Preparation method of whole leech meal rich in high-activity hirudins

A technology of hirudin and high activity, which is applied in the field of preparation of leech powder, can solve problems such as allergic reactions, achieve mild conditions and improve anticoagulant activity

Inactive Publication Date: 2015-09-16
广东益安生物股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hirudin can be taken orally, and high-purity hirudin can also be used for intravenous injection, but intravenous injectio...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1. Get 20 g of fresh Hirudo phenanthae, rinse with clear water to remove impurities, immerse in 100 ppm chlorine dioxide aqueous solution for disinfection for 3 minutes, drain and set aside; put Hirudo phenanthae sterilized into a homogenizer, add 40 g of sterile water Homogenize; pour the homogenate into a beaker, add 0.22g of chitinase and 0.1g of collagenase, and hydrolyze for 1.0 h at a pH value of 5.0 and a temperature of 37°C. Inactivate the enzyme at 90°C for 10 minutes. After cooling, adjust the pH value to 7 with 2mol / L NaOH solution, pre-cool at -30°C for 240 minutes, and then carry out vacuum freeze-drying to obtain a whole powder of leeches rich in highly active hirudin. Packaged in a thin tin bag. The anticoagulant activity of the powder was detected to be 580AT-U / g.

Embodiment 2

[0018] Embodiment 2, get 20 g of fresh Hirudo phenanthae, rinse with clear water to remove impurities, immerse in 50 ppm chlorine dioxide aqueous solution for disinfection for 5 minutes, drain and set aside; put the sterilized Hirudo phenanthae into a homogenizer, add 60 g of sterile Homogenize with water; pour the homogenate into a beaker, add 0.25g of chitinase and 0.12g of collagenase, and hydrolyze for 2 hours at a pH value of 4.0 and a temperature of 40°C. Inactivate the enzyme at 90°C for 10 minutes. After cooling, adjust the pH value to 7 with 2mol / L NaOH solution, pre-cool at -30°C for 180 min, and then vacuum freeze-dry to obtain a whole leech powder rich in highly active hirudin. Packaged in a thin tin bag. The anticoagulant activity of the powder was detected to be 560AT-U / g.

Embodiment 3

[0019] Embodiment 3, get 20 g of fresh Hirudo phenanthae, rinse with clear water to remove impurities, immerse in 75ppm chlorine dioxide aqueous solution for disinfection for 4min, drain and set aside; put the sterilized Hirudo phenanthae into a homogenizer, add 50 g of sterile Homogenate with water; Pour the homogenate into a beaker, add 0.20g of chitinase and 0.12g of collagenase, and hydrolyze for 1.5h at a pH value of 4.0 and a temperature of 35°C, and the body of B. phenanthrene will dissolve. Heat up to 90°C to inactivate the enzyme for 10 minutes. After cooling, adjust the pH value to 7 with 2mol / L NaOH solution, pre-cool at -30°C for 240 minutes, and then carry out vacuum freeze-drying to obtain a whole powder of leeches rich in highly active hirudin. Packaged in a thin tin bag. The anticoagulant activity of the powder was detected to be 552AT-U / g.

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Abstract

The invention discloses a preparation method of whole leech meal rich in high-activity hirudins. The preparation method comprises the following steps: by taking fresh poecilobdella manillensis as a raw material, washing by clear water to remove impurities, soaking in 50-100ppm of chlorine dioxide aqueous solution and disinfecting for 3-5min, adding sterile water which is 2-3 times of the weight of poecilobdella manillensis and homogenizing, then hydrolyzing using chitinase and collagenase for 1-2h under the conditions of a pH value of 4.0-5.0 and a temperature of 35-40 DEG C until poecilobdella manillensis bodies are dissolved, heating to 90 DEG C and carrying out enzyme deactivation for 10min, cooling and then adjusting the pH value to 7, then carrying out vacuum freeze drying to obtain the whole leech meal. By using the double-enzyme method, the whole bodies comprising salivary glands are hydrolyzed, so that all hirudins can become free states, all the hirudins in leech bodies are released, and the absorption and utilization of human body on the hirudins are prompted. Meanwhile, the whole leech meal contains other active ingredients in the various tissues and saliva of leeches, and improvement for the anti-coagulant activity of the hirudins is promoted.

Description

technical field [0001] The invention relates to a preparation method of leech whole powder rich in highly active hirudin. Background technique [0002] Leeches are a traditional Chinese medicine with a long history. Chinese medicine believes that leeches have the functions of breaking blood, removing stasis, and stimulating menstruation. Since hirudin, a substance with anticoagulant effect, was isolated from medical leeches in the 1950s, extensive and in-depth pharmacological studies have been carried out at home and abroad. Hirudin is the strongest thrombin inhibitor, and can prevent fibrin from coagulating, and is effective for various thrombosis diseases. [0003] Natural hirudin is a single-chain small-molecule acidic single-chain polypeptide with 65 or 66 amino acid residues and no glycosylation extracted from the secretion of leech salivary glands. At present, it has been found that there are more than ten variants of natural hirudin, and there are only a few amino a...

Claims

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Application Information

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IPC IPC(8): A61K35/62A61K38/58A61K9/19A61P7/02
Inventor 方练方卓榕余以刚
Owner 广东益安生物股份有限公司
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