Chapman University Chapman University Digital Commons Pharmaceutical Sciences (MS) Theses Dissertations and Theses Summer 8-2019 Proteomics and Biological Evaluation of Marshmallow (Althea officinalis) Seeds Mahshid Amini Chapman University, [email protected] Follow this and additional works at: https://digitalcommons.chapman.edu/ pharmaceutical_sciences_theses Part of the Other Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Amini M. Proteomics and Biological Evaluation of Marshmallow (Althea officinalis) Seeds. [master's thesis]. Irvine, CA: Chapman University; 2019. https://doi.org/10.36837/chapman.000091 This Thesis is brought to you for free and open access by the Dissertations and Theses at Chapman University Digital Commons. It has been accepted for inclusion in Pharmaceutical Sciences (MS) Theses by an authorized administrator of Chapman University Digital Commons. For more information, please contact [email protected]. Proteomics and Biological Evaluation of Marshmallow (Althea officinalis) Seeds A Thesis by Mahshid Amini Chapman University Irvine, CA School of Pharmacy Submitted in partial fulfillment of the requirements for the degree of Master of Science in Pharmaceutical Sciences August 2019 Committee in charge: Dr. Aftab Ahmed, Ph.D., Chair Dr. Sherif Elshahawi, Ph.D. Dr. Rakesh Tiwari, Ph.D. Proteomics and Biological Evaluation of Marshmallow (Althea officinalis) Seeds Copyright © 2019 by Mahshid Amini iii ACKNOWLEDGMENTS I want to express my special appreciation and thanks to my advisor Dr. Aftab Ahmed, Research Associate Professor, Chapman University School of Pharmacy, for the continuous support of my Master study and research, for his patience, motivation, enthusiasm, and immense knowledge. His guidance and his support helped me in all the time of research and writing of this thesis. He has provided me extensive personal and professional guidance and taught me a great deal about both scientific research and life in general. I would like to thank you for encouraging my research and for allowing me to grow as a research scientist. I want to extend my sincere gratitude to my thesis committee members, Dr. Sherif Elshahawi for his valuable and constructive suggestions during this research and his help for performing Anti- Microbial assay, and Dr. Rakesh Tiwari for his encouragement, insightful comments, and his support. My sincere thanks also go to Syed Basir, Laboratory Specialist, CUSP Analytical Core Facility, for his help in LC/MS/MS data analysis. A special word of gratitude for Yamna Khurshid and Taibah Aldakhil, Mekdes Megeressa, Saud Alshammari for their help in the cytotoxic analysis and other laboratory techniques. I am grateful to all of those with whom I have had the pleasure to work during this project with administrative and research staff, and other members of Chapman University School of Pharmacy for all of their help. Many thanks to my colleagues and friends for all the fun and collaboration. Last but not least, I would like to express my very special thanks to my family for always supporting me throughout my life and always being there for me. This journey would not have been possible without your support. Thank you for everything! Mahshid Amini iv This thesis is dedicated to my parents for their endless love, support, and encouragement. My sister and brother for their love I appreciate everything that you have done for me. v ABSTRACT Proteomics and Biological Evaluation of Marshmallow (Althea officinalis) Seeds by Mahshid Amini Marshmallow (Althaea officinalis) belongs to family Malvaceae, a medicinal plant used in traditional and alternative medicine in various parts of the world. Marshmallow is one of the seven medicinal plants that are collectively known as Joshanda. It is a herbal preparation which has been used traditionally as a home remedy for the treatment of common cold, coughs, nasal congestion, and fever. Previous studies showed that crude extract and/or purified compounds from flowers, root, and leaves from Marshmallow have anti-microbial, anti-inflammatory, and other pharmacological effects. However, there is no data reported on the biological activity and the structure of proteins isolated from the seeds. Therefore, this study is carried out to purify and characterize proteins and peptides from Marshmallow seeds as well as to evaluate their antimicrobial activity against Salmonella enterica, Staphylococcus aureus, Saccharomyces cerevisiae and cytotoxic activities in MCF-7 breast cancer cell line. Extraction of proteins was performed in 20 mM Tris/HCl pH 8.0, then purified by the combination of gel filtration and reverse-phase HPLC. Purified proteins were analyzed by SDS-PAGE gel electrophoresis. The partial amino acid sequence of 22 kDa protein was established up to 25 residues by N-terminal amino acid sequencing. The data suggest that Marshmallow RP-HPLC purified protein is a monomeric protein of 22 kDa. The amino acid sequence alignment using BLASTp revealed similarity in 22/25 amino acid residues with Thaumatin from Corchorus capsularis. The MTT assay of Marshmallow crude protein led to a dose-dependent cytotoxic effect in MCF-7 cells with an IC50 value of 163.29 µg/ml. The gel filtration pooled fractions 5-8, 9-11, and 12-13 showed 58%, 46% and 32% inhibition respectively. The antimicrobial activity of crude protein extract vi showed 69% inhibitory activity against Staphylococcus aureus. Proteomic evaluation of crude Marshmallow protein tryptic peptides achieved by LC-MS/MS and bioinformatic analysis using SwissProt Viridiplanta database and Mascot search engine. The De novo protein/peptide sequence search by Peaks Studio-X identified 76 proteins belonging to 9 protein groups. vii TABLE OF CONTENTS Chapter 1 1 INTRODUCTION Chapter 2 8 METHODS Chapter 3 17 RESULTS and DISCUSSION Chapter 4 40 CONCLUSION References 41 viii LIST OF TABLES Table 1. List of chemicals for gel electrophoresis 11 Table 2. Protein ID by MASCOT SwissProt Database 30 Table 3. Statistics of filtered result 34 Table 4. Cytotoxicity Assay against MCF 7 Human breast cancer cell line 38 ix LIST OF FIGURES Figure 1. Picture of Joshanda (Herbal Medicine) 03 Figure 2. Picture of Marshmallow (Althaea officinalis) seeds 05 Figure 3. Schematic of the workflow of Marshmallow seeds protein 10 Figure 4. SDS-PAGE profile of crude Marshmallow protein extract 18 Figure 5. Elution profile of Marshmallow crude proteins by gel filtration chromatography 19 Figure 6. SDS-PAGE profile of gel filtration fractions 20 Figure 7. 2D-LC elution profile of Marshmallow seeds pooled gel filtration fraction 5-8 21 Figure 8. SDS-PAGE profile of 2D RP-HPLC fractions 22 Figure 9. Reverse Phase HPLC profile of crude Marshmallow protein extract 23 Figure 10. SDS-PAGE profile of RP-HPLC purified Marshmallow fractions 24 Figure 11. Amino acid sequence of RP-HPLC fraction 51 26 Figure 12. Tryptic digest LC/MS profile of Marshmallow protein 28 Figure 13. Q-TOF MS/MS sample acquisition parameters 29 Figure 14. Protein hits in Mascot search engine using SwissProt database 31 Figure 15. False discovery rate (FDR) curve by PEAKS software 32 Figure 16. Profile of Marshmallow crude tryptic digest PSM score distribution 33 Figure 17. De novo only searches by Peaks Studio 35 Figure 18. Proteins identified against NCBI nr database De novo peptide sequencing 36 Figure 19. Cytotoxicity analysis of MCF-7 cell lines 37 Figure 20. Cell inhibition of MCF-7 breast cancer cells treated with doxorubicin 38 Figure 21. Anti-microbial Assay of Marshmallow extract in organic solvents and proteins 39 x Abbreviation AO Althea officinalis DMSO Dimethyl Sulfoxide DI H2O Deionized Water EDTA Ethylenediaminetetraacetic Acid EtOH Ethanol MeOH Methanol PBS Phosphate Buffered Saline PEG Polyethylene Glycol? SDS Sodium Dodecyl Sulfate TEMED N,N,N’,N’- tetramethylethylenediamine Tris Tris-(hydroxymethyl) amino-methane HCl Hydrochloric Acid THF Tetrahydrofuran HPLC High-Performance Liquid Chromatography RP-HPLC Reverse-phase HPLC FPLC Fast protein liquid chromatography MSP Sodium phosphate monobasic DSP Sodium phosphate dibasic AMS Ammonium sulfate AM Acrylamide BME 2-mercaptoethanol TPCK Trypsin Tosyl-phenylalanyl-chloromethyl-ketone xi MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide PVDF Polyvinylidene difluoride GFD Glass fiber discs FDR False discovery rate PSM Peptide spectrum match RT Retention time m/z Mass to charge ratio ALC Average local confidence NCBI National Center for Biotechnology Information FDA Food and Drug Administration xii Chapter 1 INTRODUCTION 1.1. Botanicals (Herbal Medicine): Herbal medicine is used to treat disease and enhance general health for centuries. Herbal medicine (Natural remedies) may seem like a new concept, but it is the oldest form of medicine in history. Studies in human subjects also confirm the specific therapeutic effects of particular herbs (Al- Snafi et al., 2013). Plants have been the basis for medical treatments in a different part of the world. Parts of the plant such as leaves, flower, fruit, root, and seed have been in use as a traditional and folk remedy to treat many different health conditions. Echinacea is most commonly used for the treatment of the common cold, Ginseng is used to improve energy and is found in many drinks and tonics, Garlic is used for many medicinal properties, but it most affects on cholesterol levels, and Ginger is commonly used as a treatment for nausea. (Bent 2008) Herbal supplements come in all forms: dried, chopped, powdered, capsule, or liquid. Numerous