Anti-inflammatory and antioxidant activities of constituents isolated from Pueraria lobata roots

In order to evaluate the anti-inflammatory and antioxidant activities of Pueraria lobata roots and its active components, in vitro inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) protein expression, and tert-butylhydroperoxide (t-BHP)-induced reactive oxygen species (ROS) generation in RAW 264.7 cells, as well as in vitro scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH), peroxynitrite (ONOO-), nitric oxide (NO center dot), superoxide anion (center dot O-2 (-)) and total ROS, and inhibitory activities against ONOO--mediated tyrosine nitration, were determined. Repeated column chromatography was performed to isolate four known compounds from the anti-inflammatory and antioxidant EtOAc fraction: daidzein; genistein; puerarin; (+)-puerarol B-2-O-glucopyranoside; four known compounds from the anti-inflammatory n-hexane fraction: lupenone; lupeol; puerarol; coumestrol; seven known compounds from the antioxidant n-BuOH fraction: allantoin; 3'-hydroxypuerarin; daidzein 8-C-apiosyl-(1 -> 6)-glucoside; puerarin; genistin; 3'-methoxypuerarin; daidzin. Among these compounds, lupenone and lupeol reduced NO production, as well as iNOS and COX-2 protein levels in LPS-stimulated RAW 264.7 cells. Furthermore, lupeol showed significant inhibitory activity against intracellular ROS generation by t-BHP. Meanwhile, 3'-hydroxypuerarin showed marked ONOO-, NO center dot, total ROS scavenging activities, and weak center dot O-2 (-) scavenging activity, while 3'-methoxypuerarin showed ONOO- scavenging activity and weak NO center dot and O-2 (-) scavenging activities, suggesting that existence of the 3'-hydroxyl group in puerarin plays an important role in the scavenging of ONOO-, NO center dot, and total ROS, as well as inhibiting the ONOO--mediated tyrosine nitration mechanism. These results indicate that P. lobata roots and its constituents may be a useful therapeutic and preventive approach to various inflammatory diseases and oxidative stress-related disease.