臺灣博碩士論文加值系統

樟芝又稱牛樟菇學名為(Antrodia camphorata)，近幾年樟芝已被廣泛研究得知
其生物活性具有抗腫瘤、抗發炎、抗氧化、增強免疫力等特性，是一種被認知具
有保健效果之藥用菌菇。超臨界流體被廣泛應用於天然物及食品的萃取與精製，
因其具有密度高、黏度低、高擴散係數等特性以達到高萃取率的效果，而超臨界
二氧化碳技術具有低溫萃取特性，能減低熱不穩定物質之破壞，又其化學安定性
佳、無毒、無腐蝕性、來源取得容易，因此本篇研究以固態培養樟芝菌絲體經不
同壓力下之超臨界二氧化碳萃取其產物再經Dimethyl sulfoxide (DMSO)溶解之生
物活性探討。由結果顯示單純以DMSO 溶解之固態培養樟芝及經不同壓力之超臨
界流體萃取後的固態培養樟芝DMSO 溶解物皆能有效抑制乳癌細胞
(MDA-MB-231 及MCF-7)的生長，亦在流式細胞儀中發現乳癌細胞經樟芝DMSO
溶解物及超臨界樟芝DMSO 溶解物作用後會使得乳癌細胞產生Reactive oxygen
species (ROS)，促使乳癌細胞發生早期凋亡現象而導致細胞凋亡，又進一步探討細
胞凋亡相關因子，由結果可發現當癌細胞經樣品作用會增加Bax/Bcl-2 蛋白質比值
的表現量，也會增加caspase-9、caspas-3 活化態蛋白質的表現量；在抗轉移試驗中，
利用樣品作用於人類纖維瘤細胞(HT-1080)，在明膠電泳試驗(Gelatin Zymography
Assay)結果中亦可發現樣品作用HT-1080 細胞後能夠有效抑制細胞分泌於
conditioned medium 中的Matrix metallopeptidase 9 (MMP-9)、MMP-2 蛋白活性表現
量，而在RT-PCR 結果可發現能夠有效抑制MMP-9-mRNA 表現量。在免疫調節試
驗結果中可發現，樣品能夠刺激老鼠巨噬細胞Raw 264.7 分泌細胞激素抑制乳癌細
胞增長，而在抗發炎試驗結果方面可發現，樣品能夠抑制由Lipopolysaccharides
(LPS)誘導Raw 264.7 產生的NO 表現量。綜合上述結果顯示，DMSO 溶解之固態
培養樟芝及經不同壓力之超臨界流體萃取後的固態培養樟芝DMSO 溶解物能夠有
效抑制腫瘤存活率並且增加乳癌細胞中的ROS 使癌細胞走向凋亡，亦能抑制轉移
蛋白MMP-9 之表現，又能增加免疫反應及具有抗發炎反應，其中以超臨界二氧化
碳萃取樟芝菌絲體DMSO 溶解物較未經超臨界二氧化碳萃取之樟芝菌絲體DMSO
溶解物效果好。

Cinnamomum kanehirai Hayata is one of the unique indigenous species in Taiwan,
Antrodia camphorata is a saprophyte and it lives in the inner wall of the C. kanehirai
Hayata in the field. Recently many studies have shown that A. camphorata have some
bioactivities including anti-tumor, anti-inflammatory, anti-oxidation, and immunity,
therefore it has been known as a medicinal mushrooms. The supercritical fluid
extraction (SFE) technique has been applied to natural product and food extraction. The
high extraction rate can be achieved due to its features of high density, low viscosity and
high diffusion capacity. Furthermore, the process of the supercritical carbon dioxide
technology is under lower temperature condition, therefore it has some benefits such as
reducing the destruction of thermal- instability substances, maintaining chemical
stability, non-toxic, and non-corrosiveness. In order to investigate the bioactivities of A.
camphorata, samples from A. camphorata were extracted by the supercritical fluid
extraction under different pressures and then dissolved in dimethyl sulfoxide (DMSO).
The results showed that the DMSO-dissolved A. camphorata from SFE significantly
inhibited the growth of the breast carcinoma cells (MDA-MB-231 and MCF-7)
evaluated by MTT assay. Moreover, the growth-inhibition effect on breast carcinoma
cells was due to apoptosis evaluated by flow cytometer. In addition, the apoptosis factor
such as Bax and Bcl-2 was evaluated by Western Blot assay RT-PCR assay. Our results
showed that A. camphorata dissolved in DMSO extracted from SFE not only increased
the ratio of Bax to Bcl-2 expression but also increased cleaved caspase-9 and caspase-3
expression which lead the breast carcinoma cells tended to apoptosis. In the gelatin
zymography assay, the results showed that A. camphorata dissolved in DMSO extracted
from SFE effectively and significantly inhibited the matrix metalloproteinase-9 and the
matrix metalloproteinase-2 secretion on HT-1080 cell. According to anti-inflammatory
assay, the results showed that the A. camphorata dissolved in DMSO extracted from
SFE could effective inhibit nitrite generation. Compared to the data from water
extraction, our data had showed that A. camphorata dissolved in DMSO extracted from
SFE has better effect on anti-tumor, anti-metastasis, anti-inflammatory, and
immunomodulatoy.

致謝
中文摘要
英文摘要
第一章 研究動機
第二章 文獻回顧
一、樟芝(Antrodia camphorata)
1.1 樟芝簡介
1.2 樟芝生理活性
1.3 樟芝應用
二、超臨界流體(Supercritical fluid, SCF)
2.1 超臨界流體發展
2.2 超臨界流體萃取(Supercritical Fluid Extraction，SFE)技術原理與應用
2.3 超臨界二氧化碳
三、乳癌簡介
四、癌細胞凋亡與轉移
4.1 癌細胞凋亡
4.2 癌細胞氧化壓力
4.3 癌細胞轉移
五、免疫反應
第三章 實驗架構
第四章 材料與方法
一、實驗材料
1.1實驗細胞株
二、實驗方法
2.1 樣品製備
2.1.1 樟芝DMSO溶解法
2.1.2 樟芝超臨界二氧化碳DMSO溶解法
2.1.3 樟芝熱水溶解法
2.1.4 樟芝超臨界二氧化碳熱水溶解法
2.2 乳癌細胞存活率試驗(MTT assay)
2.3 乳癌細胞凋亡試驗(Annexin V/PI stain)
2.4 乳癌細胞氧化壓力試驗(DCFH-DA)
2.5 反轉錄聚合酶連鎖反應(Reverse Transcription Polymerase Chain Reaction, RT-PCR)
2.5.1 抽取mRNA
2.5.2 反轉錄聚合酶連鎖反應(RT-PCR)
2.6 西方墨點法(Western Blot)
2.7 Gelatin Zymography Assay
2.7.1 細胞溶解液(cell lysate)與Conditioned medium
2.7.2 蛋白質含量測定
2.7.3 電泳分析法
2.8 免疫調節
2.8.1 抑制癌細胞生長測試
2.8.2 抗發炎反應
2.9 抗氧化能力試驗
2.9.1 螯合亞鐵能力
2.9.2 抗氧化能力(DPPH)
2.10 統計分析
第五章 結果
一、抑癌試驗
1.1 抑制腫瘤細胞生長試驗
1.2 AC、SFE-250 bar與SFE-400 bar對乳癌細胞凋亡影響
1.3 AC、SFE-250 bar與SFE-400 bar對乳癌細胞氧化壓力影響
1.4 AC、SFE-250 bar與SFE-400 bar對乳癌細胞BAX、BCL2及caspase-3 mRNA表現影響
1.5 AC、SFE-250 bar與SFE-400 bar對乳癌細胞Bax、Bcl-2、caspase-9以及caspase-3蛋白質表現的影響
二、轉移酵素試驗
2.1 AC、SFE-250 bar與SFE-400 bar對人類癌化纖維母細胞MMP-9及MMP-2蛋白活性表現影響
2.2 AC、SFE-250 bar與SFE-400 bar對人類癌化纖維母細胞MMP-9及MMP-2 mRNA表現影響
三、調節免疫能力試驗
3.1 間接免疫試驗
3.2 抗發炎試驗
四、抗氧化能力試驗
4.1 螯合亞鐵能力
4.2 抗氧化能力(DPPH)
第六章 討論
一、抑制癌細胞生長試驗
二、樟芝萃取物對癌細胞氧化壓力試驗
三、細胞凋亡因子探討
四、基質金屬蛋白酶活性試驗
五、調節免疫試驗
5.1 間接免疫
5.2 抗發炎試驗
六、抗氧化能力試驗
6.1 螯合亞鐵能力
6.2 抗氧化能力(DPPH)
第七章 結論
第八章 未來計畫
第九章 參考文獻

http://digiku.nmns.edu.tw/fungi_web/FungiUnit/image/0707_picture_1.jpg
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