JP5144112B2 - Brain protectant - Google Patents

Description

  The present invention relates to a brain protective agent comprising as an active ingredient a component extracted from a mycelium culture of basidiomycetes and / or ascomycetes.

  Nerve cells are particularly vulnerable to ischemia, and the brain is susceptible to damage from ischemia. For example, cerebral infarction caused by ischemia due to cerebrovascular occlusion is a serious lifestyle-related disease that is the third leading cause of death in Japan, and the number of patients is also increasing. In addition, even in the case of cerebral infarction, even if the life is saved, there is a high possibility that serious sequelae such as paralysis remain due to irreversible damage of nerve cells.

  A transient ischemic attack is a reversible neurological symptom that often appears prior to cerebral infarction, but it is said that brain cells may not recover from injury even after several minutes of ischemia. In addition, blood reperfusion after ischemia is said to significantly worsen damage to brain cells due to the influence of active oxygen and the like generated during reperfusion. Therefore, it is said that if brain cells can be protected from ischemia or ischemia / reperfusion injury, it is possible to avoid severe sequelae such as paralysis and speech impairment.

  For example, edaravone (trade name: “Radicut”) has been approved as a brain protective agent used to improve neurological symptoms associated with the acute phase of cerebral infarction, impairment of daily living, functional impairment, and the like.

On the other hand, cultures obtained by culturing mycelium of basidiomycetes in a medium containing plant fiber raw materials have been reported to have various physiological activities, and have an antioxidant function enhancing effect (Patent Document 1 below). ), Physiological activities such as blood glucose level increase inhibitory effect (Patent Document 2 below) and antihypertensive effect (Patent Document 3 below) have been reported.
Japanese Patent No. 3284097 Japanese Patent Laying-Open No. 2005-213211 JP 2006-265179 A

  Drugs used as conventional brain protective agents require sufficient caution when used, and various side effects have been reported. For this reason, there was a problem from the viewpoint of safety, and it could not be taken easily in daily life.

  Therefore, an object of the present invention is to provide an easy and safe brain protective agent that can improve brain cell damage caused by ischemia or ischemia / reperfusion, for example, by daily intake.

The present inventors have found that a medium containing vegetable fiber material was subjected to various studies on basidiomycete and / or child components extracted from cultures of the mycelium obtained by cultivation of Basidiomycetes, these components Was found to have an effect of improving brain cell damage caused by ischemia or ischemia / reperfusion, and the present invention was completed.

That is, cerebral protective agent of the present invention, the feature that an active ingredient extracted component from the culture obtained by culturing mycelia of Ganoderma mushroom is basidiomycete in a medium containing a plant fiber material To do.

  In the brain protective agent of the present invention, the extract component preferably contains a saccharide, a protein, and a water-soluble lignin.

  In the brain protective agent of the present invention, it is preferable that the plant fiber raw material is prepared from a scallop.

  In the brain protective agent of the present invention, the plant fiber raw material is preferably one or more selected from bagasse, corn stover, wheat bran, rice bran, rice straw, rice bran, bear cocoon, and bamboo. .

Which is the active ingredient of the present invention, the cellulose component extracted from a culture obtained by culturing mycelia of Ganoderma mushroom is basidiomycete in a medium containing a plant fiber material is contained in the plant fiber in the raw material Pentose-based proteoglycan produced by digestion, degradation, and condensation by enzymes such as cellulase, phenol oxidase, laccase, peroxidase, and protease produced by basidiomycetous mycelium during the culture period In addition, it is composed of a substance in which a modified water-soluble lignin modified by an enzyme and rendered water-soluble is bound in a complex manner.
This extract component is a component derived from natural materials, and since safety has been confirmed by extensive dietary experience, there is no risk of side effects, and by ingestion, ischemia or ischemia・ Cerebro-neuropathy at the time of reperfusion can be reduced, cerebral infarction volume can be reduced, and the occurrence of cerebral infarction and severe motor dysfunction can be suppressed.

As a basidiomycete used in the present invention, mannenpox is used.

In the present invention, the mycelium of the basidiomycetes, cultured with plant fibrous material, to extract the active ingredient from the culture. As the medium, either solid culture or liquid culture can be used.

  As the plant fiber raw material used for the medium, those prepared from plants containing lignin are preferably used. Examples of plants containing lignin include scallops. For example, bagasse (a fibrous component of sugar cane), corn stover, wheat bran, rice bran, rice straw, and straw are preferably used. In addition, bears, bamboo, etc. can be used.

  In the present invention, as the culture medium, a culture medium containing bagasse, one kind selected from corn stalks and corn stalks, and rice bran is particularly preferably used. Moreover, you may make a culture medium contain a yeast extract, dry yeast, chlorella, spirulina, corn meal, okara etc. as a nutrient component as needed.

The mycelium of the basidiomycete is cultured by inoculating the mycelium or spore of the basidiomycete in a medium containing the plant fiber raw material as described above .

  In the case of solid culture, the water content is adjusted to 60 to 80%, sterilized by autoclaving according to a conventional method, then inoculated with mycelia, for example, 3 to 6 in a culture room conditioned at 18 to 25 ° C. Incubate for months. The medium in which the mycelium has spread is desirably transferred to the temperature treatment chamber and subjected to a temperature change treatment. In the temperature change treatment, for example, the mixture is first heated at 30 to 34 ° C. for 24 to 48 hours, and then transferred to a low temperature chamber and treated for 3 to 5 days. After that, when it is moved to a culture room, the generation of fruiting bodies starts. At this point, the culture is terminated and the culture is crushed with a crusher.

After completion of the culture, the mycelium is preferably self-digested and the culture is extracted using the mycelium-external enzyme produced by the mycelium. As a preferable method, in the case of a solid medium, the medium after culturing is crushed, a small amount of water is added as necessary, and the mycelium is self-digested by enzymatic action at 30 to 60 ° C. for 3 to 6 hours. . Subsequently, this crushed material is infiltrated with warm water or hot water of 50 ° C. or higher, and an active ingredient is extracted. Extraction can also be performed under a high pressure such as 120 ° C. under a pressurized vapor pressure of 1 kg / cm ² , for example. The extract suspension thus obtained is preferably filtered or centrifuged to collect a filtrate or supernatant, which contains a medium degradation product, a mycelium metabolite, a mycelium cell degradation product, and the like. An extract can be obtained.

  In the case of a liquid medium, the plant raw material is finely pulverized, and if necessary, other nutrients such as rice bran are added, and the medium is prepared so that the raw material is 5 to 20% by weight, and then aeration stirring culture or By shaking culture, culture is preferably performed at a temperature of 20 to 28 ° C. for about 1 week to 2 months. The culture is terminated when the pH of the medium is lowered to 3.5 to 5 and the mycelium is prevalent in the medium.

After completion of the culture, preferably, the whole culture is treated at 30 to 60 ° C. for 3 to 6 hours to self-digest the mycelium to obtain a liquid suspension culture. Next, water is added as necessary, and the extract is collected by heating at 50 ° C. or higher, possibly under high pressure conditions (for example, under a pressurized vapor pressure of 1 Kg / cm ² ). The extract is filtered or centrifuged as necessary, and the filtrate or supernatant is collected to obtain an extract containing a medium degradation product, a mycelium metabolite, a mycelium cell degradation product, and the like. Can be obtained.

  The brain protective agent of the present invention can be obtained by subjecting the extract obtained by the above method as it is or by concentrating it into a liquid product, and further pulverizing the extract by a method such as freeze drying or spray drying. You can also When the extract is dried, a fine powder is obtained, which can be further pulverized into ultrafine particles.

  The thus obtained cerebral protective agent of the present invention can be commercialized as a powder, granule, tablet or capsule by a conventional method. Moreover, it can also be commercialized as a liquid and jelly-like drink by adding an extract. Furthermore, it can also be used by adding to various foods and drinks.

  Such foods and drinks are not particularly limited. For example, fresh food such as meat, seafood, vegetables, fruits, etc .; processed livestock products such as ham and sausage; processed marine products such as hampen and kamaboko; jam, dried Processed fruits such as fruits; processed vegetables such as pickles; dairy products such as milk, butter, cream, cheese; oils and fats such as rapeseed oil, palm oil, sunflower oil, shortening; processed soybean foods such as tofu, fried chicken, natto; Beverages such as coffee, cocoa and soft drinks; seasonings such as soy sauce, miso, sauce and ketchup; breads and cakes; confections such as Japanese confectionery and Western confectionery; noodles such as udon, soba, somen and spaghetti.

  The cerebral protective agent of the present invention comprises a component derived from a natural material as an active ingredient, and since safety has been confirmed by abundant eating experience, there is no fear of side effects. Incidentally, in the single oral administration test of the brain protective agent of the present invention, the minimum lethal dose was 22500 mg / Kg or more for both sexes in rats and 2000 mg / Kg or more for both sexes in mice. In the rat 3-month repeated administration test, the maximum no-effect level was 3610 mg / Kg for males and 4190 mg / Kg for females.

  The effective dose of the brain protective agent of the present invention is 1 to 10 g per day for an adult when taken orally. If the dose is smaller than this, a sufficient effect is hardly obtained, and if the dose is larger than this, loose stool or abdominal bloating may occur. However, there is no problem in safety even if the dose is larger than the above.

<Example 1> (Solid culture of Bacillus mannen using bagasse)
90% bagasse and 10% defatted rice bran were blended and adjusted to a moisture content of 70% to form a solid medium, which was then packed in a polypropylene bag. Then, after sterilizing at 121 ° C. for 40 minutes, inoculated with Mannen spp. And cultured at 22 ° C. ± 1 ° C. for 4 months.
After completion of the culture, the medium was crushed and kept at 5 ° C. for 5 hours, and then extracted with warm water at 60 ° C. for 16 hours. The obtained extract was roughly filtered, then filtered through a membrane having a diameter of 0.45 μm, concentrated and spray-dried to obtain an extract powder of a mannen mycelium culture.
The components of this powder were carbohydrate: 36.1%, protein: 13.2%, lignin: 9.9%, and inorganic matter: 13.5%.
For each component analysis, the sugar was measured using the phenol sulfate method, the protein was measured using the semi-micro Kjeldahl method, the lignin was measured using the ionization differential spectrum method, and the inorganic material was measured using the direct ashing method.

<Test Example 1> (Normal rat)
Male SD rats (10 weeks old) were divided into a control group and a test group as 10 mice per group. Water was administered to the control group, and the substance of Example 1 (1 g / Kg / day) was forcibly orally administered to the test group for 2 weeks.
After the breeding period, under the halothane anesthesia, 4-0 monofilament thread (emboli thread) with a rounded tip was inserted from the common carotid artery to occlude the middle cerebral artery (middle cerebral artery occlusion: MCAO) did. After ischemia, motor dysfunction was evaluated by a 6-step evaluation, and after 2 hours, the embolic thread was withdrawn to restore blood flow (reperfusion) (MCAO / Re). After reperfusion, motor dysfunction was measured and evaluated by a 6-level evaluation as in the case of ischemia, and used as a neurological score. The neurological score after ischemia is shown in FIG. 1, and the neurological score after reperfusion is shown in FIG. Motor dysfunction was evaluated and scored in 6 stages as follows.
・ Score 0: No obstacle ・ Score 1: Symptoms such as decreased grip strength on the forefoot ・ Score 2: Mild disability with spontaneous rotation when grabbing the tail ・ Score 3: Spontaneous Moderate disability with rotational movement ・ Score 4: Severe disability with involuntary behavior and decreased consciousness ・ Score 5: death and reperfusion of rats 2 hours after obstruction of middle cerebral artery The brain was removed 24 hours after reperfusion, and a brain section was prepared. This was stained with TTC (2% triphenyltetrazolium chloride), the infarct was analyzed by NIH Image, and the cerebral infarction volume was measured. Infarct NIH Image is shown in FIG. 3, and cerebral infarction volume is shown in FIG.

From the results shown in FIG. 1, motor function impairment was observed in both the control group and the test group due to ischemia. However, as shown in FIG. 2, this motor dysfunction was caused by the increase in neurological score from 1.4 to 2.3 due to reperfusion in the control group, and the motor dysfunction was exacerbated. In contrast, in the test group, the neurological score, which was 1.3 at the time of ischemia, was not significantly changed to 1.3 at the time of reperfusion, which was significant compared to the control group (P <0.05). ).
In addition, from the results of FIG. 3, in the normal rat brain removed after ischemia / reperfusion, although a clear infarct is not recognized only by ischemia, in the control group, a clear infarction ( A white part in the photo) appeared. In contrast, in the test group, almost no infarct (white area) was observed.
Further, from the results of FIG. 4, the cerebral infarction volume after ischemia / reperfusion is 29% in the control group, whereas the test group is 15%, which is significant compared to the control group (P <0. 05).

<Test Example 2> (Diabetic rat)
Streptozotocin (STZ) 60 mg / Kg was intraperitoneally administered to 20 male SD rats (5 weeks of age) and reared for 5 weeks to prepare type 1 diabetes model rats. The diabetic model rats were randomly selected into a control group and a test group, 10 rats per group. Then, as in Test Example 1, water was forcibly administered to the control group and the substance of Example 1 (1 g / Kg / day) was forcibly orally administered to the test group for 2 weeks.
Normal rats not treated with STZ were also randomly selected from 10 groups per control group and test group, water for the control group and 2 substances of Example 1 (1 g / Kg / day) for the test group. Oral gavage was administered weekly.
Blood was collected from 12-week-old rats, and blood glucose levels were measured with Dexter ZII (Bayer Medical). As a result, as shown in Table 1, SD rats showed significant (P <0.01) hyperglycemia compared to STZ non-administered rats (normoglycemic rats) by STZ administration.

After the breeding period, in the same manner as in Test Example 1, each group of rats was inserted into the common carotid artery with a 4-0 monofilament thread (emboli thread) with a rounded tip under halothane anesthesia to occlude the middle cerebral artery ( MCAO). After ischemia, motor dysfunction was evaluated and scored on a 6-point scale. Subsequently, the embolic thread was withdrawn after 2 hours to restore blood flow (reperfusion) (MCAO / Re). After reperfusion, motor dysfunction was measured and evaluated by 6-level evaluation and scored as in the case of ischemia. FIG. 5 shows the neurological score after ischemia, and FIG. 6 shows the neurological score after reperfusion.
Then, the brain of a rat reperfused 2 hours after the middle cerebral artery was occluded was removed 24 hours after the reperfusion, and a brain section was prepared. This was stained with TTC (2% triphenyltetrazolium chloride), the infarct was analyzed by NIH Image, and the cerebral infarction volume was measured. The infarct NIH Image is shown in FIG. 7, and the cerebral infarction volume is shown in FIG.

From the results of FIG. 5, the neurological score increased and motor dysfunction was recognized due to ischemia. In the control group, the normal rat of Test Example 1 was 1.4 (see FIG. 1), whereas in the diabetic model rat of Test Example 2, 1.85 (see FIG. 5), It has increased greatly. In contrast, in the test group, the neurological score of the normal rat of Test Example 1 was 1.3 (see FIG. 1), while that of the diabetic model rat of Test Example 2 was 1. As low as 2 (see FIG. 5), no change in functional impairment was observed.
In addition, as shown in FIG. 6, this motor dysfunction is markedly increased in neurological score from 1.85 to 3.5 by reperfusion in the control group, and the dysfunction is remarkably deteriorated. On the other hand, in the test group, the neurological score that was 1.2 at the time of ischemia was 1.5, even at the time of reperfusion, and there was almost no deterioration in dysfunction. And was significantly suppressed (P <0.01).
Further, from the results of FIG. 7, in the brain of diabetic rats removed after ischemia / reperfusion, in the control group, the infarct was spread more extensively than in the normal rats, and almost no in the brain during normal rat ischemia. The infarct that was not observed was also clear. In particular, diabetic rats reperfused after ischemia showed a more extensive infarct. In contrast, in the test group to which the substance of Example 1 was administered, the cerebral infarction lesion after reperfusion was significantly reduced.
Further, from the results of FIG. 8, the cerebral infarction volume after ischemia / reperfusion is 17.5% in the test group administered with the substance of Example 1 compared to 66% in the control group. In the test group, the cerebral infarction was significantly suppressed (P <0.01) compared to the control group.

It is a figure showing the neurological score after ischemia in a normal rat. It is a figure showing the neurological score after reperfusion in a normal rat. Infarct focus NIH Image in normal rats. It is a figure showing the cerebral infarction volume after the reperfusion in a normal rat. It is a figure showing the neurological score after ischemia in a diabetic rat. It is a figure showing the neurological score after reperfusion in a diabetic rat. Infarct focus NIH Image in diabetic rats. It is a figure showing the cerebral infarction volume after the reperfusion in a diabetic rat.

Claims (4)

Neuroprotectant agents for a component extracted from a culture mycelia were obtained by culture of Ganoderma mushroom as an active ingredient a basidiomycete in a medium containing a plant fiber material.   The brain protective agent according to claim 1, wherein the extraction component contains a sugar, a protein, and a water-soluble lignin.   3. The brain protective agent according to claim 1 or 2, wherein the plant fiber raw material is prepared from a plant of the genus Honcho.   The brain protective agent according to claim 1 or 2, wherein the plant fiber raw material is one or more selected from bagasse, corn stover, wheat bran, rice bran, rice bran, rice bran, kumabushi, and bamboo.