Use of gamma-aminobutyric acid for preventing and / or improving gum damage caused by cigarette smoke and method for achieving the use

Abstract

The application discloses use of gamma-aminobutyric acid for preventing and / or improving gum damage caused by cigarette smoke and a method for realizing the use, the gamma-aminobutyric acid can effectively prevent and / or improve the gum damage caused by cigarette smoke, and the application range of the gamma-aminobutyric acid is expanded. Further, the gamma-aminobutyric acid can effectively prevent and / or improve gum atrophy and / or gum inflammation caused by cigarette smoke, and the gamma-aminobutyric acid can be applied to oral care products and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of personal care products, in particular to the use of gamma-aminobutyric acid in preventing and / or improving gum damage caused by cigarette smoke and a method for achieving the use. BACKGROUND

[0002] Gums refer to the light red structure close to the neck of the tooth and adjacent alveolar bone, which is composed of stratified squamous epithelium and lamina propria. It is part of the oral mucosa, rich in blood vessels, light red, tough and elastic, and directly connected with the periosteum, so the gums cannot be moved. The condition that the gum margin position retreats to the root side and exposes the tooth root is called gum atrophy. After the gum atrophy, the exposed heel surface is easy to have caries, and after the thin dentin on the heel surface is mechanically ground off, it is easy to have wedge-shaped defects or dentin sensitivity, and even cause pulp hyperemia and degeneration due to long-term stimulation; the recession of the interdental papilla increases the interproximal space, which is easy to cause food impaction and plaque accumulation; gum fissure and thickened gum margin also hinder the removal of plaque, and secondary more severe inflammation and hyperplasia.

[0003] However, the gum damage caused by cigarette smoke is different from other gum damage. Two main phases are found in the whole cigarette smoke, one is tar phase, and the other is gas phase, which is a complex mixture of more than 7000 compounds. Studies have shown that cigarette smoke extract (CSE) has the ability to degrade collagen and can cause alveolar type II epithelial cell damage, nuclear factor-kappa B (NF-κB) activation and increased secretion of tumor necrosis factor (TNF-α). And smoking reduces the binding of fibroblasts and the production of collagen and bone morphogenetic protein, and increases the level of tissue destruction enzymes (MMPs), so the gum damage caused by cigarette smoke is multiple and complex damage.

[0004] The gum damage caused by cigarette smoke is also different from the skin damage. In the cavity, the epithelial cells on the mucosal surface are the first line of defense against environmental harmful stimuli such as cigarette smoke. The tissues such as gums in the oral cavity are directly contacted with cigarette smoke to produce a series of oral diseases. Epidemiological studies have confirmed that smoking is a recognized risk factor for periodontal disease, and in patients with periodontitis, smoking is associated with attachment loss and bone resorption. Smoking also has a negative impact on the response of periodontal tissue to treatment. Studies have found that cigarette smoke affects gum blood flow, cytokine production, cell morphology of connective tissue cells (such as fibroblasts), cell migration, proliferation and attachment, and protein synthesis and secretion. SUMMARY

[0005] In order to effectively protect the gum health and reduce the damage caused by external adverse factors, the present application finds through research that gamma-aminobutyric acid can effectively protect the gums, especially the gums of smokers, thereby completing the present application.

[0006] The specific technical solutions of the present application are as follows:

[0007] 1. Use of gamma-aminobutyric acid for preventing and / or improving gum damage caused by cigarette smoke.

[0008] 2. The use according to item 1, wherein the gum damage comprises gum atrophy and / or gum inflammation.

[0009] 3. The use according to item 1 or 2, wherein the gum damage comprises gum fibroblast damage.

[0010] 4. A method for preventing and / or improving gum damage caused by cigarette smoke, the method comprising administering gamma-aminobutyric acid in the oral cavity.

[0011] 5. The method according to item 4, wherein the gum damage comprises gum atrophy and / or gum inflammation.

[0012] 6. The method according to item 4 or 5, wherein the gum damage comprises gum fibroblast damage.

[0013] Inventive effect

[0014] Gamma-aminobutyric acid can effectively prevent and / or improve gum damage caused by cigarette smoke, thus expanding the application range of gamma-aminobutyric acid.

[0015] Further, gamma-aminobutyric acid can effectively prevent and / or improve gum atrophy and / or gum inflammation caused by cigarette smoke, thus gamma-aminobutyric acid can be applied to oral care products and the like. BRIEF DESCRIPTION OF DRAWINGS

[0016] The accompanying drawings are used to better understand the present application and do not constitute an improper limitation on the present application. Among them:

[0017] Figure 1 Relative content of ROS in cells of different groups in Example 1;

[0018] Figure 2 Relative content of TNF-α of different groups in Example 2;

[0019] Figure 3 Fluorescence photos of collagen type I of different groups in Example 3. DETAILED DESCRIPTION

[0020] The following description of certain examples of the application will be made with reference to which various details are set forth in order to provide a thorough understanding of the examples. They are intended to merely be exemplary and those of ordinary skill in the art will therefore appreciate that various modifications are possible and can be made to the examples described herein without departing from the scope and spirit of the application. Likewise, descriptions of one or preferred implementation alternatives do not imply that other alternatives are not possible. So that the applicant does not lose the right to later claim such alternatives, the applicant has not restricted the application to a single preferred implementation alternative. For the sake of clarity, descriptions of well-known functions and constructions are omitted so as to not unnecessarily obscure the application in detail.

[0021] The present application provides new uses of gamma-aminobutyric acid, and methods for achieving the uses.

[0022] In one aspect, the present application provides uses of gamma-aminobutyric acid for preventing and / or ameliorating gum damage caused by cigarette smoke.

[0023] The gum refers to the light red structure close to the neck of the tooth and adjacent to the alveolar bone, which is composed of stratified squamous epithelium and lamina propria, and is part of the oral mucosa. The gum described in the present application conforms to the general definition in the art.

[0024] The gamma-aminobutyric acid (GABA) refers to gamma-aminobutyric acid, the chemical name of which is 4-aminobutyric acid. It is an amino acid widely existing in vertebrates, plants and microorganisms, and is also an important inhibitory neurotransmitter. GABA was first discovered in mammalian brain extracts by Florey and Robert in 1950. Subsequent studies have shown that GABA can reduce the sensitivity of neurons and prevent nerve cells from overexcitation in mammalian nerves, and has the effects of calming, relaxing and eliminating nervous tension. In recent years, studies have also shown that GABA can quickly penetrate the skin, release wrinkles, fade fine lines, and enhance the relaxation function of muscles themselves, thereby achieving the effect of stress relief and beauty.

[0025] Cigarette smoke can cause gum damage. The present application has found that gamma-aminobutyric acid can effectively prevent and / or ameliorate gum damage caused by cigarette smoke, and thus can be used in products for preventing and / or ameliorating gum damage caused by cigarette smoke, for example, it can be used in personal care products to alleviate gum damage caused by cigarette smoke. The personal care products can be oral preparations or external preparations. The present application does not limit the specific type of preparation, and those skilled in the art can select from the prior art according to the needs of use, for example, the oral preparations can be powders, granules, capsules, liquid preparations, suspensions, etc., and the external preparations can be patches, sprays, creams, liquid application preparations, etc.

[0026] In some embodiments, the present application provides uses of gamma-aminobutyric acid in the preparation of products / personal care products for preventing and / or ameliorating gum damage caused by cigarette smoke.

[0027] The dosage of gamma-aminobutyric acid in the product / personal care product is not limited in the present application, and can be selected by those skilled in the art according to the conventional dosage of gamma-aminobutyric acid, for example, can be 0.01% to 10% by weight, and in some embodiments, the concentration of gamma-aminobutyric acid is 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, etc.

[0028] In some embodiments, the product comprises mouthwash, toothpaste, powder, lozenge or oral film. The preparation method of the mouthwash, toothpaste, powder, lozenge or oral film is not limited in the present application, and can be prepared by conventional preparation method in the art.

[0029] In some embodiments, the product further comprises one or more than two of antibacterial agent, anticaries agent, antiallergic agent, anticalculus agent, anti-inflammatory agent, whitening agent, moisturizing agent.

[0030] For example, the gamma-aminobutyric acid can be used in the product together with any one or any two or any three or any four of the antibacterial agent, anticaries agent, antiallergic agent, anticalculus agent, anti-inflammatory agent, whitening agent, moisturizing agent, or all of the above.

[0031] In the present application, the antibacterial agent is not limited, and can be conventional antibacterial agent in the art, for example, the antibacterial agent can be stannous chloride, tetrahydrocurcumin, triclosan, etc.

[0032] In the present application, the anticaries agent is not limited, and can be conventional anticaries agent in the art, for example, the anticaries agent can be calcium phosphate, sodium trimetaphosphate, magnesium glycerophosphate, lactophosphate, etc.

[0033] In the present application, the antiallergic agent is not limited, and can be conventional antiallergic agent in the art, for example, the antiallergic agent can be dipotassium glycyrrhizinate, potassium fluoride, potassium chloride, etc.

[0034] In the present application, the anticalculus agent is not limited, and can be conventional anticalculus agent in the art, for example, the anticalculus agent can be pyrophosphate, tripolyphosphate, citrate, etc.

[0035] In the present application, the anti-inflammatory agent is not limited, and can be conventional anti-inflammatory agent in the art, for example, the anti-inflammatory agent can be metronidazole, tinidazole, ornidazole, etc.

[0036] In this application, no restrictions are placed on the whitening agent, which can be a whitening agent commonly used in the art, such as peroxide bleaching agent, papain, glucose oxidase, etc.

[0037] In this application, no restrictions are placed on the use of moisturizers, which can be commonly used moisturizers in the art, such as glycerin, propylene glycol, sorbitol, xylitol, hyaluronic acid, etc.

[0038] In some embodiments, the personal care product further includes one or more of a pH adjuster, a thickener, and an osmotic pressure adjuster.

[0039] The pH adjuster can be an acid, alkali, inorganic salt, etc., available in the art; the thickener can be hydroxyethyl cellulose, carboxymethyl cellulose and its salts, xanthan gum, etc.; the osmotic pressure adjuster can be an inorganic salt, etc., available in the art.

[0040] Furthermore, this application provides the use of γ-aminobutyric acid for non-therapeutic purposes in preventing and / or improving gingival damage caused by cigarette smoke.

[0041] The so-called non-therapeutic purpose means that it is not for the diagnosis or treatment of the disease, but can be for the purpose of prevention, improvement, relief, reduction, or slowing down, etc.

[0042] Furthermore, this application provides a non-therapeutic method for preventing and / or improving gingival damage caused by cigarette smoke, the method comprising applying γ-aminobutyric acid (GABA) into the oral cavity. The application method can be any method within the art, including but not limited to application, sublingual administration, nebulization, oral administration, injection, etc.

[0043] This application does not limit the dosage of γ-aminobutyric acid (GABA) administered into the cavity. Those skilled in the art can select the dosage based on the conventional dosage of GABA, for example, it can be 0.01% to 10% by weight. In some embodiments, the concentration of GABA is 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, etc.

[0044] The gingival damage described in this application includes, but is not limited to, one or more of the following: gingival bleeding, gingival oxidation, gingival pain, gingival swelling, gingival recession, and gingival inflammation. In one specific embodiment, the gingival damage includes gingival recession and / or gingival inflammation.

[0045] The gingival injury described in this application includes, but is not limited to, one or more of the following: fibroblast injury, lymphocyte injury, plasma cell injury, and macrophage injury in the gingiva. In one specific embodiment, the gingival injury includes gingival fibroblast injury.

[0046] This application provides the use of γ-aminobutyric acid in the preparation of products for preventing or inhibiting gingival damage caused by smoking.

[0047] In some embodiments of this application, γ-aminobutyric acid is used as the sole active ingredient in the preparation of products / personal care products for preventing or improving cell damage caused by smoking / cigarette smoke.

[0048] In some embodiments of this application, γ-aminobutyric acid (GABA) is used as the sole active ingredient for the prevention and / or improvement of gingival damage caused by cigarette smoke.

[0049] In some embodiments of this application, γ-aminobutyric acid is used as the sole active ingredient for non-therapeutic purposes to prevent and / or improve gingival damage caused by cigarette smoke.

[0050] This application utilizes γ-aminobutyric acid (GABA) in the preparation of personal care products for the prevention and / or improvement of gingival damage caused by cigarette smoke, or for non-therapeutic purposes, to significantly inhibit the increase in reactive oxygen species (ROS) content in gingival fibroblasts after oxidative stimulation by cigarette smoke extracts. Compared to cells not treated with GABA, the results showed a highly statistically significant difference, indicating that GABA can inhibit gingival oxidative damage induced by cigarette smoke extracts. Simultaneously, it can also inhibit the increase in tumor necrosis factor (TNF) content after stimulation by cigarette smoke extracts, again showing a highly statistically significant difference compared to cells not treated with GABA. This demonstrates that GABA can inhibit the increase in gingival inflammatory factor content induced by cigarette smoke extracts and alleviate gingival inflammation.

[0051] The novel uses of GABA and the methods for achieving these uses provided in this application can improve oral health, alleviate oral discomfort caused by smoking, prevent the formation of oral diseases such as oral tumors, and protect the oral cavity, thus having broad application prospects.

[0052] Example

[0053] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0054] Experimental Example 1: The effect of GABA on ROS generation induced by CSE

[0055] Weigh out γ-aminobutyric acid (hereinafter referred to as GABA, purchased from Bloomage Biotechnology), mix and dissolve it in HGF-1 complete cell culture medium (containing 10% FBS and 1% penicillin and streptomycin), and prepare 10 μg / mL and 100 μg / mL GABA solutions respectively.

[0056] Preparation of Cigarette Smoke Extract (CSE): A gas collecting bottle was sterilized and filled with HGF-1 cell culture medium. A cigarette of a certain brand was fixed to the long end of the collecting bottle, and the short end of the tube was connected to a vacuum pump. The vacuum pump was turned on and the cigarette was lit, allowing the cigarette smoke to enter the culture medium from the long end of the tube. Cigarette smoke extract was prepared in this manner.

[0057] Test method: Prepare a 10 μM DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) solution using phenol red-free medium.

[0058] HGF-1 cells were seeded at a density of 30,000 cells / well in 96-well plates and cultured at 37°C and 5% CO2. After 24 hours of cell attachment, blank, control, and experimental groups containing 1, 10, and 100 μg / mL LGABA were established (100 μL / well). After 24 hours of incubation, 200 μL of 5% CSE solution was added to each well (blank and control groups only received culture medium). After 3 hours of incubation, the supernatant was aspirated, and the cells were washed with DPBS. 200 μL of 10 μM DCFH-DA working solution was added, and the cells were incubated for 30 minutes, followed by washing with DPBS. Fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

[0059] Blank group: No GABA is added, and no CSE processing is performed.

[0060] Control group: No GABA added, treated with CSE.

[0061] Experimental group: Add 1, 10, or 100 μg / mL GABA and perform CSE treatment.

[0062] Experimental results are as follows Figure 1 As shown, according to Figure 1Data analysis, comparing the blank group and the control group, showed that CSE significantly stimulated an increase in ROS content in cells. Simultaneously, observation of the experimental and control groups revealed that 10 μg / mL and 100 μg / mL GABA inhibited the increase in ROS content after CSE oxidative stimulation, indicating that it can inhibit oxidative damage in HGF-1 cells, and the results were statistically significant.

[0063] The data was analyzed using Graphpad Prism statistical software. Pairwise comparisons were performed using t-tests. *P < 0.05 indicated a statistically significant difference, **P < 0.01 indicated a highly significant difference, and ***P < 0.001 indicated an extremely significant difference.

[0064] Experimental Example 2: The effect of GABA on TNF-α production induced by CSE

[0065] Inflammation and tumors are closely related. Tumor necrosis factor-α (TNF-α) is one of the main inflammatory cytokines involved in inflammation and is highly expressed in various chronic inflammatory diseases and tumor environments. TNF-α can induce the release of other inflammatory factors, amplifying the inflammatory response, and can also directly induce epithelial cell carcinogenesis and promote tumorigenesis by releasing oxygen and nitrogen mediators. This experiment uses the effect of GABA on TNF-α production induced by CSE to verify the influence of GABA on cigarette-induced inflammation.

[0066] Weigh out γ-aminobutyric acid (hereinafter referred to as GABA, purchased from Bloomage Biotechnology), mix and dissolve it in HGF-1 complete cell culture medium (containing 10% FBS and 1% penicillin and streptomycin), and prepare GABA solutions of 1 μg / mL, 10 μg / mL and 100 μg / mL respectively.

[0067] Preparation of Cigarette Smoke Extract (CSE): A gas collecting bottle was sterilized and filled with HGF-1 cell culture medium. A cigarette of a certain brand was fixed to the long end of the collecting bottle, and the short end of the tube was connected to a vacuum pump. The vacuum pump was turned on and the cigarette was lit, allowing the cigarette smoke to enter the culture medium from the long end of the tube. Cigarette smoke extract was prepared in this manner.

[0068] Assay Method: HGF-1 cells were seeded at a density of 30,000 cells / well in 96-well plates and cultured at 37°C with 5% CO2. After 24 hours of cell attachment, blank, control, and experimental groups containing 1, 10, and 100 μg / mL LGABA were established (100 μL / well). After 24 hours of incubation, 200 μL of 5% CSE solution was added to each well (blank and control groups only received culture medium) for 24 hours. The supernatant was collected, and the secretion of TNF-α was detected using an ELISA kit. Simultaneously with the ELISA assay, cell lysis buffer was added to the 96-well plate and centrifuged. The supernatant was used for BCA protein quantification. After homogenization, the relative TNF-α content was expressed as a percentage (%), with the control group as the baseline.

[0069] Blank group: No GABA is added, and no CSE processing is performed.

[0070] Control group: No GABA added, treated in a CSE incubator.

[0071] Experimental group: Add 1, 10, or 100 μg / mL GABA and perform CSE treatment.

[0072] Experimental results are as follows Figure 2 As shown, according to Figure 2 Data analysis, comparing the blank group and the control group, showed that CSE significantly stimulated the increase of TNF-α content in cells. Simultaneously, observation of the experimental group and the control group revealed that 1 μg / mL, 10 μg / mL, and 100 μg / mL of GABA inhibited the increase of TNF-α content after CSE stimulation, indicating that it can suppress inflammatory stimulation, and the results were statistically significant.

[0073] The data was analyzed using Graphpad Prism statistical software. Pairwise comparisons were performed using t-tests. *P<0.05 indicates a statistically significant difference, **P<0.01 indicates a statistically significant difference, and ***P<0.001 indicates an extremely significant statistical difference.

[0074] Experimental Example 3: Effect of GABA on Type I Collagen Production under CSE Induction

[0075] The tissues that make up the gums and tooth roots are mostly composed of collagen. Loss of collagen in the gums easily leads to dental diseases such as cavities, gingival recession, periodontal disease, loose teeth, tooth loss, pain, sensitivity, and weakened biting power. Furthermore, collagen loss slows down the renewal rate of new cells in the gum tissue, causing pigments to deposit on the tooth surface, resulting in yellowing and blackening of the teeth. This experiment uses the effect of GABA on type I collagen expression induced by CSE to verify the use of GABA in preventing and / or improving gingival damage caused by cigarette smoke.

[0076] HGF-1 cells were cultured in DMEM medium in T75 culture flasks until the cell density reached approximately 80%. They were then seeded into 24-well plates with coverslips and cultured at 37°C and 5% CO2. After 24 hours, the supernatant was aspirated. Experimental groups were incubated with 100 μg / mL LGABA and 5% CSE. Control and blank groups were also provided. Cells were incubated at 37°C and 5% CO2 for 72 hours. The supernatant was aspirated, and the cells were washed twice. Cells were then fixed with ice-cold methanol at -20°C. Type I collagen primary antibody was added, and the cells were incubated overnight at 4°C. The next day, the cells were washed three times with PBS, and the corresponding secondary antibody was added. Cells were then incubated at room temperature in the dark for 1.5 hours. Nuclear staining with DAPI and mounting were performed. The cells were then observed and photographed under a fluorescence microscope. Finally, the fluorescence data of the photographs were semi-quantitatively analyzed using ImageJ software.

[0077] Blank group: No GABA is added, and no CSE processing is performed.

[0078] Control group: No GABA added, treated with CSE.

[0079] Experimental group: Add 100 μg / mL GABA and perform CSE treatment.

[0080] Experimental results are as follows Figure 3 As shown, according to Figure 3 Analysis showed that, compared to the control group, the fluorescence intensity of type I collagen (fluorescent area in the above figure) decreased after CSE stimulation, indicating that CSE treatment affected the content of type I collagen. Meanwhile, the addition of 100 μg / mL GABA to the experimental group significantly enhanced the expression level of type I collagen, resulting in stronger fluorescence. Therefore, this indicates that GABA effectively inhibits the reduced expression of type I collagen caused by CSE and has a strong anti-smoking effect.

[0081] Although the embodiments of this application have been described above in conjunction with the specific embodiments described, this application is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Those skilled in the art can make many other forms based on the teachings of this specification and without departing from the scope of protection of the claims of this application, and these are all within the scope of protection of this application.

Claims

1. Use of γ-aminobutyric acid in the preparation of products for the prevention and / or improvement of gingival damage caused by smoking / cigarette smoke.

2. The use according to claim 1, wherein the gingival injury includes gingival recession and / or gingival inflammation.

3. The use according to claim 1 or 2, wherein the gingival injury includes gingival fibroblast injury.

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