CN111214385B - A kind of nanoparticle emulsion loaded with skin nutrition agent and preparation method thereof - Google Patents

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

A kind of nanoparticle emulsion loaded with skin nutrition agent and preparation method thereof

technical field

The invention relates to the technical field of nanoparticle emulsion preparation, in particular to a nanoparticle emulsion loaded with skin nutrient and a preparation method thereof.

Background technique

With the development of science and technology and the improvement of people's beauty awareness, more and more beauty ingredients are used in cosmetics, but many raw materials have problems such as difficulty in dissolution and easy discoloration. As a new carrier, nano-formulation technology has begun to be used in cosmetics, but the current nano-particle emulsions mostly use chemical surfactants, which have a certain impact on the skin barrier.

Spider silk is a natural protein fiber with mechanical properties such as high strength, high elasticity, and high fracture energy, as well as biological properties such as remarkable degradability and histocompatibility. All have significant potential application value. The main component of spider silk is protein, so it has good compatibility with biological tissues. The molecular weight of spider silk proteins found so far is about 100-750 kDa. Spider drag silk protein is a highly repetitive fibrin, rich in alanine and glycine. The alanine-rich region can form a hydrophobic β-sheet crystalline structure, while the hydrophilic glycine-rich region forms an α-helix. Since spidroin has both the amino end and the hydrophobic end of the hydrophilic end, it has a certain emulsifying ability.

There have been many studies on the techniques of optimizing the mechanical properties of spider silk proteins, such as recombinant acquisition, but there are few reports on how to optimize the emulsifying properties and emulsion stability of spider silk proteins. Chinese invention patent CN200680035440.4 discloses a method for producing nanocapsules and microcapsules of spider silk protein, but solvents such as guanidine thiocyanate and toluene are used, which are not very friendly to the environment and skin. And it is directly coated with macromolecular spider silk protein, which has poor emulsifying power, and spider silk protein is not acid and alkali resistant, and the produced nanocapsules have poor stability, and the encapsulation efficiency and permeability are not good enough.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a nanoparticle emulsion with high encapsulation rate and good stability for carrying skin nutritional agents.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions: a nanoparticle emulsion loaded with skin nutrient, characterized in that: it comprises the following components by weight: 0.1%-50% of spidroin, 0.1%-10% % of bis-hydroxyethyl trimethylolmethylamine, 0.1% to 40% of skin nutrient, 0.1% to 85% of hydroalcoholic mixture, the molecular weight of the spider silk protein is 80 to 100 kDa.

Described spider silk protein adopts following method to prepare:

A. Pretreatment of spider silk: Weigh 100g spider silk and place it in a sodium carbonate solution with a weight percentage of 0.8% at 80-85°C and stir at 600 r/min for 20 minutes, then degumming for the first time. After the degumming is completed, pour it out Solution, take out the spider silk and repeatedly wash it with distilled water for 10 minutes, then place it in a sodium carbonate solution with a weight percentage of 0.8% at 80~85 ° C and stir at 600 r/min for 20 minutes, carry out the second degumming, put it in 90 ℃ oven, drying to obtain spider silk fibers;

B, take by weighing the 50g spider silk fiber gained from step A, place it in a calcium chloride solution with a weight percentage of 30% and stir at 600 r/min rotating speed at 80 to 85 ° C until the spider silk fiber is completely dissolved, to obtain spider silk protein chloride For the calcium mixture, first filter the above-mentioned spider silk protein calcium chloride mixture with gauze, and then put it into a 100-750 kDa dialysis bag, and dialyze it with distilled water for 36 hours to remove the calcium chloride ions to obtain the spider silk protein solution;

C. Enzymatic hydrolysis of spider silk protein: take an appropriate amount of the spider silk protein solution obtained in step B, add subtilisin for enzymatic hydrolysis, wherein the amount of subtilisin is 28000 U/g, and the enzymatic hydrolysis time is 45-65 min. pH: 6.5~7.5, the enzymolysis temperature is 40℃~60℃, and the enzymolysis mixture is obtained after the enzymolysis is completed;

D, adopt gel filtration chromatography to carry out ultrafiltration purification: take 100mg of the enzymatic hydrolysis mixture obtained in step C, dissolve in 3ml of phosphate buffer, the pH of the phosphate buffer is 7.0, and the phosphate concentration is 10 mmol/L; then added to the chromatography column, eluted with phosphate buffer as the eluent, the eluent flow rate was 1ml/min, and a tube was collected every 2 minutes; the chromatography column was connected to a liquid chromatography separation chromatograph The absorbance of the eluate of the chromatography column was detected, and the gel imaging system was used for analysis and processing to obtain spider silk proteins with a molecular weight of 80-100 kDa.

The spider silk in the step A can be the pulling silk, the frame silk, the radius silk and the rivet silk in the large jug-shaped silk glands of the Jingzhao tassels, Hainan tarantula, and Huwen tarantula. In the present invention, the traction silk of the tiger-pattern tarantula is preferred, and the commercially available products include the traction silk of the tiger-pattern tarantula of Guangxi Dongfang Breeding Co., Ltd.

The gel filtration chromatography system was purchased from Huamei Company, and the model is Sephadex G-100.

The gel imaging system was purchased from UVP Company in the United States, and the model is UV Transilluminator.

The high-performance liquid chromatograph was purchased from Shimadzu Corporation, Japan, model LC-2010C.

The subtilisin protease was purchased from Shanghai Xinyu Biotechnology Co., Ltd., and the enzyme activity was ≥4000 units/mg.

A preparation method of a nanoparticle emulsion loaded with skin nourishing agent is characterized in that adopting the following steps:

A. Weigh 0.1%~50% spider silk protein, 0.1%~10% bis-hydroxyethyl trimethylolmethylamine, 0.1%~40% skin nutrient, 0.1%~50% according to the following mass ratios % hydroalcoholic mixture, the molecular weight of the spider silk protein is 80-100 kDa;

B, the skin nourishing agent weighed in step A is heated to 70～80 ℃, stirring 10～20 min under the rotating speed of 600～1200 r/min to uniform; Described skin nourishing agent is water-soluble skin nourishing agent or oil-soluble skin Nutrients or mixtures thereof;

C, the spider silk protein, bis-hydroxyethyl trimethylolmethylamine, and the water-alcohol mixture weighed in step A are mixed and heated to 70~80 ° C, and stirred for 10~20 min under the rotating speed of 1000~1500 r/min to uniformity to obtain a carrier solution;

D, the skin nourishing agent obtained in step B is added to the carrier solution obtained in step C, first, under normal pressure and normal temperature environment with a homogenizer, homogenize at 2000-3000 r/min rotation speed for 10-18 min; then at 100- Under the conditions of 160 MPa pressure and 65-80 ℃ temperature, 2000-3000 r/min rotation speed is circulated and homogenized for 5-8 times, and cooled to room temperature to obtain a nanoparticle emulsion loaded with skin nutrients.

The bis-hydroxyethyl trimethylolmethylamine is a pH buffer, and the CAS number is 6976-37-0, which was purchased from Suzhou Luosen Chemical Co., Ltd.

Described skin nutrition agent is water-soluble skin nutrition agent or oil-soluble skin nutrition agent or their mixture, can specifically be resveratrol, tetrahydrocurcumin, astaxanthin, curcumin, quercetin, phloretin, Aloe Verain, Limonin, Silymarin, Lutein, Riboflavin, Scutellarin, Anthocyanin, Lycopene, Phenethyl Resorcinol, and Honokiol, Paeonol, Eugenol, Hinokiol, tea polyphenols, hesperidin, naringin, astragalus saponins, asiaticoside, aesculin, stevioside, vitamin E, bisabolol, cholesterol, cholesterol, soy sterol, rice bran Sterols, Dihydrosphingosine, Retinol, Salicylphytosphingosine, Phytosphingosine, Tetrahydromagnolol, Magnolol, Quercus Roche Seed Oil, Norway Spruce Leaf Extract, White Willow Bark One of the extract, Echinacea purpurea extract or a mixture of them in any ratio.

The hydroalcoholic mixture is a mixture of water and polyol, wherein the polyol can be one of propylene glycol, glycerol, 1,3-butanediol, dipropylene glycol, diglycerol, polyethylene glycol-8, pentaerythritol or them. The ratio of water and polyol is 0.5~3:1.

In the nanoparticle emulsion loaded with skin nutrients prepared by the invention, the spider silk protein is first treated with subtilisin, and then purified by gel filtration chromatography to obtain spider silk protein with moderate molecular weight, which is conducive to the formation of smaller particle size and The uniform nanoparticle emulsion is also conducive to the formation of a denser interface film, which increases the emulsion stability. After the molecular weight of the spidroin is reduced, the specific surface area increases, the encapsulation efficiency becomes higher, and the particle size of the nanoparticles formed at the same time increases. It is smaller and the permeability is increased; the present invention adds bis-hydroxyethyl trimethylol methylamine as an acid-base buffer to avoid excessive sensitivity of the spider silk protein to pH; and no organic solvent is added in the production process, which is more green and environmentally friendly .

In order to verify the properties of the nanoparticle emulsion loaded with skin nutrient agents obtained in the present invention, the present invention has carried out the following tests.

Encapsulation assay experiment

The encapsulation efficiency of the nanoparticle emulsion of Example 1 was measured by ultrafiltration centrifugation, 500 uL of the nanoparticle emulsion of Example 1 was placed in a 10ml volumetric flask, an appropriate amount of anhydrous ethanol was added to dissolve and disperse, and then ultrasonic demulsification was performed; Water ethanol is carried out to constant volume, the absolute ethanol solution of the nanoparticle emulsion after constant volume is diluted 10 times, the spectrophotometric value is measured by an ultraviolet spectrophotometer, and then calculated according to the standard curve of the skin nutrient resveratrol in Example 1 The total content of skin nutrients in the sample solution: W _Total .

Take 1ml of the nanoparticle emulsion of Example 1, dilute it 10 times with ultrapure water, take about 300 uL of the diluted nanoparticle emulsion dispersion in an ultrafiltration centrifuge tube, and freeze and centrifuge it for 40 min at 10,000 rpm to obtain clarification. Precisely absorb the centrifuge, add absolute ethanol to dissolve to volume, shake well to obtain the centrifuge for test, measure the absorbance value by UV spectrophotometer, and also calculate the diluted nanoparticle carrier aqueous dispersion according to the standard curve equation Content of skin nutrient: W _Free .

Calculated encapsulation rate = [1-(W _Free /W _Total )]*100%

Table 1 Comparison of encapsulation efficiency of nanoparticle emulsions

Sample A is the nanoparticle emulsion prepared in Example 1; Sample B is a nanoparticle emulsion wrapped with the same skin nourishing agent as Example 1 according to the method of Patent No. CN200680035440.4.

It can be seen from the experiments that the nanoparticle emulsion loaded with skin nutrient of the present invention has better encapsulation efficiency.

Penetration test

Franz diffusion cell method was used to investigate the absorption and penetration effects of the nanoparticle emulsion of Example 1 and other spider silk protein nanoparticle emulsions.

The artificial skin was fixed between the drug supply pool and the receiving pool of the Franz diffusion cell, the stratum corneum of the skin faced the drug supply pool, the effective penetration area was 4.15 cm ² , the volume of the receiving pool was 30 ml, and the dermis side of the skin was in contact with the receiving fluid. The air bubbles were removed, the receiving liquid was an aqueous solution of 30% ethanol nanoparticle emulsion, the water bath was circulated outside the Franz diffusion cell at (37.0±0.5) °C, and the inside of the receiving cell was stirred at a constant speed of 350 r.min ^-1 .

After the temperature of the system was constant, 2.0 ml of the above test solution was gently injected into the medicine supply pool. Stir at a constant speed and start timing. At 1, 2, 3, 4, 6, 8, 12, 18, and 24 h, 1.0 ml of the receiving solution was extracted from the sampling tube, and an equal volume of isothermal 30% ethanol aqueous solution of nanoparticle emulsion was added in time after each sampling. And remove air bubbles in the receiving cell. The collected samples of the receiving fluid were placed in a stoppered glass test tube, sealed at a low temperature and protected from light, and the measurement was completed within 24 hours. The sample solution was filtered through a microporous membrane with a pore size of 0.45 microns for high performance liquid chromatography (HPLC) analysis. The samples whose peak area exceeds the working curve are appropriately diluted with blank control receiver solution for determination. Calculate the concentration of skin nutrient in the sample based on the working curve.

Table 2 Comparison of cumulative permeation of nanoparticle emulsions

Sample A is the nanoparticle emulsion prepared in Example 1; Sample B is a nanoparticle emulsion wrapped with the same skin nourishing agent as Example 1 according to the method of Patent No. CN200680035440.4.

It can be seen from Table 2 that the nanoparticle emulsion loaded with skin nutrient of the present invention has better absorption and permeability.

Stability Test Experiment

(1) The particle size and distribution of the nanoemulsion were determined by ZETASIZER3000 dynamic light scattering analyzer from MALVERN, UK. The experimental operating conditions were as follows: the temperature was 25 °C, the scattered light intensity measurement angle was 90°, the laser wavelength was 640 nm, and the particle size and distribution of the nanoemulsion were measured.

(2) The potential of the nanoparticle emulsion was measured by the ZETASIGER 2000 potentiometer of MALVERN Company, and the measurement was carried out under the conditions of pH 7 and emulsion mass concentration of 0.1 mg/mL, the sample volume was 1 mL, and the measurement temperature was 25 °C. The measurement was repeated 3 times, and the average value was calculated as the measured value of the Zeta potential of the sample.

Table 3 Particle size and Zeta potential of nanoparticle emulsions prepared from different spider silk proteins

Sample A is the nanoparticle emulsion prepared in Example 3; Sample B is a nanoparticle emulsion wrapped with the same skin nourishing agent as Example 3 according to the method of Patent No. CN200680035440.4.

It can be seen from the above Table 3 that after the spider silk protein is treated with subtilisin, the obtained nanoparticle emulsion loaded with skin nutrient has better stability.

Acid and alkali resistance test experiment

Add 0.1%, 0.2%, 0.5%, 1% citric acid and 0.1%, 0.2%, 0.5%, 1% sodium hydroxide to the nanoparticle emulsion obtained in Example 4 and other spider silk protein nanoemulsions, respectively. The tolerance to acid and alkali is shown in Table 4 below.

The influence of table 4 acid and alkali on other spider silk protein nanoemulsions and nanoemulsions of Example 1

Sample A is the nanoparticle emulsion prepared in Example 4; Sample B is a nanoparticle emulsion wrapped with the same skin nourishing agent as Example 4 according to the method of Patent No. CN200680035440.4.

As can be seen from Table 4 above, other spider silk protein nanoemulsions are very sensitive to acid and alkali, and are easy to break demulsification and precipitate, while the nanoparticle emulsion loaded with skin nutrient provided by the present invention has a certain resistance to acid and alkali.

To sum up, the nanoparticle emulsion loaded with skin nutrition agent obtained by the present invention has the advantages of high encapsulation rate, good permeability, low irritation, good stability, acid and alkali resistance, and environmental protection.

Detailed ways

The present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to these embodiments.

Example 1:

A kind of nanoparticle emulsion of skin nourishing agent and preparation method thereof, it is characterized in that adopting the following steps:

A. Pretreatment of spider silk: Weigh 100 g of spider silk and place it in a sodium carbonate solution with a weight percentage of 0.8% at 81°C for 20 minutes with stirring at 600 r/min for the first degumming. After the degumming is completed, pour out the solution. The spider silk was taken out and repeatedly washed with distilled water for 10 minutes, and then placed in a sodium carbonate solution with a weight percentage of 0.8% for 20 minutes at 81 °C with a rotating speed of 600 r/min, and then degummed for the second time. dried spider silk fibers;

B, take by weighing the gained 50g spider silk fiber of step A, be placed in the calcium chloride solution that the percentage by weight is 30% at 81 ° C and stir until the spider silk fiber is completely dissolved with a rotating speed of 600 r/min, to obtain the spider silk protein calcium chloride mixed First, the above-mentioned spider silk protein calcium chloride mixture was filtered and crudely extracted with gauze, then put into a 100-750 kDa dialysis bag, and dialyzed with distilled water for 36 h to remove calcium chloride ions to obtain spider silk protein solution;

C. Enzymolysis of spider silk protein with subtilisin: adding subtilisin to the spider silk protein solution obtained in step B for enzymolysis, wherein the consumption of subtilisin is 28000 U/g, and the enzymolysis time: 50 min , enzymatic hydrolysis pH: 6.8, enzymatic hydrolysis temperature: 45 ℃, the enzymatic hydrolysis is completed and the enzymatic hydrolysis mixture is obtained;

D. Use gel filtration chromatography to carry out ultrafiltration purification: take 100 mg of the enzymatic hydrolysis mixture obtained in step C, dissolve it in 3 ml of phosphate buffer, the pH of the phosphate buffer is 7.0, and the phosphate concentration is 10 mmol/L; then added to the chromatography column, eluted with phosphate buffer as the eluent, the eluent flow rate was 1 ml/min, and a tube was collected every 2 min; the chromatography column was connected to the liquid chromatography separation layer The absorbance of the eluate of the chromatographic column was detected by an analyzer, and the spidroin with a molecular weight of 80-100 kDa was obtained by analyzing and processing it with a gel imaging system;

E, take by weighing 8% spider silk protein by following mass ratio, the bis-hydroxyethyl trimethylol amine of 0.5%, the skin nourishing agent of 15%, the water alcohol mixture of 76.5%;

F, the skin nourishing agent weighed in step E is heated to 75°C, and stirred for 15 min to uniform at a rotating speed of 750 r/min; the skin nourishing agent is resveratrol;

G. Mix the spider silk protein, bis-hydroxyethyl trimethylolmethylamine, and the water-alcohol mixture weighed in step E and heat to 72 ° C, and stir at 1200 r/min for 15 min until uniform to obtain a carrier solution ; The hydroalcoholic mixture is a mixture of 61.5% water, 10% 1,3-propanediol and 10% glycerol;

H. Add the skin nourishing agent obtained in step F into the carrier solution obtained in step G, first use a homogenizer under normal pressure and normal temperature environment, homogenize at 2300 r/min speed for 13 min; then under 120 MPa pressure, 70 ℃ Under the condition of temperature, circulate and homogenize 6 times at 2500 r/min, and cool to room temperature to obtain nanoparticle emulsion loaded with skin nutrient.

Example 2:

A kind of nanoparticle emulsion of skin nourishing agent and preparation method thereof, it is characterized in that adopting the following steps:

A. Pretreatment of spider silk: Weigh 100 g of spider silk and place it in a sodium carbonate solution with a weight percentage of 0.8% at 82°C for 20 minutes with stirring at 600 r/min. The first degumming is performed. After the degumming is completed, the solution is poured out. The spider silk was taken out and repeatedly washed with distilled water for 10 minutes, and then placed in a sodium carbonate solution with a weight percentage of 0.8% at 82 °C for 20 minutes at a speed of 600 r/min, and then degummed for the second time. dried spider silk fibers;

B, take by weighing the gained 50g spider silk fibers of step A, be placed in the calcium chloride solution that the percentage by weight is 30% and stir at 600 r/min rotating speed at 82 DEG C until the spider silk fibers are completely dissolved, and obtain spider silk protein calcium chloride mixed First, the above-mentioned spider silk protein calcium chloride mixture was filtered and crudely extracted with gauze, then put into a 100-750 kDa dialysis bag, and dialyzed with distilled water for 36 h to remove calcium chloride ions to obtain spider silk protein solution;

C. Enzymolysis of spider silk protein with subtilisin: adding subtilisin to the spider silk protein solution obtained in step B for enzymolysis, wherein the consumption of subtilisin is 28000 U/g, and the enzymolysis time: 52 min , enzymatic hydrolysis pH: 6.6, enzymatic hydrolysis temperature: 48 ℃, the enzymatic hydrolysis is completed and the enzymatic hydrolysis mixed solution is obtained;

D. Use gel filtration chromatography to carry out ultrafiltration purification: take 100 mg of the enzymatic hydrolysis mixture obtained in step C, dissolve it in 3 ml of phosphate buffer, the pH of the phosphate buffer is 7.0, and the phosphate concentration is 10 mmol/L; then added to the chromatography column, eluted with phosphate buffer as the eluent, the eluent flow rate was 1 ml/min, and a tube was collected every 2 min; the chromatography column was connected to the liquid chromatography separation layer The absorbance of the eluate of the chromatographic column was detected by an analyzer, and the spidroin with a molecular weight of 80-100 kDa was obtained by analyzing and processing it with a gel imaging system;

E. Weigh 10% of spider silk protein, 0.8% of bis-hydroxyethyl trimethylolamine, 15% of skin nutrient, and 74.2% of water-alcohol mixture according to the following mass ratios;

F, the skin nourishing agent that step E is weighed is heated to 73 ℃, stirring 14 min under 780 r/min rotating speed to uniform; Described skin nourishing agent is 5% resveratrol, 3% phenethyl resorcinol , a mixture of 2% soy sterol, 3% retinol, 2% Norway spruce leaf extract;

G. The spider silk protein, bis-hydroxyethyl trimethylolmethylamine and water alcohol mixture weighed in step E are mixed and heated to 72 ° C, and stirred for 16 min at a rotating speed of 1450 r/min until uniform to obtain a carrier solution ; The water-alcohol mixed solution is a mixture of 56.2% water, 5% butylene glycol, 5% propylene glycol and 8% glycerin;

H. Add the skin nourishing agent obtained in step F into the carrier solution obtained in step G, first use a homogenizer to homogenize at 2200 r/min for 16 min under normal pressure and normal temperature environment; then under 140 MPa pressure, 66 ℃ Under the condition of temperature, 2600 r/min speed was circulated and homogenized for 7 times, and cooled to room temperature to obtain nanoparticle emulsion loaded with skin nutrient.

Example 3:

A kind of nanoparticle emulsion of skin nourishing agent and preparation method thereof, it is characterized in that adopting the following steps:

A. Pretreatment of spider silk: Weigh 100 g of spider silk and place it in a sodium carbonate solution with a weight percentage of 0.8% at 84°C and stir at 600 r/min for 20 minutes to carry out the first degumming. After the degumming is completed, pour out the solution. The spider silk was taken out and repeatedly washed with distilled water for 10 minutes, and then placed in a sodium carbonate solution with a weight percentage of 0.8% at 84 °C for 20 minutes at a speed of 600 r/min, and then degummed for the second time. dried spider silk fibers;

B, take by weighing the gained 50g spider silk fibers of step A, be placed in the calcium chloride solution that the percentage by weight is 30%, and stir at 600 r/min rotating speed at 84 ° C until the spider silk fibers are completely dissolved, and obtain spider silk protein calcium chloride mixed First, the above-mentioned spider silk protein calcium chloride mixture was filtered and crudely extracted with gauze, then put into a 100-750 kDa dialysis bag, and dialyzed with distilled water for 36 h to remove calcium chloride ions to obtain spider silk protein solution;

C. Enzymolysis of spider silk protein with subtilisin: adding subtilisin to the spider silk protein solution obtained in step B for enzymolysis, wherein the consumption of subtilisin is 28000 U/g, and the enzymolysis time: 63 min , enzymatic hydrolysis pH: 7.1, enzymatic hydrolysis temperature: 56 ℃, the enzymatic hydrolysis is completed and the enzymatic hydrolysis mixture is obtained;

D. Use gel filtration chromatography to carry out ultrafiltration purification: take 100 mg of the enzymatic hydrolysis mixture obtained in step C, dissolve it in 3 ml of phosphate buffer, the pH of the phosphate buffer is 7.0, and the phosphate concentration is 10 mmol/L; then added to the chromatography column, eluted with phosphate buffer as the eluent, the eluent flow rate was 1 ml/min, and a tube was collected every 2 min; the chromatography column was connected to the liquid chromatography separation layer The absorbance of the eluate of the chromatographic column was detected by an analyzer, and the spidroin with a molecular weight of 80-100 kDa was obtained by analyzing and processing it with a gel imaging system;

E, take by weighing 25% of spider silk protein, 0.6% of bis-hydroxyethyl trimethylolamine, 25% of skin nutrient, 49.4% of water-alcohol mixture according to the following mass ratios;

F, the skin nourishing agent weighed in step E is heated to 73 ℃, stirring 15 min under 700 r/min rotating speed to uniform; Described skin nourishing agent is 5% astaxanthin, 1% limonin, 3% silymarin, 2% anthocyanins, 1% honokiol, 2% tea polyphenols, 3% madecassoside, 3% vitamin E, 1% bisabolol, 2% rice bran sterol, 3% white chia seed oil mixture;

G. The spider silk protein, bis-hydroxyethyl trimethylolmethylamine and water alcohol mixture weighed in step E are mixed and heated to 76 ° C, and stirred for 18 min at a rotating speed of 1200 r/min until uniform to obtain a carrier solution ; Described hydroalcoholic mixed solution is the mixed solution of 32.4% water, 5% propylene glycol and 12% glycerin;

H. Add the skin nourishing agent obtained in step F to the carrier solution obtained in step G, first use a homogenizer under normal pressure and normal temperature environment, and homogenize at 2650 r/min for 14 min; then under 155 MPa pressure, 67 ℃ Under the condition of temperature, circulate and homogenize at 2850 r/min for 8 times, and cool to room temperature to obtain nanoparticle emulsion loaded with skin nutrient.

Example 4:

A preparation method of a nanoparticle emulsion loaded with skin nourishing agent is characterized in that adopting the following steps:

A. Pretreatment of spider silk: Weigh 100g of spider silk and place it in a sodium carbonate solution with a weight percentage of 0.8% at 85°C for 20 minutes with stirring at 600 r/min. The first degumming is performed. After the degumming is completed, the solution is poured out. The spider silk was taken out and repeatedly washed with distilled water for 10 minutes, and then placed in a sodium carbonate solution with a weight percentage of 0.8% at 85 °C for 20 minutes at a speed of 600 r/min, and then degummed for the second time. dried spider silk fibers;

B, take by weighing the gained 50g spider silk fiber of step A, be placed in the calcium chloride solution that the percentage by weight is 30% at 85 DEG C with 600 r/min rotating speed stirring until spider silk fiber is completely dissolved, obtain spider silk protein calcium chloride mix First, the above-mentioned spider silk protein calcium chloride mixture was filtered and crudely extracted with gauze, then put into a 100-750 kDa dialysis bag, and dialyzed with distilled water for 36 h to remove calcium chloride ions to obtain spider silk protein solution;

C. Enzymolysis of spider silk protein with subtilisin: adding subtilisin to the spider silk protein solution obtained in step B for enzymolysis, wherein the consumption of subtilisin is 28000 U/g, and the enzymolysis time: 57 min , enzymatic hydrolysis pH: 6.9, enzymatic hydrolysis temperature: 59 ℃, enzymatic hydrolysis is completed to obtain enzymatic hydrolysis mixture;

D. Use gel filtration chromatography to carry out ultrafiltration purification: take 100 mg of the enzymatic hydrolysis mixture obtained in step C, dissolve it in 3 ml of phosphate buffer, the pH of the phosphate buffer is 7.0, and the phosphate concentration is 10 mmol/L; then added to the chromatography column, eluted with phosphate buffer as the eluent, the eluent flow rate was 1 ml/min, and a tube was collected every 2 min; the chromatography column was connected to the liquid chromatography separation layer The absorbance of the eluate of the chromatographic column was detected by an analyzer, and the spidroin with a molecular weight of 80-100 kDa was obtained by analyzing and processing it with a gel imaging system;

E. Weigh 22% spider silk protein, 0.75% bis-hydroxyethyl trimethylolamine, 18% skin nutrient, and 59.25% hydroalcoholic mixture according to the following mass ratios;

F, the skin nourishing agent weighed in step E is heated to 74 ° C, and stirred for 18 min to uniform under 850 r/min rotating speed; the skin nourishing agent is 3% resveratrol, 3% anthocyanin, 3% tea A mixture of polyphenols, 5% astragalus saponins, 1% magnolia alcohol, 3% Roche quercetin oil;

G. The spider silk protein, bis-hydroxyethyl trimethylolmethylamine, and the water-alcohol mixture weighed in step E are mixed and heated to 79 ° C, and stirred for 11 min at a rotating speed of 1300 r/min until uniform to obtain a carrier solution ; The water-alcohol mixed solution is a mixed solution of 43.25% water, 6% butanediol mixture, and 10% glycerin;

H. Add the skin nourishing agent obtained in step F to the carrier solution obtained in step G, first use a homogenizer to homogenize at 2800 r/min under normal pressure and normal temperature environment for 16 min; then under 130 MPa pressure, 75 ° C Under the condition of temperature, circulate and homogenize 6 times at 2600 r/min, and cool to room temperature to obtain nanoparticle emulsion loaded with 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.