JPH08319240A - Enzyme-inhibiting agent and food product having enzyme-inhibitory action - Google Patents

Abstract

PURPOSE: To obtain a food product having an enzyme inhibitory activity effective for geriatric dementia of Alzheimer type from a kind of mushroom, Grifola frondosa in Polyporeae. CONSTITUTION: This food product contains an extract from a mushroom, Grifola frondosa or a crude product or a purified product therefrom in an amount of about 0.1-1wt.%. The extract is obtained from the one selected from its fruits, stems, mycelia and culture medium such as saw dust and the extraction and purification are customarily carried out. Since the active ingredient obtained from the mushroom inhibits proline endopeptidase, it can be used as an enzyme inhibitor, particularly as a proline endopeptidase inhibitor. It can be given by injection and, in addition, can be formulated to a wide range of food products such as noodles, cereal food products, oil and fat products, soybean products, meat product, sea food products, fruit products, vegetable products, confectionery and the like.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an enzyme inhibitor and a food product which are produced from Maitake mushrooms of the family Anodontaceae and can be used for the prevention and treatment of Alzheimer's dementia.

[0002]

2. Description of the Related Art Maitake mushrooms are usually used for food because they are delicious, but the ingredients extracted from Maitake mushrooms are:
It has been confirmed to have various medicinal effects, and as a result, studies on maitake mushrooms are being conducted for medicinal purposes. Although it has been known that maitake has many pharmacological effects, it has never been known to have prolyl endopeptidase inhibitory activity which is considered to be effective for Alzheimer's dementia.

[0003] Further, although quite a number of research reports have been made on the pharmacological effect of basidiomycetes other than Maitake mushroom, no pharmacological effect on Alzheimer's dementia has been reported.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to extract a prolyl endopeptidase inhibitory active ingredient from Maitake mushroom and to utilize it to provide an enzyme inhibitor and a food.

[0005]

[Means for Solving the Problems] The inventors of the present invention have studied the medicinal components contained in mushrooms, and found that the hot water extract of Maitake and its sawdust medium can treat and prevent Alzheimer's dementia. The present invention was completed as a result of discovering that an effective ingredient exists in the above and conducting various studies.

That is, the present invention is an enzyme inhibitor containing an extract from Maitake mushroom or a crudely purified product or purified product of the extract as an active ingredient. Further, the present invention is a food having an enzyme-inhibiting activity, which comprises an extract from Maitake mushrooms or a crudely purified product or a purified product of the extract. Furthermore, the present invention provides the enzyme inhibitor and the food having enzyme inhibitory activity, wherein the inhibiting enzyme is prolyl endopeptidase.

Furthermore, the present invention is characterized in that the extract from Maitake is obtained from at least one selected from fruiting bodies, stone tips and hyphae of Maitake, and a medium in which Maitake is cultured. And a food having an enzyme inhibiting activity. Hereinafter, the present invention will be described in detail. The enzyme inhibitor of the present invention contains an extract from Maitake mushroom or a crudely purified product or purified product of the extract as an active ingredient. The extract from Maitake mushrooms includes not only those obtained from fruiting bodies, stone tips and hyphae of Maitake mushrooms, but also those obtained from the medium after Maitake culture.

The maitake mushroom may be of any kind as long as it contains a prolyl endopeptidase inhibitory active ingredient, and for example, maitake mushroom ( Grifola frondo).
sa ), Shilomaitake ( Grifola albicans ), Tonbimaitake ( Grifola gigantea ) and all of the genus Grifola genus. As the medium of Maitake,
For example, it is possible to use sawdust, dough and corn bran (corn dregs) in a volume ratio of 3: 10: 2.5. In this medium, it is preferable to mix and stir each component, and then add water and further stir. The water content of this medium is preferably 55 to 68%, and when the amount of maitake produced per medium is taken into consideration, it is particularly preferably around 65%.

As the sawdust, sawdust of broad-leaved trees such as beech, chestnut, oak, and oak, which are used for cultivating nameko, can be used. To increase the porosity of sawdust, 30 to 40% of chip dust may be mixed, and even if the particles of sawdust are fine, the air permeability in the medium can be kept good by the mixing. Although fresh rice bran is generally used as a nutrient source, the use of corn blanc can reduce the rate of dying from the formation of primordia to the growth of fruiting bodies.

Sterilization of the medium may be carried out by a conventional method, for example, 12
It can be carried out by high-pressure sterilization at 0 ° C for 120 minutes. In Maitake cultivation, it is preferable to completely sterilize harmful bacteria in the medium because the medium preparation is an extremely important point. Further, since the medium has poor heat conduction, it is preferable to extend the sterilization time when sterilizing a large amount of medium. It is preferable to immediately transfer the sterilized medium to a cooling chamber and cool the medium temperature to 20 ° C. or lower, and then inoculate in a sterilized sterile chamber. Since maitake inoculum has a poorer initial bite than other mushrooms, the higher the inoculum, the higher the success rate of culturing. The inoculation volume is 1 bottle (1,000 ml) per bottle (1,
40 to 50 bottles (000 ml volume) and 15 to 20 single-sided bags (3,000 ml volume) are standard.

The temperature of the culture chamber is preferably about 18 to 20 ° C. When mycelium spreads, the temperature inside the medium rises by 2 to 3 ° C., which is a temperature convenient for the growth of maitake hyphae. Since Maitake mushrooms have the property of forming primordia in sequence during culturing, the shelving system is suitable for culturing administratively. In addition, it is preferable that the light has a brightness of 50 lux or more during the culture, and such a brightness facilitates the formation of primordia. Of particular importance is ventilation during culture, which is an important operation in the process of culture management. It is preferable to increase the number of times of ventilation as much as possible so that oxygen can be smoothly supplied to the medium.

The extraction may be carried out by a conventional method, but as one method for extracting the heat stable component, for example, finely crushed Maitake fruit bodies and water are mixed in equal amounts and the mixture is heated at 90 to 95 ° C. for 2-3 times. After heating for a period of time, it is allowed to stand until it reaches room temperature and then filtered. Extraction from the medium can be performed in the same manner. The extract thus obtained has the following physicochemical properties and is characterized by high thermal stability.

Color and Liquid A dark brown liquid with a unique scent of Maitake. Molecular Weight (Dialyzability) The Maitake extract was placed in a dialysis cellulose tube (manufactured by Sanko Junyaku Co., Ltd., dialysis membrane 27/32) and dialyzed against deionized water at 4 ° C. for 24 hours. When the activities of the outer solution and the inner solution were examined, the activity was detected in both solutions. From this result, it is highly possible that both low molecular weight substances and relatively high molecular weight substances are involved in the active ingredient.

The pH of the solution The pH of the solution is 6-7. Stability to heat It was confirmed that the substance was quite heat-resistant because it was active even after heat treatment at 121 ° C for 15 minutes in an autoclave during extraction.

The crude purification and purification of the extract may be carried out by a conventional method, for example, adsorption and elution with an adsorbent, chromatography and the like may be appropriately combined. The active ingredient obtained as described above (extract from Maitake, its crudely purified product and purified product) has a prolyl endopeptidase inhibitory activity as will be apparent from the examples described later, and therefore an enzyme inhibitor, particularly It can be used as a prolyl endopeptidase inhibitor. This prolyl endopeptidase inhibitor is effective for the prevention and treatment of Alzheimer's dementia.

This enzyme inhibitor can be administered, for example, as an injection. When used as an injection, a pH adjusting agent, a buffer, a stabilizer, an excipient and the like may be added to the present active ingredient. It can also be used as a freeze-dried injection by freeze-drying it by a conventional method. Furthermore,
A pH adjusting agent, a buffering agent, a stabilizing agent, an isotonic agent, a local anesthetic, etc. may be added to the present active ingredient to prepare a subcutaneous or intramuscular or intravenous injection in the form of a solution or suspension by a conventional method. Can be used.

The extract from Maitake, the crudely purified product and the purified product thereof can also be used in foods. The extract from Maitake, the crudely purified product, and the purified product thereof can be orally ingested as they are, but can also be used by adding them to other foods. Foods include noodles (Udon, buckwheat, Chinese noodles, etc.), bread, cooked rice, processed grain products such as rice cakes, margarine,
Processed oils and fats such as mayonnaise and dressings, processed soybeans such as tofu, miso and natto, processed meats such as ham, bacon and sausages, processed fish products such as kamaboko, chikuwa, hanpen, fish sausages and processed fish eggs, Dairy products such as yogurt, butter, cheese, ice cream, fruit processed products such as jams, vegetable processed products such as pickles, chocolates, biscuits, cakes, caramels, candies, chewing gum, jellies, confectionery such as rice crackers, etc. Including food. Further, not only foods generally referred to but also various drinks such as juice, coffee, tea, green tea, oolong tea, carbonated drink, sports drink, milk, and seasonings such as soy sauce, sauce and mirin can be included.

The amount of the extract of Maitake mushroom, its crudely purified product and purified product added to food is preferably about 0.1 to 1.0% by weight. By ingesting this food,
It is possible to prevent and treat Alzheimer's dementia.

[0019]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples. (Example 1) (1) Preparation of maitake heat extract The maitake heat extract was prepared by the procedure shown in the flow sheet of FIG.

(2) Enzymes used Originally, when measuring the inhibitory activity of prolyl endopeptidase, human brain-derived prolyl endopeptidase should be the target enzyme, but human brain is difficult to obtain. A partially purified enzyme derived from brain was used. Partial purification of the enzyme was performed as follows. Buffer solution for bovine brain [10 mM EDTA and 10 mM
20 mM Tris-HCl (pH containing mM 2-mercaptoethanol
7.0)] was added and the mixture was homogenized with a mixer and then centrifuged, and the resulting supernatant was subjected to ammonium sulfate fractionation. Then, the active fraction was dissolved in a buffer [20 mM Tris-HCl (pH 7.0) containing 10 mM EDTA and 10 mM 2-mercaptoethanol] and dialyzed, followed by DEAE cellulose column chromatography (column manufactured by Iwaki Glass Co., Ltd.). Partially used).

(3) Method for measuring inhibitory activity In a test tube, 20 mM Tris-HCl containing 40 μl of a crude enzyme solution derived from bovine brain, 10 mM EDTA and 10 mM 2-mercaptoethanol was added.
After adding 850 μl of buffer solution (pH 7.0) and 10 μl of Maitake mushroom hot water extract <A> and preincubating at 37 ° C. for 30 minutes, 100 μl of 1 mM Z-Gly-Pro-pNA as a substrate was added to start the reaction. After reacting at 37 ℃ for 15 minutes, 1M
The reaction was stopped by adding 1000 μl of Acetate buffer (pH 4.0). To detect the enzyme activity, the absorption at 410 nm derived from pNA (p-nitroaniline) was measured using a spectrophotometer, and the inhibitory activity was calculated in comparison with the control. As a blank, buffer solution [10 mM EDTA and
20 mM Tris-HCl (p containing 10 mM 2-mercaptoethanol
H7.0)] was used.

(4) Prolyl endopeptidase (PEP) inhibitory activity of Maitake hot water extract The PEP inhibitory activity was measured by the above-mentioned method.
The P inhibition rate was 40 to 60%. From these results, it was suggested that the enzyme inhibitor of the present invention is effective as a preventive and therapeutic agent for Alzheimer's dementia.

Example 2 An animal experiment was actually conducted using the enzyme inhibitor of the present invention. The problem here is that the amnesia and human dementia caused by the animal experiment occur. Is the difference. However, since there is no stochastic animal model for dementia, it is inevitable to judge the possibility based on the results of various methods. In general, it is known that administration of scoboramine to humans causes symptoms similar to dementia, so this time, using the passive avoidance learning method using experimental amnestic rats with scoboramine, the maitake extract The antiamnestic effect of was investigated.

The experimental apparatus used was one in which electrodes (grids) were laid on the bottom of the box, voltage was applied alternately and repeatedly from one end, and a table was installed at one end. . When the rat is placed on the table, the rat will make an exploratory action and descend to the floor due to its habit. At that time, I feel an electric shock when I touch the grid, and I return to the table to avoid it. Most rats, once learned, will no longer land on the floor. However, when 3 mg of scoboramine was administered to the rat per 150 g of body weight, the rat forgot the fear of the previous electric shock and came to the floor. Therefore,
Rats were given 2 ml / day of maitake extract concentrated sample <B> in advance, and how much the memory loss of this rat was prevented was determined by the time until the rat fell to the floor (latenc
y). The results are shown in Figure 2. As is clear from FIG. 2, a significant difference was observed in the rats fed the concentrated sample of Maitake mushroom extract, and the enzyme inhibitor of the present invention is effective as a preventive and therapeutic agent for Alzheimer's dementia. It was suggested.

[0025]

INDUSTRIAL APPLICABILITY According to the present invention, an enzyme inhibitor effective for Alzheimer's dementia and a food having an enzyme inhibitory activity can be obtained.

[Brief description of drawings]

FIG. 1 is a flow sheet showing a method for preparing a hot water extract of Maitake and a concentrated liquid thereof.

FIG. 2 is a graph showing the time until the amnestic rats fed with the concentrated extract of Maitake mushroom (test rats) and the amnestic rats not fed with the concentrated concentrate of Maitake extract (control rat) go down to the floor. .

Claims (6)

[Claims] 1. An enzyme inhibitor comprising an extract from Maitake mushroom or a crudely purified product or purified product of the extract as an active ingredient. 2. The enzyme inhibitor according to claim 1, wherein the inhibiting enzyme is prolyl endopeptidase. 3. Enzyme inhibitor according to claim 1, characterized in that the extract from Maitake is obtained from at least one selected from fruiting bodies, stone tips and hyphae of Maitake, and a medium in which Maitake is cultivated. . 4. A food having an enzyme-inhibiting activity, which comprises an extract from Maitake mushroom or a crudely purified product or a purified product of the extract. 5. The enzyme inhibitor according to claim 4, wherein the inhibiting enzyme is prolyl endopeptidase. 6. The enzyme-inhibiting activity according to claim 4, characterized in that the extract from Maitake is obtained from at least one selected from fruiting bodies, stone tips and hyphae of Maitake and a medium in which Maitake is cultured. Having food.