Hydrogen peroxide-induced oxidative damage and apoptosis in cerebellar granule cells: protection by Ginkgo biloba extract.

The ability of oxidative stress to induce apoptosis and the protective effects of Ginkgo biloba extract (EGb761) against this induction were studied in cultures of rat cerebellar granule cells. Cells were exposed to oxidative stress by treatment with 50 microm hydrogen peroxide+100 microm ferrous sulphate which generates hydroxyl radicals by Fenton reaction. Both morphological observation and biochemical analysis revealed that H(2)O(2)/FeSO(4)treatment induced apoptotic cell death in cerebellar granule cells, which was characterized by chromatin condensation and DNA fragmentation. During this process, the fluidity of the cell membrane decreased markedly, and the conformation of membrane proteins altered significantly. Pretreating cerebellar granule cells with the antioxidant EGb761 (Ginkgo biloba extract) effectively attenuated oxidative damage induced by H(2)O(2)/FeSO(4), and prevented cells from apoptotic cell death. The results suggested that EGb761 might be used as a potential drug for neuronal diseases associated with the excessive production of reactive oxygen species.     

The effect of ginkgo biloba extract on free radical production in hypoxic rats.

In the present study, we assayed the antioxidant properties of Ginkgo biloba (Gb) extract on rats submitted to 21 d of chronic hypoxia. Doses of 25 and 50 mg/kg were examined. Oxygenated free radical production measured by the chemiluminescence technique was significantly decreased in treated rats compared to control rats placed in similar experimental conditions, and this effect was more significant at the 50 mg/kg dose. On the other hand, no antioxidant enzyme activities of the drug were observed towards red blood cells. These results suggest that ginkgo biloba extract has a free radical scavenging action. These antioxidant properties could explain the beneficial hematological properties of Gb extract.     

Ginkgolide B, a constituent of Ginkgo biloba, facilitates glutamate exocytosis from rat hippocampal nerve terminals

Although previous studies have demonstrated that Ginkgo biloba extract has modest effects in the improvement of memory and cognitive function of the Alzheimer's disease patients, the mechanism(s) underlying its beneficial effects remain(s) unclear. In this study, the effect of ginkgolide B, one of the major constituents of Ginkgo biloba extract, on the release of endogenous glutamate from rat hippocampal nerve terminals (synaptosomes) was studied. Ginkgolide B facilitated the Ca2+-dependent release of glutamate evoked by 4-aminopyridine in a concentration-dependent manner. The facilitatory action of ginkgolide B was not due to it increasing synaptosomal excitability because ginkgolide B did not alter the 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential. Rather, examination of the effect of ginkgolide B on cytosolic free Ca2+ concentration revealed that the facilitation of glutamate release could be attributed to an enhancement of presynaptic voltage-dependent Ca2+ influx. Consistent with this, the ginkgolide B-mediated facilitation of glutamate release was significantly prevented in synaptosomes pretreated with a wide spectrum blocker of N-, P-, and Q-type Ca2+ channels, omega-conotoxin MVIIC. Moreover, the facilitation produced by ginkgolide B was completely abolished by the protein kinase A inhibitor, but not by the protein kinase C inhibitor. These results suggest that ginkgolide B effects a increase in protein kinase A activation, which subsequently enhances the Ca2+ entry through voltage-dependent N- and P/Q-type Ca2+ channels to cause a increase in evoked glutamate release from rat hippocampal nerve terminals. In addition, glutamate release elicited by Ca2+ ionophore (ionomycin) was also facilitated by ginkgolide B, which suggests that ginkgolide B may have a direct effect on the secretory apparatus downstream of Ca2+ entry. These actions of ginkgolide B may provide some information regarding the beneficial effects of Ginkgo biloba in the central nervous system.     

Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents

Previous studies have already demonstrated that some constituents of an extract of Ginkgo biloba (EGb), such as ginkgolide B and bilobalide, protect cultured neurons from hypoxia- and glutamate-induced damage. This prompted us to investigate whether they were also able to inhibit neuronal apoptosis. We induced apoptosis in cultured chick embryonic neurons as well as in mixed cultures of neurons and astrocytes from neonatal rat hippocampus by serum deprivation and staurosporine. The increase in the percentage of apoptotic chick neurons from 12% in controls to 30% after 24 h of serum deprivation was reduced to control level by EGb (10 mg/l), ginkgolide B (10 microM), ginkgolide J (100 microM) and bilobalide (1 microM). After treatment with staurosporine (200 nM) for 24 h we observed 74% apoptotic chick neurons. This percentage of apoptotic neurons was reduced to 24%, 62% and 31% in the presence of EGb (100 mg/l), ginkgolide J (100 microM) and ginkgolide B (10 microM), respectively. Bilobalide (10 microM) decreased apoptotic damage induced by staurosporine treatment for 12 h nearly to the control level. In mixed neuronal/glial cultures, the extract of EGb (100 mg/l) and bilobalide (100 microM) rescued rat neurons from apoptosis caused by serum deprivation, whereas, bilobalide (100 microM) and ginkgolide B (100 microM) reduced staurosporine-induced apoptotic damage. Ginkgolide A revealed no anti-apoptotic effect in either serum-deprived or staurosporine-treated neurons. Our results suggest that EGb and some of its constituents possess anti-apoptotic capacity and that bilobalide is the most potent constituent.     

Ginkgolide A contributes to the potentiation of acetaminophen toxicity by Ginkgo biloba extract in primary cultures of rat hepatocytes

The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long-Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5-25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations > or =75 mug/ml and > or =750 mug/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 mug/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [(14)C]-leucine incorporation. At the level present in a modulating concentration (50 mug/ml) of the extract, ginkgolide A (0.55 mug/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A.     

Standardized extracts of flavonoids increase the viability of PC12 cells treated with hydrogen peroxide: effects on oxidative injury

Oxidative stress plays an important role in cell death associated with many diseases. In the present study, concentration-dependence of hydrogen peroxide on rat pheochromocytoma (PC12) cell viability was studied. Preventive effects of antioxidants on the viability of these cells treated with 2 mM hydrogen peroxide were compared. Trolox and Stobadine, as chain-breaking antioxidants were studied in comparison with standardized extracts of flavonoids of Ginkgo biloba and Pycnogenol, known as agents effective in several diseases. All antioxidants increased the viability of hydrogen peroxide-treated PC12 cells. Flavonoid extracts were more effective than Trolox and Stobadine. Antioxidants were most effective if present after the oxidative treatment. As expected, the preloading with antioxidants was without effect on cell viability. Correlations between viability increase induced by antioxidants, and content of oxidation products of proteins and lipids were studied at concentrations of antioxidants mostly effective in preventing cell death: Trolox (10 microM), Stobadine (30 microM), Ginkgo biloba (160 microg/ml), Pycnogenol (100 microg/ml). In these concentrations, antioxidants did not statistically significantly decrease the content of protein carbonyls, with exception of Stobadine, which had no effect. Ginkgo biloba, Trolox and Stobadine intensively decreased the content of malondialdehyde, a product of lipid peroxidation. Pycnogenol was without any preventive effect. Concentrations of antioxidants with a large effect on viability of PC12 cells were not effective in preventing oxygen radical-induced injury of proteins. Antioxidants prevented the oxidative injury of lipids more effectively than that of proteins.     

Inhibition of intracellular calcium elevation and blunting of vasopressor response due to serotonin by ginkgolide B

The vascular effects of each individual composition in the extract of Ginkgo biloba have not been clarified. In this work, we have investigated whether ginkgolide B (GKB), a terpene lactone component from Ginkgo biloba, modulates intracellular calcium ([Ca (2+)] i) of vascular smooth muscle cells (smc) and how it influences the vasopressor response in vitro caused by serotonin (5-HT). GKB (3.2 - 9.6 microM) selectively decreased serotonin-induced [Ca (2+)] i elevations in cultured smc from bovine aorta in dose-dependent manner, while it had no effects on both resting [Ca (2+)] i and potassium-elicited [Ca (2+)] i elevations. On the other hand, GKB in a concentration of 3.2 - 9.6 microM moderately decreased the maximal pressor efficacy of serotonin by 14 - 45 % in rat mesenteric vascular beds, and the EC50 remained unchanged. The comparison study also showed GKB had no effects on noradrenaline-elicited pressor reactions. These results implicate that GKB may selectively inhibit serotonin-mediated [Ca (2+)] i mobilization in vascular smc and non-competitively alleviate the vasopressor effect of serotonin in vitro.     

Alpha 2-adrenoceptor changes during cerebral ageing. The effect of Ginkgo biloba extract.

[3H]Rauwolscine binding to alpha 2-adrenoceptors in cerebral cortex and hippocampus membranes of young (4 months) and aged (24 months) Wistar rats has been investigated. In aged rats, Bmax values of [3H]rauwolscine binding were significantly reduced (25-32%) in the cerebral cortex and hippocampus, as compared with the number of alpha 2-adrenoceptors found in young rats. Chronic treatment with Ginkgo biloba extract did not alter [3H]rauwolscine binding in the hippocampus of young rats, but significantly increased (28%) the [3H]rauwolscine binding density in aged rats. These data confirm the previously described age-related noradrenergic alteration and suggest that noradrenergic activity in aged rats is more susceptible to Ginkgo biloba extract treatment.     

银杏叶提取物对大鼠心肌缺血-再灌注损伤的保护作用

目的探讨银杏叶提取物对大鼠心肌缺血-再灌注损伤引起的氧化应激损伤的保护作用。方法将SD大鼠随机分为3组,假手术(Sham)组、缺血-再灌注损伤(MIR)组和银杏叶提取物组,每组10只。建立心肌缺血-再灌注模型。银杏叶提取物在造模前给药14d。再灌注结束后采血,测定丙二醛(MDA)、超氧化物歧化酶(SOD)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)、乳酸脱氢酶同工酶1(LDH1),测量心肌梗死面积。结果银杏叶提取物能明显降低血浆中AST、LDH、LDH1和MDA的含量,提高SOD活性,减少心肌梗死面积。结论银杏叶提取物对大鼠心肌缺血-再灌注损伤引起的氧化应激损伤的有保护作用。     

Oxidative derangement in rat synaptosomes induced by hyperglycaemia: restorative effect of dehydroepiandrosterone treatment

Central nervous system damage in diabetes is caused by both cerebral atherosclerosis and the detrimental effect of chronic hyperglycaemia on nervous tissue. Hyperglycaemia is the primer of a series of cascade reactions causing overproduction of free radicals. There is increasing evidence that these reactive molecules contribute to neuronal tissue damage. Dehydroepiandrosterone (DHEA) has been reported to possess antioxidant properties. This study evaluates the oxidative status in the synaptosomal fraction isolated from the brain of streptozotocin-treated rats and the antioxidant effect of DHEA treatment on diabetic rats. Hydroxyl radical generation, hydrogen peroxide content, and the level of the reactive oxygen species was increased (P<0.05) in synaptosomes isolated from streptozotocin-treated rats. The derangement of the oxidative status was confirmed by a low level of reduced glutathione and alpha-tocopherol. DHEA treatment (4 mg per day for 3 weeks, per os) protected the synaptosomes against oxidative damage: synaptosomes from diabetic DHEA-treated rats showed a significant decrease in reactive species (P<0.05) and in the formation of end products of lipid peroxidation, evaluated in terms of fluorescent chromolipid (P<0.01). Moreover, DHEA treatment restored the unsaturated fatty acid content of the membrane and the reduced glutathione and alpha-tocopherol levels to normal levels and restored membrane NaK-ATPase activity close to control levels. The results demonstrate that DHEA supplementation greatly reduces oxidative damage in synaptosomes isolated from diabetic rats and suggest that this neurosteroid may participate in protecting the integrity of synaptic membranes against hyperglycaemia-induced damage.     

Evaluation of antioxidant activity of Brazilian plants.

In this work, 22 alcoholic extracts, obtained from 14 species of plants belonging to four families, used for different food and medicinal purposes in Brazil, were evaluated for their capacity to inhibit the reduction of the free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and to protect Saccharomyces cerevisiae cells, an eukaryotic cell model, against the lethal oxidative stress caused by tert-butylhydroperoxide (TBH). Five extracts, two from Lamiaceae family (ethanol and butanol extracts from aerial parts of Hyptis fasciculata) and three from Palmae family (Copernicia cerifera leaves and mesocarp of fruits and the endocarp/mesocarp of fruits from Orbignya speciosa) were able to increase the tolerance of S. cerevisiae to TBH and showed to be active as DPPH radical scavengers, thus indicating that these plant extracts could be considered as potential sources of antioxidants. With the exception of ethanol extract of H. fasciculata, the remainder four extracts exhibited a DPPH radical scavenging activity higher than that obtained from Ginkgo biloba, a reference plant with well documented antioxidant activity. Interestingly, the ethanol extract of G. biloba were not effective for yeast cell protection, reinforcing the antioxidant potential of these extracts.     

银杏内酯提取物中微量成分的LC/DAD/ESI/MS分析及结构鉴定

目的　银杏内酯提取物中有关微量成分的鉴别。方法　采用LC DAD ESI MS联用技术。结果　鉴别了已知的银杏内酯A ,B ,C ,J,M以及白果内酯,并发现了3个微量未知化合物。结论　根据UV ,IR ,MS和NMR光谱数据,鉴定了其中两个新化合物的结构,分别为1,10-二羟基-3,14-二去氢银杏内酯和10-羟基3,14-二去氢银杏内酯,并命名为GK和GL。     

Paradoxical effects of ginkgolide B on cardiomyocyte contractile function in normal and high-glucose environments

AIM: Ginkgo biloba extract is a natural product used widely for cerebral and cardiovascular diseases. It is mainly composed of terpene lactones (ginkgolide A and B) and flavone glycosides (eg quercetin and kaempferol). To better understand the cardiac electromechanical action of Ginkgo biloba extract in normal and diabetic states, this study was designed to examine the effect of ginkgolide B on cardiomyocyte contractile function under normal and high-glucose environments. METHODS: Isolated adult rat ventricular myocytes were cultured for 6 h in a serum-free medium containing either normal (NG; 5.5 mmol/L) or high (HG; 25.5 mmol/L) glucose with or without ginkgolide B (0.5-2.0 microg/mL). Mechanical properties were evaluated using the IonOptix MyoCam system. Contractile properties analyzed included peak shortening (PS), maximal velocity of shortening/relengthening (+/-dl/dt), time-to-PS (TPS) and time-to-90% relengthening (TR90). Levels of essential Ca(2+) regulatory proteins sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban (PLB) and Na(+)-Ca(2+) exchanger (NCX) were assessed by Western blotting. RESULTS: Ginkgolide B nullified HG-induced prolongation in TR90. However, ginkgolide B depressed PS, +/-dl/dt and shortened TPS in NG and HG cells. Ginkgolide B also prolonged TR90 in NG cells. Western blot analysis revealed that HG upregulated SERCA2a and downregulated PLB expression without affecting that of NCX. Ginkgolide B disrupted the NG-HG response pattern in SERCA2a and NCX without affecting that of PLB. CONCLUSION: Ginkgolide B affects cardiomyocyte contractile function under NG or HG environments in a paradoxical manner, which may be attributed to uneven action on Ca(2+) regulatory proteins under NG and HG conditions.     

Liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry of terpene lactones in plasma of animals

Liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry (LC/APCI-ITMS) was applied to evaluate the bioavailibility of two different forms (free and complexed with soy phospholipids) of pure bilobalide and ginkgolide B in rats after acute administration. The same technique was used to measure the levels of ginkgolide A, B and bilobalide in plasma of guinea pigs fed Ginkgo biloba extract enriched in terpene lactones after chronic administration. The ratio R(P)/R(A) increased two to four times after the administration in the phytosomic form, where R(P) and R(A) represent the percentage ratio between the concentration of each terpene lactone in plasma and in the administrated form, respectively.     

Ginkgo biloba extract protects against mercury(II)-induced oxidative tissue damage in rats.

Mercury(II) is a highly toxic metal which induces oxidative stress in the body. In this study we aimed to investigate the possible protective effect of Ginkgo biloba (EGb), an antioxidant agent, against experimental mercury toxicity in rat model. Following a single dose of 5mg/kg mercuric chloride (HgCl(2); Hg group) either saline or EGb (150mg/kg) was administered for 5days. After decapitation of the rats trunk blood was obtained and the tissue samples from the brain, lung, liver, and kidney were taken for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen contents. Formation of reactive oxygen species in the tissue samples was monitored by chemiluminescence (CL) technique. BUN, creatinin, ALT, and AST levels and tumor necrosis factor-alpha (TNF-alpha) and lactate dehydrogenase (LDH) activity were assayed in serum samples. The results revealed that HgCl(2) induced oxidative damage caused significant decrease in GSH level, significant increase in MDA level, MPO activity and collagen content of the tissues. Treatment of rats with EGb significantly increased the GSH level and decreased the MDA level, MPO activity, and collagen contents. Similarly, serum ALT, AST and BUN levels, as well as LDH and TNF-alpha, were elevated in the Hg group as compared to control group. On the other hand, EGb treatment reversed all these biochemical indices. Our results implicate that mercury-induced oxidative damage in brain, lung, liver, and kidney tissues protected by G. biloba extract, with its antioxidant effects.     

Identification of kaempferol as a monoamine oxidase inhibitor and potential Neuroprotectant in extracts of Ginkgo biloba leaves

The effects of Ginkgo biloba leaf extract on rat brain or livermonoamine oxidase (MAO)-A and -B activity, biogenic amine concentration in nervous tissue, N-methyl-D-aspartate (NMDA)- and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)-induced neurotoxicity and antioxidant activity was investigated to determine the effects of the extract on monoamine catabolism and neuroprotection. Ginkgo biloba leaf extract was shown to produce in-vitro inhibition of rat brain MAO-A and -B. The Ginkgo biloba extract was chromatographed on a reverse-phase HPLC system and two of the components isolated were shown to be MAO inhibitors (MAOIs). These MAOIs were identified by high-resolution mass spectrometry as kaempferol and isorhamnetin. Pure kaempferol and a number of related flavonoids were examined as MAOIs in-vitro. Kaempferol, apigenin and chrysin proved to be potent MAOIs, but produced more pronounced inhibition of MAO-A than MAO-B. IC50 (50% inhibition concentration) values for the ability of these three flavones to inhibit MAO-A were 7 x 10(-7), 1 x 10(-6) and 2 x 10(-6) M, respectively. Ginkgo biloba leaf extract and kaempferol were found to have no effect ex-vivo on rat or mouse brain MAO or on concentrations of dopamine, noradrenaline, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. Kaempferol was shown to protect against NMDA-induced neuronal toxicity in-vitro in rat cortical cultures, but did not prevent DSP-4-induced noradrenergic neurotoxicity in an in-vivo model. Both Ginkgo biloba extract and kaempferol were demonstrated to be antioxidants in a lipid-peroxidation assay. This data indicates that the MAO-inhibiting activity of Ginkgo biloba extract is primarily due to the presence of kaempferol. Ginkgo biloba extract has properties indicative of potential neuroprotective ability.     

应用圆二色谱法验证银杏内酯的绝对构型及银杏提取物的化学成分

目的验证银杏内酯A、B的绝对构型,并对银杏提取物的化学成分进行研究。方法应用圆二色谱法并依据Klyne内酯八区-扇形区律对银杏内酯A、B的绝对构型进行分析讨论;采用反复硅胶柱色谱、聚酰胺柱色谱、Sephadex LH-20柱色谱、高效液相色谱、重结晶等方法分离化学成分,1H-NMR1、3C-NMR等方法进行结构鉴定。结果从银杏提取物中分离得到11个化合物,分别鉴定为:芫花素(genkwanin,1)、异鼠李素(isorhamnetin,2)、山奈酚(kaempferol,3)、槲皮素(quercetin,4)、白果内酯(bilobalide,5)、银杏内酯A(ginkgolide A,6)、银杏内酯B(ginkgolide B,7)、银杏内酯C(ginkgolide C,8)、芦丁(rutin,9)、β-谷甾醇(-βsitosterol,10)、胡萝卜苷(daucosterol,11)。结论银杏内酯A、B的绝对构型与文献报道相符。     

What is the combined effect of intense intermittent exercise and Ginkgo biloba plant on the brain neurotrophic factors levels,and learning and memory in young rats?

BackgroundThe present study was conducted to investigate the effect of intense intermittent exercise and Ginkgo biloba on the hippocampal levels of brain‐derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) and also memory and learning in young rats.MethodsForty two eight-week-old rats were randomly divided into six groups including control, low dose of Ginkgo biloba (65 mg/kg), high dose of Ginkgo biloba (100 mg/kg), exercise, exercise + low dose of Ginkgo biloba, exercise + high dose of Ginkgo biloba. The exercise protocol or Ginkgo biloba administration was six days a week for six weeks. The hippocampal levels of BDNF and NT-4 were measured by ELISA method, and learning and memory were evaluated by Morris water maze test in all groups. Data were analyzed using SPSS software.ResultsIncrease in hippocampal levels of BDNF and NT-4 appeared following exercise (p < 0.01). The levels do not change following exercise + Ginkgo biloba administration. However, the NT-4 level decreased in the high dose of Ginkgo biloba group (p < 0.01). Disorder in learning and memory was indicated following the use of low dose of Ginkgo biloba or exercise + low dose Ginkgo biloba administration (p < 0.001). Learning elevated in the exercise group (p < 0.05).ConclusionsExercise in young rats may increase brain neurotrophin levels and lead to improved learning. The preventative or protective role of Ginkgo biloba against some diseases has been suggested, but its consumption in young athletes is recommended with caution.     

银杏叶提取物对CYP酶的影响及与其他药物的相互作用

综述了近年来银杏叶提取物(Ginkgo biloba extract)与其他药物相互作用的研究进展。研究表明银杏叶提取物在大鼠体内和人体内外对CYP酶的影响不一致。人体内银杏叶提取物对药物代谢酶CYP 2C19和CYP 2E1有诱导作用,分别增强了美芬妥因、奥美拉唑和氯唑沙宗的代谢。银杏叶提取物通过降低血中超氧化物歧化酶水平而发挥抗氧化作用,提高了抗精神病药物的疗效。银杏叶提取物还可能与阿司匹林、华法林和曲唑酮等发生药效学方面的相互作用,在临床应用中要引起重视。因此,需要进一步在人体内研究银杏叶提取物与其他药物的相互作用。