1100 series liquid chromatograph

HPLC analysis was executed according to (Sati et al., 2020 (link)) with slight modifications using an Agilent Technologies 1100 series liquid chromatograph equipped with an autosampler (Table 1).

The HPLC analysis was performed as described previously [36 (link)], with slight modifications, using an Agilent Technologies 1100 series liquid chromatograph equipped with an autosampler. The analytical column was Agilent Eclipse XDB C18 (100 × 4.6 μm; 3.5 μm particle size). The diode array detector was set to a scanning range of 180–420 nm. The mobile phase consisted of methanol (solvent A) and 0.1% formic acid (v/v) (solvent B). The flow rate was kept at 0.4 mL min^− 1 and the gradient program was as follows: 10% A - 90% B (0–5 min); 20% A - 80% B (5–10 min); 30% A - 70% B (10–15 min); 50% A - 50% B (15–20 min); 70% A - 30% B (20–25 min); 90% A -10% B (25–30 min); 50% A -50% B (30–35 min); and 10% A - 90% B (35–36 min). A 5 min post-run was used for reconditioning. The injection volume was 10 μL and peaks were monitored simultaneously at 280, 320 and 360 nm for the benzoic acid and cinnamic acid (Sigma, St. Louis, MO, USA) derivatives and flavonoid compounds, respectively. All samples were filtered through a 0.45 μm Acrodisc syringe filter (Gelman Laboratory, MI) before injection. Peaks were identified based on congruent retention times and UV spectra and compared with those of the standards (Sigma, St. Louis, MO, USA).

High-performance liquid chromatography (HPLC) analysis was carried out according to Kim et al. (2006 (link)) using Agilent Technologies 1100 series liquid chromatograph equipped with an autosampler and a diode-array detector. The analytical column was an Eclipse XDB-C18 (150 × 4.6 µm; 5 µm) with a C18 guard column (Phenomenex, Torrance, CA). The mobile phase consisted of acetonitrile (solvent A) and 2% acetic acid in water (v/v) (solvent B). The flow rate was kept at 1 mL/min for a total run time of 60 min and the gradient program was as follows: 100 to 85% B in 30 min, 85 to 50% B in 20 min, 50 to 0% B in 5 min, and 0 to 100% B in 5 min. The injection volume was 20 µL and peaks were monitored simultaneously at 280 and 320 nm for the benzoic acid and cinnamic acid derivatives, respectively, as well as 360 nm for flavonoids. All samples were filtered through a 0.45 µm acrodisc syringe filter (Gelman Laboratory, MI) before injection. Peaks were identified by congruent retention times and UV spectra and compared with those of the standards.

HPLC analysis was carried out following the method of Kim et al.^{27 (link)} with minor modification, using 95% ethanolic extract of the aerial parts. "Agilent Technologies 1100 series liquid chromatograph equipped with an autosampler and a diode-array detector" was established for the chromatographic separation. Eclipse column XDB-C18 (150 × 4.6 µm; 5 µm) connected with C18 guard column (Phenomenex, Torrance, CA) was used along with 0.45 µm acrodisc syringe filter (Gelman Laboratory, MI). The mobile phase consisted of solvent A (acetonitrile) and solvent B (2% v/v acetic acid in water).
Gradient elution was applied following the technique "100% B to 85% B in 30 min, 85% B to 50% B in 20 min, 50% B to 0% B in 5 min and 0% B to 100% B in 5 min" with a total run time of 60 min. The injection volume was 50 µl at a flow rate of 0.8 ml/min. Peaks were detected at 280 nm (for benzoic acid derivatives), 320 nm (for cinnamic acid derivatives) and 360 nm (for flavonoids). Peaks identification was performed by congruent retention times and UV spectra and compared with those of the standards.
Three independent injections were performed to calculate SD.