CN109096408A - A kind of plant source polysaccharide and the cosmetics containing the plant source polysaccharide - Google Patents
A kind of plant source polysaccharide and the cosmetics containing the plant source polysaccharide Download PDFInfo
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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
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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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
- A61K2800/10—General cosmetic use
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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
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
Abstract
The invention discloses a kind of plant source polysaccharide and the cosmetics containing the plant source polysaccharide.Known positive control arbutin is widely used in skin-lightening cosmetic, it is derived from the natural active matter of green plants, collection " green plants, safe and reliable " and " efficient decolorizing " three, which close, humorous is unified in integrated skin bleaching component, it can rapidly permeate into skin, effectively inhibit the activity of the tyrosinase in skin, block the formation of melanin, accelerate the decomposition and excretion of melanin, it is current popular the most safely and effectively whitening raw materials to reducing Skin pigmentation, dispelling stain and freckle.The study found that enzymolysis polypeptide 1 provided by the invention, enzymolysis polypeptide 2, enzymolysis polypeptide 4 and purified polysaccharide 1, purified polysaccharide 3, purified polysaccharide 4 have extremely wide application prospect with tyrosinase inhibitory activity more superior than arbutin in skin-lightening cosmetic.
Description
Technical field
The invention belongs to polypeptides and polysaccharide chemistry field, are related to plant source polypeptide, plant source polysaccharide and in cosmetics
Using.
Background technique
The color of skin is related with the type and quantity of pigment present in skin, and melanocyte influences skin color maximum.
Melanocyte is formed in the melanocyte in epidermal basal portion.Its process is the tyrosine in melanocyte under the action of tyrosinase
Generate DOPA, DOPA quinone ultimately forms melanocyte.Melanocyte is transferred in basal cell, as migrating for epidermal cell is brought to epidermis
Holostrome, the finally depigmentation with falling off for keratinocyte.Tyrosinase is the key enzyme of melanogenesis, controls the formation of melanocyte
Journey, level of activity plays a major role to the deposition of pigment, and (Qiu Ping Yi, cosmetic chemistry and technology are complete works of, Chinese light industry
Industry publishing house, 1997:884-888).Many whitenings, nti-freckle product are all to inhibit tyrosinase to reach whitening function in the market.
Natural products refers to animal, plant extracts (referred to as plant and mention) or insect, marine organisms and intracorporal group of microorganism
At ingredient or its metabolite, wherein mainly including protein, polypeptide, amino acid, nucleic acid, various enzymes, monosaccharide, oligosaccharides, more
Sugar, glycoprotein, resin, colloid substances, lignin, vitamin, fat, grease, wax, alkaloid, volatile oil, flavones, glycoside, terpene
Class, Phenylpropanoid Glycosides class, organic acid, phenols, quinones, lactone, steroidal compounds, tannins, antibiotics etc..The study found that including
The chemical component of these naturally occurring including polypeptide and polysaccharide is smaller to the side effect of human body.
The tyrosinase inhibitor screened from the native chemicals ingredient such as polypeptide and polysaccharide can be not only used for preparing
Skin-lightening cosmetic, whitening effect is good, and highly-safe, is always the research hotspot of major research institution chased.
Summary of the invention
The purpose of the present invention is to provide a kind of plant source polypeptide, plant source polysaccharide and the applications in cosmetics.
Object of the present invention is to what is be achieved through the following technical solutions:
Enzymolysis polypeptide 1:
A kind of polypeptide is made by the steps to obtain:
Step S1, enzymatic hydrolysis:
Defatted wheat germ is ground into germ flour, 10g germ flour is taken to be placed in 50mL ultrapure water, papain is added
With flavor protease compounding than being that the compound protease of 2:1 is digested, the additive amount of compound protease is substrate germ flour weight
The 0.38% of amount, enzymatic hydrolysis condition are as follows: pH value, 7.0；Hydrolysis temperature, 42 DEG C；Enzymolysis time, 10 hours；
After enzymatic hydrolysis, boiling water bath makes compound protein enzyme-deactivating, is cooled to room temperature；
After cooling, 10000rpm is centrifuged 20 minutes, is collected supernatant, is freeze-dried to obtain wheat germ polypeptide freeze-dried powder；
Step S2, purifying:
Freeze-dried powder is dissolved with ultrapure water, then successively with the ultrafiltration membrane that molecular cut off is 10000u, 7000u to gained
To enzymolysis polypeptide separated, molecular weight obtained by ultrafiltration is freeze-dried between the component of 10000u~7000u to obtain the final product.
Preferably, papain enzyme activity is 50 × 104U/g。
Preferably, flavor protease enzyme activity is 2 × 104U/g。
Preferably, 1g freeze-dried powder adds 10mL ultrapure water in step S2.
Preferably, ablation method is boiling water bath 10 minutes.
Preferably, ultrafiltration obtained component is freeze-dried to water content≤5%.
Aforementioned polypeptides are used as the purposes of tyrosinase inhibitor.
A kind of skin whitener contains above-mentioned polypeptide.
A kind of whitening water, contains above-mentioned polypeptide.
A kind of whitening mask contains above-mentioned polypeptide.
Enzymolysis polypeptide 2:
A kind of polypeptide is made by the steps to obtain:
Step S1, enzymatic hydrolysis:
Defatted wheat germ is ground into germ flour, 10g germ flour is taken to be placed in 50mL ultrapure water, papain is added
With flavor protease compounding than being that the compound protease of 2:1 is digested, the additive amount of compound protease is substrate germ flour weight
The 0.38% of amount, enzymatic hydrolysis condition are as follows: pH value, 7.0；Hydrolysis temperature, 42 DEG C；Enzymolysis time, 10 hours；
After enzymatic hydrolysis, boiling water bath makes compound protein enzyme-deactivating, is cooled to room temperature；
After cooling, 10000rpm is centrifuged 20 minutes, is collected supernatant, is freeze-dried to obtain wheat germ polypeptide freeze-dried powder；
Step S2, purifying:
Freeze-dried powder is dissolved with ultrapure water, then successively with the ultrafiltration membrane that molecular cut off is 7000u, 5000u to gained
To enzymolysis polypeptide separated, molecular weight obtained by ultrafiltration is freeze-dried between the component of 7000u~5000u to obtain the final product.
Preferably, papain enzyme activity is 50 × 104U/g。
Preferably, flavor protease enzyme activity is 2 × 104U/g。
Preferably, 1g freeze-dried powder adds 10mL ultrapure water in step S2.
Preferably, ablation method is boiling water bath 10 minutes.
Preferably, ultrafiltration obtained component is freeze-dried to water content≤5%.
Aforementioned polypeptides are used as the purposes of tyrosinase inhibitor.
A kind of skin whitener contains above-mentioned polypeptide.
A kind of whitening water, contains above-mentioned polypeptide.
A kind of whitening mask contains above-mentioned polypeptide.
Enzymolysis polypeptide 4:
A kind of polypeptide is made by the steps to obtain:
Step S1, enzymatic hydrolysis:
Defatted wheat germ is ground into germ flour, 10g germ flour is taken to be placed in 50mL ultrapure water, papain is added
With flavor protease compounding than being that the compound protease of 2:1 is digested, the additive amount of compound protease is substrate germ flour weight
The 0.38% of amount, enzymatic hydrolysis condition are as follows: pH value, 7.0；Hydrolysis temperature, 42 DEG C；Enzymolysis time, 10 hours；
After enzymatic hydrolysis, boiling water bath makes compound protein enzyme-deactivating, is cooled to room temperature；
After cooling, 10000rpm is centrifuged 20 minutes, is collected supernatant, is freeze-dried to obtain wheat germ polypeptide freeze-dried powder；
Step S2, purifying:
Freeze-dried powder is dissolved with ultrapure water, then successively with the ultrafiltration membrane that molecular cut off is 3000u, 1000u to gained
To enzymolysis polypeptide separated, molecular weight obtained by ultrafiltration is freeze-dried between the component of 3000u~1000u to obtain the final product.
Preferably, papain enzyme activity is 50 × 104U/g。
Preferably, flavor protease enzyme activity is 2 × 104U/g。
Preferably, 1g freeze-dried powder adds 10mL ultrapure water in step S2.
Preferably, ablation method is boiling water bath 10 minutes.
Preferably, ultrafiltration obtained component is freeze-dried to water content≤5%.
Aforementioned polypeptides are used as the purposes of tyrosinase inhibitor.
A kind of skin whitener contains above-mentioned polypeptide.
A kind of whitening water, contains above-mentioned polypeptide.
A kind of whitening mask contains above-mentioned polypeptide.
Purified polysaccharide 1:
A kind of polysaccharide is made by the steps to obtain:
Step S1, the extraction of Thick many candies:
Defatted wheat germ is ground into germ flour, with 90 DEG C of extraction temperature, extraction time 3h, extracting times 3 times for work
The water extraction of skill parameter extracts；Extracting solution centrifugation, collects supernatant, 1/5 volume is concentrated into, with Sevag method removing protein, 3000r/
Min is centrifuged 10min and removes intermediate organic phase and water phase intersection emulsion layer, merges supernatant, the dehydrated alcohol of 4 times of volumes is added
Precipitate polysaccharides are stood, and precipitating is collected by centrifugation in 3000r/min centrifugation 10min, and the sediment of collection freezes dry after being redissolved with distilled water
It is dry to obtain wheat germ Thick many candies freeze-dried powder；
Step S2, the purifying of Thick many candies:
The above-mentioned wheat germ Thick many candies freeze-dried powder of 100mg is weighed to be completely dissolved in 10mL sterile water, the Thick many candies prepared are molten
Loading is all added dropwise to DEAE-Sepharose Fast Flow (3.6cm × 20cm) cellulose anion displacement chromatography column in liquid
In, 8 column volumes are successively eluted respectively with the NaCl solution of 0,0.1mol/L, collect the NaCl solution eluent of 0.1mol/L,
Freeze-drying；It weighs 0.1mol/LNaCl solution eluent freeze-dried powder 20mg and is dissolved in 4mL ultrapure water, loading to SephacrylS-
It in 200HR gel column (1.6cm × 70cm), distills water elution, collects the 3-4 column volume eluent, be freeze-dried to obtain the final product.
Preferably, the solid-to-liquid ratio that step S1 is extracted is 1:8.
Preferably, extracting solution centrifugal condition is that 5000r/min is centrifuged 10min.
Preferably, step S1 static conditions are stood for 24 hours under the conditions of being 4 DEG C.
Preferably, anion exchange chromatography elution flow rate is 1mL/min.
Preferably, gel column elution flow rate is 0.4mL/min.
Above-mentioned polysaccharide is used as the purposes of tyrosinase inhibitor.
A kind of skin whitener contains above-mentioned polysaccharide.
A kind of whitening water, contains above-mentioned polysaccharide.
A kind of whitening mask contains above-mentioned polysaccharide.
Purified polysaccharide 3:
A kind of polysaccharide is made by the steps to obtain:
Step S1, the extraction of Thick many candies:
Defatted wheat germ is ground into germ flour, with 90 DEG C of extraction temperature, extraction time 3h, extracting times 3 times for work
The water extraction of skill parameter extracts；Extracting solution centrifugation, collects supernatant, 1/5 volume is concentrated into, with Sevag method removing protein, 3000r/
Min is centrifuged 10min and removes intermediate organic phase and water phase intersection emulsion layer, merges supernatant, the dehydrated alcohol of 4 times of volumes is added
Precipitate polysaccharides are stood, and precipitating is collected by centrifugation in 3000r/min centrifugation 10min, and the sediment of collection freezes dry after being redissolved with distilled water
It is dry to obtain wheat germ Thick many candies freeze-dried powder；
Step S2, the purifying of Thick many candies:
The above-mentioned wheat germ Thick many candies freeze-dried powder of 100mg is weighed to be completely dissolved in 10mL sterile water, the Thick many candies prepared are molten
Loading is all added dropwise to DEAE-Sepharose Fast Flow (3.6cm × 20cm) cellulose anion displacement chromatography column in liquid
In, 8 column volumes are successively eluted respectively with 0,0.1,0.2,0.3mol/L NaCl solution, collect the NaCl solution of 0.3mol/L
Eluent, freeze-drying；
It weighs 0.3mol/LNaCl solution eluent freeze-dried powder 20mg and is dissolved in 4mL ultrapure water, loading to SephacrylS-
It in 200HR gel column (1.6cm × 70cm), distills water elution, collects the 5-6 column volume eluent, be freeze-dried to obtain the final product.
Preferably, the solid-to-liquid ratio that step S1 is extracted is 1:8.
Preferably, extracting solution centrifugal condition is that 5000r/min is centrifuged 10min.
Preferably, step S1 static conditions are stood for 24 hours under the conditions of being 4 DEG C.
Preferably, anion exchange chromatography elution flow rate is 1mL/min.
Preferably, gel column elution flow rate is 0.4mL/min.
Above-mentioned polysaccharide is used as the purposes of tyrosinase inhibitor.
A kind of skin whitener contains above-mentioned polysaccharide.
A kind of whitening water, contains above-mentioned polysaccharide.
A kind of whitening mask contains above-mentioned polysaccharide.
Purified polysaccharide 4:
A kind of polysaccharide is made by the steps to obtain:
Step S1, the extraction of Thick many candies:
Defatted wheat germ is ground into germ flour, with 90 DEG C of extraction temperature, extraction time 3h, extracting times 3 times for work
The water extraction of skill parameter extracts；Extracting solution centrifugation, collects supernatant, 1/5 volume is concentrated into, with Sevag method removing protein, 3000r/
Min is centrifuged 10min and removes intermediate organic phase and water phase intersection emulsion layer, merges supernatant, the dehydrated alcohol of 4 times of volumes is added
Precipitate polysaccharides are stood, and precipitating is collected by centrifugation in 3000r/min centrifugation 10min, and the sediment of collection freezes dry after being redissolved with distilled water
It is dry to obtain wheat germ Thick many candies freeze-dried powder；
Step S2, the purifying of Thick many candies:
The above-mentioned wheat germ Thick many candies freeze-dried powder of 100mg is accurately weighed to be completely dissolved in 10mL sterile water, it is thick more by what is prepared
Loading is all added dropwise to DEAE-Sepharose Fast Flow (3.6cm × 20cm) cellulose anion displacement chromatography in sugar juice
In column, 8 column volumes are successively eluted respectively with 0,0.1,0.2,0.3,0.5mol/L NaCl solution, collect 0.5mol/L's
NaCl solution eluent, freeze-drying；
It weighs 0.5mol/LNaCl solution eluent freeze-dried powder 20mg and is dissolved in 4mL ultrapure water, loading to SephacrylS-
It in 200HR gel column (1.6cm × 70cm), distills water elution, collects the 4-5 column volume eluent, be freeze-dried to obtain the final product.
Preferably, the solid-to-liquid ratio that step S1 is extracted is 1:8.
Preferably, extracting solution centrifugal condition is that 5000r/min is centrifuged 10min.
Preferably, step S1 static conditions are stood for 24 hours under the conditions of being 4 DEG C.
Preferably, anion exchange chromatography elution flow rate is 1mL/min.
Preferably, gel column elution flow rate is 0.4mL/min.
Above-mentioned polysaccharide is used as the purposes of tyrosinase inhibitor.
A kind of skin whitener contains above-mentioned polysaccharide.
A kind of whitening water, contains above-mentioned polysaccharide.
A kind of whitening mask contains above-mentioned polysaccharide.
The utility model has the advantages that
Known positive control arbutin is widely used in skin-lightening cosmetic, is derived from the natural active matter of green plants,
Collection " green plants, safe and reliable " and " efficient decolorizing " three close it is humorous be unified in integrated skin bleaching component, it can be seeped rapidly
Enter skin, effectively inhibit the activity of the tyrosinase in skin, block the formation of melanin, accelerates decomposition and the row of melanin
It lets out, to reduce Skin pigmentation, dispelling stain and freckle are current popular the most safely and effectively whitening raw materials.It grinds
Study carefully discovery, enzymolysis polypeptide 1, enzymolysis polypeptide 2, enzymolysis polypeptide 4 and purified polysaccharide 1 provided by the invention, purified polysaccharide 3, purifying are more
Sugar 4 has tyrosinase inhibitory activity more superior than arbutin, has extremely wide application prospect in skin-lightening cosmetic.
Detailed description of the invention
Fig. 1 is IC50 value of each sample to tyrosinase inhibitory action.
Specific embodiment
It is specific with reference to the accompanying drawings and examples to introduce essentiality content of the present invention, but guarantor of the invention is not limited with this
Protect range.
Embodiment 1: the preparation of wheat germ polypeptide
One, experimental material
Defatted wheat germ is purchased from Shandong Guangming Industrial Co., Ltd..
Papain (50 × 104U/g), flavor protease (2 × 104U/g) it is purchased from Sigma Co., USA.
Two, experimental method
1, enzyme solution
Defatted wheat germ is ground into germ flour, 10g germ flour is taken to be placed in 50mL ultrapure water, papain is added
It is digested with flavor protease compounding than the compound protease that (weight ratio) is 2:1, the additive amount of compound protease is substrate
The 0.38% of germ flour weight, enzymatic hydrolysis condition are as follows: pH value, 7.0；Hydrolysis temperature, 42 DEG C；Enzymolysis time, 10 hours；
After enzymatic hydrolysis, makes compound protein enzyme-deactivating within boiling water bath 10 minutes, be cooled to room temperature；
After cooling, 10000rpm is centrifuged 20 minutes, is collected supernatant, is freeze-dried to obtain wheat germ polypeptide freeze-dried powder.
2, purification process
Freeze-dried powder ultrapure water is dissolved into (1g freeze-dried powder adds 10mL ultrapure water), is then successively with molecular cut off
The ultrafiltration membrane of 10000u, 7000u, 5000u, 3000u and 1000u separate obtained enzymolysis polypeptide, by ultrafiltration institute
Component freeze-drying (water content≤5%) is obtained up to the enzymolysis polypeptide of different molecular weight size.
CO is controlled in ultra-filtration process2Pressure make its be less than 0.25MPa.
Three, experimental result
Collection obtains the enzymolysis polypeptide of following molecular weight ranges:
Enzymolysis polypeptide 1, molecular weight is between 10000u and 7000u；
Enzymolysis polypeptide 2, molecular weight is between 7000u and 5000u；
Enzymolysis polypeptide 3, molecular weight is between 5000u and 3000u；
Enzymolysis polypeptide 4, molecular weight is between 3000u and 1000u.
Embodiment 2: the preparation of wheat germ polysaccharide
One, experimental material
Defatted wheat germ is purchased from Shandong Guangming Industrial Co., Ltd..
Two, experimental method and result
1, the extraction of Thick many candies
Defatted wheat germ is ground into germ flour, with solid-to-liquid ratio 1:8,90 DEG C of extraction temperature, extraction time 3h, extraction time
The number 3 times water extractions for technological parameter extract；Extracting solution 5000r/min is centrifuged 10min, collects supernatant, is concentrated into 1/5 body
Product, with Sevag method removing protein, 3000r/min is centrifuged 10min and removes intermediate organic phase and water phase intersection emulsion layer, in merging
Clear liquid is added the dehydrated alcohol precipitate polysaccharides of 4 times of volumes, stands for 24 hours under the conditions of 4 DEG C, and 3000r/min is centrifuged 10min centrifugation and receives
Collection precipitating, the sediment of collection are freeze-dried to obtain wheat germ Thick many candies freeze-dried powder after being redissolved with distilled water.
2, the purifying of Thick many candies
The above-mentioned wheat germ Thick many candies freeze-dried powder of 100mg is accurately weighed to be completely dissolved in 10mL sterile water, it is thick more by what is prepared
Loading is all added dropwise to DEAE-Sepharose Fast Flow (3.6cm × 20cm) cellulose anion displacement chromatography in sugar juice
In column, 8 column volumes, flow velocity 1mL/min, difference are successively eluted respectively with 0,0.1,0.2,0.3,0.5mol/L NaCl solution
0.1,0.2,0.3,0.5mol/L NaCl solution eluent is collected, freeze-drying obtains different polysaccharide components.
0.1mol/LNaCl solution eluent freeze-dried powder 20mg is weighed respectively and is dissolved in 4mL ultrapure water, and loading is extremely
In SephacrylS-200HR gel column (1.6cm × 70cm), water elution is distilled, flow velocity 0.4mL/min collects the 3-4 column
Volume eluent, freeze-drying obtain purified polysaccharide 1；
0.2mol/LNaCl solution eluent freeze-dried powder 20mg is weighed respectively and is dissolved in 4mL ultrapure water, and loading is extremely
In SephacrylS-200HR gel column (1.6cm × 70cm), water elution is distilled, flow velocity 0.4mL/min collects the 2-3 column
Volume eluent, freeze-drying obtain purified polysaccharide 2；
0.3mol/LNaCl solution eluent freeze-dried powder 20mg is weighed respectively and is dissolved in 4mL ultrapure water, and loading is extremely
In SephacrylS-200HR gel column (1.6cm × 70cm), water elution is distilled, flow velocity 0.4mL/min collects the 5-6 column
Volume eluent, freeze-drying obtain purified polysaccharide 3；
0.5mol/LNaCl solution eluent freeze-dried powder 20mg is weighed respectively and is dissolved in 4mL ultrapure water, and loading is extremely
In SephacrylS-200HR gel column (1.6cm × 70cm), water elution is distilled, flow velocity 0.4mL/min collects the 4-5 column
Volume eluent, freeze-drying obtain purified polysaccharide 4.
Embodiment 3: the inhibiting effect of wheat germ polypeptide and wheat germ polysaccharide to tyrosinase
One, experimental material
L-tyrosine (Sigma company)；Arbutin (Shanghai source leaf biology)；95% ethyl alcohol, dimethyl sulfoxide (DMSO), phosphorus
Acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, concentrated hydrochloric acid are that commercially available analysis is pure；Experimental water is deionized water.
Fresh potato is purchased from Nanjing China Resources Su Guo supermarket.
The enzymolysis polypeptide 1-4 of wheat germ is prepared according to 1 method of embodiment.
The purified polysaccharide 1-4 of wheat germ is prepared according to 2 method of embodiment.
Two, experimental method
1, preparation of reagents
Sodium phosphate buffer (1/15M, pH=6.8): 1.0002g sodium dihydrogen phosphate, 1.1865g phosphoric acid hydrogen two are accurately weighed
Sodium after a small amount of deionized water dissolving is added, is settled to 500mL, and 4 DEG C of refrigerators save backup.
L-tyrosine solution (7.5mmol/L): l-tyrosine 0.2725g accurately is weighed, few drops of concentrated hydrochloric acids are first added, add
Ionized water about 50mL after low-grade fever is completely dissolved, with sodium hydroxide solution tune pH to 7.0, adds deionized water to be settled to 200mL.
By test solution: accurately weighing enzymolysis polypeptide 1-4, each 0.1g of purified polysaccharide 1-4 of wheat germ, be dissolved in 20mL deionization respectively
Water, obtains the prepare liquid of 5mg/mL, then two-fold dilution is to 2.5,1.25,0.625,0.3125mg/mL and 0.15625mg/mL.
Positive control: 0.1g arbutin accurately is weighed, is dissolved in the deionized water of 20mL, obtains the positive control of 5mg/mL
Mother liquor, then two-fold dilution is to 2.5,1.2500,0.6250,0.3125mg/mL and 0.15625mg/mL.
2, the preparation of tyrosine enzyme solution
Tyrosine enzyme solution, concrete operations are produced with fresh intact potato are as follows:
Potato is cleaned, in 4 DEG C of pre-cooling 4h or so.Peeling, is cut into about 1 × 1 × 1cm3Ding shape was freezed in -20 DEG C
Night.The sodium phosphate buffer of 4 DEG C of pre-coolings is added in the ratio of 1:1 (w:v), homogenate, 3 layers of yarn are made with tissue mashing machine for weighing
Cloth filtering, filtrate are centrifuged 10min in 4000r/min, and supernatant is tyrosinase crude enzyme liquid, and 4 DEG C save, and are finished in 2h.
3, inhibition of the sample to tyrosinase activity
Overall reaction system is 5mL, design such as table 1.When wherein, with spectrophotometer measurement light absorption value, " by test solution ", " mark
Quasi- control ", " positive control " are respectively with " negative control 1 ", " negative control 2 " and " negative control 3 " zeroing.
The design of 1 test system of table
In this system, by test solution (including positive control arbutin) final concentration (mg/mL) gradient be 0.015625,
0.03125,0.0625,0.125,0.25 and 0.5.
When experiment, sequentially added into test tube phosphate buffer, various concentration gradient it is (including positive right by test solution
According to) and enzyme solution, in 30 DEG C of water-bath 10min.Then substrate l-tyrosine is added, immediately begins to timing.When measurement reaction 20min
Light absorption value under 475nm wavelength.Using corresponding negative control as reference when measurement, calculated with following equation by test solution (including sun
Property control) to the inhibiting rate of tyrosinase, and according to concentration-enzyme inhibition rate curve estimation half-inhibitory concentration (IC50) approximation
Value.
Inhibiting rate (%)=(A-B) ÷ A × 100%
Wherein, " A " is the light absorption value of standard control, and " B " is the light absorption value by test solution (or positive control).Each experiment is done
3 parallel.Inhibiting rate height shows that it is high to the inhibition strength of tyrosinase activity.
Three, experimental result
Positive control and enzymolysis polypeptide 1-4, purified polysaccharide 1-4 are shown in Table 2 and figure to the IC50 value of tyrosinase inhibitory action
1。
IC50 value of the table 2 to tyrosinase inhibitory action
Known positive control arbutin is widely used in skin-lightening cosmetic, is derived from the natural active matter of green plants,
Collection " green plants, safe and reliable " and " efficient decolorizing " three close it is humorous be unified in integrated skin bleaching component, it can be seeped rapidly
Enter skin, without affecting the cell proliferation concentration, the activity of the tyrosinase in skin can be effectively inhibited, block black
The formation of element, is directly binded to tyrosinase by itself, accelerates the decomposition and excretion of melanin, so that it is heavy to reduce skin pigment
Product, dispelling stain and freckle, and do not generate the side effects such as toxic, irritation, sensitization to melanocyte, while also
It sterilizes, the effect of anti-inflammatory.It is the current popular the most safely and effectively desired skin of whitening raw materials and 21st century
Whitening active agent.The study found that enzymolysis polypeptide 1, enzymolysis polypeptide 2, enzymolysis polypeptide 4 and embodiment 2 that embodiment 1 provides mention
Purified polysaccharide 1, purified polysaccharide 3, the purified polysaccharide 4 of confession have tyrosinase inhibitory activity more superior than arbutin, in whitening
There is extremely wide application prospect in cosmetics.
A kind of embodiment 4: skin whitener
Be made of the ingredient of following weight percentage: enzymolysis polypeptide 1, enzymolysis polypeptide 2, the enzymatic hydrolysis of the preparation of embodiment 1 are more
Purified polysaccharide 1, purified polysaccharide 3, purified polysaccharide 4 prepared by peptide 4 or embodiment 2,0.1%；Saualane, 10%；Propylene glycol, 3%；
Sodium stearyl sulfate, 4%；Laruyl alcohol PCA ester, 3%；Hyaluronic acid, 4%；Cocounut oil, 7%；Surplus is water.
A kind of embodiment 5: whitening water
It is made of the ingredient of following parts by weight: enzymolysis polypeptide 1, enzymolysis polypeptide 2, the enzymolysis polypeptide 4 or real of the preparation of embodiment 1
Apply purified polysaccharide 1, the purified polysaccharide 3,4,0.5 part of purified polysaccharide of the preparation of example 2；Glycerol, 20 parts；Triethanolamine, 5 parts；Sodium lactate,
5 parts；5 parts of hydroxymethyl cellulose；75 parts of deionized water.
A kind of embodiment 6: whitening mask
It is made of the ingredient of following weight percentage: peony seed oil, 10%；Fat soluble vitamin E, 3%；Emulsifier,
5%；Organic Bulgarian Rosa rugosa hydrosol, 5%；Enzymolysis polypeptide 1, enzymolysis polypeptide 2, enzymolysis polypeptide 4 or implementation prepared by embodiment 1
Purified polysaccharide 1, the purified polysaccharide 3, purified polysaccharide 4 of the preparation of example 2,0.8%；Glycerol, 10%；Low molecule sodium hyaluronate, 3%；Aloe
Glue, 12%；Antibacterial agent, 0.2%, surplus is deionized water.
The effect of above-described embodiment is specifically to introduce essentiality content of the invention, but those skilled in the art should know
Protection scope of the present invention should not be confined to the specific embodiment by road.
Claims (10)
1. a kind of polysaccharide, which is characterized in that be made by the steps to obtain:
Step S1, the extraction of Thick many candies:
Defatted wheat germ is ground into germ flour, is joined with 90 DEG C of extraction temperature, extraction time 3h, extracting times 3 times for technique
Several water extractions extracts；Extracting solution centrifugation, collects supernatant, 1/5 volume is concentrated into, with Sevag method removing protein, 3000r/min
It is centrifuged 10min and removes intermediate organic phase and water phase intersection emulsion layer, merge supernatant, the dehydrated alcohol that 4 times of volumes are added is heavy
Shallow lake polysaccharide is stood, and precipitating is collected by centrifugation in 3000r/min centrifugation 10min, and the sediment of collection is freeze-dried after being redissolved with distilled water
Obtain wheat germ Thick many candies freeze-dried powder；
Step S2, the purifying of Thick many candies:
The above-mentioned wheat germ Thick many candies freeze-dried powder of 100mg is accurately weighed to be completely dissolved in 10mL sterile water, the Thick many candies prepared are molten
Loading is all added dropwise to DEAE-Sepharose Fast Flow (3.6cm × 20cm) cellulose anion displacement chromatography column in liquid
In, 8 column volumes are successively eluted respectively with 0,0.1,0.2,0.3,0.5mol/L NaCl solution, collect the NaCl of 0.5mol/L
Solution eluent, freeze-drying；
It weighs 0.5mol/L NaCl solution eluent freeze-dried powder 20mg and is dissolved in 4mL ultrapure water, loading to SephacrylS-200
It in HR gel column (1.6cm × 70cm), distills water elution, collects the 4-5 column volume eluent, be freeze-dried to obtain the final product.
2. polysaccharide according to claim 1, it is characterised in that: the solid-to-liquid ratio that step S1 is extracted is 1:8.
3. polysaccharide according to claim 1, it is characterised in that: extracting solution centrifugal condition is that 5000r/min is centrifuged 10min.
4. polysaccharide according to claim 1, it is characterised in that: step S1 static conditions are stood for 24 hours under the conditions of being 4 DEG C.
5. polysaccharide according to claim 1, it is characterised in that: anion exchange chromatography elution flow rate is 1mL/min.
6. polysaccharide according to claim 1, it is characterised in that: gel column elution flow rate is 0.4mL/min.
7. the purposes that any polysaccharide of claim 1-6 is used as tyrosinase inhibitor.
8. a kind of skin whitener, it is characterised in that: contain any polysaccharide of claim 1-6.
9. a kind of whitening water, it is characterised in that: contain any polysaccharide of claim 1-6.
10. a kind of whitening mask, it is characterised in that: contain any polysaccharide of claim 1-6.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713552A (en) * | 2019-10-26 | 2020-01-21 | 陈敏 | Ginger-flavored grass polysaccharide and application thereof in preparing whitening and spot-lightening cosmetics |
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2018
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713552A (en) * | 2019-10-26 | 2020-01-21 | 陈敏 | Ginger-flavored grass polysaccharide and application thereof in preparing whitening and spot-lightening cosmetics |
| CN110713552B (en) * | 2019-10-26 | 2021-08-27 | 浙江宝韵生物科技有限公司 | Ginger-flavored grass polysaccharide and application thereof in preparing whitening and spot-lightening cosmetics |
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Application publication date: 20181228 |