Introduction

Premna serratifolia Linn., (Verbenaceae) is an important plant belonging to the family Verbenaceae, and is one of the most widespread large shrubs in the forests of India, usually occurring in deciduous forests. The whole plant possesses medicinal properties, useful in the treatment of cardiovascular diseases, skin diseases, inflammatory diseases, arthritis, gonorrhea, rheumatism, anorexia and jaundice. It is an important Ayurvedic medicinal herb and its synonym is Premna integrifolia. It is popularly known as “Munney” in Tamil, and “Agnimantha” in Ayurvedic system of medicine. Root forms an ingredient in well known Ayurvedic formula-tion “Dasamula” which is used for variety of affections (1). It is widespread throughout Micronesia and much of the tropical Pacific and tropical Asia. It is common along the Indian and Andaman coasts. Infusion of the leaves is administered with pepper in cold and fever. Leaves are used to cure "weakness of limbs" and the leaves and leaf sap were used to alleviate headache (2).
Premna serratifolia Linn., has cardiotonic (3), anti-coagulant (4), anti-inflammatory (5), anti hyperglycaemic (6), anti-parasitic (7), antioxidant (8) and antimicrobial (9) properties. Most of the plant parts of Premna serratifolia Linn., have been used in the traditional system of medicine in India to treat various infectious diseases. Adjuvant induced arthritis is a chronic crippling, skeleton-muscular disorder having nearest approximation to human rheumatoid arthritis for which there is presently no medicine available effecting a permanent cure. The modern drugs both steroidal and non-steroidal anti-inflammatory drugs are used for the amelioration of the symptoms of the disease, however they offer only temporary relief and also produce severe side effects (10). In the Ayurvedic system of medicine, this plant has been used in the treatment of rheumatoid arthritis (1).
Literature survey revealed that there is no systematic study regarding the anti-arthritic activity of the wood of Premna serratifolia. Hence in the present study, an attempt has been made to evaluate the anti-arthritic activity of the ethanol extract of wood of Premna serratifolia Linn., using adjuvant induced arthritis in rats.

Materials and Methods

Plant material
Fresh wood (without bark) of Premna serratifolia Linn., was procured from The Indian Medical Practitioners Cooperative Pharmacy and Stores (IMPCOPS) garden, Thiruvanmiyur, Chennai, Tamil Nadu. The plant was identified (11), authenticated by Botanist, Dr. P. Jayaraman, Plant Anatomical Research Centre (PARC), Tambaram, Chennai and the voucher specimen (PARC/ 2007/ 71) have been kept in the Department of Pharmacognosy, Madras Medical College, Chennai, for future reference. Care was taken to select the healthy plants and for normal organs.

Extraction
The freshly collected wood was chopped, shade dried and coarsely powdered (40 mesh size). The powder was defatted with petroleum ether (60 - 80 ?C) and then extracted with 90% ethanol in a Soxhlet extractor. The extract was dried under reduced pressure using a rotary vacuum evaporator and the percentage yield was 7.90% w/w. The obtained ethanol extract was suspended in 5% gum acacia for the pharmacological screening.

Phytochemical screening
Phytochemical screening (12) of the extract was performed using the following reagents and chemicals: Alkaloids with Dragendorff’s reagent, flavonoids with magnesium and hydrochloric acid, tannins with ferric chloride and potassium dichromate, Trim-hill test for iridoid glycosides, Libermann-Burchard test for steroids and for phenolic compounds with ferric chloride.

Animals
For acute toxicity and anti-arthritic activities, Wistar albino rats weighing between 150 - 200 gm were selected. The animals were acclimatized to the standard laboratory conditions (temperature 25±2 ?C) and maintained on 12 hr light, 12 hr dark cycle. The animals were fed with standard diet and water ad libitum. The animals were maintained as per the norms of CPCSEA and the experiments were cleared by CPCSEA and the institutional ethics committee.

Acute toxicity studies
Acute toxicity study was performed for methanol extract according to the acute toxic classic method as per OECD guidelines (13). Female albino rats were used for acute toxicity study. The animals were kept fasting for overnight providing only water, after which the extract was administered orally at the dose of 300 mg/kg and observed for 14 days. If mortality was observed in two animals out of three animals, then the dose administered was assigned as toxic dose. If the mortality was observed in one animal, then the same dose was repeated to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher doses such 50, 200 and 2000 mg/kg body weight. The animals were observed for toxic symptoms such as behavioral changes, locomotion, convulsions and mortality for 72 hr.

Anti-arthritic activity
Freud's adjuvant induced arthritis (14) model was used to assess the anti-arthritic activity in albino rats. Animals were divided into three groups of six animals each. Group I served as control, which received 5% gum acacia suspension, Group II served as reference standard, which received 10 mg/kg body weight IP of indomethacin, and Group III served as test, which received the ethanol extract of Premna serratifolia Linn., wood at the dose of 300mg/kg body weight PO, respectively.
Arthritis was induced by injecting 0.05 ml of suspension of killed Mycobacterium tuberculosis bacteria (0.5% w/w) homogenized in liquid paraffin into the left hind paw. Drug treatment was started from the initial day i.e. from the day of adjuvant injection (0 day), 30 min before adjuvant injection and continued till 21st day. Paw volume was measured on 4th, 8th, 14th and 21st day with the help of Plethysmometer.
The mean changes in injected paw edema with respect to initial paw volume, were calculated on respective days and percentage inhibition of paw edema with respect to untreated group (control) was calculated using the formula: Percentage inhibition of paw edema = [1- (mean change in paw volume of treated rat/ mean change in paw volume of untreated rat)] x 100.

The changes in body weight were recorded daily. On the 22^nd day, blood was withdrawn through retro-orbital vein puncture of all groups by anaesthetizing the animals with diethyl ether and the biochemical parameters such as hemoglobin content, total WBC count, ESR and RBC were analyzed.

Statistical analysis

Results were expressed as mean ± SD. The significance of difference among the groups was assessed using One way analysis of vari-ance (ANOVA) followed by Dunnet's test. P<0.05 was considered significant.

Result

The results of the preliminary phytochemical screening of the ethanol extract of Premna serratifolia Linn., revealed the presence of phytoconstituents such as alkaloids, steroids, flavonoids, phenolic compounds, tannins and glycosides specifically iridoid glycosides.
In acute toxicity studies, the ethanol extract of Premna serratifolia Linn., did not produce any toxic symptoms or mortality up to the dose level of 2000 mg/kg body weight in rats, and hence the extract was considered to be safe and non-toxic for further pharmacological screening. In adjuvant induced arthritis model, rats developed a chronic swelling in multiple joints with the influence of inflammatory cells, erosion of joint cartilage and bone destruction and remodeling.
These inflammatory changes ultimately result in the complete destruction of joint integrity and functions in the affected animal (15). The ethanol extract inhibited the rat paw edema by 68.32%, which is comparable with standard drug, indomethacin 74.87% inhibition of rat paw edema after 21 days (Tables 1 and 2). As shown in table 3 standard drug, indomethacin and the ethanol extract have shown to increase the hemoglobin content when compared to control group. The total WBC counts were remarkably increased in adjuvant-induced rats (Table 3-control group).
However, Premna serratifolia Linn., wood ethanol extract and the standard drug treated groups significantly decreased (p<0.01) the total WBC count. The ESR count, which drastically increased in arthritic control group, has been remarkably counteracted by the standard drug; indomethacin and ethanol extract, restoring it back to normal, thus justifying its significant roles in the severe arthritic conditions. The loss of body weight observed during the arthritis condition, which is in the arthritic control group, the standard drug and the ethanol extract treatment has significantly increased the body weight further confirming the significant anti-arthritic activity of the ethanol extract of Premna serratifolia Linn., wood and it is shown in the table 4.

Discussion

In the present study, the rats were selected to induce arthritis because rats develop a chronic swelling in multiple joints with the influence of inflammatory cells, erosion of joint cartilage and bone destruction. It has close similarities to human rheumatoid disease (16). The determination of rat paw swelling is apparently simple, sensitive and one of the quick procedures for evaluating the degree of inflammation and the therapeutic effects of drugs. The chronic inflammation involves the release of number of mediators like cytokines, GM-CSF, interferons and PGDF. These mediators are responsible for the pain, destruction of bone and cartilage that can leads to severe disability (17). However, the standard drug, indomethacin and the ethanol extract significantly suppressed the swelling of the rat paws.