ANTI-INFLAMMATORY ACTIVITY OF THE FERN, Pyrrosia heterophylla (L.) M.G. Price (Polypodiaceae): LEAF EXTRACTS ATTENUATE LIPOPOLYSACCHARIDE INDUCED INFLAMMATION IN RAW 264.7 MACROPHAGES VIA DUAL COX/LOX INHIBITION

Inflammation involves a tightly regulated immunological process in response to microbial infections, injury and trauma. Sustained, uncontrolled inflammation results in many diseases such as rheumatoid arthritis, inflammatory bowel disease, atherosclerosis and even cancer. Present study was directed at the scientific validation of the traditional use of Pyrrosia heterophylla as an anti-inflammatory agent. RAW 264.7 cells were pretreated with different concentrations of the leaf extracts in hexane (PHH), ethyl acetate (PHE) and methanol (PHM). After one hour the cells were stimulated with lipopolysaccharide (LPS) and after 18 hrs the cells and culture medium were analysed for different markers of inflammation namely cyclooxygenase-2 (COX-2), 5- lipooxygenase (5-LOX), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and cellular nitrite levels. Expression of the most important proinflammatory cytokine TNF-α was estimated through ELISA. All the three extracts exhibited a dose dependent inhibition of the inflammatory mediator enzymes. At the highest tested concentration (100 µg/ml), the extracts PHE, PHM & PHH inhibited COX-2 activity by 73.66 ± 0.86 %; 70.63 ± 1.89 % and 44.84 ± 1.75% respectively; while 5-LOX was inhibited by 63.27 ± 2.17 %, 58.38 ± 2.24 % and 47.45 ± 1.08% respectively with reference to control. The most potent extract, PHE could significantly inhibit the expression of TNF-α in LPS stimulated RAW 264.7 cells. TNF-α level in normal cells (24.13± 2.55 pg/ml) was significantly elevated by LPS treatment (757.61 ± 16.32 pg/ml). Pretreatment with PHE (100 µg/ml) resulted in significant reduction of TNF-α levels (501.13 pg/ml). PHE and PHM exhibited dual inhibition of COX-2 and 5-LOX, two most important enzymes involved in synthesis of prostaglandins and leucotrienes, respectively. Therefore, these two herbal extracts could be utilized for development of herbal formulations as a safer treatment option against inflammatory conditions.