丹參毛狀根液態培養生產丹參酮之探討

Studies on production of tanshinone from hairy root of Salvia miltiorrhiza in liquid culture medium

羅子庭(Tzu-ting Lo)

指導教授 ： 詹効松 ； 夏奇鈮

朝陽科技大學/理工學院/生物技術與化學工程研究所碩士班/碩士(2010年)

摘要

丹參(Salvia miltiorrhiza)為唇形科(Lamiaceae)鼠尾草屬多年生草本植物，其根部含二?類的丹參酮化合物，對治療心血管疾病有良好之功效。本研究以農桿根群菌(Agrobacterium rhizogenes) R1601感染丹參葉片所得之毛狀根為材料，比較以單因子試驗設計，調整培養基中單一成分及其濃度與反應曲面試驗設計法調整培養基內之多種組成分及其濃度，對提高毛狀根之生質量及丹參酮產量之影響。試驗以B5基本鹽類配方為基礎培養基，首先進行培養基內各別成分之最佳濃度測試，進一步利用反應曲面試驗設計法(Response Surface Methodology, RSM)，進行培養基內多種成分及其濃度的同時調整，尋求對生質量、丹參酮(隱丹參酮、丹參酮I、丹參酮IIA)含量及三種丹參酮總產量最佳之培養基組成。
在單一成分試驗中對生質量之影響，結果顯示降低B5培養基中硫酸銨濃度至0 mg/L，毛狀根之最佳生質量為10.25 g/L；磷酸二氫鈉濃度提高至300 mg/L時，可得最佳生質量為9.45 g/L；硫酸銅濃度提高至75 μg/L時，可得最佳生質量為10.03 g/L。在丹參酮的含量及產量方面，調整培養基中之蔗糖、硫酸銨及硝酸鉀的濃度可提高毛狀根的丹參酮含量及產量，其中提高蔗糖濃度至60 g/L，丹參酮產量為56.93 mg/L；提高硝酸鉀濃度至3750 mg/L，丹參酮產量為53.42 mg/L；降低硫酸銨濃度至67 mg/L，丹參酮產量為60.94 mg/L。
選取六種培養基成分以反應曲面設計試驗進行培養基組成最適化研究，六種成分分別為硝酸鉀、硫酸銨、硫酸銅、氯化鈷、磷酸二氫鈉及蔗糖，由於在反應曲面設計試驗最終之中心混成試驗中未能預估出個別成分之最適值，因此減少試驗之因子數量，選擇以影響較顯著之硝酸鉀、硫酸銨及蔗糖三個因子，重新進行反應曲面試驗設計。所得之結果顯示，毛狀根生質量之預估最佳值為10.55 g/L，三個因子之最佳濃度分別為硝酸鉀5.9 g/L、硫酸銨0 mg/L以及蔗糖48.3 g/L。

關鍵字

丹參酮 ； 反應曲面試驗設計法 ； 毛狀根 ； 丹參

並列摘要

Salvia miltiorrhiza Bunge, a perennial plant belongs to Lamiaceae Family. Its roots contain diterpene compounds of tanshinones, which are important medicine for cardiovascular disease. Hairy roots derived from leaves of S. miltiorrhiza infected by Agrobacterium rhizogenes were used to produce tanshinone compounds. A salt formula of B5 medium was taken as basal culture medium and statistic methods of single factor experimental design for single medium component and response surface methodology (RSM) design for multiple medium components were conducted separately on optimization medium composition for tanshinone (cryptotanshinone, tanshinone I, tanshinone IIA) production in this study.
In single factor design experiments, decreasing ammonium sulfate concentration (0 mg/L), increasing sodium dihydrogen phosphate (300 mg/L) and sulfate copper concentration (75 μg/L) of the B5 medium enhanced hairy root biomass production to 10.25 g/L, 9.45 g/L and 10.03 g/L, respectively. Meanwhile, increasing concentration of sucrose (60 g/L), ammonium sulfate (67 g/L) and potassium nitrate (3750 mg/L) was found to enhance tanshinone content and three tanshinones production of hairy root was up to 56.93 mg/L, 60.94 mg/L, and 53.42 mg/L, respectively.
Six components of potassium nitrate, ammonium sulfate, copper sulfate, cobalt chloride, sodium dihydrogen phosphate and sucrose were tested in proceeding of optimization medium composition using response surface methodology. Since the of central composite design failed to estimate the optimal value, three components factors of potassium nitrate, ammonium sulfate and sucrose which with more influential on tanshinone production were chosen to conduct RSM again. According to the mathematic calculation, the optimal hairy root biomass was 10.55 g/L, and the best concentration of potassium nitrate, ammonium sulfate and sucrose was 5.9 g/L, 0 mg/L and 48.3 g/L, respectively.

並列關鍵字

response surface methodology ； hairy root ； tanshinone ； Salvia miltiorrhiza

參考文獻

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