CN114632136A

CN114632136A - Skin external composition with enhanced anti-eczema effect

Info

Publication number: CN114632136A
Application number: CN202011477554.2A
Authority: CN
Inventors: 张梦璐; 王莎莎
Original assignee: Zhejiang Yangshengtang Institute of Natural Medication Co Ltd
Current assignee: Zhejiang Yangshengtang Institute of Natural Medication Co Ltd
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2022-06-17

Abstract

The present invention relates to a skin external composition having an enhanced anti-eczema effect, which comprises (A) birch juice and (B) one or more selected from the group consisting of mung bean extract, ginger root extract, terpinen-4 ol, willow flower/leaf/stem extract, inulin, alpha glucan fructose, brassinosteroids and hydrolyzed rice protein.

Description

Skin external composition with enhanced anti-eczema effect

Technical Field

The invention relates to an anti-eczema skin external composition which comprises (A) birch juice and (B) one or more of mung bean extract, ginger root extract, terpinen-4 alcohol, willow flower/leaf/stem extract, inulin, alpha glucan fructose, rape sterol and hydrolyzed rice protein.

Background

Eczema is an inflammatory skin disease with obvious exudation tendency caused by various internal and external factors, and has the characteristics of obvious pruritus, abnormal skin barrier function, easy relapse and the like.

Genetic defects in the barrier function of the skin promote the entry of allergens and microbial pathogens into the skin. Allergens induce the production of IgE by B cells, and binding of allergens to IgE activates Dendritic Cells (DCs) and mast cells. The TSLP and pathogen-derived molecules secreted by keratinocytes also activate dendritic cells. Activation of DCs further induced Th2 polarization. Th2 is helper T cell, and its main Th2 cytokines IL-4, IL-5, IL-13 may down-regulate the expression of TLRs (Toll-like receptor) and AMPs in keratinocytes, further aggravating disease. IL-22 secreted by Th22 lymphocytes is also involved in the development of AD, and IL-22 down-regulates profilaggrin and enzymes responsible for processing and producing natural moisturizing factors, and up-regulates the expression of S100 family proteins. Th1 and Th17 play important roles in skin inflammation mainly through IFN-gamma and IL-17 in the chronic stage of AD. Mast cells produce histamine, trypsin and other inflammatory mediators, causing itch associated with AD. Itching also damages the skin barrier while increasing skin water loss (TEWL) induces skin dryness while exacerbating AD. In addition, mast cells produce IL-4 and IL-13, which exacerbates AD. IL-5 causes eosinophil activation, promoting an inflammatory response. The mechanism of eczema can discover that five ways of anti-inflammation, moisture retention, antibiosis, pruritus resistance and barrier repair are closely related to eczema.

Birch is a deciduous tree of the betulinaceae family, and currently, there are about 100 varieties worldwide, mainly distributed in the northern temperate zone and the cold temperate zone. Wherein, there are about 29 varieties in China, and the varieties are mainly distributed in the northeast, northwest and southwest. It has great effect on preventing water and soil loss, improving environment, preventing wind and sand. Birch trees are mostly grown in remote mountainous areas with less human intervention and no industrial pollution. The birch sap is fresh sap obtained by cutting birch bark or drilling trunk, and has no color or light yellow, no precipitate or impurity, and light birch fragrance. The health-care tea is rich in nutrition, and can form factors beneficial to health after being ingested by a human body through metabolism, so that the health-care effect on sub-health people is achieved, the health-care tea has good nutrition and health-care effects, and the health-care tea is often used for making beverages, wines and other beverages. The birch sap contains large amount of saccharides, amino acids, vitamins, biotin, cytokinin, trace mineral elements, aromatic oil, betulin, saponin, etc., and has good effects of keeping moisture, resisting eczema, etc.

Disclosure of Invention

In one aspect, the invention relates to a kit comprising (a) birch juice, and (B) one or more of mung bean extract, ginger root extract, terpinen-4-ol, willow flower/leaf/stem extract, inulin, alpha glucan fructose, rape sterols, and hydrolyzed rice protein.

In yet another aspect, the present invention relates to the use of the kit in a skin external composition having enhanced anti-eczema efficacy.

In still another aspect, the present invention relates to a skin external composition having enhanced anti-eczema efficacy, comprising (a) birch juice, and (B) one or more of a mung bean extract, a ginger root extract, terpinen-4 ol, a willow flower/leaf/stem extract, inulin, alpha glucan fructose, brassinosteroids and hydrolyzed rice protein.

The birch juice as component (A) may be birch juice stock solution, concentrated birch juice or fermented birch juice.

Birch juices contemplated in the present invention are derived from Betula genus of betulinaceae family, and may be derived from four varieties of white birch (Betula alba), Betula luminifera (Betula pubescens), Betula Pendula (Betula Pendula) and Betula asiatica (Betula platyphylla). The birch juice is colorless, transparent, precipitate-free and impurity-free juice which is obtained by manually drilling and collecting at the base of a trunk of the birch between thawing and early spring leaf emergence and has birch faint scent and rich nutrition. The birch juice is commercially available and used as is (i.e., stock solution), for example, from greater than wild berry development, llc in greater Khingan.

The concentrated birch sap is obtained by concentrating the above commercial products. Concentration methods are known in the art, such as heat concentration, low temperature vacuum concentration, membrane concentration, and the like. In the present invention, the concentration is preferably performed by a low-temperature freeze concentration or membrane concentration process, for example, commercially available birch juice stock solution is fed into a low-temperature drying device, cooled to about-40 ℃ to-70 ℃, and subjected to low-temperature vacuum concentration by vacuumizing to about 0.1-30Pa, so as to obtain concentrated birch juice with different concentration times. In the present invention, the concentration of the concentrated birch juice is about 1.05 to 8 times, preferably about 1.1 to 4 times, and more preferably about 1.2 to 2 times.

The fermented birch juice may be an inonotus obliquus fermented birch juice, such as the fermented birch juice disclosed in application No. CN201910689097.4, the entire content of which is incorporated herein by reference.

Unexpectedly, the inventor finds that compared with the single use of birch juice, mung bean extract, ginger root extract, terpinen-4-ol, willow flower/leaf/stem extract, inulin, alpha glucan fructose, rape sterol or hydrolyzed rice protein, the combination of the birch juice, the mung bean extract, the ginger root extract, the terpinen-4-ol, the willow flower/leaf/stem extract, the inulin, the alpha glucan fructose, the rape sterol and/or the hydrolyzed rice protein has a significantly better anti-eczema effect, is far higher than the function superposition effect of the two, is even significantly better than that of a positive medicine, and shows that the synergistic effect is generated between the two medicines.

The content of the birch sap of the component (a) is about 18 to 98% by weight, preferably about 20 to 95% by weight, more preferably about 22 to 90% by weight, most preferably about 30 to 90% by weight, based on the total weight of the skin external composition.

The component (B) mung bean extract, ginger root extract, terpinen-4-ol, willowherb flower/leaf/stem extract, inulin, alpha glucan fructose, rape sterol and hydrolyzed rice protein are all known in the art and are commercially available.

The total content of the component (B) is about 0.01 to 10% by weight, preferably about 0.1 to 6% by weight, more preferably about 0.5 to 5% by weight, based on the total weight of the composition for external application to the skin.

The skin external composition includes a pharmaceutical composition or a cosmetic composition.

The composition for external application to the skin does not contain any added water, but does not exclude moisture inherently contained in the respective components.

In a preferred embodiment, the composition for external application to the skin does not contain a chelating agent such as EDTA salt, sodium polyphosphate, sodium metaphosphate, gluconic acid, or the like.

The skin external composition may optionally include, in addition to the components (a) and (B), component (C), ingredients commonly used in skin external compositions, including but not limited to vehicles, active ingredients, adjuvants, and the like. Component (C) is known in the art, and the type and amount thereof can be selected by those skilled in the art as desired, for example, the total content of component (C) is usually about 2 to 80% based on the total weight of the skin external composition.

The vehicle includes, for example, diluents, dispersants or carriers and the like, examples of which include, but are not limited to, ethanol, dipropylene glycol, butylene glycol, and the like. The content of the vehicle in the skin external preparation is known in the art, and for example, it is generally about 0.5 to 20% by weight of the total weight of component (C).

Such actives include, for example, emollients, humectants, anti-eczema actives and the like.

Examples of such emollients include, but are not limited to, olive oil, macadamia nut oil, sweet almond oil, grape seed oil, avocado oil, corn oil, sesame oil, soybean oil, peanut oil, meadowfoam seed oil, safflower seed oil, rosa canina oil, argan oil, jojoba oil, sunflower seed oil, oil of mauritika, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutene, isohexadecane, isododecane, diethylhexyl carbonate, dioctyl carbonate, isopropyl lauroyl sarcosinate, isononyl isononanoate, hydrogenated polydecene, tris (ethylhexanoate), cetyl ethylhexanoate, bis-diethoxydiol cyclohexane 1, 4-dicarboxylate, caprylic/capric triglyceride, oleyl erucate, octyldodecanol myristate, octyldodecanol, polydimethylsiloxane, polymethyloctylsiloxane, One or more of cetyl dimethicone, cyclopentadimethicone, and the like. Examples of solid emollients include, but are not limited to, one or more of cetyl alcohol, stearyl alcohol, cetostearyl alcohol, behenyl alcohol, batyl alcohol, lauric acid, myristic acid, palmitic acid, stearic acid, beeswax, candelilla wax, carnauba wax, lanolin, ozokerite wax, jojoba seed wax, paraffin wax, microcrystalline wax, hydrogenated rice bran wax, hydrogenated coconut oil glycerides, glyceryl behenate/eicosanoate, myristyl myristate, bis-diglycerol polyacyladipate-2, shea butter, mugwort palm seed fat, and the like. The content of the emollient in the skin external preparation is known in the art, and for example, it is usually about 1 to 50% by weight based on the total weight of component (C).

Examples of such humectants include, but are not limited to, diglycerin, butylene glycol, propylene glycol, 1, 3-propanediol, dipropylene glycol, 1, 2-pentanediol, polyethylene glycol-8, polyethylene glycol-32, methyl gluceth-10, methyl gluceth-20, PEG/PPG-17/6 copolymer, glyceryl polyether-7, glyceryl polyether-26, glyceryl glucoside, PPG-10 methyl glucose ether, PPG-20 methyl glucose ether, PEG/PPG/polytetramethylene glycol-8/5/3 glycerol, sucrose, trehalose, rhamnose, mannose, raffinose, betaine, erythritol, xylitol, urea, glyceryl polyether-5 lactate, sodium hyaluronate, hydrolyzed sodium hyaluronate, sodium acetylated hyaluronate, sodium polyglutamate, sodium salt of a fatty acid, sodium glutamate, sodium glycerophosphate, sodium glutamate, and sodium glutamate, and sodium glutamate, sodium glutamate, One or more of hydrolyzed sclerotium rolfsii gum, pullulanase, tremella polysaccharide, sour bean seed polysaccharide, 1, 2-hexanediol, natural moisturizing factor, ceramide 2, ceramide 3, cholesterol, phospholipid and the like. The content of the moisturizer in the skin external preparation is known in the art, and for example, it is usually about 1 to 30% by weight based on the total weight of component (C).

Examples of the anti-eczema active ingredient include, but are not limited to, one or more of dipotassium glycyrrhizinate, purslane (PORTULACA OLERACEA) extract, bioglycan-1, beta-glucan, levan, SCUTELLARIA BAICALENSIS (scutelaria BAICALENSIS) root extract, AESCULUS HIPPOCASTANUM (AESCULUS hippopacanum) extract, 4-tert-butylcyclohexanol, ceramide 3, GLYCYRRHIZA GLABRA (GLYCYRRHIZA GLABRA) extract, royal jelly protein hydrolysate, oryzanol, phytosphingosine, quercetin (quercetin), ginger root extract, rosemary leaf extract, and the like. The content of the anti-eczema active ingredient in the skin external preparation is known in the art, and for example, it is generally about 0.01 to 10% by weight based on the total weight of the component (C).

Such adjuvants include, for example, emulsifiers, thickeners, preservatives, fragrances and the like.

Examples of such emulsifiers include, but are not limited to, cetearyl olivate, sorbitan olivate, polysorbate-60, polysorbate-80, methylgluco-sesquistearate, PEG-20 methylgluco-sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-Butanethol-26, PEG-4 polyglyceryl-2 stearate, PEG-60 hydrogenated castor oil, steareth-2, steareth-21, PPG-13-decyltetraeth-24, cetearyl glucoside, PEG-100 stearate, glyceryl stearate SE, coco glucoside, ceteareth-25, PEG-40 stearate, polyglyceryl-3 methylgluco distearate, sorbitan esters, glyceryl esters, sorbitan esters, ceteareth-25, PEG-40 stearate, polyglyceryl esters, glyceryl esters, sorbitan esters, glyceryl esters, and the salts of esters, and the salts of the compounds of the, One or more of glyceryl stearate citrate, polyglyceryl-10 stearate, polyglyceryl-10 myristate, polyglyceryl-10 dioleate, polyglyceryl-10 laurate, polyglyceryl-10 isostearate, polyglyceryl-10 oleate, polyglyceryl-10 diisostearate, polyglyceryl-6 laurate, polyglyceryl-6 myristate, sucrose stearate, sucrose polystearate, and the like. The content of the emulsifier in the skin external preparation is known in the art, and for example, it is usually about 0.5 to 10% by weight based on the total weight of component (C).

Examples of the thickener include, but are not limited to, one or more of carbomers, acrylates and derivatives thereof, xanthan gum, acacia, polyethylene glycol-14M, polyethylene glycol-90M, succinoglycan, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, and other high molecular polymers. The content of the thickener in the skin external preparation is known in the art, and for example, it is usually about 0.1 to 10% by weight based on the total weight of component (C).

Examples of such preservatives include, but are not limited to, one or more of methylparaben, propylparaben, phenoxyethanol, benzyl alcohol, phenylethyl alcohol, bis (hydroxymethyl) imidazolidinyl urea, potassium sorbate, sodium benzoate, chlorphenesin, sodium dehydroacetate, caprylhydroxamic acid, 1, 2-hexanediol, 1, 2-pentanediol, p-hydroxyacetophenone, capryl glycol, glyceryl undecylenate, sorbitan caprylate, ethylhexylglycerin, peony root extract, and the like. The content of the preservative in the skin external composition is known in the art, and for example, it is usually about 0.01 to 2% by weight based on the total weight of the component (B).

The skin external composition having enhanced anti-eczema effect of the present invention may be prepared by any suitable method known in the art. For example, it is prepared by using a dissolving tank, an emulsifying pan, a disperser, a transfer pump, etc., which are generally used in the field of cosmetics. The preparation method comprises putting water soluble substance into water phase dissolving kettle, putting oil soluble substance into oil phase dissolving kettle, heating the two kettles to about 80 deg.C, wherein the raw material easy to agglomerate can be pre-dispersed with disperser. And after the dissolution is finished, conveying the oil phase and the water phase into an emulsifying pot, and homogenizing and emulsifying for about 5-15 minutes. After emulsification is finished, the temperature of the material body is reduced to normal temperature, optional essence, preservative and the like are added, and the pH value of the product is adjusted according to needs. After the relevant detection indexes are qualified, the products can be filled and delivered.

The preparation method can be deleted or adjusted according to the requirements of dosage forms. The skin external composition can be made into various dosage forms such as cream, milky lotion, essence, spray, gel, etc. according to need.

Examples

The present invention will be described in further detail with reference to examples. However, it should be understood that these examples and comparative examples are only for illustrating the present invention in more detail, and should not be construed as limiting the scope of the appended claims of the present invention in any way.

Example 1: composition of birch juice and mung bean extractive liquid or willow flower/leaf/stem extract has effect on atopic dermatitis mice

In this example, 2, 4-Dinitrofluorobenzene (DNFB) sensitization and excitation are used to establish an eczema model of a mouse, 1% hydrocortisone ointment is given as a positive control, and the influence of the combination of birch juice and mung bean extractive solution or willow flower/leaf/stem extract on the content of inflammatory factors TSLP, IL-1 β, Th2 cytokines (IL-4, IL-5, IL-13 and IL-17), Th1 cytokine IFN- γ and the key indicator of eczema IgE is examined.

The experimental method is as follows:

1. experimental animals: ICR mouse, male.

2. Experimental materials: 0.5% DNFB acetone solution, depilatory cream, dust mite extract, birch juice, mung bean extract, willow flower/leaf/stem extract, and 1% hydrocortisone ointment.

3. The experimental procedure was as follows: ICR mice were randomly grouped by body weight, 12 mice per group, normal group, model group, and sample group. The skin hair was removed from the area of approximately 2x2cm on the back of each group of mice with depilatory cream 1 day before molding. Determining the experimental range of the back of the mouse, and repeatedly sticking the transparent wide adhesive tape for 8 times to remove the horny layer. All experimental animals were then sensitized by injection of dust mite extract on days 1,3, 5, and 7 of week 1 of the experiment. Injecting dust mite extracting solution to sensitize on 10 th and 13 th days of the 2 nd week of the experiment; in 3-6 weeks of experiment, the dust mite extract solution is injected 1 time per week for sensitization, and simultaneously acetone patch induction is carried out. Acetone (DNCB) was diluted to 0.5% acetone solution using double distilled water, and the back of the mice was contacted with 0.5% DNCB fixed as described above every other day on week 2 for 6 weeks, 100 μ L each, and 1 every 2 days from week 2 and 70 μ L each.

The drug administration is started 12 hours after the molding is successful, different formulas are uniformly sprayed on the back of the experimental mouse by using an aerosol bottle, 3 times a day, and the drug administration is continuously carried out for 7 days. The model group was sprayed with distilled water and the positive group given hydrocortisone for 7 consecutive days, 3 times per day.

The formula of the sample to be tested is as follows:

wherein the 1.2 times of birch juice concentrated solution is obtained by concentrating birch juice stock solution, and the concentration process comprises the following steps: fresh birch juice stock solution purchased from Daxingan mountain surpassing wild berry development Limited company is input into a low-temperature drying device, cooled to-65 ℃, vacuumized to 0.1Pa, and concentrated to 1.2 times.

The fermented birch sap was prepared by fermentation according to the method described in example 1 of application No. CN 201910689097.4.

4. And (4) investigation indexes are as follows:

after 7 days of administration, serum of the mice is taken, and the content change conditions of inflammatory factors TSLP, IL-1 beta, Th2 cytokines (IL-4, IL-5, IL-13 and IL-17), Th1 cytokines (IFN-gamma) and key indicators IgE of the eczema of the serum of the mice are detected by using an ELISA kit.

The results obtained are shown in the following table:

	IL-4(pg/ml)	IL-13(pg/ml)	IL-17(pg/ml)	IL-5(pg/ml)
					normal group	21.53±0.35	12.60±0.23	34.14±0.93	19.23±0.82
Model set	60.38±1.43	38.76±1.49	60.39±1.42	53.59±1.34
					Hydrocortisone group	25.12±0.82	15.18±0.63	35.01±1.49	25.24±1.64
Formulation 1	25.59±2.42	15.23±0.92	37.12±1.92	25.73±1.73
					Formulation 2	26.19±1.42	15.92±2.83	38.33±0.89	26.14±1.46
Formulation 3	35.19±1.84	26.73±1.40	43.12±0.91	36.12±1.47
					Formulation 4	39.12±2.38	28.39±1.48	46.23±2.34	41.35±1.73
Formulation 5	22.47±1.38	13.12±1.94	35.91±1.03	24.19±2.81
					Formulation 6	23.19±0.82	13.92±1.42	35.10±1.93	22.23±2.35
Formulation 7	21.93±1.49	12.98±1.38	34.94±1.24	20.19±1.36
					Formulation 8	22.14±2.10	12.91±2.13	35.29±2.10	22.35±1.63
Formulation 9	43.48±1.49	34.39±2.19	49.12±1.99	40.19±2.10
					Formulation 10	46.21±2,55	38.22±1.68	48.14±2.10	46.82±2.23
Formulation 11	34.19±1.84	23.20±2.04	45.01±1.58	35.77±1.04

	IgE(pg/ml)	TSLP(pg/ml)	TNF-α(pg/ml)	IL-1β(pg/ml)
					Normal group	51.12±0.73	41.35±0.92	46.27±1.33	29.25±0.58
Model set	91.24±1.52	91.24±1.42	72.53±1.94	69.83±1.43
					Hydrocortisone group	59.29±1.84	43.13±1.35	50.29±1.37	35.16±1.83
Formulation 1	60.24±1.63	43.53±0.89	51.63±1.64	37.59±1.93
					Formulation 2	59.27±1.94	45.24±1.35	53.25±1.83	36.19±1.47
Formulation 3	73.83±1.37	56.25±1.45	61.21±1.64	49.20±2.49
					Formulation 4	78.65±1.48	59.32±2.01	61.63±2.82	47.26±1.30
Formulation 5	55.27±2.84	41.31±1.59	49.23±1.92	35.42±2.04
					Formulation 6	53.86±2.01	42.12±1.93	47.15±2.01	34.95±1.02
Formulation 7	53.24±1.29	42.41±1.42	46.92±2.49	32.49±0.91
					Formulation 8	52.14±1.73	41.90±2.41	47.52±1.24	30.49±2.49
Formulation 9	80.29±2.12	61.49±2.02	69.66±1.93	58.39±2.78
					Formulation 10	83.22±2.99	65.33±2.33	70.39±1.77	60.77±1.88
Formulation 11	75.01±1.03	50.28±3.01	58.38±2.01	49.30±2.09

The results show that the combination of birch juice and mung bean extractive solution or willow flower/leaf/stem extract shows remarkable anti-eczema effect. Further, compared with the birch juice stock solution, the 1.2 times of concentrated solution or fermented birch juice and the mung bean extract or the willow flower/leaf/stem extract show better anti-eczema effect when used in combination.

Example 2: effect of composition of birch sap and campesterol or hydrolyzed rice protein on expression of protein related to moisture retention and barrier repair

This example investigated the effect of birch sap in combination with canola sterols or hydrolysed rice protein on cutin transglutaminase TGM1, epidermal tight junction protein (ZO-1 and CLDN1), silk fibroin FLG and aquaporin AQP 3.

The experimental method is as follows:

1. an experimental instrument: a fluorescent quantitative PCR instrument (Roche), a super clean bench (Sujing), a carbon dioxide incubator (Binder), a microplate reader (BIO-TEK) and a micro oscillator.

2. Experiment reagent and consumable: keratinocytes, 6-well plates, cell culture solution, RNA extraction kit, reverse transcription kit, Trizol lysate and the like.

3. The keratinocyte-based gene expression analysis procedure was as follows:

(1) inoculation: cells were seeded into 6-well plates at a seeding density of 2E 5/well at 37 ℃ and 5% CO₂Incubating in an incubator overnight;

(2) preparing liquid: the specific test formulations are shown in the following table:

wherein the 1.2 times of birch juice concentrated solution is obtained by concentrating stock solution of birch juice, and the concentration process comprises: inputting fresh birch juice stock solution purchased from Daxingan Ling surpassing wild berry development Limited company into low-temperature drying equipment, cooling to-65 deg.C, vacuumizing to 0.1Pa, and concentrating to 1.2 times;

the fermented birch juice is prepared by fermenting according to the method described in example 1 of application No. CN 201910689097.4.

(3) Administration: when the cell plating rate in the 6-hole plate reaches about 50%, adding the test substances of each group, and arranging 4 multiple holes in each group;

(4) collecting a sample: at 37 ℃ and 5% CO₂After 24 hours in the incubator, discarding the culture solution, adding 1mL Trizol into each hole, blowing and beating the lysed cells, and collecting the samples;

(5) and (3) PCR detection: extracting RNA, carrying out reverse transcription to cDNA, and carrying out fluorescent quantitative PCR detection;

(6) and (3) analysis: by using 2^-△△CTThe method carries out result calculation and adopts a T-Test method for statistical analysis.

The results obtained are shown in the following table:

the results in the above table show that when birch sap is used in combination with rape sterols or hydrolysed rice protein, the expression of moisturizing and barrier repair related genes such as cutin transglutaminase TGM1, epidermal tight junction proteins (ZO-1 and CLDN1), silk fibroin FLG and aquaporin AQP3 can be significantly increased. Further, the 1.2-fold concentrated solution or fermented birch sap showed better anti-eczema effect when used in combination with campestanols or hydrolyzed rice protein, compared to the birch sap stock solution.

Example 3: effect of composition of birch juice and ginger root extract or terpinen-4-ol on degranulation of mast cells of zebra fish

This example uses a zebrafish juvenile allergy model to test the effect of birch juice in combination with ginger root extract or terpinen-4 ol on mast cell degranulation, thereby verifying that the anti-inflammatory efficacy of the combination of birch juice with ginger root extract or terpinen-4 ol is much better than that of ginger root extract or terpinen-4 alcohol used alone.

The experimental method is as follows:

1. experimental animals: zebra fish.

2. The experimental procedure was as follows: AB wild-type zebrafish embryos were collected and cultured in E3 buffer to 5dpf (days post fertilization) in a 28.5 ℃ incubator with daily changes. Randomly transferring 5dpf zebra fish juvenile fish into 48-hole cell culture plates according to the number of 10 tails per hole, and grouping, wherein each group comprises 4 multiple holes, and the grouping condition is as follows:

model group: RO Water + 15. mu.g/ml SP

A positive drug group: ketotifen + 15. mu.g/ml SP

The formula of the sample to be tested is as follows:

corresponding to each group added with SP for inducing degranulation, a negative control group (without SP) is additionally arranged, and each group has 4 multiple wells; a background control group (not containing zebrafish juvenile fish) was additionally set corresponding to the SP degranulation induction group and the SP-free negative control group, with 2 replicates per group. The residual E3 buffer in each well was blotted and 250. mu.l of the corresponding solution was added to each well and reacted in an incubator at 28.5 ℃ for 60 minutes in the absence of light. After 60 minutes, 200. mu.l of each group of supernatants was placed in a 96-well cell culture plate, and then enzyme reaction substrate BAPNA was added to make the concentration to 400. mu.g/ml. And (3) covering the 96-well plate in a dark place, putting the 96-well plate into an incubator at 28.5 ℃ for reacting for 2 hours, measuring the light absorption value of the whole plate at 405nm after 2 hours, wherein the value reflects the tryptase release condition of the zebra fish mast cells.

The results of the efficacy experiments of the zebra fish juvenile fish mast cell protection model are recorded in the following table:

the results in the above table show that birch juice shows a significant effect of inhibiting mast cell degranulation when used in combination with ginger root extract or terpinen-4-ol. Further, the 1.2-fold concentrated solution or fermented birch juice showed better efficacy when used in combination with ginger root extract or terpinen-4-ol, compared to the birch juice stock solution.

Example 4: composition of birch juice and mung bean extractive solution or willow flower/leaf/stem extract has effect on histamine release of RBL-2H3 cells

The experimental principle is as follows: compound 48/80 (a polymer produced by the condensation of N-methyl-p-methoxyphenethylamine and formaldehyde) is a tool drug that causes the degranulation reaction of mast cells by a mechanism that acts on the mast cell membrane, causing an increase in intracellular calcium ions that alter the amounts of the second messengers cAMP and cGMP, resulting in degranulation of mast cells and the release of histamine.

Experimental materials: the RBL-2H3 cell strain is provided by animal center of Zhejiang medical science institute; the formulations of the test samples are shown in the table below, and the reagents include fetal bovine serum, benchtop salt, compound 48/80, histamine Elisa kit.

The experimental process comprises the following steps:

1. solution preparation:

(1) preparing a desktop liquid: one portion of table salt is diluted by 80ml of sterilized deionized water, the volume is adjusted to 100ml to prepare table liquid with 10 times of concentration, and the table liquid is autoclaved.

(2) Compound 48/80 solution preparation: 48/80 powder (1.0 mg) is weighed and diluted with 1ml of bench-top solution, 50ul is diluted to 1000ul, namely 48/80 solution with the concentration of 50ug/ml, which needs to be prepared for use.

2. The experimental steps are as follows:

(1) cell treatment: RBL-2H3 cells in logarithmic phase are paved on the bottom of a culture flask, then the cells are digested by digestive juice containing 0.25 percent of trypsin and 0.03 percent of EDTA, and centrifugation is carried out for 5 minutes at 1200 rpm; 1 times the desktop solution was counted, diluted to 1 x 10⁴Per ml; the supernatant was centrifuged again at 1200rpm for 5 minutes.

(2) Grouping and testing of samples: taking 700ul of cell suspension from the model group, centrifuging the cell suspension, carrying out background liquid resuspension, and placing the cell suspension for 30 minutes at 37 ℃; taking 700ul of cell suspension, centrifuging, performing background liquid resuspension, standing at 37 ℃ for 30 minutes, mixing the samples of the formulas 1-11 uniformly, and subpackaging into three tubes with 200ul of each tube; 50ul of 48/80 solution was added to the cell suspension to a final concentration of 10ug/ml in the model and sample groups, and left at 37 ℃ for 20 minutes, with 3 replicates in each group.

(3) Histamine determination: the reaction was terminated by placing each of the above-mentioned groups of samples in an ice box and cooling for 10 minutes, and the reaction-terminated samples were centrifuged (4 ℃, 1500rpm, 30 minutes) to pellet the cells at the bottom of the EP tube, and the supernatant was transferred to a new centrifuge tube. Suspending the precipitated cells in 250ul of 1-fold desk type liquid, heating in a 90 ℃ water bath, taking out after 5 minutes, immediately placing in an ice box, taking out after the sample is completely cooled, placing in a 90 ℃ water bath again for heating, repeatedly freezing and thawing for several times, and crushing the cells. The supernatant and the cell sap were diluted 10 times, and the stock solution was stored in a refrigerator at-20 ℃. The diluted samples were subjected to histamine determination using an imported histamine kit and calculated. The histamine release rate is calculated by the formula: histamine release rate (%). extracellular histamine release/(extracellular histamine release + intracellular histamine release). 100%.

3. The experimental results are as follows: the results of the test for histamine release in the cell model are shown below:

group of	Release amount%	P value (vs model)	P value
				Model set	70.1±1.3	——	——
Formulation 1	30.1±2.4	<0.001	——
				Formulation 2	29.8±1.6	<0.001	——
Formulation 3	43.2±2.5	<0.001	(vs formulation 1) P ═ 0.019
				Formulation 4	46.3±3.1	<0.001	(vs formulation 2) P ═ 0.008
Formulation 5	25.1±4.5	<0.001	——
				Formulation 6	23.2±2.8	<0.001	——
Formulation 7	20.1±3.3	<0.001	——
				Formulation 8	17.3±2.7	<0.001	——
Formulation 9	56.2±1.9	0.005	(vs formulation 1) P<0.001
				Formulation 10	60.8±2.2	0.007	(vs formulation 2) P<0.001
Formulation 11	44.3±2.9	<0.001	——

The results in the above table show that the birch juice combined with the mung bean extract or the willow flower/leaf/stem extract significantly reduces the release amount of histamine, indicating that it has a synergistic effect. Further, the 1.2 times concentrated solution or fermented birch juice has better efficacy when combined with mung bean extract or willow flower/leaf/stem extract compared with birch juice raw solution.

Example 5: anti-eczema face cream composition

The formula of the anti-eczema cream composition is as follows:

the anti-eczema cream composition is prepared as follows:

1. uniformly dispersing the raw material 4 and the raw material 11;

2. heating and dissolving the raw material 7 and the raw material 10;

3. putting the raw materials 1 and 25 into a water phase pot, scattering the raw material 12 while stirring, adding the raw materials 2, 3, 4, 5, 6, 7, 9, 10 and 11 after the raw material No. 12 is completely swelled, and heating to 80 ℃;

4. putting raw materials 8, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 into an oil phase pot, and heating to 80 ℃;

5. pumping the raw materials in the water phase pot into an emulsifying pot, and homogenizing at high speed for 5 minutes;

6. pumping the raw materials in the oil phase pot into an emulsifying pot, homogenizing at high speed for 5 minutes, and keeping the temperature for 10 minutes;

7. cooling to 50 deg.C while stirring, adding raw materials 23 and 24, and slowly homogenizing for 3 min;

8. cooling to 40 ℃ while stirring;

9. discharging after the inspection is qualified.

In this example, 20 children with mild eczema between 3 and 15 years old and 20 children with moderate eczema were selected for 3 weeks of treatment in the child's period. Wherein 10 mild and 10 moderate eczema children were applied with the anti-eczema cream 2-3 times daily, each time 100 g, and the other patients were applied with skin cream without birch sap (control product, formulation of which is identical to that in the above table, but all birch sap was replaced with water), and disease degree scoring was performed for three weeks, day 14.

The results show that subjects in the group of anti-eczema creams significantly improved skin condition after application of the anti-eczema cream at 14 days of administration (P <0.05), significantly reduced Eczema Area and Severity Index (EASI) (P <0.01), and significantly reduced atopic dermatitis score index (SCORAD) (P <0.05) compared to pre-administration. Meanwhile, compared with the control group product, the anti-eczema cream group also improves the skin condition (P <0.05), the Eczema Area and Severity Index (EASI) is obviously reduced (P <0.01), and the atopic dermatitis integral index (SCORAD) is obviously reduced (P < 0.05). The eczema condition is further improved when the drug is administrated for 21 days. The experimental results show that the anti-eczema cream has the obvious effect of treating mild to moderate eczema.

Example 6: anti-eczema essence composition

The formula of the anti-eczema essence composition is as follows:

the anti-eczema essence composition is prepared as follows:

1. uniformly dispersing the raw material 3 and the raw material 11;

2. putting the raw material 1 into an emulsifying pot, scattering the raw materials 14 and 15 while stirring, adding the raw materials 2,4, 5, 6, 7, 8, 9, 10, 12 and 13 and the raw material 21 after the raw materials 14 and 15 are completely swelled, heating to 80 ℃ while stirring, homogenizing at high speed for 5 minutes, and keeping the temperature for 10 minutes;

3. cooling to 50 ℃ while stirring, adding the raw materials 16 and 17 and the raw material 18, and homogenizing at high speed for 5 minutes;

4. cooling to 50 ℃ while stirring, and adding the raw materials 19 and 20;

5. cooling to 40 ℃ while stirring;

6. discharging after the inspection is qualified.

In this example, 20 children with mild eczema at 3-15 years of age and 20 children with moderate eczema were selected for 3 weeks of treatment. Wherein 10 children with mild eczema and 10 children with moderate eczema use the anti-eczema essence liquid, and are smeared for 2-3 times every day, and each time is 100-200g, and the other patients use essence liquid without birch juice (control product, the formula is completely the same as in the above table, but all birch juice is replaced by water), and disease degree scoring is carried out continuously for three weeks and 14 days.

The results show that the anti-eczema essence group improves the skin condition (P <0.05), the Eczema Area and Severity Index (EASI) is significantly reduced (P <0.01), and the atopic dermatitis integral index (SCORAD) is significantly reduced (P <0.05) compared with the control group product. The eczema condition is further improved when the drug is administrated for 21 days. The experimental results show that the anti-eczema essence has the obvious effect of treating mild to moderate eczema.

Example 7: anti-eczema emulsion composition

The formula of the anti-eczema emulsion composition is as follows:

serial number	Composition (I)	Weight percent
			1	1.2 times concentrated birch juice	76
2	Hydrolyzed sodium hyaluronate	0.05
			3	Willow flower/stem/leaf extract	4.05
4	Xanthan gum	0.1
			5	Hydroxy phenyl methyl ester	0.2
6	Polysorbate-60	0.3
			7	PEG-60 hydrogenated Castor oil	0.5
8	Hydrolyzed rice protein	1
			9	Dipropylene glycol	4
10	Inulin powder	4
			11	Glycerol	7.1
12	Carbomer	0.2
			13	Phytosterol/octyldodecanol lauroyl glutamate	1
14	Tromethamine	0.1
			15	PEG/PPG-17/6 copolymer	1
16	Phenoxyethanol	0.4

The anti-eczema emulsion composition is prepared as follows:

1. uniformly dispersing the raw material 4 and the raw material 9;

2. putting the raw material 1 into an emulsifying pot, scattering the raw material 11 while stirring, and putting the raw materials 2, 3, 5, 6, 7, 8 and 10 after the raw material 11 is completely swelled;

3. heating to 80 ℃ while stirring, homogenizing at high speed for 5 minutes, and keeping the temperature for 10 minutes;

4. adding the raw materials 12, homogenizing at high speed for 5 minutes, and keeping the temperature for 10 minutes;

5. cooling to 60 ℃ while stirring, and adding the raw material 13;

6. cooling to 50 ℃, adding the raw materials 14, 15 and 16, and slowly homogenizing for 3 minutes;

7. cooling to 40 ℃ while stirring;

8. discharging after the inspection is qualified.

In this example, 20 children with mild eczema at 3-15 years of age and 20 children with moderate eczema were selected for 3 weeks of treatment. Wherein 10 mild and 10 moderate eczema children were administered the anti-eczema lotion 2-3 times daily at 100 g each, and the remaining patients were administered the lotion without the anti-eczema active (control product, formulation identical to that in the above table, but all birch sap, willow flower/stem/leaf extract, hydrolysed rice protein and inulin were replaced by water) for three weeks with disease severity scoring at day 14.

The results show that the anti-eczema lotion group improved skin condition (P <0.05), the Eczema Area and Severity Index (EASI) was significantly reduced (P <0.01), and the atopic dermatitis score index (SCORAD) was significantly reduced (P <0.05) compared to the control group product. The eczema condition is further improved after 21 days of administration. The experimental results show that the anti-eczema lotion has the obvious effect of treating mild to moderate eczema.

Claims

1. A skin external composition with enhanced anti-eczema effect comprises (A) birch juice, and (B) one or more of mung bean extract, ginger root extract, terpinen-4-ol, willow flower/leaf/stem extract, inulin, alpha glucan fructose, campesterol and hydrolyzed rice protein.

2. The composition for skin external use according to claim 1, wherein the (a) birch juice is a birch juice stock solution, a concentrated birch juice or a fermented birch juice.

3. The composition for external skin application according to claim 2, wherein the concentrated birch sap is concentrated about 1.05 to 8 times, preferably about 1.1 to 4 times, more preferably about 1.2 to 2 times.

4. The topical skin composition of claim 2, wherein the fermented birch sap is a birch sap fermented by inonotus obliquus.

5. The composition for skin external use according to any one of claims 1 to 4, wherein the (A) birch sap is contained in an amount of about 18 to 98% by weight, preferably about 20 to 95% by weight, more preferably about 22 to 90% by weight, most preferably about 30 to 90% by weight, based on the total weight of the composition for skin external use.

6. The composition for skin external application according to any one of claims 1 to 5, wherein the total content of one or more of (B) mung bean extract, ginger root extract, terpinen-4-ol, willow flower/leaf/stem extract, inulin, alpha glucan fructose, rape sterol and hydrolyzed rice protein is about 0.01 to 10% by weight, preferably about 0.1 to 6% by weight, more preferably about 0.5 to 5% by weight, based on the total weight of the composition for skin external application.

7. The composition for external application to skin as claimed in any one of claims 1 to 6, wherein the composition for external application to skin is a pharmaceutical composition or a cosmetic composition.

8. The reagent bag comprises (A) birch juice and (B) one or more of mung bean extract, ginger root extract, terpinene-4 alcohol, willow flower/leaf/stem extract, inulin, alpha glucan fructose, rape sterols and hydrolyzed rice protein.

9. Use of the kit of claim 8 in a skin external composition having enhanced anti-eczema efficacy.