Gallic acid (purity ≥ 98%), ascorbic acid (purity ≥ 99%), and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) were purchased from Merck (Budapest, Hungary). Analytical grade methanol and acetic acid were purchased from Molar Chemicals (Halásztelek, Hungary). Gradient-grade acetonitrile was from Fisher Scientific (Pittsburgh, PA) and pure water was from a Millipore Direct-Q 3 UV System (Merck).

The wild-growing macrobasidiomycetous mushrooms, poplar fieldcap (Cyclocybe cylindracea) and slate bolete (Leccinum duriusculum) were collected in Eger and Buda Hills, respectively, in Hungary. The fresh fruiting bodies were cleaned, sliced, frozen at −18 °C, and carefully dried using a freeze dryer (Alpha-1–2 D, Christ, Osterode am Harz, Germany). The dried fruit bodies were pulverized (Bosch MKM6000, Stuttgart, Germany) and kept in a hermetically sealed sample holder at room temperature until analysis. The mushroom samples (total mass of the dried mushroom samples 1 g) were extracted with methanol (concentration: 1 mL per 100 mg dried mushroom sample) by ultrasonication for 1 h [20], and the supernatant was filtered through filter paper (Macherey–Nagel, MN 85/70 BF; Düren, Germany). The extraction was repeated twice with the residual material, and the resulting filtrates were combined. The merged extracts were filtered through a 0.22-μm pore diameter polytetrafluoroethylene (PTFE) syringe filter (Nantong FilterBio Membrane Co., Zhongnan, P.R. China) and stored in vials at 5 °C for further use. The concentrations of mushroom extracts were set to 10 mg/mL with methanol (for dry weight). Methanol solutions of gallic acid and ascorbic acid were prepared at concentrations of 10 μg/mL for HPTLC–DPPH and 100 μg/mL for the microplate-based DPPH assay.

The HPTLC–DPPH method was implemented on HPTLC silica gel 60 F254 10 × 10 cm glass-backed plates (art. no. 5629, Merck, Darmstadt, Germany). The mushroom extracts were applied on the HPTLC layer by an automatic TLC sampler (ATS 3, CAMAG, Muttenz, Switzerland) with the following parameters: a 5.0 mm band length, a 4.0 mm distance between tracks, and applied volumes of the mushroom extracts of 8, 10, 12, and 14 μL, with an 8.0 and 10 mm distance from the bottom and left/right sides, respectively. The first (application at 7.5 mm) and last (application at 92.5 mm) tracks were used for calibration series of the standards, gallic acid and ascorbic acid. The HPTLC layers were developed with acetonitrile–water–acetic acid (75:25:3, V/V) up to 75 mm in a twin-trough chamber (CAMAG) without saturation [21]. After development, the plates were dried with a stream of cold air using a hair dryer and the standards were applied with volumes of 1, 2, 4, 6, 8, 10, and 12 μL and at heights of 8, 18, 28, 38, 48, 58, and 68 mm. Then, the plates were documented under an ultraviolet (UV) lamp (CAMAG) at 254 and 366 nm by a digital camera (DSC-WX350, Sony, Tokyo, Japan). After documentation, the plates were dipped into the 1 mM DPPH solution (19.7 mg of the DPPH was dissolved in 50 mL methanol) and dried quickly with a stream of cold air. Then the antioxidant activity was measured based on scavenging the stable artificial radical molecule (DPPH). The zones with an antioxidant effect appeared as yellow spots on the dark purple background and were documented by the camera in visible light after the plates dried (2 min). Images of the autograms were processed by ImageJ [22] software [National Institutes of Health, Laboratory for Optical and Computational Instrumentation (LOCI), University of Wisconsin, Madison, WI]. Initially, the images were converted to black and white, inverted, and denoised (reduction of the background noise). First, the rectangular selection tool was chosen and a small part of the chromatogram background was marked to provide the median value that was used for background subtraction (Process/Math/Subtract). Each track was designated as rectangles with the same size. Then, line profile plots (videodensitograms) were generated and the peak areas were determined, including the peaks at the application zones. Developments and treatments were performed in triplicate on separated plates, and these results are expressed as the mean ± standard deviation (mean ± SD). As the results of the HPTLC–DPPH–densitometry were appropriately repeatable for plate by plate, which was also indicated by the low standard deviation values, for quantification, the calibration curves were constructed by plotting averages of the peak areas obtained from the three plates versus the amount of gallic acid or ascorbic acid. Quantitative results were expressed as a gallic acid equivalent (GAE, mg of GA/g of extract) or ascorbic acid equivalent (AAE, mg of AA/g of extract).

The standards and the mushroom extracts were tested using a microplate-based DPPH assay. Ascorbic and gallic acid (0.1 mg/mL) and the extracts (10 mg/mL) were prepared for twofold serial dilutions. Ascorbic acid or gallic acid or extracts (50 μL) were mixed with 150 μL of methanol and 50 μL of a 1 mM DPPH solution in wells of a 96-well microplate in triplicate. The concentrations in the wells were as follows: 2, 1, 0.5, 0.25, 0.125, 0.063, and 0.031 mg/mL (extracts) and 20, 10, 5, 2.5, 1.25, 0.625, and 0.31 µg/mL (standards). After loading, the microplate was kept at room temperature in the dark for 30 min. After that, the absorbance at 517 nm was measured by a microplate reader (LabSystems Multiskan MS 4.0, Thermo Fischer Scientific, Budapest, Hungary). The capability to scavenge the DPPH radical was calculated using this equation:

$$}\,\left( \% \right) = \left( }_}}} }_}}} } \right)} \mathord}_}}} }_}}} } \right)} }_}}} }}} \right. \kern-0pt} }_}}} }}} \right) \times 00$$

(1)

where Ablank is the absorbance of the control (the reaction mixture contains 150 μL of methanol and 50 μL of DPPH solution, plus 50 μL of methanol instead of the mushroom extract) and Asample is the absorbance of the mixture with mushroom extracts or ascorbic/gallic acid. Each sample/standard in the microplate experiments was measured three times (in three microplates), each time in triplicate. The results were reported as the mean ± standard deviation (mean ± SD). The calibration curves (radical scavenging activity against the concentrations of the standards at 5, 2.5, 1.25, 0.625, and 0.31 µg/mL) provided the basis for calculating the amounts of antioxidants in the crude extracts and the half maximal inhibitory concentration (IC50) values of the extracts.