Identification of anti-inflammatory fraction & isolation of active compound formononetin from root of Astragalus membranaceus

The present study aimed to identify active fraction(s) and compound(s) from AR (root of Astragalus membranaceus) aqueous extract that are responsible for the anti-inflammatory effect using in vitro bioassay-guided fractionation. The effect was monitored by inhibition of nitric oxide (NO) released from lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells after treated with AR extract, sub-fraction or isolated component(s). Two active fractions (P2–3-2–2-2 and P2–3-2–2-3) were found to significantly inhibit NO production. Formononetin was isolated from P2–3-2–2-3 and was shown to inhibit NO production. Besides, the anti-inflammatory effect of combined fraction (P2–3-2–2-2 and P2–3-2–2-3 in 1:1 ratio) was evaluated by measuring (i) release of prostaglandin E₂ (PGE₂)_, interleukins (IL-1β, IL-6) and tumor necrosis factor (TNF-α) using ELISA; (ii) expression of inducible nitric oxide synthase (iNOS) by RT-PCR; (iii) expression of iNOS, cyclooxygenase-2 (COX-2) and mitogen-activated protein kinase (MAPK) pathway by Western blot; (iv) activation of nuclear factor kappa B (NFκB) translocation by electrophoretic mobility shift assay. The combined fraction could significantly inhibit LPS-induced expression of iNOS, COX-2, MAPK and NFκB, as well as production of PGE₂, IL-1β, IL-6 and TNF-α. In conclusion, two major anti-inflammatory AR fractions were identified and formononetin was one of the active components.