK under salt stress. The superoxide dismutase (SOD) activity was higher in Ze1 under control condition and its peroxidase (POD) activity increased significantly under salt stress. With Fr1 catalase (CAT) activity was higher under salt stress. The higher activity of these antioxidant enzymes may contribute to the enhanced salt tolerance of the four plant lines.     

Aneuploid and alloplasmic lines as tools for the study of nuclear and cytoplasmic control of culture ability and regeneration of scutellar calli from common wheat

Summary Twenty four B genome aneuploid lines (di-telosomics, nullisomic-tetrasomics and tetrasomics) of Triticum aestivum cv Chinese Spring were used in an analysis of the culture ability and regeneration capability of scutellar calli. Several correlations were found between the presence or absence of specific chromosomes and chromosomal arms of the B genome of common wheat and the growth and differentiation capabilities of these calli. The rate of callus growth decreased only when the long arm of chromosome 6B was not present. The absence of chromosomes 3B and 7B did not result in an apparent change in morphogenetic capability, while the absence of other B genome chromosomes was significantly correlated to changes in the frequency of calli that regenerated plants. The presence of the short arm of chromosome 1B was negatively correlated with regeneration, whereas its long arm is probably required to counteract this effect and to maintain the normal ratio of regeneration. The presence of the chromosomal arm 2BS seemed to be essential for differentiation to shoots. In the absence of the short arms of chromosomes 4B and 5B, the rate of regeneration was slightly reduced. In the absence of the long arm of chromosome 6B there was a marked reduction of the ability of scutellar calli to regenerate plants. The use of additional aneuploid lines belonging to homoeologous group 6 revealed that only calli derived from lines having chromosome 6D in their complement regenerated plants similarly to the euploid control. Culture ability and regeneration capability were also analysed with alloplasmic lines of T. aestivum cv Chris. The lines were derived from five species, representing plasma-types of different phylogenetic distances from plasma-type B of T. aestivum. The results showed that when the endogenous cytoplasm (B-type) was exchanged with T. timopheevii cytoplasm (G-type) there was a significant increase in the regeneration of shoots from the scutellar calli.     

Behaviour of antioxidant defense system in the adaptive response to salt stress in Helianthus annuus L. cells

A relationship between the antioxidant defense system and salt tolerance in two types of sunflower calli differing in salt sensitivity was studied. No reduction in growth occurred in the NaCl-salt-adapted cell line (T) when grown on 175 mM NaCl but growth of the salt-stressed cell line (S) was reduced by 83%. Lipid peroxidation and protein oxidation increased during acute stress of salt stressed cells at 14 and 28 d of the experiment, while salt-adapted calli (T) remained similar to non-shocked (C) values. The antioxidant defense system of callus adapted to growth under NaCl responded differently to 175 mM of salt compared with the corresponding controls under shock treatment. Salt-adapted and salt-stressed calli showed a similar pattern in GSH content at day 14 but at day 28 in S calli, GSH content was increased 100% over the non-shocked calli, while T calli returned to the initial values. In the salt-stressed calli, a general decrease in all the antioxidant enzymes studied (except for glutathione reductase and dehydroascorbate reductase activities) was observed at day 28. Except for catalase, the antioxidant enzymes were elevated constitutively in adapted calli as compared to stressed cells, when both were grown in the absence of NaCl (time 0), and remained unaltered until 28 d after the beginning of the experiment. These results suggest the involvement of an enzymatic antioxidant defense system in the adaptive response to salt stress in Helianthus annuus L. cells.