Methods: Chemical profiling of P. blanda was carried out using gas chromatography mass spectrometry (GCMS) followed by isolation of bioactive compounds by column chromatography. DPPH• and FRAP assays were used to evaluate antioxidant activity and the MTT assay was performed to estimate the cytotoxicity activity against three cancer cell lines, namely MCF-7, HL-60 and WEHI-3, and three normal cell lines, MCF10A, WRL-68 and HDFa.
Results: X-ray crystallographic data for peperomin A is reported for the first time here and N,N'-diphenethyloxamide was isolated for the first time from Peperomia blanda. Methanol and dichloromethane extracts showed high radical scavenging activity with an IC50 of 36.81 ± 0.09 µg/mL, followed by the dichloromethane extract at 61.78 ± 0.02 µg/mL, whereas the weak ferric reducing activity of P. blanda extracts ranging from 162.2 ± 0.80 to 381.5 ± 1.31 µg/mL were recorded. In addition, petroleum ether crude extract exhibited the highest cytotoxic activity against all the tested cancer cell lines with IC50 values of 9.54 ± 0.30, 4.30 ± 0.90 and 5.39 ± 0.34 µg/mL, respectively. Peperomin A and the isolated mixture of phytosterol (stigmasterol and β-sitosterol) exhibited cytotoxic activity against MCF-7 and WE-HI cell lines with an IC50 of (5.58 ± 0.47, 4.62 ± 0.03 µg/mL) and (8.94 ± 0.05, 9.84 ± 0.61 µg/mL), respectively, compared to a standard drug, taxol, that has IC50 values of 3.56 ± 0.34 and 1.90 ± 0.9 µg/mL, respectively.
Conclusion: The activities of P. blanda extracts and isolated compounds recorded in this study underlines the potential that makes this plant a valuable source for further study on anticancer and antioxidant activities.
METHODS: α-Mangostin (AM) was isolated from C. arborescens and its cell death mechanism was investigated. AM-induced cytotoxicity was observed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Acridine orange/propidium iodide staining and annexin V were used to detect cells in early phases of apoptosis. High-content screening was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential, and cytochrome c release. The role of caspases-3/7, -8, and -9, reactive oxygen species, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. To determine the role of the central apoptosis-related proteins, a protein array followed by immunoblot analysis was conducted. Moreover, the involvement of nuclear factor-kappa B (NF-κB) was also analyzed.
RESULTS: Apoptosis was confirmed by the apoptotic cells stained with annexin V and increase in chromatin condensation in nucleus. Treatment of cells with AM promoted cell death-transducing signals that reduced MMP by downregulation of Bcl-2 and upregulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase-9 followed by the executioner caspase-3/7 and then cleaved the PARP protein. Increase of caspase-8 showed the involvement of extrinsic pathway. AM treatment significantly arrested the cells at the S phase (P<0.05) concomitant with an increase in reactive oxygen species. The protein array and Western blotting demonstrated the expression of HSP70. Moreover, AM significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus.
CONCLUSION: Together, the results demonstrate that the AM isolated from C. arborescens inhibited the proliferation of MDA-MB-231 cells, leading to cell cycle arrest and programmed cell death, which was suggested to occur through both the extrinsic and intrinsic apoptosis pathways with involvement of the NF-κB and HSP70 signaling pathways.