Hypericum perforatum extract demonstrates antioxidant properties against elevated rat brain oxidative status induced by amnestic dose of scopolamine

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Abstract

This study was designed to investigate if the impairment of learning and memory induced by acute administration of scopolamine (1.4 mg/kg ip) in rats is associated with altered brain oxidative stress status. The passive avoidance paradigm was used to assess retrieval memory of rats after scopolamine treatment. Following retrieval testing, biochemical assessments of malondialdehyde (MDA), glutathione peroxidase (GSHPx), glutathione (GSH), and superoxide dismutase (SOD) levels/activities as oxidative stress indices were performed. This study also investigated the effect of acute administration of Hypericum perforatum extract (4.0, 8.0, 12.0, and 25.0 mg/kg ip), containing flavonoids with documented antioxidant activity, on brain oxidative status of naı̈ve rats treated with amnestic dose of scopolamine. Results showed that administration of 1.4 mg/kg of scopolamine impaired retrieval memory of rats and that such amnesia was associated with elevated MDA and reduced GSH brain levels. In naı̈ve rats, which have not been exposed to conditioned fear, scopolamine administration also increased MDA and reduced GSH levels, although with an increase in brain GSHPx activity. Pretreatment of the animals with Hypericum extract (4, 8, and 12 mg/kg) resulted in an antioxidant effect through altering brain MDA, GSHPx, and/or GSH level/activity. Since oxidative stress is implicated in the pathophysiology of dementia, the findings of this study may substantiate the value of scopolamine-induced amnesia in rats as a valid animal model to screen for drugs with potential therapeutic benefit in dementia. Exposure of animals to conditioned fear may be suggested to impair the balance between the rate of lipid peroxidation and the activation of GSHPx as a compensatory antioxidant protective mechanism. It is also concluded that low doses of Hypericum extract, demonstrating antioxidant activity, may be of value for demented patients exhibiting elevated brain oxidative status. Since depression commonly coexists with dementia, Hypericum extract as a drug with documented antidepressant action may also be a better alternative than several other antidepressant medications that have not been evaluated to test their effect on brain oxidative status during amnesia.

Introduction

Partially reduced forms of oxygen are produced in the brain during cellular respiration and, at accelerated rates, during brain insults. This increase in production of free radicals has been reported to cause damage to cell membranes, enzymes, DNA, lipids, and proteins, impairing their function (Gu et al., 1998). Oxidative stress is a disparity between the rates of free radical production and elimination through endogenous antioxidant mechanisms such as the enzymes superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase, as well as the low molecular weight reductants alpha-tocopherol, glutathione (GSH), and ascorbate (Wilson, 1997). This imbalance is initiated by numerous factors including acidosis, transition metals, nitric oxide, dopamine, glutamate, amyloid beta-peptide, and uncouplers of mitochondrial electron transport. Lipid peroxidation is thought to be a prominent and especially deleterious form of neuronal oxidative injury damaging membranes and generating several secondary products, both from fission and endocyclization of oxygenated fatty acids that possess neurotoxic activity (Bassett and Montine, 2003). Increased level of malondialdehyde (MDA) as one of the reactive oxidative species (ROS) has been shown to be a reliable index of in vivo lipid peroxidation (Ilic et al., 1999). On the other hand, the tripeptide GSH as a redox regulator participates in the maintenance of oxidant homeostasis and the cellular detoxification of ROS in brain cells (Cruz et al., 2003). GSH depletion has been shown to affect mitochondrial function probably via selective inhibition of mitochondrial complex I activity (Bharath et al., 2002). Therefore, compromised GSH system in the brain has been considered as a relevant index of neuronal oxidative stress (Dringen and Hirrlinger, 2003). Enhanced expression/activity of the endogenous antioxidant enzymes SOD and GSHPx has also been commonly used as relevant indices of brain oxidative stress Ilic et al., 1999, Maier and Chan, 2002.

Several clinical research findings implicated oxidative stress in the pathophysiology of dementia among other age-related neurodegenerative disorders Cruz et al., 2003, Floyd, 1999. Impairment of learning and memory, as the most characteristic manifestation of dementia, could be induced chemically in experimental animals by administration of scopolamine, a cholinergic antagonist known to interfere with acetylcholine transmission in the central nervous system (Misane and Ogren, 2003). This experimental animal model of scopolamine-induced amnesia has been extensively used in research to screen for drugs with potential therapeutic value in dementia Bejar et al., 1999, de Angelis and Furlan, 1995, Hiramatsu et al., 1998b, Mishima et al., 2003, Rubaj et al., 2003. Nevertheless, brain oxidative status in this experimental animal model of scopolamine-induced amnesia has yet to be evaluated. Therefore, this study aimed at investigating whether such impaired cognition due to scopolamine administration is associated with altered oxidative stress indices. A step-through passive avoidance paradigm was used to evaluate the effect of scopolamine (1.4 mg/kg ip) on memory function. Following retrieval testing, biochemical assessments of brain MDA, GSH, GSHPx, and SOD levels/activities, as oxidative stress indices, were conducted.

Hypericum perforatum extract contains flavonoids such as rutin, quercetin, and quercitrin, which demonstrated a free radical scavenging activity in a model of autooxidation of rat cerebral membranes (Saija et al., 1995). An antioxidant activity of quercetin was also demonstrated by inhibition of brain lipid peroxidation, as manifested by lowering MDA while elevating phospholipid contents in a rat model of endotoxemia (Abd El-Gawad and Khalifa, 2001). Therefore, Hypericum extract, with a potential antioxidant activity, may be of value in dementia among other disorders of senility in which free radical generation is implicated. In addition, since depression commonly coexists with dementia, there is need to test the effect of different antidepressant medications on elevated brain oxidative status during amnesia Bassuk et al., 1998, Gallassi et al., 2001, Palsson et al., 1999. Nevertheless, studies investigating the effect of antidepressants, with potential antioxidant activity, on elevated brain oxidative status are lacking. Hypericum perforatum extract has been used as an antidepressant in folk medicine for over 2000 years (Maidment, 2000). Today, it is best known for its use in the treatment of mild to moderately severe depressive disorders (Barnes et al., 2001). Hypericum extract has been always referred to have a benign side-effect profile compared to tricyclic antidepressants and serotonin-specific reuptake inhibitors (Vitiello, 1999). There has not been a single fatal intoxication of the extract as a monotherapy reported in the literature (Kasper, 2001). Hypericum extract, as an efficacious antidepressant medication with a benign side effect profile together with a potential antioxidant activity, was therefore hypothesized to be a better alternative to other antidepressants for depressed demented elderly patients exhibiting elevated oxidative stress status. Therefore, this study also aimed at investigating the effect of Hypericum extract, in doses equivalent to the ones used clinically for depression, on brain MDA, GSH, GSHPx, and SOD levels/activities in naı̈ve rats treated with amnestic dose of scopolamine.

Section snippets

Drugs and animals

Chinese Hypericum perforatum was grown in Hebei province and was harvested in August. The aboveground parts (leaves, flowers, and stem) were dried before extraction with 80% ethanol (vol/vol). The herb-to-extract ratio is 12:1 for a 100% native extract. The dried extract was obtained from China National Corporation of Traditional & Herbal Medicine, Beijing, China. The extract solutions were prepared according to Good Manufacturing Practice rules and the quality and identity of the constituents

Experiment 1

Mann–Whitney U test showed that scopolamine administration (1.4 mg/kg) before retrieval testing resulted in shorter latency to step-through during the test session compared to the control group (U=2.0, P<.001) (Fig. 1). Student's t test showed that in animals exposed to conditioned fear such amnestic dose of scopolamine caused about twofold increase in brain MDA level while reducing GSH level by 69%. Nevertheless, scopolamine administration did not affect either brain GSHPx or SOD activities

Discussion

In this study, administration of scopolamine (1.4 mg/kg ip) 30 min before retrieval testing induced amnesia reflected by reducing step-through latencies of animals beyond their normal controls. The resultant impairment of memory function after administration of 1.4 mg/kg of scopolamine substantiate previous research finding of induced amnesia in rats intraperitoneally injected with the same dose of scopolamine (Wanibuchi et al., 1994). In animals exposed to conditioned fear, scopolamine

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