Isolation and Purification of Anti-inflammatory Compounds from Needles of Pinus densiflora

Abstract

Pinus densiflora sieb. et zucc. contains several phenolic compounds that have various biological activities such as antimicrobial, antioxidant and antihypertension. However, the health beneficial effects of these compounds have rarely been reported. Inflammatory response protects the host against tissue injury, microbial invasion but uncontrolled inflammation cause disease such as cardiovascular disease, autism, cancer and autouimmune disease. RAW264.7 murine macrophage cell line play a major role in inflammatory response. In this study, we seperated n-Hexane, EtOAc n-BuOH and H2O layer from methanolic extract of pinus densiflora and tested anti-inflammation activity on LPS-induced inflammation in RAW264.7 murine macrophage cell line. EtOAc layer was not cytotoxic in RAW 264.7 and nitric oxide (NO) production decreased in LPS-stimuated RAW264.7 more than other layers. We fractionated EtOAc layer by silica gal (63-200um) open column chromatography and eluted with gradient mixtures of methylen chloride-MeOH (70:1 to 0:1) to yield nine fractions. We tested cytotoxicity, anti-inflammation activity. Most of activity fraction 5 (F.5) treatment reduced mRNA level of cytokines (IL-6) and pro-inflammatory genes such as iNOS and COX-2 in LPS-stimulated macrophage activation in a dose-dependent manner.