Application of a recombinant short peptide in anti-wrinkle, whitening, brightening skin tone and anti-oxidation

Abstract

The present application relates to the technical field of protein engineering, and specifically discloses the application of a recombinant short peptide in anti-wrinkle, whitening, brightening skin tone, and anti-oxidation. The recombinant short peptide includes an amino acid sequence as shown in SEQ ID NO.2. The present application extracts the amino acid sequence from microorganisms isolated from seawater, and obtains the recombinant short peptide by fermentation and culture of Pichia pastoris, further proving that the recombinant short peptide does have anti-wrinkle, whitening, brightening skin tone, and anti-oxidation effects, and can remove harmful free radicals in the human body.

Description

Technical Field

[0001] The present application relates to the technical field of protein engineering, and more specifically, to the application of a recombinant short peptide in anti-wrinkle, whitening, brightening skin tone, and anti-oxidation. Background Art

[0002] With the development of society and the improvement of living standards, people pay more and more attention to personal image and skin health. As the largest organ of the human body, the skin not only protects internal tissues from external environmental damage, but also is the external manifestation of beauty and health. However, due to the influence of aging, ultraviolet radiation, environmental pollution and many other factors, the skin will have problems such as wrinkles, spots, dullness and oxidative damage. Therefore, the development of safe and effective skin care ingredients for anti-wrinkle, whitening, brightening skin tone and anti-oxidation has always been the focus of cosmetics and skin care research.

[0003] Currently, there are many anti-wrinkle and whitening products on the market, which usually contain various natural extracts, vitamins, antioxidants and other active ingredients. However, these ingredients often have problems such as poor stability, low transdermal absorption efficiency, limited effects or side effects. In view of this, scientists have been looking for new active ingredients that are more efficient, safe and have good stability.

[0004] As a product of bioengineering technology, recombinant short peptides have shown great potential in the field of skin care in recent years due to their small molecular weight, easy transdermal absorption, high activity and good safety. Through genetic engineering technology, short peptides with specific amino acid sequences can be designed and synthesized. These short peptides can specifically affect the physiological processes of skin cells, thereby achieving the purpose of improving skin problems. Summary of the invention

[0005] The present application provides an application of a recombinant short peptide in anti-wrinkle, whitening, brightening skin tone and anti-oxidation.

[0006] In the first aspect, the present application provides an application of a recombinant short peptide in anti-wrinkle, whitening, brightening skin tone, and anti-oxidation, using the following technical solution:

[0007] A recombinant short peptide is used in anti-wrinkle, whitening, brightening skin tone and anti-oxidation. The recombinant short peptide comprises an amino acid sequence as shown in SEQ ID NO.2.

[0008] Optionally, when the concentration of the recombinant short peptide is 50%, the DPPH free radical scavenging rate reaches 20%.

[0009] Optionally, when the concentration of the recombinant short peptide is 0.2%, the whitening, skin-lightening and anti-oxidation effects reach 50%.

[0010] In the second aspect, the present application provides an application of a recombinant short peptide in the preparation of products for anti-wrinkle, whitening, brightening skin tone, and anti-oxidation, using the following technical solution:

[0011] A recombinant short peptide is used in the preparation of products for anti-wrinkle, whitening, brightening skin tone and anti-oxidation. The recombinant short peptide comprises an amino acid sequence as shown in SEQ ID NO.2.

[0012] In a third aspect, the present application provides a composition comprising the above-mentioned recombinant short peptide.

[0013] In a fourth aspect, the present application provides a gene sequence. The composition includes the above-mentioned recombinant short peptide.

[0014] In a fifth aspect, the present application provides a recombinant vector. The composition includes the above gene sequence.

[0015] In a sixth aspect, the present application provides a recombinant cell. The composition includes the above gene sequence.

[0016] In summary, this application has the following beneficial effects:

[0017] The recombinant short peptide provided in this application has obvious anti-wrinkle, whitening, brightening and anti-oxidation effects, and can achieve the regeneration of its own coenzyme NADH. It can also be absorbed and utilized by the human body as a nutrient to improve the body's immunity and resistance, and is of great significance for anti-aging and cell repair. However, under the same conditions, the freckle removal effect is not obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the anti-wrinkle effect test result of the recombinant short peptide of the present application (the concentration of the recombinant short peptide in the test sample is 50%).

[0019] Figure 2 These are the test results of the whitening, brightening and antioxidant effects of the recombinant short peptide of the present application (A. normal control group; B. recombinant short peptide concentration 0.05%; C. recombinant short peptide concentration 0.2%).

[0020] Figure 3 The results of the freckle removal efficacy test of the recombinant short peptide of the present application (A. normal control group; B. recombinant short peptide concentration 0.05%; C. recombinant short peptide concentration 0.2%). DETAILED DESCRIPTION

[0021] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0022] The present application provides a recombinant short peptide for use in anti-wrinkle, whitening, brightening skin tone, and anti-oxidation, wherein the recombinant short peptide comprises an amino acid sequence as shown in SEQ ID NO. 2. When the concentration of the recombinant short peptide is 50%, the DPPH free radical scavenging rate reaches 20%. When the concentration of the recombinant short peptide is 0.2%, the whitening, brightening, and anti-oxidation effects reach 50%.

[0023] The present application also provides an application of a recombinant short peptide in the preparation of a product for anti-wrinkle, whitening, brightening skin tone, and anti-oxidation. The recombinant short peptide comprises an amino acid sequence as shown in SEQ ID NO. 2. Specifically, the product may be a composition comprising the recombinant short peptide.

[0024] The present application also provides a gene sequence for synthesizing the above-mentioned recombinant short peptide, as well as a recombinant vector and a recombinant cell comprising the gene sequence.

[0025] In order to make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application are clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application.

[0026] The present application is further described in detail below in conjunction with the embodiments and test results.

[0027] Example

[0028] Example 1

[0029] This embodiment provides a recombinant short peptide.

[0030] The preparation process of the recombinant short peptide is as follows:

[0031] (1) Strain isolation and detection

[0032] The red strain obtained from the coral symbiotic strain was used as a sample and identified as marine red yeast through electron microscopy. Through comparison with the NCBI database, the gene information corresponding to the marine red yeast was found, and it was found that it included the SOD gene. Therefore, we used the SOD gene to design primers and amplified a sequence from the red yeast by PCR amplification. After gene sequencing, a new gene fragment was obtained and named TXOD. Finally, the re-obtained TXOD was obtained by gene recombination. The specific gene recombination method is the same as the commonly used method in the relevant technology, and the expressed strain is obtained and saved for later use.

[0033] The obtained TXOD was tested using a ROS kit (cell reactive oxygen species detection kit), and the test method was referred to the instructions of the kit. According to the test results, a gene fragment with antioxidant activity was screened from TXOD designed with different primers, and was named TXOD-1. The gene sequence includes the nucleotide sequence shown in SEQ ID NO.1.

[0034] (2) Preparation of recombinant short peptides

[0035] The above gene sequence is connected to a recombinant expression vector (vector pPIC9K), and then the recombinant expression vector is introduced into Pichia pastoris (can be completed by electroporation, chemical methods (such as LiCl method) or using a yeast transformation kit), and the positive clone strains are screened and the positive clone strains obtained by screening are subjected to a small amount of expression test to evaluate the expression level and activity of the target protein. The positive clone strains with good performance are expanded and cultured. The target protein obtained by culture is extracted and purified, and sent to a third-party detection structure for detection, and the amino acid sequence shown in SEQ ID NO.2 is obtained, which is a recombinant short peptide.

[0036] Example 2

[0037] In this example, the anti-wrinkle efficacy of the recombinant short peptide provided in Example 1 was tested.

[0038] The specific analysis is as follows:

[0039] 1. Experimental principles and basis

[0040] According to the free radical theory, excessive free radical production is the main cause of natural skin aging and photoaging, which leads to wrinkles. Therefore, whether a product has the ability to scavenge free radicals is one of the important indicators for evaluating anti-aging and anti-wrinkle cosmetics (raw materials).

[0041] This test is carried out in accordance with the laboratory method (free radical scavenging method): QTTZ-XZ-37 "Detailed Rules for Verification and Testing of Anti-wrinkle Efficacy of Cosmetics". The DPPH free radical scavenging rate test results of the test sample are compared with those of the negative control. If the scavenging rate of the test sample is higher than that of the negative control and there is a significant difference (P < 0.05), it can be considered that the test sample has a certain anti-wrinkle effect.

[0042] 2. Materials and Methods

[0043] (1) Instruments and equipment

[0044] Thousandth electronic balance: Shanghai Puchun Measuring Instrument Co., Ltd., JA2003.

[0045] 1 / 10,000 electronic balance: Shanghai Puchun Measuring Instrument Co., Ltd., FA2004.

[0046] Microplate reader: Thermo Fisher (Shanghai) Instrument Co., Ltd., Multiskan FC.

[0047] (2) Reagents

[0048] DPPH:McLean,C12998355.

[0049] Ascorbic acid: Tianjin Damao Chemical Reagent Factory, 20210715.

[0050] Anhydrous ethanol: Tianjin Damao Chemical Reagent Factory, 20211004.

[0051] (3) Experimental procedures

[0052] Pre-treat the sample (recombinant short peptide): add 2 mL of the sample with different concentrations to 2 mL of DPPH working solution, mix well and react at room temperature in the dark for 30 minutes. The concentration of the recombinant short peptide in the sample is 5%, 10%, 30%, 50% and 100%;

[0053] Working solution preparation: Use 2 mL of 0.2 mM DPPH solution as the working solution. The working solution is prepared by dissolving DPPH in 95% ethanol and set aside.

[0054] The sample to be tested reacts with the DPPH reagent, and the OD value is measured at 517 nm after the reaction.

[0055] 3. Detection indicators

[0056] Clearance rate (%) = (A2 + A3 - A1) / A2 × 100.

[0057] Among them, A1 is the OD value of the reaction system with DPPH in the sample solution; A2 is the OD value of the reaction system with DPPH in anhydrous ethanol; A3 is the OD value of the reaction system without DPPH (with anhydrous ethanol as a substitute).

[0058] The test results are characterized by the free radical scavenging rate of the sample. If the test sample has a higher scavenging rate than the negative control, and there is a significant difference between the test sample and the negative control, it means that the test product has anti-wrinkle effect, and to ensure the accuracy of the test system, the average scavenging rate of the positive control group should be greater than 25%. The significant difference was statistically analyzed by SPSS software, and when the significant factor P < 0.05, it was considered to be significantly different.

[0059] 4. The test results are shown in Table 1 and Figure 1 shown.

[0060] Table 1 Clearance determination results

[0061]

[0062] Figure 1 It is the comparison result between the negative control and the positive control when the concentration of the recombinant short peptide in the test sample is 50%.

[0063] From Table 1 and Figure 1 It can be seen that according to the laboratory method (free radical scavenging method): QTTZ-XZ-37 "Detailed Rules for Verification and Testing of Cosmetic Anti-Wrinkle Efficacy", the anti-wrinkle efficacy of the sample was tested by the laboratory method (free radical scavenging method). The test results show that: under laboratory conditions, when the concentration of the recombinant short peptide in the sample to be tested is in the range of 5-100%, the DPPH free radical scavenging rate is greater than 11.83±0.70%, which is significantly different from the negative control (P<0.05); in particular, when the concentration of the recombinant short peptide in the sample to be tested is 50%, the average DPPH free radical scavenging rate is 19.52±1.40%, which is significantly different from the negative control (P<0.05), and the positive control DPPH free radical scavenging rate is>25%, the reaction system is effective, indicating that the sample has anti-wrinkle efficacy.

[0064] Example 3

[0065] In this example, the recombinant short peptide provided in Example 1 was tested for its whitening, skin-lightening and antioxidant effects.

[0066] The specific analysis is as follows:

[0067] 1. Method name: Zebrafish whitening efficacy test method.

[0068] 2. Method source: "Standard operating procedures for zebrafish whitening efficacy evaluation experiment".

[0069] 3. Principle and method: Zebrafish are transparent in the early stages of development. Melanin begins to grow from the retinal epithelium when the embryo develops to 24 hours. Pigment cells originate from a group of cells differentiated from the dorsal ectoderm - neural crest cells, which then proliferate, migrate and differentiate into pigment mother cells. Intervention in the process of melanin formation can inhibit the formation of melanin. The whiteness of zebrafish skin is used to evaluate whether the sample has whitening effect.

[0070] 4. Volume and sample size: Fish strain used: wild-type AB strain zebrafish; Zebrafish age: 6 hours after fertilization (6hpf); Experimental sample size for each group: 15 fish (N=10): Adult fish rearing and breeding methods: in accordance with laboratory standard rearing and breeding methods, in line with the requirements of international AAALAC certification (certification number: 001458).

[0071] 5. Experimental Procedure

[0072] (1) Zebrafish were randomly selected and placed in a 6-well plate, with 15 zebrafish in each well.

[0073] (2) Water-soluble samples were administered and a normal control group was set up at the same time. The volume of each well was 3 mL.

[0074] (3) Incubate at 28°C in the dark for 45 h.

[0075] (4) Ten zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Advanced image processing software was used to analyze and collect data. The melanin signal intensity (S) of the zebrafish head was analyzed, and the whitening effect of the sample was calculated according to the formula to determine whether it had whitening effect.

[0076] Whitening effect (%) = (S (normal control group) - S (sample group)) / S (normal control group) × 100.

[0077] (6) Judgment basis: Statistical analysis p < 0.05, efficacy value ≥ 20%, judged as effective.

[0078] 6. The test results are shown in Table 2 and Figure 2 shown.

[0079] Table 2 Results of whitening, brightening and anti-oxidation effects

[0080] Serial number Detection concentration (%) Whitening, brightening skin tone, anti-oxidant effect (%) P-value Test results 1 0.05 28 ＞0.05 Not obvious 2 0.2 50 ＜0.001 efficient

[0081] From Table 2 and Figure 2 It can be seen that the head melanin signal intensity of the recombinant short peptide provided by the present application is significantly reduced compared with the normal control group, revealing that the recombinant short peptide has whitening, brightening and antioxidant effects. In particular, when the concentration of the recombinant short peptide provided by the present application is 0.2%, it has obvious whitening, brightening and antioxidant effects.

[0082] Example 4

[0083] In this example, the recombinant short peptide provided in Example 1 was tested for its freckle removal efficacy.

[0084] The specific analysis is as follows:

[0085] 1. Method name: Zebrafish freckle removal efficacy test method.

[0086] 2. Source of the method: "Standard Operating Procedure for Evaluation of Zebrafish Freckle Removal Efficacy".

[0087] 3. Principle and method: The formation of spots is related to the deposition of melanin in the skin. The spot removal effect is essentially the removal of melanin. Zebrafish are transparent in the early stages of development. Melanin begins to grow from the retinal epithelium at 24 hours of embryonic development, and stable and visible melanin spots gradually form at 48 hours. The degree of melanin reduction is used to evaluate whether the sample has the effect of spot removal.

[0088] 4. Volume and sample size: Fish strain used: wild-type AB strain zebrafish; Zebrafish age: 2 days after fertilization (2 dpf); Experimental sample size for each group: 15 fish (N=10): Adult fish rearing and breeding methods: in accordance with laboratory standard rearing and breeding methods, in line with the requirements of international AAALAC certification (certification number: 001458).

[0089] 5. Experimental Procedure

[0090] (1) Zebrafish were randomly selected and placed in a 6-well plate, with 15 zebrafish in each well.

[0091] (2) Water-soluble samples were administered and a normal control group was set up at the same time. The volume of each well was 3 mL.

[0092] (3) Incubate at 28°C in the dark for 24 h.

[0093] (4) Ten zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Advanced image processing software was used to analyze and collect data. The melanin signal intensity (S) of the zebrafish head was analyzed, and the freckle removal effect of the sample was calculated according to the formula to determine whether it had freckle removal effect.

[0094] Anti-freckle effect (%) = (S (normal control group) - S (sample group)) / S (normal control group) × 100.

[0095] (6) Judgment basis: Statistical analysis p < 0.05, efficacy value ≥ 20%, judged as effective.

[0096] 6. The test results are shown in Table 3 and Figure 3 shown.

[0097] Table 3 freckle removal efficacy test results

[0098] Serial number Detection concentration (%) Whitening effect (%) P-value Test results 1 0.05 6 ＞0.05 Not obvious 2 0.2 12 ＞0.05 Not obvious

[0099] From Table 3 and Figure 3 It can be seen that the melanin signal intensity of the head of the recombinant short peptide provided in the present application is similar to that of the normal control group, revealing that the freckle removal effect of the recombinant short peptide is not obvious.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A use of a recombinant short peptide in the preparation of a product for anti-wrinkle, whitening, brightening skin tone, and anti-oxidation, characterized in that: The recombinant short peptide is an amino acid sequence as shown in SEQ ID NO. 2; When the concentration of the recombinant short peptide is 5-100%, the DPPH free radical scavenging rate reaches more than 10%; When the concentration of the recombinant short peptide is not less than 0.2%, the whitening, brightening and anti-oxidation effects reach more than 50%.

2. The use according to claim 1, characterized in that: When the concentration of the recombinant short peptide is 50%, the DPPH free radical scavenging rate reaches 20%.

3. The use according to claim 1, characterized in that: When the concentration of the recombinant short peptide is 0.2%, the whitening, brightening and anti-oxidation effects reach 50%.

4. A composition, characterized in that The composition comprises the recombinant short peptide according to claim 1.

5. A gene, characterized in that The gene is used to encode the recombinant short peptide described in claim 1; specifically, the nucleotide sequence shown in SEQ ID NO.

1.

6. A recombinant vector, characterized in that: The recombinant vector comprises the gene according to claim 5.

7. A recombinant cell, characterized in that The recombinant cell comprises the gene of claim 5.

Images