Polypeptides, peptide-containing products, and uses

Abstract

The embodiments of the present application disclose a polypeptide, a peptide-containing product and use, the polypeptide has the effect of resisting glycosylation, and 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.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of peptides, in particular to polypeptides, peptide-containing products and uses. BACKGROUND

[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, resulting in advanced glycation end products (AGEs), which are the products of the combination of excess sugars and proteins.

[0003] Studies have found that AGEs can accelerate the aging of the human body and lead to the occurrence of many chronic degenerative diseases, producing adverse effects. The effects of glycosylation on aging may include impaired protein function, oxidative stress, inflammation, disrupted cell signaling and impaired skin barrier function.

[0004] KR-12 (amino acid sequence KRIVQRIKDFLR) is an 18-29 residue of human LL-37 antibacterial peptide, which is also a short peptide with the property of being easily absorbed by organisms, and therefore has a wide application prospect, for example, it can be applied to the field of skin care products.

[0005] Currently, there have been new developments in the study of KR-12 polypeptides, and some improvement studies have been made based on KR-12, but most of them focus on the functions of antibiosis and immunity. In these improvement studies, no application in anti-glycosylation has been found. SUMMARY

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

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

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

[0009] In a first aspect, the embodiments of the present application provide 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 embodiments of the present application provide a peptide-containing product, comprising:

[0013] The polypeptide of the first aspect.

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

[0015] In another aspect, the embodiments of the present application provide use of the polypeptide of the first aspect in the preparation of a peptide-containing product.

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

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

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

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

[0020] In another aspect, the embodiments of the present application provide use of the polypeptide of the first aspect in the preparation of an anti-glycation preparation.

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

[0022] The polypeptide provided by the embodiments of the present application has a specific amino acid sequence and has an anti-glycation effect.

[0023] In some embodiments, the polypeptide of the embodiments of the present application can be used in the preparation of a skin care product. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A diagram showing one AGEs concentration in Experimental Example 2 of the present application. DETAILED DESCRIPTION

[0025] The present application will be further described in conjunction with the accompanying drawings, so that those skilled in the art can implement the present application according to the description in the specification.

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

[0027] Furthermore, the terms "comprising" and "containing" and variations thereof as used herein are intended to be synonymous with the term "including" and are therefore meant to be construed in an inclusive sense and not in an exclusive sense. For example, a process, method, system, product, or apparatus that comprises a list of steps or elements is not necessarily limited to only those steps or elements but can include other steps or elements not expressly listed or inherent to such process, method, system, product, or apparatus.

[0028] It is also to be noted that when referring to amino acid sequences, unless explicitly stated and limited otherwise, 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; Y represents tyrosine.

[0029] In addition to the above, it is still emphasized that the reference herein to "embodiments" means that the particular features, structures, or characteristics described in connection with the embodiments can be included in at least one of the embodiments of the present application. The appearance of the phrase in various places in the specification does not necessarily all refer to the same embodiments, nor is it necessarily independent or alternative embodiments to 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] <Polypeptide>

[0031] The embodiments of the present application provide a polypeptide, which 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 NO: 1-2 can also be referred to the sequence listing.

[0035] The polypeptide shown in any one of SEQ ID NO: 1-2 has an anti-glycosylation effect.

[0036] In addition, the polypeptide provided by the embodiments of the present application has an anti-glycosylation effect, so when it acts on the skin, it can play a protective effect on the skin.

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

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

[0039] In addition, glycosylation can also affect photoaging, so the polypeptide provided in the embodiment of the application has the use of resisting photoaging.

[0040] The polypeptide can be prepared according to a solid-phase peptide synthesis (SPPS) method.

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

[0042] Accordingly, the embodiment of the application provides a preparation method S10 of a polypeptide, including the following steps:

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

[0044] Step S200: remove the protecting group Fmoc of the α-amino group of the first amino acid with an alkaline solution (for example, a hexahydropyridine DMF solution), at this time, the first amino acid with the exposed α-amino group remains on the resin;

[0045] Step S300: add an excess of the second amino acid protected with Fmoc at the α-amino group and with the corresponding acid-sensitive protecting group at the side chain group, and let it react with the first amino acid connected to the resin through a condensing agent;

[0046] Step S400: detection, which is generally performed by detecting the exposed α-amino group of the last amino acid, and is generally detected with ninhydrin, if the 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, and the next amino acid is continuously subjected to the condensation reaction, until all the amino acids are condensed, and then the Fmoc at the α-amino group of the last amino acid is removed (the method is the same as step S200);

[0047] Step S500: cut the peptide chain from the resin with an acid (commonly used is trifluoroacetic acid), and at the same time, because the side chain protecting group of the amino acid is selected to be an acid-sensitive group, the protecting group on the side chain is also removed at this step, so that the required polypeptide is obtained.

[0048] <Peptide-containing product>

[0049] Based on the polypeptide described in the foregoing embodiments, the embodiments of the present application further provide a peptide-containing product comprising the polypeptide described in the foregoing embodiments.

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

[0051] <Use of polypeptide>

[0052] Based on the polypeptide described in the foregoing embodiments, the embodiments of the present application further provide the following use of the polypeptide.

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

[0054] In some embodiments, the embodiments of the present application further provide the use of the polypeptide described for 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 an external skin preparation.

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

[0058] It can be understood that the polypeptide provided by the embodiments of the present application has a wide application scenario in cosmetics due to having one or more of the above uses.

[0059] <Preparation example of polypeptide>

[0060] Embodiment 1

[0061] The polypeptide shown in the sequence of SEQ ID NO: 1, i.e., KR12-N1 polypeptide, is prepared according to the polypeptide solid-phase synthesis method.

[0062] Embodiment 2

[0063] The polypeptide shown in the sequence of SEQ ID NO: 2, i.e., KR12-N2 polypeptide, is prepared according to the polypeptide solid-phase synthesis method.

[0064] Comparative Example 1

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

[0066] Comparative Example 2

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

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

[0069] The polypeptide needs to be dissolved in many cases before being administered, and thus the exertion of its efficacy is related to the solubility. However, any improvement of KR12 does not necessarily result in a polypeptide with good solubility, and thus the properties of the polypeptides of the embodiments of the present application are discussed below through solubility experiments.

[0070] Experimental method: the polypeptides such as KR12-N1-N4 corresponding to Examples 1-2 and Comparative Examples 1-2 were mixed in phosphate buffer to form a peptide solution (polypeptide concentration of 50 μM) with a concentration of 50 μM (μmol / L), and the solubility of the polypeptides in each peptide solution was detected under the condition of 4 degrees Celsius.

[0071] Results and analysis of experimental example 1

[0072] KR12-M5-M6 of Comparative Examples 1-2 became turbid liquid within 12 hours, showing protein flocculent precipitation, while the remaining polypeptides could still maintain clear protein solution within 7 days. It is shown that KR12-N1-N2 corresponding to Examples 1-4 have good solubility in phosphate buffer, while KR12-N3-N4 of Comparative Examples 1-2 have very poor solubility and are not stable.

[0073] Due to poor solubility, KR12-N3-N4 is difficult to be prepared into a solution type preparation for use, and it is even more difficult to exert the effect of polypeptides in anti-photoaging. Unlike this, the polypeptides of Examples 1-2 of the present application have excellent solubility and better stability.

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

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

[0076] The experimental procedure of experimental example 2 comprised 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, respectively, and each group is configured with a corresponding culture hole on a 6-hole culture plate.

[0078] Step S102, cell plating:

[0079] The cell liquid of the hacat cells (cell density of 2.5 x 105 Each group of culture holes is added with cell solution, 2 mL of cell solution is added to each hole; then, it is placed in a CO2 incubator and cultured at 37°C and 5% CO2 for 20-24 hours.

[0080] Step S103, cell treatment:

[0081] The corresponding culture solution is added to each group of culture holes to obtain a treated culture plate, and the culture solution is shown in Table 1; then, the treated culture plate is placed in a 37°C, 5% CO2 incubator for continuous culture for 48 hours to incubate and culture each group.

[0082] Table 1, culture solution of each group

[0083]

[0084]

[0085] Step S104, supernatant collection:

[0086] After the incubation and culture, 1 mL of cell supernatant of each group of culture holes of the 6-hole culture plate is collected in a 1.5 mL sterile centrifuge tube and stored in an ultra-low temperature freezer at -80°C.

[0087] Step S105, cell counting:

[0088] The 6-hole culture plate after collecting the supernatant is washed twice with 2 mL of PBS, 500 uL of trypsin is added to each hole, and the digestion is performed for 8 minutes (mins), followed by the addition of 1 mL of high-sugar DMEM medium to terminate the digestion; then, 1.5 mL of cell suspension of each group is respectively taken into the corresponding EP tube and centrifuged at 1000 revolutions per minute (rpm) for 10 minutes; then, the supernatant of each group of EP tubes is discarded, 1 mL of PBS is added to each group of EP tubes for resuspension, and centrifuged again at 1000 rpm for 10 minutes; then, 1 mL of PBS is added for resuspension to obtain the cell resuspension of each group, 10 uL of which is taken into a counting plate for counting and recording the cell amount of each group.

[0089] Step S106, AGES content detection:

[0090] The cell supernatant of each group in step S104 is taken as a sample;

[0091] According to the kit (kit is CUSABIO / Huawmei Biology CSB-E09412h-48T human advanced glycation end products (AGEs) enzyme-linked immunoassay kit) instructions, dilute the standard (AGEs pure product in the kit) by gradient, then the standard and sample according to the instructions for ELISA (enzyme-linked immunosorbent assay) operation, finally in the OD450 wavelength measurement 6 hole culture plate in each group corresponding to the culture hole absorbance.

[0092] Step S107, statistics and mapping:

[0093] Make a standard curve, and then get the AGEs content of each group, and then divide the cell amount of each group to get the relative AGEs concentration (Concentrations of AGEs), and finally statistical analysis and column chart.

[0094] Results and analysis of experimental example 2

[0095] The experimental results are shown in Figure 1 , Figure 1 An example of AGEs concentration for the blank control group, the model group, the positive control group, the KR-12 group, the KR-12-N1 group and the KR-12-N2 group.

[0096] In Figure 1 , 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 the results of Figure 1 , the AGEs concentration of the model group is higher than that of 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, however, after kojic acid treatment, the AGEs concentration decreased significantly, indicating that kojic acid has anti-glycation activity.

[0099] Comparing the results of the peptide experimental group and the positive control group, the model group, the KR-12 group, the KR-12-N1 group and the KR-12-N2 group also showed anti-glycation activity; among them, the AGEs concentration of the KR-12-N1 group and the KR-12-N2 group was significantly lower than that of the KR-12 group, indicating that the KR12-N 1 polypeptide and the KR12-N2 polypeptide have good anti-glycation 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 medium. Compared with MEM, the amount of various components is increased, and meanwhile, it 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 medium selected in the above experimental examples contains 4500 mg / L of sugar.

[0102] PBS (English full name: phosphate buffered saline) refers to a phosphate buffered saline solution.

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

[0104] VE refers to Vitamin E (Vitamin E).

[0105] The above specific embodiments have been described in detail, but these do not constitute a limitation on the present application. The protection scope of the present application is not limited to the above-mentioned embodiments, but any equivalent modifications or changes made by those skilled in the art according to the disclosed content of the present application shall 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 set forth 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 of claim 1 in the manufacture of a skin care product, wherein, The skin care product is an external skin preparation.

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

4. A peptide-containing product, characterized in that, The peptide-containing product is a skin care product, which is an external skin preparation, and the peptide-containing product comprises: The polypeptide of claim 1.

5. The peptide-containing product of claim 4, wherein, The concentration of the polypeptide in the peptide-containing product is 1-200 µM.

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