2.3.1. Total Phenolic Content

The TPC of the HI extracts was assessed by Folin–Ciocâlteu (FC) spectrophotometric method as previously reported [15,16]. A Synergy HT multi-detection microplate reader with 96-well plates (BioTek Instruments, Inc., Winooski, VT, USA) was used for high sample throughput. At first, 20 μL of each sample (HI extracts diluted five times) were mixed with 100 μL of FC reagent (diluted 1:10) and, after 3 min, 80 μL of sodium carbonate solution (7.5% *w*/*v*) was added. The mixtures were incubated in the dark at room temperature for 30 min. The absorbance was measured at 760 nm against a solvent blank. Gallic acid was used as a reference standard, and the TPC was expressed as gallic acid equivalents (GAE) per dry weight (dw) of involucre (mg GAE/g dw).
