Anti-Inflammatory Mechanisms of Phlorotannins derived from Eisenia bicyclis and Their Inhibitory Effects on Matrix Metalloproteinases.
- Alternative Title
- 대황으로부터 분리한 Phlorotannins의 염증저해기전 및 기질금속단백질분해효소 저해효과
- Abstract
- Eisenia bicyclis (Kjellman. E. bicyclis) Setchell is a perennial brown alga, belonging to the family Laminariaceae. In present study, antioxidant, anti-inflammatory and matrix Metalloproteinases (MMPS) inhibitory effects of E. bicyclis were investigated. The antioxidant effects of methanolic extract of E. bicyclis and its organic solvent soluble fractions including dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (n-BuOH) and water (H2O) fractions were measured through their free radical scavenging activities in non-cellular and cellular systems in vitro. Several antioxidant assays, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, superoxide anion and peroxynitrite radicals scavenging activities using the electron spin resonance spectrometry (ESR) technique and intracellular reactive oxygen species (ROS) by 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) method in mouse macrophages cell line (RAW 264.7 cell) were evaluated. The antioxidant activities of the each fractions were in the order of ethyl acetate (EtOAc) > n-butanol (n-BuOH) > dichloromethane (CH2Cl2) > and water (H2O) fraction. Moreover, the order of total poly-phenolics contents of each fraction showed the same order in accordance with the radical scavenging activities. Five phlorotannins were isolated and characterized from EtOAc fraction of E. bicyclis, which showed strongest antioxidant activity among the fractions. According to comprehensive spectral analysis of MS and NMR data, phloroglucinol, Fucofuroeckol-A (FF), Dioxinodehydroeckol (DD), Eckol (EK) and Dieckol (DE) were isolated. DD and FF were isolated from E. bicyclis for the first time, and furthermore, no biological reports of FF have been published up to now. In cellular and non-cellular oxidative systems, EK and FF showed significant activities compared to other phlorotannins.
To evaluate the anti-inflammatory effects of phlorotannins derived from E. bicyclis on RAW264.7 mouse macrophage, FF and EK, which have no cytotoxicity and previous reports regarding anti-inflammatory activity, were selected for further study. To identify the effects of FF and EK on inflammation, mediated by reactive oxygen species (ROS), The anti-inflammatory effects of these phlorotannins on lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage was investigated and their anti-inflammatory mechanisms were elucidated. The results showed that FF and EK suppress LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) dose-dependently. Enzyme-linked immunosorbent (ELISA) and cytometric bead array assay (CBA) clearly demonstrated that FF and EK significantly reduced the productions of pro-inflammatory cytokines such as, interleukin (IL)-6 and tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1. Moreover, these phlorotannins reduced nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) activation. These results strongly suggest that the inhibitory effects of FF and EK on LPS-induced NO and PGE2 production might be due to the suppression of NF-κB and MAPKs signaling pathway. Moreover, above intracellular antioxidant activities of FF and EK supports that their anti-inflammatory effects might be attributable to scavenging ROS in RAW264.7 mouse macrophage.
Consequently, we evaluated the inhibitory effects of FF and EK on matrix metalloproteinase (MMP) -2 and 9 in HT1080 human fibrosarcoma cell line. In MMPs inhibitory assay, FF and EK showed strong direct inhibition on both MMP-2 and 9 dose-dependently. FF and EK also inhibited protein expression of MMP-2 and 9. Especially, FF stimulates the expression of tissue inhibitor of matrix metalloproteinase (TIMP) -2 and this may cause inhibition on MMPs activities via direct binding. Moreover, FF and EK suppressed the cell migration, and cell invasion in 3D culture model on HT1080 cells.
Therefore, these results suggested that FF and EK have remarkable antioxidant activities and strong potential as valuable natural anti-inflammatory and cancer chemopreventive agents to develop nutraceuticals and pharmaceuticals.
- Issued Date
- 2010
- Awarded Date
- 2010. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- Alternative Author(s)
- Sang-Hoon Lee
- Affiliation
- 부경대학교 화학과
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Abstract i
Table of contents iv
List of table ix
List of figures x
List of abbreviations xiv
Chapter.Ⅰ. General introduction 1
Chapter.Ⅱ. Isolations of phlorotannins derived from brown alga Eisenia bicyclis and their antioxidative activities 4
1. Introduction 5
2. Materials and Methods 8
2.1. Materials and chemicals 8
2.2. Extraction, isolation and purification of phlorotannins from E. bicyclis 8
2.3. Quantification of total phenol contents 10
2.4. DPPH radical scavenging activity assay 10
2.5. Hydroxyl radical scavenging activity assay 11
2.6. Superoxide radical scavenging activity assay 11
2.7. Peroxynitrite scavenging activity assay 12
2.8. Cell cultures 12
2.9. Cell viability assay 13
2.10. Myeloperoxidase activity 13
2.11. Measurement of intracellular reactive oxygen species (ROS)-scavenging activities using DCFH-DA labeling 14
2.12. Statistical analysis 14
3. Results and discussion 15
3.1. Total phenolic contents of the MeOH extract and its solvent fractions 15
3.2. DPPH radical scavenging activity of the MeOH extract and its solvent fractions 16
3.3. Hydroxyl radical scavenging activity MeOH extract and its solvent fractions 16
3.4. Superoxide anion scavenging activity of the MeOH extract and its solvent fractions 19
3.5. Peroxynitrite scavenging activity of the MeOH extract and its solvent fractions 19
3.6. Viability of MeOH extract and its fractions in RAW264.7cell lines 20
3.7. Effects of MeOH extract and its solvent fractions in cellular reactive oxygen species (ROS) 20
3.8. Isolation of phlorotannins from E. bicyclis 22
3.9. Spectroscopic properties of compounds 1-5 isolated from the EtOAc fraction 24
3.10. Identification of compounds isolated from E. bicyclis 28
3.11. Radical scavenging activity of the phlorotannnins derived from
E. bicyclis 31
3.12. Cellular reactive oxygen species (ROS) determination of the phlorotannnins derived from E. bicyclis using DCFH-DA assay 34
3.13. Effects of phlorotannnins derived from E. bicyclis on MPO activities 34
4. Summary 42
Chapter. Ⅲ. Anti-Inflammatory mechanisms of phlorotannins derived from Eisenia bicyclis 45
1. Introduction 46
2. Materials and Methods 51
2.1. Materials and chemicals 51
2.2. Cell culture 51
2.3. Cell viability assay 51
2.4. Nitrite assay 52
2.5. Determination of TNF-α and IL-1β using Enzyme-linked immunosorbent assay 52
2.6. Determination of prostaglandin E2 (PGE2) using prostaglandin E2 direct assay kit 53
2.7. Determination of IL-6 and MCP-1 using cytometric bead array (CBA) method 54
2.8. Western blot 54
2.9. RT-PCR 55
2.10. Electrophoretic Mobility Shift Assay (EMSA) 56
2.11. NF-κB/p65 nuclear localization image using confocal Laser Scanning Microscopy 57
2.12. Statistical analysis 58
3. Results and discussion 59
3.1. Cell cytotoxic effects of FF and EK on RAW264.7 cell line 59
3.2. Effects of FF and EK on NO production in LPS-stimulated RAW264.7 cell line 59
3.3. Effects of FF and EK on productions of TNF-α, IL-6 and MCP-1 in LPS-stimulated RAW264.7 cell line 63
3.4. Effects of FF and EK on productions of prostaglandin E2 (PGE2) in LPS-stimulated RAW264.7 cell line 70
3.5. Effects of FF and EK on productions of iNOS and COX-2 in LPS-stimulated RAW264.7 cell line 70
3.6. Effects of FF and EK on productions of TNF-α, IL-6 and IL-1β in LPS-stimulated RAW264.7 cell line 75
3.7. Effects of FF and EK on LPS-induced Nuclear translocation of NF-κB and I-κBα 78
3.8. Effects of FF and EK on the phosphorylation of MAPKs in LPS-stimulated RAW264.7 cell line 83
4. Summary 87
Chapter Ⅳ. Inhibitory effects of phlorotannins derived from brown alga Eisenia bicyclis on MMP-2 and -9 expressions in HT1080 cell line 91
1. Introduction 92
2. Materials and Methods 96
2.1. Materials and chemicals 96
2.2. Cell culture 96
2.3. Cell viability assay 96
2.4. In vitro wound migration assay 97
2.5. In vitro three-dimensional (3D) culture of HT1080 cell line 97
2.6. MMP-2 and 9 enzyme inhibitory assay 98
2.7. Western blot analysis 98
2.8. Statistical analysis 99
3. Results and discussion 100
3.1. Cell cytotoxic effects of FF and EK on HT1080 cell line 101
3.2. In vitro effects of FF and EK on the migration of HT1080 cell line 101
3.3 Effects of FF and EK on the 3D culture in HT1080 cell line 101
3.4 Effects of FF and EK on MMP-2 and -9 activities 107
3.5 Effects of FF and EK on the protein levels of MMP-2 and 9 on HT1080 cell line 110
3.6 Effects of FF and EK on the protein levels of TIMP-1 and 2 on HT1080 cell line 110
3.7 Effects of FF and EK on transcriptional regulation of NF-κB and C-fos on HT1080 cell line 113
4. Summary 119
Chapter Ⅴ. Conclusion 122
References 126
Abstract in Korean 143
Acknowledgements 145
- Degree
- Doctor
- Files in This Item:
-
-
Download
Anti-Inflammatory Mechanisms of Phlorotannins derived from Eisenia bicyclis and Their Inhibitory Eff.pdf
기타 데이터 / 2.7 MB / Adobe PDF
-
Items in Repository are protected by copyright, with all rights reserved, unless otherwise indicated.