臺灣鉤藤活性成分及 statin 新穎衍生物之神經細胞保護活性研究

Neurocytoprotective effects on the constituents of Uncaria hirsuta Haviland and novel statin derivatives

林怡慈(Yi-Tzu Lin)

指導教授 ： 李美賢

臺北醫學大學/藥學院/生藥學研究所/碩士(2013年)

摘要

帕金森氏症 (Parkinson’s disease) 為常見的神經退化性疾病之一，乃由於黑質體多巴胺神經元受損所造成，主要發生在老年人身上。本研究中，本論文以六-羥基多巴胺 (6-hydroxydopamine) 處理 PC12 細胞株作為體外試驗模式，探討中藥材-臺灣鉤藤 (Uncaria hirsuta Haviland) (茜草科) 及 statin 新穎衍生物之神經保護作用。臺灣鉤藤為中藥材鉤藤的來源之一，本論文以其帶鉤莖枝及葉部，分別利用 95% 酒精及水加熱迴流萃取，再以乙酸乙酯、正丁醇及水進行部份劃分。利用薄層色層分析法 (TLC) 及高效能液相層析法 (HPLC) 進行化學指紋圖譜分析。以具有較佳的神經細胞保護活性之正丁醇層萃取物，利用高效液相層析儀-固相萃取-核磁共振儀串連分析技術 (LC-SPE-NMR) 及各種管柱層析進行化學成分之分離純化，得到三個化合物，經理化及光譜相關數據與參考文獻比對後，確認結構為 5β-carboxystrictosidine (1)、chlorogenic acid (2)、uncarine A (3)，其中 5β-carboxystrictosidine (1) 及 chlorogenic acid (2) 具有顯著的神經細胞保護活性。本論文並以紅麴菌的二次代謝產物 lovastatin 進行結構修飾，合成一系列的 statin 類新穎衍生物 (S1~S12)，探討其神經保護活性及作用機制，結果顯示 S10 於 100 μM 濃度下，具有顯著的神經保護活性且具有劑量依存性，EC50 為 63.6 μM。S10 可抑制六-羥基多巴胺誘導的 PC12 細胞凋亡，具抑制細胞內 caspase-3 及 caspase-8 活性，但對細胞內 ROS含量無影響。期望透過本研究在臺灣鉤藤及statin 衍生物之研究，提供未來於發展治療或預防帕金森氏症藥物之參考。

關鍵字

帕金森氏症 ； 神經保護 ； 臺灣鉤藤 ； PC12 細胞 ； statin

並列摘要

Parkinson’s disease is one of the most common neurodegenerative diseases affecting the elders. It mainly results from the damages of dopaminergic neurons in the substantia nigra. In the present study, we investigated the neurocytoprotective effects of Uncaria hirsuta Haviland (Rubiaceae), one of Chinese herbal medicines, and novel statin derivatives using 6-hydroxydopamine (6-OHDA)-treated nerve growth factor (NGF)-differentiated PC12 cells as an in vitro model. U. hirsuta was used as one of the sources of the Chinese herbal medicine, “Gou-teng”. In this study, hooks and leaves of U. hirsuta were extracted with 95% ethanol and water, respectively, and then partitioned with a sequence of ethyl acetate (EA), n-butanol (n-BuOH) and water. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were represented as chemical chromatographic fingerprints. n-Butanol extract exhibited greater neurocytoprotective activity and was isolated and purified by high-performance liquid chromatography with post-column solid-phase extraction to nuclear magnetic resonance spectroscopy (LC-SPE-NMR) and series of columns. Three compounds were obtained and their structures were identified as 5β-carboxystrictosidine (1), chlorogenic acid (2) and uncarine A (3) by physical and spectroscopic characterizations. Among these compounds, 5β-carboxystrictosidine (1) and chlorogenic acid (2) showed significant neurocytoprotective activity. Lovastatin is a secondary metabolite of Anka. We also evaluated the neurocytoprotective effects and mechanisms of semi-synthetic novel lovastatin derivatives (S1~S12) obtained from Dr. Huang. S10 showed neurocytoprotective effects on 6-hydroxydopamine (6-OHDA)-treated NGF-differentiated PC12 cells with EC50 of 63.6 μM. S10 also inhibited the apoptosis, caspase-3, and caspase-8 activation caused by 6-OHDA, but it did not affect intracellular reactive oxygen species (ROS) levels. The present results imply that U. hirsuta and lovastatin derivertives may be useful in treating or preventing Parkinson's disease in the future.

並列關鍵字

Parkinson’s disease ； neurocytoprotective ； Uncaria hirsuta ； PC12 cell ； statin

參考文獻

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