Sonic 6d

The lyophilized powdered sample (approx. 1 g for fruits and 0.5 g for leaves) was taken for the determination of biological in vitro activity. The sample was mixed with 7 mL of methanol:water: 37% hydrochloric acid (80:19:1, v/v/w), sonicated for 20 min (Sonic 6D; Polsonic, Warsaw, Poland) and incubated overnight (4 °C). Next, after 24 h, the slurry was centrifuged (MPW-55; Warsaw, Poland) at 19,000× g at 4 °C for 10 min to obtain extract for all biological in vitro analysis.
ABTS and FRAP were performed using UV-2401 PC spectrophotometer (Shimadzu; Kyoto, Japan), and the remaining assays were performed using a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). All tests were performed in triplicate.

Dried powdered samples of roots and leaves were extracted with 50% methanol. The extraction was performed twice via an incubation for 20 min under sonication with shaking (Sonic 6D, Polsonic, Warsaw, Poland), then the sample was centrifuged at 19,000× g for 10 min, and the supernatant was filtered through a hydrophilic PTFE 0.20 μm membrane (Millex Samplicity Filter, Merck, Darmstadt Germany) before analysis. The content of iridoids and polyphenolics was determined by means of ultra-performance liquid chromatography-photodiode array detector-mass spectrometry (UPLC-PDA-QTof-MS/MS) method. All extractions were carried out in triplicate.

In order to prepare the stock solution, the method described by Kopjar et al. [20 (link)], with slight modifications, was used. 1 g of hydrogel beads was dissolved in 5 mL of 80% methanol acidified with HCl. The samples were placed in an ultrasonic bath (Sonic 6D, Polsonic, Warsaw, Poland) for 15 min at 20 °C and then placed in a refrigerator for 24 h. After this time, extracts were centrifuged at 3600 rpm for 20 min, and the supernatants were filtered through a hydrophilic PTFE 0.20 µm membrane (Millex Samplicity Filter, Merck, Darmstadt, Germany) and used for analysis.

To obtain an extract rich in polyphenols of P. armeniaca L. leaf powder after freeze-drying was mixed with 50% ethanol, sonicated for 20 min (Sonic 6D; Polsonic, Warsaw, Poland), occasionally mixed, and supernatant was separated by centrifuged for 10 min at 19,000× g (MPW-55; Warsaw, Poland). The residue was reextracted 3–4 times and all obtained fractions were mixed and ethanol was evaporated at 40 °C by a scale rotary evaporator (Hei-VAP Expert, Heidolph; Schwabach, Germany). The obtained aqueous fraction was loaded into a glass column (50 cm; Ø 6 cm) filled with Amberlite polymeric XAD-16 resin previously washed with water. The absorbed aqueous fraction was washed with water (1 mL/min) to remove sugars, organic acids or other ballast substances and then polyphenolic compounds were eluted with ethanol (80 and 100%; 1 mL/min). Collected fractions were mixed and, after evaporation (Hei-VAP Expert, Heidolph; Schwabach, Germany) of alcohol at 40 °C, the obtained fraction was freeze-dried to obtain the powder of polyphenol-rich apricot leaf extract (PrALe).

First, 0.2 g of plant material was extracted with 15 mL of 80% MeOH for 30 min using an ultrasonic bath (Sonic-6D, Polsonic, Warsaw, Poland). The extract was then centrifuged at 5000× g for 30 min. The supernatant was collected and evaporated under a vacuum at 40 °C (Hei-VAP Precision, Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). The residue was suspended in water and applied onto an SPE (solid phase extraction) column, equilibrated with water. SPE was carried out with a Visiprep™ SPE Vacuum Manifold (Sigma-Aldrich, Poznan, Poland), using a Chromabond C18ec column (Macherey-Nagel GmbH & Co. KG, Dueren, Germany). The column was washed with H₂O and the phenolic acids were eluted with methanol.

Rye bread (1 g) was mixed with 80% of methanol and water (10 mL) + 1% hydrochloric acid, and incubated for 20 min under sonication (Sonic 6D, Polsonic, Warsaw, Poland). Next, the slurry was centrifuged at 19,000× g for 10 min, and the supernatant was filtered through a hydrophilic PTFE 0.20 μm membrane (Merck, Darmstadt, Germany) and used for analysis.
The ABTS method was performed according to the method described by Re et al. [22 (link)]. ABTS^•+ was generated by oxidation of ABTS with potassiumpersulphate. The ABTS^•+ solution was diluted to an absorbance of 0.7 ± 0.05 at 734 nm. Then, 0.03 mL of extract was mixed with 2.97 mL of ABTS^•+ solution and left for 6 min at 25 °C. Next, the absorbance was measured at 734 nm using the UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). The results of antiradical capacity were expressed as Trolox equivalents in µmol per g d.s.
The DPPH method was carried out with the method described by Yen and Chen [23 (link)]. Then, 0.50 mL of extract was mixed with ethanol (1.5 mL) and DPPH^•+ solution (0.5 mL), and left for 10 min at 25 °C. Next, the absorbance was measured at 517 nm using the UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). The results of antiradical activity were expressed as Trolox equivalents in µmol per g d.s.

The analysis of organic acids and carbohydrate was performed as described previously by Wojdyło et al. [14 (link)], using HPLC-PDA (Waters Co.; Milford, CT, USA) and HPLC-ELSD (PL-ELS 1000; Merck; Hitachi, Japan), respectively. The sample (approximately 3 g of fruit) was mixed with distilled water, exposed to ultrasounds (Sonic 6D; Polsonic, Warsaw, Poland) for 15 min, heated at 90–100 °C for 30 min, and finally centrifuged (MPW-55; Warsaw, Poland) at 12,000× g for 10 min at 4 °C. The supernatant (2.5 mL) was injected into a Sep-Pak C-18 cartridge (1 g, Millipore Waters, Milford, MA, USA) and eluted with H₂O into Eppendorf tubes. Before analysis, the extract was filtered through a hydrophilic PTFE membrane (0.20 µm; Millex Simplicity filters; Merck, Germany). The organic acids were analyzed on Polymex IEX H column (8 μm, 250 × 8 mm, Watrex; Prague, Czech Republic) using isocratic elution with 0.9 M sulfuric acid in H₂O for 20 min. The carbohydrates were analyzed on Alltech^® Prevail^TM Carbohydrate ES HPLC Column-W 250 × 4.6 mm, 5 µm (Columbia, MD, USA) using isocratic elution with 70% acetonitrile in H₂O for 20 min. The results were expressed in g per 100 g of d.w.