Ginkgo biloba extract (Egb 761) inhibits beta-amyloid production by lowering free cholesterol levels

Ginkgo biloba extract (EGb 761) can improve cognitive function in patients with Alzheimer's disease, but the molecular mechanisms underlying this effect remain undefined. Because free cholesterol may be involved in the production of β-amyloid precursor protein and amyloid β-peptide, key events in the development of Alzheimer's disease, we examined EGb 761 in relation to cholesterol and amyloidogenesis. In aging rats, EGb 761 treatment lowered circulating free cholesterol and inhibited the production of brain β-amyloid precursor protein and amyloid β-peptide. Exposure of PC12 cells to EGb 761 decreased the processing of β-amyloid precursor protein and abolished cholesterol-induced overproduction of this protein. Exposure of human NT2 cells to EGb 761 decreased free cholesterol influx and increased free cholesterol efflux. Our findings indicate that free circulating and intracellular cholesterol levels affect the processing of β-amyloid precursor protein and amyloidogenesis. Our findings also provide the first demonstration that EGb 761 can influence these mechanisms.     

EGb761 Provides a Protective Effect against Aβ1-42 Oligomer-Induced Cell Damage and Blood-Brain Barrier Disruption in an In Vitro bEnd.3 Endothelial Model

Alzheimer’s disease (AD) is the most common form of senile dementia which is characterized by abnormal amyloid beta (Aβ) accumulation and deposition in brain parenchyma and cerebral capillaries, and leads to blood-brain barrier (BBB) disruption. Despite great progress in understanding the etiology of AD, the underlying pathogenic mechanism of BBB damage is still unclear, and no effective treatment has been devised. The standard Ginkgo biloba extract EGb761 has been widely used as a potential cognitive enhancer for the treatment of AD. However, the cellular mechanism underlying the effect remain to be clarified. In this study, we employed an immortalized endothelial cell line (bEnd.3) and incubation of Aβ_1–42 oligomer, to mimic a monolayer BBB model under conditions found in the AD brain. We investigated the effect of EGb761 on BBB and found that Aβ_1–42 oligomer-induced cell injury, apoptosis, and generation of intracellular reactive oxygen species (ROS), were attenuated by treatment with EGb761. Moreover, treatment of the cells with EGb761 decreased BBB permeability and increased tight junction scaffold protein levels including ZO-1, Claudin-5 and Occludin. We also found that the Aβ_1–42 oligomer-induced upregulation of the receptor for advanced glycation end-products (RAGE), which mediates Aβ cytotoxicity and plays an essential role in AD progression, was significantly decreased by treatment with EGb761. To our knowledge, we provide the first direct in vitro evidence of an effect of EGb761 on the brain endothelium exposed to Aβ_1–42 oligomer, and on the expression of tight junction (TJ) scaffold proteins and RAGE. Our results provide a new insight into a possible mechanism of action of EGb761. This study provides a rational basis for the therapeutic application of EGb761 in the treatment of AD.     

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.     

EGb 761 is a neuroprotective agent against beta-amyloid toxicity.

Beta-amyloid (Abeta) deposition likely plays a causal role in the lesions that occur in Alzheimer's disease (AD). The Ginkgo biloba extract EGb 761 is widely prescribed in the treatment of cognitive deficits that are associated with normal and pathological brain aging such as AD. We have investigated here the potential effectiveness of EGb 761 against cell death produced by Abeta fragments on primary cultures of hippocampal cells, these cells being severely damaged in AD. A co-treatment with EGb 761 protected cells against toxicity induced by Abeta fragments in a concentration dependent manner. The effect of EGb 761 was even significant if added up to 8 hr to cells and was shared by its flavonoid fraction CP 205, whereas the terpenes bilobalide and ginkgolide B were ineffective. EGb 761 also displayed protective effects against toxicity produced by either H2O2 or nitric oxide, two neurotoxic agents that possibly mediate Abeta toxicity. Moreover, EGb 761, and to a lesser extent CP 205, completely blocked Abeta-induced events, such as reactive oxygen species accumulation and apoptosis. Taken together, these results and those obtained by other groups highlight the neuroprotective abilities of EGb 761 against dysfunction and death of neurons caused by Abeta deposits.     

Induction of heme oxygenase 1 by Ginkgo biloba in neuronal cultures and potential implications in ischemia.

Ginkgo biloba extract (EGb 761) is a standardized extract originating in traditional Chinese medicine. Ginkgo biloba dried leaves have been used for centuries to treat various neurological conditions. The constituents from the extract are likely to have synergistic effects that have been shown to be protective against oxidative stress injury. However, the cellular mechanisms of protection afforded by Ginkgo biloba are still unclear. The cascade leading to neuronal cell death in acute and chronic neurodegenerative conditions, such as cerebral ischemia and Alzheimer's disease, has been postulated to be mediated by free radical damage. We tested the hypothesis that the neuroprotective action of EGb 761 could be due partially to an induction of heme oxygenase I (HO1). We and others have previously reported that modulation of HO total activity may well have direct physiological implications in stroke and in Alzheimer's disease. Heme oxygenase acts as an antioxidant enzyme by degrading heme into iron, carbon monoxide, and biliverdin which is rapidly converted into bilirubin. Through the use of primary neuronal cultures, we demonstrated that EGb 761 induces HO1 in a dose-dependent manner (0, 10, 50, 100 and 500 microg/ml) and time-dependent manner with a maximal induction at 8 hr. We are proposing that several of the protective effects of EGb 761 in ischemia could be mediated through beneficial actions of heme degradation and its metabolites.     

Protective effects of Ginkgo biloba extract (EGb761) and its constituents quercetin and ginkgolide B against β-amyloid peptide-induced toxicity in SH-SY5Y cells

Ginkgo biloba extract EGb761 has been shown to protect against β-amyloid peptide (Aβ)-induced neurotoxicity but the specific mechanisms remain unclear. In the present study, effects of EGb761 and two of its constituents, quercetin and ginkgolide B, on the cytotoxic action of Aβ (1-42) were tested with human neuroblastoma SH-SY5Y cells. We found that EGb761 was able to block Aβ (1-42)-induced cell apoptosis, reactive oxygen species (ROS) accumulation, mitochondrial dysfunction and activation of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt signaling pathways. Both quercetin and ginkgolide B may be involved in the inhibitory effects of EGb761 on JNK, ERK1/2 and Akt signaling pathways. Ginkgolide B also helped to improve mitochondrial functions but quercetin failed to show this effect. Additional experiments suggest that, protective effects of EGb761 against Aβ toxicity may be associated with its antioxidant and platelet activating factor (PAF) antagonist activities. Quercetin but not ginkgolide B is one of the constituents responsible for the antioxidant action of EGb761. Both quercetin and ginkgolide B may be involved in the PAF antagonist activity of EGb761. Overall, actions of individual EGb761 components provide further insights into direct mechanisms underlying the neuroprotective effects of EGb761.     

Effect of a short- and long-term treatment with Ginkgo biloba extract on amyloid precursor protein levels in a transgenic mouse model relevant to Alzheimer's disease

Several clinical trials have reported beneficial effects of the Ginkgo biloba extract EGb761 in the prevention and therapy of cognitive disorders including Alzheimer’s disease (AD). The aim of the present long-term feeding trial was to study the impact of dietary EGb761 on Amyloid precursor protein (APP) metabolism in mice transgenic for human APP (Tg2576). Tg2576 mice were fed diets with and without EGb761 (300 mg/kg diet) for 1 and 16 months, respectively. Long-term treatment (16 months) with EGb761 significantly lowered human APP protein levels by ∼50% as compared to controls in the cortex but not in the hippocampus. However, APP levels were not affected by EGb761 in young mice. Current data indicate that APP seems to be an important molecular target of EGb761 in relation to the duration of the Ginkgo biloba treatment and/or the age of the animals. Potential neuroprotective properties of EGb761 may be, at least partly, related to its APP lowering activity.     

Mechanisms of A beta mediated neurodegeneration in Alzheimer's disease

Development of a comprehensive therapeutic treatment for the neurodegenerative Alzheimer's disease (AD) is limited by our understanding of the underlying biochemical mechanisms that drive neuronal failure. Numerous dysfunctional mechanisms have been described in AD, ranging from protein aggregation and oxidative stress to biometal dyshomeostasis and mitochondrial failure. In this review we discuss the critical role of amyloid-beta (A beta) in some of these potential mechanisms of neurodegeneration. The 39-43 amino acid A beta peptide has attracted intense research focus since it was identified as a major constituent of the amyloid deposits that characterise the AD brain, and it is now widely recognised as central to the development of AD. Familial forms of AD involve mutations that lead directly to altered A beta production from the amyloid-beta A4 precursor protein, and the degree of AD severity correlates with specific pools of A beta within the brain. A beta contributes directly to oxidative stress, mitochondrial dysfunction, impaired synaptic transmission, the disruption of membrane integrity, and impaired axonal transport. Further study of the mechanisms of A beta mediated neurodegeneration will considerably improve our understanding of AD, and may provide fundamental insights needed for the development of more effective therapeutic strategies.     

Dosage effects of EGb761 on hydrogen peroxide-induced cell death in SH-SY5Y cells

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, is one of the most popular herbal supplements, taken for its multivalent properties. In this study, dosage effects of EGb761 on hydrogen peroxide (H₂O₂)-induced apoptosis of human neuroblastoma SH-SY5Y cells were investigated. It was found that H₂O₂-induced apoptotic cell death in SH-SY5Y cells, which was revealed in DNA fragmentation, mitochondrial membrane potential depolarization, and activation of Akt, c-Jun N-terminal kinases (JNK) and caspase 3. Low doses of EGb761 (50–100 μg/ml) inhibited H₂O₂-induced cell apoptosis via inactivation of Akt, JNK and caspase 3 while high doses of EGb761 (250–500 μg/ml) enhanced H₂O₂ toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments revealed that H₂O₂ decreased intracellular GSH content, which was also inhibited by low concentrations of EGb761 but enhanced after high concentrations of EGb761 treatment. This further suggests to us that dosage effects of EGb761 on apoptotic signaling proteins may be correlated with regulation of cell redox state. Therefore, treatment dosage may be one of the vital factors that determine the specific action of EGb761 on oxidative stress-induced cell apoptosis. To understand the mechanisms of dosage effects of EGb761 may have important clinical implications.     

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.     

EGb 761 (Ginkgo biloba) protects cochlear hair cells against ototoxicity induced by gentamicin via reducing reactive oxygen species and nitric oxide-related apoptosis

Gentamicin is an effective and powerful antibiotic. Extended use or excessive dosages of which can result in irreversible damage to the inner ear. The development of otoprotective strategies is a primary and urgent goal in research of gentamicin ototoxicity. Ginkgo biloba leaves and their extracts are among the most widely used herbal products and/or dietary supplements in the world. We investigated the protection of EGb 761 (a standardized preparation of EGb) on gentamicin ototoxicity and the involvement of reactive oxygen species (ROS) and nitric oxide (NO)-related mechanisms using in vitro organ cultures and an in vivo animal model. Gentamicin induced hair cell damage in cochlear cultures that could be prevented by EGb 761. EGb 761 also significantly reduced gentamicin-induced ROS and NO production. Furthermore, EGb 761 inhibited cellular apoptosis in cultured cochleae treated with gentamicin. In guinea pigs with gentamicin application onto the round window membrane, the mean auditory brain stem response threshold, ratio of cochlear hair cell damage and apoptosis were significantly elevated compared with those in the control group, and this could be prevented by oral administration of EGb 761. Individual EGb 761 components quercetin, bilobalide, ginkgolide A and ginkgolide B, but not kaempferol, significantly prevented gentamicin-induced hair cell damage. These results indicate that EGb 761 has a protective effect against gentamicin ototoxicity through a reduction in the formation of ROS and NO and subsequent inhibition of hair cell apoptosis in the cochlea.     

Biphasic effect of EGb761 on simulated ischemia-induced rat BMSC survival in vitro and in vivo

AimsThe standardized extract from the leaves of Ginkgo biloba (EGb761) is applied as a phyto-pharmacon in therapy of diverse cardiovascular disorders. However, the effects of EGb761 on bone-marrow mesenchymal stem cells (BMSCs) transplanted into the ischemic myocardium currently remain uncertain. In this study, the dosage-effects of EGb761 on BMSC survival in vitro and in vivo were investigated.Main methodsThe ischemic microenvironment of rat BMSCs was simulated by hypoxia/serum deprivation (SD) and the rat myocardial infarction model was established. The rat BMSCs were cultured under hypoxia/SD or transplanted into the animal ischemic heart. The BMSC apoptosis was determined by FACS and TUNEL assay. Each apoptotic signal molecule's activity was assayed by immunoblot.Key findingsEGb761 showed a biphasic effect on the hypoxia/SD-induced BMSC apoptosis. Low concentration of EGb761 (10–100 μg/ml) aggravated hypoxia/SD-induced apoptosis via Akt inactivation and an enhancement of caspase-9 and caspase-3 expressions, whereas high concentration of EGb761 (500–2000 μg/ml) significantly prevented hypoxia/SD-induced BMSC apoptosis via the activated Akt and the inactivated caspase-9 and caspase-3. The animal study also indicated that the apoptotic index (AI) in the high concentration of EGb761 group was significantly lower than the low concentration of EGb761 group.SignificanceThe biphasic effect of EGb761 is closely related to the PI3K-Akt and caspase-9 signaling pathways. The therapeutic concentration of EGb761 may be one of the vital factors determining the specific action of EGb761 on cell apoptosis. It is of significant clinical implication to investigate the mechanisms of the biphasic effect of EGb761.     

Clearing amyloid through the blood-brain barrier

According to the amyloid hypothesis, accumulation of amyloid beta-peptide (A beta) in the brain is the primary pathogenic event in Alzheimer's disease (AD). Recent evidence indicates that A beta within the intravascular space is linked to A beta deposited in the brain suggesting that transport of A beta between the brain, blood and cerebrospinal fluid, and across the blood-brain barrier, regulates brain A beta. Thus, understanding A beta exchanges between brain and blood, and vice versa, and developing transport-based systemic A beta-lowering strategies may provide new important insights into pathogenesis and therapeutic control of AD.     

Ginkgo biloba extract protects human melanocytes from H2O2‐induced oxidative stress by activating Nrf2

Vitiligo is a common skin depigmenting disorder characterized by the loss of functional melanocytes. Its pathogenesis is complicated and oxidative stress plays a critical role in the development of vitiligo. Thus, antioxidant therapy is a promising therapeutic strategy to prevent or even reverse the progression of depigmentation. Ginkgo biloba extract EGb761 has been confirmed to have protective effects on neurons against oxidative stress. Notably, several clinical trials have shown that patients with stable vitiligo achieved repigmentation after taking EGb761. However, the exact mechanism underlying the protective effects of EGb761 on melanocytes against oxidative stress has not been fully elucidated. In the present study, we found that EGb761 effectively protected melanocytes against oxidative stress‐induced apoptosis and alleviated the excessive accumulation of reactive oxygen species (ROS) and lipid peroxidation by enhancing the activity of antioxidative enzymes. Furthermore, the antioxidative effect of EGb761 was achieved by activating Nrf2 and its downstream antioxidative genes. In addition, interfering Nrf2 with siRNA abolished the protective effects of EGb761 on melanocytes against oxidative damage. In conclusion, our study proves that EGb761 could protect melanocytes from H₂O₂‐induced oxidative stress by activating Nrf2. Therefore, EGb761 is supposed to be a potential therapeutic agent for vitiligo.     

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.     

Alzheimer's disease therapeutics: new approaches to an ageing problem

Abnormal proteinaceous deposits are found in the brain of patients with many different neurodegenerative diseases. In many of these diseases, the production of the deposits is probably associated with disease pathogenesis. In Alzheimer's disease (AD), the amyloid protein (A beta), is produced by the action of enzymes known as secretases, which cleave the beta-amyloid protein precursor. A beta is secreted from cells in the brain, after which it oligomerizes and is deposited in the extracellular compartment of the brain to form amyloid plaques and amyloid angiopathy. Targeting the production of A beta and its aggregation is now a key strategy in the development of novel therapeutic agents for the treatment of AD. This review examines the potential of immunization strategies, cholesterol-lowering drugs, protease inhibitors and nicotinic drugs for the treatment of AD.     

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.