以茭白筍黑粉菌及醱酵糟生產stigmastane-3,6-dione之條件探討

惡性腫瘤的抗藥性為癌症病人的死因之一，癌症的抗藥性機制有很多，目前最著名的就屬P-醣蛋白，其為跨膜蛋白、可以協助癌細胞排除抗癌藥物。近年來研究指出從茭白筍可以萃取出豆甾烷3,6二酮stigmastane-3,6-dione，豆甾烷3,6二酮stigmastane-3,6-dione不僅可以顯著降低P-醣蛋白的表現，同時亦可抑制肝癌細胞及抑制破骨細胞的活性。目前豆甾烷3,6二酮stigmastane-3,6-dione是以生物半合成法製成，故其價格極其昂貴，本研究以醱酵槽培養菰黑粉菌試圖改變接種量及攪拌速度，探討養菌的條件對產生豆甾烷3,6二酮stigmastane-3,6-dione的影響。醱酵培養部份的結果為:第一次培養條件:140 rpm，300萬的接種量，25℃的培養，第5天以TLC分析豆甾烷3,6二酮stigmastane-3,6-dione量較多，菌絲球中等且數量多。因期望菌絲球大顆故第二次培養條件只降低接種量為100萬顆孢子，培養過程中可觀察到菌絲球比第一次大顆且數量少，以TLC分析在培養到第7天時豆甾烷3,6二酮stigmastane-3,6-dione量較多。在140 rpm的攪拌速率下，部份菌絲沉積在醱酵槽的底部，故第三次培養時只調降攪拌速率為100 rpm其餘因子不變，在第6天以TLC分析豆甾烷3,6二酮stigmastane-3,6-dione量較多、菌絲球中等且數量多。為增大菌絲球，第四次培養條件只改變接種量為30萬、其餘條件不變，以TLC分析第5天豆甾烷3,6二酮stigmastane-3,6-dione量較多菌絲球中等且數量多。且經本次實驗以氯仿萃取豆甾烷3,6二酮stigmastane-3,6-dione較過去研究指出的甲醇效果較為佳，最後以第二次醱酵的條件將3 L培養基萃取後經TLC分析，並取可能含有豆甾烷3,6二酮stigmastane-3,6-dione的TLC部份，分別用一次、二次質譜分析，驗證豆甾烷3,6二酮stigmastane-3,6-dione的存在。

關鍵字

豆甾烷3,6二酮；醱酵培養；菰黑粉菌；腫瘤耐藥性

並列摘要

Cancer drug resistance is a major cause of death for cancer patients. Many mechanisms of drug resistance have been known. The most famous cancer drug resistance protein is p-glycoprotein, which is a transmembrane protein and may help cancer cells to exclude anti-cancer drugs. Recently, the studies indicated that stigmastane-3 ,6-dione extracted from water bamboo can not only significantly reduce the expression of P-glycoprotein but also inhibit the proliferation of liver cancer cells and osteoclast activity. Stigmastane-3,6-dione is a semi-synthesized so the price is extremely expensive. This study tested the possibility of using a fermentor to culture Ustilago esculenta and to produce stigmastane-3, 6-dione by exploring the conditions such as the inoculum and the stirring speed. We found that stigmastane-3, 6-dione dissolved better in chloroform than in methanol and acetonitrile. Followed by TLC separation and MS analyses, stigmastane-3, 6-dione was identified in the extract of culture medium. Therefore fermentation culture of Ustilago esculenta could be one way to produce stigmastane-3, 6-dione in large scale.