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Sample Preparation Methods and Pre-Harvest Factors SAMPLE PREPARATION METHODS AND PRE-HARVEST FACTORS INFLUENCING THE CONTENTS OF BIOACTIVE COMPOUNDS AND ANTIOXIDANT ACTIVITY IN PEPPERS A Dissertation by HAE JIN BAE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2011 Major Subject: Horticulture SAMPLE PREPARATION METHODS AND PRE-HARVEST FACTORS INFLUENCING THE CONTENTS OF BIOACTIVE COMPOUNDS AND ANTIOXIDANT ACTIVITY IN PEPPERS A Dissertation by HAE JIN BAE Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Co-Chairs of Committee, Bhimanagouda S. Patil John Jifon Committee Members, G.K. Jayaprakasha Kevin Crosby Tom Cothren Head of Department, Leland Pierson December 2011 Major Subject: Horticulture iii ABSTRACT Sample Preparation Methods and Pre-harvest Factors Influencing the Contents of Bioactive Compounds and Antioxidant Activity in Peppers. (December 2011) Hae Jin Bae, B. S., Sangmyung University; M. S., University of California, Davis Co-Chairs of Advisory Committee: Dr. Bhimanagouda S. Patil Dr. John Jifon Peppers are a rich source of diverse bioactive compounds with potential health- promoting properties. The levels of bioactive compounds and antioxidant activity can be affected by analytical methods, pre-harvest factors, and the quality of peppers. In order to understand the nutrient composition and antioxidant activity in peppers, determination of factors influencing the contents of bioactive compounds is important. The overall objectives were to determine the efficient conditions of sample preparation and the impact of pre-harvest factors affecting bioactive compounds and antioxidant activity. Optimal extraction procedures were developed, and HPLC methods were validated for bioactive compounds in peppers. The highest flavonoids were extracted in ethanol, while myricetin was extracted using N-N-dimethylformamide. Optimized conditions for flavonoids were obtained during 3 h of extraction time and hydrolysis in 3 M HCl for 60 min at 95 °C. Capsaicinoids and ascorbic acid were simultaneously separated and extracted using a solvent mixture consisting of 3% metaphosphoric acid: ethanol (2:8) after 30 min of sonication. To determine the relationship between bioactive iv compounds and antioxidant activities in pepper extracts from different solvent properties, bioactive compounds were analyzed, and the antioxidant activities were assayed by 2,2,- Diphenyl-1-picryl hydrazyl (DPPH), reducing power, and degradation of deoxyribose. Hexane extracts had the highest levels of capsaicinoids and carotenoids, while methanol extracts had the highest levels of flavonoids. Strong DPPH scavenging activity and reducing power were found in lipophilic extracts, while hydrophilic extracts were appropriate for inhibition of deoxyribose degradation. Variation in content of ascorbic acid, capsaicinoids, and flavonoids was evaluated at immature and mature stages of pepper cultivars in different locations over two years. Mature peppers contained the highest levels of capsaicinoids and ascorbic acid. Flavonoids were variable at different maturity stages. Interactions between pre-harvest factors and bioactive compounds were highly significant. This study demonstrated the efficient sample preparation methods and simultaneous separation of bioactive compounds, which reduces analysis time and leads to reduced cost. The antioxidant properties were strongly associated with the concentration of bioactive compounds based on selective pepper extracts. The pepper quality can be improved by using appropriate pre-harvest conditions that increase the levels of bioactive compounds in peppers. v DEDICATION This dissertation is dedicated to my father-in-law vi ACKNOWLEDGMENTS I would like to thank my committee co-chairs, Dr. Bhimanagouda Patil and Dr. John Jifon, for their invaluable and comprehensive help throughout my doctoral study. Dr. Patil has been generous in his support of me from the start of my research and with inspiring numerous discussions. I gratefully acknowledge my supervisor, Dr. Jifon, for sharing his knowledge and guidance, and for his critical reading and valued comments on my manuscripts. I express deep gratitude to Dr. G. K. Jayaprakasha, who provided strategic guidance and excellent technical assistance in all aspects of my work. I owe a special thanks to Dr. Kevin Crosby for the peppers I used in my experiments and for his helpful corrections to my manuscript. I also sincerely appreciate Dr. Tom Cothren‟s encouraging words and sound advice. Finally, my deepest appreciation goes to my husband, who always stands beside me to offer a helping hand with his unconditional love and support. My family deserves as much credit for my doctoral degree as I do. I express sincere appreciation to my parents and parents-in-law for their love, support, and encouragement. vii TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi TABLE OF CONTENTS .......................................................................................... vii LIST OF FIGURES ................................................................................................... x LIST OF TABLES .................................................................................................... xii CHAPTER I INTRODUCTION AND LITERATURE REVIEW ............................ 1 Peppers and Health Benefits .......................................................... 1 Bioactive Compounds in Peppers .................................................. 2 Analytical Methods for Bioactive Compounds .............................. 4 Antioxidants in Peppers ................................................................. 5 Pre-harvest Factors Influencing Bioactive Compounds ................. 6 Rationale ......................................................................................... 6 Objectives ....................................................................................... 7 II EXTRACTION EFFICIENCY AND HPLC METHOD FOR FLAVONOID ANALYSIS FROM PEPPERS .................................... 9 Introduction .................................................................................... 9 Materials and Methods ................................................................... 10 Results and Discussion ................................................................... 18 viii CHAPTER Page III SIMULTANEOUS EXTRACTION AND SEPARATION OF CAPSAICINOIDS AND ASCORBIC ACID ...................................... 33 Introduction .................................................................................... 33 Materials and Methods ................................................................... 36 Results and Discussion ................................................................... 39 IV EVALUATION OF SOLVENT PROPERTY AFFECTING ANTIOXIDANT ACTIVITY AND EXTRACTION OF BIOACTIVE COMPOUNDS IN PUNGENT PEPPERS .......................................... 49 Introduction .................................................................................... 49 Materials and Methods ................................................................... 50 Results and Discussion ................................................................... 56 V DETERMINATION OF EXTRACTION EFFICIENCY FOR THE LEVELS OF BIOACTIVE COMPOUNDS AND ANTIOXIDANT ACTIVITY IN NON-PUNGENT PEPPERS ...................................... 66 Introduction .................................................................................... 66 Materials and Methods ................................................................... 68 Results and Discussion ................................................................... 72 VI IMPACT OF CULTIVAR, YEAR, MATURITY AND THEIR INTERACTIONS WITH BIOACTIVE COMPOUNDS IN GREENHOUSE-GROWN PEPPERS ................................................. 81 Introduction .................................................................................... 81 Materials and Methods ................................................................... 82 Results and Discussion ................................................................... 85 VII IMPACT OF PRE-HARVEST FACTORS AND THEIR INTERACTIONS WITH BIOACTIVE COMPOUNDS IN FIELD-GROWN PEPPERS ................................................................. 94 Introduction .................................................................................... 94 Materials and Methods ................................................................... 95 Results and Discussion ................................................................... 100 VIII SUMMARY AND CONCLUSION ..................................................... 110 ix Page REFERENCES .......................................................................................................... 112 VITA ......................................................................................................................... 127 x LIST OF FIGURES FIGURE Page 1 Extraction of flavonoids from paprika using EtOH; (A) different homogenization time, (B) sample to solvent ratio, and (C) extraction time.
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