Evaluation of Anti-diabetic and Anti-oxidant Activity of Centratherum anthelmintica in STZ – induced Diabetic Rats
J Shah, M Patel, K Patel, T Gandhi
Keywords
diabetes, streptozotocin stz.
Citation
J Shah, M Patel, K Patel, T Gandhi. Evaluation of Anti-diabetic and Anti-oxidant Activity of Centratherum anthelmintica in STZ – induced Diabetic Rats. The Internet Journal of Pharmacology. 2007 Volume 6 Number 1.
Abstract
The present work is carried out to study the effect of
Introduction
Diabetes mellitus is a chronic metabolic disorder characterized by a high blood glucose concentration - hyperglycemia (fasting plasma glucose>126 mg/dL, or plasma glucose >200 mg/dL 2 hrs after a meal) - caused by insulin deficiency, often combined with insulin resistance [1]. In diabetic patients, oxidative stress also has been found to be mainly due to increased production of oxygen free radicals and sharp reduction of anti-oxidant defense [2]. Hypoinsulinemia associated with diabetes increased the activity of enzyme, fatty acyl Coenzyme A oxidase which initiates the β-oxidation of the fatty acids, resulting in lipid peroxidation. Increased lipid peroxidation impairs membrane function by decreased membrane fluidity and changing the activity of the membrane-bound enzyme and receptors. Its products (lipid radices and lipid peroxides) are harmful to the cell in the body and associated with atherosclerosis and brain damage [3]. Diabetes Mellitus is associated with abnormalities in carbohydrates and lipid metabolism that results in excessive production of reactive oxygen species [ROS] and oxidative stress. [4,5,6,7,8,9]
Diabetes mellitus is one of the disease for which a satisfactory treatment is not available in modern allopathic system of medicine. Therefore the search for an ideal drug for the treatment of diabetes containing and has been extended to herbs.
Material and methods
Plant materials and prepatration of methanolic extracts
The fresh seeds of
1 kg of powder of shade dried seeds of
Animals
Healthy Male Wister rats weighing 200-250 gm were used for the study [11]. The animals were housed in a group of 3 rats per cage under well-controlled conditions of temperature (22 2 C), humidity (55 ± 5%) and 12hrs/12hrs light-dark cycle. Animals had free access to laboratory diet and tap water
Induction of type I diabetes mellitus
Diabetes was induced with Streptozotocin (50 mg/kg; i.p.; once) dissolved in citrate buffer (pH 4.5) in 0.9% normal saline solution under light ether anesthesia. Normal Control animals were injected with an equivalent volume of citrate buffer in 0.9% normal saline solution. Animals were divided in to four groups. After 24-48 hours, extent of glycosuria was checked using Diastix (Bayer Diagnostics, India). Animals showing glycosuria (>2%) were considered as diabetic. In addition, the groups III and group IV were treated with a methanolic extract of
Methanolic extract of
Group I: normal control treated with normal saline (once);
Group II: diabetic control treated with Streptozotocin (50 mg/kg/ i.p./ once);
Group III: diabetic treated with
Group IV: diabetic treated with Glibenclamide (025 mg/ kg/ o.p. / daily)
Evaluation of Biochemical Parameters
At the end of the treatment blood samples were collected from the retro orbital plexuses under light ether anesthesia. The serum was separated by centrifugation. The serum levels parameters were analyzed spectrophotometrically by using double beam UV-Visible spectrophotometer (Shimadzu UV- UV-Visible spectrophotometer, model 1601). Estimation of serum glucose level (GOD-POD method), serum insulin level (RIA method), serum cholesterol (enzymatic method), HDL cholesterol (enzymatic method), serum triglyceride (enzymatic method), serum urea (berthelot method) and creatinine (alkaline picrate method) was done. VLDL-cholesterol and LDL- cholesterol were calculated as per Friedewald' equation[12].
VLDL-cholesterol = total serum triglycerides/5
LDL-cholesterol= Total serum cholesterol- total serum triglycerides/5-HDL-C
Evaluation of antioxidant Parameters
Animal were scarified at the end of 28 days treatment. The liver of animal was dissected out, rinsed with ice cold distilled water followed by sucrose solution (0.25 M). One gm of liver tissue was homogenized in 10 ml ice cold Tris hydrochloride buffer. The prepared homogenates were centrifuged and used for the determination of antioxidant parameters like Malondialdehyde (MDA) [13], Superoxide dismustase (SOD) [14], catalase [15], Reduced Glutathione (GSH) [16] levels and total protein estimation [17]. Liver and kidney were isolated from one animal of each group and used for histopathology.
Statistical analysis
Results were presented as mean SEM. Statistical differences between the means of the various groups were evaluated using one-way analysis of variance followed by tukey's multiple parametric tests. Data were considered statistically significant at P value ≤ 0.05 and highly significant at P 0.001. Statistical analysis was performed using Sigma stat statistical software (Ver.2.03).
Results
Body weight, Food intake and Water intake
Intraperitoneal injection of 50 mg/kg STZ in adult rats produced cardinal signs of type I diabetes i.e., loss of body weight, polyphagia, and polydipsia (table 1). Chronic treatment with methanolic extract of CA (100mg/kg/p.o./28 days) was found to prevent the loss of body weight (205.98
Figure 1
Serum glucose and insulin levels
STZ-diabetic rats were found to be significant hyperglycemic with a corresponding hypoinsulinaemia as compared to the group I. Treatment with methanolic extract of
Figure 2
Various biochemical parameters
STZ-diabetic rats (group II) were found to have significantly increased serum cholesterol, LDL-cholesterol, VLDL-cholesterol, and triglycerides, levels as compared to group I. HDL-cholesterol was also reduced significantly in diabetic rats. Treatment of methanolic extract of
Figure 3
Estimation of anti-oxidant parameters
STZ-diabetic rats (group II) were found to exhibit significantly decreased glutathione level (GSH) and SOD level as well as significantly increased Malondialdehyde (MDA) level as compared to group I. Treatment with methanolic extract of
Figure 4
Histopathological Changes
Liver sections of group I rats showed normal hepatocytes, central vein of liver lobules and normal sinusoid (Fig 1 a). In contrast hepatocytes of group II rats showed more lesions and reduction in sinusoid (Fig 1 b). The group II and group III showed reduced severity of morphological changes and fewer lesions in hepatocytes and sinusoid compared with the group II. But significant improvement was observed with group IV (Fig 1 c, d).
Histopathological examination of sections of kidney from group I rats showed no pathological changes (Fig. 2 a). Group I rats had normal renal glomerular morphology. In contrast the glomeruli of group II rats were enlarged due to diabetic exudative and diffuse lesion (Fig. 2 b). The group III
Figure 5
Figure 6
Discussion
Diabetes mellitus (DM) is an endocrine disorder in which glucose metabolism is impaired because of total loss of insulin after destruction of pancreatic beta cell (type I diabetes mellitus) or because of inadequate release of insulin from the pancreatic beta cells or insensitivity of target tissue to insulin. The fundamental mechanism underlying hyperglycemia involved over-production (excessive hepatic glyconeolysis and gluconeogenesis) and decreased utilization of glucose by the tissue [18]. Persistence hyperglycemia, the common characteristics of diabetes can cause most diabetic complications.
The antidiabetic activity of
STZ induces type I diabetes through significant increase in glucose levels associated with decrease in insulin levels. Similar results were observed in our study. Treatment with CA reversed the STZ induced changes suggested that the CA has anti diabetic potential.
STZ diabetic animals exhibits most of the diabetic complications like myocardial, gastrointestinal, nervous, vas deferens, and kidney and urinary bladder dysfunction, through oxidative stress [19]. Hypertriglyceridemia is also associated with metabolic consequences of hypercoagulability, hyperinsulinemia, insulin resistance and insulin intolerance [20]. Shirwaikar et al, (2004) [21] demonstrated marked increase in triglycerides level and decrease in insulin levels in STZ induced diabetic rats. Results of the present study were in accordance showing increase in triglyceride levels and decrease in insulin level in STZ diabetic rats.
The observed hypolipidemic effect may be because of decreased cholestrogenesis and fatty acid synthesis [21]. In diabetic rats there is decrease in lipoprotein lipase activity [23] resulting in impaired clearance of VLDL and chylomicrons from plasma thus increasing the LDL and VLDL levels. In our study, treatment with methanolic extract
The morphological abnormalities in kidney in type 1 diabetic rats were associated with a significant elevation in serum creatinine and urea levels indicating impaired renal function of diabetic animals. Chronic treatment with methanolic extract of
STZ induced liver and kidney damage as seen in histopathology which was reversed on CA treatment. In our study, administration of methanolic extract of
Oxidative stress plays a major role in the induction of diabetes I and II and, as such, antioxidants may have a role in the elevation of diabetes [23] Streptozotocin produces oxygen free radicals in the body, which cause pancreatic injury and could be responsible for increased blood sugar seen in animals [24]. Recent studies have indicated that high glucose levels caused oxidative stress [25]. Furthermore, enhanced oxidative stress due to diabetes may also result from a dysfunction in the defense system against free radicals, such as reduction in glutathione or inactivation of superoxide dismutase [26].
In the present study, malondialdehyde levels were significantly higher in the STZ treated diabetic rats. The treatment with the CA showed significant decrese in the malondialdehyde (MDA) levels as compared to the normal control rats suggesting that lipid peroxidation reduced by STZ was reversed by CA treatment.
Studies have been reported on the reduction of hepatic SOD and CAT activities in STZ-induced diabetic rats when compared with the normal rats [27, 28]. SOD has been reported as one of the most important enzymes in the enzymatic anti-oxidant defense system. The superoxide anion has been known to inactivate CAT, which is involved in the detoxification of hydrogen peroxide [29]. SOD scavenges the supeoxide anion to form hydrogen peroxide, hence diminishing the toxic effect caused by this radical. Wohaibe et al (1987) [29] had suggested that the reactive oxygen free radicals could inactivate and reduced the hepatic SOD and CAT activities [27]. In the present study, it was observed the methanolic extract of
GSH has a multiple role in anti-oxidant defense. It is a direct scavenger of the free radicals as well as a co-substrate for peroxide detoxification by glutathione peroxidases [31]. Loven et al (1986) [32] had suggested that the decrease in hepatic GSH could be result of decreased synthesis or increased degradation of GSH by oxidative stress in diabetes. The treatment with CA.improve the reduced GSH level as compare to STZ diabetic rats suggesting strengthening of anti oxidant defenses.
The CA seeds are known to have 12,13-epoxy-9-octadecenoic, Linolenic acid, Myristic acid, Oleic acid, Palmitic acid, Stearic acid, Vernoleic acid, Brassicasterol, Stigmasterol an elemanolide lactone, Vernodalol which possess anti oxidant activity [33]. The anti oxidant action of CA might be because of the presence of above constituents which may be responsible for anti diabetic activity.
Conclusion
From these results, we conclude that methanolic extract of
Correspondence to
J. G. shah Department of Pharmacology Anand pharmacy college, Anand, Gujarat. E-mail: jigneshshah_rx@yahoo.co.in