Optimizing an Enzymatic Extraction Method for the Flavonoids in Moringa (Moringa oleifera Lam.) Leaves Based on Experimental Designs Methodologies.

Moringa oleifera Lam. is known to have significant antioxidant properties. Because of this, the development of an optimal extraction method is crucial to obtain pharmacological products based on the bioactive compounds produced by this tree. Through a Plackett-Burman and a Box-Behnken design, enzymatic extraction conditions (temperature, agitation, solvent pH and composition, sample-to-solvent ratio, enzyme-to-sample ratio and extraction time) have been optimized using normalized areas (UA/g) as response variable and relative mass (mg/g) as quantification variable. Extractions were performed in an incubator, where all the extraction conditions could be digitally controlled. Thus, 58.9 °C, 50 rpm, 4.0 pH, 32.5% EtOH, 0.2 g sample in 15 mL solvent and 106 U/g were established as the optimal extraction conditions for the extraction with a mix of pectinases coming from Aspergillus niger. Under these optimal conditions, two-minute extractions were performed and evaluated through a single factor design. The enzymatic extraction method demonstrated its suitability to produce extracts with good antioxidant power (antioxidant activity 4.664 ± 0.059 mg trolox equivalent/g sample and total phenolic compounds 6.245 ± 0.101 mg gallic acid equivalent/g sample). The method was also confirmed to have good repeatability (1.39%) and intermediate precision (2.37%) levels.

Short and long-term exposure to the pesticides fipronil and 2,4-D: Effects on behavior and life history of Daphnia magna.

The high levels of contamination in aquatic ecosystems caused by pesticides and the organisms' consequent continuous exposure to it has made them vulnerable to damage. However, mobile organisms can avoid this continued exposure to contaminants by moving to less disturbed habitats. Therefore, through the use of the Heterogenous Multi-Habitat Assay System (HeMHAS), our objective was to evaluate the ability of Daphnia magna to detect and avoid habitats contaminated by fipronil and 2,4-D, in a spatially connected landscape. Further, the role of contamination by these pesticides, isolated and in mixtures, concerning the colonization of habitats by daphnids was also evaluated. Given that not all organisms successfully escape contamination, the chronic toxicity of the same pesticides using different parameters for D. magna (maternal survival, fecundity and maternal body length) was also evaluated. When evaluating the avoidance response by D. magna exposed to pesticides, there was no preference for the less contaminated areas for both compounds. However, organisms did not move to contaminated zones in the colonization experiments, with no immigration of daphnids to the zones with intermediate and the highest levels of fipronil, nor to the highest concentration of 2,4-D. Finally, the colonization by daphnids was significantly prevented when exposed to a mixture of the pesticides, in which the areas with the highest combinations of pesticide concentrations were not colonized by D. magna. Regarding the long-term chronic effects, negative consequences were observed, particularly for maternal body length, fecundity and maternal survival, due to the exposure to fipronil. Considering that pesticides can limit the areas colonized by organisms by making them unattractive, the risk of local population extinction may be underestimated if only standard endpoints involving forced exposure are studied.

Avoidance responses by Danio rerio reveal interactive effects of warming, pesticides and their mixtures.

Temperature variations and thermal extremes events caused by climate change can have profound implications for the toxicity of pesticides in aquatic organisms. Using an innovative system (Heterogeneous Multi-Habitat Test System - HeMHAS) that allows the simulation of different scenarios within a spatially heterogeneous landscape, the effects on the habitat selection of Danio rerio fish caused by the pesticides fipronil and 2,4-D were studied as single compounds and in mixture and integrated with air temperature variation (20, 24 and 28 °C). As a result, D. rerio detected and avoided both pesticides at air temperatures of 20 and 24 °C; however, at 28 °C no significant difference was observed in habitat choice by fish. Additionally, when pesticides were mixed in a heterogeneously contaminated landscape, it was observed that D. rerio detected contamination and preferred the clean zone at 20 and 24 °C; however, at 28 °C the potential to escape from the most contaminated areas was impaired. Thus, contamination by both pesticides made the habitat selection behavior of fish at 20 and 24 °C more noticeable. In addition, the association between pesticides and temperature showed negative effects on the response of fish to detect and escape from contaminated environments, suggesting the influence of temperature in altering the ability of the organism to provide an efficient response to stress.

Optimization through a Box-Behnken Experimental Design of the Microwave-Assisted Extraction of the Psychoactive Compounds in Hallucinogenic Fungi (Psylocibe cubensis).

Hallucinogenic fungi, mainly those from the Psilocybe genus, are being increasingly consumed even though there is no control on their culture conditions. Due to the therapeutic potential as antidepressants and anxiolytics of the alkaloids that they produce (psilocin and psilocybin), some form of control on their production would be highly recommended. Prior to identifying their optimal culture condition, a methodology that allows their study is required. Microwave-assisted extraction method (MAE) is a technique that has proven its efficiency to extract different compounds from solid matrices. For this reason, this study intends to optimize a MAE method to extract the alkaloids found in Psylocibe cubensis. A surface-response Box-Behnken design has been employed to optimize such extraction method and significantly reduce time and other resources in the extraction process. Based on the Box-Behnken design, 50 °C temperature, 60% methanol as extraction solvent, 0.6 g:10 mL sample mass:solvent ratio and 5 min extraction time, were established as optimal conditions. These mild conditions, combined with a rapid and efficient UHPLC analysis result in a practical and economical methodology for the extraction of psilocin and psilocybin from Psylocibe cubensis.