ANTI- INFLAMMATORY ACTIVITY OF VITEX TRIFOLIA LINN. (VERBANEACEAE) LEAVES EXTRACTS

ANTI- INFLAMMATORY ACTIVITY OF VITEX TRIFOLIA LINN. (VERBANEACEAE) LEAVES EXTRACTS

1. A. Kulkarni*

Department of Pharmacology, KLES College of Pharmacy, JNMC Campus, Nehru Nagar, Belgaum-10, Karnataka, India

ABSTRACT

Objectives: The present study was carried out to evaluate the anti-inflammatory effect of extracts of leaves of Vitex trifolia Linn. (Verbaneaceae).

Methods: The anti inflammatory activity of plant extracts at different dose intervals was evaluated by using two models Carrageenan induced rat paw edema and Xylene induced ear edema in Mice. Acute inflammation in rats was induced by injecting Carrageenan (0.1 ml of 1% suspension in 0.9% saline) in subplantar region and topical application of xylene (0.03ml)to the anterior and posterior surface of mice ear. The mean paw volume and percent reduction in ear edema were evaluated.

Results: Significant reduction in Paw volume (P<0.01) and ear edema were observed in all treated groups. Alcoholic extract showed more activity than aqueous extract and also in dose dependent manner.

Conclusion: The study showed that the leaves of Vitex trifolia Linn. (Verbaneaceae) possess an anti-inflammatory activity.

Inflammation

INTRODUCTION: Inflammation is a complex localized response of living mammalian tissue to foreign substances such as bacteria, trauma chemicals and heat. It is characterized by pain, swelling and redness. These characteristic features are brought about by complex actions of various inflammogens like Histamines, Bradykinins, Prostaglandins, and Leukotrines etc. By inhibiting these mediators, the inflammatory response could be suppressed ¹.

However, the use of antagonists of some of these mediators to suppress inhibition may not be feasible clinically. Antihistaminics are rarely used as anti-inflammatory agents probably due to their clinical inefficacy, while NSAIDS are clinically effective but are not completely devoid of adverse effects such as gastric or intestinal mucosal damage ². Vitex trifolia Linn. (Verbaneaceae)  is well known as ‘Pani- ki- Sanbhalu’, ‘Sufed- Sanbhalu’. It is stout aromatic shrub or a small tree, found from the foot of Himalayas southwards throughout greater part of India and in Western ghats ^{3, 4}.

The leaves are oppositely arranged along the stems and are usually compound, composed of 3 linear leaflets which range between 1 -12 cm in length ⁵. Leaves and twig yields 0.11-0.28% of essential oil of spicy odor and flavones ^{6, 7}.

The plant is used as fungicidal, cytotoxic and insecticidal ⁸. Leaves showed inhibitory action against Mycobacterium tuberculosis ⁹ and also used as an anti-inflammatory and sedative for headache, rheumatism and in common cold in Asian countries ⁶. However no scientific data regarding anti-inflammatory effect of said plant is available.

In this study, the anti-inflammatory effect of alcoholic and aqueous extracts of Vitex trifolia leaves was evaluated by using carrageenan induced paw edema in rats; xylene induced ear edema in mice as an inflammatory models.

MATERIALS AND METHODS:

Animals: Swiss Albino Mice of Either Sex (20-25gm) and Male Albino Wistar rats (150-200gm) were housed in a group of 5 in clean polyacrilic cages and were maintained under the natural day night cycle. Animals were acclimatized for one week before experiment.

Plant material: The leaves of Vitex trifolia Linn. were collected from the local areas of Belgaum, and were authenticated from Dr. R. S. Gaudar, Botanist & Head, Department of Botany, R.L.S. Institute, Belgaum. The drug was later powdered and used for the extraction process. The ethanolic extract and aqueous extract of leaves of Vitex trifolia Linn. obtained were used as the test drug for the evaluation of anti inflammatory activity.

Preparation of plant extract: Hundred grams of dried, coarse powdered leaves were continuously extracted for 48 h with 90% ethanol in a Soxhlet’s apparatus. The extract was filtered and concentrated in a rotatory evaporator, at 30-40^oC, to obtain semi-solid material. The viscous residue thus obtained was kept in a vacuum desiccator to obtain a completely dry solid mass. The extract yield was 12 %. Aqueous extract was prepared by maceration process (Yield 8 %). For screening purpose, the leaves extracts were weighed and triturated with tween 80 (1%) and then was suspended in distilled water quantity sufficient to produce a suspension of suitable strength. The extracts were administered at two different doses (100mg/kg b.w, 200mg/kg b.w.p.o.)

Drugs and chemicals: Carrageenan (Himedia Ltd. Mumbai, India), Xylene, and Diclofenac sodium as a reference standard were used in the study.

Methodology:

 Model 1- Carrageenan induced rat paw edema ^{10, 11}: The rats were divided into six groups each containing 5 animals. Group 1 served as Control, Group 2 Standard drug Diclofenac (100 mg/kg) treated, Group 3, 4, 5 & 6 served as treatment with the extracts at two different doses. Acute inflammation in rats was induced by injecting Carrageenan (0.1 ml of 1% suspension in 0.9% saline) in subplantar region and the paw volume was measured at 0, 1, 2 & 3 hrs with Plethysmometer. A mark was put on the leg at the mallelous region to facilitate the dipping of the leg to the same level. The mean paw volume at different time interval was measured and compared to control and the percentage inhibition was then calculated by following formulas:

% Inhibition = (1- Ct/Co) X 100

Where, Co= edema volume in Control group, Ct= edema volume in Drug treated group.

Model 2 - Xylene induced ear edema in Mice ¹²: Mice were divided into six groups, each containing 5 animals. After 30 min of administration of standard drug (i.p) and tests compounds (p.o), 0.03 ml of xylene was applied to the anterior and posterior surface of right ear. Left ear was remained untreated. Mice were killed after 2 hrs of application and both ears were removed. Circular disks were taken by using a cork borer with a diameter of 7 mm and weighed. The increase in weight was measured by subtracting the weight of untreated left ear to that of treated right ear.

Data analysis: Data are expressed as MEAN ± SEM and were analyzed by one way ANOVA followed by Dunnet’s post test for multiple comparisons. P<0.01 was considered as significant.

RESULTS: The result of anti-inflammatory activity of leaves extracts of Vitex trifolia Linn. against carrageenan induced paw edema is shown in Table 1. Paw volume was significantly reduced (P<0.01) in all treated groups as compared to control group. Alcoholic extract at dose 200mg/kg showed more inhibition of edema at 3^rd hr (43%) than other treated groups. However the anti inflammatory activity showed by two different doses of both the extracts were found to be less effective than reference standard compound (59.49%). Alcoholic extract showed more activity than aqueous extract and also in dose dependent manner. The percentage inhibition of edema after 3hr for aqueous extract (100, 200mg/kg), alcoholic extract (100 mg/kg) was found to be 14.55%, 36.07% and 32.91% respectively.

Significant activity by both the extracts was observed against acute inflammation in the xylene ear edema test (Table 2). The alcoholic and aqueous extracts (at 100mg/kg dose) showed 40.74%and 34.56% reduction in the edema respectively. In higher doses (200mg/kg), the aqueous (46.91%) and ethanolic (60.49%) extracts showed significant activity (P<0.0001) against the acute inflammatory response which is less than that of reference standard (70.27%).

TABLE 1: EFFECT OF VARIOUS TREATMENTS ON CARRAGEENAN INDUCED PAW EDEMA IN RATS AT DIFFERENT TIME INTERVAL

N=5, Data expressed as MEAN±SEM. One way ANOVA followed by Dunnet’s test. **P<0.01, ***P<0.0001 Vs Control

TABLE 2: EFFECT OF VARIOUS TREATMENTS ON XYLENE INDUCED EAR EDEMA IN MICE

Values are expressed as MEAN ± SEM. Student's t-test (N = 5). **P<0.01, ***P<0.0001 Vs Control

DISCUSSION: The plant extracts of Vitex trifolia Linn. were studied against Carrageenan induced rat paw edema and Xylene induced ear edema formation in mice. Carrageenan induced paw edema is the most widely used screening method for anti-inflammatory agents¹³.Carrageenan is said to mediate its action through the mobilization of PG synthesis. There are three distinct phases in edema, namely an initial phase (Histamine release), a second phase mediated by Kinins and finally third phase mediated by Prostaglandins ¹⁴. Xylene is used as a pholgestic agent in the present study because Xylene causes instant irritation to mouse ear which leads to edema and fluid accumulation which are responsible for acute inflammation and the suppression of these indicates antiphlogestic effect ¹⁵.

In the present study, we observed that plant extracts significantly reduced paw edema after 3^rd hr, alcoholic extract being more prominent than aqueous at two different doses. This suggests that the extracts modify the effects of prostaglandins which are released into third phase of inflammation. The extracts also showed antiphlogestic effect in xylene induced ear edema in mice. Diclofenac sodium is used as a reference standard. The drug is one of the most extensively used NSAID’s which acts by inhibiting prostaglandin synthesis ¹⁶.

The preliminary phytochemical investigation showed the presence of Carbohydrates, Phenolic compounds, Flavonoids and tannins. It could be suggested that the anti-inflammatory effects of leaves extracts may be due to presence of flavonoids and tannins. Other studies have demonstrated that various flavonoids such as rutin, quercetin, luteolin, and hesperidin produced significant anti-inflammatory activity ^{17, 18}.

Flavonoids inhibit both inflammatory and allergic reaction as well as offer some protection in ulcer development by increasing capillary resistant and improving microcirculation which renders the cell less injurious to precipitating factors ¹⁹.

CONCLUSION: These findings concluded that the leaves of Vitex trifolia Linn.(Verbaneaceae) possess anti-inflammatory activity which is due to the presence of flavonoids, which may derive from inhibition of prostaglandins synthesis, inhibition of increased vascular permeability, which may enhance tissue repair and healing.

ACKNOWLEDGEMENT: I would like to extend my sincere gratitude to KLE college of Pharmacy, Belgaum, for providing all the laboratory facilities to conduct the proposed work.

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