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Senescence mediated redox imbalance in cardiac tissue: Antioxidant rejuvenating potential of green tea extract
Nutrition 25 (2009) 847–854
Basic nutritional investigation
www.nutritionjrnl.com
Senescence mediated redox imbalance in cardiac tissue: Antioxidant rejuvenating potential of green tea extract Vadivel Senthil Kumaran, M.Sc., Karpagavinayagam Arulmathi, M.Sc., and Periandavan Kalaiselvi, Ph.D.* Department of Medical Biochemistry, DR.ALM Post-Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, India Manuscript received July 13, 2008; accepted February 16, 2009
Abstract
Objective: The activities and capacities of antioxidant systems of tissue cells are declined during aging, leading to the gradual loss of pro-oxidant/antioxidant balance and accumulation of oxidative damage. Hence, the present study evaluated the role of green tea extract (GTE), rich in polyphenols, in combating age-associated macromolecular damage in rat cardiac tissue. Methods: The antioxidant defense system, lipid peroxidation, protein oxidation, and redox status in heart tissue were studied using young and aged rats. Results: Significant increases in lipid peroxidation, protein carbonyls, and marked decreases in glutathione redox status, protein thiols, and activities of enzymatic and non-enzymatic antioxidants were observed in aged rats compared with young rats. Supplementation of GTE (100 mg/kg of body weight per day) orally for 30 days ameliorated these changes significantly. Conclusion: This study accredits GTE’s antioxidant rejuvenating potency and its role in the amelioration of senescence-mediated redox imbalance in aged rat cardiac tissue. Ó 2009 Elsevier Inc. All rights reserved.
Keywords:
Aging; Green tea extract; Heart; Oxidative stress; Antioxidants; Redox status
Introduction Aging, an aspect of almost all living organisms, is a multifactorial process of enormous complexity and is characterized by impairment of various cellular functions. Cardiac senescence, or cardiac aging, is referred to as a dramatic decline in cardiac pump function and contractile reserve with advanced age. It is an irreversible biological process that contributes to high cardiovascular mortality [1,2]. Several rationales have been speculated for cardiac aging, of which accumulation of oxidative macromolecular damage may lead to abnormal cardiac contractile function [2–4]. The accumulation of oxidative derivatives is because of the inefficient antioxidative defense system to scavenge reactive
This work was supported by a Junior Research Fellowship awarded in the form of TNJRF by the University of Madras, Chennai, Tamil Nadu, India. *Corresponding author. Tel.: þ91-44-2454-1767; fax: þ91-44-24540709. E-mail address: [email protected] (P. Kalaiselvi). 0899-9007/09/$ – see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.02.005
oxygen species (ROS) [5]. There is strong evidence to link the unfavorable accumulation of ROS and the resulting oxidative damages with the aging process and human diseases [6,7]. Oxidative stress, an apoptosis-inducing signal, may also increase in the aging heart. Several defense methods have evolved within the organism to recognize the levels of ROS and to limit the damage they inflict. The defenses include enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and non-enzymatic antioxidants such as ascorbate, a-tocopherol, and glutathione (GSH) [8]. The defense against the oxidant declines during the aging process [9,10] and it can be enhanced by supplementation of antioxidants. Green tea is the one, derived from the plant, Camellia sinensis, that has attracted significant attention recently in the scientific and consumer communities for its extensive health benefits for many disorders. Historically, green tea has been consumed by the Japanese and Chinese populations for centuries and probably is the most consumed beverage, besides water, in Asian society. There are several polyphenolic catechins present in green tea extract (GTE)
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such as ()-epicatechin (EC), ()-epicatechin-3-gallate (ECG), ()-epigallocatechin (EGC), and ()-epigallocatechin-3-gallate (EGCG). The protective effect of green tea in cardiovascular diseases is thought to stem from its antioxidant activity [11,12]. With all this background, this study was designed to evaluate the role of GTE in augmenting the function of antioxidants and to assess its potency to mitigate the macromolecular damage in aged rat heart tissue. Materials and methods Fresh green tea leaves were obtained from Nilgiris, Tamil Nadu, India. The leaves were dried in the shade and ground well into a fine powder in a mill with the temperature of the container maintained at lower than 50 C. Then the extract was prepared as described by Yang et al. [13], with slight modification. Tea powder was extracted with 95% ethanol (1:10, w/v) for 2 d with constant stirring. Ethanol was used to extract catechin because it has been reported to be an ideal solvent for the high recovery of tea catechins. Suspensions were filtered through Whatman No. 1 filter paper to retain the clear solution. The residue was extracted again. The pooled tea solution was vacuum evaporated at lower than 50 C and the concentrate was dried well, lyophilized, and stored at 4 C. Analysis of GTE The percentage of the catechin content of the GTE was analyzed using High Performance Liquid Chromatography (Column C18 phenomenex; Gemini, Tomance, CA, USA) according to the method of Anandh Babu et al. [14], with slight modifications. The column was C18 and the mobile phase consisted of phosphate buffer (pH adjusted to 3.5 with acetic acid) and methanol in the ratio of 97:3. Twenty microliters of the sample were injected into the column and the flow rate was maintained at 0.8 mL/min. The elution was detected by an ultraviolet-visible detector (SPD 20A) at 254 nm. Chemicals Bovine serum albumin (BSA) was purchased from Sigma (St. Louis, MO, USA). All other chemicals used were of analytical grade and were obtained from Sisco Research Laboratory (Mumbai, India). Animals Male albino rats of Wistar strain, weighing approximately 130 to 160 g (young, 3–4 mo old) and 380 to 410 g (aged, 24 mo old, maximum lifespan is 3ˇ1/2 y), used in this study were procured from the central animal house facility, Taramani Campus, University of Madras. They were healthy animals and maintained in a clean rodent room. 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/dark cycle throughout the experimental period. Experimental animals were handled according to university and institutional legislation, regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India, and approved by the institutional animal ethical committee (IAEC No. 02/079/07). Experimental design The animals were divided into four groups consisting of six animals in each group: group I, control young rats (received vehicle alone); group II, young rats (received GTE); group III, control aged rats (received vehicle alone); and group IV, aged rats (received GTE). Green tea extract (100 mg/kg of body weight per day) was dissolved in physiological saline (0.89%) and administered for 30 d by oral gavage. On completion of the experimental period, the animals were maintained in a fasting condition for 12 h and thereafter all animals were sacrificed by cervical decapitation. The heart was excised immediately and immersed in ice-cold physiological saline. A 10% homogenate was prepared using 0.01 M Tris HCl buffer, pH 7.4, and stored at 80 C until analyses were performed. Biochemical assays Lipid peroxidation (LPO) was determined by making use of the procedure of Ho¨gberg et al. [15]. One of the major secondary products of LPO is malondialdehyde (MDA). Malondialdehyde and other by-products react with thiobarbituric acid (TBA) to generate a colored product that absorbs at 532 nm, representing the color produced by all thiobarbituric acid-reactive substances (TBARS). The ferrous sulfate (FeSO4) and ascorbate-induced LPO system contained 10 mM FeSO4 and 0.2 mM ascorbate as inducers [16]. The amount of thiobarbituric acid-reactive substances was calculated using a molar extinction coefficient of 1.56 3 105 $ M1 $ cm1. The results were expressed as nanomoles of malondialdehyde released per milligram of protein. Protein carbonyl content was analyzed as described by Levine et al. [17] using 2,4-dinitrophenylhydrazine (DNPH). The difference in absorbance between the 2,4-dinitrophenylhydrazine–treated and the HCl-treated samples are determined at 366 nm and the results were expressed as nanomoles of carbonyl groups per milligram of protein. The protein carbonyl content was calculated based on a molar extinction coefficient of 22 000 $ M1 $ cm1 for 2,4-dinitrophenylhydrazine. The level of thiols was evaluated by the method of Sedlak and Lindsay [18] and expressed as micrograms per milligram of protein. The level of ascorbic acid was estimated by the method of Omaye et al. [19] and expressed as micrograms per milligram of protein. The level of a-tocopherol was measured by the method of Desai [20] and expressed as micrograms per
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milligram of protein. The level of GSH was measured by the method of Moron et al. [21]. Oxidized glutathione (GSSG) was measured by the method of Asensi et al. [22]. The redox index was calculated as (GSH þ 2 3 GSSG)/(2 3 GSSG 3 100), as reported by Oztu¨rk and Gu¨mu¨slu¨ [23]. SOD (E.C. 1.15.1.1) activity was measured by the method reported by Marklund and Marklund [24]. The degree of inhibition of the auto-oxidation of pyrogallol at an alkaline pH by SOD was used as a measurement of enzyme activity. The final results were expressed as units per minute per milligram of protein. CAT (E.C. 1.11.1.9) activity was measured by the method of Sinha [25] and expressed as unit micromoles of H2O2 consumed per minute per milligram of protein. GPx (E.C. 1.11.1.6) activity was assayed as described by Rotruck et al. [26]. The kinetic change was recorded at 420 nm immediately and the activity was expressed as units, which is defined as micromoles of GSH oxidized per minute per milligram of protein. Glutathione reductase (GR; E.C. 1.6.4.2), which utilizes reduced nicotinamide adenine dinucleotide phosphatase (NADPH) to convert GSSG to GSH, was assayed by the method of Staal et al. [27] and expressed as nanomoles of NADPH oxidized per minute per milligram of protein. The activity of glucose-6-phosphate dehydrogenase (G6PDH; E.C. 1.1.1.49) was estimated by the method of Ellis and Kirkman [28] and expressed as micrograms of NADP reduced per minute per milligram of protein. Aspartate transaminase (AST) was determined and expressed in terms of micromoles of pyruvate liberated per minute per milligram of protein at 37 C [29]. The protein content was determined according to the method of Lowry et al. [30] using bovine serum albumin as a standard. Statistical analysis Values were expressed as mean 6 standard deviation for six rats in each group. Statistical significance of changes in different groups was evaluated by one-way analysis of variance using SPSS 7.5 for Windows (SPSS Inc., Chicago, IL, USA). Post hoc testing was performed for intergroup comparisons using the least significant difference test. Statistical significance was set at P < 0.001, P < 0.01, and P < 0.05.
Results Composition of GTE Green tea extract contains volatile oils, vitamins, minerals, and caffeine but the primary constituents of interest are the polyphenols, particularly catechins. The percentage of catechins present in GTE was obtained from high-performance liquid chromatographic analysis and is presented in Figure 1. High-performance liquid chromatographic analysis showed that catechins are the major component of GTE. Nearly 72% of the extract was a mixture of catechins, namely EC, ()-epicatechin-3-gallate, ()-epigallocatechin, and EGCG,
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Fig. 1. Major components of green tea extract were determined as a percentage of the weight of the extract. All values are means 6 SDs of three replicates. EC, ()-epicatechin; ECG, ()-epicatechin-3-gallate; EGC, ()-epigallocatechin; EGCG, ()-epigallocatechin-3-gallate.
of which EGCG concentration is high compared with other catechins. Body weight Table 1 lists the body weight of young and aged rats before and after supplementation of GTE. No significant difference in body weight was detected between initial and after 30-d treatment with GTE in all four groups. Lipid peroxidation To evaluate the possible consequences of oxidative stress, LPO (basal, FeSO4, and ascorbate induced) was analyzed. Figure 2A shows the effect of GTE on levels of LPO in young and aged rats. There was a significant (P < 0.001) increase in LPO levels in aged rats compared with young rats. Aged control rats showed 2.5-fold increase in basal LPO, 1.8-fold increase for FeSO4-induced LPO, and 1.9-fold increase in ascorbate-induced LPO. GTE administration lowered basal LPO, FeSO4-induced, and ascorbate-induced LPO. There were no remarkable changes in young rats treated with GTE. Protein carbonyl and total thiols Figure 2B shows the levels of cardiac protein carbonyls and total thiols of young and aged experimental rats. An increase in protein carbonyl level (1.8-fold) was found in aged rats compared with young rats. With GTE supplementation, the protein carbonyl level was found to be lower in aged rats compared with aged control rats. Total thiol status was found to be decreased during the aging process. With GTE administration, the status of total thiols was found to be significantly (P < 0.001) increased by 1.5-fold. Non-enzymatic antioxidants and redox index Measurements of GSH and GSSG levels have been used to estimate the redox environment of a cell. Table 2 presents
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Table 1 Body weight of young and aged rats before and after supplementation of green tea extract* Body weight (g)
Group I
Group II
Group III
Group IV
Initial After 30 d
151 6 12 170 6 10
145 6 11 167 6 14
392 6 23 408 6 15
390 6 19 404 6 22
Group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract * Values are expressed as mean 6 SD for six rats in each group.
the effect of GTE on non-enzymatic antioxidants GSH, GSSG, GSH/GSSG ratio, and GSH redox index in the hearts of young and aged rats. A significant (P < 0.001) decrease was observed in levels of GSH, GSH/GSSG ratio, and GSH redox index in aged rats compared with young rats. A 1.8-fold increase in levels of GSSG was observed in aged rats compared with their young counterparts. These alterations were reversed significantly (P < 0.001) in cardiac tissue of GTE-administered aged rats. Figure 3 presents the status of ascorbate and a-tocopherol in cardiac tissue of young and aged rats. A significant decrease (P < 0.001) was observed in levels of ascorbate and a-tocopherol in aged rats compared with young rats.
GTE-supplemented aged rats showed elevated levels of ascorbate and a-tocopherol. Enzymatic antioxidants Biochemical antioxidants and a host of enzymic reactions supply the necessary reduction potential to maintain cells in a state of redox balance. Antioxidant enzyme activities such as SOD, CAT, GPx, GR, G6PDH, and AST in cardiac tissue of young and aged rats are presented in Table 3. During the aging process the activities of these enzymes were found to be decreased except AST. AST enzyme activity increased in aged rats. GTE-administered rats appeared capable of restoring antioxidant enzyme activities toward young control values, with the increased values being 1.5-fold for SOD, 1.3-fold for CAT, 1.5-fold for GPx, 1.5-fold for GR, and 1.4-fold for G6PDH. Moreover, AST activities was brought back to near normal in GTE-supplemented aged rats. There were no marked changes observed in young rats treated with GTE. Discussion
Fig. 2. Effect of green tea extract on levels of (A) lipid peroxidation and (B) protein carbonyls (nanomoles per milligram of protein) and total thiols (micrograms per milligram of protein) in cardiac tissue of control and experimental rats. Values are means 6 SDs for six rats in each group. aGroup III compared with group I; bgroup IV compared with group III. $Significant at P < 0.001. Group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract; TBARS, thiobarbituric acid-reactive substances.
Aging is defined as a highly complex biological process associated with a progressive decline in the performance of most organs. The heart is highly vulnerable to age-related changes [31] because of its high rate of oxygen utilization and consequent ROS-induced damage. Hence, treatment measures that aim to slow down the aging process should be able to curtail macromolecular damage and strengthen the antioxidant status of the system such that it can effectively combat the oxidative stress. Significantly higher levels of basal, FeSO4, and ascorbateinduced LPO were observed in aged rats compared with young rats. Increased LPO during aging has been well documented [32–34]. Increased free radical production [35] during aging may be the cause for increased LPO. This study shows that supplementation of GTE results in a lower level of LPO in aged rats. Green tea catechins can act as scavengers of ROS and prevent free radical damage. In addition to directly quenching ROS, tea flavonoids can chelate iron and copper to prevent their participation in Fenton and HaberWeiss reactions [36]. Glutathione is vital to antioxidant defenses. Besides serving as a substrate in the GPx reaction, it also acts as a free radical scavenger and helps in the regulation of thiol disulfide concentration of a number of glycolytic enzymes and
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Table 2 Effect of GTE on the level of GSH, GSSG, GSH/GSSG, and redox index in heart of young and aged rats* Parameter GSH (nmol/mg protein) GSSG (nmol/mg protein) GSH/GSSG Redox index
Group I 27.1 6 3.0 0.72 6 0.07 37.6 6 3.5 0.20 6 0.023
Group II 27.4 6 2.8 0.69 6 0.07 39.7 6 3.8 0.21 6 0.02
Group III
Group IV y,x
17.2 6 1.5 1.12 6 0.12y,x 15.4 6 1.2y,x 0.08 6 0.01y,x
26.3 6 4.1z,x 0.75 6 0.06z,x 35.1 6 2.9z,x 0.19 6 0.02z,x
Group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract; GSH, glutathione; GSSG, oxidized glutathione * Values are expressed as mean 6 SD for six rats in each group. y Group III compared with group I. z Group IV compared with group III. x Significant at P < 0.001.
Ca2þ-adenosine triphosphatases, thus indirectly maintaining intracellular Ca2þ homeostasis. Moreover, GSH regenerates other scavengers and antioxidants such as ascorbic acid and a-tocopherol. This study shows that GSH level was lower in aged rats compared with young rats and this is corroborated with an earlier study [37]. GSH depletion affects mainly GSH-dependent enzymes and the redox status of different structural proteins and makes cells more susceptible to any further challenge. An increased GSH content was observed in GTE-treated aged rats. Tea and tea polyphenols have prevented or attenuated decreases in levels of tissue GSH in various animal models [38]. A recent study has suggested that EGCG, one of the major constituents of GTE, attenuates oxidative stress by increasing the level of cellular GSH in passaged rat hepatic stellate cells. It has been reported that pure flavonoids stimulate glutamate cysteine lyase catalytic subunit (a key rate-limiting enzyme in GSH synthesis) gene expression through antioxidant response elements in the gene promoter [39]. In this study, lower levels of the GSH/GSSG ratio and the redox index and an elevated level of GSSG with advancing age were found. The decrease in the redox index with age is related to decreased GSH levels and increased
Fig. 3. Effect of green tea extract on ascorbate and a-tocopherol levels in hearts of control and experimental rats. Values are means 6 SDs for six rats in each group. aGroup III compared with group I; bgroup IV compared with group III. $Significant at P < 0.001. Group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract.
GSSG levels and correlate well with previous studies [23,40]. The reversal of these alterations are observed in GTE-treated aged rats. The changes in the GSH/GSSG ratio of aged rats treated with GTE can be linked more plausibly to increased antioxidant production. Yumei et al. [39] reported that an EGCG dose dependently improved the ratio of GSH/GSSG. Ascorbate and a-tocopherol are non-enzymatic antioxidants and scavengers of free radicals. Lower levels of ascorbate and a-tocopherol were observed in aged rats compared with young rats. van der Loo et al. [41] reported that tissue and plasma levels of ascorbate decrease during aging. Rajasekaran et al. [42] reported that depletion of GSH leads to lower levels of ascorbate and a-tocopherol. In the present study higher levels of ascorbate and a-tocopherol were observed in aged rats treated with GTE. Feeding catechins have been shown to improve plasma and liver a-tocopherol in rats [43]. The loss of atocopherol prevented by catechins, by repairing tocopheryl radicals and affording protection to the hydrophilic antioxidant ascorbate, also repairs this radical [44]. Higher GSH levels with GTE administration might have another possible mechanism responsible for the elevated levels of ascorbate and a-tocopherol. The concentrations of the small molecular-weight antioxidants and the activities of the free radical scavengers are altered and insufficient in the aging condition [45]. In this study, a significantly lower activity of the enzymes SOD, CAT, and GPx was observed during the aging process and the reduction in the activities of these enzymes in aging has been well documented [46–48]. SOD, a family of enzymes, protects against oxygen free radicals by catalyzing the removal of the superoxide radical. This radical damages the membrane and biological structures. During oxidative stress an increased intracellular Ca2þ ion concentration induces the irreversible conversion of xanthine dehydrogenase to xanthine oxidase, which in turn catalyzes the oxidation of xanthine to provide a source of superoxide radicals. These reactions and the decline in SOD activity during aging may lead to the overloading of oxygen radicals. CAT and GPx, the two antioxidant enzymes, played a crucial role in the detoxification of H2O2. Aged rats that received GTE showed significantly higher
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Table 3 Effect of green tea extract on enzymatic antioxidants levels and AST in hearts of young and aged rats* Parameter
Group I 1
1
y
SOD (U $min $ mg protein) CAT (mmol H2O2 consumed $ min1 $ mg1 protein) GPx (mg GSH consumed $ min1 $ mg1 protein) GR (nmol NADPH oxidized $ min1 $ mg1 protein) G6PDH (mg NADP reduced $ min1 $ mg1 protein) AST (mmol 3 102 pyruvate liberated/min)
6.93 6 0.72 63.25 6 5.60 21.42 6 1.97 1.56 6 0.25 4.14 6 0.50 0.26 6 0.03
Group II 7.01 6 0.63 64.90 6 5.10 22.75 6 3.60 1.7 6 0.16 4.33 6 0.37 0.25 6 0.03
Group III
Group IV z jj
4.32 6 0.44 45.64 6 7.40z jj 13.07 6 1.80z jj 1.04 6 0.11z jj 2.93 6 0.53z jj 0.42 6 0.04z jj
6.65 6 1.20x jj 60.07 6 5.00x jj 20.19 6 2.06x jj 1.52 6 0.15x jj 3.96 6 0.36x { 0.27 6 .04x jj
AST, aspartate transaminase; CAT, catalase; G6PDH, glucose-6-phosphate dehydrogenase; GPx, glutathione peroxidase; GR, glutathione reductase; group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract; GSH, glutathione; NADPH, reduced nicotinamide adenine dinucleotide phosphatase; SOD, superoxide dismutase * Values are expressed as mean 6 SD for six rats in each group. y One unit is equal to the amount of enzyme that inhibits pyrogallol auto-oxidation by 50%. z Group III compared with group I. x Froup IV compared with group III. jj Significant at P < 0.001. { Significant at P < 0.01.
activities of these enzymes. The enhanced antioxidant defense system by green tea in an animal model [49] and human subjects [50] has been well documented. It has been reported that EGCG causes increases in the activities of SOD, CAT, and GPx and decreases LPO in the liver and skeletal muscle [51]. The direct scavenging effect of GTE on H2O2 and superoxide radicals may be one of the possible reasons as reported by Hong et al. [52]. In our study, G6PDH and GR activities were found to be decreased in aged rats. The decreased GR and G6PDH activities in the skeletal muscle and heart of aged rats associated with age-related depletion of GSH was previously reported by Kumaran et al. [40]. The enzyme G6PDH is the regulatory enzyme of hexose monophosphate shunt and catalyzes the reaction that generates reducing equivalents, e.g., NADPH from NADPþ. GR at the expense of this NADPH reduces GSSG to GSH. Oxidative inactivation of G6PDH might be the possible reason for the lower level of NADPH. Lower activity of G6PDH leads to a decrease in the activity of GR, which requires NADPH as a cofactor, for the conversion of GSSG to GSH. Supplementation of GTE to aged rats increased the activities of G6PDH and GR. A higher level of cellular GSH, which is substrate availability, might be responsible for the increase in the activity of GPx, which in turn increases GR in GTE-administered aged rats. The oxidative damage to proteins is reflected by an increase in protein carbonyl and a decreased level of thiols. This study shows the elevated level of protein carbonyls in aged rats compared with young rats. The protein carbonyl modifications are good indicators of oxidized proteins that increase with advancing age [53,54]. Protein carbonyl level was reverted back to near control values in GTE-supplemented aged rats. It has been proved that GTE administration decreases protein carbonyl levels [55]. Polyphenols function as in vivo antioxidants by virtue of their ability to directly scavenge ROS [47]. This might be a possible reason for the lower level of protein carbonyl levels in GTE-administered aged rats. Decreased levels of protein thiols in aged rats were observed in this study. It has been shown that
increased oxidant production can cause macromolecular anomalies, especially loss of protein thiol groups by oxidation during aging [56]. In this study, an elevated level of thiols was observed in GTE-supplemented aged rats compared with control aged rats. Hydroxyl and superoxide radicals are scavenged and the activity of a free radical-generating enzyme, xanthine oxidase, was also inhibited by GTE [57]. This might lead to decreased oxidative stress and thereby the oxidation of proteins, resulting in elevated levels of thiols in GTE-treated aged rats. Because the antioxidant status is basically normal in young rats, there were no significant changes observed with GTE supplementation. However, GTE administration was found to be very effective in reducing LPO and protein carbonyl and improving enzymatic and non-enzymatic antioxidants and redox status in aged rats. Conclusion The role of GTE in the modulation of GSH may be significant in the restoration of cellular redox status and minimizing cell damage. We suggest that GTE alleviates the indices of oxidative stress associated with aging. References [1] Lakatta EG, Mitchell JH, Pomerance A, Rowe GG. Human aging: changes in structure and function. J Am Coll Cardiol 1987;10: 42A-7. [2] Lakatta EG. Cardiovascular aging in health. Clin Geriatr Med 2000; 16:419–44. [3] Kass DA, Shapiro EP, Kawaguchi M, Capriotti AR, Scuteri A, deGroof RC, Lakatta EG. Improved arterial compliance by a novel advanced glycation endproduct crosslink breaker. Circulation 2001; 104:1464–70. [4] Lakatta EG. Cardiovascular aging research: the next horizons. J Am Geriatr Soc 1999;47:613–25. [5] Meng Q, Wong YT, Chen J, Ruan R. Age-related changes in mitochondrial function and antioxidative enzyme activity in fischer 344 rats. Mech Ageing Dev 2007;128:286–92. [6] Dro¨ge W. Free radicals in the physiological control of cell function. Physiol Rev 2002;82:47–95.
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Basic nutritional investigation
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Senescence mediated redox imbalance in cardiac tissue: Antioxidant rejuvenating potential of green tea extract Vadivel Senthil Kumaran, M.Sc., Karpagavinayagam Arulmathi, M.Sc., and Periandavan Kalaiselvi, Ph.D.* Department of Medical Biochemistry, DR.ALM Post-Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, India Manuscript received July 13, 2008; accepted February 16, 2009
Abstract
Objective: The activities and capacities of antioxidant systems of tissue cells are declined during aging, leading to the gradual loss of pro-oxidant/antioxidant balance and accumulation of oxidative damage. Hence, the present study evaluated the role of green tea extract (GTE), rich in polyphenols, in combating age-associated macromolecular damage in rat cardiac tissue. Methods: The antioxidant defense system, lipid peroxidation, protein oxidation, and redox status in heart tissue were studied using young and aged rats. Results: Significant increases in lipid peroxidation, protein carbonyls, and marked decreases in glutathione redox status, protein thiols, and activities of enzymatic and non-enzymatic antioxidants were observed in aged rats compared with young rats. Supplementation of GTE (100 mg/kg of body weight per day) orally for 30 days ameliorated these changes significantly. Conclusion: This study accredits GTE’s antioxidant rejuvenating potency and its role in the amelioration of senescence-mediated redox imbalance in aged rat cardiac tissue. Ó 2009 Elsevier Inc. All rights reserved.
Keywords:
Aging; Green tea extract; Heart; Oxidative stress; Antioxidants; Redox status
Introduction Aging, an aspect of almost all living organisms, is a multifactorial process of enormous complexity and is characterized by impairment of various cellular functions. Cardiac senescence, or cardiac aging, is referred to as a dramatic decline in cardiac pump function and contractile reserve with advanced age. It is an irreversible biological process that contributes to high cardiovascular mortality [1,2]. Several rationales have been speculated for cardiac aging, of which accumulation of oxidative macromolecular damage may lead to abnormal cardiac contractile function [2–4]. The accumulation of oxidative derivatives is because of the inefficient antioxidative defense system to scavenge reactive
This work was supported by a Junior Research Fellowship awarded in the form of TNJRF by the University of Madras, Chennai, Tamil Nadu, India. *Corresponding author. Tel.: þ91-44-2454-1767; fax: þ91-44-24540709. E-mail address: [email protected] (P. Kalaiselvi). 0899-9007/09/$ – see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.02.005
oxygen species (ROS) [5]. There is strong evidence to link the unfavorable accumulation of ROS and the resulting oxidative damages with the aging process and human diseases [6,7]. Oxidative stress, an apoptosis-inducing signal, may also increase in the aging heart. Several defense methods have evolved within the organism to recognize the levels of ROS and to limit the damage they inflict. The defenses include enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and non-enzymatic antioxidants such as ascorbate, a-tocopherol, and glutathione (GSH) [8]. The defense against the oxidant declines during the aging process [9,10] and it can be enhanced by supplementation of antioxidants. Green tea is the one, derived from the plant, Camellia sinensis, that has attracted significant attention recently in the scientific and consumer communities for its extensive health benefits for many disorders. Historically, green tea has been consumed by the Japanese and Chinese populations for centuries and probably is the most consumed beverage, besides water, in Asian society. There are several polyphenolic catechins present in green tea extract (GTE)
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such as ()-epicatechin (EC), ()-epicatechin-3-gallate (ECG), ()-epigallocatechin (EGC), and ()-epigallocatechin-3-gallate (EGCG). The protective effect of green tea in cardiovascular diseases is thought to stem from its antioxidant activity [11,12]. With all this background, this study was designed to evaluate the role of GTE in augmenting the function of antioxidants and to assess its potency to mitigate the macromolecular damage in aged rat heart tissue. Materials and methods Fresh green tea leaves were obtained from Nilgiris, Tamil Nadu, India. The leaves were dried in the shade and ground well into a fine powder in a mill with the temperature of the container maintained at lower than 50 C. Then the extract was prepared as described by Yang et al. [13], with slight modification. Tea powder was extracted with 95% ethanol (1:10, w/v) for 2 d with constant stirring. Ethanol was used to extract catechin because it has been reported to be an ideal solvent for the high recovery of tea catechins. Suspensions were filtered through Whatman No. 1 filter paper to retain the clear solution. The residue was extracted again. The pooled tea solution was vacuum evaporated at lower than 50 C and the concentrate was dried well, lyophilized, and stored at 4 C. Analysis of GTE The percentage of the catechin content of the GTE was analyzed using High Performance Liquid Chromatography (Column C18 phenomenex; Gemini, Tomance, CA, USA) according to the method of Anandh Babu et al. [14], with slight modifications. The column was C18 and the mobile phase consisted of phosphate buffer (pH adjusted to 3.5 with acetic acid) and methanol in the ratio of 97:3. Twenty microliters of the sample were injected into the column and the flow rate was maintained at 0.8 mL/min. The elution was detected by an ultraviolet-visible detector (SPD 20A) at 254 nm. Chemicals Bovine serum albumin (BSA) was purchased from Sigma (St. Louis, MO, USA). All other chemicals used were of analytical grade and were obtained from Sisco Research Laboratory (Mumbai, India). Animals Male albino rats of Wistar strain, weighing approximately 130 to 160 g (young, 3–4 mo old) and 380 to 410 g (aged, 24 mo old, maximum lifespan is 3ˇ1/2 y), used in this study were procured from the central animal house facility, Taramani Campus, University of Madras. They were healthy animals and maintained in a clean rodent room. 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/dark cycle throughout the experimental period. Experimental animals were handled according to university and institutional legislation, regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India, and approved by the institutional animal ethical committee (IAEC No. 02/079/07). Experimental design The animals were divided into four groups consisting of six animals in each group: group I, control young rats (received vehicle alone); group II, young rats (received GTE); group III, control aged rats (received vehicle alone); and group IV, aged rats (received GTE). Green tea extract (100 mg/kg of body weight per day) was dissolved in physiological saline (0.89%) and administered for 30 d by oral gavage. On completion of the experimental period, the animals were maintained in a fasting condition for 12 h and thereafter all animals were sacrificed by cervical decapitation. The heart was excised immediately and immersed in ice-cold physiological saline. A 10% homogenate was prepared using 0.01 M Tris HCl buffer, pH 7.4, and stored at 80 C until analyses were performed. Biochemical assays Lipid peroxidation (LPO) was determined by making use of the procedure of Ho¨gberg et al. [15]. One of the major secondary products of LPO is malondialdehyde (MDA). Malondialdehyde and other by-products react with thiobarbituric acid (TBA) to generate a colored product that absorbs at 532 nm, representing the color produced by all thiobarbituric acid-reactive substances (TBARS). The ferrous sulfate (FeSO4) and ascorbate-induced LPO system contained 10 mM FeSO4 and 0.2 mM ascorbate as inducers [16]. The amount of thiobarbituric acid-reactive substances was calculated using a molar extinction coefficient of 1.56 3 105 $ M1 $ cm1. The results were expressed as nanomoles of malondialdehyde released per milligram of protein. Protein carbonyl content was analyzed as described by Levine et al. [17] using 2,4-dinitrophenylhydrazine (DNPH). The difference in absorbance between the 2,4-dinitrophenylhydrazine–treated and the HCl-treated samples are determined at 366 nm and the results were expressed as nanomoles of carbonyl groups per milligram of protein. The protein carbonyl content was calculated based on a molar extinction coefficient of 22 000 $ M1 $ cm1 for 2,4-dinitrophenylhydrazine. The level of thiols was evaluated by the method of Sedlak and Lindsay [18] and expressed as micrograms per milligram of protein. The level of ascorbic acid was estimated by the method of Omaye et al. [19] and expressed as micrograms per milligram of protein. The level of a-tocopherol was measured by the method of Desai [20] and expressed as micrograms per
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milligram of protein. The level of GSH was measured by the method of Moron et al. [21]. Oxidized glutathione (GSSG) was measured by the method of Asensi et al. [22]. The redox index was calculated as (GSH þ 2 3 GSSG)/(2 3 GSSG 3 100), as reported by Oztu¨rk and Gu¨mu¨slu¨ [23]. SOD (E.C. 1.15.1.1) activity was measured by the method reported by Marklund and Marklund [24]. The degree of inhibition of the auto-oxidation of pyrogallol at an alkaline pH by SOD was used as a measurement of enzyme activity. The final results were expressed as units per minute per milligram of protein. CAT (E.C. 1.11.1.9) activity was measured by the method of Sinha [25] and expressed as unit micromoles of H2O2 consumed per minute per milligram of protein. GPx (E.C. 1.11.1.6) activity was assayed as described by Rotruck et al. [26]. The kinetic change was recorded at 420 nm immediately and the activity was expressed as units, which is defined as micromoles of GSH oxidized per minute per milligram of protein. Glutathione reductase (GR; E.C. 1.6.4.2), which utilizes reduced nicotinamide adenine dinucleotide phosphatase (NADPH) to convert GSSG to GSH, was assayed by the method of Staal et al. [27] and expressed as nanomoles of NADPH oxidized per minute per milligram of protein. The activity of glucose-6-phosphate dehydrogenase (G6PDH; E.C. 1.1.1.49) was estimated by the method of Ellis and Kirkman [28] and expressed as micrograms of NADP reduced per minute per milligram of protein. Aspartate transaminase (AST) was determined and expressed in terms of micromoles of pyruvate liberated per minute per milligram of protein at 37 C [29]. The protein content was determined according to the method of Lowry et al. [30] using bovine serum albumin as a standard. Statistical analysis Values were expressed as mean 6 standard deviation for six rats in each group. Statistical significance of changes in different groups was evaluated by one-way analysis of variance using SPSS 7.5 for Windows (SPSS Inc., Chicago, IL, USA). Post hoc testing was performed for intergroup comparisons using the least significant difference test. Statistical significance was set at P < 0.001, P < 0.01, and P < 0.05.
Results Composition of GTE Green tea extract contains volatile oils, vitamins, minerals, and caffeine but the primary constituents of interest are the polyphenols, particularly catechins. The percentage of catechins present in GTE was obtained from high-performance liquid chromatographic analysis and is presented in Figure 1. High-performance liquid chromatographic analysis showed that catechins are the major component of GTE. Nearly 72% of the extract was a mixture of catechins, namely EC, ()-epicatechin-3-gallate, ()-epigallocatechin, and EGCG,
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Fig. 1. Major components of green tea extract were determined as a percentage of the weight of the extract. All values are means 6 SDs of three replicates. EC, ()-epicatechin; ECG, ()-epicatechin-3-gallate; EGC, ()-epigallocatechin; EGCG, ()-epigallocatechin-3-gallate.
of which EGCG concentration is high compared with other catechins. Body weight Table 1 lists the body weight of young and aged rats before and after supplementation of GTE. No significant difference in body weight was detected between initial and after 30-d treatment with GTE in all four groups. Lipid peroxidation To evaluate the possible consequences of oxidative stress, LPO (basal, FeSO4, and ascorbate induced) was analyzed. Figure 2A shows the effect of GTE on levels of LPO in young and aged rats. There was a significant (P < 0.001) increase in LPO levels in aged rats compared with young rats. Aged control rats showed 2.5-fold increase in basal LPO, 1.8-fold increase for FeSO4-induced LPO, and 1.9-fold increase in ascorbate-induced LPO. GTE administration lowered basal LPO, FeSO4-induced, and ascorbate-induced LPO. There were no remarkable changes in young rats treated with GTE. Protein carbonyl and total thiols Figure 2B shows the levels of cardiac protein carbonyls and total thiols of young and aged experimental rats. An increase in protein carbonyl level (1.8-fold) was found in aged rats compared with young rats. With GTE supplementation, the protein carbonyl level was found to be lower in aged rats compared with aged control rats. Total thiol status was found to be decreased during the aging process. With GTE administration, the status of total thiols was found to be significantly (P < 0.001) increased by 1.5-fold. Non-enzymatic antioxidants and redox index Measurements of GSH and GSSG levels have been used to estimate the redox environment of a cell. Table 2 presents
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Table 1 Body weight of young and aged rats before and after supplementation of green tea extract* Body weight (g)
Group I
Group II
Group III
Group IV
Initial After 30 d
151 6 12 170 6 10
145 6 11 167 6 14
392 6 23 408 6 15
390 6 19 404 6 22
Group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract * Values are expressed as mean 6 SD for six rats in each group.
the effect of GTE on non-enzymatic antioxidants GSH, GSSG, GSH/GSSG ratio, and GSH redox index in the hearts of young and aged rats. A significant (P < 0.001) decrease was observed in levels of GSH, GSH/GSSG ratio, and GSH redox index in aged rats compared with young rats. A 1.8-fold increase in levels of GSSG was observed in aged rats compared with their young counterparts. These alterations were reversed significantly (P < 0.001) in cardiac tissue of GTE-administered aged rats. Figure 3 presents the status of ascorbate and a-tocopherol in cardiac tissue of young and aged rats. A significant decrease (P < 0.001) was observed in levels of ascorbate and a-tocopherol in aged rats compared with young rats.
GTE-supplemented aged rats showed elevated levels of ascorbate and a-tocopherol. Enzymatic antioxidants Biochemical antioxidants and a host of enzymic reactions supply the necessary reduction potential to maintain cells in a state of redox balance. Antioxidant enzyme activities such as SOD, CAT, GPx, GR, G6PDH, and AST in cardiac tissue of young and aged rats are presented in Table 3. During the aging process the activities of these enzymes were found to be decreased except AST. AST enzyme activity increased in aged rats. GTE-administered rats appeared capable of restoring antioxidant enzyme activities toward young control values, with the increased values being 1.5-fold for SOD, 1.3-fold for CAT, 1.5-fold for GPx, 1.5-fold for GR, and 1.4-fold for G6PDH. Moreover, AST activities was brought back to near normal in GTE-supplemented aged rats. There were no marked changes observed in young rats treated with GTE. Discussion
Fig. 2. Effect of green tea extract on levels of (A) lipid peroxidation and (B) protein carbonyls (nanomoles per milligram of protein) and total thiols (micrograms per milligram of protein) in cardiac tissue of control and experimental rats. Values are means 6 SDs for six rats in each group. aGroup III compared with group I; bgroup IV compared with group III. $Significant at P < 0.001. Group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract; TBARS, thiobarbituric acid-reactive substances.
Aging is defined as a highly complex biological process associated with a progressive decline in the performance of most organs. The heart is highly vulnerable to age-related changes [31] because of its high rate of oxygen utilization and consequent ROS-induced damage. Hence, treatment measures that aim to slow down the aging process should be able to curtail macromolecular damage and strengthen the antioxidant status of the system such that it can effectively combat the oxidative stress. Significantly higher levels of basal, FeSO4, and ascorbateinduced LPO were observed in aged rats compared with young rats. Increased LPO during aging has been well documented [32–34]. Increased free radical production [35] during aging may be the cause for increased LPO. This study shows that supplementation of GTE results in a lower level of LPO in aged rats. Green tea catechins can act as scavengers of ROS and prevent free radical damage. In addition to directly quenching ROS, tea flavonoids can chelate iron and copper to prevent their participation in Fenton and HaberWeiss reactions [36]. Glutathione is vital to antioxidant defenses. Besides serving as a substrate in the GPx reaction, it also acts as a free radical scavenger and helps in the regulation of thiol disulfide concentration of a number of glycolytic enzymes and
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Table 2 Effect of GTE on the level of GSH, GSSG, GSH/GSSG, and redox index in heart of young and aged rats* Parameter GSH (nmol/mg protein) GSSG (nmol/mg protein) GSH/GSSG Redox index
Group I 27.1 6 3.0 0.72 6 0.07 37.6 6 3.5 0.20 6 0.023
Group II 27.4 6 2.8 0.69 6 0.07 39.7 6 3.8 0.21 6 0.02
Group III
Group IV y,x
17.2 6 1.5 1.12 6 0.12y,x 15.4 6 1.2y,x 0.08 6 0.01y,x
26.3 6 4.1z,x 0.75 6 0.06z,x 35.1 6 2.9z,x 0.19 6 0.02z,x
Group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract; GSH, glutathione; GSSG, oxidized glutathione * Values are expressed as mean 6 SD for six rats in each group. y Group III compared with group I. z Group IV compared with group III. x Significant at P < 0.001.
Ca2þ-adenosine triphosphatases, thus indirectly maintaining intracellular Ca2þ homeostasis. Moreover, GSH regenerates other scavengers and antioxidants such as ascorbic acid and a-tocopherol. This study shows that GSH level was lower in aged rats compared with young rats and this is corroborated with an earlier study [37]. GSH depletion affects mainly GSH-dependent enzymes and the redox status of different structural proteins and makes cells more susceptible to any further challenge. An increased GSH content was observed in GTE-treated aged rats. Tea and tea polyphenols have prevented or attenuated decreases in levels of tissue GSH in various animal models [38]. A recent study has suggested that EGCG, one of the major constituents of GTE, attenuates oxidative stress by increasing the level of cellular GSH in passaged rat hepatic stellate cells. It has been reported that pure flavonoids stimulate glutamate cysteine lyase catalytic subunit (a key rate-limiting enzyme in GSH synthesis) gene expression through antioxidant response elements in the gene promoter [39]. In this study, lower levels of the GSH/GSSG ratio and the redox index and an elevated level of GSSG with advancing age were found. The decrease in the redox index with age is related to decreased GSH levels and increased
Fig. 3. Effect of green tea extract on ascorbate and a-tocopherol levels in hearts of control and experimental rats. Values are means 6 SDs for six rats in each group. aGroup III compared with group I; bgroup IV compared with group III. $Significant at P < 0.001. Group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract.
GSSG levels and correlate well with previous studies [23,40]. The reversal of these alterations are observed in GTE-treated aged rats. The changes in the GSH/GSSG ratio of aged rats treated with GTE can be linked more plausibly to increased antioxidant production. Yumei et al. [39] reported that an EGCG dose dependently improved the ratio of GSH/GSSG. Ascorbate and a-tocopherol are non-enzymatic antioxidants and scavengers of free radicals. Lower levels of ascorbate and a-tocopherol were observed in aged rats compared with young rats. van der Loo et al. [41] reported that tissue and plasma levels of ascorbate decrease during aging. Rajasekaran et al. [42] reported that depletion of GSH leads to lower levels of ascorbate and a-tocopherol. In the present study higher levels of ascorbate and a-tocopherol were observed in aged rats treated with GTE. Feeding catechins have been shown to improve plasma and liver a-tocopherol in rats [43]. The loss of atocopherol prevented by catechins, by repairing tocopheryl radicals and affording protection to the hydrophilic antioxidant ascorbate, also repairs this radical [44]. Higher GSH levels with GTE administration might have another possible mechanism responsible for the elevated levels of ascorbate and a-tocopherol. The concentrations of the small molecular-weight antioxidants and the activities of the free radical scavengers are altered and insufficient in the aging condition [45]. In this study, a significantly lower activity of the enzymes SOD, CAT, and GPx was observed during the aging process and the reduction in the activities of these enzymes in aging has been well documented [46–48]. SOD, a family of enzymes, protects against oxygen free radicals by catalyzing the removal of the superoxide radical. This radical damages the membrane and biological structures. During oxidative stress an increased intracellular Ca2þ ion concentration induces the irreversible conversion of xanthine dehydrogenase to xanthine oxidase, which in turn catalyzes the oxidation of xanthine to provide a source of superoxide radicals. These reactions and the decline in SOD activity during aging may lead to the overloading of oxygen radicals. CAT and GPx, the two antioxidant enzymes, played a crucial role in the detoxification of H2O2. Aged rats that received GTE showed significantly higher
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Table 3 Effect of green tea extract on enzymatic antioxidants levels and AST in hearts of young and aged rats* Parameter
Group I 1
1
y
SOD (U $min $ mg protein) CAT (mmol H2O2 consumed $ min1 $ mg1 protein) GPx (mg GSH consumed $ min1 $ mg1 protein) GR (nmol NADPH oxidized $ min1 $ mg1 protein) G6PDH (mg NADP reduced $ min1 $ mg1 protein) AST (mmol 3 102 pyruvate liberated/min)
6.93 6 0.72 63.25 6 5.60 21.42 6 1.97 1.56 6 0.25 4.14 6 0.50 0.26 6 0.03
Group II 7.01 6 0.63 64.90 6 5.10 22.75 6 3.60 1.7 6 0.16 4.33 6 0.37 0.25 6 0.03
Group III
Group IV z jj
4.32 6 0.44 45.64 6 7.40z jj 13.07 6 1.80z jj 1.04 6 0.11z jj 2.93 6 0.53z jj 0.42 6 0.04z jj
6.65 6 1.20x jj 60.07 6 5.00x jj 20.19 6 2.06x jj 1.52 6 0.15x jj 3.96 6 0.36x { 0.27 6 .04x jj
AST, aspartate transaminase; CAT, catalase; G6PDH, glucose-6-phosphate dehydrogenase; GPx, glutathione peroxidase; GR, glutathione reductase; group I, control young rats; group II, young rats treated with green tea extract; group III, control aged rats; group IV, aged rats treated with green tea extract; GSH, glutathione; NADPH, reduced nicotinamide adenine dinucleotide phosphatase; SOD, superoxide dismutase * Values are expressed as mean 6 SD for six rats in each group. y One unit is equal to the amount of enzyme that inhibits pyrogallol auto-oxidation by 50%. z Group III compared with group I. x Froup IV compared with group III. jj Significant at P < 0.001. { Significant at P < 0.01.
activities of these enzymes. The enhanced antioxidant defense system by green tea in an animal model [49] and human subjects [50] has been well documented. It has been reported that EGCG causes increases in the activities of SOD, CAT, and GPx and decreases LPO in the liver and skeletal muscle [51]. The direct scavenging effect of GTE on H2O2 and superoxide radicals may be one of the possible reasons as reported by Hong et al. [52]. In our study, G6PDH and GR activities were found to be decreased in aged rats. The decreased GR and G6PDH activities in the skeletal muscle and heart of aged rats associated with age-related depletion of GSH was previously reported by Kumaran et al. [40]. The enzyme G6PDH is the regulatory enzyme of hexose monophosphate shunt and catalyzes the reaction that generates reducing equivalents, e.g., NADPH from NADPþ. GR at the expense of this NADPH reduces GSSG to GSH. Oxidative inactivation of G6PDH might be the possible reason for the lower level of NADPH. Lower activity of G6PDH leads to a decrease in the activity of GR, which requires NADPH as a cofactor, for the conversion of GSSG to GSH. Supplementation of GTE to aged rats increased the activities of G6PDH and GR. A higher level of cellular GSH, which is substrate availability, might be responsible for the increase in the activity of GPx, which in turn increases GR in GTE-administered aged rats. The oxidative damage to proteins is reflected by an increase in protein carbonyl and a decreased level of thiols. This study shows the elevated level of protein carbonyls in aged rats compared with young rats. The protein carbonyl modifications are good indicators of oxidized proteins that increase with advancing age [53,54]. Protein carbonyl level was reverted back to near control values in GTE-supplemented aged rats. It has been proved that GTE administration decreases protein carbonyl levels [55]. Polyphenols function as in vivo antioxidants by virtue of their ability to directly scavenge ROS [47]. This might be a possible reason for the lower level of protein carbonyl levels in GTE-administered aged rats. Decreased levels of protein thiols in aged rats were observed in this study. It has been shown that
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