Yves Christen

The ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by β-amyloid

Substantial evidence suggests that the accumulation of β‐amyloid (Aβ)‐derived peptides, and to a lesser extent free radicals, may contribute to the aetiology and/or progression of Alzheimers disease (AD). Ginkgo biloba extract (EGb 761) is a well‐defined plant extract containing two major groups of constituents, i.e. flavonoids and terpenoids. It is viewed as a polyvalent agent with a possible therapeutic use in the treatment of neurodegenerative diseases of multifactorial origin, e.g. AD. We have investigated here the potential effectiveness of EGb 761 against toxicity induced by (Aβ)‐derived peptides (Aβ25−35, Aβ1−40 and Aβ1−42) on hippocampal primary cultured cells, this area being severely affected in AD. A co‐treatment with EGb 761 concentration‐dependently (10–100 μg/mL) protected hippocampal neurons against toxicity induced by Aβ fragments, with a maximal and complete protection at the highest concentration tested. Similar, albeit less potent protective effects were seen with the flavonoid fraction of the extract (CP 205), while the terpenes were ineffective. Most interestingly, EGb 761 (100 μg/mL) was even able to protect (up to 8 h) hippocampal cells from a pre‐exposure to Aβ25−35 and Aβ1−40. EGb 761 was also able to both protect and rescue hippocampal cells from toxicity induced by H2O2 (50–150 μm), a major peroxide possibly involved in mediating Aβ toxicity. Moreover, EGb 761 (10–100 μg/mL), and to a lesser extent CP 205 (10–50 μg/mL), completely blocked Aβ‐induced events, e.g. reactive oxygen species accumulation and apoptosis. These results suggest that the neuroprotective effects of EGb 761 are partly associated with its antioxidant properties and highlight its possible effectiveness in neurodegenerative diseases, e.g. AD via the inhibition of Aβ‐induced toxicity and cell death.

Oxidative damage and protection by antioxidants in the frontal cortex of Alzheimer’s disease is related to the apolipoprotein E genotype

A great number of epidemiological studies have demonstrated that the frequency of the epsilon4 allele of the apolipoprotein E gene (APOE) is markedly higher in sporadic and in familial late onset Alzheimer disease (AD). In the frontal cortex of AD patients, oxidative damage is elevated. We address the hypothesis that the APOE genotype and reactive oxygen-mediated damage are linked in the frontal cortex of AD patients. We have related the APOE genotype to the levels of lipid oxidation (LPO) and to the antioxidant status, in frontal cortex tissues from age-matched control and AD cases with different APOE genotypes. LPO levels were significantly elevated in tissues from Alzheimers cases which are homozygous for the epsilon4 allele of APOE, compared to AD epsilon3/epsilon3 cases and controls. Activities of enzymatic antioxidants, such as catalase and glutathione peroxidase (GSH-PX), were also higher in AD cases with at least one epsilon4 allele of APOE, while superoxide dismutase (SOD) activity was unchanged. In the frontal cortex, the concentration of apoE protein was not different between controls and AD cases, and was genotype independent. The Ginkgo biloba extract (EGb 761), the neurosteroid dehydroepiandrosterone (DHEA) and human recombinant apoE3 (hapoE3rec) were able to protect control, AD epsilon3/epsilon3 and epsilon3/epsilon4 cases against hydrogen peroxide/iron-induced LPO, while hapoE4rec was completely ineffective. Moreover, EGb 761 and DHEA had no effect in homozygous epsilon4 cases. These results demonstrate that oxidative stress-induced injury and protection by antioxidants in the frontal cortex of AD cases are related to the APOE genotype.

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.

EGb 761 enhances adult hippocampal neurogenesis and phosphorylation of CREB in transgenic mouse model of Alzheimer’s disease

Standardized Ginkgo biloba extract EGb 761 exhibits beneficial effects to patients with Alzheimers disease (AD). It was previously demonstrated that EGb 761 inhibits amyloid beta (Aβ) oligomerization in vitro, protects neuronal cells against Aβ toxicity, and improves cognitive defects in a mouse model of AD (Tg 2576). In this study, the neurogenic potential of EGb 761 and its effect on cAMP response element binding protein (CREB) were examined in a double transgenic mouse model (TgAPP/PS1). EGb 761 significantly increases cell proliferation in the hippocampus of both young (6 months) and old (22 months) TgAPP/PS1 mice, and the total number of neuronal precursor cells in vitro in a dose‐dependent manner. Furthermore, Aβ oligomers inhibit phosphorylation of CREB and cell proliferation in the hippocampus of TgAPP/PS1 mice. Administration of EGb 761 reduces Aβ oligomers and restores CREB phosphorylation in the hippocampus of these mice. The present findings suggest that 1) enhanced neurogenesis by EGb 761 may be mediated by activation of CREB, 2) stimulation of neurogenesis by EGb 761 may contribute to its beneficial effects in AD patients and improved cognitive functions in the mouse model of AD, and 3) EGb 761 has therapeutic potential for the prevention and improved treatment of AD.—Tchantchou, F., Xu, Y., Wu, Y., Christen, Y., Luo, Y. EGb 761 enhances adult hippocampal neurogenesis and phosphorylation of CREB in transgenic mouse model of Alzheimers disease. FASEB J. 21, 2400–2408 (2007)

Ginkgo Biloba Extract Neuroprotective Action Is Dependent on Heme Oxygenase 1 in Ischemic Reperfusion Brain Injury

Background and Purpose— Ginkgo biloba extracts are now prescribed in several countries for their reported health benefits, particularly for medicinal properties in the brain. The standardized Ginkgo extract, EGb761, has been reported to protect neurons against oxidative stress, but the underlying mechanisms are not fully understood. Methods— To characterize the oral consumption of EGb761 in transient ischemia, we performed the middle cerebral artery occlusion (MCAO) filament model in wild-type and heme oxygenase 1 (HO-1) knockouts. Mice were pretreated for 7 days before the transient occlusion or posttreated acutely during reperfusion; then neurobehavioral scores and infarct volumes were assessed. Furthermore, primary cortical neuronal cultures were used to investigate the contribution of the antioxidant enzyme HO-1 in the EGb761-associated cytoprotection. Results— Mice that were pretreated with EGb761 had 50.9±5.6% less neurological dysfunction and 48.2±5.3% smaller infarct volumes than vehicle-treated mice; this effect was abolished in HO-1 knockouts. In addition to the prophylactic properties of EGb761, acute posttreatment 5 minutes and 4.5 hours after reperfusion also led to significant reduction in infarct size (P<0.01). After our previous demonstration that EGb761 significantly induced HO-1 levels in a dose- and time-dependent manner in neuronal cultures, here we revealed that this de novo HO-1 induction was required for neuroprotection against free radical damage and excitotoxicity as it was significantly attenuated by the enzyme inhibitor. Conclusion— These results demonstrate that EGb761 could be used as a preventive or therapeutic agent in cerebral ischemia and suggest that HO-1 contributes, at least in part, to EGb761 neuroprotection.

Amyloid precursor protein metabolism is regulated toward alpha-secretase pathway by Ginkgo biloba extracts

Clinical trials report that Ginkgo biloba extracts (e.g., EGb761) reduce cognitive symptoms in age-associated memory impairment and dementia, including Alzheimer disease (AD). However, the mechanisms behind their neuroprotective ability remain to be fully established. In this study, the effect of EGb761 on the amyloid precursor protein (APP) metabolism has been investigated by both in vitro and in vivo models. To this aim, alpha-secretase, the enzyme regulating the non-amyloidogenic processing of APP and the release of alphaAPPs, the alpha-secretase metabolite, were studied in superfusates of hippocampal slices after EGb761 incubation, and in hippocampi and cortices of EGb761-treated rats. PKC translocation state was evaluated as well. EGb761 increases alphaAPPs release through a PKC-independent manner. This effect is not accompanied by a modification of either APP forms or alpha-secretase expression. Moreover, EGb761 influence on alphaAPPs release was strictly dependent on treatment dosage. Our findings suggest that the benefit of EGb761 reported by previous clinical studies is underscored by a specific biological mechanism of this compound on APP metabolism, directly affecting the release of the non-amyloidogenic metabolite. Additional research will be needed to clearly define the effective clinical relevance, thus considering EGb761 as a possible supplementary treatment in dementing diseases.

Differential oxidation of apolipoprotein E isoforms and interaction with phospholipids.

Accumulation of oxidized proteins has been demonstrated in the brain of patients suffering from Alzheimers disease (AD). Among the proteins found in cerebral amyloid deposits, apolipoprotein (apo) E is a polymorphic protein which one specific isoform, apo E4, has been widely associated with AD. Apo E may be linked with AD by its isoform-specific interaction with lipids or other proteins in amyloid plaques. Using the myeloperoxidase oxidative system, we report that oxidation of the three recombinant apo E isoforms is differential (as estimated using immunoblot and high-performance liquid chromatography analysis), with apo E4 being more susceptible than apo E3, which in turn is much more susceptible than apo E2. In addition, susceptibility to thrombin proteolysis is reduced when apo E is oxidized, and oxidation of apo E decreases its incorporation into phospholipid discs by approximately 50%. Oxidation of apo E may contribute to inefficient lipid recycling in the brain, particularly regarding apo E4 and E3. Our results link and strengthen both the E4 allele linkage with AD and the role of protein oxidation in AD. The cerebral mechanisms underlying apo E oxidation and/or myeloperoxidase functions in vivo remain to be assessed.

Effect of long-term treatment with EGb 761 on age-dependent structural changes in the hippocampi of three inbred mouse strains

Female mice of the inbred strains C57BL/6J, BALB/cJ and DBA/2J were used to determine the possible existence of a genetically-based differential susceptibility to the effects of treatment with an extract of Ginkgo biloba (EGb 761). Timms silver-sulphide staining method was used to visualize and determine changes in the areas of the hippocampal structures of aged subjects, and more specifically on the projection fields of the mossy fibers which appear to decrease as a function of ageing. Experiments were begun when the animals were 15 months old. Treated animals received EGb 761 (50 mg/kg/day, p.o.) for 7 months in their drinking water. Inter-strain differences existed for the areas of the whole regio inferior, stratum pyramidale, stratum lacunosum moleculare and hilus (CA4) and for the projection field of intra- and infrapyramidal mossy fibers (iipMF) in the CA3 region of the hippocampus. Chronic treatment with EGb 761 significantly increased the projection field of iipMF and significantly reduced the area of the stratum radiatum, as compared with control mice. No differential sensitivity to EGb 761 existed among the mouse strains tested. Antioxydint properties of EGb 761 may explain its neuroprotective and neurotrophic actions on the hippocampus, and might explain certain improvements in memory and other cognitive functions in both humans and experimental animals.

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.

The Ginkgo biloba extract, EGb761, increases synaptosomal uptake of 5‐hydroxytryptamine: in‐vitro and ex‐vivo studies

Abstract— The Ginkgo biloba extract (EGb 761) added to a synaptosomal fraction prepared from mice cerebral cortex modified [3H]5‐hydroxytryptamine ([3H]5‐HT) uptake in a biphasic manner. Between 4 and 16 μg mL−1 EGb 761 increased significantly the [3H]‐5‐HT uptake (maximum + 23%). A similar increase was also obtained when synaptosomes were prepared from the cortex of mice treated orally with EGb 761, either acutely (100 mg kg−1, 14 h and 2 h before death) or semi‐chronically (2× 100 mg−1 kg daily for 4 consecutive days). The in‐vitro increase in [3H]5‐HT uptake induced by EGb 761 was not observed in the presence of 10−6 m clomipramine, a 5‐HT‐uptake inhibitor. EGb 761 did not increase [3H]dopa‐mine uptake by synaptosomes prepared from striatum of mice. We investigated different fractions of EGb 761 in order to determine the compounds inducing the increase in [3H]5‐HT uptake. The BN 52063 extract (corresponding to the EGb 761 devoid of flavonoid substances) did not increase [3H]5‐HT uptake. The Cp 202 extract (corresponding to the EGb 761 devoid of terpenic substances and containing mostly flavonoid substances) increased [3H]5‐HT uptake. Among the flavonoids, quercetin has been tested and had no effect on the [3H]5‐HT uptake. Since at the usual therapeutic doses of EGb 761, the effective concentrations of the components responsible for this increase are likely to be reached in the brain, one may suggest that this effect could contribute to the therapeutic effect of EGb 761.