INTRODUCTION:

Plants have a great potential for producing new drugs for human benefit. Plants used in traditional medicine contain a vast array of substances that can be used to treat chronic and even infectious diseases. According to a report of World Health Organization, more than 80% of world’s populations depend on traditional medicine for their primary health care needs [1]. The demand for more and more drugs from plant sources is continuously increasing. It is therefore essential for systematic evaluation of plants used in traditional medicine for various ailments. Hence, there is need to screen medicinal plants for promising biological activity. In our study, we choose the leaf portion of Vitex negundo (common name: Sarsa, Samalu, Chasta tree, Nirgundi) to evaluate its biological activity. Vitex negundo (Family-Verbenaceae) chiefly occurring throughout India [2, 3] is widely distributed in Similipal Biosphere Reserve, Orissa. Though, almost all parts of V negundo are used, the leaves and the barks are the most important in the field of medicine [4].

The decoction of leaves is considered as tonic, vermifuge and is given along with long pepper in catarrhal fever [4]. Water extract of mature fresh leaves exhibited anti-inflammatory, analgesic and antihistamine properties [5]. Leaves of this plant have been shown mosquito repellent effects [6] as well as antiulcerogenic [7], antiparasitic [8], antimicrobial [9] and hepatoprotective [10] potentials.

The methanolic root extract possessed potent snake venom (Viper russellii and Naja kaouthia) neutralizing capacity [11]. Satpuda hill region in India, Juice of fresh leaves was applied externally to treat joint pain [12]. In the present Study an attempt has been made to evaluate the anti-arthritic activity of petroleum ether and fresh aqueous crude extract of leaves of Vitex negundo Linn. against formalin induced arthritis in Wister albino rats of either sex.

MATERIAL AND METHODS:

Plants Collection and Authentification

The leaves of Vitex negundo Linn. have been collected from the local area of Nandurbar (Maharashtra). This plant is authentifying by Dr. Santosh K. Tayade, Dept. of Botany, Art’s, Science and Commerce College, Lonkheda, Shahada, Dist-Nandurbar (MS). The voucher specimen has been preserved in the laboratory for future reference.

Plant Extraction:

The leaves were dried at room temperature for 10 days and coarsely powdered with help of a hand-grinding mill and the powdered was passed through sieve no 40. The powered of leaves of Vitex negundo Linn. was extracted separately by continuous hot extraction process using soxhelet apparatus with petroleum ether and macerated with fresh aqueous extract (chloroform: water) [13,14]. Each extract was concentrated in rotary flash evaporator under vacuum and dried over anhydrous sodium sulphate.

Animals:

Wister albino rats (150–250 g each) of either sex and of approximately same age, weighing about 150–250 g were used for the study. They were housed in polypropylene cages and fed with standard diet and water ad libitum. The animals were exposed to alternate cycle of 12 h of darkness and light. Before each test, the animals were fasted for at least 12 h. The experimental protocols were subjected to the scrutinization of the Institutional Animal Ethical Committee and cleared by the same.

Acute Toxicity Studies :

The acute oral toxicity study was carried out as per OECD guidelines. The LD₅₀ cut-off dose was found to be 2000 mg/kg body weight for both the extracts; hence, 1/10^th of LD₅₀ cut-off dose was taken as therapeutic doses (i.e. 200 mg / kg b.w). [15]

Evaluation of Anti-arthritic Activity [16]:

Formalin induced Arthritis model was used to assess the anti-arthritic activity in albino rats. The acclimatized animals were kept fasting for 24 hrs with water ad libitum. Animals were randomly divided into four groups of six animals each (n=6). First group (negative control) received 1ml of normal saline, second group received 13.5mg / kg p.o. Diclofenac sodium, third and fourth group received petroleum ether and fresh aqueous extracts of leaves Vitex negundo Linn.

Arthritis was induced by injecting a 0.1ml of 2% formaldehyde solution in Normal saline to the sub-planter region of left hind paw of weight on the first and third days of the experiment. Drug treatment was started from the initial day i.e. from the day of formaldehyde injection (0 day), continued till 10^th day. The size of hind Paws were measured daily and compared with the controls. Daily change in paw edema was measured by digital Plethysmometer (UGO Basile 7140 Plethysmometer). The percentage inhibitions of paw volume of injected paw over vehicle control were calculated by using following formula. (Control edema was assumed to be 100%)

% edema=N׳×100/N

% edema inhibition = (N-N׳)/N×100

Where,

N= is the edema value of the control group (calculated by subtracting the Paw value measured on the 10^th day from the initial paw diameter measurement).

N׳= is the edema value of the experimental group.

The result of daily change in paw edema, percentage of edema inhibition has been shown in Table 1.

Statistical analysis:

The experimental results are represented as mean ± SEM. The data were statistical analyzed by one-way Anova followed by Dunnet’s test. P values < 0.01 were considered significant value. [17].

RESULT AND DISCUSSION:

The results obtained with petroleum ether and fresh aqueous extracts of leaves of Vitex negundo on formalin induced arthritis rat in left hind paw edema (Table 1). The result indicated that aqueous extract showed more significant (p<0.001) anti-arthritic activity (4.05±0.18) on day 10 of treatment compared to control (5.25±0.05). The result was comparable with reference standard Diclofenac sodium i.e.3.82±0.10. From graph 1, aqueous extract showed 56.75 % edema inhibition, which is comparable with standard i.e.71.89 %. Petroleum ether extracts showed moderate anti-arthritic activity.

The experimental model chosen here is formalin induced arthritis. It is known that inhibition of formalin induced paw edema is on of the suitable procedures to screen anti-arthritic and anti-inflammatory agents, as it closely resembles to human rheumatoid arthritis. [18] The activity linked with presence of alkaloids and saponins in the extract. On preliminary Phytochemical screening of leaves of Vitex negundo showed the presence of alkaloids and saponins as major chemical constituents. Alkaloids was found in many plants have been shown to have good anti-arthritic and anti-inflammatory property.[19]

Thus, this study concludes that aqueous extract of leaves of Vitex negundo has significantly posses anti-arthritic property in rats. Further study is involving the purification of the chemical constituents of leaves of Vitex negundo and investigation on the biochemical pathways may result in the development of a potent anti-arthritic agent with a low toxicity and higher therapeutic index.

Table 1:- Percentage edema inhibition by various extracts of leaves of Vitex negundo Linn.

Sr. No.	Groups	Day-0	Day-10	Difference	% Edema	% Edema Inhibition
1	Control	3.40±0.14	5.25±0.05	1.85	100	-
2	Standard	3.30±0.12	3.82±0.10	0.52	28.10	71.89**
3	Petroleum ether Extract	3.35±0.16	4.23±0.18	0.88	47.56	52.43
4	Aqueous Extract	3.25±0.19	4.05±0.18	0.80	43.24	56.75**

**Highly Significant; p<0.01 (Comparison of control with standard and both extracts)

ACKNOWLEDGEMENT:

Authors are thankful to P. S. G. V. P. M’s College of Pharmacy, Shahada, District- Nandurbar. (M.S) for providing necessary support for research purposed.

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Received on 15.05.2012 Modified on 30.05.2012

Asian J. Research Chem. 5(7): July, 2012; Page 843-845