MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY PATRICK A BLAMO, JR EVALUATION OF TYROSINASE AND ANTIOXIDANT ACTIVITIES OF BROMINATED COMPOUNDS FROM VIETNAMESE RED SEAWEEDS MASTER THESIS KHANH HOA - 2020 MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY PATRICK A BLAMO, JR EVALUATION OF TYROSINASE AND ANTIOXIDANT ACTIVITIES OF BROMINATED COMPOUNDS FROM VIETNAMESE RED SEAWEEDS MASTER THESIS Food Technology Major: Topic allocation Decision Decision on establishing the Committee: September 19, 2020 Defense date: Supervisors: Dr The Han Nguyen Dr Hong Ngoc Thuy Pham Chairman: Faculty of Graduate Studies: KHANH HOA - 2020 UNDERTAKING I declare that the thesis entitled: ―Evaluation of Tyrosinase and Antioxidant Activities of Brominated Compounds from Vietnamese Red Seaweeds‖ is my own work The work has not been presented elsewhere for assessment until the time this thesis is submitted 09 2020 Patrick A Blamo, Jr (Author) iii ACKNOWLEDGMENT My health, strength and the opportunity to be part of this amazing family, I owe it to God I would like to appreciate my professors and the entire staff in the Department of Food Technology, Nha Trang University Your sacrifices, knowledge impacted and the amicable environment created to study and successfully complete this master's thesis, cannot go unappreciated My profound gratitude to Dr The Han Nguyen and Dr Hong Ngoc Thuy Pham, supervisor and co-supervisor, respectively Your guidance, support, patience and continuous courage fueled my enthusiasm Your vast knowledge and experience, created a confident roadmap that has led to the success of this thesis Finally, and importantly, my family: my mother and partner whose prayers never fainted, my uncle whose support never quenched, you are all the reason this is possible…thank you! 09 2020 Patrick A Blamo, Jr iv CONTENT UNDERTAKING iii ACKNOWLEDGMENT iv CONTENT .v LIST OF SYMBOLS ix LIST OF ABBREVIATIONS x LIST OF TABLES xi LIST OF FIGURES xii ABSTRACT xiv CHAPTER INTRODUCTION 1.1 Introduction 1.2 Background 1.3 Statement of Problem 1.4 Justification of the Study .6 1.5 Research Question 1.6 Hypothesis .7 1.7 Objective 1.7.1 General Objective 1.7.2 Specific Objective 1.8 Scope of Study .8 1.9 Limitation of the study CHAPTER LITERATURE REVIEW .10 2.1 Introduction 10 2.2 Scope 10 2.3 Overview of Seaweeds 11 2.4 Significance of Investigating Seaweeds .12 2.5 Biological Potential of Seaweeds (Laurencia) 13 v 2.5.1 Total Phenolic Profile 13 2.5.2 Tyrosinase Inhibitory Property 13 2.5.2.1 Influence of Enzymatic discoloration .13 2.5.2.2 Role of Tyrosinase in Melanin Biosynthesis 14 2.5.3 Oxidative Damages and Control 16 2.5.3.1 Effect of Reactive Oxygen Species 16 2.5.3.2 Antioxidant 17 2.5.3.3 Antioxidant Properties 18 2.5.4 Brominated Compounds 19 2.5.4.1 Definition .19 2.5.4.2 Brominated compounds from Laurencia .19 2.6 Health Effect of Synthetic Compound 20 2.7 Effect of extraction methods and parameters on the biological activity of seaweeds 21 2.7.1 Extraction method 21 2.7.2 Extraction solvent .22 2.7.3 Extraction time .23 2.7.4 Extraction temperature 23 2.7.5 Solute-solvent (volume/mass) ratio .24 2.8 Mechanism of Ultrasound-Assisted Extraction 24 2.9 Response Surface Methodology (RSM) .26 2.10 Modern Application of Seaweeds .26 CHAPTER MATERIALS AND METHODS 29 3.1 Materials .29 3.1.1 Seaweed samples 29 3.1.2 Chemicals and Reagent 29 3.2 Methods 29 3.2.1 Experimental Procedure .29 vi 3.2.2 Screening test to select the starting sample 30 3.2.3 Single factor test 31 3.2.4 Experimental optimization Design .31 3.2.5 Ultrasound Assisted Extraction (UAE) 31 3.2.6 Fractionation, Isolation and characterization of compounds from L intermedia Yamada 32 3.2.6.1 General experimental procedures 32 3.2.6.2 Extraction, fractionation and isolation 32 3.2.6.3 Characterization of isolated compounds 33 3.2.7 Determination of total phenolic content, antioxidant and tyrosinase inhibitory activities .34 3.2.7.1 Total phenolic content (TPC) 34 3.2.7.2 DPPH radical scavenging assay (DRSC) 34 3.2.7.3 Ferric reducing antioxidant power (FRAP) 34 3.2.7.4 Tyrosinase Inhibition Assay (TIA) 35 3.2.8 Statistical analysis 35 CHAPTER RESULTS AND DISCUSSION 36 4.1 Screening 36 4.2 Single factor optimization 36 4.3 Optimization .40 4.3.1 Fitting the models 40 4.3.2 Influence of extraction parameters on TPC and antioxidant capacity 45 4.3.3 Optimization and validation of the models 53 4.4 Correlation between the total phenolic content and antioxidant activities 54 4.5.Total phenolic content and biological activities of the fractions and isolated compounds 55 4.6 Purification and structural identification of aplysistatin and palisadin b 56 CHAPTER CONCLUSION recommendation AND FUTURE PERSPECTIVE .63 vii 5.1 Conclusion 63 5.2 Recommendation 63 5.3 Future Perspective 64 REFERENCE 65 APPENDICES I Appendices A I Appendices B II viii LIST OF SYMBOLS % : percent g : gram g : milligram GAE : Gallic Acid Equivalent AAE : Ascorbic Acid Equivalent m : mass M : molar nm : Nanometer °C : degree Celsius TE : Trolox Equivalent ix LIST OF ABBREVIATIONS ANOVA : Analyzes of Variance BBD : Box-Behnken Design DPPH : 1,1-Diphenyl-2-picrylhydrazyl DRSC : DPPH Radical scavenging capacity EtOAc : Ethyl Acetate EtOH : Ethanol FRAP : Ferric Reducing Antioxidant Power HCl : Hydrochloric Acid HPLC : High-Performance Liquid Chromatography MeOH : Methanol Na2CO3 : Sodium carbonate NMR : Nuclear Magnetic Resonance R2 : R-square (determined coefficient) RMSE : Root Mean Square Error ROS : Reactive Oxygen Species RSM : Response Surface Methodology TIA : Tyrosinase Inhibitory Activity TLC : Thin layer chromatography TPC : Total Phenolic Content TPTZ : 2,4,6-tris(2-pyridyl)-s-triazine UAE : Ultrasound-assisted extraction UV : Ultraviolet Radiation x 98 Mandrekar VK, Gawas UB, Majik MS Brominated molecules from marine algae and their pharmacological importance Studies in Natural Products Chemistry 2018 Jan;6:461–490 DOI: 10.1016/B978-0-444-64183-0.00013-0 99 https://pubchem.ncbi.nlm.nih.gov/compound/laurinterol#section=3D-Conformer 100 It N, Hirose M Antioxidants-carcinogenic and chemopreventive properties Advances in Cancer Research 1989;53:247-302 DOI: 10.1016/s0065- 230x(08)60283-3 101 Raja RR, Sreenivasulu M, Reddy SR, Indra CK, Reddy SK, Royal PT, et al Synthetic Cosmetics-An Overview RA Journal of Applied Research 2016 Sept;2(9) https://journals.indexcopernicus.com/api/file/viewByFileId/135981.pdf 102 Wu H Effect of different light qualities on growth, pigment content, chlorophyll fluorescence, and antioxidant enzyme activity in the red alga Pyropia haitanensis (Bangiales, Rhodophyta) BioMed Research International 2016;2016:1–8 https://doi.org/10.1155/2016/7383918 103 Xu P, Tan H, Jin W, Li Y, Santhoshkumar C, Li P, et al Antioxidative and antimicrobial activities of intertidal seaweeds and possible effects of abiotic factors on these bioactivities Journal of Oceanology Limnology 2018 Nov;36(6):2243–2256 https://doi.org/10.1007/s00343-019-7046-z 104 Michiels JA, Kevers C, Pincemail J, Defraigne JO, Dommes J Extraction conditions can greatly influence antioxidant capacity assays in plant food matrices Food Chemistry 2012 Feb;130(4):986–993 https://doi.org/10.1016/j.foodchem.2011.07.117 105 Aboshora W, Lianfu Z, Dahir M, Qingran M, Qingrui S, Jing L, et al Effect of Extraction Method and Solvent Power on Polyphenol and Flavonoid Levels in Hyphaene Thebaica L Mart (Arecaceae) (Doum) Fruit, and its antioxidant and antibacterial activities Tropical Journal of Pharmaceutical Research 2015 Jan;13(12):2057 DOI: 10.4314/tjpr.v13i12.16 77 106 Do QD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, Soetaredjo FE, Ismadji S, et al Effect of extraction solvent on total phenol content, total flavonoid content, and antioxidant activity of Limnophila aromatica Journal of Food and Drug Analysis 2014 Sep;22(3):296–302 https://doi.org/10.1016/j.jfda.2013.11.001 107 Mokrani A, Madani K Effect of solvent, time and temperature on the extraction of phenolic compounds and antioxidant capacity of peach (Prunus persica L.) fruit Separation and Purification Technology 2016 Apr;162:68–76 https://doi.org/10.1016/j.seppur.2016.01.043 108 Pagni R Solvents and Solvent Effects in Organic Chemistry, Third Edition (Christian Reichardt) Journal of Chemical Education 2005 Mar;82(3):382 DOI: 10.1021/ed082p382.2 109 Hansen CM Hansen Solubility Parameters: A user‘s handbook, second edition Hoboken: Taylor and Francis 2007;2(2):544–545 Doi: 10.1201/9781420006834 110 O‘Sullivan AM, O‘Callaghan YC, O‘Grady MN, Hayes M, Kerry JP, O‘Brien NM The effect of solvents on the antioxidant activity in Caco-2 cells of Irish brown seaweed extracts prepared using accelerated solvent extraction Journal of Functional Foods 2013 Apr;5(2):940–948 https://doi.org/10.1016/j.jff.2013.02.007 111 Hwang E-S, Nhuan DT Effects of extraction and processing methods on antioxidant compound contents and radical scavenging activities of laver (Porphyra tenera) Preventive Nutrition and Food Science 2014 Mar 31;19(1):40–48 doi: 10.3746/pnf.2014.19.1.040 112 Gope PS, Dutta AK, Makhnoon S, Banik S, Siddiquee MA, Kabir Y Effect of solvent and extraction time on the antioxidant properties of Citrus macroptera 2014 Apr;6(2):28–35 78 113 Navarro AE, Portales RF, Sun-Kou MR, Llanos BP Effect of pH on phenol biosorption by marine seaweeds Journal of Hazardous Materials 2008 Aug;156(1–3):405–411 DOI: 10.1016/j.jhazmat.2007.12.039 114 Navarro A, Hernandez-Vega A, Masud M, Roberson L, Diaz-Vázquez L Bioremoval of phenol from aqueous solutions using native caribbean seaweed Environments 2016 Dec;4(1):1 https://doi.org/10.3390/environments4010001 115 Gaffney JS, Marley NA Chemistry of environmental systems: fundamental principles and analytical methods First edition Hoboken, NJ: Wiley; 2019;1 ISBN-10: 1119313406 116 Khaw K-Y, Parat M-O, Shaw PN, Falconer JR Solvent supercritical fluid technologies to extract bioactive compounds from natural sources: A Review Molecules 2017 Jul;22(7):1186 doi: 10.3390/molecules22071186 117 Roohinejad S, Nikmaram N, Brahim M, Koubaa M, Khelfa A, Greiner R Potential of novel technologies for aqueous extraction of plant bioactives In: Water extraction of bioactive compounds Elsevier 2017 Nov:399–419 https://doi.org/10.1016/B978-0-12-809380-1.00016-4 118 Wang L, Weller CL Recent advances in extraction of nutraceuticals from plants Trends in Food Science & Technology 2006 Jun;17(6):300–312 https://doi.org/10.1016/j.tifs.2005.12.004 119 Reedijk J Reference module in chemistry, molecular sciences and chemical engineering Elsevier; 2014 http://www.sciencedirect.com/science/referenceworks/9780124095472 120 Pacheco-Fernández I, González-Hernández P, Rocío-Bautista P, TrujilloRodríguez MJ, Pino V Main uses of microwaves and ultrasounds in analytical extraction schemes: An overview Analytical Separation Science 2015 Nov;5:1469–1502 DOI: 10.1002/9783527678129.assep054 79 121 Pacheco-Fernández I, Pino V Extraction With ionic liquids-organic compounds In: Liquid-phase extraction Elsevier 2020;2020:499–537 DOI: 10.1016/B9780-12-816911-7.00017-7 122 Panzella L, Moccia F, Nasti R, Marzorati S, Verotta L, Napolitano A Bioactive phenolic compounds from agri-food wastes: An update on green and sustainable extraction methodologies Frontiers in Nutrition 2020 May;7:60 https://doi.org/10.3389/fnut.2020.00060 123 Box GEP, Wilson KB On the experimental attainment of optimum conditions journal of the royal statistical society: Series B (Methodological) 1951 Jan;13(1):1–45 DOI: 10.2307/2983966 124 Montgomery DC, Runger GC Applied statistics and probability for engineers 3rd ed New York: Wiley; 2003;706 125 Andrade PB, Barbosa M, Matos RP, Lopes G, Vinholes J, Mouga T, et al Valuable compounds in macroalgae extracts Food Chemistry 2013 Jun;138(2– 3):1819–1828 https://doi.org/10.1016/j.foodchem.2012.11.081 126 Gujar A Cui H, Ji C, Kubar S, & Li R Development Development, production and market value of microalgae products Applied Microbiology 2019 Oct;5(2): DOI: 10.35248/2471-9315.19.5.162 127 Jun J-Y, Jung M-J, Jeong I-H, Yamazaki K, Kawai Y, Kim B-M Antimicrobial and antibiofilm activities of sulfated polysaccharides from marine algae against dental plaque bacteria Marine Drugs 2018 Aug;16(9):301 DOI: 10.3390/md16090301 128 Gandhi AD, Vizhi DK, Lavanya K, Kalpana VN, Devi Rajeswari V, Babujanarthanam R In vitro anti- biofilm and anti-bacterial activity of Sesbania grandiflora extract against Staphylococcus aureus Biochemistry and Biophysics Reports 2017 Dec;12:193–197 doi: 10.1016/j.bbrep.2017.10.004 80 129 Kim JK, Kottuparambil S, Moh SH, Lee TK, Kim Y-J, Rhee J-S, et al Potential applications of nuisance microalgae blooms Journal of Applied Phycology 2015 Jun;27(3):1223–1234 https://doi.org/10.1007/s10811-014-0410-7 130 Baghel RS, Trivedi N, Gupta V, Neori A, Reddy CRK, Lali A, et al Biorefining of marine macroalgal biomass for production of biofuel and commodity chemicals Green Chemistry 2015;17(4):2436–2443 https://doi.org/10.1039/C4GC02532F 131 Milledge JJ, Nielsen BV, Bailey D High-value products from macroalgae: the potential uses of the invasive brown seaweed, Sargassum muticum Rev Environ Sci Biotechnol 2016 Mar;15(1):67–88 https://doi.org/10.1007/s11157-015-9381-7 132 Bwapwa JK, Jaiyeola AT, Chetty R Bioremediation of acid mine drainage using algae strains: A review South African Journal of Chemical Engineering 2017 Dec;24:62–70 https://doi.org/10.1016/j.sajce.2017.06.005 133 Zeraatkar AK, Ahmadzadeh H, Talebi AF, Moheimani NR, McHenry MP Potential use of algae for heavy metal bioremediation, a critical review Journal of Environmental Management 2016 Oct;181:817–831 https://doi.org/10.1016/j.jenvman.2016.06.059 134 De Almeida CLF, De S Falcão H, De M Lima GR, De A Montenegro C, Lira NS, De Athayde-Filho PF, et al Bioactivities from Marine Algae of the Genus Gracilaria International Journal of Molecular Sciences 2011 Jul;12(7):4550– 4573 doi: 10.3390/ijms12074550 135 Morton DW A-KS Cosmeceuticals derived from bioactive substances found in marine algae Oceanography 2013;01(02): DOI: 10.4172/2332-2632.1000106 136 Kim S-K, editor Marine cosmeceuticals: trends and prospects Boca Raton: CRC Press 2012;412 https://doi.org/10.1201/b10120 81 137 Uysal O, Uysal FO, Ekinci K Evaluation of microalgae as microbial fertilizer EJSD 2015 Jun;4(2):77-82 https://doi.org/10.14207/ejsd.2015.v4n2p77 138 Haq SH, Al-Ruwaished G, Al-Mutlaq MA, Naji SA, Al-Mogren M, Al-Rashed S, et al Antioxidant, anticancer activity and phytochemical analysis of green algae, chaetomorpha collected from the Arabian Gulf Scientific Reports 2019 Dec;(1):18906 https://doi.org/10.1038/s41598-019-55309-1 139 Sridhar, K R., & Vidyavathi, N (1991) Antimicrobial Activity of Seaweeds Acta Hydrochimica et Hydrobiologica, 1991;19(5):455–496 doi:10.1002/aheh.19910190502 140 Martínez Andrade K, Lauritano C, Romano G, Ianora A Marine Microalgae with anti-cancer properties Marine Drugs 2018 May;16(5):165 doi: 10.3390/md16050165 141 Liu X, Wang S, Cao S, He X, Qin L, He M, et al Structural characteristics and anticoagulant property in vitro and in vivo of a seaweed sulfated Rhamnan Marine Drugs 2018 Jul;16(7):243 doi: 10.3390/md16070243 142 Magalhaes KD, Costa LS, Fidelis GP, Oliveira RM, Nobre LTDB, DantasSantos N, et al Anticoagulant, Antioxidant and Antitumor Activities of Heterofucans from the Seaweed Dictyopteris delicatula IJMS 2011 May;12(5):3352–3365 143 Kim S-K, Wijesekara I Anticoagulant effect of marine algae In: Advances in food and nutrition research Elsevier 2011;(64):235–244 DOI: 10.1016/B9780-12-387669-0.00018-1 144 Lin H-TV, Lu W-J, Tsai G-J, Chou C-T, Hsiao H-I, Hwang P-A Enhanced antiinflammatory activity of brown seaweed Laminaria japonica by fermentation using Bacillus subtilis Process Biochemistry 2016 Dec;51(12):1945–1953 https://doi.org/10.1016/j.procbio.2016.08.024 82 145 García Delgado N, Frías Vázquez AI, Cabrera Sánchez H, Soto del Valle RM, Sierra Gómez Y, Suárez Alfonso AM Anti-inflammatory and antinociceptive activities of methanolic extract from red seaweed Dichotomaria obtusata Brazilian Journal of Pharmaceutical Sciences 2013 Mar;49(1):65–74 https://doi.org/10.1590/S1984-82502013000100008 146 Arumugam, N., Chelliapan, S., Kamyab, H., Thirugnana, S., Othman, N., & Nasri, N (2018) Treatment of Wastewater Using Seaweed: A Review International Journal of Environmental Research and Public Health, 15(12), 2851 https://doi.org/10.3390/ijerph15122851 147 Bourgougnon N, Bedoux G, Sangiardi A, Stiger V Las algas: potencial nutritivo y aplicaciones cosméticas 2016 Aug;15(1):512-519 148 Lee K-Y, Jeong M-R, Choi S-M, Na S-S, Cha J-D Synergistic effect of fucoidan with antibiotics against oral pathogenic bacteria Archives of Oral Biology 2013 May;58(5):482–492 DOI: 10.1016/j.archoralbio.2012.11.002 149 Marsh PD Dental plaque as a biofilm and a microbial community – implications for health and disease BMC Oral Health 2006 Jun;6(S1):S14 DOI: 10.1186/1472-6831-6-S1-S14 150 Gomez-Zavaglia A, Prieto Lage MA, Jimenez-Lopez C, Mejuto JC, SimalGandara J The potential of seaweeds as a source of functional ingredients of prebiotic and antioxidant value Antioxidants 2019 Sep;8(9):406 doi: 10.3390/antiox8090406 151 Holdt SL, Kraan S Bioactive compounds in seaweed: functional food applications and legislation Journal of Applied Phycology 2011 Jun;23(3):543–597 https://doi.org/10.1007/s10811-010-9632-5 152 Kim JK, Kraemer GP, Yarish C Field scale evaluation of seaweed aquaculture as a nutrient bioextraction strategy in Long Island Sound and the Bronx River Estuary Aquaculture 2014 Sep;433:148–156 DOI: 10.1016/j.aquaculture.2014.05.034 83 153 Ibañez E, Cifuentes A Benefits of using algae as natural sources of functional ingredients: Algae as sources of functional ingredients Journal of Science Food and Agriculture 2013 Mar;93(4):703–709 DOI: 10.1002/jsfa.6023 154 Mtolera MSP, Semesi AK Antimicrobial activity of extracts from six green algae from Tanzania 1996 Jan;8:211-217 155 Senguttuvan J, Paulsamy S, Karthika K Phytochemical analysis and evaluation of leaf and root parts of the medicinal herb, Hypochaeris radicata L for in vitro antioxidant activities Asian Pacific Journal of Tropical Biomedicine 2014 May;4(35):9–67 doi: 10.12980/APJTB.4.2014C1030 156 Yuan YV, Walsh NA Antioxidant and antiproliferative activities of extracts from a variety of edible seaweeds Food and Chemical Toxicology 2006 Jul;44(7):1144–1150 https://doi.org/10.1016/j.fct.2006.02.002 157 Pham H, Nguyen V, Vuong Q, Bowyer M, Scarlett C Effect of extraction solvents and drying methods on the physicochemical and antioxidant properties of Helicteres hirsuta Lour Leaves Technologies 2015 Dec 7;3(4):285–301 https://doi.org/10.3390/technologies3040285 158 Pham HNT, Tang Nguyen V, Van Vuong Q, Bowyer MC, Scarlett CJ Bioactive compound yield and antioxidant capacity of Helicteres hirsuta Lour Stem as affected by various solvents and drying methods: Impact of solvents and drying on H hirsuta Stem Journal of Food Processing and Preservation 2017 Feb;41(1):e12879 https://doi.org/10.1111/jfpp.12879 159 Luisi G, Stefanucci A, Zengin G, Dimmito M, Mollica A Anti-oxidant and tyrosinase inhibitory in vitro activity of amino acids and small peptides: New hints for the multifaceted treatment of neurologic and metabolic disfunctions Antioxidants 2018 Dec;8(1):7 doi: 10.3390/antiox8010007 160 Wang F, Guo XY, Zhang DN, Wu Y, Wu T, Chen ZG Ultrasound-assisted extraction and purification of taurine from the red algae Porphyra yezoensis 84 Ultrasonics Sonochemistry 2015 May;24:36–42 https://doi.org/10.1016/j.ultsonch.2014.12.009 161 Santos JS, Deolindo CTP, Esmerino LA, Genovese MI, Fujita A, Marques MB, et al Effects of time and extraction temperature on phenolic composition and functional properties of red rooibos (Aspalathus linearis) Food Research International 2016 Nov;89:476–487 https://doi.org/10.1016/j.foodres.2016.08.041 162 Wardhani DH, Sari DK, Prasetyaningrum A Ultrasonic-assisted extraction of antioxidant phenolic coumpounds from Eucheuma cottonii Reaktor 2014 Dec 9;14(4):291–297 DOI: 10.14710/reaktor.14.4.291-297 163 Predescu NC, Papuc C, Nicorescu V, Gajaila I, Goran GV, Petcu CD, et al The influence of solid-to-solvent ratio and extraction method on total phenolic content, flavonoid content and antioxidant properties of some ethanolic plant extracts Revista de Chimie -Bucharest 2016 Oct;67(10):1922–1927 164 Wang L, Wang Z, Li X Optimization of ultrasonic-assisted extraction of phenolic antioxidants from Malus baccata (Linn.) Borkh using response surface methodology: Sample Preparation Journal of Separation Science 2013 May;36(9–10):1652–1658 DOI: 10.1002/jssc.201300062 165 Ahmed MI, Xu X, Sulieman AA, Mahdi AA, Na Y Effect of extraction conditions on phenolic compounds and antioxidant properties of koreeb (Dactyloctenium aegyptium) seeds flour Food Measure 2020 Apr;14(2):799– 808 https://doi.org/10.1007/s11694-019-00328-9 166 Bamba B, Shi J, Tranchant C, Xue S, Forney C, Lim L-T Influence of extraction conditions on ultrasound-assisted recovery of bioactive phenolics from blueberry pomace and their antioxidant activity Molecules 2018 Jul 11;23(7):1685 doi: 10.3390/molecules23071685 167 Topuz OK, Gokoglu N, Yerlikaya P, Ucak I, Gumus B Optimization of antioxidant activity and phenolic compound extraction conditions from red 85 seaweed (Laurencia obtuse) Journal of Aquatic Food Product Technology 2016 Apr 2;25(3):414–422 https://doi.org/10.1080/10498850.2013.868844 168 Hacke ACM, Marques JA, Vellosa JCR, Boligon AA, da Silva FD, de Souza D, et al Ethyl acetate fraction of Cymbopogon citratus as a potential source of antioxidant compounds New J Chem 2018;42(5):3642–3652 https://doi.org/10.1039/C7NJ04352J 169 Belhaoues S, Amri S, Bensouilah M Major phenolic compounds, antioxidant and antibacterial activities of Anthemis praecox Link aerial parts South African Journal of Botany 2020 Jul;131:200–205 https://doi.org/10.1016/j.sajb.2020.02.018 170 Mariem S, Hanen F, Inès J, Mejdi S, Riadh K Phenolic profile, biological activities and fraction analysis of the medicinal halophyte Retama raetam South African Journal of Botany 2014 Sep;94:114–121 https://doi.org/10.1016/j.sajb.2014.06.010 171 Zubia M, Fabre MS, Kerjean V, Lann KL, Stiger-Pouvreau V, Fauchon M, et al Antioxidant and antitumoural activities of some Phaeophyta from Brittany coasts Food Chemistry 2009 Oct;116(3):693–701 https://doi.org/10.1016/j.foodchem.2009.03.025 172 Ishii T, Hisada W, Abe T, Kikuchi N, Suzuki M A new record of the marine red alga Laurencia snackeyi from Japan and its Chemotaxonomic Significance Records of Natural Products 2019 Nov 26;14(2):150–153 DOI: 10.25135/rnp.145.19.06.1310 173 Kuniyoshi M, Marma MS, Higa T, Bernardinelli G, Jefford CW New bromoterpenes from the red alga Laurencia l uzonensis Journal of Natural Product 2001 Jun;64(6):696–700 https://doi.org/10.1021/np000638o 174 Dagani MJ, Barda HJ, Benya TJ, Sanders DC Bromine Compounds In: WileyVCH Verlag GmbH & Co KGaA, editor Ullmann‘s Encyclopedia of Industrial Chemistry [Internet] Weinheim, Germany: Wiley-VCH Verlag GmbH & Co 86 KGaA; 2000 [cited 2020 Sep 25] p a04_405 Available from: http://doi.wiley.com/10.1002/14356007.a04_405 175 Winter JM, Moore BS Exploring the Chemistry and Biology of Vanadiumdependent Haloperoxidases J Biol Chem 2009 Jul 10;284(28):18577–81 https://doi.org/10.1074/jbc.R109.001602 176 Neidleman SL, Geigert J Biohalogenation: principles, basic roles, and applications Chichester, West Sussex, England: New York: E Horwood  ; Halsted Press; 1986 203 p (Ellis Horwood series in organic chemistry) 177 Fusetani N, editor Drugs from the Sea [Internet] S Karger AG; 2000 [cited 2020 Sep 25] Available from: http://www.karger.com/Book/Home/223792 DOI: 10.1159/isbn.978-3-318-00599-8 87 APPENDICES Appendices Content Page A Calibration Curves 88 B Equipment List 89 APPENDICES A Calibration curves for the assays applied, were develop to determine the unknown biological activities of the seaweeds sample by interpolation Standard Curve (DRSC) Standard Curve (TPC) 1.20 1.00 0.80 y = 0.0113x - 0.0448 R² = 0.9998 0.60 0.40 0.20 0.00 Absorbance (515nm) Absorbance (765nm) 1.20 1.00 0.80 0.60 y = 0.0012x + 0.0172 R² = 0.9988 0.40 0.20 0.00 50 100 Concentration (ug/ml) 150 500 Concentration (ug/ml) (a) 1000 (b) Standard Curve (FRAP) Absorbance (593nm) 2.00 1.50 y = 0.0023x + 0.0204 R² = 0.9981 1.00 0.50 0.00 200 400 600 800 Concentration (ug/ml) 1000 (c) Figure of Calibration curve for Total Phenolic Compounds (a) DPPH Radical Scavenging Capacity (b) Ferric Reducing Antioxidant Power and (c) I APPENDICES B Equipment List Branson 2510 Digital Ultrasonic Cleanser 2510R-DTH UV–VIS spectrophotometer (Biochrom Libra S50, Biochemical Ltd Heidolph Rotary Evaporator, Laborota 4010 II Mega 17R HIGH SPEED Refrigerated Centrifuge Bruker Avance III 500 FT-NMR Orbital Shaking Water Bath (VS – 1205 SW2) III X500R QToF-MS (Sciex, USA), APCI-MS/ESI-MS Shimadzu IR Affinity 1-S (Japan) IV ... kingdom from the most complex to the simplest of species, both aquatic and terrestrial Evaluation of antioxidant and tyrosinase inhibitory activities of brominated compounds from Vietnamese red seaweeds, ...MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY PATRICK A BLAMO, JR EVALUATION OF TYROSINASE AND ANTIOXIDANT ACTIVITIES OF BROMINATED COMPOUNDS FROM VIETNAMESE RED SEAWEEDS MASTER... this study, evaluation of tyrosinase and antioxidant activities of brominated compounds from Vietnamese red seaweeds, Laurencia intermedia Yamada (L intermedia Yamada), which belongs to red algae