Investigation on the potential anti-Alzheimer’s disease and anti-aging edible plant materials using Aβtreated PC-12 cells and Caenorhabditis elegans model systems

• Main Authors: Cheng-Chang Ho, 何承璋
• Other Authors: 孫璐西
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/959z2t

博士 === 國立臺灣大學 === 食品科技研究所 === 97 === Abstract Aging of population and the accompanying degenerative disorders are the common problems in developed countries. “Aging” is part of normal human development. It is defined as the progressive accumulation of changes with time that are associated with or responsible for the ever-increasing susceptibility to disease and death which accompanies advancing of age. Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder which is the most common cause of dementia in people aged over 65. The aim of this study is to evaluate the potential of the traditional Chinese herbal medicines to delay aging or the progress of AD. We used the “ Inhibition of aggregated Aβ-induced PC-12 cells death” as the “anti-Alzheimer’s disease” model, and the “Extension of nematode''s life-span” as the “anti-aging” model. In the anti-Alzheimer’s disease model, we used the standardized extract from the leaves of the Ginkgo biloba tree（EGb 761） and salivianolic acid B（Sal B）as the positive controls. In the anti-aging model, we used Eukarion-8 (EUK-8) as the positive control. The Chinese herbal medicines under investigation were Panax ginseng C. A. Mey.、Gastrodia elata Blume、Glycyrrhiza uralensis Fisch.、Angelica sinensis (Oliv.) Diels、Paeonia veitchii Lynch、Lycium chinense Miller、Sesamum indicum L.、Polygonum multiflorum Thunb. and Morus alba L. In the initial screening, Angelica sinensis, Polygonum multiflorum and Gastrodia elata showed superior biological activity. The ground powders of these three traditional Chinese herbal medicines were extracted with methanol. Then the methanol extracts were partitioned between ethyl acetate / water and n-butanol / water in succession, ethyl acetate, n-butanol and aqueous fractions of each methanol extract were obtained. After removing the solvents under vacuum, each solvent fraction was tested for its activities by “Inhibition of aggregated Aβ-induced differentiated PC-12 (dPC-12) cells death ” and “Extension of nematode''s life-span ” models. EGb 761/Sal B and EUK-8 were again used as the positive controls, respectively. We found that ethyl acetate fraction of the methanol extract of Angelica sinensis (AS-M-EA) was the most biologically active fraction. AS-M-EA was separated by silica gel column chromatography with n-hexane (H) / ethyl acetate (EA) mixture as the gradient elution solvent system. I obtained three extremely active fractions, fraction 3 (F3), fraction 4 (F4) and fraction 9 (F9), which inhibited approximately half of the cytotoxicity induced by Aβ at a concentration of 100 μg/ml (inhibited 50.5, 50.0 and 49.5% agg Aβ1-40-induced cytotoxicity, respectively). The possible mechanisms of these fractions to reduce agg Aβ1-40-induced cytotoxicity were further investigated. Our results showed that F3, F4 and F9 significantly reversed the agg Aβ1-40-induced neurotoxicity by attenuating the elevation of ROS level and intracellular [Ca2+], and the decline of mitochondrial membrane potential. We found these fractions also destroyed the structure of Aβ aggregates in thioflavin T and atomic force microscopy (AFM) assays, and the disaggregation effects decreased in the following order: F4＞F9＞F3. These three fractions could extend the life-span of N2 (wild type C. elegans), mev-1 mutant and daf-2 mutant. But only F9 elevated SOD activity in the filial generation. In daf-16 mutant, only F9 showed a significant lifespan-extending effect. This may suggest that the mechanism of F3 and F4 to prolong nematode''s life-span is through insulin/IGF-1 signaling pathway（IIS pathway）and is DAF-16 involved. Bioassay-guided fractionation and purification processes were employed to identify the active compounds from the root of Angelica sinensis. Four compounds showing potent activities were identified by comparing spectral data (UV, NMR, and ESI-MS) with literature values to be z-ligustilide, 11-angeloylsenkyunolide F, coniferyl ferulate and ferulic acid. They were found to significantly inhibit Aβ1-40 toxicity on dPC-12 cells at lower concentrations (1~10 µg/mL), but they were toxic to the dPC-12 cells at high concentrations (> 50 µg/mL). Coniferyl ferulate and ferulic acid were found in F9 and displayed a strong effect on antioxidant and Aβ disaggregation activities. Z-ligustilide and 11-angeloylsenkyunolide F, which existed in F4, displayed a strong inhibition effect on Aβ-induced cytotoxicity but weak effect on antioxidant and Aβ disaggregation activities. Thus the mechanism of these phthalides to reduce Aβ1-40-induced toxicity in dPC-12 cells needs further investigation.