Neuroscience Letters 383 (2005) 295300

Rejuvenation of antioxidant system in central nervous system of aged rats by grape seed extract
Muthaiya Balu, Purushotham Sangeetha, Dayalan Haripriya, Chinnakannu Panneerselvam
Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, India Received 11 February 2005; received in revised form 13 April 2005; accepted 13 April 2005

Abstract Oxidative stress is considered as a major risk factor that contributes to age-related increase in lipid peroxidation and declined antioxidants in the central nervous system during aging. Grape seed extract, one of the bioavonoid, is widely used for its medicinal properties. In the present study, we evaluated the role of grape seed extract on lipid peroxidation and antioxidant status in discrete regions of the central nervous system of young and aged rats. Male albino rats of Wistar strain were divided into four groups: Group Icontrol young rats, Group IIyoung rats treated with grape seed extract (100 mg/kg body weight) for 30 days, Group IIIaged control rats and Group IVaged rats supplemented with grape seed extract (100 mg/kg body weight) for 30 days. Age-associated increase in lipid peroxidation was observed in the spinal cord, cerebral cortex, striatum and the hippocampus regions of aged rats (Group III). Activities of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and levels of non-enzymic antioxidants like reduced glutathione, Vitamin C and Vitamin E were found to be signicantly decreased in all the brain regions studied in aged rats when compared to young rats. However, normalized lipid peroxidation and antioxidant defenses were reported in the grape seed extract-supplemented aged rats. These ndings demonstrated that grape seed extract enhanced the antioxidant status and decreased the incidence of free radical-induced lipid peroxidation in the central nervous system of aged rats. 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Aging; Central nervous system; Free radicals; Lipid peroxidation; Antioxidant; Grape seed extract

Aging is the accumulation of changes responsible for the sequential alterations that accompany with advancing age and the associated progressive increase in the chance of disease and death. The free radical theory of aging is one of the most popular, single mechanistic theories of aging, which discloses increased generation of free radical as the major cause of cellular damage. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an important role in neurodegenerative disorders by oxidizing the macromolecules like proteins, DNA and lipids leading to the common nal pathway for cell death [13,37]. Such free radical-mediated damages are prevalent during aging which leads to age-associated diseases as like Alzheimers disease and Parkinsons disease [32].

Corresponding author. Tel.: +91 44 24480767; fax: +91 44 24926709. E-mail address: biobalu@gmail.com (C. Panneerselvam).

Brain is highly vulnerable to oxidative damage due to high utilization of inspired oxygen, the large amount of easily oxidizable polyunsaturated fatty acids, the abundance of redoxactive transition metal ions, and relative dearth of antioxidant defense system, etc. There are numerous endogenous antioxidants that constitute the bodys own natural defenses and limits free radical damage in neuronal tissues [26]. The increased oxidative damage during aging might be due to the insufciency of antioxidants [30]. Lipid peroxidation (LPO), a marker for oxidative damage, is associated with a progressive loss in membrane uidity, reduction in membrane potential, increase in membrane permeability to ions which nally leads to cellular damage [41]. Elevated levels of LPO are responsible for the decreased physiological performance and increased susceptibility to disease and death [39]. Brain accessible phytochemicals potentially defend against oxidative damage during aging [6]. Polyphenols of

0304-3940/$ see front matter 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.042

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grape seed extract have long been recognized to possess many properties, including antioxidant, anti-inammatory, anticarcinogenic, platelet aggregation inhibiting, and metal chelating properties, etc. [3]. Yamakoshi et al. showed that grape seed extracts are non-toxic to rats [40]. Interestingly, epidemiological studies pointed out moderate consumption of red wine, an alcoholic beverage, containing huge amount of polyphenols (proanthocyanin, reservatrol) reduces the incidence of certain age-related neurological disorders including macular degeneration and dementia [5]. Various reports have also shown that long-term dietary supplementation of poly phenols improved cognitive performance in aged rats [16]. In recent years, the French paradox has stimulated new interest to investigate whether grape seed polyphenols offer antioxidant benet to aging brain. In this study, we have investigated the role of grape seed extract on age-related changes in LPO and antioxidant system in various brain regions such as spinal cord, cerebral cortex, striatum, and hippocampus. Bovine serum albumin, malondialdehyde and reduced glutathione were purchased from Sigma chemical company (St. Louis, MO, USA). All other chemicals used were of analytical grade and were obtained from Glaxo Laboratories, CDH division, Mumbai, India and Sarabhai M.Chemicals, Baroda, India. Grapes, as large clusters with red berries, were bought from a local supermarket in Chennai and identied as Vitis vinifera. Grape seeds were removed from the grapes, air dried for 1 week and milled to a particle size of <0.4 mm. The grape seed powder (100 g) was macerated for 12 h at room temperature three times with 800 ml of 100 mM acetate buffer, pH 4.8, in water/acetone (30:70, v/v), each time. The three macerates were combined and concentrated until no acetone was left using a rotary evaporator under reduced pressure and a water bath temperature <35 C. The concentrated solution was extracted four times with 200 ml of ethyl acetate each time. The four ethyl acetate extracts were combined, evaporated to remove ethyl acetate and grape seed polyphenols was obtained as a lyophilized powder [42]. Healthy male albino rats of Wistar strain used in this study maintained and bred for more than two decades at Kings Institute of Preventive Medicine, Chennai. The animals were housed in large spacious cages and were given food and water ad libitum. The animal room was well ventilated with a 12 h light/12 h dark cycle, throughout the experimental period. Experimental animals were handled according to the regulations
Table 1 Effects of grape seed extract on LPO level in brain regions of aged rats Brain regions Spinal cord Cerebral cortex Striatum Hippocampus Group I 0.44 0.04 0.68 0.07 0.89 0.084 0.59 0.043 Group II

of the University and Institutional legislation, controlled by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. The animals were divided into four groups consisting of six animals each. Group I rats served as vehicle-treated young controls (34 months old; weighing about 140 20 g). Group II (young) rats received grape seed extract (100 mg/kg body weight) dissolved in saline by oral gavage for 30 days. Group III rats served as vehicle-treated aged rats (above 24 months old weighing about 400 20 g). Group IV (aged) rats received grape seed extract (100 mg/kg body weight) for 30 days by oral gavage. The rats were not fed with diluted grape seed extract primarily due to the fact that the preparation otherwise would confer a typical bitter taste. On completion of 30 days experimental period, all animals were killed by cervical decapitation. Brain was excised immediately and immersed in ice-cold physiological saline. Brain regions were separated according to the method of Glowinski and Iverson [12]. A 10% homogenate of the brain regions were prepared and used for biochemical analysis. Protein estimation was carried out as suggested by Lowry et al. [20]. LPO was assessed by the method of Okawa et al. [27]. The superoxide dismutase (SOD) activity was measured by the method reported by Marklund and Marklund [22]. Catalase (CAT) was measured by the method of Sinha [35] and glutathione peroxidase (GPx) was by the method of Rotruck et al. [31]. GSH was estimated by the method of Moron et al. [24] and ascorbic acid was assayed by protocols suggested by Omaye et al. [29]. Vitamin E estimation was performed using the method proposed by Desai [8]. Values were expressed as mean S.D. for six rats in each group. Statistical signicance of changes in different groups was evaluated by one-way ANOVA using SPSS software package for Windows. The statistical signicance at P < 0.001, P < 0.01 and P < 0.05 was considered to be significant. The body weight and food intake were not altered in the study animals that were treated with grape seed extract. Table 1 shows the levels of LPO in control and grape seed extract-treated young and aged rats. The spinal cord, cerebral cortex, striatum and the hippocampus regions of aged rats showed a highly signicant (P < 0.001) increase in the LPO. The levels of LPO were signicantly decreased (P < 0.001) on grape seed extract administration. Grape seed extract

Group III 0.58 0.05a,*** 0.94 0.09a,*** 1.29 0.11a,*** 0.81 0.08a,***

Group IV 0.48 0.04b,*** 0.75 0.07b,*** 0.83 0.08b,*** 0.64 0.06b,***

0.40 0.04 0.64 0.06 0.82 0.07 0.55 0.05

Values are expressed as mean S.D. for six rats in each group. Unit: LPO: nmol of MDA released/mg protein. a Comparisons are made between Group I and Group III. b Comparisons are made between Group III and Group IV. P < 0.001.

M. Balu et al. / Neuroscience Letters 383 (2005) 295300 Table 2 Effects of grape seed extract on activities of enzymic antioxidants in brain regions of aged rats Enzymic antioxidants Superoxide dismutase Spinal cord Cerebral cortex Striatum Hippocampus Catalase Spinal cord Cerebral cortex Striatum Hippocampus Glutathione peroxidase Spinal cord Cerebral cortex Striatum Hippocampus Group I 1.61 0.14 1.34 0.12 0.86 0.08 1.42 0.14 3.56 0.35 3.48 0.35 2.30 0.25 2.70 0.28 3.44 0.33 2.04 0.17 1.75 0.17 2.50 0.24 Group II 1.71 0.15 1.42 0.13 0.91 0.09 1.54 0.13 3.80 0.33 3.60 0.38 2.56 0.21 2.91 0.29 3.62 0.37 2.17 0.19 1.70 0.14 2.68 0.22 Group III 1.12 0.12a,** 0.85 0.08a,** 0.48 0.04a,** 0.96 0.09a,** 2.86 0.28a,*** 2.64 0.26a,*** 1.59 0.15a,*** 1.76 0.18a,*** 2.71 0.26a,** 1.44 0.15a,** 1.13 0.10a,** 1.88 0.19a,** Group IV

297

1.45 0.13b,** 1.28 0.12b,** 0.71 0.07b,** 1.26 0.14b,** 3.36 0.36b,** 3.23 0.31b,** 2.10 0.26b,** 2.30 0.21b,* 3.22 0.30b,* 1.77 0.17b,* 1.64 0.14b,* 2.39 0.23b,*

Values are expressed as mean S.D. for six rats in each group. Units: SOD: units/mg protein (1 U = amount of enzyme that inhibits the autooxidation of pyrogallol by 50%); CAT: micromoles of H2 O2 consumed/min/mg protein; Gpx: micromoles of GSH oxidized/min/mg protein. a Comparisons are made between Group I and Group III. b Comparisons are made between Group III and Group IV. P < 0.05. P < 0.01. P < 0.001.

administration did not bring any marked change in the levels of LPO in young rats. Table 2 represents the activities of enzymatic antioxidants in brain regions of young and aged rats before and after supplementation with grape seed extract. The status of SOD activity was found to be signicantly decreased in the spinal cord, cerebral cortex, striatum and the hippocampus (30%, 36%, 44% and 32%, respectively) regions of aged rats compared to young rats. Administration of grape seed extract for 30 days signicantly augmented the SOD activity in aged rats (29%, 50%, 47% and 31%, respectively) when compared to aged control rats. The activity of CAT was signicantly decreased in the spinal cord, cerebral cortex, striatum and the hippocampus regions of aged rats (19%, 24%, 30% and 34%, respectively) when compared to young rats. The grape seed extract increased the CAT activity in aged rats in a significant manner (17%, 22%, 30% and 30%, respectively). A marked decline in the activity of GPx observed in the spinal cord, cerebral cortex, striatum and the hippocampus regions of aged rats (21%, 29%, 35% and 24%, respectively) when compared to young rats. Supplementation of grape seed extract increased the GPx activity in aged rats compared to that of young control rats. The increase being 18% in spinal cord, 22% in cerebral cortex, 45% in striatum and 27% in the hippocampus when compared with aged rats. Table 3 shows the levels of non-enzymic antioxidants in the brain regions of control and experimental rats. The level of GSH was found to be signicantly decreased (1.31-, 1.35-, 1.52- and 1.32-fold) in the spinal cord, cerebral cortex, striatum and the hippocampus regions of aged rats when compared to young rats. A signicant (1.26-, 1.16-, 1.29- and 1.2-fold, respectively) increase of GSH level was observed

in grape seed-administered aged rats. The level of Vitamin C was found to be signicantly decreased (30%, 24%, 35% and 45%, respectively) in the spinal cord, cerebral cortex, striatum and the hippocampus regions of aged rats. Administration of grape seed extract for 30 days signicantly (31%, 24%, 33% and 47%, respectively) improved the Vitamin C level in aged rats when compared to aged control rats. There was a signicant (P < 0.001) decrease in the level of Vitamin E in the spinal cord, cerebral cortex, striatum and the hippocampus regions of aged rats when compared to young rats. Aged rats supplemented with grape seed extract showed a marked elevation (P < 0.01, P < 0.001, P < 0.001 and P < 0.001, respectively) in the level of Vitamin E compared to control aged rats. Supplementation of grape seed extract to young rats did not bring any signicant alteration in any of the parameters investigated. Aging is often dened as the loss of function that accompanies with advance of chronological years and for many it is the loss of physiological and motor function that causes the greatest concern. Oxidative stress is considered as a risk factor and contributing to age-related increase in oxidized lipids and proteins in the central nervous system during aging that ultimately leads to cellular damage [33]. LPO is a marker of oxidative stress and also one of the prime factors involved in cellular damage caused by free radicals. The results of the present study showed that the LPO levels were signicantly increased in the discrete brain regions of aged rats when compared with young rats. Mitochondria act like a timer that ticks all the way through the aging process. Under normal physiological conditions, a small fraction of oxygen consumed by mitochondria is constantly converted to

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Table 3 Effects of grape seed extract on activities of non-enzymic antioxidants in brain regions of aged rats Non-enzymic antioxidants Reduced glutathione Spinal cord Cerebral cortex Striatum Hippocampus Vitamin C Spinal cord Cerebral cortex Striatum Hippocampus Vitamin E Spinal cord Cerebral cortex Striatum Hippocampus Group I 0.059 0.005 0.046 0.005 0.026 0.003 0.033 0.003 0.50 0.04 0.33 0.03 0.37 0.03 0.29 0.03 3.07 0.29 2.29 0.22 1.41 0.13 1.42 0.15 Group II 0.065 0.052 0.029 0.039 0.53 0.35 0.41 0.031 3.27 2.47 1.53 1.49 0.006 0.005 0.003 0.003 0.05 0.04 0.036 0.03 0.33 0.21 0.14 0.14 Group III 0.045 0.005a,*** 0.034 0.004a,*** 0.017 0.002a,*** 0.025 0.002a,*** 0.35 0.4a,*** 0.25 0.02a,** 0.24 0.02a,*** 0.17 0.02a,*** 2.14 0.26a,** 1.70 0.16a,*** 0.97 0.11a,*** 0.88 0.09a,*** Group IV 0.057 0.005b,** 0.042 0.004b,* 0.022 0.002b,** 0.030 0.003b,* 0.46 0.04b,* 0.31 0.027b,** 0.32 0.03b,** 0.25 0.31b,** 2.71 0.27b,** 2.08 0.21b,*** 1.25 0.12b,*** 1.27 0.12b,***

Values are expressed as mean S.D. for six rats in each group. Units: GSH, Vitamin C, Vitamin E: g/mg protein. a Comparisons are made between Group I and Group III. b Comparisons are made between Group III and Group IV. P < 0.05. P < 0.01. P < 0.001.

superoxide anions, hydrogen peroxide, hydroxyl radicals and other ROS [17]. The observed difference in the levels of LPO products in distinct brain regions may be due to the differences in their oxygen consumption rate, which inuences the generation of ROS. Though an aged brain as a whole would be susceptible to injury by free radicals, the cerebral cortex and striatum are more prone to oxidative damage due to a higher oxygen consumption rate in those regions [9]. The presently observed reduction of LPO by grape seed extract administration in aged rats suggests the free radical scavenging role of avonoids and their capacity to chelate redox-active metals [1]. SOD plays a key role in detoxifying superoxide anions, which otherwise damages the cell membranes and macromolecules. The activity of this enzyme showed a signicant reduction with aging in the regions that are vulnerable to oxidative damage such as cortex and striatum. This might be due to the increased iron content in these regions hence it becomes feasible to propose that superoxide radicals form via the Fenton reaction [28]. Baek et al. [2] previously showed a close relationship between increased ROS production and decreased SOD activity in brain regions. Declined SOD activity in aged tissue was brought back to near normal level upon grape seed extract administration. This observation divulges that grape seed extract may act as a potent scavenger of superoxide radicals and metal chelator [3]. CAT has the capability to detoxify H2 O2 radicals. Release of H2 O2 promotes the formation of numerous other oxidant species that greatly contributes for oxidative stress leading to the pathogenesis of aging. The over expression of CAT in neuronal cells provides protection against H2 O2 -induced toxicity that subsequently leads to the activation of endonu-

clease digestion of DNA into oligosomes [21]. Decrease in CAT activity was observed signicantly in the striatum and the hippocampus regions of aged rats. The accelerated oxidation of dopamine by monoamine oxidase in the nerve terminals of the striatal neurons has been suggested to increase H2 O2 production in these neurons [38]. This may lead to decline in the CAT activities in these regions. The grape seed extract-supplemented group showing an increase in CAT activity may be explained by the free radical quenching action of di-OH (catechol) structure in the B ring of proanthocyanin present in the grape seed extract [15]. The most important antioxidant enzyme, GPx efciently protects the cell membrane from LPO. It catalyzes the reaction of hydroperoxides with GSH to form glutathione disulphide. GPx, which uses GSH as a proton donor, converts H2 O2 to water and molecular oxygen; in this process GSH is oxidized to GSSG. In our present study, the activity of this enzyme was found to be turn down in aged rats. The decrease in the activity of GPx in the striatum of aged brain could be attributed to declined GSH concentrations in this region [4]. Increase in the GSH availability may be responsible for the increase in the activity of GPx in grape seed extract-treated aged rats. Apart from enzymic antioxidants, various non-enzymic antioxidants also play an important role in preventing cells from oxidative threats. In the present study, the levels of nonenzymic antioxidants like GSH, Vitamin E and Vitamin C were signicantly decreased in aged rats. GSH, a tripeptide composed of l-glutamate, l-cysteine and l-glycine may act as a redox modulator of ionic receptors that serve as a neuroprotectant in glutamatergic excitotoxicity, and as neurotransmitters. The protective capacity of GSH is due to the reactive

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sulfhydryl cysteine moiety, which can bind to electrophilic sites on xenobiotics and endogenous toxins [23]. The observed decrease in GSH concentration in striatum of aged rat may be due to the enhanced dopamine turnover, which associated with increased formation of GSSG [34]. Grape seed extract treatment signicantly increased the level of GSH in aged rats. This could be explained by the expression of glutamyl cysteine synthetase, the rate-limiting enzyme for GSH synthesis in avonoids [25] present in grape seed extract. Ascorbate, belonging to the water-soluble class of vitamins, readily donates electrons to break the chain reaction of LPO. Furthermore, ascorbic acid regulates the activity of some neurons within the brain. Some of these functions include neurotransmitter membrane receptor synthesis, and neurotransmitter dynamics [18]. In the present study, we found that ascorbic acid concentration was signicantly reduced with age particularly in striatum and hippocampus since these areas are particularly vulnerable to oxidative stress. For example, an increase in 8-oxo-deoxyguanosine, and an increase in nitrotyrosine have been shown in the hippocampus [7,36]. Similarly increased oxidative DNA damage has been shown in the striatum of aged animals [11]. Our results show that grape seed extract is capable of reversing age-associated decline of Vitamin C in the aged rats. The possible reason for the observed increase in the levels of ascorbate are (i) increase in ascorbic acid absorption, (ii) stabilization of ascorbic acid, (iii) reduction of dehydroascorbate to ascorbate, [14] and (iv) metabolic sparing of ascorbic acid by avonoids [43]. The only membrane-bound lipid-soluble antioxidant, Vitamin E plays a key role in preventing cellular injury from oxidative stress associated with premature aging. At the cellular level, Vitamin E deciency promotes increased LPO, making cells more vulnerable to oxidative injury [10]. The observed decrease in Vitamin E concentration in hippocampus of aged rats may be due to the decreased concentration of Vitamin C in this region. Supplementation of grape seed extract enhanced the level of -tocopherol in aged rats. Phenolics and some avonoids, owing to their intermediate redox potential and physiochemical characteristics, can possibly act an interface between ascorbate and tocopherol. Ascorbic acid has been shown to directly regenerate the tocopherol from -tocopherol radicals, and is thus an integral part of the redox antioxidant-recycling network [19]. Hence, grape seed extract brought back the levels of Vitamin E by improving the levels of ascorbic acid in aged rats. To conclude, the results of the present study clearly demonstrate an age-linked increase in LPO and decline in the activities of enzymic and non-enzymic antioxidants in central nervous system of aged rats. This study also divulges that dietary supplementation of grape seed extract enhances antioxidant status in aged rats by reducing the levels of LPO end products, whose accumulation otherwise, would play a key role in brain aging.

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