INTRODUCTION:

Diabetes mellitus is a disorder of carbohydrate metabolism in which sugars in the body are not oxidized to produce energy due to lack of pancreatic hormone (insulin). The accumulations of the sugars leads to it’s appearance in the blood (hyperglycemia), then in urine. The symptoms those are associated with diabetes include fatigue, excessive thirst, frequent urination, blurred vision, mood changes, and sexual problems. Diabetes that starts in childhood or adolescence (type 1) is usually more severe than that in beginning, in middle or old age (type 2) but may also develop in younger people. The pancreas retains some ability to produce insulin but it is inadequate for the body needs; alternatively, the body becomes resistant to the effects of insulin and may require treatment with oral hyperglycemic drugs or insulin ^1-3.

Anacardium occidentale (Anacardiaceae) is an important traditional shrub, native to India and Africa but also found in other parts of the world. Inhabitants of these areas have been using Anacardium occidentale for the treatment of diabetes, wounds, ulcers, fever, sore throats and snakebites. Leaves are green in color, rich in flavonoids, triterpenoids and other phytochemicals.^4-6

The aim of the present study was to demonstrate the antihyperglycemic effects of ethanol leaf extract of Anacardium occidentale in normal and alloxan induced diabetic rats.

MATERIAL AND METHODS:

Chemicals and instruments used:

The following chemicals were used in the study: Alloxan Monohydrate (Sigma, St. Louis,M.O), Glibenclamide ( Cadilla Pharmaceuticals) , Sodium CMC.

Plant material:

Leaf samples were collected from Paneer, Managalore, India during March and April 2008 and authentified by a taxonomist Dr.Gopalkrishna Bhat, Dept. of Botany, Poornaprajna college, Udupi. The specimen sample No.72 is kept in the department, NGSM institute of pharmaceutical sciences, Paneer, Deralakatte, Mangalore.

Preparation of plant extracts:

Dried leaves (5 kg) were ground in a waring blender and sifted through a wire screen (mesh size 2 mm × 2 mm). The powdered leaves (500 gm) were exhaustively extracted with 70 % ethanol. The extracts were filtered and concentrated on a rotary evaporator.

Animals:

Wistar rats of both sexes (150-200 g) were maintained under standard animal house conditions, fed standard pellet diet (Hindustan Lever Ltd. Bombay) and allowed water ad libitum.

Fasted animals were deprived of food for at least 16 hr, but were allowed free access to water. The study was approved by the Institutional Animal Ethical Committee of KSHEMA, Mangalore, India.

Table 1. Effect of ethanolic extract on serum glucose level (mg/dL) in glucose tolerance test in glucose loaded rats.

Group	Treatment	0 min	30 min	60 min	90 min	120 min
1	Control (Vehicle )	89.2 ± 8.9	108.3± 6.1	103.7± 9.1	100.2*± 5.4	96.3*± 3.6
2	Ethanolic extract (100mg/kg)	80.4± 3.1	92.7± 4.1	86.7± 10.1	83.6± 7.8	79.8± 6.3
3	Ethanolic extract (200mg/kg)	84.3± 5.7	97.4± 3.2	90.5± 3.5	84.2*± 7.2	81.4*± 4.3
4	Ethanolic extract (400mg/kg)	81.9± 4.1	78.7± 5.4	83.7± 3.2	77.3*± 3.5	70.2± 5.2
5	Glibenclamide (500 µg/kg)	83.4± 3.5	79.5± 2.1	80.1± 6.7	75.1*± 8.1	73.6± 3.9

Values are expressed as mean ±SEM; n=6 in duplicate for each treatment; *statistically significant difference from the corresponding zero time value; p<0.05;

Table 2. Effect of different ethanolic extracts on serum glucose level (mg/dL) in normal fasted animals

Groups	Dose	Day 0	Day 4	Day 8	Day 12
1	Control (Vehicle )	78.4± 7.3	76.1± 8.6	79.4± 5.2	76.3± 2.4
2	Ethanolic extract (100 mg/kg)	82.1± 2.6	80.3± 3.3	79.8*± 3.7	77.5*± 4.3
3	Ethanolic extract (200 mg/kg)	74.4± 6.3	70.6± 2.4	67.3± 8.6	61.8*± 7.3
4	Ethanolic extract (400 mg/kg)	77.2± 3.4	69.1± 5.3	61.4*± 2.5	56.2± 3.6
5	Glibenclamide (500 µg/kg )	80.1± 2.3	73.2± 3.9	66.7*± 3.2	59.6*± 4.7

Values are expressed as mean ±SEM; n=6 in duplicate for each treatment; *statistically significant difference from the corresponding zero time value; p<0.05

Table 3 : Effect of different ethanolic extracts on serum glucose level (mg/dL) in alloxan diabetic rats

Groups	Dose	Day 0	Day 4	Day 8	Day 12
1	Control (Vehicle )	249.6± 17.3	250.2± 18.6	248.7± 11.2	250.1± 12.4
2	Ethanolic extract (100 mg/kg)	258.4± 12.6	247.2± 10.3	235.6± 13.7	222.7± 14.3
3	Ethanolic extract (200 mg/kg)	267.1± 16.3	249.4*± 12.4	229.3± 18.6	212.3± 17.3
4	Ethanolic extract (400 mg/kg)	260.2± 13.4	225.2*± 15.3	183.7*± 12.5	149.3*± 13.6
5	Glibenclamide (500 µg/kg)	255.2± 10.3	198.5*± 10.9	166.3± 11.2	137.2*± 11.7

Values are expressed as mean ±SEM; n=6 in duplicate for each treatment; *statistically significant difference from the corresponding zero time value; p<0.05

Acute toxicity and selection of doses:

The acute toxicity studies were carried out in adult female albino rats weighing about 150-200 g, by up and down method as per OECD 425 guidelines ⁷. Over night fasted animals received test drug at a dose of 2000 mg/kg body weight orally. Then the animals were observed continuously once in half an hour for next 4 hrs and then after 24 hrs for general behavioral, neurological, autonomic profiles and to find out mortality. The extract was found safe up to a dose of 2000mg/kg body weight.

Oral glucose tolerance test:

The oral glucose tolerance test was performed in overnight fasted normal animals ⁸.Rats divided into four groups (n=6) were administered 2% gum acacia solution, ethanolic extract 200 mg/kg, 400 mg/kg and glibenclamide (500 µg/kg), respectively. Glucose (2 g/kg) was fed 30 min after the administration of ethanolic extract. Blood was withdrawn from the retro-orbital sinus

at 0, 30, 60, 90, and 120 min of ethanolic extract administration. Fasting serum glucose levels were estimated by Radio Immuno Assay Kit (BRAC, Mumbai).

Normoglycemic study:

For, normoglycemic study rats were divided into five groups (n=6) and were administered 2% gum acacia solution, ethanolic extract 100 mg/kg, 200 mg/kg and 400 mg/kg and glibenclamide (500 µg/kg) respecitively ⁹. Blood glucose levels were estimated on days 0, 4, 8 and 12.

Induction of experimental diabetes:

The diabetes was induced in overnight fasted animals by a single intraperitoneal injection of 60 mg/kg ¹⁰ of alloxan monohydrate. Hyperglycemia was confirmed by the elevated blood sugar levels determined at 72 h and on day 7 of the injection. Only rats confirmed with permanent NIDDM were used in the antidiabetic study.

Experimental and design:

The animals were divided into five groups of six animals in each group:

Group I: - Normal healthy control;

Group II: - Diabetic control (Alloxan 60 mg/kg i.p)

Group III: - Diabetic + Exrtact (100 mg/kg body weight, orally)

Group IV: - Diabetic + Exrtact (200 mg/kg body weight, orally)

Group V: - Diabetic + Exrtact (400 mg/kg body weight, orally)

Group VI: - Diabetic + Glibenclamide (500 µg/kg body weight, orally)

Sample collection:

Blood sampling :At the end of day 12, blood samples were collected from the inner canthus of the eye under light ether anesthesia using capillary tubes (Micro Hemocrit Capillaries, Mucaps). Blood was collected in to fresh vials containing anticoagulant antiserum was separated in a centrifuge at 2000 rpm for 2 min. Serum insulin levels were estimated by the Radio Immuno Assay Kit issued by the Board of Radiation and Isotope Research, Bhaba Atomic Research Centre (BRAC), Mumbai, India.

Statistical analysis:

Data were statistically evaluated by the use of one-way ANOVA, followed by post hoc Scheffe’s test using 7.5 version of SPSS computer software. The values were considered significant when p<0.05.

RESULTS:

Acute toxicity studies revealed the non-toxic nature of ethanolic extract at the three dose levels tested. No lethality or toxic reactions were observed until the end of the study.

At the 30 min of initiating glucose tolerance test, blood glucose concentration was higher than at zero time but decreased significantly from 30 min to 120 min (Table 1). Ethanolic extracts significantly decreased the level of blood glucose at a dose of 100, 200, 400 mg/kg and glibenclamide at the dose of 20 mg/kg from 30 min to 120 min.

In normoglycemic rats the doses of 100, 200, 400 mg/kg reduced hyperglycemia after 4, 8 and 12 days of treatment (Table 2). A significant hypoglycemic activity was found at 12^thday with 100, 200, 400 mg/kg doses. The extract significantly reduced blood glucose level at 12^thday as compared to zero day for each treatment.

After oral administration of 100, 200, 400 mg/kg of the ethanolic extract of Anacardium occidentale , a significant reduction was observed in blood glucose level of alloxan induced diabetic rats. The doses of 100, 200, 400 mg/kg decreased the level of the blood glucose throughout the study period (Table 3). Blood sugar level was also determined before and after the glibenclamide treatment. Glibenclamide a known hypoglycemic agent, reduced blood sugar level.

DISCUSSION:

In the present study, the hyperglycemic activity of ethanolic leaf extract of Anacardium occidentale was assessed in normal and alloxan induced diabetic rats. Oral administration of a single dose of ethanolic leaf extract of Anacardium occidentale caused a significant decrease in serum glucose level in normal rats. Adose of 400 mg/kg of ethanolic extract produced maximum glucose lowering effect, whereas 100 and 200 mg/kg of ethanolic extract showed a significant hypoglycemic effect throughout the study period (Tables 1-3). In the oral glucose tolerance test, the Anacardium occidentale leaf extract showed significant reduction of serum glucose levels and these effects were dose dependent. The extract of Anacardium occidentale leaf displaved a significant hypoglycemic effects in normal rats; the main mechanism by which the extracts bring the hypoglycemic effects most probably involves stimulation of peripheral glucose consumption. Furthermore the glycemia profile observed in the glibenclamide group indicates that the extract of Anacardium occidentale acts on the liver or on peripheral glucose consumption (11).

The glibenclamide effects on glucose can be attributed to the enhanced activity of the β cells of the pancreas, resulting in secretion of the large amount of insulin (12). Although alloxan treatment causes permanent effects, these effects could be retrieved by the action of alcoholic plant extracts. These results revealed that the some drugs may also be effective in insulin independent diabetes. The significant hypoglycemic effects of Anacradium occidentale leaf in diabetic rats indicate that this effect can be ediated by stimulation of glucose utilization by peripheral tissues.

It is concluded that the leaf extract reduced serum glucose level in normal and alloxan induced diabetic rats, however the effects of 400 mg/kg ethanolic extract was more pronounced in diabetic rats. Therefore, additional studies are needed for isolation and separation of bioactive compounds from the leaves of this important medicinal plant.

ACKNOWLEDGEMENT:

The authors are thankful to Nitte Education Trust for providing necessary facilities and are also thankful to Sophisticated Analytical Instrumentation Facility, Chandigarh for providing the spectras.

REFERENCES:

1. Bell GI. Molecular defects in diabetes mellitus. Daibetes, 1991; 40: 413-417.

2. Shalev A. Hope for insulin mimetic oral antidiabetic drugs. Eur J Endocrinol 1999; 8: 561-562.

3. Shweta S, Subhash B, Ramesh B, Mohan V. Combinative therapeutic approach

better blood sugar level control in alloxan diabetic mice. Int J Diabetes and Metab olism, 2006; 12: 104-105.

4. Kirtikar KR, Basu BD, Indian medicinal plant, vol.II Delhi Periodical express; 1975. pp. 142-43.

5. Khare CP,Rational western therapy. Ayurvadec and other traditional usage. Encyclopedia of Indian medicinal plant.Botny.Sprenger; 2000. pp. 207-208.

6. Kurian JC. Plant That Heal. 7^th ed. Pune Oriental Watchman Publishing House.vol.І. 2004. pp. 18.

7. OECD 425 guidelines. OECD guidelines for testing of chemicals. 2001 Dec; 1/26:1-26.

8. Boner-weir S, Dery D, John LL, Gordon CW. Compensatory growth of pancreatic β cells in adult rats after short-term glucose infusion. Diabetes 1989; pp. 38:49-53.

9. Rakesh B, Sanjay J, Deep Q, Amit J, Girraj ST, Ravi G. Antidiabetic activity of aqueous root extract of Ichnocarpus frutescens in streptozotocin-nicotinamide induced type-II diabetes in rats. Indian J of Pharmaco. 2008 (40); 1: 19-22.

10. Chacko MS, Sabitha T, Kuttan R. Amelioration of alloxan induced hyperglycemia by aloe arborescens miller and it’s possible mechanism. Pharmacologyonline 2007; 3: 571-584.

11. Stanely P, Prince /m, Menon VP. Hypoglycemic and other related actions of Tinospora cordofolia roots in alloxan induced diabetic rats. J Ethnopharmacol 2000; 70: 9-15.

12. Ozturk Y, Altan VM, Yildizoglu A. Effect of experimental diabetes and insulin on smooth muscle functions. Pharmacol Rev 1996; 10: 69-112.

Received on 10.04.2009 Modified on 27.05.2009

Asian J. Research Chem. 2(3): July-Sept. 2009 page 262-265

Groups	Treatment	Initial (g)	Final (g)
1	Control (Vehicle )	178.6 ± 17.3	153.3 ± 18.6
2	Ethanolic extract (100 mg/kg)	158.4 ± 12.6	139.2 ± 19.3
3	Ethanolic extract (200 mg/kg)	172.1 ± 16.3	157.4 ± 12.4
4	Ethanolic extract (400 mg/kg)	165.2 ± 13.4	148.2 ± 15.3
5	Glibenclamide (500 µg/kg)	187.2 ± 10.3	164.5 ± 10.9