Antioxidant and Anti-Inﬂammatory Activity of Five Medicinal Mushrooms of the Genus Pleurotus

: Within the group of higher fungi, edible medicinal mushrooms have a long history of being used as food and in folk medicine. These species contain biologically active substances with many potential beneﬁcial effects on human health. The Pleurotus genus is representative of medicinal mushrooms because Pleurotus ostreatus is one of the most commonly cultivated culinary mushrooms. In our study, we focused on lesser-known species in the genus Pleurotus and measured their antioxidant and anti-inﬂammatory activity. We prepared extracts of the mushrooms and analyzed them using HPLC − HRMS, GC − MS, and 1 H-NMR. Signiﬁcant differences in biological activities were found among the Pleurotus spp. extracts. A MeOH extract of P. ﬂabellatus was the most active as a radical scavenger with the highest ORAC, while a chloroform extract had signiﬁcant anti-inﬂammatory COX-2 activity. The 80% MeOH extract of P. ﬂabellatus contained the highest amounts of ergosterol, ergothioneine, and mannitol. The 80% MeOH extract of P. ostreatus Florida was the most active in the NF- κ B inhibition assay and had the highest content of β -glucans (43.3% by dry weight). Given the antioxidant and anti-inﬂammatory properties of P. ﬂabellatus , the potential therapeutic usefulness of this species is worth evaluating through in-depth investigations and conﬁrmation by clinical trials.


Introduction
Edible mushrooms have been a significant part of human culture since ancient times [1] and those with medicinal properties have had a long tradition in the treatment of various human diseases [2]. In addition to their high content of micro and macronutrients [1], mushrooms also produce a wide array of secondary metabolites including biologically active compounds [2]. Out of the approximately 14,000 known mushroom species, about 700 are considered pharmacologically active [3]. Some of these mushroom species are consumed directly or are employed as food supplements and functional foods [4]. In view of their remarkable usefulness as both a food and a medicine, the new term, 'mushroom nutraceutical', was introduced to describe these edible medicinal mushrooms [5].

Samples and Cultivation
The following species and varieties were chosen from the collection of the Research Institute of Crop Production in Prague: P. flabellatus 5013, P. pulmonarius KZ50, P. opuntiae 5012, P. ostreatus Sylvan Ivory, and P. ostreatus 5175 Florida. Pleurotus spp. mushrooms were cultivated and harvested by the Department of Horticulture at CZU Prague on wheat straw pellets substrate. Two thousand five hundred g of substrate was pasteurized (90 • C for 24 h), moistened with water (67-69% humidity), and placed in polypropylene bags. Three percent grain spawn from selected Pleurotus samples was added into the cooled substrate. The samples were mixed thoroughly with the substrate and cultivated at 24 • C for 21 days. The samples were then switched to 17 • C in a relative humidity of 85-90% and a light of 1000 lux for the next 21 days and then harvested.

Sample Preparation
The harvested mushrooms were freeze dried for three days, ground, and stored at room temperature in a dark place. A modified procedurewas used for sample extraction [31]. The sample (one gram) was extracted separately with (a) 12 mL of 80% methanol (80% MeOH) and (b) 10 mL of chloroform (CHL) plus 1 mL of distilled water for 30 min on an orbital shaker at 210 RPM. The extract was sonicated for 1 min at room temperature (Sonorex Digitec DT 255 H, 160/640 W, Bandelin, Berlin, Germany) and centrifuged at 24,400× g for 10 min at room temperature (Rotanta 460R, Hettich, Germany). The supernatant was transferred into an evaporation flask and the rest matrix was re-extracted using the same procedure. The supernatants were mixed and evaporated at 30 • C on a rotary evaporator (Büchi AG, Flawil, Switzerland). After evaporation, the flask containing the evaporated extracts was weighed and the extract were resuspended with the corresponding extraction solvent: (a) 10 mL of 80% MeOH or (b) 10 mL CHL. One mL of resuspended extract was collected for HPLC−HRMS analysis; the rest of the extract was evaporated, and then the flask was weighed again. These residual extracts were resuspended in dimethyl sulfoxide (DMSO) and collected in order to assay anti-inflammatory and antioxidant activities. For GC−MS analysis, the procedure was the same as the previous CHL procedure but the extraction solvent was hexane:diethyl ether (3:1, v/v) and the final resuspending volume was 2 mL. All extracts were prepared in triplicate and stored at −18 • C. For 1 H-NMR spectroscopy, the extraction was performed based on a modified protocol proposed by Kim, Choi, and Verpoorte (2010) [32]. Each 50.0 mg of dried sample was extracted with 600 µL of 80% MeOH for 30 min with shaking. The extracts were sonicated for 5 min at room temperature and centrifuged at 24,400× g for 10 min to separate the extract from the matrix. After collecting the supernatants, the extraction process was repeated with 600 µL of the same solution. Both supernatants were mixed and evaporated using a stream of nitrogen gas. The residue was dissolved in 400 µL of deuterated methanol-D 4 and 400 µL of KH 2 PO 4 buffer in deuterated water (pH 6.0) containing 0.1% TSP-D 4 (trimethylsilylpropionic acid, sodium salt-D4, wt/wt). The mixture was centrifuged at 24,400× g for 10 min at room temperature. Supernatants (600 µL) were transferred to 5 mm NMR tubes and subjected to 1 H-NMR analyses (see Section 2.2.3).

Liquid Chromatography−Mass Spectrometry (LC−MS) Analysis
A non-targeted screening analysis of the crude extracts (80% MeOH) from the five Pleurotus species was performed using the LC-HRMS system consisting of the Ultimate 3000 UPLC chromatograph Thermo Fisher Scientific (Waltham, MA, USA) with a Q-TOF highresolution mass spectrometer (Impact II, Bruker Daltonic, Bremen, Germany). An Acclaim RS-LC 120 C18 column (2 µm, 2.1 × 100 mm, Thermo Scientific, Waltham, MA, USA) was used for chromatographic separation. The column temperature was set at 35 • C. Two types of polar phases (A1-5 mM ammonium formate (COONH 4 ) and A2-0.1% formic acid (HCOOH) in water) were chosen as the mobile phase due to the differing ionization properties of the molecules. Methanol (MeOH) was used as the organic phase (B). In order to ensure the widest possible range of polarity of the analytes contained in the extracts, a gradient ranging from 2% to 100% organic phase (MeOH) over 26 min and then isocratic for 10 min with 100% MeOH, was used for analysis. Each sample was analyzed with both mobile phase systems (A1/B, A2/B). The flow rate of the mobile phase was 0.25 mL/min and the injection volume of the sample was 5 µL. The m/z range of the monitored masses was between 60 and 1500. For the non-target analysis, an electrospray ionization (ESI) in both positive and negative mode data was applied. The data were collected in ddMS 2 mode in order to contain fragmentation spectra of the most important ions for later identification.
The content of ergosterol was determined using the same LC−MS system with APCI ionization in positive mode. The mobile phase consisted of 0.2% formic acid (polar phase A) and methanol (organic phase B). An elution gradient was used: for the first minute the mobile phase was kept on 60% B, then the percentage of B grew to 100% in 6 min, and remained on this level for the next 10 min. Then, the level of B decreased to the starting conditions (60% B) and was kept at this level for 5 min to achieve system equilibration. The same chromatographic column that was used in the non-target analysis was used. The injection volume of samples was 5 µL. The flow rate of the mobile phase was set to 0.35 mL/min and the column temperature was set to 35 • C. Ergosterol was identified by the commercially available standard (Pharmaceutical Secondary Standard; Certified Reference Material, Sigma-Aldrich, St. Louis, MO, USA). Detailed mass spectrometry measurement parameters for both ion source analyses are shown in Table S1. The identification of ergosterol was based on retention time and the exact mass of the ion [M + H] + = 397.3470, which was also used as a quantification ion, and [M-H 2 O] + = 379.3365, which was used as a confirmation ion. The following values were calculated for ergosterol: LOD = 121.8 ng/mL, LOQ = 406.1 ng/mL, and a precision (RSD) of 2.34%. Ten points of the concentration range 20-2500 ng/mL were used for calibration curve construction and the R 2 of the curve was 0.9991. A carryover effect was monitored by measuring solvent blanks between the samples. The quantification of analytes was performed using TASQ 2.2 software (Bruker Daltonik, Bremen, Germany).
In the non-target analysis, the data acquisition and the first step of data processing was performed by Otof Control 5.2, Bruker Compass Hy Star 5.1 and DataAnalysis 5.2 software (all Bruker Daltonik, Bremen, Germany). The results were then processed using XCMS (Scripps Research) and Mzmine software (authors: Matej Orešič, Mikko Katajamaa) and based on these outputs, sets of difference signals (features) were identified for each type of crude extract. The total number of identified features (about 120,000) was subsequently reduced to the resulting several hundred potential metabolites by applying criteria like intensity, peak shape, m/z and retention time, and exclusion of isotopic ions and adducts. The reduced dataset (5000 features) with the highest importance was normalized (Pareto scaling), cleaned using principal component analysis (PCA) in the online free software MetaboAnalyst (v 5.0, Xia Lab, Ste. Anne de Bellevue, QC, Canada) by selection of the most important features responsible for the biggest differences among samples. The final number of features was then reduced to 330 by removing in-source fragment ions and other artifacts.

Gas Chromatography Coupled to Mass Spectrometry (GC−MS) Analysis
For GC−MS, a modified sample preparation protocol by Pedneault et al. (2007) was used [33]. GC Agilent Technologies 7890A, MS 5975C (Palo Alto, CA, USA) was used for the analysis of non-polar compounds extracted with hexane:diethyl ether (3:1, v/v). One µL of extract was injected in split mode (1:12) into a system equipped with an Agilent J&W DB-5MS column (Palo Alto, CA, USA). The separation was performed using a temperature gradient program, starting at a temperature of 60 • C, which then increased to 280 • C in 3 • C increments. Mass spectra were collected in TIC mode.

Proton Nuclear Magnetic Resonance ( 1 H-NMR) Analysis
The 1 H-NMR parameter for spectral acquisition was used according to the protocol by Mascellani et al. (2021) [34]. All spectra were recorded at 298 K (25 • C) on a Bruker Avance III HD spectrometer equipped with a broadband fluorine observation (BBFO) SmartProbe™ with z-axis gradients (Bruker BioSpin GmbH, Rheinstetten, Germany), operating at a 1 H-NMR frequency of 500.18 MHz. The spectrometer transmitter was locked to deuterated MeOH and all spectra were recorded with the Bruker pulse sequence 'noesypr1d' for presaturation of the water signal at 4.704 ppm. Each sample was collected in 64 k data points after 128 scans and 4 dummy scans using a spectral width of 8000 Hz. The receiver gain was set to 18, the relaxation delay was 1 s, the acquisition time was 4 s, and the mixing time was 0.1 s. The free induction decay was multiplied by 0.3 Hz (line broadening) before Fourier transformation. TSP was used to calibrate to 0.0 ppm. The acquired 1 H-NMR spectra were phased and corrected for baseline using Chenomx NMR suite 9 software professional edition (Chenomx Inc., Edmonton, AB, Canada). The assignment of signals was performed using spiked samples, in-house, and built-in databases. The spiking for ergothioneine was performed using the commercially available standard (purity ≥ 98.0%, Sigma-Aldrich, St. Louis, MO, USA) to confirm the annotation. The ability of the tested samples to inhibit DPPH (2,2-diphenyl-1-picrylhydrazyl; Sigma-Aldrich, St. Louis, MO, USA) radicals was determined using the method described by Sharma and Bhat (2009) [35] with modification. Initially, two-fold serial dilutions of each sample were prepared in analytical grade MeOH (VWR, Radnor, PA, USA) in 96-well microtiter plates. Subsequently, 100 µL of freshly prepared 0.25 mM DPPH in MeOH was added to each well and mixed with the samples, creating a concentration range of 512 to 8 µg/mL. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control at concentrations of 64, 32, 16, 8, 4, 2, and 1 µg/mL, with MeOH used as a blank. The absorbance was measured at 517 nm using a Synergy H1 multi-mode reader (BioTek, Winooski, Winooski, VT, USA). Results were expressed as half-maximal inhibitory concentrations (IC 50 in µg/mL) and Trolox equivalents (mg TE/g extract).

Oxygen Radical Absorbance Capacity (ORAC) Assay
The sample's ability to retard the AAPH-induced oxidant decay of fluorescein (FL) was measured by the method of Ou and Hampsch-Woodill (2001) [36] with modification. All samples and reagents were prepared and diluted in 75 mM phosphate buffer (pH adjusted to 7.0 with HCl). Inorganic acids and salts used for buffer preparation included monopotassium phosphate (KH 2 PO 4 , Lach-ner, Neratovice, Czech Republic), dipotassium phosphate (K 2 HPO 4 , Sigma-Aldrich, St. Louis, MO, USA), and hydrochloric acid (Penta, Czech Republic). The outer wells of black absorbance 96-well microtiter plates were filled with 200 µL of distilled water in order to provide better thermal mass stability, as suggested by Held (2005) [37]. Afterwards, 25 µL of each sample was transferred to the plates, diluted with 150 µL of fluorescein (54 nmol/L) (Sigma-Aldrich, St. Louis, MO, USA), and incubated at 37 • C for 10 min. To initiate the reaction, 25 µL of 153 mmol/L AAPH (2,2 -azobis(2-amidinopropane) dihydrochloride, Sigma-Aldrich, St. Louis, MO, USA) was added to each well. Fluorescence decay was measured at one-minute intervals for two hours with excitation and emission wavelengths set at 494 and 519 nm. Trolox at 8, 4, 2, 1, and 0.5 µg/mL was used as a positive control with the buffer used as a blank. Samples were tested at a final concentration of 1024 µg/mL. The ORAC values were calculated as the area under the curve (AUC) values and compared with the calibration curve of Trolox, as previously suggested by Ou and Hampsch-Woodill (2001) [36]. Results were expressed as Trolox equivalents (mg TE/g extract).

COX-2 Anti-Inflammatory Activity Assay
Human recombinant cyclooxygenase-2 (COX-2; Sigma-Aldrich, St. Louis, MO, USA) was used in an in-vitro enzymatic assay. COX-2 (0.5 unit/reaction) was added to 180 µL of 100 mM Tris buffer (pH 8.00) containing 5 µM hematin (porcine), 18 mM (L)-(−)epinephrine, and 50 µM Na 2 EDTA. Mushroom extracts in 80% MeOH were evaporated and dissolved in DMSO (VWR, Radnor, PA, USA). The final concentration of the extracts in the reaction was 10 µg/mL. Pure DMSO was used as a blank and (S)-(+)-ibuprofen (Sigma-Aldrich, St. Louis, MO, USA) was used as a reference inhibitor (positive control). Aliquots of 10 µL of extract were incubated with the reaction mixture for 5 min at room temperature. The reaction was then initiated by the addition of 5 µL of 10 µM arachidonic acid and incubated for 20 min at 37 • C. The reaction was stopped by the addition of 20 µL of 10% formic acid (v/v). The inhibitory activity was calculated as the percentage inhibition of prostaglandin E 2 (PGE 2 ) production compared to the blank. The concentration of PGE 2 was quantified using a prostaglandin E 2 ELISA kit (Enzo Life Sciences, Farmingdale, NY, USA).
Samples were diluted (1:15) and incubated according to the manufacturer's instructions. Absorbance was measured at 405 nm using a Tecan Infinite M200 microplate reader (Tecan Group, Männedorf, Switzerland). The results were expressed as the percent inhibition of the sample compared to the blank.

Cell-Based Assays
The THP-1-XBlue™-MD2-CD14 cell line was purchased from Invivogen (San Diego, CA, USA) and was cultured as reported previously by Hosek et al. (2011) [38]. In more detail, the cells were cultivated at 37 • C in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere containing 5% CO 2 . The WST-1 assay was used to test the effects of extracts on the viability of using the THP-1-XBlue™-MD2-CD14 cells according to the manufacturer's instructions. The antiproliferative activity of the test extracts from P. flabellatus 5013, P. opuntiae 5012, P. ostreatus 5175 Florida, and P. pulmonarius KZ50 was tested at five concentrations ranging from 0.25 to 20 µg/mL. Based on these results, the non-toxic extract concentration of 10 µg/mL was used for subsequent cell-based assays.

Cellular Antioxidant Activity (CAA) Assay
The antioxidant activity of test extracts was measured using the method of Wolfe and Liu (2007) [39] with some modifications, as reported by Malanik et al. (2020) [40]. THP-1-XBlue™-MD2-CD14 cells were pre-incubated for 1 h in a serum-free RPMI 1640 medium containing 25 µM 2 ,7 -dichlorodihydrofluorescein-diacetate (DCFH 2 -DA; Sigma-Aldrich, St. Louis, MO, USA) dissolved in DMSO [the final concentration of DMSO in the medium was 0.1% (v/v)] at 37 • C. After that, the cells were centrifuged, washed with phosphatebuffered saline, re-suspended in serum-free RPMI 1640 medium, and placed into 96-well plates in triplicates-60,000 cells/well. The cells were then incubated with the extracts for 1 h and after that, 2,2 -azobis(2-methylpropionamidine) dihydrochloride (AAPH; Sigma-Aldrich, St. Louis, MO, USA) was added (at a final concentration of 600 µM) to induce the generation of ROS. The plate was immediately placed into a FLUOstar Omega microplate reader (BMG Labtech) tempered at 37 • C. The level of oxidized fluorescent 2 ,7dichlorfluorescein (DCF) was measured every 5 min for 1 h (excitation wavelength at 485 nm; emission at 538 nm). Each plate included triplicate control and blank wells: control wells contained cells treated with DCFH-DA and AAPH; blank wells contained cells treated with the dye and serum-free RPMI 1640 medium without AAPH. Quercetin (Koch-Light Laboratories, Haverhill, UK) was used as a positive control at the same concentration as the test compounds at a concentration of 10 µg/mL. The solvent (80% MeOH) was used as the negative control (NC).
After the blank was subtracted from the fluorescence readings, the area under the curve of fluorescence versus time was integrated to calculate the CAA values of the test compounds: CAA unit = 100 − ( SA/ CA) × 100, where SA is the integrated area under the sample fluorescence versus time curve and CA is the integrated area obtained from the control curve.

Detection of the Activation of NF-κB/AP-1
The anti-inflammatory activity of the extracts was also investigated in terms of their effect on NF-κB/AP-1 signaling in lipopolysaccharide (LPS)-stimulated THP-1-XBlue™-MD2-CD14 cells (Invivogen, San Diego, CA, USA). The cell line expresses an NF-κB/AP-1inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. The cells were incubated for 24 h with test extracts dissolved in 80% MeOH at a concentration of 10 µg/mL. Solvent alone was used as a negative control (NC) and 10 µM prednisone was used as a positive control (PC).

Determination of the Content of Glucans
The "MUSHROOM and YEAST β-GLUCAN" K-YBGL 07/11 analytical set (Megazyme International, Wicklow, Ireland) was used for the determination of the total αand βglucans [41]. The assay determines the difference between the glucose content after total acidic hydrolysis of glucans and specific enzymatic hydrolysis of starch-like α-glucans. Samples were solubilized in concentrated hydrochloric acid (37%; 10 N) and then hydrolyzed with 1.3 mol/L hydrochloric acid at 100 • C for 2 h. Total hydrolysis was completed via incubation with a mixture of exo-(1-3)-β-glucanase and β-glucosidase. Starch-like α-glucans were solubilized in 2 mol/L potassium hydroxide and the mixture was neutralized with an excess of 1.2 mol/L sodium acetate buffer (pH 3.8). The dissolved glucans were then hydrolyzed with amyloglucosidase. The β-glucan, or non-starch glucan, content was calculated as the difference between the glucose content after total acidic hydrolysis of glucans and specific enzymatic hydrolysis of α-glucans. All measurements were made in triplicate.

Statistical Analysis
Statistical analyses were carried out using Statistica (TIBCO Software, CA, USA) and MetaboAnalyst v. 5.0 (Xia Lab, Ste. Anne de Bellevue, Quebec, Canada). The data are presented as means ± SD. To determine the significance of sample differences, one-way ANOVA, followed by a post-hoc Tukey test (α = 0.01) was used. For cell-based assays, the statistical analyses were carried out using IBM SPSS for Windows, software version 26.0 (Armonk, NY, USA). The data were graphed as a mean ± SD. Comparisons between groups were made using a Kruskal-Wallis test, followed by pair-wise comparison with Bonferroni correction. Table 1 shows the extraction yields from mushroom samples. There was a significant difference in the extraction yield between the extraction solvents. The yield of 80% MeOH samples ranged between 28.4 and 32.9% and was higher than the yield of CHL extracts, which ranged from 2.5-4.5%.

Extraction Yield of Mushroom Extracts
The difference may have been caused by the low content of lipids and nonpolar compounds in the mushroom samples. Eighty percent MeOH has a high extraction power for a broad spectrum of the polar or slightly nonpolar compounds present in mushrooms. According to Alam et al. [42], dried mushrooms of three Pleurotus species were determined to contain 4.4-4.6% lipids, 38.8-42.8% carbohydrates, 20.6-24.6% proteins, 8.3-9.4% ash, and 22.9-24.3% fiber. According to Chaiharn et al. [43], the extraction yield of two P. ostreatus samples was 16.00% and 22.25% for water and 6% and 10% for MeOH. The a-c within a column means that significance was determined by one-way ANOVA followed by post-hoc Tukey test (p < 0.01).

LC−MS Analysis of Mushroom Extracts
Preliminary identification based on the screening data obtained from chloroform extracts indicated the presence of a number of lipids such as phospholipids and acylglycerol-and steroid-type compounds. Higher molecular weight compounds, such as oligosaccharides, peptides, and their conjugates with smaller molecules, were found in 80% MeOH extracts, as indicated by the collected MS 2 spectra. Untargeted HPLC−HRMS analysis by PCA score plot based on 330 of the most important features ( Figure S1) showed specific differences between P. flabellatus and other samples, which was confirmed by hierarchical clustering (Figure S2). Subsequent tentative identification of the most important features using MS 2 analysis showed that most of them were related to higher molecular structures, especially oligosaccharides and peptides.
An example of an identified low molecular weight metabolite with possible biological activity in these extracts was ergothioneine, whose identity was also confirmed by the fragment spectrum ( Figure S3). Another abundant compound identified in the analysis was ergosterol ( Table 2). The a,b within a column means that significance was determined by one-way ANOVA followed by post-hoc Tukey test (p < 0.01).
The highest ergosterol content was found in P. flabellatus 5013 and the lowest content was in P. ostreatus Sylvan Ivory. The ergosterol level in selected mushroom samples ranged from 214.5-163.7 mg/kg of d.w. powder and 538.5-805.3 µg/g of dry extract.

GC−MS Analysis of Mushroom Extracts
The predominant compound in all measured extracts was ergosterol, a precursor of vitamin D 2 . The other most abundant compounds were isomers of 2-butyl-2-octanol, dodecanol, and 2-hexyl-1-octanol derivatives, which are common in mushrooms [44]. The P. ostreatus 5175 Florida showed the greatest difference among the samples because of the presence of octadecadienoic acid derivatives ( Figure S4). Table 3 shows the results of the 1 H-NMR spectroscopy of selected analytes. The most abundant were the saccharides trehalose and mannitol, followed by specific amino acids, organic acids, and amines including choline and ergothioneine. The highest content of ergothioneine was detected in P. flabellatus 5013. In general, the ergothioneine content was higher in lesser-known species compared to P. ostreatus samples. The most abundant substance was trehalose (105.0-347.7 mg/g of dry extract); however, in P. flabellatus 5013, the mannitol content was higher than trehalose. The content of mannitol ranged from 18.14 to 144.3 mg/g of the extract and the lowest mannitol level was found in P. ostreatus 5175 Florida. Since all tested mushrooms were cultivated in similar conditions, this variability may be mainly caused by genetic differences and subsequent changes in metabolic pathways. According to Lin et al.'s [45] phylogenetic analysis of Pleurotus species, P. ostreatus and P. flabellatus are the most distinct among the tested species, which is in agreement with the results of our metabolomic and bioactivity investigations, wherein usually these two species are the most distinct. The a-d within a column means that significance was determined by one-way ANOVA followed by post-hoc Tukey test (p < 0.01).

Content of Glucans
The total glucan content (Table 4) ranged from 35.8 ± 0.3% to 49.0 ± 0.3% of d.w. and P. opuntiae 5012 had the highest content of α-glucans (4.5-9.8%). The range of β-glucan content was 31.53 ± 0.6% to 43.3 ± 0.6% w/w and P. ostreatus 5175 Florida had the highest β-glucan content of all the samples (43.3 ± 0.6%, w/w). According to Avni et al. [46], the total glucan content ranged from 20.25 ± 0.52% to 48.27 ± 0.68% in Pleurotus spp. and the α-glucan content was 0.47 ± 0.02% to 4.57 ± 0.06%. Lam and Okello [47] determined that the total glucan content of P. ostreatus PL 132 was 27.1% (w/w) and the β-glucan content was 23.9% (w/w). The differences in glucan levels may have been caused by genetic factors. The a-d within a column means that significance was determined by one-way ANOVA followed by post-hoc Tukey test (p < 0.01). The highest values in bold.

Antioxidant Activity
There was a difference in ORAC and DPPH antioxidant activity between the different solvent extracts ( Table 5). The IC 50 of CHL extracts was <0.68 mg of TE (Trolox equivalent)/g extract, which is considered very low (data not shown). The activity of 80% MeOH extracts ranged from 4.3 to 24.9 mg of TE/g extract in the DPPH assay and 21.7 to 63.9 mg of TE/g extract in the ORAC assay. P. flabellatus 5013 was the most active species in both assays. The DPPH and ORAC results are expressed as means ± SD for four independent experiments measured in triplicate, while the CAA assay results are given as means ± SE for four independent experiments measured in triplicate. Abbreviations: n.d. = not determined; TE = Trolox equivalent. Values representing the highest activity in bold. The a-d within a column means that significance was determined by one-way ANOVA followed by post-hoc Tukey test (p < 0.01).
The advantage of CAA assays over ORAC and DPPH assays is the involvement of the cellular environment in the activity [48]; however, our results in the CAA assay did not confirm the promising activity of the P. flabellatus 5013 extract. None of the tested extracts had significantly higher activity than the quercetin control. The observed discrepancy might be explained by the different mechanisms tested in chemical-based antioxidant assays (e.g., ORAC and DPPH) and CAA, a cell-based assay. For an extract to be active in the CAA assay, first, the cellular membrane must be crossed by the active constituents. Secondly, the antioxidants must then scavenge various types of peroxyl radicals and other ROS formed in a cellular environment, which are obviously different from those formed in ORAC or DPPH assays. The tested extracts proved inactive in either one of these two aspects or both [48].
Bakir et al. [49] measured the DPPH antioxidant activity in P. ostreatus stored for 24 h at 20 • C and obtained an IC 50 of 321 µg/mL, while a sample stored at −40 • C had significantly decreased antioxidant activity with an IC 50 of 3486 µg/mL. The IC 50 range in our study was 200.4 to 1134.0 µg/mL. We used three-day lyophilized mushroom samples, stored frozen and our IC 50 results are similar to those of Bakir et al. [49]. The antioxidant activity of mushroom extract may be linked to ergothioneine (ERG), a water-soluble thiol derivative of histidine, which was previously shown to be associated with antioxidant activity in vitro and in vivo [50]. Many studies confirmed the presence of ergothioneine in various Pleurotus species [29,[51][52][53].
According to Halliwell, Cheah, and Tang (2018), the ERG content in food is quite low, yet the human body can accumulate it efficiently. ERG is transported in the blood via a specific transporter (OTCN1) and ERG levels appear to decline during neurodegenerative and cardiovascular disorders, which are associated with oxidative stress. This suggests that ERG may play an important role as a dietary antioxidant. The ERG content in various foods is variable. P. eryngi, for example, was found to contain 541.7 mg of ERG per kg dry weight and in other mushrooms, it ranged from 5.8 to 1812.4 mg/kg d.w [50]. The ERG content of P. cintronopileatus was 3.94 mg/g d.w. compared to 1.21 mg/g d.w. in P. ostreatus [52], which indicated that some lesser-known species of Pleurotus could contain a higher concentration than P. ostreatus. A few non-mushroom foods such as tempeh have a high ERG content (2011.3 mg/kg), but foods like rice, milk, nuts, beans, spices, and vegetables have significantly lower ERG levels at about 49.2 mg/kg d.w. Only fungi are considered to have the necessary biosynthetic apparatuses for ERG biosynthesis and its presence in tempeh is perhaps the result of culturing with Rhizopus fungi [50]. Dubost et al. [54] determined that P. ostreatus contained 2010 mg of ERG per kg d.w. and P. eryngii had 1720 mg ERG/kg d.w., which is comparable to our results.
Tsiapali et al. [55] showed that polysaccharides, such as glucans, have only weak in vitro free radical scavenging ability and may actually stimulate free radical activity in murine macrophages, increasing the level of reactive oxygen species associated with immunomodulation. Trehalose, a disaccharide found in our samples in large quantities, was also proven to decrease oxidative stress in vivo by enhancing the expression of antioxidant genes [56]. According to Radbakhsh et al. [57], trehalose was effective in increasing the total antioxidant capacity and thiol groups in serum in an experimental type 2 diabetes rat model. J.-H. Liu et al. [58] claimed that mannitol acted like a free-radical scavenging agent in vivo against peroxyl radicals and Pelle et al. [59] showed that mannitol was able to protect DNA against UV damage in vivo but was not effective against oxygen radicals, as determined by increased malondialdehyde as a marker level in vivo. Results published by Meza-Menchaca et al. [60] demonstrated that ergosterol peroxide may also be linked to antioxidant activity. The analysis of antioxidant activity in the presence of 80% MeOH extract constituents showed a high correlation of ERG content with IC 50 (R = −0.71%) and nicotinate content (−0.90%) (the lower IC 50 means higher antioxidant activity). Nicotinate or niacin (vitamin B 3 ) is part of an important redox agent in the NAD(P) + /H system [61] and, therefore, has the ability to prevent oxidative stress [62,63] and act as an anti-inflammatory agent [63].

Anti-Inflammatory Activity
In the COX-2 inhibitory assay, the non-polar CHL extracts were more active than the 80% MeOH extracts (Table 6), which were virtually inactive and did not even reach the 50% inhibition level. The CHL extract activity was 43.6% to 85.0% of the control. The most active extract in the COX-2 assay was from P. flabellatus 5013. In the NF-κB/AP-1 inhibition assay, the extract activity ranged from 72.7% to 83.4% of the NC, while the inhibitory effect of prednisone (PC) was 79.2 ± 8.0%. The most active sample was P. pulmonarius KZ50. Each assay involves a different mechanism, so the results are not fully comparable. 100.0 ± 12.2 b The results are expressed as means ± SD for three (*) or four (**) independent experiments measured in triplicate. Abbreviations: n.d. = not determined. The a,b within a column indicates significance as determined by one-way ANOVA followed by post-hoc Tukey test (p < 0.01).
As reported in previous studies, ergosterol may also be linked to anti-inflammatory and immunomodulatory effects. Ergosterol decreased p-NF-κB and COX-2 expression in a mouse model of diabetic nephropathy [64]. Ergosterol is one of the most abundant metabolites in many fungal species and it was detected in the mushroom extracts in our study as well as in other studies [65][66][67][68]. Previous studies suggested that ergosterol, a provitamin form of vitamin D 2 whose mechanism of effect is unclear, is beneficial to human health by decreasing the risk of inflammatory disease. According to clinical studies, vitamin D deficiency is associated with inflammatory health risks [69].
The work of Du et al. [70] dealt with different types of glucans, including those that possess immunomodulatory and anti-inflammatory effects, mediated through cytokines such as TNF-α. The extract from P. ostreatus 5175 Florida was determined to contain the highest amount of total glucans and β-glucans and this may explain why it was the most active in inhibiting NF-κB/AP-1 activity. According to Deo et al. [71], an anti-inflammatory assay of P. ostreatus extract showed over 90% inhibition of TNF-α in the LPS-stimulated mouse macrophage cell 264.7.
Trehalose, which was also assayed in our study, was found to suppress NF-κB pathway activation in cultured cells [72]. The extract of P. ostreatus 5175 Florida was most effective at inhibiting NF-κB (72.2 ± 7.7% of NC), while the lowest activity was recorded for P. flabellatus 5013 extract, which correlated with the lowest measured trehalose level. However, it was reported that increasing the dietary level of trehalose by adding it to food may increase the risk of severe Clostridium infection in the gut [73].
Jedinak et al. [11] presented evidence that the P. ostreatus Florida extract had antiinflammatory activity as determined by an NF-κB/AP-1 assay in murine splenocytes and it may be effective as a dietary supplement for the reduction of inflammation. In the study, mushroom extracts were added to LPS-activated RAW264.7 mouse macrophages and an extract concentration of 100 µg/mL decreased TNF-α levels from 15.4 to 5.87 ng/mL, IL-6 from 16.1 to 1.16 ng/mL, and IL-12 from 108.8 to 39.2 ng/mL compared controls without extract.
An in vivo study of Wistar rats using the carrageenan-induced paw edema model of inflammation compared the anti-inflammatory activity of dried powdered P. ostreatus with an acetone extract of the same mushroom. The powdered sample at doses of 250-1000 mg/kg exerted a significant inhibition of rat paw edema, but the effect was not dose-dependent. The acetone extract of P. ostreatus (500 mg/kg) showed a maximum 87% inhibition of late phase inflammation, and this was also not dose-dependent [74].

Conclusions
In our study, we examined the biological activities of several species of mushrooms of the Pleurotus genus, including lesser-known varieties, and attempted to identify the compounds that were responsible for the observed effects. There is evidence that the less common varieties and species may have greater potential for antioxidant and anti-inflammatory properties than the conventional varieties. In the measurement of the total and β-glucans, the highest content was found in P. ostreatus, but there was relatively little difference in the content among the other species. P. flabellatus 5013 contained the highest level of ergosterol and mannitol was the most abundant compound determined. The results of the in vitro assays were confirmed by in vivo assays. Pleurotus flabellatus 5013 extract was the most effective in the antioxidant assay, which may be linked to the higher measured level of ergothioneine. In other samples, the most abundant compound was trehalose. In antioxidant assays, the 80% MeOH extracts displayed activity, while the CHL extracts did not. These results may also be linked to the ergothioneine content, as confirmed by 1 H-NMR and LC−MS analysis of the extracts. The COX-2 anti-inflammatory assay showed that CHL extracts were more active than 80% MeOH extracts. The ergosterol content determined in our study may be associated with this activity. The P. ostreatus 5175 Florida extract was the most active in the NF-κB inhibition assay, and this may be linked to the fact that it contained the highest total glucan content. On the other hand, P. flabellatus 5013, which had the highest antioxidant activity, was found to contain the lowest amount of total and β-glucans. A clinical trial to test the potential therapeutic effects of Pleurotus extracts ought to be conducted to confirm the results of this study.

Data Availability Statement:
The data presented in this study are available in Tables 1-6 and S1 and Figures S1-S4.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Sample Availability: Samples of the compounds are available from the authors.