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Separating method of isoflavone derivatives and application thereof

A separation method and technology of dimethoxyisoflavones, which are applied in the field of medicine and can solve problems such as activity detection

Inactive Publication Date: 2018-12-21
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no literature on the activity detection of 5-hydroxy-4,7'-dimethoxyisoflavones

Method used

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  • Separating method of isoflavone derivatives and application thereof
  • Separating method of isoflavone derivatives and application thereof
  • Separating method of isoflavone derivatives and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Separation and structure confirmation of isoflavones

[0076] Red clover 2kg, with 8 times the amount of 75% ethanol as solvent, heated and refluxed for 3 extractions. Each time for 1.5 hours, filter and combine the filtrates, concentrate to obtain 200 g of ethanol extract; add water to suspend and dissolve, sequentially extract with petroleum ether and ethyl acetate, and extract 3 times respectively to obtain 45 g of ethyl acetate. Add appropriate amount of silica gel to the ethyl acetate fraction, mix the sample, perform silica gel column chromatography, and use gradient elution of dichloromethane-methanol system. The combined 9 fractions are recorded as Fr.1-Fr.9 according to the peak eluting time. Fr4 was chromatographed on a silica gel column, eluted with a dichloromethane-methanol gradient from 95:5 to 80:20, and colorless crystals were precipitated, which were designated as Fr4A. The structure was confirmed by NMR method, and its 1H-NMR spectral data ...

Embodiment 2

[0079] Example 2: Evaluation of NQO1-inducing activity of isoflavone 5-hydroxy-4,7'-dimethoxyisoflavone

[0080] (1) Culture of mouse hepatocarcinoma hepa 1c1c cell line

[0081] The mouse hepatocarcinoma hepa 1c1c cell line was purchased from the American Type Culture Collection (ATCC) and cultured in MEM medium containing 10% fetal bovine serum (FBS) in a 37°C, 5% CO2 incubator.

[0082] (2) NQO1-inducing activity test

[0083] The hepa 1c1c cells were inoculated on a 96-well plate, and different concentrations of isoflavones (confirmed in Example 1) were added after the cells adhered to the wall, treated for 24 hours, and the cells were lysed with 0.8% digitonin solution, and the detection solution (1.0mL 0.5M Tris-hydrochloric acid (Tris-HCl), 15 mg bovine serum albumin, 6 mg MTT, 150 μL Tween-20, 150 μL 150 mMD-glucose-6-phosphate, 15 μL 7.5 mM flavin adenine dinucleotide, 27 μL of 50 mM nicotinamide adenine dinucleotide phosphate, 20 μL of 50 mM menadione) were left fo...

Embodiment 3

[0085] Embodiment 3: Isoflavone 5-hydroxyl-4,7'-dimethoxyisoflavone increases Nrf2 protein stability

[0086] (1) Culture of human normal epidermal Beas-2B cells

[0087] Human normal lung bronchial epithelial Beas-2B cells were purchased from the American Type Culture Collection (ATCC), using 1640 medium, and adding 10% fetal bovine serum (FBS) and 5% glutamine to it, and placed at 37°C , cultured in a 5% CO2 incubator.

[0088] (2) Western blot analysis to detect the half-life of Nrf2 protein

[0089] Inoculate Beas-2B cells in a 35mm diameter petri dish, culture until the density reaches 70%-80%, add isoflavones for 8 hours, then add cycloheximide, and time them at 0, 10, 20, 30, 40 minutes respectively Proteins were harvested, detected by Western blot analysis, and quantified using Image J software.

[0090] Result: if figure 2 As shown, the half-life of Nrf2 protein in Beas-2B cells in the blank group was 14.7 minutes, and after being treated with isoflavones for 8 h...

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Abstract

The invention belongs to the field of medicines, and relates to a separating method of isoflavone derivatives and application thereof. The invention discloses application of isoflavone in preparationof medicines for treating oxidative stress-induced diseases. A chemical formula of the isoflavone is shown in the attached figure. The isoflavone can be used for regulating up the protein levels of Nrf2 (nuclear factor-erythroid 2-related factor 2) and Nrf2-regulated II phase detoxification enzyme NQO1 and anti-oxidizing enzyme GCS; an activation mechanism is achieved by increasing the protein stability of Nrf2 to inhibit the protein degrading of Nrf2; an arsenic-induced lung bronchial epithelium Beas-2B cell injury model is used for evaluating the cell protecting function of the isoflavone; proofed by results, the isoflavone can increase the GSH level of cell endogenous antioxidant, and the injury and withering of arsenic-induced Beas-2B cell are inhibited.

Description

technical field [0001] The invention belongs to the field of medicine, and relates to the application of isoflavones in the preparation of medicines, health care products or cosmetics for preventing or treating oxidative stress-induced diseases. Background technique [0002] Population expansion and the rapid development of industrial technology have led to increasing environmental pollution problems. A variety of pollutants, such as carbon sulfur nitrogen oxides (CO, SO 2 , NO 2 etc.), heavy metals (lead, chromium, arsenic, etc.), particulate matter, electromagnetic radiation, chemical organic reagents, etc., continuously invade human tissues and organs through air, water, food and skin contact, causing oxidative stress. A large number of lipid peroxidation products produced by the body and excessive endogenous active oxygen and active nitrogen act on cells, directly or indirectly causing damage, mutation and functional inhibition of intracellular proteins, lipids, nuclei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D311/36C07D311/40A61P11/00A61P17/00A61P3/10A61P35/00A61P25/28A23L33/105A61K8/49A61Q19/00A61Q19/08
CPCA23L33/105A23V2002/00A61K8/498A61K2800/10A61P3/10A61P11/00A61P17/00A61P25/28A61P35/00A61Q19/00A61Q19/08C07D311/36C07D311/40A23V2200/314A23V2200/318A23V2200/322A23V2250/2116
Inventor 沈涛李妍茹向兰王小宁娄红祥
Owner SHANDONG UNIV
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