JPS645576B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a novel fibrinolytic active substance from earthworms. Specifically, a fibrinolytic active substance is a crude or purified enzyme protein obtained by adding an aqueous solvent with a pH of 5 to 10 to earthworms, holding the mixture at 5 to 60°C for at least 3 minutes to perform extraction, and concentrating or drying the extract. In recent years, various diseases caused by blood coagulation have been attracting attention as they frequently occur in the elderly and elderly.
For example, myocardial infarction, cerebral thrombosis, disseminated intravascular coagulation syndrome, etc. are well known. It is well known that human urokinase and streptokinase are used as therapeutic agents for these diseases. However, human urokinase has drawbacks such as difficulty in obtaining the raw material (human urine), streptokinase has antigenic properties, and both treatments are administered intravenously, causing great pain to patients. That's a drawback. In order to alleviate and improve the above-mentioned drawbacks, the inventors of the present invention have conducted intensive studies with the aim of eliminating the difficulty in obtaining raw materials and avoiding intravenous infusion. The present invention was completed after learning that fibrinolytic active substances, which are enzyme proteins, can be produced at low cost. The fibrinolytic active substance obtained by the production method of the present invention can be administered orally, parenterally, or as a suppository, but oral administration is particularly preferred. That is, as shown below, it was demonstrated that this fibrinolytic active substance exhibits excellent fibrinolytic activity effects when orally administered to humans. Since ancient times, earthworms have been called worms and earthworms in Eastern countries and have been used as analgesics, hypothermics, diuretics, etc. Furthermore, it has been suggested that fibrinolytic active substances exist in earthworms. However, earthworms have an optimal pH of 8 to 10 and a stable pH of 5 to 10 (especially PH7.5 to 9.0).
Stable when treated at 50℃ for 30 minutes. ), a crude or purified enzyme protein that is inhibited by Trasylol, Transamine, soybean trypsin inhibitor and serum, has plasminogen activation and fibrinolytic activity, and has no fibrinogenolytic activity. There are no reports of separation and purification of dissolved active substances. Next, the method by which the present inventors produce a fibrinolytic active substance from earthworms will be explained in detail. The raw earthworms are fresh raw earthworms, earthworm bodies and their contents after the contents have been removed from fresh raw earthworms, earthworm powders heated, decompressed or freeze-dried, or defatted powders thereof, etc. But it can be used. Most preferred is freeze-dried powder or freeze-dried defatted powder of live earthworms. Aqueous solvent has pH5~
An aqueous solvent with a pH of 6 to 8 is most preferred. For example, water, physiological saline, buffer solutions, preparation solutions or aqueous solvents of one or more thereof, or a small amount of a polar organic solvent such as ethanol, methanol, propanol, acetone, ether, dioxane, etc. Aqueous solvents can be used. The most preferred aqueous solvent is physiological saline, buffer. Buffer solutions include phosphoric acid, acetic acid,
PH5-10 such as boric acid, citric acid, Tris-hydrochloric acid, etc.
Preferably, one or more buffer solutions having various compositions having a pH of 6 to 8 can be used. The prepared liquid having a pH of 5 to 10, preferably 6 to 8, according to the present invention is a water-soluble inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, lactic acid, citric acid, or succinic acid, or an organic acid and an alkali such as sodium or potassium. A dilute aqueous solution prepared in-house from metal hydroxides, carbonates and ammonia. The extraction time is at least 3 minutes or more, particularly preferably from 30 minutes to about 30 days. The extraction time is preferably at a temperature of 60°C or lower, most preferably from 5°C to 40°C. The ratio of the aqueous solvent used to the raw material earthworm is 1 to 100 times, most preferably 5 to 30 times. The extraction method, which involves adding an aqueous solvent to raw earthworms and holding them at an appropriate temperature for an appropriate time, is a particularly useful method for destroying earthworm tissue (cells), in addition to the usual extraction methods such as stirring, shaking, and countercurrent. Preferred is an extraction method in which a suspension (homogenate) of earthworm cell components is prepared and incubated using equipment such as a homogenizer, blender, sonication, pressurized cell disruption device, or crusher. Next, the resulting extract is filtered as it is or kept at an appropriate temperature for an appropriate time, and then concentrated using a concentration method such as reduced pressure, heating, or ultrafiltration, and is subjected to fibrinolysis using a drying method such as reduced pressure or freezing. This is a method for producing a crude active substance. It is preferable to add a small amount of a known preservative during the extraction or incubation of the extract. From this point of view, as shown above, it is meaningful to use an aqueous solvent to which a small amount of a polar organic solvent is added, and the extraction effect can be enhanced. Next, as a method for fractionating and purifying the fibrinolytic active substance from a solution obtained by dissolving the earthworm extract concentrate or its dried product in a small amount of an aqueous solvent, for example, a general method for purifying high molecular weight proteins can be used. A preferred method for fractionating and purifying the fibrinolytic active substance of the present invention is as follows. A solution obtained by dissolving the extracted concentrate or dried product obtained by the above operation in a small amount of an aqueous solvent is used as an adsorbent, a polar organic solvent, salting out, ultrafiltration, ion exchange chromatography, gel filtration, or affinitake. Impurities are removed by one of treatments such as chromatography, hydrophobic chromatography, etc., or an appropriate combination thereof. As the adsorbent used in the method of the present invention, activated carbon, acid clay, activated clay, synthetic resin-based synthetic adsorbents such as Amberlite XAD (trade name), etc. can be used. Further, as the polar organic solvent used for fractional precipitation in the method of the present invention, ethanol, methanol, propanol, acetone, ether, dioxane, etc. can be used. Most preferred are ethanol, acetone and propanol. Salts used for salting out include ammonia sulfate, sodium sulfate, magnesium sulfate, potassium phosphate,
Sodium chloride, potassium chloride, sodium citrate, etc. can be used, but ammonia sulfate is most preferred. Ion exchangers that can be used for ion exchange chromatography are cellulose,
It is based on hydrophilic polysaccharides such as dextran and agarose. In particular, diethylaminoethylcellulose (hereinafter referred to as DEAE-cellulose), triethylaminoethylcellulose (hereinafter referred to as DEAE-cellulose),
It is called TEAE-cellulose. ), aminoethylcellulose (hereinafter referred to as AE-cellulose), carboxymethylcellulose (hereinafter referred to as CM-cellulose), phosphorus-phosphocellulose (hereinafter referred to as P-cellulose), fluorophoremethylcellulose (hereinafter referred to as PPM-cellulose), diethylaminoethylcellulose Ein (hereinafter referred to as DEAE-cellulofine) and the like are preferred. In addition, examples of preferred ion exchange resins by trade name include weakly acidic cation exchange resins such as Amberlite IRC-50, Amberlite IRC-75, Amberlite IRC-84, Dowex CCR-2, and Amberlite.
These are weakly basic anion exchange resins such as IR-4B, IR-45, IRA-400, and Dowex 3. Preferred examples of gel filtration include, by trade name, Cephadex, Cepharose, Biogel, Toyopearl Ultra Gel, Cellulofine, and the like. Furthermore, various types of affinite chromatography using Sepharose or Toyopearl as a support can be used. For hydrophobic chromatography, agarose or cellulose adsorbents having aliphatic, alicyclic, aromatic compounds having 2 to 20 carbon atoms, and compounds having an amino group attached thereto as hydrophobic groups are used. Table 1 shows the manufacturing method of the present invention in the form of a flowchart.
~As shown in Table 16.

【table】
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Claims (1)

[Claims] 1. Add an aqueous solvent with a pH of 5 to 10 to earthworms to
Extract by holding at ℃ for at least 3 minutes, concentrating the extract, and drying.
PH is 8-10, stable PH is 5-10, inhibited by Trasylol (registered trademark), Transamine (registered trademark), soybean trypsin inhibitor and serum, and has plasminogen activation effect and 1. A method for producing a fibrinolytic active substance, which comprises obtaining a crude enzyme protein fraction having various properties such as having a fibrinolytic action and not having a fibrinogen dissolving action. 2 Add an aqueous solvent with a pH of 5 to 10 to the earthworms to
Extraction is carried out by holding at ℃ for at least 3 minutes, and after concentrating the extract, adsorbent, polar organic solvent, salting out, ultrafiltration, ion exchange chromatography,
By combining gel filtration, affinity chromatography, or hydrophobic chromatography, the optimum pH is 8 to 10, the stable pH is 5 to 10, and Trasylol (registered trademark) Transamine (registered trademark name), an enzyme protein fraction that is inhibited by soybean trypsin inhibitor and serum, has plasminogen activation and fibrinolytic activity, but has no fibrinogenolytic activity. A method for producing a fibrinolytic active substance, characterized by obtaining a minute amount.