Skin care composition added with recombinant III type collagen as well as preparation method and application of skin care composition

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a skin care composition added with recombinant III-type collagen as well as a preparation method and application of the skin care composition. The skin care composition is prepared from the following components in percentage by mass: 2.4 to 5.6 percent of trehalose, 3.1 to 4.5 percent of 1, 2-hexanediol, 0.8 to 1.4 percent of arginine, 0.6 to 0.9 percent of 1, 2-pentanediol, 1.5 to 2.8 percent of sodium chloride, 2.3 to 4.7 percent of herba centellae extract, 1.3 to 2.5 percent of acetyl hexapeptide-8, 1.7 to 3.2 percent of soluble collagen and the balance of water. The soluble collagen is recombinant III type collagen; the amino acid sequence of the recombinant III type collagen is as shown in SEQ ID NO. 1. The recombinant III-type collagen has no cytotoxicity, and the skin care composition added with the recombinant III-type collagen has the effects of obviously improving wrinkles and improving skin elasticity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a skin care composition added with recombinant type III collagen as well as a preparation method and application thereof. BACKGROUND

[0002] With the continuous growth of age, the metabolism of epidermal cells will slow down, the epidermal layer will become thin, and it is easy to produce dry lines and wrinkles, which directly affects the appearance and function of the skin. At present, most of the anti-wrinkle products on the market have a relatively single approach in improving wrinkles, and it is difficult to achieve a rapid, continuous and effective improvement effect. With the increasing improvement of people's living standards, the quality requirements for beauty and skin care products are also getting higher and higher.

[0003] Collagen is an important functional protein in the dermis, which provides sufficient nutrition and strength for the dermis, and is closely related to skin regeneration. It is widely added to skin care products such as anti-aging, moisturizing and repairing damaged skin. At present, at least 28 different types of collagen are known, among which types I, II, III and IV are the most common in the body. Type III collagen is a triple helix structure formed by three identical polypeptide chains spirally wound, has a highly conserved amino acid sequence, is essential for maintaining skin elasticity, promoting wound healing and maintaining vascular integrity, and is an important structural protein. At present, the production of collagen mainly uses acid and alkali methods to process animal-derived tissues to extract collagen. However, the collagen product obtained by the traditional method has complex components, is mainly used for feed additives and microbial fermentation medium, and when applied to the human body, it can easily cause immune rejection, thereby limiting the application of collagen in medicine.

[0004] With the development of biotechnology, the production mode of directed production of collagen by microorganisms using DNA recombination technology has been applied. The recombinant collagen produced by DNA recombination technology has higher controllability and safety, improves the usability of collagen, and has a wide application prospect in the field of biomedical research. Based on the above background, the present application provides a preparation method and application of recombinant type III collagen, which has good safety and can be widely applied in the fields of skin care products, biological materials, artificial skin, health foods and the like. SUMMARY

[0005] The first object of the present application is to provide a skin care composition added with recombinant type III collagen, which has no cytotoxicity and good safety.

[0006] In order to achieve the above object, the technical scheme adopted by the present application is:

[0007] A skincare composition containing recombinant type III collagen, the skincare composition comprising the following components in weight fractions: trehalose 2.4-5.6%, 1,2-hexanediol 3.1-4.5%, arginine 0.8-1.4%, 1,2-pentanediol 0.6-0.9%, sodium chloride 1.5-2.8%, Centella asiatica extract 2.3-4.7%, acetyl hexapeptide-8 1.3-2.5%, soluble collagen 1.7-3.2%, and the balance being water; wherein the soluble collagen is recombinant type III collagen; and the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO.1.

[0008] Furthermore, the nucleotide sequence encoding the recombinant type III collagen is shown in SEQ ID NO.2.

[0009] Furthermore, the specific preparation process of the recombinant type III collagen is as follows:

[0010] (1) The nucleotide sequence encoding recombinant type III collagen was ligated to the vector to obtain a recombinant plasmid;

[0011] (2) Transform the recombinant plasmid into bacteria to construct a recombinant strain;

[0012] (3) The recombinant strain constructed in step (2) was expressed. The bacterial precipitate was collected, the supernatant was obtained by breaking it, and the recombinant type III collagen was obtained by purification.

[0013] Further, the expression process described in step (3) is as follows: the recombinant strain is inoculated into LB medium containing kanamycin and shaken overnight, and IPTG is added to induce the expression of recombinant type III collagen.

[0014] Furthermore, the concentration of kanamycin is 50-100 μg / mL.

[0015] Furthermore, the IPTG induction concentration is 0.3-0.5 mmol / L, and the IPTG induction time is 5-10 h.

[0016] The second objective of this invention is to provide a method for preparing a skincare composition containing recombinant type III collagen.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0018] The preparation method of the skin care composition with added recombinant type III collagen described above includes the following specific steps:

[0019] Trehalose, 1,2-hexanediol, 1,2-pentanediol, arginine, and water are mixed in the specified proportions and stirred until homogeneous to obtain a mixture. Sodium chloride, Centella asiatica extract, acetyl hexapeptide-8, and soluble collagen are added to the mixture and stirred until homogeneous at 30-40°C to obtain the final product.

[0020] The third objective of this invention is to provide an application of skincare products containing recombinant type III collagen in the preparation of skincare products.

[0021] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0022] The application of the skin care composition containing recombinant type III collagen in the preparation of skin care products, wherein the skin care composition containing recombinant type III collagen has a mass percentage of 2-5% based on the total mass of the skin care products.

[0023] Compared with the prior art, the main advantages of the present invention are as follows:

[0024] This invention uses gene recombination technology to construct and express a recombinant type III collagen. This recombinant type III collagen is non-cytotoxic and has good safety. Skin care compositions containing recombinant type III collagen can promote the absorption of collagen in the skin, improve skin elasticity, and significantly improve wrinkles, showing good application prospects. Attached Figure Description

[0025] Figure 1 This is an expression diagram of the purified recombinant type III collagen prepared in this invention;

[0026] Figure 2 A bar chart showing the cytotoxicity of the recombinant type III collagen prepared in this invention;

[0027] Figure 3 The percentage increase in skin elasticity achieved by the skincare products prepared according to this invention. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0029] Example 1

[0030] A skincare composition containing recombinant type III collagen, the skincare composition comprising 2.4% trehalose, 3.1% 1,2-hexanediol, 0.8% arginine, 0.6% 1,2-pentanediol, 1.5% sodium chloride, 2.3% centella asiatica extract, 1.3% acetyl hexapeptide-8, 1.7% recombinant type III collagen, with the balance being deionized water.

[0031] The preparation method of the recombinant type III collagen specifically includes the following steps.

[0032] (1) By analyzing the composition and structure of human type III collagen sequence, the stability of peptide segments, and their ability to bind to receptors on cells, short peptides with amino acids at positions 234-440 were screened, and the tail of the short peptides was modified with GNPGAPGSP to obtain recombinant type III collagen COL1. The recombinant collagen has multiple important functional sites of human type III collagen, has high biological activity, and its amino acid sequence is shown in SEQ ID NO.1. Based on the amino acid sequence of recombinant type III collagen COL1 SEQ ID NO.1, the gene sequence was reverse-engineered and the codons were optimized to obtain the nucleotide sequence SEQ ID NO.2 encoding recombinant type III collagen. The nucleotide sequence of recombinant type III collagen was ligated with the pET28a vector, and the recombinant plasmid pET28a-COL1 was obtained after identification and sequencing.

[0033] SEQ ID NO.1:

[0034] GRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGS PGAKGEVGPAGPSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGNPGAPGSPGNPGAPGSP;

[0035] SEQ ID NO.2:

[0036] GGCCGTCCGGGCCGTCCAGGTGAACGTGGCCTGCCAGGTCCTCCGGGTATTAAAGGCCCGGCTGGCATCCCGGGTTTCCCGGGTATGAAGGGCCACCGTGGTTTCGACGGTCGTAATGGCGAGAAGGGCGAGACCGGTGCGCCGGGTCTGAAGGGCGAAAACGGTCTGCCGGGCGAAAACGGTGCACCTGGTCCGATGGGTCCGCGTGGCGCTCCGGGTGAACGTGGCCGTCCAGGTCTGCCGGGTGCAGCCGGTGCCCGCGGTAACGACGGTGCGCGTGGCTCCGATGGCCAGCCTGGCCCGCCAGGCCCGCCGGGTACTGCCGGTTTCCCGGGCTCTCCAGGCGCGAAAGGTGAAGTGGGTCCAGCCGGTAGCCCGGGTAGCAACGGTGCACCGGGTCAGCGTGGTGAACCTGGTCCGCAGGGCCACGCTGGTGCTCAAGGTCCGCCGGGTCCGCCGGGTATTAACGGCTCCCCAGGTGGCAAGGGCGAAATGGGCCCGGCAGGCATTCCAGGCGCTCCTGGCCTGATGGGTGCGCGTGGCCCTCCTGGTCCGGCAGGTGCTAATGGCGCACCTGGCCTGCGCGGTGGTGCAGGTGAACCGGGCAAAAACGGCGCTAAAGGTAACCCGGGTGCGCCGGGCTCTCCGGGTAACCCGGGTGCGCCAGGTTCTCCA。

[0037] (2) Transfer the recombinant plasmid pET28a-COL1 constructed in step (1) into competent E. coli BL21(DE3) cells, spread the cells on an LB plate containing 100 μg / mL kanamycin and incubate overnight in an inverted position in a 37 °C incubator to obtain a recombinant strain named pET28a-COL1 / BL21;

[0038] (3) Positive monoclonal strain pET28a-COL1 / BL21 was selected and inoculated into 5 mL of LB medium containing 100 μg / mL kanamycin, and cultured at 37℃ with shaking at 220 rpm for 12 h. The bacterial culture was then inoculated into 50 mL of LB medium containing 100 μg / mL kanamycin at a ratio of 1:100, and cultured until the OD600 reached approximately 0.8. 0.4 mmol / L IPTG was then added to induce recombinant type III collagen expression for 8 h. The bacterial cells were collected by centrifugation, dispersed in buffer solution, and the supernatant was collected after cell lysis. Recombinant type III collagen was purified by affinity chromatography, and the purified recombinant type III collagen was detected by SDS-PAGE gel electrophoresis. The results are as follows: Figure 1 As shown, M represents the protein marker, and band 1 represents the expression result of purified recombinant type III collagen.

[0039] This embodiment also provides a method for preparing the above-mentioned skin care composition with added recombinant type III collagen, specifically including the following steps:

[0040] Trehalose, 1,2-hexanediol, 1,2-pentanediol, arginine, and deionized water were mixed in proportion and stirred until dissolved to obtain a mixture. Sodium chloride, Centella asiatica extract, acetyl hexapeptide-8, and recombinant type III collagen were added to the mixture and stirred at 30°C for 15 minutes to obtain the skin care composition with added recombinant type III collagen.

[0041] Example 2

[0042] A skincare composition containing recombinant type III collagen, the skincare composition comprising 4.0% trehalose, 3.8% 1,2-hexanediol, 1.1% arginine, 0.7% 1,2-pentanediol, 2.1% sodium chloride, 3.5% centella asiatica extract, 1.9% acetyl hexapeptide-8, 2.4% recombinant type III collagen, with the balance being deionized water.

[0043] The preparation method of the recombinant type III collagen is the same as in Example 1;

[0044] This embodiment also provides a method for preparing the above-mentioned skin care composition with added recombinant type III collagen, specifically including the following steps:

[0045] Trehalose, 1,2-hexanediol, 1,2-pentanediol, arginine, and deionized water were mixed in proportion and stirred until dissolved to obtain a mixture. Sodium chloride, Centella asiatica extract, acetyl hexapeptide-8, and recombinant type III collagen were added to the mixture and stirred at 35°C for 25 minutes to obtain the skin care product composition with added recombinant type III collagen.

[0046] Example 3

[0047] A skincare composition containing recombinant type III collagen, the skincare composition comprising 5.6% trehalose, 4.5% 1,2-hexanediol, 1.4% arginine, 0.9% 1,2-pentanediol, 2.8% sodium chloride, 4.7% centella asiatica extract, 2.5% acetyl hexapeptide-8, 3.2% recombinant type III collagen, with the balance being deionized water.

[0048] The preparation method of the recombinant type III collagen is the same as in Example 1;

[0049] This embodiment also provides a method for preparing the above-mentioned skin care composition with added recombinant type III collagen, specifically including the following steps:

[0050] Trehalose, 1,2-hexanediol, 1,2-pentanediol, arginine, and deionized water were mixed in proportion and stirred until dissolved to obtain a mixture. Sodium chloride, Centella asiatica extract, acetyl hexapeptide-8, and recombinant type III collagen were added to the mixture and stirred at 35°C for 25 minutes to obtain the skin care product composition with added recombinant type III collagen.

[0051] Comparative Example 1

[0052] Comparative Example 1 provides a skincare composition that differs from Example 1 in that it omits recombinant type III collagen, while all other aspects are the same as in Example 1.

[0053] Comparative Example 2

[0054] Comparative Example 2 provides a skincare composition that differs from Example 1 in that the recombinant type III collagen in Example 1 is replaced with human type III collagen, while all other aspects are the same as in Example 1.

[0055] Experimental Example 1

[0056] Safety evaluation of recombinant type III collagen:

[0057] HeLa cells that have grown to 80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin to achieve a cell density of 4 × 10⁻⁶ cells / year. 5Cell suspension was prepared at 100 μL / mL. 100 μL of cell suspension was seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator. After 24 hours of culture, the culture medium was aspirated. Cells were divided into experimental and control groups. The experimental group contained different concentrations of recombinant type III collagen (0.005-1.0 mg / mL, with 3 replicates per concentration) diluted in high-glucose DMEM medium. The control group contained high-glucose DMEM medium without recombinant type III collagen. The blank group contained cell-free DMEM medium. Cells were cultured for another 24 hours at 37°C in a 5% CO2 incubator. 10 μL of LCK-8 reagent was added to each group, and the cells were incubated for 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader, and cell viability was calculated based on the absorbance of each group.

[0058] Depend on Figure 2 It can be seen that, compared with the control group, the cell viability of the experimental group with added recombinant type III collagen was improved, and all were above 95%. The different concentrations of recombinant type III collagen prepared by the invention have no cytotoxicity and good safety, and can be used in the preparation and use of cosmetics.

[0059] Experimental Example 2

[0060] Testing of the anti-wrinkle effects of the skincare compositions in Examples 1-3 and Comparative Examples 1-2 of this invention:

[0061] A skin care product comprising the following ingredients in weight percentages: 1.5% carbomer, 0.4% PEG-100 stearate, 0.01% fragrance, 4% of the above-mentioned skin care composition, with the balance being water.

[0062] The anti-wrinkle effect of the skincare products obtained by adding the skincare compositions of Examples 1-3 and Comparative Examples 1-2 was tested, and the specific process is as follows:

[0063] One hundred test subjects, aged 45-55, were randomly divided into 5 groups. The outer corner of the eye was the target area (same region and area). Test subjects applied skincare products containing the skincare compositions of Examples 1-3 and Comparative Examples 1-2 twice daily, morning and evening. After 4 weeks of use, the PROMIS system was used to analyze and evaluate the area, depth, and percentage reduction in the number of wrinkles at the outer corner of the eye, and the average values ​​for each group were obtained. The results are shown in Table 1.

[0064] Table 1

[0065]

[0066] As shown in Table 1, compared with Comparative Examples 1 and 2, the skincare products prepared in Examples 1-3 significantly improved the depth, area, and number of wrinkles around the eyes. This indicates that the skincare composition containing recombinant type III collagen can promote the absorption of collagen in the skin and has a significant effect on improving wrinkles.

[0067] Experimental Example 3

[0068] Skincare product efficacy test for improving skin elasticity:

[0069] One hundred test subjects, aged 45-55 years, were randomly selected and randomly divided into 5 groups. The cheeks were the target area (same region and area). Test subjects applied skincare products prepared with the skincare compositions of Examples 1-3 and Comparative Examples 1-2 twice daily, morning and evening. The percentage increase in skin elasticity on the cheeks was measured using an ELASTIMETER ELM1086 at 7, 14, and 28 days. The results are as follows: Figure 3 As shown.

[0070] Depend on Figure 3 It can be seen that, compared with Comparative Examples 1 and 2, the skin care products prepared in Examples 1-3 can significantly improve skin elasticity. This indicates that the skin care composition with added recombinant type III collagen can promote the absorption of collagen in the skin and has a significant effect on improving skin elasticity.

[0071] In summary, the skincare composition containing recombinant type III collagen prepared in this invention has the effect of significantly improving wrinkles and enhancing skin elasticity.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A skincare composition containing recombinant type III collagen, characterized in that, The skincare composition comprises the following components in the indicated mass fractions: trehalose 2.4-5.6%, 1,2-hexanediol 3.1-4.5%, arginine 0.8-1.4%, 1,2-pentanediol 0.6-0.9%, sodium chloride 1.5-2.8%, centella asiatica extract 2.3-4.7%, acetyl hexapeptide-8 1.3-2.5%, soluble collagen 1.7-3.2%, and the balance being water; wherein the soluble collagen is recombinant type III collagen; and the amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO.

1.

2. The skincare composition with added recombinant type III collagen according to claim 1, characterized in that, The nucleotide sequence encoding the recombinant type III collagen is shown in SEQ ID NO.

2.

3. The skincare composition with added recombinant type III collagen according to claim 1, characterized in that, The specific preparation process of the recombinant type III collagen is as follows: (1) The nucleotide sequence encoding recombinant type III collagen was ligated to the vector to obtain a recombinant plasmid; (2) Transform the recombinant plasmid into bacteria to construct a recombinant strain; (3) The recombinant strain constructed in step (2) was expressed. The bacterial precipitate was collected, the supernatant was obtained by breaking it, and the recombinant type III collagen was obtained by purification.

4. The skincare composition with added recombinant type III collagen according to claim 3, characterized in that, The expression process described in step (3) is as follows: the recombinant strain is inoculated into LB medium containing kanamycin and shaken overnight, and IPTG is added to induce the expression of recombinant type III collagen.

5. The skincare composition with added recombinant type III collagen according to claim 4, characterized in that, The concentration of kanamycin is 50-100 μg / mL.

6. The skincare composition with added recombinant type III collagen according to claim 4, characterized in that, The IPTG induction concentration was 0.3-0.5 mmol / L, and the IPTG induction time was 5-10 h.

7. The method for preparing the skincare composition with added recombinant type III collagen according to any one of claims 1-6, characterized in that, The specific steps include: Trehalose, 1,2-hexanediol, 1,2-pentanediol, arginine, and water are mixed in the specified proportions and stirred until homogeneous to obtain a mixture. Sodium chloride, Centella asiatica extract, acetyl hexapeptide-8, and soluble collagen are added to the mixture and stirred until homogeneous at 30-40°C to obtain the final product.

8. The application of the skincare composition with added recombinant type III collagen according to claim 1 in the preparation of skincare products.

9. The application of the skin care composition with added recombinant type III collagen according to claim 8 in the preparation of skin care products, wherein the skin care composition with added recombinant type III collagen has a mass percentage content of 2-5% based on the total mass of the skin care products.

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