Firstly, polyphenols from the garlic samples were extracted. The peeled garlic cloves (fresh and black) were homogenized, and 5 g was weighed into a glass tube. Then, 50 mL of 50% methanol was added. Additionally, the mixture was thoroughly homogenized using a laboratory mixer (Ultra-Turrax T25, IKA-Werke GmbH & Co. Kg, Staufen, Germany) for 30 s. Then, the tube was put into an ultrasound bath and was extracted for 15 min. Afterwards, an aliquot was pipetted into a centrifuge tube and was centrifuged at 4000 rpm for 15 min. The supernatant was stored at −18 °C until analysis. One extraction per group was conducted, and the extract was analyzed three times (n = 3).
The total polyphenolic content was spectrophotometrically determined according to the method of Lachman et al. [19 ]: 35 µL of the sample extract was pipetted into a disposable 4.5 mL plastic cuvette; then, 175 µL of Folin–Ciocâlteu reagent, 3465 µL of water, and 525 µL of 20% sodium carbonate were added. This reaction mixture was then incubated at room temperature for two hours in the dark. The absorbance was read at 765 nm, and gallic acid was used as a standard for the preparation of a calibration curve. The results are expressed as mg of gallic acid equivalent (GAE) per kg of sample DM (g GAE/kg DM).
The total polyphenolic content was spectrophotometrically determined according to the method of Lachman et al. [19 ]: 35 µL of the sample extract was pipetted into a disposable 4.5 mL plastic cuvette; then, 175 µL of Folin–Ciocâlteu reagent, 3465 µL of water, and 525 µL of 20% sodium carbonate were added. This reaction mixture was then incubated at room temperature for two hours in the dark. The absorbance was read at 765 nm, and gallic acid was used as a standard for the preparation of a calibration curve. The results are expressed as mg of gallic acid equivalent (GAE) per kg of sample DM (g GAE/kg DM).