Dipeptide compound and use of the same in preparation of anti-complement drugs
A compound and anti-complement technology, applied in drug combination, antipyretics, anti-inflammatory agents, etc., can solve problems such as compounds that have not yet seen complement inhibition
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Embodiment 1
[0020] Embodiment 1. Preparation of dipeptide compounds
[0021] Take 20kg of dried Viola chinensis, crush it, soak it in 95% ethanol at room temperature (50L×5 times), combine the extracts and concentrate until there is no alcohol smell, add water to dilute the extract to 2.5L, and then add an equal volume of petroleum ether (60-90°C), ethyl acetate, n-butanol extraction (each 2.5 L x 3 times), combined ethyl acetate extracts and concentrated to dryness to obtain 180 g of ethyl acetate extract. The ethyl acetate extraction part was separated by silica gel (200-300 mesh) column chromatography, followed by petroleum ether-acetone (petroleum ether, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1 ) gradient elution to obtain 7 fractions (Fr1-7), of which fraction Fr.4 (21.5g) was subjected to silica gel column chromatography (petroleum ether-ethyl acetate as eluent, 30:1, 20:1 , 15:1, 10:1, 5:1) and Sephadex LH-20 (chloroform-methanol, 1:1) were repeatedly purified, and two compounds were isol...
Embodiment 2
[0024] Example 2. Anti-complement classical pathway test in vitro
[0025] Take 0.1ml of complement (guinea pig serum), add barbiturate buffer solution (BBS) to prepare a 1:5 solution, and double-dilute with BBS to 1:10, 1:20, 1:40, 1:80, 1: 160, 1:320 and 1:640 solutions. Take 1:1000 hemolysin, 0.1ml of each concentration of complement and 2% sheep red blood cells (SRBC) and dissolve them in 0.3ml of BBS, mix well, put them in a low-temperature high-speed centrifuge at 5000rpm, 4°C after 30min in 37°C water bath Centrifuge for 10 min under conditions. Take 0.2ml of the supernatant from each tube and place it in a 96-well plate, and measure its absorbance at 405nm. A full hemolysis group (0.1ml 2% SRBC dissolved in 0.5ml triple distilled water) was also set up in the experiment. The absorbance of three-distilled water lysed blood vessels was used as the standard of total hemolysis, and the hemolysis rate was calculated. Taking the dilution of complement as the X-axis, the ...
Embodiment 3
[0026] Example 3. Anti-complement alternative pathway test in vitro
[0027] Take 0.2ml of complement (human serum), add AP to dilute (barbital buffer, pH=7.4, containing 5mM Mg 2+ , 8mM EGTA) solution was prepared into a 1:5 solution, and double-diluted into 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 and 1:640 solutions. Take 0.15ml of complement of each concentration, 0.15ml of AP diluent and 0.20ml of 0.5% rabbit erythrocytes (RE), mix well, put in a low-temperature high-speed centrifuge after 30 minutes in a 37°C water bath, and centrifuge at 5000rpm and 4°C for 10 minutes. Take 0.2ml of the supernatant from each tube and place it in a 96-well plate, and measure the absorbance at 405nm. A full hemolysis group (0.20ml 0.5% RE dissolved in 0.3ml triple distilled water) was also set up in the experiment. The absorbance of three-distilled water lysed blood vessels was used as the standard of total hemolysis, and the hemolysis rate was calculated. Taking the dilution of complement...
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