Ultrasound bath

Commercial raisins (V. vinifera L.) of three different types and origins—(i) Thompson seedless (from Chile), (ii) Muscat (from Spain), and (iii) sultanas (from Turkey)—were purchased in a local supermarket in Barcelona, Spain. The extraction was performed following a previously reported procedure with minor modifications [55 (link)].
A sample of each variety of raisin (0.5 g, n = 3) was homogenized with an ULTRA-TURRAX^® (IKA, Staufen, Germany) for 30 seconds, and then vortexed for 1 min with 4 mL ethanol/water (80:20, v/v). Later, they were sonicated in an ultrasound bath (Bandelin electronic GmbH&Co.KG, Berlin, Germany) during 10 min and centrifuged at 4000 RPM for 5 min at 4 °C. The supernatants were collected and the extraction procedure was repeated twice. The supernatants obtained were combined and evaporated with a sample concentrator (MIVAC^®, FisherScientific, UK) at room temperature under a stream of nitrogen gas, and the residue was reconstituted into 0.1% of aqueous formic acid (4 mL). The raisin extracts were filtered through a 13 mm, 0.45 µm PTFE filter into an amber vial and stored at −20 °C until analysis by LC-LTQ-Orbitrap.

The freeze-dried PLGA NP were resuspended in distilled water and centrifuged at 20,000 rpm (14 °C, 30 min) to remove D-mannitol and excess PVA. Then the supernatant solution was discarded, and the precipitated nanoparticles were resuspended in the same amount of distilled water using Vortex (Velp Scientifica, Deer Park, NY, USA) and ultrasound bath (Bandelin Electronic GmbH & Co. KG, Berlin, Germany). The drop of PLGA NP suspension (diluted 5-fold) was placed on a sample table, air-dried, and then sputtered with a layer of platinum for 6 to 30 s which produces a surface layer of Pt with a thickness of 13 nm. The table was then placed in the JSM-7600F Schottky field emission scanning electron microscope (JEOL, Nieuw-Vennep, Japan), and the sample was imaged in the secondary electron mode (planar). Capturing mode: high vacuum, accelerating voltage up to 15 kV, detection of secondary electrons (planar).

To prepare extracts, 3 g of dried powdered leaves or lyophilised fruits were extracted with 80 mL of methanol or water in an ultrasound bath (Bandelin Sonorex, Berlin, Germany) at 50 °C for 60 min. Samples were vortexed (Vortex 3, IKA, Staufen, Germany) every 15 min. The extracts were filtered through a 0.45 µm PTFE membrane filter (Supelco, Bellefonte, PA, USA) after cooling to room temperature. Obtained extract aliquots were used to determine the total phenolic content and antiradical activity.
To determine the phenolic profile by UHPLC-LTQ OrbiTrap MS, aliquots of water and methanolic extracts were lyophilised and dissolved in ultrapure water for analysis.