KR100648739B1 - Process for preparing protease powder from Lumbricus rubellus using an organic solvent - Google Patents

Abstract

The present invention includes a step of fractional precipitation and ultrafiltration of crude extracts of lupus erythematosus ( Lumbricus rubellus ) with a water-soluble organic solvent, which have antithrombotic and thrombolytic activity. The present invention relates to a method for preparing a protease powder.

Description

Process for preparing protease powder from Lumbricus rubellus using an organic solvent

1 is a photoelectrophoresis of glomerular protease powder and lumpbrokinase prepared according to the present invention.

The present invention relates to a method for producing a protease powder having thrombolytic ability from Guin ( Lumbricus rubellus ). More specifically, the present invention relates to a method for highly purifying only protease by fractional precipitation and ultrafiltration of a crude crude extract with a water-soluble organic solvent.

Diseases caused by thrombi are broadly classified into heart disease (eg myocardial infarction), cerebrovascular disease (eg cerebral hemorrhage) and venous disease (eg deep vein thrombosis, pulmonary embolism). This circulatory disease caused by blood clots is the third leading cause of death in the United States, and the mortality rate is rising in East Asia, where food is becoming westernized (George J. H. Science & Medicine, 8-17 (1999)).

The most commonly used thrombolytic agents currently in the market include the urokinase (UK) and the tissue-type plasminogen activator (tPA), all of which use blood clots, or fibrins. Rather than dissolving directly, plasminogen (plasminogen) present in the human body has a mechanism of action to dissolve blood clots (plasmin) by converting it. In addition, UK or tPA is an enzyme derived from the human body has the advantage that there is no immune response, but there are side effects such as bleeding, the body half-life is very short (5 to 10 minutes), fibrin-rich blood clots are good, while the brain It has a common problem that the effect is very poor on platelet-rich blood clots, such as blood clots or blood clots that are reconstituted after thrombolytic treatment (see Higgins, DL et al. Ann. Rev. Pharmacol. Toxicol. 30: 91-121). , 1990; Harris, TJR Protein Eng. 1: 449-458, 1987; Collen, D. et al. Ann. Rev. Med. 39: 405-423, 1988). Therefore, research is being actively conducted in various countries in order to develop the next generation of thrombolysis which does not have such a problem.

On the other hand, job offer has been used as a medicine since ancient times, especially in the Chinese Pharmacopoeia (1977 edition, Part 1, pp. 197-198) and Chinese Medicinal Dictionary (Kangcho New Academy of Medicine, District Publisher, 1986 p.2111). There are two types of job openings , Lumbricus Kwang tungesis and Lumbricus nativus , which are effective as antipyretic, anti- cursive , blood-promoting, anti-incompetent, joint, urinary, bronchial asthma, and hypertension. Is described. These effects have been recently discovered scientifically, and oral administration of gut extracts has been reported to have anti-thrombotic and thrombolytic effects in a concentration-dependent manner (Kim YS et al. Arch. Pharm. Res. 21 (4). 374-377, 1998; Chang CS et al. Yakhak Hoeji 39 666-670, 1995; Hahn BS et al. Arch Pharm. Res. 20 (1) 17-23, 1997). Also, Mihara et al. (Mihara, H. et al. US Pat. No. 4,568,545) isolated six fibrin degrading enzymes from Job and named it lumbrokinase. Lumbrokinase has a very wide optimum pH range and dissolves fibrin and blood clots without plasminogen, and is known to be particularly effective during oral administration.

However, the extract from the existing gut is a crude extract made by simply grinding the grout, and it is difficult to completely exclude the aversibility of the 'earthworm extract', and the problem of containing hemolysin and heavy metals. In addition, a commonly used method for purifying proteins uses ammonium sulfate fractional precipitation, which requires considerable time and cost.

In this regard, the present inventors conducted a continuous study to obtain a gut extract having excellent antithrombotic and thrombolytic activity. As a result, the present inventors found that the proteinase contained in the gut was very stable to the water-soluble organic solvent, and that only the proteinase could be highly purified by fractionating and prefiltering the crude crude extract with the water-soluble organic solvent. It was found and completed the present invention.

First, the present invention

Viii ) Lumbricus rubellus was mixed with water to pulverize and autolysis to obtain an aqueous layer from a solution obtained.

Ii) fractional precipitation of the aqueous layer with a water-soluble organic solvent to obtain a precipitate;

Iii) ultrafiltration of the precipitate,

Provided is a method of preparing a protease fraction from the gut.

In step ii) of the process according to the invention, the water soluble organic solvent comprises ethanol, acetone, methanol, tetrahydrofuran (THF), dimethylformamide (DMF), acetonitrile, isopropanol (IPA) or 1,4-dioxane But it is not limited thereto. More specifically, the aqueous layer is treated with 40% ethanol, 60% acetone, 40% methanol, 60% tetrahydrofuran, 50% dimethylformamide, 50% acetonitrile, 40% isopropanol or 70% 1,4-dioxane. After primary fractional precipitation, the supernatant was diluted with 80% ethanol, 80% acetone, 100% methanol, 80% tetrahydrofuran, 80% dimethylformamide, 80% acetonitrile, 70% isopropanol or 90% 1,4-dioxane. Secondary fractionation can be done. In this case, it is preferred that the aqueous layer is subjected to primary fractional precipitation with 40% ethanol, and then the supernatant is subjected to secondary fractional precipitation with 80% ethanol.

In step iii) of the process, it is preferable to filter the precipitate obtained in step ii) with a superfiltration device which passes only molecular weight 10,000 Daltons or less.

The filtrate obtained in the step iii) is lyophilized, so that the proteinase can be obtained in powder form.

Second, the present invention provides a proteolytic enzyme fraction having antithrombotic and thrombolytic activity prepared according to the above production method. The protease fraction according to the present invention was confirmed to have excellent antithrombotic and thrombolytic activity in animal experiments.

Third, the present invention provides an antithrombotic and thrombolytic composition containing the above-described protease fraction. The composition may be formulated in oral preparations, in particular in tablet form.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are only intended to aid the understanding of the present invention and are not intended to limit the scope of the present invention.

Example 1 Preparation of Sump Protease Powder 1

100 kg of Gum ( Lumbricus rubellus ) living body was washed with water, and then put in 100 L of distilled water and wet-pulverized with a homomixer. Self-decomposition was carried out for 5 hours while keeping the temperature constant at 50 ° C. The resulting solution was rapidly cooled to 0 ° C. to freeze, left at −20 ° C. for about one day, and then the temperature was raised to room temperature again to thaw to separate the insoluble layer and the aqueous layer. The separated aqueous layer was recovered and centrifuged at 5,000 rpm for 1 hour. The centrifuged supernatant was continuously filtered through diatomaceous earth filtration and 0.45 μm membrane filter to prepare aseptic extract. The extract was treated with 40% ethanol and centrifuged to recover the supernatant, which was then treated with 80% ethanol and centrifuged to recover the precipitate. The recovered precipitate was filtered by an ultrafiltration device having a pore size of less than 10,000 Daltons in molecular weight. The obtained filtrate was frozen at −30 ° C. and then dried under a 0.2 mmHg vacuum for 20 hours at 0 ° C., 15 hours at 20 ° C., 12 hours at 20 ° C. and finally at 35 ° C. for 5 hours to contain 60% protein. 0.71 kg was obtained.

Example 2 Preparation of Sump Protease Powder 2

Example 1 uses 60% acetone, 40% methanol, 60% THF 50% DMF, 50% acetonitrile, 40% IPA and 70% 1,4-dioxane instead of 40% ethanol and 80 instead of 80% ethanol According to the same method as in Example 1, except that% acetone, 100% methanol, 80% THF, 80% DMF, 80% acetonitrile, 70% IPA and 90% 1,4-dioxane were used, Protease powder was prepared.

Experimental Example 1: Determination of proteolytic activity of globular protease powder 1

The proteolytic activity of the enzyme powder prepared in Example 1 was intended to be expressed by casein cleavage titer. The titer was measured as follows. That is, 2 ml of 4% α-casein solution (pH 7.4), 1.6 ml of 67 mM sodium phosphate buffer (pH 7.4) and 0.4 ml of sample (1 mg / ml) were mixed and reacted at 37 ° C for 30 minutes. Thereafter, 6.0 ml of 15% trichloroacetic acid (TCA) was added to terminate the reaction, and the absorbance of the reaction solution was measured at 280 nm. The amount of enzyme releasing 450 μg TCA-soluble tyrosine for 1 hour was defined as 1 unit.

Table 1 shows the protein recovery and proteolytic activity of each step of Example 1.

Protein Recovery and Casein Degradation Titer for Each Step of Example 1 Manufacturing stage Weight (kg) Titer per unit weight (unit / g) Total potency (unit) Potency recovery rate (%) Protein content (%) Freshman 100 - - - - Crude extract  12    870 10,444,000 100 22 Ethanol Fractionation Precipitation  4  2,360  9,444,000  90 28 Ultrafiltration     0.71 12,700  9,017,000  86 60

As shown in Table 1, the protease powder prepared by the present invention has a titer per unit weight of about 15 times and a protein content of about 3 times higher, based on the crude extract prepared by a known method. The recovery rate was 86%, so the loss of potency due to manufacturing was not so large.

In order to determine the protein composition of the prepared protease powder, electrophoresis was performed by loading 20 μg into each lane of a 10% polyamide gel, and the results are shown in FIG. 1 (lane A: low molecular weight standard labeled protein (top) From molecular weight 101,000, 83,000, 506,000, 355,000, 291,000 Daltons), B: Lumbrokinase fraction F-1 (LKF-1), C: Lumbrokinase fraction F-2 (LKF-2), D: Lumbrokinase fraction F-3 (LKF-3), E: Lumbrokinase fraction F-4 (LKF-4), F: Lumbrokinase fraction F-5 (LKF-5), G: Lumbrokinase fraction F-6 (LKF -6), H: 6 fractions of lumbrokinase combined in equal weight, I: protease powder, J: polymer standard labeled protein).

As can be seen from Figure 1, the prepared protease powder contained all of the number 1 to 6 of the room bromase fraction, in particular was confirmed to contain a large amount of 3. On the other hand, a single band in the polymer and four bands in the low molecule were identified in addition to the roombrokinase.

Therefore, when applying the proteolytic enzyme powder to the clinical application according to the present invention, the dosage is reduced compared to the crude extract administration (7.5 mg / kg) (0.5-2 mg / kg), which is easy and economical to take in the capsules used in the past The change to phosphorus purification became possible. In addition, most of them are made of protein, which can reduce side effects caused by substances other than proteins (for example, heavy metals and hemolytic factors).

Experimental Example 2: Determination of proteolytic activity of globular protease powder 2

According to the method described in Experimental Example 1, the casein degradation titers of the protease powders obtained in Example 2 were measured, and the results are shown in Table 2.

Casein Degradation Activity of Protease Powders Purified with Water Soluble Organic Solvents Water soluble organic solvent Weight (g) Titer per unit weight (unit / g) Total potency (unit) ethanol One 12,700 12,700 Acetone     0.952 12,194 11,609 Methanol     0.936  3,172  2,969 THF     0.574  8,944  5,134 DMF     0.541  2,600  1,407 Acetonitrile     0.184  3,653   672 IPA     0.900 10,972  9,875 1,4-dioxane     0.554 10,569  5,855

As shown in Table 2, with the exception of ethanol, the acetone was the highest and the acetonitrile was the lowest in the weight recovery, and the acetone was the highest in the titer per unit weight and the DMF was the lowest. Total titers were ethanol, acetone, IPA, 1,4-dioxane, THF, methanol, DMF, acetonitrile.

Experimental Example 3 Measurement of Inhibitory Effect of Thrombus Formation by Oral Administration Before Thrombus Induction

Healthy male Sprague-Dawley rats weighing 250-300 g were used as experimental animals. Proteolytic enzyme powder, a test substance, was administered orally at a dose of 50 mg / 10 ml / kg from 2 or 4 weeks before thrombus induction. The normal control group underwent surgery without thrombus induction, and distilled water was administered at a 10 ml / kg / day dose. The thrombus control group induced thrombus and distilled water was administered at a 10 ml / kg / day dose. Experimental groups were performed for thrombosis-induced surgery and observed for 3, 7, 14, and 28 days, respectively. The venous thrombosis model was created using a modified method of Friedman et al. (1960). Briefly, after the animals were fasted one night before surgery and anesthetized with cecobarbital (30 mg / kg, intraperitoneally), the normal control group was incised without inserting a wire coil (diameter 0.22 mm, Dentaurnm, Germany). Sutures were made immediately after. The thrombus control group and the protease-treated group were incised 2 to 3 cm from the abdominal midline and inserted into the vein while turning the wire coil (23 pitch, about 1.5 cm) to the left branch of the inferior vena cava and about 2 pitches. It was. After the wire coil was inserted to induce thrombus, the incision was closed and the surgical site was sterilized with iodine tincture. The thrombus size was measured by the total protein content of the thrombi separated from the wire coil. Briefly, the portion of the inferior vena cava in which the wire coil was inserted was removed without damaging blood vessels and washed well with saline. The blood vessels were excised and carefully removed from the blood vessel wall to remove the blood, and then completely removed.Then, the tube was placed in a tube containing 1 ml of a solution of 2% Na ₂ CO _{3 in} 0.1 N NaOH and boiled for 3 minutes. After dissolving the thrombi, the total protein mass of the solution was measured with a protein-quantity analysis kit of Bio-Rad by Bradford (1976). All measurements were expressed as mean ± standard error (n = 10) and the significance between each group was tested as an unpaired Student's t-test to determine that the p-value was 0.05 or less. The results are shown in Table 3.

Antithrombotic Effects by Oral Administration of Protease Powders on Thrombus Formation in Experimental Inferior Vena cava Induced Rats Experimental group Thrombus (total protein, mg) Thrombosis-only group Thrombus induced after protease powder administration (2 weeks) Thrombosis after protease powder administration (4 weeks) 3 days thrombus 1.0 ± 0.0 0.4 ± 0.1 * 0.5 ± 0.1 * 7 days thrombus 0.8 ± 0.1 0.3 ± 0.1 * - 14 day thrombus 1.0 ± 0.1 0.8 ± 0.1 * 0.8 ± 0.1 * 28 days thrombus 1.2 ± 0.1 0.7 ± 0.1 * 0.5 ± 0.0 *

As shown in Table 3, first, when the wire coil was inserted into the inferior vena cava before administration of the protease powder, the thrombus increased as time passed. In the group on the 3rd day of thrombus (day 3 thrombi), proteolytic enzyme powder was administered from 2 weeks and 4 weeks before thrombus induction, respectively, and the total thrombus production amount was 0.4 ± 0.1 and 0.5 ± 0.1. Could. On the other hand, the thrombus inhibitory effect was the strongest in the group on the 7th day of thrombus formation (day thrombus group) by administration of protease powder (2 weeks before treatment) (0.3 ± 0.1 mg, p <0.05). In addition, the blood clot production was significantly inhibited by protease powder pretreatment in 14 and 28 day thrombi groups. From these experiments, it was concluded that the administration of protease powder inhibited the formation of experimentally induced thrombi.

Experimental Example 4: Measurement of the Thrombolysis Effect by Oral Administration after Induction of Thrombosis

As the experimental animals, male Sprague-Dawley rats were fed and purified for a certain period of time, and then only healthy animals of about 250 to 300 g in weight were selected for the experiment. Venous thrombosis model was prepared in the same manner as described in Experimental Example 3. The protease powder was orally administered daily at a dose of 50 mg / 10 ml / kg. The proteolytic enzyme powder was first administered 6 hours after the thrombotic surgery. The normal control group underwent surgery without thrombus induction, and distilled water was administered at a 10 ml / kg / day dose. The thrombus control group induced thrombus and distilled water was administered at a 10 ml / kg / day dose. Thrombus induction period was 3, 7, 14, 28 days were observed by setting each experimental group. The results are shown in Table 4 (n = 10).

Thrombolytic Effects of Oral Administration of Protease Powders on Thrombosis in Experimental Inferior Vena cava Induced Rats Experimental group Thrombus amount (total protein amount, mg) Thrombosis-only group Protease powder administration group after thrombus induction 3 days thrombus 1.0 ± 0.0 0.5 ± 0.1 * 7 days thrombus 0.8 ± 0.1 0.3 ± 0.0 * 14 day thrombus 1.0 ± 0.1 0.7 ± 0.1 * 28 days thrombus 1.2 ± 0.1 0.7 ± 0.1 *

As shown in Table 4, administration of protease powder (50 mg / kg / day) in the three-day thrombi group significantly reduced the thrombus amount compared to the thrombus amount of the control group (thrombosis control group: 1.0 ± 0.0 mg, Protease powder administration group: 0.5 ± 0.1 mg, p <0.05). In addition, a significant decrease in the amount of thrombosis was observed in the group treated with the protease powder in the 7-day thrombi group (thrombosis control group: 0.8 ± 0.1 mg, protease powder administration group: 0.4 ± 0.0 mg, p <0.05). In the 14- and 28-day thrombi groups, the thrombi was significantly reduced to 30% and 42%, respectively, by the administration of protease powder. These results suggest that the administration of protease powder dissolves the experimentally induced thrombi.

The globular protease powder according to the present invention exhibits remarkably superior antithrombotic and thrombolytic activity as compared to the existing globular crude extracts, so that the dosage can be reduced during clinical application to enable simple and economical formulation. In addition, since most of the protein is composed of a protein can reduce the side effects of the substances contained.

Claims (10)

i) Lumbricus rubellus Mixed with water, ground and incubated, ii) freeze and thaw twice to remove insoluble materials and then filter to obtain an aqueous layer, iii) a group consisting of 40% ethanol, 60% acetone, 40% methanol, 60% tetrahydrofuran, 50% dimethylformamide, 50% acetonitrile, 40% isopropanol and 70% 1,4-dioxane After primary fractional precipitation with a water soluble organic solvent selected from, the supernatant is 80% ethanol, 80% acetone, 100% methanol, 80% tetrahydrofuran, 80% dimethylformamide, 80% acetonitrile, 70% isopropanol and 90 Fractional precipitation with a water-soluble organic solvent selected from the group consisting of% 1,4-dioxane to obtain a precipitate, iv) ultrafiltration of the precipitate, the method of producing a protease fraction from the globose. delete delete 2. The method of claim 1, wherein the aqueous layer is first fractionated with 40% ethanol and the supernatant is secondarily fractionated with 80% ethanol. The method according to claim 1, which is filtered by an ultrafiltration device which passes only molecular weight 10,000 Daltons or less. The method of claim 1, further comprising lyophilizing the filtrate to powder. delete delete delete delete

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