Mixed birch sap and its use in skin care cosmetic compositions

Abstract

The present invention relates to a mixed birch sap comprising birch sap from two or more different origins. The present invention also relates to use of the mixed birch sap in a skin care cosmetic composition and to a skin care cosmetic composition comprising the mixed birch sap, in particular a moisturizing cosmetic composition, a whitening cosmetic composition, an anti-wrinkle cosmetic composition, and an anti-inflammatory cosmetic composition.

Description

Technical Field

[0001] The present invention relates to a mixed birch sap and its use in a skin care cosmetic composition. Background Art

[0002] Birch sap is the sap exuded from Betula plants in the Betulaceae family. There are approximately 100 species of Betula, primarily found in the northern temperate zone, with a few species found in the Arctic. Birch sap is rich in essential polysaccharides, amino acids, vitamins, biotin, cytokinins, and minerals. It has excellent moisturizing, whitening, and anti-wrinkle properties, promoting the expression of proteins in skin cells related to moisturizing, whitening, and anti-wrinkle, reducing transepidermal water loss, brightening the skin, and improving skin elasticity.

[0003] Studies have shown that the nutritional components of birch sap are affected by many factors. Its variety, origin, growth environment, collection method, etc. can lead to significant differences in its composition, which in turn leads to significant differences in efficacy. For example, different origins can lead to differences in the composition of birch sap. For example, Wang Yunfang et al. reported that the types and contents of trace elements in birch sap collected from different regions vary greatly. This is related to the water and soil quality of its growth environment, or to the ability of the birch tree itself to enrich certain elements. Therefore, birch sap is often used as an indicator plant for biological prospecting (see Wang Yunfang, Xiang Yingmei, Xu Guoying et al., Study on the Chemical Composition of Xinjiang Birch Sap, Arid Zone Research, 1995(1):40-44). Summary of the Invention

[0004] The inventors have found through research that mixed birch sap formed by compounding birch sap from different origins can significantly enhance the various skin care effects of birch sap, which indicates that birch sap from different origins can synergistically enhance the efficacy.

[0005] Thus, in one aspect, the present invention provides a blend of birch sap comprising birch sap from two or more different origins.

[0006] Birch sap (also commonly referred to as white birch sap) is produced in Northeast China, North China, Northwest China, Southwest China, Japan, North Korea, Mongolia, Russia, Finland, Norway, Sweden, Estonia, Lithuania, Poland, Belarus, Ukraine, the United States, and Canada. In the present invention, the preferred production areas are Northeast China (hereinafter referred to as Northeast China), Finland, Belarus, and Russia. Herein, birch sap from Northeast China is referred to as Northeast China birch sap, birch sap from Finland is referred to as Finnish birch sap, and so on.

[0007] In a preferred embodiment, the mixed birch sap comprises Northeast birch sap and Finnish birch sap. In a further preferred embodiment, the mixed birch sap comprises 20-80%, preferably 30-70%, more preferably 40-60% of Northeast birch sap and 80-20%, preferably 70-30%, more preferably 60-40% of Finnish birch sap, based on the total weight of the mixed birch sap.

[0008] In a preferred embodiment, the mixed birch sap comprises Northeast birch sap and Belarus birch sap. In a further preferred embodiment, the mixed birch sap comprises 20-80%, preferably 30-70%, more preferably 40-60% of Northeast birch sap and 80-20%, preferably 70-30%, more preferably 60-40% of Belarus birch sap, based on the total weight of the mixed birch sap.

[0009] In a preferred embodiment, the mixed birch sap comprises Finnish birch sap and Belarusian birch sap. In a further preferred embodiment, the mixed birch sap comprises 20-80%, preferably 30-70%, more preferably 40-60% of Finnish birch sap and 80-20%, preferably 70-30%, more preferably 60-40% of Belarusian birch sap, based on the total weight of the mixed birch sap.

[0010] In a preferred embodiment, the mixed birch sap comprises Northeast birch sap and Russian birch sap. In a further preferred embodiment, the mixed birch sap comprises 20-80%, preferably 30-70%, more preferably 40-60% of Northeast birch sap and 80-20%, preferably 70-30%, more preferably 60-40% of Russian birch sap, based on the total weight of the mixed birch sap.

[0011] In another aspect, the present invention also relates to use of the mixed birch sap in a skin care cosmetic composition.

[0012] In another aspect, the present invention relates to a skin care cosmetic composition comprising the mixed birch sap (A).

[0013] The content of the mixed birch sap (A) in the above-mentioned skin care cosmetic composition is about 18-98% by weight, preferably about 20-95% by weight, more preferably about 22-90% by weight, and most preferably about 30-90% by weight, based on the total weight of the skin care cosmetic composition.

[0014] Preferably, the skin care cosmetic composition of the present invention does not contain any added water, but does not exclude the moisture inherently contained in the various components.

[0015] In a preferred embodiment, the skin care cosmetic composition of the present invention does not contain chelating agents such as EDTA salts, sodium polyphosphate, sodium metaphosphate, and gluconic acid.

[0016] In one embodiment, the skin care cosmetic composition is a moisturizing cosmetic composition, wherein the moisturizing cosmetic composition significantly increases the skin moisture content of the subject due to the presence of the mixed birch sap.

[0017] In one embodiment, the skin care cosmetic composition is a whitening cosmetic composition, wherein, due to the presence of the mixed birch sap, the whitening cosmetic composition significantly improves the skin brightness of the subject and reduces the content of melanin and red pigment in the skin.

[0018] In one embodiment, the skin care cosmetic composition is an anti-wrinkle cosmetic composition, wherein the anti-wrinkle cosmetic composition significantly reduces the number, size, and depth of wrinkles in a subject due to the presence of the blended birch sap.

[0019] In one embodiment, the skin care cosmetic composition is an anti-inflammatory cosmetic composition, wherein the anti-inflammatory cosmetic composition significantly reduces inflammation in a subject due to the presence of the mixed birch sap.

[0020] In the present invention, birch sap can be used in the form of a raw liquid or a concentrated liquid, for example, in the form of a concentrated liquid of about 1.05-8 times, preferably about 1.1-4 times (abbreviated as about 1.05-8 times, preferably about 1.1-4 times concentrated birch sap).

[0021] The birch sap is commercially available, for example, Northeast birch sap can be purchased from Daxinganling Chaoyue Wild Berry Development Co., Ltd., Finnish birch sap can be purchased from MySeTo Oy Co., Ltd., Belarusian birch sap can be purchased from Jiulu Fruit and Vegetable Factory, and Russian birch sap can be purchased from Healthy Stream Co., Ltd.

[0022] Concentrated birch sap is obtained by concentrating the commercially available product. Concentration methods are known in the art, such as heating concentration, low-temperature vacuum concentration, and membrane concentration. In the present invention, concentration is preferably performed by low-temperature freeze concentration or membrane concentration. For example, commercially available birch sap stock solution is fed into a low-temperature drying device, cooled to -40°C to -70°C, and vacuumed to 0.1-30 Pa for low-temperature vacuum concentration, thereby obtaining concentrated birch sap of varying concentration ratios.

[0023] In addition to the birch sap mixed with component (A), the skin care cosmetic composition may optionally further comprise component (B) commonly used ingredients in skin care cosmetics, including various commonly used ingredients known in the field of skin care cosmetics, examples of which include but are not limited to vehicles, active ingredients, and excipients. Those skilled in the art can select the type and amount of component (B) as needed. For example, the content of component (B) is generally about 2-82% by weight, based on the total weight of the cosmetic composition.

[0024] The vehicle is known in the art, such as a diluent, a dispersant, and a carrier, and examples thereof include but are not limited to ethanol, dipropylene glycol, butylene glycol, etc. The content of the vehicle in the cosmetic composition is known in the art, for example, it generally accounts for 0.5-20% of the total weight of component (B).

[0025] The active ingredients are those known in the art, and examples thereof include, but are not limited to, emollients, moisturizers, whitening active ingredients, anti-wrinkle active ingredients, anti-inflammatory active ingredients, and the like.

[0026] Examples of the emollient include, but are not limited to, one or more of 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 fruit oil, argania spinosa kernel oil, jojoba seed oil, sunflower seed oil, palm oil, squalane, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutene, isohexadecane, isododecane, diethylhexyl carbonate, dicaprylyl carbonate, isopropyl lauroyl sarcosinate, isononyl isononanoate, hydrogenated polydecene, triethylhexanoin, cetyl ethylhexanoate, bis-diethoxydiglycol cyclohexane 1,4-dicarboxylate, caprylic / capric triglyceride, oleyl erucate, octyldodecyl myristate, octyldodecanol, dimethicone, caprylyl methicone, cetyl dimethicone, cyclopentasiloxane, and the like. Examples of solid emollients include, but are not limited to, one or more of cetyl alcohol, stearyl alcohol, cetearyl alcohol, behenyl alcohol, batyl alcohol, lauric acid, myristic acid, palmitic acid, stearic acid, beeswax, candelilla wax, carnauba wax, lanolin, ozokerite, jojoba seed wax, paraffin, microcrystalline wax, hydrogenated rice bran wax, hydrogenated coconut glycerides, glyceryl behenate / eicosate, myristyl myristate, bis-diglyceryl polyacyl adipate-2, shea butter, and Murumuru seed butter. The content of the emollient in the cosmetic composition is known in the art, for example, it generally accounts for about 1-50% of the total weight of component (B).

[0027] Examples of the moisturizing agent include, but are not limited to, one or more of glycerin, 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, glycereth-7, glycereth-26, glyceryl glucoside, PPG-10 methyl gluceth, PPG-20 methyl gluceth, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol, sucrose, trehalose, rhamnose, mannose, raffinose, betaine, erythritol, xylitol, urea, glycereth-5 lactate, sodium hyaluronate, hydrolyzed sodium hyaluronate, acetylated sodium hyaluronate, sodium polyglutamate, hydrolyzed sclerotium gum, budding pullulan polysaccharide, tremella polysaccharide, and sour bean seed polysaccharide. The content of the humectant in the composition is known in the art, for example, it generally accounts for about 1-30% of the total weight of component (B).

[0028] The whitening active ingredients include but are not limited to kojic acid, ascorbyl glucoside, arbutin, tranexamic acid, niacinamide, phytosterols, phytosterol / behenyl alcohol / octyldecyl lauroyl glutamate, phenylethyl resorcinol, turmeric root extract, birch bark extract, ceramide 2, ceramide 3, acetyl phytosphingosine, resveratrol, palmyra palm bark extract, coleus forskohlii root extract, pepper seed extract, ubiquinone, cholesterol, cholesterol stearate, ascorbic acid, ascorbyl dipalmitate, tocopherol (vitamin E), tocopheryl acetate, bisabolol, ascorbyl tetraisopalmitate, pyridoxine dioctanoate, pyridoxine dipalmitate, retinyl palmitate, phytosterol / octyldodecanol lauroyl glutamate, bis-behenyl alcohol / isostearyl alcohol / phytosteryl dimer linoleyl dimer linoleate, phytosteryl macadamia nut oleate, various peptides, various plant extracts, etc. One or more. The amount of the anti-aging ingredient in the composition is known in the art. Typically, in the composition of the present invention, the skin conditioning agent comprises about 0.01-50% by weight of the total weight of component (B).

[0029] Examples of the anti-wrinkle active ingredients include, but are not limited to, tocopherol (vitamin E), retinol, retinyl palmitate, hydrolyzed collagen, hydrolyzed elastin, allantoin, yeast extract, oryzanol, tetrahydrocurcumin, ellagic acid, ubiquinone, whey protein, acetyl hexapeptide-8, palmitoyl pentapeptide-4, salicyloyl phytosphingosine, birch sap concentrate, silymarin, sericin, sodium tocopheryl phosphate, ribonucleic acid (RNA), dipeptide diaminobutyryl benzylamide diacetate, palmitoyl tripeptide-5, oligopeptide-1, hexapeptide-9, palmitoyl oligopeptide, palmitoyl tetrapeptide-7, grape (VITIS VINIFERA) seed extract, PTEROCARPUS MARSUPIUM bark extract, tea (CAMELLIA SINENSIS) polyphenols, wine extract, apple seed extract, Fagus sylvatica bud extract, hydrolyzed baobab (ADANSONIA DIGITATA) extract, Artemia (ARTEMIA) extract, Iris florentina) root extract, hesperidin, ginsenosides, Salvia miltiorrhiza) extract, niacinamide, ursolic acid, sodium hyaluronate, acetylated sodium hyaluronate, hydrolyzed sodium hyaluronate, lycopene, coffee (COFFEA ARABICA) extract, dipeptide-2, lactic acid, superoxide dismutase (SOD), evening primrose (OENOTHERA BIENNIS) oil, ceramide, dipalmitoyl hydroxyproline, hydroxystearic acid, salicylic acid, ergothioneine, lysolecithin, carnosine, decarboxylated carnosine HCL, lipoic acid, adenosine, glycogen, resveratrol, ferulic acid, bifid yeast fermentation product lysate, lactic acid bacteria fermentation lysate, etc. The content of the anti-aging active ingredient in the cosmetic composition is known in the art, for example, it generally accounts for about 0.01-10% by weight of the total weight of component (B).

[0030] Examples of the anti-inflammatory active ingredients include, but are not limited to, one or more of dipotassium glycyrrhizate, purslane (PORTULACAOLERACEA) extract, oat (AVENA SATIVA) kernel extract (avenous ylanthranilic acid), panthenol, allantoin, biosaccharide gum-1, β-glucan, fructan, scutellaria baicalensis root extract, horse chestnut (AESCULUS HIPPOCASTANUM) extract, bisabolol, 4-tert-butylcyclohexanol, ceramide 3, hydrogenated lecithin, glycyrrhiza glabra extract, hydrolyzed royal jelly protein, oryzanol, phytosphingosine, quercetin, ginger root extract, rosemary leaf extract, chamomile extract, calendula extract, Centella asiatica extract, naringin, hesperidin, and the like. The content of the anti-inflammatory active ingredient in the cosmetic composition is known in the art, for example, it generally accounts for about 0.01-10% by weight of the total weight of component (B).

[0031] Examples of the auxiliary materials include, but are not limited to, emulsifiers, thickeners, preservatives, flavors, and the like.

[0032] Examples of the emulsifier include, but are not limited to, cetearyl olivate, sorbitan olivate, polysorbate 60, polysorbate 80, methyl glucose sesquistearate, PEG-20 methyl glucose sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-buteth-26, PEG-4 polyglyceryl-2 stearate, PEG-60 hydrogenated castor oil, steareth-2, steareth-21, PPG-13-decyltetradeceth-24, cetearyl glucoside, PEG-100 stearate, glycerin. Stearate, glyceryl stearate SE, coco-glucoside, ceteareth-25, PEG-40 stearate, polyglyceryl-3 methylglucose distearate, 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, etc. One or more of the following. The content of the emulsifier in the cosmetic composition is known in the art, for example, it generally accounts for about 0.5-10% of the total weight of component (B).

[0033] Examples of the thickener include, but are not limited to, one or more of carbomers, acrylic acid (esters) and their derivatives, xanthan gum, gum arabic, polyethylene glycol-14M, polyethylene glycol-90M, succinoglycan, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, and other high molecular weight polymers. The content of the thickener in the cosmetic composition is known in the art, for example, it generally accounts for about 0.1-10% of the total weight of component (B).

[0034] Examples of the preservative 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, caprylyl glycol, glyceryl caprylate, glyceryl undecylenate, sorbitan caprylate, ethylhexylglycerin, peony root extract, etc. The content of the preservative in the cosmetic composition is known in the art, for example, it generally accounts for about 0.01-2% of the total weight of component (B).

[0035] The skin care cosmetic composition of the present invention can be prepared by any suitable method known in the art. For example, it can be prepared using equipment commonly used in the cosmetics field, such as dissolution tanks, emulsifying pots, dispersers, and delivery pumps. During preparation, the water-soluble substance is first put into the water phase dissolution kettle, and the oil-soluble substance is put into the oil phase dissolution kettle. The temperature of the two kettles is heated to about 80°C. For raw materials that are prone to caking, they can be pre-dispersed with a disperser. After the dissolution is completed, the oil phase and the water phase are transferred to the emulsifying pot and homogenized and emulsified for about 5-15 minutes. After the emulsification is completed, the temperature of the material body is reduced to room temperature, and optional flavors, preservatives, etc. are added, and the pH of the product is adjusted as needed. The product can be filled and shipped only after all relevant test indicators are qualified.

[0036] The above preparation method can be deleted or adjusted according to the dosage form requirements. The cosmetic composition can be prepared into various dosage forms such as ointment, cream, lotion, essence, etc. as needed. Example

[0037] The present invention will be further described in detail below with reference to the examples. However, it should be understood that these examples and comparative examples are merely for illustrating the present invention in detail and should not be construed as limiting the scope of the appended claims in any form.

[0038] Example 1: Moisturizing Effects of Birch Sap Mixtures from Different Origins

[0039] This example aims to investigate the differences in moisturizing efficacy of birch sap from different origins, and examines the effects of birch sap mixtures on moisturizing-related genes, proteins, and cell proliferation.

[0040] Specifically, the inspection methods are as follows:

[0041] 1. Moisturizing-related gene expression test

[0042] Experimental instruments: fluorescence quantitative PCR instrument (Roche), clean bench (Sujing), carbon dioxide incubator (Binder), microplate reader (BIO-TEK), micro-oscillator.

[0043] Experimental reagents and consumables: human primary keratinocytes, 6-well plates, keratinocyte culture medium, RNA extraction kit, reverse transcription kit, Trizol lysis buffer, etc.

[0044] The steps for keratinocyte-based gene expression analysis are as follows:

[0045] (1) Seeding: Seed cells into 6-well plates at a seeding density of 5E5 / well and incubate overnight in a 37°C, 5% CO2 incubator;

[0046] (2) Drug administration: When the cell plating rate in the 6-well plate reaches about 60%, add the test substance of each group, with 6 replicate wells for each group;

[0047] (3) Sample collection: After 24 h in a 37°C, 5% CO2 incubator, discard the culture medium, add 1 mL of Trizol to each well, pipette and lyse the cells, and then collect the samples;

[0048] (4) PCR detection: RNA was extracted, reverse transcribed into cDNA, and then fluorescence quantitative PCR was performed;

[0049] (5) Analysis: Using 2 -△△CT Methods The results were calculated and statistically analyzed using the T-Test method.

[0050] The test results are shown in Table 1.

[0051] Table 1

[0052]

[0053]

[0054] * indicates that the difference was significant compared with 100% Northeast birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Northeast birch sap, with a P value of less than 0.01.

[0055] a. The 50% concentration of Finnish birch sap or Northeastern birch sap refers to the original Finnish birch sap or Northeastern birch sap diluted to 50% with pure water.

[0056] b. Double concentrated Finnish birch sap or double concentrated Northeastern birch sap means that the original liquid of Finnish birch sap or Northeastern birch sap is input into low temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 2 times.

[0057] The results in Table 1 show that compared with Finnish birch sap or Northeast birch sap alone, a mixture containing 30-70% Finnish birch sap and 70-30% Northeast birch sap can significantly increase the expression of genes related to moisturizing in keratinocytes, especially when the mixed birch sap contains 40-60% Finnish birch sap and 60-40% Northeast birch sap, the gene expression levels of keratin transglutaminase (TGM1) related to the skin barrier, epidermal tight junction proteins Z0-1 and CLDN, filaggrin (FLG) related to the skin's water-locking ability, and aquaporin (AQP3) related to the skin's water absorption ability are all significantly higher than 100% Northeast birch sap and 100% Finnish birch sap.

[0058] In addition, the results in Table 1 also show that when the Finnish and Northeast birch sap is concentrated twice and then mixed in a ratio of 4:6, it can significantly increase the expression of genes related to moisturizing in keratinocytes; but when the Finnish and Northeast birch sap is diluted once with pure water and then mixed in proportion, the expression of genes related to moisturizing decreases significantly.

[0059] 2. Moisturizing-related protein expression test

[0060] Experimental instruments: clean bench (Sujing), plate washer (BIO-RAD), microplate reader (BIO-TEK), carbon dioxide incubator (Binder)

[0061] Experimental reagents and consumables: human primary keratinocytes, 12-well plates, keratinocyte culture medium, ELISA detection kits for different indicators, etc.

[0062] The test steps are as follows:

[0063] (1) Seeding: The cells were seeded into 12-well culture plates at a seeding density of 2E5 / well and cultured in a 37°C, 5% CO2 incubator. The culture medium was changed every two days.

[0064] (2) Drug administration: When the cell fusion reaches more than 60% again, add different groups of test substances, with 6 replicate wells for each group;

[0065] (3) Sample collection: After 48 h in a 37°C, 5% CO2 incubator, discard the culture medium, add 1 mL of Trizol to each well, pipette and lyse the cells, and then collect the samples;

[0066] (4) Detection: Measure the indicators according to the ELISA kit method;

[0067] (5) Analysis: T-Test method was used for statistical analysis.

[0068] The test results are shown in Table 2.

[0069] Table 2

[0070]

[0071]

[0072] * indicates that the difference was significant compared with 100% Finnish birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Finnish birch sap, with a P value of less than 0.01.

[0073] a. Finnish birch sap or Belarusian birch sap with a concentration of 50% means that the original Finnish birch sap or Belarusian birch sap is diluted to 50% with pure water.

[0074] b. Double concentrated Finnish birch sap or double concentrated Belarusian birch sap means that the original liquid of Finnish birch sap or Belarusian birch sap is input into low temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 2 times.

[0075] The results in Table 2 show that compared with Finnish or Belarusian birch sap alone, a mixture containing 30-70% Finnish birch sap and 70-30% Belarusian birch sap can significantly increase the expression of proteins related to moisturizing in keratinocytes, especially when the mixed birch sap contains 40-50% Finnish birch sap and 60-50% Belarusian birch sap, the protein expression levels of keratin transglutaminase (TGM1) related to the skin barrier, epidermal tight junction proteins Z0-1 and CLDN, filaggrin (FLG) related to the skin's water-locking ability, and aquaporin (AQP3) related to the skin's water absorption ability are all significantly higher than those of 100% Finnish birch sap and 100% Belarusian birch sap.

[0076] In addition, the results in Table 2 also show that concentrating Finnish and Belarusian birch sap twice and then mixing them in a ratio of 4:6 can significantly increase the expression of moisturizing-related proteins in keratinocytes; however, when Finnish and Belarusian birch sap is diluted once with pure water and then mixed in proportion, the expression of moisturizing-related proteins decreases significantly.

[0077] 3. Keratinocyte proliferation and differentiation ability

[0078] Experimental instruments: clean bench (Sujing), plate washer (BIO-RAD), microplate reader (BIO-TEK), carbon dioxide incubator (Binder)

[0079] Experimental reagents and consumables: human primary keratinocytes, 96-well plates, keratinocyte culture medium, MTT kit.

[0080] Experimental method: Logarithmically growing cells were routinely digested and seeded into 96-well plates at a seeding density of 2E3 / well. The plates were cultured in a 37°C, 5% CO2 incubator for 24 hours. The test sample was added, and a negative control group (culture medium + cells) was set up. After incubation for 48 hours, the culture medium was aspirated and the plates were washed three times with PBS. The PBS was aspirated and 100 μl of 1 mg / ml MTT solution was added to each well. The plates were further cultured in a 37°C, 5% CO2 incubator for 4 hours. The supernatant was discarded and 100 μl of DMSO was added to each well. The absorbance (A) was measured at 570 nm using a microplate reader. The cell proliferation capacity was calculated according to the following formula:

[0081] Cell proliferation rate (%) = (A treatment group - A negative control group) / A negative control group * 100%.

[0082] The results are shown in Table 3.

[0083] Table 3

[0084]

[0085] * indicates that the difference was significant compared with 100% Northeast birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Northeast birch sap, with a P value of less than 0.01.

[0086] a. Belarusian birch sap or Northeastern birch sap with a concentration of 50% refers to the original Belarusian birch sap or Northeastern birch sap diluted to 50% with pure water.

[0087] b. Double concentrated Belarusian birch sap or double concentrated Northeastern birch sap means that the original Belarusian birch sap or Northeastern birch sap is fed into a low-temperature drying device, cooled to -65°C, vacuumed to 0.1Pa, and concentrated to 2 times.

[0088] The results in Table 3 show that compared with either Belarusian or Northeastern birch sap alone, a mixture containing 30-70% Belarusian birch sap and 70-30% Northeastern birch sap can significantly enhance the proliferation of keratinocytes, enabling them to rapidly replenish the epidermis after the stratum corneum is shed to form a barrier, reduce water loss, and achieve a moisturizing effect.

[0089] In addition, the results in Table 3 also show that concentrating Belarusian and Northeast birch sap by 2 times and then mixing them in a ratio of 4:6 can significantly improve the proliferation ability of keratinocytes; but when Belarusian and Northeast birch sap is diluted by half with pure water and then mixed in proportion, the proliferation ability of keratinocytes decreases significantly.

[0090] Example 2: Whitening Effects of Birch Sap Mixtures from Different Origins

[0091] This example aims to investigate the differences in whitening efficacy of birch sap from different origins, and examines the effects of birch sap mixtures on whitening-related genes, proteins, melanocytes, and a 3D melanin skin model.

[0092] Specifically, the inspection methods are as follows:

[0093] 1. Melanin synthesis and transport related gene expression test

[0094] Experimental instruments: fluorescence quantitative PCR instrument (Roche), clean bench (Sujing), carbon dioxide incubator (Binder), microplate reader (BIO-TEK), micro-oscillator.

[0095] Experimental reagents and consumables: human primary melanocytes, 6-well plates, melanocyte culture medium, RNA extraction kit, reverse transcription kit, Trizol lysis buffer, etc.

[0096] The steps for melanocyte-based gene expression analysis are as follows:

[0097] (1) Seeding: Seed cells into 6-well plates at a seeding density of 5E5 / well and incubate overnight in a 37°C, 5% CO2 incubator;

[0098] (2) Drug administration: When the cell plating rate in the 6-well plate reaches about 60%, add the test substance of each group, with 6 replicate wells for each group;

[0099] (3) Sample collection: After 24 h in a 37°C, 5% CO2 incubator, discard the culture medium, add 1 mL of Trizol to each well, pipette and lyse the cells, and then collect the samples;

[0100] (4) PCR detection: RNA was extracted, reverse transcribed into cDNA, and then fluorescence quantitative PCR was performed;

[0101] (5) Analysis: The 2-△△CT method was used to calculate the results, and the T-Test method was used for statistical analysis.

[0102] The results are shown in Table 4.

[0103] Table 4

[0104]

[0105]

[0106] * indicates that the difference was significant compared with 100% Northeast birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Northeast birch sap, with a P value of less than 0.01.

[0107] a. Northeast birch sap or Finnish birch sap with a concentration of 50% refers to the original birch sap of Northeast birch sap or Finnish birch sap diluted to 50% with pure water.

[0108] b 1.5 times concentrated Northeast birch sap or 1.5 times concentrated Finnish birch sap means that the Northeast birch sap or Finnish birch sap concentrate is fed into a low-temperature drying device, cooled to -65°C, vacuumed to 0.1Pa, and concentrated to 1.5 times

[0109] The results in Table 4 show that compared with the Northeast birch sap or Finnish birch sap alone, the mixture containing 30-70% Northeast birch sap and 70-30% Finnish birch sap can significantly inhibit the expression of genes related to melanin synthesis and transport, especially when the mixed birch sap contains 40-60% Northeast birch sap and 60-40% Finnish birch sap, the expression levels of melanin synthesis-related genes TYR, TYR1, TYR2, PMEL 17, MIFT M, MIFT B and melanin transport-related genes Rab-27a, Myosin va are all significantly lower than those of 100% Northeast birch sap and 100% Finnish birch sap.

[0110] In addition, the results in Table 4 also show that concentrating Northeast birch sap and Finnish birch sap 1.5 times and then mixing them in a 1:1 ratio can significantly inhibit the expression of genes related to melanin synthesis and transport; but when Northeast birch sap and Finnish birch sap are diluted by half with pure water and then mixed in proportion, the expression of genes related to melanin synthesis and transport is significantly increased.

[0111] 2. Protein expression test related to melanin synthesis and transport

[0112] Experimental instruments: clean bench (Sujing), plate washer (BIO-RAD), microplate reader (BIO-TEK), carbon dioxide incubator (Binder)

[0113] Experimental reagents and consumables: human primary melanocytes, 12-well plates, melanocyte culture medium, ELISA detection kits for different indicators, etc.

[0114] The test steps are as follows:

[0115] (1) Seeding: The cells were seeded into 12-well culture plates at a seeding density of 2E5 / well and cultured in a 37°C, 5% CO2 incubator. The culture medium was changed every two days.

[0116] (2) Drug administration: When the cell fusion reaches more than 60% again, add different groups of test substances, with 6 replicate wells for each group;

[0117] (3) Sample collection: After 48 h in a 37°C, 5% CO2 incubator, discard the culture medium, add 1 mL of Trizol to each well, pipette and lyse the cells, and then collect the samples;

[0118] (4) Detection: Measure the indicators according to the ELISA kit method;

[0119] (5) Analysis: T-Test method was used for statistical analysis.

[0120] The test results are shown in Table 5.

[0121] Table 5

[0122]

[0123]

[0124] * indicates that the difference was significant compared with 100% Northeast birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Northeast birch sap, with a P value of less than 0.01.

[0125] a. Northeast birch sap or Belarus birch sap with a concentration of 50% refers to the original juice of Northeast birch sap or Belarus birch sap diluted to 50% with pure water.

[0126] b 1.5 times concentrated Northeast birch sap or 1.5 times concentrated Belarus birch sap means that the original liquid of Northeast birch sap or Belarus birch sap is put into low temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 1.5 times

[0127] The results in Table 5 show that compared with the Northeast birch sap or Belarus birch sap alone, the mixture containing 30-70% Northeast birch sap and 70-30% Belarus birch sap can significantly inhibit the expression of proteins related to melanin synthesis and transport, especially when the mixed birch sap contains 40-60% Northeast birch sap and 60-40% Belarus birch sap, the expression levels of melanin synthesis-related proteins TYR, TYR1, TYR2, PMEL 17, MIFT M, MIFT B and melanin transport-related proteins Rab-27a, Myosin va are all significantly lower than those of 100% Northeast birch sap and 100% Belarus birch sap.

[0128] In addition, the results in Table 5 also show that concentrating Northeast birch sap and Belarusian birch sap 1.5 times and then mixing them in a 1:1 ratio can significantly inhibit the expression of proteins related to melanin synthesis and transport; but when Northeast birch sap and Belarusian birch sap are diluted by half with pure water and then mixed in proportion, the expression of proteins related to melanin synthesis and transport is significantly increased.

[0129] 3. Melanin production test in melanocytes

[0130] Experimental instruments: clean bench (Sujing), carbon dioxide incubator (Binder), water bath, microplate reader (BIO-TEK).

[0131] Experimental reagents and consumables: human primary melanocytes, 12-well plates, melanocyte culture medium, NaOH lysis solution.

[0132] Experimental methods:

[0133] (1) Inoculation: The cells were seeded into 12-well culture plates at a density of 2E5 / well and cultured in a 37°C, 5% CO2 incubator. The culture medium was changed every two days.

[0134] (2) Drug administration: When the cell fusion reaches more than 60% again, different groups of test substances are added, and a negative control group (no drug) is set up at the same time. Each group has 6 replicate wells.

[0135] (3) Sample collection: After 48 hours in a 37°C, 5% CO2 incubator, discard the culture medium, wash three times with PBS, then add 100 μl of 1 mol / L NaOH and incubate in an 80°C water bath for 1 hour.

[0136] (4) Detection: After the water bath, centrifuge at 10,000 rpm for 10 minutes, aspirate the supernatant, and measure the absorbance (A) at 460 nm using a microplate reader. Calculate the melanin inhibition rate according to the following formula: Melanin inhibition rate (%) = (A negative control group - A treatment group) / A negative control group * 100%

[0137] (5) Analysis: T-Test method was used for statistical analysis.

[0138] The test results are shown in Table 6.

[0139] Table 6: Effects of birch sap concentrate and birch sap concentrate on melanin production in melanocytes

[0140]

[0141] * indicates that the difference was significant compared with 100% Northeast birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Northeast birch sap, with a P value of less than 0.01.

[0142] a. Northeast birch sap or Russian birch sap with a concentration of 50% refers to the original juice of Northeast birch sap or Russian birch sap diluted to 50% with pure water.

[0143] b 1.5 times concentrated Northeast birch sap or 1.5 times concentrated Russian birch sap means that the Northeast birch sap or Russian birch sap concentrate is fed into a low-temperature drying device, cooled to -65°C, vacuumed to 0.1Pa, and concentrated to 1.5 times

[0144] The results in Table 6 show that a mixture comprising 30-70% of Northeast birch sap and 70-30% of Russian birch sap can significantly inhibit melanin production in melanocytes compared to either Northeast birch sap or Russian birch sap alone. In particular, when the birch sap mixture comprises 30-60% of Northeast birch sap and 70-40% of Russian birch sap, melanin production in melanocytes can be very significantly inhibited.

[0145] In addition, the results in Table 6 also show that concentrating Northeast birch sap and Russian birch sap 1.5 times and then mixing them in a 1:1 ratio can significantly inhibit the production of melanin in melanocytes; but when the Northeast birch sap and Russian birch sap are diluted by half with pure water and then mixed in proportion, the melanin inhibition rate is significantly reduced.

[0146] 4. Melanin production test in 3D melanocyte model

[0147] Experimental instruments: clean bench (Sujing), carbon dioxide incubator (Binder), UVB irradiator, water bath, microplate reader (BIO-TEK).

[0148] Experimental reagents and consumables: 3D melanin skin model (laboratory-made), model culture medium, NaOH lysis solution.

[0149] Experimental methods:

[0150] (1) 3D model construction: 3D skin model is constructed using keratinocytes and melanocytes.

[0151] (2) Modeling and drug administration: On the 0th day after the model leaves the factory, UVB irradiation treatment (UVB: 50mJ / cm2) is carried out every day, and then a certain amount of sample is coated on the corresponding model surface. The model control group is only coated with model culture medium. Each group has 6 replicates, and the sample is coated once a day for a total of 4 days.

[0152] (3) Sampling and detection: After the sample reaction is completed, the model is removed, washed three times with PBS, and placed in a centrifuge tube. Then, 100 μl of 1 mol / L NaOH is added and the tube is incubated in a water bath at 80°C for 1 hour. After the water bath, the tube is centrifuged at 10,000 rpm for 10 minutes. The supernatant is aspirated and the absorbance (A) is measured at 460 nm using a microplate reader. The melanin inhibition rate is calculated according to the following formula:

[0153] Melanin inhibition rate (%) = (A model control group - A drug group) / A model control group * 100%

[0154] (4) Analysis: T-Test method was used for statistical analysis.

[0155] The test results are shown in Table 7.

[0156] Table 7: Effect of birch sap on melanin production in 3D melanocyte model

[0157]

[0158] * indicates significant difference compared with 100% Finnish birch sap, with a P value less than 0.05; ** indicates extremely significant difference compared with 100% Finnish birch sap, with a P value less than 0.01

[0159] a. Finnish birch sap or Belarusian birch sap with a concentration of 50% means that the original Finnish birch sap or Belarusian birch sap is diluted to 50% with pure water.

[0160] b 1.5 times concentrated Finnish birch sap or 1.5 times concentrated Belarusian birch sap means that the original liquid of Finnish birch sap or Belarusian birch sap is put into low temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 1.5 times

[0161] The results in Table 7 show that a mixture containing 30-70% Finnish birch sap and 70-30% Belarusian birch sap can significantly inhibit melanin production in the 3D melanocyte model compared to either Finnish birch sap or Belarusian birch sap alone. In particular, when the birch sap mixture contains 30-60% Finnish birch sap and 70-40% Belarusian birch sap, melanin production in the 3D melanocyte model can be very significantly inhibited.

[0162] In addition, the results in Table 7 also show that concentrating Finnish birch sap and Belarusian birch sap by 1.5 times and then mixing them in a 1:1 ratio can significantly inhibit the production of melanin in melanocytes; but when Finnish birch sap and Belarusian birch sap are diluted by half with pure water and then mixed in proportion, the melanin inhibition rate is significantly reduced.

[0163] Example 3: Effects of birch sap mixtures from different origins on anti-wrinkle and anti-aging effects

[0164] This example aims to investigate the differences in the anti-wrinkle and anti-aging effects of birch sap from different origins, and examines the effects of birch sap mixtures on anti-wrinkle-related genes, proteins, and 3D skin models.

[0165] Specifically, the inspection methods are as follows:

[0166] 1. Aging-related gene expression test

[0167] Experimental instruments: fluorescence quantitative PCR instrument (Roche), clean bench (Sujing), carbon dioxide incubator (Binder), microplate reader (BIO-TEK), micro-oscillator.

[0168] Experimental reagents and consumables: human primary fibroblasts, 6-well plates, fibroblast culture medium, RNA extraction kit, reverse transcription kit, Trizol lysis buffer, etc.

[0169] The steps for fibroblast-based gene expression analysis are as follows:

[0170] (1) Seeding: Seed cells into 6-well plates at a seeding density of 5E5 / well and incubate overnight in a 37°C, 5% CO2 incubator;

[0171] (2) Drug administration: When the cell plating rate in the 6-well plate reaches about 60%, add the test substance of each group, with 6 replicate wells for each group;

[0172] (3) Sample collection: After 24 h in a 37°C, 5% CO2 incubator, discard the culture medium, add 1 mL of Trizol to each well, pipette and lyse the cells, and then collect the samples;

[0173] (4) PCR detection: RNA was extracted, reverse transcribed into cDNA, and then fluorescence quantitative PCR was performed;

[0174] (5) Analysis: Using 2 -△△CT Methods The results were calculated and statistically analyzed using the T-Test method.

[0175] The test results are shown in Table 8.

[0176] Table 8

[0177]

[0178] * indicates that the difference was significant compared with 100% Northeast birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Northeast birch sap, with a P value of less than 0.01.

[0179] a. Northeast birch sap or Finnish birch sap with a concentration of 50% refers to the original birch sap of Northeast birch sap or Finnish birch sap diluted to 50% with pure water.

[0180] b 1.2 times concentrated Northeast birch sap or 1.2 times concentrated Finnish birch sap means that the Northeast birch sap or Finnish birch sap stock solution is input into low temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 1.2 times

[0181] The results in Table 8 show that compared with either Northeast birch sap or Finnish birch sap alone, a mixture comprising 30-70% Northeast birch sap and 70-30% Finnish birch sap can significantly increase the expression of aging-related genes in basal fibroblasts. In particular, when the mixed birch sap contains 40-60% Northeast birch sap and 60-40% Finnish birch sap, the gene expression levels of collagen I, collagen III, collagen IV, collagen VII, elastin, and laminin are all significantly higher than those of 100% Northeast birch sap and 100% Finnish birch sap.

[0182] In addition, the results in Table 8 also show that concentrating Northeast birch sap and Finnish birch sap by 1.2 times and then mixing them in a ratio of 6:4 can significantly increase the expression of aging-related genes in basal fibroblasts; but when Northeast birch sap and Finnish birch sap are diluted by half with pure water and then mixed in proportion, the expression of their aging-related genes increases significantly.

[0183] 2. Aging-related protein expression test

[0184] Experimental instruments: clean bench (Sujing), plate washer (BIO-RAD), microplate reader (BIO-TEK), carbon dioxide incubator (Binder)

[0185] Experimental reagents and consumables: human primary fibroblasts, 12-well plates, fibroblast culture medium, ELISA detection kits for different indicators, etc.

[0186] The test steps are as follows:

[0187] (1) Seeding: The cells were seeded into 12-well culture plates at a seeding density of 2E5 / well and cultured in a 37°C, 5% CO2 incubator. The culture medium was changed every two days.

[0188] (2) Drug administration: When the cell fusion reaches more than 60% again, add different groups of test substances, with 6 replicate wells for each group;

[0189] (3) Sample collection: After 48 h in a 37°C, 5% CO2 incubator, discard the culture medium, add 1 mL of Trizol to each well, pipette and lyse the cells, and then collect the samples;

[0190] (4) Detection: Measure the indicators according to the ELISA kit method;

[0191] (5) Analysis: T-Test method was used for statistical analysis.

[0192] The test results are shown in Table 9.

[0193] Table 9

[0194]

[0195] * indicates that the difference was significant compared with 100% Northeast birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Northeast birch sap, with a P value of less than 0.01.

[0196] a. Northeast birch sap or Belarus birch sap with a concentration of 50% refers to the original juice of Northeast birch sap or Belarus birch sap diluted to 50% with pure water.

[0197] b 1.2 times concentrated Northeast birch sap or 1.2 times concentrated Belarusian birch sap means that the original liquid of Northeast birch sap or Belarusian birch sap is input into low-temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 1.2 times.

[0198] The results in Table 9 show that compared with either Northeast birch sap or Belarusian birch sap alone, a mixture comprising 30-70% Northeast birch sap and 70-30% Belarusian birch sap can significantly increase the expression of senescence-related proteins in basal fibroblasts, especially when the mixed birch sap contains 40-60% Northeast birch sap and 60-40% Belarusian birch sap, the expression levels of collagen I, collagen III, collagen IV, collagen VII, elastin, and laminin are all significantly higher than those of 100% Northeast birch sap and 100% Belarusian birch sap.

[0199] In addition, the results in Table 9 also show that concentrating Northeast birch sap and Belarusian birch sap 1.2 times and then mixing them in a ratio of 6:4 can significantly increase the expression of aging-related proteins in basal fibroblasts; but when Northeast birch sap and Belarusian birch sap are diluted once with pure water and then mixed in proportion, the expression of their aging-related proteins increases significantly.

[0200] 3. Reconstruction of 3D full-thickness skin model

[0201] Experimental instruments: clean bench (Sujing), carbon dioxide incubator (Binder), UVA and UVB irradiators, microplate reader (BIO-TEK), and frozen microtome (Leica).

[0202] Experimental reagents and consumables: 3D full-thickness skin model (laboratory-made), ELISA detection kits for different indicators, MTT kit, etc.

[0203] The test steps are as follows:

[0204] (1) 3D full-thickness model construction: 3D skin models were constructed using keratinocytes and fibroblasts;

[0205] (2) Modeling and drug administration: When the model was shipped from the factory, i.e., day 0, SSUV irradiation treatment (UVA: 30 J / cm2; UVB: 50 mJ / cm2) was performed every day. Then, a certain amount of sample was applied to the corresponding model surface. The model control group was only coated with model culture medium. Each group had 6 replicates, and the treatment was performed once a day for a total of 4 days.

[0206] (3) Sampling and testing: After the sample reaction was completed, the culture medium was collected and MMP-1 (matrix metalloproteinase 1) was measured using an ELISA kit. Three models in each group were used to test the cell proliferation activity using an MTT kit to obtain the MTT value. The remaining three models were fixed and the content of collagen IV at the dermal-epidermal junction was measured using IHC staining.

[0207] (4) Analysis: T-Test method was used for statistical analysis.

[0208] The test results are shown in Table 10.

[0209] Table 10

[0210]

[0211] * indicates that the difference was significant compared with 100% Finnish birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Finnish birch sap, with a P value of less than 0.01.

[0212] a. Finnish birch sap or Belarusian birch sap with a concentration of 50% means that the original Finnish birch sap or Belarusian birch sap is diluted to 50% with pure water.

[0213] b 1.2 times concentrated Finnish birch sap or 1.2 times concentrated Belarusian birch sap means that the original liquid of Finnish birch sap or Belarusian birch sap is input into low-temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 1.2 times.

[0214] The results in Table 10 show that, compared to either Finnish birch sap or Belarusian birch sap alone, a mixture containing 30-70% Finnish birch sap and 70-30% Belarusian birch sap significantly improved tissue viability in the reconstructed 3D full-thickness skin model, promoted the relative expression of collagen IV at the dermal-epidermal junction, and significantly reduced the expression of MMP-1 in the extracellular matrix. In particular, when the birch sap mixture contained 40-60% Finnish birch sap and 60-40% Belarusian birch sap, the tissue viability, collagen IV, and MMP-1 levels in the 3D full-thickness skin model were significantly different from those in the 100% Finnish birch sap and 100% Belarusian birch sap groups.

[0215] In addition, the results in Table 10 also show that concentrating Finnish birch sap and Belarusian birch sap by 1.2 times and then mixing them in a ratio of 6:4 can significantly improve the tissue vitality of the reconstructed 3D full-thickness skin model, promote the relative expression of collagen IV at the dermal-epidermal junction, and reduce the expression of MMP-1 in the extracellular matrix; however, when Finnish birch sap and Belarusian birch sap are diluted by half with pure water and then mixed in proportion, the tissue vitality and collagen IV expression of the 3D full-thickness skin model are significantly reduced, and the expression of MMP-1 is significantly increased.

[0216] Example 4: Anti-inflammatory effects of birch sap mixtures from different origins

[0217] This example aims to investigate the differences in anti-inflammatory efficacy of birch sap from different origins, and examines the effects of birch sap mixtures on animal models related to inflammation.

[0218] Specifically, the inspection methods are as follows:

[0219] 1. Zebrafish Mast Cell Allergy Model Testing

[0220] Experimental animals: zebrafish 5 days post fertilization (dpf)

[0221] Evaluation index: tryptase release

[0222] Experimental methods:

[0223] AB wild-type zebrafish embryos were collected and cultured in E3 buffer in a 28.5°C incubator until 5 dpf, with daily medium changes. 5 dpf zebrafish larvae were randomly transferred into 48-well cell culture plates at a rate of 10 per well, with four replicates per group.

[0224] Model group: RO water (pure water) + 15 μg / ml substance P (SP);

[0225] Positive drug group: 60 μg / ml ketotifen + 15 μg / ml SP;

[0226] Test sample group: a mixture of Northeast birch sap and Finnish birch sap in different proportions + 15 μg / ml SP.

[0227] In addition to the groups corresponding to the above-mentioned addition of SP to induce degranulation, a negative control group (without SP) and a background control group (without zebrafish larvae) were set up.

[0228] Aspirate the remaining E3 buffer from each well and add 250 μl of the corresponding solution. Incubate in a 28.5°C incubator in the dark for 60 minutes. After 60 minutes, transfer 200 μl of the supernatant from each group to a 96-well cell culture plate. Add the enzyme substrate, Na-benzoyl-DL-arginine-p-nitroamide hydrochloride (BAPNA), to a concentration of 400 μg / ml. Cover the 96-well plate and place it in a 28.5°C incubator in the dark for 2 hours. After 2 hours, measure the absorbance of the entire plate at 405 nm. The value reflects the release of tryptase from zebrafish mast cells.

[0229] The test results are shown in Table 11.

[0230] Table 11

[0231] Mast cell protection rate Normal group 100％ Model Group 0％ 100% Northeastern birch sap 64.9％ 70% Northeast birch sap + 30% Finnish birch sap 82.6％* 60% Northeast birch sap + 40% Finnish birch sap 91.2％** 50% Northeast birch sap + 50% Finnish birch sap 100％** 40% Northeast birch sap + 60% Finnish birch sap 93.7％** 30% Northeast birch sap + 70% Finnish birch sap 86.2％* 100% Finnish birch sap 69.7％ 50% Northeast birch sap aqueous solution (concentration 50%) + 50% Finnish birch sap aqueous solution (concentration 50%)a 57.40％ 50% Northeast birch sap + 50% 4 times concentrated Finnish birch sap 100％**c

[0232] * indicates that the difference was significant compared with 100% Northeast birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Northeast birch sap, with a P value of less than 0.01.

[0233] a. Northeast birch sap or Finnish birch sap with a concentration of 50% refers to the original birch sap of Northeast birch sap or Finnish birch sap diluted to 50% with pure water.

[0234] b. 4 times concentrated Northeast birch sap or 4 times concentrated Finnish birch sap means that the original Northeast birch sap or Finnish birch sap is input into low-temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 4 times.

[0235] c Mast cell protection rates exceeding 100% were counted as 100%.

[0236] The results in Table 11 show that in the zebrafish mast cell anaphylactoid model, a mixture containing 30-70% Northeast birch sap and 70-30% Finnish birch sap significantly inhibited zebrafish mast cell degranulation compared to either Northeast birch sap or Finnish birch sap alone. In particular, when the birch sap mixture contained 40-60% Northeast birch sap and 60-40% Finnish birch sap, its mast cell degranulation inhibition rate was significantly superior to that of 100% Northeast birch sap and 100% Finnish birch sap.

[0237] 2. Irritant Contact Dermatitis Model Test in Mice

[0238] Experimental animals: ICR mice, male

[0239] Investigation indicators: serum inflammatory factor interleukin-1a (IL-1a)

[0240] Test reagents: 10% sodium dodecyl sulfate solution (SDS), 1% hydrocortisone ointment, depilatory cream

[0241] Experimental Methods: ICR mice were randomly divided into 12 groups based on body weight and divided into a normal group, a model group, a positive group, and each test sample group. All mice were depilatory, with an area of ​​approximately 2 x 2 cm of skin and hair removed from their abdomens using a depilatory cream. The normal group received no other treatment, while the model, positive, and test sample groups were treated with a 10% sodium lauryl sulfate solution for 5 consecutive days to establish the model. Following model establishment, the positive group received a 1% hydrocortisone ointment applied to their skin three times daily for two consecutive days; the model group received no treatment; and each test sample group was sprayed with the corresponding birch sap mixture three times daily for two consecutive days. On day 7 of the experiment, the skin condition of each group of mice was observed, and serum levels of the inflammatory cytokine IL-1a were measured.

[0242] The test results are shown in Table 12.

[0243] Table 12

[0244]

[0245]

[0246] * indicates that the difference was significant compared with 100% Northeast birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Northeast birch sap, with a P value of less than 0.01.

[0247] a. Northeast birch sap or Belarus birch sap with a concentration of 50% refers to the original juice of Northeast birch sap or Belarus birch sap diluted to 50% with pure water.

[0248] b. 4 times concentrated Northeast birch sap or 4 times concentrated Belarusian birch sap means that the original liquid of Northeast birch sap or Belarusian birch sap is input into low-temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 4 times.

[0249] The results in Table 12 show that in a mouse irritant contact dermatitis model, a mixture containing 30-70% Northeast birch sap and 70-30% Belarusian birch sap significantly inhibited the expression of the inflammatory factor IL-1a compared to either Northeast birch sap or Belarusian birch sap alone. In particular, when the birch sap mixture contained 30-60% Northeast birch sap and 70-40% Belarusian birch sap, the expression of IL-1a was significantly lower than that of 100% Northeast birch sap or 100% Belarusian birch sap.

[0250] 3. Mouse Allergic Contact Dermatitis Model Test

[0251] Experimental animals: ICR mice, male

[0252] Investigation indicators: ear thickness index, ear weight, inflammatory indicators interferon-γ (IFN-γ), interleukin-4 (IL-4)

[0253] Test materials: 1% 2,4-dinitrofluorobenzene (DNFB) acetone olive oil solution (4:1), 1% hydrocortisone ointment, depilatory cream

[0254] Experimental Methods: ICR mice were randomly divided into 12 groups based on body weight, each consisting of a normal group, a model group, a positive group, and each test sample group. Each experimental group had their abdominal hair removed using a depilatory cream covering an area approximately 2 x 2 cm. The abdomen and ears of mice in the normal group were evenly coated with an acetone-olive oil solution as a control. Allergic contact dermatitis models were established in mice by sensitization and challenge with an acetone-olive oil solution containing 1% DNFB in the model, positive, and test sample groups. Following challenge, the model group was sprayed with distilled water; the positive group was applied with 1% hydrocortisone ointment three times daily for two consecutive days; and each test sample group was sprayed with the corresponding birch sap mixture three times daily for two consecutive days.

[0255] The test results are shown in Table 13.

[0256] Table 13

[0257]

[0258] * indicates that the difference was significant compared with 100% Finnish birch sap, with a P value of less than 0.05; ** indicates that the difference was extremely significant compared with 100% Finnish birch sap, with a P value of less than 0.01.

[0259] a. Finnish birch sap or Belarusian birch sap with a concentration of 50% means that the original Finnish birch sap or Belarusian birch sap is diluted to 50% with pure water.

[0260] b. 4 times concentrated Finnish birch sap or 4 times concentrated Belarusian birch sap means that the original liquid of Finnish birch sap or Belarusian birch sap is input into low temperature drying equipment, cooled to -65℃, vacuumed to 0.1Pa, and concentrated to 4 times.

[0261] The results in Table 13 show that in the mouse allergic contact dermatitis model, compared with Finnish birch sap or Belarusian birch sap alone, the mixture containing 30-70% Northeast birch sap and 70-30% Belarusian birch sap has a significant anti-inflammatory effect. In particular, when the mixed birch sap contains 40-60% Finnish birch sap and 60-40% Belarusian birch sap, the ear thickness, ear weight and serum IL-4 / IFN-γ ratio representing the inflammatory effect are significantly better than those of Finnish birch sap and Belarusian birch sap.

[0262] Example 5: Moisturizing Cream Composition

[0263] The formula of the moisturizing cream composition is shown in the following table:

[0264] Serial number Element weight% 1 Mixed birch sap (40% Finnish birch sap + 60% Northeast birch sap) 62.2 2 Sodium polyglutamate 0.1 3 Hydrolyzed sodium hyaluronate 0.1 4 Xanthan gum 0.2 5 Allantoin 0.2 6 Methylparaben 0.2 7 hydrogenated lecithin 0.5 8 Panthenol 0.5 9 Betaine 3 10 Butanediol 4 11 glycerin 6 12 Carbomer 0.2 13 Propylparaben 0.1 14 Phytosteryl / Octyldodecyl Lauroyl Glutamate 1 15 microcrystalline wax 1 16 C10-18 fatty acid triglycerides 2 17 Cetearyl Glucoside, Cetearyl Alcohol 2 18 Cetearyl Alcohol 2 19 PEG-100 Stearate, Glyceryl Stearate 3 20 Caprylic / capric triglyceride 4 21 Coco-Caprylate / Caprate 5 22 Polydimethylsiloxane 2 23 Tromethamine 0.2 24 Phenoxyethanol 0.5

[0265] The above-mentioned moisturizing cream composition is prepared as follows:

[0266] 1. Disperse raw material 4 and raw material 11 evenly.

[0267] 2. Heat and dissolve raw material 7 and raw material 10.

[0268] 3. Put raw material 1 into the water phase pot, sprinkle in raw material 12 while stirring, and after raw material 12 is completely swollen, add raw materials 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, and raise the temperature to 80℃.

[0269] 4. Put raw materials 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 into the oil phase pot and heat it to 80℃.

[0270] 5. Pump the raw materials in the water phase pot into the emulsification pot and homogenize at high speed for 5 minutes.

[0271] 6. Pump the raw materials in the oil phase pot into the emulsifying pot, homogenize at high speed for 5 minutes, and keep warm for 10 minutes.

[0272] 7. Cool down to 50°C while stirring, add raw materials 23 and 24, and homogenize slowly for 3 minutes.

[0273] 8. Cool down to 40℃ while stirring.

[0274] 9. Discharge the material after passing the inspection.

[0275] In this example, the skin moisture content of 20 volunteers was tested using a Corneometer (Courage & Khazaka, Germany). The test method is as follows:

[0276] Approximately 0.02 grams of each composition sample was dropped onto a pre-marked square area on the volunteer's arm once in the morning and evening each day, and the sample was evenly applied to the area. The skin's epidermal moisture content (unit: au) was measured using an instrument before sample application (baseline) and after 14 days of application. Each area was tested three times and the average value was calculated.

[0277] Test results showed that the average skin hydration level of the subjects increased by 49% compared to the placebo.

[0278] Example 6: Whitening Essence Composition

[0279] The formula of the whitening essence composition is shown in the following table:

[0280]

[0281]

[0282] The above-mentioned whitening essence composition is prepared as follows:

[0283] 1. Heat phases A and B to 80 degrees and dissolve evenly;

[0284] 2. Maintaining the temperature at 80°C, slowly add phase B to phase A while stirring. Stir for 10 minutes, then homogenize at 10,000 rpm for 5 minutes.

[0285] 3. After defoaming, slowly stir and cool to 60 degrees, add the pre-dispersed phase C to phase AB, and homogenize at 10000 rpm for 3 minutes;

[0286] 4. Add phase D to phase ABC and cool to 50 degrees while stirring;

[0287] 5. Add the pre-mixed phase E to phases ABCD, stir evenly and cool to 40 degrees before discharging.

[0288] Using a half-face comparison test method, 20 volunteers were tested before and after 8 weeks of use:

[0289] 1) Use a Colorimeter to measure the L* of the facial skin;

[0290] 2) Use a Maxmeter MX18 reflectance spectrometer to measure the changes in melanin and red pigment in the skin at the same location.

[0291] 3) VISIA-CR was used to collect facial images of the subjects under different light sources at different time periods. IPP software was used to analyze the skin gloss, whiteness value L*, and visible spot area of ​​the same measurement area on the subjects' faces at different test time points.

[0292] The results showed that compared with the placebo group, the use of the whitening essence composition for 8 weeks can increase the facial skin brightness L* value by 3.2%, reduce the melanin content by 14.5%, reduce the hemoglobin content by 17.4%, and increase skin brightness by 16.9%. The facial pigmentation area of ​​13 out of 20 subjects was significantly reduced.

[0293] Example 7: Anti-wrinkle cream composition

[0294] The formula of the anti-wrinkle cream composition is shown in the following table:

[0295]

[0296] The above anti-wrinkle cream composition is prepared as follows:

[0297] 1. Oil phase: Add raw materials 4, 6, 7, 8, 9, 11, 12, 13, 15, and 21 to the oil phase pot, heat to 80°C, dissolve, and mix evenly;

[0298] 2. Mix raw materials No. 2, 16 and 18 evenly at room temperature;

[0299] 3. Mix raw materials No. 10, 14, 17, and 20 evenly at room temperature;

[0300] 4. Water phase: Heat raw materials 1, 3, and 5 to 80°C, add the mixture from step 2, dissolve, and mix well;

[0301] 5. Emulsification: Add the water phase and oil phase into the emulsification tank, keep the temperature at 80℃, homogenize and emulsify at 3000rpm for 5 minutes, and add raw material 19 after emulsification is completed;

[0302] 6. When the temperature is cooled to 40° C. by stirring, add the mixture in step 3, stir evenly and then discharge the mixture to obtain the skin care cream composition.

[0303] Using a half-face comparison test method, 20 volunteers were tested as follows before and after 4 weeks of use:

[0304] 1) Use Primos to take photos of the outer canthi of the left and right eyes of the volunteers, and use software to calculate wrinkle parameters, including wrinkle number, wrinkle area, wrinkle depth, etc.

[0305] 2) Use a Corneometer to test the skin moisture content at the left and right corners of the eyes.

[0306] The results showed that the moisture content of the skin at the corners of the eyes of 18 out of 20 subjects increased significantly, and the wrinkles at the corners of the eyes of 15 of them became significantly lighter, with the wrinkle area and wrinkle depth reduced by 25% and 40% respectively.

[0307] Example 8: Anti-inflammatory spray composition

[0308] The formula of the anti-inflammatory spray composition is as follows:

[0309]

[0310]

[0311] The above anti-inflammatory spray composition is prepared as follows:

[0312] 1. Add raw materials 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 into the emulsifying pot, heat to 80°C while stirring, and keep warm for 10 minutes;

[0313] 2. Cool down to 45°C while stirring;

[0314] 3. Add raw material 11 and stir for 10 minutes;

[0315] 4. Discharge the material after passing the inspection.

[0316] A half-face control test was conducted on 18 volunteers who self-rated their skin as sensitive for 4 weeks:

[0317] 1) Use Periscan PIM 3 to measure the blood perfusion values ​​of the left and right cheeks of volunteers;

[0318] 2) The current sensory thresholds on the left and right cheeks of the volunteers were measured using Neurometer CPT;

[0319] 3) VISIA-CR was used to collect facial images of volunteers under polarized light conditions before and after use, and IPP software was used to analyze the erythema area in the same measurement area on the left and right cheeks of the volunteers at different test time points.

[0320] The results showed that compared with the side using A2 formula, the blood perfusion value of the cheek area using A1 anti-inflammatory skin care spray was significantly decreased, the erythema area was significantly reduced, and the current sensation threshold was significantly increased.

[0321] The technical solutions of the above embodiments are preferred implementations of the present invention. Several improvements and changes can be made without departing from the principles of the present invention. These improvements and changes should also be considered to be within the scope of protection of the present invention.

Claims

1. A mixed birch sap selected from a mixture of 40-60% of Northeast birch sap and 60-40% of Finnish birch sap, Belarusian birch sap or Russian birch sap, based on the total weight of the mixed birch sap.

2. The mixed birch sap according to claim 1, wherein the birch sap is birch sap concentrate or concentrated birch sap with a concentration ratio of 1.05 to 8 times.

3. The mixed birch sap according to claim 2, wherein the concentrated birch sap is concentrated by a factor of 1.2 to 4.

4. Use of the mixed birch sap according to any one of claims 1 to 3 in the preparation of a skin care cosmetic composition.

5. A skin care cosmetic composition comprising the mixed birch sap according to any one of claims 1 to 3.

6. The skin care cosmetic composition according to claim 5, wherein the mixed birch sap is present in an amount of 18 to 98% by weight based on the total weight of the skin care cosmetic composition.

7. The skin care cosmetic composition of claim 5, which does not contain any added water, but does not exclude the moisture inherently contained in the components.

8. The skin care cosmetic composition according to any one of claims 5 to 7, which is a moisturizing cosmetic composition, a whitening cosmetic composition, an anti-wrinkle cosmetic composition or an anti-inflammatory cosmetic composition.

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