Isolation and Biological Activities of Secondary Metabolites from Marine-derived Fungus, Microsporum sp.
- Alternative Title
- 해양유래 곰팡이 Microsporum sp.의 이차 대사산물
- Abstract
- Recently, a great deal of interest has been paid by the consumers towards bioactive compounds from natural resources as functional ingredients in nutraceuticals, cosmeceuticals, and pharmaceuticals. Among natural resources, marine-derived endophytic fungi are valuable sources of structurally diverse bioactive compounds. Marine-derived fungi have been shown in recent years to produce a plethora of new bioactive secondary metabolites, which are of interest as potential lead structures for the food, cosmeceutical, and pharmaceutical industries. In the present study, a marine-derived algicolous fungus identified as Microsporum sp. was mass cultivated and three secondary metabolites including a prenylated indole alkaloid, neoechinulin A (34.3 mg) and two anthraquinone derivatives, such as chrysophanol (17.5 mg) and physcion (11.8 mg) were isolated from the culture broth extract (1710 mg). The structures of the above isolated three secondary metabolites were determined by 1H, 13C, UV, IR, HMQC, HMBC, and MS spectroscopic data. Furthermore, free radical scavenging effects of neoechinulin A, chrysophanol, and physcion were analyzed by various in vitro methods, such as reducing power, DPPH scavenging, hydroxyl radical scavenging, superoxide radical scavenging, and Fe2+ chealting assays as well as intracellular free radical scavenging assays, such as reactive oxygen species (ROS) scavenging, DNA oxidation, membrane protein oxidation and membrane lipid oxidation assays. Among three isolated secondary metabolites, neoechinulin A has shown moderate antioxidant effect in vitro and intracellular free radical scavenging in murine microglial BV2 cells.
Moreover, cosmeceutical effects, such as antityrosinase activity, and cellular melanin formation inhibitory effect on mouse melanoma B16F1 cells were analyzed. Chrysophanol and physcion have shown potent skin whitening effect and chrysophanol has stronger activity than physcion. Tyrosinase protein expression has down regulated by chrysophanol in a dose-dependent manner.
Induction of apoptosis is a useful approach in cancer therapies and many efforts have been made to discover novel anticancer drugs through the isolation of apoptosis-inducing agents from marine-derived fungi. Furthermore, cancer chemoprevention, antiproliferative, and apoptosis induction effects of neoechinulin A, chrysophanol, and physcion were evaluated. The quinone reductase (QR) activity in mouse hepatoma 1c1c7 cells was induced by neoechinulin A, chrysophanol, and physcion and shown potent cancer chemopreventive effect. Moreover, neoechinulin A and physcion have reduced the cell viability in human cervical carcinoma HeLa cells. According to the flow cytometry analysis, it has proven that physcion increased the level of ROS and depleted the cell number in HeLa cells. Furthermore, Western blot analysis confirmed that both neoechinulin A and physcion could significantly induce cell apoptosis through down-regulating Bcl-2 expression, up-regulating Bax expression, and activating the caspase-3 pathway. Collectively, these results suggest that neoechinulin A, chrysophanol, and physcion could be potential candidates in the utilization of nutraceutical, cosmeceutical, and pharmaceutical fields, respectively.
- Issued Date
- 2012
- Awarded Date
- 2012. 2
- Type
- Dissertation
- Keyword
- Microsporum
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 화학과
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Table of Contents
Abstract i
Table of Contents iii
List of Tables viii
List of Figures ix
List of Abbreviations xiii
Chapter 1. Introduction 1
1. Introduction to marine-derived bioactive functional ingredients 2
2. Nutraceuticals and functional foods from marine organisms 4
3. Cosmeceuticals from marine organisms 7
4. Chemopreventive and anticancer pharmaceuticals from marine resources
12
5. The value of marine-derived fungi as a source of functional ingredients 14
6. Scope of the study 16
Chapter 2. Extraction and isolation of bioactive secondary metabolites from marine-derived fungus, Microsporum sp. 18
1. INTRODUCTION 19
2. MATERIALS AND METHODS 21
2.1. Instruments, chemicals and reagents 21
2.2. Isolation and culture of marine-derived fungus Microsporum sp .22
2.3. Extraction, isolation, and structure elucidation of bioactive secondary metabolites from Microsporum sp. 22
2.4. Statistical analysis 23
3. RESULTS AND DISCUSSION 24
3.1. Identification of the marine-derived fungus Microsporum sp. 24
3.2. Extraction of bioactive secondary metabolites from marine-derived fungus Microsporum sp. 24
3.3. Secondary metabolites from marine-derived fungus Microsporum sp. 30
3.4. Spectroscopic properties of compounds isolated from the marine-derived fungus Microsporum sp. 31
4. SUMMARY 50
Chapter 3. Antioxidant effects of neoechinulin A, chrysophanol, physcion from marine-derived fungus, Microsporum sp. 51
1. INTRODUCTION 52
2. MATERIALS AND METHODS 55
2.1. Chemicals and reagents 55
2.2. Cell culture and cytotoxicity determination 55
2.3. Reducing power assay (FRAP assay) 56
2.4. DPPH radical scavenging assay 57
2.5. Superoxide radical scavenging assay 57
2.6. Hydroxyl radical scavenging assay 58
2.7. Fe2+ ion chelating assay 58
2.8. Genomic DNA isolation 59
2.9. Genomic DNA oxidation assay 60
2.10. Determination of intracellular formation of ROS using fluorescence labeling 60
2.11. Membrane protein oxidation assay 61
2.12. Membrane lipid peroxidation assay by DPPP fluorescence method 62
2.13. Statistical analysis 63
3. RESULTS AND DISCUSSION 64
3.1. Cell viability 64
3.2. Reducing power 64
3.3. DPPH radical scavenging effect of neoechinulin A, chrysophanol, and physcion 65
3.4. Superoxide radical scavenging effect of neoechinulin A, chryophanol, and physcion 66
3.5. Hydroxyl radical scavenging effect of neoechinulin A, chrysophanol, and physcion 66
3.6. Fe2+ chelating effect of neoechinulin A, chrysophanol, and physcion 67
3.7. Intracellular free radical scavenging effect of neoechinulin A 75
3.8. Membrane protein oxidation prevention by neoechinulin A 76
3.9. Membrane lipid oxidation prevention by neoechinulin A 77
3.10. Cellular DNA oxidation prevention by neoechinulin A 78
3.11. Free radical scavenging pathways of neoechinulin A, chrysophanol, and physcion 84
4. SUMMARY 87
Chapter 4. Cosmeceutical effects of neoechinulin A, chrysophanol, and physcion from marine-derived fungus, Microsporum sp. 89
1. INTRODUCTION 90
2. MATERIALS AND METHODS 94
2.1. Materials 94
2.2. Cell culture and cytotoxicity determination 94
2.3. Tyrosinase inhibitory assay in vitro 95
2.4. Determination of cellular melanin content 95
2.5. Western blot analysis 96
2.6. Statistical analysis 97
3. RESULTS AND DISCUSSION 98
3.1. Cell viability 98
3.2. Inhibition of tyrosinase activity in vitro 98
3.3. Inhibition of cellular melanin content in B16 melanoma cells 100
3.4. Expression level of tyrosinase in α-MSH induced melanoma cells 101
4. SUMMARY 108
Chapter 5. Chemopreventive and anticancer effects of neoechinulin A, chrysophanol, and physcion from marine-derived fungus, Microsporum sp. 109
1. INTRODUCTION 110
2. MATERIALS AND METHODS 113
2.1. Materials 113
2.2. Cell culture and cytotoxicity determination 113
2.3. Quinone reductase (QR) induction assay 114
2.4. Detection of intracelluar ROS and O2•- levels 116
2.5. Acridine orange/Ethidium bromide staining 116
2.6. DNA fragmentation assay 117
2.7. Hoechst staining 118
2.8. Mitochondrial membrane potential (ΔΨm) by JC-1 staining 118
2.9. Western blot analysis 118
2.10. Statistical analysis 119
3. RESULTS AND DISCUSSION 121
3.1. Cell viability and cytotoxicity of neoechinulin A, chrysophanol, and physcion on 1c1c7 cells 121
3.2. Cell viability and cytotoxicity of neoechinulin A, chrysophanol, and physcion on HeLa cells 121
3.3. Cancer chemopreventive and quinone reuctase induction effect of neoechinulin A, chrysophanol, and physcion on 1c1c7 cells 122
3.4. Intracellular ROS and O2•¯ formation induction by physcion 127
3.5. DNA fragmentation effect of physcion in HeLa cells 128
3.6. FACS analysis of physcion induced ROS formation in HeLa cells
129
3.7. Observation of nuclear DNA damage by Hoechst staining 130
3.8. Mitochondrial membrane potential (ΔΨm) reduction 130
3.9. Induction of apoptosis by neoechinulin A and physcion 130
3.10. Modulation of Bcl-2 and p53 family by neoechinulin A and
physcion 131
4. SUMMARY 147
Conclusions 149
References 152
Acknowledgements 168
List of Publications 171
List of Tables
Table 1. Nutraceuticals and functional food ingredients from marine bioresources 07
Table 2. Marine-derived cosmeceuticals 08
List of Figures
Figure 1. Natural bioactive functional ingredients as nutraceuticals, cosmeceuticals, and pharmaceuticals 03
Figure 2. Potential health benefits of cosmeceuticals on human skin 11
Figure 3. Molecular targets of dietary agents (Aggarwal & Shishodia, 2006) 13
Figure 4. Habitats of marine-derived fungi in the marine environment (Bugni & Ireland, 2004) 14
Figure 5. Bioactive compounds from marine-derived fungi with habitats (Bugni & Ireland, 2004) 15
Figure 6. Morphology of the marine-derived fungus Microsporum sp. 25
Figure 7. Cellular fatty acid composition analysis of Microsporum sp. 26
Figure 8. Cellular fatty acid analysis by the Korean Culture Center of the Microorganisms (KCCM) 27
Figure 9. Extraction of the marine-derived fungus, Microsporum sp. (MFS-YL) 28
Figure 10. Isolation of compounds 1-3 from the broth extract of marine-derived fungus, Microsporum sp. 29
Figure 11. Structures of the compounds 1-3 isolated from marine-derived fungus, Microsporum sp., compound 1 (Neoechinulin A, 34.3 mg), compound 2 (Chrysophanol, 17.5 mg), and compound 3 (Physcion, 11.8 mg) 30
Figure 12. Purification of compound 1 (Neoechinulin A): HPLC chromotogramme (a), TLC (b), and purified compound (c) 33
Figure 13. LREI-MS spectrum of Neoechinulin A in CD3OD 34
Figure 14. 1H-NMR (a) and 13C-NMR (b) spectrums of Neoechinulin A in CD3OD 35
Figure 15. DEPT NMR (a) and HMQC NMR (b) spectrums of Neoechinulin A in CD3OD 36
Figure 16. HMBC NMR (a) and COSY NMR (b) spectrums of Neoechinulin A in CD3OD 37
Figure 17. IR spectrum of Neoechinulin A 38
Figure 18. UV spectrum of Neoechinulin A 39
Figure 19. LREI-MS spectrum of Chrysophanol in CHCl3 41
Figure 20. 1H-NMR (a) and 13C-NMR (b) spectrums of Chrysophanol in CDCl3 42
Figure 21. HMQC (a) and HMBC (b) spectrums of Chrysophanol in CDCl3 43
Figure 22. COSY spectrum of Chrysophanol in CDCl3 44
Figure 23. LREI-MS spectrum of Physcion in CHCl3 46
Figure 24. 1H-NMR (a) and 13C-NMR (b) spectrums of Physcion in CDCl3 47
Figure 25. HMQC (a) and HMBC (b) spectrums of Physcion in CDCl3 48
Figure 26. COSY spectrum of Physcion in CDCl3 49
Figure 27. Effects of neoechinulin A on cell viability of murine microglia BV-2 cells. 69
Figure 28. Reducing power of neoechinulin A, chrysophanol, and physcion. Vitamin C was used as positive control. 70
Figure 29. DPPH radical scavenging activity of neoechinulin A, chrysophanol, and physcion. 71
Figure 30. Superoxide radical scavenging activity of neoechinulin A, chrysophanol, and physcion. 72
Figure 31. Hydroxyl radical scavenging activity of neoechinulin A, chrysophanol, and physcion. 73
Figure 32. Fe2+ ion chelating activity of neoechinulin A, chrysophanol, and physcion. EDTA was used as positive control. 74
Figure 33. ROS scavenging effect of neoechinulin A on BV-2 cells. 80
Figure 34. Membrane protein oxidation preventive effect of neoechinulin A on BV-2 cells. 81
Figure 35. Cellular lipid perxidation preventive effect of neoechinulin A on BV-2 cells. 82
Figure 36. DNA oxidation preventive effect of neoechinulin A on BV-2 cells. 83
Figure 37. Pathways of ROS-induced damage prevention by neoechinulin A, chrysophanol, and physcion from marine-derived fungus Microsporum sp. 86
Figure 38. Melanin biosynthesis (King et al., 1995). 92
Figure 39. Cellular melanin biosynthesis signaling pathways (Park et al., 2009).
93
Figure 40. Cell viability and cytotoxicity of neoechinulin A, chrysophanol, and physcion on mouse melanoma B16F1 cells. 102
Figure 41. Tyrosinase enzyme activity inhibition in vitro effect of neoechinulin A, chrysophanol, and physcion. Kojic acid was used as positive control. 103
Figure 42. Inhibitory effect of α-MSH stimulated melanin formation in B16F1 cells. 104
Figure 43. α-MSH induced cellular melanin formation in B16 melanoma cells inhibition assay. 105
Figure 44. Effect of chrysophanol on expression of tyrosinase in α-MSH induced melanin synthesis in B16F melanoma cells. 106
Figure 45. Skin-whitening effect of chrysophanol from marine-derived fungus Microsporum sp. 107
Figure 46. Two mechanisms of Phase II xenobiotic-metabolizing enzyme induction by Phase II inducers (Tan & Spivack, 2009). 112
Figure 47. Relative cell viability of neoechinulin A, chrysophanol, and physcion on mouse hepatoma 1c1c7 cells. 124
Figure 48. Cell viability and cytotoxicity of neoechinulin A, chrysophanol, and physcion on human cervical carcinoma HeLa cells. 125
Figure 49. Realative quinone reductase induction activity of neoechinulin A, chrysophanol, and physcion on mouse hepatoma 1c1c7 cells. 126
Figure 50. Intracellular ROS formation induced by physcion. 134
Figure 51. Apoptosis cell staining in control and physcion treated (5 µM) HeLa cells with acridin orange/ethidium bromide. 135
Figure 52. DNA fragmentation effect of physcion on HeLa cells. 136
Figure 53. FACS analysis of ROS formation induction in HeLa cells by physcion. 137
Figure 54. Comparison of FACS analysis of intracellular ROS formation induction in HeLa cells by physcion. 138
Figure. 55. Hoechst staining of HeLa cells treated with neoechinulin A. 139
Figure. 56. Hoechst staining of HeLa cells treated with physcion. 140
Figure. 57. Visualization of the reduction of mitochondrial membrane potential via JC-1 staining. 141
Figure 58. Western blot analysis of the activation of caspase-3, and -9 in HeLa cells by physcion. 142
Figure 59. Western blot analysis of the activation of caspase-3, and -9 in HeLa cells by neoechinulin A. 143
Figure 60. Western blot analysis of the effect of physcion on the expression of p53, p21, Bcl-2, and Bax in HeLa cells. 144
Figure 61. Western blot analysis of the effect of neoechinulin A on the expression of p53, p21, Bcl-2, and Bax in HeLa cells. 145
Figure 62. Proposed mechanism with pathways of apoptosis induction effect of neoechinulin A and physcion from marine-derived fungus Microsporum sp. on HeLa cells. 146
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