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研究生:温峰昌
研究生(外文):Feng-Chang Wen
論文名稱:牛樟芝成份分析、生物活性及其 對人類肝癌細胞Hep G2抑制之研究
論文名稱(外文):Anticancer effects of human liver Hep G2 from Antrodia cinnamomea and its analysis of compositions and bioactivities
指導教授:廖信昌廖信昌引用關係顏宏愷施玟玲
指導教授(外文):Sin-Chung LiaoHung-Kai YenWen-Ling Shih
學位類別:碩士
校院名稱:美和科技大學
系所名稱:生技科技系健康產業碩士班
學門:商業及管理學門
學類:醫管學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:51
中文關鍵詞:牛樟芝高壓液相層析儀樟芝酸細胞存活檢測人類肝癌細胞抗氧化能力
外文關鍵詞:Antrodia cinnamomeaHPLCAntcinMTT assayHepG2DPPH
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牛樟芝(Antrodia cinnamomea)為台灣特有種藥用真菌菇類,已多篇研究報告證實牛樟芝因含有三萜類及多醣體等成份故具有解毒、消炎、治療肝臟疾病及抗癌等功用,據說白牛樟芝為白色變種牛樟芝是更稀少的菌種、功效更佳及價格更昂貴,為了確定紅、白牛樟芝的成份及藥效,故本研究進行椴木白牛樟芝BWAC(Basswood White AC)、自製白牛樟芝HMWAC(Homemade medium white AC)、自製白牛樟芝HMWAC+P1 (Homemade medium white AC+P1)、市售白牛樟芝CMWAC(Commercial medium white AC)、自製紅牛樟芝(15cm dish)HMRAC(Homemade medium Red AC +15cm dish)、自製野生紅牛樟芝HMNTAC+P1(Homemade medium Native Type AC)、野生紅牛樟芝NTAC(Native Type AC)等上述自製改良培養基及與市售培養基培育紅牛樟、白牛樟,並以野生型紅牛樟芝及椴木白牛樟芝做樟芝酸指標成份之比較及進一步萃取各式培育牛樟芝之成份分析測量包括總三萜類、多醣體、總多酚類及抗氧化力及抗人類肝癌細胞HepG2細胞存活檢測(MTT assay)效果比較,以尋找出最佳之生產方式以滿足產業需求。本研究結果發現皿培牛樟芝確可得到相當量的三萜類,而多醣體含量自製培養基之紅白牛樟均高於市售培養基,而總多酚類為皿培牛樟芝高於椴木白牛樟,在抗氧化力比較亦如此。另外研究發現皿培培養基添加重要元素P1的確可增加牛樟芝之指標成份如Antcin A、B、H、DeSA和DeEA等含量。在抗肝癌細胞試驗發現皿培牛樟芝在62.5ug/ml即有約50%抑制效果,而野生紅牛樟濃度高於125ug/ml以上,有超過80%抑制效果,本研究結果顯示野生紅牛樟有不錯抑癌效果,然而取得不易及培育時間長等缺點,故研發適合的配方以生產皿培牛樟芝產品對B肝病人的健康保護是相當具有潛力生技健康產品。
Antrodia cinnamomea (AC) is a specially and locally medicinal fungus in Taiwan. Many studies have been confirmed that AC is rich in triterpenoids, polysaccharides and other nutrients that may carry functional properties like detoxification, anti-inflammatory, improve liver function and anti-cancer. It is said that white AC is relative rare and more expensive and more functional contents than regular form of AC. Thus, studies to develop the best culture medium, comparing the bioactive compounds include total triterpenes, polysaccharides, total polyphenols and medicinal functions like to free radicals(DPPH) and anti-B liver cancer (MTT assay) on the regular form(red AC), white variant of AC, wild type(red AC) and various AC species in petri-dish culture medium (HMWAC、HMWAC+P1、CMWAC、HMRWAC、HMNTAC+P1 and NTAC) and basswood AC(BWAC) should be conducted. This study results showed that the homemade medium can enhance crude triterpenoid of mycelia and basidiomatal formation of AC in petri-dish solid-state fermentation. The polysaccharides content of homemade medium for regular and white AC were higher than the commercial medium. The total polyphenols of AC in the petri-dish culture was more than basswood white AC and DPPH was also the case. In addition, it was found that the addition of P1 element to the culture medium could increase the contents of Antcin A 、B、H 、DeSA and DeEA. In the anti- liver cancer cell HepG2 assay showed that about 50% inhibition was achieved at 62.5 μg / ml. The wild type AC (NTAC) fruiting bodies extracted concentration was higher than 125μg/ml, and the anti-liver cancer cell effect was over 80%. The results showed that NTAC also had good anti-cancer effects. But it’s not easy to obtain and need long-time about 2-3 years to culture and other shortcomings like infection issues to overcome. This study demonstrates that the development of culture medium formula to produce solid–state fermentation of AC products on the B liver health protection is considerable potential for health products and is the best making way to fit the needs of the industry.
謝誌 I
中文摘要 II
ABSTRACT III
目錄 V
表目錄 VII
圖目錄 VIII
第一章 前言 1
1.1 研究背景 1
1.2 研究動機 2
第二章 文獻回顧 4
第三章 材料與方法 6
3.1 實驗架構 6
3.2 藥品試劑與設備 6
3.2.1 藥品試劑 7
3.2.2 設備儀器 8
3.3 牛樟芝菌種製備 8
3.4 牛樟芝菌種之培養基製備 8
3.5 培養基固態發酵牛樟芝子實體之育成 9
3.6 利用HPLC 定量分析牛樟芝樟芝酸相對含量分析 9
3.7 育成牛樟芝總三萜類含量測定: 9
3.8 培養基育成牛樟芝總多醣體含量定量 10
3.9 牛樟芝總多酚含量定量: 10
3.10 各種培養基育成牛樟芝抗氧化能力測定 10
3.11 肝癌細胞培養 11
3.12 MTT 反應分析 11
3.13 統計分析(Statistical analysis) 12
第四章 結果 13
4.1 紅牛樟芝與白牛樟芝之培育 13
4.2 白牛樟芝之椴木培養 14
4.3 建立HPLC樟芝酸八大指標成分分析圖譜 15
4.4 白牛樟芝指標成分分析 16
4.5 樟芝酸成分統計分析 18
4.6 牛樟芝總三萜類含量之測定 19
4.7 各式牛樟芝試驗樣品之總多醣體含量之測定 21
4.8 各式牛樟芝試驗樣品總酚類之測定 22
4.9 各式牛樟芝試驗樣品抗氧化力測定 23
4.10 牛樟芝萃取物對HepG2細胞株之細胞毒殺試驗 25
第五章 討論 32
第六章 參考文獻 35
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