KR100447101B1 - A thrombus solublizer comprising extract of Cordyceps militaris and Paecilomyces japonica and their enzyme - Google Patents

Abstract

The present invention relates to Cordyceps sinensis extract having a thrombolytic action and a thrombolytic enzyme isolated therefrom. In the present invention, it is found that Cordyceps sinensis, in particular, the extracts of Cordyceps militaris and Paecilomyces japonica have excellent thrombolytic action. Based on these findings, the present invention provides a Cordyceps sinensis extract and fibrinolytic enzyme isolated therefrom that exhibit fibrinolytic activity obtained from Cordyceps militaris or Paecilomyces japonica or mixtures thereof.

Description

Cordyceps sinensis extract with thrombolytic action and thrombolytic enzyme isolated from it {A thrombus solublizer comprising extract of Cordyceps militaris and Paecilomyces japonica and their enzyme}

The present invention relates to a Cordyceps sinensis extract having a thrombolytic action and a thrombolytic enzyme isolated therefrom.

The blood must be fluid in normal blood vessels, and if the blood vessels are damaged, they must coagulate quickly to stop blood loss from the damaged blood vessels. In the blood coagulation phenomenon, that is, hemostasis, a thrombus, which is an aggregate of platelets and fibrin, is formed. In a normal state, when the hemostatic process is completed and the tissues of the damaged area are regenerated, thrombolytic action is activated. The liquidity is restored. In order for the vascular system to function properly, the formation and decomposition of blood clots must be balanced to maintain homeostasis. When this balance is broken due to various factors, thrombosis is a pathological phenomenon of the normal hemostasis mechanism. do.

Thrombosis is the formation of clots in the active cardiovascular system. These clots are called thrombus. Thrombus and blood clot should be distinguished, which is formed by the interaction of blood vessel walls and tangible components of the blood, especially platelets and plasma coagulation factors, and clots only involve coagulation. Thrombus causes ischemic damage to tissues or organs by reducing blood flow or blocking blood flow, and when it comes out of the blood vessel to which the thrombus is attached, it becomes an embolus, which is transported far into the bloodstream. In general, embolism is a generic term for lumps (including solids, liquids, and gases) that occlude blood vessels, and most of them are derived from blood clots, and the term thromboembolism is closely related to thrombosis. Infarcts refer to sites of ischemic necrosis, and infarcts usually occur due to thromboembolic obstruction in the blood vessels. The death rate from heart, lung, and brain infarction is currently known to be higher than that from cancer or infectious disease.

Blood clots produced in veins and blood clots produced in arteries differ in composition and clinical characteristics. Venous thromboembolism is relatively common and can cause edema and inflammatory reactions in veined tissues, deep vein thrombosis and pulmonary embolism. Pulmonary embolism is one of the leading causes of death, and deep thrombosis also causes morbidity such as phlebitis of the legs. Arterial thromboembolism in the arteries can block peripheral blood vessels, causing problems (e.g. stroke) in the blood vessels of the legs or brain, and are a common cause of acute myocardial infarction.

The normal hemostatic process in which blood coagulates when the vascular system is damaged is induced by the conversion of fibrinogen to fibrin by thrombin, and the clots and clots formed during the hemostasis process are recovered by plasminogen after wound recovery. Is dissolved by a fibrinolytic enzyme such as). However, as described above, when the balance between the formation and decomposition of the thrombus is broken, the thrombus is excessively accumulated in the body or the thrombolytic mechanism does not work smoothly, causing thrombosis.

As thrombosis is known as one of the main causes of vascular disease, research and interest in thrombolysis is increasing. Currently used thrombolytic agents include plasminogen activator streptokinase, urokinase and tissue-type plasminogen activator (tPA) (Reed et. al., 1995).

Streptokinase is a relatively large protein extracted from β-hemolytic streptococci and is one of the most commonly used thrombolytic agents. Streptokinase stably binds plasminogen 1: 1 to enable plasmin to be degraded by activating plasmin, and circulating fibrinogen produces anticoagulants. Oral administration is not possible, but only intravenous injection. Like most thrombolytics, side effects are bleeding, and allergic reactions or treatment-resistant anaphylactic reactions may occur. The half life is about 40 to 80 minutes.

The streptokinase-plasminogen complex is a complex of streptokinase, which can be injected intravenously faster than streptokinase (over 4-5 minutes), and is selective so that only plasminogen that is bound to fibrin can be activated quickly. Side effects of bleeding are reduced. However, allergic reactions are more likely and more expensive than streptokinase.

Tissue plasminogen activator (tissue-type plasminogen activator (tPA) is normally present in humans and is an activator of plasma plasminogen. It activates plasminogen bound to fibrin and thus exhibits less systemic anticoagulant action, but it is expensive and is mainly used for people who are ineffective or allergic to streptokinase. The half life is 5 to 10 minutes.

Eurokinase is a proteolytic agent produced in the human kidney. The mechanism of action, like the above drugs, activates plasminogen to plasmin. It is not as selective as streptokinase and can lead to systemic lysis and is as expensive as t-PA. The half life is 15 to 20 minutes. Single chain urokinase known to show selectivity is currently in clinical trials.

Currently, the method of activating the fibrinolytic system of the human body by intravenous injection of the thrombolytic agent, ie, plasminogen activators, is most commonly used for the removal of thrombi. However, these thrombolytic agents have large side effects such as systemic hemorrhage, and are expensive. Most of them are not oral except for urokinase.

Therefore, in recent years, various studies on the more economical and orally administrable thrombosis therapeutics have been attempted, and unlike the conventional thrombolytic agents that activate plasminogen and indirectly dissolve blood clots, a new method of directly dissolving blood clots Thrombolytic agents have also been reported.

Recent studies have reported the separation of worms (Lumbricus lubellus), which contains six thrombolytic enzymes, and enzymes that directly dissolve thrombi from snake venom for oral administration. In addition, many attempts have been made to find and develop new thrombolytic substances from various living organisms.

Among these, research on thrombolytic enzymes in fermented foods, especially in Korea and Japan, has attracted attention because it targets foods that can be consumed directly. Natto (natto), a traditional soybean fermented food, and shiokara (pickled food), respectively, are used to isolate thrombolytic enzymes nattokinase and katchuokinase that can enhance thrombolytic activity in vivo when administered orally. It has been reported that katsuwokinase has been purified. In Korea, Bacillus strains with thrombolytic ability were isolated and purified from Cheonggukjang, a traditional fermented food made from soybeans similar to Japanese natto. Also, Korean Patent Application No. 96-75523 is a Korean traditional soybean fermented food. Strains that produce two to three times higher thrombolytic enzymes than those isolated from natto and cheonggukjang from phosphorus doenjang and thrombolytic proteins produced by these strains are described.

On the other hand, Cordyceps sinensis is a fungus that parasitic to insects and forms fruiting bodies in the host of insects, and its name is derived from the insects in winter and grass in summer. Cordyceps sinensis belongs to the genus Cordyceps sinensis in the Aspergillus fungus and is widely distributed in Korea, China and Japan. Cordyceps sinensis (Cordyceps militaris), Cicada Cordyceps (C. sobolifera), Beetlewormweed (C. sphecocephala).

The record of Cordyceps sinensis is that medicinal herb life (1757) of Qing Dynasty was first or already used medicinally. In 1765, Cho Hak-min's "Herbaceous Herbal Milk" contains a relatively detailed description of Cordyceps sinensis. Cordyceps sinensis is known to be gentle in the body and to improve the constitution after the disease, Oui Nak's herbal life (1757) has been shown to be effective in pulmonary tuberculosis and bronchial asthma, and Cho Hak-min's "herbal river milk milk" in lung It is said to be effective and has a tonic effect.

As the mysterious efficacy of Cordyceps sinensis is known, a lot of research has recently been done to scientifically identify the efficacy of Cordyceps sinensis. According to recent studies, Cordyceps sinensis has been shown to be effective against kidney disease (Kwang Guk-dong, "Lianning magazine", 1995.2.), To support the function of the spleen, and to have an excellent effect on hair growth ("The Journal of Dermatology", 1986. , Japan), reported to be effective in lowering blood sugar levels ("Pharmaceutical Bulletin", 1993., Japan).

Meanwhile, many edible mushrooms are known to have various pharmacological effects while being directly consumed by a simple dish. Korean mushrooms are classified into about 990 species, of which 100 are known to be edible. Recently, among the various studies on the efficacy of mushrooms, there have been cases of selecting mushrooms with thrombolytic activity and reporting the specificity of activity by fibrin plate measurement method (Korean Patent Publication No. 2001-10739, Korean Patent Publication No. 2000) -30891).

The present inventors have found a new efficacy of Cordyceps sinensis, the thrombolytic action, and completed the present invention.

It is an object of the present invention to provide a thrombolytic extract extracted from Millyris cordyceps ( Cyceyceps militaris ) and snowflake cordyceps ( Paecilomyces japonica ) and its use.

It is also an object of the present invention to provide a thrombolytic enzyme isolated from the Cordyceps sinensis extract.

Other objects and advantages of the invention will be described below, and will be better understood by practice of the invention.

1 is a result of searching the thrombolytic ability of Cordyceps sinensis extract according to the fibrin plate measuring method.

Figure 2 is the result of measuring the thrombolytic activity of Cordyceps sinensis protein separated by 70% ethanol according to the fibrin plate measurement method.

Figure 3 shows the thrombolytic ability of Cordyceps sinensis protein isolated with 70% ethanol through azocaine analysis.

Figure 4 is the result of azocaine analysis of the fraction obtained after passing through the ion exchange resin (CM cellulose) column.

Figure 5 is a result of measuring the resolution of the fibrinolytic fractionation by performing the gel separation fibrin plate method.

Figure 6 is the result of measuring the molecular weight of the thrombolytic enzyme using electrophoresis.

7 is a result of examining the effect of metal ions on the enzyme isolated from Militaris cordyceps,

Figure 8 is the result of examining the effect of metal ions on the enzyme isolated from the Snow Cordyceps.

In the embodiment of the present invention, it is found that Cordyceps sinensis, in particular, the extracts of Cordyceps militaris and Paecilomyces japonica have excellent thrombolytic action.

Based on the results of this study, the present invention provides a thrombolytic agent comprising Cordyceps sinensis extract having a thrombolytic action, that is, Cordyceps sinensis extract showing fibrin soluble activity as an active ingredient. The Cordyceps sinensis extract can be extracted from mycelia as well as the fruiting bodies of Militaris and Snow Cordyceps sinensis, enabling mass production.

In addition, the present invention provides a 66 kDa thrombolytic enzyme isolated from the Cordyceps Sinensis extract.

In addition, the present invention provides a thrombolytic agent containing the enzyme as an active ingredient. The thrombolytic agent may include a pharmaceutically acceptable conventional excipient or carrier.

The composition may preferably have oral dosage forms (eg, powders, tablets, capsules, granules, solutions, pills, dispersants, syrups, etc.).

In addition, the present invention provides a functional food comprising the Cordyceps sinensis extract and the thrombolytic enzyme isolated therefrom as an active ingredient. The functional food may be provided, for example, in the form of a health drink, dairy products such as beverages, yogurt, health tea, and the like.

The effective dose of the Cordyceps sinensis extract is 0.1 µg to 100 µg / kg, preferably 10 to 20 µg / kg, on an adult's daily basis.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited by the following examples, and those skilled in the art to which the present invention pertains should be within the equivalent scope of the technical concept of the present invention and the claims to be described below. Of course, various modifications and variations are possible.

Example 1

Sample Preparation

Cordyceps sinensis and edible mushrooms used in the experiments of the present invention were grown directly in the laboratory and Iksan University using a biological sample and lyophilized samples. Millyceris Cordyceps militaris and Paecilomyces japonica were used in the experiments, and azocasein, trichloroacetic acid, plasmin and CM cellulose were manufactured by Sigma. Was used to purchase. Sephadex G-150 was purchased from Pharmacia (phamacia), and other reagents were used as a special reagent.

Example 2

Preparation of Extract

The same amount of sterilized water was added to 100 g of each of the fruiting bodies of Militaris Cordyceps and Snowflower Cordyceps, homogenized and centrifuged at 600 × g. The supernatant was recovered, and the same amount of 100% EtOH was added and stirred at 4 ° C for 1 hour. After the supernatant was recovered and centrifuged at 1200 x g with a total EtOH concentration of 70%, the resulting precipitate was used by dissolving in 10 mM citrate pH 6.0 buffer. In the case of mycelium, sonication was used.

Example 3

Protein Quantitation

Enzyme proteins showing platelet aggregation inhibitory activity were isolated and purified from each sample extract of Example 2. Protein quantification was performed using BCA protein assay kit (BCA protein assay kit: Pierce co. USA) using bicincroninic acid, and bovine serum albumin was used as a standard protein. Absorbance at OD 562 was calculated and quantified by protein standard curve.

Example 4

Exploration of thrombolytic activity

The thrombolytic ability of each extract obtained in Example 2 was explored. To 5 ml of a solution of fibrinogen completely dissolved in 50 mM phosphate buffer solution (pH 7.4, 0.15 M NaCl) to a final concentration of 0.3%, 5 ml of the same amount of 2% agarose was added and mixed. 0.1 ml of thrombin (100 NIH unit / mL) was added to the mixed solution, followed by sufficient mixing, immediately poured into a saare, and solidified at room temperature for 1 hour to prepare a fibrin plate. For the activity measurement, a 5 mm diameter hole was made with a Pasteur pippet in a fibrin plate, 10 μg of each extract was injected, and reacted at 37 ° C. for 12 hours. As a negative control, 10 mM citrate pH 6.0 buffer solution was used, and as a positive control, purified thrombolytic enzyme plasmin (0.1 unit / ml, 12.6 µg) was used.

The reaction result is shown in FIG. 1. In Figure 1 lane 1 negative control (10mM citrate pH 6.0), lane 2 Militaris cordyceps extract, lane 3 Militaris cordyceps mycelium extract, lane 4 snow Cordyceps sinensis extract, lane 5 Snow Cordyceps fungus mycelium extract As can be seen from Figure 1 all showed a significant thrombolytic action compared to the negative control.

Example 5

Enzyme Activity Measurement

Protease activity was measured for the extracts of Militaris and Snow Cordyceps sinensis. Activity was measured from crude extract to ethanol (50%, 70%, 85%) purification.

Activity measurements were determined by measuring the amount of acid-souble material from azocasein. Add 50 µl of each fraction to 300 µl of 1% azocaine (50mM Tris-HCl, pH 7.0), incubate at 37 ° C for 20 minutes, and then ice-cold 10% (W / V) 600 µl of trichloroacetic acid. Was added and mixed immediately. The samples were kept in ice for 10 minutes and then centrifuged at 4 ° C., 15,000 rpm for 15 minutes, and the supernatant was separated and measured at OD 366 nm. One unit of protease was incubated at 37 ° C. for 20 minutes, and the amount of acid solubles was determined from azocaine, which exhibited an absorbance of 0.1 at 366 nm. Azocaine analysis results (unit / mg) are shown in Table 1 below.

refine Militaris Cordyceps Sinensis Snowflake Cordyceps Crude extract 455 49.5 EtOH 50% 664 189 EtOH70% 940 282 EtOH85% 202 61

As can be seen from Table 1, the protease activity of Militaris cordyceps was higher than that of snowhopper.

Example 6

Measurement of thrombolytic activity over time

The thrombolytic activity of 70% ethanol extracts of Militaris and Snow Cordyceps was measured over time. 20 ㎍ of crude extract (crude extract) of each sample was added to the fibrin plate and reacted in a 37 ℃ incubator and the results were confirmed. The results are shown in Table 2 below. Observation was carried out for 5 hours, and the result was shown, with + representing the reaction occurring, ± representing the reaction starting to occur, and-representing the non-reacting.

sample Time-lapse Militaris Cordyceps Sinensis Snowflake Cordyceps 1 hours + ± 2 hours + ± 3 hours + + 4 hours + + 5 hours + +

Example 7

Thrombolytic activity measurement

Thrombolytic activity was measured using 10 μg of protein separated by 70% ethanol from Militaris and Snowfly Indigenous plants, respectively. The thrombolytic activity was measured in the same manner as in Example 4, and the results are shown in FIG. In Figure 2 lane 1 is negative control (10mM citrate pH6.0), lane 2 is positive control (plasmin), lane 3 is snow extract Cordyceps sinensis, lane 4 is Militaris cordyceps extract. In addition, the thrombolytic ability of Cordyceps sinensis protein isolated with 70% ethanol is shown in FIG. 3 through azocaine analysis.

From the above results, the area of the transparent ring was calculated, and the thrombolytic activity of each enzyme protein was calculated by converting the ratio of the dissolution area of the sample to the dissolution area of the positive control. The results are shown in Table 3 below.

division area Μg / μl Area / μg activation Plasmin 50.24 1.26 39.89 One Snowflake Cordyceps 50.24 2 25.1 0.62 Militaris Cordyceps Sinensis 176.6 2 88.3 2.21

As shown in Table 3, the Militaris cordyceps (C / m) was about 2,2 times more active than the positive control plasmin.

Example 8

Ion exchange resin column

After passing through the Millitaris Cordyceps Sinensis extract (separated with 70% ethanol) through a cation exchange resin (CM cellulose) column, each fraction was analyzed by azocaine. As a result, it was confirmed that thrombolytic enzyme was separated at about 80 mM.

CM cellulose ion exchange resins were purchased from Sigma, USA. In order to activate CM cellulose, 0.1 normal NaOH and 0.1 normal hydrochloric acid were used, equilibrated to 10 mM citrate pH6.0, and charged into a 5 cm x 12 cm column, and a 200 mM citrate (pH 6.0) solution of 10 mM citrate (pH 6.0) was used. Each fraction was separated using a concentration gradient. The results are shown in FIG.

Example 9

Gel filtration

After passing through the cation exchange resin column, gel filtration was performed on the active fractions to obtain thrombolytic enzymes. Sephadex G-150 was purchased from Pharmacia USA. After activating Sephadex G-150 for 5 hours at 90 ° C., it was charged into a 1 cm × 120 cm column, equilibrated with a buffer solution consisting of 10 mM citrate pH6.0 and 0.1 M NaCl, and fractionated at 0.5 ml / min. In the same manner as in Example 8, the fraction having activity was identified to purify the thrombolytic enzyme. Purified enzyme was checked for thrombolytic activity in the fibrin plate in the same manner as in Example 4, the results are shown in FIG. Lane 1 represents the negative control group, lanes 2 to 4 represent the thrombolytic enzyme purified by the gel filtration method, lane 5 positive control group (plasmin).

Example 10

Molecular Weight Measurement of Thrombolytic Enzyme by SDS-PAGE

The enzyme purified in Example 9 was purified using SDS-PAGE 10% gel and the molecular weight was confirmed. The results are shown in FIG. Lane 1 is a protein marker and lane 2 is an isolated gel fraction. As a result of measuring the molecular weight of the thrombolytic enzyme using protein electrophoresis, the enzyme protein isolated from Militaris cordyceps was found to have a molecular weight of 66 kDa.

Example 11

In order to confirm the temperature stability of the separated protein enzymes were reacted for 10 minutes at 20 ℃, 30 ℃, 40 ℃, 50, ℃ 60 ℃ each was analyzed for azocaine. As a result, protein enzymes were found to lose their activity at about 42 ℃ to 45 ℃.

Example 12

The effect of metal ions on the isolated protein enzyme was confirmed. Copper, zinc, cobalt, iron ions, and EDTA were used to make 2 mM each, and the same amount of enzyme was treated, and the final concentration was 1 mM. The activity was measured by azocaine assay. Results for the protein isolated from Militaris cordyceps sinensis is shown in Figure 7, for the protein isolated from Snow Cordyceps The results are shown in FIG.

In the above embodiment, the thrombus resolution was determined by using Militaris Cordyceps sinensis and Snow Cordyceps sinensis. Experiment and isolate the enzyme, but in addition to Cicada cordyceps (C. sobolifera), Beetlewormweed (C. sphecocephalaIt is apparent to those skilled in the art that thrombolytic enzymes can be obtained from other common Cordyceps sinensis in the same manner.

As can be seen from the above examples, Cordyceps sinensis extract and the enzyme of the present invention exhibits excellent thrombolytic action, can be administered orally and is easy to administer, and has no side effects of conventional thrombolytic agents such as systemic bleeding. Therefore, the Cordyceps sinensis extract and the isolated thrombolytic enzyme of the present invention can be used as safe thrombolytic and functional foods, health supplements, etc., in particular, since they are extracted and separated from the edible materials that have already been used, concern about side effects. It can be administered in a routine manner without the use can also be used for the prevention of vascular diseases.

Claims (7)

A thrombolytic agent comprising Cordyceps sinensis extract as an active ingredient, wherein the extract is obtained from Militaris cordyceps militaris or Paecilomyces japonica or a mixture thereof, and exhibits fibrinolytic activity. The thrombolytic method of claim 1, further comprising a conventional pharmaceutically acceptable excipient or carrier. The thrombolytic agent according to claim 1 or 2, wherein the extract is extracted from a mycelium. Enzyme protein having a molecular weight of 66 kDa exhibiting fibrinolytic activity isolated from Millyris cordyceps or Cordyceps militaris or Paecilomyces japonica or mixtures thereof. Thrombolysis comprising the enzyme protein of claim 4 as an active ingredient. 6. The thrombolytic method of claim 5, further comprising a conventional pharmaceutically acceptable excipient or carrier. delete