CN114886830B - Scalp/hair/skin repairing composition using seawater pearl as raw material, preparation method and application thereof - Google Patents
Scalp/hair/skin repairing composition using seawater pearl as raw material, preparation method and application thereof Download PDFInfo
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- CN114886830B CN114886830B CN202210235950.7A CN202210235950A CN114886830B CN 114886830 B CN114886830 B CN 114886830B CN 202210235950 A CN202210235950 A CN 202210235950A CN 114886830 B CN114886830 B CN 114886830B
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- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 1
- 229960001052 streptozocin Drugs 0.000 description 1
- 229910052712 strontium Inorganic materials 0.000 description 1
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 210000000106 sweat gland Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000003664 tensile strength of the hair Effects 0.000 description 1
- 238000009864 tensile test Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 201000010653 vesiculitis Diseases 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
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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
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/987—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of species other than mammals or birds
-
- 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
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- 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
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q5/00—Preparations for care of the hair
- A61Q5/002—Preparations for repairing the hair, e.g. hair cure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q5/00—Preparations for care of the hair
- A61Q5/12—Preparations containing hair conditioners
-
- 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
- A61K2800/41—Particular ingredients further characterized by their size
- A61K2800/412—Microsized, i.e. having sizes between 0.1 and 100 microns
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- 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
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- 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
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- 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
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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Abstract
The invention discloses a scalp/hair/skin repair composition taking seawater pearl as a raw material, a preparation method and application thereof, wherein the scalp/hair/skin repair composition comprises the steps of preparing pearl powder, preparing seawater pearl extract, preparing hydrolyzed seawater pearl, preparing hydrolyzed fibroin, and preparing pearl powder: seawater pearl extract: hydrolyzing seawater pearl: the hydrolyzed fibroin is mixed according to the mass ratio of (0.01-2): 8-12): 4-6. The invention combines pearl powder, seawater pearl extract, hydrolyzed seawater pearl and hydrolyzed fibroin to complement various functional components, has stronger damaged hair restoration function, and has the functions of regulating scalp sebaceous gland activity and inhibiting 5 alpha-reductase, has obvious antioxidation and anti-inflammatory effects, and simultaneously has the effects of moisturizing and restoring skin barrier.
Description
Technical Field
The invention belongs to the technical field of cosmetic compositions, and particularly relates to a scalp/hair/skin repair composition taking seawater pearls as raw materials, a preparation method and application thereof.
Background
The pearl medicine has more than 2000 years history in China, and the pearl in the Ming dynasty of plum places more importance on the pharmacological action of the pearl, and the medicine effect of the pearl is considered to be skin-beautifying, so the pearl medicine is particularly written in the 'materia medica schema': "Pearl taste sweet and cold without toxicity, and the heart-relieving and eyesight-improving effects: the pearl is coated with the surface, so that the color is well-finished. Applying to hands and feet, removing skin and reversing skin: sputum, facial speckle and infantile convulsion heat are removed, an Hunbo: stopping seminal emission, removing acne and treating toxin. The pearl powder is white and various methods for pearl medicine are also recorded. When the Ming dynasty Chen Jiru turns to "Du Zhi", tang Wuzong Li Yan is in place, chancellor Li Deyu takes decoction of Jewelry powder, realgar and Cinnabaris as thick soup, and about three tens of thousands of money are consumed per cup, and the residue is discarded after three decoction. In the current popular refining, people think that pearl powder, realgar and other substances can grow and old after being extracted and taken, and the young people can feel like a crane.
The seawater pearl powder contains rich fatty alcohol, is easily absorbed by human epidermal cells, enhances cell activity and promotes cell metabolism. The pearl powder can promote the collagen cell growth of human body, when trauma and wound exist, the collagen cell growth can be promoted to fill up gaps, the connection tissue can promote the skin regeneration, the skin is kept tender and moist and white, the seawater pearl necklace is clung to the neck of the human body, the seawater pearl necklace has a certain absorption capacity on body fluid secretion, the skin also has absorption on pearl medicine components and even affects local affected parts, the anti-inflammatory treatment effect is achieved, the pearl powder has multiple elements such as calcium carbonate, calcium oxide, calcium phosphate, calcium sulfate, magnesium, manganese, copper, iodine, phosphorus, strontium, lead, sodium, potassium and the like and more than ten amino acids, and the pearl powder has the effects of arresting convulsion, clearing heat and damaging yin, improving eyesight and detoxifying, promoting tissue regeneration and the like in medicine.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The invention provides a preparation method of a scalp/hair/skin repair composition taking seawater pearl as a raw material, which comprises the following steps:
Preparing pearl powder: pulverizing seawater pearl into coarse powder with particle size of 10-15 microns by an ultrafine pulverizer, pulverizing into seawater pearl powder with average particle size of 2-3 microns by air flow, placing the seawater pearl powder into a pressurizing device, adding water with 15-25% of the weight of the powder, pressurizing, instantly depressurizing, taking out, mixing the obtained pearl powder with water to form pearl slurry, carrying out wet grinding on the obtained pearl powder, filtering and drying to obtain bright white pearl powder;
Preparing seawater pearl extract: adding the pearl powder into a lactic acid aqueous solution with the mass concentration of 3-5% for soaking for 1-3 h, filtering, collecting filter residues, repeatedly washing with distilled water, carrying out wet grinding, drying, and carrying out secondary grinding by a high-speed grinder to obtain a seawater pearl extract;
preparing hydrolyzed seawater pearl: preparing a streptozotocin solution with the pH value of 7.0-7.5 and 0.1-0.4 mg/mL, adding the pearl powder, carrying out enzymolysis for 1-3 hours at the temperature of 40-45 ℃ and the temperature of 0.1-0.2% of the mass of the pearl powder, heating to inactivate enzyme, adjusting the pH value to 5.5-6.5, and adding a complex enzyme with the mass of 0.1-0.2% of the mass of the pearl powder, wherein the complex enzyme is debitterizing protease and bromelain, and the mass of the complex enzyme is 1-2: 1, carrying out enzymolysis for 2-3 hours at 50-55 ℃, heating to inactivate enzyme, decompressing, evaporating and concentrating, and freeze-drying to obtain hydrolyzed seawater pearl;
Preparing hydrolyzed fibroin:
Pearl powder: seawater pearl extract: hydrolyzing seawater pearl: the hydrolyzed fibroin is mixed according to the mass ratio of (0.01-2): 8-12): 4-6.
The preparation method of the scalp/hair/skin repair composition using seawater pearl as raw material comprises the following steps: the preparation method of the pearl powder comprises the steps of putting seawater pearl into an ultrafine grinder to be ground into coarse powder with the particle size of 10-15 microns, then grinding the coarse powder into seawater pearl powder with the average particle size of 2-3 microns through air current, putting the seawater pearl powder into a pressurizing device, adding water accounting for 20% of the weight of the powder, pressurizing to 0.5MP, maintaining for 4-5 hours, instantly decompressing, taking out, and mixing the obtained pearl powder with water according to the mass ratio of 1: 4-5, grinding by wet method to obtain pearl powder with particle size of 10-100 nm, filtering and drying to obtain bright white pearl powder.
The preparation method of the scalp/hair/skin repair composition using seawater pearl as raw material comprises the following steps: the seawater pearl extract is prepared by adding pearl powder into a lactic acid aqueous solution with the mass concentration of 4% for soaking for 2-3 hours, filtering, collecting filter residues, repeatedly washing with distilled water, carrying out wet grinding, drying, and carrying out secondary grinding by a high-speed grinder to obtain the bright white seawater pearl extract.
The preparation method of the scalp/hair/skin repair composition using seawater pearl as raw material comprises the following steps: the seawater pearl extract is prepared by preparing 0.2mg/mL of streptogramin solution by using phosphate buffer solution with pH of 7.5, adding pearl powder, carrying out enzymolysis for 2-3 hours at 40 ℃ with streptogramin accounting for 0.16% of the mass of the pearl powder, heating to inactivate enzyme, regulating pH to 6, and adding complex enzyme accounting for 0.2% of the mass of the pearl powder, wherein the complex enzyme is debitterizing proteinase and bromelain according to the weight of 2:1, after enzymolysis for 3-4 hours at 55 ℃, heating to inactivate enzyme, decompressing, evaporating and concentrating, and freeze-drying to obtain the hydrolyzed seawater pearl.
The preparation method of the scalp/hair/skin repair composition using seawater pearl as raw material comprises the following steps: the hydrolyzed fibroin is prepared by putting silk into a sodium carbonate aqueous solution with the mass fraction of 1-2% to be boiled for 30-40 min, removing sericin, filtering and drying, and mixing the silk after the sericin removal with a 0.1mol/L phosphoric acid aqueous solution with the pH of 6.5 according to the following g/ml: 20-1:40, adding 800U/g neutral protease, hydrolyzing at 45deg.C for 4 hr, filtering to adjust pH=7 to obtain hydrolyzed fibroin, concentrating by evaporation under reduced pressure, freeze drying, and hydrolyzing fibroin.
The preparation method of the scalp/hair/skin repair composition using seawater pearl as raw material comprises the following steps: pearl powder: seawater pearl extract: hydrolyzing seawater pearl: the hydrolyzed fibroin is mixed according to the mass ratio of 0.02-1:10:10:5.
As another aspect of the invention, the invention also provides the scalp/hair/skin repair composition prepared by the method and using seawater pearls as raw materials.
As another aspect of the present invention, the present invention also provides the use of the scalp/hair/skin care composition using seawater pearl as a raw material in cosmetics: the composition is used for preparing cosmetics comprising one or more of shampoo, hair conditioner, scalp care solution, emulsion, face cream, bath lotion, facial mask and face cleaning products, has a strong damaged hair restoration function, can regulate scalp sebaceous gland activity and inhibit 5 alpha-reductase, and has obvious effects of resisting oxidation and inflammation, and moisturizing and restoring skin barriers.
The invention has the beneficial effects that: the invention combines pearl powder, seawater pearl extract, hydrolyzed seawater pearl and hydrolyzed fibroin to complement various functional components, has stronger damaged hair restoration function, can regulate scalp sebaceous gland activity and inhibit 5 alpha-reductase, has obvious effects of resisting oxidation and inflammation, and simultaneously has the effects of moisturizing and restoring skin barrier.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
fig. 1 is a graph showing the change in scalp moisture content before and after use of the composition of example 1.
FIG. 2 is a graph showing changes in scalp transdermal water loss (g/m 2 h) before and after use of example 1.
Fig. 3 is a graph of scalp sensitivity of a portion of volunteers before and after use of the example 1 composition.
FIG. 4 is a graph showing skin moisturization efficacy test of various example products
FIG. 5 shows scalp 5α -reductase inhibition test of each example product.
FIG. 6 is a graph showing the results of DPPH clearance test.
Fig. 7 is a graph showing the tensile strength of hair after use of the product of example 1.
FIG. 8 is a graph showing the expression level of IL-6.
FIG. 9 is a graph showing the change in the percutaneous loss of water in comparative example 7.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
Preparing a component A: pearl powder: selecting pollution-free seawater pearl, removing dead pearl and impurities, cleaning the pearl with clear water, filtering, drying at low temperature, putting the pearl into an ultrafine grinder, grinding the pearl into coarse powder with the particle size of 10-15 microns, grinding the coarse powder into seawater pearl powder with the average particle size of 2-3 microns by air flow, putting the seawater pearl powder into a supercharging device, adding water accounting for 20% of the weight of the powder, supercharging to 0.5MP, maintaining for 4 hours, instantly decompressing, taking out, and mixing the obtained pearl powder with water according to a proportion of 1:4 (m/m) to prepare a pearl slurry, carrying out wet grinding to obtain pearl powder with the particle size of 10-100nm, and filtering and drying to obtain a component A: bright white pearl powder;
Preparing a component B: seawater pearl extract: adding the pearl powder into a lactic acid aqueous solution with the mass concentration of 4% for soaking for 2 hours, filtering, collecting filter residues, repeatedly washing with distilled water, carrying out wet grinding, drying, and carrying out secondary grinding by a high-speed grinder to obtain a component B: a bright white seawater pearl extract;
Preparing a component C: hydrolyzing seawater pearl: preparing a 0.2mg/mL streptokinase solution from a phosphate buffer solution of pH7.5, adding the pearl powder, carrying out enzymolysis for 2 hours at 40 ℃ and accounting for 0.16% of the mass of the pearl powder, heating to inactivate enzyme, regulating pH to 6, and adding a complex enzyme accounting for 0.2% of the mass of the pearl powder, wherein the complex enzyme is debitterizing protease and bromelain according to the following weight ratio of 2:1, after enzymolysis for 3 hours at 55 ℃, heating to inactivate enzyme, decompressing, evaporating and concentrating, and freeze-drying to obtain a component C: hydrolyzing seawater pearl;
Preparing a component D: hydrolyzed fibroin: boiling silk in 1% sodium carbonate aqueous solution for 30min, removing sericin, filtering and drying, mixing the silk after sericin removal with 0.1mol/L phosphoric acid aqueous solution with pH of 6.5 according to the ratio of 1:20-1:40 (g: ml) mixing, adding 800U/g neutral protease, hydrolyzing at 45deg.C for 4h, filtering to adjust pH=7 to obtain hydrolyzed fibroin, concentrating by evaporation under reduced pressure, and freeze drying to obtain component D: hydrolyzed fibroin.
The components A, B, C, D are mixed according to the mass ratio of 1:10:10:5, mixing uniformly to obtain scalp/hair/skin repairing composition. The scalp/hair/skin care compositions were formulated as solutions according to table 1.
Table 1 composition formulation
| Raw materials | Mass fraction (wt%) |
| Deionized water | To 100 |
| Component A | 1 |
| Component B | 10 |
| Component C | 10 |
| Component D | 5 |
| Carbomer 940 | 0.3 |
The testing method comprises the following steps:
DPPH radical scavenging experiments: preparing DPPH liquid: dissolving 1mg DPPH in small amount of absolute ethanol, adding distilled water to 1L, keeping in dark, adding a certain amount of substance to be measured into 2.5mL of 50 μg ∈ -
In mL of DPPH absolute ethanol solution, the absorbance of the solution was measured at 515nm after being left at room temperature for 30 min. The scavenging effect was calculated from the following formula:
S(％)＝[A0-(A-A1)]×100％/A0
Wherein: s is the clearance of DPPH free radical, A 0 is the absorbance of the DPPH absolute ethanol solution at 515nm wavelength; a is absorbance of the mixed solution after being cleared for 30min at 515nm wavelength; a 1 is the absorbance of the sample itself at a wavelength of 515 nm.
5 Alpha-reductase inhibition assay: 5α -reductase is a membrane protein that localizes on the microsomes of target cells and plays an important role in the development of androgenic alopecia. After androgen binding to the receptor, complex enzymatic reactions occur, indirectly altering hair growth processes. There are 3 isozymes for 5α -reductase, i.e., types I, II, III. Type i 5 alpha-reductase is present in skin tissues such as sebaceous glands, sweat glands, papilla cells, fibroblasts, epidermal keratinocytes, hair follicle keratinocytes, etc. And type II 5 alpha-reductase is mainly present in tissues such as the prostate, reproductive skin, epididymis, seminal vesicles, etc. The conversion of testosterone to Dihydrotestosterone (DHT) depending on reduced coenzyme ii, high levels of dihydrotestosterone are prone to androgen-dependent diseases, and down-regulation of androgen levels by inhibiting 5α -reductase activity is an important method for treating androgenic alopecia. 5 alpha-reductase inhibitors have the advantage of selectively blocking dihydrotestosterone synthesis without affecting normal testosterone levels and physiological function. The 5 alpha-reductase inhibitors currently studied can be classified into natural plant-derived components, chemically synthesized steroids, pyridines, quinolinones, phenoxybutanoic acid compounds. Can screen high-efficiency inhibitor by taking 5 alpha-reductase activity as a target spot.
Preparation of 5α -reductase: SPF-class mice/rats are killed after being fasted without water forbidding overnight, livers and epididymis (fat tissues are stripped) are rapidly taken, weighed, rinsed with normal saline, sheared, added with 5 times of precooled homogenate [0.32mol/l sucrose, 1mmol/l DTT,1mmol/l EDTA,20mmol/l PBS (liver pH=7.5; epididymis pH=5.5) ] and homogenized in a glass homogenizer, the homogenate is centrifuged for 10min at 4 ℃ and 3000g, supernatant is centrifuged for 60min at 4 ℃ and 10000g, the supernatant is crude enzyme, split charging is carried out, and the obtained product is stored in an ultralow temperature refrigerator at-80 ℃ and taken out before use, so that repeated freeze thawing is avoided.
The protein content is measured by using a Lowry method protein content detection kit or the Coomassie brilliant blue experiment is quantified, the total protein content in crude enzyme is used for representing the concentration of 5 alpha-reductase, and the operation is carried out according to the specification of the kit.
Preparation of coomassie brilliant blue G250 solution: 100mg of Coomassie brilliant blue G250 was precisely weighed and 50ml of 95% ethanol solution was added. The solution was blue, stirred to dissolve, then 100ml of 85% (W/V) phosphoric acid was added to stir, the solution was reddish in blood, finally deionized water was used to fix the volume to 1000ml, the solution turned brown, stirred on a magnetic heating stirrer overnight, and filtered with filter paper for use. BSA protein standards were gradient diluted to 0.0, 2.5, 5.0, 10.0, 20.0, 40.0, 50.0. Mu.g/ml with PBS. Sequentially adding 50 μl of BSA solution with each concentration into the 96-well plate, sequentially adding 150 μl of Coomassie brilliant blue G250 solution into each well, placing into a multifunctional enzyme-labeling instrument, shaking for 5min, and measuring absorbance at 595 nm. The Bradford protein standard curve is plotted on the abscissa with BSA concentration and on the ordinate with absorbance.
The crude enzyme extract was diluted to an appropriate multiple and the protein mass concentration of the crude enzyme extract was measured by the Bradford method and considered as the 5 a-reductase content.
Inhibition activity of active substance on type II 5α -reductase: adding samples according to the following reaction system, incubating for a certain time at 37 ℃, adding 2ml of precooled methanol to terminate the reaction, uniformly mixing, centrifuging for 5min at 10000r/min, taking supernatant, filtering with a 0.22 mu m filter membrane, and measuring the absorbance value of each sample hole by using a multifunctional enzyme-labeled instrument; or detecting the residual testosterone concentration in the individual samples.
Table 15 alpha-reductase inhibitory Activity research reaction System
| Reagent(s) | Blank control tube | Sample tube | Positive control tube | Negative control tube |
| Phosphate buffer/. Mu.l | 300 | 300 | 300 | 300 |
| 5 Alpha-reductase crude enzyme solution/. Mu.l | 500 | 500 | 500 | 500 |
| Testosterone/. Mu.l | 50 | 50 | 50 | 50 |
| 75% Ethanol/. Mu.l | 50 | / | 50 | / |
| Sample solution/. Mu.l | / | 50 | / | / |
| Finasteride/. Mu.l | / | / | 50 | / |
| NADPH/μl | 100 | 100 | 100 | 100 |
| Duration of reaction/t | t | t | t | t |
| Methanol (precooling) | 2000 | 2000 | 2000 | 2000 |
Volunteer experiment: the enrolled group of volunteer subjects were enrolled as required, and written informed consent was signed. Prior to the group entry, subjects were asked a series of questions regarding disease history, health status, etc. based on inclusion and exclusion criteria, etc., and prior to the use of the product scalp condition evaluation and image capture were performed on the inclusion subjects and recorded.
The test included 30 scalp sensitive subjects aged 20-50 years and the number of final effective subjects was 30. The volunteers enter a constant temperature and humidity evaluation room to sit still for 30min for testing, 0.2ml of pearl stock solution is taken every day and smeared on scalp for fixing a region of 2 x 2cm 2; the same evaluation and test was again carried out on the product for 14 days, and the experimental results are: fig. 1 is a graph showing changes in scalp moisture content before and after use, indicating significant differences from before use, p <0.05, p <0.001. FIG. 2 is a graph showing the change in the percutaneous water loss before and after use (g/m 2 h). Fig. 3 is a graph of scalp sensitivity of a portion of volunteers before and after application of the composition of the present invention. After use of the samples, 30 subjects can draw the following conclusions compared to before use of the samples: 1) After 14 days of using the sample, the moisture content of the scalp cuticle is obviously improved, the difference is obvious, the scalp percutaneous water loss condition is obviously reduced, and the instant water supplementing effect is realized. 2) The test sample had the effect of soothing scalp reddening, and the scalp reddening of the volunteer was significantly reduced after 14 days of use of the sample (see fig. 3).
Comparative example 1:
Preparing a component A: pearl powder: selecting pollution-free seawater pearl, removing dead pearl and impurities, cleaning the pearl with clear water, filtering, drying at low temperature, putting the pearl into an ultrafine grinder, grinding the pearl into coarse powder with the particle size of 10-15 microns, grinding the coarse powder into seawater pearl powder with the average particle size of 2-3 microns by air flow, putting the seawater pearl powder into a supercharging device, adding water accounting for 20% of the weight of the powder, supercharging to 0.5MP, maintaining for 4 hours, instantly decompressing, taking out, and mixing the obtained pearl powder with water according to a proportion of 1:4 (m/m) to prepare a pearl slurry, carrying out wet grinding to obtain pearl powder with the particle size of 10-100nm, and filtering and drying to obtain a component A: pearl powder;
Preparing a component B: seawater pearl extract: adding the pearl powder into a lactic acid aqueous solution with the mass concentration of 4% for soaking for 2 hours, filtering, collecting filter residues, repeatedly washing with distilled water, carrying out wet grinding, drying, and carrying out secondary grinding by a high-speed grinder to obtain a component B: seawater pearl extract;
Preparing a component C: hydrolyzing seawater pearl: preparing a 0.2mg/mL solution of pronase by using a pH7.5 phosphate buffer solution, adding the pearl powder, carrying out enzymolysis for 2 hours at 40 ℃ and accounting for 0.16% of the mass of the pearl powder, heating to inactivate enzyme, regulating the pH to 6, and adding a complex enzyme accounting for 0.2% of the mass of the pearl powder, wherein the complex enzyme is the debitterizing protease and the bromelain according to the following weight of 2:1, after enzymolysis for 3 hours at 55 ℃, heating to inactivate enzyme, decompressing, evaporating and concentrating, and freeze-drying to obtain a component C: hydrolyzing seawater pearl;
Preparing a component D: commercially available keratin.
The components A, B, C, D are mixed according to the mass ratio of 1:10:10:5 are mixed uniformly and prepared into solutions according to table 1 of example 1.
Comparative example 2:
Preparing a component A: pearl powder: selecting pollution-free seawater pearl, removing dead pearl and impurities, cleaning the pearl with clear water, filtering, drying at low temperature, putting the pearl into an ultrafine grinder, grinding the pearl into coarse powder with the particle size of 10-15 microns, grinding the coarse powder into seawater pearl powder with the average particle size of 2-3 microns by air flow, putting the seawater pearl powder into a supercharging device, adding water accounting for 20% of the weight of the powder, supercharging to 0.5MP, maintaining for 4 hours, instantly decompressing, taking out, and mixing the obtained pearl powder with water according to a proportion of 1:4 (m/m) to prepare a pearl slurry, carrying out wet grinding to obtain pearl powder with the particle size of 10-100nm, and filtering and drying to obtain a component A: pearl powder;
Preparing a component B: seawater pearl extract: adding the pearl powder into a lactic acid aqueous solution with the mass concentration of 4% for soaking for 2 hours, filtering, collecting filter residues, repeatedly washing with distilled water, carrying out wet grinding, drying, and carrying out secondary grinding by a high-speed grinder to obtain a component B: seawater pearl extract;
Preparing a component C: hydrolyzing seawater pearl: preparing papain into a papain solution with the concentration of 0.2mg/mL by using phosphate buffer solution with the pH of 6.0, adding the pearl powder, carrying out enzymolysis on the papain pearl powder at the mass of 0.36 percent and 55 ℃ for 5 hours, heating to inactivate enzyme, evaporating and concentrating under reduced pressure, and freeze-drying to obtain a component C: hydrolyzing seawater pearl;
Preparing a component D: hydrolyzed fibroin: boiling silk in 1% sodium carbonate aqueous solution for 30min, removing sericin, filtering and drying, mixing the silk after sericin removal with 0.1mol/L phosphoric acid aqueous solution with pH of 6.5 according to the ratio of 1:20-1:40 (g: ml) mixing, adding 800U/g neutral protease, hydrolyzing at 45deg.C for 4h, filtering to adjust pH=7 to obtain hydrolyzed fibroin, concentrating by evaporation under reduced pressure, and freeze drying to obtain component D: hydrolyzed fibroin.
Comparative example 3:
the hydrolyzed seawater pearl of the component C of the example 1 is replaced by polyglutamic acid, and other preparation methods are the same as the example 1.
Comparative example 4:
The procedure of example 1 was repeated except that the seawater pearl extract of example 1 was replaced with conchiolin powder.
Skin moisturizing efficacy test of the products of each example: the test was carried into 30 volunteers, and the number of final effective subjects was 30. Volunteers enter a constant temperature and humidity evaluation room to clean the arm and sit still for 30min for testing, each person takes 0.2ml of sample to smear the left forearm for 2 x 2cm2 area, and the test is carried out after smearing for 1, 2, 4 and 8 hours respectively. No adverse reactions were reported by 30 volunteers. The results of the experiment are shown in FIG. 4, and all of the 5 samples had moisturizing ability, wherein the moisturizing effect of example 1 performed optimally in all samples.
The scalp 5α -reductase inhibition test results of each example are shown in fig. 5. The 5. Alpha. -reductase inhibition ratio of the composition of example 1 was 79.01%, and the 5. Alpha. -reductase inhibition ratio of comparative example 4 was 68.34% (P < 0.001).
DPPH clearance experiment: as shown in FIG. 6, the composition of example 1 had a DPPH clearance of 93.7%, and the composition of comparative example 1 had a DPPH clearance of 80.67% (P < 0.01) and a DPPH clearance of 76.17% (P < 0.01) and a DPPH clearance of comparative example 3.
Hair strength and toughness test:
experimental hair tresses: HIGHLY DAMAGED CHINESE (Chinese severely damaged hair tresses, multiple bleaching), 18cmx 1g, shanghai lautu company; human hair wig piece 40cm x 25g, hangzhou Beiyufei trade company.
The equipment is used: XS (08) XT-3 carbon fiber tester, shanghai Xuesai instruments Co., ltd; HF5 constant temperature and humidity unit, available from Suzhou Co., ltd.
Environmental conditions: test results observations should be made in an environment with a temperature of 21.+ -. 1 ℃ and a relative humidity of 50.+ -. 10% RH, and visual evaluations should be made under constant illumination conditions (fluorescent tubes or LED illumination with a color temperature of 5500-6500K).
The experimental method comprises the following steps: the severely damaged hair tresses are selected and stored under the conditions of temperature (25+/-2) DEG C and humidity (50+/-5)% constant temperature and humidity. The hair bundles of the experimental group are smeared with quantitative products for three minutes and washed away by warm water, each warm water washing is performed for ten times by using a similar force line, and then the hair bundles are dried under the condition of constant temperature and constant humidity, thus the hair bundles are operated once. Each set of hair was treated five times with the product. The control group was untreated hair tresses. Tensile testing was performed after 12h storage at constant temperature and humidity. The diameter of the hairline is measured by using an SN-1200W high-definition camera, and the specific method is to average the diameters of 3 positions in the middle section. The filament strength test was performed by taking 30 hairs with a diameter difference within 10 μm from the hair bundle using a filament strength tester, and the tensile strength of the hairs of the control group and each sample group was calculated and compared by the following formula:
σ＝Fb/So
Where σ is the tensile strength, fb is the maximum force experienced by the specimen when it breaks, and So is the original cross-sectional area of the specimen. As shown in FIG. 7, after the products of each example 1 are used, the toughness of the hairline is increased by 22.13% compared with that of the control group, and the significant difference exists between P and 0.01, so that the composition has the effect of improving the toughness of the hairline.
Comparative example 5:
Preparing a component A: pearl powder: selecting pollution-free seawater pearl, removing dead pearl and impurities, cleaning pearl with clear water, filtering, drying at low temperature, pulverizing pearl into coarse powder with particle diameter of 10-15 μm by an ultrafine pulverizer, pulverizing into seawater pearl powder with average particle diameter of 2-3 μm by air flow, mixing the obtained powder with water according to a ratio of 1:4 (m/m) to prepare a pearl slurry, carrying out wet grinding to obtain pearl powder, and filtering and drying to obtain a component A: light purple pearl powder with slight fishy smell;
Preparing a component B: seawater pearl extract: adding the pearl powder into a lactic acid aqueous solution with the mass concentration of 4% for soaking for 2 hours, filtering, collecting filter residues, repeatedly washing with distilled water, carrying out wet grinding, drying, and carrying out secondary grinding by a high-speed grinder to obtain a component B: a light purple seawater pearl extract.
The pearl powder and the seawater pearl extract obtained in comparative example 5 are light purple and have slight fishy smell, the technical scheme of example 1 increases the specific surface area of seawater pearl particles, the prepared pearl powder is fine and porous, the color of the seawater pearl powder is obviously improved, the fishy smell of the seawater pearl is removed, and the pearl powder obtained in example 1 has no fishy smell.
Comparative example 6:
Component A of example 1 was replaced with taurine, and the other preparation methods were the same as in example 1, except that component A, B, C, D was used in a mass ratio of 0.02:10:10:5 are mixed uniformly and prepared into a solution according to table 1.
Comparative example 7:
the seawater pearl of example 1 was replaced with fresh water pearl, and the preparation method was the same as that of example 1. The components A, B, C, D are mixed according to the mass ratio of 1:10:10:5 are mixed uniformly and prepared into a solution according to table 1.
LPS induces Hacat cells to release inflammatory factors: incubating cells for 24 hours by using a sample to be tested with the volume fraction of HaCat cells of 0.5%o, then adding 1 mug/ml LPS to act cells for 6 hours, incubating for 24 hours, collecting cell culture supernatant, and respectively measuring the content of IL-6 inflammatory factors in the supernatant by using a kit. As shown in FIG. 8, the sample of example 1 has an inhibitory effect on LPS-induced inflammatory factor IL-6. The IL-6 secretion of example 1 was reduced by 34% (P < 0.001), the IL-6 secretion of control 6 was reduced by 21% (P < 0.01), and the IL-6 secretion of control 7 was reduced by 19% (P < 0.01) compared to the model group.
30 Volunteers enter a constant temperature and humidity evaluation room to clean the arm and sit still for 30min for testing, each person takes 0.2ml of sample to smear the left forearm for 2 x 2cm2 area, and the test is carried out after smearing for 1,2, 4 and 8 hours respectively. No adverse reactions were reported by 30 volunteers. The rate of change of the cutaneous percutaneous water loss indication is shown in figure 9.
Compared with fresh water pearl, the seawater pearl contains about 0.02% of taurine and other functional components, contains various proteins such as shell keratin, various amino acid active components such as tyrosine, arginine, aspartic acid, lysine, glycine, L-cysteine and the like, contains mineral macro-elements such as calcium carbonate, sodium, potassium, phosphorus and the like, and various microelements required by human bodies such as iron, zinc, iodine, selenium, germanium and the like, and the pearl powder, the seawater pearl extract, the hydrolyzed seawater pearl and the hydrolyzed fibroin are compounded.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (5)
1. The application of scalp/hair/skin repair composition with seawater pearl as raw material and functions of inhibiting 5 alpha-reductase, repairing damaged hair, regulating scalp sebaceous gland activity, resisting oxidation, resisting inflammation, moisturizing and repairing skin barrier in preparing cosmetics is characterized in that: the composition is prepared by the following steps:
preparing pearl powder: adding seawater pearl into a superfine pulverizer to pulverize to coarse powder with the particle size of 10-15 microns, then carrying out jet milling to obtain seawater pearl powder with the average particle size of 2-3 microns, putting the seawater pearl powder into a pressurizing device, adding water accounting for 20% of the weight of the powder, pressurizing to 0.5MP, maintaining for 4-5 hours, instantly decompressing, taking out, and mixing the obtained pearl powder with water according to the mass ratio of 1: 4-5, preparing a pearl slurry, carrying out wet grinding to obtain pearl powder with the particle size of 10-100 nm, and filtering and drying to obtain bright white pearl powder;
preparing seawater pearl extract: adding the pearl powder into a lactic acid aqueous solution with the mass concentration of 3-5% for soaking for 1-3 hours, filtering, collecting filter residues, repeatedly washing with distilled water, carrying out wet grinding, drying, and carrying out secondary grinding by a high-speed grinder to obtain a seawater pearl extract;
Preparing hydrolyzed seawater pearl: preparing a streptokinase solution with the pH value of 7.0-7.5 and 0.1-0.4 mg/mL, adding the pearl powder, carrying out enzymolysis for 1-3 hours at the temperature of 40-45 ℃ and the temperature of 0.1-0.2% of the mass of the pearl powder, heating to inactivate enzyme, adjusting the pH value to 5.5-6.5, and adding a complex enzyme with the mass of 0.1-0.2% of the mass of the pearl powder, wherein the complex enzyme is debitterized proteinase and bromelain, and the weight of the complex enzyme is 1-3: 1, carrying out enzymolysis for 2-3 hours at 50-55 ℃, heating to deactivate enzyme, decompressing, evaporating and concentrating, and freeze-drying to obtain hydrolyzed seawater pearl;
Preparing hydrolyzed fibroin: boiling silk in a sodium carbonate aqueous solution with the mass fraction of 1-2% for 30-40 min, removing sericin, filtering and drying, and mixing the silk after sericin removal with a 0.1 mol/L phosphoric acid aqueous solution with the pH of 6.5 according to the following g: ml is 1:20-1:40, adding 800-900U/g neutral protease, hydrolyzing at 45deg.C for 4 hr, filtering to adjust pH=7, evaporating under reduced pressure, concentrating, and freeze drying to obtain hydrolyzed fibroin;
pearl powder: seawater pearl extract: hydrolyzing seawater pearl: mixing the hydrolyzed fibroin according to the mass ratio of (0.01-2): 8-12): 4-6;
The cosmetic comprises one or more of shampoo, hair conditioner, scalp care solution, emulsion, facial cream, bath lotion, facial mask and facial cleanser.
2. The use according to claim 1, characterized in that: the seawater pearl extract is prepared by adding pearl powder into a lactic acid aqueous solution with the mass concentration of 4% for soaking for 2-3 hours, filtering, collecting filter residues, repeatedly flushing with distilled water, carrying out wet grinding, drying, and carrying out secondary grinding by a high-speed grinder to obtain the bright white seawater pearl extract.
3. Use according to claim 1 or 2, characterized in that: the seawater pearl extract is prepared by preparing 0.2mg/mL of streptogramin solution by using phosphate buffer solution with pH of 7.5, adding pearl powder, carrying out enzymolysis for 2-3 hours at 40 ℃ with streptogramin accounting for 0.16% of the mass of the pearl powder, heating to inactivate enzyme, regulating pH to 6, and adding complex enzyme accounting for 0.2% of the mass of the pearl powder, wherein the complex enzyme is debitterizing proteinase and bromelain according to the weight of 1-3: 1, carrying out enzymolysis for 3-4 hours at 55 ℃, heating to inactivate enzyme, decompressing, evaporating and concentrating, and freeze-drying to obtain the hydrolyzed seawater pearl.
4. Use according to claim 1 or 2, characterized in that: pearl powder: seawater pearl extract: hydrolyzing seawater pearl: the hydrolyzed fibroin is mixed according to the mass ratio of 0.02-1:10:10:5.
5. A use according to claim 3, characterized in that: the complex enzyme is debittering proteinase and bromelain according to 2: 1.
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