CN117942276A - Compound peptide for realizing percutaneous absorption promotion of fibroblast proliferation under low concentration and preparation method thereof - Google Patents

Compound peptide for realizing percutaneous absorption promotion of fibroblast proliferation under low concentration and preparation method thereof Download PDF

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CN117942276A
CN117942276A CN202410355951.4A CN202410355951A CN117942276A CN 117942276 A CN117942276 A CN 117942276A CN 202410355951 A CN202410355951 A CN 202410355951A CN 117942276 A CN117942276 A CN 117942276A
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compound peptide
compound
essence
peptide
low concentration
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CN117942276B (en
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查建生
童莉
郑莹
郑云云
孙翠娥
王会玉
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Nanjing Sbaike Biotechnology Co ltd
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Nanjing Sbaike Biotechnology Co ltd
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Abstract

The invention discloses a compound peptide for realizing percutaneous absorption promotion of fibroblast proliferation under low concentration and a preparation method thereof, comprising the following steps: acetyl hexapeptide-8, dipeptide diamino Ding Xianbian-yl amide diacetate, glutaminyl ethyl imidazole, hydrophilic silica aerogel, glycerol, preservative and the balance water; the preparation method comprises stirring glycerol, antiseptic and water to obtain a uniform clear solution; and (3) when the system is cooled to room temperature, adding acetyl hexapeptide-8, dipeptide diamino Ding Xianbian-yl amide diacetate and glutaminyl ethyl imidazole, stirring uniformly, and finally adding hydrophilic silica aerogel, and mixing uniformly. The compound peptide can permeate into the dermis layer under low concentration, promote fibroblast proliferation, promote collagen self-generation from inside to outside, wake up fibroblasts, stimulate the expression of elastin genes, thereby promoting the synthesis of collagen, regulating the normalization of biological clock law and adapting to the change of day and night environment and biological clock.

Description

Compound peptide for realizing percutaneous absorption promotion of fibroblast proliferation under low concentration and preparation method thereof
Technical Field
The invention belongs to the field of compound peptide preparation, and in particular relates to a compound peptide capable of realizing percutaneous absorption under low concentration and promoting fibroblast proliferation and a preparation method thereof.
Background
Elastin is an important element that makes up the dermis layer, accounting for about 5% of the body, but governs 95% of skin elasticity. However, with age, elastin production is stopped, the elastin component in the skin is reduced, and the associated reduction provides network support for maintaining a firm skin, with the result that aging phenomena such as skin sagging, blurring contours, fine lines on the skin, etc. occur.
Acetyl hexapeptide-8 is used as a component of a well-known anti-aging product, and is mainly used for fading expression lines in cosmetics. It also belongs to neurotransmitter inhibiting peptide, and can reduce muscle contraction by inhibiting the release of nerve conduction element acetylcholine, thereby reducing the generation of dynamic lines and expression lines. It has a mechanism of action similar to that of botulinum neurotoxin, it mimics the N-terminal structure of the small associated protein and competes with the small associated protein for its position in the SNARE complex, thereby modulating SNARE complex formation, reducing acetylcholine release, and achieving muscle relaxation. Compared with botulinum neurotoxin, acetyl hexapeptide-8 can interfere with the formation and stability of the complex, does not cause irreversible damage, and shows great advantage of its extremely low toxicity (2000 mg/kg), high safety, and can be used daily, so it becomes a star component of anti-wrinkle products.
However, to be effective against the skin, the acetyl hexapeptide-8 must reach the target site in sufficient concentration, and the skin itself has considerable barrier properties limiting its penetration in the skin, the efficiency of the polypeptide's action to reach the target site of the skin is reduced.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing a compound peptide, which can realize transdermal penetration into a dermis layer on the basis of low-concentration and low-content compound polypeptide so as to promote collagen self-generation, wake up fibroblasts and stimulate the expression of elastin genes, thereby promoting the synthesis of collagen, regulating the normalization biological clock law, adapting to the day-night environment and biological clock change and achieving the effects of resisting the senium and the aging.
The technical scheme is as follows: the invention realizes the compound peptide which is absorbed transdermally under low concentration and promotes fibroblast proliferation, and comprises the following components in percentage by mass: acetyl hexapeptide-8.005-1.0%, dipeptide diamino Ding Xianbian-yl amide diacetate 0.005-0.1%, glutaminyl ethyl imidazole 0.005-0.1%, hydrophilic silicon dioxide aerogel 0.015-1.5%, glycerin 1-10%, preservative 0.1-1.5% and the balance of water.
According to the invention, the hydrophilic silica aerogel is used as an active component carrier, based on the porous network structure of the hydrophilic silica aerogel, the polypeptide substances acetyl hexapeptide-8, dipeptide diamino Ding Xianbian-yl amide diacetate and glutaminyl ethyl imidazole are compounded and loaded on the carrier, when the hydrophilic performance of the carrier is used for a skin layer, the water content of the skin horny layer can be increased, the cells (especially the cells of the basal horny layer) of the layer are expanded (namely the volume is increased), so that the active polypeptide substances loaded on the hydrophilic silica aerogel penetrate into the dermis to form an inside-outside promotion effect, and the compound peptides of the acetyl hexapeptide-8, the dipeptide diamino Ding Xianbian-yl amide diacetate and the glutaminyl ethyl imidazole can promote collagen to be self-produced at a low content concentration, promote fibroblast proliferation and stimulate the expression of elastin genes, thereby promoting the synthesis of collagen, regulating the normalization biological clock law, adapting to the change of day and night environment and biological clock, achieving the effects of resisting aging and resisting aging, and effectively solving the technical problem that the prior art can be realized only under the high target concentration; in addition, by virtue of the hydrophilic silica aerogel, the compound polypeptide component can be stabilized in the system, the stability of the compound peptide liquid is improved, and the compound peptide liquid can be kept to stand for a long time, still presents colorless, transparent and uniform liquid with fluidity and is not layered.
Further, the hydrophilic silica aerogel adopted by the compound peptide has the porosity of 95-99%, the pore diameter of 10-50nm, the specific surface area of 20-1000m 2/g, the density of 3-300kg/m 3 and the diameter of colloid particles forming a network of less than 100nm.
Further, the preservative adopted by the compound peptide is prepared by compounding 60-80% of octyl glycol and 20-40% of ethylhexyl glycerol.
The essence prepared based on the compound peptide comprises, by mass, 1-10% of the compound peptide, 1-10% of trehalose, 0.05-0.15% of L-2-oxo-thiazolidine-4-carboxylic acid, 2-6% of butanediol, 0.15-0.2% of xanthan gum, 0.1-0.2% of acrylic acid (esters)/C10-30 alkanol acrylate crosslinked polymer, 0.1-0.2% of allantoin, 0.5-1% of 1, 2-hexanediol, 0.5-1% of p-hydroxyacetophenone, 0.1-0.3% of dipotassium glycyrrhizinate, 0.15-0.2% of essence and the balance of water.
The gel prepared from the compound peptide comprises, by mass, 1-10% of the compound peptide, 1-10% of trehalose, 0.05-0.15% of L-2-oxo-thiazolidine-4-carboxylic acid, 2-6% of butanediol, 0.15-0.2% of xanthan gum, 0.4-0.5% of acrylic acid (esters)/C10-30 alkanol acrylate crosslinked polymer, 0.5-1% of 1, 2-hexanediol, 0.5-1% of p-hydroxyacetophenone, 0.4-0.5% of a solubilizer, 0.1-0.2% of essence and the balance of water. Preferably, the solubilizing agent comprises 51-57% PPG-26-butanol polyether-26, 34-38% PEG-40 hydrogenated castor oil and the balance water.
The emulsion prepared from the compound peptide comprises, by mass, 1-10% of the compound peptide, 1-10% of trehalose, 0.05-0.15% of L-2-oxo-thiazolidine-4-carboxylic acid, 2-6% of butanediol, 0.15-0.2% of xanthan gum, 0.1-0.2% of allantoin, 1-3% of tea seed oil, 1-3% of ethylhexyl palmitate, 1-2% of polydimethylsiloxane, 1-2% of dioctyl carbonate, 1-3% of shea butter, 0.5-1% of glycerol stearate, 1-3% of caprylic/capric triglyceride, 1-3% of squalane, 0.2-0.4% of tocopheryl acetate, 0.5-1% of ammonium acryloyldimethyl taurate/VP copolymer, 0.5-1% of phenoxyethanol, 0.1-0.3% of dipotassium glycyrrhizinate, 0.1-0.2% of essence and the balance water.
The cream prepared from the compound peptide comprises, by mass, 1-10% of compound peptide, 1-10% of trehalose, 0.05-0.15% of L-2-oxo-thiazolidine-4-carboxylic acid, 2-6% of butanediol, 0.1-0.2% of xanthan gum, 0.1-0.2% of allantoin, 1-3% of tea seed oil, 1-3% of ethylhexyl palmitate, 1-2% of polydimethylsiloxane, 0.2-0.4% of tocopheryl acetate, 1-2% of dioctyl carbonate, 1-3% of cetostearyl alcohol, 0.5-1% of glycerol stearate, 1-3% of butter fruit, 1-3% of squalane, 1-3% of caprylic/capric acid triglyceride, 1-3% of hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, 0.5-1% of caprylic glycol triglyceride, 0.1-0.2% of essence and water.
The method for preparing the compound peptide comprises the following steps: stirring glycerol, antiseptic and water at 80-90deg.C to obtain a uniform clear solution; and (3) when the system is cooled to room temperature, adding acetyl hexapeptide-8, dipeptide diamino Ding Xianbian-yl amide diacetate and glutaminyl ethyl imidazole, stirring uniformly, and finally adding hydrophilic silica aerogel, and mixing uniformly.
The beneficial effects are that: compared with the prior art, the invention has the remarkable advantages that: the compound peptide can permeate into the dermis layer at low concentration and low content, so as to promote collagen self-generation and wake up fibroblasts from inside to outside, promote fibroblast proliferation, stimulate the expression of elastin genes, promote the synthesis of collagen, regulate the normalization biological clock law, adapt to the change of day and night environment and biological clock, and achieve the effects of resisting the primary aging and resisting the aging.
Drawings
FIG. 1 is a graph showing the stability of the compound peptide prepared in example 1 of the present invention;
FIG. 2 is a graph showing cell viability of the compound peptide prepared in example 1 of the present invention;
FIG. 3 is a diagram showing the morphology of the compound peptide cell prepared in example 1 of the present invention;
FIG. 4 is a histogram of PERI gene expression level of the compound peptide prepared in example 1 of the present invention;
FIG. 5 is a graph of cell viability of monomeric GAI (1%);
FIG. 6 is a diagram of the morphology of monomeric GAI (1%) cells;
FIG. 7 is a histogram of the expression level of the PERI gene of monomeric GAI (1%);
Fig. 8 shows changes in wrinkle volume, wrinkle area ratio, wrinkle depth, immediate elasticity, skin roughness at various times after using the essence prepared in example 2 of the present invention (< 0.05, <0.01, <0.001 vs 0 d);
FIG. 9 is a graph showing the comparison of wrinkles of 3D forehead at different time periods after using the essence prepared in example 2 of the present invention;
fig. 10 is a graph showing skin texture comparison at various periods of time after using the essence prepared in example 2 of the present invention;
FIG. 11 is a graph showing the comparison of time periods of the skin-sealing patch test (after 30 minutes of patch removal) using the essence prepared in example 2 of the present invention;
FIG. 12 is a graph showing a comparison of time periods of a skin-sealing patch test (after removing a patch for 24 hours) using the essence prepared in example 2 of the present invention;
Fig. 13 is a comparison graph of time periods of a skin-sealing patch test (after 48 hours of patch removal) using the essence prepared in example 2 of the present invention.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and examples.
The raw materials used in the present invention are commercially available. Wherein, the preservative adopted by the invention is compounded by 60-80% of octyl glycol and 20-40% of ethylhexyl glycerol. The solubilizer comprises 51-57% PPG-26-butanol polyether-26, 34-38% PEG-40 hydrogenated castor oil and the rest water. The purchased hydrophilic silica aerogel has the porosity of 95-99%, the pore diameter of 10-50nm, the specific surface area of 20-1000m 2/g, the density of 3-300kg/m 3, and the diameter of colloid particles forming a network is less than 100nm.
Example 1
The raw material components and contents of the compound peptide of this example are shown in table 1 below.
TABLE 1 raw material composition and content of the Compound peptide of example 1
Sequence number Raw materials Content/%
1 Acetyl hexapeptide-8 0.025
2 Dipeptide diamino Ding Xianbian-ylamide diacetate 0.025
3 Glutaminyl ethyl imidazole 0.05
4 Hydrophilic silica aerogel 0.1
5 Glycerol 5
6 Preservative agent 1.0
7 Water and its preparation method To 100
The preparation method of the compound peptide of the embodiment comprises the following steps: stirring glycerol, antiseptic and water at 80-90deg.C to obtain a uniform clear solution; and (3) when the system is cooled to room temperature, adding acetyl hexapeptide-8, dipeptide diamino Ding Xianbian-yl amide diacetate and glutaminyl ethyl imidazole, uniformly stirring, and finally adding hydrophilic silica aerogel, and uniformly stirring.
Performance test 1 stability test
The compound peptide prepared in example 1 was allowed to stand for stability detection, and the results obtained are shown in FIG. 1 below. As can be seen from the figure 1, the initial appearance of the compound peptide prepared by the invention is colorless, transparent and uniform fluid; and after 24h, 3d, 7d, 14d, 30d, 60d and 90d, the appearance of the sample is not changed obviously under the 5 conditions of 4 ℃/RT/-18 ℃/illumination/40 ℃. Therefore, the compound peptide prepared by the invention has strong stability.
Performance test 2 promotion of collagen secretion Property
1. Experimental materials
(1) Cell line: mouse embryonic fibroblasts (NIH/3T 3);
(2) Culture medium: 10% new born calf serum+90% dmem medium;
(3) Detection reagent: PBS buffer, 0.25% pancreatin (EDTA), MTT, DMSO, collagen ELISA assay kit.
2. Experimental procedure
NIH/3T3 cell fusion reaches about 80%, the cells are digested by pancreatin, cell density is regulated, the cells are uniformly inoculated in 96 well plates, the number of cells in each well is 1 multiplied by 104, the cells are placed in a 37 ℃ and 5% CO 2 incubator for culture overnight, old culture medium is sucked, 0.2 mL of culture medium containing samples with different concentrations is respectively added into each well, blank control is arranged, and culture is continued for 48 h.48 After h, each cell was collected separately and the effect of the sample on the collagen content of NIH/3T3 cells was detected using a collagen ELISA assay kit. The results obtained are shown in table 2 below.
TABLE 2 intracellular collagen content and promotion rate under the action of different concentrations of the Compound peptide
Concentration (mg/mL) Blank control group 20 10 5 2.5
Collagen content (ng/mL) 220.5±15.4 290.5±20.6 263.6±0.6 257.3±3.2 246.4±1.9
Promotion rate (%) / 31.8 19.6 16.7 11.8
As shown in Table 2, the compound peptide prepared by the invention has a promotion effect on the secretion of fibroblast collagen, and when the concentration is 20 mg/mL, the promotion rate can reach 31.8%, and the promotion effect shows concentration dependency. Therefore, the compound peptide prepared by the invention can permeate into the dermis layer, and has promotion effect on collagen regeneration in the dermis layer.
Performance test 3 fibroblast proliferation
1. Experimental materials
(1) Cell line: mouse embryonic fibroblasts (NIH/3T 3);
(2) Culture medium: 10% new born calf serum+90% dmem medium;
(3) Detection reagent: PBS buffer, 0.25% pancreatin (EDTA), MTT, DMSO.
2. Experimental procedure
The cell fusion degree reaches about 80%, the cell density is adjusted to be 1X 105/mL by digestion of pancreatin, and the cells are uniformly inoculated into 96-well plates with 0.1mL per well. After 24h old medium was aspirated and then the cells were synchronized by changing to 0.4% serum DMEM medium for further incubation for 24 h. Old medium was aspirated and 0.1mL of 0.4% serum DMEM medium containing samples at different concentrations was added while solvent blank wells (solvent-only medium, cell-free) were established. After 24h of incubation, 20. Mu.L of MTT solution (5 mg/mL) was added to each well and incubation was continued for 3h. If the drug reacts with MTT, the culture broth may be discarded first, carefully rinsed 2-3 times with PBS, and then the MTT-containing culture broth may be added. After the culture is finished, carefully sucking the culture solution in the holes, adding 150 mu L of dimethyl sulfoxide into each hole, and putting the mixture on a shaking table to shake for 10min at a low speed so as to fully dissolve crystals. The absorbance of each well was measured at 490nm on a microplate reader. Cell viability% = [ OD (sample) -OD (blank) ]/[ OD (solvent control) -OD (blank) ]x100%. The results obtained are shown in table 3 below.
TABLE 3 cell viability on exposure to different concentrations of the formulations and their active ingredients
Concentration (mg/mL) Blank control group 20 10 5
24H cell viability (%) 100±6.8 117.7±18.1 102.8±5.9 99.0±7.5
48H cell viability (%) 100±5.8 159.0±11.0 156.7±9.2 136.8±9.4
As can be seen from Table 3, the 20 mg/mL formulation increased the fibroblast viability by 59.0% at 48 hours. And the lifting of the compound of 5mg/mL can reach about 36%, namely the compound can lift the activity of the fibroblast under low concentration.
Performance detection 4 Complex peptide rhythmic Gene expression level detection
The detection method is carried out according to the detection method of PER1 gene expression quantity based on keratinocyte.
1. Test system
The cells used in this test were keratinocytes, lot number: ep23082101, available from Guangdong Boxi Biotechnology Inc.
2. Main reagent
KcGrowth culture (Guangdong Boxi organism), PBS (Soy treasure), MTT (Sigma), DMSO (Sigma), RNAiso Plus (Ai Kerui organism), reverse transcription kit (Ai Kerui organism), and fluorescent dye (Ai Kerui organism).
3. Main equipment
CO 2 incubator (Thermo, 150I), ultra clean bench (Suzhou Antai, SW-CJ-1F), microplate reader (BioTek, epoch), ordinary PCR instrument (Bori), fluorescent quantitative PCR instrument (BioRad, CFX-96), inverted microscope (Olympus, CKX 53).
4. Cytotoxicity test
(1) Cell viability test method
1) Cell inoculation: after resuscitating the cells, when the plating rate reaches about 60%, the cells are inoculated into a 96-well plate and incubated overnight in a CO 2 incubator (37 ℃, 5% CO 2);
2) Test grouping: the test set was littering, solvent control, positive control and sample. In the sample group, 8 concentrations are set for each sample, and 3 duplicate wells are set for each concentration;
3) Preparing liquid: preparing sample working solutions with different concentrations according to a test concentration setting table 4;
4) Administration: administration was performed when the cell plating rate in 96-well plates reached 50-60%. A culture rate of 200. Mu.L of 10% DMSO per well was added to the solvent control group; 200 mu L of culture solution containing samples with corresponding concentrations is added into each hole of the sample group; the litters were inoculated without cells and 200. Mu.L of cell culture medium was added. After the completion of the administration, the 96-well plate was placed in a CO 2 incubator (37 ℃, 5% CO 2) and cultured for 24 hours;
5) And (3) detection: after the cells are incubated for 24 hours, the supernatant is discarded, MTT working solution (0.5 mg/mL) is added, the cells are incubated for 4 hours at 37 degrees away from light, after the incubation is finished, the supernatant is discarded, 150 mu LDMSO is added to each hole, and OD value is read at 490 nm;
6) Cell relative viability calculation: according to the formula, the relative cell viability (%) = (sample well OD-littering well OD)/(solvent control well OD-littering well OD) ×100%.
Table 4 test concentration profile
(2) Cell morphology test method
1) Cell inoculation: after resuscitating the cells, when the plating rate reaches about 60%, the cells are inoculated into a 96-well plate and incubated overnight in a CO 2 incubator (37 ℃, 5% CO 2);
2) Test grouping: the test sets a solvent control group and a sample group. In the sample group, 5 concentrations are set for each sample;
3) Administration: administering when the cell plating rate in the 24-well plate reaches 50-60%; adding 1mL of culture solution into each hole of the solvent control group; adding 1mL of culture solution containing a sample to be tested with corresponding concentration into each hole of the sample group; after the completion of the administration, the 24-hour well plate was placed in a CO 2 incubator (37 ℃, 5% CO 2) and cultured for 24 hours;
4) Photographing: after the incubation, the supernatant was discarded and photographed under an inverted microscope.
(3) Test results
The cytotoxicity test results are shown in Table 5 below. The results of the cell viability assay are shown in FIG. 2 below. The cell morphology is shown in FIG. 3 below.
TABLE 5 MTT assay results for the Compound peptides
From the MTT and morphological results, it is clear that the formulated peptide prepared according to the invention does not show significant cytotoxicity in the concentration range of 0.3125% based on keratinocytes.
5. Gene expression level test
(1) Test method
1) Cell inoculation: after resuscitating the cells, when the plating rate reached about 60%, inoculating the cells to a 6-well plate, incubating in a CO 2 incubator (37 ℃, 5% CO 2) overnight;
2) Preparing liquid: preparing test object working solutions according to the test components (table 6);
3) Administration: according to the test components, when the cell plating rate in the 6-hole plate reaches 40-60%, grouping drug administration is carried out, and 3 compound holes are arranged in each group; adding 2mL of culture solution into each hole of a blank control group, adding 2mL of culture solution containing samples with corresponding concentrations into each hole of a sample group, and placing a 6-hole plate into a CO 2 incubator (37 ℃ C., 5% CO 2) for culturing for 6h and 24h respectively after the administration is completed;
4) Collecting cells: after incubation, the old liquid is sucked and removed, PBS is used for cleaning twice, 1mLRNAiso Plus is added into each hole, and after the lysed cells are blown, the sample is collected;
5) And (3) gene expression detection: extracting RNA, carrying out reverse transcription to cDNA, carrying out fluorescent quantitative PCR detection, and carrying out result calculation by adopting a2 -ΔΔCT method;
6) And (5) calculating an up-regulation rate: up-regulation (%) = (sample group-blank group)/blank group x 100%;
7) Results statistical analysis: plotted using GRAPHPAD PRISM, the results are expressed as mean±sd; the comparison among the groups adopts t-test statistical analysis; statistical analysis was double tailed. P <0.05 was considered to have significant differences and P <0.01 was considered to have very significant differences.
TABLE 6 test Components
(2) Test results
Based on the test method, samples were collected after RNAiso Plus treatments were performed on keratinocytes, and RNA extraction, reverse transcription, i.e., fluorescent quantitative PCR operations were performed according to the kit instructions, and the detection results are shown in table 7 and fig. 4 below.
TABLE 7 PER1 Gene test results summary table
As can be seen from the results of table 7 and fig. 4, the PER1 gene expression level was significantly reduced when the compound peptide was administered for 24 hours, compared with the BC group; at 6h of administration, there was no significant change in PER1 gene expression.
Namely, based on keratinocytes, compared with a control group, the compound peptide prepared by the invention has no obvious change in PER1 gene expression amount when being administered for 6 hours at the concentration of 0.3125 percent; at this concentration, PER1 gene expression was significantly down-regulated when administered for 24 h. Therefore, the compound peptide prepared by the invention can reduce the expression of PER1 genes, regulate and control the disordered genes and regulate biological clocks.
Performance detection 5 glutaminyl ethyl imidazole (GAI) Gene expression level detection
In order to verify that the regulation of the compound peptide of the invention under low content and low concentration is normalized to biological clock law, a comparison test is designed, and the basic component formula is the same as that of example 1, except that the content of polypeptide components and glutaminyl ethylimidazole is shown in the following table 8.
Table 8 GAI (1%) raw material composition and content
Sequence number Raw materials Content/%
1 Acetyl hexapeptide-8 -
2 Dipeptide diamino Ding Xianbian-ylamide diacetate -
3 Glutaminyl ethyl imidazole 1
4 Hydrophilic silica aerogel 1
5 Glycerol 5
6 Preservative agent 1.0
7 Water and its preparation method To 100
The detection is performed according to the detection method of the performance detection 4. The concentration gradients are shown in table 9 below. The MTT assay results are shown in Table 10 below. The results of the cell viability assay are shown in FIG. 5 below. The cell morphology is shown in FIG. 6 below.
TABLE 9 test concentration setting table
MTT assay results of Table 10 GAI (1%)
From the MTT and morphological results, it is clear that the GAI (1%) prepared by this property does not show significant cytotoxicity in the concentration range of 0.625% based on keratinocytes.
The GAI (1%) was subjected to gene expression level test, and the test packets and PER1 gene test results are shown in Table 11, table 12 and FIG. 7, respectively.
Table 11 test components
TABLE 12 PER1 Gene test results summary table
Compared with the BC group, GAI (1%) -0.625% has significantly down-regulated PER1 gene expression level at 24h of administration; at 6h of administration, there was no significant change in PER1 gene expression. And the comparative performance detection 4 shows that when the PER1 gene detection is carried out on the single-component GAI, the content of the GAI needs to be 1 percent and the concentration of the GAI is 0.625 percent, but the compound peptide disclosed by the invention only needs to be 0.05 percent, and the total compound peptide concentration can achieve the rhythm under the condition of only 0.3125 percent. Therefore, the compound peptide provided by the invention can synergistically promote the rhythmic action.
Comparative example 1
The basic procedure was the same as in example 1, except that different monomers were used to prepare solutions, the component contents of which are shown in Table 13 below.
TABLE 13 component content tables of the Complex peptides and different monomers
Performance test 6 promotion of collagen secretion Property
The comparative example 1 was subjected to collagen secretion performance test using the test method of performance test 2, and the obtained results are shown in table 14 below.
TABLE 14 content and promotion rate of intracellular collagen under the action of Compound peptide and monomer at different concentrations
As is clear from Table 14, the promotion rates of the compound peptide and the active ingredient on the collagen secretion of the fibroblast were 31.8%, 8.7%, 30.1% and 1.4%, respectively, and the promotion effects exhibited concentration dependence at the concentration of 20 mg/mL. Overall, the compound peptide (AH 8P) > (GAI) > (SKE-P).
Performance test 7 fibroblast proliferation
The comparative example 1 was subjected to fibroblast proliferation performance test by the test method of performance test 3, and the obtained results are shown in Table 15 below.
TABLE 15 cell viability on exposure to different concentrations of the formulations and their active ingredients
According to the experimental results, the compound peptide of 20 mg/mL improves the activity of the fibroblast by 59.0% at 48 hours. And as can be seen from the table 15, the ability of the compound prepared by the invention to promote the activity of the fibroblast is far better than that of the monomer at the high concentration of 20 mg/mL under the low concentration of 5 mg/mL. Therefore, the compound disclosed by the invention is based on compounding the three monomers, so that not only can the activity of the fibroblasts be improved, but also the activity of the fibroblasts at low concentration can be realized.
Comparative example 2
The basic procedure was the same as in example 1, except that the hydrophilic silica aerogel was not used as a carrier, as the component contents are shown in Table 16 below.
Table 16 component content table of the Compound peptide
Performance test 8 transdermal absorption Properties
The transdermal absorption rates of the compound prepared in example 1 and the compound prepared in comparative example 2 were evaluated.
1. Test materials
Transdermal diffusion instrument RYJ-12B, test tube, syringe, surgical scissors, piglet skin (3-7 days).
2. Test procedure
The permeation diffusion device is formed by combining an upper cylindrical glass and a lower cylindrical glass, the skin is clamped between the upper chamber and the lower chamber, the upper chamber is a diffusion chamber, the lower chamber is a receiving chamber, and a sampling tube is arranged at the bottom of the receiving chamber for taking liquid. The osmotic diffusion area was about 2.2cm 2. The osmotic diffusion device is placed in a constant temperature water bath, the temperature is maintained at 37+/-1 ℃, and the dynamic environment of the receiving chamber is maintained by a multifunctional stirrer. The pretreated pigskin is fixed between two chambers, and 2mL of sample is added into a diffusion chamber for fixation. The receiving chamber was filled with purified water, the volume recorded, and stirring was continued, and 1.0mL (1.0 mL of purified water was immediately added) was sampled at 1h,2h,4h,6h,8h, and 24h, respectively. The sampling is determined according to high performance liquid chromatography (2020 edition of Chinese pharmacopoeia, four-part rule 0512) and the sample concentration Cn in the receiving chamber at different sampling times is calculated.
The cumulative permeation quantity per unit area Qn of the different sampling times was calculated according to the following formula:
；
Where Qn is the cumulative permeation per unit area (μg/cm 2), cn is the drug concentration measured at the nth time point (μg/mL), ci is the drug concentration measured at the ith time point (μg/mL), V is the total volume of the receiving cell (mL), vs is the volume per sample (1 mL), and S is the permeation diffusion area (cm 2).
The same set of diffusion experiments used as much skin as possible at the same location, because the skin locations differ, the stratum corneum thickness differs, and the drug penetration is proportional to the thickness. After the diffusion cell is installed, the skin should be stretched, otherwise the diffusion area is increased. When the ointment or cream is filled, a small amount of the ointment or cream can be firstly coated on the surface of the skin, and then the ointment or cream can be continuously filled so as to ensure that the ointment is tightly contacted with the skin, and an air chamber is not formed between the ointment and the skin. After the diffusion chamber is installed, the ointment should be applied to the skin evenly by tapping down a few days. After the receiving chamber is filled with the receiving liquid, the air in the chamber should be completely removed. The magnetic stirring speed is suitable for uniformly mixing liquid, vortex can be formed when the magnetic stirring speed is too high, the diffusion area is reduced, and the upper layer solution and the lower layer solution of the receiving chamber are difficult to uniformly mix when the magnetic stirring speed is too low.
3. Test results
The test results obtained are shown in table 17 below.
Table 17 transdermal absorption rate of the compound peptides prepared in example 1 and comparative example 2
From this table 17, it is clear that the percutaneous absorption rate of comparative example 2 was 34.0% and the comparative absorption rate of example 1 was 45.0% at 24 hours. From this, it is understood that the hydrophilic silica gel used in the present invention can effectively promote permeation.
Example 2 essence of Compound peptide
Based on the compound peptide of the example 1, the compound peptide is prepared into essence, and the anti-aging performance is detected. The formulation of the essence is shown in the following table 18.
Table 18 essential oil component formulations formulated based on the compound peptides of example 1
Sequence number Raw materials Content/%
1 Complex peptides 5
2 Trehalose 5
3 L-2-oxo-thiazolidine-4-carboxylic acid 0.08
4 Butanediol (butanediol) 5
5 Xanthan gum 0.18
6 Acrylic acid (esters) C10-30 alkanol acrylate cross-linked polymers 0.15
7 Allantoin 0.15
8 1, 2-Hexanediol 0.8
9 Para hydroxy acetophenone 0.8
10 Dipotassium glycyrrhizinate 0.2
11 Essence 0.18
12 Water and its preparation method to 100
Performance test 9 anti-aging Properties
1. Testing basic information
(1) The subject: female 20;
(2) Age range: 20-55 years old;
(3) Test part: a face;
(4) Test period: 0d,14d,28d,56d;
(5) Test instrument: visioFace 1000D (facial image analyzer), ELASTIMETER (skin elasticity tester), C-CUBE (multifunctional skin imaging system).
2. Test procedure
(1) Cleaning the face, and resting for 20min in a constant-temperature (20-25 ℃) constant-humidity (40-60%) environment;
(2) Collecting a 0D facial image and testing skin elasticity;
(3) The face cleaning cream is uniformly smeared on the face after cleaning the face every morning and evening, and is lightly pressed until absorption;
(4) Facial images were taken and skin elasticity was tested at 14d,28d,56d, respectively (taken after cleaning the face and sitting still for 20 min).
The results obtained are shown in fig. 8 to 10 below. After 14 days of use, the subject typically had a 12.21% decrease in wrinkle volume, 11.40% decrease in area, 12.13% decrease in area ratio, 15.17% decrease in depth, 2.99% decrease in skin roughness, and 9.71% increase in immediate elasticity value. After 28 days of use, the subject typically had a reduced wrinkle volume at the site of 23.35%, an area reduction of 23.02%, an area ratio reduction of 22.12%, a depth reduction of 29.16%, a skin roughness reduction of 6.87% and an immediate elasticity value increase of 17.94%. After 56 days of use, the subject typically had a reduced wrinkle volume of 39.78%, an area of 43.18%, an area ratio of 42.20%, a depth of 39.41%, a skin roughness of 20.05% and an immediate elasticity value of 28.24%.
Performance test 10 stimulatory performance
The essence prepared in example 2 was subjected to skin irritation detection, and the results obtained are shown in fig. 11 to 13 below. The experiments of the closed type patch of the skin in the figures 11 to 13 show that the compound peptide prepared by the invention is negative and does not cause adverse reaction of the skin.
In addition, the compound of the invention can be prepared into different products according to actual requirements, such as essence, gel, emulsion, cream and the like, and the difference is that the adopted auxiliary agents are different, and the specific examples are shown in tables 19 to 22 below.
Table 19 component content table of essence prepared by using the compound peptide of the present invention
Sequence number Raw materials Content of the first group/% Content of second group/% Content of the third group/% Content of group IV/%
1 Complex peptides 5 8 1 10
2 Trehalose 5 8 1 10
3 L-2-oxo-thiazolidine-4-carboxylic acid 0.08 0.1 0.05 0.15
4 Butanediol (butanediol) 5 4 2 6
5 Xanthan gum 0.18 0.16 0.15 0.2
6 Acrylic acid (esters) C10-30 alkanol acrylate cross-linked polymers 0.15 0.18 0.1 0.2
7 Allantoin 0.15 0.18 0.1 0.2
8 1, 2-Hexanediol 0.8 0.7 0.5 1
9 Para hydroxy acetophenone 0.8 0.7 0.5 1
10 Dipotassium glycyrrhizinate 0.2 0.15 0.1 0.3
11 Essence 0.18 0.17 0.15 0.2
12 Water and its preparation method to 100 to 100 to 100 to 100
TABLE 20 component content Table for gel formulation using the Compound peptides of the invention
Sequence number Raw materials Content of the first group/% Content of second group/% Content of the third group/% Content of group IV/%
1 Complex peptides 5 8 1 10
2 Trehalose 5 8 1 10
3 L-2-oxo-thiazolidine-4-carboxylic acid 0.08 0.1 0.05 0.15
4 Butanediol (butanediol) 4 5.5 2 6
5 Xanthan gum 0.18 0.17 0.15 0.2
6 Acrylic acid (esters) C10-30 alkanol acrylate cross-linked polymers 0.45 0.42 0.4 0.5
7 1, 2-Hexanediol 0.8 0.6 0.5 1
8 Para hydroxy acetophenone 0.8 0.6 0.5 1
9 Solubilizer 0.45 0.48 0.4 0.5
10 Essence 0.15 0.18 0.1 0.2
11 Water and its preparation method to 100 to 100 to 100 to 100
TABLE 21 component content Table of emulsion prepared from the Compound peptide of the present invention
Sequence number Raw materials Content of the first group/% Content of second group/% Content of the third group/% Content of group IV/%
1 Complex peptides 5 8 1 10
2 Trehalose 5 8 1 10
3 L-2-oxo-thiazolidine-4-carboxylic acid 0.08 0.1 0.05 0.15
4 Butanediol (butanediol) 4 3 2 6
5 Xanthan gum 0.18 0.16 0.15 0.2
6 Allantoin 0.15 0.18 0.1 0.2
7 Tea seed oil 2 1.5 1 3
8 Ethylhexyl palmitate 2 1.5 1 3
9 Polydimethylsiloxane 1.5 1.8 1 2
10 Dioctyl carbonate 1.5 1.8 1 2
11 Butter fruit tree fruit fat 2 2.5 1 3
12 Glycerol stearate 0.6 0.8 0.5 1
13 Caprylic/capric triglyceride 2 2.5 1 3
14 Squalane (Squalene) 2 2.5 1 3
15 Tocopheryl acetate 0.3 0.25 0.2 0.4
16 Acryloyldimethyl taurate ammonium/VP copolymer 0.6 0.8 0.5 1
17 Phenoxyethanol 0.6 0.8 0.5 1
18 Dipotassium glycyrrhizinate 0.2 0.15 0.1 0.3
19 Essence 0.15 0.18 0.1 0.2
20 Water and its preparation method to 100 to 100 to 100 to 100
Table 22 component content table of creams formulated with the compound peptides of the invention
Sequence number Raw materials Content/%of first group component Content of the second group of components/%
1 Complex peptides 5 8
2 Trehalose 1 10
3 L-2-oxo-thiazolidine-4-carboxylic acid 0.05 0.15
4 Butanediol (butanediol) 2 6
5 Xanthan gum 0.1 0.2
6 Allantoin 0.1 0.2
7 Tea seed oil 1 3
8 Ethylhexyl palmitate 1 3
9 Polydimethylsiloxane 1 2
10 Tocopheryl acetate 0.2 0.4
11 Dioctyl carbonate 1 2
12 Cetostearyl alcohol 1 3
13 Glycerol stearate 0.5 1
14 Butter fruit tree fruit fat 1 3
15 Squalane (Squalene) 1 3
16 Caprylic/capric triglyceride 1 3
17 Hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer 1 3
18 Octyl glycol 0.5 1
19 Essence 0.1 0.2
20 Water and its preparation method To 100 To 100
In addition to the above examples, the compound peptide of the present invention may comprise 0.005-1.0% of acetyl hexapeptide-8, 0.005-0.1% of dipeptide diamino Ding Xianbian-yl amide diacetate, 0.005-0.1% of glutaminyl ethyl imidazole, 0.015-1.5% of hydrophilic silica aerogel, 1-10% of glycerol, 0.1-1.5% of preservative and the balance water. The compound peptide within the content range of the components can permeate into the dermis layer at low concentration, promote fibroblast proliferation, promote collagen self-generation from inside to outside, wake up the fibroblast, stimulate the expression of elastin genes, thereby promoting the synthesis of collagen, regulating the normalization biological clock law, adapting to the change of day and night environment and biological clock and achieving the effects of resisting the primary aging and resisting the aging.

Claims (8)

1. The compound peptide for realizing percutaneous absorption promotion of fibroblast proliferation under low concentration is characterized by comprising the following components in percentage by mass: acetyl hexapeptide-8.005-1.0%, dipeptide diamino Ding Xianbian-yl amide diacetate 0.005-0.1%, glutaminyl ethyl imidazole 0.005-0.1%, hydrophilic silicon dioxide aerogel 0.015-1.5%, glycerin 1-10%, preservative 0.1-1.5% and the balance of water.
2. The compound peptide for promoting fibroblast proliferation by percutaneous absorption at low concentration according to claim 1, wherein the porosity of the hydrophilic silica aerogel is 95-99%, the pore diameter is 10-50nm, the specific surface area is 20-1000m 2/g, the density is 3-300kg/m 3, and the diameter of colloidal particles forming a network is less than 100nm.
3. The compound peptide for promoting fibroblast proliferation by percutaneous absorption at low concentration according to claim 1, wherein the preservative is prepared by compounding 60-80% of octaethylene glycol and 20-40% of ethylhexyl glycerol.
4. An essence prepared based on the compound peptide of claim 1, characterized in that: the essence comprises, by mass, 1-10% of compound peptide, 1-10% of trehalose, 0.05-0.15% of L-2-oxo-thiazolidine-4-carboxylic acid, 2-6% of butanediol, 0.15-0.2% of xanthan gum, 0.1-0.2% of acrylic acid (esters)/C10-30 alkanol acrylate cross-linked polymer, 0.1-0.2% of allantoin, 0.5-1% of 1, 2-hexanediol, 0.5-1% of p-hydroxyacetophenone, 0.1-0.3% of dipotassium glycyrrhizinate, and 0.15-0.2% of essence and the balance water.
5. A gel prepared based on the compound peptide of claim 1, characterized in that: the gel comprises, by mass, 1-10% of compound peptide, 1-10% of trehalose, 0.05-0.15% of L-2-oxo-thiazolidine-4-carboxylic acid, 2-6% of butanediol, 0.15-0.2% of xanthan gum, 0.4-0.5% of acrylic acid (esters) or C10-30 alkanol acrylate cross-linked polymer, 0.5-1% of 1, 2-hexanediol, 0.5-1% of p-hydroxyacetophenone, 0.4-0.5% of solubilizer, 0.1-0.2% of essence and the balance of water.
6. An emulsion prepared based on the compound peptide of claim 1, characterized in that: the emulsion comprises, by mass, 1-10% of compound peptide, 1-10% of trehalose, 0.05-0.15% of L-2-oxo-thiazolidine-4-carboxylic acid, 2-6% of butanediol, 0.15-0.2% of xanthan gum, 0.1-0.2% of allantoin, 1-3% of tea seed oil, 1-3% of ethylhexyl palmitate, 1-2% of polydimethylsiloxane, 1-2% of dioctyl carbonate, 1-3% of shea butter, 0.5-1% of glycerol stearate, 1-3% of caprylic/capric triglyceride, 1-3% of squalane, 0.2-0.4% of tocopheryl acetate, 0.5-1% of ammonium acryloyldimethyl taurate/VP copolymer, 0.5-1% of phenoxyethanol, 0.1-0.3% of dipotassium glycyrrhizinate, and 0.1-0.2% of essence.
7. A cream prepared based on the compound peptide of claim 1, characterized in that: the cream comprises, by mass, 1-10% of compound peptide, 1-10% of trehalose, 0.05-0.15% of L-2-oxo-thiazolidine-4-carboxylic acid, 2-6% of butanediol, 0.1-0.2% of xanthan gum, 0.1-0.2% of allantoin, 1-3% of tea seed oil, 1-3% of ethylhexyl palmitate, 1-2% of polydimethylsiloxane, 0.2-0.4% of tocopheryl acetate, 1-2% of dioctyl carbonate, 1-3% of cetylstearyl alcohol, 0.5-1% of glyceryl stearate, 1-3% of shea butter, 1-3% of squalane, 1-3% of caprylic/capric triglyceride, 1-3% of hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, 0.5-1% of caprylyl glycol, 0.1-0.2% of essence and the balance of water.
8. A method of preparing the compound peptide of claim 1, comprising the steps of: stirring glycerol, antiseptic and water at 80-90deg.C to obtain a uniform clear solution; and (3) when the system is cooled to room temperature, adding acetyl hexapeptide-8, dipeptide diamino Ding Xianbian-yl amide diacetate and glutaminyl ethyl imidazole, stirring uniformly, and finally adding hydrophilic silica aerogel, and mixing uniformly.
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