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In vivo and in vitro Anti-inflammatory activities of sulfated polysaccharide, a fucoidan isolated from Sargassum horneri

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Abstract
Sargassum horneri China strain is an edible brown alga that grows in the subtidal zone as an annual species along the coasts of Korea, China and Japan. Recently, an extreme amount of S. horneri was moved into the coasts of Jeju, Korea from east coast of China and need to utilize this large biomass in order to protect environmental stability along the shores of Jeju Island. In addition, recent studies suggest that polysaccharide separated from S. horneri China strain has promising anti-inflammatory activities in vitro conditions. However, underlying mechanisms and or polysaccharide responsible for its anti-inflammatory activities not yet discovered. Therefore, the present study was designed to isolate the polysaccharide responsible for anti-inflammatory activities via enzyme assistance extraction from the brown seaweed S. horneri by using five food-grade enzymes (AMG, Celluclast, Viscozyme, Alcalase and Protamax) and FPLC system. Bioactivity-guided polysaccharide isolation lead to a separate fucose-rich sulfated polysaccharide (SH fucoidan) from cellular enzymatic extraction. The levels of nitric oxide (NO) and pro-inflammatory were measured by Gris sassy and enzyme-linked immunosorbent assay respectively. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) were measured using western blot in LPS-Stimulated RAW 264.7 cells. In addition image analysis software used to quantify cell death, NO and ROS production of LPS-exposed zebrafish embryo by using acridine orange, DFA-FM-DA and DCF-DA respectively.
SH fucoidan inhibited the NO production in LPS-stimulated RAW 264.7 cells with the IC50 value: 40.0 μg/m. in addition SH fucoidan down-regulated the LPS-stimulated iNOS and COX-2 protein levels as well as the production of inflammatory cytokine, tumor necrosis factor (TNF)-α in a dose-dependent manner. Furthermore, SH fucoidan inhibited the activation of NF-κB p50, p65 and the phosphorylation of MAPKs, including p38 and extracellular signal-regulated kinase (ERK), in LPS-stimulated RAW 264.7 cells. In addition SH fucoidan also down-regulate the LPS-induced heart beating rate, cell death, NO production and ROS production in the LPS-exposed zebrafish embryo. A Fourier transform infrared spectroscopy (FT-IR) analysis showed that the FT-IR spectrum of SH fucoidan is similar to that of commercial fucoidan. In Conclusion, our results showed that SH fucoidan which is likely a fucoidan has anti-inflammatory activities and is a potential candidate for the formulation of a functional food ingredient or/and drug to treat inflammatory diseases.
Author(s)
Kalu Kapuge Asanka Sanjeewa
Issued Date
2016
Awarded Date
2016. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007779
Alternative Author(s)
Sanjeewa, Kalu Kapuge Asanka
Department
대학원 해양생명과학과
Advisor
전유진
Table Of Contents
Acknowledgments i
Summary iv
List of figures vi
List of Tables ix
1. Background 1
2. Materials and Methods 7
2.1 Chemicals and reagents 7
2.2 Sample collection and preparation 7
2.3 Preparation of enzymatic extracts from seaweeds 8
2.4 Crude polysaccharide separation 10
2.6 Anion exchange chromatography 12
2.7 Analytical procedures 12
2.8 Quantification of neutral sugar composition on purified polysaccharides 13
2.9 Fourier Transform Infrared (FT-IR) Spectrophotometer analysis 13
2.10 In vitro experiments 14
2.11 In vivo Zebrafish experiments 18
2.12 Statistical analysis 21
3. Result and Discussion 22
3.1 Proximate chemical composition of S. horneri 22
3.2 Yield and general components of the S. horneri extracts. 24
3.3 In vitro cytotoxic effect and NO inhibitory properties of enzymatic digests separated from S. horneri on LPS-Stimulated RAW 264.7 cells 26
3.4 In vitro cytotoxic effect and NO inhibitory properties of crude polysaccharides separated from S. horneri on LPS-Stimulated RAW 264.7 cells 28
3. 5 Isolation and Characterization of anti-inflammatory polysaccharides from S. horneri 31
3.6 Effect of F1 and F2 on cell viability and NO production in LPS-stimulated RAW 2647 cells. 33
3.7 Effects of F2 on iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 cells. 35
3.8 Inhibitory effect of F2 on PGE2 secretion in LPS-stimulated RAW 264.7 cells 37
3.9 Effect of F2 on secretion of pro-inflammatory cytokines from LPS-stimulated RAW 264.7 cells.1 38
3.10 Effects of F2 on the NFB and MAPK signaling pathway in LPS-stimulated RAW 264.7 cells 40
3.11 In Vivo toxicity of LPS and F2 in zebrafish model 43
3.12 Effects of F2 on LPS-exposed heart-beating rate of zebrafish model 44
3.13 Protective effect of F2 against LPS-induced cell death in in vivo zebrafish model. 46
3.14 protective effect of F2 on LPS –induced ROS production in in vivo zebrafish model. 48
3.15 protective effect of F2 on LPS –induced NO production in in vivo zebrafish model. 50
3.16 FT-IR spectrum of F2 fraction and its monosaccharide composition 52
4. Conclusion 55
Reference 56
Degree
Master
Publisher
제주대학교 대학원
Citation
Kalu Kapuge Asanka Sanjeewa. (2016). In vivo and in vitro Anti-inflammatory activities of sulfated polysaccharide, a fucoidan isolated from Sargassum horneri
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General Graduate School > Marine Life Sciences
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