Phloretin

Chemicals and reference standards were obtained from Sigma-Aldrich (Steinheim, Germany): caffeic acid (CAA), (+)-catechin (CAT), 3,4-dihydroxybenzoic acid (DBA), (−)-epicatechin (EPC), ferulic acid (FEA), gallic acid (GAA), 4-hydroxybenzoic acid (HBA), hesperetin (HES), kaempferol (KAE), morin (MOR), naringenin (NAN), p-coumaric acid (PCA), phloridzin (PHD), phloretin (PHT), quercetin-3-D-galactoside (QGA3), quercetin-3-D-glucoside (QGU3), quercetin-7-D-glucoside (QGU7), quercetin (QUR), sinapic acid (SIA), siringic acid (SRA), taxifolin (TAF) and FC reagent. The standard reference narirutin (NAR) was obtained from K&J Scientific (Marbach am Neckar, Germany) and isorhamnetin (IRT) and naringin (NAG) from Carl Roth (Karlsruhe, Germany). Stock solutions were prepared by dissolving the reference standards in analytical grade absolute ethanol and diluting each of them in seven steps for the measurements.
The reducing capacity was determined by spectrophotometry as orignally described [28 ] with slight modifications. Briefly, 20 μL of each sample (reference standard dilution or ethanol) was mixed with 100 μL demineralized water (H ${}_2$ O ${}_{\mathrm{demin}}$ ) and 100 μL FC reagent and were incubated for 3 $\min$ in the dark. We then added 1580 μL of H ${}_2$ O ${}_{\mathrm{demin}}$ and 200 μL 7.5% (w/v) sodium carbonate and incubated for another 30 $\min$ in the dark. The absorption of the samples was measured at 765 $\mathrm{n}$ $\mathrm{m}$ using a Specord 210 plus spectrophotometer (Analytik Jena, Jena, Germany). The slope was determined dy linear regression and presented as the reducing capacity.
For statistical analysis, Sigma Plot (Systat Software, San Jose, CA, USA) was used for one-way analysis of variance (ANOVA) corresponding to an unpaired t-test. If there was a significant difference, an additional pairwise test was carried out using the Holm–Šidák method. The significance level for both tests was 0.05. Statistical analysis was always carried out with all significant decimal places.

For determination of polyphenolics and HMF, the powdered samples (approx. 1 g) were taken and 5 mL of methanol/water/ascorbic acid mixture (30:68:1, v/v/m) with 1% hydrochloric acid was added to each sample before and after incubated overnight time (4 °C) and sonicated (Sonic 6D; Polsonic, Warsaw, Poland) for 20 min [33 (link)]. Then, the extract was centrifuged (MPW-55; Warsaw, Poland) at 19,000× g for 10 min at 4 °C. Finally, before analysis, the extract was filtered through a 0.20 μm hydrophilic PTFE membrane (Millex Simplicity Filter; Merck; Darmstadt, Germany) and analyzed by UPLC.
The analysis of polyphenols provided by UPLC-PDA (Aquity, Waters; Milford and Taunton, Millford, MA, USA) was provided as described previously Wojdyło et al. [34 (link)]. analysis of total polyphenols expresses as sum of dihydrochalcones (sum of phloretin and phloridzin at 280 nm), flavan-3-ols (sum of monomer, dimer, trimer at 280 nm), phenolic acid (chlorogenic acid at 320 nm), flavonols (as sum of quercetin derivatives at 360 nm) and anthocyanins (as sum of cyanidins derivatives at 520 nm). The analysis of HMF was made at 284 nm. Prior to the measurements, the equipment was calibrated using a standard quercetin-3-O-glucoside, (−/+)-(epi)catechin, procyanidins B1, B2, chlorogenic acid, cyanidin-3-O-glucoside and phloretin-2-O-glucoside and HMF at 1 to 5 mg/L (r² = 0.999–0.997). Data are the mean of three replicates, and expressed as mean value as mg/kg dry weight (dw).
For polyphenolic and HMF quantification, 5 μL of each sample was analyzed an BEH C18 column (2.1 × 100 mm, 1.7 μm; Waters Corp., Dublin, Ireland) at 30 °C with gradient elution at a flow rate of 0.42 mL/min for 15 min. The mobile phase was composed of solvent A (2.0% formic acid) and solvent B (acetonitrile) as 1% to 25% solvent B until 12 min, and then held constant to wash and re-equilibrate the column.
Analysis of polymeric procyanidins was provide by UPLC-FL using phloroglucinolysis method as described previously by Wojdyło et al. [15 (link)]. Approx. 0.05 g were precisely measured into 2 mL Eppendorf vials and freeze-dried (24 h; Alpha 1-4 LSC; Martin Christ GmbH, Osterode am Harz, Germany), then 0.8 mL of the methanolic solution of phloroglucinol (75 g/L) and ascorbic acid (15 g/L) was added. After the addition of 0.4 mL of methanolic HCl (0.3 mol/L), the vials were closed and incubated for 30 min at 50 °C with continuous vortexing using a thermo shaker (TS-100; BIOSAN., Riga, Latvia). The reaction was stopped by placing the vials in an ice bath with drawing 0.5 mL of the reaction medium and diluting with 0.5 mL of 0.2 mol/L sodium acetate buffer. Next, the vials were cooled in ice water and centrifuged immediately at 20,000× g for 10 min at 4 °C. The analysis of polymeric procyanidins was carried out on a UPLC-FL Acquity system (Waters Corp., Waters Corp., Dublin, Ireland) and detection was recorded at an emission wavelength of 360 nm and excitation wavelength of 278 nm. Injection of 5 μL of each sample was analyzed on an BEH C18 RP column (2.1 × 5 mm, 1.7 μm; Waters Corporation, Milford, MA, USA) at 15 °C with gradient elution at a flow rate of 0.42 mL/min for 10 min. The mobile phase was composed of solvent A (2.5% acetic acid) and solvent B (acetonitrile) as 2% B initially until 0.6 min, 9% B until 7.3 min and then held constant to wash and re-equilibrate the column until 10 min. Prior to the measurements, the equipment was calibrated using a standard (+)-catechin, (−)-epicatechin and procyanidin B1. Data are the mean of three replicates, and expressed as mean value as mg/kg dry weight (dw).

Commercial standards of derivatives of anthraquinones (aloe-emodin, aloe-emodin-8-Glu, emodin, emodin-8-Glu, rhein, rhein-8-Glu, chrysophanol, chrysophanol-8-Glu, physcion, sennoside-A, sennoside-B, sennoside-C, sennoside-D), stilbenes (resveratrol, pterostilbene, pinostilbene, piceatannol, astringin, polydatin, rhapontigenin, rhaponticin, isorhapontigenin, isorhaponticin, deoxyrhapontigenin, deoxyrhaponticin), phenolic acids (glucogallin, gallic-acid), flavones (vicenin-II, vicenin-III, apigenin-7-Glu), flavanones (pinocembrin, pinocembroside), chalcones (phloretin, phloridzin, trilobatin), and catechins (catechin, epicatechin, gallocatechin, procyanidin-B1, procyanidin-B2, procyanidin-B3, and procyanidin-C1), acetonitrile LC-MS grade, formic acid MS-grade, and tert-butanol were purchased from Merck (Darmstadt, Germany). Methanol, n-hexane, and n-butanol, all of analytical grade, were purchased from Fisher Chemical (Loughborough, UK). Ultrapure water was prepared using a Milli-Q water purification system (MerckMillipore). General reagents for bioassays were purchased from Sigma-Aldrich (a part of Merck KGaA, Darmstadt, Germany) and Cayman Chemicals (Ann Arbor, MI, USA). Reagents specific to cell culture, gene expression analyses, and cytokine profiling have been indicated below in the descriptions of the applied methods.

UHPLC chromatographic system Acquity UPLC (Waters, Milford, MA, USA) comprising a binary pump, an autosampler, a column oven, and a diode array detector (DAD) was used for the determination of active compounds in the extracts. The system control, data acquisition, and data evaluation were executed using the Empower software (Waters, Milford, MA, USA). The reversed-phase Triart ExRS C18 (150 × 3 mm; 1.9 µm) column preceded by a guard column Ascentis Express C18 (5 × 4.6 mm) packed with 5 µm particles was used for the separations. The mobile phases consisted of aqueous acetic acid with pH 2.8 (A) and acetonitrile (B) with a flow rate of 0.35 mL/min. The gradient elution enabled the separation of the active compounds. The gradient started with 10% B in A as the initial conditions. Then, B was ramped to 22% in 8 min and 28% in the next 2.2 min. Then, a steep increase to 40% in 30 s followed by a further increase to 50% in 3 min and held for 0.2 min. Finally, the percentage of B was decreased to initial conditions in 0.2 min and held for 3.4 min to equilibrate the system. The temperature of the column was 30 °C. The sample was cooled at 6 °C in an autosampler and 2 µL were injected into the UHPLC system. The separation of all tested phenolic compounds lasted 15 min.
The analytes summarized in Table S1 were detected by DAD at 4 different wavelengths: (i) 254 nm selected for guaiaverin, hirsutrin, hyperoside, reynoutrin, quercitrin; (ii) 280 nm selected for gallic acid, epicatechin, catechin, phloridzin, phloretin; (iii) 320 nm applied for chlorogenic and caffeic acid; and (iv) 354 nm selected for rutin and quercetin. The 3D record was also recorded for wavelengths in the range of 210–400 nm to collect UV spectra for all detected peaks. Obtained UV spectra, together with retention times, were used for the identification of individual active compounds by comparing standard solutions and extracts.
Concentration levels for each analyte were calculated from the respective integrated peak areas compared to the standard (10 µg/mL) while within the framework of the previous measurements for the determination of phenolics in apples (unpublished results). Calibrations of all analytes proved to be linear in the whole tested range starting from 0.1 µg/mL.