In a Soxhlet continuous reflux system, the dried lichen was ground into a powder; 20 g of dried lichen was extracted for 8 h with 150 mL solvent: ethyl acetate, methanol (Chemical Company, Iasi, Romania), acetone, ethanol (Chimreactiv S.R.L., Bucharest, Romania), and water [46 (link)]. The extraction temperature was different for each extract, being around the boiling point of each solvent (Table 1 ). After filtration with filter paper, UBW was concentrated on a Rotavapor R-215 with a vacuum controller V-850 (BÜCHI Labortechnik AG, Flawil, Switzerland), and lyophilized with a freeze-dryer Christ Alpha 1-2L (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) connected to a vacuum pump RZ 2.5 (VACUUBRAND GmbH, Wertheim, Germany) [46 (link)]. For UBEA, UBA, UBE, and UBM, the rotary evaporator TurboVap 500 (Caliper Life Sciences Inc, Hopkinton, MA, USA) evaporated the correspondent solvents [41 (link)]. Next, each extract was kept for 16 h in a chemical exhaust hood for optimal solvent evaporation. All obtained U. barbata dry extracts were transferred to sealed-glass containers and stored in the freezer (Sirge® Elettrodomestici—S.A.C. Rappresentanze, Torino, Avigliana (TO) Italy) at −24 °C until processing [46 (link)].
V 850
Manufactured by Büchi
Sourced in Switzerland
The V-850 is a laboratory rotary evaporator from Büchi. It is a device used for the evaporation and concentration of solvents from liquid samples.
Automatically generated - may contain errors
Lab products found in correlation
V 700, by Büchi (2 mentions)
Rotavapor r 215, by Büchi (1 mentions)
R 200, by Büchi (1 mentions)
Uflc system, by Shimadzu (1 mentions)
Lc 6a system, by Shimadzu (1 mentions)
Lh 20, by Merck Group (1 mentions)
B 491, by Büchi (1 mentions)
Frc 10a, by Shimadzu (1 mentions)
Lc solution, by Shimadzu (1 mentions)
Avance 3 500 mhz spectrometer, by Bruker (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Silica gel, by Merck Group (1 mentions)
Cbm 20a, by Shimadzu (1 mentions)
Silica gel plate, by Merck Group (1 mentions)
Ud 100 sonicator, by TOMY (1 mentions)
Vortex genie 2, by Scientific Industries (1 mentions)
Fz compact, by Labconco (1 mentions)
Rzr 2020, by Heidolph (1 mentions)
Rotavapor r 210, by Büchi (1 mentions)
12 protocols using v 850
1
Soxhlet Extraction of U. barbata Lichen Compounds
2
Normoxic Low Altitude Simulation
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For normoxic low altitude simulation treatment, animals were placed inside of a custom constructed cylindrical 20.1 x 26.67 cm chamber (S1 Fig ), sealable to maintain a lowered barometric pressure. A control pressure was established on the pressure sensor in relation to the barometric pressure denoted by the meteorological service of Canada (Edmonton, Alberta station). Low altitude simulation corresponding to a pressure of 714 mmHg, approximately 40 mmHg below the barometric pressure of the laboratory room was maintained through a vacuum controller (Buchi V-850; New Castle, DE) for a period of 3 hours daily with the experimental animals enclosed. This pressure was chosen to reflect a condition of low altitude, without inducing the effects of hypoxia on the animals.
3
Synthesis of DES-BA Multicomponent Crystal
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The equimolar mixture of DES and BA was dissolved in methanol at 35 °C until a clear solution was obtained. The resulting solution was then evaporated using a Buchi Rotavapor R-215 (Flawil, Switzerland) at 50 °C, followed by a Buchi Heating Bath B-491 (Flawil, Switzerland) and sucked at a pressure of 208 mbar using the Buchi V-850 vacuum controller. The powder obtained from the multicomponent crystal of DES–BA salt was collected and stored at room temperature for further analysis.
4
Anthocyanin Purification using AB-8 Resin
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Anthocyanin was extracted in our previous study in the condition of 19 min of ultrasound assistance, a liquid‐to‐solid ratio of 1:22 (g/mL), and hydrochloric acid volume fraction of 0.63% (Chen et al., 2022 ). In this experiment, AB‐8 macroporous resin was used to purify anthocyanin after the extracting solution was concentrated by a rotary evaporator (BUCHI V‐850, Switzerland). Before use, macroporous resin was pretreated according to the previously described methods (Lin et al., 2012 ; Wang et al., 2019 (link)).
5
Normoxic Low-Altitude Simulation for Mice
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For normoxic low‐altitude simulation treatment, animals were placed in a specially constructed cylindrical 20.1 × 26.67 cm chamber (Figure S1 ; https://doi.org/10.6084/m9.figshare.7859972.v2 ) that could be sealed to maintain a given barometric pressure. A baseline pressure was noted with a pressure sensor relative to the barometric pressure denoted by the meteorological service of Canada (Edmonton, Alberta). With the experimental animals enclosed in the chamber, low‐altitude simulation corresponding to a pressure of 714 mmHg (approximately 40 mmHg below the barometric pressure of the laboratory room) was maintained through a vacuum controller (Buchi V‐850; New Castle, DE). This pressure was chosen carefully to simulate low altitude without inducing the effects of hypoxia in the mice.
6
Isolation and Structural Elucidation of Natural Compounds
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A rotary evaporator, R-200 (Büchi, Flawil, Switzerland), a vacuum pump V-700 (Büchi), a vacuum controller V-850 (Büchi), and a heating bath B-491 (Büchi) were used for evaporating solvents from crude extract and subsequent fractions. Various chromatographic techniques were used for separating fractions and isolating compounds, for example, a Sephadex LH-20 (Sigma, St. Louis, MO, USA), 270 × 45 mm glass chromatographic column, silica gel (60 Å pore size, Merck, Germany), and rotating disc chromatography (RDC) (1 mm). Further separations and isolation of compounds were performed by HPLC, consisting of a two-pumps LC-8A unit (Shimadzu, Kyoto, Japan), by using Activon Gold pack normal phase (NP)-semi-preparative (25 × 1 cm, silica) HPLC columns on a Shimadzu LC-6A system and reverse phase (RP) analytical HPLC column (250 × 4.60 mm, 3 µ, C18) on Shimadzu UFLC system with a UV/VIS detector SPD-20A (Shimadzu), a communication bus module CBM-20A (Shimadzu), fraction collector FRC-10A (Shimadzu), software LC Solution (Shimadzu). For identifying the structures of compounds, NMR data (both one- and two- dimensional spectra) were obtained on a Brüker Avance III 500 MHz spectrometer.
7
Lipid Extraction and Analysis Protocol
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To analyze the lipids accumulating in cells, samples were prepared as follows. Cells were collected from the main cultivation and freeze-dried. Twenty milligrams of dried cells were transferred into a 15-mL glass tube, to which 2 mL of a solvent (chloroform:methanol = 2:1) was added, and lipids extracted using the UD-100 sonicator (TOMY, Tokyo, Japan: set at an output of 70 for 10 min). The resultant samples were left overnight in a cool dark place to improve extraction. Subsequently, they were centrifuged at 3,000 × g for 10 min and the supernatants were collected. To wash the samples, equal amount of 0.9 % aqueous KCl solution was added to the supernatant, mixed, and subjected to centrifugation at 2,000 × g for 5 min. Approximately 2 mL of the bottom fraction was collected as the organic layer, transferred to a 15-mL glass tube, and placed in a rotary vacuum evaporator (V-850: BUCHI, Flawil, Switzerland). The resultants were dissolved in 200 μL chloroform, and eventually used for TLC. A 4-μL aliquot of each sample was spotted onto the origin (ori) of a silica gel plate (#60F254: Merck Millipore, Darmstadt, Germany). Lipids on the plate were developed with an eluent (hexane:diethylether:acetic acid = 80:20:1) and stained with 5 % (v/w in EtOH) phosphomolybdic acid solution (#32186-00: Kanto Kagaku Co., Ltd., Tokyo, Japan).
8
Cashew Peduncle Powder Extraction
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The peduncles were sliced and freeze-dried (LioBras K105, São Carlos, SP, Brazil) for 96 h for the complete removal of water. Subsequently, the samples were ground in an electric grinder (Cadence MDR302-127, Navegantes, SC, Brazil) to obtain a fine powder.
For each extraction, 0.5 g of powder was weighed, and 9 mL of methanol was added. Five sequential extractions were performed on the cashew peduncle powder to extract the AnAc from the samples completely. Each extraction was performed for 20 min using an Ultrasonic Cleaner 1400 (THORTON/UNIQUE) at room temperature, after which the mixture was centrifuged (Kindly KC5, São Paulo, SP, Brazil) for 10 min at 2950× g. The supernatant obtained from each extraction was filtered through filter paper and evaporated by vacuum distillation (Buchi V-850, Valinhos, SP, Brazil). Each supernatant was analyzed separately to obtain the percentage extraction for every stage to ensure the complete extraction of the target analytes.
For each extraction, 0.5 g of powder was weighed, and 9 mL of methanol was added. Five sequential extractions were performed on the cashew peduncle powder to extract the AnAc from the samples completely. Each extraction was performed for 20 min using an Ultrasonic Cleaner 1400 (THORTON/UNIQUE) at room temperature, after which the mixture was centrifuged (Kindly KC5, São Paulo, SP, Brazil) for 10 min at 2950× g. The supernatant obtained from each extraction was filtered through filter paper and evaporated by vacuum distillation (Buchi V-850, Valinhos, SP, Brazil). Each supernatant was analyzed separately to obtain the percentage extraction for every stage to ensure the complete extraction of the target analytes.
9
Extraction and Quantification of Microalgal Lipids
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The green- or red-stage D3–1 cells (50 mL culture) grown under SCC for 1 or 3 months were harvested and freeze-dried using a freeze dryer (FZ-Compact: Labconco Co. Ltd., Kansas City, USA). The DCW was measured, and the samples were subjected to cell disruption with glass beads (φ = 1 ± 0.1 mm) and a mixture of diethyl ether:chloroform:methanol (400 μL, 1:2:1) using Vortex-Genie 2 (Scientific Industries, Inc., New York, USA). The supernatants were obtained via centrifugation (8,000 × g, 10 min). This extraction was repeated four times and approximately 1.6 mL of the extract was prepared. Notably, the cell disruption rate was measured using an optical microscope. These rates were considered to calculate the yield of the extracts. The resultant samples were subjected to a rotary evaporator (V-850; BUCHI, Flawil, Switzerland), and the dried extracts were weighed (approximately 10−30 mg per sample) and stored at −30 °C until use.
10
Rhamnolipid Extraction and Quantification
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The recovery of the rhamnolipids was performed first by acid precipitation of the supernatant I obtained from the centrifugation as commented in topic 2.2. The cell-free broth was acidified to pH 2.0 using HCl (6M) and stored at 4 °C overnight. The sample was then centrifuged at 1370 x g for 10 minutes. The supernatant from that centrifugation was discarded and 5 mL of distilled water and petroleum ether in the ratio of 1: 1 (v/v) were added to the precipitate. This procedure was repeated three times and at each repetition the emulsion formed by the ether and the rhamnolipids were removed and stored. Finally, the organic phase obtained from the last step was taken to the rotary evaporator V-850 (Büchi, Switzerland). Ten mL of distilled water was added to the obtained concentrate and stored (Peng et al., 2012) . The quantification of the rhamnolipids was performed by the thioglycolic colorimetric method according to Oliveira et al. (2013) .
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