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Extraction, Purification and Characterization of a Pure Peptide from Soybean to Demonstrate Anti-Proliferation Activity on Human

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Extraction, Purification and Characterization of a Pure Peptide from Soybean to Demonstrate Anti-Proliferation Activity on Human University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 12-2015 Extraction, Purification and Characterization of a Pure Peptide from Soybean to Demonstrate Anti- Proliferation Activity on Human Cancer Cells and Test the Ability of Soy Peptide Fractions in Reducing the Activity of Angiotensin-I Converting Enzyme Srinivas Jairam Rayaprolu University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Food Processing Commons Recommended Citation Rayaprolu, Srinivas Jairam, "Extraction, Purification and Characterization of a Pure Peptide from Soybean to Demonstrate Anti- Proliferation Activity on Human Cancer Cells and Test the Ability of Soy Peptide Fractions in Reducing the Activity of Angiotensin-I Converting Enzyme" (2015). Theses and Dissertations. 1343. http://scholarworks.uark.edu/etd/1343 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected]. Extraction, Purification and Characterization of a Pure Peptide from Soybean to Demonstrate Anti-Proliferation Activity on Human Cancer Cells and Test the Ability of Soy Peptide Fractions in Reducing the Activity of Angiotensin-I Converting Enzyme A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Science by Srinivas Rayaprolu Osmania University Bachelor of Science in Farm Science and Rural Development, 1997 Pondicherry Central University Master of Foreign Trade, 2002 University of Bridgeport Master of Science in Human Nutrition, 2005 December 2015 University of Arkansas This dissertation is approved for recommendation to the Graduate Council. ____________________________________ Dr. Navam Hettiarachchy (Dissertation Director) ____________________________________ __________________________________ Dr. Timothy Kral Dr. Pengyin Chen (Committee member) (Committee member) ____________________________________ ____________________________________ Dr. Phil Crandall Dr. Jerald Foote (Committee member) (Committee member – ex-officio) ____________________________________ Dr. Andy Mauromoustakos (Committee member) Abstract Proteins are of major interest for nutritional value, functionality and sensory characteristics in foods, and biological activity as health-promoting ingredients. A large number of potential biological activities are encrypted within the primary structures of animal and plant proteins. This research has been based on utilizing a legume by-product, soybean meal, to obtain value-added protein products. Forty four Arkansas grown soybean lines were subjected to analysis for protein and amino acid (profile) content to select lines that have high protein attributes. Three soybean lines, R95-1705 (high yielding, non-transgenic and high protein), N98- 445A and S03-543CR (bred for high oleic acid - monounsaturated fatty acid) were selected for further study. Seeds from the three soybean lines were ground and defatted to prepare the meals. The protein isolates (SPI) with highest (90-93%) purity prepared from the three meals were hydrolyzed using a food grade enzyme, Alcalase 2.4L, under optimal hydrolysis conditions to prepare protein hydrolysates (pools of peptides). The protein hydrolysates were tested using simulated gastric and intestinal juices to determine the gastro-intestinal (GI) resistance of the peptides. The GI resistant hydrolysates were fractionated to collect distinct molecular size cut- offs of <5, 5-10, 10-50 and >50kDa using ultra-filtration technique. These peptide fractions were tested for biological activities including inhibition of ACE-I activity and cancer cell proliferation inhibition using specific assays. Research highlights include demonstration of ACE-I activity inhibition and cancer cell anti-proliferation activity by the GI-resistant peptides from the soybean lines in comparison to positive controls, Captopril (anti-hypertensive drug) and Genistein (anti- cancer agent). A comprehensive screening provided definitive selection criteria for choosing the 10-50kDa fraction from N98-4445A for further purification to separate a single pure peptide with enhanced biological activity. A purified peptide with a sequence of 158 amino acids and molecular weight of 18kDa from the 10-50kDa fraction showed nearly 75 and 80% inhibition of blood and colon cancer cells respectively. Hence, the importance of this investigation is in the potential ability of a peptides obtained from soybean meal protein to sustain a progressive impact on human health condition. Acknowledgements Foremost, I thank the Almighty for the blessings bestowed upon me which helped in achieving the important goals in my life. I wish to extend my deepest gratitude to Dr. Navam Hettiarachchy for giving me an opportunity to work under her advisement. I appreciate her consistent guidance and input throughout my research, in publications and towards the preparation of this Ph.D. dissertation. I am grateful to Dr. Phil Crandall, Dr. Timothy Kral, Dr. Pengyin Chen, Dr. Andy Mauromoustakos and Dr. Jerald Foote for serving on my advisory committee and for their immense support. I am thankful for the training and instruction received from Drs. Kannan, Eswaranandam and Horax who served as Research Associates for Dr. Hettiarachchy, and would like to acknowledge the help I received from Dr. Rohana Liyanage with the peptide characterization. Being a vital part of my life and for encouraging me to pursue a field of interest away from home, I am eternally indebted to my Parents and Family. I extend my heartfelt appreciation to my friends, fellow researchers and colleagues who have always helped and supported me. I would also like to thank the staff and faculty in the Department of Food science, University of Arkansas for their help and encouragement. Last but not the least, I would like to acknowledge the funding provided by Arkansas Soybean Promotion Board, without which this research might not have been possible. Dedication This Ph D. Dissertation is dedicated to my grandfather, late Mr. Ramakrishniah Rayaprolu. Table of Contents CHAPTER 1: Introduction ................................................................................................. 1 CHAPTER 2: Literature Review ........................................................................................ 6 References………………………………………………………………………………..43 CHAPTER 3: Protein content and amino acid profiling of 44 soybean lines developed in Arkansas ............................................................................................................................ 57 References……………………………………………………………………………..…66 CHAPTER 4: Preparation of gastro-intestinal resistant peptide fractions from N98- 4445A, R95-1705 and S03-543CR soybean lines, and their ACE-I inhibitory activities.77 References………………………………………………………………………………..89 CHAPTER 5: Peptides Derived from High Oleic Acid Soybean Meals Inhibit Colon, Liver and Lung Cancer Cell Growth. ............................................................................... 99 References……………………………………………………………………………....108 CHAPTER 6: Bioactivity of soybean protein fractions against human blood, breast and prostate cancer cell lines ................................................................................................. 116 References…………………………………………………………………………..…..126 CHAPTER 7: Purification and characterization of single peptides with anticancer activities…………………………………………………………………………….......135 References…………………………………………………………………………..…..145 Conclusion ...................................................................................................................... 153 List of Figures Figure 1.1. Illustration of soybean seed parts.……………………………….……………1 Figure 2.1. Breakdown of proteins into peptides and amino acids by enzyme hydrolysis. ................................................................................................................................... 16 Figure 3.1. One-way analysis of amino acid profile among the 44 soybean lines. .......... 76 Figure 4.1. The prediction profiler of the JMP showing the optimized conditions. ......... 92 Figure 4.2. The ACE-I inhibitory activity of soybean peptide fractions at 500µg/mL concentration. ............................................................................................................ 93 Figure 4.3. Dose response study of 5-10kDa and <5kDa peptide fractions from R95-1705 and N98-4445A soybean lines on ACE-I inhibitory activity .................................... 94 Figure 4.4. Purification of the 5-10kDa peptide fraction from R95-1705 using HPLC method ....................................................................................................................... 95 Figure 5.1. Caco-2 colon cancer cell line growth inhibition by gastro-intestinal resistant peptides from the three soybean lines. .................................................................... 111 Figure 5.2. HCT-116 colon cancer cell line growth inhibition by GIPs from the three soybean lines……….……………………………………………………………...112 Figure 5.3. HepG-2 Liver cancer cell line growth inhibition by GIPs from the three soybean lines……………………………………………………………………... 113 Figure 5.4. NCL-H1299 lung cancer cell line growth inhibition by gastro-intestinal resistant peptides from the three soybean lines………………………………...… 114 Figure 5.5. Dose
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