建立粉防己毛狀根培養生產粉防己鹼之研究

Establishment of Hairy Root Culture in Stephania tetrandra S. MOORE for the Production of Tetrandrine

劉選頡(Hsuan-Chieh Liu)

指導教授 ： 詹効松

朝陽科技大學/理工學院/生化科技研究所/碩士(2010年)

摘要

粉防己鹼(Tetrandrine)為bisbenzylisoquinoline類之生物鹼，萃取自中草藥植物粉防己(Stephania tetrandra S. MOORE)的根，具有抗發炎、抗氧化及降血壓等功效，並能有效抑制結腸癌細胞生長與和血小板凝集之作用；本研究利用農桿根群菌(Agrobacterium rhizogenes)菌株ATCC 15834及 ATCC 43057轉殖粉防己所誘導的毛狀根(Hairy roots)，於液態培養基中建立二次代謝產物粉防己鹼之生產系統，提供了有效生產二次代謝產物的另一途徑。所獲得毛狀根經PCR鑑定證實為轉殖根系，挑選高生質量及高粉防己鹼含量根系HR10-1, HR11-1經固態培養基培養一個月後，其生質量分別為 0.137, 0.068 g dw/petri-dish，經HPLC分析其粉防己鹼含量分別為 8.57, 11.66 mg/g，其值高於市售藥材(4.81 mg/g)；比較毛狀根在不同基礎鹽類配方(MS, 1/2MS, B5, WPM, N6)之固態培養基中生長狀況，可發現WPM培養基最適合粉防己毛狀根生長；粉防己毛狀根於WPM液態培養之生長曲線試驗，於第五週可達最高生質量為 6.5 g/L，粉防己鹼產量為 68.69 mg/L；以WPM鹽類配方為基礎培養基，進一步利用反應曲面試驗設計法(Response Surface Methodology, RSM)，針對培養基內蔗糖、硝酸銨和硝酸鈣等組成進行調整，尋求其對生質量、粉防己鹼含量及產量之最佳濃度組成，於二階層試驗結果顯示，提高硝酸銨與硝酸鈣可得較高之粉防己鹼含量與產量，分別為控制組的1.8倍與1.5倍，針對生質量的陡升路徑試驗中，結果於第5組的處理，可得最高之生質量，中心混成之生質量實驗設計試驗之結果顯示，三個因子最佳濃度分別為硝酸銨 564.34 mg/L、硝酸鈣 624.14 mg/L、蔗糖 41.24 g/L，毛狀根生質量之預估最佳值為 8.09 g/L，此外，以中心混成之粉防己鹼產量實驗設計試驗之結果顯示，三個因子最佳濃度分別為硝酸銨555.48 mg/L、硝酸鈣880.41 mg/L、蔗糖29.32 g/L，毛狀根粉防己鹼產量之預估最佳值為 65.74 mg/L。毛狀根利用氣泡塔生物反應器及噴霧滴流式生物反應器培養於WPM培養基五週後，結果顯示，粉防己毛狀根之生質量、粉防己鹼含量及產量經氣泡塔生物反應器培養都可得較高值。
關鍵字：粉防己、毛狀根、粉防己鹼、反應曲面試驗設計法、生物反應器

關鍵字

粉防己鹼 ； 反應曲面試驗設計法 ； 毛狀根 ； 粉防己 ； 生物反應器

並列摘要

Stephania tetrandra S. MOORE is an important Chinese medicinal herb and it has been traditionally used as anti-allergic, anti-inflammatory, anti-cancer and against coronary diseases. Tetrandrine, a bis-benzylisoquinoline alkaloid has been identified as an active ingredient in S. tetrandrae and it is mainly exists in the root. Extraction of these metabolites from plants grown in the natural habitat is tedious and several factors can alter their yield. In vitro hairy root culture can be the probable solution to extract this alkaloid. The present study describes the consistent production of tetrandrine, achieved by Agrobacterium mediated transformation using A. rhizogenes strains ATCC 15834 and ATCC 43057. WPM was the most suitable media for in vitro hairy root culture. The integration of foreign gene into the genome was confirmed by PCR using rolB and rolC gene specific primers in different transgenic lines. There was variation in the growth rate and alkaloid content within different transgenic hairy root lines. Two transgenic lines HR10-1 and HR11-1 were selected and their HPLC analysis showed that the content of tetrandrine was 8.57 mg g-1 and 11.66 mg g-1, respectively. The tetrandrine contents were higher than that in the marketed crude drug (4.81 mg g-1). Furthermore, the HR10-1 transgenic line produced maximum biomass (6.5 g L-1) and tetrandrine (68.69 mg L-1) after 5 weeks cultivation. Affect three additives total biomass Response Surface Methodology (RSM). For media optimization were used to the higher biomass by alterity three different component of WPM media ie (NH4NO3, Ca(NO3)2 and sucrose). According to the central composite design experiment and mathematic calculation, the optimal concentration of NH4NO3, Ca(NO3)2 and sucrose was 564.34 mg L-1, 624.14 mg L-1, 41.24 g L-1, respectively, and the best hairy root biomass was 8.09 g L-1.Our results indicated that transgenic lines of S. tetrandra hairy root culture exhibited potential for mass production of tetrandrine.
Key words: Stephania tetrandra S. MOORE, Hairy root, Tetrandrine, RSM, Bioreactor

並列關鍵字

Stephania tetrandra S. MOORE ； Hairy root ； RSM ； Tetrandrine ； Bioreactor

參考文獻

林李昌、李鎮宇、林昆彥、葉茂生、蔡新聲。2006。何首烏毛狀根誘導與 Emodin 及 Physcion 之成分分析。作物、環境與生物資訊3: 265-274。

陳威臣、詹効松、李秋儀、曹進義、劉禎祺、李艷琪、夏奇鈮。2008。丹參毛狀根培養建立與其丹參酮產量之研究。台灣農業研究。57: 305–316。

林珠鳳。2006。建立丹參轉殖毛狀根系統及其二次代謝產物生產之研究。國立中興大學農藝學系碩士論文。台中，台灣。

陳菀茹。2007。建立高氏柴胡轉殖毛狀根系統之研究。國立中興大學農藝學系碩士論文。台中，台灣。

常振鎧。2004。建構絞股藍毛狀根培養系統生產類人參皂甙之研究。國立台灣大學微生物與生化學研究所博士論文。台北，台灣。