Preparation method of protein active peptide of limulus blood hemocytes

A protein activity and blood cell technology, which is applied in the field of preparation of Limulus blood cell protein active peptides, can solve the problems of failing to provide experimental data on the inhibition rate of acetylcholinesterase, failing to produce acetylcholinesterase, and unable to implement specific applications, etc. The effect of promoting application value, easy mass production, and not cumbersome steps

Active Publication Date: 2018-12-04
湛江博康海洋生物有限公司
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AI Technical Summary

Problems solved by technology

The limulus blood proteolytic peptide obtained by this method has higher DPPH free radical scavenging capacity and total antioxidant capacity, but fails to produce acetylcholinesterase or fails to provide experimental data on the inhibition rate of acetylcholinesterase, and does not provide specific Peptide yield, so it cannot be applied to drugs and health products for Alzheimer's disease or specific drug applications

Method used

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  • Preparation method of protein active peptide of limulus blood hemocytes
  • Preparation method of protein active peptide of limulus blood hemocytes

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Experimental program
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Effect test

Embodiment 1

[0028] 1. Limulus blood cells were frozen at -18°C for 12 hours, then thawed at 4°C, and repeated freezing and thawing three times.

[0029] 2. Stir the Limulus blood cells after freezing and thawing three times at 4°C for 10 minutes to cause the Limulus blood cells to rupture.

[0030] 3. Keep the temperature at 4°C, centrifuge the ruptured Limulus blood cells at a low temperature of 4000 rpm for 20 minutes, collect the solid precipitate, and wash it with distilled water several times to obtain the Limulus blood cell membrane tissue.

[0031] 4. Use trypsin powder with an enzyme activity of 4000u / g to enzymolyze the Limulus blood cell membrane tissue obtained above. Enzymolysis conditions: pH 7.0, 70°C, enzyme concentration 2.2 mg / mL, substrate concentration 4.0 mg / mL, enzyme Solution 5h.

[0032] 5. Place the above enzymolysis solution in a low-temperature refrigerated centrifuge, keep the temperature at 4°C, and centrifuge at a speed of 4000rpm for 20min at a low temperatu...

Embodiment 2

[0036] 1. Limulus blood cells were frozen at -18°C for 11 hours, then thawed at 4°C, and repeated freezing and thawing three times.

[0037] 2. Stir the Limulus blood cells after freezing and thawing three times at 4°C for 10 minutes to cause the Limulus blood cells to rupture.

[0038] 3. Keep the temperature at 4°C, centrifuge the ruptured Limulus blood cells at a low temperature of 4500 rpm for 18 minutes, collect the solid precipitate, and wash it with distilled water several times to obtain the Limulus blood cell membrane tissue.

[0039] 4. Enzymolyze Limulus blood cell membrane tissue with papain, enzymolysis conditions: pH 7.0, 70°C, enzyme concentration 2.2 mg / mL, substrate concentration 4.0 mg / mL, enzymolysis 5h.

[0040] 5. Place the above enzymolysis solution in a low-temperature refrigerated centrifuge, keep the temperature at 4°C, and centrifuge at a speed of 4500rpm / min for 18min at a low temperature to obtain a supernatant.

[0041] 6. Keep the temperature at ...

Embodiment 3

[0044] 1. Limulus blood cells were frozen at -18°C, then thawed at 4°C, and repeated freezing and thawing three times.

[0045] 2. Magnetically stir the Limulus blood cells after freezing and thawing three times at 4°C for 10 minutes to cause the Limulus blood cells to rupture.

[0046] 3. Keep the temperature at 4°C, centrifuge the ruptured Limulus blood cells at a low temperature of 3800 rpm for 25 minutes, collect the solid precipitate, and wash it with distilled water several times to obtain the Limulus blood cell membrane tissue.

[0047] 4. Use trypsin to enzymolyze the Limulus blood cell membrane tissue, enzymolysis conditions: pH 7.0, 70°C, enzyme concentration 2.2 mg / mL, substrate concentration 4.0 mg / mL, enzymolysis 5h.

[0048] 5. Place the above enzymolysis solution in a low-temperature refrigerated centrifuge, keep the temperature at 4°C, and centrifuge at a speed of 3800rpm for 25min at a low temperature to obtain a supernatant.

[0049] 6. Keep the temperature at...

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Abstract

The invention relates to a preparation method of a protein active peptide of limulus blood hemocytes, wherein the preparation method particularly comprises the following steps: placing the limulus blood hemocytes at low temperature of -18 DEG C, freezing for 12 h, thawing at the temperature of 4 DEG C, repeating three times, and carrying out magnetic stirring at the temperature of 4 DEG C for 10 min until the hemocytes are ruptured; centrifuging at low temperature of 4 DEG C with the rate of 4000 rpm, to obtain a solid precipitate, and repeatedly flushing the solid precipitate with distilled water to obtain a limulus hemocyte membrane tissue; carrying out enzymolysis of the limulus hemocyte membrane tissue with pancreatin powder having the enzyme activity of 4000 u / g, wherein the enzymolysis conditions comprise that the pH is 7.0, the temperature is 60 DEG C-80 DEG C, the enzyme concentration is 2.2 mg / mL, the substrate concentration is 4.0 mg / mL and enzymatic hydrolysis is performed for 5 h; and carrying out centrifugation separation of the enzymatic hydrolysate at low temperature, to obtain a supernatant, and freeze-drying the supernatant, to obtain the protein active peptide ofthe limulus blood hemocytes. According to the method, a residual waste hemocyte membrane tissue for preparing a limulus reagent is used as a raw material, the utilization rate of the raw material is improved, the application scope of limulus blood is enlarged, scavenging of hydroxyl free radicals and acetylcholinesterase is effectively inhibited, and the protein active peptide can be used for treatment and health care of patients with Alzheimer's disease. The preparation method belongs to the technical field of extraction of natural active substances.

Description

technical field [0001] The invention relates to the technical field of extracting natural active substances, in particular to a preparation method of Limulus hemocytoprotein active peptide. Background technique [0002] With the aging of the population, the incidence of Alzheimer's disease (AD) is increasing year by year, seriously endangering the physical and mental health and quality of life of the elderly, and bringing a heavy burden to the family and society. It has become a serious social problem, causing The general concern of the governments and medical circles of various countries. The exact pathogenic mechanism of Alzheimer's disease (AD) is not yet very clear, and the cholinesterase inhibitors (donezil, galantamine and rivastigmine) that have been developed based on the "cholesterin hypothesis" ) is an important drug in the current treatment and the main means of AD drug treatment. Huperzine A is also commonly used in China as a drug for the treatment of Alzheimer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K2/00A61K38/01A61P25/28
CPCA61K38/00A61P25/28C07K2/00C12P21/06
Inventor 邓春梅洪鹏志康信煌何兰珍张国光
Owner 湛江博康海洋生物有限公司
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