Freeze-dried powder capable of rapidly generating copper-coated peptides and preparation method thereof
阅读说明：本技术 一种能够快速生成蓝铜肽的冻干粉及其制备方法 (Freeze-dried powder capable of rapidly generating copper-coated peptides and preparation method thereof ) 是由 丁文锋 于 2020-12-30 设计创作，主要内容包括：本发明公开一种能够快速生成蓝铜肽的冻干粉及其制备方法,包括冻干粉部分、溶媒部分,所述冻干粉部分由以下原料组成：三肽-1、甘露糖醇、海藻糖、去离子水,所述溶媒部分是铜离子溶液。所述冻干粉部分和溶媒部分隔开存储于同一双腔瓶中,双腔瓶的两个腔室通过连接部组合在一起,连接部具有可开启的连接通道。临用前开启连接部的连接通道,冻干粉部分与溶媒部分接触,摇匀,即快速生成蓝铜肽。本发明所述能够快速生成蓝铜肽的冻干粉稳定性好,制备工艺简单,生产成本低；具有优异的皮肤修复效果,还能应用于防脱生发方面；具有变色指示作用,能够给消费者指示蓝铜肽的生成,同时带来良好感观,增加消费者使用兴趣。(The invention discloses a freeze-dried powder capable of rapidly generating copper-coated peptides and a preparation method thereof, wherein the freeze-dried powder comprises a freeze-dried powder part and a solvent part, and the freeze-dried powder part comprises the following raw materials: tripeptide-1, mannitol, trehalose and deionized water, wherein the solvent part is a copper ion solution. The freeze-dried powder part and the solvent part are stored in the same double-cavity bottle in a separated mode, two cavities of the double-cavity bottle are combined together through a connecting part, and the connecting part is provided with an openable connecting channel. Before use, the connecting channel of the connecting part is opened, the freeze-dried powder part is contacted with the solvent part, and the mixture is shaken up, so that the copper-coated peptide is quickly generated. The freeze-dried powder capable of rapidly generating the copper-coated peptides has good stability, simple preparation process and low production cost; has excellent skin repairing effect and can be applied to the aspects of preventing hair loss and growing hair; the indicator has a color change indicating effect, can indicate the generation of the blue copper peptide to consumers, brings good perception and increases the use interest of the consumers.)
1. The freeze-dried powder capable of rapidly generating the bluecopper peptide comprises a freeze-dried powder part and a solvent part, and is characterized in that the freeze-dried powder part is composed of the following raw materials: tripeptide-1, mannitol, trehalose, deionized water, wherein the solvent part is a copper ion solution, the freeze-dried powder part and the solvent part are separately stored in the same double-cavity bottle, and two cavities of the double-cavity bottle are combined together through a connecting part which is provided with an openable connecting channel.
2. The freeze-dried powder capable of rapidly generating the bluecopper peptide according to claim 1, wherein the freeze-dried powder is partially prepared from the following components in percentage by mass:
tripeptide-1: 1.5% -3.5%;
mannitol: 4.0% -8.0%;
trehalose: 0.5% -2.5%;
deionized water: 88.0 to 92.0 percent.
3. The lyophilized powder capable of rapidly producing a bluecopper peptide according to claim 1, wherein the copper ion solution is a copper gluconate solution or a copper chloride solution.
4. The lyophilized powder capable of rapidly producing a bluecopper peptide according to claim 3, wherein the copper ion solution is a copper gluconate solution.
5. The freeze-dried powder capable of rapidly generating the bluecopper peptide according to claim 4, wherein the copper gluconate solution is prepared from the following components in percentage by mass:
copper gluconate: 0.5% -2.0%;
betaine: 1.0% -4.0%;
panthenol: 0.1% -1.0%;
allantoin: 0.1% -0.5%;
hexanediol: 0.1% -1.0%;
p-hydroxyacetophenone: 0.1% -1.0%;
sodium hyaluronate: 0.01% -0.1%;
glycerol: 1.0% -3.0%;
deionized water: 90.0 to 95.5 percent.
6. The method for preparing a lyophilized powder capable of rapidly generating a bluepatin peptide according to claim 1, wherein the method for preparing comprises a method for preparing a lyophilized powder part and a method for preparing a solvent part.
7. The method for preparing lyophilized powder capable of rapidly generating a bluepatin peptide according to claim 6, wherein the method for preparing the lyophilized powder comprises the following steps:
(1) weighing the raw materials according to the mass percentage, putting tripeptide-1, mannitol, trehalose and deionized water into a mixing container, stirring for 10-15 min at the rotating speed of 500-800 rpm, mixing and dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a filtered solution;
(2) filling the filtered solution into a bottom cavity of a double-cavity bottle, wherein the filling amount is 0.2-0.5 mL, so as to obtain a filled solution;
(3) pre-starting a vacuum freeze dryer, reducing the temperature of a freeze-drying plate layer to-45 to-40 ℃, and preserving the temperature for 2 to 3 hours;
(4) putting the filled solution into a vacuum freeze dryer, vacuumizing, maintaining the vacuum degree at 30Pa, heating to-5 ℃, preserving heat for 10-15 h, then heating to 10 ℃, and preserving heat for 1-3 h;
(5) and maintaining the vacuum degree at 30Pa, continuously heating to 30 ℃, preserving heat for 1-3 h, heating to 38 ℃, preserving heat for 8-12h, and completing freeze drying to obtain the freeze-dried powder part.
8. The method for preparing lyophilized powder capable of rapidly generating a bluepatin peptide according to claim 6, wherein the method for preparing the solvent part comprises the following steps:
(1) weighing the raw materials according to the mass percentage, adding deionized water, glycerol, sodium hyaluronate and allantoin into an emulsifying pot, heating to 80-85 ℃, and uniformly stirring;
(2) cooling to 60 ℃, adding p-hydroxyacetophenone and hexanediol, stirring for 10-20 min, and completely dissolving;
(3) continuously cooling to 40-45 ℃, adding panthenol, betaine and copper gluconate, stirring for 5-10 min, and filtering by using a 0.22 mu m filter membrane to obtain a filtered copper ion solution;
(4) and filling the filtered copper ion solution into a bottle body cavity of a double-cavity bottle, wherein the filling amount is 1.5-2.0 mL, so as to obtain the solvent part.
9. The method for preparing a lyophilized powder for rapid production of a bluepatin peptide according to any of claims 6-8, wherein the lyophilized powder portion and the vehicle portion are combined together through a connecting portion to obtain a lyophilized powder preparation in which the lyophilized powder portion and the vehicle portion are separately stored in the same double-chamber bottle.
10. The method for preparing the freeze-dried powder capable of rapidly generating the bluecopper peptide according to the claim 9, wherein the connecting channel of the connecting part is opened before the freeze-dried powder preparation is used, the freeze-dried powder part is contacted with the solvent part, and the shaking is carried out uniformly, so that the bluecopper peptide is rapidly generated.
Technical Field
The invention belongs to the technical field of beauty polypeptide, and particularly relates to freeze-dried powder capable of quickly generating copper-coated peptide and a preparation method thereof.
Background
The polypeptide is a compound formed by connecting amino acids together through peptide bonds, and plays an important role in various skin repair processes such as proliferation of skin tissue cells, cell chemotaxis and migration, collagen synthesis and secretion, tissue repair and regeneration, angiogenesis and reconstruction, pigment formation and removal, regulation of inflammation-related factors, improvement of cell growth environment and the like. The polypeptide can remarkably improve various skin problems, so the polypeptide is widely concerned by the medical cosmetology industry.
Tripeptide-1 and copper-blue peptide are commonly used anti-aging repair polypeptides. The tripeptide-1 is a tripeptide consisting of glycine, histidine and lysine, and has a sequence of GHK. Tripeptide-1 is a Matrikine signal peptide, can regulate the activities of Matrix Metalloproteinases (MMPs) and tissue inhibitory factors (TIMPs) for metalloproteinases, promotes the synthesis of extracellular matrices such as collagen and glycosaminoglycans, and is an important regulator for skin repair and wound healing. Tripeptide-1 has strong affinity with copper ions to form tripeptide-1 copper (GHK-Cu) complex, specifically, the tripeptide-1 sequence is complexed by nitrogen atom on imidazole ring of histidine, nitrogen atom at amino terminal of glycine and nitrogen atom on peptide bond formed by glycine and histidine, and the 3 nitrogen atoms are complexed with copper. The tripeptide-1 copper appears blue and is therefore also referred to as a blue copper peptide. Copper ions are a very important component in wound healing and many enzymatic reactions in the human body and on the skin, and also exert a signaling function that can affect the behavior and metabolism of cells. The tripeptide-1 is complexed with copper to form the blue copper peptide, which can effectively promote the proliferation of fibroblast, promote the synthesis of collagen, elastin and glycosaminoglycan, increase the growth of blood vessels and the oxidation resistance, repair skin barriers, stimulate hair follicles and promote hair growth.
In the traditional synthesis process of the blue copper peptide, tripeptide-1 is synthesized by liquid phase, and is complexed with copper after being purified and recrystallized to obtain the blue copper peptide. The tripeptide-1 and copper complex has poor process reproducibility, a plurality of influencing factors and high production cost. Due to different contents of acid, salt and water in different batches of tripeptide-1, the obtained blue copper peptide crystals have large differences in shape, color and quality under the same feeding amount, and different appearance properties can directly influence the visual perception of consumers, thereby greatly influencing the sale of the blue copper peptide. Different pH conditions also have an effect on the synthesis of the bluecopper peptide. In addition, white precipitates may appear during the standing process after the tripeptide-1 is complexed with copper.
In order to overcome the above disadvantages, it is necessary to develop a product which can rapidly generate the blue copper peptide through the tripeptide-1, has low preparation cost and high stability, can bring good perception to consumers, and is convenient to use.
Disclosure of Invention
Aiming at the defects of the prior art, the tripeptide-1 is prepared into freeze-dried powder, the copper ion solution is used as a solvent, the tripeptide-1 and the copper ion solution are independently coexisted in the same double-cavity bottle and are mixed before use to immediately generate the copper-blue peptide, and the copper-blue peptide is prepared for use at present, has good stability, simple preparation process and low cost and is convenient to use. Meanwhile, the light green copper ion solution is mixed with the tripeptide-1 freeze-dried powder to change color rapidly to form light blue or blue solution, the color change can be visually felt by consumers to indicate the generation of the blue copper peptide, and the process is carried out in the same double-cavity bottle, so that the problems of solvent leakage, operation process pollution and the like caused by independent packaging of the freeze-dried powder and the solvent are avoided, and the invention is completed.
Therefore, the technical problem to be solved by the invention is to provide the freeze-dried powder capable of rapidly generating the blue copper peptide and the preparation method thereof, the freeze-dried powder has a better skin repair effect than the traditional tripeptide-1 freeze-dried powder, the application range is wider, the production cost is lower than that of the traditional blue copper peptide freeze-dried powder, and the generation of the blue copper peptide can be indicated to consumers.
The invention relates to a freeze-dried powder capable of rapidly generating a blue copper peptide, which comprises a freeze-dried powder part and a solvent part, wherein the freeze-dried powder part is composed of the following raw materials: tripeptide-1, mannitol, trehalose, deionized water, wherein the solvent part is a copper ion solution, the freeze-dried powder part and the solvent part are separately stored in the same double-cavity bottle, and two cavities of the double-cavity bottle are combined together through a connecting part which is provided with an openable connecting channel.
The freeze-dried powder part is prepared from the following components in percentage by mass:
tripeptide-1: 1.5% -3.5%;
mannitol: 4.0% -8.0%;
trehalose: 0.5% -2.5%;
deionized water: 88.0 to 92.0 percent.
The copper ion solution is a copper gluconate solution or a copper chloride solution.
The copper ion solution is a copper gluconate solution.
The copper gluconate solution is prepared from the following components in percentage by mass:
copper gluconate: 0.5% -2.0%;
betaine: 1.0% -4.0%;
panthenol: 0.1% -1.0%;
allantoin: 0.1% -0.5%;
hexanediol: 0.1% -1.0%;
p-hydroxyacetophenone: 0.1% -1.0%;
sodium hyaluronate: 0.01% -0.1%;
glycerol: 1.0% -3.0%;
deionized water: 90.0 to 95.5 percent.
The preparation method of the freeze-dried powder capable of rapidly generating the copper-clad peptides comprises a preparation method of a freeze-dried powder part and a preparation method of a solvent part.
The preparation method of the freeze-dried powder part comprises the following steps:
(1) weighing the raw materials according to the mass percentage, putting tripeptide-1, mannitol, trehalose and deionized water into a mixing container, stirring for 10-15 min at the rotating speed of 500-800 rpm, mixing and dissolving, and filtering by using a 0.22 mu m filter membrane to obtain a filtered solution;
(2) filling the filtered solution into a bottom cavity of a double-cavity bottle, wherein the filling amount is 0.2-0.5 mL, so as to obtain a filled solution;
(3) pre-starting a vacuum freeze dryer, reducing the temperature of a freeze-drying plate layer to-45 to-40 ℃, and preserving the temperature for 2 to 3 hours;
(4) putting the filled solution into a vacuum freeze dryer, vacuumizing, maintaining the vacuum degree at 30Pa, heating to-5 ℃, preserving heat for 10-15 h, then heating to 10 ℃, and preserving heat for 1-3 h;
(5) and maintaining the vacuum degree at 30Pa, continuously heating to 30 ℃, preserving heat for 1-3 h, heating to 38 ℃, preserving heat for 8-12h, and completing freeze drying to obtain the freeze-dried powder part.
The preparation method of the solvent part comprises the following steps:
(1) weighing the raw materials according to the mass percentage, adding deionized water, glycerol, sodium hyaluronate and allantoin into an emulsifying pot, heating to 80-85 ℃, and uniformly stirring;
(2) cooling to 60 ℃, adding p-hydroxyacetophenone and hexanediol, stirring for 10-20 min, and completely dissolving;
(3) continuously cooling to 40-45 ℃, adding panthenol, betaine and copper gluconate, stirring for 5-10 min, and filtering by using a 0.22 mu m filter membrane to obtain a filtered copper ion solution;
(4) and filling the filtered copper ion solution into a bottle body cavity of a double-cavity bottle, wherein the filling amount is 1.5-2.0 mL, so as to obtain the solvent part.
The freeze-dried powder part and the solvent part are combined together through the connecting part to obtain the freeze-dried powder preparation which is stored in the same double-cavity bottle in a manner that the freeze-dried powder part and the solvent part are separated.
The freeze-dried powder preparation is opened before use, the freeze-dried powder part is contacted with the solvent part, and the mixture is shaken up, so that the copper-coated peptide is quickly generated.
Compared with the prior art, the invention has the advantages that:
1. the tripeptide-1 is prepared into the freeze-dried powder, the copper ion solution is used as a solvent, the tripeptide-1 and the copper ion solution are independently coexisted in the same double-cavity bottle and are mixed before use to immediately generate the copper-coated peptide, and the copper-coated peptide is ready to use, good in stability, simple in preparation process and low in cost.
2. By adjusting the feeding proportion of the tripeptide-1 and the copper ion solvent, the method can generate 2: 1 blue copper peptide (GHK: Cu ═ 2: 1), whereas traditionally prepared 1: 1 (GHK: Cu is 1: 1), so the technical scheme of the invention can achieve better skin repair effect than the traditional blue copper peptide.
3. The method uses the light green copper ion solution and the tripeptide-1 freeze-dried powder to mix, and quickly changes color to form light blue or blue solution, the color change effect can bring good perception to consumers, and simultaneously can indicate the generation of the blue copper peptide to the consumers, and the process is carried out in the same double-cavity bottle, thereby avoiding the problems of troublesome operation, solvent leakage, operation process pollution and the like caused by independent packaging of the freeze-dried powder and the solvent during use and dissolution.
4. The tripeptide-1 freeze-dried powder and the copper ion solution are mixed before use to quickly generate the copper-coated peptide, so that the tripeptide-1 freeze-dried powder has a better skin repairing effect than the traditional tripeptide-1 freeze-dried powder, is wider in application range, and can be applied to the aspects of hair loss prevention and hair growth besides being used for skin repairing.
5. The tripeptide-1 freeze-dried powder and the copper ion solution are mixed before use to quickly generate the copper-blue peptide, and after the tripeptide-1 is complexed with the copper ions, the resistance to enzyme can be improved, the problem that the traditional tripeptide-1 freeze-dried powder is degraded by protease in the using process is solved, so that the tripeptide-1 freeze-dried powder has better use stability than the traditional tripeptide-1 freeze-dried powder and is more beneficial to the exertion of effects.
Drawings
FIG. 1 results of cell migration experiment
FIG. 2 Experimental results of hair growth promotion
Detailed Description
For a better understanding of the present invention, the following detailed description of the present invention is given in conjunction with examples, test examples, and drawings, however, it should be understood that these examples, test examples, and drawings are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 preparation of lyophilized powder capable of rapidly generating a blue copper peptide
1.1 preparation of tripeptide-1 lyophilized powder
(1) Placing 2.5% tripeptide-1, 6% mannitol, 1.5% trehalose, and 90% deionized water in a mixing container, stirring at 700rpm for 12min, mixing and dissolving, and filtering with 0.22 μm filter membrane to obtain filtered solution;
(2) filling the filtered solution into a bottom cavity of a double-cavity bottle, wherein the filling amount is 0.3mL, so as to obtain a filled solution;
(3) starting a vacuum freeze dryer in advance, reducing the temperature of a freeze-drying plate layer to-45 ℃, and preserving the heat for 2.5 hours;
(4) putting the filled solution into a vacuum freeze dryer, vacuumizing, maintaining the vacuum degree at 30Pa, heating to-5 ℃, keeping the temperature for 13h, heating to 10 ℃, and keeping the temperature for 2 h;
(5) and keeping the vacuum degree at 30Pa, continuously heating to 30 ℃, keeping the temperature for 2h, heating to 38 ℃, keeping the temperature for 10h, and completing freeze drying to obtain the tripeptide-1 freeze-dried powder.
1.2 preparation of copper gluconate solvent
(1) Adding 91.75% of deionized water, 2% of glycerol, 0.05% of sodium hyaluronate and 0.2% of allantoin into an emulsifying pot, heating to 80-85 ℃, and uniformly stirring;
(2) cooling to 60 deg.C, adding 0.5% p-hydroxyacetophenone and 0.5% hexanediol, stirring for 15min, and dissolving completely;
(3) continuously cooling to 40-45 ℃, adding 0.5% of panthenol, 2.5% of betaine and 2.0% of copper gluconate, stirring for 10min, and filtering by using a 0.22-micron filtering membrane to obtain a filtered copper ion solution;
(4) and filling the filtered copper ion solution into a body cavity of a double-cavity bottle, wherein the filling amount is 1.5mL, so as to obtain the copper gluconate solvent.
1.3 combining the freeze-dried powder obtained in the steps 1.1 and 1.2 with a solvent through a connecting part to obtain a freeze-dried powder preparation, wherein the freeze-dried powder part and the solvent part are separated and stored in the same double-cavity bottle, opening a connecting channel of the connecting part before use, contacting the freeze-dried powder part with the solvent part, and shaking up to quickly generate the copper-coated peptide (GHK: Cu ═ 1: 1).
Example 2 preparation of lyophilized powder capable of rapidly generating blue copper peptide
2.1 preparation of tripeptide-1 lyophilized powder
(1) Placing 2.5% tripeptide-1, 6% mannitol, 1.5% trehalose, and 90% deionized water in a mixing container, stirring at 700rpm for 12min, mixing and dissolving, and filtering with 0.22 μm filter membrane to obtain filtered solution;
(2) filling the filtered solution into a bottom cavity of a double-cavity bottle, wherein the filling amount is 0.3mL, so as to obtain a filled solution;
(3) starting a vacuum freeze dryer in advance, reducing the temperature of a freeze-drying plate layer to-45 ℃, and preserving the heat for 2.5 hours;
(4) putting the filled solution into a vacuum freeze dryer, vacuumizing, maintaining the vacuum degree at 30Pa, heating to-5 ℃, keeping the temperature for 13h, heating to 10 ℃, and keeping the temperature for 2 h;
(5) and keeping the vacuum degree at 30Pa, continuously heating to 30 ℃, keeping the temperature for 2h, heating to 38 ℃, keeping the temperature for 10h, and completing freeze drying to obtain the tripeptide-1 freeze-dried powder.
2.2 preparation of copper gluconate solvent
(1) Adding 92.75% of deionized water, 2% of glycerol, 0.05% of sodium hyaluronate and 0.2% of allantoin into an emulsifying pot, heating to 80-85 ℃, and uniformly stirring;
(2) cooling to 60 deg.C, adding 0.5% p-hydroxyacetophenone and 0.5% hexanediol, stirring for 15min, and dissolving completely;
(3) continuously cooling to 40-45 ℃, adding 0.5% of panthenol, 2.5% of betaine and 1.0% of copper gluconate, stirring for 10min, and filtering by using a 0.22-micron filtering membrane to obtain a filtered copper ion solution;
(4) and filling the filtered copper ion solution into a body cavity of a double-cavity bottle, wherein the filling amount is 1.5mL, so as to obtain the copper gluconate solvent.
2.3 combining the freeze-dried powder obtained in the steps 2.1 and 2.2 with a solvent through a connecting part to obtain a freeze-dried powder preparation, wherein the freeze-dried powder part and the solvent part are separated and stored in the same double-cavity bottle, opening a connecting channel of the connecting part before use, contacting the freeze-dried powder part with the solvent part, and shaking up to quickly generate the copper-coated peptide (GHK: Cu ═ 2: 1).
Comparative example 1 preparation of conventional tripeptide-1 lyophilized powder
1.1 preparation of tripeptide-1 lyophilized powder
(1) Placing 2.5% tripeptide-1, 6% mannitol, 1.5% trehalose, and 90% deionized water in a mixing container, stirring at 700rpm for 12min, mixing and dissolving, and filtering with 0.22 μm filter membrane to obtain filtered solution;
(2) filling the filtered solution into a bottom cavity of a double-cavity bottle, wherein the filling amount is 0.3mL, so as to obtain a filled solution;
(3) starting a vacuum freeze dryer in advance, reducing the temperature of a freeze-drying plate layer to-45 ℃, and preserving the heat for 2.5 hours;
(4) putting the filled solution into a vacuum freeze dryer, vacuumizing, maintaining the vacuum degree at 30Pa, heating to-5 ℃, keeping the temperature for 13h, heating to 10 ℃, and keeping the temperature for 2 h;
(5) and keeping the vacuum degree at 30Pa, continuously heating to 30 ℃, keeping the temperature for 2h, heating to 38 ℃, keeping the temperature for 10h, and completing freeze drying to obtain the tripeptide-1 freeze-dried powder.
1.2 preparation of conventional vehicle
(1) Adding 93.75% of deionized water, 2% of glycerol, 0.05% of sodium hyaluronate and 0.2% of allantoin into an emulsifying pot, heating to 80-85 ℃, and uniformly stirring;
(2) cooling to 60 deg.C, adding 0.5% p-hydroxyacetophenone and 0.5% hexanediol, stirring for 15min, and dissolving completely;
(3) continuously cooling to 40-45 ℃, adding 0.5% of panthenol and 2.5% of betaine, stirring for 10min, and filtering by using a 0.22-micron filtering membrane to obtain a filtered solution;
(4) and filling the filtered solution into a body cavity of a double-cavity bottle, wherein the filling amount is 1.5mL, so as to obtain the conventional solvent.
1.3 combining the lyophilized powder obtained in steps 1.1 and 1.2 with solvent via connecting part to obtain traditional tripeptide-1 lyophilized powder.
Comparative example 2 preparation of conventional Blueptide lyophilized powder
1.1 preparation of lyophilized powder of copper peptide
(1) Placing 2.5% of copper-coated peptide, 6% of mannitol, 1.5% of trehalose and 90% of deionized water in a mixing container, stirring at 700rpm for 12min, mixing and dissolving, and filtering with a 0.22 μm filter membrane to obtain a filtered solution;
(2) filling the filtered solution into a bottom cavity of a double-cavity bottle, wherein the filling amount is 0.3mL, so as to obtain a filled solution;
(3) starting a vacuum freeze dryer in advance, reducing the temperature of a freeze-drying plate layer to-45 ℃, and preserving the heat for 2.5 hours;
(4) putting the filled solution into a vacuum freeze dryer, vacuumizing, maintaining the vacuum degree at 30Pa, heating to-5 ℃, keeping the temperature for 13h, heating to 10 ℃, and keeping the temperature for 2 h;
(5) and maintaining the vacuum degree at 30Pa, continuously heating to 30 ℃, keeping the temperature for 2h, heating to 38 ℃, keeping the temperature for 10h, and completing freeze drying to obtain the bluecopper peptide freeze-dried powder.
1.2 preparation of conventional vehicle
(1) Adding 93.75% of deionized water, 2% of glycerol, 0.05% of sodium hyaluronate and 0.2% of allantoin into an emulsifying pot, heating to 80-85 ℃, and uniformly stirring;
(2) cooling to 60 deg.C, adding 0.5% p-hydroxyacetophenone and 0.5% hexanediol, stirring for 15min, and dissolving completely;
(3) continuously cooling to 40-45 ℃, adding 0.5% of panthenol and 2.5% of betaine, stirring for 10min, and filtering by using a 0.22-micron filtering membrane to obtain a filtered solution;
(4) and filling the filtered solution into a body cavity of a double-cavity bottle, wherein the filling amount is 1.5mL, so as to obtain the conventional solvent.
1.3 combining the freeze-dried powder obtained in the steps 1.1 and 1.2 with a solvent through a connecting part to obtain the traditional bronze peptide freeze-dried powder.
Test example 1 cell proliferation-promoting experiment
1.1 reagents and materials
Fetal Bovine Serum (FBS), DMEM medium, PBS buffer, MTT, DMSO, 96 well plates.
1.2 instruments
Enzyme-linked immunosorbent assay (MD) and CO2Incubator (Shanghai Yi Heng), super clean bench (Suzhou purification).
1.3 cell lines
Mouse skin fibroblasts, NIH3T3, were purchased from shanghai cell bank, china academy of sciences.
1.4 samples to be tested
Administration group: example 1, example 2, comparative example 1, comparative example 2.
Control group: PBS blank control.
1.5 test methods
And (3) taking the same passage cell, inoculating the cell into a 96-well plate at the density of 5000 cells/well, wherein 200 mu L of cell suspension liquid is placed in each well, and after the cell is attached to the wall, replacing a culture medium containing 0.5-1% FBS to maintain the cell for 24h so as to synchronize the cell. And then adding 20 mu L of sample to be detected, respectively setting 5 multiple holes, adding 20 mu L of PBS to the control group, reacting for 24-72 h, adding 20 mu L of MTT reagent, reacting for 4h, centrifuging to remove supernatant, adding DMSO to each hole to fully dissolve crystals, and measuring OD value by an enzyme-labeling instrument at 490 nm.
Cell activity (%). RTM OD value of administration group/OD value of control group X100%
1.6 test results
The OD values obtained in step 1.5 were calculated according to the formula for cell activity, and the effects of different samples at the same concentration on the proliferation of fibroblast NIH3T3 were obtained, and the results are shown in Table 1.
TABLE 1 Effect of test samples on fibroblast NIH3T3 proliferation
As is clear from the results in table 1, the growth of fibroblasts was promoted in all of examples 1 and 2, comparative examples 1 and 2, and the fibroblasts were allowed to act on the blank control group. Fibroblasts play an important role in wound healing and skin repair processes, and carry out tissue repair by cell proliferation, synthesis and secretion of extracellular matrices such as collagen, elastin, glycosaminoglycan and the like. Compared with comparative example 1, the proliferation promoting effect of example 1 on fibroblasts is more obvious, which shows that tripeptide-1 is mixed with a copper gluconate solvent to instantly generate a mixture of 1: 1 blue copper peptide (GHK: Cu ═ 1: 1) can achieve better skin repair effect than the traditional tripeptide-1 freeze-dried powder. Compared with the comparative example 2, the proliferation promoting effect of the tripeptide-1 and the copper gluconate solvent on fibroblasts is equivalent, so that the tripeptide-1 is mixed with the copper gluconate solvent to instantly generate the copper-blue peptide (GHK: Cu ═ 1: 1), the skin repairing effect similar to that of the traditional copper-blue peptide (GHK: Cu ═ 1: 1) freeze-dried powder can be achieved, the technical scheme of the example 1 can reduce the production cost, and the accompanying color change phenomenon from light green to light blue can indicate the generation of the copper peptide, is beneficial to the identification of consumers and increases the use interest of the consumers. Example 2 can generate 2: 1 blue copper peptide (GHK: Cu ═ 2: 1), whereas traditionally prepared 1: 1-bluecuprinopeptide (GHK: Cu ═ 1: 1), example 2 had a higher activity of promoting proliferation of fibroblasts than comparative example 2, and thus example 2 had a better skin repair effect than comparative example 2. In addition, in the examples 1 and 2, after the GHK is complexed with the copper ions in the copper gluconate solvent, the glucose in the solution can also play a certain conditioning effect on the skin.
Test example 2 cell migration promoting experiment
2.1 cell lines
The HaCaT cell strain of human immortalized keratinocytes was purchased from Kunming cell Bank, Chinese academy of sciences.
2.2 samples to be tested
Administration group: example 1, example 2, comparative example 1, comparative example 2.
Control group: PBS blank control.
2.3 test methods
Cells are inoculated in a 24-well plate at the density of 15 ten thousand per well, 2mL of cell suspension liquid is added in each well, and after the cells are attached to the wall, a culture medium containing 0.5-1% FBS is replaced to maintain the cells for 24h so as to synchronize the cells. Scratching is carried out by using a sterilized gun head, PBS is used for washing off dropped cells, medicine-containing culture media are added, 5 multiple holes are arranged respectively, the same amount of PBS is added into a control group, after 24 hours of action, cell migration from the scratched position to the central growth is observed under a microscope, and pictures are taken.
The results of the experiment were analyzed using ImageJ, and the cell mobility (%) was calculated according to the following formula.
Cell mobility (%) - (scratch width of 0 h-scratch width after 24h incubation)/scratch width of 0h × 100% 2.4 test results
The cell states of example 1, example 2, comparative example 1, comparative example 2 and blank control at 0h and 24h are shown in FIG. 1.
According to the cell migration pictures of 0h and 24h, the cell migration rate is calculated according to the formula, and the result is shown in table 2.
TABLE 2 Effect of test samples on migration of human immortalized keratinocytes HaCaT cells
As is clear from the results of fig. 1 and table 2, each of examples 1 and 2, comparative examples 1 and 2, after acting on keratinocytes, promoted the migration of keratinocytes compared to the blank control group. The keratinocyte migrates to the center of the wound surface, which is beneficial to the healing of the wound surface. Compared with comparative example 1, example 1 can promote keratinocyte migration more obviously, and shows that tripeptide-1 is mixed with a copper gluconate solvent to form a new compound 1: 1 blue copper peptide (GHK: Cu ═ 1: 1), can obtain the better effect of accelerating wound healing than the traditional tripeptide-1 freeze-dried powder. Compared with the comparative example 2, the effect of the tripeptide-1 and the copper gluconate solvent on promoting the migration of the keratinocytes is equivalent, so that the tripeptide-1 is mixed with the copper gluconate solvent to generate the copper-blue peptide immediately, the wound healing accelerating effect similar to that of the traditional copper-blue peptide freeze-dried powder (GHK: Cu 1: 1) can be achieved, the technical scheme of the example 1 can reduce the production cost, and the accompanying color change phenomenon from light green to light blue can indicate the generation of the copper peptide, is beneficial to the identification of consumers and increases the use interest of the consumers. Example 2 can generate 2: 1 blue copper peptide (GHK: Cu ═ 2: 1), whereas traditionally prepared 1: 1-bluecupreotide (GHK: Cu ═ 1: 1), example 2 had higher cell mobility than comparative example 2, and thus example 2 had a better wound healing promoting effect than comparative example 2. In addition, in the examples 1 and 2, after the GHK is complexed with the copper ions in the copper gluconate solvent, the glucose in the solution can also play a certain conditioning effect on the skin.
Test example 3 Hair growth promoting animal experiment
3.1 Experimental animals
C57BL/6 mice, SPF grade, female, 6-8 weeks old, 18-20g body weight, pink skin, hair at telogen.
The experimental animal is purchased from the medical experimental animal center of Guangdong province, and has the license number SCXK (Guangdong) 2018-.
3.2 reagents and materials
Chloral hydrate (Michael), sodium sulfide (Dache chemical reagent, Tianjin), 75% medical alcohol.
3.3 test methods
Induction of hair growth period: mice were anesthetized with 4% chloral hydrate, shaved with an electric razor, 6% Na2And S, unhairing and washing with warm water. Avoiding damaging the skin. Inducing hair to enter anagen phase from telogen phase. After depilation, the depilation area was disinfected with 75% alcohol.
Grouping experimental animals: the day after depilation, 8 drugs were administered in groups, randomly. Respectively 5 groups: normal saline control group, example 1, example 2, comparative example 1, comparative example 2. The depilatory area of each group of mice was smeared with the corresponding substance.
The administration method comprises the following steps: the administration is started the next day after depilation, and the application amount is 0.2 ml/piece, 2 times/day, and about 0.1 ml/piece. The day of application was recorded as day 1 and the application was completed until the end of the experiment.
Mice were observed daily for new hair growth for 18 consecutive days.
At day 18, 10 hairs were randomly plucked from the same site in the area of each mouse where the drug was applied (to exclude broken hairs), and their lengths were measured with a vernier caliper and averaged. The average length of 10 hairs was taken as the new hair length per mouse.
3.4 test results
The skin color of each group of mice was observed to change from pink to black, and then to hair growth. Compared with the control group, the skin color of the C57BL/6 mice after depilation is obviously shortened by the comparison of example 1, example 2 and comparative example 2. A comparison of hair growth between the 0 day and 18 day groups is shown in FIG. 2.
After 18 days of use, the new hair length was measured and the results are shown in Table 3 below.
Table 3 test sample fresh hair length after 18 days of use
As is clear from the results of fig. 2 and table 3, examples 1, 2, and 2 significantly promoted the hair growth of mice compared to the control group and comparative example 1. The new hair lengths of the example 1 group and the comparative example 2 were measured by using the example 1 and the comparative example 2 respectively and were close to each other on day 18, which shows that the tripeptide-1 mixed with the copper gluconate solvent instantly generates the blue copper peptide (GHK: Cu ═ 1: 1), and can achieve the hair growth promoting effect similar to that of the conventional blue copper peptide freeze-dried powder (GHK: Cu ═ 1: 1), but the technical scheme of the example 1 has lower production cost. Comparing the results of example 2 and comparative example 2, the hair growth promoting effect of example 2 was more significant, and after 18 days of use, the mice grew thicker hairs and the new hairs were longer, indicating that the tripeptide-1 mixed with the copper gluconate solvent instantly produced the blue copper peptide (GHK: Cu ═ 2: 1), and the hair growth promoting effect was better than that of the conventional blue copper peptide lyophilized powder (GHK: Cu ═ 1: 1). On the other hand, after 18 days of use, the difference between the new hair lengths of the comparative example 1 and the control group is measured to be small, the hair growth promoting effect of the comparative example 1 is weak, the technical schemes of the examples 1 and 2 can obviously promote the hair growth, and the examples 1 and 2 have wider application range than the traditional tripeptide-1 freeze-dried powder, so that the freeze-dried powder capable of rapidly generating the copper-nickel peptide can be applied to the aspects of preventing hair loss and promoting hair growth besides being used for skin repair.
Test example 4 aging skin repair test
4.1 subjects
150 healthy women, 50-65 years old, were randomized into 5 groups, with an average of 30 people per group.
4.2 test specimens
Example 1, example 2, comparative example 1, comparative example 2, placebo as a blank.
4.3 test Instrument
Cutomer skin elasticity tester (Germany CK company)
4.4 evaluation method
The blank value of 30min after the face area is cleaned is measured, then the test sample is uniformly coated on the face, one side is coated with a placebo, the other side is coated with the test sample, the test sample is continuously used for 4 weeks twice a day in the morning and evening without using other cosmetics, the skin mechanical properties of the test area before and after administration are respectively tested and recorded, and simultaneously questionnaire investigation is carried out on the acceptance degree and the use effect of the product of a subject. The test of the same subject is performed by the same measuring person.
4.5 test results
4.5.1 questionnaire results
The product properties of example 1, example 2, comparative example 1, comparative example 2, placebo are shown in table 4 below.
Questionnaires were performed on the subjects before and after 4 weeks of product use, and the results are shown in table 5 below.
TABLE 4 Properties of the test samples
TABLE 5 results of the subject questionnaire
Questionnaire results show that subjects prefer to continue using the products of examples 1 and 2 after the test is completed. Example 1 can achieve better effects in improving skin firmness and skin elasticity than comparative example 1, and the skin repair effect is equivalent to comparative example 2. Example 2 had a better skin repair effect than comparative examples 1 and 2. In addition to the consideration of the use effect, the visual perception of the product acceptance of the subject is considered, and the mixing of the products of the examples 1 and 2 immediately before use to generate the blue-copper peptide can increase the use interest of the consumer along with the process of changing the light green color into the light blue color or the blue color, so that the acceptance of the products of the examples 1 and 2 by the subject is higher.
4.5.2 skin repair test results
And testing indexes of skin tightness, skin tension, skin viscoelasticity and skin plasticity of each sample before and after use by using a Cutomer so as to evaluate the mechanical performance of the skin and judge the repairing effect of the test sample on the aged skin.
The percent improvement in skin firmness, skin tone, skin viscoelasticity, and skin plasticity of the test samples after 4 weeks of use was calculated and the results are given in table 6 below.
Table 6 percent improvement in skin firmness, skin tone, skin viscoelasticity and skin plasticity (%), administered to each group for 4 weeks
As can be seen from the data in the table, examples 1-2 and comparative examples 1-2 all showed improvement in skin firmness, skin tension, skin viscoelasticity and skin plasticity measured after 4 weeks of use in the presence of the active polypeptide relative to the placebo group. Compared with comparative example 1, example 1 has a better skin repair effect, which shows that the tripeptide-1 is mixed with a copper gluconate solvent to instantly generate copper-blue peptide (GHK: Cu ═ 1: 1), and the skin repair effect is better than that of the traditional tripeptide-1 freeze-dried powder. Compared with the comparative example 2, the skin repairing effect of the embodiment 1 is equivalent to that of the comparative example 2, which shows that the tripeptide-1 is mixed with the copper gluconate solvent to instantly generate the blue copper peptide (GHK: Cu ═ 1: 1), so that the skin repairing effect similar to that of the traditional blue copper peptide freeze-dried powder (GHK: Cu ═ 1: 1) can be achieved, the technical scheme of the embodiment 1 can reduce the production cost, and the accompanying color change phenomenon from light green to light blue can indicate the generation of the copper peptide, is beneficial to the identification of consumers and increases the use interest of the consumers. Example 2 can generate 2: 1 blue copper peptide (GHK: Cu ═ 2: 1), whereas traditionally prepared 1: 1-bluecupeptide (GHK: Cu ═ 1: 1), therefore example 2 had superior skin repair effect than comparative example 2. In addition, in the examples 1 and 2, after the GHK is complexed with the copper ions in the copper gluconate solvent, the glucose in the solution can also play a certain conditioning effect on the skin.
Test example 5 protease degradation experiment
5.1 test methods
1.5mg of papain was added to the solution of example 1, comparative example 1. The resulting solution was incubated at 40 ℃. After papain addition, samples were taken at 0h, 1h, 3h and 24h, respectively, centrifuged at 1400rpm for 1min, the supernatant filtered using a 0.45pm filter, and analyzed by HPLC.
5.2 test results
The stability results of example 1 and comparative example 1 under papain are shown in table 7 below.
TABLE 7 stability of the test samples under the action of proteases
The results in table 7 show that example 1 can better resist the degradation of protease, and after 24 hours under the action of papain, the solution still contains high-content polypeptide, while the polypeptide in comparative example 1 is completely degraded by papain, which indicates that in example 1, the tripeptide-1 lyophilized powder is mixed with the copper ion solution immediately before use to rapidly generate copper-blue peptide, and after the tripeptide-1 is complexed with the copper ions, the resistance to protease can be improved, and the problem that the conventional tripeptide-1 lyophilized powder is degraded by protease in the use process is avoided, so that the tripeptide-1 lyophilized powder has better use stability than the conventional tripeptide-1 lyophilized powder, and is more beneficial to the exertion of the effect.
The foregoing is a more detailed description of the present invention in connection with specific preferred embodiments thereof, and is not intended to limit the invention to the particular forms disclosed. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.