Sipunculus nudus plasmin and preparation method thereof

A technology of phalanx and plasmin, which is applied in biochemical equipment and methods, enzymes, pharmaceutical formulas, etc., can solve the problems of cumbersome extraction process, complex components of snake venom, slow onset of action, etc., and achieve simple extraction process and preparation Low cost, good effect

Inactive Publication Date: 2010-11-24
GUANGXI MEDICAL UNIVERSITY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

The first and second-generation drugs for thrombolysis dissolve thrombus by activating plasminogen, converting it into plasmin, hydrolyzing fibrin, the main component of thrombus, and generating fibrin degradation products, but in the long-term formation of blood clots And contractile blood clots, such as peripheral arterial occlusion and deep vein thrombosis, the lack of plasminogen in the clot, the thrombolytic effect is limited
The third-generation thrombolytic drug defibrase, which is currently mainly used clinically, is a serine protease extracted from the venom of Agkistrodon akistrodon, also known as thrombin-like enzyme, which can decompose plasma fibrinogen and generate fibrin It is soluble and non-cross-linked, and achieves anticoagulant and thrombolytic effects, but the composition of snake venom is very complex, and the hemorrhagic toxin and neurotoxin contained in it may not be completely removed during the production and preparation process, which will be brought into the product and become a cause of death. Main causes of various side effects
Lumbrokinase, another commonly used third-generation thrombolytic drug, is a serine protease with kinase and plasmin activities. Since the activity of the enzyme varies greatly with the type of earthworm and the separation process, its extraction process is very different. It is cumbersome and difficult to develop into intravenous injections. At present, it is mainly capsule preparations made of a variety of isozyme mixtures, such as Puenfu, Bio, Bolock, Thrombolytic Capsules, etc., which have weak effects and slow onset of action

Method used

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  • Sipunculus nudus plasmin and preparation method thereof
  • Sipunculus nudus plasmin and preparation method thereof

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preparation example Construction

[0021] The preparation process steps of P. kaleidoscope fibrinolytic enzyme are as follows:

[0022] a. Separation and extraction of plasmin from A. chevron:

[0023] Wash the fresh starworm, take the body cavity fluid and visceral tissue, add 2 times the volume of pre-cooled pH 6-8 buffer solution, homogenate at high speed, centrifuge, take the supernatant, and select the condensate with a molecular weight separation range of 27,000 to 35,000. Gel filter packing and anion exchange packing capable of adsorbing plasminus plasmin in the pH range of 6 to 8 were used for chromatographic separation, and the components with plasmin activity were collected; the purity and Molecular weight, isoelectric point determined by isoelectric focusing electrophoresis.

[0024] B. Determination of plasmin activity and its fibrinolytic property identification with fibrin plate method: take urokinase as reference substance, and fibrin plate method measures plasmin activity and vitality; Urokinas...

Embodiment 1

[0028] 1. Homogenate: Take 500 grams of fresh Starworm, wash, take body cavity fluid and visceral tissue, add 2 times the volume of pre-cooled pH7.0, 0.02mol L -1 Na 2 HPO 4 -NaH 2 PO 4 The buffer solution was placed in a tissue homogenizer for homogenization, and the homogenate was centrifuged in a refrigerated centrifuge for 20 minutes (4°C, 8000rm), the precipitate was discarded, and the supernatant was collected.

[0029] 2. Column chromatography: take the above supernatant on Sephadex G-75 column, use pH7.00.02mol L -1 Na 2 HPO 4 -NaH 2 PO 4 Equilibrium elution with buffer solution to obtain 4 fractions, collect the second fraction with fibrinolytic activity on DEAE-Sepharose CL-6B column, use pH7.0 0.02mol·L -1 Na 2 HPO 4 -NaH 2 PO 4 Equilibrate elution with buffer solution, and then use 0.05~0.5mol·L -1 The NaCl buffer was used for linear gradient elution, and 6 fractions were collected. The fourth fraction had fibrinolytic activity. -1 Na 2 HPO 4 -N...

Embodiment 2

[0034] 1. Homogenate: Take 500 grams of fresh Starworm, wash, take body cavity fluid and visceral tissue, add 2 times the volume of pre-cooled pH7.5, 0.02mol L -1 Na 2 HPO 4 -NaH 2 PO 4 The buffer solution was placed in a tissue homogenizer for homogenization, and the homogenate was centrifuged in a refrigerated centrifuge for 20 minutes (4°C, 8000rm), the precipitate was discarded, and the supernatant was collected.

[0035] 2. Column chromatography: take the above supernatant on Sephacryl S-100HR column, use pH7.50.02mol L -1 Na 2 HPO 4 -NaH 2 PO 4 Equilibrium elution with buffer solution to obtain 4 fractions, collect the second fraction with fibrinolytic activity on DEAE-Sepharose F.F. column, use pH7.50.02mol·L -1 Na 2 HPO 4 -NaH 2 PO 4 Equilibrate elution with buffer solution, and then use 0.05~0.5mol·L -1 The NaCl buffer was used for linear gradient elution, and 7 fractions were collected. The fifth fraction had fibrinolytic activity. -1 Na 2 HPO 4 -...

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Abstract

The invention relates to sipunculus nudus plasmin which is extracted from sipunculus nudus and has double activities of directly dissolving fibrin and kinase. The sipunculus nudus has the materialization performances that the molecular weight ranges from 27000 to 35000, the isoelectric point ranges from 4.5 to 7.0, the dissolving activity of fibrin varies little within the temperature range of 30 to 70 degrees centigrade, and the stability range of the pH value ranges from 5.0 to 9.0. The sipunculus nudus plasmin has double activities of directly dissolving the fibrin and the kinase, simple extraction process, low preparation cost and easily obtained materials, can be developed into vein injection, contains the plasmin for directly degrading thrombus and is expected to have better effect on treating periphery arterial occlusion and deep vein thrombosis in a conduit-assisted thrombolytic therapy.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular fibrinolytic enzyme of the spirochete and its preparation method. Background technique [0002] Thromboembolic disease is one of the diseases that seriously threaten human health and even life today. This type of disease is closely related to blood coagulation factors and coagulated fibrin. Drug thrombolysis is currently the most widely used and most effective treatment in clinical practice. There have been three generations of thrombolytic drugs currently in clinical use. The first generation of antifibrinolytic and thrombolytic drugs are represented by streptokinase SK and urokinase UK, which have strong effects but lack specificity. Fibrinogen degrades blood fibrinogen at the same time, leading to serious adverse reactions such as bleeding; the typical representative of the second-generation thrombolytic drug is tissue plasminogen activator (t-PA), the main advantage is that it intera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/68A61K38/48A61P7/02
Inventor 雷丹青廖共山李肖肖唐景财
Owner GUANGXI MEDICAL UNIVERSITY
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