The EtOAc soluble fraction (152.6 mg) was subjected to column chromatography (CC) over a sephadex LH-20 column (Sigma-Aldrich., St. Louis, Mo, USA)) and eluted with a CHCl3/MeOH (8:2) system. Fractions were combined based on their TLC pattern to yield subfractions designated E1–E6. Fraction E2 (46.2 mg) was purified by sephadex LH-20 CC (CHCl3/MeOH = 9:1) to yield four subfractions (E21–E24). Subfraction E22 (21.6 mg), containing α-spinasterol-3-O-β-D-glucopyranoside, was purified by silica gel CC (CHCl3/MeOH, 20:1 → 15:1), and finally by silica gel CC (CHCl3/MeOH, 18:1), to yield α-spinasterol-3-O-β-D-glucopyranoside (5.0 mg).
Sephadex lh 20 column
Manufactured by Merck Group
Sourced in United States, Germany
Sephadex LH-20 is a size-exclusion chromatography medium used for the purification and separation of molecules based on their size and molecular weight. It is a gel-filtration resin composed of cross-linked dextran beads that provides efficient separation of a wide range of biomolecules, including proteins, peptides, nucleic acids, and other small organic compounds. The porous structure of the Sephadex LH-20 matrix allows for the size-based fractionation of the sample components, enabling the isolation and purification of the desired analytes.
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Lab products found in correlation
Co2 incubator, by Thermo Fisher Scientific (3 mentions)
Methanol, by Xilong (3 mentions)
Amberlite xad 7 resin, by Merck Group (1 mentions)
Rotary evaporator, by Heidolph (1 mentions)
Sep pak c18 cartridge, by Waters Corporation (1 mentions)
Kieselgel 60, by Merck Group (1 mentions)
Polyamide, by Merck Group (1 mentions)
Silica gel, by Merck Group (1 mentions)
Microplate spectrophotometer reader, by Thermo Fisher Scientific (1 mentions)
Acquity tqd system, by Waters Corporation (1 mentions)
Acquity uplc beh c18, by Waters Corporation (1 mentions)
Texas red, by Abcam (1 mentions)
Interleukin 6 (il 6), by Elabscience (1 mentions)
Il 1β, by Elabscience (1 mentions)
Tnf α, by Elabscience (1 mentions)
Nalgene rapid flow filter, by Thermo Fisher Scientific (1 mentions)
Hematoxylin and eosin h e, by Merck Group (1 mentions)
Diaion hp 20 gel, by Merck Group (1 mentions)
C18 silica gel, by Merck Group (1 mentions)
Agilent 1260 series, by Agilent Technologies (1 mentions)
24 protocols using sephadex lh 20 column
1
Isolation of α-Spinasterol-3-O-β-D-Glucopyranoside
2
Trichoderma Secondary Metabolite Extraction
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For secondary metabolite extraction, Trichoderma species were grown in 500 mL flasks containing 100 g (dry weight) solid substrate (wheat bran to white rice (2:1 w/w)) supplemented with 1% (v/v) glycerol (98.9%) and 1% (w/v) (NH4)2SO4) (99.5%) to moisten the substrate [16 (link)]. The flasks were sterilized at 121°C for 15 min and, after cooling, inoculated with 20 mL spore suspension (1 × 107 spores/mL) followed by incubation at 28°C for 21 days [16 (link)]. The cultures were homogenized by adding methanol, followed by centrifugation at 5000 rpm for 15 min. The supernatant was collected and dried under a vacuum; the obtained methanolic extracts was purified using Sephadex LH-20 column (Sigma-Aldrich, St. Louis, MO) and stored at -20°C until use for oral administration in mice and GC-MS analysis.
3
Extraction and Purification of Anthocyanins
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B. calycina petals (100 g) were blanched with 1 L of 0.3 M aqueous HCl and the red extracts were collected and filtered through qualitative filter paper (Grade 1, medium flow, Whatman®, Sigma-Aldrich). The filtrate was then subjected to Amberlite XAD-7 resin (Sigma-Aldrich) column (2.5 × 30 cm) chromatography according to Zhang (2004) [33 (link)]. Fractions with the highest A510 nm were pooled and concentrated by removing the methanol at 50 °C in a rotary evaporator (Heidolph, Schwabach, Germany). The concentrated anthocyanins were then loaded onto a Sephadex LH-20 column (1.0 × 90 cm, Sigma-Aldrich), eluted with 10% (v/v) aqueous FA (formic acid) at a flow rate of 0.3 mL/ min. Fractions (3 mL/tube) were collected and monitored by the absorbance at 510 nm. The fractions in the major peaks of the A510 nm elution profile were pooled and subjected to Amberlite XAD-7 resin to remove the residual FA and then the elution was concentrated as purified anthocyanins. The purified anthocyanins were filtered through the 0.22 μm PVDF membrane and transferred to a vial before analysis.
4
Extraction and Identification of Bioactive Compounds from Scutellaria baicalensis
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The dried aerial parts of Scutellaria baicalensis Georgi (1.2 Kg) were cut into pieces and extracted with methanol (3 × 2 L) in an ultrasonic apparatus at room temperature after evaporation of the solvent. The methanol extract was suspended in H2O and successively partitioned into chloroform, ethyl acetate, n-butanol, and water fractions after removal of the solvents under vacuum. Then, these fractions were subjected to a sulforhodamine B (SRB)-based antiviral activity assay (Table 1 ), and we found that the chloroform fraction had antiviral activity against CVB3. Next, the chloroform fraction was subjected to C18 column silica gel column adsorption chromatography (40–63 μm, 300 g) (Merck & Co., Kenilworth, NJ, USA), and eluted with a gradient consisting of methanol:water (3:7, 4:6, 6:4, 8:2, and 10:0; 2 × 500 ml). The fraction was separated on a Sephadex LH-20 column (Sigma-Aldrich, St. Louis, MO, USA) using 100% methanol, and norwogonin, oroxylin A, and mosloflavone were obtained. The structures of norwogonin, oroxylin A, and mosloflavone were identified using electrospray ionization mass spectrometry, 1H-nuclear magnetic resonance (NMR), and 13C-NMR.
5
Isolation and Characterization of POP from Rice Bran
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POP was isolated from RBO. The RBO was extracted from rice bran (Ochang Rice Processing Center, Chungbuk, Korea) as described previously [20 ] and an active fraction of RBO was purified by silica gel column chromatography extracted with hexane-ethyl acetate (10:1, v/v), followed by Sephadex LH-20 column chromatography (Sigma-Aldrich, St. Louis, MO, USA) using chloroform-methanol (1:1, v/v). The active component POP was purified with a silica Sep-Pak cartridge extracted with hexane-ethyl acetate (10:1, v/v) and identified by the mass measurement and nuclear magnetic resonance (NMR) spectroscopic method. In brief, electron ionization-mass measurement and NMR spectra, including 1H NMR, 13C NMR, 1H-1H COSY (correlation spectroscopy), HMQC (Heteronuclear Multiple Quantum Coherence), and HMBC (Heteronuclear Multiple Bond Coherence) spectra confirmed the presence of one glycerol, two palmitoyl, and one oleoyl moieties. The presence of two oxygenated carbon peaks in glycerol moiety demonstrated that this compound should have the symmetrical structure of POP (Figure 1 ).
6
Extraction and Purification of Luteolin
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The above residue (16 g) was suspended in chloroform and then extracted thrice with the same solvent. The chloroform soluble fraction (CSF) was purified through column chromatography using silica gel at an elution rate of 2 mL/min flow with a total elution of 200 mL and a gradient of chloroform:methanol (0.9:0.1) to acquire the fractions CSF1 (0.7 g), CSF2 (0.5 g), CSF3 (0.3 g), CSF4 (0.5 g), CSF5 (0.2 g), CSF6 (0.5 g), CSF7 (0.9 g), CSF8 (1.1 g), and CSF9 (1.0 g). Subsequently, CSF8 was yet again subjected to silica gel column chromatography using a column measuring 50 cm in length and 3 cm in diameter, with the elution rate adjusted to 1 mL/min. The total elution carried out was for 100 mL using the linear gradients of chloroform:acetone (90:10; 80:20; 70:30; 60:40; 50:50; 20:80; v/v), to obtain 6 sub-fractions. Sub-fraction 3 was further separated by silica gel CC using chloroform:acetone (70:30) followed by re-chromatography on a Sephadex LH-20 column (Sigma-Aldrich, Texas, USA) with methanol as the eluting solvent, yielding luteolin (332.1 mg). The structure elucidation of the luteolin by 1H NMR and mass spectral techniques is shown in Figures S1 and S2 .
7
Pectolinarigenin Isolation via HSCCC
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After successive cycles of HSCCC using SFCO2E, the collected samples were grouped into 11 fractions according to the similarity of elution pattern and retention factor (Rf) observed on the TLC plates. HEMWat 1:1:1:1 was used as the solvent system again as described above. Fractions 15–29 showed the presence of yellow bands from which pectolinarigenin was isolated. Fractions 15–29 were put together and submitted to Gel-filtration Chromatography using a Sephadex LH-20 column obtained from Sigma-Aldrich (Dublin, Ireland) and eluted with methanol at an average flow rate of 0.5 mL/min. A complete purification was achieved allowing for the isolation of Pectolinarigenin at 98% purity.
8
Isolation of Antifungal Compounds from Strain G341
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In preliminary experiment, TSB medium was used as the optimal medium for the production of antifungal substances. Strain G341 was cultured in TSB medium (4 l) at 37°C and 150 rpm for 3 days. Bacterial cells were removed after centrifugation at 8,000 rpm for 6 min. The culture supernatant was successively partitioned twice with equal volumes of ethyl acetate (EtOAc) and n-butanol (BuOH). Each layer was concentrated by drying and then subjected to in vitro antifungal activity against M. oryzae. Among four organic fractions obtained from culture broth of strain G341, BuOH layer (17.2 g) was found to be the most active layer, followed by the EtOAc layer (10.2 g). The BuOH layer was applied onto a silica-gel column (3.6 (i.d.) × 60 cm, Kiesel gel 60, 400 g, 70–230 mesh; E. Merck, Darmstadt, Germany). It was then eluted with a mixture of chloroform-methanol-water (30:9:1 and 65:25:4, v/v/v). Active fractions showing inhibitory activity against M. oryzae were collected and applied onto a Sephadex LH-20 column (2.8 (i.d.) × 45 cm, 200 g; Sigma, MO, USA) after concentration. Active fractions eluted from the Sephadex LH-20 column with methanol were evaporated to dryness and loaded onto a Sep-Pak C18 cartridge (10 g) (Waters, Milford, MA, USA) with methanol-water. Using this protocol, 220 mg of compound 1 and 24 mg of compound 2 were obtained from 4 l of culture supernatant.
9
Polyphenol Enrichment from Methanolic Plant Extract
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The methanolic extract was resuspended in DMSO using ultrasonication and subjected to solid-phase extraction using a preconditioned RP-C18 column (100 × 80 mm i.d.; Cosmosil 140C18-PREP, 140 µm), followed by the exclusion of compounds with high polarity (using solutions of 1% MeOH v/v and 50% MeOH v/v, respectively). Subsequently, a polyphenolic-rich fraction was eluted with a solution containing 85% MeOH and 0.1% formic acid. This fraction was then further purified by elution with 100% MeOH using a Sephadex LH-20 column (970 × 34 mm i.d., Sigma-Aldrich, Steinheim, Germany). After performing this separation, four major fractions (Fr 1–4) were collected. The chemical composition of these fractions was monitored through LC-MS techniques.
10
Phytochemical Investigation of Litsea australis
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Air-dried, powdered pericarp of L. australis (1.75 kg) was extracted with 70% ethanol (5 L) by cold maceration for 72 h, and then filtered through filter paper. The marc was re-extracted again until exhaustion, and the collected extracts was dried under vacuum at 45 °C to afford the crude extract (340 g). A part of the dried extract (200 g) was fractionated using n-hexane, dichloromethane and ethyl acetate to give 22, 14 and 6 g, respectively. The n-hexane fraction (6 g) was chromatographed on a silica gel (E-Merck, Germany) column using hexane with 10% ethyl acetate increments to afford one major compound 1 (500 mg). The ethyl acetate fraction (5 g) was chromatographed on a polyamide (E-Merck, Germany) column starting with 100% water as the eluent, then with decreasing polarity by 20% MeOH to afford five sub-fractions. The sub-fraction eluted with 20% water (500 mg) was re-chromatographed on a Sephadex LH-20 column using methanol to afford compounds 2 and 3 (25, 10 mg, respectively). The sub-fraction eluted with 60% water (100 mg) was re-chromatographed on a Sephadex LH-20 column (Sigma-Aldrich, USA) using methanol as an eluent to afford three compounds 4–6 (15, 12, 20 mg, respectively).
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