Flavone composition for preventing and improving atopic dermatitis and use thereof

Abstract

The application discloses a flavone composition for preventing and improving atopic dermatitis and application thereof, and belongs to the fields of medicines and cosmetics. It is proved by cell morphology observation and beta-amino hexosidase detection that apigenin and chrysin have good effects of inhibiting degranulation of mast cell strains. The scratch experiment proves that kaempferol has a good effect of promoting gap closure of human keratinocytes. Then, the expression of atopic dermatitis related factors is detected, and it is found that the apigenin, chrysin and kaempferol have the potential of enhancing the soothing effect. Further, human safety evaluation and efficacy experiment are carried out to screen the best ratio of soothing effect. The application explores the effects of flavone compounds on preventing and improving atopic dermatitis from three aspects of anti-allergy, anti-inflammation and barrier repair, and multiple target points, and expands the application and development value of the flavone compounds in soothing cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of plant composition application technology, specifically relating to a flavonoid composition that can prevent and improve atopic dermatitis and its application. Background Technology

[0002] Atopic dermatitis (AD) is a common skin disease characterized by its chronic, recurrent, and inflammatory nature. It typically begins in infancy and can persist into adulthood or even old age in severe cases. Infants and young children with AD often also have other atopic diseases, including asthma and allergic rhinitis. The main clinical symptoms of AD are eczema-like lesions accompanied by dry skin and intense itching; some patients may also experience skin fissures and crusting. Due to the long treatment period and recurrent nature of AD, patients bear a significant economic and psychological burden, severely impacting their quality of life.

[0003] The pathogenesis of Alzheimer's disease (AD) is complex, mainly involving skin microbiota imbalance, immune dysregulation, and skin barrier dysfunction. Allergens are a significant trigger for AD. After allergens penetrate the skin barrier and enter the body, they bind to IgE antibodies on the surface of mast cells, activating mast cells and causing degranulation. This releases various inflammatory mediators, such as interleukin-4 (IL-4), interleukin-13 (IL-13), and histamine. IL-4 and IL-13 are two major cytokines related to the pathogenesis of AD. They not only promote Th2 immune responses and increase eosinophil infiltration, but also induce neurogenic pruritus, increasing scratching behavior and further damaging the skin barrier. This makes it easier for external irritants and allergens to penetrate the skin, exacerbating AD symptoms.

[0004] Currently, commonly used drugs for treating atopic dermatitis (AD) are mainly divided into topical and oral medications. Topical medications, such as corticosteroids, are currently first-line clinical drugs for AD treatment, but long-term, high-dose use can cause adverse reactions such as skin atrophy and telangiectasia. Oral medications, such as antihistamines and immunosuppressants, can damage the digestive system and liver and kidney function. In recent years, many novel biologics targeting AD have been marketed, such as dupilumab, which can specifically bind to and block the common receptor subunits of IL-4 and IL-13, inhibiting IL-4 and IL-13 signal transduction, and has shown significant efficacy in the treatment of atopic dermatitis. However, its high price has limited its widespread application to some extent. Therefore, developing safe, effective, and inexpensive multi-target therapeutic drugs has become a research hotspot for AD.

[0005] Flavonoids are natural products widely distributed in plants in nature. There are many types, and they have been proven to have a variety of pharmacological activities such as antioxidant, anti-inflammatory, and anti-cancer effects. Compared with traditional therapeutic drugs, flavonoids have a relatively mild metabolic process in the body and have a high safety profile.

[0006] Currently, research on the application of flavonoids in treating inflammation is primarily focused on single-component approaches. However, atopic dermatitis is a complex pathological process involving multiple interactions, including immune imbalance, excessive release of inflammatory factors, and impaired skin barrier function, making it difficult to address the problem with a single component. Existing flavonoid compositions for treating atopic dermatitis are mostly plant extracts, with complex compositions and challenging quality control. Differences in composition between different batches affect the stability of therapeutic effects, and their mechanisms of action are relatively difficult to study, making it hard to determine which specific components play a key role in the treatment process. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the object of the present invention is to overcome the shortcomings of the prior art and provide a flavonoid composition that can prevent and improve atopic dermatitis.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a flavonoid composition for preventing and improving atopic dermatitis, comprising,

[0011] The mixture contains apigenin, salinomycin, and kaempferol; wherein the mass ratio of apigenin, salinomycin, and kaempferol is 1–5:1–5:1–5.

[0012] In a preferred embodiment of the flavonoid composition of the present invention, the mass ratio of apigenin, kaempferol and kaempferol is 1:2 to 4:1.

[0013] In a preferred embodiment of the flavonoid composition of the present invention, the mass ratio of apigenin, kaempferol and kaempferol is 1:4:1.

[0014] Another object of the present invention is to overcome the shortcomings of the prior art and provide an application of a flavonoid composition in the preparation of products for the prevention and improvement of atopic dermatitis.

[0015] In a preferred embodiment of the application described in this invention, the product is a health product, a pharmaceutical product, or a cosmetic product.

[0016] As a preferred embodiment of the application described in this invention, the dosage form of the medicine or cosmetic includes ointment, paste, patch, toner, essence, lotion, and cream.

[0017] In a preferred embodiment of the application described in this invention, the cosmetic product comprises a soothing face cream, wherein...

[0018] The soothing face cream comprises skin conditioning agents, emulsifiers, thickeners, moisturizers, skin-nourishing agents, preservatives, colorants, fragrances, flavonoid compositions, and water;

[0019] Based on the weight percentage of raw materials, the skin conditioning agent is 0.5-2%, the emulsifier is 2-5%, the thickener is 0.5-1.5%, the moisturizer is 10%-20%, the skin emollient is 8%-14%, the preservative is 0.1-0.6%, the colorant is 0.05-0.2%, the fragrance is 0.01-0.1%, the flavonoid composition is 0.05-0.2%, and the water balance is made up to 100%.

[0020] In a preferred embodiment of the application described in this invention, the skin conditioning agent is a saccharide isomer; the emulsifier is one or more of sucrose polystearate, glyceryl stearate, sodium stearoyl lactylate, and myristic acid.

[0021] The thickener includes one or more of the following: ammonium acryloyl dimethyl taurate / VP copolymer, sodium polyacryloyl dimethyl taurate, sodium hyaluronate, xanthan gum, glyceryl polyacrylate, acrylate copolymer, sodium chloride, and potassium chloride;

[0022] The moisturizer includes one or more of the following: glycerin polyether-26, glycerin, propylene glycol, butylene glycol, pentylene glycol, hexanediol, sorbitol, panthenol, hyaluronic acid, ceramide, niacinamide, allantoin, and tremella polysaccharide.

[0023] The emollients include one or more of the following: squalane, hydrogenated ethylhexyl olive oil, hydrogenated unsaponifiables, murum palm oil, babassu seed oil, cetearyl alcohol, and C10-18 triglycerides.

[0024] The preservatives include one or more of phenoxyethanol, ethylhexylglycerin, and p-hydroxyacetophenone;

[0025] The colorant is comfrey oil.

[0026] In a preferred embodiment of the application described in this invention, the method for preparing the soothing face cream includes,

[0027] Weigh out the emulsifier, emollient, and colorant, heat to 80-85°C, stir until all components are completely dissolved and homogeneous, and prepare phase A, which is an oil phase, and keep it warm for later use.

[0028] Weigh out the moisturizer, thickener, skin conditioning agent, preservative and the remaining water, heat to 80-85℃, stir until all components are completely dissolved and homogeneous, prepare phase B, which is an aqueous phase, and keep warm for later use;

[0029] Weigh out the flavonoid composition, butylene glycol and an appropriate amount of water according to the formula, heat to 40-60℃, stir and mix well to prepare phase C;

[0030] Slowly pour the dissolved B phase into the A phase, cool to 60°C, then slowly add the C phase, and stir evenly while hot.

[0031] Stop stirring; the soothing face cream is now ready.

[0032] Beneficial effects of this invention:

[0033] (1) This invention verifies through experiments that apigenin and apigenin have a good inhibitory effect on degranulation of rat basophilic leukemia cells without affecting cell proliferation and morphology.

[0034] (2) The present invention has verified through experiments that kaempferol can promote the migration of human keratinocytes without affecting cell proliferation and morphology.

[0035] (3) The flavonoid composition of the present invention is a compound preparation made by using kaempferol, apigenin and apigenin as raw materials and in a suitable ratio. The determination experiment of inflammatory factors related to atopic dermatitis proved its synergistic effect, which is far higher than that of single components, greatly enhancing the application and development value of flavonoids in anti-allergy, promoting tissue repair and improving atopic dermatitis.

[0036] (4) The present invention also provides a soothing face cream containing a flavonoid composition. The addition of the flavonoid composition to the face cream makes it able to relieve skin itching and reduce the degree of allergic redness. This is of great significance for further expanding the application of flavonoids in cosmetics. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0038] Figure 1The figure shows the experimental results of the effect of different types of flavonoids on the degranulation morphology of RBL-2H3 cells in Example 1 of this invention.

[0039] Figure 2 The figure shows the experimental results of the effect of different types of flavonoids on the release of β-aminohexosidase in RBL-2H3 cells in Example 1 of the present invention.

[0040] Figure 3 The figure shows the experimental results of the effects of different types of flavonoids on HaCaT cell migration in Example 2 of the present invention.

[0041] Figure 4 The figure shows the experimental results of the effect of different ratios of flavonoids on the expression of the inflammatory factor IL-4 in Example 3 of the present invention.

[0042] Figure 5 The figure shows the experimental results of the effect of different ratios of flavonoids on the expression of the inflammatory factor IL-13 in Example 3 of the present invention.

[0043] Figure 6 The figure shows the experimental results of the effect of different ratios of flavonoids on the expression of the inflammatory factor histamine in Example 3 of the present invention. Detailed Implementation

[0044] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0045] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0046] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0047] The flavonoids used in this invention were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0048] Apigenin (product number A106675, batch number C2329727);

[0049] Populin (product number C110078, batch number F1812032);

[0050] Kaempferol (product number K107144, batch number G2318065).

[0051] Example 1

[0052] The rat basophilic leukemia cell (RBL-2H3) degranulation model is an important tool for screening anti-allergy drugs. By observing the inhibitory effect of drugs on cell degranulation, potential anti-allergy drugs can be discovered.

[0053] 1) Resuscitation, culture, passage, and cryopreservation of RBL-2H3 cells

[0054] (1) Cell revival: Remove the cryovial from the liquid nitrogen tank, accelerate thawing in a 37°C water bath, disinfect with 75% alcohol and transfer to a clean bench. Aspirate the liquid from the cryovial and transfer it to a centrifuge tube containing about 6 mL of complete culture medium. Centrifuge at 1000 r / min for 5 min, discard the supernatant, resuspend in 1 mL of fresh culture medium and transfer to a culture dish containing 6 mL of culture medium.

[0055] (2) Cell culture: RBL-2H3 cells were cultured in EMEM complete medium (15% FBS + 1% double antibiotics) at 37°C in a 5% CO2 constant temperature incubator.

[0056] (3) Cell passage: When the cell density is 80-90%, discard the supernatant, wash twice with PBS, digest with 0.25% trypsin + 0.03% EDTA, let stand in the incubator for 2-3 min, centrifuge at 1000 r / min for 5 min, and passage at 1:3.

[0057] (4) Cell cryopreservation: Collect cells, add serum-free unprogrammed cell cryopreservation solution, transfer to cryopreservation tubes, label with name, number, time and name, and store the cryopreservation tubes in a -80℃ freezer. The next day, transfer the cryopreservation tubes to a liquid nitrogen tank for long-term storage.

[0058] This invention evaluates and compares the effects of various flavonoids on the degranulation of RBL-2H3 cells by detecting morphological changes and β-hexosaminidase release in RBL-2H3 cells (purchased from Beina Chuanglian Biotechnology Research Institute), and verifies the excellent anti-allergic effects of apigenin and apigenin.

[0059] Using dimethyl sulfoxide (DMSO) as a solvent, working solutions of the flavonoids to be tested (luteolin, apigenin, salicumin, quercetin, kaempferol, and rutin) were prepared at a molar concentration of 50 mM. These solutions were aliquoted and stored at -20°C to avoid repeated freeze-thaw cycles. Before use, the working solutions were thawed at room temperature and serially diluted with EMEM basal medium. After each dilution, the solutions were sonicated for 30 min to ensure complete dissolution. Finally, the flavonoids were prepared into 5 μM dilutions for samples 1-6 to inhibit RBL-2H3 degranulation.

[0060] 2) Effects of flavonoids on degranulation of RBL-2H3 cells

[0061] 1. Morphological changes in RBL-2H3 cells:

[0062] (1) Take RBL-2H3 cells in the logarithmic growth phase, prepare a cell suspension, and use 1*102 4 Cells were seeded at a density of 1 cell per well in 96-well plates and incubated in a 5% CO2, 37°C incubator for 12 hours in EMEM complete medium until the cells adhered and reached 60% confluence.

[0063] (2) Remove the supernatant, rinse carefully twice with PBS, add EMEM basal medium containing 0.5 μg / mL DNP-IgE to the model group and sample group, add fresh EMEM basal medium to the control group, and continue to culture overnight;

[0064] (3) Remove the supernatant, rinse carefully twice with PBS, add EMEM basal medium containing 5 μM flavonoids 1-6 to the sample group, and add equal amounts of fresh EMEM basal medium to the control group and model group, and continue to culture for 10 h.

[0065] (4) DNP-BSA (final concentration 0.5 μg / mL) was added to the model group and the sample group, and an equal amount of fresh EMEM basal medium was added to the control group. The culture was continued for 4 h.

[0066] (5) Place in an ice bath for 10 min to terminate the reaction, remove the culture medium, and carefully rinse twice with PBS;

[0067] (6) Add 1 mL of 4% paraformaldehyde to each well for 20 min to fix the cells. Remove the supernatant, add 100 μL of toluidine blue staining agent for 10 min, wash away excess staining agent with PBS, add 1 mL of PBS, and immediately observe the cell morphology and take pictures for recording using an optical microscope.

[0068] Experimental results are as follows Figure 1As shown, the RBL-2H3 cells in the blank group are spindle-shaped with neat edges and full shape. After stimulation, they degranulate, become swollen, round, and irregular in shape. The cytoplasm becomes loose, stains lighter, and vacuolar structures appear.

[0069] Apigenin and apigenin showed the best inhibitory effect on the morphological changes of RBL-2H3 cells and had a good inhibitory effect on the degranulation of RBL-2H3 cells.

[0070] 2. Colorimetric determination of β-aminohexosidase release from RBL-2H3 cells.

[0071] 1) Preparation of substrate and stop solution:

[0072] (1) Weigh 4-nitrophenyl N-acetyl-β-glucosinolate (17.1 mg), dissolve it in citrate-sodium citrate buffer, and bring the volume up to 50 mL. The final concentration is 1 mM. Under sterile conditions, filter the solution through a filter membrane to remove bacteria.

[0073] (2) Weigh out 529.5 mg of anhydrous calcium carbonate and 420.6 mg of sodium bicarbonate, dissolve them in a small amount of deionized water, and finally make up to 100 mL, with a final concentration of 0.1 M.

[0074] 2) Cell processing and supernatant collection:

[0075] (1) Take RBL-2H3 cells in the logarithmic growth phase, prepare a cell suspension, and use 2*10 5 Cells were seeded at a density of 1 cell per well in 48-well plates and incubated in a 5% CO2, 37°C incubator for 24 hours in EMEM complete medium until the cells adhered and reached 80% confluence.

[0076] (2) Remove the supernatant, rinse carefully twice with PBS, add EMEM basal medium containing 0.5 μg / mL DNP-IgE to the model group and sample group, add fresh EMEM basal medium to the control group, and continue to culture overnight;

[0077] (3) Remove the supernatant, rinse carefully twice with PBS, add EMEM basal medium containing 5 μM of the flavonoids to be tested to the sample group, and add the same amount of fresh EMEM basal medium to the control group and the model group, and continue to culture for 10 h.

[0078] (4) DNP-BSA (final concentration 0.5 μg / mL) was added to the model group and the sample group, and an equal amount of fresh EMEM basal medium was added to the control group. The culture was continued for 4 h.

[0079] (5) Stop the reaction by placing the cell supernatant in an ice bath for 10 minutes. Collect the cell supernatant in a new EP tube and centrifuge at 4°C for 5 minutes at 14,000 rpm. Take the supernatant for testing.

[0080] (6) Take 50 μL of cell supernatant and 50 μL of substrate into a 96-well plate, incubate at 37°C for 1.5 h, add 100 μL of stop solution to terminate the reaction, and measure the absorbance OD value at 405 nm using an ELISA reader.

[0081] 3) Data processing:

[0082] Cell viability values ​​measured in the model group represent 100% degranulation.

[0083] β-Aminohexosidase release rate (%) = (OD 样品组、对照组 / OD 模型组 )×100%,

[0084] Release rates were calculated for each group. Statistical analysis was performed using GraphPad One-way ANOVA. ### p < 0.01 compared to the control group, *** p < 0.01 compared to the model group.

[0085] Experimental results are as follows Figure 2 As shown, apigenin and apigenin showed the best inhibitory effect on the release of β-aminohexosidase from RBL-2H3 cells, and had a good inhibitory effect on RBL-2H3 cell degranulation.

[0086] In summary, apigenin and apigenin showed the best inhibitory effect on RBL-2H3 cell degranulation, demonstrating their good anti-allergic effect.

[0087] Example 2

[0088] Following skin tissue injury, immortalized human keratinocytes (HaCaT cells) can rapidly migrate to the site of injury. Observing the effects of drugs on cell scratch healing can help identify potential tissue repair agents.

[0089] 1) HaCaT cell resuscitation, culture, passage, and cryopreservation:

[0090] (1) Cell thawing: Remove the cryovial from the liquid nitrogen tank, accelerate thawing in a 37°C water bath, disinfect with 75% alcohol, aspirate the liquid from the cryovial into a centrifuge tube containing about 6 mL of complete culture medium, centrifuge at 1000 r / min for 5 min, discard the supernatant, resuspend in 1 mL of fresh culture medium, and then transfer to a culture dish containing 6 mL of culture medium.

[0091] (2) Cell culture: HaCaT cells were cultured in DMEM complete medium (10% FBS + 1% double antibiotics) at 37°C in a 5% CO2 constant temperature incubator.

[0092] (3) Cell passage: When the cell density is 80-90%, discard the supernatant, wash twice with PBS, digest with 0.25% trypsin + 0.03% EDTA, let stand in the incubator for 5-6 min, centrifuge at 1200 r / min for 5 min, and passage at 1:2.

[0093] (4) Cell cryopreservation: Collect cells, add serum-free unprogrammed cell cryopreservation solution, transfer to cryopreservation tubes, label with name, number, time and name, and store the cryopreservation tubes in a -80℃ freezer. The next day, transfer the cryopreservation tubes to a liquid nitrogen tank for long-term storage.

[0094] This invention verifies the excellent effect of kaempferol in promoting wound healing by detecting the gap closure speed of HaCaT cells (purchased from BeiNa Chuanglian Biotechnology Research Institute) and evaluating and comparing the effects of various flavonoids on cell migration.

[0095] Using dimethyl sulfoxide (DMSO) as a solvent, working solutions of the flavonoids to be tested (luteolin, apigenin, salicylic acid, quercetin, kaempferol, and rutin) were prepared to a molar concentration of 50 mM. These solutions were aliquoted and stored at -20°C to avoid repeated freeze-thaw cycles. Before use, the working solutions were thawed at room temperature and serially diluted with DMEM basal medium. After each dilution, the solutions were sonicated for 30 min to ensure complete dissolution. Finally, the flavonoids were prepared into 5 μM dilutions, which were used as samples 1-6 for scratch testing.

[0096] 2) Cell scratch assay:

[0097] (1) Insert fixation: Carefully pick up the ibidi culture-inserts (2 wells) with sterile forceps and fix them in the center of each well of the 24-well plate.

[0098] (2) Cell seeding: HaCaT cells in the logarithmic growth phase were seeded at 5.0 × 10⁻⁶ cells per cell line. 5 Inoculate the cells at a density of 70 μL / mL (cultured in DMEM complete medium) into the insert, with each well containing 70 μL.

[0099] (3) Grouped drug administration: The group was divided into a control group, a model group, and a sample group, with three parallel groups in each group;

[0100] After each group of cells has been incubated, carefully remove the insert using sterile forceps, which will reveal uniform 500μm scratch-like blank spaces. Discard the supernatant and carefully wash 2-3 times with PBS buffer to remove any residual cells or floating dead cells at the scratch sites.

[0101] Control group: Fresh DMEM basal medium was added, and the culture was continued for 12h and 24h.

[0102] Model group: Add DMEM basal medium (containing 10% FBS and 1% penicillin antibiotics) containing IL-4, IL-13 (final concentration 50 ng / mL) and TNF-α (final concentration 100 ng / mL), and culture for 12 h and 24 h.

[0103] Sample group: Add DMEM basal medium containing flavonoids 1-6 (final concentration 5μM) and IL-4, IL-13 (final concentration 50ng / mL) and TNF-α (final concentration 100ng / mL), and culture continuously for 12h and 24h.

[0104] (4) Microscopic observation: Immediately observe and record the scratch area at 0h using an optical microscope, and take and save scratch images at 12h and 24h.

[0105] (5) Data processing: ImageJ software was used to calculate the scratch area of ​​each group at each time point and to calculate the cell migration rate of each group.

[0106] Statistical analysis was performed using GraphPad T-tests, with p < 0.01 for the model group.

[0107] Experimental results are as follows Figure 3 As shown, kaempferol has the best effect on promoting the migration of human keratinocytes, proving that it has a good effect on promoting wound healing and repairing skin damage.

[0108] Example 3

[0109] To investigate the effects of different ratios of flavonoids on the expression of factors related to atopic dermatitis.

[0110] Sample preparation: Using dimethyl sulfoxide (DMSO) as solvent, the flavonoids (apigenin, kaempferol, and kaempferol) were prepared into working solutions with a molar concentration of 50 mM. These solutions were aliquoted and stored at -20°C to avoid repeated freeze-thaw cycles. Before use, the working solutions were thawed at room temperature and serially diluted with EMEM basal medium. After each dilution, the solutions were sonicated for 30 min to ensure complete dissolution, resulting in 5 μM dilutions of the flavonoids. Flavonoid compositions with the same molar concentration but different ratios were added, as shown in the following samples (1-11):

[0111] No. 1 (apigenin: apigenin: kaempferol ratio of 1:0:0);

[0112] No. 2 (apigenin: apigenin: kaempferol ratio of 0:1:0);

[0113] No. 3 (apigenin: apigenin: kaempferol ratio of 0:0:1);

[0114] No. 4 (apigenin: apigenin: kaempferol ratio of 1:1:1);

[0115] No. 5 (apigenin: apigenin: kaempferol ratio of 1:2:1);

[0116] No. 6 (apigenin: apigenin: kaempferol ratio of 1:4:1);

[0117] No. 7 (apigenin: apigenin: kaempferol ratio of 2:1:1);

[0118] No. 8 (apigenin: apigenin: kaempferol ratio of 4:1:1);

[0119] No. 9 (apigenin: apigenin: kaempferol ratio of 1:5:0);

[0120] No. 10 (Apigenin:Pyrrolin:Kaempferol ratio of 1:0:5) ；

[0121] No. 11 (apigenin: apigenin: kaempferol ratio of 0:5:1) 。

[0122] ELISA was used to measure the expression of IL-4, IL-13, and histamine in RBL-2H3 cells.

[0123] IL-4 and IL-3 play a central role in the pathogenesis of atopic dermatitis.

[0124] In terms of immune regulation, they promote the polarization of Th0 cells into Th2 cells, making the immune response more humoral.

[0125] Regarding the skin barrier function, they disrupt tight junction proteins, leading to barrier damage, and interfere with mucin production, resulting in dry, flaky skin.

[0126] In inflammatory responses, these two cytokines can recruit eosinophils, whose toxic proteins can damage tissues. They also induce keratinocytes and other cells to release inflammatory mediators, attracting more immune cells to aggregate and exacerbating inflammation.

[0127] Histamine plays a key role in pruritus in atopic dermatitis. It can directly stimulate the sensory nerve endings of the skin, transmit pruritus signals, and trigger patients to scratch, forming a vicious cycle of pruritus-scratching.

[0128] Therefore, inhibiting the expression of IL-4, IL-13 and histamine can improve the symptoms of atopic dermatitis and relieve skin itching.

[0129] 1) Cell processing and supernatant collection:

[0130] (1) Take RBL-2H3 cells in the logarithmic growth phase, prepare a cell suspension, and use 2*10 5 Cells were seeded at a density of 1 cell per well in 48-well plates and incubated in a 5% CO2, 37°C incubator for 24 hours in EMEM complete medium until the cells adhered and reached 80% confluence.

[0131] (2) Remove the supernatant, rinse carefully twice with PBS, add EMEM basal medium containing 0.5 μg / mL DNP-IgE to the model group and sample group, add fresh EMEM basal medium to the control group, and continue to culture overnight;

[0132] (3) Remove the supernatant, rinse carefully twice with PBS, add EMEM basal medium containing test samples 1-11 to the sample group, and add equal amounts of fresh EMEM basal medium to the control group and model group, and continue culturing for 10 h.

[0133] (4) DNP-BSA (final concentration 0.5 μg / mL) was added to the model group and the sample group, and an equal amount of fresh EMEM basal medium was added to the control group. The culture was continued for 4 h.

[0134] (5) Stop the reaction by placing the cell supernatant in an ice bath for 10 minutes. Collect the cell supernatant in a new EP tube and centrifuge at 4°C for 5 minutes at 14,000 rpm. Take the supernatant for testing.

[0135] 2) Use the ELISA kit according to the instructions to detect the levels of IL-4, IL-13 and histamine in the cell supernatant.

[0136] 3) Data processing

[0137] Graphpad was used for significant difference analysis (One-way ANOVA Tukey's Method).

[0138] Experimental results are as follows Figures 4-6 As shown, all groups of samples could inhibit the expression of IL-4, IL-13 and histamine. Among them, the ratio of apigenin: apigenin: kaempferol was 1:2:1 and 1:4:1, which showed the best effect in inhibiting multiple inflammatory factors.

[0139] Example 4

[0140] Preparation of a soothing face cream containing a flavonoid composition (apigenin, kaempferol, and genistein):

[0141] Includes the following steps:

[0142] (1) Preparation of solution: Weigh 4g glyceryl stearate, 4g sodium stearoyl lactylate, 6g squalane, 10g babassu seed oil, 3g cetearyl alcohol, 4g hydrogenated ethylhexyl olive oil, 4g hydrogenated olive oil unsaponifiables, 0.2g comfrey oil and 0.06g fragrance, stir evenly to prepare phase A, which is the oil phase, and heat for later use;

[0143] Weigh 1.4g sodium polyacrylamide dimethyl taurate, 2g sugar isomer, 4g glycerol polyether-26, 10g butanediol, 6g nicotinamide and 130g water, stir and mix well to prepare phase B, which is an aqueous phase, and heat for later use.

[0144] Weigh 0.25g of a combination of apigenin, kaempferol, and 5g of butylene glycol and 10g of water, stir and mix well to prepare phase C for later use;

[0145] (2) Slowly pour the dissolved B phase into the A phase, stir evenly while hot, add the C phase after cooling, and stir evenly.

[0146] (3) Stop stirring to obtain a soothing face cream containing a flavonoid (apigenin, apigenin, kaempferol) composition.

[0147] Example 5

[0148] Safety and efficacy evaluation of soothing and repairing face creams containing flavonoid compositions:

[0149] Human patch experiment:

[0150] In accordance with the "Cosmetic Safety Technical Specifications", 30 volunteers who meet the experimental requirements will be recruited to conduct human patch experiments, including two experiments: open patch and closed patch. Informed consent forms will be signed with the volunteers before the experiments.

[0151] Sample: The face cream prepared in Example 4 of this invention was used for the experiment, wherein...

[0152] Sample No. 1 – The ratio of apigenin: apigenin: kaempferol is 1:2:1;

[0153] Sample No. 2 – The ratio of apigenin: apigenin: kaempferol was 1:4:1;

[0154] Reference Standard No. 3 – Composition without added flavonoids.

[0155] Closed patch test:

[0156] Wipe the test site with 70% ethanol, dry it, add 20 μL of sample into the small chamber of the patch applicator, and attach the patch applicator to the inside of the arm with tape.

[0157] Remove the patch after 24 hours, mark the test site with a marker, and observe the skin reaction at 30 minutes, 12 hours, and 24 hours. Record the results according to the skin reaction grading standard in the "Cosmetic Safety Technical Specifications".

[0158] Judgment criteria:

[0159]

[0160] Test results:

[0161]

[0162]

[0163] Open patch assay:

[0164] The test area was selected on the flexor side of the forearm, measuring approximately 3*3cm. 2 Aside from applying the sample, volunteers should avoid contact with any reagents as much as possible. Volunteers are instructed to apply the sample evenly to the test area twice daily for seven consecutive days, and to observe the degree of skin reaction, scoring it according to a grading standard.

[0165] Scoring criteria:

[0166]

[0167] Test results:

[0168]

[0169] None of the samples caused redness or swelling at the application sites in the 30 volunteers; all results were negative. Therefore, it can be concluded that neither of the two flavonoid compositions will irritate human skin.

[0170] 2. Lactic acid stinging test

[0171] Test environment requirements: constant temperature and humidity environment, set temperature: 20℃, set humidity: 50%;

[0172] Test Method: A randomized, blinded, self-controlled method was adopted. A 10% lactic acid solution was used. One drop was applied to either side of the nasolabial fold using a cotton swab, with an equal volume of ultrapure water used as a negative control on the other side. Subjects were asked about their subjective symptoms at 2.5 minutes and 5 minutes, and scored on a 4-point scale (0 for no stinging, 1 for mild stinging, 2 for moderate stinging, and 3 for severe stinging). The two scores were then added together; a total score of ≥3 was considered a positive lactic acid stinging reaction.

[0173] Volunteers applied the product to the designated areas according to the instructions, ensuring even application. The product was used twice daily, morning and evening, with each application consisting of 0.5g (the size of two peanuts). The treatment period lasted four weeks, during which similar cosmetic products were discontinued.

[0174] Data collection: Two data collection sessions were conducted, one for initial data collection and the other for data collection 4 weeks after using the soothing product. Volunteers cleaned the test area and sat quietly for 20 minutes before the test.

[0175] Test results:

[0176]

[0177] If there is a statistically significant difference (P<0.05) in the subjective pain or itching scores between the sample group and the control group, then the product is considered to have a soothing effect on skin irritation caused by lactic acid.

[0178] Test results showed that the mean pain score of sample No. 2 (the ratio of apigenin, apigenin and kaempferol was 1:4:1) was significantly lower than that of the control group, proving that it had a better soothing effect.

[0179] This invention combines multiple flavonoid components and, based on a synergistic mechanism, improves the inflammation of atopic dermatitis at a lower dose through multi-target synergistic action. This reduces the need for high doses of single components and improves the overall safety and tolerability of the medication. Existing flavonoid compositions for treating atopic dermatitis are mostly plant extracts, which are complex in composition and difficult to control in terms of quality. The differences in composition between different batches affect the stability of the therapeutic effect.

[0180] This article selects flavonoid compositions formed by combining flavonoid monomers. This allows for the selection of the most effective flavonoid monomers for combination based on the specific pathological mechanisms and treatment needs of atopic dermatitis, improving the accuracy and effectiveness of treatment. Due to the relatively simple composition, quality control between batches is easier, ensuring the stability and reproducibility of treatment effects, which is more conducive to drug development and clinical application.

[0181] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A flavonoid composition for preventing and improving atopic dermatitis, characterized in that: include, The mixture contains apigenin, salinomycin, and kaempferol; wherein the mass ratio of apigenin, salinomycin, and kaempferol is 1:4:

1.

2. The use of the flavonoid composition according to claim 1 in the preparation of products for the prevention and improvement of atopic dermatitis, characterized in that: The product in question is a pharmaceutical or cosmetic product.

3. The application as described in claim 2, characterized in that: The dosage form of the medicine or cosmetic is a paste, patch, toner, essence, lotion, or cream.

4. The application as described in claim 3, characterized in that: The cosmetic product is a soothing face cream, wherein... The soothing face cream comprises skin conditioning agents, emulsifiers, thickeners, moisturizers, skin-nourishing agents, preservatives, colorants, fragrances, flavonoid compositions, and water; Based on the weight percentage of raw materials, the skin conditioning agent is 0.5-2%, the emulsifier is 2-5%, the thickener is 0.5-1.5%, the moisturizer is 10%-20%, the skin-nourishing agent is 8%-14%, the preservative is 0.1-0.6%, the colorant is 0.05-0.2%, the fragrance is 0.01-0.1%, the flavonoid composition is 0.05-0.2%, and the water balance is made up to 100%.

5. The application as described in claim 4, characterized in that: The skin conditioning agent is a saccharide isomer; the emulsifier is one or more of sucrose polystearate, glyceryl stearate, sodium stearoyl lactylate, and myristic acid; The thickener includes one or more of the following: ammonium acryloyldimethyl taurate / VP copolymer, sodium polyacryloyldimethyl taurate, sodium hyaluronate, xanthan gum, glyceryl polyacrylate, acrylate copolymer, sodium chloride, and potassium chloride.

6. The application as described in claim 5, characterized in that: The moisturizer includes one or more of the following: glycerin polyether-26, glycerin, propylene glycol, butylene glycol, pentylene glycol, hexanediol, sorbitol, panthenol, hyaluronic acid, ceramide, niacinamide, allantoin, and tremella polysaccharide. The emollients include one or more of the following: squalane, hydrogenated ethylhexyl olive oil, hydrogenated unsaponifiables, murum palm oil, babassu seed oil, cetearyl alcohol, and C10-18 triglycerides. The preservatives include one or more of phenoxyethanol, ethylhexylglycerin, and p-hydroxyacetophenone; The colorant is comfrey oil.

7. The application as described in claim 4, characterized in that: The method for preparing the soothing face cream includes, Weigh out the emulsifier, emollient, and colorant, heat to 80-85°C, stir until all components are completely dissolved and homogeneous, and prepare phase A, which is an oil phase, and keep it warm for later use. Weigh out the flavonoid composition, butylene glycol and appropriate amount of water according to the formula, heat to 40~60℃, stir and mix well to prepare phase B; Weigh out the moisturizer, thickener, skin conditioning agent, preservative and the remaining water, heat to 80~85℃, stir until all components are completely dissolved and homogeneous, prepare phase C, which is an aqueous phase, and keep warm for later use; Slowly pour the dissolved C phase into the A phase, cool to 60°C, then slowly add the C phase, and stir evenly while hot. Stop stirring; the soothing face cream is now ready.

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