An innovative insecticidal approach based on plant protease inhibitor and Bt protoxins inhibits trypsin-like activity in zebrafish.

The Leucaena leucocephala trypsin inhibitor (LTI) + Bacillus thuringiensis (Bt) protoxins mix has been proposed as a novel larvicide agent in order to control the vector mosquito of dengue virus, Aedes aegypti, in their aquatic breeding sites. However, use of this insecticide formulation has raised concerns about its impacts on aquatic biota. In this context, this work aimed to assess the effects of LTI and Bt protoxins, separately or in combination, in zebrafish, in regard to the evaluation of toxicity at early life stages and to the presence of LTI inhibitory effects on intestinal proteases of this fish. Results showed that LTI and Bt concentrations (250 mg/L, and 0.13 mg/L, respectively), and LTI + Bt mix (250 mg/L + 0.13 mg/L) - 10 times superior to those with insecticidal action - did not cause death nor did it induce morphological changes during embryonic and larval development (3 to 144 h post-fertilization) of zebrafish. Molecular docking analyses highlighted a possible interaction between LTI and zebrafish trypsin, especially through hydrophobic interactions. In concentrations near to those with larvicidal action, LTI (0.1 mg/mL) was able to inhibit in vitro intestinal extracts of trypsin in female and male fish by 83 % and 85 %, respectively, while LTI + Bt mix promoted trypsin inhibition of 69 % in female and 65 % in male ones. These data show that the larvicidal mix can potentially promote deleterious effects to nutrition and survival in non-target aquatic organisms, especially those with trypsin-like dependent protein digestion.

Toxicological assessment of a bioactive extract from Triplaris gardneriana Wedd. seeds using alternative models.

The Triplaris gardneriana Wedd. seeds extract has great therapeutic potential due to numerous biological activities such as antioxidant, antibacterial and anti-inflammatory, which are associated with phenolic content. Although this herbal preparation has shown many benefits, recently their toxicity profile has begun to be explored. In this present study, the toxic effects of T. gardneriana seeds ethanolic extract (EETg) on biological systems of different taxonomical groups and levels of complexity (from cell culture to lower vertebrates) were assessed, through a variety of viability and toxicological assays. It was found that EETg did not impair the Saccharomyces cerevisiae growth at the highest tested concentration (200 µg/mL), and no toxicant evidence was observed in Aedes aegypti larvae or in Drosophila melanogaster adult stage. Contrarily, the extract reduced the viability of undifferentiated Caco-2 cells (250 µg/mL, 40% of viable cells), but did not affect differentiated ones. The embryotoxicity in Danio rerio model showed a LC50 of 7.41 mg/L (95% confidence interval, 4.78 - 11.49 mg/L). EETg did not show signs of toxicity in the majority of the models used, but lethality and malformations in zebrafish embryos occurred. Further analyses are needed to better understand the selective toxicity mechanism of EETg on zebrafish, as well as whether the toxic effects happen in higher vertebrates.

Triplaris gardneriana seeds extract exhibits in vitro anti-inflammatory properties in human neutrophils after oxidative treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Triplaris gardneriana Wedd. (Polygonaceae family) is a plant species from Brazilian semiarid region which is used in local traditional medicine for the treatment of inflammatory conditions such as hemorrhoids. AIM OF THE STUDY: In this study, the in vitro anti-inflammatory activity of different concentrations of ethanolic extract from T. gardneriana seeds (EETg) was performed in order to contribute to the knowledge about etnomedicinal use of this plant species. MATERIALS AND METHODS: The anti-inflammatory properties were evaluated through different approaches, such as in vitro protein anti-denaturation test, scavenging of reactive oxygen species (ROS) and myeloperoxidase (MPO) inhibition in human neutrophils activated by phorbol-12-myristate-13-acetate (PMA). Besides that, molecular docking was performed to provide new insights about the interaction between the major phenolic components in the plant extract and MPO. RESULTS: EETg was characterized showing a total phenol content of 153.5 ± 6.3 µg gallic acid equivalent/mg extract, ability to remove hydrogen peroxide (H2O2) in a concentration-dependent manner and had a spectroscopic profile which suggests the presence of hydroxyl groups. EETg was able to prevent protein denaturation ranging from 40.17 to 75.09%. The extract, at 10 and 20 µg/mL, was able to modulate neutrophils pro-inflammatory functions, such as degranulation and burst respiratory. In both assays, the EETg had anti-inflammatory effect comparable to nonsteroidal anti-inflammatory drugs. Among the main phenolic compounds of EETg, quercitrin, quercetin and catechin showed the highest binding affinity in silico to MPO. CONCLUSION: This study demonstrated, for the first time, that the anti-inflammatory effect of T. gardneriana seeds occurs due to its modulatory effect on human neutrophil degranulation and free-radical scavenging activity.