제주대학교 Repository

Cosmeceutical effect of fucoidan isolated from brown seaweed, Hizikia fusiforme collected from Jeju Island

Metadata Downloads
Abstract
In human, skin is the largest organ of the integumentary system. Skin is outer covering of the body and plays an important immunity role because of its protective effect against pathogens. Skin undergoes chronological aging like other organs. In addition, it undergoes aging as a consequence of environmental damage because it is direct exposure to the outside environment. The environmental factors, such as chemicals, air pollution, fine dust particles, and ultraviolet (UV) irradiation, which bring toxicity or stress to human skin and induce skin aging. In these environmental factors, UV irradiation from sunlight is the primary environmental factor that causes human skin aging and results in pigment accumulation and wrinkle formation. UV can be classified into three subtypes of UVA, UVB, and UVC, based on the wavelength. UVB has a medium wavelength and is thought to bring more cellular stress to humans compared to the other two subtypes. UVB is known to be associated with human health through stimulating reactive oxygen species (ROS) generation. The excessive ROS induce oxidative stress and apoptosis, as well as damage cellular components including proteins, lipids, and DNA. In addition, ROS activate cell signaling pathways including nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), and mitogen‐activated protein kinases (MAPKs). The activated pathways will stimulate the expression of the relative protein including matrix metalloproteinases (MMPs) and pro-inflammatory cytokines, which subsequently lead to skin wrinkling and inflammation. Therefore, a ROS scavenger, NF-κB, AP-1, and MAPKs blocker, and MMPs inhibitor that can scavenges ROS, blocks NF-κB, AP-1, and MAPKs
pathways, inhibits MMPs without toxicity may potentially against UVB-induced skin inflammatory and photo-damage. Natural compounds such as vitamins, polysaccharides, polyphenols, and proteins isolated from terrestrial or aquatic natural resources, including plants, animals, and microorganisms, have been used medicinally throughout human history. Compounds isolated from seaweeds, such as polyphenols, polysaccharides, and pigments, possess antioxidant, anti-inflammation, anticancer, UV protection, and anti-wrinkle effect. In particular, polysaccharides isolated from seaweeds have been reported to possess strong bioactivities. Sargassum fulvellum, Codium fragile, and Hizikia fusiforme are the most popular edible seaweeds in Asian countries such as Korea, China, and Japan. S. fulvellum, C. fragile, and H. fusiforme are abundantly distributed in Jeju Island. It is a rich and potential natural resource could be used as an ingredient in functional food and medicine, industries. Many reports support that S. fulvellum, C. fragile, and H.
fusiforme contain various natural bioactive compounds and possess the potential to develop functional food and medicine. However, the cosmeceutical effects of these seaweeds had not been investigated so far. Therefore, in the present study, the cosmeceutical effects of these three edible seaweeds have been investigated. In the present study, S. fulvellum, C. fragile, and H. fusiforme had extracted by water, ethanol, and enzymes. The composition of seaweed extracts were analysis. In addition, the free radical scavenging activities and the commercial tyrosinase, collagenase, and elastase inhibitory effects of seaweed extracts were determined. The results indicate that enzyme-assisted extraction could improve the extraction yield comparing to water and ethanol extraction. And all seaweed extracts possess strong free radical scavenging activity, especially on alkyl and hydroxyl radicals. Furthermore, the Celluclast-assisted extract of H. fusiforme contains the highest polysaccharides content and possesses strong free radical scavenging activity as well as strong tyrosinase, collagenase, and elastase inhibitory effects. Therefore, H. fusiforme was selected as target seaweed to separate polysaccharides and evaluate the cosmeceutical effects. The crude polysaccharides from Celluclast-assisted extract of H. fusiforme (HFCPS) were prepared by Celluclast-assisted hydrolysis and ethanol precipitation. HFCPS possesses potent cosmeceutical effects including antioxidant, anti-inflammation,
whitening, UV protection, and anti-wrinkle effect. Furthermore, the Hizikia fucoidan (HFCPSF4) was separated from HFCPS. HFCPSF4 contains 99.01±0.61% sulfated polysaccharides, which comprise fucose (97.20%), rhamnose (2.09%), mannose (18.32%), and arabinose (0.38%). The cosmeceutical effects of HFCPSF4 were measured including antioxidant activity, anti-inflammatory activity, whitening effect,
protective effect, and anti-wrinkle effect. The results indicate that HFCPSF4 significant reduces hydrogen peroxide (H2O2)-induced oxidative stress in monkey kidney fibroblast cells (Vero cells) and in zebrafish; remarkable attenuates lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages and in zebrafish; inhibits alpha-melanocyte stimulating hormone (α-MSH)-stimulated melanogenesis in B16F10
melanoma cells; against UVB-induced photo-damage in keratinocytes (HaCaT cells) and in zebrafish; protects UVB-induced skin wrinkling in human dermal fibroblasts (HDF cells). These results demonstrate that HFCPSF4 possesses potent cosmeceutical effects, and can be a potential ingredient in the cosmetic industry. In a conclusion, the present study demonstrates the fucoidan (HFCPSF4) isolated from crude polysaccharides from Celluclast-assisted extract of Hizikia fusiforme (HFCPS) possesses strong cosmeceutical effects including antioxidant, anti-inflammation, whitening, UV protection, and anti-wrinkle activity. It can be used as an ingredient in pharmaceutical and cosmeceutical industries.
Author(s)
Wang, Lei
Issued Date
2019
Awarded Date
2019. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000008806
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
전유진
Table Of Contents
SUMMARY i
LIST OF TABLE iv
LIST OF FIGUREv
Part Ⅰ. 1
Extraction and bioactivity scavenging of seaweeds1
ABSTRACT 2
INTRODUCTION 4
1. Materials and Methods 7
1.1. Reagents and chemicals 7
1.2. Collection and process of seaweed samples 7
1.3. Water and ethanol extraction 10
1.4. Enzyme-assisted extraction 10
1.5. Determination of chemical composition of seaweed extracts 14
1.5.1. Analysis of polysaccharide content of seaweed extracts 14
1.5.2. Analysis of total phenolic content of seaweed extracts 14
1.5.3. Analysis of protein content of seaweed extracts 15
1.6. Evaluation of the free radical scavenging activity of seaweed extracts 15
1.7. Measurement of mushroom tyrosinase inhibitory effects of seaweed extracts 15
1.8. Measurement of collagenase inhibitory effects of seaweed extracts 15
1.9. Measurement of elastase inhibitory effects of seaweed extracts 16
2. Results and Discussion 17
2.1. Extraction yield and proximate composition of seaweed extracts 17
2.2. Free radical scavenging activities of seaweed extracts 23
2.3. Tyrosinase inhibitory effects of seaweed extracts 29
2.4. Collagenase inhibitory effects of seaweed extracts 31
2.5. Elastase inhibitory effects of seaweed extracts 33
CONCLUSION 35
Part Ⅱ. 37
Preparation of crude polysaccharides of Hizikia fusiforme (HFCPS) and evaluation of its bioactivities 37
ABSTRACT 37
INTRODUCTION 38
Section 1: Celluclast-assisted extraction of Hizikia fusiforme and separation of polysaccharides from Celluclast-assisted extract of Hizikia fusiforme40
Abstract 40
1. Materials and Methods 41
1.1. Reagents and chemicals 41
1.2. Preparation of polysaccharides from H. fusiforme 41
1.3. Chemical analysis of HFC and HFCPS 41
2. Results and Discussion 43
2.1. Yield, total carbohydrate, phenolic, and sulfate content of HFC and HFCPS 43
2.2. Natural sugar composition of HFC and HFCPS 43
3. Conclusion 47
Section 2: Antioxidant activity of polysaccharides from Celluclast-assisted extract of Hizikia fusiforme in vitro in Vero cells and in vivo in zebrafish 48
Abstract 48
1. Materials and methods 49
1.1. Reagents and Chemicals 49
1.2. Evaluation of free radical scavenging activities of HFC and HFCPS 49
1.3. Cell culture 49
1.4. Determination of the cytotoxicity of HFCPS on Vero cells 50
1.5. Determination of the protective effect of HFCPS against H2O2-induced intracellular ROS generation in Vero cells 50
1.6. Measurement of the protective effect of HFCPS against H2O2-induced cytotoxicity in Vero cells 50
1.7. Nuclear staining with Hoechst 33342 50
1.8. Maintenance of zebrafish 51
1.9. Application of HFCPS and H2O2 to zebrafish embryos 51
1.10. Determination of heart-beating rate, ROS generation, and cell death in zebrafish 51
1.11. Statistical analysis52
2. Results and Discussion 53
2.1. Free radical scavenging activities of HFC and HFCPS 53
2.2. Cytotoxicity of HFCPS on Vero cells 53
2.3. Protective effect of HFCPS against H2O2-induced oxidative stress in Vero cells 56
2.4. Protective effect of HFCPS against H2O2-induced apoptosis 56
2.5. HFCPS improve survival rate and reduce heart-beating rate in H2O2-induced zebrafish 59
2.6. Protective effect of HFCPS against H2O2-induced ROS generation and cell death in zebrafish 59
3. Conclusion 63
Section 3: Anti-inflammatory effect of polysaccharides from Celluclast-assisted extract of Hizikia fusiforme 64
Abstract 64
1. Materials and methods 65
1.1. Reagents and Chemicals 65
1.2. Cell culture 65
1.3. Measurement of NO production and cell viability 65
1.4. Measurement of PGE2 and pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) production 66
1.5. Western blot analysis 66
1.6. Statistical analysis67
2. Results and Discussion 68
2.1. The effect of HFCPS on LPS-induced NO generation and cytotoxicity in RAW 264.7 macrophages 68
2.2. HFCPS decreased PGE2 and pro-inflammatory cytokines release in LPS-induced RAW 264.7 macrophages 68
2.3. HFCPS inhibited the expression of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages 71
3. Conclusion 73
Section 4: Whitening effect of polysaccharides from Celluclast-assisted extract of Hizikia fusiforme 74
Abstract 74
1. Materials and methods 75
1.1. Reagents and Chemicals 75
1.2. Measurement of the mushroom tyrosinase inhibitory effect of HFCPS 75
1.3. Cell culture 75
1.4. Cytotoxicity assay76
1.5. Determination of cellular melanin content 76
1.6. Measurement of intracellular tyrosinase activity 76
1.7. Western blot analysis 77
1.8. Statistical analysis77
2. Results and Discussion 78
2.1. Tyrosinase inhibitory activity of HFCPS 78
2.2. Cytotoxicity of HFCPS 78
2.3. Effect of HFCPS on melanin synthesis in α-MSH-stimulated B16F10 cells 78
2.4. Effect of HFCPS on intracellular tyrosinase activity in α-MSH-stimulated B16F10 cells 79
2.5. Effect of HFCPS on tyrosinase, TRP-1, TRP-2, and MITF expression in α-MSH-stimulated B16F10 cells 83
3. Conclusion 85
Section 5: UV protective effect of polysaccharides from Celluclast-assisted extract of Hizikia fusiforme 86
Abstract 86
1. Materials and methods 87
1.1. Reagents and Chemicals 87
1.2. Cell culture and UVB irradiation 87
1.3. Determination of cytotoxicity of HFCPS on HaCaT cells 87
1.4. Measurement of intracellular ROS generation in UVB-irradiated HaCaT cells 88
1.5. Determination of cell viability 88
1.6. Measurement of apoptosis body formation 88
1.7. Maintenance of zebrafish 89
1.8. Application of HFCPS and UVB to zebrafish 89
1.9. Statistical Analysis 90
2. Results and Discussion 91
2.1. Cytotoxicity of HFCPS on HaCaT cells 91
2.2. Effect of HFCPS on intracellular ROS generation and cell death in UVB-irradiated HaCaT cells 91
2.3. Effect of HFCPS on apoptosis formation in UVB-irradiated HaCaT cells . 94
2.4. Effect of HFCPS on ROS generation, cell death, NO production, and lipid peroxidation in UVB-irradiated zebrafish 96
3. Conclusion 101
Section 6: Anti-wrinkle effect of polysaccharides from Celluclast-assisted extract of Hizikia fusiforme 102
Abstract 102
1. Materials and methods 103
1.1. Reagents and Chemicals 103
1.2. Measurement of collagenase inhibitory effect of HFCPS 103
1.3. Measurement of elastase inhibitory effect of HFCPS 104
1.4. Cell culture and UVB-irradiation 104
1.5. Determination of cytotoxicity of HFCPS and UVB irradiation on HDF cells 104
1.6. Determination of the effect of HFCPS against UVB-induced HDF cell damage 105
1.7. Determination of relative intracellular elastase and collagenase activities on UVB-irradiated HDF cells 105
1.8. Determination of collagen synthesis level and MMPs expression levels on UVB-irradiated HDF cells 106
1.9. Western blot analysis 106
1.10. Statistical Analysis 107
2. Results and Discussion 108
2.1. HFCPS inhibits collagenase from Clostridium Histolyticum and elastase from porcine pancreas 108
2.2. HFCPS promotes HDF cell proliferation and UVB irradiation damages HDF cells 110
2.3. HFCPS improves cell viability and reduces intracellular ROS in UVB-irradiated HDF cells 112
2.4. HFCPS inhibits intracellular collagenase and elastase activities in UVB-irradiated HDF cells 112
2.5. HFCPS protects collagen synthesis and reduces MMPs expression levels in UVB-irradiated HDF cells 115
2.6. HFCPS inhibits NF-κB activation, reduces AP-1 phosphorylation, and suppress MAPKs activation in UVB-induced HDF cells 115
3. Conclusion 119
CONCLUSION 120
Part Ⅲ. 122
Isolation of fucoidan from crude polysaccharides of Hizikia fusiforme and evaluation of its bioactivities 122
ABSTRACT 122
INTRODUCTION 123
Section 1: Isolation fucoidan from crude polysaccharides from Celluclast-assisted extract of Hizikia fusiforme 125
Abstract 125
1. Materials and Methods 126
1.1. Reagents and chemicals 126
1.2. Separation of fucoidan from HFCPS 126
1.3. Chemical analysis of fractions separated from HFCPS126
1.4. Evaluation of the free radical scavenging activity of seaweed extracts 127
1.5. Measurement of mushroom tyrosinase inhibitory effects of seaweed extracts 127
1.6. Measurement of collagenase inhibitory effects of seaweed extracts 128
1.7. Measurement of elastase inhibitory effects of seaweed extracts 128
1.8. Characterization of the purified fucoidan from HFCPS by Fourier transform infrared (FT-IR) spectroscopy 128
2. Results and Discussion 129
2.1. Total carbohydrate, phenolic, and sulfate content of fractions separated from HFCPS 129
2.2. Natural sugar composition of fractions separated from HFCPS 129
2.3. Free radical scavenging activities of fractions separated from HFCPS 132
2.4. Tyrosinase inhibitory effects of fractions from HFCPS 134
2.5. Collagenase inhibitory effects of fractions from HFCPS 134
2.6. Elastase inhibitory effects of fractions from HFCPS 134
2.7. FT-IR analysis of purified fucoidan 135
3. Conclusion 140
Section 2: Antioxidant activity of Hizikia fucoidan in vitro in Vero cells and in vivo in zebrafish 141
Abstract 141
1. Materials and methods 142
1.1. Reagents and Chemicals 142
1.2. Cell culture 142
1.3. Determination of the protective effect of HFCPSF4 against H2O2-induced intracellular ROS generation in Vero cells 142
1.4. Measurement of the protective effect of HFCPSF4 against H2O2-induced cytotoxicity in Vero cells 143
1.5. Nuclear staining with Hoechst 33342 143
1.6. Western blot analysis 143
1.7. Application of HFCPSF4 and H2O2 to zebrafish embryos 144
1.8. Determination of heart-beating rate, ROS generation, cell death, and lipid peroxidation in zebrafish 144
1.9. Statistical analysis144
2. Results and Discussion 145
2.1. Protective effect of HFCPSF4 against H2O2-induced oxidative stress in Vero cells 145
2.2. Protective effect of HFCPSF4 against H2O2-induced apoptosis 145
2.3. HFCPSF4 improves the expression of catalase and SOD-1 via regulating Nrf2/HO-1 pathway in H2O2-induced Vero cells 148
2.4. HFCPSF4 improves survival rate and reduces heart-beating rate in H2O2-induced zebrafish 152
2.5. Protective effect of HFCPSF4 against H2O2-induced ROS generation, cell death, and lipid peroxidation in zebrafish 152
3. Conclusion 157
Section 3: Anti-inflammatory effect of Hizikia fucoidan in vitro in RAW 264.7 macrophages and in vivo in zebrafish 158
Abstract 158
1. Materials and methods 159
1.1. Reagents and Chemicals 159
1.2. Cell culture 159
1.3. Measurement of NO production and cell viability 159
1.4. Measurement of PGE2 and pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) production 160
1.5. Western blot analysis 160
1.6. Application of HFCPSF4 and LPS to zebrafish embryos 160
1.7. Determination of heart-beating rate, ROS generation, cell death, and NO generation in zebrafish 161
1.8. Statistical analysis161
2. Results and Discussion 162
2.1. The effect of HFCPSF4 on LPS-induced NO generation and cytotoxicity in RAW 264.7 macrophages 162
2.2. HFCPSF4 decreased PGE2 and pro-inflammatory cytokines release in LPS-induced RAW 264.7 macrophages 162
2.3. HFCPSF4 inhibited the expression of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages 165
2.4. HFCPSF4 inhibited NF-κB in LPS-induced RAW 264.7 macrophages 165
2.5. HFCPSF4 improved survival rate and reduced heart-beating rate in LPS-induced zebrafish 169
2.6. Protective effect of HFCPSF4 against LPS-induced ROS generation, cell death, and NO production in zebrafish 169
3. Conclusion 174
Section 4: Whitening effect Hizikia fucoidan isolated from HFCPS in vitro in B16F10 melanoma cells175
Abstract 175
1. Materials and methods 176
1.1. Reagents and Chemicals 176
1.2. Cell culture 176
1.3. Cytotoxicity assay176
1.4. Determination of cellular melanin contents177
1.5. Western blot analysis 177
1.6. Statistical analysis177
2. Results and Discussion 179
2.1. Cytotoxicity of HFCPSF4 179
2.2. Effect of HFCPSF4 on melanin synthesis in α-MSH-stimulated B16F10 cells 179
2.3. Effect of HFCPSF4 on tyrosinase, TRP-1, TRP-2, and MITF levels and ERK-MAPK phosphorylation in α-MSH-stimulated B16F10 cells 182
3. Conclusion 186
Section 5: UV protective effect of Hizikia fucoidan isolated from HFCPS in vitro in HaCaT cells and in vivo in zebrafish 187
Abstract 187
1. Materials and methods 188
1.1. Reagents and Chemicals 188
1.2. Cell culture and UVB irradiation 188
1.3. Measurement of intracellular ROS generation in UVB-irradiated HaCaT cells 188
1.4. Determination of cell viability 189
1.5. Measurement of apoptosis body formation 189
1.6. Western blot analysis 189
1.7. Application of HFCPSF4 and UVB to zebrafish190
1.8. Statistical Analysis 190
2. Results and Discussion 191
2.1. Effect of HFCPSF4 on intracellular ROS generation and cell death in UVB-irradiated HaCaT cells 191
2.2. Effect of HFCPSF4 on apoptosis formation in UVB-irradiated HaCaT cells 191
2.3. Effect of HFCPSF4 on Bax/Bcl-xL, PARP, and cleaved caspass-3 levels in UVB-irradiated HaCaT cells 194
2.4. Effect of HFCPSF4 on ROS generation, cell death, NO production, and lipid peroxidation in UVB-irradiated zebrafish 197
3. Conclusion 202
Section 6: Anti-wrinkle effect of Hizikia fucoidan isolated from HFCPS in vitro in human dermal fibroblasts 203
Abstract 203
1. Materials and methods 204
1.1. Reagents and Chemicals 204
1.2. Cell culture and UVB-irradiation 204
1.3. Determination of the effect of HFCPSF4 against UVB-induced HDF cell damage 205
1.4. Determination of relative intracellular elastase and collagenase activities on UVB-irradiated HDF cells 205
1.5. Determination of collagen synthesis level, MMPs expression levels, pro-inflammatory cytokines levels on UVB-irradiated HDF cells 206
1.6. Western blot analysis 206
1.7. Statistical Analysis 207
2. Results and Discussion 208
2.1. HFCPSF4 improves cell viability and reduces intracellular ROS in UVB-irradiated HDF cells 208
2.2. HFCPSF4 inhibits intracellular collagenase and elastase activities in UVB-irradiated HDF cells 208
2.3. HFCPSF4 protects collagen synthesis and reduces MMPs and pro-inflammatory cytokines expression levels in UVB-irradiated HDF cells 211
2.4. HFCPSF4 inhibits NF-κB activation, reduces AP-1 phosphorylation, and suppresses MAPKs activation in UVB-irradiated HDF cells 211
3. Conclusion 217
CONCLUSION 218
REFERENCES 219
ACKNOWLEDGEMENT 235
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Wang, Lei. (2019). Cosmeceutical effect of fucoidan isolated from brown seaweed, Hizikia fusiforme collected from Jeju Island
Appears in Collections:
General Graduate School > Marine Life Sciences
Authorize & License
  • AuthorizeOpen
Files in This Item:

Items in Repository are protected by copyright, with all rights reserved, unless otherwise indicated.