A composition for inhibiting inflammatory repigmentation and uses thereof

Abstract

This invention relates to a composition for inhibiting inflammation and hyperpigmentation, and its application. The composition comprises the following components: tranexamic acid, oat β-glucan, Schizophyllum commune fermentation product filtrate, and plant extracts. By using a combination of cost-effective components, this invention achieves multi-dimensional anti-inflammatory and repairing effects, resulting in highly effective hyperpigmentation prevention.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a composition for inhibiting inflammation and hyperpigmentation and its application. Background Technology

[0002] Post-inflammatory hyperpigmentation (PIH) refers to increased pigmentation following various acute and chronic inflammations of the skin, hence it is also known as post-inflammatory melanosis. The intensity of PIH is closely related to skin color, the degree and depth of inflammation, the extent of basement membrane damage, and melanocytes. The pathogenesis of PIH may involve increased melanin production and / or abnormal melanin distribution caused by inflammation, resulting from the action of endogenous or exogenous inflammatory factors: ① Stimulation by inflammatory factors leads to active melanin synthesis in melanocytes, resulting in epidermal melanin deposition; ② Melanocytes are damaged by inflammation, causing melanin granules to detach from the epidermis into the dermis, resulting in dermal melanin deposition.

[0003] In the above process, limiting the expression of inflammatory mediators and inflammatory cytokines and preventing inflammation are crucial. After cosmetic procedures, the skin is in a "low-protection" state of barrier damage, accompanied by erythema, burning, and stinging, making it highly susceptible to various external stimuli. Therefore, wound repair, moisturizing, and anti-inflammation are of paramount importance within the first 48 hours. Preventing heat-induced damage, targeting and protecting the proteome, inhibiting tyrosinase and inflammatory factors, and photoprotection are also necessary. This includes increasing the expression of the autophagy regulator AMBRA1, reducing melanin expression, reducing lipofuscin expression after oxidative stress, increasing the expression of lipofuscin, and brightening the skin. Reducing the production and secretion of melanin in cells under the influence of nitric oxide (NO) and α-melanocyte-stimulating hormone (α-MSH) is also important. One of the main mechanisms of UV-induced skin darkening is the increase and delay of melanosome transport, which leads to the synthesis of large amounts of melanin and causes pigmentation. This also helps prevent photoaging.

[0004] When developing new products, how to achieve efficient anti-blackening while maintaining cost-effectiveness and plant-based claims is a technical problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a composition for inhibiting inflammation and hyperpigmentation, and its application. By selecting cost-effective components, this invention achieves multi-dimensional anti-inflammatory and repairing effects, resulting in highly efficient hyperpigmentation prevention.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a composition for inhibiting inflammatory hyperpigmentation, the composition comprising the following components: tranexamic acid, oat β-glucan, Schizophyllum commune fermentation product filtrate, and plant extracts.

[0007] In this invention, tranexamic acid stabilizes the melanin matrix, inhibiting the production and transport of melanin from the source, effectively reducing pigmentation caused by ultraviolet rays and free radicals, and has significant anti-inflammatory properties, preventing the proper synthesis of melanin and thus preventing dull skin.

[0008] In this invention, oat β-glucan is a flexible high-molecular-weight polysaccharide that can significantly enhance the activation ability of EGFR, a key target for human repair and anti-aging, stimulate the skin's endogenous repair and anti-aging power, and achieve the repair of the scarce "bricks" in the skin barrier's "brick wall-mud" structure. Its repair effect on people with damaged skin barriers is comparable to that of EGF. In this invention, the fermentation product filtrate of Schizophyllum commune helps lock in skin moisture, repair the skin barrier, inhibit the expression of inflammatory factors, and achieve anti-pigmentation. More preferably, Xishiyin® SPG Marubi uses gene sequencing technology and a unique process optimization system to ensure the stability of the strain. By monitoring the quality of fermentation raw materials through microbial metabolomics technology, a high-efficiency repairing Schizophyllum commune extract with stable quality can be obtained.

[0009] The above three ingredients, combined with plant extracts that have repairing and brightening effects, can work synergistically to inhibit inflammation and darkening.

[0010] Preferably, the composition comprises, by weight, 0.1-3 parts of tranexamic acid (e.g., 0.1, 0.5, 1, 1.5, 2, 2.5, 3, etc.), 0.02-15 parts of oat β-glucan (e.g., 0.02, 0.1, 0.5, 1, 2, 5, 8, 10, 15, etc.), 1-100 parts of Schizophyllum commune fermentation product filtrate (e.g., 1, 10, 20, 50, 80, 100, etc.), and 40-410 parts of plant extract (e.g., 40, 80, 100, 200, 300, 400, 410, etc.).

[0011] Preferably, the plant extract includes any one or a combination of at least two of the following: birch bark extract, *Phyllostachys pubescens* root extract, yarrow extract, *Ganoderma lucidum* extract, *Sophora flavescens* root extract, *Tribulus terrestris* fruit extract, or *Cornus officinalis* fruit extract. More preferably, it is a combination of birch bark extract, *Phyllostachys pubescens* root extract, yarrow extract, *Ganoderma lucidum* extract, *Sophora flavescens* root extract, *Tribulus terrestris* fruit extract, and *Cornus officinalis* fruit extract.

[0012] In this invention, birch bark extract and Sophora flavescens root extract are extracted from plants at low temperatures. They can effectively inhibit the mitosis of keratinocytes and melanocytes, and have a strong effect on removing pigmentation. They can also quickly penetrate the dermis and effectively destroy the growth and synthesis of melanin. Sophora flavescens root extract not only inhibits tyrosinase, but also has anti-photoaging, repair and anti-inflammatory effects.

[0013] In this invention, yarrow extract prevents heat stress-induced damage, targets and protects the proteome, inhibits tyrosinase and inflammatory factors, and also has a photoprotective effect.

[0014] In this invention, the extract of Ganoderma lucidum protects the skin's microecological balance and has excellent anti-inflammatory effects. It is preferred to use a product of compound fermentation of lactic acid bacteria and yeast.

[0015] In this invention, Sophora flavescens root extract, Tribulus terrestris fruit extract, and Cornus officinalis fruit extract can solve facial redness caused by various factors and soothe the skin in a timely manner.

[0016] When the above-mentioned plant extracts are used in combination, the components work synergistically to achieve a more superior effect in inhibiting inflammation and hyperpigmentation.

[0017] Preferably, the plant extracts, by weight, include 0.03-50 parts of birch bark extract (e.g., 0.03, 0.1, 1, 5, 10, 20, 40, 50 parts, etc.), 0.03-50 parts of *Symplocos buergeriana* root extract (e.g., 0.03, 0.1, 1, 5, 10, 20, 40, 50 parts, etc.), 0.03-10 parts of yarrow extract (e.g., 0.03, 0.5, 2, 6, 8, 10 parts, etc.), and 35-300 parts of *Ganoderma lucidum* extract (e.g., ...). The extracts are available in quantities of 35, 50, 100, 150, 200, 250, 300, etc.), Sophora flavescens root extract 0.03-27 parts (e.g., 0.03, 0.1, 1, 3.6, 5, 10, 15, 20, 27, etc.), Tribulus terrestris fruit extract 0.02-18 parts (e.g., 0.02, 0.1, 2.4, 5, 8, 12, 18, etc.), and Cornus officinalis fruit extract 0.02-9 parts (e.g., 0.01, 0.1, 1.2, 3, 6, 8, 9, etc.).

[0018] In a second aspect, the present invention provides the application of the composition for inhibiting inflammatory hyperpigmentation according to the first aspect in cosmetics.

[0019] Preferably, the cosmetic includes toner, serum, lotion, cream, or mask.

[0020] Thirdly, the present invention provides an essence for inhibiting inflammatory hyperpigmentation, the essence comprising the following components: an active ingredient, a moisturizer, a thickener, a skin emollient, a chelating agent, a pH adjuster, and water; the active ingredient comprising the composition for inhibiting inflammatory hyperpigmentation according to the first aspect.

[0021] Preferably, the essence comprises the following components by weight percentage: 1-20% active ingredients (e.g., 1%, 5%, 10%, 15%, 20%, etc.), 1-20% moisturizer (e.g., 1%, 5%, 10%, 15%, 20%, etc.), 1-20% thickener (e.g., 1%, 5%, 10%, 15%, 20%, etc.), 1-20% emollient (e.g., 1%, 5%, 10%, 15%, 20%, etc.), 0.01-5% chelating agent (e.g., 0.01%, 0.05%, 0.1%, 1%, 2%, 5%, etc.), 0.01-5% pH adjuster (e.g., 0.01%, 0.05%, 0.1%, 1%, 2%, 5%, etc.), and 60-80% water (e.g., 60%, 65%, 70%, 75%, 80%, etc.).

[0022] Preferably, the moisturizer comprises any one or a combination of at least two of butylene glycol, sodium hyaluronate, panthenol, 1,2-pentanediol, glycerin, 1,2-hexanediol, or diphenylpolydimethylsiloxane.

[0023] Preferably, the thickener comprises any one or a combination of two of acrylate / C10-30 alkanol acrylate crosspolymers or maltodextrin.

[0024] Preferably, the emollient comprises any one or a combination of at least two of glyceryl tri(ethylhexanoate), polyglycerol-10 myristate, or PEG / PPG-14 / 7 dimethyl ether.

[0025] Preferably, the chelating agent comprises disodium EDTA.

[0026] Preferably, the pH adjuster comprises aminomethylpropanol.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects: This invention combines tranexamic acid, oat β-glucan, Schizophyllum commune fermentation product filtrate, and plant extracts. The components work synergistically to exert their effects from multiple dimensions, achieving anti-inflammatory, anti-photoaging, and highly effective anti-darkening effects. Moreover, the composition is mild and non-irritating, making it suitable for various cosmetic types. Detailed Implementation

[0028] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0029] The raw materials used in the following examples are from the following sources: The trade name of oat beta-glucan is Miaokexiu, and the manufacturer is Beijing Dongfang Miaosen. The commercial name of the fermentation product filtrate of Schizophyllum commune is Xishiyin, and the purchaser is Baister Pharmaceutical (Guangzhou). The trade name of birch bark extract and blue ear grass root extract is Keban-BH70. The mass ratio of the two ingredients is 1:1. The manufacturer is Boyue Fine Chemical. The yarrow extract is marketed under the name Supervisome EPH and was purchased from Symrise Fragrance & Flavor. The extract of Tibetan Ganoderma lucidum contains lactic acid bacteria fermentation products and yeast fermentation products in a mass ratio of 1:1. The manufacturer is Baister Pharmaceutical (Guangzhou). The product name of the extracts of Sophora flavescens root, Tribulus terrestris fruit, and Cornus officinalis fruit is Shuyidan. The mass ratio of the three ingredients is 3:2:1. The manufacturer is Beijing Dongfang Miaosen.

[0030] Example 1 This embodiment provides a composition for inhibiting inflammation and hyperpigmentation. The composition comprises, by weight, 1 part tranexamic acid, 10 parts oat β-glucan, 50 parts Schizophyllum commune fermentation product filtrate, 2.5 parts birch bark extract, 2.5 parts Podophyllum oleraceum root extract, 0.03 parts yarrow extract, 188 parts Ganoderma lucidum extract, 9 parts Sophora flavescens root extract, 6 parts Tribulus terrestris fruit extract, and 3 parts Cornus officinalis fruit extract.

[0031] Example 2 This embodiment provides a composition for inhibiting inflammation and hyperpigmentation. The composition comprises, by weight, 0.1 parts tranexamic acid, 15 parts oat β-glucan, 1 part Schizophyllum commune fermentation product filtrate, 50 parts birch bark extract, 50 parts Podophyllum spp. root extract, 0.5 parts yarrow extract, 300 parts Ganoderma lucidum extract, 0.6 parts Sophora flavescens root extract, 0.4 parts Tribulus terrestris fruit extract, and 0.2 parts Cornus officinalis fruit extract.

[0032] Example 3 This embodiment provides a composition for inhibiting inflammation and hyperpigmentation. The composition comprises, by weight, 3 parts tranexamic acid, 0.02 parts oat β-glucan, 100 parts Schizophyllum commune fermentation product filtrate, 0.03 parts birch bark extract, 0.03 parts Podophyllum spp. root extract, 2 parts yarrow extract, 35 parts Ganoderma lucidum extract, 3 parts Sophora flavescens root extract, 2 parts Tribulus terrestris fruit extract, and 1 part Cornus officinalis fruit extract.

[0033] Example 4 This embodiment provides a composition for inhibiting inflammation and hyperpigmentation. The only difference between this composition and that of Example 1 is that it does not contain birch bark extract and blue ear grass root extract. The dosage of these components is determined according to the proportions of yarrow extract, Tibetan lingzhi extract, sophora flavescens root extract, tribulus fruit extract, and cornus fruit extract. Other aspects are the same as in Example 1.

[0034] Example 5 This embodiment provides a composition for inhibiting inflammation and hyperpigmentation. The only difference between this composition and Example 1 is that it does not contain yarrow extract. The amount of yarrow extract is allocated according to the proportion of birch bark extract, blue ear grass root extract, Tibetan lingzhi extract, sophora flavescens root extract, tribulus fruit extract and cornus fruit extract. Other aspects are the same as in Example 1.

[0035] Example 6 This embodiment provides a composition for inhibiting inflammation and hyperpigmentation. The only difference between this composition and that of Example 1 is that it does not contain Ganoderma lucidum extract. The amount of this composition is allocated according to the proportions of birch bark extract, Podophyllum heliotropium root extract, Achillea millefolium extract, Sophora flavescens root extract, Tribulus terrestris fruit extract, and Cornus officinalis fruit extract. Other aspects are the same as in Example 1.

[0036] Example 7 This embodiment provides a composition for inhibiting inflammation and hyperpigmentation. The only difference between this composition and Example 1 is that it does not contain Sophora flavescens root extract, Tribulus terrestris fruit extract, and Cornus officinalis fruit extract. The dosage of these components is determined according to the proportions of birch bark extract, Podophyllum oxypetalum root extract, Achillea millefolium extract, and Ganoderma lucidum extract. Other aspects are the same as in Example 1.

[0037] Comparative Example 1 This comparative example provides a composition for inhibiting inflammatory hyperpigmentation. The only difference between this composition and Example 1 is that it does not contain tranexamic acid, and its dosage is allocated according to the proportion of the remaining components. Other aspects are the same as in Example 1.

[0038] Comparative Example 2 This comparative example provides a composition for inhibiting inflammatory hyperpigmentation, which differs from Example 1 only in that it does not contain oat β-glucan, and its dosage is allocated according to the proportion of the remaining components, otherwise referring to Example 1.

[0039] Comparative Example 3 This comparative example provides a composition for inhibiting inflammatory hyperpigmentation. The only difference between this composition and Example 1 is that it does not contain the fermentation product filtrate of Schizophyllum commune, and its dosage is allocated according to the proportion of the remaining components. Other aspects are the same as in Example 1.

[0040] Comparative Example 4 This comparative example provides a composition for inhibiting inflammation and hyperpigmentation. The only difference between this composition and Example 1 is that it does not contain birch bark extract, Sophora flavescens root extract, yarrow extract, Ganoderma lucidum extract, Sophora flavescens root extract, Tribulus terrestris fruit extract, and Cornus officinalis fruit extract. The amounts of these components are allocated according to the proportions of the remaining components. Other aspects are the same as in Example 1.

[0041] Application Example 1 This application example provides an essence that inhibits inflammation and hyperpigmentation, comprising the following components: Preparation method: (1) Mix phase A evenly, stir, heat to 80℃, and keep warm for 10 minutes; (2) Keep stirring, cool down to 40°C, add phase B, and stir until the material is uniform.

[0042] Application Examples 1-7, Comparative Application Examples 1-4 The above application examples and comparative application examples each provide an essence that inhibits inflammation and hyperpigmentation. The only difference between them and application example 1 is that the composition provided in example 1 is replaced in equal amounts with the compositions provided in examples 2-7 and comparative examples 1-4. Other aspects are the same as in application example 1.

[0043] Test case (1) Cell proliferation test 1.1 Preparation of cell suspension Human dermal fibroblasts in good growth condition were removed from the CO2 incubator (cell morphology and cell adhesion were observed under a microscope) and placed in a clean bench. The old culture medium was carefully aspirated, and the cells were rinsed with PBS using a pipette. After rinsing, the cells were carefully aspirated, and trypsin-EDTA was added for digestion (25cm). 2 Add 1 mL of digestion solution, enough to cover the cells. Immediately place the container in a CO2 incubator for 5 minutes. Observe under a microscope to see if the cells begin to flow with the digestion solution. Quickly add culture medium to neutralize. Pipe the cells a few times to disperse them evenly. Then, transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add culture medium, and obtain the cell suspension.

[0044] 1.2 Cell suspension counting Adherent cells: Take 20 μL of cell suspension and inject it into two counting chambers of a cell counting chamber. Place the chamber under a microscope, adjust the objective magnification appropriately, and count the four large squares in each counting chamber (top left, bottom left, top right, and bottom right) in a clockwise direction. Count the cells at the left and top edges, but not the cells at the bottom and right edges. Take the average value and calculate the original cell concentration according to the formula. At the same time, dilute the cell suspension to a concentration of 1% with culture medium.

[0045] Suspension cells: Take 20 μL of cell suspension and inject it into two counting chambers of a cell counting chamber. Place the chamber under a microscope, adjust the objective lens magnification appropriately, and count the four large squares in each counting chamber (top left, bottom left, top right, and bottom right) in a clockwise direction. Count the cells at the left and top edges, but not the cells at the bottom and right edges. Take the average value and calculate the original cell concentration according to the formula. At the same time, dilute the cell suspension to the appropriate concentration with culture medium.

[0046] Cell stock solution concentration (cells / mL) = average cell count × dilution factor × 1 × 10 4 1.3 Paving Prepare the cell plate, pour the cell suspension into the sample loading well, add 100 μL of the cell suspension to the cell plate, and add an equal amount of PBS to the outermost part of the cell plate. Incubate in a CO2 incubator for 12 h.

[0047] 1.4 Starvation Treatment Once the cells have adhered to the plate and the cell plating area is approximately 30%, the culture medium is aspirated, and 100 μL of serum-free culture medium is added to each well. The plates are then incubated in a CO2 incubator for 16 hours.

[0048] 1.5 Sample Preparation After filtration, the sample was mixed with the culture medium. A quantitative amount of the sample was dissolved in a quantitative amount of dimethyl sulfoxide, vortexed, and centrifuged (4500 rpm, 10 min). The clear liquid at the bottom was filtered and mixed with the culture medium.

[0049] 1.6 Cell proliferation effect detection After incubation, remove the culture medium from the wells using a pipette in a clean bench. Turn off the lights in the clean bench to keep it in a dark environment. Dilute the CCK-8 solution 10 times with the culture medium and pour it into the sample tank. Use a multi-channel pipette to add 100 μL to the cell plate. Record the time accurately. Incubate the cell plate in a CO2 incubator for 0.5 h. Open the lid and place it in a microplate reader to measure the absorbance at a wavelength of 450 nm.

[0050] The procedure for cytotoxicity assays is as follows: 1.7 Data Analysis and Processing Cell viability (%) = OD 试验 ×100 / OD 空白 Where: OD 试验 — Absorbance of test wells or solvent control wells OD 空白 ———Absorbance value of the blank aperture.

[0051] (2) Nematode resistance to photoaging test 2.1 Culture of Escherichia coli OP50 Streak OP50 culture onto an LB agar plate, pick a single colony and add it to 10 mL of LB liquid medium. Incubate at 37°C and 200 rpm with shaking for 12 h until OD50 is reached. 600 Equal to 0.4, used for inoculating NGM-fed normal group nematodes.

[0052] 2.2 E. coli OP50 coating Add an appropriate amount of bacterial suspension to each NGM plate (150 μL for a 60 mm diameter plate), and spread the suspension evenly on the NGM plate using a sterile spreader. The NGM plates with the bacteria spread can be used after incubating overnight at room temperature (21–25°C).

[0053] 2.3 Nematode Synchronization Wash the young adult nematodes on the NGM plate twice with 1 mL of M9 buffer solution, then transfer them to a sterile 2 mL centrifuge tube. Add 1 mL of freshly prepared lysis buffer, shake thoroughly for 5 min, and centrifuge at 3000 rpm for 1 min, discarding the supernatant. Wash the nematodes again with 1 mL of M9 buffer and centrifuge twice under the same conditions, discarding the supernatant, leaving 0.4 mL of egg-containing buffer. Gently pipette the eggs to mix them, and drop approximately 100 μL of the egg-containing buffer onto the sterile area near OP50 on the NGM plate. Wait until the fertilized nematode eggs have basically developed into L4 stage larvae, completing synchronization. Select L4 stage larvae and transfer them to a drug-treated plate for further experiments.

[0054] 2.4 Preparation of sample containing NGM: Sample group: Dissolve the sample in M9 buffer or sterile water to prepare a stock solution of a certain concentration, and take... E. coli The OP50 bacterial culture and the sample were mixed thoroughly at a ratio of 90:10. The final effective concentration of the sample can be determined based on preliminary experiments.

[0055] Blank group: Take E. coli Gently pour the OP50 bacterial suspension into the center of an NGM plate, let it air dry in the dark, and then seal it. Store at ℃ for later use. Use a pipette to draw 150 ml of the solution. A μL of bacterial suspension was gently poured into the center of each NGM plate.

[0056] Survival rate (%) = 100% - Cumulative number of deaths / Sample size × 100% Differences between different treatment groups were analyzed using t-tests, and p < 0.05 was generally considered statistically significant.

[0057] Only when the survival rate of a sample is positive and significant compared to the blank control can it be said that the sample has a significant anti-photoaging effect.

[0058] (3) Tyrosinase inhibition test 3.1 Preparation of cell suspension Remove well-grown cells from the CO2 incubator (observe cell morphology and cell adhesion under a microscope) to a clean bench, carefully aspirate the old culture medium, rinse with PBS using a pipette, carefully aspirate after rinsing, and add an appropriate amount of trypsin-EDTA for digestion (25cm). 2 Add 1 mL of digestion solution, enough to cover the cells. Immediately place in a CO2 incubator for 5 minutes. Observe under a microscope to see if the cells begin to flow with the digestion solution. Quickly add culture medium to neutralize. Disperse the cells evenly by pipetting a few times, then transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add culture medium, and prepare a cell suspension. After evenly dispersing the cell suspension, count the cells using a cell counter. Perform three counts and calculate the average. Dilute the cell suspension with culture medium to the required concentration of 8 × 10⁻⁶. 4 cfu / mL.

[0059] 3.2 Decking Logarithmic growth phase B16 cells were seeded in 96-well plates at a cell density of 8 × 10⁶ cells / well. 4 The concentration of cfu / mL was 100 μL per well, and the mixture was incubated in a carbon dioxide incubator for 24 h.

[0060] 3.3 Sample Preparation After 24 hours of incubation, carefully aspirate the supernatant from the cell plate and add the following sample at 100 μL / well, with four replicates for each concentration. After adding the sample, incubate in a CO2 incubator for 72 hours. A positive control (α-arbutin) and a blank control (serum-free medium without the drug) are also required. Water-soluble samples: Dilute the sample to a 1% concentration using serum-free culture medium.

[0061] 3.4 Data Detection After incubation for 72 h, the supernatant was discarded from the cell plate, and 150 μL of 1% Triton X-100 solution was added to each well. The plate was then quickly frozen at -80 ℃ for 30 min, followed by thawing at room temperature to completely rupture the cells. After pre-warming at 37 ℃ for 5 min, 60 μL of 0.5% L-DOPA solution was added, and the reaction was carried out at 37 ℃ for 2 h. The absorbance (OD) value was measured at 490 nm using a microplate reader.

[0062] 3.5 Data Analysis and Processing Calculate the tyrosinase activity inhibition rate using the following formula: Tyrosinase activity inhibition rate (%) = (1 - average absorbance of sample / average absorbance of blank) × 100.

[0063] (4) Anti-melanin production test 4.1 Preparation of cell suspension Remove well-grown cells from the CO2 incubator (observe cell morphology and cell adhesion under a microscope) to a clean bench, carefully aspirate the old culture medium, rinse with PBS using a pipette, carefully aspirate after rinsing, and add trypsin-EDTA for digestion (25cm). 2 Add 1 mL of digestion solution, enough to cover the cells. Immediately place in a CO2 incubator for 5 minutes. Observe under a microscope to see if the cells begin to flow with the digestion solution. Quickly add culture medium to neutralize. Disperse the cells evenly by pipetting a few times, then transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add culture medium, and prepare a cell suspension. After evenly dispersing the cell suspension, count the cells using a cell counter. Perform three counts and calculate the average. Dilute the cell suspension to the required concentration of 2 × 10⁻⁶ cells / mL with culture medium. 4 cfu / mL.

[0064] 4.2 Decking Log-phase B16 cells were seeded into 6-well plates at a cell density of 2 × 10⁶ cells / well. 4 The concentration of cfu / mL was 2 mL per well, and the mixture was incubated in a carbon dioxide incubator for 24 h.

[0065] 4.3 Sample Preparation Weigh 1g of sample and mix with 1g of culture medium, then filter. Weigh 1g of sample and dissolve in 1g of dimethyl sulfoxide. Vortex and centrifuge (4500 rpm, 10 min). Take the clear liquid at the bottom, filter, and mix with the culture medium.

[0066] 4.4 First fluid change After the prepared 6-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours, the cell culture medium was replaced with culture medium containing the test sample, 2 mL per well, and the plates were incubated for another 48 hours. Note: The negative control group contained only cell culture medium, while the positive control group required the addition of cell culture medium containing the positive control substance.

[0067] 4.5 Second fluid change Forty-eight hours after the first medium change, repeat the first medium change procedure, replacing the cell culture medium and adding 3 mL of culture medium containing the corresponding sample to each well. Continue culturing in a 37°C, 5% CO2 incubator.

[0068] 4.6 Third fluid change 72 hours after the second medium change, repeat the first medium change procedure, replacing the cell culture medium and adding 3 mL of culture medium containing the sample to each well, and continue culturing in a 37°C, 5% CO2 incubator.

[0069] 4.7 Detection 72 hours after the third medium change (when cell fusion reached over 90%), the melanin content of the cells was measured. The cells were washed twice with 1×PBS, the PBS was removed, and 200 μL of 0.25% trypsin was added to each well to digest the cells. The cells were then incubated in a CO2 incubator for 5 minutes. Two 2 mL PBS solutions were added and the cells were agitated by pipetting. The cells were collected in centrifuge tubes and centrifuged at 4000 rpm for 5 minutes. 200 μL of melanin extraction solution was added to each tube, shaken well, and heated in an 80°C water bath for 1 hour. After cooling, the droplets on the tube wall were centrifuged moderately, agitated, and 150 μL of the solution from each centrifuge tube was transferred to a 96-well plate. The absorbance at 405 nm was measured using a microplate reader.

[0070] 4.8 Data Analysis and Processing Calculate the melanin inhibition rate of B16 cells using the following formula: B16 cell melanin inhibition rate (%) = (1 - average absorbance of sample / average absorbance of blank) × 100.

[0071] (5) Cellular anti-inflammatory assay 5.1 Preparation of cell suspension Remove well-grown cells from the CO2 incubator (observe cell morphology and cell adhesion under a microscope) to a clean bench. Carefully aspirate the old culture medium, rinse with PBS using a pipette, and carefully aspirate the rinsed cells. Add an appropriate amount of trypsin-EDTA for digestion (25cm recommended). 2Add 1 mL of digestion solution, enough to cover the cells. Immediately place in a CO2 incubator for 5 minutes. Observe under a microscope to see if the cells begin to flow with the digestion solution. Quickly add culture medium to neutralize. Disperse the cells evenly by pipetting a few times, then transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add culture medium, and prepare a cell suspension. After evenly dispersing the cell suspension, count the cells using a cell counter. Perform three counts and calculate the average. Dilute the cell suspension with culture medium to the required concentration of 4 × 10⁻⁶. 5 cfu / mL.

[0072] 5.2 Decking Log-phase RAW264.7 cells were seeded in 24-well plates at a cell density of 3 × 10⁻⁶ cells / well. 5 0.5 mL per well, incubated in a CO2 incubator for 24 h.

[0073] 5.3 Sample Preparation After incubating the cells for 24 h, the supernatant was aspirated, and 0.5 mL of the following samples were added respectively: ① Positive control group: 1% Ominol + LPS (1 μg / mL), cultured normally for 24 h; ② Solvent control group: cells were treated with DMEM medium (2% FBS; 1% penicillin antibody) for 24 h; ③ LPS inflammation model group: RAW264.7 cells were continuously stimulated with 1 μg / mL LPS for 24 h; ④ Experimental group: cells were treated with diluted sample + LPS (1 μg / mL) for 24 h.

[0074] 5.4 Detection of inflammatory factors in cell supernatant After 24 h of cell sample treatment, the supernatant was transferred to a centrifuge tube using a pipette, centrifuged at 1000 r / min for 5 min, the precipitate was discarded, and the supernatant was collected. The changes in the amount of inflammatory factors in the supernatant were detected using a TNF-α and IL-6 inflammatory factor detection kit, and the detection method was strictly in accordance with the instructions on the detection kit.

[0075] 5.5 Data Analysis and Processing According to the detection method on the test kit, a standard curve is prepared using inflammatory factor standards. The content of inflammatory factors can be obtained by inserting the absorbance of the test sample into the standard curve formula.

[0076] The test results of the best practices provided by Application Examples 1-7 and Comparative Application Examples 1-4 are shown in Table 1.

[0077] Table 1 Test results: (1) As can be seen from Application Examples 1 to 7, the present invention, through the compounding of tranexamic acid, oat β-glucan, Schizophyllum commune fermentation product filtrate and plant extracts, allows each component to exert its effect from multiple dimensions, achieving anti-inflammatory, anti-photoaging, and highly effective anti-melanin effects. This composition is mild and non-irritating and can be applied to a variety of cosmetics. Specifically, the inhibition rate of tyrosine at a 1wt% concentration is 4.20-14.91%, the inhibition rate of melanocyte synthesis at a 1wt% concentration is 6.88-16.98%, the inhibition rate of inflammatory factor release at a 1wt% concentration is 85-100%, the cell proliferation survival rate at a 1wt% concentration is 96.052-138.177%, and the survival rate of anti-photoaging nematodes at a 0.5wt% concentration after 168 hours is 8.54-17.72%.

[0078] (2) By comparing application example 1 and application examples 4-7, it can be seen that by combining specific plant extracts, the components of the present invention can play a synergistic role, which can further enhance the anti-inflammatory, anti-photoaging and highly effective anti-blackening effects.

[0079] (3) By comparing application example 1 and comparative application examples 1-4, it can be seen that the four components of this invention, namely tranexamic acid, oat β-glucan, filtrate of Schizophyllum commune fermentation product and plant extract, are indispensable in their function. When any one of them is missing, the effect is significantly reduced.

[0080] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A composition for inhibiting inflammatory repigmentation, characterized by, The composition comprises the following components: tranexamic acid, oat β-glucan, Schizophyllum commune fermentation product filtrate, and plant extracts.

2. The composition of claim 1, wherein, The composition comprises, by weight, 0.1-3 parts tranexamic acid, 0.02-15 parts oat β-glucan, 1-100 parts filtrate of Schizophyllum commune fermentation product, and 40-410 parts plant extract.

3. The composition according to claim 1 or 2, characterized in that, The plant extracts include any one or a combination of at least two of the following: birch bark extract, *Phyllostachys pubescens* root extract, yarrow extract, *Ganoderma lucidum* extract, *Sophora flavescens* root extract, *Tribulus terrestris* fruit extract, or *Cornus officinalis* fruit extract, preferably a combination of birch bark extract, *Phyllostachys pubescens* root extract, yarrow extract, *Ganoderma lucidum* extract, *Sophora flavescens* root extract, *Tribulus terrestris* fruit extract, and *Cornus officinalis* fruit extract.

4. The composition of claim 3, wherein, The plant extracts, by weight, include 0.03-50 parts of birch bark extract, 0.03-50 parts of *Symplocos buergeriana* root extract, 0.03-10 parts of *Achillea millefolium* extract, 35-300 parts of *Ganoderma lucidum* extract, 0.03-27 parts of *Sophora flavescens* root extract, 0.02-18 parts of *Tribulus terrestris* fruit extract, and 0.02-9 parts of *Cornus officinalis* fruit extract.

5. The use of a composition for inhibiting inflammatory hyperpigmentation according to any one of claims 1-4 in cosmetics.

6. Use according to claim 5, characterized in that, The cosmetics include toners, serums, lotions, creams, or masks.

7. An essence for inhibiting inflammation rebounding, characterized by, The essence comprises the following components: active ingredients, moisturizers, thickeners, emollients, chelating agents, pH adjusters, and water; the active ingredients include the composition for inhibiting inflammatory hyperpigmentation according to any one of claims 1-4.

8. The serum of claim 7, wherein the serum is characterized by, The essence comprises the following components by weight percentage: 1-20% active ingredients, 1-20% moisturizer, 1-20% thickener, 1-20% emollient, 0.01-5% chelating agent, 0.01-5% pH adjuster, and 60-80% water.

9. The serum according to claim 7 or 8, characterized in that, The moisturizer includes any one or a combination of at least two of the following: butylene glycol, sodium hyaluronate, panthenol, 1,2-pentanediol, glycerin, 1,2-hexanediol, or diphenylpolydimethylsiloxane. Preferably, the thickener comprises any one or a combination of two of acrylate / C10-30 alkanol acrylate crosspolymers or maltodextrin.

10. The serum according to any one of claims 7-9, characterized in that, The emollients include any one or a combination of at least two of the following: triglyceride tri(ethylhexanoate), polyglycerol-10 myristate, or PEG / PPG-14 / 7 dimethyl ether. Preferably, the chelating agent comprises disodium EDTA; Preferably, the pH adjuster comprises aminomethylpropanol.

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