Lichrospher rp 18

Analyses were carried out on an Agilent 1200 RR HPLC instrument with a DAD detector (Agilent, Waldbronn, Germany) using a reversed-phase LiChrospher RP-18 (Agilent) analytical column (250 × 4 mm i.d.; 5 µm particle size). The mobile phase consisted of solvent A (1% v/v solution of orthophosphoric acid in water) and solvent B (acetonitrile). Separation was achieved according to the following scheme: 90–80% A 0–5 min, 80% A 5–10 min, 80–70% A 10–20 min, 70–30% A 20–30 min, 30–0% A 30–35 min. Detection wavelengths were set at 280 and 330 nm, and the flow rate was 1 mL/min. The injection volume was 5 μL, while the column temperature was maintained at 25 °C. The compounds 4′-O-methylhypolaetin-7-O-[6‴-O-acetyl-β-d-allopyranosyl (1→2)-β-d-glucopyranoside (HYP), isoscutellarein 7-O-[6‴-O-acetyl-ß-d-allopyranosyl (1→2)]-ß-d-glucopyranoside (ISC 1) and 4′-O-methylisoscutellarein-7-O-[6‴-O-acetyl-β-d-allopyranosyl-(1→2)]-β-d-glucopyranoside (ISC 2) were previously isolated in our laboratory [2 ]. The concentrations of compounds in the investigated extracts were determined using calibration curves (r > 0.9997). The results are presented as milligrams per gram of dry weight (mg/g dw).

Analyses of individual polyphenolic compounds were carried out on an Agilent 1200 RR system (Agilent, Waldbronn, Germany) with a diode array detector. A reverse-phase Lichrospher RP-18 (Agilent) column (250 mm × 4 mm, 5 μm) was used, and the column temperature was maintained at 25°C. The mobile phase consisted of solvent A (10%, v/v solution of formic acid in water) and solvent B (acetonitrile), using gradient elution as follows: 1% B, 0–0.5 min; 1–7% B, 0.5–1 min; 7% B, 1–4 min; 7–10% B, 4–7.5 min; 10–14% B, 7.5–11.5 min; 14–25% B, 11.5–15.5 min; 25–40% B, 15.5–18.5 min; 40–75% B, 18.5–22 min; 75% B, 22–25 min. The injection volume was 10 μL, the flow rate was 1 mL/min and the detection wavelengths were set at 290, 350 and 520 nm. The contents of the compounds were calculated using calibration curves. The results are presented as milligrams per gram of dried extract (mg/g DE). Of the detected polyphenolic compounds, eight of them were quantified by measuring peak areas against a standard curve constructed with commercial standards, including: cyanidin-3-galactoside, cyanidin-3-sambubioside (calculated based on a standard curve for cyanidin-3-glucoside), cyanidin-3-glucoside, kaempferol derivative 1, kaempferol derivative 2 (both calculated based on a standard curve for kaempferol) and chlorogenic acid.

The quantification of the dominant anthocyanin compound cyanidin-3-O-glucoside was performed by HPLC analysis according to the procedure described in European Pharmacopoeia 10.0 (2019) (monograph 04/2019:02394) with slight modifications [35 ]. All extract samples were diluted with deionized water (1:1 v/v) immediately before chromatographic analysis and filtered using a 0.45 μL syringe filter. An Agilent series 1200 HPLC with a diode array detector and a reverse phase analytical column Lichrospher RP-18 (250 × 4 mm i.d., 5 µm particle size, Agilent) were used. The mobile phase was composed of solution A (anhydrous formic acid and water, 8.5:91.5 v/v) and solution B (anhydrous formic acid, acetonitrile, methanol, and water, 8.5:22.5:22.5:41.5 v/v/v/v). The sample was injected at a volume of 20 µL and separated at a flow rate of 1 mL/min by the following elution program: starting with the solution B 7%, from 7% to 25% on 35 min, from 25% to 65% on 45 min, and from 65% to 100% on 46 min and maintained during 46–50 min. The column was thermostated at 30 °C and the detection wavelength was set at 535 nm. The cyanidin-3-O-glucoside peak was identified based on the position in the chromatogram and the UV spectrum of the authentic standard. Its amount was determined using the calibration curve of the standard and is expressed as mg per g of dried drug weight (mg/g DW).