갈근의 항염증 및 항산화 활성성분

Abstract

The Pueraria lobata (Willd.) Ohwi (Leguminosae) is widely distributed in temperate regions of Far Eastern Asia including Korea, Japan, China, and India and is one of the earliest and most important edible crude herbs used in traditional Oriental medicine as a muscle relaxant, antipyretic, and antidysenteric, and for the treatment of cardiovascular diseases. It is popular for consumption in the treatment of hypertension and alcoholism with antioxidant and has shown anti-dipsotropic activities. The genus Pueraria contains high amounts of isoflavonoids, especially puerarin and daidzein. Isoflavonoids exhibit a wide range of biological activities; they have anti-inflammatory, antithrombotic, antihypertensive, antiarrhythmic, spasmolytic, and cancer chemopreventive properties. It is well known that the beneficial effects on health of isoflavonoids are due to their antioxidant and phytoestrogenic properties. In this study, anti-inflammatory and antioxidant activities of the methanolic (MeOH) extract and its soluble fractions, such as the n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (n-BuOH) and water (H2O) from P. lobata roots were investigated. Intracellular anti-inflammatory capacities were determined via inhibitory activities of lipopolysaccharide (LPS)-induced nitric oxide (NO) production, as well as expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 cells. Antioxidant activities were evaluated by in vitro scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH), peroxynitrite (ONOO-), nitric oxide (NO•), superoxide anion (•O2-), and reactive oxygen species (ROS), as well as inhibitory activities against ONOO--mediated tyrosine nitration. In the antioxidant assays, the MeOH extract exhibited scavenging activities of DPPH and ONOO- with IC50 values of 83.30 and 58.61 μg/ml, respectively. Among the tested fractions, the EtOAc fraction exhibited highest scavenging activities in both DPPH and ONOO- assays with respective IC50 values of 7.90 and 37.31 μg/ml followed by the n-BuOH fraction, of which corresponding respective IC50 values of 41.55 and 54.69 μg/ml. Although the CH2Cl2 fraction showed DPPH radical scavenging activity with an IC50 value of 31.05 μg/ml, it showed weak ONOO- scavenging activity with an IC50 value of 117.51 μg/ml. Conversely, the H2O fraction showed no activities in both of the antioxidant assays at the concentration tested. In the anti-inflammatory assay, the MeOH extract showed inhibition of LPS-induced NO production with an IC50 value of 31.80 μg/ml in RAW 264.7 cells. The n-hexane and EtOAc fractions exhibited higher NO inhibitory activities over other fractions with respective IC50 values of 5.88 and 0.13 μg/ml, and n-BuOH fraction showed weak NO inhibitory activity (IC50 = 101.64 μg/ml) in LPS-stimulated RAW 264.7 cells. Although, the EtOAc fraction having numerous isoflavonoids such as daidzin, daidzein, genistin, and genistein, exhibited strong NO inhibitory activity, it is well known that isoflavonoids might attribute to inhibition of NO production, thus detailed phytochemical investigations and anti-inflammatory activity studies on the n-hexane fraction were carried out. Repeated column chromatography of the n-hexane fraction was accomplished to yield lupenone, lupeol, puerarol, coumestrol, and glyceryl-1-tetracosanoate. These compounds were identified by direct comparisons of their spectral data with the reported ones. Among these compounds, lupenone and lupeol reduced NO production (IC50 value = 10.81 and 64.65 μM, respectively) as well as iNOS and COX-2 expression in LPS-stimulated RAW 264.7 cells. Furthermore, lupeol showed significant inhibitory activity against intracellular ROS generation by tert-butylhydroperoxide (t-BHP) (IC50 = 122.48 μM). Meanwhile, bioactivity-guided isolation of the antioxidant active the n-BuOH fraction resulted in the isolation of allantoin, 3'-hydroxypuerarin, 3'-methoxypuerarin, daidzein 8-C-apiosyl-(1→6)-glucoside, daidzin, genistin, and ononin. The structural identification of these compounds was performed by spectroscopic analysis. Among them, 3'-hydroxypuerarin demonstrated marked ONOO-, NO• and total ROS scavenging activities (IC50 = 1.36, 1.13, and 6.51 μM, respectively), and weak •O2- scavenging activity (IC50 = 211.66 μM). Moreover, 3'-hydroxypuerarin showed inhibitory activity of ONOO--mediated tyrosine nitration. On the other hand, 3'-methoxypuerarin showed ONOO- scavenging activity (IC50 = 1.94 μM) and weak NO•, •O2- scavenging activities (IC50 = 68.43 and 247.35 μM, respectively). These results suggest that the existence of 3'-hydroxyl group in puerarin is proved to play an important role in the scavenging of ONOO-, NO•, and total ROS as well as inhibiting of 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.