Quantifying Procyanidin Content in Extracts

In order to determine procyanidin contents in the extracts, the vanillin-H₂SO₄ methodology was conducted with some modifications [17 (link)]. Two hundred and fifty µL of the extract was reacted with 250 µL of 1% vanillin (w/v, dissolved in methanol), followed by 250 µL of the 25% sulfuric acid solution (v/v, dissolved in methanol) and incubated at a temperature of 30 °C for 15 min. Absorbance was read at 500 nm using a Synergy HTX Multi-Mode microplate reader (Biotek, Rochester, VT, USA). Total tannin contents were estimated using a calibration curve with catechin (A₅₀₀ = 1.9461 (catechin) −0.0007, R² = 0.9990) using nine concentrations of catechin (10–100 µg/mL). Condensed tannin contents were expressed as milligrams of catechin equivalents per gram of dry extract (mg CE/g DE).