C18 column

The content of tricin in the ETZL was determined by HPLC using an Agilent Infinity 1200 series system with a diode array detector (Agilent Technologies, Palo Alto, CA, USA). The separation of tricin was conducted at 30 °C using a SUPELCO Discovery^® C18 column (4.6 × 250 mm, 5 μm, Merck KGaA, Darmstadt, Germany). The mobile phase composition was as follows: A, 0.15% phosphoric acid in water; B, methanol. The gradient elution conditions were as follows: 0–3 min, 20% B; 3–8 min, 20–50% B; 8–20 min, 50–55% B; 20–30 min, 55–85% B; 30–45 min, 85–20% B. The flow rate was 1.0 mL/min and the injection volume was 10 μL. The chromatograms were obtained at 350 nm and UV-Vis spectra were acquired in 190–400 nm (Figure 1). The content of tricin was 1.09 ± 0.01 mg/g in the ETZL.

EE was solubilized in methanol (Tedia) (1 mg/mL), and 10 μL of this sample was analyzed using LaChrom Elite HPLC system (Hitachi, Tokyo, Japan) liquid chromatograph equipped with L2130 pump, auto-sampler L2200, L2300 column oven was set at 25 °C and an L2455 diode array detector (DAD) (Hitachi, Tokyo, Japan). The C-18 column (5 μm, 150 mm × 4.6 mm) was used in combination with an appropriate guard column (4.0 mm × 4.0 mm; 5 μm of particle size) (Merck, Darmstadt, Germany). The analysis was performed at a wavelength fixed at 354 nm. The eluents used were aqueous phosphoric acid (Merck, Darmstadt, Germany) (1%) (solvent A) and acetonitrile (J. T. Baker, Leicestershire, UK) (solvent B). The gradient employed was 90% A and 10% B for 0 min, 70% A and 30% B for 40 min, 50% A and 50% B for 50 min, 90% A and 10% B for 51 min, and 90% A and 10% B for 55 min at a flow rate of 0.6 mL/min. Data acquisition was performed using ExChrom Elite software (version 3.3.2 SP1) (Scientific Software Inc., Santa Clara, CA, USA). The compounds present in the extract were compared according to their UV–Vis spectra (similarity index > 0.99) and retention times with commercial standards, as previously described [44 ].

Plumericin detection in extracts and in standard samples was performed using an HPLC-DAD device (ELITE LaChrom). A gradient system developed in the lab was applied: solvent A (0.05% Trifluoroacetic acid (TFA) in water milli-Q, and solvent B (acetonitrile) for 30 min on a C18 column (Merck©, France; column temperature 27 °C) at a flow rate of 1.0 mL/min. The Elution profile: 0 to 7 min, 40 to 45% B (linear gradient); 7 to 12 min, 45% B (isocratic); 12 to 13 min, 45 to 50% B (linear gradient); 13 to 15 min, 50 to 70% B (linear gradient); 15 to 17 min, 70 to 100% B (linear gradient); 17 to 28 min (column washing), 100% B (linear gradient). Plumericin detection and quantification were established at 230 nm wavelength.