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Nanomaterials 2020, 10, 422 12 of 16 strategy for the efficient control of C. albicans. To our knowledge, this was the first report to identify the MIC value of a combination between a plant extract and its biosynthesized AgNPs against C. albicans. Previously, the anti-candidal action of AgNPs was found to be improved using a combination with commercially available amphotericin b against five Candida species [23]. Thus, our data identified C. gigantea leaf extract as a source of bioactive antifungal compounds that could contribute in the extracellular synthesis of AgNPs, and has a stronger antifungal action than the previously designated antifungal effect [14,56,57]. The phenotypic switching between yeast and hyphal forms has been considered as one of the most significant virulent factors in C. albicans [58]. The development of hyphae is an interesting property of C. albicans that plays a vital role in adherence and biofilm formation, which is certainly crucial for colonization and pathogenesis [59,60]. The blocking of morphogenesis from yeast to a filamentous form would mean preventing the infection. Our data demonstrated the blocking of hyphal growth using AgNPs/CG. Similarly, several studies demonstrated the suppression of the C. albicans morphogenesis using AgNPs [6,8,53,61] in a mechanism that involved targeting the Ras-mediated signal transduction pathways in C. albicans through the downregulation of the expression of cell elongation gene (Ece1), hyphal inducer gene (Tec), and yeast to hyphal transition genes (Tup1 and Rfg1), which are key genes for the morphological transition [62]. The prevention of blastospore adhesion and differentiation into a filamentous form using AgNPs appears to provide an excellent therapeutic choice [51,63–65]. Our results showed that AgNPs/CG was more efficient at suppressing the adhesion and biofilm formation of C. albicans than plant extract or AgNPs alone. Similar to our finding, several other studies demonstrated the inhibitory effect of AgNPs on C. albicans biofilm formation [8,61,66]. In addition, AgNPs biosynthesized by Dodonaea viscosa and Hyptis suoveolens leaf extracts were shown to suppress the biofilm formation of Candida spp. from 79% to 88% at 10 μg/mL [51]. The mechanism underlying the suppression of biofilm formation using AgNPs was reported to include the anti-adhesive action of AgNPs that regulate the growth of living microbial cells and the suppression of microbial adhesions gene expression [27,67,68]. In addition, AgNPs were shown to suppress blastospores and to disrupt the cell wall of both the yeast and the filamentous forms in order to cause the inhibition of biofilm formation in Candida [61,66]. C. albicans has developed enzymatic antioxidant defense mechanisms to reduce the harmful effects of ROS produced by phagocytes during the infection process. Our data demonstrated the inhibitory effect of AgNPs/CG on the production of antioxidant enzymes by C. albicans. Supporting our data, C. gigantea was found to exert an antioxidant activity in other studies [69–71]. AgNPs were also reported to display antioxidant activity via stimulating C. albicans to express genes encoding for antioxidants, such as catalase, glutathione peroxidase, superoxide dismutase, and components of the glutathione/glutaredoxin and thioredoxin systems [55,72]. The stimulatory effect of AgNPs on oxidative stress as a mechanism of toxicity in C. albicans resulted in the shifting of the total redox balance to oxidation and thus causing the functional destruction of cells [73–75]. Our data demonstrated the safety of using AgNPs/CG at the concentration of (200 μg/mL plant extract + 50 μg/mL AgNPs) in both primary and transformed cultured animal cells, suggesting the plausible application of AgNPs/CG in vivo in a preclinical study. The toxicity of AgNPs to human cells was found to be attributed to the type of the reducing agent used in the biosynthesis [76]. In this regard, the plant extracts could act as a stabilizing agent to stabilize the particles of AgNPs against dissolution, subsequently reducing the toxicity of AgNPs. In addition, the antioxidant and the anti-inflammatory activities of C. gigantean extracts might contribute toward reducing the cytotoxicity of AgNPs [69–71]. Thus, the combination of AgNPs and C. gigantean extracts provide a novel strategy for developing biosynthesized-based drug for preventing candidiasis.PDF Image | Inhibition of Candidiasis Calotropis Silver Nanoparticles
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