Inhibition of fatty acid synthase by ginkgolic acids from the leaves of Ginkgo biloba and their cytotoxic activity

Fatty acid synthase (FAS) has been proposed to be a new drug target for the development of anticancer agents because of the significant difference in expression of FAS between normal and tumour cells. Since a n-hexane-soluble extract from Ginkgo biloba was demonstrated to inhibit FAS activity in our preliminary test, we isolated active compounds from the n-hexane-soluble extract and evaluated their cytotoxic activity in human cancer cells. Three ginkgolic acids 1–3 isolated from the n-hexane-soluble extract inhibited the enzyme with IC₅₀ values 17.1, 9.2 and 10.5 µM, respectively, and they showed cytotoxic activity against MCF-7 (human breast adenocarcinoma), A549 (human lung adenocarcinoma) and HL-60 (human leukaemia) cells. Our findings suggest that alkylphenol derivatives might be a new type of FAS inhibitor with cytotoxic activity.     

Effects of Ginkgo biloba extract (EGb 761) on learning and possible actions on aging

A study of the effect of Ginkgo biloba extract (EGb 761) has shown enhancing effects on training in adult and aged Swiss mice. An analysis of inbred mice has confirmed this sensitivity to EGb 761, but depending on the strains, with different effects at different ages. The most interesting results are related to improvements in performances observed with aged mice of the DBA/2J strain. The results obtained with inbred strains in the study of the mossy fibers of the hippocampus make it possible to suggest a link between the improvements in training and the histological structure of the hippocampus. This possibility, which can be confirmed by further studies, is presented here.     

Quantitative analysis of ginkgolic acids from Ginkgo leaves and products using 1H-NMR

The determination of ginkgolic acids in Ginkgo products is one of the principal components of quality control. However, a number of ginkgolic acids with different side chains may be present and this makes their analysis by conventional chromatographic methods more complex. In this study, 1H-NMR spectrometry was applied to the analysis of the total content of ginkgolic acids in leaves of Ginkgo biloba and in six types of commercial Ginkgo products in the absence of chromatographic purification. For this analysis, protons H-3, H-4, and H-5, which are well separated in the range 8 (ppm) 6.5-7.5 in the 1H-NMR spectrum, were utilised. For further confirmation, the correlations of H-3, H-4 and H-5 were examined by 1H-1H COSY spectra in all extracts. The quantity of the compounds was calculated from the relative ratio of the integral of each peak to the integral of the peaks of a known amount (100 microg) of anthracene used as an internal standard. The quantitative results obtained by 1H-NMR analysis were compared with those obtained by GC, which showed that the 1H-NMR method allows a simple quantification of total ginkgolic acids in Ginkgo extracts without any pre-purification steps.     

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min.2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region.3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group.4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare.5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.     

Decreased Electroconvulsive Shock-Induced Diacylglycerols and Free Fatty Acid Accumulation in the Rat Brain by Ginkgo biloba Extract (EGb 761): Selective Effect in Hippocampus as Compared with Cerebral Cortex

Abstract: The effect of Ginkgo biloba extract (EGb 761) treatment (100 mg/kg/day, per os, for 14 days) on electroconvulsive shock (ECS)-induced accumulation of free fatty acids (FFA) and diacylglycerols (DAG) was analyzed in rat cerebral cortex and hippocampus. EGb 761 reduced the FFA pool size by 33% and increased the DAG pool by 36% in the hippocampus. These endogenous lipids were unaffected in cerebral cortex. During the tonic seizure (10 s after ECS) the fast accumulation of FFA, mainly 20:4, was similar in sham- and EGb 761 -treated rats, in both the cerebral cortex and hippocampus. However, further accumulation of free 18:0 and 20:4, observed in the hippocampus of sham-treated rats during clonic seizures (30 s to 2 min after ECS), did not occur in EGb 761-treated animals. The rise in DAG content triggered in the cortex and hippocampus by ECS was delayed by EGb 761 treatment from 10 s to 1 min, when values similar to those in sham animals were attained. Moreover, in the hippocampus the size of the total DAG pool was decreased by 19% during the tonic seizure. At later times, DAG content showed a faster decrease in EGb 761-treated rats. By 2 min levels of all DAG acyl groups decreased to values significantly lower than in sham animals in both cortex and hippocampus. This study shows that EGb 761 treatment affects, with high selectivity, lipid metabolism and lipid-derived second messenger release and removal in the hippocampus, while affecting to a lesser extent the cerebral cortex.     

银杏种子和叶的蛋白质分析

采用分级提最方法提取银杏（Ginkgo biloba L.）种子和银杏叶蛋白质,并进行各组分蛋白质含量测定和银杏种子后熟过程中蛋白质含量动态变化的分析。结果表明,银杏种子以水溶性和盐溶性蛋白质为主,银杏叶以醇溶性蛋白质为主。银杏叶中主要蛋白质在HPLC柱中的保留时间为3.457min,相对含量达70%以上。银杏叶蛋白质含有丰富的亮氨酸、缬氨酸、赖氨酸和色氨酸,其含量分别为20.23%、13.35%、4.81%和3.73%。萌发种子胚体中的蛋白质主要是醇溶性蛋白质和谷蛋白类蛋白质。在种子萌发过程中,胚乳蛋白质含量明显增加,播种后第3周和萌发时总蛋白质含量达到高峰。     

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.     

生长调节剂对离体银杏叶苯丙氨酸解氨酶活性的影响

用6种生长调节剂诱导离体银杏(Ginkgo biloba Linn.)叶苯丙氨酸解氨酶(PAL)活性,结果表明:300mg·L-1ETP诱导作用最强,其动态诱导在处理4 h后最高;40 mg·L-1 2,4-D诱导效果最强,并有2个明显的诱导高峰;200mg·L-1CCC诱导作用最强,在处理8 h后出现诱导高峰;除100mg·L-1IAA处理组的酶活性比对照低外,其他浓度的IAA处理均比对照高,诱导作用最强的是70 mg·L-1IAA处理;除了100mg·L-1ABA处理使酶活性略有降低外,其他浓度的ABA处理都能诱导酶活性升高,以75 mg·L-1ABA诱导能力最强,在处理4 h后酶活性最大;4个浓度的GA处理中,仅75 mg·L-1GA处理组的酶活性高于对照,处理8 h后酶活性达到最大.结果说明诱导效果从高至低依次为:乙烯利(ETP)、2,4-D、矮壮素(CCC)、吲哚乙酸(IAA)、脱落酸(ABA)、赤霉素(GA).     

Insecticidal activities of Ginkgo biloba seed coat extracts against Spodoptera exigua (Lepidoptera: Noctuidae)

The present study relates to a methanol extract of the seed coat of Ginkgo biloba, and tested particularly on the third instar larvae of Spodoptera exigua. The extract was found to have an inhibitory effect on the growth of the larvae besides bringing a change in the nutrient reserves in the body of the insect. Topical application of five different doses of the methanol extract resulted in a mortal effect to third instar larvae of S. exigua that is very much dependent on the dose as well as duration of exposure. Lower doses revealed lower mortality after 24 h of application. At doses of 1.00, 2.00, 4.00, 8.00 and 16.00 ng/larva, mortalities were 9.25, 26.07, 50.32, 56.28 and 92.44%, respectively. The dose for 50% mortality (LD₅₀) of methanol extracts by applied by a topical method with 1 µL of acetone solution was 1.92 ng/larva. Nutrient reserves like protein, glycogen and lipid are known to regulate pupation and adult emergence. These reserves have been found to be lower in treated larvae, indicating the insecticidal role of methanol extracts from G. biloba against third instar larvae of S. exigua.     

高效液相色谱分析银杏萜内酯的含量

用高效液相色谱法测定银杏叶及提取物中银杏内酯Ａ、Ｂ、Ｃ和白果内酯的含量。采用Ｃ１８分离柱,示差检测器,甲醇：水（３３：６７）为流动相,方法回收率平均达９７％以上,变异系数（ＣＶ）为２．１％。     

银杏叶多酚的机械力辅助提取及其体外抗氧化活性研究

为优化银杏叶多酚提取工艺,通过单因素试验考察填充率、球磨转速、球磨时间、乙醇浓度、料液比、提取温度、提取时间七个因素对机械力辅助提取银杏叶多酚得率的影响,以银杏叶多酚得率为响应值,采用Box-Benhnken三因素三水平响应面设计优化工艺,同时比较了4种提取方法对银杏叶多酚提取得率和抗氧化活性的差异。结果表明,机械力辅助提取银杏叶多酚的最佳工艺条件为:填充率26%、球磨转速为400rpm、球磨时间为15min。在此条件下,银杏叶多酚的得率为7.33%。机械力辅助乙醇提取银杏叶多酚得率低于碱水提取法,但是抗氧化活性高于碱水法提取的银杏叶多酚;抗氧化活性与乙醇回流法提取的银杏叶多酚相当,但是提取得率高于乙醇回流法。此提取工艺高效可行,具有一定的参考价值。     

Photoinhibition and Photoprotection in Ginkgo biloba leaves: Influence of Temperature,CO2 and O2

In midday ginkgo ( Ginkgo biloba L. ) leaves have to bear photon flux density over 1 400 μmol·m-2·s-l in combination with high temperatures around 35℃ at natural habitat. They show typical midday depression of stomatal conductance and of CO2 assimilation rate. The zeaxanthin changes with light intensity during the day. The influence of the combination of strong light and temperature on photoinhibition was also examined in the laboratory. A low CO2 internal conductance (31 mmol· m- 2·s- 1 ) was found in ginkgo leaves, which had been exposed to excessive light at temperature between 15 ℃ and 35 ℃ with reduced CO2 (80 μL·L-l) or oxygen (2%) for 2 h, causing a low CO2 concentration at the carboxylation site and a high proportion of photorespimtion. The ratio of electron transport to CO2 fixation was rather high in ginkgo ( 16 e- /CO2 at 25 ℃ ) as compared with other plants. It increased with temperature also in 2% 02 which could not be explained solely as due to change of photorespimtion. The reduction of oxygen in 340 or 80 μL·L- 1 CO2 had no effect on the extent of photoinhibition at all temperatures, which indicated that eleetron flow caused by photorespiration in excess light was negligible in protective effect in ginkgo leaves. However, a decreased CO2 coneentration increased photoinhibition, especially at high temperature. It is concluded that the dissipation of excessive excitation energy in the PS II antennae through the xanthophyll cycle may be the major protective mechanism to preventing from the deteriorated effects of strong light in ginkgo leaves.     

银杏精细胞结构及受精作用的细胞学研究

采用常规石蜡制片技术和环氧树脂半薄切片技术,对银杏的精细胞结构及受精过程进行了研究。结果表明,临近受精前,精原细胞分裂形成两个半球形的精细胞,每个精细胞内含液泡状结构、生毛体和纤维性颗粒体各一个。两精细胞的液泡状结构其位置可同时或分别位于近极面和远极面,这种位置的变动可能是鞭毛摆动导致精细胞的旋转所引起,这说明银杏的精细胞在花粉管内也许即可以旋转运动。在银杏受精前后,珠孔端颈卵器室附近的珠心组织表现出向上隆起、出现受精滴、皱褶等规律性变化,这些现象是判断银杏受精时期的良好形态特征。在银杏受精时带有鞭毛的完整精细胞进入颈卵器,随后鞭毛带及精细胞质遗留于颈卵器口下方、卵细胞上面,仅精核进入卵细胞。进入卵细胞的精核直径约30μm,小于成熟精细胞的精核直径（约40μm）。这些对探讨银杏的系统地位及裸子植物的生殖演化具有一定意义。     

银杏枝、花、种子在树冠上的分布格局及其相互关系研究

采用分层调查的方法研究了１４年生银杏嫁接植株树冠上枝、花、种子的分布格局及其相互关系。结果表明,长枝、短枝、短果枝和花主要分布在树冠中部,且分布比较均匀,树冠上从上到下,长枝平均年龄逐渐增大,分枝角度逐渐升高,种子主要集中分布在距地面１８０－３２０ｃｍ的体积冠层中,这一冠层中的种子数占植株种子总数的５１．３％,不同冠层中枝、花、种子数量之间密切相关     

沈阳市区不同环境下银杏水力特征和非结构性碳水化合物含量

银杏为重要的城市绿化树种,但在城市环境中常出现生长不良和受害的现象,作为行道树时该树种衰退死亡现象尤为多发,相关生理机制还有待深入研究.本研究选取生长在沈阳市区行道和公园两个不同生境的银杏,对其受胁迫伤害程度、水力功能性状及非结构性碳水化合物含量(NSC)等进行了对比分析.结果表明:行道银杏枝条及叶片相比公园中的银杏受...     

Antidepressant-like effect of a Ginkgo biloba extract (EGb761) in the mouse forced swimming test: role of oxidative stress

EGb761 is a well-defined mixture of active compounds extracted from Ginkgo biloba leaves. This extract is used clinically due to its neuroprotective effects, exerted probably via its potent antioxidant or free radical scavenger action. Previous studies suggest that oxidative stress, via free radical production, may play an important role in depression and animal models for depression-like behavior. Preclinical studies have suggested that antioxidants may have antidepressants properties. The aim of this study was to investigate the antidepressant-like of EGb761 due to its antioxidant role against oxidative stress induced in the forced swimming test, the most widely used preclinical model for assessing antidepressant-like behavior. Male BALB/c mice were pretreated with EGb761 (10 mg/kg, ip) daily for 17 days followed by the forced swimming test and spontaneous locomotor activity. Animals were sacrificed to evaluate lipid peroxidation, different antioxidant enzyme activities, serotonin and dopamine content in midbrain, hippocampus and prefrontal cortex. EGb761 significantly decreased the immobility time (39%) in the forced swimming test. This antidepressant-like effect of EGb761 was associated with a reduction in lipid peroxidation and superoxide radical production (indicated by a downregulation of Mn-superoxide dismutase activity), both of which are indicators of oxidative stress. The protective effect of EGb761 is not related to excitatory or inhibitory effects in locomotor activity, and was also associated with the modulation of serotonergic and dopaminergic neurotransmission. It is suggested that EGb761 produces an antidepressant-like effect, and that an antioxidant effect against oxidative stress may be partly responsible for its observed neuroprotective effects.     

Reduction of rise in blood pressure and cortisol release during stress by Ginkgo biloba extract (EGb 761) in healthy volunteers.

The standardized extract of Ginkgo biloba (EGb 761) was found not only to improve memory and aging associated cognitive deficits but also to exert beneficial effects on mood. An antistress action of the extract has been suggested but not directly proven. The present study was aimed to evaluate the effects of EGb 761 on salivary cortisol and blood pressure responses during stress in healthy young volunteers (n = 70) in a double blind placebo controlled design. A stress model involving a combination of static exercise (handgrip) and mental stimuli was used. Single treatment with EGb 761 (120 mg) reduced stress-induced rise in blood pressure without affecting the heart rate. Salivary cortisol responses showed differences with respect to the gender and the time of day of the stress exposure, with the activation only in male subjects in the afternoon. This activation was absent if they were treated with EGb 761. The performance in a short memory test with higher scores achieved by women remained unaffected by EGb 761 treatment. Thus, this study provides evidence that EGb 761 has an inhibitory action on blood pressure and it may influence cortisol release in response to some stress stimuli.     

Bilobalide, a component of the Ginkgo biloba extract (EGb 761), protects against neuronal death in global brain ischemia and in glutamate-induced excitotoxicity.

In this study, the effect of bilobalide, a purified terpene lactone component of the Ginkgo biloba extract (EGb 761), and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death was compared. In the case of ischemic injury, neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in the hippocampal regions of gerbils was measured. A significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons at 7-days of reperfusion after 5 min of transient global forebrain ischemia. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected hippocampal CA1 neurons against ischemia-induced neuronal death and reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death [effective concentration (EC50) = 5 microg/ml (12 microM) forbilobalide and 100 microg/ml for EGb 761]. These results suggest thatboth EGb 761 and bilobalide protect against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.     

Cytokinins in the leaves of Ginkgo biloba. II. Metabolism and transport of 8 (14C) zeatin applied to shoot explants

Irrespective of their age, leaves of Ginkgo biloba metabolised applied 8 (¹⁴C) zeatin to compounds of similar chromatographic properties. Glucosylation is apparently not a normal feature of cytokinin metabolism in immature leaves. However, the application of zeatin to these leaves did result in the formation of metabolites which co-chromatographed with glucosylated cytokinins. As far as cytokinin metabolism is concerned therefore, this application of excess zeatin allowed immature leaves to behave as mature or senescing leaves. Overall metabolism was fastest in immature leaves. From the metabolites formed it would appear as if oxidation, which resulted in the formation of a metabolite which co-eluted with N-(purin-6-yl)glycine, was also important in immature leaves. In senescing leaves glucosylation was the major form of metabolism. Extraction and re-application of the polar metabolites (formed from zeatin) to mature leaves resulted in the formation of compounds which co-chromatographed with zeatin. This suggests that these compounds could serve as precursors for zeatin or could be hydrolysed to form zeatin.Very little of the applied radioactivity was exported from the leaves irrespective of their physiological age. When the metabolites, obtained after zeatin application to mature leaves, were extracted and reapplied to the leaves, export of radioactive material was much improved. The results suggest that should cytokinins such as zeatin be translocated to mature leaves of this deciduous gymnosperm their export from the leaves would be unlikely unless first metabolised. In all probability the metabolites concerned are cytokinin glucosides.The financial support of the C.S.I.R., Pretoria, is gratefully acknowledged.