粉防己（Stephania tetrandra S. MOORE）組織培養之研究：粉防己癒合組織培養及其成分測定

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Title:	粉防己（Stephania tetrandra S. MOORE）組織培養之研究：粉防己癒合組織培養及其成分測定
Other Titles:	Studies on Tissue Culture of Stephania tetrandra S. MOORE : Callus Culture of Stephania tetrandra S. MOORE and Determination of its Constituents
Authors:	張君儀;Chun-Yi Chang
Contributors:	中國醫藥大學：中國藥學研究所
Keywords:	粉防己;組織培養;癒合組織;粉防己鹼;防己諾林鹼;Stephania tetrandra;tissue culture;callus;tetrandrine;fangchinoline
Date:	2008-07-18
Issue Date:	2009-08-10 18:02:16 (UTC+8)
Abstract:	防己為常用中藥，粉防己為防己之基原植物。粉防己(Stephania tetrandra S. MOORE)為防己科(Menispermaceae)千金藤屬(Stephania LOUR.)之多年生藤本植物，有利水消腫，祛風止痛之功效，故常用於降血壓、抗心律失常、抗纖維化、抗腫瘤與消炎等，與粉防己活性成分防己諾林鹼(fangchinoline)及粉防己鹼(tetrandrine)有關。本研究利用植物組織培養技術，建立粉防己癒合組織之誘導及繼代培養系統，結果發現在MS基礎鹽類含2 mg/L 2,4-D及0.5 mg/L BA之固體培養基中，葉片培殖體可獲得最高誘導率，而粉防己癒合組織之最佳繼代培養條件為MS基礎鹽類添加1 mg/L BA、0.5 mg/L TDZ、3% sucrose、0.9% Difco agar及pH 5.7 ± 0.1之培養基，在25 ± 1 ℃之恆溫下進行暗培養，以30天繼代一次為宜，添加2 g/L 蛋白凍或500 mg/L 水解酪蛋白或10% 椰子汁均對癒合組織之生長有利，其中尤以蛋白凍效果最佳，添加馬鈴薯則呈反效果。在MS基礎鹽類添加1 mg/L BA、3% sucrose、1% Difco agar及pH 5.7 ± 0.1之培養基，在25 ± 1 ℃之恆溫下進行暗培養，有利於粉防己二次代謝物fangchinoline及tetrandrine之生合成。另外，添加1 g/L 蛋白凍或250 mg/L 水解酪蛋白或10% 椰子汁均可有利於tetrandrine的生成，其中又以10% 椰子汁最具效果，癒合組織經35天培養可獲得0.392 mg/g dry wt。培養基中添加4 g/L 蛋白凍或750 mg/L水解酪蛋白或20% 椰子汁均可有利於fangchinoline的生成，其中又以750 mg/L水解酪蛋白效果最佳，癒合組織經35天培養可獲得 0.660 mg/g dry wt。經由本研究粉防己癒合組織之誘導、最佳繼代培養條件之建立及有機成分之添加，未來將能達成大量生產其活性成分之目的。 Stephaniae Tetrandrae Radix, a common-used herb in tradition Chinese medicine, is scientifically known as Stephania tetrandra S. MOORE (Menispermaceae). Normally it is used for reducing blood pressure, edema and pain, and has anti-hypertensive, anti-arrhythmia, anti-fibrotic, anti-tumor and anti-inflammatory effects. Fangchinoline and tetrandrine are the two major active compounds of S. tetrandra. The main objective of this research is to establish an efficient protocol for induction and proliferation of its callus by tissue culture techniques. It was found that the best explant for callus initiation was leaf cultured on MS basal medium supplemented with 2 mg/L 2,4-D, 0.5 mg/L BA. The best cultural condition for callus proliferation was subculturing callus every 30 days on MS basal medium supplemented with 1 mg/L BA, 0.5 mg/L TDZ, 3% sucrose and 0.9% Difco agar at pH 5.7 ± 0.1 and incubated in the dark at 25 ± 1 °C. Medium containing 2 g/L peptone or 500 mg/L casein hydrolysate (CH) or 10% coconut milk (CM) could promote callus growth. A culture medium containing MS basic salts supplemented with 1 mg/L BA, 3% sucrose and 1% Difco agar at pH 5.7 ± 0.1 in the dark at 25 ± 1 °C enhanced fangchinoline and tetrandrine production. By adding 1 g/L peptone or 250 mg/L CH or 10% CM could promote tetrandrine production, especially 10% CM containing medium possessed the highest tetrandrine content (0.392 mg/g dry wt). Whereas, adding 4 g/L peptone or 750 mg/L CH or 20% CM into medium increased the fangchinoline production, the content of fangchinoline (0.660 mg/g dry wt) from 750 mg/L CH containing medium was found to be the highest among three medium tested. In this study, we provided an efficient induction and proliferation protocol which could be used for large production of active compounds of S. tetrandra.
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