ANTI-INFLAMMATORY PROPERTIES OF ACEROLA (MALPIGHIA EMARGINATA) LEAF AND RIPE FRUIT GENOTYPES FOR PROTECTION AGAINST LPS-INDUCED INFLAMMATION IN MACROPHAGE CELLS AND THEIR SELECTIVITY TO CYCLOOXYGENASE-2 (COX-2) ACTIVITY A Thesis by PRERNA BHARGAVA Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Chair of Committee, Luis Cisneros Zevallos Committee Members, Joseph Awika Chaodong Wu Head of Department, Boon Chew May 2015 Major Subject: Food Science and Technology Copyright 2015 Prerna Bhargava ABSTRACT Conventionally, NSAID has been proposed to have inhibitory action against COX and therefore has traditionally been used for treatment of acute and chronic inflammation. This study aims at exploring putative anti-inflammation mechanism of acerola. Previous studies have illustrated that phytochemicals like alkaloids, terpenoids, flavonoids, curcumin and phenolics have COX inhibitory activities as well. However, a natural occurring selective inhibitor of COX-2 that can modulate inflammation and can overcome the limitations of drugs like aspirin is still a priority. Aspirin is known to form an irreversible and non-competitive binding to COX which proves to be a potent cardiovascular protective agent. On the other hand, irreversible binding has implications by initiating inhibition of blood platelet aggregation. Acerola has been earlier studied for its antioxidant, antimicrobial, anti- inflammatory, anticancer, antigenotoxic and antihyperglycemic properties. In the present study, anti-inflammatory properties of acerola have been established where different genotypes of acerola fruit and leaf fractions were studied for their biological properties. A comparative study using TLC, LC-MS and bioassays using macrophages is employed to identify which groups of phytochemicals are responsible for scavenging and inflammation inhibitory effect of acerola. Initially, phytochemicals were extracted using methanolic and methanolic/acetone/water solvents which isolated different groups of compounds in two fractions, including polyphenols and a mixture of polyphenols/terpenoids, respectively. The two fractions were explored to elucidate mode of action for different acerola genotypes. Results indicated that the methanolic fractions ii of acerola showed higher activity exhibited suppression of ROS and partial decrease of nitric oxide levels in LPS-stimulated RAW264.7 macrophage cell line. This fraction also demonstrated inhibition of enzyme expression of COX-1/2. Moreover, BRS-238, a ripe fruit genotypes of acerola had a selective action against COX-2 - confirming the hypothesis that acerola’s mode of anti-inflammatory action is through selective inhibition of COX-2. iii DEDICATION To my parents and brother iv ACKNOWLEDGEMENTS First and foremost, I would like to take this opportunity to express my sincere gratitude to my adviser Dr. Luis Cisneros-Zevallos, for his continuous mentoring, patience, enthusiasm, motivation and immense knowledge that have been instrumental in completion of this project. Without his valuable guidance and encouragement this project would not have materialized. I would also like to thank my committee members, Dr. Joesph Awika and Dr. Chaodong Wu, for their constant support and valuable time- to-time comments. This research project imprints support and guidance of many other people. I would like to show my greatest appreciation to Dr. Woo Young Bang, for his guidance, persistence, and cooperation throughout this two year journey. He not only mentored me on different molecular biological techniques but also showed me the right direction in several research related phases. I would like to thank Dr. Vimal Nair, for guiding me through chemical analysis and the chemistry involved in the project. Besides, I really appreciate the support from my fellow lab mates Elisa Schreckinger, Fang Mian Chang, Facundo Ibanez and Dr. Ricardo Elesbão Alves for their constant motivation, stimulating discussions and enormous fun get togethers throughout this journey. I am grateful to them for their continued support and help. A special note of thanks goes out to my family and friends. I cannot express in mere words how much grateful I am to have them in my life. My parents Dr. Anuraag Bhargava and Mrs. Richa Bhargava for their unconditional trust, care and faith that v escorted me emotionally throughout. An exclusive note of gratitude to my brother Mr. Prateek Bhargava for his supportive nature, edifying thoughts and constant counseling sessions that shepherded me and kept me motivated. This acknowledgement will be incomplete if I do not mention my friends who stood by me like family to help me accomplish this research project. vi TABLE OF CONTENT Page ABSTRACT ....................................................................................................................... ii DEDICATION .................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................... v TABLE OF CONTENT ................................................................................................... vii LIST OF FIGURES........................................................................................................... ix LIST OF TABLES ............................................................................................................. x CHAPTER I INTRODUCTION ....................................................................................... 1 Macrophages and inflammation ......................................................................... 2 Macrophages infiltration and activation ............................................................. 3 Extravasation in case of endothelial inflammation ............................................. 4 Role of redox molecules in inflammation .......................................................... 5 Approaches utilized to quantify antioxidant contents of polyphenols ................ 6 Acerola as a source of anti-inflammatory compounds ....................................... 7 Objectives ........................................................................................................... 8 Hypothesis .......................................................................................................... 8 CHAPTER II P HYTOCHEMICAL ANALYSIS OF ACEROLA LEAVES AND FRUIT GENOTYPES...................................................................................................... 10 Synopsis ............................................................................................................ 10 Introduction ....................................................................................................... 11 Material and Methods ....................................................................................... 13 Results and Discussion ..................................................................................... 17 Conclusions....................................................................................................... 21 CHAPTER III ANTI-INFLAMMATORY PROPERTIES OF ACEROLA LEAVES AND FRUIT GENOTYPES .............................................................................................23 Synopsis ............................................................................................................ 23 vii Introduction ....................................................................................................... 24 Materials and Methods ..................................................................................... 27 Results and Discussion ..................................................................................... 31 Conclusion ........................................................................................................ 37 CHAPTER IV SUMMARY AND CONCLUSIONS....................................................... 39 Future Recommendations.................................................................................. 41 REFERENCES ................................................................................................................. 43 APPENDIX ...................................................................................................................... 51 viii LIST OF FIGURES Page Figure 2.1. Systematic flowchart for TLC ....................................................................... 51 Figure 2.2. HPLC/MS profile of acerola leaves genotypes .............................................. 53 Figure 2.3. HPLC/MS profile of acerola fruits genotypes ............................................... 55 Figure 3.1. Effect of acerola on cell viability in macrophage cells .................................. 57 Figure 3.2. Effect of acerola on LPS-induced ROS production for 4 h. .......................... 59 Figure 3.3. Effect of acerola on LPS-induced ROS production for 19 h. ........................ 61 Figure 3.4. Effect of acerola on LPS-induced NO production ......................................... 63 Figure 3.5. Effect of acerola on mitochondrial activity after LPS stimulation ................ 66 Figure 3.6. Effect of acerola on COX-2 enzyme activity ................................................. 67 Figure 3.7. Effect of acerola on COX-1 enzyme activity ................................................. 68 Figure 3.8. Proposed