CN114668683B - Nano gold doped self-assembled polypeptide active matter and preparation method thereof - Google Patents
Nano gold doped self-assembled polypeptide active matter and preparation method thereof Download PDFInfo
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Abstract
The application discloses a nano gold doped self-assembled polypeptide active substance and a preparation method thereof, wherein the nano gold doped self-assembled polypeptide active substance is prepared from a self-assembled polypeptide active substance and a nano gold solution in a weight ratio of (50-100): 1; the self-assembled polypeptide mixture is obtained by self-assembling active polypeptide and Thermus thermophilus fermentation products; the active polypeptide comprises one or more of acetyl hexapeptide-8, palmitoyl pentapeptide-4 and glutathione. The nano gold doped self-assembled polypeptide active matter prepared by the application has stable performance and good effects of skin repair, water retention, water locking, wrinkle resistance, DPPH free radical removal and the like.
Description
Technical Field
The application relates to the field of bioactive raw materials of cosmetics, in particular to a nano-gold doped self-assembled polypeptide active substance and a preparation method thereof.
Background
Self-assembled polypeptides are nanomaterials that achieve self-assembly with non-covalent interactions between peptide molecules as the driving force. The non-covalent effect mainly comprises hydrogen bonding effect, pi-pi stacking effect, electrostatic effect, hydrophobic interaction and the like, and the driving force endows the polypeptide with excellent self-assembly capability and controllability of self-assembly, and when the self-assembly driving force reaches dynamic balance, the structure of the polypeptide falls into metastable state, so that a regular self-assembly structure such as fiber, gel, nano-tube, nano-sphere, strip and the like can be formed.
The self-assembled polypeptide has excellent biocompatibility, biodegradability and cell targeting, so that the self-assembled polypeptide has wide application in wound repair, tissue repair, skin care, cosmetics, cell culture, drug carrier and the like. However, when used as a skin care product component, the self-assembled polypeptide is easily denatured due to poor stability, resulting in poor use.
Disclosure of Invention
The application provides a nano-gold doped self-assembled polypeptide active substance and a preparation method thereof, wherein the self-assembled polypeptide active substance has high stability and outstanding skin water-retaining and repairing effects.
The application provides a nano gold doped self-assembled polypeptide active substance, which is prepared from the self-assembled polypeptide active substance and a nano gold solution in a weight ratio of (50-100): 1; the self-assembled polypeptide mixture is obtained by self-assembling active polypeptide and Thermus thermophilus fermentation products; the active polypeptide comprises one or more of acetyl hexapeptide-8, palmitoyl pentapeptide-4 and glutathione.
In the technical scheme, the gold nanoparticle has the advantages of larger specific surface area, good biocompatibility, small particle size and the like, can penetrate into the deep layer of the epidermis layer, and promotes the accumulation of active substances in the skin. By adding nano gold into the self-assembled polypeptide system, the sulfhydryl group in the self-assembled polypeptide molecule can be combined with the nano gold, so that the self-assembled polypeptide can be utilized to carry out surface modification on the nano gold, and the nano gold doped self-assembled polypeptide active substance is formed. On one hand, the penetration of the self-assembled polypeptide to the deep layer of the epidermis layer can be promoted, the content of the active substance of the self-assembled polypeptide in the skin can be improved, and the effects of repairing the skin, retaining water, resisting wrinkles and the like of the self-assembled polypeptide can be effectively improved; on the other hand, after the nano gold is combined with the self-assembled polypeptide, the stability of the self-assembled polypeptide structure can be improved, and the phenomenon of efficacy reduction or even loss caused by polypeptide denaturation can be reduced.
Preferably, the dosage ratio of the thermophilic thermus fermentation product to the active polypeptide is (90-99.8) (0.1-2).
In the polypeptide self-assembly process, the application adopts the fermentation product of the active polypeptide and thermophilic thermus as an assembly substrate, wherein the active polypeptide comprises acetyl hexapeptide-8, palmitoyl pentapeptide-4 and glutathione which have acetyl, palmitoyl and other modification groups, on one hand, the application is beneficial to cooperatively promoting the self-assembly of the polypeptide and obtaining self-assembly polypeptide with more abundant types; on the other hand, acetyl and palmitoyl have better lipophilicity, are favorable for the penetration of self-assembled polypeptide active substances into deep layers of skin, and improve the effects of repairing, water retention, wrinkle resistance and the like.
The thermophilic thermus fermentation product is obtained by fermenting and crushing thermophilic thermus under high temperature condition, and the lysate mainly contains nucleic acid, protein, lipid, polysaccharide, secondary metabolite and various polypeptides. The natural polypeptide components contained in the polypeptide comprise valyl-phenylpropyl-tyryl-isoleucyl, cereal-lysyl-spermine-propyl-isoleucyl-propyl, glycyl-prolyl-leucyl-phenylpropyl-valyl-cereal and the like, and meanwhile, the polypeptide contains a proper amount of glutathione and amino acids with different charges and different hydrophobicity, so that the non-covalent interaction of the peptide can be changed, the self-assembly of the peptide is promoted, the diversity of self-assembly products is enriched, and finally, the effects of repairing, water retention, wrinkle resistance and the like are effectively improved.
Preferably, the active polypeptide adopts acetyl hexapeptide-8, palmitoyl pentapeptide-4 and glutathione with the mass ratio of (8-20) to 1:1.
The added glutathione and the nano gold have higher affinity, can be combined with the surface of the nano gold through sulfhydryl groups, so that the stability of a self-assembled polypeptide system is improved, and the repairing effect of the nano gold doped self-assembled polypeptide active substance is ensured.
Preferably, the average particle size of the nano gold in the nano gold solution is 10-20 nm.
By adopting the technical scheme, the reduction of the granularity of the nano gold is beneficial to the improvement of the permeability of the nano gold; however, too small a particle size will be detrimental to increasing its loading on the self-assembled polypeptide, thereby affecting the repair effect of the nanogold doped self-assembled polypeptide active.
Preferably, the thermophilic thermus fermentation product is obtained by fermenting and culturing a strain with the preservation number of BAA-163 at 50-80 ℃ and crushing the strain for 2-12 hours.
By adopting the technical scheme, thermophilic thermus strain is fully fermented in a high-temperature environment, and the fermentation product of the thermophilic thermus strain has rich polypeptide, so that the diversity of self-assembled polypeptides is improved.
It should be noted that the thermophilic thermus strain of the present application is not unique, and other thermophilic thermus strain fermentation products have the effect of promoting the self-assembly of the polypeptide. The fermentation culture temperature of the thermophilic thermus strain is 50-80 ℃ and the fermentation time is 2-12 h.
Preferably, the nano-gold solution is prepared according to the following method:
s101: heating a gold ion solution to boiling, then adding a reducing agent for oxidation-reduction reaction, and obtaining a nano gold crude solution when the solution is reddish wine;
s102: and adding butanediol into the crude gold nanoparticle solution, stirring uniformly, and adding polyvinylpyrrolidone to obtain the gold nanoparticle solution.
According to the application, the reduction method is adopted to prepare the nano gold, the color development of the reaction solution gradually changes from transparent to black along with the reduction reaction, and when the solution is further changed into wine red, the reaction is finished, so that the crude solution containing the nano gold is obtained. Under the condition of full stirring, the nano gold has better dispersibility in butanediol, and by adding polyvinylpyrrolidone (PVP), the agglomeration trend of the nano gold can be effectively inhibited, and the stability of the nano gold solution is improved.
Preferably, in step S102, a modifier is added together with polyvinylpyrrolidone, and the modifier is one or more of tetramethoxysilane, tetraethoxysilane, trimethoxysilane and triethoxysilane.
By adopting the technical scheme, the surface of the nano gold is modified by adopting the organosiloxane, a protective layer with a Si-O-Si main chain can be formed on the surface of the nano gold, the steric hindrance effect is achieved, the dispersibility and the uniformity of the nano gold particles can be further improved, and the formation of the nano gold doped self-assembled polypeptide active substance is ensured.
Preferably, the reducing agent adopts sodium citrate and sodium ascorbate with the mass ratio of (2-3): 1.
By adopting the technical scheme, the reducing agent obtained by compounding sodium citrate and sodium ascorbate has better reducibility. Wherein, the sodium citrate also has the function of a stabilizer, which is beneficial to controlling the reaction speed and ensuring the average granularity of the obtained nano gold particles to be between 10 and 20nm.
In a second aspect, the application provides a method for preparing a nanogold-doped self-assembled polypeptide active substance, which comprises the following steps:
s201: adding acetyl hexapeptide-8, palmitoyl pentapeptide-4 and glutathione into a thermophilic thermus fermentation product according to a proportion, and uniformly mixing to obtain a reaction solution;
s202: adjusting the pH value of the reaction solution to 3-6, and performing self-assembly reaction at the reaction temperature of 25-50 ℃ to obtain a self-assembled polypeptide active substance;
s203: and mixing the self-assembled polypeptide active substance with the nano gold aqueous solution according to the proportion to obtain the nano gold doped self-assembled polypeptide active substance.
By adopting the technical scheme, the obtained nano gold doped self-assembled polypeptide active substance has rich polypeptide self-assembled components and high permeability, can be effectively accumulated in the deep layer of the skin, and has more remarkable skin repairing effect and water retention effect.
In summary, the application has the following beneficial effects:
1. according to the application, the nano gold is mixed with the self-assembled polypeptide active substance to obtain the nano gold doped self-assembled polypeptide active substance with excellent stability and permeability, so that the DPPH (digital versatile disc) cleaning, fine grain fading, water locking, water retention and other effects of the self-assembled polypeptide active substance are remarkably improved.
2. According to the application, the thermophilic thermus fermentation product is added into the substrate of the self-assembled polypeptide, so that the variety of the polypeptide and amino acid in the substrate is effectively increased, and meanwhile, the variety of the self-assembled polypeptide product is enriched and the stability of the self-assembled polypeptide is improved by introducing the polypeptide substrate with acetyl, palmitoyl and other modification groups. Finally, the skin repair effect of the obtained nano gold doped self-assembled polypeptide active substance is improved.
3. According to the application, the surface of the gold nanoparticle is modified by adopting polyvinylpyrrolidone and organosiloxane molecules, so that the stability of the gold nanoparticle in a solution is obviously improved, the agglomeration phenomenon is reduced, and the stability of the gold nanoparticle doped self-assembled polypeptide active substance is further ensured.
Drawings
FIG. 1 is a chart of periocular wrinkles before and after use of the nano-gold doped self-assembled polypeptide active of example 1.
Detailed Description
Preparation example of Thermus thermophilus fermentation product
Preparation example 1-1, a Thermus thermophilus fermentation product, was prepared as follows:
step 1: inoculating thermophilic Thermus (strain number BAA-163) into a culture medium for fermentation culture, wherein the fermentation temperature is 55+ -5deg.C, the fermentation time is 12h, and the culture medium comprises the following components: 60g peptone, 4g wort, 7g anhydrous magnesium sulfate, 2.4g monopotassium phosphate, 40g ammonium sulfate, 1g anhydrous ferric chloride, 5g sodium chloride, pH was adjusted to 7.0 with acid or alkali, and water was added to prepare 20L culture broth.
Step 2: after fermentation, the thalli is crushed by adopting ultrasonic, and the obtained thalli lysate is the thermophilic thermus fermentation product.
Through detection, the content of the reduced glutathione in the Thermus thermophilus fermentation product is 20 mu mol/L; the polypeptide content in the obtained fermentation product is 0.35g/L.
Preparation examples 1-2, a Thermus thermophilus fermentation product, was prepared as follows:
step 1: inoculating thermophilic Thermus (strain number is CICC 2489) into a culture medium for fermentation culture, wherein the fermentation temperature is 75+/-5 ℃, the fermentation time is 2 hours, and the culture medium comprises the following components: 60g peptone, 4g wort, 7g anhydrous magnesium sulfate, 2.4g monopotassium phosphate, 40g ammonium sulfate, 1g anhydrous ferric chloride, 5g sodium chloride, pH was adjusted to 7.0 with acid or alkali, and water was added to prepare 20L culture broth.
Step 2: after fermentation, the thalli is crushed by adopting ultrasonic, and the obtained thalli lysate is the thermophilic thermus fermentation product.
Through detection, the content of the reduced glutathione in the Thermus thermophilus fermentation product is 15 mu mol/L; the polypeptide content in the obtained fermentation product is 0.3g/L.
Preparation example of gold nanoparticle solution
Preparation example 2-1, a nano gold solution, was prepared as follows:
s101: 90ml of purified water is added into a round bottom flask, and then gold chloride solution (1 wt percent) accounting for 3 percent of the weight of the purified water is added into the round bottom flask and heated to boiling; then adding a reducing agent (sodium citrate and sodium ascorbate with the mass ratio of 2:1) which is 1.5 times of the gold chloride solution, carrying out oxidation-reduction reaction, observing the color change of the solution, turning off a heating power supply after the solution is developed from transparent to black to wine red, stopping heating, and taking out the round-bottom flask; and cooling the nano gold to normal temperature to obtain a nano gold crude solution.
S102: adding butanediol into the crude gold nanoparticle solution, stirring for 18h, adding purified water to a volume of 100ml, and adding polyvinylpyrrolidone accounting for 0.1% of the total weight of the reaction system and tetraethoxysilane accounting for 0.05% of the total weight of the reaction system (modifier) to obtain the gold nanoparticle solution. The average particle size of the obtained nano-gold was about 10nm as measured by transmission electron microscopy.
Preparation example 2-2, a nano gold solution, was prepared as follows:
s101: 90ml of purified water was added to the round bottom flask, and a solution of gold chloride (1.5 wt%) was added in an amount of 5wt% based on the weight of the purified water, heated to boiling; then adding reducing agent (sodium citrate and sodium ascorbate with the mass ratio of 3:1) accounting for 2 times of the gold chloride solution, carrying out oxidation-reduction reaction, observing the color change of the solution, turning off a heating power supply after the solution is developed from transparent to black to wine red, stopping heating, and taking out the round-bottomed flask; and cooling the nano gold to normal temperature to obtain a nano gold crude solution.
S102: adding butanediol into the crude gold nanoparticle solution, stirring for 16h, adding purified water to a volume of 100ml, and adding polyvinylpyrrolidone accounting for 0.1 percent of the total weight of the system and trimethoxysilane accounting for 0.05 percent of the total weight of the system (modifier) to obtain the gold nanoparticle solution. The average particle size of the obtained nano gold is about 20nm through detection.
Preparation example 2-3, a nanogold solution, differs from preparation example 2-1 in that polyvinylpyrrolidone is not added in step S102; the average particle size of the obtained nano gold is about 24nm through detection.
Preparation example 2-4, a nano-gold solution, differs from preparation example 2-1 in that no modifier is added in step S102; the average particle size of the obtained nano gold is about 26nm through detection.
Preparation example 2-5, a nano gold solution, differs from preparation example 2-1 in that in step S102, polyvinylpyrrolidone and modifier are not added; the average particle size of the obtained nano gold is about 33nm through detection.
Preparation example 2-6, a nano-gold solution, differs from preparation example 2-1 in that sodium borohydride is used as a reducing agent in step S102; the average particle size of the obtained nano gold is about 6nm through detection.
Examples
Example 1, a nanogold-doped self-assembled polypeptide active, was prepared as follows:
s201: adding 0.12 kg of three polypeptide substrates of acetyl hexapeptide-8, palmitoyl pentapeptide-4 and glutathione into 108 kg of thermophilic thermus fermentation products (prepared in preparation example 1-1) according to the mass ratio of 10:1:1, and uniformly mixing to obtain a reaction solution;
s202: adjusting the pH value of the reaction solution to 3, controlling the reaction temperature to 45+/-5 ℃, simultaneously assisting in proper ultrasonic dispersion (the power is 1000W), and stirring for 2 hours to enable the substrate raw material to carry out self-assembly reaction to obtain a self-assembly polypeptide active substance;
s203: according to the proportion, the self-assembled polypeptide active substance and the nano-gold solution (prepared in preparation example 2-1) are mixed and stirred for 1h, and the nano-gold doped self-assembled polypeptide active substance is obtained.
Example 2, a nanogold-doped self-assembled polypeptide active, was prepared as follows:
s201: adding 0.22 kg of three polypeptide substrates of acetyl hexapeptide-8, palmitoyl pentapeptide-4 and glutathione into 10.98 kg of thermophilic thermus fermentation products (prepared in preparation examples 1-2) according to the mass ratio of 20:1:1, and uniformly mixing to obtain a reaction solution;
s202: adjusting the pH value of the reaction solution to 6, controlling the reaction temperature to 30+/-5 ℃, simultaneously assisting in proper ultrasonic dispersion (the power is 1000W), and stirring for 3 hours to enable the substrate raw material to carry out self-assembly reaction to obtain a self-assembly polypeptide active substance;
s203: according to the proportion, the self-assembled polypeptide active substance and the nano-gold solution (prepared in preparation example 2-2) are mixed and stirred for 0.5h, and the nano-gold doped self-assembled polypeptide active substance is obtained.
Example 3, a nanogold-doped self-assembled polypeptide active, is different from example 1 in that the nanogold solution prepared in preparation examples 2-3 is used in step S203.
Example 4, a nanogold-doped self-assembled polypeptide active, is different from example 1 in that the nanogold solution prepared in preparation examples 2-4 is used in step S203.
Example 5, a nanogold doped self-assembled polypeptide active, is different from example 1 in that the nanogold solution prepared in preparation examples 2-5 is used in step S203.
Example 6, a nanogold-doped self-assembled polypeptide active, is different from example 1 in that the nanogold solution prepared in preparation examples 2-6 is used in step S203.
Comparative example
Comparative example 1, a nanogold doped self-assembled polypeptide active, differs from example 1 in that the substrate material of the self-assembled polypeptide mixture (step S202) was not supplemented with a thermophilic thermus fermentation product.
Comparative example 2, a nanogold doped self-assembled polypeptide active, differs from example 1 in that no active polypeptide was added to the substrate material of the self-assembled polypeptide mixture (step S202).
Comparative example 3, a nanogold doped self-assembled polypeptide active, differs from example 1 in that equal amounts of glutathione are used in place of acetyl hexapeptide-8 and palmitoyl pentapeptide-4 in the substrate material of the self-assembled polypeptide mixture (step S202).
Comparative example 4, a nanogold doped self-assembled polypeptide active, differs from example 1 in that the substrate material of the self-assembled polypeptide mixture (step S202) was replaced with an equivalent amount of acetyl hexapeptide-8 instead of glutathione.
Comparative example 5, a nanogold-doped self-assembled polypeptide active, differs from example 1 in that no nanogold solution was added to the starting material of the nanogold-doped self-assembled polypeptide active (step S202).
Performance test
Test 1: safety test
The nano gold doped self-assembled polypeptide active substances prepared in all the examples and the comparative examples are subjected to a human body safety patch test with the concentration of 20%, and the test method refers to the human body skin patch test in 2015 cosmetic safety technical Specification.
The method for the skin closed patch test comprises the following steps: 30 people with age of 18-60 years are selected, and the selected area is not more than 50mm 2 The qualified plaque test equipment with the depth of about 1mm is prepared by putting 0.020mL of the nano gold doped self-assembled polypeptide active substance prepared in the examples and the comparative examples into a plaque test chamber, wherein a control hole is a blank control (no substance is placed), applying the plaque test with the nano gold doped self-assembled polypeptide active substance on the bent side of the forearm of a subject by using a hyposensitization adhesive tape, and uniformly applying the patch on the skin by using palm light pressure for 24 hours. Skin reactions were observed according to the table 1 standard for 30min (after the disappearance of the indentation), 24h and 48h after removal of the nanogold-doped self-assembled polypeptide active spot tester, respectively, and the observations were recorded. Nano gold doped self assembled polypeptide active matterThe patch test results are shown in Table 2.
TABLE 1 skin response grading Standard for skin seal Patch test
TABLE 2 human safety test results
| Sample of | 30min | 24h | 48h | Total result |
| Example 1 | 0 | 0 | 0 | 30 people have no positive reaction |
| Example 2 | 0 | 0 | 0 | 30 people have no positive reaction |
| Example 3 | 0 | 0 | 0 | 30 people have no positive reaction |
| Example 4 | 0 | 0 | 0 | 30 people have no positive reaction |
| Example 5 | 0 | 0 | 0 | 30 people have no positive reaction |
| Example 6 | 0 | 0 | 0 | 30 people averageNo positive reaction |
| Comparative example 1 | 0 | 0 | 0 | 30 people have no positive reaction |
| Comparative example 2 | 0 | 0 | 0 | 30 people have no positive reaction |
| Comparative example 3 | 0 | 0 | 0 | 30 people have no positive reaction |
| Comparative example 4 | 0 | 0 | 0 | 30 people have no positive reaction |
| Comparative example 5 | 0 | 0 | 0 | 30 people have no positive reaction |
Experimental results show that all the nano-gold doped self-assembled polypeptide active substances prepared in the examples and the comparative examples with the concentration of 20% pass the human body patch safety test.
Test 2: skin wrinkle repair Performance test
The nanogold doped self-assembled polypeptide active substances prepared in all the examples and the comparative examples are subjected to human skin wrinkle test, wherein a test instrument is a skin surface texture analysis system VisioScan VC20plus, and the test parameter is SEw. The testing method comprises the following steps: 30 healthy females are selected as each group of nano gold doped self-assembled polypeptide active matter dilution samples, the ages of the samples are 35+/-2 years, the samples are smeared on cheeks and corners of eyes, the skin wrinkle degree is tested by an instrument before the samples are smeared and after the samples are used for four weeks, the samples are tested for 3 times in parallel, the average value is obtained, and SEw values are recorded.
Wherein the change rate is the change rate relative to the change rate before use, and the calculation formula is as follows:
delta (difference) =l after four weeks 4 -L 0
Values of SEw for skin after four weeks of use versus before use
Wherein L is 0 SEw value before cosmetic application to the test area.
L 4 SEw values after four weeks of cosmetic application to the test area.
N—number of subjects.
The test results are shown in Table 3, wherein the SEw value represents the arithmetical average roughness of skin, the lower the value, the lower the degree of skin wrinkles, the higher the change rate of SEw value, and the better the repairing effect of the sample on skin wrinkles.
TABLE 3 skin wrinkle test results
Test 3: skin texture testing
The nanogold doped self-assembled polypeptide active substances prepared in all the examples and the comparative examples are subjected to human skin texture value test, the test instrument is a skin rapid optical imaging analysis system DermaTOP, and the arithmetic average roughness Ra value of the skin is analyzed by applying corresponding software. The testing method comprises the following steps: the diluted samples of the polypeptide self-assembled products participated by the gold nano-particles of each group are selected from 30 healthy females with the age of 30+/-2 years, the samples are smeared on cheeks, skin roughness tests are carried out by an instrument before the samples are smeared and after the samples are used for four weeks, the samples are tested for 3 times in parallel, an average value is obtained, and an Ra value is recorded.
Wherein the change rate is the change rate relative to the change rate before use, and the calculation formula is as follows:
delta (difference) =m after four weeks 4 -M 0
Skin roughness value after four weeks of application relative to before application
Wherein M0-the Ra value of the test zone before cosmetic use.
M4-Ra value after four weeks of cosmetic application in the test area.
N—number of subjects.
The test results are shown in Table 4, and the Ra value represents the arithmetical average roughness of the skin, the lower the numerical value is, the lower the skin texture degree is, the higher the change rate of the Ra value is, and the better the repairing effect of the sample on the skin roughness is.
Table 4 skin texture test results
Test 4: human body percutaneous water loss rate test
The nanogold-doped self-assembled polypeptide active material prepared in all the above examples and comparative examples was diluted with a certain solvent and then subjected to a human body percutaneous moisture loss test, and the test instrument was a skin surface moisture loss tester tewatemeter (TM 300) (CK company, germany).
The testing method comprises the following steps: 30 healthy females with ages of 30+/-2 years are selected as each group of nano gold doped self-assembled polypeptide active matter dilution samples, samples are smeared on cheeks, and the samples are smeared before smearing (A 0 ) And percutaneous water loss rate (A) was measured with an instrument after four weeks of sample use 4 ) The measurement was performed 3 times in parallel, an average value was taken, and the percutaneous water loss rate was recorded.
The sample of the polypeptide self-assembly product participated by each group of gold nano-particles is diluted by water, and the preparation proportion is nano-gold doped self-assembly polypeptide active matter: water=1:5.
Wherein the change rate is the change rate relative to the change rate before use, and the calculation formula is as follows:
delta (difference) =a after four weeks 4 -A 0
Percutaneous water flow rate values after four weeks of use relative to before use
Wherein A is 0 Transdermal water flow before cosmetic application in the test areaLoss rate value.
A 4 Transdermal water flow rate values after four weeks of cosmetic application to the test area.
N—number of subjects.
The smaller the percutaneous water loss rate is, the less the water is lost, the stronger the water locking capability is, and the better the skin barrier is; the greater the rate of change of the percutaneous water flow rate value is, the better the water-retaining and water-locking effect of the sample is. The results of the percutaneous moisture loss statistics are shown in Table 5.
TABLE 5 results of human percutaneous Water loss test
Test 5: radical scavenging test
The nano gold doped self-assembled polypeptide active substances prepared in the examples and the comparative examples are subjected to an antioxidation test of DPPH free radical clearance, and a common antioxidant Vc is selected as a control for more intuitively observing the antioxidation capability of the nano gold doped self-assembled polypeptide active substances. The specific method comprises the following steps:
the experimental steps are as follows: adding 2mL of DPPH ethanol solution and 2mL of sample to be detected (nano gold doped self-assembled polypeptide active substance and Vc solution) into a test tube, shaking and uniformly mixing, then carrying out light-shielding reaction for 30min, measuring a light absorption value A at 517nm, simultaneously measuring a light absorption value A0 of 2mL of DPPH ethanol solution and 2mL of absolute ethanol mixed solution, and measuring a light absorption value Ab of 2mL of absolute ethanol and 2mL of sample mixed solution to be detected, and calculating DPPH free radical clearance:
DPPH radical clearance (%) = (A0- (A-Ab))/a0×100
The test results are shown in table 6, and the higher the DPPH clearance of the self-assembled product, the stronger the oxidation resistance of the nano-gold doped self-assembled polypeptide active.
TABLE 6 free radical scavenging test results
| Sample of | Clearance of DPPH (%) |
| Vc solution (control group) | 71 |
| Example 1 | 78 |
| Example 2 | 75 |
| Example 3 | 73 |
| Example 4 | 72 |
| Example 5 | 67 |
| Example 6 | 73 |
| Comparative example 1 | 65 |
| Comparative example 2 | 67 |
| Comparative example 3 | 68 |
| Comparative example 4 | 63 |
| Comparative example 5 | 62 |
Application example
The application example provides repair emulsion containing the nano gold doped self-assembled polypeptide active substance provided in the example 1, and the specific formula is as follows:
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the product of example 1 was selected and applied to the volunteer's eyes for 4 weeks, and the change in periocular fine lines is shown in fig. 1.
In fig. 1, from left to right, the subject was photographed for periocular wrinkles before use, 2 weeks of use, and 4 weeks of use, respectively (instrument was VisioScan VC98 (Courage & Khazaka, germany). The white (light) areas in fig. 1 represent periocular fine lines, and it can be seen that there is a significant reduction in periocular fine lines at weeks 2 and 4.
Analysis of test results:
(1) By combining examples 1 to 6 and comparative examples 1 to 4 and combining tables 3 to 6, the application can effectively improve the effects of repairing, water retention, wrinkle resistance and the like of products obtained by self-assembly by adopting thermophilic thermus fermentation products and active polypeptides such as acetyl hexapeptide-8, palmitoyl pentapeptide-4, glutathione and the like as substrates of self-assembly polypeptides.
The reason for this may be that, for the thermophilic thermus fermentation product, it contains nucleic acid, protein, lipid, polysaccharide, secondary metabolite, and natural polypeptide such as glutathione, and amino acid with different charges and different hydrophobicity, so that the non-covalent interaction between the active peptides can be changed, the self-assembly of the active peptides is promoted, the diversity of the self-assembly product and the utility is enriched, and finally, the effects of the product on skin repair, water retention, anti-wrinkle and the like are effectively improved.
For active polypeptides such as acetyl hexapeptide-8, palmitoyl pentapeptide-4, glutathione and the like, the acetyl hexapeptide-8, palmitoyl pentapeptide-4 and the glutathione have acetyl, palmitoyl and other modification groups, on one hand, the self-assembly of the polypeptides is promoted in a synergistic way, and the self-assembled polypeptides with more abundant types are obtained; on the other hand, acetyl and palmitoyl have better lipophilicity, are favorable for the penetration of self-assembled polypeptide active substances into deep layers of skin, and improve the effects of repairing, water retention, wrinkle resistance and the like.
(2) It can be seen from the combination of examples 1 to 6 and comparative example 5 and the combination of tables 3 to 6 that the addition of the nanogold to the self-assembled polypeptides can effectively improve the effects of repairing, retaining water, resisting wrinkles and the like of the self-assembled product. The reason for this may be that the nanogold has higher affinity with glutathione, and the active groups on the surface of the nanogold can be combined with sulfhydryl groups in polypeptide molecules, so that the stability of a self-assembled polypeptide system is increased, the possibility of denaturation of the self-assembled polypeptide is reduced, and the repair effect of the active substances of the self-assembled polypeptide doped with the nanogold is ensured.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (5)
1. The nano gold doped self-assembled polypeptide active matter is characterized by being prepared from the self-assembled polypeptide active matter and nano gold solution in a weight ratio of (50-100): 1; the self-assembled polypeptide mixture is obtained by self-assembling active polypeptide and Thermus thermophilus fermentation product with the dosage ratio of (0.1-2) (90-99.8); the active polypeptide adopts acetyl hexapeptide-8, palmitoyl pentapeptide-4 and glutathione with the mass ratio of (8-20) of 1:1; the average granularity of the nano gold in the nano gold solution is 10-20 nm; the thermophilic thermus fermentation product is obtained by fermenting and culturing a strain with the preservation number of BAA-163 at 50-80 ℃ and crushing the strain for 2-12 h.
2. The nanogold-doped self-assembled polypeptide active according to claim 1, wherein the nanogold solution is prepared according to the following method:
s101: heating a gold ion solution to boiling, then adding a reducing agent for oxidation-reduction reaction, and obtaining a nano gold crude solution when the solution is reddish wine;
s102: and adding butanediol into the crude gold nanoparticle solution, stirring uniformly, and adding polyvinylpyrrolidone to obtain the gold nanoparticle solution.
3. The nanogold-doped self-assembled polypeptide active material according to claim 2, wherein in the step S102, a modifier is added together with polyvinylpyrrolidone, and the modifier is one or more of tetramethoxysilane, tetraethoxysilane, trimethoxysilane or triethoxysilane.
4. The nanogold-doped self-assembled polypeptide active material according to claim 2, wherein the reducing agent is sodium citrate and sodium ascorbate with a mass ratio of (2-3): 1.
5. The method for preparing the nanogold-doped self-assembled polypeptide active material according to any one of claims 1 to 4, which is characterized by comprising the following steps:
s201: adding acetyl hexapeptide-8, palmitoyl pentapeptide-4 and glutathione into a thermophilic thermus fermentation product according to a proportion, and uniformly mixing to obtain a reaction solution;
s202: adjusting the pH value of the reaction solution to 3-6, and performing self-assembly reaction at the reaction temperature of 25-50 ℃ to obtain a self-assembled polypeptide active substance;
s203: and mixing the self-assembled polypeptide active substance with the nano gold solution according to the proportion to obtain the nano gold doped self-assembled polypeptide active substance.
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