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Bioactive natural products from marine algae and soft corals

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Abstract
Natural products drug discovery from marine sources has received a remarkable attention in recent years given their potential biological activities. Second to none, natural products from marine origin exert a fascinating functionality owing to their wide range of structural diversity. Based on the literature, marine organisms including sponges, soft corals, cyanobacteria, microalgae, macroalgae, bryozoans, cnidaria, platyhelminthes, hoplonomertea, polychaetes, and hemichordate have extensively been studied for identifying numerous bioactive natural products those which are the secondary metabolites of these organisms. Some extensive reviews discuss the significance of marine natural products as bioactive agents [1, 2]. However, given the vast biological diversity of marine organisms widespread throughout the globe in different environmental habitats facing different physical and chemical environmental conditions, marine natural products remain under-explored. With recent developments which enable the exploration of marine habitats, the world has gone one foot forward in exploring fascinating marine organisms. Current series of studies merely represent a hay in a haystack contributing to the vast knowledge base of natural product drug discovery. The source of these studies were seaweeds harvested from the coast of Sri Lanka and soft corals harvested from ocean bordering Jeju island of the Republic of Korea. Following a general methodology of natural product drug discovery, extraction methods were designed to obtain targetted compound categories. Regarding seaweeds, the studies were focused upon the purification of bioactive polysaccharides and polyphenolic compounds. In the case of soft corals, the objectives were set to purify bioactive terpenoids and their derivatives. All selected fractions and purified compounds indicated potential functionality towards tested bioactivities unraveling their potential to be used in manufacturing a wide variety of consumer products. The studies incorporate the use of different chromatographic purification techniques, spectroscopic techniques, computational methods of molecular geometry optimization, spectroscopic analysis, and molecular docking and a variety of biological and chemical assays based on colorimetry, spectroscopy, western blot analysis and in vivo zebrafish model. The content of the thesis is divided into four parts to provide the reader a clear understanding of the workflow of current studies, which spread their branches to a variety of different areas. The first part of this thesis is devoted for the extraction, screening of bioactivities and purification of natural products from Sri Lankan marine algae. This section provides a general introduction to the importance of exploring marine natural products of seaweed featuring green, brown, red and microalgae and their significance in facilitating physiological wellbeing of man. In the first study crude polysaccharides were obtained from eleven different seaweed species by hot water extraction and ethanol precipitation. Among the investigated algae, the brown algae Chnoospora minima were found to be having better alkyl and DPPH radical scavenging activities and better intracellular ROS scavenging potential against both H_(2)O_(2) and AAPH induced ROS levels in "Chang" cells. FTIR analysis and monosaccharide composition of these crude polysaccharides were used to confirm its structural characteristics. During the interpretation of FTIR, a novel approach that combines the use of computational quantum chemical calculations in combination with predesignated structural information was followed. The calculations were done using density functional theory (DFT) calculations at RB3LYP/6-31G(d,p) level. Finally, the major constituent of the crude polysaccharides of Chnoospora minima was identified as fucoidan. In the next study, the green extraction approach that combines the use of enzymes to obtain a better yield of bioactive constituents were utilized in obtaining the water-based algae extracts. Ten different commercial food grade enzymes consisting of 5 carbohydrases (Viscozyme, Celluclast, AMG, Termamyl, and Ultraflo) and five proteases (Protamex, Kojizyme, Flavozyme, Alcalase, and Neutrase) were utilized in obtaining the extracts provided their optimum extraction conditions. Celluclast extracts gave the highest yield for all the algae samples with higher amounts of polyphenolic contents. The antioxidant and anti-inflammatory properties of the Celluclast extracts of Sargassum polycystum and Chnoospora minima were identified superior compared to other extracts using both in-vivo and in-vitro analysis. As for the third study, the olysaccharides rich in fucoidans were precipitated out from the Celluclast extract of C. minima using ethanol and further purification was done via a DEAE-Sepharose fast flow column. Four fractions were collected from the column purification. One of the purified fraction demonstrated excellent anti-inflammatory effects compared to other three fractions in-vivo and in-vitro. The selected fraction was found to be a fucoidan with a higher fucose content and degree of sulfation via FTIR characterization and monosaccharide analysis. The results further demonstrated the variation of anti-inflammatory properties with the degree of sulfation of the polysaccharide backbone. In the next study, we optimized a method to obtain fucoidan-rich polysaccharides from S. polycystum and C. minima following depigmentation, phenol polymerization, enzyme-assisted extraction, removal of alginate impurities by CaCI_(2) addition and ethanol precipitation. The obtained polysaccharides demonstrated the characteristics of fucoidan evidenced via both FTIR analysis and monosaccharide composition analysis. Further, they demonstrated superior antioxidant properties for DPPH and Alkyl radical scavenging activities. Also, the fucoidan-rich polysaccharides demonstrated desirable bioactive properties such as anti-inflammatory, tyrosinase inhibition, skin whitening, elastase and collagenase inhibition abilities making them much desirable natural ingredients to be used in the formulation of cosmeceutical products. Apart from studying about the fascinating bioactive polysaccharides of algae, 70% ethanol extracts of some selected algae were evaluated for their bioactive properties. The algae (Ahnfeltiopsis pygmaea, Gracilaria corticata var. ramalinoides, Chnoospora minima, and Caulerpa racemosa) extracts obtained in such manner was found to composed of a higher level of polyphenolic content when compared to their water based extracts described previously. Among the extracts, C. minima, and C. racemose demonstrated superior antioxidant and antiinflammatory effects. Also, the C. minima indicated potential anti-cancer activities against HL-60 cell line. The crude of selected C. minima and C. racemose extracts were dissolved in water and fractionated between inert organic solvents in order of increasing their polarity following hexane, Chloroform, and ethyl acetate. The ethyl acetate fraction of C. minima and C. racemose demonstrated strong antioxidant and anti-inflammatory properties whereas the hexane fraction indicated anti-inflammatory activity. Chloroform fractions of C. racemose indicated potential anti-cancer properties against HL-60 (human leukemia) and MCF-7 (human breast cancer) cells. Further purification of the hexane fraction guided by bioassays (anti-inflammatory activity) resulted in the isolation of Squalene, which was confirmed based on its molecular weight, and NMR data. The purified compound indicated a desirable anti-inflammatory effect on LPS-induced RAW 264.7 murine macrophages by the reduction in NO production and down-regulation of pro-inflammatory cytokines and inflammatory regulators. Further, the antiinflammatory implications of the isolated compound were confirmed in the LPS-induced in vivo zebrafish embryo model. Investigation of soft corals harvested from Jeju describes the next part of the study. Soft corals are species belongs to the order of Alcyonacea, which defines corals that do not produce a calcium carbonate skeleton. They primary inhabit nutrition rich tropical or subtropical waters favoring habitats with less light and warm seawaters. Based on the estimates from 2010 to 2011, 22% of the total new marine natural products have been discovered from soft corals [3]. Natural products from soft corals are considered as a primary source of a new array of therapeutics. Surprisingly, most of the natural products identified from soft corals have reported demonstrating a spectrum of biological activities including antitumor, antiviral, antifouling and anti-inflammatory effects [4]. Given their evolutionary perspectives, these compounds many of which are toxic to other organisms assist the soft corals in protecting themselves from predators [5]. The soft corals belong to the genus Dendronephthya that was central to current studies encompasses nearly 248 species and are highly prolific and widely distributed throughout the Indian Ocean, the Red Sea, Pacific Ocean and Southeast Asia [6]. Especially the sterols isolated from soft corals have shown promising bio functionalities. A review about marine sterols published in 1993 report the discoveries been done on more than 200 different types of mono hydroxy sterols from marine organisms [7]. Discovery of these various metabolites will shape the future of natural product drug development with a broad perspective of counteracting a broad range of disease conditions. Ten different soft coral species including Dendronephthya gigantea, Dendronephthya spinulosa, Dendronephthya puetteri, Dendronephthya castanea, Dendronephthya aurea, Dendronephthya suensoni, Scleronephthya gracillimum, Chromonephthea hirotai and two unknown Dendronephthya species designated Dendronephthya sp1 and Dendronephthya sp2 were extracted using 70% ethanol for the initial evaluation of its bioactive properties. The 70% ethanol extracts of Dendronephthya gigantea, Dendronephthya aurea, Dendronephthya puetteri, Chromonephthea hirotai, and Scleronephthya gracillimum indicated promising antiinflammatory effects against LPS-induced NO production, down-regulation of proinflammatory cytokines and inflammatory mediators. Among the screened soft corals Dendronephthya gigantea was selected for further studies to purify bioactive principals responsible for the observed anti-inflammatory and anti-cancer effects. The solvent/solvent fractionation of the 70% ethanol extract obtained by 6.00 Kg of the Dendronephthya gigantea dry powder indicated the accumulation of the anti-inflammatory and anti-cancer effects in the hexane fraction. Following the bioassays, the hexane fraction was further purified by two consecutive silica open columns to obtain a subfraction with highest potential bioactive properties. The selected fraction indicated a remarkable anti-inflammatory activity towards LPS-induced RAW 264.7 macrophages evidenced via the reduced NO production, downregulation of pro-inflammatory cytokines and inflammatory mediators. Effects were also displayed in LPS-induced in vivo zebrafish model. The active principals in the purified fraction were identified as a mixture of eight 3β-hydroxy-Δ5-steroidal congeners via GC-MS/MS analysis. Following the impressive biological functionality of the purified fraction, attempts were taken to isolate the individual compounds in the sterol mixture. The separation of sterols is a tricky task that needs both accuracy and precision. For the further separation, a long silica column was used with gradually increasing amounts of ethyl acetate in hexane mixture. Column eluates were collected into 130 test tubes and grouped into five fractions. Based on its TLC analysis patterns, the selected test tubes were mixed with each other and further developed on a preparative TLC plate. The separated bands were collected from the TLC plates and analyzed via GC-MS/MS and NMR. The isolated sterols are remaining to be screened for some possible bioactivities including anti-inflammatory, anti-cancer, and anti-diabetic. Hence, the progressions in marine bioresource technology would provide a new scope and a platform for the developments in natural product chemistry, functional food, cosmeceutical, and pharmaceutical industry.
Author(s)
페르난도 일레쿠티게 프리안 샤누라
Issued Date
2017
Awarded Date
2017. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000008120
Alternative Author(s)
Fernando, I. P. Shanura
Affiliation
제주대학교 일반대학원
Department
대학원 해양생명과학과
Advisor
전유진
Table Of Contents
SUMMARY xi
LIST OF FIGURES xvii
LIST OF TABLES xxix
PART I; NATURAL PRODUCTS FROM SRI LANKAN MARINE ALGAE 2
SECTION 1; HOT WATER EXTRACTION OF BIOACTIVE POLYSACCHARIDES SCREENING OF BIOACTIVITIES AND CHARACTERISATION 2
ABSTRACT 2
1. INTRODUCTION . 3
1.1. A historical overview 3
1.2. Natural products of marine algae 4
1.2.1. Polyphenolic compounds of marine algae 5
1.2.2. Bioactive polysaccharides of marine algae 7
1.3. Sri Lankan marine algae 10
1.4. Commercial and industrial applications of marine algae 12
2. MATERIALS AND METHODS . 12
2.1. Collection of algae samples. 13
2.2. Extraction of hot water soluble crude polysaccharides . 17
2.3. Evaluation of chemical composition . 18
2.3.1. Analysis of polysaccharide content using phenol-sulfuric acid colorimetric assay 18
2.3.2. Analysis of polyphenol content by Folin-Ciocalteu assay. 18
2.4. Analysis of radical scavenging antioxidant activity of samples by electron spin resonance (ESR) spectroscopy. 19
2.5. Functional group analysis of crude polysaccharides using FTIR spectroscopy. . 19
2.5.1. Interpretation of FTIR spectra using computational calculations of constructed disaccharide models 20
2.6. Mainte nance of cell lines. 20
2.6.1. Evaluation of intracellular ROS scavenging effects . 21
2.7. Statistical analysis . 21
3. RESULTS AND DISCUSSION 21
3.1. Proximate composition of algae material 21
3.2. Yields of polysaccharide precipitates obtained by adding ethanol . 24
3.3. Radical scavenging activities of the algae crude polysaccharides from hot water extracts of algae. 26
3.4. Sample toxicity of crude polysaccharides upon normal cells . 28
3.5. Intracellular ROS scavenging activity and cytoprotective effects 28
3.6. Structural characterization of crude polysaccharides 31
3.6.1. FTIR analysis via computational calculations and predefined peak characteristics 31
3.6.2. Analysis of the monosaccharide composition 38
4. Conclusions 40
SECTION 2; ENZYME-ASSISTED EXTRACTION, SCREENING OF BIOACTIVITIES, PURIFICATION AND CHARACTERISATION OF BIOACTIVE POLYSACCHARIDES. 41
ABSTRACT . 41
1. INTRODUCTION . 43
1.1. Enzyme-assistant extraction 43
1.2. Antioxidants from marine algae 44
1.3. Anti-inflammatory agents from marine algae . 44
1.4. Fucoidans; a sulfated polysaccharide from brown algae possessing desirable biofunctional properties 45
2. MATERIALS AND METHODS . 46
2.1. Analysis of mineral constituents by inductively coupled plasma optical emission spectrometry (ICP-OES). . 46
2.2. Enzyme-assisted extraction (EAE) of marine algae 46
2.3. Precipitation of polysaccharides of selected C. minima and S. polycystum extracts obtained via EAE of depigmented raw material. . 49
2.4. Separation of polysaccharides via anion-exchange chromatography . 50
2.5. Characterization of the polysaccharides by FTIR and monosaccharide analysis . 50
2.6. Analysis of antioxidant activities of the samples using colorimetric methods . 50
2.7. Maintenance of cell lines. 51
2.7.1. Evaluation of anti-inflammatory activity in LPS-stimulated RAW 264. 7 macrophages. 51
2.7.2. Western blot analysis of the expression levels of inflammatory mediators. 52
2.7.3. Evaluation of the expression levels of PGE2 and pro-inflammatory cytokines . 52
2.8. Maintenance of zebrafish and obtaining embryos. 53
2.8.1. In vivo evaluation of the anti-inflammatory activity of algae polysaccharides by zebrafish embryos. 55
2.8.2. Statistical analysis . 55
3. RESULTS AND DISCUSSION 56
3.1. Mineral content of algae material 56
3.2. Extraction yields algae extracts obtained via enzyme-assisted extraction. . 58
3.3. Antioxidant activities of the algae crude polysaccharides from enzyme assisted extracts of algae. 60
3.4. Sample toxicity of enzyme-assisted extracts of algae upon normal cells . 64
3.5. Anti-inflammatory effects of the algal extracts obtained by enzyme assisted extraction 64
3.6. Effect of Celluclast extract of C. minima upon LPS-induced iNOS, COX-2, PGE2 and TNF-α protein expression in LPS-induced RAW cells 67
3.7. Protective effect of Celluclast extract of S. polycystum against H2O2-induced oxidative stress and cell death in zebrafish 69
3.8. Anti-inflammatory activity of Celluclast extract of C. minima against LPS-induced NO production, oxidative stress and cell death in zebrafish. 71
3.9. Yield and chemical composition of polysaccharides obtained from selected S. polycystum (SPP) and C. minima (CMP) samples. 73
3.10. Anion exchange chromatography separation of CMP gave 4 different fractions . 75
3.11. FTIR characterization of the polysaccharides indicated the presence of fucoidan in subfractions 77
3.12. NMR analysis of F4 indicated the structural characteristics of a fucoidan 79
3.13. Monosaccharide content analysis indicated the abundance of fucose in F4 . 81
3.13. The fraction F4, efficiently inhibited the LPS induced NO production in RAW 264. 7 macrophages. 84
3.14. The fraction F4 could downregulate the expression of iNOS, COX-2, PGE2 and proinflammatory cytokines in RAW 264.7 macrophages. 86
3.15. In vivo anti-inflammatory effects of F4 indicated the ability to inhibit NO production, ROS production, and cell death in LPS stimulated zebrafish embryos. 88
4. CONCLUSIONS 90
SECTION 3; SCREENING BIOACTIVITIES OF 70% ETHANOL EXTRACTS OF SRILANKAN ALGAE AND PURIFICATION OF BIOACTIVE PRINCIPALS 92
ABSTRACT . 92
1. INTRODUCTION . 93
1.1. Bioactive phenolic metabolites of marine algae . 93
1.2. Bioactive terpenoid derivatives . 95
1.3. Bioactive polyunsaturated fatty acids . 95
2. MATERIALS AND METHODS . 96
2.1. Extraction of algae using 70% EtOH 96
2.2. Solvent/solvent fractionation and purification of the selected 70% ethanol extracts of the algae . 96
2.3. HPLC and GC-MS/MS analysis . 97
2.4. Silica open column chromatographic purification 97
2.5. NMR analysis 98
2.6. Evaluation of anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophages 98
2.7. Evaluation of antiproliferative effects of algae material against carcinoma cells 98
2.8. Observation of apoptotic/necrotic body formation. 99
2.9. Flow cytometric analysis of the cell cycle 99
2.10. Evaluation of the expression levels of proteins by western blot analysis. 100
2.11. In vivo evaluation of the NO, ROS levels and cell death in zebrafish embryo model 101
2.12. Statistical analysis . 101
3. RESULTS AND DISCUSSION 102
3.1. Extraction yields of 70% ethanol extracts and their proximate chemical composition 102
3.2. Radical scavenging activities of the algae 70% ethanol extracts. . 104
3.3. Anti-inflammatory activity and sample toxicity of extracts obtained by 70% ethanol 106
3.4. Anti-Cancer Effects . 108
3.5. Yields of solvent fractions of the selected algae extracts and their chemical composition 110
3.6. Free radical scavenging activity of the solvent fractions of selected algae 112
3.7. Cytotoxicity of the obtained solvent fractions 112
3.8. Anti-inflammatory activity of the solvent fractions against LPS-induced NO production and protective effects against LPS-induced cytotoxicity. 115
3.9. Anti-cancer activity of the solvent fractions . 117
3.10. Identification and purification of bioactive constituents . 119
3.11. Further purification of CRH using silica open column chromatography and preparative thin-layer-chromatography. 123
3.12. Structural characterization of the isolated compounds by GC-MS/MS and NMR analysis . 125
3.13. Squalene isolated from C. racemose could inhibit the inflammatory responses in RAW 264.7 murine macrophages via mediating protein expression. 130
4. CONCLUSIONS 132
PART II; NATURAL PRODUCTS FROM JEJU SOFT CORALS 134
SECTION 4; EXTRACTION, SCREENING AND IDENTIFICATION OF ANTIINFLAMMATORY ACTIVE PRINCIPALS. 135
ABSTRACT . 135
1. INTRODUCTION . 136
2. MATERIALS AND METHODS . 137
2.1. Sample collection, identification, and extraction 137
2.2. Solvent/solvent fractionation and further purification 138
2.3. GC-MS/MS analysis . 139
2.4. Analysis of the Proximate composition 139
2.5. Cell culture 139
2.6. Western blot analysis 140
2.7. Evaluation of PGE2 and pro-inflammatory cytokine production 140
2.8. Statistical analysis . 141
3. RESULTS AND DISCUSSION 142
3.1. Proximate chemical composition of the specimens 142
3.2. Mineral composition indicated high levels of Ca2+ . 142
3.3. Extraction yields and chemical composition of the soft coral ethanolic extracts . 145
3.4. Anti-inflammatory activity of soft coral ethanol extracts . 145
3.5. Mediation of anti-inflammatory activity in LPS-stimulated RAW macrophages 148
3.6. Large-scale extraction, solvent/solvent fractionation, and chromatographic purification processes. 150
3.7. Characterization of chemical constituents in DGEH21' . 154
3.8. Anti-inflammatory potential against NO production in LPS stimulated RAW 264. 7 macrophages. 158
3.9. Regulation of inflammatory mediators . 160
3.10. In vivo anti-inflammatory effects of DGEH21' as a measure of inhibiting NO production, ROS production, and cell death in LPS stimulated zebrafish embryos 162
4. CONCLUSIONS 164
SECTION 5; SCREENING OF ANTI-CANCER ACTIVITIES, PURIFICATION AND ISOLATION OF BIOACTIVE STEROLS WITH ANTI-CANCER PROPERTIES FROM THE SOFT CORAL Dendronephthya gigantea. 165
ABSTRACT . 165
1. INTRODUCTION . 166
2. MATERIALS AND METHODS . 167
2.1. Sample collection and extraction 168
2.2. Fractionation and further purification . 168
2.3. GC-MS/MS analysis . 169
2.4. Cell culture 169
2.5 Apoptotic and necrotic body formation . 169
2.6. Cell cycle analysis . 170
2.7. Western blot analysis 170
2.8. NMR analysis 171
2.9. Statistical analysis . 171
3. RESULTS AND DISCUSSION 172
3.1. Cytotoxicity of the soft coral extracts . 172
3.2. Anti-proliferative Effects of the soft coral ethanol extracts on HL-60 cells . 174
3.3. Evaluation of nuclear morphology 174
3.4. Anti-cancer activity of the solvent extracts of selected soft corals on HL-60 and MCF- 7 cells. 177
3.5. Cytotoxicity and anti-cancer activity of DGEHF2 column fractions on Vero, MCF-7, and HL-60 cell lines. 179
3.6. Chemical composition of the eluates from the second open column 181
3.7. Nuclear morphology of cells indicating apoptotic body formation induced by DGEHF21' in MCF-7 and HL-60 cell lines. . 183
3.8. Flow-cytometric analysis of the proportion of apoptotic cells in Sub-G1 186
3.9. DGEHF21' could regulate the expression of apoptosis-related proteins. . 188
3.10. Further purification and isolation of bioactive constituents from DGEHF21' . 191
3.11. Characterization of purified and isolated compounds . 193
3.12. Evaluating anti-cancer activities of purified fractions by PTLC separation . 195
3.13. Evaluating Nuclear morphology of HL-60 and MCF-7 cells for the determination of apoptotic body formation. 197
3.14. Flow-cytometric analysis of the proportion of apoptotic cells in Sub-G1 199
4. CONCLUSIONS 201
ACKNOWLEDGEMENTS . 203
REFERENCES 205
Degree
Doctor
Publisher
제주대학교 일반대학원
Citation
페르난도 일레쿠티게 프리안 샤누라. (2017). Bioactive natural products from marine algae and soft corals
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