建立新疆雪蓮癒合組織生產有效成份之研究

Establishment of Saussurea involurata callus cultures for the production of active ingredients.

黃楚朋(Chu-Peng Huang)

指導教授 ： 蔡新聲

朝陽科技大學/理工學院/應用化學系生化科技碩士班/碩士(2014年)

摘要

新疆雪蓮 (Saussurea involucrata Kar. et Kir.) 為菊科鳳毛菊屬多年生草本植物，又名雪荷花，株高15~30 cm左右；葉叢生，呈長橢圓形，邊緣有鋸齒；花苞多層，呈蓬座狀，具管狀、紫紅色花冠。新疆雪蓮全株所含化學成分有生物鹼、多醣類、lignans與guaianolides等，主要以黃酮類為主，如懈皮素 (quercetin) 及蘆丁 (rutin)，而木質素則以紫丁香苷 (syringetin) 為主。新疆雪蓮所含之黃酮類化合物，研究發現在抗風濕、消炎鎮痛、擴張血管、降血壓、防止心血管疾病、增強免疫、延緩衰老、抗癌及抗疲勞等具明顯療效。近年來，野生新疆雪蓮因生長緩慢、繁殖困難、過度採挖以致藥材短缺，故利用藥材來萃取雪蓮有效成分有應用上之困難，而利用植物組織培養即為解決方法之一。本研究主要培養新疆雪蓮之癒合組織，再經由添加誘引劑誘導成分表現，大量生產黃酮類化合物。本研究根據基礎鹽類、培植體部位、凝膠物質、植物生長調節劑、培養溫度及誘引劑等變因，進行新疆雪蓮癒合組織誘導、增殖及有效成分表現的試驗設計，並將所得之有效成分結果與新疆雪蓮瓶苗與市售藥材進行比較。結果顯示，誘導新疆雪蓮癒合組織以葉片為最佳培植體，經1/2 MS基礎鹽類添加0.4% gelrite搭配0.5 mg/L BA，0.5 mg/L 2,4-D或是1.0 mg/L BA，0.5 mg/L 2,4-D之試驗組可得最高誘導率達80.0%。癒合組織之最佳增殖條件以1/2 MS基礎鹽類添加0.4% gelrite添加0.5 mg/L BA，0.5 mg/L NAA及3.0 mg/L 離層酸 (Abscisic acid)，於20℃並進行透氣處理條件下培養4週，每0.2 g葉片癒合組織之鮮重可增加7.7倍至1.740 g。在添加誘引劑使新疆雪蓮葉片癒合組織累積有效成分試驗中，將培植體培養在20℃的環境下，對照組和培養在3 mg/L苯丙氨酸 (phenylalanine) 之癒合組織，分別測得最高的syringetin (13.524 mg/g) 和rutin (0.374 mg/g) 含量。以HPLC分析不同的培植體材料和市售藥材，發現在syringetin和rutin之含量，分別具有不同程度之差異。在培養8週組培苗之基部測得到最高的syringetin含量 (11.235 mg/g)，而野生藥材葉片之rutin含量 (2.556 mg/g) 為最高。本研究結果證實，利用組織培養技術所生產之新疆雪蓮組培苗與癒合組織其有效成分syringetin和rutin化合物之含量，並不輸給野生新疆雪蓮植株。

關鍵字

蔡新聲 ； 癒合組織 ； 蘆丁 ； 懈皮素 ； 紫丁香苷 ； 黃楚朋 ； 新疆雪蓮

並列摘要

Saussurea involucrata Kar.et Kir (Family Asteraceae) is a perennial herb. The herb is also known as snow S. involucrata, since it mainly grows on snowy mountains. The plant is about 15 to 30 cm in height; leaf in clusters, elongated oval, with serrated edges, enclosing the bud; flower with a tubular purple corolla. S. involucrata contain polysaccharides lignans, guaianolides, and flavonoids, such as quercetin and rutin and syringetin. These flavonoids have been reported to be anti-rheumatic, anti-inflammatory, dilate blood vessels, lower blood pressure, prevent cardiovascular disease, and enhance immunity, anti-aging, anti-cancer and anti-fatigue. In recent years, due to excessive collection of S. involucrata plants in the natural habitat, and their slow growth in wild, the species has become endangered, resulting in short supply of plant materials and scarcity of medicine. Owing to this, the direct use of herbs to extract the active ingredients is a serious limitation. Hence, it is very important to apply tissue culture technique to obtain the active compounds. In the present study, different plant parts (leaf, petiole and root) of S. involucrata were used as explants to induce callus. Experiments were carried out to optimize the callus induction and its proliferation using different physical (temperature, container closure type) and chemical factors (different basal media, plant growth regulators, gelling substances and elicitors).
Induction of callus in leaf, petiole and roots explants of S. involucrate varied among the five different basal media. The maximum leaf explants (80%) induced callus on 1/2 MS basal medium supplemented with 0.4% Gelrite, 0.5 mg/L BA, 0.5 mg/L 2,4-D or 0.4% Gelrite, 1.0 mg/L BA, 0.5 mg/L 2,4-D. The highest callus proliferation was achieved in S. involucrata leaf callus culture on 1/2 MS basal medium supplemented with 0.5 mg/L BA, 0.5 mg/L NAA, 0.4% Gelrite. In the experiment with temperature, the maximum callus proliferation was recorded at 20℃. Sealing of culture vessels with 3 layers of Dispense papers supported the maximum callus proliferation (1.036 g) on medium supplemented with 0.5 mg/L BA and 0.5 mg/L NAA. In the experiment on effect of elicitors on syringetin and rutin content in callus, we obtained the maximum syringetin (13.524 mg/g) and rutin (0.374 mg/g) contents on control and medium with phenylalanime (3.0 mg/L) respectively, when cultures were incubated at 20℃. The HPLC analysis of different in vitro plant materials and commercially available crude drug had different levels of syringetin and rutin contents in them. The highest syringetin (11.235 mg/g) and rutin (2.556 mg/g) content were recorded in petioles of two month old in vitro plantlets and leaves of crude drug, respectively. Interestingly, leaf callus in an experiment with elicitors induced a still higher syringetin content (13.524 mg/g) in control (on 1/2 MS basal medium supplemented with 0.5 mg/L BA, 0.5 mg/L NAA, 3% sucrose, 0.4% Gelrite and without any elicitor.).
In summary, the present results demonstrate that tissue culture technique can be used successfully to obtain the syringetin and rutin compounds in Saussurea involucrate without sacrificing the plants in the wild.

並列關鍵字

rutin ； quercetin ； syringetin ； Chu-Peng Huang ； callus ； Dr. Hsin-Sheng Tsay ； Saussurea involurata

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