Polypeptide metal chelate and use thereof in field of cosmetics

Abstract

The present invention relates to the field of polypeptide compositions, and specifically relates to a polypeptide metal chelate and the use thereof in the field of cosmetics. The polypeptide metal chelate is formed by means of the chelation of a polypeptide compound with a metal ion compound. A structure of the polypeptide compound is: [γ-Glu]n-AA, wherein n≥1, and AA is selected from glycine, alanine, valine, leucine, isoleucine, methionine (Met), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine. The polypeptide zinc chelate prepared by the present invention has significant oil-controlling and anti-inflammatory effects, and can be compounded with other raw materials to prepare cosmetics with oil control and acne removal effects.

Description

Polypeptide metal chelate and its application in the field of cosmetics TECHNICAL FIELD

[0001] The present application relates to the field of polypeptide composition, in particular to a polypeptide metal chelate and its application in the field of cosmetics. BACKGROUND

[0002] Metal ions are widely used in the field of cosmetics, and there are many kinds of metal salts in the "Catalogue of Cosmetic Ingredients (2021 Edition)", including zinc ions, magnesium ions, copper ions, iron ions, etc. However, inorganic metal salts usually have small non-toxic doses, which limits their use, while organic metal ions can effectively increase their non-toxic doses, thereby increasing the use range of metal salts, such as zinc hyaluronate, zinc gluconate, and zinc amino acid. Polypeptides also have a relatively wide application in the field of cosmetics, and there are as many as 79 kinds in the "Catalogue", involving moisturizing, whitening, sunscreen, soothing and other functions. However, there are only a few polypeptide chelated metal ion cosmetic raw materials, such as blue copper peptide, which is a copper peptide complex (Copper Peptide Complex™) GHK compound, where GHK is a glycyl-L-histidyl-L-lysine peptide, which has the functions of repair, anti-aging, wrinkle removal and hair growth. This shows that peptide chelated metal ions as cosmetic functional raw materials have great development and application value.

[0003] Polypeptide metal ion chelate refers to a bioactive substance formed by the coordination of carboxyl, phosphate, amide and amino acid groups on small molecular polypeptides obtained by proteolysis, which has the advantages of high bioavailability, high safety and high biological activity. The research on the chelation process of polypeptides and metal elements mainly focuses on the optimization of the chelation conditions of polypeptides and metal ions in order to obtain a higher metal chelation rate. Factors related to chelation rate include proteolysis degree, chelation temperature, ratio of polypeptide to metal ion, pH of the solution, chelation time, etc. In addition, other technical means can also be used to improve the chelation efficiency, such as the process for preparing fish skin collagen polypeptide chelated zinc by ultrasonic method disclosed in the patent publication CN103626867, in which the inventor first adds 0.2-0.4% NaHCO3 solution to the prepared fish skin collagen polypeptide solution, then adds zinc salt after heating, and keeps the reaction solution under ultrasonic conditions for 10-15 min. The chelate is prepared by precipitating the reaction solution with ethanol. The patent CN201410078823 discloses a method for preparing whey protein peptide chelated metal ions, in which metal ions are added during the enzymatic hydrolysis process, and then a complex separation and purification process such as ion exchange chromatography, gel filtration chromatography and RP-HPLC-C18 reverse phase high performance liquid chromatography separation is used to obtain metal chelated peptides.

[0004] However, due to the complexity of proteolytic peptides, in addition to polypeptides, other impurities are also contained, and the metal chelation rate is not high. At the same time, the prepared polypeptide metal chelate also has the following problems:

[0005] 1) Low bioavailability: The bioavailability of polypeptide metal chelate may be low due to its large molecular weight and complex structure. This means that the prepared part of the polypeptide metal chelate may not be sufficient in terms of absorption in the body, reducing the therapeutic effect.

[0006] 2) Toxicity problem: Some polypeptide metal chelates may have certain toxicity, which may be related to the toxicity of metal ions, or may be related to the biological activity of polypeptides themselves.

[0007] 3) Complex preparation process: The preparation of polypeptide metal chelate usually requires complex synthesis process and purification steps, which may result in high production cost, limiting its large-scale application. SUMMARY

[0008] In order to solve the above problems, the purpose of the present application is to develop a polypeptide metal chelate with high bioavailability, low toxicity and convenient preparation process, so that it can be applied to large-scale production application; further, the polypeptide metal chelate prepared by the present application has significant oil control and anti-inflammatory effects, and can be compounded with other raw materials to form oil control and acne-removing cosmetic.

[0009] In order to achieve the above purpose, the first aspect of the present application discloses a polypeptide metal chelate, which is formed by chelating a polypeptide compound with a metal ion compound;

[0010] The polypeptide compound has the structure of [γ-Glu] n-AA, wherein n≥1, and 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.

[0011] Preferably, AA in the polypeptide compound is glutamine.

[0012] Preferably, the metal ion compound is a zinc ion compound.

[0013] The toxicity of the metal ion in the prepared polypeptide metal chelate is greatly reduced.

[0014] The second aspect of the present application discloses a preparation method of the above-mentioned polypeptide metal chelate, and the specific steps are as follows: according to the mass ratio of peptide to zinc 2:1, ZnSO4 is added to the polypeptide solution, mixed, the pH is adjusted to 5.5, constant temperature water bath oscillation reaction for 30 min, then 80℃ reaction for 20 min, polypeptide chelated zinc is obtained.

[0015] The pH adjustment uses 0.1 mol / L HCl or 0.1 mol / L NaOH.

[0016] The third aspect of the present application discloses the use of the polypeptide metal chelate in the preparation of cosmetics.

[0017] The cosmetics are used for oil control and / or acne removal of the skin.

[0018] The present application has the following advantages:

[0019] The present application uses polypeptide compounds with relatively clear structures for metal chelation, and the prepared polypeptide chelated zinc has high chelation rate, and subsequent experiments have verified that it has high bioavailability and low toxicity; the preparation process of the polypeptide chelated zinc is convenient, and is suitable for large-scale industrial production; at the same time, the prepared polypeptide 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 DRAWINGS

[0020] Figure 1 shows the effect of zinc sulfate, zinc hyaluronate and polypeptide chelated zinc (WIJ-RM001Y) on cell proliferation in Example 3;

[0021] Figure 2 shows the effect of polypeptide chelated zinc and zinc hyaluronate on cell proliferation in Example 3;

[0022] Figure 3 shows the effect of polypeptide chelated zinc on cell lipid synthesis in Example 4;

[0023] Figure 4 shows the effect of polypeptides with different concentrations on cell lipid synthesis in Example 4;

[0024] Figure 5 shows the effect of polypeptide chelated zinc on inflammatory factor NO in Example 5;

[0025] Figure 6 shows the effect of polypeptide chelated zinc on inflammatory factor IL-6 in Example 5. DETAILED DESCRIPTION

[0026] The technical solutions of the present application will be described in detail below in combination with the drawings and examples, but the present application is not limited in the scope of the described examples. The process parameters not mentioned in the examples of the present application can be carried out according to conventional methods, and the raw materials used can be obtained through commercial channels.

[0027] Example 1

[0028] Preparation of polypeptides

[0029] The present application synthesizes a polypeptide compound [γ-Glu] n -Gln, wherein n≥1, Gln can be replaced by other 19 amino acids, and the content of the polypeptide compound is about 30%.

[0030] Preparation of polypeptide chelated zinc

[0031] According to the mass ratio of peptide zinc 2:1, ZnSO4 is added to the polypeptide solution, mixed, the pH is adjusted to 5.5 by 0.1 mol / L HCl or 0.1 mol / L NaOH, and then oscillated in a constant temperature water bath for 30 min, and then reacted at 80℃ for 20 min, to obtain polypeptide chelated zinc, which is abbreviated as WIJ-RM001Y.

[0032] Example 2: Detection of zinc content in polypeptide chelate

[0033] The second method of national standard GB5009.268-2016, inductively coupled plasma optical emission spectrometry (ICP-OES) is used to detect the zinc content in WIJ-RM001Y prepared in Example 1. From the table, the zinc content of the chelate WIJ-RM001Y is about 1%.

[0034]

[0035] The conversion content mg / kg calculation formula: instrument reading × constant volume × dilution factor / sample mass

[0036] mg / kg unit conversion wt.% formula: conversion content divided by 10000 is wt.%, that is, 10000 mg / kg = 1 wt.%

[0037] Detection of nitrogen and zinc in polypeptide chelated zinc and calculation of chelation rate

[0038] First, the total nitrogen content in the polypeptide chelated zinc solution is detected by Kjeldahl determination instrument, which is 9.65%, and the zinc content is detected by the second method of national standard GB5009.268-2016, inductively coupled plasma optical emission spectrometry (ICP-OES), which is 10.1 g / kg. After adding 4 times volume of ethanol to the polypeptide chelated zinc solution, precipitating, and taking the supernatant to detect the total nitrogen content 0.11% and the zinc content 0.58 mg / kg. Because only the peptide and zinc involved in chelation are 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%.

[0039] Example 3: Cell efficacy experiment of polypeptide chelated zinc

[0040] After recovery of the frozen SZ95 cells, they were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 1 x 10 5 U / L penicillin, 100 mg / L streptomycin at 37 °C in a 5% carbon dioxide cell incubator. When the cells adhered and grew, the confluence was more than 80%, and the cells were passaged by trypsin containing 0.25% EDTA. The logarithmic growth phase cells were used for experiments.

[0041] Cell proliferation experiments of peptide chelated zinc and zinc sulfate and zinc hyaluronate

[0042] WIJ-RM001Y, zinc hyaluronate, and zinc sulfate were dissolved in water and diluted with 10% FBS DMEM to the test concentration. The cells were inoculated in a 96-well plate, and after 24 h of culture at 37 °C, 5% CO2, the supernatant was removed, and 200 μL of the aforementioned solution of 001Y, zinc hyaluronate, zinc sulfate, and the medium control group was added to each well. Incubate in a 37 °C, 5% CO2 incubator for 24 h. Discard the supernatant, wash once with PBS, and add 200 μL of 0.5 mg / mL MTT solution to each well and incubate for 3 h. After incubation, discard the supernatant, add 100 μL of DMSO, and shake for 15 min. Measure the OD value at 490 nm. Calculate the relative viability of the cells according to the following formula: Cell viability (%) = (experimental group OD value / blank group OD value) x 100%

[0043] The results are shown in Figures 1-2. When the zinc content is used as the standard, the maximum non-toxic dose of zinc sulfate (ZnSO4) is less than the peptide chelated zinc (WIJ-RM001Y) and zinc hyaluronate (HA-Zn) developed in this project when the zinc content is the same. Compared with zinc hyaluronate, the non-toxic dose of WIJ-RM001Y is smaller.

[0044] Example 4 Nile red staining method for detecting neutral lipids in SZ95 cells

[0045] The peptide chelated zinc was diluted with deionized water to obtain dilutions with mass fractions of 1%, 2%, and 5%. The above dilutions and the composition were diluted 500 times with DMEM medium to obtain a stock solution. In the same way, a polypeptide stock solution without chelated metal ions was prepared. Linoleic acid (LA) was dissolved in DMSO and diluted with DMEM medium to obtain a 20 μg / mL LA working solution. The 20 μg / mL LA solution was mixed with the above stock solution or medium in equal volumes to obtain a working solution of LA mixed with different samples and pure LA, respectively, which was used for the following experiments.

[0046] The logarithmic growth phase SZ95 cells were digested and counted, and the cell suspension was adjusted to 1 x 10 4The samples were transferred into each sample well of a black edge 96-well plate in an amount of 100 μL per well. The next day, the medium in the well plate was aspirated, and washed once with PBS. Then, 100 μL of the working solution described above was added as the experimental group, and the medium alone was added as the blank control group. After incubation for 48 h, the DMEM complete medium was discarded, and the cells were washed twice with PBS. Then, 100 μL of 10 μg / mL Nile red was added to each well, and the cells were incubated in a dark environment at 37 °C for 15 min. The fluorescence intensity was detected by taking a photograph under a fluorescence microscope, and by using a multifunctional enzyme label meter with a fluorescence function, with an excitation wavelength of 485 nm and an absorption wavelength of 565 nm. There were 6 replicate wells in each group. The proportion of lipid synthesis of the SZ95 cells was calculated according to formula (1-1).

[0047] Lipid synthesis rate (%) = (FIT / FIB) x 100% (1-1)

[0048] In the formula, FIT and FIB are the average fluorescence intensities of the experimental group and the control group, respectively

[0049] The results show that the photograph under the fluorescence microscope (Figure 3) shows that 0.005% peptide-chelated zinc has a significant inhibitory effect on lipid synthesis, while the polypeptide without chelated metal ions has a smaller effect on lipid synthesis (Figure 4).

[0050] Example 5: Cell anti-inflammatory experiment of peptide-chelated zinc

[0051] 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 composition solutions were diluted 500 times with DMEM medium to obtain stock solutions. LPS was dissolved in DMEM medium and configured into an LPS working solution with a concentration of 2.0 μg / mL. The LPS solution with a concentration of 2.0 μg / mL was mixed with the above stock solutions or medium in equal volumes to obtain working solutions of LPS mixed with different samples and pure LPS, respectively.

[0052] After the cryopreserved RAW 264.7 macrophages were recovered, they were cultured in DMEM high-sugar medium containing 10% fetal bovine serum, 1 x 10 5 U / L penicillin, 100 mg / L streptomycin at 37 °C in a cell incubator with 5% carbon dioxide. When the cells were adherently growing and the confluence was more than 80%, they were passaged by trypsinization with 0.25% EDTA, and the logarithmically growing cells were used for experiments.

[0053] Anti-inflammatory efficacy detection The logarithmically growing cells were collected and the density of the cells was adjusted to 1 x 10 5The cell suspension was added to 500 μL per well in a 24-well cell culture plate, and after 24 hours of culture, the above working solution was added as the experimental group, and the medium alone was added as the blank control group. Put into the incubator and continue to culture for 24 h. According to the operation of the NO kit and the IL-6 kit, 3 wells were repeated for each sample, and the OD value was measured at 540 nm using a microplate reader and the NO content was calculated. The OD value was measured at 450 nm and 540 nm using a microplate reader and the IL-6 content was calculated.

[0054] The results show that the peptide chelated zinc has a significant inhibitory effect on the inflammatory factors NO (Figure 5) and IL-6 (Figure 6) produced by LPS-induced RAW 264.7, and the inhibitory effect is more significant as the concentration increases.

[0055] In summary, the present study developed a simple and practical method for polypeptide chelated metal ions, and the efficacy experiment proved that the peptide chelated zinc has oil control and anti-inflammatory efficacy, and compared with zinc hyaluronate, it has less cytotoxicity, and can be used for oil control, acne removal and other types of cosmetics by combining with other efficacy raw materials.

[0056] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A polypeptide metal chelate characterized in that, formed by chelation of a polypeptide compound with a metal ion compound; the polypeptide compound has a structure of [γ-Glu] n-AA, wherein n≥1, and AA is selected from glycine, alanine, valine, leucine, isoleucine, 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, wherein, Preferably, AA in the polypeptide compound is glutamine.

3. A polypeptide metal chelate according to claim 1, wherein, Preferably, the metal ion compound is a zinc ion compound.

4. The method for preparing the polypeptide metal chelate according to any one of claims 1-3, characterized in that, The specific steps are as follows: according to the mass ratio of peptide to zinc of 2:1, ZnSO4 is added into the polypeptide solution, mixed, the pH is adjusted to 5.5, constant temperature water bath oscillation reaction is carried out for 30 min, and then 80℃ reaction is carried out for 20 min, to obtain the polypeptide chelated zinc.

5. The method for preparing a polypeptide metal chelate as described in claim 4, characterized in that, 0.1 mol / L HCl or 0.1 mol / L NaOH is used for pH adjustment.

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

7. The polypeptide metal chelate according to any one of claims 1-3 for use in the preparation of a cosmetic product.

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

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