곰피의 항당뇨 및 항동맥경화 효과

Abstract

Ecklonia stolonifera OKAMURA belonging to the perennial brown alga, is a member of the family Laminariaceae. It usually grows well in subtidal zones at 2-10 m depth and commonly distributed in the mid-Pacific coast around Korea and Japan. It is frequently consumed as a foodstuff, together with Undaria pinnatifida and Laminaria japonica. This brown alga has been also used as phlorotannin-rich raw materials and phlorotannins are secondary metabolites polymerizing phloroglucinol (1,3,5-trihydroxybenzene) through an ether, phenyl, or 1,4-dibenzodioxin linkage. Phlorotannins were found to have a variety of bioactivities, such as anti-diabetic complications, antioxidant, tyrosinase inhibitory, angiotensin-converting enzyme inhibitory, antimutagenic, nitrite-scavenging, algicidal, anti-inflammatory, nitrite-scavenging, anti-skin aging, and anti-allergic. However, protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase inhibitory activities of phlorotannins isolated from E. stolonifera and their antioxidative effects on human low density lipoprotein (human LDL) have not been investigated yet. Increased PTP1B enzyme related insulin resistance and α-glucosidase that catalyze the final step in the digestive process of carbohydrates have been implicated in the pathogenesis of many diabetic complications containing atherosclerosis, cardiac dysfunction, retinopathy, and nephropathy. The diabetic patients have a higher risk of developing atherosclerotic diseases than non-diabetic. Oxidation of LDL has been represented as one of the main reason for atherosclerosis and has been reported that dietary antioxidants and free radical scavenger are able to prevent LDL oxidation which can reduce the risk of atherosclerosis in diabetes. The present work investigated the anti-diabetic and anti-atherosclerotic effects of the methanol (MeOH) extract and different solvent soluble fractions including dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (n-BuOH), and water (H2O) of the edible brown alga, E. stolonifera together with its isolated phlorotannins via the inhibition of α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), the inhibition of human low density lipoprotein (LDL) oxidation and the conjugated diene formation in human LDL under in vitro conditions, respectively. The MeOH extract showed promising inhibitory activities of both PTP1B and α-glucosidase, with IC50 values of 6.39 ± 0.18 μg/ml against PTP1B and 2.82 ± 0.63 μg/ml against α-glucosidase, respectively. Among several fractions, the EtOAc fraction and n-BuOH fraction exhibited potential inhibitory activities in PTP1B with IC50 values of 0.26 ± 0.06 μg/ml and 0.23 ± 0.07 μg/ml and in human LDL oxidation with IC50 values of 3.04 ± 0.13 μg/ml and 4.54 ± 0.02 μg/ml, respectively. On the other hand n-BuOH fraction showed weak α-glucosidase inhibitory activities with IC50 value of 4.59 ± 0.57 μg/ml than EtOAc fraction with IC50 value of 1.15 ± 4.59 μg/ml. Since the EtOAc fraction showed significant inhibitory activities in PTP1B, α-glucosidase, human LDL oxidation, it was selected for chromatographic separation of active compounds using silica gel, Sephadex LH-20, RP-18 column chromatographies to isolate six phlorotannins, phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofurofucoeckol–A (4), dieckol (5), and 7-phloroeckol (6). Phlorotannins 3-6 were found as the potent and non-competitive PTP1B inhibitors with respective Ki values of 2.86, 1.43, 1.22, and 2.90 µM and with IC50 values ranging from 0.56 to 2.64 µM. Phlorotannins 4-6 exhibited the most potent α-glucosidase inhibitory activity with IC50 values ranging from 1.37 to 6.13 µM. Interestingly, phlorotannins 4 and 6 were non-competitive with Ki values of 0.45 and 0.56 µM, while phlorotannins 5 exhibited competitive inhibition with a Ki value of 0.27 µM against α-glucosidase enzyme. Furthermore, phlorotannins 3-5 showed the strongest inhibitory activity against human LDL oxidation with IC50 value of 7.47 ± 0.05 μM, 4.34 ± 0.07 μM, 3.10 ± 0.24 μM and significantly reduced the lag time (117.48 ± 0.15 min, 105.42 ± 0.59 min, 137.61 ± 0.39 min at concentration 10 μM) of conjugated diene formation, respectively. These results demonstrated that the MeOH extract and individual fractions of E. stolonifera as well as its isolated phlorotannins exhibited the potent anti-diabetic and anti-atherosclerotic effects via the inhibition of PTP1B, α-glucosidase, and human LDL oxidation, conjugated diene formation. Therefore, the extract and individual fractions of E. stolonifera as well as its isolated phlorotannins have anti-diabetic and anti-atherosclerotic effects in vitro conditions, which can be explored further for developing pharmaceutical drugs or functional foods to treat diabetes and atherosclerosis.