Leech extract and preparation method and use thereof
A leech extract and leech technology, applied in the direction of drug combination, medical raw materials derived from leeches/worms, extracellular fluid diseases, etc., can solve the problems of unclear active ingredients, loss of active ingredients, complex ingredients, etc., to improve activity and quality controllability, improved utilization, and strong biological activity
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Embodiment 1
[0030] Embodiment 1: prepare leech extract, the steps are as follows:
[0031] (1) Get the dried leeches and grind them into 100 mesh powder, add 8 times the amount of water, and homogenate;
[0032] (2) Take the homogenate, adjust the pH to 8.5, add trypsin at a ratio of 5000U of enzyme per 1g of substrate, control the temperature at 50°C, enzymatically hydrolyze for 6 hours, and cool to room temperature;
[0033] (3) Add 2 times the volume of acetone to the enzymolysis solution for refrigerated precipitation, filter, take the filtrate to recover under reduced pressure below 60°C, add water to dissolve the residue, and filter through a 0.45 micron microporous membrane;
[0034] (4) passing the above-mentioned medicinal liquid through an ultrafiltration membrane with a molecular cut-off of 5000Da, and collecting the part with a molecular weight less than 5000Da;
[0035] (5) Adjust the pH of the ultrafiltrate to 8.0, pass through Q Sepharose resin, rinse with water first, dis...
Embodiment 2
[0036] Embodiment 2: prepare leech extract, the steps are as follows:
[0037] (1) Get fresh and alive whole body of leeches, add 2.5 times the amount of water, and homogenate;
[0038] (2) Take the homogenate, adjust the pH to 8.5, add trypsin and trypsin at a ratio of 5000 U of enzyme per 1 g of substrate, wherein trypsin provides protease activity accounting for 2% of the total protease activity, control the temperature at 50°C, and perform enzymatic hydrolysis 8 hours, cooled to room temperature;
[0039] (3) Add 3 times the volume of ethanol refrigerated precipitation to the enzymolysis solution, filter, take the filtrate and recover it under reduced pressure until it has no alcohol smell, add 2 times the volume of acetone refrigerated precipitation, filter, take the filtrate below 60 °C and recover under reduced pressure, the residue Add water to dissolve, filter with 0.45 micron microporous membrane;
[0040] (4) passing the above-mentioned medicinal liquid through an u...
Embodiment 3
[0042] Embodiment 3: prepare leech extract, the steps are as follows:
[0043] (1) Get fresh and alive whole body of leeches, add 2.5 times the amount of water, and homogenate;
[0044] (2) Take the homogenate, adjust the pH to 8.0, add trypsin at a ratio of 10000U of enzyme per 1g of substrate, control the temperature at 50°C, enzymatically hydrolyze for 4 hours, and cool to room temperature;
[0045] (3) Add 3 times the volume of ethanol to the enzymatic hydrolysis solution to refrigerate and precipitate, filter, take the filtrate and recover it under reduced pressure below 60°C, add water to dissolve the residue, and filter through a 0.45 micron microporous membrane;
[0046] (4) passing the above-mentioned medicinal liquid through an ultrafiltration membrane with a molecular cut-off of 5000Da, and collecting the part with a molecular weight less than 5000Da;
[0047] (5) Adjust the pH of the ultrafiltrate to 7.9, pass through DE-52 resin, wash with pH7.9, 0.02mol / L Tris-h...
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