Ten grams of finely grated cheese were suspended in 45 ml of 0.1 N HCl. (L,L)-phenylalanyl-phenylalanine (Phe-Phe) was added as an internal standard (2.5 ml of a 1 mM solution). The suspension was homogenized for 1 min using an Ultra-Turrax homogenizer (IKA Werke GmbH & Co. KG, Staufen, Germany) and then centrifuged at 3,400 × g for 30 min at 4°C. The solution was filtered through paper filters, and extracted 3 times with 40 ml of ethyl ether. Diethyl ether residues were removed using a Rotavapor (Buchi, New Castle, DE, United States); then, the aqueous solution was filtered through a 0.45 μm filter. 0.5 ml of 0.1% of formic acid solution were added to 1.5 ml of filtered extract, and the solution was passed through Vivaspin 2 Ultrafilter (Sartorius, Gottingem, Germany) with polyether sulfone (PES) membrane (nominal MWCO 10 kDa) previously washed and conditioned following manufacturer instructions. The filtration lasted for 45 min at 4,930 × g, using a centrifuge Hettich Universal 320R (Kirchlengern, Germany) at 23°C. Further washing steps of the ultrafilters were performed (3 times using acidified water at 0.1% formic acid). Final extracts were dried using a Rotavapor (Buchi, New Castle, DE, United States). The filtrate was dried under nitrogen, dissolved in 250 μL of 0.1% HCOOH in H2O, and analyzed by UPLC/ESI-MS. Each sample was extracted and analyzed in triplicate.
Rotavapor
Manufactured by Büchi
Sourced in Switzerland, Germany, China, United States, France, United Kingdom, Italy
The Rotavapor is a laboratory evaporator used for the gentle and efficient removal of solvents from samples. It allows for the controlled evaporation of liquids under reduced pressure, enabling the recovery of solid or liquid residues.
Automatically generated - may contain errors
Lab products found in correlation
Whatman, by Cytiva (32 mentions)
No 1 filter paper, by Cytiva (4 mentions)
Whatman no 1 filter paper, by Cytiva (3 mentions)
Whatman filter paper number 1, by Cytiva (3 mentions)
Vc750, by Sonics (2 mentions)
Ethyl acetate, by Merck Group (2 mentions)
Formic acid, by Avantor (2 mentions)
R 220se, by Büchi (2 mentions)
Cholesterol, by Merck Group (2 mentions)
Sephadex lh 20, by Merck Group (2 mentions)
Silica gel 60 f254, by Merck Group (2 mentions)
Kieselgel 60h, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Christ gamma 2 16 lscplus, by Martin Christ (2 mentions)
Ultra turrax, by IKA Group (1 mentions)
Whatman no 1, by Cytiva (1 mentions)
Orbital shaker, by Gallenkamp (1 mentions)
Buchi mini spray dryer b 290, by Büchi (1 mentions)
Freezone 2.5 model 76530 lyophilizer, by Labconco (1 mentions)
Inova 500 spectrometer, by Agilent Technologies (1 mentions)
188 protocols using rotavapor
1
Cheese Peptide Extraction and Analysis
2
Blueberry Extract Preparation Protocol
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A 50/50 (w/w) blend of freeze-dried Tifblue/Rubel blueberries was used as a phytophenol extraction source, and extractions were conducted as described by Grace et al. (33 (link)). Briefly, blueberries were blended (Waring, Inc., Torrington, CT, USA) at a ratio of 1:12 blueberry powder to acidified 70% methanol (0.5% acetic acid) w/v for 2 min. After centrifuging the mixture for 20 min at 4,000 rpm, the supernatant was transferred to a round-bottom flask. This extraction of the pellet was repeated two more times and the extracts were combined. The blueberry extract was neutralized and evaporated via Rotavapor (Buchi Rotavapor, New Castle, DE, USA), frozen at −80°C overnight, then lyophilized (Labconco, Kansas City, MO, USA). Blueberry extract powder was subsequently resuspended in PBS, aliquoted into black microcentrifuge tubes (Argos Technologies, Vernon Hills, IL, USA), and stored at −80°C until use.
3
Ethanol Extraction of Medicinal Plant
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Fresh leaves of the experimental plant were gathered from the field in Woliso, 113 km West of Addis Ababa, Ethiopia. The plant was identified and authenticated in the National Herbarium, Department of Plant Biology and Biodiversity Management, Addis Ababa University, Ethiopia. A reference number of the specimen (MS 001) was allocated for future utilization. Extraction was carried out as previously discussed by Abebe et al. [17 (link)] in line with Zhang et al. [18 (link)]. Briefly, the leaves were cleaned, shade dried, coarsely crushed by a grinder and blended with 70% ethanol and kept on an orbital shaker (Gallenkamp, UK) for 24 h at 130 g. The mixture was then filtered (Whatman No. 1, Whatman Ltd. England), and the filtrate concentrated using Rota Vapor (Büchi Rota Vapor R-205, Switzerland) at a temperature not exceeding 40 °C and stored in a refrigerator at −4 °C until use [19 ].
4
Preparation of SDM-4-ASA Cocrystal Polymorphs
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All cocrystals were prepared using a 1:1 molar ratio (SDM:4-ASA), as previously described (Serrano et al., 2015b) .
Briefly, cocrystal Polymorph I (PI) was obtained by liquid-assisted co-milling which was carried out in a planetary ball mill (Retsch PM100, Haan, Germany) as previously described (Grossjohann et al., 2015) . Cocrystal Polymorph II-Habit I (PII-HI) was obtained by crystallization from ethanol using a rotavapor (Buchi, Flawil, Switzerland) at 250 mbar pressure and 55 °C as previously described (Serrano et al., 2015b) . Cocrystal Polymorph II-Habit II (PII-HII) was obtained by crystallization from acetone also using a rotavapor (Buchi, Flawil, Switzerland) at 250 mbar pressure and 55 °C as previously described (Serrano et al., 2015b) . Cocrystal Polymorph II-Habit III (PII-HIII) was obtained by spray-drying ethanolic solutions of SDM:4-ASA (1:1 molar ratio) using a Buchi B-290 Mini Spray Dryer (Buchi, Flawil, Switzerland) operating in the open-mode, as previously described [12] . Cocrystal Polymorph II-Habit IV (PII-HIV) was obtained from polymorph II-Habit I after dry milling in a planetary ball mill (Retsch PM100, Haan, Germany) as previously described (Serrano et al., 2015b ). An equimolar physical mixture (PM) of 4-ASA and SDM was prepared by mixing the raw components in a mortar and pestle.
Briefly, cocrystal Polymorph I (PI) was obtained by liquid-assisted co-milling which was carried out in a planetary ball mill (Retsch PM100, Haan, Germany) as previously described (Grossjohann et al., 2015) . Cocrystal Polymorph II-Habit I (PII-HI) was obtained by crystallization from ethanol using a rotavapor (Buchi, Flawil, Switzerland) at 250 mbar pressure and 55 °C as previously described (Serrano et al., 2015b) . Cocrystal Polymorph II-Habit II (PII-HII) was obtained by crystallization from acetone also using a rotavapor (Buchi, Flawil, Switzerland) at 250 mbar pressure and 55 °C as previously described (Serrano et al., 2015b) . Cocrystal Polymorph II-Habit III (PII-HIII) was obtained by spray-drying ethanolic solutions of SDM:4-ASA (1:1 molar ratio) using a Buchi B-290 Mini Spray Dryer (Buchi, Flawil, Switzerland) operating in the open-mode, as previously described [12] . Cocrystal Polymorph II-Habit IV (PII-HIV) was obtained from polymorph II-Habit I after dry milling in a planetary ball mill (Retsch PM100, Haan, Germany) as previously described (Serrano et al., 2015b ). An equimolar physical mixture (PM) of 4-ASA and SDM was prepared by mixing the raw components in a mortar and pestle.
5
Grape Pomace Polyphenol Extraction and Antibacterial Evaluation
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Phenol compounds were extracted from whole and skin pomaces. Whole grape pomace and skins separated from dried grape pomace were freezed in liquid nitrogen and grinded with a blender until a fine powder was obtained. In order to evaluate the antibacterial activity of pomaces, whole and skin samples (1 g) were extracted in 10 mL of ethanol:water (40:60, v:v) at room temperature for 16 h in the dark under continuous stirring. Extraction mixtures were centrifuged (4000× g) for 5 min and the supernatants were dealchoolized by Rotavapor (Buchi, Rotavapor R205, Switzerland) and freeze-dried by a Freezone® 2.5 model 76530 lyophilizer (Labconco Corp., Kansas City, MO, USA) and stored at −20 °C until biological activity analysis. The extracts of whole and skin pomace (WP and SP respectively), of grape cultivars Negramaro (N) and Fiano (F) were lyophilized and suspended in phosphate-buffered saline (PBS, 16.9 mM K2HPO4, 33.1 mM KH2PO4) and then analysed for their antioxidant activity (TEAC and ORAC) and the total phenol content (TP). The powder obtained was weighed and suspended in PBS, at a concentration of 500 µg/mL total polyphenols (Gallic acid equivalents). This stock solution was used for antibacterial assays.
6
Extraction and Fractionation of Plant Compounds
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Preparation of leaf, bark extracts, fractions, and extraction of seed oil was undertaken as per our earlier studies [19 (link)]. In this study, approximately 1 kg of dried powder of leaf and bark was macerated (5 L × 3 times) at room temperature using concentrations of 80% ethanol: water for 12 h. Filtered samples were then evaporated in a Rotavapor (Buchi, R-2010) at 45 °C under low pressure and the solvent was removed. Further, the dried ethanol aqueous extracts of the leaf and bark were sequentially fractionated using different solvents based on their polarity, i.e., n-hexane, ethyl acetate, n-butanol, and water. The compounds were isolated from the ethyl acetate and n-butanol fractions using the column chromatography method.
The seeds were dried for 15 days and the seed coat was taken out. The fat layer was removed using hot water treatment until the appearance of black kernels. The kernels were dried (1 kg) and macerated in n-hexane solvent (3 L × 4 times) at room temperature. The eluted solvent was evaporated under Rotavapor (Buchi, R-2010) at 45 °C under low pressure, which yielded 1 L of seed oil. The leaf and bark ethanol aqueous extracts, fractions, and seed oil were kept at less than 4 °C until further analysis.
The seeds were dried for 15 days and the seed coat was taken out. The fat layer was removed using hot water treatment until the appearance of black kernels. The kernels were dried (1 kg) and macerated in n-hexane solvent (3 L × 4 times) at room temperature. The eluted solvent was evaporated under Rotavapor (Buchi, R-2010) at 45 °C under low pressure, which yielded 1 L of seed oil. The leaf and bark ethanol aqueous extracts, fractions, and seed oil were kept at less than 4 °C until further analysis.
7
Preparation of Ibuprofen-Loaded Liposomes
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Plain liposomes were prepared by using the thin-film hydration method described earlier by Berginc et al. [16 (link)]. Briefly, 200 mg of S100 soybean lecithin lipids and 20 mg of ibuprofen were dissolved in a mix of methanol and chloroform (2:4 v/v). The solvent was evaporated using a Büchi rotavapor with a vacuum controller (Büchi Labortechnik, Flawil, Switzerland) for a total of 3 h at 60 mBar and 45 °C. The resulting thin film was hydrated using 10 mL of distilled water, and the liposomal dispersion was manually mixed until a homogeneous liposomal suspension was obtained. The liposomal formulation was stored in the refrigerator (4–6 °C) overnight prior to a size reduction. The size of the liposomes was reduced stepwise by extruding through polycarbonate membranes with pore sizes of 0.8, 0.4, and 0.2 µm (Nuclepore Track-Etch Membran, Whatman House, Maidstone, UK). Four extrusion cycles were carried out for each pore size. As a control, liposomes without drugs were also prepared in the same manner (i.e., empty plain liposomes).
8
Carotenoid Extraction from Arthrobacter arilaitensis
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After 7 d of growth, A. arilaitensis cells were harvested by centrifugation at 6000 Â g (25 C) for 15 min. The cell pellets were washed with deionised water, frozen at À80 C for 48 h, and then lyophilised to dryness (COSMOS-80 manufactured by CRYOTEC, vacuum below 200 mtorr, temperature of the shelf increased to À20 C for overnight primary drying then to 20 C for 2 h for the secondary drying phase). Lyophilised cells (1 g) were extracted with 8 mL of 99% methanol (Carlo Erba, Peypin, France) including a sonication step (Transsonic T420 00 homogeniser, Elma, Singen, Germany, HF-frequency 35 kHz, 10 min, on ice), at room temperature for 90 min. The mixture was centrifuged at 6000 Â g (25 C) for 15 min to separate the cell debris from the carotenoid-containing supernatant. The cell debris were re-extracted two times with 8 mL of methanol and twice with 8 mL of methyl tert-butyl ether (MTBE; Carlo Erba) until complete bleaching of the biomass was obtained. The extracts were combined and subsequently evaporated to dryness under vacuum at 55 C using a Büchi Rotavapor (BÜCHI Labortechnik AG, Flawil, Switzerland). The residue was dissolved in 1 mL methanol/MTBE (1:1, v/v), filtered through Millex-GV 0.2-mm hydrophilic membrane (Millipore, Molsheim, France), and stored at À20 C in an amber vial prior to HPLC analysis.
9
Preparation and Characterization of Solid Dispersions
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Solid dispersions were prepared according to previously defined experimental plan (Table 1 ). Neusilin® UFL2 was dispersed in ethanolic solution of CBZ and Kollidon® VA64 under stirring on a magnetic stirrer (RCT basic, IKA Labortechnik, Staufen, Germany). Evaporation of ethanol from the prepared dispersions was performed using rotary vacuum evaporator (Büchi Rotavapor®, Büchi Labortechnik AG, Flawil, Switzerland) at a temperature of 70 °C. After evaporation, precipitated material was scrapped off and stored for 48 h in a desiccator. Samples were further pulverized in a mortar with a pestle and afterwards sieved through 300 µm sieve. Until further analysis, samples were kept in sealed glass vials, away from light and moisture.
10
Extraction and Isolation of Anthocyanins
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ACN extraction was carried out following the methodology described by Rodriguez-Saona and Wrolstad [46 (link)] with minor modifications. Black carrot color powders were prepared for extraction by reconstituting them in water. Berberis boliviana berries were deseeded manually, soaked in water, and frozen with liquid nitrogen before extraction. Then, the frozen berries and reconstituted black carrot color powder were each blended with 0.01% HCl acetone (v/v) using a Waring laboratory blender. The slurry was filtered through an N° 4 Whatman filter paper, and the cake was re-extracted using 0.01% HCl aqueous acetone (70% v/v) until a faint pink solution was obtained. The filtrate was then partitioned using chloroform for a final acetone:chloroform proportion of 1:2 (v/v) in a separatory funnel and stored overnight at 4 °C. The upper aqueous layer containing ACNs and other phenolic compounds was collected, and residual solvents were evaporated using a Büchi rotavapor at 45 °C (Büchi, Flawil, Switzerland).
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