Blood Glucose Lowering Potential of Chinese Violet (Asystasia gangetica (L.) T. Anderson) in Normal and Streptozotocin Induced Diabetic Rats

Kavitha S¹, Ravi D², Vijayabharathi V², Parthasarathy R², Rajeshwari S¹

¹Department of Biotechnology, School of Life Sciences,

Karpagam University, Coimbatore-641021, Tamil Nadu, India.

²Bioprocess Lab, Department of Botany, Government Arts College, Coimbatore, Tamil Nadu, India -641018

*Corresponding Author E-mail:dravi.botany@gmail.com

ABSTRACT:

Objective: The present study was undertaken to investigate the glucose reducing level by Asystasia gangetica flowers with various doses (125, 250 and 500 mg/kg bw) in normal and streptozotocin induced diabetic rats. Methods: Acute and sub-acute toxicity of A. gangetica was evaluated in rats with various doses and its and serum parameters were analyzed. Diabetes was induced by giving streptozotocin (45 mg/kg bw) to the rats and after conformation of diabetes, A. gangetica flower ethanol extract (AGFEE) was administrated to the normal and diabetic rats for a period of 14 days. Body weight and glucose levels were determined after 7^th and 14^th day of the study. Glucose tolerance test was also carried out after administration of glucose at the rate of 2 g/kg bw till 14^th day. Results: The LD₅₀ of extract found to be higher than 2000 mg/kg bw and the extract did not show any significant changes in serum parameters. In blood glucose reducing activity the three doses showed significant lowering glucose levels, when compared with each doses at the dose of 250 mg/kg bw the AGFEE showed maximum activity. In diabetic control rats there was a significant elevation in blood glucose levels and reduction in body weight was produced and continued till the end of the study. No alterations were found in control and extract alone treated rats. Results showed AGFEE had exhibited significant hypoglycemic (glucose lowering) activity. Conclusion: Present in vivo model indicating that the plant extract contain active principle compound for the management of hyperglycemia.

KEYWORDS: Asystasia gangetica Flowers, Streptozotocin, Glucose tolerance, Hyperglycemia.

1. INTRODUCTION

Asystasia gangetica (L.) T. Anderson (Acanthaceae) is a fast growing, spreading, perennial herb is used as ethno medicine for the treatment of heart pains, stomach pains and rheumatism, while in Nigeria, the leaves are popularly used in the treatment of asthma [1]. In India, the plant is being used for rheumatism and the sap is applied to swellings and skin allergies [2, 3]. The plant is recognized as a potential food source because of biologically active substances such as proteins, amino acids, minerals, sugars, lipids, and fiber present in it [4]. In the traditional medicine of East Africa (Kenya), Asystasia gangetica is used as an anthelmintic and leaves decoction drunk as a cure for intestinal worms [5].

Diabetes mellitus is a chronic metabolic disorder resulting from lack of insulin, characterized by hyperglycemia, altered metabolism of protein, carbohydrates, lipids it is increased risk of vascular complication [6, 7].

In vivo studies provide valuable clues in understanding the underlying pathological mechanisms of diabetes and are useful for the screening of drugs for the prevention and treatment of diabetes. Currently induced models have gained widespread acceptance for pathogenesis and drug screening research due to their rapid induction of diabetes [8]. Experimental diabetes mellitus has been induced in laboratory animals by several methods. The generally effective method is injecting drugs such as alloxan or Streptozotocin (STZ). Streptozotocin is a commonly used chemical to generate diabeties in animals in the laboratory for insulin-dependent diabetes mellitus characterized by high fasting blood glucose levels and drastic reduction in plasma insulin concentration [9].

Many synthetic drugs have been used for the treatment of diabetes, although, these drugs have limits in terms of efficacy and side effects [10]. Therefore much interest is going on for searching antidiabetic drugs from medicinal plants. Herbal medicine is an alternative method for the treatment of diabetes due to their perceived effectiveness, affordability, safety, and acceptability, with least side effects in clinical experience, and relatively low cost [11]. The present study was assessed for hypoglycemic potentiality of A. gangetica in normal and STZ induced rats.

2. MATERIAL AND METHODS

2.1 Extraction of plant sample

A. gangetica’s flowers were collected in Coimbatore surrounding areas in Tamilnadu, India and identiﬁed by Botanical survey of India, Coimbatore, India. Flowers were washed and dried at room temperature for 2 weeks and ground into fine powder. Dry powder (200 g) was dissolved in 200 ml of ethanol and kept in rotary shaker for 72 hr. Extract was ﬁltered; the ﬁltrate was concentrated in an evaporator at 40◦C for 24 hr, and stored at 4◦C for further use.

2.2 Chemicals

Streptozotocin (STZ) was purchased from Sigma chemicals, St. Louis, Mo, USA. All other chemicals and solvents used were of analytical grade.

2.3 Animals

Rats weighing (150-200 g) were maintained under standard conditions of humidity and temperature (28 ± 2ºC) and light (12 hr light/dark). The animals were housed in polypropylene cages (45×24×15cm) and were handled according to the university and institutional legislation, regulated by the ethical committee. The study was approved by Institutional Animal Ethical Committee constituted for the purpose of CPCSEA.

2.4 Acute and sub-acute toxicity study

Healthy rats were randomly assigned five groups of six rats.

Group I: Control rats

Group II: rats + AGFEE (250 mg/kg bw)

Group III: rats + AGFEE (500 mg/kg bw)

Group IV: rats + AGFEE (1000 mg/kg bw)

Group V: rats + AGFEE (2000 mg/kg bw)

Rats were fasted overnight (12 hr) with free access to water prior to administration of single doses (250, 500, 1000 and 2000 mg/kg bw) of the extract dissolved in distilled water and continued for 14 days (acute toxicity) 28 days (sub-acute toxicity). The animals of all the groups were observed for physiological, behavioral responses and mortality. Food consumption and water intake were checked daily. At the end of the sub-acute toxicity study, all the animals were anesthetized using chloroform and bled via inferior vena cava puncture. The blood samples were collected in plastic test tubes and allowed to stand for complete clotting. The clotted blood samples were centrifuged at 3000 rpm for 15 min and serum samples were aspirated off and frozen at −80◦C. Serum samples were analyzed for the estimation of glucose by ortho-toluidine method [12], urea [13], creatinine [14], total cholesterol [15], albumin and total protein [16], Assay of alanine transaminase and Assay of aspartate transaminase [17] and alkaline phosphatase [18].

2.6 Glucose lowering effect

Animals were randomly divided into nine groups of six animals each:

Group I: Control rats

Group II: Diabetic rats STZ (45 mg/kg bw)

Group III: Diabetic rats + AGFEE (125 mg/kg bw)

Group IV: Diabetic rats + AGFEE (250 mg/kg bw)

Group V: Diabetic rats + AGFEE (500 mg/kg bw)

Group VI: rats + AGFEE (125 mg/kg bw)

Group VII: rats + AGFEE (250 mg/kg bw)

Group VIII: rats + AGFEE (500 mg/kg bw)

Group IX: Diabetic rats + glibenclamide (5 mg/kg bw)

2.7 Experimental induction of diabetes

The male albino Wister rats weighing (150-180 g) were made diabetic by intraperitoneal injections of STZ. The animals were allowed to fast for 24 hr and were given STZ injection (45 mg/kg bw), with freshly prepared aqueous solution of citrate buffer as vehicle, pH 4.5. The control animals received buffer alone. STZ treated animals were allowed to drink 5% glucose solution over night to overcome drug. After 72 h of STZ induction, diabetes was confirmed by measuring fasting blood glucose concentration above 250 mg/dl.

2.8 Oral glucose tolerance test (OGTT)

After the treatment period, on the 14^th day, rats were fasted overnight with free access to water. Fasting blood samples were collected from the tail vein of the rats. Four additional blood samples were collected at 30, 60, 90 and 120 min intervals after administration of glucose at a concentration of 2 g/kgbw. The blood samples were collected for the estimation of glucose.

2.9 Statistical analysis

All quantitative measurements were expressed as mean ± SD for control and experimental animals. The data were analyzed using one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT) by using statistical package of social science (SPSS) Version 10.0 for Windows. A difference in the mean values of P<0.05 was considered to be statistically significant.

3. RESULTS

3.1 Acute and sub-acute toxicity study

In the acute and sub-acute toxicity evaluation, rats given AGFEE various dose (250, 500, 1000 and 2000 mg/kg bw) showed no mortality and all rats did not produce any symptoms of toxicity. Thus, the results suggested that the oral lethal dose (LD₅₀) of A. gangetica is greater than 2000 mg/kg bw, and can be classified as a low toxicity extract according to the Organisation for Economic Co-operation and Development, 2008. There were no changes observed in all estimated serum parameters of control and extract treated rats (Table 1). AGFEE did not show any significant difference in the levels of creatinine and urea, probably indicate that the extract did not interfere with the renal capacity to excrete the metabolite. Total protein measurements can reflect nutritional status and may be used to screen and diagnose kidney/liver diseases and many other conditions.

3.2 Glucose lowering effect

Fig. 1 shows Glucose lowering effect obtained in STZ diabetic rats after administration of AGFEE for 14 days. In the present study the fasting blood glucose estimation and body weight measurement were done on 1, 7 and 14^th day. Blood glucose levels were measured by using the glucose oxidase-peroxidase reactive strips and a glucometer. STZ induced rats showed extreme significant increase (P<0.05) in fasting blood glucose level compared to normal rats. Treatment with AGFEE at the doses of 125, 250 and 500 mg/kg bw and glibenclamide at the dose of 5 mg/kg bw showed significant glucose lowering effect in normal and diabetic rats. The difference between the treated groups and the diabetic control rats in the levels of fasting plasma glucose was significant (P<0.05). Ethanol extract of A. gangetica at the dose of 250 mg/kg bw showed extremely significant decrease (P<0.05) in blood glucose level after 14^th day of treatment. The higher dose (500 mg/kg) did not produce any stronger effect when compared to other doses. In normal rats, the extract showed minimum glucose lowering effect, but in diabetic rats the dose of 250 mg/kg bw was found to be most effective.

Table 1. Effect of AGFEE on serum parameters in control and experimental rats

Parameters	Group I	Group II	Group III	Group IV	Group V
Glucose (mg/dl)	112.84 ± 1.52	107.24 ± 0.8	108.66 ± 1.22	100.57 ± 1.80	105.38 ± 1.45
Urea (mg/dl)	38.65 ± 1.22	33.00 ± 1.53	37.35 ± 1.18	41.50 ± 1.0	34.00 ± 1.25
Creatinine (mg/dl)	0.952 ± 0.06	0.874 ± 0.04	0.926 ± 0.06	1.08 ± 0.05	0.984 ± 0.02
Cholesterol	64.33 ± 1.54	65.82 ± 1.82	67.00 ± 1.65	64.58 ± 1.0	68.65 ± 1.52
Albumin (g/dl)	2.87 ± 0.44	2.92 ± 0.67	3.34 ± 0.59	2.74 ± 0.75	3.15 ± 0.57
Total protein (g/dl)	6.32 ± 0.62	5.67 ± 0.12	6.85 ± 0.40	5.94 ± 0.11	6.14 ± 0.25
AST(IU/L)	86.55 ± 0.15	89.30 ± 0.62	84.56 ± 0.24	91.40 ± 0.46	82.16 ± 0.22
ALP(IU/L)	68.17 ± 0.67	71.35 ± 0.73	65.11 ± 0.11	67.58 ± 0.44	70.40 ± 0.26
ALT(IU/L)	74.94 ± 0.88	77.26 ± 0.47	81.55 ± 0.35	79.14 ± 0.24	76.42 ± 0.85

Values are expressed as mean ± SD of 6 rats in each group. No significant difference was observed in any parameter.

Table 2: Effect of AGFEE on body weight in control and experimental rats (dose determination test)

Groups	Initial day	7^th day	14^th day
Control	161.16 ± 1.61	164.53 ± 1.26^e	166.35 ± 1.40^e
Diabetic control	155.26 ± 1.04	143.60 ± 2.18^a	131.55 ± 2.83^a
Diabetic + AGFEE 125 mg/kg	157.95 ± 1.28	146.25 ± 3.61^a	147.62 ± 1.41^c
Diabetic + AGFEE 250 mg/kg	154.63 ±1.43	147.58 ± 1.55^b	151.77 ± 2.47^c
Diabetic + AGFEE 500 mg/kg	155.03 ± 1.24	142.09 ± 1.20^a	146.35 ± 4.50^b
AGFEE 125 mg/kg	160.11 ± 1.55	162.22 ± 4.80^c	165.29 ± 5.47^d
AGFEE 250 mg/kg	158.80 ± 1.36	161.22 ± 2.34^c	164.22 ± 1.65^d
AGFEE 500 mg/kg Diabetic + Glibenclamide 5 mg/kg	155.52 ± 1.08 162.17 ± 1.64	157.82 ± 3.58^d 156.55 ± 1.37^d	158.32 ± 1.16^d 158.58 ± 4.02^d

Values are expressed as mean ± SD (n=6)

4. DISCUSSION:

A. gangetica is a traditional medicinal plant of South Africa and it is used for various alignments in India also. The current study was carried out to determine the glucose lowering (hypoglycemic) effect of A. gangetica flowers in STZ-induced diabetic rats. Acute and sub-acute toxicity showed the LD₅₀ is more than 2000 mg/kg bw therefore, it can be considered to be relatively safe [19]. Serum parameters were estimated and there were no significant changes in total protein in rats treated with AGFEE, which suggested that there was no sign of impaired renal function [20]. Both the control and treated groups appeared uniformly healthy at the end of the experiment. Several previous studies have investigated the acute effects of high and lower dosages in animals including the dosage potentially usable in humans [21].

Streptozotocin induced diabetes has been described as a useful experimental model to study the activity of hypoglycaemic agents and it is selectively destroys the pancreatic insulin secreting β-cells, leaving less active cells and resulting in a diabetic state [22]. The continuous treatment of AGFEE at dose levels of 125, 250 and 500 mg/kg bw for 14 days produced a significant (P<0.05) decrease in blood glucose level in diabetic rats when compared to diabetic control rats. Comparing the results of 125, 250 and 500 mg/kg bw AGFEE in diabetic rats, it was found that the extract at a dose of 250 mg/kg bw showed significant (P<0.05) decrease in blood glucose levels when compared to other doses. An increase in blood glucose observed in the oral glucose tolerance test was significantly greater in the diabetic rats than in the normal rats. Oral administration of AGFEE 250 mg/kg bw significantly improved the impaired glucose tolerance in the diabetic rats. The observed hypoglycemic activity of AGFEE might be attributed to an enhancement of peripheral metabolism of glucose, besides the possible increased release of insulin caused by its chemical constituents [23]. STZ induced diabetes is characterized by severe weight loss [24, 25] which is due to increased muscle wasting and loss of tissue proteins [26, 27]. The reversal of weight loss in extract treated diabetic group indicates that the protective effect of AGFEE in controlling muscle wasting i.e. reversal of gluconeogenesis [28]. Moreover, the ability to protect body weight loss seems to be the result of its ability to reduce hyperglycemia.

Considering the above results, the hypoglycemic effect of the plant may involve in reducing hyperglycemia through insulin secretion from β-cells and augmented glucose transport and utilization. In preliminary phytochemical screening AGFEE showed the presence of phenols, flavonoids, terpenoids, glycosides and alkaloids, such compounds were earlier implicated as having antidiabetic effects [29, 30, 31, 32]. A number of plants have been shown to exert hypoglycemic activity through stimulation of insulin release [33] like glibenclamide that is reported to enhance the activity of beta cells of pancreas resulting in increased secretion of large amount of insulin which in turn brings down blood glucose level [34].

5. CONCLUSION:

The present study can be concluded that AGFEE in a dose of 250 mg/kg bw produced an appropriate glucose lowering (hypoglycemic) effect comparable to that of glibenclamide, which suggests that flowers of A. gangetica may produce the effect by a mechanism similar to that of glibenclamide, so it could serve as good example to other oral hypoglycemic agents and seems to be promising for the development of phytomedicines for diabetes mellitus.

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Received on 19.08.2014 Modified on 25.09.2014

Res. J. Pharmacognosy & Phytochem. 6(4):Oct. - Dec.2014; Page 170-175