Composition beneficial to skin health and application thereof

Abstract

The invention provides a composition for sensitive skin. The composition is prepared from the following components in parts by weight: 4 to 40 parts of ceramide, 100 to 300 parts of grape seed extract, 100 to 300 parts of quercetin and 200 to 250 parts of citrus flavone. The composition has a remarkable effect on preventing, relieving and / or treating sensitive skin, atopic dermatitis, xerodermia and the like due to the synergistic effect of all the components. Specifically, in an LPS (lipopolysaccharide) induced macrophage inflammation model, a DNFB (2, 4-dinitrofluorobenzene) specific dermatitis animal model and an AEW (acetone-ether) skin barrier injury animal model, the composition provided by the invention can improve the skin barrier, immune inflammatory response, nerve response and skin blood vessel couperose skin of a mouse, and compared with the application of a single component, the composition provided by the invention has the advantages that the effect of improving the skin barrier, immune inflammatory response, nerve response and skin blood vessel couperose skin can be achieved; the obvious effect and the synergistic effect are realized.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, health food or cosmetics, and in particular to a composition beneficial to skin health and application thereof. Background Art

[0002] The skin is the largest organ covering the human body. It has the important mission of resisting harmful external environments and protecting the body's health. It also plays the role of sensing changes in the external environment and transmitting signals to enable the body to make adaptive changes. In recent years, with the rapid development of the domestic economy and the accelerated pace of life of residents, internal and external factors such as air pollution, staying up late to work overtime, life pressure, anxiety, excessive skin care, plastic surgery, and irritating diet have caused damage to the human skin barrier and induced a series of skin health problems, such as sensitive skin, atopic dermatitis, and dry skin. Skin health problems have become common social problems that affect people's daily lives. Taking sensitive skin as an example, according to the "Guidelines for Clinical Diagnosis and Treatment of Sensitive Skin in China (2024 Edition)", more than 50% of adults have experienced the process of sensitive skin.

[0003] Sensitive skin is a special type of skin, a highly reactive state of the skin under physiological or pathological conditions, manifested by burning, stinging, itching and tightness when stimulated by physical, chemical, mental and other factors. Atopic dermatitis (AD) is a chronic inflammatory skin disease related to genetic allergies and other factors, manifested by repeated scratching and severe itching, polymorphic skin lesions and exudative tendency. Skin xerosis is a common skin disease, which refers to any condition where the skin is dry beyond the normal state, and is the initial manifestation of skin barrier damage. Its clinical manifestations are mainly dry skin, bran-like desquamation, accompanied by systemic itching. Sensitive skin, atopic dermatitis and xerosis have many similarities in pathogenesis and clinical manifestations. Most of them are related to skin barrier dysfunction in pathogenesis, and most of them are manifested in dry and tight skin, skin inflammation, skin itching, skin redness and abnormal transepidermal water loss in clinical manifestations.

[0004] Previous studies have shown that gentle care, while taking into account skin barrier repair, reducing neurovascular hyperreactivity, controlling inflammatory responses, and moisturizing, is the overall idea for solving the above skin problems. Taking the intervention of sensitive skin as an example, according to the "Chinese Expert Consensus on the Diagnosis and Treatment of Sensitive Skin", skin sensitivity can be improved by improving skin barrier function, regulating skin immune inflammation, nerve reactions, and red blood reactions. At present, the main treatment options for sensitive skin, atopic dermatitis, etc. are oral antihistamines and topical hormones, all of which have side effects such as drug dependence and cannot be used for a long time. Therefore, it is of great significance to develop a composition and product that is beneficial to skin health and solve related skin problems. Summary of the invention

[0005] Based on this, the present invention provides a composition that is beneficial to skin health. The composition screens a scientific formula, and through multi-indicator and multi-level experimental research, proves that various ingredients are reasonably coordinated and synergistic, to solve skin health problems, effectively prevent, alleviate and / or treat sensitive skin, atopic dermatitis, dry skin and other related skin diseases or symptoms. Specifically, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a composition, which comprises the following components in parts by weight:

[0007] 4-40 parts of ceramide, 100-300 parts of grape seed extract, 100-300 parts of quercetin, and 200-250 parts of citrus flavonoids.

[0008] In certain specific embodiments, the composition comprises the following components in parts by weight:

[0009] 4-40 parts of ceramide, 200 parts of grape seed extract, 200 parts of quercetin, 225 parts of citrus flavonoids.

[0010] In certain specific embodiments, the composition comprises the following components in parts by weight:

[0011] 18 parts of ceramide, 200 parts of grape seed extract, 200 parts of quercetin, 225 parts of citrus flavonoids.

[0012] In certain specific embodiments, the composition may further include 50-200 parts of Centella asiatica extract, 20-80 parts of Haematococcus pluvialis astaxanthin, and 2500-5000 parts of collagen peptide.

[0013] The second aspect of the present invention provides use of the composition of the first aspect in preparing a product beneficial to skin health.

[0014] In certain embodiments, the benefit to skin health refers to preventing, alleviating and / or treating diseases and / or symptoms associated with skin barrier dysfunction.

[0015] In certain embodiments, the skin barrier dysfunction-related diseases and / or symptoms are selected from one or more of sensitive skin, atopic dermatitis and xeroderma.

[0016] In certain specific embodiments, the symptoms associated with skin barrier dysfunction are selected from one or more of dry and tight skin, skin inflammation, skin itching, skin redness and abnormal transepidermal water loss.

[0017] In certain specific embodiments, the prevention, alleviation and / or treatment of diseases and / or symptoms associated with skin barrier dysfunction includes preventing skin barrier damage, repairing skin barrier damage, reducing transepidermal water loss, improving skin immune response and inflammatory infiltration, and / or reducing skin vascular neurosensitivity.

[0018] In certain embodiments, the skin is human facial skin.

[0019] The third aspect of the present invention provides a food, which comprises the composition of the first aspect and an auxiliary material acceptable in food.

[0020] In certain specific embodiments, the acceptable excipients in the food include fillers, lubricants, disintegrants, thickeners, acidulants, sweeteners, and preservatives.

[0021] In certain specific embodiments, the food is a health food.

[0022] The fourth aspect of the present invention provides a cosmetic comprising the composition of the first aspect and an additive acceptable in cosmetics.

[0023] In certain specific embodiments, the cosmetic is essence water, essence milk, sleeping mask, body lotion, cleanser, pressed powder, massage cream, loose powder, sunscreen or hand cream.

[0024] In certain specific embodiments, the additives include any one or more combinations of surfactants, thickeners, conditioners, moisturizers, preservatives, chelating agents, fragrances and water.

[0025] The fifth aspect of the present invention provides a method for preparing the composition, comprising the following steps: weighing the components, mixing, grinding, and sieving to obtain the composition.

[0026] In some embodiments, the mixing may be combined with stirring to ensure uniform mixing of the ingredients.

[0027] In certain embodiments, the mixing is performed at 20-30°C; preferably 25°C.

[0028] In some embodiments, the mixing is performed in a vacuum or inert gas environment.

[0029] Advantageous Effects of the Invention

[0030] Compared with the prior art, the composition of the present application has at least the following beneficial effects:

[0031] The present invention provides a composition for benefiting skin health, wherein the components of the composition work synergistically and have significant efficacy in preventing, alleviating and / or treating skin diseases or symptoms such as sensitive skin, atopic dermatitis, and dry skin. Specifically, in the LPS (lipopolysaccharide)-induced macrophage inflammation model, the DNFB (2,4-dinitrofluorobenzene) atopic dermatitis animal model, and the AEW (acetone-ether) skin barrier damage animal model, the composition of the present invention can improve the skin barrier damage, immune inflammatory response, neural response, and skin vascular red blood streaks in mice, and has significant efficacy and synergistic effect compared to the use of a single component. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The back skin appearance of mice in different intervention groups on days 1, 7, and 14 in the 2,4-dinitrofluorobenzene (DNFB)-induced skin inflammation model is shown;

[0033] Figure 2 H&E staining of the back skin of mice in different intervention groups in the 2,4-dinitrofluorobenzene (DNFB)-induced skin inflammation model is shown;

[0034] Figure 3 H&E staining of the auricle skin of mice in different intervention groups in the 2,4-dinitrofluorobenzene (DNFB)-induced skin inflammation model is shown;

[0035] Figure 4 Shows the staining of mast cells in the back skin of mice in different intervention groups in the 2,4-dinitrofluorobenzene (DNFB)-induced skin inflammation model;

[0036] Figure 5 The IL-4 and IL-5 levels in the skin of mice in different intervention groups in the 2,4-dinitrofluorobenzene (DNFB)-induced skin inflammation model are shown, where "#" indicates that the difference between the model group and the blank group is statistically significant (P<0.05), and "***" indicates that the difference between the experimental group and the model group is statistically significant (P<0.001).

[0037] Figure 6The IL-4 and IL-5 levels in the skin of mice in different intervention groups in the 2,4-dinitrofluorobenzene (DNFB)-induced skin inflammation model are shown, where "#" indicates that the difference between the model group and the blank group is statistically significant (P<0.05), and "**, ***" indicates that the difference between the experimental group and the model group is statistically significant (P<0.01, P<0.001).

[0038] Figure 7 The ET-1 content in the back skin of mice is shown. “#” indicates that the difference between the model group and the blank group is statistically significant (P<0.05), and “*”, “**” indicate that the difference between the model group and the experimental group is statistically significant (P<0.05, P<0.01).

[0039] Figure 8 TRPV1 content in the back skin of mice, “#” indicates that the difference between the model group and the blank group was statistically significant (P<0.05), “*,” “*,” “**” indicates that the difference between the model group and the experimental group was statistically significant (P<0.05, P<0.01, P<0.001).

[0040] Fig. 9 IgE content in the back skin of mice, “##” indicates that the difference between the blank group and the model group is statistically significant (P<0.01), “###” indicates that the difference between the model group and the blank group is statistically significant (P<0.001), “*”, “*” indicates that the difference between the treatment group and the model group is statistically significant (P<0.05, P<0.01).

[0041] Fig.10 The back skin appearance of mice in different intervention groups in the acetone ether (AEW)-induced dry and itchy skin model on days 1, 7, and 14 is shown;

[0042] Fig.11 HE staining of the back skin of mice in different intervention groups in the acetone ether (AEW)-induced dry skin itching model is shown;

[0043] Fig.12 The LOR content of the back skin of mice in different intervention groups of the acetone ether (AEW)-induced dry and itchy skin model is shown, where "####" indicates that the difference between the blank group and the model group is statistically significant (P<0.0001).

[0044] Fig.13The FLG content in the back skin of mice in different intervention groups of the acetone ether (AEW)-induced dry and itchy skin model is shown. "####" indicates that the difference between the blank group and the model group is statistically significant (P<0.0001), and "*" indicates that the difference between the experimental group and the model group is statistically significant (P<0.05).

[0045] Fig.14 The AQP3 content in the back skin of mice in different intervention groups of the acetone ether (AEW)-induced dry and itchy skin model is shown. "#" indicates that the difference between the blank group and the model group is statistically significant (P<0.05). Specific implementation methods

[0047] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0048] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0049] As used herein, the optional scope of the terms "and / or", "or / and", and "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, said any and all combinations including any two related listed items, any more related listed items, or a combination of all related listed items.

[0050] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0051] As used herein, "the first aspect", "the second aspect", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "the first", "the second", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0052] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0053] In the present application, the percentage contents involved, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0054] As used herein, the term "sensitive skin" refers to a highly reactive state of the skin under physiological or pathological conditions, mainly occurring on the face, and clinically manifested as subjective symptoms such as paroxysmal or periodic burning, stinging, flushing, itching, and tightness when stimulated by physical, chemical, mental, etc. factors, with or without objective signs such as erythema, scaling, and capillary dilation. The main methods for treating sensitive skin are to prevent skin barrier damage, repair skin barrier damage, improve skin immune response and inflammatory infiltration, and / or reduce skin vascular neurosensitivity.

[0055] As used herein, the term "atopic dermatitis (AD)" is a chronic, recurrent, inflammatory skin disease with recurrent chronic eczematous rashes as the main clinical manifestation, accompanied by significant dry skin and itching. Symptoms include dry skin, itching and inflammation, which are common in young children, but can also occur at any age. Repairing skin barrier damage and improving skin immune response and inflammatory infiltration are the main methods for treating atopic dermatitis.

[0056] As used herein, the term "skin xerosis" is a common skin disease, which generally refers to any condition where the skin is dry beyond normal, and is the initial manifestation of skin barrier damage. The water content of the normal stratum corneum is 15% to 20%. When the water content in the stratum corneum drops below 10%, the skin becomes rough, causing skin discomfort and dry appearance. Its clinical manifestations are mainly dry skin, bran-like desquamation, accompanied by systemic itching, etc. Repairing the skin barrier is the main method to improve dry skin.

[0057] As used herein, the term "skin vascular neurosensitivity" refers to a state in which the skin's reactivity to external stimuli is enhanced, the protective ability of the skin nerve endings is weakened, the density of nerve fibers is increased, and the reactivity of sensory nerves is increased. Specifically, it is manifested as increased reactivity to external stimuli (such as physical, chemical, and mental factors), with symptoms such as burning, stinging, flushing, itching, and tightness, which may or may not be accompanied by persistent erythema.

[0058] As used herein, the term "ceramide" is a class of compounds formed by the combination of long-chain fatty acids and sphingosine through amide bonds, mainly including ceramide-1, ceramide-2, ceramide-3, and ceramide-4. It plays an important structural and functional role in cell membranes and the stratum corneum of the skin, especially in maintaining the skin barrier, moisturizing and anti-aging. Ceramide can be derived from konjac, wheat germ, rice bran, and rice, and is obtained by extraction and purification.

[0059] As used herein, the term "grape seed extract (GSE)" is a natural substance extracted from grape seeds, which mainly contains polyphenols, including proanthocyanidins, catechins, epicatechins, gallic acid, etc., of which proanthocyanidins account for more than 95% by mass. Grape seed extract has a strong ability to scavenge free radicals and resist oxidation.

[0060] As used herein, the term "quercetin" is a flavonol compound, and its chemical name is 3,3',4',5,7-pentahydroxyflavone. Quercetin exists in many plants, including Sophora japonica, Sophora japonica, French maritime pine and larch, and can be extracted and purified from the above plants. There is no difference in the material composition and function of quercetin from different plant sources.

[0061] As used herein, the term "citrus flavonoids" is a natural substance extracted from the peel of citrus fruits, which mainly contains flavonoid components, including naringin, hesperidin, neohesperidin, etc., of which naringin accounts for more than 20% by weight, hesperidin accounts for more than 2% by weight, and neohesperidin accounts for more than 10% by weight. The main plant source of citrus flavonoids is bitter orange.

[0062] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references.

[0063] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0064] The ceramide used in the examples of the present application was purchased from Nanjing Geneo Biological Products Co., Ltd.; the grape seed extract used in the implementation of the present application was purchased from Ningbo Traditional Chinese Medicine Pharmaceutical Co., Ltd.; the quercetin used in the implementation of the present application was purchased from Ningbo Traditional Chinese Medicine Pharmaceutical Co., Ltd.; the citrus flavonoids used in the implementation of the present application were purchased from Shaanxi Huike Plant Development Co., Ltd.; the Centella asiatica extract used in the examples of the present application was purchased from Indena SAS of Italy; the green tea extract used in the comparative example was purchased from Shanghai Nord Bio-Industry Co., Ltd.

[0065] Example 1

[0066] A composition comprises: 40 parts of ceramide, 200 parts of grape seed extract, 200 parts of quercetin and 225 parts of citrus brass.

[0067] Example 2

[0068] A composition comprises: 4 parts of ceramide, 200 parts of grape seed extract, 200 parts of quercetin and 225 parts of citrus flavonoids.

[0069] Example 3

[0070] A composition comprises: 18 parts of ceramide, 200 parts of grape seed extract, 200 parts of quercetin and 225 parts of citrus flavonoids.

[0071] Example 4

[0072] A composition comprises: 18 parts of ceramide, 200 parts of grape seed extract, 200 parts of quercetin, 225 parts of citrus flavonoids and 150 parts of Centella asiatica extract.

[0073] Comparative Example 1

[0074] A composition comprises: 200 parts of grape seed extract, 200 parts of quercetin and 225 parts of citrus flavonoids.

[0075] Comparative Example 2

[0076] A composition comprising: 40 parts of ceramide.

[0077] Comparative Example 3

[0078] A composition comprising 4 parts of ceramide.

[0079] Comparative Example 4

[0080] A composition comprises: 200 parts of green tea extract, 200 parts of quercetin and 225 parts of citrus flavonoids.

[0081] Comparative Example 5

[0082] A composition comprises: 40 parts of ceramide, 200 parts of green tea extract, 200 parts of quercetin and 225 parts of citrus flavonoids.

[0083] Test Example 1 Cell Test

[0084] Compared with normal skin, skin barrier dysfunction can lead to relatively poor skin defense against exogenous stimuli and allergens, and it is easy to produce a series of inflammatory reactions due to irritation or allergies. Therefore, the detection of various inflammatory factors is also the main detection target of repairing ingredients for diseases and / or symptoms related to skin barrier dysfunction. The LPS (lipopolysaccharide)-induced inflammatory macrophage model can be used to evaluate the inhibitory effect of raw materials on inflammation.

[0085] 1. Test indicators

[0086] An LPS-induced inflammatory macrophage model was constructed, and three indicators were detected, specifically the NO content and the expression levels of inflammatory factors (COX-2, IL-6).

[0087] 2. Group settings

[0088] The experiment set up a blank control group, a model control group, and test groups 1-7 (corresponding to the compositions of Examples 1-2 and Comparative Examples 1-5, respectively)

[0089] 3. Experimental methods

[0090] 3.1. Specific methods and steps of NO (nitric oxide) content test experiment

[0091] RAW264.7 cells in the logarithmic growth phase were taken and 2x10 6 The cells were inoculated at a density of 100 / mL on a 6-well plate and incubated in an incubator for 24 hours before administration. The cells were divided into two experimental groups: a blank group, a lipopolysaccharide (LPS) model group, and a sample group. Complete culture medium was added to the blank group, LPS with a final mass concentration of 1 mg / L was added to the LPS model group, and samples with different final mass concentrations and 1 mg / L LPS were added to the drug administration group, respectively, so that the total volume of each group was 3 mL, and each group was set up with 3 replicates. After continued culture in a cell culture incubator for 24 hours, the cell supernatant was collected, and the content of NO in the supernatant was determined according to the instructions of the nitric oxide (NO) detection kit.

[0092] 3.2. qPCR testing of IL-6 and COX-2

[0093] RAW264.7 cells in the logarithmic growth phase were taken and 2x10 6The cells were inoculated on a 6-well plate at a density of 100 / mL and incubated in an incubator for 24 hours before administration. The cells were divided into 9 groups, namely, a blank group, a lipopolysaccharide LPS model group, and a sample group. Complete culture medium was added to the blank group, LPS with a final mass concentration of 1 mg / L was added to the LPS model group, and samples with different final mass concentrations and 1 mg / L LPS were added to the drug administration group, respectively, so that the total volume of each group was 3 mL, and each group was set up with 3 replicates. After being placed in a cell culture incubator and continued to be cultured for 24 hours, the cells were collected. RNA was extracted from the cells, reverse transcribed into mRNA, primers were designed, and the relative expression levels of IL-6 and COX2 mRNA levels were detected and calculated.

[0094] 4. Calculation formula

[0095] NO inhibition rate (%) = [(Tc-Tx) / (Tc-T0)] ╳ 100; Tx: NO content in the sample group, μM; T0: NO content in the blank group, μM; Tc: NO content in the model group, μM;

[0096] COX-2 relative expression inhibition rate (%) = [(Ac-Ax) / (Ac-A0)] ╳ 100; Ax: relative expression of COX-2 in the sample group; A0: relative expression of COX-2 in the blank group; Ac: relative expression of COX-2 in the model group;

[0097] IL-6 expression inhibition rate (%) = [(Bc-Bx) / (Bc-B0)] ╳ 100; Bx: relative expression of IL-6 in the sample group; B0: relative expression of IL-6 in the blank group; Bc: relative expression of IL-6 in the model group;

[0098] 5. Experimental results

[0099] Table 1 NO content and inhibition rate

[0100] Group concentration Content (μM) Inhibition rate (%) blank \ 1.25±0.13 / Model \ <![CDATA[17.67±1.04 #### ]]> / Experimental Group 1 0.03325mg / mL 9.26±0.28*** 51.19±1.72 Experimental Group 2 0.03145mg / mL 9.61±0.59*** 49.06±3.58 Experimental Group 3 0.03125mg / mL 14.05±0.11** 22.04±0.67 Experimental Group 4 0.002mg / mL 15.11±0.13* 15.59±0.82 Experimental Group 5 0.0002mg / mL 15.60±0.23* 12.64±1.39 Experimental Group 6 0.03125mg / mL 16.10±0.11 9.58±0.64 Experimental Group 7 0.03325mg / mL 13.28±0.47** 26.74±2.85

[0101] Note: “####” indicates that the difference between the blank group and the model group was statistically significant (P<0.0001), and “**, *, *” indicate that the difference between the treatment group and the model group was statistically significant (P<0.001, P<0.01, P<0.5), respectively.

[0102] Table 2 Relative expression of COX-2

[0103] Group Group Relative expression Inhibition rate (%) blank \ 1.00±0.00 / Model \ <![CDATA[104.72±0.68 #### ]]> / Experimental Group 1 0.03325mg / mL <![CDATA[72.84±0.44 **** ]]> 30.74±0.43 Experimental Group 2 0.03145mg / mL <![CDATA[71.57±1.81 **** ]]> 31.96±1.75 Experimental Group 3 0.03125mg / mL <![CDATA[97.95±0.60 *** ]]> 6.53±0.58 Experimental Group 4 0.002mg / mL <![CDATA[89.60±0.63 **** ]]> 14.58±0.61 Experimental Group 5 0.0002mg / mL <![CDATA[90.67±0.57 **** ]]> 13.55±0.55 Experimental Group 6 0.03125mg / mL <![CDATA[93.90±1.10 *** ]]> 10.43±1.06 Experimental Group 7 0.03325mg / mL <![CDATA[81.42±0.69 **** ]]> 22.47±0.66

[0104] Note: “####” indicates that the difference between the blank group and the model group was statistically significant (P<0.0001), and “****, ***” indicates that the difference between the treatment group and the model group was statistically significant (P<0.0001, P<0.001), respectively.

[0105] Table 3 Relative expression of IL-6

[0106]

[0107]

[0108] Note: “####” indicates that the difference between the blank group and the model group was statistically significant (P<0.0001), and “****, ***” indicates that the difference between the treatment group and the model group was statistically significant (P<0.0001, P<0.001), respectively.

[0109] As shown in Tables 1-3, each test group has the effect of inhibiting the expression of inflammatory factors NO, COX-2 and IL-6 to a certain extent. The interaction between the components in the composition was further calculated according to the Jin Zhengjun Q value method, and the formula is as follows:

[0110] Q=E[a+b] / [Ea+(1-Ea)*Eb]

[0111] E[a+b] is the inhibition rate when formula a and formula b are used together; Ea and Eb are the inhibition rates when formula a and formula b are used alone. When q < 0.85, it means that the two formulas have antagonistic effects; when q > 1.15, it means that the two formulas have synergistic effects.

[0112] From the experimental results in Tables 1-3, it can be seen from the inhibition rates of NO, COX-2 and IL-6 of test group 1 (ceramide, grape seed extract, quercetin, citrus flavonoids), test group 3 (grape seed extract, quercetin, citrus flavonoids) and test group 4 (ceramide) that the Q values ​​of the combined use of grape seed extract, quercetin, citrus flavonoids and ceramide in inhibiting NO, COX-2 and IL-6 reached 1.50, 1.52 and 1.56 respectively, all greater than 1.15, so the combination of grape seed extract, quercetin, citrus flavonoids and ceramide has a synergistic effect.

[0113] From the inhibition rates of NO, COX-2 and IL-6 of experimental group 7 (ceramide, green tea extract, quercetin, citrus flavonoids), experimental group 6 (green tea extract, quercetin, citrus flavonoids) and experimental group 4 (ceramide), it can be seen that the combined Q values ​​of green tea extract, quercetin, citrus flavonoids and ceramide in inhibiting NO and COX-2 were 1.12 and 0.96, respectively, both less than 1.15, and no synergistic effect was shown; the combined Q value of the inhibition rate of IL-6 was 0.61, less than 0.85, showing an antagonistic effect.

[0114] Test Example 2 Animal Experiment (2,4-Dinitrofluorobenzene (DNFB)-Induced Skin Inflammation Model)

[0115] 1. Experimental Grouping

[0116] The experiment set up a blank group, a model group, a positive group (dexamethasone (DXMS) about 0.06 mg / day), and a test group (the composition of Example 3 was used to set up three low, medium and high dose groups, and the dosages were 643, 1286, and 1929 mg / day respectively).

[0117] 2. Experimental Animals

[0118] SPF grade 6-8 week old BALB / c mice, 18-22 g, male, 6 mice per group, were provided by Guangdong Provincial Animal Center.

[0119] 3. Experimental steps

[0120] ① Adaptive training: All mice were kept in the animal room of the Experimental Animal Center of the School of Biomedicine of Guangdong University of Technology. The indoor temperature of the animal room was maintained between 20-25℃, the humidity was 45-65%, and a 12-hour light / dark cycle lighting system was used. They were allowed to eat and drink freely for 24 hours and were adaptively fed for 1-2 weeks.

[0121] ② Model construction: The back hair of BALB / c mice was removed, and the model was established two days later, that is, on Day 1. 100 μL of 0.15% DNFB was applied to the back and 25 μL to the right ear. The blank group was applied with acetone olive oil (v:v=3:1) and once on Day 4, for a total of two times; stimulation began on Day 7, and 100 μL of 0.2% DNFB was applied to the back and once on Day 10 and Day 13, for a total of three times, to establish an atopic dermatitis model.

[0122] ③Administration and observation: Administration began on Day 1. The model group and the blank group were gavaged with normal saline, and the combination group and the positive drug group were gavaged with the corresponding doses (low dose, medium dose, high dose) of the formula product, with a gavage volume of 200uL, and the concentration was as shown in the group, once a day for a total of 14 days. The back skin of BALB / c mice was photographed every day, and the degree of skin lesions was scored, and the thickness of the auricle was tested.

[0123] ④End and sampling: Day 15 experimental techniques, routine indicators of body weight, skin photography and transcutaneous water loss test, and then the mice were killed. Blood was collected by removing the eyeballs, and the blood was left to stand at room temperature for 2 hours, centrifuged at 1500g for 15 minutes at room temperature, and the serum was divided into two tubes and stored at -80℃; the back skin was taken, and the local skin of the same part (about 22cm 2 The skin was fixed in tissue fixative (4% paraformaldehyde) and the remaining skin was stored at -80℃ for later use.

[0124] 4. Index detection

[0125] (1) Detection of apparent indicators:

[0126] ① Mouse weight: Use a special container and place it on an electronic scale. Before measuring, lay the electronic scale flat and adjust the zero point to ensure that the scale is in a stable state. Gently place the mouse on the tray of the scale to avoid causing unnecessary pressure or harm to the mouse. After the value stabilizes, record the weight of each mouse.

[0127] ② Back skin photography: To photograph the back skin, the mouse was fixed, and the back was kept stretched and exposed. The same equipment, 30 cm shooting height and the same shooting environment were required for each photograph.

[0128] ③ Lesion scoring: The mouse lesion scoring method includes three indicators: erythema, scaling and lesion degree. The standard is erythema: 0 points for no erythema, 1 point for light red erythema, 2 points for red erythema, 3 points for dark red erythema, and 4 points for extremely deep erythema. Scaling: 0 points for no scaling on the surface, 1 point for scaling on the surface of some lesions, 2 points for scaling on the surface of most lesions, 3 points for almost all lesions covered with scaling, and 4 points for all lesions covered with scaling. Lesion hypertrophy: 0 points for lesions flush with normal skin, 1 point for lesions slightly higher than normal skin surface, 2 points for moderately raised lesions, 3 points for hypertrophy of lesions with obvious protrusion, and 4 points for highly hypertrophic lesions with obvious protrusion. The three scores of erythema, scaling and hypertrophy of lesions are added together to obtain the total score, ranging from 0 to 12 points. Each mouse was scored according to the standard, and the scores were statistically analyzed.

[0129] ④H&E staining (back and auricle): Take the corresponding tissues and prepare the corresponding tissue sections according to the SOP of the Sevier pathological tissue sampling, fixation, embedding, paraffin sectioning, frozen sectioning, etc. The sections were placed in environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and washed with tap water. The frozen sections were taken out of the -20° refrigerator and restored to room temperature. After fixing with tissue fixative for 15 minutes, they were rinsed with running water. The sections were treated with high-definition constant staining pretreatment solution for 1 minute. The sections were dyed with hematoxylin staining solution for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue return solution, and rinsed with running water. The sections were dehydrated in 95% alcohol for 1 minute and stained in eosin staining solution for 15 seconds. The slices were placed in absolute ethanol I for 2 min, absolute ethanol II for 2 min, absolute ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, xylene II for 2 min, and then transparent and sealed with neutral gum. The slices were examined under a microscope and images were collected and analyzed.

[0130] ⑤ Mast cell staining and calculation of average optical density: Take the corresponding tissues and prepare the corresponding tissue sections according to the SOP of the Sevier pathological tissue sampling, fixation, embedding, paraffin sectioning, frozen sectioning, etc. The sections were placed in environmentally friendly dewaxing solution I for 20 minutes, environmentally friendly dewaxing solution II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and washed with tap water. The frozen sections were taken out of the -20° refrigerator and restored to room temperature. After fixing with tissue fixative for 15 minutes, they were rinsed with running water. The tissue sections were placed in toluidine blue dye solution for 2-5 minutes, washed with water, slightly differentiated with 0.1% glacial acetic acid, and terminated by tap water washing. The degree of differentiation was controlled under a microscope. After washing with tap water, the sections were placed in an oven to dry. The sections were placed in clean xylene for 10 minutes and sealed with neutral gum. Microscope examination, image acquisition and analysis.

[0131] Each mast cell staining image was imported into the imgae-Pro Plus6 software, and the average optical density of the test area for evaluating mast cells was tested.

[0132] (2) ELISA test:

[0133] ① Skin tissue immune targets (IL-4, IL-6), skin tissue vascular regulation-related targets (ET-1, TRPV1)

[0134] Take an appropriate amount of mouse back skin tissue placed at -80℃ and freeze-thaw on ice. Add 1mL of pre-cooled tissue lysis solution for every 100mg of mouse skin tissue, grind the tissue using a tissue grinder, and centrifuge at 10000g for 10 minutes at 4℃. All test samples are uniformly calibrated for protein using the BCA method. The quantitative skin tissue protein solution is used to determine the target protein content according to the requirements of the ELISA kit.

[0135] ②Serum IgE

[0136] Blood was collected from mice by removing the eyeballs, placed in an EP tube without anticoagulant, and placed at room temperature for 30 min to 1 h (no more than 2 h), then centrifuged at 4000 g for 15 min at 4 °C to separate serum, which was packaged as needed and stored in a -80 °C refrigerator. The IgE enzyme-linked immunosorbent assay (ELISA) was then measured according to the experimental instructions.

[0137] 5. Experimental results

[0138] 5.1. Changes in mouse body weight

[0139] Table 4 Changes in body weight of mice

[0140] Group Day 0 Day 5 Day 10 Day 15 Control 20.32±1.03 19.92±0.96 20.49±0.95 20.66±1.14 Model 20.21±1.39 20.36±0.68 19.84±0.64 19.33±1.24 DXMS 20.72±0.88 20.15±1.09 19.17±1.02 17.83±1.19 ZL 20.64±0.88 20.53±0.68 20.88±0.83 21.28±0.90 ZM 21.00±0.55 19.35±0.89 19.52±1.21 21.33±1.07 ZH 20.99±1.09 20.74±0.72 20.58±0.53 20.44±0.70

[0141] Note: Control: blank group; Model: model group; DXMS: dexamethasone group; ZL: low-dose group of the combination; ZM: medium-dose group of the combination; ZH: high-dose group of the combination; the same below.

[0142] The weight of mice was tested on days 0, 7, 10, and 15, and the results are shown in Table 1. The weight of mice in the model group, blank group, positive group, and drug administration group changed little during the experimental period. This indicates that the composition has no negative effect on the growth of mice.

[0143] 5.2. Apparent changes and skin lesion scores of the back skin of mice

[0144] The skin lesions on the backs of the mice were photographed on days 1, 7, and 14. Figure 1 As shown in Table 5, compared with the blank group, the skin of the mice in the model group had visible damage and the skin was accompanied by large areas of papules, erythema, and scab-like changes. Among them, there was a significant difference between the model group and the blank group, indicating that the model was successfully established; and after treatment with low, medium, and high doses of the positive drug and the composition, the skin lesion-like symptoms of the mice were significantly improved. After administration of the composition in different dose groups, there was no significant difference in the macroscopic evaluation of the skin.

[0145] Table 5 Macroscopic evaluation of skin of samples and control groups (skin lesion scores)

[0146] Group Day 14 Skin Macroscopic Evaluation Cotrol 0.00±0 Model <![CDATA[11.20±1.10 #### ]]> DXMS <![CDATA[3.40±0.89 **** ]]> ZL <![CDATA[4.67±0.52 **** ]]> ZM <![CDATA[5.33±1.37 **** ]]> ZH <![CDATA[4.60±0.89 **** ]]>

[0147] Note: “####” indicates that the difference between the blank group and the model group is statistically significant (P<0.0001), and “****” indicates that the difference between the treatment group and the model group is statistically significant (P<0.0001).

[0148] 5.3 HE staining of mouse back skin

[0149] HE staining can be used to observe the thickening and tissue morphology of the mouse epidermis. Figure 2 As shown in Table 6, after DNFB modeling, the epidermis of the skin proliferated abnormally and the thickness increased significantly. Among them, the blank group and the model group had significant differences, confirming the successful modeling. After 14 days of intragastric administration, the positive group can effectively inhibit the increase of epidermal thickness. After 14 days of intragastric administration, the low, medium and high dose groups of the composition all had the effect of significantly inhibiting the increase of epidermal thickness, and the effects of different dose groups were comparable to those of the positive drug group.

[0150] Table 6 Epidermal thickness of the back skin of mice in each group

[0151] Group Epidermal thickness / μm Cotrol 17.01±2.15 Model <![CDATA[95.99±11.09 #### ]]> DXMS <![CDATA[59.76±15.31 *** ]]> ZL <![CDATA[60.59±10.63 ** ]]> ZM <![CDATA[59.11±10.38 *** ]]> ZH <![CDATA[59.02±4.28 *** ]]>

[0152] Note: “####” indicates that the difference between the blank group and the model group is statistically significant (P<0.0001), and “**, ***” indicates that the difference between the model group and the experimental group is statistically significant (P<0.01, P<0.001).

[0153] 5.4 HE staining of mouse ear

[0154] The swelling and pathological changes of the auricle can reflect the degree of inflammation and nerve response of the mouse skin. By HE staining the mouse auricle tissue, the thickness changes of the mouse auricle can be observed and calculated to evaluate the degree of swelling. Figure 3 As shown in Table 7, after DNFB modeling, the auricle swelled and thickened. The blank group and the model group were significantly different, confirming that the modeling was successful. After 14 days of intragastric administration, the low, medium and high dose groups of the composition all showed a trend of inhibiting the thickness of the auricle epidermis, and all showed different degrees of relief of ear swelling. The high dose group had the best effect in inhibiting auricle swelling.

[0155] Table 7 Swelling thickness of the auricle skin of mice in each group

[0156] Group Swelling thickness / μm Cotrol 180.72±7.53 Model <![CDATA[392.69±9.40 ### ]]> DXMS <![CDATA[236.85±0.14 ** ]]> ZL 361.44±6.12 ZM <![CDATA[278.68±21.42 * ]]> ZH <![CDATA[273.28±18.42 * ]]>

[0157] Note: “###” indicates that the difference between the blank group and the model group is statistically significant (P<0.001); “*”, “*” indicates that the difference between the model group and the experimental group is statistically significant (P<0.05, P<0.01).

[0158] 5.5. Mast cell staining and mean optical density analysis

[0159] Mast cell staining experiments can be used to observe the infiltration of inflammatory factors (blue) in the skin. Figure 4 As shown in Table 8, after DNFB modeling, the infiltration of inflammatory factors in the model group increased. Among them, the model group had a significant difference compared with the blank group, indicating that the modeling was successful; the positive group could effectively inhibit the expression of inflammatory factors. In the sample group, relative quantitative analysis by average optical density showed a trend of inhibiting the expression of inflammatory factors. Among them, the low, medium and dose groups had a significant inhibitory effect in the average optical density analysis results, and the high dose group had the best effect. Improve the infiltration of inflammatory factors, thereby improving the symptoms caused by sensitive skin inflammation.

[0160] Table 8 Inflammatory factor infiltration in each group of mice

[0161] Group Average optical density of inflammatory factor infiltration Cotrol 0.005±0.001 Model <![CDATA[0.067±0.013 ### ]]> DXMS <![CDATA[0.004±0.003 *** ]]> ZL <![CDATA[0.025±0.016 * ]]> ZM <![CDATA[0.023±0.011 * ]]> ZH <![CDATA[0.016±0.011 ** ]]>

[0162] Note: ###" indicates that the difference between the blank group and the model group is statistically significant (P<0.001); ", *" indicates that the difference between the model group and the experimental group is statistically significant (P<0.05, P<0.01).

[0163] 5.6. Detection of inflammatory factors (IL-4, IL-6) levels

[0164] In skin problems such as sensitive skin and atopic dermatitis, the levels of immune-related inflammatory indicators in skin tissue will increase accordingly, and the inhibition of inflammatory factors by the IL-4 and IL-6 reactive combination was tested. Figure 5 and 6 As shown, after DNFB modeling, the levels of inflammatory factors IL-4 and IL-6 in the model group increased significantly, among which the model group had significant differences compared with the blank group, indicating that the modeling was successful; from the experimental results, it can be seen that the low, medium and high dose groups of the composition can inhibit the effects of IL-4 and IL-6 inflammatory factors; this indicates that the composition can reduce the level of inflammatory factors, thereby improving the inflammatory response of the skin.

[0165] 5.7. Tissue vascular regulation-related targets (ET-1, TRPV1)

[0166] In skin problems such as sensitive skin, atopic dermatitis, and dry skin, ET-1 induces the secretion of TNF-α and IL-6, and promotes the production of VEGFA, which increases vascular reactivity and causes vasodilation. TRPV-1 is widely expressed on skin nociceptive sensory nerve endings, keratinocytes, and mast cells. High temperature, acidic environment, capsaicin, ultraviolet rays, and endogenous and exogenous inflammatory mediators activate TRPV-1, thermal and chemical stimulation sensory input, causing symptoms such as burning, paroxysmal redness, pain, and itching. Inhibiting the production of ET-1 and TRPV-1 can improve the various skin symptoms caused by them.

[0167] like Figure 7-8 As shown, after DNFB modeling, the levels of ET-1 and TRPV1 in the model group increased significantly, and the model group had significant differences compared with the blank group, indicating that the modeling was successful; the experimental results showed that the positive drug and the low, medium and high dose groups of the drug administration group could significantly inhibit the expression of ET-1 protein; the low, medium and high dose groups of the drug administration group had the effect of significantly inhibiting the expression of TRPV1 protein. This indicates that the composition may be able to improve symptoms such as burning, paroxysmal redness, pain, itching, etc. caused by sensitive skin, atopic dermatitis, dry skin, etc.

[0168] 5.8. Serum IgE

[0169] Detect the changes in the content of immune proteins in the blood and characterize the changes in serum IgE caused by modeling in mice. In the experiment, blood was collected and the content of immune proteins was detected. Fig. 9As shown in the results, the content of IgE in the DNFB model increased significantly. The low and medium doses of the composition had a significant inhibitory effect, with an inhibition rate between 13.04% and 17.44%. However, the high dose group of the composition had no obvious inhibitory effect. This shows that under medium and low dose conditions, the composition can balance the body's immune response in sensitive skin, atopic dermatitis, etc.

[0170] Experimental Example 3 Animal Experiment (Acetone Ether (AEW)-Induced Dry Skin Itch Model)

[0171] 1. Experimental Grouping

[0172] The experiment set up a blank group, a model group, a positive group (dexamethasone DXMS, about 0.06 mg / day), and a test group (the composition of Example 3 was used to set up three low, medium and high dose groups, and the dosages were 643, 1286, and 1929 mg / day, respectively).

[0173] 2. Experimental Animals

[0174] SPF 8-10 week old BALB / c mice, 15-25 g, male, 10 mice per group, were provided by Guangdong Provincial Animal Center.

[0175] 3. Experimental steps

[0176] ① Adaptive feeding: All mice were kept in the animal room of the Experimental Animal Center of the School of Biomedicine of Guangdong University of Technology. The indoor temperature of the animal room was maintained between 20-25℃, the humidity was 45-65%, and a 12-hour light / dark cycle lighting system was used. They were allowed to eat and drink freely for 24 hours and were adaptively fed for 1-2 weeks.

[0177] ② Model construction: On Day 1, the hair on the back of BALB / c mice was removed. On Day 7, the model was established. The back of the mice was soaked with a cotton ball in a mixture of acetone / ether (1:1) for 30 seconds, and then soaked with a cotton ball in distilled water for 30 seconds. The blank group was soaked with a cotton ball in distilled water for 60 seconds, twice a day (9:00 and 17:00), and the application and soaking were continued for 7 days to establish a dry itchy allergy model in mice.

[0178] ③Administration and observation: Gavage administration began 1 week in advance of modeling. The model group and the blank group were gavaged with normal saline, and the combination group and the positive drug group were gavaged with the corresponding dose of the combination (low, medium, and high doses), once a day, with a gavage volume of 200uL, and the concentration was shown in the group, for a total of 14 days until the end of the experiment. Every other day after modeling, the back skin of BALB / c mice was photographed to observe the degree of skin lesions, and the transcutaneous water loss of the back skin of mice was measured by a transcutaneous water loss instrument.

[0179] (5) Sampling: The experiment was ended on Day 15. The body weight was measured, the percutaneous water loss test was performed, the appearance was photographed, and the number of scratches within 1 minute was recorded. The mice were then killed. The eyeballs were removed to collect blood. The blood was allowed to stand at room temperature for 2 hours and centrifuged at 1500g for 15 minutes. The serum was divided into two tubes and stored at -80°C. The back skin was taken, and the local skin of the same part (about 22cm 2 The skin was fixed in tissue fixative (4% paraformaldehyde) and the remaining skin was stored at -80℃ for later use.

[0180] 4. Index detection:

[0181] (1) Apparent detection indicators:

[0182] ① Mouse weight: Use a special container and place it on an electronic scale. Before measuring, lay the electronic scale flat and adjust the zero point to ensure that the scale is in a stable state. Gently place the mouse on the tray of the scale to avoid causing unnecessary pressure or harm to the mouse. After the value stabilizes, record the weight of each mouse.

[0183] ②Photographing and scoring of skin lesions on the dry back:

[0184] The mice were fixed, with their backs stretched and exposed. Each photo was taken with the same equipment, 30 cm shooting height and the same shooting environment.

[0185] The scoring method for mouse skin lesions includes three indicators: erythema, scaling, and degree of lesions. The standard is erythema: 0 points for no erythema, 1 point for light red erythema, 2 points for red erythema, 3 points for dark red erythema, and 4 points for extremely deep erythema. Scaling: 0 points for no scaling on the surface, 1 point for scaling on the surface of some lesions, 2 points for scaling on the surface of most lesions, 3 points for almost all lesions covered with scaling, and 4 points for all lesions covered with scaling. Lesion hypertrophy: 0 points for lesions flush with normal skin, 1 point for lesions slightly higher than normal skin surface, 2 points for moderately raised lesions, 3 points for hypertrophic lesions with obvious protrusions, and 4 points for highly hypertrophic lesions with obvious protrusions. The three scores of erythema, scaling, and hypertrophic lesions are added together to obtain the total score, ranging from 0 to 12 points. Each mouse was scored according to the standard, and the scores were statistically analyzed.

[0186] ③H&E staining of back skin (epidermal thickness):

[0187] Take the corresponding tissues and prepare the corresponding tissue sections according to the SOP of the Sevier pathological tissue sampling, fixation, embedding, paraffin sectioning, frozen sectioning, etc. Put the sections into environmentally friendly dewaxing liquid I for 20 minutes - environmentally friendly dewaxing liquid II in turn

[0188] 20min—anhydrous ethanol I 5min—anhydrous ethanol II 5min—75% alcohol 5min, washed with tap water. Take the frozen sections out of the -20° refrigerator and restore them to room temperature. Fix them with tissue fixative for 15min and then rinse with running water. Put the sections into high-definition constant staining pretreatment solution for 1min. Slices are dyed with hematoxylin staining solution for 3-5min, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue reversion solution, and rinsed with running water. Slices are dehydrated in 95% alcohol for 1min and stained in eosin staining solution for 15s. Slices are sequentially placed in anhydrous ethanol I 2min-anhydrous ethanol II 2min-anhydrous ethanol III 2min-n-butanol I 2min-n-butanol II 2min-xylene I 2min-xylene II 2min to make them transparent and seal with neutral gum. Microscope examination, image acquisition and analysis.

[0189] ④ Back skin transepidermal water loss test:

[0190] The transepidermal water loss rate of the mouse back skin was measured using an instrument. The TM300-Germany CK skin water loss (TEWL) tester was used. The test environment was maintained at a constant temperature and humidity of 20-22°C and 50%-70% for 30 minutes before measurement. The probe was kept in contact with the mouse back skin and the value was recorded after it stabilized.

[0191] (2) ELISA test indicators:

[0192] ① Skin tissue barrier protein detection (FLG, LOR, AQP3):

[0193] Take an appropriate amount of mouse back skin tissue placed at -80℃ and freeze-thaw on ice. Add 1mL of pre-cooled tissue lysis solution for every 100mg of mouse skin tissue, grind the tissue using a tissue grinder, and centrifuge at 10000g for 10 minutes at 4℃. All test samples are uniformly calibrated for protein using the BCA method. The quantitative skin tissue protein solution is used to determine the target protein content according to the requirements of the ELISA kit.

[0194] 5. Experimental results:

[0195] 5.1. Changes in mouse body weight

[0196] Table 9 Changes in body weight of mice

[0197] Group Day 0 Day 5 Day 10 Day 15 Control 18.54±1.32 18.59±2.33 18.62±2.04 18.62±1.03 Model 18.98±1.91 19.15±1.74 19.55±2.06 19.55±1.84 DXMS 18.85±0.86 19.48±0.96 19.13±0.98 19.13±1.63 ZL 18.19±2.29 17.90±3.23 17.90±2.75 17.90±2.81 ZM 20.36±1.05 20.24±1.22 20.42±1.45 20.42±1.64 ZH 20.06±1.96 20.00±2.54 21.41±2.12 21.41±0.78

[0198] The weight of mice was tested on days 0, 7, 10, and 15, and the weight of mice in the model group, blank group, positive group, and drug administration group changed little during the experimental period, indicating that the composition did not have a negative effect on the growth of mice.

[0199] 5.2. Apparent changes of dryness on the back of mice and skin lesion scores

[0200] like Fig.10 As shown in Table 10, oral administration began on d1 after acetone ether modeling, oral administration and modeling began on d7, and the experiment ended on d14. After modeling, the skin on the back of the mice was dry and partially scaly, and the apparent difference between the groups was not obvious. After 14 days of oral administration of the low, medium, and high dose groups of the positive drug and the combination, it can be observed that the dryness and scaly skin on the back of the mice were improved. There was no significant difference between the different dose groups.

[0201] Table 10 Macroscopic evaluation of mouse back skin (skin lesion score)

[0202]

[0203]

[0204] Note: "####" indicates that the difference between the blank group and the model group is statistically significant (P<0.0001); "****" indicates that the difference between the model group and the experimental group is statistically significant (P<0.0001).

[0205] 5.3 HE staining and epidermal thickness measurement

[0206] like Fig.11 And Table 11, corresponding to the dry appearance of the back of the mouse, HE staining can observe the epidermal morphology, and after acetone ether modeling, the epidermis of the skin abnormally proliferates and the thickness increases significantly. Among them, the blank is significantly different from the model, proving that the modeling is successful. The low, medium and high dose groups of the formula can reduce the thickness of the skin epidermis to varying degrees, among which the medium and high dose groups are significantly different. Improving the skin barrier is an important part of the treatment or prevention of sensitive skin, atopic dermatitis and dry skin. The experimental results confirm the protective effect of the composition on skin damage and the improvement of the skin barrier, indicating that it has a certain improvement effect on the above-mentioned skin problems.

[0207] Table 11 Epidermal thickness of the back skin of mice in each group

[0208] Group Epidermal thickness / μm Cotrol 22.65±2.00 Model <![CDATA[55.92±4.28 ### ]]> DXMS 39.77±5.98 ZL 55.04±13.88 ZM <![CDATA[38.36±10.66 * ]]> ZH <![CDATA[40.20±4.22 * ]]>

[0209] Note: “###” indicates that the difference between the blank group and the model group is statistically significant (P<0.001), and “*” indicates that the difference between the model group and the experimental group is statistically significant (P<0.05).

[0210] 5.4. Determination of transepidermal water loss in mice

[0211] As shown in Table 12, the transepidermal water loss rate of the skin increased significantly during the acetone ether modeling process, and the blank and the model were significantly different, indicating that the modeling was successful. Among them, the positive group can effectively inhibit the increase in transepidermal water loss rate. The administration groups with different doses all had a significant inhibitory effect on the transepidermal water loss rate. After 14 days of gavage, the inhibition rate was 62.28%-66.02%, and the difference between the dose groups was not obvious. The results confirmed that the composition has a significant effect on skin barrier repair.

[0212] Table 12 Changes in TEWL in mice

[0213] Group Day 6 Day 10 Day 14 Control 8.98±2.42 9.65±1.17 11.28±3.09 Model 7.58±1.26 12.17±4.89 41.30±4.73#### DXMS 6.90±0.84 10.55±3.06 9.13±1.46**** ZL 7.96±2.08 9.58±3.82 14.58±3.37**** ZM 6.15±1.25 15.95±6.89 15.30±1.43**** ZH 9.22±2.32 10.87±2.85 14.03±1.28****

[0214] Note: “####” indicates that the difference between the blank group and the model group is statistically significant (P<0.0001), and * indicates that the difference between the experimental group and the model group is significant, among which *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0215] 5.5. Mouse skin tissue barrier proteins (FLG, LOR, AQP3)

[0216] In the skin barrier, the keratin capsule is a special structure covering the outer surface of keratinocytes. FLG is an important molecule that connects keratin fibers. The amino acid small molecules formed by the degradation of FLG in the epidermal stratum corneum have moisturizing function, so they are called "natural moisturizing factors". Loricrin (LOR) is its key component and plays a role in strengthening the skin's physical barrier. AQP3 is a water channel protein that is responsible for regulating the water permeability of the skin and acts as a mechanical barrier to prevent microbial infection, ultraviolet rays, damage, cold and heat and other environmental insults.

[0217] The mouse back skin was collected, ground and centrifuged to extract the supernatant, and the protein concentration was detected using a BCA kit and then diluted to the same protein concentration.

[0218] The experimental results showed that the low, medium and high dose groups of the formula had a tendency to improve the expression of LOR and AQP3 proteins, but the results were not significant, which may be caused by the relatively large detection error. The low and medium dose groups of the formula had a tendency to improve the expression of FLG protein, and the high dose group could significantly increase the expression of FLG protein.

[0219] The present invention constructs a DNFB-induced skin allergy model and an AEW-induced skin barrier dryness model to comprehensively evaluate the efficacy of the composition. Based on the results of cell experiments and animal experiments, the composition can synergistically inhibit the production of inflammatory factors, improve immune response and inflammatory infiltration, inhibit neurovascular response, and repair the skin barrier by improving transepidermal water loss.

Claims

1. A composition, characterized in that The composition comprises the following components in parts by weight: 4-40 parts of ceramide, 100-300 parts of grape seed extract, 100-300 parts of quercetin, and 200-250 parts of citrus flavonoids.

2. The composition according to claim 1, characterized in that The composition comprises the following components in parts by weight: 4-40 parts of ceramide, 200 parts of grape seed extract, 200 parts of quercetin, and 225 parts of citrus flavonoids; Preferably, the composition comprises the following components in parts by weight: 18 parts of ceramide, 200 parts of grape seed extract, 200 parts of quercetin, and 225 parts of citrus flavonoids.

3. Use of the composition according to claim 1 or 2 in preparing products beneficial to skin health.

4. The use according to claim 3, characterized in that: The benefit to skin health refers to the prevention, alleviation and / or treatment of diseases and / or symptoms related to skin barrier dysfunction.

5. The use according to claim 4, characterized in that: The skin barrier dysfunction-related diseases and / or symptoms are selected from one or more of sensitive skin, atopic dermatitis and xeroderma.

6. The application according to claim 4, characterized in that: The skin barrier dysfunction-related symptoms are selected from one or more of dry and tight skin, skin inflammation, skin itching, skin redness and abnormal transepidermal water loss.

7. The use according to claim 3, characterized in that: The prevention, alleviation and / or treatment of diseases and / or symptoms related to skin barrier dysfunction includes preventing skin barrier damage, repairing skin barrier damage, reducing skin transepidermal water loss, improving skin immune response and inflammatory infiltration, and / or reducing skin vascular neurosensitivity.

8. A food comprising the composition according to any one of claims 1 to 2 and an adjuvant acceptable in food; preferably, The food is a health food.

9. A cosmetic comprising the composition according to any one of claims 1 to 2 and an auxiliary material acceptable in cosmetics; Preferably, the cosmetic is essence water, essence milk, sleeping mask, body milk, cleanser, pressed powder, massage cream, loose powder, sunscreen or hand cream; Preferably, the acceptable auxiliary materials in the cosmetics include any one or more combinations of surfactants, thickeners, conditioners, moisturizers, preservatives, chelating agents, fragrances and water.

10. A method for preparing the composition according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: taking each component by weight, mixing, grinding and sieving to obtain the composition; Preferably, a stirring operation can be added during the mixing process to ensure that the ingredients are mixed evenly; Preferably, the mixing is performed at a temperature of 20-30° C. in a vacuum or inert gas environment.

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