Polypeptides, peptide-containing products and uses

Abstract

The embodiment of the invention discloses a polypeptide, a peptide-containing product and application, the polypeptide has an anti-glycosylation effect, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2: SEQ ID NO: 1: KRIVQRIKDFIR; and the SEQ ID NO: 2: KRIVWRIKDYLR is shown as SEQ ID NO: 2.

Description

Technical Field

[0001] The present invention relates to the field of peptide technology, in particular to polypeptides, peptide-containing products and uses. Background Art

[0002] Glycosylation involves the covalent binding of sugar molecules to proteins, lipids or nucleic acids, usually through the reaction of reducing sugars such as glucose and fructose with free amino groups to produce advanced glycation end products (AGEs). Advanced glycation end products are the products of excess sugar and protein binding.

[0003] Studies have found that AGEs can accelerate human aging and cause many chronic degenerative diseases, producing adverse effects. The effects of glycation on aging may include impaired protein function, oxidative stress, inflammation, cell signaling disruption, and impaired skin barrier function.

[0004] KR-12 (amino acid sequence: KRIVQRIKDFLR) is the 18-29 residues of the human LL-37 antimicrobial peptide. It is also a short peptide that is easily absorbed by organisms. Therefore, it has many application prospects, for example, it can be applied to the field of skin care products.

[0005] At present, there are new developments in the research of KR-12 peptides. Some improvement studies have been conducted based on KR-12, but most of them focus on antibacterial and immune functions. Among these improvement studies, no application in anti-glycosylation has been found. Summary of the invention

[0006] One purpose of the embodiments of the present application is to solve at least one problem in the above background and provide corresponding beneficial effects.

[0007] Another object of the embodiments of the present application is to provide a polypeptide, a peptide-containing product and a use that can be used for anti-glycosylation.

[0008] The embodiments of the present application mainly achieve the above objectives through the following technical solutions.

[0009] In a first aspect, the present application provides a polypeptide, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2:

[0010] SEQ ID NO: 1: KRIVQRIKDFIR;

[0011] SEQ ID NO:2: KRIVWRIKDYLR.

[0012] In a second aspect, the present application provides a peptide-containing product, including:

[0013] The polypeptide described in the first aspect.

[0014] In some technical embodiments, the concentration of the polypeptide in the peptide-containing product is 1-200 μM.

[0015] In another aspect, an embodiment of the present application provides a use of the polypeptide described in the embodiment of the first aspect in preparing a peptide-containing product.

[0016] In another aspect, the present application provides a use of the polypeptide described in the first aspect embodiment in the preparation of a skin care product.

[0017] In some technical solutions, the concentration of the polypeptide in the skin care product is 1-200 μM.

[0018] In some technical solutions, the skin care product is an external skin preparation.

[0019] In some technical solutions, the skin care product has anti-glycation function.

[0020] In another aspect, the embodiments of the present application provide a use of the polypeptide described in the first aspect for preparing an anti-glycosylation preparation.

[0021] The beneficial effects of the embodiments of the present invention include:

[0022] The polypeptide provided in the examples of the present application has a specific amino acid sequence and has an anti-glycosylation effect.

[0023] In some embodiments, the polypeptides of the embodiments of the present application can be used to prepare skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of AGEs concentration in Experimental Example 2 of this application. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0026] The terms "first", "second", "third", etc. in the embodiments of the present application are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second", etc. may explicitly or implicitly include at least one of the features.

[0027] Furthermore, the terms "comprises", "comprising", "including", "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products or apparatuses.

[0028] It should also be noted that when it comes to amino acid sequences, unless otherwise clearly specified and limited, R represents arginine, Q represents glutamine, K represents lysine, D represents aspartic acid, I represents isoleucine, V represents valine, L represents leucine, F represents phenylalanine, W represents tryptophan; and Y represents tyrosine.

[0029] In addition to the above, it is still necessary to emphasize that the reference to "embodiment" in this article means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0030] <Peptide>

[0031] The present application provides a polypeptide that can be used for anti-glycosylation. The polypeptide is selected from any one of KR12-N1 and KR12-N2; wherein the amino acid sequence of the KR12-N1 polypeptide is shown in SEQ ID NO:1, and the amino acid sequence of the KR12-N2 polypeptide is shown in SEQ ID NO:2:

[0032] SEQ ID NO: 1: KRIVQRIKDFIR;

[0033] SEQ ID NO:2: KRIVWRIKDYLR.

[0034] The amino acid sequences shown in SEQ ID NOs: 1 to 2 can also be found in the sequence listing.

[0035] The polypeptide represented by any one of the above SEQ ID NOs: 1 to 2 has an anti-glycosylation effect.

[0036] In addition, the polypeptide provided in the examples of the present application has an anti-glycosylation effect, so when acting on the skin, it can protect the skin.

[0037] In addition to the above effects, the polypeptide has good solubility, which is beneficial to the performance of the polypeptide function.

[0038] In addition, compared with the long LL-37 peptide, the polypeptide of the embodiment of the present application, as a short peptide, has a lower production cost.

[0039] Furthermore, glycosylation may also affect photoaging, so the polypeptide provided in the examples of the present application has the use of anti-photoaging.

[0040] The polypeptide can be prepared by solid-phase peptide synthesis (SPPS).

[0041] Illustratively, the solid phase synthesis sequence can be synthesized from the C-terminus (carboxyl terminus) to the N-terminus (amino terminus).

[0042] Accordingly, the present embodiment provides a method S10 for preparing a polypeptide, comprising the following steps:

[0043] Step S100: linking the first amino acid at the C-terminus of the polypeptide to a specific resin through a chemical bond, protecting the α-amino group of the amino acid with Fmoc, and protecting the side chain group with a corresponding acid-sensitive protecting group;

[0044] Step S200: removing the protective group Fmoc of the α-amino group of the first amino acid with an alkaline solution (e.g., piperidine DMF solution). At this time, the first amino acid with the α-amino group exposed remains on the resin;

[0045] Step S300: adding an excess amount of a second amino acid whose α-amino group is protected by Fmoc and whose side chain group is protected by a corresponding acid-sensitive protecting group, and allowing the second amino acid to undergo a condensation reaction with the first amino acid connected to the resin by means of a condensing agent;

[0046] Step S400: Detection. Detection is generally performed by detecting the exposed α-amino group of the previous amino acid, generally using ninhydrin. If an exposed α-amino group is detected, it indicates that the reaction is not complete, and the third step is repeated. If the reaction is complete, steps S200, S200, and S300 are repeated to continue the condensation reaction with the next amino acid until all amino acids are condensed, and then the Fmoc on the α-amino group of the last amino acid is removed (the method is the same as step S200);

[0047] Step S500: Use acid (commonly trifluoroacetic acid) to cut the peptide chain from the resin. At the same time, because the amino acid side chain protecting group is an acid-sensitive group, the protecting group on the side chain is also removed in this step to obtain the desired polypeptide.

[0048] <Products containing peptides>

[0049] Based on the polypeptides described in the preceding embodiments, the present application provides a peptide-containing product, including the polypeptides described in the preceding embodiments.

[0050] In some embodiments, the concentration of the polypeptide in the peptide-containing product is 1-200 μM (μmol / L), optionally 1-100 μM.

[0051] <Uses of polypeptides>

[0052] Based on the polypeptides described in the foregoing embodiments, the present application also provides the following uses of the polypeptides.

[0053] In some embodiments, the present application provides a use of the polypeptide for preparing an anti-glycosylation preparation.

[0054] In some embodiments, the present application also provides a use of the polypeptide in preparing a peptide-containing product.

[0055] In some embodiments, the peptide-containing product is a skin care product.

[0056] In some embodiments, the skin care product is a topical skin preparation.

[0057] In some embodiments, the peptide-containing product has anti-glycosylation function.

[0058] It is understandable that the polypeptides provided in the embodiments of the present application have a wide range of application scenarios in cosmetics because they have one or more of the above uses.

[0059] <Example of Preparation of Polypeptide>

[0060] Example 1

[0061] The polypeptide represented by the sequence of SEQ ID NO: 1, namely, KR12-N1 polypeptide, was prepared according to the polypeptide solid phase synthesis method.

[0062] Example 2

[0063] The polypeptide represented by the sequence of SEQ ID NO: 2, namely, KR12-N2 polypeptide, was prepared according to the polypeptide solid phase synthesis method.

[0064] Comparative Example 1

[0065] The KR12-N3 polypeptide was prepared according to the polypeptide solid phase synthesis method. The sequence of KR12-N3 is as follows: WRIVWRIKDFLR.

[0066] Comparative Example 2

[0067] The KR12-N 4 polypeptide was prepared according to the polypeptide solid phase synthesis method. The sequence of KR12-N 4 is as follows: WRICRRFKLFLR.

[0068] <Experimental Example 1, Peptide Stability Experiment>

[0069] In many cases, polypeptides need to be dissolved before administration, so their efficacy is related to solubility. However, any improvement to KR12 may not necessarily result in a polypeptide with better solubility. Therefore, the properties of the polypeptides in the embodiments of the present application are discussed below through solubility experiments.

[0070] Experimental method: Take the polypeptides KR12-N1~N4 corresponding to Examples 1~2 and Comparative Examples 1~2, respectively, mix them in phosphate buffer to form a peptide solution with a concentration of 50 μM (μmol / L) (the concentration of the polypeptide is 50 μM), and detect the solubility of the polypeptide in each peptide solution at 4 degrees Celsius.

[0071] Results and analysis of Experimental Example 1

[0072] KR12-M5-M6 of Comparative Examples 1-2 turned into turbid liquid within 12 hours, showing protein flocculent precipitation, while the remaining polypeptides could still maintain clear protein solutions within 7 days. This shows that KR12-N1-N2 corresponding to Examples 1-4 have good solubility in phosphate buffer, while KR12-N3-4 of Comparative Examples 1-2 have very poor solubility and are unstable.

[0073] Due to poor solubility, KR12-N3~4 are difficult to prepare into solution preparations for use, and it is even more difficult to exert the effect of the polypeptide in anti-photoaging. In contrast, the polypeptides of Examples 1~2 of the present application have excellent solubility and better stability.

[0074] <Experimental Example 2, Anti-glycosylation Assay>

[0075] Therefore, the effect of the polypeptides of the examples of the present application on anti-glycosylation was tested by detecting the production of AGEs.

[0076] The experimental method of Experimental Example 2 includes the following steps: :

[0077] Step S101, setting a blank control group, a model group (MODEL), a positive control group and three peptide experimental groups, wherein the three peptide experimental groups are KR-12 group, KR-12-N1 group and KR-12-N2 group, and configuring corresponding culture wells for each group on a 6-well culture plate.

[0078] Step S102, cell plating:

[0079] Take the cell fluid of Hacat cells (cell density is 2.5×105 ), add cell solution to the culture wells of each group, 2 mL of cell solution to each well; then place in a CO2 incubator and culture at 37°C and 5% CO2 for 20-24 hours.

[0080] Step S103: Cell processing:

[0081] The corresponding culture medium was added to the culture wells of each group to obtain a treated culture plate. The culture medium is specifically shown in Table 1. Then, the treated culture plate was placed at 37° C. and 5% CO 2 for 48 hours to incubate each group.

[0082] Table 1. Culture medium of each group

[0083]

[0084]

[0085] Step S104, supernatant collection:

[0086] After the incubation, 1 mL of the cell supernatant from each group of culture wells on the 6-well culture plate was collected into a 1.5 mL sterile centrifuge tube and stored in a -80°C ultra-low temperature refrigerator.

[0087] Step S105: cell counting:

[0088] The 6-well culture plate after collecting the supernatant was washed twice with 2 mL of PBS, 500 uL of trypsin was added to each well, digested for 8 minutes (mins), and then 1 mL of high-glucose DMEM medium was added to terminate the digestion; then 1.5 mL of the cell suspension of each group was respectively aspirated into the corresponding EP tube, and centrifuged at 1000 rpm for 10 minutes; then the supernatant of each group of EP tubes was discarded, and then 1 mL of PBS was added to the EP tube of each group for resuspending, and centrifuged again at 1000 rpm for 10 minutes; then 1 mL of PBS was added for resuspending to obtain the cell resuspension of each group, and 10 uL was taken in the counting plate for counting and the cell amount of each group was recorded.

[0089] Step S106: AGES content detection:

[0090] Taking the cell supernatant of each group in step S104 as a sample respectively;

[0091] According to the instructions of the kit (the kit is CUSABIO / Huamei Biotechnology CSB-E09412h-48T human advanced glycation end products (AGEs) enzyme-linked immunosorbent assay kit), the standard (pure AGEs in the kit) was diluted in a gradient manner, and then the standard and samples were subjected to ELISA (enzyme-linked immunosorbent assay) according to the instructions. Finally, the absorbance in the culture wells corresponding to each group of the 6-well culture plate was measured at an OD450 wavelength.

[0092] Step S107: Statistical data and drawing:

[0093] A standard curve was prepared and the AGES content of each group was obtained by substituting the data into the standard curve. The relative concentration of advanced glycation end products (Concentrations of AGEs) was then obtained by dividing the cell amount of each group. Finally, statistical analysis was performed and a bar graph was prepared.

[0094] Experimental results and analysis of Experimental Example 2

[0095] The experimental results are shown in Figure 1 , Figure 1 A schematic diagram of AGEs concentrations in the blank control group, model group, positive control group, KR-12 group, KR-12-N1 group, and KR-12-N2 group.

[0096] exist Figure 1 In the figure, the unit of advanced glycation end product concentration is μg / mL; in the figure, A represents the blank control group, B represents the model group, C represents the positive control group, D represents the KR-12 group, E represents the KR-12-N1 group, and F represents the KR-12-N2 group.

[0097] according to Figure 1 The results showed that the AGEs concentration in the model group exceeded that in the blank control group, indicating that methylglyoxal (MGO) can induce the production of AGEs.

[0098] Comparing the results of the positive control group and the model group, it can be seen that after kojic acid treatment, the concentration of AGEs decreased significantly, indicating that kojic acid has anti-glycation activity.

[0099] Comparing the results of the peptide experimental group with the positive control group and the model group, it can be seen that the KR-12 group, KR-12-N1 group, and KR-12-N2 group also showed anti-glycosylation activity; among them, the AGEs concentrations of the KR-12-N1 group and the KR-12-N2 group were significantly lower than that of the KR-12 group, indicating that the KR12-N1 polypeptide and the KR12-N2 polypeptide have good anti-glycosylation activity, and the KR12-N2 polypeptide is even better than the KR-12 polypeptide before modification.

[0100] It should also be noted that:

[0101] DMEM is a culture medium containing amino acids and glucose, which is developed on the basis of MEM culture medium. Compared with MEM, it has increased the dosage of various components and is divided into high-sugar type (higher than or equal to 4500 mg / L) and low-sugar type (lower than 1000 mg / L). The high-sugar DMEM culture medium selected in the above experimental example has a sugar content of 4500 mg / L.

[0102] PBS (phosphate buffered saline) refers to phosphate buffered saline.

[0103] EP (eppendorf) tube is a centrifuge tube.

[0104] VE refers to Vitamin E.

[0105] The above specific embodiments have been described in detail for the present invention, but these do not constitute limitations on the present invention. The protection scope of the present invention is not limited to the above embodiments, and any equivalent modifications or changes made by ordinary technicians in the field based on the content disclosed by the present invention should be included in the protection scope recorded in the claims.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO: 2: SEQ ID NO: 1: KRIVQRIKDFIR; SEQ ID NO:2: KRIVWRIKDYLR.

2. Use of the polypeptide according to claim 1 in preparing skin care products.

3. The use according to claim 2; wherein The concentration of the polypeptide in the skin care product is 1-200 μM.

4. The use according to claim 2; wherein The skin care product is an external skin preparation.

5. The use according to claim 2; wherein The skin care product has the function of anti-glycation.

6. Use of the polypeptide according to claim 1 for preparing an anti-glycosylation preparation.

7. A peptide-containing product, characterized in that include: The polypeptide according to claim 1.

8. The peptide-containing product according to claim 7, characterized in that The concentration of the polypeptide in the peptide-containing product is 1-200 μM.

9. The peptide-containing product according to claim 7, characterized in that The peptide-containing product is a skin care product.

10. The peptide-containing product according to claim 9, characterized in that The skin care product is an external skin preparation.

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