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Antioxidant and Cytotoxic Effects of Dillenia Suffruticosa and Eugenia Polyantha Water Extracts Against Nasopharyngeal Carcinoma Cells

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Antioxidant and Cytotoxic Effects of Dillenia Suffruticosa and Eugenia Polyantha Water Extracts Against Nasopharyngeal Carcinoma Cells Antioxidant and cytotoxic effects of Dillenia suffruticosa and Eugenia polyantha water extracts against nasopharyngeal carcinoma cells by NUR DIYANA BINTI MUSA A thesis submitted in fulfilment of the requirements for the degree of Master of Science (Research) Faculty of Engineering, Computing and Science SWINBURNE UNIVERSITY OF TECHNOLOGY 2020 ABSTRACT Nasopharyngeal carcinoma (NPC) is one of the cancers that is silently prevalent in the east. In Sarawak, it is commonly detected among the local Chinese and Bidayuh ethnics, especially in males. Sarawak is home to a wide variety of flora species that may possess anticancer properties. In this study, the edible plants that were analysed were; (i) Dillenia suffruticosa (Griff.) Martelli or locally known as “daun simpoh/buan” and (ii) Eugenia polyantha Wight or “daun bungkang”. For D. suffruticosa, only the edible upper young shoots were selected as the sample while for E. polyantha, the middle leaves (var. a) and young shoots (var. b) were used. Crude water extracts were prepared from freeze-dried powder of the plant samples. The extract samples were then subjected to total polyphenolic assays; total phenolic content (TPC) and total flavonoid content (TFC). The antioxidant capacities of the extracts were determined using DPPH and ABTS assays. The anticancer potential of the extracts was assessed based on cell viability and cell migration rate in the presence of the extract samples. The cell lines used were nasopharyngeal cancer cells, NPC/HK1 and normal keratinocyte cells, HaCaT for comparisons. Cell viability was determined using MTS cell proliferation assays. The half maximal inhibitory concentration (IC50) value was determined based on the cell viability dose-response curve of the cells incubated with the plant extracts for 72 h. The cell migration rate was estimated based on the closure of linearly scratched zones on a 6-wells plate at 0 h, 7 h and 24 h periods (scratch assay). E. polyantha var. b showed the highest total phenolic content value out of the three samples (6.62 ± 1.6 mg GAE/ 100 mg) as well as having the strongest antioxidant capacity potential based on the DPPH (EC50 = 60.9 ± 15.5 mg/L) and ABTS assays (EC50 = 24.15 ± 1.34 mg/L). Cytotoxicity assay on NPC/HK1 cells showed that extracts of E. polyantha var. b had the most cytotoxic effect on the cells (IC50 = 61.5 ± 17.1 mg/L) and it had also prevented the migration of the cancer cells in the scratch assay. In contrast, E. polyantha var. a showed comparatively insignificant value as compared to E. polyantha var. b and D. suffruticosa. D. suffruticosa leaves showed the highest total flavonoid content (0.45 ± 0.79 mg QE/ 100 mg) and it also showed cytotoxic effects on the NPC/HK1 nasopharyngeal cancer cells (IC50 = 145.3 ± 14.6 mg/L). Scratch migration assay shows that at IC25 concentration, the aqueous extracts were able to prevent migration of the assay mainly through the cytotoxic effect on the cells. In conclusion, the results from the study suggested that the young shoot of E. polyantha (var. b) is a very good source of phenolic compounds and antioxidant, as well as a potential anticancer agent. Further research needs to be done on E. polyantha so that it can be utilised in the growing health industry. ii ACKNOWLEDGEMENT I would like to give my sincerest, heartfelt gratitude and respect to my supervisor, Dr. Irine Runnie Henry Ginjom for giving her many supports throughout my laboratory work and thesis writing. Thank you guiding me through the many new laboratory techniques and assays that were new to me and having so much patience as I go through them with many setback and failures. I am also in gratitude to my co-supervisor, Dr. Hwang Siaw San for also supporting my research especially with the cell-based assays and for giving support in terms of ideas and laboratory materials that were unbeknown to me. I would also like to thank fellow postgraduate colleagues, Lee Boon Kiat, Reagen Entigu, Kong Ee Ling, Diana Choo and Vivian Lee from the cancer team. Their insights were very helpful and thank you helping as well guiding throughout the laboratory works. With their teachings and guidance, I was able to successfully perform was laboratory works in short period of time. My gratitude also for my previous senior, Isuriy Adasuriya for providing samples that have my laboratory work much simpler and easier. Thank you for still communicating with me in the early part of the studies when everything was still new to me. Thank you as well to Phoebe Li Lingyun, a fellow postgraduate colleague with me under Dr. Irine, for helping me with extraction process of the plants and helping me to sometimes to take care of the cells. I would also like to thank the Swinburne laboratory staffs, for giving me help when I am not familiar with the equipment in the laboratory and for being patient as I do my assays which could sometimes go to late evenings or night. Thank you as well to Swinburne School of Research (SoR) for providing me with the fee waiver that I was able to continue with the studies and for putting up with me as I can be quite forgetful. I would like to express my gratitude to my parents, Aishah Ahmad and Musa Brahim for giving me their support throughout my whole project. And lastly to my friends, Fiona Chung, Mertensia Kho and Cindy Chung for giving me moral support and helping me out throughout the study. iii DECLARATION I hereby declare that the thesis presented, contains no material which has been accepted for the award to the candidate of any other degree or diploma, except where due reference is made in the text of the examinable outcome. To the best of my knowledge, the document does not contain material previously published or written by another person except where due reference is made in the text of this thesis. (NUR DIYANA BINTI MUSA) DATE: 11/9/2019 In my capacity as the Principal Supervisor of the candidate’s thesis, I hereby certify that the above statements are true to the best of my knowledge. (IRINE RUNNIE ANAK HENRY GINJOM) DATE: 11/9/2019 iv TABLE OF CONTENTS ABSTRACT ................................................................................................................................ II ACKNOWLEDGEMENT ........................................................................................................ III DECLARATION....................................................................................................................... IV LIST OF FIGURES ............................................................................................................... VIII LIST OF TABLES .................................................................................................................... XI 1 INTRODUCTION .............................................................................................................. 1 1.1 Background of study................................................................................................... 1 1.2 Problem statement ...................................................................................................... 2 1.3 Research hypothesis ................................................................................................... 3 1.4 Research aims and objectives ..................................................................................... 3 1.5 Thesis outline ............................................................................................................. 4 1.6 Significance of the study ............................................................................................ 4 1.7 Limitations of the study .............................................................................................. 5 2 LITERATURE REVIEW .................................................................................................. 6 2.1 Introduction ................................................................................................................ 6 2.2 Plants in the medicine world ...................................................................................... 8 2.2.1 Eugenia polyantha Wight or Syzygium polyanthum (Wight) Walp .............. 8 2.2.2 Dilennia suffruticosa (Griff.) Martelli .......................................................... 9 2.2.3 Health benefits of other plants .................................................................... 12 2.3 Plant secondary metabolites ..................................................................................... 13 2.3.1 Terpenoids ................................................................................................... 13 2.3.2 Phenols ........................................................................................................ 15 2.3.3 Alkaloids ..................................................................................................... 19 2.4 Polyphenol assay ...................................................................................................... 21 2.4.1 Total Phenolic Content assay (TPC) ........................................................... 21 2.4.2 Total Flavonoid Content assay (TFC) ......................................................... 22 2.5 Antioxidant assay ..................................................................................................... 22 2.5.1 DPPH assay (2,2-diphenyl-1-1-picrylhydrazyl) .......................................... 23 2.5.2 ABTS assay (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) ........ 24 2.5.3 CUPRAC ....................................................................................................
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