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

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY ABOSEDE, FUNMILOLA FANIFOSI EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF SAPONINS FROM COCOA POD HUSK (Theobroma Cacao L.) MASTER THESIS KHANH HOA - 2020 MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY ABOSEDE, FUNMILOLA FANIFOSI EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF SAPONINS 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 17/09/2020 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 undertake that the thesis entitled ―Extraction and assessment of antioxidant and antimicrobial activities of saponins 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 20th August 2020 Fanifosi Abosede Funmilola iii ACKNOWLEDGMENTS Firstly, I want to express my sincere gratitude to God Almighty for protection, provision and help through this program I also would like to express my gratitude to the following organizations and persons for their invaluable support, help and encouragement to me in conducting this study: Faculty of Food Technology, Nha Trang University in VLIR-UOS program for giving me the opportunity to study and conduct this Master thesis on ―Extraction and assessment of antioxidant and antimicrobial activities of saponins from cocoa pod husk (Theobroma cacao L.)‖ Secondly, I would like to express special appreciation to my supervisors Dr Nguyen Van Tang and Dr Tran Thi My Hanh whom I have learned a lot from their guidance, useful advices and valuable comments throughout the period of study My special thank is given to 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‖ Finally, I would like to express my sincere gratitude to my parent (Engr Olaniyi and Mrs Kikelomo Fanifosi), my uncle and aunt (Mr Olufemi and Mrs Opeyemi Fanifosi), my bachelor‘s degree thesis supervisor (Dr Olugbenga Awolu), friends, classmates and colleagues for their love, encouragement and help Nha Trang, 20th August 2020 Fanifosi Abosede Funmilola iv TABLE OF CONTENTS UNDERTAKING iii ACKNOWLEDGMENTS iv TABLE OF CONTENTS v LIST OF ABBREVIATIONS viii LIST OF TABLES ix LIST OF FIGURES x ABSTRACT .xii CHAPTER : INTRODUCTION 1.1 Aims of the research 1.1.1 Overall aim 1.1.2 Specific aims 1.2 Contents of research CHAPTER : LITERATURE REVIEW 2.1 Cocoa 2.1.1 Cocoa pod husk 2.1.2 Composition (chemical) of CPH 2.2 Bioactive compounds 2.2.1 Polyphenols 2.2.2 Flavonoids 2.2.3 Saponin 2.2.4 Alkaloids 2.2.5 Extraction method 10 2.2.6 Conventional extraction method 10 2.2.7 Pressurized liquid extraction (PLE) 11 2.2.8 Ultrasound-assisted extraction (UAE) 11 2.2.9 Enzyme-assisted extraction (EAE) 11 2.2.10 Microwave-assisted extraction (MAE) 12 v 2.2.11 Freeze drying 12 2.2.12 Fractionation and identification methods 14 2.2.13 Column chromatography (CC) 14 2.2.14 Fourier-transform infrared spectroscopy (FTIR) 15 2.2.15 Thin layer chromatography (TLC) 16 2.2.16 High performance liquid chromatography (HPLC) 16 2.2.17 Anti-oxidation 17 2.2.18 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) 18 2.2.19 Cupric reducing antioxidant capacity (CUPRAC) 18 2.2.20 FRAP (ferric reducing antioxidant power) 18 2.2.21 Antimicrobial activity 19 CHAPTER : MATERIALS AND METHODS 21 3.1 Overall experimental design 21 3.2 Study area 21 3.3 Preparation of dried sample for extraction 21 3.4 Extraction 24 3.5 Preparation of crude saponin-enriched powder 26 3.6 Identification of saponin-enriched extract and powder 28 3.6.1 Fourier-transform infrared spectroscopy (FTIR) 28 3.6.2 Thin-layer chromatography (TLC) 28 3.6.3 High performance liquid chromatography (HPLC) 30 3.7 Purification/fractionation of crude saponin-enriched extract 31 3.7.1 Preparation of column 31 3.7.2 Preparation of sample 31 3.7.3 Sample fractionation 32 3.8 Analytical methods 32 3.8.1 Determination of total saponin content 32 3.8.2 Total phenolic content (TPC) determination 33 3.8.3 Determination of total flavonoid content (TFC) 33 vi 3.8.4 DPPH radical scavenging capacity (DRSC) determination 33 3.8.5 Ferric reducing antioxidant power (FRAP) determination 34 3.8.6 Determination of cupric reducing antioxidant capacity assay (CUPRAC) 34 3.8.7 Determination of antimicrobial activity 34 3.9 Statistical Analysis 35 CHAPTER : RESULTS AND DISCUSSION 36 4.1 Physicochemical properties of crude saponin extract from cocoa pod husk 36 4.2 Bioactive compounds of crude saponin extract and fractions from CPH 37 4.2.1 Total phenol content (TPC) of crude saponin extracts and fractions of CPH 37 4.2.2 Total flavonoid content (TFC) of crude saponin extract and fractions from CPH 38 4.2.3 Saponin content (SC) of crude saponin extract and fractions from CPH 39 4.3 Antioxidant activity of crude saponin extract and fractions from CPH 39 4.3.1 DPPH radical scavenging capacity (DRSC) of crude saponin extract and fractions from CPH 39 4.3.2 Ferric reducing antioxidant power (FRAP) of crude saponin extract and fractions from CPH 41 4.3.3 Cupric reducing antioxidant capacity (CUPRAC) of crude saponin extract and fractions from CPH 41 4.4 Identification of phytochemicals in extract and fractions from CPH 42 4.4.1 TLC spectrum of crude saponin-enriched extract 42 4.4.2 FTIR spectrum of crude saponin-enriched powder 43 4.4.3 HPLC results of mixed standards and saponin fractions 46 4.4.4 HPLC result of re-fractionated fractions from fractions and 52 4.5 Antimicrobial activity of saponin-enriched powder from CPH 55 CHAPTER : CONCLUSIONS AND RECOMMENDATION 57 5.1 Conclusions 57 5.2 Recommendations 57 APPENDICES I vii LIST OF ABBREVIATIONS CPH : Cocoa pod husk CUPRAC : Cupric reducing antioxidant capacity DPPH : 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl DRSC : DPPH radical scavenging capacity FRAP : Ferric reducing antioxidant capacity HPLC : High performance liquid chromatography MAE : Microwave-assisted extraction SC : Saponin content TFC : Total flavonoid content TPC : Total phenolic content viii LIST OF TABLES Table 2.1: Composition of cocoa pods based on structure Table 4.1: Physicochemical properties of crude saponin extract from CPH 36 Table 4.2: Bioactive compounds of crude saponin extract and fractions from CPH 37 Table 4.3: Antioxidant capacity of crude saponin extract and fractions from CPH 40 Table 4.4: FTIR peaks of saponin-enriched extract from CPH 45 Table 4.5 Antimicrobial activity of saponin-enriched powder from cocoa pod husk 56 ix LIST OF FIGURES Figure 2.1: Schematic diagram of a simple chromatographic system 15 Figure 3.1: Fresh Cocoa pod husk stored in Styrofoam boxes 21 Figure 3.2: Preparation, purification and assessment of biological activity of saponins from cocoa pod husk (CPH) 22 Figure 3.3: Cutting Cocoa pod husk into thin slices 23 Figure 3.4: Dried cocoa pod husk 23 Figure 3.5: Milled cocoa pod husk 24 Figure 3.6: MAE set-up 25 Figure 3.7: Crude saponin extract 25 Figure 3.8: Filtering crude saponin extract 26 Figure 3.9: Condensed crude saponin extract 27 Figure 3.10: Sample prepared for freeze drying 27 Figure 3.11: Crude saponin-enriched powder after freeze drying 28 Figure 3.12: TLC plate undergoing 29 Figure 3.13: TLC plate under UV light 30 Figure 3.14: Colum set up for fractionation 32 Figure 4.1: TLC of saponin-enriched extract from cocoa pod husk using 80% methanol as solvent 43 Figure 4.2: FTIR spectrum of standard (-)-epigallocatechin gallate 43 Figure 4.3: FTIR spectrum of standard theophylline, theobromine and caffeine 44 Figure 4.4: FTIR spectrum of saponin-enriched extract from CPH 45 Figure 4.5: HPLC chromatogram of standards (4, 6, and 8) acquired at 272 nm 47 Figure 4.6: HPLC chromatogram of standards (1, 2, and 5) acquired at 272 nm 47 Figure 4.7: HPLC chromatogram of fraction acquired at 272 nm 48 x (Theobroma cacao) though chemical and biological treatments for ruminant feeding: In vitro and in vivo evaluation Asian Australasian Journal of Animal Science, 28(3), 343–350 Lee, J B., Kim, H Y., Jang, Y M., Song, J Y., Woo, S M., Park, M S., … Kim, M (2010) Determination of malachite green and crystal violet in processed fish products Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment, 27(7), 953–961 https://doi.org/10.1080/19440041003705839 Lee C, Oh H, Han S, Lim S (2012) Effects of hot air and freeze drying methods on physicochemical properties of citrus ‗hallabong‘ powders Food Sci Biotechnol 21(6):1633–9 Levi, G., & Karel, M (1995) Volumetric shrinkage (collapse) in freeze-dried carbohydrates above their glass transition temperature Food Research International, 28(2), 145–151 doi: 10.1016/0963-9969(95)90798-f Levin IW, Bhargava R (2005) Fourier transform infrared vibrational spectroscopic imaging: integrating microscopy and molecular recognition Ann Rev Phys Chem 56:429–474 Lianou, A., Panagou, E Z., & Nychas, G J E (2016) Microbiological Spoilage of Foods and Beverages In P Subramaniam (Ed.), The stability and shelf life of food (Second Edition, pp 3–42) Duxford, UK: Woodhead Publishing Lister, E., & Wilson, P (2001) Measurement of total phenolics and ABTS assay for antioxidant activity (personal communication) Crop Research Institute, Lincoln, New Zealand Litvin, S.; Mannheim, C.H.; Miltz, J Dehydration of carrots by a combination of freeze drying, microwave heating and air or vacuum drying Journal of Food Engineering 1998, 36 (1), 103–111 Liu, Y., Zhao, Y., & Feng, X (2008) Exergy analysis for a freeze-drying process Applied Thermal Engineering, 28(7), 10.1016/j.applthermaleng.2007.06.004 67 675–690 doi: Liu, R.H., 2013 Dietary bioactive compounds and their health implications Journal of Food Science 78 (S1),A18–A25 https://doi.org/10.1111/1750-3841.12101 Lobo, V., Patil, A., Phatak, A., & Chandra, N (2010) Free radicals, antioxidants and functional foods: Impact on human health Pharmacognosy Reviews, 4(8), 118–126 https://doi.org/10.4103/0973-7847.70902 Lu, F., Rodriguez-Garcia, J., Van Damme, I., Westwood, N J., Shaw, L., Robinson, J S., Charalampopoulos, D (2018) Valorisation strategies for cocoa pod husk and its fractions Current Opinion in Green and Sustainable Chemistry, 14, 80– 88 https://doi.org/10.1016/j.cogsc.2018.07.007 Magnúsdóttir, E V (2002) [Phytoestrogens and human health.] In Laeknabladid (Vol 88) Manach, C., Scalbert, A., Morand, C., Re me sy, C & Jime nez, L.(2004) Polyphenols: food sources and bioavailability.AmericanJournal of Clinical Nutrition,79, 727–747 Manach, C., Williamson, G., Morand, C., Scalbert, A & Re me sy, C.(2005) Bioavailability and bioefficacy of polyphenols in humans I.Review of 97 bioavailability studies.American Journal of ClinicalNutrition,81(Suppl), 230S–242S Martin, C.M., 2007 Chronic disease and illness care Canadian Family Physician 53, 2086–2091 Martín Ortega, A M., & Segura Campos, M R (2019) Bioactive Compounds as Therapeutic Alternatives In Bioactive Compounds https://doi.org/10.1016/b978-012-814774-0.00013-x Mendiola, J A., Herrero, M., Cifuentes, A., & Ibañez, E (2007) Use of compressed fluids for sample preparation: Food applications Journal of Chromatography A, 1152(1-2), 234–246 doi: 10.1016/j.chroma.2007.02.046 Morais, A R D V., Alencar, É D N., Xavier Júnior, F H., Oliveira, C M De, Marcelino, H R., Barratt, G., … Elaissari, A (2016) Freeze-drying of emulsified systems: A review International Journal of Pharmaceutics, 503(1–2), 102–114 https://doi.org/10.1016/j.ijpharm.2016.02.047 68 Nguyen, V T., Vuong, Q V., Bowyer, M C., van Altena, I A., & Scarlett, C J (2015b) Effects of different drying methods on bioactive compound yield and antioxidant capacity of Phyllanthus amarus Drying Technology, 33: 1006–1017 Nguyen, V T., Pham, H N T., Bowyer, M C., van Altena, I A., & Scarlett, C J (2016b) Influence of solvents and novel extraction methods on bioactive compounds and antioxidant capacity of Phyllanthus amarus Chemical Papers, 70: 556–566 Nguyen, V T., Bowyer, M C., Van Altena, I A., & Scarlett, C J (2017): Microwave assisted extraction as an advanced technique for optimization of saponin yield and antioxidant potential from Phyllanthus amarus, Separation Science and Technology, DOI: 10.1080/01496395.2017.1374972 Nychas, G J E., & Panagou, E (2011) Microbiological spoilage of foods and beverages In D Kilcast & P Subramaniam (Eds.), Food and beverage stability and shelf life (pp 3–28) Cambridge, UK: Woodhead Publishing Oddoye, E O., Agyente-Badu, C K., & Gyedu-Akoto, E (2013) Cocoa and its by-products: Identification and utilization In R Watson, V Preedy, & S Zibadi (Vol Eds.), Chocolate in health and nutrition Nutrition and health Vol Odeyemi, O A., Alegbeleye, O O., Strateva, M., & Stratev, D (2020) Understanding spoilage microbial community and spoilage mechanisms in foods of animal origin Comprehensive Reviews in Food Science and Food Safety, 19(2), 311– 331 https://doi.org/10.1111/1541-4337.12526 Odongo, G A., Schlotz, N., Herz, C., Hanschen, F S., Baldermann, S., Neugart, S., … Lamy, E (2017) The role of plant processing for the cancer preventive potential of Ethiopian kale (Brassica carinata) Food & Nutrition Research, 61(1), 1271527 doi: 10.1080/16546628.2017.1271527 Oliveira, R N., Mancini, M C., de Oliveira, F C S., Passos, T M., Quilty, B., Thiré, R M da S M., & McGuinness, G B (2016) Análise por FTIR e quantificaỗóo de fenúis e flavonúides de cinco produtos naturais disponớveis comercialmente utilizados no tratamento de feridas Revista https://doi.org/10.1590/S1517-707620160003.0072 69 Materia, 21(3), 767–779 Omodanisi, E., Aboua, Y., & Oguntibeju, O (2017) Assessment of the AntiHyperglycaemic, Anti-Inflammatory and Antioxidant Activities of the Methanol Extract of Moringa Oleifera in Diabetes-Induced Nephrotoxic Male Wistar Rats Molecules, 22(4), 439 doi: 10.3390/molecules22040439 Os, A., Sp, D., Rajeshkumar, R., & Mvnl, C (2017) Antioxidant activities of Hydro-ethanol and Saponin extracts of Terminalia schimeperiana root 47–53 Retrieved from https://www.scholarsresearchlibrary.com/abstract/antioxidant- activities-of-hydroethanol-and-saponin-extracts-of-terminalia-schimeperiana-root13270.html Osbourn, A.; Goss, R J M.; Field, R A (2011) The saponins–polar isoprenoids with important and diverse biological activities Natural Product Report, 28: 12611268 ệzyỹrek, M., Gỹỗlỹ, K., Tỹtem, E., Bakan, K S., Erỗa , E., Esin Çelik, S., Apak, R (2011) A comprehensive review of CUPRAC methodology Analytical Methods, 3(11), 2439–2453 https://doi.org/10.1039/c1ay05320e Pagliaro, B., Santolamazza, C., Simonelli, F., Rubattu, S., 2015 Phytochemical compounds and protection from cardiovascular diseases: a state of the art BioMed Research International 2015, 1–17 https://doi org/10.1155/2015/918069 Payne, M J., Hurst, W J., Stuart, D A., Ou, B., Fan, E., Ji, H., & Kou, Y (2010) Determination of Total Procyanidins in Selected Chocolate and Confectionery Products Using DMAC Journal of AOAC INTERNATIONAL, 93(1), 89–96 doi: 10.1093/jaoac/93.1.89 Pham, H., Nguyen, V., Vuong, Q., Bowyer, M., & Scarlett, C (2015) Effect of Extraction Solvents and Drying Methods on the Physicochemical and Antioxidant Properties of Helicteres hirsuta Lour Leaves Technologies, 3(4), 285–301 https://doi.org/10.3390/technologies3040285 Pisoschi, A M.; Negulescu, G P (2011) Methods for total antioxidant activity determination: a review Biochemistry and Analytical Biochemistry, 1: 1–10 Pronyk, C., & Mazza, G (2009) Design and scale-up of pressurized fluid extractors for food and bioproducts Journal of Food Engineering, 95(2), 215–226 doi: 10.1016/j.jfoodeng.2009.06.002 70 Quinto, E J., Caro, I., Villalobos-Delgado, L H., Mateo, J., De-Mateo-silleras, B., & Redondo-Del-río, M P (2019) Food safety through natural antimicrobials Antibiotics, 8(4), 1–30 https://doi.org/10.3390/antibiotics8040208 Raisa, K., Farhana, M., & Muhammad, J (2015) Antioxidant evaluations of polar and non-polar fractions of Cajanus cajan seeds Journal of Medicinal Plants Research, 9(6), 193–198 https://doi.org/10.5897/jmpr2014.5456 Ramkumar Ponnuraj (2015) Formulation and Characterization of Epigallocatechin Gallate Nanoparticles Indo American Journal of Pharmaceutical Research , 5(01), 387–399 Ramos, L., Kristenson, E., & Brinkman, U (2002) Current use of pressurised liquid extraction and subcritical water extraction in environmental analysis Journal of Chromatography A, 975(1), 3–29 doi: 10.1016/s0021-9673(02)01336-5 Rao, A V., & Gurfinkel, D M (2000) The bioactivity of saponins: Triterpenoid and steroidal glycosides Drug Metabolism and Drug Interactions, 17(1– 4), 211–235 https://doi.org/10.1515/DMDI.2000.17.1-4.211 Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay Free Radical Biology and Medicine, 26(9-10), 1231-1237 doi:10.1016/s0891-5849(98)00315-3 Rohman, A., Che Man, Y B., Ismail, A., & Hashim, P (2010) Application of FTIR spectroscopy for the determination of virgin coconut oil in binary mixtures with olive oil and palm oil JAOCS, Journal of the American Oil Chemists’ Society, 87(6), 601–606 https://doi.org/10.1007/s11746-009-1536-7 Riccioni, G., Gammone, M.A., Tettamanti, G., Bergante, S., Pluchinotta, F.R., Orazio, N.D., August 2015 Resveratrol and anti-atherogenic effects International Journal of Food Sciencies and Nutrition 1–8 https://doi.org/10.3109/09637486.2015.1077796 Rigacci, S., & Stefani, M (2016) Nutraceutical Properties of Olive Oil Polyphenols An Itinerary from Cultured Cells through Animal Models to Humans International Journal of Molecular Sciences, 17(6), 843 doi:10.3390/ijms17060843 71 Sagdic, O., Ozturk, I., Yilmaz, M T., & Yetim, H (2011) Effect of Grape Pomace Extracts Obtained from Different Grape Varieties on Microbial Quality of Beef Patty Journal of Food Science, 76(7) doi:10.1111/j.1750-3841.2011.02323.x Sajid, M., Khan, M R., Shah, S A., Majid, M., Ismail, H., Maryam, S., Younis, T (2017) Investigations on anti-inflammatory and analgesic activities of Alnus nitida Spach (Endl) stem bark in Sprague Dawley rats Journal of Ethnopharmacology, 198, 407–416 doi: 10.1016/j.jep.2017.01.041 Sakugawa, K., Ikeda, A., Takemura, A., & Ono, H (2004) Simplified method for estimation of composition of alginates by FTIR Journal of Applied Polymer Science, 93(3), 1372–1377 https://doi.org/10.1002/app.20589 Sánchez-Moreno, C., Larrauri, J A., & Saura-Calixto, F (1998) A procedure to measure the antiradical efficiency of polyphenols Journal of the Science of Food and Agriculture, 76(2), 270–276 https://doi.org/10.1002/(sici)1097- 0010(199802)76:23.0.co;2-9 Sarker, S D., Nahar, L., & Kumarasamy, Y (2007) Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals Methods, 42(4), 321–324 Sasidharan, S., Chen, Y., Saravanan, D., Sundram, K., & Latha, L (2010) Extraction, Isolation and Characterization Of Bioactive Compounds From Plants‘ Extracts African Journal of Traditional, Complementary and Alternative Medicines, 8(1) doi: 10.4314/ajtcam.v8i1.60483 Sendra JM, Sentandreu E, Navarro JL (2006) Reduction kinetics of the free stable radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH•) for determination of the antiradical activity of citrus juices Eur Food Res Technol 223:615–624 Shaw, O M., Hurst, R D., & Harper, J L (2016) Boysenberry ingestion supports fibrolytic macrophages with the capacity to ameliorate chronic lung remodeling American Journal of Physiology-Lung Physiology, 311(3) doi: 10.1152/ajplung.00309.2015 72 Cellular and Molecular Sobamiwa, O., & Longe, O G (1994) Utilization of cocoa pod pericarp fractions in broiler chick diets Animal Feed Science and Technology, 47(3–4), 237–244 Somani, S.J., Modi, K.P., Majumdar, A.S., Sadarani, B.N., 2015 Phytochemicals and their potential usefulness in inflammatory bowel disease Phytotherapy Research 29 (3), 339–350 https://doi.org/10.1002/ptr.5271 Spanos, G A., & Wrolstad, R E (1990) Influence of processing and storage on the phenolic composition of Thompson seedless grape juice Journal of Agricultural & Food Chemistry, 38, 1565–1571 Stewart, B.W., Wild, C.P., 2014 World Health Organization World cancer report 2014 https://doi.org/9283204298 Strumillo, C.; Adamiec, J Energy and quality aspects of food drying Drying Technology 1996, 14 (2), 423–448 Sun, X., Yang, X., Xue, P., Zhang, Z., & Ren, G (2019) Improved antibacterial effects of alkali-transformed saponin from quinoa husks against halitosisrelated bacteria BMC Complementary and Alternative Medicine, 19(1), 1–10 https://doi.org/10.1186/s12906-019-2455-2 Tamang, J P., Watanabe, K., & Holzapfel, W H (2016) Review: Diversity of microorganisms in global fermented foods and beverages Frontiers in Microbiology, 7(MAR) https://doi.org/10.3389/fmicb.2016.00377 Tan, S P., Vuong, Q V., Stathopoulos, C E., Parks, S E., & Roach, P D (2014) Optimized Aqueous Extraction of Saponins from Bitter Melon for Production of a Saponin-Enriched Bitter Melon Powder Journal of Food Science, 79(7) https://doi.org/10.1111/1750-3841.12514 Tao, Z.; Wu, H.; Chen, G.; Deng, H Numerical simulation of conjugate heat and mass transfer process within cylindrical porous media with cylindrical dielectric cores in microwave freeze-drying International Journal of Heat and Mass Transfer 2005, 48, 561–572 73 Taylor, P., Duan, X., Zhang, M., Mujumdar, A S., Wang, R., Duan, X., … Wang, R (n.d.) Drying Technology  : An International Journal Trends in Microwave-Assisted Freeze Drying of Foods Trends in Microwave-Assisted Freeze Drying of Foods (November 2012), 37–41 https://doi.org/10.1080/07373931003609666 Temple, N J (2000) Antioxidants and disease: More questions than answers Nutrition Research, 20(3), 449–459 doi: 10.1016/s0271-5317(00)00138-x Tenore, G C., Caruso, D., Buonomo, G., Davino, M., Campiglia, P., Marinelli, L., & Novellino, E (2017) A Healthy Balance of Plasma Cholesterol by a Novel Annurca Apple-Based Nutraceutical Formulation: Results of a Randomized Trial Journal of Medicinal Food, 20(3), 288–300 doi: 10.1089/jmf.2016.0152 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(6–7), 669–675 https://doi.org/10.1016/j.jfca.2006.01.003 Ullah, F., Iqbal, N., Ayaz, M., Sadiq, A., Tradit, A J., Altern, C., Sasidharan, S., Chen, Y., Saravanan, D., Sundram, K M., … Nasi, B A (2007) Proper Actions Lecture Notes in Mathematics, 1902, 121– 130 https://doi.org/10.1007/978-3-540-69153-2_6 Tang, X., Pikal, M.J., 2004 Design of freeze-drying processes for pharmaceuticals: practical advice Pharm Res 21, 191-200 Ullah, I., Ahmad, S., & Imran, M (2017) DPPH, ABTS free radical scavenging, antibacterial and phytochemical evaluation of crude methanolic extract and subsequent fractions of Chenopodium botrys aerial parts Pakistan Journal of Pharmaceutical Sciences, 30(3), 761–766 Valadez-Carmona, L., Plazola-Jacinto, C P., Hernández-Ortega, M., Hernández-Navarro, M D., Villarreal, F., Necoechea-Mondragón, H., Ceballos-Reyes, G (2017) Effects of microwaves, hot air and freeze-drying on the phenolic compounds, antioxidant capacity, enzyme activity and microstructure of cacao pod 74 husks (Theobroma cacao L.) Innovative Food Science & Emerging Technologies, 41, 378–386 Verraes, C., Van Boxstael, S., Van Meervenne, E., Van Coillie, E., Butaye, P., Catry, B., Herman, L (2013) Antimicrobial resistance in the food chain: A review International Journal of Environmental Research and Public Health, 10(7), 2643– 2669 https://doi.org/10.3390/ijerph10072643 Vriesmann, L C., Renata Dias De Mello Castanho Amboni, & Petkowicz, C L D O (2011) Cacao pod husks (Theobroma cacao L.): Composition and hot-watersoluble pectins Industrial Crops and Products, 34(1), 1173–1181 doi: 10.1016/j.indcrop.2011.04.004 Vriesmann, L C., Teófilo, R F., & Lúcia de Oliveira Petkowicz, C (2012) Extraction and characterization of pectin from cacao pod husks (Theobroma cacao L.) with citric acid LWT - Food Science and Technology, 49(1), 108–116 https://doi.org/10.1016/j.lwt.2012.04.018 Vuong, Q V.; Hirun, S.; Roach, P D.; Bowyer, M C.; Phillips, P A.; Scarlett, C J (2013) Effect of extraction conditions on total phenolic compounds and antioxidant activities of Carica papaya leaf aqueous extracts Journal of Herbal Medicine, 3: 104–111 Wang, W (2000) Lyophilization and development of solid protein pharmaceuticals International Journal of Pharmaceutics, 203(1-2), 1–60 doi: 10.1016/s0378-5173(00)00423-3 Wang, L., & Weller, C L (2006) Recent advances in extraction of nutraceuticals from plants Trends in Food Science & Technology, 17(6), 300–312 doi: 10.1016/j.tifs.2005.12.004 Wang, W., Chen, M., & Chen, G (2012) Issues in Freeze Drying of Aqueous Solutions Chinese Journal of Chemical Engineering, 20(3), 551–559 doi: 10.1016/s1004-9541(11)60218-8 Wang, X., Yu, H., Xing, R., & Li, P (2017) Characterization, Preparation, and Purification of Marine Bioactive Peptides BioMed Research International, 2017, 1– 16 75 Wijngaard, H., Hossain, M B., Rai, D K., & Brunton, N (2012) Techniques to extract bioactive compounds from food by-products of plant origin Food Research International, 46(2), 505–513 https://doi.org/10.1016/j.foodres.2011.09.027 World Agriculture (2011) Systematics, anatomy and morphology of cacao Monday August 22, 2011 www.agrotechnomarket.com Wu, Y., Ding, Y., Tanaka, Y., Zhang, W., 2014 Risk factors contributing to type diabetes and recent advances in the treatment and prevention International Journal of Medical Sciences 11, 1185–1200 https://doi.org/10.7150/ijms.10001 Yadav, B S., Yadav, R., Yadav, R B., & Garg, M (2016) Antioxidant activity of various extracts of selected gourd vegetables Journal of Food Science and Technology, 53(4), 1823–1833 https://doi.org/10.1007/s13197-015-1886-0 Yashin, Y I., & Yashin, A Y (2004) Analysis of food products and beverages using high-performance liquid chromatography and ion chromatography with electrochemical detectors Journal of Analytical Chemistry, 59(12), 1121–1127 https://doi.org/10.1023/B:JANC.0000049711.43423.a1 Yeo, J., Wang, X C., & Hammond, M C (2017) c-di-GMP Signaling Methods and Protocols C-Di-GMP Signaling, 1657, 111–130 https://doi.org/10.1007/978-1-4939-7240-1 Zhang, Z., Pang, X., Xuewu, D., Ji, Z., & Jiang, Y (2005) Role of peroxidase in anthocyanin degradation in litchi fruit pericarp Food Chemistry, 90(1-2), 47-52 doi:10.1016/j.foodchem.2004.03.023 76 APPENDICES ALPHA FTIR spectrometer used for identification I Chromaster HPLC system used for identification II Gallic acid standard curve for TPC analysis Catechin standard curve for TFC analysis III Escin standard curve for saponins analysis Trolox standard curve for DPPH analysis IV Trolox standard curve for FRAP analysis Trolox standard curve for CUPRAC analysis V ... concentration of 200 g/mL The mixed standard solutions (gallic acid, theobromine, theophylline and caffeine; and (+)catechin, ( -)- epicatechin, ( -)- epigallocatechin and ( -)- epigallocatechingallate) were... entitled ? ?Extraction and assessment of antioxidant and antimicrobial activities of saponins from cocoa pod husk (Theobroma cacao L. )? ?? is my own work The work has not been presented elsewhere for assessment. .. directly linked to free radicals and active oxygen (Naumovski, 201 4), and this may distort biological macromolecules and damage cells, which leads to aging of cell and long-term ailments, like