Antioxidant Properties, Bioactive Compounds Contents, and Chemical Characterization of Two Wild Edible Mushroom Species from Morocco: Paralepista flaccida (Sowerby) Vizzini and Lepista nuda (Bull.) Cooke

Mushrooms have been consumed for centuries and have recently gained more popularity as an important source of nutritional and pharmaceutical compounds. As part of the valorization of mushroom species in northern Morocco, the current study aimed to investigate the chemical compositions and antioxidant properties of two wild edible mushrooms, Paralepista flaccida and Lepista nuda. Herein, the bioactive compounds were determined using spectrophotometer methods, and results showed that the value of total phenolic content (TPC) was found to be higher in P. flaccida (32.86 ± 0.52 mg) than in L. nuda (25.52 ± 0.56 mg of gallic acid equivalents (GAEs)/mg of dry methanolic extract (dme)). On the other hand, the value of total flavonoid content (TFC) was greater in L. nuda than in P. flaccida, with values of 19.02 ± 0.80 and 10.34 ± 0.60 mg of (+)-catechin equivalents (CEs)/g dme, respectively. Moreover, the ascorbic acid, tannin, and carotenoids content was moderate, with a non-significant difference between the two samples. High-performance liquid chromatography–mass spectrometry (HPLC-MS) analysis allowed the identification and quantification of thirteen individual phenolic compounds in both P. flaccida and L. nuda, whereas p-Hydroxybenzoic acid was recognized as the major compound detected, with values of 138.50 ± 1.58 and 587.90 ± 4.89 µg/g of dry weight (dw), respectively. The gas chromatography–mass spectrometry (GC-MS) analysis of methanolic extracts of P. flaccida and L. nuda revealed the presence of sixty-one and sixty-six biomolecules, respectively. These biomolecules can mainly be divided into four main groups, namely sugars, amino acids, fatty acids, and organic acids. Moreover, glycerol (12.42%) and mannitol (10.39%) were observed to be the main chemical compositions of P. flaccida, while L. nuda was predominated by linolelaidic acid (21.13%) and leucine (9.05%). L. nuda showed a strong antioxidant property, evaluated by DPPH (half maximal effective concentration (EC50) 1.18–0.98 mg/mL); β-carotene bleaching (EC50 0.22–0.39 mg/mL); and reducing power methods (EC50 0.63–0.48 mg/mL), respectively. These findings suggested that both mushrooms are potential sources of various biomolecules, many of which possess important biological activities which are interesting for the foods and pharmaceuticals industry.


Introduction
Mushrooms have been consumed for centuries due to their nutritional and medicinal benefits. In terms of nutritional value, the fruiting bodies of mushrooms are known

Extraction Yield
As presented in Table 1, the extraction yields of methanolic extracts of P. flaccida (30.32 %) and L. nuda (31.69 %) were statistically similar to each other's, while lower than the previous yield in the Portuguese Lepista inversa (39%) which was reported by Heleno et al. [29].  1 Values are expressed as means ± SD of three independent measurements. * p < 0.05 indicates that the mean value of at least one component differs from the others. For each mushroom sample, means within a line with different letters differ significantly (p < 0.05).

Estimation of Bioactive Compounds
The bioactive compound contents in the studied mushroom samples were estimated using a UV-Visible spectrophotometer, and the results are presented in Table 1.
Total phenolic contents in the methanolic extract were observed to be significantly important in both tested species, although P. flaccida was shown to have a higher amount, with the value of 32.86 mg GAE/g of dme, which is higher than the previous studies by Heleno et al., (3.60 mg) [29] and Vaz et al., (10.8 mg in ethanolic extract) [30] in Lepista inversa. Similarly, L. nuda content (25.52 mg) was noted to be higher than several works from Morocco, Turkey, Portugal and Turkey, with values of 11.83, 7.7, 6.31, and 4.18 mg GAE/g of dme, respectively [25,[31][32][33], which was close to the amount in the given results in the Argentinian (27.34 mg) [34] and the Indian mushrooms (23.77 mg) [35].
Concerning total flavonoid contents, the methanolic extract of L. nuda was given, statically, as a more important content than P. flaccida, with the values of 19.02 and 10.34 mg CE/g of dme, respectively. However, Barros et al. [32] (3.36 mg CE/g dme) and Sharma et al. [35] (2.47 mg quercetin equivalent/g dme) found lower flavonoid contents in L. nuda than in the present study.
Regarding ascorbic acid content, the fruiting body of samples presented a moderate result and there were no significant differences between P. flaccida (1.27 mg/g) and L. nuda (1.31 mg/g). The amount of ascorbic acid in L. nuda was observed to be higher than the values of 0.34 and 0.23 mg/g obtained in the previous studies [32,35], respectively. However, ascorbic acid was not detected in the work by Lkay Koca et al. [31].
The amount of tannin content in P. flaccida was found to be significantly similar to L. nuda, with the values of 2.67 and 2.26 mg CE/g of dw, respectively. To our knowledge, there were no previous studies on the tannin content of both samples.
As shown in Table 1, β-carotene and lycopene contents were observed to be present statistically in small quantities in comparison with the premier bioactive compounds. However, the values of β-carotene and lycopene from L. nuda were higher than the previous study from India [35] (0.39 and 0.20 µg/100 g), while smaller than the one reported in the Portuguese sample [32] (2.52 and 0.98 µg/g).
Overall, the content of bioactive compounds determined in the studied wild edible mushrooms from Morocco was very important, although these compounds have been previously estimated in many other mushrooms and are known for their strong antioxidant capacity [5].

Phenolic Compounds by HPLC-MS Analysis
The identification and quantification of individual phenolic compounds in fruiting body extracts of P. flaccida and L. nuda were performed using the HPLC-MS technique. The chromatogram illustrating the phenolic compounds peaks in P. flaccida and L. nuda is shown in Figure 1 and Figure S1, respectively, whereas Table 2 gives the amounts of the thirteen compounds identified and quantified by using standards and their mass spectra. The HPLC-MS results showed that p-hydroxybenzoic acid was recognized as the major phenolic compound identified and quantified in both mushrooms P. flaccida and L. nuda, with values of 138.50 and 587.90 µg/g dw, respectively. Chlorogenic acid (136.30 µg/g) was classified as the second main compound in P. flaccida, followed by gallic acid (132 µg/g) and cinnamic acid (124.20 µg/g), while catechin (400.20 µg/g), ellagic acid (362.60 µg/g) and chlorogenic acid (327.60 µg/g) were listed as the second, the third and the fourth main phenolic compounds detected in L. nuda extract, respectively. The lowest component that had been detected was syringic acid for both P. flaccida and L. nuda, with values of 11.25 and 8.57 µg/g dw, respectively. However, the phenolic compounds rutin, vanillin, rosmarinic acid, salicylic acid and quercetin were not detected in either sample. Statistically, and except gallic acid, all phenolic compounds characterized in the current work showed a significant difference in the comparison between the two tested mushrooms (Table 2).
There have been a few investigations on phenolic compounds of the studied mushrooms, whereas P. flaccida phenolics characterization was only reported by the study of Vaz et al. [36] under the name Lepista inversa, without detecting any compounds in their samples; however, L. nuda individual phenolic compounds were analyzed in three previous research works from two countries (Portugal and Argentina). Herein, from Portugal, Pinto et al. [37] identified two compounds, p-hydroxybenzoic (sample from wild pine forest: 100 µg/g dw and from the wild oak forest: 150 µg/g) and cinnamic acids (from wild pine forest: trace and from the wild oak forest: 10 µg/g), with their concentrations significantly lower than the present work, while the study by Barros at al. [38] detected three phenolic acids which are protocatechuic: p-hydroxybenzoic and p-coumaric acids, with the values of 33.57, 29.31 and 3.75 µg/g dw, respectively. Regarding Argentina, Toledo et al. [34] did not find any of the phenolic compounds analyzed (gallic, p-hydroxybenzoic and p-coumaric acids) in their L. nuda. The phenolic compounds from mushrooms have already been studied in several species, and it was reported that these compounds have been attributed to different biological activities such as antioxidant, antimicrobial and antitumor activities [5,39].
al. [34] did not find any of the phenolic compounds analyzed (gallic, p-hydroxybenzoic and p-coumaric acids) in their L. nuda. The phenolic compounds from mushrooms have already been studied in several species, and it was reported that these compounds have been attributed to different biological activities such as antioxidant, antimicrobial and antitumor activities [5,39].    1 Each value is expressed as means ± SD of three independent measurements. * p < 0.05 indicates that the mean value of at least one component differs from the others. For each mushroom sample, means within a line with different letters differ significantly (p < 0.05). nd = not detected.

Biomolecules by GC-MS Analysis
The chemical compositions of the fruiting bodies' methanolic extracts after their derivatization were established by GC-MS, a powerful tool for qualitative and quantitative analysis of various compounds present in natural products and the technique widely used in medical, biological, and food research [40]; the summarized results of this analysis are represented in Tables S1-S5. The GC-MS chromatogram of P. flaccida ( Figure S2) and L. nuda ( Figure 2) revealed the presence of sixty-one and sixty-six biologically active compounds, respectively. The identified biomolecules can be mainly divided into five main groups of constituents of each sample, namely sugars, amino acids, fatty acids, organic acids, and the five composed of rest groups, whereas sugars (52.51%) and fatty acids (29.72%) were observed to be the main chemical group in P. flaccida and L. nuda, respectively (Table 3).
in medical, biological, and food research [40]; the summarized results of this analysis are represented in Tables S1-S5. The GC-MS chromatogram of P. flaccida ( Figure S2) and L. nuda ( Figure 2) revealed the presence of sixty-one and sixty-six biologically active compounds, respectively. The identified biomolecules can be mainly divided into five main groups of constituents of each sample, namely sugars, amino acids, fatty acids, organic acids, and the five composed of rest groups, whereas sugars (52.51%) and fatty acids (29.72%) were observed to be the main chemical group in P. flaccida and L. nuda, respectively (Table 3). Glycerol (12.42%), mannitol (10.39%) and linoleic acid (9.67%) were recognized as major chemical compositions of P. flaccida, while L. nuda was predominated by linolelaidic acid (21.13%), leucine (9.05%) and mannitol (5.05%). The two main compounds detected in this study, mannitol and linoleic acid, were previously considered antioxidants [41,42]. Alongside nutritional values, the biomolecules identified in both mushrooms could be responsible for various pharmacological actions such as antioxidant, antiinflammatory, antimicrobial, antiviral and antitumor activities.  As presented in Table S1, the contents of sugar compositions of methanolic extracts of the two analyzed mushrooms were strong and diverse. P. flaccida extract contained 21 compounds which were dominated by glycerol (12.42%), mannitol (10.39%) and trehalose (8.58%). Likewise, L. nuda methanolic extract was composed of 16    As presented in Table S1, the contents of sugar compositions of methanolic extracts of the two analyzed mushrooms were strong and diverse. P. flaccida extract contained 21 compounds which were dominated by glycerol (12.42%), mannitol (10.39%) and trehalose (8.58%). Likewise, L. nuda methanolic extract was composed of 16 components, in which mannitol (5.16%), threitol (4.16%) and trehalose (4.13%) were the most abundant sugar compounds detected. Heleno et al. previously reported the presence of two sugar compounds, trehalose and mannitol, in L. inversa [43]. The presence of the two main sugars in L. nuda, mannitol and trehalose, were also observed in several previous studies [31,38,41]. Moreover, glucose, rhamnose, mannose, and xylose were the four monosaccharides quantified in L. nuda from India without detecting galactose and fructose [35]. Trehalose, a naturally occurring nontoxic disaccharide, functions as an antioxidant and may be useful to treat many chronic diseases, involving oxidative stress [44].
Concerning fatty acids, L. nuda methanolic extract contained the major diversity (11) of compounds, representing 29.72% of the total of compounds identified, whereas linoelaidic (21.13%), palmitic (4.49%) and stearic acids (1.78%) were the major fatty acids detected (Table S2). In contrast, only four fatty acids were detected in P. flaccida, which were  (Table S2). Several previous works studied the fatty acids of L. nuda, and all of them reported that linoleic acid was the main compound identified, which was not detected in our mushroom [34,35,37,45]. According to these previous studies on L. nuda and our P. flaccida result, two studies from Bulgaria and Portugal also found linoleic acid as the main fatty acid determined in the species' inversa form (Lepista inversa) [28,43].
Regarding amino acids, the major diversity of amino acids was observed in P. flaccida (15 amino acids), while there was less in L. nuda, with eight compounds (Table S3). Gammaaminobutyric acid (3.04%), glutamine (1.99%) and threonine (1.42%) were classified as the main amino acids detected in P. flaccida. For L. nuda, leucine, threonine and alanine represented the majority of the amino acids identified, with percentages of 9.05%, 2.69% and 2.15%, respectively. To our knowledge, there have been no previous studies on the amino acids of P. flaccida or of inversa form; however, one study was performed on L. nuda from India with the identification of four amino acids, namely aspartic acid, arginine, tyrosine and proline [35].
For organic acids, GC-MS analysis of the derivatized methanolic extracts showed the presence of eight compounds in P. flaccida and eleven compounds in L. nuda (Table S4). The P. flaccida was predominated by 3,4-dihydroxybutanoic (2.59%), malic (2.26%), succinic (1.85%) and citric (1.83%) acids, while acetoacetic (2%), oxalic (1.66%), maleic (1.44%) and lactic (1.32%) acids were observed to be the highest presented organic acids in L. nuda. Three analyses were realized on organic acids in L. nuda, and the results demonstrated that quinic and oxalic acids were listed as the main compounds in the three studies [34,37,46]. Contrary to our work, quinic and fumaric acids were not detected in L. nuda; citric acid was not detected in a study from Portugal [46], and citric and malic acids were not identified in the previous work from Argentina [34]. Organic acids may have a protective role against various diseases due to their antioxidant activity (such as in the case of tartaric, malic, citric or succinic acids), being able to chelate metals or to delocalize the electronic charge coming from free radicals [46].
Alongside sugars, fatty acids, amino acids and organic acids, the GC-MS analyses of derivatized methanolic extracts of the studied mushrooms showed that the samples also contained many other biologically active compounds belonging to the group of alcohols, steroids, nucleic acids, lipids, glycerides, etc. (Table S5). Ergosterol was noted to represent 1.97% and 1.61% of total biomolecules in P. flaccida and L. nuda, respectively. This biomolecule is the most abundant sterol found in mushrooms, and it has several biological activities including antioxidant, anti-inflammatory, anti-hyperlipidemic, anti-tyrosinase and antimicrobial activities [47,48].

Antioxidant Activity
Natural antioxidants have become scientifically interesting compounds due to their many benefits for human health [49]. There are numerous methods available to determine the antioxidant capacity of extracts or pure compounds. Herein, the antioxidant activity of methanolic extracts of the two Moroccan mushrooms P. flaccida and L. nuda were evaluated spectrophotometrically using three different assays: DPPH radical scavenging, β-carotene/linoleate, and Ferricyanide/Prussian blue activity. The antioxidant results are expressed in EC 50 values, as summarized in Table 4. In addition, the results have been graphically represented in Figures S3-S5. The methanolic extracts of P. flaccida and L. nuda showed a strong antioxidant capacity, which was in agreement with the important amount of phenolic compounds and other bioactive compounds found in both mushrooms. These important results were significantly different with Trolox, a standard that was used as a control. On the other hand, the strongest antioxidant capacity was observed in P. flaccida extract using a β-carotene bleaching inhibition assay with the value of 0.22 mg/mL (lower EC 50 value), and in the same mushroom the lowest antioxidant activity by using DPPH radical-scavenging activity with the value of 1.18 mg/mL (higher EC 50 value) was noted. The results are presented as mean ± SD (n = 3). * p < 0.05 indicates that the mean value of at least one component differs from the others. For each mushroom sample, means within a line with different letters differ significantly (p < 0.05).
Concerning DPPH radical-scavenging activity ( Figure S3), the results showed that the two studied samples exhibited significant free radical reducing capacity. Herein, the methanolic extract of L. nuda gave higher antioxidant capacity than P. flaccida extract, with EC 50 values of 0.98 and 1.18 mg/mL, respectively. A previous study was released on L. nuda from Morocco, and the antioxidant activity was 10.60 mg of Trolox equivalent per gram of lyophilized mushroom. Moreover, several works from other countries, namely Portugal, Argentina, Turkey and India, evaluated the DPPH radical-scavenging activity of L. nuda extracts and the EC 50 values ranged between 2.16 and 16.20 mg/mL, which were significantly higher than our values [31,32,34,35,37]. Another work from Portugal, by Heleno et al., noted that L. inversa gave the EC 50 value of 10.57 mg/mL, which was highly different from our results [29]. This important radical-scavenging activity is due to the high content of total phenolic and flavonoids found in the studied mushrooms [5].
Regarding the β-carotene-linoleate bleaching assay ( Figure S4), the biomolecules existing in the methanolic extract of the two mushrooms were able to inhibit the discoloration of β-carotene and have demonstrated strong antioxidant properties. The methanolic extract of P. flaccida revealed significantly higher antioxidant activity than the L. nuda extract, with values of 0.22 and 0.39 mg/mL, respectively, which were more effective than L. inversa (1.80 mg/mL) from Portugal reported previously by Heleno et al. [29]. Furthermore, recent studies have also demonstrated the antioxidant activity of L. nuda extract using β-carotenelinoleate bleaching assay and the results were observed to be lower than our samples, with higher EC 50 values which were between 3.53 and 14.24 mg/mL [32,34,37]. These important β-carotene-linoleate bleaching results could be due to the high quantity of carotenoids and other major biomolecules found in the methanolic extract of P. flaccida and L. nuda.
For reducing power by Ferricyanide/Prussian blue assay ( Figure S5), the natural antioxidant compounds exiting in methanolic extracts of the two edible Moroccan mushrooms were able to convert Fe 3+ into Fe 2+ and, therefore, exhibited high reducing power with EC50 values of 0.48 mg/mL for the L. nuda and 0.63 mg/mL for P. flaccida. Our extracts have given a strong reducing power in comparison with previous results by Heleno et al. in L. inversa (2.9 mg/mL) [29], and with the ones reported in various works on L. nuda extracts, in which their EC 50 values ranged between 0.75 and 4.21 mg/mL [32,34,35,37]. This finding of reducing power could be related to the ability of biomolecules found in the samples to reduce Fe 3+ [50].

Mushroom Material
The edible mushrooms P. flaccida and L. nuda were harvested from Koudiat Taifour  These identifications were made according to the two determination keys [51,52]. Voucher specimens were deposited at the herbarium of the BEDD laboratory, Department of Matter and Life Sciences, ENS of Tetouan, Morocco. The fruiting bodies were immediately cleaned, weighed, cut into small pieces, air-dried, and reduced to a fine powder (20 mesh).

Preparation of Crude Methanolic Extracts
The methanol extraction was carried out following the previous work by Barros et al. [32], with some modifications. A total of 1 g of fine-dried mushroom power (20 mesh) was extracted by stirring with 20 mL of methanol at 25 • C at 150 rpm for 24 h and filtered through Whatman N • 4 paper. The residue from the filtration was extracted again, twice, using the procedure described earlier. The combined methanolic extracts were evaporated at 40 • C to dryness. Then, the dried extracts were weighed and stored at −81 • C for further use. The extraction yield was calculated for each studied species. This preparation and all the further works were carried out at the Faculty of Sciences of the University of Porto, Portugal.

Estimation of Bioactive Compounds
The contents of bioactive compounds, including total phenolic compound content (TPC), total flavonoid content (TFC), total ascorbic acid content (TAAC), total tannin content (TTC), and total carotenoids contents (β-carotene (Tβ-CC) and lycopene (TLC)), in fruiting bodies of P. flaccida and L. nuda were determined by spectrophotometry using the same conditions, equipment and procedures described previously by Erbiai et al. [26].
TPC was determined by Folin-Ciocalteu assay. Briefly, one ml of extract methanolic solution was mixed with 5 mL of Folin-Ciocalteu reagent and 4 mL of sodium carbonate solution (7.5%). The tubes were vortex mixed for 15 s and allowed to stand for 30 min at 40 • C in the dark. Then, the absorbance of the solution was measured at 765 nm against the blank. The results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry methanolic extract (dme). TFC was determined by using an aluminum chloride colorimetric method, based on the formation of a complex between aluminum chloride and the C-4 keto group and either the C-3 or C-5 hydroxyl group of flavones and flavonols. The intensity of the pink color was measured at 510 nm using a UV-Visible spectrophotometer against the blank, which contained all reagents except extract samples. The results were expressed as mg of (+)-catechin equivalents (CEs) per gram of dme.
TAAC was determined using a method based on the reaction of ascorbic acid existing in the extract with the reagent 2,6 dichlorophenolindophenol. Meta-phosphoric acid (1%) was used for ascorbic acid extraction. The absorbance was measured at 515 nm against a blank. The results were expressed as mg of L -ascorbic acid equivalents (AAEs) per gram of dw.
TTC of the sample powder was assayed by the Vanillin-HCL method, which is a method specific to dihydroxyphenols and particularly sensitive to molecules containing meta-substituted, di-and tri-hydroxybenzene. The absorbance of color developed was measured at 500 nm against the blank. The TTC was expressed as mg of (+)-catechin equivalents per gram (CEs/g) of dme.
Tβ-CC and TLC were determined following a method based on the mixture of methanol extract and acetone-hexane (4:6). The solution absorbance (A) was measured at 453, 505, 645, and 663 nm using a UV-Vis spectrophotometer. Tβ-CC and TLC were calculated according to the following equations: Lycopene

Phenolic Compounds Analysis by HPLC-MS
The extraction and analysis of individual phenolic compounds of P. flaccida and L. nuda were carried out following the same procedure, conditions and HPLC equipment used in our previous published work [26]. Briefly, the phenolic extract was analyzed by highperformance liquid chromatography-mass spectrometry (HPLC-MS). Chromatographic separation was accomplished using Acclaim™ 120 reverse phase C18 columns (3 µm 150 × 4.6 mm) thermostatted at 35 • C, and peaks were detected at 280 nm as the preferred wavelength. The mobile phase used was composed of 1% acetic acid and 100% acetonitrile. The identification of phenolic compounds in the samples was characterized according to their UV-Vis spectra and identified by their mass spectra and retention times in comparison with commercial standards. Quantification was made from the areas of the peaks recorded at 280 nm by comparison with calibration curves obtained from the standard of each compound. The results were expressed in µg per gram of dry weight (dw).

Biomolecules Analysis by GC-MS
Before GC-MS analysis, the crude methanolic extracts of each mushroom (10 mg) were derivatized by adding 100 µL of anhydrous pyridine and 100 µL BSTFA, and the mixture was heated at 80 • C for 25 min, then the mixture was diluted with 200 µL chloroform [53,54]. The derivatized solution was analyzed by using Gas Chromatography (GC) (Trace 1300 gas chromatography; Thermo Fisher Scientific, Waltham, MA, USA) linked to a mass spectrometry (MS) system (ISQ single quadrupole mass spectrometer; Thermo Fisher Scientific) and automatic injector. The GC separation was conducted with a TG5-MS capillary column (60 m × 0.25 mm i.d.; 0.25 µm film thickness) with a non-polar stationary phase (5% Phenyl 95% dimethylpolysiloxane). The injection and detector temperature were made at 300 • C using splitless injection mode (1:10). Helium was used as a carrier gas at a flow rate of 1.2 mL/min. The oven temperature was programmed from 40 • C (2 min) to 200 • C at a rate of 6 • C/ min (2 min), and then at a rate of 6 • C/min (6 min) up to 300 • C. The total run time was 65 min. MS conditions were: electron ionization mass spectra were set at 70 eV, the mass ranged from 50 to 650 amu, and the ion source temperature was 300 • C. Retention indices were calculated for all components, using a homologous series of known standards of alkanes mixture (C 8 -C 20 and C 21 -C 40 ) injected in conditions equal to sample ones. Identification of components of mushroom extracts was based on retention indices (RI) relative to alkanes, with those of authentic compounds and with the spectral data obtained from the databases of the National Institute Standard and Technology (NIST) and PubChem Libraries of the corresponding compounds. Data acquisition was operated by Software Thermo XcaliburTM 2.2 SP1.48, and data analysis was performed using NIST MS Search 2.2 Library 2014.

Evaluation of Antioxidant Activity
The antioxidant activity of methanolic extracts from the two edible mushrooms P. flaccida and L. nuda was evaluated by three different assays, including DPPH radicalscavenging, reducing power, and β-carotene bleaching inhibition assay, and by following the same procedures, equipment and conditions used previously by Heleno et al. [29]. The extract concentration providing 50% of antioxidant capacity or 0.5 of absorbance (EC 50 ) was calculated from the graphs of antioxidant activity percentages (DPPH, and β-carotene/linoleate assays) or absorbance at 690 nm (ferricyanide/Prussian blue assay) against extract concentrations. Trolox was used as a reference standard.
DPPH radical-scavenging activity (RSA) of the samples was determined using the stable free radical DPPH (1.1-diphenyl-2-picrylhydrazyl). The absorbance was measured at 517 nm using a UV-Vis spectrophotometer against a blank. The RSA was calculated as a percentage of DPPH discoloration using the equation: RSA (%) = [(A DPPH − A Sample )/A DPPH ] × 100, where A DPPH is the absorbance of the DPPH solution and A Sample is the absorbance of the test extract.
For the β-carotene-linoleate bleaching assay, the antioxidant activity of the methanolic extracts was carried out using the β-carotene linoleate model system, in which the presence of antioxidants in the extracts and their capacity to neutralize the linoleate free radicals avoids β-carotene bleaching. The absorbance was measured immediately at zero-time at 470 nm against a blank, and measured for the second time at 120 min. A control containing methanol instead of the extract was realized in parallel. β-carotene bleaching inhibition was calculated using the following formula: (%) = (β-carotene content after 2 h of the assay/initial β-carotene content) × 100.
Reducing power by Ferricyanide/Prussian blue assay, the methodology of which is based on the capacity to convert Fe 3+ into Fe 2+ , the absorbance of the solution was measured at 690 nm using a UV-Vis spectrophotometer against a blank containing the same solution mixture without mushroom extract.

Statistical Analysis
Three samples were used, and all assays were carried out in triplicate. Extraction yield, bioactive compounds, and antioxidant activity values were expressed as mean ± standard deviation (SD). The statistical significance of the data was made with a one-way analysis of variance (ANOVA), followed by post hoc Tukey's multiple comparison tests with α = 0.05 using GraphPad Prism 8.0.1 software (San Diego, CA, USA).

Conclusions
This research work constitutes the first report on the chemical characterizations and antioxidant properties of the two wild edible mushrooms P. flaccida and L. nuda from southern Mediterranean countries and, in particular, from Morocco. The fruiting bodies of the studied samples demonstrated an important content of bioactive compounds, namely phenolic compounds (individual and total contents), ascorbic acid and carotenoids. In addition, the GC-MS analysis of P. flaccida and L. nuda extracts revealed the presence of more than sixty biologically active compounds for each. On the other hand, the two edible mushrooms showed strong antioxidant properties by using three assays: DPPH radical scavenging activity, inhibition of β-carotene bleaching, and ferric-reducing power. The highly considered antioxidant capacity of the samples could be related to their richness of bioactive compounds. In general, the findings may encourage more people from southern Mediterranean countries to consume edible mushrooms as food due to their benefits on human health. They may also allow researchers to make the valorization of mushrooms from these regions an interesting objective for their investigation to open up new perspectives in nutritional and pharmaceutical research, and to contribute to discovering novel antioxidant agents and medicaments which can be used for the treatment of many diseases.

Supplementary Materials:
The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/molecules28031123/s1, Figure S1: HPLC-MS chromatogram of phenolic compounds in Lepista nuda extract detected at 280 nm, Figure S2: GC-MS chromatogram of P. flaccida derivatized methanolic extract, Table S1: Sugar compositions of the derivatized methanolic extract by GC-MS analysis, Table S2: Fatty acids of the derivatized methanolic extract by GC-MS analysis, Table S3: Amino acids of the derivatized methanolic extract by GC-MS, Table S4: Organic acids of the derivatized methanolic extract by GC-MS analysis, Table S5: Rest of the biomolecule constituents of the derivatized methanolic extract by GC-MS analysis, Figure S3: Radical-scavenging activity on DPPH radicals. Each value is expressed as mean ± SD (n = 3), Figure S4: Lipid peroxidation inhibition measured by the β-carotene bleaching inhibition. Each value is expressed as mean ± SD (n = 3), Figure S5: Reducing power. Each value is expressed as mean ± SD (n = 3).