PC12 세포에서 아밀로이드 베타 펩타이드로 유도된 독성에 대한 식용 갈조류 대황의 신경보호 효과

Abstract

Alzheimer’s disease (AD) is the most general neurodegenerative disease in humans that is characterized by neuronal degeneration and senile plaques. Amyloid beta peptide (Aβ) induced neurocytotoxicity is the important pathway of neuronal cell death which might play a crucial role in the development of AD. The mechanism by which Aβ induces cell death is not clear yet. However, it has been suggested that one of the mechanisms is oxidative stress produced by reactive oxygen species (ROS), reactive nitrogen species (RNS). Aβ induced toxicity is also related with the disturbance of calcium (Ca2+) homeostasis. Eisenia bicyclis (Kjellman) Setchell is a perennial brown alga, belonging to the family Laminariaceae. It is widely distributed in Korea and Japan and especially abundantly produced in Ulleung and Dok Island in Korea. This alga is frequently used as a dietary alternative of Laminaria japonica, along with Ecklonia stolonifera and Ecklonia cava. This species has a lot of bioactive components, including phlorotannins, polysaccharides, pyropheophytin, lipids, tripeptides, and oxylipins. Particularly phlorotannins, produced by polymerization of phloroglucinol (1, 3, 5-trihydroxybenzene), have been reported for various biological activities such as antioxidant, anti-diabetic complication, antitumor, hepatoprotective, anti-plasmin inhibitory, tyrosinase inhibitory, anti-inflammatory, nitrite-scavenging, anti-skin aging, anti-cholinesterase, anti-hyperlipidemic, anti-allergic, angiotensin converting enzyme-I inhibitory, β-secretase1 inhibitory, protein tyrosine phosphatase 1B, and α-glucosidase inhibitory activities. However, the neuroprotective effects of E. bicyclis on Aβ induced neurocytotoxicity had not been investigated. In the present study, the neuroprotective effects of methanolic (MeOH) extract and its solvent soluble fractions including dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (n-BuOH) and water (H2O) from E. bicyclis were investigated against Aβ induced neurocytotoxicity, as well as inhibitory activities of intracellular ROS, peroxynitrite (ONOO¯) and Ca2+ in PC12 cells. Exposure of Aβ to PC12 cells caused significant cell death and increased the number of apoptotic cells. Treatment of 2.5 μM Aβ alone induced about 50% cell death compared with control. However, the addition of 20 μg/ml MeOH extract and its two subfractions, EtOAc and n-BuOH fractions from E. bicyclis increased cell viability to 75.06, 104.30 and 82.76%, respectively. On the other hand, the CH2Cl2 and H2O fractions had no obvious effects. In particular, the EtOAc fraction restored cell viability to almost the normal level, indicating that the EtOAc fraction showed the highest neuroprotective effects against Aβ induced neurocytotoxicity among the tested fractions and has neuroprotective constituents. Therefore, the EtOAc fraction was selected and chromatographed over a silica, RP-18, and Sephadex LH-20 column to yield active compounds. Six phlorotannins, including phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofucofuroeckol-A (4), dieckol (5), and 7-phloroeckol (6) were isolated from the EtOAc fraction of E. bicyclis and their chemical structures were elucidated by spectral analysis. Among six phlorotannins, 4, 5, and 6 significantly decreased Aβ induced cell death. 4, 5, and 6 with no obvious toxic concentration (5, 20, and 30 μM, respectively) increased cell viability by 15, 20, and 40%, respectively. The MeOH extract and its fractions from E. bicyclis, mainly EtOAc fraction, and isolated phlorotannins from active EtOAc fraction also inhibited the level of intracellular ROS and ONOO¯, indicating the neuroprotective effects may be mediated through reduced intracellular ROS and ONOO¯ generation. The EtOAc fraction manifested potent t-BHP-induced intracellular ROS and ONOO¯ generation inhibitory activity with IC50 values of 1.85 ± 0.03 and 3.93 ± 0.51 μg/ml, respectively, followed by the n-BuOH fraction with IC50 values of 2.25 ± 0.11 and 7.49 ± 0.58 μg/ml, respectively. Among the compounds from EtOAc fraction, 4, 5, and 6 significantly decreased t-BHP-induced intracellular ROS generation. 5 and 6 also significantly decreased t-BHP-induced intracellular ONOO¯ generation, while 4 had cytotoxicity in tested concentrations. Incubation of PC12 cells with the fractions and the compounds from E. bicyclis which have relatively high neuroprotective effects also significantly inhibited Aβ-induced ROS generation. Furthermore, since the mechanism of toxicity of Aβ may be mediated by elevations of Ca2+, the study examined whether the phlorotannins affected the Aβ-induced increase of Ca2+. 3, 4, and 6 decreased the elevations of Ca2+ in a dose dependent manner, and especially pretreatment with 6 (30 μM) reduced intracellular Ca2+ to the normal level. Thus, the results of the present study imply that E. bicyclis and its active components attenuated the oxidative stress and reduced neuronal cell death, suggesting that it may be used as a dietary neuroprotective agent in AD.