Saccharomyces cerevisiae

After the 2 h incubation at 37 °C, H₂O₂ production by PBMCs and neutrophils was measured using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as an indicator [41 (link),42 (link)]. A stock solution of DCFH-DA (2.05 mM) in ethanol was prepared and stored at −20 °C until analysis. Hanks’ Balanced Salts Medium was prepared in a relation of 9.8 g/L water. H₂O₂ production in PBMCs and neutrophils was measured before and after stimulation with lipopolysaccharide (LPS) (100 µg/mL PBS) from Escherichia coli (Sigma-Aldrich, St. Louis, MO, USA) or Zymosan A (ZYM) (1 mg/mL PBS) from Saccharomyces cerevisiae (Sigma-Aldrich). A total of 50 µL of LPS or ZYM prepared in PBS was added to the wells. DCFH-DA in ethanol was diluted in Hanks’ Balanced Salts Medium (relation 30 µL DCFH-DA/mL Hanks’) and added to a 96-well microplate containing 50 µL of cell suspension (containing about 6 × 10⁵ cells). The fluorescence (Ex, 480 nm; Em, 530 nm) was recorded at 37 °C for 60 min in FLx800 Microplate Fluorescence Reader (Bio-tek Instruments, Inc.) by punctual ultraviolet light exposures and emission readings were recorded every minute (60 total readings). H₂O₂ concentration was calculated by measuring fluorescence of a standard curve of known H₂O₂ concentration after its reaction with DCFH-DA, in the same conditions as the samples.

The activity of both enzymes was determined by the production of ADP which was coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase^{58 (link)}. The assays were performed in a final volume of 100 µl 96-well deep-well plates and spectrophotometrically monitored (Omega multimode microplate reader) at 360 nm at 35 °C. KH₂PO₄ (100 mM) at pH 7.0, supplemented with 1.5 mM MgCl₂ and 100 mM KCl, was used as a buffer. For NADH, a molar extinction coefficient of 4,546.7 cm⁻¹ M⁻¹ was experimentally determined for the above-named conditions. To the buffer, 1 mM NADH, 2.5 mM Na₂SO₄, 1 mM phosphoenolpyruvate (PEP), 2 mM ATP, 2 U inorganic pyrophosphatase (Saccharomyces cerevisiae, 10108987001, Sigma-Aldrich), 1.1 U ml⁻¹ lactate dehydrogenase, 0.8 U ml⁻¹ pyruvate kinase (rabbit muscle, P0294, Sigma-Aldrich) and 0.5 mg ml⁻¹MtAPSK (all final concentrations) were added. The reaction was started by the addition of 0.5 mg ml⁻¹MtATPS. Addition of 0.02 mM Na₂MoO₄ did not affect activity (0.116 ± 0.027 µmol of oxidized NADH min⁻¹ mg⁻¹), but the addition of 2 mM Na₂MoO₄ resulted in a decrease (0.068 ± 0.019 µmol of oxidized NADH min⁻¹ mg⁻¹). All assays were performed in triplicates.

α-Glucosidase inhibitions were determined following a modified spectroscopic method [87 (link)]. A solution of α-glucosidase (obtained from Saccharomyces cerevisiae (Sigma-Aldrich, Munich, Germany)) was prepared in phosphate buffer (pH 6.8, 100 mM). Wells of the 96-well microliter plates were filled with test sample solutions (12.5 μL) prepared in DMSO. After this, enzyme (0.5 U/mL, 40 μL) and then phosphate buffer (100 mM, 120 μL) were also mixed with the test sample solutions. Incubation at 37 °C for 5 min and then addition of 40 μL of the substrate, p-nitrophenyl-α-D-glucopyranoside (PNPG) (5 nM, Sigma-Aldrich, Munich, Germany), was performed. Mixtures were again incubated for 30 min, and absorbance was measured at 405 nm and 37 °C for the released 4-nitrophenol in each mixture. Acarbose (Sigma-Aldrich, Munich, Germany) and DMSO worked as standard and negative inhibitors, respectively. Each experiment was carried out thrice, and percentage inhibitions were determined with the help of following expression, followed by the determination of IC₅₀ values.

where A is absorbance.