CN110731922B - Enhanced hair loss prevention and hair growth promoting composition - Google Patents
Enhanced hair loss prevention and hair growth promoting composition Download PDFInfo
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Abstract
The invention provides an enhanced hair loss prevention and hair growth promotion composition, which comprises (A) concentrated birch juice and (B) a combination of any one or more of ginger extract, polygonum multiflorum root extract, ginseng extract, green tea extract, platycladus orientalis extract, angelica extract, astragalus, eclipta alba, birch bud extract, birch leaf extract, adenosine, nicotinamide and tripeptide-1 copper, wherein the concentration multiple of the concentrated birch juice is about 1.2-8 times, preferably about 1.5-6 times, and more preferably about 1.5-5 times.
Description
Technical Field
The invention relates to an enhanced hair loss prevention and hair growth composition, which comprises (A) concentrated birch juice and (B) a combination of any one or more of ginger extract, polygonum multiflorum root extract, ginseng extract, green tea extract, platycladus orientalis extract, angelica extract, astragalus, eclipta alba, birch bud extract, birch leaf extract, adenosine, nicotinamide and tripeptide-1 copper, wherein the concentration multiple of the concentrated birch juice is about 1.2-8 times, preferably about 1.5-6 times, and more preferably about 1.5-5 times.
Background
With the acceleration of the pace of life of modern society and the gradual deterioration of the environment, more and more people have sub-health problems of different degrees under the conditions of high mental stress, overtime, overnight stay, haze and the like, wherein the problems include the increased secretion of scalp grease, scalp inflammation, hair follicle atrophy, hair shedding and the like caused by the problems. Alopecia is a topic of increasing concern for modern people, especially young people. The main solution to the problem of hair loss prevention and hair growth today is hair growth promoting medicine and surgery. The main drug therapy is finasteride and minoxidil, so that the side effects are serious, drug dependence is easy to form, and once the drug is stopped, alopecia is serious; the operation treatment cost is high, the wound is generated, and a certain recovery period is needed. Therefore, the development of natural anti-alopecia and hair-growing raw materials for solving the problem of alopecia has important significance.
Disclosure of Invention
In one aspect, the present invention relates to the use of a combination of (a) concentrated birch juice and (B) any one or more of ginger extract, polygonum multiflorum root extract, ginseng extract, green tea extract, arborvitae extract, angelica extract, astragalus, eclipta alba, birch sprout extract, birch leaf extract, adenosine, nicotinamide, tripeptide-1 copper in an enhanced hair loss prevention and hair growth composition, wherein the concentrated birch juice is concentrated about 1.2-8 times, preferably about 1.5-6 times, more preferably about 1.5-5 times.
In another aspect, the present invention provides an enhanced hair loss prevention and growth promotion composition comprising (a) a concentrated birch juice, and (B) any one or more of ginger extract, polygonum multiflorum root extract, ginseng extract, green tea extract, platycladus orientalis extract, angelica sinensis extract, astragalus membranaceus, eclipta alba, birch bud extract, birch leaf extract, adenosine, nicotinamide, tripeptide-1 copper, wherein the concentration of the concentrated birch juice is about 1.2 to 8 times, preferably about 1.5 to 6 times, more preferably about 1.5 to 5 times.
Hair loss prevention hair growth involves two very critical aspects: one is the vitality of the 5 alpha-reductase, inhibits the vitality of the 5 alpha-reductase, can effectively reduce the generation of dihydrotestosterone and avoid the atrophy of hair follicles; secondly, the vitality of hair papilla cells is strong, the proliferation vitality of the hair papilla cells can influence the state of the whole hair follicle, so that the hair follicle is kept in the growth phase, the hair regeneration is promoted, and the hair growth time is prolonged. The birch juice can inhibit activity of 5 alpha-reductase, regulate proliferation and differentiation of hair papilla cells, prevent alopecia, and promote hair growth, and promote transcription of related gene and protein expression.
Unexpectedly, the inventor finds that compared with the single use of the concentrated birch juice or the ginger extract, the fleece-flower root extract, the ginseng extract, the green tea extract, the arborvitae extract, the angelica extract, the astragalus root, the eclipta alba, the birch bud extract, the birch leaf extract, the adenosine, the nicotinamide and the tripeptide-1 copper, the combination of the concentrated birch juice and any one or more of the ginger extract, the fleece-flower root extract, the ginseng extract, the green tea extract, the arborvitae extract, the angelica extract, the astragalus root, the eclipta alba, the birch bud extract, the birch leaf extract, the adenosine, the nicotinamide and the tripeptide-1 copper has the advantages of obviously better alopecia preventing and hair growing effect, is far higher than the function superposition effect of the two, shows that the activity of 5 alpha-reductase is better inhibited, the transcription of hair papilla and genes related to the alopecia preventing and the expression of proteins are adjusted, promoting the proliferation and differentiation of hair papilla cells and improving the depilation area of androgen model mice, which shows that the synergistic effect is generated between the concentrated birch juice and the substances.
Birch juices involved in the present invention are obtained from Betula genus of betulinaceae family, which may be from four varieties of white birch (Betula alba), Betula luminifera (Betula pubescens), Betula pendula (Betula pendula) and birch asiana (Betula platyphylla). The birch juice is colorless, transparent, precipitate-free and impurity-free, and has birch fragrance and rich nutrition, and is collected by manually drilling holes at the base of the birch trunk between thawing and early spring leaf emergence. The birch juice is commercially available and used as such, for example from greater Khingan over wild berry development, LLC.
The concentrated birch sap of the present invention is obtained by concentrating the above-mentioned commercially available products. Concentration methods are known in the art, such as heat concentration, low temperature vacuum concentration, membrane concentration, and the like. In the present invention, the concentration is preferably performed by a low-temperature freeze concentration or membrane concentration process, for example, commercially available birch juice stock solution is fed into a low-temperature drying device, cooled to-40 ℃ to-70 ℃, and vacuumized to 0.1 to 30Pa to perform low-temperature vacuum concentration, so as to obtain concentrated birch juice with different concentration times.
Further, the inventor also finds that the anti-hair loss and hair growth efficacy of the concentrated birch juice and the concentration degree are not simply linear relationship, but show a trend of increasing and then decreasing with the increase of the concentration factor. Therefore, it is critical to control the concentration of the birch sap, and in the present invention, the concentration of the birch sap is controlled to be about 1.2 to 8 times, preferably about 1.5 to 6 times, and more preferably about 1.5 to 5 times.
The content of the concentrated birch sap is about 10-98%, preferably about 20-98%, more preferably about 30-97%, based on the total weight of the enhanced hair loss prevention composition.
The component (B) ginger extract, polygonum multiflorum root extract, ginseng extract, green tea extract, thuja extract, angelica extract, astragalus root, eclipta alba, birch bud extract, birch leaf extract, adenosine, nicotinamide and tripeptide-1 copper are known in the art, and they are all commercially available as such and used in the present invention. The total content of the component (B) is about 0.0005 to 30%, preferably about 0.001 to 10%, more preferably about 0.1 to 5%, most preferably about 0.5 to 3%, based on the total weight of the hair loss prevention and hair growth promotion composition.
The hair loss prevention and hair growth promotion composition does not contain any added water, but does not exclude moisture inherently contained in the components.
Preferably, the hair loss preventing and hair growing composition does not contain a chelating agent such as EDTA salt, sodium polyphosphate, sodium metaphosphate, gluconic acid and the like.
The hair loss preventing and growing composition may optionally comprise, in addition to the above components (a) and (B), component (C), which is a component commonly used in hair care and shampoo compositions. Examples of the component (C) include, but are not limited to, vehicles, active ingredients, and adjuvants, and the like. These ingredients are known in the art and the type and amount of component (C) may be selected by those skilled in the art as desired, for example, the content of component (C) is generally about 0 to 70% based on the total weight of the hair loss prevention and hair growth promotion composition.
Such vehicles are known in the art and include, for example, diluents, dispersants or carriers and the like, examples of which include, but are not limited to, ethanol, dipropylene glycol, butylene glycol, and the like. The amount of vehicle in the composition is known in the art, and for example, it is generally 0.01 to 20% by weight of the total weight of component (C).
Such active ingredients are known in the art and include, for example, hair loss preventing hair restorers, moisturizers, hair conditioners and the like.
Examples of the hair loss prevention and hair growth agent include, but are not limited to, pubescent angelica root, angelica, ledebouriella root, fennel fruit, cnidium fruit, whiteflower hogfennel root, notopterygium root, perilla fruit, baikal skullcap root, peppermint, patchouli, catnip, lavender, sage, lime, chrysanthemum, inula flower, xanthium fruit, atractylodes rhizome, white atractylodes rhizome, saussurea involucrate, elecampane, chamomile, climbing groundsel, galangal, ginger, zedoary, turmeric, curcuma root, mace nutmeg, root of herbaceous peony, moutan bark, cimicifuga foetida, ash bark, dittany bark, dried orange peel, citrus grandiflora, finger citron, hawthorn leaf, hairyvein agrimony, asarum, madder, gardenia, sweet orange, garter, veratrum, thyme, artemisia, valerian, prunella vulgaris, purslane, hiraute, taxillus, giant knotweed, privet, cedar, sophora fruit, grape seed, clove, magnolia bark, magnolia flower, star anise, cinnamon, lindera root, piper, piper, Myrrh, frankincense, hot pepper, blackberry lily, paniculate swallowwort root, common lilac flower bud, dogwood fruit, lily, benzoin, storax, dragon's blood, chastetree fruit, lithospermum, aloe, celandine, horse chestnut, great burdock achene, lawn pennywort herb, tuber fleeceflower root, white mustard seed, Chinese lobelia herb, borneol, clematis root, common andrographis herb, large-leaf beautyberry, lamiophlomis rotata, red grass, shinyleaf pricklyash root, pyrrosia lingua, weeping forsythia capsule, ampelopsis grossedentata, amur corktree bark, wild buckwheat rhizome, caragana, anemarrhena asphodeloides bunge, nettle, peach seed, dodder, sea-buckthorn, ginseng, swertia japonica, motherwort herb, tea leaf and the like. The anti-hair loss hair restorer is present in the composition in an amount known in the art, and is, for example, usually 0.01 to 50% by weight based on the total weight of component (C).
Examples of such humectants include, but are not limited to, glycerol, diglycerol, butylene glycol, propylene glycol, 1, 3-propanediol, dipropylene glycol, 1, 2-pentanediol, polyethylene glycol-8, polyethylene glycol-32, methyl gluceth-10, methyl gluceth-20, PEG/PPG-17/6 copolymer, glyceryl polyether-7, glyceryl polyether-26, glyceryl glucoside, PPG-10 methyl glucose ether, PPG-20 methyl glucose ether, PEG/PPG/polytetramethylene glycol-8/5/3 glycerol, sucrose, trehalose, rhamnose, mannose, raffinose, betaine, erythritol, xylitol, urea, glyceryl polyether-5 lactate, sodium hyaluronate, hydrolyzed sodium hyaluronate, acetylated sodium hyaluronate, sodium polyglutamate, sodium alginate, sodium hyaluronate, sodium alginate, hydrolyzing one or more of sclerotium rolfsii gum, pullulanase, tremella polysaccharide, sour bean seed polysaccharide, etc. The humectant is present in the composition in amounts known in the art, for example, typically from 0.01 to 30% by weight of the total weight of component (C).
Examples of such hair conditioning agents include, but are not limited to, fatty alcohols, sterols, fatty acids, monoglycerides, triglycerides, lanolin, sarcosinates, fatty acid esters, white oil, squalane, polyquaternium-10, polyquaternium-7, polyquaternium-39, polyquaternium-52, polyquaternium-73, guar hydroxypropyltrimonium chloride, behenyltrimethylammonium chloride, dimethicone, PEG-60 almond oil glycerides, one or more of panthenol, polyvinylpyrrolidone, gelatin, pectin, Gleditsia sinensis extract, Saviae Miltiorrhizae radix extract, radix Angelicae sinensis extract, butanediol, Polygoni Multiflori radix extract, folium Platycladi extract, rhizoma Zingiberis recens extract, Scutellariae radix extract, radix Sophorae Flavescentis extract, radix Glycyrrhizae Inflatae extract, allantoin, Notoginseng radix extract, fructus Chaenomelis extract, and oat polypeptide. The hair conditioning agents are present in the compositions in amounts known in the art, for example, typically from 0.01 to 50% by weight based on the total weight of component (C).
Such adjuvants are known in the art and include, for example, foaming agents, surfactants, emulsifiers, thickeners, preservatives, fragrances and the like.
Examples of such foaming agents include, but are not limited to, one or more of sodium laureth sulfate, sodium trideceth sulfate, decyl glucoside, sodium lauroyl sarcosinate, sodium C14-16 olefin sulfonate, coconut monoethanolamide, coconut diethanolamide, cocoyl propyl betaine, ammonium lauryl sulfate, sodium cocoamphoacetate, and the like. The amount of said blowing agent in said composition is known in the art and is, for example, generally comprised between 0.1 and 50% of the total weight of component (C).
Examples of such surfactants include, but are not limited to, cocamidopropyl betaine, sodium laureth sulfate, PEG-150 distearate, ethylene glycol distearate, ammonium laureth sulfate, sodium laureth sulfate, palmitamidopropyl betaine, cocamidodiethanol amine, lauryl ether sulfate, ammonium lauryl sulfate (K12A), sodium fatty alcohol polyoxyethylene ether sulfate (AES), ammonium fatty alcohol polyoxyethylene ether sulfate (AESA), ammonium lauryl ether sulfate, ammonium lauryl alcohol sulfate, sodium lauryl alcohol sulfate, ammonium laureth sulfate, ammonium lauryl alcohol sulfate, cocoyl monoethanolamide, N-fatty acyl amino acid salts, lauramidopropyl betaine, sodium cocoamphoacetate, sodium lauroamphoacetate, sodium laureth sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, sodium laureth sulfate, sodium lauryl ether sulfate, sodium lauryl ether sulfate, sodium lauryl ether sulfate, sodium lauryl ether sulfate, sodium, One or more of sodium dodecyl sulfate, alkyl glycoside, dodecyl dimethyl betaine, disodium imidazoline amphodiacetate, sodium cocoamphoacetate, ammonium polyquatemate-7, ammonium polyquatemate-10, ammonium polyquatemate-37, hydroxypropyl guar hydroxypropyl trimethyl ammonium chloride, hydroxypropyl trimethyl ammonium chloride guar gum, linden seed extract and the like. The surfactant is present in the composition in amounts known in the art, for example, generally from 0.1 to 50% by weight based on the total weight of component (C).
Examples of such emulsifiers include, but are not limited to, cetearyl olivate, sorbitan olivate, polysorbate-60, polysorbate-80, methylgluco-sesquistearate, PEG-20 methylgluco-sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-Butanethol-26, PEG-4 polyglyceryl-2 stearate, PEG-60 hydrogenated castor oil, steareth-2, steareth-21, PPG-13-decyltetraeth-24, cetearyl glucoside, PEG-100 stearate, glyceryl stearate SE, coco glucoside, ceteareth-25, PEG-40 stearate, polyglyceryl-3 methylgluco distearate, sorbitan esters, glyceryl esters, sorbitan esters, ceteareth-25, PEG-40 stearate, polyglyceryl esters, glyceryl esters, sorbitan esters, glyceryl esters, and the salts of esters, and the salts of the compounds of the, One or more of glyceryl stearate citrate, polyglyceryl-10 stearate, polyglyceryl-10 myristate, polyglyceryl-10 dioleate, polyglyceryl-10 laurate, polyglyceryl-10 isostearate, polyglyceryl-10 oleate, polyglyceryl-10 diisostearate, polyglyceryl-6 laurate, polyglyceryl-6 myristate, sucrose stearate, sucrose polystearate, and the like. The content of said emulsifier in said composition is known in the art, and it is, for example, generally from 0.05 to 30% by weight, based on the total weight of component (C).
Examples of the thickener include, but are not limited to, one or more of carbomers, acrylates and derivatives thereof, xanthan gum, acacia, polyethylene glycol-14M, polyethylene glycol-90M, succinoglycan, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, and other high molecular polymers. The thickening agent is present in the composition in amounts known in the art, for example, generally from 0.1 to 30% by weight based on the total weight of component (C).
Examples of such preservatives include, but are not limited to, one or more of methylparaben, ethylparaben, propylparaben, phenoxyethanol, benzyl alcohol, phenethyl alcohol, bis (hydroxymethyl) imidazolidinyl urea, potassium sorbate, sodium benzoate, chlorphenesin, sodium dehydroacetate, and the like. The content of the preservative in the hair loss preventive composition is known in the art, and for example, it is usually 0.01 to 30% by weight based on the total weight of the component (C).
The anti-alopecia hair growth composition of the present invention can be prepared by any suitable method known in the art. For example, it is prepared using a dissolving tank, an emulsifying pot, a disperser, a transfer pump, etc., which are generally used in the art. The preparation method comprises putting water soluble substance into water phase dissolving kettle, putting oil soluble substance into oil phase dissolving kettle, heating the two kettles to about 80 deg.C, wherein the raw material easy to agglomerate can be pre-dispersed with disperser. After the dissolution is finished, the oil phase and the water phase are conveyed into an emulsifying pot, and homogenized and emulsified for about 5-15 minutes. After emulsification is finished, the temperature of the material body is reduced to normal temperature, optional essence, preservative and the like are added, and the pH of the product is adjusted according to needs. After the relevant detection indexes are qualified, the products can be filled and delivered. The preparation method can be deleted or adjusted according to the requirements of dosage forms.
The hair loss preventing and growing composition of the present invention may be in the form of a shampoo for washing off, or a hair tonic for leave-on, and it may be prepared in various forms such as cream, milky lotion, liquid, etc., as required.
Examples
The present invention will be described in further detail with reference to examples. However, it should be understood that these examples and comparative examples are only for illustrating the present invention in more detail, and should not be construed as limiting the scope of the appended claims of the present invention in any way.
Example 1: inhibition of 5 alpha-reductase activity
In this example, the effect of concentrated birch juice, ginger extract, polygonum multiflorum root extract, ginseng extract, green tea extract, thuja extract, and combinations thereof on 5 α -reductase activity was examined and compared.
1. Concentration of birch sap
Fresh birch sap stock solution purchased from Daxingan mountain surpassing the company Limited for wild berry development is introduced into a low-temperature drying device, cooled to-65 ℃, vacuumized to 0.1Pa, and concentrated by 1.5 and 10 times.
2. Testing
An experimental instrument: balance, wall breaking machine, constant temperature oscillator, high speed freezing centrifuge, enzyme mark appearance.
Experimental reagents and consumables:
finasteride: shanghai modern pharmaceuticals, Inc. (tablets 5 mg/tablet);
reduced coenzyme ii (NADPH): roche, USA (powder 10 mg);
testosterone Elisa kit: wuhan Youerson company (Cat: CEA458 Ge);
5 alpha-reductase crude enzyme: self-contained;
PBS: wuhan doctor de Corp;
phenylmethylsulfonyl fluoride (PMSF): sigma, USA;
dithiothreitol (DTT): sigma, USA;
1M Tris-HCl: wuhan doctor de Corp.
Sample loading information:
the loading amount of a single raw material is the whole raw material; the compound raw material is half of birch juice raw material and half of 0.1% of other active raw materials.
The in vitro 5 alpha-reductase activity impact assay procedure was as follows:
(1) preparing a crude enzyme: taking 5 normal healthy mice, dislocating and killing the neck, opening the abdominal cavity, taking testis, dividing into several parts, placing in a 2mL EP tube, adding a proper amount of crude enzyme extract according to a ratio of 1:4, crushing by a wall breaking machine at 4 ℃ to prepare homogenate, freezing and centrifuging at 4 ℃ at a high speed (10000 rpm/min, 10 min), and taking supernatant and storing at 4 ℃. The BCA method is used for determining the protein concentration, and the subsequent experiment can be carried out when the protein concentration is more than 1 mg/mL;
(2) measuring blank control, namely respectively taking 2 groups of 200 mu L EP tubes (4 in each group), adding 5 alpha-reductase crude enzyme, PBS (pH 7.4) and reduced coenzyme II, mixing one group, immediately putting into boiling water for boiling for 5 minutes, centrifuging, and absorbing 50 mu L of liquid for subsequent Elisa detection to obtain the initial testosterone content of the blank control group; the other group is mixed and placed in a constant temperature shaking table to be mixed for 60 minutes, the mixture is placed in boiling water to be boiled for 5 minutes, after centrifugation, 50 mu L of liquid is absorbed to carry out subsequent Elisa detection, and the difference value between the initial content and the initial content is the content of the testosterone after the conversion of the blank control group;
(3) respectively taking 2 groups of 200 mu L EP tubes (4 in each group), adding 5 alpha-reductase crude enzyme, PBS solution (pH 7.4), reduced coenzyme II and test raw materials, mixing one group, immediately putting into boiling water for boiling for 5 minutes, centrifuging, sucking 50 mu L of liquid for subsequent Elisa detection to obtain the initial testosterone content of the inhibitor group; and the other group is mixed and placed in a constant temperature shaking table to be mixed for 60 minutes, the mixture is placed in boiling water to be boiled for 5 minutes, after centrifugation, 50 mu L of liquid is absorbed to be subjected to subsequent Elisa detection, and the difference value between the initial content and the initial content is the content of the testosterone after the inhibitor group is converted. Experiments were performed in 4 replicates each time.
The 5 alpha-reductase inhibition rate is calculated according to the following formula:
I％＝(△A 0 -△A n )/△A 0 *100％
wherein:
△A 0 control testosterone reduction;
△A n testosterone reduction in the inhibitor group.
The test results are shown in the following table.
The results show that the effect of the combination of the 1.5 times of concentrated birch juice and the ginger extract, the polygonum multiflorum root extract, the ginseng extract, the green tea extract and the arborvitae extract is obviously better than the effect of the combination of the single birch juice stock solution, the 1.5 times of concentrated birch juice and the ginger extract, the polygonum multiflorum root extract, the ginseng extract, the green tea extract and the arborvitae extract in the aspect of inhibiting the 5 alpha-reductase, and simultaneously the effect of the combination of the birch juice stock solution and the ginger extract, the polygonum multiflorum root extract, the ginseng extract, the green tea extract and the arborvitae extract is also obviously better than the effect of the combination of the birch juice stock solution and the ginger extract, the polygonum multiflorum root extract, the ginseng extract, the green tea extract and the arborvitae extract. When the birch juice is concentrated to 10 times, the effect of combining the birch juice with other active matters is obviously weakened or even not as good as the effect of combining the stock solution birch juice with other active matter raw materials.
Example 2: influence on the expression of hair loss prevention and hair growth related genes and proteins
In this example, the effects of concentrated birch juice, angelica extract, astragalus, eclipta alba, birch bud extract, birch leaf extract, and combinations thereof on the expression of hair loss prevention and hair growth related genes and proteins were examined and compared.
1. Concentration of birch sap
Fresh birch sap stock solution purchased from Daxingan mountain surpassing the company Limited for wild berry development is introduced into a low-temperature drying device, cooled to-65 ℃, vacuumized to 0.1Pa, and concentrated by 1.5 and 10 times.
2. Testing
The dermal papilla cells used in this experiment were produced by Guangdong Boxi Biotech limited.
An experimental instrument: CO 2 2 Incubator (Thermo), clean bench (Sujing Antai), inverted microscope (Olympus), micro-oscillator (Linbel), enzyme labeling instrument (BioTek), incubator (Tester), fluorescent quantitative PCR instrument (BioRad), general PCR instrument (Bori).
The experimental reagent: mesenchymal Stem Cell Medium (ScienCell), PBS (Philid), DHT (Sigma), VEGF ELISA kit (Abcam), RNAiso Plus (Takara), reverse transcription kit (Takara), fluorescent dye (Takara).
Sample loading information: the loading amount of a single raw material is the whole raw material; the compound raw material is half of birch juice raw material and half of 0.1% of other active raw materials.
The steps of analysis of gene and protein expression based on hair papilla cells were as follows:
(1) inoculation: inoculating into 6-well plate at 37 deg.C and 5% CO 2 Incubating in an incubator overnight;
(2) preparing liquid: the test article working solution was prepared according to the following experimental design table.
The experimental design is as follows:
(3) administration: according to the specific design of the experiment in the table above, when the plating rate of the cells in the 6-well plate reaches 40-50%, the administration amount of each well is 2mL, and each group is provided with 3 multiple wells. 37 ℃ and 5% CO 2 The incubator continues to incubate for 24 hours.
(4) Cell supernatants and cells were collected separately:
a. collecting cell supernatant: after culturing for 24 hours, collecting cell culture supernatant in an EP tube, and after collection, placing a sample for VEGF content detection in a refrigerator at-80 ℃ for freezing and storing;
b. collecting cells: after 24 hours of culture, cell supernatants were collected, washed twice with 1 mL/well PBS, 1mL of RNAioso Plus was added to each well, lysed cells were aspirated, and samples were collected.
(5) And (3) detection of VEGF content: the detection assay was performed according to the instructions of the VEGF ELISA kit.
(6) Detection of AR gene expression: extracting RNA, reverse transcribing to cDNA, and fluorescent quantitative PCR detection with 2 -△△CT The method performs a result calculation.
3. Statistical analysis of results
The plots were generated using GraphPad Prism Program software and statistically analyzed between groups using T-test.
The test results are shown in the following table.
| Sample (I) | VEGF protein (pg/mL) | AR Gene (relative transcription amount) |
| Negative control | 706±6 | 1.00±0.05 |
| Stock solution of birch juice | 814±6 | 0.82±0.03 |
| 1.5 times concentrated birch juice | 839±12 | 0.64±0.04 |
| Concentrated birch juice 10 times | 828±8 | 0.74±0.05 |
| Angelica sinensis extract | 765±17 | 0.88±0.07 |
| Astragalus membranaceus | 728±23 | 0.91±0.06 |
| Herba Ecliptae | 789±19 | 0.86±0.05 |
| Birch bud | 744±25 | 0.83±0.06 |
| White birch leaf | 775±18 | 0.88±0.04 |
| Birch juice stock solution and radix Angelicae sinensis extract | 933±14 | 0.78±0.03 |
| Birch juice stock solution and radix astragali | 956±22 | 0.75±0.06 |
| Birch juice stock solution and eclipta alba | 978±16 | 0.74±0.05 |
| Stock solution of birch juice and birch bud | 991±21 | 0.76±0.07 |
| Birch juice stock solution and birch leaves | 948±18 | 0.75±0.01 |
| Concentrated birch juice 1.5 times and radix Angelicae sinensis extract | 1265±45 | 0.38±0.04 |
| 1.5 times of concentrated birch juice and radix astragali | 1309±31 | 0.37±0.03 |
| 1.5 times of concentrated birch juice and eclipta alba | 1257±28 | 0.41±0.05 |
| 1.5 times of concentrated birch juice and birch bud | 1319±34 | 0.35±0.02 |
| 1.5 times of concentrated birch juice and birch leaf | 1301±27 | 0.43±0.04 |
| Concentrated birch juice 10 times and radix Angelicae sinensis extract | 887±36 | 0.79±0.03 |
| 10 times of concentrated birch juice and astragalus root | 843±28 | 0.78±0.06 |
| 10 times of concentrated birch juice and eclipta alba | 865±27 | 0.74±0.05 |
| Concentrated birch juice 10 times and birch bud | 896±41 | 0.80±0.04 |
| Concentrated birch juice 10 times and birch leaf | 870±38 | 0.75±0.07 |
The above results show that compared with the use of the birch juice stock solution, the 1.5-fold concentrated birch juice, the angelica sinensis extract, the astragalus membranaceus, the eclipta alba, the birch bud and the birch leaves alone, and the combination of the birch juice stock solution and the angelica sinensis extract, the astragalus membranaceus, the eclipta alba, the birch bud or the birch leaves, the use of the 1.5-fold concentrated birch juice in combination with the angelica sinensis extract, the astragalus membranaceus, the eclipta alba, the birch bud or the birch leaves significantly increases the expression level of VEGF protein (vascular endothelial growth factor) related to the microcirculation of blood vessels around papillae, and effectively inhibits the transcription of AR (androgen receptor) genes related to androgen signal transmission. When the birch juice is concentrated to 10 times, the effect of combining the birch juice with other active matters is obviously weakened or even not as good as the effect of combining the stock solution birch juice with other active matter raw materials.
Example 3: effect on the proliferation and differentiation Capacity of Hair papilla cells
In this example, the effect of various fold-concentrated birch sap, adenosine, nicotinamide, tripeptide-1 copper, and their combinations on the proliferation and differentiation capacity of hair papilla cells was examined and compared.
1. Concentration of birch sap
Fresh birch sap stock solution purchased from Daxingan mountain surpassing the company Limited for wild berry development is introduced into a low-temperature drying device, cooled to-65 ℃, vacuumized to 0.1Pa, and concentrated by 1.5 and 10 times.
2. Test method
The dermal papilla cells used in this experiment were produced by Guangdong Boxi Biotech limited.
An experimental instrument: CO 2 2 Incubator (Thermo), super clean bench (Sujing an)Tai), inverted microscope (Olympus), micro-oscillator (its linbel), microplate reader (BioTek), and incubator (tester).
Experimental reagents and consumables: mesenchymal Stem Cell Medium (science Cell), PBS (Phd.), MTT (Sigma), DMSO (Sigma).
Sample loading information: the loading amount of a single raw material is the whole raw material; the compound raw material is half of birch juice raw material and half of 0.1% of other active raw materials.
The experimental method comprises the following steps:
(1) preparing a cell suspension: digesting cells in logarithmic growth phase, inoculating the cells into a 96-well plate, and inoculating the cells into the 96-well plate at 37 ℃ and 5% CO 2 Incubating in an incubator overnight;
(2) administration: when the cell plating rate in the 96-well plate reaches 20-30%, the prepared culture solution with the sample to be detected is administered in groups, the administration amount of each hole is 200 mu L, and each group is provided with 3 multiple holes. MTT assay was performed on 0 hour-grouped cells simultaneously, and at other time points, the cells were incubated at 37 ℃ in 5% CO 2 Continuously culturing in an incubator;
(3) and (3) detection: the cells were incubated for 48 hours, the supernatant was discarded, MTT working solution was added, and incubation was carried out at 37 ℃ in the dark. Discarding the supernatant after 4 hours, adding 150 mu L DMSO into each hole, and reading the OD value by an enzyme-labeling instrument at 490 nm;
(4) calculating the formula:
the test results are shown in the following table.
| Sample (I) | Rate of hair papilla cell proliferation |
| Stock solution of birch juice | 18％±2％ |
| Concentrated birch juice 1.5 times | 33％±7％ |
| Concentrated birch juice 10 times | 22％±3％ |
| Adenosine (I) | 6％±3％ |
| Nicotinamide | 13％±5％ |
| Tripeptide-1 copper | 8％±3％ |
| Birch juice stock solution + adenosine | 23％±4％ |
| Birch juice stock solution and nicotinamide | 24％±6％ |
| Birch juice stock solution + tripeptide-1 copper | 26％±5％ |
| 1.5 times of concentrated birch juice + adenosine | 52％±4％ |
| 1.5 times of concentrated birch juice + nicotinamide | 48％±7％ |
| 1.5 times of concentrated birch juice and tripeptide-1 copper | 49％±4％ |
| 10 times concentrated birch juice + adenosine | 22％±4％ |
| 10 times of concentrated birch juice and nicotinamide | 22％±3％ |
| 10 times concentrated birch juice + tripeptide-1 copper | 23％±3％ |
MTT value test results show that compared with the single use of birch juice stock solution, 1.5 times of concentrated birch juice, adenosine, nicotinamide and tripeptide-1 copper and the combination of the birch juice stock solution and the adenosine, the nicotinamide and the tripeptide-1 copper, the combination of the 1.5 times of concentrated birch juice and the tripeptide-1 copper obviously improves the proliferation and differentiation capacity of hair papilla cells. When the birch juice is concentrated to 10 times, the effect of combining the birch juice with other active matters is obviously weakened or even not as good as the effect of combining the stock solution birch juice with other active matter raw materials.
Example 4: comparison of Effect on mouse models
1. Concentration of birch sap
Fresh birch sap stock solution purchased from Daxingan mountain surpassing the company Limited for wild berry development is introduced into a low-temperature drying device, cooled to-65 ℃, vacuumized to 0.1Pa, and concentrated by 1.5 and 10 times.
2. Experimental methods
Experimental animals: c57BL/6 mice, 6-7 weeks old, male; the animal is from Beijing vitamin Tonglihua.
Sample loading information: the loading amount of a single raw material is the whole raw material; the compound raw material is half of birch juice raw material and half of 0.1% of other active raw materials.
The determination method comprises that about 15 mice C57BL/6 are used in each group, and after 1 week of acclimatization, the mice are treated according to the bodyIndifferent packets are repeated. Removing hair from back with depilator, and uniformly applying depilatory cream to back at a speed of about 3x4cm 2 After several minutes, the hair follicles were washed off and induced to grow into the anagen phase, at which time the skin of the mice appeared pink. On the next day after depilation, testosterone propionate was subcutaneously injected at a dose of 5 mg/kg/day at both sides (one site was taken) of each of the animals except for the normal control group, 1 time per day, and soybean oil was subcutaneously injected at an equal volume to the normal control group. The drug was administered while molding, and each group was smeared with 0.1 mL. The area of the mice's back depilated was measured after 2 months.
The test results are shown in the following table.
| Sample (I) | Depilatory area (Pixel) |
| Blank space | 10183±92 |
| Stock solution of birch juice | 9105±47 |
| Concentrated birch juice 1.5 times | 7466±90 |
| Concentrated birch juice 10 times | 9005±85 |
| Polygonum multiflorum root extract | 9819±43 |
| Green tea extract | 9836±64 |
| Birch juice stock solution and radix Polygoni Multiflori extract | 7967±59 |
| Birch juice stock solution and green tea extract | 8165±39 |
| Concentrated birch juice 1.5 times and radix Polygoni Multiflori extract | 5173±93 |
| Concentrated birch juice 1.5 times and green tea extract | 4032±75 |
| Concentrated birch juice 10 times and radix Polygoni Multiflori extract | 8467±59 |
| Concentrated birch juice 10 times and green tea extract | 8565±39 |
The experimental results of mouse models show that compared with the single use of the birch juice stock solution, the 1.5-time concentrated birch juice, the polygonum multiflorum root extract and the green tea extract, and the combination of the birch juice stock solution and the polygonum multiflorum root extract or the green tea extract, the combined use of the 1.5-time concentrated birch juice and the polygonum multiflorum root extract or the green tea extract obviously reduces the depilation area of mice and promotes the rapid growth of new hair. When the birch juice is concentrated to 10 times, the effect of combining the birch juice with other active matters is obviously weakened or even not as good as the effect of combining the stock solution birch juice with other active matter raw materials.
Example 5: hair loss preventing and hair growing liquid
The formula of the anti-hair loss and hair growth liquid is shown in the following table:
the preparation method of the hair restorer comprises the following steps:
1. firstly, adding the phase A components into a main pot according to the mass ratio, heating to 80 ℃, and mixing;
2. adding the components of the phase C into a side pot according to the mass ratio, heating to melt and uniformly stirring, putting the mixture into the phase A, and uniformly stirring and mixing the mixture with the phase A;
3. sequentially adding the amino acid, the nutritional supplement, the oil control agent and the nicotinamide in the C phase into the main pot, and uniformly stirring;
4. sequentially adding the diaminopyrimidine oxide in the phase D and the solvent in the phase D into the main pot, and uniformly stirring;
5. cooling the main kettle to 50 ℃, sequentially adding the alcohol of the E phase and the solvent of the E phase, and uniformly stirring;
6. cooling the main kettle to 45 ℃, adding the tripeptide-1 copper of the F phase, and uniformly stirring;
7. mixing Tween-20 and essence in the G phase, pre-dissolving, stirring to transparent, adding into the main pot, and stirring;
8. and finally, adding the H phase into the main pot, uniformly stirring, cooling to room temperature, and discharging after the detection is qualified to obtain the hair growth liquid for preventing alopecia.
Clinical human body test, 120 hair loss patients (the hair loss number is more than 100 per day) are selected and randomly divided into six groups, and the six shampoos are respectively used for half each group of men and women. The subjects applied to the scalp daily and massaged and absorbed, combed hair 2 times in the morning and evening, collected shed hair and counted. Once weekly, for 4 weeks, the following results were obtained:
the above results of the anti-sloughing solution show that the combination of the concentrated birch juice of 1.5 times and the tripeptide-1 copper has more remarkable anti-sloughing effect compared with the single use of the birch juice stock solution, the concentrated birch juice of 1.5 times, the tripeptide-1 copper and the combination of the birch juice stock solution and the tripeptide-1 copper.
Example 6: hair loss preventing and hair growing shampoo
The formula of the hair loss preventing and hair growing shampoo is shown in the following table:
the preparation method of the shampoo comprises the following steps:
deionized water or birch sap was added in the proportions indicated in the table and heated to 80 c for half an hour. Sequentially adding sodium lauryl alcohol ether sulfate, sodium cocoyl glutamate, guar gum and birch sprout into water, continuously stirring, and keeping for 10 minutes. Homogenizing the mixture for 20-30 minutes; mixing 1, 2-pentanediol, 1,3-CG, D-panthenol, menthol and eucalyptus leaf oil in advance, adding the mixture into the system when the temperature of the homogeneous mixture is reduced to room temperature, and stirring for 10 minutes. Citric acid and sodium chloride were added. The pH was adjusted to 5-7 and finally deionized water was added to 100% of the recipe weight. Filtering with 1200 mesh, and bottling to obtain shampoo.
Clinical human body test: 120 alopecia patients (the number of alopecia is more than 100 per day) are selected, randomly divided into six groups, and the six shampoos are respectively used, wherein each group is half of male and female. Subjects washed their hair every other day, combed hair 2 times in the morning and evening, and shed hair was collected and counted. Once every 15 days, for 3 months, the following results were obtained:
the use results show that compared with the single use of the birch sap stock solution, the 1.5-time concentrated birch sap, the birch bud and the combination of the birch sap stock solution and the birch bud, the combination of the 1.5-time concentrated birch sap and the birch bud has more remarkable anti-dropping effect.
The technical solutions of the above-described embodiments are preferred embodiments of the present invention, and several modifications and changes can be made without departing from the principle of the present invention, and these modifications and changes should also be considered as being within the protection scope of the present invention.
Claims (6)
- Use of (a) concentrated birch juice in combination with (B) any one or more of ginger extract, polygonum multiflorum root extract, ginseng extract, green tea extract, arborvitae extract, birch bud extract, adenosine and tripeptide-1 copper for the preparation of an anti-alopecia hair growth composition, wherein the concentrated birch juice is concentrated in a multiple of 1.5-6, and wherein the anti-alopecia hair growth composition does not comprise any added water.
- 2. The use according to claim 1, wherein the concentrated birch sap has a concentration factor of 1.5-5.
- 3. An anti-alopecia and hair-growing composition comprising (a) a concentrated birch juice, and (B) any one or more of a ginger extract, a polygonum multiflorum root extract, a ginseng extract, a green tea extract, a thuja extract, a birch bud extract, adenosine, and tripeptide-1 copper, wherein the concentration ratio of the concentrated birch juice is 1.5-6, wherein the anti-alopecia and hair-growing composition does not contain any additional water.
- 4. The composition of claim 3, wherein the concentrated birch sap has a concentration factor of 1.5-5.
- 5. The composition of claim 3, wherein the content of the component (A), the concentrated birch sap, is 10-98% based on the total weight of the alopecia preventing and growing composition.
- 6. The composition according to any one of claims 3 to 5, wherein the content of the component (B) is 0.0005 to 30% based on the total weight of the alopecia preventing composition.
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| CN110731924B (en) * | 2019-07-30 | 2022-05-17 | 浙江养生堂天然药物研究所有限公司 | Enhanced anti-aging cosmetic compositions |
| CN111110618B (en) * | 2020-02-11 | 2022-09-27 | 张运适 | A composition for promoting hair growth |
| CN114632049B (en) * | 2020-12-15 | 2023-10-31 | 浙江养生堂天然药物研究所有限公司 | Hair loss preventing and hair growing composition |
| CN113069375A (en) * | 2021-04-20 | 2021-07-06 | 广州博裕生物科技有限公司 | Hair growth liquid containing high-content adenosine and derivatives thereof, and preparation method and application thereof |
| CN113208966A (en) * | 2021-05-19 | 2021-08-06 | 广州环亚化妆品科技有限公司 | Hair loss prevention and hair growth scalp care essence containing hinokitiol and preparation method and application thereof |
| CN116077400A (en) * | 2021-11-08 | 2023-05-09 | 厦门本草真源生物医药科技有限公司 | Plant formula with antioxidant and anti-aging effects and application thereof |
| CN114042022A (en) * | 2021-11-16 | 2022-02-15 | 滁州植兀生物科技有限公司 | Anti-dropping composition, anti-dropping washing and protecting product and preparation method |
| CN114681365B (en) * | 2022-04-29 | 2023-10-27 | 福建片仔癀化妆品有限公司 | Shampoo with oil control and mite removal effects and preparation method thereof |
| CN114939095A (en) * | 2022-06-22 | 2022-08-26 | 烟台新时代健康产业日化有限公司 | Oil-control and hair loss-prevention composition, preparation method thereof, hair-care spray and shampoo |
| CN115068390A (en) * | 2022-08-01 | 2022-09-20 | 广州发妍化妆品制造有限公司 | Multifunctional composition for shampoo, preparation method and application |
| CN115737512B (en) * | 2022-11-18 | 2024-09-20 | 深圳市熙雨中医药研究有限公司 | Essence with hair loss prevention and hair growth functions and preparation method thereof |
| CN117017883B (en) * | 2023-08-25 | 2024-02-20 | 广州帝臣生物科技股份有限公司 | Deoiling shampoo and preparation method thereof |
| CN117050923B (en) * | 2023-10-13 | 2023-12-08 | 山东福瑞达生物股份有限公司 | Lactobacillus rhamnosus LR06 and application thereof in fermentation of chenopodium album |
| CN118141736A (en) * | 2024-05-11 | 2024-06-07 | 威莱(广州)日用品有限公司 | Scalp oil control maintenance composition and preparation method thereof |
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