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  • The Internet Journal of Pharmacology
  • Volume 6
  • Number 1

Original Article

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 Centratherum anthelminticum wild. kuntze (Asteaceae) on blood glucose level and antioxidant enzymes level in streptozotocin induced diabetic rats. Streptozotocin (STZ 50 mg/kg, i.p) induced diabetic rats were treated with methanolic extract of Centratherum anthelminticum (100 mg/kg; p.o.) for 28 days. Methanolic extract of Centratherum anthelminticum wild. Kuntze significantly prevented loss of body weight, decrease food and water intake. It showed significant prevention in elevation of glucose and significantly increased insulin level and showed prevention in increase in the cholesterol, triglyceride, LDL cholesterol, VLDL cholesterol levels in serum of diabetic rats. The treatment with methanolic extract of Centratherum anthelminticum also significantly prevented decrease in the HDL-cholesterol level and increase in urea and creatinine levels in diabetic rats compared to that of diabetic control. The chronic treatments with methanolic extract of CA significantly prevented the increase of malondialdehyde and prevent reduction of superoxide dismutase, catalase, and reduced glutathione levels. Histopathological study revealed that Centratherum anthelminticum extract provided significant protection against STZ induced degeneration in liver and kidney. This confirms the antidiabetic and antioxidant activity of Centratherum anthelminticum wild. kuntze in streptozotocin induced diabetic rats.

 

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. C. anthelminticum is an important drug of indigenous system of medicine and has been known for a number of medicinal properties in Ayurveda. C. anthelminticum is used as folk-medicine for diabetes in Rayalaseema of Andhra Pradesh of india[10].And therefore the present study was undertaken to evaluate anti-diabetic and anti-oxidant activity of Centratherum anthelminticum in streptozotocin induced diabetic rats.

Material and methods

Plant materials and prepatration of methanolic extracts

The fresh seeds of Centratherum anthelminticum (Wild.) Kuntze; fimily Asteaceae were obtained from the Commercial sources and identified and authentified by Dr. G. C. Jadeja, Professor and Head, Department of Agricultural Botany, B. A. College Of Agriculture, Anand Agriculturural University, Anand, Guajrat, India.

1 kg of powder of shade dried seeds of C. Anthelminticum were extracted in round botton flask with 95% methanol for 48 hours with vigorous shaking at the regular intervals. The alcoholic extract was filtered and solvent was removed under vaccume the resulting extract was air dried, weighed (30 % ) and stored in cool and dry place.

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 ad libitum.

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 Centratherum anthelminticum (100 mg/kg; p.o.) and standard drug Glibenclamide (0.25 mg/kg;p.o.) respectivly for 28 days after induction of diabetes.

Methanolic extract of Centratherum anthelminticum (CA) was suspended in 0.5% NaCMC solution before administration. The food intake, water intake and body weight of animals were recorded in all groups daily. The groups were as follows:

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 Centratherum anthelminticum (100 mg/ kg/ p.o. / daily);

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 ± 1.93 gms/animal/day group II Vs 198.66 ± 2.9 gms/animal/day in group III) and significantly decreased the food intake (20.59 ± 0.42 gms/animal/day group III Vs 40.76 ± 0.67 gms/animal/day in group II) and water intake (59.69 ± 0.50 ml/animal/day in group III Vs 75.89 ± 1.13 ml/animal/day in group II) as compared to group II (Table 1).

Figure 1
Table 1: Effect of methanolic extract of treatment on general features of diabetic rats.

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 CA (100mg/kg; p.o.) produced significant decrease in serum glucose level, and increased serum insulin level as compared to group II. Decreased in blood glucose level was more with treatment of Glibenclamide (0.25 mg/kg/day/p.o.) as compared to CA. (Table 2).

Figure 2
Table 2: Effect of methanolic extract of treatment on biochemical parameters of diabetic rats.

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 CA or glibencamide significantly preventing the increase in serum cholesterol, LDL-cholesterol, and VLDL-cholesterol, serum triglycerides levels as compared to group decreased non-significantly in group II. Treatment with the methanolic extract of CA or glibenclamide produced significant increase in HDL-Cholesterol levels as compared to in group II. STZ-diabetic rats exhibited significantly higher serum urea level and serum creatinine level as compared to those of group I. Chronic treatment with methanolic extract of CA and glibenclamide significantly prevented this increase in serum urea and serum creatinine levels of group II (Table 3).

Figure 3
Table 3: Effect of methanolic extract of treatment on biochemical parameters of diabetic rats.

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 CA produced significant increase in glutathione level, and significant decrease in MDA level but SOD level was not significantly changed. But glibenclamide showed highly significant change in MDA and glutathione level. The catalase activity in the group II were found to be significantly decreased as compared to that of the group I. Treatment with the methanolic extract of CA increased the catalase activity but it was not significantly different than group II. The Super Oxide Dismutase and catalase levels were found to be increased with glibenclamide as compared with the group III in STZ-treated diabetic rats (Table 4).

Figure 4
Table 4: Effect of methanolic extract of C. anthelminticum on antioxidant parameters in STZ induced diabetic rats.

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 or group IV showed reduced severity of morphological changes and lesion compared with the group II rats (Fig 2 c,d).

Figure 5
Figure 1: Histopathology of liver

Figure 6
Figure 2: Histopathology of kidney

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 Centratherum anthelminticum was reported as a folklore medicine in India [10]. The characteristics of diabetes like polyphagia, polyuria and polydipsia were also improved by the treatment of CA (100 mg/kg/28 days) in diabetic animals which was comparable to normal control.

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 C. anthelminticum produced similar decrease in serum cholesterol level in diabetic rats. There were also significant decrease in serum LDL and VLDL level and significant increase in serum HDL level by treatment with methanolic extract C. anthelminticum compared to diabetic control.

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 C. anthelminticum significantly preventing in increase serum creatinine level in diabetic rats which indicated its beneficial effect on kidney. But the decrease with the Glibenclamide was highly significant and which was evidenced by histopathology study of kidney. We found significant protective effect on kidney with Glibenclamide, similar results were not observed with the test drug.

STZ induced liver and kidney damage as seen in histopathology which was reversed on CA treatment. In our study, administration of methanolic extract of C. anthelminticum to the STZ induced diabetic rats protect STZ induced liver and kidney damage.

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 C. antehlminticum caused the increase in the hepatic SOD and CAT activities of the STZ-induced diabetic rats. This means that the methanolic extract of CA can reduce not only the reactive oxygen free radicals but also and improve the activities of the hepatic anti-oxidant enzymes.

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 Centratherum antehlminticum decreased glucose levels, increase insulin levels and decreased oxidative stress suggesting its use as antidiabetic and antioxidants.

Correspondence to

J. G. shah Department of Pharmacology Anand pharmacy college, Anand, Gujarat. E-mail: jigneshshah_rx@yahoo.co.in

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Author Information

J.G. Shah, M. Pharm.
Department of pharmacology,, Anand Pharmacy College

M.S. Patel, M. Pharm.
Department of pharmacology,, Anand Pharmacy College

K.V. Patel, M. Pharm.
Department of pharmacology,, Anand Pharmacy College

T.R. Gandhi, M. Pharm., Ph.D.
Department of pharmacology,, Anand Pharmacy College

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