JP2006131551A - Pharmacological composition having fibrinolysis enhancement activity derived from basidiomycete - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a composition having thrombolytic action derived from Basidiomycetes. <P>SOLUTION: The pharmacological composition for thrombolysis is obtained from fruit bodies of Lyophyllum decastes and has thrombolytic action. The pharmacological composition contains a crushed product, a suspension, an extract solution or an extract of the fruit bodies obtained from the fruit bodies of Lyophyllum decastes and exhibiting thrombolytic action as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a thrombolytic pharmacological composition having plasminogen activator activity obtained from Hatake shimeji fruiting bodies. Specifically, a thrombolytic pharmacological composition comprising an extract or extract having plasminogen activator activity obtained from Hatake shimeji fruiting body as an active ingredient and exhibiting a thrombolytic action, and the like The present invention relates to a food / beverage product or a medicine for prevention and / or symptom improvement of contained thrombosis.

  Plasminogen activator is currently widely used as an instillation agent in the clinical setting as a thrombosis therapeutic agent, but the composition of the present invention obtained from Hatake-shimeji is only used as a raw material. It is safe to eat and effective for prevention.

  In Europe and the United States as well as in Japan, cerebral infarction and myocardial infarction are the leading cause of death after overcoming cancer. In recent years, this type of disease, such as transient cerebral ischemic attack, cerebral infarction (cerebral thrombosis, cerebral embolism), ischemic angina, myocardial infarction, arterial thrombosis, venous thrombosis, deep vein Patients with various diseases caused by thrombosis such as thrombosis, peripheral arterial occlusion, peripheral venous occlusion, pulmonary thrombosis, pulmonary embolism, etc. frequently occur and become a problem.

  The removal of blood clots mainly involves a fibrinolytic system (fibrinolytic system) via plasminogen and plasmin systems. When a thrombus occurs, plasminogen binds to fibrin, which is the main component of the thrombus, together with plasminogen activator, and is activated to plasminogen activator on the thrombus. The resulting plasmin breaks down fibrin and dissolves the thrombus. The plasmin produced by activation further activates other plasminogen molecules and prourokinase molecules, respectively, and is activated by Lys plasminogen (plasminogen activator faster than the original plasminogen). ), And urokinase (which also has the activity of activating plasminogen and converting it to plasmin). Thus, once plasminogen is activated, it is known that the thrombolytic system is activated in an autocatalytic manner (edited by Takeo Nomura et al., Illustrated Blood Cell-Physiology / Pathology / Clinical Platelet, Upper Blood, Coagulation, fibrinolysis, Chugai Pharmaceutical, 1994).

  Since thrombolysis in vivo is such a mechanism, plasminogen activator is considered to be effective to dissolve thrombus. In fact, there is a thrombolytic therapy using plasminogen activator as a method for dissolving and improving the thrombus in the clinic. Until now, two types of plasminogen activator were known, urokinase obtained from human urine and tissue plasminogen activator (t-PA) produced by cell culture such as melanoma (cancer). Since there is only a very small amount, and because it is an intravenous injection, it is necessary to completely purify it, so it was extremely expensive at 10,000 yen or more per 1 mg. In addition, since the half-life in blood is as short as 3 to 20 minutes, it is indispensable to continuously infuse intravenously, and there is a drawback that it causes great pain to patients. Streptokinase that indirectly activates plasminogen is produced by the culture of β-hemolytic streptococci, but it is also essential to be intravenously infused and has problems such as antigenicity.

  On the other hand, lumbrokinase (earthworm extract), nattokinase (natto extract) and the like have been reported as those having a thrombolytic effect when taken orally (Japanese Patent No. 3037355, Chemistry and Biology 29: 119). -123, 1991).

  Lumbrokinase is a serine protease consisting of six types having a molecular weight of 24 to 43 kDa, and these are homologous to plasmin, a type of serine protease that dissolves thrombus in vivo. In vitro tests have revealed that these enzymes have fibrinolytic activity and plasminogen activator activity. When humans eat 300-600 mg of lumbrokinase (mixture) per day, it significantly increases endogenous t-PA levels, significant transient increases in fibrin degradation products, and significant plasma fibrinolytic activity. An increase has been confirmed.

  Nattokinase contains a serine protease homologous to plasmin with a molecular weight of 27 kDa. In vitro tests have shown that it has fibrinolytic activity and prourokinase activation ability. When a person eats nattokinase equivalent to 2 packs of natto per day, a significant increase in endogenous t-PA, a transient increase in significant fibrin degradation products, and a significant increase in plasma fibrinolytic activity were confirmed. Yes.

  In addition, reports on the effects of mushrooms on the thrombus coagulation-fibrinolysis system (Fibrinolysis 8: sup1 1,74, 1994, Nippon Shokuhin 43: 318-321, 1996, J. Home Econ. Jpn. 50: 683-688). , 1999) and a patent application (Japanese Patent Laid-Open No. 2003-116535). Among them, plasminogen activator activity has been reported to be weak against oyster mushroom autolysates. According to this report, it has been reported that the autolysis product has an increase in the area of the dissolution window by the standard fibrin plate method about 2-3 times that of the original oyster mushroom.

  In addition, a report on a thrombolytic enzyme extracted from bonito salted (pig stew) (Japanese Patent Laid-Open No. 6-46851), a report on a thrombolytic enzyme using a semi-alkaline protease derived from Aspergillus or a modified polyethylene glycol thereof (Japanese Patent Laid-Open No. Sho 61) No. -215331), and a thrombus dissolving agent which is a krill aqueous extract or an extracted protease (Japanese Patent Laid-Open No. 61-30527).

  However, although earthworms have dietary experience in China and Korea as a herbal medicine or health food, there is a problem that modern people have considerable resistance to eating it. Natto is liking and disliked by people and contains a lot of vitamin K, so there is a problem that patients taking warfarin, a blood coagulation inhibitor, may not be able to take it because there is a risk of competing effects. Oyster mushrooms (Pleurotus osteatus) are known to have an adverse effect on feeding suppression when eaten raw due to the lectin mushroom toxin (Mushroom science, Tatsuyuki Sugawara, Asakura Shoten, Biochimica et Biophysica Acta 1474 (2000) 299-308) . According to the report, as a result of a 7-day dietary test with 5% oyster mushroom added, food consumption was significantly reduced to 13.7% compared to the control diet, and body weight was also significantly reduced from the start of the test. Has been confirmed. Thus, there is a problem that it is virtually impossible to eat oyster mushrooms live. Other edible mushrooms also have an anti-feeding effect when eaten raw by type. Skipjack salty salt contains a lot of salt, and if you eat it in large quantities to increase its effect, it will cause excessive intake of salt, which increases the risk of high blood pressure.

  In addition to the fact that there is an artificial thrombolytic effect in vitro instead of in vivo, Ganoderma lucidum metal protease inhibits thrombin that forms thrombus and inhibits thrombus. There is a report (Mycologia, 92 (3), 2000, pp. 545-552) having an effect of preventing the formation of. In addition, there is a report (Biosci. Biotechnol. Biochem., 63 (12), 2130-2136, 1999) that metal protease extracted from the mushroom Armillariella mellea decomposes fibrinogen and fibrin. However, these proteases are not the thrombolytic mechanism through plasminogen activator activity that is most effective in activating the fibrinolytic system, and the effect when taken orally is not clarified at all.

  In this way, among the things that modern people can easily obtain, procure and eat, especially in edible mushrooms with high protease activity, the fibrinolytic system in vivo is mediated through plasminogen activator activity. There are no known mushrooms that can be activated with confidence and can be eaten with peace of mind without causing food intake suppression, and there is a problem that cannot be put into practical use as a thrombolytic food. As a result of extensive examination of various mushrooms, the present inventor has unexpectedly found that the fruit body itself, suspension, extract, or extract of Hatake shimeji (Lyophyllum decastes (Fr.) Sing) has been used in in vitro tests. It was found that there is a very strong plasminogen activator activity.

  In addition, laboratory animals such as rats are used as an effective means for evaluation and screening of pharmaceuticals and foods having a bioregulatory function because they are easy to breed and require a small amount of administration sample. . However, rodents, including rats, including rabbits, have very low plasma plasminogen levels compared to humans (according to Millstone's report, Journal of Immunology 42, 109-116, 1941). Is described as having no "fibrinolytic activator".) Even if a certain test food has plasminogen activator activity, the amount of plasmin produced by activation is very small. There was a problem that plasminogen activator activity was hardly detectable. In fact, according to a report by Murata et al. (J. Home. Econ. Jpn. 50 (7) 689-694, 1999), a rat plasma euglobulin fraction was prepared and fibrinolytic activity was measured on a standard fibrin plate. That is, even when EFA was measured, the activity was weak, and a significant difference could not be confirmed between the target group and the test group.

  For the above reasons, there has been no method for evaluating the effects and activities of foods having plasminogen activator activity except for feeding them to humans. Therefore, in the case of a material with little food experience, there was a problem that it had to be evaluated using humans with risk. In addition, it is necessary to prepare a large amount of administration sample for the evaluation, and there is a problem that many test foods cannot be evaluated quickly and easily. Thus, as a result of intensive studies, the present inventors have developed a specific plasma plasminogen activator activity evaluation method using rats by creating an environment similar to that of humans. Furthermore, by using this evaluation method, when a rat smashed fruit body (for example, powder), suspension, extract or extract is orally ingested into a rat, it does not adversely affect eating suppression. It has been found that plasma plasminogen activator activity is increased, and the present invention has been completed.

  As for the pharmacological effect of Hatake-shimeji, the improvement effect on hypertension, hyperlipidemia and obesity has been reported so far (Japanese Patent Laid-Open No. 2003-73299), but the fibrinolytic system enhancing activity has not been known.

Japanese Patent No. 3037355 JP 2003-116535 A JP-A-6-46851 JP-A-61-215331 JP 61-30527 A JP 2003-73299 A Chemistry and Biology 29, 119-123, 1991 Fibrinolysis 8: sup1 1,74,1994 Japanese Restaurant Journal 43, 318-321, 1996 J. et al. Home Econ. Jpn. 50, 683-688, 1999 Mushroom science edited by Tatsuyuki Sugawara Asakura Shoten (1997) Biochimica et Biophysica Acta 1474, 299-308, 2000 Mycologia 92 (3), 545-552, 2000 Biosci. Biotechnol. Biochem. 63 (12), 2130-2136, 1999 Journal of Immunology 42, 109-116, 1941 J. et al. Home.Econ. Jpn. 50 (7), 689-694, 1999 Takeo Nomura et al., Illustrated Blood cell-Physiology / Pathology / Clinical Platelet, Upper Blood, Coagulation, Fibrinolysis, Chugai Pharmaceutical, 1994

  The object of the present invention is to obtain a thrombolytic agent having plasminogen activator activity which is obtained from Hatake shimeji mushrooms which have been eaten since ancient times as a result of searching many edible mushrooms and which can be mass-produced and is highly safe under the oral environment. It is to provide a pharmacological composition.

  In the present invention, among basidiomycetes, plasminogen activator activity has been recognized in Hatake-Shimeji, which has recently been successfully artificially cultivated all year long and has come to market, and it is suspended in water or extracted with water, for example. To use. The raw materials are inexpensive, have been taken from the mouth for many years, and even when eaten raw, there is no adverse effect of eating suppression like oyster mushrooms, so it is excellent in safety under the oral cavity.

The present invention is summarized as follows.
In a first aspect, the present invention provides a thrombolytic pharmacological composition characterized in that it is obtained from the fruit body of Hatake Shimeji and has a thrombolytic action.

The fruiting body refers to the entire mushroom including the reproductive organs that produce spores.
The thrombolytic action refers to thrombolysis caused by plasminogen activator-dependent or independent fibrinolytic activity.

  In addition, the pharmacological composition refers to a processed product of a fruit body of Hatake shimeji mushroom, which includes the active ingredient of the present invention, or a composition including the processed product. Here, the treated product refers to a substance obtained by performing artificial treatment such as drying, pulverization, and extraction.

  In an embodiment of the present invention, the pharmacological composition may contain, as an active ingredient, a pulverized product, suspension, extract, or extract of a fruiting body that exhibits a thrombolytic action obtained from the fruiting body of Hatake Shimeji.

  In another embodiment, the extract or extract is an extract or extract obtained by water extraction of Hatake shimeji fruiting bodies.

  In still another embodiment, a dried product of the fruit body can be used as the fruit body.

  In another embodiment, the thrombolytic action is a thrombolytic action due to plasminogen activator activity at least in part.

  In the second aspect, the present invention provides a food or drink for the prevention of thrombosis and / or symptom improvement containing the above-mentioned pharmacological composition.

  As one of the embodiments, the container containing the food or drink of the present invention can be labeled as being used for prevention of thrombosis and / or symptom improvement.

  In a third aspect, the present invention provides a pharmaceutical agent for preventing thrombosis and / or ameliorating symptoms, comprising the above pharmacological composition.

  As described above, the present invention is characterized by a thrombolytic pharmacological composition obtained from the fruit body of Hatake Shimeji and having a thrombolytic action. The composition of the present invention is inexpensive, has no side effects such as suppression of feeding, is highly safe and has a thrombolytic action comparable to or exceeding that of oyster mushrooms. The product can be used as a food or as a medicine for the prevention of thrombosis and / or the improvement of symptoms.

(Hatake shimeji)
Hatake shimeji fruiting body used in the present invention may be either a natural fruiting body naturally occurring in the natural world or an artificially cultivated fruiting body, but an artificially cultivated hatake shimeji fruiting body with a stable quality such as component content. Is preferred. In the case of natural Hatake shimeji fruiting bodies that naturally grow in nature, the content of ingredients varies depending on the habitat area and climate, etc., and it is not possible to harvest stably throughout the year. It is not affected by climate and climate, and can be harvested stably year-round. Moreover, although it does not specifically limit as an artificial cultivation method, The fungus bed cultivation which can harvest the bamboo shimeji mushroom with stable quality, such as a component content, cheaply and year-round stably is preferable. Here, fungal bed cultivation refers to a method of inoculating a material composed of a water retaining body and a nutrient source with an inoculum, and cultivating under an environment in which temperature, humidity, illuminance, and the like are controlled. For example, a method as described in JP-A-2001-131082 can be given as an example, but is not limited thereto.

  The Hatakeshimeji strains used in the present invention, in any strains including the strain resulting from the commercial strain and nature as long as a strain belonging to Hatakeshimeji containing the ingredients shown the effect of dissolving thrombus from fruiting bodies of Hatakeshimeji Good.

(Pharmacological composition and production method)
The pharmacological composition of the present invention may be any composition as long as it contains a component having an action of dissolving a thrombus obtained from Hatake shimeji fruiting body, for example, a thrombus obtained from Hatake shimeji fruiting body is dissolved. In addition to an extract or extract having an action, or a composition containing such an extract or extract as an active ingredient, a pulverized product obtained by pulverizing Hatake shimeji fruit bodies as they are or after air drying, hot air drying or freeze drying, Examples thereof include granulated, encapsulated, tableted and pastes by conventional methods. Further, the suspension or suspension of the pulverized product thus pulverized may have any form as long as it contains the action of dissolving the thrombus.

  The extract or extract showing the action of dissolving the thrombus in the present invention can be obtained by a known method. For example, Hatake shimeji fruiting body is water at 0 ° C. to 100 ° C., preferably 0 to 40 ° C., more preferably 4 ° C. to room temperature, an appropriate buffer, an appropriate organic solvent (for example, alcohols such as methanol and ethanol), or Contains an activity of dissolving a thrombus by extraction with a mixed solution of an appropriate organic solvent and water (for example, ethanol water), preferably water, preferably for 5 minutes or more, preferably about 60 minutes, and then centrifuged or filtered. An extract can be obtained. The amount of the extraction liquid at the time of extraction is not particularly limited, but it is preferable to use 10 to 20 times the amount of water with respect to the dry mushroom weight in terms of cost, extraction efficiency, and the like. As the extraction solvent, a solvent such as ethanol can be added as appropriate, but it is desirable to extract only with water from the viewpoint of cost and ease of handling at the production site. Cellulase and peptidase enzymes can also be used to improve the recovery rate during extraction.

  The fruiting body may be extracted as it is, but it is preferable that the fruiting body is extracted from the fruiting body that can be preserved by drying because an extract or extract having excellent quality can be obtained. The extract is obtained by further processing the extract, and includes liquid or solid forms such as concentrates, lyophilized products, and partially purified products. The drying method is not particularly limited as long as it is a known drying method such as hot air drying, air drying, spray drying, vacuum (or reduced pressure) drying, freeze drying method, etc. Then, the freeze-drying method is more preferable. In addition, it is preferable to extract the fruit body after drying and then pulverizing it, since the extraction rate is improved. For example, mushrooms or pulverized mushrooms are prepared at 0 ° C. to room temperature, preferably near 0 ° C., a suitable buffer solution, a suitable organic solvent (for example, alcohols such as methanol and ethanol), or an organic solvent-water mixture (for example, Suspended in ethanol water), stirred as it is, or disrupted by sonication, grinding, French press, etc., followed by solid-liquid separation using solid-liquid separation operations such as centrifugation and filtration alone or in combination. Then, if necessary, the extract can be prepared by subjecting the extract to a treatment such as concentration, lyophilization, or purification using a method such as concentration, lyophilization, or purification. Further, the extraction operation can be performed a plurality of times to improve the extraction efficiency.

  An active substance that dissolves thrombus present in an extract such as cold water extraction may be further purified. In this case, known separation and purification methods can be combined appropriately. Here, it is presumed that the active substance (or active ingredient) is probably an enzyme because at least part of it has plasminogen activator activity. Methods that can be used for further purification of the active substance include, for example, methods utilizing solubility such as salt precipitation and organic solvent precipitation, methods utilizing differences in molecular weight such as dialysis, ultrafiltration, gel filtration, A method utilizing a difference in charge such as ion exchange chromatography, a method utilizing a specific affinity such as affinity chromatography, a method utilizing a difference in hydrophobicity such as hydrophobic chromatography and reverse phase chromatography, Further, there is a method using a difference in isoelectric point such as isoelectric focusing. Further, the concentration after extraction and purification may be any method as long as it is a known method, and specific examples include ultrafiltration and vacuum heating concentration. Further, the drying after extraction and purification may be any known method as long as it is a known method, and specific examples include air drying, heat drying, spray drying, and freeze drying.

  The plasminogen activator activity of the composition exhibiting the action of dissolving the thrombus thus obtained is measured by adding plasminogen and using a standard fibrin plate as shown in detail in the Examples. Can do.

  At least a part of the pharmacological composition of the present invention has a thrombolytic action due to the enhancement of the fibrinolytic system caused by a strong plasminogen activator activity equivalent to or higher than that of known basidiomycetes. The composition of the present invention may further contain plasminogen-independent fibrinolytic activity as an active ingredient derived from Hatake shimeji mushroom. That is, the pharmacological composition of the present invention may contain at least two active substances having plasminogen-dependent and independent fibrinolytic activity. Further, these active substances may be separated by the above purification method and included as separate components in the composition. Furthermore, no adverse effects such as inhibition of feeding and weight loss due to lectins reported in mushrooms such as oyster mushrooms were observed in the Hatake shimeji of the present invention, and plasma plasminogen activator activity was enhanced. Can take active ingredients such as plasminogen activator derived from Hatake Shimeji without adverse effects such as suppression of feeding or weight loss, and exerts its effect on prevention of thrombosis and / or improvement of symptoms Can be fully expected.

  Therefore, the pharmacological composition of the present invention is used as a thrombosis-preventing and / or symptom-improving agent, and a food or drink (for example, health food, functional food, etc.) or a pharmaceutical for thrombosis-preventing and / or symptom-improving. It can be used as an active ingredient.

  The pharmacological composition containing an ingredient that dissolves a thrombus according to the present invention usually has a thrombolytic action by ingesting an effective amount within a range of about 1 mg to about 20 g / kg body weight / day in terms of dry fruiting body. However, the intake can be adjusted as appropriate according to symptoms, sex, age and the like. In addition, when used as a pharmaceutical for the prevention and / or symptom improvement of thrombosis, for example, a fruit body pulverized product (eg, powder), suspension, extract, extract, or extract thereof is further purified. Or a mixture containing an active ingredient, or a product purified to be substantially single, and mixed with an excipient or carrier generally used in pharmaceutics. It can be used in the form of orally administered drugs such as soft or hard capsules, tablets, solutions, powders, suspensions, emulsions, pastes and the like.

  The composition can also contain other additives such as disintegrants, colorants, binders, pH adjusters, flavoring agents, stabilizers, preservatives, wetting agents, lubricants. Etc. can be selectively included as appropriate.

  Examples of the carrier and excipient include lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, gum arabic, olive oil, sesame oil, cocoa butter, ethylene glycol, etc. A plurality can be selected and contained, but is not limited thereto.

  Examples of additives include lactose, mannitol, glucose, dextrin, hydroxypropyl cellulose, methyl cellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone, magnesium aluminate metasilicate, magnesium carbonate, magnesium stearate, calcium glycolate, Examples include, but are not limited to, glutamic acid, aspartic acid, sucrose, gelatin, hydroxypropylmethylcellulose phthalate, talc, tragacanth, gastric or enteric substances.

  The pharmacological composition of the present invention can also be used as a food or drink for preventing thrombosis and / or improving symptoms. In this case, such a composition can be used as a part of the raw material for food and drink, or can be added and blended after the production process or after production. Or the extract obtained by making it above can also be added to a drink as it is. The container containing the food / beverage product of the present invention can be labeled as being used for prevention of thrombosis and / or symptom improvement.

  Such foods and drinks are not limited to the following, for example, yogurt, drink yogurt, juice, milk, soy milk, alcoholic beverages (alcoholic beverages), coffee, tea, sencha, oolong tea, sports beverages, etc. Cookies, bread, cakes, baked confectionery such as rice crackers, Japanese confectionery such as mutton, frozen confectionery such as pudding, jelly and ice cream, confectionery such as chewing gum and candy, snacks such as crackers and chips, udon and soba Noodles, fish paste products such as kamaboko, ham, fish sausage, seasonings such as miso, soy sauce, tsukemono, vinegar, alcohol, dressing, mayonnaise, sweetener, tofu, konnyaku, other boiled fish, dumplings, croquettes Specific examples such as salads, soups, stews, etc. That. The compounding amount of the active ingredient is the same as in the case of the above pharmaceutical product.

  The blending amount of the extract at the time of preparing various beverages using the extract obtained by the above method as a raw material can be appropriately determined so as to have an excellent flavor, but it has a sufficient effect of dissolving the thrombus of Hatake Shimeji. In view of expectation, it is preferable to set so as to contain about 0.1 g to about 20 g of an extract from Hatake shimeji mushroom in terms of dry fruit body weight per beverage in a container. In the preparation of various beverages, any sugars, fragrances, food additives, etc. that are used in the formulation design of various ordinary beverages can be used. In addition, ginseng extract, sorghum extract, eucalyptus extract, tochu tea You may use together with other functional materials, such as an extract. Moreover, about the manufacturing method etc. of various drinks, the existing reference books, for example, "revised new edition, soft drinks" (Korin Co., Ltd.) etc. can be referred. Hereinafter, typical beverages will be described, but the beverage of the present invention is not limited to the following beverages.

  (Tea-based beverage) Hatake-shimeji mushroom extract is easy to match with any tea such as green tea, semi-fermented tea (oolong tea), fermented tea (tea), especially Chinese tea, especially golden katsura or jasmine tea leaves And the special flavor of mushrooms is masked. In addition, the above tea leaves may be used alone, or in combination with tea leaves that can be expected to promote health, such as Tochu tea, Kashiwa leaf, Kumakuma tea, Gabaron tea, corn tea, hub tea, chrysanthemum tea, etc. Also good. The method for producing the tea leaf extract is not particularly limited, but it is desirable to use 0.6% by weight or more of tea leaves in order to mask the flavor unique to mushrooms. The mushroom extraction step and the tea leaf extraction step are preferably separate steps from the viewpoint of efficient extraction of the active ingredient that dissolves the thrombus. For example, a bamboo shimeji extract separately prepared in the tea leaf extract Is preferably added and blended.

  (Nutrition drink) An energy drink can be produced by adding and blending a bamboo shimeji extract simultaneously with or before or after the ingredients normally used in the production of the energy drink. Mushroom flavor is masked by the body feeling due to and the characteristic flavor due to vitamin mix and healthy drink flavor.

  (Fruit / vegetable drinks) Fruit / vegetable drinks can be selected from orange, apples, grapes, peaches, strawberries, bananas, lemons and other fruit juices, and tomatoes, carrots, cabbage, celery and other vegetable juices. After selecting two or more kinds, it can be manufactured by adding and blending a bamboo shimeji mushroom extract. Such a mushroom shimeji mushroom extract-containing fruit / vegetable beverage has a mushroom flavor that matches well with fruit juice or vegetable juice. It becomes easy to drink.

  (Carbonated drinks) Carbonated drinks can be produced by adding and blending Hatake shimeji mushroom extract simultaneously with or before and after the ingredients normally used in the production of carbonated drinks. Carbonation is the same as ordinary carbonated drinks. What is necessary is just to make it a carbonic acid pressure (0.1-0.4Mpa). Carbonated beverages are generally characterized by a sharp aftertaste, and the mushroom flavor can be masked by making the carbonated beverage a mushroom flavor that feels a relatively long aftertaste.

  (Low-calorie beverages) High-sugar beverages have the image of causing obesity, and in order to dispel the image, high-sweetness sweeteners such as stevia, aspartame, and sucralose, and sugar alcohols such as erythritol and maltitol A low calorie beverage having a low calorie content is prepared using a low calorie sweetener. Addition and blending of the bamboo shimeji mushroom extract can be performed in an arbitrary process, and the mushroom odor can be masked by selecting a flavor. Preferable examples of such flavor include ume flavor and lychee flavor.

  (Alcoholic beverages) Alcoholic beverages can be produced by adding and blending Hatake shimeji and Hatake shimeji mushroom extracts in any process of producing alcoholic beverages such as happoshu, whiskey and liqueurs. It is preferable to produce alcoholic beverages such as whiskeys and liqueurs by adding and blending Hatake shimeji mushroom extract. Such a bamboo shimeji mushroom extract-containing alcoholic beverage has a mushroom flavor masked and is easy to drink.

(Measurement of plasma plasminogen activator activity in rats)
The measurement of plasminogen activator activity in plasma using rats was originally based on the fact that plasminogen activator activity by standard fibrin plates is not as it is because rats have very little plasma plasminogen activity. Although it is almost impossible to detect or to be measured, there are many errors and comparative evaluation is almost impossible, but the present inventors have a substantially sufficient amount of plasminogen corresponding to the substrate of plasminogen activator. This time, it was found that plasminogen activator activity in rat plasma can be evaluated by adding together. This method makes it possible to search for foods having an activity of enhancing (enhancing) plasma plasminogen activator activity when taken orally by rats.

  Specifically, in the method for evaluating the activity of a test sample presumed to contain plasminogen activator activity by oral administration to a rat, the measurement method comprises administering the test sample to a rat and then collecting the test sample from the rat. Adding plasminogen to the treated plasma and assessing plasminogen activator activity through measurement of fibrinolytic activity.

  As the plasminogen that can be used in this method, for example, plasminogen as a reagent derived from bovine or human can be used. At this time, even if plasminogen contains a trace amount of plasmin and has fibrinolytic activity, it is possible to examine the plasminogen activator activity of the test sample by subtracting it. Therefore, there is no restriction on the presence or absence of trace amounts of plasmin. In addition, the amount of plasminogen added at this time may be any amount as long as it can detect a difference in the amount of plasma plasminogen activator, but is preferably 0.001 to 0.25 U / A specimen, more preferably 0.0025 to 0.05 U / sample is preferred. Here, 1 U of plasminogen is converted into plasmin which is active by urokinase, and then soluble in perchloric acid contained in 5 ml of a reaction solution with α-casein at pH 7.5 and 37 ° C. for 20 minutes. Is defined as the amount of enzyme that increases the absorbance at 275 nm by 1.0.

  The plasminogen concentration in human blood is approximately 2 μM (= approximately 180 μg / ml, thrombus formation and thrombolysis, edited by Osamu Matsuo, Koji Suzuki, Yasuo Ikeda, Japan Science and Technology Association, published in February 1991). When converted to bovine-derived plasminogen (manufactured by Sigma), it corresponds to 0.026 U / specimen, and it is considered that the human blood concentration is almost reproduced.

  Furthermore, plasminogen activator activity contained in a test plasma sample is evaluated by measuring plasma fibrinolytic activity using a standard fibrin plate. It is preferable to evaluate the fibrinolytic activity of the rat, which is originally weak and hardly detectable, or has a large error and is not suitable for evaluation. However, when the concentration is excessively increased, the area of the dissolution window is excessively increased, and an error is increased to determine the intensity of plasminogen activator activity. Therefore, from the viewpoint of sensitivity and ease of discrimination, the plasma euglobulin fraction is preferably prepared at a concentration about twice as high as that of the original plasma, more preferably about 4 times as high. .

  The fibrinolytic activity can be measured using a standard fibrin plate and the area of the dissolution window as an index. However, any method using solidified fibrin may be used. For example, the fibrin degradation activity was prepared in a test tube. Method to measure time to dissolve artificial thrombus (coagulated fibrin) (clot lysis time), Method to measure time to dissolve artificial thrombus when using Euglobulin fraction (euglobulin lysis time) From the viewpoint of simplicity, a measurement method using a standard fibrin plate is preferable. Plasminogen activator activity is measured in the absence of plasminogen from the value obtained by subtracting control (sample-free MQ water) from the fibrinolysis window area measured in the presence of plasminogen for the same concentration of sample. It is expressed as a difference value obtained by subtracting the melting window area value. This is because the plasminogen used contained a slight amount of active plasmin, so that a slight dissolution window was generated even when the control sample was not added (MQ water). In addition, fibrinolytic activity in the absence of plasminogen is independent of plasminogen activator and is distinguished from plasminogen activator activity.

  The concentration of fibrin is preferably 0.1 to 0.7%. However, if the concentration is too dilute, solidification becomes insufficient. Conversely, if the concentration is too high, the time until fibrin is dissolved increases and the error increases. Therefore, the concentration of fibrin is more preferably 0.2 to 0.4%.

  In the standard fibrin plate, plasminogen is mixed in the fibrin used, but this amount of plasminogen is not sufficient to detect plasminogen activator activity in plasma. It is necessary to add plasminogen as in the method developed this time.

  The present invention will now be described in further detail in the following examples. In addition, this invention is not limited at all by the said Example.

Preparation of extract from Hatake shimeji fruiting body 1 part by weight (1.5 g) of freeze-dried pulverized product of Hatake shimeji fruiting body purchased at a supermarket in Gunma Prefecture is placed in a test tube, and 20 parts by weight (30 ml) of distilled water at about 4 ° C. Was added and stirred to prepare a suspension. This suspension was centrifuged at 10000 × g, 4 ° C. for 10 minutes to obtain an extract as a yellowish brown supernatant.

  For comparison, an extract was also obtained from freeze-dried pulverized oyster mushroom fruit bodies in the same manner. Furthermore, an extract was obtained in the same manner from the oyster mushroom suspension that had been self-digested at room temperature for 24 hours.

Fibrinolytic activity and plasminogen activator activity of Hatake shimeji mushroom extract Standard fibrin plates were prepared with a bovine fibrin concentration of 0.3% (Astrup & Mullertz method; (Astrup & Mullertz method; Arch. Biochem. Biophys. 40, 346). -351, 1952)). Each mushroom extract of Example 1 was diluted with MQ water (Millipore) so that the concentrations shown in Table 1 were obtained. As a positive control having plasminogen activator activity, a commercially available health food containing lumbrokinase (product name: Lumbrengold, manufactured by Waki Pharmaceutical Co., Ltd., containing 23.5% earthworm extract per solid) is also available. Similarly diluted as shown in the table. Bovine plasminogen (manufactured by Sigma) was dissolved in 0.1 M sodium phosphate buffer pH 7.4 to give 135 μg protein / ml (0.67 U / ml).

  30 μl of this mushroom extract and plasminogen solution mixed 1: 1 (the amount of plasminogen in this is 0.01 U), or mushroom extract and 0.1 M sodium phosphate buffer pH 7. 30 μl of 4 mixed with 1: 1 was added dropwise to the prepared fibrin plate and incubated at 37 ° C. for 18 hours.

At that time, the area (mm ² ) of a clear zone (dissolution window) formed by dissolving fibrin was measured. The results are shown in Table 1 and FIG.

  As can be seen from these results, the lumbrokinase-containing health food measured as a positive control had an increased dissolution window in the case where plasminogen was added, compared to the case where plasminogen was not added.

Lumbrokinase-containing health foods have a strong activity of directly degrading fibrin, but by separately activating plasminogen, the activity of degrading fibrin can be further enhanced, that is, plasminogen activator activity is increased. Something was confirmed. That is, since lumbrokinase originally has fibrinolytic activity, especially when the addition concentration is high, the fibrin degradation activity is too strong and it is difficult to distinguish the net plasminogen activator activity. when .1%, dissolution window area without plasminogen (38.5 mm ^2), and plasminogen only (i.e. MQ water, 56.7mm ²⁾ a dissolution window area, plasminogen added (113 mm ²⁾ The value 17.8 mm ² subtracted from is the dissolution window area produced by the net plasminogen activator activity when 0.1% was added.

Similarly, Hatake shimeji has an increased dissolution window for those with plasminogen added compared to those without plasminogen added. Hatake shimeji has a strong fibrin-degrading activity, but it has been found that activating plasminogen has a fibrin-degrading activity, that is, plasminogen activator activity. For example, when the addition concentration is 0.1%, the dissolution window area caused by the net plasminogen activator activity of Hatake shimeji is 25.7 mm ^2, which is a high value compared to the value of lumbrokinase. I knew that there was. The plasminogen used contained a slightly active form of plasmin, so plasminogen alone produced a slight dissolution window, but subtracting it also has these plasminogen activator activities. I understand that.

  Hatake shimeji was also found to have strong fibrinolytic activity independent of plasminogen activator (see FIG. 1 or Table 1). In the comparison at 1% concentration, even if calculated simply, Hatake shimeji has an activity about 1.6 times greater than that of Lumbrokinase.

  For comparison, oyster mushrooms and oyster mushrooms at room temperature for 24 hours were similarly measured for fibrin degrading activity and plasminogen activator activity. These results are shown in Table 2 and FIG.

Thus, oyster mushroom plasminogen activator activity is very weak, in other words, when the addition concentration is 1%, the dissolution window area produced by oyster mushroom net plasminogen activator activity is only 2.2 mm. ² and the activity of the oyster mushrooms was not enhanced even after self-digestion.

Fibrinolytic enzyme activity of Hatake shimeji mushroom extract with synthetic substrate As a positive control, a commercially available lumbrokinase-containing health food and an extract of Hatake shimeji mushroom prepared by the method of Example 1 whose activity was confirmed in vitro were subjected to synthetic substrate degradation. Activity was measured.

  As synthetic substrates, Pyr-Gly-Arg-MCA, a substrate for urokinase purchased from Peptide Institute, HD-Ile-Pro-Arg-pNA, a substrate for tPA purchased from Sigma, and Sigma HD-Val-Leu-Arg-pNA and HD-Val-Leu-Lys-pNA, which are substrates for plasmin, purchased from the company, were used.

  The final concentration of each substrate is 0.25 mM, mixed with each sample, and reacted at 37 ° C. for 10 minutes. After completion of the reaction, 1.5 M acetic acid is added twice as much as the reaction solution and stirred to stop the reaction. Of these, 160 μl was transferred to a microplate, and 405 nm and fluorescence (Ex370, Em440) were measured to determine the relative reactivity to each lumbrokinase-containing health food. The results are shown in Table 3.

  Thus, it was found that Hatake shimeji has stronger plasmin activity, almost no urokinase activity, and tPA activity than lumbrokinase.

Examination of evaluation method of plasminogen activator enhancement activity by single administration of SD rat. MQ water was dissolved in MQ water as a control and commercially available lumbrokinase-containing health food as 2.5% as a positive control. The supernatant was orally administered to SD rats (14 weeks old, male, 1 each) at 10 ml / kg (250 mg / kg) with a stomach tube. Two hours after administration, anesthesia was performed with a pentobarbitur tool, and blood was collected from the abdominal aorta in a blood collection test tube containing heparin sodium. This was centrifuged at 3000 × g for 15 minutes to obtain plasma.

  A plasma euglobulin fraction was prepared from this plasma (modified method of MJ Gallimore et al., Thrombos. Diathes. Haemorr. 26, 295-310, 1971). That is, under ice-cooling, 19 times the amount of ice-cold water was added, 0.25% acetic acid was added to adjust the pH to 5.70, and after 10 minutes, centrifuged at 3000 × g for 10 minutes. The precipitate was separated. The resulting precipitate was dissolved by adding 0.12 M sodium acetate, 0.34 mM citrate buffer pH 7.4 so as to be equivalent to 1, 2 or 4 times the original concentration. The plasma euglobulin fraction thus prepared was adjusted to 0.1 M sodium phosphate buffer pH 7.4 so that bovine plasminogen (manufactured by Sigma) was 270 μg protein / ml (1.34 U / ml). 45 μl of the dissolved solution mixed to 2: 1 (the amount of plasminogen in this is 0.02 U) is prepared and added dropwise to a standard fibrin plate prepared in the same manner as in Example 2. Similarly, fibrinolytic activity was measured.

  For comparison, a measurement was also performed using a 0.1 M sodium phosphate buffer pH 7.4 mixed in a similar manner to 2: 1 instead of the plasminogen solution.

  As a result, as shown in FIG. 3, the difference between the lumbrokinase-administered rat and the MQ-administered rat, which was a positive control, was clearer in the plasminogen-added rat than in the buffer-added one. Furthermore, it was found that the difference between the plasma euglobulin fraction and the concentration obtained by doubling and doubling the plasma euglobulin fraction became clearer as compared with the solution obtained by dissolving the euglobulin fraction at a 1-fold concentration. Thus, when the concentration of plasma euglobulin fraction is increased, plasminogen is added, and fibrinolytic activity is measured with a fibrin plate, it is difficult to measure plasminogen activator activity due to the low amount of plasma plasminogen. It was also found that plasminogen activator activity can be clearly measured in normal rats.

Plasminogen activator-enhancing activity when SD rats are mixed with diet AIN-93G standard feed as a control diet, a diet obtained by adding 5% of lyophilized powder of Hatake shimeji mushroom to the standard diet, and commercially available as a positive control Each of the SD rats (14 weeks old, male, 8 animals in each group) was allowed to freely ingest a diet supplemented with 2% lumbrokinase-containing health food, and anesthetized 9 days later with a pentobarbitool Then, blood was collected from the abdominal aorta in a blood collection test tube containing heparin sodium. This was centrifuged at 3000 × g for 15 minutes to obtain plasma.

  A plasma euglobulin fraction was prepared from this plasma according to the method of Example 3 so as to be equivalent to 4 times the original concentration. To this plasma euglobulin fraction, a solution obtained by dissolving bovine plasminogen (manufactured by Sigma) in 0.1 M sodium phosphate buffer pH 7.4 so as to be 270 μg protein / ml (1.34 U / ml) Prepare 45 μl of 2: 1 mixture (the amount of plasminogen in this is 0.02 U), drop it onto a standard fibrin plate prepared in the same way as in Example 2, and measure fibrinolytic activity in the same way. did.

  As a result, as shown in FIG. 4, the lumbrokinase mixed-fed group that is a positive control and the Hatake-shimeji mixed-fed group that is the test subject are both significantly less than the standard diet group that is a control at a risk rate of 2% or less. It was found that plasminogen activator activity was enhanced. The enhancement rate was 165% for Lumbrokinase as compared to the control, whereas Hatake shimeji was 191%, which was higher than Lumbrokinase. In this way, it was found that Hatake shimeji significantly enhances plasma plasminogen activator activity.

  In addition, when food intake, water intake, and body weight gain were examined in this test, as shown in FIGS. There was almost no difference in the amount of water, and there was no statistically significant difference. On the other hand, as for the weight gain, 8 out of 8 controls accidentally had a lower weight gain until the 2nd day, so the control seemed to be lower, but the tendency of the increase after that was the control and Hatake Shimeji diet group It was confirmed that there was no statistical difference, and therefore there was no feeding suppression.

[Comparative Example 1]
Fibrinolytic activity in the absence of plasminogen Plasma Euglobulin fraction prepared by the method of Example 4 to the original 1-fold concentration and 4-fold concentration (method of MJ Gallimore et al .; Trombos. Diathes. Haemorr. 26 , 295-310, 1971), respectively, instead of plasminogen, 0.1 μM sodium phosphate buffer pH 7.4 was mixed at a ratio of 2: 1 to prepare 45 μl, and prepared in the same manner as in Example 2. The fibrinolytic activity was measured in the same manner by dropping on a standard fibrin plate.

  As a result, as shown in FIG. 8, when the fibrinolytic activity was measured without adding plasminogen at both the 1-fold concentration and 4-fold concentration, the dissolution window was small and it was difficult to confirm the fibrinolytic activity. It was confirmed that there was no statistically significant difference between the Lumbrokinase and Hatake Shimeji administration groups compared to the control group. That is, it was found that this method cannot detect the effect of administration of lumbrokinase and hatake shimeji, which have enhanced plasminogen activator activity.

[Comparative Example 2]
Plasminogen activator activity of various mushrooms According to the same procedure as in Example 2, the fibrinolytic activity of maitake, numerisugitake, nameko and tamagotake was measured in the presence or absence of plasminogen (PG), and plasminogen activator Activity was evaluated. The results are shown in Table 4 and FIG.

  As is apparent from these results, none of the mushrooms tested have plasminogen activator activity.

A comparison of the fibrinolytic activity of Hatake shimeji, lumbrokinase and oyster mushroom in the presence or absence of plasminogen is shown. A comparison of the fibrinolytic activity of oyster mushrooms and oyster mushroom autolysates in the presence or absence of plasminogen is shown. Fig. 2 shows the influence of plasma euglobulin fraction concentration on the measurement of fibrinolytic activity in rats. The measurement result of the fibrinolytic activity of the plasma at the time of administration of Hatakeshimeji or lumbrokinase mixed diet in rats in the presence of plasminogen is shown. Error bars represent SE. The influence which the food intake of Hatake-Shimeji or Lumbrokinase in rats gives to the food intake is shown. The influence which the food intake of Hatake-Shimeji or Lumbrokinase in rats gives to the water intake of rats is shown. Fig. 2 shows the influence of administration of Hatake shimeji or lumbrokinase diet on rat weight gain in rats. The measurement result of the fibrinolytic activity of the plasma at the time of administration of Hatake shimeji or lumbrokinase mixed diet in the rat in the absence of plasminogen is shown. Error bars represent SE. A comparison of the fibrinolytic activity of maitake, numerisutake, nameko and tamagotake in the presence or absence of plasminogen is shown.

Claims (7)

  A pharmaceutical composition for thrombolysis obtained from the fruit body of Hatake-shimeji and having a thrombolytic action.   The pharmacological composition according to claim 1, wherein the pharmacological composition contains, as an active ingredient, a pulverized product, suspension, extract or extract of a fruiting body that exhibits a thrombolytic action obtained from the fruiting body of Hatake Shimeji. Composition.   The pharmacological composition according to claim 2, wherein the extract or extract is an extract or extract obtained by water extraction of Hatake shimeji fruiting bodies.   The pharmacological composition according to any one of claims 1 to 3, wherein a dried product of the fruiting body is used as the fruiting body.   The pharmaceutical composition according to any one of claims 1 to 4, wherein at least a part of the thrombolytic action is a thrombolytic action by plasminogen activator activity.   A food or drink for preventing thrombosis and / or improving symptoms, comprising the pharmacological composition according to any one of claims 1 to 5.   A pharmaceutical for preventing and / or improving symptoms of thrombosis comprising the pharmacological composition according to any one of claims 1 to 5.

Images