Phascolosoma esculenta plasmin and preparation method thereof

A technology of Astrocystis and plasmin, which is applied in the field of plasmin and its preparation, can solve the problems of cumbersome extraction process, complex components, strong hemorrhagic toxicity, etc., and achieve low preparation cost and high extraction The effect of simple process

Inactive Publication Date: 2011-04-06
GUANGXI MEDICAL UNIVERSITY
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Problems solved by technology

Thrombolytic drugs currently used in clinical practice mainly include kinases, defibrinolytics, and direct fibrinolytics. Kinases are represented by streptokinase SK and Urokinase UK. The zymogen converts it into plasmin, hydrolyzes fibrin, the main component of thrombus, and generates fibrin degradation products, thereby dissolving thrombus; its effect is strong, but lacks specificity. Peripheral arterial occlusion and deep vein thrombosis, lack of plasminogen in blood clots, limited thrombolytic effect, easily lead to serious adverse reactions such as bleeding
Defibranase is represented by snake venom defibrase (thrombin-like), which can decompose plasma fibrinogen and generate non-cross-linked soluble fibrin to achieve anticoagulant effect, but the thrombolytic effect is weak; and the composition of snake venom is relatively complex, and its The hemorrhagic toxin and neurotoxin contained in it may not be completely removed during the production and preparation process, and thus brought into the product and become the main cause of various toxic and side effects
Direct fibrinolytics are represented by snake venom fibrinolytic enzyme and lumbrokinase, which have not been used clinically due to their strong hemorrhagic toxicity; lumbrokinase is a serine protease with kinase and plasmin activity. The activity of different earthworms varies greatly with the different types of earthworms and the separation process. Its extraction process is very cumbersome, and it is difficult to develop into intravenous injections. At present, it is mainly capsule preparations made of a variety of isozyme mixtures, such as Puenfu, BIO, Bollock, Thrombolytic Capsules, etc. have weak effects and slow onset

Method used

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  • Phascolosoma esculenta plasmin and preparation method thereof
  • Phascolosoma esculenta plasmin and preparation method thereof
  • Phascolosoma esculenta plasmin and preparation method thereof

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preparation example Construction

[0026] The preparation process steps of Dermatosus plasmolytic enzyme are:

[0027] a. Separation and extraction of plasminase from Derma cystis:

[0028] Wash the fresh Dermocystus, take body cavity fluid and visceral tissue, add pre-cooled pH 6-8 buffer solution according to the conventional ratio of extracting plasmin, homogenate at high speed, centrifuge, take supernatant, select the molecular weight separation range at 2.7 ~35,000 gel filtration fillers and anion exchange fillers that can absorb P. plasminus in the pH range of 6~8 were used for chromatographic separation to collect components with plasminase activity; through SDS-polyacrylamide coagulation The purity and molecular weight were determined by gel electrophoresis, and the isoelectric point was determined by isoelectric focusing electrophoresis.

[0029] B. Determination of plasmin activity and its fibrinolytic property identification with fibrin plate method: take urokinase as reference substance, and fibrin...

Embodiment 1

[0037] 1. Homogenate: Take 100 grams of fresh Asteroides and wash, take body cavity fluid and visceral tissue, add 2 times the volume of pH8.0, 0.02mol L -1 Na 2 HPO 4 -NaH 2PO 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.

[0038] 2. Column chromatography: take the above supernatant and put it on the QAE-Sephadex A-25 column, use pH8.0 0.02mol L -1 Na 2 HPO 4 -NaH 2 PO 4 Equilibrium elution with buffer, and then with 1.0mol·L -1 The NaCl buffer was eluted, and the fraction containing fibrinolytic activity was collected on a Sephacryl S300 column with a pH of 8.00.02mol L -1 Na 2 HPO 4 -NaH 2 PO 4 Buffer for elution, collect the second fraction containing fibrinolytic activity on the QAE-Sephadex A-25 column, use pH8.00.02mol·L -1 Na 2 HPO 4 -NaH 2 PO 4 Equilibrate elutio...

Embodiment 2

[0043] 1. Homogenate: Take 100 grams of fresh Dermocystus and wash, take visceral tissue, add 2 times the volume of 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.

[0044] 2. Column chromatography: put the above supernatant on a DEAE-Sepharose CL-6B column, and use pH7.5 0.02mol L -1 Na 2 HPO 4 -NaH 2 PO 4 Equilibrium elution with buffer, and then with 1.0mol·L -1 Elute with NaCl buffer, collect the fibrinolytic active fraction on Sephadex G-100 column with pH7.5 0.02mol L -1 Na 2 HPO 4 -NaH 2 PO 4 Buffer for elution, collect the second fraction containing fibrinolytic activity on the DEAE-Sepharose CL-6B column, use pH7.5 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 Th...

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Abstract

The invention provides phascolosoma esculenta plasmin, which is extracted from phascolosoma esculenta, and has double activities of directly dissolving fibrin and kinase. The phychemical performance of the plasmin is that: the molecular weight is between 28,000 and 32,000, the isoelectric point is between 4.6 and 6.6, the dissolution activity of fibrin does not change greatly under a temperature range between 30 and 70 DEG C, and the stability range to the pH value is between 5.0 and 9.0. The phascolosoma esculenta plasmin has the direct fibrin and kinase dissolving activities, has simple extracting process, low preparation cost and easily obtained raw materials, can be developed into intravenous injection, contains the plasmin capable of directly dissolving thrombus, and is expected to obtain better effect in treating catheter assisted thrombolysis treating peripheral arterial occlusion and deep venous thrombosis.

Description

technical field [0001] The invention belongs to the field of biomedicine, especially the fibrinolytic enzyme of Dermatocystis 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. Thrombolytic drugs currently used in clinical practice mainly include kinases, defibrinolytics, and direct fibrinolytics. Kinases are represented by streptokinase SK and Urokinase UK. The zymogen converts it into plasmin, hydrolyzes fibrin, the main component of thrombus, and generates fibrin degradation products, thereby dissolving thrombus; its effect is strong, but lacks specificity. Peripheral arterial occlusion and deep vein thrombosis, lack of plasminogen in blood clots, limited thrombolytic e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/68
Inventor 廖共山雷丹青唐景财李肖肖
Owner GUANGXI MEDICAL UNIVERSITY
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