Abstract: The present study was aimed to evaluate the oxidants, enzymatic and nonenzymatic antioxidants, glycoproteins and membrane adenosine triphosphatases (ATPases). The increased levels of oxidants and decreased levels of enzymatic, nonenzymatic antioxidants, glycoproteins and ATPases were observed in erythrocytes of aged rats. After administration of Terminalia chebula, the levels were reverted to normal in aged rats. These results indicate that the administration of T. chebula acts as a free radical scavenger with potential antioxidant effects in erythrocytes of aged rats.
INTRODUCTION
Aging is the accumulation of changes responsible for the sequential alterations that accompany with advancing age and the age associated progressive increase in the chance of disease and death (Harman, 1992). Also, aging has been proposed to be a result of continuous reactions of the cell components with oxygen free radicals throughout the life span (Reznick et al., 1992). These oxygen free radicals can modify DNA, proteins and membrane lipids, leading to cellular degeneration (Olanow, 1992). Erythrocytes have been used as a biological probe in exploring the aging process (Bernabucci et al., 2002), which is associated with a loss of ability at the molecular, cellular and whole organism levels (Hall et al., 2000). The high polyunsaturated fatty acid content of the erythrocyte membrane and the continuous exposure to high concentrations of oxygen and iron in heamoglobin are factors that make erythrocytes very sensitive to oxidative injury (Bernabucci et al., 2002). However, during aging an oxidant challenge exceeds the capacity of the cell’s defense system, membrane damage may occur. During such conditions, dietary intake of antioxidants gains immense importance.
Consumption of fruits and vegetables containing high amounts of antioxidative nutraceuticals have been associated with the balance of the free radicals/antioxidants status, which helps to minimize the oxidative stress in the body and to reduce the risk of diseases (Kaur and Kapoor, 2001). Chebulic myrobalan (Terminalia chebula Retz.) belonged to the family Combretaceae, a native plant in India and found in the deciduous forests. Its dried ripe fruit tissues have traditionally been used to treat various ailments in Asia. It is a carminative, deobstruent, astringent and expectorant agent (Anonymous, 1978). Its principles constituents contain chebulagic, chebulinic acid and corilagin (Harborne et al., 1999). Chebulic myrobalan is highly nutritious and could be an important source of dietary supplement in vitamin C, energy, protein, amino acids and mineral nutrients (Bharthakur and Arnold, 1991). T. chebula has been reported to exhibit a variety of biological activity, including anticancer (Saleem et al., 2002), antidiabetic (Sabu and Kuttan, 2002), antimutagenic (Kaur et al., 2002), antibacterial (Malekzadeh et al., 2001) activities. Hence, the present study was designed to examine the effect of T. chebula on erythrocyte oxidation products level, antioxidant status, membrane ATPases and glycoproteins in aged rats compared with young rats.
MATERIALS AND METHODS
Chemicals
1-chloro-2, 4-dinitro benzene (CDNB), 5,5’-dithio-bis (2-nitrobenzoic
acid), glutathione (reduced), glutathione (oxidized), 2-thiobarbituric acid
were obtained from Sigma (St. Louis, MO, USA). All other chemicals were analytical
grade marketed by Sisco Research Laboratories Pvt. Ltd., Mumbai and SD Fine
Chemicals Ltd., Mumbai, India.
Preparation of T. chebula aqueous Extract
The fully ripe fruits of Terminalia chebula were collected from Kolli
hills, Tamilnadu, India during the month of January 2005. Hundred grams of dried
fruit skins were hammered in to small pieces followed by extraction with 800
mL distilled water for 24 h in water bath at 40°C and repeated for two times.
The extracts were then combined, concentrated and finally lyophilized to dry.
The final yield of the water extracts was 43.7 g. The extract was re-dissolved
in distilled water for further experiments.
Animals and Treatment
Young (3-4 months, 120-150 g) and aged (22-24 months, 380-410 g) Wistar
male albino rats were used for the experiments. The rats were housed in polypropylene
cages on a 12L:12D cycle and fed ad libitum on commercial laboratory
food pellets and water. The animals were divided into four groups namely, Group
I: Control young rats were received water only. Group II: Young rats were treated
with T. chebula aqueous extract, 200 mg dose kg-1 body weight
in 1.5 mL of water through gastric incubations for 4 weeks. Group III: Control
aged rats were received water only. Group IV: Aged rats were treated with T.
chebula aqueous extract, 200 mg dose kg-1 body weight in 1.5
mL of water through gastric incubations for 4 weeks.
Preparation of Erythrocyte and Erythrocyte Membranes
On completion of the experimental period, animals were anaesthetized with
Thiopentone sodium (50 mg kg-1) and the blood was collected with
EDTA as anticoagulant. The erythrocyte membrane was isolated according to the
procedure of Dodge et al. (1963) with a change in buffer according to
Quist (1980). Preparation of the haemolysate was carried out as described by
Beutler (1984).
Determination of Oxidation Products in Erythrocyte Membranes
Lipid peroxidation was assessed biochemically by determining the level of
malondialdehyde (MDA) (Beuge and Aust, 1978). MDA levels were expressed as nmoles
of MDA formed/mg protein using 1,1,3,3-tetraethoxypropane as standard. The protein
carbonyl (PCO) content was analyzed using 2,4-dinitrophenylhydrazine (DNPH)
as described by Levine et al. (1990).
Assay of Enzymatic Antioxidants in Erythrocyte Lysate
Copper, zinc superoxide dismutase (Cu, Zn-SOD) activity was measured by
the method of Kakkar et al. (1984) using NADH-PMS-NBT. Catalase (CAT)
activity was measured according to the method of Beers and Sizer (1952). Glutathione
peroxidase (GPx) was estimated by Rotruck et al. (1973), glutathione
reductase (GR) activity by the procedure of Staal et al. (1969), glucose-6-phosphate
dehydrogenase (G6PDH) by Korenberg et al. (1955) and glutathione-s-transferase
(GST) according to Habig et al. (1974).
Estimation of Non-enzymatic Antioxidants in Erythrocyte Lysate
Reduced glutathione (GSH) was measured as described by Ellman and Archs
(1959) using 5, 5-dithiobis- (2-nitrobenzoic acid) (DTNB) reagent. Oxidized
glutathione (GSSG) was measured by masking GSH with 2-vinylpyridine by the same
procedure of GSH estimation. Redox state was determined by the redox index:
(GSH+2xGSSG)/(2xGSSGx100). Ascorbic acid (vitamin C) and α-tocopherol (vitamin
E) contents were assayed according to Omaye et al. (1979) and Desai
(1984), respectively.
Determination of Glycoproteins in Erythrocyte Membranes
In the erythrocyte membrane pellets the lipids were extracted according
to the method of Folch et al. (1957) using chloroform-methanol mixture
(2:1 v/v). The resulting defatted residue was suspended in sodium acetate buffer
(containing 2 mg cysteine HCl mL-1, final pH 7.0) and deproteinized
by 4-5 volumes of ethanol, evaporated to dryness in the cold under a vaccum
and subjected to hydrolysis by heating with 2 mL of 4 N HCl for 4-6 h. The hydrolyzed
material was neutralized with 4 N sodium hydroxide and used for estimating erythrocyte
sialic acid (Warren, 1959), hexose (Niebes, 1972) and hexosamine (Wagner, 1979).
Determination of Membrane ATPases in Erythrocyte Membranes
Sodium potassium ATPase (Na+ K+-ATPase), calcium ATPase
(Ca2+-ATPase) and magnesium ATPase (Mg2+-ATPase) were
determined by the method of Bonting (1970), Hjerten and Pan (1983) and Ohnishi
et al. (1982), respectively. In all the three cases the enzyme activity
was expressed as a function of inorganic phosphorous liberated, which is due
to the breakdown of ATP.
Estimation of Haemoglobin
Haemoglobin content was determined using the cyanmethemoglobin method (Fairbanks
and Klee, 1999).
Statistical Analysis
The values are expressed as mean±Standard Deviation (SD). The results
were computed statistically (SPSS software package) using one-way analysis of
variance. Post hoc testing was performed for intergroup comparisons using the
least significance (LSD) test. A p-value<0.05 was considered significant.
RESULTS
The results obtained in erythrocytes showed that the levels of MDA and PCO were found significantly increased 43.75 and 31.73%, respectively in aged control rats as compared with young control rats (Table 1). In T. chebula treatment to aged rats, levels of MDA and PCO were significantly decreased (p<0.001, p<0.01) with comparison of age-matched controls. In drug treated young rats showed 22.22% (p<0.05) decrease in MDA levels.
The activities of the enzymatic antioxidants SOD, CAT, GPx, GR, G6PDH and GST were significantly (p<0.001) decreased in erythrocytes of aged control rats when compared with young control rats. T. chebula aqueous extract treatment to aged rats showed an increase (p<0.001) in these erythrocytes antioxidant enzymes at 24.79, 40.56, 26.45, 42.29, 43.59 and 31.90%, respectively (Table 2). In the young drug treated rats, T. chebula showed an increase in SOD, CAT and GPx activities at 17.28, 11.32 and 11.25% as compared with young control rats.
A significant decrease (43.13%) in GSH level with increase (25%) of GSSG levels in erythrocytes of aged rats, these alterations decreased (p<0.001) the GSH/GSSG ratio and redox ratio in erythrocytes of aged rats. T. chebula treatment increased the GSH content in erythrocytes of young and aged rats at 14.55 and 42.34%, respectively and decreased (p<0.001) the GSSG level with increased GSH/GSSG ratio (51.33% in aged and 14.56% in young) and redox ratio at p<0.001 (Table 3).
| Table 1: | Effect of T. chebula treatment on oxidation products in erythrocytes of young and aged rats |
| Values are expressed as mean±SD of six rats; Units: MDA nmoles of MDA formed mg-1 protein; PCO μmoles of DNPH mg-1 protein; Statistical comparison: a: Compared with Group I; b: Compared with Group II; *: p<0.05, **: p<0.01, ***: p<0.001 | |
| Table 2: | Effect of T. chebula treatment on enzymatic antioxidants in erythrocytes of young and aged rats |
| Values are expressed as mean±SD of six rats; Units: SOD: 50% nitroblue tetrazolium reduction min-1 mg-1 Hb; CAT: μmoles of H2O2 consumed min-1 mg-1 Hb; GPx: μmoles of GSH consumed min-1 mg-1 Hb; GR: μmoles of NADPH oxidized min-1 mg-1 Hb; G6PDH: U mg-1 Hb; GST: mmoles of GSH-CDNB conjugated min-1 mg-1 Hb; Statistical comparison: a: Compared with Group I; b: Compared with Group II; *: p<0.05, **: p<0.01, ***: p<0.001 | |
| Table 3: | Effect of T. chebula treatment on non-enzymatic antioxidants in erythrocytes of young and aged rats |
| Values are expressed as mean±SD of six rats; Units: GSH and GSSG- μmol g-1 Hb; Vitamin C and E- μmol d L-1; Statistical comparison: a: Compared with Group I; b: Compared with Group II; *: p<0.05, **: p<0.01, ***: p<0.001 | |
| Fig. 1: | Effect of T. chebula treatment on glycoproteins in erythrocytes of young and aged rats. Each bar represents the mean±SD of six rats. Statistical comparison: a: Compared with Group I; b: Compared with Group II; *: p<0.05, **: p<0.001 |
The levels of sialic acid, hexose and hexosamine in aged control rats showed significant decrease in p<0.001 (Fig. 1). T. chebula administration to aged rats showed a significant increase in 18.16, 13.69 and 30.05%, respectively. The increased level of sialic acid in young drug treated rats also observed at 7.14% (p<0.05).
Figure 2 shows that the levels of membrane ATPases such as Na+K+ATPase (25.73%), Ca2+ATPase (35.55%) and Mg2+ATPase (53.04%) were decreased in erythrocytes of aged control rats. Administration of T. chebula to aged rats increased the levels of Na+K+ATPase, Ca2+ATPase and Mg2+ATPase, respectively at 20.93, 31.93 and 47.78%.
| Fig. 2: | Effect of T. chebula treatment on membrane ATPases in erythrocytes of young and aged rats. Each bar represents the mean±SD of six rats. Statistical comparison: a: Compared with Group I; b: Compared with Group II; *: p<0.05, **: p<0.001 |
DISCUSSION
According to the free radical theory of ageing, ageing is caused by the accumulation in the cell of macromolecules, such as DNA, proteins and lipids, that have been damaged by free radicals leading to the common final pathway for cell death (Sohal et al., 1995). Because of continuous generation of free radicals by the oxidation of hemoglobin, erythrocytes are exposed to continuous oxidative stress (Irene et al., 1992). There were positive correlations between the oxidative markers-MDA and PCO levels and aging. Nohl (1991) reported that accumulation of lipid peroxidation products increased during aging. These results are parallel with present findings. Upon T. chebula treatment, a decrease in MDA and PCO levels were observed. This may be due to reducing the amount of hydroxyl radicals and also as a scavenger of peroxide and superoxide radicals by several thiols (Haenen et al., 1989).
Imbalance between radical production and catabolism of oxidant during aging would shift the cells towards oxidative stress resulting in alterations of membrane properties and cell dysfunction. Cu, Zn-SOD is one of the major antioxidant enzymes in erythrocytes where superoxide radials (O2•G) are continuously generated by the autooxidation of hemoglobin (Bernabucci et al., 2002). The decrement in Cu, Zn-SOD activity in the present study may be due to the inactivation of this enzyme by its product H2O2 (Ceballos-Picot et al., 1992). The increase in SOD activity in erythrocytes of young and aged rats on T. chebula treatment may be due to the potential quenching of free radicals by its phenoxyl radial, which significantly decrease the superoxide radicals level (Rice-Evans et al., 1996). CAT catalyses the reduction of H2O2 to H2O and O2 (Ceballos-Picot et al., 1992). According to present results, a significant decrease in CAT activity in erythrocytes with aging could be the decrease in NADH, since NADH is necessary for the conversion of CAT from its inactive state. Treatment with T. chebula increased the CAT activity in erythrocytes via the increase of G6PDH activity, which produces NADH by pentose pathway in erythrocytes. A major function of GPX in erythrocytes may be the disposal of organic peroxides and the maintenance of protein thiols in their reduced states (Mueller et al., 1997). In the present study the GPX activity was decreased in erythrocytes of aged rats, this may be the accumulation of the superoxide anion which inactivates GPX by reacting with the selenium at the active site of the enzyme (Prabhu, 2002). T. chebula administration increased the GPX activity by the protection of sulphydryl groups in glutathione from oxidative damages.
GR is an important enzyme for maintaining the intracellular concentration of reduced glutathione. The lowered activity of GR in erythrocytes of aged rats might be attributed to the excessive production of oxidized glutathione, which fails to match the capacity of GR, to reduce oxidized glutathione (Sen et al., 1993). Oxidized glutathione is produced when peroxides are detoxified by GPX and is recycled back to the reduced form by GR at the expense of NADPH (Mc Intyre and Curthoys, 1980). After administration of T. chebula aqueous extract, the GR activity was restored, which is indicative reduced oxidative stress. Also, the most important enzyme in the defense of the erythrocyte against oxidative attack is G6PDH, which catalyses the initial step of the pentose phosphate pathway and whose most important function is the reduction of nicotinaide adenine dinucleotide phosphate (NADH+) to NADPH (Lord-Fontaine and Averill-Bates, 2002). In this study, the G6PDH activity was decreased in aged rats may be due to the oxidation of the active site of G6PDH, which contains an essential lysine residue (Naylor et al., 1996). T. chebula supplementation increased the activity of G6PDH in aged rats by producing more reducing equivalents and by the reduction of oxidized glutathione to reduced glutathione. The multifunctional enzyme, GSTs are performs several roles in the detoxification of broad spectrum of electrophilic reactive substances and drug metabolize (Jodynis-Liebert et al., 2000). GST catalyzes the transformation of peroxides to less toxic products conjugating them to reduced glutathione, it can be considered an antioxidant enzyme as well (Yi, 1990). In the present study the GST activity was lowered in erythrocytes of aged rats might be attributed to increased lipid peroxidation and depletion of glutathione status (Huang et al., 2003). GST activity was increased in T. chebula treated aged rats by inhibiting lipid peroxidation and increased availability of GSH from GSSG by the enzyme GPX.
GSH plays a pivotal role in defending against oxidative haemolyses. Conditions that perturb intracellular levels of GSH have been shown to result in significant alterations in cellular metabolism. In the present study, the decreased levels of GSH may be the result of an increased oxidation of GSSG, increased degradation or decreased synthesis. T. chebula administration increase the levels by providing NADPH for the reduction of oxidized glutathione into reduced glutathione, catalyzed by GR and G6PDH. GSSG acts as a physiological indicator of the intracellular defense system against free radicals (Ding et al., 2002). An increase in GSSG levels in erythrocytes of aged rats was supported that enhanced oxidative challenge, such as by lipid peroxides, would be expected to result in depletion of the cellular GSH pool and a corresponding increase in GSSG (Aw, 1999). Administration with T. chebula decreased the GSSG levels by recycling of GSH from GSSG by the enzyme GR using NADPH as a cofactor. Measurement of GSH/GSSG levels has been used to estimate the redox environment of a cell (Schafer and Buettner, 2001). We observed a significant decrease in the redox index in aging, which may be related to the decreased GSH levels and increased GSSG concentrations. T. chebula treatment reverted to near normalcy in aged rats.
The decrease in the levels of vitamin C and E in aged control rats may be increased oxidative stress with aging. The decrease of ascorbate damages the erythrocyte membrane, since they are involved in regeneration of tocopherols, the lipophilic membrane antioxidant from its oxidized form. T. chebula reversed the decline of vitamins due to the increased ascorbic acid absorption, reduction of dehydroascorbate to ascorbic acid and preventing the involvement of hemoglobin iron in lipid peroxidation processes (Maffei Facino et al., 1996).
Oxidative stress increases MDA (Halliwell and Chirico, 1993) and the cytotoxic effects of MDA are well known than can induce a reduction of membrane fluidity (Chen and Yu, 1994) and increase erythrocyte membrane fragility (Spickett et al., 1998). A decreased erythrocyte membrane glycoproteins content were found in the present study in aged subjects compared with the young and a significant negative correlation was found between erythrocyte glycoproteins content and subject age. Sialylated glycoproteins are responsible for the negative charge of the erythrocyte membrane surface (Izumida et al., 1991). Therefore, the reduction in glycoproteins content might lead to decreased intercellular electrostatic repulsion and enhanced erythrocyte aggregation in the aging. T. chebula administration restored the glycoproteins in erythrocytes of aged rats might be due to either enhanced sialic acid synthesis or decreased sialidase activity.
Maintenance of membrane fluidity within narrow limits is a perquisite for proper functioning of RBCs and lipids and membrane-bound ATPases play a key role in this connection. Erythrocyte membrane bound enzymes are sensitive indices of altered cellular environment and responsible for unidirectional activated transport of Ca2+ and Mg2+ ions. Na+ ions are extruded against the concentration gradient. Due to peroxidation of membrane lipids, the osmotic stability of the erythrocytes is altered in the presence of the divalent metal. The causative factor for the shortened survival and decreased deformability can be closely related to inhibition of the membrane-bound ATPases. Aging has been shown to reduce ATPases activity and K+ transport and to produce membrane changes in red cells. These changes may be more damaging to the cell and a more important cause of haemolysis than haemoglobin denaturation. Erythrocyte Ca2+-ATPase is also reported to be potentially vulnerable to auto-oxidative modification because it has free thiol groups and resides in close proximity to unsaturated membrane lipids (Hebbel et al., 1986). Consequently this conclusion may be pertinent to Na+K+ATPase that has an environment in common with Ca2+ATPase. In the present study, the inhibition of Na+K+ATPase, Mg2+ATPase and Ca2+ATPase was observed in the erythrocytes of aged control animals. Administration of T. chebula brought about a significant increase in the activities of these enzymes and this might be due to the ability of T. chebula to act as a reducing agent, thus helping to maintain membrane thiols essential for the activity of these enzymes in their reduced state.
All these results suggest that T. chebula is highly protective against oxidative damage and ageing. It balances the antioxidant system and stimulates metabolism of oxidative wastes. Thereby, Terminalia chebula can be acts as a potent drug for preventing age and age related degenerative diseases and improving the normal life.