결명자의 안지오텐신 변환 효소 저해작용과 그 안트라퀴논 성분

Abstract

The renin-angiotensin system plays a relevant role in the regulation of an organism's water, electrolytes and blood. Angiotensin Converting Enzyme (ACE) is a zinc-containing enzyme, and playsan important physiological role in regulating blood pressure. This enzyme increases blood pressure by hydrolyzing the decapeptide angiotensinⅠto potentvasoconstrictor, angiotensin Ⅱ, which exercise a powerful vasoconstrictive action and stimulates the secretion of aldosterone, which promotes sodium and water retention in the kidneys and the consequent increase in artery pressure. Therefore, inhibition of ACE results in an overall anti-hypertensive effect. As food and natural herbal plants are rich sources of bioactive chemicals, and are mostly free from harmful side effects. The objective of this study was investigate the inhibition of ACE activity by Cassia tora which consumed as tea in Korea. The dried or roasted seeds of Cassia tora L. (Leguminosae) are used in Chinese herbal medicine to improve vision and it is also reputed for its medicinal value as an aperient, diuretic and antihypertension. In the present studies, the ACE inhibitory activity of the MeOH extract and its organicsolvent soluble fractions such as CH₂Cl₂, EtOAc, n-BuOH fraction and H₂O layer from dried or roasted seeds of C. tora were evaluated. The MeOH extracts from raw and roasted seed of the C. torahad potent inhibitory effects on ACE activity and the inhibitory activities increased with the increase of extract concentration.They were partitioned successively with several solvents, and their ACE inhibitory properties were further assessed at concentration of between 32.69 to 169.72 g/ml. The EtOAc soluble fractions obtained from raw and roasted MeOH extracts evidenced significant inhibitory activities with the IC_(50) values of 94.65 ± 0.92 g/mL and 74.94 ± 0.08 g/mL, respectively.Further detailed phytochemical investigations of the ACE inhibitory activities of the EtOAc fractions were conducted by the repeated chromatography over silica gel, Sephadex LH-20 and RP-18 gel columns, and led to the isolation of seven antraquinones, [questin (1), 2-hydroxyemodin 1-methylether (2), emodin (3), alaternin (4), glucoobtusifolin (5), gluco-aurantioobtusin (7), cassitoroside (8), toralactone gentiobioside (9)], two naphtopyrone glycosides [cassiaside (6), chrysophanol triglucoside (10)]and a naphthalene glycoside [cassitoroside (8)]. The structural identifications of these compounds were performed by analysis of 1D(¹H-NMR,(13)^1H-NMR,13C-NMR-NMR) and 2D NMR(HMQC and HMBC), FAB-Mass spectral data, and by comparison with published spectral data. The ACEinhibitory effects of the ten isolated compounds, as well as obtusifolin (5a) and aurantioobtusin (7a) were obtained from 5 and 7 by acid hydrolysis, respratively. Among the tested compounds 1, 5, 6, 8-10 did not show ACE inhibitory activity within tested concentrations and 2-4, 7 exhibited ACE inhibitory activity. EspeciallyACE inhibitory properties of gluco-aurantioobtusin (7) was ranked quite high, with IC_(50) values of 30.24±0.20μM, which manifested in a concentration-dependent manner. In these ACE assay 2-4 manifested marginal inhibitory activity, with IC_(50) values of 219.66 ± 1.43, 236.11 ± 6.61 and 841.75 ± 0.36μM, respectively. The inhibition kinetics for the 7 was analyzed by Lineweaver-Burk plots. The results indicated that 7exhibited a competitive-type of inhibition with respect to the substrate(FAPGG). The equilibrium constant for inhibitor binding, Ki value of 7 was estimated to be 8.3 x 105 M. Because of ACE has been reported to be implicated in cell oxidative stress byaugmenting the generation of both reactive oxygen species (ROS) and peroxynitrite, the inhibitory and scavenging activities of 7 on total ROS generation and on ONOO^(-) were also evaluated. The result that 7 evidenced marked inhibitory and scavenging activities, with IC_(50) value of 49.64 ± 0.37M for total ROS generation, and 4.60 ±1.12 M for ONOO^(-). According to the results of the peroxynitrite and ACE assays, C. tora and its isolated anthraquinones may take cein parts onthe ACE-related mechanism in rennin-angiotensin systems, associated with the pathogenesis of diabetes, hypertension, thrombis, and ROS generation.