Biological Activities of Novel Synthetic Tri-peptide Conjugated Chitosan Oligosaccharides
- Abstract
- Nature has been serving as an invaluable source of templates for the most important inventions and discoveries. Chitosan and chitosan derived compounds are marine byproducts which have been shown to exhibit bioactivities including antibacterial, antioxidant, antidiabetic and anti-HIV. Proteins are among the most potent and selective molecules offering an endless potential of different structure and sequence preferences. In this study, we investigated the antidiabetic, antiobesity and anti-HIV properties of small size (90% deacetylated, MW below 1 kD) Chitooligosaccharide-tripeptide (pep-COS) conjugates. The tripeptides which consist of Tryptophan (W), Methionine (M) and Glutamine (Q) have been synthesized. The structure of successfully conjugated chitooligosaccharides was determined by carbon and proton NMR, elemental analysis and spectrometry. The newly synthesized compounds were compared with nascent COS. Initially, cytotoxicity of the compounds were determined using AGS, 3T3L1, C8166, IEC-6 and RAW264.7 cells and all the compounds were found to be safe. Subsequently, the compounds were used to screen anti-HIV, antiobesity and antidiabetic activities. QMW-COS and WMQ-COS protected C8166 cells from cell-lytic effects of HIV-1 RF strain. Furthermore, these two compounds inhibited HIV-induced syncytia formation. We confirmed the decrease in viral-load in cell culture using p24 ELISA assay. To determine the specific mode of action of QMW-COS and WMQ-COS to inhibit HIV, we checked the inhibitory action of these compounds on viral reverse transcriptase and protease enzymes. However, we could not detect any inhibitory activity of conjugated compounds on recombinant reverse transcriptase and protease enzyme in vitro. When we co-cultured HIV-infected and uninfected C8166 cells, these two compounds actively inhibited the syncytia formation upon co-culture assay. Time-dependent addition of compounds and finally gp120-CD4 ELISA assay revealed that QMW-COS and WMQ-COS are bioactive compounds inhibiting HIV-induced cytopathic effects via exerting their effects on HIV entry stage.
Besides, three derivatives of chitooligosaccharides (COS) were tested for their mechanism on antiobesity and antidiabetic effects where nascent COS shows a slight inhibition of adipogenesis. Peptide conjugated COS treatment remarkably decreased lipid accumulation, an indicator for adipogenesis in 3T3-L1 adipocyte cells, which shows that peptide conjugation of COS increased adipogenesis inhibitory effect compared to nascent COS. Also mRNA analysis of adipogenic factors such as peroxisome proliferator-activated receptor (PPAR) gamma and sterol regulatory element-binding protein (SREBP) 1 were considerably decreased when compared between COS and its derivatives. Protein levels of PPAR-gamma and CCAAT/enhancer-binding protein (C/EBP) alpha, the key adipogenic factors, downregulated with COS derivative treatment, Moreover, only MWQ-COS moderately inhibited alpha-glucosidase and alpha-amylase activity which are important enzymes for controlling blood glucose in diabetic conditions. Further assays clearly showed that MQW-COS were able to enhance the insulin facilitation through increasing secreted insulin levels in pancreatic cells and insulin effectiveness as indicated by glucose uptake assays. Among all, QWM-COS also protected pancreatic beta-cells from high glucose-induced deterioration which is the main cause of beta-cell loss in diabetic conditions. Additionally, WQM-COS were also able to prevent protein glycation according to advanced glycation end product assay which promotes WQM-COS as a promising lead molecule to prevent diabetic complications caused in later stages of disease.
As a result, this studies clearly exhibited strong bioactivities for six pep-COS derivatives. While QMW-COS and WMQ-COS were notable able to prevent HIV-1 infection therefore related damages to human T-cells, the remaining four samples showed their potential on diabetes and related fields such as insulin secretion and adipogenesis prevention. Overall, these six pep-COSs were proved to be promising lead molecules to be used in drug development or several industries including functional food and nutraceuticals.
- Issued Date
- 2013
- Awarded Date
- 2013. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 대학원
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Abstract i
Table of Contents iv
List of Figures ix
List of Tables xii
List of Abbreviation xiii
Chapter I. Peptide-Chitooligosasccharide Conjugation and Screening of Bioactivities
1. Introduction 2
1.1. Chitooligosaccharides and their bioavailability 2
1.2. Chitooligosaccharide derivatization 6
1.3. Potential usage of peptide as bioactivity enhancer 7
1.4. Conjugation of amino acids and peptides 8
2. Materials and Methods 10
2.1. Materials and chemicals for synthesis 10
2.2. Cell lines and chemicals for screening methods 10
2.3. Synthesis of peptide conjugated COSs 11
2.4. Instrumental analysis 13
2.5. Determination of toxicity of the samples 16
2.6. Anti-inflammatory effect screening 17
2.6.1. Nitric oxide production assay 17
2.7. Anti-cancer effect screening 17
2.7.1. Anti-proliferative effect 17
2.8. Anti-HIV effect screening 18
2.8.1. Syncytia formation assay 18
2.8.2. Cell viability assay 18
2.9. Screening anti-diabetes activity 19
2.9.1. Glycerol assay 19
2.10. Statistical analysis 20
3. Results and Discussion 21
3.1. Synthesis of amino-acid conjugated COSs 23
3.2. Determination of the toxicity of the samples 32
3.3. Anti-inflammation activity 32
3.4. Anti-cancer activity 37
3.5. Anti-HIV activity 37
3.6. Anti-diabetes activity 43
4. Conclusion 45
References 46
Chapter II. Investigation Anti-Diabetes and Anti-Obesity Mechanism
1. Introduction 54
1.1. Enzyme inhibitors 58
1.2. Protein glycation and diabetes 59
1.3. Role of obesity in type 2 diabetes (insulin resistance) 61
1.4. Obesity 62
1.5. Chitooligosaccharides and peptides 64
2. Materials and Methods 65
2.1. Materials and chemicals 65
2.2. Cell culture 65
2.3. Differentiation of adipocytes 65
2.4. Oil red O staining 66
2.5. Triglyceride assay 67
2.6. RNA extraction 68
2.7. Reverse transcription polymerase chain reaction (RT-PCR) 68
2.8. Western blot 71
2.9. α -glucosidase enzyme assay 71
2.10. α -amylase enzyme assay 72
2.11. Protein glycation assay 73
2.12. Glucose uptake assay 73
2.13. Statistical analysis 74
3. Results and Discussion 75
3.1. Differentiation of adipocytes 75
3.2. Effect of COS and its derivative on lipid accumulation of adipocyte cells 75
3.3. Inhibition of adipogenic transciption factor expression 83
3.4. Effect of QWM-COS and MWQ-COS on protein glycation 86
3.5. Effect of WQM-COS and MQW-COS on activity of α-glucosidase and
α-amylase 88
3.6. Effect of QWM-COS and MWQ-COS on glucose uptake 90
4. Conclusion 94
References 95
Chapter III. Elucidation of the action mechanism against HIV-1 infection
1. Introduction 101
1.1. Acquired immunodeficiency syndrome (AIDS) and HIV 101
1.2. Course of infection 103
1.3. Human immunodeficiency virus type 1 106
1.4. Genes and structure 107
1.5. HIV-1 replication cycle 109
1.6. Viral entry 109
1.7. Reverse transcription, nuclear import, and integration 110
1.8. Gene expression 111
1.9. Budding and maturation 112
1.10. Anti-HIV therapy 114
1.11. Antiretroviral drugs 116
1.11.1. Protease inhibitors 116
1.11.2. Reverse transcriptase inhibitors 118
1.11.3. Integrase inhibitors 119
1.11.4. Entry inhibitors 120
1.11.5. Fusion inhibitors 120
1.11.6. Coreceptor antagonists 121
1.12. Current status of HIV/AIDS in Korea 122
2. Materials and Methods 126
2.1. Materials and chemicals 126
2.2. Cells and viruses 126
2.3. Cell Viability assay 127
2.4. Inhibition of syncytia formation 127
2.5. Inhibition of cytopathic effect of HIV-1 128
2.6. Western blot analysis 128
2.7. p24 antigen capture ELISA 129
2.8. Delayed addition of pep-COSs to HIV-1IIIB infected cells 130
2.9. Effect of pep-COSs on cell-cell fusion 130
2.10. Luciferase gene reporter assay 130
2.11. CD4-gp41 ELISA 131
2.12. X-Gal staining 132
2.13. Polymerase chain reaction 132
2.14. Statistical analysis 133
3. Results and Discussion 134
3.1. Cell viability assay 134
3.2. Inhibition of syncytia formation 134
3.3. Inhibition of cytopathic effect of HIV-1 137
3.4. p24 antigen capture ELISA 139
3.5. Luciferase gene reporter assay 141
3.6. Delayed addition of pep-COSs to HIV-1IIIB infected cells 141
3.7. Effect of pep-COSs on cell-cell fusion 143
3.8. CD4-gp41 ELISA 144
3.9. X-gal staining 149
3.10. Polymerase chain reaction 149
4. Conclusion 152
References 143
Acknowledgement 162
- Degree
- Doctor
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