Multiskan go microplate spectrophotometer

The antifungal activity of the pyrazole derivatives 4a-j against Candida spp. was evaluated with the CLSI M27-A3 microdilution method [75 (link)]. Fluconazole 20 (the antifungal reference drug) and the pyrazole 4a-j and dihydrooxazole derivatives 6a-j were examined at concentrations of 6.4 to 0.0125 μg/mL. The diluent was RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) for 20 and DMSO for the two series of test compounds. To avoid an inhibitory effect by DMSO, it was employed at less than 10% of the total volume. For the preparation of the inoculum of Candida spp., the optical density was adjusted on a spectrophotometer to 620 nm, followed by a 1:1000 dilution with RPMI medium. The 96-well microplates were inoculated with 100 µL of yeast suspension. RPMI served as the sterility control and DMSO without any antifungal compound as the growth control. The microplates were incubated at 37 °C for 24 h, and upon completion of this, the time growth was quantified by optical density on a Multiskan™ GO microplate spectrophotometer at 620 nm. The reported values of yeast growth are expressed as the averages of three independent assays.

The tyrosinase inhibitory activity was determined according to the modified method using L-3.4-dihydroxyphenylalanine (L-DOPA) as substrate, as previously described [21 ], with some modifications. An aliquot of 25 μL of extract solution (1 mg/mL) was mixed with tyrosinase (40 μL, Sigma Aldrich- USA) and a buffering solution (100 μL, pH 6.8) in 96 wells of microplates (Multiskan GO Microplate Spectrophotometer–Finland) and incubated for 15 min at 25°C. The reaction was initiated with the addition of L-DOPA (40 μL, Sigma Aldrich—USA). Similarly, a bleach was prepared by adding the extract solution to all reagents except for the enzyme solution (tyrosinase). The sample absorbance and bleaching (Kojic acid, Sigma Aldrich—USA) were read at 492 nm after incubation at 25°C. The results were compared with the control (DMSO). The tyrosinase inhibitory activity was calculated based on the formula:
$\mathrm{T}\mathrm{y}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{o}\mathrm{r}\mathrm{y}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{\%}=\left[\mathrm{A}0‑\mathrm{A}1/\mathrm{A}0\right]\mathrm{x}100$
Where: A0 is the absorbance at 492 nm with DMSO, replacing the sample, and A1 is the absorbance at 492 nm with the sample under analysis.

In vitro hemolysis assay of NCS was performed as described previously (Jamil et al., 2016a (link)). Briefly human whole blood sample was collected in Li–heparin as anti-coagulant vacutainer. Blood sample was incubated with 1% of nano-antimicrobials' solution at 37°C for 45 min. Unexposed samples were taken as negative control, whereas sodium dodecyl sulfate (1% SDS) solution was added as positive control in the blood sample. After incubation, samples were centrifuged at 14,000 rpm for 5 min. After centrifugation, supernatant was collected from each samples and OD was measured at 540 nm by Multiskan™ GO microplate spectrophotometer. Percentage hemolysis was calculated relative to the untreated control. All assay values were observed in duplicate.

To determine the in vitro minimum inhibitory concentrations (MIC) for fluconazole (8) and the 2-amino-3-cyano-4H-chromenes 4a–i and 6a–h on six Candida species (C. albicans, C. dubliniensis, C. glabrata, C. kefyr, C. krusei, and C parapsilosis), the standard guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) were employed, with an RPMI of 1640 (Sigma) buffered with 0.165 MMOPS (Sigma) as the test medium [64 (link)]. The MIC value was defined as the lowest concentration of a compound that generated a culture with turbidity less than or equal to 100% inhibition when compared with the growth of the control. The 4H-chromene derivatives 4a–i and 6a–h were dissolved in DMSO solvent and serially dripped into the growth medium. The concentration gradient of 8 was from 64–0.033 µg/mL and for the derivatives 160–0.078 µg/mL. The inoculum was prepared by resuspending colonies (from a yeast culture of 24 h growth) in a tube with saline solution (0.85% NaCl), and then adjusting them to an optical density 0.5 McFarland. Subsequently, they were incubated at 37 °C in an incubator. The MIC results were quantified in a Multiskan™ GO microplate spectrophotometer by agitation of the plates, followed by a spectrophotometric reading at 450 nm. The value is expressed as the average of three replicates.