Spd m20a

The fingerprinting of the extracts was done on Shimadzu Prominence Series coupled with photodiode array (PDA) detector SPD-M20A using either reversed-phase or normal phase settings:

Reversed-phase, Chromolith HighResolution RP-18 endcapped 100–4.6 mm (Merck): the mobile phase used was solvent A, acetonitrile and solvent B, water with a standard flow rate of 1.0 mL/min, and injection volume of 20 μL of 10 mg/mL extract; the gradient of the mobile phase was as follows: 0% to 50% A (0–45 min).

Normal phase, Phenomenex Luna Silica column (250 × 4.6 mm, 100 Å, 5 μm): the mobile phase used consists of solvent A, hexane and solvent B, 2-propanol with a flow rate of 1.0 mL/min, and injection volume of 20 μL of 10 mg/mL extract; the gradient program was as follows: 100% A (0–5 min), 100% to 0% A (5–25 min).

HPLC analysis was carried out according to a previously described method [14 (link)] with few modifications. In brief, aqueous extract was fractionated with ethyl acetate and was dissolved in methanol (1 mg/mL) before subjecting to analysis. The HPLC system (Shimadzu, Japan) consisted of dual pump LC-20AD binary system, PDA detector SPD-M20A; Merck C₁₈ reversed-phase column (I.D. 4.6 mm × 250 mm, 5 μm). Separation was carried out using a two-pump linear gradient program for pump A (40% acetonitrile) and pump B (5% acetonitrile). Elution was initiated with a gradient of 12% A changing to 40% in 20 min, 100% in 45 min and finally to 12% in 55 min followed by washing for 35 min. Injection volume was 20 μL and flow rate was maintained at 1 mL/min throughout the process.