Polypeptide metal chelate and application thereof in field of cosmetics

Abstract

The invention relates to the field of polypeptide compositions, in particular to a polypeptide metal chelate and application thereof in the field of cosmetics, and the polypeptide metal chelate is formed by chelating a polypeptide compound and a metal ion compound. The structure of the polypeptide compound is [gamma-Glu] n-AA, n is greater than or equal to 1, and AA is selected from glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, asparaginic acid, glutamic acid, lysine, arginine or histidine; the polypeptide chelated zinc prepared by the invention has remarkable oil control and anti-inflammatory effects, and can be compounded with other raw materials to form cosmetics with oil control and acne removal effects.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide compositions, and in particular to a polypeptide metal chelate and its application in the field of cosmetics. Background Art

[0002] Metal ions are widely used in the cosmetics field, and there are many types of metal salts in the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)", including zinc ions, magnesium ions, copper ions, iron ions, etc. However, inorganic metal salts usually have a small non-toxic dose, which limits their use. Organic metal ions can effectively increase their non-toxic dose, thereby increasing the scope of use of metal salts, such as zinc hyaluronate, zinc gluconate, amino acid zinc, etc. Peptides are also widely used in the cosmetics field. There are as many as 79 types in the "Catalogue", involving moisturizing, whitening, sun protection, soothing and other functions. However, there are only a handful of cosmetic raw materials that chelate metal ions with peptides, such as blue copper peptide, which is a copper-bonded amino acid (Copper Peptide Complex™) GHK complex, in which GHK is a glycyl-L-histidyl-L-lysine peptide, which has repair, anti-aging, wrinkle removal and hair growth effects. This shows that peptide chelated metal ions have great development and application value as cosmetic functional raw materials.

[0003] Peptide metal ion chelate refers to the carboxyl, phosphate, amide and amino acid on the small molecule polypeptide obtained by protein hydrolysis, and the bioactive substance formed by coordination has the advantages of high bioavailability, high safety and high biological activity. The research on the chelation process of polypeptide and metal elements mainly focuses on the optimization of the chelation conditions of polypeptide and metal ions, in order to obtain a higher metal chelation rate. Factors related to the chelation rate include the degree of protein hydrolysis, chelation temperature, the ratio of polypeptide and metal ion, the pH of the solution, the chelation time, etc. In addition, there are other technical means to improve the chelation efficiency, such as the process of preparing fish skin collagen polypeptide chelated zinc by ultrasonic method in application publication number CN103626867. The inventor first added 0.2-0.4% NaHCO3 solution to the prepared fish skin collagen polypeptide solution, then heated and added zinc salt, kept warm for 10-15min under ultrasonic conditions, and the reaction solution was precipitated by ethanol to obtain a chelate. Patent CN201410078823 discloses a method for preparing whey protein peptides chelated with metal ions, which includes adding metal ions during enzymatic hydrolysis and then obtaining metal chelated peptides through complex separation and purification steps such as ion exchange chromatography, gel filtration chromatography, and RP-HPLC-C18 reverse phase high performance liquid chromatography.

[0004] However, since protein hydrolyzed peptides usually have complex components and contain other impurities in addition to peptides, the metal chelation rate is not high. At the same time, the prepared peptide metal chelates also have the following problems: 1) Low bioavailability: Due to their large molecular weight and complex structure, peptide metal chelates may have low bioavailability. This means that some of the prepared peptide metal chelates may not be adequately absorbed in the body, thus reducing the therapeutic effect.

[0005] 2) Toxicity issues: Some peptide metal chelates may have certain toxicity, which may be related to the toxicity of metal ions or the biological activity of the peptide itself.

[0006] 3) Complex preparation process: The preparation of peptide metal chelates usually requires complex synthesis processes and purification steps, which may lead to high production costs and limit their large-scale application. Summary of the invention

[0007] In order to solve the above problems, the purpose of the present invention is to develop a polypeptide metal chelate with high bioavailability, low toxicity and convenient preparation process, so that it can be suitable for large-scale production and application; further, the polypeptide metal chelate developed and prepared by the present invention has significant oil control and anti-inflammatory effects, and can be compounded with other raw materials to form cosmetics with oil control and acne removal effects.

[0008] In order to achieve the above-mentioned object, the first aspect of the present invention discloses a polypeptide metal chelate, which is formed by chelating a polypeptide compound with a metal ion compound; The structure of the polypeptide compound is: [γ-Glu] n -AA, wherein n≥1, AA is selected from glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine.

[0009] In the polypeptide compound, AA is preferably glutamine.

[0010] The metal ion compound is preferably a zinc ion compound.

[0011] Among them, the toxicity of metal ions in the prepared polypeptide metal chelate is greatly reduced.

[0012] The second aspect of the present invention discloses a method for preparing the above-mentioned polypeptide metal chelate, which specifically comprises the following steps: adding ZnSO4 to the polypeptide solution at a peptide-zinc mass ratio of 2:1, mixing, adjusting the pH to 5.5, reacting in a constant temperature water bath with shaking for 30 min, and then reacting at 80°C for 20 min to obtain polypeptide chelated zinc; The pH is adjusted using 0.1 mol / L HCl or 0.1 mol / L NaOH.

[0013] The third aspect of the present invention discloses the use of the above polypeptide metal chelate in the preparation of cosmetics.

[0014] The cosmetic is used for oil control and / or acne removal of the skin.

[0015] The beneficial effects of the present invention are: The present invention utilizes a polypeptide compound with a relatively clear structure for metal chelation. The prepared polypeptide chelated zinc has a high chelation rate, and subsequent experiments have verified that it has high bioavailability and low toxicity. In addition, the preparation process of the polypeptide chelated zinc is convenient and suitable for large-scale industrial production. At the same time, the prepared peptide chelated zinc has significant oil control and anti-inflammatory effects, and can be compounded with other raw materials to form cosmetics with oil control and acne removal effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effects of zinc sulfate, zinc hyaluronate and polypeptide chelated zinc (WIJ-RM001Y) on cell proliferation in Example 3 are shown.

[0017] Figure 2 The effects of polypeptide chelated zinc and hyaluronate zinc on cell proliferation are shown in Example 3.

[0018] Figure 3 The effect of polypeptide chelated zinc in Example 4 on cellular lipid synthesis is shown.

[0019] Figure 4 The effect of different concentrations of the polypeptide in Example 4 on cellular lipid synthesis is shown.

[0020] Figure 5 The effect of polypeptide chelated zinc in Example 5 on the inflammatory factor NO is shown.

[0021] Figure 6 The effect of polypeptide chelated zinc in Example 5 on the inflammatory factor IL-6 is shown. DETAILED DESCRIPTION

[0022] The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the embodiments. The process parameters not specified in the embodiments of the present application can be carried out according to conventional methods, and the raw materials used can be obtained through commercial channels.

[0023] Example 1 Peptide preparation The present invention uses amino acids as raw materials and synthesizes the polypeptide compound [γ-Glu] by fermentation (the prepared polypeptide compound is consistent with the polypeptide compound obtained in Chinese patent application CN202410645442.5). n -Gln, where n≥1, Gln can be replaced by 19 other amino acids, with a polypeptide compound content of about 30%.

[0024] Preparation of polypeptide chelated zinc According to the peptide-zinc mass ratio of 2:1, ZnSO4 was added to the peptide solution, mixed, and the pH was adjusted to 5.5 with 0.1 mol / L HCl or 0.1 mol / L NaOH. The mixture was shaken in a constant temperature water bath for 30 min, and then reacted at 80°C for 20 min to obtain peptide chelated zinc, referred to as WIJ-RM001Y.

[0025] Example 2 Detection of zinc content in polypeptide chelates The second method inductively coupled plasma optical emission spectrometry (ICP-OES) in the national standard GB5009.268-2016 was used to detect the zinc content in WIJ-RM001Y prepared in Example 1. As can be seen from the table, the zinc content of the chelate WIJ-RM001Y is about 1% by mass.

[0026]

[0027] The calculation formula for converting the content to mg / kg is: instrument reading × constant volume × dilution factor / sampling mass The formula for converting mg / kg to wt.% is: the converted content divided by 10000 is wt.%, i.e. 10000 mg / kg = 1 wt.% Detection of nitrogen and zinc in peptide chelated zinc and calculation of chelation rate First, the total nitrogen content of the peptide chelated zinc solution was detected by Kjeldahl nitrogen analyzer, which was 9.65%, and the zinc content was detected by the second method of inductively coupled plasma emission spectrometry (ICP-OES) in the national standard GB5009.268-2016, which was 10.1 g / kg. Four volumes of ethanol were added to the peptide chelated zinc solution, and after precipitation, the supernatant was taken to measure the total nitrogen content of 0.11% and the zinc content of 0.58 mg / kg. Since only the peptides and zinc involved in chelation will be precipitated by ethanol, it can be calculated that the precipitation rate of the peptide is 94.4%, and the chelation degree of zinc is close to 100%.

[0028] Example 3 Cellular efficacy experiment of peptide chelated zinc After the cryopreserved SZ95 cells were revived, they were cultured with 10% fetal bovine serum and 1×10 5 The cells were cultured in a DMEM high-glucose medium containing 100 U / L penicillin and 100 mg / L streptomycin at 37 °C in a cell culture incubator with 5% carbon dioxide. When the cells adhered to the wall and the confluence exceeded 80%, they were digested and passaged with trypsin containing 0.25% EDTA, and the cells in the logarithmic growth phase were used for experiments.

[0029] Cell proliferation assay with peptide-chelated zinc, zinc sulfate and zinc hyaluronate Dissolve WIJ-RM001Y, zinc hyaluronate, and zinc sulfate in water and dilute to the test concentration with 10% FBS DMEM. Inoculate the cells in a 96-well plate, culture at 37 °C, 5% CO2 for 24 h, remove the supernatant, add the above solutions of 001Y, zinc hyaluronate, zinc sulfate, and the culture medium control group, 200 μL per well. Incubate in a 37 °C, 5% CO2 incubator for 24 h. Aspirate the supernatant, wash once with PBS, add 200 μL of MTT solution with a mass concentration of 0.5 mg / mL to each well and incubate for 3 h. After incubation, discard the supernatant, add 100 μL DMSO, shake and incubate for 15 min, and measure the OD value at 490 nm. Calculate the relative cell viability according to the following formula: Cell viability (%) = (OD value of experimental group / OD value of blank group) × 100% The results are as follows Figure 1-2 As shown in the figure, when the zinc content is the same, the maximum non-toxic dose of zinc sulfate (ZnSO4) is less than that of the peptide chelated zinc (WIJ-RM001Y) and hyaluronate zinc (HA-Zn) developed in this project. Compared with hyaluronate zinc, the non-toxic dose of WIJ-RM001Y is smaller.

[0030] Example 4 Detection of neutral lipids in SZ95 cells by Nile red staining The peptide chelated zinc was diluted with deionized water to obtain dilutions with mass fractions of 1%, 2%, and 5%, respectively. The above dilutions and compositions were diluted 500 times with DMEM culture medium to obtain a stock solution. In the same way, a stock solution of a peptide without chelated metal ions was prepared. Linoleic acid (LA) was dissolved in DMSO and diluted with DMEM culture medium to a 20 μg / mL LA working solution. The 20 μg / mL LA solution was mixed evenly with an equal volume of the above stock solution or culture medium to obtain working solutions of LA mixed with different samples and pure LA, and these working solutions were used for the following experiments.

[0031] SZ95 cells in the logarithmic growth phase were digested and counted, and the cell suspension was adjusted to 1×10 4 / mL, and transfer 100 μL per well to each sample well of a black-edged 96-well plate. The next day, aspirate the culture medium in the well plate and rinse once with PBS. Then add 100 μL of the above working solution as the experimental group, and only add the culture medium as the blank control group. After incubation for 48 h, discard the DMEM complete culture medium, carefully rinse twice with PBS, and add 100 μL of 10 μg / mL Nile Red to each well, and let it stand in the dark at 37 ℃ for 15 min. Take pictures with a fluorescence microscope, and use a multifunctional microplate reader with fluorescence function to detect the fluorescence intensity with an excitation wavelength of 485 nm and an absorption wavelength of 565 nm. There are 6 replicates in each group. The lipid synthesis ratio of SZ95 cells is calculated according to formula (1-1).

[0032] Lipid synthesis rate (%) = (FIT / FIB) × 100% (1-1) Where FIT and FIB are the average fluorescence intensities of the experimental group and the control group, respectively. The results showed that the fluorescence microscopy results ( Figure 3 ) showed that 0.005% peptide-chelated zinc had a significant inhibitory effect on lipid synthesis, while peptides without chelated metal ions had little effect on lipid synthesis ( Figure 4 ).

[0033] Example 5 Anti-inflammatory Experiment of Peptide Chelated Zinc Cells The peptide chelated zinc was diluted with deionized water to obtain peptide chelated zinc dilutions with mass fractions of 1%, 2%, 5% and 10%, respectively. The above dilutions and the combination solution were diluted 500 times with DMEM culture medium to obtain a stock solution. LPS was dissolved with DMEM culture medium and prepared into a 2.0 μg / mL LPS working solution. The 2.0 μg / mL LPS solution was evenly mixed with the above stock solution or culture medium in equal volumes to obtain working solutions of LPS mixed with different samples and pure LPS, respectively.

[0034] After the cryopreserved RAW 264.7 macrophages were revived, they were incubated with 10% fetal bovine serum and 1×10 5 The cells were cultured in a DMEM high-glucose medium containing 100 mg / L penicillin and 100 mg / L streptomycin at 37 °C in a cell culture incubator with 5% carbon dioxide. When the cells adhered to the wall and the confluence exceeded 80%, they were digested and passaged with trypsin containing 0.25% EDTA, and cells in the logarithmic growth phase were used for experiments.

[0035] Anti-inflammatory efficacy detection: Collect cell pellets in the logarithmic growth phase and adjust the cell density to 1×10 5 / mL, add 500 μL of cell suspension to each well of a 24-well cell culture plate. After culturing for 24 hours, add the above working solution as the experimental group, and add only culture medium as the blank control group. Place in an incubator and continue to culture for 24 hours. According to the NO kit and IL-6 kit, repeat 3 wells for each sample, use an enzyme reader to measure the OD value at 540 nm and calculate the NO content. Use an enzyme reader to measure the OD value at 450 nm and 540 nm and calculate the IL-6 content.

[0036] The results showed that peptide chelated zinc had an effect on the production of inflammatory factor NO ( Figure 5 ) and IL-6 ( Figure 6 ) has a significant inhibitory effect, and the inhibitory effect becomes more significant as the concentration increases.

[0037] In summary, this study developed a simple and practical method for peptide chelation of metal ions. Efficacy experiments have shown that peptide chelated zinc has oil control and anti-inflammatory effects. Compared with zinc hyaluronate, it has lower cytotoxicity and can be used in cosmetics such as oil control and acne removal when combined with other functional raw materials.

[0038] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A polypeptide metal chelate, characterized in that: It is formed by the chelation of polypeptide compounds and metal ion compounds; The structure of the polypeptide compound is: [γ-Glu] n -AA, wherein n≥1, AA is selected from glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine.

2. A polypeptide metal chelate according to claim 1, characterized in that: In the polypeptide compound, AA is preferably glutamine.

3. The polypeptide metal chelate according to claim 1, characterized in that: The metal ion compound is preferably a zinc ion compound.

4. The method for preparing the polypeptide metal chelate according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: add ZnSO4 to the polypeptide solution at a peptide-zinc mass ratio of 2:1, mix well, adjust the pH to 5.5, oscillate in a constant temperature water bath for 30 minutes, and then react at 80°C for 20 minutes to obtain polypeptide chelated zinc.

5. The method for preparing a polypeptide metal chelate according to claim 4, characterized in that: The pH is adjusted using 0.1 mol / L HCl or 0.1 mol / L NaOH.

6. Use of the polypeptide compound according to any one of claims 1 to 3 in reducing the toxicity of high-dose metal ions.

7. Use of the polypeptide metal chelate according to any one of claims 1 to 3 in the preparation of cosmetics.

8. The use according to claim 7, characterized in that: The cosmetic is used for oil control and / or acne removal of the skin.

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