Biological Activities of Tropical Green Algae from Australia

A thesis in fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY By

NA WANG School of Chemical Engineering Faculty of Engineering April 2016

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Wang

First name: Na Other name/s:

Abbreviation for degree as given in the University calendar:

School: Chemical Engineering Faculty: Engineering

Title: Biological Activities of Tropical Green Algae from Australia

Abstract

Macroalgae are rich in bioactive components such as carotenoids, phenolic compounds and proteins/peptides, which may play a significant role in the prevention of diseases like cancer, obesity and diabetes. The aim of this thesis was to examine the in vitro biological activities of phenolic compounds, carotenoids and protein hydrolysates from three edible green macroalgae (Ulva ohnoi, Derbesia tenuissima and Oedogonium intermedium) cultured in tropical Australia. The phenolic components were extracted with 60% aqueous ethanol and their antioxidant activities were determined by four different assays (ABTS, DPPH, FRAP and ORAC). The extracts exhibited moderate levels of antioxidant activities. However, analysis of the extracts by HPLC-PDA, GC-MS, LC-MS and 1H NMR failed to detect any phenolic components, while a number of free amino acids, fatty acids and sugars were found, which were likely responsible for the measured antioxidant activities. Carotenoids were extracted from the algae by dichloromethane, and the extracts exhibited significant antioxidant activities, as well as potent inhibitory effects against several metabolically important enzymes including α-amylase, α-glucosidase, pancreatic lipase and hyaluronidase. However, the carotenoid extracts were poor inhibitors of angiotensin-converting enzyme (ACE). The extracts were analysed by LC-MS, which resulted in the identification of nine major carotenoids in the algae: siphonaxanthin, neoxanthin, 9’-cis-neoxanthin, loroxanthin, violaxanthin, lutein, siphonein, α-carotene and β-carotene. Proteins were extracted from the algae by alkaline solution. The extracted proteins were subjected to in vitro simulated human digestion and the resultant hydrolysates were fractionated by ultrafiltration. The hydrolysates showed markedly increased antioxidant activities and inhibition effects against α-amylase, α-glucosidase and ACE over the undigested proteins. Most of the peptides in the hydrolysates were extensively hydrolysed with MW less than 3 kDa. LC-MS/MS analysis identified a large number of peptides in the MW <3 kDa fraction of the hydrolysates and most of them contained peptides with known antioxidant, antidiabetic or antihypertensive activities as reported in the BIOPEP database. Overall, this thesis demonstrated that consumption of the algae could confer significant health benefits and the algae could be developed into bioactive ingredients with potential applications in functional food, nutraceutical and pharmaceutical products.

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ACKNOWLEDGEMENT

Firstly, I would like to express my appreciation and gratitude to my supervisor Associate Professor Jian Zhao. You have been a tremendous mentor and have given me this wonderful opportunity to conduct postgraduate research study in the School of Chemical Engineering, UNSW. I also would like to thank you for encouraging my research with your innovative research direction, valuable advice, and enthusiastic supervision and for allowing me to grow as a research scientist. Secondly, I would like to thank my co-supervisor Dr. Nicholas Paul for his valuable advice, knowledge of algae and providing algae samples throughout my study.

Sincere thanks to the following for all their support in analytical instruments: Professor Tatsuya Sugawara and Mr Yuki Manabe from Marine Bioproducts Technology, Kyoto University, Dr. Martin Bucknall and Ms Sydney Liu Lau from Bioanalytical Mass Spectrometry Facility, UNSW, Dr. James Hook and Dr. Donald Thomas from Nuclear Magnetic Resonance Facility, UNSW.

I wish to express my profound appreciation to Mr. Camillo Taraborrelli, for his support in providing all laboratory equipment, and Dr. Robert Chan for technical assistance.

My sincere gratitude goes to my family for their ongoing encouragement and beliefs in me. Financial support from my parents thought my PhD study. Special thanks to Wuxuan Liu and Wenda Wang for their endless support. Also, I would like to thank Kitty Tang for all her help throughout my PhD.

Many thanks to all my friends-for brightening up my PhD life! Without you, my PhD life would not have been complete.

Table of Contents

LIST OF TABLES ...... VI

LIST OF FIGURES ...... VIII

ABBREVIATIONS ...... X

ABSTRACT ...... XII

CHAPTER 1 INTRODUCTION ...... 1

CHAPTER 2 LITERATURE REVIEW ...... 7

2.1. Algae...... 7 2.1.1. Classification of macroalgae ...... 8 2.1.2. Distribution ...... 9

2.2. Chemical composition of macroalgae ...... 10

2.3. Current and potential utilization of macroalgae ...... 12 2.3.1. Seaweed as food and food ingredients ...... 14 2.3.2. Seaweed in agriculture ...... 17 2.3.3. Seaweed in environmental management ...... 18 2.3.3.1. Wastewater treatment ...... 18 2.3.3.2. Energy and bio-fuels ...... 18 2.3.4. Seaweed as bioactive and functional products ...... 19

2.4. Major bioactive components in algae and their health-related biological activities ...... 19 2.4.1. Phytochemicals in macroalgae ...... 20 2.4.2. Carotenoids ...... 21 2.4.2.1. Major algal carotenoids and their biological function activities ...... 23 2.4.2.2. Fucoxanthin ...... 23 2.4.2.3. Siphonaxanthin ...... 24 2.4.2.4. Astaxanthin ...... 24 2.4.2.5. Other carotenoids in algae ...... 25 2.4.3. Phenolic compounds ...... 26 2.4.4. Protein, peptides and amino acid ...... 30 2.4.4.1. Bioactive peptides...... 34 2.4.4.2. Bioactivities of protein hydrolysates and peptides from macroalgae ...... 35 2.4.5. Polysaccharides ...... 40 2.4.6. Fatty acid/Lipid ...... 42 2.4.7. Minerals ...... 43 2.4.8. Vitamins ...... 44

2.5. Algal species investigated in this study...... 45 I

2.6. Conclusion ...... 49

CHAPTER 3 PHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITIES OF GREEN MACROALGAE CULTURED IN THE TROPICS ...... 50

3.1. Introduction ...... 50

3.2. Material and methods ...... 51 3.2.1. Chemicals and reagents ...... 51 3.2.2. Algal sample collection and storage ...... 52 3.2.3. Preparation of seaweed extracts ...... 53 3.2.3.1. Preparation of crude extracts ...... 53 3.2.3.2. Preparation of purified ethanolic extracts ...... 54 3.2.3.3. Preparation of defatted ethanolic extracts ...... 54 3.2.3.4. Preparation of hydrolysed crude ethanolic extracts ...... 54 3.2.3.5. Preparation of hydrolysed purified ethanolic extracts ...... 55 3.2.3.6. Preparation of hydrolysed defatted ethanolic extracts ...... 55 3.2.4. Assays of phenolic content and antioxidant activities of seaweed extracts ...... 55 3.2.4.1. Total phenolic content (TPC) ...... 55 3.2.4.2. Total Flavonoid content (TFC) ...... 56 3.2.4.3. ABTS-radical scavenging assay ...... 56 3.2.4.4. DPPH- radical scavenging assay ...... 57 3.2.4.5. Ferric reducing antioxidant power (FRAP) assay ...... 57 3.2.4.6. Oxygen radicals absorbance capacity (ORAC) assay ...... 57 3.2.5. Identification of chemical compounds in phenolic extracts of algae ...... 60 3.2.5.1. High Performance Liquid Chromatography-Photodiode Array Detector analysis (HPLC-PDA) ...... 60 3.2.5.2. Gas Chromatography-Mass Spectrometry (GC-MS) analysis ...... 60 3.2.5.3. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) ...... 61 3.2.5.4. Nuclear magnetic resonance (NMR) spectroscopy ...... 62 3.2.6. Statistical analysis ...... 62

3.3. Results and discussion ...... 63 3.3.1. Extraction yields ...... 63 3.3.2. Total phenolic content (TPC) and total flavonoid content (TFC) of the algal species ...... 64 3.3.3. Antioxidant capacities of the algal species ...... 66 3.3.3.1. DPPH free radical scavenging capacity ...... 67 3.3.3.2. ABTS free radical scavenging capacity ...... 68 3.3.3.3. Ferric reducing antioxidant power (FRAP) ...... 68 3.3.3.4. Oxygen radicals absorbance capacity (ORAC) ...... 69 3.3.3.5. Correlation analysis ...... 70 3.3.4. Identification of chemical compounds in the phenolic extracts of algae ...... 71 3.3.4.1. HPLC-PDA analysis ...... 71 3.3.4.2. GC-MS analysis ...... 76 3.3.4.3. LC-MS ...... 81 3.3.4.4. 1H NMR analysis ...... 83

3.4. Conclusion ...... 87

II

CHAPTER 4 EXTRACTION, IDENTIFICATION AND BIOLOGICAL ACTIVITIES OF CAROTENOIDS FROM ULVA, DERBESIA AND OEDOGONIUM ...... 89

4.1. Introduction ...... 89

4.2. Materials and methods ...... 91 4.2.1. Chemicals and reagents ...... 91 4.2.2. Seaweed sample collection and storage ...... 92 4.2.3. Extraction of carotenoids from seaweeds ...... 92 4.2.3.1. Extraction methods ...... 92 4.2.3.2. Determination of total carotenoids ...... 93 4.2.4. Identification and quantification of carotenoids ...... 93 4.2.4.1. HPLC- PDA analysis of carotenoids ...... 93 4.2.4.2. HPLC-PDA-APCI-IT-TOT-MS analysis of carotenoids ...... 94 4.2.4.3. HPLC-PDA quantification of carotenoids ...... 95 4.2.5. Assays of antioxidant capacity ...... 95 4.2.6. Assays of inhibitory activity against metabolically important enzymes ...... 95 4.2.6.1. In vitro pancreatic lipase inhibition assay ...... 95 4.2.6.2. In vitro α-amylase inhibition assay ...... 96 4.2.6.3. In vitro α-glucosidase inhibition assay ...... 97 4.2.6.4. Hyaluronidase inhibition assay ...... 98 4.2.6.5. ACE inhibition assay ...... 99 4.2.7. Statistical analysis ...... 99

4.3. Results and discussion ...... 100 4.3.1. Total carotenoids content ...... 100 4.3.2. Identification of carotenoids in algal extracts ...... 102 4.3.2.1. Optimisation of extraction and HPLC procedures for the identification of carotenoids ...... 102 4.3.2.2. LC-MS identification of carotenoids in algal extracts ...... 106 4.3.2.3. Quantification of carotenoids in Ulva, Derbesia and Oedogonium ...... 112 4.3.3. Antioxidant activities of the carotenoid extracts of the three green macroalgae ...... 115 4.3.3.1. ABTS free radical scavenging capacity ...... 115 4.3.3.2. DPPH free radical scavenging capacity ...... 116 4.3.3.3. FRAP ...... 118 4.3.3.4. Correlation of total carotenoid contents with the antioxidant capacity of the three algae ...... 119 4.3.4. Inhibitory effects of algal carotenoid extracts against metabolically significant enzymes ... 120 4.3.4.1. In vitro pancreatic lipase inhibition by carotenoid extracts of algae ...... 120 4.3.4.2. In vitro antidiabetic effects ...... 123 4.3.4.3. α-Amylase inhibitory activities...... 124 4.3.4.4. α-Glucosidase inhibitory activity ...... 126 4.3.4.5. Hyaluronidase inhibitory activity ...... 128 4.3.4.6. ACE inhibitory activities ...... 131

4.4. Conclusion ...... 132

CHAPTER 5 BIOLOGICALLY ACTIVE PEPTIDES PRODUCED BY IN VITRO SIMULATED HUMAN DIGESTION OF GREEN ALGAE PROTEINS ...... 134

III

5.1. Introduction ...... 134

5.2. Materials and methods ...... 136 5.2.1. Chemicals and reagents ...... 136 5.2.2. Seaweed sample collection and storage ...... 136 5.2.3. Preparation of crude seaweed protein extracts ...... 136 5.2.4. Phenol extraction of protein for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis ...... 137 5.2.5. Determination of protein ...... 138 5.2.5.1. Total nitrogen by the Kjeldahl method ...... 138 5.2.5.2. BCA Protein Assay (Lowry method)...... 138 5.2.6. Analysis of amino acid composition ...... 138 5.2.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)...... 139 5.2.8. In vitro simulated gastrointestinal digestion of algal protein ...... 139 5.2.9. Fractionation of algal protein hydrolysates ...... 140 5.2.10. Determination of degree of hydrolysis ...... 140 5.2.11. Assay of bioactive activities ...... 141 5.2.11.1. Assays of antioxidant capacity ...... 141 5.2.11.2. Assays of α-amylase and α-glucosidase inhibition activity ...... 141 5.2.11.3. Assay of ACE inhibition activity ...... 141 5.2.12. Liquid chromatography-tandem mass spectrometry analysis of peptide sequences ...... 142 5.2.13. Statistical analysis ...... 143

5.3. Results and discussion ...... 143 5.3.1. Protein content of the three green algae ...... 143 5.3.2. Amino acid composition ...... 144 5.3.3. Protein extractability of the three green algae ...... 146 5.3.3.1. SDS-PAGE analysis of the algal proteins ...... 148 5.3.3.2. Protein content in the extract ...... 152 5.3.4. In vitro simulated gastrointestinal digestion of algal proteins ...... 153 5.3.4.1. Degree of protein hydrolysis ...... 153 5.3.5. Antioxidant activity of in vitro simulated digests of algae proteins ...... 157 5.3.5.1. ABTS radical scavenging activities ...... 157 5.3.5.2. Ferric reducing antioxidant power (FRAP) ...... 158 5.3.6. Inhibitory activity of algal protein hydrolysates against starch digesting enzymes ...... 160 5.3.6.1. α-Amylase inhibitory activities...... 160 5.3.6.2. α-Glucosidase inhibitory activity ...... 161 5.3.6.3. ACE inhibition activities of algal protein hydrolysates ...... 163 5.3.7. Fractionation of the bioactive peptides ...... 165 5.3.7.1. Antioxidant activities of the peptide fractions ...... 165 5.3.7.2. α-Glucosidase and α-amylase inhibition activities of the peptide fractions ...... 168 5.3.7.3. ACE-inhibition activities of the peptide fractions ...... 170 5.3.8. Identification of bioactive peptides in the hydrolysates of algal proteins ...... 171 5.3.8.1. Identification antioxidant peptides...... 172 5.3.8.2. Identification of potential ACE-inhibitory peptides ...... 176 5.3.8.1. Identification of potential antidiabetic peptides ...... 177

5.4. Conclusion ...... 186

CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER STUDIES ...... 187

IV

REFERENCES ...... 192

APPENDICES ...... 218

V

LIST OF TABLES

Table 1. Chemical characteristics of macroalgae (% w/w on dry basis) ...... 11 Table 2. Mineral composition of seaweeds (mg/100g DW)...... 12 Table 3. Algae species used as food or in food products ...... 15 Table 4. Major application of hydrocolloids ...... 16 Table 5. Chemical structure of major carotenoids compounds identified in algae ...... 22 Table 6. Natural pigments identified in algae species and their biological functions .... 26 Table 7. Protein levels of selected individual seaweed species ...... 31 Table 8. Amino acid profile of seaweed species (mg/100mg of algae protein) ...... 33 Table 9. Biologically active protein and peptides in naturally occurring in food ...... 35 Table 10. Bioactive peptides and their bioactivities from protein hydrolysates of marine algae ...... 38 Table 11. The extraction yield, total phenolic content and total flavonoid content of Ulva, Derbesia and Oedogonium ...... 64 Table 12. Reported levels of total phenolic/flavonoid content in selected marine algae and some antioxidant rich foods ...... 65 Table 13. ORAC capacity obtained from Ulva, Derbesia and Oedogonium for both hydrophilic and lipophilic extracts ...... 69 Table 14. Pearson’s correlation (r) values between the levels of phenolic compounds and antioxidant capacity for three different algae species ...... 71 Table 15. Compounds identified in the three green algae by GC-MS ...... 79 Table 16. Compounds identified in the three algae by NMR analysis ...... 87 Table 17. Chlorophyll a, chlorophyll b and carotenoid content of Ulva, Derbesia and Oedogonium ...... 102 Table 18. UV-vis spectral data for carotenoids identified in Ulva, Derbesia and Oedogonium ...... 110 Table 19. MS spectral data for carotenoids identification in Ulva, Derbesia and Oedogonium ...... 111 Table 20. Carotenoid contents in three green algae determinate by HPLC ...... 113 Table 21. Relationship between the total carotenoid content and antioxidant capacity for crude and saponified extracts of Ulva, Derbesia and Oedogonium ...... 120 Table 22. Lipase inhibitory activity of carotenoid extracts of three algal species ...... 123 Table 23. Angiotensin converting enzyme (ACE) inhibitory activities of carotenoid extracts from three green algae ...... 132 Table 24. Typical amino acid profiles of Oedogonium, Ulva, and Derbesia ...... 146 Table 25. Protein content in the extracts of Ulva, Derbesia and Oedogonium ...... 153 Table 26. The degree of hydrolysis (%) of proteins from three algal species at different stages of simulated human digestion ...... 154 Table 27. Comparison of ACE inhibition potency of protein hydrolysates from Ulva, Derbesia and Oedogonium with other algal species reported in literature ...... 164

VI

Table 28. List of peptides identified in the MW <3kDa fraction of the protein hydrolysates from three algal species and the potential antioxidative sequences identified using the BIOPEP database ...... 174 Table 29. List of peptides identified in the MW <3kDa fraction of the protein hydrolysates of three algal species and the potential antihypertensive sequences identified using the BIOPEP database ...... 178 Table 30. List of peptides identified in the MW <3kDa fraction of the protein hydrolysates of three algal species and the potential antidiabetic sequences identified using the BIOPEP database ...... 1812

VII

LIST OF FIGURES

Figure 1. Distribution of different macroalgae in Australia (A) green algae, (B) red algae and (C) brown algae ...... 10 Figure 2. The current and potential utilization of macroalgae...... 13 Figure 3. Basic structures of flavonoids...... 28 Figure 4. Chemical structure of main phenolic acids ...... 29 Figure 5. Specimen photos of Derbesia tenuissima (a and b), Ulva ohnoi (c and d) and Oedogonium intermedium (e and f) showing growth habit in culture and cellular ...... 46 Figure 6. The total phenolic content (TPC) and antioxidant activities for Ulva, Derbesia and Oedogonium...... 67 Figure 7. HPLC chromatograms of standard phenolic compounds...... 72 Figure 8. HPLC chromatograms of crude extracts from (A) Ulva, (B) Derbesia and (C) Oedogonium...... 74 Figure 9. HPLC chromatograms of purified algae extracts from (A) Ulva, (B) Derbesia and (C) Oedogonium...... 75 Figure 10. Mass spectral confirmation of trimethylsilyl-derivatised palmitic acid (RT 23.68) using the NIST library (top) and the head to tail matching with reference standard (bottom)...... 78 Figure 11. LC-MS total ion current (TIC) chromatogram (A) of Ulva using atmospheric pressure chemical ionisation with mass range between m/z 120-700. A [M-H]- corresponded to peaks at retention time 21.48 (B), 22.00 (C), and 26.67 (D) min...... 82 Figure 12: 1H NMR spectra of crude (A) and purified (B) ethanolic extracts of Ulva. . 84 Figure 13: 1H NMR spectra of crude (A) and purified (B) ethanolic extracts of Derbesia...... 85 Figure 14: 1H NMR spectra of crude (A) and purified (B) ethanolic extracts of Oedogonium...... 86 Figure 15. HPLC profile of carotenoids isolated from Ulva with (A) extraction procedure 1 and HPLC method 1, (B) extraction procedure 2 and HPLC method 1 ... 104 Figure 16. HPLC-PDA chromatograms of carotenoid extracts of three algal species at 450 nm...... 107 Figure 17. UV-Vis absorption spectra of (A) reference standard of violaxanthin and (B) peak 5 in Ulva...... 108 Figure 18. Mass spectra of (A) reference standard of violaxanthin, and (B) peak 5 in Ulva...... 108 Figure 19. ABTS scavenging activities of crude and saponified carotenoid extracts obtained from Ulva, Derbesia and Oedogonium...... 116 Figure 20. DPPH scavenging activities of crude and saponified carotenoid extracts obtained from Ulva, Derbesia and Oedogonium...... 117 Figure 21. FRAP activities of crude and saponified carotenoid extracts obtained from Ulva, Derbesia and Oedogonium...... 118

VIII

Figure 22. Dose-dependent effects of pancreatic lipase inhibition activity of crude (A) and saponified (B) carotenoid extracts obtained from Ulva, Derbesia and Oedogonium...... 122 Figure 23. Dose-dependent effects of α-amylase inhibition activity of crude (A) and saponified (B) carotenoid extracts obtained from Ulva, Derbesia and Oedogonium.. . 125 Figure 24. Dose-dependent effects of inhibition α-glucosidase activity of crude (A) and saponified (B) carotenoid extracts obtained from Ulva, Derbesia and Oedogonium.. . 127 Figure 25. Dose-dependent effects of hyaluronidase inhibition of crude (A) and saponified (B) carotenoid extracts obtained from Ulva, Derbesia and Oedogonium.. . 130 Figure 26. Influence of extraction conditions on protein yield from Ulva, Derbesia and Oedogonium...... 147 Figure 27. SDS-PAGE separation of Ulva, Derbesia and Oedogonium protein prepared using different extraction method (A) without sonication, (B) with ultrasound...... 151 Figure 28. SDS-PAGE separation of Ulva, Derbesia and Oedogonium protein prepared using phenol extraction...... 152 Figure 29. The degree of hydrolysis of sample controls of in vitro human simulated digestion...... 156 Figure 30. The degree of hydrolysis of sample controls of in vitro human simulated digestion...... 157 Figure 31. ABTS scavenging activities of protein from Ulva, Derbesia and Oedogonium before and after in vitro simulated digestion...... 158 Figure 32. FRAP values of proteins from Ulva, Derbesia and Oedogonium before and after in vitro simulated digestion...... 159 Figure 33. Inhibition of α-amylase activity by proteins from Ulva, Derbesia and Oedogonium before and after in vitro simulated digestion...... 161 Figure 34. Inhibition of α-glucosidase activity by proteins from Ulva, Derbesia and Oedogonium before and after in vitro simulated digestion...... 163 Figure 35. FRAP reducing power of ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion...... 167 Figure 36. ABTS scavenging activities of ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion...... 168 Figure 37. α-Amylase inhibition by ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion...... 169 Figure 38. α-Glucosidase inhibition by ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion...... 170 Figure 39. ACE inhibitory activity of ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion...... 171

IX

ABBREVIATIONS

4-MUO 4-methylumbelliferyl oleate AAPH 2, 2’-azobis(2-amidinopropane)dihydrochloride ABTS 2, 2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt ACE Angiotensin I-converting enzyme ANOVA Analysis of variance APCI Atmospheric pressure chemical ionisation BSA Bovine serum albumin BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide

D2O Deuterium oxide water DH Degree of hydrolysis DHA Docosahexaenoic acid DMAB 4-dimethylaminobenzaldehyde DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPPH Di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium DPP-IV Dipeptidy peptidase-IV DW Dry weight EPA Eicosapentaenoic acid ESI Electrospray FL Fluorescein FRAP Ferric reducing antioxidant power G Gram GAE Gallic acid equivalent GC-MS Gas Chromatography-Mass Spectrometry H Hour HA Hippuric acid HHL N-Hippuryl-his-Leu HIV human immunodeficiency virus HPLC High Performance Liquid Chromatography HPLC-DAD High Performance Liquid Chromatography-Diode Array Detector HPLC-PDA High Performance Liquid Chromatography-Photodiode Array Detector HPVs Human papillomavirus 1H NMR Proton Nuclear Magnetic Resonance

IC50 Half maximal inhibitory concentration X i.d. Internal diameter JCU James Cook University kDa Kilo Dalton LC-MS Liquid Chromatography-Mass Spectrometry LC-MS/MS Liquid Chromatography-Tandem Mass Spectrometry LCMS-IT-TOF Liquid Chromatography iron trap-time of flight mass spectrometer LDL Low density lipoprotein m/z Mass-to-charge ratio MAF Microalgae Food min Minute mL Millilitre MTBE Methyl-tert-butyl ether n.d. not detectable

N2 Nitrogen NMR Nuclear Magnetic Resonance ORAC Oxygen radicals absorbance capacity pNP p-nitrophenol p-NPG p-nitrophenyl-β-ᴅ-glucopyranoside PTFE polytetrafluoroethylene PUFA Polyunsaturated fatty acid RE Rutin hydrate equivalent RMCD Randomly methylated betacyclodextrin ROS Reactive oxygen species RT Retention time SD Standard deviation SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis TE Trolox equivalent TFC Total Flavonoid content TPC Total phenolic content TPTZ 2,4,6-tripyridin-2-yl-1,3,5-triazine v/v Volume per volume w/w Weight per weight

XI

ABSTRACT

Macroalgae are rich in bioactive components such as carotenoids, phenolic compounds and proteins/peptides, which may play a significant role in the prevention of diseases like cancer, obesity and diabetes. The aim of this thesis was to examine the in vitro biological activities of phenolic compounds, carotenoids and protein hydrolysates from three edible green macroalgae (Ulva ohnoi, Derbesia tenuissima and Oedogonium intermedium) cultured in tropical Australia. The phenolic components were extracted with 60% aqueous ethanol and their antioxidant activities were determined by four different assays (ABTS, DPPH, FRAP and ORAC). The extracts exhibited moderate levels of antioxidant activities. However, analysis of the extracts by HPLC-PDA, GC- MS, LC-MS and 1H NMR failed to detect any phenolic components, while a number of free amino acids, fatty acids and sugars were found, which were likely responsible for the measured antioxidant activities. Carotenoids were extracted from the algae by dichloromethane, and the extracts exhibited significant antioxidant activities, as well as potent inhibitory effects against several metabolically important enzymes including α- amylase, α-glucosidase, pancreatic lipase and hyaluronidase. However, the carotenoid extracts were poor inhibitors of angiotensin-converting enzyme (ACE). The extracts were analysed by LC-MS, which resulted in the identification of nine major carotenoids in the algae: siphonaxanthin, neoxanthin, 9’-cis-neoxanthin, loroxanthin, violaxanthin, lutein, siphonein, α-carotene and β-carotene. Proteins were extracted from the algae by alkaline solution. The extracted proteins were subjected to in vitro simulated human digestion and the resultant hydrolysates were fractionated by ultrafiltration. The hydrolysates showed markedly increased antioxidant activities and inhibition effects against α-amylase, α-glucosidase and ACE over the undigested proteins. Most of the peptides in the hydrolysates were extensively hydrolysed with MW less than 3 kDa. LC-MS/MS analysis identified a large number of peptides in the MW <3 kDa fraction of the hydrolysates and most of them contained peptides with known antioxidant, antidiabetic or antihypertensive activities as reported in the BIOPEP database. Overall, this thesis demonstrated that consumption of the algae could confer significant health benefits and the algae could be developed into bioactive ingredients with potential applications in functional food, nutraceutical and pharmaceutical products.

XII

Chapter 1 Introduction

In recent decades, the functional food and nutraceutical market has grown enormously. It is estimated that the market is currently worth $168 billion and is projected to grow to $305 billion by the year 2020 (Research and Markets, 2014). The principal drivers for the growth of the functional food market are current population and health trends. Across the globe, life expectancy continues to rise and populations are rapidly aging in many countries. Concurrent to the aging population is the rising incidence of a number of non-communicable diseases, such as cancer, heart diseases, diabetes and obesity, throughout the world. World Health Organization (WHO) estimated that there are more than 1.9 billion overweight adults worldwide in 2014 and of these, 13% are clinically obese (WHO, 2015); 8.5% the world’s adult population are diabetes and 1.5 million deaths were directly caused by diabetes in 2012 (WHO, 2016). About 25% of the world’s adult population suffers hypertension, which is estimated to increase to 29% by 2025 (Mittal and Singh, 2010). In 2011-2012, there are around 60% of Australian adults who were classified as overweight or obese (National Health and Medical Research Council, 2015), 5% having diabetes (Australian Institute of Health and Welfare, 2016) and 31.6% having hypertension (Australian Bureau of Statistics, 2013). It has become increasingly evident that medical service alone is neither able nor the best strategy to cope with these chronical disorders. Rather, life style change, including the consumption of health foods and nutraceuticals with disease-preventing functions, should play a major role in national health strategies (Cencic and Chingwaru, 2010). For this reason, there has been a growing interest in bioactive food components with health- promoting properties that can be used in functional food, nutraceutical, and pharmaceutical products.

The major sources of bioactive components are plant based foods, especially fruits, vegetables and herbs, and the main components are phenolics, carotenoids, carbohydrates, lipids, proteins and peptides. Some plants are well known for their bioactive components. For example, green tea contains epigallocatechin gallate that might have anticancer properties (Fujiki and Suganuma, 2012); berries such as blueberry and raspberry are rich in flavonoids that are potent natural antioxidants (Bowen-Forbes et al., 2010); red wine contains resveratrol that may benefit heart health

1

(Chong et al., 2015); and tomatoes contain lycopene that has potential anticancer properties (Kris-Etherton et al., 2002).

In recent years, edible macroalgae have emerged as another major source of bioactive components comparable or even superior to fruit and vegetables. Algae are simple chlorophyll-containing organisms composed of either single cells or multiple cells grouped together in colonies (Kim, 2012). Over 80% of the world’s plant and animal species are found in the ocean environments, including more than 150,000 macroalgae, commonly known as seaweeds (de Almeida et al., 2011). Macroalgae can be classified into three classes based on their pigmentation: red (rhodophyta), brown (phaeophyta) and the green (chlorophyta) algae (El Gamal, 2010). Historically, seaweeds have been used as food mainly in Asian countries, particularly China, Japan and Korea, where they are consumed in various forms after being made into raw salads, cookies, jam, jellies and soups or as ingredients in a variety of dishes (Ruperez, 2002). In modern industries, seaweeds are mainly used as a source of phycocolloids (e.g., alginate, carrageenan and agar), which are manufactured into thickening and gelling agents for various industrial applications, including uses in foods (Jimenez-Escrig and Sanchez-Muniz, 2000). However, to date, there are only about 145 species of red, brown or green seaweeds that are used worldwide as food (Pereira et al., 2009, Zemke-White and Ohno, 1999). Australian waters are home to at least 12,000 marine and fresh macroalgal species, but only around 35 have been used in food applications; hence, there is a huge potential food resource remaining to be explored (Orchard and McCarthy, 2007).

Macroalgae possess a number of unique properties that make them an attractive source for food and bioactive compounds. Owning to their simple structure and reproductive process, they grow rapidly and can be easily cultivated in large scale all year round. Moreover, it has been recently demonstrated that the production of some bioactive compounds can be controlled by manipulating the culturing conditions (Magnusson et al., 2015). From a nutritional perspective, edible seaweeds are rich in a number of nutrients including protein, minerals, vitamins and unsaturated fatty acids. Macroalgae contain 10-47% protein in a dry biomass basis with high proportions of essential amino acids and are an excellent source of vitamins A, B1, B12, C, D, and E (Rajapakse and Kim, 2011). They contain up to 50% carbohydrates with the vast majority of which

2 being dietary fiber, and are low in lipids and energy content. Such nutrient profile would clearly qualify them as healthy foods.

Furthermore, because algae are organisms that live in complex habitats exposed to extreme conditions (e.g., salinity, temperature, UV irradiation, etc.), they have evolved special traits to survive and rapidly adapt to the environmental stresses. Part of the mechanisms to alleviate the environmental stresses is the production of a great variety of secondary metabolites, many of which are not found in other organisms (Beaulieu et al., 2015). For example, macroalgae have strong antioxidant systems to protect themselves from oxidative damage from radicals that are formed during photosynthesis and other physiological activities. The antioxidant activity of phenolic compounds from several seaweeds has been well documented (Chew et al., 2008, Kelman et al., 2012, Lopez et al., 2011, Matanjun et al., 2008, Sabeena and Jacobsen, 2013). A number of phenolic compounds, including phenolic acids and flavonoids, have been identified from algal extracts (Lopez et al., 2011, Rodriguez-Bernaldo de Quiros et al., 2010, Santoso et al., 2002). However, the research to date has largely focused on commercially cultivated brown and red seaweeds prevalent in cold waters, while information is limited on phenolic antioxidants of green algae, especially those grown in tropical waters (Yuan et al., 2005c).

As photosynthetic organisms algae also synthesise chlorophylls and carotenoids. Dunaliella salina and Haematococcus pluvialis are well known to accumulate β- carotene and astaxanthin, and are already used as food colouring agents (Hu et al., 2008). Muriellopsis has been used commercially to produce lutein due to its high lutein content and high growth rate (Ahmed et al., 2014). Chlorella vulgaris has also been reported as an efficient producer of lutein with commercial potentials (Cha et al., 2008). Several algal carotenoids have received particular attention as they are structurally different from those found in terrestrial plants. Astaxanthin from red seaweeds (e.g., Dunaliella salina), siphonaxanthin from green macroalgae (e.g., Codium fragile) and fucoxanthin from brown seaweeds (e.g., Undaria pinnatifida) are examples of such carotenoids. These carotenoids have been found to exhibit a number of significant pharmacological effects, e.g., inhibiting the growth and inducing apoptosis in human cancer cells as well as possessing antioxidant, antidiabetic, anti-obesity and anti- inflammatory properties (Peng et al., 2011, Ambati et al., 2014, Sugawara et al., 2014).

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However, these carotenoids are only reported for a limited number of algal species, and more studies are needed to find additional algal sources of these carotenoids, especially those species that are already commercially cultivated, so that should carotenoids of interest are found, fast track production in commercial scale can be achieved.

The high protein content of macroalgae means that they could also be a source for the production of bioactive protein hydrolysates and peptides. Several studies have found that hydrolysates or peptides generated from algal proteins possessed a number of health beneficial biological activities including antioxidant (Karawita et al., 2007, Kang et al., 2011, Kim et al., 2006b, Sheih et al., 2009b), antihypertensive (Qu et al., 2010, Sato et al., 2002a, Sato et al., 2002b, Suetsuna and Nakano, 2000, Suetsuna et al., 2004, Suetsuna and Chen, 2001, Sheih et al., 2009a), immunostimulating (Morris et al., 2007), anticancer (Sheih et al., 2010), and hepeto-protective (Kang et al., 2012, Hwang et al., 2008) effects. However, the number of algal species investigated is limited, and a large number of edible macroalgal species are yet to be explored. Furthermore, the protein hydrolysates and peptides in the aforementioned studies were produced using single proteolytic enzyme digestion (pepsin, pancreatin, pronase, trypsin or chymotrypsin). Multi-proteolytic enzyme system data, which more closely resemble the actual digestion environment in vivo, on seaweed proteins are very scant.

Three green macroalgae (chlorophyta) species were selected for study in this thesis. They are the marine macroalgae Derbesia tenuissima and Ulva ohnoi and the freshwater macroalga Oedogonium intermedium. Ulva ohnoi belongs to a familiar genus Ulva, but it is a tropical variety of the blade-form and the biological activities of which has not been well investigated. Derbesia tenuissima and Oedogonium intermedium are two new semi-commercial species that have only recently been domesticated for the first time. These species were selected based on their high productivity in land-based culture, resistance to contamination and a high tolerance to environmental fluctuations (Neveux et al., 2014b). For these reasons, they are considered to have a good potential as natural sources of bioactive ingredients. These species have also been shown to have a low toxicity, and therefore can be used for extraction of bioactive compounds for application in functional food, nutraceutical and pharmaceutical products (Hiraoka et al., 2004, Gosch et al., 2015).

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In light of the above discussion, the overall aim of this PhD project was to expand the knowledge on the phenolic, carotenoid and protein/peptide compositions and health- related biological activities of Derbesia tenuissima, Ulva ohnoi and Oedogonium intermedium. The specific objectives of the study were:

1. To investigate the major health-related bioactivities of these three green macroalgae, including antioxidant capacity and inhibitory activities against several key metabolically significant enzymes (α-glucosidase, α-amylase, angiotensin converting enzyme (ACE), lipase and hyaluronidase), using in vitro methods; 2. To identify the main phenolic and carotenoid compounds in the algae that are responsible for the biological activities using a series of analytical techniques, including high performance liquid chromatography-photodiode array detector (HPLC-PDA), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy; and 3. To investigate the biological activities of protein hydrolysates of the three algae produced by in vitro simulated human digestion using a multiple enzyme system, and identify the active peptide sequences by HPLC-MS/MS and the bioactive peptide database BIOPEP.

The thesis begins with a general introduction (Chapter 1), followed by a review of the relevant literature focusing on biological activities of algae and the bioactive components published in the last two decades (Chapter 2). This is followed by three results chapters presented in a journal paper format as the content of the investigation and methodologies used in each chapter are largely independent from each other. Chapter 3 is the first results chapter, which describes the extraction of phenolic compounds from the three green macroalgae, investigation of their antioxidant activities using four in vitro assays, and identification of the phenolic compounds by using HPLC-DAD, GC-MS, LC-MS and NMR. Chapter 4 describes the extraction of carotenoid compounds from the three green macroalgae, identification of the carotenoids by HPLC-PDA and LC-MS, and assessment of their biological activities

5 including antioxidant capacity and inhibitory effects against several key enzymes involved in metabolic syndromes, namely ACE, α-glucosidase, α-amylase, pancreatic lipase and hyaluronidase. Chapter 5 describes the extraction of algal proteins, hydrolysis of the proteins by in vitro simulated human digestion, fractionation of the hydrolysates, analysis of the bioactivities of the hydrolysates, and identification of peptides in the most active fractions. Finally, chapter 6 outlines the major conclusions obtained in this thesis and provides some recommendations for future research in this area.

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Chapter 2 Literature Review

2.1. Algae

Algae are simple chlorophyll-containing organisms composed of either single cells or multiple cells grouped together in colonies, sometimes forming simple tissues (Kim, 2012). Algae are ubiquitous, found virtually everywhere on Earth: in the sea, rivers and lakes, on soil and walls, and in animals and plants.

Algae are commonly divided into macro- and microalgae based on the size of the organisms. Microalgae are microscopic algae which can be found in both benthic, littoral habitats and also as phytoplankton all through the ocean waters (Kim, 2012). Microalgae include organisms such as blue-green algae (Cyanophyta), diatoms (Bacillariophyta) and dinoflagellates (Dinophyta) (El Gamal, 2010). These microalgae play a significant role in the ecosystems of the ocean as they constitute the basis of the marine food chain (El Gamal, 2010).

Macroalgae are multicellular organisms with a great diversity of forms and sizes grown predominantly in marine environment (commonly known as seaweed), but also in fresh waters. They are fast growing plants that can grow to considerable sizes (up to 70m in length) (Hillson, 1977) and are one of the most ecologically and economically important resources of the ocean. There are about 14,000 species of macroalgae that have been described to date (Michael, 2012, Diaz-Pulido and McCook, 2008). These macroalgae play important roles in the marine ecosystems as a basic food source for herbivores in the creation of habitats or structures, and by acting as a form of biological purifiers in coastal ecosystems. For the latter role, they work by filtering nutrients coming from industrial discharge and stormwater runoffs prior to reaching sensitive habitats such as coral reefs, as well as by absorbing carbon dioxide and releasing oxygen into the water (Harrison and Hurd, 2001).

In addition to the environmental roles, many macroalgae also have commercial applications as sources of food, gelling agents, pharmaceutical ingredients, fodder, fertiliser and raw materials for cosmetic and other industrial products. While it is not widespread amongst macroalgae, some species have a high concentration of energy-rich oils that can be exploited as a source of biofuel (Gosch et al., 2012, Gosch et al., 2015). 7

In recent years, microalgae have also been explored as a potential source of bioactive ingredients for functional food and nutraceutical products. The food and health related properties of macroalgae have been the subject of several reviews, including those on the chemical composition and nutritional values of edible seaweeds (Rajapakse and Kim, 2011, Mendis and Kim, 2011, MacArtain et al., 2007) and on the biological activities of natural pigments (Pangestuti and Kim, 2011) and proteins (Samarakoon and Jeon, 2012). This review focuses on the major bioactive components in macroalgae and their health- related biological activities.

2.1.1. Classification of macroalgae

Macroalgae can be classified into three classes: green algae (chlorophyta), brown algae (phaeophyta) and red algae (rhodophyta) (El Gamal, 2010). The name of the different algae comes from their colour, and the different colours of algae are the result of different photosynthetic pigments prevalent in them. In addition to the different pigmentation, they also differ considerably in many ultrastructural and biochemical features including storage compounds, composition of cell walls and the fine structure of the chloroplasts (Michael, 2012).

Green algae (chlorophyta) have a green pigmentation which is mainly due to the presence of chlorophyll a and b in roughly the same amounts as in high plants (El Gamal, 2010). There are about 1600 species of green algae that have been discovered and 90% of these are freshwater ones (Michael, 2012, Hillson, 1977, Diaz-Pulido and McCook, 2008). Green algae are mostly tiny unicellular organisms or simple filaments. Some species of green algae, such as Caulerpa spp. (sea grapes), Ulva spp. (sea lettuce), Palmaria palmata (dulse) and Undaria pinnatifida (wakame) are consumed directly by human, either in salads or as marine vegetables (Hillson, 1977).

Red algae (rhodophyta) have red colour mainly due to the pigment of phycoerythrin and phycocyanin, which mask the other pigments, such as chlorophyll a, beta-carotenes and a number of unique xanthophylls (El Gamal, 2010). There are around 9500 species and about 90% of them are marine species (Michael, 2012, Hillson, 1977, Diaz-Pulido and McCook, 2008). These are found in all oceans, but most commonly in warm temperature and tropical climates, where they may occur at great depths of up to 250 m

8 due to their ability to absorb light in the blue wavelength area (Hillson, 1977). Some of the red algae contain a high amount of agar and carrageenan, polysaccharides with specific physicochemical properties that have applications in many products (El Gamal, 2010). At the present, red algae are cultured and utilised more extensively than other algal classes (Diaz-Pulido and McCook, 2008, Orchard and McCarthy, 2007).

The brown colour of brown algae (Phaeophyta) results from the dominance of the brown coloured xanthophyll pigments and fucoxanthin. These pigments mask the other pigments, such as chlorophyll a and c, β-carotenes, and other xanthophylls (El Gamal, 2010). There are approximately 2100 species of brown algae and almost all are marine species (Michael, 2012, Diaz-Pulido and McCook, 2008). Most brown algae are found in the colder oceans of the world, many in tidal zones. Brown algae can potentially be used as food because they are typically rich in complex polysaccharides and higher alcohols, as well as laminaran that can act as carbohydrate (Kim, 2012).

2.1.2. Distribution

Algae can grow almost anywhere and they are found in virtually all parts of the earth. They are distributed not only in fresh water and marine environments, but also in the atmosphere as free-floating organisms, in precipitation or mixed with dust. Some algae grow in ice or snow, in moist areas on the ground, in rocks and even on turtles. Research shows that algae also grow on mosquito larvae antennae, in hot springs, on ducks' feet, on sulfur belly whales, on polar bears (turning them green) and on tropical sloths. For Australia, this means that algae are found in all parts of the country and because the land is surrounded by three different oceans, the Indian, Pacific and Southern Oceans, there is a diverse range of algal species available. As seen from Figure 1, green algae are distributed almost everywhere in Australia including both the coastline and mainland, whereas most of the red algae are distributed in the coastline of Australia.

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(A) (B)

(C)

Figure 1. Distribution of different macroalgae in Australia (A) green algae, (B) red algae and (C) brown algae. Source: The Atlas of Living Australia (www.ala.org.au) (ALA, 2015).

2.2. Chemical composition of macroalgae

Like most terrestrial plants, marine algae are a rich source of carbohydrates, proteins, lipid, minerals and vitamins. Table 1 shows the proximate composition of some seaweeds and it is clear that their nutrient composition varies considerably depending on the species, season and location (Peng et al., 2015). Compared with common vegetables, edible seaweeds generally have a high level of fibre, minerals, omega-3 polyunsaturated fatty acids (PUFA), and moderate concentrations of lipid and proteins, which make them a potentially important food source for human and animals.

Seaweed contains a large amount of carbohydrate ranging from 20% to 76% of the dry biomass depending on the species (Rajapakse and Kim, 2011). Importantly, most of the carbohydrate of seaweed is fibre, which is not digested to any significant extent in the gut. For example, Porphyra, which is normally processed into “Nori” sheets, contains a 10 much higher level of fibre than bananas (a commonly cited fibre source) in direct weight comparison (49.8 g versus 9.65 g per 100 g of dry weight (DW), respectively) (Dawczynski et al., 2007, Liao and Hung, 2015). The main components of carbohydrate in seaweeds are alginates in brown seaweed, carrageenan and agar in red seaweed and cellulose in green seaweed. With regards to protein, red seaweed contains the highest content of the nutrient at 30-40% DW, which is comparable to legumes, while brown and green seaweeds contain about 15 and 30% of protein, respectively (Rajapakse and Kim, 2011). In general, seaweed proteins contain all the essential amino acids, but are especially rich in glycine, arginine, alanine and glutamic acid. However, they are deficient in lysine and cysteine when compared with other major food protein sources. Seaweed generally has a low lipid content, ranging from 1% to 5% DW, and much of this content is made up of polyunsaturated fatty acids (PUFA) (Rajapakse and Kim, 2011).

Table 1. Chemical characteristics of macroalgae (% w/w on dry basis)

Dietary Specie Moisture Ash Protein Lipid Carbohydrate Reference fibre Green algae Ulva lactuca 12.6 11.0 27.2 0.3 61.5 60.5 (Ortiz et al., 2006) Ulva lactuca 14.94 19.59 8.46 7.87 - 54.9 (Yaich et al., 2011) E. intestinalis 8.5 22.4 10.5 2.9 35.5 - Red algae Porphyra 3.66 8.78 32.16 1.96 53.44 - (Hwang et al., tenera 2013) Porphyra 6.74 9.07 36.88 2.25 45.06 - (Hwang et al., haitanensis 2013)

Brown algae Undaria 10.7 - 19.8 4.5 - 45.9 (Dawczynski pinnatifida et al., 2007) Laminaria sp. 9 - 7.5 1.0 - 36.0 (Dawczynski et al., 2007) Hizikia 10.6 - 11.6 1.4 - 62.3 (Dawczynski fusiforme et al., 2007) Durvillaea 72.3 17.9 10.4 0.8 70.9 71.4 (Ortiz et al., antarctica 2006) “-” : data not available.

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Mineral content of seaweed can account for up to 36% of its dry mass and they include calcium, potassium, magnesium, sodium, copper, iron, iodine and zinc (Table 2). In addition, seaweed contains several vitamins, including both water-soluble vitamins such as B and C and lipid soluble ones such as A and E. Some seaweed species, such as purple laver, nori, spirulina and wakame are relatively rich in which is deficient in most land plants (Rajapakse and Kim, 2011).

Table 2. Mineral composition of seaweeds (mg/100g DW)

Species Ca K Mg Na Cu Fe Zn Reference

Chlorophyta (Green)

Ulva lactuca - 515.6 79.1 - 0.34 46.4 1.6 (Rohani-Ghadikolaei et al., 2012)

Enteromorpha intestinalis - 589.3 61.7 - 0.43 25.4 2.1 (Rohani-Ghadikolaei et al., 2012)

Rhodophyta (Red)

Porphyra/Pyropia (Nori) 390 3500 565 3627 <0.5 10.3 2.21 (Ruperez, 2002)

Chondrus 420 3184 732 4270 <0.5 3.97 7.14 (Ruperez, 2002)

Phaeophyta (Brown)

Fucus 938 4322 994 5469 <0.5 4.2 3.71 (Ruperez, 2002)

Laminaria 1005 11579 659 3818 <0.5 3.29 1.77 (Ruperez, 2002)

Undaria (wakame) 931 8699 1181 7064 <0.5 7.56 1.74 (Ruperez, 2002)

“-” : data not available.

2.3. Current and potential utilization of macroalgae

Macroalgae are economically valuable resources. Many macroalgal species have been used as sources of food, medicine, fodder, fertiliser and materials for many other industrial applications in many countries. They can also be used as wastewater treatment agents and sources of alternative fuels. Research in recent decades has found

12 that macroalgae are rich in a number of bioactive phytochemicals such as carotenoids, and have the potential to be developed into bioactive ingredients for functional foods, nutraceutical supplements and even pharmaceutical products (Rajapakse & Kim, 2011). The broad areas of current and potential utilisation of seaweeds are shown in Figure 2.

Marine vegetable

Food products Hydrocolloid foods

Condiments

Manure Agriculture Fodder

Biomass/Fuel Environment Wastewater treatment

Textile products MACROALGAE AS RESOURCE AS MACROALGAE Industry application Rubber products

Pharmaceutical Products Paper products or Functional foods

Figure 2. The current and potential utilization of macroalgae.

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2.3.1. Seaweed as food and food ingredients

Many seaweed species have high nutritional value, being rich in proteins, amino acids, fibre, minerals and vitamins, and have been used traditionally and commercially for food (Michael, 2012). The utilisation of these plants as food occurs mainly in Asia, particularly Japan, Korea and China, where seaweeds are traditionally consumed in various forms after being made into salads, jams, jellies and soups or as ingredients in a variety of dishes (Ruperez, 2002). In western countries the principal use of seaweeds has been as a source of phycocolloids (alginate, carrageenan and agar), which are manufactured into thickening and gelling agents for various industrial applications, including uses in foods. Worldwide, about 221 species of algae are currently utilised in significant quantity, 125 of which being red, 64 brown, and 32 green species. Of these, about 145 species are used directly for food, 101 species in the phycocolloid industry, 24 species in traditional medicine and about 25 species in agriculture as animal feed and fertilisers (Zemke-White and Ohno, 1999, Pereira et al., 2009). Some seaweed species currently used for food are summarised in Table 3. The top three species that are available commercially in the market are kombu, wakame and nori.

Edible seaweeds are high in protein, soluble dietary fibre and a range of vitamins including A, B1, B2, B6, B12, niacin and C (Peng et al., 2015) and minerals including iodine, potassium, iron, magnesium and calcium (Ruperez, 2002). Several seaweeds are consumed in their raw form as marine vegetables in various kinds of salads. Seaweed consumed as marine vegetables include sea grapes/green caviar (Caulerpa lentillifera), sea lettuces (Ulva spp.), winged kelp (Alaria esculenta), and dulse (Palmaria palmata). Another example in this category is nori or purple laver (Porphyra spp.), which is commonly used as warp-up vegetables to make sushi (Cha et al., 2008).

Another common food use of seaweeds is as condiments to flavour broths and stews. Seaweeds such as kombu in Japanese or haidai in Chinese (Saccharina japonica, previously Laminaria japonica) are used for this purpose. It is also a major ingredient in some popular snacks such as sea sedge, as well as a savoury ingredient or garnish for many dishes (Plaza et al., 2008).

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Table 3. Algae species used as food or in food products

Phylum Common name Scientific name Product/Application

Chlorophyta Sea grapes or green caviar Caulerpa lentillifera; Fresh salad

(Green) Caulerpa racemose; Sea lettuces Ulva prolifera; Fresh salad

Ulva intestinalis; cooked in soup

Aonori or Green laver Monostroma latissimum Roasted seaweed

Rhodophyta Dulse Palmaria palmata Dried sea vegetable

(Red) Irish moss or carrageenan moss Chondrus crispus Salad; Sources of carrageenan

Nori Porphyra umbilicalis Nori sheets/Roasted nori; used as wrap for sushi Porphyra yezoensis

Hizikia fusiforme

Ogo, ogonori or sea moss Gracilaria verrucosa (source of thickener agar)

Phaeophyta Kombu Laminaria japonica Dried sea vegetable; pickled; (Brown) Source of alginates Laminaria longissimi

Laminaria angustata

Laminaria coriacea

Laminaria ochotensis

Wakame Undaria pinnatifida Dried seaweed; served in soups and salads; miso soup

Mozuku Cladosiphon okamuranus Source of fucoidan

Winged kelp Alaria esculenta Eat fresh or cooked

Ecklonia cava Herbal remedy in form of an Seanol (polyphenolic) and Ventol (Phlorotannin)

Arame Eisenia bicyclis Dried seaweed

Ascophyllum nodosum Alginic acid

Sea spaghetti Himanthalia elongate Salad

Microalgae Spirulina Arthrospira platensis Dietary supplement

Arthrospira maxima

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One of the major modern industrial uses of seaweeds is as phycocolloids, which has stemmed from the rich content of polysaccharides in many macroalgal species. Thus, alginic acids are extracted from brown algae while agar and carrageenan are prepared from red algae. The usefulness of these phycocolloids mainly lies in their ability to emulsify, stabilise and thicken. These phycocolloids are used widely in products as diverse as ice cream, dry ice cream mix, instant puddings, chocolate, toddy, sherbet, chocolate milk, sterilized cream, cheese, dental impression material, wound dressings and pharmaceuticals (Jimenez-Escrig and Sanchez-Muniz, 2000). Table 4 provides an overview of the application of algal phycocolloids in these products. Agar is able to bind with large amounts of water to form a gel often called “elastic jelly”. Alginates have similar applications as agar but tend to be more widely used due to their broader gelling properties. Carrageenan (E 407) is a group of phycocolloids mainly derived from the Chondrus crispus, and it is extensively used in a variety of industrial products as gelling, thickening, and stabilising agents, especially in food products such as canned foods, dessert mousses, salad dressings, bakery fillings, ice cream, instant desserts and canned pet foods (Campo et al., 2009).

Table 4. Major application of hydrocolloids

Hydrocolloids Characteristics Species Application

Agar Stabiliser Pastries Gelidium sp. Jellies Gracilaria sp. Ice cream Processed meats Culture medium for medical and plant tissue

Alginates Stabiliser Ascophyllum nodosum Sauces Laminaria sp. Jellies Macrocystis sp. Meats Sargassum sp. Pastry Durvillea sp. Ice cream Ecklonia sp. Textile and paper industries Biotechnology industry (slow-release of medicines and drugs) Carrageenan emulsifier Chondrus crispus 70% of the applications are for meat products and the other 30% are for dairy products. Toothpaste, hand creams, paints, inks and cosmetics Source: (Campo et al., 2009, Jimenez-Escrig et al., 2011). 16

2.3.2. Seaweed in agriculture

Apart from human food, seaweed is also used to produce seaweed meal as a feed additive for many farm and pet animals, including goats, cattle, horses, dogs and cats, in many different countries such as Australia, Canada, America and United Kingdom (Stengel et al., 2011). Ascophyllum nodosum is the main raw material for seaweed meal, but Schizochytrium sp., Laminaria sp. and Durvillea sp. are also used. Seaweed meal is believed to be a good additive of animal feed as it contains some nutrients, such as certain vitamins and minerals (e.g., iodine, vitamin B12 and vitamin E) which are poor in land plants. Research shows that some components of seaweed, such as polysaccharides and oligosaccharides, can act as prebiotic substances to beneficial bacteria in animal guts, which, in turn, can help improve the immune system and provide other probiotic benefits to the host (O’Sullivan et al., 2010).

In marine environment, seaweeds help maintain the ecology by providing food resources for fish stock in the surrounding area. In aquaculture, seaweeds are used to manufacture fodder for shrimp, abalone, juvenile crabs and fishes (McHugh, 2003). The red seaweeds Hypnea spinella, Hypnea musciformis and Gracilaria cornea, for example, have been used as a feed for abalone (Aitken and Senn, 1965).

In agriculture, macroalgae are traditionally used as a nutrient-rich fertiliser as they are rich in several major fertiliser elements including nitrogen and potassium, while relatively low in phosphorus compared with traditional animal manures (Aitken and Senn, 1965). They also have the advantage of high growth rates and being environmental friendly. Additionally, they can increase the water-holding capacity of soil and are rich sources of some of the microelements (e.g., iron, chlorine, manganese, zinc, copper and boron) required for the plant growth. These properties make macroalgae a good source of raw material for producing useful organic fertilisers and commercial manufacture of seaweed fertilisers is well established around the world (McHugh, 2003). For example, seaweeds like Sargassum sp. and Gracilaria sp. are used as manure for coconut plantation in India. Moreover, seaweed species such as Ascophyllum nodosum, Ecklonia Laminaria and Fucus spp. are used to produce seaweed meal which is sold commercially as soil additives that function as both a fertiliser and a soil conditioner (Yuan et al., 2005b).

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2.3.3. Seaweed in environmental management 2.3.3.1. Wastewater treatment

Macroalgae can potentially be used in wastewater treatment as an efficient and biodegradable sorbent biomaterial for removing pollutants such as toxic and heavy metal ions from industrial wastewater. Research has shown that Catenella repens and Ulva spp. have unique absorbing properties for Zn(II), Cu(II), Fe(II), and Cd(II) etc. in aquatic medium (Cha et al., 2008). Macroalgal species such as Ecklonia maxima, Lessonia flavicans and Durvillaea potatorum are found to absorb Cu(II), Ni(II), Pb(II), Zn(II) and Cd(II) from wastewater (McHugh, 2003). In addition, Chara sp. is reported to have the potential for biological treatment of industrial dye wastes, such as Malachite Green which is a cationic dye in the textile industry and is a major pollutant in many developing countries where textile is a major industry (Khataee et al., 2010).

Moreover, macroalgae may be used as bio-filters for improving water quality due to their ability to reduce nitrogen and phosphorus containing compounds in aquaculture, agricultural and domestic wastewaters before they are released into the ocean or rivers (Paul and de Nys, 2008, Castine et al., 2013). A number of studies have investigated G. caudata and A. franciscana for this purpose and they are found to perform well in absorbing nitrogen-containing compounds, such as ammonium, and use them as source of nitrogen for their growth (Marinho-Soriano et al., 2011).

2.3.3.2. Energy and bio-fuels

Macroalgae are considered as a promising source of biofuel due to their fast rate of growth, good environmental and economic feasibility and, crucially, their low impact on food security. Currently, the major raw materials used for commercial biofuel production are all food stocks such as corn, potato, sugar cane and edible oils etc., which have a direct impact on the world's food supply. The issue is that there is unlikely a sufficient supply of these food stocks to meet the rising energy demands without a significant negative effect on the food security of the growing population of the world. In addition, crop farming uses significant amounts of pesticides and nitrogen fertilisers, which in turn can cause degradation of arable land and the environment. Therefore, macroalgae are emerging as a promising alternative source for biofuel. Apart from the

18 very fast cultivation cycle (in tropical climates, they can be harvested 4-6 times annually), they also have the advantages of not requiring any costly resources (e.g., freshwater, fertilisers etc.) to grow and their impact on the environment is much less and, sometimes, can even be beneficial. However, a great amount of research and changes in the market value of oil is needed to realise the potential of algae in biofuel production. More imminent applications of macroalgae should focus on bioactives, specifically those that relate to human and animal health as the potential areas of most valuable application.

2.3.4. Seaweed as bioactive and functional products

Macroalgae contain many biologically active compounds which have been reported to provide potential health beneficial properties such as antioxidant, anticancer, anti- diabetic, anti-hypertension, antimicrobial and anti-inflammatory effects (Mendis and Kim, 2011). In addition to these more “traditional” health functionalities, more recent studies have also explored other health potentials of algal bioactive components. For example, the macroalgal species Agardhiella subulata is reported to be potentially useful in the treatment of the human immunodeficiency virus (HIV) and Undaria pinnatifida may have an effect in preventing breast cancer (Mendis and Kim, 2011). Palmaria palmata contains kainoid amino acid, which may be useful in the treatment of Alzheimer’s and Parkinson’s disease and epilepsy. Alginates from Sargassum sp. and Turbinaria sp. have been studied as controlled release agent to prolong the period of activity for certain drugs. Carrageenans from Hypnea sp. and Acanthophora sp. have been trialled in ulcer therapy. Given the vast number and diversity of algal species, there is a strong interest to discover more biological compounds in different macroalgal species with nutritional and health functional properties. As this is the focus of this thesis, it will be discussed further in a separate section (section 2.4).

2.4. Major bioactive components in algae and their health-related biological activities

Macroalgae have a long history of use as food in Asia and lately have become popular in many western countries as well. Many of these seaweeds are consumed not only for 19 their special taste, texture and other sensory properties, but also for their perceived nutritional and health benefits. In recent decades, the micronutrients and bioactive components in macroalgae have attracted considerable amount of research attention, and many bioactive compounds have been isolated and identified from them. Macroalgae are found to contain a diverse array of biologically active compounds including proteins, fatty acids, polysaccharides, polyphenols, carotenoids, minerals and vitamins (Mendis and Kim, 2011). A number of studies have investigated the potential health-promoting properties of these compounds in pure form or in mixtures with other components of macroalgae in the form of extracts. These studies have reported that extracts or components of macroalgae possess a wide range of biological properties including antioxidant, antitumor, anticancer, anti-diabetic and hypocholesterolemic activities (Rajapakse and Kim, 2011). In addition, a number of brown algae contain a high level of polysaccharides, which are dietary fibre and can promote intestinal health as well as provide a number of other health functions that dietary fibre is known to confer (Wijesinghe and Jeon, 2012). Therefore, macroalgae have a great potential to be developed into bioactive ingredients for application in functional food, nutraceutical and pharmaceutical products.

2.4.1. Phytochemicals in macroalgae

Phytochemicals are biologically active, non-nutritive secondary metabolites naturally synthesised in plants grown both on land and in water (Bellik et al., 2012). They are largely responsible for the huge diversity of colors in the plant kingdom. Many phytochemicals are toxic to insects and microorganisms and form a crucial part of the defense mechanism of plants against pests and diseases caused by fungi, bacteria and viruses (Johnson and Williamson, 2003). Plants consist of a diverse range of phytochemicals which are generally classified into five main groups: phenolic compounds, carotenoids, alkaloids, nitrogen-containing compounds and organosulfur compounds (Bellik et al., 2012). In recent decades, the bioactivities and health benefits of phytochemicals have been extensively studied, especially those in fruit, vegetables and medicinal plants. Phytochemicals have been found to provide a number of significant health benefits, particularly in relation to the alleviation and prevention of many of the chronic disorders prevalent in industrialized societies, including 20 cardiovascular disease, stroke, obesity, cancer, Alzheimer disease, cataracts, chronic inflammation, allergy and skin lightening (Nadpal et al., 2016, Ikram et al., 2015, Zhu et al., 2015).

2.4.2. Carotenoids

Carotenoids are a major class of phytochemicals and also one of the most important groups of natural pigments, because of their widespread distribution, structural diversity and numerous functions. They are lipid-soluble pigments responsible for the beautiful colours of many flowers, fruits, vegetables as well as birds, insects and animals. Chemically, carotenoids can be divided into two groups: the first group is carotenes which are non-oxygenated molecules such as α-carotene, β-carotene and lycopene, and the second group is xanthophylls, molecules containing oxygen, with examples such as lutein and zeaxanthin. To date, there are more than 700 carotenoids that have been isolated from plants, animals, fungi, and microorganisms (Johnson and Williamson, 2003, Saini et al., 2015). Table 5 lists the major carotenoids that have been identified in algae.

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Table 5. Chemical structure of major carotenoids compounds identified in algae

Carotenoids Chemical structures

α-carotene

β-carotene

Astaxanthin

Lutein

Zeaxanthin

Fucoxanthin

Siphonaxanthin

Violaxanthin

Neoxanthin

Loroxanthin

Source: (Oliver and Palou, 2000, Raposo et al., 2015).

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2.4.2.1. Major algal carotenoids and their biological function activities

Numerous observational studies have found that increased intake of carotenoids rich food, such as fruits and vegetables, have a protective effect against several chronic human diseases (Pangestuti and Kim, 2011, Fernandez-Garcia et al., 2012). Carotenoids can act as potent free radical quenchers, singlet oxygen scavengers, and lipid antioxidants (Rao et al., 2006, Murthy et al., 2005, Raposo et al., 2015). Carotenoids have also been successfully used for many years in the treatment of individuals suffering from photosensitivity disease, such as erythropoietic protoporphyria (Mathews-Roth, 1993). Algae are a good source of carotenoids as they have been shown to contain several major carotenoids, including lutein, zeaxanthin, violaxanthin, neoxanthin, α-carotene and β-carotene during their normal growth phase (Inbaraj et al., 2006). Significantly, they also contain very high amounts of secondary carotenoids, such as astaxanthin in red algae, siphonaxanthin in green algae and fucoxanthin in brown algae, which are only found in a limited number of foods (Peng et al., 2011, Ambati et al., 2014, Sugawara et al., 2014). Table 6 shows the major carotenoids identified in various algal species and their biological functions.

2.4.2.2. Fucoxanthin

Fucoxanthin is one of the major carotenoids found mainly in marine brown macroalgae (Saccharina japonica, Undaria pinnatifida, Sargassum siliquastrum and Myagropsis myagroides) and some microalgae (Phaeodactylum tricornutum) (Yu et al., 2011, Heo and Jeon, 2009, Das et al., 2010, Kim et al., 2012). The chemical structure of fucoxanthin consists of an allenic bond and oxygenic functional groups, including hydroxyl, epoxy, carbonyl and acetyl groups, in addition to its polyene chain (structure shown above in Table 5). Fucoxanthin has been reported to exhibit several potential beneficial effects related to its anti-cancerous (Kotake-Nara et al., 2005), anti-oxidative (Sasaki et al., 2008, Yan et al., 1999, Ayyad et al., 2011), anti-inflammatory and anti- obesity (Maeda et al., 2005, Maeda et al., 2007a, Maeda et al., 2007b) properties. Moreover, fucoxanthin induces uncoupling protein (CUP1) in the mitochondria of abdominal white adipose tissue, leading to the oxidation of fatty acids (Gammone and D’Orazio, 2015). On the other hand, it is found that fucoxanthin and its deacetylated product, fucoxanthinol, could effectively suppress angiogenesis, implying that 23 fucoxanthin would be useful in preventing angiogenesis-related diseases such as cancer and diabetic retinopathy (Sugawara et al., 2006).

2.4.2.3. Siphonaxanthin

Siphonaxanthin is a specific keto-carotenoid found mainly in green algae, which helps the organisms absorbing available green and blue green light in underwater conditions (Akimoto et al., 2007, Ganesan et al., 2010). Codium fragile, Caulerpa lentillifera and Umbraulva japonica have been reported to contain this compound (Ganesan et al., 2011, Li et al., 2015). Its biological properties are not yet well understood, but some studies indicated that it can potently inhibit the viability of human leukemia HL-60 cells via induction of apoptosis (Ganesan et al., 2011). Siphonaxanthin has also been found to play a role in preventing angiogenesis related diseases and exhibited strong inhibitory effect on human umbilical vein endothelial cells (Ganesan et al., 2010). A recent study has reported that compared with other carotenoids, siphonaxanthin has much greater potence in inhibiting adipocyte differentiation because it can effectively regulates adipogenesis in 3T3-L1 cells (Li et al., 2015). Oral administration of siphonaxanthin to diabetic KK-Ay mice significantly reduced the total weight of white adipose tissue (Li et al., 2015).

2.4.2.4. Astaxanthin

The use of astaxanthin as a nutritional supplement has been rapidly growing in functional foods, feeds, nutraceuticals and pharmaceuticals (Ambati et al., 2014). Astaxanthin is a xanthophyll carotenoid, found in Haematococcus pluvialis, Dunaliella salina, Chlorococcum humicola, and Botryococcus braunii. The chemical structure of astaxanthin consists of two terminal rings joined by a polyene chain. This molecule has two asymmetric carbons located at the 3, 3’ positions of the β-ionone ring with hydroxyl group (-OH) on either end of the molecule. The biological properties and potential health effects of astaxanthin has been studied extensively, and the consumption of astaxanthin has been reported to prevent or reduce the risk of several disorders in humans and animals (Yamashita, 2015, Ambati et al., 2014). Astaxanthin has been found to exhibit very strong antioxidant activities, being about 10 times higher

24 than those of lutein, zeaxanthin, canthaxanthin, and β-carotene and 100 times greater than that of α-tocopherol (Rao et al., 2010, Rao et al., 2013b, Rao et al., 2013a, Miki, 1991). Apart from its strong antioxidant activity, this carotenoid is also reported to exhibit a number of potential health benefitting effects, including anti-inflammation (Park et al., 2013), anti-diabetic (Chan et al., 2012, Dong et al., 2013), cardiovascular disease preventing (Nakao et al., 2010) and anticancer (Rao et al., 2013b, Huangfu et al., 2013, Maoka et al., 2012) effects. Some clinical studies have shown that astaxanthin protects human dermal fibroblasts from UV induced damage, resulting in visual wrinkle reduction and elasticity enhancement and improvement in age spot, skin texture and coenocyte condition (Tominaga et al., 2012). Astaxanthin is also reported to enhance lipid metabolism resulting in a reduction in fat accumulation (Ikeuchi et al., 2007).

2.4.2.5. Other carotenoids in algae

Apart from the above discussed carotenoids, algae also contain a number of other carotenoids, including β-carotene, lutein and zeaxanthin. These carotenoids, however, are also found in numerous other sources, especially fruit and vegetables, and their health functions are well studied and documented. For example, lutein and its coexistent isomer, zeaxanthin, are the primary pigments for the yellow colour of plants and their health benefits are well reviewed (Hahn and Mang, 2008, Koushan et al., 2013, Sarmadi and Ismail, 2010, Ribaya-Mercado and Blumberg, 2004, Abdel-Aal et al., 2013, Roberts et al., 2009). Lutein is believed to play a vital role in the maintenance of normal visual function of the human macula as well as having a number of other biological properties, such as reducing the risk of certain types of cancer, particularly breast and lung cancers and potential prevention of heart disease and stroke. β-Carotene is a well- known strong antioxidant and is believed to help mediate the harmful effects of free radicals implicated in various disorders such as many forms of gastrointestinal cancer (Stahl et al., 1998, Stahl and Sies, 2005, El Baz et al., 2002). The biological functions and health effects of β-carotene have been well reviewed (Goralczyk, 2009, Kasperczyk et al., 2014, Stahl and Sies, 2005).

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Table 6. Natural pigments identified in algae species and their biological functions

Natural Health benefit Algae References pigments effects β-carotene Anti-mutagenic Porphyra tenera (Okai et al., 1996) Chlorococcum humicola (Bhagavathy et al., 2011) Skin protection Dunaliella salina (Stahl et al., 1998) Antioxidant Dunaliella salina (El Baz et al., 2002) α-carotene Antioxidant Dunaliella salina (Hu et al., 2008) Astaxanthin Antioxidant Haematococcus pluvialis, (Rao et al., 2010, Rao et al., 2013a, Botryococcus braunii Rao et al., 2013b, Regnier et al., Dunaliella salina 2015) Anti-mutagenic Chlorococcum humicola (Bhagavathy et al., 2011) Anti-skin cancer Haematococcus pluvialis (Rao et al., 2013b, Huangfu et al., 2013, Maoka et al., 2012) Anti-inflammation - (Park et al., 2013) Anti-diabetic - (Chan et al., 2012, Dong et al., 2013) Cardiovascular disease - (Nakao et al., 2010) prevention Fucoxanthin Antioxidant Hijikia fusiforme, (Sasaki et al., 2008, Yan et al., 1999, Undaria pinnatifida, Ayyad et al., 2011) Fucus serratus, Padina tetrastromatic, Sargassum elegans Anti-cancer Undaria pinnatifida (Kotake-Nara et al., 2005) Anti-tumor Undaria pinnatifida (Yu et al., 2011) Anti-inflammatory Myagropsis myagroides (Heo et al., 2010) Anti-obesity Undaria pinnatifida (Maeda et al., 2005, Maeda et al., 2007a, Maeda et al., 2007b) Anti-angiogenic Undaria pinnatifida (Sugawara et al., 2006) Neuroprotective Hijikia fusiformis (Okuzumi et al., 1990) Prevent osteoporosis Laminaria japonica (Das et al., 2010) Photoprotective Laminaria japonica Sargassum siliquastrum (Heo and Jeon 2009) Siphonaxanthin Anticancer Codium fragile (Ganesan et al., 2011) Anti-angiogenic Codium fragile (Ganesan et al., 2010, Li et al., 2015) Apoptosis-inducing Codium fragile (Ganesan et al., 2011, Li et al., 2015) activities -----anti- cancer Anti-degranulation Codium fragile (Manabe et al., 2014) Lutein Antimutagenic Porphyra tenera (Okai et al., 1996) Chlorococcum humicola (Bhagavathy et al., 2011) Antioxidant Dunaliella salina (Hu et al., 2008) Eye Health - (Abdel-Aal et al., 2013) zeaxanthin Antioxidant Dunaliella salina (Hu et al., 2008) Eye Health - (Abdel-Aal et al., 2013)

2.4.3. Phenolic compounds

Phenolic compounds are a huge and diverse group of phytochemicals that are synthesised by all plants as secondary metabolites (Dai and Mumper, 2010). They are an important group of natural antioxidants associated with several significant health functions such as preventing heart disease, lessening inflammation, lowering the incidence of cancer and diabetes, as well as reducing the rate of mutagenesis in human 26 cells (Khoddami et al., 2013). Chemically, all phenolic compounds share a common structural feature: an aromatic ring bearing at least one hydroxyl (-OH) substituent, i.e. a phenol (Stalikas, 2007). Approximately 8,000 naturally occurring phenolic compounds have been identified. They are widespread constituents of plant foods such as fruits, vegetables, herbs, cereals and chocolate as well as beverages such as tea, coffee, bear and wine (Dai and Mumper, 2010) and often play a crucial role in the sensory qualities of the products. For example, tannins from black tea tend to have a bitter taste and astringent properties, while tea flavonoids including flavonols, flavones, isoflavones and anthocyanins contribute to the colour of the tea as well as its taste. Plant phenolic compounds are classified into simple phenols and polyphenols, based exclusively on the number of phenol subunits present (Khoddami et al., 2013). Thus, plant phenolics encompass simple phenols, phenolic acids, flavonoids, coumarins, stilbenes, hydrolysable and condensed tannins, lignans and lignins.

Algal polyphenols are mostly derived from polymerised phloroglucinol units (Tierney et al., 2013). Green and red seaweeds have a low concentration of phenolic components compared to brown seaweed species, which have a high concentration of the phenolic compound group phlorotannin. Phenolic content of marine macroalgae varies from <1% to 14% of dry seaweed biomass.

Flavonoids are some of the most common phenolics, widely distributed in plant tissues, and often responsible for the orange, red, blue and purple colours of many fruits and vegetables such as apples, berries and onions. Almost all flavonoids exhibit a distinctive triple-ring structure as illustrated on Figure 3. Flavonoids are divided into six subgroups: flavones, flavonols, flavanols, flavanones, isoflavones and anthocyanins (Khoddami et al., 2013). Some of the most common flavonoids include catechin from tea, quercetin from onion and anthocyanin from various berries and plums. The functional significance of common flavonoids has been reviewed (Agati et al., 2012, Yao et al., 2004). Phenolic compounds (catechol, rutin and hesperidin) were identified in the crude extract of Porphyra dentate (an edible red seaweed), and the extract was found to inhibit the production of nitric oxide in lipopolysaccharides-stimulated RAW 264.7 cells. Catechol was a more potent suppressor of the up-regulation of iNOS promoter and nuclear factor-kappaB enhancer than rutin while hesperidin failed to inhibit either activity (Kazlowska et al., 2010).

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Figure 3. Basic structures of flavonoids.

Phenolic acids can be divided into two classes: hydroxycinnamic acids such as ferulic, caffeic, p-coumaric and sinapic acids, and hydroxybenzoic acids such as gallic, vanillic, syringic and protocatechuic acids (Figure 4). Caffeic acid is the most abundant phenolic acid in many fruits and vegetables, frequently esterified with quinic acid as in chlorogenic acid, which is the major phenolic compound in coffee. Another common phenolic acid is ferulic acid, which is present in cereals and is esterified to non-starch polysaccharides such as pentosans and hemicelluloses in the cell wall. Several recent reviews are available on the occurrence, chemical characterisation and bioactivities of phenolic acids in foods (Khoddami et al., 2013, Dai and Mumper, 2010). Phenolic acids were found in two freshwater algae (Anabaena doliolum and Spongiochloris spongiosa) and two marine macroalgae Porphyra tenera (nori) and Undaria pinnatifida (wakame), which showed moderate levels of antioxidant activities (Onofrejova et al., 2010). Phenolic acids identified in the algae were protocatechuic, p-hydroxybenzoic, 2,3- dihydroxybenzoic, chlorogenic, vanillic, caffeic, p-coumaric, salicylic, and cinnamic acids. Hydroxybenzaldehydes (p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde) and vanillin were also identified.

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Ferulic acid Gallic acid

Caffeic acid Vanillic acid

p-coumaric acid Syringic acid

Sinapinic acid Protocatechuic acid

Figure 4. Chemical structure of main phenolic acids (Stalika, 2007).

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Phlorotannins are a group of phenolic compounds produced by plant secondary metabolism in many Phaeophyceae (brown algae) (Kim and Himaya, 2011, Li et al., 2011), which are not found in terrestrial plants. They constitute an extremely heterogeneous group of molecules in structure and the degree of polymerisation providing a wide range of potential biological activities, such as antioxidant (Sathya et al., 2013, Shin et al., 2014), antimicrobial (Eom et al., 2012), and anti-diabetic (Lee and Jeon, 2013) activities. Two recent reviews have examined and categorised the reports on phlorotannins that have shown strong bioactivities (Li et al., 2011, Kim and Himaya, 2011). In addition, the potential anti-proliferative activities of this group of phenolic compounds have been highlighted in other studies (Macias-Sanchez et al., 2007, Zubia et al., 2009, Nwosu et al., 2011). Moreover, phlorotannin from Ecklonia cava has been shown to enhance the inhibitory effect of cisplatin against tumour growth and ameliorated cisplatin-induced nephrotoxicity in vivo (Yang et al., 2015).

2.4.4. Protein, peptides and amino acid

Protein is a vital nutrient required for the normal growth and maintenance of both human and animals (Cerna, 2011). Edible macroalgae provide a significant amount of protein, which ranges from 5% to 47%, DW, depending on species, growth conditions, season and the methods used for determination (Cerna, 2011, Dawczynski et al., 2007). Red algae contain the greatest amount of protein with values reaching up to 47%, DW (Galland-Irmouli et al., 1999); green seaweeds contain moderate amounts (9-30%, DW) (Ramos et al., 2000), while brown algae have much lower amounts of protein (3-15%, DW) (Dawczynski et al., 2007, Cerna, 2011, Rajapakse and Kim, 2011). The protein levels of red seaweeds are comparable to those found in high-protein food sources such as soybeans (about 35%) (Liu, 2004, Taylor et al., 2015). The protein levels reported for individual seaweed species are summarised in Table 7. In some red seaweeds, such as Palmaria palmata (dulse) and Porphyra tenera (nori), protein levels can be as high as 47.5% DW (Galland-Irmouli et al., 1999). The green algae Ulva spp. are also relatively rich in protein, containing up to 29% of this nutrient in their dry biomass (Yaich et al., 2011, Fleurence, 1999).

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Table 7. Protein levels of selected individual seaweed species

Species Protein (% DW) References

Chlorophyta (Green) Caulerpa veravelensis 7.77 (Kumar et al., 2011) Caulerpa scalpelliformis 10.50 (Kumar et al., 2011) Caulerpa racemosa 12.8-18.4 (Pangestuti and Kim, 2015) Caulerpa lentillifera 10-13 (Pangestuti and Kim, 2015) Ulva sp. 10-26 (Yaich et al., 2011, Fleurence, 1999) Ulva lactuca 7.06-29 (Yaich et al., 2011, Wong and Cheung, 2000) Ulva rigida 18-19 (Pangestuti and Kim, 2015) Rhodophyta (Red) Pyropia columbina 30 (Cian et al., 2012) Palmaria palmata (Dulse) 13.5-21.9 (Galland-Irmouli et al., 1999) Porphyra tenera (Nori) 47.5 (Galland-Irmouli et al., 1999) Kappaphycus alvarezii (Doty) 18.16 (Kumar et al., 2014) Phaeophyceae (Brown) Laminaria japonica 13.1 (Zhou et al., 2015) Alaria esculenta 9-20 (Pangestuti and Kim, 2015) Undaria pinnatifida 12-23 (Pangestuti and Kim, 2015) Fucus spiralis 10.77 (Patarra et al., 2011) Hypnea charoides 19.0 (Wong and Cheung, 2000)

Among seaweed proteins, there are two functionally active members, lectins and phycobiliproteins, which are not found in terrestrial plants (Pangestuti and Kim, 2015, Lange et al., 2015). Lectins are glycoproteins, proteins which bind with carbohydrates and participate in many biological processes such as intercellular communication (Pangestuti and Kim, 2015). They have been successfully isolated from a number of seaweeds including the green species Caulerpa cupressoides (da Conceicao Rivanor et al., 2014, Vanderlei et al., 2010), Bryopsis hypnoides (Niu et al., 2009) and Ulva pertusa (Wang et al., 2004) and the red seaweeds Kappaphycus striatum (Hung et al., 2011), Gracilaria ornata (Leite et al., 2005) and Amansia multifida (Neves et al., 2007). This class of protein has been reported to have a number of potential health related functions including anti-nociceptive (da Conceicao Rivanor et al., 2014, Leite et al., 2005, Hung et al., 2011, Vanderlei et al., 2010, Neves et al., 2007, Wang et al., 2004),

31 anti-inflammatory (da Conceicao Rivanor et al., 2014, Vanderlei et al., 2010), anti-HIV (Singh et al., 2015, Sato et al., 2011), antiviral (Barrientos et al., 2003, Bewley et al., 2004), anti-cancer (Sugahara et al., 2001), and anti-tumour (Omokawa et al., 2010) properties.

Phycobiliproteins, also known as phycocyanins and allophycocyanins, are a family of reasonably stable and highly soluble fluorescent proteins found in red seaweeds. They have long been used as natural food colouring in products such as chewing gums, dairy products, ice sherbets and jellies. In recent years, these proteins have also been reported to have health related functional effects including antioxidant, anti-inflammatory, liver protecting, antiviral, antitumor and anti-atherosclerosis activities, as well as the capacity to inhibit pancreatic lipase activity, thus reducing lipid absorption, and to obstruct the absorption of environmental pollutants into the body (Sekar and Chandramohan, 2008).

Although the structure and biological properties of algal proteins are still relatively poorly documented, the amino acid composition of several species of algae has been published (Table 8). In general, most algae proteins contain all of the essential amino acids. Barbarino and Lourenco (2005), Pangestuti and Kim (2015), and Beaulieu et al. (2015) studied the amino acid profiles of 21 species of marine algae. They, as well as previous researchers, found that aspartic acid and glutamic acid are the most abundant amino acids in most seaweed species (Angell et al., 2015). Interestingly, these two amino acids are known to have significant flavour characteristics, with glutamic acids being a main component that elicits the taste sensation of “umami” (Pangestuti and Kim, 2015). This is undoubtedly one of the major reasons why some seaweed species are often used as a condiment in Asian cooking. However, compared with other protein rich food sources, seaweed is deficient in tryptophan and cysteine. With respect to the amino acid composition, the essential amino acid indices of red seaweeds were higher than those of brown and green seaweeds (Pangestuti and Kim, 2015).

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Table 8. Amino acid profile of seaweed species (mg/100mg of algae protein)

Green seaweed Red seaweed Brown seaweed

Amino Acid Caulerpa Caulerpa Ulva Ulva Codium Palmaria Porphyra Pyropia Pterocladiella Sargassum Saccharina racemosa lentillifera fasciata reticulata decoriticatu palmata acanthophora columbina capillacea vulgare longicruris m

Essential amino acid Threonine 5.7 6.4 5.1 5.4 6 3.6 5.8 7.7 5.2 4.8 1.72 Valine 5.7 0.9 5.7 6.3 6.2 6.9 6.4 2.6 5.5 5.8 1.66 Methionine 1 – 0.9 – 0.7 2.7 1.1 0.2 1.1 2.2 0.35 Isoleucine 4.1 5 3.9 4.2 3.8 3.7 4.1 1.1 3.7 4.8 1.08 Leucine 8.3 1 7.6 7.9 8.4 7.1 8.1 3.5 6.8 8.5 1.75 Phenylalanine 5.4 4.9 5.1 5.3 5 5.1 4.7 0.8 5.3 5.3 0.99 Histidine 2.9 0.7 2.4 1.1 3.3 0.5 3 9.7 3.5 2.1 0.55 Lysine 6.5 6.6 5.1 6 6.3 3.3 6.3 4.1 7.9 5.4 1.11 Non-Essential amino acid Aspartic acid 9.9 11.6 13 12.5 10.7 18.5 12.5 15.5 11.6 10.9 4.29 Serine 5.4 0.8 5.8 6.4 5 6.3 5.3 2.5 5.7 5.1 1.52 Glutamic acid 14.6 14.4 12.6 12.1 12 9.9 12.9 17.6 14.7 17.6 3.48 Proline 4.6 0.6 4.6 5.1 4.8 1.8 4.6 8.6 4.9 4.6 1.34 Glycine 6.8 0.9 6.5 6.5 7.2 13.3 7.1 2.6 6 5.7 2.63 Alanine 6.5 0.9 8.5 8.1 8.8 6.7 8.8 15.3 7.2 7.2 3.24 Tyrosine 2.6 3.9 3.3 3.6 2.1 3.4 2.4 1.2 3.7 2.2 0.47 Arginine 5.1 7 5.6 8.7 5.1 5.1 4.8 4.7 5.6 4.3 0.53 References (Lourenco (Arporn (Louren (Arporn (Barbarino (Galland- (Barbarino (Cian et al., (Barbarino (Lourenco (Beaulieu et al., and co et and and Irmouli et and 2015) and et al., 2002) et al., 2015) 2002) Chirapart, al., Chirapart, Lourenco, al., 1999) Lourenco, Lourenco, 2006) 2002) 2006) 2005) 2005) 2005)

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2.4.4.1. Bioactive peptides

Apart from being a vital nutrient required for normal growth and maintenance of both human and animals, food proteins also contain sequences of bioactive peptides that can exert a physiological effect in the body. Bioactive peptides usually contain about 3-40 amino acid residues, and their activities are based on the amino acid composition and sequence (Aneiros and Garateix, 2004). These short chains of amino acids are inactive within the sequence of the parent protein, but can be released during gastrointestinal digestion, food processing or fermentation (Cian et al., 2015). Many of these bioactive peptides have been shown to possess significant biological activities that are beneficial to human health (Table 9). Bioactive peptides may come from three sources: 1) peptides naturally present in foods, including those produced naturally in fermented foods; 2) peptides produced by enzymatic hydrolysis of food proteins; and 3) peptides produced during the digestive process in the gastrointestinal tract. Because it is rather difficult to isolate peptides from the digestive tract, in vitro simulated digestion is often used as a substitute, where proteins are subjected to hydrolysis by single or multiple proteases under conditions mimicking those of the human digestive tract.

Of the major food protein sources, milk and milk products are the first and most extensively studied source of bioactive peptides. Milk contains approximately 3.5% protein, of which 80% is casein and 20% whey proteins (Mohanty et al., 2015). Casein is heat stable and easy to digest, and its hydrolysates have been found to exhibit a number of biological activities, including antioxidant, angiotensin I-converting enzyme (ACE) inhibitory and opioid activities (Suetsuna et al., 2000, Hernandez-Ledesma et al., 2004b, Jarmolowska et al., 2007). Many bioactive peptides are also found in, or produced from, other animal and plant protein sources, including egg, fish, oyster, cereal (rice, wheat, soybeans, buckwheat, barley and corn) and soybean, and the subject has been well reviewed (Malaguti et al., 2014, Sarmadi and Ismail, 2010, Garcia et al., 2013, Ngo et al., 2012, Singh et al., 2014, Nongonierma and FitzGerald, 2015, Mohanty et al., 2015).

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Table 9. Biologically active protein and peptides in naturally occurring in food

Source Bioactive peptides Bioactivities References Milk Casein Antioxidant (Suetsuna et al., 2000) Antihypertensive (Hernandez-Ledesma et al., 2004a, Hernandez-Ledesma et al., 2007) Opioid (Jarmolowska et al., 2007) Lactoferrin Antimicrobial, Antibacterial (Jenssen and Hancock, 2009) Lycozyme Antimicrobial (Benkerroum, 2008) lactoperoxidase Antibacterial (Bjorck et al., 1975) Egg Ovotransferrin Binds iron (Giansanti et al., 2012) Ovoinhibitor Serine proteinase inhibitor (Bourin et al., 2011) Ovomucin Antioxidant, (Chang et al., 2013) Antihypertensive Antiviral, Antitumor (Omana et al., 2010) Ovalbumin Antihypertensive (Miguel et al., 2006, Fujita et al., 2000) Cystatin Cysteine proteinase (Wesierska et al., 2005) inhibitor, Antimicrobial Meat Glutathione Antihypertensive (Remond, 2008) Carnosine Antioxidant (Decker et al., 2001) Rice Albumin Ileum contracting (Takahashi et al., 1996) Immunostimulation Antioxidant Soybean Glycinin Antioxidant (Singh et al., 2014) Conglycinin Antihypertensive (Shin et al., 2001) Lunasin Anticancer (Kim et al., 2000)

2.4.4.2. Bioactivities of protein hydrolysates and peptides from macroalgae

The possible roles of seaweed-derived bioactive peptides in reducing the risk of diseases have been reported. Several studies have investigated bioactive peptides naturally present in algae biomass. For example, Suetsuna et al. (2004) examined the ACE inhibitory activity of a hot water extract of Undaria pinnatifida (wakame). Ten naturally present dipeptides were isolated from the extract by multiple steps of chromatography. The amino acid sequences of the peptides were identified to be Tyr- His, Lys-Trp, Lys-Tyr, Lys-Phe, Phe-Tyr, Val-Trp, Val-Phe, Ile-Tyr, Ile-Trp, and Val- Tyr. Both single and repeated oral administration of synthetic Tyr-His, Lys-Tyr, Phe- Tyr and Ile-Tyr were found to significantly decrease blood pressure in spontaneously hypertensive rats. 35

However, the vast majority of algal bioactive peptides are obtained by controlled hydrolysis of algal proteins using various types of protease preparations. Table 10 summarises seaweed bioactive peptides obtained by such methods. These peptides exhibit several major biological activities significant to human health, including antioxidant (Karawita et al., 2007, Kim et al., 2006b), anti-diabetic, anti-microbial, anti- hypertension (Jimsheena and Gowda, 2011), anti-human immunodeficiency virus (anti- HIV), antithrombotic, antiviral and mineral binding activities (Mendis and Kim, 2011). These peptides are inactive in the amino acid sequence of the parental protein but become active upon release through the enzymatic digestion.

A number of researchers have investigated hydrolysates and peptides produced by hydrolysis of algal proteins with commercial proteases. Ko et al. (2012) hydrolysed protein from the marine algal species Chlorella ellipsoidea using several commercial protease preparations, namely Protamax, Kojizyme, Neutrase, Flavourzyme and Alcalase. Alcalase hydrolysate exhibited the highest ACE inhibitory activity and a potent ACE inhibiting tetrapeptide, Val-Glu-Gly-Tyr (IC50 128.4 μM) was isolated from the extract. Qu et al. (2010) produced hydrolysates from Porphyra yezoensis protein using five commercial enzymes (Alcalase, Neutrase, Flavourzyme, Protamax, and papain) and examined their ACE-inhibitory activities. Alcalase was found to be the most effective for hydrolysis and the hydrolysate of glutelin produced by this protease exhibited the highest antihypertensive activity (IC50 1.6g/L). Harnedy et al. (2014) has investigated the antioxidant, ACE inhibitory and Dipeptidy peptidase-IV (DPP-IV, an enzyme linked with glycemic regulation) inhibitory activities of protein hydrolysates of Palmaria palmata harvested at different seasons. The hydrolysates were prepared with a mixture of Alcalase and Coralase, and the results show that the hydrolysates inhibited

DPP-IV (IC50: 1.60-4.24 mg/mL) and ACE (IC50: 0.14-0.35 mg/mL) with considerable potency. In a follow up study, the group subjected an aqueous Palmaria palmata protein extract to hydrolysis with Corolase. Thirteen peptides were identified from the hydrolysates and three of them, Ile-Leu-Ala-Pro, Leu-Leu-Ala-Pro and Met-Ala-Gly-

Val-Asp-His, were novel DPP-IV inhibitory peptides with IC50 values in the range 43- 159 µM (Harnedy et al., 2015). Cian et al. (2015) investigated the ACE inhibitory and antioxidant activity of Pyropia columbina protein hydrolysates prepared with a mixture of an alkaline protease and Flavourzyme, and the hydrolysates exhibited a moderate level of ACE-inhibitory activity. 36

Other researchers have used physiological digestive enzymes to produce hydrolysates from algal proteins and studied the biological activities of the hydrolysates. Potent ACE-I inhibitory effect was observed from pepsin hydrolysates of Chlorella vulgaris, spirulina platensis and Undaria pinnatifida protein (Suetsuna and Nakano, 2000, Suetsuna and Chen, 2001). Several potent ACE-I inhibiting peptides were isolated from the hydrolysates, including Ile-Val-Val-Glu, Ala-Phe-Leu, Phe-Ala-Leu, Ala-Glu-Leu and Val-Val-Pro-Pro-Ala from C. vulgaris, Ile-Ala-Glu, Phe-Ala-Leu, Ala-Glu-Leu, Ile- Ala-Pro-Gly, and Val-Ala-Phe from S. platensis (Suetsuna and Chen, 2001), and Ala- Ile-Tyr-Lys, Tyr-Lys-Tyr-Tyr, Lys-Phe-Tyr-Gly, and Tyr-Asn-Lys-Leu from U. pinnatifida (Suetsuna and Nakano, 2000). Sheih et al. (2009a) isolated a peptide with the amino acid sequence of Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe, with a strong ACE inhibitory activity (IC50 29.6 μM) from the pepsin hydrolysate of algal protein waste of C. vulgaris obtained as an industrial by-product. In another study, they isolated an antioxidant peptide of VECYGPNRPQF from Chlorella vulgaris protein hydrolysed by pepsin with high efficiency in quenching a variety of free radicals, including hydroxyl radical, superoxide radical, peroxyl radical, DPPH radical and ABTS radicals (Sheih et al., 2009b). In a recent study, proteins from the macroalga Saccharina longicruris were extracted and hydrolysed with trypsin in order to recover antibacterial peptides (Beaulieu et al., 2015). The >10kDa protein hydrolysate fraction exhibited activity against the bacterium Staphylococcus aureus with a significant decrease in the maximum specific growth rate.

The in vitro digestion of algal proteins discussed above was carried out using single digestive enzymes. To date, there are very limited reports that have employed multiple enzyme systems to digest seaweed proteins, although the process is more comparable to the actual digestive environment in vivo. The in vitro simulated gastrointestinal digestion normally involves three different enzymes: a-amylase, pepsin and pancreatin, which mimic the protein digestion process from the mouth where α-amylase is secreted with saliva, through to the stomach (pepsin digestion under low pH), and finally, the lumen of the small intestine (pancreatin digestion) (Cerna, 2011). In this thesis, such in vitro simulated gastrointestinal digestion was used to hydrolyse algal proteins, followed by characterisation of the biological activities of the hydrolysates.

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Table 10. Bioactive peptides and their bioactivities from protein hydrolysates of marine algae

Peptide/Amino acid Product Enzyme Bioactivities Reference sequence Green algae Chlorella Pepsin ACE inhibition Ile-Val-Val-Glu (IC50, 315.3 (Suetsuna vulgaris µM), and Chen,

Ala-Phe-Leu (IC50 63.8 μM), 2001)

Phe-Ala-Leu (IC50 26.3 μM),

Ala-Glu-Leu (IC50 57.1 μM),

Val-Val-Pro-Pro-Ala (IC50 79.5 μM) Chlorella Pepsin Antioxidant VECYGPNRPQF (Sheih et al., vulgaris Val-Glu-Cys-Iyr-Gly-Pro-Asn- 2009b)

Arg-Pro-Glu-Phe (IC50 7.5 μM) Chlorella Pepsin ACE inhibition Val-Glu-Cys-Tyr-Gly-Pro-Asn- (Sheih et al., vulgaris Arg-Pro-Gln-Phe (IC50. 29.6 2009a) μM) Chlorella Pepsin Anti- Val-Glu-Cys-Tyr-Gly-Pro-Asn- vulgaris proliferation Arg-Pro-Gln-Phe (IC50 70.7 μM)

Chlorella Protamex, ACE inhibition Val-Glu-Gly-Tyr (IC50 128.4 (Ko et al., ellipsoidea Kojizyme, µM) VEGY 2012) Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin and papain Red algae Pyropia Pepsin, pancreatin Antioxidant, Asp, Glu, Ala (Cian et al., columbina Antiplatelet 2015) aggregation, ACE I inhibition Pyropia Trypsin, Antihypertensive, Asp, Ala, Glu, (Cian et al., columbina Alcalase, Antioxidant 2012)

Palmaria Corolase Dipeptidyl Ile-Leu-Ala-Pro (IC50 43.4 μM), (Harnedy et palmata peptidase (DPP)- Leu-Leu-Ala-Pro (IC50 53.67 μM) al., 2015) IV inhibitory Met-Ala-Gly-Val-Asp-His-Ile (IC50 159.37 μM) Palmaria Papain Anti- IPP, VPP (Fitzgerald et palmata athersclerotic al., 2013) Porphyra Alcalase, Trypsin, ACE inhibition Glutelin, albumin, and gliadin (Qu et al., yezoensis Neutrase, 2010) Flavourzyme, Protamex, Papain, Pepsin

*IC50, inhibitor concertation at which 50% of the enzymatic activity was inhibited.

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Table 10. (Continued) Bioactive peptides and possible bioactivities with the IC50 values from protein hydrolysates of the marine algae

Product Enzyme Bioactivities Peptide/Amino acid sequence Reference Brown Algae Saccharina Trypsin Antibacterial TITLDVEPSDTIDGVK, (Beaulieu et longicruris activity SGLIYEETR, al., 2015) MALSSLPR, ILVLQSNQIR, SAILPSR, IGNGGELPR, LPDAALNR, EAESSLTGGNGCAK, QVHPDTGISK Undaria Water ACE inhibition 10 dipeptides being the most active: (Suetsuna et pinnatifida extract Tyr-His (IC50 5.1 μM), al., 2004)

(Wakame) Lys-Tyr (IC50 7.7 μM),

Phe-Tyr (IC50 3.7 μM),

Ile-Tyr (IC50 2.7 μM)

Undaria Pepsin ACE inhibition Ala-Ile-Tyr-Lys (IC50 213 μM), (Suetsuna and pinnatifida Tyr-Lys-Tyr-Tyr (IC50 64.2 μM), Nakano, 2000)

(Wakame) Lys-Phe-Tyr-Gly (IC50 90.5 μM),

Tyr-Asn-Lys-Leu (IC50 21 μM)

Wakame Protease S ACE inhibition Val-Tyr (IC50 35.2 μM); (Sato et al.,

Ile-Tyr (IC50 6.1 μM), 2002a)

Ala-Trp (IC50 18.8 μM),

Phe-Tyr (IC50 42.3 μM),

Val-Trp (IC50 3.3 μM),

Ile-Trp (IC50 1.5 μM),

Leu-Trp (IC50 23.6 μM) IW Microalgae

Spirulina Pepsin ACE inhibition Ile-Ala-Glu (IC50 34.7 μM), (Suetsuna and platensis Phe-Ala-Leu (IC50 26.2 μM), Chen, 2001)

Ala-Glu-Leu (IC50 57.1 μM),

Ile-Ala-Pro-Gly (IC50 11.4 μM),

Val-Ala-Phe (IC50 35.8 μM) Spirulina Trypsin, α- anti- Amino acids sequences (Vo et al., maxima chymotrypsi inflammatory LDAVNR, MMLDF, 2013) n, Pepsin Navicula Pepsin, , α- Antioxidant Acidic amino acids; Glu-, Asp-, Lys-, (Kang et al., incerta chymotrypsi Arg- 2011) n, Neutrase

*IC50, inhibitor concertation at which 50% of the enzymatic activity was inhibited.

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2.4.5. Polysaccharides

Edible seaweeds are extremely rich in polysaccharides, with the level of total fibre greater than most land plants, and therefore can be a good source of dietary fibre (Gomez-Ordonez et al., 2010, Urbano and Goni, 2002). Like their land plant counterpart, dietary fibre in marine algae can be classified into two types, insoluble type such as cellulose, mannans and xylan, and water soluble dietary fibre such as ulvan in green algae, agar and carrageenan in red algae, and fucoidan, alginate and laminarin in brown algae (Misurcova et al., 2012). Agar, carrageenan and alginate have well defined thickening and gelling properties, and are mainly used as stabilisers, thickeners and emulsifiers in the food industry (Campo et al., 2009). These sulphated polysaccharides have been found to have a number of significant biological functions, such as antioxidant, antiviral, anticancer, anticoagulant, and immunomodulating activities (Jiao et al., 2011, Lahaye and Robic, 2007).

Carrageenans are a group of sulphated linear polysaccharides of D-galactose and 3,6- anhydro-D-galactose. This structure allows carrageenan to dissolve in water, forming highly viscous solutions and remaining stable over a wide pH range. From a human health perspective, antiviral properties of carrageenans from several species have been studied extensively including Stenogramme interrupta, Chondrus ocellatus, Gelidium cartilagineum, Gigartina skottsbergii and Chondrus cripus (Caceres et al., 2000, Zhou et al., 2004, Carlucci et al., 1997, Pujol et al., 2006). Carrageenan exhibits anti-HIV activity (Haaland et al., 2012, Buck et al., 2006) as well as inhibitory effects against herpes viruses types 1 and 2 of k/I-carrageenan (including acyclovir- resistant variants and clinical isolates) (Carlucci et al., 2004, Carlucci et al., 1997). Also, carrageenan has been shown to be an extremely potent infection inhibitor against a broad range of sexually transmitted human papillomavirus (Buck et al., 2006). Apart from its potential antiviral effect, carrageenan is also reported to confer several other health effects including antitumor effect by promoting tumour immune response (Luo et al., 2015, Yuan and Song, 2005, Yuan et al., 2006), and antioxidant properties (Yuan et al., 2005a). Luo et al. (2015), for example, found that intratumoral injection of λ- carrageenan could inhibit tumour growth in B16-F10 and 4T1 bearing mice and enhance tumour immune response by increasing the number of tumour-infiltrating M1 + + macrophages, DCs and more activated CD4 CD8 T lymphocytes in spleen. It is also reported that carrageenan could significantly inhibit the growth of transplantable 40 sarcoma S180 and increase macrophage phagocytosis (the form of antibody secreted by spleen cells), spleen lymphocyte proliferation, natural killer cells activity, interleukin (IL-2) and tumour necrosis factor-α (TNF-α) level in S180-bearing mice (Yuan et al., 2006).

Laminarin, a storage carbohydrate composed of (1, 3)-β-D-glucan and some β-(1, 6)- intrachain links, is commercially extracted mainly from brown algae such as Laminaria and Saccharina species (Kim et al., 2006a). Laminarin has been extensively investigated for its potential biological activities. The extraction, structure and health functional properties of laminarin from brown algae have recently been reviewed by Kadam et al. (2015). Major reported bio-functional activities of laminarin include anti- tumour, anti-inflammatory, immunostimulatory (Lee et al., 2012), anticoagulant (Alban et al., 1992) and antioxidant (Choi et al., 2011, Cheng et al., 2011) activities. For example, laminarin has been found to induce apoptosis in human colon cancer cells through a mitochondrial pathway (Ji et al., 2012). Lee et al. (2012) and Yin et al. (2014) suggested that laminarin has immunostimulatory properties and can strengthen immune reactions via the TF pathway in macrophages, because it was found to significantly increase the release of calcium, hydrogen peroxide, nitric oxide, monocyte chemotactic protein-1, vascular endothelial growth factor, leukemia inhibitory, and granulocyte- colony stimulating factor with enhanced expression of Signal Transducer and Activator of Transcription 1 (STAT1), STAT3, c-Jun, c-Fos, and cyclooxygenase-2 mRNA in RAW 264.7 cells.

Ulvan is a laminarin obtained from members of the ulvales, such as U. pertusa and U. lactuca. Lahaye and Robic (2007) have reviewed the functional properties of ulvan, which include anti-tumour, antithrombotic, anticoagulant and immune modulation activities. Recently, ulvan has been reported to exhibit significant antioxidant activities in vivo (Qi et al., 2012). It was also found to be effective in the protection of liver tissue from damage in rats fed with a cholesterol-rich diet, which led to the suggestion that ulvan may be used as an antihyperlipidemic agent (Qi and Sun, 2015).

Fucoidans are another group of sulphated polysaccharides primarily composed of sulphated L-fucose with <10% of other monosaccharides (Peng et al., 2015). They are only found in brown algae and not found in higher plants (Berteau and Mulloy, 2003). This group of algal polysaccharides have been reported to possess a number of 41 significant biological activities important to health, including anticoagulant, antithrombotic, immunomodulation, anticancer and anti-proliferative activities, which have been reviewed by Wijesinghe and Jeon (2012). Since then, a more recent study reported that fucoidans can delay spontaneous apoptosis of human neutrophils and induce their production of pro-inflammatory cytokines (Jin and Yu, 2015). These two critical effects of fucoidan on human neutrophils are achieved in a PI3K/AKT- dependent manner. This suggests that fucoidans may have therapeutic potentials to control neutrophil homeostasis and neutropenia. Thuy et al. (2015) showed that fucoidans inhibited HIV-1 infection when they were pre-incubated with the virus (viral pseudotype particles) but not with the U373-CD4-CXCR4 cells or after infection, whereby blocking the early steps of HIV entry into target cells.

Alginate and alginic acid are another important group polysaccharides obtained from brown seaweed. It is a linear copolymer consisted of homopolymeric blocks of (1-4)- linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues, covalently linked together in different sequences or blocks. From a nutritional perspective, alginate can act as a dietary fibre as it is hydrolysed during digestibility test (Jimenez-Escrig and Sanchez-Muniz, 2000). Alginate has been reported to exhibit antioxidant activities (Kelishomi et al., 2016, Falkeborg et al., 2014). Houghton et al. (2015) found that alginate incorporated in bread had pancreatic lipase-inhibition activity, which was maintained after cook and digestion. They suggested that adding alginate to bread may have a potential role in the treatment of obesity.

2.4.6. Fatty acid/Lipid

Seaweed has a low lipid content of about 1-5% of the dry biomass (Dawczynski et al., 2007, Gosch et al., 2012). ). The different levels of lipid content of seaweeds are due to the influences of species, growth temperature and other environmental conditions (Mendis and Kim, 2011). One of the main characteristics of lipids in macroalgae is their high proportion of polyunsaturated fatty acids (PUFAs), which account for almost half of the lipids and which are rich in omega-6 and omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These fatty acids are linked to a range of biochemically and physiologically important functions in the human body (Mendis and Kim, 2011, Rajapakse and Kim, 2011). A large amount of scientific 42 evidence supports the efficacy of omega-3 PUFAs as agents possessing anti- arteriosclerotic (Siegel and Ermilov, 2012), anti-inflammatory (Kellogg et al., 2015), and immunoregulatory effects (Mendis and Kim, 2011). Several studies have also shown that consumption of sufficient quantity of PUFAs may be beneficial to rheumatoid arthritis and heart disease (Stengel et al., 2011, Harris et al., 2008), although the evidence is not conclusive.

Macroalgae also contain a small amount of phospholipids, which account for about 4-10% of the total lipids (Peng et al., 2015). Phospholipids in the diet act as natural emulsifiers and facilitate the digestion and absorption of fatty acids, thus enhancing the nutritive value of the food. Like many terrestrial plants, macroalgae also contain some phytosterols, which belong to the phytosteroid group of compounds that all have a fused four-ring core structure. Phytosterols act as hormones and signalling molecules in plants and thus play a crucial role in plant physiology (Kim and Ta, 2012). From a nutritional perspective, phytosterols have been shown to provide a number of important health benefits, most notably its role in reducing serum low density lipoprotein (LDL)- cholesterol (Kim and Ta, 2011). They have also been shown to exhibit anti- inflammatory, antibacterial, antioxidant, anti-cancer, anti-diabetic and antifungal activities (Kim and Ta, 2012). The main phytosterols found in microalgae are fucosterol and its derivatives in brown algae, cholesterol analogues in red algae and ergosterol and 24-ethylcjolesterol in green algae (Machado et al., 2004).

2.4.7. Minerals

Edible marine seaweeds contain a high level of minerals because they absorb minerals in their ionic form from seawater. Generally, marine seaweeds contain 8-40% minerals in the dry biomass, depending on the type of seaweed, season, the growing environment, geographical location, and physiological state and the mineralisation methods used (Mendis and Kim, 2011, Ruperez, 2002). Ruperez (2002) confirms that marine algae contain significant amount of nutritionally important trace elements including iodine, sodium, potassium, calcium, magnesium, iron and manganese, which play important functions in human health. The mineral content of seaweed is about ten times higher than land plants, and can be an important source of dietary minerals (Ruperez, 2002).

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Seaweed is a primary source of iodine, and in some seaweed, iodine content exceeds its minimum dietary requirement and the upper limit of 3 mg/day (150 μg/day) (Ruperez, 2002, Zava and Zava, 2011). The highest iodine content is found in brown algae, which ranges from 500 to 8000 µg/g dry mass (Zava and Zava, 2011). ). Iodine is an essential element for the healthy function of the body's thyroid hormones, which control a number of physiological functions including the growth of tissue for the central nervous system, hair, nails, bones and skin (Cha et al., 2008). It is also believed to be responsible for the low prevalence of breast cancer in some high dietary seaweed countries (Mendis and Kim, 2011).

Interestingly, seaweed contains considerably higher amounts of iron (2.8 mg/100g) compared to food sources renowned to contain this mineral such as pork meat (1.4 mg/100g) (Tomovic et al., 2015) and spinach (2.7 mg/100 g) (Reddy et al., 1995). In addition, a normal portion size of brown seaweed, which includes species such as Laminaria and Undaria, provides more than 50% of the recommended daily intake of magnesium. Therefore, seaweed can be used as a food supplement to complement many of the important mineral requirements of the body.

However, some of heavy metals such as As, Cd, Cu, Hg, Pb and Zn which are toxic to human, are also present in algae, but their content is generally below the toxic limits allowed (Ruperez, 2002).

2.4.8. Vitamins

Vitamins are essential substances which cannot be synthesised by the body or only in limited quantities (Skrovankova, 2011); therefore they must be obtained from the diet. Edible seaweeds are very good sources of a number of vitamins including water soluble

B1, B2, B6, B12, and C and the lipid soluble A, and E (Skrovankova, 2011). Seaweeds contain relatively high amounts of vitamins B and C, e.g., Vitamin B12, which is only found in marine plants and not in land plants or vegetables (Cha et al., 2008). The nutritional value and health benefits, including antioxidant activities of vitamins such as C and E are well documented (Skrovankova, 2011).

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2.5. Algal species investigated in this study

It is clear from the preceding discussion that there is a huge range of algal species as well as a great diversity of biological compounds that are potentially present in them with significant biological activities. Furthermore, the amount of these bioactive components may change in relation to the environment in which the algae are grown. This presents a dilemma for a researcher who seeks to investigate the biological activities of algae. On the one hand, there are virtually endless possibilities for screening novel bioactive compounds and their health beneficial properties from a vast reservoir of algal species. On the other hand, when faced with such a vast number of choices, a researcher can be baffled at what species to select for study. In this thesis, it was decided to focus on a few emerging species of commercial algae, as these are currently cultivated for purposes other than their capacity for producing bioactive components, and hence their health-related biological activities have not been investigated to date. Furthermore, being commercially cultivated means that if bioactive components of potentials were found, scale up production could be more readily realised than species grown naturally.

Therefore, three species of green macroalgae (chlorophyta) were selected for study in this thesis, namely the marine macroalgae Derbesia tenuissima and Ulva ohnoi and the freshwater macroalga Oedogonium intermedium. Ulva ohnoi belongs to a familiar genus Ulva, but it is a tropical variety of the blade-form, whose biological activities have not been well investigated. Derbesia tenuissima and Oedogonium intermedium are two totally new commercial species that have only recently been domesticated for the first time. These species were selected based on their high productivity in land-based culture, resistance to contamination and a high tolerance to environmental fluctuations (Neveux et al., 2014b). For these reasons, they have been considered to have a good potential as natural sources of functional ingredients. Also, an important factor to consider here was the low toxicity of the selected varieties, assuming that any bioactive ingredient obtained from them would be used for future development of new functional foods (Hiraoka et al., 2004, Gosch et al., 2015). Figure 5 presents photographs of these algal species showing their growth habit in culture and cellular structure.

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Figure 5. Specimen photos of Derbesia tenuissima (a and b), Ulva ohnoi (c and d) and Oedogonium intermedium (e and f) showing growth habit in culture and cellular. Courtesy of Neveux et al. (2014a).

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Derbesia tenuissima (bryopsidales, chlorophyta) is a filamentous marine macroalga found in temperate to tropical waters including the North Atlantic, Mediterranean and South Pacific (Gosch et al., 2015). Derbesia tenuissima is found to be suitable for intensive land-based production (Mata et al., 2015, Neveux et al., 2014b) and is considered a promising species for development of bio-products and functional food applications as it is rich in bioactive components and has high biomass productivity. It is a species rich in lipids (>12% dry weight) with a content of total fatty acids greater than 5% dw, of which 40% are in the form of omega-3 PUFAs (Magnusson et al., 2014, Gosch et al., 2012). Due to the high content of lipid, it is also being investigated for biofuel production (Neveux et al., 2014b). Magnusson et al. (2014) quantified the effects of temperature, light and nitrogen availability on the growth and fatty acid level of Derbesia tenuissima and the effects of inducing different degrees of light stress by managing culture conditions on its biomass, total phenolic content and antioxidant productivity. However, research conducted to date on this seaweed species is mainly concerned with their cultivation and culture conditions to improve biomass, antioxidant and fatty acid content. There is very little information regarding the health related biological activities of the seaweed, and little is known about the components responsible for these activities.

Ulva ohnoi, also known as green laver or sea lettuce, is a group of edible green algae composed of smooth, lobed and ruffled disks attached to rocks, shells or hard bottom by a single holdfast, or are free floating (Lawton et al., 2013b). This kind of green algae have no true roots present and the leaves can be bright, flat, thin, broad, and often rounded or oval shaped. Ulva can grow rapidly under nutrient rich water with intense lighting. The genus of Ulva consists of a wide variety of species that are commonly distributed worldwide. In Australia, they are mainly located in the Queensland and Western Australia coastline. It has been a benchmark for land-based cultivation of marine macroalgae (Mata et al., 2015). Currently, Ulva is cultivated in commercial scales to remediate waste waters from the commercial production of abalone, and the produced biomass is used as animal feed (Bolton et al., 2008) and the same approach has been recently adopted by the Australian algae company MBD Energy Ltd at the Pacific Reef prawn farm in Ayr, North Queensland. Ulva has also been found to have a biofiltering function for dissolved inorganic nitrogen discharged from coastal fish farming (Yokoyama and Ishihi, 2010). It has been suggested as a candidate for 47 production of biofuels (Bruhn et al., 2011, Neveux et al., 2014b). However, Ulva biomass also has the potential as a resource for high value bio-products. One of the more extensively studied components of Ulva is ulvan, a group of soluble, sulphated polysaccharides with unique biomedical functionalities that have been investigated for use in tissue engineering, regenerative medicine and drug delivery (Coste et al., 2015, Lahaye and Robic, 2007). Ulvan has also shown strong antioxidant activity in both in vitro and in vivo assays (Qi and Sun, 2015) and antihyperlipidemic activity on triglyceride and low density lipoprotein cholesterol (Qi et al., 2012). Yaich et al. (2011) investigated the chemical composition and processing functional properties (hydration and oil holding capacity) of Ulva lactuca. Celikler et al. (2009) investigated the anti- hyperglycemic, anti-oxidative and genotoxic/antigenotoxic capacity of Ulva rigida in vivo, while Garcia-Casal et al. (2009) studied the antioxidant capacity and polyphenol content of an unnamed Ulva species. However, no study has investigated Ulva ohnoi for its bioactive components and health related biological activities.

Oedogonium intermedium (or TSV1 Strain Genbank) belongs to a genus of unbranched filamentous green algae that grow in freshwater (Lawton et al., 2013a). It is commonly found in calm waters where it can attach to other plants or form floating mats. The cells are cylindrical, frequently wider at one end than the other, and contain a netlike chloroplast which usually fills the cell with pyrenoids and a large central vacuole. Proximate, biochemical and ultimate analyses have been performed on the dry biomass of Oedogonium (Neveux et al., 2014b). Some research has shown that the Oedogonium can be used as bio-sorbent to remove harmful metals such as As, Cr, Cu, Se, Zn, Pb, Cd and Ni (Bakatula et al., 2014, Gupta and Rastogi, 2008, Roberts et al., 2015), to produce Fe-biochar to remove the oxidised form of selenium, arsenic and molybdenum from wastewater (Johansson et al., 2016), and as a fertiliser and soil conditioner (Lawton et al., 2015). It has also been considered as a feedstock biomass for bioenergy applications since it has a high biocrude yield compared with many other algal species (Neveux et al., 2014b, Lawton et al., 2015, Lawton et al., 2013a). To date, there has been no reported research that investigated its biological activities and related chemical components.

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2.6. Conclusion

In conclusion, there is a vast number of macroalgae widely distributed over the world. Many of the algae are rich in nutrients required by humans, animals and crops and have been used as food, fodder and fertilisers. Macroalgae also contain a diverse of range of chemical components that have found applications in medicine, cosmetics and a wide range of other industrial products. Research conducted in recent decades has found that microalgae also contain a plethora of bioactive compounds, including carotenoids, phenolics, carbohydrates and proteins and peptides that can confer a wide array of biological activities with potential health benefits. These bioactive compounds may play a significant role in the prevention and mitigation of many disorders prevalent in our societies such as obesity, diabetes, hypertension, chronic inflammation, cardiovascular diseases and cancer. Several of these bioactive compounds are found only in substantive quantities in macroalgae. To date, most of the research in this area has focused on brown and red algal species because they are the traditional seaweed species utilised as food. On the other hand, studies on the biological activities of green algae have been limited, especially for the domesticated species cultivated in commercial scale. These species are currently cultivated mainly for the production of fodder. However, they are also a potential and readily scalable source of bioactive components that should be explored. This PhD project was thus conceived to partially bride this gap of knowledge by investigating the biological activities of the major bioactive components of three green macroalgal species currently cultured in tropical Australian environment.

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Chapter 3 Phenolic compounds and antioxidant activities of green macroalgae cultured in the tropics

3.1. Introduction

Reactive oxygen species (ROS), such as hydroxyl, superoxide and peroxyl radicals, are formed in human cells by endogenous factors and can react non-enzymatically with cellular molecules such as membrane lipids, proteins and DNA (Pangestuti and Kim, 2011, Chandini et al., 2008). These reactions result in extensive oxidative damage, which is directly implicated in pathogenesis of many diseases or disorders, including cancer, mutagenesis, Alzheimer’s disease, diabetes mellitus and aging (Pangestuti and Kim, 2011, Orcic et al., 2011). Many commercial synthetic antioxidants are currently in use to limit disease pathogenesis including butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate (PG). These antioxidants act by retarding the oxidation and peroxidation processes. However, synthetic antioxidants have higher toxicity than natural compounds (Kahl and Kappus, 1993, Kahl, 1984). Therefore there is a considerable interest in the research and development of antioxidants from natural sources.

The primary natural sources of antioxidants that are readily available are terrestrial plants including fruits, vegetables, herbs and tea, and their application in food systems to prevent oxidation has been extensively reviewed (Al-Jaber et al., 2011, Dai and Mumper, 2010, Shori, 2015). The antioxidant activities in these plants are largely attributed to phenolic compounds, such as flavonoids and phenolic acids, which are known as safe and non-toxic antioxidants. More recently, aquatic plants have also been considered as a potential source of antioxidants. For example, many seaweeds (marine macroalgae) are traditionally served as human food, such as kombu, wakame, nori and dulse (Plaza et al., 2008), but may also have strong antioxidant systems to protect themselves from oxidative damage from free radicals that are formed during their growth. The antioxidant activity of phenolic compounds from several seaweeds has been studied (Chew et al., 2008, Kelman et al., 2012, Lopez et al., 2011, Matanjun et al., 2008, Sabeena and Jacobsen, 2013) and these natural antioxidants play an important role in preventing lipid peroxidation. A series of phenolic compounds such as gallic

50 acid, protocatechuic acid, gentisic acid, catechins (e.g. gallocatechin, epicatechin and catechin gallate) and flavonols have been identified from algal extracts (Sabeena and Jacobsen, 2013, Rodriguez-Bernaldo de Quiros et al., 2010, Lopez et al., 2011, Santoso et al., 2002). Furthermore, other chemical components of the seaweeds, including dietary fibre, fatty acids, free amino acids, proteins and vitamins may also have biological activities (Mendis and Kim, 2011, Plaza et al., 2008).

Although many aspects of the nutritional components of seaweeds have been studied, including the antioxidant and phenolic components more specifically, the focus is largely on brown and red seaweeds, as well as the brown seaweeds/kelps that are prevalent in cold waters (Yuan et al., 2005c). There is limited information on the phenolic components and antioxidant activities of green algae, especially those grown in tropic waters. In this chapter, three emerging commercial species of green macroalgae that are currently cultured at large but pre-commercial scales in tropical Australia were investigated. They were Ulva ohnoi, an edible seaweed also known as sea lettuce or “aosa”, Derbesia tenuissima, a filamentous algal genus which is siphonous in structure and Oedogonium intermedium, a freshwater filamentous alga. The objectives of the this chapter were: 1) to estimate the total phenolic content and total flavonoid content of the three algal species; 2) to evaluate their antioxidant activities using a number of different assay procedures; and 3) to identify their phenolic components using a series of analytical techniques including HPLC-PDA, GC-MS, LC- MS and NMR.

3.2. Material and methods

3.2.1. Chemicals and reagents

AAPH (2,2'Azobis(2-methylpropion-amidine) dihydrochloride (97%)), ABTS (2,2’- azino-bis(3-ethylbenzothiazoline-6 -sulphonic acid)), caffeic acid, chlorogenic acid, р- coumaric acid, trans-cinnamic acid, deuterium oxide water (D2O), DPPH (2,2-diphenyl- 1-(2,4,6-trinitrophenyl) hydrazyl), ferulic acid, Folin-Ciocalteu’s phenol reagent, hesperetin, myricetin, naringenin, protocatechuic acid, pyridine, quercetin, rutin hydrate, trolox ((+-)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), vanillic acid, and XAD-7 Amberlite resin were purchased from Sigma-Aldrich (Sydney, Australia). 51

Acetic acid, acetone, ethanol, hexane, hydrochloric acid (32%) (HCl), iron (III) chloride, hexahydrate (FeCl3·6H2O), methanol, potassium acetate, potassium persulfate (K2S2O8), sodium acetate trihydrate (NaOAc·3H2O), sodium carbonate, and sodium potassium tartrate tetrahydrate were purchased from Ajax Finechem Pty Ltd (Sydney, Australia). Fluorescein (FL), formic acid, and TPTZ (2,4,6- Tris(2-pyridyl)-s-triazine) were purchased from Fluka Analytical (Sydney, Australia). Aluminium chloride, anhydrous

(AlCl3) was purchased from Merck KGaA, (Darmstadt, Germany). BSTFA (N, O-bis (trimethylsilyl) trifluoroacetamide) was purchased from Supelao, Bellefonte (PA, USA). Gallic acid was purchased from TCI (Tokyo, Japan). Randomly methylated betacyclodextrin (RMCD), technical grade, was purchased from Trappsol, CTD Holding, Inc. (FL, USA). Syringic acid was purchased from Aldrich Chemical Company, Inc. (Sydney, Australia). Methanol (HPLC grade) was purchased from Burdick & Jackson, SK Chemicals (Sydney, Australia). All chemicals were of at least analytical grade unless stated otherwise. Water used in all experiments was purified by reverse osmosis using the MilliQ RO system (referred to as MilliQ water).

3.2.2. Algal sample collection and storage

Three species of green macroalgae (chlorophyta) were selected for study in this thesis; they were the two marine algal species Ulva ohnoi (hereafter Ulva) and Derbesia tenuissima, (hereafter Derbesia) and the freshwater macroalga Oedogonium intermedium (hereafter Oedogonium). Samples were harvested in December 2012 from stock cultures held in outdoor tanks at James Cook University (JCU), Townsville, Queensland, Australia. Ulva was cultured in 10,000 L parabolic tanks, stocked at densities between 0.5-3 g/L, with daily additions of nutrient-rich waste water from Barramundi broodstock tanks and additional enrichment of 7 mg/L nitrogen and 3 mg/L phosphorous (Mata et al., 2015). Derbesia, a filamentous marine green alga and siphonous in structure, was cultured in 2,000 L tanks using filtered sea water (35 ppt) enriched with MAF (Microalgae Food) nutrients to maintain a nitrogen concentration of 7 mg/L (Magnusson et al., 2015). The biomass was harvested after seven days, from tanks stocked at 0.25 g/L and 0.5 g/L. Oedogonium (Cole et al., 2013), a freshwater filamentous alga that naturally grows under eutrophic conditions in streams and drainage ditches, was cultured at the JCU/MBD research facility in two 10,000 L 52 parabolic tanks, using de-chlorinated tap water, enriched with Guillard’s F2 nutrient medium to 1 g/L. Biomass was harvested weekly to maintain the optimal stocking density of 0.5 g/L.

Biomass from each species was harvested through a combination of large hand nets and pumps which pumped the culture medium through nylon bags, and the biomass was collected. The algal biomass was then centrifuged, frozen to -80°C and subsequently freeze-dried (Labogene ScanVac Coolsafe 110-4 Pro Freeze Dryer, Lynge, Denmark) for 48 h. The freeze-dried biomass was vacuum packed immediately in plastic bags and air freight to our laboratory where they were stored at -80°C until use.

3.2.3. Preparation of seaweed extracts

3.2.3.1. Preparation of crude extracts

Solid/liquid extraction was employed to extract phenolic components from each of the algal samples. 60% ethanol was used as extraction solvent. For the extraction, samples of the freeze dried algal biomass were ground (Pulverisette 14, Fritsch GmbH) to pass through a 0.5 mm sieve to afford a uniform powder. The powered biomass was mixed with the extraction solvent at the ratio of 1: 20 w/v to counter the high viscosity of the material extracted (Lawton et al., 2013b). The extraction was conducted in an ultra- sonication bath (Ultrasonic cleaner, 50 Hz, Unisonics Pty. Ltd. Sydney, Australia) for 30 min (Taha et al., 2011), then centrifuged (Sorvall TC-6, MedWoW, Wilmington, DE, USA) for 10 min at 3500 g, and the supernatant collected. The pellet was re-extracted with the same amount of fresh solvents two more times to maximise the extraction of phenolic compounds (Chew et al., 2008). The combined supernatants were filtered with a 13 mm x 0.45 polytetrafluoroethylene (PTFE) syringe filter (Grace Davison Discovery Scences, VIC, Australia) and stored at -20°C. These crude extracts were used directly for analysis of their antioxidant activities without further treatments. Analysis of the extracts was usually conducted within 3 days after extraction.

To obtain extraction yield, aliquots of the combined supernatants was subjected to vacuum rotary evaporation at 35°C (Orme Scientific Ltd., Manchester, UK) to remove the organic solvent. The remaining extract was then freeze-dried (Labogene ScanVac Coolsafe 110-4 Pro Freeze Dryer, Lynge, Denmark) at -109°C, 0.015 kPa to remove the

53 remaining water and the lyophilised extract was weighed. The extraction yield was calculated as a percentage of the original seaweed sample in dry biomass basis.

3.2.3.2. Preparation of purified ethanolic extracts

Purified extracts were prepared from the crude extracts, obtained as described in Section 3.2.3.1, after the combined supernatants were vacuum evaporated to remove the solvents, but before the remaining aqueous extracts were freeze dried. The remaining concentrated aqueous extract was purified by reversed phase liquid chromatography using an Amberlite XAD-7 resin column (300 x 60 mm i.d.). Briefly, the aqueous extract (20 mL) was loaded onto a glass column filled with the Amberlite resin (the resin had previously been washed with water until bobble less, and then socked in 80% ethanol overnight before use). The column was washed with MilliQ water by gravity until impurities had passed through and only clean water came out. Phenolic compounds were then eluted out with 80% ethanol. The eluate was collected and evaporated under reduced pressure at 35°C using a rotary evaporator. Then, the concentrated aqueous extract was lyophilised at -109°C and 0.015 kPa. These fine lyophilised powders were taken as purified extracts and stored at -20°C prior to the analysis.

3.2.3.3. Preparation of defatted ethanolic extracts

Defatted ethanolic extracts of algal biomass were prepared in the same way as for non- defatted extracts except that prior to extraction, the algae samples was treated with hexane (1:4, w/v) 3 times, each for 10 min to remove the fat (Wikandari et. al., 2015). The defatted biomass was then dried overnight under a fume hood and extracted with ethanol as described in Section 3.2.3.1.

3.2.3.4. Preparation of hydrolysed crude ethanolic extracts

Hydrolysed extracts were prepared from the crude extracts, obtained as described in Section 3.2.3.1. Aliquots of the combined supernatants were hydrolysed with 0.1% (v/v)

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HCl at room temperature; then the supernatant was subjected to vacuum rotary evaporation at 35°C to remove the organic solvent, and the remaining extract was lyophilised and stored at -20°C prior analysis.

3.2.3.5. Preparation of hydrolysed purified ethanolic extracts

Hydrolysed extracts were also prepared from the purified extracts, obtained as described in Section 3.2.3.2, and the procedure used was exactly the same as described in Section 3.2.3.4.

3.2.3.6. Preparation of hydrolysed defatted ethanolic extracts

Hydrolysed extracts were also prepared from defatted extracts, obtained as described in Section 3.2.3.3, and the procedure used was exactly the same as described in Section 3.2.3.4.

3.2.4. Assays of phenolic content and antioxidant activities of seaweed extracts

3.2.4.1. Total phenolic content (TPC)

Total phenolic content of the extracts was determined by the Folin-Ciocalteu assay as described by Li et al. (2008) with some modifications. Aliquots (30 μL) of crude ethanolic extracts (Section 3.2.3.1) were mixed with 140 μL of Folin-Ciocalteu reagent (1:10 diluted) in a 96 well microplate (Greiner bio-one, Germany; the same was used throughout the thesis), which was allowed to stand at room temperature for 5 min. Then, 30 μL of 20% (w/v) sodium carbonate was added to the mixture and incubated in the darkness at room temperature for 2 h. After that, absorbance was measured at 765 nm using a microliter plate spectrometer (SpectroMax, Molecular Devices, Australia). The determination of total phenolic content was carried out in triplicate. A calibration curve of gallic acid (100-500 µM) was prepared (R2=0.999), and the results, determined by the regression equation of the calibration curve, were expressed as milligrams of gallic acid equivalents per 100 g dry weight (mg GAE/100 g DW).

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3.2.4.2. Total Flavonoid content (TFC)

The total flavonoids content (TFT) of each extract was determined using the aluminium chloride colorimetry methods modified from Moghaddam and Mehdizadeh (2015). In brief, 20 μL of crude ethanolic extract samples (Section 3.2.3.1) and 20 μL of 10% (w/v) aluminium chloride were mixed in a 96 well microplate, and the mixtures were incubated at room temperature for 5 min. Subsequently, 20 μL of 1 M potassium acetate and 180 μL of MilliQ water were added and the incubation continued for another 30 min. Following that, the absorbance was read at 415 nm using the same plate reader described above. A calibration curve of rutin hydrate (25-500 µg/mL) was prepared (R2=0.999), and the total flavonoid content was expressed as mg rutin hydrate equivalent per g dry weight of algal biomass (mg RE/g DW).

3.2.4.3. ABTS-radical scavenging assay

ABTS assay followed the method of Thaipong et al. (2006) with some modifications. The working ABTS reagent was prepared by mixing 3.5 mL of 10 mM ABTS·+ and 1.5 mL of 8.17 mM K2S2O8 solutions, and allowing them to react overnight at room temperature in the dark (the time required for formation of the radical). For the actual assay, 1 mL ABTS·+ solution was diluted with 39 mL MilliQ water to obtain an absorbance of 1.2±0.01 units at 734 nm using the spectrometer. Ten µL of crude ethanolic extracted samples (Section 3.2.3.1) or Trolox standard was added in triplicate to each well of a 96-well microliter plate, and 190 µL ABTS reagent was then added using a multichannel pipette. The reaction mixtures were incubated for 5 min at room temperature and the absorbance read at 734 nm. Trolox solutions at the concentration range of 65-1000 µM were used to construct a standard calibration curve (R2=1.000). The results are expressed as mg Trolox equivalent per 100 g dry weight (mg TE/100g DW). A new standard curve was prepared for each plate to counter any possible variations in results between plates.

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3.2.4.4. DPPH- radical scavenging assay

DPPH assay was done according to the method of Fukumoto and Mazza (2000) with some modifications. For the modified procedure, a 200 μM solution of DPPH· was prepared in 80% methanol, then stored at -20°C for 15 min until needed. The crude ethanolic extract samples (40 μL) were allowed to react with 160 μL of the DPPH· solution for 30 min in the dark and the absorbance was taken at 517 nm. The standard curve of gallic acid was linear between 10 and 100 μM (R2=0.995). Results are expressed as mg gallic acid equivalents (GAE)/100g dry algae. A new standard curve was prepared for each plate to counter any possible variations in results between plates. The assay was carried out in triplicates.

3.2.4.5. Ferric reducing antioxidant power (FRAP) assay

The assay was done according to the method of Kelman et al. (2012) with minor modifications in the use of Trolox as the standard rather than the FeSO4. The working FRAP reagent was made by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution and 20 mM FeCl3·6H2O in a 10:1:1 ratio and heating to 37°C prior to use. The

300 mM acetate buffer was prepared by mixing 3.1 g of NaOAc·3H2O with 16 mL glacial acetic acid and made to 1 L with MilliQ water. The TPTZ solution was prepared by mixing equal volumes of 10 mM TPTZ with 40 mM HCl. For the assay, 150 µL of FRAP reagent was added to each well of a 96 well microliter plate. A blank reading was taken at 595 nm using a microliter plate spectrometer. To each well 20 µL of crude ethanolic extract of algae samples in triplicate was then added; the plate was incubated for 8 min at room temperature and the absorbance read at 595 nm. The standard curve of Trolox was linear between 7 and 250 μM (R2=1.000). Results are expressed in mg Trolox equivalents per 100 g dry weight (mg TE/100 g DW). A new standard curve was prepared for each plate to counter any possible variations in results between plates.

3.2.4.6. Oxygen radicals absorbance capacity (ORAC) assay

ORAC assay was used to determine the antioxidant activities of both the hydrophilic and lipophilic compounds in the algal biomass in separate assays. These components 57 were first extracted from the algal biomass prior to performance the assays. To extract hydrophilic components, aliquots (250 mg) of the ground algal samples were mixed with 5 mL of 80% aqueous methanol. The samples were sonicated for 10 min, centrifuged (10 min, 4000 g), and the supernatant collected. The pellet was re-extracted two more times under the same conditions and the supernatants combined. Aliquots of the combined supernatants (15 mL) were filtered with a 13 mm × 0.45 µm PTFE membrane, and were used for ORAC assay without further treatments. The extraction was carried out in triplicate for each sample.

For the extraction of lipophilic components, aliquot (250 mg) of the ground samples were mixed with 10 mL of cold acetone (4°C). The mixtures were shaken on an orbital shaker (Bioline Global, NSW, Australia) at 175 rpm for 20 min, centrifuged (10 min, 4000 g), and the supernatants collected. The pellet was re-extracted two more times under the same conditions and the supernatants combined. Freshly prepared aliquots of the combined supernatants (30 mL) were filtered with a 13 mm × 0.45 µm PTFE filter membrane and immediately analysed. The extraction was carried out in triplicate for each sample.

ORAC assay for hydrophilic compounds of algae

ORAC assay on the hydrophilic extracts was conducted according to Konczak et al. (2010) in FIA 200 black 96 well, flat bottom and medium binding microliter plates, untreated (FLUOTRAC, Germany). As the ORAC assay is extremely sensitive, samples must be diluted appropriately before analysis to avoid interference. In this case, optimal dilutions, which were obtained by trial and error in preliminary experiments, were 1:50, 1:100 and 1:100 for Ulva, Derbesia and Oedogonium, respectively. The diluted samples (in triplicate) were mixed with a FL (70 nM) solution and a solution of AAPH (140 mM), both in phosphate buffered saline (PBS, 75 mM, pH 7.0). Both AAPH and FL solution were prepared daily and warmed to 37°C prior to use. A stock solution of FL (700 μM) was prepared in PBS, and stored in complete darkness under refrigeration conditions. The working solution (70 nM) was prepared daily by diluting the stock solution in PBS. The AAPH radical (140 mM) was prepared daily by dissolving 379.8 mg of AAPH and making it up to 10 mL with PBS. The automated ORAC assay was carried out on a Polar-Star OPTIMA microplate fluoro-meter (BMG Labtech, Germany) with fluorescence filters for an excitation wavelength of 485 nm and an emission

58 wavelength of 520 nm. Data points were acquired over 120 min, in 1 min kinetic cycles with a 5 s medium intensity orbital shaking between cycles. A calibration curve was constructed daily by plotting the calculated differences of area under the FL decay curve between the blank and the sample for a series of standards of Trolox solutions in the range of 10-100 µg/L prepared daily in PBS (R2=0.998). The results were expressed as µmol Trolox equivalents per 100 g dry weight (µmol TE/100 g DW). A new standard curve was prepared for each plate to counter any possible variations in results between plates.

ORAC assay for lipophilic compounds of algae

ORAC assays on the lipophilic extracts were conducted according to Davis et al. (2010) with some modifications. All the reagents used were prepared in the same way as described for the ORAC assay of hydrophilic compounds. In addition, a solution of 7% randomly methylated betacyclodextrin (RMCD) was prepared from 50% (v/v) acetone/water mixture and was shaken for 1 h at room temperature on an orbital shaker at 175 rpm prior to use. Trolox standards were prepared (10-100 µM) also in this 7% RMCD. The lipophilic extracts were diluted using 7% RMCD (Ulva 1:50, Derbesia 1:150, and Oedogonium 1:150) so that the readings would fall within the linear region of the Trolox standard curve. Diluted sample extracts and standards, both at 25 µL, were added to the wells followed by 120 µL of the FL solution, which was automatically added by the instrument. Following incubation in the instruments for 15 min at 37°C, 80 µL of the AAPH solution was then added automatically by the machine. Data points were acquired over 180 min, 1 min kinetic cycles with a 5 s medium intensity orbital shaking between cycles. A calibration curve was constructed daily by plotting the calculated differences of area under the FL decay curve between the blank and the sample for a series Trolox solutions in the range of 10-100 µg/L (R2=0.892). The results were expressed as µmol TE/100 g DW.

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3.2.5. Identification of chemical compounds in phenolic extracts of algae

3.2.5.1. High Performance Liquid Chromatography-Photodiode Array Detector analysis (HPLC-PDA)

Identification of phenolic compounds in extracts was carried out by HPLC-PDA using a Shimadzu Prominence UFLC (Shimadzu, Kyoto, Japan) system that consisted of a vacuum degasser (DGU-20A5), two pumps (LC-20AD), a photodiode array detector (PDA) (SPD-M20A), a column oven (CTO-20A), and an auto sampler (SIL-20A HT), equipped with the LC solution software. The separation was performed with a reverse phase Luna C18 column (250 x 4.6 mm i.d., 5μ, Phenomenex, NSW, Australia) using a gradient elution system. Eluent A was water containing 0.1% formic acid and eluent B was pure methanol. The eluting conditions applied were: 0-5 min, 20% B; 5-30 min, 60% B; 30 -35 min, 60% B; 35-40 min, 20% B. Finally, the column was washed and reconditioned. The eluent solvents were filtered through a 0.45 μm PTFE membrane before use. The flow rate was 1.0 mL/min, injection volume was 60 µL of extracts and 20μL for phenolic standards, and the system was operated at 27°C, with monitoring at 280 nm. These conditions were adapted from a previous study with some modifications (Lopez et al., 2011). For analysis run, the crude ethanoic extract (Section 3.2.3.1) and the purified ethanolic extract (Section 3.2.3.2) were filtered through a 0.45 μm PTFE syringe filter and directly injected to HPLC without further treatment. The phenolic standards (gallic acid, protocatechuic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, coumaric acid, ferulic acid, rutin hydrate, myricetin acid, cinnamic acid, quercetin, naringenin and hesperetin) were prepared in 60% ethanol, and also filtered prior to injection to HPLC.

The peaks of phenolic components in the samples were identified by comparing their retention times and overlaying of UV spectra with those of standard compounds. Spiking of the samples with standards was used to confirm or disapproves the identity of peaks.

3.2.5.2. Gas Chromatography-Mass Spectrometry (GC-MS) analysis

The following samples were analysed by GC-MS to identify phenolic compounds in them: a) crude ethanolic extracts (Section 3.2.3.1), b) purified ethanolic extracts 60

(Section 3.2.3.2), c) defatted ethanolic extracts (Section 3.2.3.3), d) crude ethanolic extracts with hydrolysis (Section 3.2.3.4), e) purified ethanolic extracts with hydrolysis (Section 3.2.3.5), and f) defatted ethanolic extracts with hydrolysis (Section 3.2.3.6). Prior to GC-MS analysis, the algal extracts were derivatised in order to obtain their volatile alkyl silylated derivatives. Briefly, the freeze-dried extract (1 mg) was treated by adding 160 μL of BSTFA + TMCS 1% anhydrous and pyridine (1:1, v/v), the mixture was heated at 60-65°C for 1 h in the dark to ensure complete reaction. The phenolic standards were also derivatised in the same way. The derivatised samples and phenolic standards were subjected to GC-MS analysis as described below.

GC-MS analysis was performed using a Thermo Electron Corporation (San Jose, CA, USA) Trace DSQ mass spectrometer, equipped with a TR-5MS SQC capillary non polar column (15m x 250μm, ID, film thickness 2.5μm). The inject temperature was set at 280°C while the oven temperature was initially at 70°C (held for 0.7 min), then increased to 150°C at 20°C/min for 50 min, and finished at 300°C at 4°C/min (held for 2 min). This last increase was to clean the column from possible residues. One µL of each derivatised sample extract or standard solution was injected into system.

The mass spectrometer was operated in the positive electron ionisation (EI) mode with the ionisation energy of 70 eV. The ion source temperature was 200°C. Detection was performed in selective ion monitoring (SIM) mode and total ion capture (TIC) mode. The peaks were identified by comparing their retention times and mass spectra with those of reference standards and in the NIST Mass Spectral Database.

3.2.5.3. Liquid chromatography-high resolution mass spectrometry (LC-

HRMS)

Identification of phenolic compounds was also carried out using liquid chromatography high resolution mass spectrometry (LC-HRMS) on an Orbitrap LTQ XL system (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an Accela 900 UPLC system. A Phenomenex Synergi column (250 mm x 1 mm i.d., 4 μm Hydro-RP) was used and was maintained at 45°C. The mobile phase consisted of 5 mM ammonium formate in water, pH 7.4, adjusted with ammonium hydroxide (solvent A) and 5 mM ammonium formate in 90% methanol, pH 7.4 (solvent B). The method was based upon

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Tang et al. (2016). A flow rate of 150 μL/min was used with a gradient elution program: 20% B (0 min), 30% B (2 min), 30% B (4.5 min), 45% B (5.5 min), 45% B (7 min), 75% B (12min), 90% B (14 min), and 20% B (14.1 min), 20% B (19 min). The crude ethanolic extracts (20 μL, Section 3.2.3.1) were injected to the system and the analysis was repeated at least twice for each sample. Atmospheric pressure chemical ionisation (APCI) source was used in negative mode to generate ions from the analyte in solution, recorded over the mass range of m/z 120-1000 (full scan mode) at a resolution of 60,000 over 20 min. The limit of detection (LOD) was set at the signal-to-noise ratio of 20. LC-HRMS was calibrated at the start of each run using positive and negative calibration solutions (as per instrument manual) on each day of analysis using direct infusion into the HESI source. The LOD for LC-HRMS was 0.625 ng rosmarinic acid.

3.2.5.4. Nuclear magnetic resonance (NMR) spectroscopy

Identification of chemical components in the algal extracts was also performed with NMR on a Bruker Avance III HD 600 MHz (Rheinstetten, Germany) with a cryoprobe.

Lyophilised sample extracts were dissolved (2 mg/mL) in 200 μL of D2O in NMR Shigemi tubes and a mixture of 10 μL each of sodium azide (0.02 mg/mL), imidazole solution (0.34 mg/mL), and d-TMSP solution (8.6 mg/mL) was added. Topspin 3.2 (Bruker, Rheinstetten, Germany) software was used for data analysis, and NMR suite 7 (Chenomx Inc., Canada), a NMR spectra database, was also used to facilitate identification and quantification of compounds. The following samples were analysed by NMR: a) crude ethanolic extracts (Section 3.2.3.1) and b) purified ethanolic extracts (Section 3.2.3.2). Phenolic standards, namely syringic, caffeic, ferulic, protocatechuic, р-hydroxybenzoic, chlorogenic, salicylic, trans-cinnamic, vanillic, p-coumaric and caffeic acids, were used to spike the samples to facilitate the identification of these compounds in the crude ethanolic samples.

3.2.6. Statistical analysis

The whole experiment, starting from the extraction of phenolic compounds, was done twice and each analysis was performed usually in triplicate (n≥3). Results were expressed as mean ± standard deviation (SD) with three independent experiments. One 62 way analysis of variance (ANOVA) was carried out using SPSS version 22 (IBM, USA) to determine whether significant differences exist among treatments and Duncan triplicates range test was used to separate significant differences between the means at the 5% level. A p value <0.05 was considered as statistically significant. All graphs were drawn by Graphpad prism 6 (Graphpad Software Inc, Australia).

3.3. Results and discussion

3.3.1. Extraction yields

The yield of solvent extraction of phenolic components depends on a number of factors, including the type of solvents with varying polarities, pH, extraction time, temperature as well as the chemical composition of the sample matrix (Lopez et al., 2011). When extraction conditions, such as time and temperature, are equal, the solvent and the chemical properties of the sample becomes two most important factors. In previous studies, a number of different solvents, including methanol, ethanol, butanol, acetone, chloroform and water, have been used for the extraction of phenolic compounds from seaweeds (Lopez et al., 2011, Chew et al., 2008, Matanjun et al., 2008, Sabeena and Jacobsen, 2013). The commonly used solvents are ethanol, methanol and acetone in various concentrations of aqueous mixtures. In this study, a number of solvent systems were trialled in preliminary experiments and 60% (v/v) ethanol was found to give the best extraction yields from the three algal samples. Consequently, this solvent system was used for extracting the phenolic compounds in subsequent experiments and the results are shown in Table 11. Of the three algal samples, Derbesia gave highest yield, followed by Ulva, while the fresh water alga Oedogonium gave the lowest extraction yield, which was less than half of the yield for Derbesia.

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Table 11. The extraction yield, total phenolic content and total flavonoid content of Ulva, Derbesia and Oedogonium

Extraction yield (%)1 TPC2 TFC3

Ulva 34.1±3.0b 111.6±3.4a 371.8±32.7a

Derbesia 45.3±2.1c 309.6±2.6b 2130±68.3c

Oedogonium 20.5±0.9a 388.6±8.2c 1154.0±11.5b All values are mean ± SD for triplicate experiments (n=3). Different letters within the same column indicate significant difference between the values (p<0.05). 1Extraction yield (%) calculated as g of dried extract/100 g DW. 2TPC: represent total phenolic content (mg of GAE/100 g DW). 3TFC: represent total flavonoid content (mg RE/100 g DW).

3.3.2. Total phenolic content (TPC) and total flavonoid content (TFC) of the algal species

Table 11 also shows the TPC and TFC of the three algal samples. The amount of TPC differed substantially among the three algal samples with Oedogonium having the highest level, followed by Derbesia, while the TPC level in Ulva was the lowest, being less than a third of that in Oedogonium. Considering the much lower extraction yield of Oedogonium compared with the two marine algal species, this means that the concentration of phenolic compounds in the extract of Oedogonium would be much higher than in those of the other two algal extracts. Table 12 also shows the reported levels of TPC and TFC in selected marine algae and some antioxidant rich food. TPC levels reported in literature ranged from 198.2 to 508.0 mg GAE/100 g DW for green algae, and from 35.4 to 1045.0 mg GAE/100 g DW for brown algae (Sabeena and Jacobsen, 2013, Farasat et al., 2014). The TPC levels found in the three algae of the present study were generally higher than those of green algae but lower than some of the brown species. They are also lower than the traditional high phenolic plants including some berries, herbs and spices (Amensour et al., 2010, Gurnani et al., 2015) as shown in Table 12.

The TFC levels in the three algae also differed markedly with Derbesia having the highest TFC level, followed by Oedogonium, while Ulva had the lowest TFC level by far. Derbesia and Oedogonium had comparable levels of TFC to other Ulva species reported in the literature, ranged from 946.2 - 3309.4 mg RE/100 g DW; however, the 64

TFC level in the Ulva species of the present study was lower than other Ulva species and was much lower than those in the high antioxidant products such as berries and red seeds (Farasat et al., 2014, Gurnani et al., 2015, Amensour et al., 2010) as shown in Table 12.

Table 12. Reported levels of total phenolic/flavonoid content in selected marine algae and some antioxidant rich foods

Biomass Solvents TPC1 TFC2 Reference Green algae Enteromorpha Ethanol 265.8 - (Sabeena and Jacobsen, 2013) intestinalis Ulva lactuca Ethanol 236.5 - (Sabeena and Jacobsen, 2013) Ulva clathrata 80% 508.0 3309.4 (Farasat et al., 2014) Methanol Ulva linza 80% 199.6 1043.1 (Farasat et al., 2014) Methanol Ulva intestinalis 80% 198.2 2531.6 (Farasat et al., 2014) Methanol Ulva flexuosa 80% 267.4 946.2 (Farasat et al., 2014) Methanol Red algae Palmaria Ethanol 76.9 - (Sabeena and Jacobsen, 2013) palmata Chondrus Ethanol 113.6 - (Sabeena and Jacobsen, 2013) crispus Brown Algae Fucus Ethanol 1045.0 - (Sabeena and Jacobsen, 2013) vesiculosus Saccharina Ethanol 35.4 - (Sabeena and Jacobsen, 2013) latissima Other antioxidant rich product Myrtus Methanol, 781-3556 2137- (Amensour et al., 2010) communis L. Ethanol, 12996 (Berry) Ethyl acetate, water Capsicum Chloroform, 4293- 2506- (Gurnani et al., 2015) frutescens L n-Hexane 29288 14381 (red chilli seeds) 1TPC: represent total phenolic content (mg of GAE/100 g DW). 2TFC: represent total flavonoid content (mg RE/100 g DW).

Correlation analysis between extraction yield on one hand, and TPC and TFC on the other, was performed (data shown on Appendix A). No correlation was found between extraction yield and TPC, nor between the yield and TFC, which agrees with findings in

65 some previous reports (Chandini et al., 2008, Lopez et al., 2011, Sabeena and Jacobsen, 2013). This indicates that the extracts contained more than just phenolic compounds or flavonoids. Other substances, such as sugars, soluble proteins, peptides and amino acids, fatty acids, and pigments may also be present in the extracts. It needs to be pointed out that the Folin-Ciocalteu reagent used for determining TPC basically measures the reducing capacity of the sample, and it is not specific for phenolic compounds. Many non-phenolic substances with reducing ability, such as sugars and proteins, peptides and amino acids, also react with this reagent (de Oliveira et al., 2012). This may explain the discrepancy between extraction yields and TPC in the algal samples. On the other hand, it has been shown that the aluminium chloride reagent used for the determination of total flavonoids could be interfered with by non-flavonoid impurities, leading to a significant bathochromic shift and a lowering of the measured TFC level in the sample (Joubert et al., 2008). This could be partly responsible for the non-correlation between the extraction yields and TFC levels in the algal extracts.

3.3.3. Antioxidant capacities of the algal species

Natural antioxidants such as flavonoids and phenolic acids have a positive effect on human health as they can act as electron donors, singlet oxygen quenchers, reducing agents and inhibitors of peroxyl radicals, thus preventing cell damages by reactive oxygen species (ROS). These ROS can react non-enzymatically with cellular molecules such as membrane lipids, proteins and DNA, with these reactions being collectively termed “oxidative stress”, which is believed to be linked with many health disorders such as cancer, diabetes mellitus, aging and neurodegenerative diseases (Pangestuti and Kim, 2011, Orcic et al., 2011). There are many methods that can be used to determine antioxidant capacity. These methods differ in terms of their assay principles and experimental conditions, and they may not always measure the same aspects of the total antioxidant potential of the sample in question. Therefore, it is usually necessary to use more than one method to assess the antioxidant capacity of a sample to ensure that a fuller picture of its antioxidant capacity is obtained. In this study, the antioxidant activities of the seaweed extracts were tested using four different in vitro assays, ABTS (scavenging activity), DPPH (scavenging activity), FRAP (ferric reducing capacity) and ORAC (inhibition of peroxyl-radical induced oxidation) assays, which are some of the

66 most commonly used methods for assessing the antioxidant capacity of foods. Figure 6 shows the antioxidant capacities of the three algal samples measured by the first three assays.

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Figure 6. The total phenolic content (TPC) and antioxidant activities for Ulva, Derbesia and Oedogonium. Results are measured as mg GAE/100 g DW for TPC and DPPH scavenging activity, while mg TE/100 g DW for ABTS scavenging activity and FRAP values. All values are mean ± SD of triplicate experiments. Different letters within the same assay indicate significant difference between the values (p<0.05).

3.3.3.1. DPPH free radical scavenging capacity

This assay is based on the reduction of DPPH· radical in the presence of a hydrogen- donating antioxidant due to the formation of the non-radical form (DPPH-H) in the reaction (Fukumoto and Mazza, 2000). As shown in Figure 6, Derbesia was found to have the highest DPPH scavenging capacity (61.76 mg GAE/100 g DW), followed by Oedogonium (46.29 mg GAE/100 g DW), while Ulva possessed the lowest antioxidant capacity (23.68 mg GAE/100 g DW). Reported values of DPPH radical scavenging capacities for algal species varied considerably, ranging from 19.45 to 1069.30 µM TE/100 g DW (Ferraces-Casais et al., 2012). In the present study, gallic acid was used 67 as reference standard instead Trolox, as it gave a standard calibration curve with greater linearity. It is clear that, similar to the situation with terrestrial plants, antioxidant capacity can also vary greatly among different algal species. Furthermore, as previous studies have found, cultivars, growing conditions and maturity stage at harvest, as well as analytical conditions such as sample storage conditions, time elapsed before the algae were analysed, and sample preparation methods could also influence the results (Addai et al., 2013). The DPPH radical scavenging capacity of Derbesia obtained in this study was comparable to that reported by Magnusson et al. (2015) for the same species.

3.3.3.2. ABTS free radical scavenging capacity

ABTS radical scavenging assay works on similar antioxidant mechanisms as those for DPPH scavenging assay, and therefore is frequently used to confirm results obtained by the latter. As shown in Figure 6, similar data trend to that of DPPH assay was obtained with ABTS assay although for the latter, Oedogonium, rather than Derbesia (as the case with DPPH assay) was found to have the highest antioxidant capacity (519.61 mg TE/100 g DW), but this was followed very closely by Derbesia (479.63 mg TE/100 g DW). As was the case with DPPH assay, Ulva exhibited considerably lower activities (199.17 mg TE/100 g DW) in comparison with the other two algal samples. Highly different values of ABTS scavenging capacities have been reported for other algal species, ranging from a low value of 45 mg TE/100 g DW) for Undaria pinnatifida and a very high value of 351 mg TE/100 g DW) for himanthalia elongate (Cofrades et al., 2010). The values found for the three algal species in this study were comparable with the reported values.

3.3.3.3. Ferric reducing antioxidant power (FRAP)

Unlike the free radical scavenging activity assays, the FRAP assay is based on the ability of antioxidants to reduce ferric tripyridyltriazine complex (Fe(III)-TPTZ) to ferrous complex (Fe(II)- TPTZ) by accepting an electron from an antioxidant. This reduction depends on the hydroxylation pattern and the degree of polymerisation (Kelman et al., 2012). As shown in Figure 6, Derbesia had the highest FRAP value by

68 far at 500.12 mg TE/100 g, DW), followed by Oedogonium (280.53 mg TE/100 g, DW), which was about 45% less than that of Derbesia. Similar to the results obtained by DPPH and ABTS assays, Ulva gave the lowest FRAP value, which was only about 23% of that for Derbesia. These values were higher or comparable with those reported in the literature for various algal species. For examples, FRAP values of 339.5 and 172.7 mg TE/100 g DW have been reported for P.umbilicalis (nori) and Laminaria ochroleuca (Kombu), respectively (Cofrades et al., 2010). However, the values were lower than traditional high antioxidant products such as berries (Kahkonen et al., 1999).

3.3.3.4. Oxygen radicals absorbance capacity (ORAC)

The ORAC assay is recognised as one of the in vitro antioxidant assays that most closely approximate the physiological conditions in nature, because it utilises a biologically relevant radical source (MacDonald-Wicks et al., 2006), and it is the only method that combines both inhibition time and degree of inhibition into a single quantity in the measurement (Cao and Prior, 1999). It is also a method that can be used to measure the antioxidant capacity of both hydrophilic and hydrophobic components. The ORAC values for the phenolic components (hydrophilic and hydrophobic) of the three algae samples are shown in Table 13.

Table 13. ORAC capacity obtained from Ulva, Derbesia and Oedogonium for both hydrophilic and lipophilic extracts ORAC-H1 ORAC-L2 Ulva 102.3±4.0a 1.3±0.1a Derbesia 354.0±8.7b 178.6±2.8c Oedogonium 350.8±1.3b 34.3±0.6b All values are mean ± SD for triplicate experiments (n=3). Different letters within the same column indicate significant difference between the values (p <0.05). 1 ORAC of hydrophilic extract, 2 ORAC of lipophilic extract. Results are expressed as µmol TE/g DW.

It is clear that the hydrophilic components of the algal extracts had markedly higher ORAC values than their lipophilic counterparts for all three algal species. This is especially so for Oedogonium and Ulva, where the ORAC values of the hydrophilic components were about 78 and 102 times higher than those of their lipophilic 69 counterparts, respectively. For Derbesia, the ORAC value of the hydrophilic components was about twice of that of the lipophilic fraction. Of the three species, the hydrophilic components of Derbesia and Oedogonium had similar (not significantly different) ORAC values, while the value for Ulva was considerably lower, being than less 30% of the other two species. For the lipophilic components, Derbesia had a relatively high ORAC value of 178.6 µmol TE/g DW, followed by Oedogonium at 34.3 µmol TE/g DW, while the value for Ulva was very low at 1.3 µmol TE/g DW.

Wang et al. (2009) reported the ORAC activities of 10 seaweeds extracted with aqueous acetone and water, which varied from 4 to 2567 µmol of TE/g DW, with the brown algal species having the highest ORAC values. The ORAC values obtained in the present study were lower than those of the brown algal species, but comparable with those of the green alga Ulva lactuca (80 and 300 µmol of TE/g DW in the water and 70% acetone extracts, respectively). The ORAC values of the three species investigated in the current study were also lower than some of the native Australian fruit and herbs (Tang et al., 2016, Konczak et al., 2010).

3.3.3.5. Correlation analysis

Correlation analysis was conducted between TPC, TFC and antioxidant capacity values obtained by the different assays and the resultant Pearson’s correlation coefficients are presented in Table 14. The total phenolic content in the seaweed extracts showed significant correlations only with ABTS (r=0.985, p<0.01) and ORAC-H (r=0.958, p<0.01) values, but not with results by the other antioxidant assays. The total flavonoid content only showed significant correlations with ORAC-L (r=0.957, p<0.01) and FRAP (r=0.848, p<0.05) values, but not with results by the other assays. The results of ABTS and ORAC-H assays were strongly correlated (r=0.993, p<0.001), so were the results by DPPH and FRAP (r=0.980, p<0.01) and those by FRAP and ORAC-L (r=0.965, p<0.01). Previous studies have shown a positive correlation between TPC and antioxidant activities, especially DPPH radical scavenging capacity of different seaweed extracts (Matanjun et al., 2008, Wang et al., 2009, Machu et al., 2015). However, Zhao et al. (2007) stated that the ferrous ion-chelating ability of malting barley extracts exhibited poor correlations with both TPC and other antioxidant activities, such as

70

DPPH, ABTS and ORAC (r ranging from 0.17 to 0.46). The generally poor correlations between TPC and TFC on the one hand, and antioxidant activities on the other, appeared to suggest that there might be non-phenolic compounds present in the algal extracts, such as low molecular weight polysaccharides, proteins or peptides, that have interfered with the various assays as suggested by some researchers previously (Wang et al., 2009, Chew et al., 2008).

Table 14. Pearson’s correlation (r) values between the levels of phenolic compounds and antioxidant capacity for three different algae species TPC TFC ABTS DPPH FRAP ORAC-H ORAC-L

TPC 1 0.126ns 0.985** 0.773ns 0.633ns 0.958** 0.407ns

TFC 1 0.297ns 0.727ns 0.848* 0.405ns 0.957**

ABTS 1 0.872* 0.758ns 0.993*** 0.560ns

DPPH 1 0.980** 0.922** 0.894*

FRAP 1 0.829* 0.965**

ORAC-H 1 0.652ns

ORAC-L 1

Significant correlation at p <0.05, 0.01 and 0.001 are indicated by *, ** and ***, respectively; ns = not significant.

3.3.4. Identification of chemical compounds in the phenolic extracts of algae

3.3.4.1. HPLC-PDA analysis

A number studies have used HPLC-PDA techniques to identify phenolic compounds in biological samples (Tang et al., 2016, Parys et al., 2007). This technique is most useful where the types of phenolic compounds in the sample are, at least to some extent, known so that appropriate reference standards can be obtained for comparison. Although this is not necessarily the case with phenolic components in algae, it was nevertheless considered that HPLC-PDA would be a good preliminary technique to determine the complexity of phenolic components in the samples. A number of mobile

71 phases and gradient eluting systems were trailed to optimise resolution of sample peaks, and the eventual elution system obtained was able to achieve a good separation of sample peaks as well as a number of major phenolic standards. In all, 14 phenolic standards were used (gallic, protocatechuic, chlorogenic, vanillic, caffeic, syringic, coumaric, cinnamic and ferulic acids, rutin hydrate, myricetin, quercetin, naringenin, and hesperetin), and they were well resolved in the HPLC system (Figure 7). These standards were selected because they have been reportedly found in seaweeds (Onofrejova et al., 2010, Lopez et al., 2011, Parys et al., 2007, Sabeena and Jacobsen, 2013).

11

12 7 13 8 5 6 3 9 10 14 1 2 4

Figure 7. HPLC chromatograms of standard phenolic compounds. peak 1=gallic acid (5.4min), peak 2 = protocatechuic acid (9.8 min), peak 3 = chlorogenic acid (16.1 min), peak 4 = vanillic acid (18.3 min), peak 5 = caffeic acid (18.8 min), peak 6 = syringic acid (19.5 min), peak 7 = coumaric acid (23.9 min), peak 8 = ferulic acid (24.8 min), peak 9 = rutin hydrate (28.5 min), peak 10 = myricetin acid (30.9 min), peak 11 = cinnamic acid (34.6 min), peak 12 = quercetin (35.1 min), peak 13 = naringenin (35.5 min), and peak 14 = hesperetin (36.3 min).

Figure 8 and Figure 9 show the chromatograms of crude and purified ethanolic extracts from the three algal species, respectively. A two-step process was used to identify the peaks in the chromatograms. The first step was to compare the retention time (RT) and UV spectra of peaks in the extract with those of the standard compounds. The second step was to spike the algal extracts with the suspect phenolic standards. When the retention times and UV spectra of the peaks were compared with those of the phenolic standards, only one peak (RT = 30.9 min) was found to have similar retention time and

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UV spectra as a standard (myricetin, peak 10) for Ulva, one peak (RT = 35.2 min, quercetin, peak 12) for Derbesia, while no peak in Oedogonium matched any of the standards. To confirm the presence of myricetin and quercetin in Ulva and Derbesia, reference standards of these two compounds were spiked into the respective algal extracts. However, in subsequent HPLC runs of the spiked samples, the peaks of the standards did not overlap with the suspect peaks in the chromatograms, and the presence of these two phenolic compounds were thus ruled out.

Initially, it was thought that the extracts might contain large amounts of impurities such as sugars and proteins, or the phenolic compounds might be bounded with sugars in glycosides, which would affect the separation. However, analysis of the purified extracts did not yield further information on the identity of the peaks in the samples although the number of peaks was fewer and peak intensities were lower coampred with the crude extracts. Previous studies have reported that green algae in the Ulva genus had a low polyphenolic content compared to species of red and brown algae and for that reason, identifications of the phenolic compounds were not carried out further (Nwosu et al., 2011, Celikler et al., 2009, Garcia-Casal et al., 2009). However, Sabeena and Jacobsen (2013) reported that gallic, protocatechuic and gentisic acids were found in the ethanolic extract from Ulva lactuca. Since HPLC-PDA was unable to identify the phenolic compounds in the three algal species investigated in the present study, GC-MS analysis was carried out to identify them in the algal samples.

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A

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C

Figure 8. HPLC chromatograms of crude extracts from (A) Ulva, (B) Derbesia and (C) Oedogonium.

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A

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Figure 9. HPLC chromatograms of purified algae extracts from (A) Ulva, (B) Derbesia and (C) Oedogonium.

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3.3.4.2. GC-MS analysis

Gas chromatography mass spectrometry (GC-MS) is also a commonly used technique for identifying phenolic compounds in food samples. Because phenolic compounds are generally non-volatile, a derivatisation step must be carried out on the samples first to volatilise them. In the present study, the algal extracts were derivatised with BSTFA, which attached a trimethylsilyl group to the functional group of the analytes, thus making them volatile. Six different types of ethanolic extracts of the three algae were subjected to GC-MS analysis to identify the phenolic compounds present in them: (a) crude extracts, (b) hydrolysed crude extracts, (c) purified extracts, (d) hydrolysed purified extracts, (e) defatted extracts, and (f) hydrolysed defatted extract. The GC chromatograms for these samples are showed in Appendix C. Peaks in the chromatograms were analysed using the NIST software which contains a database library of reference mass spectra, which provided a reverse match value for each compound. The reverse match value compares the mass spectra of the unknown compound back-to-back with those from the NIST library. An example of such comparison is shown in Figure 10. Phenolic standards were also analysed in the same way and their mass spectra were compared with the sample, which provided a percentage matching for each suspect compound.

The list of compounds identified by GC-MS analysis is given in Table 15. As can be seen, the compounds from the extracts contained several amino acids (serine, L-aspartic acid, L-proline and L-tryptophan), some organic compounds (glutaconic acid, butanedioic acid, fumaric acid, malic acid and trimethyl silane), fatty acids (palmitic, linolenic, oleic acid and linoleic acids), and sugars (L-threonic acid, D-(-)- fructofuranose, D-(+)-galactopyranose, α-D-mannopyranose, D-glucose, maltose and sucrose), but no phenolic compounds were found. This was the case even after the purification process where most of the impurities were thought to have been removed. These results once again demonstrated that the green algae studied in this thesis had low levels of phenolic compounds. Importantly, a number of these non-phenolic compounds are known to have antioxidant and other biological activities. The amino acids L-Serine and L-aspartic acid, for example, have been reported to exhibit significant antioxidant activities (Maralani et al., 2012, Liu et al., 2004). Palmitic, linolenic, oleic and linoleic acids are also found to exhibit antioxidant, as well as antibacterial and antifungal

76 properties (Seidel and Taylor, 2004, Agoramoorthy et al., 2007, Cai et al., 2011). Since the algal samples were low in phenolic components, it is therefore highly probable that most of their antioxidant activities might be derived from other compounds such as amino acids, proteins, fatty acids and even some reducing sugars. It is also worth noting that some of the fatty acids present in the algal samples have been reported to exert several significant health effects. For example, Palmitic acid has been shown to be cytotoxic to human leukemic cell line and it also shows in vitro antitumor activity in mice (Harada et al., 2002). Linoleic acid and its geometrical and positional isomers, conjugated linoleic acid (CLA) have been shown to reduce oxidative stress (Beeharry et al., 2003, Ha et al., 1990). CLA also exhibits potent anticarcinogenic and antiatherogenic effects in animal models (Ip et al., 1994, Ip et al., 1996), immune modulating activity characterised by increased blastogenesis and macrophage killing ability (Cook et al., 1993) as well as the capacity to reduce body fat content and increase lean body mass in pigs and rodents (Dugan et al., 1997). The presence of these components in the algal species could therefore potentially confer them those health beneficial properties.

77

313 100 73

117

O Si O

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69 328 61 83 97 185 201 269 285 30 159 171 213 227 243 257 299 0 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 (w9n11) Hexadecanoic acid, trimethylsilyl ester

73 117 100 75 313 50 43 129 55 145 45 69 61 83 89 97 105 159 171 185 201 213 227 243 257 269 285 328 0 49 177 191 237 297 308 30 89 95 105 159 171 185 213 227 243 255 285 299 45 61 69 83 201 269 328 55 145 50 43 129

75 117 100 73 313 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 Na_TMS_S02#2745-2751 RT: 23.33-23.36 AV: 7 SB: 16 23.26-23.30 , 23.41-23.45 Head to Tail MF=932 RMF=937 Hexadecanoic acid, trimethylsilyl ester

Figure 10. Mass spectral confirmation of trimethylsilyl-derivatised palmitic acid (RT 23.68) using the NIST library (top) and the head to tail matching with reference standard (bottom). 78

Table 15. Compounds identified in the three green algae by GC-MS

Ulva Derbesia Oedogonium Compounds Retention Time 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

Amino acid Serine 8.51 Y Y L-Aspartic acid 11.46 Y Y L-Proline 14.25 Y Y Y L-Tryptophan 30.01 Y Organic compounds Glutaconic acid 8.75 Y Y Y Butanedioic acid 9.10 Y Fumaric acid 9.38 Y Malic acid 10.91 Y Y Y Trimethyl silane 20.12 Y Y Sugars L-Threonic acid 11.11 Y D-(-)-Fructofuranose 14.65 Y D-(+)-Galactopyranose 16.82 Y α-D-mannopyranose 17.06 Y D-Glucose 18.93 Y Y Y Y Y Maltose 25.09 Y Glyceryl-glycoside 25.89 Y Y Y Y Y Y Y Y Y Y Y 1: Crude ethanolic extract; 2: Purified ethanolic extract; 3: Defatted ethanolic extract; 4: Crude ethanolic extract with acid hydrolysis; 5: Purified ethanolic extract with acid hydrolysis; 6: Defatted ethanolic extract with acid hydrolysis; Y: available in sample extracts.

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Table 15. (Continued) Compounds identified in the three green algae by GC-MS

Ulva Derbesia Oedogonium Compounds Retention Time 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

Sucrose 31.25 Y Y Y Y Y Melibiose 39.45 Y Y Vitamin Inositol 19.81 Y Y Fatty acid Palmitic acid 23.68 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Linolenic acid 24.38 Y Y Y Y Y Y Y Y Oleic acid 28.05 Y Linoleic acid 28.13 Y - Y Y Y Y Y Y α-Linolenic acid 29.06 Y Y Y Y Y Y Y Y Y Y Y Y Y Nucleoside Adenosine 37.53 Y Y Y Y Y Y Y Inosine 36.47 Y 1: Crude ethanolic extract; 2: Purified ethanolic extract; 3: Defatted ethanolic extract; 4: Crude ethanolic extract with acid hydrolysis; 5: Purified ethanolic extract with acid hydrolysis; 6: Defatted ethanolic extract with acid hydrolysis. Y: available in sample extracts.

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3.3.4.3. LC-MS Because neither HPLC-PDA nor GC-MS analyses were able to detect any phenolic compounds in the algal samples, LC-MS was subsequently employed to analyse the crude extracts as it is a very sensitive technique able to detect very low concentrations of compounds. For LC-MS analysis, the choice of ionisation method is important and depends on the nature of the analyte molecules. Atmosphere pressure chemical ionisation (APCI) was the ion source chosen in this study because it can detect compounds with moderate polarity and molecular mass (Tang et al., 2016). The total ion chromatograms of Ulva extract using APCI source are displayed in Figure 11. As can be seem, there were no substantial peaks in the first 20 min in the chromatogram, and the abundance shown were likely from the instrument noise. There were three major peaks at 21.48, 22.00 and 26. 67 min; however, their mass-to-charge ratio (m/z) were all greater than 700, which meant that the compounds were not phenolic compounds, as the mass range of phenolic compounds was at m/z of 120-700. Similar results of LC-MS analysis were also obtained for the other two species Derbesia and Oedogonium (Appendix D). It was therefore concluded that the levels of phenolic compounds in the three green algal species, if present, would be so low that they could not be detected by LC-MS. These results appeared to be in conflict with those in Section 3.2.4.1, which showed reasonable amounts of TPC in the algal extracts. One possible explanation for this discrepancy is that most of the “measured TPC” by the Folin-Ciocalteu reagent were not phenolic compounds. This is because the Folin- Ciocalteu assay for TPC basically measures the reducing capacity of the sample, and it is not specific for phenolic compounds. Many non-phenolic substances with reducing ability, such as sugars and proteins, peptides and amino acids, also react with this reagent (de Oliveira et al., 2012). As shown in Table 15, many compounds belonging to these groups were indeed present in the algal extracts.

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C:\Users\...\LC-MS\141126_APCI_Neg_U 26/11/2014 3:35:42 PM

RT: 0.00 - 34.02 21.48 NL: 100 1.01E8 TIC F: - p APCI 90 C:\Users\...\LC-MS\141126_APCI_Neg_U 26/11/2014 3:35:4222.00 PM 26.67 Q1MS [100.000- 80 21.05 RT: 0.00 - 34.02 26.07 27.19 700.000] MS 70 21.48 141126_APCI_ NL: 100 27.54 Neg_U 1.01E8 TIC F: - p APCI 6090 27.88 22.00 Q1MS 20.70 26.67 [100.000- 5080 21.05 28.49 26.07 27.1931.87 700.000] MS 70 141126_APCI_ 40 13.08 27.54 Neg_U 1.31 3.56 5.03 6.41 9.10 11.35 14.03 17.76

60 27.88 RelativeAbundance 30 20.70 50 28.49 (A) 31.87 20 40 13.08 1.31 3.56 5.03 6.41 9.10 11.35 14.03 17.76 RelativeAbundance 1030 C:\Users\...\LC-MS\141126_APCI_Neg_U 26/11/2014 3:35:42 PM 200 0 5 10 15 20 25 30 RT: 0.0010 - 34.02 Time (min) 21.48 NL: 0 100 1.01E8 0 5 10 15 20 25 30 141126_APCI_Neg_U #2447-2523 RT: 21.22-21.82 AV: 8 NL: 2.84E5 TIC F: - p APCI F: - p90 APCI Q1MS [100.000-700.000] Time (min) 22.00 26.67 Q1MS 106.23 [100.000- 141126_APCI_Neg_U10080 #1 RT: 0.01 AV: 1 NL: 5.65E5 21.05 T: - p APCI Q1MS [100.000-700.000] 26.07 27.19 700.000] MS 90 141.08 141126_APCI_ 10070 27.54 Neg_U 806090 118.97 27.88 20.70 7080 50 28.49 31.87 70 60 40 223.18 13.08 60 1.31 3.56 5.03 6.41 9.10 11.35 14.03 17.76

RelativeAbundance 50 3050 157.39 213.03 40 212.82

2040 223.11 RelativeAbundance RelativeAbundance 3030 10 232.98 (B) 149.27188.19 315.01 C:\Users\...\LC-MS\141126_APCI_Neg_U2020 241.03238.93 315.4326/11/2014 3:35:42 PM 0 355.67 389.83 414.26 366.03 547.17 10 400.89 630.94 10 0 5 10 15 20 462.69447.2225 574.0430 654.74 RT: 0.00 - 34.02 504.75 553.33 616.25 692.96 0 Time (min) NL: 0 21.48 100 150 200 250 300 350 400 450 500 550 1.01E8600 650 700 150 200 250 300 350 400 450 500 550 600 650 700 m/z TIC F: - p APCI 141126_APCI_Neg_U90 #2641 RT: 22.86 AV: 1 NL: 1.42E6 m/z F: - p APCI Q1MS [100.000-700.000] 22.00 26.67 Q1MS 277.35 [100.000- 10080 21.05 26.07 27.19 700.000] MS 141126_APCI_ 9070 27.54 Neg_U 8060 27.88 20.70 7050 28.49 31.87

6040 13.08 1.31 3.56 5.03 6.41 9.10 11.35 14.03 17.76 RelativeAbundance 5030

4020

RelativeAbundance 695.40 3010 231.51 20 0 118.90 176.08 293.94 391.72 0 5 10 15 20450.51 25 533.80 583.0730 10 350.91 499.22 643.75 (C) Time (min) 0 141126_APCI_Neg_U150 #3011-3087200 250RT: 26.07-26.67300 AV:3508 NL: 2.52E5400 450 500 550 600 650 700 F: - p APCI Q1MS [100.000-700.000] m/z 106.23 100

90

80

70

60 223.04 50

40

RelativeAbundance 30 156.83 277.28 20 313.47 363.09 390.95 529.39 10 448.76 497.61 (D) 567.46 638.29 679.80 0 150 200 250 300 350 400 450 500 550 600 650 700 m/z

Figure 11. LC-MS total ion current (TIC) chromatogram (A) of Ulva using atmospheric pressure chemical ionisation with mass range between m/z 120-700. A [M-H]- corresponded to peaks at retention time 21.48 (B), 22.00 (C), and 26.67 (D) min.

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3.3.4.4. 1H NMR analysis

The ethanolic extracts were also subjected to 1H NMR analysis to identify potential phenolic compounds. The proton NMR spectra of the crude and purified ethanolic extracts of Ulva, Derbesia, and Oedogonium are displayed in Figure 12, Figure 13 and Figure 14, respectively. It is clear that there were compounds with aromatic ring present in the extracts as the aromatic region of the spectrum being at 6.5-7.5 ppm (Kutyshenko et al., 2015). There was also a huge amount of sugars (sugar region of the spectrum being 2.5-6.5 ppm) (Kutyshenko et al., 2015) and a small amount of amino acid (free amino acid region being 0.9-2.5 ppm) (Kunyanga et al., 2012). By comparing the NMR spectra of the crude and purified ethanolic extracts, it can be seen that the purification step had removed most of sugars and amino acids, as well as some of the aromatic compounds.

When the standard phenolic compounds (syringic, caffeic, ferulic, protocatechuic, and p-hydroxybenzoic acids) were spiked into the crude ethanolic extracts, their peaks did not overlap with any of the peaks in the spectra of the extracts (Appendix E). Therefore, it was hypothesised that the aromatic ring detected in sample extracts might not from phenolic compounds; rather, it could come from free amino acids with an aromatic ring in their structures. To test this hypothesis, the SDBS (Spectral Database) for organic compounds library database was searched to compare the spectra with those of the algal extracts (Appendix F). This resulted in the identification of a number of amino acids and sugars in the samples, which are shown in Table 16. As can be seen, the compounds from the extracts contained several aromatic amino acids (e.g., asparagine, proline, tyrosine, valine, and arginine), sugars (e.g., maltose, glucose, sucrose, fucose and galactose) and organic compounds, which broadly agreed with the results obtained by GC-MS, as discussed in Section 3.2.5.2. Combining all the results, it was concluded that the three green algal species studied in this thesis have very low amounts of phenolic compounds, and the moderate antioxidant activities of the ethanolic (phenolic) extracts were likely attributable to free amino acids, fatty acids and reducing sugars present in them.

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(A)

(B)

Figure 12: 1H NMR spectra of crude (A) and purified (B) ethanolic extracts of Ulva.

84

(A)

(B)

Figure 13: 1H NMR spectra of crude (A) and purified (B) ethanolic extracts of Derbesia.

85

(A)

(B)

Figure 14: 1H NMR spectra of crude (A) and purified (B) ethanolic extracts of Oedogonium.

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Table 16. Compounds identified in the three algae by NMR analysis

Ulva Oedogonium Derbesia

Compounds C * Compounds C * Compounds C*

Asparagine 2.2 4-Aminobutyrate 3.5 Alanine 4.1 Carnitine 0.1 Alanine 7.9 Arabinitol 4.1 Dimethylamine 2.0 Asparagine 2.5 Arginine 7.3 Fucose 0.1 Formate 1.0 Betaine 11. 9 Glutamate 1.1 Fumarate 1.2 Formate 0.3 myo-Inositol 2.6 Galactose 5.3 Galactose 0.2 N- 0.1 Glucose 5.1 Glucose 1.1 Acetylglucosamin e N-Acetylserotonin 0.1 Glutamine 4.7 Glutamate 3.6 Pantothenate 0.1 Glycerol 5.1 Glutamine 1.2 Propylene glycol 8.4 Isoleucine 1.5 Glycerol 2.8 Sucrose 0.4 Malate 5.6 Glycylproline 0.8 Trehalose 0.2 Maltose 1.3 Inosine 0.2 N-Acetylglucosamine 0.4 Lactate 3.6 Phenylalanine 0.4 Maltose 0.7 Proline 3.5 O- 6.2 Phosphoethanolamine Sucrose 1.2 Tyrosine 0.8 Uridine 0.6 Valine 3.7 Xanthurenate 0.5 *C: Concentration (mmol/L of crude extract)

3.4. Conclusion

The most important conclusion that can be drawn from the results of this chapter is that the three green macroalgal species examined in this thesis, Ulva, Oedogonium and Derbesia, had very low levels of phenolic compounds. After exhaustive analysis by HPLC-PDA, GC-MS, LC-MS and 1H NMR, no phenolic compounds could be identified from the ethanolic extracts of any of the algal samples. Rather, a number of other organic compounds, including free amino acids, fatty acids and sugars, were identified from the algal extracts. The ethanolic extracts contained moderate levels of total phenolic content as measured by the Folin-Ciocalteu procedure. However, since Folin-Ciocalteu reagent is known to react with many non-phenolic compounds with reducing capacities, it was concluded that the measured total phenolic content in the extracts came mostly from these non-phenolic impurities such as free amino acids, fatty 87 acids and sugars. Furthermore, since many of these non-phenolic compounds are also known to exhibit antioxidant activities, it was concluded that they would also be the major contributors to moderate levels of the antioxidant capacities of the algal extracts measured by the four different assays. Finally, since many published studies on the phenolic content and antioxidant capacity of algae were carried out without identifying the phenolic components, it was uncertain as to which components were responsible for the measured antioxidant activities. In light of the findings of the present study, it is worthwhile to revisit these studies to ascertain the presence and/or absence of phenolic compounds in these algal species.

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Chapter 4 Extraction, identification and biological activities of carotenoids from Ulva, Derbesia and Oedogonium

4.1. Introduction

Carotenoids are one of the most important groups of natural pigments, because of their wide distribution, structural diversity and numerous functions. These pigments are widely distributed in nature, from plants through to microorganisms, with more than 700 different molecular structures identified (Saini et al., 2015). However, only about 40 of these are present in a typical human diet. Of those 40, about 20 carotenoids have been identified in human blood and tissues. Close to 90% of the carotenoids in the diet and human body is represented by α-carotene, β-carotene, lycopene, lutein and cryptoxanthin (Rao and Rao, 2007).

Carotenoids are lipophilic compounds and can be broadly divided into carotenes and xanthophylls based upon their chemical structure. Carotenes are hydrocarbon carotenoids that are made only of C and H, while xanthophylls are oxidised carotenoids that contain some O-substituent groups such as hydroxyl, keto and epoxy groups. The spectrophotometric features of the carotenoids are produced by the conjugated double bond system and these same functional groups provide protection to the photosynthetic apparatus in plants by dissipating excess energy (Oliver and Palou, 2000). Carotenoids are excellent scavengers of singlet oxygen, which mainly arises from sunlight absorption by chromophores and thus protect chlorophylls, lipids, proteins and deoxyribonucleic acid (DNA) from oxidative damage. They are widely used as food colorant and functional ingredients in functional foods, nutraceuticals, cosmetics and other products (Rao et al., 2006, Murthy et al., 2005, Raposo et al., 2015). Carotenoids also exhibit protective actions against several human health disorders such as certain cancers and eye conditions (e.g., cataracts) (Koushan et al., 2013), skin texture (e.g., macular degeneration) (Tominaga et al., 2012), heart disease and cardiovascular disease (Nakao et al., 2010), improve the function of the immune system and enhance gap- junction communication (Oliver and Palou, 2000). For the structural identification of carotenoids, most researchers use high performance liquid chromatography (HPLC) coupled to mass detectors using electrospray or atmospheric pressure chemical

89 ionisation (Bhagavathy et al., 2011, Bhagavathy and Sumathi, 2012, Regnier et al., 2015, Matsuura et al., 2012, Rao et al., 2013a).

Algae are a good source of carotenoids, and many of which are extracted commercially. For example, the microalgae Dunaliella salina and Haematococcus pluvialis accumulate β-carotene and astaxanthin that are used as commercial food colouring agents (Hu et al., 2008). Muriellopsis has been used commercially to produce lutein due to its high lutein content and high growth rate (Ahmed et al., 2014). Chlorella vulgaris has also been reported as a high producer of lutein with commercial potentials (Cha et al., 2008). Recently, there has been a growing interest in astaxanthin from red alga (e.g., Dunaliella salina), siphonaxanthin from green seaweed (e.g., Codium fragile) and fucoxanthin from brown seaweed (e.g., Undaria pinnatifida). These carotenoids have been found to possess a number of potential health functions including antioxidant, antidiabetic, anti-obesity and anti-inflammatory properties as well as ability to inhibit cell growth and induce apoptosis in human cancer cells, and are generally lacking in terrestrial plants (Peng et al., 2011, Ambati et al., 2014, Sugawara et al., 2014).

Despite algae being a rich source of carotenoids, research on the biological activities algal carotenoids is still limited. Most of the research in this field is concerned with the identification and isolation of individual carotenoids from fruit, vegetables and some algae, and characterisation of their biological activities. However, information on the biological activities of carotenoid extracts of algae is very limited, particularly for macroalgae which are the primary targets for mass production with greater than 15 million tonnes produced annually around the world (Paul et al., 2012). This is significant for two reasons. For edible algae, the biological activities of the carotenoid extracts are likely more reflective of those of the algae than the individual carotenoids. For the production of algae sourced bioactive ingredients, carotenoid extracts would be simpler and less costly to produce than pure carotenoids.

The overall aim of this chapter is to investigate the carotenoid content, composition and health-related biological activities of Ulva ohnoi, Derbesia tenuissima and Oedogonium intermedium. The specific objectives are to:

1) Determine the carotenoid content of the three green macroalgae using both spectrophotometric and HPLC methods;

90

2) Identify and quantify carotenoids in the three algal species by HPLC and LC- MS; and 3) Investigate the antioxidant capacities of the carotenoid extracts, as well as their in vitro inhibitory activities against several metabolically important enzymes including α-amylase α-glucosidase, pancreatic lipase, hyaluronidase and angiotensin-converting enzyme (ACE).

4.2. Materials and methods

4.2.1. Chemicals and reagents

4-Methylumbelliferyl oleate (4-MUO), 4-nitrophenyl-a-D-glucopyranoside (p-NPG), Acarbose, angiotensin converting enzyme (ACE) from rabbit lung, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), hyaluronic acid sodium salt from Streptococcus equi, hyaluronidase from bovine testes, pancreatic lipase from Candida rugose, N-Hippuryl-his-Leu (HHL), sodium citrate, sodium potassium tartrate tetrahydrate, α-amylase from Bacillus subtilis (380U/mg) and α-glucosidase from Saccharomyces cerevisiae (100U/mg) were purchased from Sigma-Aldrich (Sydney, Australia). Acetonitrile, 3,5-dinitrosalicylic acid, ammonium acetate, boric acid, citric acid, potassium carbonate, sodium chloride, and potato starch were purchased from Ajax Finechem Pty Ltd (Sydney, Australia). 4-Dimethylaminobenzaldehyde (DMAB) was purchased from BDH Chemicals Ltd (Poole, UK). Dichloromethane was purchased from Merck KGaA (Germany). Methyl-tert-butyl ether (MTBE) was purchased from Fisher Scientific (Sydney, Australia). Orlistat was purchased from Tokyo Chemical Industry (Tokyo, Japan). Potassium hydroxide and sodium hydroxide were purchased from Chem-Supply Pty Ltd (Sydney, Australia). Suppliers for all other solvents and chemicals (methanol, hexane, acetone, acetic acid, hydrochloride acid (32%), DPPH, gallic acid, trolox, ABTS, TPTZ, sodium phosphate diabasic, sodium phosphate monobasic, potassium persulfate and iron (III) chloride hexahydrate) were given in Chapter 3 Section 3.2.1. All chemicals were of at least analytical grade unless stated otherwise. Solvents used in HPLC were of liquid chromatography grade and water used in all experiments was purified by reverse osmosis using the MilliQ RO system (referred to as MilliQ water).

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4.2.2. Seaweed sample collection and storage

The same three green macroalgal species, Ulva ohnoi, Derbesia tenuissima and Oedogonium intermedium are used here. The collection, treatment and storage of the algal biomass are described in Chapter 3, Section 3.2.2.

4.2.3. Extraction of carotenoids from seaweeds

4.2.3.1. Extraction methods

There is no standard extraction procedure for carotenoids because of the wide variety of food products and biological samples containing these compounds, and the great range of carotenoids that can be found in these matrices (Amaya, 2010). To optimise extraction of carotenoids from the algal biomass, two published extraction procedures were trialled. Procedure one was a method described by Hu et al. (2008). Under nitrogen gas, 200 mg of freeze-dried samples was extracted with 6 mL of hexane/acetone/ethanol (2:1:1, v/v/v) at ambient temperature for 24 h followed by addition of 270 μL of 40% methanolic KOH for saponification at ambient temperature for 8 h. After that, the mixture was centrifuged for 10 min at 3500 g (Sorvall TC-6, MedWoW, Wilmington, DE, USA). The supernatant was transferred to a separation funnel, washed 5 times, each time with 6 mL of MilliQ water to remove the alkali, and the solvent was removed with a rotary evaporator (Orme Scientific Ltd., Manchester, UK) at 30°C. The extract was then dissolved in 1 mL of methanol/dichloromethane (1/1, v/v) for carotenoid analysis. The entire extraction procedure was carried out under dim lighting. Saponification was employed to hydrolyse the carotenoid esters, remove fatty material and destroy chlorophyll. This step facilitated the subsequent separation, identification and quantification of the carotenoids.

Extraction procedure 2 was a modified Bligh-Dyer method (Rao et al., 2013a). Briefly, 15 mg of freeze-dried samples was mixed with 1.2 mL of MilliQ water, 3 mL of methanol, and 1.5 mL of dichloromethane, followed by vigorous mixing. After 10 min incubation at room temperature, the mixture was added with 1.5 mL each of distilled water and dichloromethane, and was mixed vigorously again. The organic layer was separated by centrifugation (10 min at 3500 g) and collected. The extraction step was repeated twice more using 3 mL of dichloromethane each time. The solvent was dried 92 with nitrogen (N2) gas, and the resultant extract was dissolved in 1 mL of methanol or DMSO for further analysis. The crude extract was then saponified with 10% methanolic KOH and kept overnight in the room temperature at dark; after which, the carotenoid extract was washed with water to remove the alkali, and dried over N2 gas.

For both extraction procedures, precautions were taken to avoid or minimise potential loss of carotenoids due to light, heat and oxidation. These included conducting the extraction procedures under dim lighting, use of low temperatures, evaporation of solvents with nitrogen streams where applicable and performing each operation in the shortest possible times.

As will be described in detail in the results section, procedure 2 was found to be superior to procedure 1, and extracts obtained by procedure 2 was used in all subsequent experiments.

4.2.3.2. Determination of total carotenoids

Determination of total carotenoids was carried out spectrophotometrically according to Lichtenthaler (1987). The dried extract (15 mg dry algal biomass weight equivalent) was re-dissolved in 5 mL of methanol, and the absorbance of the extracts was measured at 470, 665.2 and 652.4 nm by a Spectramax Plus M2 spectrophotometer (Molecular Devices, Australia). The pigment contents (chlorophyll a and chlorophyll b and total carotenoids) were calculated using the Lichtenthaler equations (Lichtenthaler, 1987):

4.2.4. Identification and quantification of carotenoids

4.2.4.1. HPLC- PDA analysis of carotenoids

To optimise separation of carotenoids, two HPLC procedures were tested. Both HPLC procedures were carried out using a Shimadzu Prominence UFLC system (Shimadzu,

Kyoto, Japan) that consisted of a vacuum degasser (DGU-20A5), two pumps (LC- 20AD), a photodiode array detector (PDA) (SPD-M20A), a column oven (CTO-20A), and an auto sampler (SIL-20A HT), connected to LC solution software. Carotenoids

93 were separated on a C30 column (250 x 4.6 mm, 5 µm particle size, YMC, Kyoto, Japan).

The elution program of procedure 1 was adapted from Mertz et al. (2010) with some modifications. The mobile phases consisted of three eluents, with methanol as eluent A, MTBE as eluent B and MilliQ water as eluent C. The flow rate was fixed at 1 mL/min and column temperature was set at 25°C. The gradient program was as follows: 0-2 min, 60% A/40% C, isocratic elution; 2-5 min, 80% A/20% C; 5-10 min, 81% A/15% B/4% C; 10-60 min, 11% A/85% B/4% C; 60-71 min, 100% A; 71-72 min, back to the initial conditions for re-equilibration. The injection volume was 20 µL and peaks were monitored at 450 nm.

For HPLC procedure 2, the mobile phases consisted of two eluents with acetonitrile/methanol/water (75/15/10, v/v/v) as eluent A, and methanol/ethyl acetate (70/30, v/v) as eluent B. The flow rate was fixed at 1 mL/min and column temperature was set at 25°C. The gradient program was: 0 to 5 min 0% B; 5 to 20 min, 0 to 100% B linear, 20 to 35 min, 100% B; 35 to 40 min, 100 to 0% B linear; 40 to 45 min, 0% B (back to the initial conditions for re-equilibration). The injection volume was 20 µL and peaks were monitored at 450 nm.

4.2.4.2. HPLC-PDA-APCI-IT-TOT-MS analysis of carotenoids

The analysis was performed using a Prominence LC system (Shimadzu, Kyoto, Japan) connected to a PDA detector (SPM-M20A, Shimadzu, Japan) followed by an ion trap- time of flight mass spectrometer (LCMS-IT-TOF, Shimadzu, Japan) equipped with an atmospheric pressure chemical ionisation (APCI) source. The extract was separated on a TSK gel ODS-80TsQA column (2.0 × 250 mm, 5μm, Tosoh, Tokyo, Japan) at 40°C. The injection volume was 5 μL and flow rate was 0.2 mL/min. The mobile phase consisted of acetonitrile/methanol/water (75/15/10, by vol.) containing 0.1% of ammonium acetate (A) and methanol/ethyl acetate (70/30, by vol.) containing 0.1% of ammonium acetate (B). The gradient elution was performed as follows: 0 to 5 min 0% B; 5 to 20min, 0 to 100% B linear, 20 to 35 min, 100% B; 35 to 40 min, 100 to 0% B linear; 40 to 45 min, 0% B. To quantify the carotenoids, absorbance at 450 nm was monitored. Mass spectra were recorded in positive ion mode with a detection voltage of

94

1.7 kV, APCI temperature of 400°C, curved desolation line of 200°C and block temperature of 200°C. Carotenoids were identified by comparing their retention times and mass spectra with those of reference standards and the mass spectra published in the literature.

4.2.4.3. HPLC-PDA quantification of carotenoids

Quantification of the detected carotenoids was carried out by HPLC-PDA using the external standard method by constructing standard curves of reference carotenoids. Due to the high costs of neoxanthin, loroxanthin and α-carotene, these three carotenoids were quantified based on the standard curves of 9’-cis-neoxanthin, lutein and β-carotene, respectively.

4.2.5. Assays of antioxidant capacity

The N2 dried carotenoid extracts were dissolved in 1 mL of methanol, and the in vitro antioxidant activities were determined by three methods: ABTS-radical scavenging assay, DPPH- radical scavenging assay and ferric reducing antioxidant power (FRAP) assay. The procedures for these assays were the same as those described in Chapter 3, Section 3.2.4. A standard curve of Trolox or gallic acid was constructed for each assay and the regression coefficient (R2) was greater than 0.98 for all three assays. Each assay was done in triplicates.

4.2.6. Assays of inhibitory activity against metabolically important enzymes

4.2.6.1. In vitro pancreatic lipase inhibition assay

The inhibition of pancreatic lipase activity was determined following the method described by Sakulnarmrat and Konczak (2012) with slight modifications. Initially, stock solutions of the sample extracts were prepared at a concentration of 15 mg dry algal biomass weight equivalent/mL (henceforth expressed as dry weight equivalent) in DMSO, and working sample solutions were prepared by diluting the stock extract solutions in DMSO to 10, 5 and 1 mg dry weight equivalent/mL. Pancreatic lipase from 95

Candida rugose (65 mg/mL in Mcllvaine’s buffer (0.1 M citric acid and 0.2 M disodium phosphate mixed in appropriate portions to arrive at pH 7.4) and 0.1 mM 4- MUO in DMSO served as the reaction enzyme and fluorogenic substrate, respectively. Prior to use, the enzyme solution was centrifuged at 3500g for 10 min, and the supernatant was collected. For the assay, a mixture of 100 μL of the substrate and 50 μL of sample solution, orlistat (positive control) or solvent blank (negative control) in DMSO was incubated at 37°C for 5 min, followed by addition of 50 μL of enzyme solution in a 96 well microplate. After reaction at 37°C for 20 min, 1 mL of 0.1N HCl was added to terminate the reaction followed by the addition of 2 mL of 0.1 M sodium citrate. The amount of 4-methylumbelliferone released by the pancreatic lipase was measured fluorometrically at an emission wavelength of 460 nm and excitation wavelength of 320 nm using a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, USA). Orlistat was used to plot a standard calibration curve with R2=0.957. The pancreatic lipase inhibition activity was calculated using the following formula:

(퐹 − 퐹 ) % inhibition = 1 − 푆 푆퐵 × 100 (퐹퐶 − 퐹퐶퐵)

Where FS and FC were the values of samples and negative control measured fluorometrically, and FSB and FCB were the fluorescence readings of sample and control blank, respectively.

4.2.6.2. In vitro α-amylase inhibition assay

This activity was determined as previously described by Lordan et al. (2013). Starch solution (1% w/v) was used as the substrate, which was prepared by mixing 0.5 g starch in 50 mL of 20 mM sodium phosphate buffer (pH 6.9 with 6 mM sodium chloride), heating to boil and maintaining the solution at boiling temperature for 15 min. Afterwards, the starch solution was cooled naturally to room temperature with stirring, and then made to the original volume with water. For the assay, equal volumes (100 µL) of extract (1, 5, 10 and 15 mg dry weight equivalent/mL) and the starch solution were incubated in eppendorf tubes at 25°C for 10 min. A volume of 100 μL of α-amylase (0.5 mg/mL) was added to each tube and the reaction mixtures were incubated at 25°C for a

96 further 10 min. The reaction was stopped with the addition of 200 μL of colour reagent solution (prepared by mixing 8 mL of sodium potassium tartrate solution (12.0 g in 2M NaOH) and 20 mL of 96 mM 3,5-dinitrosalucyclic acid solution) and incubation at 100°C for 5 min. Once the mixtures had cooled to room temperature, 50 μL was removed from each tube and transferred to the wells of 96 well microplates. The reaction mixture was diluted by adding 200 μL of MilliQ water to each well and absorbance was measured at 540 nm. The pharmacological inhibitor, Acarbose, was included as a positive control and a standard calibration curve was plotted with R2=0.958. Blank readings (no enzyme) were substrate from each well and results were comparted to the control. The activity of amylase inhibition was calculated as follows:

(퐴 − 퐴 ) − (퐴 − 퐴 ) % inhibition = 퐶 퐶퐵 푆 푆퐵 × 100 (퐴퐶 − 퐴퐶퐵)

Where AC and AS were the absorbance readings of the control and sample, and where

ACB and ASB were the absorbance readings of the control and sample blank, respectively.

4.2.6.3. In vitro α-glucosidase inhibition assay

This activity was determined following the method described by Rengasamy et al. (2013). Briefly, α-glucosidase (0.1 U/mL) was dissolved in 0.1 M potassium phosphate buffer (pH 6.8) which was used as the enzyme solution. The substrate, 0.375 mM of p- nitrophenyl-α-D-glucopyranoside (p-NPG), was prepared in the same buffer and the sample extracts were dissolved in DMSO at the concentration of 1, 5, 10 and 15 mg dry weight equivalent/mL. Aliquots of (30 µL) each sample or Acarbose (positive control) were mixed with the enzyme solution (30 μL) in 96 well microplates and the reaction was initiated by adding 60 μL of the substrate. The reaction mixture was incubated at 37°C for 40 min in darkness; after that, 120 μL of 0.2 M sodium carbonate in 0.1 M potassium phosphate buffer (pH 6.8) was added to each well to quench the reaction. The amount of p-nitrophenol (pNP) released was quantified using a 96-well microplate reader at 405 nm. Acarbose was used as a positive control and a standard calibration curve was plotted with R2=0.941. The determinations were carried out in triplicate. The percentage inhibition (%) of α-glucosidase was calculated as follows:

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퐴 − 퐴 % inhibition = 퐶 푆 × 100 퐴퐶

Where AC and AS were the absorbance readings of the control and sample.

4.2.6.4. Hyaluronidase inhibition assay

The colorimetric Morgan-Elson assay was used to determine the inhibitory activity of algal extracts against hyaluronidase following the procedure described by Muckenschnabel et al. (1998) with some modifications. The extracts 5, 10, 15 and 30 mg dry weight equivalent/mL or quercetin (0.1 mg/mL, positive control) dissolved in DMSO (27 μL), were mixed with a reagent mixture that contained 300 μL of citrate- phosphate buffer (pH 5.0) (0.1 M Na2HPO4/0.1M NaCl and 0.1M citric acid/0.1M NaCl mixed in appropriate portions to arrive at pH 5.0), 75 μL BSA solution (0.2 mg BSA/mL water), 75 μL substrate hyaluronic acid (HA) solution (5 mg/mL in water), 129 μL MilliQ water and 75 μL of the enzyme solution (100U). The mixture was incubated at 37°C for 1 h. After that, the enzymatic reaction was stopped by the addition of 160 μL of alkaline borate solution and heating for 4.5 min in a boiling water bath. The alkaline borate solution was prepared by combining 10 volumes of borate solution (2.8 M H3BO4/1.4 M KOH) and 1 volume of 5.8 M potassium carbonate solution. After cooling on ice for at least 2 min, 90 µL of the reaction mixture was transferred to a 96 well microplate and mixed with 110 μL of Ehrlich’s reagent. The mixture was incubated at 37°C for 20 min, and the absorbance of the coloured product was measured at 590 nm on a microliter plate reader. The Ehrlich’s reagent was prepared immediately before use by diluting 1 volume of 1.3 M DMAB in 32% HCl/glacial acetic acid (1:4, v/v) with 4 volumes of glacial acetic acid.

Inhibition of the enzyme activity was calculated according to the equation:

(퐴 − 퐴 ) Enzyme activity % = 푆 푆퐵 × 100 (퐴퐶 − 퐴퐶퐵)

Where AS was the absorbance of the incubation mixture containing the extract, AC was the absorbance without the extract (extract solution replaced with DMSO); ASB was the absorbance of the incubation mixture containing the inhibitor without the enzyme

(enzyme solution replaced with buffer) and ACB was that in the absence of both the 98 enzyme and extract (enzyme solution replaced with the buffer and extract solution replaced with DMSO).

4.2.6.5. ACE inhibition assay

ACE-inhibitory activity of the algal carotenoid extracts was assayed according to the method of Boschin et al. (2014) using HHL as the substrate, freshly prepared daily, and HPLC-PDA to detect hippuric acid (HA), the product of the enzymatic reaction. A volume of 100 µL of 2.5 mM HHL in 100 mM Tris buffer (containing 300 nM NaCl, pH 8.3) was mixed with 30 µL of the sample extract (10 mg dry weight equivalent/mL). The mixture was pre-incubated at 37°C for 15 min, and then 15 µL of ACE solution, corresponding to 3 mU of enzyme in the same buffer, was added. The mixture was incubated for 60 min at 37°C, and the reaction was stopped with the addition of 125 µL of 0.1 M HCl. The reaction mixture was extracted twice each with 600 µL of ethyl acetate. The solvents were combined and evaporated under nitrogen gas, and the resultant residue was dissolved in 500 µL of the same buffer and analysed by HPLC.

HPLC analyses were performed with the same HPLC system as described in Section 4.2.4.1. Hippuric acid (HA) was detected on a Luna C18 column (250 x 4.6 mm i.d., 5μ) column (Phenomenex, Sydney, Australia). Water and acetonitrile were used as eluents with the following gradient program: 0 min 5% acetonitrile, 10 min 60% acetonitrile, 12 min 60% acetonitrile, 15 min 5% acetonitrile. The flow rate was fixed at 0.5 mL/min. The injection volume was 10 µL and peaks were monitored at 228 nm. The retention time of hippuric acid (HA) was 4.2 min. The detector response for standard HA was linear in the range from 1-100 µg/mL with R2 = 0.999.

4.2.7. Statistical analysis

The whole experiment, starting from the extraction of carotenoid, was conducted twice and each analysis was performed usually in triplicate (n≥3). The results were presented as mean ± standard deviation (SD). One way analysis of variance (ANOVA) was carried out using SPSS version 22 (IBM, USA), to determine whether significant differences exist among treatments and Duncan triplicates range test was used to

99 separate significant differences between the means at the 5% level. A p-value <0.05 was considered as statistically significant. All graphs were drawn by Graphpad prism 6 (Graphpad Software Inc, Australia).

4.3. Results and discussion

4.3.1. Total carotenoids content

The crude and saponified extracts obtained from Ulva, Derbesia and Oedogonium were analysed for chlorophylls and total carotenoids (Table 17). The chlorophyll concentrations in saponified extracts were considerably lower (p<0.05) than in crude extracts for all the species. This was especially so for chlorophyll a, for which the amounts in the saponified extracts were less than 10% of those in the crude extracts, and in the case of Ulva and Oedogonium, they were less than 5%. The amounts of chlorophyll b in the saponified extracts were also significantly lower than in the crude extracts, although the magnitudes of the differences were not as pronounced. This demonstrated that saponification was very effective for the removal of chlorophylls from the carotenoid extracts. Compared with the huge loss of chlorophylls by saponification, the loss of total carotenoids by this treatment was much less. The amounts of carotenoids in the saponified extracts were around 85% of those in the crude extracts.

The inclusion of a saponification step in the extraction of carotenoids was mainly aimed at removing saponifiable impurities such as lipids and chlorophylls, which could interfere with the chromatographic separation and detection of carotenoids as well as the assay of their biological activities. While most lipids can easily be hydrolysed under the alkaline conditions of saponification, chlorophylls can also be saponified and removed, together with the saponified lipids, in the aqueous phase from the organic phase which contained the carotenoids (Larsen and Christensen, 2005, Bijttebier et al., 2014). The majority of carotenoids are stable towards alkaline treatment. However, alkaline treatment may cause isomerisation and several other unwanted reactions of carotenoids which could cause destruction or structural transformation of carotenoids (Britton et al., 1994). For this step to succeed, it needs to achieve a substantial removal of chlorophylls while retaining most of the carotenoids in the extracts. In the present

100 study, this objective can be regarded as having been achieved as the saponification removed more than 90% of the chlorophyll a and also most of the chlorophyll b while more than 85% of the carotenoids were retained. Loss of some carotenoids during saponification has been reported in the literature. Divya et al. (2012) compared the concentration of β-carotene before and after saponification and observed a loss of 20-30% of this compound and over 50% of other carotenes during saponification of coriander extracts. Biehler et al. (2010) also reported that saponification led to significant carotenoid losses by 12.6% in a variety of fruits and vegetables.

Of the three algal species, Oedogonium had the highest amounts of total carotenoids at 3411.2 and 2929.6 µg/g for the crude and saponified extracts, respectively. These were followed closely by Derbesia, while the carotenoid contents in Ulva were markedly less at 224.5 and 195.8 µg/g for the crude and saponified extracts, respectively. The levels of carotenoids in Oedogonium and Derbesia were comparable with those reported for the acetone extract of Botryococcus braunii (Rao et al., 2006).

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Table 17. Chlorophyll a, chlorophyll b and carotenoid content of Ulva, Derbesia and Oedogonium

Content (μg/g)

Species Crude Saponified

Chlorophyll a

Ulva 1774.8±2.4aB 81.2 ±0.9aA

Derbesia 10906.2 ±8.2bB 1019.0 ±14.8cA

Oedogonium 21877.0 ±16.8cB 688.7 ±19.2bA

Chlorophyll b

Ulva 1126.8 ±0.4aB 560.4 ±5.0aA

Derbesia 11604.4 ±6.5cB 1986.1 ±19.4cA

Oedogonium 10258.1 ±10.2bB 1871.5 ±9.0bA

Total carotenoids content

Ulva 224.5 ±1.0aB 195.8 ±2.0aA

Derbesia 2775.5 ±16.5bB 2347.8 ±3.4bA

Oedogonium 3411.2 ±20.7cB 2929.6 ±5.9cA

All values are mean ± SD for triplicate experiments (n=3). Different lower case letters within the same content for different species indicate significant difference between the values (p<0.05). Different capital letters within the same row indicate significant difference between the values (p<0.05).

4.3.2. Identification of carotenoids in algal extracts

4.3.2.1. Optimisation of extraction and HPLC procedures for the identification of carotenoids

Carotenoids in algae are diverse molecules in a complex biological matrix and there are no standard procedures for their extraction. However, most extraction methods follow a common path involving the release of carotenoids from their matrices by disrupting tissues, followed by a saponification step to remove the unwanted components. Two different extraction procedures were tested in this study, which differed mainly in the polarities of the extraction solvents. The polarities of the carotenoids and the structure of the analytical matrix and its components are key factors influencing the selection of extraction solvents. Usually, polar solvents, such as ethanol and acetone, are more

102 appropriate for polar carotenoids (e.g., xanthophylls), while non-polar solvents, such as hexane, are good choice for non-polar (e.g., carotenes) or esterified carotenoids (Carrilho et al., 2014). Because the types of the carotenoids present in the three algal species of the present study are unknown, both types of solvents were used to compare their extraction efficacy.

Furthermore, the carotenoids obtained by the two different extraction procedures were analysed by two different HPLC methods to compare their separation capacity. This combination of different extraction and HPLC procedures provided a basis to identify the optimal extraction and HPLC gradient programs for analysing carotenoids in the algal samples. The HPLC chromatograms so obtained for Ulva are shown in Figure 15, while the chromatograms for Derbesia and Oedogonium are shown in Appendix G.

Figure 15 (A) and (B) were chromatograms of Ulva extracts obtained by extraction procedures 1 and 2, respectively and analysed by HPLC method 1. Most of the compounds were separated with the retention times ranging from 15 to 45 min. However, Figure 15 (A) highlighted a greater abundance of peaks than Figure 15 (B), although both achieved good resolution of the peaks. Therefore, it was concluded that the combination of extraction procedure 1 and HPLC method 1 (Figure 15 (A)) yielded the better results. Similarity, when Figures 15 (C) and (D), which were chromatograms of the same Ulva extracts but analysed by HPLC method 2, were compared, the data indicates that extraction procedure 2 was more suited to HPLC analysis method 2. When Figure 15 (A) and Figure 15 (D) were compared, it was revealed that the former had two major HPLC peaks and several minor ones resolved at baseline, while the latter produced four well resolved HPLC peaks as well as several minor ones. Furthermore, the runtime of HPLC procedure 2 was about one third shorter than procedure 1. For these reasons, extraction procedure 2 and HPLC method 2 were concluded to be the best for extracting and separating carotenoids from the three green algal samples, and were used in all subsequent experiments, including LC-MS analysis.

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(A) 1

2

(B) 1 mAU 7.5

5.0

2.5

15.0 20.0 25.0 min

Figure 15. HPLC profile of carotenoids isolated from Ulva with (A) extraction procedure 1 and HPLC method 1, (B) extraction procedure 2 and HPLC method 1. Extraction procedure 1 used hexane/acetone/ethanol as solvent, while extraction procedure 2 used dichloromethane/water/methanol. For HPLC method 1, the mobile phase was methanol, MTBE and water, while for method 2, it was acetonitrile/methanol/water and methanol/ethyl acetate. Peak 1 and 2 are major peaks.

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(C) 1 3

2

2 mAU(x10) (D) 3.0

2.0

1.0

0.0

17.5 20.0 22.5 25.0 min

1 3

Figure 15. (Continued) HPLC profile of carotenoids isolated from Ulva with (C) extraction procedure 1 and HPLC method 2, and (D) extraction procedure 2 and HPLC method 2. Extraction procedure 1 used hexane/acetone/ethanol as solvent, while extraction procedure 2 used dichloromethane/water/methanol. For HPLC method 1, the mobile phase was methanol, MTBE and water, while for method 2, it was acetonitrile/methanol/water and methanol/ethyl acetate. Peak 1, 2 and 3 are major peaks.

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4.3.2.2. LC-MS identification of carotenoids in algal extracts

LC-MS analysis is particularly useful for studies of carotenoids obtained from natural sources, since these compounds are usually present in trace quantities coexisting with other compounds in a complex mixture. The sensitivity, specificity and selectivity of LC-MS make it an essential tool for the characterisation and identification of carotenoids. LC-MS has been used for the identification of carotenoids in several food samples, such as Chinese herbs (Kao et al., 2012), papaya (Sancho et al., 2011), mango (Ornelas-Paz et al., 2008), capsicum (Giuffrida et al., 2013) and cabbage (Watanabe et al., 2011).

The LC-MS chromatographic profiles of carotenoids in Ulva, Derbesia and Oedogonium are shown in Figure 16. The algal extracts contained three classes of pigments, namely xanthophylls, chlorophylls, and hydrocarbon carotenoids, which were separated on a C18 column within 35 min using a gradient elution system. Ammonium acetate was added to the elution solvents to improve the recovery of carotenoids from the column. Due to the low polarity nature of carotenoids, the mass spectra of carotenoid standards and unknown peaks were obtained using the atmospheric pressure chemical ionisation (APCI) source with positive mode for detection of [M+H]+ ions. These pigments were first tentatively identified by comparison of their retention times and UV-Vis absorption spectra with those of the respective reference standards. Confirmation of the chemical structures of the peaks was made by comparison of their mass spectra with those of reference standards as well the mass spectrometric data published in the literature.

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Ulva sp. 6 * 9 2’ 100 3 5 mAU

0. 5. 10. 15. 20. 25. 30. 35. 40. 45.0 0 0 0 0 0 0 0 0 0 min

* Derbesia sp. 7 6’ 8’ 1 2’ 3

0. 5. 10. 15. 20. 25. 30. 35. 40. 45.0 0 0 0 0 0 0 0 0 0 min

Oedogonium sp. *

4 5 6 2 3 8 9

0. 5. 10. 15. 20. 25. 30. 35. 40. 45.0 0 0 0 0 0 0 0 0 0 min

Figure 16. HPLC-PDA chromatograms of carotenoid extracts of three algal species at 450 nm. Peak denotation: peak 1, siphonaxanthin; peak 2, neoxanthin; peak 3, 9’-cis-Neoxanthin; peak 4, loroxanthin; peak 5, violaxanthin; peak 6, lutein; peak 7, siphonein; peak 8, α-carotene; peak 9, β-carotene; peak 2’, a mixture of neoxanthin and some unknown pigments; Peak 6’, mixture of lutein and some unknown pigments; peak 8’, mixture of α-carotene and chlorophylls. *, chlorophylls.

For example, peak 5 was initially identified tentatively as violaxanthin, based on comparison of its retention time and UV absorption spectra with the reference standard, as shown in Figure 17. The UV-Vis absorption spectral characteristics of peak 5, i.e.

λmax at 326, 410, 434, and 465 nm, were virtually identical to those of the reference standard violaxanthin and also agreed to those reported by Inbaraj et al. (2006) for the same compound. The mass spectra of peak 5 and violaxanthin reference standard are shown in Figure 18. As can be seen, they were identical with characteristic molecular

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and fragment ions [M+H] + 601, [M+H-18] + 583, [M+H-18-18] + 565 and [M+H-92] + 509, which were the same as those reported by (Inbaraj et al., 2006) and (Kao et al.,

2011) for violaxanthin. UV-Vis absorption spectral λmax comparisons of all other peaks with reported data of reference standards are shown in Table 18, while comparison of MS spectral data obtained for the peaks with reference standards and those reported in literature are presented in Table 19. The raw data of UV-Vis and mass spectra for the detectable carotenoids from Ulva, Derbesia and Oedogonium are shown in Appendix H, and the raw data of UV-Vis and mass spectra for the carotenoid standards are shown in Appendix I. Using these techniques, detectable carotenoids in the three algal species were identified and confirmed to be comprised of siphonaxanthin (peak 1), neoxanthin (peak 2), 9’cis-Neoxanthin (peak 3), loroxanthin (peak 4), violaxanthin (peak 5), lutein (peak 6), siphonein (peak 7), α-carotene (peak 8) and β-carotene (peak 9). The chemical structures of these carotenoids are shown in Appendix J.

mAU

175 13.77/ 1.00

438 468 150 (A) (B)

125

415 456 100 423

75

50

25

319 339 0

300 350 400 450 500 550 nm

Figure 17. UV-Vis absorption spectra of (A) reference standard of violaxanthin and (B) Inten.(x10,000,000)peak 5 in Ulva. (A) 601.4601 (B) 2.0

1.0 583 565 0.0 509 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z Figure 18. Mass spectra of (A) reference standard of violaxanthin, and (B) peak 5 in Ulva. 108

There is no published information on the carotenoids of Ulva, Derbesia and Oedogonium, and this is the first report on the carotenoids of these commercially significant green algal species from Australia. Analysis of carotenoids has been done on a few other green algal species including Chlorococcum humicola (Bhagavathy et al., 2011, Bhagavathy and Sumathi, 2012), Botryococcus braunni (Rao et al., 2006), Haematococcus pluvialis (Ranga et al., 2009) and Dunaliella salina (Hu et al., 2008), using similar analytical techniques. The carotenoids found in the green algae of the current study were similar to those in the literature with the major compounds being violaxanthin, lutein, α-carotene, β-carotene, astaxanthin and zeaxanthin. The first four compounds were also found in the three green algae of the present study, but the last two carotenoids were not. However, siphonaxanthin and siphonein were found in Derbesia and loroxanthin was found in Oedogonium, which were not reported for the other green algae. Other major differences among the different algae appear to be in the levels at which the various carotenoids are present. These differences may be related to species variations and environmental factors (Goiris et al., 2012).

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Table 18. UV-Vis spectral data for carotenoids identified in Ulva, Derbesia and Oedogonium

a a Peak no Compound λmax (on-line)/nm λmax (standard)/nm λmax (reported)/nm

Ulva 2 Neoxanthin - - 441 468 - 412 435 464 - 418 440 468c 3 9’-cis-Neoxanthin 338 411 434 464 n.a n.a n.a n.a 328 414 436 464c 5 Violaxanthin 343 415 438 468 327 415 438 468 - 416 440 468c 6 Lutein 347 421 444 472 330 420 444 472 332 423 446 470e 9 β-Carotene - - 449 481 - - 450 474 350 430 458 482c Derbesia 1 Siphonaxanthin - - - 449 - - - 452 - - 333 449b 2 Neoxanthin - - 441 468 - 412 435 464 - 418 440 468c 3 9’-cis-Neoxanthin 338 411 434 464 n.a n.a n.a n.a 328 414 436 464c 6 Lutein - - 447 474 330 420 444 472 332 423 446 470e 7 Siphonein - - - 458 - - - 458 - - - 452f 8 α-Carotene 341 416 444 472 n.a n.a n.a n.a 344 426 449 476c Oedogonium 2 Neoxanthin - - 439 467 - 412 435 464 - 418 440 468c 3 9’-cis-Neoxanthin 327 412 435 464 n.a n.a n.a n.a 328 414 436 464c 4 Loroxanthin - - 444 471 n.a n.a n.a n.a - 423 447 475d 5 Violaxanthin 326 415 438 468 327 415 438 468 - 416 440 468c 6 Lutein - - 444 472 330 420 444 472 332 423 446 470e 8 α-Carotene - - 444 472 n.a n.a n.a n.a 344 426 449 476c 9 β-Carotene - - 450 475 - - 450 474 350 430 458 482c n.a: standards were not available. aA gradient mobile phase of acetonitrile/methanol/water (75/15/10, v/v/v) containing 0.1% of ammonium acetate and methanol/ethyl acetate (70/30, v/v) containing 0.1% of ammonium acetate. bA mobile phase of acetonitrile, methanol and water (75:15:10, v/v/v) containing 0.1% (w/v) ammonium acetate was used by (Ganesan et al., 2010). cA mobile phase of methanol/acetonitrile-water (84/14/2,v/v/v) and methylene chloride was used by (Kao et al., 2011). dA mobile phase of methanol: acetonitrile: aqueous pyridine solution (0.025 M pyridine, pH adjusted to 5.0 with acetic acid) in the proportions 50:25:25 (v/v/v), and acetonitrile: acetone (80:20 v/v) (Garrido et al., 2009). eA mobile phase of methanol/acetonitrile-water (84/14/2,v/v/v) and methylene chloride was used by (Inbaraj et al., 2006). fBased on a reference by (Ricketts, 1971).

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Table 19. MS spectral data for carotenoids identification in Ulva, Derbesia and Oedogonium

Peak Compound [M+H]+ [M+H]+ [M+H]+ Fragment ions Fragment Fragment ions (m/z) reported (m/z) (m/z) (m/z) (m/z) found ions (m/z) found standard reported standard Ulva 2 Neoxanthin 601 601 601b 583, 565 583,565 583, 565, 547, 509, 491, 221b 3 9’-cis-Neoxanthin 601 601 601b 583, 565 n.a 583, 565, 547, 509, 393, 221b 5 Violaxanthin 601 601 601b 583,565,509 583,565 583, 565, 509, 491, 221b 6 Lutein 569 569 569b 551, 533 551,533 551, 533, 495, 477, 463, 459b 9 β-Carotene 537 537 537b 444 444 444b Derbesia 1 Siphonaxanthin 601 601 601a 583, 565 583 583, 565, 547a 2 Neoxanthin 601 601 601b 583, 565 583,565 583, 565, 547, 509, 491, 221b 3 9’-cis-Neoxanthin 601 601 601b 583, 565 n.a 583, 565, 547, 509, 393, 221b 6 Lutein 569 569 569b 551, 533 551,533 551, 533, 495, 477, 463, 459b 7 Siphonein 763 763 763d 628, 593 628, 593 628, 593, 583d 8 α-Carotene 537 537 537b 481 n.a 481, 444b Oedogonium 2 Neoxanthin 601 601 601b 583, 565 583,565 583, 565, 547, 509, 491, 221b 3 9’-cis-Neoxanthin 601 601 601b 583,565 n.a 583, 565, 547, 509, 393, 221b 4 Loroxanthin 567 567 567c 549,531 n.a 549, 531c 5 Violaxanthin 601 601 601b 583,565 583,565 583, 565, 509, 491, 221b 6 Lutein 569 569 569b 551,533 551,533 551, 533, 495, 477, 463, 459b 8 α-Carotene 537 537 537b 481 n.a 481, 444b 9 β-Carotene 537 537 537b 444 444 444b n.a: standards were not available. aBased on a reference by (Ganesan et al., 2010). bBased on a reference by (Kao et al., 2011). cBased on a reference by (Garrido et al., 2009). dBased on a reference by (Ricketts, 1971).

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4.3.2.3. Quantification of carotenoids in Ulva, Derbesia and Oedogonium

The detected carotenoids were quantified by using the external standard method where standard curves were constructed. Due to the high costs of carotenoid standards, neoxanthin, loroxanthin and α-carotene were quantified based on the standard curves of 9’-cis-neoxanthin, lutein and β-carotene, respectively. The carotenoid content for each algal species are shown in Table 20. As can be seen, Oedogonium had the highest amounts of total carotenoids, followed by Derbesia and Ulva, which agrees with the trend of the total carotenoids reported earlier (Table 17). However, for Derbesia, the total carotenoid content quantified by HPLC (1277.2 µg/g) was much lower than that by the spectrophotometric method (2347.8 µg/g), partly because three of the detectable compounds (neoxanthin, lutein and α-carotene) could not be quantified by HPLC due to contaminations of the peaks. Also, the total amount of carotenoids quantified by HPLC in Oedogonium equated to 77% of that determined by the spectrophotometric method. For Ulva, the total amount of carotenoids content quantified by HPLC (300.0 µg/g) was higher than measured earlier (195.8 µg/g). It has been reported that spectrophotometric methods tend to overestimate the carotenoid content when compared to HPLC quantification. This is mainly due to the former measuring not just carotenoids, but also certain impurities such as carotenoid degradation products (Kimura et al., 2007) and also chlorophyll degradation products, e.g., chlorophyllides (Almela et al., 2000), which also absorb UV-Vis light at similar wavelengths. Biehler et al. (2010) stated that the average carotenoid concentration obtained by the Lichtenthaler method was about 21% higher than that by HPLC.

Four carotenoids were present in Ulva at appreciable concentrations, and among which the xanthophyll, lutein, was the large component accounting for 59.8% of the total carotenoids. β-Carotene was the second most abundant carotenoid, accounting for 22.1%, while 9’-cis-neoxanthin and violaxanthin were present in similar but much lower amounts compared with the other two compounds. Only three carotenoids were present in detectable amounts in Derbesia, with siphonaxanthin being the most abundant (44.2%), followed by siphonein (31.3%) and 9’-cis-neoxanthin (24.5%). Other three detectable compounds cannot be quantified due to contamination of the peaks. Oedogonium was not only the alga with the highest amount of total carotenoids, it also contained the greatest diversity of carotenoids, with several of them present in similar

112 amounts. The most abundant compound was lutein (18.4%), followed closely by violaxanthin (18.1%), and 9’-cis-neoxanthin (17.2%). Neoxanthin (602 nmol/g), loroxanthin (526 nmol/g), and β-carotene (534 nmol/g) were also present in appreciable amounts, while α- carotene (143 nmol/g) was the least abundant carotenoid.

Hydroxylation of hydrocarbon carotenoids is known to be responsible for the formation of 3-hydroxy cyclic carotenoids and epoxy carotenoids (Rao et al., 2013a). The presence of relatively low levels of α-carotene and β-carotene in Ulva and Oedogonium may therefore be related to the conversion of these compounds to lutein. This may be a reason for the higher content of lutein in these two algae (Rao et al., 2006). Interestingly, Derbesia contained no detectable amount of lutein, but a very high level of siphonaxanthin, which is a member of keto-carotenoids mostly found in siphonaceous green algae (Sugawara et al., 2014). Derbesia can therefore be considered as a new source for siphonaxanthin (0.05% of dry algal biomass). Previously, siphonaxanthin has only be reported in marine green algae such as Codium fragile, Caulerpa lentillifera and Umbraulva japonica, and the content is approximately 0.03%-0.1% of the dry weight (Li et al., 2015, Ganesan et al., 2011). Oedogonium contained a reasonable amount of loroxanthin, which had been detected in certain green algae but, to date, this carotenoid is found only in a small number of algal species (Aitzetmuller et al., 1969).

Table 20. Carotenoid contents in three green algae determinate by HPLC nmol/g dry weight or% Ulva Derbesia Oedogonium nmol/g % nmol/g % nmol/g % Siphonaxanthin (peak 1) n.d. n.d. 859 44.2 n.d. n.d. Neoxanthin (peak 2) # - # - 602 15.5 9’-cis-Neoxanthin (peak 3) 45.9 8.7 475 24.5 671 17.2 Loroxanthin (peak 4) n.d. n.d. n.d. n.d. 526 13.5 Violaxanthin (peak 5) 49.7 9.4 n.d. n.d. 705 18.1 Lutein (peak 6) 316 59.8 # # 715 18.4 Siphonein (peak 7) n.d. n.d. 608 31.3 n.d. n.d. α-Carotene (peak 8) n.d. n.d. # # 143 3.7 β-Catotene (peak 9) 117 22.1 n.d. n.d. 534 13.7 Total 528.6 1942 3896 Neoxanthin, loroxanthin, and α-carotene were quantified based on standard curves of 9’-cis-neoxanthin, lutein, and β-carotene, respectively. n.d., not detectable. #, not quantifiable due to co-elution with contaminants.

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Carotenoids have been shown to have a preventive effect against a number of diseases, such as arteriosclerosis, cardiovascular disease, cancer (liver, medulla and prostate tumours), macular degeneration and neurodegenerative diseases like Alzheimer’s disease (Tominaga et al., 2012, Nakao et al., 2010, Oliver and Palou, 2000). For example, siphonaxanthin has been shown to potently inhibit the viability of human leukemia HL-60 cells via induction of apoptosis (Ganesan et al., 2011). It has also been found to play a role in preventing angiogenesis related diseases and exhibit strong inhibitory effect on human umbilical vein endothelial cells (Ganesan et al., 2010). A recent study has reported that compared with other carotenoids, siphonaxanthin has much greater potence in inhibiting adipocyte differentiation because it can effectively regulates adipogenesis in 3T3-L1 cells (Li et al., 2015). Oral administration of siphonaxanthin to diabetic KK-Ay mice significantly reduced the total weight of white adipose tissue (Li et al., 2015). Lutein and its coexistent isomer, zeaxanthin, play a vital role in the maintenance of normal visual function of the human macula as well as a number of other biological properties, such as reducing the risk of certain types of cancer, particularly those of the breast and lung, and potential prevention of heart disease and stroke (Hahn and Mang, 2008, Koushan et al., 2013, Sarmadi and Ismail, 2010, Ribaya-Mercado and Blumberg, 2004, Abdel-Aal et al., 2013, Roberts et al., 2009). Violaxanthin has been shown to exhibit a strong anti-proliferative activity on a human mammary cancer cell line (Pasquet et al., 2011). β-Carotene is a well-known strong antioxidant and is believed to mitigate the harmful effects of free radicals implicated in various disorders such as many forms of gastrointestinal cancer (Stahl et al., 1998, Stahl and Sies, 2005, El Baz et al., 2002), and lung cancer (Goralczyk, 2009). Also, β-carotene may help prevent symptoms of Alzheimer’s disease (AD) through inhibition of amyloid beta formation, deposition and fibril formation either by reducing the levels of p35 or inhibiting corresponding enzymes (Obulesu et al., 2011). Neoxanthin was found to induce apoptosis through caspase-3 activation in PC-3 human prostate cancer cells (Kotake-Nara et al., 2005). The presence of these carotenoids in the three green macroalgae in relatively high levels, especially siphonaxanthin, which is found only in a few green algal species, implies that they have the potential to be new sources for these carotenoids, or be developed into bioactive ingredients for functional food, nutraceutical and pharmaceutical products. It also implies that consumption of

114 these algae directly or as a food ingredient may provide significant health benefits to the consumer.

4.3.3. Antioxidant activities of the carotenoid extracts of the three green macroalgae

Antioxidants have a positive effect on human health as they can protect the human body from damage by ROS, which attack macromolecules such as membrane lipids, proteins and DNA, leading to many health disorders such as cancer, diabetes mellitus, aging and neurodegenerative diseases (Amaya, 2010). Although carotenoids are widely used as colorants, they also play an important role as powerful antioxidants. In this study, the antioxidant activities of crude and saponified extracts of Ulva, Derbesia and Oedogonium were assessed using three different in vitro assays, ABTS scavenging capacity, DPPH scavenging capacity and FRAP (ferric reducing capacity) assays.

4.3.3.1. ABTS free radical scavenging capacity

In this assay, ABTS radical was generated by chemical reaction with potassium persulfate. When antioxidants were added, the ABTS radical, which has a blue-green colour, is reduced to ABTS (colourless). Different de-coloration abilities indicate different ABTS scavenging capacities (Thaipong et al., 2006). ABTS is soluble in both hydrophilic and hydrophobic solvents, and can therefore be applied to a wide range of phytochemicals including the hydrophobic carotenoids (Yoo and Moon, 2016).

Figure 19 shows the ABTS radical scavenging activities of the crude and saponified extracts of Ulva, Derbesia and Oedogonium. The crude extracts produced higher ABTS scavenging values than the saponified extracts for all three algal species, but especially for Derbesia and Oedogonium. This result is expected as the saponification step removed considerable amount of chlorophylls (Table 17), which are known to exhibit antioxidant activities (Le Tutour et al., 1998). Saponification also caused some loss of carotenoids and probably some other components such as lipids, which would otherwise contribute to the antioxidant activities (Granado et al., 1992, Khachik et al., 1986).

115

Of the three algal species, Oedogonium extracts showed the highest ABTS scavenging activity for both the crude and saponified extracts. For the crude extracts, Derbesia gave the second highest activity while Ulva exhibited the lowest activity. For the saponified extracts, Derbesia and Ulva showed statistically significant but numerically similar activities. The high ABTS scavenging activities of Oedogonium extracts were clearly attributable to its higher carotenoid content (3411.2 µg/g for the crude and 2929.6 µg/g for the saponified extracts). This is also true for the other two species, where the low antioxidant activities of Ulva was reflected in it having the lowest carotenoid contents (224.5 µg/g for the crude and 195.8 µg/g for the saponified extracts.

1 0

y U lv a

t i

c c ,B

a D e rb e sia

8

p a

) O e d o g o n iu m

C

W

g b ,B D

6

n

i

g

/

g

E

n T

c ,A

e g

v 4

m ( a b ,A

c a ,B a ,A

S

S 2

T

B A

0 C ru d e Sa p o n ifie d

Figure 19. ABTS scavenging activities of crude and saponified carotenoid extracts obtained from Ulva, Derbesia and Oedogonium. Results are measured as mg Trolox equivalent per gram dry weight (mg TE/g DW). Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate the values being significantly different for the same extraction condition with p<0.05 (n=3). Different capital letters indicate the values being significantly different for the same species with p<0.05 (n=3).

4.3.3.2. DPPH free radical scavenging capacity

DPPH is a stable free radical containing an odd electron in its structure and the degree of decrease in the absorbance of DPPH indicates the radical scavenging potential of the antioxidant. The DPPH radical scavenging assays is frequently used to confirm results

116 obtained with ABTS assay because both possess similar antioxidant mechanisms. Figure 20 shows the DPPH scavenging activities of the crude and saponified extracts of Ulva, Derbesia and Oedogonium. Overall, the data trend was similar to the ABTS radical scavenging results. The crude extracts produced higher DPPH scavenging values than the saponified extracts for all three algal species. Of the three species, Oedogonium showed the highest DPPH scavenging activity for the crude extract, followed by Derbesia. For the saponified extracts, Derbesia and Oedogonium showed statistically significant but numerically similar values in their DPPH scavenging activities. Ulva extracts exhibited the lowest activity for both the crude and saponified extracts. As with ABTS scavenging activity, the high DPPH scavenging activity of Oedogonium extracts were clearly attributable to its high total carotenoid content.

1 . 0 c , B

y U l v a

t

i c

a D e r b e s i a

0 . 8 b , B

p a ) O e d o g o n i u m

C b , A

W b , A

g D

0 . 6

n

g

i

/

g

E

A

n

G

e

v

g 0 . 4

a

m

(

c S

a , A a , A

H 0 . 2

P

P D

0 . 0 C r u d e S a p o n i f i e d

Figure 20. DPPH scavenging activities of crude and saponified carotenoid extracts obtained from Ulva, Derbesia and Oedogonium. Results are expressed as mg gallic acid equivalent per gram dry weight (mg GAE/g DW). Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate the values being significantly different for the same extraction condition with p<0.05 (n=3). Different capital letters indicate the values being significantly different for the same species with p<0.05 (n=3).

117

4.3.3.3. FRAP

Unlike the free radical scavenging assays, the FRAP assay measures the capacity of antioxidants to reduce ferric ion to ferrous iron (Kelman et al., 2012). Figure 21 shows the FRAP values of the crude and saponified extracts of Ulva, Derbesia and Oedogonium. The same trend as in the case of ABTS and DPPH radical scavenging assays was observed where the crude extracts produced higher FRAP values than the saponified extracts for all three algal species. Of the three algal species, Oedogonium extracts showed the highest antioxidant capacity for both the crude and saponified extracts, followed by Derbesia, while Ulva exhibited the lowest activity.

1 0 U lv a c ,B D e rb e sia 8 b ,B

s O e d o g o n iu m )

e c ,A

W

u l

D 6

a

g /

v b ,A

E

T

P

g a ,B

A 4

m

R

( F

2 a ,A

0 C ru d e Sa p o n ifie d

Figure 21. FRAP activities of crude and saponified carotenoid extracts obtained from Ulva, Derbesia and Oedogonium. Results are expressed as mg Trolox equivalent per gram dry weight (mg TE/g DW). Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate the values being significantly different for the same extraction condition with p<0.05 (n=3). Different capital letters indicate the values being significantly different for the same species with p<0.05 (n=3).

There are no previous reports on the antioxidant activities of carotenoid extracts of algae; therefore direct comparison of the current results with those in the literature is not possible. The antioxidant activities of algae published in the literature are usually reported on extracts obtained by aqueous alcohols or acetone, and the results varied considerably among different algal species (Ferraces-Casais et al., 2012, Cofrades et al., 2010, Ahmed et al., 2014). This diversity was also reflected in the results of the present

118 study. Furthermore, the antioxidant activities of the carotenoid extracts of the three algal species investigated in this study were generally higher than their respective phenolic extracts reported in Chapter 3. This is probably a reflection of the relatively high carotenoid but low phenolic contents of the algal species.

Many carotenoids are known to be excellent antioxidants, which make them important in human nutrition and health as they play an important role in mitigating oxidative stress in the body. Ahmed et al. (2014) screened 12 algae species for their carotenoid profiles and in vitro antioxidant capacity (ORAC) and showed that the algae exhibited a high ORAC values (45-577 µmol/g DW) comparable to some high antioxidant fruits such as blueberry (46 µmol/g DW), and strawberry (540 µmol/g DW). The carotenoids in the algae, including violaxanthin, astaxanthin, lutein, zeaxanthin, neoxanthin, α- carotene and β-carotene, were believed to be major contributors to the antioxidant activity. The strong antioxidant activities of many of the carotenoids have been well documented and are believed to be responsible for the antioxidant activities of a number of algal species (Murthy et al., 2005, Rao et al., 2006, Sies and Stahl, 1995). The presence of these compounds in the three green macroalgae investigated in the present study no doubt contributed significantly to the strong antioxidant activities of their extracts, especially the saponified extracts where carotenoids were the predominate antioxidants.

4.3.3.4. Correlation of total carotenoid contents with the antioxidant capacity of the three algae

Correlation analysis was performed between the total carotenoid contents of the three algal species and their antioxidant capacities as measured by the ABTS, DPPH and FRAP assays. The resultant Pearson’s correlation coefficients are presented in Table 21. For the crude extracts, no significant correlation was found between the total carotenoid content (TCC) and the antioxidant capacity measured by any of the assays. In contrast, significant correlation was found between TCC and the antioxidant capacity measured by all three assays in the saponified extracts, where significant correlated also existed between the three antioxidant activity assays. These results indicate that the carotenoid compounds might not be the only major contributors to the antioxidant capacities of the

119 crude “carotenoid” extracts of the algae. Rather, non-carotenoid substances, such as chlorophylls, may also contribute significantly to the antioxidant capacity of the crude extracts. On the other hand, these non-carotenoid materials were largely removed by saponification, leaving carotenoids primarily responsible for the antioxidant activity of the saponified extracts.

Table 21. Relationship between the total carotenoid content and antioxidant capacity for crude and saponified extracts of Ulva, Derbesia and Oedogonium

TCC ABTS DPPH FRAP Crude extracts TCC 1 0.796ns 0.477ns 0.685ns ABTS 1 0.553ns 0.747ns DPPH 1 0.967** FRAP 1 Saponified extracts TCC 1 0.941** 0.999* 0.971** ABTS 1 0.957** 0.995* DPPH 1 0.982** FRAP 1 Significant correlation at p<0.05 and 0.01 are indicated by * and **, respectively; ns = not significant.

4.3.4. Inhibitory effects of algal carotenoid extracts against metabolically significant enzymes

4.3.4.1. In vitro pancreatic lipase inhibition by carotenoid extracts of algae

Obesity and overweight are becoming one of the major health issues worldwide. World Health Organization (WHO) estimated that there are more than 1.9 billion overweight adults worldwide in 2014; of these, 13% are clinically obese (WHO, 2015). Obesity can lead to several adverse chronic conditions, especially cardiovascular disease (Hubert et al., 1983, Lavie et al., 2014), diabetes mellitus (Tobias et al., 2014), certain cancers (Hursting et al., 2007), hypertension (Rahmouni et al., 2005) and sleep-breathing disorders (Vitelli et al., 2015). An imbalance between calorie intake and metabolic expenditure is a central factor in many cases of obesity and reductions in the intake of energy dense fats is one of the major strategies in the reduction of body weight. On the metabolic side, pancreatic lipase is the key enzyme in the digestion and utilisation of

120 dietary fats. It hydrolyses triglyceride into monoglycerides and fatty acids which can be absorbed in the human digestive system. Orlistat, a pancreatic lipase inhibitor, is one of the standard prescription drugs used to treat obesity and intestinal fat absorption is reportedly reduced by as much as 30% by oral administration of this drug (120 mg with main meals) (Guerciolini, 1997).

The pancreatic lipase inhibitory effect of crude and saponified carotenoid extracts of the three algal species was assessed and a dose-dependent inhibition effect was observed for all the extracts (Figure 22 (A)). Among the crude extracts, at each concentration tested, Oedogonium gave the highest pancreatic lipase inhibitory activity with IC50 of

3.69 mg/mL, followed by Derbesia (IC50 = 9.09 mg/mL) and Ulva (IC50 = 28.3 mg/mL) (Table 22). Similarly, saponified carotenoid extracts of the algae also exhibited a dose dependent inhibition of pancreatic lipase (Figure 22 (B)), and the activities were higher than their crude counterparts. At the concentration of 10 mg/mL, Derbesia had the greatest inhibitory activity of 82.1%, followed by Oedogonium (74.3%), while the activity of Ulva was the lowest at 49.3%. In terms of IC50 values, however, the saponified extracts of Oedogonium and Derbesia had the same values of 2.08 mg/mL, while the value for Ulva was considerably higher at 16.02 mg/mL (Table 22). The IC50 value for the standard obesity treatment drug, orlistat, is 0.71 mg/mL, which is comparable in magnitude to the carotenoid extracts, especially those from Oedogonium and Derbesia. Quantitatively, the results meant that consumption of 1 mg of orlistat was equivalent in the pancreatic lipase inhibition effect to that of about 3 mg of saponified extract Derbesia, the most effective extract across all the samples.

There are no reports on the pancreatic lipase inhibition effect by carotenoid extracts of algae, and this thesis is the first study on this aspect of biological activities of algae. However, a number of studies have investigated the preventative effect of individual carotenoids on obesity. For example, siphonaxanthin is shown to dramatically inhibit lipid accumulation during 3T3-L1 adipocytes differentiation, and lower a series of key adipogenesis gene expression, such as C/EBPα, PPARγ, FABP4, and SCD1. The anti- obesity effect of siphonaxanthin was confirmed with an oral administration study on obese mice, in which it was found to decrease adipose tissue weight, due to limited lipid biosynthesis and stimulated fatty acid oxidation (Li et al., 2015). Neoxanthin and fucoxanthin, which have a similar structure, have been found to significantly reduce

121 lipid droplets and mRNA expression of C/EBPα and PPARγ (Okada et al., 2008). Derbesia is rich in siphonaxanthin while Oedogonium is rich in neoxanthin. This, coupled with the high pancreatic lipase inhibitory activities of the algal extracts, indicates that these green macroalgae could be developed into functional food ingredients with the potential to play a part in the management of body weight. Direct consumption of the algae or products derived from them could also play a role in reducing fat absorption and weight gains.

1 0 0 (A ) 1 m g / m l

5 m g / m l 8 0 d ,C c ,C

0 .6 m g / m l 1 0 m g / m l

%

n d ,B o

i 1 5 m g / m l

t b ,C

i 6 0 c , B

b

i

h

n

i

e

s 4 0

a d ,A

p i

L b ,B

c ,A a ,C a , B 2 0 b ,A a ,A

0 O rlista t U lv a D e rb e s ia O e d o g o n iu m

1 0 0 (B ) 1 m g / m l

d ,C 5 m g / m l 8 0 d ,B c , B

0 .6 m g / m l c , B 1 0 m g / m l

%

n o

i 1 5 m g / m l t b ,B b ,B

i 6 0 d ,A

b i

h c ,A a , B a , B

n

i

e

s 4 0 b ,A

a

p i

L a ,A

2 0

0 O rlista t U lv a D e rb e s ia O e d o g o n iu m

Figure 22. Dose-dependent effects of pancreatic lipase inhibition activity of crude (A) and saponified (B) carotenoid extracts obtained from Ulva, Derbesia and Oedogonium. Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate values being significantly different for the same species but different concentrations 122 with p<0.05 (n=3). Different capital letters indicate values being significantly for different species at the same concentration with p<0.05 (n=3).

Table 22. Pancreatic lipase inhibitory activity of carotenoid extracts of three algal species

* Inhibiting agent Pancreatic lipase IC50 (mg/mL) Orlistat 0.71±0.01a

Crude

Ulva 28.31±0.11f

Derbesia 9.09±0.07d

Oedogonium 3.69±0.04c

Saponified

Ulva 16.02±0.07e

Derbesia 2.08±0.01b

Oedogonium 2.08±0.02b

* IC50: half maximum inhibitory concentration. Data represent the mean ± SD of at least three independent experiments (n=3). Different letters within the same column indicate that the values are significant different (p<0.05).

4.3.4.2. In vitro antidiabetic effects

Diabetes mellitus has become an epidemic occurring in adults and, increasingly children also, throughout the world and is the leading cause of kidney failure, heart attack, blindness and lower limb amputation (Tabish, 2007). WHO estimated that 9% the world’s adult population are diabetes and 1.5 million deaths were directly caused by diabetes in 2012 (WHO, 2016). It is the fourth main cause of death in most developed countries. Effective management of diabetes mellitus, especially the non-insulin- dependent Type II, involves preventing excessive rise of the blood glucose level through the inhibition of starch digestive enzymes in the digestive system (Tundis et al., 2010). The inhibition of intestinal α-glucosidase and pancreatic α-amylase are two of the mostly commonly used in vitro assays for determining the anti-type II diabetic

123 potential of a substance. Inhibition of the enzymes would delay the degradation of starch and prolong its overall digestion time. In recent years, natural sources of α- amylase and α-glucosidase inhibitors have received a lot of interest due to the side effects associated with synthetic enzyme inhibitors such as acarbose, sulfonylureas, biguanides, glinides, metformin and orlistat (Kunyanga et al., 2012).

4.3.4.3. α-Amylase inhibitory activities

α-Amylase is a key enzyme involved in starch digestion. It catalyses the hydrolysis of α-1,4-glucosidic linkages of starch and glycogen to oligosaccharides, which, in turn, are further broken down by α-glucosidase to glucose, ready for absorption by the body. Therefore, inhibition of α-amylase is regarded as one of the most effective approaches for diabetic care.

The inhibition of α-amylase was observed over four different concentrations of the crude and saponified extracts of all three algal species (Figure 23). With increases in the concentration of crude extracts, the inhibition of α-amylase increased. Derbesia extract was found to exhibit the strongest inhibition activity. At the concentration of 10mg/mL, Derbesia gave 53.6% inhibition, followed by Oedogonium (49.2%) while Ulva gave the lowest inhibition (41.7%). For saponified extracts, the inhibition of α- amylase generally increased with increases in the concentration of the extracts although the trend was not as clear as that with the crude extracts. At the concentration of 10 mg/mL, Oedogonium extract gave the highest inhibition at 39.3%, followed by Ulva 37.4% and Derbesia 36.3%. Surprisingly, the α-amylase inhibition activities of the saponified extracts were lower than their crude counterparts in most cases, especially at the higher concentration range. This appeared to indicate that some of the impurities in the crude extracts that were removed by saponification also contributed to the inhibitory activity against α-amylase. Significantly, the α-amylase inhibitory activities of the crude and saponified extracts were comparable to that of Acarbose, one of the standard prescription drugs for the management of diabetes. At the concertation of 10 mg/mL or higher, the inhibitory activities of the extracts were comparable or higher than that of Acarbose at 5 mg/mL.

124

8 0 ( A ) 1 m g / m l

d , C 5 m g / m l

c , B % 6 0

n 1 0 m g / m l

0 . 5 m g / m l d , A c , C o i b , B b , B

t b , B i

c , A 1 5 m g / m l

b

i

h

n i

4 0

e 0 . 1 m g / m l

s a

l b , A a , C y

m a , B

a a , A

- 2 0 

0 A c a r b o s e U l v a D e r b e s i a O e d o g o n i u m

8 0 ( B ) 1 m g / m l

5 m g / m l

d , B % 6 0 n 1 0 m g / m l

0 . 5 m g / m l

o

i t

i d , A

b b , A 1 5 m g / m l i c , B

h c , B b , B

n 0 . 1 a , C a , A

i a , B

4 0 m g / m l a , B b , A

e

s a

l a , A

y

m a

- 2 0 

0 A c a r b o s e U l v a D e r b e s i a O e d o g o n i u m

Figure 23. Dose-dependent effects of α-amylase inhibition activity of crude (A) and saponified (B) carotenoid extracts obtained from Ulva, Derbesia and Oedogonium. Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate values being significantly different for the same species but at different concentrations with p<0.05 (n=3). Different capital letters indicate values being significantly for different species at the same concentration with p<0.05 (n=3).

125

4.3.4.4. α-Glucosidase inhibitory activity

α-Glucosidase is a membrane-bound enzyme located in the epithelium of the small intestines and it hydrolyses complex carbohydrate (oligosaccharides and disaccharides) into simpler sugars, which are readily available for intestinal absorption (Lee and Jeon, 2013). Hence, inhibition of α-glucosidase activity reduces the release and subsequent uptake of glucose. Studies have shown that the retardation of α-glucosidase by inhibitors could be one of the most effective ways to control Type II diabetes (Kunyanga et al., 2012).

Figure 24 presents the α-glucosidase inhibitory activities of the carotenoid extracts of the various green algae at four different concentrations. Similar to the case with inhibition on α-amylase, the inhibition activities of the extracts were compared with Acarbose. In general, the same trend as the inhibition of α-amylase was observed for crude extracts, i.e., the inhibitory activities increased with increasing concentrations of the extracts, with Derbesia showing the highest activity, followed by Oedogonium and Ulva. For example, at the concentration of 10 mg/mL, Derbesia caused a complete inhibition of the enzyme while Oedogonium and Ulva gave an inhibition of 73.98% and 69.5%, respectively. At the concentration of 1 mg/mL, Ulva and Oedogonium did not show detectable inhibition towards α-glucosidase. Furthermore, at concentrations higher than 10 mg/mL, the crude extracts showed higher inhibitions than Acarbose at 5 mg/mL for all three algae, demonstrating the potency of them as an inhibitor of this enzyme.

With regards to saponified carotenoid extracts, their inhibition on α-glucosidase was also examined at four concentrations; however, all the extracts showed very low activities compared with their crude counterparts. This appeared to indicate that it is primarily the non-carotenoid components in the extract that were responsible for the α- glucosidase inhibitory activity.

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( A ) 1 5 0 1 m g / m l

5 m g / m l

%

n

d , C c , A 1 0 m g / m l

o i

t d , A

i b , C d , A

b 1 0 0

i 1 5 m g / m l

a , B

h

n i

c , B

e 1 m g / m l c , A

s

a

d

i

s o

c 5 0 u

l b , B

g -

 b , A

a , A a , A 0 A c a r b o s e U l v a D e r b e s i a O e d o g o n i u m

( B ) 8 0 1 m g / m l

1 m g / m l

5 m g / m l

%

n 6 0

o 1 0 m g / m l

i

t

i b

i 1 5 m g / m l

h

n

i

e 4 0

s

a

d

i

s

o

c

u l

g 2 0

-  d , C c , C c , B c , C d , A a , A b , A a , A a , A b , B a , A b , B 0 A c a r b o s e U l v a D e r b e s i a O e d o g o n i u m

Figure 24. Dose-dependent effects of inhibition α-glucosidase activity of crude (A) and saponified (B) carotenoid extracts obtained from Ulva, Derbesia and Oedogonium. Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate values being significantly different for the same species but at different concentration with p<0.05 (n=3). Different capital letters indicate values being significantly for different species at the same concentration with p<0.05 (n=3).

To date, there is no report on the α-amylase and α-glucosidase inhibition effects of carotenoid extracts of algae. This study is the first to investigate this aspect of the 127 biological activities of algal carotenoid extracts, and they were found to be potent inhibitors of these metabolically important enzymes for starch digestion, comparable in potency to that of Acarbose, one of the standard prescription drugs for diabetic care. This implies that consumption of these green algae could be beneficial to diabetic patients, and that the extracts have the potential to be developed into nutraceutical supplements as alternatives to Acarbose for the treatment of diabetes.

The antidiabetic effects of several carotenoids have been well documented. For example, several studies have shown that dietary β-carotene intake has an inverse association with type 2 diabetes (Montonen et al., 2004, Sluijs et al., 2015, Arnlov et al., 2009). Montonen et al. (2004) reported that β-cryptoxanthin intake was significantly associated with a reduced risk of type 2 diabetes. The presence of these carotenoids in the three algal species further demonstrates their potential role in diabetic prevention as well as management.

4.3.4.5. Hyaluronidase inhibitory activity

Hyaluronidase is an enzyme which predominantly hydrolyses hyaluronic acid by splitting glucosaminidic bonds to yield oligosaccharides, and it is found both in organs and in body fluids (Zeng et al., 2015). This enzyme plays an important role in angiogenesis (Liu et al., 1996), carcinogenesis (Menzel and Farr, 1998), type I allergic reactions (El Maradny et al., 1997) and inflammatory (Edelstam et al., 1992) diseases, and inhibition of its activity is regarded as an effective anti-allergic and anti- inflammatory therapy (Gautam and Jachak, 2009).

To evaluate the potential anti-allergic and anti-inflammatory activities of crude and saponified carotenoid extracts of the three algae, their inhibitory effect on hyaluronidase was investigated. The hyaluronidase activities in the presence of various concentrations of crude and saponified extracts are shown in Figure 25. With the increase in crude and saponified extract concentration, hyaluronidase activities decreased significantly (P<0.05). For the crude extract, the inhibitory effect of Ulva increased continuously with increasing concentrations of the extract. However, the inhibitory effects of Derbesia and Oedogonium reached a plateau when the extract concentration reached 15 mg/mL. At the highest extract concentration of 30 mg/mL, Ulva gave the greatest

128 inhibition at 19.4%, followed closely by Derbesia (18.9%) and Oedogonium (15.6%) (Figure 25 (A)). For the saponified extracts, the inhibitory effects increased with increasing extract concentrations for all three algae. At the highest extract concentration of 30 mg/mL, Derbesia gave the greatest inhibition followed closely by Oedogonium, while Ulva gave the smallest inhibition (Figure 25 (B)). At the highest extract concentration, the inhibitory effects of the saponified extracts were smaller than the crude extracts, although the differences were not substantial except for Ulva. These results indicated that the anti-hyaluronidase effects of the extracts were mainly due to the carotenoids present although the non-carotenoid impurities removed by saponification also had a small contribution. The hyaluronidase inhibition effects of the algal extracts were considerably lower than quercetin, which is a flavonoid with well reported anti-inflammatory activity.

There are no reports on the hyaluronidase inhibition effects of carotenoid extracts of algae, and therefore comparison of the present results with literature is not possible. However, several carotenoids have been reported to exhibit hyaluronidase inhibition activities. For example, astaxanthin, β-carotene, fucoxanthin and zeaxanthin have been found to exhibit significant inhibitory activities against antigen-induced degranulation of rat basophilic leukemia RBL-2H3 cells and bone marrow-derived mast cells (Sakai et al., 2009). These carotenoids also exerted an anti-inflammatory effect by suppressing mast cell degranulation in vivo, whereby significantly inhibited ear swelling and reduced the contents of TNF-α and histamine in the DHFB-treated mice (Ganesan et al., 2011). β-Carotene and lutein have also been reported to have a beneficial effect for the treatment of oxidative stress-mediated gastric inflammation, because they showed inhibitory effects in H2O2-induced increase in intracellular reactive oxygen species levels, activation of NF-KB, and IL-8 expression in AGS cells (Kim et al., 2011). Siphonaxanthin also exhibited inhibitory effects on antigen-induced degranulation of mast cells (Sugawara et al., 2014). The presence of these components in the carotenoid extracts of the algae samples may explain their hyaluronidase inhibition activity and also indicate that these extracts or the algae themselves have the potential to be developed into functional ingredients for application in functional food, nutraceutical and pharmaceutical products for management of inflammation-related disorders.

129

( A ) 2 5 5 m g / m l 0 . 1 m g / m l

1 0 m g / m l

% d , C

2 0 c , C c , B

n o

i 1 5 m g / m l

t i

c , B c , A

b i

h 3 0 m g / m l

1 5

n

i

e

s

a d i 1 0

n b , C

o c , A r

u b , B

l

a y

5 a , C H a , B

a , A b , A 0 Q u e r c e t i n U l v a D e r b e s i a O e d o g o n i u m

( B ) 2 5 5 m g / m l 0 . 1 m g / m l

1 0 m g / m l

%

2 0 c , C

n o

i 1 5 m g / m l t

i d , B

b i c , C 3 0 m g / m l

h 1 5

n

i

e s

a b , B d , A b , C

b , B d

i 1 0 n

o a , C

r c , A

u l

b , A a

y 5 H a , B a , A

0 Q u e r c e t i n U l v a D e r b e s i a O e d o g o n i u m

Figure 25. Dose-dependent effects of hyaluronidase inhibition of crude (A) and saponified (B) carotenoid extracts obtained from Ulva, Derbesia and Oedogonium. Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate values being significantly different for the same species but different concentration with p<0.05 (n=3). Different capital letters indicate values being significantly for different species at the same concentration with p<0.05 (n=3).

130

4.3.4.6. ACE inhibitory activities

Hypertension is a major risk factor for a number of disorders including cardiovascular disease, kidney failure and stroke (Fernandez, 2007). It has become another major public health problem with about 25% of the world’s adult population suffering the condition, which is estimated to increase to 29% by 2025 (Mittal and Singh, 2010). Over expression of the ACE enzyme plays a key role in the increase of blood pressure, and identification and incorporation of natural ACE-inhibitors into functional food could be a key component in the overall strategy in combating the hypertension epidemic.

The crude and saponified extracts of the three green algae were assessed for their ACE inhibitory activity at the concentration of 10 mg/mL, and the results are shown in Table 23. Low ACE inhibition was observed for both the crude and saponified extracts of all three algal species. Of the three algal species, Oedogonium extracts showed highest inhibition for both the crude and saponified extracts and the purified extract showed a higher inhibition than the crude extract. But even for this extract, the inhibition of ACE activity was less than 8%. When the concentrations of the extracts were increased, no significant increases in the activity were observed. It is therefore concluded that the carotenoid extracts of the three green algae were poor inhibitors of ACE. There is no report on the ACE-inhibitory effect of carotenoid extracts of algae in the literature. Some carotenoids have been reported to exhibit anti-hypertensive activities. For example, the administration of astaxanthin at the dose 50 mg/kg for 5 weeks caused a significant reduction in the arterial blood pressure in spontaneously hypertensive rats (SHR), and also delayed the incidence of stroke in the stroke prone SHR (Hussein et al., 2005). Lycopene supplementation at the rate of 15 mg per day for 8 weeks significantly decreased systolic blood pressures from the baseline value of 144 mmHg to 134 mmHg in mildly hypertensive subjects (Li and Xu, 2013). However, these carotenoids were not found in the three green algal species, which might explain their low ACE-inhibitory effects. Furthermore, ACE-inhibition is only one of several routes through which lowering of blood pressure can be achieved. It is possible that these carotenoids produced the anti-hypertensive effects through other mechanisms.

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Table 23. Angiotensin converting enzyme (ACE) inhibitory activities of carotenoid extracts from three green algae

Inhibition (%) Crude extracts Ulva 0.40±0.01a Derbesia n.d. Oedogonium 4.48±0.02d Saponified extracts Ulva 1.90±0.01c Derbesia 1.47±0.01b Oedogonium 7.37±0.02f Captopril (µg/mL) 200 6.15±0.01e ACE inhibition was evaluated at the extract concentration of 10 mg/mL. Data represent the mean ± SD of at least three independent experiments. Mean with different letters were significantly different at the level (p<0.05); n=3. n.d. = not detected.

4.4. Conclusion

A number of major conclusions can be drawn from the results of this chapter. The three green macroalgae contained substantial amounts of carotenoids, but the content varied significantly among the three species. Oedogonium had the highest carotenoid content, which was about 20% higher than that of Derbesia, while the amount in Ulva was 12-15 folds lower than the other two species. Furthermore, the spectrophotometric method for measuring total carotenoids was found to overestimate the carotenoid content when compared to HPLC method, due to interferences of impurities with the measurements.

There are nine carotenoids compounds that were found by LC-MS in the three green algal species: siphonaxanthin, neoxanthin, 9’-cis-neoxanthin, loroxanthin, violaxanthin, lutein, siphonein, α-carotene and β-carotene. Lutein was the most abundant carotenoid in Ulva; siphonaxanthin was most abundant in Derbesia, while Oedogonium contained several major carotenoids, namely neoxanthin, 9’-cis-Neoxanthin, loroxanthin violaxanthin, lutein and β-carotene, in similar amounts. The carotenoid extracts exhibited significant antioxidant capacities, as well as potent inhibitory effects against

132 several metabolically important enzymes including α-amylase, α-glucosidase, pancreatic lipase and hyaluronidase. However, the algal extracts were found to be poor inhibitors of ACE. Overall, the results of this chapter demonstrated that the carotenoid extracts of these algae, especially Derbesia and Oedogonium, have the potential to be developed into bioactive ingredients for application in functional food or nutraceutical products to supplement or replace drugs such as Acarbose and Orlistat in the management of diabetes and for control of weight gain and mitigation of inflammation. Direct consumption of these algae as foods could also provide those potential health benefits. However, these results are obtained by in vitro methods, and animal model studies or clinical trials are needed confirm the results before real health benefits can be claimed.

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Chapter 5 Biologically active peptides produced by in vitro simulated human digestion of green algae proteins

5.1. Introduction

Protein is a fundamental nutrient that is needed for growth and maintenance of physiological functions. Traditional research on food protein is focused on its production (e.g., sources and extraction), nutrition (e.g., amino acid balance and bioavailability) and processing functions (e.g., foaming, emulsifying, water binding and viscosity modifying properties) (Hefnawy and Ramadan, 2011). In recent decades, the research focus on food protein has gradually shifted towards health related biological activities of proteins and bioactive peptides derived from them. Bioactive peptides, which usually consist of 2 to 20 amino acid residues, are defined as the specific protein fragments that possess beneficial pharmacological properties in the human body beyond normal and adequate nutrition (Ryan et al., 2011). These peptides are inactive within the sequence of the parent proteins, but can be released from them during gastrointestinal digestion or in vitro proteolysis by the action of hydrolytic enzymes (Erdmann et al., 2008). In recent years, it has been acknowledged that dietary protein is a good source of bioactive peptides with a broad spectrum of biological activities, including antioxidant, antihypertensive, antithrombotic, anti-adipogenic, antimicrobial, anti-inflammatory, opioid and immune-stimulative effects (Sarmadi and Ismail, 2010, de Castro and Sato, 2015, Ngo et al., 2012, Ahn et al., 2015, Biziulevicius et al., 2006, Tavares et al., 2011, Zhang et al., 2010, Alashi et al., 2014).

Studies on bioactive peptides have so far mainly focused on animal and plant food proteins. Animal proteins from egg, milk, fish and meat have been extensively studied as sources of bioactive peptides (Mohanty et al., 2015, Chakrabarti et al., 2014, Senevirathne and Kim, 2012). Plant protein sources investigated are generally grains including wheat, rice, oats, rye, millet, sorghum and corn, but also some legumes such as soy, peas and chickpeas (Kitts and Weiler, 2003, Malaguti et al., 2014).

Edible algae are a rich source of protein. The amount of protein in seaweeds varies greatly among different species, with brown seaweeds generally having a lower level (3-15%, dry weight) than green (9-26%) and red (10-47%) seaweeds (Fleurence, 1999, 134

Harnedy and FitzGerald, 2011). These levels are generally comparable or higher than plant and animal protein sources. A number of studies have examined the biological activities of algal proteins and hydrolysates or peptides derived from them. Algal protein hydrolysates and peptides have been reported to exhibit antioxidant (Kang et al., 2011, Karawita et al., 2007, Kim et al., 2006b, Sheih et al., 2009b), antihypertensive (Suetsuna et al., 2004, Suetsuna and Chen, 2001, Qu et al., 2010, Sato et al., 2002a, Sato et al., 2002b, Suetsuna and Nakano, 2000, Sheih et al., 2009a), immunostimulating (Morris et al., 2007), anticancer (Sheih et al., 2010), and hepatoprotective (Kang et al., 2012, Hwang et al., 2008) properties. However, compared with the large amount of studies on the biological properties of plant and animal proteins, such research on algal proteins is rather limited. Furthermore, most of the studies on algal protein used single proteolytic enzymes, such as pepsin, pancreatin, pronase, trypsin or chymotrypsin to hydrolyse the proteins and produce bioactive peptides. Multi-proteolytic enzyme system data, which more closely resembles the actual digestion environment in vivo, on algal proteins are limited. Additionally, most of the studies are concerned with brown and red algae grown naturally in the marine environment. There is very little information on the bioactive peptides from green algae, especially those produced by controlled cultivation.

In this context, the objectives of this chapter were to: 1) investigate the health-related biological activities of protein hydrolysates of the three green algae; and 2) to identify the peptides in the most active fractions of the hydrolysates. Proteins extracted from the three algae were subjected to hydrolysis by a multi-proteolytic enzyme system using simulated in vitro human digestion and the hydrolysates generated were fractionated by ultrafiltration. The crude hydrolysates and the ultrafiltered fractions were analysed for their antioxidant, and α-amylase, α-glucosidase and ACE-inhibitory activities. Finally, the peptides in the fractions with the highest bioactivities were identified using LC- MS/MS. The resulting data were further mined by using the BIOPEP database to identify potential bioactive peptide sequences.

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5.2. Materials and methods

5.2.1. Chemicals and reagents

Albumin from bovine serum (minimum 96%), captopril, N-[3-(2-Furyl)acryloyl]-Phe- Gly-Gly (FAPGG), pepsin from porcine gastric mucosa (3,200U/mg), picrylsulfonic acid (TNBS), sucrose, trizma base and β-mercaptoethanol were purchased from Sigma- Aldrich (Sydney, Australia). Pierce bicinchoninic (BCA) protein assay kit was purchased from Thermo Scientific (Sydney, Australia). Calcium chloride dihydrate

(CaCl2·2H2O), magnesium chloride (MgCl2), phenol, potassium chloride (KCl), sodium hydrogen carbonate (NaHCO3), and trichloroacetic acid (TCA) were purchased from Ajax Finechem Pty Ltd (Sydney, Australia). 10x Tris/Glycine/SDS Buffer, coomassie brilliant blue R-250, laemmli sample buffer, precision plus protein dual xtra standard (5000-250,000 da), sodium dodecyl sulfate (SDS), and tris were purchased from Bio- Rad (Sydney, Australia). Hog bile extract and pancreatin (porcine pancreas at 3x USP activity) were purchased from MP Biomedicals (Seven Hills, Australia). L-Leucine was purchased from Fluka Analytical (Sydney, Australia). Suppliers of methanol, acetone, acetic acid, hydrochloride acid (32%), trolox, ABTS, TPTZ, potassium persulfate, ion (III) chloride hexahydrate and sodium carbonate are given in Chapter 3, Section 3.2.1; p-NPG, Acarbose, ammonium acetate, sodium hydroxide, sodium chloride, α-amylase, α-glucosidase and ACE are given in Chapter 4, Section 4.2.1. All chemicals were of at least analytical grade unless stated otherwise. Water used in all experiments was purified by reverse osmosis using the MilliQ RO system (referred to as MilliQ water).

5.2.2. Seaweed sample collection and storage

The same three green macroalgal species, Ulva ohnoi, Derbesia tenuissima and Oedogonium intermedium are used here. The collection, treatment and storage of the algal biomass are described in Chapter 3, section 3.2.2.

5.2.3. Preparation of crude seaweed protein extracts

Proteins were extracted from the algal biomass using a combination of methods published elsewhere (Fleurence et al., 1995, Barbarino and Lourenco, 2005, Wong and Cheung, 2001, Kumar et al., 2014) with modifications. In brief, 5 g of seaweed powder 136 was suspended in Milli-Q water (1: 20 w/v) to induce cell lysis by osmotic shock which facilitates subsequent protein extraction (Wong and Cheung, 2001). This suspension was gently stirred (120 rpm) in an orbital shaking incubator (Bioline Global, NSW, Australia) overnight at 35°C, which was found to be the optimal temperature for seaweed protein solubility (Wong and Cheung, 2001). After incubation, the pH of the mixture was adjusted to 7 or 12 with 5 N NaOH. The mixture was sonicated for 20 min at 70 MHz with the sonication temperature kept below room temperature, and then gently shaken at 25°C for 2 h in an orbital shaker before centrifugation at 15, 000 g for 30 min at 4°C (Avanti JE centrifuge, Beckma Coulter, USA). The supernatant was adjusted to the isoelectric point pH 4.0 (Ursu et al., 2014) and left at room temperature for 2 h for protein precipitation. After that, the mixture was centrifuged at 15,000 g for 30 min at 4°C, the supernatant discharged, the precipitate washed with MilliQ water and adjusted to pH 7.0 using 0.1 M NaOH before being freeze dried. The lyophilised protein concentrates were stored at -20°C before use. Extraction yield (%) was calculated as mg of protein concentrate per mg of dry algal biomass (dry weight).

5.2.4. Phenol extraction of protein for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis

Phenol extraction of proteins was carried out using the protocol described by Wang et al. (2006). Briefly, lyophilised algal sample (1 g) was washed with three different washing solvents: 10% TCA in acetone, 80% methanol in 0.1 M ammonium acetate and acetone. For each washing solvent, the washing was repeated three times, each time by mixing 10 mL of the solvent with the sample by vortexing vigorously, followed by centrifugation at 15,000 g for 3 min at 4°C and removal of the supernatant. The pellet obtained after the final washing step was air-dried at room temperature for 2 h to remove the residual acetone. After that, the pellet was mixed well, by vortexing, with 2 mL of an emulsion of 50% w/v phenol in 0.1 M Tris HCl, pH 8.0 and SDS buffer (30% sucrose and 2% SDS in the same Tris buffer containing 5% -mercaptoethanol) in equal proportions. The mixture was centrifuged at 15, 000 g for 3 min and the top phenol phase was pipetted out. The protein was precipitated by mixing with 4 volumes of methanol containing 0.1 M ammonium acetate and storing the mixture at 20°C for 2 h.

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The mixture was then centrifuged at 15, 000 g for 5 min at 4°C, the supernatant discarded and the white pellet washed with 100% methanol and again with 80% acetone. The final pellet was air dried and dissolved in 1 mL Laemmli buffer for SDS PAGE analysis.

5.2.5. Determination of protein

5.2.5.1. Total nitrogen by the Kjeldahl method

The crude protein content in the algal biomass was determined by the Kjeldahl method using the using a LECO TruSpec CN Analyser (LECO Corporation, St Joseph, MI, USA) according to the approved AACC (46-30, 2000) standard procedures. The nitrogen conversion factors used were 4.8 for Derbesia, 4.6 for Ulva and 4.7 for Oedogonium (Neveux et al., 2014c).

5.2.5.2. BCA Protein Assay (Lowry method)

The soluble protein and peptide concentration in protein hydrolysates was determined by the Lowry method using the Pierce BCA protein assay kit according to the manufacturer’s instructions, with bovine serum albumin (BSA) as the standard. Working BCA reagent was prepared by mixing 50 parts of Reagent A with 1 part of reagent B. Samples (25 μL) of protein hydrolysates were mixed with 200 μL of the BCA reagent in microplates (Greiner bio-one, Germany), which were incubated at 37°C for 30 min prior to measurement of absorbance at 562 nm, which was then compared against the BSA standard curve with R2 = 0.991.

5.2.6. Analysis of amino acid composition

The amino acid profile of the three algae was analysed. Algal biomass samples (15 mg) were mixed with 1.5 mL of 6 M HCl and the solutions were sealed in tubes under nitrogen and incubated in an oven at 110°C for 24 h for complete hydrolysis. The amino acids generated were quantified using the Water AccQTag Ultra chemistry kit at the

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Australian Proteome Analysis Facility (Sydney, Australia). All the samples were analysed in duplicate.

5.2.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Algae protein concentrates and phenol protein extracts, obtained as described in 5.2.3 and 5.2.4, respectively, were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine their protein profile. The protocol described by Uraipong and Zhao (2015) was followed. Briefly, SDS-PAGE was performed using the Mini-Protean TGX Precast Gels (8-16%) and Precision Plus Protein Dual xtra standard (Bio-Rad Laboratories, Sydney, Australia). Samples of the protein concentrates (20 µ, 10 mg/mL) were mixed with 20 µl of Laemmli sample buffer and β- mercaptoethanol (19/1, v/v), boiled for 5 min, cooled down, centrifuged at 10,000 g for 3 min, and the upper clear supernatant (20 µL) was loaded into the gel. The gel was run at 200 V for 30 min and washed three times with distilled water for 5 min each. The gel was stained with Coomassie Blue (125 mg in 500 mL of water/methanol/acetic acid (95/4/1, v/v/v)) overnight, followed by de-staining with a mixture of acetic acid/methanol/MilliQ (1/1/8, v/v/v). The gel was visualised with the ChemiDoc MP image analyser (Bio-Rad Laboratories, USA). Image LabTM software version 4.0 (Bio- Rad Laboratories, Inc. Hercules, CA) was used to analyse the electrophoresis pattern.

5.2.8. In vitro simulated gastrointestinal digestion of algal protein

The in vitro gastrointestinal digestion of algal protein mimicking the physiological situation in the upper tract (mount, stomach and small intestine) was adapted from Durak et al. (2013) with some modifications. The digestion consisted of three stages: α- amylase digestion, pepsin digestion and pancreatin digestion. Briefly, 50 mg of crude algal protein extract was mixed with 500 µL of 250 U α-amylase solution for 5 min at 37°C. The α-amylase solution was prepared by dissolving 8.5 mg α-amylase in 5 mL of carbonate buffer. After that, 5 mL of simulated stomach juice (1% pepsin in 20 mM HCl, pH 2) was added and the mixture was incubated at 37°C in an orbital shaker (Bioline Global, NSW, Australia) for 2 h. After pepsin digestion, the pH of the digest

139 was adjusted to 7 with 1 M NaOH. Finally, 5 mL of pancreatin+bile extract solution (4 mg/mL pancreatin and 25 mg/mL bile extract in sodium acetate buffer, pH 6) was added and the mixture was incubated at 37°C for 1 h. The digestion was immediately stopped by heating in a 95°C water bath (Edwards Group Pty Ltd, NSW, Australia) for 5 min, and the mixture was cooled down at room temperature. The hydrolysates were clarified by centrifugation (15,000 g for 15 min at 4°C) to remove the residues and the supernatants, which were taken as protein hydrolysates, were freeze-dried and stored at -20°C. The lyophilised hydrolysates were later re-dissolved in 1 mL of 50 mM Tris buffer, pH 8.2 containing 300 mM NaCl for determination of the total soluble peptides (Section 5.2.5.2) and characterisation of bioactivities.

5.2.9. Fractionation of algal protein hydrolysates

The algal protein hydrolysates were fractionated using the Amicon ultra-15 centrifugal membrane filter units (Merck Millipore, Melbourne, Australia) with molecular weight (MW) cut-off of 3 and 10 kDa. The hydrolysate samples (5 mL) were first loaded into the filter with 10 kDa MW cut-off, which was centrifuged at 4,500 g and 4°C for 15 min to obtain the fraction with MW > 10 kDa as the retentate. The resulting filtrate was then passed through another filter with 3 kDa MW cut-off by centrifugation at 10,000 g and 4°C for 15 min. The retentate was collected as the fraction with MW 3-10 kDa and the resulting filtrate was collected as the fraction with MW <3 kDa. The obtained protein hydrolysate fractions were freeze-dried and stored at -20°C until use. The hydrolysate fractions were assayed for their antioxidant and α-glucosidase, α-amylase and ACE-inhibitory activities.

5.2.10. Determination of degree of hydrolysis

Degree of hydrolysis (DH) was determined by measuring the amount of free amino groups in the sample using the TNBS colorimetric method described by Uraipong and Zhao (2015). Briefly, hydrolysate samples before freeze-drying or L-leucine standards (0.5 mL) were mixed with 250 µL of 0.01% (w/v) TNBS solution followed by incubation in the dark at 37°C for 120 min and the reaction was stopped by the addition of 125 µL of 1 N HCl. Absorbance was then measured at 335 nm and compared with a L-leucine standard curve (0.25-2 amino meq/g in sodium phosphate buffer, pH 7.9) to 140 obtain the concentration of free amino groups. The total number of amino groups in the sample (htot) was determined by completely hydrolysing the peptide bonds in the samples first with 6 M HCl at 120°C for 24 h, followed by the same colorimetric procedure described above. The degree of hydrolysis, defined as the percent ratio of the number of peptide bonds broken (h) to the total number of bonds per unit weight (htot), was calculated as DH=(h/htot)×100%.

5.2.11. Assay of bioactive activities

5.2.11.1. Assays of antioxidant capacity

The antioxidant activities of the algal proteins, their hydrolysates and the ultrafiltered fractions were determined by the ABTS radical scavenging and ferric reducing antioxidant power (FRAP) assays. The procedures for these assays were the same as described in Chapter 3, Section 3.2.4. Samples (protein, hydrolysate or fraction) were prepared as 10 mg/mL solution in 50 mM Tris-HCl (pH 8.2) containing 300 mM NaCl for the assays. A standard curve of Trolox or gallic acid was constructed for each assay and the regression coefficient (R2) was greater than 0.97 for both assays. Each assay was done in triplicates.

5.2.11.2. Assays of α-amylase and α-glucosidase inhibition activity

The α-amylase and α-glucosidase inhibition activity of the algal proteins, their hydrolysates and the ultrafiltered fractions were determined. The procedures for these assays were the same as described in Chapter 4, Section 4.2.6.2 and 4.2.6.3. Samples (protein, hydrolysate or fraction) were prepared as 10 mg/mL solution in 50 mM Tris- HCl (pH 8.2) containing 300 mM NaCl for the assays. A standard calibration curve of Acarbose (100-1000 µg/mL) was constructed for α-amylase (R2=0.972) and α- glucosidase (R2=0.977) inhibition activity. The results were expressed as mg of Acarbose equivalents per g of protein. Three independent assays were performed for each sample.

5.2.11.3. Assay of ACE inhibition activity

ACE inhibition activity of the algal proteins, their hydrolysates and the ultrafiltered fractions was assayed according to the method described by Uraipong and Zhao (2015). 141

Briefly, algal protein extracts or hydrolysates (10 mg) were re-dissolved in 1 mL of 50 mM Tris-HCl (pH 8.2) containing 300 mM NaCl to prepare the sample solution. Ten µL of ACE solution (250 mU/mL in MilliQ water) and 30 µL of samples were mixed in a 96 well microplate, which was incubated at 37°C for 5 min, and 150 µL of FAPGG (0.88 mM in the same Tris buffer) was added to each well within 30 s. The decay in absorbance at 340 nm due to the degradation of FAPGG by ACE was monitored for 50 min. ACE activity was expressed as the slope of the decrease in the absorbance at 340 nM over the linear interval from the 10th to 35th min. The pharmacological inhibitor, Captopril, was included as positive control. ACE-inhibitory activity was calculated using the following equation% ACE inhibition = (1-Ainhibitor/Acontrol) ×100, where

Ainhibitor and Acontrol are the slopes of the samples and the control, respectively.

5.2.12. Liquid chromatography-tandem mass spectrometry analysis of peptide sequences

The ultrafiltered fractions of algal protein hydrolysates with the highest bioactivities were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the peptide sequences. The LC-MS/MS system consisted of an Ultimate 3000 HPLC (Dionex, Amsterdam, Netherlands) coupled to a LTQ-Orbitrap Velos (Thermo Electron, Bremen, Germany) mass spectrometer equipped with an electrospray source (Thermo Electron, Bremen, Germany). A fritless nano-LC column (75 μm x 12 cm) containing C18 media (1.9 μm, 120 Å ReproSil-Pur 120 C18-AQ, Dr Maisch GmbH) was used and maintained at 45°C. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in 80% acetonitrile (solvent B). A flow rate of 0.2 µL/min was used with a gradient elution program: 0-4 min 2% B, 4-36 min 2% to 45% B, 36-37 min 45% to 80% B, 37-37.5 min isocratic with 80% B, 37.5-39 min 80% to 2% B, 39 -52min isocratic with 2% B.

High voltage (2000 V) was applied to a low volume tee (Upchurch Scientific) and the column tip was positioned at 0.5 cm from the heated capillary (T=275°C) of the mass spectrometer. Positive ions were generated by heated electrospray and the mass spectrometer operated in data dependent acquisition mode (DDA). Full scan MS spectra were acquired (m/z 350-1750) at a resolution of 30,000. The 15 most abundant ions 142

(>5,000 counts) with charge states ≥+2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q=0.25 and activation time of 10 ms at a target value of 30,000 ions; m/z rations selected for MS/MS were dynamically excluded for 35 seconds.

Samples were analysed in duplicate, and peak lists of MS/MS data were generated using Mascot Darmon/extract_msn (Matrix Science, London, England, Thermo) and data were processed using a search program Mascot version 2.5 (Matrix Science, Inc., Boston, MA, USA). Peptides were identified by matching against the protein database for Viridiplantae (Green Plants) from the National Centre for Biotechnical Information (NCBI) (October 2015). Precursor tolerances were 4 ppm and product ion tolerances were ±0.4 Da. Modifications accounted for are Carbamidomethyl (C) and Oxidation (M), with no enzyme specificity. No decoy database was employed. The BIOPEP database from the University of Warmia and Mazury in Olsztyn, Poland was used to determine the potential bioactive fragments within a peptide sequence.

5.2.13. Statistical analysis

The whole experiment, starting from the extraction of protein, was conducted twice and each analysis was performed usually in triplicate (n≥3). The results were presented as mean ± standard deviation (SD). One way ANOVA was carried out using SPSS version 22 (IBM, USA), to determine whether significant differences exist among treatments and Duncan triplicates range test was used to separate significant differences between the means at the 5% level. A p value <0.05 was considered as statistically significant. All graphs were drawn by Graphpad prism 6 (Graphpad Software Inc, Australia).

5.3. Results and discussion

5.3.1. Protein content of the three green algae

The Kjeldahl method for protein estimation requires a nitrogen to protein conversion factor, which varies among different types of samples and proteins. Algae usually contain a higher concentration of non-protein nitrogenous compounds, such as pigments

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(chlorophyll and phycoerythrin), nucleic acids, free amino acids, non-protein amino acids, amines, amides, nitrites, vitamins, phospholipids and inorganic nitrogen compounds, and the amount of these compounds varies among different algal species (Barbarino and Lourenco, 2005). Therefore, although the average values of conversion factors used for green, brown, and red seaweeds are 5.13, 5.18, and 4.92, respectively, the value for each seaweed genus should be determined individually to obtain a reliable result (Lourenco et al., 2002, Peng et al., 2015). The nitrogen to protein conversion factor for Ulva, Derbesia, and Oedogonium has been previously determined to be 4.6, 4.8 and 4.7, respectively (Neveux et al., 2014c), which were significantly smaller than the average value for green algae. These conversion factors were used in the present study.

The crude protein content of Ulva, Derbesia and Oedogonium was 15.8±0.1%, 28.4±0.1% and 22.3±0.1% of dry weight of algal biomass (henceforth expressed as DW) respectively. These levels of crude protein were comparable with other green algae, such as Caulerpa scalpelliformis and Caulerpa racemosa (10.50 and 12.88% DW, respectively) (Kumar et al., 2011). The protein content of the Ulva species in this study was within the 3.3-27.2% levels reported for other Ulva spp. (Cerna, 2011, Fleurence et al., 1995). As expected, the protein contents were lower than the 30-48% levels of red seaweed (Fujiwara-Arasaki et al., 1984, Morgan et al., 1980). The crude protein contents of these green algae are slightly lower than high-protein plant foods such as soybean but higher than cereal grains (Capriotti et al., 2015). Therefore, they can be considered as an alternative source of protein.

5.3.2. Amino acid composition

Table 24 shows the amino acid profiles and the essential amino acid scores of Ulva, Derbesia and Oedogonium. These green macroalgae contained all the essential amino acids (excluding tryptophan), which accounted for 38.92-43.35% of the total amino acid contents. These values were comparable to other green seaweeds such as Ulva lactuca and Ulva clathrate, in which the essential amino acids accounted for 40-42% of the total amino acids (Yaich et al., 2011, Pena-Rodriguez et al., 2011). The three green algae were quite rich in leucine, valine, lysine and threonine, which also agreed with previous reports (Yaich et al., 2011, Wong and Cheung, 2001). They exhibited similar non- 144 essential amino acid patterns to many other types of seaweed in that aspartic and glutamic acids were the predominant types, accounting for approximately 24.32-26.13% of the total amino acids, respectively. This observation was in accordance with previous reports on other seaweeds such as Pyropia columbina (Cian et al., 2015), Ulva lactuca (Yaich et al., 2011), and Ulva clathrate (Pena-Rodriguez et al., 2011). The high levels of aspartic and glutamic acids are well known to be responsible for the savoury taste of seaweeds (Yaich et al., 2011, Pena-Rodriguez et al., 2011). Previous studies also revealed that these two amino acids possess strong antioxidant activities, due to the presence of excessive electrons that can be donated when they interact with free radicals (Udenigwe and Aluko, 2011, Siow and Gan, 2013). Moreover, alanine, arginine and glycine were the third, fourth and fifth abundant amino acids in the three algae, respectively. They also can act as radical scavengers and protect tissues from oxidative stress (Udenigwe and Aluko, 2011). Apart from those amino acids, tyrosine, methionine, histidine and lysine have also been suggested to be potent antioxidants (Udenigwe and Aluko, 2011).

Of the three algal species, Oedogonium had the highest amount of total amino acids (224.9±0.9 g/kg) and also the highest amount of essential amino acids (97.5±1.4 g/kg). These were followed very closely by Derbesia while Ulva had the lower level of the total (163.4±0.8 g/kg) as well as essential amino acids (63.6±0.7 g/kg). The amounts of savoury and antioxidant amino acids generally followed the same pattern of the total and essential amino acids in the three algal species.

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Table 24. Typical amino acid profiles of Oedogonium, Ulva, and Derbesia

Amino acid Oedogonium Ulva Derbesia g/kg % g/kg % g/kg % Aspartic acid & 25.3±0.5 11.2 22.7±0.3 13.9 23.0±0.4 10.6 Asparagine Glutamic acid & 29.4±0.6 13.1 20.0±0.4 12.2 33.0±0.4 15.3 Glutamine Histidinea 4.6±0.1 2.1 2.8±0.0 1.7 4.7±0.1 2.2 Serine 11.4±0.1 5.1 9.4±0.1 5.8 11.2±0.2 5.2 Arginine 13.2±0.2 5.9 10.3±0.1 6.3 12.6±0.2 5.8 Glycine 12.4±0.0 5.5 9.5±0.1 5.8 12.4±0.2 5.7 Threoninea 12.3±0.1 5.5 9.1±0.1 5.6 11.2±0.2 5.2 Alanine 16.2±0.3 7.2 13.7±0.3 8.4 14.7±0.2 6.8 Proline 11.5±0.2 5.1 8.5±0.1 5.2 10.0±0.1 4.6 Lysinea 15.2±0.5 6.8 8.8±0.1 5.4 14.8±0.2 6.8 Tyrosine 8.0±0.1 3.6 5.7±0.0 3.5 8.4±0.1 3.9 Methioninea 4.3±0.1 1.9 2.6±0.1 1.6 4.6±0.0 2.1 Valinea 14.6±0.2 6.5 10.7±0.1 6.6 14.3±0.2 6.6 Isoleucinea 10.7±0.0 4.8 7.4±0.1 4.5 10.2±0.1 4.7 Leucinea 21.8±0.3 9.7 12.0±0.1 7.3 18.1±0.2 8.4 Phenylalaninea 14.0±0.1 6.2 10.2 ± 0.1 6.2 13.1±0.2 6.1 Essential amino acidsa 97.5±1.4 43.4 63.6±0.7 38.9 91.0±1.2 42.1 Total amino acids 224.9±0.9 - 163.4±0.8 - 216.3±0.8 - aessential amino acids for humans (except tryptophan)

5.3.3. Protein extractability of the three green algae

Extraction of proteins from seaweed is recognised to be a difficult process due to the presence of large amounts of polyanionic or neutral polysaccharides in the form of cell wall mucilage (Wong and Cheung, 2001). The cell wall mucilage can form highly viscous solutions and interfere with the extraction and purification of proteins (Wong and Cheung, 2001, Fleurence, 1999). A number of solvents, such as water, salt solution, alcohols and alkaline or acid solution, can potentially be used for extracting proteins. In this study, water and alkaline extraction was compared for their efficiency in extracting proteins from the algal biomass. Water generally only extracts water soluble proteins

146 while alkali are capable of solubilise many types of proteins, thus improving the extraction yield.

Figure 26 shows the protein extraction yields of the three algal species obtained by different extraction methods. The yields varied significantly among the three algae with Oedogonium giving the biggest yield (5.7-59.8%) followed by Derbesia (2.1-31.9%) while the yields for Ulva were rather low at 1.1-7.9%. The highest yield obtained for Derbesia and Oedogonium were higher than those reported in the literature for Ulva rigida (9.4-26.8%) and Ulva rotundata (13.8-36.1%) (Fleurence et al., 1995); however, the yield of the Ulva species of the present study was lower than the literature values.

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r p H 1 2 w ith o u t so n ica tio n p

d ,C

l 6 0 a

t p H 7 w ith so n ica tio n

o t

p H 1 2 w ith so n ica tio n m

o c ,C

r 4 0 f

d ,B d

e b ,C

t

c

a r

t 2 0

x c , B e c ,A s b ,B a ,C

n b ,A

i a ,A a ,A a , B e

t 0 o

r U lv a D e r b e sia O e d o g o n iu m P

Figure 26. Influence of extraction conditions on protein yield from Ulva, Derbesia and Oedogonium. Results are expressed as% of total protein; values are mean ± SD for triplicate experiments. Different lower case letters within the same species indicate the values being significantly different (p<0.05). Different capital letters with the same extraction condition indicate the values being significantly different (p<0.05).

As expected, solvent pH had a significant influence on the protein extraction yield. Increasing extraction solvent pH from 7 to 12 resulted in markedly higher yields for all three algal species. For example, the protein yield for Oedogonium more than tripled from 11.7±0.1% to 37.9±0.1% when the pH was increased from 7 to 12, in the absence of ultrasound treatment. Similar trends were also observed for the other two algae, and also reported by previous researchers (Ursu et al., 2014). The results are expected as

147 alkaline solutions are known to solubilise many proteins in natural matrices and are commonly used to extract proteins from sources such as soybean for making protein isolates.

In the present study, ultrasound was tested for its effect on facilitating protein extraction from algae. Ultrasound generates mechanical, cavitation and thermal effects which can result in disruption of cell walls, particle size reduction and enhanced mass transfer across cell membranes. The implosion of cavitation bubbles generates micro-turbulence, high velocity inter-particle collisions and perturbation in micro-porous particles, resulting in enhanced extraction yield (Shirsath et al., 2012). For these reasons, ultrasound-assisted extraction has been applied to the extraction of many components such as protein, lipid and polyphenolics from biological matrices including algae (Carrera et al., 2012, Araujo et al., 2013, Tang et al., 2010). In this study, ultrasound was found to substantially increase the extraction yield of protein from all three algal samples under both neutral and alkaline conditions (Figure 26). For example, at pH 12, protein yield from Derbesia almost tripled from 11.68 ± 0.02% to 31.92 ± 0.03 with the application of ultrasound. At pH 7, the protein yield from the same species increased from 5.73 ± 0.01% to 13.40 ± 0.01% with ultrasound treatment. Similar trends were also observed for the other two algal samples. This finding also agrees with data reported for the extraction of rapeseed protein (Dong et al., 2011), perilla seed protein (Zhu and Fu, 2012), and peanut protein (Li et al., 2013). Li et al. (2013) further showed that ultrasound increased the solubility of peanut proteins, enhanced cleavage of peptide bonds and loosened the peanut kernel structure. Similar structural changes were likely to also occur in the algae, whereby facilitating the extraction of protein from them.

5.3.3.1. SDS-PAGE analysis of the algal proteins

The SDS-PAGE profiles of proteins extracted from the three algal samples using various methods are shown in Figure 27. Figure 27 (A) shows proteins extracted at pH 7 and pH 12. As can be seen, lanes 4-6 had a strong background, which made the bands hard to differentiate. Lanes 1 and 2 were Ulva protein extracted at pH 7 and pH 12, respectively, which showed similar bands; however, the bands in lane 2 were much more intense than those in lane 1, indicating greater protein content obtained at pH 12. Figure 27 (B) shows the SDS-PAGE profiles of proteins extracted under the same 148 conditions but with the assistance of ultrasound treatment. The bands were broadly similar to those obtained without ultrasound as shown in Figure 27 (A).

It is clear that the proteins extracted by both the alkaline and aqueous methods had a poor quality in that they were poorly resolved by electrophoretic separation. This is most likely due to the co-extraction of non-protein cellular components that could affect protein migration during electrophoresis. These interfering compounds may include polyphenols, pigment, terpenes, polysaccharides and organic acids, which can accumulate in vacuoles abundantly in biological materials such as algae (Wang et al., 2003).

In order to obtain a better understanding of the proteins in the algal species, another protein extraction procedure was used, in which TCA, acetone and methanol washes were employed, coupled with phenol extraction, to remove polysaccharides, lipids, pigments, phenolics and other substances from the biomass. With this extraction procedure, proteins obtained gave much better electrophoretic separation as shown in Figure 28, where most of the protein bands were well resolved with low background interference. Lanes 1, 2 and 3 were Ulva proteins applied at increasing concentration (5 µl, 10 µl and 15 µl, respectively), which showed similar band profile but with increasing intensity. Similar trends were also observed for the other two algal species, but Oedogonium gave much more intense bands than Ulva and Derbesia.

The SDS-PAGE profiles indicated that the algal proteins had molecular weights ranging from 15 to 50 kDa. Many studies have shown that phenol extraction gave satisfactory results in recalcitrant plant tissues rich in components which can interfere with electrophoresis. Examples include high levels of polyphenols in olive leaves (Wang et al., 2003), high sugar content in banana (Song et al., 2006), high acidity in grape (Vincent et al., 2006), and high pigment content in sea grass (Migliore et al., 2007). Results obtained in this study confirmed these previous findings. However, this procedure also has drawbacks. The TCA/acetone washing step needs to be carried out until the biomass becomes colourless, which is time-consuming. Also, extended exposure to low pH may lead to protein degradation or modifications. Furthermore, protein obtained from TCA precipitation is hard to be completely re-solubilised (Wang et al., 2008). Finally, phenol extraction is not suitable to produce protein for food use as it has high toxicity. For this reason, the phenol/acetone extraction was only used for the 149

SDS-PAGE analysis of algal proteins. The alkaline extraction procedure, as described in the previous section, was used to obtain proteins for analysis of biological activities.

150

kDa (A) kDa (B)

M 1 2 3 4 5 6 M 1 2 3 4 5 6

Figure 27. SDS-PAGE separation of Ulva, Derbesia and Oedogonium protein prepared using different extraction method (A) without sonication, (B) with ultrasound. Line M indicates protein mass markers. Line 1-2, Ulva protein extracted at pH 7 (line 1) and 12 (line 2). Line 3-4, Derbesia protein extracted at pH 7(line 3) and 12 (line 4). Line 5-6, Oedogonium protein extracted at pH 7(line 5) and 12 (line 6). Protein molecular mass standards (in kDa) are indicated on theleft.

151

kDa

M 1 2 3 4 5 6 7 8 9

Figure 28. SDS-PAGE separation of Ulva, Derbesia and Oedogonium protein prepared using phenol extraction. Line 1-3, Ulva protein loaded at 5 µL (line 1), 10 µL (Line 2) and 15 µL (line 3). Line 4-6, Derbesia protein loaded at 5 µL (line 4), 10 µL (Line 5) and 15 µL (line 6). Line 7-9, Oedogonium loaded at 5 µL (line 7), 10 µL (Line 8) and 15 µL (line 9). Protein molecular mass standards (line M) with sizes (in kDa) are indicated on the left.

5.3.3.2. Protein content in the extract

Protein content in the protein extracts was estimated by the Lowry method and the results are shown in Table 25. The protein contents in the extracts were generally low, ranging from non-detectable levels in the water extracts (pH 7) of Ulva to 24.53 mg/g extract in the alkaline extract (pH 12) of Derbesia. Extraction with alkaline at pH 12 gave higher protein contents than extraction with water at pH 7 except for Oedogonium, for which a higher protein content was obtained at pH 7 with sonication. Sonication generally did not have a remarkable effect on protein content, although the protein content values were statistically different. However, since extraction with sonication gave considerably higher extraction yields, as shown in the previous section, extraction with alkaline solution at pH 12 with sonication was selected as the method of protein extraction in all subsequent experiments. 152

Table 25. Protein content in the extracts of Ulva, Derbesia and Oedogonium

Without sonication With sonication

BCA pH 7 pH 12 pH 7 pH 12

Ulva NA 19.75±0.10bB NA 17.93±0.17aB

Derbesia 17.11±0.01bA 23.83±0.09cC 16.53±0.10aA 24.53±0.30dC

Oedogonium 19.69±0.03cB 18.24±0.10bA 20.16±0.11dB 15.92±0.16aA

All values are mean ±SD for triplicate experiments (n=3). Results are measured as mg of BSA/g of dry crude protein extract. Different lower case letters within the same row indicate significant difference between the values (p<0.05). Different capital letters within the same column indicate significant difference between the values (p<0.05). NA: too low to detect.

5.3.4. In vitro simulated gastrointestinal digestion of algal proteins

The proteins extracted from the three algal species were subjected to in vitro simulated human gastrointestinal digestion where α-amylase, pepsin and pancreatin preparations were added sequentially under conditions and for durations that mimic the human digestive process. The digests (hydrolysates) obtained at the end of the digestion were analysed for the degree of protein hydrolysis, antioxidant capacities and inhibitory activities against enzymes that associated with several important metabolic syndromes.

5.3.4.1. Degree of protein hydrolysis

Table 26 shows the DH under simulated human digestion conditions for proteins from the three algal species. For proteins from all three algal samples, the DH increased at every stage of digestion, with the DH values rising to 31.7-40.1%, 53.5-62.8% and 75.6-78.6% after amylase, pepsin and pancreatin digestions, respectively. The rate of protein hydrolysis for the three algal species did not show great variations, and at the end of the digestion process, very similar DH values (75.6-78.6%) were observed for proteins from all three sources.

153

Table 26. The degree of hydrolysis (%) of proteins from three algal species at different stages of simulated human digestion

Algal species Before Enzyme digestion α-Amylase Pepsin Pancreatin

Ulva 12.7 ± 0.2aA 31.7 ± 1.2bA 62.8 ± 1.9cC 75.6 ± 3.5dA

Derbesia 19.4 ± 1.2aB 40.1 ± 1.9bC 58.5 ± 1.8cB 78.6 ± 3.3dC

Oedogonium 22.1 ± 1.5aC 38.8 ± 1.7bB 53.5 ± 3.5cA 76.1 ± 1.8dB

All values are mean ±SD for triplicate experiments (n=3). Different lower case letters within the same row indicate significant difference between the values (p<0.05). Different capital letters within the same column indicate significant difference between the values (p<0.05).

When the hydrolysis of protein at each digestion stage was examined closely, it was found that protein hydrolysis occurred more or less equally over each stage, although there were notable variations to this general observation. The hydrolysis of protein during pepsin (an endopeptidase) and pancreatin (a mixture of trypsin, chymotrypsin, elastase and carboxypeptidases) digestions were expected as the enzymes involved are well known proteases present in the human digestive system. The action of pepsin occurs in the stomach where it hydrolyses proteins at various cleaving points to smaller polypeptides (Goodman, 2010). Pancreatin acts in the small intestinal where the various proteolytic enzymes function jointly to cleave the polypeptides into oligopeptides and amino acids (Goodman, 2010). However, the significant hydrolysis during the amylase digestion stage was unexpected as this enzyme is not proteolytic. There are two possible explanations for this unexpected result: 1) contamination of the α-amylase preparation by proteases; and 2) presence of indigenous proteases in the algal protein extracts. The α-amylase preparation used in this study was from the microorganism Bacillus subtilis. This organism is also commonly used for the production of proteases in the fermentation industry (Oyeleke et al., 2011, Mukhtar and Ikram-Ul, 2012). Therefore, it is possible that during the extraction of α-amylase from the cell culture used for its production, some proteases were co-extracted, especially given the difficulties in the fractionation and purification of proteins. On the other hand, it is also highly possible that the algal biomass used in the study also contained proteases, some of which were extracted alongside the other proteins. To test these possibilities, a sample control experiment was conducted where the protein samples were subjected to the same 154 simulated digestion, except that no digestive enzymes were used at any of the stages (Figure 29). It was clear that protein hydrolysis occurred in the sample controls in the absence of the digestive enzymes including α-amylase, thus demonstrating that the algal protein extracts contained proteases. To further verify this finding, another control experiment was conducted where the protein samples were subjected to the same simulated digestion process, except that the protein samples were heat treated at a 100°C water bath for 10 min before the digestion to inactivate indigenous proteases (Figure 30). As can be seen, the DH values after α-amylase digestion was slightly higher than those before digestion, although the differences were statistically significant. Furthermore, the overall DH values after pancreatin digestion were slightly lower than 60%, which were about 16-20% lower than the DH values shown in Table 26 where the indigenous proteases in the algal protein extracts were not subjected to heat inactivation. The differences in the DH values between the two experiments were very close to those caused by the indigenous proteases as shown in Figure 29. These results clearly demonstrated that contamination of the algal proteins by indigenous proteases was the main, if not the only, cause of protein hydrolysis occurred in the α-amylase digestion stage.

To date, there have been no reported studies on simulated human digestion of algal proteins using multiple proteolytic enzymes as in this thesis. However, several studies have used individual digestive enzymes to hydrolyse algal proteins and the results are generally comparable to those in the present study. For examples, Wong and Cheung (2001) hydrolysed Hypnea and Ulva protein with trypsin and obtained DH vaules of 85.7 to 88.9%. Ryu et al. (1982) achieved a DH of 78.5% for the hydrolysis of red and green seaweed proteins from Korea using chymotrypsin and peptidase enzymes. Wong and Cheung (2001) also stated that the in vitro protein digestibility of the red seaweed protein extracts was significantly higher than that of the green algae, which appeared to agree with the results obtained in this study. These differences are attributed to several factors including the digestibility of proteins in different algal species as well as the non-proteins contaminants in the extracts, such as polysaccharides, phenolic compounds, etc., and their different ability to interfere with protein digestion (Wong and Cheung, 2001).

155

B e fo re d ig e stio n P e p s in s ta g e

 -a m y la s e s ta g e P a n c re a tin s ta g e 5 0

) d ,C %

( 4 0 d ,B

d ,A s

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y b ,B

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y a ,C h

a , B f

o 2 0

e

e a ,A

r g

e 1 0 D

0 U lv a D e r b e sia O e d o g o n iu m

Figure 29. The degree of hydrolysis of sample controls of in vitro human simulated digestion. The digestion was conducted in the absence of the digestive enzymes; all other conditions were as described in section 5.2.8. Data represent the mean ± SD of at least three independent experiments. Different lower case letters within the same species at different digestive stages indicate significant difference between the values (p<0.05). Different capital letters at the same digestive stages for different species indicate significant difference between the values.

156

B e fo re d ig e stio n P e p s in s ta g e

 -a m y la s e s ta g e P a n c re a tin s ta g e

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% (

d ,C s 6 0 d ,B

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Figure 30. The degree of hydrolysis of sample controls of in vitro human simulated digestion. Prior to digestion, the sample protein extracts were heated at 100°C for 10 min to inactivate indigenous proteases of algae. All other conditions were as described in section 5.2.8. Data represent the mean ± SD of at least three independent experiments. Different lower case letters within the same species at different digestive stages indicate significant difference between the values (p<0.05). Different capital letters at the same digestive stages for different species indicate significant difference between the values (p<0.05).

5.3.5. Antioxidant activity of in vitro simulated digests of algae proteins

5.3.5.1. ABTS radical scavenging activities

ABTS assay was used to measure the antioxidant activities of in vitro simulated gastrointestinal digests of algal proteins before and after the digestion process (Figure 31). The ABTS scavenging activities of the protein samples before digestion ranged from 686.7 to 991.0 µmol TE/g of protein, which increased markedly (1208.1-2525.8 µmol TE/g protein) after digestion. The increases in ABTS scavenging activities were particularly pronounced for the Derbesia and Oedogonium protein samples where the activities more than doubled after the digestion. This broadly agrees with the findings of Cian et al. (2015) that the ABTS scavenging activity of Pyropia columbina proteins increased by more than 4 times after digestion with pepsin and pancreatin. Similar 157 increases in ABTS values have also been reported for other proteins, e.g., peanut protein, after gastric and gastrointestinal digestion (Zheng et al., 2013).

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f

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1 0 0 0

c

l o

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m

S



T

(

B A

0 N o n -h y d r o ly se d H y d r o ly se d

Figure 31. ABTS scavenging activities of protein from Ulva, Derbesia and Oedogonium before and after in vitro simulated digestion. All values are mean ± SD for triplicate experiments. Different lower case letters within the same treatment indicate significant difference between the values (p<0.05). Different upper case letters for the same species indicate significant difference between the values (p<0.05).

5.3.5.2. Ferric reducing antioxidant power (FRAP)

FRAP assay was also used to measure the antioxidant activities of in vitro simulated digests of algal proteins before and after the digestion process (Figure 32). The data trends were similar to those obtained by the ABTS assay. FRAP values of the proteins were very low (0.5- 1.2 µmol TE/g of protein) prior to digestion. After the protein was hydrolysed, substantially higher FRAP values were observed (2.75-13.75 µmol TE/g protein) for all the protein digests, but especially for Derbesia and Oedogonium proteins where the values increased by more than 10 times. The FRAP values obtained for the protein hydrolysates in the present study were comparable with those reported for Palmaria palmate protein hydrolysates (8.9-19.9 µmol TE/g) (Harnedy et al., 2014). Similar increases in FRAP values have been reported for a number of food proteins hydrolysed with digestive and other commercial proteases (Lin et al., 2012, Siow and Gan, 2013).

158

1 5 c , B

b ,B U lva

)

n i

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t o

r O e d o g o n iu m

s

p

e

f 1 0

o

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l

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a

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P

o

l

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o R

r 5

F

T

l

o a , B

m 

( b ,A b ,A a ,A 0 N o n -h y d r o ly se d H y d r o ly se d

Figure 32. FRAP values of proteins from Ulva, Derbesia and Oedogonium before and after in vitro simulated digestion. All values are mean ± SD for triplicate experiments. Different lower case letters within the same treatment indicate significant difference between the values (p<0.05). Different upper case letters within the same species indicate significant difference between the values (p<0.05).

The improvement in the antioxidant activities of algal and other proteins as a result of proteolysis is not surprising as proteins contain many amino acids that have been shown to have antioxidant activities. For example, aspartic and glutamic acids, which were two of the most abundant amino acids in the three algal species studied in this thesis (Table 24), possess strong antioxidant activities, due to the presence of excessive electrons that can be donated when they interact with free radicals (Udenigwe and Aluko, 2011, Siow and Gan, 2013). Moreover, alanine, arginine and glycine acids, which also exist abundantly in the algal species, are also potent antioxidants, as are tyrosine, methionine, histidine and lysine (Udenigwe and Aluko, 2011). Hydrolysis would cause the release of these amino acids and exposure of their electron-dense side groups (Zheng et al., 2013), thus leading to an increase in antioxidant activities of the protein digests. Furthermore, previous studies have shown that after pepsin treatment, more hydrophobic side chain groups were exposed, and when the pepsin digest was further hydrolysed with pancreatin, the digest became more hydrophilic with the accumulation of shorter peptides and amino acids, which could readily react with the water-soluble ABTS radical (Zheng et al., 2013).

159

5.3.6. Inhibitory activity of algal protein hydrolysates against starch digesting enzymes

Diabetes mellitus has become an epidemic occurring in adults and, increasingly, children also, throughout the world and is the leading cause for kidney failure, heart attack, blindness and lower limb amputation (Tabish, 2007). α-Amylase and α- glucosidase are two of the enzymes closely involved in the digestion of starch. The former is an endo-enzyme, acting on α-1,4-glucosidic bonds of starch, producing predominantly oligosaccharides. The latter is an exo-enzyme that also cleaves α-1,4- glucosidic bonds, but breaking down starch and oligosaccharides into glucose that can be readily absorbed by the body (Lee and Jeon, 2013). Postprandial increase in blood glucose level after a starch meal can be dangerous for diabetic patients as their body does not produce enough insulin to metabolise the sugar (Tundis et al., 2010). Research has also shown that repeated high postprandial blood glucose levels may lead to insulin resistance, with potential development of type 2 diabetes (Tundis et al., 2010). Inhibition of the enzymes would delay the degradation of starch and prolong its overall digestion time, thus reducing the postprandial glucose rush. Oral administration of α- amylase and α-glucosidase inhibitors, such as Acarbose, is a major strategy in the health management of type 2 diabetes. Hence, testing of the inhibitory activity of a substance against α-glucosidase and α-amylase are two of the mostly commonly used in vitro assays for determining its potential antidiabetic activities. In this study, the anti-diabetic potential of the hydrolysates produced by the in vitro simulated digestion of proteins from the three green algae were investigated using these two in vitro assays, which has not been attempted for algal proteins previously.

5.3.6.1. α-Amylase inhibitory activities

Figure 33 shows the α-amylase inhibitory activities of the algal proteins before and after in vitro simulated digestion. The crude proteins before in vitro digestion showed very little α-amylase inhibitory activity. Digestion of the proteins resulted in drastic increases in their α-amylase inhibitory activities, which were 7-10 times higher than the original proteins. The activities of the algal protein hydrolysates ranged from 9.0 to 35.4 mg Acarbose equivalent/g protein (peptide), with the hydrolysate of Oedogonium

160 protein exhibiting the highest activity, followed very closely by Derbesia, while Ulva exhibited the lowest activity. These results essentially mean that consumption of 144-36 mg of the algae protein, which would gave rise to 111-28 mg of the protein digest, would be equivalent to the oral administration of one mg of Acarbose in the effect of α- amylase inhibition.

5 0

c , B U lva

) n

e D e rb e sia d

o 4 0

i

i

t

t i

p b ,B e

b O e d o g o n iu m

i

p

h

g 3 0

n

/

i

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e

s

s o

a a , B b

l 2 0

r

y

a

c

m

a

A g

- 1 0

m

 ( b ,A b ,A a ,A 0 N o n -h y d r o ly se d H y d r o ly se d

Figure 33. Inhibition of α-amylase activity by proteins from Ulva, Derbesia and Oedogonium before and after in vitro simulated digestion. Data represent the mean ± SD of at least three independent experiments. Different lower case letters for the same algal species but different treatment indicate significantly different values (p<0.05). Different uppercase letters for the same treatment but different algal species indicate significantly different values (p<0.05).

5.3.6.2. α-Glucosidase inhibitory activity

α-Glucosidase is a key enzyme in starch digestion as it converts starch fragments produced by a-amylase into glucose which is readily absorbed by the body. Therefore, blocking the action of α-glucosidase can effectively reduce the impact of starchy foods on the level of blood sugar in diabetic patients (Kunyanga et al., 2012). Figure 34 shows the α-glucosidase inhibitory activities of the algal proteins before and after the simulated digestion, and essentially the same data trends as for α-amylase inhibition were observed. The undigested proteins showed very little α-glucosidase inhibitory activities, but their digests exhibited substantially increased activities ranging from 37 to 76 mg Acarbose equivalent/g protein digest, which were 10-11 times higher than the

161 original undigested proteins. This essentially means that consumption of 35-17 mg of algae protein, which would gave rise to 27-13 mg of the protein digest, would be equivalent to the oral administration of one mg of Acarbose in the effect of α- glucosidase inhibition. The hydrolysates of Oedogonium and Derbesia proteins had virtually the same a-glucosidase inhibitory activity, while the activity of Ulva protein hydrolysate was considerably lower, being slightly less than half of the other two proteins.

This thesis is the first to report on the α-amylase and α-glucosidase inhibition effects of algal protein hydrolysates produced under in vitro simulated human digestion conditions, and there are no similar studies on algae proteins to compare with. Studies on these activities of other protein digests are reported in the literature, but the activities reported are usually expressed as percentage of inhibition or IC50 values. To facilitate comparison, some of the reported activities were converted to Acarbose equivalents as in the current study. Reported inhibitory activities against α-amylase from other proteins ranged from 5 mg Acarbose/g protein for bioactive peptides derived from cumin seeds (Siow and Gan, 2016) to 26 mg Acarbose/g protein for Pinto bean protein digested using Protamex (Ngoh and Gan, 2016), while activities against α-glucosidase ranged from 13.6 mg Acarbose/g protein for whey protein (Lacroix and Li-Chan, 2013) to 172 mg Acarbose per g protein for egg yolk protein (Zambrowicz et al., 2015). The activities of the hydrolysates of the three algal proteins fall within these values.

162

1 0 0

b ,B U lva

n )

o b ,B

i

n i t D e rb e sia

i 8 0

e

t

b

i o

r O e d o g o n iu m

h

p

n

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6 0

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d a , B

i b

r 4 0

s

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c

c

a

u

g l

G 2 0

m

- (

b ,A b ,A  a ,A 0 N o n -h y d r o ly se d H y d r o ly se d

Figure 34. Inhibition of α-glucosidase activity by proteins from Ulva, Derbesia and Oedogonium before and after in vitro simulated digestion. Data represent the mean ± SD of at least three independent experiments. Different lower case letters for the same algal species but different treatment indicate significantly different values (p<0.05). Different uppercase letters for the same treatment but different algal species indicate significantly different values (p<0.05).

Some researchers have reported antidiabetic activities of algal protein hydrolysates using different assay procedures. For example, Harnedy et al. (2015) examined the dipeptidyl peptidase (DPP)-IV inhibitory activities of an aqueous Palmaria palmate protein extract, and the results showed that peptides derived from the extract exhibited the activity. This has led the researchers to suggest that the algal extract may have the potential to be developed into functional food ingredients for the prevention and management of type 2 diabetes. Clearly, proteins from the three green algae investigated in this thesis also have such potentials. The results obtained in this study also imply that consumption of the algal proteins or the algae directly may confer significant health benefits to the consumer in the prevention and management of diabetes.

5.3.6.3. ACE inhibition activities of algal protein hydrolysates

The capacity of the in vitro simulated digests of algae proteins to inhibit ACE was determined to assess their antihypertensive potentials (Table 27). The ACE-inhibitory activity of the protein digests was expressed as captopril equivalent, which is one of the 163 standard prescription drugs for managing hypertension (Odaka and Mizuochi, 2000). Results showed that the protein hydrolysates had a moderate capacity to inhibit ACE with effects ranging from 78.5 to 122.9 nmol captopril/g protein, whereas the non- hydrolysed samples did not show any ACE-inhibitory effects (data not shown). Of the three algal species, the hydrolysate of Ulva protein exhibited the highest ACE- inhibitory activity, followed by Oedogonium, while Derbesia exhibited the lowest activity.

Table 27. Comparison of ACE-inhibition potency of protein hydrolysates from Ulva, Derbesia and Oedogonium with other algal species reported in literature

Source Digestion IC50 Captopril References enzyme Equivalent1

Captopril 4.73 ng/mL - Current study

Ulva NA 122.9±2.3 Current study

Derbesia NA 76.4±1.3 Current study

Oedogonium NA 78.5±0.7 Current study

Palmaria Alcalase 140 µg/mL 155.5 (Furuta et al., 2016) palmata

Undaria Protease S 70-219 µg/mL 99.4-311.0 (Sato et al., 2002a, Sato pinnatifida “Amano” et al., 2002b)

Porphyra 1520-3210 µg/mL 6.8-14.32 (Suetsuna, 1998b) yezoensis

1Captopril equivalent are measured as nmol captopril/g protein.

Table 27 also shows the ACE-inhibition activities of protein hydrolysates of three other algal species published in the literature for the purpose of comparison. The protein digests of the three algae of the present study exhibited higher activity than Porphyra yezoensis (a red alga), but generally lower activities than Undaria pinnatifida and Palmaria palmate. Siow and Gan (2013) suggested that large protein polypeptide chains 164 usually do not possess ACE inhibitory activity due to the long and bulky structure that prevents them from accessing the binding site of ACE.

5.3.7. Fractionation of the bioactive peptides

It has been widely reported that molecular sizes of peptides in protein hydrolysates affect their bioactivities and fractionation of the peptides according to their molecular weight (MW) has generally led to improvement in the biological activities of particular fractions (Fan et al., 2012). In the present study, the protein hydrolysates from the three algal species were fractionated using ultrafiltration (UF) membranes. To guide the selection of membranes with appropriate molecular cut-offs, SDS-PAGE was performed on the protein hydrolysates, and it revealed that there were no definitive bands in the electrophoretogram (Appendix L), indicating that the digestion resulted in the formation of a large number of peptides of various sizes. This led to the selection membranes with two MW cut-offs, 3 kDa and 10 kDa, and the fractionation was expected to result in three peptide fractions: MW > 10kDa, MW 3-10 kDa and MW <3kDa.

When the collected fractions were analysed, it was found that for the Ulva protein hydrolysate, 70% of the hydrolysates passed through the membrane with 10 kDa cut-off, and for Derbesia and Oedogonium, 50% of them passed through. Interestingly, virtually all of the MW <10 kDa peptides (those passed through the 10 kDa membrane) also passed through the 3 kDa cut-off membrane, which meant that no MW 3-10 kDa fraction was collected due to the small quantities. Consequently, only two fractions were collected, the MW >10kDa fraction and the MW <3 kDa fractions. It was thought that the former may consist of some proteins which were resistant to hydrolysis, as well as impurities such as polysaccharides, while the latter consisted of small peptides resulted from the digestion. The fractions were analysed for their antioxidant, and α- amylase, α-glucosidase and ACE-inhibitory activities.

5.3.7.1. Antioxidant activities of the peptide fractions

The antioxidant activities of the ultrafiltered fractions of the algal protein digests were analysed by both the FRAP and the ABTS free radical scavenging assays, and the results are shown in Figure 35 and Figure 36, respectively. Of the two fractions, the 165

MW <3 kDa peptides had significantly higher FRAP and ABTS values than the other fraction (p<0.05) for all three algal hydrolysates, indicating that peptides with smaller molecular weights conferred greater antioxidant activities. For the MW <3 kDa fraction of the three algal species, Oedogonium exhibited the highest ABTS scavenging activity, which was nearly twice that of Ulva, while Derbesia had the second highest activity, but it was substantially lower than that of Oedogonium. On the other hand, the MW <3 kDa fraction of Derbesia had the highest FRAP value, followed closely by Oedogonium, while the FRAP value of Ulva was considerably lower than the other two species.

When the antioxidant activities of the fractions were compared with those of the crude (non-ultrafiltered) hydrolysates of respective species, it was found that the former were significantly lower than the latter for all three algal species, and for both the FRAP and ABST assay results. The antioxidant activities of the MW <3 kDa fractions were generally lower than half of the crude hydrolysates. These results appear to suggest that there might be some synergistic effects between the proteins and peptides in the algal hydrolysates, and separation of the peptides interrupted the synergy with resultant reduction in their antioxidant activities.

The results obtained in this study are in general agreement with those in the literature (Alashi et al., 2014, Ren et al., 2008, Zhang et al., 2014, Ngoh and Gan, 2016). These researchers also reported that peptides with molecular weight less than 3kDa exhibited higher antioxidant activities compared to the larger MW fractions. These findings are not surprising because peptides exhibit antioxidant activities through their electron- dense side groups that can donate electrons when they interact with free radicals (Udenigwe and Aluko, 2011, Siow and Gan, 2013). The smaller peptides are expected to have more of these side groups exposed than the large ones. Furthermore, the MW <3 kDa fraction is also expected to contain some free amino acid released during protein hydrolysis. As discussed previously, many of these amino acids possess antioxidant activities and would contribute to the overall antioxidant activities of this fraction. Because the MW <3 kDa fraction had the higher antioxidant activities, it was selected for identification of the antioxidative peptides present in it.

166

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Figure 35. FRAP reducing power of ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion. Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate significantly different values with p<0.05.

167

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Figure 36. ABTS scavenging activities of ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion. Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate significantly different values with p<0.05.

5.3.7.2. α-Glucosidase and α-amylase inhibition activities of the peptide fractions

The α-amylase and glucosidase inhibition activities of the ultrafiltered fractions of algal protein digests are presented in Figure 37 and Figure 38, respectively. Similar to the antioxidant results, the MW <3 kDa fractions showed higher α-glucosidase and α- amylase inhibitory activities than the MW > 10 kDa fractions for all three algal species. Of the three species, Oedogonium hydrolysates exhibited the highest α-glucosidase and the α-amylase activities for both fractions. This was followed very closely by Derbesia hydrolysates, while Ulva hydrolysates gave the lowest activities, which were substantially lower than those of the other two green algae. Furthermore, the inhibitory activities of the fractions on the enzymes were also lower than the crude hydrolysates

168 without ultrafiltration, although the differences were not very pronounced between the crude and the MW <3 kDa fraction.

These results demonstrated two findings. First, it is the extensively hydrolysed peptides with relatively small molecular masses that are largely responsible for the α-glucosidase and α-amylase inhibitory activity of the hydrolysates. This agrees with the finding that α-glucosidase and α-amylase inhibition activities of the algal proteins increased markedly after in vitro simulated digestion as shown in previous sections of this chapter, and is consistent with reports on other protein hydrolysates (Ngoh and Gan, 2016). Second, some degree of synergism existed among the peptides in the hydrolysates and separation of the peptides into fractions disrupted this synergy with resultant decrease in their inhibitory activity towards these two enzymes.

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Figure 37. α-Amylase inhibition by ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion. Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate significantly different values with p<0.05.

169

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Figure 38. α-Glucosidase inhibition by ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion. Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate significantly different values with p<0.05.

5.3.7.3. ACE-inhibition activities of the peptide fractions

Figure 39 shows the ACE-inhibitory activities of the ultrafiltered fractions of the in vitro simulated digests of algal proteins. The data trends are generally similar to those for the antioxidant and α-glucosidase activities, i.e., the MW <3 kDa fraction of algal protein hydrolysates exhibited higher ACE-inhibitory activities than the MW > 10 kDa fraction. This result is in agreement with the reports of Tagliazucchi et al. (2016) and Mirzaei et al. (2015) who also found that low MW peptides usually exhibit higher ACE-inhibitory activities than high MW ones. However, for the MW <3 kDa fraction, the Ulva hydrolysate exhibited the highest ACE activity, which was nearly twice that of Derbesia which had the second highest activity, while Oedogonium showed the lowest activity, which was substantially lower than that of Derbesia. This trend was the

170 complete opposite to those of the antioxidant and α-amylase and α-glucosidase inhibitory activities. The ACE-inhibitory activity of the non-ultrafiltered crude hydrolysate was higher than the MW <3 kDa fractions for all three algal species; however, the differences were not very pronounced, a trend that was similar to that for α-amylase and α-glucosidase inhibitory activities. Because the MW <3 kDa fraction gave the highest activity for all the bioactivities tested, this fraction was subjected to LC-MS analysis to determine the peptides present.

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Figure 39. ACE inhibitory activity of ultrafiltered fractions of algal protein hydrolysates produced by in vitro simulated gastrointestinal digestion. Data represent the mean ± SD of at least three independent experiments. Different lower case letters indicate significantly different values with p<0.05.

5.3.8. Identification of bioactive peptides in the hydrolysates of algal proteins

The results described in the previous sections showed that the MW <3 kDa fraction obtained by ultrafiltration of the in vitro simulated digests of algal proteins had the 171 highest bioactivities. Therefore, this fraction was collected and subjected to analysis by LC-MS/MS to identify the peptide sequences present. The peptides identified were then searched against the peptide bioactivity database, BIOPEP, for amino acid sequences with known antioxidant, antidiabetic or ACE-inhibitory activities. In recent literature, the BIOPEP database has been used more often than other peptide bioactivity analysis tools for food protein as it is a database that focuses on peptides of food origin (Huang et al., 2015).

In total, 28, 16 and 23 different peptide sequences were found in the MW <3 kDa fraction of Ulva, Derbesia and Oedogonium, respectively (Table 28). Some of these peptide sequences have previously been identified in other algal species such as Ulva pertusa, Monoraphidium neglectum, Oedogonium cardiacum and Coccomyxa subellipsoidea (Brouard et al., 2008, Bogen et al., 2013, Blanc et al., 2012). The relatively large number of peptides in the hydrolysates was in agreement with the results of SDS-PAGE analysis that there was no distinct band from the peptides in the hydrolysates. It also indicated the random nature of the proteolytic actions on the algal proteins during the in vitro simulated digestion.

The molecular weight of the peptides was between 350 and 1400 Da. Many peptides with MW within this range have been shown to exhibit various biological activities. For example, It has reported that the molecular weight of the majority of antioxidant peptides falls in the range between 500 and 1800 Da (Jun et al., 2004, Kim et al., 2007, Klompong et al., 2009). Also, Xin et al. (2003) reported that peptides with molecular weights from 400 to 1500 Da have strong ACE inhibition activities with significant antihypertensive potentials.

5.3.8.1. Identification antioxidant peptides

When the algal peptides were searched against the BIOPEP database, six of the amino acid sequences (VKL, PHN, RHV, IR, EL and FD) from Ulva protein hydrolysates, four (IR, KD, EL and RHV) from Derbesia, and eleven sequences (AH, HL, KD, KP, LH, LHL, LK, MY, PHL, PW and PWQ) from Oedogonium, are found in the BIOPEP database as antioxidant peptides (Table 28). All these antioxidant sequences consisted of two or three amino residues. Several peptides, such as IR, KD, PHN and VKL,

172 occurred multiple times across several peptides. PHN was the most frequently repeated active peptide in Ulva, which was found 8 times in different peptide sequences (Table 28). The antioxidant activities of peptides are believed to be due mainly to the presence of amino acid residues such as lysine (K), methionine (M), histidine (H), tryptophan (W) and tyrosine (Y) (Aluko, 2015). These residues are frequently found in the identified peptides. However, the number of antioxidant peptides was relatively small, especially when compared with the much larger numbers of peptide sequences with potential antidiabetic and antihypertensive activities.

Several of the antioxidant peptide sequences have been reported in other proteins or their hydrolysates. For example, AH was found in egg white albumin (Davalos et al., 2004). HL, a dipeptide derived from milk was found to exhibit scavenging activity towards superoxide radicals (Nongonierma et al., 2013). LH, produced from soybean hydrolysis, was found to exhibit good antioxidant activity against the peroxidation of linoleic acid (Chen et al., 1996). Finally, VKL and KD derived from dried bonito and synthetic PHN and RHV also exhibit antioxidant activities (Saito et al., 2003, Kunio, 1999). These peptides are expected to make a contribution to the antioxidant activities of the simulated digest of algae protein.

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Table 28. List of peptides identified in the MW <3kDa fraction of the protein hydrolysates from three algal species and the potential antioxidative sequences identified using the BIOPEP database

Protein Observed mass Peptide sequences Potential antioxidative origin (Da) peptides* Ulva 418.7309 KMTITVQ none 435.2163 INNAGFPH none 516.2721 VKLGGDDGSLA VKL 558.2862 NAGFPHNIVF PHN 572.8152 AIVKLGGDDGSL VKL 598.3153 INNAGFPHNIV PHN 608.3329 AAIVKLGGDDGSL VKL 651.7703 DEDEVPAGVDADA none 671.849 FINNAGFPHNIV PHN 671.8491 INNAGFPHNIVF PHN 686.8361 IEFINNAGFPHN PHN 745.3847 FINNAGFPHNIVF PHN 867.4187 FINNAGFPHNIVFDE PHN 1370.1472 INNAGFPHNIVFDEDEVPAGVDADAI PHN 501.2541 RSLGQNPTE None 600.3141 AELRHVMTNL EL RHV 632.283 GEKLTDEEVDE None 699.2966 DADGNGTIDFPEF None 699.3579 KVFDKDGNGFISA KD 705.7868 ADVDGDGQVNYEE None 748.8312 VDADGNGTIDFPEF None 755.8384 DADGNGTIDFPEFL None 759.8838 LGTVMRSLGQNPTE None 849.3759 MIREADVDGDGQVNY IR 904.8998 IREADVDGDGQVNYEE IR 935.3948 QDMINEVDADGNGTIDF None 1121.9769 QDMINEVDADGNGTIDFPEF None 976.1428 FDKDGDGTITTKELGTVMRSLGQNPTE EL KD Derbesia 454.2128 VDEMIRE IR 539.7395 AELQDMINE EL 563.7812 KVFDKDGNGF KD 585.2701 FDKDGDGTITT KD 601.2944 RSLGQNPTEAE None 632.2833 GEKLTDEEVDE None 713.84 FDKDGDGTITTKE KD *Sequences with potential antioxidant activities were those that match the antioxidant sequences in the BIOPEP database.

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Table 28. (Continued) List of peptides identified in the MW <3kDa fraction of the protein hydrolysates from three algal species and the potential antioxidative sequences identified using the BIOPEP database

Protein Observed mass Peptide sequences Potential antioxidative origin (Da) peptides* Derbesia 755.8395 DADGNGTIDFPEFL None 767.845 MIREADVDGDGQVN IR 904.9011 IREADVDGDGQVNYEE IR 938.9774 TKELGTVMRSLGQNPTE EL 972.4687 FDKDGDGTITTKELGTVM EL KD 978.4188 MIREADVDGDGQVNYEE IR 657.9993 AELRHVMTNLGEKLTDE RHV EL 700.6802 FDKDGDGTITTKELGTVMR EL KD 976.1417 FDKDGDGTITTKELGTVMRSLGQNPTE EL KD Oedogonium 352.696 HVAHAGL AH 362.7211 VSGVLHL LH HL LHL 439.2794 ILLPHLAT LLPH HL PHL 460.7449 QGPTGLGKY none 513.2306 GGKDQESTGF KD 535.2525 YLMRSPTGE none 539.78 SAQGPTGLGKY none 571.3254 LVRDQRLGAN none 628.3439 NKLKNDIQPW PW LK 706.3991 VRIITNPTTNPSV none 709.8683 EKGIDRFNEPTL none 762.9418 VRIITNPTTNPSVI none 783.4033 FEKGIDRFNEPTL none 802.4267 LGANVASAQGPTGLGKY none 812.8943 NGTLTLGGKDQESTGF KD 834.9458 NKLKNDIQPWQER PWQ PW LK 836.4759 VRIITNPTTNPSVIF none 864.9865 VRIITNPTTNPSVIFG none 888.9169 EVAHFVPEKPMYEQG AH MY KP 983.4897 SAQGPTGLGKYLMRSPTGE none 1068.5446 VASAQGPTGLGKYLMRSPTGE none 1125.5658 NVASAQGPTGLGKYLMRSPTGE none 831.0926 LGANVASAQGPTGLGKYLMRSPTGE none *Sequences with potential antioxidant activities were those that match the antioxidant sequences in the BIOPEP database.

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5.3.8.2. Identification of potential ACE-inhibitory peptides

Compared with the relatively small number of antioxidant peptide sequences in the hydrolysates, a large number of peptide sequences with potential antihypertensive activities were found in the hydrolysates. In total, 21 potential antihypertensive sequences were found in the hydrolysate of Ulva, 18 from that of Derbesia and 24 from that of Oedogonium, with most of which being di-peptides and a few tri-peptides (Table 29). Many of the amino acid sequences occurred multiple times across several peptides from the three algal species. For Ulva, DG, GF and AG were the most repeated sequences, which were found in thirteen, ten and ten peptides, respectively, while ITT, IE and EK were found just once. Similarly for Derbesia and Oedogonium, several amino acid sequences appeared multiple times in many different peptide sequences. For example, DG was also the most repeated sequence for Derbesia and it was found in ten peptides, while PT and TG were the most repeated sequence for Oedogonium and were found also in ten peptides. Siow and Gan (2013) suggested that the presence of amino acids proline (P), tyrosine (Y) and phenylalanine (F) at the carboxyl-terminal (C- terminal) and branched-chain aliphatic amino acids such as glycine (G), alanine (A), valine (V), isoleucine (I) and leucine (L) at the amino-terminal of a peptide sequence is highly favoured for ACE inhibition. Besides, ACE is a zinc-containing peptidyl dipeptide hydrolase and its active site binds to the guanidine group of arginine (R) (Bunning, 1983), hence peptides that contain R at the C-terminal potentially facilitate peptide binding to ACE as an inhibitor (Li et al., 2006). Several peptides identified in the algal protein hydrolysates, such as IR, RF and RL, have these characteristics, indicating that these peptides could bind to the catalytic sites on ACE. A number of the antihypertensive peptides are also present in other food proteins and their hydrolysates. For example, the peptides AG, IVF and DG were isolated from soybean (Wu and Ding, 2002), NKL, KL, KY and NK from wakame (Sato et al., 2002a, Sato et al., 2002b), ITT, TNP and TTN from porcine myosin (Arihara et al., 2001), NY from gallic (Suetsuna, 1998a), GP from Alaska pollock (Byun and Kim, 2001), PT from α-zein (Yano et al., 2014) and garlic (Suetsuna, 1998a), and RF was isolated from sake lees (Saito et al., 1994). In these studies, the peptide sequences are reported as ACE inhibitors with the ability to bind to the active site of ACE. Huang et al. (2015) also reported IR and GP possessing ACE-inhibitory activity. Therefore, the presence of these peptide sequences

176 would be expected to contribute to the ACE-inhibitory activities of the algal protein hydrolysates.

5.3.8.1. Identification of potential antidiabetic peptides

Using the BIOPEP database, a large number of potential antidiabetic peptides were also identified in the algal protein hydrolysates. In total, 32 potential antidiabetic peptides were found in the hydrolysate of Ulva, 21 in Derbesia and 35 in Oedogonium, with most of which being di-peptides and a tri-peptide (Table 30). Many of the sequences occurred multiple times across several peptides from the three algal species. FP, AD, AG, GF and IN were the most frequently occurring sequences in Ulva, GQ, TI, KE, TK, and TT were the most repeated sequences in Derbesia, while PT, TG, GL, QG, and GP were the most frequently found sequences in Oedogonium. However, it needs to be pointed out that the antidiabetic peptide sequences in the BIOPEP database were based on their capacity to inhibit the dipeptidyl peptidase IV (DPP-IV) activity, while the assay of potential antidiabetic properties in the present study was based on the capacity of the hydrolysates or their fractions to inhibit α-glucosidase and α-amylase. The peptides FL, TV, GG and SL found in Ulva, TV in Derbesia and HL, EK, GG, GP, VA and GL in Oedogonium have been reported as human DPP-IV inhibitors, providing an effective strategy for the treatment of type 2 diabetes (Nongonierma et al., 2013, Lan et al., 2015, Gallego et al., 2014). The presence of these peptide sequences in the in vitro simulated digests of the algal proteins is expected to contribute to their α-amylase and α-glucosidase inhibitory activities and further demonstrates the potential of algal protein as a natural precursor for the generation of potential antidiabetic peptides in the human digestive tract.

Finally, it needs to be pointed out that the LC-MS/MS systems used in the present study did not allow identification of peptides smaller than 6 amino acid residues. However, it is likely that such smaller peptides were present in the 3 kDa permeate, and some of them could also possess antioxidant and α-amylase, α-glucosidase and ACE-inhibitory activities and make a contribution to the bioactivities of the algal protein hydrolysates.

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Table 29. List of peptides identified in the MW <3 kDa fraction of the protein hydrolysates of three algal species and the potential antihypertensive sequences identified using the BIOPEP database

Protein Observed mass Peptide sequences Potential ACE inhibitory peptides origin (Da) Ulva 418.7309 KMTITVQ none 435.2163 INNAGFPH FP GF AG PH 516.2721 VKLGGDDGSLA VK LA GS GG LG GD DG KL 558.2862 NAGFPHNIVF VF FP GF AG PH IVF 572.8152 AIVKLGGDDGSL VK GS GG LG GD DG KL 598.3153 INNAGFPHNIV FP GF AG PH 608.3329 AAIVKLGGDDGSL VK AA GS GG LG GD DG KL AI 651.7703 DEDEVPAGVDADA VP AG DA GV EV 671.849 FINNAGFPHNIV FP GF AG PH 671.8491 INNAGFPHNIVF VF FP GF AG PH IVF 686.8361 IEFINNAGFPHN FP GF AG IE PH 745.3847 FINNAGFPHNIVF VF FP GF AG PH IVF 867.4187 FINNAGFPHNIVFDE VF FP GF AG PH IVF 1370.1472 INNAGFPHNIVFDEDEVPAGVDADAI VF FP VP GF AG DA GV EV PH AI IVF 501.2541 RSLGQNPTE GQ LG TE PT 600.3141 AELRHVMTNL none 632.283 GEKLTDEEVDE GE KL EV EK 699.2966 DADGNGTIDFPEF FP DA GT NG DG 699.3579 KVFDKDGNGFISA VF GF NG DG 705.7868 ADVDGDGQVNYEE GQ GD DG NY 748.8312 VDADGNGTIDFPEF FP DA GT NG DG 755.8384 DADGNGTIDFPEFL FP DA GT NG DG 178

Table 29. (Continued) List of peptides identified in the MW <3 kDa fraction of the protein hydrolysates of three algal species and the potential antihypertensive sequences identified using the BIOPEP database

Protein Observed Peptide sequences Potential ACE inhibitory peptides origin mass (Da) Ulva 759.8838 LGTVMRSLGQNPTE GQ GT LG TE PT 849.3759 MIREADVDGDGQVNY IR GQ GD EA DG NY 904.8998 IREADVDGDGQVNYEE IR GQ GD EA DG NY 935.3948 QDMINEVDADGNGTIDF DA GT NG DG EV 1121.9769 QDMINEVDADGNGTIDFPEF FP DA GT NG DG EV 976.1428 FDKDGDGTITTKELGTVMRSLGQNPTE GQ GT LG GD ITT DG TE PT KE Derbesia 454.2128 VDEMIRE IR 539.7395 AELQDMINE LQ 563.7812 KVFDKDGNGF VF GF NG DG 585.2701 FDKDGDGTITT GT GD ITT DG 601.2944 RSLGQNPTEAE GQ LG EA TE PT 632.2833 GEKLTDEEVDE GE KL EV EK 713.84 FDKDGDGTITTKE GT GD ITT DG KE 755.8395 DADGNGTIDFPEFL FP DA GT NG DG 767.845 MIREADVDGDGQVN IR GQ GD EA DG 904.9011 IREADVDGDGQVNYEE IR GQ GD EA DG N 938.9774 TKELGTVMRSLGQNPTE GQ GT LG TE PT KE 972.4687 FDKDGDGTITTKELGTVM GT LG GD ITT DG KE 978.4188 MIREADVDGDGQVNYEE IR GQ GD EA DG NY 657.9993 AELRHVMTNLGEKLTDE GE LG KL EK

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Table 29. (Continued) List of peptides identified in the MW <3 kDa fraction of the protein hydrolysates of three algal species and the potential antihypertensive sequences identified using the BIOPEP database

Protein Observed Peptide sequences Potential ACE inhibitory peptides origin mass (Da) Derbesia 700.6802 FDKDGDGTITTKELGTVMR GT LG GD ITT DG KE 976.1417 FDKDGDGTITTKELGTVMRSLGQNPTE GQ GT LG GD ITT DG TE PT KE Oedogonium 352.696 HVAHAGL GL AG AH 362.7211 VSGVLHL HL GV SG 439.2794 ILLPHLAT LLP LA HL PH 460.7449 QGPTGLGKY GP GL GK QG LG TG KY PT 513.2306 GGKDQESTGF GF GK GG TG 535.2525 YLMRSPTGE YL GE TG PT 539.78 SAQGPTGLGKY GP GL GK QG LG TG KY PT 571.3254 LVRDQRLGAN RL LVR GA LG VR 628.3439 NKLKNDIQPW NKL KL NK IQP 706.3991 VRIITNPTTNPSV VR TNP TTN PT 709.8683 EKGIDRFNEPTL RF GI KG PT EK 762.9418 VRIITNPTTNPSVI VR TNP TTN PT 783.4033 FEKGIDRFNEPTL RF GI KG PT EK 802.4267 LGANVASAQGPTGLGKY GP GA GL GK QG LG TG KY PT 812.8943 NGTLTLGGKDQESTGF GF GK GT GG LG TG NG 834.9458 NKLKNDIQPWQER GF GK GT GG LG TG NG NKL KL NK IQP 836.4759 VRIITNPTTNPSVIF IF VR TNP TTN PT 864.9865 VRIITNPTTNPSVIFG IF FG VR IFG TNP TTN PT

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Table 29. (Continued) List of peptides identified in the MW <3 kDa fraction of the protein hydrolysates of three algal species and the potential antihypertensive sequences identified using the BIOPEP database

Protein Observed Peptide sequences Potential ACE inhibitory peptides origin mass (Da) Oedogonium 888.9169 EVAHFVPEKPMYEQG MY VP QG KP EV AH EK FVP 983.4897 SAQGPTGLGKYLMRSPTGE YL GP GL GK GE QG LG TG KY PT 1068.5446 VASAQGPTGLGKYLMRSPTGE YL GP GL GK GE QG LG TG KY PT 1125.5658 NVASAQGPTGLGKYLMRSPTGE YL GP GL GK GE QG LG TG KY PT 831.0926 LGANVASAQGPTGLGKYLMRSPTGE YL GP GA GL GK GE QG LG TG KY PT

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Table 30. List of peptides identified in the MW <3 kDa fraction of the protein hydrolysates of three algal species and the potential antidiabetic sequences identified using the BIOPEP database

Protein Observed Peptide sequences Potential DPPV inhibitor peptides origin mass (Da) Ulva 418.7309 KMTITVQ TI TV VQ 435.2163 INNAGFPH FP AG GF IN NA NN PH 516.2721 VKLGGDDGSLA LA SL GG VK 558.2862 NAGFPHNIVF FP AG GF NA PH VF 572.8152 AIVKLGGDDGSL SL GG VK 598.3153 INNAGFPHNIV FP AG GF IN NA NN PH 608.3329 AAIVKLGGDDGSL SL AA GG VK 651.7703 DEDEVPAGVDADA PA VP AD AG EV GV VD 671.849 FINNAGFPHNIV FP AG GF IN NA NN PH 671.8491 INNAGFPHNIVF FP AG GF IN NA NN PH VF 686.8361 IEFINNAGFPHN FP AG GF IN NA NN PH 745.3847 FINNAGFPHNIVF FP AG GF IN NA NN PH VF 867.4187 FINNAGFPHNIVFDE FP AG GF IN NA NN PH VF 1370.1472 INNAGFPHNIVFDEDEVPAGVDADAI PA VP FP AD AG EV GF GV IN NA NN PH VD VF 501.2541 RSLGQNPTE GQ NP SL PT QN TE 600.3141 AELRHVMTNL AE HV NL RH TN VM 632.283 GEKLTDEEVDE EK EV GE LT TD VD 699.2966 DADGNGTIDFPEF FP AD NG TI 699.3579 KVFDKDGNGFISA GF KV NG VF 705.7868 ADVDGDGQVNYEE GQ AD NY QV VD VN YE 748.8312 VDADGNGTIDFPEF FP AD NG TI VD

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Table 30. (Continued) List of peptides identified in the MW <3 kDa fraction of the protein hydrolysates of three algal species and the potential antidiabetic sequences identified using the BIOPEP database

Protein Observed Peptide sequences Potential DPPV inhibitor peptides origin mass (Da) Ulva 755.8384 DADGNGTIDFPEFL FP FL AD NG TI

759.8838 LGTVMRSLGQNPTE GQ NP SL MR PT QN TE TV VM 849.3759 MIREADVDGDGQVNY GQ AD IR MI NY QV VD VN 904.8998 IREADVDGDGQVNYEE GQ AD IR NY QV VD VN YE 935.3948 QDMINEVDADGNGTIDF AD EV IN MI NE NG QD TI VD 1121.9769 QDMINEVDADGNGTIDFPEF VD FP AD EV IN MI NE NG QD TI 976.1428 FDKDGDGTITTKELGTVM GQ NP SL KE MR PT QN TE TI TK TT TV VM RSLGQNPTE Derbesia 454.2128 VDEMIRE IR MI VD 539.7395 AELQDMINE AE IN MI NE QD 563.7812 KVFDKDGNGF GF KV NG VF 585.2701 FDKDGDGTITT TI TT 601.2944 RSLGQNPTEAE GQ NP SL AE PT QN TE 632.2833 GEKLTDEEVDE EK EV GE LT TD VD 713.84 FDKDGDGTITTKE KE TI TK TT 755.8395 DADGNGTIDFPEFL FP FL AD NG TI 767.845 MIREADVDGDGQVN GQ AD IR MI QV VD VN 904.9011 IREADVDGDGQVNYEE GQ AD IR NY QV VD VN YE 938.9774 TKELGTVMRSLGQNPTE GQ NP SL KE MR PT QN TE TK TV VM 978.4188 MIREADVDGDGQVNYEE GQ AD IR MI NY QV VD VN YE 657.9993 AELRHVMTNLGEKLTDE EK AE GE HV LT NL RH TD TN VM 183

Table 30. (Continued) List of peptides identified in the MW <3 kDa fraction of the protein hydrolysates of three algal species and the potential antidiabetic sequences identified using the BIOPEP database

Protein Observed Peptide sequences Potential DPPV inhibitor peptides origin mass (Da) Derbesia 700.6802 FDKDGDGTITTKELGTVMR KE MR TI TK TT TV VM 976.1417 FDKDGDGTITTKELGTVMRS GQ NP SL KE MR PT QN TE TI TK TT TV VM LGQNPTE Oedogonium 352.696 HVAHAGL VA HA GL AG AH HV 362.7211 VSGVLHL HL GV LH VL VS 439.2794 ILLPHLAT LA LP LL HL AT IL PH 460.7449 QGPTGLGKY GP GL KY PT QG TG 513.2306 GGKDQESTGF DQ ES GF GG QE TG 535.2525 YLMRSPTGE SP GE LM MR PT TG YL 539.78 SAQGPTGLGKY GP GL KY PT QG TG 571.3254 LVRDQRLGAN GA VR DQ LV RL 628.3439 NKLKNDIQPW QP IQP IQ ND PW 706.3991 VRIITNPTTNPSV NP VR II PS PT RI SV TN TT 709.8683 EKGIDRFNEPTL EP EK DR FN GI KG NE PT TL 762.9418 VRIITNPTTNPSVI NP VR II PS PT RI SV TN TT VI 783.4033 FEKGIDRFNEPTL EP EK DR FN GI KG NE PT TL 802.4267 LGANVASAQGPTGLGKY GP VA GA GL AS KY NV PT QG TG 812.8943 NGTLTLGGKDQESTGF DQ ES GF GG LT NG QE TG TL 834.9458 NKLKNDIQPWQER QP WQ IQP IQ ND PW QE

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Table 30. (Continued) List of peptides identified in the MW <3 kDa fraction of the protein hydrolysates of three algal species and the potential antidiabetic sequences identified using the BIOPEP database

Protein Observed Peptide sequences origin mass (Da) Oedogonium 836.4759 VRIITNPTTNPSVIF NP VR II PS PT RI SV TN TT VI 864.9865 VRIITNPTTNPSVIFG NP VR II PS PT RI SV TN TT VI 888.9169 EVAHFVPEKPMYEQG VA VP KP EK AH EV HF MY PM QG YE 983.4897 SAQGPTGLGKYLMRSPTGE GP SP GL GE KY LM MR PT QG TG YL 1068.5446 VASAQGPTGLGKYLMRSPTGE GP VA SP GL AS GE KY LM MR PT QG TG YL 1125.5658 NVASAQGPTGLGKYLMRSPTGE GP VA SP GL AS GE KY LM MR NV PT QG TG YL 831.0926 LGANVASAQGPTGLGKYL GP VA SP GA GL AS GE KY LM MR NV PT QG TG MRSPTGE

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5.4. Conclusion

This chapter investigated the biological activities of the hydrolysates and peptides derived from in vitro simulated digestion of proteins from three green algae, and the following major conclusions can be drawn from the results obtained. First, in vitro simulated digestion was able to hydrolyse most of the algal proteins, with the degree of hydrolysis greater than 75% for protein from all three species. No major variations in protein hydrolysis were found among the three species as the degree of protein hydrolysis varied only slightly among them. Second, the protein hydrolysates had drastically higher antioxidant, and α-amylase, α-glucosidase and ACE-inhibitory activities than the undigested proteins. The α-amylase and α-glucosidase inhibitory activities of the protein hydrolysates were especially remarkable. Digestion of 144 to 36 mg of the algal protein, which would gave rise to 111 to 28 mg of the protein digest, would be equivalent to the oral administration of one mg of Acarbose, one of the standard drugs used in the management of diabetes. Considering the amount of algal protein that can be safely consumed, this means that consumption of the algal protein, or the algae directly, could be a potential alternative to the administration of this drug for diabetic care. Third, most of the peptides in the hydrolysates were extensively hydrolysed with MW less than 3 kDa. Fourth, a large number of peptides were identified in the MW <3 kDa fractions of the hydrolysates and most of them contained peptide sequences with known antioxidant, antidiabetic and antihypertensive activities. The antidiabetic and antihypertensive peptide sequences were especially abundant in the hydrolysates, while the antioxidative peptides were relatively deficient in comparison. However, the predicted number of peptides may not always correlate with the actual biological activities as this prediction is based on reported bioactive peptides in the BIOPEP database, which does not consider the potency of the individual peptide sequence. Overall, it can be concluded that consumption of the algal proteins, or the algae, may bring significant health benefits to the consumer, and that the algal proteins have the potential to be developed into bioactive ingredients for application in functional food and nutraceutical products. Finally, it needs to be appointed out that these results were obtained by in vitro methods, and thus the conclusions can only be regarded as tentative. Animal model and clinic studies are needed to confirm the results before claims over the health beneficial properties can be made, which should be a major direction for future study. 186

Chapter 6 Conclusions and recommendations for further studies

The overall aim of this thesis was to investigate the health relevant biological activities of three green macroalgal species, Derbesia tenuissima, Ulva ohnoi and Oedogonium intermedium, and determine the bioactive components in the algae that are responsible for the activities. The project began with the examination of biological activities of the phenolic extracts of the algae and identification of the phenolic compounds in them. The following main conclusions with respect to this phase of the study were obtained:

 After exhaustive analysis by HPLC-PDA, GC-MS, LC-MS and 1H NMR, no phenolic compounds could be identified from the ethanolic extracts of any of the three algal species. Rather, a number of other organic compounds, including free amino acids, fatty acids and sugars, were identified from the algal extracts. It is thus concluded that phenolic compounds, if present, occur in extremely low concentrations in the algae.  The ethanolic extracts of the three algal samples contained moderate levels of total phenolic content as measured by the Folin-Ciocalteu procedure and moderate levels of total flavonoid content as measured by the aluminium chloride colourimetric method. Since no phenolic compounds could be identified, it was concluded that the measured total phenolic content in the extracts came mostly from non-phenolic impurities such as free amino acids, fatty acids and sugars, because the Folin-Ciocalteu reagent is known to react with many non-phenolic compounds with reducing capacities.  The ethanolic extracts of the three green algae exhibited moderate levels of antioxidant activities as assessed by four different assays (ABTS scavenging, DPPH scavenging, FRAP and ORAC assays). Derbesia and Oedogonium had comparable antioxidant capacities, while Ulva exhibited the lowest antioxidant activity. These antioxidant activities, however, were not due to phenolic compounds which were not detected in the algae, but were attributed to compounds such as amino acids, fatty acids and reducing sugars in the extracts, which are known to also exhibit antioxidant activities.

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 The total “phenolic” content of the algae was correlated only with the antioxidant capacities measured by the ABTS and hydrophilic ORAC assays, but not with those obtained by DPPH, FRAP and lipophilic ORAC assays. The total flavonoid content in the ethanolic extracts showed correlation with lipophilic ORAC and FRAP antioxidant values, but not with those determined by ABTS, DPPH and hydrophilic ORAC assays. This was also concluded to be caused by the presence of a variety of non-phenolic phytochemicals in the extracts. Overall, it was concluded that the three green macroalgae are a poor source of phenolic compounds.

The second phase of the study was concerned with the evaluation of biological activities of the carotenoid extracts of the three algae and identification of the carotenoids present. The following major conclusions were drawn with respect to these aspects of the study:

 The three algae contained substantial amounts of carotenoids, but the content varied significantly among the three species. Oedogonium had the highest carotenoid content, which was about 20% higher than that of Derbesia, while the amount in Ulva was 12-15 folds lower than the other two species.  The spectrophotometric method used for measuring total carotenoids was found to overestimate the carotenoid content by a substantial margin when compared to the HPLC method. The overestimation was likely due to interferences with the measurement by impurities such as residual chlorophyll and their degradation products formed during saponification. The HPLC method for measuring total carotenoids, however, also had shortcomings, and chief among them was the co-elution of non-carotenoid impurities that interfered with the quantification of the carotenoids. Therefore, neither methods are perfect for the quantification of carotenoids, and further improvements in the procedures are needed.  Nine carotenoids were found by LC-MS in the three green algal species: siphonaxanthin, neoxanthin, 9’-cis-neoxanthin, loroxanthin, violaxanthin, lutein, siphonein, α-carotene and β-carotene. Lutein was the most abundant carotenoid in Ulva; siphonaxanthin was the most abundant in Derbesia, while Oedogonium contained several major carotenoids, namely neoxanthin, 9’-cis-neoxanthin, loroxanthin, violaxanthin, lutein and β-carotene, in similar amounts. The

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relatively high levels of siphonoxanthin in Derbesia and loraxanthin in Oedogonium meant that these two green algae could be an important new source for the respective carotenoids, which have significant health beneficial properties and are lacking in terrestrial plants.  The carotenoid extracts exhibited significant antioxidant capacities, as well as potent inhibitory effects against several metabolically important enzymes including α-amylase, α-glucosidase, pancreatic lipase and hyaluronidase. However, the algal extracts were found to be poor inhibitors of ACE.  The crude carotenoid extracts exhibited higher antioxidant activities than the saponified extracts. Furthermore, no significant correlation existed between the total carotenoid content and the antioxidant capacity of the crude extracts, whereas significant correlation was found between the two parameters in the saponified extracts. This is likely attributable to the removal of non-carotenoid compounds such as lipids, protein and chlorophylls by saponification, which would also contribute to the biological activities.

In the final phase of the study, proteins extracted from the algae were subjected to in vitro simulated human digestion using a multiple enzyme system to determine whether peptides with biological activities are produced during such a hydrolytic process. The following main conclusions were obtained:

 In vitro simulated human digestion of algae proteins led to substantial hydrolysis of them, with the degrees of protein hydrolysis greater than 75% for proteins from all three species. The protein hydrolysates produced possessed significant antioxidant activities, as well as strong inhibitory effects against α-amylase, α- glucosidase and ACE.  The hydrolysis generated two major peptide fractions, a MW >10 kDa fraction and a MW <3 kDa fraction, with the latter being more abundant. The molecular sizes of the peptides produced by the hydrolysis strongly affected their biological activities. The peptides with MW <3 kDa exhibited much greater biological activities than the larger peptide fraction for all the bioactivities tested without exception. However, the biological activities of the peptide fractions were lower than the unfractionated crude hydrolysates, suggesting that some

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form of synergism may exist among the peptides of different molecular weight when exerting their biological effects.  A large number of peptides were identified from the most active fraction (MW <3 kDa) of the algal protein digests by LC-MS/MS analysis, and many of these peptides contained amino acid sequences that are known to have antioxidant, antidiabetic and ACE-inhibitory activities. Several of the bioactive sequences appeared multiple times in different peptides. Sequences with antidiabetic and ACE-inhibitory activities were especially abundant, while sequences with antioxidant activities were relatively deficient in comparison.  Apart from the above major conclusions, several minor conclusions are also worth mentioning with respect to the protein properties of the three algae. First, the three green macroalgae contained all the essential amino acids (excluding tryptophan), which accounted for 38.92-43.35% of the total amino acid contents, but were especially rich in leucine, valine, lysine and threonine. The algae were also rich in the savoury tasting aspartic and glutamic acids, demonstrating that the algae not only possessed good nutritional value but also could be used for their savoury characteristics, similar to many other edible algae. Second, the algae were rather difficult matrices for protein extraction, and the protein extraction yield was strongly affected by the pH of the extraction medium. Alkaline extraction was much more efficient than extraction at neutral pH. Furthermore, application of mechanic shearing by sonication could greatly improve the extraction efficiency. Third, proteins extracted from the algae were contaminated with indigenous proteases, which interfered with the in vitro simulated human digestion of algal proteins. Heat treatment at 100°C for 10 min, however, was sufficient to inactivate the indigenous proteases. Finally, the extracted algal proteins had molecular weights ranging from 15 to 50 kDa.

Overall, this thesis demonstrated that the green macroalgae Ulva, Derbesia and Oedogonium have the potential to be developed into bioactive ingredient for application in functional food or nutraceutical products to supplement or replace drugs such as Acarbose and Orlistat in the management of diabetes and for control of weight gain and mitigation of inflammation. Moreover, consumption of the algae or their proteins can potentially lead to generation of bioactive peptides in the digestive tract with substantial 190 health benefits. Consumption of the algae could also bring significant health benefits through the health-promoting bioactivities provided by their carotenoids. However, these results are obtained by in vitro methods, and the same conclusions cannot be extrapolated to in vivo conditions. Therefore, in vivo studies using animal models or human volunteers are needed to confirm the results before real health benefits can be claimed. This could be a major direction for future study.

Several other recommendations can also be made with respect to future research in this area. This study has found nine carotenoid compounds in the three algae. Of these, two of them are rather uncommon in terrestrial plants and even in algae, which were siphonaxanthin from Derbesia and loroxanthin from Oedogonium. While there have been considerable amount of research conducted on the biological activities of siphonaxanthin, there is a lack of similar studies on loroxanthin. Therefore, isolation of loroxanthin and detailed examination of its biological activities should be a major direction for future study. This thesis demonstrated that carotenoids and proteins of the three algae could potentially confer significant health benefits to the consumer. Investigation of the digestibility and bioaccessibility of the carotenoids and proteins were beyond the scope of this study. However, digestibility and bioaccessibility are essential questions that need to be addressed if the health benefits are to be realised, and this should be another major direction for further study. Finally, the scope of this thesis does not include the study of the biological activities of carbohydrates. However, carbohydrates are a major component of algae and many of which have unique chemical structures that confer specific biological activities. Elucidation of the structure of carbohydrates in the three algae and investigation of their bioactivities would be another major area that warrants future studies.

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References

Abdel-Aal, E. S. M., Akhtar, H., Zaheer, K. & Ali, R. (2013). Dietary sources of lutein and zeaxanthin carotenoids and their role in eye health. Nutrients, 5, 1169-1185. Addai, Z. R., Abdullah, A. & Mutalib, S. A. (2013). Effect of extraction solvents on the phenolic content and antioxidant properties of two papaya cultivars. Journal of Medicinal Plants Research, 7, 3354-3359. Agati, G., Azzarello, E., Pollastri, S. & Tattini, M. (2012). Flavonoids as antioxidants in plants: location and functional significance. Plant Science, 196, 67-76. Agoramoorthy, G., Chandrasekaran, M., Venkatesalu, V. & Hsu, M. J. (2007). Antibacterial and antifungal activities of fatty acid methyl esters of the blind-your-eye mangrove from India. Brazilian Journal of Microbiology, 38, 739-742. Ahmed, F., Fanning, K., Netzel, M., Turner, W., Li, Y. & Schenk, P. M. (2014). Profiling of carotenoids and antioxidant capacity of microalgae from subtropical coastal and brackish waters. Food Chemistry, 165, 300-306. Ahn, C. B., Cho, Y. S. & Je, J. Y. (2015). Purification and anti-inflammatory action of tripeptide from salmon pectoral fin byproduct protein hydrolysate. Food Chemistry, 168, 151-156. Aitken, J. B. & Senn, T. L. (1965). Seaweed products as a fertilizer and soil conditioner for horticultural crops. Botanica Marina, 8, 144-147. Aitzetmuller, K., Strain, H. H., Svec, W. A., Grandolfo, M. & Katz, J. J. (1969). Loroxanthin, a unique xanthophyll from Scenedesmus obliquus and Chlorella vulgaris. Phytochemistry, 8, 1761-1770. Akimoto, S., Tomo, T., Naitoh, Y., Otomo, A., Murakami, A. & Mimuro, M. (2007). Identification of a new excited state responsible for the in vivo unique absorption band of siphonaxanthin in the green alga Codium fragile. Journal of Physical Chemistry B, 111, 9179-1981. Al-Jaber, N. A., Awaad, A. S. & Moses, J. E. (2011). Review on some antioxidant plants growing in Arab world. Journal of Saudi Chemical Society, 15, 293-307. Atlas of Living Australia (ALA). (2015). Atlas of Living Australia [Online]. Available: http://www/ala.org.au [Accessed 05/01/2013]. Alashi, A. M., Blanchard, C. L., Mailer, R. J., Agboola, S. O., Mawson, A. J., He, R., Girgih, A. & Aluko, R. E. (2014). Antioxidant properties of Australian canola meal protein hydrolysates. Food Chemistry, 146, 500-506. Alban, S., Kraus, J. & Franz, G. (1992). Synthesis of laminarin sulfates with anticoagulant activity. Arzneimittel Forschung, 42, 1005-1008. Almela, L., Fernandez-Lopez, J. A. & Roca, M. a. J. (2000). High-performance liquid chromatographic screening of chlorophyll derivatives produced during fruit storage. Journal of Chromatography A, 870, 483-489. Aluko, R. E. (2015). Amino acids, peptides, and proteins as antioxidants for food preservation A2. In: SHAHIDI, F. (ed.) Handbook of Antioxidants for Food Preservation. Cambridge: Woodhead Publishing. Amaya, R. D. B. (2010). Quantitative analysis, in vitro assessment of bioavailability and antioxidant activity of food carotenoids—A review. Journal of Food Composition and Analysis, 23, 726-740. Ambati, R., Phang, S. M., Ravi, S. & Aswathanarayana, R. (2014). Astaxanthin: sources, extraction, stability, biological activities and its commercial applications—A Review. Marine Drugs, 12, 128-152. Amensour, M., Sendra, E., Abrini, J., Perez-Alvarez, J. A. & Fernandez-Lopez, J. (2010). Antioxidant activity and total phenolic compounds of myrtle extracts actividad antioxidante y contenido de compuestos fenolicos totales en extractos de myrtus. CyTA - Journal of Food, 8, 95-101.

192

Aneiros, A. & Garateix, A. (2004). Bioactive peptides from marine sources: pharmacological properties and isolation procedures. Journal of Chromatography B, 803, 41-53. Angell, A. R., Mata, L., Nys, R. & Paul, N. A. (2015). The protein content of seaweeds: a universal nitrogen-to-protein conversion factor of five. Journal of Applied Phycology, 28, 511-524. Araujo, G. S., Matos, L. J. B. L., Fernandes, J. O., Cartaxo, S. J. M., Goncalves, L. R. B., Fernandes, F. a. N. & Farias, W. R. L. (2013). Extraction of lipids from microalgae by ultrasound application: Prospection of the optimal extraction method. Ultrasonics Sonochemistry, 20, 95-98. Arihara, K., Nakashima, Y., Mukai, T., Ishikawa, S. & Itoh, M. (2001). Peptide inhibitors for angiotensin I-converting enzyme from enzymatic hydrolysates of porcine skeletal muscle proteins. Meat Science, 57, 319-24. Arnlov, J., Zethelius, B., Riserus, U., Basu, S., Berne, C., Vessby, B., Alfthan, G. & Helmersson, J. (2009). Serum and dietary beta-carotene and alpha-tocopherol and incidence of type 2 diabetes mellitus in a community-based study of Swedish men: report from the uppsala longitudinal study of adult men (ULSAM) study. Diabetologia, 52, 97-105. Arporn, R. P. & Chirapart, A. (2006). Nutritional evaluation of tropical green seaweeds Caulerpa lentillifera and Ulva reticulata. Kasetsart Journal :Natural Science, 40, 75-83. Australia Bureau of Statistics. (2013). Hypertension [Online]. Available: http://www.abs.gov.au [Accessed 10/07/2016]. Australia Institute of Health and Welfare. (2006). Diabetes [Online]. Available: http://www.aihw.gov.au [Accessed 10/07/2013]. Ayyad, S. E. N., Ezmirly, S. T., Basaif, S. A., Alarif, W. M., Badria, A. F. & Badria, F. A. (2011). Antioxidant, cytotoxic, antitumor, and protective DNA damage metabolites from the red sea brown alga Sargassum sp. Pharmacognosy research, 3, 160-165. Bakatula, E. N., Cukrowska, E. M., Weiersbye, I. M., Mihaly-Cozmuta, L., Peter, A. & Tutu, H. (2014). Biosorption of trace elements from aqueous systems in gold mining sites by the filamentous green algae (Oedogonium sp.). Journal of Geochemical Exploration, 144, 492-503. Barbarino, E. & Lourenco, S. (2005). An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae. Journal of Applied Phycology, 17, 447-460. Barrientos, L. G., O'keefe, B. R., Bray, M., Sanchez, A., Gronenborn, A. M. & Boyd, M. R. (2003). Cyanovirin-N binds to the viral surface glycoprotein, GP1,2 and inhibits infectivity of Ebola virus. Antiviral Research, 58, 47-56. Beaulieu, L., Bondu, S., Doiron, K., Rioux, L. E. & Turgeon, S. L. (2015). Characterization of antibacterial activity from protein hydrolysates of the macroalga Saccharina longicruris and identification of peptides implied in bioactivity. Journal of Functional Foods, 17, 685-697. Beeharry, N., Lowe, J. E., Hernandez, A. R., Chambers, J. A., Fucassi, F., Cragg, P. J., Green, M. H. & Green, I. C. (2003). Linoleic acid and antioxidants protect against DNA damage and apoptosis induced by palmitic acid. Mutation Research, 530, 27-33. Bellik, Y., Boukraa, L., Alzahrani, H., Bakhotmah, B., Abdellah, F., Hammoudi, S. & Iguer- Ouada, M. (2012). Molecular mechanism underlying anti-inflammatory and anti- allergic activities of phytochemicals. Molecules, 18, 322. Benkerroum, N. (2008). Antimicrobial activity of lysozyme with special relevance to milk. Journal of Biotechnology, 7, 4856-4867. Berteau, O. & Mulloy, B. (2003). Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide. Glycobiology, 13, 29r-40r. Bewley, C. A., Cai, M., Ray, S., Ghirlando, R., Yamaguchi, M. & Muramoto, K. (2004). New carbohydrate specificity and HIV-1 fusion blocking activity of the cyanobacterial

193

protein MVL: NMR, ITC and sedimentation equilibrium studies. Journal of Molecular Biology, 339, 901-14. Bhagavathy, S. & Sumathi, P. (2012). Purification and characterization of carotenoids from green algae Chlorococcum humicola by HPLC-NMR and LC-MS-APCI. Biomedicine & Preventive Nutrition, 2, 276-282. Bhagavathy, S., Sumathi, P. & Madhushree, M. (2011). Antimutagenic assay of carotenoids from green algae Chlorococcum humicola using iTA98, TA100 and TA102. Asian Pacific Journal of Tropical Disease, 1, 308-316. Biehler, E., Mayer, F., Hoffmann, L., Krause, E. & Bohn, T. (2010). Comparison of 3 spectrophotometric methods for carotenoid determination in frequently consumed fruits and vegetables. Journal of Food Science, 75, C55-61. Bijttebier, S., D’hondt, E., Noten, B., Hermans, N., Apers, S. & Voorspoels, S. (2014). Tackling the challenge of selective analytical clean-up of complex natural extracts: The curious case of chlorophyll removal. Food Chemistry, 163, 147-153. Biziulevicius, G. A., Kislukhina, O. V., Kazlauskaite, J. & Zukaite, V. (2006). Food-protein enzymatic hydrolysates possess both antimicrobial and immunostimulatory activities: a "cause and effect" theory of bifunctionality. FEMS Immunology Medical Microbiology, 46, 131-138. Bjorck, L., Rosén, C. G., Marshall, V. & Reiter, B. (1975). Antibacterial activity of the lactoperoxidase system in milk against pseudomonads and other gram-negative bacteria. Applied Microbiology, 30, 199-204. Blanc, G., Agarkova, I., Grimwood, J., Kuo, A., Brueggeman, A., Dunigan, D. D., Gurnon, J., Ladunga, I., Lindquist, E., Lucas, S., Pangilinan, J., Proschold, T., Salamov, A., Schmutz, J., Weeks, D., Yamada, T., Lomsadze, A., Borodovsky, M., Claverie, J. M., Grigoriev, I. V. & Van Etten, J. L. (2012). The genome of the polar eukaryotic microalga Coccomyxa subellipsoidea reveals traits of cold adaptation. Genome Biology, 13, 1-12. Bogen, C., Al-Dilaimi, A., Albersmeier, A., Wichmann, J., Grundmann, M., Rupp, O., Lauersen, K. J., Blifernez-Klassen, O., Kalinowski, J., Goesmann, A., Mussgnug, J. H. & Kruse, O. (2013). Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production. BMC Genomics, 14, 1-18. Bolton, J. J., Robertson-Andersson, D. V., Shuuluka, D. & Kandjengo, L. (2008). Growing Ulva (Chlorophyta) in integrated systems as a commercial crop for abalone feed in South Africa: a SWOT analysis. Journal of Applied Phycology, 21, 575-583. Boschin, G., Scigliuolo, G. M., Resta, D. & Arnoldi, A. (2014). ACE inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes. Food Chemistry, 145, 34-40. Bourin, M., Gautron, J., Berges, M., Attucci, S., Le Blay, G., Labas, V., Nys, Y. & Rehault- Godbert, S. (2011). Antimicrobial potential of egg yolk ovoinhibitor, a multidomain Kazal-like inhibitor of chicken egg. Journal of Agricultural and Food Chemistry, 59, 12368-74. Bowen-Forbes, C. S., Zhang, Y. & Nair, M. G. (2010). Anthocyanin content, antioxidant, anti- inflammatory and anticancer properties of blackberry and raspberry fruits. Journal of Food Composition and Analysis, 23, 554-560. Britton, G., Liaaen-Jensen, S. & Pfander, H. (1994). Carotenoids: Isolation and Analysis, Basel, Germany, Birkhauser Boston. Brouard, J.-S., Otis, C., Lemieux, C. & Turmel, M. (2008). Chloroplast DNA sequence of the green alga Oedogonium cardiacum (Chlorophyceae): Unique genome architecture, derived characters shared with the Chaetophorales and novel genes acquired through horizontal transfer. BMC Genomics, 9, 1-16. Bruhn, A., Dahl, J., Nielsen, H. B., Nikolaisen, L., Rasmussen, M. B., Markager, S., Olesen, B., Arias, C. & Jensen, P. D. (2011). Bioenergy potential of Ulva lactuca: Biomass yield, methane production and combustion. Bioresource Technology, 102, 2595-2604.

194

Buck, C. B., Thompson, C. D., Roberts, J. N., Müller, M., Lowy, D. R. & Schiller, J. T. (2006). Carrageenan is a potent inhibitor of papillomavirus Infection. PLoS Pathogens, 2, e69. Bunning, P. (1983). The catalytic mechanism of angiotensin converting enzyme. Clinical and Experimental Hypertension A, 5, 1263-1275. Byun, H. G. & Kim, S. K. (2001). Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin. Process Biochemistry, 36, 1155-1162. Caceres, P. J., Carlucci, M. J., Damonte, E. B., Matsuhiro, B. & Zuniga, E. A. (2000). Carrageenans from chilean samples of Stenogramme interrupta (Phyllophoraceae): Structural analysis and biological activity. Phytochemistry, 53, 81-86. Cai, S., Huang, C., Ji, B., Zhou, F., Wise, M. L., Zhang, D. & Yang, P. (2011). In vitro antioxidant activity and inhibitory effect, on oleic acid-induced hepatic steatosis, of fractions and subfractions from oat (Avena sativa L.) ethanol extract. Food Chemistry, 124, 900-905. Campo, V. L., Kawano, D. F., Silva Jr, D. B. D. & Carvalho, I. (2009). Carrageenans: Biological properties, chemical modifications and structural analysis - A review. Carbohydrate Polymers, 77, 167-180. Cao, G. & Prior, R. L. (1999). Measurement of oxygen radical absorbance capacity in biological samples. Methods Enzymology, 299, 50-62. Capriotti, A. L., Caruso, G., Cavaliere, C., Samperi, R., Ventura, S., Zenezini Chiozzi, R. & Lagana, A. (2015). Identification of potential bioactive peptides generated by simulated gastrointestinal digestion of soybean seeds and soy milk proteins. Journal of Food Composition and Analysis, 44, 205-213. Carlucci, M. J., Pujol, C. A., Ciancia, M., Noseda, M. D., Matulewicz, M. C., Damonte, E. B. & Cerezo, A. S. (1997). Antiherpetic and anticoagulant properties of carrageenans from the red seaweed Gigartina skottsbergii and their cyclized derivatives: correlation between structure and biological activity. International Journal of Biological Macromolecules, 20, 97-105. Carlucci, M. J., Scolaro, L. A., Noseda, M. D., Cerezo, A. S. & Damonte, E. B. (2004). Protective effect of a natural carrageenan on genital herpes simplex virus infection in mice. Antiviral research, 64, 137-141. Carrera, C., Ruiz-Rodriguez, A., Palma, M. & Barroso, C. G. (2012). Ultrasound assisted extraction of phenolic compounds from grapes. Analytica Chimica Acta, 732, 100-104. Carrilho, A. K. T., Cepeda, A., Fente, C. & Regal, P. (2014). Review of methods for analysis of carotenoids. TrAC Trends in Analytical Chemistry, 56, 49-73. Castine, S., Mckinnon, A. D., Paul, N. A., Trott, L. A. & De Nys, R. (2013). Wastewater treatment for land-based aquaculture improvements and value-adding alternatives in model systems from Australia. Aquaculture Environment Interactions, 4, 285-300. Celikler, S., Tas, S., Vatan, O., Ziyanok-Ayvalik, S., Yildiz, G. & Bilaloglu, R. (2009). Anti- hyperglycemic and antigenotoxic potential of Ulva rigida ethanolic extract in the experimental diabetes mellitus. Food and Chemical Toxicology, 47, 1837-1840. Cencic, A. & Chingwaru, W. (2010). The role of functional foods, nutraceuticals, and food supplements in intestinal health. Nutrients, 2, 611-625. Cerna, M. (2011). Seaweed proteins and amino acids as nutraceuticals. Advances in Food and Nutrition Research, 64, 297-312. Cha, K. H., Koo, S. Y. & Lee, D. U. (2008). Antiproliferative effects of carotenoids extracted from Chlorella ellipsoidea and Chlorella vulgaris on human colon cancer cells. Journal of Agricultural and Food chemistry, 56, 10521-10526. Chakrabarti, S., Jahandideh, F. & Wu, J. (2014). Food-derived bioactive peptides on inflammation and oxidative stress. BioMed Research International, 2014. Chan, K. C., Pen, P. J. & Yin, M. C. (2012). Anticoagulatory and antiinflammatory effects of astaxanthin in diabetic rats. Journal of food Science, 77, H76-80. Chandini, S. K., Ganesan, P. & Bhaskar, N. (2008). In vitro antioxidant activities of three selected brown seaweeds of India. Food Chemistry, 107, 707-713.

195

Chang, O. K., Ha, G. E., Han, G. S., Seol, K. H., Kim, H. W., Jeong, S. G., Oh, M. H., Park, B. Y. & Ham, J. S. (2013). Novel antioxidant peptide derived from the ultrafiltrate of ovomucin hydrolysate. Journal of Agricultural and Food Chemistry, 61, 7294-7300. Chen, H. M., Muramoto, K., Yamauchi, F. & Nokihara, K. (1996). Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. Journal of Agricultural and Food Chemistry, 44, 2619-2623. Cheng, D., Liang, B., Li, M. & Jin, M. (2011). Influence of laminarin polysaccahrides on oxidative damage. International Journal of Biological Macromolecules, 48, 63-66. Chew, Y. L., Lim, Y. Y., Omar, M. & Khoo, K. S. (2008). Antioxidant activity of three edible seaweeds from two areas in South East Asia. LWT - Food Science and Technology, 41, 1067-1072. Choi, J. I., Kim, H. J. & Lee, J. W. (2011). Structural feature and antioxidant activity of low molecular weight laminarin degraded by gamma irradiation. Food Chemistry, 129, 520- 523. Chong, E., Chang, S. L., Hsiao, Y. W., Singhal, R., Liu, S. H., Leha, T., Lin, W. Y., Hsu, C. P., Chen, Y. C., Chen, Y. J., Wu, T. J., Higa, S. & Chen, S. A. (2015). Resveratrol, a red wine antioxidant, reduces atrial fibrillation susceptibility in the failing heart by PI3K/AKT/eNOS signaling pathway activation. Heart Rhythm, 12, 1046-56. Cian, R. E., Garzon, A. G., Ancona, D. B., Guerrero, L. C. & Drago, S. R. (2015). Hydrolyzates from Pyropia columbina seaweed have antiplatelet aggregation, antioxidant and ACE I inhibitory peptides which maintain bioactivity after simulated gastrointestinal digestion. LWT - Food Science and Technology, 64, 881-888. Cian, R. E., Martinez-Augustin, O. & Drago, S. R. (2012). Bioactive properties of peptides obtained by enzymatic hydrolysis from protein byproducts of Porphyra columbina. Food Research International, 49, 364-372. Cofrades, S., Lopez-Lopez, I., Bravo, L., Ruiz-Capillas, C., Bastida, S., Larrea, M. T. & Jimenez-Colmenero, F. (2010). Nutritional and antioxidant properties of different brown and red Spanish edible seaweeds. Food Science and Technology International, 16, 361-370. Cole, A. J., Mata, L., Paul, N. A. & De Nys, R. (2013). Using CO2 to enhance carbon capture and biomass applications of freshwater macroalgae. GCB Bioenergy, 6, 637-645. Cook, M. E., Miller, C. C., Park, Y. & Pariza, M. (1993). Immune modulation by altered nutrient metabolism: nutritional control of immune-induced growth depression. Poultry Science, 72, 1301-1305. Coste, O., Malta, E. J., Lopez, J. C. & Fernandez-Diaz, C. (2015). Production of sulfated oligosaccharides from the seaweed Ulva sp. using a new ulvan-degrading enzymatic bacterial crude extract. Algal Research, 10, 224-231. Da Conceicao Rivanor, R. L., Chaves, H. V., Do Val, D. R., De Freitas, A. R., Lemos, J. C., Rodrigues, J. a. G., Pereira, K. M. A., De Araújo, I. W. F., Bezerra, M. M. & Benevides, N. M. B. (2014). A lectin from the green seaweed Caulerpa cupressoides reduces mechanical hyper-nociception and inflammation in the rat temporomandibular joint during zymosan-induced arthritis. International Immunopharmacology, 21, 34-43. Dai, J. & Mumper, R. J. (2010). Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules, 15, 7313-52. Das, S. K., Ren, R., Hashimoto, T. & Kanazawa, K. (2010). Fucoxanthin induces apoptosis in osteoclast-like cells differentiated from RAW264.7 cells. Journal of Agricultural and Food Chemistry, 58, 6090-6095. Davalos, A., Miguel, M., Bartolome, B. & Lopez-Fandino, R. (2004). Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis. Journal of Food Protection, 67, 1939-44. Davis, J. P., Dean, L. L., Price, K. M. & Sanders, T. H. (2010). Roast effects on the hydrophilic and lipophilic antioxidant capacities of peanut flours, blanched peanut seed and peanut skins. Food Chemistry, 119, 539-547.

196

Dawczynski, C., Schubert, R. & Jahreis, G. (2007). Amino acids, fatty acids, and dietary fibre in edible seaweed products. Food Chemistry, 103, 891-899. De Almeida, C. L. F., Falcao, H. D. S., Lima, G. R. D. M., Montenegro, C. D. A., Lira, N. S., De Athayde-Filho, P. F., Rodrigues, L. C., De Souza, M. D. F. V., Barbosa-Filho, J. M. & Batista, L. M. (2011). Bioactivities from marine algae of the genus Gracilaria. International Journal of Molecular Sciences, 12, 4550-4573. De Castro, R. J. S. & Sato, H. H. (2015). Biologically active peptides: Processes for their generation, purification and identification and applications as natural additives in the food and pharmaceutical industries. Food Research International, 74, 185-198. De Oliveira, A. M. F., Pinheiro, L. S., Pereira, C. K. S., Matias, W. N., Gomes, R. A., Chaves, O. S., De Souza, M. D. F. V., De Almeida, R. N. & De Assis, T. S. (2012). Total phenolic content and antioxidant activity of some malvaceae family species. Antioxidants, 1, 33-43. Decker, E. A., Ivanov, V., Zhu, B. Z. & Frei, B. (2001). Inhibition of low-density lipoprotein oxidation by carnosine and histidine. Journal of Agricultural and Food Chemistry, 49, 511-516. Diaz-Pulido, G. & Mccook, L. J. (2008). Environmental Status: Macroalgae (seaweeds). In: CHIN, A. (ed.) The State of the Great Barrier Reef On-line. Townsville: Great Barrier Reef Marine Park Authority. Divya, P., Puthusseri, B. & Neelwarne, B. (2012). Carotenoid content, its stability during drying and the antioxidant activity of commercial coriander (Coriandrum sativum L.) varieties. Food Research International, 45, 342-350. Dong, L. Y., Jin, J., Lu, G. & Kang, X. L. (2013). Astaxanthin attenuates the apoptosis of retinal ganglion cells in db/db mice by inhibition of oxidative stress. Marine Drugs, 11, 960- 974. Dong, X. Y., Guo, L. L., Wei, F., Li, J. F., Jiang, M. L., Li, G. M., Zhao, Y. D. & Chen, H. (2011). Some characteristics and functional properties of rapeseed protein prepared by ultrasonication, ultrafiltration and isoelectric precipitation. Journal of the Science of Food and Agriculture, 91, 1488-1498. Dugan, M. E. R., Aalhus, J. L., Schaefer, A. L. & Kramer, J. K. G. (1997). The effect of conjugated linoleic acid on fat to lean repartitioning and feed conversion in pigs. Canadian Journal of Animal Science, 77, 723-725. Durak, A., Baraniak, B., Jakubczyk, A. & Swieca, M. (2013). Biologically active peptides obtained by enzymatic hydrolysis of Adzuki bean seeds. Food Chemistry, 141, 2177- 2183. Edelstam, G. a. B., Laurent, U. B. G., Lundkvist, O. E., Fraser, J. R. E. & Laurent, T. C. (1992). Concentration and turnover of intraperitoneal hyaluronan during inflammation. Inflammation, 16, 459-469. El Baz, F. K., Aboul-Enein, A. M., El-Baroty, G. S., Youssef, A. M. & Abdel-Baky, H. H. (2002). Accumulation od antioxidant vitamins in Dunaliella salina. Journal of Biological Sciences, 2, 220-223. El Gamal, A. A. (2010). Biological importance of marine algae. Saudi Pharmaceutical Journal, 18, 1-25. El Maradny, E., Kanayama, N., Kobayashi, H., Hossain, B., Khatun, S., Liping, S., Kobayashi, T. & Terao, T. (1997). The role of hyaluronic acid as a mediator and regulator of cervical ripening. Human Reproduction, 12, 1080-1088. Eom, S. H., Kim, Y. M. & Kim, S. K. (2012). Antimicrobial effect of phlorotannins from marine brown algae. Food and Chemical Toxicology, 50, 3251-3255. Erdmann, K., Cheung, B. W. Y. & Schroder, H. (2008). The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. The Journal of Nutritional Biochemistry, 19, 643-654. Falkeborg, M., Cheong, L. Z., Gianfico, C., Sztukiel, K. M., Kristensen, K., Glasius, M., Xu, X. & Guo, Z. (2014). Alginate oligosaccharides: enzymatic preparation and antioxidant property evaluation. Food Chemistry, 164, 185-194.

197

Fan, J., He, J., Zhuang, Y. & Sun, L. (2012). Purification and identification of antioxidant peptides from enzymatic hydrolysates of Tilapia (Oreochromis niloticus) frame protein. Molecules, 17, 12836-12850. Farasat, M., Khavari-Nejad, R. A., Nabavi, S. M. B. & Namjooyan, F. (2014). Antioxidant activity, total phenolics and flavonoid contents of some edible green seaweeds from northern coasts of the persian gulf. Iranian Journal of Pharmaceutical Research 13, 163-170. Fernandez-Garcia, E., Carvajal-Lerida, I., Jaren-Galan, M., Garrido-Fernandez, J., Perez-Galvez, A. & Hornero-Mendez, D. (2012). Carotenoids bioavailability from foods: From plant pigments to efficient biological activities. Food Research International, 46, 438-450. Fernandez, M. L. (2007). The metabolic syndrome. Nutrition Reviews, 65, S30-4. Ferraces-Casais, P., Lage-Yusty, M. A., De Quiros, A. R. & Lopez-Hernandez, J. (2012). Evaluation of bioactive compounds in fresh edible seaweeds. Food Analytical Methods, 5, 828-834. Fitzgerald, C., Gallagher, E., O’connor, P., Prieto, J., Mora-Soler, L., Grealy, M. & Hayes, M. (2013). Development of a seaweed derived platelet activating factor acetylhydrolase (PAF-AH) inhibitory hydrolysate, synthesis of inhibitory peptides and assessment of their toxicity using the Zebrafish larvae assay. Peptides, 50, 119-124. Fleurence, J. (1999). Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends in Food Science & Technology, 10, 25-28. Fleurence, J., Le Coeur, C., Mabeau, S., Maurice, M. & Landrein, A. (1995). Comparison of different extractive procedures for proteins from the edible seaweeds Ulva rigida and Ulva rotundata. Journal of Applied Phycology, 7, 577-582. Fujiki, H. & Suganuma, M. (2012). Green tea: an effective synergist with anticancer drugs for tertiary cancer prevention. Cancer Letters, 324, 119-25. Fujita, H., Yokoyama, K. & Yoshikawa, M. (2000). Classification and antihypertensive activity of angiostensin I-converting enzyme inhibitory peptides derived from food proteins. Journal of Food Science, 65, 564-569. Fujiwara-Arasaki, T., Mino, N. & Kuroda, M. (1984). The protein value in human nutrition of edible marine algae in Japan. Hydrobiologia, 116, 513-516. Fukumoto, L. R. & Mazza, G. (2000). Assessing antioxidant and prooxidant activities of phenolic compounds. Journal of Agricultural and Food Chemistry, 48, 3597-3604. Furuta, T., Miyabe, Y., Yasui, H., Kinoshita, Y. & Kishimura, H. (2016). Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata. Marine Drugs, 14, 32-42. Galland-Irmouli, A. V., Fleurence, J., Lamghari, R., Lucon, M., Rouxel, C., Barbaroux, O., Bronowicki, J. P., Villaume, C. & Gueant, J. L. (1999). Nutritional value of proteins from edible seaweed Palmaria palmata (dulse). The Journal of Nutritional Biochemistry, 10, 353-359. Gallego, M., Aristoy, M. C. & Toldra, F. (2014). Dipeptidyl peptidase IV inhibitory peptides generated in Spanish dry-cured ham. Meat Science, 96, 757-761. Gammone, M. A. & D’orazio, N. (2015). Anti-obesity activity of the marine carotenoid fucoxanthin. Marine Drugs, 13, 2196-2214. Ganesan, P., Matsubara, K., Ohkubo, T., Tanaka, Y., Noda, K., Sugawara, T. & Hirata, T. (2010). Anti-angiogenic effect of siphonaxanthin from green alga, Codium fragile. Phytomedicine, 17, 1140-1144. Ganesan, P., Noda, K., Manabe, Y., Ohkubo, T., Tanaka, Y., Maoka, T., Sugawara, T. & Hirata, T. (2011). Siphonaxanthin, a marine carotenoid from green algae, effectively induces apoptosis in human leukemia (HL-60) cells. Biochimica Biophysica Acta (BBA), 1810, 497-503. Garcia-Casal, M. N., Ramirez, J., Leets, I., Pereira, A. C. & Quiroga, M. F. (2009). Antioxidant capacity, polyphenol content and iron bioavailability from algae (Ulva sp., Sargassum sp. and Porphyra sp.) in human subjects. British Journal of Nutrition, 101, 79-85.

198

Garcia, M. C., Puchalska, P., Esteve, C. & Marina, M. L. (2013). Vegetable foods: A cheap source of proteins and peptides with antihypertensive, antioxidant, and other less occurrence bioactivities. Talanta, 106, 328-349. Garrido, J. L., Rodriguez, F. & Zapata, M. (2009). Occurrence of loroxanthin, loroxanthin decenoate, and loroxanthin dodecenoate in tetraselmis species (prasinophyceae, chlorophyta). Journal of Phycology, 45, 366-374. Gautam, R. & Jachak, S. M. (2009). Recent developments in anti-inflammatory natural products. Medicinal Research Reviews, 29, 767-820. Giansanti, F., Leboffe, L., Pitari, G., Ippoliti, R. & Antonini, G. (2012). Physiological roles of ovotransferrin. Biochimica et Biophysica Acta (BBA), 1820, 218-25. Giuffrida, D., Dugo, P., Torre, G., Bignardi, C., Cavazza, A., Corradini, C. & Dugo, G. (2013). Characterization of 12 Capsicum varieties by evaluation of their carotenoid profile and pungency determination. Food Chemistry, 140, 794-802. Goiris, K., Muylaert, K., Fraeye, I., Foubert, I., De Brabanter, J. & De Cooman, L. (2012). Antioxidant potential of microalgae in relation to their phenolic and carotenoid content. Journal of Applied Phycology, 24, 1477-1486. Gomez-Ordonez, E., Jimenez-Escrig, A. & Ruperez, P. (2010). Dietary fibre and physicochemical properties of several edible seaweeds from the northwestern Spanish coast. Food Research International, 43, 2289-2294. Goodman, B. E. (2010). Insights into digestion and absorption of major nutrients in humans. Advances in Physiology Education, 34, 44-53. Goralczyk, R. (2009). Beta-carotene and lung cancer in smokers: review of hypotheses and status of research. Nutrition and Cancer, 61, 767-74. Gosch, B. J., Lawton, R. J., Paul, N. A., De Nys, R. & Magnusson, M. (2015). Environmental effects on growth and fatty acids in three isolates of Derbesia tenuissima (Bryopsidales, Chlorophyta). Algal Research, 9, 82-93. Gosch, B. J., Magnusson, M., Paul, N. A. & De Nys, R. (2012). Total lipid and fatty acid composition of seaweeds for the selection of species for oil-based biofuel and bioproducts. GCB Bioenergy, 4, 919-930. Granado, F., Olmedilla, B., Blanco, I. & Rojas-Hidalgo, E. (1992). Carotenoid composition in raw and cooked spanish vegetables. Journal of Agricultural and Food Chemistry, 40, 2135-2140. Guerciolini, R. (1997). Mode of action of orlistat. International Journal of Obesity and Related Metabolic Disorders, 21 S12-23. Gupta, V. K. & Rastogi, A. (2008). Biosorption of lead(II) from aqueous solutions by non- living algal biomass Oedogonium sp. and Nostoc sp.—A comparative study. Colloids and Surfaces B: Biointerfaces, 64, 170-178. Gurnani, N., Gupta, M., Mehta, D. & Mehta, B. K. (2015). Chemical composition, total phenolic and flavonoid contents, and in vitro antimicrobial and antioxidant activities of crude extracts from red chilli seeds (Capsicum frutescens L.). Journal of Taibah University for Science. Ha, Y. L., Storkson, J. & Pariza, M. W. (1990). Inhibition of benzo(a)pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Research, 50, 1097-1101. Haaland, R. E., Chaowanachan, T., Evans-Strickfaden, T., Van De Wijgert, J. H., Kilmarx, P. H., Mclean, C. A. & Hart, C. E. (2012). Carrageenan-based gel retains limited anti- HIV-1 activity 8-24 hours after vaginal application by HIV-infected Thai women enrolled in a phase I safety trial. Journal of Acquired Immune Deficiency Syndromes, 61, e71-3. Hahn, A. & Mang, B. (2008). Lutein and eye health--current state of discussion. Medozomosche Monatsschrift Pharmazeuten, 31, 299-308. Harada, H., Yamashita, U., Kurihara, H., Fukushi, E., Kawabata, J. & Kamei, Y. (2002). Antitumor activity of palmitic acid found as a selective cytotoxic substance in a marine red alga. Anticancer Research 22, 2587-90.

199

Harnedy, P. A. & Fitzgerald, R. J. (2011). Bioactive proteins, peptides, and amino acids from macroalgae. Journal of Phycology, 47, 218-232. Harnedy, P. A., O’keeffe, M. B. & Fitzgerald, R. J. (2015). Purification and identification of dipeptidyl peptidase (DPP) IV inhibitory peptides from the macroalga Palmaria palmata. Food Chemistry, 172, 400-406. Harnedy, P. A., Soler-Vila, A., Edwards, M. D. & Fitzgerald, R. J. (2014). The effect of time and origin of harvest on the in vitro biological activity of Palmaria palmata protein hydrolysates. Food Research International, 62, 746-752. Harris, W. S., Miller, M., Tighe, A. P., Davidson, M. H. & Schaefer, E. J. (2008). Omega-3 fatty acids and coronary heart disease risk: clinical and mechanistic perspectives. Atherosclerosis, 197, 12-24. Harrison, P. & Hurd, C. (2001). Nutrient ohysiology of seaweeds: application of concepts to aquaculture. Cahier De Biologie Marine, 42, 78-82. Hefnawy, H. T. M. & Ramadan, M. F. (2011). Physicochemical characteristics of soy protein isolate and fenugreek gum dispersed systems. Journal of food science and technology, 48, 371-377. Heo, S. J. & Jeon, Y. J. (2009). Protective effect of fucoxanthin isolated from Sargassum siliquastrum on UV-B induced cell damage. Journal of Photochemistry and Photobiology B: Biology, 95, 101-107. Heo, S. J., Yoon, W. J., Kim, K. N., Ahn, G. N., Kang, S. M., Kang, D. H., Affan, A., Oh, C., Jung, W. K. & Jeon, Y. J. (2010). Evaluation of anti-inflammatory effect of fucoxanthin isolated from brown algae in lipopolysaccharide-stimulated RAW 264.7 macrophages. Food and Chemical Toxicology, 48, 2045-2051. Hernandez-Ledesma, B., Amigo, L., Ramos, M. & Recio, I. (2004a). Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion. Journal of Agricultural and Food Chemistry, 52, 1504-1510. Hernandez-Ledesma, B., Amigo, L., Ramos, M. & Recio, I. (2004b). Release of angiotensin converting enzyme-inhibitory peptides by simulated gastrointestinal digestion of infant formulas. International Dairy Journal, 14, 889-898. Hernandez-Ledesma, B., Quiros, A., Amigo, L. & Recio, I. (2007). Identification of bioactive peptides after digestion of human milk and infant formula with pepsin and pancreatin. International Dairy Journal, 17, 42-49. Hillson, C. J. (1977). Seaweeds, a color-coded, illustrated guide to common marine plants of the east coast of the United States, University Park, Pennsylvania State University Press. Hiraoka, M., Shimada, S., Uenosono, M. & Masuda, M. (2004). A new green-tide-forming alga, Ulva ohnoi Hiraoka et Shimada sp. nov. (Ulvales, Ulvophyceae) from Japan. Phycological Research, 52, 17-29. Houghton, D., Wilcox, M. D., Chater, P. I., Brownlee, I. A., Seal, C. J. & Pearson, J. P. (2015). Biological activity of alginate and its effect on pancreatic lipase inhibition as a potential treatment for obesity. Food Hydrocolloids, 49, 18-24. Hu, C. C., Lin, J. T., Lu, F. J., Chou, F. P. & Yang, D. J. (2008). Determination of carotenoids in Dunaliella salina cultivated in Taiwan and antioxidant capacity of the algal carotenoid extract. Food Chemistry, 109, 439-446. Huang, B. B., Lin, H. C. & Chang, Y. W. (2015). Analysis of proteins and potential bioactive peptides from tilapia (Oreochromis spp.) processing co-products using proteomic techniques coupled with BIOPEP database. Journal of Functional Foods, 19, 629-640. Huangfu, J., Liu, J., Sun, Z., Wang, M., Jiang, Y., Chen, Z.-Y. & Chen, F. (2013). Antiaging effects of astaxanthin-rich alga Haematococcus pluvialis on fruit flies under oxidative stress. Journal of Agricultural and Food Chemistry, 61, 7800-7804. Hubert, H. B., Feinleib, M., Mcnamara, P. M. & Castelli, W. P. (1983). Obesity as an independent risk factor for cardiovascular disease: A 26-year follow-up of participants in the Framingham Heart Study. Circulation, 67, 968-977.

200

Hung, L. D., Sato, Y. & Hori, K. (2011). High-mannose N-glycan-specific lectin from the red alga Kappaphycus striatum (Carrageenophyte). Phytochemistry, 72, 855-861. Hursting, S. D., Nunez, N. P., Varticovski, L. & Vinson, C. (2007). The obesity-cancer link: Lessons learned from a fatless mouse. Cancer Research, 67, 2391-2393. Hussein, G., Nakamura, M., Zhao, Q., Iguchi, T., Goto, H., Sankawa, U. & Watanabe, H. (2005). Antihypertensive and neuroprotective effects of astaxanthin in experimental animals. Biological and Pharmaceutical Bulletin, 28, 47-52. Hwang, E. S., Ki, K. N. & Chung, H. Y. (2013). Proximate composition, amino acid, mineral, and heavy metal content of dried Laver. Preventive Nutrition and Food Science, 18, 139-144. Hwang, H. J., Kim, I. H. & Nam, T. J. (2008). Effect of a glycoprotein from Hizikia fusiformis on acetaminophen-induced liver injury. Food and Chemical Toxicology, 46, 3475-3481. Ikeuchi, M., Koyama, T., Takahashi, J. & Yazawa, K. (2007). Effects of astaxanthin in obese mice fed a high-fat diet. Bioscience, Biotechnology, and Biochemistry, 71, 893-899. Ikram, E. H. K., Stanley, R., Netzel, M. & Fanning, K. (2015). Phytochemicals of papaya and its traditional health and culinary uses - A review. Journal of Food Composition and Analysis, 41, 201-211. Inbaraj, B. S., Chien, J. T. & Chen, B. H. (2006). Improved high performance liquid chromatographic method for determination of carotenoids in the microalga Chlorella pyrenoidosa. Journal of Chromatography A, 1102, 193-199. Ip, C., Briggs, S. P., Haegele, A. D., Thompson, H. J., Storkson, J. & Scimeca, J. A. (1996). The efficacy of conjugated linoleic acid in mammary cancer prevention is independent of the level or type of fat in the diet. Carcinogenesis, 17, 1045-1050. Ip, C., Singh, M., Thompson, H. J. & Scimeca, J. A. (1994). Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat. Cancer Research, 54, 1212-1515. Jarmolowska, B., Szlapka-Sienkiewicz, E., Kostyra, E., Kostyra, H., Mierzejewska, D. & Marcinkiewicz-Darmochwal, K. (2007). Opioid activity of Humana formula for newborns. Journal of the Science of Food and Agriculture, 87, 2247-2250. Jenssen, H. & Hancock, R. E. W. (2009). Antimicrobial properties of lactoferrin. Biochimie, 91, 19-29. Ji, Y. B., Ji, C. F. & Zhang, H. (2012). Laminarin induces apoptosis of human colon cancer LOVO cells through a mitochondrial pathway. Molecules, 17, 9947-60. Jiao, G., Yu, G., Zhang, J. & Ewart, H. S. (2011). Chemical structures and bioactivities of sulfated polysaccharides from marine algae. Marine Drugs, 9, 196-233. Jimenez-Escrig, A., Gomez-Ordonez, E. & Ruperez, P. (2011). Seaweed as a Source of Novel Nutraceuticals: Sulfated Polysaccharides and Peptides. Advances in Food and Nutrition Research, 64, 325-337. Jimenez-Escrig, A. & Sanchez-Muniz, F. J. (2000). Dietary fibre from edible seaweeds: Chemical structure, physicochemical properties and effects on cholesterol metabolism. Nutrition Research, 20, 585-598. Jimsheena, V. K. & Gowda, L. R. (2011). Angiotensin I-converting enzyme (ACE) inhibitory peptides derived from arachin by simulated gastric digestion. Food Chemistry, 125, 561-569. Jin, J. O. & Yu, Q. (2015). Fucoidan delays apoptosis and induces pro-inflammatory cytokine production in human neutrophils. International Journal of Biological Macromolecules, 73, 65-71. Johansson, C. L., Paul, N. A., De Nys, R. & Roberts, D. A. (2016). Simultaneous biosorption of selenium, arsenic and molybdenum with modified algal-based biochars. Journal of Environmental Management, 165, 117-123. Johnson, L. & Williamson, G. (2003). Phytochemical functional foods, Cambridge, England, Woodhead publishing limited. Joubert, E., Manley, M. & Botha, M. (2008). Evaluation of spectrophotometric methods for screening of green rooibos (Aspalathus linearis) and green honeybush (Cyclopia

201

genistoides) extracts for high levels of Bio-active compounds. Phytochemical Analysis, 19, 169-178. Jun, S. Y., Park, P. J., Jung, W. K. & Kim, S. K. (2004). Purification and characterization of an antioxidative peptide from enzymatic hydrolysate of yellowfin sole ( Limanda aspera) frame protein. European Food Research and Technology, 219, 20-26. Kadam, S. U., Tiwari, B. K. & O'donnell, C. P. (2015). Extraction, structure and biofunctional activities of laminarin from brown algae. International Journal of Food Science & Technology, 50, 24-31. Kahkonen, M. P., Hopia, A. I., Vuorela, H. J., Rauha, J.-P., Pihlaja, K., Kujala, T. S. & Heinonen, M. (1999). Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural and Food Chemistry, 47, 3954-3962. Kahl, R. (1984). Synthetic antioxidants: Biochemical actions and interference with radiation, toxic compounds, chemical mutagens and chemical carcinogens. Toxicology, 33, 185- 228. Kahl, R. & Kappus, H. (1993). Toxicology of the synthetic antioxidants BHA and BHT in comparison with the natural antioxidant vitamin E. Zeitschrift fur Lebensmittel Untersuchung und Forschung, 196, 329-338. Kang, K. H., Qian, Z. J., Ryu, B., Kim, D. & Kim, S. K. (2012). Protective effects of protein hydrolysate from marine microalgae Navicula incerta on ethanol-induced toxicity in HepG2/CYP2E1 cells. Food Chemistry, 132, 677-685. Kang, K. H., Qian, Z. J., Ryu, B. & Kim, S. K. (2011). Characterization of growth and protein contents from microalgae Navicula incerta with the investigation of antioxidant activity of enzymatic hydrolysates. Food Science and Biotechnology, 20, 183-191. Kao, T. H., Chen, C. J. & Chen, B. H. (2011). Carotenoid composition in Rhinacanthus nasutus (L.) Kurz as determined by HPLC-MS and affected by freeze-drying and hot-air-drying. Analyst, 136, 3194-3202. Kao, T. H., Loh, C. H., Inbaraj, B. S. & Chen, B. H. (2012). Determination of carotenoids in Taraxacum formosanum by HPLC-DAD-APCI-MS and preparation by column chromatography. Journal of Pharmaceutical and Biomedical Analysis, 66, 144-153. Karawita, R., Senevirathne, M., Athukorala, Y., Affan, A., Lee, Y. J., Kim, S. K., Lee, J. B. & Jeon, Y. J. (2007). Protective effect of enzymatic extracts from microalgae against DNA damage induced by H2O2. Journal of Marine Biotechnology, 9, 479-490. Kasperczyk, S., Dobrakowski, M., Kasperczyk, J., Ostalowska, A., Zalejska-Fiolka, J. & Birkner, E. (2014). Beta-carotene reduces oxidative stress, improves glutathione metabolism and modifies antioxidant defense systems in lead-exposed workers. Toxicology and Applied Pharmacology, 280, 36-41. Kazlowska, K., Hsu, T., Hou, C. C., Yang, W. C. & Tsai, G. J. (2010). Anti-inflammatory properties of phenolic compounds and crude extract from Porphyra dentata. Journal of Ethnopharmacology, 128, 123-130. Kelishomi, Z. H., Goliaei, B., Mahdavi, H., Nikoofar, A., Rahimi, M., Moosavi-Movahedi, A. A., Mamashli, F. & Bigdeli, B. (2016). Antioxidant activity of low molecular weight alginate produced by thermal treatment. Food Chemistry, 196, 897-902. Kellogg, J., Esposito, D., Grace, M. H., Komarnytsky, S. & Lila, M. A. (2015). Alaskan seaweeds lower inflammation in RAW 264.7 macrophages and decrease lipid accumulation in 3T3-L1 adipocytes. Journal of Functional Foods, 15, 396-407. Kelman, D., Posner, E. K., Mcdermid, K. J., Tabandera, N. K., Wright, P. R. & Wright, A. D. (2012). Antioxidant activity of hawaiian marine algae. Marine Drugs, 10, 403-416. Khachik, F., Beecher, G. R. & Whittaker, N. F. (1986). Separation, identification, and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography. Journal of Agricultural and Food Chemistry, 34, 603-616. Khataee, A. R., Dehghan, G., Ebadi, A., Zarei, M. & Pourhassan, M. (2010). Biological treatment of a dye solution by Macroalgae Chara sp.: Effect of operational parameters,

202

intermediates identification and artificial neural network modeling. Bioresource Technology, 101, 2252-2258. Khoddami, A., Wilkes, M. A. & Roberts, T. H. (2013). Techniques for analysis of plant phenolic compounds. Molecules, 18, 2328-75. Kim, K. H., Kim, Y. W., Kim, H. B., Lee, B. J. & Lee, D. S. (2006a). Anti-apoptotic activity of laminarin polysaccharides and their enzymatically hydrolyzed oligosaccharides from Laminaria japonica. Biotechnology Letters, 28, 439-446. Kim, K. N., Heo, S. J., Song, C. B., Lee, J., Heo, M. S., Yeo, I. K., Kang, K. A., Hyun, J. W. & Jeon, Y. J. (2006b). Protective effect of Ecklonia cava enzymatic extracts on hydrogen peroxide-induced cell damage. Process Biochemistry, 41, 2393-2401. Kim, S. E., Kim, H. H., Kim, J. Y., Kang, Y. I., Woo, H. J. & Lee, H. J. (2000). Anticancer activity of hydrophobic peptides from soy proteins. BioFactors, 12, 151-155. Kim, S. K. (2012). Handbook of marine macroalgae: biotechnology and applied phycology, Chichester, John Wiley & Sons. Kim, S. K. & Himaya, S. W. A. (2011). Medicinal effects of phlorotannins from marine brown algae. Advances in Food and Nutrition Research, 64, 97-109. Kim, S. K. & Ta, Q. V. (2011). Potential Beneficial Effects of Marine Algal Sterols on Human Health. Advances in Food and Nutrition Research, 64, 191-198. Kim, S. K. & Ta, Q. V. (2012). Bioactive sterols from marine resources and their potential benefits for human health. Advances in Food and Nutrition Research, 65, 261-268. Kim, S. M., Jung, Y. J., Kwon, O. N., Cha, K. H., Um, B. H., Chung, D. & Pan, C. H. (2012). A potential commercial source of fucoxanthin extracted from the microalga Phaeodactylum tricornutum. Applied Biochemistry Biotechnology, 166, 1843-1855. Kim, S. Y., Je, J. Y. & Kim, S. K. (2007). Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion. The Journal of Nutritional Biochemistry, 18, 31-38. Kim, Y., Seo, J. H. & Kim, H. (2011). Beta-carotene and lutein inhibit hydrogen peroxide- induced activation of NF-kappaB and IL-8 expression in gastric epithelial AGS cells. Journal of Nutritional Science and Vitaminology, 57, 216-223. Kimura, M., Kobori, C. N., Rodriguez-Amaya, D. B. & Nestel, P. (2007). Screening and HPLC methods for carotenoids in sweetpotato, cassava and maize for plant breeding trials. Food Chemistry, 100, 1734-1746. Kitts, D. D. & Weiler, K. (2003). Bioactive proteins and peptides from food sources. Applications of bioprocesses used in isolation and recovery. Current Pharmaceutical Design, 9, 1309-1323. Klompong, V., Benjakul, S., Yachai, M., Visessanguan, W., Shahidi, F. & Hayes, K. D. (2009). Amino acid composition and antioxidative peptides from protein hydrolysates of yellow stripe Trevally (Selaroides leptolepis). Journal of Food Science, 74, 126-133. Ko, S. C., Kang, N., Kim, E. A., Kang, M. C., Lee, S. H., Kang, S. M., Lee, J. B., Jeon, B. T., Kim, S. K., Park, S. J., Park, P. J., Jung, W. K., Kim, D. & Jeon, Y. J. (2012). A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats. Process Biochemistry, 47, 2005-2011. Konczak, I., Zabaras, D., Dunstan, M. & Aguas, P. (2010). Antioxidant capacity and phenolic compounds in commercially grown native Australian herbs and spices. Food Chemistry, 122, 260-266. Kotake-Nara, E., Asai, A. & Nagao, A. (2005). Neoxanthin and fucoxanthin induce apoptosis in PC-3 human prostate cancer cells. Cancer Letters, 220, 75-84. Koushan, K., Rusovici, R., Li, W., Ferguson, L. R. & Chalam, K. V. (2013). The role of lutein in eye-related disease. Nutrients, 5, 1823-39. Kris-Etherton, P. M., Hecker, K. D., Bonanome, A., Coval, S. M., Binkoski, A. E., Hilpert, K. F., Griel, A. E. & Etherton, T. D. (2002). Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. American Journal of Medicine, 113, 71s-88s.

203

Kumar, K. S., Ganesan, K., Selvaraj, K. & Rao, P. V. S. (2014). Studies on the functional properties of protein concentrate of Kappaphycus alvarezii (Doty) Doty – An edible seaweed. Food Chemistry, 153, 353-360. Kumar, M., Gupta, V., Kumari, P., Reddy, C. R. K. & Jha, B. (2011). Assessment of nutrient composition and antioxidant potential of Caulerpaceae seaweeds. Journal of Food Composition and Analysis, 24, 270-278. Kunio, S. (1999). Separation and identification of antioxidant peptides from proteolytic digest of dried bonito. Nippon Suisan Gakkaishi, 65, 92-96. Kunyanga, C. N., Imungi, J. K., Okoth, M. W., Biesalski, H. K. & Vadivel, V. (2012). Total phenolic content, antioxidant and antidiabetic properties of methanolic extract of raw and traditionally processed Kenyan indigenous food ingredients. LWT - Food Science and Technology, 45, 269-276. Kutyshenko, V. P., Budantsev, A. Y. & Uversky, V. N. (2015). Analysis of seasonal changes in plants by high-resolution NMR spectroscopy: Looking at the aqueous extracts from different plant tissues. Journal of Nature and Science, 1, 88. Lacroix, I. M. & Li-Chan, E. C. (2013). Inhibition of dipeptidyl peptidase (DPP)-IV and alpha- glucosidase activities by pepsin-treated whey proteins. Journal of Agricultural and Food Chemistry, 61, 7500-7506. Lahaye, M. & Robic, A. (2007). Structure and functional properties of ulvan, a polysaccharide from green seaweeds. Biomacromolecules, 8, 1765-1774. Lan, V. T., Ito, K., Ohno, M., Motoyama, T., Ito, S. & Kawarasaki, Y. (2015). Analyzing a dipeptide library to identify human dipeptidyl peptidase IV inhibitor. Food Chemistry, 175, 66-73. Lange, K. W., Hauser, J., Nakamura, Y. & Kanaya, S. (2015). Dietary seaweeds and obesity. Food Science and Human Wellness, 4, 87-96. Larsen, E. & Christensen, L. P. (2005). Simple saponification method for the quantitative determination of carotenoids in green vegetables. Journal of Agriculture and Food Chemistry, 53, 6598-6602. Lavie, C. J., Mcauley, P. A., Church, T. S., Milani, R. V. & Blair, S. N. (2014). Obesity and cardiovascular diseases: Implications regarding fitness, fatness, and severity in the obesity paradox. Journal of the American College of Cardiology, 63, 1345-1354. Lawton, R. J., Carl, C., De Nys, R. & Paul, N. A. (2015). Heritable variation in growth and biomass productivity in the clonal freshwater macroalga Oedogonium. Algal Research, 8, 108-114. Lawton, R. J., De Nys, R. & Paul, N. A. (2013a). Selecting reliable and robust freshwater macroalgae for biomass applications. PLoS One, 8, e64168. Lawton, R. J., Mata, L., De Nys, R. & Paul, N. A. (2013b). Algal bioremediation of waste waters from land-based aquaculture using ulva: selecting target species and strains. PLoS One, 8, e77344. Le Tutour, B., Benslimane, F., Gouleau, M. P., Gouygou, J. P., Saadan, B. & Quemeneur, F. (1998). Antioxidant and pro-oxidant activities of the brown algae, Laminaria digitata, Himanthalia elongata, Fucus vesiculosus, Fucus serratus and Ascophyllum nodosum. Journal of Applied Phycology, 10, 121-129. Lee, J. Y., Kim, Y. J., Kim, H. J., Kim, Y. S. & Park, W. (2012). Immunostimulatory effect of laminarin on RAW 264.7 mouse macrophages. Molecules, 17, 5404-5411. Lee, S. H. & Jeon, Y. J. (2013). Anti-diabetic effects of brown algae derived phlorotannins, marine polyphenols through diverse mechanisms. Fitoterapia, 86, 129-136. Leite, Y. F. M. M., Silva, L. M. C. M., Amorim, R. C. D. N., Freire, E. A., De Melo Jorge, D. M., Grangeiro, T. B. & Benevides, N. M. B. (2005). Purification of a lectin from the marine red alga Gracilaria ornata and its effect on the development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae). Biochimica et Biophysica Acta (BBA) - General Subjects, 1724, 137-145.

204

Li, G. H., Wan, J. Z., Le, G. W. & Shi, Y. H. (2006). Novel angiotensin I-converting enzyme inhibitory peptides isolated from alcalase hydrolysate of mung bean protein. Journal of Peptides Science, 12, 509-514. Li, H., Yu, J., Ahmedna, M. & Goktepe, I. (2013). Reduction of major peanut allergens Ara h 1 and Ara h 2, in roasted peanuts by ultrasound assisted enzymatic treatment. Food Chemistry, 141, 762-768. Li, H. B., Wong, C. C., Cheng, K. W. & Chen, F. (2008). Antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants. LWT - Food Science and Technology, 41, 385-390. Li, X. L. & Xu, J. H. (2013). Lycopene supplement and blood pressure: an updated meta- analysis of intervention trials. Nutrients, 5, 3696-3712. Li, Y. X., Wijesekara, I., Li, Y. & Kim, S. K. (2011). Phlorotannins as bioactive agents from brown algae. Process Biochemistry, 46, 2219-2224. Li, Z. S., Noda, K., Fujita, E., Manabe, Y., Hirata, T. & Sugawara, T. (2015). The green algal carotenoid siphonaxanthin inhibits adipogenesis in 3T3-L1 preadipocytes and the accumulation of lipids in white adipose tissue of KK-Aymice. Journal of Nutrition, 145, 490-498. Liao, H. J. & Hung, C. C. (2015). Chemical composition and in vitro starch digestibility of green banana (cv. Giant Cavendish) flour and its derived autoclaved/debranched powder. LWT - Food Science and Technology, 64, 639-644. Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods in Enzymology. Academic Press. Lin, S. J., Tian, W. N., Li, H., Cao, J. K. & Jiang, W. B. (2012). Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin, trypsin, alcalase and flavourzyme. International Journal of Food Science & Technology, 47, 2045-2051. Liu, D., Pearlman, E., Diaconu, E., Guo, K., Mori, H., Haqqi, T., Markowitz, S., Willson, J. & Sy, M. S. (1996). Expression of hyaluronidase by tumor cells induces angiogenesis in vivo. Proceedings of the National Academy of Sciences of the United States of America, 93, 7832-7837. Liu, D. Z., Lin, Y. S. & Hou, W. C. (2004). Monohydroxamates of aspartic acid and glutamic acid exhibit antioxidant and angiotensin converting enzyme inhibitory activities. Journal of Agricultural and Food Chemistry, 52, 2386-1390. Liu, K. S. (2004). Soybeans as a powerhouse of nutrients and phytochemicals. In: LIU, K. S. (ed.) Soybeans as Functional Foods and Ingredients. AOCS Publishing. Lopez, A., Rico, M., Rivero, A. & Suarez De Tangil, M. (2011). The effects of solvents on the phenolic contents and antioxidant activity of Stypocaulon scoparium algae extracts. Food Chemistry, 125, 1104-1109. Lordan, S., Smyth, T. J., Soler-Vila, A., Stanton, C. & Ross, R. P. (2013). The α-amylase and α- glucosidase inhibitory effects of Irish seaweed extracts. Food Chemistry, 141, 2170- 2176. Lourenco, S. O., Barbarino, E., De-Paula, J. C., Pereira, L. O. D. S. & Marquez, U. M. L. (2002). Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds. Phycological Research, 50, 233-241. Luo, M., Shao, B., Nie, W., Wei, X. W., Li, Y. L., Wang, B. L., He, Z. Y., Liang, X., Ye, T. H. & Wei, Y. Q. (2015). Antitumor and adjuvant activity of λ-carrageenan by stimulating immune response in cancer immunotherapy. Scientific Reports, 5, 11062. Macartain, P., Gill, C. I. R., Brooks, M., Campbell, R. & Rowland, I. R. (2007). Nutritional value of edible seaweeds. Nutrition Reviews, 65, 535-543. Macdonald-Wicks, L. K., Wood, L. G. & Garg, M. L. (2006). Methodology for the determination of biological antioxidant capacity in vitro: a review. Journal of the Science of Food and Agriculture, 86, 2046-2056.

205

Machado, S. D. I., Lopez-Hernandez, J., Paseiro-Losada, P. & Lopez-Cervantes, J. (2004). An HPLC method for the quantification of sterols in edible seaweeds. Biomedical Chromatography, 18, 183-190. Machu, L., Misurcova, L., Vavra Ambrozova, J., Orsavova, J., Mlcek, J., Sochor, J. & Jurikova, T. (2015). Phenolic content and antioxidant capacity in algal food products. Molecules, 20, 1118. Macias-Sanchez, M. D., Mantell, C., Rodriguez, M., Martinez De La Ossa, E., Lubian, L. M. & Montero, O. (2007). Supercritical fluid extraction of carotenoids and chlorophyll a from Synechococcus sp. The Journal of Supercritical Fluids, 39, 323-329. Maeda, H., Hosokawa, M., Sashima, T., Funayama, K. & Miyashita, K. (2005). Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues. Biochemical and Biophysical Research Communications, 332, 392-397. Maeda, H., Hosokawa, M., Sashima, T., Funayama, K. & Miyashita, K. (2007a). Effect of medium-chain triacylglycerols on anti-obesity effect of fucoxanthin. Journal of oleo science, 56, 615-621. Maeda, H., Hosokawa, M., Sashima, T. & Miyashita, K. (2007b). Dietary combination of fucoxanthin and fish oil attenuates the weight gain of white adipose tissue and decreases blood glucose in obese/diabetic KK-A y mice. Journal of Agricultural and Food Chemistry, 55, 7701-7706. Magnusson, M., Mata, L., De Nys, R. & Paul, N. A. (2014). Biomass, lipid and fatty acid production in large-scale cultures of the marine macroalga Derbesia tenuissima (Chlorophyta). Marine Biotechnology, 16, 456-464. Magnusson, M., Mata, L., Wang, N., Zhao, J., De Nys, R. & Paul, N. A. (2015). Manipulating antioxidant content in macroalgae in intensive land-based cultivation systems for functional food applications. Algal Research, 8, 153-160. Malaguti, M., Dinelli, G., Leoncini, E., Bregola, V., Bosi, S., Cicero, A. F. & Hrelia, S. (2014). Bioactive peptides in cereals and legumes: agronomical, biochemical and clinical aspects. International journal of Molecular Sciences, 15, 21120-21135. Manabe, Y., Hirata, T. & Sugawara, T. (2014). Suppressive effects of carotenoids on the antigen-induced degranulation in RBL-2H3 rat basophilic leukemia cells. Journal of Oleo Science, 63, 291-294. Maoka, T., Tokuda, H., Suzuki, N., Kato, H. & Etoh, H. (2012). Anti-oxidative, anti-tumor- promoting, and anti-carcinogensis activities of nitroastaxanthin and nitrolutein, the reaction products of astaxanthin and lutein with peroxynitrite. Marine Drugs, 10, 1391- 1399. Maralani, M. N., Movahedian, A. & Javanmard Sh, H. (2012). Antioxidant and cytoprotective effects of L-Serine on human endothelial cells. Research in Pharmaceutical Sciences, 7, 209-215. Marinho-Soriano, E., Azevedo, C. a. A., Trigueiro, T. G., Pereira, D. C., Carneiro, M. a. A. & Camara, M. R. (2011). Bioremediation of aquaculture wastewater using macroalgae and Artemia. International Biodeterioration & Biodegradation, 65, 253-257. Mata, L., Magnusson, M., Paul, N. A. & De Nys, R. (2015). The intensive land-based production of the green seaweeds Derbesia tenuissima and Ulva ohnoi: biomass and bioproducts. Journal of Applied Phycology, 1-11. Matanjun, P., Mohamed, S., Mustapha, N., Muhammad, K. & Ming, C. (2008). Antioxidant activities and phenolics content of eight species of seaweeds from north Borneo. Journal of Applied Phycology, 20, 367-373. Mathews-Roth, M. M. (1993). Carotenoids in erythropoietic protoporphyria and other photosensitivity diseases. Annals of the new york Academy of Sciences, 691, 127-138. Matsuura, H., Watanabe, M. & Kaya, K. (2012). Echinenone production of a dark red-coloured strain of Botryococcus braunii. Journal of Applied Phycology, 24, 973-977. Mchugh, D. J. (2003). A guide to the seaweed industry, Canberra, FAO Corporate Document Repository.

206

Mendis, E. & Kim, S. K. (2011). Present and future prospects of seaweeds in developing functional foods. Advances in Food and Nutrition Research, 64, 1-15. Menzel, E. J. & Farr, C. (1998). Hyaluronidase and its substrate hyaluronan: Biochemistry, biological activities and therapeutic uses. Cancer Letters, 131, 3-11. Mertz, C., Brat, P., Caris-Veyrat, C. & Gunata, Z. (2010). Characterization and thermal lability of carotenoids and vitamin C of tamarillo fruit (Solanum betaceum Cav.). Food Chemistry, 119, 653-659. Michael, G. (2012). A catalogue of Irish Seaweeds, Germany, Koeltz Scientific books. Migliore, L., Rotini, A., Randazzo, D., Albanese, N. N. & Giallongo, A. (2007). Phenols content and 2-D electrophoresis protein pattern: a promising tool to monitor Posidonia meadows health state. BMC Ecology, 7, 1-8. Miguel, M., Aleixandre, M. A., Ramos, M. & Lopez-Fandino, R. (2006). Effect of simulated gastrointestinal digestion on the antihypertensive properties of ACE-inhibitory peptides derived from ovalbumin. Journal of Agricultural and Food Chemistry, 54, 726-731. Miki, W. (1991). Biological functions and activities of animal carotenaoids. Pure and Applied Chemistry, 63, 141-146. Mirzaei, M., Mirdamadi, S., Ehsani, M. R., Aminlari, M. & Hosseini, E. (2015). Purification and identification of antioxidant and ACE-inhibitory peptide from Saccharomyces cerevisiae protein hydrolysate. Journal of Functional Foods, 19, Part A, 259-268. Misurcova, L., Skrovankova, S., Samek, D., Ambrozova, J. & Machu, L. (2012). Health benefits of algal polysaccharides in human nutrition. Advances in Food and Nutrition Research, 66, 75-145. Mittal, B. V. & Singh, A. K. (2010). Hypertension in the developing world: challenges and opportunities. American Journal of Kidney Diseases, 55, 590-598. Moghaddam, M. & Mehdizadeh, L. (2015). Variability of total phenolic, flavonoid and rosmarinic acid content among Iranian basil accessions. LWT - Food Science and Technology, 63, 535-540. Mohanty, D. P., Mohapatra, S., Misra, S. & Sahu, P. S. (2015). Milk derived bioactive peptides and their impact on human health Saudi Journal of Biological Sciences, 9. Montonen, J., Knekt, P., Jarvinen, R. & Reunanen, A. (2004). Dietary antioxidant intake and risk of type 2 diabetes. Diabetes Care, 27, 362-366. Morgan, K. C., Wright, J. L. C. & Simpson, F. J. (1980). Review of chemical constituents of the red alga Palmaria palmata (dulse). Economic Botany, 34, 27-50. Morris, H. J., Carrillo, O., Almarales, A., Bermúdez, R. C., Lebeque, Y., Fontaine, R., Llaurado, G. & Beltrán, Y. (2007). Immunostimulant activity of an enzymatic protein hydrolysate from green microalga Chlorella vulgaris on undernourished mice. Enzyme and Microbial Technology, 40, 456-460. Muckenschnabel, I., Bernhardt, G., Spruss, T. & Buschauer, A. (1998). Pharmacokinetics and tissue distribution of bovine testicular hyaluronidase and vinblastine in mice: an attempt to optimize the mode of adjuvant hyaluronidase administration in cancer chemotherapy. Cancer Letters, 131, 71-84. Mukhtar, H. & Ikram-Ul, H. (2012). Concomitant production of two proteases and alpha- amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor. Brazilian Journal of Microbiology, 43, 1072-1079. Murthy, K. N. C., Vanitha, A., Rajesha, J., Swamy, M. M., Sowmya, P. R. & Ravishankar, G. A. (2005). In vivo antioxidant activity of carotenoids from Dunaliella salina — a green microalga. Life Sciences, 76, 1381-1390. Nadpal, J. D., Lesjak, M. M., Sibul, F. S., Anackov, G. T., Cetojevic-Simin, D. D., Mimica- Dukic, N. M. & Beara, I. N. (2016). Comparative study of biological activities and phytochemical composition of two rose hips and their preserves: Rosa canina L. and Rosa arvensis Huds. Food Chemistry, 192, 907-914. Nakao, R., Nelson, O. L., Park, J. S., Mathison, B. D., Thompson, P. A. & Chew, B. P. (2010). Effect of astaxanthin supplementation on inflammation and cardiac function in BALB/c mice. Anticancer Research, 30, 2721-2725.

207

National Health and Medical Research Council. (2015). Obesity and overweight [Online]. Available: http://www.nhmrc.gov.au [Accessed 10/07/2016] Neves, S. A., Freitas, A. L., Sousa, B. W., Rocha, M. L., Correia, M. V., Sampaio, D. A. & Viana, G. S. (2007). Antinociceptive properties in mice of a lectin isolated from the marine alga Amansia multifida Lamouroux. Brazilian Journal of Medical and Biological Ressearch, 40, 127-134. Neveux, N., Magnusson, M., Maschmeyer, T., De Nys, R. & Paul, N. A. (2014a). Comparing the potential production and value of high energy liquid and protein from marine and freshwater macrolagae. GCB Bioenergy, 7, 673-689. Neveux, N., Yuen, A. K. L., Jazrawi, C., He, Y., Magnusson, M., Haynes, B. S., Masters, A. F., Montoya, A., Paul, N. A., Maschmeyer, T. & De Nys, R. (2014b). Pre- and post-harvest treatment of macroalgae to improve the quality of feedstock for hydrothermal liquefaction. Algal Research, 6, 22-31. Neveux, N., Yuen, A. K. L., Jazrawi, C., Magnusson, M., Haynes, B. S., Masters, A. F., Montoya, A., Paul, N. A., Maschmeyer, T. & De Nys, R. (2014c). Biocrude yield and productivity from the hydrothermal liquefaction of marine and freshwater green macroalgae. Bioresource Technology, 155, 334-341. Ngo, D. H., Vo, T. S., Ngo, D. N., Wijesekara, I. & Kim, S. K. (2012). Biological activities and potential health benefits of bioactive peptides derived from marine organisms. International Journal of Biological Macromolecules, 51, 378-383. Ngoh, Y. Y. & Gan, C. Y. (2016). Enzyme-assisted extraction and identification of antioxidative and α-amylase inhibitory peptides from Pinto beans (Phaseolus vulgaris cv. Pinto). Food Chemistry, 190, 331-337. Niu, J., Wang, G., Lu, F., Zhou, B. & Peng, G. (2009). Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides. Chinese Journal of Oceanology and Limnology, 27, 502-512. Nongonierma, A. B. & Fitzgerald, R. J. (2015). The scientific evidence for the role of milk protein-derived bioactive peptides in humans: A Review. Journal of Functional Foods, 17, 640-656. Nongonierma, A. B., Mooney, C., Shields, D. C. & Fitzgerald, R. J. (2013). Inhibition of dipeptidyl peptidase IV and xanthine oxidase by amino acids and dipeptides. Food Chemistry, 141, 644-653. Nwosu, F., Morris, J., Lund, V. A., Stewart, D., Ross, H. A. & Mcdougall, G. J. (2011). Anti- proliferative and potential anti-diabetic effects of phenolic-rich extracts from edible marine algae. Food Chemistry, 126, 1006-1012. O’sullivan, L., Murphy, B., Mcloughlin, P., Duggan, P., Lawlor, P. G., Hughes, H. & Gardiner, G. E. (2010). Prebiotics from marine macroalgae for human and animal health applications. Marine Drugs, 8, 2038-2064. Obulesu, M., Dowlathabad, M. R. & Bramhachari, P. V. (2011). Carotenoids and Alzheimer’s Disease: An insight into therapeutic role of retinoids in animal models. Neurochemistry International, 59, 535-541. Odaka, C. & Mizuochi, T. (2000). Angiotensin-converting enzyme inhibitor captopril prevents activation-induced apoptosis by interfering with T cell activation signals. Clinical and Experimental Immunology, 121, 515-522. Okada, T., Nakai, M., Maeda, H., Hosokawa, M., Sashima, T. & Miyashita, K. (2008). Suppressive effect of neoxanthin on the differentiation of 3T3-L1 adipose cells. Journal of Oleo Science, 57, 345-351. Okai, Y., Higashi-Okai, K., Yano, Y. & Otani, S. (1996). Identification of antimutagenic substances in an extract of edible red alga, Porphyra tenera (Asadusa-nori). Cancer Letters, 100, 235-240. Okuzumi, J., Nishino, H., Murakoshi, M., Iwashima, A., Tanaka, Y., Yamane, T., Fujita, Y. & Takahashi, T. (1990). Inhibitory effects of fucoxanthin, a natural carotenoid, on N-myc expression and cell cycle progression in human malignant tumor cells. Cancer Letters, 55, 75-81.

208

Oliver, J. & Palou, A. (2000). Chromatographic determination of carotenoids in foods. Journal of Chromatography A, 881, 543-555. Omana, D. A., Wang, J. & Wu, J. (2010). Ovomucin – a glycoprotein with promising potential. Trends in Food Science & Technology, 21, 455-463. Omokawa, Y., Miyazaki, T., Walde, P., Akiyama, K., Sugahara, T., Masuda, S., Inada, A., Ohnishi, Y., Saeki, T. & Kato, K. (2010). In vitro and in vivo anti-tumor effects of novel Span 80 vesicles containing immobilized Eucheuma serra agglutinin. International Journal of Pharmaceutics, 389, 157-167. Onofrejova, L., Vasckova, J., Klejdus, B., Stratil, P., Misurcova, L., Kracmar, S., Kopecky, J. & Vacek, J. (2010). Bioactive phenols in algae: The application of pressurized-liquid and solid-phase extraction techniques. Journal of Pharmaceutical and Biomedical Analysis, 51, 464-470. Orchard, A. E. & Mccarthy, P. M. (2007). Algae of Australia : introduction, Melbourne, CSIRO Publishing. Orcic, D. Z., Mimica-Dukic, N. M., Franciskovic, M. M., Petrovic, S. S. & Jovin, E. D. (2011). Antioxidant activity relationship of phenolic compounds in Hypericum perforatum L. Chemistry Central Journal, 5, 34-44. Ornelas-Paz, J. D. J., Yahia, E. M. & Gardea, A. A. (2008). Changes in external and internal color during postharvest ripening of ‘Manila’ and ‘Ataulfo’ mango fruit and relationship with carotenoid content determined by liquid chromatography–APCI+- time-of-flight mass spectrometry. Postharvest Biology and Technology, 50, 145-152. Ortiz, J., Romero, N., Robert, P., Araya, J., Lopez-Hernandez, J., Bozzo, C., Navarrete, E., Osorio, A. & Rios, A. (2006). Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds Ulva lactuca and Durvillaea antarctica. Food Chemistry, 99, 98-104. Oyeleke, S. B., Oyewole, O. A. & Egwin, E. C. (2011). Production of protease and amylase from Bacillus subtilis and Aspergillus niger using Parkia biglobossa (Africa locust beans) as substrate in solid state fermentration. Advances in Life Sciences, 1, 49-53. Pangestuti, R. & Kim, S. K. (2011). Biological activities and health benefit effects of natural pigments derived from marine algae. Journal of Functional Foods, 3, 255-266. Pangestuti, R. & Kim, S. K. (2015). Seaweed proteins, peptides, and amino acids. In: TIWARI, B. K. & TROY, D. (eds.) Seaweed Sustainability. San Diego: Academic Press. Park, J. S., Mathison, B. D., Hayek, M. G., Zhang, J., Reinhart, G. A. & Chew, B. P. (2013). Astaxanthin modulates age-associated mitochondrial dysfunction in healthy dogs. Journal of Animal Science, 91, 268-275. Parys, S., Rosenbaum, A., Kehraus, S., Reher, G., Glombitza, K. W. & Konig, G. M. (2007). Evaluation of quantitative methods for the determination of polyphenols in algal extracts. Journal of Natural Products, 70, 1865-1870. Pasquet, V., Morisset, P., Ihammouine, S., Chepied, A., Aumailley, L., Berard, J. B., Serive, B., Kaas, R., Lanneluc, I., Thiery, V., Lafferriere, M., Piot, J. M., Patrice, T., Cadoret, J. P. & Picot, L. (2011). Antiproliferative activity of violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts. Marine Drugs, 9, 819-831. Patarra, R., Paiva, L., Neto, A., Lima, E. & Baptista, J. (2011). Nutritional value of selected macroalgae. Journal of Applied Phycology, 23, 205-208. Paul, N. A. & De Nys, R. (2008). Promise and pitfalls of locally abundant seaweeds as biofilters for integrated aquaculture. Aquaculture, 281, 49-55. Paul, N. A., Tseng, C. K. & Borowitzka, M. (2012). Seaweed and microlage. In: LUCUS, J. S. & SOUGHGATE, P. C. (eds.) Aquaculture: Farming Aquatic Animal and Plants. Hoboken, NJ: Wiley Blackwell. Pena-Rodriguez, A., Mawhinney, T. P., Ricque-Marie, D. & Cruz-Suarez, L. E. (2011). Chemical composition of cultivated seaweed Ulva clathrata (Roth) C. Agardh. Food Chemistry, 129, 491-498.

209

Peng, J., Yuan, J. P., Wu, C. F. & Wang, J. H. (2011). Fucoxanthin, a marine carotenoid present in brown seaweeds and diatoms: Metabolism and bioactivities relevant to human health. Marine Drugs, 9, 1806-1828. Peng, Y., Hu, J., Yang, B., Lin, X. P., Zhou, X. F., Yang, X. W. & Liu, Y. H. (2015). Chemical composition of seaweeds. In: TIWARI, B. K. & TROY, D. (eds.) Seaweed Sustainability. San Diego: Academic Press. Pereira, L., Critchley, A., Amado, A. & Ribeiro-Claro, P. A. (2009). A comparative analysis of phycocolloids produced by underutilized versus industrially utilized carrageenophytes (Gigartinales, Rhodophyta). Journal of Applied Phycology, 21, 599-605. Plaza, M., Cifuentes, A. & Ibanez, E. (2008). In the search of new functional food ingredients from algae. Trends in Food Science & Technology, 19, 31-39. Pujol, C. A., Scolaro, L. A., Ciancia, M., Matulewicz, M. C., Cerezo, A. S. & Damonte, E. B. (2006). Antiviral activity of a carrageenan from Gigartina skottsbergii against intraperitoneal murine herpes simplex virus infection. Planta Medica, 72, 121-125. Qi, H., Liu, X., Zhang, J., Duan, Y., Wang, X. & Zhang, Q. (2012). Synthesis and antihyperlipidemic activity of acetylated derivative of ulvan from Ulva pertusa. International Journal of Biological Macromolecules, 50, 270-272. Qi, H. & Sun, Y. (2015). Antioxidant activity of high sulfate content derivative of ulvan in hyperlipidemic rats. International Journal of Biological Macromolecules, 76, 326-329. Qu, W., Ma, H., Pan, Z., Luo, L., Wang, Z. & He, R. (2010). Preparation and antihypertensive activity of peptides from Porphyra yezoensis. Food Chemistry, 123, 14-20. Rahmouni, K., Correia, M. L. G., Haynes, W. G. & Mark, A. L. (2005). Obesity-associated hypertension: New insights into mechanisms. Hypertension, 45, 9-14. Rajapakse, N. & Kim, S. K. (2011). Nutritional and digestive health benefits of seaweed. Advances in Food and Nutrition Research, 64, 17-28. Ramos, M. V., Monteiro, A. C. O., Moreira, R. A. & Carvalho, A. D. F. F. U. (2000). Amino acid composition of some Brazilian seaweed species. Journal of Food Biochemistry, 24, 33-39. Ranga, R., Sarada, A. R., Baskaran, V. & Ravishankar, G. A. (2009). Identification of carotenoids from green alga Haematococcus pluvialis by HPLC and LC-MS (APCI) and their antioxidant properties. Journal of Microbiology and Biotechnology, 19, 1333- 1341. Rao, A. R., Baskaran, V., Sarada, R. & Ravishankar, G. A. (2013a). In vivo bioavailability and antioxidant activity of carotenoids from microalgal biomass — A repeated dose study. Food Research International, 54, 711-717. Rao, A. R., Raghunath Reddy, R. L., Baskaran, V., Sarada, R. & Ravishankar, G. A. (2010). Characterization of microalgal carotenoids by mass spectrometry and their bioavailability and antioxidant properties elucidated in rat model. Journal of Agricultural and Food Chemistry, 58, 8553-8559. Rao, A. R., Sarada, R., Baskaran, V. & Ravishankar, G. A. (2006). Antioxidant activity of Botryococcus braunii extract elucidated in vitro models. Journal of Agricultural and Food Chemistry, 54, 4593-4599. Rao, A. R., Sindhuja, H. N., Dharmesh, S. M., Sankar, K. U., Sarada, R. & Ravishankar, G. A. (2013b). Effective inhibition of skin Cancer, tyrosinase, and antioxidative properties by astaxanthin and astaxanthin esters from the green alga Haematococcus pluvialis. Journal of Agricultural and Food Chemistry, 61, 3842-3851. Rao, A. V. & Rao, L. G. (2007). Carotenoids and human health. Pharmacological Research, 55, 207-216. Raposo, M., De Morais, A. & De Morais, R. (2015). Carotenoids from marine microalgae: A valuable natural source for the prevention of chronic diseases. Marine Drugs, 13, 5128- 5155. Reddy, N. S., Khan, T. N., Malewar, V. G. & Dudde, K. B. (1995). Trace elements in spinach (Spinacia oleracea) cultivated in soil fortified with graded levels of iron. Plant Foods for Human Nutrition, 47, 357-360.

210

Regnier, P., Bastias, J., Rodriguez-Ruiz, V., Caballero-Casero, N., Caballo, C., Sicilia, D., Fuentes, A., Maire, M., Crepin, M., Letourneur, D., Gueguen, V., Rubio, S. & Pavon- Djavid, G. (2015). Astaxanthin from Haematococcus pluvialis prevents oxidative stress on human endothelial cells without toxicity. Marine Drugs, 13, 2857-2874. Remond, D. (2008). Nutritional properties of meat peptides and proteins: impact of processing. Sciences des aliments, 28, 389-401. Ren, J., Zhao, M., Shi, J., Wang, J., Jiang, Y., Cui, C., Kakuda, Y. & Xue, S. J. (2008). Purification and identification of antioxidant peptides from grass carp muscle hydrolysates by consecutive chromatography and electrospray ionization-mass spectrometry. Food Chemistry, 108, 727-736. Rengasamy, K. R. R., Aderogba, M. A., Amoo, S. O., Stirk, W. A. & Van Staden, J. (2013). Potential antiradical and alpha-glucosidase inhibitors from Ecklonia maxima (Osbeck) Papenfuss. Food Chemistry, 141, 1412-1415. Research and Markets. (2014). Global functional food and nutraceuticals market (2014-2020)- By type (foods, beverages, supplements); benefits (health and wellness, disease prevention, fitness, beauty); origin & ingredient [Online]. Available: www.researchandmarkets.com [Accessed 18/04 2016]. Ribaya-Mercado, J. D. & Blumberg, J. B. (2004). Lutein and zeaxanthin and their potential roles in disease prevention. The Journal of the American College of Nutrition, 23, 567- 587. Ricketts, T. R. (1971). The structures of siphonein and siphonaxanthin from Codium fragile. Phytochemistry, 10, 155-160. Roberts, D. A., Paul, N. A., Cole, A. J. & De Nys, R. (2015). From waste water treatment to land management: Conversion of aquatic biomass to biochar for soil amelioration and the fortification of crops with essential trace elements. Journal of Environmental Management, 157, 60-68. Roberts, R. L., Green, J. & Lewis, B. (2009). Lutein and zeaxanthin in eye and skin health. Clinics Dermatology, 27, 195-201. Rodriguez-Bernaldo De Quiros, A., Lage-Yusty, M. A. & Lopez-Hernandez, J. (2010). Determination of phenolic compounds in macroalgae for human consumption. Food Chemistry, 121, 634-638. Rohani-Ghadikolaei, K., Abdulalian, E. & Ng, W. K. (2012). Evaluation of the proximate, fatty acid and mineral composition of representative green, brown and red seaweeds from the Persian Gulf of Iran as potential food and feed resources. Journal of Food Science and Technology, 49, 774-780. Ruperez, P. (2002). Mineral content of edible marine seaweeds. Food Chemistry, 79, 23-26. Ryan, J. T., Ross, R. P., Bolton, D., Fitzgerald, G. F. & Stanton, C. (2011). Bioactive Peptides from Muscle Sources: Meat and Fish. Nutrients, 3, 765-791. Ryu, H. S., Satterlee, L. D. & Lee, K. H. (1982). Nitrogen conversion factors and in vitro protein digestibility of some seaweeds. Bull. Korean Fish, Soc, 15, 263-270. Sabeena, F. K. H. & Jacobsen, C. (2013). Phenolic compounds and antioxidant activities of selected species of seaweeds from Danish coast. Food Chemistry, 138, 1670-1681. Saini, R. K., Nile, S. H. & Park, S. W. (2015). Carotenoids from fruits and vegetables: Chemistry, analysis, occurrence, bioavailability and biological activities. Food Research International, 76, Part 3, 735-750. Saito, K., Jin, D. H., Ogawa, T., Muramoto, K., Hatakeyama, E., Yasuhara, T. & Nokihara, K. (2003). Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry. Journal of Agricultural and Food Chemistry, 51, 3668-3674. Saito, Y., Wanezaki, K., Kawato, A. & Imayasu, S. (1994). Structure and activity of angiotensin I converting enzyme inhibitory peptides from sake and sake lees. Bioscience,Biotechnology and Biochemistry, 58, 1767-1771. Sakai, S., Sugawara, T., Matsubara, K. & Hirata, T. (2009). Inhibitory effect of carotenoids on the degranulation of mast cells via suppression of antigen-induced aggregation of high affinity IgE receptors. The Journal of Biological Chemistry, 284, 28172-28179.

211

Sakulnarmrat, K. & Konczak, I. (2012). Composition of native Australian herbs polyphenolic- rich fractions and in vitro inhibitory activities against key enzymes relevant to metabolic syndrome. Food Chemistry, 134, 1011-1019. Samarakoon, K. & Jeon, Y.-J. (2012). Bio-functionalities of proteins derived from marine algae — A review. Food Research International, 48, 948-960. Sancho, L. E. G.-G., Yahia, E. M. & Gonzalez-Aguilar, G. A. (2011). Identification and quantification of phenols, carotenoids, and vitamin C from papaya (Carica papaya L., cv. Maradol) fruit determined by HPLC-DAD-MS/MS-ESI. Food Research International, 44, 1284-1291. Santoso, J., Yoshie, Y. & Suzuki, T. (2002). The distribution and profile of nutrients and catechins of some Indonesian seaweeds. Fisheries science, 68, 1647-1648. Sarmadi, B. H. & Ismail, A. (2010). Antioxidative peptides from food proteins: A review. Peptides, 31, 1949-1956. Sasaki, K., Ishihara, K., Oyamada, C., Sato, A., Fukushi, A., Arakane, T., Motoyama, M., Yamazaki, M. & Mitsumoto, M. (2008). Effects of fucoxanthin addition to ground chicken breast meat on lipid and colour stability during chilled storage, before and after cooking. Asian-Australasian Journal of Animal Sciences, 21, 1067-1072. Sathya, R., Kanaga, N., Sankar, P. & Jeeva, S. (2013). Antioxidant properties of phlorotannins from brown seaweed Cystoseira trinodis (Forsskal) C. Agardh. Arabian Journal of Chemistry. Sato, M., Hosokawa, T., Yamaguchi, T., Nakano, T., Muramoto, K., Kahara, T., Funayama, K., Kobayashi, A. & Nakano, T. (2002a). Angiotensin I-converting enzyme inhibitory peptides derived from wakame (Undaria pinnatifida) and their antihypertensive effect in spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry, 50, 6245-6252. Sato, M., Oba, T., Yamaguchi, T., Nakano, T., Kahara, T., Funayama, K., Kobayashi, A. & Nakano, T. (2002b). Antihypertensive effects of hydrolysates of wakame (Undaria pinnatifida) and their angiotensin-I-converting enzyme inhibitory activity. Annals of Nutrition and Metabolism, 46, 259-267. Sato, Y., Hirayama, M., Morimoto, K., Yamamoto, N., Okuyama, S. & Hori, K. (2011). High mannose-binding lectin with preference for the cluster of alpha1-2-mannose from the green alga Boodlea coacta is a potent entry inhibitor of HIV-1 and influenza viruses. The Journal of Biological Chemistry, 286, 19446-19458. Seidel, V. & Taylor, P. W. (2004). In vitro activity of extracts and constituents of Pelagonium against rapidly growing mycobacteria. International Journal of Antimicrobial Agents, 23, 613-619. Sekar, S. & Chandramohan, M. (2008). Phycobiliproteins as a commodity: Trends in applied research, patents and commercialization. Journal of Applied Phycology, 20, 113-136. Senevirathne, M. & Kim, S. K. (2012). Development of bioactive peptides from fish proteins and their health promoting ability. Advances in Food and Nutrition Ressearch, 65, 235- 248. Sheih, I. C., Fang, T. J. & Wu, T. K. (2009a). Isolation and characterisation of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from the algae protein waste. Food Chemistry, 115, 279-284. Sheih, I. C., Fang, T. J., Wu, T. K. & Lin, P. H. (2010). Anticancer and antioxidant activities of the peptide fraction from algae protein waste. Journal of Agricultural and Food Chemistry, 58, 1202-1207. Sheih, I. C., Wu, T.-K. & Fang, T. J. (2009b). Antioxidant properties of a new antioxidative peptide from algae protein waste hydrolysate in different oxidation systems. Bioresource Technology, 100, 3419-3425. Shin, T., Ahn, M., Hyun, J. W., Kim, S. H. & Moon, C. (2014). Antioxidant marine algae phlorotannins and radioprotection: A review of experimental evidence. Acta Histochemica, 116, 669-674.

212

Shin, Z. I., Yu, R., Park, S. A., Chung, D. K., Ahn, C. W., Nam, H. S., Kim, K. S. & Lee, H. J. (2001). His-His-Leu, an angiotensin I converting enzyme inhibitory peptide derived from Korean soybean paste, exerts antihypertensive activity in vivo. Journal of Agricultural and Food Chemistry, 49, 3004-3009. Shirsath, S. R., Sonawane, S. H. & Gogate, P. R. (2012). Intensification of extraction of natural products using ultrasonic irradiations—A review of current status. Chemical Engineering and Processing: Process Intensification, 53, 10-23. Shori, A. B. (2015). Screening of antidiabetic and antioxidant activities of medicinal plants. Journal of Integrative Medicine, 13, 297-305. Siegel, G. & Ermilov, E. (2012). Omega-3 fatty acids: Benefits for cardio-cerebro-vascular diseases. Atherosclerosis, 225, 291-295. Sies, H. & Stahl, W. (1995). Vitamins E and C, beta-carotene, and other carotenoids as antioxidants. The American Journal of Clinical Nutrition, 62, 1315-1321. Singh, B. P., Vij, S. & Hati, S. (2014). Functional significance of bioactive peptides derived from soybean. Peptides, 54, 171-179. Singh, R. S., Thakur, S. R. & Bansal, P. (2015). Algal lectins as promising biomolecules for biomedical research. Critical Reviews in Microbiology, 41, 77-88. Siow, H. L. & Gan, C. Y. (2013). Extraction of antioxidative and antihypertensive bioactive peptides from Parkia speciosa seeds. Food Chemistry, 141, 3435-3442. Siow, H. L. & Gan, C. Y. (2016). Extraction, identification, and structure–activity relationship of antioxidative and α-amylase inhibitory peptides from cumin seeds (Cuminum cyminum). Journal of Functional Foods, 22, 1-12. Skrovankova, S. (2011). Seaweed vitamins as nutraceuticals. Advances in Food and Nutrition Research, 64, 357-369. Sluijs, I., Cadier, E., Beulens, J. W. J., Van Der A, D. L., Spijkerman, A. M. W. & Van Der Schouw, Y. T. (2015). Dietary intake of carotenoids and risk of type 2 diabetes. Nutrition, Metabolism and Cardiovascular Diseases, 25, 376-381. Song, J., Braun, G., Bevis, E. & Doncaster, K. (2006). A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis. Electrophoresis, 27, 3144-3151. Stahl, W., Heinrich, U., Jungmann, H., Von Laar, J., Schietzel, M., Sies, H. & Tronnier, H. (1998). Increased dermal carotenoid levels assessed by noninvasive reflection spectrophotometry correlate with serum levels in women ingesting betatene. Journal of Nutrition, 128, 903-907. Stahl, W. & Sies, H. (2005). Bioactivity and protective effects of natural carotenoids. Biochimica et Biophysica Acta (BBA) 1740, 101-107. Stalikas, C. D. (2007). Extraction, separation, and detection methods for phenolic acids and flavonoids. Journal of Separation Science, 30, 3268-3295. Stengel, D. B., Connan, S. & Popper, Z. A. (2011). Algal chemodiversity and bioactivity: Sources of natural variability and implications for commercial application. Biotechnology Advances, 29, 483-501. Suetsuna, K. (1998a). Isolation and characterization of angiotensin I-converting enzyme inhibitor dipeptides derived from Allium sativum L (garlic). The Journal of Nutritional Biochemistry, 9, 415-419. Suetsuna, K. (1998b). Purification and identification of angiotensin I-converting enzyme inhibitors from the red alga Porphyra yezoensis. Journal of Marine Biotechnology, 6, 163-167. Suetsuna, K. & Chen, J. R. (2001). Identification of antihypertensive peptides from peptic digest of two microalgae, Chlorella vulgaris and Spirulina platensis. Marine Biotechnology, 3, 305-309. Suetsuna, K., Maekawa, K. & Chen, J. R. (2004). Antihypertensive effects of Undaria pinnatifida (wakame) peptide on blood pressure in spontaneously hypertensive rats. The Journal of Nutritional Biochemistry, 15, 267-272.

213

Suetsuna, K. & Nakano, T. (2000). Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida). The Journal of Nutritional Biochemistry, 11, 450-454. Suetsuna, K., Ukeda, H. & Ochi, H. (2000). Isolation and characterization of free radical scavenging activities peptides derived from casein. Journal of Nutritional Biochemistry, 11, 128-131. Sugahara, T., Ohama, Y., Fukuda, A., Hayashi, M., Kawakubo, A. & Kato, K. (2001). The cytotoxic effect of Eucheuma serra agglutinin (ESA) on cancer cells and its application to molecular probe for drug delivery system using lipid vesicles. Cytotechnology, 36, 93-99. Sugawara, T., Ganesan, P., Li, Z., Manabe, Y. & Hirata, T. (2014). Siphonaxanthin, a green algal carotenoid, as a novel functional compound. Marine Drugs, 12, 3660-3668. Sugawara, T., Matsubara, K., Akagi, R., Mori, M. & Hirata, T. (2006). Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, fucoxanthinol. Journal of Agricultural and Food Chemistry, 54, 9805-9810. Tabish, S. A. (2007). Is diabetes becoming the biggest epidemic of the twenty-first century? International Journal of Health Sciences, 1, V-VIII. Tagliazucchi, D., Shamsia, S. & Conte, A. (2016). Release of angiotensin converting enzyme- inhibitory peptides during in vitro gastro-intestinal digestion of camel milk. International Dairy Journal, 56, 119-128. Taha, F. S., Mohamed, G. F., Mohamed, S. H., Mohamed, S. S. & Kamil, M. M. (2011). Optimization of the extraction of total phenolic compounds from sunflower meal and evaluation of the bioactivities of chosen extracts. American Journal of Food Technology, 6, 1002-1020. Takahashi, M., Moriguchi, S., Ikeno, M., Kono, S., Ohata, K., Usui, H., Kurahashi, K., Sasaki, R. & Yoshikawa, M. (1996). Studies on the ileum-contracting mechanisms and identification as a complement C3a receptor agonist of oryzatensin, a bioactive peptide derived from rice albumin. Peptides, 17, 5-12. Tang, D. S., Tian, Y. J., He, Y. Z., Li, L., Hu, S. Q. & Li, B. (2010). Optimisation of ultrasonic- assisted protein extraction from Brewer's spent grain. Caech Journal of Food Sciences, 28, 9-17. Tang, K. S. C., Konczak, I. & Zhao, J. (2016). Identification and quantification of phenolics in Australian native mint (Mentha australis R. Br.). Food Chemistry, 192, 698-705. Tavares, T. G., Contreras, M. M., Amorim, M., Martin-Alvarez, P. J., Pintado, M. E., Recio, I. & Malcata, F. X. (2011). Optimisation, by response surface methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained with cardoon extract. International Dairy Journal, 21, 926-933. Taylor, S. L., Remmington, B. C., Panda, R., Goodman, R. E. & Baumert, J. L. (2015). Detection and control of soybeans as a food allergen. In: FLANAGAN, S. (ed.) Handbook of Food Allergen Detection and Control. Woodhead Publishing. Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L. & Hawkins Byrne, D. (2006). Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. Journal of Food Composition and Analysis, 19, 669- 675. Thuy, T. T. T., Ly, B. M., Van, T. T. T., Van Quang, N., Tu, H. C., Zheng, Y., Seguin-Devaux, C., Mi, B. & Ai, U. (2015). Anti-HIV activity of fucoidans from three brown seaweed species. Carbohydrate Polymers, 115, 122-128. Tierney, M. S., Smyth, T. J., Rai, D. K., Soler-Vila, A., Croft, A. K. & Brunton, N. (2013). Enrichment of polyphenol contents and antioxidant activities of Irish brown macroalgae using food-friendly techniques based on polarity and molecular size. Food Chemistry, 139, 753-761. Tobias, D. K., Pan, A., Jackson, C. L., O'reilly, E. J., Ding, E. L., Willett, W. C., Manson, J. E. & Hu, F. B. (2014). Body-mass index and mortality among adults with incident type 2 diabetes. New England Journal of Medicine, 370, 233-244.

214

Tominaga, K., Hongo, N., Karato, M. & Yamashita, E. (2012). Cosmetic benefits of astaxanthin on humans subjects. Acta Biochimica Polonica, 59, 43-47. Tomovic, V., Jokanovic, M., Sojic, B., Skaljac, S., Tasic, T. & Ikonic, P. (2015). Minerals in pork meat and edible offal. Procedia Food Science, 5, 293-295. Tundis, R., Loizzo, M. R. & Menichini, F. (2010). Natural products as alpha-amylase and alpha-glucosidase inhibitors and their hypoglycaemic potential in the treatment of diabetes: an update. Mini Reviews in Medicinal Chemistry, 10, 315-331. Udenigwe, C. C. & Aluko, R. E. (2011). Chemometric analysis of the amino acid requirements of antioxidant food protein hydrolysates. International Journal of Molecular Sciences, 12, 3148-3161. Uraipong, C. & Zhao, J. (2015). Rice bran protein hydrolysates exhibit strong in vitro α- amylase, β-glucosidase and ACE-inhibition activities. Journal of the Science of Food and Agriculture, 96, 1101-1110. Urbano, M. G. & Goni, I. (2002). Bioavailability of nutrients in rats fed on edible seaweeds, Nori (Porphyra tenera) and Wakame (Undaria pinnatifida), as a source of dietary fibre. Food Chemistry, 76, 281-286. Ursu, A. V., Marcati, A., Sayd, T., Sante-Lhoutellier, V., Djelveh, G. & Michaud, P. (2014). Extraction, fractionation and functional properties of proteins from the microalgae Chlorella vulgaris. Bioresource Technology, 157, 134-139. Vanderlei, E. S., Patoilo, K. K., Lima, N. A., Lima, A. P., Rodrigues, J. A., Silva, L. M., Lima, M. E., Lima, V. & Benevides, N. M. (2010). Antinociceptive and anti-inflammatory activities of lectin from the marine green alga Caulerpa cupressoides. International Immunopharmacology, 10, 1113-1118. Vincent, D., Wheatley, M. D. & Cramer, G. R. (2006). Optimization of protein extraction and solubilization for mature grape berry clusters. Electrophoresis, 27, 1853-1865. Vitelli, O., Tabarrini, A., Miano, S., Rabasco, J., Pietropaoli, N., Forlani, M., Parisi, P. & Villa, M. P. (2015). Impact of obesity on cognitive outcome in children with sleep-disordered breathing. Sleep Medicine, 16, 625-630. Vo, T. S., Ryu, B. M. & Kim, S. K. (2013). Purification of novel anti-inflammatory peptides from enzymatic hydrolysate of the edible microalgal Spirulina maxima. Journal of Functional Foods, 5, 1336-1346. Wang, L., Liu, S., Manson, J. E., Gaziano, J. M., Buring, J. E. & Sesso, H. D. (2006). The consumption of lycopene and tomato-based food products is not associated with the risk of type 2 diabetes in women. Journal of Nutrition, 136, 620-625. Wang, S., Zhong, F. D., Zhang, Y. J., Wu, Z. J., Lin, Q. Y. & Xie, L. H. (2004). Molecular characterization of a new lectin from the marine alga Ulva pertusa. Acta Biochimica Biophysica Sinica (Shanghai), 36, 111-117. Wang, T., Jonsdottir, R. & Olafsdottir, G. (2009). Total phenolic compounds, radical scavenging and metal chelation of extracts from Icelandic seaweeds. Food Chemistry, 116, 240-248. Wang, W., Scali, M., Vignani, R., Spadafora, A., Sensi, E., Mazzuca, S. & Cresti, M. (2003). Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds. Electrophoresis, 24, 2369-2375. Wang, W., Tai, F. & Chen, S. (2008). Optimizing protein extraction from plant tissues for enhanced proteomics analysis. Journal of Separation Science, 31, 2032-2039. Watanabe, M., Musumi, K. & Ayugase, J. (2011). Carotenoid pigment composition, polyphenol content, and antioxidant activities of extracts from orange-colored Chinese cabbage. LWT - Food Science and Technology, 44, 1971-1975. Wesierska, E., Saleh, Y., Trziszka, T., Kopec, W., Siewinski, M. & Korzekwa, K. (2005). Antimicrobial activity of chicken egg white cystatin. World Journal of Microbiology and Biotechnology, 21, 59-64. World Health Organization (WHO). (2015). Obesity and overweight [Online]. Available: http://www.who.int [Accessed 20/09 2015].

215

World Health Organization (WHO). (2016). Diabetes [Online]. Available: http://www.who.int [Accessed 01/04 2016]. Wijesinghe, W. A. J. P. & Jeon, Y. J. (2012). Biological activities and potential industrial applications of fucose rich sulfated polysaccharides and fucoidans isolated from brown seaweeds: A review. Carbohydrate Polymers, 88, 13-20. Wikandari, R., Nguyen, H., Millati, R., Niklasson, C. and Taherzadeh, M.J. (2015). Improvement of biogas production from orange peel waste by leaching of Limonene. Biomed research international. Wong, K. H. & Cheung, P. C. K. (2000). Nutritional evaluation of some subtropical red and green seaweeds: Part I. proximate composition, amino acid profiles and some physico- chemical properties. Food Chemistry, 71, 475-482. Wong, K. H. & Cheung, P. C. K. (2001). Nutritional evaluation of some subtropical red and green seaweeds Part II. In vitro protein digestibility and amino acid profiles of protein concentrates. Food Chemistry, 72, 11-17. Wu, J. & Ding, X. (2002). Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides. Food Research International, 35, 367-375. Xin, Z. H., Wu, S. Y., Ma, H. L. & Dai, C. H. (2003). Study on processing antihypertensive peptides derived from wheat germ protein. Food Science, 24, 120-123. Yaich, H., Garna, H., Besbes, S., Paquot, M., Blecker, C. & Attia, H. (2011). Chemical composition and functional properties of Ulva lactuca seaweed collected in Tunisia. Food Chemistry, 128, 895-901. Yamashita, E. (2015). Let astaxanthin be thy medicine. PharmaNutrition, 3, 115-122. Yan, X., Chuda, Y., Suzuki, M. & Nagata, T. (1999). Fucoxanthin as the major antioxidant in Hijikia fusiformis, a common edible seaweed. Bioscience, Biotechnology and Biochemistry, 63, 605-607. Yang, Y. I., Ahn, J. H., Choi, Y. S. & Choi, J. H. (2015). Brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity. Gynecologic Oncology, 136, 355-364. Yano, S., Suzuki, K. & Funatsu, D. (2014). Isolation from a-Zein of thermolysin peptides with angiotensin I-converting enzyme inhibitory activity. Bioscience,Biotechnology and Biochemistry, 60, 661-663. Yao, L. H., Jiang, Y. M., Shi, J., Barberan, T. F. A., Datta, N., Singanusong, R. & Chen, S. S. (2004). Flavonoids in food and their health benefits. Plant Foods for Human Nutrition, 59, 113-122. Yin, G., Li, W., Lin, Q., Lin, X., Lin, J., Zhu, Q., Jiang, H. & Huang, Z. (2014). Dietary administration of laminarin improves the growth performance and immune responses in Epinephelus coioides. Fish & Shellfish Immunology, 41, 402-406. Yokoyama, H. & Ishihi, Y. (2010). Bioindicator and biofilter function of Ulva spp. (Chlorophyta) for dissolved inorganic nitrogen discharged from a coastal fish farm — potential role in integrated multi-trophic aquaculture. Aquaculture, 310, 74-83. Yoo, K. M. & Moon, B. (2016). Comparative carotenoid compositions during maturation and their antioxidative capacities of three citrus varieties. Food Chemistry, 196, 544-549. Yu, R. X., Hu, X. M., Xu, S. Q., Jiang, Z. J. & Yang, W. (2011). Effects of fucoxanthin on proliferation and apoptosis in human gastric adenocarcinoma MGC-803 cells via JAK/STAT signal pathway. European Journal of Pharmacology, 657, 10-19. Yuan, H. & Song, J. (2005). Preparation, structural characterization and in vitro antitumor activity of kappa-carrageenan oligosaccharide fraction from Kappaphycus striatum. Journal of Applied Phycology, 17, 7-13. Yuan, H., Song, J., Li, X., Li, N. & Dai, J. (2006). Immunomodulation and antitumor activity of κ-carrageenan oligosaccharides. Cancer Letters, 243, 228-234. Yuan, H., Zhang, W., Li, X., Lü, X., Li, N., Gao, X. & Song, J. (2005a). Preparation and in vitro antioxidant activity of κ-carrageenan oligosaccharides and their oversulfated, acetylated, and phosphorylated derivatives. Carbohydrate Research, 340, 685-692.

216

Yuan, Y. V., Bone, D. E. & Carrington, M. F. (2005b). Antioxidant activity of dulse (Palmaria palmata) extract evaluated in vitro. Food Chemistry, 91, 485-494. Yuan, Y. V., Carrington, M. F. & Walsh, N. A. (2005c). Extracts from dulse (Palmaria palmata) are effective antioxidants and inhibitors of cell proliferation in vitro. Food and Chemical Toxicology, 43, 1073-1081. Zambrowicz, A., Pokora, M., Setner, B., Dabrowska, A., Szoltysik, M., Babij, K., Szewczuk, Z., Trziszka, T., Lubec, G. & Chrzanowska, J. (2015). Multifunctional peptides derived from an egg yolk protein hydrolysate: isolation and characterization. Amino Acids, 47, 369-380. Zava, T. T. & Zava, D. T. (2011). Assessment of Japanese iodine intake based on seaweed consumption in Japan: A literature-based analysis. Thyroid Research, 4, 1-7. Zemke-White, W. L. & Ohno, M. (1999). World seaweed utilisation: An end-of-century summary. Journal of Applied Phycology, 11, 369-376. Zeng, H. J., Hu, G. Z., You, J., Yang, R. & Qu, L. B. (2015). Spectroscopic and molecular modeling investigation on the interactions between hyaluronidase and baicalein and chrysin. Process Biochemistry, 50, 738-745. Zhang, L., Li, J. & Zhou, K. (2010). Chelating and radical scavenging activities of soy protein hydrolysates prepared from microbial proteases and their effect on meat lipid peroxidation. Bioresource Technology, 101, 2084-2090. Zhang, M., Mu, T. H. & Sun, M. J. (2014). Purification and identification of antioxidant peptides from sweet potato protein hydrolysates by Alcalase. Journal of Functional Foods, 7, 191-200. Zhao, X., Xue, C. H. & Li, B. F. (2007). Study of antioxidant activities of sulfated polysaccharides from Laminaria japonica. Journal of Applied Phycology, 20, 431-436. Zheng, L., Ren, J., Su, G., Yang, B. & Zhao, M. M. (2013). Comparison of in vitro digestion characteristics and antioxidant activity of hot- and cold-pressed peanut meals. Food Chemistry, 141, 4246-4252. Zhou, A. Y., Robertson, J., Hamid, N., Ma, Q. L. & Lu, J. (2015). Changes in total nitrogen and amino acid composition of New Zealand Undaria pinnatifida with growth, location and plant parts. Food Chemistry, 186, 319-325. Zhou, G., Sun, Y., Xin, H., Zhang, Y., Li, Z. & Xu, Z. (2004). In vivo antitumor and immunomodulation activities of different molecular weight lambda-carrageenans from Chondrus ocellatus. Pharmacological Research, 50, 47-53. Zhu, J. F. & Fu, Q. (2012). Optimization of ultrasound-assisted extraction process of perilla seed meal proteins. Food Science and Biotechnology, 21, 1701-1706. Zhu, Q., Nakagawa, T., Kishikawa, A., Ohnuki, K. & Shimizu, K. (2015). In vitro bioactivities and phytochemical profile of various parts of the strawberry (Fragaria × ananassa var. Amaou). Journal of Functional Foods, 13, 38-49. Zubia, M., Fabre, M. S., Kerjean, V., Lann, K. L., Stiger-Pouvreau, V., Fauchon, M. & Deslandes, E. (2009). Antioxidant and antitumoural activities of some phaeophyta from Brittany coasts. Food Chemistry, 116, 693-701.

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Appendices

Appendix A: Correlation between extraction yield with TPC and TFC

12 U lv a

5 0 0 (A ) 12 D e rb e s ia

12

) O e d o g o n iu m

W 4 0 0

D

1

-

g 0

0 3 0 0

1

E

A G

2 0 0

g

m

(

C

P 1 0 0 T

0 0 1 0 2 0 3 0 4 0 5 0 E x tra c tio n y ie ld (% )

12 U lv a (B ) 12 D e rb e s ia 2 5 0 0 12 O e d o g o n iu m

) 2 0 0 0

W

D

1

- g

1 5 0 0

n

i

t

u

R

g 1 0 0 0

m

(

C F

T 5 0 0

0 0 1 0 2 0 3 0 4 0 5 0 E x tra c tio n y ie ld (% )

Figure A1. Correlation between (A) TPC and extraction yield, (B) TFC and extraction yield.

218

Appendix B: ORAC-Hydrophilic and lipophilic

Figure B1. ORAC-Hydrophilic.

219

Figure B2. ORAC-Lipophilic.

220

Appendix C: GC-MS chromatograms

RT: 0.00 - 52.24 30.93 NL: 100 3.32E9 (A) TIC MS 95 Na_TMS_S 02 90

85

80

75

70

65

60 25.57

55

50

45 RelativeAbundance 40

35

30

25

20 8.51

15 19.81

10 13.59 11.11 23.35 31.40 5 10.39 14.26 30.03 39.44 17.06 18.63 23.14 37.21 13.02 28.73 33.00 34.01 39.93 42.23 43.94 46.74 48.86 50.37 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.24 25.63 NL: 100 8.01E9 TIC MS 95 Na_TMS_S (B) 06 90

85

80

75

70

65

60

55

50

45 RelativeAbundance 40

35 18.64 30

25 17.06 30.93 20 28.74

23.35 15 9.09 14.65 14.24 24.37 39.45 10

5 13.47 15.59 12.24 28.10 37.21 30.03 31.40 21.23 26.13 32.18 36.14 37.44 41.11 42.31 45.80 46.77 50.34 51.78 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

(C)

Figure C1. GC-MS Chromatograms for crude ethanolic extracts of (A) Ulva, (B) Derbesia, and (C) Oedogonium.

221

RT: 0.00 - 52.21 17.05 NL: 100 5.30E8 TIC MS 95 Na_TMS_S (A) 03 90 8.50

85

80

75

70

65 25.57 60 18.63 55

50

45 RelativeAbundance 40 19.81 35

30 11.08 14.07 25 14.37 13.58 14.65 20 8.24 10.33 15.93 15 11.46 24.79 10 9.69 12.15 30.93 5 23.34 36.05 21.23 29.17 25.74 33.01 34.49 37.21 39.45 42.39 44.36 46.26 48.91 50.11 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.25 8.21 NL: 100 1.05E10 TIC MS 95 Na_TMS_S (C) 11 90

85

80

75

70

65

60

55 28.79

50 10.91 45

8.51 RelativeAbundance 40

35

30 23.37

25 11.47 16.84 28.11 20 9.10 24.38 14.25 25.58 15 18.64

10 35.00 15.94 21.25 29.13 38.58 39.44 5 13.57 20.11 27.70 33.00 34.02 31.31 21.53 26.41 38.00 39.68 42.31 45.49 47.26 49.38 51.78 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

Figure C2. GC-MS Chromatograms for crude ethanolic extracts of (A) Ulva and (C) Oedogonium with acid hydrolysis.

222

RT: 0.00 - 52.22 28.80 NL: 100 6.61E9 95 TIC MS Na_TMS_S 90 01

85

80

75 (A)

70

65 23.40 60

55 28.13 50

45 24.37 25.60 37.24

RelativeAbundance 40

35 27.88 30 12.84

25

20 19.88 34.01 15 35.27 38.37 10 20.74 31.52 18.88 25.71 14.54 18.60 33.49 5 38.61 9.33 10.54 11.46 21.53 40.20 41.75 43.40 46.62 50.37 51.39 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.24 (B) 28.89 NL: 24.52 100 1.29E10 95 TIC MS Na_TMS_S 90 05

85

80

75

70

65

60

55

50 23.43

45

RelativeAbundance 40 28.14 35

30 37.24

25

20 25.22 15 32.83 12.83 32.12 10 18.89 19.88 33.66 5 8.16 20.75 30.81 14.53 18.73 25.60 35.90 38.42 9.09 10.69 39.50 41.10 43.11 44.78 47.27 50.35 51.79 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.22 (C) 28.94 NL: 100 1.41E10 TIC MS 95 Na_TMS_S 08 90

85

80

75 24.47 70

65

60 28.18

55

50

45

23.42 RelativeAbundance 40

35

30

25 35.02 20

25.62 15 34.44 32.86 10 27.73 16.36 29.19 38.58 8.16 5 37.21 39.45 12.83 14.40 18.23 19.87 32.09 8.91 12.55 20.12 27.27 36.07 41.78 42.31 42.69 47.29 50.48 51.16 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

Figure C3. GC-MS Chromatograms for purified ethanolic extracts of (A) Ulva, (B) Derbesia and (C) Oedogonium.

223

RT: 0.00 - 52.24 11.49 NL: 100 3.06E9 95 TIC MS Na_TMS_S 90 04

85

80 75 (A) 70

65

60 30.82

55 14.28

50

45 37.23

RelativeAbundance 40 38.37 35

30

25 36.14

20 10.55 12.85 23.36 28.74 15 33.01 25.57 33.80 39.38 10 39.76 20.74 27.86 30.93 41.74 5 8.53 19.88 29.67 41.96 8.24 14.55 18.72 25.44 23.23 27.34 44.48 45.95 50.38 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.22 28.81 NL: 100 7.30E9 TIC MS 95 Na_TMS_S 07 90 24.43 (B)

85

80

75 23.41

70

65

60

55

50

45 RelativeAbundance 40

35

30 28.12 25

20 25.21 15 30.08 27.71 8.16 10 14.23 30.31 19.88 21.54 33.66 12.83 32.26 37.21 18.70 27.19 5 14.36 9.09 10.17 17.43 34.56 37.44 42.32 38.57 42.53 45.09 47.26 50.37 51.78 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.24 28.80 NL: 100 7.89E9 TIC MS 95 Na_TMS_S (C) 10 90

85

80

75

70

65 24.41 60

55

50

45 RelativeAbundance 40

35 35.01 30 23.37 28.12 29.68 25

20 19.89 8.16 15 12.83 34.43 42.32 10 18.71 20.74 38.58 9.09 18.24 27.68 5 16.35 21.53 30.80 25.19 32.84 38.02 12.34 39.11 40.69 42.69 45.50 47.28 50.46 51.14 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

Figure C4. GC-MS chromatograms for purified ethanolic extracts of (A) Ulva, (B) Derbesia and (C) Oedogonium with acid hydrolysis.

224

RT: 0.00 - 52.24 31.25 NL: 100 4.37E9 TIC MS 95 Na_TMS_S 01 90 25.89

85 80 (A) 75

70

65

60 18.93 55

50 11.74

45

RelativeAbundance 17.36 40 20.12 14.55 35 37.53 30 23.68

25

20 8.75 10.80 25.09 29.06 15 28.07 10 33.64 15.93 27.98 29.55 35.56 39.75 9.41 21.03 5 13.91 21.88 40.55 42.95 40.79 43.62 45.12 46.88 50.36 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.22 24.77 NL: 100 29.14 6.42E9 95 TIC MS Na_TMS_S 90 05 (B) 85 23.73

80

75

70 28.53 65 37.56 60

55

50

45

RelativeAbundance 40

35

30 32.43 36.47 25 25.52

20 25.89 32.57

15 29.25 33.15 26.74 33.96 39.80 8.40 22.50 30.64 10 19.20 34.63 14.55 16.65 9.34 38.31 5 8.07 15.96 19.01 21.56 11.39 13.91 41.41 42.65 50.37 43.07 45.13 47.04 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.22 29.24 NL: 100 7.22E9 95 TIC MS Na_TMS_S (C) 90 09 23.78 28.51 85

80 24.75

75

70

65 25.93

60

55

50

45 28.05

RelativeAbundance 40 16.67

35

30 33.17 35.32 25 8.40

20 30.03 15 26.76 34.33 38.89 10 37.52 19.20 20.43 32.52 5 8.54 11.16 14.55 21.44 36.39 42.65 8.08 43.47 44.71 47.73 50.36 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

Figure C5. GC-MS Chromatograms for defatted ethanolic extracts of (A) Ulva, (B) Derbesia and (C) Oedogonium.

225

RT: 0.00 - 52.24 31.26 NL: 100 4.52E9 95 TIC MS 25.90 Na_TMS_S 90 02 (A) 85

80

75 23.70 70 11.75 65

60

55 20.14 14.57 50

45

RelativeAbundance 40

35

30 29.07 25 10.81 20 29.41 33.65 25.10 28.19 37.53 15 9.35 19.17 27.99 17.36 21.56 35.57 10 8.76 38.65 8.48 31.83 12.63 14.94 21.89 26.03 39.75 5 8.09 42.95 43.21 45.15 47.21 50.90 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.23 25.91 NL: 100 9.36 5.14E9 95 TIC MS Na_TMS_S (B) 90 06

85

80

75

70

65

60

55 36.47

50 14.55 45

RelativeAbundance 40 11.16 35 31.24 30 18.94

25 8.40 23.68 37.53 20 15.44 17.36 29.40 15 13.76 33.95 39.80 16.65 25.66 29.07 10 21.56 8.08 12.94 20.79 27.52 5 34.55 37.75 40.39 41.14 43.14 45.55 47.33 50.36 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

RT: 0.00 - 52.24 29.12 NL: 100 5.84E9 95 TIC MS Na_TMS_S (C) 90 10

85

80

75

70

65

60

55

50

45 23.69 RelativeAbundance 40

35 28.43 30.01 8.40 30 24.70 25 9.38 20 35.31 15 11.16 16.66 33.64 10 27.99 38.88 14.55 19.01 33.16 5 12.28 21.88 25.88 35.64 8.08 9.60 20.09 39.75 40.56 44.47 46.39 50.36 51.70 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 Time (min)

Figure C6. GC-MS Chromatograms for defatted ethanolic extracts of (A) Ulva, (B) Derbesia and (C) Oedogonium with acid hydrolysis.

226

C:\Users\...\LC-MS\141126_APCI_Neg_DAppendixC:\Users\...\LC-MS\141126_APCI_Neg_D D: LC-MS26/11/2014 total26/11/2014 2:24:47 PM 2:24:47 ion PM current (TIC) chromatograms

RT:RT:0.000.00 - 34.02 - 34.02 22.86 22.86 NL: NL: 100 100 8.35E7 8.35E7 TIC F: - p APCI TIC F: - p APCI 9090 Q1MS Q1MS [100.000- [100.000- 8080 24.51 24.51 700.000] MS 700.000] MS 20.79 20.79 7070 141126_APCI_141126_APCI_ (A) Neg_D Neg_D 6060 2.522.52 2.782.78 5050 3.73 9.70 3.73 6.50 6.50 9.70 12.30 14.3812.30 14.38 0.870.87 18.27 32.30 32.30 18.27 27.89 27.89

4040 31.18 31.18 RelativeAbundance RelativeAbundance 3030

2020

1010

0 0 0 0 5 5 10 10 15 15 20 20 25 25 30 30 Time (min)Time (min) C:\Users\...\LC-MS\141126_APCI_Neg_D 26/11/2014 2:24:47 PM

141126_APCI_Neg_D141126_APCI_Neg_D #2396-2421 #1566 RT:RT: 13.5620.79-20.96AV: 1AV:NL:3 9.21NL: 4.86E5 RT: 0.00 - 34.02 F:T:- p- APCI p APCI Q1MS SRM [100.000-700.000] ms2 197.000 [122.850-123.350] 22.86 NL: 100 140.94 123.25 100100 8.35E7 TIC F: - p APCI 9090 90 Q1MS [100.000- 8080 24.51 80 700.000] MS 20.79 141126_APCI_ 7070 213.24 70 Neg_D 60 6060 2.52 2.78 5050 50 3.73 6.50 9.70 12.30 14.38 0.87 18.27 32.30 (B) 27.89 4040 31.18

40 123.00 123.10 RelativeAbundance

RelativeAbundance 156.69

RelativeAbundance 30 30 30 233.19 20 187.28 2020 239.21 314.94 10 346.78 493.76 387.10 422.93 666.43 1010 523.09 606.87 0 0 0 122.95 123.00 123.05 123.10 123.15 123.20 123.25 123.30 150 200 250 300 350 400 450 500 550 600 650 700 0 5 10 15 20 m/z 25 30 C:\Users\...\LC-MS\141126_APCI_Neg_D 26/11/2014Time (min) 2:24:47m/z PM

141126_APCI_Neg_DRT: 0.00 - 34.02 #2641 RT: 22.86 AV: 1 NL: 1.70E6 F: - p APCI Q1MS [100.000-700.000] 22.86 NL: 100 277.42 8.35E7 100 TIC F: - p APCI 90 90 Q1MS [100.000- 80 24.51 80 700.000] MS 20.79 70 141126_APCI_ 70 Neg_D 60 2.52 60 2.78 50 50 3.73 6.50 9.70 12.30 14.38 (C) 0.87 18.27 32.30 27.89 31.18

40 RelativeAbundance RelativeAbundance 30 695.61 20 118.90 231.02 141.22 10 193.44 325.16 406.28 439.03 571.80 476.76 535.13 612.33 655.37 0 0 150 5 200 25010 300 15350 400 20 450 50025 550 30 600 650 700 Time (min)m/z

141126_APCI_Neg_D #2789-2814 RT: 24.16-24.34 AV: 3 NL: 2.48E5 F: - p APCI Q1MS [100.000-700.000] 106.44 100 118.83 90

80 141.22 70

60

50 223.39 159.07 40 247.82

RelativeAbundance 30 289.46 (D) 213.45 363.51 409.50 445.19 313.75 487.81 677.35 20 562.14 502.16 621.78 10 631.64

0 150 200 250 300 350 400 450 500 550 600 650 700 m/z

Figure D1. LC-MS chromatogram (A) of Derbesia using atmospheric pressure chemical ionisation with mass range between m/z 120-700. A [M-H]+ corresponded to peaks at retention times 20.79 (B), 22.86 (C), and 24.51 (D) min.

227

C:\Users\...\LC-MS\141126_APCI_Neg_O 26/11/2014 4:46:34 PM C:\Users\...\LC-MS\141126_APCI_Neg_O 26/11/2014 4:46:34 PM

RT: 0.000.00 - 34.02- 34.02 21.91 21.91 NL: NL: 100 100 1.03E8 1.03E8 TIC F: - p APCI TIC F: - p APCI 9090 21.65 22.8621.65 22.86 Q1MS Q1MS [100.000- [100.000- 8080 25.37 26.67 25.37 26.67 21.31 21.31 700.000] MS 700.000] MS 27.54 27.54141126_APCI_ 7070 25.03 25.03 141126_APCI_ Neg_O Neg_O 60 27.88 60 20.78 27.88 20.78 28.23 50 28.23 50 28.75 32.82 28.75 32.82 40 40 5.46 1.74 5.03 5.466.41 10.40 11.43 14.90 18.88 RelativeAbundance 1.74 5.03 6.41 10.40 11.43 14.90 18.88 (A) RelativeAbundance 30 30 20 20 10 10 C:\Users\...\LC-MS\141126_APCI_Neg_O0 26/11/2014 4:46:34 PM 00 5 10 15 20 25 30 0 5 10 Time (min)15 20 25 30 RT: 0.00 - 34.02 Time (min)21.91 NL: 141126_APCI_Neg_O100 #2531 RT: 21.91 AV: 1 NL: 4.85E5 1.03E8 F:141126_APCI_Neg_O- p APCI Q1MS [100.000-700.000] #1 RT: 0.01 AV: 1 NL: 4.12E5 TIC F: - p APCI 90 106.51 21.65 22.86 T: 100- p APCI Q1MS [100.000-700.000] Q1MS 141.01 [100.000- 10080 25.37 26.67 90 21.31 700.000] MS 27.54 141126_APCI_ 7090 25.03 80 Neg_O 6080 27.88 70 20.78 141.01 213.03 28.23 605070 223.18 28.75 32.82 50 4060 5.46 1.74 5.03 223.536.41 10.40 11.43 14.90 18.88 RelativeAbundance 284.98 403050 159.63178.88 299.12 RelativeAbundance 251.39 302040 349.02 402.50 512.52 233.12 415.79 557.45 593.99 484.18 631.36 (B) RelativeAbundance 20 1030 681.55 315.64 403.13 10 263.15 200 370.93 C:\Users\...\LC-MS\141126_APCI_Neg_O0 0 5 10 1526/11/2014 4:46:3420 PM 25 30 10 150 200 250 300 350Time (min)400 450 420.76500 472.70550 522.46600 650 700 635.63 673.22 548.78 RT: 0.000 - 34.02 m/z 21.91 NL: 141126_APCI_Neg_O150 #2641 RT:20022.86 AV:2501 NL: 1.35E6300 350 400 450 500 550 600 650 700 100 1.03E8 F: - p APCI Q1MS [100.000-700.000] m/z TIC F: - p APCI 90 277.21 21.65 22.86 100 Q1MS [100.000- 80 25.37 26.67 90 21.31 700.000] MS 27.54 141126_APCI_ 8070 25.03 Neg_O 7060 27.88 20.78 28.23 6050 28.75 32.82 50 40 5.46 1.74 5.03 6.41 10.40 11.43 14.90 18.88 RelativeAbundance 40 (C) 30 694.92

RelativeAbundance 30 20 106.16 20 244.39 10 184.34 299.82 343.92 370.86 534.99 623.53 10 434.76 462.13 560.11 0 687.85 0 0 5 10 15 20 25 30 150 200 250 300 350Time (min)400 450 500 550 600 650 700 m/z 141126_APCI_Neg_O #3091 RT: 26.76 AV: 1 NL: 3.30E5 F: - p APCI Q1MS [100.000-700.000] 118.97 100

90

80 140.94 70 211.63

60 201.00 334.26 50 319.91 158.86 223.53 278.05 364.77 427.48 40 249.71 547.52 (D) RelativeAbundance 30 518.26 390.04 453.59 633.81 695.96 20 577.19

10 666.92 0 150 200 250 300 350 400 450 500 550 600 650 700 m/z Figure D2. LCMS chromatogram (A) of Oedogonium using atmospheric pressure chemical ionisation with mass range between m/z 120 -700. A [M-H]+ corresponded to peaks at retention time 21.91 (B), 22.86 (C), and 26.67 (D) min.

228

Appendix E: NMR analysis

(A)

(B)

(C)

(D)

(E)

Ulva

Figure E1. NMR standard (A) syringic acid, (B) caffeic acid, (C) ferulic acid, (D) protocatechuic acid, and (E) p-hydroxybenzoic acid spiked in Ulva crude extract.

229

(A)

(B)

(C)

(D)

(E)

Derbesia

Figure E2. NMR standard (A) p-hydroxybenzoic acid, (B) protocatechuic acid, (C) formate, (D) ferulic acid, and (E) caffeic acid spiked in Derbesia crude extract.

230

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

Oedogonium

Figure E3. NMR standard (A) p-hydroxybenzoic acid, (B) protocatechuic acid, (C) ferulic acid, (D) caffeic acid, (E) tyrosine, (F) valine, (G) proline, and (H) formate spiked in Oedogonium crude extract. 231

Appendix F: NMR SDBS simulated spectra with standard match

(A)

(B)

(C)

Figure F1. The NMR Spectral Database (SDBS) for organic compounds was used to compare the simulated spectra with those of the algae extracts (A) Ulva, (B) Derbesia, and (C) Oedogonium. Black line indicates the sample spectrum. Red line indicates simulated spectrum from library.

232

Appendix G: HPLC-PDA chromatograms for carotenoid identification

(A)

(B) mAU(x10)

7.5

5.0

2.5

0.0 15.0 20.0 25.0 min

Figure G1. HPLC profile of carotenoids isolated from Derbesia with (A) extraction procedure 1 and HPLC method 1, (B) extraction procedure 2 and HPLC method 1. Extraction procedure 1 used hexane/acetone/ethanol as solvent, while extraction procedure 2 used dichloromethane/water/methanol. For HPLC method 1, the mobile phase was methanol, MTBE and water, while for method 2, it was acetonitrile/methanol/water and methanol/ethyl acetate.

233

(C)

mAU(x100) (D) 2.0 1.5

1.0

0.5

0.0

20.0 25.0 min

Figure G2. HPLC profile of carotenoids isolated from Derbesia with (C) extraction procedure 1 and HPLC method 2, and (D) extraction procedure 2 and HPLC method 2. Extraction procedure 1 used hexane/acetone/ethanol as solvent, while extraction procedure 2 used dichloromethane/water/methanol. For HPLC method 1, the mobile phase was methanol, MTBE and water, while for method 2, it was acetonitrile/methanol/water and methanol/ethyl acetate.

234

(A)

mAU(x10) (B) 7.5

5.0

2.5

0.0

20.0 25.0 30.0 min

Figure G3. HPLC profile of carotenoids isolated from Oedogonium with (A) extraction procedure 1 and HPLC method 1, (B) extraction procedure 2 and HPLC method 1. Extraction procedure 1 used hexane/acetone/ethanol as solvent, while extraction procedure 2 used dichloromethane/water/methanol. For HPLC method 1, the mobile phase was methanol, MTBE and water, while for method 2, it was acetonitrile/methanol/water and methanol/ethyl acetate.

235

(C)

mAU(x100) (D) 1.5

1.0

0.5

0.0

20.0 25.0 min

Figure G4. HPLC profile of carotenoids isolated from Oedogonium with (C) extraction procedure 1 and HPLC method 2, and (D) extraction procedure 2 and HPLC method 2. Extraction procedure 1 used hexane/acetone/ethanol as solvent, while extraction procedure 2 used dichloromethane/water/methanol. For HPLC method 1, the mobile phase was methanol, MTBE and water, while for method 2, it was acetonitrile/methanol/water and methanol/ethyl acetate.

236

Appendix H: HPLC-PDA chromatograms, UV-Vis absorption and mass spectra of carotenoid extracts of three algal species *

6

2’ 9 3 5

0. 5. 10. 15. 20. 25. 30. 35. 40. 45.0 0 0 0 0 0 0 0 0 0 min Figure H1. HPLC-PDA chromatogram of Ulva carotenoid extract at 450 nm. Peak denotation: peak 2, neoxanthin; peak 3, 9’-cis-neoxanthin; peak 5, violaxanthin; peak 6, lutein; and peak 9, β-carotene. Peak 2’, a mixture of neoxanthin and some unknown pigments. *, chlorophylls.

Peak 2’ Peak 3 Peak 5 Peak 6 Peak 9

Figure H2. Mass and UV-Vis absorption spectra for peaks corresponding to HPLC chromatogram of Ulva.

237

*

7 8’ 1 6’ 2’ 3

0. 5. 10. 15. 20. 25. 30. 35. 40. 45.0 0 0 0 0 0 0 0 0 0 min Figure H3. HPLC-PDA chromatograms of Derbesia carotenoid extracts at 450 nm. Peak denotation: peak 1, siphonaxanthin; peak 2, neoxanthin; peak 3, 9’-cis-Neoxanthin; peak 6, lutein; peak 7, siphonein, and peak 8, α-carotene; peak 2’, a mixture of neoxanthin and some unknown pigments; Peak 6’, mixture of lutein and some unknown pigments; peak 8’, mixture of α-carotene and chlorophylls. *, chlorophylls.

Peak 2 Peak 3 Peak 6 Peak 7 Peak 8

mAU mAU 19.07/ 1.00/bgnd(Ch1) 29.49/ 1.00/bgnd(Ch1)

90 458 40 444

80 416 35 472

70 429 461 30 60

50 25

40 20 30 15 20

10

334

310 320

10 349 341

0 5 526 300 350 400 450 500 550 nm 0 300 350 400 450 500 550 nm

Figure H4. Mass and UV-Vis absorption spectra for peaks corresponding to HPLC chromatogram of Derbesia.

238

*

6 4 5 3 9 2 8

0. 5. 10. 15. 20. 25. 30. 35. 40. 45.0 0 0 0 0 0 0 0 0 0 min Figure H5. HPLC-PDA chromatograms of carotenoid extracts of Oedogonium at 450 nm. Peak denotation: peak 2, neoxanthin; peak 3, 9’-cis-Neoxanthin; peak 4, loroxanthin; peak 5, violaxanthin; peak 6, lutein; peak 8, α-carotene, and peak 9, β-carotene. *, chlorophylls.

Peak 2 Peak 3 Peak 4 Peak 5

mAU mAU mAU mAU 11.53/ 1.00/bgnd(Ch1) 12.19/ 1.00/bgnd(Ch1) 70 12.49/ 1.00/bgnd(Ch1) 13.61/ 1.00/bgnd(Ch1)

80

438

444 435

439 50 468 30 464 60

45 70

471 467

40 461

25 50 60 415

456 35 412

50 424 456 420 40 20 30 451 40 25 30 15 20 30

15 20 10 20 10

10 10

328

343 319

5 5 319

339

303

326

339

344 302 0 0 565 0 0 565 300 350 400 450 500 550 nm 300 350 400 450 500 550 nm 300 350 400 450 500 550 nm 300 350 400 450 500 550 nm Peak 6 Peak 8 Peak 9

mAU mAU mAU 17.48/ 1.00/bgnd(Ch1) 29.43/ 1.00/bgnd(Ch1) 35 29.77/ 1.00/bgnd(Ch1)

12.5

444 450 110 444

30 472

100 472

475 467 10.0

90 461

460 25 80

70 7.5 20 60

50 15 5.0 40 10 30 2.5

20 5

331

339

355

353

308 301

10 331

345 303 0.0 0 560 0 565

-10 300 350 400 450 500 550 nm 300 350 400 450 500 550 nm 300 350 400 450 500 550 nm Figure H6. Mass and UV-Vis absorption spectra for peaks corresponding to HPLC chromatogram of Oedogonium.

239

Appendix I: Chromatogram of carotenoid standard, with UV spectral and mass spectra

mAU 450nm,4nm (1.00) 225

200 4 5

175

150

125 3 100

75 1 6 50 2 25

0

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min Figure I1. HPLC-PDA chromatograms of carotenoid standards at 450 nm. Peak denotation: peak 1, siphonaxanthin; peak 2, neoxanthin; peak 3, violaxanthin; peak 4, lutein; peak 5, siphonein, and peak 6, β-carotene.

Inten.(x10,000,000) Peak 1 Inten.(x10,000,000) Peak 2 Inten.(x10,000,000)Peak 3 1.5 583.4 583.4 601.4 2.0 1.0 1.0 1.0 0.5 509.3 623.4 0.0 0.0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z 300 400 500 600 0.0 700 800 900 1000 1100 1200 1300 1400 m/z 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z mAU mAU mAU 90 12.37/ 1.00 13.77/ 1.00

9.26/ 1.00 120 175

438

435 452

80 110 468 464 150 100 70 90 125

60 80

415

412

456 423 70 420 100 50 451 60 75 40 50 40 30 50 30 20 20 25

10 318

319 339

10 334

339

312

318 347 0 0 0 -10 300 350 400 450 500 550 nm 300 350 400 450 500 550 nm 300 350 400 450 500 550 nm Inten.(x10,000,000)Peak 4 Inten.(x10,000,000) Peak 5 Inten.(x1,000,000)Peak 6 551.4 2.0 565.4 537.4 2.0 5.0 1.0 1.0 2.5 763.5 0.0 0.0 300 400 500 600 700 800 900 1000 1100 566.5 1200 1300 1400 m/z 300 400 500 600 700 800 900 1000 1100 0.0 1200 1300 1400 m/z 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z mAU mAU mAU 17.55/ 1.00 18.86/ 1.00 100 29.80/ 1.00

250

458 444 225 90 450

225

472 474 200 80 468 200

460 175 70 175 150 60 150 125 50 125 100 40 100

75 75 30

50 50 20

337

334

359 312 319 10

25 25 350

307

330

346 303

0 569 0 0

300 350 400 450 500 550 nm 300 350 400 450 500 550 nm 300 350 400 450 500 550 nm Figure I2. Mass and UV-Vis absorption spectra for peaks corresponding to HPLC chromatogram of carotenoid standards. 240

Appendix J: Chemical structures of carotenoids present in Ulva, Derbesia and Oedogonium

Carotenoids Chemical structure α-carotene

β-carotene

Lutein

Siphonaxanthin

Siphonein

Violaxanthin

Neoxanthin

Loroxanthin

Figure J1. Chemical structures of carotenoids present in Ulva, Derbesia and Oedogonium.

241

Appendix K: Ultra-sonication temperature and frequency control.

Black line: Frequency control

Red line: Temperature control

Figure K1. Ultra-sonication temperature and frequency control.

242

Appendix L: SDS-PAGE separation of protein hydrolysis

M 1 2 3

Figure L1. SDS-PAGE separation of Ulva, Derbesia and Oedogonium protein hydrolysis. Line M indicates protein mass markers. Line 1, Derbesia protein hydrolysis. Line 2, Oedogonium protein hydrolysis. Line 3, Ulva protein hydrolysis. Protein molecular mass standards (in kDa) are indicated on the left

243