香杉芝菌絲體及其固態發酵產品之乙醇萃取物誘導癌細胞凋亡之作用

Apoptotic effect of the extract from Antrodia salmonea mycelim and solid-state fermented products on cancer cells

林雨潔

香杉芝 ； 固態發酵 ； 細胞凋亡 ； 細胞週期 ； Sk-Hep1 ； Caco2 ； Antrodia salmonea ； solid-state fermentation ； apoptosis ； cell cycle ； SK-Hep1 ； Caco2

中興大學食品暨應用生物科技學系所學位論文

2015年

碩士

毛正倫

繁體中文

香杉芝 (Antrodia salmonea) 為寄生於香杉木上的台灣特有真菌，於 2004 年被鑑定出為薄孔菌屬之新種。香杉芝子實體與菌絲體萃取物具有抗氧化、抗動脈硬化、和抗發炎等功效，且對人類白血球細胞具有抗氧化作用。本實驗之研究目的為探討香杉芝菌絲體及香杉芝穀物 (蕎麥、燕麥) 固態發酵產品之 70% 乙醇萃取物，誘導大腸癌細胞與肝癌細胞凋亡的特性。 在生理活性物質方面，香杉芝菌絲體之麥角硫因含量為 168.06 µg/g 高於香杉芝燕麥 (ASFO) 138.70 µg/g 和香杉芝蕎麥 (ASFB) 118.64 µg/g。香杉芝固態發酵產品總多酚及類黃酮含量均高於香杉芝菌絲體。總多酚類含量依序為 ASFB (37.58 µg/mL)、ASFO (24.09 µg/mL) 及香杉芝菌絲體 (13.49 µg/mL)；類黃酮含量依序為 ASFB (1.37 µg/mL)、ASFO (0.36 µg/mL) 及香杉芝菌絲體 (0.11 µg/mL)。 由流式細胞儀分析細胞週期分布與細胞凋亡結果可知，人類大腸癌細胞 (Caco2)、人類肝癌細胞 (Sk-Hep1) 細胞增生能力，會隨著萃取物濃度升高及時間增加而被抑制；此外，萃取物不會對人類正常腎臟上皮細胞 (293T) 細胞株有抑制生長之效果。 接著探討誘導細胞凋亡之機制。經流式細胞儀偵測其細胞週期，實驗結果發現香杉芝菌絲體、ASFB、ASFO 可透過使細胞之 G0/G1 期上升和增加 subG1 來提升細胞凋亡比率；然而， 293T在細胞凋亡比率雖然會隨著作用濃度上升，但於 24 小時與控制組沒有顯著差異。僅 ASFB 組別作用 48 小時，濃度為 250 µg/mL 時，細胞凋亡比率為 3.83%，與控制組有顯著差異。另由 JC-1 之方法測定粒線體膜電位變化 (△Ψm），結果發現濃度為 250 µg/mL 時，粒線體膜電位有明顯下降的趨勢。此外，在細胞凋亡的訊息傳導路徑中，發現香杉芝菌絲體、 ASFB、 ASFO 均可使 Caspase-3,8,9活性顯著增加。 綜上所述，香杉芝菌絲體、ASFB 及 ASFO 具有誘導人類大腸癌細胞 (Caco2)、人類肝癌細胞 (Sk-Hep1) 凋亡的能力，推測其誘導凋亡路徑可由上升 G0/G1 期、subG1 期、降低粒線體膜電位及增加 Caspase-3,8,9 活性啟動細胞內一系列的凋亡。由本研究可知，香杉芝穀物之固態發酵產品具有誘導肝癌細胞及大腸癌細胞凋亡的潛力，可提供在抗癌應用上新的選擇。

Antrodia salmonea, which grows on the empty rotten trunk of Cunninghamia konishii in Taiwan, is a new species of the genus Antrodia as a basidiomycete idenfied in 2004. Many studies have suggested that the extract of A. salmonea, both fruiting body and mycelium, shows the abilities of anti-oxidative, anti-atherosclerotic, anti-inflammatory, and also have antioxidative ability in human leukocytes. We investigates the apoptotic cells of the 70% ethanol extract from A. salmonea mycelium, A. salmonea fermented buckwheat (ASFB) and A. salmonea fermented oat (ASFO) in Sk-Hep1cells and Caco2 cells. The physiological activity in ergothioneine, total phenol and flavonoids were studied. The total ergothioneine cotent of A. salmonea mycelium (168.06 µg/g) was higher than ASFO (138.70µg/g) and ASFB (118.64µg/g). Both the total phenol and tota flavonoids of A. salmonea mycelium solid state fermented products (ASF and ASFB) were higher than A. salmonea mycelium. Contents of total phenol was ASFB (37.58 µg/mL)、ASFO (24.09 µg/mL) and A. salmonea mycelium (13.49 µg/mL). Contents of tota flavonoids.was ASFB (1.37 µg/mL)、ASFO (0.36 µg/mL) and A. salmonea mycelium (0.11 µg/mL)。 We used MTT assay to measured that the cell viability of Sk-Hep1 cells and Caco2 cells, and the results exhibited dose- and time- dependent response, but no effect on 293T cells. The main mechanism of the apoptotic effect were included to arrest G1/G0 phase, subG1 phase. While the proportion apoptotic cells of 293T cells have concentration- and time- dependent response, but showed no significantly different in 24h. After 48h incubation.only the apoptotic cells of ASFB (250 µg/mL) was 3.83%, which have significantly different compare with control. We evalutated the mitochondrial membrane potential (△Ψm) by JC-1 stain. Results reveled that 70% ethanol extracts from A. salmonea mycelia, ASFB and ASFO caused a significantly loss of mitochondrial membrane potential (△Ψm) at 250 µg/mL. The results exhibited the up-regulated expression of Caspase-3,8,9 in 70% ethanol extract from A. salmonea mycelia , ASFB and ASFO. In conclution, we demonstrated that 70% ethanol extract from A. salmonea mycelia and A. salmonea fermented products (ASFB and ASFO) might suppress the proliferation in Sk-Hep1 cells and Caco2 cells by inducing apoptosis. A. salmonea mycelia、ASFB and ASFO triggered apoptosis activities by arresting G1/G0 phase, subG1 phase, a significantly loss of mitochondrial membrane potential, and promoting activities of caspase-3, caspase-8 and caspase-9.. Thses data indicated that the apoptosis-inducing effects of A. salmonea mycelia and A. salmonea solid-state fermented products (ASFB, ASFO) might have the new choice as a potential chemotherapeutic agents.

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1. 吳宣慧(2018)。香杉芝菌絲體乙醇萃取物對癌細胞侵襲與轉移之影響。中興大學食品暨應用生物科技學系所學位論文。2018。1-82。