Extraction and assessment of antioxidant and antimicrobial activities of phenolics from cocoa pod husk (theobroma cacao l )

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY UWIHAYE FESTUS EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF PHENOLICS FROM COCOA POD HUSK (Theobroma cacao L.) MASTER THESIS KHANH HOA – 2020 MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY UWIHAYE FESTUS EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF PHENOLICS FROM COCOA POD HUSK (Theobroma cacao L.) MASTER THESIS Major Food Technology Topic allocation decision 192/QD-DHNT on 03/03/2020 Decision in establishing the Committee 899/QD-DHNT on 04/09/2020 Defense date 19/09/2020 Supervisors Dr Nguyen Van Tang (Principal supervisor) Dr Tran Thi My Hanh (Co-supervisor) Chairman Assoc Prof Dr Huynh Nguyen Duy Bao Department of Graduate Studies KHANH HOA – 2020 UNDERTAKING I declare that the thesis entitled “Extraction and assessment of antioxidant and antimicrobial activities of phenolics from cocoa pod husk (Theobroma cacao L.)” is my own work The work has not been presented elsewhere for assessment until the time this thesis is submitted 3rd September, 2020 Uwihaye Festus iii ACKNOWLEDGEMENTS Many thanks and praises to God Almighty that he has been with me throughout my study period in Vietnam Firstly, I would highly acknowledge Dr Nguyen Van Tang, my principal supervisor, and Dr Tran Thi My Hanh, my co-supervisor for their help and guidance, they gave me an amazing experience I also would like to thank the financial support for my Master thesis through the research project funded by the Ministry of Education and Training, Vietnam entitled “Extraction of some bioactive compounds from cocoa pod husk for potential application in the functional foods” Secondly, I also wish to appreciate my country, Rwanda through the Ministry of Labour as it accepted me to join in Master program at the Nha Trang University, Vietnam Thirdly, I am grateful to the supports from VLIR, Graduate Studies Department, and Faculty of Food Technology, Nha Trang University At large, I can not forget the Nha Trang University’s entire teaching staff and my classmates for providing me with a good and favorable working environment Lastly but not least, my special acknowledgment goes to my wife, Ugwaneza Grace and my children, Jayden, Joanna and Jordan for their undeniable love, encouragement, patience and understanding 3rd September, 2020 Uwihaye Festus iv TABLE OF CONTENTS UNDERTAKING iii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS .v LIST OF ABBREVIATIONS ix LIST OF TABLES .x LIST OF FIGURES xi CHAPTER INTRODUCTION 1.1 PROBLEM STATEMENT 1.2 RESEARCH SIGNIFICANCE 1.3 RESEARCH AIMS 1.3.1 Overall aim 1.3.2 Specific aims .4 CHAPTER REVIEW OF THE LITERATURE 2.1 COCOA AND COCOA BY-PRODUCTS AS WASTES AND VALUE ADDITION 2.1.1 Husk of cocoa pod 2.1.2 Cocoa bioactive compounds with emphasis on phenolics .6 2.1.3 CPH applications and phenolic compounds 2.2 IMPACT OF PROCESSING METHODS ON PHENOLIC COMPOUNDS AND ANTIOXIDATION POTENTIAL OF CPH 12 2.3 FRACTIONATION, PURIFICATION, CHARACTERIZATION AND QUANTIFICATION OF PHENOLIC COMPOUNDS .16 2.4 EVALUATION OF ANTIOXIDANTION POTENTIAL OF PHENOLICS 17 2.5 COCOA PHENOLIC COMPOUNDS AND BIOLOGICAL ACTIVITIES 18 CHAPTER MATERIALS AND METHODS 20 3.1 EXPERIMENTAL PROCEDURE 20 v 3.2 MATERIAL AND CHEMICALS 21 3.2.1 Fresh cocoa pod husk 21 3.2.2 Analytical reagents .21 3.3 EXPERIMENTAL METHODS .21 3.3.1 Preparation of powder from crude CPH extract 21 3.3.2 Fractionation of crude extract from CPH by column chromatography .22 3.3.3 Preparation of solutions from crude CPH extract and derived fractions for evaluation of phenolic content, antioxidation potential and antimicrobial activity 23 3.3.4 Phenolic compounds identification in CPH extract 23 3.3.5 Analysis of physicochemical properties of powdered extract from CPH 25 3.3.6 Analysis of bioactive compounds .26 3.3.7 Evaluation of biological activities .27 3.3.8 Statistical analysis .29 CHAPTER RESULTS AND DISCUSSIONS 30 4.1 PHYSICOCHEMICAL PROPERTIES OF CRUDE CPH POWDER 30 4.2 IDENTIFICATION OF PHENOLIC COMPOUNDS BY THIN-LAYER CHROMATOGRAPHY 32 4.3 IDENTIFICATION OF PHENOLIC COMPOUNDS BY FTIR SPECTROSCOPY .33 4.4 IDENTIFICATION OF PHENOLIC COMPOUNDS BY HPLC 35 4.5 BIOACTIVE COMPOUNDS OF DERIVED CPH FRACTIONS 42 4.5.1 Total phenolic content (TPC) 42 4.5.2 Total flavonoids content (TFC) 44 4.5.3 Saponins .45 4.6 ANTIOXIDANT ACTIVITIES OF CPH EXTRACT AND DERIVED FRACTIONS .46 4.6.1 DPPH radical scavenging capacity (DRSC) .46 4.6.2 Cupric reducing antioxidant capacity (CUPRAC) 47 vi 4.6.3 Ferric reducing antioxidant power (FRAP) 48 4.7 ANTIMICROBIAL ACTIVITY OF CRUDE CPH EXTRACT 49 CHAPTER CONCLUSIONS AND FUTURE PERSPECTIVES .52 5.1 CONCLUSIONS 52 5.2 FUTURE PERSPECTIVES 52 REFERENCES I vii LIST OF SYMBOLS g : Gram : Minute mL : Millilitre mm : Millimetre nm : Nanometre s : Second W : Watt  : Micro viii LIST OF ABBREVIATIONS CBS : Cocoa bean shells CE : Catechin equivalents CPH : Cocoa pod husk CUPRAC : Cupric reducing antioxidant capacity DPPH : 2,2-diphenyl-1-picrylhydrazyl DRSC : DPPH radical scavenging capacity EE : Escin equivalents EtOH : Ethanol FRAP : Ferric reducing antioxidant power FTIR : Fourier-transform infrared spectroscopy GAE : Gallic acid equivalents HPLC : High performance liquid chromatography MAE : Microwave assisted extraction MeOH : Methanol MIC : Minimum inhibition concentration ORAC : Oxygen radical absorbance capacity RE : Rutin equivalents SC : Saponins content SFE : Supercritical fluid extraction TAA : Total antioxidant activity TE : Trolox equivalents TFC : Total flavonoid content TLC : Thin layer chromatography TPC : Total phenolic content WSI : Water soluble index ix LIST OF TABLES Table 2.1 Total phenolic content (TPC) of cocoa by-products 10 Table 2.2 MAE characteristics 15 Table 4.1 Physiochemical properties of crude CPH powder 31 Table 4.2 Bioactive compounds of crude extract and fractions from CPH 46 Table 4.3 Antioxidant capacity of crude extract and fractions from CPH 49 Table 4.4 Antimicrobial activity of phenolic-enriched powder from CPH 51 x 105 Goranova Z, Baeva M, Vrancheva R, Petrova T, Stefanov S Antioxidant properties and color characteristics of sponge cakes containing functional components Ukr Food J 2019;8(2):260–70 106 Todorovic V, Milenkovic M, Vidovic B, Todorovic Z, Sobajic S Correlation between Antimicrobial, Antioxidant Activity, and Polyphenols of Alkalized/Nonalkalized Cocoa Powders J Food Sci 2017;82(4):1020–7 107 Hasanuddin A, Anwar C, Mappatoba M, Hafsah H Antibacterial and Antioxidant Activities of Cocoa Pod That Associated in Maltodextrin in Various Concentration Agrol Agric Sci J 2018;5(2):123 108 Pham HNT, Sakoff JA, Bond DR, Vuong Q Van, Bowyer MC, Scarlett CJ In vitro antibacterial and anticancer properties of Helicteres hirsuta Lour leaf and stem extracts and their fractions Mol Biol Rep [Internet] 2018;45(6):2125–33 Available from: http://dx.doi.org/10.1007/s11033-018-4370-x 109 Ariza BTS, Mufida DC, Fatima NN, Hendrayati TI, Wahyudi T, Misnawi In vitro antibacterial activity of cocoa ethanolic extract against Escherichia coli Int Food Res J 2014;21(3):935–40 110 Yuanita T, Mooduto L, Lina RC, Muttaqin FA, Tangdan I, Marpaung REI, et al Minimum inhibitory concentration of cocoa pod husk extract in Enterococcus faecalis extracellular polymeric substance biofilm thickness Dent J (Majalah Kedokt Gigi) 2019;52(4):215 111 Yuanita T, Oktavianti RA, Suryani DF, Rukmo M, Kunarti S, Kusuma AH The inhibitory ability of cocoa pod husk extract on enterococcus faecalis glucosyltransferase enzyme activity J Contemp Dent Pract 2020;21(3):271–6 112 Rakholiya K, Kaneria M, Chanda S Inhibition of microbial pathogens using fruit and vegetable peel extracts Int J Food Sci Nutr [Internet] 2014;7486:1–7 Available from: http://informahealthcare.com/ijf 113 Ogunremi O, Ishola O, Ogunedina H Phytochemical screening and in vitro antimicrobial activity of aqueous and ethanol extracts from Mucuna Pruriens husks against some foodborne microorganisms Food Environ Saf [Internet] 65 2018;XVII(2):233–40 Available from: www.fia.usv.ro/fiajournal 114 Arora DS, Onsare JG In vitro antimicrobial evaluation and phytoconstituents of Moringa oleifera pod husks Ind Crops Prod [Internet] 2014;52:125–35 Available from: http://dx.doi.org/10.1016/j.indcrop.2013.09.016 115 Hong Ngoc Thuy Pham, Jennette A Sakoff, Quan Van Vuong, Michael C Bowyer CJS Phytochemical, antioxidant, anti‑proliferative and antimicrobial properties of Catharanthus roseus root extract, saponin‑enriched and aqueous fractions Springer Nature B.V.; 2019 p 3265–3273 116 Johnson IM, Prakash H, Prathiba J, Raghunathan R, Malathi R Spectral Analysis of Naturally Occurring Methylxanthines (Theophylline, Theobromine and Caffeine) Binding with DNA PLoS One 2012;7(12) 117 Ponnuraj R, Janakiraman K, Gopalakrishnan S, Senthilnathan K, Saravanan P Formulation and characterization of Epigallocatechin Gallate Nanoparticles Indo Am J Pharm Res 2015;5(01):387–99 66 APPENDICES Appendix I: Figure of columns used in column chromatography (A) Column with fractions elution level and (B) Column with fractions elution levels Glass column; One fraction elution distance (3 cm); All fractions elution distance also solvent (mobile phase level (18 cm and 24 cm for A and B, respectively); Solvent layer above silica gel (prevent dryness); Silica gel (solid phase) distance (21 cm equal 9.4 g packed) for both A and B; Tube for fraction collection; Cotton wool to hold silica gel and allow solvent to flow; Column stand; Solvent level (9 cm) for sample draining through solid phase (silica gel) I Appendix II: Figure of glass chamber used in the TLC analysis II Appendix III: Figure of ALPHA FTIR spectrometer III Appendix IV: Figure of Chromaster HPLC system IV Appendix V: Figure of Gallic acid standard curve for TPC Appendix VI: Figure of Catechin standard curve for TFC V Appendix VII: Figure of Escin standard curve for saponins Appendix VIII: Figure of Trolox standard curve for DPPH VI Appendix IX: Figure of Trolox standard curve for FRAP Appendix X: Figure of Trolox standard curve for CUPRAC VII Appendix XI: Table of recorded CPH extract absorbances for bioactive and antioxidation activities Activity/compound ABS1 ABS2 ABS3 DPPH (Control) 1.289 1.311 1.312 DPPH (Sample) 1.238 1.255 1.304 FRAP 0.506 0.49 0.534 CUPRAC 0.251 0.25 0.243 TPC 0.147 0.145 0.164 TFC 0.006 0.008 0.006 SC 0.118 0.114 0.113 Appendix XII: Table of recorded DPPH absorbances of CPH extract fractions Activity Sample ABS1 ABS2 ABS3 DPPH Control 1.240 1.213 1.214 Fraction1 1.187 1.190 1.177 Fraction 1.172 1.177 1.146 Fraction 1.129 1.138 1.123 Fraction 1.186 1.191 1.196 Fraction 1.181 1.182 1.181 Fraction 1.162 1.093 1.132 Fraction 1.148 1.000 1.202 Fraction 1.192 1.215 1.238 VIII Appendix XIII: Table of recorded FRAP absorbances of CPH extract fractions Activity Sample ABS1 ABS2 ABS3 FRAP Fraction -0.07 -0.080 -0.118 Fraction 0.065 0.067 0.143 Fraction 0.112 0.138 0.171 Fraction 0.114 0.044 0.152 Fraction -0.105 -0.080 -0.020 Fraction 0.112 0.053 0.094 Fraction -0.044 -0.055 - Fraction -0.14 -0.060 - Appendix XIV: Table of recorded CUPRAC absorbances of CPH extract fractions Activity Sample ABS1 ABS2 ABS3 CUPRAC Fraction 0.097 0.079 0.120 Fraction 0.086 0.085 0.079 Fraction 0.063 0.053 0.097 Fraction 0.077 0.079 0.086 Fraction 0.138 0.140 0.139 Fraction 0.186 0.184 0.186 Fraction 0.072 0.067 0.074 Fraction 0.067 0.095 0.051 IX Appendix XV: Table of recorded TPC absorbances of CPH extract fractions Compound Sample ABS1 ABS2 ABS3 TPC Fraction 0.111 0.166 0.141 Fraction 0.177 0.138 0.14 Fraction 0.164 0.168 0.147 Fraction 0.195 0.187 0.174 Fraction 0.166 0.177 0.153 Fraction 0.254 0.269 0.285 Fraction 0.186 0.128 0.126 Fraction 0.022 0.043 0.035 Appendix XVI: Table of recorded TFC absorbances of CPH extract fractions Compound Sample ABS1 ABS2 ABS3 TFC Fraction 0.005 0.005 0.003 Fraction 0.011 0.001 0.006 Fraction 0.006 0.007 0.005 Fraction 0.007 0.001 0.006 Fraction 0.008 0.005 0.007 Fraction 0.013 0.007 0.012 Fraction 0.016 0.010 0.019 Fraction -0.001 -0.003 _ X Appendix XVII: Table of recorded SC absorbances of CPH extract fractions Compound Sample ABS1 ABS2 ABS3 SC Fraction _ 0.126 0.175 Fraction 0.171 0.212 0.194 Fraction 0.102 0.076 0.136 Fraction 0.098 0.06 _ Fraction 0.188 0.158 0.177 Fraction 0.18 0.206 0.2 Fraction 0.199 _ 0.196 Fraction 0.034 0.082 0.022 XI Appendix XVIII: Figure of FTIR spectrum of standard theophylline, theobromine and caffeine Source: Johnson et al.[116] XII Appendix XIX: Figure of FTIR spectrum of standard (-)-epigallocatechingallate (-)-epigallocatechingallate Source: Ponnuraj, R.et al [117] XIII ...MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY UWIHAYE FESTUS EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF PHENOLICS FROM COCOA POD HUSK (Theobroma cacao L.) MASTER... propagation like black pod rot [4] and with ches' bloom [7] Around 6.4 (1.5 times cocoa beans) [2] and 55 million tons (13 times cocoa beans) [8] of cocoa pod shell and cocoa pod husk are world annual... (TPC, TFC and SC), and evaluation of biological activities (Antioxidation and antimicrobial) Figure 3.1 Overall experimental procedure for extraction and assessment of biological activity of phenolics