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Identification, Quantification and Investigation of Anti-Inflammatory Effects of Echinacea Purpurea Constituents

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Identification, Quantification and Investigation of Anti-Inflammatory Effects of Echinacea Purpurea Constituents KANDHI, VAMSIKRISHNA, Ph.D. Identification, Quantification and Investigation of Anti-inflammatory Effects of Echinacea purpurea Constituents. (2012) Directed by Dr. Nadja B. Cech. 263 pp. Echinacea is among the top selling botanical medicines in the United States. Clinical trials have yielded contradictory reports on the efficacy of Echinacea preparations for treatment of colds, influenza, and inflammation, perhaps due to inconsistency in quality control and lack of information as to which constituents are responsible for their activity. Our overall goal with this research was to develop and apply mass spectrometry techniques to better understand the relationship between chemical composition of Echinacea extracts and their biological activity. This objective was achieved through three separate aims. (1) To determine whether in vitro anti-inflammatory activity of Echinacea purpurea extracts could be correlated with the presence of the alkylamides or caffeic acid derivatives, which have previously been reported to be bioactive. The ability of these extracts to inhibit production of TNF-alpha and PGE2 from influenza A-infected RAW 264.7 macrophage-like cells was assessed. Chemical analysis of the extracts revealed variations in levels of alkylamides, caftaric acid, and cichoric acid. The biological activity of these extracts, however, did not correlate with concentrations of any of these compounds. (2) To investigate the effects of Echinacea purpurea preparations and their constituents on protein expression by immune cells in vitro. Stable isotope labeling with amino acid in cell culture (SILAC), a quantitative proteomics approach, was employed to measure changes in protein expression by activated RAW 264.7 macrophage- type cells exposed to an Echinacea purpurea extract and several of its constituents. The extract and several isolated alkylamides were observed to block expression of a number of proteins, some of which were directly or indirectly involved in inflammation. (3) To develop new analytical methods to analyze bacterial lipoproteins. Lipoproteins are commonly found in aqueous Echinacea extracts and have pronounced immunostimulatory activity in vitro. These compounds are large and polydisperse, making them very difficult to characterize analytically. A new liquid chromatography – mass spectrometry (LC- MS) analytical method was developed and employed to analyze lipoproteins from E. coli and from an endophyte isolated from Echinacea purpurea seeds. IDENTIFICATION, QUANTIFICATION AND INVESTIGATION OF ANTI- INFLAMMATORY EFFECTS OF ECHINACEA PURPUREA CONSTITUENTS by Vamsikrishna Kandhi A Dissertation Submitted to the Faculty of The Graduate School at The University of North Carolina at Greensboro in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Greensboro 2012 Approved by _______________________ Committee Chair To my Wife, Thank you for your love, encouragement, and support. ii APPROVAL PAGE This dissertation has been approved by the following committee of the Faculty of The Graduate School at The University of North Carolina at Greensboro. Committee Chair Dr. Nadja B. Cech, Ph.D. Committee Members Dr. Scott M. Laster, Ph.D. Dr. Gregory M. Raner, Ph.D. Dr. Norman H. L. Chiu, Ph.D. __________________________________ Dr. Vincent C. Henrich, Ph.D. 10/26/2012 Date of Acceptance by Committee 10/26/2012 Date of Final Oral Examination iii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to Dr. Nadja B. Cech for her support through my work at UNCG. I learned valuable lessons from Dr. Cech and I will always be grateful for her guidance. I would also like to thank my other committee members Dr. Gregory M. Raner, Dr. Scott M. Laster Dr. Vincent C. Henrich and Dr. Norman H. L. Chiu for their valuable comments and assistance through my graduate work. I would also like to thank all the Professors who have helped me at UNCG. Without my family and friends, I would not be where I am today. Thanks go to my parents, and my sister and my brother. They were there every step of the way supporting me and helping and for always being there for me. iv TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................... viii LIST OF FIGURES ............................................................................................... x CHAPTER I. INTRODUCTION .................................................................................... 1 1.1 Introduction to Echinacea .......................................................... 1 1.2 Review of Literature .................................................................. 2 1.3 Activity of Constituents from Echinacea In Vitro ........................ 3 1.4 Activity of Crude Echinacea Extracts In Vitro ............................ 4 1.5 Activity of Crude Echinacea Extracts In Vivo............................. 4 1.5.1 Structures of compounds present in Echinacea .......... 5 1.6 Mass Spectrometry ................................................................... 5 1.6.1 Nano-Electrospray Ionization (Nano-ESI) .................... 6 1.6.2 Linear quadrupole ion trap coupled to orbitrap mass spectrometer (LTQ-Orbitrap) ........................... 7 1.7 Mass Spectrometry Based Proteomics ..................................... 9 1.7.1 Proteomics .................................................................. 9 1.7.2 Shotgun proteomics ................................................... 11 1.8 Quantitative Proteomics .......................................................... 12 1.8.1 Absolute quantitation ................................................. 12 1.8.2 Relative quantitation .................................................. 13 1.8.2.1 Chemical labeling .......................................... 13 1.8.2. 2 Metabolic labeling ........................................ 16 1.8.3. Label free quantitation .............................................. 18 1.9 Protein Identification and Database Searching ....................... 19 1.10 Chromatography Used in Shotgun Proteomics ..................... 20 II. IDENTIFY AND QUANTIFY CONSTITUENTSRESPONSIBLE FOR THE ANTI-INFLAMMATORY ACTIVITY OF ECHINACEA PURPUREA CONSTITUENTS .................................... 21 2.1 Abstract ................................................................................... 21 2.2 Introduction ............................................................................. 23 2.3 Experimental ........................................................................... 24 2.3.1 Preparation of extracts .............................................. 24 2.3.2 LC-MS method .......................................................... 25 v 2.3.3 Anti-inflammatory activity ........................................... 26 2.4 Results and Discussion ........................................................... 27 2.5 Conclusion .............................................................................. 29 III. QUANTITATIVE PROTEOMICS USING SILAC TO INVESTIGATE THE IMMUNOMODULATORY EFFECTS OF ECHINACEA PURPUREA .......................................................... 34 3.1 Abstract ................................................................................... 34 3.2 Introduction ............................................................................. 37 3.3 Materials and Methods ............................................................ 39 3.3.1 Cell culture media ...................................................... 40 3.3.2 Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) ........................................... 40 3.3.3 Extraction of protein ................................................... 42 3.3.4 Quantitation by Bicinchonic Acid Assay (BCA) .......... 43 3.3.5 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and coomassie G-250 staining ....................................................... 46 3.3.6 In-gel digestion .......................................................... 46 3.3.7 Nano-LC-NSI-MS/MS analysis .................................. 47 3.3.8 Lock mass internal calibration ................................... 48 3.3.9 Data analysis ............................................................. 49 3.3.10 Preparation of Echinacea extracts ........................... 50 3.4 Experimental Design ............................................................... 51 3.5 Results and Discussion ........................................................... 54 3.5.1 Identification of proteins by proteomics ..................... 57 3.5.2 Effect of lipopolysaccharide on RAW 264.7 cells ..................................................... 59 3.5.3 Effects of Echinacea extract 7 on RAW 264.7 cells ..................................................... 62 3.5.4 Effects of Echinacea extract 7 on LPS induced proteins in RAW 264.7 cells ................................... 62 3.5.5 Effects of individual alkylamides on LPS-induced proteins in RAW 264.7 cells ................................... 66 3.6 Conclusion .............................................................................. 68 3.7 Tables of Protein Expression Data .......................................... 69 IV. METHOD DEVELOPMENT FOR ANALYSIS OF COMPLEX PROTEIN SAMPLES USING OFFLINE LC-MALDI MS/MS .......... 222 4.1 Introduction ........................................................................... 222 4.2 Identification Without Prior Separation .................................. 222 vi 4.3 Reduction and Alkylation
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