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.     

Bilobalide, a component of the Ginkgo biloba extract (EGb 761), protects against neuronal death in global brain ischemia and in glutamate-induced excitotoxicity.

In this study, the effect of bilobalide, a purified terpene lactone component of the Ginkgo biloba extract (EGb 761), and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death was compared. In the case of ischemic injury, neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in the hippocampal regions of gerbils was measured. A significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons at 7-days of reperfusion after 5 min of transient global forebrain ischemia. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected hippocampal CA1 neurons against ischemia-induced neuronal death and reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death [effective concentration (EC50) = 5 microg/ml (12 microM) forbilobalide and 100 microg/ml for EGb 761]. These results suggest thatboth EGb 761 and bilobalide protect against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.     

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

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

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.     

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor G?-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or G?-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.     

Nitric oxide-induced capacitation of cryopreserved bull spermatozoa and assessment of participating regulatory pathways

The effect of nitric oxide (NO*) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from four bulls were incubated in TALP medium in the presence of heparin (10 IU/ml) or sodium nitroprusside (SNP, 0.05-100 microM), a NO* donor. The participation of NO* was confirmed by the use of scavengers, i.e. methylene blue (50,100 microM) and hemoglobin (20-40 microg/ml). The role of nitric oxide synthase in heparin-induced capacitation was evaluated using enzyme inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME) and Nomega-nitro-L-arginine (L-NA) in concentrations ranging from 1 to 500 microM. The effects of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK), on NO*-induced capacitation were evaluated by incubation with specific inhibitors of these enzymes (H-89, 50 microM; bisindolylmaleimide I, 0.1 microM and genistein, 3 microM). The role of hydrogen peroxide or superoxide anion in NO*-induced capacitation was evaluated by incubation with catalase (20-100 microg/ml) or superoxide dismutase (SOD, 0.05-0.5 mg/ml), respectively. Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC). SNP concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of the 0.05 microM SNP treatment (31 +/- 5.15%) were similar to those of heparin treated samples (33 +/- 4.27%). Inhibitors of nitric oxide synthase (NOS) diminished capacitation percentages in a dose-dependent manner as did the addition of NO*- scavengers (P <0.05). The presence of PKA, PKC and PTK inhibitors likewise decreased capacitation percentages (6.25 +/- 0.71, 12.75 +/- 1.41, 9.00 +/- 1.41%, respectively). The presence of catalase or SOD in the incubation medium had no effect on capacitation percentages. These results indicate that NO* may be generated by a sperm NOS during heparin-induced capacitation and that exogenous NO* acts as a capacitation inducer and involves the participation of PKA, PKC and PTK as part of the intracellular mechanisms that lead to capacitation in cryopreserved bull spermatozoa.     

Involvement of monoamine oxidase inhibition in neuroprotective and neurorestorative effects of Ginkgo biloba extract against MPTP-induced nigrostriatal dopaminergic toxicity in C57 mice.

The present study investigated the neuroprotective and neurorestorative effects of Ginkgo biloba extract (EGb 761) and its two components ginkgolides A (BN52020) and B (BN52021) in mice. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg/d i.p. for six days) significantly reduced striatal dopamine (DA) levels in C57 mice measured by high performance liquid chromatography with electrochemical detection (HPLC-EC). When C57 mice were pretreated with EGb 761 (20, 50, 100 mg/kg/d i.p.) for 7 days and then treated with the same extract 30 min before MPTP injection for 6 days, the neurotoxic effect of MPTP was antagonized in a dose-dependent fashion. Similar treatment with ginkgolides A and B (5, 10, 50 mg/kg/d i.p.) showed no protective effect. When C57 mice were treated with EGb 761 (50 mg/kg/d i.p.) after MPTP-lesion, the recovery of striatal dopamine (DA) levels was accelerated. However, similar treatment with ginkgolides A or B (10 mg/kg/d i.p.) did not show any effect. EGb 761, but not ginkgolides A and B, nonselectively inhibited mouse brain MAO activity in vitro (IC50 = 36.45 +/- 1.56 microg/ml) tested by an improved fluorimetric assay. The results demonstrate that EGb 761 administered before or after MPTP treatment effectively protects against MPTP-induced nigrostriatal dopaminergic neurotoxicity and that the inhibitory effect of EGb 761 on brain MAO may be involved in its neuroprotective effect.     

Different effects of the constituents of EGb761 on apoptosis in rat cerebellar granule cells induced by hydroxyl radicals

The present study was conducted to evaluate the different effects of the constituents of EGb761 (Ginkgo biloba Extract) on apoptosis in cerebellar granule cells induced by hydroxyl radicals. The total flavonoid component of EGb761, two pure EGb761 components (rutin and quercetin), and a mixture of flavonoids and terpenes protected cerebellar granule cells from oxidative damage and apoptosis induced by hydroxyl radicals. ESR(electron spin resonance) results showed that the IC50 of the flavonoids for scavenging hydroxyl radicals was almost the same as that of EGb761, even though flavonoids make up only 24% of EGb761, implying that other constituents of EGb761 besides flavonoids can scavenge hydroxyl radicals. Total terpenes of EGb761 did not protect against apoptosis. Flavonoids and terpenes did not show a synergistic effect in this regard. Terpenes did not scavenge hydroxyl radicals directly, which might be related to their "cage-like" structures.     

Ginkgo biloba prevents transient global ischemia-induced delayed hippocampal neuronal death through antioxidant and anti-inflammatory mechanism

We have previously reported neuroprotective properties of Ginkgo biloba/EGb 761® (EGb 761) in transient and permanent mouse models of brain ischemia. In a quest to extend our studies on EGb 761 and its constituents further, we used a model of transient global ischemia induced delayed hippocampal neuronal death and inflammation. Mice pretreated with different test drugs for 7 days were subjected to 8-min bilateral common carotid artery occlusion (tBCCAO) at day 8. After 7 days of reperfusion, mice brains were dissected out for TUNEL assay and immunohistochemistry. In situ detection of fragmented DNA (TUNEL staining) showed that out of all test drugs, only EGb 761 (13.6% ± 3.2) pretreatment protected neurons in the hippocampus against global ischemia (vs. vehicle, 85.1% ± 9.9; p < 0.05). Immunofluorescence-based studies demonstrated that pretreatment with EGb 761 upregulated the expression levels of heme oxygenase 1 (HO1), nuclear factor erythroid 2-related factor 2 (Nrf2), and vascular endothelial growth factor (VEGF) as compared to the vehicle group. In addition, increased number of activated astrocytes and microglia in the vehicle group was observed to be significantly lower in the EGb 761 pretreated group. Together, these results suggest that EGb 761 is a multifunctional neuroprotective agent, and the protection is in part associated with activation of the HO1/Nrf2 pathway, upregulation of VEGF and downregulation of inflammatory mediators such as astrocytes and microglia.     

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.     

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

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

Protective effect of keishi-bukuryo-gan and its constituent medicinal plants against nitric oxide donor-induced neuronal death in cultured cerebellar granule cells.

Keishi-bukuryo-gan (Gui-Zhi-Fu-Ling-Wan) (KBG) is a traditional Chinese/Japanese medical (Kampo) formulation that has been administered to patients with "Oketsu" (blood stagnation) syndrome. In the process of neuronal cell death induced by brain ischemia, excessive generation of nitric oxide (NO) free radicals is implicated in the neurotoxicity. In the present study, we examined the protective effects of KBG and its constituent medicinal plants against NO donors, sodium nitroprusside (SNP) and 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC18)-induced neuronal death in cultured rat cerebellar granule cells (CGCs). MTT assay showed cell viability to be significantly increased by the addition of KBG extract (KBGE) (100 microg/ml), Cinnamomi Cortex extract (CCE) (3, 10 and 30 microg/ml), Paeoniae Radix extract (PRE) (100 microg/ml) and Moutan Cortex extract (MCE) (10 and 30 microg/ml) compared with exposure to SNP (30 microM, 24 h) only. Also, cell viability was significantly increased by the addition of KBGE (100 and 300 microg/ml), CCE (30 and 100 microg/ml), PRE (100 and 300 microg/ml) and MCE (30 and 100 microg/ml) compared with exposure to NOC 18 (100 microM, 48 h) only. Persicae Semen extract and Hoelen extract did not protect against NO donor-induced neuronal death. These results suggest that KBG has protective effect against NO-mediated neuronal death in cultured CGCs and that it is derived from Cinnamomi Cortex, Paeoniae Radix and Moutan Cortex.     

EGb 761 protects liver mitochondria against injury induced by in vitro anoxia/reoxygenation.

The present study investigated the protective effects of Ginkgo biloba extract (EGb 761) on rat liver mitochondrial damage induced by in vitro anoxia/reoxygenation. Anoxia/reoxygenation was known to impair respiratory activities and mitochondrial oxidative phosphorylation efficiency. ADP/O (2.57 +/- 0.11) decreased after anoxia/reoxygenation (1.75 +/- 0.09, p < .01), as well as state 3 and uncoupled respiration (-20%, p < .01), but state 4 respiration increased (p < .01). EGb 761 (50-200 microg/ml) had no effect on mitochondrial functions before anoxia, but had a specific dose-dependent protective effect after anoxia/reoxygenation. When mitochondria were incubated with 200 microg/ml EGb 761, they showed an increase in ADP/O (2.09 +/- 0.14, p < .05) and a decrease in state 4 respiration (-22%) after anoxia/reoxygenation. In EPR spin-trapping measurement, EGb 761 decreased the EPR signal of superoxide anion produced during reoxygenation. In conclusion, EGb 761 specially protects mitochondrial ATP synthesis against anoxia/reoxygenation injury by scavenging the superoxide anion generated by mitochondria.     

Protective effect of ginkgo biloba against gossypol-induced apoptosis in human lymphocytes

Ginkgo biloba (EGb 761) is a well-defined plant extract that directly scavenges hydroxyl radicals. It is a potent antioxidant that inhibits apoptosis in cultured cells and is effective in treating mild-to-moderate dementia in Alzheimer patients. Apoptosis is an active process of cell destruction and it plays an important role in pathological processes. The aim of this study is to investigate the anti-apoptotic effect of EGb 761 in gossypol-treated human lymphocytes. Pretreatment of lymphocytes with 10 microg/ml EGb 761 for 30 min or 1h decreased the percentage of apoptosis to 17.5% and 20%, respectively. EGb 761 treatment (25-150 microg/ml) decreased the level of apoptosis to a plateau between 8 and 10% of the control values. We conclude that EGb 761 reduces gossypol-induced apoptosis in human lymphocytes.     

Nitric oxide and platelet energy metabolism

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.     

Inhibition of cPLA2 activation by Ginkgo biloba extract protects spinal cord neurons from glutamate excitotoxicity and oxidative stress-induced cell death

Ginkgo biloba extract (EGb761) has been shown to be neuroprotective; however, the mechanism by which EGb761 mediates neuroprotection remains unclear. We hypothesized that the neuroprotective effect of EGb761 is mediated by inhibition of cytosolic phospholipase A(2) (cPLA(2)), an enzyme that is known to play a key role in mediating secondary pathogenesis after acute spinal cord injury (SCI). To determine whether EGb761 neuroprotection involves the cPLA(2) pathway, we first investigated the effect of glutamate and hydrogen peroxide on cPLA(2) activation. Results showed that both insults induced an increase in the expression of phosphorylated cPLA(2) (p-cPLA(2)), a marker of cPLA(2) activation, and neuronal death in vitro. Such effects were significantly reversed by EGb761 administration. Additionally, EGb761 significantly decreased prostaglandin E(2) (PGE(2)) release, a downstream metabolite of cPLA(2). Moreover, inhibition of cPLA(2) activity with arachidonyl trifluromethyl ketone improved neuroprotection against glutamate and hydrogen peroxide-induced neuronal death, and reversed Bcl-2/Bax ratio; notably, EGb761 produced greater effects than arachidonyl trifluromethyl ketone. Finally, we showed that the extracellular signal-regulated kinase 1/2 signaling pathway is involved in EGb761's modulation of cPLA(2) phosphorylation. These results collectively suggest that the protective effect of EGb761 is mediated, at least in part, through inhibition of cPLA(2) activation, and that the extracellular signal-regulated kinase 1/2 signaling pathway may play an important role in mediating the EGb761's effect.