JP2022007698A - Composition for improving brain functions - Google Patents

Abstract

To provide a composition for improving brain functions that has excellent action of improving brain functions.SOLUTION: A composition for improving brain functions contains pentapeptide (Val-Tyr-Thr-Pro-Gly). The brain function improvement may be cognitive function improvement.SELECTED DRAWING: None

Description

The present invention relates to a composition for improving brain function containing a specific peptide as an active ingredient.

It is known that a peptide consisting of a specific sequence exerts various functions when ingested orally.
For example, as disclosed in Patent Document 1, it is known that a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly has various physiological actions. Patent Document 1 discloses a composition containing a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly, which is used for improving glucose metabolism, suppressing an increase in blood glucose level, and for antidiabetes.

Japanese Unexamined Patent Publication No. 2020-2018

An object of the present invention is to provide a composition for improving brain function, which is excellent in improving brain function.

As a result of research to solve the above-mentioned problems, the present inventors have found that the pentapeptide consisting of SEQ ID NO: 1 exhibits an excellent effect of improving brain function.
In order to achieve the above object, the composition for improving brain function according to one aspect of the present invention is characterized by containing a pentapeptide consisting of SEQ ID NO: 1.

In the composition for improving brain function, the improvement in brain function may be an improvement in cognitive function.
The composition for improving brain function may be an oral composition.
The food composition or pharmaceutical composition of another aspect of the present invention is characterized by containing the composition for improving brain function.

According to the present invention, an excellent brain function improving effect can be exhibited.

Hereinafter, an embodiment embodying the composition for improving brain function (hereinafter, also referred to as a composition) of the present invention will be described. The composition included in this embodiment contains a specific pentapeptide (SEQ ID NO: 1: Val-Tyr-Thr-Pro-Gly). Further, in the present specification, a pentapeptide having an amino acid sequence: Val-Tyr-Thr-Pro-Gly may be referred to as "VYTPG peptide".

The VYTPG peptide can be prepared by chemical synthesis using a known peptide synthesis method. Examples of the peptide synthesis method include peptide synthesis methods such as an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DDC method, an active ester method, a carboimidazole method, and an oxidation-reduction method. .. These peptide synthesis methods can be carried out by either a solid phase synthesis method or a liquid phase synthesis method.

In the above-mentioned peptide synthesis method, it is preferable to protect the amino group, the carboxy group, and / or the side chain functional group (for example, the guanidino group of arginine (Arg)) with a protecting group. As the protecting group, a known protecting group can be used without particular limitation, for example, a benzyloxycarbonyl group (Cbz), a tert-butoxycarbonyl group (Boc), a fluorenylmethoxycarbonyl group (Fmoc), a benzyl group. (Bz), p-toluenesulfonyl group (p-Ts) and the like can be mentioned.

Further, after synthesizing the VYTPG peptide by the above-mentioned peptide synthesis method, if necessary, a product purified by a known method can be used as the VYTPG peptide.
Furthermore, the VYTPG peptide may be obtained by hydrolyzing a protein with a protease or the like and purifying or crudely purifying it. The VYTPG peptide is also produced, for example, by treating rice bran with a thermolysin. In other words, the composition obtained by treating rice bran with a thermolysin (rice bran thermolysin-treated composition) contains a VYTPG peptide. Therefore, the rice bran thermolysin-treated composition may be purified or crudely purified by, for example, HPLC to obtain a VYTPG peptide. Further, a rice bran thermolysin-treated composition which has been treated as a raw material to increase the concentration of VYTPG peptide may be used.

In the thermolysin treatment of rice bran, the rice bran used is not particularly limited, but it is preferably defatted rice bran. Further, it is preferable that the rice bran is degreased by pressing (squeezed degreased rice bran). In particular, the pressed degreased rice bran obtained by inactivating lipase by heating the rice bran (raw rice bran) obtained after polishing and further pressing the rice bran is preferable. Further, it is preferable to use defatted rice bran having a lipid content of 5 to 20% by mass. A defatted rice bran having a lipid content in this range is preferably obtained by squeezing defatting (particularly squeezing defatting after lipase deactivation treatment).

The lipase deactivation treatment is carried out for the purpose of preventing oxidative deterioration of rice bran (raw rice bran) containing about 18 to 20% by mass of lipid. Usually, it is carried out by heating and roasting raw rice bran at about 70 to 130 ° C. The squeezing treatment is performed by a known squeezing method, for example, rice bran that has been heat-roasted and has reached about 100 to 115 ° C. is squeezed by a low-temperature continuous squeezing machine (for example, a miracle chamber sold by TechnoSigma Co., Ltd.). conduct. The squeezing is preferably carried out until the lipid content in the defatted rice bran after squeezing is about 5 to 20% by mass. Further, the drying treatment may be performed for the purpose of reducing the water content of the rice bran before the lipase deactivation treatment and the degreased rice bran after the oil extraction. Further, it is preferable that the defatted rice bran obtained by the pressing treatment is further pulverized and classified by a known method to form a powder. At this time, the average particle size of the powder is preferably 5 to 200 μm, more preferably 10 to 150 μm, and most preferably 40 to 100 μm. The lipid content in the defatted rice bran is preferably 5 to 20% by mass, more preferably 7 to 15% by mass, as described above. It is also possible to purchase and use a commercially available degreased rice bran as described above. Examples of such commercially available degreased rice bran include Hi-Bleff (manufactured by Saint-Blanc Co., Ltd.).

The thermolysin used in the present invention is a known enzyme and has an EC number of 3.4.24.27. The thermolysin can be used, for example, by purchasing a commercially available product. For example, it can be purchased from Peptide Institute Co., Ltd. (thermolysin), Amano Enzyme Co., Ltd. (samoase), and the like.

The thermolysin treatment conditions for obtaining the rice bran thermolysin-treated composition are not particularly limited as long as the thermolysin activity can be obtained, and can be appropriately set. For example, the treatment can be carried out by mixing rice bran and thermolysin with water and reacting them at about 37 ° C. with gentle stirring for about 10 to 30 hours. The thermolysin may be inactivated after the treatment. The deactivation treatment of thermolysin can be performed, for example, by heating (for example, treatment at 100 ° C. for about 10 to 20 minutes). After the treatment, purification may be performed by, for example, filtering and collecting the filtrate.

As a method for increasing or purifying the concentration of VYTPG peptide using the rice bran thermolysin-treated composition as a raw material, a known method, for example, HPLC or the like can be used.
The composition containing the VYTPG peptide exerts an excellent effect of improving brain function, especially when ingested orally. The composition of the present embodiment is applied for improving cognitive disorders such as brain dysfunction, particularly memory disorder, attention disorder, executive dysfunction, and social behavior disorder. Specifically, it is used for the treatment, improvement, and prevention of dementia that develops cognitive impairment.

Specific examples of dementia include Alzheimer-type dementia, vascular dementia, Lewy body dementia, frontotemporal dementia, and alcohol-related dementia. Improvement of dementia includes improvement of core symptoms of dementia such as memory disorder, attention disorder, executive dysfunction, and social behavior disorder mentioned above, as well as delusion, depression, wandering, sleeplessness, hallucinations, and decreased motivation. It also includes improvement of peripheral symptoms.

The application form of the composition is not particularly limited, and is applied as, for example, a food composition or a pharmaceutical composition. The VYTPG peptide may be used as it is as a material for a food composition or a pharmaceutical composition, or may be used as a product after appropriately blending a carrier, an additive or the like that is pharmaceutically or food hygiene acceptable.

When used as a food composition, it may contain other components as needed in addition to the VYTPG peptide as described above. The other ingredients are not particularly limited and can be appropriately selected according to the purpose. For example, bases, carriers, additives, and other ingredients / materials that can be used as foods, etc., which are acceptable in terms of food hygiene. Can be exemplified.

When used as a food composition, its ingestion form is oral ingestion. In this case, the form of the composition of the present embodiment is not particularly limited, and it can be used for general foods, foods with health claims, and foods for special purposes. Examples thereof include foods for specified health use, foods with nutritional function, foods with functional claims, foods for the sick, foods for people with swallowing difficulties, health supplements, dietary supplements, hospital foods, nursing foods, processed foods, beverages and the like. These can be prepared by a conventional method. When labeling the use of a food composition, the labeling specified by various laws, enforcement regulations, guidelines, etc. can be mentioned. In addition to labeling on packages such as packaging and containers, labeling of uses includes labeling on advertising media such as pamphlets.

In addition to the indication of improvement or prevention of brain function, the indication of the use of the composition of the present embodiment includes the indication of improvement or prevention of the above-mentioned core symptom and peripheral symptom. It also includes labeling suggesting these improvements or preventions. For example, display such as maintenance, support, or improvement of memory, attention, judgment, and spatial cognition, which are part of cognitive function. More specifically, it memorizes information about things that you see and hear, such as words, numbers, figures, colors, position information, and faces, and maintains or supports the ability to remember (memory), helps or improves the accuracy of information, and is short-term. Expressions such as maintaining or supporting memory (ability to retain or process information in a short time), maintaining attention (speed and accuracy of office work), and maintaining efficiency of calculation work can be mentioned.

The dosage form of the food composition includes, for example, hard capsules, soft capsules, supplements, chewable tablets, beverages, powdered beverages, granules, films, etc., and when used as food and drink, for example, tea-based beverages, sports. Beverages, beauty beverages, fruit juice beverages, carbonated beverages, alcoholic beverages, soft beverages, jelly beverages, beverages such as concentrated type beverages diluted with water or hot water, carbonated water, etc. Powders and granules, dried solids such as tablets, tablet confectionery, jellies, snacks, baked confectionery, fried confectionery, cakes, chocolate, gum, candy, gummy and other confectionery, soups, noodles, rice, It can also be in the form of food such as cereals. Of these, in normal life, supplement types, chewable tablets, one-shot drink types and the like are preferable, and when ingested for the purpose of enhancing exercise effects, beverage forms such as sports drinks are also preferable. In particular, in the case of foods with health claims, health supplements, dietary supplements, etc., in order to facilitate continuous ingestion, for example, powders, granules, pastes, capsules, tablets and tablets (chewable agents). Etc.), beverages (beverage powders, drinks, etc.), jelly agents, etc. are preferable.

When the composition is used as a food additive composition or a food material composition, its form is not particularly limited, and examples thereof include liquid, powder, flake, granule, and paste. Be done. More specifically, seasonings (sweeteners, salt substitute compositions, soy sauce, vinegar, miso, sauces, ketchup, dressings, spices, herbs, etc.), flakes (sprinkle, rice cooking additives, etc.), roasted meat sauce, roux paste (Ketchup paste, etc.)), premixed ingredients, etc.

When the composition is used as a food composition, the blending amount of the VYTPG peptide in the food composition is not particularly limited as long as it exhibits the above-mentioned action of the VYTPG peptide, and can be appropriately set.

Further, when the composition of the present invention is used as a food composition, the intake amount, intake interval, intake target and the like are not particularly limited and can be appropriately set. For example, the intake amount can be appropriately set according to the age, sex, weight, health condition of the target, and other conditions of the target. For example, the intake interval may be once or multiple times (preferably 2 to 3 times) per day, or once or multiple times every few days to several weeks when the above-mentioned amount is ingested. In addition to humans, the ingestion target may be domestic animals such as pets or livestock, for example, dogs, cats, cows, horses, sheep, llamas, rats, mice and the like.

In particular, when the administration target is a human, the dose of VYTPG peptide administration (ingestion) is preferably, for example, about 10 to 5000 μg, more preferably about 100 to 4000 μg per day for an adult (60 kg). The administration (ingestion) period is not particularly limited, and examples thereof include 3 days to 12 weeks, but may be longer.

The human to be ingested is not particularly limited, but a human having a desire or need for improvement or prevention of brain function is preferable.
The form, intake amount, intake interval, etc. when the intake target is a domestic animal can be appropriately set with reference to the case where a human is an intake target.

Pharmaceutical compositions include pharmaceuticals and quasi-drugs. When the composition of the present embodiment is used as a pharmaceutical composition, the administration method is not particularly limited, and administration (oral ingestion), enteral administration, enteral administration (particularly intravenous injection) and the like can be adopted. Is. When applied as a pharmaceutical composition, it may contain other components as needed, in addition to the VYTPG peptide as described above. The other components are not particularly limited and may be appropriately selected depending on the intended purpose. For example, pharmaceutically acceptable bases, carriers and / or additives (for example, solvents, dispersants, emulsifiers, etc.). Buffering agents, stabilizers, excipients, binders, disintegrants, lubricants, etc.) and the like. The blending amount of the other components can be appropriately set according to the purpose.

The dosage form of the pharmaceutical composition is not particularly limited, and for example, tablets such as orally disintegrating tablets, chewable tablets, effervescent tablets, dispersion tablets, dissolving tablets; lotions; powders; suspensions; emulsions; elixirs; Limonade; syrup; lotion; granules; capsules such as hard capsules and soft capsules; creams; ointments; suppositories; paps, tapes, microneedles, iontophoresis, electroporation, etc. Percutaneous / mucosal administration; aerosols and the like. These forms can be prepared by a conventional method in combination with the VYTPG peptide and, if necessary, the other components described above.

The blending amount of the VYTPG peptide in the pharmaceutical composition is not particularly limited as long as it exhibits the above-mentioned action of the VYTPG peptide, and can be appropriately set.
When used as a pharmaceutical composition, the dose, administration interval, administration target, etc. are not particularly limited and can be appropriately set. For example, the dose can be appropriately set according to the age, gender, body weight, health condition of the subject, and other conditions of the subject. Further, for example, the administration interval may be once or a plurality of times (preferably 2 to 3 times) per day, or may be once or a plurality of times every several days to several weeks. Further, the administration target may be not only humans but also domestic animals such as pets or livestock, for example, dogs, cats, cows, horses, sheep, llamas, rats, mice and the like.

In particular, when the administration target is a human, the dose of VYTPG peptide administration (ingestion) is preferably, for example, about 10 to 5000 μg, more preferably about 100 to 4000 μg per day for an adult (60 kg). The administration (ingestion) period is not particularly limited, and examples thereof include 3 days to 12 weeks, but may be longer.

When the administration target is a domestic animal, the administration form, dosage form, dose, administration interval and the like can be appropriately set with reference to the case where a human is the administration target.
The effect of the composition for improving brain function of this embodiment will be described.

(1) In this embodiment, it is configured to contain a VYTPG peptide. Therefore, it can exert an excellent effect of improving brain function. In particular, it has an excellent effect on improving cognitive function. Furthermore, in the case of lifestyle-related decline in cognitive function such as a high-fat diet, short-term ingestion can contribute to the recovery of cognitive function.

The above embodiment may be changed as follows.
-The composition of the above embodiment is not only applied to patients for the purpose of improving symptoms related to brain function, but also for the purpose of preventing the deterioration of symptoms, delaying the deterioration of symptoms, and preventing the deterioration of brain function in healthy subjects. Ingestion is also included.

-In the composition of the above embodiment, the amount of VYTPG peptide contained in the food composition or the pharmaceutical composition is not particularly limited as long as the effect of the present invention can be obtained, and is, for example, 0.1 to 100% by mass in the solid content. It can be configured to contain, preferably 1 to 90% by mass.

-In addition, in this specification, "includes" also includes "consisting essentially" and "consisting of" (The term "comprising" includes "consisting essentially of" and "consisting of."). The present invention also includes all combinations of the constituent elements described herein.

In addition, the various properties (property, structure, function, etc.) described for each embodiment of the present invention described above may be combined in any way in specifying the subject included in the present invention. That is, the present invention includes all subjects consisting of any combination of each of the combinable properties described herein.

Hereinafter, the above-described embodiment will be described in more detail with reference to test examples, but the present invention is not limited thereto.
(Synthesis of VYTPG peptide)
Production Example 1: Synthesis of Pentapeptide by Fmoc Method A pentapeptide consisting of Val-Tyr-Thr-Pro-Gly was solid-phase synthesized by the Fmoc method. After purifying the obtained VYTPG peptide by HPLC, the sequence was analyzed by a protein sequencer, and as a result, it was confirmed that it was a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly.

Production Example 2: Synthesis of Peptide Peptide by Boc Method We requested Peptide Institute Co., Ltd. to synthesize a pentapeptide consisting of Val-Tyr-Thr-Pro-Gly by the Boc method. The delivered VYTPG peptide was confirmed by RP-HPLC, mass spectrometry and amino acid analysis to be a pentapeptide composed of Val-Tyr-Thr-Pro-Gly.

Production Example 3: Purification of Pentapeptide from Rice Bran Enzyme-treated Product First, rice bran was enzymatically treated to obtain an enzyme-treated composition as follows.
As the enzyme, thermolysin (Peptide Institute, Ltd .; Code3504) was used.

As the rice bran, high bluff (manufactured by Saint-Blanc Co., Ltd.) was used. High bluff is a squeezed degreased rice bran that has been degreased by squeezing the rice bran.
First, rice bran was mixed with ultrapure water so as to be 30 g / L, and thermolysin was added to this so as to be 0.4 g / L. Then, the mixture was placed in a 37 ° C. incubator using a stirrer at 250 r. p. m. Incubated for 20 hours with stirring. Further, it was immersed in a boiling water bath and treated for 15 minutes to inactivate the enzyme. 15 ° C., 7150r. p. m. Centrifugation was performed in (8000 × g) 15 minutes, and the pH of the obtained supernatant was adjusted to 7.0 using NaOH, and the filter paper No. Filtration was carried out using 2 (manufactured by Advantech), and the obtained filtrate was lyophilized. The freeze-dried product was used as a rice bran enzyme-treated composition. The rice bran enzyme-treated composition may be hereinafter referred to as "HB-T".

Next, the VYTPG peptide was purified from HB-T as follows.
The HB-T aqueous solution adjusted to 50 mg / mL with ultrapure water was fractionated with a Preparative HPLC apparatus (1525 binary pump, 2487 spectroscope). A C18 (Cosmosil 5C18-AR-II 4.6 × 250 mm) column was used as the column. The HPLC conditions were as shown in Table 1 below.

得られたＨＰＬＣチャートのうちピーク５３を分画後濃縮遠心し、４００μＬの超純水に溶解した。このうち１００μＬを下記表２記載のａｎａｌｙｔｉｃａｌＨＰＬＣの条件で分画した。カラムはＰＥカラム（Cosmosil 5PE-MS 4.6×250mm）を用いた。

Of the obtained HPLC chart, peak 53 was fractionated, concentrated and centrifuged, and dissolved in 400 μL of ultrapure water. Of this, 100 μL was fractionated under the conditions of analytical HPLC shown in Table 2 below. A PE column (Cosmosil 5PE-MS 4.6 × 250 mm) was used as the column.

得られたチャートのピーク５３－１３を分取後濃縮遠心し、１００μＬの超純水に溶解した。当該サンプルを再度ａｎａｌｙｔｉｃａｌＨＰＬで分画した。注入量は５０μＬとし、カラムはＣＮカラム（Cosmosil 5CN-MS 4.6×250mm）を用いた。得られたチャートのピーク５３３を精製した。得られた精製サンプルを質量分析装置およびプロテインシーケンサで解析したところ、ＶＹＴＰＧペプチドであった。

The peaks 53-13 of the obtained chart were separated, concentrated and centrifuged, and dissolved in 100 μL of ultrapure water. The sample was fractionated again with an analytical HPL. The injection volume was 50 μL, and a CN column (Cosmosil 5CN-MS 4.6 × 250 mm) was used as the column. The peak 533 of the obtained chart was purified. When the obtained purified sample was analyzed by a mass spectrometer and a protein sequencer, it was a VYTPG peptide.

(Measurement of cognitive function)
The test method was based on the mouse object recognition test method (Marianne. Leger et al., Nature Protocols 8 (12), 2531-2537, 2013).

The mice used in the test were 11-week-old male ddY mice, which were fed with a high-fat diet for 1 week and acclimatized, and then divided into two populations. Both groups were bred on a high-fat diet for one week (free feeding / free drinking). One group (VYTPG peptide group) was orally administered with Teflon sonde to 1 mg / kg of VYTPG peptide prepared with physiological saline, and the same amount of physiological saline was orally administered to the other control group for 1 week. Of these, oral administration was performed on the 5th, 6th, and 7th days.

As the high-fat diet, HFD-60 (manufactured by Oriental Bio Co., Ltd.) composed of the components shown in Table 3 was used.

飼育６日目に、各個体群中のマウス１匹を所定の大きさの箱の中に入れ、５分間環境に順応させた（馴化１）。２４時間後、２個の物質が左右１箇所ずつ設置された箱の中にマウスを入れ、両方の物質探索時間の合計が２０秒になるまで行動させた後にその箱から取り出し、ケージに戻した（馴化２）。この際、箱に入っている物質は２個とも同じもので積み木又はフラスコである。さらに１時間後に２個の物質のうち片方を別の形の物質で置き換えた箱の中にマウスを入れ、両方の探索時間の合計が２０秒になるまでマウスを行動させ、そのうち置き換えた新規物質の方の探索時間の割合（％）を求めた。

On the 6th day of breeding, one mouse in each population was placed in a box of a predetermined size and acclimatized to the environment for 5 minutes (acclimation 1). Twenty-four hours later, the mouse was placed in a box containing two substances, one on each side, and the mice were allowed to act until the total search time for both substances reached 20 seconds, then removed from the box and returned to the cage. (Familiarization 2). At this time, both substances contained in the box are the same and are blocks or flasks. After another hour, the mouse was placed in a box in which one of the two substances was replaced with another substance, and the mouse was allowed to act until the total search time of both substances reached 20 seconds, of which the new substance was replaced. The ratio (%) of the search time for the mouse was calculated.

Whether the new substance was on the right side or the left side was tested so as to be completely random. The test box was placed in a compartment separated by a partition, and the examiner observed the behavior from outside the partition through a video camera installed above the box so that it could not be seen by the mouse. All of this test was conducted in a facility set at 23 ° C., which was set to have a 12-hour light period and a 12-hour dark period.

Mean ± standard deviation was calculated for each test group population. The results of each population are shown in Table 4. There was no significant difference in body weight between the populations during the test period. * Indicates vs. Control: p <0.05 (tukey's multiplex test).

表４に示されるように、ＶＹＴＰＧペプチドを高脂肪食に添加した個体群においては、Ｃｏｎｔｒｏｌ群に対して有意に新規物質の探索時間の割合の上昇が認められた。

As shown in Table 4, in the population to which the VYTPG peptide was added to the high-fat diet, a significant increase in the ratio of the search time for the new substance was observed with respect to the Control group.

Claims (4)

A composition for improving brain function containing a pentapeptide consisting of SEQ ID NO: 1. The composition for improving brain function according to claim 1, wherein the improvement of brain function is improvement of cognitive function. The composition for improving brain function according to claim 1 or 2, which is an oral composition. A food composition or a pharmaceutical composition containing the composition for improving brain function according to any one of claims 1 to 3.