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研究生:林怡伶
研究生(外文):Yi-Ling Lin
論文名稱:探討天然發酵諾麗果汁於高脂飲食與慢性酒精攝取下其心血管與肝臟保護功效
論文名稱(外文):Cardioprotection and Hepatoprotection of naturally fermented noni (Morinda citrifolia L.) juice in a high fat diet and chronic alcohol consumption
指導教授:曾博修曾博修引用關係陳億乘
學位類別:博士
校院名稱:中山醫學大學
系所名稱:生化暨生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:126
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It has been summarized that a western-type dietary habit or chronic alcohol consumption easily results in hyperlipidemic, cardiovascular, and liver disorders. Meanwhile, chronic liver disease and cirrhosis are serious diseases facing to either worldwide or Taiwan. One-year naturally fermented noni juice (NJ) contains polyphenols, polysaccharides, and some trace minerals. Hence, this dissertation involved three independent studies on the protective effects of NJ against hyperlipidemia, high-fat-diet induced fatty liver, and alcoholic fatty livers, respectively.
In the first study of this dissertation, NJ supplementation decreased (p<0.05) serum triacylglycerol (TAG), cholesterol (TC), atherogenic index, triobarbituric acid reactive substances (TBARS) values, and hepatic lipids in high-fat/cholesterol diet (HFCD) fed hamsters, whereas serum trolox equivalent antioxidant capacity (TEAC), glutathione (GSH), and fecal lipids were increased (p<0.05) by NJ supplementation. Besides, NJ supplementation downregulated (p<0.05) sterol regulator element binding protein-1c in HFCD fed hamsters, but upregulated (p<0.05) hepatic peroxisome proliferator-activated receptor-alpha and uncoupling protein 2 gene expressions in HFCD hamsters.
In the second study, after a 6-week feeding period, increased (p<0.05) sizes of liver and visceral fat in high-fat dietary hamsters were ameliorated (p<0.05) by NJ supplementation compared to the normal fat dietary hamsters (Control group). NJ also decreased (p<0.05) serum/liver lipids but enhanced (p<0.05) daily faecal lipid/bile acid outputs in high-fat dietary hamsters. Meanwhile, high-fat dietary hamsters co-treated with NJ enhanced (p<0.05) liver antioxidant capacities but lowered (p<0.05) liver iNOS, COX-2, TNF-α, and IL-1β expressions, and gelatinolytic levels of MMP9 and serum ALT values compared to those without NJ.
The last study was to assess the hepatoprotective effects of NJ against a chronic alcohol consumption. NJ supplementation decreased (p<0.05) serum AST, ALT and liver lipids, as well as increased (p<0.05) daily fecal lipid outputs in alcohol-diet fed mice. Besides, NJ supplementation not only downregulated (p<0.05) hepatic lipogenesis but also upregulated (p<0.05) fatty acid-β-oxidation-related gene expressions in alcohol-diet fed mice. Alcohol-diet fed mice cotreated with NJ had higher (p<0.05) hepatic TEAC and GSH levels but lower (p<0.05) TBARS value, TNF-α, and IL-1β expressions compared to those without NJ.


TABLE OF CONTENTS

ACKNOWLEDGEMENTS Ⅰ
LIST OF TABLES Ⅴ
LIST OF FIGURES Ⅵ
ABSTRACT Ⅶ
CHAPTER
I. INTRODUCTION 1
II. LITERATURE REVIEW 4
2.1 Cardiovascular disease (CVD) 4
2.2 The fatty liver 10
2.2.1 Epidemiology and Risk Factors by Fatty Liver 10
2.2.2 Pathogenesis by Fatty Liver… 11
2.3 Lipid homeostasis 14
2.4 Antioxidant Systems 20
2.4.1 Enzymatic antioxidants 21
2.4.1.1 Superoxide dismutase (SOD). 21
2.4.1.2 Glutathion peroxidase (GSH-Px). 21
2.4.1.3 Catalase 22
2.4.2. Non-enzymatic antioxidants 23
2.4.2.1 Glutathione. 23
2.4.2.2 Vitamin C (Ascorbic acid). 23
2.4.2.3 Vitamin E (Tocopherols). 24
2.4.2.4 Phytochemicals. 24
2.5 Inflammatory signaling pathways 28
2.6 Alcohol metabolism 33
2.7 Noni juice 37

III. HYPOLIPIDEMIC AND ANTIOXIDATIVE EFFECTS OF NONI (Morinda citrifolia L.) JUICE ON HIGH-FAT/CHOLESTEROL-DIETARY HAMSTERS 41

Abstract 41
3.1 Introduction 42
3.2 Materials and methods 44
3.2.1 Noni Juice material 44
3.2.2 Determination of phytochemicals in noni juice (NJ) 44
3.2.3 Animal and diets 45
3.2.4 Collection of serum, liver, visceral fat, and feces 45
3.2.5 Determination of serum lipid parameters and fecal lipids/bile acids 46
3.2.6 Hepatic mRNA Expressions of LDL Receptor, SREBP2, HMG-CoA Reductase, CYP7A1, SREBP1c, FAS, PPAR-alpha, UCP2, and GAPDH 46
3.2.7 Determination of serum 2-thiobarbituric acid reactive substances (TBARS),Glutathione (GSH), and trolox equivalent antioxidant capacity (TEAC) 48
CHAPTER Page
3.2.8 Statistical analysis 49
3.3 Results and discussion 50
3.3.1 Phytochemical contents in noni Juice (NJ) 50
3.3.2 Growth performance of experimental hamsters 51
3.3.3 Serum lipids profiles as well as liver and fecal triacylglycerol and cholesterol levels and bile acid contents of experimental hamsters 52
3.3.4 Gene expressions related to lipid homeostasis in experimental hamsters 53
3.3.5 Serum antioxidant status of experimental hamsters 55
3.4 Conclusion 57

IV. BENEFICIAL EFFECTS OF NONI (Morinda citrifolia L.) JUICE ON LIVERS OF HIGH-FAT DIETARY HAMSTERS 64

Abstract 64
4.1 Introduction 65
4.2 Materials and methods 68
4.2.1 Noni juice manufacture 68
4.2.2 Polyphenol compounds of NJ 68
4.2.3 Animal and diets 68
4.2.4 Determination of serum biochemical values, hepatic lipids, and fecal lipids /bile acids 69
4.2.5 Preparation of liver homogenate 70
4.2.6 Determination of liver lipid peroxidation level and antioxidant capacity 70
4.2.7 Determination of hepatic tumor necrosis factor-alpha (TNF-α) and
interleukin-1β (IL-1β) levels 71
4.2.8 Determination of liver inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels, and gelatinolytic levels of matrix metalloproteinase (MMP) 2 and 9 72
4.2.9. Histopathological analysis 73
4.2.10 Statistical analysis 73
4.3 Results and discussion 74
4.3.1 Polyphenol compounds of NJ 74
4.3.2 Effects of NJ on growth performance, serum/liver lipids, daily fecal lipid /bile acid outputs, and liver histological examination 75
4.3.3 Effects of NJ on antioxidative capacities of livers 76
4.3.4 Effects of NJ on liver damage and inflammatory responses 78
4.4 Conclusion 80

V. HEPATOPROTECTION ON NONI JUICE AGAINST CHRONIC ALCOHOL CONSUMPTION: LIPID HOMEOSTASIS, ANTIOXIDATION, AND ANTIINFLAMMATION 87

Abstract 87
5.1 Introduction 88
5.2 Materials and methods 90
5.2.1 Noni juice manufacture 90
CHAPTER Page
5.2.2 Phytochemicals and mineral analysis of NJ 90
5.2.3 Animal and diets 91
5.2.4 Determination of serum biochemical values, hepatic lipids, and daily fecal lipid /bile-acid outputs 91
5.2.5 Preparation of liver homogenate 92
5.2.6 Determination of liver lipid peroxidation level and antioxidant capacity 92
5.2.7 Determination of hepatic tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) levels 93
5.2.8 RNA preparation and quantitative real-time PCR 93
5.2.9 Histopathological analysis 95
5.2.10 Statistical analysis 96
5.3 Results and discussion 97
5.3.1 Effects of NJ on growth performance, serum biochemical values,
liver lipids, daily fecal lipid/bile acid outputs 97
5.3.2 Effects of NJ on de novo lipogenesis of livers 99
5.3.3 Effects of NJ on lipid peroxidation and antioxidant capacities in livers 100
5.3.4 Effects of NJ on liver damage and inflammatory responses 100
5.4 Conclusion 102

VI. SUMMARY AND CONCLUSION 110
REFFERNCE 113






















LIST OF TABLES
TABLE Page
2.1 Previous criteria proposed for clinical diagnosis of metabolic syndrome 7
3.1 Total ascorbic acid and crude polysaccharide contents, as well as contents of
flavonoids and phenolic acids in noni juice 47
3.2 Growth performance, as well as relative size of heart, liver, and visceral fat
of the experimental hamsters 48
3.3 Liver and fecal lipid, and fecal bile acid levels of the experimental hamster 50
4.1 Growth performance, as well as relative size of liver and visceral fat in the
experimental hamsters 67
4.2 Liver damage indices, liver peroxidation and antioxidant capacities of the
experimental hamsters 70
5.1 Phytochemical and mineral contents in noni juice 85
5.2 Growth performance, relative sizes of organs and visceral fat, and serum
biochemical values of experimental mice 86
5.3 Liver antioxidant capacities and cytokine excretion of experimental mice 89





















LIST OF FIGURES
FIGURE Page
2.1 Recruitment of monocytes and formation of foam cells 8
2.2 Initiation and progression of atherosclerosis 8
2.3 The primary pathways for the metabolism of human plasma lipoproteins are summarized 16
2.4 Antioxidative system 22
2.5 Antioxidant groups and actions 22
2.6 Sources of reactive oxygen species generation and their effects on signaling systems in chronic disease 26
2.7 Summary of interactions leading to NFκB activation 26
2.8 Ethanol metabolism pathway 29
2.9 Photographs of M. citrifolia fruits 32
3.1 Serum triacylglycerol (A), total cholesterol (B), serum HDL-C (C), and
atherogenic index (D) of experimental hamsters 49
3.2 Low-density-lipoprotein receptor (LDL receptor), sterol regulator element
binding protein-2 (SREBP2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), cholesterol 7-α hydroxylase (CYP7A1), sterol regulator element binding protein-1c (SREBP1c), fatty acid synthase (FAS), peroxisome proliferator-activated receptor-alpha (PPAR-alpha), and uncoupling protein-2 (UCP2) mRNA expressions of experimental hamsters 51
3.3 Serum TBARS (A), GSH (B), and TEAC (C) levels of experimental hamsters 52
4.1 HPLC chromatograms of flavonoids and phenolic acids in noni juice. 71
4.2 Serum (A), liver (B), fecal lipid (C), and fecal bile acid (D) contents of the
experimental hamsters 68
4.3 H&E stain under microscope (200X) of liver tissues in the experimental
hamsters. 69
4.4 (A) Protein levels of iNOS and COX-2 and (B) MMP2/9 activities in livers
of the experimental hamsters. 71
5.1 The mRNA expressions of SREBP-1c, LXR-α, ACC, FAS, DGAT, RXR-α,
PPAR-α, and UCP2 in livers of experimental mice 87
5.2 Liver lipid contents (A), and daily fecal lipid (B) and fecal bile acid (C)
outputs of experimental mice. 88
5.3 Liver TNF-α (A) and IL-β (B) levels of the experimental mice 90
5.4 H & E stain of liver tissues 91








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