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研究生:謝景汝
研究生(外文):Jing-Ru Hsieh
論文名稱:樟芝超臨界萃取物對癌細胞生長與蝕骨細胞分化之抑制活性
論文名稱(外文):Antrodia cinnamomea supercritical fluid extract inhibits cancer cell growth and cell differentiation of osteoclasts
指導教授:洪哲穎洪哲穎引用關係
指導教授(外文):Jer-Yiing Houng
學位類別:碩士
校院名稱:義守大學
系所名稱:化學工程學系暨生物技術與化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:46
中文關鍵詞:樟芝有效活性成分癌細胞毒殺蝕骨細胞分化超臨界萃取
外文關鍵詞:Antrodia cinnamomeabioactive ingredientscancerosteoclastsupercritical fluid extraction
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樟芝為台灣特有種的藥用真菌,生長在台灣山區高海拔的牛樟樹幹腐朽之內壁,許多研究已證實樟芝具有調節血壓、血糖、血脂、保肝、抗發炎與抗癌等功效,樟芝的許多活性物質,包括多醣體、腺苷及三萜等化合物,也已被報導;本研究則擬針對樟芝的抗癌及抑制蝕骨細胞分化等活性篩選中低極性活性小分子化合物。首先,針對牛樟芝超臨界萃取物(ACS)依序以MeOH、EA 與n-Hexane進行分層萃取,分別得到甲醇層萃取物(ME)、乙酸乙酯層萃取物(EE)與正己烷層萃取物(HE)。其次,針對EE 層萃取利用薄層分析法進行分離。將分離出之9個區分物,進行抗癌與抑制蝕骨細胞分化活性之檢測,進而從中找出有效之區分物,再以氣相層析質譜儀 (GC-MS) 鑑定其化學組成。
ACS 對HepG2 肝癌細胞的抑制活性較對A549 肺癌細胞更為顯著。其分層萃取物中以EE 對癌細胞的毒殺效果較佳,對HepG2 的抑制活性亦較對 A549 更為顯著。EE 的九個區分物中,以Fr. E1、Fr. E2、Fr. E3、Fr. E5、Fr. E7 等五個樣品具有顯著抑制A549、HepG2 生長之效果,其中尤以Fr. E7 最為顯著。在抑制蝕骨細胞分化方面,ACS 在1.25 μg/mL 時即有顯著抑制效果;分層萃取物中,除高極性ME 層外,EE 與HE 皆無抑制效果。然而,EE 的九個區分物中Fr. E3與Fr. E5 則有顯著的抑制效果。在使用GC-MS 進行Fr. E1、Fr. E2、Fr. E3、Fr. E5、Fr. E7 等五個區分物化學組成分析方面發現,此五個區分物成份以脂肪酸為主,未來可以繼續針對這些區分物中不同脂肪酸成分對癌細胞與蝕骨細胞分化之抑制作用進行更深入之探討。綜上所述,ACS 具有抑制癌細胞生長與蝕骨細胞分化之應用潛力,未來可望從中找出有效之化學成分進行更深入的探討,以期開發成保健食品或臨床輔助用藥。

Niuchangchih (Antrodia cinnamomea) is a medical fungus grown on Cinnamomum kanehirai Hay tree in Taiwan. A. cinnamomea has been shown to exhibit a wide range of health-promoting benefits for the protection of liver,
cardiovascular systems, anti-inflammatory and anti-cancer. A few of bioactive ingredients of A. cinnamomea, such as polysaccharides, adenosines, triterpenes, have
been reported. This study attempts to screen the low-polarity bioactive small molecules that possess the anti-cancer and inhibitory activity against cell differentiation of osteoclasts. At first, the supercritical extract of A. cinnamomea (ACS)
was isolated and partitioned by methanol, ethyl acetate and n-hexane. Three extracts,ME, EE and HE, were obtained. Secondly, the EE extract was separated by thin layer chromatography, and the isolated fraction were determined their cytotoxicity on cancer cells and the inhibitory activity on the cell differentiation of osteoclasts. Furthermore, their chemical compositions were also examined by GC-MS analysis.
Compared to the inhibitory activity against A549 lung cancer cells, ACS exhibited more significant inhibitory activity against HepG2 hepatoma cells. Among the partitioned extracts, EE extract had the highest cytotoxicity on cancer cells. The cytotoxicity against HepG2 cells was higher than that against A549 cells. Among the nine fractionated extracts, five fractions Fr. E1, Fr. E2, Fr. E3, Fr. E5 and Fr. E7 had significant cytotoxicity on HepG2 and A549 cancer cells. Especially, Fr. E7 had the highest cytotoxicity. In terms of the inhibition on cell differentiation of osteoclasts,
ACS at the concentration as low as 1.25 μg/mL had the significant inhibitory effect. Among the partitioned extracts, only ME had inhibitory activity. Although EE had no inhibitory effect, Fr. E3 and Fr. E5 showed potent activity. From the chemical composition analysis on Fr. E1, Fr. E2, Fr. E3, Fr. E5 and Fr. E7 by GC-MS, the main
ingredients were fatty acids. The inhibitory effects of these fatty acids on the cytotoxicity against cancer cells and the cell differentiation of osteoclasts will be study in the future. In summary, ACS has the potential to become healthy food or complementary agents for treating cancer and bone diseases associated with osteoclast formation.

摘 要 I
Abstract II
致謝 III
目 錄 IV
圖目錄 VI
表目錄 VII
1. 緒 論 1
1.1肺癌 2
1.2肝癌 3
1.3蝕骨細胞(Osteoclast) 4
1.4天然藥物 5
1.5超臨界萃取(Supercritical fluid extraction) 6
1.6牛樟芝 7
1.7研究目的 8
2. 實驗材料與方法 11
2.1材料與萃取物之製備 11
2.1.1 材料 11
2.1.2 試劑 11
2.1.3 儀器 11
2.1.4 樟芝酒精萃取物之製備 11
2.1.5 樟芝超臨界萃取物之分層物製備 11
2.1.6 樣品分層物之分離純化 12
2.2細胞培養實驗 12
2.2.1 試劑 12
2.2.2 磷酸鹽緩衝溶液配製 12
2.2.3 細胞培養 13
2.2.4 繼代培養 13
2.2.5 細胞數目計數 13
2.3細胞存活率之測定-MTT assay 14
2.3.1 試劑 14
2.3.2 實驗原理 14
2.3.3 實驗步驟 14
2.4蝕骨細胞-抗酒石酸鹽酸性磷酸酶(TRAP)總量活性測試 15
2.4.1 誘導RAW264.7細胞分化為蝕骨細胞 15
2.4.2 藥品 15
2.4.3 實驗原理 15
2.4.4 實驗步驟 15
2.5 HPLC圖譜分析 16
2.6 GC-MS成分分析 16
2.7數據分析 16
3. 結果與討論17
3.1 樟芝超臨界萃取物之分層與區分物分離 17
3.1.1 樟芝超臨界萃取分層物製備 17
3.1.2 利用TLC薄層分析法進行ACS-EE之區分分離 17
3.2 樟芝不同萃取物之癌細胞毒殺活性 18
3.2.1 超臨界萃取物與其他分層物之癌細胞毒殺活性 18
3.2.2 ACS-EE之區分物的癌細胞活性 21
3.3 樟芝不同萃取物對蝕骨細胞分化之抑制活性 22
3.3.1 超臨界萃取物與其他分層物之TRAP抑制活性 22
3.3.2 ACS-EE之區分物之TRAP抑制活性 22
3.4 ACS-EE之區分物成分分析 24
4. 結論 32
參考文獻 33


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