Inhibition of radical-mediated oxidation of cellular biomolecules and NF-kB expression by gallic acid conjugated chitooligosaccharides with molecular weight 3-5 kDa
- Alternative Title
- Gallic acid 키토산올리고당(3-5 kDa)의 세포 내 라디칼 저해 및 NF-kB 발현 억제 효과
- Abstract
- The aim of this study was to explain the novel possibility of the involvement of gallic acid conjugated chitooligosaccharides with molecular weight 3-5 kDa (G-COS) in improving cellular antioxidant potential and thereby controlling oxidative damage that could be effective for its therapeutic potential in osteoarthritis. The free radicals scavenging G-COS ability was assessed by using electron spin resonance (ESR) technique. In addition, the inhibition of reactive oxygen species (ROS), membrane lipid peroxidation, membrane protein oxidation and DNA oxidation were assessed on mouse macrophages (RAW264.7 cells) and human chondrosarcoma (SW1353 cells). Moreover, the antioxidative mechanisms of G-COS were evaluated by protein expression and gene expression levels of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione (GSH). More importantly, nuclear factor-kappa B (NF-B) western blot analysis revealed that G-COS inhibits radical mediated expression and activation of NF-B proteins (transcription factor in the regulation of the expression of a number of genes related to osteoarthritis). This study demonstrated that G-COS can be used as a potential compound-based natural antioxidant in functional foods and pharmaceutical industries.
- Issued Date
- 2010
- Awarded Date
- 2010. 8
- Type
- Dissertation
- Keyword
- Chitooligosaccharides gallic acid antioxidant radical scavenging mouse macrophages (RAW 264.7) human chondrosarcoma (SW1353)
- Publisher
- 부경대학교
- Affiliation
- Department of Chemistry, The Graduate School Pukyong National University
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Abstract i
Table of contents ii
List of tables iv
List of schemes and figures v
List of Abbreviations ix
Introduction 1
1. Antioxidant enzymes 1
2. Chitin, chitosan and applications 2
3. Gallic acid 4
4. Reactive oxygen species (ROS) 4
5. Membrane lipid, membrane protein and DNA oxidation 5
6. NF-B signal in antioxidant activity 7
Materials and methods 12
1. Materials 12
2. Synthesis of gallic acid conjugated chitooligosaccharides (G-COS) 12
3. Infrared spectroscopy 13
4. Instrumental analysis 13
5. Electron spin resonance (ESR) spectrometer 13
5.1. Hydroxyl radical (OH) assay 14
5.2. DPPH radical (DPPH) assay 14
5.3. Superoxide radical anion (O2) assay 14
5.4. Carbon-centered radical (R) assay 14
6. Cell culture and cytotoxicity determination 15
7. Determination of intracellular formation of reactive oxygen species (ROS) using fluorescence labeling 15
8.Genomic DNA isolation 16
9. DNA oxidation assay 16
10. Protein oxidation 17
11. Membrane lipid peroxidation assessment by DPPP fluorescence method 17
12. Membrane lipid peroxidation assessment by TBARS method 18
13. RNA isolation 18
14. Reverse transcription polymerase chain reaction (RT-PCR) analysis 19
15. Extraction of nuclear and plasma protein 20
16. Western blot analysis 20
17. Statistical analysis 21
Results 22
1. Structural characterization of G-COS 22
2. Radical scavenging potency in cell-free systems 24
2.1. Hydroxyl radical (OH) scavenging capacity 24
2.2. DPPH scavenging capacity 27
2.3. Superoxide radical anion (O2) scavenging capacity 27
2.4. Carbon-centered radical (R) scavenging capacity 28
3. Cell viability 28
4. Cellular radical scavenging ability 31
5. Inhibition of radical mediated DNA damage 34
6. Cell membrane protein oxidation inhibition 34
7. Inhibition of membrane lipid peroxidation 38
8. Effect on antioxidant enzymes of G-COS 43
9. Expression and nuclear translocation of NF-B in the presence of G-COS 43
Discussion 48
Conclusion 52
References 53
Acknowledgement 66
- Degree
- Master
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