Metabolomic and biodirected phytochemical study of Hechtia glomerata, evaluation of its antibacterial and cytotoxic activity, and determination of the mechanism of action of one active compound

Stefani, Tommaso (2020) Metabolomic and biodirected phytochemical study of Hechtia glomerata, evaluation of its 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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Purpose of the study: The pharmaceutical industry has left the active research and development of new antibiotics, and the lack of new molecules caused the antibacterial resistance to become an even more threatening issue. At the same time cancer resistance is also thriving, making old chemotherapeutic drugs useless. Therefore, there is the absolute need of new bioactive molecules to contrast these pathologies. Plants’ natural products have always been a source for bioactive and structurally complex compounds and could represent a solution to these emerging health problems. The majority of the world still relies on the use of ethnomedicine for the treatment and prevention of diseases. This is why in this work a Mexican medicinal plant: Hechtia glomerata Zucc. known as Guapilla was investigated for its curative properties. The goal of this study was to find out the actual antibacterial and cytotoxic activity of this plant’s extracts and its adequacy as source for biologically active compounds. Also, to isolate its major compounds and deeply investigate its secondary metabolites production. Finally, the mechanism of action of one of its bioactive constituents was investigated by microarray assay. Results and conclusions: The major constituents of H. glomerata’s leaf organic extracts (Hex and C/M) were isolated and characterized. Also, a metabolomic study on all H. glomerata’s leaf extracts was performed by UPLC-QTOF-MS analysis. This allowed the identification of the chemical profile of the plant accounting for a total of 226 compounds comprising a 38% of flavonoids and stilbenes, 31% of terpenes and steroids, a 15% of phenolic acids and derivatives, 10% of fatty acids and hydrocarbons, 6% of nitrogen containing compounds. The statistical analysis of the date permitted the visualization of the main differences among the extracts. It was also performed a pathway analysis showing which ones are the most predominant biosynthetic routes of H. glomerata. The antibacterial and cytotoxic activity of both leaf and root extracts was investigated. The MIC values for the extracts against resistant bacteria were ³ 500 µg/mL and were considered weak. Meanwhile of the isolated compounds only β-sitosteryl acetate had a MIC value of 100 µg/mL against carbapenem resistant Acinetobacter baumannii. The CHCl3/MeOH leaf extract had instead activity against two sensitive bacterial strains: Staphylococcus aureus and Enterobacter faecium with MIC values of 125 and 62.5 µg/mL respectively. Therefore, a bioassay-guided directed study was performed, testing against these two bacteria the fractions of CHCl3/MeOH extract. Fractions 1, 3 and 8 resulted to be the active ones, the most active being the fraction 8 with a MIC of 100 and 50 µg/mL against S. aureus and E. faecium respectively. The three active fractions were analyzed in order to identify their active components with GC-MS and/or HPLC-QTOF-MS. The extracts were also tested against cancer cells and only the organic leaf extracts resulted active (IC50: 24-32 µg/mL). The most active was the Hex extract which accounted for the higher number in cell lines inhibited. These results highlighted the lack in specificity of this extract and also its major constituent (β-sitosterol) was already widely studied in the literature. For these reasons it was decided to focus on the CHCl3/MeOH extract, which presented activity against PC3 and was slightly active against MCF7 cell lines. A bioassay-guided directed study was also conducted on this extract showing fractions 3 and 4 as the most active ones. Hence their constituents were investigated giving a high similarity in their composition, especially they shared many hydroxycinnamic acid derivatives like p-coumaric and caffeic acid. Since these two compounds were found so abundant and were already isolated and characterized in the same extract, it was decided to test them against the two cancer cell lines. For the stability in culture medium of p-coumaric acid was selected for the microarray assay on PC3 cells. The results gave the up- and down-regulation of many genes involved in cancer, some of them specific for prostate cancer (PI3K/AKT, AMPK, MAPK, p53, RAS, and TNF among others). Some proteins of the pathways affected by pcoumaric acid treatment were modeled in silico and their probable binding sites identified by molecular docking. Finally, the complex ligand-proteins were submitted to molecular dynamic simulation and their binding energy calculated. The interactions leading to a negative binding energy confirm the spontaneous binding of the p-coumaric acid to its targets: MAPK8, MDM2, CCNB2, NTRK3, SLC2A4, MDM4 and the protein complex STK11-STRADCAB39.

Tipo de elemento: Tesis (Doctorado)
Información adicional: Doctor en Ciencias con Orientación en Farmacia
Materias: Q Ciencia > QD Química
R Medicina > RS Farmacia y Materia Médica
Divisiones: Ciencias Químicas
Usuario depositante: Editor Repositorio
Fecha del depósito: 14 Oct 2020 12:46
Última modificación: 29 Ene 2021 23:59

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