식용 갈조류 다시마의 최종당화산물과 흰쥐 망막 알도즈 환원효소에 대한 억제효과

Abstract

The sea tangle, Laminaria japonica Areschoung, is a brown alga, which belongs to the family Laminariaceae and is widely distributed throughout Korea, Japan, China and France. This alga has long been used as common seafood as well as traditional medicines to promote maternal health. Previously, it has been reported for several biological activities such as scavenging activity against free radicals, antimutagenic activity, down-regulation of blood glucose in diabetic rats, inhibitory effects on oxidative stress and xanthine oxidase activity in streptozotocin-induced diabetic rat liver and protective effects on high glucose-induced oxidative stress in human umbilical vein endothelial cells. However, inhibitory activities of L. japonica on advanced glycation end products (AGEs) formation and rat lens aldose reductase (RLAR) had not been investigated. Increased aldose reductase related polyol pathway and formation and accumulation of AGEs have been implicated in the onset of many diabetic complications, including atherosclerosis, cardiac dysfunction, retinopathy, neuropathy and nephropathy. In the present study, the effects of methanolic (MeOH) extract and its fractions including dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (n-BuOH) and water (H2O) from L. japonica against diabetic complications via in vitro asaays, such as RLAR and AGEs formation inhibitory activities were evaluated. The MeOH extract showed promising inhibitory activities on both AGEs formation and RLAR, with IC50 value of 1,206.08 ± 21.29 ㎍/ml against AGEs formation and inhibition percentage of 27.44 ± 1.13% at 100 ㎍/ml against RLAR. Among several fractions, the EtOAc fraction manifested potent AGEs formation inhibitory activity with IC50 value of 150.32 ± 2.84 ㎍/ml. Furthermore, the EtOAc fraction showed the highest inhibitory activity on RLAR, with inhibition percentage of 50.83 ± 0.12% at a concentration of 100 ㎍/ml. In addition, the CH2Cl2 fraction exhibited noticeable inhibitory activity of AGEs formation with IC50 value of 167.65 ± 1.76 ㎍/ml. The CH2Cl2 fraction also inhibitied the RLAR at a concentration of 100 ㎍/mll by 30.24 ± 0.38%. Though the CH2Cl2 fraction was less active than EtOAc fraction in both RLAR and AGEs formation assays, it showed higher yield than EtOAc fraction. Therefore, the CH2Cl2 fraction was selected for chromatographic separation of active compounds using silica gel, RP-18 and Diaion HP-20 column chromatographies to yield two compounds 1 (pheophorbide a) and 2 (pheophytin a). The chemical structures of both compounds were elucidated by 1D NMR (1H & 13C-NMR), and confirmed by comparing with their published spectral data. Both 1 and 2 were isolated from L. japonica for the first time. The present study, the inhibitory effects of 1 and 2 from L. japonica against AGEs formation and RLAR were measured. 1 exhibited potent inhibitory activities against both AGEs formation and RLAR, with IC50 values of 49.4 and 12.3 μM, respectively. On the other hand, 2 was found to be active against AGEs formation, with IC50 228.7 μM. For the further elucidation of the structure-inhibitory activity relationship of porphyrin derivatives, the inhibitory activities of commercially available four porphyrin derivatives and two active porphyrin derivatives isolated from L. japonica on both AGEs formation and RLAR were compared. Among the commercially available porphyrin derivatives, protoporphyrin IX exhibited moderate activity on RLAR, with the IC50 value of 55.5 μM, compared with 1 (12.31 μM) and quercetin (1.17 μM). On the other hand, hemin showed marginal inhibitory activity with on RLAR an IC50 value of 101.3 μM. In contrast, chlorophyll a and hematin showed no activity toward RLAR. In AGEs formation assays, not only 1 and 2 from L. japonica but also commercially available porphyrin derivatives have inhibitory effect on AGEs formation greater than positive control, aminoguanidin (IC50 735.7 μM). Protoporphyrin IX and chlorophyll a exhibited potent activity, evidencing the IC50 values of 42.1 and 69.5 μM, respectively. On the other hand, hemin and hematin showed marginal inhibitory activity with the IC50 values of 280.9 and 299.14 μM, respectively. Taken together, 1 and protoporphyrin IX exhibited potent significant effects against both AGEs formation and RLAR, indicating that the presence of carboxyl group and the absence of phytyl group at the C-17 position of porphyrin derivatives seem to play key roles on the inhibitory effects of AGEs formation and RLAR. In order to determine the optimized extraction conditions of 1 and 2, L. japonica were extracted with different kinds of solvents such as MeOH, CH2Cl2 and acetone, and then quantitative analysis of 1 and 2 in the various extract of L. japonica was simultaneously performed using HPLC-UV detector system. The peaks of 1 and 2 were revealed at approximately 16.00 and 45.85 min in the three extracts. The contents of 1 and 2 in the MeOH extract were 0.3 and 2.24 mg/g extract, respectively. In the CH2Cl2 extract, content of compound 1 and 2 were 1.13 and 14.3 mg/g extract, respectively. The contents of 1 and 2 in acetone extract were 3.15 and 42.93 mg/g extract, respectively. These results indicated that acetone is optimized extracting solvent for extraction of these active constituents. In the present study, these results demonstrated that the MeOH extract and its CH2Cl2 fraction as well as pheophorbide a and pheophytin a from L. japonica exhibited potent effects of anti-diabetic complications via inhibition of AGEs formation and RLAR, suggesting that L. japonica and pheophorbide a and pheophytin a from L. japonica might potential utilization of functional food resource to prevent for diabetic complications.