Method for extracting high-anticoagulation-activity hirudin from natural leeches

A technology of hirudin and leech, which is applied to the preparation method of peptides, from leech inhibitors, chemical instruments and methods, etc., can solve the problem of unstable activity index, inability to form batch production extraction, extraction technology art restricts the wide application of hirudin, etc. problem, to achieve the effect of high raw material utilization

Inactive Publication Date: 2011-02-16
南宁市和兰记生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The yield of the obtained product is generally below 20%, the anticoagulant activity is maintained at about 100AT-U / g, and the activity index is unstable, and it cannot be extracted in batch production. It can only provide some research data for a small number of clinical applications and laboratories.
The backwardness of extraction technology has seriously restricted the wide application of hirudin in the field of medicine

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Dissolve 5g of sodium chloride, 0.3g of acetylcysteine ​​hydrochloride monohydrate, 5g of lysine, 100U of neutral protease, and 150U of papain in 1000mL of deionized water to prepare into a stimulating fluid. The live leech is weighed and placed in a container, and the stimulating solution is added to 1.25 times the weight of the leech, and left for 1 hour to stimulate the leech to secrete the secretion. After the stimulation was completed, the living leeches were fished out and continued to be reared so as to be reused. The secretions are successively subjected to centrifugal separation at 20,000r / min, organic membrane ultrafiltration with a molecular weight of 10,000 Daltons, and reverse osmosis nanofiltration to concentrate, and finally add medicinal starch according to the weight-to-volume ratio of solid to liquid at a ratio of 1:6 to 10, and spray Dry to get hirudin finished product.

Embodiment 2

[0015] Dissolve 15g of sodium chloride, 0.2g of acetylcysteine ​​hydrochloride monohydrate, and 300U of papain in 1000mL of deionized water to prepare a stimulating solution. The live leech is weighed and placed in a container, and the stimulating solution is added to 1.25 times the weight of the leech, and left for 1 hour to stimulate the leech to secrete the secretion. After the stimulation was completed, the living leeches were fished out and continued to be reared so as to be reused. The secretions are successively subjected to centrifugal separation at 18,000r / min, ultrafiltration of an organic membrane with a molecular weight of 7,000 Daltons, and concentration by reverse osmosis nanofiltration. Finally, medicinal starch is added according to the weight-to-volume ratio of solid to liquid at a ratio of 1:6 to 10. Spray drying to obtain hirudin finished product.

Embodiment 3

[0017] Fully dissolve 25g of sodium chloride, 0.1g of acetylcysteine ​​hydrochloride monohydrate, 0.5g of lysine, and 250U of neutral protease in 1000mL of deionized water to prepare a stimulating solution. The live leech is weighed and placed in a container, and the stimulating solution is added to 1.25 times the weight of the leech, and left for 1 hour to stimulate the leech to secrete the secretion. After the stimulation was completed, the living leeches were fished out and continued to be reared so as to be reused. The secretions are successively separated by centrifugation at 17000r / min, organic membrane ultrafiltration with a molecular weight of 4000 Daltons, and concentration by reverse osmosis nanofiltration. Finally, medicinal starch is added according to the weight-to-volume ratio of solid to liquid at a ratio of 1:6 to 10. After spraying Dry to get hirudin finished product.

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Abstract

The invention relates to a method for extracting high-anticoagulation-activity hirudin from natural leeches, which comprises the steps that two kinds or a plurality of kinds of substances in sodium chloride, acetyl cysteine hydrochloride monohydrate, lysine, neutral protease and papain are combined and dissolved in deionized water to be prepared into stimulation liquid; the weight percentage concentration of each substance in the stimulation liquid is 0.01 to 5%, wherein the enzyme concentration is 100U to 300U/ml; the living leeches are weighed and placed into a container; the stimulation liquid is added according to the weight and volume ratio of the leeches to the stimulation liquid of 1 to (1-5); the leeches secrete exudate after the stimulation is carried out for 3 hours; the exudateis separated to obtain the hirudin crude product through 16000 to 20000r/min centrifugation; the hirudin crude product is micro-filtered through a stainless steel membrane with the thickness of 0.2 micrometer; the micro-filtration is carried out through an organic membrane with the molecular weight of 10000 to 3000 Dalton; the reverse osmosis, the nano-filtration and the concentration are carriedout; starch for drugs is added according to the weight and volume ratio of the solid to the liquid of 1 to (6-10) to allocate the mixture; and the spray drying is carried out to obtain the leech finished product. Finally, the extraction yield of the hirudin is above 30%, and the activity unit of the product is stabilized between 600AT-U/g to 1000AT-U/g.

Description

technical field [0001] The invention relates to a method for extracting hirudin with high anticoagulant activity from natural leeches. Background technique [0002] Hirudin is a single-chain polypeptide composed of 65 amino acid residues, with a relative molecular weight of 7000 and an isoelectric point of 3.9. It is soluble in water, normal saline and pyridine, but insoluble in ethanol, ether, acetone and benzene. Therefore, the extraction, separation and purification of hirudin can be carried out according to its physical and chemical properties and the basic principles of general protein purification. According to the characteristics of molecular size, solubility, adsorption, charge, and ligand special affinity, the crude product is extracted by physiological saline, ethanol fractional precipitation and dialysis, and the crude product is extracted by gel chromatography, ion exchange chromatography, and high performance liquid. Phase chromatography, isoelectric focusing a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/34C07K14/815
Inventor 苏国强
Owner 南宁市和兰记生物科技有限公司
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