ANTIHYPERLIPIDEMIC EFFECT OF TINOSPORA CORDIFOLIA EXTRACT IN STREPTOZOTOCIN INDUCED DIABETIC RATS

ANTIHYPERLIPIDEMIC EFFECT OF TINOSPORA CORDIFOLIA EXTRACT IN STREPTOZOTOCIN INDUCED DIABETIC RATS

K. Vetrivadivelan *¹, K.V. Venkateswaran ¹, S. Selvasubramanian ¹ and P.S.L. Sesh ²

Department of Veterinary Pharmacology and Toxicology ¹, Madras Veterinary College, Chennai – 600 007, Tamil Nadu, India

Department of Veterinary Biochemistry ², Madras Veterinary College, Chennai – 600 007, Tamil Nadu, India

ABSTRACT

The present study was aimed to evaluate the antihyperlipidemic effect of aqueous extract of stem of Tinospora cordifolia in streptozotocin induced diabetic and diet induced hyperlipidemic rats. Diabetes mellitus was induced in male albino Wistar rats by intraperitoneal injection of streptozotocin (45 mg/kg b.w.). Hyperlipidemia was induced in diabetic rats by feeding 0.5% cholesterol and 0.1% cholic acid on diet basis in arachis oil (10 ml/kg b.wt) orally throughout the experimental period and simultaneously, the treatment groups received T. cordifolia (100 and 200 mg/kg b.w.) orally for 14 days. Glibenclamide (600 µg/kg b.w) was used as a standard antidiabetic drug and atorvastatin (1.2 mg/kg b.w.) was used as a standard antihyperlipidemic drug. Serum glucose levels were determined at weekly intervals and serum cholesterol, triglycerides, HDL-C, creatine kinase and free fatty acids were analyzed at the end of the experiment. Aqueous extract of T. cordifolia at 200 mg/kg b.w. dose decreased serum glucose, cholesterol, triglycerides, creatine kinase, and free fatty acids to normal level when compared to that of standard drug. Thus the study revealed that the aqueous extract of T. cordifolia was found to have potential effective in controlling the diabetes associated hyperlipidemic conditions effectively.

Hyperlipidemia

INTRODUCTION: The relationship between diabetes and hyperlipidemia is a well-recognized phenomenon. Diabetes mellitus is a syndrome that is characterized by chronic hyperglycemia and disturbances of carbohydrate, fat and protein metabolism.

Accumulation   of   lipids   in   diabetes is mediated  through a variety of derailment in metabolic and regulatory processes, especially insulin deficiency, thereby  rendering  the  diabetic  patient  more  prone  for  hypercholesterolemia  and  hypertriglyceridemia. The major pathogenesis of derailed lipid metabolism in diabetes is increased mobilization of fatty acids from adipose tissue and secondary elevation of free fatty acid level in the blood ¹.

Hyperlipidemia is characterized by elevated serum total cholesterol and         low-density and very low-density lipoprotein levels. Hyperlipidemia associated lipid disorders are considered to be the cause for atherosclerotic cardiovascular disease ². Hyperlipidemia represents a determinant for the development of atherosclerosis and an important risk factor for cardiovascular disease. When hyperlipidemia is present in the context of the insulin resistance syndrome or metabolic syndrome, it predisposes a particular high risk condition.

This constellation, often referred to as the ‘lipid triad’, is particularly inducing atherogenic changes in the arterial wall and to promote plague instability, thus predisposing acute cardiovascular events ³.

Liver, an insulin-dependent tissue, that plays a pivotal role in glucose and lipid homeostasis, is severely affected during diabetes. Liver participates in the uptake, oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol, phospholipids and triglycerides. Pharmacological treatment of hyper-lipidemia in particular with statin drugs, was shown to greatly influence cardiovascular morbidity and mortality. A body of evidence also underlines the need for a multidisciplinary approach, integrating non-pharmacological life style and diet interventions, as well as treatment of concomitant diseases like diabetes and hyperlipidemia.

Since the currently available hypolipidemic agents lack desired properties of an ideal drug, research is aimed to find out an effective, safe and less expensive drug ⁴. Medicinal plants have been reported to be useful in diabetes worldwide and used empirically as antidiabetic and antihyperlipidemic remedies ⁵. Herbal therapy for hypercholesterolemia has no side effects and will be economically effective in reducing the lipid levels. Tinospora cordifolia (Guduchi) has been used extensively in the Indian system of medicine since ancient times. It is a glabrous climbing succulent shrub, commonly found in hedges. It is native to India and thrives easily on tropical region ⁶ and possess a variety of pharmacological activities.

In view of the pathogenic mechanisms involved in the progression of hyperlipidemia and plieotropic effects of T. cordifolia, the present study was carried out to evaluate the antihyperlipidemic effect of T. cordifolia aqueous extract in STZ induced diabetic rats.

MATERIALS AND METHODS:

Chemicals: Streptozotocin (STZ) was purchased from Sigma Chemicals Co. (USA). Atorvastatin and glibenclamide were provided by Blue Cross Laboratories Limited and Aventis Pharma Limited, India as gratis, respectively. Cholesterol and cholic acid were purchased from Himedia Laboratories Private Limited (India). All other chemicals used were of analytical grade.

Plant material collection and preparation of extract: Fresh, mature Tinospora cordifolia stem portions were obtained as gratis from M/s SKM Siddha & Ayurveda Nature’s Wealth, Erode District, Tamilnadu, India. The plant was identified and authenticated by the Botanist in the Department of Botany, Pachaiyappa’s College, University of Madras, Chennai, India. The specimen sample was assessed and validated for its quality by the M/s Natural Remedies Private Limited, Bangalore, India.

The stem portions of the plant were shade-dried at room temperature for 10 days and powdered.  The aqueous extract was prepared by soaking the powder in distilled water at the ratio of 1:2 (w/v) with constant stirring for 12-18 hrs and then filtered. The aqueous portion of the filtrate was evaporated by using rotary evaporator (Heidolph, Laborota 4000 efficient) at 40–50°C under reduced pressure, to yield dark brown coloured pasty product. The yield from this process was 4 per cent (w/w).

Experimental animals: Male albino Wistar rats, weighing about 150 - 200 grams were used for the present study. The animals were maintained in cages under controlled animal house conditions with 12/12 hours light/dark cycle. The animals were fed with standard rat feed (M/s Tetragon Chemie Private Limited, Bangalore, India) and provided water ad libitum throughout the experimental period. The animals were acclimatized under experimental conditions for one week prior to the start of experiment. All the animal procedures were carried out as per CPCSEA norms and the study was approved by Institutional Animal Ethics Committee (IAEC) of Madras Veterinary College, Chennai (India).

Induction of Diabetes mellitus: Mature normoglycemic rats (blood glucose level of 50-70 mg/dl) ⁷ were used for the present study.  Diabetes was induced in overnight fasted rats by single intraperitoneal injection of STZ (45 mg/kg b.wt.) in 0.1 M cold citrate buffer (pH 4.5). Diabetes was confirmed 3 days (72 hours) after STZ injection by determining blood glucose concentration.

Only animals with blood glucose levels above 250 mg/dl were considered as diabetic.

Experimental design and induction of Hyperlipidemia: Rats were divided into nine groups with 6 animals in each. Hyperlipidemia was induced in STZ induced diabetic animals by oral administration of 0.5 % cholesterol and 0.1 % cholic acid on diet basis in arachis oil (10 ml/kg body weight) along with standard rat feed for 14 days. The composition of the feed fed to animals is as follows:

Carbohydrate – 70.72%; Fat (EE) – 13.77%; Crude protein – 26.29%; Total energy – 2800 Kcal/kg

All the treatments were started on the fourth day after STZ injection and continued for 14 days. Blood glucose levels and body weight were measured at weekly intervals during the treatment period. At the end of the experimental period, animals were anaesthetized and blood samples were collected from the heart. Serum was separated and stored at -20ºC until assay. After blood collection, the animals were sacrificed and the different tissues (heart, liver, pancreas, and kidney) were excised and stored in 10% formalin for histopathological studies.

Biochemical Analysis: Serum glucose and total cholesterol, triglycerides, HDL-C and creatine kinase were estimated using commercial standard kits (Merck diagnostics, India) using semi auto analyzer.

Acute oral Toxicity Study: Acute oral toxicity of T. cordifolia extract in Wistar Female rats was performed as per OECD Guideline 425. Two groups of three rats each were used for the study. Group I served as control and received distilled water. Group II received single oral dose of T. cordifolia extract (2000 mg/kg). The animals were observed for gross behavioral, neurological, autonomic and toxic effects at short intervals for 24 h and then daily for 14 days. Feed intake was recorded daily and body weight was recorded weekly. On 14^th day, animals were sacrificed and gross pathological examination performed.

Statistical Analysis: The results were expressed as mean ± S.E. All the data were analyzed by one way analysis of variance followed by Duncan’s test multiple comparison test ⁸. A value of p<0.05 was considered statistically significant.

RESULTS:

Effect of Tinospora cordifolia extract on Serum Glucose Level: A significant reduction (P < 0.05) in blood glucose levels was observed at the end of first week (day 7) of treatment with T. cordifolia extract in diabetic rats; this was further reduced after 14 days of treatment. Maximum reduction in blood glucose was observed in group VII with T. cordifolia extract at 200 mg/kg compared to glibenclamide and the other treatment (Table 1).

TABLE 1. EFFECT OF TINOSPORA CORDIFOLIA EXTRACT ON SERUM GLUCOSE LEVEL

Means bearing different superscripts (a,b,c,d,e) differ significantly between groups (P<0.05); Means bearing different superscripts (1,2,3,4) differ significantly between periods (P<0.05)

Effect of Tinospora cordifolia extract on Serum Triglycerides and Serum Total Cholesterol: The rats in groups II and III showed a marked increased in serum total cholesterol, triglycerides, LDL-C and a fall in HDL-C levels when compared to normal control rats indicating the induction of hyperlipidemia. Treatment with atorvostatin, T. cordifolia extract alone or in combination with atorvostatin significantly decreased serum total cholesterol, triglycerides when compared to group II and III. Maximum decrease in serum total cholesterol and triglycerides was observed in group treated with T. cordifolia extract at 200 mg/kg and atorvostatin. There was no significant difference between group IX and normal control group.

There was a significant increase in serum HDL-C level in groups IV, V, VI, VII, VIII and IX when compared to rats in group II and III. Treatment with T. cordifolia extract at 200 mg/kg alone or in combination with atorvastatin caused significant decrease in serum LDL-C level when compared to diabetic rats and levels of LDL-C were comparable to normal control rats. T. cordifolia extract at 200 mg/kg was found to be more effective in reducing LDL-C when compared to atorvastatin alone (Table 2).

TABLE 2. EFFECT OF TINOSPORA CORDIFOLIA EXTRACT ON SERUM TRIGLYCERIDES, SERUM TOTAL CHOLESTEROL, SERUM HDL-C AND SERUM LDL-C

Means bearing different superscripts differ significantly between groups (p<0.05)

Effect of Tinospora cordifolia extract on serum creatine kinase - MB and Serum Free Fatty Acids: Serum creatine kinase - MB level was significantly increased (P<0.05) diabetic rats when compared to normal control and these levels were further increased in hyperlipidemic rats (Group III). The serum creatine kinase - MB level was significantly decreased in groups VI, VII, VIII and IX when compared to that of diabetic control group and hyperlipidemic rats. The levels of serum creatine kinase-MB in plant extract treated groups did not differ significantly when compared to that of normal control. Table 3 shows the alterations in the free fatty acid levels in serum of control and experimental groups of rats. There is a significant elevation in serum free fatty acids levels observed in diabetic and group III rats and these altered free fatty acid levels were restored by treatment with standard drugs and plant extract (Table 3).

Effect of Tinospora cordifolia on Atherogenic Index: The atherogenic index values were showed a significant increase in diabetic group and group III when compared to normal control group. The atherogenic index values were nearly similar in groups V and VI. There is no significant difference between high doses of plant extract administered groups (groups VII and IX) and normal control group (Table 3).

TABLE: 3 EFFECT OF TINOSPORA CORDIFOLIA EXTRACT ON SERUM CREATINE KINASE-MB, SERUM FREE FATTY ACID LEVELS AND ATHEROGENIC INDEX

Means bearing different superscripts differ significantly between groups (p<0.05)

Effect of Tinospora cordifolia on Vital Organs: STZ induced diabetic control rats showed marked microscopical changes like degeneration of islet cell in pancreas (Fig. 1B) and tubular epithelial cell degeneration with interstitial nephritis in kidney (Fig. 2B) when compared to that of normal control rats.

But this degenerative changes in pancreas and kidney were restored back to near normal in 200 mg/kg b.wt. of Tinospora cordifolia extract treated group when compared to diabetic control group (Fig.1C & Fig. 2C).

Histopathological examination of liver and heart showed no remarkable changes.

DISCUSSION: STZ was widely used for inducing type I diabetes in a variety of animals ⁹. In the present study, STZ (45 mg/kg, i.p.) produced significant hyperglycemia and body weight loss. The hyperglycemia was persistent throughout the experimental period, since STZ caused degeneration of islets of Langerhans of pancreas ¹⁰.

TCAE showed significant and consistent reduction in fasting blood glucose levels and also it significantly improved the body weight loss at different intervals throughout the period of experiment as compared to the vehicle treated diabetic controls indicating its potent antidiabetic activity. Since T. cordifolia extract could not exert any effect on normoglycemic animals, but reduced the blood glucose level, it may exert its mode of action on extrapancreatic pathways rather than stimulating insulin secretion like glibenclamide. It includes stimulation of peripheral glucose utilization or retarding gluconeogenesis or decreasing the intestinal absorption of glucose ¹¹.

However, histological examination revealed a recovery from degenerative changes in islets brought out by the T. cordifolia, indicating its capacity to regenerate the damaged cells. This may augment the release of insulin and reversal of hyperglycemia as that of glibenclamide.

The rise in the level of serum triglycerides and total cholesterol is attributed due to accumulation of lipids and insulin deficiency induced derangements in lipid metabolism. These observations were observed on first and throughout the period of experiment ^{12, 13, 14}. Hypercholesterolemia in the present study was high on 14^th day in STZ induced diabetic group which may be due to inhibited actions of lipolytic hormones on fat depot and phospholipids along with cholesterol and triglycerides were discharged from liver to blood ¹¹.

However, T. cordifolia extract at 200 mg/kg b.w. could produce better effect than glibenclamide. According to Randle’s glucose-fatty acid cycle, increased triglycerides will elevate free fatty acids availability and oxidation that impair insulin action, via glucose utilization leading to development of hyperglycemia ¹⁴. Therefore, the reduction of triglyceride following T. cordifolia extract treatment might facilitate better utilization of lipid along with reduction in hyperglycemia. The restoration of lipoprotein lipase activity will bring back triglyceride level to normal and T. cordifolia extract was able to correct the high triglyceride to normal, there by indicating its possible action on lipase ¹.

In the present study, T. cordifolia extract at both doses produced a remarkable increase in serum HDL-C and the effect was comparable to that of atorvastatin alone and atorvastatin combined with glibenclamide treated rats. An increase in total lipids in liver and kidney in STZ induced diabetic rats indicate an increased synthesis of lipids and storage capacity which in turn increase the triglyceride and phospholipids. HDL serves as acceptor of lipids for ultimate excretion in bile. The ability of T. cordifolia to increase HDL-C is largely attributed to its central function in the reverse cholesterol transport, a process whereby excess cell cholesterol is taken up and processed by HDL particles for further delivery to the liver for metabolism ^{15, 16}.

Therefore it is logical that an increase in HDL-C level can contribute to lower risk of atherosclerosis ¹⁷. It is observed from the present study that T. cordifolia extract is capable of increasing the serum level of good cholesterol i.e., Serum HDL-C in the treated rats compared to diabetic rats, thereby indicating its usefulness as a potent therapeutic agent ¹⁸.It is widely accepted that elevation of plasma LDL-C levels is the major risk factor for CVD. Direct correlation exists between LDL-C and atherosclerosis and also the reversibility of the related pathological events by lowering the serum LDL-C level ¹⁹.

In our study, T. cordifolia extract at 200 mg/kg b.w. dose independently increased HDL-C level to near normal. Our result indicates that, high concentration of LDL-C in diabetic and hyperlipidemic rats was significantly reduced by oral administration of T. cordifolia extract. Therefore, T. cordifolia might constitute a good candidate for the treatment of CVD and atherosclerosis by lowering serum LDL-C which is responsible for formation of lipid peroxidase that specifically leads to atherogenesis. STZ induced diabetic rats showed an increase in serum creatine kinase - MB.  T. cordifolia extract in both doses decreased the serum creatine kinase - MB level. Increased serum creatine kinase level in diabetic rats indicates cardiac muscular damage ²⁰. The quantity of enzyme released from the damaged tissue is a measure of the number of necrotic cells ²¹. This indicates possible correlation between this enzyme with cardiomyopathy in diabetes. The extract of T. cordifolia extract was able to reverse the level of creatine kinase in treatment groups, there by showing its ability to protect STZ induced cardiomyopathy caused by STZ.

The significant increase of free fatty acid may be due to breakdown of membrane phospholipids by the action of oxygen derived free radicals induced during hyperlipidemia or increased activity of phospholipase ²². In the present study, T. cordifolia extract was able to reduce the free fatty acid level, thereby indicating its antioxidant properties during hyperlipidemic conditions. A decrease in atherogenic index by administering T. cordifolia extract in diabetic rats indicates its antihyperlipidemic and antiatherogenic potentials.

The pancreas of diabetic control rats showed islet cell degeneration because of action of STZ on the islet cells. In the present study, T. cordifolia extract at 200 mg/kg b.w. caused regeneration of damaged cells. The kidney of diabetic rats showed tubular epithelial degeneration and interstitial nephritis suggestive of diabetic nephropathy because of altered metabolism of lipids and carbohydrates ²³.

Treatment of T. cordifolia extract at 200 mg/kg b.w. ameliorated the degenerative changes and nephritic conditions, thereby making the kidneys returns to near normalcy. The reversal of serum glucose and lipid profiles and also the normalization of renal nephropathic changes after the administration of T. cordifolia extract are suggestive of a beneficial role of T. cordifolia in STZ induced nephropathy.

The proposed study revealed that aqueous extract of T. cordifolia can be considered as an important addition to the therapeutic armamentarium for the treatment of hyperlipidemia associated with diabetes. Further studies at the cellular and molecular level, may give clues regarding its mechanism in detail.

ACKNOWLEDGEMENTS: The research was approved for the Post graduate degree by the Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007 and the facilities provided by the University is duly acknowledged.

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