Inhibition of the norA gene expression and the NorA efflux pump by the tannic acid.

The NorA efflux pump of Staphylococcus aureus is known to play a major role in the development of resistance against quinolone drugs by reducing their concentration inside target pathogens. The objective of this study was to evaluate the ability of tannic acid to inhibit the gene expression of the NorA efflux pump in Staphylococcus aureus and to evaluate the in silico effect on the pump. Efflux pump inhibition was evaluated by fluorimetry. The checkerboard method evaluates the effect of the test substance in combination with an antimicrobial at different concentrations. To gene expression evaluation NorA the assay was performed using: a sub-inhibitory concentration preparation (MIC/4) of the antibiotic; a sub-inhibitory concentration preparation (MIC/4) of the antibiotic associated with tannic acid at a sub-inhibitory concentration (MIC/4). In this study, docking simulations were performed by the SWISSDOCK webserver. The ability of tannic acid to inhibit the NorA efflux pump can be related to both the ability to inhibit the gene expression of this protein, acting on signaling pathways involving the ArlRS membrane sensor. As well as acting directly through direct interaction with the NorA protein, as seen in the approach and in silico and in vitro per checkerboard method and fluorimetry of bromide accumulated in the cell.

In vitro and in silico inhibitory effects of synthetic and natural eugenol derivatives against the NorA efflux pump in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium responsible for a number of diseases and has demonstrated resistance to conventional antibiotics. This study aimed to evaluate the antibacterial activity of eugenol and its derivatives allylbenzene, 4-allylanisole, isoeugenol and 4-allyl-2,6-dimethoxyphenol against the S. aureus NorA efflux pump (EP) in association with norfloxacin and ethidium bromide. The antibacterial activity of the compounds was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC). A reduction in the MIC of ethidium bromide (a substrate for several efflux pumps) or norfloxacin was used as a parameter of EP inhibition. Molecular modeling studies were used to predict the 3D structure and analyze the interaction of selected compounds with the binding pocket of the NorA efflux pump. Except for 4-allylanisole and allylbenzene, the compounds presented clinically effective antibacterial activity. When associated with norfloxacin against the SA 1199B strain, 4-allyl-2,6-dimethoxyphenol eugenol and isoeugenol caused significant reduction in the MIC of the antibiotic, demonstrating synergistic effects. Similar effects were observed when 4-allyl-2,6-dimethoxyphenol, allylbenzene and isoeugenol were associated with ethidium bromide. Together, these findings indicate a potential inhibition of the NorA pump by eugenol and its derivatives. This in vitro evidence was corroborated by docking results demonstrating favorable interactions between 4-allyl-2,6-dimetoxypheno and the NorA pump mediated by hydrogen bonds and hydrophobic interactions. In conclusion, eugenol derivatives have the potential to be used in antibacterial drug development in strains carrying the NorA efflux pump.

Astronium fraxinifolium Schott Exerts Leishmanicidal Activity by Providing a Classically Polarized Profile in Infected Macrophages.

BACKGROUND: Leishmania braziliensis is prevalent in Latin American countries, including Brazil. It causes cutaneous and mucocutaneous leishmaniasis, leading to high morbidity, and has a low cure rate. Treatment is based on pentavalent antimonials; nonetheless, there are problems related to high toxicity, high cost, and parasitic resistance. Discovery of new leishmanicidal drugs without these limitations and that stimulate the cellular immune response is necessary. PURPOSE: The present work evaluates whether Astronium fraxinifolium Schott exerts leishmanicidal activity against L. braziliensis by providing a classically polarized profile in infected macrophages. METHODS: For the evaluation of the A. fraxinifolium Schott leishmanicidal activity, amastigote cell death was demonstrated in infected RAW 267.4 macrophages treated with an ethanolic extract from the plant sapwood (EEAF). For the evaluation of the EEAF capacity in providing a classically polarized profile in infected macrophages, the following analyses were done: detection of LAMP-1 protein by the baculovirus technology, measurement of superoxide anion by the NBT testing, quantification of TNF-α, IL-12p40, IL-10, IL-4, and TGF-ß by sandwich-type enzyme immune assays, and iNOS and COX-2 expression by RT-PCR technique. RESULTS: The EEAF significantly reduced amastigote counts inside the cells. Vacuoles were visualized in infected and treated cells before and after May-Grünwald-Giemsa staining. A strong LAMP-1 protein fluorescence revealed phagosome maturation in infected cells treated with the EEAF. No production of superoxide was visualized in infected cells treated with the plant material. Nonetheless, high levels of TNF-α, IL-12p40, and IL-10 were found in cell supernatants, but reduced levels of TGF-ß and no IL-4 production. We identified augmented mRNA expression for COX-2, but no expression of iNOS mRNA. CONCLUSION: Our results demonstrated that A. fraxinifolium induced a classically polarized profile in infected macrophages but also provided a less harmful environment by stimulating the production of certain anti-inflammatory mediators, such as IL-10.

Photoprotective potential of medicinal plants from Cerrado biome (Brazil) in relation to phenolic content and antioxidant activity.

Photooxidative damage affects cellular lipids, proteins and DNA in addition to being involved in the pathobiochemistry of erythema formation, premature skin aging, photodermatoses development and skin cancer. Phenolic compounds, flavonoids and hydroxycinnamic acid esters protect plant tissues against harmful UV-radiation. This study aimed to evaluate the sun protection factor of several Brazilian plant extracts in relation to UVB radiation absorption, which causes skin cancer, and to correlate the findings with their antioxidant activity, as well as with total phenol and flavonoid content. The antioxidant activity of the extracts were evaluated using the DPPH radical scavenging test. The photoprotective effect was evaluated using the methodology developed by Mansur. The antioxidant activity from the extracts showed IC50 values ranging from 4.91 to 132.24 µg/mL when compared to the standard quercetin with an IC50 of 5.01 µg/mL; the phenolic content varied from 3.77 to 57.14 mg GAE/g extract while flavonoid content varied from 1.80 to 5.89 mg EQ/g extract. Almost all extracts showed a potential for UVB radiation absorption in accordance with the ANVISA (Agência Nacional de Vigilância Sanitária) technical regulation for cosmetic sunscreens. The extracts with greater sun protection factors were those originating from Lippia microphylla (SPF = 26.82) and Dimorphandra gardneriana (SPF = 20.12). A sun protection factor of 15 or higher contributes to protect the skin, where the presence of sakuranetin flavonoids and quercetin glycosides contribute to this action.