蔣丙煌臺灣大學：食品科技研究所蘇振強Su, Chen-ChiangChen-ChiangSu2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/56263三七(Panax notoginseng (Burk.) F. H. Chen)乃五加科人參屬，為中國傳統名貴藥材之一；目前已被證實具有鎮痛、護肝、抗發炎、抗血栓、降血壓、保護心肌與抗腫瘤等生理功效，其主要生理活性物質為多醣（一次代謝產物）和皂苷（二次代謝產物）。文獻指出皂苷可透過微生物的代謝提升其生理活性，故本實驗以三七為基質，利用乳酸菌醱酵進行生物性轉化，探討最佳醱酵條件(包括醱酵時間、溫度和pH值)，再藉由體外和體內試驗評估醱酵產品抑制肝癌細胞株生長之功效，並分析皂苷成分變化，期望提升三七在抗肝癌方面之效用。本研究之體外實驗結果發現，以6.6 公升醱酵槽執行醱酵時，在37℃、不控制pH值，醱酵48小時後可得到具最佳抑制肝癌細胞功效之醱酵液（Hep 3B與Hep G2之IC50分別為226 μg/ml、149 μg/ml）。同時，將最具生理活性效果之三七乳酸菌醱酵液以甲醇萃取後，再以LC–ESI-MS/ MS結合CID進行其結構分析與鑑定，可得到protopanaxtriol、ginsenoside Rh1、notoginsenoside R2與ginsenoside Rf四種皂苷；其中protopanaxtriol與ginsenoside Rh1之抗癌效果更已被證實。此部分結果可作為日後純化分離得到更具抑制肝癌細胞增生、降低對正常細胞毒性之產品之參考。後續將以動物模式進一步探討醱酵液抗肝癌之功效。Panax notoginseng (Burk.) F. H. Chen, is one of the most famous traditional Chinese medicines. It belongs to the Araliaceae family. Its medicinal properties are sweet and slightly bitter in flavor and warm in nature. The main bioactive compound was regarded as saponins, and was used for treatment of diseases of cardiovascular, central nervous, and hematopoietic systems. Cancer prevention and hepatoprotection by saponins have received increasing attention. It has been found that the ginsenosides could be transformed by human intestinal bacteria to metabolites with improvement of anti-carcinogenic activities. Biotransformation of saponins of P. notoginseng by lactic acid bacteria (LAB) is a possible way to enhance its anti-hepatoma activity. The objectives of this study are (1) to establish the optimal conditions, such as fermentation time, temperature and pH value, for the lactic acid fermentation of P. notoginseng, to enhance the anti-hepatoma function of the fermented products; (2) to investigate the bioactive components in the fermentation products which are responsible for the anti-hepatoma activity; (3) to elucidate the inhibitory effect of the fermented products on the growth of hepatoma cells by means of in vitro and in vivo studies. The present study established the optimal fermentation conditions for the inhibitory effects of fermentative broth on hepatoma cells. The best inhibitory effects were reached after fermentation with LAB for 48 hours at 37℃, and leaving the pH value uncontrolled when carrying out the fermentation in a 6.6 liter bioreactor. The IC50 of Hep 3B and Hep G2 were 226 μg/ml and 149 μg/ml, respectively. Meanwhile, the identification of the saponins in the fermentative broth of P. notoginseng was elucidated by using LC–ESI-MS/ MS and CID. Based on matching their detection of the saponin molecular weight, and the fragment ions of the molecular ion obtained in the CID experiments with data reported in the literature. Further study will focus on the animal experiments.第一章 前言...……………………………………………..………..…….1 第二章 文獻回顧……………………………………………….….…..…2 一、肝癌簡介…………………………....……………………....………2 二、三七之簡介……………………………..……………..……………5 三、三七之活性成分及其藥理作用…………………..………………..7 四、皂苷之水解、吸收代謝及抗癌作用..…………..…………………14 五、乳酸菌與抗癌之關係………………..……………………………28 六、微生物醱酵炮製…………..………………………………………30 第三章 研究目的…………..……………..………………………..……31 第四章 實驗架構………………………………………………..………33 第一部份 乳酸菌醱酵三七最適醱酵條件及其機能性與化學分析之研究………………………………………….………..….33 第二部份 三七乳酸菌醱酵液對小鼠移植人類肝腫瘤生長影響…..34 第五章 材料與方法………………………………………………....…..35 第一部份 乳酸菌醱酵三七最適條件之建立………………….....….35 第二部份 三七乳酸菌醱酵液之物化性質分析…………………..…39 第三部份 三七乳酸菌醱酵液機能性之探討…………………..……43 第一節 肝癌細胞生長抑制離體實驗( in vitro )…………..………43 第二節 肝癌細胞生長抑制活體實驗( in vivo )……………..….....48 第三節 統計分析……………….……………………………..…....54 第六章 結果與討論…………………………………………..……....…55 第一節 6.6公升醱酵槽培養………….…………………………...….55 一、不同天數、溫度之醱酵液對抑制人類肝腫瘤生長之影響........55 二、不同天數、pH值之醱酵液對抑制人類肝腫瘤生長之影響….59 第二節 結構分析與鑑定………………………………...…………..81 第三節 動物實驗結果探討………………………………..…..……..81 第七章 結論……………………...………………………………..…….89 參考文獻……………………………………………….…..…...………..90 附錄…………………………………………………………..…………1061143676 bytesapplication/pdfen-US三七乳酸菌皂苷抗肝腫瘤SCID micePanax notoginsengLABsaponinsanti-hepatoma[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56263/1/ntu-95-R93641003-1.pdf