Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without prior permission of the Author. Development of a novel functional yogurt containing anti-inflammatory bioactive compounds Akshay Bisht 2019 Development of a novel functional yogurt containing anti-inflammatory bioactive compounds A thesis submitted in partial fulfilment of the requirements for the degree of Master of Food Technology Massey University Albany, New Zealand Akshay Bisht June 2019 i Abstract The consumption of bioactive compounds is increasingly becoming popular due to their beneficial effects on health and wellbeing. The anti-inflammatory properties of bioactives such as curcumin are well established. However, curcumin has low bioavailability, hence it is frequently consumed in capsules to enable the delivery of the required dosage to achieve optimum health benefits. Synergistic effects may be achieved by combining curcumin with other anti-inflammatory bioactive compounds. Recent investigations on lupeol and chlorogenic acid (CGA) have reported that these bioactive compounds show similar therapeutic benefits to curcumin. Furthermore, delivery of bioactives via a food matrix, such as fermented coconut yogurt, may improve bioavailability. Thus, this research investigated the potential of an anti-inflammatory combination of curcumin with CGA or lupeol with the objective of developing coconut yogurt to deliver the combined bioactives to humans. This research was performed in two parts. In part 1, the anti-inflammatory potential of three bioactive compounds (curcumin, CGA and lupeol), individually and in combination, was investigated using an in vitro model of human THP-1 macrophages stimulated with LPS. Differentiated THP-1 cells were treated with variable concentrations of curcumin, CGA and lupeol and their effects on the production of TNF-α, a pro-inflammatory cytokine, and cell viability was measured using ELISA and MTT assays, respectively. Curcumin alone significantly (p≤0.05) suppressed TNF-α production in a dose dependent manner. Curcumin in combination with lupeol gave an additional 15-35 % reduction in TNF-α level. However, the reduction in TNF-α production by curcumin + lupeol was accompanied by cell death. In contrast, treatment with CGA appeared to protect the THP-1 cells from LPS toxicity and its co-administration with curcumin at a 1:1 ratio reduced TNF-α production without impacting cell viability. Further, it is proposed that the latter combination showed anti-inflammatory activity by reducing mRNA expression of pro-inflammatory cytokines and COX-2 enzyme via suppressing NF-κB, IκB-β-kinase and TLR-4 receptor. Thus, a 1:1 combination of curcumin with CGA was selected to be delivered in coconut yogurt. In part 2, coconut yogurt enriched with turmeric and coffee to deliver the benefits of curcumin and CGA, respectively, was developed. Addition of 100 mg of each bioactive compound to 150 g coconut cream did not have any significant (p≤0.05) effect on the viable cell counts of the yogurt culture, pH and titratable acidity during fermentation. However, slight changes in pH, titratable acidity, viable cell counts and colour were noted during ii refrigerated storage of the yogurt for 15 days; no changes in syneresis was observed in the control and bioactive added samples. By the end of the storage period, 63.31±3.20 % and 84.81±3.17 % of curcumin and CGA, respectively, were retained in the yogurt samples. The yogurt samples with added bioactive compounds were well accepted by consumer sensory evaluation panellists. Thus, from the obtained data it can be concluded that coconut yogurt may be a potential delivery medium for health promoting curcumin and CGA to consumers. iii Acknowledgement I am indebted to many individuals for their help and encouragement rendered while conducting this research work. Firstly, I would like to express my gratitude to the Riddet Institute, Palmerston North, New Zealand for giving me the opportunity to work on this project by providing financial support. I express my profound thanks to Dr. Harjinder Singh (Director, Riddet Institute) for his valuable comments during conceptualizing of the project. This work would not have been possible without the constant guidance of all my thesis advisors. I am especially indebted to my supervisor, Dr. Tony N Mutukumira (Senior Lecturer, School of Food and Nutrition, College of Sciences, Massey University) for believing in me and proving his valuable comments, guidance, and encouragement starting from project conceptualization, conducting trials and to submission of the final thesis. I am also grateful to Dr. Mutukumira‘s open door policy that makes him more accessible and allowed me to run into his office whenever I was into trouble. Thank you Dr. Mutukumira for letting me freely explore research environment and identify my niche in that. I would also like to express my gratitude to Dr. Kay Rutherfurd-Markwick (Associate Professor, School of Health Sciences, College of Health, Massey University) and Dr. Martin Dickens (Senior Lecturer, School of Health Sciences, College of Health, Massey University) for their help while drafting the project and working with cell culture. Dr. Rutherfurd- Markwick and Dr. Dickens are also thanked for helping with the interpretation of the results and sharing their valuable knowledge. Dr. Rohith Thota (Research Officer, Riddet Institute, Massey University) is thanked for his guidance in conceptualizing the work. Furthermore, I want to thank PC Tong (Human Nutrition Laboratory Manager, School of Sport, Exercise and Nutrition, Massey University) for always being there to help me and solving all my childish problems while handling cells. I am also grateful to Rachel Liu (Food Microbiology Laboratory Manager, Institute of Food Science and Technology, Massey University) and Negah Nikanjam (Institute of Food Science and Technology, Massey University) for helping with the procurement of the required materials and making me acquaint with various equipment‘s available in their respective labs. I also express my profound thanks to my Dr. Kaio Vitzel (Lecturer, School of Health Sciences, College of Health, Massey University) for his inputs during qRT-PCR. iv I would also like to thank all my friends for their moral support and encouragement during the tough times. All the members of G Gang and Sevenzzz are deeply acknowledged as they had been a motivational force at some or the other time in my journey. Special thanks to Ghoda, Saummya, Jaini, Sanju, Yathu, and Kunal for bearing all my tantrums and helping me during all thick and thin. You people are the safety net that gave me enough strength to aim high. I am grateful to Saummya, Gautam and Preetha for helping me with editing my work despite the fact that they didn‘t understand it most of the time. I am also thankful to my batch-mates Arthur Jonathan, Lisa Ning and Doris for always finding time to engage with me in random discussions. I am thankful to Nitesh, Aditya, and Sanjay for making a home away from home. Lastly, I would like to thank my sister Diksha for always being my spine. v Dedicated to all the sacrifices made by my parents. Thank you for believing in my dreams and letting me fly. ! औ ! Translated by Neelabh Utkarsh vi Table of Content Content Page No. Abstract i Acknowledgement iii Table of content vi List of figures x List of tables xiii Abbreviation and terminology xiv CHAPTER 1: INTRODUCTION 1 1.1 Background 1 1.2 Aim and objectives 4 CHAPTER 2: LITERATURE REVIEW 6 2.1 Introduction 6 2.2 What is inflammation? 6 2.3 Events during inflammation 7 2.4 Cell mediators of inflammation 9 2.4.1 Cytokines 9 2.4.2 Reactive oxygen species (ROS) and reactive nitrogen species (RNS) 10 2.4.3 Arachidonic acid metabolites 12 2.5 Bioactive compounds and inflammation 13 2.5.1 Curcumin 20 2.5.1.1 Curcumin and inflammation 22 Evidence for the anti-inflammatory properties of curcumin Evidence for in vitro studies Evidence for animal studies Evidence for clinical trials 2.5.1.2 Bioavailability and metabolism of curcumin 31 2.5.1.3 Safety of curcumin 31 2.5.1.4 Co-administration of curcumin with other bioactives 32 2.5.2 Chlorogenic acid 33 2.5.2.1 Chlorogenic acid and inflammation 35 Evidence for the anti-inflammatory properties of chlorogenic acid Evidence for in vitro studies Evidence for animal studies Evidence for clinical trials 2.5.2.2 Metabolism of chlorogenic acid 40 2.5.2.3 Safety of chlorogenic acid 40 2.5.3 Lupeol 41 vii 2.5.3.1 Lupeol and inflammation 43 Evidence for the anti-inflammatory properties of lupeol Evidence for in vitro studies Evidence for animal studies 2.5.3.2 Safety of lupeol 47 2.6 Delivery of bioactive compounds via food matrices 47 CHAPTER 3: RESEARCH METHODOLOGY 49 3.1 Introduction 49 3.2 Part 1: In vitro studies 49 3.2.1 Cell line 49 3.2.2 Medium and solutions 50 3.2.2.1 Cell work 50 3.2.2.2 Preparation of bioactive compounds 51 3.2.2.3 Enzyme-linked immunosorbent assay (ELISA) 51 3.2.2.4 Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) 52 3.2.3 Methods for in vitro studies 53 3.2.3.1 Culturing of THP-1 cells 53 3.2.3.2 Differentiation of THP-1 Cells 53 3.2.3.3 Treatment of THP-1 cells with bioactive compounds and LPS 54 3.2.3.4 MTT assay 54 3.2.3.5 Quantification of TNF-α using ELISA 55 3.2.3.6 Relative gene expression using two-step qRT-PCR 59 (a) Extraction and purification of total RNA 59 (b) Complementary DNA (cDNA) synthesis from RNA 59 (c) qRT-PCR 59 3.3 Part 2: Preparation of coconut cream yogurt with added bioactive compounds 61 3.3.1 Ingredients in yogurt 61 3.3.2 Stage 1: Quantification and optimisation of bioactive compounds added to coconut cream yogurt 63 3.3.2.1 Quantification of bioactive compound in coffee and turmeric 63 (a) Extraction of curcumin and CGA 63 (b) HPLC analysis for CGA 63 (c) HPLC analysis for curcumin 64 3.3.2.2 Preparation of yogurt 64 3.3.2.3 Sensory evaluation 66 3.3.2.4 Fermentation of yogurt in the presence of CGA and curcumin 66 (a) pH and titratable acidity 66 (b) Colour 66 (c) Microbiological analysis 67 3.3.3 Stage 2: Stability of coconut yogurt with added bioactive compounds during storage at 4°C for 15 days 67 viii 3.3.3.1 Analysis of yogurt during storage 68 (a) pH, titratable acidity, colour and microbiological count 68 (b) Syneresis 68 (c) Texture profile analysis 68 (d) Quantification of bioactive compounds using HPLC 68 (e) Consumer sensory evaluation 68 3.4 Data analysis 69 CHAPTER 4: INVESTIGATION OF ANTI-INFLAMMATORY ACTIVITY OF BIOACTIVE COMPOUNDS USING AN IN VITRO MODEL 70 4.1 Introduction 70 4.2 Results and discussion 71 4.2.1 PMA induced differentiation of THP-1 cells 71 4.2.2 LPS induced inflammation in THP-1 macrophages 73 4.2.2.1 Optimisation of dilution factor for TNF-α measurement from LPS stimulated cells 74 4.2.2.2 Optimisation of LPS dose-response for stimulation of THP-1 macrophages 74 4.2.2.3 Optimisation of LPS incubation time 76 4.2.3 Anti-inflammatory potential of bioactive compounds in LPS stimulated THP-1 cells 77 4.2.3.1 Effect of different doses of curcumin, lupeol and CGA 77 (a) Treatment with curcumin 77 (b) Treatment with lupeol 79 (c) Treatment with CGA 80 4.2.3.2 Effect of carrier vehicle on LPS stimulated THP-1 macrophages 81 4.2.3.3 Effect of combined treatment of curcumin + lupeol and curcumin + CGA on LPS stimulated THP-1 macrophages 82 4.2.4 Effect of curcumin, CGA and their combination on the NF-κB signalling pathway 85 4.3 Summary 90 CHAPTER 5: DEVELOPMENT OF COCONUT CREAM YOGURT FORTIFIED WITH CURCUMIN AND CHLOROGENIC ACID 91 5.1 Introduction 91 5.2 Results and discussion 92 5.2.1 Stage 1: Optimising the amount of bioactive compounds added to coconut cream yogurt 92 5.2.1.1 Quantification of bioactive compounds present in coffee and turmeric 92 5.2.1.2 Sensory characteristics of coconut cream yogurt with added CGA and curcumin 93 5.2.1.3 Effect of bioactive compounds on fermentation of coconut cream 95 ix (a) Growth of L. bulgaricus and S. thermophilus 95 (b) Development of acidity 96 (c) Change in colour 97 5.2.2 Stage 2: Stability of coconut yogurt with added bioactive compounds during storage at 4±1°C for 15 days 100 (a) Survival of L. bulgaricus and S. thermophilus 100 (b) Acidity 101 (c) Colour 102 (d) Syneresis 103 (e) Firmness 105 (f) Retention of curcumin and CGA in yogurt 106 (g) Consumer sensory evaluation 107 5.3 Summary 109 CHAPTER 6: CONCLUSION 110 CHAPTER 7: RECOMMENDATIONS 111 References 112 Appendices 156 x List of Figures Figure No. Title Page No. Figure 2.1 Key events during inflammatory response of body and migration of leukocytes. 8 Figure 2.2 Cyclooxygenase (COX) and lipoxygenase (LOX) pathways for production of arachidonic acid metabolites. 12 Figure 2.3 Keto-enol isomers of curcumin. 20 Figure 2.4 Degradation of curcumin (A) at alkaline pH; (B) autoxidation in solvent; (C) photo-oxidation when in crystalline or aqueous state; and (D) photo-oxidation when in specific solvent like isopropanol. 21 Figure 2.5 Various molecules targeted by curcumin. 23 Figure 2.6 Chemical structure of different isomers of chlorogenic acid. 34 Figure 2.7 (A) Chemical structure of lupeol, and (B) key steps of mevalonate (MVA) biosynthesis pathway of lupeol in plant cells. 42 Figure 3.1 Treatment of THP-1 cells with bioactives and LPS, and cytotoxicity analysis using MTT assay. 56 Figure 3.2 Summary of protocol used for the detection of TNF-α using sandwiched ELISA. 58 Figure 3.3 Experimental design used for the development of coconut cream yogurt enriched with chlorogenic acid (CGA) and curcumin. 62 Figure 3.4 Production of coconut yogurt containing curcumin and chlorogenic acid (CGA). 65 Figure 4.1 Effect of PMA concentration on differentiation of THP-1 cells. 72 Figure 4.2 Optimum dilution factor for supernatant containing TNF-α. 75 Figure 4.3 Effect of lipopolysaccharide (LPS) concentration on (A) TNF-α production and (B) cell viability. 76 Figure 4.4 Effect of lipopolysaccharide (LPS) incubation time on (A) TNF- α production and (B) cell viability. 77 xi Figure 4.5 Effect of curcumin dose on (A) TNF-α production and (B) cell viability. 78 Figure 4.6 Effect of lupeol dose on (A) TNF-α production and (B) cell viability. 79 Figure 4.7 Effect of chlorogenic acid (CGA) dose on (A) TNF-α production and (B) cell viability. 81 Figure 4.8 Effect of carrier vehicles on (A,C) TNF-α production and (B,D) cell viability. 83 Figure 4.9 Effect of curcumin + lupeol on (A) TNF-α production and (B) cell viability. 84 Figure 4.10 Effect of curcumin + chlorogenic acid (CGA) on (A) TNF-α production and (B) cell viability. 85 Figure 4.11 Effect of curcumin, chlorogenic acid (CGA) and their combination on mRNA expression of several inflammatory biomarkers and NF-κB signalling pathway. 87 Figure 4.12 Response of curcumin, chlorogenic acid (CGA) and their combination on mRNA expression of NF-κB signalling pathway. 89 Figure 5.1 Viable cell counts of (A) L. bulgaricus and (B) S. thermophilus in coconut cream yogurt (with or without bioactive compounds) during fermentation at 42±1ºC for 8 h. 96 Figure 5.2 Changes in (A) pH and (B) titratable acidity of coconut cream yogurt (with or without bioactive compounds) during fermentation at 42±1ºC for 8 h. 97 Figure 5.3 Visual changes in the colour of coconut cream yogurt (with or without bioactive compounds) during fermentation at 42±1ºC for 8 h. 98 Figure 5.4 Changes in (A) L*, (B) a* and (C) b* values of coconut cream yogurt (with or without bioactive compounds) during fermentation at 42±1ºC for 8 h. 99 Figure 5.5 Viable cell counts of (A) L. bulgaricus and (B) S. thermophilus in coconut cream yogurt (with or without bioactive compounds) during storage at 4±1ºC for 15 days. 100 Figure 5.6 Changes in (A) pH and (B) titratable acidity of coconut cream 102 xii yogurt (with or without bioactive compounds) during storage at 4±1ºC for 15 days. Figure 5.7 Changes in (A) L*, (B) a* and (C) b* values of coconut cream yogurt (with or without bioactive compounds) during storage at 4±1ºC for 15 days. 103 Figure 5.8 Syneresis (%) in coconut cream yogurt (with or without bioactive compounds) during storage at 4±1ºC for 15 days. 105 Figure 5.9 Firmness (g) (A) of coconut cream yogurt (with or without bioactive compounds) during storage at 4±1ºC for 15 days. (B) Typical texture profile plot for yogurt with added curcumin and CGA at day 5. 106 Figure 5.10 (A) Retention (%) of curcumin and CGA in coconut cream yogurt during storage at 4±1ºC for 15 days. HPLC chromatogram for (B) curcumin and (C) CGA. 107 Figure 5.11 Spider web showing the average sensory score for (A) control (B) curcumin and CGA added yogurt during storage at 4±1ºC for 15 days. 108 xiii List of Tables Table No. Title Page No. Table 2.1 Major pro- and anti-inflammatory cytokines produced during inflammation. 11 Table 2.2 Bioactive compounds and their associated health benefits. 14 Table 2.3 In vitro and animal evidences of anti-inflammatory properties of bioactive compounds. 15 Table 2.4 Pre-clinical and clinical studies showing the anti-inflammatory effects of curcumin. 27 Table 2.5 Pre-clinical and clinical studies showing the anti-inflammatory effects of chlorogenic acid (CGA). 37 Table 2.6 Pre-clinical and clinical studies showing the anti-inflammatory effects of lupeol. 44 Table 3.1 Primer sequences used for qRT-PCR. 60 Table 3.2 List of ingredients used in the production of coconut cream yogurt. 61 Table 5.1 Pugh decision matrix for screening coconut yogurt fortified with curcumin and chlorogenic acid (CGA). 95 xiv Abbreviation and Terminology Abbreviation Terminology ANOVA Analysis of variance Cfu/g Colony forming unit per gram CGA Chlorogenic acid CO2 Carbon dioxide COX Cyclooxygenase ºC Degree Celsius DNA Deoxyribonucleic acid DMSO Dimethyl sulfoxide ELISA Enzyme-linked immunosorbent assay FBS Fetal bovine serum FAO Food and agriculture organisation g Gram h Hour iNOS Inducible nitric oxide synthase IL Interleukin L. bulgaricus Lactobacillus bulgaricus LPS Lipopolysaccharide LOX Lipoxygenase L Litre mRNA Messenger ribonucleic acid µM Micromoles ml Millilitre mg Milligram min Minute MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor nm Nanometre NO Nitric oxide NF-κB Nuclear transcription factor kappa-B % Percentage xv PBS Phosphate buffered saline pH Potential of hydrogen PMA Phorbol 12-myristate 13-acetate PGE-2 Protagladin-E2 qRT-PCR Quantitative reverse transcriptase polymerase chain reaction rcf Relative centrifugal force ROS Reactive oxygen species SDS Sodium dodecyl sulfate SPSS Statistical package for the social sciences S. thermophilus Streptococcus thermophilus TNF- α Tumor necrosis factor-alpha UV-vis Ultraviolet–visible WHO World health organisation 1 CHAPTER 1 Introduction 1.1 Background Today, food is not only consumed to curtail hunger and provide necessary macro- and micro- nutrients but also to achieve additional mental and physiological health benefits (Betoret, Betoret, Vidal, & Fito, 2011; Galanakis, 2017; Sun‐Waterhouse, 2011; Szakály, Szente, Kövér, Polereczki, & Szigeti, 2012). Traditionally, therapeutic effects were derived naturally from a diet composed of fruits, vegetables, cereals, dairy, and meat. However, today‘s busy lifestyle has led to an increase in the consumption of processed food over fresh food (Mozaffarian, Hao, Rimm, Willett, & Hu, 2011). To compensate for inadequate intake of nutrients and therapeutic non-nutritive compounds from processed food, there is an increase in the intake of dietary supplements (Gahche et al., 2011). Hence, there is a demand from the modern health-conscious consumer who wants food with balanced calories that can improve their health, leading to the development of contemporary so-called ―functional foods‖ (Bech- Larsen & Scholderer, 2007; Galanakis, 2017). The increasing demand is the result of a number of factors such as urbanisation, busy lifestyle, increasing healthcare cost, growing awareness, surge in life expectancy and the desire to improve the quality of life (Kaur & Das, 2011; Roberfroid, 2000; Siro, Kápolna, Kápolna, & Lugasi, 2008). Therefore, the development of functional foods is one of the most growing research area (Silva, Barreira, & Oliveira, 2016). The term ―functional food‖ was first introduced in Japan in the mid-1980s and was referred to as ―food for specified health uses (FOSHU)‖ (Ohama, Ikeda, & Moriyama, 2008). The American Dietetic Association defines functional food as ―foods that are in forms of whole, fortified, enriched or enhanced foods that provide a functional advantage and/or health benefits beyond basic nutrition, when consumed at an effective level on a regular basis‖ (Thomson et al., 1999). In Europe, there is no legal definition but a working definition explains functional food as ―a food that beneficially affects one or more target functions in the body beyond adequate nutritional effects in a way that is relevant to either an improved state of health and well-being and/or reduction of risk of diseases, and it is consumed as part 2 of a normal food pattern (not a pill, a capsule or any form of dietary supplement)‖ (European Commission, 2010). Despite disparities in the legal definition of function food, its market worth is increasing. The functional food market was worth approximately USD 200 billion in 2013 and is expected to increase above USD 300 billion by 2020 (Santeramo et al., 2018). A large number of functional foods are commercially available such as vitamin and mineral fortified fruit juice, vitamin D and calcium fortified milk, vitamin and mineral fortified bread, catechin enriched green tea, cereals with soluble fibres, Omega-3 enriched eggs and yogurt with probiotics and prebiotics. The health of an individual is greatly affected by their diet and lifestyle. The World Health Organisation (WHO) and the Food and Agriculture Organisation (FAO) have reported several dietary patterns and lifestyle habits that can lead to the development of chronic diseases (WHO, 2003). Chronic diseases were the leading cause of about 60 % mortality across the globe in 2005 which further increased to approximate 68 % in 2012 (Tsai, Lin, & Wu, 2016; WHO, 2014). Chronic diseases are also an economic burden on society and are estimated to cost about USD 7 trillion during 2011-25 in low- and middle-income countries (WHO, 2014). Chronic diseases such as obesity, type-2 diabetes, arthritis, asthma, bronchitis, pancreatitis, cardiovascular, neurodegenerative, colitis, multiple sclerosis, and metabolic diseases as well as some types of cancer are the onsets lead by long-term uncontrolled or chronic inflammation (He et al., 2015; Hewlings & Kalman, 2017; Panahi et al., 2016). During chronic inflammatory responses several complex cellular signalling pathways are activated in the body, resulting in increased levels of inflammatory biomarkers including transcription factors such as nuclear transcription factor kappa-B (NF-κB); inflammatory cytokines and chemokines such as tumour necrosis factor-alpha (TNF-α), and interleukin (IL- 6); and inflammatory enzymes such as cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) (Franceschi & Campisi, 2014; He et al., 2015; Lin & Tang, 2008; Prasad & Aggarwal, 2014). To control inflammatory responses, one needs to combat the stimulating pathogen and/or reduce the production of cell mediator‘s (Liang & Kitts, 2015). Several anti-inflammatory drugs have been developed to control chronic inflammation but long-term consumption of these drugs may result in side effects such as bleeding in the stomach and predisposition to 3 ulcers (Laine, 2001). Therefore, there is a growing interest in alternative treatments that are more natural and can be a part of the diet (Khan, Grigor, Winger, & Win, 2013). Traditionally plant-based diets and its derivatives have been used to prevent inflammation and thereby related chronic diseases (Mueller, Hobiger, & Jungbauer, 2010). The therapeutic effect of food is primarily associated with the presence of bioactive compounds (Arvanitoyannis & Van Houwelingen-Koukaliaroglou, 2005; Day, Seymour, Pitts, Konczak, & Lundin, 2009; Herrero, Plaza, Cifuentes, & Ibáñez, 2010; Vicentini, Liberatore, & Mastrocola, 2016). Bioactive compounds are present in fruits, vegetables, and whole grains and can exert physiological effects on consumer‘s body (Astley & Finglas, 2016; Galanakis, 2017). Certain bioactive compounds can suppress inflammatory biomarkers and reduce oxidative stress in cells, thereby help in averting chronic inflammation-related diseases (Astley & Finglas, 2016; Calixto, Otuki, & Santos, 2003). For example, a study by Ha, Park, Eom, Kim, and Choi (2012), reported a decrease in expression of iNOS and COX-2 following administration of the narirutin fraction of citrus peels in lipopolysaccharide (LPS) stimulated macrophages. Similar results have been reported in other in vitro and in vivo studies testing a variety of different bioactive compounds (Joseph, Edirisinghe, & Burton- Freeman, 2016; Zhang & Tsao, 2016; Zhu, Du, & Xu, 2018). Curcumin is one such bioactive that has been extensively studied for its health benefits. It is the main phenolic compound that is extracted from the rhizome of the turmeric plant (Wilken, Veena, Wang, & Srivatsan, 2011). Its therapeutic benefits include anti-oxidant, antiseptic, anti-tumour, anti-malarial, analgesic, anti-obesity and anti-inflammatory properties (Amalraj, Pius, Gopi, & Gopi, 2017). It can exert anti-inflammatory properties by acting on multiple biomarkers including transcription factors, enzymes, pro-inflammatory cytokines and chemokines, and free radical (Lozada-García et al., 2017; Yunes Panahi et al., 2015). However, the low bioavailability of curcumin has been a challenge. Due to the low bioavailability of curcumin high dose is required to deliver its intended benefits in humans. Previous studies have reported a minimum dosage of 3.6 g/day of curcumin is required to achieve measurable plasma levels in humans (Anand, Kunnumakkara, Newman, & Aggarwal, 2007; Cui et al. 2009). It is possible that this high dose requirement may be compensated by co-administrating curcumin with other bioactive compounds that may result in a synergistic effect. 4 Although less studied than curcumin, chlorogenic acid (CGA) and lupeol are two bioactive compounds obtained primarily from coffee and several fruits, respectively, which have been reported to have similar therapeutic effects. Initial studies reported that CGA and lupeol can downregulate multiple inflammatory pathways, similar to curcumin, principally by suppressing oxidative stress and the activation of NF-κB (Liang & Kitts, 2015; Salminen, Lehtonen, Suuronen, Kaarniranta, & Huuskonen, 2008). It is hypothesised that administration of curcumin with CGA or lupeol may result in a synergistic anti-inflammatory effect. High dose requirement can also be reduced by enhancing the bioavailability of bioactive compounds. Presently, most of the bioactive compounds are consumed as dietary supplements in the form of pills and capsules but bioavailability can be improved by delivering these compounds via appropriate food matrices (Rodríguez-Roque et al., 2016). Milk and milk products may form a suitable delivery vehicle as they can solubilise hydrophobic bioactive compounds (such as curcumin, CGA and lupeol) in their oil phase (Cuomo et al., 2011; Jakobek, 2015; Rege & Momin, 2017). In addition, the acidity of the delivery medium can improve the stability of bioactive compounds. Hence, coconut milk yogurt, a fermented product with a final pH ≈4.5-4.6 (Fazilah, Ariff, Khayat, Rios-Solis, & Halim, 2018), may be an effective vehicle to deliver bioactive compounds for human consumption. 1.2 Aim and objectives The aim of this work was to identify the most promising anti-inflammatory combination of curcumin with CGA and/or lupeol and thereby develop a coconut cream yogurt to deliver the combination of bioactive compounds for human consumption. To achieve the targeted aim, the experimental investigations were divided into three major objectives:  Objective 1: To investigate the effective synergistic combination of curcumin with CGA and lupeol on reducing TNF-α secretion; The anti-inflammatory effects of curcumin in combination with different amounts of CGA and lupeol were studied using an in vitro model of human TPH-1 monocyte cell line by assessing changes in the production of the pro-inflammatory cytokine TNF-α.  Objective 2: To investigate the effect of the most promising synergistic combination of bioactive compounds on other inflammatory pathways; 5 The synergistic combination was studied for its effect on mRNA expression of inflammatory biomarkers such as IL-6, IL-10, TNF-α, NF-κB, iNOS, and COX-2.  Objective 3: To develop a coconut cream yogurt containing the synergistic combination of bioactive compounds suitable for human consumption. The effective concentration of bioactives were added to coconut cream yogurt and their effect on the fermentation process was studied. The resulting yogurt was analysed for its sensory attributes, physical-chemical and microbiological stability. 6 CHAPTER 2 Literature Review 2.1 Introduction Lifestyle changes have increased the occurrence of ―modern life disorders‖ including chronic inflammation, which is occurring over a wide range from children to adults. Chronic inflammation is important as it may play a key role in triggering other deadly diseases such as asthma, arthritis, type-2 diabetes, heart disease and several forms of cancers (Franceschi & Campisi, 2014; Meirow & Baniyash, 2017). Many food-derived bioactive compounds such as epigallocatechin-3-gallate (EGCG), gingerol, naringenin, quercetin, resveratrol, silymarin, tocopherol, β-carotene, genistein, chrysin, curcumin, lupeol and chlorogenic acid (CGA) have been documented for their anti- inflammatory properties (Fernández-Mar, Mateos, García-Parrilla, Puertas, & Cantos-Villar, 2012; Gil-Cardoso et al., 2016; Prabhala, Pai, & Prabhala, 2013; Yu, Bi, Yu, & Chen, 2016; Zhu, Du, & Xu, 2018). Therefore, recent research has focused on the potential of these naturally occurring bioactive compounds to aid in the prevention or treatment of chronic inflammation. This review focuses on outlining the events that occur during inflammation and the mechanisms of bioactive compounds such as curcumin, lupeol, and CGA in suppressing uncontrolled inflammatory responses. 2.2 What is inflammation? Inflammation is the first complex biological response by the host to any stimuli such as invasion by a foreign body like microbes, toxins, dirt or burns or cuts or tissue necrosis or radiation (Cavaillon, 2017a; Prabhala et al., 2013). According to Kumar, Abbas, and Aster (2012c) page 29: “Inflammation is a protective response involving host cells, blood vessels, and proteins and other mediators that is intended to eliminate the initial cause of cell injury, as well as the necrotic cells and tissues resulting from the original insult, and to initiate the process of repair” 7 The inflammatory response can be: (i) rapid and short-term called acute inflammation, (ii) or be for longer duration called chronic inflammation (Ward, 2010). Acute inflammation can last for a few minutes to a few days and is predominantly the response of neutrophilic leukocytes to mild and self-limiting tissue injuries resulting in prominent local and systemic signs (Ward, 2010). Chronic inflammation is the result of severe and progressive injury, predominantly due to macrophages responses that can last for several days (Ward, 2010). Chronic inflammation can be caused by: (i) continuous infection caused by microbes that are difficult to eliminate, such as Mycobacterium tuberculosis, fungi and viruses; (ii) auto-immune responses resulting in diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease; (iii) allergies like bronchial asthma; and (iv) persistent contact with toxic elements e.g. inhalation of silica causing silicosis: a chronic inflammatory response in the lungs (Hnizdo & Vallyathan, 2003; Kumar et al., 2012c; Shacter & Weitzman, 2002). Uncontrolled chronic inflammation may also contribute to the onset of diseases such as Alzheimer, type-2 diabetes, metabolic syndrome and several forms of cancer that traditionally were not considered to be related to inflammation (Franceschi & Campisi, 2014; Kumar et al., 2012c). 2.3 Events during inflammation Any stimuli entering beyond the first line of defence (the skin and mucus layer) triggers multiple events at the cellular level. The immune response is the combined result of a number of different cells including monocytes, mast cells, dendritic cells, and T, B and NKT lymphocytes and is not limited to only one cell type (Prabhala et al., 2013). The cellular events during inflammation can be summarised as shown in Figure 2.1. Stimuli such as bacteria express a certain molecular pattern of proteins on their surface, called the pathogen- associated molecular pattern (PAMP), which can be detected by the receptors such as toll-like receptors (TLR) present on the cells, particularly on mast cells and macrophages (Cavaillon, 2017b; Kumar et al., 2012c). On recognising the pathogen, the mast cell releases histamine stored in its granules (Amin, 2012), while macrophages produce cytokines such as TNF-α and IL-6 which as a result initiate the events in blood vessels (Dunster, 2016). The main reason for changes in vessels is to increase the blood flow at the site of action. Histamine and cytokines leads to vasodilation and increased blood flow (Benly, 2015). This allows more blood cells and protein to be delivered to the place of injury. Histamine also 8 Figure 2.1: Key events during inflammatory response of body and migration of leukocytes. 1: Invasion of bacteria in body; 2: mast cells and macrophages detect bacteria; 3: secretion of histamine and cytokines from their respective cells; 4: vasodilation and increase in blood vessel permeability resulting in fluid leakage. 1‘: leukocyte moving towards blood vessel wall; 2‘: rolling of leukocyte to form weak adhesion; 3‘: strong adhesion of leukocyte with endothelial cells; 4‘: leukocyte squeezes between endothelial cells; 5‘: leukocyte migrates towards the site of action; 6‘: bacteria engulfed by leukocyte; and 7‘: bacteria ingested by leukocyte and release of cytokines, reactive oxygen species (ROS) and nitric oxide (NO) (Created using Microsoft PowerPoint, 2016) 9 causes contraction of the endothelial cells which increases vessel permeability (Ashina et al., 2015), and aids in moving the fluid carrying proteins and blood cells into extra-vascular tissues so that repair mechanisms can be established (Benly, 2015). During vasodilation, cytokines and histamine also activate the adhesion molecules on the endothelial cells that facilitate the migration of leukocytes out from the vessel (Shalova, Saha, & Biswas, 2017). The type of leukocyte leaving the blood vessels will depend on the duration of the inflammatory response. During acute inflammation neutrophils are dominant because these cells are more abundant in the blood than monocytes, hence they respond more rapidly to mediators (Kumar et al., 2012c). However, after entering the tissues, neutrophils die within 24-48 h while macrophages (developed from monocytes) can survive longer (Kumar et al., 2012c). Hence macrophages dominate during chronic inflammation. The leukocytes move towards the site of action by following a chemical gradient by a process called chemo-taxis (Shalova et al., 2017). At the site of infection, the leukocytes can bind to microbes, damaged cells, and foreign bodies and results in phagocytosis (Shalova et al., 2017). Leukocytes then produce substances like reactive oxygen species (ROS), nitric oxide (NO) and liposomal enzyme to degrade the engulfed material. 2.4 Cell mediators of inflammation The complex cascades of cellular events during inflammation involve the production of enzymes, pro-inflammatory cytokines and chemokine, and other chemical mediators that eventually eliminates the pathogen and heal the injured tissues. Macrophages, mast cells and other cell involved in inflammatory response can produce a number of cell mediators as described below. 2.4.1 Cytokines Cytokines are small proteins which are synthesised and secreted by immune cells (principally from macrophages and lymphocytes) to commence, amplifies, prolong and proliferate inflammation process (Holdsworth & Gan, 2015; Prabhala et al., 2013). Cytokines can act on their producer cells (autocrine) or neighbouring cells (paracrine) or on cells away from their production site (endocrine) (Kumar et al., 2012b; Zhang & An, 2007). Broadly, cytokines are classified as pro-inflammatory and anti-inflammatory (Table 2.1). Pro-inflammatory cytokines such as TNF-α and IL-6 upregulate inflammatory responses while anti- 10 inflammatory cytokines such as IL-10 downregulate and have a negative feedback on pro- inflammatory cytokine responses (Corwin, 2000). An imbalance between productions of pro- and anti-inflammatory cytokines may be responsible for chronic inflammatory responses in the body resulting in the damage to host cells (Holdsworth & Gan, 2015). 2.4.2 Reactive oxygen species (ROS) and reactive nitrogen species (RNS) After macrophage engulf bacteria, two independent antibacterial pathways are stimulated involving (i) nicotinamide adenine dinucleotide phosphate (NADPH) phagocyte oxidase (or NADPH oxidase) and (ii) inducible nitric oxide synthase (iNOS), which results in the production of ROS and NO respectively (Mittal, Siddiqui, Tran, Reddy, & Malik, 2014; Swindle & Metcalfe, 2007). Both ROS and NO are free radicals that can react with organic and inorganic chemicals to kill the bacteria (Kumar, Abbas, & Aster, 2012a). However, during chronic inflammation high amounts of ROS and NO are produced that may damage the host cells (Soufli, Toumi, Rafa, & Touil-Boukoffa, 2016). During inflammation, NADPH phagocyte oxidase can produce ROS in the cytoplasm and phagosome of macrophage. NADPH phagocyte oxidase once activated by pro-inflammatory cytokines like TNF-α, will utilise NADPH and oxygen (O2) to produce superoxide (O2 •- ) by the process called the oxidative bust or respiratory bust (Mittal et al., 2014). O2 •- has very low half-life, therefore it rapidly converts to hydrogen peroxide (H2O2), which is then converted to highly reactive hypochlorous acid (HOCl) by the enzyme myeloperoxidase (MPO) or the eosinophil peroxidase (EPO) (Swindle & Metcalfe, 2007). In the presence of metal ions such as iron (Fe 2+ ) and copper (Cu 2+ ), H2O2 can undergo the Fenton or Haber Weiss reaction to produce hydroxyl radical (OH • ) and hydroxyl anion (OH - ) (Mittal et al., 2014). NO is the principle reactive specie of nitrogen produced de novo by oxidation of an amino acid L-arginine in the presence of NADPH (Kumar et al., 2012a). During inflammation, this reaction is carried out in macrophages or other inflammatory cells by the iNOS (Fang, 2004; Forrester, Kikuchi, Hernandes, Xu, & Griendling, 2018; Soufli et al., 2016). NO can further react with O2 to produce nitrogen dioxide (NO2 • ), which can then react with itself to yield one molecule of dinitrogen tetraoxide (N2O4) (Swindle & Metcalfe, 2007). The presence of PAMP and cytokines such as TNF-α, and IL-6 can trigger cascades like NF-κB and Janus- activated kinase-signal transducer and activator of transcription (JAK-STAT) leading to transcription of iNOS (Fang, 2004). 11 Table 2.1: Major pro- and anti-inflammatory cytokines produced during inflammation. Cytokine Principal Cell Source Major Activity Pro-inflammatory TNF-α Macrophages, mast cells and dendritic cells Stimulate adhesion of leukocytes to endothelial cells and the production of other pro-inflammatory cytokines e.g. IL-1, IL-2 and IL-6, induces death of host cells resulting in pain and fever IFN-γ T cells and NK cells Activates macrophages, influence B cell proliferation, increases expression of antigens and induce death of host cells MIP-1α Macrophages Chemo-taxis TGF-β T cells and monocytes Chemo-taxis and synthesis of IL-1 and IgA IL-1α and IL-1β Macrophages, neutrophils, B cells, endothelial cells and other cell types Recruitment of macrophages and neutrophils to inflammation site, stimulates the production of IL-6, increases vessel permeability and promotes tumor development IL-5 Mast cells and T cells Stimulates growth and functioning of B cells and eosinophils IL-6 Macrophages, T cells and fibroblasts Stimulates proliferation of B cells, works synergistically with TNF and IL-1, activates and recruits macrophages to inflammation site, activates acute-phase proteins, acts on hypothalamus to induce sickness behaviors e.g. fever and anorexia IL-8 Macrophages Increases vessel permeability and recruits neutrophils to inflammation site via chemo-taxis IL-12 Macrophages, B cells and dendritic cells Induces proliferation of NK cells, stimulates IFN production Anti-inflammatory IL-4 Mast cells, T cells and NK cells Stimulates growth and functioning of B cells, suppresses production of IL-1 and TNF IL-10 T cells, B cells and macrophages Stimulates proliferation of B cells, suppresses IL-1 synthesis in macrophages IL-11 Bone marrow Promotes growth of bone marrow cells, activates acute-phase proteins IL-13 T cells Down-regulates the production of pro-inflammatory cytokines similar to IL-4 TNF: tumour necrosis factor; IFN: interferon; NK: natural killer; MIP: macrophage inflammatory proteins; TGF: transforming growth factor; IL: interleukin; IgA: immunoglobulin A. Adapted from: (Corwin, 2000; Neurath, 2014; Zhang & An, 2007) 12 2.4.3 Arachidonic acid metabolites Arachidonic acid is a polyunsaturated fatty acid which is present in the human body as an esterified cell membrane phospholipid. Production of cytokines such as TNF-α and IL-6 and ROS and NO triggers the release of free arachidonic acid from the membrane phospholipids by activating enzyme phospholipase A2 (Li, Gao, Du, Cheng, & Mao, 2018). Free arachidonic is metabolised by two enzymatic pathways: (i) cyclooxygenase (COX) or (ii) lipoxygenase (LOX) to synthesise various classes of eicosanoids (Figure 2.2) (Meirer, Steinhilber, & Proschak, 2014; Prabhala et al., 2013), that result in an increase in vascular permeability, vasodilation, vasocontraction and bronchoconstriction (Prabhala et al., 2013). Figure 2.2: Cyclooxygenase (COX) and lipoxygenase (LOX) pathways for production of arachidonic acid metabolites. PG(G2, H2, D2, I2, F2αa): prostaglandin (G2, H2, D2, I2, F2αa); TXA2: thromboxane A2; 5- HPETE: 5-hydroperoxyeicosatetraenoic acid; 5-HETE: 5-hydroxyeicosatetraenoic acid; LT(A4, B4, C4, D4, E4): leukotriene (A4, B4, C4, D4, E4); LX(A4, B4) : lipoxin (A4, B4). Adapted from: (Kumar et al., 2012c; Meirer et al., 2014) (Created using Microsoft PowerPoint, 2016) 13 COX initially downregulate arachidonic acid to produce unstable prostaglandin G2 (PGG2) which is further transformed into more stable prostaglandin H2 (PGH2) (Meirer et al., 2014). COX has two isoforms, of which COX-1 is always expressed to maintain prostaglandin homeostasis while COX-2 on other hand is expressed as a result of pro-inflammatory cytokines, bacteria or growth factors (Funk, 2001; Li et al., 2018; Prabhala et al., 2013). Both COX-1 and COX-2 function similarly to produce PGH2. Further downregulation of PGH2, produces various prostaglandins and thromboxane (Meirer et al., 2014). Of all the prostaglandins produced, prostaglandin E2 (PGE2) is the most critical during inflammation as it is responsible for inflammatory symptoms of pain and fever (Funk, 2001; Hwang, Wecksler, Wagner, & Hammock, 2013). LOX-5 is the key enzyme involved in arachidonic acid metabolism in neutrophils. LOX-5 oxidises arachidonic acid to produce intermediary 5-hydroperoxyeicosatetraenoic acid (5- HPETE) which is then oxidised to produce a series of leukotriene‘s resulting in inflammatory responses. 5-HPETE can also be transformed by 12-LOX to produce lipoxin A4 (LXA4) and lipoxin B4 (LXB4). Both the lipoxins are anti-inflammatory in nature and can inhibit neutrophil chemo-taxis and adhesion to the endothelial surface (Kumar et al., 2012c; Meirer et al., 2014). 2.5 Bioactive compounds and inflammation In addition to the macro- and micro- nutrients, food also contain bioactive compounds which are significant for maintaining the well-being of humans (Galanakis, 2017). Bioactive compounds are naturally occurring extra-nutritional compounds that can affect the metabolic process by targeting whole body or specific tissues or cells, thus, impacting on the function and wellness of the body (Astley & Finglas, 2016; Galanakis, 2017; Torres-Fuentes, Schellekens, Dinan, & Cryan, 2015). Table 2.2 summarises the sources and health benefits of some bioactive compounds. These compounds can have positive biological effects such as anticancer, antibacterial, antifungal, antioxidant, anticoagulant, anti-obesity and anti- inflammatory (Astley & Finglas, 2016; D‘Orazio et al., 2012; Elias, Kellerby, & Decker, 2008; Gooda Sahib et al., 2012; Korhonen & Pihlanto, 2003; Phelan & Kerins, 2011; Torres- Fuentes et al., 2015; Wang et al., 2014). Currently, the role of bioactive compounds are being investigated to develop a novel strategy for the alleviation of chronic inflammation. Some of the bioactive compounds with reported 14 anti-inflammatory effect are shown in Table 2.3. Bioactives can reduce the stress signals produced by cells in response to stimuli thereby control inflammatory responses in the body (Prabhala et al., 2013). Table 2.2: Bioactive compounds and their associated health benefits. Class Bioactive Compounds Major Food Source Health Benefits Bioactive peptide Various peptides Milk, meat and fish Antihypertensive, antioxidant properties or immunomodulatory activities Carotenoids Curcumin, lutein and β- carotenr Carrots, tomato, spinach, apricot, pepper and citrus Prevention against cardio vascular disease, cancer and age related degeneracy Essential Oil Cardamom oleoresin, eugenol and eugenyl acetate Citrus fruit skin, cardamom, marjoram, clove oil and basil oil Antidepressant, antispasmodic and anti- inflammatory properties Prevention against cardio vascular disease, cancer Fatty acid Omega 3 and omega 6 Flex seeds, vegetable oils, fish oil, nuts and egg Anti-inflammatory, anti-carcinogenic and immunomodulatory properties Phenol Caffeine, catechins, chlorogenic acid, ellagic acid, gallic acid, isoflavone, mangiferin, naringenin, quercetin, vanillin, resveratrol, rutin and anthocyanins Fruits like apple, cherries, berries, plums and grapes, legumes, green coffee extract, green tea, olive oil, broccoli and onion Prevention against cancer, neurodegenerative, cardio vascular and metabolic disease Reduce oxidative stress Protein Albumin, hirudin and papain Egg, peas, grains and papaya Antioxidant, age related degeneracy, immunity booster and weight and blood pressure management Organic acid Citric acid and hydroxycitric acid Citrus fruits, malabar tamarind and hibiscus sabdariffa Antioxidant, anti-ageing and weight management Vitamin and mineral Water and fat soluble vitamins, iron, zinc, magnesium, calcium Majorly in all fruits and vegetables, milk and meat Benefits ranging from improved vision and gums to development of bones, and teeth‘s They even play important role as co-factors and co-enzymes in metabolic reactions Adapted from: (Augustin & Sanguansri, 2015; Dias, Ferreira, & Barreiro, 2015; Onwulata, 2013) 15 T a b le 2 .3 : In v it ro a n d a n im a l ev id en ce s o f a n ti -i n fl a m m a to ry p ro p er ti es o f b io a ct iv e co m p o u n d s. S a fe ty f o r h u m a n co n su m p ti o n G R A S (F D A , 2 0 1 3 ) G R A S (F D A , 2 0 1 8 ) D eb at ab le (B o d e & D o n g , 2 0 1 1 a) R ep o rt ed s af e b u t la ck su ff ic ie n t d at a to co n cl u d e sa fe (O n ak p o y a, S p en ce r, T h o m p so n , & H en eg h an , 2 0 1 4 ) E v id en ce s o f a n ti -i n fl a m m a to ry e ff ec ts ( d o se u se d ) A n im a l tr ia l R es u lt (s ) ↓ I L -6 ↓ I L -8 ↓ I L -1 0 ↓ I L -1 8 ↓ T N F -α ↓ C O X ‐2 ↓ T N F -α ↓ I L -6 ↑ I L -1 0 ↓ N O ↓ N O ↓ i N O S E x p er im en t m o d el K 2 C r 2 O 7 i n d u ce d a d u lt m al e w is ta r ra ts ( 2 5 0 m g /k g ) (A l Ja m ei l et a l. , 2 0 1 7 ) 1 6 2 S p ra g u e- D aw le y ( S D ) m al e ra ts f ed w it h h ig h f at d ie t (0 .0 5 % ) (C h o ia e t al ., 2 0 0 6 ) S ep ti c fe m al e S p ra g u e- D aw le y r at s (D em ir b il ek e t al ., 2 0 0 4 ) L P S i n d u ce d m al e IC R m ic e (5 -5 0 m g /k g ) (Z h an g e t al ., 2 0 1 0 ) In v it ro t ri a l R es u lt (s ) ↓ T N F -α ↓ S u p er o x id e an io n ↓ T N F -α ↓ N O ↓ i N O S ↓ P G E 2 ↓ N F -κ B ↓ I L -1 β ↓ i N O S ↓ C O X ‐2 ↓ P G E 2 ↓ N O ↓ T N F -α ↓ I L -6 ↓ i N O S ↓ C O X ‐2 ↓ N O ↓ N F -κ B E x p er im en t m o d el In fl am m at io n i n d u ce d i n fe m al e B A L B /c m ic e b y L P S a n d e ff ec t o f as co rb ic ac id o n c o ll ec te d c el ls ( 6 0 % ly m p h o cy te s an d 4 0 % m ac ro p h ag es ) (0 .0 0 1 -2 .5 m M ) (V ic to r, G u ay er b as , P u er to , M ed in a, & F u en te , 2 0 0 0 ) In fl am m at io n i n d u ce d i n R A W 2 6 4 .7 c el ls s ti m u la te d w it h L P S ( 1 0 -5 0 m M ) (B ai e t al ., 2 0 0 5 ) L P S s ti m u la te d p er it o n ea l m ac ro p h ag es ( 1 0 – 5 0 µ M ) (K im a et a l. , 2 0 0 3 ) In fl am m at io n i n d u ce d i n R A W 2 6 4 .7 c el ls s ti m u la te d b y L P S ( 2 -2 0 µ M ) (H w an g , K im , P ar k , L ee , & K im , 2 0 1 4 ) F o o d s o u rc es P ap ay a, b ro cc o li , k iw i fr u it , m an g o , ca u li fl o w er , st ra w b er ri es , p in ea p p le C ar ro ts , sw ee t p o ta to es , p u m p k in , sp in ac h , ap ri co ts Ja la p eñ o p ep p er s, ca y en n e p ep p er s C o ff ee , te a, eg g p la n t, p o ta to es N a m e A sc o rb ic a ci d β -c ar o te n e C ap sa ic in C h lo ro g en ic ac id 16 T a b le 2 .3 ( co n t. ) L ac k s u ff ic ie n t d at a S af e (E u ro p ea n F o o d S af et y A u th o ri ty e t al ., 2 0 1 8 ) L ac k s u ff ic ie n t d at a (K an g , B u ck n er , S h ay , G u , & C h u n g , 2 0 1 6 ) L ac k s u ff ic ie n t d at a G R A S (B o d e & D o n g ., 2 0 1 1 b ) ↓ T N F -α ↓ I L -1 β ↓ I L -6 ↓ i N O S ↓ N O ↓ N F -κ B p 6 5 ↓ T N F -α ↓ I L -1 β ↓ i N O S ↓ C O X -2 ↓ C O X -2 ↓ i N O S ↓ P G E 2 ↓ T N F -α ↓ I L -1 β ↓ T N F -α ↓ I L -6 ↓ N F -κ B ↓ T N F -α ↓ N F -κ B ↓ i N O S ↓ C O X -2 A d u lt w is ta r ra ts w it h s p in al co rd i n ju ry ( 3 0 -1 0 0 m g /k g ) (J ia n g , G o n g , Z h ao , & L i, 2 0 1 4 ) M al e ad u lt S p ar g u e- D aw le y ra ts w it h s p in al c o rd i n ju ry (5 0 m g /k g ) (K h al at b ar y & A h m ad v an d , 2 0 1 1 ) C ar ra g ee n an -i n d u ce d a d u lt m al e w is ta r ra ts p aw e d em a m o d el ( 1 -3 0 m g /k g ) (M an so u ri e t al ., 2 0 1 5 ) Z y m o sa n -i n d u ce d f em al e C 5 7 B L /6 J m ic e (1 0 -1 5 m g /k g ) (F an e t al ., 2 0 1 7 ) 1 % t w ee n 8 0 i n d u ce d m al e w is ta r ra ts ( 2 5 m g /k g ) (A lg an d ab y e t al ., 2 0 1 6 ) ↓ C O X ‐2 ↓ N O ↓ i N O S ↓ I L -6 ↓ I L -8 ↓ M C P -1 ↓ G -C S F ↓ G M -C S F ↓ R O S ↓ N F κ B ↓ A P -1 ↑ L O X -1 ↓ R O S ↓ i N O S ↓ N O ↓ C O X -2 ↓ P G E 2 ↓ i N O S ↓ N O ↓ T N F -α ↓ I L -1 β ↓ I L -6 ↓ P G E 2 ↓ i N O S ↓ C O X -2 In fl am m at io n i n d u ce d i n R A W 2 6 4 .7 c el ls s ti m u la te d b y L P S ( 0 .0 1 -1 0 0 µ M ) (C h o , 2 0 0 4 ) In fl am m at io n i n d u ce d i n H C E p iC c el ls b y I L -1 β (0 .3 – 3 0 µ M ) (C av et , H ar ri n g to n , V o ll m er , W ar d , & Z h an g , 2 0 1 1 ) In fl am m at io n i n d u ce d i n h u m an u m b il ic al v ei n en d o th el ia l ce ll s b y o x L D L (o x id iz ed l o w -d en si ty li p o p ro te in ) (5 -2 0 µ M ) (L ee e t al ., 2 0 1 0 ) H y p o x ia i n d u ce d in fl am m at io n i n R A W 2 6 4 .7 ce ll s (0 .3 -3 μ M ) (L iu e t al ., 2 0 0 9 ) L P S i n d u ce d i n fl am m at io n in R A W 2 4 6 .7 c el ls ( 5 0 - 3 0 0 μ g /m l) (L ia n g , S an g , L iu , Y u , & W an g , 2 0 1 8 ) B lu e p as si o n fl o w er , p ro p o li s, h o n ey G re en t ea R as p b er ri es , p o m eg ra n at e, b la ck b er ri es , ch er ri es , p ec an s, w al n u ts E v o d ia p la n t G in g er C h ry si n E p ig al lo ca te c- h in g al la te (E G C G ) E ll ag ic a ci d E v o d ia m in e G in g er o l 17 T a b le 2 .3 ( co n t. ) R ep o rt ed s af e b u t la ck su ff ic ie n t d at a to co n cl u d e sa fe (M ar in i et a l. , 2 0 1 2 ) R ep o rt ed s af e b u t la ck su ff ic ie n t d at a to co n cl u d e sa fe (T ak eu ch i et a l. , 2 0 0 7 ) R ep o rt ed s af e b u t la ck su ff ic ie n t d at a to co n cl u d e sa fe (S id d iq u e & S al ee m , 2 0 1 1 ) G R A S ( if f ro m to m at o es ) (D ev ar aj e t al ., 2 0 0 8 ; T ru m b o , 2 0 0 5 ) L ac k s u ff ic ie n t d at a ↓ I L -6 ↓ T N F -α ↓ i N O S ↓ C O X -2 ↓ N F -κ B ↓ T L R 4 ↓ R O S ↓ N rf 2 ↓ T N F -α ↓ N F -κ B ↓ C O X -2 ↓ N F -κ B ↓ i N O S ↓ T N F -α ↓ N O ↓ T N F -α ↓ I L -6 ↓ n N O S ↓ N O ↓ C O X -2 ↓ M P O L P S i n d u ce d m al e al b in o w is ta r ra ts ( 1 0 -1 0 0 m g /k g ) (M ir ah m ad i et a l. , 2 0 1 8 ) 2 -4 -6 -t ri n it ro b en ze n su lf o n ic a ci d ( T N B S ) in d u ce d S p ra g u e- D aw le y m al e ra ts ( 4 5 0 m g /k g ) (H as sa n e t al ., 2 0 1 0 ) C ar ra g ee n an -i n d u ce d m al e S w is s m ic e p aw e d em a m o d el ( 1 0 -5 0 m g /k g ) (L u ce tt i, L u ce tt i, B an d ei ra , V er as , S il v a, L ea l, L o p es , A lv es , S il v a, B ri to , et a l. , 2 0 1 0 ) E n d o to x in -i n d u ce d ad u lt S p ra g u e- D aw le y r at s (1 0 m g /k g ) (G o n cu e t al ., 2 0 1 6 ) A d u lt m al e S p ra g u e– D aw le y r at s l is ch em ia /r ep er fu si o n i n ju ry (5 m g /m l) (P ei & C h eu n g , 2 0 0 4 ) ↓ I L -6 ↓ N F -κ B ↓ R O S ↓ I C A M -1 ↓ I L -6 ↓ T N F -α ↓ I L -1 β ↓ N F -κ B ↓ P P A R γ ↓ P G E 2 ↓ C O X -2 ↓ i N O S ↓ N O ↓ i N O S ↓ C O X -2 ↓ N O ↓ P G E 2 ↓ T N F -α ↓ I L -1 β ↓ I L -8 ↓ i N O S ↓ C O X -2 ↓ S IR T 1 H o m o cy st ei n e in d u ce d in fl am m at io n i n e n d o th el ia l ce ll s (E C V -3 0 4 ) (1 0 -1 0 0 μ M ) (H an , W u , L i, & G ao , 2 0 1 5 ) In fl am m at io n i n d u ce d i n T H P -1 b y L P S ( 0 .5 -1 0 0 µ M ) (Z h ao e t al ., 2 0 0 5 ) In fl am m at io n i n d u ce d i n R A W 2 6 4 .7 b y L P S ( 1 -8 µ M ) (C h en e t al ., 2 0 1 2 ) L P S i n d u ce d i n fl am m at io n in R A W 2 4 6 .7 c el ls ( 2 .5 -1 0 µ M ) (R af i, Y ad av , & R ey es , 2 0 0 7 ) In fl am m at io n i n d u ce d i n C H O N -0 0 1 b y H 2 O 2 ( 0 .1 - 1 0 0 n g ) (L im e t al ., 2 0 1 2 ) S o y b ea n s, f av a b ea n s F la x se ed o il , w al n u ts , ra p es ee d o il , so y b ea n o il M an g o , ca rr o t, cu cu m b er T o m at o es , g u av as , p ap ay a, g ra p ef ru it T o m at o es , g in g er , p o m eg ra n at e, ri ce , o li v es , al m o n d s G en is te in α -l in o le n ic a ci d L u p eo l L y co p en e M el at o n in 18 T a b le 2 .3 ( co n t. ) R ep o rt ed s af e b u t la ck su ff ic ie n t d at a to co n cl u d e sa fe (N g u y en , S ta u b ac h , T am ai , & L an g g u th , 2 0 1 5 ) S af e (a d eq u at e in ta k es ≈ 1 g /d ay ) (N at io n al I n st it u te s o f H ea lt h U S A , 2 0 1 8 ) G R A S ( if d er iv ed fr o m S a cc h a ro m yc es ce re vi si a e) o th er w is e re p o rt ed s af e b u t la ck su ff ic ie n t d at a to co n cl u d e sa fe (M ei , L iu , W an g , W an g , & D ai , 2 0 1 5 ; T o m é- C ar n ei ro e t al ., 2 0 1 3 ) L ac k s u ff ic ie n t d at a o n l o n g t er m s tu d ie s at h ig h d o se ↓ T N F -α ↓ I L -6 ↓ N F -κ B ↓ C O X -2 ↓ M IP -2 ↓ i N O S ↓ T N F -α ↓ I L -6 ↓ I L -1 β ↓ o x id at iv e st re ss ↓ C O X -2 ↓ P G E 2 ↓ T N F -α ↓ I L -6 ↓ C O X -2 ↓ P G E 2 ↓ N F -κ B A d u lt m al e a lb in o w is ta r ra ts w it h e th an o l in d u ce d li v er i n ju ry ( 5 0 m g /m l) (J ay ar am an , Je su d o ss , M en o n , & N am as iv ay am , 2 0 1 2 ) H y p er th y ro id is m -i n d u ce d h ep at ic d y sf u n ct io n a d u lt m al e w is ta r A lb in o r at s (3 g /k g ) (G o m aa & A b d E l- A zi z, 2 0 1 6 ) D ex tr an s u lf at e so d iu m (D S S ) in d u ce d m al e F is ch er F 3 4 4 r at s (1 m g /k g ) (L ar ro sa e t al ., 2 0 0 9 ) L P S i n d u ce d m al e B A L B /c m ic e (5 0 m g /k g ) (Q i, X u , Y an , L i, & L i, 2 0 1 6 ) ↓ T N F -α ↓ N O ↓ M C P -1 ↓ T N F ↓ I L -1 β ↓ I L -6 ↓ N F -κ B ↓ C O X -2 ↓ R O S ↓ N O ↓ E R K 1 /2 ↓ J N K - p h o sp h o ry la ti o n ↓ N F -κ B ↓ A P -1 In fl am m at io n i n d u ce d i n c o - cu lt u re d R A W 2 6 4 .7 c el ls an d 3 T 3 -L 1 p re ad ip o cy te st im u la te d b y L P S ( 2 5 -2 0 0 µ M ) (H ir ai e t al ., 2 0 0 7 ) L P S i n d u ce d i n fl am m at io n in T H P -1 c el ls ( 1 0 0 µ M ) (W el d o n , M u ll en , L o sc h er , H u rl ey , & R o ch e, 2 0 0 7 ) L P S i n d u ce d i n fl am m at io n in m ac ro p h ag es f ro m m u ri n e. ( 0 .3 -3 0 µ M ) (M ar ti n ez a & M o re n o a, 2 0 0 0 ) In fl am m at io n i n d u ce d i n m u ri n e m ic ro g li a ce ll l in e B V 2 s ti m u la te d b y L P S ( 0 .5 - 2 µ M ) (B ra n d en b u rg , K ip p , L u ci u s, P u fe , & W ru ck , 2 0 1 0 ) O ra n g es , le m o n s, g ra p ef ru it F is h o il , fl ax se ed , w al n u ts P ea n u ts , re d w in e, p is ta ch io s, b lu eb er ri es , g ra p es B ro cc o li , ca u li fl o w er , ca b b ag e N ar in g en in O m eg a- 3 f at ty ac id s R es v er at ro l S u lf o ra p h an e 19 T a b le 2 .3 ( co n t. ) G A R S (A g g ar w al & N es ar et n am , 2 0 1 2 ) G R A S ( h ig h ly p u ri fi ed f o rm , u p t o 2 0 0 m g /d ay ). (A n d re s et a l. , 2 0 1 8 ) T N F -α : tu m o r n ec ro si s fa ct o r- al p h a; I L : in te rl eu k in ; N O : n it ri c o x id e; C O X : cy cl o o x y g en as e; N O S : n it ri c o x id e sy n th as e; P G E 2 : p ro ta g la d in -E 2 ; N F -κ B : n u cl ea r tr an sc ri p ti o n f ac to r k ap p a- B ; R O S : re ac ti v e o x y g en s p ec ie s; N rf 2 : n u cl ea r fa ct o r (e ry th ro id -d er iv ed 2 )- li k e 2 ; M C P : m o n o cy te c h em o ta ct ic p ro te in ; G -C S F : g ra n u lo cy te co lo n y -s ti m u la ti n g fa ct o r; G M -C S F : g ra n u lo cy te -m ac ro p h ag e co lo n y -s ti m u la ti n g fa ct o r; A P : ac ti v at o r p ro te in ; L O X : li p o x y g en as e; P P A R γ: p er o x is o m e p ro li fe ra to r- ac ti v at ed r ec ep to r g am m a; M P O : m y el o p er o x id as e; S IR T : si rt u in ; M IP : m ac ro p h ag e in fl am m at o ry p ro te in ; E R K : ex tr ac el lu la r si g n al -r eg u la te d k in as e; J N K : Ju n N -t er m in al k in as e; L P S : li p o p o ly sa cc h ar id e; G R A S : g en er al ly r ec o g n iz ed a s sa fe ↓ T N F -α ↓ I L -6 ↓ I L -1 β ↓ N F -κ B ↓ C O X -2 ↓ P G E 2 ↓ T N F -α ↓ M IP -2 L P S i n d u ce d a d u lt m al e B A L B /c m ic e (2 0 m g ) (G o d b o u t, B er g , K rz y sz to n , & J o h n so n , 2 0 0 5 ) C ar ra g ee n an i n d u ce d m al e w is ta r ra ts w ei g h in g ( 1 0 m g /m l) (M o ri k aw a et a l. , 2 0 0 3 ) ↓ T N F -α ↓ N F -κ B ↓ T N F -α ↓ C O X -2 ↓ N O S -2 In fl am m at io n i n d u ce d i n K u p ff er c el ls b y L P S ( 5 0 µ M ) (F o x , B ro w er , B el le zz o , & L ei n g an g , 1 9 9 7 ) In fl am m at io n i n d u ce d i n R A W 2 6 4 .7 c el ls b y P o rp h yr o m o n a s g in g iv a li s fi m b ri ae ( 1 2 .5 -1 0 0 µ M ) (M u ra k am i, K aw at a, I to , K at ay am a, & F u ji sa w a, 2 0 1 5 ) S u n fl o w er o il , al m o n d s, s p in ac h T o m at o es , ap p le sk in s, o n io n s, g ra p es , re d w in e α -T o co p h er o l Q u er ce ti n 20 2.5.1 Curcumin Turmeric, a member of the ginger family (Zingiberaceae), is obtained from the rhizome of the Curcuma longa plant (Alappat & Awad, 2010; Nelson et al., 2017). Turmeric is a popular spice and a key ingredient in traditional Chinese and India Ayurvedic medicines for wound healing, scabbing of pox, reducing discomfort from insect bites, and treatment of colds, coughs, stomach disorders and cancers (Kim, Lee, & Shin, 2015; Nelson et al., 2017). Turmeric has been used in the food industry for its natural yellow colour and distinct flavour due to the essential oils and oleoresins. Nutritionally, turmeric typically contains 60-70 % carbohydrate, 5-10 % fat, 6-8 % protein, 3-7 % mineral, 2-7 % fiber, 3-7 % essential oil and 6-13 % moisture (Nelson et al., 2017; Prasad, Gupta, Tyagi, & Aggarwal, 2014). Today turmeric is increasingly being used as a therapeutic agent, with its benefits primarily due to the presence of curcuminoids (Raina, Srivastava, & Syamsundar, 2005; Ramirez et al., 2018; Zeng et al., 2015). The term curcuminoids refer to a group of compounds, namely curcumin (accounting for ≈ 60-70 %), demethoxycurcumin and bis-demethoxycurcumin and a trace amount of secondary metabolites (Nelson et al., 2017; Priyadarsini, 2014). Together, curcuminoids make-up approximately 2-9 % of turmeric on a dry matter basis (Tsuda, 2018). Structurally, curcumin (also known as diferuloyl methane) is C12H20O6 with IUPAC name 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (Kocaadam & Şanlier, 2017; PubChem Open Chemistry Data Base, 2018). The presence of a β-diketone moiety is responsible for keto-enol isomers of curcumin (Figure 2.3), however, the enol isoform is reported to be more stable (Payton, Sandusky, & Alworth, 2007; Shen & Ji, 2007). Figure 2.3: Keto-enol isomers of curcumin. Source: (Wanninger et al., 2015) (Reproduced from an open access article) Curcumin is a hydrophobic molecule, therefore, it is insoluble in water but soluble in solvents like ethanol, methanol, chloroform and dimethyl sulfoxide (DMSO) (Hatcher, Planalp, Cho, Torti, & Torti, 2008; Priyadarsini, 2014). Its solubility can further be improved by mixing 21 solvent with serum such as fetal calf serum (FCS) or bovine serum albumin (BSA). This technique is commonly used for in vitro studies (Klawitter et al., 2012; Quitschke, 2008). However, Wang et al. (1997) reported 50 % degradation of curcumin dissolved in 10 % FBS for 8 h. Although the solubility of curcumin is enhanced at alkaline pH (up to pH ≈ 10.2), it is relatively unstable and degrades quickly (about 90 % in 30 min) in neutral or alkaline pH to produce compounds like vanillin and ferulic acid (Figure 2.4a) (Nelson et al., 2017). In aqueous solution curcumin is mainly degraded by autoxidation. Free radical species initiate a series of degradation steps, producing several unstable intermediary products, resulting in a stable cyclic compound called bicyclopentadione (Figure 2.4b) (Gordon, Luis, Sintim, & Schneider, 2015). Curcumin is also sensitive to light and degradation occurs rapidly in both crystalline and aqueous states. Powdered curcumin exposed to sunlight converts into vanillin, vanillic acid Figure 2.4: Degradation of curcumin (a) at alkaline pH; (b) autoxidation in solvent; (c) photo-oxidation when in crystalline or aqueous state; and (d) photo-oxidation when in specific solvent like isopropanol. Source: (Nelson et al., 2017) (Reproduced with permission) 22 and ferulic aldehyde like compounds (Figure 2.4c) (Griesser et al., 2011). Curcumin dissolved in a solvent and exposed to light produces several solvent-depended products, e.g. production of guaiacol derivative when dissolved in isopropanol (Figure 2.4d) (Nelson et al., 2017). Curcumin is relatively stable at high temperature, but above 90ºC the β-diketone linkage break and leads to degradation (Peram, Jalalpure, Palkar, & Diwan, 2017). Despite poor water solubility and low stability, curcumin has found wide applications as a therapeutic agent. The α and β-diketo moiety is a good chelating agent, therefore curcumin can reduce the toxicity of heavy metals such as lead and cadmium by forming strong complexes with them (Akram et al., 2010; Priyadarsini, 2014). The phenolic group of curcumin can scavenge free radicals by trapping the radical and thereby inhibiting the chain reaction, hence act as an anti-oxidant (Barzegar, 2012; Chen, Xue, & Mu, 2014). It can act on a wide spectrum of microorganism showing antibacterial, antifungal, anti-parasitic and anti- HIV activities (Nelson et al., 2017). Curcumin is also reported to be effective against several infections such as malaria, sexually transmitted disease and chronic inflammation-related diseases such as arthritis, type-2 diabetes, osteoporosis, psoriasis, bronchitis, depression, obesity and several cancers (Hewlings & Kalman, 2017; Kocaadam & Şanlier, 2017; Mullaicharam & Maheswaran, 2012). 2.5.1.1 Curcumin and inflammation Curcumin has been extensively studied for its anti-inflammatory properties, which helps to explain some of its multiple therapeutic benefits (Hewlings & Kalman, 2017; Marchiani, Rozzo, Fadda, Delogu, & Ruzza, 2014). Curcumin can influence the activity of multiple pathways by blocking certain enzymes (e.g. COX-2, 5-LOX and iNOS), growth factors (e.g. transforming growth factor-β1: TGF-β1), receptors (e.g. integrin receptor: IR and interleukin 8-receptor: IL-8-R), kinases (e.g. mitogen-activated protein kinase: MAPK and Janus kinase: JAK), inflammatory cytokines (e.g. TNF-α, IL-6 and IL-8) and transcriptional factors (e.g. NF-κB and nuclear factor 2-related factor: Nrf-2) (Anand, Sundaram, Jhurani, Kunnumakkara, & Aggarwal, 2008; Jurenka, 2009; Lozada-García et al., 2017). The different molecular targets reported to be affected by curcumin are illustrated in Figure 2.5. Of these targets, the suppression of inflammatory cytokines and transcriptional factors are key contributors to the anti-inflammatory property of curcumin. 23 Figure 2.5: Various molecules targeted by curcumin. EGR-1: early growth response gene-1; AP-1: activating protein1; CREB-BP: CREB-binding protein; WT-1: Wilms‘ tumour gene-1; NF-κB: nuclear transcription factor kappa-B; STAT: signal transducers and activators of transcription; HIF-1: hypoxia inducible factor-1; ERE: electrophile response element; Nrf-2: nuclear factor 2-related factor; PPAR-γ: peroxisome proliferator-activated receptor-gamma; MCP: monocyte chemoattractant protein; MIP: macrophage inflammatory protein; IL: interleukin; TNF-α: tumour necrosis factor alpha; MaIP: macrophage inflammatory protein; IR: integrin receptor; ER-α: estrogen receptor alpha; H2-R: histamine (2)- receptor; HER-2: human epidermal growth factor-2; LDL-R: low density lipoprotein receptor; Fas-R: Fas receptor; EPC-R: endothelial protein C-receptor; AR: androgen receptor; EGF-R: epidermal growth factor-receptor; IL-8-R: interleukin 8-receptor; CXCR4: alpha-chemokine receptor; AHR: aryl hydrocarbon receptor; DR-5: death receptor- 5; AATF-1: arylamine N-acetyltransferases-1; COX-2: cyclooxygenase-2; NQO-1: NADPH- quinoneoxidoreductase-1; TMMP-3: tissue inhibitor of metalloproteinase-3; Src-2: src homology-2 domain containing tyrosine phosphatase 2; PhpD: phospholipase D; GCL: glutamate cysteine ligase; MMP: matrix metalloproteinase; iNOS: inducible nitric oxide 24 oxidase; LOX, lipoxygenase; DNA pol: DNA polymerase; GST: glutathione S-transferase; FPT: farnesyl protein transferase; ODC: ornithine decarboxylase; HGF: hepatocyte growth factor; CTGF: connective tissue growth factor; FGF: fibroblast growth factor; NGF: nerve growth factor; PDGF: platelet-derived growth factor; TGF-β1: transforming growth factor- β1; EGF: epidermal growth factor; VEGF: vascular endothelial growth factor; TF: tissue factor; FAK: focal adhesion kinase; AAPK: autophosphorylation-activated protein kinase; Pp60c-tk: a non-receptor protein tyrosine kinase c-Src; EGFR-K: EGF receptor-kinase; Ca 2+ PK, Ca 2+ -dependent protein kinase; PTK: protein tyrosine kinase; MAPK: mitogen activated protein kinase; PKB: protein kinase B; PKA: protein kinase A; JAK: janus kinase; ERK: extracellular receptor kinase; PhK: phosphorylase kinase; JNK: c-jun N-terminal kinase; uPA: urokinase-type plasminogen activator; Bcl-2: beta-cell lymphoma protein; Bcl- xL: beta-cell lymphoma extra-large; VCAM-1: vascular cell adhesion molecule- 1; ICAM-1: intracellular adhesion molecule-1; ELAM-1: endothelial leukocyte adhesion molecule-1; IAP: inhibitory apoptosis protein; HSP-70: heat-shock protein 70; MDRP: multi-drug resistance protein; DFF-40: DNA fragmentation factor 40-kd subunit. Adapted from: (Anand et al., 2008; Noorafshan & Ashkani-Esfahani, 2013) and created using Microsoft PowerPoint. NF-κB is the principal regulator influencing the expression of more than 500 inflammation- related genes (Buhrmann et al., 2011). NF-κB protein is present in the cytoplasm of the cell which on activation is translocated into the nucleus (Jobin et al., 1999). In its in-activated form, the heterodimer structure of NF-κB is sequestered by binding with IκB, an NF-κB inhibitory protein (Surh et al., 2001). When cells are exposed to an external stimuli e.g. bacteria, inflammatory cytokines, ROS, ultraviolet radiation or viral proteins, NF-κB is functionally activated (Hatcher et al., 2008; Jobin et al., 1999; Surh et al., 2001). The exposure to the external stimulus will activate various kinases such as NF-κB inducing kinase (NIK) and IκBα kinase (IKK) resulting in phosphorylation and degradation of inhibitor κB (IκB), thereby activating NF-κB (Surh et al., 2001). The exposure to the external stimulus can also activate mitogen-activated protein kinases (MAPK, a family of serine/threonine kinase) signalling pathways leading to phosphorylation and activation of NF-κB (Hatcher et al., 2008; Kaminska, 2005; Liang et al., 2015). Activated NF-κB travel into the nucleus where it binds to the promoter region on DNA and result in increased transcription of various inflammation-related genes responsible for enhancing the expression of cytokines e.g. TNF-α and IL-6 and enzymes e.g. COX-2 and iNOS (Li, Suwanwela, & Patumraj, 2017; Surh et al., 2001). The pro-inflammatory cytokines, free radicals and products formed during arachidonic acid metabolism can further promote activation of NF-κB and result in prolonging inflammatory response (He et al., 2015). Curcumin can suppress the secretion of pro- inflammatory cytokines, quenches ROS and RNS and inhibits TNF-α mediated phosphorylation and degradation of the IκB signalling pathway. Thus, it can help control 25 prolonged inflammation (Alappat & Awad, 2010; Hatcher et al., 2008; Jobin et al., 1999; Panahi et al., 2015; Singh & Aggarwal, 1995; Surh et al., 2001). Evidence for the anti-inflammatory properties of curcumin Studies from many in vitro, animal and clinical trials have reported the anti-inflammatory effect of curcumin, some of which are summarised in Table 2.4. Evidence for in vitro studies As previously discussed, NF-κB inhibition is an important pathway involved in the anti- inflammatory effects of curcumin. Several studies (Table 2.4) have shown that curcumin can suppress NF-κB activation and NF-κB-regulated gene expression in both animal and human cell lines. Pan, Lin-Shiau, and Lin (2000) reported that curcumin could down-regulate iNOS secretion and transcription in LPS induced inflammation in RAW264.7 cell. Further, these authors reported that curcumin decreases the activity of IκB kinase-1/2 (IKK-1/2 or IKK-α/β), thereby inhibiting NF-κB activation. Ben et al. (2011) confirmed that curcumin suppresses iNOS secretion and transcription, and proposed that curcumin inhibits extracellular signal- regulated kinase-1/2 (ERK-1/2) activation and subsequently iNOS secretion. Other studies on LPS treated RAW 264.7 cells showed that treatment with curcumin decreased secretion of pro-inflammatory cytokines (e.g. TNF-α and IL-6) and enzymes (e.g. COX-2 and iNOS) (Murakami et al., 2008; Sun et al., 2010). Curcumin treatment of LPS induced inflammation in BV2 cell resulted in a decrease in the secretion of NO, PGE2, TNF-α, IL-6 and IL-1β and downregulation of expression of iNOS and COX-2 (Jin, Lee, Park, Choi, & Kim, 2007). In another study, 3T3-L1 preadipocyte, isolated from male C57BL/6 mice fed with high-fat diet for 3 months, were cultured along with RAW 264.7 and a decrease in monocyte chemoattractant protein-1 (MCP-1, responsible for chemo-taxis in adipose tissues) was reported on treatment with 10 µM curcumin for 6 h (Woo et al., 2007). Curcumin has elicited similar effects on human cells. For example, administration of curcumin to U937 and A293 cells stimulated with TNF-α to induce inflammation, resulted in a dose-dependent decrease in the expression of NF-κB-regulated genes like COX-2, IκB-α and IKK (Aggarwal et al., 2006). Vascular smooth muscle cells, administrated with curcumin, suppressed expression of TNF-α, NO, MCP-1 and iNOS by reducing NF-κB activation via downregulation of MAPK pathways (Meng, Yan, Deng, Gao, & Niu, 2013). 26 Curcumin incubated with human umbilical vein endothelial cells led to a decrease in high mobility group box 1 (HMGB-1) (Kim, Lee, & Bae, 2011), which is responsible for the secretion of pro-inflammatory cytokines and neutrophil adhesion and migration. Evidence for animal studies The molecules shown to be targeted by curcumin in in vitro studies are also reported to be affected in animal trials (Table 2.4). Curcumin administered for 6 h at a rate of 35 mg/kg/h decreased the level of TNF-α and IL-6 in Sprague-Dawley rats with induced pancreatitis by blocking IκB mediated NF-κB activation (Gukovsky, Reyes, Vaquero, Gukovskaya, & Pandol, 2003). In another study, 100 mg/kg curcumin administered for 20 days prior to inducing acute pancreatitis in male Wistar-Albino rats decreased TNF-α, IL-6, iNOS and NO, (Gulcubuk et al., 2013). Curcumin is also effective in reducing obesity-induced inflammation as shown by Weisberg, Leibel, and Tortoriello (2008) and Shao et al. (2012). In these two studies, dietary curcumin was administrated via high-fat diet in male C57BL/6J mice and reported inhibition of NF-κB activation resulting in a decrease in oxidative stress and macrophage infiltration in white adipose tissues. Incubation of mice with curcumin (200 µl of 0.2 mg/ml/day for 12 days) reduces TNF-α level and also speeds up the wound healing process by increasing collagen level (Yen et al., 2018). Similar results were reported by Kant et al. (2014) in wounded diabetic adult male Wistar rats on treatment with curcumin for 19 days. Kant et al. (2014) also reported an increase in anti-inflammatory cytokine IL-10 and enzyme superoxide dismutase (SOD) on exposure with curcumin. Curcumin downregulates pro-inflammatory cytokines e.g. IL-1β, TNF-α and IL-6, and upregulates anti-inflammatory cytokines e.g. TGF- α and IL-10 in hippocampus, cortex, hypothalamus and spleen of Wistar rats affected with chronic mild stress (You et al., 2011). Evidence for clinical trials A clinical trial is the best way to understand the effectiveness of a compound in performing the intended function in the human body. However, due to high cost, longer duration, withdrawal of participants and high precision requirement there is limited clinical evidence supporting the anti-inflammatory effects of curcumin. Some of the clinical trials are summarised in Table 2.4. 27 Table 2.4: Pre-clinical and clinical studies showing the anti-inflammatory effects of curcumin. Researcher(s) (reference) Experiment Design Dose and Duration Key Observations Mechanism Purposed/ Comment (if any) In vitro studies Pan et al. (2000) LPS (100 ng/ml) induced inflammation in RAW 264.7 (mouse monocyte) cells 10 µM for 6 h Decrease in iNOS secretion and transcription Inhibition of IKK1 and IKK2 activity Curcumin down- regulate NF-κB activation by inhibiting IKK Aggarwal et al. (2006) TNF-α (0.1 and 1 nM) induced inflammation in U937 (human myeloid leukemia) and A293 (human embryonic kidney) cells 10-50 µM for 2-24 h Downregulate COX-2, cyclin D1, IAP-1, IAP-2, Bcl-2, Bcl-xL, Bfl-1/A1, p65, IκB-α, IKK and Akt in dose dependent manner Curcumin suppress TNF-α induced NF-κB activation and NF-κB- regulated gene expression by inhibiting IKK and Akt activation Bachmeier et al. (2007) TNF-α (10 ng/ml) induced inflammation in MDA-MB-231 (human breast cancer) cell line 25 µM for 2-24 h Reduces ΙκΒ, p65 phosphorylation, MMP and AP-1 Curcumin silences NF- κB functioning Woo et al. (2007) 3T3-L1 preadipocyte were isolated from mice fed with high-fat diet and were cultures along with RAW 264.7 10 µM for 6 h Decrease in TNF-α, NO and MCP-1 Curcumin can suppress obesity-induced inflammatory responses by suppressing MCP-1 release from adipocytes Jin et al. (2007) LPS (0.5 μg/mL) induced inflammation in BV2 (mouse murine microglial) cell line 5-20 µM for 24 h Decrease in secretion of NO, PGE2, TNF-α, IL-6 and IL-1β Downregulate expression of iNOS and COX-2 Curcumin reduce pro- inflammatory cytokines by suppressing NF-κB Murakami et al. (2008) LPS (100 ng/ml) induced inflammation in RAW 264.7 cells 0.2-20 µM for 3 h Decrease in COX-2 and NF-κB expression Chen, Nie, Fan, and Bian (2008) LPS (1 µg/ml) induced inflammation in RAW 264.7 cells 5-30 µmol/l for 8 h Decrease in TNF-α and IL-1β Curcumin downregulate NF-κB dependent transcription factors Sun et al. (2010) LPS (50 ng/ml) induced inflammation in RAW 264.7 cells 20 µmol/l for 7 h Decrease in TNF-α and IL-6 Kim et al. LPS (100 ng/ml) 2.5-100 Decrease in HMGB-1 Curcumin inhibit 28 (2011) induced inflammation in human umbilical vein endothelial cells (HUVEC) µM for 6 h release and neutrophil adhesion and migration HMGB-1-mediated pro- inflammatory response Ben et al. (2011) LPS (100 ng/ml) induced inflammation in RAW 264.7 cells 20 µM for 12 h Decrease in iNOS secretion and transcription Curcumin inhibits ERK 1/2 activation and subsequently suppressed iNOS enzyme activity Meng et al. (2013) LPS (1 µg/ml) induced inflammation in vascular smooth muscle cells (VSMCs) of rats 5-30 µmol/l for 24 h Decrease in TNF-α, NO, MCP-1, iNOS, IκBα and NF-κB Curcumin suppresses MAPK and NF-κB pathways Youn, Kwon, Ju, Choi, and Park (2013) THP-1 (human monocyte) cells were cultured alone and in combination with HaCaT (immortalized human keratinocyte) cells in presence of TNF-α 1-30 µM for 1-24 h Decrease in ICAM-1, Nrf2 and HO-1 Curcumin suppress the TNF-α-induced ICAM-1 expression and subsequent THP-1 adhesion via expression of HO-1 in the HaCaT Animal trials Gukovsky et al. (2003) Ethanol and CCK-8 induce pancreatitis in Sprague-Dawley rats 200 mg/kg for 6 h Downregulate TNF-α, IL- 6, IκB and AP-1 Curcumin blocks NF- κB and AP-1 activation Banerjee, Tripathi, Srivastava, Puri, and Shukla (2003) Arthritis induced in male Sprague Dawley rats via Freund‘s adjuvant (10 mg/ml) 100 mg/kg every day for 35 days Decrease in TNF-α, IL- 1β and NO Weisberg et al. (2008) Wild-type and ob/ob male obese C57BL/6J mice 3 % dietary curcumin per day for 35 days Decrease in macrophage infiltration of white adipose tissue Curcumin downregulate NF-κB activity Gupta et al. (2011) Streptozotocin induced diabetic Wistar albino rats 1 g/kg every day for 16 weeks Downregulate SOD, TNF-α and VEGF Shao et al. (2012) Male C57BL/6J mice fed with high fat diet Dietary curcumin for 28 weeks Decrease in oxidative stress Curcumin inhibit NF- κB or pJNK levels Jiang et al. (2013) Chronic mild stress induced in male Wistar 10 mg/kg per day for Inhibit TNF-α and IL-6 at both mRNA and protein Curcumin reduced the activation of NF-κB 29 rats 21 days level Gulcubuk et al. (2013) Sodium taurocholate induced acute pancreatitis in male Wistar-Albino rats 100 mg/kg for 20 days before pancreatitis Downregulation of TNF- α, IL-6 and iNOS and NO (but not at all time points) Curcumin inhibit NF- κB and AP-1 Kant et al. (2014) Wounded diabetic adult male Wistar rats 400 µl applied on wounds every day for 19 days Decrease in pro- inflammatory cytokine TNF-α and IL-1β Upregulation of anti- inflammatory cytokine IL-10 and enzyme SOD Curcumin have thicker collagen deposition at wounded area Wang et al. (2014) LPS (0.83 mg/kg) induced depression in adult male Kun-Ming mice 50 mg/kg per day for 7 days Decrease in TNF-α, IL- 1β, iNOS and COX-2 Curcumin have anti- depressive effect by inhibiting NF-κB pathway Zhang, Li, Jia, and He (2015) Ovalbumin (40 µg/kg) induced allergy in female BALB/c and C57BL/6 mice 100 and 200 mg/kg per day for 3 days Downregulate TNF-α, IL- 1β, IL-6, IL-8, ERK, p38, JNK, IκBα and NF-κB Curcumin have anti- allergic effect by inhibiting MAPK/NF- κB pathway Kaur, Patro, Tikoo, and Sandhir (2015) PTZ induced chronic epilepsy in adult male Wistar rats 100 mg/kg every day for 30 days Decrease in secretion and transcription of IL-1β, IL- 6, TNF-α and MCP-1 Zhong et al. (2016) LPS (5 mg/kg) injected male wild-type C57BL/6 mice 20-80 mg/kg every day for 28 days Decrease in serum level of TNF-α, IL-1β and IL- 18 Downregulate O2 •- , H2O2, ROS and NO in liver Curcumin inhibit PI3K/Akt and CYP2E/Nrf2/ROS signaling Li et al. (2017) Stroke‑induced male Wistar rats 300 mg/kg 30 min after operation Decrease in NF‑κB, ICAM‑1 and MMP‑9 Yang et al. (2018) DSS induced inflammatory bowel disease in male ICR mice 0.1 or 0.25 mmol/kg for 7 days Decrease in NF‑κB, COX-2 and iNOS Curcumin inhibit STAT-3 pathway Clinical trials Dhillon et al. (2008) Nonrandomized, open- label, phase II trial with patients confirmed adenocarcinoma of the pancreas (n=21) 8 g/day until disease progression Downregulate NF‑κB and COX-2 peripheral blood mononuclear cells from patients No toxicity observed Khajehdehi et al. (2011) Randomized double- 500 mg turmeric Decrease in serum level of TGF-β and IL-8 No adverse effects related to the turmeric 30 blind and placebo- controlled trail with patients suffering from type 2 diabetic nephropathy (n=20) capsule trice/day for 2 months supplementation Each capsule contain 22.1 mg of the active ingredient curcumin Ganjali et al. (2014) Randomized double- blind crossover trial with obese patients (n=28) 500 mg capsules twice/day for 30 days (each capsule contains 5 mg bioperine) Downregulate IL-1β, IL- 4 and VEGF No effect on serum level of IL-1α, IL-2, IL-6, IL- 8, IL-10, IFNγ, EGF, MCP-1, and TNFα Panahi et al. (2015) Randomized double- blind placebo- controlled trial with male participants suffering from chronic sulfur mustard (n=40) 500 mg capsules /day for 4 weeks Decrease in TNF-α, IL-6, TGF-β, hs-CRP, CGRP and MCP-1 No toxicity observed Sciberras et al. (2015) Effect on inflammatory cytokine after 2 h exercise in randomized double-blind cross-over trail with male athletes (n=11) 500 mg capsule with midday meal for 3 days and 1 capsule just before exercise Statistically insignificant decrease in serum level of IL-1, IL-6 and IL-10 Panahi et al. (2016) Randomized double- blind placebo- controlled trial with a parallel-group design having males and females diagnosed with metabolic syndrome (n=50) 500 mg capsules twice/day for 8 weeks (each capsule contains 5 mg piperine) Decrease in serum level of TNF-α, IL-6, TGF-β and MCP-1 No toxicity observed LPS: lipopolysaccharide; iNOS: inducible nitric oxide synthases; IKK: IκB kinase; NF-κB: nuclear transcription factor kappa-B; TNF-α: tumour necrosis factor alpha; COX-2: cyclooxygenase-2; IAP: inhibitor of apoptosis protein; Bcl-2: beta-cell lymphoma protein; Bcl-xL: beta-cell lymphoma extra- large; MMP: matrix metalloproteinases; AP-1: activating-protein-1; NO: nitric oxide; MCP-1: monocyte chemoattractant protein-1; PGE2: prostaglandin E2; IL: interleukin; VCAM-1: vascular cell adhesion molecule- 1; ICAM-1: intracellular adhesion molecule-1; HMGB-1: high mobility group box 1; ERK: extracellular signal-regulated kinase; MAPK: mitogen activated protein kinase; Nrf2: nuclear factor 2-related factor 2; HO-1: heme oxygenase-1; mRNA: messenger ribonucleic acid; SOD: superoxide dismutase; VEGF: vascular endothelial growth factor; JNK: c-jun N-terminal kinase; PTZ: pentylenetetrazole; O2 •- : superoxide; H2O2: hydrogen peroxide; ROS: reactive oxygen species; NO: nitric oxide; PI3K/Akt: phosphatidylinositol 3-kinase/protein kinase B; CYP2E: cytochrome P450- 2E1; DSS: dextran sulfate sodium; IFNγ: interferon γ; EGF: epidermal growth 31 factor; TGF-β: transforming growth factor-β; hs-CRP: high-sensitivity C-reactive protein; CGRP: calcitonin gene related peptide. Panahi, Ghanei, Bashiri, Hajihashemi, and Sahebkar (2015) conducted a randomized double- blind placebo-controlled trial on male participants suffering from chronic sulfur mustard. Participants were given 500 mg capsules of curcuminoids per day for 4 weeks. 40 individuals completed the trial and authors reported a decrease in TNF-α, IL-6, MCP-1, transforming growth factor-β (TGF-β), high-sensitivity C-reactive protein (hs-CRP) and calcitonin gene- related peptide (CGRP). Similar results have been reported in another randomized double- blind placebo-controlled trial in people diagnosed with metabolic syndrome (Panahi et al., 2016). A decrease in serum levels of TNF-α, IL-6, TGF-β and MCP-1 was reported following administration of 500 mg curcumin consumed twice a day for 8 weeks. In contrast, consumption of 500 mg curcuminoids twice a day for 30 days did not downregulate IL-1α, IL-2, IL-6, IL-8, IL-10, interferon γ (IFNγ), epidermal growth factor (EGF), MCP-1, and TNF-α in obese individuals (Ganjali et al., (2014). 2.5.1.2 Bioavailability and metabolism of curcumin Curcumin is known to have poor bioavailability in both humans and animals. The small portion of curcumin that is absorbed in the intestine quickly degrades in the blood and liver (Liu et al., 2016), leading to the formation of curcumin glucuronide and curcumin sulphate via two different pathways (Mahran, Hagras, Sun, & Brenner, 2017). Curcumin can undergo a series of reduction reactions, where four bonds of curcumin are broken to form octa-hydro- curcumin. During this conversion, intermediates such as hexahydrocurcimin glucuronide and hexahydrocurcimin sulphate are produced (Hassaninasab, Hashimoto, Tomita-Yokotani, & Kobayashi, 2011; Mahran et al., 2017). Curcumin, via the second pathway, can form conjugates with monoglucuronide and monosulfate to produce curcumin glucuronide and curcumin sulphate which are more prominent in the blood (Cuomo et al., 2011; Ghosh, Banerjee, & Sil, 2015). The unabsorbed fraction of curcumin which may be as high as 75 % is excreted directly in faeces (Yadav, Sah, Jha, Sah, & Shah, 2013). 2.5.1.3 Safety of curcumin Turmeric is listed as generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) (Nelson et al., 2017) and curcumin has been reported safe for human 32 consumption. Curcumin dose of 0-3 mg/kg body weight have been reported as adequate daily intake (ADI) by the Joint FAO/WHO Expert Committee on Food Additives (2004) (JECFA) and European Food Safety Authority (2014) (EFSA). However, some studies have used curcumin doses of up to 8,000-12,000 mg/day and reported adverse effects such as headache, diarrhoea, rash, nausea and yellow stool (Hewlings & Kalman, 2017; Kocaadam & Şanlier, 2017; Nelson et al., 2017). Such high doses are used because of poor bioavailability of curcumin in the human body as its therapeutic effects are limited at lower doses. 2.5.1.4 Co-administration of curcumin with other bioactives Different methods are being considered to improve the bioavailability of curcumin such as the development of nanoparticles, nanoemulsions and liposomes (Cuomo et al., 2011; Jin, Lu, & Jiang, 2016; Mahran et al., 2017; Prasad et al., 2014). Curcumin is also being investigated in combination with other bioactive compounds that may enhance its activity and thereby its therapeutic effects at lower doses (Mahran et al., 2017). Some of the bioactive compounds listed in Table 2.2 have been studied in combination with curcumin and their synergistic effects reported. Piperine, a bioactive compound from black pepper, has been extensively studied in combination with curcumin and it is reported to improve the absorption of curcumin in the body by 2000 % (Hewlings & Kalman, 2017). In a phase III randomized double-blind placebo-controlled trial with a parallel-group design on 59 subjects diagnosed with metabolic syndrome treated with a daily dose of 1 g curcuminoids mixed with 10 mg of piperine for 8 weeks, the authors (Panahi et al., 2015) reported a significant increase in serum concentration of SOD in comparison with the control. Curcumin was also studied in combination with resveratrol (a bioactive compound from grapes, berries and wine). In combination, curcumin (10 µM) and resveratrol (10 µM) synergistically downregulated MAPK pathway in human articular chondrocytes (Shakibaei, Mobasheri, & Buhrmann, 2011). However, in a double-blind, crossover, randomized study in obese adults treatment with a combination of curcumin (100 mg) and resveratrol (200 mg) was not effective in significantly reducing serum concentration of IL-6, IL-8, MCP-1 or VCAM-1 (Vors et al., 2018). Furthermore, there was no difference in NF-κB gene expression in comparison with the control. 33 2.5.2 Chlorogenic acid Chlorogenic acid (CGA) is a less studied bioactive compound. It is one of the principle polyphenols in the human diet as it is abundant in many food sources such as fruits (e.g. apples, grapes, pears, peaches, plums, kiwi fruit, mangoes, blueberries, and blackberries) and vegetables (e.g. cabbage, eggplants, carrots, potatoes, tomatoes and coriander) (Clifford, 2000; Furrer, Cladis, Kurilich, Manoharan, & Ferruzzi, 2017; Liang & Kitts, 2015; Naso et al., 2014; Santana-Gálvez, Cisneros-Zevallos, & Jacobo-Velázquez, 2017; Upadhyay & Mohan Rao, 2013). However, the amount available varies according to the maturity, storage conditions and processing steps applied to different food sources. For example, CGA in potato is reported to increase slowly during storage in the presence of light (Onyeneho & Hettiarachchy, 1993). CGA is distributed throughout the plant including roots, seeds, leaves, flowers, and tubes, as well as products developed from them, such as coffee, juice and even wine (Gil & Wianowska, 2017). Green coffee beans are key source of CGA containing about 6-12 % CGA on a dry weight basis (Gil & Wianowska, 2017). Chemically, CGA is a group of polyphenols derived from esterification reactions between cinnamic acids derivatives and quinic acid (QA) (Tajik, Tajik, Mack, & Enck, 2017). CGA is the whole hydroxyl-cinnamic acid family containing caffic acid (CA), ferulic acid (FA) and p-coumaric acid with quinic acid (Naveed et al., 2018). Further, these subgroups have several isomeric forms which are distributed in food sources. The chemical structures of CGA which are dominant in food are shown in Figure 2.6. Coffee beans for instance consist of several CGA isoforms, including 3-caffeoylquinic acid (3-CQA), 4-caffeoylquinic acid (4-CQA), 5- caffeoylquinic acid (5-CQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), 3,5-dicaffeoylquinic) (Liang & Kitts, 2015; Matei, Jaiswal, & Kuhnert, 2012). In coffee beans, 5-CQA is present in the largest quantity