Preparation method and application of LLVV-Hemorphin-6 peptide
A peptide chain and protease technology, applied in the field of medicinal chemistry, can solve the problems of low yield, different active peptides in enzymolysis time, and uncertain enzymolysis products, and achieve simple extraction and purification process, enhanced controllability and reproducibility. Good results
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Embodiment 1
[0029] (1) Activation of protease: Protease clostripain 3mg / mL was dissolved in tris (Tris) buffer solution containing dithiothreitol and calcium chloride, activated at 4°C;
[0030] (2) Hemoglobin was dissolved in Tris buffer solution with a concentration of 20 mg / mL. Mix hemoglobin with activated protease solution and hydrolyze at 55°C for 4 hours; analyze the components and molecular weight in the hydrolyzate by HPLC to determine the yield of the target peptide peak time, see figure 1 .
[0031] (3) The LLVV-Hemorphin-6 peptide sample was extracted from the hydrolyzate, and the product was freeze-dried to obtain the product. According to HPLC analysis, the content of LLVV-Hemorphin-6 in the system was 82%.
Embodiment 2
[0033] (1) Activation of protease: Protease clostripain8mg / mL was dissolved in Tris buffer solution containing dithiothreitol and calcium chloride, activated at 25°C;
[0034] (2) Hemoglobin was dissolved in Tris buffer solution at a concentration of 1 mg / mL, mixed with the activated protease solution, and enzymatically hydrolyzed at 4°C for 30 hours.
[0035] (3) The LLVV-Hemorphin-6 peptide sample was extracted from the hydrolyzate, and the product was obtained by freeze-drying. According to HPLC analysis, the content of LLVV-Hemorphin-6 in the system was 85%.
Embodiment 3
[0037] (1) Activation of protease: Protease clostripain 0.5mg / mL was dissolved in Tris buffer solution containing dithiothreitol and calcium chloride, activated at 30°C;
[0038] (2) Hemoglobin was dissolved in Tris buffer solution at a concentration of 40 mg / mL, and the hemoglobin and activated protease solution were mixed for enzymatic hydrolysis at 37°C for 15 hours.
[0039] (3) The LLVV-Hemorphin-6 peptide sample was extracted from the hydrolyzate, and the product was obtained by freeze-drying. According to HPLC analysis, the LLVV-Hemorphin6 content in the system was 87%.
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