探討當歸甲醇萃出物及其活性成分對大鼠腦皮質神經細胞之保護效果

Neuroprotective Effects of the Methanol Extract of Angelica sinensis and Its Bioactive Components Against Aβ-induced Toxicity in Rat Cortical Neurons

陳逸婷(Ying-Ting Chen)

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(2012年)

https://doi.org/10.6342/NTU.2012.01716

摘要

阿茲海默症 (Alzheimer’s disease , AD) 是一漸進式神經退化性疾病，也是最常見的一種失智症。病徵包括記憶力喪失、理解及語言能力下降、行走及吞嚥困難，最終將喪失獨立生活的能力。由神經病理學來看，AD患者腦中會形成類澱粉斑塊( amyloid plaque ) 及神經糾結 ( neurofibrillary tangles ) 的沉積。近年來許多研究指出，分子小而具水溶性的β-類澱粉胜肽寡聚合物 ( Aβ1-42 oligomers ) 是導致AD的主因。由於目前並沒有根治AD的方法，因此本研究以「Aβ-42寡聚合物誘發神經細胞毒性」為模式，希望能研究出有效保護神經細胞抗Aβ-42寡聚合物毒性進而預防AD的天然中草藥。根據本研究室過去實驗指出，當歸 ( Anglica sinensis ) 甲醇萃出物 ( As-M ) 在dPC 12細胞模式具有抗Aβ1-40寡聚合物毒性之效果。文獻指出當歸中所含主要的活性成分Z-藳本內酯 ( Z-ligustilide, Z-LIG ) 以及阿魏酸 ( Ferulic acid, FA ) 皆具有保護神經細胞的效果。因此本實驗一方面製備純化的Z-LIG，並以HPLC分析當歸甲醇萃出物( As-M )中Z-LIG及FA含量，同時以初代大鼠腦皮質細胞為模式，以As-M、Z-LIG和FA，等樣品進行抗Aβ-42寡聚合物毒性效果之探討，並研究其可能機制。實驗結果顯示，抑制Aβ-42寡聚合物毒性效果最好的樣品是75 μM FA，其抑制率為28.6%；其次是25 μM Z-LIG，抑制率為24.0%；而As-M在10 ppm時保護效果最好，但與Aβ-42寡聚合物處理組相比沒有顯著差異。為探討兩者是否具協同作用，進一步將Z-LIG與FA混和後，進行抑制Aβ1-42寡聚合物毒性之試驗，結果顯示兩者不具協同作用。續以Aggrregated human Aβ ELISA kit分析有效樣品是否對Aβ1-42寡聚合物的產生有抑制效果，發現Z-LIG可有效降低(51 )% Aβ1-42寡聚合物的產生，而FA則可降低23.3% ，與Aβ1-42寡聚合物組相比沒有顯著差異。雖Z-LIG可有效降低(51 )% Aβ1-42寡聚合物的產生，但在高劑量下對保護神經細胞抗Aβ1-42寡聚合物毒性之效果較差，推測由於在培養液中溶解度甚低，致使無法有效保護神經細胞；而FA雖能有效抗Aβ1-42寡聚合物毒性，但卻無法有效減少Aβ1-42寡聚合物之產生，推測其對神經細胞之保護效果，可能來自其他機制。

關鍵字

阿茲海默症 ； β-類澱粉寡聚合物 ； 當歸 ； Z-藁本內酯 ； 阿魏酸

並列摘要

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline in memory, orientation, judgment, and reasoning; it is also the most common cause of dementia among the elderly. Neuropathologically, it is characterized by the accumulation of extracellular plaques and intracellular neurofibrillary tangles (NFTs). Plaques are mainly comprised of a small peptide called amyloid-β (Aβ),and neurofibrillary tangles are consisting of aggregates of hyperphosphorylated tau protein. Recent researches suggested that soluble oligomers may be the aggregating species responsible for Aβ neurotoxicity. Because there is no known cure for AD, developing a prevention of AD is important. Therefore, the objective of this study is to find potential phytochemicals that can protect the neuron against Aβ-induced toxicity. Methanol extract of Anglica sinensis (As-M) was proven to have protective potential in dPC12 cell model against Aβ-induced toxicity in our laboratory. Z-ligustilide (Z-LIG) is thought to be the most biologically active component in Anglica sinensis. Ferulic acid is a phenolic compound contained in Angelica sinensis, has been demonstrated that long-term administration of ferulic acid (FA) induces resistance to Aβ1-42 toxicity in the brain. We plan to use As-M and mixtures of the two compounds to evaluate their potential neuroprotective effect against Aβ-induced toxicity in primary rat cortical neurons by assessing the cell viability with MTT colorimetric assay and further investigate their mechanism of action. FA (75 μM) and Z-LIG (25 μM) showed significant protective effect to primary rat cortical neurons against Aβ1-42 toxicity, their inhibitory ratios were 28.6% and 24.0%, respectively. But As-M did not show significant protective effect against Aβ1-42 toxicity. Therefore, we mixed FA and Z-LIG to test their protective effect to primary rat cortical neurons. Our results suggested that Z-LIG and FA did not show a synergistic effect in this regard. In aggrregated human Aβ ELISA assay, EGb761, Z-LIG and FA were found to reduce the production of Aβ1-42 oligomers, indicating that these compounds may protect neuron through accelerating Aβ assembly.

並列關鍵字

Alzheimer’s disease (AD) ； amyloid-β (Aβ) ； primary rat cortical neuron ； Angelica sinensis ； Z-ligustilide ； ferulic acid

參考文獻

Sakai, S.; Kawamata, H.; Kogure, T.; Mantani, N.; Terasawa, K.; Umatake, M.; Ochiai, H. Inhibitory effect of ferulic acid and isoferulic acid on the production of macrophage inflammatory protein-2 in response to respiratory syncytial virus infection in RAW264.7 cells. Mediat Inflamm. 1999, 8, 173-175.

蔡依潔。2011。數種天然物對大鼠初代皮質神經細胞的保護功效。國立台灣大學食品科技研究所。碩士論文。台北市。

羅喬文。2008。食藥材萃出物及stilbenoisds化合物對Aβ聚集所導致的PC-12細胞凋亡之影響。國立台灣大學食品科技研究所。碩士論文。台北市。

侯怡卉。2010。數種天然物對於Aβ1-40引起的PC12及分化後PC12細胞死亡的保護功效。國立台灣大學食品科技研究所。碩士論文。台北市。

Ahmed, M.; Davis, J.; Aucoin, D.; Sato, T.; Ahuja, S.; Aimoto, S.; Elliott, J.I.; Van Nostrand, W.E.; Smith, S.O. Structural conversion of neurotoxic amyloid-beta(1-42) oligomers to fibrils. Nat Struct Mol Biol. 2010 , 17, 561-7.