The samples were extracted following the methods of Babero et al. (2008) (link) and Ma et al. (2009) (link). Samples of 25g lyophilized edible fruits of Vitis vinifera, Punica granatum, Citrus aurantium and Opuntia ficus indica were macerated at room temperature for 24 h with 500 mL methanol/water (70/30, v/v) along with ultrasound extraction for 30 min (Fisher scientific fb 15046, Leicestershire, England) (>20 kHz in frequency). After filtration, the remaining vegetal material was re-extracted twice applying same procedure. The filtrates were combined and concentrated in a rotavap (Buchi, Switzerland) under vacuum at low temperature (<40 °C). The extracts were lyophilized (Christ Alpha 2–4 LD plus, Osterode am Harz, Germany) and stored at under low temperature (-25 °C) for future use.
Rotavap
Manufactured by Büchi
Sourced in Switzerland
The Rotavap is a laboratory equipment used for the efficient and gentle removal of solvents from samples through evaporation under reduced pressure. It is a key tool for applications such as purification, concentration, and drying of various chemical and pharmaceutical substances.
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11 protocols using rotavap
1
Extraction of Bioactive Compounds from Edible Fruits
2
Concentrating and Fractionating Yeast Conditioned Media
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Fifteen BY4741 NR or CR cultures (10 ml SC each) were grown to saturation for 5 days at 30 °C in 18 mm glass test tubes with loose-fitting metal caps. Tubes were used instead of a single flask to maintain the same incubation conditions as our typical CLS assays. The glassware was acid washed with 0.1 N HCl before use. Cells were pelleted by centrifugation and the conditioned media pooled. The pooled media was then concentrated in a Büchi Rotavap from 150 ml down to approximately 2.5 ml (60-fold). The concentrates were centrifuged at 4000 rpm (2987 RCF) for 10 min in 15 ml conical tubes to remove any solid precipitates. Two milliliter of the clarified concentrate was loaded onto a 1 x 26 cm Sephadex G-10 column and fractionated with double-distilled water. Two milliliter fractions were collected by gravity flow in a Pharmacia fraction collector and filter sterilized through 0.22 μm syringe filters. The fractions were then added to new 5 ml CLS cultures at a 1:5 ratio (ml concentrate: ml culture) and semiquantitative CLS assays performed. NaCl (100 mM) was eluted through the column before and after the media concentrates to determine the gel size retention fractions as measured by electrical conductivity.
3
Synthesis of pH-Responsive Hydroxyapatite Nanoparticles
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The Pip loading ratio was 1:3 (drug: nanoparticle); 300 mg Pip (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 20:80 ultrapure water:ethanol (Fisher Scientific, Loughborough, UK) at room temperature, followed by 900 mg of HAPs and HAP-Ps. The pH adjusted to either 7.2 or 9.3. The mixture solution was stirred for 24 h, and then evaporated at 60 °C (Rotavap; Büchi, Flawil, Switzerland). The dried mixture was resuspended in ultrapure water several times to remove unloaded molecules before drying in a 60 °C oven for 12 h. The obtained product was named HAP-Pip7.2, HAP-Pip9.3, HAP-P-Pip7.2, and HAP-P-Pip9.3. These materials were intended for coating with GA in subsequent steps.
4
Extraction of H. sibthorpioides Compounds
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Fresh whole plants of H. sibthorpioides, including the aerial parts, were collected and identified to be of the correct species (reference number SBID: 001/09) by botanists from the Forest Research Institute Malaysia (FRIM). The plants were washed under running tap water and finally rinsed with distilled water. The plants were then air dried for 48 h, homogenized into a fine powder and stored in air-tight plastic containers. The powder was then extracted in a soxhlet extractor for 24 h, using either methanol or water. Both types of extract were evaporated to dryness under pressure and in a controlled temperature using a rotavap (Buchi, Flawil, Switzerland). Finally, the extracts were freeze-dried and stored until use.
5
Isolation and Characterization of Methyl Gamma Linolenate
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All chemicals used were purchased from Fluka chemicals. Their purity was checked by GC. All solvents were purified by distillation using Rotavap (Buchi R120) and if necessary residual water was removed. The components of solvents and elements are given in volume ratios of the components. Methyl gamma linolenate was isolated and purified by using Isolera Flash chromatography System (Biotage INC) and identified using different spectral and HPTLC techniques. The standard methyl gamma linolenate and routine were purchased from Sigma-Aldrich uses thin layer chromatography. The 1H NMR and 13C NMR were recorded on Bruker DRX-300 (300 MHz FT-NMR) using deuterated chloroform as solvent and TMS as internal standard. The mass spectra of compounds were recorded on JEOL GCMATE II GC-MS.
6
Phenolic Compound Extraction by Alkaline Hydrolysis
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Covalently linked phenolic compounds were extracted through alkaline hydrolysis (1 g powder: 15 mL 2 M NaOH) by stirring under vacuum in darkness, at 4°C for 2 h, according to Vaidyanathan and Bunzel (18 (link)). Afterward, the pH was adjusted to 1.5 by the addition of 6 M HCl. Three successive extractions with 25 mL of ethyl acetate were performed, and then the supernatants were combined, and the solvent was evaporated at low pressure (Büchi rotavap, Germany). The residue was dissolved by the addition of 1,000 μl of methanol, filtrated through a 0.45-μm membrane, and analyzed by reversed-phase HPLC-PAD and HPLC-ESI-MS/MS, as explained earlier.
7
Methanolic Extraction of Salvadora persica
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The methanolic extract of S. persica plant was prepared at the Faculty of Pharmacy under the Pharmaceutical Consulting and Research Unit at King Abdulaziz University (KAU), Jeddah, Saudi Arabia. Fresh arak roots (Salvadora persica) (1.5 kg) were obtained from a traditional store, Jeddah city, Saudi Arabia, which were collected by an expert from the western region. The arak roots were minced and dried, pulverized, and extracted with methanol (3 × 1 L, Sigma-Aldrich, Chemie GmbH, Germany) using an ULTRA-TURRAX disperser (T 50 basic IKA-Werke, Germany) for 3–5 min to ensure an equal distribution of the methanol. The pooled methanolic extract was filtered until a clear solution was obtained and then evaporated under vacuum at 45 °C and 70–100 rpm using a rotary evaporator (Rotavap, BUCHI, Switzerland). The extracted product was then dried using a lyophilized freeze-dryer (Alpha 3-4 LSCbasic, CHRIST, Germany) at −80 °C for 3 days to obtain the crude extract. The extract was sterilized with UV light for 1 h.
8
Guayule Shrub Resin Extraction
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Resin (defined by acetone extract) is extracted from ground guayule shrub samples using an Accelerated Speed Extractor (ASE) (Buchi Speed Extractor E-914).
40 mL stainless steel extraction cells with metal frits are filled with three layers: 2 g inert sand (Fat free quartz sand 0.3–0.9 mm (Buchi)) a mix of 5 g of a dry sample and 25 g of sand and 2 g of sand (Fig.2 ). Bottom glass fiber filters (Buchi) are inserted into stainless steel extraction cells prior to loading samples into the cells and top cellulose filters are added on the top after.
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40 mL stainless steel extraction cells with metal frits are filled with three layers: 2 g inert sand (Fat free quartz sand 0.3–0.9 mm (Buchi)) a mix of 5 g of a dry sample and 25 g of sand and 2 g of sand (Fig.
Filling scheme of an ASE cell.
9
Pithecellobium dulce Seed Extraction
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Seeds were collected from Pithecellobium dulce tree (Hail region, Saudi Arabia) and washed with sterile distilled water. Around 400 grams of P. dulce seed was ground to a coarse powder using an electrical grinding mill and extracted by cold maceration in 80% methanol (Sigma-Aldrich, MO, USA) at room temperature [19 (link)]. It was then left standing for 3-4 days with an occasional shaking to produce the crude extract. Later, the crude extract was filtered using Whatman® qualitative filter paper (grade 1, 45 mm) (Wagtech International Ltd., England). Then, filtered product was concentrated using a rotary vacuum evaporator (Buchi Rotavap) at reduced pressure (45 rpm) and temperature (40°C). After drying, around 6 grams of the powder extract was produced and stored at 4°C.
10
Yeast Conditioned Media Fractionation
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Fifteen BY4741 NR or CR cultures (10 ml SC each), were grown to saturation for 5 days at 30°C in 18 mm glass test tubes with loose fitting metal caps. The glassware was acid washed with 0.1N HCl before use. Cells were pelleted by centrifugation and the conditioned media pooled.
The pooled media was then concentrated in a Büchi Rotavap from 150ml down to approximately 2.5 ml. The concentrates were centrifuged at 4,000 rpm (2,987 RCF) for 10 minutes in 15 ml conical tubes to remove any solid precipitates. Two ml of the clarified concentrate was loaded onto a 1 x 26 cm Sephadex G-10 column, and fractionated with double distilled water. 2 ml fractions were collected by gravity flow in a Pharmacia fraction collector and filter sterilized through 0.22µm syringe filters. The fractions were then added to new 5 ml CLS cultures at a 1:5 ratio (ml concentrate: ml culture) and semiquantitative CLS assays performed. NaCl (100 mM) was eluted through the column before and after the media concentrates to determine the gel size retention fractions as measured by electrical conductivity.
The pooled media was then concentrated in a Büchi Rotavap from 150ml down to approximately 2.5 ml. The concentrates were centrifuged at 4,000 rpm (2,987 RCF) for 10 minutes in 15 ml conical tubes to remove any solid precipitates. Two ml of the clarified concentrate was loaded onto a 1 x 26 cm Sephadex G-10 column, and fractionated with double distilled water. 2 ml fractions were collected by gravity flow in a Pharmacia fraction collector and filter sterilized through 0.22µm syringe filters. The fractions were then added to new 5 ml CLS cultures at a 1:5 ratio (ml concentrate: ml culture) and semiquantitative CLS assays performed. NaCl (100 mM) was eluted through the column before and after the media concentrates to determine the gel size retention fractions as measured by electrical conductivity.
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