Wizard advanced ir vortex mixer

Freeze-dried garlic (100 mg) was defatted and deproteinized with acetonitrile (Pol-Aura Chemical Reagents, Zabrze, Poland), and then washed with dichloromethane (Pol-Aura Chemical Reagents, Zabrze, Poland). The samples were weighed into 50 mg screw cap tubes, and 5 mL of 1.2 M HCl in 50% methanol (Pol-Aura Chemical Reagents, Zabrze, Poland) was added. Samples with the extraction mixture were vortexed (Wizard Advanced IR Vortex Mixer, VELP Scientifica Srl, Usmate, Italy) for 1 min, incubated (IKA KS 4000 Control, IKA^® Poland Ltd., Warsaw, Poland) for 3 h (90 °C, shaking every 30 min), and then cooled. Samples were diluted with methanol to a volume of 10 mL and then centrifuged (MPW-380R, MPW Med. Instruments, Poland, Warsaw) (10 min, 5 °C, 6000 rpm). A clear supernatant was used to determine the antioxidant activity and bioactive ingredients.

Defatted and deproteinized with acetonitrile (Pol-Aura Chemical Reagents, Zabrze, Poland), 100 mg of the freeze-dried plant material was washed with dichloromethane (Pol-Aura Chemical Reagents, Zabrze, Poland). Then, 50 mg of the sample was weighed into a tube with a screw cap and 5 mL of the extraction mixture (1.2 M HCl in 50% methanol) (Pol-Aura Chemical Reagents, Zabrze, Poland) was added. The samples were vortexed (Wizard Advanced IR Vortex Mixer, VELP Scientifica Srl, Usmate, Italy) for 1 min and then incubated (IKA KS 4000 Control, IKA^® Poland Ltd., Warsaw, Poland) for 3 h at 90 °C with shaking performed every 30 min. After incubation the samples were cooled, then diluted to 10 mL with methanol and centrifuged (MPW-380R, MPW Med. Instruments, Poland, Warsaw) (10 min, 5 °C, 6000 rpm). The obtained clear supernatants were used for the determination of bioactive ingredients and antioxidant activity.

Antioxidant activity in Matcha green tea was determined using the cation radical ABTS^+• (2,2‣-azino-bis 3-ethylbenzothiazolin-6-sulfonic acid) (Sigma-Aldrich, Poznań, Poland) according to the Re et al. (1999) [69 (link)] method. The solution (1.5 mL of diluted supernatant, 3.0 mL radical cations ABTS^+• in PBS solution (PBS—phosphate buffer solution)) was vortexed (Wizard Advanced IR Vortex Mixer, VELP Scientifica Srl, Usmate, Italy) and incubated for 6 min (20 °C), and the absorbance was measured in a spectrophotometer (UV/Vis UV-6100A, Metash Instruments Co., Ltd., Shanghai, China) at λ = 734 nm. After taking into account the dilutions used, the obtained results of absorbance measurements were recalculated based on the standard curve (y = –5.6067x + 0.7139, R² = 0.9998) for Trolox (Sigma-Aldrich, Poznań, Poland) as a standard substance and the antioxidant activity was expressed as µM TEAC/g d.m. (TEAC—Trolox equivalent antioxidant capacity, dm.—dry matter).