Optimization of Phenolic Compounds Extraction and Antioxidant Activity from Inonotus hispidus Using Ultrasound-Assisted Extraction Technology

The use of ultrasound-assisted extraction (UAE) of bioactive compounds has been increasing because it is a good alternative to the conventional extraction methods. UAE was used to maximize total polyphenol content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity, and ferric reducing antioxidant power (FRAP) of the mushroom Inonotus hispidus using response surface methodology (RSM). Firstly, the effect of 40% (v/v) ethanol and 80% (v/v) methanol on the TPC, DPPH scavenging capacity, and FRAP was evaluated. The ethanolic extracts showed a significantly higher (p < 0.0001) TPC, DPPH scavenging capacity, and FRAP than the methanolic extracts. The best condition to produce an extract with the higher TPC and antioxidant activity was achieved when using 40% (v/v) ethanol, a ratio of 75 mL/g, and an extraction time of 20 min. The chromatographic profile of the extract obtained in the optimized condition revealed that hispidin is the main polyphenol present in the extracts of I. hispidus, representing, together with hispidin-like compounds, the majority of the phenolic compounds (159.56 µg/g DW out of 219.01 µg/g DW). The model allowed us to optimize the conditions to maximize the extraction of phenolic compounds with antioxidant activity from I. hispidus, demonstrating its potential as a source of antioxidant compounds, with possible industrial, pharmaceutical, and food applications.


Mushroom Material
I. hispidus sporocarps were collected in an apple orchard, located in Lamego, North of Portugal, in October 2021. After taxonomic identification at the Laboratory of Mycology of the University of Trás-os-Montes and Alto Douro, the sporocarps were cut into small pieces, dried in a drying oven (Termaks, Nordic Labtech AB, Kungsbacka, Sweden) at 40 • C, and then ground to a fine powder. The samples were kept in the dark in hermetically sealed plastic bags up to analyses.

UAE Methodology
The UAE method was used to extract the bioactive compounds of I. hispidus, which is based on the principles of the breakdown of cell walls using ultrasound waves [25,27,29]. Methanol 80% (v/v) was used as solvent since it is often used for extracting phenolic compounds, partly due to being more economical. Ethanol was also used as a solvent and the choice of a concentration of 40% (v/v) was based on previous studies carried out in our laboratory, whose extraction of phenolic compounds and antioxidant activity of the extract proved to be better than the other tested concentrations. Firstly, 1 g of dried powder was mixed with 50 mL of 80% (v/v) methanol or 40% (v/v) ethanol. The extractions were performed in a pulsed mode of 5 s on/5 s off cycles for 40 min (contact time) using the Hielscher ultrasonic processor device (Hielscher UP400St, Berlin, Germany), with a sonotrode of 14 mm diameter, 400 Watts, 24 kHz, and adjustable amplitude (1:2.55). During the sonication process, the samples were placed in an ice bath to maintain a sample temperature in the range of 40 to 50 • C to avoid thermodegradation of the phenolic compounds [30]. After completion of the extraction, the samples were centrifuged (4500× g for 20 min at 4 • C), the supernatants were filtered using a Whatman no. 4 filter paper, collected, and stored at −20 • C until analyses. All the experiments were performed in triplicate.

Experimental Design
For the previously selected extraction solvent, new experimental assays were carried out using different contact time conditions and solvent-to-solid ratio conditions (Table 1). These variables were combined based on a central composite design. After sonication, each extract was centrifuged and filtered as previously described, and the supernatants were stored until analyses began. The effect of two independent numeric variables, contact time (min, X 1 ) and solvent-to-solid ratio (mL/g, X 2 ), on the efficiency of the extraction of phenolic compounds, DPPH scavenging capacity, and ferric reducing antioxidant power (FRAP) in I. hispidus was evaluated according to a central composite design [31,32]. Variables were coded at the levels −2, −1, 0, 1, and 2 (Table 2), and thirteen runs were established under specific conditions. The content of total phenolics, the radical scavenging capacity (DPPH assay), and FRAP were assessed in the obtained extracts. The model included five central points. To lessen the effect of natural variability on the response, sampling was done randomly. The optimized condition was determined using RSM, while the determination of the significance of primary variable effects, variable interaction, and the model was evaluated using Analysis of Variance (ANOVA).

Evaluation of TPC
The Folin-Ciocalteu method was used to evaluate the content of phenolic compounds in the extracts as previously described [33], with some modifications. Briefly, 20 µL of each sample extract, 100 µL of Folin-Ciocalteu reagent (10%, v/v), and 80 µL of aqueous sodium carbonate (7.5%, w/v) were mixed in a microplate and then incubated at 42 • C during 30 min protected from the light. The absorbance was then measured at 750 nm (Multiskan FC Microplate Photometer, Thermo Fisher Scientific, Vantaa, Finland). The standard curve was obtained using gallic acid (5 to 200 mg/L). Values were expressed as mg of gallic acid per gram of dry weight of raw material (mg GA/g DW).

In Vitro Antioxidant Capacity
The DPPH assay was used to determine the free radical scavenging activity as previously reported [34], with slight modifications. The measurements were performed on a microscale using a 96-well microplate reader (Multiskan FC Microplate Photometer, Thermo Fisher Scientific, Vantaa, Finland). In the DPPH assay, 10 µL of the samples were added to 190 µL of DPPH solution (8.87 mM). The plate was allowed to rest in the dark and the absorbances were read at 520 nm after 15 min of incubation. The antioxidant capacity of the extracts was determined by using the calibration curve with Trolox in a concentration range varying from 0.156 up to 2.500 mM. Values were expressed as millimoles of Trolox per gram of DW of raw material (mmol Trolox/g DW). The FRAP assay was performed according to Mena et al. [34] with some modifications. A FRAP working solution was prepared by mixing 10-volumes of 300 mM acetate buffer (pH 3.6), 1-volume of 10 mM TPTZ (dissolved in hydrochloric acid), and 1-volume of 20 mM ferric chloride (prepared in distilled water). Samples (20 µL) were placed in a 96-well microplate, and then 280 µL of FRAP working solution (warmed at 37 • C for 10 min) was added. The reaction was incubated at 37 • C for 30 min and the absorbance was read at 593 nm. A calibration curve with Trolox (0.039 to 1.25 mM) was used, and the values were expressed as millimoles of Trolox per gram of DW of raw material (mmol Trolox/g DW).

HPLC Analysis
The profile and content of phenolic compounds from the extract obtained at the optimized condition were analyzed in triplicate by HPLC-DAD, as previously described [35]. Sample extracts (10 µL), in triplicate, were injected into a C18 column (250 × 4.6 mm, 5 µm particle size; ACE HPLC Columns, Advanced Chromatography Technologies Ltd., Abeerden, Scotland, UK) with an eluent composed of water with 0.1% TFA (solvent A) and acetonitrile with 0.1% TFA (solvent B). The elution was performed at a flow rate of solvent of 1 mL/min, with a gradient starting from 0% solvent B at 0 min, 0% solvent B at 5 min, 20% solvent B at 15 min, 50% solvent B at 30 min, 100% solvent B at 45 min, 100% solvent B at 50 min, 0% solvent B at 55 min, and 0% solvent B at 60 min. Chromatograms were recorded in a range of 200-600 nm: 254 and 280 nm for benzoic acids and flavan-3-ols, 320 nm for cinnamic acids, and 370 nm for flavonoids. Phenolics were identified using peak retention time, UV spectra, and UV maximum absorbance band, and by comparison with the literature. Naringin (internal standard) was prepared at a concentration of 2.0 mg/mL in 70% (v/v) methanol (methanol:water) and run simultaneously with the samples. The amount of each compound was calculated using the internal standard method, and the results were expressed as µg/g DW.

Statistical Analysis
Firstly, the Mann-Whitney U test (GraphPad Prism 7 Software, Inc.) was used to verify the effect of each solvent on the extraction of polyphenolic compounds with antioxidant capacity from I. hispidus. All the assays were carried out in triplicate and the results were expressed as mean ± standard deviation (n = 3). Secondly, statistical analysis of the design, to determine the conditions able to maximize the extraction results, was performed with the software Design Expert (version 13.0, Stat-Ease Inc., Minneapolis , MN, USA). The Pearson correlation coefficient for selected pairs of parameters was also estimated. Differences were considered significant when p < 0.05.

Determination of the Extraction Solvent
As observed in Table 3, extracts with phenolic compounds and antioxidant capacity were achieved with both ethanol and methanol. These data are consistent with previous works that report ethanol and methanol as the most efficient organic solvents to extract phenolic compounds from different raw materials [28,[36][37][38][39][40]. According to our results, ethanol produced extracts with higher and statistically different (p < 0.0001) TPC, DPPH, and FRAP values than methanol. In fact, ethanol has been successfully used to extract antioxidant compounds [41], including from mushrooms [42], and due to its lower toxicity compared to methanol, it has been chosen as an organic solvent in different extraction procedures using different raw materials. Accordingly, ethanol was the selected solvent for the subsequent analysis of the study. Data presented as mean ± SD of three replicates. The means of both solvents were compared by Mann-Whitney test, **** p < 0.0001. DPPH, 2,2-diphenyl-1-picrylhydrazyl; DW, dry weight of raw material; FRAP, ferric reducing antioxidant power; GA, gallic acid; TPC, total polyphenol content.

Optimization of the Extraction Conditions
Once the extraction solvent has been selected, different extraction conditions were tested to maximize the recovery of antioxidant phenolic compounds from I. hispidus. It is well known that various factors affect the antioxidant activity of the extracts and the kinetic of phenolic compounds released from the solid matrix. Those critical variables are directly related to the yield of the extracts and include, for instance, the extraction procedure, the solvent type and concentration, the extraction time, the solvent-to-solid ratio, and the temperature at which the extraction is performed, among others. Using the UAE methodology and based on an experimental design, Ianni et al. [43] described how different variables affect the yield of the extraction process to obtain phenolic compounds from Pleurotus ostreatus. In our study, to optimize the extraction conditions to obtain the greatest extraction yields, the contact time employed in the UAE method and the solvent-to-solid ratio used were the variables tested. Table 4 presents the conditions of each experimental assay and the respective measured and predicted values of TPC, DPPH scavenging capacity, and FRAP. The values were analyzed by multiple regression to fit a second-order polynomial equation, and quadratic models describing the variations of the responses as a function of the significant process variables (contact time, X 1 ; solvent-to-solid ratio, X 2 -coded values) were established ( Table 5). The square coefficient of determination (R 2 ) was used to assess the quality of fit, which was 0.95 for total phenolic compounds, 0.87 for DPPH antiradical activity, and 0.86 for FRAP. These findings suggest a highly significant agreement between the results experimentally obtained and those predicted by the equations for TPC, DPPH, and FRAP values, which can adequately predict the experimental results ( Figure 1A-C). The model F values of 29.00 (TPC), 9.11 (DPPH), and 8.73 (FRAP) and the associated lower p value (TPC p < 0.001, DPPH p < 0.01, and FRAP p < 0.01) mean that the generated model is meaningful.
Based on the regression model constructed, a 2D-contour line (Figures 2A-4A) and 3D-response graphs (Figures 2B-4B) were plotted for each of the responses under analysis. These figures show the similarity between the maximized responses of TCP, DPPH, and FRAP. The results in Table 4 indicate that the highest level of TPC was reached at a ratio of 75 mL/g with a sonication time of 20 min. Likewise, concerning the DPPH and FRAP assays, the highest efficiency was obtained when using those same conditions. As observed, both the content of phenolic compounds as well as the values of antioxidant activity by the DPPH and FRAP assays increased as the solvent-to-solid ratio increased. Likewise, other authors also described, in studies performed with ethanolic extracts from mushrooms [43] and fruits [44], that TPC increased as the solvent-to-solid ratio increased. These results are in accordance with the mass transfer principle where a high solvent-to-solid ratio promotes an increasing concentration gradient. This will further increase the diffusion rate, resulting in a higher extraction of solids by solvent [26,45]. On the other hand, the time of extraction in our study did not significantly affect any of the responses evaluated, which implies that 20 min of sonication could be sufficient for the extraction of the compounds of interest. Ballesteros et al. [31] also aimed to optimize different variables for the ethanolic extraction of antioxidant phenolic compounds from coffee silverskin and found that the time of extraction did not influence either the TPC or the antioxidant activity (evaluated by FRAP and DPPH assays). From an economic perspective, using a shorter extraction time is important as it reduces energy consumption.   Table 4 indicate that the highest level of TPC was reached at a ratio of 75 mL/g with a sonication time of 20 min. Likewise, concerning the DPPH and FRAP assays, the highest efficiency was obtained when using those same conditions. As observed, both the content of phenolic compounds as well as the values of antioxidant activity by the DPPH and FRAP assays increased as the solvent-to-solid ratio increased. Likewise, other authors also described, in studies performed with ethanolic the extraction of the compounds of interest. Ballesteros et al. [31] also aimed to optimize different variables for the ethanolic extraction of antioxidant phenolic compounds from coffee silverskin and found that the time of extraction did not influence either the TPC or the antioxidant activity (evaluated by FRAP and DPPH assays). From an economic perspective, using a shorter extraction time is important as it reduces energy consumption.  different variables for the ethanolic extraction of antioxidant phenolic compounds from coffee silverskin and found that the time of extraction did not influence either the TPC or the antioxidant activity (evaluated by FRAP and DPPH assays). From an economic perspective, using a shorter extraction time is important as it reduces energy consumption.    In order to confirm the best extraction condition capable of enhancing the responses under analysis, an overlay plot of TPC, DPPH, and FRAP responses was generated. Thus, the following criteria to find the optimal extraction conditions were used: TPC ≥ 58.67 mg GA/g DW, DPPH ≥ 0.6 mmol Trolox/g DW, and FRAP ≥ 0.55 mmol Trolox/g DW. The resulting overlaying plot ( Figure 5), obtained by the quadratic polynomial regression model, displayed an area where all the demanded conditions were fulfilled. In this area, an optimum point was chosen corresponding, as expected, to a contact time of 20 min and a ratio of 75 mL/g. The model predicts an extraction of TPC of 104.45 mg GA/g DW, a DPPH value of 0.85 mmol Trolox/g DW, and a FRAP value of 0.96 mmol Trolox/g DW under these conditions. In order to confirm the best extraction condition capable of enhancing the responses under analysis, an overlay plot of TPC, DPPH, and FRAP responses was generated. Thus, the following criteria to find the optimal extraction conditions were used: TPC ≥ 58.67 mg GA/g DW, DPPH ≥ 0.6 mmol Trolox/g DW, and FRAP ≥ 0.55 mmol Trolox/g DW. The resulting overlaying plot ( Figure 5), obtained by the quadratic polynomial regression model, displayed an area where all the demanded conditions were fulfilled. In this area, an optimum point was chosen corresponding, as expected, to a contact time of 20 min and a ratio of 75 mL/g. The model predicts an extraction of TPC of 104.45 mg GA/g DW, a DPPH value of 0.85 mmol Trolox/g DW, and a FRAP value of 0.96 mmol Trolox/g DW under these conditions. After determining the best conditions for the two independent variables, extractions were performed in triplicate under those conditions to validate the model. The data obtained for TPC (104.61 ± 13.82 mg GA/g DW), DPPH (0.86 ± 0.15 mmol Trolox/g DW), and FRAP (1.01 ± 0.08 mmol Trolox/g DW) showed a close agreement with the results predicted (TPC, 104.45; DPPH, 0.85; and FRAP, 0.96) by the statistical analysis (Table 6).  After determining the best conditions for the two independent variables, extractions were performed in triplicate under those conditions to validate the model. The data obtained for TPC (104.61 ± 13.82 mg GA/g DW), DPPH (0.86 ± 0.15 mmol Trolox/g DW), and FRAP (1.01 ± 0.08 mmol Trolox/g DW) showed a close agreement with the results predicted (TPC, 104.45; DPPH, 0.85; and FRAP, 0.96) by the statistical analysis (Table 6).

Correlation between the Evaluated Responses of I. hispidus Extracts
The correlation between phenolic compounds and the antioxidant activity of extracts from different raw materials has been investigated [46,47]. In the present study, the degree or strength between the results obtained for TPC, DPPH, and FRAP was analyzed by Pearson's correlation coefficient (r). As suggested by Evans [48], the correlation was divided into categories based on the value of the strength to which the two variables are related: very strong (1.0-0.80), strong (0.79-0.60), moderate (0.59-0.40), weak (0. 39-0.20), and very weak (0.19-0.00). Correlation analysis charts were plotted ( Figure 6) and revealed that DPPH scavenging capacity and FRAP were directly proportional and very strongly correlated with TPC (r = 0.89, p < 0.0001 and r = 0.88, p < 0.0001, respectively). Moreover, a statistically significant correlation between DPPH and FRAP values was also observed (r = 0.84, p < 0.0003). Our results are consistent with other investigations that have demonstrated in mushroom extracts a general correlation between a larger amount of phenolics and a higher antioxidant activity [43,49], which means that phenolic compounds are directly related to the antioxidant potential. However, other studies reported a poor correlation between TPC and DPPH values, which may be due to the presence of other compounds in addition to phenols that could also have DPPH radical scavenging activity [43,50]. Similarly, despite our very strong correlation between DPPH and FRAP values, other authors have reported a weak correlation between them [43,51]. A correlation between DPPH and FRAP means that both assays share a similar mechanism of action, such as electron transfer from antioxidant to oxidant [52]. lation between TPC and DPPH values, which may be due to the presence of other compounds in addition to phenols that could also have DPPH radical scavenging activity [43,50]. Similarly, despite our very strong correlation between DPPH and FRAP values, other authors have reported a weak correlation between them [43,51]. A correlation between DPPH and FRAP means that both assays share a similar mechanism of action, such as electron transfer from antioxidant to oxidant [52].

HPLC Analysis of Phenolic Compounds
The profile and content of phenolic compounds from the extract obtained in optimal condition (20 min and 75 mL/g liquid/solid ratio) was analyzed using HPLC-DAD. The obtained results are in Table 7. Hispidin and hispidin-like compounds (compounds with hispidin-like UV spectrum) accounted for the majority of phenolic compounds in this research (159.56 µg/g out of 219.01 µg/g). Hispidin is a yellow polyphenol pigment known as styrylpyrone, which was isolated and identified for the first time in I. hispidus [53]. This compound displays a large number of biological effects, such as anti-cancer, anti-platelet, anti-oxidative, anti-diabetic, anti-inflammatory, and antiviral activities [53,54]. Hispidin has the capacity to neutralize free radicals [17,53,55,56], and a recent study performed in Phenllinus mushroom extracts showed that this compound, but not polysaccharides or flavonols, determines its antioxidant and antitumor properties [57]. Gründemann et al. [58] identified hispidin in methanolic extracts of I. hispidus as the most prevalent compound. In our investigation, the HPLC profile also demonstrated the presence of other compounds to a lesser extent, such as flavonols (quercetin, myricetin, and isorhamnetin), flavones (luteolin-7-O-glucoside, luteolin-4 -O-glucoside, and diosmetin), isoflavones (glycitin), phenolic acids (hydroxybenzoic acid), and hydroxycinnamic acids (caffeic acid). According to our results, it is plausible that the antioxidant and reducing power observed in the extract of I. hispidus at the optimized condition is mainly due to the presence of hispidin and hispidin-like compounds.

Conclusions
In this work, the UAE extraction process of phenolic compounds from I. hispidus was optimized by RSM design. Ethanol at 40% (v/v) was an effective solvent for the extraction of phenolics with antioxidant capacity in the ratio of 75 mL/g DW, during 20 min of sonication. I. hispidus is an underexplored mushroom, and for the first time, the best conditions to maximize the extraction of antioxidant phenolic compounds were successfully established. These findings are of great relevance because these compounds have vast functional properties and potential cosmeceutical, pharmaceutical, and food applications. However, although the extract of I. hispidus has favorable in vitro potential, the pharmacokinetics and toxicity of the extract compounds must be determined through in vivo toxicological studies. Funding: This work was funded by the Fungi4Health project (no. NORTE-01-0247-FEDER-070171), financed by the European Regional Development Fund (ERDF) through NORTE 2020 (North Regional Operational Program 2014/2020) and by National Funds by FCT-Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020.

Informed Consent Statement: Not applicable.
Data Availability Statement: Dataset will be provided upon reasonable request. Data is not publicly available due to privacy.

Conflicts of Interest:
The authors declare no conflict of interest.