Method for screening and separating hypoglycemic functional compound in plant
A screening method and compound technology, applied in chemical instruments and methods, material separation, drug combination, etc., can solve problems such as waste and efficiency reduction
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Embodiment 1
[0058] This example provides a screening method for compounds with hypoglycemic functions in rock cabbage.
[0059] 1. Extraction of samples
[0060] Soak 5 kg of rock cabbage medicinal material (collected from Pakistan), and extract with 2×12 L of 80% ethanol for 24 hours, repeating 3 times. All the extracts were combined and evaporated to dryness at 40°C to obtain 256 g of crude extract, which was stored in a refrigerator at 4°C.
[0061] 2. Nanofiltration screening of hypoglycemic components
[0062] Rock cabbage extract (1.0 mg / mL, 250 μL) was incubated with 50 μL of 0.5 U / mL concentration of α-glucosidase in pH 6.8 acetate buffer at 37° C. for 30 minutes. After incubation, pass the enzyme and the mixture through an ultrafiltration membrane (molecular weight cut-off of 30,000 Da) and centrifuge at room temperature at 10,000×g for 15 minutes, wash with buffer three times, and centrifuge to remove unbound components. Add 100 μL of methanol / water (50:50, v / v, pH 3.30) to t...
Embodiment 2
[0071] This example provides the lower K under different solvent components D value.
[0072] The K of the target compound of table 1 different solvent systems D value
[0073]
[0074] Choosing the right two-phase solvent system for countercurrent chromatographic separation is a very critical step, and a suitable two-phase solvent system can successfully complete the separation experiment. In this study, various biphasic mixed solvent systems including two, three or four solvent mixtures were tested at different ratios. A good solvent system can provide ideal partition coefficients (K D )value. K D The value describes the ratio of solute distribution between two-phase solvent systems in mutual equilibrium. The biphasic solvent systems studied in our experiments included n-hexane / ethyl acetate / methanol / water (1:9:1:9, v / v), ethyl acetate / n-butanol / water (1:4: 5, v / v). In addition, different ratios of biphasic solvent system tert-butyl methyl ether / n-butanol / methanol...
Embodiment 3
[0076] This example provides the hypoglycemic test experiment of the compounds β-arbutin (1) and 6-O-galloylarbutin (2) screened in Example 1.
[0077] 1. HepG2 cell viability test
[0078] The cytotoxicity of the crude extract and monomeric compounds i.e. total extract, β-arbutin (1), 6-O-galloylarbutin (2) was analyzed in HepG2 (human liver cancer cell line) cells according to MTT Determination. First, HepG2 cells were cultured in DMEM supplemented with penicillin (100 U / mL) / streptomycin (100 μg / mL) and 10% FBS. Then place the cells in 5% CO 2 Incubate in the presence at 37°C. Trypsin solution was used to digest HepG2 cells in log phase. The cell density was then adjusted to 5 × 10 5 / mL. Incubate the cells in a volume of 100 µL / well at 37 °C and 5% CO 2 seeded into 96-well cell culture plates. Afterwards, the seeded cells were treated with appropriate concentrations of test samples for 24 hours. The wavelength of absorbance was kept at 570nm to determine cell viabi...
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