JP6944240B2 - An agent for maintaining or increasing the content of fibrous structural proteins in living tissues containing GABA as an active ingredient. - Google Patents

Description

The present invention relates to a technical field relating to maintenance or increase of the content of fibrous structural protein, which is an extracellular matrix of living tissue. In particular, it relates to maintaining or increasing the content of fibrous structural proteins, which are extracellular matrices of living tissues using GABA.

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that is widely distributed in nature and is an inhibitory neurotransmitter that is abundant in the central nervous system of mammals. The 2001 revision of the food category has made it possible to use it as a food, and a wide range of physiological activities such as blood pressure lowering action (Non-Patent Document 1-3) have been reported.

Elastin, on the other hand, is an insoluble protein that is a major constituent of elastic fibers that are widely distributed in various elastic tissues in the body such as the aorta, nuchal ligament, skin, and lungs. Elastin in the skin and blood vessels is known to decrease or decrease in function with aging. A substance that promotes elastin production in skin fibroblasts that produce elastin in the skin is considered to have potential as a skin improving agent. For example, Patent Documents 1 to 3 propose elastin production promoters containing plant extracts.

In addition, it was reported that elastin derived from pigs and horses had an improving effect and a whitening effect on the skin (see Patent Document 4), and that elastin derived from animals (bovine nuclei) improved the condition of blood vessels (see Patent Document 4). Patent Document 5) and the like.
Further, in Patent Document 6, 79 to 84% of the amino acids constituting elastin are composed of proline, glycine, alanine and valine, 2 to 3% are composed of aspartic acid and glutamic acid, and 0.7 to 1.3% are composed of glutamic acid. Disclosed is an arteriosclerosis inhibitor containing low molecular weight water-soluble elastin having a molecular weight of about 130,000, which is composed of lysine, histidine, and arginine and 0.2 to 0.4% of desmosine and isodesmosine. ..

The skin is one of the largest organs in the human body, and its condition and appearance are partially determined by the amount and condition of elastin contained in the skin. Elastin is a substrate protein, composed of tropoelastin monomers and cross-linked to form larger tertiary structure. Elastin provides elasticity, suppresses or stops the dynamic deformation of tissue due to stretching under load, and allows the tissue to return to its original arrangement after the load is removed. Loss of skin suppleness and elasticity, joint stiffness and loss of flexibility are associated with deterioration of elastin and alteration of tissue binding structures.
Many compositions that improve the properties of the skin are not drugs for the treatment of diseases, but modify the normal condition of the skin, such as wrinkles and sagging (dents). Such normal skin conditions are often associated with the deterioration (or alteration, degradation) of elastin, an extracellular protein.

Patent Document 7 contains an elastin peptide extracted from fish arterial spheres as an active ingredient, and the total content of glycine, alanine, valine and proline per 1000 residues is 650 residues or more, and the aspartic acid and aspartic acid contents. The total of 10 to 35 residues, the total content of glutamine and glutamine is 20 to 50 residues, the total content of lysine, histidine and arginine is 20 to 50 residues, and the total content of desmosin and isodesmosin. Skin improving agents and vascular improving agents having a total of 0.3 residues or more and a hydroxyproline content of 10 residues or less are disclosed.

Elastin is also abundant in ligaments and tendons such as the nuchal ligament and knee ligament, and is expected to play an important role in mechanical and physiological functions in the knee ligament, for example. Similar to the skin and the like, it is considered that the functional deterioration of the tendons and ligaments due to the decrease in the elastin content due to aging and the like contributes to the joint pain of the knees and the like. Therefore, increasing the proliferative capacity of tendon cells and ligament cells and promoting gene expression of proteins that make up tendons and ligaments can prevent locomotive syndromes such as knee arthritis, tendon and ligament damage caused by sports, and tendons and ligaments. It is expected to lead to improvement of tendon and ligament functions such as strengthening.

As a composition for improving, promoting or preventing healing, improving or strengthening tendon or ligament, for example, a degradation product obtained by decomposing collagen or gelatin with collagenase, and having an amino acid sequence. Peptide represented by (Gly-XY) _n (in the formula, Gly represents a glycine residue, X and Y represent any amino acid residue other than glycine, and n represents a positive integer). Those containing the active ingredient (see, for example, Patent Document 8) have been proposed. Further, Patent Document 9 describes a platelet-derived growth factor and a pharmaceutically acceptable solid carrier at a concentration in the range of about 0.1 mg / mL to about 1.0 mg / mL in a pharmaceutically acceptable liquid carrier. Disclosed are methods of promoting the growth of bone, periodontium, ligament or cartilage by applying the composition comprising to bone, periodontium, ligament or cartilage.

Japanese Unexamined Patent Publication No. 2002-293747 Japanese Unexamined Patent Publication No. 2005-22993 Japanese Unexamined Patent Publication No. 2005-60341 Japanese Unexamined Patent Publication No. 2002-205913 Japanese Unexamined Patent Publication No. 2007-045722 JP-A-2007-405722 (Claim 6 etc.) Japanese Unexamined Patent Publication No. 2010-155820 Japanese Unexamined Patent Publication No. 2005-281186 Japanese Unexamined Patent Publication No. 2009-195739

Eastern Medicine 2001; 17: 1-7. Pharmacology and Treatment 2002; 30: 963-72. Health and Nutrition Food Research 2003; 6: 51064. "Evaluation and Development of Skin-Beautiful Food Materials", 1st Edition, supervised by Tetsuro Yamamoto, (CMC Publishing, Tokyo), pp. 113-118 (2013). 12 J. Jpn. Soc. Food Sci. Technol. 2016; 63: 306-311 "Evaluation and Development of Skin-Beautiful Food Materials", CMC Publishing, 11 (2013) Funct. Food, 2,358-365 (2009) Experimental Medicine Vol. 20, No. 4, p. 570 (currently Kansai Medical University, by Tomoyuki Nakamura) Int J Food Sci Nutr. 2012; 63; 689-698

In an open human study (conducted from February to April) in which GABA was ingested at 180 mg per day for 8 consecutive weeks, it was suggested that the skin elasticity was improved before and after ingestion (Hideki Sozono of Non-Patent Document 4, the main material). Evaluation and development). However, skin condition is sensitive to the seasons and should be evaluated by a randomized controlled trial.
Therefore, first, the present inventors conducted a placebo-controlled, double-blind, parallel-group study on women who regularly feel rough skin, fatigue, and sleep disorders, and investigated the effect of GABA intake on the skin condition. .. That is, in order to evaluate the effect of GABA intake on skin quality and sleep quality, a double-blind parallel group was targeted for adult women who are aware of rough skin and are feeling tired or sleep disorder. An interim test was conducted. As a result, the cheek skin elasticity index was significantly improved in the GABA intake group as compared with the placebo intake group. From the results of this study, it was shown that ingestion of GABA may suppress the deterioration of viscoelasticity of the cheeks of adult women who are aware of rough skin and feel fatigue and sleep disorders on a daily basis (Non-Patent Document 5).
In the study of Non-Patent Document 5, GABA taken orally did not affect the water content of the skin. However, it was mainly the water content of the stratum corneum that was measured there, and the effect on the water content in the dermis is unknown. It was suggested that GABA intake may contribute to the improvement of skin firmness and elasticity.
However, in addition to the indirect effects of stress relief and sleep quality improvement on the skin condition, it is possible that GABA intake has a direct effect on the skin, and GABA taken orally is the skin (dermis / epidermis). Whether or not to reach the above is a subject of study of the present invention.
An object of the present invention is to study the effect on collagen and elastin, which are components related to the elasticity of skin tissue, and to clarify the mechanism of action of the GABA ingestion effect.

The present invention promotes the synthesis of fibrous structural proteins such as collagen, elastin, and fibrillin, which are extracellular matrices of living tissues, such as the elastin production promoter containing plant extracts in Patent Documents 1 to 3. To provide an agent for maintaining or increasing the content of fibrillinous structural protein, which is an extracellular matrix, for example, an agent for maintaining or increasing the content of skin elastin, vascular elastin, and ligament elastin. Make it a solution.
A solution that enables improved elasticity of skin, tendons or blood vessels by promoting the synthesis of fibrous structural proteins, which are extracellular matrices of living tissues such as elastin, collagen, fibrillin-1, fibrillin-2, and fibrillin-5. Providing means to produce cosmetics, drug compositions, especially dermatological compositions, foods and drinks, which are effective in treating, reducing and suppressing the appearance (or manifestation) of skin aging, as well as ligaments and Drugs (tendon and ligament injury therapeutic agents), foods, etc. that have the effects of promoting recovery of ligament injury, preventing tendon and ligament injury due to sports, etc., and strengthening tendon and ligament function of elderly people, athletes, racehorses, etc. To provide feed, and also to provide medicines, foods, and feeds that have effects such as suppressing the risk of vascular diseases and abnormalities and strengthening the function of vascular such as keeping the vascular healthy.

The present inventors have found that GABA maintains or increases the elastin content of living tissues, and based on this discovery, as a cosmetic agent for promoting elastin synthesis by dermal fibroblasts. This is the result of completing the present invention originating from the use of GABA.

The present invention is for promoting the synthesis of collagen, elastin, fibrillin-1, fibrillin-2, and fibrillin-5, which are extracellular matrices of living tissues consisting of the following (1) to ( 5) skin or ligaments. The gist is the drug.
(1) Collagen, elastin, fibrillin, which are extracellular matrices of these tissues, without promoting the proliferation of fibroblasts, in living tissues consisting of skin or ligaments, which are characterized by containing GABA as an active ingredient. 1. An agent for promoting the synthesis of fibrillin-2 and fibrillin-5.
(2) Further, the agent according to (1) above, for suppressing the synthesis of matrix metalloproteinase-1 having a collagen-degrading action.
(3) The agent according to (1) or (2) above, which is a preparation to be orally administered.

(4) The agent according to any one of (1) to (3 ) above, wherein the daily intake of GABA is 10 to 3000 mg.
(5) The agent according to any one of (1) to (4 ) above, which is a food or drink.

According to the present invention, the content of the proteolytic enzyme matrix metalloprotease-1 by promoting the synthesis of elastin, collagen, fibrillin-1, fibrillin-2, fibrillin-5 and / or having an extracellular matrix degrading effect. As a solution that enables improvement of skin, tendon or vascular elasticity by suppressing the increase in collagen, the content of fibrillinous structural protein, which is an extracellular matrix of living tissue, containing GABA as an active ingredient is maintained or Agents for increasing can be provided.
It also produces cosmetics, drug compositions, especially dermatological compositions, foods and drinks that are effective in treating, reducing and suppressing the appearance (or manifestation) of skin aging, and for the recovery of tendon and ligament injuries. To provide medicines (tendon and ligament injury therapeutic agents), foods, and feeds that have the effects of promoting, preventing tendon and ligament injuries caused by sports, etc., and strengthening tendon and ligament functions of elderly people, athletes, racehorses, etc. Furthermore, it is to provide pharmaceuticals, foods, and feeds that have effects such as strengthening the function of blood vessels such as suppressing the risk of vascular diseases and abnormalities and keeping the blood vessels healthy.

From the examples of the present invention, it was revealed that GABA has an effect of increasing the mRNA expression levels of tropoelastin, fibrillin-1, 2 and fibrin-5 (see FIG. 1).
In addition, in immunostaining of elastin, it was confirmed that the GABA-exposed group produced about twice as much elastin as the control group, so that it may promote the synthesis of elastin not only at the gene level but also at the protein level. It was suggested (see Figure 7).
Therefore, it was suggested that GABA improves the elasticity of the skin by promoting elastin synthesis in the skin tissue.
Similar to skin, it is expected to lead to prevention of locomotive syndrome such as knee osteoarthritis, damage to tendons and ligaments caused by sports, and improvement of tendon and ligament functions such as strengthening of tendons and ligaments.
In addition, as described in Patent Document 5 that elastin improved the state of blood vessels, it is expected to lead to suppression of arteriosclerosis.

The conceptual diagram of this invention is shown. The main constituents of the present invention are promotion of the expression of the trapoelastin gene (ELN) in living tissues, promotion of the expression of the elastin synthesis-related substances fibrillin-1 and fibrillin-2 genes (FBN-1, FBN-2), and elastin synthesis-related. The promotion of the expression of the substance fibrillin-5 gene (FBRN-5) is a pathway / index leading to the effect of improving the elasticity of the skin, and has a ripple effect of preventing arteriosclerosis, ligament damage, and rupture. The results of toxicity tests at 0.01, 0.1, 1, 10, 100 μg / ml as GABA in normal human dermal fibroblasts of Example 1 are shown in terms of proliferation rate. The results obtained by determining the ELN mRNA expression level at a concentration (GABA: 0.01, 0.1, 1, 10 μg / ml) that does not significantly reduce the growth of Example 1 as a ratio to the GAP DH mRNA expression level are shown. The results obtained by determining the FBN-1 mRNA expression level at 0.01, 0.1, 1, 10 μg / ml as GABA of Example 1 as a ratio to the GAP DH mRNA expression level are shown. The results obtained by determining the FBN-2 mRNA expression level at 0.01, 0.1, 1, 10 μg / ml as GABA of Example 1 as a ratio to the GAP DH mRNA expression level are shown. The results obtained by determining the expression level of FBRN-5 mRNA at 0.01, 0.1, 1, 10 μg / ml as GABA of Example 1 as a ratio to the expression level of GAP DH mRNA are shown. Photomicrograph of normal human dermal fibroblasts used in this test of Example 1 when GABA was exposed and immunostaining was performed. Areas other than black indicate areas stained as elastin. The area ratio of the elastin-stained area is shown in the lower row. * Indicates a significant difference. The results of toxicity tests at 0.01, 0.1, 1, 10, 100 μg / ml as GABA in normal human dermal fibroblasts of Example 2 are shown in terms of proliferation rate. The results obtained by determining the COL1A1 mRNA expression level at a concentration (GABA: 0.01, 0.1, 1, 10 μg / ml) that does not significantly reduce the growth of Example 2 as a ratio to the GAP DH mRNA expression level are shown. The results obtained by determining the COL1A2 mRNA expression level at 0.01, 0.1, 1, 10 μg / ml as GABA of Example 2 as a ratio to the GAP DH mRNA expression level are shown. The results obtained by determining the MMP-1 mRNA expression level at 0.01, 0.1, 1, 10 μg / ml as GABA of Example 2 as a ratio to the GAP DH mRNA expression level are shown. This is an excerpted and modified drawing of Experimental Medicine Vol. 20, No. 4, p. 570 (currently written by Tomoyuki Nakabe, Kansai Medical University), and is a drawing explaining the biosynthetic process of elastin.

GABA ingested from outside the body in order to maintain or increase the content of elastin and the like in the living tissue of the present invention is an abbreviation for γ-amino butyric acid (γ-aminobutyric acid). The abbreviation GABA, which is an acronym for the English name, is generally widely used. It is a type of amino acid that is widely distributed in animals and plants, and is an inhibitory neurotransmitter that exists in the brain and spinal cord of mammals. GABA improves cerebral blood flow, increases oxygen supply, and enhances cerebral metabolism, so it is applied to the treatment of stroke, head trauma sequelae, headache due to cerebral artery sequelae, tinnitus, decreased desire, etc. .. In addition, as other physiological effects, improvement of learning ability and activation of renal function are known. GABA is mainly present in the brain pulp of the living body, is involved as a neurotransmitter of the central nervous system, is known as a major inhibitory transmitter of nerves, activates blood flow in the mesencephalon, and activates the metabolic function of brain cells. It is used for the purpose of enhancing and relaxing the autonomic nerves caused by stress. It is known that it is a substance that does not cross the blood-brain barrier, and even if GABA is ingested from outside the body, it is not used as it is as a neurotransmitter.
One embodiment of the agent for maintaining or increasing the content of the fibrous structural protein, which is the extracellular matrix of the living tissue of the present invention, is for an oral composition, an external composition, a food (supplement), a food additive. It is preferable that it is used for at least one of the uses. Compositions used for the body, such as the pharmaceutical or cosmetic compositions of the present invention, are excipients with agents for maintaining or increasing the content of fibrous structural proteins, which are the extracellular matrix of the biological tissues of the present invention. Is preferably contained. In that case, an external composition for maintaining or increasing the content of fibrous structural protein, which is the extracellular matrix of living tissue, or for maintaining or increasing the content of fibrous structural protein, which is the extracellular matrix of living tissue. It is preferable to use the oral composition of. Tablets are preferred as one embodiment of the pharmaceutical composition of the present invention.
A composition for pharmaceuticals or cosmetics containing at least one excipient together with an agent for maintaining or increasing the content of fibrous structural protein, which is an extracellular matrix of living tissue containing GABA as an active ingredient. In the case, such a composition is not particularly limited in the dosage form, and can be any composition for oral use or external use. External compositions such as eye drops, nasal drops, ear drops, oral medicines (gargles, sprays), suppositories (suppositories, ointments, enemas) are liquid preparations by blending known ingredients that are usually used. , Solid agents, semi-solid agents, etc. can be prepared in various dosage forms according to the purpose of use. Preferred compositions include, for example, lotions, ointments, gels, creams, sprays, patches, powders and the like. For oral administration or ingestion, oral compositions such as tablets, powders, granules, capsules and liquids are preferred. The oral composition may be a sublingual drug (not only tablets, but also sheets and pastes such as wafers), jellies, and drinks in which fine powder is suspended. Various ingredients and manufacturing methods for producing compositions such as pharmaceuticals or cosmetics of various dosage forms including those exemplified above are known in the fields related to the production of pharmaceuticals and cosmetics, and those skilled in the art are required. It can be appropriately selected accordingly. The pharmaceutical composition is not limited to humans, and includes pharmaceutical compositions for mammals such as dogs and cats raised as pets and livestock. Further, the cosmetic composition includes not only cosmetics but also various quasi-drugs under the Pharmaceutical Affairs Law, medicated cosmetics and the like.

The GABA in the present invention is GABA extracted from vegetables, fruits, grains and the like, GABA produced from fermented foods, and GABA produced from organic synthesis.
The vegetables, fruits, and grains mentioned above refer to pumpkin, eggplant, tomato, cucumber, rice, brown rice, malt, soybean, etc., and fermented foods include kimchi, pickles, fermented milk, natto, etc. derived from lactic acid bacteria, yeast, and natto bacteria. Fermented food. It can be obtained by fermenting germ rice, green tea, and rice bran with lactic acid bacteria, and fermenting glutamic acid with lactic acid bacteria. Furthermore, using naturally occurring glutamic acid decarboxylase (GAD), glutamic acid and / or sodium glutamate is used as a raw material. It may be the one obtained by enzymatic conversion, or the one prepared by isolating the bacteria in the fermented food and preparing it in the culture solution.

The fermented solution and the extracted solution described above may be appropriately dried by freeze-drying or spray-drying and pulverized. Although not particularly limited, the GABA content is 0.1% to 30% in the liquid state and 0.1% to 99% in the powder form. The daily intake of GABA for humans is 1 to 3000 mg, preferably 10 mg to 3000 mg. More preferably, it is 20 mg to 1000 mg, and even more preferably 50 mg to 200 mg. If the GABA is less than 10 mg, no effect can be expected, and if it is 3000 mg or more, it becomes difficult to take a single intake.

GABA ingested from outside the body to maintain or increase the elastin content of the living tissue of the present invention increases the elasticity of the skin, reduces the rate of increase of the inelasticity of the skin of the subject, and / or It is intended for people who need to maintain the elasticity of the subject's skin, and is used to improve the elasticity of the skin. Collagen, fibrillin-1, fibrillin-2, and / or fibrillin-5 are the same as elastin above.
It is also intended for people who need to prevent locomotive syndromes such as knee joint disease, tendon and ligament injuries caused by sports, and improve tendon and ligament functions such as strengthening tendons and ligaments. And used to improve ligament function. In addition, it is intended for people with reduced elastin function who are at high risk of vascular diseases and abnormalities due to aging, etc., and is used to keep blood vessels healthy.
GABA may be in the form of food and drink containing abundant amounts of it. The form of the food or drink is not particularly limited, but may be formed into powder, granules, capsules, or tablets. In addition, other forms may be food materials, food additives, syrups, suspending agents, drinks, liquid foods, soft drinks, milk drinks, lactic acid bacteria drinks, functional seasonings, gel-like foods. , Pudding, yogurt, confectionery / cakes, breads, noodles, pasta, chocolate, candy, chewing gum and the like.
An agent for maintaining or increasing the content of fibrous structural protein, which is an extracellular matrix of living tissue, which comprises GABA of the present invention as an active ingredient, alone or as another food additive, etc. In combination with various physiologically acceptable ingredients, it can be used as a food additive for addition to foods such as confectionery, health foods, preserved foods, and processed foods. The food and drink is not limited to humans, and includes feed for mammals such as dogs and cats raised as pets and livestock. In addition to ordinary foods and drinks, the concept of foods and drinks includes beverages, so-called supplements, health foods, enterotrophic foods, special-purpose foods, nutritionally functional foods, foods for specified health uses, and the like.

The daily intake of GABA of the present invention is 10 to 3000 mg, and the food or drink containing GABA as an active ingredient may contain 10 to 90% by weight, preferably 25 to 50% by weight of GABA as an active ingredient. .. The daily intake of the food or drink per adult is in the range of 10 to 5000 mg, preferably 250 to 3500 mg. If the daily intake is less than 10 mg, the effect of maintaining or increasing the content of elastin or the like in the living tissue cannot be obtained, and if the daily intake is 3000 mg or more, it becomes difficult to take a single intake. In addition, the effect produced does not change much even if the amount is more than 5000 mg. The intake amount of the food and drink can be appropriately determined within the above-mentioned range in consideration of the subject's elastin and the like decrease state.
The effective dose of the pharmaceutical composition varies depending on the type and degree of the disease or symptom to be treated or prevented, the condition of the administration target (including age, gender, physical condition, etc.), the dosage form, etc., but is human (body weight 60 kg). The oral dose to (adults) can be in the range of 10 to 5000 mg for the above daily intake.
In the case of an external composition, an external preparation containing GABA of about 1 to 400 mg / ml (0.1 to 40%) is applied 1 to several times a day, although it depends on the area and site of the skin to which it is applied. Can be applied. The application method is not limited to coating, and for example, if the composition is in the form of a liquid, it may be sprayed, and if it is in the form of a film, it may be appropriately selected such as sticking. Since GABA is extremely safe and there is no concern about side effects, there is no problem even if the intake amount or the applied amount exceeds the above range as long as other ingredients are appropriately selected to prepare the composition.

For foods and drinks that effectively enhance the maintenance or increase of the content of elastin and the like in the living tissue of the present invention, it is preferable to take 350 mg per adult per day in 1 to 3 divided doses, but 1050 mg per day after dinner. May be taken at once.

The fibrotic structural protein, which is an extracellular matrix, will be described.
Fibroblasts present in the dermis are involved in the elasticity of the skin. Fibroblasts produce the extracellular matrix type I collagen, hyaluronic acid and elastin.
Elastin plays a role of supporting collagen fibers and is also called an elastic fiber, and exhibits a structure in which the epidermis is stretched and fixed like rubber (Hitoshi Masaki of Non-Patent Document 6, Chapter 1 Biological). Understanding the skin). It has also been reported that UVB irradiation of animal skin causes a change in the microstructure of elastin rather than a decrease in collagen and elastosis at the initial stage of wrinkle formation (Imokawa of Non-Patent Document 7). Collagen, skin aging and functional foods 3. Mechanism of aging / photoaging (wrinkles)). In other words, it is considered that wrinkles and sagging of the skin can be improved and prevented by promoting the biosynthesis of elastin.
The biosynthetic process of elastin will be described with reference to FIG. 12, which is an excerpted and modified drawing from Non-Patent Document 8.
First, the elastin precursors tropoelastin and fibrillin are synthesized intracellularly and released extracellularly. Extracellularly, using fibrillin as a scaffold, tropoelastin aggregates and deposits to form elastic fibers. Fibrillin is a glycoprotein, and there are two types, fibrillin 1 and fibrillin 2.
In addition, mutation of the fibrillin-1 gene reduces the elasticity of connective tissue, resulting in Marfan Syndrome (MFS), which is a disease that causes malformation abnormalities. In addition, mutations in the fibrillin-2 gene cause similar congenital contracture arachnodactynia. In other words, fibrillin is an important component for the elasticity of connective tissue.
Fibrin-5 is a protein also called DANCE, which is secreted from fibroblasts and binds to elastic fibers while still bound to the cells, assembling the constituents and stabilizing the fibrous structure. DANCE gene-deficient mice showed hypoplasia of elastic fibers throughout the body and showed phenotypes such as sagging skin, emphysema, and hardening of arteries as seen in aging. In other words, it is said to be an essential component for elastic fiber synthesis.
In Example 1, it was revealed that GABA has an effect of increasing the mRNA expression levels of tropoelastin, fibrillin-1, 2 and fibrin-5. In addition, in immunostaining of elastin, it was confirmed that the GABA-exposed group produced about twice as much elastin as the control group, so that it may promote the synthesis of elastin not only at the gene level but also at the protein level. It was suggested. Therefore, it was suggested that GABA improves skin elasticity by promoting elastin synthesis in dermal fibroblasts derived from normal human adults.

In addition, type I collagen forms a grid-like fibrous layer in the dermis, supports the epidermis, and gives elasticity to the skin.
However, type I collagen is easily reduced by nutritional deficiency, oxidative stress, ultraviolet rays, etc., and causes wrinkles.
Type I collagen is composed of one α1 chain and two α2 chains. In Example 2, GABA significantly increased the expression of α1 chain mRNA, and α2 chain increased it in a dose-dependent manner, although there was no significant difference.
On the other hand, MMP-1 is an enzyme that decomposes type I collagen, and its production increases due to ultraviolet irradiation and physical aging. It is said that the mixture of soybean peptide and vitamin C promoted the production of both type I collagen and MMP-1 in fibroblasts (Tokudome Y et al. Effects of Soyben and collagen of Non-Patent Document 9).
peptide on collagen synthesis in normal
human dermal fibroblasts. ).
Therefore, in Example 2, when the effect of GABA on MMP-1 mRNA was confirmed, GABA significantly suppressed the expression of MMP-1 mRNA.
From the above, it was suggested that GABA may improve skin elasticity by controlling type I collagen at the genetic level.

In addition, when used in pharmaceuticals, oral administration agents such as powders, granules, capsules, pills and tablets can be mentioned without particular limitation.

GABA as an agent for maintaining or increasing the content of elastin and the like in the living tissue of the present invention can be taken orally as a food additive, as an orally administered preparation, or as a food or drink containing them. Used for

Next, specific examples of the present invention will be described with reference to the following examples, but the present invention is not limited to these examples.

Effect of GABA on elastin synthesis in normal human dermal fibroblasts Previously, when GABA 180 mg / day was ingested for 8 weeks in an open human test, skin elasticity increased compared to the control group (Non-Patent Document 4). ).
After that, when GABA 100 mg / day was ingested for 8 weeks in a double-blind human study, the skin elasticity of the control group decreased during the period, whereas the skin elasticity of the GABA intake group decreased. Not (Non-Patent Document 5).

[Test method]
Normal human dermal fibroblasts (NHDF, AD) were purchased from Kurabo Industries, and DMEM + FBS 0.5% was used as the medium. Samples were lysed in PBS (−) to the desired concentration and exposed to cells.

[Toxicity test]
A cell proliferation test was performed using Cell couting kit-8 (Dojin Chemical Laboratory Co., Ltd.). The cell suspension prepared at 5 × 10 ⁴ Cells / ml was seeded on a 96-well flat-bottomed microplate (FALCON, Becton Dickinson) at a rate of 100 μl / well. As a blank, only the medium was added dropwise at a rate of 100 μl / well. Then, the cells were cultured at 37 ° C. in the presence of 5% carbon dioxide gas. After 24 hours of culturing, PBS (−) was added to the control group, and GABA solution was added to the test group by 10 μl / well. After culturing at 37 ° C. in the presence of 5% carbon dioxide gas for 24 hours, 10 μl / well of Cell couting kit-8 solution was added, and the mixture was reacted at 37 ° C. in the presence of 5% carbon dioxide gas for 1 hour. After completion of the reaction, the absorbance at 450 nm was measured with a microplate reader (PerkinElmer). The values related to cell proliferation were calculated by subtracting the measured values of blank wells from the measured values of wells in which cells were seeded.
In addition, a measurement test of each mRNA expression level was carried out at a concentration at which proliferation did not significantly decrease.

[Real-time PCR]
Primer: The primer shown in Table 2 was used.
Twenty-four hours after the sample was added, the cells were peeled off and mRNA was extracted.
Total RNA was extracted from cells using TRIzol Plus RNA Purification Kit 50preps (Life Technologies). Then, cDNA was synthesized using SuperScriptIII First-Strand Synthesis System for RT-PCR (Life Technologies). Further, the mRNA was prepared by Fast Start Essential DNA Green master (Roche), and the expression level of each mRNA was measured by the real-time-PCR method using Light Cycler Nano. The PCR reaction was initially denatured at 95 ° C. for 600 seconds and then carried out by a 3step Amplification, Melting program.
The mRNA expression level was determined as a ratio to the GAP DH mRNA expression level.

[Immunostaining]
Cells were seeded at ^{20000 cells / cm 2} in a Labtech II chamber slide system 154534JP. GABA 10 μg / ml was exposed, and after 7 days the supernatant was removed and exposed to 10% formalin solution for 15 minutes. The cells were washed 3 times with PBS and blocked with blocking one Histo (nacalai tesque, Japan) for 5 minutes. It was washed 3 times with PBS and permeated with 0.1% Triton X-100 for 5 minutes. The primary antibody polyclonal rabbit anti-alpha elastin (Bio-Rad, America) was added, and after 1 hour, the cells were washed 3 times with PBS. Then, a secondary antibody, gout antibody-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor 488 conjugate (Thermo Fisher, USA) was added and left for 30 minutes. The cells were washed 3 times with PBS, and Nuclear were stained with DAPI (4,6-diamino-2-phenyl iodine) was added to perform nuclear staining and prevention of fading.
The completed slide was observed with a fluorescence microscope.

[result]
1) Figure 2 shows the results of the toxicity test.
As shown in FIG. 2 , GABA of 10 μg / ml or less had no effect on cell proliferation, but 100 μg / ml significantly reduced the proliferation. Therefore, subsequent genetic experiments and immunostaining were conducted at 10 μg / ml or less.
2) The results of real-time PCR are shown in FIGS. 3 to 6.
3) The result of immunostaining is shown in FIG.

[Discussion]
In this study, it was clarified that GABA has an effect of increasing the mRNA expression levels of tropoelastin, fibrillin-1, 2 and fibrillin-5.
In addition, in immunostaining of elastin, it was confirmed that the GABA-exposed group produced about twice as much elastin as the control group, so that it may promote the synthesis of elastin not only at the gene level but also at the protein level. It was suggested.
Therefore, it was suggested that GABA improves skin elasticity by promoting elastin synthesis in dermal fibroblasts derived from normal human adults.

Effect of GABA on type I collagen regulation in normal human dermal fibroblasts
[Test method] Normal human dermal fibroblasts (NHDF) were purchased from Kurabo Industries, and DMEM + FBS 0.5% was used as a medium. Samples were lysed with PBS (−) to the desired concentration and exposed to cells.

[Toxicity test]
A cell proliferation test was performed using Cell couting kit-8 (Dojin Chemical Laboratory Co., Ltd.). The cell suspension prepared at 5 × 10 ⁴ Cells / ml was seeded on a 96-well flat-bottomed microplate (FALCON, Becton Dickinson) at a rate of 100 μl / well. As a blank, only the medium was added dropwise at a rate of 100 μl / well. Then, the cells were cultured at 37 ° C. in the presence of 5% carbon dioxide gas. After 24 hours of culturing, PBS (−) was added to the control group, and GABA solution was added to the test group by 10 μl / well. After culturing at 37 ° C. in the presence of 5% carbon dioxide gas for 24 hours, 10 μl / well of Cell couting kit-8 solution was added, and the mixture was reacted at 37 ° C. in the presence of 5% carbon dioxide gas for 1 hour. After completion of the reaction, the absorbance at 450 nm was measured with a microplate reader (PerkinElmer). The values related to cell proliferation were calculated by subtracting the measured values of blank wells from the measured values of wells in which cells were seeded.
In addition, a measurement test of each mRNA expression level was carried out at a concentration at which proliferation did not significantly decrease.

[Real-time PCR]
Primer: The primer shown in Table 2 was used.
Twenty-four hours after the sample was added, the cells were peeled off and mRNA was extracted. Total RNA was extracted from cells using TRIzol Plus RNA Purification Kit 50preps (Life Technologies). Then, cDNA was synthesized using SuperScriptIII First-Strand Synthesis System for RT-PCR (Life Technologies). It was prepared by FastStart Essential DNA Green master (Roche), and the expression level of each mRNA was measured by the real-time-PCR method using LightCyclor Nano. The PCR reaction was initially denatured at 95 ° C. for 600 seconds and then carried out by a 3step Amplification and Melting program.
The mRNA expression level was determined as a ratio to the GAP DH mRNA expression level.

[result]
1) The results of the toxicity test are shown in FIG.
As shown in FIG. 8, GABA 10 μg / ml or less did not affect the cell proliferation, but 100 μg / ml significantly reduced the proliferation. Therefore, in the subsequent gene experiments, the test was conducted at 10 μg / ml or less.
2) The results of real-time PCR are shown in FIGS. 9 to 11.

[Discussion]
Fibroblasts are involved in skin elasticity and produce type I collagen, hyaluronic acid, and elastin.
Type I collagen forms a grid-like fibrous layer in the dermis, supports the epidermis, and gives elasticity to the skin.
Type I collagen is composed of one α1 chain and two α2 chains. In this study, GABA significantly increased the expression of α1 chain mRNA, and α2 chain increased it in a dose-dependent manner, although there was no significant difference.
On the other hand, MMP-1 is an enzyme that decomposes type I collagen, and its production increases due to ultraviolet irradiation and physical aging. Therefore, when the effect of GABA on MMP-1 mRNA was confirmed, GABA significantly suppressed the expression of MMP-1 mRNA.
From the above, it was suggested that GABA may improve skin elasticity by controlling type I collagen at the genetic level.

While there is a need for measures to maintain or increase the content of elastin in living tissues, attention is focused on the function of maintaining or increasing the content of elastin in living tissues of GABA as a dietary approach. It is expected to have the effect of GABA administration, which is expected to not only improve the elasticity of the skin but also improve the function of tendons and ligaments such as strengthening tendons and ligaments and suppress arteriosclerosis. Cosmetics, pharmaceuticals, foods and feeds It can be used for such purposes.

Claims (5)

Collagen, elastin, fibrillin-1, fibrillin, which are extracellular matrices of these tissues, without promoting the proliferation of fibroblasts, in living tissues consisting of skin or ligaments, which are characterized by containing GABA as an active ingredient. -2, and an agent for promoting the synthesis of fibrillin-5. The agent according to claim 1, further for suppressing the synthesis of matrix metalloproteinase-1 having a collagen-degrading action. The agent according to claim 1 or 2, which is a preparation to be orally administered. The agent according to any one of claims 1 to 3 , wherein the daily intake of GABA is 10 to 3000 mg. The agent according to any one of claims 1 to 4 , which is a food or drink.

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