곰피 플로로탄닌 성분의 콜린 에스테라제와 렌즈 알도즈 리닥타제 저해작용과 타크린 유도 간독성 및 고지혈증 모델 흰쥐에 대한 보호효과

Abstract

Ecklonia stolonifera OKAMURA is a perennial brown alga, belonging to the family Laminariaceae. It is frequently used as a foodstuff, along with Laminaria japonica and Undaria pinnatifida. This alga was found to have antioxidant, antimutagenic acitivities and feeding-deterrent effect. The beneficial effects of E. stolonifera are thought to be due to its phlorotannin constituents. Phlorotannins, phenolic secondary metabolites of brown algae, are produced entirely by polymerization of phloroglucinol (1,3,5- trihydroxybenzene). Recently, phlorotannins have been shown to have many positive physiological effects including potent feeding deterrent, nitrite-scavenging, antimutagenic, antioxidant, antidiabetic complication, tyrosinase inhibitory, angiotensin-converting enzyme inhibitory, algicidal, antiallergic, antiviral and anti-skin aging activities. However, cholinesterase and len aldose reductase inhibitory activities of phlorotannins from E. stolonifera and their protective effects on tacrine-induced hepatotocixity and hyperlipidemic rat models had not been investigated. The bioactivities of the ethanolic extracts from Korean seaweeds were screened using in vitro assays, such as cholinesterase (acetylcholinesterase and butyrylcholinesterase) and lens aldose reductase inhibitory activities, and hepatoprotective effect on tacrine-induced hepatototoxicity in Hep G2 cells. Among them, E. stolonifera exhibited promising inhibitory properties against cholinesterase and lens aldose reductase, as well as potent hepatoprotective effect. Moreover, the organic solvent-soluble fractions, such as n-hexane, dichloromethane (CH₂Cl₂), ethylacetate (EtOAc), n-butanol (n-BuOH), and H₂O fractions derived from E. stolonifera, were evaluated via acetylcholinesterase and butyrylcholinesterase, and lens aldose reductase inhibitory activities and tacrine-induced hepatoprotective activity. Among the five fractions tested, the EtOAc soluble fraction had noticeable inhibitory activity towards acetylcholinesterase and lens aldose reductase, and potent hepatoprotective acitivity. Conversely, the n-hexane fraction had profound inhibitory activity against butyrylcholinesterase. Bioassay-guided isolation of the active n-hexane and EtOAc soluble fractions, obtained from the ethanolic extract of E. stolonifera, resulted in the isolation of twelve compounds (1-12). These compounds have been identified by direct comparisons of their spectral data with the reported ones: two sterols [fucosterol (1) and 24-hydroperoxy 24-vinylcholesterol (2)] from the n-hexane fraction, and ten phlorotannins [phloroglucinol (3), dioxinodehydroeckol (4), eckol (5), phlorofucofuroeckol-A (6), dieckol (7), triphlorethol-B (8), 2-phloroeckol (9), 7- phloroeckol (10), diphlorethol (11), and fucofuroeckol-A (12)] from the EtOAc fraction. Among these compounds, compounds 8-11 were isolated from E. stolonifera for the first time and compound 12 was first isolated as free form from natural sources, although hepta-acetate of compound 12 has been previously isolated from Eisenia arborea. The molecular structure of these compounds was elucidated by 1D (¹H, ¹³C, DEPT) and 2D NMR (HMQC, HMBC) spectral data, and by comparison of their spectral data with those previously reported. The inhibitory activities of cholinesterase and lens aldose reductase of compounds 1-12, and the protective effect on tacrine-induced hepatotoxicity were also evaluated. Of the isolated compounds, compounds 4-7, 9, and 10 exhibited potent acetylcholinesterase inhibitory activities, with IC_(50) values of 42.66 ± 8.48, 20.56 ± 5.61, 4.89 ± 2.28, 17.11 ± 3.24, 38.13 ± 4.95, and 21.11 ± 4.16 μM, respectively. On the other hand, compound 2 showed marginal inhibitory activity, with an IC_(50) value of 389.10 ± 2.29 μM. Conversely, compounds 1, 2, 4, and 6 exhibited moderate inhibitory activities against butyrylcholinesterse, with IC_(50) values of 421.72 ± 1.43, 176.46 ± 2.51, 230.27 ± 3.52, and 136.71 ± 3.32 μM, respectively. However, compounds 3 and 8 showed no activity toward either acetylcholinesterase or butyrylcholinesterase. These results suggest that the degree of polymerization and closed-ring structure of phlorotannins may play key roles in the inhibitory potential of phlorotannins toward the cholinesterase (ChEs). The E. stolonifera-derived phlorotannins exhibited lens aldose reductase inhibitory activity with except of compounds 8, 11, and 12. The IC_(50) values of compounds 4 and 10 were 21.95 and 8.51 mM, respectively, the most active in the lens aldose reductase assay. The IC_(50) values of compounds 5 and 7 followed, at 54.68 and 42.39 mM, respectively. Compound 6 exhibited marginal activity in the lens aldose reductase assay (IC_(50) 125.45 mM). Among the isolated compounds, compounds 4 and 6 showed hepatoprotective activities with the EC_(50) values of 62.0 and 79.2 μg/ml, respectively on tacrine-induced cytotoxicity in human liver-derived Hep G2 cells. The hepatoprotective activity of compound 4 is comparable with that (EC_(50) = 50.0 μg/ml) of a positive control, silybin. However, compounds 3, 5, and 7 did not show any hepatoprotective activity. Because phlorotannins isolated from E. stolonifera showed potent activities in vitro assays, further experiment in vivo was conducted to clarify the bioactivity of E. stolonifera and their phlorotannins using the hyperlipidemic rat models. The hypolipidemic effects of E. stolonifera and its major phlorotannin constituents were investigated using the hyperlipidemic rats induced by poloxamer 407 and hypercholesterolemic rats fed a high cholesterol diet. The hyperlipidemic rats induced by poloxamer 407, a hydrophilic non-toxic surface active agent, had remarkably high serum levels of triglyceride (TG), total and low density lipoprotein cholesterol (TC and LDL-C), and significantly increased atherogenic index (A.I.), while they were significantly decreased by the administration of ethanolic extract or phlorotannins rich EtOAc and n- BuOH fractions of E. stolonifera. In addition, high density lipoprotein cholesterol (HDL C) level in serum was reduced in poloxamer 407-induced hyperlipidemic control rats. However, the 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 effects of compounds 5 and 7, the main compounds isolated from active EtOAc fraction, were also investigated using hyperlipidemic and hypercholesterolemic rat models. The elevated TC, TG and LDL-C levels, and A.I. values in poloxamer 407-induced hyperlipidemic rats were significantly decreased after the oral administration of these compounds, while the level of HDL-C led to a significant increase. Moreover, the serum lipid levels, such as TC, TG and LDL-C, were elevated in high cholesterol diet fed rats, while lipid levels were significantly decreased in the rats administered by compound 7. Furthermore, A.I. value was lowered by administration of compound 7. However, compound 5 did not show any protective effect in hypercholesterolemic rats. In the present study, compound 7 exhibited stronger protective activities on hyperlipidemia than compound 5, which was indicated that the degree of polymerization of phlorotannins seem to play key roles in the serum lipid level lowering potential. The effect of E. stolonifera and E. stolonifera-derived phlorotannins on the ChEs, lens aldose reductase, hepatoprotective, and hypolipidemic activities were assayed with in vitro and in vivo assay in the present study to demonstrate for a potential utilization of functional food resource to treat or protect for Alzheimer’s disease, diabetic complications, hepatotoxicity, and hyperlipidemia.