A copper peptide composition and its application

Abstract

The present invention discloses a copper peptide composition and its application, belonging to the technical field of skin care. Specifically, it relates to a copper peptide composition composed of a copper peptide and N-acetylneuraminic acid. The structure of the copper peptide is that copper is linked to glycyl-L-histidyl-L-lysine. The copper peptide and N-acetylneuraminic acid are mixed and used in a mass ratio of 1:0.1-1. Further, phenylalanine derivatives can be added for compounding. When the obtained copper peptide composition of the present invention is applied to cells, the cell survival rate is high, the non-enzymatic glycosylation inhibition rate is good, and the expressions of Elastin, Col-I, and Col-IV mRNA in the cells are high. Therefore, the present invention is a copper peptide composition with a high cell survival rate, a good non-enzymatic glycosylation inhibition rate, and an improvement in the expressions of Elastin, Col-I, and Col-IV mRNA in cells, and its application.

Description

Technical Field

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

[0002] The main components of the dermal extracellular matrix (ECM) are collagen fiber network, elastic fiber network and proteoglycan. Collagen and elastin are the main extracellular components, mainly including type I collagen and type III collagen. Newly synthesized type I procollagen is secreted into the extracellular space of the dermis, and after metabolism by related enzymes, it forms a triple helical spatial complex, which combines with other extracellular matrix proteins (for example: small molecule proteoglycans) to form a collagen fiber bundle with regular structure, providing toughness and tensile resistance for the skin. Skin aging problems caused by environmental stimulation are mainly manifested as: the secretion of type I collagen by dermal cells decreases, cells lack collagen support, and the stratum corneum and epidermis are inward to form wrinkles. There are two main reasons for the extracellular matrix (ECM): on the one hand, human skin fibroblasts first synthesize and secrete procollagen, and then the procollagen cleaves part of the peptide chain to form mature collagen. External factors lead to a decrease in skin collagen components by promoting the degradation of collagen, and can also lead to a decrease in skin collagen components by inhibiting the synthesis of procollagen. External factors including light cause a decrease in collagen synthesis in the skin, and the collagen fiber network also degenerates and disintegrates. Studies have also found that fibroblasts exposed to large amounts of degenerated collagen have reduced proliferation and collagen synthesis capabilities. Elastin is the main component of elastic fibers. Elastic fibers are mainly found in ligaments and vascular walls. Elastic fibers coexist with collagen fibers, giving tissues elasticity and tensile strength. Although elastin accounts for only 2% of the total dermal protein, it plays an important role in skin elasticity.

[0003] In terms of anti-inflammatory, the proportion of Chinese consumers with sensitive skin is increasing. The manifestations on the face are mainly redness, burning, stinging, itching, peeling, etc. The causes of these sensitive symptoms are closely related to external environmental pollution, extreme climate changes, improper personal care methods, etc. In the face of these stimuli, the skin's immune system will start signals, releasing various inflammatory factors and histamine, etc., causing facial discomfort, and the majority of consumers are also looking for skin care solutions for these skin problems. 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 solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] Use of a copper peptide and N-acetylneuraminic acid in the preparation of an anti-aging drug and / or an anti-inflammatory drug or an anti-aging and / or anti-inflammatory cosmetic. The structure of the copper peptide is that copper is linked to glycyl-L-histidyl-L-lysine. The copper peptide and N-acetylneuraminic acid are used in a mass ratio of 1:0.1 - 1. The copper peptide (Cu-GHK) acts in the extracellular matrix and is released in wounds or inflammation to support healing. It acts as a signaling and carrier peptide, promoting the synthesis of conventional collagen, elastin, proteoglycans, and glycosaminoglycans, and providing anti-inflammatory and antioxidant responses. In cosmetic applications, Cu-GHK is used in anti-aging, anti-wrinkle, post-sun, skin renewal, skin moisturizing, and hair growth stimulation products. N-acetylneuraminic acid is an important component in biological membranes, mostly bound to membrane proteins, distributed on the surface of the plasma membrane, forming a polysaccharide-protein complex. N-acetylneuraminic acid has a negative charge on its surface and is an important carrier in the process of information conduction. Therefore, negatively charged N-acetylneuraminic acid is called the antenna on the cell surface, which helps to enhance cell activity. The lack of N-acetylneuraminic acid will lead to a decrease in the lifespan of blood cells and enzyme proteins in metabolism, and thus lead to cell aging. N-acetylneuraminic acid can also scavenge reactive oxygen free radicals generated in the human body, and thus play an antioxidant and protective role. By compounding the copper peptide with N-acetylneuraminic acid, the present invention has higher efficacy and is superior to the use of the copper peptide or N-acetylneuraminic acid in some properties.

[0007] Use of a copper peptide, N-acetylneuraminic acid, and a phenylalanine derivative in the preparation of an anti-aging drug and / or an anti-inflammatory drug or an anti-aging and / or anti-inflammatory cosmetic. The structure of the copper peptide is that copper is linked to glycyl-L-histidyl-L-lysine. The copper peptide, N-acetylneuraminic acid, and the phenylalanine derivative are used in a mass ratio of 1:0.1 - 1:0.1 - 0.3. The phenylalanine derivative is made from phenylalanine and monoethyl oxalyl chloride or malonyl chloride. By preparing a phenylalanine derivative and then compounding it with the copper peptide and N-acetylneuraminic acid, through the non-bonding interaction forces in its molecule, they are combined with each other, improving the compounding effect of the composition, increasing the cell survival rate, increasing the inhibition rate of non-enzymatic glycosylation, and increasing the expression of Elastin, Col-Ⅰ, and Col-Ⅳ mRNA in cells.

[0008] A copper peptide composition comprising at least a composite of a copper peptide and N-acetylneuraminic acid. The structure of the copper peptide is that copper is linked to glycyl-L-histidyl-L-lysine. The copper peptide and N-acetylneuraminic acid are used in a mass ratio of 1:0.1 - 1.

[0009] Preferably, the composition contains a phenylalanine derivative, which is made from phenylalanine and monoethyl oxalyl chloride or malonyl chloride.

[0010] Preferably, the copper peptide GHK, N-acetylneuraminic acid and the phenylalanine derivative are mixed and used in a mass ratio of 1:0.1 - 1:0.1 - 0.3.

[0011] Preferably, in the preparation of the phenylalanine derivative, the usage amount of monoethyl oxalyl chloride is 20 - 60 wt% of phenylalanine.

[0012] Preferably, in the preparation of the phenylalanine derivative, the usage amount of malonyl chloride is 20 - 60 wt% of phenylalanine.

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

[0014] Preferably, malonyl chloride is dissolved in anhydrous tetrahydrofuran to prepare a malonyl chloride solution.

[0015] Preferably, in the preparation of phenylalanine derivative 1, an alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, then the monoethyl oxalyl chloride solution is added, and the mixture is stirred and reacted for 6 - 18 h under ice bath conditions. 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.

[0016] More preferably, in the preparation of phenylalanine derivative 1, the alkaline reagent is sodium hydroxide, and the usage amount of the alkaline reagent is 70 - 90 wt% of phenylalanine.

[0017] More preferably, in the preparation of phenylalanine derivative 1, the usage amount of phenylalanine is 5 - 25 wt% of distilled water.

[0018] More preferably, in the preparation of phenylalanine derivative 1, the monoethyl oxalyl chloride solution is made by mixing monoethyl oxalyl chloride and anhydrous tetrahydrofuran, and the monoethyl oxalyl chloride solution contains 10 - 20 wt% of monoethyl oxalyl chloride.

[0019] More preferably, in the preparation of phenylalanine derivative 1, the usage amount of the monoethyl oxalyl chloride solution is based on the amount of monoethyl oxalyl chloride therein, and the usage amount of monoethyl oxalyl chloride is 20 - 60 wt% of phenylalanine.

[0020] Preferably, in the preparation of phenylalanine derivative 2, an alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, then the malonyl chloride solution is added, and the mixture is stirred and reacted for 6 - 18 h under ice bath conditions. 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.

[0021] More preferably, in the preparation of phenylalanine derivative 2, the basic reagent is sodium hydroxide, and the usage amount of the basic reagent is 70-90 wt% of phenylalanine.

[0022] More preferably, in the preparation of phenylalanine derivative 2, the usage amount of phenylalanine is 5-25 wt% of distilled water.

[0023] 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.

[0024] More preferably, in the preparation of phenylalanine derivative 2, the usage amount of the malonyl chloride solution is based on the amount of malonyl chloride therein, and the usage amount of malonyl chloride is 20-60 wt% of phenylalanine.

[0025] The present invention discloses a blue copper peptide composition, which is compounded by blue copper peptide and N-acetylneuraminic acid, and the amount of N-acetylneuraminic acid is 10-100 wt% of blue copper peptide.

[0026] Preferably, the blue copper peptide composition is compounded by blue copper peptide, N-acetylneuraminic acid and phenylalanine derivative 1, the amount of N-acetylneuraminic acid is 10-100 wt% of blue copper peptide, and the amount of phenylalanine derivative 1 is 5-20 wt% of blue copper peptide.

[0027] Preferably, the blue copper peptide composition is compounded by blue copper peptide, N-acetylneuraminic acid and phenylalanine derivative 2, the amount of N-acetylneuraminic acid is 10-100 wt% of blue copper peptide, and the amount of phenylalanine derivative 2 is 5-20 wt% of blue copper peptide.

[0028] Preferably, the blue copper peptide composition is compounded by blue copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1 and phenylalanine derivative 2, the amount of N-acetylneuraminic acid is 20 wt% of blue copper peptide, the amount of phenylalanine derivative 1 is 5 wt% of blue copper peptide, and the amount of phenylalanine derivative 2 is 5 wt% of blue copper peptide.

[0029] Preferably, thiamine hydrochloride can be added to the blue copper peptide composition for compounding, and the usage amount of thiamine hydrochloride is 2-6 wt% of blue copper peptide. In the compounding of blue copper peptide and N-acetylneuraminic acid, and after using phenylalanine derivative 1 and phenylalanine derivative 2, and further using thiamine hydrochloride, the usage effect of the composition is better, while improving the cell survival rate, increasing the inhibition rate of non-enzymatic glycosylation, and increasing the expression of Elastin, Col-Ⅰ, Col-Ⅳ mRNA in cells.

[0030] The present invention is composed of a combination of copper peptide and N-acetylneuraminic acid. The structure of the copper peptide is that copper is linked to glycyl-L-histidyl-L-lysine. The copper peptide and N-acetylneuraminic acid are mixed and used in a mass ratio of 1:0.1 - 1. Further, phenylalanine derivatives can be added for compounding. Thus, it has 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 is a copper peptide composition with a high cell survival rate, good non-enzymatic glycosylation inhibition rate, and capable of increasing the expression of Elastin, Col-Ⅰ, and Col-Ⅳ mRNA in cells, as well as its application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the infrared diagram of phenylalanine derivatives;

[0032] Figure 2 It is the cell survival rate diagram;

[0033] Figure 3 It is the non-enzymatic glycosylation inhibition rate diagram;

[0034] Figure 4 It is the expression diagram of Elastin mRNA;

[0035] Figure 5 It is the expression diagram of Col-Ⅰ mRNA;

[0036] Figure 6 It is the expression diagram of Col-Ⅳ mRNA. DETAILED DESCRIPTION OF THE INVENTION

[0037] The technical solutions of the present invention will be further described in detail below in combination with the specific embodiments and the drawings:

[0038] The cells and peptides used in the present invention are as follows:

[0039] HFF-1 human skin fibroblasts are from Zhejiang Meisen Cell Technology Co., Ltd.; the copper peptide is from Zhejiang Peptide Biological Co., Ltd.

[0040] The phenylalanine used in the present invention is

[0041] Example 1:

[0042] A copper peptide composition comprising a copper peptide and N-acetylneuraminic acid.

[0043] The copper peptide composition is compounded from a copper peptide and N-acetylneuraminic acid, and the amount of N-acetylneuraminic acid is 20 wt% of the copper peptide.

[0044] Example 2:

[0045] A copper peptide composition comprising copper peptide, N-acetylneuraminic acid, and phenylalanine derivative 1.

[0046] Preparation of phenylalanine derivative 1: An alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, then ethyl oxalyl chloride solution is added, and the mixture is stirred and reacted for 12 h under ice bath conditions. After the reaction is completed, the pH is adjusted to 1, filtered, washed with distilled water until neutral, dried, then washed with petroleum ether and dried to obtain phenylalanine derivative 1. The alkaline reagent is sodium hydroxide, and the usage amount of the alkaline reagent is 80 wt% of phenylalanine. The usage amount of phenylalanine is 10 wt% of distilled water. The ethyl oxalyl chloride solution is made by mixing ethyl oxalyl chloride and anhydrous tetrahydrofuran. The ethyl oxalyl chloride solution contains 15 wt% of ethyl oxalyl chloride. The usage amount of the ethyl oxalyl chloride solution is based on the amount of ethyl oxalyl chloride therein, and the usage amount of ethyl oxalyl chloride is 40 wt% of phenylalanine.

[0047] The copper peptide composition is prepared by compounding copper peptide, N-acetylneuraminic acid, and phenylalanine derivative 1. The amount of N-acetylneuraminic acid is 20 wt% of copper peptide, and the amount of phenylalanine derivative 1 is 10 wt% of copper peptide.

[0048] Example 3:

[0049] A copper peptide composition comprising copper peptide, N-acetylneuraminic acid, and phenylalanine derivative 2.

[0050] Preparation of phenylalanine derivative 2: An alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, then malonyl chloride solution is added, and the mixture is stirred and reacted for 10 h under ice bath conditions. After the reaction is completed, the pH is adjusted to 1, filtered, washed with distilled water until neutral, dried, then washed with petroleum ether and dried to obtain phenylalanine derivative 2. The alkaline reagent is sodium hydroxide, and the usage amount of the alkaline reagent is 80 wt% of phenylalanine. The usage amount of phenylalanine is 20 wt% of distilled water. The malonyl chloride solution is made by mixing malonyl chloride and anhydrous tetrahydrofuran. The malonyl chloride solution contains 10 wt% of malonyl chloride. The usage amount of the malonyl chloride solution is based on the amount of malonyl chloride therein, and the usage amount of malonyl chloride is 40 wt% of phenylalanine.

[0051] The copper peptide composition is prepared by compounding copper peptide, N-acetylneuraminic acid, and phenylalanine derivative 2. The amount of N-acetylneuraminic acid is 20 wt% of copper peptide, and the amount of phenylalanine derivative 2 is 10 wt% of copper peptide.

[0052] Example 4:

[0053] A copper peptide composition, comprising copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1, and phenylalanine derivative 2.

[0054] Preparation of phenylalanine derivative 1: An alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, then ethyl oxalyl chloride solution is added, and the mixture is stirred and reacted for 12 h under ice bath conditions. After the reaction is completed, the pH is adjusted to 1, 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 usage amount of the alkaline reagent is 80 wt% of phenylalanine. The usage amount of phenylalanine is 10 wt% of distilled water. The ethyl oxalyl chloride solution is prepared by mixing ethyl oxalyl chloride and anhydrous tetrahydrofuran. The ethyl oxalyl chloride solution contains 15 wt% of ethyl oxalyl chloride. The usage amount of the ethyl oxalyl chloride solution is based on the amount of ethyl oxalyl chloride therein, and the usage amount of ethyl oxalyl chloride is 40 wt% of phenylalanine.

[0055] Preparation of phenylalanine derivative 2: An alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, then malonyl chloride solution is added, and the mixture is stirred and reacted for 10 h under ice bath conditions. After the reaction is completed, the pH is adjusted to 1, 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 usage amount of the alkaline reagent is 80 wt% of phenylalanine. The usage amount of phenylalanine is 20 wt% of distilled water. The malonyl chloride solution is prepared by mixing malonyl chloride and anhydrous tetrahydrofuran. The malonyl chloride solution contains 10 wt% of malonyl chloride. The usage amount of the malonyl chloride solution is based on the amount of malonyl chloride therein, and the usage amount of malonyl chloride is 40 wt% of phenylalanine.

[0056] The copper peptide composition is compounded from copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1, and phenylalanine derivative 2. The amount of N-acetylneuraminic acid is 20 wt% of copper peptide, the amount of phenylalanine derivative 1 is 5 wt% of copper peptide, and the amount of phenylalanine derivative 2 is 5 wt% of copper peptide.

[0057] Example 5:

[0058] A copper peptide composition, comprising copper peptide, N-acetylneuraminic acid, and thiamine hydrochloride.

[0059] The copper peptide composition is compounded from copper peptide, N-acetylneuraminic acid, and thiamine hydrochloride. The amount of N-acetylneuraminic acid is 20 wt% of copper peptide, and the usage amount of thiamine hydrochloride is 4 wt% of copper peptide.

[0060] Example 6:

[0061] A copper peptide composition, comprising copper peptide, N-acetylneuraminic acid, thiamine hydrochloride, and phenylalanine derivative 1. Phenylalanine derivative 1 is derived from Example 2.

[0062] The copper peptide composition is prepared by compounding copper peptide, N-acetylneuraminic acid, thiamine hydrochloride, and phenylalanine derivative 1. The amount of N-acetylneuraminic acid is 20 wt% of the copper peptide, the amount of thiamine hydrochloride used is 4 wt% of the copper peptide, and the amount of phenylalanine derivative 1 used is 10 wt% of the copper peptide.

[0063] Example 7:

[0064] A copper peptide composition, comprising copper peptide, N-acetylneuraminic acid, thiamine hydrochloride, and phenylalanine derivative 2. Phenylalanine derivative 2 is derived from Example 3.

[0065] The copper peptide composition is prepared by compounding copper peptide, N-acetylneuraminic acid, thiamine hydrochloride, and phenylalanine derivative 2. The amount of N-acetylneuraminic acid is 20 wt% of the copper peptide, the amount of thiamine hydrochloride used is 4 wt% of the copper peptide, and the amount of phenylalanine derivative 2 used is 10 wt% of the copper peptide.

[0066] Example 8:

[0067] A copper peptide composition, comprising 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.

[0068] The copper peptide composition is prepared by compounding copper peptide, N-acetylneuraminic acid, thiamine hydrochloride, and phenylalanine derivative 2. The amount of N-acetylneuraminic acid is 20 wt% of the copper peptide, the amount of thiamine hydrochloride used is 4 wt% of the copper peptide, the amount of phenylalanine derivative 1 used is 5 wt% of the copper peptide, and the amount of phenylalanine derivative 2 used is 5 wt% of the copper peptide.

[0069] Example 9:

[0070] A method for constructing a cell model

[0071] After seeding HFF-1 cells and culturing for 24 h, after the cells adhered, the test substance (diluted to the administration concentration with serum-free medium) was added and treated for 24 h, and the changes in related indexes were detected. Culture conditions: 5% CO 2 , in a constant temperature incubator at 37 °C. Medium: 89% DMEM + 10% FBS + 1% double antibody (HFF-1).

[0072] Test example:

[0073] 1. Infrared analysis

[0074] Test sample: The phenylalanine derivative prepared in Example 2.

[0075] The infrared spectrum of the phenylalanine derivative 1 prepared in the present invention is as Figure 1 shown, in which, the infrared spectrum of the hydroxyl group in the carboxyl group is at 3427 cm -1 , the infrared absorption peak of nitrogen-hydrogen is at 3236 cm -1 , the infrared absorption peaks of methyl and methylene are between 2800 - 3000 cm -1 , the infrared absorption peak of the carbon-oxygen double bond is at 1671 cm -1 , the infrared absorption peaks of the benzene ring are at 1609 cm -1 and 1507 cm -1 , and the infrared absorption peak of the carbon-nitrogen bond on the amide is at 1280 cm -1 .

[0076] In this study, the effects of combined administration of polypeptide-based cosmetic raw materials and other raw materials on genes such as collagen, elastin, anti-inflammation, and repair of human dermal fibroblasts (HFF-1) were studied to evaluate the efficacy of the polypeptide raw material compounding.

[0077] 2. Detection of cell viability by MTT method

[0078] Test sample: The copper peptide blue composition of each example.

[0079] Single-cell suspensions were prepared from HFF-1 cells in the exponential growth phase. The HFF-1 cells were seeded at 5×10 3 cells / well, cultured in a medium containing 10% fetal bovine serum at 37 °C for 24 h to allow the cells to adhere, and the final culture volume was 100 μL. Then, the supernatant was aspirated, and the freshly prepared test sample (serum-free) was added. After incubation for 24 h, 10 μL of MTT with a concentration of 5 mg / mL was added to each well. After gently shaking and mixing, the cells were continued to be cultured at 37 °C. After 4 h, the 96-well culture plate was taken out, the supernatant was carefully aspirated, 100 μL of DMSO was added to each well, and the plate was shaken on a shaker for 10 min to fully dissolve the purple formazan precipitate. The absorbance OD value of each well was measured at a wavelength of 490 nm to observe the effect of the test substance on cell growth. The dosage of the test sample was characterized as 0.1 μg / mL. A blank control group was set up without adding the test sample, and single-component control groups of copper peptide blue, N-acetylneuraminic acid, phenylalanine derivative 1, phenylalanine derivative 2, and thiamine hydrochloride were set up. The dosages of the above single-component control groups were the same as those of the composition, and the concentration was 0.1 μg / mL.

[0080] The present invention tested different usage amounts of the single component of copper peptide, and found that within the range of 0.01 - 1 μg / mL of the copper peptide usage amount, the activity of HFF-1 cells increased, and the activity was the highest at 1 μg / mL. The copper peptide within the above concentration range significantly increased the activity of HFF-1 cells (p < 0.05). Then, as the concentration of the copper peptide increased, such as at 1000 μg / mL, the survival rate of HFF-1 cells decreased significantly (p < 0.01).

[0081] The present invention used the copper peptide compounded with other components into a composition to explore the influence of the composition on cell activity, and the results are as Figure 2 shown, where 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 single-component control group of copper peptide, D2 is the single-component control group of N-acetylneuraminic acid, D3 is the single-component control group of phenylalanine derivative 1, D4 is the single-component control group of phenylalanine derivative 2, D5 is the single-component control group of thiamine hydrochloride. In the present invention, the cell survival rate of the blank control group is set to 100%. The use of copper peptide can significantly improve cell activity, while the influence of the single component of N-acetylneuraminic acid on the cell activity of HFF-1 cells is not obvious. Similarly, the influence of the single component of phenylalanine derivative 1, phenylalanine derivative 2, and thiamine hydrochloride on the cell activity of HFF-1 cells is not obvious. At least it shows that the single components of N-acetylneuraminic acid, phenylalanine derivative 1, phenylalanine derivative 2, and thiamine hydrochloride will not reduce the cell activity of HFF-1 cells. After compounding one or more of the above components with copper peptide into a composition, the cell survival rate is significantly increased, and it has an excellent positive effect on cells.

[0082] 3. Use an enzyme-labeled instrument to detect the non-enzymatic glycosylation inhibition rate of the drug

[0083] Test samples: The copper peptide compositions of each example.

[0084] Dilute the test sample to a suitable concentration, prepare the BSA-fructose reaction solution according to the standard operating procedure of the non-enzymatic glycosylation inhibition test. After mixing the test sample and the reaction solution, incubate them in the dark in a constant temperature incubator at 37 °C. After 5 days, detect the fluorescence intensity of each group of samples. After mixing the test sample and the reaction solution, the concentration of the test sample is 100 ng / mL. Set up a blank control group without adding the test sample, and set up single-component control groups of cuproen, N-acetylneuraminic acid, phenylalanine derivative 1, phenylalanine derivative 2, and thiamine hydrochloride. The usage amounts of the above single-component control groups are the same as those of the composition, and the concentrations are all 100 ng / mL.

[0085] The fluorescence intensity of the sample was detected by an enzyme-labeled instrument at an excitation wavelength of 370 nm and an emission wavelength of 440 nm.

[0086] Compared with the control group or the group treated with N-acetylneuraminic acid or cuproen alone, the expression of Elastin mRNA in HFF-1 cells was significantly increased after treatment with 10% a + 50% c (p < 0.05).

[0087] In the present invention, cuproen is compounded with other components to form a composition for use, and the effect of the composition on non-enzymatic glycosylation is explored. The results are as follows Figure 3As shown, where 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 single-component control group of copper peptide, D2 is the single-component control group of N-acetylneuraminic acid, D3 is the single-component control group of phenylalanine derivative 1, D4 is the single-component control group of phenylalanine derivative 2, D5 is the single-component control group of thiamine hydrochloride. In the present invention, the inhibition rate of non-enzymatic glycosylation in the blank control group is set to 0%. The inhibitory effect of copper peptide on non-enzymatic glycosylation is lower than that of the single component of N-acetylneuraminic acid, and the inhibitory effects of the single components of phenylalanine derivative 1, phenylalanine derivative 2, and thiamine hydrochloride on non-enzymatic glycosylation are all weaker than that of the single component of N-acetylneuraminic acid. When copper peptide and N-acetylneuraminic acid are compounded into a composition, the inhibitory effect on non-enzymatic glycosylation is stronger than that of the single-component use of copper peptide, but weaker than that of the single-component use of N-acetylneuraminic acid. When phenylalanine derivative 1 or phenylalanine derivative 2 is added to the compound of copper peptide and N-acetylneuraminic acid, the inhibitory effect on non-enzymatic glycosylation is improved to a certain extent, but it is still weaker than that of the single-component use of N-acetylneuraminic acid. When phenylalanine derivative 1 and phenylalanine derivative 2 are jointly introduced into the composition, it has a good effect, stronger than the single-component use of N-acetylneuraminic acid, and after further adding thiamine hydrochloride to the composition of copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1, and phenylalanine derivative 2, the inhibition of non-enzymatic glycosylation is further improved.

[0088] 4. Detect the expression changes of genes Elastin, Col-Ⅰ, and Col-Ⅳ mRNA by qPCR method

[0089] Test samples: Copper peptide compositions of each example.

[0090] The primers used in the present invention are shown in Table 1 below:

[0091] Table 1 Primer sequences used in the experiment

[0092]

[0093] Prepare a single-cell suspension from HFF-1 cells in the exponential growth phase, at 2.4×10 5The density of cells / wells was inoculated on a 6-well plate and cultured in a medium containing 10% fetal bovine serum at 37 °C for 24 h to allow the cells to adhere. The final culture volume was 2 mL. Then, the supernatant was aspirated and discarded, and the test sample (serum-free) was added. After culturing at 37 °C for 24 h, RNA was extracted from each well according to the standard operation procedure for RNA extraction, and the expression of Col-Ⅰ, Col-Ⅳ, and Elastin mRNA in HFF-1 cells was detected by qPCR. The concentration of the test sample during use was 100 ng / mL. A blank control group was set up without adding the test sample. A single-component control group of copper peptide, a single-component control group of N-acetylneuraminic acid, a single-component control group of phenylalanine derivative 1, a single-component control group of phenylalanine derivative 2, and a single-component control group of thiamine hydrochloride were set up. The usage amounts of the above single-component control groups were the same as those of the composition, and the concentrations were all 100 ng / mL.

[0094] In the present invention, copper peptide was compounded with other components to form a composition for use, and the effect of the composition on the expression of Elastin mRNA in HFF-1 cells was explored. The results are as Figure 4 shown, where 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 single-component control group of copper peptide, D2 is the single-component control group of N-acetylneuraminic acid, D3 is the single-component control group of phenylalanine derivative 1, D4 is the single-component control group of phenylalanine derivative 2, D5 is the single-component control group of thiamine hydrochloride. In the present invention, the expression of Elastin mRNA in HFF-1 cells in the blank control group was set as 1. The use of copper peptide increased the expression of Elastin mRNA in HFF-1 cells. The use of the single component of N-acetylneuraminic acid decreased the expression of Elastin mRNA in HFF-1 cells. The use of the single components of phenylalanine derivative 1, phenylalanine derivative 2, and thiamine hydrochloride respectively decreased the expression of Elastin mRNA in HFF-1 cells. When copper peptide was compounded with N-acetylneuraminic acid to form a composition, the expression of Elastin mRNA in HFF-1 cells was significantly increased. When phenylalanine derivative 1 or phenylalanine derivative 2 was added to the compound of copper peptide and N-acetylneuraminic acid, the expression of Elastin mRNA in HFF-1 cells was increased. When phenylalanine derivative 1 and phenylalanine derivative 2 were introduced into the composition together, the effect produced was better than the use of phenylalanine derivative 1 or phenylalanine derivative 2. After further adding thiamine hydrochloride to copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1, and phenylalanine derivative 2, the expression of Elastin mRNA in HFF-1 cells was further increased.

[0095] In the present invention, by using a complex of copper peptide and other components as a composition, the effect of the composition on the expression of Col-I mRNA in HFF-1 cells was investigated. The results are as Figure 5 shown, where 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 single-component control group of copper peptide, D2 is the single-component control group of N-acetylneuraminic acid, D3 is the single-component control group of phenylalanine derivative 1, D4 is the single-component control group of phenylalanine derivative 2, D5 is the single-component control group of thiamine hydrochloride. In the present invention, the expression of Col-I mRNA in HFF-1 cells in the blank control group was set to 1. The use of copper peptide decreased the expression of Col-I mRNA in HFF-1 cells, but the effect was not significant. The use of the single-component of N-acetylneuraminic acid increased the expression of Col-I mRNA in HFF-1 cells, but the effect was not significant. The effects of the 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 differences from the expression in the blank control group were not large. When copper peptide and N-acetylneuraminic acid were complexed into a composition, the expression of Col-I mRNA in HFF-1 cells was increased. When phenylalanine derivative 1 or phenylalanine derivative 2 was added to the complex of copper peptide and N-acetylneuraminic acid, the expression of Col-I mRNA in HFF-1 cells was increased. When phenylalanine derivative 1 and phenylalanine derivative 2 were introduced into the composition together, the resulting effect was better than the use of phenylalanine derivative 1 or phenylalanine derivative 2. After further adding thiamine hydrochloride to the complex of 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.

[0096] In the present invention, by using a complex of copper peptide and other components as a composition, the effect of the composition on the expression of Col-IV mRNA in HFF-1 cells was investigated. The results are as Figure 6As shown, where 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 single-component control group of copper peptide, D2 is the single-component control group of N-acetylneuraminic acid, D3 is the single-component control group of phenylalanine derivative 1, D4 is the single-component control group of phenylalanine derivative 2, D5 is the single-component control group of thiamine hydrochloride. 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 copper peptide increases the expression of Col-IV mRNA in HFF-1 cells. The use of the single component of N-acetylneuraminic acid can also increase the expression of Col-IV mRNA in HFF-1 cells, but the effect of using the single component of N-acetylneuraminic acid is weaker than that of using copper peptide. The separate use of the single component of phenylalanine derivative 1, phenylalanine derivative 2, and thiamine hydrochloride all has a certain increase in the expression of Col-IV mRNA in HFF-1 cells. When copper peptide and N-acetylneuraminic acid are compounded into a composition, it significantly increases the expression of Col-IV mRNA in HFF-1 cells. When phenylalanine derivative 1 or phenylalanine derivative 2 is added to the compound of copper peptide and N-acetylneuraminic acid, it increases the expression of Col-IV mRNA in HFF-1 cells. When phenylalanine derivative 1 and phenylalanine derivative 2 are jointly introduced into the composition, the resulting effect is better than the use of phenylalanine derivative 1 or phenylalanine derivative 2. After further adding thiamine hydrochloride to copper peptide, N-acetylneuraminic acid, phenylalanine derivative 1, and phenylalanine derivative 2, it further increases the expression of Col-IV mRNA in HFF-1 cells.

[0097] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Those of ordinary skill in the art can also make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention. The patent protection scope of the present invention shall be defined by the claims.

Claims

1. A copper peptide composition, comprising: at least composed of a copper peptide and N-acetylneuraminic acid, and the structure of the copper peptide is that copper is connected to glycyl-L-histidyl-L-lysine; the composition contains a phenylalanine derivative, and the phenylalanine derivative is phenylalanine derivative 1 or phenylalanine derivative 2; the copper peptide, N-acetylneuraminic acid and phenylalanine derivative are used in a mass ratio of 1:0.1 - 1:0.1 - 0.3; in the preparation of phenylalanine derivative 1, an alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, then ethyl oxalyl chloride solution is added, and the reaction is stirred for 6 - 18 h under ice bath conditions. 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; the alkaline reagent is sodium hydroxide, the usage amount of the alkaline reagent is 70 - 90 wt% of phenylalanine, the usage amount of phenylalanine is 5 - 25 wt% of distilled water, the ethyl oxalyl chloride solution is made by mixing ethyl oxalyl chloride and anhydrous tetrahydrofuran, the ethyl oxalyl chloride solution contains 10 - 20 wt% of ethyl oxalyl chloride, the usage amount of the ethyl oxalyl chloride solution is based on the amount of ethyl oxalyl chloride therein, and the usage amount of ethyl oxalyl chloride is 20 - 60 wt% of phenylalanine; or, in the preparation of phenylalanine derivative 2, an alkaline reagent and phenylalanine are added to distilled water and mixed and dissolved, then malonyl chloride solution is added, and the reaction is stirred for 6 - 18 h under ice bath conditions. 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. The alkaline reagent is sodium hydroxide, the usage amount of the alkaline reagent is 70 - 90 wt% of phenylalanine, the usage amount of phenylalanine is 5 - 25 wt% of distilled water, the malonyl chloride solution is made by mixing malonyl chloride and anhydrous tetrahydrofuran, the malonyl chloride solution contains 10 - 20 wt% of malonyl chloride, the usage amount of the malonyl chloride solution is based on the amount of malonyl chloride therein, and the usage amount of malonyl chloride is 20 - 60 wt% of phenylalanine.

2. Use of the copper peptide composition according to claim 1 in the preparation of anti-aging drugs and / or anti-inflammatory drugs or in the preparation of anti-aging and / or anti-inflammatory cosmetics. The structure of the copper peptide is that copper is connected to glycyl-L-histidyl-L-lysine; the copper peptide, N-acetylneuraminic acid and phenylalanine derivative are used in a mass ratio of 1:0.1 - 1:0.1 - 0.3; the phenylalanine derivative is made from phenylalanine and ethyl oxalyl chloride or malonyl chloride, and the usage amount of ethyl oxalyl chloride is 20 - 60 wt% of phenylalanine, and the usage amount of malonyl chloride is 20 - 60 wt% of phenylalanine.

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