~ 183 ~ Journal of Pharmacognosy and Phytochemistry 2014; 3 (1): 183-189 ISSN 2278-4136 ISSN 2349-8234 JPP 2014; 3 (1): 183-189 Received: 22-03-2014 Accepted: 20-04-2014 G.S.Chhabra School of P harmacy & Technology M anagement, SV K M ’ s NM IM S, Shirpur, India. Email: gurmeetchhabra@ gmail.com Tel: + 91-9764442450 K.S.Kulkarni SP P School of Pharmacy and Technology Management, SVKM’s NMiMS, Mumbai, India Email: kalakulkarni@ gmail. com Tel: 09967689527 Correspondence: Gurmeet S.Chhabra School of Pharmacy and Technology Management, SVKM’ s NMiMS (Shirpur Campus), Babulde, Bank of Tapi River, Mumbai-Agra Road, Shirpur, Dist. Dhulia, Maharashtra- 425405, India. Email: gurmeetchhabra@ gmail.com Tel: + 91-9764442450 Evaluation of anti-inflammatory activity of Vitex agnus castus leaves. Quantitative analysis of flavonoids as possible active constituents. G.S.Chhabra, K.S.Kulkarni ABSTRACT Vitex agnus-castus L., (Verbenaceae) traditional Chinese medicinal plant recommended for various inflammatory ailments, but its mechanism of anti-inflammatory action is still unclear. The present study reports the anti-inflammatory activities of various extracts and elucidate possible mechanism(s) with the quantification of flavonoids by RP-HPLC. The petroleum ether, ethyl acetate, methanol and aqueous extracts obtained by successive solvent extraction from Vitex agnus castus leaves were subjected to standard acute, subacute and chronic models of inflammation at 200 and 400 mg/kg. Further, in vitro cytokine release estimation (IL-6 and TNF-α) using ELISA and the identification and quantification of major constituents with the RP-HPLC was done. All extracts (200 and 400 mg/kg) produced significant inhibition while methanol extract (400 mg/kg) caused a maximum inhibition of 43% in paw edema, 75% inhibition in subacute and 59.28% inhibition in the chronic inflammation model. Methanol extract (400 mg/kg) showed 77.87% and 63.34% decline in the IL-6 and TNF-α levels respectively. Kaempferol (0.24%) and luteolin (0.12%) were quantified in a bioactive methanol extract using HPLC analysis. It was concluded that the anti-inflammatory activity might be due to flavonoids in the extracts and plant regulates the inflammation by a significant decrease of TNF-α and IL-6 by macrophages, which mediates crucial events for the initiation. Information revealed that the methanol extract of Vitex agnus castus leaves can be used as effective anti-inflammatory agent. Keywords: Granuloma pouch, IL-6, TNF-α, ELISA, leaf extracts, acute toxicity, cotton pellet, inflammation, acute toxicity, carragennan, kaempferol, luteolin, p-hydroxybenzoic acid, HPLC, Cyclooxygenase, Lipooxygenase, Premenstrual syndrome. 1. Introduction Inflammation is a process involving multiple factors acting in a complex network. The ingress of leukocytes into the site of inflammation is crucial in the pathogenesis of inflammatory conditions. Neutrophils and macrophages are known to recruit and play pivotal roles in acute and chronic inflammation, respectively [1] . Recruited cells are activated to release many inflammatory responses, causing a change from the acute phase of inflammation. Therefore, inhibition of the cellular reactions is one of the targets that are generally used as an in vitro model for anti-inflammatory testing. Inflammation plays an important role in a wide variety of chronic human diseases such as cardiovascular diseases and cancer. It has been demonstrated that pro-inflammatory cytokines, cyclooxygenase-2 (COX-2) and free radical species interact in a complex manner in an inflammatory environment [2] . For example, tumor necrosis factor-α (TNF-α) has been shown to be one of the major cytokines that mediates many crucial events in the initiation of both acute and chronic inflammation through regulating production of some other cytokines, up-regulation of adhesion molecule expression, and the activation of leukocyte- specific chemotactic cytokines [3] . Interleukin-6 (IL-6) is another pro-inflammatory cytokine that promotes inflammatory events through the activation and proliferation of lymphocytes, differentiation of B cells, leukocyte recruitment and the induction of the acute-phase protein response in the liver [4] . Inhibition of the expression and production of these powerful mediators by anti-inflammatory components might represent a possible preventive or therapeutic target, and may be used to develop anti-inflammatory nutraceuticals for health promotion and disease prevention.