WO2021031750A1 - 混合的桦树汁及其在护肤化妆品组合物中的应用 - Google Patents

混合的桦树汁及其在护肤化妆品组合物中的应用 Download PDF

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Publication number
WO2021031750A1
WO2021031750A1 PCT/CN2020/102231 CN2020102231W WO2021031750A1 WO 2021031750 A1 WO2021031750 A1 WO 2021031750A1 CN 2020102231 W CN2020102231 W CN 2020102231W WO 2021031750 A1 WO2021031750 A1 WO 2021031750A1
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Prior art keywords
birch sap
sap
mixed
northeast
birch
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PCT/CN2020/102231
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English (en)
French (fr)
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王莎莎
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浙江养生堂天然药物研究所有限公司
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Publication of WO2021031750A1 publication Critical patent/WO2021031750A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/592—Mixtures of compounds complementing their respective functions
    • A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18

Definitions

  • the invention relates to a mixed birch sap and its application in skin care cosmetic compositions.
  • Birch sap is the sap that flows from plants in the Betulaceae genus Betula. There are about 100 species of birch, mainly distributed in the northern temperate zone, with a few species distributed in the Arctic region. Birch sap is rich in polysaccharides, amino acids, vitamins, biotin, cytokinins, minerals, etc., which are needed by the human body. It has good moisturizing, whitening and anti-wrinkle effects to improve the skin, and promotes skin cell neutralization, moisturizing, whitening and anti-wrinkle Related protein expression, reduce transdermal water loss, brighten skin tone, improve skin elasticity, etc.
  • the present invention provides a mixed birch sap, which contains two or more birch sap from different origins.
  • 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, Tru, Belarus, Ukraine, and the United States And Canada etc.
  • the origin is preferably Northeast China (hereinafter referred to as Northeast China), Finland, Belarus and Russia.
  • birch sap from northeast China is referred to as Northeast birch sap
  • birch sap from Finland is referred to as Finnish birch sap, and so on.
  • the mixed birch sap comprises northeastern birch sap and Finnish birch sap.
  • the mixed birch sap contains 20-80%, preferably 30-70%, more preferably 40-60% of Northeast birch sap and 80-20%, preferably 70-30%, More preferably 60-40% Finnish birch sap, based on the total weight of the mixed birch sap.
  • the mixed birch sap comprises Northeast birch sap and Camillian birch sap.
  • the mixed birch sap contains 20-80%, preferably 30-70%, more preferably 40-60% of Northeast birch sap and 80-20%, preferably 70-30%, More preferably 60-40% of administratian birch sap, based on the total weight of the mixed birch sap.
  • the mixed birch sap comprises Finnish birch sap and Camillian birch sap.
  • the mixed birch sap contains 20-80%, preferably 30-70%, more preferably 40-60% of Finnish birch sap and 80-20%, preferably 70-30%, More preferably 60-40% of administratian birch sap, based on the total weight of the mixed birch sap.
  • the mixed birch sap comprises northeastern birch sap and Russian birch sap.
  • the mixed birch sap contains 20-80%, preferably 30-70%, more preferably 40-60% of Northeast birch sap and 80-20%, preferably 70-30% , More preferably 60-40% Russian birch sap, based on the total weight of the mixed birch sap.
  • the present invention also relates to the use of the above-mentioned mixed birch sap in skin care cosmetic compositions.
  • the present invention relates to a skin care cosmetic composition, which comprises the mixed birch sap described in (A).
  • the content of the (A) mixed birch tree juice in the skin care cosmetic composition is about 18-98% by weight, preferably about 20-95% by weight, more preferably about 22-90% by weight, most preferably about 30-90 % By weight, based on the total weight of the skin care cosmetic composition.
  • the skin care cosmetic composition of the present invention does not contain any added water, but does not exclude moisture inherently contained in each component.
  • the skin care cosmetic composition of the present invention does not contain chelating agents such as EDTA salt, sodium polyphosphate, sodium metaphosphate, and gluconic acid.
  • the skin care cosmetic composition is a moisturizing cosmetic composition.
  • the moisturizing cosmetic composition significantly increases the moisture content of the subject's skin.
  • the skin care cosmetic composition is a whitening cosmetic composition.
  • the whitening cosmetic composition significantly improves the brightness of the subject's skin and reduces the content of melanin and red pigment in the skin.
  • the skin care cosmetic composition is an anti-wrinkle cosmetic composition.
  • the anti-wrinkle cosmetic composition due to the presence of the mixed birch sap, significantly reduces the number, area and depth of wrinkles in subjects.
  • the skin care cosmetic composition is an anti-inflammatory cosmetic composition.
  • the anti-inflammatory cosmetic composition significantly reduced the inflammation of the subject.
  • birch sap can be used in the form of a raw liquid or a concentrated liquid, for example, in a concentrated liquid of about 1.05-8 times, preferably about 1.1-4 times (referred to as about 1.05-8 times, preferably about 1.1- 4 times the concentrated birch sap) form to use.
  • the birch sap is commercially available.
  • the northeast birch sap can be purchased from Daxinganling Chaoyue Wild Berry Development Co., Ltd.
  • the Finnish birch sap can be purchased from MySeTo Oy Co., Ltd.
  • the Cambodian birch sap can be purchased from Jiulu Fruit and vegetable factories
  • Russian birch sap can be purchased from Healthystream Co., Ltd.
  • Concentrated birch sap is obtained by concentrating the above-mentioned commercial products. Concentration methods are known in the art, such as heating concentration, low-temperature vacuum concentration, membrane concentration and the like. In the present invention, the concentration is preferably carried out by a low-temperature freeze concentration or membrane concentration process. For example, the commercially available birch juice stock solution is input into a low-temperature drying equipment, the temperature is lowered to -40°C to -70°C, and the vacuum is applied to 0.1-30Pa. Concentrated in vacuum at low temperature to obtain concentrated birch sap with different concentration times.
  • the skin care cosmetic composition may optionally contain component (B) commonly used ingredients in skin care cosmetics, which include various commonly used ingredients known in the field of skin care cosmetics. Ingredients, 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) according to needs. For example, the content of component (B) is usually about 2-82% by weight, based on the total weight of the cosmetic composition.
  • the vehicle is those known in the art, such as diluents, dispersants and carriers, and examples thereof include, but are not limited to, ethanol, dipropylene glycol, butylene glycol, and the like.
  • the content of the vehicle in the cosmetic composition is known in the art, for example, it usually accounts for 0.5-20% of the total weight of component (B).
  • the active ingredients are those known in the art, and examples thereof include, but are not limited to, for example, emollients, moisturizers, whitening active ingredients, anti-wrinkle active ingredients, anti-inflammatory active ingredients, and the like.
  • emollients include, but are not limited to, olive oil, macadamia oil, sweet almond oil, grape seed oil, avocado oil, corn oil, sesame oil, soybean oil, peanut oil, white flower seed oil, safflower seed oil, Dogtooth rose hip oil, argan tree kernel oil, jojoba seed oil, sunflower seed oil, palm tree fruit oil, squalane, ethylhexyl palmitate, isopropyl myristate, hydrogenated polyisobutylene, isotene Hexane, isododecane, diethylhexyl carbonate, dioctyl carbonate, isopropyl lauroyl sarcosine, isononyl isononanoate, hydrogenated polydecene, glycerol tri(ethylhexanoate) , Cetyl alcohol ethyl hexanoate, bis-diethoxy diethylene glycol cyclohexane 1,
  • solid emollients include, but are not limited to, cetyl alcohol, stearyl alcohol, cetearyl alcohol, behenyl alcohol, scylitol, lauric acid, myristic acid, palmitic acid, stearic acid, beeswax, candelilla Tree wax, carnauba wax, lanolin, ozokerite, jojoba seed wax, paraffin wax, microcrystalline wax, hydrogenated rice bran wax, hydrogenated coconut oil glycerides, glyceryl behenate/eicosanate, myristyl alcohol One or more of myristate, bis-diglyceride polyacyl adipate-2, shea butter, and muluxing palm seed butter.
  • the content of the emollient in the cosmetic composition is known in the art, for example, it usually accounts for about 1-50% of the total weight of component (B).
  • moisturizer examples include, but are not limited to, glycerin, diglycerin, butylene glycol, propylene glycol, 1,3-propanediol, dipropylene glycol, 1,2-pentanediol, polyethylene glycol-8, polyethylene glycol Alcohol-32, methylglucitol-10, methylglucitol-20, PEG/PPG-17/6 copolymer, glycerol-7, glycerol-26, glycerol glucoside, PPG-10 methyl glucose ether, PPG-20 methyl glucose ether, PEG/PPG/polybutylene glycol-8/5/3 glycerin, sucrose, trehalose, rhamnose, mannose, raffinose, Betaine, erythritol, xylitol, urea, glyceryl polyether-5 lactate, sodium hyaluronate, hydrolyzed sodium hyaluronate, acetyl
  • the whitening active ingredients include, but are not limited to, kojic acid, ascorbyl glucoside, arbutin, tranexamic acid, nicotinamide, plant sterols, plant sterols/behenyl alcohol/octyldecanol lauroyl glutamate, phenyl ethyl Resorcinol, turmeric root extract, birch bark extract, ceramide 2, ceramide 3, acetyl sphingosine, resveratrol, palm tree bark extract, coleus forskohlii root Extract, pepper seed extract, ubiquinone, cholesterol, cholesterol stearate, ascorbic acid, ascorbyl dipalmitate, tocopherol (vitamin E), tocopherol acetate, bisabolol, tetraisoascorbate Palmitate, pyridoxine dicaprylate, pyridoxine dipalmitate, retinyl palmitate, phytosterol/octyl
  • anti-wrinkle active ingredients include, but are not limited to, tocopherol (vitamin E), retinol, retinol palmitate, hydrolyzed collagen, hydrolyzed elastin, allantoin, yeast extract, oryzanol, tetrahydrocurcumin , Ellagic acid, ubiquinone, whey protein, acetyl hexapeptide-8, palmitoyl pentapeptide-4, salicyl sphingosine, birch sap concentrate, silymarin, silk sericin, sodium tocopherol phosphate , Ribonucleic acid (RNA), dipeptide diaminobutyrylbenzylamide diacetate, palmitoyl tripeptide-5, oligopeptide-1, hexapeptide-9, palmitoyl oligopeptide, palmitoyl tetrapeptide-7, Grape (VITIS VINIFERA) seed extract, PTEROCARPUS (MARSU),
  • anti-inflammatory active ingredients include, but are not limited to, dipotassium glycyrrhizinate, portulaca (PORTULACA OLERACEA) extract, oat (AVENA SATIVA) kernel extract (oatyl anthranilic acid), panthenol, allantoin , Biological sugar gum-1, ⁇ -glucan, fructan, SCUTELLARIA BAICALENSIS root extract, Aesculus HIPPOCASTANUM extract, bisabolol, 4-tert-butyl cyclohexanol, nerve Amide 3, hydrogenated lecithin, licorice root (GLYCYRRHIZA GLABRA) extract, hydrolyzed royal jelly protein, oryzanol, phytosphingosine, quercetin (quercetin), ginger root extract, rosemary leaf extract, white flower One or more of chamomile extract, calendula extract, centella asiatica extract, naringin, hesperidin, etc.
  • auxiliary materials include, but are not limited to, emulsifiers, thickeners, preservatives, perfumes and the like.
  • emulsifier examples include, but are not limited to, cetearyl oleate, sorbitan oleate, polysorbate-60, polysorbate-80, methylglucose sesquistearic acid Ester, PEG-20 methyl glucose sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-butanol-26, PEG-4 polyglycerol-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 Methyl Glucose Distearate, Glyceryl stearate citrate, polyglyceryl-10 stearate, polyglyceryl-10 myristate, polyglyce
  • the thickener examples include, but are not limited to, carbomers, acrylic acid (ester) and its derivatives, xanthan gum, gum arabic, polyethylene glycol-14M, polyethylene glycol-90M, succinyl poly One or more of high molecular polymers such as sugar, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.
  • the content of the thickener in the cosmetic composition is known in the art, for example, it usually accounts for about 0.1-10% of the total weight of the component (B).
  • preservatives examples include, but are not limited to, methylparaben, propylparaben, phenoxyethanol, benzyl alcohol, phenethyl alcohol, bis(hydroxymethyl)imidazolidinylurea, potassium sorbate, sodium benzoate, chlorobenzene Glycerol, sodium dehydroacetate, caprylic hydroxamic acid, 1,2-hexanediol, 1,2-pentanediol, p-hydroxyacetophenone, caprylyl glycol, glyceryl caprylate, undecylenic acid
  • the content of the preservative in the cosmetic composition is known in the art, for example, it usually accounts for about 0.01-2% of the total weight of the component (B).
  • the skin care cosmetic composition of the present invention can be prepared by any suitable method known in the art.
  • it can be prepared by dissolving tanks, emulsifying pots, dispersers, transfer pumps and other equipment commonly used in the cosmetics field.
  • dissolving tanks When preparing, put the water-soluble substance into the water-phase dissolving kettle, and the oil-soluble substance into the oil-phase dissolving kettle, and heat the temperature of the two kettles to about 80°C.
  • a disperser Pre-dispersion. After the dissolution is completed, the oil phase and the water phase are transported to the emulsifying pot for homogenization and emulsification for about 5-15 minutes. After the emulsification is completed, the temperature of the material body is lowered to normal temperature, optional flavors, preservatives, etc. are added, and the pH of the product is adjusted as needed.
  • the products can be filled and shipped only after the relevant test indicators are qualified.
  • the above preparation methods can be deleted or adjusted according to dosage form requirements.
  • the cosmetic composition can be prepared into various dosage forms such as creams, creams, lotions, essences, etc. according to needs.
  • Example 1 The effect of birch sap mixture from different origins in moisturizing
  • This example aims to investigate the difference in moisturizing effects of birch sap from different origins. Among them, the effects of birch sap mixture on moisturizing-related genes, proteins and cell proliferation were investigated.
  • the inspection method is as follows:
  • Experimental reagents and consumables human primary keratinocytes, 6-well plates, keratinocyte culture medium, RNA extraction kit, reverse transcription kit, Trizol lysis solution, etc.
  • Inoculation Inoculate cells into a 6-well plate at a seeding density of 5E5/well, and incubate overnight in a 37°C and 5% CO 2 incubator;
  • RNA is extracted, reverse transcribed to cDNA, and then subjected to fluorescence quantitative PCR detection;
  • a Finnish birch sap or Northeast birch sap with a concentration of 50% refers to the use of pure water to dilute the original Finnish birch sap or Northeast birch sap to 50%.
  • b 2 times concentrated Finnish birch sap or 2 times concentrated Northeast birch sap refers to input Finnish birch sap or Northeast birch sap stock into low-temperature drying equipment, cool to -65°C, vacuum to 0.1Pa, and concentrate to 2 times .
  • 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 keratinocytes
  • the expression level of moisturizing-related genes especially when the mixed birch sap contains 40-60% Finnish birch sap and 60-40% Northeast birch sap, cutin transglutaminase (TGM1) associated with the skin barrier ), epidermal tight junction proteins Z0-1 and CLDN, filaggrin (FLG) related to skin water retention capacity, and aquaporin (AQP3) related to skin water absorption capacity are all significantly higher than 100 % Northeast birch sap and 100% Finnish birch sap.
  • TGM1 cutin transglutaminase
  • FLG filaggrin
  • AQP3 aquaporin
  • Experimental reagents and consumables human primary keratinocytes, 12-well plates, keratinocyte culture medium, ELISA test kits with different indicators, etc.
  • test steps are as follows:
  • Inoculation Inoculate in a 12-well culture plate at an inoculation density of 2E5/well, cultivate in an incubator at 37°C and 5% CO2, and change the medium every two days;
  • T-Test method is used for statistical analysis.
  • a Finnish birch sap or elseian birch sap with a concentration of 50% refers to the use of pure water to dilute the original sap of Finnish birch sap or elsesian birch sap to 50%.
  • b 2 times concentrated Finnish birch sap or 2 times concentrated administratian birch sap refers to the input of Finnish birch sap or elsesian birch sap stock into low-temperature drying equipment, cooling to -65°C, vacuuming to 0.1Pa, and concentrating to 2 times .
  • Experimental reagents and consumables human primary keratinocytes, 96-well plate, keratinocyte culture medium, MTT kit.
  • the cells in logarithmic growth phase were routinely digested, seeded in a 96-well plate at a seeding density of 2E3/well, incubated in a 37°C, 5% CO2 incubator for 24 hours, added test samples, and set a negative control group (medium +cells), put in the incubator for 48 hours, suck out the culture solution, wash with PBS 3 times, suck dry PBS and add 100ul of 1mg/ml MTT solution to each well, continue to incubate for 4 hours in the 37°C, 5% CO2 incubator , Discard the supernatant, add 100ul DMSO to each well, measure the absorbance value (A) at 570nm with a microplate reader, and calculate the cell proliferation capacity according to the following formula:
  • Cell proliferation rate (%) (A administration group-A negative control group)/A negative control group*100%.
  • Example 2 The effect of birch sap mixture from different origins in whitening
  • This example aims to investigate the difference in whitening effects of birch sap from different origins. Among them, the effects of birch juice mixture on whitening-related genes, proteins, melanocytes, and 3D melanin skin models were investigated.
  • the inspection method is as follows:
  • Experimental reagents and consumables human primary melanocytes, 6-well plates, melanocyte culture medium, RNA extraction kit, reverse transcription kit, Trizol lysis solution, etc.
  • Inoculation Inoculate cells into a 6-well plate at a seeding density of 5E5/well, and incubate overnight in an incubator at 37°C and 5% CO2;
  • RNA is extracted, reverse transcribed to cDNA, and then subjected to fluorescence quantitative PCR detection;
  • the results in Table 4 show that, compared with individual Northeast birch sap or Finnish birch sap, a mixture containing 30-70% Northeast birch sap and 70-30% Finnish birch sap can significantly inhibit the synthesis and transport of melanin.
  • the expression of related genes especially when the mixed birch sap contains 40-60% Northeast birch sap and 60-40% Finnish birch sap, melanin synthesis related genes TYR, TYR1, TYR2, PMEL 17, MIFT M
  • the expression levels of MIFT B, melanin transport related genes Rab-27a and Myosin va are significantly lower than 100% Northeast birch sap and 100% Finnish birch sap.
  • Experimental reagents and consumables human primary melanocytes, 12-well plates, melanocyte culture medium, ELISA test kits with different indicators, etc.
  • test steps are as follows:
  • Inoculation Inoculate in a 12-well culture plate at an inoculation density of 2E5/well, cultivate in an incubator at 37°C and 5% CO2, and change the medium every two days;
  • T-Test method is used for statistical analysis.
  • Experimental reagents and consumables human primary melanocytes, 12-well plate, melanocyte culture medium, NaOH lysis solution.
  • Inoculation Inoculate in a 12-well culture plate at an inoculation density of 2E5/well, culture in a 37°C, 5% CO2 incubator, and change the medium every two days.
  • melanin inhibition rate (% ) (A negative control group-A administration group)/A negative control group*100%
  • Table 6 show that, compared with individual Northeast birch sap or Russian birch sap, a mixture containing 30-70% Northeast birch sap and 70-30% Russian birch sap can significantly inhibit melanocytes The production of melanin. Especially when the mixed birch sap contains 30-60% Northeast birch sap and 70-40% Russian birch sap, it can significantly inhibit the production of melanin in melanocytes.
  • Experimental reagents and consumables 3D melanin skin model (made in the laboratory), model culture medium, NaOH lysis solution.
  • 3D model construction use keratinocytes and melanocytes to construct a 3D skin model.
  • UVB irradiation treatment (UVB: 50mJ/cm2) is carried out every day, and then a certain amount of samples are respectively coated on the corresponding model surface.
  • the model control group only Cloth model culture medium, each group of 6 replicate holes, smear once a day, the total duration of action is 4 days.
  • a Finnish birch sap or elseian birch sap with a concentration of 50% refers to the use of pure water to dilute the original sap of Finnish birch sap or elsesian birch sap to 50%.
  • b 1.5 times concentrated Finnish birch sap or 1.5 times concentrated administratian birch sap refers to input Finnish birch sap or elsesian birch sap raw liquid into low-temperature drying equipment, cool to -65°C, vacuum to 0.1Pa, and concentrate to 1.5 times .
  • results in Table 7 also show that the Finnish birch sap and the Cambodiaian birch sap are concentrated 1.5 times and then mixed at a ratio of 1:1, which can significantly inhibit the production of melanin in melanocytes; but when the Finnish birch sap is mixed with The Ecuadorian birch sap is diluted with pure water and then mixed in proportion, and its melanin inhibition rate is significantly reduced.
  • Example 3 The effect of birch sap mixture from different origins in anti-wrinkle and anti-aging
  • This example aims to investigate the difference in anti-wrinkle and anti-aging effects of birch sap from different origins. Among them, the effects of birch sap mixture on anti-wrinkle-related genes, proteins, and 3D skin models were investigated.
  • the inspection method is as follows:
  • Experimental reagents and consumables human primary fibroblasts, 6-well plates, fibroblast culture medium, RNA extraction kit, reverse transcription kit, Trizol lysis solution, etc.
  • Inoculation Inoculate cells into a 6-well plate at a seeding density of 5E5/well, and incubate overnight in a 37°C and 5% CO 2 incubator;
  • RNA is extracted, reverse transcribed to cDNA, and then subjected to fluorescence quantitative PCR detection;
  • the 2- ⁇ CT method is used for calculation of the results, and the T-Test method is used for statistical analysis.
  • Table 8 show that a mixture containing 30-70% Northeast birch sap and 70-30% Finnish birch sap can significantly increase the basal fibroblast sap compared with individual Northeast birch sap or Finnish birch sap.
  • the gene expression levels of VII, elastin, and laminin were significantly higher than 100% Northeast birch sap and 100% Finnish birch sap.
  • results in Table 8 also show that the concentration of Northeast birch sap and Finnish birch sap at a ratio of 6:4 after being concentrated 1.2 times can significantly increase the expression of senescence-related genes in basal fibroblasts;
  • the birch sap and Finnish birch sap were diluted with pure water and then mixed in proportion, and the expression of senescence-related genes increased significantly.
  • Experimental reagents and consumables human primary fibroblasts, 12-well plates, fibroblast culture medium, ELISA test kits with different indicators, etc.
  • test steps are as follows:
  • Inoculation Inoculate in a 12-well culture plate at an inoculation density of 2E5/well, cultivate in an incubator at 37°C and 5% CO2, and change the medium every two days;
  • results in Table 9 also show that the concentration of Northeast birch sap and Camillian birch sap at a ratio of 6:4 after being concentrated 1.2 times can significantly increase the expression of senescence-related proteins in basal fibroblasts;
  • the birch sap and Cambodiaian birch sap were diluted with pure water twice and then mixed in proportion, and the expression of senescence-related proteins was significantly increased.
  • Experimental reagents and consumables 3D full-thickness skin model (made in the laboratory), ELISA test kits with different indicators, MTT kits, etc.
  • test steps are as follows:
  • Modeling and drug administration When the model leaves the factory, that is, day 0, SSUV irradiation treatment (UVA: 30J/cm2; UVB: 50mJ/cm2) is carried out every day, and then a certain amount of samples are respectively coated on the corresponding model surface ,
  • the model control group is only coated with the model culture medium, each group has 6 multiple holes, smeared once a day, the total duration of action is 4 days;
  • T-Test method is used for statistical analysis.
  • a Finnish birch sap or elseian birch sap with a concentration of 50% refers to the use of pure water to dilute the original sap of Finnish birch sap or elsesian birch sap to 50%.
  • b 1.2 times concentrated Finnish birch sap or 1.2 times concentrated administratian birch sap refers to input Finnish birch sap or elsesian birch sap stock into low-temperature drying equipment, cool to -65°C, vacuum to 0.1Pa, and concentrate to 1.2 times .
  • This example aims to investigate the anti-inflammatory effects of birch sap from different origins. Among them, the effects of birch sap mixture on inflammation-related animal models were investigated.
  • the inspection method is as follows:
  • Test animal zebrafish 5 days after fertilization (5dpf)
  • Sample group to be tested a mixture of different proportions of Northeast birch sap and Finnish birch sap + 15 ⁇ g/ml SP.
  • Model group 0% 100% Northeast 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%
  • the zebrafish mast cell allergy model contains 30-70% Northeastern birch sap and 70-30% Finnish birch sap compared with individual Northeast birch sap or Finnish birch sap.
  • the mixture can significantly inhibit the degranulation of zebrafish mast cells.
  • the mast cell degranulation inhibition rate is significantly better than 100% Northeast birch sap and 100%.
  • Test animal ICR mouse, male
  • Test reagents 10% sodium dodecyl sulfate solution (SDS), 1% hydrocortisone ointment, depilatory cream
  • mice were randomly grouped according to their body weight, each with 12 mice, divided into normal group, model group, positive group, and each sample group to be tested. All mice used depilatory cream to remove the skin and hair of about 2*2cm area on the mouse abdomen. No other treatments were done for the normal group.
  • the model group, the positive group, and each sample group to be tested were applied with 10% sodium lauryl sulfate solution for 5 consecutive days to complete the modeling. After modeling, the positive group was smeared with 1% hydrocortisone ointment 3 times a day for 2 consecutive days; the model group was not treated; each sample group to be tested was sprayed with the corresponding mixed birch sap, 3 times a day, 2 consecutive days day. On the 7th day of the experiment, the skin conditions of the experimental mice in each group were observed, and the serum of the mice was taken to determine the content of the inflammatory factor IL-1a.
  • a 50% concentration of Northeast birch sap or elseian birch sap refers to the use of pure water to dilute the Northeast birch sap or elseian birch sap to 50%.
  • b 4 times concentrated Northeast birch sap or 4 times concentrated effetian birch sap refers to the northeast birch sap or administratian birch sap raw liquid is fed into low-temperature drying equipment, cooled to -65°C, vacuumed to 0.1Pa, and concentrated to 4 times .
  • Test animal ICR mouse, male
  • Investigation indicators ear thickness index, ear weight, inflammation index interferon- ⁇ (IFN- ⁇ ), interleukin-4 (IL-4)
  • Test materials 1% 2,4-dinitrofluorobenzene (DNFB) acetone olive oil solution (4:1), 1% hydrocortisone ointment, hair removal cream
  • mice were randomly grouped according to their body weight, each with 12 mice, divided into normal group, model group, positive group, and each sample group to be tested.
  • Each group of experimental mice used depilatory cream to remove the skin and hair in the abdomen area of about 2*2cm.
  • the abdomen and ears of mice in the normal group were evenly coated with acetone olive oil solution for control.
  • Mice in the model group, positive group, and each sample group to be tested were smeared with 1% DNFB-containing acetone olive oil solution for sensitization and stimulation to establish a mouse model of allergic contact dermatitis.
  • model group After being challenged, the model group was sprayed with distilled water; the positive group was sprayed with 1% hydrocortisone ointment for 2 consecutive days, 3 times/day; each sample group to be tested was sprayed with the corresponding mixed birch sap, 3 times a day, continuous administration 2 days.
  • a Finnish birch sap or elseian birch sap with a concentration of 50% refers to the use of pure water to dilute the original sap of Finnish birch sap or elsesian birch sap to 50%.
  • b 4 times concentrated Finnish birch sap or 4 times concentrated administratian birch sap refers to input Finnish birch sap or elsesian birch sap stock into low-temperature drying equipment, cool to -65°C, vacuum to 0.1Pa, and concentrate to 4 times .
  • the above moisturizing cream composition is prepared as follows:
  • the raw material 7 is heated and dissolved with the raw material 10.
  • a Corneometer (Courage & Khazaka, Germany) was used to test the skin moisture content of 20 volunteers.
  • the test method is as follows:
  • composition sample Approximately 0.02 grams of each composition sample was dropped into the pre-marked square area on the volunteer's arm once a day in the morning and evening, and the sample was evenly applied to the area.
  • test results showed that compared with placebo, the average moisture content of the subjects' skin increased by 49%.
  • phase ingredient weight% A Mixed birch sap (50% Northeast birch sap +50% Russian birch sap) 68.17 To Sodium hyaluronate 0.03 To Xanthan gum 0.1 To Allantoin 0.1 To Methyl paraben 0.2 To PEG-11 methyl ether polydimethylsiloxane 0.1 To Panthenol 0.3 To Cetearyl olive oil and sorbitan olive oil 1 To glycerin 4 To Butanediol 5 To Acrylic (ester) type/vinyl isodecanoate cross-linked polymer 0.3 B Squalene 0.3 To Phytosterol/octyldodecanol lauroyl glutamate 0.3 To Tocopherol (Vitamin E) 0.2 To Triglyceride (ethylhexanoate) 3 C Polymethylsilsesquioxane 0.5 To Polydimethylsiloxane 1 D Tromethamine 0.4 E ascorbic acid 2 To Mixed
  • the above whitening essence composition is prepared as follows:
  • the phases A and B are heated to 80°C to dissolve evenly;
  • phase B 1. Keep at 80°C, slowly add phase B to phase A while stirring, after stirring for 10 minutes, homogenize at 10000rpm for 5 minutes;
  • VISIA-CR Using VISIA-CR to collect facial images of subjects under different light sources in different periods, use IPP software to analyze the skin glossiness, whiteness value L* and visible stain area of the same measurement area on the subject's face at different test time points.
  • the formula of the anti-wrinkle cream composition is shown in the following table:
  • the above-mentioned anti-wrinkle cream composition is prepared as follows:
  • Oil phase add 4, 6, 7, 8, 9, 11, 12, 13, 15, 21 raw materials into the oil phase pot, and heat to 80°C, dissolve and mix well;
  • Emulsification add the water phase and the oil phase to the emulsification tank, keep it at 80°C, homogenize emulsification at 3000rpm for 5 minutes, add No. 19 raw material after emulsification is complete;
  • step 6 Add the mixture of step 3 when stirring and cooling down to 40°C, stir evenly and then discharge to obtain the skin care cream composition.
  • anti-inflammatory spray composition is prepared as follows:
  • VISIA-CR Using VISIA-CR to collect the facial images of volunteers under the condition of using front and rear polarized light, and use IPP software to analyze the erythema area of the same measurement area at different test time points on the left and right cheeks of volunteers.

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Abstract

一种混合的桦树汁，其包含两种或更多种不同产地的桦树汁。同时还涉及所述混合的桦树汁在护肤化妆品组合物中的用途以及包含所述混合的桦树汁的护肤化妆品组合物，尤其是保湿化妆品组合物、美白化妆品组合物、抗皱化妆品组合物和抗炎化妆品组合物。

Description

混合的桦树汁及其在护肤化妆品组合物中的应用 技术领域
本发明涉及一种混合的桦树汁及其在护肤化妆品组合物中的应用。
背景技术
桦树汁是从桦木科(Betulaceae)桦木属(Betula)植物中流出的汁液。桦木属植物有约100种，主要分布在北温带，少数种类分布至北极区内。桦树汁中富含人体需要的多糖、氨基酸、维生素、生物素、细胞***素、矿物质等，具有良好的保湿、美白和抗皱等改善皮肤的功效，促进皮肤细胞中与保湿、美白、抗皱相关的蛋白表达，减少经皮水分流失，提亮肤色，改善皮肤弹性等。
研究表明，桦树汁中的营养成分受多种因素的影响，其品种、产地、生长环境、采集方法等均会导致其成分上的显著差异，进而导致功效上的明显差异。例如，产地不同会导致桦树汁成分上的差异，例如，王云芳等报道不同地区采集的桦树汁微量元素的种类和含量大不相同，这与其生长环境的水质土质有关，或与白桦树自身能富集某种元素的能力有关，因此桦树汁常被作为生物找矿的指示植物(参见王云芳、向迎梅、许国英等，新疆白桦树液化学成分研究，干旱区研究,1995(1):40-44)。
发明内容
本发明人经研究发现，将不同产地的桦树汁复配形成的混合桦树汁能够显著提高桦树汁的多种护肤功效，这表明不同产地的桦树汁之间能够协同增效。
因此，一方面，本发明提供一种混合的桦树汁，其包含两种或更多种不同产地的桦树汁。
桦树汁(通常也称为白桦树汁)的产地遍布于中国东北、华北、西北、西南、日本、朝鲜、蒙古、俄罗斯、芬兰、挪威、瑞典、爱沙尼亚、立陶宛、波兰、白俄罗斯、乌克兰、美国和加拿大等。在本发明中，所述产地优选为中国东北(以下简称为东北)、芬兰、白俄罗斯和俄罗斯。在本文中，源自东北的桦树汁简称为东北桦树汁，源自芬兰的桦树汁简称为芬兰桦树汁，以此类推。
在一个优选的实施方案中，所述混合的桦树汁包含东北桦树汁和芬兰桦树汁。在进一步优选的实施方案中，所述混合的桦树汁包含20-80％、优选30-70％、更优选40-60％的东北桦树汁和80-20％、优选70-30％、更优选60-40％的芬兰桦树汁，基于混合的桦树汁的总重量。
在一个优选的实施方案中，所述混合的桦树汁包含东北桦树汁和白俄罗斯桦树汁。在进一步优选的实施方案中，所述混合的桦树汁包含20-80％、优选30-70％、更优选40-60％的东北桦树汁和80-20％、优选70-30％、更优选60-40％的白俄罗斯桦树汁，基于混合的桦 树汁的总重量。
在一个优选的实施方案中，所述混合的桦树汁包含芬兰桦树汁和白俄罗斯桦树汁。在进一步优选的实施方案中，所述混合的桦树汁包含20-80％、优选30-70％、更优选40-60％的芬兰桦树汁和80-20％、优选70-30％、更优选60-40％的白俄罗斯桦树汁，基于混合的桦树汁的总重量。
在一个优选的实施方案中，所述混合的桦树汁包含东北桦树汁和俄罗斯桦树汁。在更进一步优选的实施方案中，所述混合的桦树汁包含20-80％、优选30-70％、更优选40-60％的东北桦树汁和80-20％、优选70-30％、更优选60-40％的俄罗斯桦树汁，基于混合的桦树汁的总重量。
另一方面，本发明还涉及上述混合的桦树汁在护肤化妆品组合物中的用途。
又一方面，本发明涉及一种护肤化妆品组合物，其包含(A)所述混合的桦树汁。
所述(A)混合的桦树汁在上述护肤化妆品组合物中的含量为约18-98％重量，优选约20-95％重量，更优选约22-90％重量，最优选约30-90％重量，基于所述护肤化妆品组合物的总重量。
优选地，本发明的护肤化妆品组合物不包含任何外加的水，但不排除各组分中固有地包含的水分。
在优选的实施方案中，本发明的护肤化妆品组合物中不包含EDTA盐、多磷酸钠、偏磷酸钠、葡萄糖酸等螯合剂。
在一个实施方案中，所述护肤化妆品组合物是保湿化妆品组合物。其中，由于混合桦树汁的存在，所述保湿化妆品组合物显著提高了受试者的皮肤含水量。
在一个实施方案中，所述护肤化妆品组合物是美白化妆品组合物。其中，由于混合的桦树汁的存在，所述美白化妆品组合物显著提高了受试者的皮肤亮度，降低了皮肤中黑色素和红色素的含量。
在一个实施方案中，所述护肤化妆品组合物是抗皱化妆品组合物。其中，由于混合的桦树汁的存在，所述抗皱化妆品组合物显著减少了受试者的皱纹数量、面积和深度。
在一个实施方案中，所述护肤化妆品组合物是抗炎化妆品组合物。其中，由于混合的桦树汁的存在，所述抗炎化妆品组合物显著降低了受试者的炎症。
在本发明中，桦树汁可以原液形式使用，也可以浓缩液形式使用，例如以约1.05-8倍、优选约1.1-4倍的浓缩液(简称为约1.05-8倍、优选约1.1-4倍的浓缩桦树汁)形式使用。
所述桦树汁可商购获得，例如，东北桦树汁可购自大兴安岭超越野生浆果开发有限责任公司，芬兰桦树汁可购自MySeTo Oy股份有限公司，白俄罗斯桦树汁可购自旧路果品蔬菜厂，和俄罗斯桦树汁可购自Healthy stream股份有限公司。
浓缩桦树汁是将上述商购产品浓缩得到的。浓缩方法是本领域已知的，例如加热浓缩、低温真空浓缩、膜浓缩等。在本发明中，优选通过低温冷冻浓缩或膜浓缩工艺进行浓缩，例如，将商购的桦树汁原液输入低温干燥设备，降温至-40℃至-70℃，抽真空至0.1-30Pa而进行低温真空浓缩，从而得到不同浓缩倍数的浓缩桦树汁。
除了上述组分(A)混合的桦树汁外，所述护肤化妆品组合物还可以任选地包含组分(B)护肤化妆品中常用的成分，其包括护肤化妆品领域中已知的各种常用成分，其实例包括但不限于媒介物、活性成分和辅料等。本领域技术人员可根据需要选择组分(B)的类型和用量，例如，组分(B)的含量通常为约2-82％重量，基于所述化妆品组合物的总重量。
所述媒介物是本领域已知的那些，例如稀释剂、分散剂和载体等，其实例包括但不限于乙醇、双丙甘醇、丁二醇等。所述媒介物在所述化妆品组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的0.5-20％。
所述活性成分是本领域已知的那些，其实例包括但不限于例如润肤剂、保湿剂、美白活性成分、抗皱活性成分、抗炎活性成分等。
所述润肤剂的实例包括但不限于橄榄油、澳洲坚果油、甜杏仁油、葡萄籽油、鳄梨油、玉米油、芝麻油、大豆油、花生油、白池花籽油、红花籽油、狗牙蔷薇果油、刺阿干树仁油、霍霍巴籽油、向日葵籽油、毛瑞榈果油、角鲨烷、棕榈酸乙基己酯、肉豆蔻酸异丙酯、氢化聚异丁烯、异十六烷、异十二烷、碳酸二乙基己酯、碳酸二辛酯、月桂酰肌氨酸异丙酯、异壬酸异壬酯、氢化聚癸烯、甘油三(乙基己酸)酯、鲸蜡醇乙基己酸酯、双-二乙氧基二甘醇环己烷1,4-二羧酸酯、辛酸/癸酸甘油三酯、油醇芥酸酯、辛基十二醇肉豆蔻酸酯、辛基十二醇、聚二甲基硅氧烷、辛基聚甲基硅氧烷、鲸蜡基聚二甲基硅氧烷、环五聚二甲基硅氧烷等的一种或多种。固体润肤剂的实例包括但不限于鲸蜡醇、硬脂醇、鲸蜡硬脂醇、山嵛醇、鲨肝醇、月桂酸、肉豆蔻酸、棕榈酸、硬脂酸、蜂蜡、小烛树蜡、巴西棕榈蜡、羊毛脂、地蜡、霍霍巴籽蜡、石蜡、微晶蜡、氢化米糠蜡、氢化椰油甘油酯类、甘油山嵛酸酯/二十酸酯、肉豆蔻醇肉豆蔻酸酯、双-二甘油多酰基己二酸酯-2、牛油果树果脂、木鲁星果棕籽脂等中的一种或多种。所述润肤剂在所述化妆品组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的约1-50％。
所述保湿剂的实例包括但不限于甘油、双甘油、丁二醇、丙二醇、1,3-丙二醇、双丙甘醇、1,2-戊二醇、聚乙二醇-8、聚乙二醇-32、甲基葡糖醇聚醚-10、甲基葡糖醇聚醚-20、PEG/PPG-17/6共聚物、甘油聚醚-7、甘油聚醚-26、甘油葡糖苷、PPG-10甲基葡糖醚、PPG-20甲基葡糖醚、PEG/PPG/聚丁二醇-8/5/3甘油、蔗糖、海藻糖、鼠李糖、甘露糖、棉子糖、甜菜碱、赤藓醇、木糖醇、尿素、甘油聚醚-5乳酸酯、透明质酸钠、水解透明质酸钠、乙酰化透明质酸钠、聚谷氨酸钠、水解小核菌胶、出芽短梗酶多糖、银耳多糖、酸豆籽多糖等中的一种或多种。所述保湿剂在所述组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的约1-30％。
所述美白活性成分包括但不限于曲酸、抗坏血酸葡糖苷、熊果苷、传明酸、烟酰胺、植物甾醇、植物甾醇/山嵛醇/辛基癸醇月桂酰谷氨酸酯、苯乙基间苯二酚、姜黄根提取物、白桦树皮提取物、神经酰胺2、神经酰胺3、乙酰植物鞘氨醇、白藜芦醇、花榈木树皮提取物、毛喉鞘蕊花根提取物、胡椒籽提取物、泛醌、胆甾醇、胆甾醇硬脂酸酯、抗坏血酸、抗坏血酸二棕榈酸酯、生育酚(维生素E)、生育酚乙酸酯、红没药醇、抗坏血酸四异棕榈酸酯、吡哆素二辛酸酯、吡哆素二棕榈酸酯、视黄醇棕榈酸酯、植物甾醇/辛基十二醇月 桂酰谷氨酸酯、双-山嵛醇/异硬脂醇/植物甾醇二聚亚油醇二聚亚油酸酯、植物甾醇澳洲坚果油酸酯、各种肽类、各种植物提取物等中的一种或多种。所述抗衰老成分在所述组合物中的含量是本领域已知的。通常，在本发明组合物中，所述皮肤调理剂占组分(B)总重量的约0.01-50％重量。
所述抗皱活性成分的实例包括但不限于生育酚(维生素E)、视黄醇、视黄醇棕榈酸酯、水解胶原、水解弹性蛋白、尿囊素、酵母提取物、谷维素、四氢姜黄素、鞣花酸、泛醌、乳清蛋白、乙酰基六肽-8、棕榈酰五肽-4、水杨酰植物鞘氨醇、浓缩桦树汁、水飞蓟素、蚕丝胶蛋白、生育酚磷酸酯钠、核糖核酸(RNA)、二肽二氨基丁酰苄基酰胺二乙酸盐、棕榈酰三肽-5、寡肽-1、六肽-9、棕榈酰寡肽、棕榈酰四肽-7、葡萄(VITIS VINIFERA)籽提取物、花榈木(PTEROCARPUS MARSUPIUM)树皮提取物、茶(CAMELLIA SINENSIS)多酚、葡萄酒提取物、苹果籽提取物、欧洲水青冈(FAGUS SYLVATICA)芽提取物、水解猴面包树(ADANSONIA DIGITATA)提取物、卤虫(ARTEMIA)提取物、香根鸢尾(IRIS FLORENTINA)根提取物、橙皮苷、人参皂甙、丹参(SALVIA MILTIORRHIZA)提取物、烟酰胺、熊果酸、透明质酸钠、乙酰化透明质酸钠、水解透明质酸钠、番茄红素、咖啡(COFFEA ARABICA)提取物、二肽-2、乳酸、超氧化物歧化酶(SOD)、月见草(OENOTHERA BIENNIS)油、神经酰胺、二棕榈酰羟脯氨酸、羟基硬脂酸、水杨酸、麦角硫因、溶血卵磷脂、肌肽、脱羧肌肽HCL、硫辛酸、腺苷、糖原、白藜芦醇、阿魏酸、二裂酵母发酵产物溶胞物、乳酸菌发酵溶胞产物等中的一种或多种。所述抗衰老活性成分在所述化妆品组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的约0.01-10％。
所述抗炎活性成分的实例包括但不限于甘草酸二钾、马齿苋(PORTULACA OLERACEA)提取物、燕麦(AVENA SATIVA)仁提取物(燕麦酰基邻氨基苯甲酸)、泛醇、尿囊素、生物糖胶-1、β-葡聚糖、果聚糖、黄芩(SCUTELLARIA BAICALENSIS)根提取物、欧洲七叶树(AESCULUS HIPPOCASTANUM)提取物、红没药醇、4-叔丁基环己醇、神经酰胺3、氢化卵磷脂、光果甘草(GLYCYRRHIZA GLABRA)提取物、水解蜂王浆蛋白、谷维素、植物鞘氨醇、五羟黄酮(槲皮素)、姜根提取物、迷迭香叶提取物、白花黄春菊提取物、金盏花提取物、积雪草提取物、柚皮苷、橙皮苷等中的一种或多种。所述抗炎活性成分在所述化妆品组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的约0.01-10％。
所述辅料的实例包括但不限于乳化剂、增稠剂、防腐剂、香料等。
所述乳化剂的实例包括但不限于鲸蜡硬脂醇橄榄油酸酯、山梨坦橄榄油酸酯、聚山梨醇酯-60、聚山梨醇酯-80、甲基葡糖倍半硬脂酸酯、PEG-20甲基葡糖倍半硬脂酸酯、PEG-40氢化蓖麻油、PPG-26-丁醇聚醚-26、PEG-4聚甘油-2硬脂酸酯、PEG-60氢化蓖麻油、硬脂醇聚醚-2、硬脂醇聚醚-21、PPG-13-癸基十四醇聚醚-24、鲸蜡硬脂基葡糖苷、PEG-100硬脂酸酯、甘油硬脂酸酯、甘油硬脂酸酯SE、椰油基葡糖苷、鲸蜡硬脂醇聚醚-25、PEG-40硬脂酸酯、聚甘油-3甲基葡糖二硬脂酸酯、甘油硬脂酸酯柠檬酸酯、聚甘油-10硬脂酸酯、聚甘油-10肉豆蔻酸酯、聚甘油-10二油酸酯、聚甘油-10月桂酸酯、聚甘油-10异硬脂酸酯、聚甘油-10油酸酯、聚甘油-10二异硬脂酸酯、聚甘油-6月桂酸酯、聚甘油-6肉豆蔻 酸酯、蔗糖硬脂酸酯、蔗糖多硬脂酸酯等中的一种或多种。所述乳化剂在所述化妆品组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的约0.5-10％。
所述增稠剂的实例包括但不限于卡波姆类、丙烯酸(酯)类及其衍生物、黄原胶、***胶、聚乙二醇-14M、聚乙二醇-90M、琥珀酰聚糖、羟乙基纤维素、羟丙基纤维素、羟丙基甲基纤维素等高分子聚合物等中的一种或多种。所述增稠剂在所述化妆品组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的约0.1-10％。
所述防腐剂的实例包括但不限于羟苯甲酯、羟苯丙酯、苯氧乙醇、苯甲醇、苯乙醇、双(羟甲基)咪唑烷基脲、山梨酸钾、苯甲酸钠、氯苯甘醚、脱氢乙酸钠、辛酰羟肟酸、1,2-己二醇、1,2-戊二醇、对羟基苯乙酮、辛甘醇、甘油辛酸酯、十一碳烯酸甘油酯、山梨坦辛酸酯、乙基己基甘油、牡丹根提取物等中的一种或多种。所述防腐剂在所述化妆品组合物中的含量是本领域已知的，例如，其通常占组分(B)总重量的约0.01-2％。
本发明的护肤化妆品组合物可以通过本领域已知的任何合适的方法制备。例如，使用化妆品领域中常用的溶解槽、乳化锅、分散器、输送泵等设备制备。制备时先将水溶性物质投入水相溶解釜，将油溶性物质投入油相溶解釜，将两个釜的温度加热至约80℃，其中对于易结块的原料，可先用分散器将其预分散。待溶解完成后将油相和水相输送至乳化锅中，均质乳化约5-15分钟。乳化完成后将料体温度降至常温，加入任选的香精、防腐剂等，并视需要调节产物的pH。相关检测指标都合格后方可灌装出货。
以上制备方法可根据剂型要求进行删减或调整。可根据需要，将所述化妆品组合物制备成膏、霜、乳液、精华液等各种剂型。
实施例
以下结合实施例，对本发明进行进一步详细说明。但是，应当理解为，这些实施例、对比例仅仅是用于具体地说明本发明，而不应理解为用于以任何形式限制本发明所附权利要求书的范围。
实施例1：不同产地的桦树汁混合液在保湿方面的作用
本实施例旨在考察不同产地的桦树汁在保湿功效上的不同。其中分别考察了桦树汁混合液对保湿相关基因、蛋白、细胞增殖的影响。
具体地，考察方法如下：
1.保湿相关基因表达测试
实验仪器：荧光定量PCR仪(Roche)、超净工作台(苏净)、二氧化碳培养箱(Binder)、酶标仪(BIO-TEK)、微量振荡器。
实验试剂与耗材：人原代角质形成细胞、6孔板、角质形成细胞培养液、RNA提取试剂盒、反转录试剂盒、Trizol裂解液等。
基于角质形成细胞的基因表达分析步骤如下：
(1)接种：以5E5/孔的接种密度，接种细胞至6孔板中，在37℃和5％CO 2培养箱中孵 育过夜；
(2)给药：待6孔板中细胞铺板率达到60％左右时，加入各组别的受试物，每组设6个复孔；
(3)收样：在37℃和5％CO 2培养箱中24h后，弃掉培养液，每孔加入1mL Trizol，吹打裂解细胞后，收样；
(4)PCR检测：提取RNA，反转录至cDNA后，进行荧光定量PCR检测；
(5)分析：采用2 -△△CT方法进行结果计算，采用T-Test方法进行统计分析。
测试结果如表1所示。
表1
Figure PCTCN2020102231-appb-000001
*表示与100％东北桦树汁相比为显著，P值小于0.05；**表示与100％东北桦树汁相比为极显著，P值小于0.01。a浓度为50％的芬兰桦树汁或东北桦树汁是指用纯水将芬兰桦树汁或东北桦树汁原汁稀释至50％。b 2倍浓缩芬兰桦树汁或2倍浓缩东北桦树汁是指将芬兰桦树汁或东北桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至2倍。
表1中的结果显示，与单独的芬兰桦树汁或东北桦树汁相比，包含30-70％芬兰桦树汁以及70-30％东北桦树汁的混合物能够显著提高角质形成细胞中与保湿相关的基因的表达量，尤其是当混合桦树汁中包含40-60％的芬兰桦树汁和60-40％的东北桦树汁时，与皮肤屏障相关的角质转谷酰胺酶(TGM1)、表皮紧密连接蛋白Z0-1和CLDN，与皮肤锁水能力相 关的丝聚合蛋白(FLG)，以及与皮肤吸水能力相关的水通道蛋白(AQP3)的基因表达量均非常显著地高于100％东北桦树汁和100％芬兰桦树汁。
此外，表1中的结果也表明，当将芬兰和东北桦树汁浓缩2倍后按4：6比例混合，能够极显著提高角质形成细胞中与保湿相关的基因的表达量；但当将芬兰和东北桦树汁用纯水稀释一倍后按比例混合，其与保湿相关的基因的表达量显著下降。
2.保湿相关蛋白表达测试
实验仪器：超净工作台(苏净)、洗板机(BIO-RAD)、酶标仪(BIO-TEK)、二氧化碳培养箱(Binder)
实验试剂及耗材：人原代角质形成细胞、12孔板、角质形成细胞培养液、不同指标的ELISA检测试剂盒等。
测试步骤如下：
(1)接种：以2E5/孔的接种密度，接种于12孔培养板中，在37℃和5％CO2培养箱中培养，每两天换一次培养基；
(2)给药：待细胞融合再次达60％以上时，加入不同组别的受试物，每组设6个复孔；
(3)收样：在37℃和5％CO 2培养箱中48h后，弃掉培养液，每孔加入1mL Trizol，吹打裂解细胞后，收样；
(4)检测：按照ELISA试剂盒的测定方法进行指标测定；
(5)分析：采用T-Test方法进行统计分析。
测试结果如表2所示。
表2
Figure PCTCN2020102231-appb-000002
*表示与100％芬兰桦树汁相比为显著，P值小于0.05；**表示与100％芬兰桦树汁相比为极显著，P值小于0.01。a浓度为50％的芬兰桦树汁或白俄罗斯桦树汁是指用纯水将芬兰桦树汁或白俄罗斯桦树汁原汁稀释至50％。b 2倍浓缩芬兰桦树汁或2倍浓缩白俄罗斯桦树汁是指将芬兰桦树汁或白俄罗斯桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至2倍。
表2中的结果显示，与单独的芬兰或白俄罗斯桦树汁相比，包含30-70％芬兰桦树汁和70-30％白俄罗斯桦树汁的混合物能够显著提高角质形成细胞中与保湿相关的蛋白的表达量，尤其是当混合桦树汁包含40-50％的芬兰桦树汁和60-50％白俄罗斯桦树汁时，与皮肤屏障相关的角质转谷酰胺酶(TGM1)、表皮紧密连接蛋白Z0-1和CLDN，与皮肤锁水能力相关的丝聚合蛋白(FLG)，以及与皮肤吸水能力相关的水通道蛋白(AQP3)的蛋白表达量均非常显著地高于100％芬兰桦树汁和100％白俄罗斯桦树汁。
此外，表2中的结果也表明，将芬兰和白俄罗斯桦树汁浓缩2倍后按4：6比例混合，能够极显著提高角质形成细胞中与保湿相关的蛋白的表达量；但当将芬兰和白俄罗斯桦树汁用纯水稀释一倍后按比例混合，其与保湿相关的蛋白的表达量显著下降。
3.角质形成细胞增殖和分化能力
实验仪器：超净工作台(苏净)、洗板机(BIO-RAD)、酶标仪(BIO-TEK)、二氧化碳培养箱(Binder)
实验试剂及耗材：人原代角质形成细胞、96孔板、角质形成细胞培养液、MTT试剂盒。
实验方法：取对数生长期细胞常规消化，以2E3/孔的接种密度接种于96孔板，37℃，5％CO2培养箱中培养24小时，加入测试样品，同时设置阴性对照组(培养基+细胞)，置于培养箱中作用48小时后吸出培养液，并用PBS清洗3次，吸干PBS每孔加1mg/ml MTT溶液100ul，继续在37℃，5％CO2培养箱中培养4小时，弃上清，每孔加100ul DMSO，用酶标仪在570nm下测定吸光度值(A)，并根据下式计算细胞的增殖能力：
细胞增殖率(％)＝(A给药组-A阴性对照组)/A阴性对照组*100％。
结果如表3所述。
表3
Figure PCTCN2020102231-appb-000003
*表示与100％东北桦树汁相比为显著，P值小于0.05；**表示与100％东北桦树汁相比为极显著，P值小于0.01。a浓度为50％的白俄罗斯桦树汁或东北桦树汁是指用纯水将白俄罗斯桦树汁或东北桦树汁原汁稀释至50％。b 2倍浓缩白俄罗斯桦树汁或2倍浓缩东北桦树汁是指将白俄罗斯桦树汁或东北桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至2倍。
表3中的结果表明，与单独的白俄罗斯或东北桦树汁相比，包含30-70％白俄罗斯桦树汁以及70-30％东北桦树汁的混合物，能够显著提高角质形成细胞的增殖能力，使其能在皮肤角质层脱落后快速补充表皮形成屏障，减少水分外逸，实现保湿功效。
此外，表3中的结果也表明，将白俄罗斯和东北桦树汁浓缩2倍后按4：6比例混合，能够显著提高角质形成细胞的增殖能力；但当将白俄罗斯和东北桦树汁用纯水稀释一倍后按比例混合时，其角质形成细胞的增殖能力显著下降。
实施例2：不同产地的桦树汁混合液在美白方面的作用
本实施例旨在考察不同产地的桦树汁在美白功效上的不同。其中分别考察了桦树汁混合液对美白相关基因、蛋白、黑素细胞、3D黑色素皮肤模型的影响。
具体地，考察方法如下：
1.黑色素合成、转运相关基因表达测试
实验仪器：荧光定量PCR仪(Roche)、超净工作台(苏净)、二氧化碳培养箱(Binder)、酶标仪(BIO-TEK)、微量振荡器。
实验试剂与耗材：人原代黑素细胞、6孔板、黑素细胞培养液、RNA提取试剂盒、反转录试剂盒、Trizol裂解液等。
基于黑素细胞的基因表达分析步骤如下：
(1)接种：以5E5/孔的接种密度，接种细胞至6孔板中，在37℃和5％CO2培养箱中孵育过夜；
(2)给药：待6孔板中细胞铺板率达到60％左右时，加入各组别的受试物，每组设6个复孔；
(3)收样：在37℃和5％CO2培养箱中24h后，弃掉培养液，每孔加入1mL Trizol，吹打裂解细胞后，收样；
(4)PCR检测：提取RNA，反转录至cDNA后，进行荧光定量PCR检测；
(5)分析：采用2-△△CT方法进行结果计算，采用T-Test方法进行统计分析。
结果如表4所述。
表4
Figure PCTCN2020102231-appb-000004
Figure PCTCN2020102231-appb-000005
*表示与100％东北桦树汁相比为显著，P值小于0.05；**表示与100％东北桦树汁相比为极显著，P值小于0.01。a浓度为50％的东北桦树汁或芬兰桦树汁是指用纯水将东北桦树汁或芬兰桦树汁原汁稀释至50％。b 1.5倍浓缩东北桦树汁或1.5倍浓缩芬兰桦树汁是指将东北桦树汁或芬兰桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至1.5倍
表4中的结果显示，与单独的东北桦树汁或芬兰桦树汁相比，包含30-70％东北桦树汁和70-30％芬兰桦树汁的混合物能够显著抑制与黑色素合成及转运相关基因的表达，尤其是当混合桦树汁中包含40-60％的东北桦树汁和60-40％的芬兰桦树汁时，黑色素合成相关基因TYR、TYR1、TYR2、PMEL 17、MIFT M、MIFT B以及黑色素转运相关基因Rab-27a、Myosin va的表达量均非常显著地低于100％东北桦树汁和100％芬兰桦树汁。
此外，表4中的结果也表明，将东北桦树汁和芬兰桦树汁浓缩1.5倍后按1：1比例混合，能够显著抑制与黑色素合成及转运相关基因的表达；但当将东北桦树汁和芬兰桦树汁用纯水稀释一倍后再按比例混合，其与黑色素合成及转运相关的基因表达量明显增加。
2.与黑色素合成和转运相关的蛋白表达测试
实验仪器：超净工作台(苏净)、洗板机(BIO-RAD)、酶标仪(BIO-TEK)、二氧化碳培养箱(Binder)
实验试剂及耗材：人原代黑素细胞、12孔板、黑素细胞培养液、不同指标的ELISA检测试剂盒等。
测试步骤如下：
(1)接种：以2E5/孔的接种密度，接种于12孔培养板中，在37℃和5％CO2培养箱中培 养，每两天换一次培养基；
(2)给药：待细胞融合再次达60％以上时，加入不同组别的受试物，每组设6个复孔；
(3)收样：在37℃和5％CO2培养箱中48h后，弃掉培养液，每孔加入1mL Trizol，吹
打裂解细胞后，收样；
(4)检测：按照ELISA试剂盒的测定方法进行指标测定；
(5)分析：采用T-Test方法进行统计分析。
测试结果如表5所示。
表5
Figure PCTCN2020102231-appb-000006
*表示与100％东北桦树汁相比为显著，P值小于0.05；**表示与100％东北桦树汁相比为极显著，P值小于0.01。a浓度为50％的东北桦树汁或白俄罗斯桦树汁是指用纯水将东北桦树汁或白俄罗斯桦树汁原汁稀释至50％。b 1.5倍浓缩东北桦树汁或1.5倍浓缩白俄罗斯桦树汁是指将东北桦树汁或白俄罗斯桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至1.5倍
表5中的结果显示，与单独的东北桦树汁或白俄罗斯桦树汁相比，包含30-70％东北桦树汁和70-30％白俄罗斯桦树汁的混合物能够显著抑制与黑色素合成及转运相关蛋白的表达，尤其是当混合桦树汁中包含40-60％东北桦树汁和60-40％白俄罗斯桦树汁时，黑色素合成相关蛋白TYR、TYR1、TYR2、PMEL 17、MIFT M、MIFT B以及黑色素转运相关蛋白Rab-27a、Myosin va的表达量均非常显著地低于100％东北桦树汁和100％白俄罗斯桦树汁。
此外，表5中的结果也表明，将东北桦树汁和白俄罗斯桦树汁浓缩1.5倍后按1：1比例混合，能够显著抑制与黑色素合成及转运相关蛋白的表达；但当将东北桦树汁和白俄罗斯桦树汁用纯水稀释一倍后再按比例混合，其与黑色素合成及转运相关的蛋白表达量明显增加。
3.黑素细胞中黑色素生成测试
实验仪器：超净工作台(苏净)、二氧化碳培养箱(Binder)、水浴锅、酶标仪(BIO-TEK)。
实验试剂及耗材：人原代黑素细胞、12孔板、黑素细胞培养液、NaOH裂解液。
实验方法：
(1)接种：以2E5/孔的接种密度，接种于12孔培养板中，37℃，5％CO2培养箱培养，每两天换一次培养基。
(2)给药：待细胞融合再次达60％以上时，加入不同组别的受试物，同时设置阴性对照组(不加药)每组设6个复孔。
(3)收样：37℃，5％CO2培养箱48h后，弃掉培养液，用PBS液洗3遍，然后加入100ul浓度1mol/L NaOH，80度水浴1小时。
(4)检测：水浴结束后在10000rpm条件下离心10分钟，吸取上清液用酶标仪在460nm下测定吸光值(A)，并根据下式计算黑素抑制率：黑素抑制率(％)＝(A阴性对照组-A给药组)/A阴性对照组*100％
(5)分析：T-Test方法进行统计分析。
测试结果表6所示。
表6：桦树汁原液和浓缩桦树汁对黑素细胞中黑色素生成的影响
Figure PCTCN2020102231-appb-000007
*表示与100％东北桦树汁相比为显著，P值小于0.05；**表示与100％东北桦树汁相比为极显著，P值小于0.01。a浓度为50％的东北桦树汁或俄罗斯桦树汁是指用纯水将东北桦树汁或俄罗斯桦树汁原汁稀释至50％。b 1.5倍浓缩东北桦树汁或1.5倍浓缩俄罗斯桦树汁是指将东北桦树汁或俄罗斯桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至1.5倍
表6中的结果表明，与单独的东北桦树汁或俄罗斯桦树汁相比，包含30-70％东北桦树汁和70-30％俄罗斯桦树汁的混合物能够显著地抑制黑素细胞中黑色素的生成。尤其是当混合桦树汁中包含30-60％东北桦树汁和70-40％俄罗斯桦树汁时，能够非常显著的抑制黑素细胞中黑色素的生成。
此外，表6中的结果也表明，将东北桦树汁和俄罗斯桦树汁浓缩1.5倍后按1：1比例混合，能够极显著抑制黑素细胞中黑色素的生成；但当将东北桦树汁和俄罗斯桦树汁用纯水稀释一倍后再按比例混合，其黑色素抑制率显著降低。
4.3D黑素细胞模型中黑色素生成测试
实验仪器：超净工作台(苏净)、二氧化碳培养箱(Binder)、UVB辐照仪、水浴锅、酶标仪(BIO-TEK)。
实验试剂及耗材：3D黑素皮肤模型(实验室自制)，模型培养液、NaOH裂解液。
实验方法：
(1)3D模型构建：用角质细胞与黑素细胞构建3D皮肤模型。
(2)造模及给药：在模型出厂即0天时，每天进行UVB辐照处理(UVB：50mJ/cm2)，然后将一定量的样品分别涂布于相应的模型表面，模型对照组只涂布模型培养液，每组6个复孔，每天涂抹一次，总作用时长4天。
(3)收样与检测：样品作用结束后，取出模型，用PBS清洗3次后置于离心管中，然后加入100ul浓度1mol/L NaOH，80度水浴1小时，水浴结束后在10000rpm条件下离心10分钟，吸取上清用酶标仪在460nm下测定吸光值(A)，并根据下式计算黑素抑制率：
黑素抑制率(％)＝(A模型对照组-A给药组)/A模型对照组*100％
(4)分析：T-Test方法进行统计分析。
测试结果如表7所示。
表7：桦树汁对3D黑素细胞模型中黑色素生成的影响
Figure PCTCN2020102231-appb-000008
*表示与100％芬兰桦树汁相比为显著，P值小于0.05；**表示与100％芬兰桦树汁相比为极显著，P值小于0.01。a浓度为50％的芬兰桦树汁或白俄罗斯桦树汁是指用纯水将芬兰桦树汁或白俄罗斯桦树汁原汁稀释至50％。b 1.5倍浓缩芬兰桦树汁或1.5倍浓缩白俄罗斯桦树汁是指将芬兰桦树汁或白俄罗斯桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至1.5倍。
表7中的结果表明，与单独的芬兰桦树汁或白俄罗斯桦树汁相比，包含30-70％芬兰桦树汁和70-30％白俄罗斯桦树汁的混合物能够极显著地抑制3D黑素细胞模型中黑色素的生成。尤其是当混合桦树汁中包含30-60％芬兰桦树汁和70-40％白俄罗斯桦树汁时，能够非常显著的抑制3D黑素细胞模型中黑色素的生成。
此外，表7中的结果也表明，将芬兰桦树汁和白俄罗斯桦树汁浓缩1.5倍后按1：1比例混合，能够显著抑制黑素细胞中黑色素的生成；但当将芬兰桦树汁和白俄罗斯桦树汁用纯水稀释一倍后再按比例混合，其黑色素抑制率显著降低。
实施例3：不同产地的桦树汁混合液在抗皱抗衰方面的作用
本实施例旨在考察不同产地的桦树汁在抗皱抗衰功效上的不同。其中分别考察了桦树汁混合液对抗皱相关基因、蛋白、3D皮肤模型上的影响。
具体地，考察方法如下：
1.衰老相关基因表达测试
实验仪器：荧光定量PCR仪(Roche)、超净工作台(苏净)、二氧化碳培养箱(Binder)、酶标仪(BIO-TEK)、微量振荡器。
实验试剂与耗材：人原代成纤维细胞、6孔板、成纤维细胞培养液、RNA提取试剂盒、反转录试剂盒、Trizol裂解液等。
基于成纤维细胞的基因表达分析步骤如下：
(1)接种：以5E5/孔的接种密度，接种细胞至6孔板中，在37℃和5％CO 2培养箱中孵育过夜；
(2)给药：待6孔板中细胞铺板率达到60％左右时，加入各组别的受试物，每组设6个复孔；
(3)收样：在37℃和5％CO 2培养箱中24h后，弃掉培养液，每孔加入1mL Trizol，吹打裂解细胞后，收样；
(4)PCR检测：提取RNA，反转录至cDNA后，进行荧光定量PCR检测；
(5)分析：采用2 -△△CT方法进行结果计算，采用T-Test方法进行统计分析。
测试结果如表8所示。
表8
Figure PCTCN2020102231-appb-000009
Figure PCTCN2020102231-appb-000010
*表示与100％东北桦树汁相比为显著，P值小于0.05；**表示与100％东北桦树汁相比为极显著，P值小于0.01。a浓度为50％的东北桦树汁或芬兰桦树汁是指用纯水将东北桦树汁或芬兰桦树汁原汁稀释至50％。b 1.2倍浓缩东北桦树汁或1.2倍浓缩芬兰桦树汁是指将东北桦树汁或芬兰桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至1.2倍。
表8中的结果显示，与单独的东北桦树汁或芬兰桦树汁相比，包含30-70％东北桦树汁和70-30％芬兰桦树汁的混合物能够显著提高基底成纤维细胞的衰老相关基因的表达量，尤其是当混合桦树汁中包含40-60％的东北桦树汁和60-40％芬兰桦树汁时，胶原蛋白I、胶原蛋白III、胶原蛋白IV、胶原蛋白VII、弹性蛋白、层粘连蛋白的基因表达量均非常显著地高于100％东北桦树汁和100％芬兰桦树汁。
此外，表8中的结果也表明，将东北桦树汁和芬兰桦树汁浓缩1.2倍后按6：4比例混合，能够显著提高基底成纤维细胞的衰老相关基因的表达量；但当将东北桦树汁和芬兰桦树汁用纯水稀释一倍后再按比例混合，其与衰老相关基因的表达量显著增加。
2.衰老相关蛋白表达测试
实验仪器：超净工作台(苏净)、洗板机(BIO-RAD)、酶标仪(BIO-TEK)、二氧化碳培养箱(Binder)
实验试剂及耗材：人原代成纤维细胞、12孔板、成纤维细胞培养液、不同指标的ELISA检测试剂盒等。
测试步骤如下：
(1)接种：以2E5/孔的接种密度，接种于12孔培养板中，在37℃和5％CO2培养箱中培养，每两天换一次培养基；
(2)给药：待细胞融合再次达60％以上时，加入不同组别的受试物，每组设6个复孔；
(3)收样：在37℃和5％CO 2培养箱中48h后，弃掉培养液，每孔加入1mL Trizol，吹打裂解细胞后，收样；
(4)检测：按照ELISA试剂盒的测定方法进行指标测定；(5)分析：采用T-Test方法进行统计分析。
测试结果如表9所示。
表9
Figure PCTCN2020102231-appb-000011
*表示与100％东北桦树汁相比为显著，P值小于0.05；**表示与100％东北桦树汁相比为极显著，P值小于0.01。a浓度为50％的东北桦树汁或白俄罗斯桦树汁是指用纯水将东北桦树汁或白俄罗斯桦树汁原汁稀释至50％。b 1.2倍浓缩东北桦树汁或1.2倍浓缩白俄罗斯桦树汁是指将东北桦树汁或白俄罗斯桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至1.2倍。
表9中的结果显示，与单独的东北桦树汁或白俄罗斯桦树汁相比，包含30-70％东北桦树汁以及70-30％白俄罗斯桦树汁的混合物能够显著提高基底成纤维细胞的衰老相关蛋白的表达量，尤其是当混合桦树汁中包含40-60％的东北桦树汁和60-40％白俄罗斯桦树汁时，胶原蛋白I、胶原蛋白III、胶原蛋白IV、胶原蛋白VII、弹性蛋白、层粘连蛋白的表达量均非常显著地高于100％东北桦树汁和100％白俄罗斯桦树汁。
此外，表9中的结果也表明，将东北桦树汁和白俄罗斯桦树汁浓缩1.2倍后按6：4比例混合，能够显著提高基底成纤维细胞的衰老相关蛋白的表达量；但当将东北桦树汁和白俄罗斯桦树汁用纯水稀释一倍后再按比例混合，其与衰老相关蛋白的表达量显著增加。
3.重组3D全层皮肤模型
实验仪器：超净工作台(苏净)、二氧化碳培养箱(Binder)、UVA及UVB辐照仪、酶标仪(BIO-TEK)，冰冻切片机(Leica)。
实验试剂及耗材：3D全层皮肤模型(实验室自制)，不同指标的ELISA检测试剂盒，MTT试剂盒等。
测试步骤如下：
(1)3D全层模型构建：用角质细胞与成纤维细胞构建3D皮肤模型；
(2)造模及给药：在模型出厂即0天时，每天进行SSUV辐照处理(UVA：30J/cm2；UVB：50mJ/cm2)，然后将一定量的样品分别涂布于相应的模型表面，模型对照组只涂布模型培养液，每组6个复孔，每天涂抹一次，总作用时长4天；
(3)收样与检测：样品作用结束后，收集培养液用ELISA试剂盒进行MMP-1(基质金属蛋白酶1)的测定，另每组取3个模型用MTT试剂盒测试细胞增值活力，得到MTT值，余下3个进行组织固定，IHC染色测定真表皮连接处胶原蛋白IV的含量。
(4)分析：采用T-Test方法进行统计分析。
测试结果如表10所示。
表10
Figure PCTCN2020102231-appb-000012
*表示与100％芬兰桦树汁相比为显著，P值小于0.05；**表示与100％芬兰桦树汁相比为极显著，P值小于0.01。a浓度为50％的芬兰桦树汁或白俄罗斯桦树汁是指用纯水将芬兰桦树汁或白俄罗斯桦树汁原汁稀释至50％。b 1.2倍浓缩芬兰桦树汁或1.2倍浓缩白俄罗斯桦树汁是指将芬兰桦树汁或白俄罗斯桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至1.2倍。
表10中的结果显示，与单独的芬兰桦树汁或白俄罗斯桦树汁相比，包含30-70％芬兰桦树汁以及70-30％白俄罗斯桦树汁的混合物能够显著提高重组3D全层皮肤模型的组织活力，促进真表皮连接处胶原蛋白IV相对表达量，同时显著减少细胞外基质中MMP-1的表达。尤其是当混合桦树汁中包含40-60％的芬兰桦树汁和60-40％的白俄罗斯桦树汁时，3D全层皮肤模型组织活力、胶原蛋白IV及MMP-1均非常显著的区别于100％芬兰桦树汁和100％白俄罗斯桦树汁。
此外，表10中的结果也表明，将芬兰桦树汁和白俄罗斯桦树汁浓缩1.2倍后按6：4 比例混合，能够显著提高重组3D全层皮肤模型的组织活力，促进真表皮连接处胶原蛋白IV相对表达量，减少细胞外基质中MMP-1的表达；但当将芬兰桦树汁和白俄罗斯桦树汁用纯水稀释一倍后再按比例混合，其3D全层皮肤模型的组织活力和胶原蛋白IV表达量显著降低，MMP-1的表达量明显增加。
实施例4：不同产地的桦树汁混合液在抗炎方面的作用
本实施例旨在考察不同产地的桦树汁在抗炎功效上的不同。其中分别考察了桦树汁混合液在炎症相关的动物模型上的影响。
具体地，考察方法如下：
1.斑马鱼肥大细胞类过敏模型测试
试验动物：受精后5天(5dpf)的斑马鱼
考察指标：类胰蛋白酶释放量
实验方法：
收集AB野生型斑马鱼胚胎，在28.5℃培养箱中用E3buffer培养至5dpf，每天换液。随机将5dpf的斑马鱼幼鱼以每孔10尾的数量转入48孔细胞培养板中分组，每组4个复孔。
模型组：RO水(纯水)+15μg/ml P物质(SP)；
阳性药组：60μg/ml酮替芬+15μg/ml SP；
待测样品组：不同比例的东北桦树汁和芬兰桦树汁的混合液+15μg/ml SP。
以及对应于上述加入SP诱导脱颗粒各组，另设阴性对照组(不含SP)和背景对照组(不含斑马鱼幼鱼)。
吸干各组孔内残余的E3buffer并加入250μl对应各个组别的溶液，在28.5℃培养箱中避光反应60分钟。60分钟后，取各组上清液200ul于96孔细胞培养板中，再分别加入酶反应底物Na-苯甲酰-DL-精氨酸-对硝基酰胺盐酸盐(BAPNA)使其浓度达到400μg/ml。将96孔板避光加盖放入28.5℃培养箱中反应2h，2h后测一次全板405nm下的光吸收值，数值大小反映了斑马鱼肥大细胞的类胰蛋白酶释放情况。
测试结果如表11所示。
表11
  肥大细胞保护率
正常组 100％
模型组 0％
100％东北桦树汁 64.9％
70％东北桦树汁+30％芬兰桦树汁 82.6％*
60％东北桦树汁+40％芬兰桦树汁 91.2％**
50％东北桦树汁+50％芬兰桦树汁 100％**
40％东北桦树汁+60％芬兰桦树汁 93.7％**
30％东北桦树汁+70％芬兰桦树汁 86.2％*
100％芬兰桦树汁 69.7％
50％东北桦树汁水溶液(浓度50％)+50％芬兰桦树汁水溶液(浓度50％)a 57.40％
50％东北桦树汁+50％4倍浓缩芬兰桦树汁b 100％**c
*表示与100％东北桦树汁相比为显著，P值小于0.05；**表示与100％东北桦树汁相比为极显著，P值小于0.01。a浓度为50％的东北桦树汁或芬兰桦树汁是指用纯水将东北桦树汁或芬兰桦树汁原汁稀释至50％。b 4倍浓缩东北桦树汁或4倍浓缩芬兰桦树汁是指将东北桦树汁或芬兰桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至4倍。c肥大细胞保护率超过100％的按100％计。
表11中的结果显示，在斑马鱼肥大细胞类过敏模型中，与单独的东北桦树汁或芬兰桦树汁相比，包含30-70％东北桦树汁以及70-30％芬兰桦树汁的混合物能够显著抑制斑马鱼肥大细胞脱颗粒。尤其是当混合桦树汁中包含40-60％的东北桦树汁和60-40％芬兰桦树汁时，其肥大细胞脱颗粒抑制率非常显著地优于100％东北桦树汁和100％芬兰桦树汁。
2.小鼠刺激性接触性皮炎模型测试
试验动物：ICR小鼠，雄性
考察指标：血清中炎症因子白介素-1a(IL-1a)
试验试剂：10％十二烷基硫酸钠溶液(SDS)、1％氢化可的松软膏、脱毛膏
实验方法：将ICR小鼠按体重随机分组，每组12只，分为正常组、模型组、阳性组、各待测样品组。所有小鼠用脱毛膏去除小鼠腹部约2*2cm区域面积皮肤毛发。正常组不做其他处理，模型组、阳性组、各待测样品组连续5天涂抹10％十二烷基硫酸钠溶液完成造模。造模后，阳性组用1％氢化可的松药膏涂抹皮肤，每天3次，连续2天；模型组不做处理；各待测样品组喷涂相应的混合桦树汁，每天3次，连续2天。实验第7天，观察各组实验小鼠皮肤状况，并取小鼠血清测定炎症因子IL-1a含量。
测试结果如表12所示。
表12
Figure PCTCN2020102231-appb-000013
*表示与100％东北桦树汁相比为显著，P值小于0.05；**表示与100％东北桦树汁相比为极显著，P值小于0.01。a浓度为50％的东北桦树汁或白俄罗斯桦树汁是指用纯水将东北桦树汁或白俄罗斯桦树汁原 汁稀释至50％。b 4倍浓缩东北桦树汁或4倍浓缩白俄罗斯桦树汁是指将东北桦树汁或白俄罗斯桦树汁原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至4倍。
表12中的结果显示，在小鼠刺激性接触性皮炎模型中，与单独的东北桦树汁或白俄罗斯桦树汁相比，包含30-70％东北桦树汁以及70-30％白俄罗斯桦树汁的混合物能够显著抑制炎症因子IL-1a的表达量。尤其是当混合桦树汁中包含30-60％的东北桦树汁和70-40％的白俄罗斯桦树汁时，其IL-1a的表达量非常显著地低于100％东北桦树汁和100％白俄罗斯桦树汁。
3.小鼠变应性接触性皮炎模型测试
试验动物：ICR小鼠，雄性
考察指标：耳厚度指数、耳重量、炎症指标γ干扰素(IFN-γ)、白介素-4(IL-4)
试验材料：1％2,4-二硝基氟苯(DNFB)丙酮橄榄油溶液(4:1)、1％氢化可的松软膏、脱毛膏
实验方法：将ICR小鼠按体重随机分组，每组12只，分为正常组、模型组、阳性组、各待测样品组。各组实验小鼠用脱毛膏去除腹部约2*2cm区域面积皮肤毛发。正常组小鼠腹部及耳部均匀涂以丙酮橄榄油溶液对照。模型组、阳性组、各待测样品组小鼠涂抹含1％DNFB的丙酮橄榄油溶液，进行致敏和激发建立小鼠变应性接触性皮炎模型。激发后，模型组喷以蒸馏水；阳性组涂以1％氢化可的松软膏，连续2天，3次/天；各待测样品组喷涂相应的混合桦树汁，每天3次，连续给药2天。
测试结果如表13所示。
表13
Figure PCTCN2020102231-appb-000014
*表示与100％芬兰桦树汁相比为显著，P值小于0.05；**表示与100％芬兰桦树汁相比为极显著，P值小于0.01。a浓度为50％的芬兰桦树汁或白俄罗斯桦树汁是指用纯水将芬兰桦树汁或白俄罗斯桦树汁原汁稀释至50％。b 4倍浓缩芬兰桦树汁或4倍浓缩白俄罗斯桦树汁是指将芬兰桦树汁或白俄罗斯桦树汁 原液输入低温干燥设备，降温至-65℃，抽真空至0.1Pa，浓缩至4倍。
表13中的结果显示，在小鼠变应性接触性皮炎模型中，与单独的芬兰桦树汁或白俄罗斯桦树汁相比，包含30-70％东北桦树汁以及70-30％白俄罗斯桦树汁的混合物具有显著的抗炎效果，尤其是当混合桦树汁中包含40-60％的芬兰桦树汁和60-40％的白俄罗斯桦树汁时，代表炎症效应的耳厚度、耳重量以及血清中IL-4/IFN-γ的比值均显著优于芬兰桦树汁和白俄罗斯桦树汁。
实施例5：保湿霜组合物
所述保湿面霜组合物的配方如下表所示：
序号 成分 重量％
1 混合桦树汁(40％芬兰桦树汁+60％东北桦树汁) 62.2
2 聚谷氨酸钠 0.1
3 水解透明质酸钠 0.1
4 黄原胶 0.2
5 尿囊素 0.2
6 羟苯甲酯 0.2
7 氢化卵磷脂 0.5
8 泛醇 0.5
9 甜菜碱 3
10 丁二醇 4
11 甘油 6
12 卡波姆 0.2
13 羟苯丙酯 0.1
14 植物甾醇/辛基十二醇月桂酰谷氨酸酯 1
15 微晶蜡 1
16 C10-18脂酸甘油三酯类 2
17 鲸蜡硬酯基葡糖苷、鲸蜡硬脂醇 2
18 鲸蜡硬脂醇 2
19 PEG-100硬脂酸酯、甘油硬脂酸酯 3
20 辛酸/癸酸甘油三酯 4
21 椰油醇-辛酸酯/癸酸酯 5
22 聚二甲基硅氧烷 2
23 氨丁三醇 0.2
24 苯氧乙醇 0.5
上述保湿面霜组合物如下制备：
1.将原料4用原料11分散均匀。
2.将原料7用原料10加热溶解。
3.将原料1投入水相锅，边搅拌边撒入原料12，待原料12号完全溶胀后加入原料2、3、4、5、6、7、8、9、10、11，升温至80℃。
4.将原料13、14、15、16、17、18、19、20、21、22投入油相锅，升温至80℃。
5.将水相锅中的原料抽入乳化锅，高速均质5分钟。
6.将油相锅中的原料抽入乳化锅，高速均质5分钟，保温10分钟。
7.边搅拌边降温至50℃，投入原料23和原料24，慢速均质3分钟。
8.边搅拌边降温至40℃。
9.检验合格后出料。
在本实施例中，采用Corneometer(Courage&Khazaka,德国)对20名志愿者的皮肤水分含量进行测试。测试方法如下：
每天早晚各一次将约0.02克的各组合物样品滴加到志愿者的手臂上事先标记的方形区域内，并将样品均匀涂抹于该区域内。用仪器测定样品使用前(基准)和使用14天的皮肤表皮的水分含量(单位：a.u.)，每一个区域重复测试三次取平均值。
测试结果显示，与安慰剂相比，受试者的皮肤平均含水量提高了49％。
实施例6：美白精华组合物
所述美白精华组合物的配方如下表所示：
相 成分 重量％
A 混合桦树汁(50％东北桦树汁+50％俄罗斯桦树汁) 68.17
  透明质酸钠 0.03
  黄原胶 0.1
  尿囊素 0.1
  羟苯甲酯 0.2
  PEG-11甲醚聚二甲基硅氧烷 0.1
  泛醇 0.3
  鲸蜡硬脂醇橄榄油酸酯和山梨坦橄榄油酸酯 1
  甘油 4
  丁二醇 5
  丙烯酸(酯)类/异癸酸乙烯酯交联聚合物 0.3
B 鲨肝醇 0.3
  植物甾醇/辛基十二醇月桂酰谷氨酸酯 0.3
  生育酚(维生素E) 0.2
  甘油三(乙基己酸)酯 3
C 聚甲基硅倍半氧烷 0.5
  聚二甲基硅氧烷 1
D 氨丁三醇 0.4
E 抗坏血酸 2
  混合桦树汁(50％垂枝桦+50％亚洲白桦) 13
上述美白精华组合物如下制备：
1.A、B相分别加热至80℃溶解均匀；
2.保持80℃，搅拌下将B相缓慢加入A相，搅拌10分钟后，在10000rpm下均质5分钟；
3.消泡后缓慢搅拌降温至60℃，将预先分散好的C相加入AB相中，在10000rpm下 均质3分钟；
4.将D相加入ABC相中，搅拌下降温至50℃；
5.将预先混合均匀的E相加入ABCD相中，搅拌均匀降温至40℃即可出料。
采用半脸对照的测试方法，在使用产品前和使用产品8周后分别对20名志愿者进行如下测试：
1)利用Colorimeter仪器测定颜面部皮肤的L*；
2)利用Maxmeter MX18反射光谱仪测量相同位置皮肤的黑色素和红色素变化情况。
3)通过VISIA-CR采集受试者不同时期不同光源下的面部图形，利用IPP软件分析受试者面部不同测试时间点同一测量区域的皮肤光泽度、白度值L*和可见色斑面积。
结果显示，与安慰剂组相比，使用该美白精华组合物8周能够提升面部皮肤亮度L*值3.2％，降低黑色素含量14.5％，降低血红素含量17.4％，提升皮肤亮度16.9％，20名受试者中有13人的面部色斑面积显著减少。
实施例7：抗皱面霜组合物
所述抗皱面霜组合物的配方如下表所示：
Figure PCTCN2020102231-appb-000015
上述抗皱面霜组合物如下制备：
1.油相：将4、6、7、8、9、11、12、13、15、21号原料加入油相锅，并加热至80℃，溶解，混合均匀；
2.将2、16、18号原料在常温下混合均匀；
3.将10、14、17、20号原料等原料在常温下混合均匀；
4.水相：将1、3、5号原料加热到80℃，加入步骤2的混合物，溶解，混合均匀；
5.乳化：将水相和油相加入乳化罐中，保温在80℃，在3000rpm速度下均质乳化5分钟，乳化完成后加入19号原料；
6.搅拌降温至40℃时加入步骤3的混合物，搅拌均匀后出料，得到所述护肤面霜组合物。
采用半脸对照的测试方法，在使用产品前和使用产品4周后分别对20名志愿者进行如下测试：
1)用primos对志愿者左右两侧外眼角拍照，用软件计算皱纹参数，其中包括皱纹数量、皱纹面积、皱纹深度等；
2)用Corneometer测试左右两侧眼角的皮肤水分含量。
结果显示，20名受试者中有18人眼角皮肤含水量显著增加，其中有15人眼角皱纹明显变浅变淡，皱纹面积和皱纹深度减少分别减少了25％和40％。
实施例8：抗炎喷雾组合物
所述抗炎喷雾组合物的配方如下表所述：
序号 成分 A1 A2
1 混合桦树汁(60％东北桦树汁+40％白俄罗斯桦树汁) 90.7 0
2 水 0 90.7
3 透明质酸钠 0.05 0.05
4 尿囊素 0.1 0.1
5 泛醇 0.5 0.5
6 羟苯甲酯 0.15 0.15
7 水解透明质酸钠 0.2 0.2
8 PPG-10甲基葡糖醚 1 1
9 甘油 3 3
10 双丙甘醇 4 4
11 苯氧乙醇 0.3 0.3
上述抗炎喷雾组合物如下制备：
1.将原料1、2、3、4、5、6、7、8、9、10投入乳化锅，边搅拌边升温至80℃，保温10分钟；
2.边搅拌边降温至45℃；
3.加入原料11，搅拌10分钟；
4.检验合格后出料。
对18名自评为敏感性皮肤的志愿者进行半脸对照测试，测试周期4周:
1)用Periscan PIM 3测试志愿者左右两侧面颊部位的血流灌注值；
2)用Neurometer CPT对志愿者左右两侧面颊部位的电流感觉阈值进行测定；
3)通过VISIA-CR采集志愿者在使用前后偏正光条件下的面部图形，利用IPP软件分析志愿者左右两侧面颊部位不同测试时间点同一测量区域的红斑面积。
结果显示，与使用A2配方的一侧相比，使用A1抗炎护肤喷雾的面颊部位的血流灌注值显著下降，红斑面积显著减少，电流感觉阈值显著提高。
以上实施例的技术方案是本发明优选实施方式，在不脱离本发明原理的前提下还可以进行若干改进和变换，这些改进和变化也应视为在本发明的保护范围内。

Claims (16)

  1. 一种混合的桦树汁，其包含两种或更多种不同产地的桦树汁。
  2. 权利要求1的混合的桦树汁，其中所述桦树汁是桦树汁原液或者浓缩倍数为1.05-8倍、优选1.2-4倍的浓缩桦树汁。
  3. 权利要求1的混合的桦树汁，其中所述产地选自中国东北、芬兰、白俄罗斯和俄罗斯。
  4. 权利要求1或2的混合的桦树汁，其包含东北桦树汁和芬兰桦树汁。
  5. 权利要求4的混合的桦树汁，其包含20-80％、优选30-70％、更优选40-60％的东北桦树汁和80-20％、优选70-30％、更优选60-40％的芬兰桦树汁，基于混合的桦树汁的总重量。
  6. 权利要求1或2的混合的桦树汁，其包含东北桦树汁和白俄罗斯桦树汁。
  7. 权利要求6的混合的桦树汁，其包含20-80％、优选30-70％、更优选40-60％的东北桦树汁和80-20％、优选70-30％、更优选60-40％的白俄罗斯桦树汁，基于混合的桦树汁的总重量。
  8. 权利要求1或2的混合的桦树汁，其包含芬兰桦树汁和白俄罗斯桦树汁。
  9. 权利要求8的混合的桦树汁，其包含20-80％、优选30-70％、更优选40-60％的芬兰桦树汁和80-20％、优选70-30％、更优选60-40％的白俄罗斯桦树汁，基于混合的桦树汁的总重量。
  10. 权利要求1或2的混合的桦树汁，其包含东北桦树汁和俄罗斯桦树汁。
  11. 权利要求10的混合的桦树汁，其包含20-80％、优选30-70％、更优选40-60％的东北桦树汁和80-20％、优选70-30％、更优选60-40％的俄罗斯桦树汁，基于混合的桦树汁的总重量。
  12. 权利要求1-11任一项的混合的桦树汁在护肤化妆品组合物中的用途。
  13. 一种护肤化妆品组合物，其包含权利要求1-11任一项的混合的桦树汁。
  14. 权利要求13的护肤化妆品组合物，其中所述混合的桦树汁在护肤化妆品组合物中的含量为18-98％重量，优选20-95％重量，更优选22-90％重量，最优选30-90％重量，基于所述护肤化妆品组合物的总重量。
  15. 权利要求13或14的护肤化妆品组合物，其不包含任何外加的水，但不排除各组分中固有地包含的水分。
  16. 权利要求13-15任一项的护肤化妆品组合物，其是保湿化妆品组合物、美白化妆品组合物、抗皱化妆品组合物或抗炎化妆品组合物。
PCT/CN2020/102231 2019-08-21 2020-07-16 混合的桦树汁及其在护肤化妆品组合物中的应用 WO2021031750A1 (zh)

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FR3121359A1 (fr) * 2021-04-02 2022-10-07 Credo Composition cosmetique comprenant au moins un extrait de bouleau et au moins un extrait de bourgeon de hetre
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