Use of ergothioneine, composition comprising same, and use thereof

Abstract

Use of ergothioneine, a composition comprising same, and use thereof. As a fibronectin (FN) inhibitor and / or an α-smooth muscle actin (α-SMA) inhibitor, ergothioneine can effectively inhibit the up-regulated expression of fibronectin and α-smooth muscle actin, thereby effectively inhibiting the process of skin cell fibrosis, and providing a new inhibitor active ingredient for skin cell fibrosis.

Description

Application of ergothioneine, composition containing ergothioneine and application thereof

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 28, 2023, with application number "202311849623.1" and invention name "Application of ergothioneine, compositions containing it and their applications", the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to the field of skin health, and in particular to the application of ergothioneine, a composition containing the same, and the application thereof. Background Art

[0003] Skin fibrosis is a condition characterized by abnormal proliferation and deposition of the extracellular matrix (ECM) in skin tissue, triggered by various internal and external factors, such as trauma, infection, circulatory disorders, and immune responses. Broadly speaking, nearly all skin-related abnormalities are associated with fibrosis. Tissue fibrosis allows for the repair and healing of tissue trauma, serving as the body's protective response against trauma. Broadly speaking, without fibrosis, there is no repair, so fibrosis has a very positive physiological significance. However, fibrotic repair is ultimately compensatory; it does not completely restore the original morphology, structure, and function of damaged tissue through regeneration. Consequently, fibrosis can lead to partial loss of normal skin function. Localized fibrosis, such as hypertrophic scars, is characterized by erythematous, raised, and itchy lesions on the healing skin. In addition to itching and pain, it can also cause cosmetic and functional issues, such as color mismatch, stiffness, and rough texture, which can impair appearance and cause psychological distress.

[0004] Drugs targeting skin fibrosis also need to be developed. For example, although troglitazone can inhibit skin fibrosis, it has certain toxic side effects. Therefore, finding a substance with little or no toxic side effects to achieve the effect of inhibiting skin fibrosis is still an urgent problem to be solved in this field. Summary of the Invention

[0005] The purpose of this application is to provide a skin fibrosis inhibitor component with high safety.

[0006] In one aspect, the present application provides the use of ergothioneine as a fibronectin (FN) inhibitor and / or an α-smooth muscle actin (α-SMA) inhibitor.

[0007] Furthermore, the ergothioneine is used to treat and / or prevent diseases and / or symptoms associated with excessive deposition of fibronectin and / or α-smooth muscle actin.

[0008] On the other hand, the present application also provides the use of ergothioneine as a skin fibrosis inhibitor.

[0009] Furthermore, the skin fibrosis includes one or more of rough skin, stretch marks, scleroderma and diseases with scleroderma-like changes, hypertrophic scars, keloid scars, hypertrophic scars, depressed scars, fibroids and / or graft-versus-host disease.

[0010] Optionally, the concentration of the ergothioneine is 0.01-10000 μg / mL.

[0011] More preferably, the concentration of the ergothioneine is 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 200 μg / mL, 30 0 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL, 2000 μg / mL, 3000 μg / mL, 4000 μg / mL, 5000 μg / mL, 6000 μg / mL, 7000 μg / mL, 8000 μg / mL, 9000 μg / mL, 10000 μg / mL, and any value therein.

[0012] This application demonstrates that ergothioneine effectively inhibits the upregulation of fibronectin (FN) and α-smooth muscle actin (α-SMA) expression while taking safety into consideration, thereby inhibiting the process of skin fibrosis.

[0013] In an optional embodiment, taking fibroblasts as an example, transforming growth factor β1 (TGF-β1) is used to treat the model, and ergothioneine at a concentration of 1-10000 μg / mL can effectively inhibit the upregulation of fibronectin (FN) and α-smooth muscle actin (α-SMA) in fibroblasts (HSF) caused by the increase of transforming growth factor β1 (TGF-β1), thereby inhibiting the process of skin cell fibrosis.

[0014] On the other hand, the present application also provides a composition for inhibiting the expression of fibronectin (FN) and / or α-smooth muscle actin (α-SMA), wherein the composition comprises ergothioneine.

[0015] Furthermore, the composition further comprises other fibronectin inhibitors, α-smooth muscle actin inhibitors and / or skin fibrosis inhibitors.

[0016] The other fibronectin inhibitors include one or more of platelet-derived growth factor (PDGF), staurosporine, and interferon.

[0017] The α-smooth muscle actin inhibitor includes one or more of PUR4 and RGDS peptides.

[0018] The other skin fibrosis inhibitors include one or more of antihistamine preparations, glucocorticoids, retinoic acid, D-penicillamine, β-aminopropionitrile, vitamin C, colchicine, estrogen / progesterone, and tumor necrosis factor.

[0019] Optionally, the concentration of ergothioneine in the composition is 0.01-10000 μg / mL.

[0020] More preferably, the concentration of the ergothioneine is 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 200 μg / mL, 30 0 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL, 2000 μg / mL, 3000 μg / mL, 4000 μg / mL, 5000 μg / mL, 6000 μg / mL, 7000 μg / mL, 8000 μg / mL, 9000 μg / mL, 10000 μg / mL, and any value therein.

[0021] Optionally, acceptable excipients may be added to the composition, and the excipients may be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.

[0022] The composition is an oral preparation, a topical preparation, or a combination of an oral preparation and a topical preparation, for example, ergothioneine is contained in the topical preparation, and other inhibitors are contained in the oral preparation; or, ergothioneine is contained in the oral preparation, and other inhibitors are contained in the topical preparation; or, ergothioneine and other inhibitors are contained in the topical preparation at the same time; or, ergothioneine and other inhibitors are contained in the oral preparation.

[0023] On the other hand, the present application also provides use of the above composition in inhibiting the expression of fibronectin (FN) and / or α-smooth muscle actin (α-SMA).

[0024] Furthermore, the composition is used in treating and / or preventing diseases and / or symptoms associated with excessive deposition of fibronectin and / or α-smooth muscle actin.

[0025] Furthermore, the ergothioneine is used to treat and / or prevent diseases and / or symptoms associated with excessive deposition of fibronectin and / or α-smooth muscle actin fibronectin.

[0026] On the other hand, the present application also provides use of the above composition in inhibiting or alleviating skin fibrosis.

[0027] Furthermore, the skin fibrosis includes one or more of rough skin, stretch marks, scleroderma and diseases with scleroderma-like changes, hypertrophic scars, keloid scars, hypertrophic scars, depressed scars, fibroids and / or graft-versus-host disease.

[0028] On the other hand, the present application also provides the use of the composition in the preparation of skin care products and / or medicines.

[0029] Furthermore, the skin care product is a skin care product that inhibits or alleviates skin fibrosis, and the medicine is a medicine that inhibits or alleviates skin fibrosis.

[0030] Furthermore, the skin fibrosis includes one or more of rough skin, stretch marks, scleroderma and diseases with scleroderma-like changes, hypertrophic scars, keloid scars, hypertrophic scars, depressed scars, fibroids and / or graft-versus-host disease.

[0031] When used in skin care products, it mainly plays the role of preventing, alleviating and improving skin fibrosis, rather than treating it. When used in medicines, it can be used as a beneficial ingredient to play a positive role in treating skin fibrosis.

[0032] This application has the following beneficial effects:

[0033] In the present application, it was found that ergothioneine can inhibit the expression of fibronectin (FN) and α-smooth muscle actin (α-SMA);

[0034] Furthermore, ergothioneine, as a cosmetic raw material with little or no toxic side effects, can effectively inhibit the expression of fibronectin (FN) and α-smooth muscle actin (α-SMA) while taking safety into consideration, thereby helping to prevent and / or alleviate the progression of skin fibrosis, providing a new active ingredient for improving skin fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0036] FIG1 is a graph showing the HSF cytotoxicity of samples at different concentrations;

[0037] Figure 2 is an electrophoretic diagram of α-SMA protein content;

[0038] Figure 3 is a grayscale analysis of α-SMA protein content;

[0039] FIG4 is an electrophoretic diagram of fibronectin content;

[0040] FIG5 is a grayscale analysis diagram of fibronectin content;

[0041] FIG6 is a graph showing fibronectin mRNA expression levels. DETAILED DESCRIPTION

[0042] In order to more clearly illustrate the overall concept of the present application, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present application. However, it is obvious to those skilled in the art that the present application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present application, some technical features well known in the art are not described.

[0043] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0044] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0045] The reagents and instruments involved in the following examples are shown in Table 1.

[0046] Table 1

[0047] In addition, the “water” mentioned in this application includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0048] In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight.

[0049] Example 1 Cytotoxicity Test

[0050] 1. HSF cell culture: Resuscitate HSF cells and inoculate them into culture flasks. Subculture them when they grow to 80%-90% of their normal size. Inoculate them when the cells are growing well.

[0051] 2. Sample processing: Sterilize the sample by filtering it through a 0.22 microporous filter membrane, and perform gradient dilution with serum-free medium according to the concentration of 10000 μg / mL, 1000 μg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL.

[0052] 3. Cytotoxicity test: HSF cells were cultured at 10 4 Cells were seeded into 96-well plates at a density of 100 cells / well and incubated in complete medium at 5% CO2 and 37°C. After 24 hours, the complete medium was removed. The blank control group was incubated with serum-free medium, and the sample group was incubated with gradient dilutions of the test sample (4 replicate wells for each concentration) at 5% CO2 and 37°C for 24 hours. CCK8 detection reagent was added according to the instructions. After incubation for 2 hours, the cells were detected using a microplate reader (450 nm wavelength) to calculate cell viability. Cell viability (%) = A 450 (Sample Group) / A 450 (blank control group)×100%.

[0053] The experimental group with cell viability ≥ 90% and no significant difference in cell morphology from the blank control group was considered to have no obvious cytotoxicity, and the concentration with no cytotoxicity was selected for the next test.

[0054] 4. Test results

[0055] Table 2 Cell viability

[0056] The cytotoxicity experiment used HSF cells as experimental cells. The experimental results are shown in Table 2 and Figure 1. Figure 1 was obtained by plotting Table 2. Among them, ergothioneine did not show obvious cytotoxicity at the administration concentration of 0.01-10000 μg / mL.

[0057] Embodiment 2 thioneine suppresses skin fibrosis test

[0058] When fibrosis reaches a severe level, hypertrophic keloids form. Gene chip analysis has shown that scar tissue exhibits altered expression of multiple gene classes compared to normal skin, including oncogenes and tumor suppressor genes, ion channels and transport proteins, cell cycle proteins and apoptosis proteins, and cytoskeletal and motor proteins. Among these, changes in the expression of cytoskeletal and motor proteins are the most representative. Among these proteins, α-smooth muscle actin (α-SMA) is a particularly prominent example. α-SMA is barely expressed in normal skin, but is expressed at high and prolonged levels in scar tissue. α-SMA forms characteristic filamentous structures in fibroblasts.

[0059] Transforming growth factor β1 (TGF-β1) is a major mediator of scarring, persistently present and overexpressed in hypertrophic scars. Following TGF-β1 stimulation, fibroblasts are activated and undergo a phenotypic transformation into myofibroblasts, key effector cells in the fibrotic state. One of the most prominent features of this phenotypic transformation is the increased production of α-SMA. Treatment of normal skin fibroblasts with TGF-β1 can establish a cell model of skin fibrosis. Troglitazone inhibits α-SMA expression in this cell model and serves as a positive control for inhibiting skin fibrosis. Increased α-SMA levels activate downstream signaling pathways, promoting the expression of other extracellular matrix proteins, including fibronectin. Decreased α-SMA expression is associated with a subsequent decrease in fibronectin expression.

[0060] 1. HSF cell culture: Resuscitate HSF cells and inoculate them into culture flasks. Subculture them when they grow to 80%-90% of their normal size. Inoculate them when the cells are growing well.

[0061] 2. Sample processing: Sterilize the sample by filtering it through a 0.22 microporous filter membrane, and dilute it with serum-free culture medium at concentrations of 1000 μg / mL, 40 μg / mL, and 8 μg / mL.

[0062] 3. Detection of α-smooth muscle actin (α-SMA) and fibronectin (FN) content: HSF cells were cultured at a rate of 5×10 5Cells were seeded into 6-well plates at a density of 100 cells / well and cultured in complete medium at 5% CO2 and 37°C. After 24 hours, the complete medium was removed and blank medium was added. After starvation for 24 hours, 200 μL of culture medium solution was added to each well (the blank group was serum-free medium, the model group was serum-free medium containing 10 ng / mL TGF-β1, the positive control group was serum-free medium containing 15 μM troglitazone + 10 ng / mL TGF-β1, and the sample group was serum-free medium containing three concentrations of the test sample + 10 ng / mL TGF-β1). After incubation for 24 hours at 5% CO2 and 37°C, the supernatant was discarded, washed with PBS, and 0.5 mL of RAPA was added to each well to lyse the cells on ice for 30 minutes. Centrifuged at 12,000 × rpm at 4°C for 15 minutes, the supernatant was collected, and the protein concentration of the supernatant was measured using a microplate reader. After quantitative dilution, the cells were added to the loading buffer and boiled for 5 minutes. Prepare a 10% polyacrylamide gel for loading, followed by electrophoresis and membrane transfer. After incubation with 0.5% skim milk powder, incubate with the primary antibody overnight. After washing, incubate with the secondary antibody for 2 hours at room temperature. After washing, add developer and develop in a chemiluminescence analyzer. Semi-quantitative analysis was performed using ImageJ.

[0063] 4. Fibronectin (FN) gene expression level detection: HSF cells were cultured at a rate of 5×10 5 Cells were seeded into 6-well plates at a density of 100 μl / well and cultured in complete medium at 5% CO2 and 37°C. After 24 hours, the complete medium was removed and blank medium was added for starvation for 24 hours. 200 μL of culture medium solution was added to each well (the blank group was serum-free medium, the model group was serum-free medium containing 10 ng / mL TGF-β1, the positive control group was serum-free medium containing 15 μM troglitazone + 10 ng / mL TGF-β1, and the sample group was serum-free medium containing 3 concentrations of the test sample + 10 ng / mL TGF-β1). After incubation for 24 hours at 5% CO2 and 37°C, the supernatant was discarded and the cells were collected after washing with PBS. RNA was extracted from each group according to the instructions of the RNA extraction kit, reverse transcription was performed according to the reverse transcription kit, and qPCR amplification was performed according to the qPCR kit. Three replicate wells were tested for each sample.

[0064] 5. Test results

[0065] 1) Protein content detection

[0066] α-SMA protein content detection:

[0067] Table 3 Relative expression of α-SMA protein Note: *: p < 0.05 (compared with the model group), **: p < 0.01 (compared with the model group), ***: p < 0.0001 (compared with the model group), ##: p < 0.01 (compared with the blank group)

[0068] The results are shown in Table 3 and Figures 2-3. Figure 3 was obtained by plotting the data in Table 3. Among them, the protein content of the modeling group was significantly higher than that of the blank group, proving that the modeling of this experiment was successful. The protein content of the positive control group was significantly lower than that of the modeling group, proving that this experimental method is effective. The α-SMA protein content of each concentration sample group was significantly lower than that of the modeling group. Therefore, at the protein expression level, ergothioneine can inhibit the increased expression of α-SMA in fibroblasts caused by TGF-β1.

[0069] Fibronectin (FN) content detection:

[0070] Table 4 Relative expression of fibronectin Note: *: **: p < 0.01 (compared with the model group), ***: p < 0.0001 (compared with the model group), ###: p < 0.001 (compared with the blank group)

[0071] The results are shown in Table 4 and Figures 4-5. Figure 5 is obtained by plotting the data in Table 4. Among them, the protein content of the modeling group was significantly higher than that of the blank group, proving that the modeling of this experiment was successful. The protein content of the positive control group was significantly lower than that of the modeling group, proving that this test method is effective. The fibronectin content of each concentration sample group was significantly lower than that of the modeling group. Therefore, at the protein expression level, ergothioneine can inhibit the increase in fibronectin expression of fibroblasts caused by TGF-β1.

[0072] 2) Gene expression level detection

[0073] Fibronectin (FN) mRNA expression level:

[0074] Table 5 Relative levels of FN mRNA Note: ***: p < 0.0001 (compared with the model group), ###: p < 0.001 (compared with the blank group)

[0075] As shown in Table 5 and Figure 6, Fig. 6 is obtained by Table 5 data mapping, and wherein, the mRNA level of modeling group (TGF-β1) is significantly higher than that of blank group (Control), proving that this experimental modeling is successful. The mRNA level of positive control group (TGF-β1+ troglitazone) is significantly lowered than modeling group, proving that this experimental method is effective. Fibronectin (FN) mRNA expression level of each concentration sample group is significantly lowered than modeling group, therefore, at the gene transcription level, thioneine can suppress fibronectin (FN) mRNA level of fibroblast (HSF) that TGF-β1 causes and raise.

[0076] 6. Conclusion

[0077] In summary, it has been demonstrated in the present application that ergothioneine can suppress skin fibrosis. Further, it has been demonstrated in the present application that the ergothioneine using a concentration of 8-1000 μg / mL can, while taking safety into account, effectively suppress the expression of fibroblast (HSF) fibronectin (FN) and alpha-smooth muscle actin (α-SMA) and increase, thereby effectively suppressing the process of skin cell fibrosis.

[0078] Example 3 Application of thioneine in suppressing skin fibrosis products

[0079] External application powder for traumatic injury:

[0080] In the present embodiment, a traumatic injury external application powder containing ergothioneine is provided. The ingredients and dosage of the traumatic injury external application powder are shown in Table 6.

[0081] Table 6

[0082] The preparation method of the above-mentioned external application powder for traumatic injuries containing thioneine is as follows: each raw material fine powder in Table 6 is fully mixed. Using method is: the powder is added with water or white vinegar etc. and is blended into a paste, and is applied to swollen and non-traumatic skin.

[0083] Common cosmetics to prevent scars - skin care water:

[0084] In the present embodiment, a skin care water containing ergothioneine is provided. The composition and dosage of the skin care water are shown in Table 7.

[0085] Table 7

[0086] The preparation method of the above-mentioned skin care water containing thioneine is as follows: fully stir the raw materials in Table 7 in water and dissolve them completely.

[0087] Dressings used on burns / scalds, after medical cosmetic procedures, or on surgical skin wounds:

[0088] In the present embodiment, a dressing containing ergothioneine is provided. The ingredients and dosage of the dressing are shown in Table 8.

[0089] Table 8

[0090] The preparation method of the above-mentioned dressing containing ergothioneine is as follows: fully stir the raw materials in Table 8 in water, dissolve them completely, and thoroughly sterilize them. Usage: Use alone, apply to damaged skin, or use in combination with membrane cloth, gauze, etc.

[0091] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. Use of ergothioneine as a fibronectin (FN) inhibitor and / or an α-smooth muscle actin (α-SMA) inhibitor.

2. The application according to claim 1, characterized in that, The ergothioneine is used for treating and / or preventing diseases and / or symptoms associated with excessive deposition of fibronectin and / or α-smooth muscle actin fibronectin.

3. The application according to claim 1, wherein The use of ergothioneine as a skin fibrosis inhibitor; Preferably, the skin fibrosis includes one or more of rough skin texture, stretch marks, scleroderma and diseases with scleroderma-like changes, hypertrophic scars, keloid scars, hypertrophic scars, depressed scars, fibromas, and / or graft-versus-host disease.

4. A composition for inhibiting the expression of fibronectin (FN) and / or alpha-smooth muscle actin (α-SMA), characterized in that, The composition includes ergothioneine.

5. The composition according to claim 4, wherein the composition further comprises other fibronectin inhibitors, α-smooth muscle actin inhibitors, and / or skin fibrosis inhibitors.

6. The composition according to claim 5, wherein the other fibronectin inhibitors include one or more of platelet-derived growth factor (PDGF), staurosporine, and interferon; The α-smooth muscle actin inhibitors include one or more of PUR4 and RGDS peptides; The other skin fibrosis inhibitors include one or more of antihistamine preparations, glucocorticoids, retinoids, D-penicillamine, β-aminopropionitrile, vitamin C, colchicine, estrogen / progesterone, and tumor necrosis factor.

7. Use of the composition according to any one of claims 4-6 in inhibiting the expression of fibronectin (FN) and / or α-smooth muscle actin (α-SMA).

8. The application according to claim 7, wherein The use of the composition in treating and / or preventing diseases and / or symptoms associated with excessive deposition of fibronectin and / or α-smooth muscle actin fibronectin.

9. The application according to claim 8, wherein The use of the composition in inhibiting or alleviating skin fibrosis; The skin fibrosis includes one or more of rough skin texture, stretch marks, scleroderma and diseases with scleroderma-like changes, hypertrophic scars, keloid scars, hypertrophic scars, depressed scars, fibromas, and / or graft-versus-host disease.

10. Use of the composition according to any one of claims 4-6 in the preparation of skin care products and / or pharmaceuticals.

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