The standardized Ginkgo biloba extract Egb-761 protects vascular endothelium exposed to oxidized low density lipoproteins.

Dietary antioxidants are frequently proposed as protective agents for the vascular endothelium during the onset of atherosclerosis. This protection may occur at two distinct levels. First, they prevent oxidative modification of atherogenic lipoproteins (LDL). Second, they can provide a cellular protection against oxidized LDL-mediated endothelium dysfunction, although this mechanism remains poorly considered in many instances. To gain insight into the mechanism underlying such cellular protection against oxidized LDL, we examined the impact of a popular traditional medicine, an extract from Ginkgo biloba with well-known antioxidant properties, on two endothelial cells properties: cell adhesion and ionic homeostasis. Cellular lipoperoxides levels were also measured as a marker of cellular oxidative stress. Human umbilical-vein endothelial cells were exposed to native (nat-) or oxidized (ox-) LDL, the latter prepared to be compatible with clinically observed levels of oxidation. Although nat-LDL had little effect, ox-LDL increased endothelial adhesive properties (35%, p<0.01) and lipoperoxidation (45%, p<0.01). Na,K-ATPase activity, a key regulator of ionic homeostasis, was significantly decreased after exposure to nat-LDL (30%, p<0.01) and dramatically depressed after exposure to ox-LDL (65%, p<0.001). The standardized preparation of Ginkgo biloba EGb-761 totally protected adhesive properties and endothelial lipoperoxide levels. Moreover, it limited the decrease in Na,K-ATPase activity induced by ox-LDL to levels similar to nat-LDL. This suggests that EGb-761 protects endothelial adhesive properties and helps prevent the disruption of ionic homeostasis. The EGb-761-mediated inhibition of ox-LDL-induced lipoperoxide levels in endothelial cells appears to be an important mechanism by which Ginkgo biloba extract protects endothelial properties.

Inhibition of type 4 phosphodiesterase by rolipram and Ginkgo biloba extract (EGb 761) decreases agonist-induced rises in internal calcium in human endothelial cells.

The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC(50)=25.1 mg/L), the isoform that is mainly present in endothelial cells, in a competitive manner (K:(i)=12.5 mg/L). Because changes in cyclic nucleotide levels may affect intracellular calcium ([Ca(2+)](i)) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca(2+)](i) levels and agonist-induced rises in [Ca(2+)](i) in single human umbilical vein endothelial cells (HUVECs) in culture. The effects of EGb 761 were compared with those of rolipram, a selective PDE4 inhibitor that increases cellular cAMP levels, and the cAMP analogue dibutyryl cAMP (db-cAMP). EGb 761 (20 and 100 mg/L), rolipram (50 micromol/L), and db-cAMP (100 micromol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca(2+)](i) increases in HUVECs without modifying resting [Ca(2+)](i) levels. Similar results were obtained by using a Ca(2+)-free bath solution. EGb 761 (100 mg/L), but not rolipram (50 micromol/L) or db-cAMP (100 micromol/L), also inhibited Ca(2+) influx into cells having thapsigargin-depleted internal Ca(2+) stores and bathed in a Ca(2+)-free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in [Ca(2+)](i) in HUVECs, mainly by inhibition of Ca(2+) mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca(2+) signaling in endothelial cells.

The protective effect of EGb 761 in isolated ischemic/reperfused rat hearts: a link between cardiac function and nitric oxide production.

This study was conducted to evaluate the effect of Ginkgo biloba extract (EGb 761) on the nitric oxide (NO) production in relation to the recovery of postischemic cardiac function in isolated working rat hearts. Rats were orally treated with various doses (25, 50, 75, and 100 mg/kg/day) of EGb 761 for 10 days. Hearts were isolated in "working mode" and subjected to 30-min ischemia followed by 120 min of reperfusion. EGb 761 inhibited NO production measured by electron spin-resonance spectroscopy (ESR), and improved the recovery of postischemic cardiac function (coronary flow, aortic flow, left ventricular developed pressure and its first derivative) in the ischemic/reperfused myocardium. Thus in rats treated with 25, 50, 75, and 100 mg/kg/day of EGb 761 and in hearts subjected to 30-min ischemia followed by 120 min of reperfusion, aortic flow was increased from its postischemic drug-free control value of 8.0+/-0.4 to 8.6+/-0.4 ml/min (NS), 17.3+/-0.9 ml/min (p<0.05), 21.5+/-1.1 ml/min (p<0.05), and 23.6+/-1.2 ml/min, respectively. The same recovery in postischemic coronary flow, left ventricular developed pressure, and its first derivative also was observed. In the initial phase of reperfusion, NO production measured by ESR was reduced by 85% in the 75 mg/ kg/day of EGb 761-treated group in comparison with the drug-free ischemic/reperfused hearts. Inducible NO synthase (iNOS) messenger RNA (mRNA) measured by reverse transcription-polymerase chain reaction (RT-PCR) also was reduced by 41 and 58% in the groups treated with 75 and 100 mg/kg/day of EGb 761, respectively. Our findings show that EGb 761 directly acts as an NO scavenger and concomitantly inhibits the expression of iNOS mRNA. Thus, EGb 761 may act as a potent inhibitor of NO production under the condition of ischemia/reperfusion, improving the recovery of postischemic cardiac function.

EGb 761 protects liver mitochondria against injury induced by in vitro anoxia/reoxygenation.

The present study investigated the protective effects of Ginkgo biloba extract (EGb 761) on rat liver mitochondrial damage induced by in vitro anoxia/reoxygenation. Anoxia/reoxygenation was known to impair respiratory activities and mitochondrial oxidative phosphorylation efficiency. ADP/O (2.57 +/- 0.11) decreased after anoxia/reoxygenation (1.75 +/- 0.09, p < .01), as well as state 3 and uncoupled respiration (-20%, p < .01), but state 4 respiration increased (p < .01). EGb 761 (50-200 microg/ml) had no effect on mitochondrial functions before anoxia, but had a specific dose-dependent protective effect after anoxia/reoxygenation. When mitochondria were incubated with 200 microg/ml EGb 761, they showed an increase in ADP/O (2.09 +/- 0.14, p < .05) and a decrease in state 4 respiration (-22%) after anoxia/reoxygenation. In EPR spin-trapping measurement, EGb 761 decreased the EPR signal of superoxide anion produced during reoxygenation. In conclusion, EGb 761 specially protects mitochondrial ATP synthesis against anoxia/reoxygenation injury by scavenging the superoxide anion generated by mitochondria.

Functional protection of photoreceptors from light-induced damage by dimethylthiourea and Ginkgo biloba extract.

PURPOSE: To investigate the functional protective effect of a synthetic (dimethylthiourea, DMTU) and a natural antioxidant (Ginkgo biloba extract, EGb 761) against light-induced retinal degeneration. METHODS: Wistar rats were exposed for 24 hours to 1700-lux light after treatment with DMTU or EGb 761. Electroretinograms were recorded before and on day (D)1, D3, D8, D15, D22, and D29 after light exposure. The b-wave amplitude was plotted against log L (ganzfeld luminance), providing the b-wave sensitivity curve. The Naka-Rushton function fitted to the sensitivity curve enabled derivation of the parameters Bmax (saturated amplitude) and K (luminance-inducing Bmax/2). In addition, rats from each group were killed for retinal morphometric analyses. RESULTS: In the untreated group, light exposure caused collapse of the b-wave sensitivity curves. Bmax was reduced by 51% at D1 without subsequent recovery. K increased temporarily, reverting to normal values 8 days later. The outer nuclear layer thicknesses decreased markedly in the superior retina. In the treated groups, light exposure had a weaker effect on sensitivity curves. The values of Bmax were not significantly different from those in the unexposed-untreated group, although K increased temporarily. Retinal morphometry was preserved. CONCLUSIONS: Dimethylthiourea and EGb 761 afford functional protection against light-induced retinal damage.

Ginkgo biloba extract (EGb 761) protects Na,K-ATPase activity during cerebral ischemia in mice.

Neuroprotective drugs such as Ginkgo biloba extract (EGb 761) could prevent the ischemia-induced impairment of the Na,K-ATPase activity. In this study, Na,K-ATPase activity and expression, contents in fatty acids and malondialdehyde, an index of lipoperoxidation, were compared in the ipsilateral (ischemic) and the contralateral (unlesioned) cortices after 1 h of unilateral focal cortices cerebral ischemia in the mouse. EGb 761 (110 mg/kg) was administered daily to half of the animals for 10 days before ischemia. Ischemia significantly reduced Na,K-ATPase activity by about 40% and increased malondialdehyde content; EGb 761 pretreatment abolished these effects. The free radical scavenger properties of EGb 761 are a potential mechanism by which Na,K-ATPase injury and lipoperoxidation are prevented.

Effects of EGb761 and superoxide dismutase in an experimental model of retinopathy generated by intravitreal production of superoxide anion radical.

BACKGROUND: A study was carried out to investigate the effect of two antioxidants--Ginkgo biloba extract (EGb761) and superoxide dismutase (SOD)--in an experimental model of vitreoretinopathy obtained by direct production of oxygen free radicals in the vitreous cavity. METHODS: Twenty-eight pigmented rabbits were used. Vitreoretinopathy was induced by intravitreal injection of 50 microliters of a mixture composed of 40 nmol of xanthine and 0.001 IU of xanthine oxidase. Rabbits were randomly distributed into four groups: Group 1 (n = 8) did not receive any treatment and served as a positive control. Groups 2 (n = 8) and 3 (n = 8) received for 1 month EGb761 given orally at a dose of 100 mg/kg/day, respectively 1 day after and 1 week before induction of retinopathy. Group 4 (n = 4) was treated by three intramuscular injections of 15,000 IU/kg of SOD, 24 h before induction and 24 and 48 h thereafter. Clinical evaluations and electroretinograms (ERG) were repeatedly performed until the animals were killed at day 28. Histological examinations and immunohistological procedures were performed to ascertain the origin and characteristics of the cellular proliferation and to compare vitreoretinal structures in the four groups. RESULTS: Intravitreal injection of xanthine-xanthine oxidase produced a strong inflammatory response with vitreous infiltrates and epiretinal membrane formation, inconstantly associated with retinal detachment. ERG showed a decrease of the a-, b- and c-waves beginning within a few hours after injection. Histologic evaluation found an intravitreal and epiretinal infiltration by leukocytes and epithelial-derived cells, dense vitreoretinal membranes and retinal detachments with occasional neovascularization. In the treated groups (groups 2-4), all clinical, electric and histologic data were significantly improved compared to the control group. However, no difference could be found among the three treated groups. CONCLUSION: This study demonstrates the strong pathologic effects of free radical production on the retina and the close relationships between free radicals, inflammatory pathways and vitreoretinal proliferative disorders. It also confirms the pharmacological interest of prevention by antioxidants and free radical scavengers.

A Ginkgo biloba extract (EGb 761) prevents mitochondrial aging by protecting against oxidative stress.

The effect of aging on indices of oxidative damage in rat mitochondria and the protective effect of the Ginkgo biloba extract EGb 761 was investigated. Mitochondrial DNA from brain and liver of old rats exhibited oxidative damage that is significantly higher than that from young rats. Mitochondrial glutathione is also more oxidized in old than in young rats. Peroxide formation in mitochondria from old animals was higher than in those from young ones. According to morphological parameters (size and complexity), there are two populations of mitochondria. One is composed of large, highly complex mitochondria, and the other population is smaller and less complex. Brain and liver from old animals had a higher proportion of the large, highly complex mitochondria than seen in organs from young animals. Treatment with the Ginkgo biloba extract EGb 761 partially prevented these morphological changes as well as the indices of oxidative damage observed in brain and liver mitochondria from old animals.

Ginkgo biloba extract (EGb 761): inhibitory effect on nitric oxide production in the macrophage cell line RAW 264.7.

The present study was conducted to evaluate the effect of Ginkgo biloba extract (EGb 761) on the synthesis of nitric oxide (NO) induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) in the mouse macrophage cell line RAW 264.7. EGb 761 inhibited nitrite and nitrate production, taken as an index for NO, in a concentration-dependent fashion. The IC50 for inhibition of nitrite production by activated macrophages was about 100 micrograms/mL EGb 761. The inducible NO synthase (iNOS) enzyme activity of cytosolic preparations from activated RAW 264.7 cells was inhibited by treatment with EGb 761. In addition, reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the expression of iNOS mRNA in activated macrophages was suppressed by high concentrations of EGb 761. However, NF-kappa B DNA binding activity induced by activation with LPS/IFN-gamma was not inhibited by EGb 761. These findings indicate that not only does EGb 761 directly act as an NO scavenger but also that it inhibits NO production in LPS/IFN-gamma-activated macrophages by concomitant inhibition of induction of iNOS mRNA and the enzyme activity of iNOS. Thus, EGb 761 may act as a potent inhibitor of NO production under tissue-damaging inflammatory conditions.

Experimental electroretinographic exploration of retinal ischemia: preventive use of free radical scavengers and anti-PAF agents.

Electroretinographic exploration is an effective approach to evaluate retinal function. In order to investigate physiopathological mechanisms and evaluate potentially protective therapies for retinal ischemia, we developed three experimental models: the first two on isolated retina, with ischemia induced by either stopping perfusion or clamping the ophthalmic artery, and the third, in vivo, with ischemia induced by ocular hypertonia. Since free radicals are implicated in the formation of post-ischemic lesions, we evaluated the protective effects of drugs known to be free radical scavengers and of an immunomediator antagonist, an anti-PAF (platelet activating factor) agent.

Direct measurement of free radicals in ischemic/reperfused diabetic rat retina.

Electron paramagnetic resonance (EPR) spectroscopy was used to directly measure free radical generation in ischemic/reperfused diabetic rat retina. Tissue was frozen at 77 degrees K after 90 min ischemia, and 90 min ischemia followed by 1 min, 3 min, 5 min, and 24 hours reperfusion, respectively. After 90 min of ischemia followed by 1 min, 3 min, 5 min, and 24 hours of reperfusion (n = 10 in each group), free radical signal intensity was increased from its diabetic nonischemic control value of 12 +/- 3 arbitrary units to 58 +/- 6 (P < 0.05), 62 +/- 7 (P < 0.05), 32 +/- 5 (P < 0.05), and 14 +/- 4 arbitrary units, respectively. The peak intensity of free radical production was observed after 90 min ischemia followed by 3 min of reperfusion; therefore, this time point was selected to study the retinal free radical production in superoxide dismutase (conjugated with polyethylene glycol, PEG-SOD) and EGb 761 (Ginkgo biloba extract)-treated groups. With 7,500, 15,000, and 30,000 U/liter of SOD, and 25, 50, and 100 mg/kg of EGb 761, a dose-dependent reduction in oxygen free radical production was detected, respectively, which may be responsible for the attenuation of abnormal postischemic function in ischemic and reperfused diabetic retina.

Effects of Ginkgo biloba extract and preconditioning on the diabetic rat myocardium.

Effects of preconditioning and Ginkgo biloba extract (EGb 761) were studied in isolated nondiabetic and diabetic ischaemic and re-perfused rat hearts. Hearts were randomly divided into five groups in both the age-matched non-diabetic and the 8-week streptozotocin-induced diabetic groups: Group I, hearts were subjected to 30 min of global ischaemia followed by 30 min of re-perfusion; Group II, one cycle of preconditioning consisting of 5 min ischaemia and 10 min re-perfusion before the induction of 30 min of ischaemia and 30 min of re-perfusion; Group III, two cycles of preconditioning; Group IV, three cycles; and Group V, four cycles before the onset of 30 min ischaemia followed by 30 min of re-perfusion. Four cycles of ischaemic preconditioning resulted in a reduction of arrhythmias in non-diabetic rats. Thus, in non-diabetics, the incidence of ventricular fibrillation and tachycardia fell from 92% and 100% (no preconditioning) to 33% (p < 0.05) and 42% (p < 0.05), respectively. Four cycles of preconditioning failed to reduce the incidence of re-perfusion arrhythmias in diabetic subjects. Preconditioning reduced the formation of oxygen free radicals measured by electron spin resonance spectroscopy, but the recovery of cardiac function was low in all non-diabetic and diabetic preconditioned groups. EGb 761 at 25 and 50 mg/kg improved cardiac function in non-preconditioned and preconditioned non-diabetic and diabetic hearts. During re-perfusion in the four-cycle preconditioned non-diabetic and diabetic groups, the amount of free radicals was reduced approximately by 50 and 70% using 25 and 50 mg/kg of EGb 761, respectively. EGb 761 improved cardiac function after ischaemia in both non-preconditioned and preconditioned non-diabetic and diabetic rats. Our data suggest that diabetes could abolish the precondition-induced protection.

Peroxynitrite cytotoxicity on bovine retinal pigmented epithelial cells in culture.

Peroxynitrite induced in vitro a dose dependent toxicity on retinal pigmented epithelial (RPE) cells. Cell death was partially mediated by apoptosis as demonstrated by nuclear fragmentation and TdT-mediated dUTP nick-end labeling assay. Peroxynitrite-induced tyrosine nitration was revealed by immunocytochemistry, both in the cytoplasm and in the nucleus of the cells. Nitration was not observed in RPE cells, producing nitric oxide (NO) after stimulation by lipopolysacharide and interferon-g (IFN-gamma), suggesting that peroxynitrite was not formed in vitro in such conditions. Peroxynitrite could be responsible for the retinal damages observed in pathological conditions in which NO has been demonstrated to be involved. In this context, EGb761, identified as a free radical scavenger, was showed herein to protect RPE cells against peroxynitrite injury.

Gingko biloba extract EGb 761 is a suppresser of AP-1 transcription factor stimulated by phorbol 12-myristate 13-acetate.

Transcription factors are activated in response to a wide variety of extracellular stimuli. The present study investigated which step Gingko biloba extract (EGb 761), a flavonoid phytochemical rich preparation can affect signal transduction during AP-1 activation. Kinetic experiments with human Jurkat T-cell lines demonstrate that it takes 1 to 1.5 h to activate AP-1 DNA binding activity. These data indicate that c-fos mRNA is expressed and then AP-1 DNA binding activity is activated. Pretreatment of Jurkat T cells with 10 micrograms/ml EGb 761 suppresses AP-1 DNA activation and c-fos mRNA expression. These results suggest that the step in the signal transduction pathway for AP-1 activation that is inhibited by EGb 761 is upstream to c-fos mRNA expression.

Effects of Ginkgo biloba extract (EGb 761) on cochlear vasculature in the guinea pig: morphometric measurements and laser Doppler flowmetry.

Ginkgo biloba extract (EGb 761) was administered orally for 4 or 6 weeks to healthy adult guinea pigs. Animals were then decapitated under deep ketamine anesthesia. Post-mortem morphometric measurements of cochlear vessels in the spiral lamina revealed a vasodilating effect of the extract in four of ten animals following 6 weeks of treatment. In vivo testing of the effect of 4 or 6 weeks of treatment with EGb 761 was monitored with laser Doppler flowmetry of the cochlear blood flow under pathological conditions. Results demonstrated that EGb 761 partly counteracted sodium salicylate-induced decreases in cochlear blood flow (CBF) and enhanced CBF increases induced by hypoxia. These findings indicate that EGb 761 may help to improve oxygenation in cochleas with compromised blood flow.

Ginkgo biloba extract (EGb 761) protects human low density lipoproteins against oxidative modification mediated by copper.

The antioxidant effects of Ginkgo biloba extract (EGb 761) on copper-mediated human low density lipoprotein (LDL) oxidative modification were evaluated by several techniques. Human LDL (0.5 mg/ml) in phosphate buffered saline, pH 7.4, was incubated with 10 microM cupric sulfate at 37 degrees C under air for 8 hours and 24 hours in the presence of varying concentrations of EGb 761. Increases in LDL apoB carbonylation, lipid peroxidation, apoB electrophoretic mobility and LDL fluorescence were all inhibited when the incubation mixture contained EGb 761 at concentrations less than 100 micrograms/ml. This inhibition was EGb 761-concentration-dependent. Thus, EGb 761 has powerful antioxidant effects on copper-mediated LDL oxidative modification.

Peroxyl radical scavenging activity of Ginkgo biloba extract EGb 761.

Antioxidant mechanisms have been proposed to underlie the beneficial pharmacological effects of EGb 761, an extract from Ginkgo biloba leaves used for treating peripheral vascular diseases and cerebrovascular insufficiency in the elderly. In vitro evidence has been reported that EGb 761 scavenges various reactive oxygen species, i.e. nitric oxide, and the superoxide, hydroxyl, and oxoferryl radicals. However, the ability of EGb 761 to scavenge peroxyl radicals (reactive species mainly involved in the propagation step of lipid peroxidation) has not been investigated. To characterize further the antioxidant action of EGb 761, we measured the protective effects of EGb 761 during: (1) the oxidation of B-phycoerythrin by peroxyl radicals generated in aqueous solution by 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH); and (2) the reaction of luminol or cis-parinaric acid with peroxyl radicals generated from 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) in liposomes or in human low density lipoprotein (LDL), respectively. To evaluate the peroxyl radical scavenging activity of EGb 761 in a more physiologically relevant model of damage to lipid-containing systems, we also analyzed the effect of the extract on the oxidation of human LDL exposed to the azo-initiators in terms of: (1) accumulation of cholesterol linoleate ester hydroperoxides, (2) depletion of alpha-tocopherol and beta-carotene, and (3) changes in intrinsic tryptophan fluorescence. EGb 761 afforded protection against oxidative damage in all the systems we analyzed; thus, it is an efficient scavenger of peroxyl radicals. This result extends the oxygen radical scavenging properties of the extract and supports the hypothesis of an antioxidant therapeutic action of EGb 761.

EGb 761 and the recovery of ion imbalance in ischemic reperfused diabetic rat retina.

We studied the effects of a free-radical scavenger, EGb 761, on electrolyte shifts (Na+, Ca2+, and K+) induced by ischemia and reperfusion in the retinas obtained from streptozotocin-induced diabetic rats. Eyes were subjected to 90 min ischemia followed by 24 h of reperfusion by clamping and releasing the central retinal artery. Ten days before the induction of ischemia and reperfusion, diabetic rats received a daily dose of 25, 50, and 100 mg/kg p.o. of EGb 761, respectively (n = 12 in each group). In the drug-free diabetic control group, 90 min ischemia followed by 24 h of reperfusion resulted in an increase in retinal Na+ and Ca2+ (measured by atomic absorption spectrophotometry) compared to nonischemic control values of 73 +/- 4 and 2.6 +/- 0.3 mumol/g dry weight to 113 +/- 5 (p < 0.05) and 5.3 +/- 0.3 mumol/g dry weight (p < 0.05), respectively. Tissue K+ content was significantly reduced compared to its nonischemic diabetic control value of 268 +/- 7 to 213 +/- 6 mumol/g dry weight (p < 0.05). EGb 761 dose-dependently reduced reperfusion-induced ion imbalance, improving the recovery of ion content in diabetic rat retina. EGb 761 did not reduce blood glucose in streptozotocin-induced diabetic rats. Therefore we may conclude that these protective effects of EGb 761 are independent of blood glucose content or of the severity of diabetes and protect against electrolyte shifts directly in retinal cells.