Multi-constituent synergism is responsible for anti-inflammatory effect of Azadirachta indica leaf extract.

Azadirachta indica A. Juss. (Meliaceaes) leaves have been used traditionally to treat swelling and rheumatism in Indian cultures. To fractionate A. indica leaf extracts using bioactivity guided manner for identification of the active anti-inflammatory principles. Polarity-gradient sequential extracts (petroleum ether, chloroform, methanol, and water) of A. indica leaves were screened for their anti-inflammatory potential using the carrageenan-induced rat paw edema model (1 g/kg). The chloroform extract was sequentially fractionated to obtain n-hexane (F-1), n-hexane-chloroform (F-2), and chloroform (F-3) fractions and their inhibitory effect on rat paw edema was evaluated (500 mg/kg). Inhibitory effect of F-2 on granuloma formation, plasma interleukin (IL-1), and tumor necrosis factor (TNF-α) was assessed at the doses of 100, 200, and 400 mg/kg using the cotton pellet assay in rats. Three sub-fractions (SF-1, SF-2, and SF-3) were obtained upon chromatography of F-2, and their inhibitory effect on cyclooxygenase was assessed at 200 µg/mL concentration. The sub-fractions were subjected to gas chromatography-mass spectrometry (GC-MS). All the extracts showed significant anti-inflammatory effect; however, chloroform extract was the most effective against paw edema (53.25% inhibition). The three fractions of chloroform extract showed significant effect, while F-2 being the most potent (51.02%). F-2 demonstrated dose-dependent inhibition of granuloma and cytokines. Interestingly, all the sub-fractions of F-2 inhibited COX-1 and COX-2 with almost equal potential. GC-MS revealed that chemically the sub-fractions were totally different from each other. Anti-inflammatory effect of A. indica is a result of cumulative and synergistic effects of diversified constituents with varying polarities that collectively exert the effect via suppression of cyclo-oxygenases and cytokines (IL-1 and TNF-α).