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

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.

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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

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 ...... 25 2.5.4 FRAP assay ...... 26 2.5.5 ORAC assay ...... 27

2.6 Nasopharyngeal cancer ...... 28 2.6.1 Background ...... 28 2.6.2 Types of cancer therapies ...... 29 2.6.3 Anticancer agent and cytotoxicity ...... 30 2.6.4 Apoptosis related proteins ...... 31 2.7 Cell culture for evaluation of anticancer properties ...... 33 2.7.1 Types of cells for NPC studies ...... 33 2.7.2 Proliferation assays ...... 33 2.7.3 Scratch assay ...... 36 2.7.4 Flow cytometry and cell cycle analysis ...... 36 2.8 Conclusion ...... 37

3 MATERIALS AND METHODS ...... 38 3.1 Sample preparation ...... 38 3.1.1 Plant sample source ...... 38 3.1.2 Preparation of plant water extract ...... 39 3.1.3 Preparation of extracts for cell culture ...... 40 3.2 Determination of polyphenol contents ...... 40 3.2.1 Chemicals ...... 40 3.2.2 Total Phenolic Content (TPC) assay ...... 41 3.2.3 Total Flavonoid Content (TFC) assay ...... 41 3.3 Determination of antioxidant properties based on in vitro methods ...... 42 3.3.1 Chemicals ...... 42 3.3.2 DPPH assay ...... 42 3.3.3 ABTS assay ...... 43 3.4 Screening of anticancer properties based on cell culture assays ...... 44 3.4.1 Chemicals, reagents and materials ...... 44 3.4.2 Cell lines and culture medium...... 45 3.4.3 Cell thawing and cell cryopreservation ...... 45 3.4.4 Cell counting ...... 46 3.4.5 Cell passaging ...... 48 3.4.6 Cell optimisation ...... 48 3.4.7 Cell viability assay with MTS ...... 49 3.4.8 Cell migration assay ...... 50 3.5 Statistical analysis ...... 53

4 RESULTS AND DISCUSSION ...... 54

4.1 Moisture content and extraction yield ...... 54 4.2 Total phenolic and total flavonoid contents in plant extracts ...... 55 4.2.1 Calibration curve ...... 55 4.2.2 Phenolic and flavonoid contents of D. suffruticosa, E. polyantha var. a and E. polyantha var. b...... 56 4.3 Antioxidant potentials of plant extracts ...... 58 4.3.1 Antioxidant activities based on DPPH assay ...... 58 4.3.2 Antioxidant activities based on ABTS assay ...... 61 4.3.3 Correlation analysis between total phenolic and antioxidant effects of selected plant extracts ...... 64 4.4 Screening of anticancer properties based on cell culture assays ...... 65 4.4.1 Cell number optimisation ...... 65 4.4.2 Cytotoxicity assay ...... 67 4.4.3 Cell migration assay - Scratch test ...... 70 4.5 Summary ...... 76

5 CONCLUSION ...... 77 5.1 Further work ...... 78

6 REFERENCES ...... 80

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LIST OF FIGURES

Figure 1: Botany illustration of E. polyantha ...... 6

Figure 2: Botany illustration of D. suffruticosa ...... 7

Figure 3: Isoprene ...... 14

Figure 4: Molecular structure of paclitaxel (Taxol) ...... 14

Figure 5: Phenol ...... 15

Figure 6: Flavonoid ...... 16

Figure 7: Anthocyanin ...... 17

Figure 8: Flavones...... 18

Figure 9: Pyridine - the most basic alkaloid structure...... 20

Figure 10: Berberine ...... 20

Figure 11: Basic antioxidant activity ...... 23

Figure 12: (a) The 2,2-diphenyl-1-1-picrylhydrazyl radical and (b) The stable form 2,2- diphenyl-1-1picrylhydrazine. Image source: (Molyneux (2004); Alam, Bristi and Rafiquzzaman (2013))...... 24

Figure 13: (a) Structure of ABTS•+ radical cation (greenish blue in colour) and (b) Structure of ABTS(H) (colourless). Image adapted from: (Gupta (2015) ...... 25

Figure 14: (a) Structure of Cu(II)-Nic (blue in colour)and (b) Structure of Cu(I)-Nic after reduction (orange in colour). Image adapted from: (Apak et al. (2014) ...... 26

Figure 15: (a) Structure of the Fe3+(ferric complex) and (b) Structure of the Fe2+ (ferrous complex). Image adapted from: (Prior, Wu and Schaich (2005) ...... 27

Figure 16: Structure of fluorescein ...... 28

Figure 17: Reduction of MTT to formazan...... 34

Figure 18: Reduction by phenazine ethyl sulfate (PES) converting MTS to water soluble formazan. Image source: Riss et al. (2016) ...... 35

Figure 19: Methodology flow chart...... 38

Figure 20: Image of leaves of plant sample used (Adasuriya (2018) with permission) ...... 39

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Figure 21: (a) Counting cells using a haemocytometer. (b) Non-viable cells i.e. dead cells are stained blue whereas viable cells are not be stained blue when observed under an inverted microscope. Image source: (Merck 2019) ...... 47

Figure 22: Diagram of how the observation area mark should be drawn on top of the lid for scratch assay...... 51

Figure 23: The diagram shows the settings of the Tscratch assay program to determine gap distance of the cells (Gebäck et al. 2009) ...... 52

Figure 24: Calibration curve of gallic acid for total phenolic content (TPC) assay: Absorbance value at 760 nm at different concentrations. All values are expressed as mean ± standard deviation (n=3)...... 55

Figure 25: Calibration curve of quercetin for total flavonoid content (TFC): Absorbance value at 415 nm at different concentrations. All values are expressed as mean ± standard deviation (n=3)...... 56

Figure 26: Dose response curve of plant extracts: (a) gallic acid, (b) Trolox, (c) D. suffruticosa, (d) E. polyantha var. a, and (e) E. polyantha var. b as determined using the DPPH assay. Data shown are mean ± standard deviation (n=3)...... 59

Figure 27: Dose response curve of plant extracts: (a) Trolox, (b) Gallic Acid, (c) D. suffruticosa, (d) E. polyantha var. b, and (e) E. polyantha var. a as determined using the ABTS assay. Data shown are mean ± standard deviation (n=3)...... 62

Figure 28: The effect of NPC/HK1 cell count on cell viability in 100 L aliquot...... 66

Figure 29: The effect of HaCaT cell count on cell viability in 100 L aliquot...... 66

Figure 30: The effect of D. suffruticosa on NPC/HK1 cell viability. All values are expressed as mean ± standard deviation (n=3) ...... 67

Figure 31: The effect of D. suffruticosa on HaCaT cell viability All values are expressed as mean ± standard deviation (n=3) ...... 67

Figure 32: The effect of D. suffruticosa incubation on cell migration. Gap distance was measured at 3 different time points; (a) 0 hour, (b) 7 h (c) 24 h (d) an example of healthy living cells (e) an example of dead cells. Results are representative for two independent experiments. Images were taken at 100X magnification and P3 phase contrast resolution light...... 71

Figure 33: The effect of E. polyantha var. a (incubation on cell migration. Gap distance was measured at 3 different time points; (a) 0 hour, (b) 7 h (c) 24 h. Results are representative for two independent experiments. Images were taken at 100X magnification and P3 phase contrast resolution light...... 72

Figure 34: The effect of E. polyantha var. b incubation on cell migration. Gap distance was measured at 3 different time points; (a) 0 hour, (b) 7 h (c) 24 h. Results are representative for two independent experiments. Images were taken at 100X magnification and P3 phase contrast resolution light...... 73

LIST OF TABLES

Table 1: The effect of different extraction solvents of D. suffruticosa leaves on different cancer cell lines...... 10

Table 2: Classes of terpenoids ...... 15

Table 3: Different types of cell line used for NPC studies ...... 33

Table 4: Summary of the moisture content and extraction yield of the plants...... 54

Table 5: Summary of total phenolic content (TPC) and total flavonoid content (TFC) of selected plant extracts...... 56

Table 6: Summary of antioxidant activities of standard references and plant extracts based on DPPH and ABTS assays...... 63

Table 7: Summary of correlation analysis of polyphenolic contents and antioxidant assays on D. suffruticosa, E. polyantha var. a and E. polyantha var. b...... 64

Table 8: Summary of cytotoxicity effect plant extracts on cancerous call line, NPC/HK1 and normal cell line, HaCaT...... 68

Table 9: Summary of migration effect of plants on cancerous cell line, NPC/HK1...... 74

LIST OF ABBREVIATIONS

ABTS 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid BA Betulinic acid BMI Body mass index CaOV3 Ovarian cancer cell COPD Chronic obstructive pulmonary disease CUPRAC Cupric reducing antioxidant capacity CVD Cardiovascular disease DCM Dichloromethane DMEM Dulbecco's modified Eagle's medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPPH 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl DW Dry Weight EBNA Epstein-Barr nuclear antigen

EC50 Half maximal effective concentration EDTA Ethylenediaminetetraacetic acid EIU Economist Intelligence Unit EtOAc Ethyl Acetate FCR Folin-Ciocalteau reagent Fe2+ Ferrous ion Fe3+ Ferric ion FRAP Ferric reducing antioxidant power FW Fresh weight HELA Cervical Cancer Cells HL60 Human Caucasian promyelocytic leukaemia cell line HSP70 Heat shock protein 70

IC50 Half maximal inhibiting concentration IPH Institute of Public Health K562 Immortalised myelogenous leukaemia cell line

LD50 Half maximal lethal dose MCF-7 Oestrogen positive breast cancer cells

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MDA-MB-231 Oestrogen negative breast cancer cells MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H- tetrazolium MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MOH Ministry of Health Malaysia NCD Non-communicable disease NHMS National health and morbidity survey OA Oleanic acid ORAC Oxygen Radical Absorbance Capacity PBS Phosphate buffer saline P53 Tumour suppressor protein ROS Reactive oxygen species TFC Total flavonoid content TPC Total phenolic content UA Ursolic acid UV Ultraviolet U937 Pro-monocytic human myeloid leukaemia cell line WHO World Health Organisation WRL-68 Hepatic cancer cell line 3T3-F442A Non-viable mouse embryo cells 3T3-L1 Normal adipose cells

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1 INTRODUCTION

1.1 Background of study

Humans have always been vulnerable to diseases. Many deaths over the centuries have been contributed by the numerous wars, diseases, malnutrition and natural death. Diseases that are not caused by infections by bacteria or viruses are also known non-communicable diseases (NCDs) or chronic diseases. According to the World Health Organization or WHO (2019), globally, NCDs causes approximately 70% of deaths every year and most of the diseases are usually due to the lifestyle choices, heredity, genetic, environmental exposure or combinations of these. Worldwide, the major contributor to deaths due to NCDs is cardiovascular diseases. In 2016, the top 3 cause of death in the world are due to NCDs with heart disease being at the top causing deaths close to 10 million of people then followed by stroke and chronic obstructive pulmonary disease (COPD).

Cancer is a disease that involves the cell in the body growing in an abnormal manner. According to WHO (2019), cancer is the second cause of death to the world population after cardiovascular diseases (CVD). Cancer usually comes about when signals during cell replication go wrong and the cells start to rapidly grow and divide uncontrollably (Cooper 2000). Based on a review done by Hanahan and Weinberg (2000), they have classified traits that a typical cancer cell would have. The six traits are namely; 1) they grow self-sufficiently, 2) they can bypass apoptosis, 3) they can perform angiogenesis, 4) they do not have any growth limit, 5) they can move to invade and attack other cells in the body and lastly, 6) they do not react to signals to stop growth.

Based on a cancer registry report of 2007 – 2011 by Azizah et al. (2016), in Malaysia, the top three cancers contributing to mortality in the region are breast cancer (17.7%), colon cancer (13.2%) and lung/trachea cancer (10.2%). Nasopharyngeal cancer (NPC) is not far behind being number 5 on the list with an incidence of 4.9% in Malaysian residents. Even though NPC is ranked the 5th most common cancer in Malaysia, this type of cancer is usually very common among Sarawakians (Devi et al. (2004). Comparing between different genders, males have a higher risk of getting NPC (8.1%) as compared to females. Not only that, the incidence risk of NPC in Malaysian males showed that Chinese are more prone to getting NPC (11.0), followed by other ethnics, which includes local ethnics in Sabah and Sarawak (9.9%), followed by the Malays (3.3). Indians have the lowest risk of getting NPC (1.1%). A recent report released by MOH (2018) on the survival of cancer patients stated that one of the main causes of death in hospitals is cancer. The report highlighted that survival rate is higher when the cancer was detected at an early stage

(e.g. Stage I). Thus, it is important for Malaysians to do health screening regularly so that detection and treatment can start early.

Malaysia is a country rich with flora and fauna and the use of traditional medicine have been practiced for a very long time by the locals. Furthermore, most of the pharmaceutical products nowadays are sourced from natural products, especially from plants. For example flavonoids, which are present in many plants like the Morinda citrifolia (local name, mengkudu or Indian mulberry) are known to be used traditionally in Malaysia to cure various illnesses (Abou Assi et al. 2015). Some more examples of alkaloids in oncotherapies are, paclitaxel derived from the barks of the Pacific yew tree, is a common drug (Taxol) used in chemotherapy (Kim & Park 2017) and also the commonly used chemo drug are vinblastine or vincristine, both derived from the plant Catharanthus roseus, locally in Malaysia as “Bunga Kemunnting Cina”. Both vinblastine and vincristine prevent mitosis of tumour cells leading to apoptosis, both are used to treat blood related diseases where the former is used to treat leukaemia in children whereas the latter is often used for the treatment Hodgkin’s lymphoma (Loh 2008).

1.2 Problem statement

Nasopharyngeal carcinoma (NPC), although not as highly known like lung, breast or blood cancers are one of the silent killers in Malaysia. They are not as highly publicised which often results in the general public overlooking their symptoms and only getting treatments at the last stages which significantly lower the patient’s survival rates.

The most commonly used drug to treat NPC is cisplatin, followed by other such as taxol, vinblastine or vincristine. However, all of these drugs also bring about side effects like hair loss, constant vomiting or extreme fatigue because they also react to the normal fast-growing normal cells.

With the change in lifestyle at the moment, the chances of people getting cancers is highly likely nowadays and that is why we must look for other new potential anticancer drugs. It is also beneficial if an anticancer drug be less harmful to normal cells, and consequently pose less side effects to healthy cells. Local plants are said to have many medicinal properties. A lot of research has been and being conducted to investigate these claims often discovered through oral information being passed from one generation to another or rom traditional medicines practitioners (e.g. traditional Chinese medicine). Due to the high numbers of plant species and

varieties in a tropical rainforest, as well as the type of specific medical conditions to be tested on, continuous efforts are needed to improve the knowledge based in this field.

Plants are highly known to contain natural antioxidants. Consumption of the right plant with these properties is suggested to aid in reducing the risk of ailments or complications related to free radical damages such as diabetes, high cholesterol, gouts and so on. Some research has also suggested that plants do bring about cytotoxicity in cancer cells, thus the general notion with plants and their anticancer activity. More research needs to be done to find new treatments especially using local plants in Malaysia as that will be highly beneficial to the country’s economy.

Sarawak, having one of the highly forested area in Malaysia, have an abundant amount of forest species containing plants that have been used for many years to treat various illnesses like diarrhoea, fever, rashes, minor cuts and bruises and many more. The uses of these plants have dated back since before the introduction of science by traditional practitioners, thus brings about the reason why research needs to continue to search, and validate the thousands of potential medicinal plants in the world especially those that are available locally, some, maybe even just from the backyard.

1.3 Research hypothesis

Research shows that many plants possess significantly high antioxidant properties, and in some, have anticancer activity. Some plants may not be edible, and the active compound would need to be extracted and isolated. In some, the active compound are located within the edible part of the plant, such as those in vegetables, fruits, and herbs. In this study, two edible young leaves of two types of local plants, namely Dillenia suffruticosa (Simpoh air) and Eugenia polyantha were analysed. These leaves were investigated for their polyphenolic contents, antioxidant activities, cytotoxic effect on normal (HaCaT cells) and cancer cells (NPC/HK1 cells); and their anti- migration effect on NPC/HK1 cells.

1.4 Research aims and objectives

The aim of the research is to determine the correlation between antioxidant values and cytotoxic activities of Dillenia suffruticosa and Eugenia polyantha (var. a and b) leaves water extracts on nasopharyngeal cancer cells.

Objectives:

(i) To determine the polyphenolic contents and in vitro antioxidant activities in the plant extracts using colorimetric-based assays; (ii) To assess the cytotoxicity effect of the plant extracts against normal cells using HaCaT keratinocyte cell line; (iii) To assess the cytotoxic and cell-migration activity of the plant extracts against nasopharyngeal cancer cells using NPC/HK1 cell line;

1.5 Thesis outline

This thesis comprised of the following chapters.

Chapter 1 provides a brief overview of the study. It presents the background of the work and the rationale behind the aim and objectives of the study.

Chapter 2 provides a summary of previous research related to the plants in the current study and on nasopharyngeal cancer.

Chapter 3 outlines the procedures involved in antioxidant analysis and cell culture assays.

Chapter 4 presents the results and discussion of this study which are the phenolic contents and antioxidant activities of the plant extracts, and their cytotoxicity effect on HaCaT (normal cells) and NPC/HK1 (cancer cells), and their effect of NPC/HK1 cell migration.

Chapter 5 concludes the thesis with a summary of the results obtained and presented, contribution of this work to the body of knowledge in the field of antioxidant therapy, followed by the recommendation for future work.

Chapter 6 list the references cited in this thesis.

1.6 Significance of the study

The study adds to the present knowledge on the potential of local plants, namely Dillenia suffruticosa (Griff.) Martelli and Eugenia polyantha (Wight) Walp against nasopharyngeal carcinoma (NPC), as examined on NPC/HKI cancer cells. The current treatment for NPC is by using a combination of radiation therapy and chemotherapy. However, the side effects of the therapies like nausea, vomiting, hair loss and immunosuppressed calls for the need to find alternatives to the current treatment. This study aims to explore potential candidates from local edible plant sources for complementary and/or treatment for NPC treatments.

1.7 Limitations of the study

There are a few limitations present in the project. The first limitation would be that all the antioxidant tests are based on in-vitro chemical analysis. They are not tested on any animals or human thus they are not biologically tested, so the bioavailability cannot be determined accurately. The second limitation involves cancer cell study, all assays are done in-vitro as well and further work can be done if the samples are to be applied in clinical trials.

2 LITERATURE REVIEW

2.1 Introduction

Eugenia polyantha Wight (Figure 1) is also known as the `Indonesian bay leaf’ or `daun salam’. In some publications, its synonym, Syzygium polyanthum (Wight) Walp is also used. Both refers to the same plant. For consistency, Eugenia polyantha or E. polyantha will be used throughout this thesis.

Figure 1: Botany illustration of E. polyantha

Image source: (Kooders & Valeton 1915)

The leaves of E. polyantha are a common culinary herb in local dishes. On top of the leaves, other parts of E. polyantha were also studied for their potential medicinal properties. Its barks and leaves were reported to have high antioxidant properties (Lelono, Tachibana & Itoh 2009). Kato

et al. (2013) reported that the isolated hydroxychavicol in E. polyantha leaves was shown to inhibit pancreatic lipase activity, which can be useful in the prevention or management of obesity (Kato et al. 2013). No research has been conducted on the effect of E. polyantha against nasopharyngeal cancer cells.

Dillenia suffruticosa (Griff.) Martelli or `simpoh air’ (Figure 2) is a common pioneer plant species seen along the Sarawak’s roadsides. Its wide leaves are commonly used to wrap foods while the young leaves can be consumed as vegetable or salad. In contrast to E. polyantha, D. suffruticosa has been widely studies for its anticancer potentials. The methanol extract of D. suffruticosa were reported to be cytotoxic against colon cancer (HT29), breast cancer (MCF-7 & MDA-MB-231), ovarian cancer (CaOV3), lung carcinoma (A549) and cervical cancer (HeLa) cells (Armania et al. 2013b). However, so far, no studies have been conducted on nasopharyngeal cancer cell lines.

Figure 2: Botany illustration of D. suffruticosa

Image source: (Griffith 1854)

Due to the lack of research for the effect of E. polyantha and D. suffruticosa against nasopharyngeal carcinoma, the current research focused on the determination of antioxidant potentials of the plants, and screening their cytotoxicity effect of the plants against NPC/HK1 cancer cell lines. Their anti-migration effect on the cancel cells was also studied.

2.2 Plants in the medicine world

With developing technologies and countless new studies on beneficial values of medicinal plants, researchers are leaning more on using plants as potential treatment of cancer. Plants contain many phytochemicals and secondary metabolites (e.g. arjunolic acid, anthocyanins and berberine) that contribute to their potential as supplements for treatment like having a cardio-protective effect in cardiotoxicity induced by in Doxorubicin in cancer treatments (Ojha et al. 2016). In terms of cancer, many plants have been looked into their potential to become an anti-cancer treatment especially plants that are already being used traditionally in the healing of ailments although unproven by science (Petrovska 2012).

2.2.1 Eugenia polyantha Wight or Syzygium polyanthum (Wight) Walp

Eugenia polyantha Wight or more commonly known as bay leaf is traditionally known to have an anti-inflammatory effect is also widely investigated for their potential to treat diabetes and obesity (Kato et al. 2013). They belong to the family of Myrtaceae, the flowers are small, white and fragrant while the leaves are often used to treat gastric ulcers, diarrhoea and as an astringent (Agus & Agustin 2008). In Sarawak, it is known as `Bungkang’ in local Iban dialect. The leaves are described as having an aromatic property and is a staple ingredient in traditional cooking as it brings out the flavour of the dish.

Some plants under the genus Eugenia has been replaced as Syzygium but are still under the same Myrtaceae family (Schmid 1972). Most Eugenia species are found at neotropics whereas Syzygium species are commonly found in Southeast Asian countries like Malaysia. Since the plant used for this experiment is acquired locally, thus it will be referred to as S. polyanthum. E. polyantha is an example of a plant that has been moved to the Syzygium genus but there are not a lot of research involving the latter and nasopharyngeal carcinoma.

Ismail et al. (2017) performed cytotoxicity assay on five different herbal plant commonly used in Asia, namely Eugenia polyantha, Cinnamon zeylanicum (Cinnamon bark), Andrographis paniculate (Hempedu Bumi), Curcuma xanthorrhiza (Temulawak), and Orthosiphon stamineus (Misai Kucing). Results of the research found that A. paniculate was the most cytotoxic to the hepatic cancer cell WRL-68 (IC50; 440 mg/L), followed by E. polyantha (IC50; 2213 mg/L), C. xanthorrhiza (IC50; 3406 mg/L), C. zeylanicum (IC50; 3691 mg/L) and finally O. stamineus (IC50; 4014 mg/L) being the least cytotoxic. Against normal adipose cell 3T3-L1, the E. polyantha ranks the best being the least cytotoxic at IC50 6690 mg/L and A. paniculate was the most cytotoxic at

IC50 649.8 mg/L. Based on the research conducted, E. polyantha would be the best sample as it

was cytotoxic to the cancer cell but the least cytotoxic to normal cells while A. paniculate, although it was the most cytotoxic to cancer cells but it was also cytotoxic to normal cells. Being toxic to normal cells would not be a preferred property because that means that there might be a chance that the sample may harm normal functioning cells as well as the cancer cells.

It was also reported that the ethanolic extract of E. polyantha was found to be able to inhibit activation of the Epstein-Barr virus in Raji cells (blood cancer cells) (Ali and Lajis (1999). It was found that the young leaves of E. polyantha were able to show significant reduction of growth in the cancer cells.

Wicaksono et al. (2013) compared E. polyantha and Piper crocatum leaves (betel leaves) against cervical cancer cell lines, HeLa CCL-2. Proliferation assay of the plants found that both plant extracts were able to prevent proliferation of the carcinoma cells. Combination of the plants, however, did not produce desirable results for preventing growth of the cancer cells. The same goes for immunocytochemical assay of the plant extracts against the nuclear factor protein complex, NFkB p65 where combination of E. polyantha and P. crocatum had resulted in an increase of the gene nuclear factor. Overexpression of NFkB p65 is not a desirable effect as it was found to promote malignancy of cancer cells (Shukla et al. 2004). Although both combination and single extracts of E. polyantha and P. crocatum was found to be able to increase expression of caspase 3 and HSP70 (Heat Shock Protein 70), both expressions prevent proliferation of cancer cells but, E. polyantha showed the best result when used alone.

E. uniflora also in the Myrtaceae family, is also used as traditional medicines to treat illnesses like fever, rheumatoid and diabetes in countries where they grow. Tannins from the leaves caused significant activity inhibition of EBV DNA polymerase activity where the IC50 values were 3.0

µM, 3.5 µM, 26.5 µM and 62.3 µM for eugeniflorin D1, eugeniflorin D2, gallocatechin and oenothein respectively (Lee et al. 2000). Compared to the IC50 value of known EBV DNA polymerase inhibitor; phosphonoacetic acid (16.4 µM), the values of eugeniflorin D1 and D2 were almost 5 times lower. This suggests that the tannins were more effective at inhibiting the synthesis which could be beneficial in the future medical field (Lee et al. 2000).

2.2.2 Dilennia suffruticosa (Griff.) Martelli

Dilennia suffruticosa belongs to Dilleniaceae family, a group of flowering plants that are traditionally used to treat many kinds of ailments. The different species and parts of the plant contributes to different healing properties. For example, the bark of D. pentagyna found in India is used for cuts and burns, pain relief and for the treatment of cancer whereas the leaf of D. indica

originating from Vietnam is used to treat intestinal diseases and malaria as well as malaria-like symptoms (Yazan & Armania 2014). Locally known as `simpoh air’ in Malay, D. suffruticosa locally found in Malaysia, identifies itself with having large oval leaves often used to wrap a local delicacy of fermented soybean (Yazan & Armania 2014). D. suffruticosa have been extensively researched on their antioxidant properties.

Studies by Armania et al. (2013a) showed that direct methanol extracts of the plant have been found to have high antioxidant activity especially from the roots as methanol is able to extract phenolic compounds of the plants. Antioxidant activities test showed that direct methanol and water extracts of D. suffruticosa roots had very high antioxidant capacity based on their DPPH (790.6 ± 1.5 & 735.9 ± 8.1 mg TEAC/g extract), ABTS (339.1 ± 5.8 & 294.6 ± 10.3 mg TEAC/g extract) and FRAP (475.8 ± 35.3 & 354.9 ± 9.3 mg TEAC/g extract); values for methanol and water extracts respectively. However, cytotoxic results showed that the exact extracts although having high antioxidant capacity values, resulted in high IC50 value (Table 1) compared to the other parts of the plant which were not favourable as it showed no sensitivity towards the cancer cells. Sequential extraction of dichloromethane (DCM) and ethyl acetate (EtOAc) D. suffruticosa extracts showed IC50 values of 19.7 ± 0.6 mg/L on HeLa cells (DCM extracts), 12.3 ± 0.6 mg/L on CaOV3 cells (EtOAc extracts) and 40.0 ± 1.7 mg/L on MCF-7 cells (DCM extracts) after 72h of incubation. However, result of the screening found that, fractionated DCM and EtOAc extracts of D. suffruticosa although, caused cytotoxicity towards HeLa, CaOV3 and MCF-7 cells, the extracts also showed cytotoxicity towards 3T3 F442A cells which were normal cells (Armania et al. 2013b).

Table 1: The effect of different extraction solvents of D. suffruticosa leaves on different cancer cell lines.

IC50 values on different cell lines (mg/L) Oestrogen Oestrogen Extracting Positive Cervical Ovarian Normal Mouse solvent Negative Breast Breast Cancer Cells Cancer Cells embryo cells Cancer cells Cancer Cells (HeLa) (CaOV3) (3T3F442A) (MDA-MB-231) (MCF-7) Water >150 >150 >150 >150 NA Hexane 45.3 ± 8.1 132.7 ± 9.0 70.0 ± 10.6 70.3 ± 8.6 66.7 ± 1.2 Methanol 132.7 ± 4.2 >150 128.0 ± 6.0 105.0 ± 1.0 >150 DCM 40.0 ± 1.7 39.0 ± 1.0 19.7 ± 0.6 44.7 ± 1.5 30.0 ± 0.0 EtOAc 50.0 ± 8.0 122.7 ± 3.1 67.3 ± 7.6 12.3 ± 0.58 80.7 ± 1.2

Data adapted from Armania et al. (2013a). Acronyms: DCM=dichloromethane; EtOAc= ethyl acetate.

As observed from Table 1, all extracts were able to inhibit proliferation of the cancer cells.

However, some of the extracts have IC50 values almost like normal cell values which may indicate that the extracts would kill cancer cells as well as the normal cells. EtOAc and methanol extracts would be a better option of further tests as the IC50 values of the normal cells were not so close to the values for the cancer cells.

Another plant belonging to the Dilleniaceae family is Dillenia indica which is common to Asia, mainly in Southeast Asia (USDA 2019). It is more commonly called as elephant apple because it is one of the main feeds for elephants. In India, the fruits, leaves and bark are used traditionally to treat diarrhoea and cancer (Sharma, Chhangte & Dolui 2001). D. indica is also a widely studied plant as they have antioxidant activities contributed by the high amount of phenolic contents, as determined using the Folin-Ciocalteu method (Abdille et al. 2005). The study reported that the methanol extract gave the highest phenolic contents (34.1%) compared to ethyl acetate (9.3%) and water (1.4%) extracts.

A study by Kumar et al. (2010) using fruits of D. indica showed that ethyl acetate fraction and methanolic extracts showed profound activity in inhibiting proliferation of leukemic cancer cell lines namely; K562, HL60 and U937. Methanolic extracts of the fruit had IC50 values of 275.4 ± 8.5 mg/L, 297.7 ± 7.3 mg/L and 328.8 ± 14.8 mg/L for K562, HL60 and U937 respectively. Then the extracts were then fractionated with ethyl acetate where the fractionated extracts showed lower

IC50 values of 241.9 ± 8.0 mg/L, 211.8 ± 5.3 mg/L and 240.0 ± 4.4 mg/L for K562, HL60 and U937 respectively.

Apart from having potential anti-leukemic cancer activity, D. indica was also found to have potential antidiabetic properties. A previous study tested ethyl acetate fractionated extract of D. indica on diabetic rats, found that daily administration of the extract reduced the blood glucose level of the rats (Kumar, Kumar & Prakash 2011).

Extracts of D. indica has also been found to have an antimicrobial effect. As previously studied by Jaiswal et al. (2014), they have found that aqueous acetone extracts of the plant were able to prevent growth of some of the common bacteria namely; Escherichia coli, Staphylococcus aureus, Bacillus cereus and Yersinia enterocolitica. Bark extracts of the D. indica had a lower and more effective concentration to produce antibacterial activity compared to extracts from the fruit.

2.2.3 Health benefits of other plants

Plants have long been used to traditionally treat minor ailments like fever, rashes or cuts and few are said to be able treat conditions like diabetes and cardiovascular problems. Treatment of cancer have been intensively researched by scientists to find cheaper alternatives for treatment or having less side effects induced by synthetic drugs like doxorubicin and cisplatin.

Portulaca oleracea, a commonly found weed was found able to induce an anti-proliferative effect on cervical carcinoma, colon carcinoma, breast carcinoma as well as nasopharyngeal carcinoma cells (Tan et al. 2013). A research conducted by Armania et al. (2013b) suggested that Dillenia suffruticosa extract was able to produce an anti-proliferative effect on breast cancer cells and antioxidants from Eugenia polyantha was also discovered to prevent cancer cell from growing, specifically colon cancer cells (Bennett et al. 2013).

In the research by Kao et al. (2015), they studied the effect of Pluchea indica root extract (PIRE), a plant used traditionally to treat ulcer, joint pain and inflammation then discovered that PIRE has the ability to inhibit cell viability and metastasis of nasopharyngeal carcinoma (NPC) cells. PIRE was screened for total phytochemicals content namely; flavonoids, tannin, phenols and alkaloids that have shown to be able to dampen the growth of cancer cells. NPC cell lines namely NPC- TW01 and NPC-TW04 were treated with the extract then were subjected to cell proliferation analysis, cell migration analysis and cell apoptosis analysis. Analysis of both cell lines showed positive results though NPC-TW04 showed more sensitivity towards the extracts as compared to NPC-TW01 cells.

Another research pertaining to plant extracts and NPC cells was carried out by Koh et al. (2015) where they used a medicinal plant Strobilantes crispa (S. crispa), commonly found in Malaysia. They tested on S. crispa leaves and stem extracts of their cytotoxic and apoptogenic effect on nasopharyngeal carcinoma (NPC) cell. Experiments performed to test the extract from different extraction methods were; cell viability assay, morphology test, doubling time determination, flow cytometry assay and caspase activity assay. The results showed that mostly non-polar extracts (i.e. hexane, chloroform and ethyl acetate) of S. crispa exert anti-cancer activity on cells where many active components like flavonoids, luteolin and catechin are found. On the other hand, methanol and water extracts as well as crude extracts showed no significant anti-cancer activity on the cells.

2.3 Plant secondary metabolites

Plant produces a variety of metabolites that can be divided into primary and secondary metabolites. The primary metabolites are those vital for the growth and development of the plants such as carbohydrates, proteins and amino acids. Secondary metabolites have no function in plant’s growth. However, they are reportedly found to play important role in reinforcement of tissue and tree body (e.g. cellulose, lignin, suberin), protection against insects, diseases, and plant regulation (plant hormones). They tend to be strain specific and produced under certain environments. The benefits of majority of these secondary metabolites to the organism itself is still unknown, and even less is known about their effects on other organisms, such as humans.

Common sub-groups of plant secondary metabolites are terpenoids, phenols and alkaloids.

2.3.1 Terpenoids

Terpenoids are made of isoprene units (Figure 3) and are found in all plants. Classification of terpenoids depends on the number carbon bonds. The common classes of terpenoids are; a) monoterpenes (C10), b) sesquiterpenes (C15), c) diterpenes (C20), d) sesterterpenes (C25), e) triterpenes (C30), tetraterpenes (C40) and polyterpenes (Withers & Keasling 2007). They are the largest group of secondary metabolites and are very volatile, which means they evaporate easily. Monoterpenoids, such as limonene gives out a citrusy fragrance (Huang et al. 2012) and often used as an essential oil. In some plants, they are used to deter pest and protect the plant against pathogens. Huang et al. (2012) reviewed many components of terpenoids have the potential of having anti-cancer properties. Terpenoids constitutes 60% of natural products (Firn 2010) and many medicinal plants are known to contain some forms of terpenoids such as ginkgolides in ginkgo plants or cinnamyl acetate in cinnamon barks. Taxol (Figure 4) is a widely used anticancer drug contains terpenoids often used in treating ovarian cancer but in small doses because it has low maximum tolerated dose (Kim & Park 2017).

Figure 3: Isoprene Figure 4: Molecular structure of paclitaxel (Taxol)

The diterpene, retinol is highly researched of their anticancer properties as it regulates many of the body biological processes like differentiation, proliferation and apoptosis (Bushue & Wan 2010). Retinol can be found in vitamin A and in food, it can be found as casbene in castor beans. A possible anticancer therapy method might be possible cancer cell can have an increase in RA turnover which might lead to growth inhibition of cancer cells. A study was done by Klaassen and Braakhuis (2002) where they researched on effect of increased in rate of turnover of all-trans- Retinoic Acid (RA) on Head and Neck Squamous Cell Carcinoma (HNSCC). Results of the findings showed that HNSCC cell lines had an average of 17-fold of RA turnover but only some cell lines showed growth inhibition following the turnover. The specific cell lines with good RA turnover with reduced HNSCC inhibition was UM-SCC-35 and VU-SCC-OE (1016.0 and 558.9 pmol/mg protein/h; 16.4 and 56.9% respectively. Klaassen had discussed that this result might be due to the high presence of CYP26A1 gene in the cell lines as normal cell lines (normal oral keratinocytes) had low levels of the genes following low levels of RA turnover.

The triterpenes, oleanic acid and ursolic acid are also potential anticancer agents. Both compounds are widely found in plants and due to having almost identical structures, they are usually found together (Jesus et al. 2015). Ursolic Acid (UA), an isomer of oleanic acid (OA) is found to have better anticancer potential. Li, Guo and Yang (2002) investigated the effect of both compounds on human colon cancer cells (HCT15) and found that cells incubated with UA resulted in a significant reduction of cell viability as compared to OA. At a dose of 20µmol/L, the IC50 value of UA was already much lower than OA and then at 80 µmol/L, the IC50 value of UA was already approximately less than 20% whereas OA was approximately less than 40%. Liu and Luo (2012) had also researched on betulinic acid (BA), a triterpenoid that was able to induce cell death in NPC/HK1 cells. BA was able to induce the activation of caspase activity specifically at cytochrome c that will then induce the release of a cascade of caspases which will result in apoptosis. Table 2 shows some of many classes of terpenoids.

Table 2: Classes of terpenoids

Terpenoids Classes Example Source

Monoterpenes Limonene Citrus fruits

Sesquiterpenes Zingeberene Ginger

Diterpenes Casbene Castor beans

Sesterterpenes Scalarester Dysidea sp. (Marine)

Triterpenes Cucurbitacin Cucumber leaves

Tetraterpenes Lycopene Naturally available in plants; gives red colour to plants

Polyterpenes Hevea bransilensis Rubber tree

2.3.2 Phenols

The phenols as seen in Figure 5, consist of a hydroxyl group (–OH) attached to an aromatic ring and they are found in nearly all parts of a plant. Phenols are generally divided according to the number of subunits attached; they can be polyphenols (like flavonoids, flavonols) or simple phenols (glucose, benzoic acid) (Robbins 2003). Other phenolic compounds also include, lignans, tannins, xanthones quinones and coumarins are also known to anticancer activities acting as free radical scavengers (Huang, Cai & Zhang 2009). Phenols are widely available in many forms like in fruits like apples and pears (Pandey & Rizvi 2009). Coffee and tea (especially green tea) are a rich source of phenolic acid (Spencer et al. 2008).

Figure 5: Phenol

Due to the huge presence of phenolic compounds in plants and vegetables, they have been studied extensively on their medicinal benefits like antioxidant and anticancer properties. Anantharaju et al. (2016) went through studies that involved phenolics and their role in natural medicine. However, they also found that phenolics can have adverse effects mainly because their ability to diffuse through cellular membranes when in free acid form which may cause systemic toxicity if overdosed. There have also been concerns on how an increase in the oestrogen level may bring

about breast cancer (Miller et al. 2001). As some phenolic compounds like isoflavones mimics oestrogen, the high consumption of food containing phenolics may induce tumour that are sensitive to oestrogen levels. The concern came from an increase in proliferation of MCF-7 due to genistein, an isoflavone meant for angiogenesis inhibition which is marketed as soy tablet supplement (Messina & Wood 2008). Clinical studies conducted on breast cancer patients as well as healthy patients however, showed no significant effect of soy tablet supplements and the development of breast cancer (Sartippour et al. 2004).

Phenols are large class of compound itself and many secondary metabolites fall under phenols. Because they are usually large compounds, they are usually called polyphenols. Example of polyphenols are flavonoids, anthocyanins and flavones.

2.3.2.1 Flavonoids

Flavonoids are the class of phenolics that are present mostly on the epidermis layer of leaves as well as on the skins o fruits. They are classified under polyphenols because of the presence of 3 aromatic rings attached to the structure (Figure 6). Flavonoids act as protection against ultraviolet light, pigmentations, resistance to disease as they act as the primary barrier to plants and vegetables (Carocho & CFR Ferreira 2013).

Figure 6: Flavonoid

Liu et al. (2011) studied the effect of flavonoids from a traditional cancer recipe called Xianhe Yanling Recipe used in China. They found that the total flavonoids from the recipe accounts to

IC50 of MCF-7 breast cancer cells (24.948), HepG-2 liver cancer cells (31.569) and ES-2 ovary cancer cells (6.923) µg/ mL. It is believed that traditional Chinese cancer medications have inhibitory effect or a mechanism that allows the immune system to be enhanced (Liu et al. 2011).

Quercetin, a well-known flavonoid is often marketed as a main ingredient in health supplements. However, it is also naturally incurring in many fruits and vegetables, as well as in nuts and honey (Cushnie & Lamb 2005). Quercetin have been studied mainly on its effect on breast cancer cells (MCF-7 cells) as it has shown promising results in the treatment of breast cancer. Choi et al. (2001)

reported of growth inhibition of MCF-7 cells from incubation with quercetin. Results from the research indicated that quercetin could prevent cell cycle progression as well as inducing apoptosis in MCF-7 cells. Quercetin was found to cause reduced cyclin B1-associated Cdc2 kinase activity after 48h and 72h of incubation. Cyclin B1-associated Cdc2 kinase reduction thus cause an arrest at the G2 phase of the cell cycle and therefore inhibits MCF-7 cell growth (Choi et al. 2001). Apoptosis was also observed through induction of p21 CIPI/WAFI which is activated by p53 gene, a tumour-suppressing gene although presence of p53 was not observable in the cells (Winters et al. 2003).

2.3.2.2 Anthocyanins

Anthocyanins (Figure 7) are from the subclass of flavonoids that has long been associated with many medicinal properties. A person can consume many anthocyanins if they take flavonoid supplements such as grape seed extract or ginkgo pills but daily consumption of anthocyanins for a person is expected to be 500 mg to 1g per day. This group is often found in teas, honey, wines, olive oil cocoa and cereals (Lila 2004). Anthocyanins are associated with protecting plants from light as well attracting pollinators with bright colours. They also have potential anti-inflammatory properties, protect DNA from damage and help with immune response (Carocho & CFR Ferreira 2013). A study by Wang and Stoner (2008) found that anthocyanin does play a role in anticancer treatment as they affect the expression of genes that leads to apoptosis but better absorptivity was needed.

Figure 7: Anthocyanin

Due to the high level of antioxidant activity of anthocyanins (Pantelidis et al. 2007), naturally the polyphenol is being researched for other medicinal properties. Faria et al. (2010) pointed out the possible use of anthocyanins from fresh blueberries (Vaccinium myrtillus) on MDA-MB-231 and MCF-7 breast carcinoma cells. The blueberries extract namely anthocyanin extract and pyruvic acid adduct extract showed anti-proliferative activities on the cancer cells through decreasing

protein synthesis which is linked to cell proliferation. Both extracts inhibited cell proliferation, but the effect was more pronounced in MDA-MB-231 cells as 65% and 70% of proliferation was inhibited by anthocyanin and pyruvic-acid respectively.

Anthocyanins from black rice have been found to be able induce apoptosis and suppress formation of new blood vessels as described by Hui et al. (2010). The black rice was extracted using 60% ethanol and 0.1 HCl and the extract was administered to immunodeficient mice implanted MDA- MB-453, MDA-MB-231 and MCF-7 breast cancer cell lines. Analysis showed the activity of caspase-3 being blocked leading to apoptosis in MDA-MB-453 cells. In vivo test also showed significant reduction of cell proliferation and reduction in tumour weights after treatment with black rice extract.

2.3.2.3 Flavones

Flavones are a subclass of flavonoids as well and structurally they are related to flavonols. A flavone compound (Figure 8) can be obtained by moving the benzene ring to C2 of a flavonoid (Figure 10). The action of flavones in a plant are more towards the protective and communication sides. Their roles in are mainly to protect the plants from harsh ultraviolet light and they interact with microbes, insects and other plants. Flavones are responsible for the antioxidant, anti- carcinogenic, antiviral, anti-inflammatory and anti-atherosclerotic properties of a plant (Carocho & CFR Ferreira 2013).

Figure 8: Flavones

A research by Li-Weber (2009) found that flavones was responsible for the cytotoxicity effect seen in tumour cells by inhibiting a few of the genes responsible for the cell cycle regulation, suppression of COX-2 gene that brings about pain and inflammation and to prevent infection of viral agent.

Luteolin is a flavone often researched for its anticancer activity. A previous study by Ong et al. (2010) on human nasopharyngeal carcinoma cells found that the compound had been able to

induce cell cycle arrest at the G1 pathway. The main pathway affected by luteolin is the regulation of Cyclin D1 which activates a cascade of events that leads to formation of cells (Alao 2007). Down-regulation of cyclin D1 in the NPC/HK1 cells inhibited further growth of the cells from the G1 phase. Although apoptosis was observed in other studies concerning luteolin anticancer properties (Cheng et al. 2005; Chiu & Lin 2008), the presence of caspase 3 was not observed between luteolin and NPC/HK1 cells (Ong et al. 2010).

Changes in structure either have a huge or a minor impact on the action of a compound. Chen et al. (2008) tested this by synthesising flavones with amide derivatives to observe its anti- proliferative effect. The synthesised extracts were tested on human lung cancer cell (HCI-H661), nasopharyngeal cancer cell (NPC-TW01) and T-cell leukaemia cell (MT-2). Results obtained from the research showed potential synthesised compound being able to bring on cell cycle arrest at the G0/G1 phase and further induce apoptosis especially in NPC-TW01 cells.

2.3.2.4 Flavonols

Flavonols are among the most easily available flavonoids found in fruits and vegetables. The most notable flavonol would be quercetin known for having high antioxidant activity as well as having the potential to prevent lipid peroxidation that may result in cardiovascular issues (Bentz 2017). Besides being an antioxidant, flavonol can be potentially marketed as a relaxant as well as a cardiovascular protectant (Woodman & Chan 2004). With all the benefits that it has, flavonols are also being studied on their anticancer potential. A study by Biscaro et al. (2013) showed that flavonol brings about apoptosis of human leukaemia cells (K562) by inhibiting a protein called HSP70 which is required for the tumour cell to survive.

The flavonol, kaempferol has been widely researched for its anticancer properties. Kaempferol was found to be able to induce cell cycle arrest in the G2/M phase in the human breast cancer cell MDA-MB-453. The main method of action of kaempferol was linked to the down-regulation cyclin-dependant kinases (CDKs) and CDK inhibitors. Cell cycle continuation depends on the complexation of CDKs and cyclin thus down-regulation of CDKs brings about inhibition of cancer cell proliferation. The gene p53 was also up-regulated causing MDA-MB-453 cells to undergo apoptosis (Choi & Ahn 2008).

2.3.3 Alkaloids

They are primarily composed of nitrogen and are widely used in medicine. The nitrogen attached to the heterocyclic ring contributes to the polar nature and water-soluble property of alkaloids

(Firn 2010). Alkaloids are present in almost all our daily consumption like in coffee and tea (Ashihara 2006) and morphine, also an alkaloid can provide an analgesic effect. However, if they are taken over the recommended dosage, they will instead be highly toxic and may cause fatality.

Figure 9: Pyridine - the most basic alkaloid structure

Figure 10: Berberine

Alkaloids are highly abundant in plants thus its meditative effects are highly investigated by researchers for potential use in treatment of illnesses. Alkaloids are classified according to their chemical structure, i.e. whether they are simple alkaloids like the basic pyridine (Figure 9) (Nitrogen (N) in a benzene ring), indoles (N is at the first position with a benzene ring attached) or tropane (N attached to a bicyclic compound).

Berberine (Figure 10), an isoquinoline alkaloid has been studied of its effect on many illnesses and cancer treatment is one of the most prominent. A recent research by Wang et al. (2017) with nasopharyngeal cells (NPC/HK1-EBV cells) showed that berberine could significantly reduce the viability of NPC/HK1-EBV cells by almost approximately 60%. IC50 value of berberine and NPC/HK1-EBV cells had also showed a reduction of 124.5 µM to 43.1 µM after 24h and 48h incubation respectively. Not only that, the study determined that berberine was also able to induce cell cycle arrest and apoptosis in the cells. Cells were found to have accumulated in G1 phase of the cell cycle and this might have been attributed the mechanism of berberine decreasing the viral gene EBNA1 expression leading to stimulation of the p53 gene which brings about the cascade of events that leads to apoptosis (Wang et al. 2017).

Another example of an alkaloid in the cancer treatment research is evodiamine, a quinolone alkaloid. Evodiamine was found to be able to cause significant anticancer effect in NCI/ADR- RES human breast cancer cell, a cell line that is resistant to many drugs. The research found that cell viability was reduced by evodiamine as compared to when it was treated with cancer drugs like paclitaxel and vincristine. Treatment with evodiamine had also caused cell cycle arrest at the G2/M phase of the NCI/ADR-RES cells (Liao et al. 2005). Based on all the study conducted on alkaloids, it shows that alkaloids have high potential in the treatment of cancer in the future.

2.4 Polyphenol assay

Polyphenols are the compounds present in edible plant food that provides such food with various properties like antioxidant capacity, anti-inflammatory or anticancer effect. Polyphenols can be found abundantly in various fruits and plants and most of the polyphenols are present as secondary metabolites. Research have found that beverages like tea, coffee and wine have high polyphenol content as well as various vegetables and cereals (Manach et al. 2004).

Polyphenol assays are usually done as the first part of an experiment to screen the plant for potential antioxidant activity. Then it will be proceeded with other tests like, anticancer activity, anti-diabetic, anti-inflammatory and so on. The most common assays to screen polyphenols are the total phenolic assay and total flavonoid assay because they are easy to repeat, quick and low cost.

2.4.1 Total Phenolic Content assay (TPC)

Another name for TPC is the Folin-Ciocalteu method because it involves the use of the Folin- Ciocalteu (FC) reagent. The methods are simple where different concentrations of the sample are prepared, and then the FC reagent is added. The initial colour will be blue which will change to colourless with increasing antioxidant activity.

Initially the FC method was developed by Folin and Ciocalteu (1927) to do protein analysis but it was later modified for total phenols assay in wine by Singleton, Orthofer and Lamuela-Raventós (1999). The mechanism of the assay involves the transfer of electrons from compounds in a sample to a generated radical. By measuring the absorbance of the sample at 760 nm, the total phenolic content can be calculated by comparing it to the standards Trolox and gallic acid (Huang, Ou & Prior 2005). The assay is a basic screening of antioxidant activity for samples thus often used the primary assay.

2.4.2 Total Flavonoid Content assay (TFC)

Another assay often used to screen for antioxidant activity is to determine the flavonoid content. Flavonoid is a class of compounds under polyphenols and it is responsible for various properties of a plant like the pigments of the flowers and fruits, protection against stress and also against ultraviolet rays (Winkel-Shirley 2002)

The modified assay (Adasuriya 2018) involves allowing the samples to react with 2% aluminium chloride. Flavonoids present in the sample will form a complex with aluminium chloride and the sample will turn yellow in colour if flavonoids are present. The sample will be measured at a wavelength of 415 nm and the total flavonoid content will be calculated against the quercetin standard.

Both TPC and TFC assays were used as a primary screening of antioxidant activity as they were quick and easy to do in a laboratory and the cost of the experiment is not high. After determining the phenolics and flavonoids content, further antioxidant assays will be tested on the samples.

2.5 Antioxidant assay

Antioxidants are essentially a property exhibited by a compound that can prevent oxidation. Oxidation is a common process occurring in almost everything but presence of highly unstable oxidative species or in other words, too much free radicals in the body may cause harm as they can attack any cells in the body to stabilise themselves. Thus, it is why there are many studies regarding preventing attacks of radical species in the body.

As shown in Figure 11, antioxidants function by scavenging the free radicals in the body and allow themselves to bind the radicals so that they would be stabilised. The use of antioxidant is common in the food, pharmaceutical and cosmetics industry as they are also used to prevent the materials from degrading (Pisoschi & Negulescu 2011).

Figure 11: Basic antioxidant activity

Antioxidant assays have been developed to allow researchers to detect antioxidant properties in a sample. Some of the most commonly used assays are the DPPH (2,2-diphenyl-1-1-picrylhydrazyl assay), ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) assay), FRAP (Ferric reducing antioxidant power assay) and CUPRAC assay (cupric reducing antioxidant capacity assay) which are colorimetric assays and assay like ORAC is a non-colorimetric assay.

The downside of antioxidant assays is that it only determines the antioxidant capacity based on chemical reactions. Therefore, it does not reflect the reactions biologically as bioavailability needs to be taken into consideration. Thus, consumption of supplements with claims of having antioxidants needs to be at a moderate level as consuming too much may be toxic as well (Huang, Ou & Prior 2005).

2.5.1 DPPH assay (2,2-diphenyl-1-1-picrylhydrazyl)

This assay involves the use of a stable DPPH radical to bind to a hydrogen donor as seen in Figure 12. The radical would be prepared as a purple colour solution where it would lose the colour once bonded and the absorbance can be measured at 517nm (Pisoschi & Negulescu 2011). The concentration can be determined as the half maximal effective concentration or EC50 (Molyneux

2004). The EC50 value will determine whether the sample can act as antioxidant and the value will be compared to the antioxidant standards Trolox and Gallic Acid.

Figure 12: (a) The 2,2-diphenyl-1-1-picrylhydrazyl radical and (b) The stable form 2,2- diphenyl-1-1picrylhydrazine. Image source: (Molyneux (2004); Alam, Bristi and Rafiquzzaman (2013)).

DPPH is often the first line assay used in antioxidant tests, especially in plants as it is simple and quick to do (Nagarajan et al. 2017). However, the assay does come with a few limitations which are making sure that the DPPH radical solution is prepared using an appropriate solvent or how much metal or hydrogen ions are present (Dawidowicz, Wianowska & Olszowy 2012) and the most important one is where the wavelength may get overlapped with other wavelengths absorbing the same range. This may occur when the plant sample itself has pigmentation like being red in colour, where anthocyanin would be present. Anthocyanins has a wavelength of 500- 550 nm which is very near to DPPH and this may cause irregularities in the result (Musa et al. 2013).

2.5.2 ABTS assay (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid))

Beside DPPH, another commonly used method is the ABTS assay method. Like DPPH, it involves dissolving the powder to generate a free radical in this case, the ABTS•+ radical (Figure 13(a)). The free radical would then be mixed with the sample and then the radical scavenging activity of the sample will be calculated (Opitz et al. 2014). The starting colour of the radical solution will be greenish blue in colour then as it interacts with the sample, the colour will fade depending on how much antioxidant activity is present. The absorbance of the sample can be read at 414 nm, 645 nm, 734 nm or 815 nm but generally most research would use 734 nm to read absorbance (Prior, Wu & Schaich 2005) (Miller et al. 1993).

Figure 13: (a) Structure of ABTS•+ radical cation (greenish blue in colour) and (b) Structure of ABTS(H) (colourless). Image adapted from: (Gupta (2015)

ABTS assay results are usually depicted as Trolox equivalent antioxidant capacity assay (TEAC) where the radical scavenging activity (RSA) of the plant sample will be compared to that of the standard, which is Trolox (Opitz et al. 2014). Additionally, the antioxidant capacity can also be depicted as EC50 like DPPH.

In this project, the both DPPH assay and ABTS assays will be used to determine the antioxidant activity of the plant samples. Both assays are quick and reliable to screen the antioxidant activity before doing further studies on the plants.

2.5.3 CUPRAC

Abbreviated from cupric reducing antioxidant capacity, it is an assay designed by Apak et al. (2004) to determine antioxidant activity especially in plant extracts. As shown on Figure 14, the assay involves the reduction of the oxidising agent (Cu (II)-NC; bis(neocuprine) copper (II)) and measuring the absorbance at 450nm. The colour of the solution will be orange and the colour intensity will vary according how much antioxidant present (Özyürek et al. 2011).

Figure 14: (a) Structure of Cu(II)-Nic (blue in colour)and (b) Structure of Cu(I)-Nic after reduction (orange in colour). Image adapted from: (Apak et al. (2014)

2.5.4 FRAP assay

Ferric reducing antioxidant power (FRAP) assay like the CUPRAC assay, is used to detect traces of metal ions in a sample (Cerretani & Bendini 2010). In this assay, the reducing power of the antioxidants in the sample is calculated when it reduces the ferric complex to ferrous complex due to electron transfer as observed in Figure 15. The assay is also a colorimetry assay as colour change will observed from yellow to blue in presence of antioxidants and absorbance will measured at 700 nm (Vijayalakshmi & Ruckmani 2016).

Figure 15: (a) Structure of the Fe3+(ferric complex) and (b) Structure of the Fe2+ (ferrous complex). Image adapted from: (Prior, Wu and Schaich (2005)

2.5.5 ORAC assay

The oxygen-radical absorbing capacity assay or the ORAC assay modified by Cao, Alessio and Cutler (1993), where they also generate radicals but measurement is based on complete oxidisation of the sample. Measurement of antioxidants is described as ORAC units (I unit is equals to net protection from 1 µM of Trolox). The method has since been modified to measure the amount of fluorescence given out by the sample from the fluorescein compound (Figure 16) added. The higher the fluorescence of the sample, the higher the amount of antioxidant is present in the sample. Antioxidant capacity is measured by comparing the sample with the standard, Trolox as unit of Trolox per 100g (unit and gram values may be different with different researchers) (Dasgupta & Klein 2014).

Figure 16: Structure of fluorescein

2.6 Nasopharyngeal cancer

2.6.1 Background

Nasopharyngeal carcinoma (NPC) is cancer arising from the epithelial cell of the nasopharynx region, the tumour commonly found at the pharyngeal recess or also known as the fossa of Rosenmüller. It is labelled as endemic because most cases are usually found in Asia (east and southeast) that accounts for about 70% of the cases and the remaining 30% are from north and east Africa (Chua et al. 2016). NPC has been reported to be more prominent in males than females with a ratio of 2:1 respectively (Ferlay et al. 2015). Three types of NPC: (1) Squamous cell carcinoma, (2) Non-keratinizing carcinoma and (3) Undifferentiated carcinoma (Chua et al. 2016). The cause of NPC is highly linked to the Epstein-Barr virus (EBV) infection. EBV is from the herpesvirus group, a DNA group that is usually the cause of animals as well as human diseases like, herpes, smallpox and Hodgkin’s disease (Weiss et al. 1987).

The symptoms of nasopharyngeal carcinoma (NPC) are often mistaken for a normal ear, nose or throat infection as they are quite similar like, ear infection, nosebleed or hearing loss. The most obvious symptom would probably be detecting a painless lump at the neck area due to metastasis of the cancer to the lymph nodes (Society 2019). The proper way to diagnose NPC is through examining the possible areas where the tumour can be found, i.e. around the head and neck especially if there is a familial history of NPC and taking a biopsy to determine if the tissue is cancerous.

Researchers are finding ways to detect NPC earlier by using biomarkers in the body where the cancer would be more prominent. One of it was by Chan et al. (2013) where they study a possible way to detect NPC using levels of Epstein-Barr Virus EBV-DNA in the body. Increased level of

EBV-DNA was shown in patients positive for NPC compared to NPC-negative patients. Studies have also been conducted on small non-coding RNA molecules that are involved in gene expression and translation of microRNAs (miRNAs) and their presence in cancer patients where miRNAs regulation are disrupted (Chen et al. 2009). Immunoglobulin A (IgA) is also a possible biomarker to diagnose NPC and is a cheaper and a non-invasive method however compared to EBV-DNA, it is less sensitive and less accurate (Song & Yang 2013).

2.6.2 Types of cancer therapies

The commonly used approaches to kill cancer cells are usually mediated through the induction of apoptosis (programmed cell death) in target cells.

2.6.2.1 Cytotoxic drugs

Cytotoxic drugs are also known as cytostatics drugs. These drugs are usually aimed to selectively kill cancer cells as they move through the body in the bloodstream. They are often consumed in combination with other cancer therapy to get maximum effect. Cytostatics act by killing proliferating cells which means although there is a possibility that it may affect normal cells, the drugs are very sensitive to malignant cells because they proliferate at a very high rate, so the normal cells are usually ignored (Hanahan & Weinberg 2011).

In the treatment of nasopharyngeal carcinoma, the usual combination of cytostatics is cisplatin/5- fluorouracil. The combination is often given to a patient together with therapy which is aimed to increase the patient’s survival rate (Kua et al. 2013).

2.6.2.2 Gamma-irradiation

G-irradiation is a type cancer treatment using gamma rays. The therapy is carried out by beaming the rays at the growth area in several treatments to kill the cancer cells. It is one of the most commonly used therapy for cancer patient to increase survivability (Baskar et al. 2014).

Radiation therapy is also often used in combination with drugs for treating NPC patients. The current preferred radiation treatment is intensity-modulated radiation therapy (IMRT) as it allows medical staff to modify the intensity according to the size or treatment area (Kaliberov & Buchsbaum 2012).

2.6.2.3 Suicide gene therapy

Suicide gene therapy involves using viral or non-viral vectors (e.g. derived from herpes simplex or retrovirus) to deliver suicide genes into the tumour site. The suicide gene is programmed to release enzymes such that when a non-cytostatic prodrug is given to the patient, the enzymes produced by the suicide gene will convert the prodrug into a cytostatic drug that can now induce apoptosis of the tumour cells (Zarogoulidis et al. 2013).

Since nasopharyngeal carcinoma revolves around the presence on EBV, suicide gene therapy has also been taken into consideration (Tao & Chan 2007). It involved using EBV C promoter gene to induce the release of thymidine kinase enzymes that can be used to convert non-toxic prodrug to cytotoxic drugs. Suicide gene therapy is a treatment option for patients but there is also a risk of getting the bystander effect which means not only the cancer cells are killed but also the cells surrounding it.

2.6.2.4 Immunotherapy

Immunotherapy is a cancer therapy by stimulation of the body immune system. An example of immunotherapy is using monoclonal antibodies to bind to the cancer cells and from there the immune system will be triggered to the bound cell and induce apoptosis (Chari 2008). An example of monoclonal antibody therapy is Herceptin (trastuzumab) for treating breast cancer (Vogel et al. 2002).

The possibility of treating NPC with immunotherapy have also been highly researched. A possible immunotherapy option is using cancer vaccines like what is being conducted with cervical cancer using human papilloma virus. Thus, it might be useful to use EBV vaccines as a method of therapy (Jain, Chia & Toh 2016).

Activation of caspase is often associated with apoptosis of cancer cells and most therapies are known to work by inducing apoptotic proteins like caspase, p53, BcL-2 and more. Amongst this, caspase activation in the most researched as any unregulated caspase activity would lead to disorder and diseases as well as development of cancer cells in the human body (McIlwain, Berger & Mak 2013).

2.6.3 Anticancer agent and cytotoxicity

Cytotoxicity literally means toxic to cells. It is often used to describe any product that is capable of being toxic to a cell which would cause the cell to die. Cytotoxicity is usually associated with

necrosis or apoptosis. Necrosis happens when cells are exposed to external trauma or toxins outside the cells which cause them to die in a manner where they swell up and rupture, then cell component to be released into the surrounding area (Proskuryakov, Konoplyannikov & Gabai 2003). Apoptosis is often described as programmed cell death or cell death by suicide (Majno & Joris 1995). Different from necrosis, it often preferred because the cells will not rupture and release its content to the surroundings but instead, when a signal is received by the cell to die, it proceeds to shrink and release a signal to phagocytes to consume the dead cells (Majno & Joris 1995).

When doing research compared to necrosis, apoptosis is a preferred way of death when dealing with cytotoxicity of cancer cells as there no inflammation and considered a more programmable function of the body (Fink & Cookson 2005). Since the cells would just shrink and die, it is also easier to view and determine cell death under microscope.

Apoptosis was observed on MCF-7 cells treated with ethyl acetate extracts of D. suffruticosa using the phospholipid phosphatidylserine (PS) as a target binding site for Annexin V-FITC. PS will be released into the outside layer of the plasma membrane for apoptotic cell detection and Annexin V-FITC which has a high binding affinity to PS will bind instead. Propidium iodide is fluorescent and it will bind to damaged cells to differentiate between apoptosis or necrosis of the cells ((Lecoeur 2002); (Tor et al. 2014) & (Armania et al. 2013b)) found that D. suffruticosa extracts caused cells to exhibit apoptotic pathway based on flow cytometry assays.

2.6.4 Apoptosis related proteins

Caspase cascade activity is one of the mechanisms that causes apoptosis in the body. Activation of caspase can be divided into two pathways; extrinsic and intrinsic pathway. The former pathway is caused by signal induction from the death receptor mediator at the plasma membrane which in turns activates the Death Inducing Signalling Complex (DISC). DISC involves the induction of the death effector domain (DED) that binds with Fas-associated death domain (FADD) adaptor proteins and brings about the activation of initiator caspase 8 protein (Fulda & Debatin 2013).

The latter pathway, which is the intrinsic pathway, starts in the membrane mitochondria instead of at the plasma membrane. In an event of cells stress or damage, the mitochondria induce the release of cytochrome c enzyme which leads to activation of apoptosome complex. The apoptosome complex consists of pro-caspase 9 and adaptor APAF-1 and once induced will cause activation of initiator caspase 9. Initiator caspases 8 and 9 once activated through each other

signalling mechanism will in turn activate executioner caspases 3, 6 and 7 that causes apoptosis of cells (Fulda & Debatin 2013).

2.7 Cell culture for evaluation of anticancer properties

2.7.1 Types of cells for NPC studies

Table 4 shows the most common cell lines used for research on nasopharyngeal cancer. Normal cells are used to compare effectiveness of sample in targeting cancer cells.

Table 3: Different types of cell line used for NPC studies

Cell Line Cell property

CNE-1 NPC epithelial cell that has overexpressed EGFR similar in cancer patients

NPC-TW 01 Epstein Barr Virus (EBV)-negative NPC cell NPC cells NPC-TW 04 EBV-negative NPC cell

NPC/HK1 Human EBV-negative NPC cell

C-666-1 Human EBV-positive NPC cell

HaCaT Normal keratin epithelial cells

Control / NRK-52E Normal rat kidney epithelial cell Normal cells NP-69 Normal nasopharyngeal epithelial cell

3T3 Normal adipocyte cells

2.7.2 Proliferation assays

Cells in the body are always multiplying at a controlled rate. Therefore, when it starts to get out of control like in the case of cancer cells, it would proliferate and multiply at a very high rate. Proliferation assays or viability assays are used to determine the growth of cells, especially cancer cells during a time period. Determining cell viability is important in a cell culture study because it would serve as a base or as a screening method to determine whether samples used for the studies work in controlling cell proliferation. Then the study can be continued to determine the exact method of action of the samples. The assays can be colorimetric like MTT (2.4.2.1) and

MTS (2.4.2.2) assays or the assay involves replacing components of cells with a detectable analogue like the BrdU assay (2.4.2.3). Being able to perform assays on cells are very important as it will help researchers to determine many cell-level activities like cell signalling and receptor binding interactions (Riss et al. 2016).

2.7.2.1 MTT

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, is one of the most common techniques used to determine cell viability. The assay works by correlating activity in the cell mitochondria to viable cell. Metabolism activity in the viable cells would reduce MTT into purple coloured formazan crystals as shown in Figure 17. In MTT assays, the crystals are the dissolved using isopropanol then the absorbance will be read at 540nm and 720nm to obtain a dose-response curve (van Meerloo, Kaspers & Cloos 2011). MTT assay kits are available commercially where the MTS reagent powder will be dissolved using a solubilization reagent to get the working MTT solution prior to addition to cells (Riss et al. 2016).

Figure 17: Reduction of MTT to formazan. Image source: Riss et al. (2016)

2.7.2.2 MTS

MTS (5-[3-(carboxymethoxy) phenyl]-3-(4,5-dimethyl-2-thiazolyl)-2-(4-sulfo-phenyl)-2H- tetrazolium inner salt. The method of action is the same with MTT but the preparation procedures are simpler as it is not required to solubilize the working solution. Compared to MTT, the formazan dye formed are water soluble thus preventing the need to solubilise the crystals prior to

reading the plate (Berridge, Herst & Tan 2005). The assay uses cell soluble electron receptor reagents like phenazine methyl sulfate (PMS) or phenazine ethyl sulfate (PES) which reduces the MTS to soluble formazan products as shown in Figure 18 (Riss et al. 2016). MTS assay is preferred as it easier and faster to do and at the same time will present less room for errors. MTS assay kits are also available commercially in the markets where the working MTS solution has already been prepared.

Figure 18: Reduction by phenazine ethyl sulfate (PES) converting MTS to water soluble formazan. Image source: Riss et al. (2016)

2.7.2.3 Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU, BUdR or BrdUrd)

Bromodeoxyuridine (5-bromo-2’-deoxyuridine, BrdU, BUdR or BrdUrd) is a synthetic nucleoside, and an analog of the DNA base thymidine. When administered to cells, BrdU can replace thymidine during DNA synthesis, making it an excellent marker for cell proliferation and

cellular apoptosis. BrdU incorporation into the newly synthesized DNA of dividing cells can be detected using anti-BrdU antibodies (Crane & Bhattacharya 2013).

2.7.3 Scratch assay

The scratch assay is also known as the wound healing assay where a scratch or a “wound” will be done on fully grown cells and the time required for the cells to move and close the scratch will be calculated. The media for the assay should also be free of any serum that would facilitate the growth of cells. The aim of the assay is to determine whether the samples tested can inhibit the cell from moving to close the wound without additional serum present. This assay should be confused with the migration assay as where the cell migration between two chambers are observed. The compound mitomycin C is used to prevent cell mitosis in the cell migration assay so the result would not be confused with proliferation and the compound is optional in the wound healing test. A brief comparison of the two assays can be observed in Table 3.

Table 3: Methods for the wound healing assay and the cell migration assay. Wound Healing Assay Cell Migration Assay • Done only well surface • Involve trans-well migration • May or may not use mitomycin C • Need to use mitomycin C to prevent proliferation • Cells need to be checked timely • Cells do not need to be checked timely

Data adapted from Jonkman et al. (2014) and Kramer et al. (2013).

2.7.4 Flow cytometry and cell cycle analysis

Flow cytometry and cell cycle analysis are techniques often used to observe and determine cell properties. In flow cytometry, the cell which is usually suspended in a liquid medium then pass through a light source. Detectors will detect the refracted light which will then correlates to the properties of a cell (Picot et al. 2012). Cell cycle analysis would then use flow cytometry to analyse the DNA content of the cell which allows researchers to distinguish the cells at different phases of the cycle. In cancer study, an accumulation of cancer cells at the sub-G1 phase is preferred because it shows that the cells are not proceeding to the synthesis and mitosis phase thus hindering growth and proliferation of the cells ('Chapter 41 - G1 Phase and Regulation of Cell Proliferation' 2017).

2.8 Conclusion

As the world continues to progress, there are always new challenges for scientists. Everything might be okay the day before and overnight, a new type of disease or a new superbug is trying to kill mankind. Cancer is one of the diseases affecting a good number of people all around the world and yet, there is still no way to completely prevent it from occurring. Sometimes the first stage of cancer is often asymptomatic thus it is often not detected early leading to preventable deaths. Thus, it is important for people to always take care of their general well-being and make sure to have a healthy and balanced lifestyle.

Antioxidants are well researched by scientists because they are well-known to have many beneficial components that can aid in treating illnesses. Plants possess many different types of components with polyphenols like phenols and flavonoid being abundant in plants which is why total quantitative assays like the total phenolic content (TPC) and the total flavonoid test (TFC) are often used in plant studies. As for antioxidant tests, two of the most commonly used assays are ABTS and DPPH which are used to test for antioxidants in plants as they are simple, cost- effective and quick to do. Free radicals are always considered to be one of the few reasons for cancer development, thus antioxidants are being studied to determine whether they can have any effects on cancer cells.

Sarawak is very rich with edible plants and there are so many possibilities that can be discovered. That is why this study uses plants that can be found easily locally so that it can open these plants to new discoveries in the future.

3 MATERIALS AND METHODS

The aim of this study was achieved by conducting a series of experiments assessing the phenolic content and antioxidant activities of the plant extract; as well as assessing the extracts’ cytotoxicity effect against cancer cell line, NPC/HK1. The experiments can be summarised as shown in Figure 19 below.

Extract preparation

Phenolic content Antioxidant activity Anticancer screening

Total phenolic content DPPH Assay Cytotoxicity assay (TPC)

ABTS Assay Total flavonoid Cell migration assay Content (TFC)

Figure 19: Methodology flow chart.

3.1 Sample preparation

3.1.1 Plant sample source

Plants samples (Figure 20) used in this study were obtained from the previous study (Adasuriya 2018). The plants were donated by the Agriculture Research Centre (ARC) Semongok, Sarawak. Identification of the plants was confirmed by a research officer in the ARC, Dr. Maclin Dayod. The plant samples were carefully selected so that only the edible part; i.e. the shoot or young leaves were used. For E. polyantha, the edible leaves were collected from two sections i.e. the middle leaves (referred as var. a) and the young shoots (var. b). Both sections are commonly used in cooking. The middle leaves are added mainly for flavouring purposes, while the soft young shoot can be consumed. For D. suffruticosa, only the young shoots section was used, as the middle leaves were too fibrous, and thus, not suitable for consumption. The leaves were immediately cleaned with distilled water upon collection and left to dry at room temperature (25℃) for 2-3 h. After that, they were chopped into small pieces. The pieces were then freeze-dried for another 36 – 48 h (Labconco 7753030 FreeZone 6L Console Freeze Dry System, Labconco Corp., Kansas

City) until consistent weight was achieved. The moisture content of the dried leaves of Dillenia suffruticosa (Griff) Martelli., Eugenia polyantha (Wight) Walp (a) and Eugenia polyantha (Wight) Walp (b) were as reported by Adasuriya (2018), which are 74.8 ± 2.2%, 67.2 % and 72.0 ± 0.3 % from respectively. The freeze-dried powder was kept in tight container in the dark at -20oC. Those samples were used in this present study.

Figure 20: Image of leaves of plant sample used (Adasuriya (2018) with permission)

3.1.2 Preparation of plant water extract

Extraction of the crude water extracts was performed freshly prior to any analysis. The extraction method was as performed by Adasuriya (2018), with a slight modification. In anticipation of the cell-culture work, this study applied water-based extraction, instead of using methanol or ethanol as the extracting solvent because using alcohol would not properly mimic how the plants are commonly consumed. In brief, 0.1 g of the powdered plant sample was weighed and transferred to a 15 mL centrifuge tube. Then 10 mL of MilliQ water was added and the mixture was then vortexed for approximately 30 seconds to 1 minute to thoroughly mix the mixture. After vortexing, the tube was then sonicated using a Bransonic 5510 ultrasonic bath (Bransonic Ultrasonics Corporation, USA) at room temperature (25℃) for 20 minutes. After sonication, the mixture was centrifuged using Eppendorf 5702 centrifuge machine (Eppendorf AG, Hamburg, Germany) at 2,500 rpm for 10 minutes. The supernatant was filtered using a Whatman No. 1 filter paper and the filtrate was transferred into a 10 mL volumetric flask. The volume in the flask was topped up with MilliQ water. This preparation gave a 10,000 mg/L or 10 mg/mL stock solution of the plant water extract (Adasuriya 2018). The stock was usually prepared freshly and used immediately.

Any remaining stock solution was kept in the dark at 5oC, and this was stable up to 2 days after its preparation.

For determination of extraction yield, 10-mL of the extract stock solution was poured into an aluminium tray pan and was subjected to drying in a drying oven (100oC) for at least 24 h or until constant weight had been achieved. The weight changes were used to estimate the extraction yield, using the equation below:

Initial weight (g) - Final weight (g) Extraction yield (%, w/w) = × 100% Sample powder weight (g)

3.1.3 Preparation of extracts for cell culture

The initial preparation of plant extracts was prepared as described in section 3.1.2. The stock solution with a concentration of 10 mg/mL was diluted as required for the cell culture experiment (for example: dilute the stock from 10 mg/mL to 2 mg/mL) to avoid pigment disturbance from the sample. The extract stock and the respective cell culture media was pre-warmed to 37℃. Then the stock extract was diluted to double the amount of initial concentration needed for example, to prepare a 2 mg/mL concentration, the stock was diluted with MilliQ water to 4 mg/mL and 125 µL was added to a well on a 96-well plate, this will first dilution. The subsequent dilutions after that was using the complete cell culture media (RPMI or DMEM) of the respective cell used (NPC/HK1 or HaCaT). To prepare the extract-media mixture, each subsequent well for dilutions was filled with 125 µL of the respective pre-warmed cell culture media. Then the first solution was diluted to half by taking 125 µL of the first solution and mixing it with the subsequent well containing pre-filled media prepared earlier. Each concentration was diluted to half until desired final lowest concentration. This mixture of extract-media will be used to replace the old media after the 24-h of seeding as described in 3.4.7.

3.2 Determination of polyphenol contents

3.2.1 Chemicals

Trolox [(±)-6-Hydroxy-2,5,7,8 tetramethyl-chromane-2-carboxylic acid] (Cat. No. 238813), quercetin (Cat. No. Q4951), aluminium chloride, were all obtained from Sigma Aldrich. Methanol AR and ethanol absolute denatured were from HmBG Chemicals, while gallic acid (Cat. No. G7384) and sodium carbonate anhydrous (Cat. No. 1613757) were from Merck. Folin-Ciocalteu phenol reagent was purchased from Nacalai Tesque (Cat. No. 37204-45).

3.2.2 Total Phenolic Content (TPC) assay

The TPC assay method used was based on a previous method by Ginjom et al. (2010) and modified to allow the assay to be run on a clear, flat-bottom 96-well plate (Eppendorf 96-well flat bottom cell culture plate, Cat. No. 0030730119). The 1,000 mg/L stock solution of the reference standard, gallic acid, was prepared with 80% methanol. The standard was diluted with MilliQ water to make 0, 2, 4, 6, 8 and 10 mg/L standard solution and the plant samples were diluted with MilliQ water to make 10, 20, 30 and 40 mg/L solution. Blank sample refers to MilliQ water. Each dilution was analysed in duplicates.

In each well, 150 µL of diluted plant extract samples (as well as the standard and blank) and 25 µL of Folin-Ciocalteu reagent were added. The plate was initially manually shaken briefly and then was left to stand for 2 minutes. After that, 75 µL of 20% sodium bicarbonate (Na2CO3) was added to the mixture and the plate was again shaken lightly to ensure complete mixing of solutions. After that, the plate was incubated at room temperature (26℃) for 2 h. The development of purple hue signifies the presence of phenolic compounds, and this was measured through the absorbance reading at 760 nm using Synergy HT Multi-Detection Microplate Reader (BioTek, USA). The phenolic content of the sample was estimated using the gallic acid calibration graph and total phenolic content in the sample was expressed as mg gallic acid equivalent per 100 mg of dry sample (mg GAE/ 100 mg).

3.2.3 Total Flavonoid Content (TFC) assay

The modified method for this TFC assay was based on a method used in a previous paper (Amado et al. 2014). Quercetin was used as the reference standard in this assay and the stock solution (1,000 mg/L) was prepared using 80% ethanol. For the assay, the quercetin stock solution was diluted to 0, 10, 20, 30, 40, 50 and 60 mg/L with distilled water. The extract stock solution was also diluted with water to 100, 200, 300 and 400 mg/L. Blank sample was MilliQ water. Each TFC analysis on each dilution was conducted in duplicates.

This assay also used clear, flat bottom 96-well plate (brand) for the sample analysis. In brief, 100 µL of the samples (diluted extract, diluted standard and blank) followed by 100 µL of 2% aluminium chloride (AlCl3) was added into each well. A colour development from colourless to yellow indicates that the presence of flavonoid forming coloured complex with aluminium chloride. To ensure that all component of the reaction mixed well, the plate was manually shaken lightly and then left to stand for 10 minutes at room temperature (26℃). The absorbance was then read at 415 nm using Synergy HT Multi-Detection Microplate Reader (BioTek, USA). Total

flavonoid content in the plant sample was expressed as mg quercetin equivalent per 100 mg dry sample (mg QE/ 100 mg).

3.3 Determination of antioxidant properties based on in vitro methods

3.3.1 Chemicals

Trolox [(±)-6-Hydroxy-2,5,7,8 tetramethyl-chromane-2-carboxylic acid] (Cat. No. 238813), quercetin (Cat. No. Q4951), DPPH [2,2-Diphenyl-1-picrylhydrazyl] (Cat. No. D9132), gallic acid (Cat. No. G7384) and ABTS (2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Cat. No. 11557) were obtained from Sigma-Aldrich (Merck). Potassium

(peroxodisulphate) persulfate (K2S2O8, Cat. No. 68882) was purchased from R&M chemicals. Methanol AR (Cat. No. C5019) and ethanol absolute denatured (Cat. No. C0314) were from HmBG Chemicals.

3.3.2 DPPH assay

The methodology for the DPPH (2,2-di[4-tert-octylphenyl]-1-picrylhydrazyl) assay used for the project was modified based on a previously used method (Ginjom et al. 2010). A colour change from purple to colourless signifies the presence of antioxidant. Two reference standards were used in this assay, namely gallic acid, a common phenolic compound in plants, and Trolox, a water- soluble analogue of vitamin E.

Gallic acid stock solution was prepared as described in 3.2.2 above. Trolox stock solution (1,000 mg/L) was also prepared in 80% methanol. On the day of analysis, the plant extract stock solution was diluted with MilliQ water to 100, 200, 300, 400 and 500 mg/L while gallic acid and Trolox were diluted with MilliQ water to 1.0, 3.0, 5.0 and 10.0 mg/L and 5, 10, 15, 20 and 25 mg/L, respectively. Control sample refers to MilliQ water. DPPH solution was prepared freshly by dissolving 4 mg of DPPH powder in 50 mL of absolute methanol. This produced a 50-mL of 80 mg/L DPPH working solution. Complete solubility of DPPH was ensured by sonicating the solution in an ultrasonic bath (Bransonic 5510, Branson Ultrasonics Corporation, USA) for about 2 minutes or until no visible powder is left on the bottom of the container. The DPPH working solution was kept in the dark or amber bottle, and if not used immediately, was kept in the fridge. The solution is only stable within 6-8 h of preparation.

As with the previous two assays, the analysis was also conducted in a 96-well plate. Each designated well was filled with 100 µL of sample (diluted extract, diluted standard and blank) and

100 µL of the working DPPH solution. After mixing, the plate was incubated in the dark for 30 minutes at room temperature (26℃) and the absorbance of the mixture was subsequently read at 517 nm using Synergy HT Multi-Detection Microplate Reader (BioTek, USA).

The purple colour of DPPH radical would diminish in the presence of an antioxidant. Thus, the paler the purple colour of the mixture after the 30-min incubation, the lower the absorbance reading, and the stronger the antioxidant activity of the sample. The antioxidant activity was expressed as Radical Scavenging Activity percentage (% RSA), and this was calculated from the absorbance values of the mixture in the absence (control) and presence (sample) or the plant extract or reference standard solutions. RSA (%) was calculated using the equation below:

�� %�� = (1 − ) × 100% ��

Graph of % RSA against sample concentration was plotted to visualise the effect of the sample concentration on antioxidant activity. The value of %RS and concentration were also used to estimate the half-maximal response (EC50) concentration, based on the dose-response function performed using GraphPad Prism version 6.01 for Windows, GraphPad Software, La Jolla California USA.

3.3.3 ABTS assay

The method for this assay was based on Re et al. (1999) for ABTS (2,2’-Azinobis [3- ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) assay, with slight modification. The green tinted solution of radical cations generated will be reduced by antioxidants present in the sample and the mixture will change to colourless.

ABTS (7 mM) stock solution was prepared the day prior to the sample analysis. Only a small volume of stock needed to be prepared per assay. For a 5-mL solution, 19.2036 mg of ABTS powder was dissolved in 5 mL of distilled water. Potassium persulfate (140 mM) was prepared by dissolving 378.5mg of potassium peroxydisulfate in 10 mL of MilliQ water. This solution can last for several months at room temperature. To make the ABTS+ radical solution, 88 µL of potassium persulfate solution was added to the 5-mL 7 mM ABTS stock solution prepared earlier. The mixture of radical solution was covered with aluminium foil and left to sit for at least 16 h in the dark at room temperature (26oC). Over the time, dark green colour developed.

On the day of analysis, and after 16 h of standing, 1 mL of the ABTS+ radical solution was diluted with approximately 80 mL of distilled water so that the absorbance reading at 734 nm is 0.70 ±

0.02. This was the ABTS+ working solution. Plant extract stock solution (10,000 mg/L) was diluted with distilled water from 0 (control) to 60 mg/L. Reference standards, Trolox and gallic acid were also diluted with water to 0 – 10 mg/L and 0 – 2.0 mg/L, respectively.

In each well of a 96-well plate, 50 µL of the test sample (control, diluted plant extract and diluted reference standard solutions) was added into designated well, followed by the addition of 100 µL of the ABTS+ working solution. The well plate was incubated in the dark at room temperature (26oC) for 30 minutes, and subsequently the absorbance intensity of the green colour of the ABTS+ radical at 734 nm was measured using Synergy HT Multi-Detection Microplate Reader (BioTek, USA). Bleaching of the green colour of ABTS+ radical suggested the effect of antioxidant from the test sample (plant extract or reference standard) scavenging the radicals. The antioxidant activity or the radical scavenging activity (%) was calculated using the following formula:

% RSA = (1 – Asample/ Acontrol) X 100

Where Asample is the absorbance of solution containing the test sample and Acontrol is the absorbance reading of solution without the test sample (contains distilled water) after 30 minutes.

Similar to DPPH assay, the % RSA value of the test sample was plotted against the concentration of test sample, the graph was used to estimate the EC50 value (in mg/L) of the test sample.

3.4 Screening of anticancer properties based on cell culture assays

3.4.1 Chemicals, reagents and materials

Roswell Park Memorial Institute medium (RPMI, GibcoTM, Cat. No. 11875093), trypsin-EDTA (Gibco™, Cat. No. 25200056), fetal bovine serum (FBS, GibcoTM, Cat. No. 10270098), Dulbecco’s Modified Eagle Medium (DMEM, GibcoTM, Cat. No. 41966029), phosphate-buffered saline pH 7.4 (PBS, GibcoTM, Cat. No. 10010023), were procured from Thermo Scientific. Cell proliferation kit (CellTiter 96 Aqueous One Solution Assay, Cat. No. G3580) was obtained from Promega, USA. Trypan blue (Merck, Cat. No. T6146) and dimethyl sulfoxide (DMSO, Merck, Cat. No. V900090) were purchased from Sigma-Aldrich (Merck). Penicillin-streptomycin mixed solution (Pen-Strep, Nacalai Tesque, Cat. No. 0936734) was purchased from Nacalai Tesque.

3.4.2 Cell lines and culture medium

NPC/HK1epithelial nasopharyngeal carcinoma cell line and HaCaT, immortalized human keratinocyte cell line were donated by Professor Dr Edmund Sim from UNIMAS (Faculty of Resource Science and Technology, FRST) for research purposes. NPC/HK1 cells were kept in RPMI 1640 media and HaCaT cells were kept in DMEM, penicillin-streptomycin was added to both culture media to prevent bacterial infection. Fetal bovine serum was filtered and added to the media at 10% volume prior to the cell culture assay. Cells were kept in an incubator (Thermo

Scientific Forma Direct Heat CO2 Class II Incubator, Model No. AC2-6E1, ESCO) at a condition of 37℃ temperature, 95% relative humidity and at 5% CO2, to imitate the conditions of a human body. Media for cell culture was prepared as 500 mL media with 10% FBS and 1% penicillin- streptomycin antibiotic solution. Serum free media was free of FBS and antibiotic solution.

3.4.3 Cell thawing and cell cryopreservation

Cells used for the assay were between passages of 15 – 25 to make sure they grow at a suitable pace. Cells with passages 15 – 25 were stored in liquid nitrogen or -80oC and when needed, was revived for culture. The cells (NPCHK1 or HaCaT) were stored using cryopreservation, where cells that grew to 80 – 90% confluency in a T75 cell culture flask (Eppendorf™, Cat. No. 30711122; Nest, Cat. No. 708003) were mixed with 2 mL of trypsin to detach the cells from the flasks’ surface. Then the flask was incubated for 5 minutes and then 3 mL of pre-warmed cell culture media (RPMI or DMEM) was added into the flask to stop the trypsin. All the mixture was removed and transferred to a 15 mL centrifuge tube and centrifuged using Eppendorf 5702 centrifuge machine (Eppendorf AG, Hamburg, Germany) at 1000 rpm for 5 minutes. After centrifugation, the supernatant was removed, leaving only the pellet. The pellet was resuspended in a cryopreserve mixture which were; 60% (Complete cell culture medium (RPMI or DMEM)), 30% fetal bovine serum (FBS) and 10% DMSO. The suspended cells were then aliquoted in 1.5mL free standing screw capped microcentrifuge tubes which were then placed in a passive cell freezing container for 24 h before placing the tubes into liquid nitrogen to avoid shock to the cells getting exposed to liquid nitrogen directly.

The stored cells were kept in a 1.5 mL free-standing screw capped microcentrifuge tubes. Prior to starting cell culture, NPC/HK1 cells were thawed for a few seconds in a water bath (37oC). The whole content of the cells was then transferred into a sterile 15-mL centrifuge tube, and then centrifuged using Eppendorf 5702 centrifuge machine (Eppendorf AG, Hamburg, Germany) at 1,000 rpm for 5 minutes. The supernatant was then removed, and the cell pellet was re-suspended in 2 mL of respective pre-warmed media (RPMI or DMEM). After that, the cells were counted

(refer to Section 3.4.4). The value was used to estimate the required number of cells needed to grow in 10 mL media. To revive cells after storage, they were seeded into a sterile T25 cell culture flask (Eppendorf™, Cat. No. 0030710029) or in a sterile cell culture dish (Corning®, 100 mm x 20 mm) to incubate, grow and multiply the cells with 10mL of respective media.

3.4.4 Cell counting

This procedure was done prior to seeding the revived or passaged cells into a new flask. Cell counting was performed to get the optimum number of seed cells required for the cytotoxicity assay. The cells were grown in a cell culture container (i.e. cell culture flasks or cell culture plates). After the cell were centrifuged and resuspended, in a 100 µL microcentrifuge tube, 10 µL of the cell were withdrawn from the 2 mL suspension and mixed with 10 µL of trypan blue. The presence of viable cells, i.e. those that were glowing under the light microscope (Nikon Eclipse Ti-S), were counted using a haemocytometer and non-viable cells which were stained blue were left out. As shown in Figure 21 below, cells in each of the four, corner square was counted and averaged.

The formula used to count the cells is as below:

�� = × �� × 104 �

�� . �� = �� × ��

��

3.4.5 Cell passaging

Cell passaging was performed to keep cells alive or to prevent them from overgrowing their T75 culture flask (Eppendorf™, Cat. No. 30711122; Nest, Cat. No. 708003). For the cytotoxicity assay, the cell passage number usually used is between P15 – P25. Cell are usually monitored for 2 – 3 days to observe their growth using an inverted microscope (Nikon Eclipse Ti inverted microscope, Nikon Corporation, Japan). Cell which have reached 80 – 90% confluency were passaged to prevent overgrowth if not used for cell seeding. To perform cell passaging, the media was first removed and thrown away, then the flask was washed with PBS to further remove any leftover media in the flask careful not to poke the bottom surface of flask where the cell are. Then, 2 mL of pre-warmed trypsin was added into the bottom edge of the flask and then the flask was gently shaken to properly mix the trypsin with the cells. The flask was again incubated for 5 minutes to allow detachment of cells from the flask. Detached cells were observed under an inverted microscope where they were able to move around on the flask surface. Then, once all cells have detached, 3 mL of the respective pre-warmed cell culture media was added into the flask to inactivate the trypsin and then whole content mixture was mixed and transferred from the flask into a 15 mL centrifuge tube. The tube was then centrifuged at 1,000 rpm for 5 minutes and then, the supernatant was removed. The pellet was re-suspended in the respective pre-warmed fresh cell culture medium. From this point, the cells were either regrown in a new flask or used for cell culture experiments.

3.4.6 Cell optimisation

Cell optimisation is a cell culture technique that is carried out by researchers especially when starting with an unknown or new cell. The objective of the procedure was to obtain the optimum amount of cell count per well so that after the period of incubation, the cells would not undergrow or overgrow the well.

The cell optimisation protocol was adapted from Bajgain et al. (2014). This procedure determined the optimum density of cell to be seeded per surface area of the well in the 96-well culture plate. The procedure was adapted to 72 h, the same duration of the incubation time of the cells and the plant sample. Optimisation does not require the addition of the samples which allowed observation of cell growth in just their respective culture media. The media was not changed because the time duration is short, and the media was monitored every day for contamination. As the procedure is performed when working with a new batch of cells or unknown cells, the initial

cell density would be 1 x 106 cells/cm2 but as growth of the NPC/HK1 cells and HaCaT cells used in this project have extensively been used, the minimum cell density was 2.5 x 104 cells/cm2 for NPC/HK1 cells and 3.5 x 104 cells/cm2 for HaCaT cells.

Basically, different cell counts per well was calculated as described in 3.4.4 and seeded into the plate which will be used for the upcoming experiment (e.g. 6, 12, 24 or 96-well plates). The cells were passaged and grown in T75 culture flask as described in 3.4.5. After the cells have reached 80 – 90% confluency, the cells would be prepared for culture and counted for 100 µL of cell per well in a 96-well flat bottom plate (Eppendorf™, Cat. No. 0030730119). The cell count range for NPC/HK1 was counted as 2500, 3000, 3500, 4500 and 5000 cell count per 100 µL (NPC/HK1 cell density, 2.5 x 104 to 5.0 x 104 cells/cm2) and HaCaT were 3500, 4000, 4500, 5000, 5500 and 6000 cell count per 100 µL (HaCaT cell density, 3.5 x 104 to 6.0 x 104 cells/cm2).

Cell seeding was started by taking 100 µL of each cell count into designated wells and then the plate was incubated at 37℃, 5% CO2 for 72 h. The cells were monitored and observed for contamination without changing the media in between. The 72-h incubation time was used as it was the incubation time used in the subsequent assays for the plant extract treatment. After 72 h, the cell viability was analysed using MTS assay as described in Section 3.4.7.

3.4.7 Cell viability assay with MTS

Cell viability assay was started by counting the cell count for seeding in each well (2,500 cell count per well for NPC/HK1 and 3,000 cell count per well for HaCaT) as described in 3.4.4. Then the amount of cell-media mixture needed to fill in the desired number of wells on a 96-pwell plate with 100 µL of the mixture was calculated. Each well was filled with 100 µL of the cell-media mixture then the plate was incubated for 24 h. After that, the plate was checked for any contamination (i.e. blurry media, presence of external dark filaments) using an inverted microscope and if there were no contamination, then the seeded cells were ready to use.

Before adding in the cell-media mixture, all media was removed from each well carefully without scratching the bottom of the well. Then the media was replaced with 100 µL of the cell-media mixture as prepared in 3.1.3. Then the plate was incubated for 72 h (37 ℃, 5% CO2) without changing the media with daily observation under the inverted microscope.

Vehicle control (10% extraction solvent and seeded cells) was also prepared. Vehicle control which was water for this project was used to rule out the possibility of the solvent causing any reaction to the cells without the extracts. The cells were then incubated together with the extracts

for 72 h. The vehicle control was prepared at 10% to mimic the concentration of extracts which were 0.1g/ 10 mL.

After 72 h, the MTS assay (CellTiter 96 Aqueous One Solution Assay, Promega, USA) was performed on the incubated cells to check for cell viability. MTS assay detects the presence of viable cells, which can produce electrons that reduce the MTS tetrazolium compounds into a coloured product called formazan. In turn, the formazan stains the viable cell bluish dark purple. The more viable cells available, the darker the colour of the solution.

In brief, the procedure was initiated by removing the old media from the wells and a mixture of 10 µL of MTS solution and 50 µL of new pre-warmed cell culture media was added back into the wells. After that, the plate was incubated for 2 h (for NPC/HK1 cells) or 4 h (for HaCaT cells) and then after that the plate was analysed using the microplate reader (Synergy HT Multi- Detection Microplate Reader, Biotek, USA) at 490 nm. Half maximal inhibitory concentration at

50% (IC50) was determined using the Dose-response function performed using GraphPad Prism version 6.01 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com.

3.4.7.1 Selectivity index (SI)

In the present study, the degree of cytotoxicity selectivity of the plant extracts against the NPC/HK1 cancer cells as compared to normal cells, HaCaT cells was calculated based on their

IC50 values. The selectivity index was calculated as below:

��50 �� (��) = ��50 �� 1 ��

SI value demonstrates the differential activity of the extract, with higher SI value suggesting a more selective extract against the cells being compared. A value greater than 2 suggests greater selectivity while those less than 2 suggest general toxicity effect.

3.4.8 Cell migration assay

The method was adapted from Kong (2018). This assay was performed on NPC/HK1 cells only as it was aimed to investigate the effect of the plant extracts on the cancer cells’ migration. Since the assay was not aimed to kill the cancer cells right away, but instead, to monitor their growth, lower plant extract concentrations were used, i.e. at their IC25, IC12.5 and IC6.25 concentrations.

The extracts were diluted with serum free RPMI media according to their IC50 value as determined

earlier (Section 3.4.7). Absence of any extract in the serum free media was used as the negative control sample.

For this assay, each well of a 24-well plate (TPP®, Cat. No. 92024) was seeded with 500 mL of 5 o RPMI cell culture media containing 8 x 10 cells. The cells were incubated at 37 C, 5% CO2 for 24h. The next day, the old media was removed, and the cells were washed with 500 µL of DBPS one time. This was followed by the addition of 500 µL of fresh pre-warmed serum-free media. A horizontal scratch was made in each well containing the cells using a 10 µL filter pipette tip. The plate was then first observed under an inverted microscope (40X magnification and P3 phase contrast resolution) and the observation area was marked on the lid as shown in Figure 22. The mark was to ensure that observation point will be at the same area during each observation.

Figure 22: Diagram of how the observation area mark should be drawn on top of the lid for scratch assay.

The scratch image was taken using Leica DMI 3000B (Leica Biosystem) at 100X magnification and phase contrast resolution at P1. The image was labelled as `0 hour’ for the respective concentration of the plant extract. The phase contrast resolution which are P1, P2 or P3 is the intensity of the light from the microscope light source with P1 with the weakest and P3 the strongest light source. The amount of light that passes through the cell would allow easy observation of the scratch image on the T-scratch application program (Gebäck et al. 2009).

For the negative control, the old incubating media in the dedicated well was removed and replaced with 500 µL of fresh serum-free media for the incubation period. For those designated for plant extract incubation, the serum-free media was removed, and replaced with the 500 µL of extract-

o serum-free media mixture. The cells were further incubated at 37 C, at 5% CO2 and examined after 7-h and 24-h. Images were taken during each observation as described earlier.

Figure 23: The diagram shows the settings of the Tscratch assay program to determine gap distance of the cells (Gebäck et al. 2009)

The image of the scratch areas were analysed using Tscratch (Gebäck et al. 2009) with default settings as shown in Figure 23. The values obtained were used to calculate the percentage of scratch area compared to the scratch area at 0 hour. The steps for using the program as shown in Figure 21; (1) Adding the files of the cell images, (2) Running the analysis and the gap distance will be automatically calculated by the program, (3) Clicking “Done” after the analysis is finished and all the gap distances are checked, and (4) Save the files.

3.5 Statistical analysis

Unless otherwise stated, all results were expressed as mean ± standard deviation of at least three different experimental replications per sample. The t-test was performed using the GraphPad QuickCalcs t-test calculator (Graphpad, https://www.graphpad.com/quickcalcs/) while the curve fitting for the EC50 and IC50 estimation based on non-linear regression fit was performed using GraphPad Prism® version 7 for Windows (GraphPad Software, la Jolla California USA). In all cases, statistical significance was set at p<0.05.

4 RESULTS AND DISCUSSION

4.1 Moisture content and extraction yield

Powdered plant samples used throughout the project was prepared using freeze-drying and the difference in weight due to the moisture loss during the drying process was used to determine the moisture content (%) of the plant sample. The extraction yield was determined when the water extract of the powder sample was dried.

As summarised in Table 5, D. suffruticosa had the highest moisture content value followed by E. polyantha var. b then lastly E. polyantha var. a. On the other hand, the extraction yield of E. polyantha var. b was the highest amongst the samples, followed by D. suffruticosa and then E. polyantha var. a. The high extraction yield of E. polyantha var. b might contribute to higher presence of phytochemical compounds that would contribute to the antioxidant and anticancer activity.

Table 4: Summary of the moisture content and extraction yield of the plants.

Plant extract Moisture content, %1 Crude Water Extract ((DW) Dry Weight), %

D. suffruticosa 74.8 ± 2.2 14.7 ± 0.6a

E. polyantha var. a 67.2* 13.1 ± 0.4b

E. polyantha var. b 72.0 ± 0.3 25.8 ± 0.2c

All values are expressed as mean ± standard deviation (n=3).1 Values were adapted from Adasuriya (2018). *value from a single determination. Means in a column followed by different letters differ significantly (P < 0.05) as analysed using GraphPad t-test calculator.

Different extraction solvent would affect the yield of the plant samples. Vieito et al. (2018) discussed in their paper that the extraction yield was higher in water/ethanol mixture (17.55 ± 0.16%), followed by ethanol only (17.08 ± 0.23%) and water solvent had the lowest yield (less than 8%). Pham et al. (2015) had also found that water extraction yielded the most solid content of their Helicteres hirsuta Lour leaves, but methanol extracted most of the flavonoid and saponins compounds.

However, methanol is toxic to human, and thus, this solvent was not used in this study. Ethanol is another good candidate as polyphenol extraction solvent, as it is less toxic compared to methanol. However, even ethanol is toxic at high concentration. Thus, water was the preferred

extraction solvent in this study, as this study mimicked the typical solvent used in the preparation and cooking of the plant samples, i.e. with the addition of water.

4.2 Total phenolic and total flavonoid contents in plant extracts

4.2.1 Calibration curve

The absorbance values (TPC: at 760 nm; TFC: at 415 nm) from each test solutions were plotted against the test samples’ concentration. For the reference standards (TPC: gallic acid, TFC: quercetin), the plot was used as a calibration curve to estimate the total phenolic content (Figure 24) and the total flavonoid content (Figure 25). Both reference standard plots showed very good linearity (R2 ≥ 0.99).

Figure 25: Calibration curve of quercetin for total flavonoid content (TFC): Absorbance value at 415 nm at different concentrations. All values are expressed as mean ± standard deviation (n=3).

4.2.2 Phenolic and flavonoid contents of D. suffruticosa, E. polyantha var. a and E. polyantha var. b.

The total phenolic content of Dillenia suffruticosa (Griff) Martelli., Eugenia polyantha (Wight) Walp (a) and Eugenia polyantha (Wight) Walp (b) leaves water extract, calculated from the calibration curve (R2 = 0.994), were 3.70 ± 0.69, 1.58 ± 0.44, and 6.62 ± 1.16 mg gallic acid equivalents/100 mg, respectively (Table 5). The total flavonoid content (R2 = 0.998) of D. suffruticosa, E. polyantha var. a and E. polyantha var. b leaves water extract, calculated from the calibration curve (R2 = 0.994), were 0.45 ± 0.69, 0.10 ± 0.22, and 0.25 ± 0.77 mg GAE/100 mg (Table 5).

Table 5: Summary of total phenolic content (TPC) and total flavonoid content (TFC) of selected plant extracts.

Plant extract Total phenolic content (mg Total flavonoid content (mg GAE/100 mg DW) QE/100 mg DW)

D. suffruticosa 3.70 ± 0.69a 0.45 ± 0.79a

E. polyantha var. a 1.58 ± 0.44b 0.10 ± 0.03a

E. polyantha var. b 6.62 ± 1.16c 0.25 ± 0.33a

All values are expressed as mean ± standard deviation (n=3). DW refers to the dry sample weight. Means in a column followed by different letters differ significantly (P < 0.05) as analysed using GraphPad t-test calculator.

As observed in Table 6, aqueous sample of E. polyantha var. b has the highest total phenolic contents followed by D. suffruticosa and E. polyantha var. b. This coincides with the results from the previous report (Adasuriya 2018) where the project had evaluated different plants of their antioxidant properties and the results showed that the alcoholic extract of E. polyantha var. b (the same plant), had the highest TPC and TFC value as compared to D. suffruticosa, P. cordifolia and E. polyantha var. a. Extraction solvent is found to have an effect on the phenolic content value as reported by Do et al. (2014) on L. aromatica plant extracts. The TPC value of the water extract was far lower (0.63 ± 0.24 mg GAE/100 mg) than those found in the 100% methanol extract (3.15 ± 1.07 mg GAE/100 mg), 100% ethanol extract (4.05 ± 0.88 mg GAE/100 mg) and 100% acetone extract (4.03 ± 0.20 mg GAE/100 mg). Similarly, on the same report, the TFC value of L. aromatica water extract was lower (0.404 mg QE/100 mg) than the other extracts prepared using other solvents - 100% methanol (1.54 mg QE/100 mg), 100% ethanol (3.11 mg QE/100 mg) and 100% acetone (3.09 mg QE/100 mg). This shows that the choice of extraction solvent influences the content of phenolic compounds extracted.

Previous studies using E. polyantha collected from Indonesia by Safriani et al. (2011) showed that TPC value to be 4.06 mg GAE/ 100 mg. The difference in values might also be linked to the fact that the same plants might be different when they are grown in different parts of the world. However, the values obtained from the current study still shows that the plant samples used were still superior in terms of TPC and TFC compared to other plants.

Tea leaves like black tea (Camellia sinensis L.), green tea (Camellia sinensis L.) and chamomile tea (Chamaemelum nobilis L.) are widely consumed as they are depicted to have high values of antioxidant capacities. Compared with formerly mentioned teas (black tea; 0.75 ± 0.9 mg GAE/100 mg, green tea; 0.77 ± 0.7 mg GAE/100 mg, chamomile tea; 0.84 ± 0.9 mg GAE/100 mg) the antioxidant values of D. suffruticosa (3.7 ± 0.69 mg GAE/100 mg) and E. polyantha (var. a; 1.58 ± 0.44 mg GAE/100 mg, var. b; 6.62 ± 1.16 mg GAE/100 mg) are still much higher (Yoo et al. 2008).

4.3 Antioxidant potentials of plant extracts

4.3.1 Antioxidant activities based on DPPH assay

DPPH assay is one of the most common assays used to determine antioxidant activity. The principal of the assay involves the scavenging of free radicals by a stable DPPH radical where it will be reduced and observable through a discolouration from purple to yellow. Thus, the higher the antioxidant capacity, the lower is the value of EC50. EC50 in this assay would be the effective concentration of the sample at to exhibit activity at 50% Radical Scavenging Activity (% RSA).

The dose-response curves from Figure 26, shows that the higher the concentration of the reference compound or plant extract, the higher the antioxidant activity as expressed as percentage radical scavenging activity, % RSA. It is also observed that the effect was not linear, as seen in the total phenolic and total flavonoid contents earlier. Thus, for the DPPH assay, the half maximal effective concentration (EC50) was used instead to evaluate the samples’ antioxidant activity. Lower EC50 value denotes samples that possess higher antioxidant activity.

Gallic acid T ro lo x

1 0 0 1 0 0

8 0 8 0

6 0 6 0

4 0

4 0 R S A (% ) R S A (% )

2 0 2 0  EC 5 0 = 3 .4 4  0 .2 4 m g /L EC 5 0 = 1 5 .9 0 0 .5 3 m g /L 0 0 0 5 1 0 1 5 0 1 0 2 0 3 0 Concentration (mg/L) Concentration (mg/L)

(a ) ( b )

D. suffruticosa E. polyantha v a r. a

1 0 0 1 0 0

8 0 8 0

6 0 6 0

4 0 4 0

R S A (% ) R S A (% )

2 0 2 0  EC 5 0 = 2 1 7 .6 0 16.41 mg/L EC 5 0 = 2 6 5 .4 0  24.56 mg/L 0 0 0 2 0 0 4 0 0 6 0 0 0 2 0 0 4 0 0 6 0 0 Concentration (mg/L) Concentration (mg/L)

( c ) ( d )

E. polyantha v a r. b

1 0 0

8 0

6 0

4 0 R S A (% )

2 0

EC 5 0 = 6 0 .8 6  15.47 mg/L 0 0 2 0 0 4 0 0 6 0 0 Concentration (mg/L)

( e )

Comparing the EC50 values by the three plant extracts as observed in Figure 27, E. polyantha var.

b exhibited the highest antioxidant activity (EC50 = 60.9 ± 15.5 mg/L), followed by D. suffruticosa

(EC50 = 217.6 ± 16.4 mg/L) and then E. polyantha var. a (EC50 = 265.4 ± 24.6 mg/L). As expected, both gallic acid and Trolox showed very strong antioxidant activities with EC50 values of 3.4 ± 0.2 mg/L and 15.9 ± 0.9 mg/L respectively. Compared to E. polyantha (b), the most potent antioxidant among the plant extracts, both reference standards were way stronger antioxidant. Gallic acid was 18 times more potent while Trolox was about 4 times more potent antioxidant than E. polyantha water extract. This is possibly due to these standard compounds in their pure forms, while the plant extracts were not, and were in fact, mixtures of different compounds.

Methanolic extracts of the same plant sample showed similar antioxidant potential trends with E. polyantha var. b being the sample with the highest antioxidant activity (EC50 = 37.8 ± 9.9 mg/L), followed by D. suffruticosa (EC50 = 40.8 ± 8.8 mg/L) and E. polyantha var. a (EC50 = 70.7 ± 8.1 mg/L) (Adasuriya 2018). Comparing the results from Adasuriya (2018), it was found that the methanol extract was 2 times more potent antioxidants than the water extract for E. polyantha var. b. For the other two samples, it was found that the water extracts of D. suffruticosa and E. polyantha var. possess 3 to 5 times less antioxidant potentials than their respective methanol extracts (Adasuriya 2018). Regardless, water extract was used in the present study to mimic the common solvent used when these plants are consumed.

Research on methanolic extract of P. cordifolia and P. serratifolia by Adasuriya (2018) which are common edible plants in Malaysia, showed to have higher EC50 value (lower antioxidant potential) than E. polyantha var. b and D. suffruticosa (188.1 ± 8.5 mg/L and 256.0 ± 8.1 mg/L respectively).

Eugenia uniflora or the Brazilian cherry tree is one of the most popular plant used as traditional medicine in Brazil. Essential oils derived from the leaves were found to have antibacterial activities especially against Bacillus cereus (Ogunwande et al. 2005). Antioxidant assays on the leaves of E. uniflora showed the DPPH EC50 value to be 833 ± 20.7 mg/L which shows that the plants used for the current research have stronger antioxidant capacity with E. polyantha var. b being 13 times stronger than E. uniflora (Victoria et al. 2012).

Research conducted by Lee et al. (2014) on plants used as traditional medicine showed that these medicinal plants does have strong antioxidant capacities. When comparing the traditional plants with E. polyantha var. b, the latter shows 2 to 18 times higher EC50 value but the D. suffruticosa and E. polyantha var. a have a lower EC50 value. This shows that E. polyantha var. b has good potential medicinal value.

Tea leaves are also good source of antioxidants and are highly consumed by the communities because they are believed to be beneficial for health. Antioxidant tests on the Sabah green and black tea leaves showed that the tea leaves are much more potent than the plants used for this experiment with EC50 values of 30.00 ± 0.03 mg/L and 30.00 ± 0.00 mg/L respectively (Izzreen & Fadzelly 2013).

4.3.2 Antioxidant activities based on ABTS assay

Apart from DPPH assay, another commonly used assay that involves scavenging of unstable free radicals is the ABTS assay. Similarly, the ABTS assay like the DPPH assay, involves the reduction of the stable radicals to bring about a colour change which in the case ABTS, was from a blue-green colour to colourless.

In this assay, Trolox and Gallic acid were used as reference standards. Like DPPH, the antioxidant activity was measured using percentage of radical scavenging activity (% RSA). Then the half maximal effective concentration (EC50) was plotted.

The antioxidant activity of the plants extracts, based on the ABTS assay was summarised in Table 6. As seen in Figure 27, based on the values the extract with the highest antioxidant activity was E. polyantha var. b (24.2 ± 1.3 mg/L), followed by D. suffruticosa (42.4 ± 3.7 mg/L) and lastly E. polyantha var. a (99.4 ± 12.7 mg/L). The value coincides with the order from the DPPH test. The antioxidant results of both DPPH and ABTS shows that E. polyantha var. b is the plant with the strongest antioxidant activity.

Previous research with similar methanol extracts of the plants showed that D. suffruticosa (68.4 ± 6.5 mg TE/100 mg) (13.0 ±1.0 mg GAE/100 mg) was more dominating in terms of ABTS activity, followed by E. polyantha var. b (64.7 ± 3.9 mg TE/100 mg) (12.3 ± 0.6 mg GAE/100 mg) and E. polyantha var. a (30.7 ± 6.2 mg TE/100 mg) (5.8 ± 1.1 mg GAE/100 mg) (Adasuriya 2018).

Comparing the DPPH activity and ABTS activity of green and black tea leaves (C. sinensis), ABTS activity of the leaves are much higher than the corresponding DPPH values (Izzreen &

Fadzelly 2013). Ethanolic extracts of the shoots showed IC50 of 170.00 ± 0.02 mg/L for green tea leaves and 180.00 ± 0.00 mg/L for black tea leaves. Both are much lower than the ABTS values of D. suffruticosa and E. polyantha var. a and (b).

ABTS activity shown by the plant samples are much higher than the ABTS activity detected in other medicinal plants used in other countries like in India, ayurvedic plants like Sida cordifilia

Linn., Cynodon dactylon Linn. and Evolvulus alsinoides Linn., have significantly lower EC50 values (172.3 mg/L, 273.6 mg/L and 342.8 mg/L respectively) (Auddy et al. 2003). This shows that the plant samples used for this research are much more potent in ABTS activity which may indicate ABTS might be more appropriate in detecting antioxidant activity (Lee et al. 2015).

T r o lo x Gallic Acid

1 0 0 1 0 0

8 0 8 0

6 0 6 0

4 0 4 0

R S A (% ) R S A (% )

2 0 EC = 4 .8 6  0 .2 8 m g /L 2 0 50 EC 50 = 0 .7 5  0 .0 4m g /L

0 0 0 5 1 0 1 5 0 .0 0 .5 1 .0 1 .5 2 .0

Concentration (mg/L) Concentration (mg/L)

(a ) ( b )

D. suffruticosa E. polyantha v a r . b

8 0 1 0 0

8 0 6 0

6 0 4 0

4 0

R S A (% ) R S A (% )

2 0 2 0  EC 50 = 4 2 .4 2 3 .7 4m g /L EC 50 = 2 4 .1 5  1 .3 4 m g /L

0 0 0 2 0 4 0 6 0 0 2 0 4 0 6 0 Concentration (mg/L) Concentration (mg/L)

( c ) ( d )

E. polyantha v a r . a

4 0

3 0

2 0 R S A (% )

1 0

EC 50 = 9 9 .3 9  1 2 .7 1 m g /L

0 0 2 0 4 0 6 0 Concentration (mg/L)

( e )

Previous research on water extract of traditional herbal plants, Eugenia polyantha, Cinnamon zeylanicum (Cinnamon bark), Curcuma xanthorrhiza (“Temulawak”), Orthosiphon stamineus (“Misai kucing”) and Andrographis paniculate (“Hempedu bumi”). C. zeylanicum had the strongest ABTS activity at EC50 78.26 mg/L followed directly behind by E. polyantha var. at EC50

94.27 mg/L. Compared to the ABTS of the water extract of plants used for this project, the EC50 of E. polyantha var. b showed the best EC50 value at 24.15 ± 1.35 mg/L and D. suffruticosa was at 42.42 ± 3.74 mg/L (Ismail et al. 2017). Though both were water extracts but Ismail et al. (2017) had used spray drying to dry the plants while this project suspended the dry powder samples in distilled water which may have an effect on the number of phytochemicals released.

Table 6: Summary of antioxidant activities of standard references and plant extracts based on DPPH and ABTS assays.

Antioxidant Activity

Sample DPPH Radical Scavenging ABTS Radical Scavenging Activity, EC50 value (mg/L) Activity, EC50 value (mg/L)

D. suffruticosa 217.6 ± 16.4a 42.4 ± 3.7a

E. polyantha var. a 265.4 ± 24.6b 99.4 ± 12.7b

E. polyantha var. b 60.9 ± 15.5c 24.2 ± 1.3c

Gallic acid 3.4 ± 0.2 0.7 ± 0.0

Trolox 15.9 ± 0.9 4.9 ± 0.3

Both assays have their limitations as they involve the use of free radicals thus there is the issue of light sensitivity, interferences from anthocyanins for DPPH as the colour spectrum is almost similar and both has yet to be proven to work in a biological system (Ratnavathi & Komala 2016). Lee et al. (2015) had also described in the paper that when comparing DPPH and ABTS with one hundred different pure chemical compounds, they discovered that ABTS seems to give a much accurate results compared to DPPH. Based on t-test analysis, it also shows that both DPPH and ABTS assays can be used because they can both produce significant results.

4.3.3 Correlation analysis between total phenolic and antioxidant effects of selected plant extracts

Correlation between the three plant extracts were done using the Pearson’s regression analysis on Microsoft® Excel. As shown in Table 8, six possible pairs were compared from the phenolic contents and the antioxidant activities. Correlation was determined using the r value where the closer the value is to r = 1, the stronger is the correlation. All parameters appeared to be positively correlated to each other, with three (which are in bold) being statistically significant whereby the p-value was less than 0.05 (p<0.05).

Table 7: Summary of correlation analysis of polyphenolic contents and antioxidant assays on D. suffruticosa, E. polyantha var. a and E. polyantha var. b.

DPPH IC50 Value Total Flavonoid Content Total Phenolic Content

(mg/mL) (mg QE/ 100 mg) (mg GAE/ 100 mg)

ABTS 0.8649 0.9587 0.9346 IC50 Value (0.6039) (0.0208) (0.0312) (mg/mL) DPPH 0.9203 0.8710 IC50 Value (0.0394) (0.2037) (mg/mL) Total Flavonoid 0.9496 Content (0.4893) (mg QE/ 100 mg)

Values are expressed as Pearson correlation coefficient (r) and p-value in brackets. Values in bold are significantly different at p<0.05.

The lowest out of the three pairs were between ABTS and the total flavonoid content (TFC) (r = 0.9587, p-value = 0.0208). The next pair was between ABTS and the total phenolic content (TPC) (r = 0.9346, p-value = 0.0312) followed by the DPPH and TFC pair (r = 0.9203, p-value = 0.0394). ABTS values are the most correlated to the polyphenol contents in plant samples as both TPC and TFC pairs showed statistically significant p-values. DPPH and ABTS pair, however, does not show significant p-value (p-value = 0.6039) and the correlation between the values are weak (r = 0.8649). The results showed that antioxidant activity of ABTS assays are significantly affected by the total flavonoid and total phenolic contents of a plant. The DPPH values was only significantly affected by the flavonoid contents in a plant.

The correlation between DPPH and TPC is also weak (r = 0.8710, p-value = 0.2037), thus suggests that phenolic contents of a plant does not strongly affect its DPPH values. The correlation between

TPC and TFC was shown to be high (r = 0.9496) but the p-value was not statistically significant (p-value = 0.4893). Therefore, it can be said that there is a positive correlation between polyphenol contents in the plant samples, but the relationship would not be significant.

To conclude, polyphenol content assays are good as initial screening for the presence of antioxidants prior to the more elaborate antioxidant assays. The data for this project would be stronger if there are more variety of antioxidant assays and more different species of plant samples that can provide more support for the relationship between the phenolic contents and their respective antioxidant activities.

4.4 Screening of anticancer properties based on cell culture assays

4.4.1 Cell number optimisation

Cell optimisation was performed to estimate suitable cell numbers for seeding to avoid overgrowth of cells and at the same time give a good growth count for cells for analysis. The experiment mimics the growth of cells similar as to how cell plating was conducted in the cell culture assay, with incubation time of 72 h. Between 80 – 90% cell confluency was preferred as a 100% confluence would result in over reproduction of cells within the incubation time, resulting in erroneous results.

4.4.1.1 NPC/HK1 cell line

The viability of NPC/HK1 cells at different cell number between 2,500 to 5,000 cells per 100 L aliquot was plotted and shown in Figure 28 below. The chart shows that the optimum cell growth was at 2,500 cell count per well. Thus, for all NPC/HK1 cell seeding, this cell count was used for each well which had a cell viability of 85.86%.

Figure 28: The effect of NPC/HK1 cell count on cell viability in 100 L aliquot.

All values are expressed as mean ± standard deviation (n=2)

4.4.1.2 HaCaT cell line

The viability of HaCaT cells at different cell number was plotted and shown in Figure 29 below. As for HaCaT cells, because normal cells do not grow as fast as tumour cells, the cell count range used was higher i.e. from 3,500 to 6,000 cells per well. However, even at 3,500, the cell growth was 92.77% which is higher than the needed cell growth. Thus, from the experiment, it was decided that the cell count of 3,000 cells per well was enough for each well.

Figure 29: The effect of HaCaT cell count on cell viability in 100 L aliquot.

All values are expressed as mean ± standard deviation (n=2)

4.4.2 Cytotoxicity assay

All plant extracts have dose-response cytotoxic effects on the NPC/HK1 cancer cells. A sample of the effect of D. suffruticosa extract on NPC/HK1 cell viability was shown in Figure 30. HaCaT cells also showed similar response to D. suffruticosa, but at different concentration range (Figure 31). The concentration of plant extract at which only 50% of the cell viability was inhibited was termed as the half maximal inhibitory concentration, IC50. This value was used to compare the cytotoxicity effect of the test sample (extracts) on the cells.

D. suffruticosa

1 0 0

8 0

6 0

4 0

Cell Viability2 (% 0 ) IC 50 : 1 4 5 .3 0  14.64 mg/L

0 05001000150020002500 E x tra c t Concentration (mg/L)

Figure 30: The effect of D. suffruticosa on NPC/HK1 cell viability. All values are expressed as mean ± standard deviation (n=3)

D. suffruticosa

1 0 0

8 0

6 0

4 0

Cell Viability2 (% 0 ) IC 50 = 4 6 8 .2 0  22.00 mg/L

0 05001000150020002500 E x tra c t Concentration (mg/L)

Figure 31: The effect of D. suffruticosa on HaCaT cell viability All values are expressed as mean ± standard deviation (n=3)

The IC50 values of the plant extracts on both NPC/HK1 and HaCaT cells were summarised in Table 8: . The order of the plant extracts ability to cause cell death or cytotoxicity effect in NPC/HK1 cells is: E. polyantha var. b > D. suffruticosa > E. polyantha var. a. The order of cytotoxicity coincides with the order of antioxidant values represented earlier with E. polyantha var. a being the sample with the most antioxidant activity. This means that the level antioxidant values have a direct effect on the cytotoxicity of the samples as higher antioxidant capacity brings about higher cytotoxicity activity. However, having a high antioxidant capacity does not necessarily mean that a plant can be very toxic to cancer cells. The reason for this is because although the experiment shows that the plant samples does cause cytotoxicity to the cells however, the values are much higher than the marketed chemotherapy drugs which only requires microgram per litre to cause cytotoxicity.

Table 8: Summary of cytotoxicity effect plant extracts on cancerous call line, NPC/HK1 and normal cell line, HaCaT. Cytotoxicity Effect

IC50 value on IC50 value on HaCaT Sample NPC/HK1 cells Selectivity Index cells (mg/L) (mg/L) D. suffruticosa 145.3 ± 14.6a 468.2 ± 22.0a 3.22 E. polyantha var. a 896.4 ± 185.3b 2347.0 ± 2760.0a 2.62 E. polyantha var. b 61.5 ± 17.1c 597.6 ± 103.5a 9.72

The three samples were also tested for cytotoxicity against HaCaT cells which were normal human keratinocytes cells. The IC50 values on HaCaT cells were expected to be much higher than the IC50 values of NPC/HK1 if the extract was aimed to cause minimal cyctotoxicity effect on normal cells.

As observed in Table 9, IC50 values of the samples were much higher for the HaCaT cells when compared to NPC/HK1 cells. This shows that the cells required a much higher concentration of similar samples to exhibit cytotoxicity. However, for this part, E. polyantha var. a was the least cytotoxic sample on the normal cell (IC50 = 2347.0 ± 2760.0 mg/L), followed by E. polyantha var. b (IC50 = 597.6 ± 103.5 mg/L) and D. suffruticosa (IC50 = 468.0 ± 22.0 mg/L). The IC50 value for E. polyantha var. a had high standard deviation value due to the non-cytotoxicity effect of the extract on some of the replicate samples.

All three plant extracts also showed selective index (SI) values between 2.62 to 9.72. SI value is used to estimate the differential activity of a test compound, the higher the value is, the more selective it is. A value above 2.0 indicates that the compound is very selective and is a good candidate for cancer drug. A value less than 2 indicates that the compound has a general toxicity, and not suitable for cancer drug as it will kill normal cells as well. All the plant samples tested had SI values above 2.0, indicating that they are all very selective against the cancer cells (Koch et al. 2005).

Compared to work done on D. suffruticosa by Armania et al. (2013a), water extracts of different parts of the plants showed to have an IC50 value of more than 150 mg/L, which is higher than the D. suffruticosa sample used for the current project (145.3 ± 14.6 mg/L). However, the roots of the sample which was analysed by Armania et al. (2013a) showed better cytotoxicity effect on

MCF-7 cells (IC50 = 68.3 ± 5.8 mg/L) and on CaOV3 cells (101.3 ± 14.7 mg/L). The study also showed that different solvent fraction showed variable cytotoxicity effects, with ethyl acetate extract giving the most potent cytotoxicity on CaOV3 cells (IC50 = 12.3 ± 0.6 mg/L) and Hela cells (IC50 = 19.7 ± 0.6 mg/L) (Armania et al. 2013a).

Tor et al. (2014) had also studied ethyl acetate extract of the roots of D. suffruticosa and found the roots to have an IC50 value of 39.0 ± 3.6 mg/L after 72 h of incubation. The value was slightly higher than Armania et al. (2013a) which was 50.0 ± 8.0 mg/L but it still shows that the root is very cytotoxic to certain cancer cells especially when extracted using ethyl acetate.

Cisplatin is one of the common chemotherapy drugs used to treat nasopharyngeal and cervical cancer. Compared to cisplatin (IC50 = 16 mg/L), the E. polyantha var. b extract was 4 times less cytotoxic against cells (Wicaksono et al. (2013). HeLa cells exposed to E. polyantha extracts was shown to have an increase in the apoptosis mediator, caspase 3 and had also reduced HSP70 (Heat Shock Protein 70). Both are good targets in chemotherapy but when E. polyantha was combined with Piper crocatum, similar results were observed but not as good as when using E. polyantha alone (Wicaksono et al. 2013). In another study (Sanubol et al. (2017), P. crocatum was claimed to be cytotoxic against HeLa cells with an IC50 of 54,580 mg/L. However, when compared to the

E. polyantha var. b extract used in this project, the IC50 value of P. crocatum is much larger, thus E. polyantha seems to be a much more potent plant extract.

Strobilanthes crispa or also known as “pokok pecah beling” is locally used as a herbal medicine to treat diabetic patients. Cytotoxic test against CNE-1 (NPC cell line) of the leaves of the plant showed IC50 values of 119.0 ± 48.1 mg/L, 123.5 ± 37.5 mg/L and 161.7 ± 20.2 mg/L for ethyl acetate, hexane and chloroform extracts respectively and no cytotoxic activity was observed from

the water extract. Although the IC50 values were quite low, in comparison with D. suffruticosa, it seems that both are more potent than S. crispa as cytotoxic effect was observed from water extracts of the plants with E. polyantha var. b having an IC50 value almost 2 times lower than S. crispa (Koh et al. 2015).

Plants are observed to have effect on cancer cells and there is a possibility that plants can also be used in conjunction with the current chemotherapy drugs. A review done by Kim et al. (2015) showed how combining chemotherapy drugs and also consuming traditional herbal medicines have a positive effect on the cancer patient. Zhang et al. (2007) reviewed studies on Chinese medicinal plants and they found that patients who consumed the herbal medicines have a much more prolonged life expectancy compared to those who just took chemotherapy drugs on their own. Based on the cytotoxicity assay, D. suffruticosa and E. polyantha var. b can be a potential addition to cancer patients.

4.4.3 Cell migration assay - Scratch test

The scratch test assay is a cell culture assay that allows investigation of a sample ability to prevent the migration of cancer cells. The concentration used for the assay will depend after a cytotoxicity assay as it will allow determination of the IC25 value to be used for the migration assay. In this study, the concentration at IC25 was used because the concentration at IC50 would be too powerful to the cells and would cause loss of cells which would deter the migration assay. For the scratch assay, three different concentrations were used namely; IC25, IC12.5 and IC6.25. the respective concentrations were based on the IC50 value determined earlier (Table 8). Negative control (no plant extract added) serves as a reference for cell migration.

The results from the migration assay (Figures 32, 33 and 34) show that even at IC6.25, the effect from the plant samples were still evident as some cells were observed to be dead, especially at

IC25 concentration. Based on the figure comparisons between plants and negative control, the negative control shows that migration of cells did occur. Table 10 shows that, after 7 h, it can be seen, that the gap distance was been reduced to only 5.37 ± 4.51% with majority of the cells still observed to be in healthy condition. Then after 24 h the gap has been closed completely (0.00 ± 0.00%) with complete cell death still unobservable.

Table 9: Summary of migration effect of plants on cancerous cell line, NPC/HK1.

Migration Assay Concentration Plant sample (Gap Distance, %) (mg/ L) 7 h vs 0 h 24 h vs 0 h

Negative control* 0 5.37 ± 4.51 0.00 ± 0.00

D. suffruticosa** 18 55.05 ± 4.40a 0.00 ± 0.00a

36 78.67 ± 2.20b 67.69 ± 2.04b

72 94.51 ± 2.50c 89.28 ± 2.50c

E. polyantha var. a** 112 81.17 ± 5.46a 58.35 ± 2.53a

225 87.45 ± 2.77a 81.82 ± 2.59b

450 98.73 ± 1.81a 93.89 ± 3.05b

E. polyantha var. b** 7 28.16 ± 1.41a 0.00 ± 0.00***a

15 62.41 ± 4.22b 0.00 ± 0.00***a

30 78.86 ± 6.89b 57.27 ± 14.52b All values are expressed as mean ± standard deviation (negative control, n=3*; samples**; n=2). *** Data cannot be analysed because the values are both 0. Means in a column followed by different letters differ significantly (P < 0.05) as analysed using GraphPad t-test calculator.

Analysis of the samples’ migration assay (Table 10), shows that E. polyantha var. a being the superior plant followed by D. suffruticosa and E. polyantha var. b. E. polyantha var. a extract showed that at IC6.25, the gap distance did not close completely and 58.35 ± 2.53% of the gap was still open after 24 h of incubation as observed in Figure 33. Then at IC25, there was no significant reduction of gap distance between 7 h (98.73 ± 1.81%) and 24 h (93.89 ± 3.05%) of incubation. Subsequently, the lowest concentration of D. suffruticosa and E. polyantha var. b extracts, showed no effect on the cell migration activity as the gap completely closes after 24 h. However, the highest concentration of D. suffruticosa; i.e. IC25, did influence the migration activity of the as the gap was still at 89.28 ± 2.50% after 24 h and E. polyantha var. b stopped only at 57.27 ± 14.52%.

However, if the analysis was based on the figures, it would be said that E. polyantha var. a is the strongest in preventing migration of cells, followed by D. suffruticosa and E. polyantha var. b. The reason for this was because as seen in Figure 34(c) at the highest concentration of plant sample, some cells were still observed to be alive. This would imply that the properties present in

E. polyantha var. b works to kill cancer cells by preventing the cells from migrating thus preventing the cells from proliferating and metastasis. D. suffruticosa and E. polyantha var. a as observed in Figure 32(c) and 33(c), showed that at the cells were dead even at IC12.5. The cells were observed to have rounded off with no more nucleus present. Thus, the reason E. polyantha var. a and D. suffruticosa appeared to be very good at preventing migration was because cell death had occurred before the cell could even migrate to close the gap.

Rubus idaeus or better known as raspberries are known to be high in antioxidant activity. Extracts of R. idaeus were observed to have an effect in preventing migration of nasopharyngeal cancer cells (Hsin et al. 2017). The concentrations for R. idaeus to show prevention of migration of NPC- 39 cells were at 75 µg/mL and 100 µg/mL after 24 h, as well as a slight prevention at 50 µg/mL. Based on this, D. suffruticosa and E. polyantha var. b looks to be more potent than R. idaeus.

Koh et al. (2015) had also researched on the effect of CNE-1 NPC cell line and found that the stems of Strobilanthes crispa was able to prevent migration cells (49.4 µg/mL for hexane extract, 148.3 µg/mL for chloroform extract and 163.5 µg/mL for ethyl acetate extract). However, there was no effect with water extract of the plant thus showing that D. suffruticosa and E. polyantha were still more potent because prevention of migration was still observed using water extracts of the plants.

Sometimes extracts do not show any effective cytotoxic effect on cancer cells which warrants extra test to observe if the extract has other properties that can affect cancer cells. Thus, the cell migration assay was done to observe whether D. suffruticosa and E. polyantha extracts affect the migration cancer cells.

The statement rings true with miR-10b (MicroRNA-10b) which is said to influence cancer cells. Cytotoxic tests done using the miRNA showed no cytotoxic effect at all on nasopharyngeal cancer cells but the wound healing assay showed that miR-10b in fact increases the time for NPC cells to move (Sun et al. 2013). Afterwards, another miRNA which was miR-100 was found to be able to prevent nasopharyngeal cancer cells from migrating which may serve as a possible biomarker for future therapy (Sun et al. 2018). The researches depict that the expression of certain genes that might influence cancer cells which may prevent or facilitate metastasis. A study done by Yu et al. (2019) discussed that the presence of exosomal microRNA’s can be used as a biomarker to detect hepatocellular cancer cells migration to aid in the prognosis and can be used to predict the migration of cancer cells in the patients’ body.

To summarise out of the three plant samples, E. polyantha var. b shows the best results in preventing migration of the cancer cells, NPC/HK1. Although D. suffruticosa also shows good result in the scratch assay but as observed in Figure 30, the extract seems to just cause cytotoxicity of the cells where the cells were just killed. Thus, the concentration range used in the study (IC6.25

– IC25) was still too cytotoxic for the migration study. Lower concentrations should be used in the future if the effect of the D. suffruticosa extract on cell migration can be meaningfully studied.

Furthermore, since E. polyantha var. b had a lower IC50 value, the concentration of the extract that was able to prevent cell migration was much lower than D. suffruticosa and E. polyantha var. a.

4.5 Summary

Three indigenous plant samples were studied in this study; D. suffruticosa, E. polyantha (var. a and b). The water extracts of the plants were subjected to polyphenol content assays, antioxidant assays, cytotoxic assay and migration assay.

Out of the three, E. polyantha var. b shows to be the most promising plant. The plant showed the highest polyphenol contents and in the antioxidant assay, E. polyantha var. b gave higher antioxidant activity readings (EC50 = 60.9 ± 15.5 mg/L for DPPH and EC50 = 24.15 ± 1.34 mg/L for ABTS). The high antioxidant activity present in the plant probably contributes to the cytotoxic activity it demonstrated on the nasopharyngeal cancer cell, NPC/HK1. Cytotoxicity assay showed that E. polyantha var. b had the lowest IC50 value which was 61.5 ± 17.1 mg/L followed by D. suffruticosa (145.3 ± 14.6 mg/L) and then E. polyantha var. a (896.4 ± 185.3 mg/L). The selectivity index of the plant samples also showed that E. polyantha var. b to be the most selective towards cancer cells which was 9.72 whereas D. suffruticosa and E. polyantha var. a had selectivity index of 3.22 and 2.62 respectively. Lastly in the migration assay, E. polyantha var. b extracts were observed to be able to slow down the migration of NPC/HK1 cancer cells at a lower concentration than D. suffruticosa and E. polyantha var. a.

5 CONCLUSION

Three aqueous plant samples were used in this project namely, D. suffruticosa and E. polyantha (var. a and b). The first objective was to estimate the polyphenolic contents and in vitro antioxidant activities in the plants using colorimetric-based assays. Colorimetric assays are the easiest, fastest and cheapest ways to screen plant extracts. The polyphenolic contents of the plant extracts used in this study was determined using the total phenolic content assay (TPC) and the total flavonoid assay (TFC). From these assays, a general quantification of the plant compounds was able to be determined which might contribute to their antioxidant and cytotoxic activities. Following the polyphenolic content assay, the antioxidant assays showed that the plants can produce some significant antioxidant activities which might contribute to their cytotoxic activity as well. Following the TPC, TFC, ABTS and DPPH assays, E. polyantha var. b was the best extract in terms of phenolic contents and antioxidant activity (TPC = 6.62 ± 1.6 mg GAE/100 mg

DW; TFC = 0.25 ± 0.33 mg QE/100 mg DW (DPPH EC50 = 60.9 ± 15.5; ABTS EC50 = 24.2 ± 1.3) followed by D. suffruticosa (TPC = 3.70 ± 0.69 mg GAE/100 mg DW; TFC = 0.45 ± 0.79 mg QE/ 100 mg DW (DPPH EC50 = 217.6 ± 16.4 mg/L; ABTS EC50 = 42.4 ± 3.7 mg/L) and E. polyantha var. a (TPC = 1.58 ± 0.44 mg GAE/100 mg DW; TFC = 0.10 ± 0.03 mg QE/100 mg

DW (DPPH EC50 = 265.4 ± 24.6 mg/L; ABTS EC50 = 99.4 ± 12.7 mg/L). Results from the study shows that polyphenolic content does play a significant role in giving plants their antioxidant activity. These plants may contain terpenoids such as betulinic acid and retinol; flavonoids such as quercetin; and alkaloids such as taxol that have been shown to possess antioxidant activities (Section 2.3 Plant Secondary Metabolites).

The second objective was to assess the cytotoxicity effect of the plant extracts on normal keratinocyte HaCaT cells. Cytotoxicity assay was determined for normal cells because it is good to compare how toxic are the plant extracts on normal non-cancerous cells as they are on cancer cells. The purpose of this assay was to make sure that the plant extracts do not harm the other cells in the human body. From the assay, the concentration to able to produce cytotoxic activity on HaCaT cells were very high (IC50 = 2347.0 ± 2760.0 mg/L, 597.0 ± 103.5 mg/L and 468.2 ± 22.0 mg/L for E. polyantha var. a then E. polyantha var.b and D. suffruticosa, respectively) which shows that the plants extracts would not cause any harms to other cells other than on cancer cells.

This study had successfully completed the last objective of this project which was to assess the cytotoxicity effect of the plant extracts against NPC/HK1 cells. Comparing plant samples used for this study and other plants studied elsewhere, the aqueous samples of plants used in this study had lower IC50 values (E. polyantha var b IC50 = mg/L; D. suffruticosa IC50 = mg/L; E. polyantha

var a IC50 = mg/L). The IC50 values determined in this study might only can be applied to NPC/HK1 cells but the values were considerably on the lower side which is desired for anticancer studies. The cell migration assay had showed that the plant extracts influenced cell migration on the cancer cells at half of the determined IC50 values of the cytotoxicity test. Thus, it can be said that the plant extracts had observable effects on nasopharyngeal cancer cells which could pave a way for more studies using D. suffruticosa and E. polyantha as suggested in the following section.

5.1 Further work

All the antioxidant tests as well cell-based tests in this study were in vitro assays. Therefore, the results are not definite yet as in living organisms, bioavailability and responses in complex system can be different. Onoja et al. (2014) studied the methanolic extract of Aframomum melegueta seed native to Africa using both in vitro DPPH and rats for in vivo studies. The study enabled the researchers to observe the effects of the sample in a living organism which may allow new breakthroughs in field. As observed in this project, E. polyantha seems to have high potential of antioxidants and cytotoxic effect and it would be interesting to observe if it has any effect in vivo. If successful, it will bring light to Sarawak with its wide variety of flora and fauna and possibly a lot to offer to the medicinal field.

Another concern, especially in cancer patients is having to go through chemotherapy. This is because there are so many side effects that comes when doing chemotherapy, as the immune systems gets very low which causes the patients to be very susceptible to immune system attacks. The possibility of using plants in cancer therapy along with chemotherapy drugs should be further investigated as it may reduce the cruciality of the side effects. This might be possible as the plants used in this study were not cytotoxic to the normal keratinocyte HaCaT cells. Mohan et al. (2013) had also studied the possibility of doing plant extracts and chemotherapy drugs combinations to reduce chemotherapy side effects.

Additionally, other than DPPH and ABTS, the samples can be subjected to many other antioxidant tests for example; CUPRAC assay (2.6.3), FRAP assay (2.6.4) or ORAC assay (2.6.5) where further antioxidant activity affected by other compounds in the plants can be determined. Alam, Bristi and Rafiquzzaman (2013) have compiled many different types of antioxidant assays in vitro and in vivo that can be done depending on the studies and budget. Other than antioxidant tests, further antibacterial tests can also be done on the samples as previously, antibacterial test was performed for the samples, but the results were less than satisfactory and there were no results

from the test. Thus, other bacteria-based assays can be performed using the samples to observe any beneficial antibacterial properties.

As for cell-based assay, there are also a wide variety of assay that can done to study the effects of the sample further. Researches that are involved in cell-based assay can use the cytotoxicity test as their preliminary test before going into other more specific tests. In this project, the cells were subjected to a migration test using the scratch migration assay. Other tests that can done are like, cell oxidative stress assay to observe the cells under the presence of reactive oxygen species or senescence assay that is linked aging. Senescence usually happens when normal cells stop dividing which is highly linked to telomere shortening that brings about aging in a person (Wang & Dreesen 2018). The plant sample in this project can be used to see whether it can reverse senescence of the cells which may help to create new anti-aging products.

There are also cell-based protein assays like the western blot or SDS-PAGE assays that can done. Protein assays allow confirmation of the proteins activated or affected by the addition of samples. These allow determination of the presence of specific proteins like caspases or Bcl-2 which causes cell death or apoptosis of cells. Apoptosis assays are also good for anticancer studies as it can give researchers an idea of how the sample affect the cell to bring about cell death. The assay also allows determination on what genes are impacted during the cell reproduction after addition of the sample. Identifying specific bioactive compounds in this study would be beneficial and would also strengthen the impact of the study.

In conclusion, the study of plants and their benefits can go through many different branches. Therefore, many researchers especially in the medicinal field are interested in plants as there are still many things that we can learn from plants. In this world of fast paced evolution, new developments are always needed and appreciated.

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