The Ginkgo biloba extract (EGb 761) protects and rescues hippocampal cells against nitric oxide-induced toxicity: involvement of its flavonoid constituents and protein kinase C

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. Numerous studies have shown that the Ginkgo biloba extract EGb 761 is a NO scavenger with neuroprotective properties. However, the mechanisms underlying its neuroprotective ability remain to be fully established. Thus, we investigated the effect of different constituents of EGb 761, i.e., flavonoids and terpenoids, against toxicity induced by NO generators on cells of the hippocampus, a brain area particularly susceptible to neurodegenerative damage. Exposure of rat primary mixed hippocampal cell cultures to either sodium nitroprusside (SNP; 100 microM) or 3-morpholinosydnonimine resulted in both a decrease in cell survival and an increase in free radical accumulation. These SNP-induced events were blocked by either EGb 761 (10-100 microg/ml) or its flavonoid fraction CP 205 (25 microg/ml), as well as by inhibitors of protein kinase C (PKC; chelerythrine) and L-type calcium channels (nitrendipine). In contrast, the terpenoid constituents of EGb 761, known as bilobalide and ginkgolide B, as well as inhibitors of phospholipases A [3-[(4-octadecyl)benzoyl]acrylic acid (OBAA)] and C (U-73122), failed to display any significant effects. Moreover, EGb 761 (50 microm) CP 205 (25 microg/ml), and chelerythrine were also able to rescue hippocampal cells preexposed to SNP (up to 1 mM). Finally, EGb 761 (100 microg/ml) was shown to block the activation of PKC induced by SNP (100 microM). These data suggest that the protective and rescuing abilities of EGb 761 are not only attributable to the antioxidant properties of its flavonoid constituents but also via their ability to inhibit NO-stimulated PKC activity.     

银杏开发造园模式的探讨

根据银杏生物学特性和生产栽培目的的不同以及目前银杏的生产状况,阐述了银杏开发的重要意义,概述了叶材兼用银杏园、果叶兼用银杏园、果材兼用银杏园的立地选择、栽培密度和抚育管理等方面的技术要求,分析了各类园的利弊,并对低效的银杏园提出了改造的建议。     

银杏无性繁殖位置效应的研究

对银杏无性繁殖的位置效应进行了研究。方差分析结果表明,位置效应在无性繁殖过程中能保持稳定,在品种之间有一定差异。随母树及接穗年龄的增长,位置效应越明显。不同无性繁殖方法有相似的位置效应,并具有显著的重现性。在自然状态下,当年生枝条生长的异质性可分为３个枝段。从嫁接到大量结实后,位置效应可经过强－弱－强阶段。采用基生复干作接穗或插条,或用修剪、复壮和扶梢等方法可以控制或消除位置效应,而且效果明显。     

Ginkgo biloba Expresses Calreticulin,the Major Calcium-Binding Reticuloplasmin in Eukaryotic Cells

Calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was characterized in Ginkgo biloba L. pollen and seeds. Electrophoretic analysis of the partly purified extracts showed the presence of two protein bands of 57 and 50kDa apparent molecular masses, which strongly cross-reacted with antibodies against plant calreticulins. Amino acid sequence comparison with other plant and animal calreticulins revealed a much higher similarity of the N-terminus of Ginkgo calreticulins with the homologue from angiosperms rather than with that from mammals. The finding of calreticulin in Ginkgo is indicative of the conservation also in gymnosperms of Ca²⁺ homeostatic mechanisms, which seem to rely on the same molecular components as all eukaryotic cells.     

Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC–MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.     

银杏叶片三种酶活性变化与抗疫霉菌关系的研究

将疫霉菌离体接种于银杏叶片,发现不同龄期银杏叶片对疫霉菌的抗病性有差异,叶龄为100d的抗病性较强,叶龄为20d的抗病性较弱。不同银杏品种叶片对疫霉菌的抗性也有差异,潮田1号和大佛手2号较抗病,大佛手1号和桂G86-1较感病。不同龄期叶片接种后,其过氧化物酶(POD)和苯丙氨酸解氨酶(PAL)的酶活性变化与抗病性成正相关,而多酚氧化酶(PPOD)的酶活性变化与抗病性没有相关性;不同银杏品种叶片接种后,品种间的抗病性与PPOD酶活性变化成正相关,而POD和PAL的酶活性变化与品种间的抗病性没有相关性。可见,POD、PPOD和PAL的酶活性变化没有明显规律性,不能作为衡量银杏叶片抗病性的生化指标。     

银杏芽中内源激素与大小年结实的关系

为探讨银杏大、小年结实现象的生理原因,于2004～2005年,用ELISA(间接酶联免疫法)对银杏大年树和小年树果芽和叶芽内的IAA,GA,ZR和ABA含量进行了对比测定。结果表明:大年树IAA、ZR和ABA含量在果芽和叶芽之间的比值均高于小年树,GA含量在果芽和叶芽之间的比值则低于小年树,有利果芽分化生长,形成大年;果芽中较高水平的ZR、IAA和ABA与较低水平的GA,以及萌芽前期较高的IAA/GA,ZR/GA和ABA/GA等比值,有利果芽分化,形成大年;叶芽中较高的ZR、IAA和较低的ABA有利于形成大年生长,尤其是ZR含量在萌芽前期或中期升高与形成大年生长关系密切。     

银杏叶提取物的微生物转化研究Ⅲ．转化产物的化学成分

为研究银杏叶提取物经微生物转化后的化学成分,采用微生物转化的方法对银杏叶提取物进行处理得到转化粗提物,采用硅胶、Sephadex^TM LH-20凝胶等色谱技术对转化粗提物进行分离纯化,通过波谱分析单体结构鉴定。结果发现,从银杏叶提取物的转化粗提物中分得5个化合物,分别为异鼠李素（isorhamnetin,1）、山奈酚（kaempferol,2）、斛皮素（quercefin,3）、山奈酚-3-O-α-L-鼠李糖苷（kaempferol-3-O-α-L-rhamnosid,4）、斛皮素-3-O-α-L-鼠李糖苷（quercetin-3-O-α-L-rhamnosid,5）。结果表明,这是首次用微生物转化的方法对银杏叶提取物进行处理。     

Chloroplast genome (cpDNA) of Cycas taitungensis and 56 cp protein-coding genes of Gnetum parvifolium: insights into cpDNA evolution and phylogeny of extant seed plants

Phylogenetic relationships among the 5 groups of extant seed plants are presently unsettled. To reexamine this long-standing debate, we determine the complete chloroplast genome (cpDNA) of Cycas taitungensis and 56 protein-coding genes encoded in the cpDNA of Gnetum parvifolium. The cpDNA of Cycas is a circular molecule of 163,403 bp with 2 typical large inverted repeats (IRs) of 25,074 bp each. We inferred phylogenetic relationships among major seed plant lineages using concatenated 56 protein-coding genes in 37 land plants. Phylogenies, generated by the use of 3 independent methods, provide concordant and robust support for the monophylies of extant seed plants, gymnosperms, and angiosperms. Within the modern gymnosperms are 2 highly supported sister clades: Cycas-Ginkgo and Gnetum-Pinus. This result agrees with both the "gnetifer" and "gnepines" hypotheses. The sister relationships in Cycas-Ginkgo and Gnetum-Pinus clades are further reinforced by cpDNA structural evidence. Branch lengths of Cycas-Ginkgo and Gnetum were consistently the shortest and the longest, respectively, in all separate analyses. However, the Gnetum relative rate test revealed this tendency only for the 3rd codon positions and the transversional sites of the first 2 codon positions. A PsitufA located between psbE and petL genes is here first detected in Anthoceros (a hornwort), cycads, and Ginkgo. We demonstrate that the PsitufA is a footprint descended from the chloroplast tufA of green algae. The duplication of ycf2 genes and their shift into IRs should have taken place at least in the common ancestor of seed plants more than 300 MYA, and the tRNAPro-GGG gene was lost from the angiosperm lineage at least 150 MYA. Additionally, from cpDNA structural comparison, we propose an alternative model for the loss of large IR regions in black pine. More cpDNA data from non-Pinaceae conifers are necessary to justify whether the gnetifer or gnepines hypothesis is valid and to generate solid structural evidence for the monophyly of extant gymnosperms.     

产黄酮银杏内共生真菌的分离及其发酵条件的优化

本文从不同年龄、不同地域的银杏叶、茎、根部组织中分离得到20株银杏内共生真菌,经过发酵复筛有5株的产黄酮能力超过5μg/m L。实验对菌株的最佳发酵产黄酮条件进行了优化,优化条件为:3%葡萄糖、0.5%蛋白胨、0.4%酵母膏、0.3%KH2PO4、0.01%Mg SO4·7H2O;起始p H值7.0、最适发酵温度28℃、摇床转速为140 r/min、装液量125 m L/250 m L。在此条件下,该系列菌株在发酵7 d左右产黄酮量可达6.4μg/m L。