KR100324254B1 - Mushroom extract with thrombolytic activity - Google Patents

Abstract

The present invention relates to a mushroom extract having a thrombolytic ability, more specifically, three kinds of fungi with thrombolytic activity, which contain thrombolytic enzymes, which can be usefully used as a therapeutic agent and health supplement for thrombosis related diseases. (Mushrooms), or cloud mushrooms (Coriolus versicolor), Sand mushrooms (Pisolithus tinctorius) And decorative brush mushrooms (Tricholomopsis decora)of Relates to an extract.

Description

Mushroom extract with thrombolytic activity

The present invention contains three types of fungi (mushrooms) having thrombolytic activity, that is, cloud mushrooms, which can be usefully used as a therapeutic agent and health supplement food for thrombosis-related diseases.Coriolus versicolor), Sand mushrooms (Pisolithus tinctorius) And decorative brush mushrooms (Tricholomopsis decora)of Relates to an extract.

When the blood vessels are damaged and the bleeding occurs, the body's defense system that coagulates blood is activated. This process is induced by the conversion of fibrinogen to fibrin by activated thrombin. Fibrin clots are solubilized by fibrinolytic enzymes, such as plasmin, after wound healing. If the resulting clots accumulate excessively in the body or the thrombolytic mechanism does not work well, Thrombosis can be triggered and cause fatal damage to the body. For example, cerebellar thrombosis, a blood clot that forms in the cerebrovascular vessels, can lead to an incapacitated stroke, and rupture of the blood vessels in the brain can lead to life-threatening damage. Paralysis may be caused.

To date, studies on the elimination of blood clots have been conducted. In particular, plasminogen activators such as streptokinase, urokinase, and t-PA (tissue-type plasminogen activator) are injected intravenously. Therapies that activate the fibrinolytic system are the most common methods. However, these substances have a large side effect such as causing systemic haemorrhage and are very expensive, and are not orally administered except for urokinase. Therefore, various economic and orally administrable thrombosis therapeutics have been tried in various ways, and agents that can increase the thrombolytic ability in the blood by direct oral administration or in combination with oral administration and vascular injection have been developed. Currently, six thrombolytic enzymes isolated from the worm ( Lumbricus lubellus ) are known as orally administrable thrombicides .

On the other hand, many attempts have been made to develop thrombolytic substances present in various organisms as natural thrombolytic agents using a wide range of screening techniques.

In the case of plants, products extracted from ginkgo biloba are known to facilitate blood circulation, and products using them have been developed and commercialized, and new plasminogen activator (Gardell, SJ,) isolated from salivary glands of vampire bats in animals. et al., Circulation 84, 244-253, 1991), as well as salmonase isolated from snake venom (Chung, KH and Kim, DS, Thromb. Haemost. 65, 953, 1991), isolated from leeches. Hementin and fibrinolytic enzyme isolated from centipedes (Kim, KY, Chung, KH, and Moon, HM, Thromb. Haemost. 69, 839, 1993) It is known.

In addition, various thrombolytic substances have been identified in microorganisms, such as streptokinase, as well as CA-7, brinorase and fibrinori, which were found in staphylokinase and aspergillus aurise, reported in 1948. God, Bacillus Fibrin degrading enzymes found in strains have been reported as thrombolytic agents.

In addition, the research on thrombolytic enzymes in fermented foods, especially in Korea and Japan, is noteworthy in that they focus on foods that can be consumed directly. Natto (natto), a traditional Japanese soybean fermented food, and shiokara (pickled food) were known to have thrombolytic enzymes, and production strains were isolated and enzymes were purified. Among them, nattokinase isolated from natto. Has been reported to increase thrombolytic capacity in vivo even when administered orally. Currently, the sales of natto are rapidly increasing in Japan as a health food with thrombolytic ability (Sumi, H., et al. , Experimentia 43 , 1110, 1997; Bioindustry 7, 725, 1990). In Korea, Bacillus strains with thrombolytic activity were isolated and purified from Chungkookjang, a traditional fermented food made from soybeans similar to Japanese natto. In addition, the present inventors isolate strains that produce thrombolytic enzymes two to three times higher than strains isolated from natto and cheonggukjang from doenjang, a traditional soybean fermented food in Korea, as well as cheonggukjang and the thrombolytic proteins produced by these strains. Has been invented and applied for a patent (Korean Patent Application No. 96-75523).

On the other hand, mushrooms can be directly ingested by simple cooking, and since the product using mushrooms has fewer side effects and oral administration, much research has been conducted on mushrooms. However, it is mainly related to metalloendopeptidase and antifungal activity, and no studies on thrombolysis have been reported.

Mushrooms have been widely used for food and medicinal purposes since ancient times. Currently, 990 kinds of Korean mushrooms are classified, and about 100 of them are identified as edible mushrooms. In addition, mushrooms can be said to be very advantageous in terms of the thrombolytic effect because the fungi themselves can be directly ingested.

Accordingly, the present inventors tried to find strains and thrombolytic enzymes excellent in thrombolytic activity in Korean mushrooms, cloud mushrooms (Coriolus versicolor), Sand mushrooms (Pisolithus tinctorius) And decorative brush mushrooms (Tricholomopsis decoraThe present invention was completed by identifying the thrombolytic enzyme protein having excellent thrombolytic activity from the extract of N.

An object of the present invention is to provide a mushroom extract having a thrombolytic ability.

Figure 1 shows the results of measuring the thrombolytic activity of the plasmin and sand mushroom ( Pisolithus tinctorius ) according to the fibrin plate assay (fibrin plate assay),

A: Sand mushroom extract solution B: Plasmin (1.0 NHI unit / ml)

Figure 2 shows the results of identifying the thrombolytic enzyme protein in the mushroom extract through the 12% SDS-fibrin zymography activity assay using fibrin as a substrate.

M: Standard Protein a: Cloud Mushroom ( Coriolus versicolor )

b: Sand mushroom c: Tricholomopsis decora

The present invention provides a mushroom extract having a thrombolytic ability. In this case, the mushrooms are preferably Coriolus versicolor , Pisolithus tinctorius, and Tricholomopsis decora .

The present invention also provides a thrombolytic enzyme protein isolated from the mushroom extract and having a molecular weight of 54 kDa, 61 kDa and 84 kDa, respectively.

The present invention also provides a pharmaceutical composition for treating thrombosis-related diseases, and a dietary supplement, comprising as an active ingredient a mushroom extract containing some or all of the thrombolytic enzyme protein. At this time, the thrombotic disease treatment agent can be administered orally, and can be applied to patients with coronary artery disease, thrombosis or cerebrovascular disease.

Hereinafter, the present invention will be described in detail.

The present invention provides a mushroom extract having a thrombolytic ability and an enzyme protein of 54 kDa, 61 kDa, 84 kDa isolated therefrom.

About 50 kinds of Korean mushrooms used in the present invention were collected from all over the country and used in experiments. Among them, three kinds of fungi (mushrooms) having thrombolytic ability, namely cloud mushrooms, sand mushrooms and decorative brush mushrooms, were selected for thrombus Lysase activity was measured and substrate specificity was investigated.

In the present invention, to measure the thrombolytic activity of the mushroom extract, the superiority of thrombolytic dissolution was compared by measuring the fibrin plate dissolution area of the extract. At this time, plasmin 1.0 NIH unit (unit) / ml well-known as a thrombolytic agent was used as a control, and the extract of the sand mushroom showed 4.71 times higher thrombolytic ability than plasmin as a control ( FIG. 1 ). This corresponds to much higher activity than that of Bacillus strains isolated from Chungkookjang (1.84 plasmin unit) and natto (1.58 plasmin unit). In addition, the present inventors showed similar activity to the activity (2-5 plasmin units) of thrombolytic strains isolated from Korean traditional doenjang (see Korean Patent Application No. 96-75523). However, unlike Bacillus strains, it is more noteworthy that the mushroom can be directly ingested.

In addition, in order to identify enzyme proteins that are presumed to have thrombolytic activity in the mushroom extracts of the present invention, new enzyme proteins were isolated and purified by subjecting the culture supernatant to a DEAE-Sephasil column by treating with ammonium sulfate. Enzyme proteins with molecular weights of 54 kDa, 61 kDa and 84 kDa could be obtained.

In order to confirm the activity of the enzyme protein purified from the above, the extract of the mushroom was centrifuged to remove the cells and obtained the supernatant, which was analyzed by SDS-fibrin zymography activity confirmation method. As a result, it was confirmed that the enzyme protein has thrombolytic activity ( FIG. 2 ).

In addition, as a result of searching for hemolytic activity to confirm whether the mushroom extract containing the enzyme protein of the present invention is suitable for clinical use, the extract of the present invention showed no hemolytic activity at all.

While conventional thrombolytic agents are almost impossible to orally administer, the mushroom extract of the present invention has the advantage that both oral or parenteral administration is possible at the time of clinical administration.

That is, the mushroom extract of the present invention may be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used Or using excipients. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which form at least one excipient such as starch, calcium carbonate, sucrose ( Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of the mushroom extract of the present invention is 10 to 1000 mg / kg, preferably 50 to 100 mg / kg.

Hereinafter, the present invention will be described in detail by way of examples.

The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

Example 1 Preparation of Mushroom Extract

About 50 kinds of Korean mushrooms used in the present invention were collected from all over the country and used in experiments, and used as fibrinogen, thrombin, agarose and control used in the manufacture of fibrin plates. The prepared plasmin and the substrate for plasmin, Np-Tosyl-Gly-Pro-Lys p-nitroanilide, were purchased from Sigma Co., and other reagents were used as a special reagent.

About 50 kinds of the classified mushrooms were immersed so that 100 g of the same weight was sufficiently submerged in 80% methanol, and left at room temperature for one month, and then the extract was filtered. The filtrate was concentrated under reduced pressure, powdered and then suspended in the same amount of methanol and filtered again. The filtrate was concentrated to 5 ml to prepare a mushroom extract.

Example 2 Selection and Activity Measurement of Mushrooms Having Thrombolytic Activity

Fibrinogen was completely dissolved in 50 mM phosphate buffer solution (pH 7.4, containing 0.15M NaCl) to a final concentration of 0.3%, and then 5 ml of this solution was mixed with an equal amount of 2% agarose. Fibrin plates were prepared by adding 0.1 ml of thrombin (100 NIH units / ml) to the mixed solution, thoroughly mixing, pouring into plates immediately, and solidifying at room temperature for 1 hour. For measuring activity, a 5 mm diameter hole was made with a pasteur pippet in a fibrin plate, and 20 µl of 50 mushroom extracts prepared in Example 1 were injected and reacted at 37 ° C. for 12 hours. The area of the transparent ring was calculated. At this time, plasmin (1.0 NIH unit / ml), which is a purified thrombolytic enzyme, was used as a control.

As a result, cloud mushrooms, sand mushrooms And decorative sol mushroom extract showed thrombolytic activity. The thrombolytic ability was calculated by measuring the dissolution area on the fibrin plate of the three mushroom extracts thus selected and converting it in a relative ratio with respect to the dissolution area of plasmin (1.0 NIH unit / ml) (Table 1). .

Confirmation of thrombolytic activity using fibrin Fungus Thrombolytic ability (plasmin 1.0 NIH unit / ml) ¹⁾ Cloud Mushroom ( Coriolus versicolor ) 4.45 Sand mushroom ( Pisolithus tinctoriius ) 4.71 Tricholomopsis decora 4.19 Thrombolytic ability ¹⁾ : Fibrin degradation area of the sample / fibrin degradation area of plasmin

As can be seen from the results of Table 1, all three mushrooms showed three to four times higher activity than plasmin 1.0 NIH unit, and among them, sand mushroom showed the highest activity (4.71 plasmin unit). (See FIG. 1 ). The thrombolytic activity of these mushroom extracts is 2-3 times higher than that of the Bacillus strain known to exhibit higher activity than plasmin.

<Example 3> Thrombolytic substrate specificity investigation

In general, fibrin plates can be easily degraded not only by plasmin but also by other proteases, thus having low specificity for plasmin. Therefore, Np-Tosyl-Gly-Pro-Lys p-nitroanilide, a chromogenic substrate (T-6140) for plasmin, was used to investigate the substrate specificity for thrombolytic enzymes in each mushroom extract. It was.

0.2 ml of 0.5 mM substrate solution (20 mM phosphate buffer solution, pH 7.0) and three mushroom extracts identified in Example 2 (3 ㎍ / 0.2 ml) were added to a 37 ° C. water bath for 1 minute, followed by 50% acetic acid solution. The reaction was stopped by adding 0.1 ml. As a control, purified plasmin (1.0 NIH unit / 0.2 ml) was used, and blank (blank) was mixed with 0.2 ml of extract and 0.2 ml of phosphate buffer solution at a measuring temperature for 1 minute, and then mixed with 0.1 ml of 50% acetic acid solution. It was prepared by. Each of the samples and blanks thus prepared were measured for absorbance at 405 nm to determine relative enzyme activity against plasmin (Table 2).

Activity measurement using plasmin-specific substrate (T-6140 ¹⁾ ) Fungus Specificity (plasmin 1.0 NIH unit / ml) ²⁾ Cloud Mushroom ( Coriolus versicolor ) 0.24 Sand mushroom ( Pisolithus tinctoriius ) 0.99 Tricholomopsis decora 1.32 T-6140 ¹⁾ : Plasmin specific substrate (Np-Tosyl-Gly-Lys pNa, specificity ²⁾ Plasmin NIH unit / ml

As can be seen from the results of Table 2, the cloud mushroom showed a very low specificity of 0.24 plasmin units, and the sand mushroom showed a high specificity of 0.99 plasmin units. In particular, decorative edible mushrooms showed enzymatic specificity for the highest fibrin multimer of 1.32 plasmin units.

Example 4 Isolation and Purification of Enzyme Protein Having Thrombolytic Activity

* The three mushroom extracts identified in Example 2 were centrifuged at 10,000 xg for 20 minutes to take a supernatant and treated with ammonium sulfate. After desalting the protein precipitate thus obtained, it was passed through a DEAE-Sephacel column (Sigma, USA) to isolate and purify the new enzyme protein, and subjected to SDS-PAGE electrophoresis with the standard protein to carry out the enzyme protein. It was confirmed that this was purified by separation.

Example 5 Identification of Enzyme Protein Having Thrombolytic Capacity

The activity and molecular weight of the enzyme protein separated and purified from Example 4 were confirmed according to SDS-fibrin zymography activity confirmation method using fibrin as a substrate. The mixture was mixed with a polyacrylamide solution so that the concentration of fibrinogen was 0.12% (w / v), followed by fibrin-polyacrylamide gel prepared by adding thrombin (1.0 NIH unit) immediately. Protein quantification was performed according to Lowry's method using bovine serum albumin.

20 ng of the separated and purified enzyme protein fractions were loaded in each lane, and electrophoresis was performed by applying a constant current of 10 mA. To reactivate the enzyme inactivated by SDS, the gel was left in Tris buffer solution (50 mM, pH 7.4) containing 2.5% Triton X-100 for 30 minutes to remove SDS, and then distilled water Triton X-100 was removed. Thrombolytic activity of the fractionated protein was reacted for 12 hours in a 37 ℃ incubator by dipping the gel in the activity reaction buffer (200 mM NaCl, 10 mM CaCl ₂ , 30 mM Tris buffer containing 0.02% NaN ₃ , pH 7.4) Let. The protein fraction having fibrin degradability decomposes fibrin on the gel by active staining to form a zona pellucida when stained with Coomassie blue, and thus it was confirmed that the protein fraction has thrombolytic activity.

In addition, all three mushrooms identified a protein having a common activity of 54 and 61 kDa in molecular weight. It was confirmed that it has a thrombolytic enzyme having a strong activity (2).

Example 6 Measurement of Hemolytic Activity

A test was conducted to determine whether the new enzyme protein produced by the strain of the present invention had hemolytic activity.

The mushroom extract obtained from Example 1 was placed on a plate and cultured after adding human blood thereto. After culturing for 24 hours and observed under a microscope, it was found that the blood is maintained without being degraded, from which the mushroom extract of the present invention was determined to have no hemolytic activity.

Mushroom extract having a thrombolytic ability of the present invention has an advantage that can be used in combination with a vascular injection because it can be administered orally as compared to conventional thrombolytics administered mainly via intravenous injection.

In addition, the mushroom extract of the present invention does not exhibit hemolytic activity, and thus can be developed as an oral dosage agent, and can be widely used as a thrombolytic therapeutic agent and health supplement.

Claims (4)

It is effective to use mushroom extracts with thrombolytic activity extracted from methanol or a mixed solvent of methanol and water from one or more mushrooms selected from the group consisting of Coriolus versicolor , Pisolithus tinctorius, and Tricholomopsis decora . Health supplements containing ingredients. One or more thrombolytic enzyme proteins selected from the group consisting of enzyme proteins of 54 kDa and 61 kDa, which separate the mushroom extract from methanol ( Coriolus versicolor) with methanol or a mixed solvent of methanol and water. Supplements containing health as an active ingredient. One or more thrombolytic enzyme proteins selected from the group consisting of enzymatic proteins of 54 kDa and 61 kDa, which separate mushroom extracts from Pisolithus tinctorius with methanol or a mixed solvent of methanol and water. Supplements containing health as an active ingredient. Mushroom extract is extracted from Tricholomopsis decora with methanol or a mixed solvent of methanol and water, and one or more selected from the group consisting of enzyme proteins of 54 kDa, 61 kDa, and 84 kDa molecular romium. Health supplement containing thrombolytic enzyme protein as an active ingredient.