This study was undertaken to evaluate the antioxidants property in the methanolic root extract of Salacia chinensis in streptozotocin (STZ) induced diabetic rats. The methanolic root extract of S. chinensis was administered orally at a dose of 250-mg/kg weight/day (28 days) to STZ-induced diabetic rats. The extract treated diabetic rats were significantly decreased the levels of blood glucose, glycosylated hemoglobin and increased the hemoglobin level. Diabetic rats treated with extract were showed decrease levels of lipid peroxidant and hydroperoxides as compared to diabetic rats. The level of reduced glutathione and activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase were significantly increased in liver and kidney tissues of diabetic treated rats. These findings demonstrated that methanolic root extract of S. chinensis possess free radical scavenging activity in STZ-induced diabetic rats.
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycaemia associated with long term dysfunction, damage and failure of various organs, particularly kidney, nerves, heart, blood vessels and eyes. The high blood glucose level due to defect in insulin secretion and insulin action (Smith and Their, 1999). In chronic complication of diabetes, oxidative stresses play an important role and it is associated with increased lipid peroxidation (Elangovan et al., 2000). The oxidative stress may constitute the key and common event in the pathogenesis of secondary diabetic complications (Ceriello, 2000). Diabetes mellitus is associated with generation of reactive oxygen species (ROS) leading to oxidative and cell membranes damage (Mohamed et al., 1999). The defense mechanisms against free radicals are reduced glutathione (GSH) and enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), there activities contribute to eliminate superoxide, hydrogen peroxide and hydroxyl radicals (Soto et al., 2003).
The oral hypoglycaemic drugs are sulphonylureas and biguanide groups have been used in the treatment of diabetes mellitus. They are lowering blood glucose levels thereby delaying or preventing the diabetic complications. The mechanisms of action of biguanides (metformin, phenformin) are not fully understood, but they may enhance the insulin receptors to increase the absorption of sugars (Uehara et al., 2001; Zangeneh et al., 2003). Sulfonylureas (e.g. glibenclamide, glipizide) stimulate the insulin secretion from the existing pancreatic β cells. Glibenclamide inhibiting the ATP sensitive K+ (KATP) channels in the plasma membrane (Ashcroft FM and Aschcroft SJH, 1992). These are leads to membrane depolarization, activation of voltage gated Ca2+ channels, a rise in cytosolic (Ca2+) and release the insulin. The STZ induced diabetes is treated by gilbenclamide and used as a stranded drug to compare the antidiabetic activity of various compounds (Ivorra et al., 1988).
Ethno botanical information indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes (Pushparaj et al., 2000), but more plants have not a Scientific scruting. Herbal medicines are used by about 80% of the world population particularly in the developing countries for the primary health care, because of better cultural acceptability, safety, efficacy, potent, inexpensive and lesser side effects (Pullaiah and Chandrasekhar Naidu, 2003). The plant drugs are considered to be less toxic when compared to the synthetic drugs (Pari and Umamaheswari, 2000). The phytochemicals such as saponins, terpenoids, flavonoids and tannins found to inhibit cancer cell proliferation, regulate inflammatory, immune response and protect against lipid peroxidation (Liu, 2003). The medicinal plants often contain substantial amounts of antioxidants including carotenoids, α-tocopherol (vitamin E), ascorbic acid (vitamin C), tannins and flavonoids, they may be an important property of medicines and used in diabetes (Larson, 1988).
Salacia chinensis Linn. , a member of the family Hippocrateaceae, is an erect or straggling tree or a woody climbing shrub found along seashore, riverbanks and forests. The roots are used in indigenous system of medicine and treated for diabetic, astringent, abortifacient, amenorrhoea, dysmenorrhoea, and veneral diseases. The aqueous methanolic (80%) stem extract of S. chinensis showed hypoglycemic effects, gastroprotective effects, α-glucosidase and aldose reductase inhibitory activites, nitric oxide production inhibitory effects and antioxidative activity (Yoshikawa et al., 2003).
The methanolic stem extract of S. chinensis showed potent anti-hyperglycemic effects in oral sucrose or maltose-loaded rats, inhibitory effects on intestinal α-glucosidase, rat lens aldose reductase, formation of amadori compounds and advanced glycation end-products, nitric oxide production from lipopolysaccharide-activated mouse peritoneal macrophage and radical scavenging activities (Yoshikawa et al., 2003).
The aim of the present study was to investigate the antioxidants potency of methanolic root extract of S. chinensis in the liver and kidney of STZ-induced diabetic rats. The results were compared with standard antidiabetic drug, glibenclamide.
Materials and Methods
The analytical grade chemicals were used for all the experiments. The streptozotocin was purchased from sigma chemicals, St.Louis, Mo., USA.
The roots of S. chinensis were collected from Madagadipet; Pondicherry. The roots were washed thoroughly with tap water, shade dried, cut into small pieces and were crushed to moderately coarse powder. It was extracted using 95% methanol in soxhlet apparatus for 6h.The extract was concentrated by using rotary evaporator at 40-50º C under reduced pressure. The root yield was 23.5%. The plant voucher specimen has been deposited in CAS in Botany, University of Madras.
Male Wistar albino rats (150-200g) were procured from Tamil Nadu Veterinary and Animal Sciences University, Chennai and were housed in polycarbonate cages in an animal room with a 12h day-night cycle with at temperature of 22 ± 2º C and humidity of 45-60 %. They were fed with a commercial pelleted rats chow and free access water during the experiment.
The experiments were designed and conducted in accordance with the ethical norms approved by Ministry of Social Justice and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (IAEC No.02/004/06) for the investigation of experimental pain in conscious animals.
Induction of experimental diabetes
Diabetes was induced by administering intrapertonial injection of a freshly prepared solution of streptozotocin (55mg/Kg b.w) in 0.1M cold citrate buffer (PH 4.5) to the over night fasted rats (Sekar, 1990). Because of the instability of streptozotocin (STZ) in aqueous media, the solution is made using cold citrate buffer (PH 4.5) immediately before administration. Control rats were injected with citrate buffer alone. The rats were allowed to drink 5% glucose solution overnight to overcome the drug-induced hypoglycemia. The blood glucose values were above 250mg/dl on the third day after STZ injection, they were considered as diabetic rats. Then the treatment was started on the 5th day after STZ injection and it was considered as 1st day of treatment.
The rats were divided into four groups with six animals for each group.
Group1: Normal control rats (nondiabetic, untreated).
Group2: Diabetic control rats (diabetic, untreated).
Group3: Diabetic rats treated with S. chinensis root extract (250mg/kg b.w/day) in aqueous solution orally for 28 days.
Group4: Diabetic rats treated with glibenclamide (600µg/kg b.w/day) in aqueous solution orally for 28 days.
At the end of the treatment (28 days), the animals were deprived of food overnight and anesthetized and sacrificed by cervical dislocation. Blood was collected in heparinised tubes and used for the estimation of hemoglobin and glycoslated hemoglobin. The liver and kidney tissue were excised and rinsed in ice-cold physiological saline. The tissue were homogenized by tissue homogenizer with a Teflon pestle at a 4 o C in 0.1 M Tris-Hcl buffer at a pH 7.4. The supernatant was kept in –20o C until for further use. The homogenate (liver and kidney) were centrifuged in a cooling centrifuge (500x g) to remove the debris and supernatant was used for the biochemical analysis. The total proteins were estimated by Lowery et al. (1951) method.
Estimation of blood glucose, hemoglobin and glycosylated hemoglobin
The blood glucose level was estimated by the method of O-toludine by Sasaki et al. (1972). Hemoglobin was estimated by Drabkin and Austin (1932) method. The method of Nayak and Pattabiraman (1981) was used for assay the glycosylated hemoglobin.
Estimation of lipid perioxidation in liver and kidney
The lipid perioxidation was estimated by the method of Okhawa et al. (1979) by using thiobarbituric acid reactive substances (TBARS). Hydroperoxides were determined spectrophotometrically by Jiang et al. (1992) method.
Determination of reduced glutathione (GSH)
For the estimation of reduced glutathione (GSH), Sedlak and Lindsay (1968) method was used.
Determination of catalase (CAT) and superoxide dismutase (SOD)
The method of Misra and Fridovich (1972) was used for the determination of superoxide dismutase (SOD). Catalase (CAT) activity was analyzed by using Takahara et al. (1960) method.
Determination of glutathione peroxidase (GPx) and glutathione-S-transferase (GST)
Glutathione peroxide (GPx) activity was determined by the method of Rotruck et al. (1973). The method of Habig et al. (1974) was used to determined glutathione-S-transferase (GST).
All data are expressed as mean ± standard deviation for six animals in each group.
All the grouped data were statistically evaluated with SPSS/7.5 software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant difference (LSD) test. P values of less than 0.05 were considered to indicate statistical significance.
The levels of blood glucose, hemoglobin and glycosylated hemoglobin in normal control and experimental rats were represented in Table-1. The levels of blood glucose and glycosylated hemoglobin were significantly increased and a concomitant decrease in the level of total hemoglobin in the diabetic rats, when compared with normal rats. The methanolic root extract of S. chinensis and glibenclamide treated diabetic rats were significantly decreased the blood glucose, glycosylated hemoglobin levels and increased the level of total hemoglobin.
The levels of TBARS and hydroperoxides in liver and kidney tissues of control and experimental groups of rats were shown in Table-2. Diabetic rats were showed a significant increase in the levels of TBARS and hydroperoxides as compared with normal control rats. The methanolic root extract of S. chinensis and glibenclamide treated diabetic rats were showed notably decrease in the levels of TBARS and hydroperoxides.
The GSH level in liver and kidney of control and experimental groups of rats were shown in Table-3. The GSH level was significantly decreased in liver and kidney of diabetic rats when compared to that of control rats. The level of GSH in the methanolic root extract of S.chinensis and glibenclamide treated diabetic rats were brought back to near normal.
The activities of SOD and CAT in the liver and kidney of control and experimental groups of rats were shown in Figure-1. There was a significant decrease in the activities of SOD and CAT in liver and kidney of diabetic rats. The SOD and CAT activities were brought back to near normalcy due to the treatment of methanolic root extract of S. chinensis and glibenclamide in diabetic rats.
The activities of GPx and GST in the liver and kidney of control and experimental groups of rats were represented in Figure-2. A significant decrease of GPx and GST activities was noticed in liver and kidney of diabetic rats when compared with corresponding control rats. The administration of methanolic root extract of S. chinensis and glibenclamide significantly increased GPx and GST activities in the diabetic rats.
The free radical and reactive oxygen species are involved in most of the diseases including diabetes. In diabetes mellitus, free radicals may play an important role in causation and complications (Baynes, 1991). The STZ-induced diabetic rats may showed a most of the diabetic complications, such as myocardial, cardiovascular, urinary bladder dysfunctions, vas deferens, nervous and kidney through oxidative stress (Ozturk et al., 1996). The treatment of medicinal plant extract to the moderate STZ-induced diabetic rats, that activated the β cells and granulation return to normal, like to be insulinogenic effect (Padmini and Chakrabarti, 1982).
The free radicals involvements in diabetes and toxic species role in lipid peroxidation and antioxidant defense system have been studied. The lipid perioxidation and antioxidant potential have been measured in various tissues such as liver and kidney of normal and experiment groups of rats. The glibenclamide is a standard hypoglycemic drug, used to compare the hypoglycemic property in experimental rats. Glibenclamide have been stimulating insulin secretion from pancreatic β cells principally by inhibiting ATP sensitive KATP channels in the plasma membrane (Ashcroft FM and Aschcroft SJH, 1992). Courtois et al., (2003) have reported that glibenclamide treated STZ-induced diabetic rats were decreased the blood glucose level. The previous report is consistent with our present findings. The methanolic root extract of S. chinensis treated STZ-induced diabetic rats were decreased the blood glucose level to near normal level. It may be considered that the mechanism of action of methanolic root extract of S.chinensis mechanism is similar to glibenclamide. The level of glycosylated hemoglobin is considered as one of the marker for oxidative stress in diabetes mellitus. The excess of glucose is present in the blood, during diabetes, which react with hemoglobin and form glycosylated hemoglobin. Glycosylated hemoglobin was found to increase in diabetic mellitus and the amount of increase is directly proportional to that of fasting blood glucose level (Sheela and Augusti, 1992). In our study, STZ-induced diabetic rats showed a high level of glycosylated hemoglobin, when compared to that of normal rats. The methanolic root extract of S. chinensis treated STZ-induced diabetic rats were decreased the level of glycoslated hemoglobin that could be due to normoglycemic activity.
The tissue lipid peroxidation in diabetic rats were increased and this may be due to increase the levels of blood glucose, with generate free radicals and auto oxidation (Halliwell and Gutteridge, 1985). Lipid peroxidation mediated tissue damage has been observed in the development of type-I and type-II diabetes mellitus and also one of the characteristic features of chronic diabetes (Feillet, 1999). Lipid radical and lipid peroxide are harmful to the body cells and associated with brain, kidney damage and atherosclerosis (Soon and Tan, 2002). In diabetic rats, increase lipid peroxidation in the tissues may be due to the observed remarkable increase in the levels of TBARS and hydroxides in the liver and kidney (Stanely et al., 2001). In the present study we observed increased levels of TBARS and hydroperoxides in liver and kidney tissues of STZ-induced diabetes, when compared to the normal control. Administration of methanolic root extract of S. chinensis and glibenclamide to STZ-induced diabetic rats were reduced the lipid peroxides to near normal level in the liver and kidney tissues. This may suggested that methanolic root extract of S. chinensis inhibit oxidative damage of liver and kidney tissues.
Glutathione is known to protect the cellular system against toxic effects of lipid peroxidant (Nicotera and Orrenius, 1986). GSH acts as an antioxidant and its level was reduced in diabetes mellitus (Baynes and Thrope, 1999). Reduced gultathione functions as a free radical scavenger and in the repair of radical caused biological damage and decrease of GSH content may alter antioxidant enzymes (Garg et al., 1996). The reduction of GSH may also reduce the GST activity, because GSH is a substrate for GST activity (Rathore et al., 2000). In our study, a significant decrease in GSH levels was observed in hepatic and renal tissues of STZ-induced diabetic rats. The methanolic root extract of S. chinensis and glibenclamide treated STZ-induced diabetic rats were increased the level of GSH in liver and kidney tissues. This suggested that the extract may increase the biosynthesis of GSH or reduce the oxidative stress or both effect. Thus our data is in agreement with other medicinal plants such as Aloe vera (Rajasekaran 2005) and Gymnema montanum (Ananthan 2004).
SOD and CAT are the two major scavenging enzymes that remove toxic free radicals (Cohen and Heikkila, 1974). The SOD and CAT activities were reduced in liver and kidney of diabetic rats has been reported (Stanley et al., 2001). The superoxide radicals were converted into H2O2 (hydrogen peroxide) and molecular oxygen by SOD (McCrod et al., 1976). CAT catalyzes the reduction of hydrogen peroxides and protects the tissues from highly reactive hydroxyl radical (Chance, 1952). The decrease of SOD and CAT activities could result from inactivation by glycation of the enzyme (Sozmen et al., 2001; Yan and Harding, 1997). The removing of O2– and OH– is probably one of the most effective defense mechanism against diseases. The methanolic root extract of S. chinensis and glibenclamide treated STZ-induced diabetic rats were increased the activities of SOD and CAT near to normal level. This result suggest that methanolic root extract of S. chinensis may contains a free radical scavenging activity and prevent pathological alteration caused by O2– and OH– .
GPx and GST along with glutathione catalyze the reduction of hydrogen peroxide into non-toxic metabolites (Bruce et al., 1982). During diabetes, decrease in the concentration of GSH which lead to reduce the activities of GPx and GST (Shanthi et al., 1994). The GST activity was decreased may result from inactivation caused by reactive oxygen species (Andallu et al., 2003). The activities of GST and GPx were normalizes in the methanolic root extract of S. chinensis treated diabetic rats.
The methanolic root extract of S. chinensis were shown to possess antioxidants activity by increase the levels of hemoglobin, GSH, CAT, SOD, GPx, GST and reducing the blood glucose, glycosylated hemoglobin levels, which reduces the glycation of the enzyme and prevent the free radical formation. Further studies will be needed to purify the bioactive compounds, which is responsible for free radical scavenging activity in the methanolic root extract of S. chinensis.
AcknowledgementsThe authors are thankful to the University Grants Commission (UGC-UWPFE Project No: HS-37/2003) for the research grant. The authors are grateful to Dr.G.Suvarnalatha, DST-women Scientist, CAS in Botany, University of Madras.
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