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Search of new bioactive components and exploitation of their health promoting potentials as functional ingredients from brown seaweed Ecklonia cava and edible sea cucumber Holothuria edulis

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Abstract
Functional foods, nutraceuticals, and dietary supplements are important for health promotion and disease risk reduction. Although a myriad of bioactive components are known to render the expected beneficial effects, the mechanisms involved are varied and may work individually or collectively in providing the effects. Hence, over the years, the biological activities of natural products could have gained a considerable research interest and studies about the extraction and isolation of active components from natural resources have attracted special attention in last recent years.
It is well known that marine organisms are not only very important resources as food, feed, and energy rich source, but they are also rich sources of structurally novel and biologically active metabolites with valuable industrial potentials. Thus, efforts at discovery of novel active components from marine bioresources over the past years have yielded a considerable amount of new active metabolites which may not available in terrestrial environment. Therefore, marine derived active components, whose immense biochemically diversity looks like to become a rich source of novel chemical entities for the use as functional ingredients in many industrial applications such as functional foods, pharmaceuticals and nutraceuticals. This report deals with the antioxidant, anti-inflammatory and anticancer potentials of biologically active components from the brown seaweed Ecklonia cave and edible sea cucumber Holothuria edulis.
The protective effect of bioactive components recovered from brown seaweed E. cava processing by-product on H2O2-mediated DNA damage in Vero cells was evaluated. E. cava processing by-product was fermented by edible yeast Candida utilis and its antioxidant activities were evaluated via radical scavenging using electron spin resonance
(ESR) spectrometer. Effective fermentation duration was discovered as 24 h prior to being extracted with 80% EtOH. Major bioactive components in the extract were phlorotannins including triphlorethol-A, eckol, dieckol and eckstolonol. The phlorotannin rich fermented E. cava processing by-product extract (FEPBE) strongly enhanced the cell viability against H2O2-induced oxidative damage in Vero cells and exhibited good protective properties against H2O2-induced cell apoptosis as demonstrated by nuclear staining with Hoechst 33342 and flow cytometric analysis.
The anti-inflammatory potential of the FEPBE was evaluated in vitro. The bioactive components recovered from E. cava processing by-product attenuate production of nitric oxide (NO) and prostaglandin-E2 (PGE2) by suppressing inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) protein expressions in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The release of pro-inflammatory cytokines, interleukin-1β (IL-1β) and interleukin-6 (IL-6) was also significantly suppressed. Hence, it could be confirmed that the bioactive components responsible for the antioxidant and anti-inflammatory activity are still remaining in the E. cava processing by-product and can be recovered as a value-added bio-mass fraction after fermentation with C. utilis.
In vitro anticancer and anti-inflammatory potentials of edible sea cucumber H. edulis were evaluated. The anticancer effect of aqueous fraction of edible sea cucumber H. edulis (ESC-AQ) on human leukemia HL-60 cells was assessed. ESC-AQ induced apoptosis in HL-60 cells as evidenced by the formation of apoptotic bodies and the accumulation of DNA in the sub-G1 phase of the cell cycle. The induced apoptosis was accompanied by down regulation of Bcl-xL, up regulation of Bax and activation of caspase-3. In this study, EtOAc solvent fraction of edible sea cucumber H. edulis (ESC-EA) modulated LPS-
induced inflammatory responses in murine macrophages. ESC-EA significantly suppressed the NO production and PGE2 production by suppressing iNOS and COX-2 protein extrusions in LPS-induced RAW 264.7 macrophages. In addition, ESC-EA suppressed the release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6. According to the results, it could be suggested that ESC-AQ and ESC-EA could be incorporated in food formulations as functional ingredients. Besides, they might be further developed as potential therapeutic agents in the particular applications.
Author(s)
자나카
Issued Date
2012
Awarded Date
2012. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000006007
Alternative Author(s)
W.A.J.P. WIJESINGHE
Affiliation
제주대학교
Department
대학원 해양생물공학과
Advisor
전유진
Table Of Contents
SUMMARY VI
LIST OF FIGURES IX
LIST OF TABLES XV
INTRODUCTION 1
PART І - Value-added fermentation of Ecklonia cava processing by-product and its antioxidant effect
ABSTRACT 13
1. INTRODUCTION 14
2. MATERIALS AND METHODS 20
2.1. Materials and reagents 20
2.2. Fermentation of E. cava processing by-product 20
2.3. Preparation of sample extract from fermented E. cava processing by-product...22
2.4. Determination of total phenolic content 22
2.5. DPPH radical scavenging assay 22
2.6. Hydroxyl radical scavenging assay 25
2.7. Alkyl radical scavenging assay 25
2.8. Determination of triphlorethol-A, eckol, dieckol and eckstolonol contents 25
2.9. Cell culture 26
2.10. Determination of intracellular ROS generation (DCHF-DA assay) 26
2.11. Determination of cell viability (MTT assay) 27
2.12. Nuclear staining with Hoechst 33342 28
2.13. Flow cytometry analysis 28
2.14. Statistical analysis 29
3. RESULTS 30
3.1. Effect of fermentation on extraction yield and TPC 30
3.2. Effect of fermentation on radical scavenging activities 30
3.3. Effect of fermentation on the phlorotannin content 31
3.4. Effect of FEPBE on intracellular ROS scavenging activity 32
3.5. Protective effect of FEPBE on H2O2-induced cell damage 32
3.6. Protective effect of FEPBE on H2O2-induced apoptosis in Vero cells 32
3.7. Effect of FEPBE on cell cycle pattern 33
4. DISCUSSION 43
5. CONCLUSION 46
PART ІІ - Anti-inflammatory activity of bioactive components recovered from Ecklonia cava processing by-product in lipopolysaccharide-stimulated RAW 264.7 macrophages
ABSTRACT 48
1. INTRODUCTION 49
2. MATERIALS AND METHODS 52
2.1. Materials and reagents 52
2.2. Fermentation of E. cava processing by-product and preparation of FEPBE 52
2.3. Cell culture 54
2.4. Determination of NO production 54
2.5. Lactate dehydrogenase (LDH) cytotoxicity assay 54
2.6. Determination of prostaglandin E2 (PGE2) production 55
2.7. Western blot analysis 55
2.8. Determination of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) production 56
2.9. Statistical analysis 56
3. RESULTS 57
3.1. Effect of FEPBE on LPS-induced NO production and LDH release in RAW 264.7 cells 57
3.2. Effect of FEPBE on LPS-induced PGE2 production 57
3.3. Effect of FEPBE on LPS-induced iNOS and COX-2 protein expression 57
3.4. Effect of FEPBE on LPS-induced pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) production 58
4. DISCUSSION 65
5. CONCLUSION 69
PART ІІІ – Investigation of the potential bioactive components from edible sea cucumber Holothuria edulis that mediate apoptosis of human HL-60 leukemia cells and suppress inflammatory responses in lipopolysaccharide-stimulated RAW 264.7 macrophages
ABSTRACT 71
1. INTRODUCTION 72
2. MATERIALS AND METHODS 75
2.1. Materials and reagents 75
2.2. Preparation of the sea cucumber extract and fractionation 75
2.3. Determination of approximate chemical composition 75
2.4. Determination of total phenolic content 76
2.5. Cell culture 76
2.6. Cell growth inhibitory assay 77
2.7. Nuclear staining with Hoechst 33342 77
2.8. Cell cycle analysis 78
2.9. Western blot analysis (anticancer) 78
2.10. Determination of NO production 79
2.11. Lactate dehydrogenase (LDH) cytotoxicity assay 79
2.12. Determination of prostaglandin E2 (PGE2) production 80
2.13. Western blot analysis (anti-inflammation) 80
2.14. Determination of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) production 81
2.15. Statistical analysis 81
3. RESULTS 82
3.1. Approximate composition and total phenolic content 82
3.2. Inhibitory effect of ESC-AQ on the growth of HL-60 cells 82
3.3. Induction effect of ESC-AQ on apoptotic bodies formation in HL-60 cells 82
3.4. Effect of ESC-AQ on sub-G1 DNA contents in cell cycle analysis 83
3.5. Effect of ESC-AQ on apoptosis-related protein expressions in HL-60 cells 83
3.6. Effect of ESC-EA on LPS-induced NO production and LDH release in murine macrophages 84
3.7. Effect of ESC-EA on LPS-induced PGE2 production 84
3.8. Effect of ESC-EA on LPS-induced iNOS and COX-2 protein expression 85
3.9. Effect of ESC-EA on LPS-induced pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) production 85
4. DISCUSSION 97
5. CONCLUSION 102
REFERENCES 103
ACKNOWLEDGEMENT 120
Degree
Doctor
Publisher
제주대학교 대학원
Citation
자나카. (2012). Search of new bioactive components and exploitation of their health promoting potentials as functional ingredients from brown seaweed Ecklonia cava and edible sea cucumber Holothuria edulis
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General Graduate School > Marine Life Sciences
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