The crude n-hexane extract recovered from phase separation during large-scale saponin extraction was dried in vacuo in a rotovapor (BUCHI). Around 2 g of the greasy yellowish crude extract was dry loaded onto a normal-phase silica column (Biotage SNAP Ultra 100g, 39 × 157 mm, 25 µm) that had been pre-equilibrated with n-hexane. Separation was carried out using Isolera One (Biotage). Chromatographic separation was carried with a gradient of 0–3% ethyl acetate (solvent B) in n-hexane (solvent A) over 60 column volumes, followed by a gradient of 3–20% solvent B over ten column volumes. Fractions (22 ml) were collected and evaluated by TLC as described earlier. Fractions with sterols of interest were pooled, and purity was assessed by GC–MS analysis as described earlier. Purified compounds were subjected to NMR in CDCl3 (Sigma-Aldrich). Compound identities were confirmed by comparing their 1H or 13C NMR data with those reported previously44 –50 .
Rotovapor
Manufactured by Büchi
Sourced in United Kingdom
The Rotovapor is a laboratory equipment designed to efficiently evaporate solvents from liquid samples. It utilizes a rotating evaporation flask and a controlled temperature water bath to facilitate the evaporation process. The Rotovapor is a versatile tool commonly used in various scientific and industrial applications that require the removal of solvents.
Automatically generated - may contain errors
4 protocols using rotovapor
1
Purification of Sterols from Hexane Extract
2
Extraction and Purification of Plant Bioactives
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As described previously8 (link),15 (link) and based on traditional knowledge obtained from the study area, fresh leaves of Rhus glutinosa, Syzygium guineensa and Albizia gumifera were harvested from their natural habitat in and around Bahirdar area including Blue Nile river basin (LATITUDE AND LONGITUDE), Amhara regional state, Ethiopia. Thereafter, the plant materials were dried in a well-aerated dark room to avoid direct sun light. Then, 500 g of dried leaves were pulverized and placed in a mixer containing 3 L of 70% acetone in water containing ascorbic acid (1 g/L) to avoid oxidation. After that, the mixture was sonicated in a water bath for 20 min. The extract was obtained from the filtered material using a filter paper. Meanwhile, the acetone was evaporated from the extract at 45°C (in vacuo) using a roto-vapor (BUCHI, England). The aqueous solution was washed four times with 500 mL methylene chloride to remove chlorophyll and lipids. A separation funnel was used for discarding the methylene chloride fraction. The remaining fractions were lyophilized (Ningbo, China) and kept refrigerated at 4°C in airtight containers until their use for biochemical and biological assays.
3
Isolation and Characterization of Sterols
4
Isolation and Characterization of R. aculeata Seed Extract
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Material and extract preparation R. aculeata fruits were acquired from local markets in the municipality of Veracruz, Mexico, in November 2021.
The taxonomic identification was confirmed by a member of the Herbarium of the Facultad de Ciencias Biologicas y Agropecuarias, Universidad Veracruzana, and deposited under the registration number JLBT2 (VER). Fruits were washed with distilled water and subsequently, each part of the fruit was separated to obtain the seeds. Seeds were cleaned and air dried for 7 days at room temperature and then pulverized. The extract was prepared at 1:10 (w/v) ratio by adding the hydroethanolic solution (EtOH-H 2 O, 80:20) to the powdered seed. Extraction was carried out by maceration at room temperature. Each 24 h for 3 days, the contents were allowed to settle. Then, the solvent was collected and filtered. The extract was reduced under vacuum using a Buchi® Roto-Vapor at 26 °C. Finally, the concentrated extracts were lyophilized and stored.
The taxonomic identification was confirmed by a member of the Herbarium of the Facultad de Ciencias Biologicas y Agropecuarias, Universidad Veracruzana, and deposited under the registration number JLBT2 (VER). Fruits were washed with distilled water and subsequently, each part of the fruit was separated to obtain the seeds. Seeds were cleaned and air dried for 7 days at room temperature and then pulverized. The extract was prepared at 1:10 (w/v) ratio by adding the hydroethanolic solution (EtOH-H 2 O, 80:20) to the powdered seed. Extraction was carried out by maceration at room temperature. Each 24 h for 3 days, the contents were allowed to settle. Then, the solvent was collected and filtered. The extract was reduced under vacuum using a Buchi® Roto-Vapor at 26 °C. Finally, the concentrated extracts were lyophilized and stored.
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