Molecular chaperone synergistic blue copper peptide and application thereof

Abstract

The invention discloses a molecular chaperone synergistic blue-copper peptide and application thereof, belongs to the technical field of skin care, and particularly relates to a blue-copper peptide composition formed by compounding blue-copper peptide and N-acetylneuraminic acid, and the structure of the blue-copper peptide is that copper is connected to glycyl-L-histidyl-L-lysine; the blue copper peptide and the N-acetylneuraminic acid are mixed according to the mass ratio of 1: (0.1-1) for use, and further, phenylalanine derivatives can be added into the mixture for compounding. When the blue copper peptide composition obtained by the invention is applied to cells, the cell survival rate is high, the non-enzymatic glycosylation inhibition rate is good, and the expression of Elastin, Col-I and Col-IV mRNA in the cells is high. Therefore, the blue copper peptide composition disclosed by the invention is high in cell survival rate, good in non-enzymatic glycosylation inhibition rate and capable of improving the mRNA expression of Elastin, Col-I and Col-IV in cells, and the application of the blue copper peptide composition.

Description

Technical Field

[0001] The invention belongs to the technical field of skin care, and particularly relates to a molecular chaperone synergistic blue copper peptide and a use thereof. Background Art

[0002] The primary components of the dermal extracellular matrix (ECM) are the collagen fiber network, the elastic fiber network, and proteoglycans. Collagen and elastin are the most important extracellular components, primarily including type I collagen and type III collagen. Newly synthesized type I procollagen is secreted into the extracellular space of the dermis. Metabolized by relevant enzymes, it forms a triple-helical complex, which then combines with other extracellular matrix proteins (such as small proteoglycans) to form regularly structured collagen fiber bundles, providing the skin with toughness and tensile strength. Skin aging caused by environmental stimuli is primarily manifested by a decrease in type I collagen secretion by dermal cells, a lack of collagen support, and the inward inward movement of the stratum corneum and epidermis, resulting in wrinkles. There are two main causes of ECM deterioration. First, human skin fibroblasts first synthesize and secrete procollagen, which then cleaves some of its peptide chains to form mature collagen. External factors can reduce skin collagen content by promoting collagen degradation or inhibiting procollagen synthesis. External factors, including light, reduce collagen synthesis in the skin, leading to degeneration and disintegration of the collagen fiber network. Studies have also found that fibroblasts exposed to large amounts of degenerated collagen have reduced proliferation and collagen synthesis. Elastin is the primary component of elastic fibers, which are primarily found in ligaments and blood vessel walls. Elastic fibers coexist with collagen fibers, imparting elasticity and tensile strength to tissues. Although elastin only accounts for 2% of the total protein content in the dermis, it plays a crucial role in skin elasticity.

[0003] When it comes to anti-inflammatory measures, a growing number of Chinese consumers suffer from sensitive skin. Facially, these symptoms primarily manifest as redness, burning, stinging, itching, and peeling. These sensitive symptoms are often linked to environmental pollution, extreme climate change, and improper personal care practices. These irritants trigger the skin's immune system, releasing various inflammatory factors and histamine, causing facial discomfort. Consumers are increasingly seeking skincare solutions for these skin issues. Summary of the Invention

[0004] The purpose of the present invention is to provide a blue copper peptide composition with high cell survival rate, good non-enzymatic glycosylation inhibition rate and improved Elastin, Col-Ⅰ and Col-Ⅳ mRNA expression in cells and its application.

[0005] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are: A copper peptide and N-acetylneuraminic acid are used in the preparation of anti-aging and / or anti-inflammatory drugs or cosmetics. The copper peptide consists of copper linked to glycyl-L-histidyl-L-lysine. The copper peptide and N-acetylneuraminic acid are mixed in a mass ratio of 1:0.1-1. The copper peptide (Cu-GHK) functions within the extracellular matrix and is released in response to wounds or inflammation to support healing. It acts as a signaling and carrier peptide, promoting the synthesis of collagen, elastin, proteoglycans, and glycosaminoglycans, while also providing anti-inflammatory and antioxidant benefits. In cosmetic applications, Cu-GHK is used in anti-aging, anti-wrinkle, after-sun, skin renewal, moisturizing, and hair growth stimulation products. N-acetylneuraminic acid is a key component of biological membranes, primarily binding to membrane proteins and distributed on the plasma membrane surface to form a polysaccharide-protein complex. N-acetylneuraminic acid has a negative charge on its surface and is an important carrier in the information transmission process. Therefore, negatively charged N-acetylneuraminic acid is called an antenna on the cell surface, which helps to improve cell activity. The lack of N-acetylneuraminic acid can lead to a reduction in blood cell lifespan and enzyme proteins in metabolism, thereby leading to cell aging. N-acetylneuraminic acid can also scavenge reactive oxygen free radicals produced in the human body, thereby playing an antioxidant and protective role. The present invention has higher efficacy by compounding blue copper peptide with N-acetylneuraminic acid, and is superior to the use of blue copper peptide or N-acetylneuraminic acid in some performance aspects.

[0006] A blue copper peptide, N-acetylneuraminic acid, and a phenylalanine derivative are used in the preparation of anti-aging and / or anti-inflammatory drugs or anti-aging and / or anti-inflammatory cosmetics. The blue copper peptide has a structure in which copper is linked to glycyl-L-histidyl-L-lysine. The blue copper peptide, N-acetylneuraminic acid, and phenylalanine derivative are mixed in a mass ratio of 1:0.1 to 1:0.1 to 0.3. The phenylalanine derivative is prepared from phenylalanine and monoethyl oxalyl chloride or malonyl chloride. The present invention prepares a phenylalanine derivative, which is then compounded with the blue copper peptide and N-acetylneuraminic acid. Through non-bonding interactions within their molecules, the compounds combine to enhance the efficacy of the compounding, improve cell survival, increase inhibition of non-enzymatic glycosylation, and increase the expression of Elastin, Col-I, and Col-IV mRNA in cells.

[0007] A blue copper peptide composition comprises: a composite of at least blue copper peptide and N-acetylneuraminic acid, wherein the structure of the blue copper peptide is copper connected to glycyl-L-histidyl-L-lysine; the blue copper peptide and N-acetylneuraminic acid are mixed in a mass ratio of 1:0.1-1.

[0008] Preferably, the composition contains a phenylalanine derivative prepared from phenylalanine and ethyl oxalyl chloride or malonyl chloride.

[0009] Preferably, the blue copper peptide and N-acetylneuraminic acid and phenylalanine derivative are mixed in a mass ratio of 1:0.1-1:0.1-0.3.

[0010] Preferably, in the preparation of phenylalanine derivatives, the amount of ethyl oxalyl chloride used is 20-60 wt % of phenylalanine.

[0011] Preferably, in the preparation of phenylalanine derivatives, the amount of malonyl chloride used is 20-60 wt % of phenylalanine.

[0012] Preferably, ethyl oxalyl chloride is dissolved in anhydrous tetrahydrofuran to prepare ethyl oxalyl chloride solution.

[0013] Preferably, malonyl chloride is dissolved in anhydrous tetrahydrofuran to prepare a malonyl chloride solution.

[0014] Preferably, in the preparation of phenylalanine derivative 1, an alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, and then a solution of monoethyl oxalyl chloride is added, and the reaction is stirred in an ice bath for 6-18 hours. After the reaction is completed, the pH is adjusted to 1-2, filtered, washed with distilled water until neutral, dried, and then washed with petroleum ether and dried to obtain phenylalanine derivative 1.

[0015] More preferably, in the preparation of phenylalanine derivative 1, the alkaline agent is sodium hydroxide, and the amount of the alkaline agent used is 70-90 wt % of phenylalanine.

[0016] More preferably, in the preparation of phenylalanine derivative 1, the amount of phenylalanine used is 5-25 wt % of distilled water.

[0017] More preferably, in the preparation of phenylalanine derivative 1, the ethyl oxalyl chloride solution is prepared by mixing ethyl oxalyl chloride with anhydrous tetrahydrofuran, and the ethyl oxalyl chloride solution contains 10-20 wt % of ethyl oxalyl chloride.

[0018] More preferably, in the preparation of phenylalanine derivative 1, the amount of ethyl oxalyl chloride solution used is based on the amount of ethyl oxalyl chloride therein, and the amount of ethyl oxalyl chloride used is 20-60 wt % of phenylalanine.

[0019] Preferably, in the preparation of phenylalanine derivative 2, an alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, and then a malonyl chloride solution is added. The reaction is stirred in an ice bath for 6-18 hours. After the reaction is completed, the pH is adjusted to 1-2, filtered, washed with distilled water until neutral, dried, and then washed with petroleum ether and dried to obtain phenylalanine derivative 2.

[0020] More preferably, in the preparation of phenylalanine derivative 2, the alkaline agent is sodium hydroxide, and the amount of the alkaline agent used is 70-90 wt % of phenylalanine.

[0021] More preferably, in the preparation of phenylalanine derivative 2, the amount of phenylalanine used is 5-25 wt % of distilled water.

[0022] More preferably, in the preparation of phenylalanine derivative 2, the malonyl chloride solution is prepared by mixing malonyl chloride with anhydrous tetrahydrofuran, and the malonyl chloride solution contains 10-20 wt % of malonyl chloride.

[0023] More preferably, in the preparation of phenylalanine derivative 2, the amount of malonyl chloride solution used is based on the amount of malonyl chloride therein, and the amount of malonyl chloride used is 20-60 wt % of phenylalanine.

[0024] The invention discloses a blue copper peptide composition, which is prepared by compounding a blue copper peptide and N-acetylneuraminic acid, wherein the amount of N-acetylneuraminic acid is 10-100 wt % of the blue copper peptide.

[0025] Preferably, the blue copper peptide composition is prepared by compounding blue copper peptide, N-acetylneuraminic acid and phenylalanine derivative 1, wherein the amount of N-acetylneuraminic acid is 10-100wt% of the blue copper peptide, and the amount of phenylalanine derivative 1 is 5-20wt% of the blue copper peptide.

[0026] Preferably, the blue copper peptide composition is prepared by compounding blue copper peptide, N-acetylneuraminic acid and phenylalanine derivative 2, wherein the amount of N-acetylneuraminic acid is 10-100wt% of the blue copper peptide, and the amount of phenylalanine derivative 2 is 5-20wt% of the blue copper peptide.

[0027] Preferably, the blue copper peptide composition is compounded by blue copper peptide and N-acetylneuraminic acid, phenylalanine derivative 1, and phenylalanine derivative 2, the amount of N-acetylneuraminic acid is 20wt% of the blue copper peptide, the amount of phenylalanine derivative 1 is 5wt% of the blue copper peptide, and the amount of phenylalanine derivative 2 is 5wt% of the blue copper peptide.

[0028] Preferably, thiamine hydrochloride can be added to the copper peptide composition, with the amount of thiamine hydrochloride being 2-6wt% of the copper peptide. When thiamine hydrochloride is further added to the copper peptide and N-acetylneuraminic acid, and after using phenylalanine derivative 1 and phenylalanine derivative 2, the composition exhibits even better efficacy, improving cell survival rate, inhibition of non-enzymatic glycosylation, and expression of Elastin, Col-I, and Col-IV mRNA in cells.

[0029] The present invention utilizes a copper peptide and N-acetylneuraminic acid as a composite. The copper peptide structure consists of copper linked to glycyl-L-histidyl-L-lysine. The copper peptide and N-acetylneuraminic acid are mixed in a mass ratio of 1:0.1-1. Furthermore, phenylalanine derivatives can be added to the mixture for compounding. This composition exhibits the following beneficial effects: high cell survival rate, good non-enzymatic glycosylation inhibition rate, and high expression of Elastin, Col-I, and Col-IV mRNA in cells. Therefore, the present invention provides a copper peptide composition and its application that exhibits high cell survival rate, good non-enzymatic glycosylation inhibition rate, and increased expression of Elastin, Col-I, and Col-IV mRNA in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the infrared image of phenylalanine derivatives; Figure 2 is the cell survival rate graph; Figure 3 is the non-enzymatic glycosylation inhibition rate diagram; Figure 4 is the expression diagram of Elastin mRNA; Figure 5 is the expression diagram of Col-Ⅰ mRNA; Figure 6 This is the expression diagram of Col-Ⅳ mRNA. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below with reference to the specific embodiments and the accompanying drawings: The cells and peptides used in the present invention were derived from the following sources: HFF-1 human skin fibroblasts were obtained from Zhejiang Meisen Cell Technology Co., Ltd.; blue copper peptide was obtained from Zhejiang Paipeptide Biological Co., Ltd.

[0032] The phenylalanine used in the present invention is .

[0033] Example 1: A blue copper peptide composition comprises blue copper peptide and N-acetylneuraminic acid.

[0034] The blue copper peptide composition is prepared by compounding blue copper peptide and N-acetylneuraminic acid, wherein the amount of N-acetylneuraminic acid is 20 wt % of the blue copper peptide.

[0035] Example 2: A blue copper peptide composition comprises blue copper peptide and N-acetylneuraminic acid and phenylalanine derivative 1.

[0036] Preparation of phenylalanine derivative 1: An alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, followed by the addition of ethyl oxalyl chloride solution. The mixture is stirred in an ice bath for 12 hours. After completion of the reaction, the pH is adjusted to 1, the mixture is filtered, washed with distilled water until neutral, dried, and then washed with petroleum ether and dried to obtain phenylalanine derivative 1. The alkaline reagent is sodium hydroxide, and the amount of the alkaline reagent used is 80% by weight of the phenylalanine. The amount of phenylalanine used is 10% by weight of distilled water. The ethyl oxalyl chloride solution is prepared by mixing ethyl oxalyl chloride with anhydrous tetrahydrofuran, containing 15% by weight of ethyl oxalyl chloride. The amount of ethyl oxalyl chloride solution used is based on the amount of ethyl oxalyl chloride therein, and the amount of ethyl oxalyl chloride used is 40% by weight of the phenylalanine.

[0037] The blue copper peptide composition is prepared by compounding blue copper peptide, N-acetylneuraminic acid and phenylalanine derivative 1, wherein the amount of N-acetylneuraminic acid is 20wt% of the blue copper peptide, and the amount of phenylalanine derivative 1 is 10wt% of the blue copper peptide.

[0038] Example 3: A blue copper peptide composition comprises blue copper peptide and N-acetylneuraminic acid and phenylalanine derivative 2.

[0039] Preparation of phenylalanine derivative 2: An alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, followed by the addition of a malonyl chloride solution. The mixture is stirred in an ice bath for 10 hours. After completion of the reaction, the pH is adjusted to 1, the mixture is filtered, washed with distilled water until neutral, dried, and then washed with petroleum ether and dried to obtain phenylalanine derivative 2. The alkaline reagent is sodium hydroxide, and the amount of the alkaline reagent used is 80% by weight of the phenylalanine. The amount of phenylalanine used is 20% by weight of distilled water. The malonyl chloride solution is prepared by mixing malonyl chloride with anhydrous tetrahydrofuran, containing 10% by weight of malonyl chloride. The amount of malonyl chloride used is based on the amount of malonyl chloride therein, and the amount of malonyl chloride used is 40% by weight of the phenylalanine.

[0040] The blue copper peptide composition is prepared by compounding blue copper peptide, N-acetylneuraminic acid and phenylalanine derivative 2, wherein the amount of N-acetylneuraminic acid is 20wt% of the blue copper peptide, and the amount of phenylalanine derivative 2 is 10wt% of the blue copper peptide.

[0041] Example 4: A blue copper peptide composition comprises blue copper peptide, N-acetylneuraminic acid, a phenylalanine derivative 1, and a phenylalanine derivative 2.

[0042] Preparation of phenylalanine derivative 1: An alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, followed by the addition of ethyl oxalyl chloride solution. The mixture is stirred in an ice bath for 12 hours. After completion of the reaction, the pH is adjusted to 1, the mixture is filtered, washed with distilled water until neutral, dried, and then washed with petroleum ether and dried to obtain phenylalanine derivative 1. The alkaline reagent is sodium hydroxide, and the amount of the alkaline reagent used is 80% by weight of the phenylalanine. The amount of phenylalanine used is 10% by weight of distilled water. The ethyl oxalyl chloride solution is prepared by mixing ethyl oxalyl chloride with anhydrous tetrahydrofuran, containing 15% by weight of ethyl oxalyl chloride. The amount of ethyl oxalyl chloride solution used is based on the amount of ethyl oxalyl chloride therein, and the amount of ethyl oxalyl chloride used is 40% by weight of the phenylalanine.

[0043] Preparation of phenylalanine derivative 2: An alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, followed by the addition of a malonyl chloride solution. The mixture is stirred in an ice bath for 10 hours. After completion of the reaction, the pH is adjusted to 1, the mixture is filtered, washed with distilled water until neutral, dried, and then washed with petroleum ether and dried to obtain phenylalanine derivative 2. The alkaline reagent is sodium hydroxide, and the amount of the alkaline reagent used is 80% by weight of the phenylalanine. The amount of phenylalanine used is 20% by weight of distilled water. The malonyl chloride solution is prepared by mixing malonyl chloride with anhydrous tetrahydrofuran, containing 10% by weight of malonyl chloride. The amount of malonyl chloride used is based on the amount of malonyl chloride therein, and the amount of malonyl chloride used is 40% by weight of the phenylalanine.

[0044] The blue copper peptide composition is prepared by compounding blue copper peptide with N-acetylneuraminic acid, phenylalanine derivative 1, and phenylalanine derivative 2. The amount of N-acetylneuraminic acid is 20wt% of the blue copper peptide, the amount of phenylalanine derivative 1 is 5wt% of the blue copper peptide, and the amount of phenylalanine derivative 2 is 5wt% of the blue copper peptide.

[0045] Example 5: A blue copper peptide composition comprises blue copper peptide, N-acetylneuraminic acid and thiamine hydrochloride.

[0046] The blue copper peptide composition is prepared by compounding blue copper peptide, N-acetylneuraminic acid and thiamine hydrochloride. The amount of N-acetylneuraminic acid is 20wt% of the blue copper peptide, and the amount of thiamine hydrochloride is 4wt% of the blue copper peptide.

[0047] Example 6: A blue copper peptide composition comprises blue copper peptide, N-acetylneuraminic acid, thiamine hydrochloride, and phenylalanine derivative 1. Phenylalanine derivative 1 is derived from Example 2.

[0048] The blue copper peptide composition is prepared by compounding blue copper peptide with N-acetylneuraminic acid, thiamine hydrochloride, and phenylalanine derivative 1. The amount of N-acetylneuraminic acid is 20wt% of the blue copper peptide, the amount of thiamine hydrochloride is 4wt% of the blue copper peptide, and the amount of phenylalanine derivative 1 is 10wt% of the blue copper peptide.

[0049] Example 7: A blue copper peptide composition comprises blue copper peptide, N-acetylneuraminic acid, thiamine hydrochloride and phenylalanine derivative 2. Phenylalanine derivative 2 is derived from Example 3.

[0050] The blue copper peptide composition is prepared by compounding blue copper peptide with N-acetylneuraminic acid, thiamine hydrochloride, and phenylalanine derivative 2. The amount of N-acetylneuraminic acid is 20wt% of the blue copper peptide, the amount of thiamine hydrochloride is 4wt% of the blue copper peptide, and the amount of phenylalanine derivative 2 is 10wt% of the blue copper peptide.

[0051] Example 8: A blue copper peptide composition comprises blue copper peptide, N-acetylneuraminic acid, thiamine hydrochloride, phenylalanine derivative 1, and phenylalanine derivative 2. Phenylalanine derivative 1 is derived from Example 2, and phenylalanine derivative 2 is derived from Example 3.

[0052] The blue copper peptide composition is prepared by compounding blue copper peptide with N-acetylneuraminic acid, thiamine hydrochloride, and phenylalanine derivative 2. The amount of N-acetylneuraminic acid is 20wt% of the blue copper peptide, the amount of thiamine hydrochloride is 4wt% of the blue copper peptide, the amount of phenylalanine derivative 1 is 5wt% of the blue copper peptide, and the amount of phenylalanine derivative 2 is 5wt% of the blue copper peptide.

[0053] Example 9: A method for constructing a cell model, HFF-1 cells were plated and cultured for 24 hours. After the cells adhered, the test substance (diluted to the dosing concentration in serum-free medium) was added for 24 hours, and changes in relevant indicators were monitored. Culture conditions: 5% CO2, 37°C constant temperature incubator. Culture medium: 89% DMEM + 10% FBS + 1% double-antibody (HFF-1).

[0054] Test example: 1. Infrared analysis Test sample: phenylalanine derivative prepared in Example 2.

[0055] The infrared spectrum of the phenylalanine derivative 1 prepared by the present invention is as follows: Figure 1 As shown, among them, 3427cm -1 The infrared spectrum of the hydroxyl group in the carboxyl group is 3236 cm -1The infrared absorption peak of nitrogen and hydrogen is at 2800-3000cm -1 The infrared absorption peaks of methyl and methylene are between 1671cm -1 The infrared absorption peak of carbon-oxygen double bond is 1609cm -1 、1507cm -1 The infrared absorption peak of the benzene ring is 1280 cm -1 The infrared absorption peak is the carbon-nitrogen bond on the amide.

[0056] This study evaluated the efficacy of the combination of peptide cosmetic raw materials by investigating the effects of co-administration of peptide cosmetic raw materials with other raw materials on the genes of collagen, elastin, anti-inflammatory and repair in skin fibroblasts (HFF-1).

[0057] 2. Detect cell viability using MTT assay Test sample: the blue copper peptide composition of each example.

[0058] HFF-1 cells in the exponential growth phase were prepared into single cell suspensions. HFF-1 cells were cultured at a density of 5×10 3 Cells were cultured at 100 µL / well in culture medium supplemented with 10% fetal bovine serum at 37°C for 24 hours to allow attachment. The final culture volume was 100 µL. The supernatant was then aspirated and freshly prepared test sample (serum-free) was added. After 24 hours of incubation, 10 µL of 5 mg / mL MTT was added to each well. After gentle shaking, the cells were mixed and incubated at 37°C. After 4 hours, the 96-well plate was removed, the supernatant carefully aspirated, and 100 µL of DMSO was added to each well. The cells were shaken on a shaker for 10 minutes to fully dissolve the blue-purple formazan precipitate. The absorbance of each well was measured at 490 nm to assess the effect of the test substance on cell growth. The test sample dosage was 0.1 µg / mL. A blank control group was set up, and no test sample was added to the blank control group. A blue copper peptide single-component control group, an N-acetylneuraminic acid single-component control group, a phenylalanine derivative 1 single-component control group, a phenylalanine derivative 2 single-component control group, and a thiamine hydrochloride single-component control group were set up. The dosage of each of the above single-component control groups was consistent with the dosage of the composition, and the concentration was 0.1 μg / mL.

[0059] The present invention tested different dosages of a single component of the blue copper peptide and found that the activity of HFF-1 cells increased when the dosage of the blue copper peptide was within the range of 0.01-1 μg / mL, with the highest activity at 1 μg / mL. The blue copper peptide within the above concentration range significantly increased the activity of HFF-1 cells ( p <0.05), and then with the increase of the concentration of blue copper peptide, such as at 1000 μg / m, the survival rate of HFF-1 cells was significantly reduced ( p <0.01).

[0060] The present invention uses a composite of blue copper peptide and other components to explore the effect of the composite on cell activity. The results are as follows Figure 2 As shown, wherein S1 is the composition of Example 1, S2 is the composition of Example 2, S3 is the composition of Example 3, S4 is the composition of Example 4, S5 is the composition of Example 5, S6 is the composition of Example 6, S7 is the composition of Example 7, S8 is the composition of Example 8, C is a blank control group, D1 is a blue copper peptide single-component control group, D2 is an N-acetylneuraminic acid single-component control group, D3 is a phenylalanine derivative 1 single-component control group, D4 is a phenylalanine derivative 2 single-component control group, and D5 is a thiamine hydrochloride single-component control group. In the present invention, the cell survival rate of the blank control group is set to 100%. The use of blue copper peptide can significantly improve cell activity, while the effect of N-acetylneuraminic acid single component on the cell activity of HFF-1 cells is not obvious. Similarly, the effects of phenylalanine derivative 1 single component, phenylalanine derivative 2 and thiamine hydrochloride single component on the cell activity of HFF-1 cells are not obvious. This at least shows that N-acetylneuraminic acid single component, phenylalanine derivative 1 single component, phenylalanine derivative 2 and thiamine hydrochloride single component will not reduce the cell activity of HFF-1 cells. After one or more of the above components are compounded with blue copper peptide into a composition, the cell survival rate is significantly improved, and it has an excellent positive effect on cells.

[0061] 3. Use a microplate reader to detect the drug non-enzymatic glycosylation inhibition rate Test sample: the blue copper peptide composition of each example.

[0062] The test sample was diluted to an appropriate concentration, and a BSA-fructose reaction solution was prepared according to the standard operating procedure for the non-enzymatic glycosylation inhibition test. The test sample and the reaction solution were mixed and incubated in a 37°C constant temperature incubator in the dark. The fluorescence intensity of each group of samples was detected after 5 days. After the test sample was mixed with the reaction solution, the concentration of the test sample was 100 ng / mL. A blank control group was set up. No test sample was added to the blank control group. A blue copper peptide single-component control group, an N-acetylneuraminic acid single-component control group, a phenylalanine derivative 1 single-component control group, a phenylalanine derivative 2 single-component control group, and a thiamine hydrochloride single-component control group were set up. The amount of each of the above single-component control groups was consistent with the amount of the composition, and the concentration was 100 ng / mL.

[0063] The fluorescence intensity of the samples was detected by a microplate reader at an excitation wavelength of 370 nm and an emission wavelength of 440 nm.

[0064] Compared with the control group or the group treated with N-acetylneuraminic acid or blue copper peptide alone, the expression of Elastin mRNA in HFF-1 cells was significantly increased after treatment with 10%a+50%c ( p <0.05).

[0065] The present invention uses a composite of blue copper peptide and other components to explore the effect of the composite on non-enzymatic glycosylation. The results are as follows Figure 3 As shown, wherein S1 is the composition of Example 1, S2 is the composition of Example 2, S3 is the composition of Example 3, S4 is the composition of Example 4, S5 is the composition of Example 5, S6 is the composition of Example 6, S7 is the composition of Example 7, S8 is the composition of Example 8, C is a blank control group, D1 is a blue copper peptide single-component control group, D2 is an N-acetylneuraminic acid single-component control group, D3 is a phenylalanine derivative 1 single-component control group, D4 is a phenylalanine derivative 2 single-component control group, and D5 is a thiamine hydrochloride single-component control group. In the present invention, the blank control group has an inhibition rate of 0% on non-enzymatic glycosylation, the blue copper peptide has an inhibitory effect on non-enzymatic glycosylation lower than that of N-acetylneuraminic acid single-component, and phenylalanine derivative 1 single-component, phenylalanine derivative 2 and thiamine hydrochloride single-component have an inhibition rate of 0% on non-enzymatic glycosylation. The inhibitory effect on glycosylation is weaker than that of N-acetylneuraminic acid alone. When blue copper peptide and N-acetylneuraminic acid are compounded into a composition, the inhibitory effect on non-enzymatic glycosylation is stronger than that of blue copper peptide alone, but weaker than that of N-acetylneuraminic acid alone. Adding phenylalanine derivative 1 or phenylalanine derivative 2 to the compound of blue copper peptide and N-acetylneuraminic acid improves the inhibitory effect on non-enzymatic glycosylation to a certain extent, but is still weaker than that of N-acetylneuraminic acid alone. When phenylalanine derivative 1 and phenylalanine derivative 2 are introduced into the composition together, a good effect is achieved, which is stronger than that of N-acetylneuraminic acid alone. After thiamine hydrochloride is further added to blue copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1, and phenylalanine derivative 2, the inhibition of non-enzymatic glycosylation is further improved.

[0066] 4. Detection of gene expression changes of Elastin, Col-Ⅰ, and Col-Ⅳ mRNA using qPCR Test sample: the blue copper peptide composition of each example.

[0067] The primers used in the present invention are shown in Table 1 below: Table 1 Primer sequences used in the experiment

[0068] The primers in the present invention were purchased from Hangzhou Qingke Biotechnology Co., Ltd.

[0069] HFF-1 cells in the exponential growth phase were prepared into a single cell suspension and cultured at 2.4×10 5Cells were seeded at a density of 10 cells / well in a 6-well plate and cultured in medium containing 10% fetal bovine serum at 37°C for 24 hours to allow attachment. The final culture volume was 2 mL. The supernatant was then aspirated and the test sample (serum-free) was added. After incubation at 37°C for 24 hours, RNA was extracted from each well according to the standard RNA extraction protocol. The expression of Col-I, Col-IV, and Elastin mRNA in HFF-1 cells was measured by qPCR. The test sample was used at a concentration of 100 ng / mL. A blank control group was established, in which no test sample was added. A single-component control group consisting of blue copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1, phenylalanine derivative 2, and thiamine hydrochloride was also established. The dosage of each single-component control group was consistent with that of the composition, at a concentration of 100 ng / mL.

[0070] The present invention uses a composite of blue copper peptide and other components to explore the effect of the composite on the expression of Elastin mRNA in HFF-1 cells. Figure 4 As shown, wherein S1 is the composition of Example 1, S2 is the composition of Example 2, S3 is the composition of Example 3, S4 is the composition of Example 4, S5 is the composition of Example 5, S6 is the composition of Example 6, S7 is the composition of Example 7, S8 is the composition of Example 8, C is the blank control group, D1 is the blue copper peptide single-component control group, D2 is the N-acetylneuraminic acid single-component control group, D3 is the phenylalanine derivative 1 single-component control group, D4 is the phenylalanine derivative 2 single-component control group, and D5 is the thiamine hydrochloride single-component control group. In the present invention, the expression of Elastin mRNA in HFF-1 cells in the blank control group is set to 1, the use of blue copper peptide increases the expression of Elastin mRNA in HFF-1 cells, the use of N-acetylneuraminic acid single-component decreases the expression of Elastin mRNA in HFF-1 cells, and the phenylalanine derivative 1 decreases the expression of Elastin mRNA in HFF-1 cells. The separate use of single components of amino acid derivative 1, phenylalanine derivative 2, and thiamine hydrochloride all reduced the expression of Elastin mRNA in HFF-1 cells. When blue copper peptide and N-acetylneuraminic acid were compounded into a composition, the expression of Elastin mRNA in HFF-1 cells was significantly increased. The addition of phenylalanine derivative 1 or phenylalanine derivative 2 to the compound of blue copper peptide and N-acetylneuraminic acid increased the expression of Elastin mRNA in HFF-1 cells. When phenylalanine derivative 1 and phenylalanine derivative 2 were introduced into the composition together, the effect was better than the use of phenylalanine derivative 1 or phenylalanine derivative 2. After thiamine hydrochloride was further added to blue copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1, and phenylalanine derivative 2, the expression of Elastin mRNA in HFF-1 cells was further increased.

[0071] The present invention uses a composite of blue copper peptide and other components to explore the effect of the composite on the expression of Col-I mRNA in HFF-1 cells. Figure 5 As shown, wherein S1 is the composition of Example 1, S2 is the composition of Example 2, S3 is the composition of Example 3, S4 is the composition of Example 4, S5 is the composition of Example 5, S6 is the composition of Example 6, S7 is the composition of Example 7, S8 is the composition of Example 8, C is the blank control group, D1 is the blue copper peptide single-component control group, D2 is the N-acetylneuraminic acid single-component control group, D3 is the phenylalanine derivative 1 single-component control group, D4 is the phenylalanine derivative 2 single-component control group, and D5 is the thiamine hydrochloride single-component control group. In the blank control group of the present invention, the expression of Col-I mRNA in HFF-1 cells is set to 1. The use of blue copper peptide reduces the expression of Col-I mRNA in HFF-1 cells, and its effect is not significant. The use of N-acetylneuraminic acid single-component increases the expression of Col-I mRNA in HFF-1 cells, and its effect is not significant. The effects of single components of phenylalanine derivative 1, phenylalanine derivative 2 and thiamine hydrochloride on the expression of Col-Ⅰ mRNA in HFF-1 cells were also not significant, and the expression levels were not much different from those of the blank control group. When blue copper peptide and N-acetylneuraminic acid were compounded into a composition, the expression of Col-Ⅰ mRNA in HFF-1 cells was increased. The addition of phenylalanine derivative 1 or phenylalanine derivative 2 to the compound of blue copper peptide and N-acetylneuraminic acid increased the expression of Col-Ⅰ mRNA in HFF-1 cells. When phenylalanine derivative 1 and phenylalanine derivative 2 were introduced into the composition together, the effect was better than the use of phenylalanine derivative 1 or phenylalanine derivative 2. After thiamine hydrochloride was further added to blue copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1 and phenylalanine derivative 2, the expression of Col-Ⅰ mRNA in HFF-1 cells was further increased.

[0072] The present invention uses a composite of blue copper peptide and other components to explore the effect of the composite on the expression of Col-Ⅳ mRNA in HFF-1 cells. Figure 6As shown, wherein S1 is the composition of Example 1, S2 is the composition of Example 2, S3 is the composition of Example 3, S4 is the composition of Example 4, S5 is the composition of Example 5, S6 is the composition of Example 6, S7 is the composition of Example 7, S8 is the composition of Example 8, C is a blank control group, D1 is a blue copper peptide single-component control group, D2 is an N-acetylneuraminic acid single-component control group, D3 is a phenylalanine derivative 1 single-component control group, D4 is a phenylalanine derivative 2 single-component control group, and D5 is a thiamine hydrochloride single-component control group. In the present invention, the expression of Col-Ⅳ mRNA in HFF-1 cells in the blank control group is set to 1. The use of blue copper peptide increases the expression of Col-Ⅳ mRNA in HFF-1 cells. The use of N-acetylneuraminic acid single-component can also increase the expression of Col-Ⅳ mRNA in HFF-1 cells, but the use of N-acetylneuraminic acid single-component The effect was weaker than that of the use of blue copper peptide. The use of single component phenylalanine derivative 1, phenylalanine derivative 2 and thiamine hydrochloride respectively improved the expression of Col-Ⅳ mRNA in HFF-1 cells to a certain extent. When blue copper peptide was compounded with N-acetylneuraminic acid into a composition, the expression of Col-Ⅳ mRNA in HFF-1 cells was significantly improved. The addition of phenylalanine derivative 1 or phenylalanine derivative 2 to the compound of blue copper peptide and N-acetylneuraminic acid increased the expression of Col-Ⅳ mRNA in HFF-1 cells. When phenylalanine derivative 1 and phenylalanine derivative 2 were introduced into the composition together, the effect was better than the use of phenylalanine derivative 1 or phenylalanine derivative 2. After thiamine hydrochloride was further added to blue copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1 and phenylalanine derivative 2, the expression of Col-Ⅳ mRNA in HFF-1 cells was further improved.

[0073] The above embodiments are intended to illustrate the present invention only and are not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also fall within the scope of the present invention, and the scope of patent protection of the present invention shall be defined by the claims.

Claims

1. A use of a blue copper peptide and N-acetylneuraminic acid in the preparation of an anti-aging drug and / or anti-inflammatory drug or anti-aging and / or anti-inflammatory cosmetic, wherein the blue copper peptide has a structure in which copper is linked to glycyl-L-histidyl-L-lysine; the blue copper peptide and N-acetylneuraminic acid are mixed in a mass ratio of 1:0.1-1.

2. A use of a blue copper peptide and N-acetylneuraminic acid and a phenylalanine derivative in anti-aging and / or anti-inflammatory drugs or anti-aging and / or anti-inflammatory cosmetics, wherein the structure of the blue copper peptide is copper linked to glycyl-L-histidyl-L-lysine; the blue copper peptide, N-acetylneuraminic acid, and the phenylalanine derivative are mixed in a mass ratio of 1:0.1-1:0.1-0.3; the phenylalanine derivative is prepared from phenylalanine and monoethyl oxalyl chloride or malonyl chloride.

3. A molecular chaperone-cooperated blue copper peptide, comprising: The invention is at least composed of a composite of a blue copper peptide and N-acetylneuraminic acid, wherein the structure of the blue copper peptide is copper connected to glycyl-L-histidyl-L-lysine; the blue copper peptide and N-acetylneuraminic acid are mixed in a mass ratio of 1:0.1-1.

4. A molecular chaperone-cooperated blue copper peptide according to claim 3, characterized in that: The composition contains a phenylalanine derivative which is prepared from phenylalanine and ethyl oxalyl chloride.

5. A molecular chaperone-cooperated blue copper peptide according to claim 4, characterized in that: The blue copper peptide, N-acetylneuraminic acid and phenylalanine derivative are mixed in a mass ratio of 1:0.1-1:0.1-0.3 for use.

6. A molecular chaperone-cooperated blue copper peptide according to claim 4, characterized in that: In the preparation of the phenylalanine derivative, the amount of ethyl oxalyl chloride used is 20-60 wt % of phenylalanine.

7. The molecular chaperone-cooperated blue copper peptide according to claim 4, characterized in that: In the preparation of the phenylalanine derivative, the amount of malonyl chloride used is 20-60 wt % of phenylalanine.

8. The molecular chaperone-cooperated blue copper peptide according to claim 4, characterized in that: The ethyl oxalyl chloride is dissolved in anhydrous tetrahydrofuran to prepare the ethyl oxalyl chloride solution.

9. The molecular chaperone-cooperated blue copper peptide according to claim 4, characterized in that: The malonyl chloride is dissolved in anhydrous tetrahydrofuran to prepare a malonyl chloride solution.

10. The molecular chaperone-cooperated blue copper peptide according to claim 4, characterized in that: The composition contains a phenylalanine derivative, which is prepared from phenylalanine and malonyl chloride.

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