Application of glucopyranogalactooligosaccharide sulfate in preparation of anticoagulant and/or antithrombotic drugs
A technology of galactooligosaccharides and antithrombotic drugs, applied in the preparation of anticoagulant and/or antithrombotic drugs, in the field of glucogalactooligosaccharide sulfate, can solve the problem of not having anticoagulant activity, etc. To achieve the effect of strong drug effect, broad application prospects, and low risk of bleeding
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Embodiment 1
[0036] Preparation of Glucogalacto-oligosaccharide Sulfate:
[0037] Weigh 1.0 g of raw galacto-oligosaccharide, transfer it to a conical flask, add 10-20 mL of DMSO, stir to dissolve, and obtain a solution of galactooligosaccharide. Pyridine and chlorosulfonic acid were mixed in a three-necked flask in a volume ratio of 3:1, and magnetically stirred in an ice-water bath for 30 min to obtain a sulfated reagent. Under stirring, the above-mentioned galacto-galacto-oligosaccharide solution was gradually added to 30 mL of sulfation reagent, the condenser tube was connected and the other two bottle caps were plugged, and the three-necked bottle was placed in a 60°C oil bath to stir and react for 3 hours. Naturally cooled to room temperature, the reaction stopped. Use 15% NaOH solution to adjust the pH of the solution to neutrality. After centrifuging at 8000rpm and 4°C to remove insoluble impurities, collect the supernatant and dialyze it with a 500-1000Da dialysis bag. After dialys...
Embodiment 2
[0040] Determination of the sulfated degree of substitution (Ds) of glucogalactooligosaccharides.
[0041] Prepare 10 mg / mL solution of glucogalacto-oligosaccharide sulfate, take 200 μL into a glass bottle, add an equal volume of 4M HCl, and conduct complete acid hydrolysis at 100 °C for 8 h. After drying with nitrogen, redissolve with 100 μL of pure water. Prepare 0.5% gelatin solution, add 0.5% BaCl when using 2 . Take 100 μL of glucogalacto-oligosaccharide sulfate solution and an equal volume of BaCl 2 - Gelatin solution, OD value measured at 360 nm. The degree of substitution is calculated by the following formula:
[0042]
[0043] The dosage of sulfation reagent, reaction time, reaction temperature and other conditions will affect the degree of substitution of sulfation. The dosage of sulfated reagents is reduced, and the degree of sulfated substitution is also reduced.
Embodiment 3
[0046] Determination of the inhibitory activity of glucogalactooligosaccharide on coagulation factor FXa.
[0047] 1. Determination of inhibitory activity
[0048] a. Mix 100 μL FXa (5nM) and 50 μL glucogalacto-oligosaccharide sulfate sample (1 mg / mL) in a 96-well plate, incubate at 37°C for 1 h, add 50 μL substrate CS-11(22) (1.77 mM), Detection at 405nm wavelength for 60min, detection once per minute.
[0049] b. Pipette 100 μL of HFXIa (human coagulation factor XIa, 1 nM) and 50 μL of glucogalacto-oligosaccharide sulfate sample (1 mg / mL) in a 96-well plate, mix well, incubate at 37°C for 1 h, add 50 μL of substrate S2366 (1 mM), and add 50 μL of substrate S2366 (1 mM). Detection at 405nm wavelength for 60min, detection once per minute.
[0050] c. Pipette 100μL of FXIIa (4nM) and 50μL of glucogalacto-oligosulfate sample (1mg / mL) into a 96-well plate, mix well, incubate at 37°C for 1h, add 50μL of substrate PefachromeFXIIa / TH5253 (1mM), and detect at 405nm for 60min. Chec...
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