Total phenolic content (TPh) in the extracts was established using the Folin–Ciocalteau spectrophotometric method adapted to microscale [11 (link)]. The sample (15 μL) was mixed with distilled water (170 μL), 2 N Folin–Ciocalteu’s reagent (12 μL) and 20% Na2CO3 (30 μL) in a plate well. Absorbance was measured after 1 h (room temperature, dark conditions) at 750 nm, using distilled water as blank. Gallic acid (GAE) was used for the calibration curve.
Chromatographic analysis for identification and quantification of phenolic compounds were carried out as recommended by Tumbas Šaponjac et al. [11 (link)], using Shimadzu Prominence HPLC, connected to an SPD-20AV UV/VIS detector (Shimadzu, Kyoto, Japan), with Luna C-18 RP column, 5 lm, 250 mm × 4.6 mm with a C-18 guard column, 4 mm × 30 mm (Phenomenex, Torrance, CA, USA). Gradient elution was applied using acetonitrile (A) and water acidified with 1% formic acid in d-water (B), at flow rates of 1 mL/min, at the following order: 10% to 25% A (0–10 min); 25% to 60% A (10–20 min); 60% to 70% A (20–30 min); 70% to 10% A (30–40 min); 10% A (5 min) (equilibration time). For hydroxybenzoic acids, chromatograms were recorded at 280 nm; for hydroxycinnamic acids at 320 nm; and for flavonoids at 360 nm. HPLC standards were dissolved in 50% methanol.
Chromatographic analysis for identification and quantification of phenolic compounds were carried out as recommended by Tumbas Šaponjac et al. [11 (link)], using Shimadzu Prominence HPLC, connected to an SPD-20AV UV/VIS detector (Shimadzu, Kyoto, Japan), with Luna C-18 RP column, 5 lm, 250 mm × 4.6 mm with a C-18 guard column, 4 mm × 30 mm (Phenomenex, Torrance, CA, USA). Gradient elution was applied using acetonitrile (A) and water acidified with 1% formic acid in d-water (B), at flow rates of 1 mL/min, at the following order: 10% to 25% A (0–10 min); 25% to 60% A (10–20 min); 60% to 70% A (20–30 min); 70% to 10% A (30–40 min); 10% A (5 min) (equilibration time). For hydroxybenzoic acids, chromatograms were recorded at 280 nm; for hydroxycinnamic acids at 320 nm; and for flavonoids at 360 nm. HPLC standards were dissolved in 50% methanol.
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