臺灣博碩士論文加值系統

迷迭香（Rosmarinus officinalis）為歐洲傳統藥用植物，其植株中所含的迷迭香酸（Rosmarinic acid；RA），已被文獻證明具有抗菌、抗氧化、抗病毒、抗發炎等生理活性，目前已經被應用於食品、藥品以及化妝品。本實驗研究目的擬以建立迷迭香懸浮細胞並利用生物反應器生產二次代謝物迷迭香酸。
利用迷迭香的葉片，以MS培養基添加植物生長調節激素NAA 1~4 mg/L，於25℃黑暗環境下均可誘導產生癒合組織（Callus）。經篩選生長較佳的細胞株進行RA含量分析為2~4 % (dw)，進而挑選出生長快速且RA含量最高的細胞株(R.O.2-2)進行液態培養研究。
接著探討於三角瓶中細胞接種量對細胞增殖與RA含量累積之相關性。結果顯示，在500 mL三角瓶內置入150 mL培養基，當其細胞接種量為10 g/L時，培養18天後可獲得細胞乾重 13.07 g/L (dw)，RA總產量最高為0.88 g/L。
進一步利用含有2 mg/L NAA和3% 蔗糖MS培養基之5 L生物反應器進行批次培養。接種量20 g/L，於25℃，通氣量0.5 vvm和攪拌速率20 rpm下，經過13天之培養，細胞乾重達到16.2 g/L，而迷迭香酸產量則為0.33 g/L。進一步進行饋料批次培養，獲得本研究之最佳結果。18天後細胞乾重為19.1 g/L，而迷迭香酸之產量為0.91 g/L。

Rosmarinus officinalis is an European traditional medicinal herb. Rosemary have been shown to accumulate rosmarinic acid (RA) in the plant tissue. RA has a number of interesting biological activities, e.g. antiviral, antibacterial, antiinflammatory and antioxidant. RA has been used as an ingredient of drug, food and cosmetic.
Leaf explants were used to induce callus with MS basal medium and 1~4 mg/L NAA under dark. RA contents were determined on cell lines which had better growth rate. For all tested samples, the RA ratios were between 2~4 % (dw). Some calli with high proliferation rates and RA contents were transferred to liquid medium to initiate suspension culture.
The purpose of this study was to establish R. officinalis suspension cell for the production of rosmarinic acid. In this study, the effect of inoculum size and RA production in the suspension cell culture system were investigated. Results indicate when inoculum size was 10 g/L, the maximum cell concentration was 13.07 g/L of the dry weight (dw) and RA productivity was 0.88 g/L.
For the cultivation of R. officinalis in 5 L stirred bioreactor, we found that a maximum cell concentration of 16.2 g/L dry weight and rosmarinic acid concentration of 0.33g/L were obtained on batch culture. However, on a maximum cell concentration of 19.1 g/L dry weight and rosmarinic acid concentration of 0.91 g/L were obtained on fed-batch culture. The results revealed that the fed-batch culture was better than batch culture for R. officinalis.

中文摘要 ………………………………………………………. I
英文摘要 ……………………………………………………….. II
目 錄 ……………………………………………………….. III
表 次 ……………………………………………………….. V
圖 次 ……………………………………………………….. VI
縮寫表 ……………………………………………………….. VIII
第一章 前言 ……………………………………………………. 1
第一節 迷迭香植株之介紹 …………………………………. 1
第二節 迷迭香所含各類成分之介紹 ………………………. 3
第三節 迷迭香酸之基本特性 ………………………………. 5
第四節 植物二次代謝物 ……………………………………. 10
第五節 植物細胞生產迷迭香酸之研究 ……………………. 14
第六節 研究動機與目的 …………………………………. 14
第二章 材料與方法 ……………………………………………. 17
第一節 迷迭香無菌植株之來源 ……………………………. 17
第二節 迷迭香癒合組織之誘導 ……………………………. 18
第三節 迷迭香酸之萃取與分析 ……………………………. 20
第四節 懸浮細胞之建立 ……………………………………. 23
第五節 形態觀察 ……………………………………………. 24
第六節 細胞株之繼代培養 …………………………………. 24
第七節 接種量對迷迭香懸浮細胞培養之影響 ……………. 24
第八節 細胞生長之測定 ……………………………………. 25
第九節 生物反應器之培養 …………………………………. 26
第十節 儀器 …………………………………………………. 29
第十一節 藥品 ………………………………………………. 30
第三章 結果與討論 ………………………...………………… 32
第一節 種子表面殺菌之結果 ………………………………. 32
第二節 迷迭香植株中各部位迷迭香酸之含量 ……………. 32
第三節 迷迭香癒合組織之誘導………………………………. 32
第四節 迷迭香酸之分析 ……………………………………. 38
第五節 迷迭香懸浮細胞之建立 ……………………………. 38
第六節 接種量對迷迭香酸懸浮細胞之影響…………………. 38
第七節 生物反應器之培養 …………………………………. 53
第四章 結 論…………………………………………………….. 68
第五章 參考文獻 ……………………………………………….. 70

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