A complex capable of stabilizing a polypeptide and a method for preparing the same

Abstract

The present application belongs to the field of biotechnology and cosmetics, and relates to a complex capable of stabilizing polypeptides and a preparation method thereof, comprising the following components in parts by weight: trehalose 3-15, lecithin 0.5-10, glycerol 65-90, water 1-10 and cosmetic polypeptide 0.03-1. Compared with the prior art, the lecithin adopted in the present application is easy to form a lamellar liquid crystal structure, can form a hollow spherical monolayer or multilayer liposome with a bilayer structure in an aqueous solution, can wrap cosmetic water-soluble or water-insoluble polypeptides in an internal hydrophilic layer or an oleophilic layer, avoids the reaction of exposed residues of the polypeptides with substances in the outside world, and leads to the disappearance of the activity of the polypeptides, and meanwhile, trehalose can be filled between phosphatidylcholine in a liquid crystal phase, can replace the position of water when the phosphatidylcholine is dehydrated, inhibits the phosphatidylcholine from changing from a liquid crystal phase to a gel phase, and protects the structure and function of the liposome.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and cosmetics, and particularly provides a polypeptide complex capable of improving the stability of polypeptides and facilitating the addition of polypeptides into cosmetics, and a preparation method thereof. BACKGROUND

[0002] With the continuous development of biotechnology and the increasing demand for functional cosmetics, the use of biologically active polypeptides in cosmetics is becoming more and more widespread, and the development of cosmetic polypeptides has gradually become a research hotspot. However, the inherent characteristics of polypeptide substances, such as low utilization rate, high enzymatic activity, and short half-life, limit their application. The main reason for its instability is the degradation of polypeptide amino acids and the easy modification of amino acid residues, which undergo a series of reactions such as oxidation, reduction, hydrolysis, deamidation, etc., thereby affecting its stability. The general solution can be chemical modification, such as glycosylation, esterification, acylation, and substitution reactions, or extending the peptide chain, modifying the ends of the molecule, or changing the straight-chain peptide to a cyclic peptide, such as the cyclic peptide without free N- and C-termini in the molecule, which greatly reduces the sensitivity of aminopeptidase and carboxypeptidase. The polypeptides used in cosmetics shown in the cosmetic raw material directory are unmodified dipeptide-1, tripeptide-3, tetrapeptide-3, and acetyl hexapeptide-8, palmitoyl tetrapeptide-7, and myristoyl pentapeptide-4, which are modified by acetyl, palmitoyl, and myristoyl. The stability of these modified peptides is relatively improved, but on the other hand, palmitoyl modification contains a long hydrophobic chain, which is difficult to dissolve in aqueous solution. Most current solutions use solubilizers such as Tween 20 to solubilize, which not only has poor skin affinity, but also has low market acceptance due to the presence of PEG, and the use of other solubilizers without PEG may not be well solubilized. SUMMARY

[0003] The purpose of the present application is to overcome the defects of the prior art and provide a complex capable of stabilizing polypeptides and a preparation method thereof.

[0004] The purpose of the present application can be achieved by the following technical solution: a complex capable of stabilizing polypeptides, characterized in that it comprises the following components by weight: trehalose 3-15, lecithin 0.5-10, glycerol 65-90, water 1-10, and cosmetic polypeptide 0.03-1.

[0005] Further, the lecithin includes one or more of lecithin, hydrogenated lecithin, soy lecithin, and hydrogenated soy lecithin, wherein the phosphatidylcholine content is greater than 70%.

[0006] Further, the cosmetic polypeptide is one or more of acetyl hexapeptide-8, acetyl octapeptide-3, a snake venom-like peptide, a conotoxin peptide, tripeptide-1 copper, palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl tetrapeptide-7, palmitoyl pentapeptide-4, palmitoyl hexapeptide-12, myristoyl pentapeptide-4, acetyl tetrapeptide-5, and an oligopeptide.

[0007] Further, the cosmetic polypeptide is a solid powder with a purity of 95%-98%.

[0008] Further, the trehalose has a purity greater than 98%.

[0009] Trehalose was first extracted from Claviceps purpureus of black wheat by Wiggers in 1832, is very stable to heat and acid, is a non-reducing disaccharide, and does not undergo Maillard reaction even when heated when coexisting with amino acids and proteins. In addition, trehalose also has the effects of preventing starch aging, protein denaturation, and inhibiting lipid peroxidation. Due to its special disaccharide molecular structure, trehalose can form a special protective film on the cell surface under high temperature, high cold, and dry and water loss states, effectively protecting the structure of biological molecules from being destroyed. Some plants and animals in nature can revive after drying, because trehalose can protect the lipids, proteins, carbohydrates, and nucleic acids of their tissues under dry conditions, so that they are not destroyed, and can be regenerated when they encounter water. In view of this, the present application coats the cosmetic polypeptide with trehalose and lecithin, which not only improves the stability of the polypeptide but also increases the transdermal absorption effect of the polypeptide into the skin.

[0010] Further, the glycerol is cosmetic glycerol with a concentration of 99.5%.

[0011] Further, the water is deionized water.

[0012] The present application also provides a preparation method of a complex capable of stabilizing a polypeptide, comprising the following steps:

[0013] (1) adding trehalose into glycerol and heating to 75-95 DEG C, and stirring to dissolve uniformly;

[0014] (2) adding lecithin into the material obtained in step (1), and continuing to heat and stir to dissolve uniformly, and cooling to 45-55 DEG C;

[0015] (3) adding a cosmetic polypeptide into the material obtained in step (2), homogenizing for 5-10 min, and continuing to stir and cool to room temperature;

[0016] (4) adding water into the material obtained in step (3), stirring uniformly, and the appearance is transparent viscous liquid.

[0017] In step (2), the temperature is continuously heated to 85-95 DEG C.

[0018] The homogenization speed in step (3) is 5000-6000 rpm.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] 1. The compound of the present application comprises the following components: trehalose, lecithin, glycerol, water and cosmetic polypeptide. The cosmetic polypeptide is mainly wrapped by trehalose and lecithin, which improves the stability of the polypeptide and increases the transdermal absorption effect of the polypeptide into the skin.

[0021] The acetyl hexapeptide-8, snake venom peptide, and conotoxin used have good effects on anti-expression line and anti-aging. Tripeptide-1 copper can promote wound healing and hair growth; palmitoyl tripeptide-1, palmitoyl tripeptide-5, palmitoyl tetrapeptide-7, palmitoyl pentapeptide-4, and palmitoyl hexapeptide-12 can promote human skin collagen synthesis; myristoyl pentapeptide-4 can stimulate human keratin gene expression, thereby promoting the growth of eyelashes; and acetyl tetrapeptide-5 has anti-edema, reduces dark circles, and improves skin elasticity. However, these polypeptides have poor stability or poor water solubility.

[0022] The main component of lecithin is phosphatidylcholine. Since its critical packing parameter (CPP) is close to 1, it is more prone to form a lamellar liquid crystal structure, and can form a double-molecular layer structure of hollow spherical single-layer or multi-layer liposomes in an aqueous solution. The cosmetic polypeptide can be wrapped in the inner hydrophilic layer or the oil layer, avoiding the reaction of the exposed residues of the polypeptide with external substances and causing the disappearance of the activity of the polypeptide. At the same time, the formed liposomes have good skin affinity due to the same structure as the skin cell membrane, and are more likely to bring the cosmetic polypeptide into the deep layer of the skin, thereby exerting its efficacy. In the compound of the present application, trehalose can be filled between phosphatidylcholine in the liquid crystal phase, and can replace the position of water when it is dehydrated, inhibiting the transition of phosphatidylcholine from the liquid crystal phase to the gel phase without destroying its structure and function, thereby well protecting the polypeptide and promoting the penetration of the polypeptide into the skin.

[0023] The method of the present application is simple. Trehalose is first dissolved in glycerol, which is close to the glycerol saturated solution of trehalose, and the long-time heating of lecithin is avoided. Then lecithin is added and dissolved at a temperature of 85-95℃, which is slightly higher than the phase transition temperature of phosphatidylcholine, so that trehalose can be filled between phosphatidylcholine in the liquid crystal phase. Then the cosmetic polypeptide is added and homogenized at high speed, so that the cosmetic polypeptide can be wrapped in the hollow spheres formed by trehalose and lecithin. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a schematic diagram of the dehydrated structure of the trehalose-containing liposome;

[0025] Figure 2 is a cosmetic lotion of liposome-encapsulated palmitoyl tetrapeptide-7;

[0026] Figure 3 is the particle size distribution of the polypeptide liposome complex of Example 1;

[0027] Figure 4 is the content test of the control palmitoyl tetrapeptide-7;

[0028] Figure 5 is the content test of palmitoyl tetrapeptide-7 of Example 2 sample at 60°C for 1 month;

[0029] Figure 6 is the content test of palmitoyl tetrapeptide-7 of Comparative Example 2 sample at 60°C for 1 month. DETAILED DESCRIPTION

[0030] The following detailed description of the embodiments of the present application is made on the premise of the technical solutions of the present application, and detailed embodiments and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0031] Examples 1-3

[0032] Examples 1-3 and Comparative Examples 1-2 are prepared according to the weight parts of Table 1 below; and then the complex is prepared by the following method:

[0033] (1) Add trehalose to glycerol and heat to 90°C, and stir to dissolve uniformly;

[0034] (2) Add lecithin to the material obtained in step (1), continue to heat and stir to dissolve uniformly, and then cool to 55°C;

[0035] (3) Add the cosmetic polypeptide to the material obtained in step (2), homogenize at 5000 rpm for 5 min, continue to stir and cool to room temperature, and defoam;

[0036] (4) Slowly add deionized water to the material obtained in step (3), stir uniformly, and the appearance is a transparent viscous liquid.

[0037] Table 1 is the selection and ratio of raw materials of Examples 1-3 and Comparative Examples 1-2

[0038] Ingredients Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Trehalose 3.00 5.00 8.00 5.00 5.00 Glycerol 89.90 86.90 77.90 91.90 91.40 Lecithin 5.00 5.00 8.00 Tween 20 0.50 Palmitoyl tetrapeptide-7 0.10 0.10 0.10 0.10 Acetyl hexapeptide-8 0.10 Water 2.00 3.00 6.00 3.00 3.00 Total 100.00 100.00 100.00 100.00 100.00

[0039] The complex obtained in Example 2 and Comparative Example 1 above is respectively mixed with the cosmetic lotion base at a mass ratio of 1:10 by stirring, and it can be clearly seen that the complex of Comparative Example 1 without lecithin has a small amount of insoluble precipitate without being encapsulated, while Example 2 can obtain a translucent clear material body without solid (palmitoyl tetrapeptide-7) precipitate, as shown in Figure 2 ​

[0040] The particle size distribution of the complex liposome was determined by dynamic light scattering nanoparticle size analyzer NanoBrook 90Plus Zeta of Brookhaven.

[0041] The above Example 1 was tested using dynamic light scattering nanoparticle size analyzer NanoBrook 90Plus Zeta, and the test results are shown in Figure 3 The average particle size of the polypeptide complex liposome was 164.91 nm, the polydispersity was 0.217, and the peak shape was unimodal, indicating that the polypeptide complex liposome was uniformly dispersed and could maintain long-term stability.

[0042] The quantification of palmitoyl tetrapeptide-7 was tested by using Agilent 1260 Infinity II high performance liquid chromatography, using an Ecosil-C18 (4.6 mm x 150 mm, 5 μm) chromatographic column with octadecylsilane-bonded silica gel as the filler, at a column temperature of 25°C, with mobile phase A being 0.1% trifluoroacetic acid aqueous solution and B being 0.1% trifluoroacetic acid acetonitrile solution, at a flow rate of 1.0 ml / min.

[0043] The sample injection volume was 5 μl, and the absorbance at a wavelength of 220 nm was used to quantify palmitoyl tetrapeptide-7, and the chromatogram was recorded for 20 minutes. The gradient conditions are as follows.

[0044] Time Mobile phase A (%) Mobile phase B (%) 0 (min) 100 0 20 (min) 95 5

[0045] Sample mass percentage calculation method:

[0046] In the formula, A X is the peak area of the test sample;

[0047] A R is the peak area of the control sample;

[0048] m X is the mass of the test sample (mg);

[0049] m R is the mass of the control sample (mg);

[0050] c X is the concentration of the test sample (mg / mg);

[0051] c R is the concentration of the control sample (mg / mg);

[0052] D X is the dilution factor of the test sample;

[0053] D R Dilution multiple of the control.

[0054] The mixture prepared in Example 2 and Comparative Example 2 was placed in an environment of 60°C for 1 month, and the palmitoyl tetrapeptide-7 content of Example 2 and Comparative Example 2 was detected, and the detection results are shown in Table 1. Figures 4-6 According to the calculation, the initial content of the palmitoyl tetrapeptide-7 polypeptide in the complex prepared in Example 2 was 0.1%, and after 1 month of high temperature of 60°C, the palmitoyl tetrapeptide-7 content was 0.061%, which was higher than 0.035% in Comparative Example 2. Therefore, it can be known that the complex capable of stabilizing polypeptides can well protect the stability of cosmetic polypeptides. Figures 4-6

[0055] Examples 4-8

[0056] Examples 4-8 were prepared according to the weight parts in Table 2; and then the complex was prepared by the following method:

[0057] (1) Trehalose was added to glycerol and heated to 70°C, and stirred to dissolve uniformly;

[0058] (2) Egg phospholipid was added to the material obtained in step (1), and after uniform dissolution under continued heating and stirring, it was cooled to 45°C;

[0059] (3) Cosmetic polypeptide was added to the material obtained in step (2), and homogenized at a speed of 6000 rpm for 10 min, and then cooled to room temperature under continued stirring and defoaming;

[0060] (4) Deionized water was slowly added to the material obtained in step (3), and stirred uniformly, and the appearance was a transparent viscous liquid.

[0061] Table 2 is the selection and ratio of raw materials of Examples 4-8

[0062]

[0063] The performance of the products obtained in Examples 4-8 was detected by the same method as Examples 1-3, and it was found that the products prepared within the range of the application could improve the stability of polypeptides.

[0064] The above examples are only for illustrating the technical concept and characteristics of the application, and the purpose is to enable those skilled in the art to understand the content of the application and to implement it, and it cannot limit the protection scope of the application. Any equivalent changes or modifications made according to the spirit and essence of the application shall be covered within the protection scope of the application.​

Claims

1. A complex capable of stabilizing a polypeptide, characterized in that, It includes the following components by weight: trehalose 3-15, lecithin 0.5-10, glycerin 65-90, water 1-10, and cosmetic peptides 0.03-1; The lecithin includes one or more of lecithin, hydrogenated lecithin, soybean lecithin, and hydrogenated soybean lecithin, wherein the phosphatidylcholine content is greater than 70%; First, trehalose is dissolved in glycerol, then lecithin is added and dissolved at 85℃-95℃, so that trehalose fills the spaces between phosphatidylcholine in the liquid crystal phase. Then, cosmetic peptides are added and homogenized at high speed, so that the cosmetic peptides are encapsulated in hollow spheres formed by trehalose and lecithin. The cosmetic polypeptide is selected from one or more of acetyl hexapeptide-8 or palmitoyl tetrapeptide-7.

2. The complex capable of stabilizing polypeptides according to claim 1, characterized in that, The cosmetic polypeptide is a solid powder with a purity of 95%-98%.

3. The complex capable of stabilizing polypeptides according to claim 1, characterized in that, The trehalose described has a purity of 98%.

4. The complex capable of stabilizing polypeptides according to claim 1, characterized in that, The glycerin mentioned is cosmetic glycerin with a concentration of 99.5%.

5. The complex capable of stabilizing polypeptides according to claim 1, characterized in that, The water mentioned is deionized water.

6. A method for preparing a complex capable of stabilizing a polypeptide as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Add trehalose to glycerol and heat to 75℃-95℃, stirring until dissolved and homogeneous; (2) Add lecithin to the material obtained in step (1), continue heating and stirring to dissolve evenly, and cool to 45℃-55℃; (3) Add the cosmetic peptides to the material obtained in step (2), homogenize for 5-10 minutes, continue stirring and cool to room temperature; (4) Add water to the material obtained in step (3) and stir evenly. The appearance is a transparent and viscous liquid.

7. The method for preparing a stable polypeptide complex according to claim 6, characterized in that, In step (2), continue heating until the temperature reaches 85℃-95℃.

8. The method for preparing a stable polypeptide complex according to claim 6, characterized in that, In step (3), the homogenization speed is 5000-6000 rpm.

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