Compound Identification and Functional Characterisation of Cane Toad Skin Extracts Author Zulfiker, Abu Hasanat Md Published 2016 Thesis Type Thesis (PhD Doctorate) School School of Medical Science DOI https://doi.org/10.25904/1912/61 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/365733 Griffith Research Online https://research-repository.griffith.edu.au Compound identification and functional characterisation of cane toad skin extracts Abu Hasanat Md. Zulfiker Master of Science (Pharmaceutical Sciences) School of Medical Science Menzies Health Institute Queensland Griffith University Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy June 2016 Dedication This thesis is dedicated to my parents Md. Abdul Zalil Sarkar and Mrs. Nurmahal Zalil who brought me to this beautiful world. Without you, I would not be here today. Thank you very much for your great support and endless care. ii Acknowledgements I have made great efforts in this project, which would not have been possible without the kind support and help of many individuals and organizations. I would like to extend my sincere thanks to all of them. First of all, I am highly indebted to my principal supervisor, Professor Ming Wei for his guidance, encouragement and patience over the last few years of my PhD journey. Thank you so much for giving me the opportunity to work in freedom, to look at research in different ways and on the whole to grow as an independent researcher. My sincere gratitude to my co-supervisor, Dr. I Darren Grice for his indispensable advice, support, constant supervision and direction regarding the project. I also greatly appreciate his immense support for proof reading of documents, which has helped me to improve my writing ability. I would like to acknowledge the valuable support of Dr. Saeed Hashimi who contributed to my lab work, protocol design, manuscript proof reading and many discussions that helped me to shape this project. Much of my experimental work would have not been completed without his kind co-operation and help. I would like to thank Ben Matthews, Laboratory Manager, SmartWater Research Centre, Griffith University for his wonderful support for my analytical work and proof reading of the analytical part of the thesis. iii My appreciation also goes to my laboratory colleagues especially Mohsen Sohrabi who gave me much support during my PhD journey. It was a great pleasure to work with them. I am thankful to Dr. Deepak Samuel Ipe for his great mental support from the very beginning of my PhD. His friendship was vital for me to keep up with my work. Thanks also to Md. Atiqur Rahman, S. M. Riajul Wahab and Md. Forhadul Islam for their support and help during my PhD. I would like to express my gratitude towards my parents and family members, my wife, mother-in-law, father-in-law and their family for the endless support, inspiration and care throughout my life. I greatly acknowledge the School of Medical Science colleagues and members of the Institute for Glycomics as well as the Smart Water Research Centre for their kind co- operation and encouragement, which has helped me to complete this project. I would like to recognize Griffith University, Griffith Graduate Research School, Griffith Health and Menzies Health Institute Queensland (MIHQ) for awarding me the scholarship as well as the grant support to finish my PhD. iv Statement of Originality This work has not previously been submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. (Signed) ---------------------------------- Abu Hasanat Md. Zulfiker v Abstract Amphibians are storehouse of bioactive compounds. Among them, the skin of toad species is rich in biologically active compounds such as peptides, proteins, steroids, alkaloids and opioids. Some of these compounds have found significant therapeutic applications, for example as antibacterials, antifungals, antiprotozoals, antidiabetics, antineoplastics, analgesics and sleep inducing agents. In Traditional Chinese Medicine (TCM) aqueous extracts of Chinese toad skins have been used for centuries to treat pain, swelling, heart failure and several types of cancer with minimal to no side effects, generating a 10 billion USD market in China. Numerous compounds have been identified from these Chinese toad skin extracts, which have reported therapeutic activities in various disease conditions, either as a single compound or as a group of compounds. In Traditional Korean Medicine, toad extracts have also been reported to show potential activity against anxiety and depression. In Australia and America cane toad skins have a history of recreational use for euphoric purposes. This information coupled with knowledge of its use in China and Korea enabled us to hypothesise that Queensland cane toad skin extracts would likely contain similar ‘biologically-active’ compounds in selective extracts. This thesis reports on research work carried out to identify such ‘biologically-active’ extracts and/or compounds, then functionally characterise these in cultured cells for investigation of their therapeutic relevance to neuropsychiatric disorders, especially in obsessive compulsive disorder (OCD) - potentially identifying potent therapeutics for future development. vi To test this hypothesis, the research work presented in chapter 3 initially focussed on designing and optimising several aqueous-based extraction systems of Australian cane toad skins. These were based on various previously reported methods used for compound identification in Chinese toad skin extracts. Extracts were generated in three different solvent systems, including aqueous, organic (60% ethanol) and acidic (acetic acid at two different pHs: 1.78 & 2.17) to attempt to obtain maximum number and amounts of constituents. Thus four extracts were prepared from collected cane toad skins for HPLC-mass spectrometry (HPLC-ESI-Q-TOF-MS/MS) analysis, producing a list of constituents in each extract. The identification of compounds was based on accurate mass measurements for molecular ions and MS/MS analysis, whereby accurate mass pseudo-molecular ions and characteristic fragment ions were compared to published reference data, including mass bank and NIST (National Institute of Standards and Technology). Analysis identified 42 compounds across the four extracts from a number of chemical classes, including alkaloids, amino acids, bufadienolides, fatty acids, nucleobases, nucleosides and vitamins. Of the 42 compounds identified in this study, 29 were identified in the aqueous extract (AE), 35 in the 60% ethanol extract (EE), 10 in the pH 1.78 acetic acid extract and 11 in the pH 2.17 acetic acid extract. Interestingly, the acetic acid extracts contained only one constituent not present in either the AE or EE extract. Therefore, the AE and EE extracts provided the greatest potential for further functional assays in cultured cells. To the best of my knowledge, this is the first comprehensive report on the identification of constituents in Australian cane toad skins. In chapter 4 functional characterisation assays investigating in vitro molecular mechanisms were developed. These assays were utilized to assess the potential of vii the cane toad skin extracts as potential therapeutics against disease conditions such as OCD. Based on recent evidence, the hypothesis was investigated whereby agonistic activation of the serotonin (5-HT)-2 receptor (likely 5- HT2A) in certain areas of brain such as the pre-frontal cortex (PFC) would improve OCD symptoms. OCD is also associated with abnormalities of dopamine neurotransmission, suggesting lower dopamine (D2/3) receptor availability, primarily in the striatum. Additionally, OCD is reportedly related to dysfunction of 5-HT2A-D2 receptor complex (heteromer) regulation. So the primary objective of the assays was to determine whether AE and EE could upregulate 5-HT2A or D2 and increase the interaction of 5-HT2A-D2 receptor heteromer formation. Thus it was demonstrated that AE (50 µg/mL), but not EE (50 µg/mL) significantly (p<0.05) upregulated both 5-HT2A and D2 receptor expression and increased the interaction of 5-HT2A-D2 receptor complex (heteromer) formation upon treatment of CLU213 cells with AE. The CLU213 cells were previously validated as expressing both 5-HT2A and D2 receptors. The classical signalling pathway (Gq11- PLCβ) was also investigated in connection with 5-HT2A mediated agonist activation. The results indicate that AE (50 µg/mL) upregulates 5-HT2A receptor expression by a mechanism that seems to involve activation of the Gq/11 G protein PLCβ signalling pathway and c-FOS transcription factor. Taken together, this molecular mechanism could provide a basis to progress further development of cane toad skin extracts as a novel therapeutic agent for OCD. In chapter 5, functional characterisation studies were extended by conducting anti- inflammatory assays on AE, EE and BT (Bufotenine-a known marker reported to be present commonly in toad skins). BT was identified in both AE and EE by HPLC-ESI- Q-TOF-MS/MS in this research. Inflammation is reported as a common condition in viii numerous diseases including OCD. This experiment was performed with AE (0.1-10 µg/mL), EE (0.1-10 µg/mL) and BT (0.1-10 nM) in a model of human monocyte cell line U937 stimulated with