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Studies on Clinacanthus Nutans Genetic, phytochemical and bioactivity studies of Clinacanthus nutans (Burm. f.) Lindau (Acanthaceae) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (Applied Biology and Biotechnology) Siat Yee Fong BSc (Biotechnology) (Monash), MAppSc (Molecular Biotechnology) (USYD) School of Applied Sciences College of Science, Engineering and Health RMIT University August 2015 Declaration I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines have been followed. Siat Yee Fong 31 August 2015 i Acknowledgements I thank my supervisors, Dr Tien Huynh, Associate Professor Terrence Piva and Dr Sylvia Urban for their supervision, advice, encouragement, help and support during my entire PhD tenure. Thank you for your insight and wisdom in manuscripts and thesis. I am also grateful to Professor Ann Lawrie, whose advice has been generously given on a number of occasions. I acknowledge TKC farm, Kui Min Liau, Vun Jhit Lim and Rattanapong Charntawong for the provision of plant materials. My sincere thanks to Robert Brkljaca and Dr Chaitali Dekiwadia for their help in HPLC and TEM work, respectively. Thanks are also extended to Layla Mehdi Alhasan, Griff D’Costa, Xian Yang Chan, Alaa Banjar and Sushil Talda for providing the cells and reagents used in this study. I am very appreciative for the provision of scholarship by the Ministry of Higher Education Malaysia and also the financial support from Universiti Malaysia Sabah. Without these, it would have been impossible for me to pursue my PhD. Last but not least, I am extremely grateful to my parents, family and friends for their unfailing love and support, mentally and spiritually throughout. Sorry I had to make you listen to all my complaints. Special thanks to Dilani Wimalasiri for the friendship. I am thankful that we could share our ups and downs together during these 3 years. ii Papers and communications arising from this study FONG, S. Y., PIVA, T., URBAN, S. & HUYNH, T. 2014. Genetic homogeneity of vegetatively propagated Clinacanthus nutans (Acanthaceae). J. Med. Plants Res., 8, 903-914. http://www.academicjournals.org/journal/JMPR/article-abstract/629A21546085. (Presented as Chapter 2). FONG, S. Y., PIVA, T., URBAN, S. & HUYNH, T. Variation in total phenolic and flavonoid contents and anticancer activity of Clinacanthus nutan (Burm. f.) Lindau (Acanthaceae) leaves against human D24 melanoma cells. (Manuscript). FONG, S. Y., PIVA, T., URBAN, S. & HUYNH, T. Effect of postharverst processes on the total phenolic and flavonoid contents and anticancer activity of Clinacanthus nutans (Burm. f.) Lindau (Acanthaceae) against selected human cancer cell lines. (Manuscript). FONG, S. Y. & HUYNH, T. Assessment of genetic variation between and within Clinacanthus nutans and Andrographis paniculata using RFLP, RAPD and microsatellite markers. RMIT School of Applied Sciences Annual Research Day, Melbourne, Australia. 14 June 2013. FONG, S. Y., BRKLJACA, R., URBAN, S., PIVA, T., & HUYNH, T. Variation in total phenolic and flavonoid content in leaves of Clinacanthus nutans. 2nd International Conference and Exhibition on Pharmacognosy, Phytochemistry & Natural Products, Beijing, China. 25-27 August 2014. FONG, S. Y., BRKLJACA, R., URBAN, S., PIVA, T. & HUYNH, T. Variation in total phenolic and flavonoid content in leaves of Clinacanthus nutans. RMIT SEH College HDR Student Conference, Melbourne, Australia. 17 October 2014. iii Table of Contents Declaration .............................................................................................................................. i Acknowledgements ................................................................................................................ ii Papers and communications arising from this study .............................................................iii List of figures ......................................................................................................................... x List of tables ......................................................................................................................... xii List of Abbreviations and acronyms ...................................................................................xiii Abstract .............................................................................................................................. xvii CHAPTER 1 ........................................................................................................................... 1 Introduction and review of literature ...................................................................................... 1 1.1 Importance of medicinal plants ........................................................................................ 1 1.2 Acanthaceae ..................................................................................................................... 3 1.2.1 Family characteristics ................................................................................................ 3 1.2.2 Important members .................................................................................................... 4 1.3 Clinacanthus nutans (Burm. f.) Lindau ........................................................................... 7 1.3.1 Vernacular names ...................................................................................................... 7 1.3.2 Geographical distribution/ecology ............................................................................ 7 1.3.3 Botanical characteristics ............................................................................................ 7 1.3.4 Propagation ................................................................................................................ 9 1.3.5 Genetic diversity ...................................................................................................... 10 1.3.6 Phytochemical constituents ..................................................................................... 16 1.3.7 Traditional medicinal uses of C. nutans .................................................................. 37 1.3.8 Pharmacological activities of C. nutans .................................................................. 38 1.3.9 Cancer ...................................................................................................................... 42 1.4 Overview and aims of study ........................................................................................... 58 CHAPTER 2 ......................................................................................................................... 61 Genetic homogeneity of vegetatively propagated Clinacanthus nutans (Burm. f.) Lindau (Acanthaceae) ....................................................................................................................... 61 2.1 Introduction .................................................................................................................... 61 2.2 Materials and methods ................................................................................................... 64 2.2.1 Plant materials ......................................................................................................... 64 2.2.2 DNA extraction........................................................................................................ 66 2.2.3 PCR-RFLP ............................................................................................................... 66 2.2.4 RAPD amplification ................................................................................................ 68 iv 2.2.5 Microsatellite amplification ..................................................................................... 68 2.2.6 Data collection and analysis .................................................................................... 69 2.3 Results ............................................................................................................................ 70 2.3.1 PCR-RFLP analysis ................................................................................................. 70 2.3.2 RAPD analysis ......................................................................................................... 71 2.3.3 Microsatellite analysis ............................................................................................. 74 2.4 Discussion ...................................................................................................................... 78 2.4.1 Genetic homogeneity in C. nutans .......................................................................... 78 2.4.2 Genetic differences between C. nutans, C. siamensis and A. paniculata ................ 80 2.4.3 Genetic variation in A. paniculata ........................................................................... 81 2.5 Conclusion ...................................................................................................................... 82 CHAPTER 3 ........................................................................................................................
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