Flow Cytometric Estimation of the Effect of Ginkgo Biloba Extract on the Content of Hydrogen Peroxide in Dissociated Mammalian Brain Neurons

Abstract

ABSTRACT-The effect of Ginkgo biloba extract (GBE) on the content of hydrogen peroxide was estimated in cerebellar neurons dissociated from rats, by means of a flow-cytometer and 2',7'-dichloro fluorescin (DCF) diacetate, a fluorescent dye for intracellular hydrogen peroxide. The GBE started to reduce the DCF fluorescence of the neuron at 0.1 ,ug/ml to 0.3 ,ug/ml. Further increases in the GBE concentration (up to 3,ug/ml) produced a dose-dependent decrease in the DCF fluorescence, suggest ing that GBE reduces the content of hydrogen peroxide or suppresses the reactive oxygen species (ROS) formation of cerebellar neurons. The present technique may be useful for preliminary evalu ations of agents affecting the ROS formation in mammalian brain neurons. Keywords:Ginkgo biloba extract, Brain neuron, Hydrogen peroxideIt is widely accepted that there are reactive oxygen species (ROS) that may be involved in ischemic and/or postischemic brain damage (1-4). Thus, the membrane phospholipids of the brain neurons undergo peroxida tion induced by the ROS because of their high content of polyunsaturated fatty acids. Therefore, agents that prevent ROS formation are expected to exert some protective actions on brain neurons subjected to ische mia. ROS such as superoxide anion, hydrogen peroxide and hydroxyl radicals are produced in biological sys tems even under normal conditions (5). However, there is no conventional technique for evaluating the effect of agents on intracellular ROS formation in dissociated mammalian brain neurons.2',7'-Dichlorofluorescin diacetate (DCFH-DA) dif fuses across the cell membrane and is then trapped by cellular deacetylation. Intracellular hydrogen peroxide oxidizes this compound to 2',7'-dichlorofluorescin (DCF), which is highly fluorescent (6). Since this prop erty of DCFH-DA has been utilized to estimate the ROS formation in neutrophils and polymorphonuclear leukocytes by measuring intracellular hydrogen perox ide (7-9), we have designed an approach to estimate the effect of the agent on the intracellular hydrogen peroxide content on dissociated mammalian brain neurons, using DCFH-DA and a flow cytometer. The present studies were designed to determine if the ex tract of Ginkgo biloba (GBE) is able to affect the hydrogen peroxide content of mammalian brain neurons, because the GBE, which contains flavone gly cosides, biobalide and ginkgolides (10), has been re ported to protect the brain against hypoxic damage, prolong the survival of rats (11) and to inhibit the ROS formation of leukocytes (12).Experiments were performed on cerebellar neurons dissociated from 1 to 2-week-old Wistar rats. The tech niques for dissociation of rat cerebellar neurons and measurement of fluorescence were previously reported (13). Briefly, the slices (0.5 mm in width) of rat cere bellum were treated with dispase (103 PU/ml; Godo Shusei Co., Japan) for 60 min in Tyrode's solution at the temperature of 35-36'C. Thereafter, the cerebellar neruons were dispersed by gentle pipetting in Tyrode's solution. Tyrode's solution ...

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“…It has been used for the treatment of various common geriatric complaints including vertigo, short term memory loss, and lack of attention or vigilance. The standardized extract of G. biloba has been shown to have effects on cerebral vascular disorders (10) and possesses anti-oxidant (11,12) and neuro-protective properties (13). Recently G. biloba has received attention as potential cognitive enhancers (14) for the treatment of Alzheimer's disease (15).…”