*2.2. Extraction Procedure*

Dried leaves and roots were ground in a laboratory mill Vitek (An-Der, Austria) by using a 0.5 mm size sieve. Methanol extract was prepared in an accelerated solvent extractor ASE 350 (Dionex, Sunnyvale, CA, USA) from 10 g of material, which was mixed with 4 g of diatomaceous earth and placed in a 66 mL extraction cell. Extraction was carried out three times at 60 ◦C temperature and 10 MPa pressure with a 15 min static and a 90 s purge time for each extraction cycle (in total, 3 cycles). Peony leaves (10 g) were also extracted in a conical flask with 200 mL methanol by using a mechanical shaker (Sklo Union LT, Teplice, Czech Republic) at room temperature and 170 rpm. Water extracts were prepared from 5 g of leaves and 2 g of roots in a conical flask by suspending plant powder in 50 mL of distilled water at 80 ◦C and continuously stirring at 400 rpm by magnetic hotplate stirrer (IKA, Wilmington, DE, USA). The extraction procedures of methanol and water were repeated three times, each lasting 24 h and 15 min, respectively. After methanol (TR) and water extraction the solids were filtered through a 0.3 μm filter (Filtrac, Niederschlag, Germany) and combined. Methanol was removed in a Rotavapor R-114 (Büchi, Flawil, Switzerland), then additionally dried in a flow of nitrogen (20 min) and finally all methanol and water extracts were freeze dried in a Maxi Dry Lyo (Hetto-Holton AIS, Allerod, Denmark). Solid residues after each extraction were also dried and stored at −18 ◦C in a freezer until further analysis. Three replicate extractions were performed for each plant, material, solvent, and method. The extracts obtained are abbreviated by the following letters: P—peony, L—leaves, R—roots, M—methanol, W—water, ASE—accelerated solvent extraction, and TR—traditional extraction.

#### *2.3. Determination of Antioxidant Potential by Single Electron Transfer Based Assays*

Fast colorimetric methods were selected for the in vitro assessment of total phenolic content (TPC), DPPH• scavenging, and ABTS•<sup>+</sup> decolourization capacity. Detailed description of these methods are provided elsewhere [14]. Briefly, for TPC, 30 μL of extract at various concentrations were mixed in a 96-well microplate with 150 μL Folin-Ciocalteu reagen<sup>t</sup> diluted in distilled water (1:10 *v*/*v*) and 120 μL 7% Na2CO3 solution. After shaking 10 s the absorbance was recorded at 765 nm in a FLUOstar Omega reader (BMG Labtech, O ffenburg, Germany). The calibration curve was prepared using 10–250 μg/mL solutions of gallic acid in water. The TPC was expressed in mg gallic acid equivalents (GAE) per dry weight of plant (DWP) and dry weight of extract (DWE (GAE/g DWP and GAE/g DWE, respectively) from four replicate measurements.

For DPPH• scavenging 8 μL of extract and 292 μL of DPPH• (6 × 10−<sup>5</sup> M) solutions in methanol were mixed in a 96-well microplate and the absorbance was recorded at 515 nm in a FLUOstar Omega reader every min during 60 min. Trolox solutions (299–699 μM/L) were used for the calibration curve, the results expressed as trolox equivalents (TE) in g of DWP and DWE from 4 replicate measurements.

For the ABTS•<sup>+</sup> decolourization reaction, working solution of ABTS•<sup>+</sup> was produced by mixing 50 mL of ABTS and 200 μL of potassium persulfate stock solutions and kept 15 h at room temperature in the dark. The absorbance was adjusted to 0.800 ± 0.020 at 734 nm with PBS (phosphate bu ffered saline) and 294 μL of ABTS•<sup>+</sup> were mixed with 6 μL of methanolic extract solutions in a 96-well microplate. The absorbance was measured in a FLUOstar Omega reader at 734 nm during 30 min at 1 min intervals. Calibration curve was constructed by using Trolox solutions (399–1198 μM/L), and the results were expressed as μM TE/g DWP and DWE from 6 replicate measurements.

The QUick, Easy, New, CHEap and Reproducible (QUENCHER) method was applied to determine the antioxidant capacity of solid materials before and after extractions in order to evaluate the effectiveness of the recovery of antioxidants. Solid materials were mixed with microcrystalline cellulose at a ratio from 1:5 to 1:100 (TPC and ABTS•<sup>+</sup>), or from 1:1 to 1:100 (DPPH•). For TPC, 5 mg of sample/blank (cellulose) were mixed with 150 μL of MeOH:H2O (1:4), 750 μL Folin-Ciocalteu's reagent, and 600 μL Na2CO3 solution, vortexed for 15 s, shaken at 250 rpm for 3 h in the dark, centrifuged (4500 rpm, 10 min), and the absorbance of the optically clear supernatant was measured at 765 nm. For the DPPH• scavenging assay, 5 mg of sample/blank were mixed with 40 μL of MeOH:H2O (1:4) and 1960 μL of DPPH• methanolic solution, vortexed 60 s, shaken at 250 rpm for 25 min in the dark, centrifuged (for 4800 rpm, 3 min), and the absorbance of the optically clear supernatant was measured at 515 nm. For ABTS•<sup>+</sup> decolorisation, 5 mg of sample/blank were mixed with 40 μL of PBS and 1960 μL of working ABTS•<sup>+</sup> solution, vortexed for 60 s, shaken at 250 rpm for 30 min in the dark, centrifuged (4800 rpm, 3 min), and the absorbance of optically clear supernatant was measured at 734 nm. The values were calculated for g DWP from 6 replicate measurements.

#### *2.4. Determination of Antioxidant Potential by Peroxyl-Radicals Inhibition Assays Based Hydrogen Atom Transfer*

Oxygen radical absorbance capacity (ORAC), hydroxyl radical antioxidant capacity (HORAC), and hydroxyl radical scavenging capacity (HOSC) assays, which are based on peroxyl-radicals inhibition (HAT), and therefore are considered as more relevant to the processes in the biological systems [15], were selected for the characterisation of peony extracts. Detailed description of these methods are provided elsewhere [14]. Briefly, in ORAC, 25 μL of trolox standards, antioxidant, and 150 μL 2 × 10−<sup>7</sup> mM of FL (fluorescein) solutions in PBS (75 mM, pH 7.4) were placed in a black 96-well microplate. Then, the mixture was preincubated in a FL800 microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT, USA) for 10 min at 37 ◦C. To start the reaction 25 μL of AAPH (153 mM), which was used as a source of peroxyl radical, were added to each well automatically through the injector coupled with the FL800 microplate reader. The fluorescence was recorded every min during 1 h at 485 ± 20 nm excitation and 530 ± 25 nm emission. Trolox solutions (5–40 μM) were used for calibration, the results expressed in μM TE/g DWP and DWE.

For HORAC, 30 μL of sample/standard/blank, 170 μL of FL (9.28 × 10−<sup>8</sup> M) and 40 μL of H2O2 (0.1990 M) solutions were pipetted into a black microplate, 60 μL of CoF2 (3.43 mM) solution was added, and the microplate was placed on a FL800 microplate fluorescence reader for 60 min at 37 ◦C using 485 nm excitation and 530 nm emission filters. The calibration curve was constructed by using 50 to 250 μM ca ffeic acid containing solutions. SPB (sodium phosphate bu ffer) (75 mM, pH 7.4) was used for hydrogen peroxide and fluorescein preparation, while acetone:Milli-Q water (50:50 *v*/*v*) was used as a blank and for the preparation of sample, calibration, and CoF2 solutions. Data was expressed as μM ca ffeic acid equivalents (CAE) per g DWP and DWE.

For the HOSC assay to 30 μL of blank/standard/sample prepared in acetone:MilliQ water (50:50 *v*/*v*), 40 μL of H2O2 (0.1990 M), and 170 μL of FL solution (9.28 × 10−<sup>8</sup> M), 60 μL FeCl3 solution (3.43 mM) were pipetted in a black microplate, which was immediately placed in the FL800 reader, and the fluorescence was recorded every min during 1 h at 37 ◦C using 485 ± 20 nm excitation and 530 ± 25 nm emission filters. FeCl3 and H2O2 solutions were prepared in ultrapure water, while SPB (75 mM, pH = 7.4) was used to prepare the solution of FL. Trolox containing solutions (5–30 μL) were used for the calibration curve, and antioxidant capacity values were expressed as μM TE/g. Mean values in all these assays were calculated for g DWP and DWE from 4 replicate measurements.

#### *2.5. Evaluation of HPLC-DPPH*• *Scavenging On-Line*

The HPLC (high performance liquid chromatography) system consisted of a Rheodyne 7125 manual injector (Rheodyne, Rohnert Park, CA, USA), a Waters 996 photodiode array detector (Milford, MA, USA), and a Waters 1525 binary pump (Milford, MA, USA). Separation of compounds was performed at 40 ◦C on a Hypersil C18 analytical column (5 μm, 250 × 0.46 cm, Thermo scientific, USA). The linear binary gradient was used at a constant flow rate 0.8 mL/min with solvent A (1% formic acid in ultrapure water) and B (acetonitrile) by using the following gradient elution order: 0 min 5% B, 0–2 min 10% B; 2–40 min 22% B; 40–55 min 100% B, 55–60 min 5%. 20 μL of the sample was injected and the spectra were recorded in the range from 210 to 450 nm. In order to detect radical scavengers, the HPLC system was coupled with an Agilent 1100 series pump (Agilent Technologies, USA) supplying freshly prepared methanolic DPPH• (5 × 10−<sup>6</sup> M) solution into a reaction coil (15 m, 0.25 mm ID) at a flow rate of 0.6 mL/min. A Shimadzu SPD-20A UV detector (Shimadzu Corporation, Kyoto, Japan) was used to record the negative peaks due to a decrease of absorbance at 515 nm after the reaction of radical scavengers with DPPH•. For the preliminary identification of compounds, a quadrupole mass detector Micromass ZQ (Waters, Milford, MA, USA) in negative ionization mode was used with the following parameters: cone voltage 30 V; cone gas flow 80 L/h; desolvation temperature 300 ◦C; desolvation gas flow 310 L/h; capillary voltage 3000 V; source temperature 120 ◦C; scanning range 100 to 1200 *<sup>m</sup>*/*<sup>z</sup>*. Chromatographic conditions were used as described above. Diluted extracts in methanol and water mixture (50:50, *v*/*v*) were analyzed.

#### *2.6. Ultra Performance Liquid Chromatography*/*Electron Spray Ionisation Quadrupole Time-of-Flight Mass Spectrometry (UPLC*/*ESI-QTOF-MS) Analysis*

UPLC/ESI-QTOF-MS analysis was carried out on a Waters Acquity UPLC system (Milford, MA) combined with a MaXis 4G Q-TOF (quadrupole time of flight) mass spectrometer, a sample manager, PDA detector, binary solvent manager, and controlled by HyStar software (Bruker Daltonic, Bremen, Germany). The Acquity C18 column (1.7 μm, 100 mm × 2.1 mm i.d. Waters, Milford, MA, USA) at a separation temperature 40 ◦C was used. The mobile phase consisting of solvent A (1% formic acid in ultrapure water) and solvent B (acetonitrile) was eluted in the following order: 95% A at 0.0 min; 0–5% B at 0 min; 5–20% B at 0–9 min; 20–50% B at 9–12 min, 50–100% B at 12–14 min, and held these condition for 1 min. Finally, the initial conditions were re-introduced over 1 min and held for 1 min. Before each run, the column was equilibrated for an additional 1 min. The following parameters were: a capillary voltage of 4 kV; an end plate offset of −0.5 kV; a flow rate of drying-gas of 10.0 L/min; a nebulizer pressure of 2.5 bar; a scanning range 79–2400 *m*/*z*; an injection volume of 1 μL; a flow rate of 0.45 mL/min. For fragmentation study, a data dependent scan was performed by deploying collision induced dissociation (CID) using nitrogen as a collision gas at 30 eV. Peaks were identified and analysed by comparing their retention times, accurate masses, and formulas by using external standards and commercial databases.

Quantitative analysis was performed by using external standards. Calibration curves were drawn using six concentrations of standard solutions and represented the dependence between the integrated chromatographic peak areas and the corresponding amounts of injected standards. According to the lowest point of the calibration curve, the LOQ (S/N = 10) and LOD (S/N = 3) were calculated.

#### *2.7. Determination of* α*-Amylase Inhibitory Activity*

An α-Amylase assay was carried out according to the procedure of Al-Dabbas et al. [16]. Briefly, 60 μL of blank (PBS)/sample/acarbose (0.02 mg/mL) solutions and 200 μL of a starch solution (400 μg/mL) were mixed in 6 Eppendorf tubes for incubation at 37 ◦C for 5 min. To start the reaction, twenty μL of α-amylase (5 mg/mL) was added to the three tubes and in the rest of three twenty μL of PBS (pH = 7.4) as a control for the sample. Afterwards, twenty μL of PBS was added to all tubes and incubated at 37 ◦C for 7.5 min. After incubation, in order to determine the degradation of the starch, 200 μL of I2 solution (0.01 M) was transferred to the Eppendorf tubes. Finally, by adding 1 mL of a distilled H2O reaction, the mixture was diluted and its absorbance was measured at 660 nm by using a GENESYS 10 spectrophotometer (Thermo Scientific, Waltham, MA, USA). For the calibration, a curve stock solution of acarbose (5 mg/mL) was used. The inhibition of enzyme activity (IC50) was expressed as mg/mL. The experiments were performed in triplicate.

#### *2.8. Cytotoxicity Assay in Caco-2 Cells*

The cells were maintained as monolayers in 175 cm<sup>2</sup> culture flasks containing an RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, at 37 ◦C in humidified air with 5% CO2. Caco-2 cells were seeded at a density of 2 × 10<sup>4</sup> cells/well in transparent 96-well plates and allowed to grow as a confluent and non-di fferentiated monolayer that can mimic the human intestinal epithelium. This cell model shares some characteristics with cryp<sup>t</sup> enterocytes, and thus it has been considered to be an accepted intestinal model widely implemented to assess the e ffect of chemical, food compounds, and nano/microparticles on the intestinal function [17]. The medium was changed every 2 days. On the day of the experiment, cells were washed twice with pre-warmed PBS at ~37 ◦C. Water and ethanol extracts of peony were solubilized in H2O and EtOH, respectively. All extracts were prepared with a final concentration of 16.7 mg/mL. Cell-based assays were performed using a maximum concentration of solvent—50% and 5% for H2O and ethanol, respectively.

Cytotoxicity was assessed by the MTS (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyltetrazolium bromide) method [17]. Prepared cells monolayers were incubated with various concentrations of peony extracts (100 μL) for 4, 24, and 48 h and washed with PBS. Their viability was determined by adding a 100 μL 10-fold diluted MTS reagen<sup>t</sup> (according to the manufacturer's guidelines), incubating for 2.5 h at 37 ◦C 5% CO2, measuring the absorbance at 490 nm in an Epoch Microplate Spectrophotometer (Bio-Tek, Instruments, Winooski, VT, USA). Data were expressed in a cellular viability percentage relative to control (%). The experiments were performed in triplicate.

#### *2.9. Cellular Antioxidant Activity Assay (CAA)*

The CAA assay was carried out by the procedure of Wolfe and Liu [18]. Cell monolayers were treated with 50 μL of PBS, sample and standard (quercetin, 2.5–20 μM) solution, and fifty μL of a DCFH-DA solution containing 50 μM of reagen<sup>t</sup> and pre-incubated at 37 ◦C, 5% of CO2 for 1 h. Afterwards, to each well containing standards/samples and tree wells containing PBS (control) was added 100 μL of AAPH (12 mM). In the rest of the six wells containing PBS (blank) 100 μL of PBS solution was added. The data were recorded every 5 min during 60 min by using a FL800 fluorescent reader (ex. 485 nm, em. 540 nm). CAA values were expressed as μM of quercetin equivalents (QE) per g of the extract from three independent experiments.

#### *2.10. Statistical Data Handling*

All results are presented as means ± standard deviation (SD), and all the experiments were completed at least three times. Significant di fferences among means were evaluated by one-way ANOVA, using the statistical package GraphPad Prism 6. Duncans' posthoc test was used to determine the significant di fference among the treatments at *p* < 0.05.
