Amelioration of oxidative stress by Tabernamontana divaricata on alloxan-induced diabetic rats.

OBJECTIVE: The purpose of this study was to evaluate the anti-diabetic activity of ethanol extract of Tabernamontana divaricata (L.) and its ameliorative effect on oxidative stress in alloxan-induced diabetic rats.
MATERIALS AND METHODS: Diabetes was induced by single intraperitoneal injection of alloxan monohydrate (140 mg/kg body weight). Methanol extract of T. divaricata was administered at the doses of 100 and 200 mg/kg body weight in diabetic induced rats including glibenclamide (3 mg/kg) as a reference drug. In the continuous 21 days treatment, fasting blood glucose level was determined on 0, 7, 14 and 21 days. On day 21, serum lipid profiles and glycosylated hemoglobin, liver antioxidant enzymes levels were estimated.
RESULTS: Experimental findings showed a significant anti-diabetic potential of the extract in terms of reduction in blood glucose levels and a correct effect on the altered biochemical parameters. Observed data were found statistically significant in correction of antioxidant enzyme level accompanied with diabetes, particularly at the dose of 200 mg/kg body weight.
CONCLUSION: Based on the results, it can be concluded that the T. divaricata is found to be effective in type 2 diabetes in rats and to have an ameliorative effect on the associated oxidative stress.

INTRODUCTION

Diabetes mellitus (DM), the state of chronic hyperglycemia, is a common disease affecting over 200 million individuals worldwide.[1] It is a progressive metabolic disease, often referred to simply as diabetes. It is a syndrome of disordered metabolism usually due to a combination of hereditary and environmental causes, resulting in abnormally high blood sugar levels.[2]

Diabetes mellitus is associated with high risk of atherosclerosis, renal, nervous system and ocular damage.[3] Uncontrolled hyperglycemia appears to be the principal biochemical abnormality that underlies the increased oxidative load in DM. Increased oxidative stress may contribute to the pathogenesis of the diabetic complications. In addition, increased oxidative injury has been implicated in the premature age-related changes in DM.[4] Multiple studies have shown that type 2 diabetes is accompanied by increased oxidative damage to all bio-molecules, especially lipids.[5] Results of studies in animal models and in humans have demonstrated that diabetes is associated with oxidative stress, which is exhibited by elevated blood levels of lipid peroxidation (LPO) products (markers of oxidative stress), especially associated with poor blood glucose control.[67] High oxidative stress can lead to microvascular cerebral diseases, e.g., stroke, cerebral hemorrhage, and brain infarction.

Diabetes is a chronic disease that occurs when the pancreas does not produce enough insulin, or alternatively when the body cannot effectively use the insulin produced. Hyperglycemia or raised blood sugar is a common effect of uncontrolled diabetes and over time leads to serious damage to many of the body's systems, especially the nerves and blood vessels. It affects all age groups of people and ethnic groups.[8]

Synthetic hypoglycemic agents can produce serious side effects and in addition, most of them are not suitable for use during pregnancy and other major disorders. Therefore, the search for more effective and safer hypoglycemic agents has continued to be an important area of active research. Extracts of various plant materials capable of decreasing blood sugar have been tested in experimental animal models, and their effects were confirmed.[9]

Tabernaemontana divaricata (syn. Ervatamia coronaria) commonly called as Crepe Jasmine is a glabrous evergreen dichotomously branched shrub mostly found in tropical regions. The plant grows up to a height of about 6-feet, bears attractive white colored sweet-scented flowers having five-petal pinwheels gathered in small clusters on the stem tips. The leaves are large, shiny, and deep green in color, and the size is about 6-inches in length and 2-inches wide.[10] The flower juice is used in the treatment of eye infection. The root is acrid and bitter in taste; the milky juice mixed with oil is applied onto the head to cure pain in the eyes. Chewing of root relieves toothache and the root paste is applied to wounds to prevent inflammation.[11] It has been used in Chinese, Ayurvedic and Thai traditional medicine for the treatment of fever, pain, and dysentery.[1213] The purpose of this study was to carry out phytochemical evaluation and to investigate the anti-diabetic effect of aerial parts of T. divaricata and its prevention of oxidative stress in alloxan-induced diabetic rats.

MATERIALS AND METHODS

Collection and authentication of plant

The plant was collected from the areas of Nagercoil and positively identified and authenticated by Prof. Mr. V. Sivanadanam, Department of Botany, Lekshmipuram College of Arts and Science, Neyyoor, Kanyakumari District, Tamil Nadu, India. The plant specimen was certified as T. divaricata of family Apocynaceae from available literature.

Preparation of extract

The shade-dried powdered plant material was successfully extracted with methanol for 48 h at 55–65°C. The extract was filtered and concentrated by distillation and solvent was recovered. The final solution was evaporated to dryness at room temperature. Then the extract was stored in the desiccators and used for subsequent experiments.

Phytochemical evaluation

The obtained extract was concentrated and subjected to various chemical tests to detect the presence of different phytoconstituents.[141516]

Animals

Healthy male Wistar albino rats, 2–3 months of age and approximately weighing between 150 and 250 g were used in the present study. Rats were housed in polypropylene cages and allowed free access to feed and tap water under strictly controlled pathogen-free conditions with room temperature 25 ± 2°C. All experiments were conducted in accordance with the internationally accepted principles for laboratory animal use and care.

Evaluation of anti-diabetic activity

Glucose tolerance test in rats

The diabetic rats were randomly divided into four groups of six animals each. The first group served as control, 1% carboxy methyl cellulose (CMC) 10 ml/kg, p.o. The second group was administered glibenclamide (3 mg/kg, p.o.), while the third and fourth groups received METD at 100 and 200 mg/kg, p.o. All groups received glucose solution (1 g/kg) 30 min after the above treatments. Blood samples were collected from the tail vein just prior to and after 30, 60 and 90 min after glucose loading.[17] Results achieved from glucose tolerance test were taken as a hypothetical reference to extrapolate the dose levels which will be used for evaluating short- and long-term effects of METD on diabetic rats.

Experimental design

Diabetes is induced in overnight fasted adult Wistar albino rats weighing 150–250 g by single i.p. injection of 140 mg/kg alloxan monohydrate dissolved in normal saline. Animals were fed with 5% glucose solution in order to prevent hypoglycemic shock for 18 h. Hyperglycemia was confirmed on observation of elevated blood glucose levels in plasma, determined at 72 h. The threshold value of fasting plasma glucose to diagnose diabetes was taken as >200 mg/dl. Only rats found with permanent diabetes were used for the anti-diabetic study.[1819]

The animals were divided into five groups containing six rats each.

Group I: Control animals were given 5% CMC 10 ml/kg body weight orally

Group II: Untreated diabetic-induced animals served as pathologic control

Group III: Diabetes-induced animals treated with glibenclamide (3 mg/kg p.o.), served as a standard group

Groups IV and V: Diabetes-induced animals were treated with METD 100 mg/kg and 200 mg/kg (p.o.), respectively.

Sample collection

Fasting blood glucose levels of all rats were determined before the start of the experiment. Blood samples were collected at weekly intervals from tail vein puncture till the end of the study. In the continuous 21 days of drug treatment, blood glucose levels of all animals were determined at the 0, 7, 14 and 21 days. On day 21, blood was collected by cardiac puncture under mild ether anesthesia from overnight fasted rats.[20] The rats were sacrificed by decapitation and relevant organs such as liver and pancreas of all the animals were dissected out and stored at −20°C.

Estimation of serum biochemical parameters

Blood samples collected were centrifuged at 3500 rpm for 15 min at room temperature for separation of serum. The clear, nonhemolyzed sera were separated using clean dry disposable plastic syringe and stored at −20°C for the estimation of total cholesterol (TC), triglycerides (TG), insulin and glycosylated hemoglobin (HbA1c) levels.[212223]

Estimation of antioxidant parameters

The liver was perfused with 0.86% cold saline to completely remove all the red blood cells. Then it was suspended in 10% (w/v) ice-cold 0.1 M phosphate buffer (pH 7.4), cut into small pieces and the required quantity was weighed and homogenized using a homogenizer. The homogenate was centrifuged at 3000 rpm for 20 min to remove the cell debris. The supernatant was used for the estimation of catalase, superoxide dismutase (SOD), lipid peroxidase and glutathione peroxidase.[2425262728]

Statistical analysis

All the results were expressed as mean ± standard error of the mean and were analyzed by analysis of variance and groups were compared by Tukey-Kramer multiple comparison test. Differences between groups were considered significant at P < 0.05 level.

Histopathology of pancreas

The pancreas was fixed in 10% formalin and embedded in paraffin wax. Sections were made using rotary microtome and hematoxylin-eosin was used as stain. Histological observations were made under light microscope.

RESULTS

Phytochemical report

The phytochemical tests showed the presence of carbohydrates, proteins and amino acid, flavonoids, glycosides, saponins, steroids, phenols, triterpenoids and alkaloids. The test shows a negative report for tannins.

Effect of METD and glibenclamide on oral glucose tolerance on rats

An immediate increase in the blood glucose level was observed in the control group after the administration of glucose. In glibanclamide and METD (200 mg/kg) treated groups, a significant reduction (P < 0.001 and P < 0.01) in the glucose level was observed at a different time interval as compared to the control groups. The blood glucose in normal rats is not disturbed by both the doses of METD. Results have been shown in Table 1.

Table 1

Effect of METD and glibenclamide on glucose tolerance on rats

Effect of METD on blood glucose level in alloxan-induced diabetic rats

The effects of METD at two dose levels (100 and 200 mg/kg, p.o.) and glibenclamide on blood glucose level in alloxan-induced diabetes have been shown in Table 2. Significant (P < 0.001) rise in blood glucose level shows the induction of diabetes by alloxan in diabetic control group. Administration of METD at 200 mg/kg dose level attenuated the increased level of glucose produced by alloxan at a different time as compared to that of glibenclamide.

Table 2

Effect of METD and glibenclamide on blood glucose level of alloxan-induced diabetic rats

Effect of METD on total cholesterol and triglycerides, insulin and glycosylated hemoglobin levels

After the administration of alloxan, there was a significant (P < 0.001) increase in the serum TC, TG, and HbA1c levels [Table 3]. Treatment with METD at 200 mg/kg, elicits a significant decrease in the cholesterol, TG (P < 0.001) and HbA1c (P < 0.01) level when compared with nontreated groups. Altered insulin level was also corrected significantly (P < 0.001) by METD at 200 mg/kg.

Table 3

Effect of METD and glibenclamide on serum TC, TG, insulin, and HbA1c level of diabetic rats

Effect of METD on antioxidant enzymes

As shown in Table 4 and Figure 1a–c, antioxidant enzyme levels such as lipid peroxidase, catalase, SOD and glutathione peroxidase were found to be altered in the diabetic control rats. Administration of METD at both the doses reversed the altered levels significantly (P < 0.01 and P < 0.001) in a dose-dependent manner.

Table 4

Effect of METD and glibenclamide on antioxidant enzymes in alloxan-induced diabetes in rats

Figure 1

(a) Effect of METD on the lipid peroxidase level in normal and diabetic rats, (b) Effect of METD on catalase level in normal and diabetic rats, (c) Effect of METD on superoxide dismutase and glutathione peroxidase level in normal and diabetic rats

Histopathology of the pancreas

In histopathological studies, the pancreas sections of control rats showed a normal pancreatic architecture. Alloxan-induced diabetes of rats shows loss of the pancreatic architecture with destruction of beta cells in the pancreas. Preadministration of METD at the dose of 100 and 200 mg/kg for 21 days reduces the pancreatic injury and necrosis which clearly indicating the protective effect of the extract, whereas glibenclamide (3 mg/kg) treated rats showing a normal pancreatic architecture with moderate and mild degree of necrosis. Histological examination of the pancreas showed a protective effect of METD on alloxan-induced diabetes at the dose level of 200 mg/kg [Figure 2].

Figure 2

Histopathological view of the pancreas of normal and treated rats: Photomicrographs showing normal structure in Group I and destructed cellular architecture in diabetic control rats (Group II). Group III showing a moderate protective effect whereas Group IV (Glibenclamide 3mg/kg) and Group V (METD 200mg/kg) trated rats showing normal cellular appearence

DISCUSSION

Alloxan is known for its selective pancreatic islet β-cell cytotoxicity and has been extensively used to induce DM in animals. Alloxan, in the presence of intracellular thiols, generates reactive oxygen species (ROS) in a cyclic reaction with its reduction product, dialuric acid. The beta cell toxic action of alloxan is initiated by free radicals formed in this redox reaction. One study suggests that the alloxan does not cause diabetes in humans.[29] In our present study, it was observed that METD can reverse the metabolic derangements occurring in alloxan-induced diabetes in rats.

To gain an understanding of the mechanism(s) by which METD elicits its hypoglycemic activity, various biochemical parameters were evaluated following sub-chronic (21 days) treatment in rats. Our experimental findings suggest a significant reduction in (i) blood glucose levels on different days, (ii) HbA1c levels and its HbA1c fraction and (iii) serum cholesterol and TG levels, in both METD and glibenclamide-treated rats. Alterations in antioxidant markers such as catalase, SOD, lipid peroxidase, and glutathione peroxidase were also rectified by the extract.

The increased level of HbA1c is directly proportional to the decreased level of total hemoglobin in diabetic control experimental rats. HbA1c is used as the most reliable marker and the standard diagnosis parameter for estimating the degree of protein glycation in DM.[30] On oral administration, METD significantly decreased the HbA1c level possibly due to normoglycemic control mechanisms in experimental rats which also reflect the decreased protein glycation condensation reactions.[2931] Hypercholesterolemia and hypertriglyceridemia have been reported to occur in alloxan diabetic rats.[23] The marked hyperlipidemia that characterizes the diabetic state may, therefore, be regarded as a consequence of the uninhibited actions of lipolytic hormones on the fat depots.[32] Our experimental findings showed a decrease in the cholesterol and TG level in the METD treated groups. It can be concluded that this effect of T. divaricata will prove useful in hyperlipidemia associated with diabetes.

Diabetes represents a state of increased oxidative stress, which is mainly based on the evidence of increased LPO, or by indirect evidence of reduced antioxidant reserve, like SOD and catalase enzymes, in animal models. Decreased antioxidant enzyme levels and enhanced LPO have been well-documented in alloxan-induced diabetes.[33] The antioxidant enzymes are the defense system of the body which protect the cell membrane and other cellular constituents against oxidative damage by free radicals (ROS).[28] Decreased serum concentration of total antioxidant enzymes in alloxan-treated diabetic rats were observed due to their utilization during inhibition or destruction of free radical species which also indicates an imbalanced ROS production and antioxidant scavenging systems. Our results also indicate that METD (100 and 200 mg/kg body weight) strongly restores antioxidant enzymes and decreases LPO in diabetic animals. A decrease in levels of liver antioxidant enzymes during diabetes may be a result of their increased utilization by the hepatic cells, possibly reflecting an attempt of counteraction against increased formation of lipid peroxides.[28] Furthermore, tissue damage mediated by lipoxygenase-derived peroxides is closely related with insulin secretion.[3435]

CONCLUSION

Tabernamontana divaricata shows significant anti-diabetic activity and also an alleviating effect the oxidative damage similar to that of the standard drug glibenclamide in rats. From the data obtained it may be stated primarily that the plant may contain some biomolecule(s) that may sensitize the insulin receptor or stimulate the β stem cells of Islets of Langerhans in pancreas in alloxan-induced diabetic rats. Reports of earlier studies suggested that the natural antioxidant constituents such as tannins, flavonoids, phenolic acids, polyphenols[36] etc., enhance the free radical scavenging activity by correcting the altered biological antioxidant enzyme systems. This may beneficial for the treatment of diabetes and its related complications. The flavonoid content present in T. divaricata may be the active constituent of plant which possess antioxidant[3537] and anti-diabetic properties. However, further comprehensive chemical and pharmacological studies are required to find out the exact mechanism and to isolate and characterize the phytoconstituent (s) responsible for the above activities.