Chromquest 5.0 chromatography

The phenolic compounds contained in the tested extract were characterized using high-performance liquid chromatography (HPLC) with a diode array detector (DAD) (Finnigan Surveyor-PDA Plus detector, Thermo Fisher Scientific, Waltham, MA, USA) and ChromQuest 5.0 chromatography software (Thermo Fisher Scientific). Separation was achieved on a Lichrospher RP 18–5 (250 mm by 4.6 mm, 5 µm packing; Hichrom, Reading, UK). The elution conditions were as follows: flow rate of 0.8 mL/min; oven temperature of 25 °C; solvent A (5% (v/v) formic acid), and solvent B (95% (v/v) acetonitrile). The injection volume was 50 µL. Detection was conducted at 280, 320, and 360 nm. The identification of compounds was carried out on the basis of the results for the standards. Gallic acid (Sigma-Aldrich, Saint Louis, MO, USA), neochlorogenic acid, chlorogenic acid (Sigma-Aldrich), epicatechin (Sigma-Aldrich), p-coumaric acid (Sigma-Aldrich), ferulic acid (Sigma-Aldrich), rosmarinic acid (Sigma-Aldrich), quercetin-3-rutinoside (Sigma-Aldrich), caffeic acid (Sigma-Aldrich), and quercetin (Sigma-Aldrich) were used as pure standard solutions. On the basis of results obtained for tested plant sample and pure standards, the individual components of mint extract were identified.

Polyphenolic compounds were quantified according to a previously published procedure [23 (link)]. Prior to analysis, the samples were filtered using 0.45 µm membrane filters. HPLC-PDA analyses were performed using a Finnigan Surveyor equipped with an autosampler and a diode array detector (Finnigan Surveyor PDA Plus Detector, Thermo Scientific), controlled with ChromQuest 5.0 chromatography software (Thermo Fisher Scientific Inc.). Separation was achieved using a Spherisorb ODS2 column (250 × 4.6 mm × 5 µm packing) (Waters), protected with a guard column of the same material. The volume of the injected sample was 50 µL, the flow rate was 0.8 mL/min, and the separation time was 60 min. The mobile phase consisted of 5% formic acid (solvent A) and 95% acetonitrile (solvent B). The samples were eluted with the following gradient: 2 min, 97% (A); 13 min, 97–85% (A); 9 min, 85–82% (A); 31 min, 82–75% (A); 5 min, 75–70% (A). Polyphenols were quantified according to calibration curves established for gallic acid (280 nm), caffeic acid (320 nm), and quercetin-glucoside (360 nm). The concentration of polyphenols was expressed as µg/g of leaves.