An active polypeptide and a method of preparation, cosmetic

Abstract

The application discloses a kind of based on stem cell exosome preparation cosmetics and preparation method thereof.The method includes the following processes:(1) preparation stem cell exosome;(2) the active polypeptide as shown in SEQ ID NO.1 is connected with sodium hyaluronate, and active polypeptide-sodium hyaluronate complex is obtained;(3) active polypeptide-sodium hyaluronate complex, stem cell exosome and adjuvant are mixed together, and the cosmetic is prepared.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cosmetic preparation, and particularly relates to a cosmetic prepared based on stem cell exosomes and a preparation method thereof. BACKGROUND

[0002] With the development of society and the pursuit of a better life by people, beauty has become an increasingly important life pursuit goal, and people have a deeper understanding of skin aging. The main influence of aging skin on appearance is dry and dull skin, roughness, dryness, wrinkles, laxity and sagging, and a significant increase in lipofuscin and age spots. Therefore, various cosmetics and drugs are widely used, but there are thousands of drugs or cosmetics for maintaining skin anti-aging and whitening, and their effects are different.

[0003] In recent years, with the development of stem cell technology research, it has become possible to use stem cells for drugs or cosmetics. Exosomes are double-membrane vesicles formed by cells through a series of regulation processes such as "endocytosis-fusion-exocytosis" and can be secreted. They do not contain DNA fragments, but contain similar cell factors, growth factors and other proteins, as well as lipids, coding or non-coding RNA and other bioactive substances, which play an important role in regulating cell physiological functions. The plasma membrane of exosomes contains abundant cholesterol, sphingomyelin, ceramide, lipid raft and phosphatidylserine; exosomes not only protect these bioactive substances from degradation and dilution in the extracellular environment, but also promote the long-distance transport of these substances through tissue fluid or blood, and the specific surface ligands on the exosome membrane allow it to bind to the receptor cells with high efficiency. However, there is still a lack of cosmetics with excellent whitening effect. SUMMARY

[0004] In view of the above deficiencies in the prior art, the present application provides a cosmetic prepared based on stem cell exosomes and a preparation method thereof.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application to solve its technical problems is:

[0006] An active polypeptide, which has an amino acid sequence as shown in SEQ ID NO. 1; the specific sequence is: HLPIAYQRRVAPFSSRG;

[0007] or an amino acid sequence as shown in SEQ ID NO. 1 after substitution, deletion or addition of one or more amino acids, which has the same function as the amino acid sequence;

[0008] or an amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO. 1.

[0009] The active polypeptide is prepared by chemical synthesis or gene function.

[0010] Further, the active polypeptide shown in SEQ ID NO. 1 is synthesized by solid phase synthesis.

[0011] A cosmetic product comprising the active polypeptide.

[0012] Further, the cosmetic product can be in the form of a cream, a water-based agent, etc. DETAILED DESCRIPTION

[0013] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0014] Example 1 Synthesis of the active polypeptide

[0015] 1. Pretreatment of Wang resin

[0016] Wang resin (0.35 mmol / g, 1 g, 0.35 mmol, 1 eq) was weighed into a 25 mL glass solid phase tube. Then, 15 mL of DCM was added to the glass solid phase tube, and the resin was allowed to swell for 15 minutes, and finally the DCM was removed by suction filtration under reduced pressure;

[0017] 2. Removal of Fmoc protecting group

[0018] 15 mL of 20% (v / v) piperidine / DMF was added to the solid phase tube, and the solid phase tube was sealed and placed on a shaker for 15 min, and the deprotection process was repeated twice. Finally, the resin was washed with MeOH, DCM, and DMF, 10 mL each, in turn;

[0019] 3. Preparation of the active peptide

[0020] The amino acids in sequence 1 were coupled to the Wang resin in turn by standard solid phase synthesis method, wherein the coupling conditions were: amino acid (1.05 mmol, 3 eq), coupling reagent HBTU (1.05 mmol, 3 eq), HOBT (1.05 mmol, 3 eq), and DIEA (2.10 mmol, 6 eq); the deprotection condition of Fmoc protecting group was 20% piperidine / DMF (v / v). After each amino acid coupling reaction and deprotection of Fmoc protecting group, the resin was washed with MeOH, DCM, and DMF, 3 times each, to obtain the active polypeptide.

[0021] Example 2 Activity polypeptide performance detection

[0022] 1. Anti-aging detection

[0023] (1) Hacat cells were inoculated into cell culture dishes containing 10% FBS 1640 medium, and when the cell density reached 85%, they were replaced with serum-free medium for culture.

[0024] (2) After 24 hours of serum-free medium culture, Hacat cells were trypsinized and counted, and diluted to 2 x 10 6 / ml with 0.25% FBS 1640 medium. In a 96-well plate, 100 μL was inoculated per well.

[0025] (3) Set the polypeptide concentration gradient to 0 nM, 0.125 nM, 0.25 nM, 0.5 nM, 1 nM and 2 nM, and incubate with cells, with a total volume of 100 μL.

[0026] (4) The OD 450 absorbance value of each well was detected using a CCK-8 kit;

[0027] (5) The absorbance value under each concentration treatment condition was calculated by taking the absorbance value at 0 nM as the standard and setting it to 100%. The results of the measurement were all expressed as mean ± standard deviation, and the inter-group t test was used. The statistical software was SPSS 17.0, P < 0.05, with significant differences, and the results are shown in Table 1.

[0028] Table 1 Cell activity

[0029]

[0030]

[0031] Among them, ** indicates P < 0.01.

[0032] As can be seen from the data in Table 1, the active polypeptide constructed by the application can effectively improve the cell activity. When the concentration is 1 nM, the relative activity of the cells reaches the highest, but with further increase in concentration, the relative activity of the cells does not further improve, but decreases.

[0033] 2. Skin repair function detection

[0034] (1) 90 healthy mice weighing about 20 g were randomly divided into three groups, 30 in each group, namely blank control group, control group and experimental group; then the control group and experimental group mice were constructed with wounds, and the blank control group was not treated.

[0035] The specific process of wound construction is as follows: first, the mice are anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.1 ml / 20 g). Then the back of the mouse is shaved clean and disinfected, and finally two symmetrical holes of about 10 mm x 10 mm in size are chiseled on both sides of the back of the mouse with a hole chisel. After the operation, the mice are placed near the heater to wake up and then returned to the normal feeding room for normal feeding.

[0036] (2) The treatment is performed on the same day of full-thickness skin excision. It is applied twice a day, with about 20 μl of sample dropped on each hole each time. The left side of the back of the mice in the experimental group is coated with normal saline, and the right side is coated with the active polypeptide repair solution (1 nM) prepared in the application; the left side of the back of the mice in the control group is coated with normal saline, and the right side is coated with Kangfuxin solution. The wound repair rate is calculated every 3 days, and the wound repair rate = (wound size before treatment - wound size after treatment) / wound size before treatment x 100%.

[0037] Table 2 Wound repair rate

[0038]

[0039]

[0040] From the data in Table 2, it can be seen that the active polypeptide constructed in the application does not have excellent wound repair efficacy.

[0041] 3. Whitening performance detection

[0042] The active polypeptide with concentration gradients of 0.125 nM, 0.25 nM, 0.5 nM, 1 nM, 2 nM and 4 nM is added to the culture solution test tube containing melanocytes (2 mL) respectively, with PBS as a control, and the concentration of melanocytes in the test tube is 100 cells / mL. Then, the cell survival rate is detected after 5 h, 10 h and 12 h of culture at 37°C respectively (3 repeats for each group, P<0.05), and the results are shown in Table 3.

[0043] Table 3 Cell viability

[0044]

[0045] From the data in Table 3, it can be seen that the active polypeptide constructed in the application has excellent inhibitory effect on melanocytes, and the best action concentration is 1 nM.

[0046] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the application and not to limit it. Although the application has been described in detail with reference to the examples, it should be understood by those skilled in the art that the technical solutions of the application can be modified or replaced equivalently without departing from the

[0047] spirit and scope of the technical solutions of the application, which should be covered in the scope of the claims of the application.

Claims

1. An active polypeptide, characterized in that The amino acid sequence of the active polypeptide is shown in SEQ ID NO.

1.

2. The method for preparing the active polypeptide according to claim 1, characterized in that: Obtained by chemical synthesis.

3. A cosmetic, characterized in that: The active polypeptide according to claim 1 or 2.

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