CN111214385B - Nanoparticle emulsion loaded with skin nutrient and preparation method thereof - Google Patents
Nanoparticle emulsion loaded with skin nutrient and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a nanoparticle emulsion loaded with a skin nutrient and a preparation method thereof, and is characterized in that: comprises the following components in percentage by weight: 0.1% -50% of spidroin, 0.1% -10% of bis-hydroxyethyl trimethylol methylamine, 0.1% -40% of a skin nutrient and 0.1% -85% of a hydroalcoholic mixture, wherein the molecular weight of the spidroin is 80-100 kDa. The nanoparticle emulsion loaded with the skin nutrient has better biocompatibility, milder property and low irritation; compared with other spider silk protein nano-emulsions, the spider silk protein nano-emulsion has the advantages of higher encapsulation efficiency, better absorption and permeability and better stability.
Description
Technical Field
The invention relates to the technical field of nanoparticle emulsion preparation, in particular to nanoparticle emulsion loaded with a skin nutrient and a preparation method thereof.
Background
With the development of science and technology and the improvement of beauty consciousness of people, more and more beauty components are used in cosmetics, but many raw materials have the problems of difficult dissolution, easy color change and the like. The nanometer preparation technology is used as a new carrier to be used in cosmetics, but the existing nanometer emulsion mostly uses chemical surfactants and has certain influence on skin barriers.
Spider silk is a natural protein fiber, has mechanical properties such as high strength, high elasticity and high breaking energy, and has remarkable biological characteristics such as degradability and histocompatibility, and has great potential application value in the fields of biomedicine, materials, textile, military equipment and the like. The major component of spider silk is protein, so that it has good compatibility with biological tissue, and the molecular weight of spider silk protein is about 100-750 kDa. Spider dragline silk protein is fibrous protein with a highly repetitive sequence and is rich in alanine and glycine, an alanine enrichment area can form a hydrophobic beta-folded crystal structure, a hydrophilic glycine enrichment area forms an alpha-helix, and the spider dragline silk protein has a certain emulsifying capacity because the spider silk protein simultaneously has an amino end and a hydrophobic end of a hydrophilic end.
The technologies of recombination acquisition and the like for optimizing the mechanical property of the spider silk protein have been studied, but the report on how to optimize the emulsifying property and the emulsifying stability of the spider silk protein is rare. Chinese invention patent CN200680035440.4 discloses a method for producing nano-and microcapsules of spider silk protein, but the method uses solvents such as guanidine thiocyanate and toluene, which are not friendly to environment and skin. And the macromolecular spider silk protein is directly used for coating, so that the emulsifying power is poor, the spider silk protein is not acid-base resistant, and the produced nano capsule has poor stability and poor encapsulation efficiency and permeability.
Disclosure of Invention
The invention aims to solve the technical problem of providing a nanoparticle emulsion loaded with a skin nutrient, which has high encapsulation rate and good stability.
In order to solve the technical problems, the invention adopts the following technical scheme: a nanoparticle emulsion loaded with a skin nutrient is characterized in that: comprises the following components in percentage by weight: 0.1% -50% of spidroin protein, 0.1% -10% of bis-hydroxyethyl trimethylol methylamine, 0.1% -40% of skin nutrient and 0.1% -85% of hydroalcoholic mixture, wherein the molecular weight of the spidroin protein is 80-100 kDa.
The spider silk protein is prepared by the following method:
A. pretreatment of spider silks: weighing 100g of spider silk, placing the spider silk in 0.8% by weight of sodium carbonate solution, stirring at 80-85 ℃ for 20 minutes at 600 r/min, degumming for the first time, pouring out the solution after degumming, taking out the spider silk, repeatedly washing the spider silk with distilled water for 10 minutes, placing the spider silk in 0.8% by weight of sodium carbonate solution, stirring at 80-85 ℃ and 600 r/min for 20 minutes, degumming for the second time, placing the spider silk in a 90 ℃ oven, and drying to obtain spider silk fiber;
B. weighing 50g of the spider silk fibers obtained in the step A, placing the spider silk fibers in 30 wt% calcium chloride solution, stirring at the rotating speed of 600 r/min at the temperature of 80-85 ℃ until the spider silk fibers are completely dissolved to obtain spider silk protein calcium chloride mixed solution, filtering and crudely extracting the spider silk protein calcium chloride mixed solution by using gauze, placing the spider silk protein calcium chloride mixed solution into a 100-plus 750 kDa dialysis bag, dialyzing for 36 hours by using distilled water, and removing calcium chloride ions to obtain the spider silk protein solution;
C. enzymolysis of spider silk protein: and (3) taking a proper amount of the spider silk protein solution obtained in the step (B), adding subtilisin for enzymolysis, wherein the dosage of the subtilisin is 28000U/g, the enzymolysis time is 45-65 min, and the enzymolysis pH is as follows: 6.5-7.5, the enzymolysis temperature is 40-60 ℃, and enzymolysis is finished to obtain an enzymolysis mixed solution;
D. performing ultrafiltration purification by adopting a gel filtration chromatography method: c, dissolving 100mg of the enzymolysis mixed liquor obtained in the step C in 3ml of phosphate buffer solution, wherein the pH value of the phosphate buffer solution is 7.0, and the phosphate concentration is 10 mmol/L; then adding the mixture into a chromatographic column, eluting by taking a phosphate buffer solution as an eluent, collecting one tube every 2 min at the flow rate of the eluent of 1 ml/min; and (3) detecting the absorbance of the eluent of the chromatographic column by connecting the chromatographic column with a liquid chromatography chromatographic instrument, and analyzing and processing by using a gel imaging system to obtain the spidroin with the molecular weight of 80-100 kDa.
The spider filaments in the step A can be drawing filaments, frame filaments, radius filaments and rivet filaments in major ampullate silk glands of Chilobrachys jingzhao spider, Aranea hainanensis and Aranea tiger veins. The invention preferably selects the traction wire of the tiger stripe bird-catching spider, and the commercial product is the traction wire of the tiger stripe bird-catching spider of Guangxi east culture Co.
The gel filtration chromatography system was purchased from Huamei corporation under the type Sephadex G-100.
Gel imaging systems are available from UVP corporation, usa under the model UV transillumiator.
The HPLC is available from Shimadzu corporation of Japan, model LC-2010C.
The subtilisin is purchased from Shanghai Xinyu biological technology limited, and the enzyme activity is more than or equal to 4000 units/mg.
A preparation method of nanoparticle emulsion loaded with a skin nutrient is characterized by comprising the following steps:
A. weighing 0.1-50% of spidroin protein, 0.1-10% of bis-hydroxyethyl trimethylol methylamine, 0.1-40% of skin nutrient and 0.1-50% of hydroalcoholic mixture according to the following mass ratio, wherein the molecular weight of the spidroin protein is 80-100 kDa;
B. heating the skin nutrient weighed in the step A to 70-80 ℃, and stirring at the rotating speed of 600-1200 r/min for 10-20 min to be uniform; the skin nutrient is a water-soluble skin nutrient or an oil-soluble skin nutrient or a mixture thereof;
C. mixing the spider silk protein, bis-hydroxyethyl trihydroxymethyl methylamine and a hydroalcoholic mixture weighed in the step A, heating to 70-80 ℃, and stirring at the rotating speed of 1000-1500 r/min for 10-20 min to be uniform to obtain a carrier solution;
D. adding the skin nutrient obtained in the step B into the carrier solution obtained in the step C, and homogenizing for 10-18 min at the rotating speed of 2000-3000 r/min by using a homogenizer under the environment of normal pressure and normal temperature; then circularly homogenizing for 5-8 times at the rotating speed of 2000-3000 r/min under the conditions of the pressure of 100-.
The bis-hydroxyethyl trimethylolmethylamine is a pH buffer having a CAS number of 6976-37-0 and is available from Suzhou Luosen chemical Co.
The skin nutrient is water soluble skin nutrient or oil soluble skin nutrient or their mixture, specifically resveratrol, tetrahydrocurcumin, astaxanthin, curcumin, quercetin, phloretin, aloesin, limonin, silymarin, lutein, riboflavin, baicalein, anthocyanidin, lycopene, phenethyl resorcinol, honokiol, paeonol, eugenol, hinokitiol, tea polyphenol, hesperidin, one or a mixture of naringin, astragalosides, asiaticoside, aescin, stevioside, vitamin E, bisabolol, cholesterol, soyasterol, furfuryl sterol, sphinganine, retinol, salicyloyl phytosphingosine, tetrahydromagnolol, magnolol, Lonicera fruit seed oil, Norway spruce leaf extract, white willow bark extract and purple coneflower extract in any ratio.
The water-alcohol mixture is a mixture of water and polyhydric alcohol, wherein the polyhydric alcohol can be one or a mixture of propylene glycol, glycerol, 1, 3-butanediol, dipropylene glycol, diglycerol, polyethylene glycol-8 and pentaerythritol, and the ratio of the water to the polyhydric alcohol is 0.5-3: 1.
according to the nanoparticle emulsion loaded with the skin nutrient, the cobweb protein is treated by using subtilisin, and is purified by using a gel filtration chromatography to obtain the cobweb protein with moderate molecular weight, so that the formation of the nanoparticle emulsion with smaller and uniform particle size is facilitated, a more compact interface membrane is facilitated, the emulsion stability is increased, the specific surface area is increased after the molecular weight of the cobweb protein is reduced, the encapsulation rate is increased, the particle size of the formed nanoparticles is smaller, and the permeability is increased; according to the invention, bis-hydroxyethyl trihydroxymethyl methylamine is added as an acid-base buffering agent, so that excessive sensitivity of spidroin to pH is avoided; and no organic solvent is added in the production process, so that the production method is more environment-friendly.
In order to verify various properties of the nanoparticle emulsion loaded with the skin nutrient, the invention was tested as follows.
Encapsulation assay
Measuring the encapsulation rate of the nanoparticle emulsion in example 1 by adopting an ultrafiltration centrifugation method, putting 500 uL of the nanoparticle emulsion in example 1 into a 10ml volumetric flask, adding a proper amount of absolute ethyl alcohol for dissolving and dispersing, and then performing ultrasonic demulsification; continuously using absolute ethyl alcohol to perform constant volume, diluting the absolute ethyl alcohol solution of the nanoparticle emulsion after constant volume by 10 times, measuring a spectrophotometer value through an ultraviolet spectrophotometer, and calculating the total content of the skin nutrient in the sample solution according to the standard curve of the skin nutrient resveratrol in the embodiment 1: wTotal。
Taking 1ml of the nanoparticle emulsion in the embodiment 1, diluting the nanoparticle emulsion by 10 times with ultrapure water, taking about 300 uL of the diluted nanoparticle emulsion dispersion liquid into an ultrafiltration centrifugal tube, and carrying out refrigerated centrifugation for 40 min at the rotating speed of 10000 rpm to obtain a clarified centrifugate; precisely absorbing the centrifugate, adding absolute ethyl alcohol to dissolve to a constant volume, shaking up to obtain a centrifugate sample, measuring by an ultraviolet spectrophotometer to obtain an absorbance value, and calculating the content of the skin nutrient in the diluted nanoparticle carrier aqueous dispersion according to a standard curve equation: wFree。
The envelope rate = [ 1- (W) is obtained through calculationFree/WTotal）］*100%
Table 1 encapsulation efficiency comparison of nanoparticle emulsions
Sample a is the nanoparticle emulsion prepared in example 1; sample B was a nanoparticle emulsion coated with the same skin benefit agent as in example 1, according to the method of patent No. CN 200680035440.4.
Experiments show that the nanoparticle emulsion loaded with the skin nutrient has better encapsulation efficiency.
Permeability determination experiment
The absorption and permeation effects of the nanoparticle emulsion of example 1 and other spidroin nanoparticle emulsions were examined using the Franz diffusion cell method.
Fixing artificial skin between the drug supply pool and the receiving pool of Franz diffusion pool, with stratum corneum facing the drug supply pool and effective permeation area of 4.15 cm2The volume of the receiving pool is 30 ml, one side of the dermis of the skin is contacted with receiving liquid, air bubbles are discharged, the receiving liquid is an aqueous solution of nanoparticle emulsion of 30 percent ethanol, the water bath circulation outside the Franz diffusion pool is (37.0 +/-0.5) DEG C, and 350 r.min in the receiving pool-1Stirring at a constant speed.
After the temperature of the system was constant, 2.0 ml of the above test solutions were gently poured into the respective reservoirs. Stirring at constant speed and timing was started. 1.0 ml of receiving liquid is extracted from the sampling tube for 1, 2, 3, 4, 6, 8, 12, 18 and 24 hours respectively, after each sampling, the equal volume of the isothermal aqueous solution of the nanoparticle emulsion of 30% ethanol is timely supplemented, and air bubbles in the receiving pool are removed. And (3) placing the collected receiving liquid sample in a glass test tube with a plug, storing the sample at low temperature in a sealed and dark manner, and completing the measurement within 24 h. And (3) filtering the sample liquid by a microporous filter membrane with the pore diameter of 0.45 micrometer, and then performing High Performance Liquid Chromatography (HPLC) determination analysis. Samples with peak areas above the working curve were measured after appropriate dilution with a blank receiver. And calculating the concentration of the skin nutrient in the sample according to the working curve.
Table 2 cumulative permeation comparison of nanoparticle emulsions
Sample a is the nanoparticle emulsion prepared in example 1; sample B was a nanoparticle emulsion coated with the same skin benefit agent as in example 1, according to the method of patent No. CN 200680035440.4.
It can be seen from table 2 that the nanoparticle emulsion loaded with the skin nourishing agent of the present invention has better absorption permeability.
Stability test experiment
(1) The particle size and distribution of the nanoemulsions were determined using a dynamic light scattering analyzer model ZETASIZER3000 from MALVERN, UK. The experimental operating conditions were: the temperature is 25 ℃, the measuring angle of the scattered light intensity is 90 ℃, the wavelength of the laser is 640nm, and the particle size and the distribution of the nano emulsion are measured.
(2) The potential of the nanoparticle emulsion was measured using a ZETASIGER 2000 potentiostat from MALVERN corporation at a loading volume of 1mL and a measurement temperature of 25 ℃ under conditions of pH 7 and an emulsion mass concentration of 0.1 mg/mL. The measurement was repeated 3 times, and the average value was calculated as a Zeta potential measurement value of the sample.
TABLE 3 particle size and Zeta potential of nanoparticle emulsions prepared with different spidroin proteins
Sample a is the nanoparticle emulsion prepared in example 3; sample B was a nanoparticle emulsion coated with the same skin nutrient as in example 3 according to the method of patent No. CN 200680035440.4.
From the above table 3, it can be seen that the nanoparticle emulsion loaded with the skin nutritional agent obtained by treating the spidroin with subtilisin has better stability.
Acid and alkali resistance test experiment
To the nanoparticle emulsion obtained in example 4 and other spider silk protein nanoemulsions were added 0.1%, 0.2%, 0.5%, 1% citric acid and 0.1%, 0.2%, 0.5%, 1% sodium hydroxide, respectively, and tested for acid-base resistance, as shown in table 4 below.
TABLE 4 influence of acid and base on other spider silk proteins nanoemulsion and example 1 nanoemulsion
Sample a is the nanoparticle emulsion prepared in example 4; sample B was a nanoparticle emulsion coated with the same skin benefit agent as in example 4, according to the method of patent No. CN 200680035440.4.
As can be seen from the above table 4, other spidroin protein nano-emulsions are very sensitive to acid and alkali, and are easy to demulsify and separate out precipitates, while the nano-emulsion loaded with the skin nutrient provided by the invention has certain tolerance to acid and alkali.
In conclusion, the nanoparticle emulsion loaded with the skin nutrient has the advantages of high encapsulation rate, good permeability, low irritation, good stability, acid and alkali resistance, environmental friendliness.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to these examples.
Example 1:
a nanoparticle emulsion loaded with a skin nutrient and a preparation method thereof are characterized by comprising the following steps:
A. pretreatment of spider silks: weighing 100g of spider silk, placing the spider silk in 0.8 wt% of sodium carbonate solution at 81 ℃ and stirring at 600 r/min for 20 minutes, carrying out first degumming, pouring out the solution after degumming, taking out the spider silk, repeatedly cleaning the spider silk with distilled water for 10 minutes, placing the spider silk in 0.8 wt% of sodium carbonate solution at 81 ℃ and stirring at 600 r/min for 20 minutes, carrying out second degumming, placing the spider silk in a 90 ℃ oven, and drying to obtain spider silk fiber;
B. weighing 50g of the spider silk fiber obtained in the step A, placing the spider silk fiber in 30 wt% calcium chloride solution, stirring at the rotating speed of 600 r/min at the temperature of 81 ℃ until the spider silk fiber is completely dissolved to obtain spider silk protein calcium chloride mixed solution, filtering the spider silk protein calcium chloride mixed solution by using gauze for crude extraction, placing the spider silk protein calcium chloride mixed solution into a 100-plus-one 750 kDa dialysis bag, dialyzing for 36 hours by using distilled water, and removing calcium chloride ions to obtain the spider silk protein solution;
C. enzymolysis of spider silk protein with subtilisin: b, adding subtilisins into the spidroin solution obtained in the step B for enzymolysis, wherein the dosage of the subtilisins is 28000U/g, and the enzymolysis time is as follows: 50 min, enzymolysis pH: 6.8, enzymolysis temperature: carrying out enzymolysis at 45 ℃ to obtain an enzymolysis mixed solution;
D. performing ultrafiltration purification by adopting a gel filtration chromatography method: c, dissolving 100mg of the enzymolysis mixed liquor obtained in the step C in 3ml of phosphate buffer solution, wherein the pH value of the phosphate buffer solution is 7.0, and the phosphate concentration is 10 mmol/L; adding into chromatographic column, eluting with phosphate buffer solution as eluent at flow rate of 1ml/min, and collecting one tube every 2 min; the chromatographic column is connected with a liquid chromatography separation chromatograph to detect the absorbance of the eluent of the chromatographic column, and a gel imaging system is used for analysis and treatment to obtain the spidroin with the molecular weight of 80-100 kDa;
E. weighing 8% of spidroin, 0.5% of bis-hydroxyethyl trimethylol methylamine, 15% of skin nutrient and 76.5% of water-alcohol mixture according to the following mass ratio;
F. e, heating the skin nutrient weighed in the step E to 75 ℃, and stirring at the rotating speed of 750 r/min for 15 min until the skin nutrient is uniform; the skin nutrient is resveratrol;
G. mixing the spidroin weighed in the step E, bis-hydroxyethyl trihydroxymethyl methylamine and a water-alcohol mixture, heating to 72 ℃, and stirring at the rotating speed of 1200 r/min for 15 min to be uniform to obtain a carrier solution; the hydroalcoholic mixture is a mixture of 61.5% water, 10% 1, 3-propanediol, and 10% glycerol;
H. adding the skin nutrient obtained in the step F into the carrier solution obtained in the step G, and homogenizing for 13 min at the rotation speed of 2300 r/min by using a homogenizer under the environment of normal pressure and normal temperature; then circularly homogenizing for 6 times at the rotating speed of 2500 r/min under the conditions of 120 MPa pressure and 70 ℃ and cooling to room temperature to obtain the nanoparticle emulsion loaded with the skin nutrient.
Example 2:
a nanoparticle emulsion loaded with a skin nutrient and a preparation method thereof are characterized by comprising the following steps:
A. pretreatment of spider silks: weighing 100g of spider silk, placing the spider silk in 0.8 wt% of sodium carbonate solution at 82 ℃ and stirring at 600 r/min for 20 minutes, carrying out first degumming, pouring out the solution after degumming, taking out the spider silk, repeatedly cleaning the spider silk with distilled water for 10 minutes, placing the spider silk in 0.8 wt% of sodium carbonate solution at 82 ℃ and stirring at 600 r/min for 20 minutes, carrying out second degumming, placing the spider silk in a 90 ℃ oven, and drying to obtain spider silk fiber;
B. weighing 50g of the spider silk fiber obtained in the step A, placing the spider silk fiber in 30 wt% calcium chloride solution, stirring at the rotating speed of 600 r/min at 82 ℃ until the spider silk fiber is completely dissolved to obtain spider silk protein calcium chloride mixed solution, filtering the spider silk protein calcium chloride mixed solution by using gauze for crude extraction, placing the spider silk protein calcium chloride mixed solution into a 100-plus 750 kDa dialysis bag, dialyzing by using distilled water for 36 hours, and removing calcium chloride ions to obtain the spider silk protein solution;
C. enzymolysis of spider silk protein with subtilisin: b, adding subtilisins into the spidroin solution obtained in the step B for enzymolysis, wherein the dosage of the subtilisins is 28000U/g, and the enzymolysis time is as follows: 52 min, enzymolysis pH: 6.6, enzymolysis temperature: carrying out enzymolysis at 48 ℃ to obtain an enzymolysis mixed solution;
D. performing ultrafiltration purification by adopting a gel filtration chromatography method: c, dissolving 100mg of the enzymolysis mixed liquor obtained in the step C in 3ml of phosphate buffer solution, wherein the pH value of the phosphate buffer solution is 7.0, and the phosphate concentration is 10 mmol/L; adding into chromatographic column, eluting with phosphate buffer solution as eluent at flow rate of 1ml/min, and collecting one tube every 2 min; the chromatographic column is connected with a liquid chromatography separation chromatograph to detect the absorbance of the eluent of the chromatographic column, and a gel imaging system is used for analysis and treatment to obtain the spidroin with the molecular weight of 80-100 kDa;
E. weighing 10% of spidroin, 0.8% of bis-hydroxyethyl trimethylol methylamine, 15% of skin nutrient and 74.2% of water-alcohol mixture according to the following mass ratio;
F. heating the skin nutrient weighed in the step E to 73 ℃, and stirring at the rotating speed of 780 r/min for 14 min to be uniform; the skin nutritional agent is a mixture of 5% resveratrol, 3% phenethyl resorcinol, 2% soyasterol, 3% retinol, and 2% taxus yunnanensis leaf extract;
G. mixing the spidroin weighed in the step E, bis-hydroxyethyl trihydroxymethyl methylamine and a water-alcohol mixture, heating to 72 ℃, and stirring at the rotating speed of 1450 r/min for 16 min to be uniform to obtain a carrier solution; the water-alcohol mixed solution is a mixture of 56.2% of water, 5% of butanediol, 5% of propylene glycol and 8% of glycerol;
H. adding the skin nutrient obtained in the step F into the carrier solution obtained in the step G, and homogenizing for 16 min at the rotating speed of 2200 r/min by using a homogenizer under the environment of normal pressure and normal temperature; and then circularly homogenizing for 7 times at the rotation speed of 2600 r/min under the conditions of 140 MPa pressure and 66 ℃, and cooling to room temperature to obtain the nanoparticle emulsion loaded with the skin nutrient.
Example 3:
a nanoparticle emulsion loaded with a skin nutrient and a preparation method thereof are characterized by comprising the following steps:
A. pretreatment of spider silk: weighing 100g of spider silk, placing the spider silk in 0.8 wt% of sodium carbonate solution, stirring at 84 ℃ for 20 minutes at 600 r/min, degumming for the first time, pouring out the solution after degumming, taking out the spider silk, repeatedly cleaning the spider silk for 10 minutes by using distilled water, placing the spider silk in 0.8 wt% of sodium carbonate solution, stirring at 84 ℃ for 20 minutes at 600 r/min, degumming for the second time, placing the spider silk in a 90 ℃ oven, and drying to obtain spider silk fiber;
B. weighing 50g of spider silk fibers obtained in the step A, placing the spider silk fibers in 30 wt% calcium chloride solution, stirring at the rotating speed of 600 r/min at 84 ℃ until the spider silk fibers are completely dissolved to obtain spider silk protein calcium chloride mixed solution, filtering and crudely extracting the spider silk protein calcium chloride mixed solution by using gauze, then placing the spider silk protein calcium chloride mixed solution into a 100-plus-750 kDa dialysis bag, dialyzing the spider silk protein calcium chloride mixed solution for 36 hours by using distilled water, and removing calcium chloride ions to obtain the spider silk protein solution;
C. enzymolysis of spider silk protein with subtilisin: b, adding subtilisins into the spidroin solution obtained in the step B for enzymolysis, wherein the dosage of the subtilisins is 28000U/g, and the enzymolysis time is as follows: 63 min, enzymolysis pH: 7.1, enzymolysis temperature: carrying out enzymolysis at 56 ℃ to obtain an enzymolysis mixed solution;
D. performing ultrafiltration purification by adopting a gel filtration chromatography method: c, dissolving 100mg of the enzymolysis mixed liquor obtained in the step C in 3ml of phosphate buffer solution, wherein the pH value of the phosphate buffer solution is 7.0, and the phosphate concentration is 10 mmol/L; adding into chromatographic column, eluting with phosphate buffer solution as eluent at flow rate of 1ml/min, and collecting one tube every 2 min; the chromatographic column is connected with a liquid chromatography separation chromatograph to detect the absorbance of the eluent of the chromatographic column, and a gel imaging system is used for analysis and treatment to obtain the spidroin with the molecular weight of 80-100 kDa;
E. weighing 25% of spidroin, 0.6% of bis-hydroxyethyl trimethylol methylamine, 25% of skin nutrient and 49.4% of water-alcohol mixture according to the following mass ratio;
F. heating the skin nutrient weighed in the step E to 73 ℃, and stirring for 15 min to be uniform at the rotating speed of 700 r/min; the skin nutritional agent is a mixture of 5% astaxanthin, 1% limonin, 3% silymarin, 2% anthocyanin, 1% honokiol, 2% tea polyphenol, 3% asiaticoside, 3% vitamin E, 1% bisabolol, 2% rice chaff sterol, 3% meadowfoam seed oil;
G. mixing the spidroin weighed in the step E, bis-hydroxyethyl trihydroxymethyl methylamine and a water-alcohol mixture, heating to 76 ℃, and stirring at the rotating speed of 1200 r/min for 18 min to be uniform to obtain a carrier solution; the water-alcohol mixed solution is a mixed solution of 32.4% of water, 5% of propylene glycol and 12% of glycerol;
H. adding the skin nutrient obtained in the step F into the carrier solution obtained in the step G, and homogenizing for 14 min at the rotation speed of 2650 r/min by using a homogenizer under the environment of normal pressure and normal temperature; then circularly homogenizing for 8 times at the rotating speed of 2850 r/min under the conditions of 155 MPa pressure and 67 ℃ and cooling to room temperature to obtain the nanoparticle emulsion loaded with the skin nutrient.
Example 4:
a preparation method of nanoparticle emulsion loaded with a skin nutrient is characterized by comprising the following steps:
A. pretreatment of spider silk: weighing 100g of spider silk, placing the spider silk in 0.8 wt% sodium carbonate solution at 85 ℃ for stirring treatment at 600 r/min for 20 minutes, carrying out first degumming, pouring out the solution after degumming is finished, taking out the spider silk, repeatedly cleaning the spider silk for 10 minutes by using distilled water, placing the spider silk in 0.8 wt% sodium carbonate solution at 85 ℃ for stirring treatment at 600 r/min for 20 minutes, carrying out second degumming, placing the spider silk in a 90 ℃ oven, and drying to obtain spider silk fiber;
B. weighing 50g of the spider silk fiber obtained in the step A, placing the spider silk fiber in 30 wt% calcium chloride solution, stirring at the rotating speed of 600 r/min at 85 ℃ until the spider silk fiber is completely dissolved to obtain spider silk protein calcium chloride mixed solution, filtering the spider silk protein calcium chloride mixed solution by using gauze for crude extraction, placing the spider silk protein calcium chloride mixed solution into a 100-plus 750 kDa dialysis bag, dialyzing by using distilled water for 36 hours, and removing calcium chloride ions to obtain the spider silk protein solution;
C. enzymolysis of spider silk protein with subtilisin: b, adding subtilisins into the spidroin solution obtained in the step B for enzymolysis, wherein the dosage of the subtilisins is 28000U/g, and the enzymolysis time is as follows: 57 min, enzymolysis pH: 6.9, enzymolysis temperature: enzymolysis is finished at 59 ℃ to obtain enzymolysis mixed liquor;
D. performing ultrafiltration purification by adopting a gel filtration chromatography method: c, dissolving 100mg of the enzymolysis mixed liquor obtained in the step C in 3ml of phosphate buffer solution, wherein the pH value of the phosphate buffer solution is 7.0, and the phosphate concentration is 10 mmol/L; adding into chromatographic column, eluting with phosphate buffer solution as eluent at flow rate of 1ml/min, and collecting one tube every 2 min; the chromatographic column is connected with a liquid chromatography separation chromatograph to detect the absorbance of the eluent of the chromatographic column, and a gel imaging system is used for analysis and treatment to obtain the spidroin with the molecular weight of 80-100 kDa;
E. weighing 22% of spidroin, 0.75% of bis-hydroxyethyl trimethylol methylamine, 18% of skin nutrient and 59.25% of water-alcohol mixture according to the following mass ratio;
F. heating the skin nutrient weighed in the step E to 74 ℃, and stirring at the rotating speed of 850 r/min for 18 min to be uniform; the skin nutritional agent is a mixture of 3% resveratrol, 3% anthocyanin, 3% tea polyphenol, 5% astragalosides, 1% magnolol and 3% Roche oak seed oil;
G. mixing the spidroin weighed in the step E, bis-hydroxyethyl trihydroxymethyl methylamine and a water-alcohol mixture, heating to 79 ℃, and stirring at the rotating speed of 1300 r/min for 11 min to be uniform to obtain a carrier solution; the water-alcohol mixed solution is a mixed solution of 43.25% of water, 6% of butanediol mixture and 10% of glycerol;
H. adding the skin nutrient obtained in the step F into the carrier solution obtained in the step G, and homogenizing for 16 min at 2800 r/min by using a homogenizer under the environment of normal pressure and normal temperature; then circularly homogenizing for 6 times at the rotation speed of 2600 r/min under the conditions of 130 MPa pressure and 75 ℃ and cooling to room temperature to obtain the nanoparticle emulsion loaded with the skin nutrient.
Claims (3)
1. A nanoparticle emulsion loaded with a skin nutrient is characterized in that: comprises the following components in percentage by weight: 0.1% -50% of spidroin protein, 0.1% -10% of bis-hydroxyethyl trimethylol methylamine, 0.1% -40% of skin nutrient and 0.1% -85% of hydroalcoholic mixture, wherein the molecular weight of the spidroin protein is 80-100 kDa, and the spidroin protein is prepared by adopting the following method:
A. pretreatment of spider silks: weighing 100g of spider silk, placing the spider silk in 0.8% by weight of sodium carbonate solution at 80-85 ℃ and stirring at 600 r/min for 20 minutes, carrying out first degumming, pouring out the solution after degumming, taking out the spider silk, repeatedly cleaning the spider silk with distilled water for 10 minutes, placing the spider silk in 0.8% by weight of sodium carbonate solution at 80-85 ℃ and stirring at 600 r/min for 20 minutes, carrying out second degumming, placing the spider silk in a 90 ℃ oven, and drying to obtain spider silk fiber;
B. weighing 50g of the spider silk fibers obtained in the step A, placing the spider silk fibers in 30 wt% calcium chloride solution, stirring at the rotating speed of 600 r/min at the temperature of 80-85 ℃ until the spider silk fibers are completely dissolved to obtain spider silk protein calcium chloride mixed solution, filtering and crudely extracting the spider silk protein calcium chloride mixed solution by using gauze, placing the spider silk protein calcium chloride mixed solution into a 100-plus 750 kDa dialysis bag, dialyzing for 36 hours by using distilled water, and removing calcium chloride ions to obtain the spider silk protein solution;
C. enzymolysis of spider silk protein: and (3) taking a proper amount of the spider silk protein solution obtained in the step (B), adding subtilisin for enzymolysis, wherein the dosage of the subtilisin is 28000U/g, the enzymolysis time is 45-65 min, and the enzymolysis pH is as follows: 6.5-7.5, the enzymolysis temperature is 40-60 ℃, and enzymolysis is finished to obtain an enzymolysis mixed solution;
D. and (3) performing ultrafiltration purification by adopting a gel filtration chromatography: c, dissolving 100mg of the enzymolysis mixed liquor obtained in the step C in 3ml of phosphate buffer solution, wherein the pH value of the phosphate buffer solution is 7.0, and the phosphate concentration is 10 mmol/L; then adding the mixture into a chromatographic column, eluting by taking a phosphate buffer solution as an eluent, collecting one tube every 2 min at the flow rate of the eluent of 1 ml/min; and (3) detecting the absorbance of the eluent of the chromatographic column by connecting the chromatographic column with a liquid chromatography chromatographic instrument, and analyzing and processing by using a gel imaging system to obtain the spidroin with the molecular weight of 80-100 kDa.
2. The skin nutrient-loaded nanoparticle emulsion according to claim 1, wherein: the spider silk is a traction silk or a frame silk or a radius silk or a rivet silk in a major huttle-shaped silk gland of a Jingzhaomao spider, a Hainan bird catching spider or a tiger-line bird catching spider.
3. The skin nutrient-loaded nanoparticle emulsion according to claim 1, wherein: the spider silk is a traction silk of a tiger-line bird-catching spider.
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| CN101309676A (en) * | 2005-08-01 | 2008-11-19 | 慕尼黑技术大学 | Method of producing nano- and microcapsules of spider silk protein |
| CN101370556A (en) * | 2006-01-20 | 2009-02-18 | 巴斯夫欧洲公司 | Use of protein microbeads in cosmetics |
| CN105648008A (en) * | 2016-01-04 | 2016-06-08 | 山东润牧生物科技有限公司 | Feeding silk antibacterial peptide preparation and preparation method thereof |
| CN108251480A (en) * | 2018-02-10 | 2018-07-06 | 海盐县凌特生物科技有限公司 | Remove the preparation method of the Functional Polypeptides of melanin in skin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101309676A (en) * | 2005-08-01 | 2008-11-19 | 慕尼黑技术大学 | Method of producing nano- and microcapsules of spider silk protein |
| CN101370556A (en) * | 2006-01-20 | 2009-02-18 | 巴斯夫欧洲公司 | Use of protein microbeads in cosmetics |
| CN105648008A (en) * | 2016-01-04 | 2016-06-08 | 山东润牧生物科技有限公司 | Feeding silk antibacterial peptide preparation and preparation method thereof |
| CN108251480A (en) * | 2018-02-10 | 2018-07-06 | 海盐县凌特生物科技有限公司 | Remove the preparation method of the Functional Polypeptides of melanin in skin |
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