Collagen production promoter

Abstract

To identify a substance that can strongly promote collagen production in cells.SOLUTION: Provided is a collagen production promoter containing an allantoin.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a collagen production promoter. [Background technology]

[0002] Collagen is part of the extracellular matrix and is the main protein that makes up skin, tendons, cartilage, connective tissue, etc. In the skin, it is important for maintaining skin elasticity, moisture retention function, skin structure, etc. As skin ages, collagen production naturally decreases, becoming the main cause of wrinkles and sagging.

[0003] Collagen is an essential component for maintaining the structure of periodontal tissues. Periodontal tissues include the gums, alveolar bone, and periodontal ligament, and collagen plays an important role in connecting cells and maintaining the shape of the tissues. A lack of collagen can weaken the gums, causing them to recede and become thinner, and teeth can become more mobile, causing them to move from their original position and affecting aesthetics.

[0004] Periodontal disease is a disease caused primarily by oral bacteria. It begins with gingivitis and, as it progresses, destroys collagen tissue, deepening periodontal pockets and reducing alveolar bone. These symptoms can ultimately lead to tooth loss. Therefore, it is believed that promoting collagen regeneration in destroyed periodontal tissues can be used to treat periodontal disease and prevent its progression.

[0005] Furthermore, the regeneration of hard tissues such as alveolar bone and cementum is also important in the treatment of periodontal disease. The regeneration of these tissues is thought to be promoted by the proliferation of periodontal ligament fibroblasts, collagen production, and calcification (hardening of bones and teeth). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2002-29953 [Non-patent literature]

[0007] [Non-Patent Document 1] Hideo Arai, Role of periodontal ligament fibroblasts in periodontal tissue regeneration, Journal of the Japanese Society of Periodontology, Vol. 38, Supplement 2, 1996, p. 53 Summary of the Invention [Problem to be solved by the invention]

[0008] There is a strong demand for ingredients that act directly on cells in skin and oral tissues to stimulate collagen production in those cells, and although many reports have been published, there is still a lack of information. In particular, there are few reports of effective ingredients that focus on situations that require immediate action, such as inflammation, and there is a strong demand from society.

[0009] Therefore, an object of the present invention is to select a component that can strongly promote collagen production in cells. An object of the present invention is, but is not limited to, to select a component that can quickly improve tissue condition based on collagen production even under inflammatory conditions. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors discovered that allantoins are components that can significantly promote collagen production in cells and improve tissue conditions, and thus completed this research.

[0011] That is, the present invention includes the following embodiments. [1] A collagen production promoter containing allantoin. [2] An agent for promoting alveolar bone formation or tooth calcification, which contains allantoins. [3] An agent for maintaining or improving periodontal tissues, containing allantoins. [4] The agent according to [3], characterized in that the periodontal tissue is measured using the degree of gum recession, the degree of gum thinning, or the degree of gum discoloration as an indicator. [5] The agent according to any one of [1] to [4], wherein the content of allantoins is 0.01% by mass to 0.55% by mass. [6] The agent according to any one of [1] to [5], which is applied to inflammation of periodontal tissues. [Effects of the Invention]

[0012] By using the present invention, it is possible to strongly stimulate collagen production in cells and maintain or improve the condition of tissue. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows the results of GO (Gene Ontology) analysis performed following the extraction of differentially expressed genes using RNA-seq. [Figure 2] Figure 2 shows the results of GO (Gene Ontology) analysis performed following the extraction of differentially expressed genes using RNA-seq. [Figure 3] FIG. 3 is a graph summarizing the increase in gene expression levels dependent on the allantoin concentration (0%, 0.3%, 0.5%). [Figure 4] FIG. 4 is a graph summarizing the increase in the expression level of each gene depending on the concentration of allantoin (0%, 0.3%, 0.5%). [Figure 5] Figure 5 shows that the gene group whose expression increased in the RNA-seq analysis of Test Example 1 was also confirmed to have increased gene expression when confirmed by other test methods (real-time PCR in Test Example 2). DETAILED DESCRIPTION OF THE INVENTION

[0014] [(A) Allantoin] Allantoin is a known compound also known as 5-ureidohydantoin. Allantoin is known to be contained in the rhizomes of Symphytum officinale. In the present invention, Symphytum officinale or an extract thereof containing allantoin can also be used.

[0015] Allantoins include allantoin, its salts, and derivatives thereof.

[0016] The salt of allantoin is not limited as long as it is a pharmaceutically acceptable salt, and examples thereof include alkali metal salts, alkaline earth metal salts, and salts with organic bases, etc., such as salts with sodium, potassium, calcium, magnesium, ammonium, or diethanolamine, ethylenediamine, etc. Further examples include salts with amines such as ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris(hydroxymethyl)aminomethane, N,N-bis(hydroxyethyl)piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N-methylglucamine, and L-glucamine; or salts with basic amino acids such as lysine, δ-hydroxylysine, and arginine. Other examples include salts with mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids such as methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, acetic acid, propionic acid, tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, valeric acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, and salicylic acid; and salts with acidic amino acids such as aspartic acid and glutamic acid. "Pharmaceutically acceptable salts" may include solvates or hydrates of the salt.

[0017] Examples of allantoins include allantoin β-glycyrrhetinic acid, allantoin dihydroxyaluminum (Aldioxa), allantoin chlorhydroxyaluminum (Alcloxa), allantoin polygalacturonic acid, allantoin ascorbic acid, allantoin acetyl-DL-methionine, allantoin DL-pantothenyl alcohol, sodium DL-pyrrolidonecarboxylate, allantoin, etc. Among these, from the viewpoint of the commonality of the basic skeleton of the chemical structure, preferred are allantoin, allantoin dihydroxyaluminum (Aldioxa), and allantoin chlorhydroxyaluminum (Alcloxa), more preferred are allantoin and allantoin chlorhydroxyaluminum (Alcloxa), and particularly preferred is allantoin.

[0018] The above components may be used alone or in any combination of two or more.

[0019] The total content of allantoins is not particularly limited and is set appropriately depending on the type and content of other ingredients, the formulation form, the method of use, etc., but from the viewpoint of significantly achieving the effects of the present invention, examples of the total content of allantoins relative to the total amount of the formulation include 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.02% by mass or more, 0.03% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.2% by mass or more, 0.3% by mass or more, 0.35% by mass or more, 0.4% by mass or more, 0.45% by mass or more, 0.5% by mass or more, etc. The total content of allantoins may be, for example, 3% by mass or less, 1% by mass or less, 0.75% by mass or less, 0.55% by mass or less, or 0.5% by mass or less. The total content of allantoins may be, for example, 0.001 to 3 mass%, 0.01 to 3 mass%, 0.1 to 3 mass%, 0.2 to 3 mass%, 0.3 to 3 mass%, 0.35 to 3 mass%, 0.4 to 3 mass%, 0.001 to 1 mass%, 0.01 to 1 mass%, 0.1 to 1 mass%, 0.2 to 1 mass%, 0.3 to 1 mass%, 0.35 to 1 mass%, 0.4 to 1 mass%, 0.001 to 0.55 Mass%, 0.01~0.55 mass%, 0.1~0.55 mass%, 0.2~0.55 mass%, 0.3~0.55 mass%, 0.35~0.55 mass%, 0.4~0.55 mass%, 0.001 ~0.5% by mass, 0.01-0.5% by mass, 0.1-0.5% by mass, 0.2-0.5% by mass, 0.3-0.5% by mass, 0.35-0.5% by mass, 0.4-0.5% by mass, etc.

[0020] In addition to the above-mentioned components, the formulation of the present invention may contain, depending on its intended use or dosage form, ingredients commonly used in the fields of pharmaceuticals, quasi-drugs, cosmetics, etc., as appropriate. The ingredients that can be added are not particularly limited, but examples of additives that can be added include surfactants, preservatives, pH adjusters, chelating agents, stabilizers, irritation reducers, colorants, dispersants, and fragrances. These ingredients can be added alone or in any combination of two or more.

[0021] (Dosage form) The formulation of the present invention is not particularly limited as long as it is in a known form as a pharmaceutical, quasi-drug, cosmetic, or food. For example, it can be formulated by a known method into the form of an ointment, gel, cream, liquid, emulsion, tablet, powder, fine granules, granules, capsule, syrup, soft capsule, injection, drink, etc. From the viewpoint of more significantly exhibiting the effects of the present application, an ointment, gel, cream, or liquid is preferred, and from the viewpoint of adhesion and cohesion to the application site of allantoin, an ointment or gel is more preferred. By formulating in such a form, it can be made into a form suitable for application to, for example, the skin or gums, and the effect can be fully exhibited. In another embodiment, the formulation of the present invention may be added to an existing pharmaceutical, quasi-drug, cosmetic, or food.

[0022] (How to use) When the formulation of the present invention is formulated in a semi-solid or solid form such as a cream, ointment, gel, or the like, it can be placed in a tube container, although this is not a limitation, allowing the optimal amount to be squeezed out when applying the formulation of the present invention, making it easier to apply directly to the affected area with fingers, etc. The viscosity can also be adjusted so that it can be applied to a narrow or wide area of ​​the affected area. After applying the formulation of the present invention to the affected area, it can also be used by spreading it out while massaging with fingers.

[0023] When the formulation of the present invention is formulated in a liquid form such as a liquid preparation, it can be placed in a resin container or the like, which is not a limitation, to allow for an optimal amount to be dispensed when applying the formulation of the present invention. The viscosity can also be adjusted so that it can be applied to a narrow or wide area of ​​the affected area. When the formulation of the present invention is formulated as a tablet, capsule, drink, or the like, it can also be taken orally, which is not a limitation.

[0024] Examples of application sites for the formulation of the present invention include the skin (including the scalp), the oral cavity, periodontal tissues, and the eye. For skin, the formulation can be preferably used in areas where dryness, wrinkles, or sagging occurs due to a natural decrease in collagen production with age. For periodontal tissue, the formulation can be used in areas such as the gums (also called gingiva or gingiva), gingival sulcus, and areas where tartar has accumulated, or areas where swelling, bleeding, sagging, thinning, or discoloration of the gums are of concern. For eye, the formulation can be used in areas such as the cornea, or areas where the transparency of the eyeball is of concern.

[0025] The dosage of the formulation of the present invention can be appropriately determined depending on the application and formulation form, but is about 0.2 to 0.5 g, preferably 0.3 to 0.4 g per administration.

[0026] The timing of application of the formulation of the present invention can be appropriately determined depending on the application and formulation form, but is exemplified by several times a day, preferably 2 to 4 times a day, and more preferably 2 times a day (for example, morning and evening). For example, when applied to the oral cavity, it is exemplified by application after brushing.

[0027] (Application) The preparation of the present invention can be used as a collagen production promoter by containing allantoins. Collagen production can be observed, for example, in the skin, periodontal tissue, or eye area, but is not limited thereto. Approximately 40% of the collagen present in the human body is present in the skin, and it is thought that collagen accounts for approximately 70% of the dermis of the skin. Collagen can affect skin functionality, such as dryness (moisture), elasticity (firmness), and wrinkles. Among the types of collagen in the skin, fibrous type I collagen is important for skin functionality. In the examples described below, it has been confirmed that the preparation of the present invention can promote the production of type I collagen.

[0028] Furthermore, collagen plays an important role in the function and structure of periodontal tissues, particularly in the gums. A lack of collagen in periodontal tissues due to aging or periodontal disease weakens the gums, causing gum recession, thinning, and changes in color. Teeth become more mobile, which can shift from their original positions, potentially affecting aesthetics and apparent age, and potentially leading to worsening periodontal disease. Therefore, since the formulations of the present invention can promote collagen production in periodontal tissues, they can be used to maintain or improve periodontal tissues. These functionalities can be measured, for example, by the degree of gum recession, the degree of gum thinning, and the degree of gum color, among other things. Furthermore, the apparent age of the gums (anti-aging effect) can also be used as an indicator. For example, the effects of the present invention can be confirmed by applying the formulations of the present invention to areas where gum recession is a concern and improving gum recession. Furthermore, the effects of the present invention can be confirmed by applying the formulations of the present invention to areas where gum thinning is a concern and improving gum thinning, among other things. For example, the effects of the present invention can be confirmed by applying the preparation of the present invention to an area where gum color (e.g., red, purple, white, etc.) is of concern and seeing an improvement in gum color (e.g., pink).Furthermore, the effects of the present invention can be confirmed by applying the preparation of the present invention to the gums and maintaining or improving the apparent age of the gums.

[0029] Furthermore, in the Examples described below, it has been confirmed that application of allantoin to periodontal ligament fibroblasts can promote collagen production by periodontal ligament fibroblasts and induce bone formation. In the human body, the periodontal ligament is a thin film 0.15 to 0.38 mm thick that directly connects the cementum on the surface of the tooth (particularly the dental root) to the alveolar bone, and is primarily composed of collagen fibers. Promoting collagen production by periodontal ligament fibroblasts improves collagen fibers in the periodontal ligament, promotes the formation of adjacent alveolar bone, and promotes tooth remineralization, thereby contributing to the maintenance or improvement of periodontal tissues.

[0030] Furthermore, in the eye, type I collagen is the main component of the corneal stroma. In the corneal stroma, collagen fibers with uniform diameters are regularly arranged, and the collagen meshwork structure is important for maintaining optical transparency. The preparation of the present invention can promote collagen production, thereby normalizing the collagen meshwork structure in the corneal stroma and contributing to maintaining optical transparency.

[0031] In the above tissues and sites, although not limited thereto, when inflammation occurs, an imbalance in the control of collagen production and degradation occurs, which may promote collagen degradation in cells. Furthermore, when chronic inflammation occurs, collagen production function may be disrupted, which may affect tissue structure. Therefore, when inflammation occurs in the skin, periodontal tissue, eye area, etc., it may be insufficient to treat it solely for the purpose of anti-inflammation, and it is also important to use a technique that promotes collagen production. Therefore, the formulation of the present invention is preferably applied to, but not limited to, tissue inflammation.

[0032] In this specification, periodontal disease refers to a condition in which the healthy state of periodontal tissues consisting of gingiva (so-called gums), cementum, periodontal ligament, and alveolar bone is impaired, and can be caused by bacterial infection or the like.

[0033] Among oral bacteria, Porphylomonas gingivalis, Treponema denticola, and Tannerella forsythia, also known as the red complex, are known to cause periodontal disease. Other bacteria, such as Aggregatibacter actinomycetemcomitans and Prevotella intermedia, are also known to cause periodontal disease. Recent research has revealed that various bacteria interact in complex ways to contribute to the progression of periodontal disease. The proliferation of these bacteria can lead to symptoms such as dental plaque formation, characteristic bad breath, and a sticky feeling in the mouth upon waking. When collagen production is disrupted and periodontal tissue structure is damaged, these bacteria can further proliferate. Therefore, by using the preparation of the present invention, it is possible to maintain or improve periodontal tissues by promoting collagen production and to suppress the proliferation of these causative bacteria.

[0034] Dental plaque releases toxins, such as lipopolysaccharide (LPS), protease, and collagenase, derived from oral bacteria. Proteases and collagenase damage the gums, and LPS acts on gum cells, causing them to secrete inflammatory cytokines. These inflammatory cytokines cause inflammation in the gums, damaging the gums and periodontal ligament. This leads to bleeding and swelling of the gums, and gum recession, making the teeth appear longer. Furthermore, inflammatory cytokines overactivate immune cells in the body, which then releases RANKL, which stimulates osteoclasts. This results in the dissolution of the alveolar bone supporting the teeth, leading to tooth mobility and eventual tooth loss. Inflammation and excessive immune cell activation cause tissue damage and destruction, as well as further inflammation, resulting in a synergistic worsening of the condition, a vicious cycle of periodontal tissue destruction.

[0035] Periodontal disease can be broadly divided into two types depending on the progression of the disease: gingivitis (gingivitis) and periodontitis (pyorrhea).

[0036] Gingivitis (periodontitis) refers to a condition in which the gums are damaged but the cementum, periodontal ligament, and alveolar bone are not destroyed. Symptoms of gingivitis (periodontitis) include redness of the gums (the gums appear red, reddish-black, blackish, or dusky), swelling (the gums feel round and swollen), edema (the gums feel limp and squishy), bleeding (the gums bleed when brushing, etc.), pain, itchy gums, a white, pus-like discharge when pressed on the gums, and tooth sensitivity (a feeling of tooth sensitivity or pain when the temperature changes or physical stimuli are applied).

[0037] Pyorrhea refers to a condition in which inflammation that initially occurs in the gums spreads to deeper periodontal tissues such as the cementum, periodontal ligament, and alveolar bone, causing damage and destruction. In addition to the symptoms caused by periodontal disease bacteria and those of gingivitis (gingivitis), symptoms of pyorrhea include receding gums, thinning gums, gaps between teeth, teeth that appear longer, objects becoming more likely to get stuck between teeth, a feeling of loose teeth, loose teeth, and so on.

[0038] In healthy gums that are not affected by periodontal disease, the roots of the teeth buried in the gums are covered with cementum, which is in turn covered by the periodontal ligament. The periodontal ligament plays a very important role in connecting the teeth to the alveolar bone, the bone that supports the teeth. Gums are naturally pale pink in color, firm, strong, and have a plump appearance, and they hardly bleed when brushed or cleaned with an interdental brush.

[0039] The formulations of the present invention act on cells and tissues with an imbalance in collagen metabolism and actively promote collagen production, thereby regenerating or repairing periodontal tissues destroyed by periodontal disease, etc., and further enhancing the regenerative or repair capacity of periodontal tissues and enhancing the firmness and elasticity of gums. As used herein, "promoting" regeneration or repair includes directly enhancing the inherent regenerative or repair capacity of periodontal tissues. Promotion of periodontal tissue regeneration or repair can also be expressed, for example, as reviving, repairing, mending, restoring, reviving, reviving, restoring, revitalizing, restoring, restoring to a healthy state, healing, firming, tightening, plumping, or providing elasticity to periodontal tissues (e.g., gums and tooth roots). Expressions such as "gum care" that describe the improvement of the condition of a periodontal tissue site can also be used. Promotion of periodontal tissue regeneration or repair can prevent, treat, or ameliorate the symptoms of gingivitis and pyorrhea described above. In another embodiment, the preparation of the present invention can also have a collagen degradation inhibitory effect on cells or tissues in which collagen degradation has been accelerated due to an imbalance in collagen metabolism.

[0040] The periodontal tissues targeted by the formulation of the present invention include, for example, the periodontal ligament, gums, gingival epithelium (the surface of the gums or the mucosa), or alveolar bone, preferably the periodontal ligament, gums, or gingival epithelium (the surface of the gums or the mucosa), and more preferably the periodontal ligament.

[0041] The periodontal tissue cells that are the target of the formulation of the present invention are, for example, cells that form the gingiva or periodontal ligament, preferably gingival epithelial cells, gingival fibroblasts, or periodontal ligament fibroblasts, and more preferably periodontal ligament fibroblasts.

[0042] Periodontal ligament fibroblasts are morphologically similar to gingival fibroblasts and are thought to be the cells responsible for maintaining the periodontal ligament tissue. They consist of a population of cells with pluripotency and different functions, making them important cells for the maintenance and regeneration of periodontal tissue.

[0043] The formulations of the present invention activate cells that form periodontal tissue. Here, "activating" cells refers to improving the state of cells, for example, by increasing the expression levels of genes involved in collagen production or bone formation, or by increasing the relevant cell capabilities. To express "activating" cells, expressions such as "reviving weakened cells," "activating cells," "revitalizing cells," "vitalizing cells," "cell anti-aging care," or "activating," "reviving," "reviving," "reviving," "rejuvenating," "regenerating," and "making healthy" can be used. Activating gingival cells can prevent or improve the symptoms of gingivitis and pyorrhea described above.

[0044] The periodontal tissues targeted by the formulation of the present invention include, for example, the periodontal ligament, gums, gingival epithelium (the surface of the gums or the mucosa), or alveolar bone, preferably the periodontal ligament, gums, or gingival epithelium (the surface of the gums or the mucosa), and more preferably the periodontal ligament.

[0045] The periodontal tissue cells that are the target of the formulation of the present invention are, for example, cells that form the gingiva or periodontal ligament, preferably oral keratinocytes, gingival fibroblasts, or periodontal ligament fibroblasts, and more preferably periodontal ligament fibroblasts.

[0046] The preparations of the present invention can prevent the above-mentioned symptoms of pyorrhea by acting on cells and tissues with an imbalance in collagen metabolism and actively promoting collagen production, and can be used to prevent or ameliorate symptoms of periodontal disease, including gingivitis and pyorrhea. More preferably, they can be used to prevent or ameliorate symptoms of pyorrhea. For example, the preparations of the present invention can also be applied to healthy gums. To express the prevention of symptoms of gingivitis and pyorrhea, expressions that include phrases indicating prevention of the various symptoms caused by the causative bacteria of gingivitis, pyorrhea, and periodontal disease (e.g., preventing bleeding gums, preventing bad breath and sticky mouth, preventing swollen and bleeding gums, etc.) can be used; expressions that describe changes to healthy gums, such as healthy gums, firm gums, maintaining pink gums, maintaining firm gums, strong gums, and plump gums; and expressions such as preventing tooth sensitivity (from eating cold foods, etc.) and clean / fresh breath can be used.

[0047] The preparation of the present invention can improve the symptoms of alveolar pyorrhea described above. To express the improvement of symptoms, expressions such as "gum care" that improve a specific site of the periodontal tissue can also be used.

[0048] The periodontal tissue to which the formulation of the present invention is applied is, for example, the periodontal ligament, gums, gingival epithelium, or alveolar bone, preferably the periodontal ligament, gums, or gingival epithelium, and more preferably the gingival epithelium.

[0049] The periodontal tissue cells to which the formulation of the present invention is applied are, for example, cells that form the gingiva or periodontal ligament, preferably oral keratinocytes, gingival fibroblasts, or periodontal ligament fibroblasts, and more preferably periodontal ligament fibroblasts.

[0050] As used herein, the term "prevention" refers to preventing or delaying the onset of a disease or condition, or reducing the risk of developing a disease or condition.

[0051] As used herein, "amelioration" refers to improving or alleviating a disease, symptom or health condition, preventing or slowing the deterioration of a disease, symptom or health condition, or reversing, preventing or slowing the progression of a disease or symptom.

[0052] As used herein, "treatment" refers to an action to improve or alleviate a disease, symptom, or health condition, or to prevent or slow the worsening of a disease, symptom, or health condition, or to reverse, prevent, or slow the progression of a disease or symptom.

[0053] [Applicable persons] The age group of subjects to whom the formulation of the present invention is applied is not particularly limited, but from the viewpoint that the number of people who have or are at risk of having gingivitis and / or alveolar pyorrhea increases with age, the subject is preferably adults, more preferably 30 years of age or older, and even more preferably middle-aged people (45 years of age or older) to elderly people (55 years of age or older).

[0054] (Manufacturing method) The preparation of the present invention can be produced by known methods, which may include a sterilization step, if necessary. [Example]

[0055] Next, the present invention will be specifically explained with reference to examples and test examples, but the present invention is not limited to the following examples and test examples.

[0056] [Test Example 1: RNA-seq analysis] (1) Data acquisition Using human periodontal ligament fibroblasts (HPLF cells), we modeled an inflammatory state by inducing inflammation and added allantoins to the cells, and then acquired RNA-seq data to examine how the cell behavior changed. RNA-seq is a method for comprehensively and quantitatively examining gene expression in a cell population. By comprehensively clarifying how the addition of allantoins changes cellular gene expression, we can elucidate the function of allantoins on tissues in an inflammatory state. Inducing inflammation using periodontal ligament fibroblasts, for example, can be used as a model for the inflammatory state of periodontal disease, enabling us to evaluate the effects on the periodontal ligament and its adjacent tissues, the tooth root and alveolar bone, as well as the health of periodontal tissues.

[0057] For data acquisition, human periodontal ligament fibroblasts were seeded in DMEM medium (Gibco, 10% FBS) on day 1, replaced with DMEM medium (0.1% FBS) on day 2, and then replaced with DMEM medium (0.1% FBS) supplemented with allantoin (Alla 0.0% (Ctrl), 0.3%, 0.5%) on day 3. Inflammation was then induced by LPS stimulation. RNA was collected 24 hours after LPS stimulation, and sequencing and data acquisition were performed.

[0058] (2) Preprocessing of RNA-seq data The resulting RNA-seq sequence data was trimmed of adapter sequences, mapped to the reference genome, and counted the number of mapped reads to quantify the gene expression levels in each sample.

[0059] The present inventors' previous research has revealed that clusters are divided according to whether inflammation is induced, and the position of points changes with the increase in allantoin concentration, and the tendency of gene expression changes according to whether inflammation is induced and the change in allantoin concentration.In addition, the direction of the change in plot position with the increase in allantoin concentration is the same regardless of whether inflammation is induced, so it has been revealed that the direction of the change in gene expression according to the change in allantoin concentration is the same regardless of whether inflammation is induced.

[0060] Genes whose expression levels were significantly elevated in the 0.5% allantoin sample compared to the 0% allantoin sample under LPS stimulation (inducing inflammation) and in the 0.5% allantoin sample compared to the 0.3% allantoin sample were extracted 24 hours after LPS stimulation. To investigate the association of these genes with cellular phenotypes, we performed GO (Gene Ontology) analysis using ClusterProfiler in R. GO analysis is an analytical method for extracting gene functions that are significantly more frequently observed in a given gene list compared to the overall gene population. Examination of the top gene clusters revealed significant hits for GO terms related to tissue "collagen production" and "bone formation," such as "ossification," "extracellular matrix tissue," and "collagen fibril tissue." Figure 1 shows the results of a comparative analysis between 0% and 0.5% allantoin concentrations, and Figure 2 shows the results of a comparative analysis between 0.3% and 0.5% allantoin concentrations.

[0061] As shown in Figures 1 and 2, it was shown that the addition of allantoin (0% to 0.5%) and increasing the amount of allantoin (0.3% to 0.5%) can induce collagen production in fibroblasts and bone formation. In skin tissue, collagen production in fibroblasts can affect skin functionality, such as dryness (moisture), elasticity (firmness), and wrinkles; in periodontal tissue, it can affect the improvement of weakened gums, receding gums, thinning gums, and changes in color; and in ocular tissue, it can affect the maintenance or improvement of optical transparency in the corneal stroma.

[0062] In inflammation of periodontal tissues, such as periodontitis, an imbalance in collagen metabolism leads to excessive destruction of gingival connective tissue due to collagen degradation. Although not limited to this, this test example used cells modeling an inflammatory state, and it was confirmed that allantoin can promote collagen production even in cells or tissues with an imbalance in collagen metabolism.

[0063] In the results of Test Example 1, genes involved in collagen degradation include MMP14, and the results of the allantoin concentration-dependent decrease in expression level of each gene (0%, 0.3%, and 0.5% from the left in the figure) are summarized in Figure 3. The vertical axis of Figure 3 is TPM (Transcripts Per Kilobase Million).

[0064] Furthermore, the above-mentioned Non-Patent Document 1 reports that "various growth factors promote functions such as proliferation, extracellular matrix synthesis, and mineralization ability of periodontal ligament fibroblasts," and it has been confirmed that the action of allantoins on periodontal ligament fibroblasts leads to collagen production and bone formation induction in tissues surrounding the periodontal ligament (promotion of alveolar bone formation and remineralization of teeth (tooth roots)).

[0065] In the results of Test Example 1, genes involved in bone regeneration, etc., include Wnt11 and FGF18, and the results of the increase in expression level of each gene depending on the allantoin concentration (0%, 0.3%, 0.5% from the left in the figure) are summarized in Figure 4. The vertical axis in Figure 4 is TPM. Although not limited to this, a particularly significant effect on the activation of periodontal ligament fibroblasts was confirmed at allantoin concentrations (0.3% to 0.5%).

[0066] (Test Example 2. Analysis of type I collagen gene expression in periodontal ligament fibroblasts) To confirm whether the gene group whose expression increased in the RNA-seq analysis of Test Example 1 would produce consistent results when other test methods were used, the expression of type I collagen gene was confirmed by real-time PCR, as an example.

[0067] To obtain these data, HPLF cells were seeded in DMEM medium (Gibco, 10% FBS) on day 1, replaced with DMEM medium (0.1% FBS) on day 2, and then replaced with DMEM medium (0.1% FBS) supplemented with 0.5% allantoin (Example 1) on day 3, after which inflammation was induced with LPS. For the control (Ctrl) sample, inflammation was induced by LPS stimulation without allantoin. 72 hours after LPS stimulation, RNA was collected, and the COL1A1 RNA level was quantified by PCR. The COL1A1 RNA level was quantified using the ΔΔCt method, with the GAPDH gene as a normalizer. Figure 5 shows the relative expression levels, with the control set at 100.

[0068] As shown in Figure 5, the gene group whose expression increased in the RNA-seq analysis of Test Example 1 was also confirmed to have increased gene expression when confirmed by other test methods, demonstrating that the results of both methods are consistent.

[0069] [Formulation example] Formulations of the present invention were prepared based on the formulations shown in the table below.

[0070] (Formulation Example 1) Allantoin 0.3g, tocopherol acetate 2g, dipotassium glycyrrhizinate 0.4g, carbazochrome 0.02g, hinokitiol 0.1g, cetylpyridinium chloride hydrate 0.05g, petrolatum, liquid paraffin, anhydrous silicic acid, white beeswax, carmellose sodium, microcrystalline wax, carboxyvinyl polymer, polyethylene, peppermint oil, l-menthol, thymol, sodium edetate, fragrance, Red No. 3 (per 100g)

[0071] (Formulation Example 2) Allantoin 0.5g, tocopherol acetate 2g, dipotassium glycyrrhizinate 0.4g, carbazochrome 0.02g, hinokitiol 0.1g, cetylpyridinium chloride hydrate 0.05g, petrolatum, liquid paraffin, paraffin, anhydrous silicic acid, white beeswax, microcrystalline wax, carboxyvinyl polymer, carmellose sodium, polyethylene, sorbitan monostearate, dibutylhydroxytoluene, pectin, peppermint oil, fennel oil, l-menthol, thymol, sodium edetate, fragrance, Red No. 3 (per 100g)

[0072] In either formulation, collagen production by cells is strongly promoted, and tissue condition can be maintained or improved.

Claims

1. A collagen production promoter containing allantoin.

2. An agent for promoting alveolar bone formation or tooth calcification, which contains allantoins.

3. An agent for maintaining or improving periodontal tissues, containing allantoins.

4. 4. The agent according to claim 3, wherein the periodontal tissue is evaluated using the degree of gum recession, the degree of gum thinning, or the degree of gum discoloration as an indicator.

5. The agent according to any one of claims 1 to 4, wherein the content of allantoins is 0.01% by mass to 0.55% by mass.

6. The agent according to any one of claims 1 to 4, which is applied to inflammation of periodontal tissues.

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