WO2017010538A1 - Composition that contains plant- or animal-derived peptide and inhibits serum carnosinase - Google Patents

Abstract

Provided are a carnosine dipeptidase 1 inhibitor composition, a use for a material that inhibits carnosine dipeptidase 1, and a method for inhibiting carnosine dipeptidase 1. The present invention demonstrates the discovery of a plant- or animal-derived peptide exhibiting an inhibitory activity against carnosine dipeptidase 1, and thus, provides a novel and effective means that contributes to the prevention or amelioration of a cognitive function decline or the like.

Description

Composition for inhibiting serum carnosine degrading enzyme containing animal and plant derived peptides

The present invention relates to a composition for inhibiting serum carnosine degrading enzyme. More specifically, the present invention relates to a composition for inhibiting carnosine dipeptidase 1 comprising a peptide derived from animals and plants as an active ingredient, the use of a peptide derived from animals and plants to inhibit carnosine dipeptidase 1, a method for inhibiting carnosine dipeptidase 1, and The present invention relates to a composition comprising an animal and plant derived peptide and carnosine.

Carnosine is a dipeptide composed of β-alanine and histidine, and is present in high concentrations in muscle and nerve tissues in mammals such as humans. Carnosine's actions include (1) proton buffering activity, (2) calcium secretion and calcium sensitivity control, (3) antioxidant action, (4) metal ion chelate action, (5) histidine / histamine extracellular donor (6) Hyperglycemia improving action, (7) Anti-inflammatory action, etc. are known. As for the action of carnosine, the production of glycated end products, the suppression of cell death due to cerebral ischemia, the accumulation of amyloid β in Alzheimer's disease (AD) model mice, the immunoregulatory action, and the like have been reported. As described above, carnosine contributes to various functions in the body. However, degradation by carnosine degrading enzyme is a problem for exerting its pharmacological action.

It is known that there are two types of carnosine-degrading enzymes: carnosine dipeptidase 1; CNDP1 and tissue carnosinase 2 (CNDP2). Among these, CNDP1 has been shown to exist only in higher primates (human and large monkeys) and not in most other mammals (Non-patent Document 1). Although these CNDP1 and CNDP2 are highly homologous proteins, their tissue distribution and enzymatic characteristics are different, and both are considered to have different functions.

Regarding CNDP2, for example, bestatin is known as an inhibitor (Non-patent Document 2), and others are β-alanine and linear chains such as Gly-L-His and L-Pro-L-His. It has been reported that dipeptides are effective in inhibiting CNDP2 (Patent Document 1). On the other hand, with regard to CNDP1, for example, phenanthroline has been reported as an inhibitor (Non-patent Document 3), but there are few known carnosine degradation inhibitors that focus on inhibition of CNDP1 activity.

As described above, since CNDP1 and CNDP2 are different proteins, the inhibitors for the respective enzymes are also considered to be different from each other. In fact, it is specified that bestatin, which is the above CNDP2 inhibitor, has no effect on the inhibition of CNDP1 (Non-patent Document 4). Moreover, although the phenanthroline shown above has CNDP1 inhibitory activity, it is known to show oral toxicity as a side effect thereof. Therefore, if a safer inhibitor of CNDP1 can be found, clinical application to diseases and symptoms related to the activity of CNDP1 is considered possible.

Regarding CNDP1, in non-patent document 5, in an animal model in which human serum carnosinase (CNDP1) is introduced into a db / db mouse, diabetes, such as fasting blood glucose level and HbA1c are higher than in younger age, indicating weight loss, etc. Admits that symptoms appear. That is, it has been suggested that enhanced carnosine degradation by serum carnosinase (CNDP1) may cause disease onset. Therefore, serum carnosine degrading enzyme (CNDP1) inhibitors can efficiently deliver L-carnosine to plasma, target organs or other organs, and against various diseases caused by diabetes, oxidative stress, and production of advanced glycation end products. It is considered as an approach to enhance the preventive effect.

Further, in a plurality of documents such as Non-Patent Document 6, there is a correlation between a specific gene polymorphism ((CTG) n) in the serum carnosinase (CNDP1) gene and the onset of diabetic nephropathy. It has been reported. In this connection, Non-Patent Document 7 reports that homozygous (CTG) 5 carriers have a low risk of developing diabetic nephropathy and low serum carnosinase activity. Therefore, suppressing serum carnosinase activity is important for maintaining carnosine concentration, and is considered to be effective in preventing or treating related diseases.

In addition, Non-Patent Document 8 can be cited as a verification example of a pharmacokinetic test after oral ingestion of carnosine in humans. According to this document, individual differences in the blood concentration of carnosine at each time after ingestion of carnosine 60 mg / kg are large, and there are some subjects in which no significant increase in blood carnosine concentration was observed compared to before intake (among 25 subjects). 17), the activity of serum carnosinase and the amount of protein were significantly lower in the group in which the increase was observed than in the group in which the increase was not. From this, it is considered that there is a high possibility that suppressing the action of serum carnosinase (CNDP1) is effective in maintaining the blood carnosine concentration.

Thus, since CNDP1 exerts various influences in the human body as a mammal, there is a strong demand for highly safe drugs for effectively inhibiting this activity.

International Publication WO 2004/064866

An object of the present invention is to provide a composition for inhibiting serum carnosine degrading enzyme (CNDP1) that has high biosafety and contributes to maintaining the blood concentration of carnosine. Another object of the present invention is to provide a use of a material for inhibiting CNDP1, a method for inhibiting CNDP1, and a composition that is highly biosafety and contributes to maintaining the blood concentration of carnosine. .

As a result of intensive studies on the above problems, the present inventors have found for the first time that collagen peptides, tea peptides and soybean peptides have an inhibitory activity on serum carnosine degrading enzyme (CNDP1). Based on this finding, the present inventors have completed the present invention.

That is, the present invention relates to the following, but is not limited thereto.
(1) A composition for inhibiting carnosine dipeptidase 1 comprising an animal or plant-derived peptide.
(2) The composition for inhibiting carnosine dipeptidase 1 according to (1), wherein the animal or plant derived peptide is derived from collagen, tea, or soybean.
(3) For prevention or improvement of various diseases, Alzheimer's disease, autism, stress, or hypertension caused by cognitive decline, diabetes, immune function decline, vascular or tissue inflammation, oxidative stress or advanced glycation end products A composition for inhibiting carnosine dipeptidase 1 according to (1) or (2).
(4) The composition for inhibiting carnosine dipeptidase 1 according to any one of (1) to (3), wherein the animal or plant derived peptide is a heat-treated product.
(5) The composition for inhibiting carnosine dipeptidase 1 according to any one of (1) to (4), which is labeled with a function exhibited by carnosine dipeptidase 1 inhibition.
(6) The function indication is “suppresses cognitive function decline”, “expects maintenance of cognitive function”, “suppresses increase in blood glucose level”, “improves immune function”, “antioxidant action” `` Expect '', `` Reduce oxidative stress '', `` Expect anti-glycation effect '', `` Reduce glycation stress '', `` Inhibit vascular inflammation '', `` Expect prevention or improvement of Alzheimer's disease '', "Expect prevention or improvement of autism", "Prevent stress", "Relieve stress", "Relieve stress", "Expect blood pressure drop", "Suppress blood pressure rise" Carnosine dipeptidase 1 inhibition according to (5), selected from the group consisting of: “slowing blood pressure rise”, “preventing hypertension”, and “helping to improve hypertension” Composition.
(7) The composition for inhibiting carnosine dipeptidase 1 according to any one of (1) to (6), wherein the composition is an agent.
(8) Use of an animal or plant-derived peptide for inhibiting carnosine dipeptidase 1.
(9) The use according to (8), wherein the animal or plant-derived peptide is a heat-treated product.
(10) A method for inhibiting carnosine dipeptidase 1 using an animal or plant-derived peptide.
(11) The method according to (10), wherein the animal or plant-derived peptide is a heat-treated product.
(12) A composition comprising an animal and plant derived peptide and carnosine,
The above-mentioned composition, wherein the animal or plant-derived peptide contains cyclophenylalanylphenylalanine [Cyclo (Phe-Phe)], and the weight ratio of cyclophenylalanylphenylalanine to carnosine is 1: 1000 to 1: 1.
(13) The composition according to (12), wherein the animal or plant-derived peptide is a soybean peptide or a heat-treated product thereof.

According to the present invention, a composition having an excellent inhibitory effect on serum carnosine degrading enzyme (CNDP1) can be provided. By using the composition of the present invention, the effect of delaying degradation of carnosine accompanying the suppression of CNDP1 function can be obtained, so that a higher concentration of carnosine can be efficiently delivered to plasma, target organ or other organs. Become. Therefore, the composition of the present invention originates from various pharmacological actions that are originally known for carnosine (cognitive decline associated with schizophrenia, diabetes, immune function decline, inflammation of blood vessels and tissues, oxidative stress, etc. It can be effective in improving various diseases onset, prevention of Alzheimer's disease, autism, stress, hypertension, and improvement effect).

The animal-and-plant-derived peptides contained in the composition of the present invention are highly safe because many of them are used as food materials, and side effects are considered to be extremely small compared to conventional pharmaceuticals.

FIG. 1 is a graph showing the pharmacokinetics of carnosine. FIG. 2 is a graph showing the inhibitory effect of heat-treated tea peptide on serum carnosine degradation. FIG. 3 is a graph showing changes in depression and depression. The vertical axis of the graph shows the T score change amount in the [Depression / Depression] item when the pre-drinking beverage (SCR) is 0, and the horizontal axis of the graph shows the questionnaire implementation time. Moreover, the data plotted in the graph shows an average value ± standard deviation. FIG. 4 is a graph showing changes in negative mood state. The vertical axis of the graph shows the amount of change in the total score of TMD (total disturbance) when the pre-drinking (SCR) is 0, and the horizontal axis of the graph shows the timing of the questionnaire. The data plotted in the graph shows the mean value ± standard deviation. FIG. 5 is a graph showing changes in blood pressure. The vertical axis of the graph indicates the amount of change (mmHg) in systolic blood pressure when the pre-beverage (SCR) is 0, and the horizontal axis of the graph indicates the blood pressure measurement timing. The data plotted in the graph shows the mean value ± standard deviation.

1. Carnosine dipeptidase 1 and carnosine dipeptidase 1 inhibition As used herein, “carnosine dipeptidase 1” refers to a serotype carnosine degrading enzyme capable of degrading carnosine (L-carnosine) into β-alanine and histidine. . Carnosine dipeptidase (carnosine degrading enzyme) can be abbreviated as CNDP (carnosine dipeptidase), and is also called carnosinase or carnosidase. Carnosine dipeptidase includes CNDP1 which is a serum (type) carnosine degrading enzyme and CNDP2 which is a tissue (type) carnosine degrading enzyme. Among these, the carnosine dipeptidase targeted in the present invention is CNDP1, which is distinguished from CNDP2.

As used herein, “carnosine dipeptidase 1 inhibition” refers to inhibiting the carnosine degradation activity of carnosine dipeptidase 1. The inhibitory action of carnosine dipeptidase 1 can be evaluated according to a known method. For example, when carnosine and carnosine dipeptidase 1 are brought into contact with each other, histidine is generated from carnosine, and histidine-specific fluorescence can be measured due to the presence of histidine. Carnosine dipeptidase can be obtained by examining the decrease in fluorescence intensity. 1 inhibitory action can be evaluated.

2. Animal and Plant Derived Peptide In the present invention, “animal and plant derived peptide” includes two concepts of an animal derived peptide and a plant derived peptide. Here, unless otherwise specified, “animal-derived peptides” are known degradation treatments of animal-derived proteins or animal tissues containing proteins (decomposition treatment with heat or pressure, degradation treatment with acids or alkalis, degradation with enzymes). It means a peptide produced by processing to reduce the molecular weight. Unless otherwise specified, “plant-derived peptide” refers to a plant-derived protein, or a known decomposition treatment (decomposition treatment with heat or pressure, decomposition treatment with acid or alkali, enzyme It means a peptide produced by reducing the molecular weight by subjecting it to a degradation treatment. Moreover, the animal and plant-derived peptide in the present invention may be a peptide obtained by adding a treatment such as heating to the peptide thus obtained.

The peptide used in the present invention may be one type of peptide available from animals or plants, or a mixture of two or more types of peptides. The number of amino acids constituting the animal or plant derived peptide is not particularly limited, but is 2 to several tens (specifically, 2 to 10, 2 to 15, 2 to 20, 2 to 25, 2 to 30). 2 to 35, or 2 to 40) are preferred, and 2 to several (specifically, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7) (2 to 8, or 2 to 9) (that is, oligopeptide) is more preferable.

In the present invention, the animal and plant derived peptides are preferably those having a high ratio of peptides having a molecular weight of 5000 or less, more preferably those having a high ratio of peptides having a molecular weight of 3000 or less, and high ratios of peptides having a molecular weight of 1000 or less. It is particularly preferable to use one. Here, “the ratio of peptides is high” means a state in which at least 50% of all the peptides derived from animals and plants correspond to the peptides. The molecular weight can be measured using a method and apparatus (such as HPLC) well known to those skilled in the art.

Although it does not specifically limit as an animal origin peptide, For example, mammals (a cow, a pig, etc.), birds (a chicken, etc.), fishes (a salmon, a salmon, a salmon, a salmon, etc.), an egg (a chicken egg etc.), milk (milk, etc.) ) And the like can be used. Specific examples of peptides obtained from these include peptides derived from collagen, albumin, casein, placenta, globulin and the like. In the present invention, a collagen-derived peptide is preferably used as the animal-derived peptide.

The plant-derived peptide is not particularly limited, and for example, plant-derived peptides such as beans, leaves, seeds, and moss can be used. Examples of beans include soybeans, red beans, and black beans. Examples of the leaves include tea (green tea, black tea, oolong tea) and the like. Examples of seeds include barley, wheat (including wheat germ), malt, sesame and rice. Examples of moss include sweet potatoes and potatoes. In the present invention, the plant-derived peptide is preferably a soybean or tea-derived peptide, and more preferably a tea-derived peptide.

In this specification, the description of “derived” may be omitted for animal and plant derived peptides (animal derived peptides and plant derived peptides). For example, in the case of “collagen-derived peptide”, this may be referred to as “collagen peptide”. For example, in the case of “soybean-derived peptide”, this may be referred to as “soybean peptide”. At this time, both are used interchangeably.

The animal and plant-derived peptide is not particularly limited, and it can be obtained by decomposing animal-derived protein or animal tissue containing protein, or plant-derived protein or plant body or plant tissue containing protein by a conventionally known method. it can. Examples of such decomposition treatment include decomposition treatment with heat or pressure, decomposition treatment with acid or alkali, and decomposition treatment with an enzyme. In any treatment, water, ethanol or the like can be used as a solvent. In the case of enzymatic degradation, various proteolytic enzymes (proteases) can be appropriately used depending on the purpose.

The animal and plant derived peptides may be prepared by themselves using known methods, or commercially available products may be used. Examples of commercially available animal-derived peptides include collagen peptides such as Nippi Peptide (Nippi Co., Ltd.), Ikuos HDL, Collagen Peptide 800F, Super Collagen Peptide SCP, Fermented Collagen Peptide LCP (Nitta Gelatin Inc.). . Examples of commercially available plant-derived peptides include soy peptides such as High Newt AM, High Newt DC, and High Newt HK (above, manufactured by Fuji Oil Co., Ltd.) and rice such as Oriza Peptide-P60 (manufactured by Oriza Oil Chemical Co., Ltd.). Examples thereof include wheat peptides such as peptides, glutamine peptide GP-1N, glutamine peptide GP-N (manufactured by Nisshin Pharma), and sesame peptides such as sesame peptide KM-20 (manufactured by KISCO).

In the present invention, the animal and plant derived peptide may be one obtained by further heat-treating the above peptide. The heat treatment can be performed using, for example, a pressure-resistant extraction device, a pressure cooker, an autoclave, or the like well known to those skilled in the art, but is not limited thereto. You may perform the heat processing of the animal and plant origin peptide in this invention with reference to the method as described in international publication 2014/200000. Moreover, the animal and plant derived peptides may have been subjected to solid-liquid separation before and / or after heat treatment. By performing the solid-liquid separation process, the liquid part can be recovered, and it can be handled only by the solid. For solid-liquid separation, means such as filtration and / or centrifugation are used. Moreover, the animal and plant derived peptide may be subjected to a purification treatment after the heat treatment. The purification treatment can be performed using a known method and apparatus. Moreover, the animal and plant derived peptides may be further clarified. The clarification treatment can be performed using a known method and apparatus, and the degree of freedom in designing a composition to which an animal or plant derived peptide is added can be increased by the treatment. In addition, the animal and plant derived peptide may be freeze-dried or powdered using a known method and apparatus.

The animal and plant derived peptide in the present invention is not particularly limited, but can be heat-treated at a temperature of 100 ° C. or higher and a pressure exceeding atmospheric pressure. The temperature is preferably 105 ° C. or higher, 110 ° C. or higher, 115 ° C. or higher, 120 ° C. or higher, 125 ° C. or higher, 130 ° C. or higher, or 135 ° C. or higher. The temperature is preferably 170 ° C. or lower, 165 ° C. or lower, 160 ° C. or lower, 155 ° C. or lower, 150 ° C. or lower, 145 ° C. or lower, or 140 ° C. or lower. In addition, this temperature shows the value which measured the exit temperature of the extraction column, when using a pressure-resistant extraction apparatus as a heating apparatus, and when using an autoclave as a heating apparatus, it is the temperature of the center temperature in a pressure vessel. The measured value is shown.

Regarding the pressure condition of the heat treatment, the numerical value is not particularly limited as long as the pressure exceeds the atmospheric pressure, but preferably 0.101 MPa or more, 0.15 MPa or more, 0.2 MPa or more, 0.25 MPa or more, or 0.3 MPa or more. It is. The pressure is preferably 0.79 MPa or less, 0.75 MPa or less, 0.7 MPa or less, 0.65 MPa or less, 0.6 MPa or less, 0.55 MPa or less, 0.5 MPa or less, or 0.48 MPa or less. .

The processing time for the heat treatment is not particularly limited. The treatment time is, for example, about 15 minutes to 600 minutes, preferably about 30 minutes to 500 minutes, and more preferably about 60 minutes to 300 minutes. In the present invention, a more suitable heat treatment condition for obtaining a heat-treated product derived from animals and plants is, for example, in a coordinate system in which the horizontal axis is time (min.) And the vertical axis is temperature (° C.) It is a heat treatment held within a range of time and temperature surrounded by i) to (vi). (i) (170 ° C, 30 min.), (ii) (150 ° C, 30 min.), (iii) (115 ° C, 180 min.), (iv) (105 ° C, 480 min.), (v ) (135 ℃, 480 min.), (Vi) (150 ℃, 180 min.)
The conditions for the heat treatment in the present invention are not particularly limited, but can be set, for example, as follows with respect to temperature, pressure and time:
(Temperature, pressure, time) =
(105 ° C to 170 ° C, 0.101 MPa to 0.79 MPa, 15 minutes to 600 minutes),
(105 ° C to 170 ° C, 0.101 MPa to 0.79 MPa, 30 minutes to 500 minutes),
(105 ° C to 170 ° C, 0.101 MPa to 0.79 MPa, 60 minutes to 300 minutes),
(105 ° C to 170 ° C, 0.15 MPa to 0.48 MPa, 15 minutes to 600 minutes),
(105 ° C to 170 ° C, 0.15 MPa to 0.48 MPa, 30 minutes to 500 minutes),
(105 ° C to 170 ° C, 0.15 MPa to 0.48 MPa, 60 minutes to 300 minutes),
(110 ° C. to 150 ° C., 0.101 MPa to 0.79 MPa, 15 minutes to 600 minutes),
(110 ° C. to 150 ° C., 0.101 MPa to 0.79 MPa, 30 minutes to 500 minutes),
(110 ° C. to 150 ° C., 0.101 MPa to 0.79 MPa, 60 minutes to 300 minutes),
(110 ° C. to 150 ° C., 0.15 MPa to 0.48 MPa, 15 minutes to 600 minutes),
(110 ° C. to 150 ° C., 0.15 MPa to 0.48 MPa, 30 minutes to 500 minutes),
(110 ° C. to 150 ° C., 0.15 MPa to 0.48 MPa, 60 minutes to 300 minutes),
(120 ° C. to 140 ° C., 0.101 MPa to 0.79 MPa, 15 minutes to 600 minutes),
(120 ° C. to 140 ° C., 0.101 MPa to 0.79 MPa, 30 minutes to 500 minutes),
(120 ° C. to 140 ° C., 0.101 MPa to 0.79 MPa, 60 minutes to 300 minutes),
(120 ° C. to 140 ° C., 0.15 MPa to 0.48 MPa, 15 minutes to 600 minutes),
(120 ° C. to 140 ° C., 0.15 MPa to 0.48 MPa, 30 minutes to 500 minutes),
(120 ° C. to 140 ° C., 0.15 MPa to 0.48 MPa, 60 minutes to 300 minutes) and the like.

Preferred embodiments of the animal and plant derived peptides in the present invention are collagen peptides, tea peptides, and soybean peptides. Hereinafter, these animal and plant derived peptides will be described.

(Collagen peptide)
As used herein, “collagen peptide” refers to a low molecular peptide obtained by subjecting collagen itself or a pulverized product of collagen to enzymatic treatment or heat treatment to lower the molecular weight of collagen. Collagen is a major protein in animal connective tissue and is the most abundant protein in mammalian bodies including humans. The obtained low molecular weight peptide may be further subjected to treatments such as filtration, centrifugation, concentration, ultrafiltration, lyophilization, and pulverization as desired.

(Tea peptide)
The term “tea peptide” as used herein refers to a low molecular weight peptide derived from tea obtained by subjecting tea itself (including tea leaves and tea shells) or tea extract to enzyme treatment or heat treatment to lower the protein. Say. As a tea leaf used as an extraction raw material, a tea leaf (scientific name: Camellia sinensis) manufactured tea leaf leaf, stem, etc. that can be extracted and used can be used. Also, the form is not limited to large leaves or powders. The harvest time of tea leaves can also be selected appropriately according to the desired flavor. The obtained low molecular weight peptide may be further subjected to treatments such as filtration, centrifugation, concentration, ultrafiltration, lyophilization, and pulverization as desired.

The tea extract used in the present invention preferably has a low by-product content and has a good flavor. From this flavor point of view, the raw tea leaves are steamed non-fermented tea (green tea) such as Sencha, Bancha, Hojicha, Gyokuro, Kabusecha, and Kochacha, Ureshino tea, Aoyagi tea, various Chinese tea, etc. It is preferable to use unfermented tea such as tea.

(Soybean peptide)
As used herein, “soy peptide” refers to a low molecular peptide obtained by subjecting soy protein itself or soy protein to enzyme treatment or heat treatment to lower the protein. Soybeans (scientific name: Glycine max) used as a raw material can be used without restriction of varieties and production areas, and can also be used in processed products such as pulverized products. The obtained low molecular weight peptide may be further subjected to treatments such as filtration, centrifugation, concentration, ultrafiltration, lyophilization, and pulverization as desired.

3. 3. Composition for inhibiting carnosine dipeptidase 1 3-1. Composition for inhibiting carnosine dipeptidase 1 containing animal and plant-derived peptide One aspect of the present invention is a composition for inhibiting carnosine dipeptidase 1 comprising an animal and plant-derived peptide as an active ingredient.

The content of the animal and plant derived peptide in the composition for inhibiting carnosine dipeptidase 1 of the present invention is not particularly limited as long as the desired effect of the present invention can be obtained in consideration of its administration form, administration method and the like. Is not to be done. For example, the content of the animal and plant derived peptide is 0.1% by weight or more, preferably 0.2% by weight or more, more preferably 0.3% by weight with respect to the total weight of the composition for inhibiting carnosine dipeptidase 1 of the present invention. % Or more. In addition, the content of animal and plant derived peptides is 30% by weight or less, preferably 20% by weight or less, more preferably 10% by weight or less, based on the total weight of the composition for inhibiting carnosine dipeptidase 1 of the present invention. Typically, the content of the peptide derived from animals and plants is 0.1 to 30% by weight, preferably 0.2 to 20% by weight, based on the total weight of the composition for inhibiting carnosine dipeptidase 1 of the present invention. %, More preferably 0.3 to 10% by weight. Unless otherwise specified, “wt%” used in the present specification means weight / volume (w / v).

3-2. Mechanism of Action As described above, inhibition of carnosine dipeptidase 1 maintains the body concentration of carnosine degraded by carnosine dipeptidase 1 in mammals such as humans, or suppresses a decrease in the concentration. Carnosine functions include proton buffering activity, calcium secretion and calcium sensitivity control, antioxidant action, metal ion chelate action, extracellular donor of histidine / histamine, hyperglycemia improvement action, anti-inflammatory action, generation of advanced glycation end products Examples thereof include suppression, suppression of cell death due to cerebral ischemia, accumulation of amyloid β, immunoregulation, anti-stress, and blood pressure reduction in Alzheimer's disease (AD) model mice. Therefore, by maintaining carnosine concentration in the body, it is possible to prevent or improve cognitive function associated with schizophrenia, Alzheimer's, or autism based on the accumulation of amyloid β. Based on this, it is possible to prevent or ameliorate the onset of various diseases caused by diabetes or oxidative stress or the production of advanced glycation end products. The prevention or improvement effect of immune function decline is obtained based on the anti-stress action, the prevention or improvement effect of stress (mental stress) is obtained based on the anti-stress action, and the prevention or improvement effect of hypertension is obtained based on the blood pressure reduction action. can get.

3-3. Other Components The composition for inhibiting carnosine dipeptidase 1 of the present invention can contain any additive and usually used components in addition to animal or plant-derived materials, depending on the form. Examples of these additives and / or ingredients include vitamins such as vitamin E and vitamin C, bioactive ingredients such as minerals, nutritional ingredients, and fragrances, as well as excipients and binders incorporated in the formulation. , Emulsifiers, tonicity agents (isotonic agents), buffers, solubilizers, preservatives, stabilizers, antioxidants, colorants, coagulants, or coating agents, but are not limited thereto. It is not something.

3-4. Use The composition for inhibiting carnosine dipeptidase 1 of the present invention is characterized by containing the above-mentioned animal or plant-derived material (that is, animal or plant-derived peptide) as an active ingredient, and the material exhibits the activity of carnosine dipeptidase 1. Inhibiting, the body concentration of carnosine decomposed by carnosine dipeptidase 1 is maintained, or a decrease in the concentration is suppressed. Carnosine is maintained at a high concentration in the body, resulting in cognitive decline, diabetes, immune function decline, vascular or tissue inflammation, oxidative stress, or various diseases caused by the production of advanced glycation end products, Alzheimer, autism, stress Alternatively, it is possible to effectively prevent or improve hypertension. Therefore, the composition of the present invention can be used for various diseases, Alzheimer's disease, autism, stress, or hypertension caused by cognitive decline, diabetes, immune function decline, vascular or tissue inflammation, oxidative stress or advanced glycation end products. It is used for prevention or improvement. Based on these uses, the composition for inhibiting carnosine dipeptidase 1 of the present invention is used for various diseases caused by cognitive decline, diabetes, immune function decline, vascular or tissue inflammation, oxidative stress or production of advanced glycation end products, Alzheimer's It can also be a composition for preventing or ameliorating autism, stress, or hypertension. In the present specification, “prevention or improvement” includes both concepts of making the current state a better state and preventing the current state from becoming worse than the current state. Terms such as treatment, recovery, alleviation, alleviation can also be included.

The composition for inhibiting carnosine dipeptidase 1 of the present invention is prepared by a known method in the form of a solid agent such as a tablet (including a coated tablet), a granule, a powder, a powder, or a capsule, a normal solution, a suspension, Alternatively, it can be formulated into a liquid such as an emulsion. These compositions can be taken with water or the like as it is. Moreover, after preparing the form (for example, powder form and granule form) which can be mix | blended easily, it can use, for example as a raw material of a pharmaceutical.

The composition for inhibiting carnosine dipeptidase 1 of the present invention can be provided in the form of an agent as an example, but is not limited to this form. The agent can be provided as a composition as it is or as a composition containing the agent. Examples of the composition of the present invention include, but are not limited to, a pharmaceutical composition, a food / beverage product composition, a food composition, a beverage composition, a cosmetic composition, and the like. Non-limiting examples of food compositions include functional foods, health supplements, functional nutrition foods, special foods, foods for specified health use, dietary supplements, diet foods, health foods, supplements, food additives, etc. Can be mentioned.

The composition for inhibiting carnosine dipeptidase 1 of the present invention can be applied to any therapeutic use (medical use) or non-therapeutic use (non-medical use). Specific examples include use as pharmaceuticals, quasi-drugs, cosmetics, and the like, and although they do not belong to these under the Pharmaceutical Affairs Law, cognitive function decline, diabetes, immune function decline, blood vessel or tissue inflammation, Examples thereof include use as a composition that explicitly or implicitly promotes prevention or improvement effects of various diseases, Alzheimer's disease, autism, stress, or hypertension caused by production of oxidative stress or advanced glycation end products.

In another aspect, the present invention relates to the composition for inhibiting carnosine dipeptidase 1, which is labeled with the function exhibited by carnosine dipeptidase 1 inhibition. Such display or function display is not particularly limited. For example, “suppress cognitive function decrease”, “expect cognitive function maintenance”, “suppress blood glucose level increase”, “enhance immune function” ”,“ Antioxidant effect ”,“ reducing oxidative stress ”,“ anti-glycation effect ”,“ reducing glycation stress ”,“ suppressing vascular inflammation ”,“ preventing Alzheimer's disease ” Or "I expect improvement", "I expect prevention or improvement of autism", "Prevent stress", "Relieve stress", "Relieve stress", "I expect lower blood pressure", " "Inhibiting blood pressure rise", "Slowing blood pressure rise", "Preventing hypertension", "Helping to improve hypertension", etc., or display or functional indication that can be equated with these It is done. In the present specification, such indications and indications such as function indications may be attached to the composition itself, or may be attached to the container or packaging of the composition.

The composition for inhibiting carnosine dipeptidase 1 of the present invention can be ingested by an appropriate method according to the form. The composition of the present invention includes, for example, oral solid preparations, oral liquid preparations such as oral solutions or syrups, and parenteral preparations such as injections, external preparations, suppositories, or percutaneous absorption agents. However, it is not limited to these. In the present specification, “ingestion” is used to include all aspects such as ingestion, taking, or drinking.

The application amount of the composition for inhibiting carnosine dipeptidase 1 of the present invention is appropriately set depending on the form, administration method, purpose of use, and age, weight and symptom of the patient or animal to be administered, and is not constant. Although the effective human intake of the composition of the present invention is not constant, for example, the weight of an animal or plant-derived material (that is, animal or plant-derived peptide) that is an active ingredient is preferably per day for a human with a body weight of 50 kg. It is 100 mg or more, more preferably 500 mg or more, still more preferably 1000 mg or more, preferably 10 g or less, more preferably 5 g or less, and even more preferably 3 g or less. Further, administration may be performed once or several times within one day within a desired dose range. The administration period is also arbitrary. The effective human intake of the composition of the present invention refers to the intake of the composition for inhibiting carnosine dipeptidase 1 of the present invention showing an effective effect in humans, and the animal and plant derived peptides contained in the composition The type of is not particularly limited.

The subject of application of the composition for inhibiting carnosine dipeptidase 1 of the present invention is preferably human, but domestic animals such as cattle, horses and goats, pet animals such as dogs, cats and rabbits, or mice, rats and guinea pigs. Or a laboratory animal such as a monkey. When a non-human animal is administered to a subject, the amount used per day for about 20 g per rat is the content of the active ingredient in the composition, the state of the subject, weight, sex, age, etc. For example, the total amount of animal or plant-derived material (ie, animal or plant-derived peptide) is preferably 100 mg / kg or more, more preferably 500 mg / kg or more, and still more preferably 1000 mg / kg or more. The amount is preferably 10 g / kg or less, more preferably 5 g / kg or less, and even more preferably 3 g / kg or less.

3-5. Combination with carnosine (combination)
The animal and plant derived peptides described above can be used in combination with carnosine. For this reason, the present invention provides, as one embodiment, a composition comprising the above-described animal and plant derived peptide and carnosine (containing the animal and plant derived peptide and carnosine) (hereinafter also referred to as “combination composition of the present invention”). can do.

By using the above-mentioned animal and plant derived peptides in combination with carnosine, the carnosine dipeptidase 1 inhibitory action of the animal and plant derived peptides delays the degradation of carnosine from carnosine dipeptidase 1 and is effective for target tissues and organs. The carnosine can be delivered. In addition to the carnosine originally present in the body, the carnosine concentration in the body can be maintained at a higher level by combining the animal and plant derived peptide and carnosine according to the present invention, effectively enhancing the action of carnosine. Can be made.

The combined composition of the present invention can be a composition for inhibiting carnosine dipeptidase 1 since it contains a peptide derived from animals and plants. The combination composition of the present invention is not particularly limited, but is preferably used for the applications described in 3-4 above from the viewpoint of enhancing the carnosine action effect. That is, the combination composition of the present invention is preferably a cognitive decline, diabetes, immune function decline, vascular or tissue inflammation, oxidative stress or various diseases caused by production of advanced glycation end products, Alzheimer, autism, stress Or a composition for preventing or improving hypertension.

Although the combination composition of the present invention is not particularly limited, it can be provided in the form of an agent (combination agent) as an example, similar to the composition for inhibiting carnosine dipeptidase 1 described above. The agent can be provided as a composition as it is or as a composition containing the agent. The combination composition of the present invention can be a pharmaceutical composition, a food / beverage product composition, a food composition, a beverage composition, a cosmetic composition, and the like, but is not limited thereto. Non-limiting examples of food compositions include functional foods, health supplements, functional nutrition foods, special foods, foods for specified health use, dietary supplements, diet foods, health foods, supplements, food additives, etc. Can be mentioned.

Carnosine in the present invention is a dipeptide composed of β-alanine and histidine and is also referred to as β-alanyl histidine. Carnosine includes all of D-form (D-carnosine), L-form (L-carnosine), and DL-form (DL-carnosine). In the present invention, preferably L-form (L-carnosine) and DL-form. The body (DL-carnosine), more preferably the L body (L-carnosine). The CAS registration number of D-form (D-carnosine) is 5853-00-9, and the CAS registration number of L-form (L-carnosine) is 305-84-0.

The method of obtaining carnosine used in the present invention is not particularly limited, and may be any natural one derived from animals or one obtained by chemical synthesis. In the present invention, commercially available carnosine is preferably used. In addition, the content of carnosine in the combination composition of the present invention is not particularly limited as long as the desired effect of the present invention is obtained in consideration of the administration form, administration method, and the like. .

The amount ratio of the animal- or plant-derived material (that is, animal or plant-derived peptide) and carnosine in the combination composition of the present invention is not particularly limited as long as the desired effect of the present invention can be obtained. Absent. For example, the ratio (animal or plant-derived material: carnosine) in the combination composition of the present invention is, as a weight ratio, 1: 300 to 300: 1, preferably 1:30 to 30: 1, more preferably 1:10. ˜10: 1, more preferably 1: 3 to 3: 1.

In the combined composition of the present invention, the quantitative ratio between the animal and plant derived peptide and carnosine can be set using one component contained in the animal and plant derived peptide as an index. Such a component is not particularly limited, and examples thereof include cyclophenylalanylphenylalanine [Cyclo (Phe-Phe)].

When the animal and plant derived peptide contains cyclophenylalanylphenylalanine, the weight ratio of the animal and plant derived peptide and carnosine is, for example, 1: 1 by weight ratio of cyclophenylalanylphenylalanine and carnosine (cyclophenylalanylphenylalanine: carnosine). It can be 1000-1: 1. The weight ratio is preferably 1: 950 to 1:50, more preferably 1: 900 to 1: 100.

The animal and plant derived peptide containing cyclophenylalanylphenylalanine is not particularly limited, but is preferably a soybean peptide heat-treated product.

4). Use of animal or plant-derived material to inhibit carnosine dipeptidase 1 One aspect of the present invention is the use of animal or plant-derived material (ie, animal or plant derived peptide) to inhibit carnosine dipeptidase 1.

The use of the present invention includes, for example, cognitive decline, diabetes, immune function decline, vascular or tissue inflammation, oxidative stress or various diseases caused by production of advanced glycation end products, Alzheimer, autism, stress, or hypertension Include, but are not limited to, the use of animal or plant-derived materials to prevent or ameliorate. In addition, the use is a use in a human or non-human animal, and may be a therapeutic use or a non-therapeutic use. Here, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by treatment.

5). Method for Inhibiting Carnosine Dipeptidase 1 One embodiment of the present invention is a method for inhibiting carnosine dipeptidase 1 using an animal or plant-derived material (ie, animal or plant-derived peptide) as an active ingredient. Moreover, another aspect regarding the method includes administering a therapeutically effective amount of an animal or plant-derived material as an active ingredient to a subject in need of inhibition of carnosine dipeptidase 1, and a method of inhibiting carnosine dipeptidase 1 It is.

In the above method, the subject requiring inhibition of carnosine dipeptidase 1 is the same as the subject of application of the composition for inhibiting carnosine dipeptidase 1 of the present invention. In the present specification, the therapeutically effective amount refers to the carnosine degradation of carnosine dipeptidase 1 when the composition for inhibiting carnosine dipeptidase 1 of the present invention is administered to the above-mentioned subject as compared to a subject not administered. The amount by which the activity is inhibited. The specific effective amount is appropriately set according to the administration form, administration method, purpose of use and age, weight, symptom, etc. of the subject and is not constant.

In the method of the present invention, the animal or plant-derived material may be administered as it is or as a composition containing the animal or plant-derived material so that the therapeutically effective amount is obtained.

According to the method of the present invention, it is possible to inhibit carnosine dipeptidase 1 without causing side effects.

Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited thereby. Those skilled in the art can use the method of the present invention with various changes and modifications, and these are also included in the scope of the present invention.

Example 1. As a prepared plant of tea peptide , tea leaf (Ichibancha tea leaf (variety: Yabukita) from Kagoshima Prefecture) was used. First, the tea was pretreated (pre-extraction three times) to reduce water-soluble protein. That is, 200 g of hot water was added to 10 g of tea, and the mixture was appropriately stirred and extracted for 5 minutes. After the completion of extraction, the mixture was filtered through 140 mesh to recover the extraction residue (tea bowl). To this tea bowl, 200 g of hot water was poured and extracted for 5 minutes to recover the tea bowl. Again, this tea bowl was similarly extracted and the tea bowl was recovered.

Next, the pre-extracted tea (teacup) was subjected to enzyme decomposition treatment. Pour 200g of 50 ° C hot water into the bowl (total amount), add 1g of protease (trade name: Protin NY100, manufactured by Daiwa Kasei Co., Ltd.), and stir with a stir bar (300rpm) in a 55 ° C water bath For 3 hours. Thereafter, the enzyme was inactivated by maintaining at 95 ° C. for 30 minutes. The enzyme-treated solution was freeze-dried to prepare a tea peptide.

Example 2 Examination of serum carnosinase (CNDP1) activity inhibitory effect Collagen peptide (HACP-50, Zerais Co., Ltd.), soybean peptide (High Newt AM, Fuji Oil Co., Ltd.) and the above-mentioned tea peptide were used for the test. Recombinant Human Carnosine Dipeptidase 1 / CNDP1 (R & D systems) was used as human serum carnosinase CNDP1. Carnosine used was made by Tokyo Chemical Industry Co., Ltd. The serum carnosinase (CNDP1) activity inhibitory effect was examined at room temperature by the following procedure.

To a 1.5 mL Eppendorf tube, add 50 μL of 2 ng / μL CNDP1 solution dissolved in buffer (50 mM Tris, pH 7.5) and 25 μL aqueous solution of various animal and plant-derived peptides, and add 25 μL of 4 mM carnosine solution also dissolved in the buffer To start the reaction. After incubating the reaction solution at room temperature for 60 minutes, 50 μL of 1% aqueous solution of Trichloroacetic acid (TCA) (Sigma) dissolved in deionized water was added, and the reaction was terminated by vortexing. To the sample after completion of the reaction, a 1.8 M sodium hydroxide aqueous solution (containing 10% DMSO) containing 5 mg / mL o-Phthaldialdehyde (OPA) (Sigma) was added, and the mixture was further incubated at room temperature for 30 minutes. An L-histidine dilution series using a buffer was prepared in the range of 15.625 to 250 μM, and TCA and OPA were similarly added, followed by incubation for 30 minutes to obtain a calibration curve solution. In addition, what added the water containing DMSO of the same content rate instead of various animal and plant origin peptides was used as control. The total amount of the sample and the calibration curve solution was added to 96-well black plate, and the fluorescence intensity at an excitation wavelength of 360 nm and a fluorescence wavelength of 460 nm was measured using a fluorometer.

For the results, the correction value is calculated by subtracting the fluorescence intensity in the sample added with buffer instead of the enzyme (CNDP1), and contains various animal and plant derived peptides when the correction value of the fluorescence intensity in the control is 100%. The correction value of the fluorescence intensity in the sample was defined as CNDP1 residual activity (%). The results are shown in Tables 1 to 3.

As shown in Tables 1 to 3, it was revealed that collagen peptide, tea peptide, and soybean peptide all have an inhibitory effect on serum carnosinase (CNDP1) activity.

Example 3 FIG. Examination of CNDP1 activity inhibitory effect of heat-treated peptide derived from animals and plants Heat-treated products of various peptides used in Example 2 were used for the test. Heat-treated products of various peptides were produced as follows.
(1) Heat-treated collagen peptide A collagen peptide heat-treated product was produced by subjecting the collagen peptide used in Example 2 to high-temperature and high-pressure treatment in a liquid. Specifically, collagen peptide was added to distilled water at 250 mg / mL, put into an autoclave (manufactured by Tommy Seiko Co., Ltd.), and subjected to high-temperature and high-pressure treatment at 135 ° C., 0.31 MPa for 10 hours.
(2) Tea Peptide Heat-treated Product The enzyme-treated solution obtained in Example 1 was subjected to heat treatment in the form of a tea liquid mixture without solid-liquid separation. The heat treatment was performed in an autoclave (manufactured by Tommy Seiko Co., Ltd.) and heat treatment with a high-temperature and high-pressure fluid at 135 ° C., 0.31 MPa for 3 hours. The treated liquid was filtered through 140 mesh to obtain a heat-treated tea peptide.
(3) Soy peptide heat-treated product The soybean peptide used in Example 2 was subjected to high-temperature and high-pressure treatment in a liquid to produce a soy peptide heat-treated product. Specifically, 15 ml of distilled water was added to 3 g of soybean peptide, respectively, and placed in an autoclave (manufactured by Tommy Seiko Co., Ltd.).

The materials other than the above-mentioned various heat-treated peptides were the same as in Example 2, and the inhibitory action of serum carnosinase (CNDP1) activity by the various heat-treated peptides was examined in the same manner as in Example 2. The results are shown in Tables 4-6. Various peptide heat-treated products used were those obtained by spray-drying the heat-treated products described in (1) to (3) above into a powder form.

As shown in Tables 4 to 6, it was revealed that the heat-treated collagen peptide, the heat-treated tea peptide, and the heat-treated soybean peptide all had an inhibitory action on serum carnosinase (CNDP1) activity.

Example 4 Examination of the inhibitory effect of serum carnosinase (CNDP1) activity by linear dipeptides The inhibitory effect of serum carnosinase (CNDP1) activity on linear dipeptides known to have tissue carnosinase (CNDP2) inhibitory activity was examined. Various linear dipeptide preparations purchased from BACHEM were used for the test. The other materials were the same as in Example 2, and the inhibitory action of serum carnosinase (CNDP1) activity by linear dipeptide was examined in the same manner as in Example 2. The results are shown in Table 7.

As shown in Table 7, the linear dipeptide known for CNDP2 inhibitory activity did not show an inhibitory effect on serum carnosinase (CNDP1) activity even when the concentration was 500 μM. From these results, it was revealed that linear dipeptides known to have CNDP2 inhibitory activity have no inhibitory action on serum carnosinase (CNDP1) activity. From the above results, it was suggested that a CNDP1 inhibitor known to have CNDP2 inhibitory activity and a tissue carnosinase (CNDP2) inhibitor differ from each other and have no relationship.

Embodiment 5 FIG. The effect of combined use of heat-treated tea peptide on the human pharmacokinetics of carnosine The following tests were carried out for the purpose of examining the blood carnosine concentration when carnosine alone or carnosine and tea peptide was heat-treated. The same heat-treated tea peptide as that used in Example 3 was used. Carnosine (test food 1) was ingested on day 0 (Day 0), and blood was collected to measure the carnosine blood concentration. From day 1 to day 6, a prescribed amount of heat-treated tea peptide (test food 2) was ingested daily. On the seventh day (Day 7), carnosine and a tea peptide heat-treated product (test food 3) were ingested, and blood was collected to measure the blood carnosine concentration. The specific test method is as follows.
(1) Subjects 10 healthy adult men and women (5 men, 5 women)
(2) Test food

(3) Method of ingesting test food Test food 1 and test food 3 were orally ingested by dissolving in 250 mL of water on the day of the test. Moreover, the capsule of test food 2 (4 capsules once) was orally ingested with 250 mL of water in the morning.
(4) Blood collection and blood treatment Blood samples were collected before taking test foods 1 and 3 and after taking 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, and 180 minutes. In order to prevent carnosine degradation in the blood after blood collection, pretreatment was performed by the following procedure at the time of blood collection. Blood was collected with an ice-cooled blood collection tube (EDTA treatment), and plasma was obtained by centrifugation (3,000 rpm / 5 minutes / 4 ° C.). Add ice-cooled 2% aqueous solution of trichloroacetic acid (TCA) to half the amount of plasma, mix, leave for 10 minutes under ice-cooling, and then centrifuge again (15,000 rpm / 10 minutes / 4 ° C). It was. The supernatant was collected and stored frozen in a freezer set at −20 ° C. until analysis.
(5) Carnosine analysis method (5) -1. Pretreatment The pretreated plasma stored frozen was thawed, 40 μL of water and 40 μL of an aqueous solution containing an internal standard substance were added to 80 μL, and the mixture was stirred. After adding 320 μL of acetonitrile and stirring again, centrifugation (15,000 rpm / 10 minutes / 4 ° C.) was performed, and then the supernatant was subjected to carnosine analysis by LC-MS / MS under the following conditions. A calibration curve for use in quantitative analysis was prepared by replacing sample plasma with blank plasma and water with carnosine standard aqueous solution.
(5) -2. HPLC condition high performance liquid chromatograph: UFLC system [prominence] (Shimadzu Corporation)
Analytical column: Scherzo SS-C18 2.0 mm ID x 100 mm, 3 μm (Imtakt)
Column temperature: 40 ° C
Mobile phase: A: Water / acetonitrile / formic acid (50/50 / 0.5)
B: 50 mM (final concentration) ammonium formate-containing acetonitrile / water (50/50)
Flow rate: 0.4 mL / min

Autosampler cleaning solution: acetonitrile / water (50:50, v / v)
Autosampler temperature: 4 ℃
Injection volume: 10μL
(5) -3. MS / MS Conditioning Material Analyzer: API5000 AB Sciex Pte. Ltd.
API interface: Turbo Spray (ESI)
Gas temperature: 600 ℃
Ionspray voltage: 5500 V
Nebulizer gas setting (GS1): 50 psi, air
Heated gas setting (GS2): 70 psi, air
Curtain gas setting: 20 psi, nitrogen
Collision gas setting: 4, nitrogen
Ionization mode: MRM mode, positive ion detection mode
MRM conditions:
Carnosine: m / z 227 → m / z 110
Internal reference material (phenytoin): m / z 206 → m / z 60
(5) -4. Other Prior to measurement of the sample, the selectivity of the peak in the chromatogram and the linearity of the added calibration curve were confirmed for the target substance. Based on the blood concentration data (Day 0, Day 7) of each subject obtained by measuring the sample, AUC (area under the plasma drug concentration-time curve) (unit: ng · hr / mL) is calculated by the trapezoidal method. did.

Figure 1 shows the AUC variation in each subject. Day 7 AUC was expressed as a ratio when Day 0 AUC was set to 1 for each subject. The analysis was performed by excluding one patient with an AUC difference of 10 times or more compared to the average of the other nine.

The AUC of Day 7 compared with Day 0 showed an increase rate of 10% or more in 9 out of 9 subjects to be analyzed, and the average value of 9 subjects increased by about 20%. There were 2 subjects with fluctuations within 10%, and 1 subject showed a decrease of more than 10%. From the above results, it was recognized that the plasma concentration of carnosine increased due to continuous administration of the heat-treated tea peptide before ingesting carnosine and the combined use of carnosine and heat-treated tea peptide. (When Paired t-test was performed, the p-value was 0.21.)

Example 6 Effect of Heat-treated Tea Peptide on Carnosine Degradation in Human Serum We investigated whether heat-treated tea peptide exhibits carnosine degradation inhibitory effect in human serum.

Test substance (tea peptide heat-treated product) prepared to a concentration 10 times the final concentration in 100 μL of human serum (manufactured by Kojin Bio) and 300 μL of buffer (50 mM Tris, pH 7.5) using a 1.5 mL Eppendorf tube ) 50 μL of the aqueous solution was added and stirred gently, followed by preincubation at 37 ° C. Next, carnosine decomposition reaction was started by adding 50 μL of a buffer containing 1000 μM carnosine (manufactured by Tokyo Kasei Kogyo Co., Ltd.) that had been kept warm at 37 ° C. and agitating lightly. A series of carnosine degradation reactions were performed at 37 ° C. 50 μL of the reaction solution was collected 0, 10 and 20 minutes after the start of the reaction, and the reaction was stopped by adding 25 μL of a 1% trichloroacetic acid (TCA) aqueous solution to an eppendorf tube previously dispensed under ice cooling. 150 μL of acetonitrile was added and centrifuged (15,000 rpm / 10 minutes / 4 ° C.), and 150 μL of the supernatant was diluted with 450 μL of 0.1% formic acid aqueous solution to obtain an analysis sample. The carnosine concentration was quantified using LC-MS / MS, and the residual rate (%) was calculated from the carnosine concentration at each time with respect to the carnosine concentration after 0 minutes of reaction. Carnosine was quantified in the same manner as in Example 5.

FIG. 2 shows the carnosine residual rate (%) at each time when the carnosine concentration at the start of the reaction (after 0 minutes of reaction) is 100% (average value ± standard deviation). The number of tests is n = 3, and the concentration of the aqueous heat-treated tea peptide product in the graph represents the final concentration during the reaction.

From this result, it was shown that carnosine was decomposed only in the presence of human serum, and that the heat-treated tea peptide inhibited the decomposition of carnosine in human serum.

Example 7 Effects of ingesting carnosine and soy peptide heat-treated products on the stress response and blood pressure The following tests were conducted for the purpose of examining the stress value lowering reaction and the effect on blood pressure when ingesting carnosine and soy peptide heat-treated products. The heat-treated soybean peptide was the same as that used in Example 3. The soybean peptide heat-treated product contains about 0.623 mg of cyclophenylalanylphenylalanine per 1 g of the soybean peptide heat-treated product. Prepare drink (test drink) containing carnosine and soybean peptide heat-treated product and drink not containing these (control drink), and evaluate stress response before ingestion, 4 weeks after ingestion, and 8 weeks after ingestion Was performed by Profile of Mood States 2nd Edition Japanese version (POMS2), and blood pressure was measured simultaneously. The specific test method is as follows.
(1) Subjects 60 adult men and women (male: 31 people, women: 29 people) who had high stress values due to POMS2 before the test were used as subjects, and 30 subjects were divided into two groups, a test beverage group and a control beverage group. All subjects were healthy except for stress values.
(2) Test beverage

(3) Method of Ingesting Test Beverage One test drink (350 mL) was orally ingested daily in the morning for 8 weeks.
(4) Evaluation of stress value reduction effect A stress questionnaire was conducted using POMS2 before ingestion (SCR), 4 weeks after ingestion (4W), and 8 weeks after ingestion (8w). For changes in depression and depression, a stratified analysis was performed on subjects with a T score of 55 or higher. The result is shown in FIG. In addition, a stratified analysis of subjects with a T score of 60 or more was performed for negative mood states. The result is shown in FIG.

As shown in FIGS. 3 and 4, subjects who took the test beverage tended to have lower stress values for depression and depression items and negative mood states than subjects who took the control beverage. It was. In addition, regarding the stress values related to other items, subjects who took the test beverage tended to have lower stress values than subjects who took the control beverage ([anger-hostility] (p = 0.25), [Confusion-Embarrassed] (p = 0.31), [Fatigue-Apathy] (p = 0.40), [Tension-Anxiety] (p = 0.17) (p values are two-way without stratified analysis) Shows the main effects between groups by analysis of variance)).

From the above results, the stress value reducing effect of the beverage containing the heat-treated soybean peptide and carnosine was confirmed, and in particular, remarkable improvement effect was observed for depression and depression and negative mood state.
(5) Evaluation of blood pressure lowering effect Systolic blood pressure and diastolic blood pressure were measured before (SCR), 4 weeks after ingestion (4W), and 8 weeks after ingestion (8w). The result regarding the blood pressure change is shown in FIG.

From the above results, a significant blood pressure lowering effect of the beverage containing the heat-treated soybean peptide and carnosine was confirmed. Such a blood pressure lowering effect has not been reported in humans of imidazole dipeptides containing carnosine. This blood pressure lowering action is considered to be obtained because the sympathetic nerve inhibitory action of carnosine was enhanced by the carnosine degradation inhibitory action of the heat-treated soybean peptide.

The present invention provides a composition for inhibiting carnosine dipeptidase 1 comprising a peptide derived from animals and plants as an active ingredient. Since the present invention provides a new means that contributes to prevention or improvement of cognitive function decline or the like, the industrial applicability is high.

Claims (10)

1. A composition for inhibiting carnosine dipeptidase 1 comprising a peptide derived from animals and plants.
2. The composition for carnosine dipeptidase 1 inhibition according to claim 1, wherein the animal or plant-derived peptide is derived from collagen, tea, or soybean.
3. Claims for prevention or improvement of various diseases, Alzheimer's disease, autism, stress, or hypertension caused by cognitive decline, diabetes, immune decline, vascular or tissue inflammation, oxidative stress or production of advanced glycation end products Item 3. The composition for carnosine dipeptidase 1 inhibition according to Item 1 or 2.
4. The composition for inhibiting carnosine dipeptidase 1 according to any one of claims 1 to 3, which is labeled with a function exhibited by carnosine dipeptidase 1 inhibition.
5. Function indications “suppress cognitive function decline”, “expect cognitive function maintenance”, “suppress blood sugar level rise”, “enhance immune function”, “antioxidant effect” , "Reduce oxidative stress", "Expect anti-glycation effect", "Reduce glycation stress", "Inhibit vascular inflammation", "Expect prevention or improvement of Alzheimer's disease", "Autism Expecting prevention or improvement of illness "," Preventing stress "," Reducing stress "," Reducing stress "," Expecting lowering blood pressure "," Suppressing increase in blood pressure "," Blood pressure " The composition for inhibiting carnosine dipeptidase 1 according to claim 4, wherein the composition is selected from the group consisting of “slowing the rise in blood pressure”, “preventing hypertension”, and “helping to improve hypertension”. .
6. The composition for carnosine dipeptidase 1 inhibition according to any one of claims 1 to 5, wherein the composition is an agent.
7. Use of animal and plant derived peptides to inhibit carnosine dipeptidase 1.
8. A method for inhibiting carnosine dipeptidase 1 using a peptide derived from animals and plants.
9. A composition comprising an animal and plant derived peptide and carnosine,
   The above-mentioned composition, wherein the animal or plant-derived peptide contains cyclophenylalanylphenylalanine [Cyclo (Phe-Phe)], and the weight ratio of cyclophenylalanylphenylalanine to carnosine is 1: 1000 to 1: 1.
10. The composition according to claim 9, wherein the animal or plant-derived peptide is a soybean peptide or a heat-treated product thereof.

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