Comparison of Different Extraction Solvents for Characterization of Antioxidant Potential and Polyphenolic Composition in Boletus edulis and Cantharellus cibarius Mushrooms from Romania

Edible mushrooms are well-known for their nutritional benefits and low energy density. In addition, mushroom extracts contain various bioactive compounds that account for their antioxidant activity; the applied extraction conditions influence the extraction efficiency of such compounds. Therefore, this study investigates the effects of four extractants on the content of polyphenols and antioxidant properties of Boletus edulis and Cantharellus cibarius mushrooms, aiming to optimize the extraction process. Powders of B. edulis and C. cibarius mushrooms were subjected to extraction with acidic water (10% CH3COOH), ethanol/water/acetic acid (15:76.5:8.5, v/v/v), hexane, and diethyl ether to measure their total phenolic content (TPC), total flavonoid content (TFC), and Trolox equivalent antioxidant capacity (TEAC). Furthermore, the level of individual polyphenolic compounds in these extracts was quantified using an HPLC-DAD-ESI-MS method. Results showed that the type of solvent significantly influenced the TPC and TEAC of mushroom powder but insignificantly influenced the TFC. A very strong positive correlation was found between TPC and TEAC, but no correlation was found between TFC and TEAC or TPC and TFC. Acidic water extracted the highest amount of polyphenolic compounds from these mushroom powders. Therefore, the aqueous extract showed the highest TPC and strongest antioxidant activity. Thus, acidic water is recommended for polyphenol analysis in B. edulis and C. cibarius mushrooms.


Introduction
The tradition of wild edible mushroom consumption has existed for centuries in many European countries [1]. Two hundred sixty-eight edible wild mushrooms are allowed for commercialization in Europe; the five top-selling edible fungi are Boletus edulis, Cantharellus cibarius, Lactarius deliciosus, Morchella esculenta, and Agaricus campestris [2]. Boletus edulis (porcini mushrooms) and Cantharellus cibarius (chanterelle) are among the most consumed wild mushrooms in Romania because of their availability in large quantities in this territory. These mushrooms are consumed fresh, dehydrated, or preserved in brine, vinegar, and oil.
In the present work, an attempt has been made to evaluate the extraction efficiency of polyphenols from Boletus edulis and Cantharellus cibarius mushrooms with aqueous, hydro-alcoholic, hexanic, and etheric solvents (Aq, Aq-EtOH, Hex, Et2O). As far as we are aware, this is the first report investigating the effects of different extraction solvents [acidic water (10% CH 3 COOH), ethanol/water/acetic acid (15:76.5:8.5, v/v/v), hexane, and diethyl ether] on total phenolic content (TPC), total flavonoid content (TFC), Trolox equivalent antioxidant capacity (TEAC), and individual polyphenols of these mushroom powders. Our findings (data not shown) revealed that mixtures of water/acetic acid and ethanol/water/acetic acid, in the ratios mentioned above, proved effective in extracting plant polyphenolic compounds. Still, their extraction yields varied with the mushroom's matrix. This study's experimental design also included the characterization of powders obtained from these mushroom species by determining their proximate composition.

Results and Discussion
Proximate analysis results of B. edulis powder revealed a mean value of 26.5 ± 0.190% for protein content, 2.2 ± 0.058% for fat content, 53.8 ± 0.097% for total carbohydrate content, 7.1 ± 0.125% for ash content, and 10.4 ± 0.097% for moisture content. Therefore, the calculated energy value was 341 kcal/100 g mushroom powder. In C. cibarius powder a similar energy value (332 kcal/100 g) was found. However, a lower protein content (19.1 ± 0.066%), similar fat content (2.7 ± 0.068%), higher carbohydrate content (57.8 ± 0.123%), higher ash content (10.5 ± 0.116%), and similar moisture content (9.9 ± 0.010%) was noticed. Table 1 shows values for total phenolic content (TPC), total flavonoid content (TFC), and Trolox equivalent antioxidant capacity (TEAC) of mushroom extracts. The highest TPC of B. edulis powder was found in Aq (3.73 mg GAE/g dw), followed by Aq-EtOH (3.42 mg GAE/g dw), Et2O (0.79 mg GAE/g dw), and Hex (0.48 mg GAE/g dw). For C. cibarius powder, the highest TPC was registered in Aq (0.79 mg GAE/g dw), followed by Aq-EtOH (0.66 mg GAE/g dw), Hex (0.58 mg GAE/g dw), and Et2O (0.29 mg GAE/g dw). Generally, total phenolic content strongly correlates with the antioxidant capacity [39]. A perfect positive relationship (r = 1.000; p < 0.001) between TPC and TEAC in B. edulis extracts was revealed, with Aq having the strongest antioxidant activity (1.90 µmol TE/g dw), followed by Aq-EtOH (1.79 µmol TE/g dw), Et2O (0.35 µmol TE/g dw), and Hex (0.23 µmol TE/g dw). In C. cibarius extracts, there was no significant correlation (p > 0.05) between TPC and TEAC; the highest TEAC value being noticed in Aq (1.12 µmol TE/g dw), followed by Aq-EtOH (1.00 µmol TE/g dw), Et2O (0.37 µmol TE/g dw), and Hex (0.28 µmol TE/g dw). It is well known that each solvent has its specific selectivity towards the extraction of different compounds which contribute toward total antioxidant activity. For instance, acidic water provides the highest extraction efficiency, but at the same time, according to the results obtained by Wu et al. [40], it does not extract melanin very well. However, it is also important to note that even if the acidic water doesn't extract melanin, it still gives the best performance compared to the other three solvents that manage to extract it. Still, these do not extract, or extract less than, other significant compounds which contribute to total antioxidant activity. Due to this diversity and variation of the extraction yield of different compounds, the performance of solvents is assessed based on the total antioxidant activity of extracts and not on the individual contribution of different compounds.
Unfortunately, it was difficult to compare our results to literature data because previous studies used different methods of preparing mushrooms, different extraction solvents and expressed results in other measurement units. To demonstrate this fact, several existing literature data on B. edulis and C. cibarius mushroom extracts were summarized in Table 2. These data indicate that, besides solvents used in the current study, there are other attractive options, like pure methanol, which gives very high yields regardless of the sample preparation method. Similarly, extraction with water proves to be very efficient in the case of freeze-dried mushroom powder. Comparing the findings of earlier reports ( Table 2) to ours (Table 1), it appears that pure methanol and acetone/water/acid mixture provide better extraction performance than acidic water, the most efficient extractant among the four solvents used in this study. Still, acidic water would be the preferred solvent, considering that it is a green solvent and mushroom extracts are intended for food industry applications. TPC-total phenolic content; TFC-total flavonoid content; DPPH radical scavenging activity-radical scavenging activity; dw-dry weight; CAE-catechin equivalents; EC 50 -half maximal effective concentration; IC 50 -half-maximal inhibitory concentration.
Regarding TFC, for B. edulis powder, the highest value was found in Aq (0.20 mg QUE/g dw), followed by Aq-EtOH (0.09 mg QUE/g dw), and equally by Hex (0.05 mg QUE/g dw) and Et2O (0.05 mg QUE/g dw). No significant correlations (p > 0.05) were detected between TFC and TEAC, respectively, between TPC and TFC in B. edulis extracts. In C. cibarius powder, the highest remarked TFC was in Et2O (0.30 mg QUE/g dw), followed by Aq-EtOH (0.28 mg QUE/g dw), Aq (0.03 mg QUE/g dw), and Hex (0.02 mg QUE/g dw).
A very strong negative correlation (r = −0.980; p < 0.05) was observed between TFC and TEAC in C. cibarius extracts, but no significant one (p > 0.05) between TPC and TFC.
Generally, extraction solvent had a significant effect on TPC (p < 0.05) and TEAC (p < 0.001) in mushroom powder but not a significant one on TFC (p > 0.05). Values of TPC and TEAC in mushroom extracts were in the following order: aqueous > hydro-alcoholic > etheric > hexanic, hence the very strong positive correlation between TPC and TEAC (r = 0.897; p < 0.005). Although near to zero as values, the order of TFC in mushroom extracts was thusly: etheric > hexanic > aqueous > hydro-alcoholic; consequently, there were no significant correlations (p > 0.05) between TFC and TPC, respectively, or TFC and TEAC.
Palacios et al. (2011) identified homogentisic acid, gallic acid, and pyrogallol as the main compounds in the methanolic extract of C. cibarius mushrooms [9]. Muszyńska et al. (2015) observed vanillic acid as the principal constituent in the acidic aqueous extract of C. cibarius powder, followed by p-hydroxybenzoic acid [38]. In the study of Islam et al. (2016) [35], gallic acid was the primary compound evidenced in the acid hydro-acetonic extract of C. cibarius mushrooms. Later, Butkhup et al. (2018) reported naringenin as the main compound in the hydro-methanolic extract of C. cibarius mushrooms, followed by catechin, epicatechin, and quercetin-3-O-rutinoside [19].
Comparing TPCs and TFCs of extracts resuspended in methanol to values of phenolic and flavonoid compounds of extracts resuspended in their extractants, the absence of flavonoid compounds was observed in hexanic and etheric extracts of the latter ones. Since values of flavonoid compounds spectrophotometrically assessed are close to zero in all extracts, both for B. edulis and C. cibarius mushroom powders, it can thus be deduced that these values were the result of measurement interferences. These findings are supported by the very strong positive relationship (r = 0.955; p < 0.001) found between phenolic compounds quantified in mushroom extracts by the HPLC method and the spectrophotometric method and the lack of a correlation (p > 0.05) between flavonoid compounds quantified by the two methods.

Solvents and Reagents
The following solvents and reagents were used: sodium sulphate anhydrous (

Raw Materials
Two types of edible wild mushrooms, Boletus edulis and Cantharellus cibarius, were purchased in July 2021 from a local vegetable-fruit collection center (S.C. Alisa Funghi S.R.L., Chichisa, Romania). Mushrooms were manually sorted based on their size and appearance, cleaned of dirt and physical impurities, washed, drained, sliced, and dried (for 24 h at 45 • C) using a dehydrator (DEH-450, Biovita S.R.L., Cluj-Napoca, Romania). Dried mushrooms were further ground (GR-020, Minimoka, Paris, France); their powders were packed in amber glass jars with Teflon-lined caps and stored in a cool and dry place until analysis.
The extraction process consisted of powder maceration with different solvents [acidic water-10% (v/v) acetic acid solution; a mixture of ethanol, water and acetic acid-15 parts of ethanol mixed with 76.5 parts distilled water and 8.5 parts acetic acid; hexane; and diethyl ether] as follows: homogenization of mushroom powder (5.0 g) with the extraction solvent (150 mL) at 21,500 rpm for 30 sec (T 18 digital Ultra-Turrax, IKA-Werke GmbH & Co. KG, Staufen, Germany), succeeded by orbital shaking at 150 rpm at room temperature for 24 h (MI01030002, Guangzhou Four E's Scientific Co., Ltd., Guangzhou, China), centrifugation at 8981 × g (9000 rpm) at 4 • C for 10 min (Universal 320 R, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany), vacuum filtration of supernatant, removal of the solvent by rotary evaporation at 55 • C for Aq and Aq-EtOH respectively at 40 • C for Hex and Et2O (Hei-VAP Expert, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany), and resuspension of dry extract in 10 mL extraction solvent (for HPLC-DAD-ESI-MS analysis of polyphenolic compounds) and 10 mL methanol (for spectrophotometric analysis of TPC, TFC and TEAC), respectively.
All extracts were stored at −18 • C until testing. These were filtered through polyamide syringe filters (0.45 µm pore size, 25 mm diameter) before chromatographic analysis and DPPH assay.

Determination of Mushroom Powders Proximate Composition
Moisture content was determined by sample drying to constant weight in an electric oven (Digitheat, J.P. Selecta S.A., Barcelona, Spain) following the instructions provided by ISO 712:2009 [41]. Samples were analyzed in triplicate. Results were expressed in percentage (%).
Protein content was determined using the Kjeldahl method described in ISO 20483:2013 [42]. It involved (1) acid digestion of the sample (DK6 Heating Digester, JP Recirculating Water Aspirator, VELP Scientifica S.R.L., Usmate Velate, Italy), (2) alkalization and steam distillation of acid digest (UDK 129 Distillation Unit, VELP Scientifica S.R.L., Usmate Velate, Italy), (3) and quantification of trapped ammonia by titration. First, the volume of 0.1 N sulfuric acid solution, used in titration, was used to calculate nitrogen content in the sample. Then, nitrogen content was multiplied by 4.38 to estimate the protein content [6]. Samples were analyzed in triplicate. Results were expressed in percentage (%).
Fat content was determined using the Soxhlet method described by Teklit (2015) [43], with minor modifications. A 1.0 g portion of the sample was weighed in a fat-free filter paper bag and loaded into a porous cellulose thimble. The thimble was immersed into an extraction cup (containing 80 mL petroleum ether) and placed into a Soxhlet extractor (SER 148 Solvent Extractor, VELP Scientifica S.R.L., Usmate Velate, Italy). The sample was extracted using the following instrument settings: hot plate temperature 110 • C, boiling time/sample immersion cycle time 30 min, sample rinse cycle time 4 h, solvent evaporation and recycle time 30 min. The extraction cup containing the extract was dried at 103 • C for 1 h in an electric oven (Digitheat, J.P. Selecta S.A., Barcelona, Spain), cooled at room temperature in a desiccator, and weighed with an analytical balance (ABJ-220-4NM, Kern & Sohn GmbH, Balingen, Germany). This step was repeated, at hourly intervals, until a constant weight was achieved. The fat content was calculated by the following Formula (1): where w f is the weight of the fat (g) and w s is the weight of the sample (g). Samples were analyzed in triplicate. Ash content was determined by sample incineration in a muffle furnace (L3/11/B170, Nabertherm GmbH, Bremen, Germany) according to the method described by Nagy et al. (2017) [44]. A 3.0 g portion of the sample was weighed and heated at 600 • C for 12 h in the muffle furnace. Next, it was cooled in a desiccator and weighed again. The ash content was calculated with the following Formula (2): where w a is the weight of the ash (g) and w s is the weight of the sample (g). Samples were analyzed in triplicate.

Determination of Total Phenolic Content
Total phenolic content in mushroom extracts was determined using the method described by Semeniuc et al. (2018) [45]. One hundred microliters of the extract were transferred into a 16-mL glass bottle with a rubber stopper; 6 mL distilled water, and 0.5 mL of 2 N Folin-Ciocalteu's phenol reagent were then added and vortexed (Vortex V-1 Plus, Biosan Ltd., Riga, Latvia). After 4 min, 1.5 mL of 0.71 M sodium carbonate solution and 1.9 mL distilled water were added and vortexed again. The mixture was kept in the dark, at room temperature, for 2 h. The absorbance value of the mixture was read at 725 nm against the blank using a double beam UV-VIS spectrophotometer (UV-1900i, Shimadzu Scientific Instruments, Inc., Columbia, MD, USA). The blank was prepared with methanol (instead of extract) and treated identically to the test sample. Samples were tested in triplicate. For the calibration curve preparation, gallic acid was used in concentrations from 0.25 to 1.25 mg/mL. Results were expressed in mg gallic acid equivalents (GAE)/g dry weight (dw).

Determination of Total Flavonoid Content
Determination of total flavonoid in mushroom extract content was carried out as previously described by Socaci et al. (2018) [46]. One hundred microliters of the extract were diluted to a final volume of 5 mL with distilled water and then transferred into a 16-mL glass bottle with a rubber stopper. Next, 300 µL of 5% (w/v) sodium nitrite solution were added and vortexed (Vortex V-1 Plus, Biosan Ltd., Riga, Latvia). After 5 min, 300 µL of 10% (w/v) aluminum chloride solution, and after another 5 min, 2 mL of 1 N sodium hydroxide solution were added and mixed using the vortex. The absorbance value of the mixture was read at 510 nm against the blank using a double beam UV-VIS spectrophotometer (UV-1900i, Shimadzu Scientific Instruments, Inc., Columbia, MD, USA). The blank was prepared with methanol (instead of aluminum chloride solution) and treated identically to the test sample. Samples were tested in triplicate. For the calibration curve preparation, quercetin was used in concentrations from 0.019 to 0.800 mg/mL. Results were expressed in mg quercetin equivalents (QUE)/g dry weight (dw).

Determination of Antioxidant Capacity by DPPH (2,2-Diphenyl-1-picrylhydrazyl) Assay
DPPH assay was performed as described by Thaipong et al. (2006) [47]. First, a stock solution was prepared by dissolving 24 mg DPPH in 100 mL methanol. Then, 24 mL of stock solution was mixed with 90 mL methanol to reach an absorbance of 1.1 ± 0.02 units at 515 nm, thus obtaining the working solution.
One hundred fifty microliters of the extract were transferred into a 16-mL glass bottle with a rubber stopper; 2850 µL of DPPH working solution was then added and vortexed (Vortex V-1 Plus, Biosan Ltd., Riga, Latvia). The mixture was kept in the dark, at room temperature, for 1 h. The absorbance value of the mixture was read at 515 nm against methanol using a double-beam UV-VIS spectrophotometer (UV-1900i, Shimadzu Scientific Instruments, Inc., Columbia, MD, USA). The blank was prepared with methanol (instead of extract) and treated identically to the test sample. The absorbance value of the blank was subtracted from that of the extract. Samples were tested in triplicate. For the calibration curve preparation, Trolox was used in concentrations from 25 to 800 µmol/L. Results were expressed in µM Trolox equivalent (TE)/g dry weight (dw).
Twenty microliters of extract were injected into the HPLC system for analysis. In the elution process, two mobile phases were employed: solution A containing 0.1% acetic acid in ultrapure water, and solution B containing 0.1% acetic acid in acetonitrile. A multi-step gradient elution model was used with the following settings: 5% B (0-2 min), from 5 to 90% B (2-20 min), 90% B (20-24 min), from 90 to 5% B (24-30 min). The flow rate was programmed to 0.5 mL/min and the column oven temperature to 25 • C. The PDA was set to scan from 200 to 600 nm. The chromatograms were acquired at 280 and 340 nm; data acquisition was performed for 30 min.
Mass spectra were recorded using the ESI source set in positive-ion mode and the MS in full scan mode (in an m/z range of 120−1200). Nitrogen was used as a drying gas. Other settings for the ESI source were as follows: drying gas temperature 350 • C, drying gas flow rate 7 L/min, nebulizer pressure 35 psi, capillary voltage 3000 V, fragmentor voltage 100 eV.
Polyphenolic compounds were tentatively identified by comparing their retention times, UV-vis spectra, and mass spectra to those of the standards analyzed under the same conditions and the data available in the literature [49,50].

Conclusions
This study reveals that B. edulis and C. cibarius mushrooms are sources of polyphenolic compounds and exhibit antioxidant activities. However, each type of tested solvent provided different extraction yields of phenolic and flavonoid compounds and different antioxidant responses. For example, mixtures of water/acetic acid (9:1, v/v) and ethanol/water/acetic acid (15:76.5:8.5, v/v/v) extracted higher amounts of polyphenolic compounds than ether or hexane and demonstrated better antioxidant activities. Therefore, acidic water (10% CH 3 COOH) is the recommended solvent for extracting these bioactive compounds from B. edulis and C. cibarius mushrooms.