Neuroinflammation Inhibitory Effects of the Secondary Metabolites isolated from Marine-derived Aspergillus sp.
- Alternative Title
- 해양 유래Aspergillus sp. 에서 분리된 이차 대사산물의Neuroinflammation 저해 효과
- Abstract
- The oxidative stress generates a wide range of reactive oxygen species that have been reported to induce oxidative damage of several components such as lipid, protein, nucleic acid and DNA, and induce inflammation or lesion on various organs. Oxidative stress also regulates the expression levels of metalloproteinases. Among the group of metalloproteinases, matrix metalloproteinases (MMPs) play a pivotal role in normal physiologic processes and neuronal development. Metalloproteinases, especially, MMPs are a class of structurally similar enzymes that contribute for the extracellular matrix degradation and play major role in normal as well as pathological tissue remodeling. Imbalance in the expression of MMPs leads to severe pathological condition that could initiate cardiac-related, cartilage-related, and neurological disorders. Three decades of endeavor for designing effective metalloproteinase inhibitors (MMPIs) with many not making unto final clinical trials seeks new resources for developing potent MMPIs. Marine environment has been the source of diverse life forms that provide varied bioactive compounds. Marine organisms, especially marine fungi have been consistently contributing with unparalleled bioactive compounds that have profound applications in cosmeceuticals and pharmaceuticals. Screening of natural products from marine organisms that could potentially inhibit the expression of metalloproteinases has gained a huge popularity and has become a popular field of research in life sciences.
In our present investigation for the isolation of bioactive substances (preferably novel) that could potentially inhibit the over expressions of MMPs, we worked on a marine-derived fungal species Aspergillus. The fungal specimen was collected at Cheongsapo, Busan, South Korea. The culturing of the fungal strain was done at large scale (10 L) tested for bioactive components from the broth and the mycelium as well. The resultant extracts were fractioned with the aid of silica gel flash chromatography that resulted in fractions that were further purified by Octadecyl functionalized silica gel (ODS) chromatography. The pure compounds from these fractions were obtained by using High-performance liquid chromatography (HPLC). The resultant pure compounds’ Electron Ionization Mass Spectrometer spectrum (EI-MS), 1-dimensional (1D) and 2-dimensional (2D) Nuclear Magnetic Resonance (NMR) spectra were recorded. The exploitation of marine fungal strain has yielded 4 compounds C1, C2, C3, and C4. Bioactive assays including cell viability and anti-oxidant properties of these secondary metabolites were also evaluated to check their potentiality for in vitro tests. Bioassays included the anti-oxidant properties, inhibitory effects on MMP-2 and MMP-9 and also the cholinesterases inhibitory effects with correlation of MMP-9 inhibition in murine microglial cells. The results have shown that C1 and C2 have exhibited potential antioxidant ability. In addition, these compounds exhibited inhibitory effects on matrix gelatinases MMP-2 and MMP-9 in murine microglia BV-2 cells in a dose dependent manner. The contribution of oxidative stress in neurological inflammation is a major concern in the eliciting of Alzheimer’s disease (AD), Parkinson’s disease (PD), Ischemia. The inhibitory effects of C1 and C2 on cholinesterases and beta-secretase are also evaluated. As the role of oxidative stress and over-expression of MMP-2 and MMP-9 is evident in the prognosis of neurological inflammations, the inhibitory effects of C1 and C2 on MMP-2 and MMP-9 expressions in LPS stimulated BV-2 cells was evaluated. In this study, C1 and C2 have inhibited MMP-2 and MMP-9 expression via the blockade of MAPKs signaling pathways thus suggesting their anti-inflammatory effects in microglia. On the other hand, these two compounds have effectively inhibited the cholinesterases and β-secretase suggesting their possible ability to control the neuroinflammatory responses pertaining to AD. Therefore these results suggest that C1 and C2 isolated from the Aspergillus sp. have effective antioxidant activities and strong potential to inhibit the neuroinflammatory responses.
- Issued Date
- 2013
- Awarded Date
- 2013. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Abstract i
Table of Contents iv
List of Tables vii
List of Figures viii
List of Abbreviations xi
Chapter 1. Oxidative Stress and Neuroinflammation 1
1.1. Oxidative stress and immune responses 2
1.2. Role of Oxidative Stress in Neuroinflammation 6
1.3. Matrix Metalloproteinases 10
1.4. Role of MMPs in Neuroinflammation 14
1.5. Inhibitors for MMP mediated inflammatory responses from natural resources 18
1.6. Marine Fungi as sources of Metalloproteinase inhibitory substances 18
Chapter 2. Isolation of secondary metabolites from marine-derived Aspergillus sp. and their cytotoxic effects 20
2.1. Introduction 21
2.2. Materials and Methods 25
2.2.1. Culture and identification of marine-derived fungus 25
2.2.2. Instruments and Chemical Reagents 25
2.2.3. Isolation and purification of secondary metabolites from marine-derived fungus, Aspergillus sp 26
2.2.4. Cell Culture 27
2.2.5. Cell viability assay 27
2.2.6. Statistical analysis 28
2.3. Results and discussion 29
2.3.1. Isolation, culture and identification of marine-derived fungal species 30
2.3.2. Fractionation of secondary metabolites from marine derives fungus, Aspergillus sp. 34
2.3.3. Isolation and purification of secondary metabolites marine-derived fungus, Aspergillus sp. 36
2.3.4. Spectroscopic properties of isolated secondary metabolites of marine-derived fungus, Aspergillus sp. 40
2.3.5. Cytotoxicity evaluation of the isolated secondary metabolites of marine-derived fungus, Aspergillus sp. 41
2.4. Summary 46
Chapter 3. Antioxidant and Neuroinflammation inhibition effects of secondary metabolites from marine-derived fungus Aspergillus sp. 48
3.1. Introduction 49
3.2. Materials and Methods 56
3.2.1. Biological chemicals and reagents 56
3.2.2. Cell Culture 56
3.2.3. Antioxidant assay 57
3.2.4. DPPH Assay 57
3.2.5. Ferric Reducing power 57
3.2.6. Cell viability determination 58
3.2.7. Preparation of protein sample and quantification 58
3.2.8. Western blot analysis 59
3.2.9. Effects of C1and C2 on the protein expressions of MMP-2, MMP-9 and MAP kinases in LPS stimulated BV-2 cells 60
3.2.10. In vitro ChEs inhibitory activity assay 60
3.2.11. In vitro β-secretase inhibitory effects 61
3.3. Results and discussion 62
3.3.1. Total antioxidant assay 63
3.3.2. DPPH Assay 65
3.3.3. Ferric-reducing antioxidant power 71
3.3.4. Effects of C1and C2 on the protein expressions of MMP-2, MMP-9 and MAP kinases in LPS stimulated BV-2 cells 71
3.3.5. In vitro ChEs inhibitory activity 83
3.3.6. In vitro β-Secretase inhibitory assay 90
3.4. Summary 93
Chapter 4. Cumulative Summary and Conclusions 96
References 106
Appendix I. Supplementary Spectroscopic Data for Compound 1 118
Appendix II. Supplementary Spectroscopic Data for Compound 2 124
Appendix III. Supplementary Spectroscopic Data for Compound 3 131
Appendix IV. Supplementary Spectroscopic Data for Compound 4 138
Acknowledgements………………………………………..…………………………………..145
Publications……………………………………………………………………………………148
- Degree
- Doctor
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