Z. Adam, M. Z.A. Mohamed Amiroudine, D. Jesus Arapoc and S. Khamis Pharmaceutical Technology Group, Medical Technology Division, Malaysian Nuclear Agency, Bangi 43000 Kajang,

INTRODUCTION METHODOLOGY Cancer remains major global health problem even though there are lots of anti-cancer drugs available. This could possibly be due to the B. Macrophylla were oven dried at 45°C and ground to powder form problems associated with the drugs, such as in-effective of the drugs and adverse effects. Therefore, research and development of Sequentially extracted with hexane, chloroform, methanol, and distilled water new anti-cancer agents with high efficacy and less adverse effects still need to be done. Bouea macrophylla Griff or locally known as Supernatant was filtered, evaporated to dryness, weighed and stored at -20ºC is one of the common fruit available in . This plant, from Anacardiaceae family is native to Southeast Asia. Our previous study showed that B. Macrophylla possess strong anti- The extracts were subjected to different test as below oxidant activity and contains high amount of phenolic compounds. Therefore, there is possibility that B. Macrophylla may possess anti- Cytotoxic evaluation Apoptosis determination cancer property. Cells were incubated with B. macrophylla Cells were incubated with B. (0-250 µg/ml) for 72 hours Macrophylla (IC50) for 24 hours OBJETIVE MTT assay Incubated with Hoechst 33342 (4 µg/m) for 30 min

To evaluate anti-cancer activity of B. Determination of IC50 value using macrophylla extracts by using Cell Reporter Enspire® Multimode Plate Reader Determination of apoptotic cells software using Cell Reporter software System and EnSpire Multimode Plate Reader. Bouea macrophylla Griff Detection modes: Applications: .Absorbance .TRF assay Applications: .Fluorescence .Enzyme assays . Apoptosis study .Lipid accumulation .ALPHA-LISA .Alpha technology .Cell cycle analysis .Hormone secretion .Label-free .Receptor-ligand .Cell differentiation .Cell proliferation .TRF .ETC .Cell viability assay .Cyto-toxicity

.Mitotic index EnSpire Multimode Plate Cell Reporter System Reader RESULTS AND DISCUSSION

Anti-proliferative activity of B. macrophylla extracts Apoptotic activity of B. macrophylla extracts

Anti-proliferative activity was determined through IC50 value. Low IC50 Apoptosis or programmed cell death is the major cell death mechanism value indicates strong anti-proliferative activity. Hexane extract exhibited induced by anti-cancer agents. It is characterized by membrane modest anti-proliferative activity against breast cancer cells, MDA-MB- blebbing, DNA condensation and nuclear fragmentation. In this study, B. 231. Meanwhile, aqueous, methanolic and chloroform extracts showed macrophylla induce apoptosis in HTB43 cells after 24-hours treatment. strong anti-proliferative activity against head and neck cancer cells, Apoptotic cells were recognised through the formation of bright signal of HTB43 cells. Among three extracts, methanolic extract showed the condensed DNA in DAPI channel after incubated with a fluorescent highest anti-proliferative activity, followed by chloroform and aqueous nucleus stain, Hoechst 33342. It was shown that methanolic extract extract. induced apoptosis the most, followed by chloroform and aqueous extract. Amount of apoptotic cells were 25.31 ± 1.38, 47.55 ± 4.15 and Cytotoxic activity of B. macrophylla seed extracts 33.76 ± 8.17 %, respectively.

IC50 values (µg/ml) Extracts HTB43 MDA-MB-231 Hexane >250 123.35±28.65 Chloroform 21.14 ± 6.97 >250 Methanolic 18.65± 2.94 >250 Aqueous 29.32 ± 5.80 >250

IC50 value was determined through a non-linear regression curve constructed using EnSpire Multimode Plate Reader software (Version 4.1) a b c IC50 value is the concentration of extracts that kill 50% of the cells Images of DNA condensation in HTB43 cells treated with aqueous (a), methanolic population. In this study, IC50 value was determine through a non-linear (b) and chloroform (c) extracts of B. macrophylla seed. regression curve constructed in EnSpire Multimode Plate Reader software version 4.1. Non- Apoptotic apop- Histogram plot Plate overview illustrates totic showed distribution of apoptotic distribution of cells and non-apoptotic total nuclear cells. The number of intensities in cells per well is the DAPI proportional to pie chart channel. diameter

Apoptotic cells in HTB43 population were determined using Cell Reporter System software. A histogram plot of total nuclear intensities Non-linear regression curve Heat map images showed was constructed and two populations of cell were gated, non-apoptotic showed the IC50 value the raw results and apoptotic. The results were also displayed in a plate overview feature, which illustrates the total number of cells per well and the CONCLUSION proportion of apoptotic cells per well. The results from this study showed that B. macrophylla possess strong anti-proliferative and apoptosis induction activity. Based ACKNOWLEDGEMENT on the positive results, we suggest that anti-cancer studies are feasible to be done by using Cell Reporter System and EnSpire The authors wish to thank Malaysian Nuclear Agency for providing Multimode Plate Reader. necessary funding and facilities to conduct this study.