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.
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.
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