山煙草試管內根培養並利用高效液相層析儀分析澳洲茄鹼含量

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Title:	山煙草試管內根培養並利用高效液相層析儀分析澳洲茄鹼含量 HPLC analysis of solasonine extracted from in vitro roots of Solanum verbascifolium L.
Authors:	高曉萱 GAO, SIAO-SYUAN
Contributors:	生物科技研究所
Keywords:	茄科固醇類生物鹼液體懸浮培養繼代週期放大培養 Solanaceae steroidal glycoalkaloids suspension culture subculture period large scale
Date:	2012-06
Issue Date:	2013-11-07 14:55:03 (UTC+8)
Abstract:	山煙草為茄科茄屬植物，含有抑制癌細胞生長之澳洲茄鹼類（solsonine）之生物活性二次代謝產物，根部為主要合成部位。故本研究利用山煙草之無菌苗，進行山煙草不定根及癒傷組織之誘導及根懸浮培養系統之建立，以分析比較山煙草懸浮根、乾材及各部位之澳洲茄鹼含量。研究結果觀察到：1.山煙草無菌苗之根、莖、葉培植體培養於含1.0 mg L-1 1-萘乙酸 (α-naphthaleneacetic acid, NAA) 與0.1 mg L-1 6-苄氨基嘌呤 (6-Benzylaminopurine, BAP) 之組合下，根部培植體增殖，莖及葉培植體可形成不定根，平均根數分別為142.4、142.4和12.15.3；2.山煙草懸浮根培養於不含生長調節劑之環境，懸浮根增殖28倍；3.繼代週期對於懸浮根生長的影響，於五週內分別每1、2、3或4週繼代一次，觀察到每隔1週或2週繼代一次，可使懸浮根增殖100與91倍；4.在量化培養方面，取初始鮮重為50、150、450 mg之懸浮根至250 mL進行放大培養，顯示初始重量為50 mg鮮重之懸浮根時，四週後可明顯增殖99.68倍；5. 利用高效液相層析儀分析500 mg乾重之山煙草根、莖、葉、果實、乾材與懸浮根之澳洲茄鹼含量，以果實為最多 (12.91.32 mg) ，依序下來為山煙草乾材 (9.741.09 mg) 和根部 (10.690.62 mg) 、山煙草懸浮根 (7.550.90 mg) ，而葉片與莖部並無偵測到澳洲茄鹼。綜觀上述，山煙草懸浮根在MS 基本培養基可大量增殖，惟澳洲茄鹼含量略低於果實、根部及乾材，因此未來將可利用生物反應器量化增殖山煙草懸浮根，從而獲取足夠的萃取材料，以利於工業之生產用途。 Solanum verbascifolium L., belonging to Solanaceae, contains biactive solasonine which has been reported to inhibit growth of liver and colon cancer cells and is mostly synthesized in roots. For establishing suspension roots culture, NAA and BAP were used to induce callus and adventitious root formation from the leave, stem, root of in vitro seedlings of S. verbascifolium L. The adventitious roots induced from stems on MS basal medium containing 1.0 mg L-1 NAA and 0.1 mg L-1 BAP can proliferate up to 28-folds in the liquid MS basal medium with suspension culture. In the subcuture period experiment, in vitro roots derived from adventitious roots of stem can proliferate up to 100-folds or 91-folds after 5 weeks of culture in every one or two weeks of subculture, respectively. For large scale culture, in vitro root of initial weight of 50, 150 and 450 mg can proliferate up to 99-folds, 71-folds, 30-folds in 250 mL flasks after 4 weeks of culture, respectively. HPLC has been applied to analyze the solasonine contents of in vitro roots, 9-month-old roots, leaves, fruits and dry marterials of S. verbascifolium L. HPLC analyses showed that the fruits contain most amount solasonine of 12.91.32 mg, roots 10.690.62 mg, dry materials 9.741.09 mg, and in vitro roots 7.550.90 mg, however, stems and leaves were not been detected. In conclusion, the in vitro root culture system will be beneficial for the sustainable utilization of S. verbascifolium L. and it will be useful for the large scale cultivation of in vitro roots as an alternative method to prevent destroying the whole plants. In vitro roots of S. verbascifolium are expected to be a valuable material for analyzing bioactive ingredients.
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