Collagen peptide with anti-aging effect and preparation method and application thereof

Abstract

This invention belongs to the field of cosmetic technology, specifically relating to a collagen peptide with anti-aging effects, its preparation method, and its application. The amino acid sequence of the collagen peptide is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. This invention constructs and expresses a collagen peptide Sa1 by recombining the effective sequences of human type III and XVII collagen. This collagen peptide Sa1 exhibits significant antioxidant effects, is non-cytotoxic, and has good safety. Skincare products containing collagen peptide Sa1 can promote the absorption of collagen in the skin, effectively exert antioxidant effects, thereby improving skin elasticity and delaying skin aging.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a collagen peptide with anti-aging effects, its preparation method, and its application. Background Technology

[0002] Collagen is a core component of the extracellular matrix (ECM), accounting for over 75% of the skin's dry weight. It not only provides mechanical support for the skin but also participates in cell signaling, tissue repair, and the maintenance of microenvironment homeostasis through dynamic remodeling. Studies have shown that with age, both the quality and quantity of the collagen network change significantly, leading to signs of aging such as skin laxity and wrinkles. Research has identified more than 28 types of collagen in human skin, which can be classified into three main categories based on their function: fibroblastic collagen, basement membrane collagen, and fibroblast-associated discontinuous collagen. Fibroblastic collagen, as a major structural component of the dermis, with type III collagen accounting for approximately 10%-15%, forms a fine network to maintain tissue elasticity and promotes cell migration, adhesion, and the synthesis of new collagen. Type XVII collagen, as a type of basement membrane collagen, maintains the adhesion and polarity of epidermal stem cells and plays a significant role in regulating hair loss, skin aging, and wound healing.

[0003] However, both natural collagen extracted from animals and single-type recombinant collagen produced by early genetic engineering have their inherent limitations: on the one hand, collagen from animal tissues may carry immunogenicity and viral contamination risks, posing safety hazards; on the other hand, different types of collagen perform different biological functions in the body, and a single type of collagen cannot simulate the complex environment of multiple collagens working synergistically in the natural extracellular matrix, thus limiting its application effects.

[0004] To address the aforementioned issues, this invention utilizes gene recombination technology to rationally design, splice, and recombine the effective active sequences from human type III and XVII collagen, constructing a novel collagen peptide, Sa1. This recombinant peptide not only overcomes the defects of natural collagen, exhibiting good safety and no cytotoxicity, but also possesses significant antioxidant properties, making it widely applicable in skincare products, biomaterials, and artificial skin. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a collagen peptide with anti-aging effects.

[0006] The second objective of this invention is to provide a method for preparing a collagen peptide with anti-aging effects.

[0007] A third objective of this invention is to provide the application of the aforementioned collagen peptides with anti-aging effects in the preparation of skin care products.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A collagen peptide with anti-aging effects, wherein the amino acid sequence of the collagen peptide is shown in SEQ ID NO.1 and the nucleotide sequence is shown in SEQ ID NO.2.

[0010] Furthermore, it includes the following steps:

[0011] (1) The effective sequences of human type III collagen and type XVII collagen were spliced ​​together to obtain the amino acid sequence of collagen peptide SEQ ID NO.1;

[0012] (2) Based on the amino acid sequence of collagen peptide SEQ ID NO.1, reverse design of gene sequence was performed, codon optimization was carried out, and the nucleotide sequence encoding collagen peptide SEQ ID NO.2 was obtained;

[0013] (3) The nucleotide sequence encoding collagen peptides was ligated to the vector pPIC9K to construct a recombinant plasmid;

[0014] (4) Electroporate the recombinant plasmid into competent cells to construct the recombinant strain;

[0015] (5) The recombinant strain constructed in step (4) was inoculated into a medium containing methanol for induced expression to obtain collagen peptides.

[0016] Further, the specific operation of step (3) is as follows: based on the nucleotide sequence SEQ ID NO.2 of the collagen peptide, design upstream and downstream primers to amplify the corresponding nucleotide sequence of the collagen peptide, clone the collagen peptide nucleotide sequence amplified by the primers into the pPIC9K vector, and construct a recombinant plasmid.

[0017] Furthermore, the upstream primer sequence of the corresponding nucleotide sequence of the collagen peptide is shown in SEQ ID NO.3; the downstream primer sequence is shown in SEQ ID NO.4.

[0018] Further, the competent cells in step (4) are Pichia pastoris GS115 competent cells.

[0019] Further, the concentration of methanol in the methanol-containing culture medium in step (5) is 0.5-1 v / v.

[0020] The above-mentioned collagen peptides with anti-aging effects are used in the preparation of skin care products.

[0021] Furthermore, the collagen peptide content in the skin care product is 4-6% by mass.

[0022] Compared with the prior art, the main advantages of the present invention are as follows:

[0023] This invention assembles a novel collagen peptide, Sa1, by splicing functional fragments from regions 520-630 and 483-498 of the α1 chain functional domain of human type III collagen, the oligopeptide fragment PRGEQG from the functional region of human type XVII collagen, and a rigid linker sequence. Sa1 exhibits significant antioxidant effects, is non-cytotoxic, and demonstrates good safety. Skincare products containing collagen peptide Sa1 can promote collagen absorption in the skin, effectively exert antioxidant effects, thereby improving skin elasticity and delaying skin aging. Attached Figure Description

[0024] Figure 1 The plasmid map of recombinant plasmid pPIC9K-Sa1;

[0025] Figure 2 This is an expression diagram of the purified collagen peptide Sa1 prepared in this invention;

[0026] Figure 3 The plasmid map of recombinant plasmid pPIC9K-Sa2;

[0027] Figure 4 The plasmid map of recombinant plasmid pPIC9K-Sa3;

[0028] Figure 5 A bar chart showing the cytotoxicity of Sa1, a collagen peptide with anti-aging effects prepared in this invention.

[0029] Figure 6 The percentage increase in skin elasticity achieved by the skincare product prepared according to this invention;

[0030] Figure 7 The percentage reduction in wrinkles achieved by the skincare products prepared according to this invention. Detailed Implementation

[0031] 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.

[0032] Example 1

[0033] A collagen peptide Sa1 with anti-aging effects, wherein the amino acid sequence of the collagen peptide Sa1 is shown in SEQ ID NO.1 and the nucleotide sequence is shown in SEQ ID NO.2.

[0034] The preparation method of the collagen peptide Sa1 with the above-mentioned anti-aging effect specifically includes the following steps:

[0035] (1) Select functional fragments from the 520-630 and 483-498 regions of the α1 chain functional domain of human type III collagen, the oligopeptide fragment PRGEQG of the functional region of human type XVII collagen, and the rigid linker sequence (GPPGPCCGGG) to splice together to form a new collagen peptide. The amino acid sequence of the new collagen peptide is shown in SEQ ID NO.1.

[0036] (2) Based on the amino acid sequence of collagen peptide SEQ ID NO.1, a gene sequence was designed in reverse, and the corresponding base sequence was codon optimized according to codon preference to obtain the nucleotide sequence encoding collagen peptide SaI1 SEQ ID NO.2. Based on the obtained collagen peptide Sa1 nucleotide sequence SEQ ID NO.2, upstream and downstream primers for amplifying the corresponding nucleotide sequence of the novel collagen peptide were designed. The upstream primer sequence is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4. The novel collagen peptide SaI1 was amplified by PCR, and the target fragment was purified using a gel extraction kit. The sequence information is shown in Table 1.

[0037] Table 1 Sequence List

[0038]

[0039] (3) The plasmid pPIC9K was double-digested using restriction endonucleases EcoRI and NotI. The double digestion reaction system is shown in Table 2. The amplified and recovered collagen peptide Sa1 was ligated to the linearized pPIC9K vector using T4 DNA ligase. The T4 DNA ligase reaction system is shown in Table 3. The constructed recombinant plasmid pPIC9K-Sa1 was transformed into E. coli DH5α competent cells and cultured in LB plates containing 100 μg / mL Amp. Single colonies were picked for colony PCR identification, and the plasmid was extracted for sequencing identification to obtain the recombinant plasmid pPIC9K-Sa1. The plasmid map of the recombinant plasmid pPIC9K-Sa1 is shown in Table 2. Figure 1 As shown.

[0040] Table 2. Double enzyme digestion reaction system

[0041]

[0042] Table 3. T4 DNA ligase reaction system

[0043]

[0044] (4) After linearizing the correct recombinant plasmid pPIC9K-Sa1 verified by sequencing with Sal I, the linearized plasmid pPIC9K-Sa1 was introduced into Pichia pastoris GS115 competent cells by electroporation. The electroporated bacterial solution was spread on MD plates and incubated upside down in a 30°C incubator until single colonies appeared. Colony PCR was used to verify the recombinant strain pPIC9K-Sa1 / GS115. At the same time, copy number screening was performed to obtain the high copy recombinant yeast strain pPIC9K-Sa1 / GS115.

[0045] (5) The high-copy recombinant yeast strain pPIC9K-Sa1 / GS115 obtained by screening was inoculated into BMGY medium and cultured at 30℃ and 250 rpm with shaking until the OD600 reached 4. The cells were collected by centrifugation and resuspended in BMGY medium containing 1% (v / v) methanol for induction expression. Methanol was added every 24 hours to a final concentration of 1%, and expression was induced at 30℃ and 250 rpm for 120 hours. The supernatant was collected by centrifugation and purified by Ni-NTA affinity chromatography to obtain collagen peptide Sa1. The purified collagen peptide Sa1 was detected by SDS-PAGE gel chromatography, and the results are as follows. Figure 2 As shown in the figure. In the figure, M represents the protein marker, and band 1 represents the expression result of the purified collagen peptide Sa1.

[0046] Comparative Example 1

[0047] A collagen peptide Sa2 with anti-aging effects, wherein the amino acid sequence of the collagen peptide Sa2 is shown in SEQ ID NO.5 and the nucleotide sequence is shown in SEQ ID NO.6.

[0048] The preparation method of the collagen peptide Sa2 with the above-mentioned anti-aging effect specifically includes the following steps:

[0049] (1) Select the functional fragment of the α1 chain functional domain of human type III collagen 483-498, the oligopeptide fragment PRGEQG of the functional region of human type XVII collagen and the rigid linker sequence (GPPGPCCGGG) and splice them together to form a new collagen peptide Sa2. The amino acid sequence of the new collagen peptide Sa2 is shown in SEQ ID NO.5.

[0050] (2) Based on the amino acid sequence of collagen peptide Sa2 (SEQ ID NO.5), a gene sequence was designed in reverse, and the corresponding base sequence was optimized according to codon preference to obtain the nucleotide sequence encoding collagen peptide Sa2 (SEQ ID NO.6). Based on the obtained nucleotide sequence of collagen peptide Sa2 (SEQ ID NO.6), upstream and downstream primers for amplifying the corresponding nucleotide sequence of the novel collagen peptide were designed. The upstream primer sequence is shown in SEQ ID NO.7, and the downstream primer sequence is shown in SEQ ID NO.8. The novel collagen peptide Sa2 was amplified by PCR, and the target fragment was purified using a gel extraction kit. The sequence information is shown in Table 4.

[0051] Table 4 Sequence List

[0052]

[0053] (3) The plasmid pPIC9K was double-digested using restriction endonucleases EcoRI and NotI. The double digestion reaction system is shown in Table 2. The amplified and recovered collagen peptide Sa2 was ligated with the linearized pPIC9K vector using T4 DNA ligase. The double digestion reaction system is shown in Table 3. The constructed recombinant plasmid pPIC9K-Sa2 was transformed into E. coli DH5α competent cells and cultured in LB plates containing 100 μg / mL Amp. Single colonies were picked for colony PCR identification, and the plasmid was extracted for sequencing identification to obtain the recombinant plasmid pPIC9K-Sa2. The plasmid map of the recombinant plasmid pPIC9K-Sa2 is shown in Table 3. Figure 3 As shown.

[0054] Steps (4) and (5) of this Comparative Example 1 are the same as those of Example 1.

[0055] Comparative Example 2

[0056] A collagen peptide Sa3 with anti-aging effects, wherein the amino acid sequence of the collagen peptide Sa3 is shown in SEQ ID NO.9 and the nucleotide sequence is shown in SEQ ID NO.10.

[0057] The preparation method of the collagen peptide Sa3 with the above-mentioned anti-aging effect specifically includes the following steps:

[0058] (1) A functional fragment from the 520-630 region of the α1 chain functional domain of human type III collagen, an oligopeptide fragment PRGEQG from the functional region of human type XVII collagen, and a rigid linker sequence (GPPGPCCGGG) were spliced ​​together to form a new collagen peptide Sa3. The amino acid sequence of the novel collagen peptide Sa3 is shown in SEQ ID NO.9. The sequence information is shown in Table 5.

[0059] Table 5 Sequence List

[0060]

[0061] (2) Based on the amino acid sequence of collagen peptide Sa3 (SEQ ID NO. 9), a gene sequence was designed in reverse, and the corresponding base sequence was optimized according to codon preference to obtain the nucleotide sequence encoding collagen peptide Sa3 (SEQ ID NO. 10). Based on the obtained nucleotide sequence of collagen peptide Sa3 (SEQ ID NO. 10), upstream and downstream primers for amplifying the corresponding nucleotide sequence of the novel collagen peptide were designed. The upstream primer sequence is shown in SEQ ID NO. 11, and the downstream primer sequence is shown in SEQ ID NO. 12. The novel collagen peptide Sa3 was amplified by PCR, and the target fragment was purified using a gel extraction kit.

[0062] (3) The plasmid pPIC9K was double-digested using restriction endonucleases EcoRI and NotI. The double digestion reaction system is shown in Table 2. The amplified and recovered collagen peptide Sa3 was ligated to the linearized pPIC9K vector using T4 DNA ligase. The double digestion reaction system is shown in Table 3. The constructed recombinant plasmid pPIC9K-Sa3 was transformed into E. coli DH5α competent cells and cultured in LB plates containing 100 μg / mL Amp. Single colonies were picked for colony PCR identification, and the plasmid was extracted for sequencing identification to obtain the recombinant plasmid pPIC9K-Sa3. The plasmid map of the recombinant plasmid pPIC9K-Sa3 is shown in Table 3. Figure 4 As shown.

[0063] Steps (4) and (5) of Comparative Example 2 are the same as those of Example 1.

[0064] Experimental Example 1

[0065] Antioxidant analysis of collagen peptides:

[0066] This invention uses DPPH as the detection index. Its ethanol solution is deep purple and exhibits strong absorption near 517 nm. When a free radical scavenger is present, the light absorption of the DPPH ethanol solution is weakened due to its pairing with a single electron, thus allowing evaluation of the test sample's ability to scavenge free radicals. Collagen peptides Sa1, Sa2, and Sa3 with anti-aging effects, prepared in Example 1 and Comparative Examples 1-2, were dissolved in ethanol to prepare samples with a mass concentration of 6.0 mg / mL. Their DPPH free radical scavenging effect was then measured. The specific steps are as follows:

[0067] (1) Add 1 mL of collagen peptides of the corresponding group to 1 mL of DPPH solution, mix quickly, and let stand at room temperature in the dark for 30 min. The absorbance value of this tube is recorded as Ax.

[0068] (2) Add 1 mL of collagen peptides of the corresponding group to 1 mL of anhydrous ethanol solution, mix quickly, and let stand at room temperature in the dark for 30 min to subtract the color of the sample itself. The absorbance value of this tube is recorded as Ab.

[0069] (3) Add 1 mL of DPPH solution to 1 mL of anhydrous ethanol solution, mix quickly, and let stand at room temperature in the dark for 30 min. This represents the initial amount of DPPH free radicals. The absorbance value of this tube is recorded as Ac.

[0070] (4) The DPPH free radical scavenging rate was calculated by the following formula: DPPH free radical scavenging rate % = [(Ac−(Ax−Ab) / Ac]×100%, where Ac is the initial absorbance of DPPH free radical; Ax is the absorbance of the sample after reacting with DPPH; and Ab is the absorbance of the blank sample. The test results are shown in 6.

[0071] Table 6. DPPH free radical scavenging rates of collagen peptides Sa1, Sa2, and Sa3

[0072]

[0073] The results are shown in Table 6, which presents the DPPH free radical scavenging rates of the collagen peptides Sa1, Sa2, and Sa3, which have anti-aging effects. As can be seen from the table, compared with Comparative Examples 1 and 2, the collagen peptide Sa1 prepared in Example 1 of this invention significantly improved the DPPH free radical scavenging rate. This indicates that the collagen peptide Sa1 provided by this invention has a significant antioxidant effect, effectively scavenging free radicals and reducing free radical damage to skin cells. Its active ingredients can serve as high-quality antioxidant ingredients in cosmetics, with anti-aging effects.

[0074] Experimental Example 2

[0075] Safety evaluation of collagen peptide Sa1:

[0076] This experiment aimed to evaluate the cytotoxicity of collagen peptide Sa1 on HeLa cells. The specific experimental procedures were as follows: Fully adherent HeLa cells were digested with 0.25% trypsin and resuspended in high-glucose DMEM medium containing 5% FBS, adjusting the cell density to 2 × 10⁵ cells / mL. 100 μL of the cell suspension was seeded into 96-well plates and cultured at 37°C in a 5% CO₂ incubator for 24 hours. After 24 hours, the culture medium was aspirated. Cells were divided into experimental and control groups. The experimental groups were treated with different concentrations of collagen peptide Sa1 (0.001, 0.005, 0.01, 0.05, 0.1, and 0.5 mg / mL, diluted in high-glucose DMEM medium, with three replicates per concentration). The control group received an equal volume of high-glucose DMEM medium without collagen peptide Sa1. The blank group received high-glucose DMEM medium without cells or collagen peptide Sa1. Continue culturing in a 37°C, 5% CO2 cell culture incubator for 24 hours. After culturing, add 10 μL of CCK-8 reagent to each group, gently shake to mix, and return to the incubator for 2 hours in the dark. Use a microplate reader to detect the absorbance value of each well at a wavelength of 450 nm. Calculate the cell viability based on the absorbance of each group. The formula is: Cell viability (%) = [(OD experimental group - OD blank group) / (OD control group - OD blank group)] × 100%.

[0077] The results are as follows Figure 5 The figure shown is a bar chart illustrating the cytotoxicity of the collagen peptide Sa1 prepared in this invention. Figure 5 As can be seen, compared with the control group, the cell viability of the collagen peptide Sa1 with anti-aging effects prepared in Example 1 of this invention was improved, and all were above 100%. This indicates that the collagen peptide Sa1 with anti-aging effects prepared by this invention has no cytotoxicity and good safety, and can be used in the preparation and use of skin care products.

[0078] Experimental Example 3

[0079] Skincare product efficacy test:

[0080] A skincare product is composed of the following ingredients in weight percentages: 5% glycerin, 5% collagen peptides, 3% caprylic / capric triglycerides, 1.5% PEG-100 stearate, 2% cetearyl alcohol, 0.5% vitamin E, 0.05% sodium hyaluronate, and the balance being deionized water. The collagen peptides are the anti-aging collagen peptides Sa1, Sa2, and Sa3 prepared in Example 1 and Comparative Examples 1-2. The skincare products prepared by adding the anti-aging collagen peptides from Example 1 and Comparative Examples 1-2 were tested for their skin elasticity-enhancing and wrinkle-reducing effects. The specific process is as follows:

[0081] Sixty women aged 45-60 with aging skin were randomly divided into three groups of 20 each. After cleansing morning and evening, they were given a treatment at 2mL / cm². 2 The dosages were applied to skincare products made from collagen peptides with anti-aging effects, as described in Example 1 and Comparative Examples 1-2, respectively. Skin elasticity on the cheek area was measured using an ELASTIMETER skin elasticity meter after 14 and 28 days. The absolute scores were statistically analyzed, and the percentage increase in skin elasticity after 14 and 28 days of use was calculated. The results are as follows: Figure 6 As shown in the figure. The VISIA skin analyzer was used to quantitatively analyze eye wrinkles at 14 and 28 days. The absolute scores were statistically analyzed, and the percentage reduction in wrinkles after 14 and 28 days was calculated. The results are shown in the figure. Figure 7 As shown.

[0082] The results are as follows Figure 6 The figure shows the percentage increase in skin elasticity achieved by the skincare product prepared according to this invention. Figure 6 It can be seen that, compared with Comparative Examples 1 and 2, the skin care product prepared in Example 1 of this invention can significantly improve skin elasticity. This indicates that the skin care product prepared by adding collagen peptide Sa1, which has anti-aging effects, can promote the absorption of collagen in the skin and has a significant effect on improving skin elasticity.

[0083] The results are as follows Figure 7 The figure shows the percentage reduction in wrinkles achieved by the skincare product prepared according to this invention. Figure 7 As can be seen, compared with Comparative Examples 1 and 2, the skin care product prepared in Example 1 of this invention exhibits significant anti-wrinkle effects. This indicates that the skin care product prepared by adding collagen peptide Sa1, which has anti-aging effects, can reduce free radical damage to skin cells, thereby delaying skin aging and improving wrinkles.

[0084] 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 collagen peptide with anti-aging effects, characterized in that, The collagen peptide is composed of a functional fragment of amino acids 520-630 and 483-498 of the human type III collagen α1 chain, an oligopeptide fragment PRGEQG of the human type XVII collagen functional region, and a rigid linker sequence GPPGPCCGGG; the amino acid sequence of the collagen peptide is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.

2.

2. The method for preparing a collagen peptide with anti-aging effects according to claim 1, characterized in that, Includes the following steps: (1) The effective sequences of human type III collagen and type XVII collagen were spliced ​​together to obtain the amino acid sequence of collagen peptide SEQ ID NO.1; (2) Based on the amino acid sequence of collagen peptide SEQ ID NO.1, reverse design of gene sequence was performed, codon optimization was carried out, and the nucleotide sequence encoding collagen peptide SEQ ID NO.2 was obtained; (3) The nucleotide sequence encoding collagen peptides was ligated to the vector pPIC9K to construct a recombinant plasmid; (4) Electroporate the recombinant plasmid into competent cells to construct the recombinant strain; (5) The recombinant strain constructed in step (4) was inoculated into a medium containing methanol for induced expression to obtain collagen peptides.

3. The method for preparing a collagen peptide with anti-aging effects according to claim 2, characterized in that, The specific operation of step (3) is as follows: based on the nucleotide sequence SEQ ID NO.2 of the collagen peptide, design upstream and downstream primers to amplify the corresponding nucleotide sequence of the collagen peptide, clone the collagen peptide nucleotide sequence amplified by the primers into the pPIC9K vector, and construct a recombinant plasmid.

4. The method for preparing a collagen peptide with anti-aging effects according to claim 3, characterized in that, The upstream primer sequence of the corresponding nucleotide sequence of the collagen peptide is shown in SEQ ID NO.3; the downstream primer sequence is shown in SEQ ID NO.

4.

5. The method for preparing a collagen peptide with anti-aging effects according to claim 2, characterized in that, The competent cells in step (4) are Pichia pastoris GS115 competent cells.

6. The method for preparing a collagen peptide with anti-aging effects according to claim 2, characterized in that, In step (5), the concentration of methanol in the methanol-containing culture medium is 0.5-1 v / v.

7. The application of the collagen peptide with anti-aging effects as described in claim 1 in the preparation of skin care products.

8. The application of the collagen peptide with anti-aging effects according to claim 7 in the preparation of skin care products, characterized in that, The collagen peptide content in the skin care product is 4-6% by mass.

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