臺灣博碩士論文加值系統

病原菌感染與其引起之發炎反應會提高罹患胃腸道疾病的風險，如常見的胃潰瘍與發炎性腸炎等。因此，如何避免或緩解細菌感染與相關之發炎反應是降低罹患胃腸道疾病的最佳策略。過去的研究已證實諾麗 (Morinda citrifolia)可以提良好的抗菌、抗發炎以及抗氧化能力。然而，諾麗果實對於胃腸道的效用仍未被發掘。在本研究中，發酵諾麗果實分別以乙醇與乙酸乙酯萃取，並探討此兩種萃取物對於幽門螺旋桿菌 (H. pylori) 與其所誘導胃上皮細胞發炎反應，以及腸道細胞發炎反應之抑制效果。首先，評估發酵諾麗果實萃取物對於幽門螺旋桿菌之抑制與抗附著效果與其引起的發炎因子，包括環氧合酶 (COX-2)、誘導型一氧化氮合酶 (iNOS)、細胞界白素8 (IL-8)、前列腺素 (PGE2)、一氧化氮 (NO)、嗜中性球趨化以及細胞毒素相關基因蛋白A (CagA)之轉運作用。萃取物對於幽門螺旋桿菌相關的傷口治癒功能與濾泡毒素A (VacA)轉運作用之效應亦被詳加觀察。另一方面，此研究亦評估發酵諾麗果實萃取物之抗氧化能力，對於腸道菌以及對於細菌脂多醣 (LPS) 與細胞界白素1β (IL-1β) 引起腸細胞發炎訊號之影響。結果顯示，兩種萃取物皆能抑制幽門螺旋桿菌之附著作用，並截斷細胞CagA轉運以及其所引起之發炎訊號(IL-8與嗜中性球趨化)，更能抑制COX-2與PGE2濃度。唯只有乙酸乙酯萃取物能抑制iNOS與NO之表現。發酵諾麗果實萃取物亦能夠藉由阻斷VacA轉運與訊號傳遞，降低細胞色素C釋放，進而促進傷口治癒。在腸道保護作用中，乙醇萃取物能夠促進益生菌腸道菌之生長 (Lactobacillus and Bifidobacterium spp.)。而乙酸已酯萃取物能夠顯著抑制LPS引起的活性氧物質(ROS)之釋放。乙酸乙酯更能夠藉由抑制NF-κB路徑來有效減緩IL-1β與LPS所引起的發炎反應 (COX-2、PGE2、IL-8與嗜中性球趨化)。進而的分析萃取物多酚類與主要貢獻物後，顯示乙醇與乙酸乙酯萃取物含有多量的多酚類成分，兩者間的多酚類物質組成亦相當迴異。而主要的多酚類物質能夠顯著的抑制幽門螺旋桿菌之附著與緩解胃細胞與腸細胞之發炎反應。綜觀結果，發酵諾麗果實萃取物能夠有效的抑制幽門螺旋桿菌感染所引起的發炎反應與促進傷口治癒，更能夠避免腸細胞陷入發炎迴圈之泥淖，而多酚類成分在此胃腸到保護作用中扮演重要角色。

Pathogen infection and its related inflammation which increase the risk of gastrointestinal disease formation, including gastric ulcer and inflammatory bowel disease. To prevent and relieve bacterial infection or inflammatory response are optimal stratagem. Noni (Morinda citrifolia) is found to possess antibacteria, anti-inflammation and antioxidative activites, but its effects on gastrointestinal protection remain unknown. In this study, the ethanol and ethyl acetate extracts of fermented noni fruit were used to evaluate the prevention and alleviation on Helicobacter pylori (H. pylori) infection and inflammatory response on gastric (AGS) and colonic (Caco-2) cells respectively. The inhibitory and antiadhesion activity of both extracts on H. pylori were evaluated. The pro-inflammatory molecules, namely cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS), interleukin-8 (IL-8), prostaglandin E2 (PGE2), nitric oxide (NO), transportation of cytotoxin associated gene A (CagA) protein and neutrophils migration were assessed after H. pylori treated in AGS cells. The wound healing and transportation of vacuolating toxin A (VacA) were estimated after H. pylori or bacterial-cultured medium treated. The inflammatory protein also be determined after lipopolysaccharide (LPS) and IL-1β treated on Caco-2 cells. Moreover, the antioxidation of both extracts on various free radicals and low-density lipoprotein (LDL) oxidation were evaluated. First, both extracts of fermented noni fruit contained abundant quercetin and coumaric acid respectively. Both extracts showed significant suppression on H. pylori adhesion, CagA transportation, inflammatory response (COX-2, iNOS, PGE2, NO), neutrophils migration and contribute to wound healing by impeding VacA signals although no inhibition on H. pylori growth. Both extract showed great antioxidative activities (trolox equivalent antioxidant capacity, reduction ability, ferrous ion chelating and LDL oxidation) and scavenging on free radicals (DPPH, superoxide anions and hydrogen peroxide), the ethyl acetate significantly suppressed the levels of intercellular reactive oxygen species (ROS) during LPS treated on Caco-2 cells. The ethanol extract promoted probiotics growth (Lactobacillus and Bifidobacterium spp.). On the contrary, the ethyl acetate extract can significantly inhibit inflammatory signals by NF-κB pathway without effecting on probiotic growth. The phenolic compounds of both extracts significantly inhibited H. pylori adhesion and inflammatory response on AGS and Caco-2 cells. In conclusion, the extracts of fermented noni fruit down-regulated inflammatory responses and promote wound healing during H. pylori infection. Ethyl acetate extract of fermented noni fruit can prevent excessive oxidation and inflammation on intestine. Phenolic compounds play key role on gastrointestinal protection.

Contents
Chinese Abstract VII

Abstract VIII

Introduction X

Abbreviation XII

Chapter I. Literature Review of Noni Fruit 1
1. Noni (Morinda citrifolia L.) 2
1.1. Plant Description and Characteristic 2
1.2. Nutritional and Phytochemical Composition of Noni Fruit 6
1.3. Biological Activity of Noni Fruit 12
1.3.1. Antioxidant Activity 13
1.3.2. Anti-inflammation and Immunomodulation 13
1.3.3. Anti-microbial Activity 17
1.3.4. Anti-aging Activity 17
1.3.5. Diabetic Prevention 17
1.3.6. Cancer Prevention 19
1.3.7. Liver Protection 19
1.3.8. Gastrointestinal Protection 20
1.3.9. Bone Protection 20
1.3.10. Renal Protection 20
1.3.11. Wound Healing 20
1.3.12. Cardiovascular Protection 21
1.3.13. Other Studies 21

Chapter II. Literature Review of Helicobacter pylori (H. pylori) 22
1. Helicobacter pylori 23
1.1. Description and Characteristic 23
1.2. Urease 27
1.3. VacA 31
1.4. LPS 35
1.5. CagA 38
1.6. Adhesins 41
2. The Prevalence and Associated-diseases of H. pylori 44
2.1. Prevalence of H. pylori 44
2.2. Associated-diseases of H. pylori 46
3. Diagnosis of H. pylori 50
4. Treatments of H. pylori 52

Chapter III. Literature Review of Intestinal Health 56
1. Intestinal Bacteria 57
1.1. Clostridium perfringens and Intestinal Diseases 57
1.2. Escherichia coli and Intestinal Diseases 60
1.3. Lactobacillus spp. and Intestinal Health . 62
1.4. Bifidobacterium spp. and Intestinal Health 62
2. Inflammation and Intestinal Diseases 65
2.1. IL-1β and Intestinal Diseases 65
2.2. IL-8 and Intestinal Diseases 67
3. ROS and Inflammation 71

Purpose 75

Study Design 77

Sample Preparation 79

Chapter IV. Antiadhesion, Anti-inflammation and Healing Effects of Noni (Morinda citrifolia) Fruit Extracts on AGS Cells During Helicobacter pylori Infection 81
Abstract 82
1. Introduction 83
2. Materials and Methods 85
2.1. Preparation of Ethanol and Ethyl acetate Extracts 85
2.2. HPLC Analysis of Phenolic Compounds Present in Noni Fruit Extracts 85
2.3. Isolation and Culture of H. pylori 86
2.4. Cell Line and Culture 86
2.5. Cell Viability Assay 86
2.6. Inhibitory Zone on H. pylori 87
2.7. Bacterial Drug Sensitivity Test 87
2.8. Antiadhesion Assay 88
2.9. CagA, iNOS and COX-2 Protein Expression Assay 88
2.10. IL-8 and PGE2 Protein Secretion Assay 89
2.11. NO Production Assay 89
2.12. Neutrophil Isolation 89
2.13. Neutrophil Chemotaxis Assay 89
2.14. Intercellular ROS Determination by 2.7-Dihydrodichlorofluorescein (H2DCF-DA) 90
2.15. Wound Healing Assay 90
2.16. Intercellular VacA Protein and Cytochrome c Determination of AGS cells 90
2.17. Statistical Analysis 91
3. Results and Discussion 91
3.1. Phenolic Profile of Both Extracts 91
3.2. The Effect of Noni Fruit Extracts on H. pylori 92
3.3. Antiadhesion of H. pylori to Gastric Epithelial Cells 93
3.4. Inhibition on IL-8 and CagA 93
3.5. Inhibition of COX-2 and iNOS Signals 94
3.6. Inhibition of Neutrophil Chemotaxis on H. pylori Infection 96
3.7. The Contribution of Phenolic Compounds on Antiadhesion 96
3.8. Intracellular ROS Inhibition 100
3.9. Wound Healing Contribution 101
4. Conclusion 103
Tables 104
Table 1. Contents of phenolic, flavonoid compounds and condensed tannin of noni fruit extracts 104
Table 2. Inhibitory zone of noni extracts and amoxicillin on H. pylori 104
Table 3. The MICs, MBCs, IC50, and BC50 of noni extracts and amoxicillin on H. pylori 105
Figures 111
Figure 1. HPLC profiles of phenolic compounds present in ethanol and ethyl acetate extracts of fermented noni fruit 106
Figure 2. The urease test, CagA protein measured and VacA production of H. pylori 107
Figure 3. Density and viability of H. pylori after noni fruit extract treatment 108
Figure 4. The cell viability of noni fruit extracts and amoxicillin on AGS cells for 24 h 109
Figure 5. The cell viability of H. pylori cultured with AGS cells for 6 h 109
Figure 6. The cell viability of noni fruit extracts and H. pylori (300:1) cocultured with AGS cells for 6 h 110
Figure 7. Adhesion of H. pylori to AGS cells after ethanol extract and ethyl acetate extract treatment 111
Figure 8. Time-dependent induction of CagA, COX-2, and iNOS protein expression in AGS cells after H. pylori treated 112
Figure 9. Time-dependent induction of IL-8 protein production from AGS cells after H. pylori treated 112
Figure 10. Transportation of CagA and expression of IL-8 induced by H. pylori after ethanol and ethyl acetate extract treatment 113
Figure 11. Expression of COX-2 and expression of iNOS induced by H. pylori after ethanol and ethyl acetate extract treatment 114
Figure 12. Production of PGE2 and production of NO induced by H. pylori after ethanol and ethyl acetate extract treatment 115
Figure 13. Effect of noni fruit extracts on neutrophil chemotaxis and quantification 116
Figure 14. Contribution of phenolic compounds on antiadhesion and quantification 117
Figure 15. Strategies to inhibit bacterial adhesion 118
Figure 16. The effects of ethanol and ethyl acetate extracts of noni fruit on H. pylori-induced ROS production from AGS cells 119
Figure 17. The procedures of wound healing test 120
Figure 18. The different ratio of H. pylori-filtered medium and cell culture medium on cell viability and wound healing 121
Figure 19. Effect of noni fruit extracts on wound healing and quantification 122
Figure 20. Effect of noni fruit extracts on cell proliferation 123
Figure 21. Noni fruit extracts on levels of intercellular VacA protein and quantification 124
Figure 22. Noni fruit extracts on levels of intercellular cytochrome c protein 125

Chapter V. Noni (Morinda citrifolia L.) Fruit Extracts on Improve Colon Microflora, Antioxidation and Exert Anti-inflammatory Activities in Caco-2 Cells 126
Abstract 127
1. Introduction 128
2. Materials and Methods 130
2.1. Preparation of Ethanol and Ethyl acetate Extracts 130
2.2. Colon Microflora Determination 130
2.3. Contents of Total Phenolics and Phenolic Profiles Assay by High Performance Liquid Chromatography (HPLC) 131
2.4. Trolox Equivalent Antioxidant Capacity (TEAC) 132
2.5. Low-density Lipoprotein (LDL) Oxidation Lag Time Assay 132
2.6. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging 132
2.7. Superoxide Anions Scavenging Activity 133
2.8. Hydrogen Peroxide (H2O2) Scavenging Activity 133
2.9. Reduction Ability 133
2.10. Ferrous ion Chelating Assay 133
2.11. Cell lines and Culture Condition 134
2.12. Cell Viability Assay 134
2.13. Intercellular ROS Determination by 2.7-Dihydrodichlorofluorescein (H2DCF-DA) 134
2.14. The Expression of COX-2, MyD88 and Phosphorylation of IRAK-1, p38 MAPK, JNK Assay 134
2.15. Nuclear Factor kappa-B (NF-κB) p65 Subunit (P65 subunit) Protein Translocation Assay 135
2.16. IL-8 and PGE2 Protein Secretion Assay 135
2.17. Neutrophil Migration Assay 135
2.18. Statistical Analysis 136
3. Results and Discussion 136
3.1. Effects on Colon Microflora 136
3.2. Phenolic Profiles in Fermented Noni Fruit Extracts 137
3.3. Antioxidative Activity and Intracellular ROS Inhibition 137
3.4. Inhibition on COX-2 Expression 139
3.5. Inhibition on IL-8 Secretion 140
3.6. Inhibition on IL-8 and COX-2 by NF-κB Pathway 140
4. Conclusion 146
Tables 147
Table1. Quantitation of phenolic compounds in extracts of fermented noni fruit 147
Table 2. Effects of fermented noni extracts on total antioxidant activity and copper-induced human LDL oxidation 147
Figures 148
Figure 1. Effects of ethanol and ethyl acetate extract on growth of Lactobacillus spp., Bifidobacterium spp., Clostridium perfringens and Escherichia coli 148
Figure 2. HPLC profiles of ethanol and ethyl acetate extracts 149
Figure 3. Chemical structures of phenolic compounds from fermented noni fruit extracts 150
Figure 4. DPPH radical, superoxide anion radical and hydrogen peroxide scavenging efficiency of extracts derived from fermented noni fruit 151
Figure 5. Reducing power and ferrous ion chelating ability of extracts derived from fermented noni fruit 152
Figure 6. Cell viability of fermented noni fruit extracts on Caco-2 cells 153
Figure 7. Effects of ethanol and ethyl acetate extracts of fermented noni fruit on LPS-induced ROS production in Caco-2 cells 154
Figure 8. Dose-dependent expression of COX-2 and IL-8 protein of Caco-2 cells after IL-1β or LPS induced 155
Figure 9. COX-2 and IL-8 expression of Caco-2 cells by IL-1β or LPS-induced after extracts-treated for 24 h 156
Figure 10. The changes of p-38 MAPK phosphorylation, JNK phosphorylation and p65 translocation after ethyl acetate extract treated for 24 h 157
Figure 11. The production of COX-2 or IL-8 after phenolic compounds treated for 24 h 158
Figure 12. Signals of COX-2 and IL-8 of Caco-2 cells cultured with treatment during IL-1β or LPS-induced for 24 h 159
Figure 13. The changes of p65 subunit translocation, IRAK-1 phosphorylation and MyD88 expression after ethyl acetate extract treated for 24 h 160

Summary 161

Conclusion and Prospect 164

References 166

Annexes 206

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