Collagen peptide composition and application thereof in recovering or promoting collagen content

Abstract

The invention relates to a collagen peptide composition and application thereof in recovering or promoting collagen content. The collagen peptide composition comprises Galvor E and a collagen peptide. Specifically, the collagen peptide composition further comprises at least one of aronia melanocarpa fruit concentrated solution and EGCG (epigallocatechin gallate). Compared with a single drug, the collagen peptide composition can more effectively recover the content of various collagens in skin cells, and also reduces the expression of senescence genes, so that the skin elasticity is recovered, and the skin elasticity is improved. The senescence is delayed.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a collagen peptide composition and its application in the preparation of a composition for restoring or promoting collagen content. Background Art

[0002] Skin aging is a complex process, among which the reduction of collagen is one of the main causes of skin sagging and wrinkle formation.

[0003] Collagen is an important protein that exists in the connective tissues of the human body, such as skin, bones, muscles and blood vessels. It has strong elasticity and toughness, and can support and protect the structure and function of various parts of the body. As we age, the collagen in the human body will gradually disappear, causing the skin to lose elasticity and firmness, and develop wrinkles and sagging. Supplementing and restoring collagen content can restore skin elasticity and delay aging.

[0004] Although some single drug ingredients have been proven to restore collagen content, the therapeutic effects of these single drugs are limited and it is difficult to achieve multi-faceted effects, which limits the comprehensiveness and advantages of the therapeutic effects. The raw materials of traditional technologies often use effective substances that have been reported, and the lack of development of new raw materials limits the innovation of the composition. In the field of collagen restoration, the development of new drugs is relatively slow, and there is a lack of innovative drug molecules, which limits the diversity and effectiveness of treatment methods.

[0005] At present, the research on collagen restoration is basically based on a single substance, and does not involve the discovery of new drugs and combination formulations. In order to improve the restoration effect of collagen, it is necessary to continuously discover new drugs and / or combination formulations and explore more effective treatment options. Summary of the invention

[0006] Based on this, one embodiment of the present application provides a collagen peptide composition having the effects of restoring collagen content and delaying aging and its use.

[0007] One of the technical solutions includes:

[0008] A collagen peptide composition, comprising GASERVO E and collagen peptide.

[0009] In one embodiment, the collagen peptide composition further comprises at least one of Aronia melanocarpa fruit concentrate and EGCG, wherein the EGCG is epigallocatechin gallate.

[0010] In one embodiment, the collagen peptide composition further comprises an aqueous solvent, and the collagen peptide composition satisfies one or more of the following conditions:

[0011] The molecular weight of the collagen peptide is 500Da~1000Da;

[0012] The concentration of the collagen peptide in the collagen peptide composition is 750 μg / mL-2500 μg / mL;

[0013] The concentration of the Jiashengwo E in the collagen peptide composition is 0.5 μM-3 μM;

[0014] The concentration of the Aronia melanocarpa fruit concentrate in the collagen peptide composition is 100 μg / mL-500 μg / mL;

[0015] The concentration of EGCG in the collagen peptide composition is 1.6 μM-3.2 μM;

[0016] The collagen peptide composition is a pharmaceutical composition;

[0017] The collagen peptide composition is an oral preparation.

[0018] The application of the collagen peptide composition in preparing products for restoring or promoting collagen content.

[0019] In one embodiment, the collagen includes one or more of type I collagen, type III collagen and type XVII collagen.

[0020] In one embodiment, the collagen peptide composition is used in the preparation of anti-aging products.

[0021] In one embodiment, the product is capable of inhibiting P21 expression.

[0022] A product for restoring collagen, the product comprising the collagen peptide composition.

[0023] In one embodiment, the product further comprises pharmaceutically acceptable excipients.

[0024] The method of promoting or restoring collagen content using the collagen peptide composition is not a diagnostic and therapeutic method.

[0025] Compared with the traditional technology, this application has at least the following beneficial effects:

[0026] The inventors of this application have discovered that single drugs such as Jiashengwo E, EGCG and black fruit glandular rib aronia fruit concentrate have potential superiority in collagen restoration. Based on the combination of single drugs, a collagen peptide composition has been developed that can restore the content of multiple collagens in skin cells more effectively than a single drug, and also reduces the expression of aging genes, thereby restoring skin elasticity and delaying aging. This application aims to provide a more effective and comprehensive drug treatment plan for protecting the skin and delaying aging. It has significant advantages in restoring collagen, delaying cell aging, new drug discovery, drug combination strategies, and therapeutic effects, and provides new possibilities and hopes for the treatment of cell aging-related diseases. Furthermore, the aforementioned collagen peptide composition can promote the recovery of collagen content by oral administration, is convenient and easy to use, has high safety, and can achieve sustained and stable effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0028] Figure 1 This is the result of the cytotoxicity test of Gashengwo E in Example 1.

[0029] Figure 2 These are the results of the single drug and the combination in Example 2 on the recovery of the collagen COL3A1 content in the DOXO-stimulated MSF cell collagen loss model.

[0030] Figure 3 The results of the effect of the composition in Example 3 on the expression of P21 in the DOXO-stimulated MSF cell aging model.

[0031] Figure 4 The results of Example 4 are as follows: the single drug and the combination restored the collagen COL1A1 content in the DOXO-stimulated MSF cell collagen loss model.

[0032] Figure 5 The results of Example 4 are as follows: the single drug and the combination restored the collagen COL3A1 content in the DOXO-stimulated MSF cell collagen loss model.

[0033] Figure 6 These are the results of Example 5 in which the single drug and the combination restored the content of collagen COL17A1 in the DOXO-stimulated HACAT cell collagen loss model.

[0034] Figure 7These are the results of the single drug and the combination in Example 6 on the recovery of the collagen COL3A1 content in the DOXO-stimulated MSF cell collagen loss model.

[0035] Figure 8 The results of the effect of the composition of Example 7 on the expression of P21 in the DOXO-stimulated MSF cell aging model.

[0036] Fig. 9 These are the results of Example 8 in which the single drug and the combination restored the collagen COL1A1 content in the DOXO-stimulated MSF cell collagen loss model.

[0037] Fig.10 These are the results of Example 8 in which the single drug and the combination restored the collagen COL3A1 content in the DOXO-stimulated MSF cell collagen loss model.

[0038] Fig.11 The results of Example 9 are as follows: the single drug and the combination restored the collagen COL17A1 content in the DOXO-stimulated HACAT cell collagen loss model.

[0039] In the figure, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. DETAILED DESCRIPTION

[0040] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0042] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0043] In the present application, "two or more" and the like, unless otherwise specified, refer to a quantity greater than 2 or equal to 2.

[0044] In this article, "ICW experiment" refers to In-Cell Western, intracellular protein immunoblotting hybridization experiment.

[0045] In this article, "COL1A1" refers to collagen type I alpha 1 chain.

[0046] Herein, “COL3A1” refers to collagen type III alpha 1 chain.

[0047] Herein, “COL17A1” refers to collagen type XVII alpha 1 chain.

[0048] The process of skin aging is the process of collagen loss, and the connection between aging and collagen is very close. With the increase of age, the synthesis rate of collagen in the human body gradually decreases, while the decomposition rate of collagen gradually accelerates. Therefore, how to effectively slow down or prevent collagen loss, restore collagen content, and thus delay aging is the primary technical problem to be solved by this application.

[0049] Although some drugs have been proven to restore collagen content, the therapeutic effects of these single drugs are limited and cannot better restore collagen content. In order to improve the recovery effect of collagen, it is necessary to continuously explore new drugs and more effective treatment options. Therefore, how to explore new collagen protection drugs and effectively combine them with existing drugs is an important technical problem to be solved in this application.

[0050] Based on this, a variety of collagen loss models and cell aging models were successfully constructed on a variety of cells, providing an important experimental basis for studying the mechanism of collagen loss, cell aging, and screening drugs for restoring collagen and delaying aging. Based on the above models, by screening and compounding a large number of medicinal and edible substances and functional food raw materials, a new substance that has not been reported before was discovered, which has potential superiority in collagen restoration. It was combined and compounded to obtain a combination that is more effective than a single drug in restoring collagen content and delaying aging.

[0051] One embodiment of the present application provides a collagen peptide composition, which includes Gashengwo E and collagen peptide. In a specific example, the concentration ratio of Gashengwo E to collagen peptide in the collagen peptide composition is 1 μM: (250-5000) μg / mL.

[0052] In a specific example, the collagen peptide composition also includes at least one of Aronia nigra fruit concentrate and EGCG. The screened single drug is combined with the new drug discovered, and it is found that the combination is more effective than the single drug in restoring the content of multiple collagens, and also reduces the expression of aging genes, and fights cell aging more comprehensively.

[0053] In a specific example, the collagen peptide composition includes Gashengwo E, collagen peptide and EGCG. Without limitation, the concentration ratio of Gashengwo E, collagen peptide and EGCG is 1 μM: (250-5000) μg / mL: (0.5-6.5) μM.

[0054] In a specific example, the collagen peptide composition includes Jiashengwo E, collagen peptides and black fruit glandular rib aronia fruit concentrate. Without limitation, the concentration ratio of Jiashengwo E, collagen peptides and black fruit glandular rib aronia fruit concentrate is 1μM: (250~5000)μg / mL: (10~1000)μg / mL. Among them, collagen peptides are a type of short-chain polypeptides made of fresh animal tissues rich in collagen (including skin, bones, tendons, tendons, scales, etc.), extracted, hydrolyzed and refined, and composed of multiple amino acids connected by peptide bonds. It has a variety of biological activities and functions. The main function of collagen peptides is to supplement collagen in human skin, bones, joints, muscles and other tissues. With age, the body's own ability to synthesize collagen gradually decreases, leading to problems such as sagging skin, increased wrinkles, and osteoporosis. Therefore, supplementing collagen peptides can help maintain the elasticity and luster of the skin, enhance the toughness and flexibility of bones and joints, and prevent and improve a variety of collagen-related diseases. The absorption rate of collagen peptides is high. Because they are small molecule peptides, they can be directly absorbed and utilized by the human intestine without going through the digestion process. In addition, collagen peptides also have multiple biological activities such as anti-oxidation, anti-inflammatory, and promoting wound healing, which can help the human body maintain a healthy and youthful state.

[0055] Epigallocatechin gallate (EGCG) is the most effective active ingredient in tea polyphenols and belongs to catechins. EGCG has antibacterial, antiviral, antioxidant, anti-atherosclerotic, anti-thrombotic, anti-angiogenic, anti-inflammatory and anti-tumor effects. It is the main active and water-soluble component of green tea and the component with the highest content among catechins, accounting for 9%-13% of the gross weight of green tea. Due to its special stereochemical structure, EGCG has very strong antioxidant activity, which is at least 100 times that of vitamin C and 25 times that of vitamin E. It can protect cells and DNA from damage, which is believed to be related to cancer, heart disease and other major diseases. These effects of EGCG are attributed to their ability to scavenge (antioxidant) oxygen free radicals. EGCG also plays an important role in anti-cancer and cardiovascular diseases. In addition, it is also used as a reversal agent for tumor multidrug resistance, which can improve the sensitivity of cancer cells to chemotherapy and reduce toxicity to the heart.

[0056] Garcinone E is a natural xanthophyll found in the peel of mangosteen. Anthocyanins and polyphenols in mangosteen peel are its main active ingredients. These ingredients can help remove free radicals in the body, reduce the formation of wrinkles and spots, promote skin metabolism, and make the skin smoother and more delicate. In addition, tannic acid and xanthones in mangosteen peel also have antibacterial and anti-inflammatory effects, which can improve skin condition and reduce inflammation of acne and wounds. Its extract Garcinone E can inhibit cancer cell migration and has anti-cancer effects on different human cancer cell lines, including colorectal cancer, breast cancer and hepatocellular carcinoma.

[0057] Aronia aroniae, also known as wild chokeberry, aronia berry, and blackchokeberry, is native to northeastern North America and later introduced to Europe. Aronia aroniae has extraordinary health benefits. The fruit and its extracts have special therapeutic effects on cardiovascular and cerebrovascular diseases such as heart disease and hypertension, and are widely used in the pharmaceutical and functional food industries in Europe and the United States. The anthocyanins, flavonoids (the content of fresh fruit is as high as 0.25%-0.35%) and polyphenols in the fruit are the highest in known plants. The fruit also contains a variety of vitamins and mineral elements. Polyphenols are very important substances for improving the structure and function of capillaries and blood vessels, and also help stimulate and improve the circulatory system. Anthocyanins and flavonoids can maintain the health of the human heart and body. Aronia aroniae is widely used in many fields such as medicine, food and beverages. It can be made into juice, fruit wine, syrup, tea and other products, and can also be used to extract natural pigments and make medicines and cosmetics. Studies have shown that the fruit of Aralia nigra has significant preventive and therapeutic effects on cardiovascular and cerebrovascular diseases, hypertension, hyperglycemia, etc., and has multiple health functions such as antioxidant, anti-inflammatory, and anti-aging.

[0058] In a specific example, the collagen peptide composition constitutes a liquid preparation, and the collagen peptide composition also includes an aqueous solvent, and the aqueous solvent can be selected from water, buffer, ethanol, etc.

[0059] In a specific example, the molecular weight of the collagen peptide is 500Da to 1000Da. Without limitation, the molecular weight of the collagen peptide can be 500Da, 600Da, 700Da, 800Da, 900Da or 1000Da.

[0060] In a specific example, the concentration of the collagen peptide in the collagen peptide composition is 750 μg / mL-2500 μg / mL, and can be optionally 750 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL, 1500 μg / mL, 2000 μg / mL or 2500 μg / mL.

[0061] In a specific example, the concentration of Gashengwo E in the collagen peptide composition is 0.5μM-3μM, which can be 0.5μM, 1μM, 1.5μM, 2μM, 2.5μM or 3μM.

[0062] In a specific example, the concentration of salidroside in the collagen peptide composition is 0.1 μM-0.5 μM, which can be 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM or 0.5 μM.

[0063] In a specific example, the concentration of the Aronia melanocarpa fruit concentrate in the collagen peptide composition is 100 μg / mL-500 μg / mL, which can be 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL or 500 μg / mL.

[0064] In a specific example, the concentration of EGCG in the collagen peptide composition is 1.6 μM-3.2 μM, and can be 1.6 μM, 2.0 μM, 2.4 μM, 2.0 μM, 2.8 μM or 3.2 μM.

[0065] In one specific example, the collagen peptide composition is a pharmaceutical composition.

[0066] In one specific example, the collagen peptide composition is an oral preparation.

[0067] One embodiment of the present application also provides a method for preparing the collagen peptide composition, comprising mixing the components with a solvent to prepare the collagen peptide composition.

[0068] In one specific example, the solvent includes one or more of culture medium and water.

[0069] In a specific example, the collagen peptide composition is used in the preparation of a product for restoring or promoting collagen content.

[0070] In a specific example, the collagen includes one or more of COL1A1, COL3A1 and COL17A1. The present application finds that the components in the composition synergistically restore the collagen content in skin cells.

[0071] One embodiment of the present application also provides the use of the collagen peptide composition in the preparation of anti-aging products.

[0072] In one specific example, the product has the characteristic of inhibiting the expression of P21.

[0073] One embodiment of the present application also provides a product for restoring collagen, which comprises the collagen peptide composition.

[0074] In one specific example, the product is a pharmaceutical collagen peptide.

[0075] In one specific example, the product includes a medication.

[0076] As used herein, "drug" includes any agent, compound, composition or mixture that provides a physiological and / or pharmacological effect in vivo or in vitro, and often provides a beneficial effect. The scope of the physiological and / or pharmacological effect produced by the "drug" in vivo is not particularly limited, and may be a systemic effect or may only produce an effect locally. The activity of the "drug" is not particularly limited, and may be an active substance that can interact with other substances, or an inert substance that does not interact.

[0077] In a specific example, the product further includes a pharmaceutically acceptable excipient.

[0078] In a specific example, pharmaceutically acceptable excipients include, but are not limited to, one or more of carriers, solvents, diluents, and excipients, and can be prepared into different dosage forms according to different requirements.

[0079] In one specific example, the product is liquid collagen peptides.

[0080] In one specific example, the product is oral collagen peptides.

[0081] One embodiment of the present application also provides a method for promoting or restoring collagen content using the collagen peptide composition.

[0082] In some specific examples, a method for promoting or restoring collagen content is provided, comprising administering an effective amount of the aforementioned collagen peptide composition to a subject.

[0083] As used herein, "effective amount" refers to the amount required to induce a biological or medical response in an individual, such as the amount of an agent that brings about a physiological and / or pharmacologically positive effect to an individual. "Physiological and / or pharmacologically positive effect" includes but is not limited to improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease, etc.

[0084] In one specific example, the method of promoting or restoring collagen content using the collagen peptide composition is not a diagnostic or therapeutic method.

[0085] In a specific example, the method of promoting or restoring collagen content using the collagen peptide composition is a diagnostic and / or therapeutic method, which comprises administering a therapeutically effective amount of the aforementioned collagen peptide composition to a subject.

[0086] "Subject" refers to an animal, preferably a mammal, and further can be a human. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs, cows, sheep, horses and humans.

[0087] In some embodiments, the subject is a human or a mouse. In some embodiments, the subject is a human.

[0088] As used herein, "therapeutically effective amount" refers to the amount required to induce a biological or medical response in an individual, such as the amount of an agent that brings about a pharmacologically positive effect in an individual. "Pharmacologically positive effect" includes but is not limited to improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease, etc.

[0089] In some specific examples, the collagen peptide composition may be administered orally to a subject.

[0090] One embodiment of the present application provides the use of collagen peptides, Gastroenterol E, EGCG or Aronia melanocarpa fruit concentrate in the preparation of products for restoring or promoting collagen content.

[0091] The new drug discovered, Jiashengwo E, has unique advantages in restoring collagen in skin cells and delaying aging. It can be used to restore collagen, delay cell aging and prevent related diseases.

[0092] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0093] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0094] The main reagents involved in the following examples include:

[0095] DOXO (Doxorubicin, Adriamycin) was purchased from MCE, CAS No. 23214-92-8, with the chemical structure formula .

[0096] Epigallocatechin gallate (EGCG) was purchased from Zhejiang Shengshi Biotechnology Co., Ltd. and has the chemical formula .

[0097] Garcinone E, purchased from MCE, CAS No. 112649-21-5, chemical structure formula: .

[0098] Aronia Juice Concentrate was purchased from Micro (Microstructure Sp. z oo), item number R695335. The solid solution content of Aronia Juice Concentrate was ≥15%, and its main components included anthocyanins, proanthocyanidins and vitamins.

[0099] CR-1-31-B, purchased from MCE, CAS No. 1352914-52-3, chemical structure is .

[0100] Salidroside, purchased from MCE, CAS No. 10338-51-9, chemical structure formula: .

[0101] Collagen peptides were purchased from Hainan Huayan Collagen Technology Co., Ltd., batch number JDA240618, with a molecular weight of 899 Da.

[0102] Example 1: Cytotoxicity test of Gastrointestinal E

[0103] 1. Cell inoculation: When MSF cells are cultured and grown to the appropriate generation and the confluence reaches 90%, the original culture medium is aspirated, washed twice with PBS, digested with 0.25% trypsin, and left in a cell culture incubator for 3 minutes until the cells shrink and become round and gradually fall off from the culture flask. Then, 2 times the volume of trypsin-containing complete culture medium is added to stop digestion, and the cell suspension is collected, centrifuged, resuspended with an appropriate amount of complete culture medium, and counted. After counting, the cells are inoculated into a 96-well plate at a density of 1W cells / well and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0104] 2. Cell dosing: When the cells reach the logarithmic growth phase, add different concentrations of Gashengwo E to treat the cells.

[0105] 3. Observe the cells the next day, discard the supernatant and add 110 μL / well of CELLTITER reagent. After incubation for 30 minutes, transfer to a white 96-well plate and detect on an ELISA reader.

[0106] The results are as follows Figure 1 The concentration of GaShengWo E used in the following examples is 0.5 μM.

[0107] Example 2 Effect of the composition on the recovery of collagen content in the collagen loss model of mouse skin fibroblasts (MSF) stimulated by DOXO

[0108] The mechanism by which DOXO induces cell aging mainly includes DNA damage and oxidative stress. After DOXO enters the cell, it will be embedded in the DNA, causing DNA damage, and then activating the DNA damage response mechanism in the cell. At the same time, DOXO will also induce oxidative stress, leading to an increase in the level of reactive oxygen species (ROS) in the cell, further promoting cell aging and leading to a decrease in the collagen content in the cell.

[0109] 1. Cell inoculation: When MSF cells are cultured and grown to the appropriate generation and the confluence reaches 90%, the original culture medium is aspirated, washed twice with PBS, digested with 0.25% trypsin, and left to stand in a cell culture incubator for 3 min until the cells shrink and become round and gradually fall off from the culture flask. Then, 2 times the volume of trypsin-containing complete culture medium is added to stop digestion, and the cell suspension is collected, centrifuged, resuspended with an appropriate amount of complete culture medium, and counted. After counting, the cells are inoculated into a black 96-well plate at a density of 1.1W cells / well and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0110] 2. Cell drug treatment: When the cell growth reaches the logarithmic growth phase, drug addition and modeling treatment are performed. Specifically, the following three drugs are involved: collagen peptide (750μg / mL), EGCG (3.2μM), and Garcinone E (0.5μM). The experiment set up a background well group, a blank group (Control), a 1μM DOXO modeling group, and a drug addition group after modeling (single drug: collagen peptide 750μg / mL group, EGCG3.2μM group, Garcinone E 0.5μM group; combination: collagen peptide 750μg / mL+EGCG 3.2μM+Garcinone E 0.5μM group). 100 μL of culture medium containing drugs of corresponding concentrations was added to each well of the modeling group and the drug-addition group after modeling, and 100 μL of DMEM complete culture medium was added to each well of the background well group and the blank group. Three replicate wells were set up in each group. After drug administration, the black 96-well plate was placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 24 hours.

[0111] 3. ICW experiment: After aspirating the complete medium in the MSF cells cultured overnight, use a multi-channel pipette to add 150μL of fixative solution to each well and let it stand at room temperature for 60min. Then aspirate the fixative solution, add 150μL of permeabilization solution (0.1% Triton X-100 solution) to each well, and incubate it on a shaker (about 50 revolutions) at room temperature for 20min. After the permeabilization, aspirate the permeabilization solution, add 150μL of blocking buffer (purchased from Gene Company) to each well, and incubate it on a shaker (about 50 revolutions) at room temperature for 1.5h. Then aspirate the blocking solution in each well, add 50μL of primary antibody working solution (anti-COL3A1 primary antibody, 1:500, 1% TBS BSA configuration, background well group plus 1% TBS BSA), and incubate it on a shaker at 4℃ overnight. The next day, the plates were washed three times with TBST, 5 min each time, and then incubated with a mixture of secondary antibody (anti-rabbit 800) and Cell Tag (purchased from Genentech) (1:500, TBST configuration, only secondary antibody was added to the background well group). After incubation at room temperature in the dark for 1 h, the plates were washed three times with TBST, 5 min each time, and the liquid was discarded. The Odyssey fluorescence imaging instrument was used to detect and analyze the data.

[0112] The results are as follows Figure 2 As shown in the figure, compared with the blank control group, the COL3A1 content in the cells after DOXO stimulation was significantly reduced, indicating that the DOXO-stimulated MSF cell collagen loss model was successfully established. The collagen peptide + EGCG + Jiashengwo E combination was used for post-injury repair. The results showed that compared with the DOXO modeling group, the COL3A1 expression was significantly increased, and was significantly better than the single drug administration in each group.

[0113] Example 3 Effect of the composition on P21 expression in the DOXO-stimulated mouse skin fibroblast (MSF) aging model

[0114] 1. Cell inoculation: When MSF cells are cultured and grown to the appropriate generation and the confluence reaches 90%, the original culture medium is aspirated, washed twice with PBS, digested with 0.25% trypsin, and left in a cell culture incubator for 3 minutes until the cells shrink and become round and gradually fall off from the culture flask. Then, 2 times the volume of trypsin-containing complete culture medium is added to stop digestion, and the cell suspension is collected, centrifuged, resuspended with an appropriate amount of complete culture medium, and counted. After counting, the cells are inoculated into a 6 cm dish at a density of 75W cells / dish, and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0115] 2. Cell drug treatment: When the cell growth reaches the logarithmic growth phase, drug addition modeling treatment is performed. Specifically, the following three drugs are involved: collagen peptide (750μg / mL), EGCG (3.2μM), and Garcinone E (0.5μM). The experiment set up a blank group (Control), a 1μM DOXO modeling group, and a post-modeling drug addition (combination) group (combination compound: collagen peptide 750μg / mL + EGCG3.2μM + Garcinone E (Garcinone E) 0.5μM group). 5mL of culture solution containing drugs of corresponding concentrations was added to each well of the modeling group and the post-modeling drug addition group, and 5mL of DMEM complete culture medium was added to each well of the blank group; after the drug administration was completed, the 6cm dish was placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 24h.

[0116] 3.qPCR experiment: The next day, scrape the cells from the culture dish with a cell spatula and collect the culture medium into a centrifuge tube. Centrifuge at 3000rpm for 5 min at room temperature and discard the supernatant. Use an RNA extraction kit (purchased from Novozymes) to extract RNA and then use a reverse kit (purchased from TIANGEN) to reverse RNA into cDNA. Then use a qPCR kit (purchased from Novozymes) to perform qPCR experiments.

[0117] The results are as follows Figure 3 As shown in the figure, compared with the blank control group, the expression of P21 in cells after DOXO stimulation was significantly increased, indicating that the DOXO-stimulated MSF cell aging model was successfully established. The collagen peptide + EGCG + Jiashengwo E combination was used for post-injury repair, and the results showed that compared with the DOXO modeling group, the expression of P21 was significantly reduced, proving that the combination has an anti-aging effect.

[0118] Example 4 Effect of the composition on the recovery of collagen content in the collagen loss model of mouse skin fibroblasts (MSF) stimulated by DOXO

[0119] 1. Cell inoculation: When MSF cells are cultured and grown to the appropriate generation and the confluence reaches 90%, the original culture medium is aspirated, washed twice with PBS, digested with 0.25% trypsin, and left in a cell culture incubator for 3 minutes until the cells shrink and become round and gradually fall off from the culture flask. Then, 2 times the volume of trypsin-containing complete culture medium is added to stop digestion, and the cell suspension is collected, centrifuged, resuspended with an appropriate amount of complete culture medium, and counted. After counting, the cells are inoculated into a 6 cm dish at a density of 75W cells / dish, and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0120] 2. Cell drug treatment: When the cell growth reaches the logarithmic growth phase, drug addition and modeling treatment are carried out. Specifically, the following five drugs are involved: collagen peptide (750μg / mL), EGCG (3.2μM), Gashengwo E (0.5μM), and two drugs with similar structures to Gashengwo E, Salidroside (0.5μM) and CR-1-31-B (0.5μM). The experiment was set up with blank group (Control), 1μM DOXO modeling group, and drug-adding group after modeling (single drug: collagen peptide 750μg / mL group, EGCG 3.2μM group, Garcinone E 0.5μM group; combination: collagen peptide 750μg / mL + EGCG 3.2μM group, collagen peptide 750μg / mL + Garcinone E 0.5μM group, EGCG 3.2μM + Garcinone E 0.5μM group, collagen peptide 750μg / mL + EGCG 3.2μM + Salidroside 0.5μM group, collagen peptide 750μg / mL + EGCG 3.2μM + CR-1-31-B 0.5μM group, collagen peptide 750μg / mL + EGCG 3.2μM+Garcinone E 0.5μM group), 5mL of culture medium containing corresponding concentration of drugs was added to each well of the modeling and sample groups, and 5mL of DMEM complete culture medium was added to each well of the blank group; after administration, the 6cm dish was placed in a constant temperature incubator at 37℃, 5% CO2 and saturated humidity for 24h.

[0121] 3. WB experiment: The next day, scrape the cells from the culture dish with a cell spatula and collect the culture medium into a centrifuge tube. Centrifuge at 3000rpm for 5 min at room temperature and discard the supernatant. Use RIPA to lyse the precipitate, ultrasonically disrupt it for 1 min after 10 min, and centrifuge to obtain the supernatant to obtain the protein sample. After measuring the protein concentration with a BCA kit (purchased from Thermo), perform WB experiment.

[0122] The results are as follows Figure 4-Figure 5As shown in the figure, compared with the blank control group, the content of COL1A1 and COL3A1 in cells after DOXO stimulation was significantly reduced, indicating that the DOXO-stimulated MSF cell collagen loss model was successfully established. The collagen peptide + EGCG + Jiashengwo E combination was used for post-injury repair. The results showed that compared with the DOXO modeling group, the expression of COL1A1 and COL3A1 was significantly increased, which was better than the single administration of each group, and better than the combination of two drugs with similar structures to Jiashengwo E.

[0123] Example 5 Effect of the composition on the recovery of collagen content in the DOXO-stimulated human keratinocyte (HACAT) collagen loss model

[0124] 1. Cell inoculation: When HACAT cells are cultured and grown to the appropriate generation and the confluence reaches 90%, the original culture medium is aspirated, washed twice with PBS, digested with 0.25% trypsin, and left in a cell culture incubator for 7 minutes until the cells shrink and become round and gradually fall off from the culture flask. Then, 2 times the volume of trypsin-containing complete culture medium is added to stop digestion, and the cell suspension is collected, centrifuged, resuspended with an appropriate amount of complete culture medium, and counted. After counting, the cells are inoculated into a 6 cm dish at a density of 250 W cells / dish, and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0125] 2. Cell drug treatment: When the cell growth reaches the logarithmic growth phase, drug addition modeling treatment is performed. Specifically, the following five drugs are involved: collagen peptide (750μg / mL), EGCG (3.2μM), Gashengwo E (0.5μM), and two drugs with similar structures to Gashengwo E, Salidroside (0.5μM) and CR-1-31-B (0.5μM). The experiment set up a blank group (Control), a 3μM DOXO modeling group, and a post-modeling drug addition group (see Example 4). 5mL of culture solution containing the corresponding concentration of drug was added to each well of the modeling group and the post-modeling drug addition group, and 5mL of DMEM complete culture medium was added to each well of the blank group; after the drug administration was completed, the 6cm dish was placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 24h.

[0126] 3. WB experiment: The next day, scrape the cells from the culture dish with a cell spatula and collect the culture medium into a centrifuge tube. Centrifuge at 3000rpm for 5 min at room temperature and discard the supernatant. Use RIPA to lyse the precipitate, ultrasonically disrupt it for 1 min after 10 min, and centrifuge to obtain the supernatant to obtain the protein sample. After measuring the protein concentration with a BCA kit (purchased from Thermo), perform WB experiment.

[0127] The results are as follows Figure 6As shown, compared with the blank control group, the COL17A1 content in cells after DOXO stimulation was significantly reduced, indicating that the DOXO-stimulated HACAT cell collagen loss model was successfully established. The collagen peptide + EGCG + Jiashengwo E combination was used for post-injury repair. The results showed that compared with the DOXO modeling group, the expression of COL17A1 was significantly increased, which was better than the single administration of each group, and better than the combination of two drugs with similar structures to Jiashengwo E.

[0128] Example 6 Effect of the composition on the recovery of collagen content in the collagen loss model of mouse skin fibroblasts (MSF) stimulated by DOXO

[0129] 1. Cell inoculation: When MSF cells are cultured and grown to the appropriate generation and the confluence reaches 90%, the original culture medium is aspirated, washed twice with PBS, digested with 0.25% trypsin, and left in a cell culture incubator for 3 minutes until the cells shrink and become round and gradually fall off from the culture flask. Then, 2 times the volume of trypsin-containing complete culture medium is added to stop digestion, and the cell suspension is collected, centrifuged, resuspended with an appropriate amount of complete culture medium, and counted. After counting, the cells are inoculated into a black 96-well plate at a density of 1.1W cells / well and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0130] 2. Cell drug treatment: When the cell growth reaches the logarithmic growth phase, drug addition and modeling treatment are performed. Specifically, the following three drugs are involved: collagen peptide (750μg / mL), black fruit glandular rib aronia fruit concentrate (100μg / mL), Garcinone E (0.5μM). The experiment set up a background well group, a blank group (Control), a 1μM DOXO modeling group, and a drug addition group after modeling (single drug: collagen peptide 750μg / mL group, black fruit glandular rib aronia fruit concentrate 100μg / mL group, Garcinone E 0.5μM group; combination: collagen peptide 750μg / mL + black fruit glandular rib aronia fruit concentrate 100μg / mL + Garcinone E 0.5μM group). 100 μL of culture medium containing drugs of corresponding concentrations was added to each well of the modeling group and the drug-addition group after modeling, and 100 μL of DMEM complete culture medium was added to each well of the background well group and the blank group. Three replicate wells were set up in each group. After drug administration, the black 96-well plate was placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 24 hours.

[0131] 3. ICW experiment: After aspirating the complete medium in the MSF cells cultured overnight, use a multi-channel pipette to add 150μL of fixative solution to each well and let it stand at room temperature for 60min. Then aspirate the fixative solution, add 150μL of permeabilization solution (0.1% Triton X-100 solution) to each well, and incubate it on a shaker (about 50 revolutions) at room temperature for 20min. After the permeabilization, aspirate the permeabilization solution, add 150μL of blocking buffer (purchased from Gene Company) to each well, and incubate it on a shaker (about 50 revolutions) at room temperature for 1.5h. Then aspirate the blocking solution in each well, add 50μL of primary antibody working solution (anti-COL3A1 primary antibody, 1:500, 1% TBS BSA configuration, background well group plus 1% TBS BSA), and incubate it on a shaker at 4℃ overnight. The next day, the plates were washed three times with TBST, 5 min each time, and then incubated with a mixture of secondary antibody (anti-rabbit 800) and Cell Tag (purchased from Genentech) (1:500, TBST configuration, only secondary antibody was added to the background well group). After incubation at room temperature in the dark for 1 h, the plates were washed three times with TBST, 5 min each time, and the liquid was discarded. The Odyssey fluorescence imaging instrument was used to detect and analyze the data.

[0132] The results are as follows Figure 7 As shown in the figure, compared with the blank control group, the COL3A1 content in the cells after DOXO stimulation was significantly reduced, indicating that the DOXO-stimulated MSF cell collagen loss model was successfully established. The collagen peptide + Aronia melanocarpa fruit + Jiashengwo E composition was used for post-injury repair. The results showed that compared with the DOXO modeling group, the COL3A1 expression was significantly increased, and was significantly better than the single-dose group.

[0133] Example 7 Effect of the composition on P21 expression in the DOXO-stimulated mouse skin fibroblast (MSF) aging model

[0134] 1. Cell inoculation: When MSF cells are cultured and grown to the appropriate generation and the confluence reaches 90%, the original culture medium is aspirated, washed twice with PBS, digested with 0.25% trypsin, and left in a cell culture incubator for 3 minutes until the cells shrink and become round and gradually fall off from the culture flask. Then, 2 times the volume of trypsin-containing complete culture medium is added to stop digestion, and the cell suspension is collected, centrifuged, resuspended with an appropriate amount of complete culture medium, and counted. After counting, the cells are inoculated into a 6 cm dish at a density of 75W cells / dish, and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0135] 2. Cell drug treatment: When the cell growth reaches the logarithmic growth phase, drug addition and modeling treatment are performed. Specifically, the following three drugs are involved: collagen peptide (750μg / mL), black fruit glandular rib aronia fruit concentrate (100μg / mL), and Gashengwo E (0.5μM). The experiment set up a blank group (Control), a 1μM DOXO modeling group, and a post-modeling drug addition (combination) group. 5mL of culture solution containing drugs of corresponding concentrations was added to each well of the modeling group and the post-modeling drug addition group, and 5mL of DMEM complete culture medium was added to each well of the blank group; after the drug administration was completed, the 6cm dish was placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 24h.

[0136] 3.qPCR experiment: The next day, scrape the cells from the culture dish with a cell spatula and collect the culture medium into a centrifuge tube. Centrifuge at 3000rpm for 5 min at room temperature and discard the supernatant. Use an RNA extraction kit (purchased from Novozymes) to extract RNA and then use a reverse kit (purchased from TIANGEN) to reverse RNA into cDNA. Then use a qPCR kit (purchased from Novozymes) to perform qPCR experiments.

[0137] The results are as follows Figure 8 As shown in the figure, compared with the blank control group, the expression of P21 in cells after DOXO stimulation was significantly increased, indicating that the DOXO-stimulated MSF cell aging model was successfully established. The collagen peptide + Aronia nigra fruit + Jiashengwo E composition was used for post-injury repair. The results showed that compared with the DOXO modeling group, the expression of P21 was significantly reduced, proving that the composition has an anti-aging effect.

[0138] Example 8 Effect of the composition on the recovery of collagen content in the collagen loss model of mouse skin fibroblasts (MSF) stimulated by DOXO

[0139] 1. Cell inoculation: When MSF cells are cultured and grown to the appropriate generation and the confluence reaches 90%, the original culture medium is aspirated, washed twice with PBS, digested with 0.25% trypsin, and left in a cell culture incubator for 3 minutes until the cells shrink and become round and gradually fall off from the culture flask. Then, 2 times the volume of trypsin-containing complete culture medium is added to stop digestion, and the cell suspension is collected, centrifuged, resuspended with an appropriate amount of complete culture medium, and counted. After counting, the cells are inoculated into a 6 cm dish at a density of 75W cells / dish, and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0140] 2. Cell drug treatment: When the cell growth reaches the logarithmic growth phase, drug addition and modeling treatment are carried out. Specifically, the following five drugs are involved: collagen peptide (750μg / mL), black fruit glandular rib aronia fruit concentrate (100μg / mL), Garsenwo E (0.5μM), and two drugs with similar structures to Garsenwo E, Salidroside (0.5μM) and CR-1-31-B (0.5μM). The experiment was set up with blank group (Control), 1μM DOXO modeling group, and drug-adding group after modeling (single drug: collagen peptide 750μg / mL group, black fruit glandular rib aronia fruit concentrate 100μg / mL group, Garcinone E 0.5μM group; combination: collagen peptide 750μg / mL + black fruit glandular rib aronia fruit concentrate 100μg / mL group, collagen peptide 750μg / mL + Garcinone E 0.5μM group, black fruit glandular rib aronia fruit concentrate 100μg / mL + Garcinone E 0.5μM group, collagen peptide 750μg / mL + black fruit glandular rib aronia fruit concentrate 100μg / mL + Salidroside 0.5μM group, collagen peptide 750μg / mL + Aronia nigra fruit concentrate 100μg / mL + CR-1-31-B 0.5μM group, collagen peptide 750μg / mL + Aronia nigra fruit concentrate 100μg / mL + Garcinone E 0.5μM group). 5mL of culture medium containing corresponding concentrations of drugs was added to each well of the modeling group and the drug-added group after modeling, and 5mL of DMEM complete medium was added to each well of the blank group; after drug administration, the 6cm dish was placed in a constant temperature incubator at 37℃, 5% CO2, and saturated humidity for 24h.

[0141] 3. WB experiment: The next day, scrape the cells from the culture dish with a cell spatula and collect the culture medium into a centrifuge tube. Centrifuge at 3000rpm for 5 min at room temperature and discard the supernatant. Use RIPA to lyse the precipitate, ultrasonically disrupt it for 1 min after 10 min, and centrifuge to obtain the supernatant to obtain the protein sample. After measuring the protein concentration with a BCA kit (purchased from Thermo), perform WB experiment.

[0142] The results are as follows Figure 9-10 As shown in the figure, compared with the blank control group, the content of COL1A1 and COL3A1 in cells after DOXO stimulation was significantly reduced, indicating that the DOXO-stimulated MSF cell collagen loss model was successfully established. The collagen peptide + Aronia melanocarpa fruit + Garsengwo E composition was used for post-injury repair. The results showed that compared with the DOXO modeling group, the expression of COL1A1 and COL3A1 was significantly increased, which was better than the single administration of each group, and better than the combination of two drugs with similar structures to Garsengwo E.

[0143] Example 9 Effect of the composition on the recovery of collagen content in the DOXO-stimulated human keratinocyte (HACAT) collagen loss model

[0144] 1. Cell inoculation: When HACAT cells are cultured and grown to the appropriate generation and the confluence reaches 90%, the original culture medium is aspirated, washed twice with PBS, digested with 0.25% trypsin, and left in a cell culture incubator for 7 minutes until the cells shrink and become round and gradually fall off from the culture flask. Then, 2 times the volume of trypsin-containing complete culture medium is added to stop digestion, and the cell suspension is collected, centrifuged, resuspended with an appropriate amount of complete culture medium, and counted. After counting, the cells are inoculated into a 6 cm dish at a density of 250 W cells / dish, and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity.

[0145] 2. Cell drug treatment: When the cell growth reaches the logarithmic growth phase, drug addition modeling treatment is performed. Specifically, the following five drugs are involved: collagen peptide (750μg / mL); black fruit glandular rib aronia fruit concentrate (100μg / mL), Gashengwo E (0.5μM), and two drugs with similar structures to Gashengwo E, Salidroside (0.5μM) and CR-1-31-B (0.5μM). The experiment set up a blank group (Control), a 3μM DOXO modeling group, and a post-modeling drug addition group (see Example 8). 5mL of culture solution containing the corresponding concentration of drug was added to each well of the modeling group and the post-modeling drug addition group, and 5mL of DMEM complete culture medium was added to each well of the blank group; after the drug administration was completed, the 6cm dish was placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 24h.

[0146] 3. WB experiment: The next day, scrape the cells from the culture dish with a cell spatula and collect the culture medium into a centrifuge tube. Centrifuge at 3000rpm for 5 min at room temperature and discard the supernatant. Use RIPA to lyse the precipitate, ultrasonically disrupt it for 1 min after 10 min, and centrifuge to obtain the supernatant to obtain the protein sample. After measuring the protein concentration with a BCA kit (purchased from Thermo), perform WB experiment.

[0147] The results are as follows Fig.11 As shown in the figure, compared with the blank control group, the COL17A1 content in the cells after DOXO stimulation was significantly reduced, indicating that the DOXO-stimulated HACAT cell collagen loss model was successfully established. The collagen peptide + black fruit glandular rib aronia fruit concentrate + Jiashengwo E composition was used for post-injury repair. The results showed that compared with the DOXO modeling group, the expression of COL17A1 was significantly increased, which was better than the single drug administration of each group, and better than the combination of two drugs with similar structures to Jiashengwo E.

[0148] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0149] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A collagen peptide composition, characterized in that: The collagen peptide composition includes Jiashengwo E and collagen peptide.

2. The collagen peptide composition according to claim 1, characterized in that The collagen peptide composition further comprises at least one of aronia melanocarpa fruit concentrate and EGCG, wherein the EGCG is epigallocatechin gallate.

3. The collagen peptide composition according to claim 2, characterized in that The collagen peptide composition constitutes a liquid preparation, and the collagen peptide composition further comprises an aqueous solvent. The collagen peptide composition satisfies one or more of the following conditions: The molecular weight of the collagen peptide is 500Da~1000Da; The concentration of the collagen peptide in the collagen peptide composition is 750 μg / mL-2500 μg / mL; The concentration of the Gastrointestinal tract E in the collagen peptide composition is 0.5 μM-3 μM; The concentration of the Aronia melanocarpa fruit concentrate in the collagen peptide composition is 100 μg / mL-500 μg / mL; The concentration of EGCG in the collagen peptide composition is 1.6 μM-3.2 μM; The collagen peptide composition is a pharmaceutical composition; The collagen peptide composition is an oral preparation.

4. Use of the collagen peptide composition according to any one of claims 1 to 3 in the preparation of a product for restoring or promoting collagen content.

5. The use according to claim 4, characterized in that: The collagen includes one or more of type I collagen, type III collagen and type XVII collagen.

6. Use of the collagen peptide composition according to any one of claims 1 to 3 in the preparation of anti-aging products.

7. The use according to claim 6, characterized in that: The product is capable of inhibiting P21 expression.

8. A product for restoring collagen, characterized in that: The product comprises the collagen peptide composition according to any one of claims 1 to 3.

9. The product according to claim 8, characterized in that The product also includes pharmaceutically acceptable excipients.

10. A method for promoting or restoring collagen content using the collagen peptide composition according to any one of claims 1 to 3, which is not a diagnostic or therapeutic method.

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