Ginkgo biloba extract EGb 761 increases stress resistance and extends life span of Caenorhabditis elegans.

EGb 761, a standardized extract of Ginkgo biloba leaves, has been used in clinical trials for its beneficial effects on brain functions. In mammals, EGb 761 has been shown to enhance cognition, stress resistance, and longevity, but its molecular and cellular mechanisms are not known. In the present investigation, we used the model organism Caenorhabditis elegans to evaluate pharmacological effects of EGb 761 on aging. We tested the theory that EGb 761 augments the natural antioxidant system of C elegans, and thus increases stress resistance and longevity. We found that treatment of the wild-type worms with EGb 761 extended their median life span by 8%. Amongst several purified components of EGb 761, the flavonoid tamarixetin showed the most dramatic effect: it extended the median life span by 25%. Furthermore, EGb 761 increased the wild type's resistance to acute oxidative and thermal stress by 33% and 25%, respectively. Treatment of the prematurely aging mutant worms mev-1 with EGb 761 increased their resistance to acute oxidative and thermal stress by 33% and 11%, respectively. It appears that oxidative stress, a major determinant of life span, as well as other types of stress, can be successfully counteracted by the Ginlkgo biloba extract EGb 761.     

Induction of glutathione synthesis in human keratinocytes by Ginkgo biloba extract (EGb761).

The objective of the present study was to characterize the action of Ginkgo biloba extract (EGb761) and its sub-fractions on glutathione homeostasis in a human keratinocyte cell culture model. Cells were incubated with EGb761, its purified flavonoid (quercetin, kaempferol, rutin) or terpenoids (gingkolides A, B, C, J, bilobalide) constituents or the vehicle for up to 72 hours. Incubation of keratinocytes with the purified flavonoids or terpenoids did not affect cellular GSH levels. However, EGb761 treatment (up to 200 microg/ml) resulted in a dose-dependent increase of cellular GSH. Western blot analysis of extracts from cells treated with EGb761 revealed increased levels of the catalytic subunit of gamma-glutamylcysteinyl synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis. The abundance of mRNA for the catalytic subunit (assayed by RT-PCR) was also increased by the treatment with EGb761. Increased levels of cellular GSH by EGb761 were also observed in other cell lines including those from human bladder and liver as well as in murine macrophages indicating that the induction of gamma-GCS mRNA, protein and GSH may be an ubiquitous effect of EGb761 in mammalian cells.     

Ginkgo biloba extract (EGb 761) prevents the ischemic brain injury-induced decrease in parvalbumin expression

Ginkgo biloba extract (EGb 761) exerts a neuroprotective effect against ischemic brain injury through an anti-apoptotic mechanism. Parvalbumin is a calcium buffering protein that plays an important role in modulating intracellular calcium concentration and regulating apoptotic cell death. The aim of this study was to investigate whether EGb 761 affects parvalbumin expression in cerebral ischemic injury. Adult male Sprague-Dawley rats were treated with vehicle or EGb 761 (100 mg/kg) prior to middle cerebral artery occlusion (MCAO) and cerebral cortex tissues were collected 24 h after MCAO. A proteomic approach revealed a reduction in parvalbumin expression in the vehicle-treated animals, whereas EGb 761 pretreatment attenuates the ischemic injury-induced decrease in parvalbumin expression. RT-PCR and Western blot analyses clearly confirmed the fact that EGb 761 prevents the injury-induced decrease in parvalbumin. Moreover, the results of immunohistochemical staining showed that the number of parvalbumin-positive cells was lower in vehicle-treated animals than in sham-operated animals, and EGb 761 averted this decrease. Thus, these results suggest that the maintenance of parvalbumin expression is associated with the neuroprotective function of EGb 761 against neuronal damage induced by ischemia.     

Specific memory effects of Ginkgo biloba extract EGb 761 in middle-aged healthy volunteers

Introduction

Recent reviews showed that Ginkgo biloba extract EGb 761¹ is effective to enhance performance in patients with cognitive impairment (e.g., dementia). The aim of this study was to investigate the effects of EGb 761 on memory and the specificity of such effects on distinct memory functions in middle-aged healthy volunteers.

Methods

A total of 188 healthy subjects aged 45-56 years were randomised to receive EGb 761 (240 mg once daily) or placebo for 6 weeks. Outcome measures were the change in memory performance in a demanding standardised free recall paradigm (list of appointments) and a less demanding standardised recognition test (driving-route). Based on previous findings we predicted superiority of EGb 761 in recall testing. Specificity in effects was assessed by separating immediate vs. delayed and quantitative vs. qualitative free recall measures.

Results

After 6 weeks, EGb 761-treated subjects improved significantly in quantity of recall, i.e., the number of correctly recalled appointments (drug-placebo differences: p = 0.038 for immediate and p = 0.008 for delayed recall). Effects on qualitative recall performance (ratio of false to correct items) were similar (drug-placebo differences: p = 0.092 for immediate and p = 0.010 for delayed recall). No superiority of Ginkgo was evident in another everyday memory test which asked for recognition of a driving route (drug-placebo differences: p > 0.10). The incidence of adverse events was low and not significantly different between treatment groups.

Discussion

EGb 761 (240 mg once daily) improves free recall of appointments in middle-aged healthy volunteers, which requires high demands on self-initiated retrieval of learned material. This function is known to be sensitive to normal aging, i.e., reduced in healthy middle-aged subjects. No effects are seen in a less demanding everyday memory task which does not tap this critical function. This ties in with previous studies which found specific patterns of benefit from EGb 761 in demanding cognitive tasks.     

Ginkgo biloba extract EGb 761 protects against mitochondrial aging in the brain and in the liver.

According to the free radical theory of aging, oxygen-derived free radicals causes the age-associated impairment at the cellular and tissue levels. The mitochondrial theory of aging points to mitochondria, and specially mitochondrial DNA, as the major targets of free radical attack upon aging. Thus, oxidative damage to mtDNA accumulate with age in human and rodent tissues and also is inversely related to maximum life span of mammals. Mitochondrial deficits, such as a decrease in mitochondrial membrane potential, occur upon aging due to oxidative damage. The age-related mitochondrial oxidative stress may be prevented by late onset administration of certain antioxidants, such as Ginkgo biloba extract EGb 761. These antioxidants may also delay the physiological impairment associated with aging.     

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

Disturbances of Na,K-ATPase activity are implicated in the pathophysiology of cerebral ischemia. Previous experiments have shown that EGb 761 protects NaK-ATPase activity against one hour of cerebral ischemia. In the brain however, the 3 isoenzymes responsible for Na,K-ATPase activity may be differentially affected by various times of ischemia. In the present study, we investigated the effect of a longer period of ischemia, and the protection provided by a pre-treatment with EGb 761 on each of the 3 cerebral NaK-ATPase isoenzymes. In control and EGb 761 pre-treated mice exposed to a 6 hr unilateral occlusion of the middle cerebral artery, Na,K-ATPase activity was decreased by 60% and lipid peroxidation was increased by 40% in the ipsilateral (ischemic) cortex compared to the contralateral one. In parallel, membrane integrity was altered. The alteration of NaK-ATPase activity, as a whole, resulted from a decrease in the activity of the 3 isoenzymes. The two isoenzymes of high ouabain affinity however, had their affinities decreased while the sensitivity of the lowest affinity isoenzyme was increased. Pre-treatment with EGb 761 abolished the differences observed between ipsi- and contralateral cortex, with the exception of the change in ouabain affinity of the low affinity isoenzyme. Ischemia also induced changes in Na,K-ATPase isoenzyme ouabain affinities in the contralateral cortex that where not prevented by EGb 761.     

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.     

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.     

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.     

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.     

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.     

UV-C irradiation-induced peroxidative degradation of microsomal fatty acids and proteins: protection by an extract of Ginkgo biloba (EGb 761).

After exposure of rat liver microsomes to UV-C irradiation, analysis of membrane fatty acids by gas chromatography confirmed that EGb 761, a drug containing a dosed and standardized extract of Ginkgo biloba, provides effective protection against free radical attack in vitro. This analysis, coupled with thiobarbituric acid (TBA) reaction, permitted qualitative and overall quantitative evaluation of radical-induced damage to polyunsaturated fatty acids (PUFA), as well as evidence of the antioxidant properties of the Ginkgo biloba extract. Assay of thiobarbituric acid reactive substances (TBARS) showed a correlation between TBARS concentration and the state of degradation of the polyunsaturated fatty acids. Mannitol (5.5 mM) did not prevent degradation of microsomal PUFA or malondialdehyde (MDA) production, nor did it prevent polymerization of membrane proteins. Low doses of EGb 761 were found to provide efficient protection of membrane PUFA regardless of individual susceptibility to peroxidation. This protection was accompanied by a decrease in the production of TBARS. EGb 761 also protected membrane proteins from the irreversible polymerization induced by these degradation products, but did not appear to prevent thiols oxidation into disulfide bonds.     

EGb761, a Ginkgo biloba extract, is effective against atherosclerosis in vitro, and in a rat model of type 2 diabetes

Background

EGb761, a standardized Ginkgo biloba extract, has antioxidant and antiplatelet aggregation and thus might protect against atherosclerosis. However, molecular and functional properties of EGb761 and its major subcomponents have not been well characterized. We investigated the effect of EGb761 and its major subcomponents (bilobalide, kaemferol, and quercetin) on preventing atherosclerosis in vitro, and in a rat model of type 2 diabetes.

Methods and Results

EGb761 (100 and 200 mg/kg) or normal saline (control) were administered to Otsuka Long-Evans Tokushima Fatty rats, an obese insulin-resistant rat model, for 6 weeks (from 3 weeks before to 3 weeks after carotid artery injury). Immunohistochemical staining was performed to investigate cell proliferation and apoptosis in the injured arteries. Cell migration, caspase-3 activity and DNA fragmentation, monocyte adhesion, and ICAM-1/VCAM-1 levels were explored in vitro. Treatment with EGb761 dose-dependently reduced intima-media ratio, proliferation of vascular smooth muscle cells (VSMCs) and induced greater apoptosis than the controls. Proliferation and migration of VSMCs in vitro were also decreased by the treatment of EGb761. Glucose homeostasis and circulating adiponectin levels were improved, and plasma hsCRP concentrations were decreased in the treatment groups. Caspase-3 activity and DNA fragmentation increased while monocyte adhesion and ICAM-1/VCAM-1 levels decreased significantly. Among subcomponents of EGb761, kaemferol and quercetin reduced VSMC migration and increased caspase activity.

Conclusions

EGb761 has a protective role in the development of atherosclerosis and is a potential therapeutic agent for preventing atherosclerosis.     

Inhibitions of vascular endothelial growth factor expression and foam cell formation by EGb 761, a special extract of Ginkgo biloba,in oxidatively modified low-density lipoprotein-induced human THP-1 monocytes cells

It has been reported that oxidatively modified low-density lipoprotein (Ox-LDL) involvement with vascular endothelial growth factor (VEGF) and foam cell formation play an important role in atherosclerosis (AS). Protective effects of Ginkgo biloba extract (EGb 761) have been identified for some cardiovascular and neurological disorders. The aim of this study was to investigate whether Ox-LDL regulates VEGF expression in human THP-1 monocytes, as well as the effect of EGb 761 on VEGF expression and the formation of foam cells. After exposure to Ox-LDL alone or in combination with EGb 761 for up to 48 h, cell viability was measured using the MTT assay. VEGF protein content in the supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA). VEGF mRNA was determined by real-time PCR. To determine the effect of EGb 761 on foam cell formation, an Ox-LDL-induced foam cell model was used. Ox-LDL inhibited the growth of THP-1 cells and EGb 761 increased the cell survival rate. Ox-LDL markedly increased VEGF expression in THP-1 cells in a time- and concentration-dependent manner, which was significantly suppressed by EGb 761. EGb 761 also inhibited monocyte/macrophage-derived foam cell formation. These results suggest that Ox-LDL is involved in the development of human AS through VEGF induction in monocytes, and that EGb 761 prevents in vitro atherogenesis, probably via downregulation of VEGF expression in monocytes and inhibition of monocyte/macrophage-derived foam cell formation. The findings suggest a mechanism for the in vivo anti-AS effect of EGb 761 and support its potential clinical use in AS.     

Effect of the extract of Ginkgo biloba (EGb 761) on the circulating and cellular profiles of polyunsaturated fatty acids: correlation with the anti-oxidant properties of the extract

Ginkgo biloba extract (EGb 761) has beneficial effects on cognitive functions in aging patients, and on various pathologies, including cardiovascular diseases. Although the extract is known to have antioxidant properties and improve membrane fluidity, the cellular mechanisms underlying these effects have not been determined. Here, we examined the in vivo effects of EGb 761 on circulating and cellular lipids. EGb 761 treatment induced significant increases in the levels of circulating polyunsaturated fatty acids (PUFAs), and a decrease in the saturation index SI (saturated/polyunsaturated species). Plasma triglycerides and cholesterol were not affected, while phospholipids were slightly increased at the higher dose of EGb 761. EGb 761 treatment also induced a significant increase in the levels of PUFAs in erythrocyte membranes, especially for the eicosapentaenoic acid (EPA omega 3), and a decrease in the saturation index. Moreover, the response of erythrocytes to oxidative stress was improved in EGb 761-treated animals (H(2)O(2)-induced cell lysis decreased by 50%). Considering that PUFAs are known to improve membrane fluidity and response to oxidative damage, and are precursors of signaling molecules such as prostaglandins, the effects of EGb 761 on circulating and cellular PUFAs may explain some of the pharmacological properties of Ginkgo biloba.