Antidiabetic Activities of Leaves and Root Extracts of Justicia Adhatoda Linn Against Alloxan Induced Diabetes in Rats

Full Length Research Paper

Antidiabetic activities of leaves and root extracts of Justicia adhatoda Linn against alloxan induced diabetes in rats

Muhammad Gulfraz*, Asma Ahmad, Muhammad Javaid Asad, Aliya sadiq, Umara Afzal, Muhammad Imran, Pervez Anwar, Asiya Zeenat, Kashif Sarfraz Abbasi, Sadaf Maqsood and Rahmat Ullah Qureshi

Department of Biochemistry, PMAS Arid Agriculture University, Rawalpindi, Pakistan.

Accepted 4 April, 2011

Justicia adhatoda Linn is a medicinal shrub and its leaves and roots have being used against diabetes in North East of Pakistan years ago. The effects of leaves and roots of J. adhatoda was studied in alloxan induced diabetic animals. The purpose of this study was to investigate the effects of leaves and root extracts of the plant on blood glucose and other parameters associated with diabetes. Oral administration of 50 and 100 mg/kg of ethanolic extracts of Justicia leaves to normal and experimental diabetic rats produced a significant (p < 0.05) reduction in blood glucose levels from 2 to 6 days of treatment as compared to the root extract of J. adhatoda (100 mg/kg) and glibenclamide (5 mg/kg). Significant effects were also observed on the glucose tolerance, glycosylated haemoglobin, serum lipid profiles and body weight of experimental animals.

Key words: Justicia adhatoda L, traditional medicine, animal model, anti diabetic.

INTRODUCTION

Diabetes mellitus is a group of syndromes characterized There is a growing interest in herbal remedies due to by hyperglycaemia, altered metabolism of lipids, carbo- the side effects associated with the therapeutic agents hydrates and proteins (Mushtaq et al., 2000). About 90% (oral hypoglycemic agents and insulin for the treatment of of diabetic patients are non-insulin-dependent diabetes diabetes mellitus). Many traditional folk medicinal and mellitus (NIDDM), insulin resistance plays a key role in herbal extracts have been used for the treatment of the development of the disease (Fuller et al., 1980; Leng diabetes mellitus, most of them have shown good effects et al., 2004). Chronic hyperglycaemia causes glycation of but some exert only little or no effect on glycemic control protein that in turn leads to secondary complications in experimental studies (Bailey and Day, 1989). effecting the eyes, kidneys, nerves and arteries (Hirsch et Justicia adhatoda Linn. (Syn. Adhatoda vasica Nees, al., 2000). These complications can be prevented, Adhatoda zeylanica Medicus) belongs to family delayed or decreased by maintaining blood glucose Acanthaceae, subclass Asteridae and specie Adhatoda values close to normal. According to WHO, over 150 (Stewart, 1972). The leaf juice is shown to cure diabetes million people suffer from diabetes worldwide and this diarrhea, dysentery and liver relevant disorders. Two figure is likely to increase to 300 million or more by the major alkaloids of J. adhatoda are vasicine and year 2025 (Sung et al., 2003). It is an increasing public vasicinone formed by the oxidation of vasicine at C-8 health problem, especially in developing countries, where position and possess interesting biological activities adequate treatment is often expensive or unavailable including antihyperglycaemic effects reported by different (WHO, 1980). authors. Therefore, the purpose of this study was to assess the possible effects of leaves and root extracts of J. adhatoda on diabetes related hematological parameters such as *Corresponding author. E-mail: [email protected] or blood glucose levels, glucose tolerance, glycosylated [email protected]. hemoglobin and serum lipid profiles on the weight of 6102 Afr. J. Biotechnol.

normal and diabetic rats. Analysis of fasting glucose, serum insulin, haemoglobin and glycosylated haemoglobin

Blood samples were collected daily, starting from the first day of MATERIALS AND METHODS treatment. Fasting blood concentration was estimated by the

method of Gaster and Hirsch (1998). Serum insulin level was Fresh root and leaves samples of J. adhatoda were collected from assayed by ELISA kit (Linco Research Inc., USA), (Holman and hilly areas of Rawalpindi (Pakistan). Samples were identified by a Turner, 1991; Bailei and Ahamd, 1981). Haemoglobin and taxonomist and deposited in the herbarium of University of Arid glycosylated haemoglobin level in blood samples was measured by Agriculture, Rawalpindi (voucher specimen no.124). Wistar rats of using the methods reported by Moller and Flier (1991) and Gaster either sex (150 to 200 g) were maintained under standard animal and Hirsch (1998). house conditions, fed with commercial rat chow (Feed Mills,

Rawalpindi) and had access to water ad libitum. Fasted animals were deprived of food for at least 16 h but allowed to have free Serum lipid (mg/dl profile) access to water. All experimental animals were carefully monitored and maintained in standard household conditions. Triglyceride and cholesterol serum levels were assayed using

commercial kits (Randox Laboratories); HDL cholesterol was

measured by using enzymatic method (Chase and Glasgow, 1976; Preparation of various doses of plants extracts for animals Kunst et al., 1984).

Different doses (50 and 100 mg/kg) of leaves and root samples were prepared in ethanol. Standard antidiabetic drug glibenclamide Body weight (g) of alloxan-diabetic rats (5 mg/kg), glucose (5 mg/kg) and insulin (5 IU) were also provided to experiment animals during the study. The animals were divided into 4 groups of 6 each (n = 6). Group 1 was designated as the diabetic control (vehicle) and received 2 ml of 1% ethanol and water mixture. The animals in groups 2 and 3 Alloxan induced diabetes were treated with 100 mg/kg each of root and leaves extract of J. adhatoda L. whereas, animals in group 4 was given glibenclamide To induce diabetes, fasted animals were given 65 mg/kg body (5 mg/kg). The changes in the body weight (g) were determined by weight of alloxan by a single dose via intravenous injection. Only the difference of initial and final weight of animals. diabetic rats (whose body weights and serum glucose levels were assessed over 6 days) were included in this study. Blood glucose concentrations (mg/dl) were measured daily from days 0 to 6 Statistical analysis (Kitaharal et al., 2002). Data were statistically evaluated by using one way ANOVA followed by Ducan‘s multiple range test (DMRT). The results were considered Administration of plant extracts to animals statistically significant if the p < 0.05.

Doses of 50 and 100 mg/kg each of ethanolic plants extracts were given to experimental animals orally by gavages. RESULTS AND DISCUSSION

Acute toxicity and selection of doses The acute toxicity studies indicated that the selected doses (50 and 100 mg/kg) of ethanolic leaves and root Acute toxicity study was conducted to select a suitable dose for extracts of J. adhatoda were non-toxic to experimental evaluation of anti diabetic activity and possible toxic effects on animals throughout the study. Treatment with different animals. doses of ethanolic extracts produced a significant reduction in blood glucose level in all animal models whereas,

Oral glucose tolerance test in the glucose tolerance test, a significant reduction in blood glucose level was observed from 30 to 120 min Animals were divided into 7 groups of 5 each (n = 5). Glucose (2 (Table 1). g/kg) was given 30 min following treatment with either roots and Effects of different doses of ethanolic plants extracts on leaves extracts of J. adhatoda L. The blood glucose concentration blood glucose levels, serum insulin, total hemoglobin, (mg/dl) was measured at 0, 30, 60 and 120 min, after the treatment glycosylated hemoglobin, changes in body weight and with plants extracts. sugar level in urine of normal and diabetic rats are presented in Tables 2 to 5. Data in Table 1 shows the Blood glucose concentrations in diabetic rats effects of different doses of root and leaves extracts of J. adhatoda on glucose tolerance in rats. Whereas, Table 2 The blood glucose concentration (mg/dl) was measured at 0 to 6 represents the effects of plant extracts in diabetic rats. It days after the treatment with ethanolic leaves and root extracts of J. was observed that 50 and 100 mg/kg of ethanolic plants adhatoda L. by using the method reported by Sung et al. (2003). At extracts successfully reduced the level of blood glucose the end of 6 days, animals were deprived of food for overnight and from day 3 to the 6 days of treatment, however, effects of blood samples were collected via the tail vein for the measurement of blood glucose levels. Animals were anesthetized with ether, 100 mg/kg of ethanolic leaves extracts were more sacrificed and liver was immediately dissected and washed in ice pronounced than glibenclamide, a standard antidiabetic cold saline, dried and weighed (Kham et al., 2003). drug ( Table 2). Gulfraz et al. 6103

Table 1. Blood glucose concentrations during oral glucose tolerance test after treatment with ethanolic plant extracts in glucose loaded rats.

Group Treatment 0 min (mg/dl) 30 min (mg/dl) 60 min (mg/dl) 90 min (mg/dl) 120 min (mg/dl) 1 Diabetic + insulin (5 IU) 278.4±1.5 162±2.1 102.1±1.2 88.3±2.5 91.2±1.3

Diabetic+ glucose 205.4 2 92.2 2 287.3±1.2 264.3±1.8 216.4±2.6 178.3±1.1 (5g/kg) ±2.1 Diabetic + root extract 3 285.2±1.2 248.1±1.5 214.2±1.5* 117.5±6.5* 107.2±2.5* (50 mg/kg)

Diabetic + root extract 4 288.4±5.2 232.4±6.5* 208.1±4.2* 112.4±3.8* 94.3±1.1* (100 mg/kg)

Diabetic+ 5 261.3±3.1 214.5 ±2.1 158.3±1.2 112.4±1.5 98.4±1.9 leaves extract (50 mg/kg)

Diabetic + leaves extract 6 284.3±3.5 210.5±4.1 142.3±1.5 104.3±1.5 92.4±1.4 (100 mg/kg)

7 Glibenclamide (5 mg/kg) 251.7±4.3 171.8±5.2 152.4±2.5 104.3±4.5 97.4±3.5

Values are expressed as mean ±SE. *significant (p < 0.05), n = 5.

Table 2. Blood glucose concentration in alloxan induced diabetic rats after treatment with ethanolic plant extracts.

Group Treatment Day 0 (mg/dl) Day 1 (mg/dl) Day 2(mg/dl) Day 4 (mg/dl) Day 6 (mg/dl) 1 Untreated Diabetic 280.4±1.2 194.2±1.6 188.4±1.6 166.2±2.1 156.2.±1.5

Diabetic control 2 272.5±4.1 236.4 ±1.5 183.5 ±2.4 129.4±2.8 122.1 ±1.6 (vehicle 2 % ethanol)

Diabetic + root extract 3 268.1±2.6 192.4±1.6 158.1±2.5* (50 mg/kg) 102.4±1.5* 105.5 ±2.1 Diabetic + root extract 4 278.1±4.2 176.4± 1.6 151.5±2.7* (100 mg/kg) 108.1±2.7* 89.1±2.5 Diabetic + leaves 5 273.1±2.8 216.2±1.9 164.5±1.6 extract (50 mg/kg) 112.6±2.4 106.4 ±2.5 Diabetic + leaves 6 285.1±2.8 236.4±1.2 147.5±1.2 extract (100 mg/kg) 116.6±2.7 98.4 ±2.6 Glibenclamide 7 272.4±2.6 182.5±1.4 118.1±2.4 (5mg/kg)

*Significant (p < 0.05). Values are expressed as mean ± SE; n = 5.

A significant increase in blood glucose and glyco- which was cleared from lower level of blood sugar after sylated haemoglobin levels and decreased in serum treatment of various doses of plants extracts. insulin and total haemoglobin levels were observed in Furthermore, in diabetic rats, the high level of sugar in alloxan induced diabetic rats when compared with normal urine (++++) was observed and after treatment with rats (Tables 3 to 4). A dose of 100 mg/kg ethanolic plant various doses of plant extracts, specially 100 mg/kg of extracts and glibenclamide were highly effective because leaves extracts of J. adhatoda, reduced sugar in urine these doses turned the parameters towards normal sides, was near normal (Table 3). The effect of ethanolic plants 6104 Afr. J. Biotechnol.

Table 3. Assessment of different parameters in normal and alloxan induced diabetic rats after treatment with ethanolic plant extracts.

Fasting blood Serum insulin Haemoglobin Glycosylated Group Treatment Urine glucose (mg/dl) (uU/ml) (g/dl) hemoglobin (mg/gHb) 1 Untreated diabetic rat 198.5 ±2.1 5.1 ±1.2 8.5± 1.5 0.98 ± 0.1 ****

Diabetic control 2 132.4±2.8 5.8 ±0.4 8.9 ±0.6 0.59 ±0.7 **** (vehicle 2% ethanol)

Diabetic + root extract 3 121.6±3.2 10.5 ±0.6 11.5 ±1.2 0.52 ±0.5 * (50 mg/kg)

Diabetic + root extract 4 128*±2.7 14.5 ± 1.2 14.1 ±2.3* 0.63±0.4* Nil (100 mg/kg)

Diabetic + leaves 5 112±1.8 9.8 ±1.4 10.8 ±0.8 0.25±0.2 ** extract (50 mg/kg)

Diabetic + leaves 6 98.1±4.1* 11.2±3.4* 11.4.±1.1* 0.42 ±0.1 * extract (100 mg/kg)

Glibenclamide (5 7 115 ±2.4 12.1 ±1.5 12.9 ±2.5* 0.45 ±0.8* Traces mg/kg)

*Significant value (p < 0.05). Values are expressed as mean ± SE; n = 5.

Table 4. Changes in different parameters in liver of normal and diabetic animals after treatment with ethanolic plant extracts.

Triglyceride Cholesterol HDL cholesterol Free fatty acids Phospholipid Group Treatment (mg/100g wet (mg/100g (mg/100g wet (mg/100 g wet (mg/100 g wet tissue) wet tissue) tissue) tissue) tissue) Untreated diabetic 1 365.7 ±1.7 318.4 ±2.1 259.2 ±5.6 778.4 ±2.5 1.7 ± 1.1 rat

Diabetic control 2 345.2 ±2.5 225.4±5.8 242.4 ±3.8 729.3 ±1.6 2.3 ± 2.1 (vehicle)

Diabetic + root 3 323.2 ±5.4* 117.4±6.1* 262.5 ±5.3* 682.1 ±2.4* 2.1 ± 1.9 extract (100 mg/kg)

Diabetic + leaves 4 368.4±5.2 358.4±6.5* 228.1±4.9* 691.4±3.1* 2.4 ±1.1* extract (100 mg/kg)

Diabetic + 5 glibenclamide 459.3 ±4.5* 338.6 ±6.4* 249.3 ± 2.4* 771.4 ±1.5 2.8 ±1.6 (5mg/kg)

*Significant (p < 0.05) as compared to control/normal animals. Values are expressed as mean ± SE n = 5.

extract on body weight of animals was significant and the were observed in the diabetic rats which was successfully weight of animals was improved after treatment with treated with plants extracts and levels of these para- plants extracts (Table 5). A significant increase in choles- meters were returned towards normal (Tables 3 to 5 ). terol, free fatty acids, triglycerides and phospholipids Alloxan is well known for its selective pancreatic islet β- Gulfraz et al. 6105

Table 5. Effect on body weight of alloxan-induced diabetic rats after treatment with ethanolic plant extracts.

Group Treatment Initial body weight (g) Final body weight 1 Diabetic control (vehicle) 291.4±7.5 298.2±1. 2 Ethanolic root extract (100 mg/kg) 290 ±7.4 289.4±7.1 3 Ethanolic leaves extracts (100 mg/kg) 286.4±5.1 290.3±9.6 4 Glibenclamide (5mg/kg) 288.1±1.6 292.5±2.1

* Significant

cell cytotoxicity and has been extensively used to induce and other similar diseases. diabetes mellitus in animals. Administration of 65 mg/kg of alloxan effectively induced diabetes in normal rats as reflected by glycosuria, hyperglycaemia, polyphagia, ACKNOWLEDGEMENT polydipsia and body loss when compared with normal rats (Tables 3 to 5). Ethanolic root and leaves extracts of The authors are grateful to PMAS Arid Agriculture plant reversed these effects in the experimental animals. University, Rawalpindi, Pakistan for providing necessary The possible mechanism by which plants extracts brings research facilities. antihyperglycaemia action maybe by potentiation of pancreatic secretion of insulin from β-cell of islets or due to enhanced transport of blood glucose to peripheral REFERENCES tissue and was obvious by increased level of insulin in diabetic rats when treated with plants extracts (Ozturk et Bailei CJ, Ahamd H (1981). Determination of plasma insulin. 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