Antioxidant Scavanger Action

The chemical structure of EGb constituents is responsible for its remarkable antioxi-dant/reactive oxygen/nitrogen species (RONS) scavenging activity. Its neuroprotec-tive function is based on its ability to eliminate free radicals, it acts as an antioxidant and a free-radical scavenger, and it reduces lipid peroxidation, which decreases tissue levels of ROSs and inhibits membrane lipid peroxidation. Both flavonoid and ginkgolide constituents are involved in the antioxidan/free-radical-scavenging effects of EGb. The flavonoids preferentially react with hydroxyl radicals [17] and chelate pro-oxidant transition heavy metal ions [18], which consequently inhibits the formation of new hydroxyl radicals. Significant antioxidant activity is consequently one of the most analysed protective effects of EGb on the CNS and the circulatory system.

An extract can significantly eliminate free oxygen radicals (superoxide, hydrogen peroxide and hydroxyl radicals) and has also been found to be an inhibitor of NADPH-oxidase. Scavenging activity against peroxide radicals of EGb has been proved also on liposomes and human lipoprotein, which are believed to be involved in lipid peroxidation and human atherogenesis [19]. In addition, several studies showed that EGb is able to reduce endogenous lipoperoxidation [20, 21] and can alter the activity of endogenous antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) in the hippocampus, striatum and substantia nigra (SN) and decrease lipoperoxidation in rat hippocampus [22]. On synaptosomal preparations from the striatum of mice, EGb761 has been shown to prevent the alteration of the neuronal dopamine uptake system and the modifications of the membrane fluidity induced by a pro-oxidant system ascorbic acid/Fe2+ [23, 24]. The flavonoid fraction of EGb761 was implicated in these protections. This efficacy was also observed in vivo. Thus dopaminergic neurons were protected against the neurotoxin MPTP when mice received EGb761 2 weeks before the neurotoxin infused peripherally through an osmotic mini-pump for 7d [24]. The inhibition of monoaminooxidase (MAO) activity by EGb761 may be involved in this protection [25]. In an experimental model of hypoxia-induced phospholipid breakdown, it was found that bilobalide, a sesquiterpene lactone constituent of EGb761, inhibited activation of NMDA-receptor-induced phospolipase A2 and choline release in hippocampal slices [26]. EGb may play a protective role in the homeostasis of inflammation and oxidative stress, in the prevention of cell membrane damage caused by free radicals, and in neurotransmission modulation [1].

As shown by Ni et al. [27], EGb761 can prevent hydroxyl-radical-induced apop-tosis in cultured neurons. Numerous studies have shown that EGb761 alone or administrated with other compounds with antioxidative properties such as vitamin E provides significant protection of membrane integrity, depresses lipoperoxidation and positively affects immune function [20]. This is one of the reasons why EGb is used for the early-stage treatment of Alzheimer's disease to interrupt the proposed oxidative stress associated with the disease [28]. In addition, it has been established by Bastianetto [29] that EGb has a protective effect against NO-induced toxicity in hippocampal cells and causes significant depression of platelet aggregation induced by the ginkgolide b fraction of EGb with a lower incidence of venous thrombosis [30].

The treatment of lumbar spinal cord ischemia may have very important clinical implications. Mechirova and Domorakova [31] have shown reasonable neuroprotective effects of EGb by histochemical analysis of NADPH-diaphorase activity (NOS-like activity) on segments of the lumbar spinal cord in rabbit subjected to 30 min ischemia and 24 h reperfusion. Reduction of reperfusion damage has been observed and was based on the scavenging activity of EGb towards free radicals produced during ischemia/reperfusion (I/R) of the spinal cord. The number of NADPH-d-positive neurons in sections of the L5 segment of the spinal cord was elevated in the ischemic spinal cord and rabbits expressed complete paraplegia. Pretreatment by EGb for 7 d caused disappearance of paraplegia, and NADPH-d activity in blood vessels and neurons was observed, similar to the controls. The authors proposed that it is the free-radical-scavenging property of EGb which decreases free radicals produced during I/R and reduces reperfusion damage [31].

Cerebral I/R insult induces inhibition of the general translation of proteins in the neocortex as well as in the highly sensitive hippocampus where, without protection, inhibition of protein synthesis persists up to the death of neurons [32]. Interestingly, the mechanism of ischemic damage protection by EGb was studied by measuring the extent to which translation inhibition occurs in rats. Thus, due to its antioxi-dant and antiradical ability, it can significantly reduce the I/R-induced inhibition of translation in the neocortex as well as in the highly sensitive hippocampus. EGb, with its ability to protect the translation mechanism, permits the newly synthesized mRNAs to be translated into functional proteins, thus allowing the altered gene expression to be effective. Rats pretretated with EGb at a dose of 40 mg/kg/d for 7 d were subjected to a 4-vessel occlusion model of transient forebrain ischemia for 20 min followed by 30 min, 4 h or 7 d of reperfusion. Ischemia induced inhibition of the protein synthesis rate. The ability to reinitiate proteosynthesis was significantly reduced in the frontal cortex and hippocampus in EGb-treated animals. Moreover, as shown by Fluoro-Jade B staining, it alleviates neurodegeneration and increases number of surviving neurons in highly sensitive hippocampus [12].

In the same paradigm of global ischemic damage, prophylactic oral administration of EGb at a dose of 40 mg/kg/d for 7 d showed efficient protection of surviving neurons, particularly in the most vulnerable CA1 pyramidal cells after 20 min of ischemia. Although ischemia is lethal for most populations of the CA1 pyramidal cell layer, pretreatment with EGb increased the number of NeuN immunoreactive cells (surviving neurons) in spite of a similar number of reactive astrocytes immuno-labelled for GFAP (glial fibrilary acidic protein) in the stratum oriens and stratum lacunosum and moleculare [33]. As reported by the authors, the inhibition of free-radical formation afforded by EGb might explain the protection of the most vulnerable CA1 pyramidal cells against ischemic damage.

In our own laboratory, we assessed the antioxidant activity of EGb761 in in vitro experiments. The formation of TBA reactive substances (TBARS) was used as an index of lipoperoxidation (LPO) in all forebrain membranes [34]. The degree of peroxidation (lipoperoxidation index LPO) increased gradually with increasing time (30 to 60 min) of incubation in the medium containing 0.1 mmol/l FeSO4/EDTA plus 1 mmol/l H2O2/mg of protein. The addition of 50 or 100 |J.l/ml EGb761 significantly protects the generation of LPO products in cerebral membranes. In addition, oxidative modifications in protein measured by the content of total sulfhydryl group level was protected up to 75 to 81% by the addition of 50 or 100 |J.l/ml EGb.

Ischemia creates several conditions that could account for the increased net production of free radicals or an impairment of cellular defenses that normally protect against such damage. The massive production of free radicals during I/R was observed in plenty of studies by indirect spin-trapping measurements and chemilu-miniscence [35]. In our laboratory, we proved that the transient global forebrain I/R induced considerable lipoperoxidation and formation of lipoperoxidation-dependent products and direct oxidative products of neural proteins. The formation of products was time dependent and culminated in a later reperfusion period where delayed neuronal death could be observed [36-38]. In the study in our laboratory conducted on all tissue membranes, forebrain ischemia caused a small but significant increase of LPO assessed by the level of thiobarbuturic reactive substances (TBARS) and by measurement of lysine conjugate fluorescence. The TBARS level from sham-operated animals treated with EGb expressed a lower level of TBARS and in the reperfusion period reached slightly depressed values (by about 11%) compared to non-treated animals. In addition, ischemic insult caused a significant increase in levels of Lys fluorescence, which was pronounced after 24 h of reperfusion. Remarkably, in vivo treatment with EGb depressed fluorescence intensity, which suggests an anti-LPO activity after ischemic insult [34].

Ischemic insult induces oxidative modifications in protein structure, as was assayed by measurement of changes in levels of total SH groups, and alterations in tryptophane (Trp) and bityrosine fluorescence. Although ischemia significantly changed the level of SH groups during reperfusion, the EGb treatment caused a remarkable reversion of the SH-group level to non-ischemic conditions. Similarly, as shown by an analysis of fluorescence intensity of Trp and bityrosine ischemia-induced changes increased with time after ischemic insult. Pretreatment with EGb induced partial but significant recovery of Trp and bityrosine fluorescence intensity in comparison with non-treated animals. These results suggest that EGb761 has potent antioxidant activity and could act as an important factor which attenuates I/R-induced protein oxidation. The protective properties of EGb761 support its potential beneficial actions against ischemia-induced brain-related pathologies that are likely associated with the deleterious effects of reactive oxygen/nitrogen species imbalance [34].

Interestingly, the oxidative changes of neural cells induced by a brief global ischemic episode appeared mainly in the later reperfusion period, which is very similar to the time course of histopathologically assessed neuronal vulnerability [39]. In addition, oxidative protein alterations also follow disturbances in the oxidant/antioxidant balance and depression of enzymatic activities of the main antioxidant enzymes such as superoxide dismutase detected at later stages after ischemic insult in both gerbils and rats [40,41]. Thus, oxidative alterations of neural proteins after IRI may at least partially explain functional postischemic disturbances of neuronal ion transport mechanisms [42] and ischemia-induced inhibition of global pro-teosynthesis [32, 43], both of which are implicated in neuronal cell damage and/or recovery from ischemic insult.

Several lines of evidence suggest that EGb allows mitochondria to maintain their respiratory activity under ischemic conditions [44] and may play a protective role in the homeostasis of inflammation and oxidative stress [22]. The antioxidant and antiradical ability is at least partially responsible for a significant reduction in the IRI-induced inhibition of translation in the neocortex and hippocampus, where, without protection, inhibition of proteosynthesis persists up to the death of neurons [32]. The scavenging-free-radical activity of EGb has been proved in different in vitro and in vivo studies [21, 45, 46].

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