BBD optimized antioxidants of Crotalaria candicans and its nanoconjugates, exert potent in vivo anti-biofilm effects against MRSA.

Crotalaria genus is extensively dispersed in tropical and subtropical provinces, and it is found to harbor antioxidant flavonoids. Response surface methodology-based optimization was carried out for the purpose of efficient extraction involving a suitable solvent which can maximize the yield along with higher total phenolic content and total flavonoid content (TFC). Optimization conditions for extraction of C.candicans flavonoids (CCF) based on variables such as solvent, solid-solvent ratio and extraction temperature were evaluated. The optimized conditions were found as Solvent i.e., Aqueous-ethanol (53.42%), Solid-solvent ratio (1:15.83 w/v) and temperature (44.42 °C) and resulted to obtain the TFC as 176.23 mg QRET/g C. candicans extract with the yield 27.42 mg CCF/g (C. candicans dry weight). LC-MS analysis of CCF, revealed the presence of seven major flavonoids. The antioxidant flavonoids were further used to functionalize the zero-valent silver (ZVAgF) and copper (ZVCuF) nanoparticles. The ZVAgF and ZVCuF were investigated using UV-Vis spectrophotometry, FT-IR spectroscopy and X-ray diffractometry to confirm the presence of the zero valent metals and possible functional groups which capped the elemental metal. Further transmission electron microscopy, dynamic light scattering method and zeta-potential studies were done to understand their respective structural and morphological properties. The efficacy of the as-prepared ZVAgF/ZVCuF as antibiofilm agents on Methicillin-resistant Staphylococcus aureus (MRSA) with the mechanism studies have been explored. The MRSA-colony count from the infection zebrafish (in vivo) model, portrayed a reduction of > 1.9 fold for ZVCuF and > twofold for ZVAgF, with no alteration in liver morphology when treated with ZVAgF, implying that the nanoparticles were safe and biocompatible.

Metal nanoparticles functionalized with nutraceutical Kaempferitrin from edible Crotalaria juncea, exert potent antimicrobial and antibiofilm effects against Methicillin-resistant Staphylococcus aureus.

Kaempferitrin (KF), a flavonol glycoside, was isolated from the edible plant Crotalaria juncea. Optimization for the synthesis of silver (AgNPs) and copper (CuNPs) nanoparticles using C. juncea extract and kaempferitrin were attempted for the first time. A detailed study on size and stability analysis have been reported. Efficacy of KF@AgNPs and KF@CuNPs against biofilm formation and planktonic mode of growth on methicillin-resistant Staphylococcus aureus (MRSA) along with possible mechanisms has been explored. Release of Cu(II) upon prolonged treatment with KF@CuNPs in the presence of MRSA was quantified through Alizarin red test, indicating the antibacterial effect is initiated by the CuNPs itself. Time kill curve depicted both the NPs have similar kill kinetics to curtail the pathogen and imaging with Crystal violet assay, Fluorescent live dead imaging and SEM analysis revealed a 60% reduction in biofilm formation at the Sub-MIC concentration of KF@AgNPs and KF@CuNPs. Furthermore, the membrane permeability and cell surface hydrophobicity were altered in the presence of both the NPs. The colony count from the in vivo infection zebrafish model in the treatment group showed a decline of > 1.8 fold for KF@AgNPs and > two fold for KF@CuNPs. Toxicity studies did not reveal any abnormality in liver and brain enzyme levels. Liver morphology images show no severe cytological alterations when treated with KF@AgNPs and were almost similar to the normal liver. Thus, KF@AgNPs was nontoxic and caused significant reduction in biofilm formation in MRSA, also reduced bacterial bioburden in the infected zebrafish, which has the potential to be explored in higher animal models.

Zero valent silver nanoparticles capped with capsaicinoids containing Capsicum annuum extract, exert potent anti-biofilm effect on food borne pathogen Staphylococcus aureus and curtail planktonic growth on a zebrafish infection model.

Food plants Hungarian wax pepper (HWP) and Green Bell pepper (GBP), belonging to Capsicum annuum were utilized for biogenic fabrication of zero valent, nano-silver (AgNPs) through a photo-mediation procedure. In the bacterial strains evaluated, HWP/GBP AgNPs demonstrated effective bacteriostatic and bactericidal effect against Staphylococcus aureus. Time kill results portrayed that HWP/GBP nano-silver exhibited comparable bactericidal potency on S. aureus. Anti-biofilm potential of HWP/GBP AgNPs displayed significant effects at sub MIC levels, by triggering 50% biofilm reduction of the food spoilage microbe S. aureus, inferring that the anti-biofilm outcome is not dependent on antibacterial result, and this was confirmed by SEM and fluorescence studies. Histopathological analyses of S. aureus infected zebrafish liver did not display any abnormality changes such as extensive cell death and degeneration, upon treatment with HWP/GBP AgNPs and the zero-valent silver nanoparticles were comparatively less toxic and more operative in restraining the bioburden in S. aureus infected zebrafish model by a >1.7 log fold. Ability of light reduced HWP/GBP AgNPs to alleviate the in vitro and in vivo planktonic mode of growth and curb the biofilm formation of S. aureus is also demonstrated.

Plant nutraceuticals (Quercetrin and Afzelin) capped silver nanoparticles exert potent antibiofilm effect against food borne pathogen Salmonella enterica serovar Typhi and curtail planktonic growth in zebrafish infection model.

Purified plant nutraceuticals afzelin and quercetrin from an edible plant- Crotolaria tetragona was employed for the fabrication of silver nanoparticles (AgNPs) by a sunlight mediated process. From among a panel of strains tested, AgNPs displayed potent bacteriostatic and bactericidal effect against P. aeruginosa and S. Typhi. Time kill studies revealed green synthesized AgNPs displayed comparable bactericidal effect with chemically synthesized AgNPs against S. Typhi. Antibiofilm potential of AgNPs showed that they were highly effective at sub MIC concentrations in causing 50% biofilm inhibition against food borne pathogen S. Typhi implying that antibiofilm effect is independent of antibacterial effect, which was evidenced by fluorescent imaging and SEM imaging. Mechanistic studies revealed that reduced cell surface hydrophobicity, decreased surface adherence, loss of membrane potential contributed to antibiofilm potential of afzelin/quercetrin AgNPs. Green synthesized afzelin/quercetrin AgNPs were also relatively less toxic and more effective in curtailing bioburden of S. Typhi in infected zebrafish by > 3 log fold. Ability of sunlight reduced afzelin/quercetrin NPs to mitigate planktonic mode of growth in vitro and in vivo and curtail biofilm formation of S. Typhi in vitro demonstrates its potential to curtail food borne pathogen in planktonic and biofilm mode of growth.