臺灣博碩士論文加值系統

肝癌是全世界最盛行的癌症之一，估計每年增加超過 1 百萬件新病例，所以肝癌發生的預防與治療是全世界主要的研究課題。過去一些天然物相關研究中，傳統用在肝臟疾病的中藥萃取物以及萃取自植物與微生物二次代謝產物中之生物活性成分皆具有廣泛的生物活性，如抗 B 型肝炎病毒，抗氧化，免疫調節與抗癌作用等。
本論文於樟芝抗酒精毒殺之保肝活性研究中，首先針對工業上醱酵量產樟芝菌絲體、胞外、胞內多醣以及三萜類等代謝產物做研究。研究中，亦探討樟芝水萃物抗酒精誘導之肝細胞毒殺的保肝效果，結果證實添加 500 mg/L 的樟芝水萃物可以有效地降低 300 mM 酒精所誘導之細胞毒殺。
此外我們進一步研究針對啤酒花萃取物 xanthohumol 對人類肝癌細胞的抑制效果，結果發現正常的肝細胞株較人類肝癌細胞株對 xanthohumol 的毒殺效果具有較高的抗性，且實驗中經由流式細胞儀、細胞核螢光染色與電泳分析 DNA 斷裂片段的結果，證實了啤酒花萃取物 xanthohumol 是經由細胞凋亡途徑來毒殺人類肝癌細胞。
最後，研究以酒精萃取自古嗜鹽菌 (Haloferax mediterranei) 的紅色色素 (hmERP) 抗肝癌細胞之作用，該色素主要由衍生於 lycopene 而被稱為 bacterioruberin 之一種類胡蘿蔔素所組成。實驗證明 hmERP 對人類肝癌細胞株 HepG2 的抗肝癌細胞活性較 lycopene 更佳。當細胞以 0.5 mg/L hmERP 處理下，可以觀察到細胞膜內層 phosphatidyl serine 外翻、核染色質濃縮、DNA 片段化以及凋亡小體形成等細胞凋亡之變化。此外經由流式細胞儀分析結果，發現 hmERP 雖誘導 HepG2 細胞凋亡但並不會誘發細胞壞死。

The hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide, with more than 1 million new cases estimated annually. Therefore, preventing HCC and HCC therapy is a major research around the world. In the past researches, some herbal extracts used traditionally in hepatic diseases and some bioactive compounds extracted from the secondary metabolites of plant and microorganism have extensive biological activities, such as anti-hepatitis B virus, antioxidation, immunomodulating and anticancer effects.
In the first instance, the objective of this study was to produce Antrodia cinnamomea mycelia and its metabolites on a large scale, such as extracellular polysaccharides (EPS), intracellular polysaccharides (IPS), and triterpenoids. The hepatoprotective effects of the water extract from the mycelia of A. cinnamomea (WAC) were also evaluated in vitro using ethanol-induced cytotoxicity on AML12 hepatocytes. The cytotoxicity to AML12 cells induced by 300 mM ethanol was reduced effectively by adding 500 mg/L of WAC.
Secondly, we also investigated the inhibitory effects of xanthohumol on the human HCC cell lines. This result indicates that normal mouse hepatocyte cell line had more resistance to xanthohumol than HCC cell lines. Besides, the inhibitory effects of xanthohumol on human HCC cell lines were attributed to apoptosis as indicated in the results of flow cytometry, fluorescent nuclear staining and electrophoresis of oligonucleosomal DNA fragments.
Finally, the anti-hepatoma effects of the ethanol-extracted red pigments from Haloferax mediterranei (hmERP) were investigated. The hmERP was composed mainly of bacterioruberin, a carotenoid derived from lycopene, and showed a more powerful anti-hepatoma activity on HepG2 cells than lycopene. When HepG2 was exposed to 0.5 mg/L hmERP, the apoptosis-associated phosphatidyl serine redistribution, chromatin condensation, DNA fragmentation, and apoptotic body formation were observed. By flow cytometry analysis, hmERP induced the apoptotic cell fraction of HepG2 without increasing the necrotic cell fraction.

ABBREVIATION LIST i
ABSTRACT v
CHINESE ABSTRACT vii
CONTENT ix
LIST OF TABLES xii
LIST OF FIGURES xiii
CHAPTER 1 INTRODUCTION 1
1.1 Epidemiology of liver cancer 1
1.1.1 Definition and classification of liver cancer 2
1.1.2 Incidence of liver cancer 4
1.1.3 Etiology of hepatocellular carcinoma 6
1.2 The mechanisms of chemical hepatotoxicity 13
1.2.1 Oxidative stress and free radical damage 13
1.2.2 Alcohol-induced oxidative stress and cell injury 15
1.2.3 The lipopolysaccharide-induced hepatic toxicity 17
1.3 The hepatoprotective activity of natural products 19
1.3.1 The hepatoprotective activity of mushrooms 19
1.3.2 The hepatoprotective activity of flavonoids 29
1.3.3 The hepatoprotective activity of carotenoids 38
1.4 Research purposes 46
CHAPTER 2 THE HEPATOPROTECTIVE ACTIVITY AGAINST ETHANOL-INDUCED CYTOTOXICITY BY AQUEOUS EXTRACT OF ANTRODIA CINNAMOMEA 49
2.1 Materials and methods 49
2.1.1 Materials 49
2.1.2 Maintenance and preculture of A. cinnamomea 50
2.1.3 Large-scale production of A. cinnamomea 50
2.1.4 Analyses of biomass, residue sugar, and polysaccharides 51
2.1.5 Preparation of mycelial water extracts 52
2.1.6 Identification of sugar components in IPS 52
2.1.7 Analysis of triterpenoids 53
2.1.8 Cell lines and culture 54
2.1.9 Lactate dehydrogenase leakage assay 54
2.1.10 Assay of hepatoprotective activity 57
2.1.11 Annexin V-EGFP apoptosis detection kit analysis 57
2.1.12 Apoptotic analysis 58
2.1.13 Statistical analysis 59
2.2 Results and discussion 60
2.2.1 Production of A. cinnamomea 60
2.2.2 Hepatoprotective effects of WAC 64
2.2.3 Inhibition of the ethanol-induced apoptosis by WAC 64
2.2.4 Components analysis of intracellular polysaccharides in WAC 66
CHAPTER 3 INHIBITORY EFFECTS OF XANTHOHUMOL FROM HOPS (HUMULUS LUPULUS L.) ON HUMAN HEPATOCELLULAR CARCINOMA CELL LINES 71
3.1 Materials and methods 71
3.1.1 Materials 71
3.1.2 Isolation of xanthohumol 72
3.1.3 Cell culture 73
3.1.4 MTS assay 73
3.1.5 Cell proliferation assay 74
3.1.6 Cell cytotoxicity assay 75
3.1.7 Flow cytometric analysis 75
3.1.8 Fluorographic observation of apoptosis-associated nuclei 76
3.1.9 Apoptotic DNA ladder kit assay 76
3.1.10 DNA gel electrophoresis 77
3.1.11 Statistical analysis 78
3.2 Results and discussion 79
3.2.1 The purity of xanthohumol 79
3.2.2 Effects of xanthohumol on immediate toxicity in mouse hepatocyte cell line 79
3.2.3 Xanthohumol inhibit proliferation in hepatocellular carcinoma cell lines 81
3.2.4 Effects of xanthohumol on immediate toxicity in hepatocellular carcinoma cell lines 86
3.2.5 Xanthohumol induces apoptosis in hepatocellular carcinoma cell lines 87
CHAPTER 4 CYTOTOXICITY OF ETHANOLIC EXTRACTS FROM HALOFERAX MEDITERRANEI IN HUMAN HEPATOMA CELLS 96
4.1 Materials and methods 96
4.1.1 Materials 96
4.1.2 Preparation of hmERP 96
4.1.3 Analysis of hmERP 97
4.1.4 Cell culture 98
4.1.5 Trypan blue exclusion method 99
4.1.6 MTT assay 100
4.1.7 Cell viability assay 102
4.1.8 Apoptotic analysis 102
4.2 Results and discussion 105
4.2.1 Analysis of hmERP 105
4.2.2 Cell inhibition by hmERP 108
4.2.3 hmERP induced apoptosis of HepG2 cell 111
CHAPTER 5 CONCLUSIONS 118
CHAPTER 6 REFERENCES 121
CHAPTER 7 APPENDIX
7.1 The hepatoprotective activity against ethanol-induced cytotoxicity by aqueous extract of Antrodia cinnamomea
7.2 Inhibitory effects of xanthohumol from hops (Humulus lupulus L.) on human hepatocellular carcinoma cell lines

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