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. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). ………………………………………………… …………………………..……………… ……….……………………...…….… Signature Witness Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: 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