Anti-Inflammatory Activity of Extracts of Leaves of Hygrophila spinosa T. Anders in Chronic Animal Models of Inflammation

Hygrophila spinosa T. Anders (Acanthaceae) is traditionally used in Indian medicine for the treatment of microbial infections, liver diseases, cancer, inflammation, rheumatism, diabetes, pain, fever etc. The aim of the present study is to evaluate the anti-inflammatory activity of chloroform and alcoholic extracts of the leaves of H. spinosa in chronic inflammation models in rats as our previous study revealed that these two extracts had anti-inflammatory activity in carrageenan induced paw oedema model. Anti-inflammatory activity was evaluated by cotton pellet-induced granuloma and Freund’s adjuvant-induced arthritis in rats. Antioxidant activity of the extracts was revealed by their scavenging activity of the stable 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical and a flavonoid compound (apigenin) was also isolated and characterized from the alcoholic extract of the plant. Chloroform and alcoholic extracts showed anti-inflammatory activity both in cotton pellet-induced granuloma and Freund’s adjuvant-induced arthritis in a dose dependent manner. The decrease in body weight due to injection of CFA was improved significantly by the above two extracts also. Both the extracts also exhibited antioxidant activity. The results demonstrated that H. spinosa has antiinflammatory activity in chronic models of inflammation which support the traditional use of H. spinosa in the treatment of rheumatism.


Plant material
H. spinosa plants were collected from Berhampur, Orissa, India and botanical identification was done through Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi (Voucher no. BITPcog. 463/07-08). Voucher specimen was preserved in the department for further verification.

Preparation of different extracts
The leaves were washed thoroughly, dried under shade and pulverized. The coarse powder was extracted successively with petroleum ether, chloroform and alcohol using soxhlet apparatus. The extracts were dried using a rotary vacuum evaporator and stored in a desiccator until further use.

Cotton pellet-induced granuloma in rats
Cotton pellet-induced granuloma in rats is used to screen drugs preventing transudative, exudative and proliferative components of chronic inflammation (Winter et al., 1957;Mizushima et al., 1972;Spector, 1969). Granuloma was induced by subcutaneous implant of a sterile cotton pellet (50 mg) in the dorsal area of anaesthetized rats. One day after the implant, the animals were divided into six groups (n=6) and were administered with vehicle/standard drug/test substances as mentioned in Table 1 for 10 days. On the 10 th day the pellets were dissected out, dried in an incubator at 60 0 C until a constant weight was obtained. The granuloma tissue formation (dry weight of granuloma) was calculated after deducting the weight of cotton pellet (50 mg) from the constant dry weight of pellet and taken as a measure of granuloma tissue formation. The anti inflammatory effect was assessed by degree of inhibition of the granuloma weight in the groups under study as compared with the control. The granuloma inhibition percentage for each group was calculated by comparison with the control group considered as 100% of inflammatory activity.

Freund's adjuvant-induced arthritis in rats
Rats with an initial body weight of 150-200 gm are used in the study. On day 1, they are injected into the sub plantar region of the left hind paw with 0.1 ml of complete Freund's adjuvant (CFA, Sigma) (Wei et al., 1986). Dosing with the test compounds or the standard is started on the same day and continued for 12 days. Paw volumes of both sides and body weight are recorded on the day of injection, whereby paw volume is measured plethysmographically. On day 5, the volume of the injected paw is measured again, indicating the primary lesion and the influence of therapeutic agents on this phase (Newbould, 1963). From day 13 to 21, the animals are not dosed with the test compound or the standard. The severity of the induced adjuvant disease is followed by measurement of the non injected paw (secondary lesions) with a plethysmometer (Walz et al., 1971). The body weight was recorded at a weekly interval upto 21 days. On day 21, the severity of the secondary lesions is evaluated visually by observing redness/inflammation/nodules in hind paws, fore paws, ears, nose and tail (Vogel, 1997

Statistical analysis
The results were expressed as mean ± standard error mean (SEM). Statistical analysis of the data was carried out using one way analysis of variance (ANOVA) followed by Student's t-test to determine the significant difference between the control and the treated groups. P < 0.05 was considered significant.

Cotton pellet-induced granuloma
The percentage inhibition of granuloma formation by chloroform and alcoholic extract (400 mg/kg) were 24.09 and 35.69% respectively (p<0.05) ( Table 1). Hence the decrease in granuloma weight indicated the anti-inflammatory activity of alcoholic and chloroform extracts of H. spinosa in the treatment related to various types of inflammatory disorders.

Freund's adjuvant-induced arthritis
Injection of complete Freund's adjuvant into the rat paw induces inflammation as primary lesion with a maximum after 3 to 5 days. Secondary lesions occur after a delay of approximately 11 to 12 days which are characterized by inflammation of non-injected sites (hind leg, forepaws, ears, nose and tail), a decrease of weight and immune responses. The decrease in body weight due to injection of CFA was improved significantly by chloroform and alcoholic extracts of H. spinosa (Fig. 1). Further, there was significant inhibition of oedema both after 5 days (only by alcoholic extract at 400 mg/kg dose) and 21 days (by both chloroform and alcoholic extract) of CFA administration ( Table 2). The percentage inhibition of paw oedema by chloroform and alcoholic extract (400 mg/kg) after 21 days of CFA treatment was 22.72 and 29.54% respectively. Further, the extent of inhibition by both the extracts was more than the standard drug, indomethacin (20.45%). The severity of secondary lesions was also decreased by both the above extracts. These findings confirm that the H. spinosa can be used for the treatment of chronic inflammation and arthritis.   Both the extracts exhibited antioxidant activity in DPPH method and the potency of different extracts are in the following order: alcoholic>chloroform (Table 3).

Discussion
The cotton pellet granuloma method has been widely employed to assess the transudative, exudative and proliferative components of chronic inflammation (Spector, 1969).
The fluid absorbed by the pellet greatly influences the wet weight of the granuloma and the dry weight correlates well with the amount of granulomatous tissue formed (Swingle and Shideman, 1972). Monocyte infilteration and fibroblast proliferation rather than neutrophil infilteration and exudation take place in chronic inflammation (Dunne, 1990). Efficacy of anti-inflammatory agents in chronic inflammatory states is indicated by their ability to inhibit the increase in the number of fibroblasts during granular tissue formation (Gupta et al., 2003). We found a dose- In summary, our results suggest that H. spinosa has considerable potency in antiinflammatory action and has prominent effects on adjuvant-induced arthritis by alleviating paw edema. Hence, the results of the present study support the traditional use of H. spinosa in the treatment of rheumatism.