Uvd100

HPLC analyses were carried out on a Dionex instrument (ThermoFisher Scientific, Milano, Italy) equipped with an Ultimate 3000 high-pressure binary pump, an ASI-100 autosampler, a TCC-100 thermostated column compartment and a UVD-100 multiple wavelength detector set at 220, 230, 240 and 260 nm. Chromeleon software (version 6.7) was used for instrument control, data acquisition, and data handling. Chiral HPLC was performed on a Phenomenex (Phenomenex, Inc. Bologna, Italy) Lux 5 μm Cellulose-1 (250 × 4.6 mm) column eluting with n-hexane/EtOH 90:10 at flow 0.5 mL/min. The composition of reaction mixtures was determined at λ 220 nm by applying the suitable response factors for sulfoxides with respect to sulfides and for sulfones with respect to sulfoxides. Peak assignment was based on the reported elution order of enantiomers of sulfoxides on the same stationary phase [43 (link),58 (link)]: t_R 17.7 min [(R)-1a] and 19.4 min [(S)-1a]; 17.2 min [(R)-2a] and 19.0 min [(S)-2a]; 21.7 min [(R)-3a] and 25.9 min [(S)-3a]; 18.1 min [(R)-4a] and 19.7 min [(S)-4a]; 22.6 min [(R)-5a] and 25.3 min [(S)-5a].

High-performance liquid chromatography (HPLC) was performed on a Dionex Chromelon TM chromatography data system with P580 and UVD100 detectors (Thermo Fisher Scientific Inc., Waltham, MA, USA). Analyzed sample (CLE) was prepared in 50% methanol at 5mg/mL. Ranges of diluted concentrations (3.125–100 µg/mL) of standard chlorogenic acid and (−)-epicatechin were dissolved in 50% methanol. Chromatographic separation was performed on a Waters 120 ODS-AP (250 × 4.6 mm², 5-µm particle size). Elution was performed with a water/acetonitrile (3:1) gradient containing 1% formic acid. The gradient was linearly increased from 5% to 90% acetonitrile over 35 min. The injection volume was 10 µL and the flow rate was 1 mL/min. Analysis of chlorogenic acid and (−)-epicatechin in CLE was validated by using the external standard method, according to the International Conference on Harmonization [56 ] (see Supplementary Materials). Linear correlations (R²) of the calibration curves for each compound were 0.999. The limit of detections (LOD) and limit of quantifications (LOQ) were less than 0.028 µg/g and 0.074 µg/g, respectively (see Supplementary Materials). Thus, established HPLC method was suitable for the quantitative analysis.

SGE was prepared at a concentration of 2 mg/mL in 50% methanol. Serial dilutions (2.5, 25, 125, 250, 500, and 1000 µg/mL) of the standard compound (kirenol) in methanol were prepared. HPLC was performed on an Ultimate 3000 LC system, using P580 and UVD100 detectors (Thermo Fisher Scientific Inc., Waltham, MA, USA). Chromatographic separation was performed on an Inno C-18 column (5 μm, 4.6 × 250 nm; Young Jin). The column temperature was 40 °C, the flow rate was 1.0 mL/min, the analysis time was 40 min, and the injection volume was 10 µL. To verify the validity of the method, the experiment was conducted based on the “Guideline for Validation of Test Methods such as Drugs” (Ministry of Food and Drug Safety).

The plant extract was prepared in 50% methanol at a concentration of 2 mg/ml. Serial dilutions (2.5, 25, 125, 250, 500, and 1000 µg/mL) of standard compounds (tannic acid, chlorogenic acid, and apigenin) were prepared in methanol. High-performance liquid chromatography (HPLC) was performed on a Dionex Chromelon TM chromatography data system with P580 and UVD100 detectors (Thermo Fisher Scientific Inc., Waltham, MA, USA). Chromatographic separation was performed on a Discovery C₁₈ (250 × 4.6 mm, 5-µm particle size). Column temperature was 25 °C; flow rate was 1.0 mL/min; injected volume was 10 µL. The condition for chromatographic separation was described in Supplementary Materials Table S1. The chemical content was quantified by determining the area of the peak in the HPLC analysis, following the formula below: $$Content \left(g/100\right)=\left(\frac{sample area}{standard area}\right)\times \left(\frac{sample dilution volume}{standard dilution volume}\times dilution factor\right)\times \left(\frac{sample amount}{standard amount}\right)\times 100$$

PASE was prepared at the concentration of 2 mg/mL in 50% methanol. Serial dilutions (2.5, 25, 125, 250, 500, and 1000 µg/mL) of the standard compound (catechin-7-O-β-D-glucopyranoside) in methanol were prepared. HPLC was performed on a Dionex Chromelon TM chromatography data system with P580 and UVD100 detectors (Thermo Fisher Scientific Inc., Waltham, MA, USA). Chromatographic separation was conducted on an Inno C-18 column (5 μm, 4.6 × 250 nm; Young Jin). The column temperature was 25 ℃, the flow rate was 1.0 mL/min, and the injected volume was 10 µL.