Metabolomic profile and Bioassay-guided Phytochemical analysis of the Stems from Cissus trifoliata, evaluation of their Antibacterial and Cytotoxic activity, and determination of the Mechanism of Action of one active compound

Méndez López, Luis Fernando (2020) Metabolomic profile and Bioassay-guided Phytochemical analysis of the Stems from Cissus trifoliata, evaluation of their Antibacterial and Cytotoxic activity, and determination of the Mechanism of Action of one active compound. Doctorado thesis, Universidad Autónoma de Nuevo León.

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Resumen

Objectives and methods: Bioprospecting the metabolic profile of medicinal plants has provided a reliable resource for drug discovery and advancements in biomedical research. Cissus trifoliata (L.) L belongs to the Vitaceae family and is an important medicinal plant used in Mexico for the management of infectious diseases and tumors. The present study aimed to identify the metabolic profile of the extracts from the stems of C. trifoliata and evaluate their antibacterial and cytotoxic activities. Additionally, to identify the molecules that contribute to their biological activity and to explore the mechanism of action of one active compound. The hexane, CHCl3-MeOH, and aqueous extracts were prepared from the stems of C. trifoliata and their metabolic profile was investigated by column chromatography, NMR, GC-MS, and LC-MS. The antibacterial activity was determined by the broth microdilution method and the cytotoxicity against cancer cell lines using the MTS proliferation assay. A bioassay-guided study of the CHCl3-MeOH extract was performed using WST-1 to identify the active constituents responsible for the antiproliferative effects against cancer cells. Additionally, microarrays were used to identify the mechanism of action of one active compound against prostate cancer cells. Contribution and conclusions: The metabolic profile of C. trifoliata stems was constituted of polyphenols (36%), terpenes (28%), fatty acids (18%), simple phenols (9%) and alkanes (9%). The pathway analysis indicated the high production of stilbenes, flavonoids, and sterols. The extracts showed no antibacterial activity (MIC > 500 µg/ml), but high cytotoxic effects against cancer cells (IC50 ≤ 30 µg/ml). The hexane and aqueous extracts showed high antiproliferative activity against cancer cells from the liver (Hep3B, HepG2) and breast (MCF7). The bioactivity of these extracts was related to the synergistic effect of the triterpenes, sterols, flavonols, and stilbenes with cytotoxic, antiproliferative, and antiestrogenic activities. The bioassay-guided study of the CHCl3-MeOH extract allowed the identification of two active fractions. Both showed significative reduction (p >0.05) of cell viability on PC3 and MCF7 cancer cells at a concentration of 100 µg/ml. The cytotoxic activity was related to the synergistic anticancer effects of the mixture of coumaric acid, kaempferol, apigenin, hydroxyursolic, ursolic, and betulinic acid plus the stilbenes resveratrol, piceatannol, and viniferin. The stilbene that characterizes the Vitaceae plants is the resveratrol, thus was selected for the study of the mechanism of action. To carry out the microarray assay, PC3 cells were exposed to a non-cytotoxic inhibitory concentration (IC25) of resveratrol. Results showed that this phenolic compound induced significative transcriptional changes (2-fold) in 847 genes. The functional analysis suggested that resveratrol influences differentiation and impair cancer stemness by induction of the transcription factors POU4F2, KLF14, Hox-A3, and repression of Nanog. Resveratrol also affected cellular metabolism by upregulation of SIRT5 and repressed genes of the cancer pathways of TGF-β, Notch, PI3K/Akt, insulin/IGF-1, and MDM4/p53. Overall, the metabolic profile and biological evaluation of the stems from C. trifoliata correlate with high anticancer activity and together with the molecular mechanism of the identified bioactive constituents explain its traditional use in the management of tumors.

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