- 學位論文
當歸萃取液保護神經細胞抗類澱粉蛋白毒性之研究
Protective effects of Angelica sinensis extracts against neuronal damage by Amyloid peptide 25-35
摘要
本研究由不同萃取條件得到的當歸萃取液，分析其機能性及風味性成分含量的變化，並探討抗氧化及保護神經細胞抗類澱粉蛋白毒性之研究，整個論文由兩部分的實驗所組成。實驗一主要建立最佳抗氧化功能之萃取條件，將當歸以水及20%酒精加熱不同時間所得到的萃取液進行機能性成分分析，同時探討各萃取液之抗氧化及抑制由脂多醣(LPS)誘導小鼠RAW 264.7巨噬細胞產生NO的活性。結果指出，不論是水萃或酒萃，總酚(Total polyphenol)於15分鐘的萃取可以得到最高的萃取量。當歸酒萃液中的揮發性成分含量明顯高於水萃液，篙本內酯(ligustilide)、正丁烯基夫內酯(butylidene phthalide) 及正丁基夫內酯(butyl phthalide)皆於酒萃30分鐘達到最高量，隨著萃取時間的延長，揮發性成分含量明顯下降。萃取液之總多醣含量顯示水萃組於各時間點皆高於酒萃組。抗氧化的結果顯示各萃取液於清除DPPH自由基、抑制脂質過氧化及抑制DNA relaxation的活性，於短時間的萃取(15 min)，就已表現出最好的效果。在抑制小鼠巨噬細胞生成NO的試驗中，各萃取條件所得到的成分，以20~200 μg/mL的作用劑量，顯示抑制NO生成具劑量關係(dose-dependent)，該結果亦顯示15分鐘的酒萃條件即具最好的活性。最後將抗氧化活性與機能性成分的含量進行相關性分析，結果顯示阿魏酸及TPA (The total peaks area of phenolic acids)含量與抗氧化活性具正相關。實驗二評估當歸萃取液對於β類澱粉蛋白(β-Amyloid peptide; Aβ)所引起之神經細胞毒性的保護作用，並探討保護由Aβ所造成之神經細胞毒性的可能機制。結果指出，Aβ25-35會造成神經母細胞瘤(mouse neuroblastoma; Neuro 2A)的細胞毒性，Aβ25-35降低Neuro 2A細胞存活率具劑量關係，其LC50為14.9μM。當歸萃取液的處理可以降低由Aβ25-35作用而上升之活性氧分子(ROS)因而提高細胞的存活率。以Aβ25-35作用的神經細胞有脂質過氧化現象(lipid peroxidation)且降低細胞中麩胱甘肽(glutathione)的含量，經加入當歸萃取液後可改善並恢復之。Aβ25-35作用的細胞明顯降低粒線體膜電位(Δψm)且經由電顯結果觀察到細胞形成自噬空泡(membrane-bound degradative vacuole)且增大細胞質量。這些現象顯示，以Aβ25-35處理細胞所引起的死亡方式是經由細胞自噬(autophage)的途徑。本實驗利用細胞自噬的特異性抑制劑bafilomycin A1及3-methyladenine作用細胞亦得到此結論的支持。當歸萃取液持有與bafilomycin A1及3-methyladenine相同的活性，這些結果指出，當歸可以有效的保護神經細胞免於由Aβ25-35造成之細胞自噬形式的毒性損傷。綜合以上結果，開發具抗氧化功能的當歸保健食品，最佳萃取條件是以20%的酒精(傳統米酒濃度)在低於30分鐘的加熱萃取為佳，同時亦顯示當歸可以有效的保護神經細胞免於由Aβ25-35造成之細胞自噬形式的毒性損傷。
並列摘要
This study consisting of two experiments investigated the proper processing procedures for preparing the Angelica sinensis (AS) products with various health-promoting functions. The first experiment investigated the effect of extraction conditions on the bioactive components and the functions of the extracts of AS. AS was extracted with water or 20% ethanol for different periods of time, and the antioxidant activity as well as volatile and non-volatile active components in the extracts were determined. The AS extracts contained significant amount of nicotinic acid, phthalic acid, p-coumaric acid, and ferulic acid, and the ferulic acid content was the highest among various phenolic acids in AS extracts. Regardless the water or alcohol extraction, most of the phenolic acids reached their maximum recoveries in 15 minutes. The contents of volatile compounds of AS were much higher in the 20% ethanol extracts than those in water extracts. In the 20% ethanol extracts, the amount of ligustilide (4.403 mg/100 ml), butylidene phthalide (0.187 mg/100 ml) and butyl phthalide (0.147 mg/100 ml) were higher in the 30-min extracts than that prepared for longer time. The inhibition of 1,1-diphenyl-2-picrylhydrazyl (DPPH), lipid peroxidation, and DNA relaxation activities by various AS extracts suggested that, a short extraction time (15 min) produced AS extracts possessing the highest antioxidant effect. The 15-min AS extracts in the concentration range of 20-200 μg/ml also showed inhibitory effects on nitric oxide (NO) production in LPS activated RAW 264.7 macrophage in a dose-dependent manner. The antioxidant activity and phenolic acid concentration for all AS extracts exhibited a positive and significant linear correlation. The second experiment investigated the protective effects of AS extracts against neuronal death by amyloid peptide 25-35 in Neuro 2A cells. Results showed that Aβ25-35 decreased viability of Neuro 2A cells in a concentration dependent manner with IC50 of 14.9 μM. AS extracts reduced Aβ25-35–induced ROS elevation and cytotoxicity in MTT assay. Aβ25-35-induced cellular lipid peroxidation and decreased glutathione levels were also rescued by AS extracts. The Aβ25-35–treated cells showed a significant reduction in the mitochondrial transmembrane potential (m) and mitochondrial dilation with fluorescence probe rhodamin 123 and NAO, respectively in flowcytometry assay. The Neuro 2A cells treated with Aβ25-35 were found to form a membrane-bound degradative vacuole and an enlargement of the mitochondrial mass under TEM observation, suggesting that Aβ25-35-induced cell death was mediated by autophagy. The autophagy-specific inhibitors bafilomycin A1 and 3-methyladenine were used in NAO assay to support the notion. This research further demonstrated that AS extracts possessed a similar activity to bafilomycin A1 and 3-methyladenine on NAO fluorescence suppression, indicating that Angelica sinensis was effective in preventing Aβ25-35 induced neurotoxicity mediated by autophagy. These findings suggested that for obtaining a better flavor product with high anti-oxidative capability and neuroprotective effect activity, we suggest an extraction condition, which was 20% ethanol and less than 30 min extraction time, and the obtained AS extracts was effective in preventing Aβ25-35 induced neurotoxicity mediated by autophagy.