JP7633679B2 - Vasodilators and their uses - Google Patents

Description

The present invention relates to a vasodilator and its use.

In recent years, it has become known that compositions containing tripeptides obtained by hydrolyzing collagen have effects such as promoting collagen production, promoting hyaluronic acid production, and promoting collagen synthesis in the skin, and are useful for improving skin beauty (Patent Document 1).

Special Publication No. 2012-501320

Collagen is a major protein that makes up the connective tissues of animals, and there is a need for further utilization of it.

Therefore, the present invention aims to provide new uses for collagen-derived tripeptides or collagen-derived dipeptides.

In order to achieve the above-mentioned objective, the vasodilator of the present invention contains at least one of a collagen-derived tripeptide and a collagen-derived dipeptide.

The periodontal disease treatment drug of the present invention contains the vasodilator of the present invention.

The oral composition of the present invention contains the vasodilator of the present invention.

The food composition of the present invention contains the vasodilator of the present invention.

According to the present invention, blood vessels can be dilated.

1(A) and (B) are photographs of the mandibular incisor gums of a rat in Example 1. 2(A) to (C) are graphs showing the change in vascular diameter after administration of the vasodilator of the present invention in Example 1, where (A) shows the results for medium-sized blood vessels, (B) shows the results for large blood vessels, and (C) shows the results for small blood vessels. 3A and 3B are schematic diagrams showing the measurement target teeth and blood flow measurement points in Example 2. FIG. 4 is a schematic diagram showing the measurement surface of the gingivitis index in Example 3.

<Vasodilators>
As described above, the vasodilator of the present invention contains at least one of a collagen-derived tripeptide (hereinafter also referred to as "tripeptide") and a collagen-derived dipeptide (hereinafter also referred to as "dipeptide"). The vasodilator of the present invention is characterized in that it contains at least one of a collagen-derived tripeptide and a collagen-derived dipeptide, and other configurations and conditions are not particularly limited. The vasodilator of the present invention can dilate blood vessels.

As a result of intensive research, the inventors discovered that collagen-derived tripeptides and collagen-derived dipeptides dilate gingival blood vessels when administered to the oral mucosa, leading to the present invention. This was the first discovery by the inventors. According to the present invention, the blood vessels of the subject can be dilated, and therefore, for example, blood flow in the subject can be improved. For this reason, the vasodilator of the present invention can also be referred to as, for example, a blood flow improver.

In the present invention, "vasodilation" means, for example, that the blood vessels of a subject are expanded. Specifically, "vasodilation" means that the blood vessels of a subject are significantly expanded compared to when the vasodilator of the present invention is not administered. The "blood vessels of a subject" include, for example, blood vessels in the gums. The blood vessels of a subject are, for example, blood vessels with a small blood vessel diameter. The "small blood vessels" are, for example, blood vessels with a blood vessel diameter of 10 μm or less. The blood vessel diameter means, for example, the inner diameter of a blood vessel. The blood vessel diameter is measured, for example, during the late vasoconstriction phase (corresponding to the late diastole phase of the heart). The blood vessel diameter can be measured, for example, as the longest diameter of the area in which blood (red blood cells) are present when the blood vessels of a subject are optically observed.

Collagen protein is composed of a polypeptide in which the amino acid sequence Gly-X-X (X is any amino acid residue) is repeated multiple times as a constituent unit. Therefore, the collagen-derived tripeptide is a peptide composed of any three consecutive amino acids in collagen protein, including a tripeptide composed of glycine and any two amino acids, and the any amino acid is an amino acid selected from the group consisting of proline, hydroxyproline, glutamic acid, alanine, and arginine. Specific examples of the collagen-derived tripeptide are not particularly limited, and include tripeptides composed of glycine, proline, and hydroxyproline, and tripeptides composed of alanine, hydroxyproline, and glycine. In each tripeptide, the order of the amino acid sequence is not particularly limited and can be any order. When the tripeptide is a tripeptide composed of glycine, proline, and hydroxyproline, the tripeptide is, for example, glycine-proline-hydroxyproline or proline-hydroxyproline-glycine from the N-terminus. When the tripeptide is a tripeptide composed of alanine, hydroxyproline, and glycine, the collagen-derived tripeptide is, for example, alanine-hydroxyproline-glycine from the N-terminus. The tripeptide is preferably glycine-proline-hydroxyproline. The collagen-derived tripeptide may be composed of one type of tripeptide or multiple types of tripeptides.

The collagen-derived dipeptide is a peptide composed of any two consecutive amino acids in a collagen protein. The dipeptide contains at least two amino acids selected from, for example, glycine, proline, hydroxyproline, glutamic acid, alanine, and arginine. Specific examples of the dipeptide include, but are not limited to, a dipeptide composed of proline and hydroxyproline, a dipeptide composed of alanine and hydroxyproline, a dipeptide composed of leucine and hydroxyproline, a dipeptide composed of isoleucine and hydroxyproline, a dipeptide composed of phenylalanine and hydroxyproline, a dipeptide composed of glycine and proline, a dipeptide composed of glycine and hydroxyproline, a dipeptide composed of glycine and glutamic acid, a dipeptide composed of glycine and alanine, a dipeptide composed of glycine and arginine, a dipeptide composed of proline and glutamic acid, a dipeptide composed of proline and alanine, a dipeptide composed of proline and arginine, a dipeptide composed of hydroxyproline and glutamic acid, a dipeptide composed of hydroxyproline and arginine, a dipeptide composed of glutamic acid and alanine, a dipeptide composed of glutamic acid and arginine, and a dipeptide composed of alanine and arginine. When the dipeptide is a dipeptide composed of proline and hydroxyproline, the dipeptide is, for example, proline-hydroxyproline from the N-terminus. When the dipeptide is a dipeptide composed of alanine and hydroxyproline, the dipeptide is, for example, alanine-hydroxyproline from the N-terminus. When the dipeptide is a dipeptide composed of leucine and hydroxyproline, the dipeptide is, for example, leucine-hydroxyproline from the N-terminus. When the dipeptide is a dipeptide composed of isoleucine and hydroxyproline, the collagen-derived dipeptide is, for example, isoleucine-hydroxyproline from the N-terminus. When the dipeptide is a dipeptide composed of phenylalanine and hydroxyproline, the dipeptide is, for example, phenylalanine-hydroxyproline from the N-terminus. The dipeptide is preferably proline-hydroxyproline. The collagen-derived dipeptide may be composed of one type of dipeptide or may be composed of multiple types of dipeptides.

In the tripeptides and dipeptides, the hydroxyproline is not particularly limited and may be, for example, 3-Hyp (3-hydroxyproline) or 4-Hyp (4-hydroxyproline).

In the present invention, the tripeptides and dipeptides may be modified. That is, the amino acid residues constituting the tripeptides and dipeptides may be natural amino acid residues or modified amino acid residues (non-natural amino acid residues). Therefore, in the present invention, one, more than one, or all of the amino acid residues constituting the tripeptides and dipeptides are natural amino acid residues or modified amino acid residues. The natural amino acid residues are alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, or tyrosine.

When the tripeptide and dipeptide are modified, each peptide can be called a modified peptide. The modified peptide is, for example, a peptide in which some or all of the amino acids constituting the tripeptide or dipeptide are modified. The modification may be, for example, a modification of the amino acids constituting the tripeptide or dipeptide, or a modification of the peptide. Specific examples of the modification include phosphorylation of serine or threonine, sulfation of tyrosine, modification of amino groups such as biotinylation, myristoylation, palmitoylation, acetylation, maleimidation, malonylation, and methylation (e.g., Lys(Me), Lys(Me) ₂ , Lys(Me) ₃ , Arg(Me), Arg(Me) _{2 ,} etc.), modification of carboxyl groups such as amidation or esterification, modification of thiol groups such as farnesylation, geranylation, and palmitoylation, glycosylation such as N-linked glycosylation to the nitrogen atom of the side chain of asparagine and O-linked glycosylation to the hydroxyl group of serine or threonine, PEG (polyethylene glycol) modification, lipidation, acylation, hydroxylation of proline or lysine, etc. The modification may be, for example, a post-translational modification. When the vasodilator of the present invention contains the modified peptide, it is preferable that the modified peptide contains, for example, hydroxyproline as a constituent amino acid.

The vasodilator of the present invention may contain only a collagen-derived tripeptide, only a collagen-derived dipeptide, or both as an active ingredient. The vasodilator of the present invention may also contain, for example, one type of peptide or multiple types of peptides.

The vasodilator of the present invention may contain, as the tripeptide or dipeptide, an isolated product, purified product, or crude product of the tripeptide or dipeptide, or may contain, for example, a proteolytic enzyme-treated product of a collagen-derived protein containing the tripeptide or dipeptide.

The collagen-derived tripeptide or collagen-derived dipeptide of the present invention is, for example, a tripeptide or dipeptide derived from collagen, and may be prepared from raw materials such as natural products or processed products thereof, or may be synthesized by a conventionally known synthesis method. The synthesis method may be, for example, a solid-phase synthesis method or a liquid-phase synthesis method, and may include a peptide synthesis method using the reverse reaction of hydrolases, a peptide synthesis method using organic chemical techniques, and a synthesis method using genetic engineering techniques.

Examples of the preparation method from the raw material include purification from the raw material (collagen) itself or from a decomposition product of the raw material. The decomposition method of the raw material is not particularly limited, and can be performed, for example, by using an acid, an alkali, or a protease. In addition, the purification method can be a conventionally known separation and purification method that utilizes, for example, differences in adsorption affinity to an adsorbent, differences in solubility in various solvents, differences in elution rate due to the molecular sieve effect, etc. In addition, extraction and concentration can be combined as appropriate. The origin of the collagen is not particularly limited, and for example, it may be derived from an animal or may be synthesized by a conventionally known synthesis method. The animal is not particularly limited, and for example, may be a human or a non-human animal. The non-human animal is, for example, a mammal other than a human, a bird, a reptile, an amphibian, a fish, etc. In terms of the risk of infection, the odor after formulation, etc., the animal is preferably, for example, a fish. In this case, the vasodilator of the present invention includes, for example, a processed product obtained by treating fish-derived collagen with a protease.

The vasodilator of the present invention may be used, for example, in vivo or in vitro . The vasodilator of the present invention may be composed of, for example, a plurality of components. In this case, the vasodilator of the present invention may be called, for example, a vasodilator composition.

The subject of administration of the vasodilator of the present invention is not particularly limited. When the vasodilator of the present invention is used in vivo , the subject of administration can be, for example, a human or a non-human animal other than a human. Examples of the non-human animal include mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, and guinea pigs. When the vasodilator of the present invention is used in vitro , the subject of administration can be, for example, a cell, a tissue, or an organ. Examples of the cell include cells collected from a living body, cultured cells, and the like, and examples of the tissue include the oral mucosa such as the gingiva. When the vasodilator of the present invention is administered to the oral mucosa, for example, the blood vessels in the gingiva can be dilated.

The conditions for use (administration conditions) of the vasodilator of the present invention are not particularly limited, and the administration form, administration timing, dosage, etc. can be appropriately set depending on, for example, the type of subject to be administered.

The dosage of the vasodilator of the present invention is not particularly limited. When the vasodilator of the present invention is used in vivo , the dosage can be appropriately determined depending on, for example, the type, symptoms, age, and administration method of the subject. As a specific example, when the vasodilator of the present invention contains the collagen-derived tripeptide, the total dosage of the tripeptide per day when administered to humans is, for example, 40 to 400 mg, 40 to 375 mg, 40 to 350 mg, 40 to 300 mg, 40 to 250 mg, 40 to 200 mg, 40 to 80 mg, or 37.5 to 75 mg. The number of administrations per day is, for example, 1 to 6 times, 1 to 4 times, 1 to 3 times, or 1 to 2 times, and preferably 1 to 3 times. When the vasodilator of the present invention contains the collagen-derived dipeptide, the total dose of the dipeptide per day when administered to humans is, for example, 40 to 400 mg, 40 to 375 mg, 40 to 350 mg, 40 to 300 mg, 40 to 250 mg, 40 to 200 mg, 40 to 80 mg, or 37.5 to 75 mg. The number of administrations per day is, for example, 1 to 6 times, 1 to 4 times, 1 to 3 times, or 1 to 2 times, and preferably 1 to 3 times. When the vasodilator of the present invention contains the collagen-derived tripeptide and the collagen-derived dipeptide, the total dose of the tripeptide and the dipeptide per day when administered to humans is, for example, 40 to 400 mg, 40 to 375 mg, 40 to 350 mg, 40 to 300 mg, 40 to 250 mg, 40 to 200 mg, 40 to 80 mg, or 37.5 to 75 mg. The number of times of administration per day is, for example, 1 to 6 times, 1 to 4 times, 1 to 3 times, or 1 to 2 times, and preferably 1 to 3 times. The content of the collagen-derived tripeptide or collagen-derived dipeptide in the vasodilator of the present invention is not particularly limited and can be appropriately set according to, for example, the above-mentioned daily dosage.

The administration form of the vasodilator of the present invention is not particularly limited. When the vasodilator of the present invention is administered in vivo , it may be administered orally or parenterally. The parenteral administration may be, for example, intravenous injection (intravenous administration), intramuscular injection (intramuscular administration), percutaneous administration, subcutaneous administration, intradermal administration, enteral administration, rectal administration, vaginal administration, nasal administration, pulmonary administration, intraperitoneal administration, local administration, etc. The vasodilator of the present invention can dilate blood vessels by penetrating, for example, the oral mucosa, so it is preferably administered by oral administration such as transmucosal administration, and more preferably by oral mucosal administration.

The dosage form of the vasodilator of the present invention is not particularly limited and can be appropriately determined according to, for example, the administration form. The dosage form can be, for example, a liquid or solid form. Specific examples of the dosage form include oral administration preparations such as release-modified preparations (enteric preparations, sustained-release preparations, etc.), capsules, oral liquids (elixirs, suspensions, emulsions, aromatic perfumes, lemonades, etc.), syrups (syrup preparations, etc.), granules (effervescent granules, fine granules, etc.), powders, tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, dissolving agents, coated tablets, etc.), pills, and oral jellies; oral application preparations such as oral tablets (gum, sublingual tablets, troches, drops, buccal tablets, adhesive tablets, etc.), oral sprays, oral semisolids, and mouthwashes; injections (implanted injections, sustained-release injections, infusions (infusion preparations, etc.), freeze-dried injections, powder injections, pre-filled syringes, preparations for bronchial and pulmonary application such as inhalants (inhalation aerosols, inhalation liquids, inhalation powders, etc.); preparations for ocular administration such as eye ointments and eye drops; preparations for ear administration such as ear drops; preparations for nasal application such as nasal drops (nasal liquids, nasal powders, etc.); preparations for rectal application such as suppositories, semisolid rectal preparations, and enemas; preparations for vaginal application such as vaginal suppositories and vaginal tablets; preparations for external application (liquor, liniments, lotions, etc.), creams, gels, solid external applications (powder external applications, etc.), sprays (aerosols for external applications, pump sprays, etc.), patches (tapes, poultices, etc.), ointments, etc. When the vasodilator of the present invention is orally administered, the dosage form may be, for example, a tablet, a coated tablet, a pill, fine granules, granules, powder, capsule, liquid, syrup, emulsion, suspension, etc. When the vasodilator of the present invention is parenterally administered, the dosage form may be, for example, a preparation for injection, a preparation for drip infusion, etc. When the vasodilator of the present invention is percutaneously administered, the dosage form may be, for example, a topical agent such as a patch, a liniment, an ointment, a cream, or a lotion.

The vasodilator of the present invention may contain, for example, additives as necessary. When the vasodilator of the present invention is used as a medicine or pharmaceutical composition, the additives preferably contain pharma- ceutically acceptable additives or pharma-ceutically acceptable carriers. The additives are not particularly limited, and examples thereof include base materials, excipients, colorants, lubricants, binders, disintegrants, stabilizers, preservatives, flavorings such as fragrances, etc. In the vasodilator of the present invention, the amount of the additives is not particularly limited as long as they do not interfere with the function of the collagen-derived tripeptide or collagen-derived dipeptide.

Examples of the excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, and dextrin; cellulose derivatives such as crystalline cellulose; organic excipients such as gum arabic; dextran; and pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate. Examples of the lubricants include metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; polyethylene glycol; silica; and hydrogenated vegetable oil. Examples of the flavoring agents include flavorings such as cocoa powder, peppermint, aromatic powder, peppermint oil, borneol, and cinnamon powder, as well as sweeteners and acidulants. Examples of the binder include hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, macrogol, etc. Examples of the disintegrant include cellulose derivatives such as carboxymethyl cellulose and calcium carboxymethyl cellulose; chemically modified starches and chemically modified celluloses such as carboxymethyl starch, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone, etc. Examples of the stabilizer include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; sorbic acid, etc.

The collagen-derived tripeptides or collagen-derived dipeptides may be commercially available or may be prepared in-house as described above.

As described above, the vasodilator of the present invention can dilate blood vessels. Therefore, the vasodilator of the present invention can improve, for example, the blood flow of a subject, and can promote the reduction and/or alleviation of symptoms involving blood circulation function. The vasodilator of the present invention can also be used, for example, as an additive in cosmetics, food, etc.

The vasodilator of the present invention exhibits a vasodilating effect, and can therefore improve, for example, blood flow. Therefore, the vasodilator of the present invention can be used as a medicine for diseases caused by vascular constriction or constriction. Examples of diseases caused by vascular constriction or constriction include periodontal disease, high blood pressure, cerebral hemorrhage, stiff shoulders, headache, cerebral circulation, blood congestion, arteriosclerosis, cerebral infarction, bedsores, itching associated with dryness, wrinkles, skin disorders caused by ultraviolet rays, atopic dermatitis, osteoporosis, tendon rupture, osteoarthritis of the knee, etc.

The vasodilator of the present invention may be contained, for example, in foods, supplements, etc. that are taken on a daily basis.

<Periodontal disease treatment drug>
As described above, the periodontal disease treatment drug of the present invention contains the vasodilator of the present invention. The periodontal disease treatment drug of the present invention is characterized by containing the vasodilator of the present invention, i.e., at least one of a collagen-derived tripeptide or a collagen-derived dipeptide, and other configurations and conditions are not particularly limited. In the periodontal disease, for example, blood vessels are constricted due to a chronic inflammatory reaction of the gums, and the alveolar bone that supports the periodontal tissue is resorbed. Since the periodontal disease treatment drug of the present invention contains the vasodilator of the present invention, it can dilate the blood vessels of the gums by administering it to the oral mucosa, and as a result, periodontal disease can be treated. The periodontal disease treatment drug of the present invention can be described in the same manner as the vasodilator of the present invention.

In the present invention, the periodontal diseases include, for example, gingivitis, periodontitis, pyorrhea, etc.

In the present invention, "treatment" may mean any of suppression or prevention of disease onset, suppression or halt of disease progression, suppression or halt of disease symptom progression, and/or improvement of disease. Therefore, the periodontal disease treatment drug of the present invention can be referred to as, for example, a periodontal disease suppressant, preventive drug, progression suppressant, progression halt drug, and/or ameliorator. Furthermore, the periodontal disease treatment drug of the present invention may (significantly) suppress the symptoms or progression of the disease compared to the absence of the periodontal disease treatment drug of the present invention (non-administration condition), and may progress the disease compared to the start (start of administration).

The administration conditions of the periodontal disease therapeutic agent of the present invention are not particularly limited, and the administration form, administration timing, administration amount, etc. can be appropriately set depending on, for example, the type of subject to be administered. The administration subjects and administration conditions of the periodontal disease therapeutic agent of the present invention can be, for example, the same as those described above for the administration subjects and administration conditions of the vasodilator of the present invention.

<Oral Composition>
As described above, the oral composition of the present invention contains the vasodilator of the present invention. The oral composition of the present invention is characterized by containing the vasodilator of the present invention, i.e., at least one of a collagen-derived tripeptide or a collagen-derived dipeptide, and other configurations and conditions are not particularly limited. The oral composition of the present invention can dilate blood vessels because it contains the vasodilator of the present invention. The oral composition of the present invention can be described in the same manner as the vasodilator of the present invention.

The oral composition of the present invention may contain, for example, other ingredients. The other ingredients are not particularly limited and may include, for example, a gum base, a sweetener, a flavoring, an acidulant, a coloring agent, etc.

<Food Composition>
As described above, the food composition of the present invention contains the vasodilator of the present invention. The food composition of the present invention is characterized by containing the vasodilator of the present invention, i.e., at least one of a collagen-derived tripeptide or a collagen-derived dipeptide, and other configurations and conditions are not particularly limited. The food composition of the present invention can dilate blood vessels because it contains the vasodilator of the present invention. The food composition of the present invention can be described in the same manner as the vasodilator of the present invention.

The food composition of the present invention can be used, for example, by adding it to food. Therefore, the food composition of the present invention can also be called, for example, a food additive. In the present invention, "food" may be liquid or solid, and can also be called, for example, a food or beverage product or a food or beverage product. There are no particular limitations on the food to which the food composition of the present invention is added.

The amount of food composition added to the food is, for example, 1 to 100% by weight, 10 to 100% by weight, or 15 to 100% by weight.

<Vasodilatory method>
As described above, the vasodilator method of the present invention includes an administration step of administering a vasodilator to a subject, and the vasodilator is the vasodilator of the present invention. The vasodilator method of the present invention is characterized by administering the vasodilator of the present invention to a subject, that is, administering at least one of a collagen-derived tripeptide or a collagen-derived dipeptide, and other steps and conditions are not particularly limited. The vasodilator method of the present invention administers the vasodilator of the present invention to a subject, and therefore can dilate the blood vessels of the subject. The vasodilator method of the present invention can be described with reference to the description of the vasodilator of the present invention.

In the vasodilator method of the present invention, the administration step may be performed, for example, in vitro or in vivo . The subjects and conditions for administration of the vasodilator of the present invention may be the same as those described above for the subjects and conditions for administration of the vasodilator of the present invention.

<Periodontal disease treatment method>
As described above, the method for treating periodontal disease of the present invention includes an administration step of administering a therapeutic drug for periodontal disease to a subject, and the therapeutic drug for periodontal disease is the therapeutic drug for periodontal disease of the present invention. The method for treating periodontal disease of the present invention is characterized by administering the therapeutic drug for periodontal disease of the present invention to a subject, i.e., administering at least one of a collagen-derived tripeptide or a collagen-derived dipeptide, and other steps and conditions are not particularly limited. The method for treating periodontal disease of the present invention administers the therapeutic drug for periodontal disease of the present invention to a subject, and therefore can treat periodontal disease in the subject. The explanation of the therapeutic drug for periodontal disease and the vasodilator of the present invention can be used for the method for treating periodontal disease of the present invention.

In the periodontal disease treatment method of the present invention, the administration step may be performed, for example, in vitro or in vivo . The subjects and conditions for administration of the periodontal disease treatment agent of the present invention may be the same as those for administration of the vasodilator of the present invention.

<Use of the agent>
The present invention relates to at least one of a collagen-derived tripeptide and a collagen-derived dipeptide, or use thereof, for vasodilatation, and at least one of a collagen-derived tripeptide and a collagen-derived dipeptide, or use thereof, for treating periodontal disease. The present invention relates to at least one of a collagen-derived tripeptide and a collagen-derived dipeptide, or use thereof, for producing a vasodilator or a therapeutic agent for periodontal disease. For example, the above-mentioned explanations of the vasodilator, the method for vasodilatation of a therapeutic agent for periodontal disease, and the method for treating periodontal disease of the present invention can be used for the present invention.

Next, examples of the present invention will be described. However, the present invention is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise indicated.

[Example 1]
It was confirmed that the vasodilator of the present invention is capable of dilating blood vessels in vivo .

(Preparation of peptide fractions)
Collagen tripeptide (CTP, FACT-P, manufactured by MC Plot Biotechnology, Inc., Gly-Pro-Hyp) was dissolved in physiological saline to give a CTP concentration of 0.5 g/ml, to prepare CTP-containing physiological saline.

After 8 weeks of age, female rats (Wistar, n = 1) were anesthetized by subcutaneous administration of urethane (Sigma-Aldrich) at 1.75 g/kg body weight. The rats were then fixed to an observation table. After the fixation, the observation table was placed under a microscope (Nikon SMZ25, Nikon). In addition, to prevent the gum surface from drying out, an ultrasonic warm air inhaler (humidifier, UN-133B, A&D) was operated in mist-free mode during the test to spray moist air (relative humidity: 95%). Figures 1(A) and (B) show photographs of the rat's mandibular incisor gingiva. Figure 1(A) is a photograph of the entire observation site, and Figure 1(B) is an enlarged photograph of the vascular observation area surrounded by a white frame in Figure 1(A). The vascular observation area included veins of the thickness described below, and the part in focus in the image was selected.

As a control, 10 μl of saline was dripped onto the gingiva of the rat's mandibular incisor, and the subcutaneous tissue in the area for vascular observation of the mandibular incisor gum (area enclosed in a square in Figure 1 (A)) was photographed and recorded under a microscope for 30 minutes after dripping. After that, after a 5-minute interval, the saline containing CTP was dripped onto the same area, and the subcutaneous tissue was similarly photographed and recorded under a microscope for 30 minutes after dripping.

Images were extracted from the obtained video, and the vascular diameter before administration of saline was measured using the scale tool in Photoshop (registered trademark, Adobe). Based on the vascular diameter before administration of saline, the size of the blood vessels was classified into three stages (thick: approximately 25 μm, medium: 15-20 μm, thin: approximately 10 μm) as shown in Figure 1 (B). The vascular diameter of blood vessels in each stage was measured every 5 minutes after administration of saline or CTP from the captured images of the video. These results are shown in Figure 2.

2(A)-(C) are graphs showing the change in vascular diameter after administration of the vasodilator of the present invention or saline, where (A) shows the results for medium-sized blood vessels (CTP1, control 1), (B) shows the results for large blood vessels (CTP2, control 2), and (C) shows the results for small blood vessels (CTP3, control 3). In Fig. 2(A)-(C), the horizontal axis indicates the time elapsed after administration, and the vertical axis indicates the vascular diameter. As shown in Fig. 2(A)-(C), the addition of control saline resulted in almost no change in vascular diameter compared to before addition. In contrast, when CTP was administered, the vascular diameter expanded at all times after administration compared to before CTP addition. Furthermore, when CTP was administered, the vascular diameter expanded regardless of the size of the blood vessel, but the degree of expansion of the vascular diameter was more pronounced in smaller blood vessels (microvessels, CTP3). In addition, when CTP is administered to the body, it is decomposed into proline-hydroxyproline. As shown in Figures 2(A) to 2(C), administration of CTP can dilate blood vessels, so it can be said that proline-hydroxyproline can also dilate blood vessels.

These results demonstrate that the vasodilator of the present invention is capable of dilating blood vessels in vivo .

[Example 2]
It was confirmed that the vasodilator of the present invention can improve blood flow in vivo .

(1) Administration of CTP Eight healthy adults (four men and four women) were orally administered the vasodilator of the present invention (CTP-containing food (CTP 37.5 mg/tablet): Kawaii Gri Collagen (registered trademark), Bioradical Research Institute) for a specified test period (14 days from October 28, 2020 to November 11, 2020), and changes in periodontal tissue blood flow before and 14 days after the start of administration were confirmed. Specifically, each subject was asked to chew the vasodilator of the present invention twice a day (after breakfast and dinner), five tablets per intake, about 10 times per tablet, allowing the CTP in the vasodilator of the present invention time to come into contact with the oral mucosa, and then swallowed. On the start date of intake (administration) (October 28, 2020), the subject was only allowed to take the vasodilator after dinner, and on the end date of intake (administration) (November 11, 2020), the subject was only allowed to take the vasodilator after breakfast. In addition, each subject was asked to clean the oral cavity with a specified oral care product (toothpaste: PC Clinica M (manufactured by Lion Corporation), toothbrush: DENT EX33 M (manufactured by Lion Corporation)) from 3 days before the start of the test period to the end of the test (October 25, 202 to November 11, 2020), and during the test period, 15 minutes after taking the vasodilator of the present invention, the subject was asked to brush his/her teeth with the oral care product. During the test period, the subject was instructed to refrain from taking supplements other than the vasodilator of the present invention. If the subject forgot to take the vasodilator of the present invention after breakfast or dinner, the subject was asked to record the fact that he/she forgot to take the vasodilator without taking any additional doses. In addition, if the participants became ill due to a cold or other illness, or if they took any medicine, they were asked to record that fact.

(2) Measurement of blood flow rate Before taking the vasodilator of the present invention (October 28, 2020) and after taking it (November 11, 2020), the microvascular blood flow rate (Flow: ml/min/100g) of the periodontal tissue of each subject was measured using a laser Doppler blood flow meter (laser blood flow meter ALF21N, manufactured by Advance Co., Ltd.). Specifically, a vinyl tube tip with a distance from the tip of the tip to the tip of the probe of the blood flow meter (laser blood flow meter probe [non-contact type] NCD type, manufactured by Advance Co., Ltd.) was first joined to the tip of the blood flow meter. Then, the tip of the probe tip was lightly brought into contact with the blood flow measurement site of each subject without pressing it against the blood flow. The measurement was performed five times for each measurement site, and the average value of the five measurements was taken as the blood flow rate at the measurement site. The tip was replaced for each subject, and when the tip was detached from the probe, the probe was lightly wiped with an alcohol swab. The measurement points of the blood flow are described with reference to FIG. 3. The teeth to be measured were the second and sixth teeth in the upper right (upper right 2, upper right 6), the fourth tooth in the lower right (lower right 4), the fourth tooth in the upper left (upper left 4), and the second and sixth teeth in the lower left (lower left 2, lower left 6), as shown in FIG. 3(A). As shown in FIG. 3(B), the measurement points were the attached gingiva located about 2 mm from the gingival-buccal transition of each measurement target tooth in the attached gingiva direction, and the intermediate part of the crown width of the target tooth mesiodistal. The measurement points were also the labial gingiva (upper right 1) in the center connecting the labial cervical part of the maxillary right central incisor and the lateral incisor. The data was collected and recorded using a data logger (insulated 10-channel handy logger midi Logger GL240, manufactured by Graphtec Corporation). The results are shown in Table 1 below.

As shown in Table 1, ingestion of the vasodilator of the present invention improved the blood flow in the periodontal tissue microvessels of each subject. Looking at the measurement sites, there was a statistically significant increase in blood flow in the upper right 1 and lower left 6. This demonstrates that the vasodilator of the present invention can improve blood flow in subjects.

[Example 3]
It has been confirmed that the vasodilator of the present invention has a therapeutic effect on periodontal disease in vivo .

In the same manner as in Example 2 (1) above, the vasodilator of the present invention was orally administered to the subjects, and before and after the specified test period, periodontal pocket examination (PD: Probing Depth (mm)), the presence or absence of gingival bleeding (BOP: Bleeding on Probing), and gingival index examination (GI: Gingival Index) were performed.

(1) Periodontal Pocket Inspection The marginal gingiva of the tooth to be measured was probed with a periodontal probe equipped with a scale to confirm the depth of the periodontal pocket (Probing Depth (mm)).

(2) Presence or absence of gingival bleeding The marginal gingiva of the tooth being measured was probed with a periodontal probe. If bleeding from the gingiva was observed within 30 seconds, it was evaluated as having gingival bleeding (Y: BOP+), and if no bleeding was observed, it was evaluated as not having gingival bleeding (N: BOP-).

(3) Gingivitis Index Test The marginal gingiva of the four surfaces (buccal/labial surface, lingual/palatal surface/mesial surface, and distal surface) shown in FIG. 4 of the teeth to be measured was scored according to the following evaluation criteria, and the average score of the four surfaces was taken as the gingivitis index (GI) of each tooth to be measured.

Gingivitis index rating: 0: Macroscopic findings were normal, and there was no bleeding on probing.
1: Mild redness and inflammation of the gingiva is visible to the naked eye, but there is no bleeding on probing.
2: Moderate redness, inflammation, and swelling of the gingiva were observed with the naked eye, and bleeding occurred on probing.
3: Severe redness, inflammation, and swelling of the gingiva are visible to the naked eye, and spontaneous bleeding is observed not only on probing but also during treatment.

The results of the periodontal pocket examination are shown in Table 2 below, the results of the presence or absence of gingival bleeding are shown in Table 3 below, and the results of the gingivitis index test are shown in Table 4 below. Note that a problem occurred during the periodontal pocket examination for subject H, so a periodontal pocket examination was not performed on subject H.

As shown in Tables 2 to 4 above, by taking the vasodilator of the present invention, each subject experienced improvement in periodontal pockets, gingival bleeding, and gingivitis index. This shows that the vasodilator of the present invention can treat gingivitis and periodontal disease in subjects.

The present invention has been described above with reference to embodiments and examples, but the present invention is not limited to the above embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

This application claims priority based on Japanese Patent Application No. 2019-216078, filed November 29, 2019, the disclosure of which is incorporated herein in its entirety.

<Additional Notes>
Some or all of the above-described embodiments and examples may be described as follows, but are not limited to the following:
(Appendix 1)
A vasodilator comprising at least one of a collagen-derived tripeptide and a collagen-derived dipeptide.
(Appendix 2)
The collagen-derived tripeptide includes a tripeptide consisting of glycine and any two amino acids,
The vasodilator described in Appendix 1, wherein the any amino acid is an amino acid selected from the group consisting of proline, hydroxyproline, glutamic acid, alanine, and arginine.
(Appendix 3)
The vasodilator according to claim 1 or 2, wherein the collagen-derived tripeptide comprises a tripeptide consisting of glycine, proline, and hydroxyproline.
(Appendix 4)
A vasodilator according to any one of claims 1 to 3, wherein the collagen-derived dipeptide contains at least two amino acids selected from glycine, proline, hydroxyproline, glutamic acid, alanine, and arginine.
(Appendix 5)
The vasodilator according to any one of claims 1 to 4, wherein the collagen-derived dipeptide comprises a dipeptide composed of proline and hydroxyproline.
(Appendix 6)
6. The vasodilator according to any one of claims 1 to 5, wherein at least one of the collagen-derived tripeptide and the collagen-derived dipeptide comprises a product obtained by treating fish-derived collagen with a protease.
(Appendix 7)
7. A vasodilator according to any one of claims 1 to 6, wherein at least one of the amino acid residues constituting the collagen-derived tripeptide and the collagen-derived dipeptide is a natural amino acid residue or a modified amino acid residue.
(Appendix 8)
The vasodilator according to any one of claims 1 to 7, wherein the vasodilator is a transmucosal vasodilator.
(Appendix 9)
The vasodilator according to any one of claims 1 to 8, wherein the vasodilator is an oral mucosal vasodilator.
(Appendix 10)
A therapeutic agent for periodontal disease comprising a vasodilator according to any one of claims 1 to 9.
(Appendix 11)
An oral composition comprising a vasodilator according to any one of claims 1 to 9.
(Appendix 12)
A food composition comprising a vasodilator according to any one of claims 1 to 9.

As described above, the vasodilator of the present invention contains at least one of a collagen-derived tripeptide and a collagen-derived dipeptide, and therefore can dilate blood vessels. Therefore, the vasodilator of the present invention can improve, for example, the blood flow of a subject, and can promote the reduction and/or alleviation of symptoms associated with blood circulation function. Therefore, the vasodilator of the present invention is extremely useful, for example, in the medical field.

Claims (7)

Contains collagen-derived tripeptides ,
A therapeutic agent for periodontal disease, comprising a vasodilator , wherein the collagen-derived tripeptide is glycine-proline-hydroxyproline . The periodontal disease treatment drug according to claim 1 , wherein the collagen-derived tripeptide comprises a product obtained by treating fish-derived collagen with a protease. The therapeutic agent for periodontal disease according to claim 1 or 2 , wherein at least one of the amino acid residues constituting the collagen-derived tripeptide is a natural amino acid residue or a modified amino acid residue. The periodontal disease treatment drug according to claim 1 , wherein the vasodilator is a transmucosal vasodilator. The periodontal disease treatment drug according to claim 1 , wherein the vasodilator is an oral mucosal vasodilator. An oral composition for treating periodontal disease, comprising the therapeutic agent for periodontal disease according to claim 1 . A food composition for treating periodontal disease, comprising the therapeutic agent for periodontal disease according to any one of claims 1 to 5 .

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