Phlorhizin

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).

Identification and quantification of the phenolic compounds in the leaf and tree bark extracts was carried out using the 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Lichrospher 100 RP 18e, 250 × 4 mm, 5 µm particle size column according to the method of Tadic et al. [28 (link)]. The concentrations of the leaf and tree bark ethanol extracts were 27.1 and 33.4 mg/mL, respectively. Prior to injection, the samples were filtered through a PTFE membrane filter. As the standard used in the investigation, the concentration were 0.15 mg/mL for isoquercetin, 0.26 mg/mL for hyperoside, 0.28 mg/mL for kaempferol-3-O-glucoside, 0.30 mg/mL for vanillic and kaempferol, 0.34 mg/mL for protocatechuic and gallic acids, 0.36 mg/mL for quercetin, 0.38 mg/mL for resveratrol, 0.40 mg/mL for rutin and epicatechin, 0.56 mg/mL for chlorogenic acid, 0.74 mg/mL for p-coumaric acid, 0.25 mg/mL for ellagic acid, 0.17 mg/mL for luteolin and morin, 0.1 mg/mL for procyanidin B1, 0.52 mg/mL for protocatechuic acid ethyl ester and 0.11 mg/mL for p-hydroxybenzoic acid and phloridzin. The volume of the standard solutions being injected, as well as for the tested sample extracts, was 4 µL.
The juice and fruit extracts were analyzed for the content of anthocyanins and anthocyanidins according to the method by Ivanovic et al. [29 (link)]. The standard solutions for the determination of anthocyanins and anthocyanidins were prepared at a final concentration of 0.2, 0.3 and 0.4 mg/mL (cyanidin chloride, cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, respectively) in methanol/HCl. The concentrations of the juice and fruit ethanol extracts were 51.0 and 49.9 mg/mL, respectively. HPLC separation of anthocyanins was achieved using a LiChrospher 100 RP 18e (5 μm), 250 × 4 mm i.d. column with a flow rate of 0.8 mL/min and mobile phase, A [500 mL of H₂O plus 9.8 mL of 85% H₃PO₄(w/w)], B (ACN), elution, a combination of gradient mode 89–75% A, 0–35 min; 75–60% A, 35–55 min; 60–35% A, 55–60 min; 35–0% A, 60–70 min. Detection was performed using a diode array detector (DAD) and chromatographs were recorded at 520 nm. The standard solutions for the determination of anthocyanins and anthocyanidins were prepared at a final concentration of 0.4, 0.3 and 0.3 mg/mL (cyanidin chloride and cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside, respectively) in methanol/HCl. The concentrations of the investigated extracts were 11.32 and 22.55 mg/mL for fresh fruit juice and Soxhlet extract of fresh fruit, respectively, in methanol/HCl. The volume of the standard solutions being injected, as well as for the tested sample extracts, was 4 μL. Prior to HPLC analysis, the samples were filtered through a 0.2 μm PTFE filter (Fisher, Pittsburgh, PA, USA).
Identification was based on the retention times and overlay curves. Once spectra matching was achieved, the results were confirmed by spiking with the respective standards to achieve a complete identification by means of the so-called peak purity test. The peaks not fulfilling these requirements were not quantified. Quantification was performed by an external standard method (taking into account the purity of the used standards) and the results were expresses as the mean value ± SD of three measurements.

Acetonitrile, water (HPLC), methylene chloride, ethyl acetate, acetone, ethyl ether, albumin standard, 1-chloro-2,4-dinitrobenzene, nicotinamide adenine dinucleotide phosphate (NADPH) and paraffin wax was obtained from Merck (Germany). Streptozotocin (STZ) was obtained from Sigma Chemicals Co (St. Louis, MO, USA). Referent HPLC standards (chlorogenic acid, vanillic acid, p-coumaric acid, rutin, hyperoside, isoquercetin, ellag-ic acid, kaempferol-3-O-glucoside, morin, resveratrol, quercetin, luteolin, kaempferol, gallic acid, cyanidin chloride, cynanidin-3-O-glucoside, cyanidin-3-O-rutinoside, pro-cyanidin B1, protocatechuic acid, protocatechuic acid ethyl ester, p-hydroxybenzoic acid, epicatechin and phloridzin) (HPLC grade, ≥99% purity) were obtained from Extrasynthese (Lyon, France). The other chemicals were purchased from Sigma-Aldrich, Schnelldorf, Germany.