Department of Biochemistry, Faculty of Science,
Annamalai University, Annamalainagar
608 002, Tamilnadu, India
Correspondence should be addressed to: K.V. PUGALENDI, M.Sc.
Submitted for publication: M.Sc
Keywords: Diabetes, Streptozotocin, Umbelliferone, Redox status, Antioxidants
Plant derived phenolic coumarins might play a role as dietary antioxidants because of their consumption in human diet as fruits and vegetables. Umbelliferone (7-hydroxycoumarin), a derivative of coumarin, is a benzopyrone in nature and it is present in the fruits of Aegle marmelos L., Anethum graveolens L. and Ruta graveolens L. (eatable fruits). Hence the present study was designed to evaluate the protective effect of Umbelliferone (UMB) on lipid peroxidation, antioxidants and lipid profile in tissues (heart and brain) of streptozotocin (STZ) diabetic rats. Adult male albino rats of Wistar strain, weighing 180-200 g, were induced diabetes by administration of STZ (40 mg/kg b.wt.) intraperitonially. The normal and diabetic rats were treated with UMB dissolved in 10% dimethyl sulfoxide (DMSO), for 45 days. Diabetic rats exhibited elevated levels of lipid peroxidation markers (thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (HP) and conjugated dienes (CD), and reduction in nonenzymic antioxidants (vitamin C and reduced glutathione (GSH) except vitamin E, and enzymic antioxidants (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and elevated levels of total cholesterol (TC), triacylglycerol (TG), free fatty acids (FFA) and phospholipids (PL) in the heart and brain tissues. Treatment with UMB brought back lipid peroxidation, antioxidants and lipid profile to near normal levels.
During hyperglycemia enhanced formation of oxygen free radicals occurs in tissues (1). Many studies have shown that increased lipid peroxides and/or oxidative stress are present in diabetic subjects, and may underlie their increased risk of cardiovascular disease (2,3) and excessive production of free radicals is believed to be involved in many diabetic complications including diabetic neuropathy in diabetes mellitus (4). Many studies have evaluated the effects of oxidative stress and antioxidant systems in the central and peripheral nervous system in diabetes mellitus (1& 5) . Oxidative stress in diabetes may partially be reduced by antioxidants and antioxidants have been prescribed to reduce the long term complications of diabetes.
Recent reports indicate that there is an inverse relationship between the dietary intake of antioxidants rich foods and the incidence of human diseases (6). Earlier study has shown that treatment with antioxidant reduces diabetic complications (7). Efforts to discover antioxidants as useful drug candidates to combat diabetic complications are going on relentlessly. In this connection, minor dietary constituents, especially plant based foods have come under serious scrutiny (8).
Plant derived phenolic coumarins might play a role as dietary antioxidants because of their consumption in the human diet in fruits and vegetables (9). Umbelliferone (7-hydroxycoumarin), a derivative of coumarin, is a benzopyrone in nature and it is present in the fruits of Aegle marmelos Correa (10) and Anethum graveolens L. (11). UMB has also been reported to have antioxidant properties (9). The parent compound coumarin has been reported to reduce blood glucose level (12). In our studies, UMB has reduced blood glucose and lipid peroxidation and elevated antioxidants’ status in plasma and liver of STZ-diabetic rats (13,14). But, no detailed study has been carried out on the effect of UMB on lipid peroxidation, antioxidants and lipid profile in the heart and brain of STZ-diabetic rats. Hence, the present study was planed to evaluate the effect of UMB on lipid peroxidation, nonenzymic and enzymic antioxidants, and lipid profile in the heart and brain of STZ- diabetic rats. The structure of UMB is depicted below.
MATERIALS AND METHODS
Male albino rats of Wistar strain with body weight 180-200 g, were procured from Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University, and were maintained in an air conditioned room (25 ± 1 °C) with a 12 h light: 12 h dark cycle. Feed and water were provided ad libitum. Studies were carried out in accordance with Indian National Law on Animal Care and Use, and the study was approved by the Ethical Committee of Rajah Muthiah Medical College and Hospital (Reg. No: 160/1999/CPCSEA*), Annamalai University, Annamalainagar, Tamilnadu, India. * Committee for the Purpose of Control and Supervision of Experiments on Animals.
Streptozotocin was purchased from Sigma-Aldrich, St. Louis, USA. UMB was procured from Carl Roth GmbH & Co, Germany. All other chemicals used were of analytical grade obtained from E. Merck and HIMEDIA, India.
Experimental induction of diabetes
The animals were rendered diabetes by a single intraperitonial injection of STZ (40 mg/kg b.wt) in freshly prepared citrate buffer (0.1M, pH 4.5) after an overnight fast. STZ injected animals were given 20 % glucose solution for 24 h to prevent initial drug-induced hypoglycemic mortality. STZ injected animals exhibited massive glycosuria (determined by Benedict’s qualitative test) and hyperglycaemia (by glucose oxidase method) within a few days. Diabetes in STZ rats was confirmed by measuring the fasting blood glucose concentration, 96 h after injection with STZ. The animals with blood glucose above 235 mg/dL were considered to be diabetic and used for the experiment.
The animals were randomly divided into 4 groups of six animals each as given below. UMB was administered intraperitonially using vehicle solution (10% DMSO).
Group I : Normal control received 10% DMSO only
Group II : Normal + UMB (30 mg/kg/b.wt. in 10% DMSO)
Group III : Diabetic control (10% DMSO)
Group IV : Diabetic + UMB (30 mg/kg/b.wt. in 10% DMSO)
After 45 days of treatment, the 12 h fasted animals were anaesthetized between 08.00 am and 09.00 am, using ketamine (24 mg/kg b.wt) (intramuscular injection) and sacrificed by cervical decapitation. Tissues (heart and brain) were collected for the estimations of lipid peroxidation, antioxidants and lipid profile.
The estimation of TBARS, HP and CD was done by the methods of Nichans and Samuelson (15), Jiang et al (16) and Klein (17) respectively. The levels of vitamin C and E and GSH were estimated by the methods of Roe and Kuether (18), Baker et al (19) and Ellman (20). The activities of SOD, CAT and GPx were measured by the methods of Kakkar et al (21), Sinha (22) and Rotruck et al (23) respectively. Tissue total lipids were extracted by the method of Folch et al (24) and the levels of TC, TG, FFA and PL were estimated by the methods of Siedel et al (25), Foster and Dunn (1973)(26) , Falholt et al (27) and Zilversmit and Davis (28) respectively.
Statistical analysis of data
Values are given as means ± S.D for six rats in each group. Data were analyzed by one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT) using SPSS-10. The limit of statistical significance was set at p < 0.05.
The levels of TBARS, HP and CD in the heart and brain tissues of diabetic rats are represented in table 1 and table 2.
In our study, the diabetic rats had elevated levels of TBARS, HP and CD in the heart and brain tissues. Treatment with UMB showed reversal of these parameters to near normal levels.
Table 3 and table 4. represent the levels of vitamin E and C and GSH in the heart and brain tissues of diabetic rats. In our study, diabetic rats exhibited an elevated level of vitamin E and decreased levels of vitamin C and GSH in the tissues. Treatment with UMB had reversed these parameters to near normal levels.
The activities of SOD, CAT and GPx in the heart and brain tissues of diabetic rats are illustrated in table 5 and table 6. . In our study, diabetic rats had increased activities of SOD, CAT and GPx in heart and brain tissues. Treatment with UMB showed reversal of these parameters to near normal levels.
Table 7 and table 8. show the levels of TC, TG, FFA and PL in the heart and brain tissues of diabetic rats. In our study, diabetic rats had elevated levels of TC, TG, FFA and PL in the heart and brain tissues. Treatment with UMB showed the reversal of these parameters to near normal levels.
Hyperglycemia of diabetics has been associated with increased reactive oxygen species (ROS) formation (29). Increased formation of glucose-derived advanced glycation end products (30) and increased glucose flux through polyol pathway are some of the known biochemical mechanisms of hyperglycemia induced tissue damage (31). Free radicals result in the consumption of antioxidant defenses which may lead to disruption of cellular functions and oxidative damage to membranes and enhance susceptibility to lipid peroxidation (32). Oxidative stress in diabetes coexists with a reduction in the antioxidants’ status (33).
In our study, the lipid peroxidation markers (TBARS, HP and CD) are elevated in diabetic rats as reported earlier (34). The increase in lipid peroxidation might be a reflection of decrease in enzymatic and nonenzymatic antioxidants of defense systems (35). Plant derived phenolic coumarins might play a role as dietary antioxidants because of their consumption in the human diet in fruits and vegetables and UMB has also been reported to have antioxidant properties (9). Treatment with UMB brought back lipid peroxidation markers to near normal levels which could be as a result of improved glycemic control and antioxidants’ status.
Endogenous antioxidant enzymes (SOD, CAT and GPx) are responsible the detoxification of deleterious oxygen radicals (36). In our study, the activities of SOD, CAT and GPx decreased in diabetic rats as reported earlier (37, 38). which could be due to increased utilization for scavenging free radicals. Treatment with UMB has reversed the activities of these enzymatic antioxidants which could be as a result of decreased utilization and/or decreased lipid peroxidation.
Vitamin E is well accepted nature’s most effective lipid soluble chain breaking antioxidant in the biological system protecting cell membrane from peroxidative damage (39). In our study, vitamin E increased in diabetic rats as reported earlier (40), which could be due to increased membrane damage by ROS. Treatment with UMB brought vitamin E to near normal levels which could be as a result of decreased membrane damage as evidenced by decreased lipid peroxidation.
Vitamin C is one of the most powerful natural antioxidants (41). It is capable of regenerating a-tocopherol from tocopheroxyl radical that is formed upon the inhibition of lipid peroxidation by vitamin E (42). Vitamin C has been reported to contribute up to 24% of the total peroxyl radical-trapping antioxidant activity (TRAP) (43). In our study, vitamin C decreased in diabetic rats as reported earlier (44). Treatment with UMB brought back vitamin C to near normal levels which could be due to decreased utilization for the recycling of vitamin E as lipid peroxidation is low.
Glutathione is widely distributed in animal tissues and provides cellular protection against oxidative damage (45). GSH is required for the recycling of vitamin C (46) and acts a substrate for GPx and GST that are involved in preventing the deleterious effect of oxygen radicals (47). In our study, diabetic rats exhibited decreased level of GSH which may be due to increased utilization. Treatment with UMB reversed GSH level in heart and brain of diabetic rats which could be due to decreased utilization, as lipid peroxidation is low.
The marked hyperlipidemia that characterizes the diabetic state may therefore be regarded as a consequence of uninhibited actions of lipolytic hormones on the fat depots (48). Lowering of serum and tissue lipids through diet or drug seems to be associated with a decrease in the risk of vascular disease (49).
Hypercholesterolemia and hypertriglyceridemia have been reported to occur in streptozotocin diabetic rats (50, 51). and a significant increase observed in our experiment was in accordance with these studies. It has been reported that an elevated plasma triglyceride level is associated with an increased risk of mortality in cerebrovascular disease (52). Diabetic rats treated with UMB brought back TC and TG to near normal levels which could be due to an increase in the utilization of glucose and thereby decreasing the mobilization of free fatty acids from the fat depots.
In our study, diabetic rats had elevated level of FFA levels, which might be associated with increased actions of lipolytic hormones on fat depots. In diabetic rats treated with UMB, the decreased level of FFA is associated with decreased actions of lipolytic hormones on fat depots and improved glycaemic control and/or decreased activity of hormone sensitive lipase.
Phospholipids are vital components of biomembrane and play an importantly role in the transport of triglycerides (53). The decreased level of phospholipids in UMB treated rats is due to decreased membrane damage as evidenced by decreased lipid peroxidation and improved antioxidants’ status.
Our results we show that UMB possesses a promising protective effect against oxidative stress in heart and brain as evidenced by decreased lipid peroxidation and increased antioxidants’ status, and decreased lipid profile in the heart and brain tissues, which may protect from the risk of diabetic complications.