Visiprep spe vacuum manifold

Honey samples (5 g) were mixed with 20 mL of deionized water adjusted to pH 2 with HCl and stirred in a magnetic stirrer for 15 min. The samples were then filtered to remove the solid particles. Extraction of phenolic compounds was performed with the Visiprep™ SPE Vacuum Manifold (Sigma-Aldrich, Saint Louis, MO, USA). The SPE cartridges used were Strata-X (500 mg) obtained from Phenomenex (Warsaw, Poland). They were conditioned by washing with 15 mL of methanol, and 20 mL acidified water. Afterwards the filtrated honey sample was passed through a cartridge, which was then washed with 20 mL of deionized water to remove all sugars and other polar constituents of honey. The adsorbed compounds were eluted with 5 mL methanol [60 (link)].

The hulled nuts were cracked manually and then the shells were removed to collect peeled kernels of black walnuts. The peeled kernels were homogenized using a coffee grinder and were extracted as described previously^{10 (link)}. Briefly, the homogenized samples (100 g, dry weight basis, 20–30 mesh) were extracted in 400 ml methanol twice and then sonicated at 10 °C for 60 min, followed by centrifugation for 10 min at 4550 g. Several extractions of different peeled kernels derived from each black cultivar were performed and utilized in at least triplicate in quantitative analyses and in vitro assays. The methanolic extracts were filtered through a 0.2 µm Whatman filter paper (GE Healthcare, Chicago, IL, USA) under SPE Vacuum Manifold (Visiprep™ SPE Vacuum Manifold, Sigma-Aldrich, St. Louis, MO, USA). The resulting supernatant was then collected and stored at − 20 °C until analysis.

An additional step to purify the extracts was necessary, before HPLC analysis. Solid-phase extraction (SPE) is a suitable technique for purifying extracts to remove co-extracted compounds. SPE was performed according to the method described by Christou et al. [8 (link)]. A vacuum manifold system with a 12-positions rack (Visiprep™ SPE Vacuum Manifold, SUPELCO, St. Louis, MO, USA) was utilized for all SPE experiments. Discovery DPA-6S (500 mg, SUPELCO, St. Louis, MO, USA) SPE polyamide cartridges were used for the purification of the extracts. The polyamide cartridges were preconditioned with 5 mL of methanol (MeOH) and equilibrated with 5 mL of acidified methanolic solution, 20:80 (v/v) MeOH: H₂O, acidified to pH 2.0 with HCl. The obtained lyophilized extracts were re-dissolved in acidified methanolic solution (20 mL) and then loaded into the cartridges. Subsequently, the cartridges were washed with 5 mL of acidified water (pH 2.0) to remove co-extracted compounds. Finally, the purified extracts were obtained after eluting the bound phenolics from the cartridges with an aqueous acetone mixture (2 × 5 mL, 80% v/v). The purified extracts were then dried using a rotary evaporator and lyophilizer. The dry residues were redissolved in an appropriate volume of MeOH, filtrated through a 0.45-μm pore size membrane filter, properly diluted, and injected into the HPLC system.

The plasma concentrations of FLU were analyzed by high-performance liquid chromatography (HPLC) with ultraviolet detection and solid-phase extraction (SPE) according to the procedure described by Ferreira et al. [22 (link)], adapted and optimized for guinea pig plasma. Plasma samples were subjected to SPE clean-up using Discovery 18-LT extraction cartridges (500 mg, 3 ml) (Supelco, USA) connected to a Visiprep SPE vacuum manifold (Supelco, USA), using acetonitrile as eluent solvent. The eluate was evaporated to dryness and reconstituted in 100 μl acetonitrile. The chromatographic separation was performed using a C18 column (Kromasil, 3.5 µm; 4.6 × 100 mm; Tedia, Rio de Janeiro, Brazil), preceded by a C18 guard column (Kromasil, 3.5 µm; 4.6 × 10 mm; Tedia, Rio de Janeiro, Brazil), both maintained at 25 °C. The mobile phase consisted of acetonitrile: water (80:20, v/v) with a flow rate of 1.0 ml/min. The UV wavelength was set at 260 nm, and the injection volume was 20 µl.