1. 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].
The genus
Pleurotus includes types of edible, wood-decaying mushroom. Various
Pleurotus species have been employed throughout history in medicine [
6]; for example, they have bene used to strengthen joints, tendons, and muscles, improve cardiovascular health [
7], and stimulate the immune system. Currently, there are 207 classified species in the genus
Pleurotus [
8]. The oyster mushroom,
P. ostreatus (Fr.)
P. Kumm, is one of the most economically significant species [
9] and the second most important commercially cultivated mushroom [
10]. It has been proven in numerous studies that
P. ostreatus offers many health-promoting benefits such as anticancer, antioxidant, antitumor, antiviral, antibacterial, antidiabetic, and anti-hypercholesteremic activities. One of the most studied biological effects is the modulation of the immune system response, which involves different anti-inflammatory mechanisms [
11]. There are several other
Pleurotus species that are less common in the edible mushroom growing industry [
12], but still possess some bioactive properties. The pink oyster mushroom (
P. flabellatus) and the Indian oyster mushroom (
P. pulmonarius) were reported to have antioxidant, anti-inflammatory, and antimicrobial activities as well [
13,
14,
15,
16,
17,
18,
19,
20,
21,
22,
23,
24].
The fruiting body of
P. ostreatus contains approximately one hundred different compounds [
9], including polysaccharide-proteins, polysaccharide-peptides, proteoglycans, functional proteins, and polysaccharides [
25]. Specific proteoglycans from
P. ostreatus have been found to have anticancer, immunomodulatory [
26], and antioxidant activity [
27]. The mushroom cell walls are rich in non-starch polysaccharides, of which β-D-glucans are the most interesting functional components [
9] because they are the most common natural macrophage activators [
26]. The α- and β-glucans and high molecular weight polysaccharides were reported to increase the production of cytokines by dendritic cells, activate natural killer cells, and increase the production of macrophages [
11]. Besides the well-studied β-D-glucans,
P. ostreatus contains other compounds, e.g., phenolic compounds like protocatechuic acid, gallic acid, homogentisic acid, rutin, myricetin, chrysin, naringin, α- and γ-tocopherol, ascorbic acid, β-carotene [
9], cinnamic acid, p-hydroxybenzoic acid [
28], and ergothioneine, many of which are potent antioxidants abundant in
P. ostreatus; these are also found in other
Pleurotus species [
29].
Some other species or varieties of
Pleurotus that are less frequently employed in the mushroom cultivation industry and have not been covered by local legislation differ in content and type of bioactive metabolites but may contain novel compounds not found in the commercially cultivated varieties of
P. ostreatus [
30]. Therefore, based on these facts, this study focused on the exploration of the less common species and varieties of the
Pleurotus genus. Our major assumption was that because the various species and varieties of the genus differ extensively in the type and abundance of their constituents, they are likely to possess some novel biological activities.
2. Materials and Methods
2.1. Materials
2.1.1. 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.
2.1.2. 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
1H-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
2PO
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
1H-NMR analyses (see
Section 2.2.3).
2.2. Chemical Analysis
2.2.1. 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 high-resolution 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 (COONH4) 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 ddMS2 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
2O]
+ = 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.
2.2.2. 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.
2.2.3. Proton Nuclear Magnetic Resonance (1H-NMR) Analysis
The
1H-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
1H-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
1H-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.
2.3. Antioxidant Activity
2.3.1. Radical Scavenging Assay Using 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
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).
2.3.2. 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
2PO
4, Lach-ner, Neratovice, Czech Republic), dipotassium phosphate (K
2HPO
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).
2.4. 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 Na2EDTA. 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 E2 (PGE2) production compared to the blank. The concentration of PGE2 was quantified using a prostaglandin E2 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.
2.5. 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.
2.5.1. 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 phosphate-buffered 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′,7′-dichlorfluorescein (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.
2.5.2. 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-1-inducible 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).
2.6. 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 α- and β-glucans [
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.
2.7. Statistical Analysis
Statistical analyses were carried out using Statistica (TIBCO Software, CA, USA) and MetaboAnalyst v. 5.0 (Xia Lab, Ste. Anne de Bellevue, QC, 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.
4. 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 1H-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.