Method for extracting single component batroxobin from Bothrops atrox poison

A technology of batroxobin and Agkistrodon halys, applied in the field of batroxobin, can solve the problems that the literature process is difficult to meet the separation and preparation requirements, it is difficult to achieve the purpose of large-scale production, and it cannot be used in actual production, so as to achieve easy control and convenience. Operation, the effect of a large amount of snake venom

Inactive Publication Date: 2009-07-01
QILU PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] To sum up, it is difficult to achieve the ideal separation and preparation requirements of the literature process, to obtain a single component product, or it is difficult to achieve the purpose of large-scale production, so it cannot be used in actual production

Method used

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  • Method for extracting single component batroxobin from Bothrops atrox poison

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The concrete steps of this embodiment are as follows:

[0041] 1. Take 3g Agkistrodon venom, dissolve it in 60ml Tris-HCL buffer solution with pH8.6 containing 1.0M NaCl, soak it in a refrigerator at 4°C to fully dissolve it, then centrifuge at 8000rpm for 10min, remove the precipitate, and collect the supernatant of the snake venom ;

[0042] 2, Benzamidine Sepharose 6B (Benzamidine Sepharose 6B) affinity chromatography column (2.6cm * 30cm) equilibrates at least 5 column volumes with the Tris-HCL buffer solution containing 1.0M NaCL of pH8.6, and above-mentioned snake venom Put the supernatant on the column and control the flow rate to 0.3ml / min;

[0043] 3. After loading the sample, use Tris-HCL buffer solution containing 1.0M NaCl at pH 8.6 to elute until the reading of the nucleic acid protein detector reaches the baseline;

[0044] 4. Connect the affinity chromatography column to Sephadex G25 desalting column (1.6cm×100cm), use pH8.6 containing 0.2M NaCL and 0.1...

Embodiment 2

[0049] The concrete steps of this embodiment are as follows:

[0050] 1. Take 5g Agkistrodon venom, dissolve it in 80ml Tris-HCL buffer solution with pH 9.0 containing 0.6M NaCl, soak it in a refrigerator at 4°C to fully dissolve it, then centrifuge at 8000rpm for 10min, remove the precipitate, and collect the supernatant of the snake venom ;

[0051] 2, Benzamidine Sepharose 6B (Benzamidine Sepharose 6B) affinity chromatography column (2.6cm * 30cm) equilibrates at least 5 column volumes with the Tris-HCL buffer solution containing 0.6M NaCL of pH9.0, and above-mentioned snake venom Put the supernatant on the column and control the flow rate to 0.3ml / min;

[0052] 3. After loading the sample, use Tris-HCL buffer solution containing 0.6M NaCl at pH 9.0 to elute until the reading of the protein detector reaches the baseline;

[0053] 4. Connect the affinity chromatography column to Sephadex G25 desalting column (1.6cm×100cm), use pH 9.0 containing 0.1M NaCL and 0.2M benzamidi...

Embodiment 3

[0058] The concrete steps of this embodiment are as follows:

[0059] 1. Take 5g Agkistrodon venom, dissolve it in 80ml Tris-HCL buffer solution with pH8.0 containing 0.6M NaCl, soak it in a refrigerator at 3°C ​​to fully dissolve it, then centrifuge at 8000rpm for 10min, remove the precipitate, and collect the supernatant of the snake venom ;

[0060] 2, Benzamidine Sepharose 4B (Benzamidine Sepharose 4B) affinity chromatography column (2.6cm * 30cm) equilibrates at least 5 column volumes with the Tris-HCL buffer solution containing 0.6M NaCL of pH8.0, and above-mentioned snake venom Put the supernatant on the column and control the flow rate to 0.3ml / min;

[0061] 3. After loading the sample, elute with pH 8.0 Tris-HCL buffer containing 0.6M NaCl until the reading of the protein detector reaches the baseline;

[0062] 4. Connect the affinity chromatography column to Sephadex G25 desalting column (1.6cm×100cm), use pH 8.0 containing 0.3M NaCL and 0.15M benzamidine hydrochlo...

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Abstract

The invention discloses a method for extracting batroxobin with fibrinolytic and thrombolytic effect or hemostatic effect from snake venom, which belongs to the technical field of medicine. The steps are as follows: the snake venom is dissolved by soaking and centrifuged; the supernatant is subjected to affinity column chromatography; the eluate is directly flowed through Sephadex G25 desalting chromatography; the eluate containing the active ingredient is embedded and concentrated; The concentrated sample is subjected to molecular sieve chromatography to obtain batroxobin with a single component. The method has the characteristics of being suitable for large-scale production, simple and convenient operation, short production cycle, high product purity and the like.

Description

technical field [0001] The invention relates to a method for extracting batroxobin with fibrinolytic, thrombolytic or hemostatic effects from the venom of two subspecies of Agkistrodon halys (Bothrops moojeni or Bothrops atrox), and belongs to the technical field of medicines. Background technique [0002] Snake venom is rich in proteolytic enzymes, and there are two main types. One is snake venom-like thrombin, which causes blood coagulation in vitro, but it is a component of defibrillation and anticoagulation in vivo; the other is snake venom fibrinolytic enzyme, which can Directly degrade blood fibers, and most of them can also degrade fibrinogen. Snake venom thrombin is a class of enzymes that have been studied earlier and have been widely used clinically. [0003] Snake venom thrombin has been used as a drug for the treatment of thrombotic diseases for more than 30 years, and it has two clinical uses. One is the defibrosis effect, namely batroxoblastase, such as Dongl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/68C12N9/74
Inventor 刘禄娟张润平于萍张明会王晶翼
Owner QILU PHARMA CO LTD
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