Hystrix brachyura bezoar (PB) has been traditionally used as an alternative medicine to treat cancer. However the anticancer effect of PB is yet to be explored. Therefore this study aims to evaluate PB anticancer potential using metabolomics approach and molecular docking. Four different PBs namely PB-A, PB-B, PB-C and PB-D were procured and used in this study to evaluate the anticancer effect. All PBs were treated with water through ultrasonication-assisted extraction technique to obtain aqueous extracts. The melanoma cells (A375) were chosen for the evaluation of further anticancer effects. A375 cells were subjected to cell proliferation, colony formation, apoptosis, cell cycle arrest, cell migration, cell invasion assays followed by molecular pathways using real time polymerase chain reaction (qPCR) and in vivo antiangiogenesis using zebrafish (Danio rerio) larvae. Additionally, all four PBs were also evaluated for their toxicity effect using zebrafish embryos. The anticancer compounds of PB were putatively identified using liquid chromatography–mass spectrometry (LC-MS) and liquid chromatography–mass spectrometry (LC-MS) based metabolomics approaches. All the compounds identified were then docked to BCL-2, cyclin B/CDK1 complex, VEGF and NM23 crystal structures to predict the ligand-protein interaction. Lastly, the study also developed a validated regression model using Fourier transform infra-red (FT-IR) spectroscopy to predict the anticancer activity of new PB extract to ensure its quality as an effective anticancer agent. All PB extracts exhibited significant cytotoxicity on A375 cells. Further analysis revealed that PB-A, PB-C and PB-D had a good inhibitory effect on cell proliferation, colony formation, cell cycle, cell migration, cell invasion, angiogenesis and apoptosis inducer. PB-A, PB-C and PB-D revealed apoptosis through intrinsic pathway, arresting the cell cycle at G2/M phase by downregulating cyclin B and CDK1. PB-A, PB-C and PB-D at molecular level were showed to suppress NM23, E-cadherin, MMP2 and MMP9. The toxicity assessment showed the morphological developmental defects caused by all four PB extracts such as deformed brain section, contorted backbone with deformities in somites and notochord, deformities in soft tissues (yolk sac, pericardial edema and swim bladder). Additionally, the PB extracts were showed to affect the cardiovascular systems via presence of heart edema, downregulating the heart rate and blood flow. The LC50 values at 96 hpf were <100 µg/mL. Chemical profiling analyses of PB extracts through GC-MS and LC-MS based metabolomics approached identified 4-androsten-4-ol-3,17-dione, acetate cholest-7-en-3-ol, gallic acid, isolongifolol, mangiferin and propafenone as the active principles. The docking results predicted the interaction of the active principles with BCL-2, cyclin B/CDK1 complex, VEGF and NM23 majorly via hydrophobic interaction with protein residues. The docking results showed good binding affinity to the crystal structures of BCL-2, cyclin B/CDK1 complex, VEGF and NM23 for 4-androsten-4-ol-3,17-dione, cholest-7-en-3beta-ol,4,4-dimethyl-, acetate and mangiferin, suggesting the roles of these compounds as the potential anticancer agents. Moreover, the study has also generated a validated statistical model to predict the anticancer activity of new PB extracts. Conclusively, the study has revealed the anticancer activity of Hystrix brachyura bezoar, active principles, in vitro mechanism of action and in vivo toxicity effect using zebrafish model for the first time. Results of this study further support the traditional claims for the use of Hystrix brachyura bezoar as an anticancer agent in Malaysia.

Cancer is a major cause of morbidity and mortality worldwide. In recent biomedical researches, the areas of cancer and infectious diseases have a leading position in utilization of medicinal plants as a source of drug discovery. Malaysia has a diversity and large quantity of underutilized fruits which are rich in phenolic compounds. The objective of study one was projected in vitro to explore natural sources of antioxidant in Artocarpus.altilis (breadfruit) extracts and antioxidant properties. The total phenolic content (TPC) was measured using the Folin-Ciocalteau method and the total flavonoid content (TFC) was determined by using aluminium chloride colorimetric method. Antioxidant properties were determined via the DPPH radical scavenging and β–carotene bleaching (βCB) assays. The various fruits parts Pulp (PU), peel (PE) and whole fruit (WF) were extracted with various solvents such as hexane, dichloromethane (DCM) and methanol. The methanol extracts obtained the highest yields among other solvents (hexane and DCM). The pulp (edible portion) had the highest yield (p<0.01). Methanol extract of pulp part revealed the highest total phenol and flavonoid content value of 781±17.32 mg (GAE)/g and 6213.33±82.24 mg (QE)/g of dry sample, respectively. IC50 values of methanol extract of pulp part in DPPH radical were obtained to be 0.05±0.00 mg/mL as compared to positive control (ascorbic acid) 0.06±0.00 and the antioxidant activity for the β-carotene bleaching assay was 88.34±0.75% of methanol extract of pulp part as compared to the positive control (Trolox) 90.02±0.87%. The objective of study two was to identify and quantify some phenolic compounds in the methanol extracts. By using the ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC/MSMS) based approach, a total of 9 compounds were detected and characterized on the basis of their chromatographic retention time, UV-vis spectra and mass spectra in the negative-ion mode and data from the literature. The results of the various parts of A. altilis fruit extracts showed promising antioxidant and potential bioactivities due to the high content of phenolic compounds. The purpose of the study three was to evaluate the cytotoxicity effects of methanol fruit extracts on four human cancer (A375, MCF-7, A549, and HT-29) cell lines. The IC50 of the samples were measured using trypan blue exclusion assay (TBEA). The methanol extract of pulp part showed the least inhibition concentration of 15.40±0.91 μg/mL on A375 cells. In the study four, the molecular mechanism of methanol extracts-induced apoptosis and cell cycle arrested in human cancer cells were investigated in a time dependent approach by using flow cytometry. The treated cells were stained with nexin to detect early and late apoptosis and with PI for cell cycle arrest associated with the DNA fragmentation, various cells arrests were occurred at G1/S, S and G2/M phases. Lastly the gene expression analysis by reverse transcription quantitative PCR (qPCR) method was carried out by analysing the expression of gene of interest for quantification of mRNA levels. Results after cells treated with IC50 were revealed by upregulating of anti-apoptotic genes/downregulated of pro-apoptotic BCL-2 gene expressions were triggered the treated cells into CASPASE-3, intrinsic and extrinsic pathways. These findings suggest that the methanol extracts of three parts of A. altilis fruit have potential chemotherapeutic activity against human cancer cell lines mainly the pulp part of the fruit.