**3. Results**

#### *3.1. Total Yield, Total Phenolic Content and Radical Scavenging Capacity of Peony Extracts*

The yields and antioxidant capacity values of extracts and plant materials are presented in Table 1. High polarity solvents, methanol, and water were compared in this study. In addition, methanol was applied in a pressurized liquid extraction (PLE). Methanol has been shown to be an e ffective solvent for polyphenolic antioxidants in numerous studies, while water is very attractive in terms of its favourable green chemistry principles and low cost. It may be observed that the total yields from leaves were remarkably higher than those from the roots in the case of both solvents, whereas PLE with methanol resulted in an approximately 10% higher yield.

Using several antioxidant activity assays is important for completing a more comprehensive evaluation of natural products. Thus, Huang et al. [15] recommended applying at least two single electron transfer (SET) and one hydrogen atom transfer (HAT) assays for this purpose. Following this recommendation, TPC (Folin-Ciocalteu), ABTS•<sup>+</sup> and DPPH• scavenging, as well as ORAC, HORAC, and HOSC assays, were used for measuring the antioxidant potential of the dried extracts (DWE) and recovery of antioxidants from the initial plant material (DWP). These values are useful for evaluating the antioxidant potential of extracts and the recovery of antioxidants from the raw material, respectively. For instance, a low yield extract may possess stronger antioxidant activity, while for high yields, a better recovery of antioxidants from the plant may be achieved, although the extracts may show lower antioxidant capacities due to the dilution of the active constituents with neutral ones.


**Table 1.** Yields, antioxidant capacity, and total phenolic content 1 of different *P. o*ffi*cinalis* extracts.

Values are represented as mean ± standard deviation (*n* = 4); different superscript letters for means down the vertical column that do not share common letters are significantly different (*p* < 0.05). The extracts isolated with methanol and water are further referred to by abbreviations composed of the first letter of the plant (P—peony), the botanical part (L—leaves, R—roots) and the solvent (M—methanol, W—water); ASE and TR mean extraction type: the accelerated solvent extraction and traditional extraction, respectively; DWE—dry weight of extract; DWP—dry weight of plant; ORAC—oxygen radical absorbance capacity; HOSC—hydroxyl radical scavenging capacity; HORAC—(hydroxyl radical antioxidant capacity).

Thus, TPC values measured for *P. o*ffi*cinalis* extracts (Table 1) were in the range of 215.7 (PRTRW)–601.1 (PLTRM) mg GAE/g DWE, while the recovery of polyphenols from the raw material was in the range of 41.42 (PRTRW)–285.9 (PLTRM) mg GAE/g DWP. Methanol extraction resulted in higher TPC values both for DWP and DWE, while the TPC in leaves was higher than that in roots. It may be observed that higher TPC values for leaves were found via conventional extraction (TR) than via PLE; most likely, this difference was due to the dilution of Folin-Ciocalteu reactive substances in the latter case (PLE gave higher yields than TR) and some other changes. Consequently, in terms of TPC, traditional methanol extraction was the most effective method.

DPPH• and ABTS•<sup>+</sup> scavenging assays, which are also based on SET (single electron transfer) reaction, gave similar results for the antioxidant capacity of extracts and the recovery of radical scavengers (Table 1). However, ABTS•<sup>+</sup> decolourisation values were remarkably higher than DPPH• scavenging values, which may be explained by the different reaction conditions [15]. Thus, DPPH• and ABTS•<sup>+</sup> scavenging values were in the ranges of 343.4 ± 6.06–2553 ± 28.40 and 886.0 ± 7.36–4610 ± 18.70 μM TE/g DWE, respectively, while antioxidant recoveries were 65.94 ± 1.16–1153 ± 11.25 and 170.1 ± 1.41–2151 ± 12.72 μM TE/g DWP, respectively. It should be noted that the TPC and ABTS•+/DPPH• scavenging capacity was not reported for *P. o*ffi*cinalis* previously. Some antioxidants may be strongly bound to the insoluble plant matrix and are not available for any solvent without pre-treatment [19]. To evaluate the recovery of the active constituents, the antioxidant capacity of peony solids was monitored by using the so-called QUENCHER procedure [20]. The results presented in Figure 1 show antioxidant capacity values of peony solids before and after extraction. It may be observed that after extraction, they are remarkably reduced, indicating that the extraction processes were quite efficient for the recovery of antioxidants. Water was slightly more efficient than methanol.

**Figure 1.** Antioxidant capacity indicators of solid substances determined by the QUENCHER (The QUick, Easy, New, CHEap and Reproducible) method. Values represented as mean ± standard deviation (*n* = 4); the mean values followed by different letters and symbols are significantly different (*p* < 0.05) (ABTS: small letters for leaves (a–c) and for roots (d–e); DPPH•: capital letters are used for leaves (A–C) and for roots (D–E) and TPC–symbols are used for leaves (\*, \*\*, \*\*\*) and for roots (#, ##). TPC is expressed in mg GAE/g DWP; DPPH•, ABTS+• in μM TE/g DWP. The residues after methanol and water extraction are further referred to by abbreviations composed of mean residue (R), peony (P), leaves or roots (L—leaves, R—roots), and solvent (M—methanol, W—water); ASE and TR mean extraction type: accelerated solvent extraction and traditional extraction, respectively.

#### *3.2. Peroxyl and Hydroxyl Radicals Inhibition in ORAC, HORAC and HOSC Assays*

Based on TPC and ABTS•+/DPPH•-scavenging values and chemical composition, methanol (PLASEM, PLTRM) and water (PLTRM) extracts were tested (Table 1). ORAC values varied in the ranges of 1232–1433 μmol TE/g DWE and 409.6–681.4 μmol TE/g DWP, HOSC in the ranges of 1957–2012 μmol TE/g DWE and 668.2–931.0 μmol TE/g DWP, and HORAC in the ranges of 1566–1891 μmol CAE/g DWE and 520.3–899.4 CAE/g DWP. These assays confirmed that methanol extracts were better antioxidants than water extracts. It may be observed that regardless of significant differences in antioxidant activity between some tested extracts, these differences were not remarkable. However, due to the differences in the extract yields, the recoveries of antioxidants from DWP were in a wider range. Thus, methanol extracted antioxidants more efficiently. For instance, for the ASE methanol extracted from 1 g of dried peony leaves, the quantity of antioxidants was equivalent to 0.17–0.23 g of Trolox.

#### *3.3. Determination of Phytochemicals by UPLC-Q*/*TOF*

In total, 23 compounds were detected and most of them were identified based on the measured accurate masses, suggested in various databases formulas, chromatographic retention times, MS/MS fragmentation, data obtained with authentic reference compounds, and various literature sources (Table 2, representative chromatogram in Figure 2A).

**Figure 2.** Representative chromatograms of methanol extract (PLASEM) of *P. <sup>o</sup>*ffi*cinalis*. (**A**) UPLC-Q-TOF chromatogram; (**B**) HPLC-UV-DPPH•-scavenging chromatogram showing 19 active compounds (negative peaks at 515 nm), which were detected by comparing their retention times with the UV chromatogram recorded at 280 nm: gallic acid derivatives (3, 4, 5, 6, 7, 10, 12, 14, 15, 16, 17, 19, 20, 21), quercetin derivatives (11, 13), paeoniflorin derivatives (9, 18), and unknown compounds (22, 23).

MS data and retention times of compounds **1** and **4** well matched the standards of quinic and gallic acids. The compound **2** with molecular ion *m*/*z* = 341 [M − H]− and fragments of 191, 149, and 89 was assigned to dihexose. The molecular ion of compound **3** (*m*/*z* = 331.0671) corresponds to C13 H15 O10, while the fragment ion ( *m*/*z* = 169.0140) fits C7H5O5, the residue of gallic acid. Based on the recorded *m*/*z* and literature data [21], the compound was tentatively identified as galloylhexose. The main ion of peak **5** (*m*/*z* = 321.0253) corresponds to C14 H9O9, while the MS/MS fragmentation, due to the loss of [M − H − 152]− and [M − H–CO2] −, gave the ions of *m*/*z* = 169 (gallic acid, C7H5O5) and *m*/*z* = 125.0240 (C6H5O5), respectively. This peak was assigned to digallic acid, while compound **6,** which gave *m*/*z* = 183.0302 ([M − H]−), was tentatively identified as methyl gallate [21]. The MS<sup>2</sup> spectrum of this compound produced characteristic ions at *m*/*z* = 168.0060, 140.0112, and 124.0166. Compound **7** gave a molecular ion [M − H]− (*m*/*z* = 635), and fragments of 483, 465, and 169, fitting C27 H23 O18, C20 H19 O14, C20 H17 O13, and C7H5O5, respectively. The loss of 152, 170, and 466 amu was attributed to the loss of [M−H–galloyl]<sup>−</sup>, [M − H–galloyl–H2O]− and [M − H–2 galloyl–hexose]<sup>−</sup> units, respectively. As a result, this compound was tentatively identified as *tri*-galloyl-hexoside. Compounds **8** (tR 4.5), **22** (tR 9.9), and **23** (tR 10.2) with *m*/*z* = 491.1768, 621.0637, and 697.0695, matching C21 H31 O13, C15 H25 O26, and C31 H21 O19, respectively, have not been identified. Compounds **9** and **18** displayed a molecular ion [M − H]− (*m*/*z* = 525, C24 H29 O13) and several fragment ions in the MS/MS mode. The ion at *m*/*z* 479.1508/4791358 (C23 H27 O11) was a basic fragment arising from the loss of [M − H–H2O–CO]− (46 mass units), which, by a further loss of CH2O, C7H6O2, and [M − H–paeonyl–CHOH–2H]<sup>−</sup>, produced the fragments at *m*/*z* 449.1448/449.1440 (C22 H25 O10), 357.1191/357.1088 (C16 H21 O9), and 283.0818/283.0714 (C13 H15 O7), respectively. The ion at *m*/*z* 327.1086/327.1075 (C15 H19 O8) can be derived by the loss of the C8H8O3 from the basic ion, with *m*/*z* = 449, or from the ion with *m*/*z* = 357 (−30 mass unit; CH2O). Finally, the fragments corresponded to the paeonyl ( *m*/*z* = 165.0556/165.0544, C9H9O3) and benzoyl ( *m*/*z* = 121.0294/121.0281, C7H5O2) units resulting from the cleavage of the paeoniflorin. The compounds shared the same *m*/*z* at 121, 165, 283, 327, 357, and 449 as previously reported for paeoniflorin [22]. Considering that molecular ion ( *m*/*z* = 479) of **9** and **18** was higher by 46 Da, which can be attributed to CO (28 Da) and H2O (18 Da), the peaks tentatively identified as carboxylated and hydratated derivatives of paeoniflorin.



Compound **10** (tR 5.9 min) gave *m*/*z* = 787.1006 (C34H27O22), which was assigned to *tetra*-galloyl-hexoside because the main fragment at *m*/*z* = 617.0793 (C27H21O17) may be obtained by the loss of gallic acid (*m*/*z* = 169.0139, C7H5O5) residue from a deprotonated parent ion (*m*/*z* = 787.1006). The other characteristic fragment (*m*/*z* = 456.0683) may result due to the loss of the [M−H–hexosyl]– moiety from *m*/*z* = 617.0793. Compound **11** was tentatively identified as quercetin-dihexoside: its molecular ion *m*/*z* = 609.1460 corresponds to C27H29O16), [M − H − 463]− indicates the loss of quercetin-hexose (C21H19O12), [M − H − 146]− is characteristic to hexose moiety, while *m*/*z* = 301.0325 represents quercetin (C15H9O7). The compound **12** with a major ion of *m*/*z* = 615.0993 was assigned to quercetin-galloyl-hexoside; the loss of 463 Da indicates quercetin hexose moiety, while the fragments of *m*/*z* = 301.0324 (C15H9O7) and *m*/*z* = 169.0132 (C7H5O5) are characteristic of quercetin and gallic acid, respectively [23]. Compound **13** was assigned to quercetin pentoside (*m*/*z* = 433.0779, [M − H]−), which in MS/MS fragmentation lost pentose (132 Da) and gave a fragment ion of *m*/*z* = 301.0340 matching molecular formula (C15H9O7) of quercetin [21]. The full scan mass spectra of compound **14** (methyl digallate) showed mainly an intense ion at *m*/*z* 335, which yielded an MS<sup>2</sup> ion at *m*/*z* 183 (methyl gallate) corresponding to the loss of a gallic acid moiety ([M − H − 152]−). Compound **15** (tR = 7.8 min) gave an [M − H]− ion with *m*/*z* = 939.1122, indicating a molecular formula of C41H31O26. Its MS<sup>2</sup> spectrum contained four fragment ions: *m*/*z* = 769.0901 (C34H25O21) showing the loss of [M–152 − H2O]− and matching *tetra*-galloyl-hexose moiety; *m*/*z* = 617.0795 (C27H21O17), showing the loss of additional galloyl moiety [M − H − 152]− and matching *tri*-galloyl-hexose moiety; *m*/*z* = 447.0569, indicating the split of [M − H − 152–H2O]− and indicating a *di*-galloyl-hexose unit; *m*/*z* = 169.0132 (C7H5O5) matching gallic acid. All these data led to the identification of *penta*-galloyl-hexoside. Compound **16** (tR 8.2 min) with the molecular ion of *m*/*z* = 629.1149 (C29H25O16) was tentatively identified as isorhamnetin-galloyl-hexoside [21]. It produced a fragment ion of *m*/*z* = 477.1046 [M − H − 152]− due to the loss of gallic acid; the fragment ion of *m*/*z* = 315.0568 indicates the loss of galloyl + hexose [M − H − 152 − 162]<sup>−</sup>, and finally the ion of *m*/*z* = 169.0141 indicates on the presence of gallic acid. Three compounds, **17**, **19**, and **20** (tR 8.4, 8.7 and 9.3 min) with the precursor ion of *m*/*z* = 545 were assigned to dihydroxybenzoic acetate-digallate derivatives based on QTOF-MS and previously reported data [21]. In addition, their spectra showed the product ions of *m*/*z* = 469.0489/469.0407/469.0381 [M − H − 76]− and 393.0466/393.0461/393.0468 [M − H − 152]<sup>−</sup>, corresponding to the neutral losses of acetyl + H2O and galloyl moieties from the parent ion (*m*/*z* = 545), respectively. Finally, a fragment ion of *m*/*z* = 169.0135/169.0139/169.0140 indicates the presence of gallic acid. Compound **21** gave a [M − H]− ion with *m*/*z* = 1091.1236 and similar to *penta*-galloyl-hexoside fragmentation pattern with a difference of 152 amu, suggesting an additional galloyl unit. Moreover, the presence of product ions of *m*/*z* = 939.1101, 769.0895, 617.0811, and 169.0143 suggests the presence of 6 galloyl groups. Consequently, compound **21** was tentatively identified as *hexa*-galloyl-hexoside.

#### *3.4. Determination of Antioxidants by the On-Line HPLC-UV-DPPH*•*-Scavenging*

HPLC-UV with the on-line DPPH•-scavenging detectors was used for the preliminary screening of antioxidant phytochemicals in paeony extracts. As it may be judged from the size of the negative peaks in the chromatogram (Figure 2B), gallic acid derivatives (**3**, **4**, **5**, **6**, **7**, **10**, **12**, **14**, **15**, **16**, **17**, **19**, **20**, **21**), quercetin derivatives (**11**, **13**), paeoniflorin derivatives (**9**, **18**), and unknown compounds (**22**, **23**) were the strongest radical scavengers in the investigated extracts. Compound **5** was detected only in the PLTRW extract (data not shown).

The constituents, for which reference compounds were available, were quantified and their amounts were expressed in mg/g DWE and mg/g DWP (Table 3). The concentration of quinic acid, gallic acid, and quercetin dihexoside in different peony fractions was in the ranges of 2.14–7.58, 0.44–12.33, and 1.04–1.64 mg/g DWE, respectively. Quinic acid was quantitatively a major constituent in *P. o*ffi*cinalis* extracts (except for PLTRW), while the amounts of quercetin *di*-hexoside and gallic acid varied depending on extract type. For instance, the PLTRW extract contained the highest amount of gallic acid (12.33 ± 0.87 mg/g DWE). Its recovery was 4.10 ± 0.25 mg/g DWP, while the best recovery of quercetin *di*-hexoside was found in case of PLTRM.


**Table 3.** Quinic acid, gallic acid, and quercetin dihexoside recovery (mg/g DWP) and their concentration in the extracts (mg/g DWE) obtained from *P. o*ffi*cinalis* by di fferent solvents.

nd—not detected; \* Based on the calibration curve obtained by using rutin; values represented as mean ± standard deviation (*n* = 3); a–d: means down the vertical column not sharing common letters are significantly different (*p* < 0.05). The extracts isolated with methanol and water are further referred to by the abbreviation composed of the first letter of the plant (P—peony), the botanical part (L—leaves, R—roots), and the solvent (M—methanol, W—water);ASEandTRmeanextractiontype:accelerated solventextractionandtraditionalextraction,respectively.

#### *3.5.* α*-Amylase Inhibitory Properties of Selected Extracts*

*P. o*ffi*cinalis* extracts applied in a concentration range of 1.25–5 mg/mL inhibited α-amylase in a dose dependent manner, whereas the di fferences for lower concentrations (0.83–1.25 mg/mL) in most cases were not significant (Figure 3). The PLTRM extract was a slightly, although significantly, stronger inhibitor of α-amylase than other leaf extracts in the concentration range of 0.83–2.5 mg/mL. The values calculated in mg/mL IC50 (inhibitory concentration) were in the following range: PLTRW (2.52 ± 0.32) > PLASEM (2.34 ± 0.18) > PLTRM (1.67 ± 0.17) > acarbose 0.3 ± 0.12 d. Consequently, the strongest α-amylase inhibitor PLTRM extract was 5.5 times less e ffective than acarbose.

**Figure 3.** The inhibitory potential of *P. o*ffi*cinalis* methanol and water extracts against porcine α-amylase activity. The values represented as a mean ± standard deviation (*n* = 3). \*–\*\*\*: different symbols indicate significant differences (*p* < 0.05) between different extracts at the same concentration. The abbreviations are composed of the first letter of the plant (P—peony), the botanical part (L—leaves, R—roots), and the solvent (M—methanol, W—water); ASE and TR mean extraction type: accelerated solvent extraction and traditional extraction, respectively.

#### *3.6. In Vitro Cytotoxic and Cellular Antioxidant Activity (CAA) Activity of Extracts*

The cytotoxic e ffect was assessed on the Caco-2 cell line using the MTS method, where results showed no cytotoxic e ffect for each peony extract (PLTRW, PLASEM, PLTRM) in the range of the applied concentrations at 4 h, 24 h, and 48 h treatment (data not shown). Therefore, all extracts could be assessed in further research work. CAA-values for the peony extracts ranged from 0.046 to 0.106 μmol QE/mg extract (Figure 4).

**Figure 4.** Antioxidant activity of *P. o*ffi*cinalis* extracts evaluated by the cellular antioxidant activity (CAA) method. \*–\*\*: the mean ± standard deviation (*n* = 3) values followed by di fferent symbols are significantly di fferent (*p* < 0.05). Other abbreviations are explained in the legend of Figure 3.
