Large unilamellar vesicles (LUVs) were prepared by mixing the lipids in chloroform in a round-bottom flask. For vesicles containing 7MC-POPE, the probes were added to the lipid in chloroform solution at a final probe concentration of 2 mol%. The solvent was rapidly evaporated using a rotary evaporator (Büchi R-3000, Flawil, Switzerland) at 60°C. The lipid film was then placed under vacuum for 4 hours and hydrated by the addition of buffer containing 20 mM MOPS, pH 7.5, 0.1 mM EGTA, 0.02% NaN3, and 100 mM KCl or appropriately modified as indicated below. The final concentration of the lipid suspension was approximately 5 mM. The suspension of multilamellar vesicles was subjected to five freeze-thaw cycles and extruded 10 × through two stacked polycarbonate filters of 0.1 μm pore size (Nuclepore, Whatman, Florham, NJ, USA), using a water-jacketed high pressure extruder (Lipex Biomembranes, Inc., Vancouver, Canada) at room temperature. Lipid concentrations were assayed as previously described (19 (link)).
R 3000
Manufactured by Büchi
Sourced in Switzerland
The R-3000 is a rotary evaporator designed for the evaporation of solvents from samples. It features a temperature-controlled heating bath and a motorized lift to control the immersion depth of the evaporation flask. The R-3000 can be used for a variety of applications that require the efficient removal of solvents from samples.
Automatically generated - may contain errors
12 protocols using r 3000
1
Preparation of Large Unilamellar Vesicles
2
Comprehensive Phytochemical Profiling of Cyperus scariosus Rhizome
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Cyperus scariosus rhizomes were separated from the plant,
washed with running tap water to remove the dust, followed by
sterilization with double distilled water, shade dried and made
into a fine powder with a blender. 500 g of the rhizome powder
were exhaustively extracted with various organic solvents such
as petroleum ether, hexane, chloroform, ethyl acetate, methanol
and ethanol with soxhlet apparatus for 12–24 h. The extracts
were filtered with Whatman filter paper (type 4) and the filtrate
was concentrated under reduced pressure on rota vapor under
vacuum (BUCHI, R-3000, Switzerland) at 400°C temperature.
The filtrate was used for analysis of phytochemical compounds,
anti-oxidant activity and for gas chromatography-mass
spectroscopy (GC-MS) studies.
washed with running tap water to remove the dust, followed by
sterilization with double distilled water, shade dried and made
into a fine powder with a blender. 500 g of the rhizome powder
were exhaustively extracted with various organic solvents such
as petroleum ether, hexane, chloroform, ethyl acetate, methanol
and ethanol with soxhlet apparatus for 12–24 h. The extracts
were filtered with Whatman filter paper (type 4) and the filtrate
was concentrated under reduced pressure on rota vapor under
vacuum (BUCHI, R-3000, Switzerland) at 400°C temperature.
The filtrate was used for analysis of phytochemical compounds,
anti-oxidant activity and for gas chromatography-mass
spectroscopy (GC-MS) studies.
3
Preparation of Magnetic Nanoemulsions
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The nanoemulsion was prepared by mixing 300 μL of extra virgin olive oil, 300 μL of DSPC, 1000 μL of PEG-DSPE and 75 µL of magnetic particles. The DSPC/PEG-DSPE molar ratio was 1.05. Then, the organic solvent was removed by evaporation at vacuum at 40 °C (Rotavapor R-3000, Büchi, Switzerland) for one hour. Once the solvent evaporated, 10 mL of Hepes buffer at 37 °C was added, followed by 15 min of agitation. The coarse emulsion obtained was sonicated in an UP200 St ultrasonic processor (Hielscher, Teltow, Germany) at a duty cycle of 70% and a frequency of 20 kHz, four times, for 5 min, with pauses of 1 min between sonications. Temperature reached in the sonication process was ≈50 °C. When the nanoemulsion contained IND, the drug dissolved in methanol (1 mg/mL) was incorporated (0.5 mL) to the mixture of surfactants and oil.
4
Preparation of Large Unilamellar Vesicles
5
Sophora interrupta Root Extraction
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Sophora interrupta roots were collected from Tirumala forest, Tirupati (Chittoor District, Andhra Pradesh, India) identified by taxonomist a voucher specimen deposited, in K L E F University, Guntur, India (voucher number KLU 1211). Shade dried and powdered roots were successively extracted from nonpolar to polar solvents such as 100% petroleum ether, n-butanol, and EtOAc and Aqueous is mixed individually with 2.5 kg of dry root powder and macerated for 48 h at room temperature. The extracts were filtered with Whatman filter paper (type 4), and the filtrate was concentrated under reduced pressure on rotavapor under vacuum (BÜCHI, R-3000, Switzerland) at 40°C temperature. The complexity of EtOAc extract on purification over a silica gel column chromatography (60–120 mesh) with step gradient systems that is, n-hexane, chloroform, EtOAc, ethanol, and methanol.
6
Fungal Metabolite Extraction Protocol
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Trichoderma harzianum (CECT 2413), A. alternata (CECT 20912) and P. aurantiogriseum (CECT 20226) from glycerol stocks were inoculated into 500 mL Erlenmeyer flasks containing 200 mL liquid Murashige and Skoog (MS) medium supplemented with 90 mM sucrose. Cultures were incubated at 28°C with constant shaking at 180 rpm on a rotatory shaker for 7 days. Next, 100 mL of the cultures were poured into 2 L Erlenmeyer flasks containing 1 L MS-sucrose medium and incubated at 28°C with constant shaking at 180 rpm for 3 days, after which the fungal mycelium was removed using Whatman #1 filter paper. The resulting filtrate was sterilized using a 0.22 μm Millipore membrane filter and stored at 4°C whilst awaiting further use. DEs were obtained by distilling the CFs at 50°C using a R3000 (BUCHI) rotavapor following manufacturer’s instructions1 .
7
Synthesis of Lipid-OA-PB Nanostructures
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A lipid structure containing OA–PB (L–OA–PB) was obtained by the thin-film hydration method [39 (link)]. Briefly, soy-bean phosphatidylcholine (S-100) was dissolved in chloroform/methanol (2:1, v/v) at 20 mM concentration. Then, 4 mL of this solution was mixed with 4 mL of the toluene solution containing OA–PB. The resulting mixture was placed in a round-bottom flask and dried in a rotary evaporator (Büchi R-3000, Switzerland) under reduced pressure at 35 °C, until a thin blue film was formed at the inner surface of the flask. Afterwards, the resulting lipid–OA–PB film was hydrated with 4.0 mL of Tris buffer (10 mM, pH 7.4). The dispersion was sonicated in bath sonicator (Transsonic Digital Bath sonifier, Elma, Singen, Germany) for 5 min. After that, the lipid suspension was constantly shaken several times (from 1 h to 60 h) at 30 rpm in a test tube rotator (L-29, Labinco, Breda, The Netherlands) at RT. The resulting lipid structures presented an intense blue color.
8
Extracting Phytochemicals from Plant Material
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The fresh plant material (leaves or flowers, 25 g) was extracted at room temperature for 3 days with small volume of diethyl ether (twice). The extracts from flowers and leaves were dried in rotary evaporator (Büchi R-3000) under vacuum at 30°C to give the following extracts: diethyl ether extracts from flowers (DEF) and leaves (DEL) and methanol extracts from flowers (MEF) and leaves (MEL).
9
Preparation and Characterization of LUVs
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Large unilamellar
vesicles (LUVs) were prepared by mixing the lipids in chloroform in
a round-bottom flask. For vesicles containing 7MC-POPE, the probes
were added to the lipid in chloroform solution at a final probe concentration
of 2 mol %. The solvent was rapidly evaporated using a rotary evaporator
(Büchi R-3000, Flawil, Switzerland) at 60 °C. The lipid
film was then placed under vacuum for 4 h and hydrated by the addition
of buffer containing 20 mM MOPS, pH 7.5, 0.1 mM EGTA, 0.02% NaN3, and 100 mM KCl or 10 mM phosphate buffer. The suspension
of multilamellar vesicles was subjected to five freeze–thaw
cycles and extruded 10× through two stacked polycarbonate filters
of 0.1 μm pore size (Nuclepore, Whatman, Florham, NJ), in a
water-jacketed high pressure extruder (Lipex Biomembranes, Inc., Vancouver,
Canada) at room temperature. Lipid concentrations were assayed by
the Bartlett phosphate method,21 modified
as previously described.22
vesicles (LUVs) were prepared by mixing the lipids in chloroform in
a round-bottom flask. For vesicles containing 7MC-POPE, the probes
were added to the lipid in chloroform solution at a final probe concentration
of 2 mol %. The solvent was rapidly evaporated using a rotary evaporator
(Büchi R-3000, Flawil, Switzerland) at 60 °C. The lipid
film was then placed under vacuum for 4 h and hydrated by the addition
of buffer containing 20 mM MOPS, pH 7.5, 0.1 mM EGTA, 0.02% NaN3, and 100 mM KCl or 10 mM phosphate buffer. The suspension
of multilamellar vesicles was subjected to five freeze–thaw
cycles and extruded 10× through two stacked polycarbonate filters
of 0.1 μm pore size (Nuclepore, Whatman, Florham, NJ), in a
water-jacketed high pressure extruder (Lipex Biomembranes, Inc., Vancouver,
Canada) at room temperature. Lipid concentrations were assayed by
the Bartlett phosphate method,21 modified
as previously described.22
10
Phenolic Compounds Extraction and Antioxidant Activity
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The extraction of the phenolic compounds was carried out following the procedures described by Capitani et al. (2012) . Ten mL of ethanol were added to 1 g of sample; then it was homogenized in Vortex for 2 min, decanted and filtered (0.25 mm nylon paper). The supernatant was then transferred into a flask and evaporated using a rotavapor apparatus (BUCHI R-3000, Germany) to concentrate the sample, which was then dissolved in 1 mL ethanol. The AA of the extracts was screened by measuring the DPPH radical scavenging activity according to Carciochi, Manrique, and Dimitrov (2014) . Aliquots (50 μL) of extracts were added to 1950 μL of a methanolic solution (100 l M) of DPPH radical. After agitation, the mixture was incubated in the dark for 30 min, and the absorbance was measured at 517 nm. AA was calculated on the basis of the percentage of DPPH radical scavenging activity.
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