以Aβ聚集導致PC-12細胞氧化損傷之抑制與線蟲壽命延長為模式開發抗氧化與抗老化食品

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

何 承 璋(Cheng-Chang Ho)

指導教授 ： 孫璐西

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.00134

摘要

摘要
人口老化與伴隨而來的退化性疾病（degenerative diseases）為已開發國家所面臨的共通問題。「老化」是人類正常發育的一個階段，乃隨著年齡增加，逐漸累積體內的一些變化，而這些變化將提高對疾病或死亡的敏感性。阿茲海默症（Alzheimer’s disease, AD）為一漸進性的神經退化性疾病，為65歲以上老年人最常發生之失智症（dementia）。
本研究之目的在於評估中國傳統食藥材對延緩老化與預防或延緩AD進程之功效。藉合適的AD研究模式--「Aβ1-40聚集導致PC-12細胞氧化凋亡之抑制」與老化研究模式--「線蟲壽命延長」，並以國內外廣被使用的銀杏葉萃出物（Ginkgo biloba extract, EGb 761）和丹參酚酸B（salivianolic acid B, Sal B）作為抗阿茲海默症模式之正對照組，以Eukarion-8 (EUK-8)作為抗老化模式之正對照組，實驗材料為中醫藥理論與現代研究中皆指向具抗老化功效之食藥用保健素材，包括人參、天麻、甘草、當歸、赤芍、枸杞子、芝麻、何首烏和桑椹等。篩選出三個具有顯著抑制Aβ的神經毒性與延緩線蟲老化功效之食藥材，分別為當歸、何首烏和天麻。接著將此三種食藥材以甲醇萃取與溶劑區分，得其甲醇粗萃物之乙酸乙酯層區分物、正丁醇層區分物以及水可溶物。以「Aβ1-40聚集導致分化的（differentiated）PC-12（dPC-12）細胞氧化凋亡之抑制」與「線蟲壽命延長」兩模式進行活性評估，同樣以EGb 761、Sal B與EUK-8作為兩模式之正對照組。選出活性最高之溶劑區分物--當歸甲醇粗萃物之乙酸乙酯層區分物（AS-M-EA），將其進行矽膠管柱層析，取得不同極性之沖提區分，以「Aβ1-40聚集導致dPC-12細胞氧化凋亡之抑制」進行活性評估，選出fraction 3 (F3)、fraction 4 (F4)與fraction 9 (F9)等三個最具抑制Aβ1-40聚集物神經毒性之管柱區分物。探究三者抑制aggregated Aβ1-40 (agg Aβ1-40)毒性之可能機制，發現F3、F4與F9三個區分，皆能減輕因agg Aβ1-40導致的dPC-12細胞內ROS與鈣離子含量上升，及粒線體膜電位下降等現象。此外，藉thioflavin T assay與atomic force microscopy (AFM) 掃描亦發現，三者皆能顯著破壞Aβ1-40聚集物的結構，且效果為F4＞F9＞F3。而F9除能降低β-sheet的結構外，本身也具有優越的抗氧化活性。
F3、F4與F9三者也能延長N2、mev-1與daf-2線蟲之平均壽命與最大壽命，其中以F4的影響最為顯著。但F3與F9對N2及mev-1線蟲體內SOD活性的影響較顯著，且只有F9能延長線蟲子代之SOD活性。進一步以daf-16線蟲探討F3、F4與F9延長線蟲壽命之機制，發現F3延長壽命的效果大幅減低，F4則無法顯著延長daf-16線蟲壽命，僅F9仍能顯著延長daf-16線蟲壽命(但影響較其對N2線蟲小)，顯示F3與F4延長線蟲壽命的機制與daf-16有密切的關係，而F9延長線蟲壽命之影響可能主要與其抗氧化活性有關。
以生物活性作為分離與純化依據 ( bioactivity guided isolation and purification)，從當歸中分離出ferulic acid與coniferyl ferulate為F9中的主要活性物質，其具有很好的抗氧化與破壞Aβ聚集結構之活性，並能顯著抑制agg Aβ1-40對dPC-12細胞之傷害。此外，F4中的兩個主要活性化合物Z-ligustilide與11-angeloylsenkyunolide F皆能顯著抑制agg Aβ1-40對dPC-12細胞之傷害，但並無法破壞Aβ聚集之結構，有關F4中主要破壞Aβ聚集物的活性成分還需再繼續研究。

關鍵字

老化 ； 阿茲海默症 ； 線蟲 ； PC-12細胞 ； 當歸

並列摘要

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.

並列關鍵字

Aging ； Alzheimer’s disease ； C. elegans ； PC-12 cells ； Angelica sinensis

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