金花石蒜組培苗與原生苗中加蘭它敏之毛細管柱氣相層析儀成分分析

Quantitative Analyses of Galanthamine of In Vitro and In Vivo Plant Bulbs of Lycoris aurea Herb. by Capillary Column Gas Chromatography

戴天佑(Taing-Yoe Dai)

指導教授 ： 黃麗春 教授 ； 楊玲玲 教授

臺北醫學大學/藥學院/生藥學研究所/碩士(2001年)

摘要

Galanthamine (加蘭它敏) 是石蒜科（Amaryllidaceae）植物中常見的生物鹼，可作乙醯膽鹼酯解脢之抑制劑，治療數種神經上疾病，也發展成治療阿滋海默氏症的臨床試驗用藥。但因化學合成的成本過高，全依賴天然界植物製造，故如何找出高產量品系植物或以人為方式提高產量，為本實驗之探討目標。 本實驗是以台灣原生種金花石蒜與金花石蒜組織培養苗作為分析材料，並利用敏感度高之毛細管柱氣相層析儀來檢測galanthamine的含量。使用Ultra-1 Capillary Column搭配NPD (Nitrogen Phosphorus Detector)，攜帶氣體為氮氣。氮氣的流速1.2 ml/min，注射器溫度260℃，偵測器溫度260℃。管柱起始溫度設定在200℃，升溫速度4℃/min ，最後溫度為250℃，並維持3分鐘。在此條件下注入1μl之甲醇溶劑，可偵測每毫升中最低galanthamine 1.5625μg 之濃度。 金花石蒜球莖或全株置於80℃之鼓風乾燥機烘乾一日，製成粉末後，取500豪克，先以6ml 0.05N HCl ，於40℃ 萃取 1 小時後，加入1ml 0.3N NaOH， 離心後再加入3ml Chloroform 繼續萃取，萃取物經40℃乾燥後，溶於100μl甲醇溶劑中。 以建立之標準條件分析台灣四月與八月宜蘭、淡水、桃園、后里、台東五個地區金花石蒜原生苗及金花石蒜組織培養苗galanthamine 之含量，得知二月生金花石蒜組織培養苗，球莖直徑1.25公分中每克乾物重含 12.47μg galanthamine，其他宜蘭等五個地區，四月與八月約3-4年生之金花石蒜球莖，每克乾物重 galanthamine之含量 為9.69 至138.85μg ，若以一年生瓶苗，其球莖直徑圍7.5公分之植株計算，培養苗全株及球莖galanthamine之含量分別為每克乾物重約含132.99μg及70.48μg。莖頂生長點培養所建立之無病毒健康苗導致galanthamine 產量增加，為一可能甚為重要的因素。

關鍵字

阿滋海默氏症 ； 乙醯膽鹼 ； 加蘭它敏 ； 氣相層析儀 ； 金花石蒜 ； 組織培養

並列摘要

Biologically Galanthamine is an alkaloid and belongs to a secondary metabolite, which is wildly distributed in Amaryllidaceae family. It exerts cholinesterase inhibitory effect and has been used in clinical trials against Alizheimer's disease pharmaceutically. In this investigation, capillary gas chromatography-NPD spectrometry was employed to detect galanthamine in Lycoris aurea plants in vitro and in vivo. This system was operated under the following conditions: an Ultra-1 Capillary Column (15m ×0.25mm i.d.×0.2mm film thickness；Hewlett Packard?) was coupled to the NPD (Nitrogen Phosphorus Detector). The carrier gas was Nitrogen at a constant flow rate of 1.2 ml/min. The temperatures were 260℃ for the injector, 260℃ for the detector, and 200℃ for initial column , The column temperature was linear increased with 4℃/min to reach to 250℃，and held for 3 minutes. The sensitivity can be detected as low as the concentration of 1.5625 ?g/ml in the standard sample. The sample preparation was first to dry the bulbs or entire plants in a oven at 80℃，then stored in the desecrator in the powder form. A mount of 500mg powder was first extracted in 6ml of 0.05N HCl solution at 40℃ for 1hour, then added 1ml 0.3N NaOH to neutralize, and partition with 3ml chloroform. Dry the extract with the aid of Nitrogen gas and dissolved in 100μl methanol. Determination the content of galanthamine in Lycoris aurea plants established in vitro and field growing in 5 areas in Taiwan from north to south., i.e., 宜蘭、淡水、桃園、后里、台東. Capillary GC analyses indicated the production of galanthamine in bulbs collected in April and August separately was not correlated in terms of bulb sizes and galanthamine yield. The galanthamine yields of tissue culture plants and bulbs were 132.99μg及70.48μg per gram dry weight respectively annually. The field growing plants in five different areas synthesized the galanthamine 9.69 to 138.85μg per gram dry weight of 3-4 years old bulbs. The superiority of galanthamine content from tissue culture plants probably was due to the shoot apical meristem established cultures or clones possessed the pathogen —free character.

並列關鍵字

Alzheimer's disease ； Acetylcholine ； Galanthamine ； Gas chromatography ； Lycoris. aurea Herb ； Tissue Culture

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