JPH0937719A - Orally ingestive substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an inhibitor of prolyl endopeptidases useful as a medicine, a functional food, a food for health, a food and drink, etc., effective in preventing and treating senile dementia by treating a rice protein with a proteolytic enzyme. SOLUTION: A rice protein such as a prolamin, a glutelin, a globulin, an albumin or a proteose or a substance containing the rice protein is treated with a proteolytic enzyme to afford an inhibitor of prolyl endopeptidases. The resultant treated substance is converted into a pasted, a dried or a diluted substance. Furthermore, brown rice, milled rice or a brewed food such as SAKE (Japanese rice wine), sweet SAKE, miso or soy sauce prepared from rice as a raw material is cited as the substance containing the rice protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a prolyl endopeptidase inhibitor orally ingested substance containing a proteolytic enzyme degradation product of rice protein (or a processed product thereof). The oral ingestion of the present invention, which can inhibit prolyl endopeptidase, is particularly effective in the prevention and treatment of senile dementia, which has recently been on the increase in medicine and functional foods, foods for specified health use, and nutrition. It can be used for food and drink such as medicines and health foods.

[0002]

2. Description of the Related Art The importance of geriatric medicine has been greatly emphasized in the aging society. Among them, prevention and treatment of dementia is a major problem not only for the person himself, but also for his family and society. Representative examples of dementia include cerebrovascular dementia and Alzheimer-type dementia, and the cause of the latter has not been completely elucidated compared to the former.
Further, Alzheimer's dementia is also called amnesia, and it causes a serious problem because it causes a heavy burden on caregiving because it does not cause any impairment in other motor abilities other than short-term memory loss. Therefore, there is an urgent need to develop therapeutic methods.
There are still many unclear points about the background and the onset mechanism,
The development of therapeutic agents is under active development, and some are even in clinical trials.

[0003] Prolyl endopeptidase (hereinafter referred to as PE
P (sometimes referred to as P) (EC 3.4.21.26)
It is a serine protease that has specificity for proline and cleaves the peptide at the carboxyl group side, and is known to have high activity in brain, testis and liver (protein nucleic acid enzyme,
25, 513-523, (1980), protein nucleic acid enzyme, 29, 127-133, (1984)). PEP is known to degrade neurotransmitter substance P and THR (thyroid stimulating hormone) and vasopressin, which are thought to be involved in memory. Vasopressin is a peptide hormone that acts on reabsorption of water in the kidney, but is also involved in learning and memory processes in the brain, and there is a fact that amnesia progresses when this hormone is degraded and inactivated . In addition, it is known that the amount of vasopressin in dementia patients is lower than that in normal persons (BI
OINDUSTRY, 4, 788-796, (198
7)).

Vasopressin is degraded by PEP into two peptides. In healthy people, PEP in the brain works normally, but if the regulatory mechanism is lost for some reason, vasopressin is degraded more than necessary, causing damage to memory retention. Therefore, studies have been conducted aiming at the treatment of dementia by inhibiting the activity of this enzyme. For example, a prolyl prolinal derivative (protein nucleic acid enzyme, 29, 127,
(1984)), N-acylpyrrolidine derivatives (JP-A 61-37764, JP-A 61-183297, JP-A 61-238775, JP-A 61-238799, JP-A 62-114957) and the like. ing. However, since these are synthetic products, there is a problem in safety, and since the structures are similar, the structure-activity relationship has already been clarified (ABC, 55, 37-43, (199).
1)).

[0005]

[Problems to be Solved by the Invention] For the prevention and treatment of senile dementia, a prolyl endopeptidase inhibitor having a safe and novel structure is required, and the present invention is particularly safe. It was made for the purpose of developing a novel PEP inhibitor with high efficacy.

[0006]

Means for Solving the Problems The present invention has been made to achieve the above object, and the present inventors have intensively searched for a prolyl endopeptidase inhibitor having no side effect in natural substances. As a result, the present inhibitor was found to be present in the degradation product of rice protein, and the substance was found to be a peptide, thus completing the present invention. In addition, since the by-products are used as raw materials, the resources can be effectively used.

As the PEP inhibitory substance of the present invention, a substance obtained by treating a rice protein or a rice protein-containing substance with a proteolytic enzyme is used. As the rice protein, not only purified products such as prolamin, glutelin, globulin, albumin, and / or proteose but also crude products can be used. Further, as the rice protein-containing material, substances containing these rice proteins are widely used, and examples thereof include brewed foods made from brown rice, white rice, bran, and rice (sake, miso, miso, soy sauce). Etc.) as well as by-products (such as bran and lees) are used.

The rice protein (containing substance) is then treated with a proteolytic enzyme. If desired, this decomposition treatment may be a known proteolytic method such as chemical decomposition or physical decomposition in addition to enzymatic decomposition, and may be used alone or in combination. Since the enzymatic degradation method is performed under mild conditions, it has the advantage that the denaturation of the produced peptide is low, etc.In addition to proteolytic enzymes of animal and plant origin such as pepsin, trypsin, chymotrypsin, and fusin, proteolytic enzymes of microbial origin are used. Just process it.

That is, the separation and purification of the prolyl endopeptidase inhibitor from rice protein is carried out by decomposing it with an acid, an alkali, or an enzyme, and if necessary, after extracting and concentrating, the difference in adsorption affinity to various adsorbents, The methods usually used for separation and purification such as the difference in solubility in various solvents and the difference in elution rate due to the molecular sieving effect, and those methods may be appropriately combined. This separation and purification is more efficient when rice protein is used as a raw material, but it is of course possible to use brown rice, white rice and bran as they are. The rice protein referred to herein can also be obtained by extracting the protein by a solvent fractionation method, or conversely by degrading starch or the like other than the protein with an enzyme. In addition, by-products such as rice bran and lees produced in the production of brewed foods made from rice (sake, miso, miso, soy sauce, etc.) can be used as they are or by applying the above solvent fractionation method as a protein source. it can. In the case of a by-product of brewed food, it may be possible that protein degradation occurs during brewing, so new degradation may not be necessary. Specifically, the hydrolysis condition is not particularly limited, but it may be determined using the prolyl endopeptidase inhibitory activity as an index.

The PEP inhibitor of the present invention is obtained by degrading rice protein (containing substance) with a proteolytic enzyme (or a processed product thereof), and contains a peptide. For example, when a pepsin degradation product of sake lees was subjected to purification treatment including reverse phase chromatography treatment, peptides of 8 to 10 residues (LLSPFWNINA, LSPFWNINA, S
PFWNINA) was isolated, and a high PEP inhibitory activity was observed especially in the 10-residue peptide.

Further, those obtained by decomposing and solubilizing alcoholic beverages with pepsin, even without isolating and purifying these peptides or their mixtures, also had high PEP inhibitory activity.

Therefore, as the PEP inhibitory substance of the present invention, rice protein (containing substance) decomposed by a proteolytic enzyme (or a processed product thereof) can be appropriately used. For example, a decomposed product of sake lees can be used, and a peptide can also be used. Can be used.

The proteolytic enzyme-decomposed product of rice protein can be used as it is, and its processed product (concentrated product, paste product, dried product, diluted product, etc.) can also be used as a PEP inhibitor.

In the present invention, the PEP inhibitory substance is used as an active ingredient and is orally ingested for PEP inhibition. The PEP-inhibiting orally ingested substance according to the present invention can be used as a pharmaceutical type and as a food and drink type.

The PEP inhibitor of the present invention has an excellent PEP inhibitory activity and is safe because it is derived from a natural product, as will be apparent from the examples described below. Even in the 10-day acute toxicity test, in the case of the decomposed product of sake lees, no death was observed even after the oral administration of 1000 mg / kg, and high safety was confirmed.

When used as a food / beverage type, the PEP inhibitor (the treated product) which is the present active ingredient can be used as it is, or can be used in combination with other foods or food ingredients in accordance with ordinary methods. Can be used. The orally ingested substance according to the present invention using the present active ingredient may be solid (powder, granules, etc.), paste, liquid or suspension, but sweeteners, sour agents, vitamins and other drink preparations. It is suitable to formulate into a health drink using various components commonly used in. In this case, there is no particular limitation on the amount used, functional foods, specific health foods, nutritional supplements, as health foods, which can be appropriately consumed and eaten, and since they are highly safe, they can be taken over a long period of time. It is possible.

When used as a pharmaceutical type composition, the active ingredient is administered in various forms. Examples of the administration form include tablets, capsules, granules, powders,
Oral administration using a syrup or the like can be mentioned. These various preparations are commonly used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, and the like, in accordance with the usual methods for the main drug. It can be formulated using known adjuvants. Although the amount used varies depending on symptoms, age, body weight, administration method, dosage form, etc., usually, about 0.1 mg to 1,000 mg can be administered once to an adult.

Since the active ingredient according to the present invention is of natural origin, it has no or very low toxicity as described above, exhibits excellent safety, and has no acute toxicity. Therefore, when used as a food-drinks type composition, there is no particular limitation on the amount of the active ingredient used for prevention, health care, or when used as a normal food or drink, and when used as a medicine. The dose may be appropriately used within the above range depending on the patient. Moreover, even if a large amount of this active ingredient is taken, no particular side effect is observed and it is extremely safe.

[0019]

Example 1 The prolyl endopeptidase inhibitory activity was measured and the inhibitory substance was prepared as follows.

1) Method for measuring PEP inhibitory activity The inhibitory activity of PEP was measured according to the method shown in Table 1 below.

[0021]

[Table 1]

That is, a PEP (from Flavobacterium genus) solution, a buffer and a sample were mixed (30 ° C., 5
Minutes). Then, a synthetic substrate dissolved in dioxane (Z-G
ly-Pro-pNA, Z-Gly-Pro-2-NN
ap and / or Z-Gly-Pro-MCA) was added and reacted at 30 ° C. for 10 minutes. After adding hydrochloric acid, the absorbance was measured at OD 410 nm.

2) Preparation of PEP inhibitor

(A) Using sake lees as a raw material Sake brewing method using a liquefied liquid (Imanasu et al.
1 (1989)) sake sake lees (water content 36%) when brewing sake from rice according to 1 (1989)) is suspended in 2 L of water, and 40 mg of pepsin (1: 60,000, Sigma) is added,
After reacting at 37 ° C and pH 1.5 for 1 hour to neutralize,
The reaction was stopped by heating in a boiling water bath for 10 minutes. 500
A lysate was obtained by centrifugation at 0 revolution for 10 minutes, and about 5 g of a decomposed product was obtained by freeze-drying.

The protein amount and prolyl endopeptidase inhibitory activity were measured. The inhibition rate was calculated by the following formula, and the inhibitor concentration IC50 (mg / L) when the inhibition rate is 50% is shown in Table 2 below. Inhibition rate = (AB) × 100 / A A = 410 nm absorption value without inhibitor B = 410 nm absorption value with inhibitor

[0026]

[Table 2]

B) Using prolamin as a raw material: 1 g of prolamin fraction obtained by fractionating rice from Osborne by a solvent fractionation method was suspended in 20 ml of water, and 20 mg of a protein-degrading enzyme (samoaase) manufactured by Daiwa Kasei Co., Ltd. was added to the suspension. In 2
After reacting for 0 hours, heat in a boiling water bath for 10 minutes,
The reaction was stopped. The supernatant was recovered by centrifugation. The IC50 of the collected supernatant was 113.2 mg / L.

[0028]

Example 2 Decomposition product powder 10 derived from sake lees obtained in Example 1
0 g, sugar 150 g, honey 15 g, ascorbic acid 1
g, 0.5 g of citric acid, and 1 kg by adding water to an appropriate amount of flavor, sterilized at 95 ° C. for 20 minutes, and aseptically filled into bottles by 100 ml, a food-drink type health drink was produced.

[0029]

Example 3 Prolamin-derived supernatant 20 obtained in Example 1
0 g, tocopherol acetate 5 g, thiamine nitrate 10 g,
Nicotinic acid amide 20 g, anhydrous caffeine 50 g, benzoate and appropriate amount of perfume and deionized water to make 30 L,
After sterilization, 30 ml of each was aseptically filled into a bottle to produce a health drink as a medicine.

[0030]

Example 4 (1) Material prepared in Example 1 50 g (2) Lactose 90 g (3) Corn starch 29 g (4) Magnesium stearate 1 g (1), (2) and (3) (however 17 g) were mixed. Then
Granulated with the paste prepared from (3) (however, 7 g). To the obtained granules, (3) (however, 5 g) and (4) were added and mixed well, and the mixture was compressed by a compression tableting machine to obtain 1000 tablets each containing 50 mg of the active ingredient (1). Manufactured.

[0031]

INDUSTRIAL APPLICABILITY According to the present invention, an excellent ingestion product for PEP inhibition can be obtained. Not only is this ingestion excellent in PEP inhibitory ability, but it is derived from a natural product and is extremely safe, so it can be ingested for a long period of time.
In particular, for the purpose of prevention and treatment of dementia, it can be advantageously used as a pharmaceutical type and / or food and drink type.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location A61K 38/55 A61K 37/64

Claims (5)

[Claims] 1. A method for producing a prolyl endopeptidase inhibitor, which comprises treating rice protein or a rice protein-containing substance with a proteolytic enzyme. 2. An orally ingested substance for inhibiting prolyl endopeptidase, which comprises a proteolytic enzyme-decomposed product of rice protein or a processed product thereof, which is obtained by the method according to claim 1. 3. The oral ingestion product according to claim 2, wherein the proteolytic enzyme degradation product of rice protein is a peptide derived from rice protein or a degradation product of sake lees. 4. The processed product is a concentrate, pasted product, dried product, and / or diluted product of a proteolytic enzyme decomposition product of rice protein, according to claim 2 or claim 3. Oral ingestion. 5. The ingested substance according to any one of claims 2 to 4, wherein the ingested substance is of a pharmaceutical type or a food and drink type.