연수의 렌즈 알도즈 리닥타제 저해작용과 당뇨병 및 고지혈증 모델 흰쥐에 대한 보호 효과

Abstract

Nelumbo nucifera Gaertn., a perennial aquatic plant, which was distributed throughout Asia, including India, China, the Middle east and Egypt. All parts, namely, the leaves, flowers, stamens, embryos and rhizomes of N. nucifera, have been used foodstuffs as well as traditional medicines in China, India and Korea. Various pharmacological activities of this plant, including psychopharmacological, hepatoprotective, antimicrobial, antibacterial, anti-HIV, antioxidant, antihyperlipidemic, antiobese, antidiabetic and antihypertensive effects have been reported. However, aldose reductase inhibitory activity of N. nucifera stamens and its protective effects on diabetic and hyperlipidemic rat models had not been investigated. Aldose reductase, one of key enzymes in the polyol pathway, has been demonstrated to play important roles not only in the cataract formation in the lens, but also in the pathogenesis of diabetic complications such as retinopathy, neuropathy and nephropathy. Much evidence suggests that aldose reductase inhibitors could be effective agent for the prevention of diabetic complications. A methanol (MeOH) extract of N. nucifera, exhibiting good aldose reductase inhibitory effect, was fractionated using several organic solvents, including dichloromethane, ethyl acetate (EtOAc) and n-butanol (BuOH). Among several fractions, the EtOAc fraction manifested potent aldose reductase inhibitory activity, and then purified via repeated column chromatography. Thirteen flavonoids [kaempferol (1), kaempferol 3-O-?-D-galactopyranoside (2), kaempferol 7-O-?-D-glucopyranoside (3), kaempferol 3-O-?-D-glucopyranoside (4), kaempferol 3-O-?-L-rhamnopyranosyl-(1→6)-?-D-glucopyranoside (5), kaempferol 3-O-?-L-rhamnopyranosyl-(1→2)-?-D-glucuronopyranoside (6), kaempferol 3-O-?-L-rhamnopyranosyl-(1→2)-?-D-glucopyranoside (7), kaempferol 3-O-?-D-glucuronopyranoside (8), kaempferol 3-O-?-D-glucuronopyranosyl methyl ester (9), myricetin 3?,5?-dimethylether 3-O-?-D-glucopyranoside (10), quercetin 3-O-?-D-glucopyranoside (11), isorhamnetin-3-O-?-D-glucopyranoside (12), isorhamnetin 3-O-?-L-rhamnopyranosyl-(1→6)-?-D-glucopyranoside (13)] and four non-flavonoid type compounds [adenine (14), myo-inositol (15), arbutin (16) and ?-sitosterol glucopyranoside (17)] were isolated from the EtOAc fraction. Among these compounds, compounds 3, 6, 7, 11-17 were isolated from N. nucifera for the first time. The chemical structures of these compounds were elucidated by 1D (1H, 13C and DEPT) and 2D NMR (HMQC and HMBC) spectral data, and confirmed by comparison of their published spectral data. Among the isolated compounds, compounds 5 and 13 exhibited the most potent aldose reductase inhibitory activity, evidencing the IC50 values of 5.6 and 9.0 ?M, respectively. Compounds 1-4, 9 and 12 were also exhibited potent aldose reductase inhibitory activities, with the IC50 values of 24, 18, 14, 11, 12 and 19 ?M, respectively. Since the MeOH extract of N. nucifera showed potent aldose reductase activities in vitro, further in vivo experiments were conducted to clarify the bioactivity of N. nucifera using the diabetic and hyperlipidemic rat models. The effects of MeOH extract of N. nucifera in the blood glucose levels, lipid profiles and hepatic antioxidant enzyme levels on streptozotocin (STZ)-induced diabetic rats were evaluated. Oral administration of MeOH extract of N. nucifera at three doses of 125, 250 and 500 mg/kg of body weight significantly and dose-dependently reduced blood glucose, triglyceride and total cholesterol levels. In addition, the MeOH extract of N. nucifera induced significant increases in the activities of superoxide dismutase, glutathione, glutathione peroxidase, glutathione reductase, catalase, and also reduced xanthine oxidase, coupled with a significant reduction in thiobarbituric acid reactive substances and lipofuscin contents in the livers of STZ-induced diabetic rats. These results evidenced that the MeOH extract of N. nucifera have an antioxidant activity exerting hepatoprotective effects against STZ-induced oxidative stresses. The antidiabetic and antioxidant effects at a dose of 500 mg/kg of body weight of the MeOH extract of N. nucifera were comparable to the effects of chlorpropamide, a positive control. Because the administration of the MeOH extract of N. nucifera showed hypoglycemic and hypolipidemic effects in STZ-induced diabetic rats, further experiment was conducted to clarify the hypolipidemic effect using the hyperlipidemic rat models. The hypolipidemic effect of the MeOH extract of N. nucifera was investigated using the hyperlipidemic rats induced by poloxamer 407. The hyperlipidemic rats induced by poloxamer 407, a hydrophilic non-toxic surface active agent, demonstrated remarkably high serum levels of triglyceride (TG), total and LDL cholesterol (TC and LDL-C) and significantly increased atherogenic index (A.I.), while they were significantly decreased by the administration of methanolic extract or the flavonoids-rich EtOAc and n-BuOH fractions of N. nucifera. In addition, serum HDL-cholesterol (HDL-C) level was reduced in poloxamer 407-induced hyperlipidemic rats. However, oral administration of the EtOAc and n-BuOH fractions significantly increased the HDL-C level. In particular, the EtOAc fraction showed more potent hypolipidemic effect than the n-BuOH fraction. The hypolipidemic effect of compound 8, the compound isolated from active EtOAc fraction, was also investigated using hyperlipidemic rat models. The elevate TC, TG and LDL-C levels and A.I. values in poloxamer 407-induced hyperlipidemic rats were significantly decreased after the oral administration of compound 8, while the level of HDL-C led to a significant increase. Therefore, the results imply that compound 8 has beneficial effects on serum lipid profiles by reducing lipid levels. From the above results, the MeOH extract, its EtOAc and BuOH fractions and its component, compound 8, had hypolipidemic effects in poloxamer 407-induced hyperlipidemic rats. In addition, compound 8 showed the same effects in high-cholesterol fed rats. These results demonstrated that N. nucifera and flavonoids rich-fractions from N. nucifera have possibility as agents in protecting hyperlipidemic, diabetic and atherosclerotic diseases.