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Characterization of bioactive components from arsenic-lower Jeju Hizikia fusiforme

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Abstract
Traditional medical application becomes potential supplement which is widely used as therapies for avoiding diseases in oriental countries [1]. Ocean is regarded as a life blood of all living creature with more than 70% of water mass surrounded on earth [2]. There are predicts 2.2 million eukaryotic species living under the water [3], hence marine product is a promising resource for medical application [4]. Seaweed, also named algae, refers to three types, Rhodophyta (red seaweed), Phaeophyta (brown seaweed), and Chlorophyta (green seaweed) [5]. Hizikia fusiforme, Hijiki, is a well-known brown seaweed that has been consumed as a marine resource for hundred years in Korea, Japan, and China [6, 7]. Hijiki is used as functional food because of its widely range of bioactivities [8, 9]. It is rich in nutrients including polysaccharides and mineral elements, and shows beneficial effects such as anticancer, immunomodulating activities, and anti-mutagenic effects [10-12]. Hijiki contains higher amounts of mineral elements than other foods [13]. At certain doses, most of mineral elements are regarded as nutrition, but some metals are human toxicants such as inorganic arsenic (iAs). iAs tend to be more toxic than organic arsenic, and the concentrations of iAs in Hijiki are as high as hundreds of ppm. It is indicated that the As concentration is greatly exceeds the limit stated under the provisional tolerable weekly intake, as advised by the World Health Organization (WHO) [14]. Chronic and acute exposure to arsenic leads to cancer, neurological disorders, liver disease, renal disease, and other health disorders [15, 16]. Acid-wash and hot water cooking are the most widely used methods to iAs removal [17-20], and the level of iAS intake allows the limits recommended by the WHO. In this work, one percentage of citric acid- and hot water processing was performed to remove AS in Hijiki. The As-lower Hijiki was used for separated and isolated different bioactive fractions depend on their polarity. There are three active fractions: polysaccharides, saringosterol acetate, and fucoxanthin, which were isolated from water, hexane, and chloroform extraction, respectively. In water extraction, polysaccharides are the high amounts and bioactivities fractions. In polysaccharides, alginic acid and fucoidan are the two major active compounds. Alginic acid is a protonized water-insoluble polysaccharide, and comprises a family of unbranched binary copolymers of (1→4) linked β-D-mannuronic acid and α-L-guluronic acid [21]. Fucoidan is a sulfate-rich polysaccharide [22], which have been investigated its biological properties such as antioxidant, antiviral, anticancer, anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-adhesive effects [23-27]. In seaweeds, the molecular weight, sulfate content, and monosaccharide composition are the three main factors linked to the biological activity of fucoidan [28]. Moreover, the structural characteristics of fucoidan differ depending on the extract method, seaweed species, harvest season, geographic area, and algal maturity [24, 29-31]. Therefore, in the presence work, the bioactivities of polysaccharides from AS-lower Hijiki were evaluated. In hexane extraction, saringosterol acetate was isolated by using centrifugal partition chromatography, and the anticancer and anti-obesity effects of saringosterol acetate were evaluated. In chloroform extraction, fucoxanthin-riched fraction was isolated and evaluated its anti-inflammatory effect against FD-induced inflammatory responses. Taken together, the aim of this study is to explore and discover the various properties of compounds of AS-lower Hijiki from different collected locations.
Author(s)
Yu Lin Dai
Issued Date
2020
Awarded Date
2020. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009405
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Jeon, You Jin
Table Of Contents
Part I. Acid- and hot water processing for arsenic-lower Hizikia fusiform 1
1 Abstract 2
2 Introduction 3
3 Materials and methods 5
3.1 Materials 5
3.2 Hijiki origin and acid- hot water processing . 5
3.3 Extraction of crude fucoidan (CF) 7
3.4 Extraction of alginic acid (HFA) 7
3.5 Fourier-transform infrared spectroscopy (FTIR) characterization . 8
3.6 Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis 8
3.7 Evaluation of heavy metal content 8
3.8 Chemical composition analysis 8
3.9 Statistical analysis 9
4 Results and Discussion 9
4.1 FTIR analysis. 9
4.2 LC-ESI-MS analysis 11
4.3 Heavy metal ion content of Hijiki. 11
4.4 Chemical compositions in processed and unprocessed Hijiki 18
5 Conclusion 18
Part II. Bioactivities of the polysaccharides from three collected-areas Hizikia fusiforme
1 Abstract 20
2 Introduction 22
3 Materials and methods 24
3.1 Materials 24
3.2 Extraction of active fractions from CF 24
3.3 Chemical analysis 25
3.3.1 Chemical composition. 25
3.3.2 Analysis of the distribution of molecular weights 25
3.3.3 FTIR characterization 25
3.3.4 Nuclear Magnetic Resonance spectroscopy (NMR) analysis 25
3.3.5 LC-ESI-MS analysis 25
3.4 Screening anticancer effect of three locations Hijiki . 26
3.4.1 Cell culture and cell viability. 26
3.4.2 Apoptotic and necrotic body formation 26
3.4.3 Apoptosis analysis by flow cytometry . 27
3.4.4 Determination of mitochondrial membrane potential . 27
3.4.5 Measurement of cytochrome c release . 27
3.4.6 Western blot analysis. 27
3.5 Antioxidant effect of JHCF4 28
3.5.1 Radical scavenging assays by using electron spin resonance (ESR) spectrometer 28
3.5.2 Cell culture 28
3.5.3 Antioxidant activity against AAPH-induced cellular damage. 28
3.5.4 Nuclear staining with Hoechst 33342 28
3.5.5 Apoptosis analysis by flow cytometry . 29
3.5.6 Western blot analysis. 29
3.5.7 Analysis of oxidative stress in AAPH treated zebrafish in 72 h post-fertilization (hpf). 29
3.6 Hepato-effective effect of JHCF4 . 30
3.6.1 Cell culture 30
3.6.2 Protective effect of JHCF4 against ethanol-induced cellular damage . 30
3.6.3 Nuclear staining with Hoechst 33342 30
3.6.4 Apoptosis analysis by flow cytometry . 31
3.6.5 Western blot analysis. 31
3.6.6 Analysis of oxidative stress in ethanol treated zebrafish (72 hpf) . 31
3.6.7 Measurement of steatosis contents in ethanol treated zebrafish (128 hpf). 32
3.6.8 Measurement of malondialdehyde (MDA), and glutathione (GSH) contents in ethanol treated zebrafish in 128 hpf 32
3.7 Anti-inflammatory effect of HFA against Fine dust (FD) 33
3.7.1 Cell culture 33
3.7.2 Measurement of cell viability and ROS production 33
3.7.3 Nuclear staining with Hoechst 33342 33
3.7.4 Evaluation of inflammatory responses. 34
3.7.5 Evaluation of heavy metal content in FD-treated keratinocytes 34
3.7.6 Enzyme immunoassay analysis 35
3.7.7 Western blot analysis. 35
3.7.8 Analysis of inflammatory responses in FD treated zebrafish in 72 hpf. 35
3.8 Statistical analysis 36
4 Results and Discussion 36
4.1 Screening anticancer effects for three locations Hijiki 36
4.2 Antioxidant effects of JHCFuc . 56
4.3 Hepato-protective effect of JHCFuc against ethanol-induced damage. 65
4.4 Anti-inflammatory potential of HFA against FD-induced inflammatory responses 75
5 Conclusion 92
Part III. Anti-inflammatory potential of the fucoxanthin-rich fraction from Hizikia fusiforme against fine dust-induced inflammatory responses in vitro and in a zebrafish model
1 Abstract 94
2 Introduction 95
3 Materials and methods 96
3.1 Preparation of FxRF. 96
3.2 Chemical analysis of FxRF. 96
3.3 Cell culture 97
3.4 Nuclear staining and ROS measurement. 97
3.5 Evaluation of inflammatory responses 97
3.6 Western blot analysis . 98
3.7 Zebrafsh embryo experiments 99
3.8 Statistical analysis 99
4 Results and Discussion 100
4.1 HPLC and RRLC-MS analysis of FxRF . 100
4.2 Measurement of FxRF against FD-induced inflammatory responses in keratinocytes 103
4.3. Protective effect of FxRF against FD-induced apoptotic body formation in keratinocytes 103
4.4 Measurement of FxRF against FD-induced inflammatory responses in RAW 264. 7 macrophages 107
4.5 Inflammatory responses in RAW 264.7 macrophages induced with a culture medium collected from FD-induced FxRF treatment keratinocytes 110
4.6 The anti-inflammatory effects of FxRF on the FD-induced zebrafish embryo model 113
5 Conclusion 117
Part IV. Isolation of saringosterol acetate from Hizikia fusiforme and its bioactivities
1 Abstract 119
2 Introduction 120
3 Materials and Methods 121
3.1 Preparation and identification of saringosterol acetate (SA) 121
3.1.1 Preparation of hexane extracts from Hijiki. 121
3.1.2 Isolation and identification of SA 121
3.2 Anticancer effect of SA against on MCF-7 cancer cells 121
3.2.1 Cell culture 121
3.2.2 Measurement of cell viability 122
3.2.3 Apoptotic and necrotic body formation 122
3.2.4 Apoptosis analysis by flow cytometry . 122
3.2.5 Western blot analysis. 122
3.3 Anti-obesity effect of SA against adipogenesis in adipocytes 123
3.3.1 Cell culture and adipocyte differentiation 123
3.3.2 Measurement of lipid accumulation. 123
3.3.3 Measurement of triglyceride contents 123
3.3.4 Western blot analysis. 124
3.4 Statistical analysis 124
4 Results and Discussion 124
4.1 Isolation and identification SA from Hijiki. 124
4.2 Anticancer effect of SA against on MCF-7 cancer cells 126
4.2.1 Cytotoxicity of SA 126
4.2.2 Apoptosis morphology of SA on MCF-7 cancer cells 126
4.2.3 Apoptosis pathway regulated by SA 126
4.3 Anti-obesity effect of SA against adipogenesis in adipocytes 131
4.3.1 SA suppresses adipocyte differentiation 131
4.3.2 SA regulates adipogenesis-related factors in adipocytes. 131
4.3.3 SA regulates AMPK and ACC factors in adipocytes 131
5 Conclusion 136
REFERENCE. 137
ACKNOWLEDGEMENT. 157
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Yu Lin Dai. (2020). Characterization of bioactive components from arsenic-lower Jeju Hizikia fusiforme
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General Graduate School > Marine Life Sciences
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