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Nanomaterials 2020, 10, 422 4 of 16 (Unisantis XMD-300, Geneva, Switzerland). The diffraction patterns were found using Cu-Kα radiation of wavelength 1.54 Å in the region of 2θ from 30◦ to 80◦ [22]. 2.4.5. Fourier Transform Infrared Spectroscopy (FTIR) The aqueous leaf extract and AgNPs were subjected to FTIR spectroscopy (Thermo Nicolet AVATAR 370, Waltham, MA, USA) to examine their spectra. The analysis was done with KBr pellets, recorded in the range of 500–4000 cm−1 [22]. 2.5. Anti-Candidal Activity A disk diffusion method was used to evaluate the anti-candidal activity of AgNPs and AgNPs/CG, as described previously [23]. Prior to use, the solution of AgNPs was prepared via dissolving AgNPs in 5% dimethylsulfoxide (DMSO, 1000 μg/mL). The sample was sonicated for 15 min and sterile filter paper disks containing 50 μg of AgNPs/disk were used for the assay. A standard antifungal agent, amphotericine b, at 5 μg/disk was used as a positive control, while 5% DMSO was used as a negative control. For the preparation of AgNPs/CG: AgNPs (50 μg/mL) and plant extract (200 μg/mL) were mixed and sonicated for 15 min at room temperature. Paper disks were prepared by adding 50 μL of the AgNPs/CG mixture solution to a 6-mm filter paper disk that contained 50 μg AgNPs and 5 μg amphotericin b. The culture of C. albicans was diluted to 1 × 106 CFU and the anti-candidal activity of the AgNPs and AgNPs/CG were evaluated by measuring the diameter of inhibition zones after 48 h of incubation at 28 ◦C. The minimum inhibitory concentration (MIC) of the AgNPs and AgNPs/CG were determined using the bi-fold serial dilution method [24]. Different concentrations of AgNPs and plant extract (6.25–400 μg/mL) were used for the MIC assay. MIC values were expressed as μg/mL. 2.6. Time-Kill Assays The anti-candidal activities of AgNPs and AgNPs/CG were evaluated at 0, 4, 8, and 10 h using a colony-counting method. At the determined time, an aliquot of 30 μL was taken from each test suspension and inoculated into SDA for quantifying the colony forming units. Sabaroud dextrose broth (SDB) without any antifungal agent was used as a control [25]. 2.7. Assay of C. albicans Hyphal Development in Liquid Media Cultures of C. albicans grown overnight were inoculated at 37 ◦C for 24 h with shaking in RPMI-1640 medium (hyphae-inducing media) supplemented with either amphotericin b, AgNPs, or AgNPs/CG. RPMI-1640 medium without any antifungal agent was used as a control. Aliquots of fungal cells were harvested after 24 h and examined using a bright field with Digital Cell Imaging System (Logos Bio Systems, Heidelberg, Germany) [26]. 2.8. Adhesion and Biofilm Formation Assays C. albicans suspensions (1 × 106 cells/mL) were inoculated in RPMI-1640 medium supplemented with 0.25% glucose and added to 96 microtiter plates (Nunc, Roskilde, Denmark). AgNPs, plant extract, and AgNPs/CG were added separately to cultured C. albicans and incubated for 2 h (to measure adhesion) and 24 h (to measure biofilm formation) at 37 ◦C under static conditions. After incubation, non-adherent cells were removed via washing and the wells were washed. Biofilm growth was measured using an MTT metabolic assay [27]. Wells without any antifungal agent were used as controls, while those without biofilms were the blanks. 2.9. Determination of Antioxidant Enzymes C. albicans was grown overnight at 37 ◦C in the presence of AgNPs/CG. Cells were harvested and homogenized in a homogenizing buffer (1 mmol/L phenylmethylsulphonyle fluoride, 250 mmol/L sucrose, 10 mmol/L Tris–HCl, pH 7.5). The cells were then disrupted at 4 ◦C using a soniprobe. ThePDF Image | Inhibition of Candidiasis Calotropis Silver Nanoparticles
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