Ott was confirmed by Prof. Emanuele Schimmenti, and a sample was deposited within the Department

Ott was confirmed by Prof. Emanuele Schimmenti, and a sample was deposited within the Department STEBICEF, University of Palermo, Italy, under identification quantity MB 548/2018. Neutral alumina (white powder; particle size: range 7090 mesh; pH variety: 7.07.5; surface region: frequent 155 m2 /g; slurry: 5 ), metakaolin (grayish powder; particle size: 400-500 mesh; pH variety: 5.0.0; surface location: 39 m2 /g; slurry: 1 ) and silica (white powder; nanoparticles; mesoporous; particle size: 19050 nm; typical pore diameter: 3.five.5 nm) have been from Sigma-Aldrich (St. Louis, MO 68178, USA); VCASTM White Pozzolans (white powder; particle size: 325 mesh; pH variety: 9.50.0; surface area: 0.60 m2 /g; slurry: 1 ) had been from Vitro Minerals, Inc (95 Pinnacle Drive, Jackson, TN 38301, USA); Lipari pumice (white porous powder; particle size: 22050 mesh; pH variety: 7.3.two; surface area: 210 m2 /g; slurry: two ) was from NOVA SPA (By way of Fossanuova 55, 55016 Porcari (LU), Italy). All the products have been used PX-478 site without any further purification. Standards for LC-ESI/LTQOrbitrap/MS evaluation have been from Sigma-Aldrich (St. Louis, MO 68178, USA).Molecules 2021, 26,9 of3.two. The Extraction Process for the Flavonoids The frozen blackberries have been lyophilized, and 50 g of dry material was blended with 400 mL of water and acetone (30:70) inside a WaringBlender (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The volume on the mixture was lowered at low pressures and finally transferred to a separating funnel. Then, 400 mL of chloroform was added. Consequently, two phases had been made, a water/blue raspberry mixture (which contained anthocyanins, phenolic compounds, sugars, organic acids and also other water-soluble compounds) and an immiscible chloroform/acetone layer (lipids, carotenoids, chlorophyll pigments and other folks non-polar compounds) [70]. The colored aqueous layer was filtered once again by an B hner flask to eliminate insoluble components. The filtered aqueous layer was subsequently purified from the presence of sugars by liquid chromatography on a column, using C18 -Reversed phase silica gel (FlukaAnalytical) (five.125 g) because the stationary phase. To cause the key elution with the sugars, two solutions were ready: acidic water (0.01 M, pH = two) and acidic methanol (0.01 M, 8 of 37 HCl in 100 mL of methanol). After the glucosidic impurities were eliminated by repeated elution using the aqueous acid option (3 14 mL), the extract containing the flavonoids was obtained by eluting it together with the acidic methanolic mixture. From 50 g of blackberries was collected 200 mL of colored extract in acidic methanol without having contaminants, containing 1.15 g of organic metabolites (yield two.30 ). 3.three. LC-ESI/LTQOrbitrap/MS/MSn Evaluation Qualitative LC-MS evaluation was performed by using an Accela HPLC technique (Thermo Scientific, Germany), equipped with a Phenomenex Luna C18 column (150 mm two.1 mm i.d., 5 ) and working at a flow rate of 0.2 mL/min, coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The Orbitrap mass analyzer was calibrated in line with the manufacturer’s directions using a mixture of caffeine, methionine-arginine phenylalanine-alanine-acetate (MRFA), sodium dodecyl sulfate, sodium taurocholate and Ultramark 1621. Data were collected and analyzed making use of the application offered by the manufacturer. Samples were prepared as 1 mg/mL (methanol/water), and five of each was injected and analyzed in unfavorable and optimistic ionization modes following a protocol PF-06454589 In Vitro previously.