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.     

Evidence that gingko biloba extract does not inhibit MAO A and B in living human brain

Extracts of Ginkgo biloba have been reported to reversibly inhibit both monoamine oxidase (MAO) A and B in rat brain in vitro leading to speculation that MAO inhibition may contribute to some of its central nervous system effects. Here we have used positron emission tomography (PET) to measure the effects of Ginkgo biloba on human brain MAO A and B in 10 subjects treated for 1 month with 120 mg/day of the Ginkgo biloba extract EGb 761, using [11C]clorgyline and [11C]L-deprenyl-D2 to measure MAO A and B respectively. A three-compartment model was used to calculate the plasma to brain transfer constant K1 which is related to blood flow, and lambdak3, a model term which is a function of the concentration of catalytically active MAO molecules. Ginkgo biloba administration did not produce significant changes in brain MAO A or MAO B suggesting that mechanisms other than MAO inhibition need to be considered as mediating some of its CNS effects.     

Natural substances and Alzheimer's disease: from preclinical studies to evidence based medicine

Over the last 10 years, the potential therapeutic effects of nutraceuticals to prevent or delay Alzheimer's disease were proposed. Among dietary antioxidants curcumin, Ginkgo biloba and carnitines were extensively studied for their neuroprotective effects. The rationale for this alternative therapeutic approach was based on several preclinical studies which suggested the neuroprotective effects for curcumin, Ginkgo biloba and acetyl-l-carnitine due to either a free radical scavenging activity or the inhibition of pro-inflammatory pathways or the potentiation of the cell stress response. However, although these are interesting premises, clinical studies were not able to demonstrate significant beneficial effects of curcumin, Ginkgo biloba and acetyl-l-carnitine in improving cognitive functions in Alzheimer's disease patients. The aim of this review is to summarize the main pharmacologic features of curcumin, Ginkgo biloba and carnitines as well as to underlie the main outcomes reached by clinical studies designed to demonstrate the efficacy of these natural substances in Alzheimer's disease patients. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Evidence for toxic effects of alkylphenols from Ginkgo biloba in the hen's egg test (HET).

Extracts from the leaves of the Gingko tree (Ginkgo biloba L.) are therapeutically used for the treatment of peripheral and cerebral vascular disorders as well as multi-infarct or Alzheimer-type dementia. As constituents with potential contact allergenic and toxic properties in crude Ginkgo extracts a group of alkylphenols (e.g., ginkgolic acids, ginkgol, bilobol) has been described. Thus, for reasons of drug safety a maximal concentration (< or = 5 ppm) of ginkgolic acids is requested by the Monograph of the Commission E of the former German Federal Health Agency (Bundesgesundheitsamt, BGA). During production of the standardized Ginkgo extract EGb 761, alkylphenols are largely eliminated as water insoluble compounds (decanter sludge) from the primary acetone extract. To further assess the adverse properties of alkylphenols, different fractions derived from the decanter sludge were evaluated for their embryotoxic effects in the hen's egg test (HET). A fraction enriched for ginkgolic acids (16%) and biflavones (6.7%) was found to induce death of 50% of the chick embryos (LD50) at a dose of 1.8 mg/egg (approximately/= 33 ppm). A similar strong lethal effect (LD50: 3.5 mg/egg; 64 ppm) was oberserved for a fraction which contained 58% ginkgolic acids but less than 0.02% biflavones. In contrast, an extreme low toxic potential (LD50: 250 mg/egg or 4540 ppm) was established for a fraction containing 16% biflavones and 1% ginkgolic acids. Thus, the present investigations confirm the high toxic potential of ginkgolic acids, although it can not be excluded that biflavones or some other constituents in the different fractions may amplify the adverse effect of these substances. Since no contribution of alkylphenols to the therapeutic efficacy of Ginkgo extracts has been confirmed and their elimination during the manufacturing process does not cause technical problems, these results further support the requirement for the completest possible removal of these compounds under toxicological considerations.     

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.     

Alzheimer's disease, the nematode Caenorhabditis elegans, and ginkgo biloba leaf extract

Alzheimer's disease (AD) is affecting larger and larger proportions of our population as lifespan increases. Thus, the means to prevent or reduce the rate of this disorder is a high priority for medical research. A standardized extract of Ginkgo biloba leaves EGb 761 is a popular dietary supplement taken by the general public to enhance mental focus and by the elderly to delay onset of age-related loss of cognitive function. EGb 761 has been used for treatment of certain cerebral dysfunctions and dementias associated with aging and AD. Substantial evidence indicates that EGb 761 has neuroprotective effects. But, mechanisms of action of the components of the extract are, unfortunately, poorly understood. Research in my laboratory focuses on understanding mechanisms of action of the components of the herbal extract EGb 761 in protection against Alzheimer's disease. We have demonstrated that EGb 761 inhibited amyloid beta aggregation in vitro and attenuates reactive oxidative species (ROS) in a model organism - the round worm Caenorhabditis elegans. Furthermore, EGb 761 eased its toxicity in the transgenic C. elegans. We also found that only a certain size of the amyloid beta aggregates is toxic to the worms. These findings suggest that EGb 761 has a clear therapeutic potential for prevention and/or treatment of AD. A better understanding of the mechanisms of neuroprotection by EGb 761 will be important for designing therapeutic strategies, for basic understanding of the underlying neurodegenerative processes, and for a better understanding of the effectiveness and complexity of this herbal medicine.     

Inhibition of platelet activating factor (PAF)-induced aggregation of human thrombocytes by ginkgolides: considerations on possible bleeding complications after oral intake of Ginkgo biloba extracts

The special Ginkgo biloba leaf extract EGb 761 is marketed for more then two decades. During this time its therapeutic efficacy and favorable safety profile have been proven in numerous clinical trails as well as by postmarketing surveillance in accordance with German drug regulations. During recent years, however, several cases of hemorrhage have been reported to occur in coincidence with the use of Ginkgo products. Although a clear causality between Ginkgo intake and bleeding could not be established, these observations have generally been explained by the platelet-activating factor (PAF)-antagonistic action of ginkgolides, which represent characteristic constituents of Ginkgo extracts. PAF was originally characterized by inducing aggregation and secretion of serotonin and histamine from rabbit platelets. We now confirmed that induction of aggregation of human platelets by PAF requires at least 200 times higher concentration when compared to rabbit cells. Under the chosen experimental conditions, PAF-mediated aggregation of human platelets was half-maximally inhibited by ginkgolide B, A, C and J at concentrations of 2.5, 15.8, 29.8 and 43.5 microg/ml, respectively. These concentrations are generally more than 100 times higher as the peak plasma values measured after oral intake of EGb 761 at recommended doses between 120 and 240 mg. As PAF is a 'weak' platelet activator, which does not appear to be of importance for primary hemostasis, our results rise serious doubts that the PAF antagonistic effect of ginkgolides could be responsible for hemorrhage in patients taking EGb 761.     

In vitro photochemical and phototoxicological characterization of major constituents in St. John's Wort (Hypericum perforatum) extracts

Extracts from St. John's Wort (SJW: Hypericum perforatum) have been used for the treatment of mild-to-moderate depression. In spite of the high therapeutic potential, orally administered SJW sometimes causes phototoxic skin responses. As such, the present study aimed to clarify the phototoxic mechanisms and to identify the major phototoxins of SJW extract. Photobiochemical properties of SJW extract and 19 known constituents were characterized with focus on generation of reactive oxygen species (ROS), lipid peroxidation, and DNA photocleavage, which are indicative of photosensitive, photoirritant, and photogenotoxic potentials, respectively. ROS assay revealed the photoreactivity of SJW extract and some SJW ingredients as evidenced by type I and/or II photochemical reactions under light exposure. Not all the ROS-generating constituents caused photosensitized peroxidation of linoleic acid and photodynamic cleavage of plasmid DNA, and only hypericin, pseudohypericin, and hyperforin exhibited in vitro photoirritant potential. Concomitant UV exposure of quercitrin, an SJW component with potent UV/Vis absorption, with hyperforin resulted in significant attenuation of photodynamic generation of singlet oxygen from hyperforin, but not with hypericin. In conclusion, our results suggested that hypericin, pseudohypericin, and hyperforin might be responsible for the in vitro phototoxic effects of SJW extract.     

Radiation-induced clastogenic factors: Anticlastogenic effect of ginkgo biloba extract

Clastogenic factors (CFs) were first described in the blood of persons irradiated accidentally or for therapeutic reasons. Work of our laboratory has shown that they occur also under other circumstances, which are characterized by oxidative stress, and that CF-induced chromosome damage is regularly prevented by superoxide dismutase (SOD). Recently we found CFs in a high percentage of salvage personnel of the Chernobyl reactor accident. These liquidators represent a high-risk population and might benefit from cancer chemoprevention by antioxidants. SOD would have to be injected and is not appropriate for longterm prophylactic treatment. In the present study, we therefore evaluated the anticlastogenic effect of the Ginkgo biloba extract EGb 761, which is known for its superoxide scavenging properties. EGb 761 was tested on CF-treated blood cultures of healthy donors. After establishing the optimal protective EGb concentration, using CFs produced by irradiation of whole blood from healthy volunteers, the extract was tested on cultures exposed to CFs from plasma of persons irradiated as liquidators. The anticlastogenic effect could be confirmed for a final concentration of 100μg/ml. In 12 consecutive experiments, CFs induced an average of 18.00 ± 4.41 aberrations/100 cells. This was reduced to 7.33 ± 3.08 in the parallel cultures receiving 100μg/ ml EGb 761 (p < .001). SOD was anticlastogenic in the same system at concentrations of 30 cytochrome C units/ml (approximately 10μg/ml). Preliminary results obtained in a small series of liquidators showed regression or complete disappearance of CFs in the plasma after 2 months of treatment with EGb 761 (3 × 40 gmg/d).     

The protective effect of Ginkgo biloba extract on CCl4-induced hepatic damage

The aim of this study was to evaluate the protective effect of Ginkgo biloba extract on CCl4-induced hepatic damage in rats. Hepatic malondialdehyde, glutathione and hydroxyproline levels and histopathologic alterations in liver specimens were assessed. 200 mg/kg/day Ginkgo biloba extract were given orally to the animals for 10 days, then a single dose of 2 ml/kg b.w. carbon tetrachloride was, administered intraperitoneally. Ginkgo biloba extract treatment reduced hepatic malondialdehyde levels significantly (p < 0.05), but did not alter glutathione (p > 0.05) and hydroxyproline levels (p > 0.05). The light and electron microscopic findings showed that Ginkgo biloba extract limited the CCl4-induced hepatocyte necrosis and atrophy. These results suggest that this extract may protect the hepatocytes from carbon tetrachloride-induced liver injury.     

Systems Pharmacology Dissection of Multi-Scale Mechanisms of Action for Herbal Medicines in Stroke Treatment and Prevention

Annually, tens of millions of first-ever strokes occur in the world; however, currently there is lack of effective and widely applicable pharmacological treatments for stroke patients. Herbal medicines, characterized as multi-constituent, multi-target and multi-effect, have been acknowledged with conspicuous effects in treating stroke, and attract extensive interest of researchers although the mechanism of action is yet unclear. In this work, we introduce an innovative systems-pharmacology method that combines pharmacokinetic prescreening, target fishing and network analysis to decipher the mechanisms of action of 10 herbal medicines like Salvia miltiorrhizae, Ginkgo biloba and Ephedrae herba which are efficient in stroke treatment and prevention. Our systematic analysis results display that, in these anti-stroke herbal medicines, 168 out of 1285 constituents with the favorable pharmacokinetic profiles might be implicated in stroke therapy, and the systematic use of these compounds probably acts through multiple mechanisms to synergistically benefit patients with stroke, which can roughly be classified as preventing ischemic inflammatory response, scavenging free radicals and inhibiting neuronal apoptosis against ischemic cerebral damage, as well as exhibiting lipid-lowering, anti-diabetic, anti-thrombotic and antiplatelet effects to decrease recurrent strokes. Relying on systems biology-based analysis, we speculate that herbal medicines, being characterized as the classical combination therapies, might be not only engaged in multiple mechanisms of action to synergistically improve the stroke outcomes, but also might be participated in reducing the risk factors for recurrent strokes.     

中药银杏制剂的心血管药理效应:机制与展望

银杏是传统的活血化瘀中药,银杏制剂是现代科技开发的植物药中最为成功的案例之一.目前研究较多的银杏制剂是银杏酮酯,其主要活性成分为银杏黄酮、银杏内酯等,具有较为广泛的心血管药理效应,对多种心血管疾病具有较好的预防和治疗效果.本文对中药银杏制剂的主要活性成分、心血管药理效应及其机制、研究展望做一述评.     

Circadian Rhythms of Leaf and Stomatal Movements in Gymnosperm Species

It is generally accepted that various physiological, morphological and gene expression phenomena are under the control of a circadian clock, and that this time keeping mechanism is universally present. Although such endogenously regulated phenomena have first been documented in plants more than 250 years ago and much work has been accumulated particularly in the past 70 years, it was not obvious from the literature whether such time keeping mechanisms exist in gymnosperms. Two prominent parameters were investigated in several gymnosperm species which have been demonstrated to be under the control of a circadian clock in many plants: (i) leaf movement and (ii) stomata movement. In young plants of Pinus sylvestris, Picea abies, Taxus baccata, Araucaria angustifolia, Araucaria heterophylla and Ginkgo biloba leaf oscillations could be recorded for about 5 days. However, compared to angiosperm plants, the amplitude was small. The period length under free running conditions (constant temperature and continuous light) was characteristic for the species. Stomatal movement was observed in Ginkgo biloba leaves by electron microscopy. Stomata were open at noon and closed at midnight under normal day/night conditions (LD) as well as under constant light conditions (LL), indicating that stomatal aperture is under circadian control in the gymnosperm Ginkgo biloba. Online recordings of stomata conductance however, exhibited diurnal but not circadian oscillations of net CO₂-exchange in G. biloba leaves. Our results show that a circadian clock controls leaf and stomatal movements in gymnosperm species indicating that endogenous time keeping mechanisms are present.     

Antiphospholipid syndrome

Antiphospholipid syndrome is diagnosed when arterial or venous thrombosis or recurrent miscarriages occur in a person in whom laboratory tests for antiphospholipid antibodies (anticardiolipin antibodies and/or lupus anticoagulant and/or anti-beta 2-glycoprotein I) are positive. Despite the strong association between antiphospho-lipid antibodies and thrombosis, their pathogenic role in the development of thrombosis has not been fully elucidated. Novel mechanisms involving both the complement pathway and micro-particles have been described. The knowledge of these new pathogenic approaches might identify novel therapeutic targets and therefore may improve the management of these patients.     

Neuroprotective effects of Ginkgo biloba extract in brain ischemia are mediated by inhibition of nitric oxide synthesis

We studied the effects of pre-treatment (15 days) with oral administration of Ginkgo biloba extract (Ph-Gb 37.5-150 mg/kg) on brain malonildialdehyde (MDA), brain edema, brain nitrite and nitrate and delayed neuronal death following transient cerebral ischemia in the Mongolian gerbil. Survival was not modified, however, pre-treatment with Ginkgo biloba significantly and in a dose-dependent way reduced post-ischemic brain MDA levels and post-ischemic brain edema. Delayed neuronal death in the CA1 of the hippocampus was attenuated by the highest dose of the extract. Increase of nitrite and nitrate was observed after cerebral ischemia in the hippocampus and it was dose-dependently reduced in animals pretreated with Ph-Gb, thus suggesting that neuroprotective effects of Ginkgo biloba may be due to an inhibitory action on nitric oxide formation.     

A Contribution to the Embryology of Ginkgo with a Discussion on the Affinity of the Ginkgoales

The ancestry of Ginkgoales or Ginkgophyta can be traced back to the Paleozoic. But today this order is represented only by a single species, Ginkgo biloba. Seward (1838) considered Ginkgo as one of the wonders of the world; it has persisted with little change until the present through a long succession of ages when the earth was inhabited by animals and plants for the most part far removed, in kind as in time, from their living descendents. Ginkgo is generally believed to be native to China, but so far it has not been found in wild state. A number of studies concerning the embryogeny of Ginkgo have been reported, on the basis of the materials accumulated during the past years and supplemented in 1978–1980, the embryogeny of Ginkgo is here described. Finally the phylogeny of Ginkgoales is discussed by comparing with the embryogeny of other groups of the living gymnosperms. Pollination usually takes place from the end of April to the first days of May and fertilization occurs around August 16–20 in the suburbs of Peking. Thus, the interval between pollination and fertilization is a few days less than four months. The embryo of Ginkgo is generally considered as suspensorless. In authors’ opinion, however, the somewhat elongated and much enlarged cells at the micropylar end of the embryo may be considered as the reduced suspensor cells though they are not the typical ones. There is no distinct demarcation between the proembryo and the young embryo in Ginkgo. In comparison with the embryogenesis in Coniferales, the tissue differentiation of the late embryo of Ginkgo is rather indistinct. Many authors such as Chamberlain (1935), Florin (1949), Delevoryas (1963), Sporne (1965) and others divide the gymnosperms into two major groups, Cycadophyta and Coniferophyta. From the point of view of morphological structure there are many significant common features shared by Ginkgoales and Coniferales. For example, Ginkgo possesses long shoot and short shoot while some conifers also have long and short shoots. The anatomical features of the stem of Ginkgo such as the well-developed secondary xylem, relatively small pith and the presence of bordered pits on the tracheids are also similar to those of the Coniferales. Not only Ginkgo has its characteristic leaf shape and the dichotomous venation but also they are quite different from the fronds of cycads. From the reproductive structure, on the other hand, Ginkgo and Cycadales are rather similar: both of them having one sulcate pollen grains, the pollen tube being of haustorial nature, the sperms being released from the base and not from the tip of the pollen tube, the presence of mulficiliate and rather large sperms, development of large female gametophyte bearing archegonia with exceptionally large eggs, more divisions of the free nuclear stage in the proembryo and less distinct differentiation of the ate embryo. All these features are primitive embryological characters. Thus, from the point of view of embryology the Ginkgo is close related to the Cycadales rather than to the Coniferales. Since Ginkgophyta are related to Cycadophyta in reproductive structures on one hand and similar to Coniferophyta in morphological and anatomical characters of the vegetative organs the Ginkgophyta are related to Cycadophyta in reproductive structures on one hand and similar to Coniferophyta in morphological and anatomical characters of the vegetative organs on the other hand, it indicates the interrelationship among these groups is clearly shown. Recently the discovery and studies on the progymnospermopsida (Beck, 1976) are worth notice, this fossil group contains the plants with certain characters of some conifers and certain characters of some cycads and this kind of plants bearing both homosporous and heterosporous forms are similar to ferns. They link the gymnosperms to the ferns. The present evidence, therefore, shows that the gymnosperms are very probably monophyletic and the progymnosperms might be the ancester of all the gymnosperms. The embryological characteristics supports the monophyletic origin of the gymnosperms.     

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.