# Antioxidant, Antibacterial, and Cosmeceutical Potential of Four Common Edible Mushrooms
## 2 Materials and Methods
### Determination of Antiradical and Antioxidant Capacities  
The antioxidant activity of sample extracts was determined using 2,2\({}^{\prime}\)-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS), 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPPH), and ferric-reducing antioxidant power (FRAP) spectrophotometric methods, as reported by [PERSON] et al. [27], with some modifications. ABTS. For the preparation of ABTS + radical cations, an ABTS stock solution (7.0 mM) was prepared with ABTS salt (Sigma Aldrich). Then, 5 mL of the ABTS stock solution was mixed with 88 \(\mu\)L of a 148 mM solution of potassium persulfate (Sigma Aldrich) and  
Figure 1: Flow chart of the experimental design. ABTS, 2,2\({}^{\prime}\)-azino-bis(3-ethylbenzothazoline-6-sulfonic acid); DPPH, 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl; FRAP, ferric-reducing antioxidant power; HPLC-DAD, high-performance liquid chromatography with diode array detector; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration. diluted to a working solution with sodium acetate buffer (20 mM, pH 4.5), showing an absorbance of 0.70 \(\pm\) 0.02 at 734 nm. Subsequently, 188 \(\mu\)L of ABTS working solution and 12 \(\mu\)L of sample dilutions or distilled water (blank) were mixed and reacted for 30 min at room temperature. The absorbance was read at 734 nm. DPPH. A DPPH solution (8.87 mM) was prepared by diluting DPPH (Sigma Aldrich) in methanol (99.9%, \(v/v\), Sigma Aldrich). Then, the DPPH solution was diluted to a working concentration with 70% (\(v/v\)) methanol to reach an absorbance of 1.000 at 520 nm. Then, 10 \(\mu\)L of sample dilutions or 70% methanol (\(v/v\), blank) was added to 190 \(\mu\)L of the DPPH solution. The plate was allowed to rest in the dark, and the absorbance was read at 520 nm after 15 min of incubation. For both assays, the inhibition percentage of the radicals was calculated using the following Equation (1):  
\[\%\;\text{Inhibition}=\frac{(\text{Abs blank}-\text{Abs samples})}{(\text{Abs blank})}\times 100 \tag{1}\]  
FRAP. To measure the reducing capacity, a FRAP working solution was prepared by mixing 10-volume acetate buffer (300 mM, pH 3.6), 1-volume 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ, Sigma Aldrich) (10 mM dissolved in hydrochloric acid), and 1-volume ferric chloride (20 mM in water, Sigma Aldrich). The solution was maintained at 37 \({}^{\circ}\)C for 10 min before use. Next, 20 \(\mu\)L of sample dilutions was added to each well of the microplate, followed by the addition of 280 \(\mu\)L of FRAP working solution. The reaction was incubated at 37 \({}^{\circ}\)C for 30 min, and the absorbance was read at 593 nm. The three antioxidant assays were adapted to microscale using 96-well microplates and a microplate reader (Multiskan GO Microplate Photometer, TermoFisher Scientific, Vantaa, Finland), using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma Aldrich) as a standard at concentrations varying from 0.016 to 0.500 mM for ABTS, from 0.156 to 1.250 mM for DPPH, and from 0.039 to 1.250 mM for FRAP. All the results were expressed as micromode of Trolox per gram of dry weight (\(\mu\)mol Trolox/g DW). Phenolic Compound Analysis Using High-Performance Liquid Chromatography with Dide Array Detector (HPLC-DAD)  
The profile and content of polyphenols of each mushroom extract were analyzed using HPLC-DAD, as previously described [28], with minor modifications. Briefly, sample extracts (10 \(\mu\)L), in triplicate, were injected into a C18 column (250 \(\times\) 4.6 mm, 5 \(\mu\)m particle size; ACE HPLC Columns, Advanced Chromatography Technologies Ltd., Aberdeen, Scotland, UK) with water with 0.1% of trifluoroacetic acid (TFA, Sigma Aldrich) (solvent A) and acetonitrile (Sigma Aldrich) with 0.1% TFA (solvent B) as eluent.