Aldose reductase inhibitor, preparation method and use thereof
A glycosyl and diabetes technology, applied to aldose reductase inhibitors, can solve problems such as undiscovered glycoside activity
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Embodiment 1
[0134] training method
[0135] The slant of actinomycetes N99-596 was inoculated in the seed medium, and after 72hr cultivation at 27°C, it was inserted into a 750ml Erlenmeyer flask with a content of 140ml medium, and shaken and cultured in the fermentation medium at 27°C for 6 days.
[0136] Separation method
[0137] Centrifuge 5000ml of N99-596 fermentation broth at 3000rpm for 15 minutes, collect the bacteria and supernatant respectively, extract the bacteria with 2000ml of acetone, evaporate the acetone, extract twice with 2500mL ethyl acetate, and wash the ethyl acetate layer through anhydrous Na 2 SO 4 After dehydration, concentration and drying, 2.2 g of brown substance was obtained.
[0138] Take 2.2g sample, dissolve it with a small amount of methanol, use silica gel column (φ2.5×25cm) chromatography to carry out further separation and purification, and the elution condition of the chromatography column is 100% CHCl 3 After elution to 100% MeOH, the combined act...
Embodiment 2
[0167] Preparation of aldose reductase
[0168] The following operations were all carried out at 0-4°C. Take about 90g of porcine lens from each batch, homogenize in three times the volume of cold NaPi buffer, and centrifuge at 10,000×g for 50 minutes to remove insoluble matter. Add (NH 4 ) 2 SO 4 to 40% saturation, stirred gently for 15 minutes, centrifuged to remove the precipitate, and then added (NH 4 ) 2 SO 4 to 50% saturation, stirred gently for 15 minutes, centrifuged to remove precipitate, added (NH 4 ) 2 SO 4 To 75% saturation, gently stirred for 15 minutes to collect the precipitate by centrifugation, dissolved with cold 0.05mol / L NaPi (containing 0.5mmol / lPMSF and 0.5mmol / lEtSH), and dissolved in 0.05mol / L NaPi solution (containing 0.5 mmol / l PMSF and 0.5mmol / l EtSH) and dialyzed overnight. After dialysis, the above enzyme solution was added to glycerol to a concentration of 40%, and stored at -20°C for future use.
[0169] Compound activity assay method:
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Embodiment 3
[0177] The cultivation method of N99-253 is identical with the cultivation method of N99-596 among the embodiment 1.
[0178] Separation method:
[0179] 5000ml of N99-253 (46) fermentation broth was centrifuged at 3000rpm for 15 minutes, and the bacteria and supernatant were collected respectively. After the bacteria were extracted with 2000ml of acetone, the acetone was evaporated, extracted twice with 2500mL ethyl acetate, and the ethyl acetate layer was Anhydrous Na 2 SO 4After dehydration, concentration and drying, 1.2g of brown substance was obtained.
[0180] Take 1.0g sample, dissolve it with a small amount of methanol, and use silica gel column (φ2.5×25cm) chromatography for further separation and purification. The elution condition of the chromatography column is CHCl 3 : MeOH 100: 0~0~100 gradient elution, the combined active components were collected, concentrated and dried to obtain 118mg of brown solid.
[0181] Get above-mentioned active substance, use ODS r...
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