Sil 40c

The compounds were dissolved in MeOH at 1 mg/mL and filtered using a 0.45 μm PVDF membrane filter (Millipore Sigma, Billerica, MA, USA) before use. Ten microliters of the compound solution were injected into the column (Cosmosil 5C₁₈-AR-II, 5 μm, 250 × 4.6 mm i.d.) and separated by HPLC (SHIMADZU SIL-40C) at 45 °C with a mobile phase of acetonitrile (ACN) and water containing 0.3% phosphoric acid. The mobile phase program was set at 5–5–15–45–100% ACN from 0–10–20–40–50 min, respectively. The flow rate was set at 1 mL/min, and the compounds were detected at 210 nm.

The hydroalcoholic extracts (Em-L, Em-A, Es-L, Es-A, and Cv-L) were analyzed through the LC–MS technique using a Shimadzu liquid chromatography system (Kyoto, Japan), composed of a CBM-20A controller, two LC-30AD dual-plunger parallel-flow pumps, a DGU-20A5R degasser, a CTO-40C column oven, a SIL-40C autosampler, an SPD-M40 photo diode array detector, and an LCMS-8050 mass spectrometer, through an ESI source (Shimadzu, Kyoto, Japan).
Separation analyses were performed on a 150 × 4.6 mm; 2.7 µm Ascentis Express RP C18 column (Merck Life Science, Merck KGaA, Darmstadt, Germany). The mobile phase was composed of two solvents, water (solvent A) and acetonitrile (solvent B), both acidified with formic acid at 0.1% v/v. The flow rate was set at 1 mL/min and a simplified linear gradient of elution program was followed: 0–5 min, 0–30% B, 5–30 min, 30–100% B, 35 min, 100% B. PDA range: 200–400; λ = 280 nm (sampling frequency: 40.0 Hz, time constant: 0.08 s).
The applied mass spectrometry conditions were as follows: scan range, m/z 100–1200; scan speed, 2500 amu/s; event time, 0.3 s; nebulizing gas (N₂) flow rate, 1.5 L/min; drying gas (N₂) flow rate, 15 L/min; interface temperature, 350 °C; heat block temperature, 300 °C; DL (desolvation line) temperature, 300 °C; DL voltage, 1 V; interface voltage, −4.5 kV.