Flavone composition for preventing and improving atopic dermatitis and application thereof

Abstract

The invention discloses a flavone composition capable of preventing and improving atopic dermatitis and application of the flavone composition, and belongs to the field of medicines and cosmetics. Cell morphology observation and beta-hexosaminidase detection prove that apigenin and chrysin have a good effect of inhibiting large cell strain degranulation; scratch experiments prove that kaempferol has a good effect of promoting interstitial closure of human keratinocytes, and then detection of atopic dermatitis related factor expression discovers that apigenin, chrysin and kaempferol are compounded to have the potential of enhancing the soothing effect. And human body safety evaluation and efficacy experiments are further carried out to screen out the optimal proportion of the soothing effect. The invention explores the multi-target synergistic prevention and improvement effect of the flavonoid compound on the aspects of allergy resistance, inflammation resistance and barrier repair, and expands the application and development value of the flavonoid compound in soothing cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of plant composition application, and specifically relates to a flavonoid composition capable of preventing and improving atopic dermatitis and application thereof. Background Art

[0002] Atopic dermatitis (AD) is a common skin disease with three major characteristics: chronicity, recurrence, and inflammation. It usually occurs in infancy and can persist into adulthood or even old age in severe cases. Infants and young children with AD often have other atopic diseases, including asthma and allergic rhinitis. The clinical symptoms of AD are mainly eczematoid lesions, accompanied by dry skin and severe itching. Some patients also have chapped skin and blood scabs. Since the treatment cycle of AD is long and recurrent, patients bear a huge burden both financially and mentally, which seriously affects their quality of life.

[0003] The pathogenesis of AD is complex, mainly involving imbalance of skin microbiota, immune disorders and skin barrier dysfunction. Allergens are one of the important triggering factors for the onset of AD. After allergens break through the skin barrier and enter the patient's body, they bind to IgE antibodies on the surface of mast cells, activate mast cells and cause degranulation reactions, and release a variety of inflammatory mediators, such as interleukin-4 (IL-4), interleukin-13 (IL-13), histamine, etc. 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, increase patients' scratching behavior, and then destroy the skin barrier, making it easier for external irritants and allergens to penetrate the skin and aggravate AD symptoms.

[0004] At present, the commonly used drugs for the treatment of AD are mainly divided into topical drugs and oral drugs. Topical drugs such as glucocorticoids are currently the first-line clinical drugs for the treatment of AD, but long-term and large-scale use will produce adverse reactions such as skin atrophy and capillary dilation. Oral drugs such as antihistamines and immunosuppressants damage the digestive system and liver and kidney function. In recent years, many new biological agents have been launched for AD, such as dupilumab, which can specifically bind to and block the co-receptor subunits of IL-4 and IL-13, inhibit the signal transduction of IL-4 and IL-13, and show significant efficacy in the treatment of atopic dermatitis. However, its high price has limited its widespread application to a certain extent. Therefore, the development of safe, efficient and low-cost multi-target therapeutic drugs has become a research hotspot for AD.

[0005] Flavonoids are a type of natural product that is widely distributed in plants in nature. There are many types of flavonoids, which have been shown to have multiple pharmacological activities such as antioxidant, anti-inflammatory, and anti-cancer. Compared with traditional therapeutic drugs, the metabolic process of flavonoids in the body is relatively mild and has a higher safety.

[0006] At present, the application research of flavonoids in treating inflammation basically stays at the level of single ingredients. However, atopic dermatitis is a complex pathological process, involving multiple interactions such as immune imbalance, excessive release of inflammatory factors, and impaired skin barrier function. It is difficult for a single ingredient to solve the problem. Existing flavonoid compositions for the treatment of atopic dermatitis are mostly plant extracts, with complex ingredients and high difficulty in quality control. The differences in ingredients between different batches affect the stability of the treatment effect, and the study of its mechanism of action is relatively difficult, making it difficult to determine which specific ingredients play a key role in the treatment process. Summary of the invention

[0007] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

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

[0009] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a flavonoid composition for preventing and improving atopic dermatitis.

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

[0011] Apigenin, chrysin and kaempferol; wherein the mass ratio of apigenin, chrysin and kaempferol is 1-5:1-5:1-5.

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

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

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide a flavonoid composition for use in preparing a product for preventing and improving atopic dermatitis.

[0015] As a preferred solution of the application of the present invention, the product is a health product, a medicine or a cosmetic.

[0016] As a preferred embodiment of the application of the present invention, the dosage forms of the medicine or cosmetic include ointment, paste, patch, toner, essence water, lotion and cream.

[0017] As a preferred embodiment of the application of the present invention, the cosmetic comprises a soothing cream, wherein:

[0018] The soothing cream includes a skin conditioner, an emulsifier, a thickener, a moisturizer, an emollient, a preservative, a colorant, a fragrance, a flavonoid composition and water;

[0019] Calculated by weight percentage of the raw materials, the skin conditioner is 0.5-2%, the emulsifier is 2-5%, the thickener is 0.5-1.5%, the moisturizer is 10%-20%, the emollient is 8%-14%, the preservative is 0.1-0.6%, the colorant is 0.05-0.2%, the flavor is 0.01-0.1%, the flavonoid composition is 0.05-0.2%, and the balance of water is supplemented to 100%.

[0020] As a preferred embodiment of the application of the present invention, the skin conditioning agent is a carbohydrate 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 ammonium acryloyldimethyltaurate / VP copolymer, sodium polyacryloyldimethyltaurate, sodium hyaluronate, xanthan gum, glycerol polypropionate, acrylic acid (ester) copolymer, sodium chloride, and potassium chloride;

[0022] The moisturizing agent includes one or more of glyceryl polyether-26, glycerin, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, sorbitol, panthenol, hyaluronic acid, ceramide, niacinamide, allantoin, and tremella polysaccharide;

[0023] The emollient includes one or more of squalane, hydrogenated ethylhexyl olive oil, hydrogenated olive oil unsaponifiable matter, Murumuru palm fat, babassu seed oil, cetearyl alcohol, and C10-18 fatty acid triglycerides;

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

[0025] The colorant is lithospermum oil.

[0026] As a preferred embodiment of the application of the present invention, the preparation method of the soothing facial cream comprises:

[0027] Weigh the emulsifier, emollient and colorant, heat to 80-85°C, stir until all components are completely dissolved and uniformly prepared into phase A, which is the oil phase, and keep warm for later use;

[0028] Weigh the moisturizer, thickener, skin conditioner, preservative and the remaining water, heat to 80-85° C., stir until all the raw materials are completely dissolved and uniformly prepared as phase B, which is the water phase, and keep warm for later use;

[0029] Weigh the flavonoid composition, butanediol and appropriate amount of water in the formula, heat to 40-60° C., stir and mix to prepare phase C;

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

[0031] Stop stirring to create a soothing cream.

[0032] Beneficial effects of the present invention:

[0033] (1) The present invention verifies through experiments that chrysin and apigenin have a good effect of inhibiting the degranulation of rat basophilic leukemia cells without affecting cell proliferation and morphology.

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

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

[0036] (4) The present invention also provides a soothing facial cream containing a flavonoid composition. The addition of the flavonoid composition to the facial cream can relieve skin itching and reduce the degree of allergic redness, which is of great significance for further expanding the application of flavonoid compounds in cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0038] Figure 1This is a graph showing the experimental results of the effects of different types of flavonoid compounds on the degranulation morphology of RBL-2H3 cells in Example 1 of the present invention.

[0039] Figure 2 This is a graph showing the experimental results of the effects of different types of flavonoid compounds on the release of β-hexosaminidase from RBL-2H3 cells in Example 1 of the present invention.

[0040] Figure 3 This is a graph showing the experimental results of the effects of different types of flavonoid compounds on HaCaT cell migration in Example 2 of the present invention.

[0041] Figure 4 This is a graph showing the experimental results of the effects of flavonoid compounds in different ratios on the expression of the inflammatory factor IL-4 in Example 3 of the present invention.

[0042] Figure 5 This is a graph showing the experimental results of the effects of flavonoid compounds in different ratios on the expression of the inflammatory factor IL-13 in Example 3 of the present invention.

[0043] Figure 6 This is a graph showing the experimental results of the effects of flavonoid compounds in different ratios on the expression of the inflammatory factor histamine in Example 3 of the present invention. DETAILED DESCRIPTION

[0044] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0045] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present 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 term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0047] The flavonoid compounds used in the experiments of the present invention were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.:

[0048] Apigenin (Product No. A106675, Batch No. C2329727);

[0049] Chrysin (Cat. No. C110078, Batch No. F1812032);

[0050] Kaempferol (Product No. K107144, Batch No. G2318065).

[0051] Example 1

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

[0053] 1) Recovery, culture, subculture and cryopreservation of RBL-2H3 cells

[0054] (1) Cell recovery: Take the cryovials out of the liquid nitrogen tank, accelerate thawing in a 37°C water bath, disinfect with 75% alcohol and move to a clean bench. Aspirate the liquid in the cryovials and transfer to a centrifuge tube containing about 6 mL of complete culture medium. Centrifuge at 1000 rpm for 5 min. Discard the upper layer of liquid, resuspend with 1 mL of new 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 antibody) in a constant temperature incubator at 37° C. and 5% CO 2 .

[0056] (3) Cell passaging: When the cell density reaches 80-90%, discard the supernatant, wash twice with PBS, digest with 0.25% trypsin + 0.03% EDTA, incubate for 2-3 min, centrifuge at 1000 rpm for 5 min, and pass the cells at a ratio of 1:3.

[0057] (4) Cell cryopreservation: Collect cells, add serum-free non-programmed cell cryopreservation solution, transfer to cryopreservation tubes, mark the name, number, time, and name, and store the cryopreservation tubes in a -80°C refrigerator. The next day, transfer the cryopreservation tubes to a liquid nitrogen tank for long-term storage.

[0058] The present invention detects the morphological changes and β-hexosaminidase release of RBL-2H3 cells (purchased from Beina Chuanglian Biotechnology Research Institute), evaluates and compares the effects of various flavonoid compounds on the degranulation of RBL-2H3 cells, and verifies the excellent effects of apigenin and chrysin in anti-allergic efficacy.

[0059] Using dimethyl sulfoxide (DMSO) as solvent, the flavonoids to be tested (luteolin, apigenin, chrysin, quercetin, kaempferol, fisetin) were prepared into working solutions with a molar concentration of 50 mM, and then packaged and stored at -20 ° C to avoid repeated freezing and thawing. The working solution was thawed at room temperature before use, and gradiently diluted with EMEM basal medium. After each dilution, it was ultrasonicated for 30 minutes to ensure complete dissolution. Finally, the flavonoids were prepared into dilutions with a molar concentration of 5 μM as samples 1-6 for the inhibition of RBL-2H3 degranulation experiment.

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

[0061] 1. RBL-2H3 cell morphology changes:

[0062] (1) Take RBL-2H3 cells in the logarithmic growth phase and prepare a cell suspension with a concentration of 1*10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and placed in a 5% CO2, 37°C incubator. They were cultured with EMEM complete medium for 12 h until the cells adhered to the wall and the confluence reached 60%;

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

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

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

[0066] (5) Incubate on ice for 10 min, 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 fixation for 20 min, remove the supernatant, add 100 μL of toluidine blue dye for staining for 10 min, wash off the excess dye with PBS, and then add 1 mL of PBS. Immediately observe the cell morphology under an optical microscope and take photos for record.

[0068] The experimental results are as follows Figure 1As shown, the RBL-2H3 cells in the blank group were spindle-shaped with neat edges and full shapes. After being stimulated, they degranulated, became swollen, rounded, and irregular in shape. The cytoplasm became loose, the staining became lighter, and vacuolar structures appeared.

[0069] Apigenin and chrysin have the best effect in inhibiting the morphological changes of RBL-2H3 cells and have a good effect in inhibiting the degranulation of RBL-2H3 cells.

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

[0071] 1) Substrate and stop solution preparation:

[0072] (1) Weigh 4-nitrophenyl N-acetyl-β-glucosamine (17.1 mg), dissolve it in citric acid-sodium citrate buffer, and make up to 50 mL with a final concentration of 1 mM. Filter and sterilize with a filter membrane under sterile conditions;

[0073] (2) Weigh 529.5 mg of anhydrous calcium carbonate and 420.6 mg of sodium bicarbonate respectively, 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 treatment and supernatant collection:

[0075] (1) Take RBL-2H3 cells in the logarithmic growth phase and prepare a cell suspension at 2*10 5 The cells were seeded at a density of 100 cells / well in a 48-well plate and placed in a 5% CO2, 37°C incubator. They were cultured with EMEM complete medium for 24 h until the cells adhered to the wall and the confluence reached 80%;

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

[0077] (3) Remove the supernatant and carefully rinse twice with PBS. Add EMEM basal medium containing 5 μM of the flavonoid compound to be tested to the sample group, and add an equal 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 sample group, and an equal amount of fresh EMEM basal medium was added to the control group, and the culture was continued for 4 h;

[0079] (5) Place the cells in an ice bath for 10 min to terminate the reaction. Collect the cell supernatant into a new EP tube and centrifuge at 4°C and 14,000 rpm for 5 min. Take the supernatant for testing.

[0080] (6) Take 50 μL of cell supernatant and 50 μL of substrate in a 96-well plate and 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 a microplate reader.

[0081] 3) Data processing:

[0082] The cell activity value measured in the model group represented 100% degranulation.

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

[0084] The release rate of each group was calculated, and the statistical analysis was performed using graphpad for One-way ANOVA analysis. ### compared with the control group p < 0.01, *** compared with the model group p < 0.01

[0085] The experimental results are as follows Figure 2 As shown, apigenin and chrysin have the best effect in inhibiting the release of β-hexosaminidase from RBL-2H3 cells, and have a good effect in inhibiting the degranulation of RBL-2H3 cells.

[0086] In summary, apigenin and chrysin have the best effect in inhibiting the degranulation of RBL-2H3 cells, proving that they have good anti-allergic effects.

[0087] Example 2

[0088] After skin tissue injury, human immortalized keratinocytes HaCaT cells can quickly migrate to the site of injury. By observing the effect of drugs on cell scratch healing, potential tissue repair drugs can be discovered.

[0089] 1) Recovery, culture, passage and cryopreservation of HaCaT cells:

[0090] (1) Cell recovery: Take the cryovials out of the liquid nitrogen tank and accelerate thawing in a 37°C water bath. After disinfection with 75% alcohol, aspirate the liquid in the cryovials into a centrifuge tube containing about 6 mL of complete culture medium and centrifuge at 1000 rpm for 5 min. Discard the upper layer of liquid, resuspend with 1 mL of new culture medium, and 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 antibody) in a constant temperature incubator at 37° C. and 5% CO 2 .

[0092] (3) Cell passaging: When the cell density reaches 80-90%, discard the supernatant, wash twice with PBS, digest with 0.25% trypsin + 0.03% EDTA, incubate for 5-6 min, centrifuge at 1200 rpm for 5 min, and pass the cells at a ratio of 1:2.

[0093] (4) Cell cryopreservation: Collect cells, add serum-free non-programmed cell cryopreservation solution, transfer to cryopreservation tubes, mark the name, number, time, and name, and store the cryopreservation tubes in a -80°C refrigerator. The next day, transfer the cryopreservation tubes to a liquid nitrogen tank for long-term storage.

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

[0095] Using dimethyl sulfoxide (DMSO) as solvent, the flavonoids to be tested (luteolin, apigenin, chrysin, quercetin, kaempferol, fisetin) were prepared into working solutions with a molar concentration of 50 mM, and then packaged and stored at -20 °C to avoid repeated freezing and thawing. The working solutions were thawed at room temperature before use, and gradiently diluted with DMEM basal medium. After each dilution, they were ultrasonicated for 30 minutes to ensure complete dissolution. Finally, the flavonoids were prepared into dilutions with a molar concentration of 5 μM as samples 1-6 for scratch tests.

[0096] 2) Cell scratch assay:

[0097] (1) Insert fixation: Use sterile tweezers to carefully pick up the ibidi culture-inserts (2 well) and fix them in the middle of each well of a 24-well plate.

[0098] (2) Cell inoculation: HaCaT cells in the logarithmic growth phase were plated at 5.0×10 5 The cells were inoculated at a density of 100 μL / mL (cultured in DMEM complete medium) in the inserts, with 70 μL per well.

[0099] (3) Grouping and dosing: divided into control group, model group and sample group, with three replicates in each group;

[0100] After each group of cells was incubated, the insert was carefully removed with sterile tweezers, and a blank gap of uniform width of 500 μm similar to a scratch appeared. The supernatant was discarded and the sample was carefully washed 2-3 times with PBS buffer to remove the residual cells or floating dead cells in the scratch.

[0101] Control group: add fresh DMEM basal medium and continue culturing for 12h and 24h;

[0102] Model group: DMEM basal medium (containing 10% FBS, 1% double antibody) containing IL-4, IL-13 (final concentration of 50 ng / mL) and TNF-α (final concentration of 100 ng / mL) was added and cultured for 12 h and 24 h;

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

[0104] (4) Observation under a microscope: Immediately observe and record the scratch area at 0 h using an optical microscope, and take photos and save the scratch images at each time point at 12 h and 24 h.

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

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

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

[0108] Example 3

[0109] To explore the effects of flavonoids in different ratios on the expression of atopic dermatitis-related factors.

[0110] Sample preparation: Using dimethyl sulfoxide (DMSO) as solvent, the flavonoid compounds to be tested (apigenin, chrysin, kaempferol) were prepared into a working solution with a molar concentration of 50mM, and the solution was divided and stored at -20°C to avoid repeated freezing and thawing. The working solution was thawed at room temperature before use, and gradiently diluted with EMEM basal medium. After each dilution, it was ultrasonicated for 30 minutes to ensure complete dissolution, and the flavonoid compounds were prepared into a dilution with a molar concentration of 5μM. Flavonoid compositions with the same molar concentration but different ratios were added, and divided into samples 1-11 as follows:

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

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

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

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

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

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

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

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

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

[0120] No. 10 (the ratio of apigenin: chrysin: kaempferol is 1:0:5) ；

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

[0122] ELISA determination of 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 to Th2 cells, making the immune response tend to humoral immunity.

[0125] Regarding the skin barrier function, they will destroy tight junction proteins, causing barrier damage, and interfere with the production of mucus proteins, making the skin dry and scaly.

[0126] In the inflammatory response, these two cytokines can recruit eosinophils, the toxic proteins released by which can damage tissues. At the same time, they can induce keratinocytes and other cells to release inflammatory mediators, attracting more immune cells to gather and aggravating inflammation.

[0127] Histamine plays a key role in itching in atopic dermatitis. It can directly stimulate the sensory nerve endings of the skin, transmit itching signals, and induce patients to scratch, forming a vicious cycle of itching and 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 treatment and supernatant collection:

[0130] (1) Take RBL-2H3 cells in the logarithmic growth phase and prepare a cell suspension at 2*10 5 The cells were seeded at a density of 100 cells / well in a 48-well plate and placed in a 5% CO2, 37°C incubator. They were cultured with EMEM complete medium for 24 h until the cells adhered to the wall and the confluence reached 80%;

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

[0132] (3) Remove the supernatant and carefully rinse twice with PBS. Add EMEM basal medium containing test samples No. 1-11 to the sample group, and add an equal amount 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 sample group, and an equal amount of fresh EMEM basal medium was added to the control group, and the culture was continued for 4 h;

[0134] (5) Place the cells in an ice bath for 10 min to terminate the reaction. Collect the cell supernatant into a new EP tube and centrifuge at 4°C and 14,000 rpm for 5 min. Take the supernatant for testing.

[0135] 2) Detect the levels of IL-4, IL-13 and histamine in the cell supernatant using ELISA kits according to the instructions.

[0136] 3) Data processing

[0137] Use graphpad to perform significant difference analysis (One-way ANOVA Tukey's Method)

[0138] The experimental results are as follows Figures 4 to 6 As shown, each group of samples can inhibit the expression of IL-4, IL-13 and histamine, among which the best effect of inhibiting multiple inflammatory factors is when the ratio of apigenin:chrysin:kaempferol is 1:2:1 and 1:4:1.

[0139] Example 4

[0140] Preparation of a soothing facial cream containing a flavonoid composition (apigenin, chrysin, kaempferol):

[0141] The steps include:

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

[0143] Weigh 1.4 g of sodium polyacryloyldimethyl taurate, 2 g of saccharide isomers, 4 g of glyceryl polyether-26, 10 g of butylene glycol, 6 g of niacinamide and 130 g of water, stir and mix to prepare phase B, which is the aqueous phase, and heat for later use;

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

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

[0146] (3) stopping stirring to prepare a soothing facial cream containing the flavonoid (apigenin, chrysin, kaempferol) composition.

[0147] Example 5

[0148] Safety and efficacy evaluation of soothing and repairing facial cream containing flavonoid composition:

[0149] Human patch test:

[0150] In accordance with the "Technical Specifications for Safety of Cosmetics", 30 volunteers who met the experimental requirements were recruited for human patch tests, including open and closed patch experiments. Informed consent forms were signed by the volunteers before the experiment.

[0151] Sample: Take the facial cream prepared in Example 4 of the present invention for experiment, wherein

[0152] Sample No. 1 - the ratio of apigenin:chrysin:kaempferol is 1:2:1;

[0153] Sample No. 2 - the ratio of apigenin:chrysin:kaempferol is 1:4:1;

[0154] Reference substance No. 3—no flavonoid composition added.

[0155] Closed patch test:

[0156] Wipe the test site with 70% ethanol, add the sample to the patch chamber after drying, with a volume of 20 μL, and attach the patch to the inside of the arm with tape;

[0157] After 24 hours, remove the patch, mark the test site with a marker, observe the skin reaction at 30 minutes, 12 hours, and 24 hours, and record the results according to the skin reaction grading standards in the "Technical Specifications for Safety of Cosmetics".

[0158] Criteria:

[0159]

[0160] Test results:

[0161]

[0162]

[0163] Open patch test:

[0164] The test area is selected on the flexor side of the forearm, with a size of about 3*3cm 2 , try to avoid any contact with any reagents except applying the samples. Ask the volunteers to apply the samples evenly on the test area, twice a day, for 7 consecutive days, and observe the degree of skin reaction and score it according to the grading standard.

[0165] Scoring criteria:

[0166]

[0167] Test results:

[0168]

[0169] All samples did not cause positive symptoms such as redness and swelling in the application site of the 30 volunteers, and the results were all negative. Therefore, it can be determined that the flavonoid compositions of the two ratios will not irritate human skin.

[0170] 2. Lactic acid sting test

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

[0172] Test method: Using the principle of randomization, blinding, and self-control, use a cotton swab to dip 1 drop of 10% lactic acid solution and apply it to either side of the volunteer's nasolabial groove, and use an equal amount of ultrapure water on the other side as a negative control. Ask the subjects about their subjective symptoms at 2.5 minutes and 5 minutes, respectively, and score them according to the 4-point method (0 for no tingling, 1 for mild tingling, 2 for moderate tingling, and 3 for severe tingling). Then add the two scores together, and those with a total score of ≥3 points are considered positive for lactic acid tingling reaction.

[0173] The volunteers used the product on the designated parts according to the instructions, and were required to apply it evenly, once in the morning and once in the evening, with a dosage of 0.5g (the size of two peanuts) each time, for a usage period of 4 weeks. During the test period, similar cosmetics were discontinued;

[0174] Data collection: A total of two data collections were conducted, divided into initial value collection and data collection after 4 weeks of using soothing products. Volunteers cleaned the test area and sat quietly for 20 minutes before the test.

[0175] Test results:

[0176]

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

[0178] The test results showed that the mean tingling sensation score in the test area of ​​sample No. 2 (the ratio of apigenin: chrysin: kaempferol was 1:4:1) was significantly lower than that of the control group, proving that it has a better soothing effect.

[0179] The present invention compounds a variety of flavonoid components, based on a synergistic mechanism, to synergistically improve atopic dermatitis inflammation at multiple targets at a lower dose, reducing the high-dose use of a single component and improving overall medication safety and tolerability; existing flavonoid compositions for the treatment of atopic dermatitis are mostly plant extracts, with complex ingredients and greater difficulty in quality control, and differences in ingredients between different batches affect the stability of the treatment effect.

[0180] The flavonoid composition formed by compounding flavonoid monomers in this paper can select the most effective flavonoid monomers for compounding according to the specific pathological mechanism and treatment needs of atopic dermatitis, thereby improving the accuracy and effectiveness of treatment. Due to the relatively simple ingredients, the quality between batches is easier to control, ensuring the stability and repeatability of the treatment effect, 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 rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A flavonoid composition for preventing and improving atopic dermatitis, characterized in that: include, Apigenin, chrysin and kaempferol; wherein the mass ratio of apigenin, chrysin and kaempferol is 1-5:1-5:1-5.

2. The flavonoid composition according to claim 1, characterized in that: The mass ratio of apigenin, chrysin and kaempferol is 1:2 to 4:

1.

3. The flavonoid composition according to claim 1 or 2, characterized in that: The mass ratio of apigenin, chrysin and kaempferol is 1:4:

1.

4. Use of the flavonoid composition according to any one of claims 1 to 3 in the preparation of a product for preventing and improving atopic dermatitis.

5. The use according to claim 4, characterized in that: The product is a health product, medicine or cosmetic.

6. The use according to claim 5, characterized in that: The dosage forms of the medicine or cosmetic include ointment, paste, patch, toner, essence water, lotion and cream.

7. The use according to claim 6, characterized in that: The cosmetic comprises a soothing cream, wherein: The soothing cream includes a skin conditioner, an emulsifier, a thickener, a moisturizer, an emollient, a preservative, a colorant, a fragrance, a flavonoid composition and water; Calculated by weight percentage of the raw materials, the skin conditioner is 0.5-2%, the emulsifier is 2-5%, the thickener is 0.5-1.5%, the moisturizer is 10%-20%, the emollient is 8%-14%, the preservative is 0.1-0.6%, the colorant is 0.05-0.2%, the flavor is 0.01-0.1%, the flavonoid composition is 0.05-0.2%, and the balance of water is supplemented to 100%.

8. The use according to claim 7, characterized in that: The skin conditioning agent is a sugar 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 ammonium acryloyldimethyltaurate / VP copolymer, sodium polyacryloyldimethyltaurate, sodium hyaluronate, xanthan gum, glycerol polypropionate, acrylic acid (ester) copolymer, sodium chloride, and potassium chloride.

9. The use according to claim 8, characterized in that: The moisturizing agent includes one or more of glyceryl polyether-26, glycerin, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, sorbitol, panthenol, hyaluronic acid, ceramide, niacinamide, allantoin, and tremella polysaccharide; The emollient includes one or more of squalane, hydrogenated ethylhexyl olive oil, hydrogenated olive oil unsaponifiable matter, Murumuru palm fat, babassu seed oil, cetearyl alcohol, and C10-18 fatty acid triglycerides; The preservative includes one or more of phenoxyethanol, ethylhexylglycerin, and p-hydroxyacetophenone; The colorant is lithospermum officinale oil.

10. The use according to claim 7, characterized in that: The preparation method of the soothing facial cream comprises: Weigh the emulsifier, emollient and colorant, heat to 80-85°C, stir until all components are completely dissolved and uniformly prepared into phase A, which is the oil phase, and keep warm for later use; Weigh the flavonoid composition, butanediol and appropriate amount of water in the formula, heat to 40-60° C., stir and mix to prepare phase B; Weigh the moisturizer, thickener, skin conditioner, preservative and the remaining water, heat to 80-85° C., stir until all the raw materials are completely dissolved and uniformly prepared as phase C, which is the water phase, and keep warm for later use; Slowly pour the dissolved phase C into phase A, cool to 60°C and slowly add phase C, stirring evenly while hot; Stop stirring to create a soothing cream.

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