Oral acute, sub-acute toxicity and phytochemical profile of Brassica carinata A. Braun microgreens ethanolic extract in Wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Currently, there is a remarkable increase in the consumption of microgreens, (young edible vegetables or herbs), as potential nutraceuticals for the management of diseases. Brassica carinata A. Braun is one of the traditional leafy vegetables cultivated in various parts of Sub- Saharan Africa. The plant is revered for its efficacy in the treatment of wounds and gastrointestinal disorders among other medicinal benefits. It is therefore crucial to characterize Brassica carinata microgreens for their phytoconstituents and ascertain their safety for use. AIM OF THE STUDY: The study evaluated the oral acute and subacute toxicity of Brassica carinata microgreens ethanol extract (BMEE) in Wistar rats and identification of its chemical composition and profile. MATERIALS AND METHODS: For acute toxicity (14 days), rats were grouped into four and received a single oral dose, the control group received distilled water, while others received 500 mg/kg, 1000 mg/kg, and 2000 mg/kg of BMEE. For the subacute toxicity (28 days), rats in four groups received daily doses of 250 mg/kg, 500 mg/kg or 1000 mg/kg and distilled water. Daily clinical observations like lethargy and mortality were conducted. Hematological, biochemical, and histopathological evaluations were performed at the end of each experiment. Phytochemical profile was determined using a UV-VIS spectrophotometer and Gas Chromatography coupled to Mass Spectrometry (GC-MS) analysis determined the potential bioactive components in the microgreens extract. RESULTS: In both acute and sub-acute toxicity studies, no mortalities, indications of abnormality, or any treatment related adverse effects were observed at doses of 2000 mg/kg, 1000 mg/kg, 500 mg/kg, and 250 mg/kg. The LD50 of BMEE was above 2000 mg/kg. No significant (p > 0.05) changes in the hematological and biochemical parameters of the treated groups compared to the control groups in both studies. Histopathological examination of the liver, kidney, lungs, and heart revealed a normal architecture of the tissues in all the treated animals. Phytochemical analyses revealed the presence of flavonoids (most abundant), phenols and alkaloids. Phytol, linoleic acid, and 9,12,15-octadecatrienoic acid, among other compounds, were identified by GC-MS analysis. CONCLUSION: The results showed that B. carinata microgreens ethanol extract is nontoxic and found to have several compounds with reported pharmacological significance suggesting safety for use.

Recombinant Actifensin and Defensin-d2 Induce Critical Changes in the Proteomes of Multidrug-Resistant Pseudomonas aeruginosa and Candida albicans.

Drug-resistant strains of Pseudomonas aeruginosa and Candida albicans pose serious threats to human health because of their propensity to cause fatal infections. Defensin and defensin-like antimicrobial peptides (AMPs) are being explored as new lines of antimicrobials, due to their broad range of activity, low toxicity, and low pathogen resistance. Defensin-d2 and actifensin are AMPs from spinach and Actinomyces ruminicola, respectively, whose mechanisms of action are yet to be clearly elucidated. This study investigated the mechanisms of action of the recombinant AMPs through label-free quantitative proteomics. The data are available at PRIDE with accession number PXD034169. A total of 28 and 9 differentially expressed proteins (DEPs) were identified in the treated P. aeruginosa and C. albicans, respectively, with a 2-fold change threshold and P values of <0.05. Functional analysis revealed that the DEPs were involved in DNA replication and repair, translation, and membrane transport in P. aeruginosa, while they were related mainly to oxidative phosphorylation, RNA degradation, and energy metabolism in C. albicans. Protein-protein interactions showed that the DEPs formed linear or interdependent complexes with one another, indicative of functional interaction. Subcellular localization indicated that the majority of DEPs were cytoplasmic proteins in P. aeruginosa, while they were of nuclear or mitochondrial origin in C. albicans. These results show that recombinant defensin-d2 and actifensin can elicit complex multiple organism responses that cause cell death in P. aeruginosa and C. albicans. IMPORTANCE AMPs are considered essential alternatives to conventional antimicrobials because of their broad-spectrum efficacy and low potential for resistance by target cells. In this study, we established that the recombinant AMPs defensin-d2 and actifensin exert proteomic changes in P. aeruginosa and C. albicans within 1 h after treatment. We also found that the DEPs in peptide-treated P. aeruginosa are related to ion transport and homeostasis, molecular functions including nucleic and amino acid metabolism, and structural biogenesis and activity, while the DEPs in treated C. albicans are mainly involved in membrane synthesis and mitochondrial metabolism. Our results also highlight ATP synthase as a potential drug target for multidrug-resistant P. aeruginosa and C. albicans.

Membrane Permeabilization and Antimicrobial Activity of Recombinant Defensin-d2 and Actifensin against Multidrug-Resistant Pseudomonas aeruginosa and Candida albicans.

Antimicrobial resistance requires urgent efforts towards the discovery of active antimicrobials, and the development of strategies to sustainably produce them. Defensin and defensin-like antimicrobial peptides (AMPs) are increasingly gaining pharmacological interest because of their potency against pathogens. In this study, we expressed two AMPs: defensin-d2 derived from spinach, and defensin-like actifensin from Actinomyces ruminicola. Recombinant pTXB1 plasmids carrying the target genes encoding defensin-d2 and actifensin were generated by the MEGAWHOP cloning strategy. Each AMP was first expressed as a fusion protein in Escherichia coli, purified by affinity chromatography, and was thereafter assayed for antimicrobial activity against multidrug-resistant (MDR) pathogens. Approximately 985 µg/mL and 2895 µg/mL of recombinant defensin-d2 and actifensin, respectively, were recovered with high purity. An analysis by MALDI-TOF MS showed distinct peaks corresponding to molecular weights of approximately 4.1 kDa for actifensin and 5.8 kDa for defensin-d2. An in vitro antimicrobial assay showed that MDR Pseudomonas aeruginosa and Candida albicans were inhibited at minimum concentrations of 7.5 µg/mL and 23 µg/mL for recombinant defensin-d2 and actifensin, respectively. The inhibitory kinetics of the peptides revealed cidal activity within 4 h of the contact time. Furthermore, both peptides exhibited an antagonistic interaction, which could be attributed to their affinities for similar ligands, as deduced by peptide-ligand profiling. Moreover, both peptides inhibited biofilm formation, and they exhibited no resistance potential and low hemolytic activity. The peptides also possess the ability to permeate and disrupt the cell membranes of MDR P. aeruginosa and C. albicans. Therefore, recombinant actifensin and defensin-d2 exhibit broad-spectrum antimicrobial activity and have the potential to be used as therapy against MDR pathogens.