EVALUATION OF ANTIDIABETIC AND ANTIHYPERLIPIDEMIC EFFECT OF VERNONIA DIVERGENS IN STREPTOZOTOCIN-INDUCED DIABETIC RATS

Objective: The current investigation for antidiabetic activity of the plant Vernonia divergens (DC.) Edgew. has not been reported till date. However, to enlighten the folkloric claim of the plant, the study was carried out on various animal models such as albino mice, albino rabbits, Wistar rats, rabbits, hamsters, dogs, and monkeys. Methods: The whole plant of V. divergens was studied on various animal models. Screening methods generally have been carried out on rodents and non-rodents, respectively. Various biochemical and hematological parameters such as serum glucose, plasma insulin, lipid profile and activities of liver enzymes, red blood cells, white blood cells, hemoglobin, and differential counts were measured to assess the antihyperglycemic and antihyperlipidemic activities as well as safety profile of the extract. Results: Among all experimental extracts of V. divergens, it was found that the aqueous and methanolic extracts had maximum control of blood glucose in diabetic Wistar rats. While comparing with normoglycemic animals, it was observed that reduction of blood sugar level and increase in plasma insulin level are maximum with test extract. Among the study, the effects of the methanolic extract of V. divergens (MEVD) and aqueous extract of V. divergens (AEVD) were done through oral route in both the models, i.e., normoglycemic and hyperglycemic animal models. The safety profile was evaluated by toxicological evaluation and observed that, even at a higher dose level of 3000 mg/kg body weight, the MEVD and AEVD were safe and retain normal physiological and behavioral effect. The whole protein, whole cholesterol, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase enzyme activity of streptozotocin-administered rats showed significantly higher than normal rats, and the test extract-treated rats significantly reduced the elevated levels. Conclusion: It is concluded that the MEVD and AEVD (DC.) Edgew. might be beneficial in effectively reducing the blood glucose concentration and managing the various complications of diabetes. However, in comparison between both the extracts, the methanol extract was found to be significantly more potent than that of the A.E. in all aspects.


INTRODUCTION
The term diabetes mellitus (DM) describes as a metabolic cum vascular syndrome of more than one etiology characterized by chronic hyperglycemia with resultant abnormalities in metabolism of protein fat and carbohydrate ensuing from impaired secretion and defected action of insulin or both. The outcomes of DM include long-term casualty, i.e., damage, dysfunction, and failure of several organs. DM may represent with attribute signs and symptoms such as polyphagia, polydipsia, polyurea, blurring of vision, and weight loss [1]. This is affecting nearly 25% of the population [2]. The prevalence of diabetes according to the World Health Organization projections is likely to increase by 35% [3]. Currently, the incidence of diabetes is nearly 150 million globally, and by the year 2025, this is likely to increase to 300 million or more [3,4]. While reviewing the statistical projection status of India, we can come across the fact that the incidence of diabetics is increasing and will rise from 15 million in 1995 to reach up to 57 million in the year 2025 which can make the country highest prevalent of diabetics worldwide [3]. Therefore, it is necessary to look for new solutions to manage this health problem. Many herbal products have been described for DM in ancient literature of Ayurveda in India [5]. The belief in traditionally used herbal drugs for curing various disease state is very strong in Nigeria and many other poor African countries [6,7].
India is one of the highest populated diabetogenic countries. In human population, DM remains one of the major contributors among all other diseases. Due to enhancing awareness now-a-days the concern of medicinal plant in health care system is increasing and interest in local plant remedies for treating of various disease is growing in many developed as well as developing countries. Vernonia divergens is commonly known as insulin plant having a potent effect against glucose reduction belonging to family Asteraceae which is used as an outstanding drug for DM described earlier [8]. Thus, the rationale of the present study was to ascertain the truth of the claim about antidiabetic activity on the plant V. divergens (DC.) Edgew. so that novel compound against the treatment of diabetes can be identified.

Maharana et al.
material (600 g) was extracted with different solvents separately of non-polar to polar, for example, petroleum ether (pet. ether), chloroform, methanol, and water by the help of Soxhlet apparatus. The liquid extracts were concentrated under vacuum to yield dry extracts and preserved in a desiccator until further experiments.

Percentage yield of the extracts
The percentage yield of the plant V. divergens was found to be 2.66%, 1.66%, 14.66%, and 12.66% with pet. ether, chloroform, methanol, and water, respectively. The percentage yield of the methanolic extract of V. divergens (MEVD) was found to be greater (14.66%) than extracts with other solvents.

Preparation of drug solution
The semisolid extracts of V. divergens were dissolved in sufficient quantity of solvent (tween and normal saline), and the aqueous extracts (A.Es) were dissolved in normal saline for administration. Glibenclamide 5 mg/kg was dissolved in a sufficient quantity of normal saline and used in the treatment [9]. Streptozotocin 40 mg/kg was dissolved in the buffer solution and used to induce diabetes in rats [10].

Selection of animals
For both toxicological and antidiabetic assessments, adult Wistar albino rats of healthy status weighing about 150-200 g were selected after getting the Institutional Animal Ethics Committee approval (Approval No-10/15/SPS/IAEC/SOAU). The selected animals have been housed in acrylic cages in well-known environmental conditions (temperature: 20-25°C and relative humidity: 45-55% under 12 h light/dark cycle). To acclimate the laboratory condition, animals were fed with standard rodent diet for 1 week and water ad libitum [11].

Induction of diabetes
In the present study, a single dose of streptozotocin 40 mg/kg in normal saline was administered intraperitoneally [10]. After 1 week assessment of gradual development, diabetes was done. For the determination of blood glucose concentration, an experiment was performed on the 7 th day. The animals with blood glucose levels between 200 and 250 mg/dl were chosen for antidiabetic screening and they were considered as diabetic rats.

Experimental procedure
The study was carried out as mentioned below to check whether the MEVD is acting as an insulin secretagogue (stimulation of insulin secretion from the pancreatic β-cell and to check the utilization) activity as well as to check whether the MEVD possesses antidiabetic activity and hypoglycemic activity.
Overnight fasted animals were grouped in six groups of six animals each group.  [12]. A significant body weight reduction is associated with diabetes hence the body weight was recorded before, during and after the treatment. Acute toxic study was performed to find out toxic symptoms of the extract and gross behavioral changes were also noticed. Sub-acute toxicity study was performed to reveal the influence of the extract on biochemical, hematological and histopathological findings. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) etc. were assayed in serum. Serum lipids like total cholesterol (TC), high density lipoprotein (HDL), Low density lipoprotein (LDL), Very LDL (VLDL) and triglyceride (TG) were estimated. Serum total protein and albumin were analyzed. Blood parameters like hemoglobin (Hb), red blood cells (RBC), white blood cells (WBC) etc. counts were determined. Clotting time was also determined.

Acute toxicity studies
To rule out any toxicity of the extracts, acute oral toxicity test was performed on 12 h fasted healthy Wistar albino rats according to the principles of Organization for economic co-operation and development (OECD) (OECD guidelines 423) [13]. This study was performed with the major aim of therapeutic index establishment and also for the purpose of primary screening. Therapeutic index meant the ratio of pharmacologically effective dose to the lethal dose. A group of 10 mice have been fasting overnight was administered the A.E. of V. divergens (AEVD) once orally at 4 dose levels (500, 1000, 2000, 3000 mg/kg). The initial observation of test drug treated mice was continuously recorded and then further reading was taken occasionally for 4 h. Final recording of overnight mortality was done [14]. In the course of duration of study, spontaneous activity reduction and behaviors of the mice were attentively observed [15].

Sub-acute toxicity studies
The purpose of this study is to determine the maximum tolerated dose and daily dose for 3-4 weeks and to indicate the nature of toxic reaction of the drug. In present study, the influence of V. divergens extract on hematological parameters, a pathological change on 21 days dosing in streptozotocin induce diabetic rats were carried out. The diabetic rats were grouped into 6 groups of 6 animals each. Group I animals received solvent, Group II animals received standard drug, Group III (100 mg/kg body weight) and Group IV (200 mg/kg body weight) was received MEVD and Group V (100 mg/kg body weight) and Group VI (200 mg/kg body weight) was received AEVD once daily for 21 days. At the end of the experimental period the blood was collected, serum was separated and subjected to hematological and biochemical examination [14].

Statistical analysis
The values are represented as mean ± standard error mean. The data obtained from various studies were subjected to one-way analysis of variance (ANOVA) [16]. Dunnett's t-test was also performed after ANOVA for establishing statistical significance.

RESULTS
In Table 1   The result expressed in Table 4 revealed that the M.E. and A.E. in the dose level of 100 mg/kg and 200 mg/kg possess 48.54% and 55.00% and 34.10% and 50.09% reduction in the blood glucose level, respectively, at the end of 8 h, while the standard drug registered 65.03% decrease in blood sugar level at the same time. Table 5 shows the hypoglycemic effect of MEVD and AEVD on multidose treated normoglycemic rats. The purpose of the study is to establish the therapeutic validity of the test extracts in long-term use. The data showed that there was a decrease in blood sugar level to the extent of 25.56% and 34.14% in case of M.E. and 19.14% and 30.09% in case of A.E. at the dose level of 100 mg/kg and 200 mg/kg, respectively, on the 21 st day of treatment.
The purpose of antidiabetic study is to confirm the antidiabetic effect of the test extracts on longer duration of treatment, and the data are shown in Table 6. The M.E. and A.Es are registered 50.68% and 57.68% and 40.55%    Table 7.
The content of Table 8 indicated that there was a decrease in percentage loss of body weight when measured on 3 rd , 7 th , 14 th , and 21 st    The data presented in Table 9 demonstrated that the glycogen content of the liver and kidney of DM animals treated with M.E. and A.Es of leaves of V. divergens and standard drug was significantly increased when compared with solvent control after the completion of 21 days of drug exposure, even the test extracts and standard drug approach the content of glycogen toward normal level.
The data observed in Table 10 present a significant (p<0.01) reduction of TC in serum in the groups treated with test extraction and standard drug when compared with solvent control. The extent of reduction in both the close level of test extracts was dose dependent. In case of TGs, there was a significant reduction with both test extract and standard drug when compared with solvent control on the 21 st day of treatment.
The value of HDL in case of test extract at tested dose levels and standard drug showed significant increase than that of solvent control group, while the LDL and VLDL levels significantly (p<0.001) decrease in all drug-treated groups when compared with solvent control.
The hematological parameters exhibited in Table 11 show that the animals treated with standard drug glibenclamide and test extracts in the tested dose levels bear normal in RBC count and Hb, whereas the clotting time slightly elevated. However, the diabetic rats treated with solvent showed a decrease value of RBC, WBC, and Hb. The neutrophil count appears to nearly equal with that of normal value. The other hematological parameters such as eosinophil, lymphocyte, and monocytes in case of the standard drug and test extract-treated animals did not show any alteration.
From Table 12, it was observed that AST and ALT enzyme activity of streptozotocin-induced diabetic rats was significantly higher than normal rats. The test extracts and treated rats at tested dose had reduced enzyme activity when compared with diabetic rats. The total protein and albumin content of serum was significantly lowered in diabetic rats when compared with solvent control, but in the drugtreated diabetic animals, it returned to nearly normal.

DISCUSSION
Diabetes is a chronic disease that occurs when either insulin deficiency or resistance or both exists. The hyperglycemic condition causes increased glycosylation leading to various complications such as neuropathy, retinopathy, nephropathy, and cardiomyopathy [17]. The present study was carried out to investigate and validate the antidiabetic activity of the plant V. divergens (DC.) Edgew. The blood glucose lowering property of MEVD and AEVD is comparable with the standard drug glibenclamide (5 mg/kg body weight). Among the tested extract, the M.E. and A.E. at the dose level of 100 mg/kg possess a marked reduction in blood glucose level through oral route. Therefore, the M.E. and A.E. were selected for the evaluation of hypoglycemic and/or antidiabetic activity. It is observed that the MEVD and AEVD effectively control the glucose concentration of blood in normoglycemic as well as hyperglycemic rats in single unit dose treated model. The background mechanism of such effect might be due to a decrease in the initial absorption rate when glucose and plant fiber given together orally [18]. Hence, it may be presumed that the glucose lowering effect   This study further supports the antidiabetic effect of the test extracts whose effectiveness persists up to 21 days and the blood sugar level decreases gradually during the observed days, which presumed that the test extracts contain some antidiabetic active principle responsible for this effect. The hypoglycemic active principle acts by initiating the release of insulin from the pancreatic cell of hyperglycemic animal (sulfonylurea like effect) [19]. The proper mechanism of action is further supported by oral glucose tolerance test, in which glucose level reduces in the response of test extract. While insulin level concerned in comparison with standard drug, it was observed that there was an increased plasma insulin level and hence concluded the insulinotropic effect [11]. Therefore, it is credible that glucose-lowering properties of the extracts strive a direct effect in hyperglycemic rat probably by a similar mechanism to insulin which was substantiated by an extrapancreatic mode of action. The experiment revealed that M.E. and A.E. of V. divergens (200 mg/kg) significantly (p<0.001) decrease the glucose level on hyperglycemic animal. The glucose lowering activity observed in the diabetic animal may be due to the stimulation of β-cells in the pancreatic islets.
It has been reported that diabetes is a heterogeneous metabolic disorder, in which one of the symptomatic characters is loss in body weight [20]. It has been stated that DM results deficiency to glucose utilization for energy leads to increased utilization & decreased storage of proteins. By the above-stated reason, depletion of protein takes place resulting in reduced body weight [21]. The results of the study presented that the decreased loss of body weight might be contributed by increased use of glucose by the tissue.
The glycogen content of the liver and kidney of DM animals treated with M.E and A.E of leaves of V. divergens and standard drug was significantly increased when compared with solvent control after the completion of 21 days of treatment, even the test extracts and standard drug approach the content of glycogen toward normal glycogen level. It was reported that hyperglycemia results in decreased glucose utilization and glycogen synthesis [22]. The glycogen content in the liver and kidney increases in the extract-treated group compared to the diabetic rats indicating participation of the extract components in glycogen synthesis similar to insulin [23]. Dyslipidemia is a known complication associated with diabetes [24]. The abnormally high concentration of serum TG, TC, LDL, and low HDL observed in diabetic rats compared to control rats is in consonant with reports from previous studies [24][25][26][27], indicating that an increase in glucose level on induction of diabetes results in an equivalent rise in blood lipids. It has been reported that, in the diabetic patients those are untreated or under treatment, hypertriglyceridemia and hypercholesterolemia often occur due to the increased generation of VLDL and deficiency of lipoprotein lipase [28]. The result revealed that the test extract reduces VLDL, TC and TG actively. Hence, it might be surmised that, similar to insulin, the test extract is the prime cause for the endowment of the transcription of lipoprotein lipase.
The result of hematological parameters evidenced that the test extracts have no significant effect on the same and is evident for the safely used at the MEVD and AEVD for a longer duration time. The experimental approach has been conducted for the test extracts to elicit tonic response over an exposure period of 21 days' alteration in the marker enzymes such as AST, ALT, and ALP. The data obtained from this study also indicated that the MEVD significantly decreased the levels of AST, ALT, and ALP. The increased activity of transaminases, which are active in the absence of insulin due to increased availability of amino acids in diabetes, is believed to be responsible for the increased gluconeogenesis and ketogenesis observed in the disease [29]. Alanine and aspartate transaminase activities are used as an indicator of hepatocyte damage [30]. Elevation of serum ALP concentration in patients with DM has been observed for several years, but the source and reasons are unknown [31].

CONCLUSION
The findings of the current study indicated that MEVD and AEVD may get a place in the treatment of DM as hypoglycemic and/or antidiabetic agent. The MEVD and AEVD (DC.) Edgew. might be beneficial in effectively reducing the blood glucose concentration and managing of various complications of diabetes. However, in comparison between both the extracts, the M.E. was found to be significantly more potent than that of the A.E. in all aspects. Clinical studies are required to establish whether the administration of MEVD and AEVD can potentiate the antidiabetic effect of conventional agents.