Neuroprotective effects of bilobalide, a component of Ginkgo biloba extract (EGb 761) in global brain ischemia and in excitotoxicity-induced neuronal death.

In this study, we compared the protective effect of bilobalide, a purified terpene lactone component of ginkgo biloba extract EGb 761, (definition see editorial) and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death. In ischemic injury, we measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. 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 CA1 neurons from death and from ischemia-induced 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 [EC (50)] = 5 microg/ml (12 microM) for bilobalide and 100 microg/ml for EGb 761. These results suggest that both EGb 761 and bilobalide are protective 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.

Antioxidant compounds EGB-761 and BN-52021 attenuate brain edema formation and hemeoxygenase expression following hyperthermic brain injury in the rat.

Role of carbon monoxide (CO) in hyperthermic brain injury induced brain pathology was examined in a rat model using immunohistochemistry of the hemeoxygenase-2 (HO-2) enzyme. Exposure of rats to 4 h heat stress at 38 degrees C resulted in profound hyperthermia, breakdown of the blood-brain barrier (BBB), brain edema formation, cell damage and expression of HO-2 in several brain regions. Pretreatment with potent antioxidant compounds EGB-761 and BN-52021 markedly reduced the HO-2 expression, BBB breakdown, brain edema formation and cell damage without attenuating the hyperthermic response. This effect was most marked in animals treated with EGB-761. These observations suggest that upregulation of HO-2 representing generation of CO plays important roles in hyperthermic brain injury, and oxidative stress seems to be one of the most important signals in inducing HO-2 expression in hyperthermia, not reported earlier.

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia.

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. 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 CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.

The Ginkgo biloba extract EGb 761 rescues the PC12 neuronal cells from beta-amyloid-induced cell death by inhibiting the formation of beta-amyloid-derived diffusible neurotoxic ligands.

beta Amyloid (Abeta) treatment induced free radical production and increased glucose uptake, apoptosis and cell death in PC12 nerve cells. Addition of the standardized extract of Ginkgo biloba leaves, EGb 761 together with the Abeta protein prevented, in a dose-dependent manner, the Abeta-induced free radical production, increased glucose uptake, apoptosis and cell death. However, pretreatment of the cells with EGb 761 did not rescue the cells from the Abeta-induced toxicity although it prevented the Abeta-induced reactive oxygen species generation. Moreover, the terpene and flavonoid-free EGb 761 extract, HE 208, although inhibited the Abeta-induced increased glucose uptake, it failed to protect the cells from apoptosis and cytotoxicity induced by Abeta. In conclusion, these results indicate that the terpenoid and flavonoid constituents of EGb 761, acting probably in combination with components present in HE 208, are responsible for rescuing the neuronal cells from Abeta-induced apoptosis and cell death; their mechanism of action being distinct of their antioxidant properties. Because pre- and post-treatment with EGb 761 did not protect the cells from Abeta-induced neurotoxicity, we examined whether EGb 761 interacts directly with Abeta. Indeed, in vitro reconstitution studies demonstrated that EGb 761 inhibits, in a dose-dependent manner, the formation of beta-amyloid-derived diffusible neurotoxic soluble ligands (ADDLs), suggested to be involved in the pathogenesis of Alzheimer's disease.

Complementary cardioprotective effects of flavonoid metabolites and terpenoid constituents of Ginkgo biloba extract (EGb 761) during ischemia and reperfusion.

Hemodynamic and electron spin resonance (ESR) analyses were performed on isolated ischemic and reperfused rat hearts to assess the cardioprotective and antioxidant effects of therapeutically relevant concentrations of Ginkgo biloba extract (EGb 761; 5, 50 or 200 microg/ml), its terpenoid constituents (ginkgolide A; 0.05 microg/ml and ginkgolide B; 0.05, 0.25 or 0.50 microg/ml), and a terpene-free fraction of EGb 761 (CP 205; 5 or 50 microg/ml). Hearts underwent 10 min of low-flow ischemia, 30 min of no-flow global ischemia, and 60 min of reperfusion. Test substances were added to the perfusion fluid during the last 10 min of control perfusion, low-flow ischemia and the first 10 min of reperfusion. A separate group of rats was treated with CP 205 (60 mg/kg/day; p.o.) for 15 days, after which the hearts were perfused with plain buffer. In ESR experiments, the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added to the perfusate to determine the effects of treatments on post-ischemic myocardial free radical generation. Results showed that in vitro exposure of hearts to EGb 761 (5 or 50 microg/ml) or to ginkgolides A and B (both at 0.05 microg/ml), or in vivo pretreatment of the rats with CP 205 delayed the onset of contracture during ischemia. The strong reperfusion-induced elevation of left ventricular end-diastolic pressure observed in untreated hearts was significantly reduced by in vitro exposure to the lowest concentrations of EGb 761, by ginkgolide A, and to a lesser extent by ginkgolide B, or by prior oral treatment with CP 205. Post-ischemic functional recovery.was significantly improved by in vivo administration of CP 205, by perfusion with 5 microg/ml of EGb 761 or with both terpenoids as compared to untreated group but in vitro CP 205 was not effective. ESR analyses revealed that DMPO-OH (the DMPO/hydroxyl radical spin-adduct) concentrations in coronary effluents were markedly decreased by all treatments, except for the lowest concentration of ginkgolide B. Perfusing 5 microg/ml EGb 761 resulted in a better inhibition of baseline DMPO-OH concentration than 5 microg/ml CP 205 (-70 % and -48 % vs. control, respectively), indicating that both terpenoid and flavonoid constituents of EGb 761 are required to produce this effect. CP 205 was significantly more efficient in reducing DMPO-OH concentration when administered in vivo than when applied in vitro, indicating that the antioxidant effect of flavonoid metabolites (formed in vivo) is superior to that of intact flavonol glycosides (present in vitro). Collectively, these findings provide the first evidence that part of the cardioprotection afforded by EGb 761 is due to a specific action of its terpenoid constituents and that this effect involves a mechanism independent of direct free radical-scavenging. Thus, the terpenoid constituents of EGb 761 and the flavonoid metabolites that are formed after in vivo administration of the extract act in a complementary manner to protect against myocardial ischemia-reperfusion injury.

Drug-induced inhibition of the peripheral-type benzodiazepine receptor expression and cell proliferation in human breast cancer cells.

The peripheral-type benzodiazepine receptor (PBR) expression and localization correlate with human breast cancer cell proliferation and aggressive phenotype expression. The standardized extract of Ginkgo biloba leaves (EGb 761) and isolated ginkgolide B (GKB) were shown to decrease PBR mRNA expression in adrenal cells. We examined the effect of EGb 761 and GKB on PBR expression and cell proliferation in human breast cancer cells. EGb 761 and GKB decreased in a time- and dose-dependent manner PBR expression and cell proliferation in the highly aggressive, rich in PBR, human breast cancer cell line MDA-231 whereas they did not affect the proliferation of the non-aggressive human breast cancer cell line MCF-7, which contains very low PBR levels. This effect was reversible and not due to the antioxidant properties of the compounds tested. Using a human cDNA expression array we determined that EGb 761 treatment altered, in addition to PBR, the expression of 36 gene products involved in various pathways regulating cell proliferation. These in vitro data were further validated in an in vivo model where EGb 761 and GKB significantly inhibited the nuclear PBR expression and growth of MDA-231 cell xenografts in nude mice. Taken together, these data suggest that the manipulation of PBR expression could be used to control tumor growth and that EGb 761 and GKB, under the conditions used, exert cytostatic properties.

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.

Antioxidant compounds EGB-761 and BN-520 21 attenuate heat shock protein (HSP 72 kD) response, edema and cell changes following hyperthermic brain injury. An experimental study using immunohistochemistry in the rat.

Influence of the extract of Gingko biloba (EGB-761) and one of its constituent Gingkolide B (BN-52021) on hyperthermia induced cellular damage and heat shock protein (HSP 72kD) response was examined in a rat model. Rats subjected to 4h heat stress at 38 degrees C in a biological oxygen demand (BOD) incubator (relative humidity 50-55%, wind velocity 20-25cm/sec) resulted in profound edema and cell injury in many parts of the cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus and brain stem. Immunostaining of HSP 72 kD showed marked upregulation in the damaged and distorted neurons located within the edematous area. Pretreatment with EGB-761 (50mg/kg/day, p.o.) and BN-520 21 (2mg/kg, p.o.) per day for 5 days significantly reduced HSP expression and attenuated cell damage. Our results show that EGB-761 and its component Gingkolide B (BN-52021) has the capacity to reduce edema and cell injury following hyperthermia and this effect of the compound is somehow associated with a reduction in cellular stress response as evidenced with a reduction in HSP expression.

[Effect of Ginkgo biloba extract (EGb 761) on rats in an experimental model of acute encephalopathy after total body irradiation]. / Effet de l'extrait de Ginkgo biloba (EGb 761) chez le rat sur un modèle expérimental d'encéphalopathie aiguë après irradiation corporelle totale.

PURPOSE: To define the therapeutic effect of Ginkgo biloba extract (EGb 761) in an experimental model of acute encephalopathy following total body irradiation in rats. MATERIAL AND METHODS: Ninety four-month-old rats received 4.5 Gy total body irradiation (TBI) at day 1 while 15 rats received sham irradiation. A behavioural study based on a conditioning test of negative reinforcement, the one-way avoidance test, was performed test, was performed after irradiation. Orally treatment was started one day (study A) or twenty two days (study B) after irradiation and repeated daily for twelve days. In the irradiated group, three subgroups were defined according to the treatment received: EGb 761 (50 mg/kg), EGb 761 (100 mg/kg), water. RESULTS: This work comprised two consecutive studies. In study A (45 rats) the one-way avoidance test was administered daily from day 7 to day 14. In study B (45 rats) the behavioural test was performed from day 28 to day 35. Study A (three groups of 15 rats): following TBI, irradiated rats treated with water demonstrated a significant delay in a learning the one-way avoidance test in comparison with sham-irradiated rats (P < 0.0002) or irradiated rats treated with EGb 761 (50 mg/kg; P < 0.0017) or EGb 761 (100 mg/kg; P < 0.0002). The irradiated rats, treated with EGb 761 (50 or 100 mg/kg) did not differ from the sham-irradiated controls. Study B (three groups of 15 rats): the irradiated rats, treated with water or EGb 761 (50 or 100 mg/kg) did not differ from the sham-irradiated controls. CONCLUSION: This study indicates that a relatively low dose of total body irradiation induces a substantial acute learning dysfunction in the rat, which persists fourteen days after TBI. This effect is prevented by the administration of EGb 761 (50 or 100 mg/kg) started twenty-four hours after irradiation.

Evaluation of a ginkgo biloba extract (EGb 761) in functional tests for monoamine oxidase inhibition.

A standardized extract of ginkgo biloba (EGb 761) was evaluated in functional tests for monoamine oxidase (MAO) inhibition in mice: l-dihydroxyphenylalanine (CAS 59-92-7,1-DOPA) potentiation, tryptamine (CAS 61-54-1) potentiation, 5-hydroxytryptophan (CAS 4350-07-6,5-HTP) potentiation and phenylethylamine (CAS 64-04-0) potentiation. The doses investigated (25, 50 and 100 mg/kg p.o once daily for 5 days) were those known to possess anti-stress properties in other animal models. In contrast to the reference substances investigated (nialamide (CAS 51-12-7), clorgyline (CAS 17780-75-5) and 1-deprenyl (CAS 14611-52-01), EGb 761 did not exhibit any activity indicative of MAO inhibition. It was concluded that MAO inhibition was not the mechanism primarily responsible for EGb 761's anti-stress activity.

Ginkgo biloba extract (EGb 761) and CNS functions: basic studies and clinical applications.

The effects of EGb 761 on the CNS underlie one of its major therapeutic indications; i.e., individuals suffering from deteriorating cerebral mechanisms related to age-associated impairments of memory, attention and other cognitive functions. EGb 761 is currently used as symptomatic treatment for cerebral insufficiency that occurs during normal ageing or which may be due to degenerative dementia, vascular dementia or mixed forms of both, and for neurosensory disturbances. Depressive symptoms of patients with Alzheimer's disease (AD) and aged non-Alzheimer patients may also respond to treatment with EGb 761 since this extract has an "anti-stress" effect. Basic and clinical studies, conducted both in vitro and in vivo, support these beneficial neuroprotective effects of EGb 761. EGb 761 has several major actions; it enhances cognition, improves blood rheology and tissue metabolism, and opposes the detrimental effects of ischaemia. Several mechanisms of action are useful in explaining how EGb 761 benefits patients with AD and other age-related, neurodegenerative disorders. In animals, EGb 761 possesses antioxidant and free radical-scavenging activities, it reverses age-related losses in brain alpha 1-adrenergic, 5-HT1A and muscarinic receptors, protects against ischaemic neuronal death, preserves the function of the hippocampal mossy fiber system, increases hippocampal high-affinity choline uptake, inhibits the down-regulation of hippocampal glucocorticoid receptors, enhances neuronal plasticity, and counteracts the cognitive deficits that follow stress or traumatic brain injury. Identified chemical constituents of EGb 761 have been associated with certain actions. Both flavonoid and ginkgolide constituents are involved in the free radical-scavenging and antioxidant effects of EGb 761 which decrease tissue levels of reactive oxygen species (ROS) and inhibit membrane lipid peroxidation. Regarding EGb 761-induced regulation of cerebral glucose utilization, bilobalide increases the respiratory control ratio of mitochondria by protecting against uncoupling of oxidative phosphorylation, thereby increasing ATP levels, a result that is supported by the finding that bilobalide increases the expression of the mitochondrial DNA-encoded COX III subunit of cytochrome oxidase. With regard to its "anti-stress" effect, EGb 761 acts via its ginkgolide constituents to decrease the expression of the peripheral benzodiazepine receptor (PBR) of the adrenal cortex.

Role of nitric oxide in blood-brain barrier permeability, brain edema and cell damage following hyperthermic brain injury. An experimental study using EGB-761 and Gingkolide B pretreatment in the rat.

The role of oxidative stress in hyperthermia induced upregulation of constitutive and inducible isoforms of nitric oxide synthase (NOS) in the central nervous system (CNS) was investigated using immunohistochemistry in a rat model. Exposure of rats to heat stress at 38 degrees C for 4 h resulted in marked upregulation of constitutive NOS (cNOS) and a mild but significant expression of inducible NOS (iNOS) in several brain regions exhibiting leakage of the blood-brain barrier (BBB), brain edema formation and cell injury. Pretreatment with the potent antioxidative compound EGB-761 or its constituent, Ginkgolide B significantly attenuated upregulation of cNOS and iNOS in the brain and also reduced the BBB permeability disturbances, brain edema and cell injury. These neuroprotective effects were most marked in the EGB-761 pretreated rats. Our observations strongly suggest that (i) EGB-761 and Ginkgolide B pretreatment offer significant neuroprotection in hyperthermic brain injury, (ii) upregulation of cNOS and iNOS are injurious to the cell and, (iii) oxidative stress plays an important role in NOS expression and cell injury.

Free radicals and lipid peroxidation do not mediate beta-amyloid-induced neuronal cell death.

"beta Amyloid (Abeta)-induced free radical-mediated neurotoxicity" is a leading hypothesis as a cause of Alzheimer's disease (AD). Abeta increased free radical production and lipid peroxidation in PC12 nerve cells, leading to increased 4-hydroxy-2-nonenal (HNE) production and modification of specific mitochondrial target proteins, apoptosis and cell death. Pretreatment of the cells with isolated ginkgolides, the anti-oxidant component of Ginkgo biloba leaves, or vitamin E, prevented the Abeta-induced increase of reactive oxygen species (ROS). Ginkgolides, but not vitamin E, inhibited the Abeta-induced HNE modification of mitochondrial proteins. However, treatment with these anti-oxidants did not rescue the cells from Abeta-induced apoptosis and cell death. These results indicate that free radicals and lipid peroxidation may not mediate Abeta-induced neurotoxicity.

Production of ginkgolides and bilobalide by Ginkgo biloba plants and tissue cultures.

The accumulation of the terpenes ginkgolides and bilobalide in Ginkgo biloba was reported in plants as well as in plant cell cultures. Several hundred plants cultivated under controlled conditions in the field have been analyzed for their terpene production over many years. Cross-pollination experiments were performed with mature trees and the terpene content of the progeny was analyzed. The age of the tree is the main factor influencing the terpene content of the leaves as the level always decreases dramatically between young and old trees. 80 cell culture strains have been established and ginkgolides analyzed by GC/MS. These cell cultures reveal very low amounts of terpenes (1 microgram g-1 D.W. or less). On the contrary, isolated in vitro root cultures accumulate terpenes at the same concentration as the young plant leaves (4 mg g-1 D.W.). Attempts to obtain rapid growing roots or even hairy-roots did not succeed but the possibility to transform Ginkgo cell strains has been demonstrated.

Anticlastogenic effects of Ginkgo biloba extract (EGb 761) and some of its constituents in irradiated rats.

In a previous study we reported that radiation-induced clastogenic factors (CF) are found in the plasma of Chernobyl accident recovery workers and that their chromosome damaging effects are inhibited by antioxidant treatment with a Ginkgo biloba extract (EGb761). In the present study, we induced CF in rats with a radiation dose of 4.5 Gy. The protective effects of the complete extract were compared to those obtained with the extract devoid of its terpene fraction (CP205), with isolated ginkgolides A+B and bilobalide at the concentrations present in EGb761. The pretreatment samples were taken at day 22 postirradiation, the posttreatment samples the day following arrest of the 3-week treatment. The adjusted clastogenic score (ACS) were reduced from 11.71+/-3.55 to 2.00+/-2.83 after treatment with 100 mg/kg and from 13.43+/-2.23 to 4.29+/-2.14 with 50 mg/kg of the complete extract (p<0.0001). Similar protective effects were observed with CP205, ginkgolides and bilobalide (p<0. 001), while the reduction of ACS in placebo-treated rats was not statistically significant (12.80+/-1.79 and 9.20+/-2.68). However, if the efficacy of the treatment was compared to placebo, only the complete extract was significantly protective. While all components exerted anticlastogenic effects at the concentrations present in the complete extract, the comparison of the different groups by analysis of variance did not reveal significant differences. This may be due to to the small number of animals available in each treatment group. The complete extract reduced the ACS by 83% at the dose of 100 mg/kg, while the lower dose of 50 mg/kg and the three components reached only 66%-68% reduction. The better protection provided by the complete extract is due to synergistic rather than to additive effects.

Effects of single intraperitoneal injections of an extract of Ginkgo biloba (EGb 761) and its terpene trilactone constituents on barbital-induced narcosis in the mouse.

A mouse model of barbital-induced narcosis was used to examine the effects of single intraperitoneal injections of an extract of Ginkgo biloba (EGb 761), an extract devoid of terpene trilactones (CP 205), and three terpene trilactone constituents of the extract (ginkgolides A and B, bilobalide). Administration of sodium barbital (180 mg/kg, IP) to the mice caused narcosis, measured as a loss in righting reflex. Single injections of EGb 761 (25 and 50 mg/kg), given 60 min prior to sodium barbital, significantly shortened barbital-induced sleeping time, whereas these same doses of CP 205 were ineffective. Single injections of ginkgolide B (1 mg/kg) and bilobalide (2 and 5 mg/kg) significantly shortened sleeping time, whereas ginkgolide A was ineffective. The effects of ginkgolide B and bilobalide were reflected as increases in latency to onset of sleep and those of EGb 761, ginkgolide B, and bilobalide were correlated with decreases in the number of mice that slept. At the behavioral level, these potent in vivo effects of EGb 761, ginkgolide B, and bilobalide resemble those of certain antidepressants. At the molecular level, it is hypothesized that interactions with the picrotoxinin/TBPT site of GABA-regulated Cl- channels of the CNS may be involved. This information appears useful in explaining the clinically observed "vigilance-enhancing" and "antidepressant-like" actions of EGb 761.

Protection of mitochondrial respiration activity by bilobalide.

Mitochondria alteration is an early event in ischemia-induced damage, and its prevention improves tissue survival upon reperfusion. Adenine translocase and complex I activities are rapidly affected by ischemia. Ginkgo biloba extract demonstrates anti-ischemic properties attributable to the terpenoid fraction, mainly due to the presence of bilobalide. The mechanism of the protection afforded by bilobalide is not yet known. In this work, the effects of bilobalide on mitochondrial respiration were investigated. Mitochondria isolated from rats treated with bilobalide (2 to 8 mg/kg) showed a dose-dependent increase in the respiratory control ratio, due to a lower oxygen consumption during state 4. Bilobalide also decreased the sensitivity of oxygen consumption to inhibition of complex I by Amytal or to inhibition of complex III by antimycin A or myxothiazol. There was no protection of complexes IV and V. It also increased the activity of complex I but not of adenine translocase. Similar effects were also obtained in vitro when control mitochondria were preincubated for 1 hr with 0.8 microg/mL bilobalide. Treatment of the rats with 8 mg/kg bilobalide also prevented the ischemia-induced decrease in state 3 of the mitochondrial respiration and thus the decrease in RCR. The protective effect of bilobalide on cellular ATP content observed under ischemic conditions can be correlated with the above observations. By protecting complex I and III activities, bilobalide allows mitochondria to maintain their respiratory activity under ischemic conditions as long as some oxygen is present, thus delaying the onset of ischemia-induced damage. This mechanism provides a possible explanation for the anti-ischemic properties of bilobalide and of Ginkgo biloba extract in therapeutic interventions.

Ginkgo biloba extract EGb761 reduces the development of amphetamine-induced behavioral sensitization: effects on hippocampal type II corticosteroid receptors.

Pretreatment of rats with the extract of Ginkgo biloba termed EGb761 reduced the behavioral sensitization induced by successive D-amphetamine administrations (0.5 mg/kg) as estimated by increasing values of locomotor activity. EGb761 pretreatment also prevented the reduced density of [3H]dexamethasone binding sites in the dentate gyrus and the CA1 hippocampal regions of D-amphetamine treated animals. These observations suggest that EGb761, by reducing glucocorticoid levels, could modulate the activity of the neuronal systems involved in the expression of the behavioral sensitization.

Effects of EGb 761 on fatty acid reincorporation during reperfusion following ischemia in the brain of the awake gerbil.

Transient cerebral ischemia (5 min) releases unesterified fatty acids from membrane phospholipids, increasing brain concentrations of fatty acids for up to 1 h following reperfusion. To understand the reported anti-ischemic effect of Ginkgo biloba extract (EGb 761), we monitored its effect on brain fatty acid reincorporation in a gerbil-stroke model. Both common carotid arteries in awake gerbils were occluded for 5 min, followed by 5 min of reperfusion. Animals were infused intravenously with labeled arachidonic (AA) or palmitic acid (Pam), and rates of incorporation of unlabeled fatty acid from the brian acyl-CoA pool were calculated by the model of Robinson et al. (1992), using quantitative autoradiography and biochemical analysis of brain acyl-CoA. Animals were treated for 14 d with 50 or 150 mg/kg/d EGb 761 or vehicle. Ischemia-reperfusion had no effect on the rate of unlabeled Pam incorporation into brain phospholipids from palmitoyl-CoA; this rate also was unaffected by EGb 761. In contrast, ischemia-reperfusion increased the rate of incorporation of unlabeled AA from brain arachidonoyl-CoA by a factor of 2.3-3.3 compared with the control rate; this factor was further augmented to 3.6-5.0 by pretreatment with EGb 761. There is selective reincorporation of AA compared with Pam into brain phospholipids following ischemia. EGb 761 further accelerates AA reincorporation, potentially reducing neurotoxic effects of prolonged exposure of brain to high concentrations of AA and its metabolites.

Effect of chronic administration of Ginkgo biloba extract or Ginkgolide on the hypothalamic-pituitary-adrenal axis in the rat.

The hypersecretion of glucocorticoids during exposure to various stressors may induce or worsen pathological states in predisposed subjects. Therefore it is of interest to evaluate drugs able to reduce glucocorticoid secretion. It has recently been shown that chronic administration of a Ginkgo biloba extract (EGb 761) inhibits stress-induced corticosterone hypersecretion through a reduction in the number of adrenal peripheral benzodiazepine receptors. The present study was designed to analyze the effect of EGb 761 and one of its components, Ginkgolide B on the biosynthesis and secretion of CRH and AVP, the hypothalamic neurohormones that regulate the pituitary-adrenal axis. Chronic administration of EGb 761 (50 or 100 mg/kg p.o. daily for 14 days) reduced basal corticosterone secretion and the subsequent increase in CRH and AVP gene expression. Under the same conditions, surgically-induced increase in CRH secretion was attenuated while the activation of CRH gene expression, ACTH and corticosterone secretion following insulin-induced hypoglycemia remained unchanged. Chronic i.p. injection of Ginkgolide B reduced basal corticosterone secretion without alteration in the subsequent CRH and AVP increase. However, the stimulation of CRH gene expression by insulin-induced hypoglycemia was attenuated by Ginkgolide B. These data confirm that the administration of EGb 761 and Ginkgolide B reduces corticosterone secretion. In addition, these substances act also at the hypothalamic level and are able to reduce CRH expression and secretion. However the latter effect appears to be complex and may depend upon both the nature of stress and substance (Ginkgolide B or other compounds of EGb 761).