Polyphenol Extraction and Quantification

Extraction of total polyphenols was carried out according to Sarker and Oba⁶⁴ using 25 mg of sample in 2.5 mL of 1.2 M HCl containing methanol (90%) at 90 °C for 2 h in a water bath. With readjusting the volume (2.5 mL), the leaf extract was centrifuged at 7,500 rpm for 20 min. The leaf extracts (100 µL) were added to the Folin–Ciocalteau reagent (2 N, 50 µL). After 5 min, 2 N Na₂CO₃ (400 µL) and water (1 mL) was added. The leaf extracts were incubated for 90 min at 37 °C. Finally, it was removed to a microplate (flat bottom). In a microplate reader, the absorbance was detected at 740 nm. We estimated the results in equivalent to gallic acid (GAE) standard µg g⁻¹ of FW.