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Characterization of bioactive components from Ishige okamurae and their biological activity

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Abstract
Seaweeds are one of the crucial marine living renewable resources as they are able to produce a great variety of secondary metabolites characterized by a broad spectrum of biological activities. Ishige okamurae (I. okamurae) is a member of the family Ishige as an edible brown alga, have attracted attention as various natural compounds with their biological activities.
In this study, new separation method was suggested to increase efficiency and improve the isolation performance using Pure C-850 FlashPrep system. In this study, Pure C-850 FlashPrep system was suggested to increase efficiency and improve the isolation of natural compounds from I. okamurae. A novel series of polyphenols, named as Ishophloroglucin; Ishophloroglucin A (IPA), Ishophloroglucin B (IPB), Ishophloroglucin C (IPC), and Ishophloroglucin D (IPD), and another two known compounds which is α/β-adenosine and Diphlorethohydroxycarmalol (DPHC) were isolated and identified. Their structure was elucidated by 1D- and 2D- Nuclear magnetic resonance (NMR), including 1H, 13C, HMBC, and COSY, as well as liquid chromatography-tandem mass spectrometry (LC-MS/MS).
As folk medicine, I. okamurae with various biological effects grows abundantly on the shores of Japan, north of China, and Korea where it is consumed as a part of daily diet. Appropriate quality control operations shall be employed to ensure that food having uniformed nutrients is suitable for human consumption. This study was conducted to establish an HPLC analysis method for determination of marker compounds as part of materials standardization for development of health functional food materials from I. okamurae. DPHC was selected as a marker compound due to it is more stable compared to IPA on the thermal stability analysis. The method was validated by system suitability, specificity, linearity, limits of detection (LOD), limits of quantification (LOQ), precision, and accuracy. The method showed high linearity of the calibration curve with a coefficient of correlation (R2) of 0.9999, and LOD and LOQ was 0.144 μg/mL and 0.435 μg/mL, respectively. Relative standard deviation values from precision was less than 0.622 %. Recovery rates of DPHC was 106.35 - 107.82 % within 100 ± 20%. An optimized method for extraction of DPHC in I. okamurae was established through diverse extraction conditions, and the validation indicated that the method is accurate and sensitive for the determination of marker compounds in I. okamurae to develop a health functional food material.
Polyphenols, a group of phloroglucinol (1,3,5-trihydroxybenzene), are the dominant polyphenolic secondary metabolites found in marine brown algae. IPA as a dominant polyphenol in I. okamurae, was investigated their anti-melanogenesis effects. Through the comparative molecular docking analysis of IPA to tyrosinase (3NM8), IPA was further investigated their inhibitory effect against tyrosinase activity and melanin formation that causes an increase anti-melanogenesis effect in both murine melanoma cells in-vitro model and zebrafish in-vivo model.
The α/β-adenosine (1:1 ratio) was firstly identified from I. okamurae, which is known as a naturally occurring substance that relaxes and dilates blood vessels in angiogenesis. To characterize the vasodilation effect of α/β-adenosine in endothelial dysfunction of angiogenesis induced by Particulate matter (PM) exposure, commercial isomer β-adenosine was also investigated as well in human endothelial cell line EA.hy926 in-vitro model and transgenic zebrafish in-vivo model.
In the last study, fucoidan is an interesting group of bioactive sulfated polysaccharides in brown seaweeds. The current study highlights the enrichment and extraction of fucoidan from I. okamurae through enzyme-assistant extraction using Celluclast enzyme. The structural characterization of fucoidan was performed using FTIR and NMR spectroscopy, their monosaccharide compositions was analyzed by HPAE-PAD, the molecular weight distribution was performed by agarose gel electrophoresis and the sulfate content was analyzed as well. The purified fucoidan and its anti-inflammatory activity was investigated in-vitro and in-vivo.
In summary, this research suggested various natural compounds from I. okamurae isolated by a flash prepare system and their potential uses as marker compounds or pharmaceutical and nutraceutical agents. Therefore, the purpose of this research is to discuss the potential health benefits of I. okamurae to be used as a functional material in industries such as functional foods, nutraceuticals, and cosmeceuticals to utilize this precious natural resource for future generations.
Author(s)
Yun Fei Jiang
Issued Date
2020
Awarded Date
2020. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009649
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
You, Jin Jeon
Table Of Contents
Background 1
Part I. Isolation and identification of polyphenols from Ishige okamurae and their uses 3
Section 1: Isolation and identification of polyphenols from Ishige okamurae 2
Abstract 3
1. Materials and methods . 4
1.1. General methods 4
1.2. Materials and chemicals 4
1.3. Extraction and fractionation of I. okamurae 4
2. Result and Discussion 5
4. Conclusion . 7
Section 2: Validation of DPHC by a HPLC method for the quality control of Ishige okamurae . 27
Abstract 28
1. Introduction 29
2. Materials and methods . 30
2.1. Chemicals 30
2.2. Analytical instruments and operation conditions 30
2.3. Sample preparation for HPLC analysis 30
2.4. Method validation 30
2.5. Statistical analysis 31
3. Results and discussion . 32
4. Conclusion . 35
Section 3: Anti-melanogenesis activity of Ishophloroglucin A (IPA) from Ishige okamurae in α-MSH-induced melanoma cells and zebrafish . 37
Abstract 38
1. Introduction 39
2. Materials and methods . 40
2.1. Chemical and reagents 40
2.2. Preparation of Ishige okamurae extract (IOE) and IPA 40
2.3. Molecular docking preparation 40
2.4. Cell assay 41
2.4.1. Determination of cell viability 41
2.4.2. Determination of melanin contents 41
2.4.3. Cellular Tyrosinase activity 42
2.5. In vivo assay 42
2.5.1. Origin and maintenance of parental zebrafish 42
2.5.2. Zebrafish pigmentation evaluating 43
2.6. Western blot analysis 43
2.7. Statistical Analysis 44
3. Results 45
3.1. Molecular Docking analysis of compounds IPA and DPHC 45
3.2. The effects of α-MSH, Arbutin, IOE and IPA on cell viability and melanin synthesis in B16F10 cells 45
3.3. Inhibitory effect of IOE and IPA on Tyrosinase activity and melanin synthesis in α-MSH-induced B16F10 cells 45
3.4. Western blotting analysis 49
3.4.1. IPA inhibits melanogenesis through downregulating the expressions of MITF, tyrosinase, TRP1, and TRP2 in α-MSH stimulated B16F10 cells 49
3.4.2. IPA inhibits melanogenesis through downregulating CREB-MITF pathway in α-MSH stimulated B16F10 cells 49
4. Discussion 54
5. Conclusion . 55
Part II. Isolation and identification of α/β-adenosine from I. okamurae and its anti-angiogenesis activity. 56
Abstract 57
1. Introduction 58
2. Materials and methods . 59
2.1. Chemicals and reagents 59
2.2. Plant material 59
2.3. Extraction and isolation 59
2.3.1. Identification of α-adenosine and β-adenosine 60
2.4. Cell culture 66
2.4.1. Cell viability 66
2.4.2. Culture Inserts for the Cell Migration Assay 66
2.4.3. Tube formation assay 66
2.4.4. Western blot analysis 67
2.4.5. Treatments of PM in zebrafish transgenic (flk: EGFP) embryos 67
2.4.6. Treatments of α/β-adenosine and β-adenosine in zebrafish transgenic (flk: EGFP) embryos and vasodilation assay 68
2.5. Statistical Analysis 68
3. Results and discussion . 69
3.1. Particulate matter (PM) induced angiogenesis in human endothelial cell line EA.hy926 69
3.2. Cell viability of the mixture of α/β-adenosine and β-adenosine on vascular endothelial cell line EA.hy926 73
3.3. Inhibitory effect of mixture α/β-adenosine and β-adenosine on PM induced cell migration 73
3.4. Effects of the mixture α/β-adenosine and β-adenosine on capillary formation in PM induced EA.hy926 Cells in Matrigel 77
3.5. Effects of the mixture α/β-adenosine on PI3K/Akt/eNOS Signal Pathway 77
3.6. Particulate matter-induced eyes vessel dilation in zebrafish embryo 77
3.7. Effects of the mixture α/β-adenosine and β-adenosine on PM-Treated Zebrafish Embryo 83
4. Conclusion . 83
Part III. Isolation of fucoidan from Ishige okamurae and its anti-inflammatory activity . 86
Abstract 87
1. Introduction 88
2. Materials and methods . 88
2.1. Materials 88
2.2. Extraction of polysaccharides 89
2.3. Separation of the IOP by anion-exchange chromatography 89
2.4. FTIR characterization and monosaccharide analysis of subfraction from IOP 90
2.5. NMR analysis 90
2.6. Cell culture 90
2.7. Determination of the PGE2 expression and pro-inflammatory cytokines expression 90
2.8. Western bolt analysis 91
2.9. In vivo zebrafish experiment 91
2.9.1 Evaluation of the inhibition of NO production, and protective effects against cell death in LPS induced zebrafish embryo model 91
2.10. Statistical analysis 91
3. Results and discussion . 92
3.1. Extraction, separation and monosaccharide analysis of IOP fractions 92
3.2. FT-IR spectroscopic and 1H NMR analysis of structural features of the polysaccharides 94
3.4. Cytotoxicity of each fraction from IOP in RAW 264.7 macrophages 98
3.5. The anti-inflammatory effect of F1-F4 fractions from IOP on the inhibition of NO in LPS stimulated RAW 264.7 macrophages 98
3.6. Pro-inflammatory cytokines production against F4 in LPS-stimulated RAW 264.7 macrophages 101
3.7. Effects of F4 on the mediation of iNOS, COX-2 in LPS-stimulated RAW 264.7 macrophages 101
3.8. In vivo evaluation of anti-inflammatory potential of F4 using zebrafish embryos 105
4. Conclusion . 105
References 106
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Yun Fei Jiang. (2020). Characterization of bioactive components from Ishige okamurae and their biological activity
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General Graduate School > Marine Life Sciences
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