Evaluation of anti-inflammatory and immunomodulatory effects of Premna integrifolia extracts and assay-guided isolation of a COX-2/5-LOX dual inhibitor.

Premna integrifolia (Agnimantha brihat) is a traditional medicinal plant with a prominent place in Ayurveda, Siddha and Unani systems of medicine. In this study we have evaluated the anti-inflammatory and immunomodulatory properties of the Premna integrifolia root extracts employing cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and 5-lipoxygenase (5-LOX) enzyme-based assays, lymphocyte proliferation assay, pro-and anti-inflammatory cytokines measurement. Petroleum ether extract (PEE) of Premna integrifolia showed potent inhibition of COX-2 and 5-LOX with IC50 values of 6.15 µg/mL and 11.33 µg/mL respectively. In in vitro studies on RAW 264.7 cell line, PEE showed inhibition in the formation of nitric oxide (NO), pro-inflammatory cytokines (IL-1ß, IL-6), prostaglandin E2 (PGE2) production, induction of anti-inflammatory cytokine (IL-2) and down-regulation of expression of COX-2, 5-LOX, TNF-α, IL-1ß and iNOS. PEE also significantly reduced carrageenan-induced paw edema in mouse model of inflammation. Further, attempts in isolating the active principle(s) involved in these anti-inflammatory effects of PEE by separation on RP-HPLC resulted in the isolation of four active peaks, H1, H2, H3 and H5, inhibiting COX-1, COX-2 and 5-LOX, out of which H3 was identified as 6- hydroxy salvinolone (6-HS). Present findings reveal that PEE of roots of Premna integrifolia exhibits potent anti-inflammatory and immunomodulatory activities, which could form a potential source for development of anti-inflammatory drugs. 6-HS, a COX-2/5-LOX dual inhibitor along with other lead molecules isolated from PEE of Premna integrifolia may form lead molecules for the development of COX-LOX dual inhibitors.

Kinetics and docking studies of a COX-2 inhibitor isolated from Terminalia bellerica fruits.

Triphala is an Ayurvedic herbal formulation consisting of equal parts of three myrobalans: Terminalia chebula, Terminalia bellerica and Emblica officinalis. We recently reported that chebulagic acid (CA) isolated from Terminalia chebula is a potent COX-2/5-LOX dual inhibitor. In this study, compounds isolated from Terminalia bellerica were tested for inhibition against COX and 5-LOX. One of the fractionated compounds showed potent inhibition against COX enzymes with no inhibition against 5-LOX. It was identified as gallic acid (GA) by LC-MS, NMR and IR analyses. We report here the inhibitory effects of GA, with an IC(50) value of 74 nM against COX-2 and 1500 nM for COX-1, showing ≈20 fold preference towards COX-2. Further docking studies revealed that GA binds in the active site of COX-2 at the non-steroidal anti-inflammatory drug (NSAID) binding site. The carboxylate moiety of GA interacts with Arg120 and Glu524. Based on substrate dependent kinetics, GA was found to be a competitive inhibitor of both COX-1 and COX-2, with more affinity towards COX-2. Taken together, our studies indicate that GA is a selective inhibitor of COX-2. Being a small natural product with selective and reversible inhibition of COX-2, GA would form a lead molecule for developing potent anti-inflammatory drug candidates.