數種天然物對於Aβ1-40引起PC12及分化後PC12細胞死亡的保護效果

Protection of PC12 and differentiated PC12 cells from β-amyloid peptide-induced cell death by some natural compounds in food

侯怡卉(I-Huei Hou)

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/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01667

摘要

阿茲海默症 (Alzheimer’s disease, AD) 為一漸進式的神經退化性疾病，常發生於65歲以上的老年人，導致患者記憶喪失及認知衰退。該病症是由於腦部類澱粉胜肽 (β-amlyoid peptide, Aβ) 斑塊的堆積，造成氧化壓力、粒線體失調、突觸萎縮，進而導致腦部功能退化。PC12細胞為一種類似神經細胞的細胞株，能表現如同神經細胞的特性，因此廣泛應用於阿茲海默症及神經相關的研究。
本研究以「Aβ1-40聚集導致naïve PC12 (nPC12) 及differentiated PC12 (dPC12) 細胞死亡之抑制」作為預防阿茲海默症之細胞模式，以銀杏葉萃出物EGb 761做為正控制組，篩選之試驗樣品包括花生芽萃出之arachidin-1、當歸中有效成分ferulic acid及z-ligustilide、廣泛存在植物中的gallic acid、龍眼花中proanthocyanidin A2 (PA2) 、芝麻油中的sesamol及薑黃代謝物tetrahydrocurcumin (THC)，目的為篩選出能有效預防或延緩阿茲海默症的天然物。
於nPC12模式中，發現20 - 40 μM (11.5 - 23.1 ppm) 的PA2及0.5 - 2 μM (0.16 - 0.62 ppm) 的arachidin-1可促進nPC12細胞生長，與100 - 200 ppm的EGb 761效果相近，且以40 μM (23.1 ppm) 的PA2及2 μM (0.62 ppm) 的arachidin-1抑制aggAβ1-40毒性效果最好，達顯著差異 (P<0.01)，與50 ppm的EGb 761效果相近。經神經生長因子刺激PC12細胞發展成更接近神經細胞的dPC12模式中，以40 μM (23.1 ppm) 的PA2、1 μM (0.31 ppm) 的arachidin-1、20 μM (3.4 ppm) 的gallic acid及10 ppm的50 % z-ligustilide抑制aggAβ1-40毒性效果最好，皆達顯著差異 (P<0.05)，其中40 μM (23.1 ppm) 的PA2抑制效果高於50 ppm的EGb 761。從Thioflavin T assay結果發現，四者皆能造成aggAβ1-40的解集，破壞aggAβ1-40的結構，進而降低aggAβ1-40的毒性，達到保護細胞之效果。PA2、arachidin-1及gallic acid在CAA及DPPH assays等抗氧化試驗中展現良好的抗氧化能力。
從實驗結果推測，PA2、arachidin-1及gallic acid能有效降低氧化壓力，並且能破壞aggAβ1-40結構，進而保護細胞；z-ligustilide則可能藉由破壞aggAβ1-40結構及其他途徑而達到保護dPC12細胞對aggAβ1-40之傷害。因此本研究推論，認為PA2、arachidin-1、gallic acid及z-ligustilide具有預防阿茲海默症之潛力。

關鍵字

阿茲海默症 ； 類澱粉胜肽氧化壓力 ； PC12 ； proanthocyanidin A2 ； arachidin-1 ； gallic acid ； z-ligustilide

並列摘要

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder in aged people. AD impairs memory and cognitive skills. The hallmark is accumulation of β-amyloid peptide in the brain, leading to elevated oxidative stress, dysfunction of mitochondrial, synapse loss and leading to destroy brain function. PC12 is a neuronal-like cell line expressed several neurobiological characteristics. PC12 cells have been extensively used as an in vitro model of AD and neuron-related research. Our research is entitled ”Inhibition of aggregated Aβ-induced naïve PC12 (nPC12) and differentiated PC12 (dPC12) cells death” as an in vitro model, by using the standardized extract from the leaves of the Ginkgo biloba extract (EGb 761) as the positive control. To investigate the possible neuroprotective effects of arachidin-1 from peanut second metabolite, ferulic acid and z-ligustilide in Angelica sinensis, naturally occurred of gallic acid, proanthocyanidin A2 from Dimocarpus longan Lour. flowers extract, sesamol from sesame oil and tetrahydrocurcumin (THC) from curcumin metabolite. From our results, 20 - 40 μM (11.5 - 23.1 ppm) PA2 and 0.5 -2 μM (0.16 - 0.62 ppm) arachidin-1 in nPC12 cells could increase cell proliferation, and the efficiency was close to 100 - 200 ppm EGb 761. And 40 μM (23.1 ppm) PA2 and 2 μM (0.62 ppm) arachidin-1showed significant potential protective effect on aggAβ1-40 induced cell death (P<0.01), and the efficiency was close to 50 ppm EGb 761. PC12 was differentiated to dPC12 under the stimulation of nerve growth factor (NGF) and growed neurite-like properties. 40 μM (23.1 ppm) PA2, 1 μM (0.31 ppm) arachidin-1, 20 μM (3.4 ppm) gallic acid and 10 ppm 50 % z-ligustilide in dPC12 cells showed significant potential protective effect on aggAβ1-40 induced cell death (P<0.05), and the efficiency of 40 μM (23.1 ppm) PA2 is better than 50 ppm EGb 761. Arachidin-1, PA2 and gallic acid had strong antioxidant effect in CAA and DPPH assays. Arachidin-1, PA2, gallic acid and z-ligustilide could disaggregate aggAβ1-40 oligomers. The results suggested that arachidin-1, PA2 and gallic acid decreased aggAβ1-40 induced cytotoxicity possibly by decreasing the oxidative stress and disaggregated activity. Z-ligustilide decreased aggAβ1-40 induced cytotoxicity possibly by disaggregated activity and other mechanisms. By these mechanisms, therefore, we suggested that arachidin-1, PA2, gallic acid and z-ligustilide had potential neuroprotective activity against aggAβ1-40 induced cytotoxicity

並列關鍵字

β-amyloid peptide ； oxidative stress ； PC12 ； proanthocyanidin A2 ； arachidin-1 ； gallic acid ； z-ligustilide

參考文獻

邱泓智。2009。巴西洋菇萃取物與數種純化合物在PC-12細胞模式中抗阿茲海默症之潛力。國立台灣大學食品科技研究所。碩士論文。

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