Collagen peptide composition and its application in restoring or promoting collagen content

Abstract

This application relates to a collagen peptide composition and its use in restoring or promoting collagen content. The collagen peptide composition includes GacE and collagen peptides. Specifically, the collagen peptide composition further includes at least one of Aronia melanocarpa fruit concentrate and EGCG (epigallocatechin gallate). The collagen peptide composition can more effectively restore the content of various collagens in skin cells than a single drug, and also reduces the expression of aging genes, thereby restoring skin elasticity and delaying aging.

Description

Technical Field

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

[0002] Skin aging is a complex process, in which the reduction of collagen is one of the main reasons for skin relaxation 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 structures and functions of various parts of the body. As people age, the collagen in the human body gradually loses, resulting in the skin losing elasticity and firmness, and problems such as wrinkles and relaxation appear. By supplementing and restoring the collagen content, the purpose of restoring skin elasticity and delaying aging can be achieved.

[0004] Although some single drug components have been proven to be able to restore collagen content, the therapeutic effects of these single drugs are limited and it is difficult to achieve multiple effects, resulting in limitations in the comprehensiveness and advantages of the therapeutic effects. The raw materials of traditional technologies often use effective substances that have been reported, lacking the development of new raw materials, which limits the innovation of the composition. In the field of collagen restoration, the research and development of new drugs is relatively slow, lacking innovative drug molecules, which limits the diversity and effectiveness of treatment methods.

[0005] Currently, the research on restoring collagen is basically based on single substances and does not involve the excavation and combination of new drugs. In order to improve the effect of collagen restoration, it is necessary to continuously excavate new drugs and / or combinations and explore more effective treatment plans. Summary of the Invention

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

[0007] One of the technical solutions includes:

[0008] A collagen peptide composition, the collagen peptide composition includes Gaxenol E and collagen peptide.

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

[0010] In one embodiment, the collagen peptide composition further includes 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 500 Da to 1000 Da;

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

[0013] In the collagen peptide composition, the concentration of Gaxianwo E is 0.5 μM - 3 μM;

[0014] In the collagen peptide composition, the concentration of the concentrated Sorbus aucuparia fruit juice is 100 μg / mL - 500 μg / mL;

[0015] In the collagen peptide composition, the concentration of EGCG 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] Use of the collagen peptide composition in the preparation of a product 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, use of the collagen peptide composition in the preparation of an anti-aging product.

[0021] In one embodiment, the product can inhibit the expression of P21.

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

[0023] In one embodiment, the product further includes a pharmaceutically acceptable excipient.

[0024] A method for promoting or restoring collagen content using the collagen peptide composition, the method is not a diagnostic and treatment method.

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

[0026] The inventors of the present application have found that single drugs such as gamma-oryzanol E, EGCG, and aronia melanocarpa fruit concentrate have potential advantages in collagen restoration. Based on the combination and compounding of single drugs, a collagen peptide composition has been developed that can more effectively restore the content of various collagens in skin cells than single drugs, and also reduces the expression of aging genes, thereby restoring skin elasticity and delaying aging. The present application aims to provide a more effective and comprehensive drug treatment plan for protecting the skin and delaying aging. It has significant advantages in aspects such as restoring collagen, delaying cell aging, new drug discovery, drug combination strategies, and treatment effects, providing new possibilities and hopes for the treatment of cell aging-related diseases. Further, the aforementioned collagen peptide composition can promote the restoration of collagen content through oral administration, is convenient to use, has high safety, and can achieve a continuous and stable effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0028] Figure 1 It is the cytotoxicity detection result of gamma-oryzanol E in Example 1.

[0029] Figure 2 It is the result of the restoration of the content of collagen COL3A1 in the collagen loss model of DOXO-stimulated MSF cells by single drugs and the composition in Example 2.

[0030] Figure 3 It is the result of the expression of P21 in the aging model of DOXO-stimulated MSF cells by the composition in Example 3.

[0031] Figure 4 It is the result of the restoration of the content of collagen COL1A1 in the collagen loss model of DOXO-stimulated MSF cells by single drugs and the composition in Example 4.

[0032] Figure 5 It is the result of the restoration of the content of collagen COL3A1 in the collagen loss model of DOXO-stimulated MSF cells by single drugs and the composition in Example 4.

[0033] Figure 6 It is the result of the restoration of the content of collagen COL17A1 in the collagen loss model of DOXO-stimulated HACAT cells by single drugs and the composition in Example 5.

[0034] Figure 7Results of the recovery of collagen COL3A1 content in the single drug and composition in Example 6 in the DOXO-stimulated MSF cell collagen loss model.

[0035] Figure 8 Results of the expression of P21 in the composition in Example 7 in the DOXO-stimulated MSF cell senescence model.

[0036] Figure 9 Results of the recovery of collagen COL1A1 content in the single drug and composition in Example 8 in the DOXO-stimulated MSF cell collagen loss model.

[0037] Figure 10 Results of the recovery of collagen COL3A1 content in the single drug and composition in Example 8 in the DOXO-stimulated MSF cell collagen loss model.

[0038] Figure 11 Results of the recovery of collagen COL17A1 content in the single drug and composition in Example 9 in the DOXO-stimulated HACAT cell collagen loss model.

[0039] Among them, * in the figure represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. Detailed implementation manners

[0040] To make the above objects, features, and advantages of the present application more obvious and understandable, the following provides a detailed description of the specific implementation manners of the present application. Many specific details are set forth in the following description to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein. Those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, 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 commonly understood by those of ordinary skill in the technical field 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] The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0043] In this application, "two or more", etc., unless otherwise specified, means greater than 2 or equal to 2 in quantity.

[0044] In this article, the "ICW experiment" is In-Cell Western, an intracellular protein immunoblot hybridization experiment.

[0045] In this text, "COL1A1" refers to Collagen Type I Alpha 1 Chain.

[0046] In this text, "COL3A1" refers to Collagen Type III Alpha 1 Chain.

[0047] In this text, "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 increases. 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 in this application.

[0049] Although some drugs have been proven to be able 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 regimens. Therefore, how to explore new collagen-protecting drugs and effectively combine them with existing drugs is an important technical problem to be solved in this application.

[0050] Based on this, various collagen loss models and cell aging models have been successfully constructed on multiple cells, providing an important experimental basis for studying the mechanisms 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 substances homologous to medicine and food and functional food raw materials, a new substance that has not been reported before is discovered, which has potential superiority in collagen restoration. After combining and compounding it, a composition that can more effectively restore collagen content and delay aging than single drugs is obtained.

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

[0052] In a specific example, the collagen peptide composition further comprises at least one of aronia berry concentrate and EGCG. By combining and compounding the selected single drug with the newly discovered drug, it is found that the composition can more effectively restore the contents of various collagens, reduce the expression of senescence genes, and more comprehensively combat cell senescence.

[0053] In a specific example, the collagen peptide composition comprises Galsenwo E, collagen peptide, and EGCG. Without limitation, the concentration ratio of Galsenwo 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 comprises Galsenwo E, collagen peptide, and aronia berry concentrate. Without limitation, the concentration ratio of Galsenwo E, collagen peptide, and aronia berry concentrate is 1 μM:(250 - 5000) μg / mL:(10 - 1000) μg / mL. Among them, collagen peptide is a kind of short-chain polypeptide formed by multiple amino acids connected by peptide bonds, which is produced by extracting, hydrolyzing, and refining with fresh animal tissues rich in collagen (including skin, bone, tendon, ligament, scale, etc.) as raw materials, and has various biological activities and functions. The main function of collagen peptide is to supplement collagen in tissues such as human skin, bone, joint, and muscle. With the increase of age, the ability of the human body to synthesize collagen by itself gradually decreases, resulting in problems such as skin relaxation, increased wrinkles, and osteoporosis. Therefore, supplementing collagen peptide can help maintain the elasticity and luster of the skin, enhance the toughness and flexibility of bones and joints, and prevent and improve various collagen-related diseases. The absorption and utilization rate of collagen peptide is relatively high because it belongs to small molecular peptide segments and can be directly absorbed and utilized by the human intestinal tract without going through the digestion process. In addition, collagen peptide also has various biological activities such as antioxidant, 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, antithrombotic, 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 in catechins, accounting for 9%-13% of the gross weight of green tea. Due to its special stereochemical structure, EGCG has very strong antioxidant activity, with antioxidant activity at least more than 100 times that of vitamin C and 25 times that of vitamin E, and 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 oxygen free radicals (antioxidation). EGCG also plays an important role in anti-cancer and cardiovascular diseases. In addition, it is also used as a reversal agent for tumor multi-drug resistance, which can improve the sensitivity of cancer cells to chemotherapy and reduce the toxicity to the heart.

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

[0057] Aronia melanocarpa fruit, also known as wild chokeberry and aronia fruit, the fruit of Aronia melanocarpa, with the English names Aronia and black chokeberry, is native to the northeastern part of North America and was later introduced to Europe. Aronia melanocarpa fruit has extraordinary health care effects. The fruit and its extracts have special curative 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 America. The content of anthocyanins, flavonoids (the fresh fruit content is as high as 0.25%-0.35%) and polyphenols in the fruit is the highest among known plants. The fruit also contains various vitamins and mineral elements and other substances. 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 melanocarpa fruit is widely used in many fields such as medicine, food and beverage. It can be made into products such as fruit juice, fruit wine, syrup, tea, etc., and can also be used for extracting natural pigments and making medicines and cosmetics. Research shows that Aronia melanocarpa fruit has significant preventive and therapeutic effects on cardiovascular and cerebrovascular diseases, hypertension, hyperglycemia, etc., and has various health care functions such as antioxidant, anti-inflammatory and anti-aging.

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

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

[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 selected from 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 Gaxianwo E in the collagen peptide composition is 0.5 μM - 3 μM, and can be selected from 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM or 3 μM.

[0062] In a specific example, the concentration of salidroside in the collagen peptide composition is 0.1 μM - 0.5 μM, and can be selected from 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 concentrated juice of Aronia melanocarpa in the collagen peptide composition is 100 μg / mL - 500 μg / mL, and can be selected from 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 selected from 1.6 μM, 2.0 μM, 2.4 μM, 2.0 μM, 2.8 μM or 3.2 μM.

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

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

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

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

[0069] In a specific example, the application of the collagen peptide composition 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. It has been found in this application that the components in the composition synergistically restore the collagen content in skin cells.

[0071] One embodiment of this application also provides the application of the collagen peptide composition in the preparation of an anti-aging product.

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

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

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

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

[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 generally provides a beneficial effect. There is no particular limitation on the range of physiological and / or pharmacological effects produced by the "drug" in vivo, which can be a systemic effect or only a local effect. There is no particular limitation on the activity of the "drug", which can 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, the 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 a specific example, the product is a liquid collagen peptide.

[0080] In a specific example, the product is an oral collagen peptide.

[0081] One embodiment of this 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, which includes 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 elicit a biological or medical response in an individual, such as a dosage amount that brings about a physiologically and / or pharmacologically positive effect in the individual. "Physiologically and / or pharmacologically positive effect" includes, but is not limited to, improving symptoms, alleviating diseases, slowing down or delaying the disease process, or preventing diseases, etc.

[0084] In a specific example, the method of using the collagen peptide composition to promote or restore collagen content is not a diagnostic and treatment method.

[0085] In a specific example, the method of using the collagen peptide composition to promote or restore collagen content is a diagnostic and / or treatment method, which includes 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 preferably 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 specific examples, 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 elicit a biological or medical response in an individual, such as a dosage amount that brings about a pharmacologically positive effect in the individual. "Pharmacologically positive effect" includes, but is not limited to, improving symptoms, alleviating diseases, slowing down or delaying the disease process, or preventing diseases, etc.

[0089] In some specific examples, the way of administering the aforementioned collagen peptide composition to a subject can be oral administration.

[0090] One embodiment of the present application provides the application of collagen peptide, gamma-oryzanol E, EGCG, or Aronia melanocarpa fruit concentrate in the preparation of products for restoring or promoting collagen content.

[0091] The newly discovered drug gamma-oryzanol E has unique advantages in restoring collagen in skin cells and delaying aging. It can be applied 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 not to limit the scope of the present application. For the experimental methods without specific conditions in the following examples, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in the art.

[0093] In the following specific embodiments, regarding the measurement parameters of raw material components, without special instructions, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

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

[0095] DOXO (Doxorubicin), purchased from MCE, CAS No. 23214 - 92 - 8, and its chemical structural formula is .

[0096] Epigallocatechin gallate (EGCG), purchased from Zhejiang Sheng's Biotechnology Co., Ltd., and its chemical structural formula is .

[0097] Garcinone E, purchased from MCE, CAS No. 112649 - 21 - 5, and its chemical structural formula is .

[0098] Aronia Juice Concentrate, purchased from Micro (Microstructure Sp. z o.o.), product number R695335. The solid content of the Aronia Juice Concentrate is ≥15%, and its main components include anthocyanins, proanthocyanidins, and vitamins.

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

[0100] Salidroside, purchased from MCE, CAS No. 10338 - 51 - 9, and its chemical structural formula is .

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

[0102] Example 1: Cytotoxicity Detection Experiment of Garcinone E

[0103] 1. Cell seeding: When the MSF cells are cultured to the appropriate passage number and the confluence reaches 90%, aspirate the original culture medium, wash twice with PBS, digest with 0.25% trypsin, let it stand in the cell culture incubator for 3 min until the cells shrink, become round, and gradually detach from the culture flask. Then add complete medium with twice the volume of trypsin to stop digestion. Collect the cell suspension, centrifuge, resuspend with an appropriate amount of complete medium, and count. After counting, seed the cells into a 96-well plate at a density of 10,000 cells / well and place it in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity.

[0104] 2. Cell drug addition: When it is observed that the cells reach the logarithmic growth phase, treat the cells with different concentrations of Gashengwo E for modeling.

[0105] 3. Observe the cells the next day, discard the supernatant, add 110 μL / well of CELLTITER reagent, incubate for 30 min, then aspirate it into a white 96-well plate and detect it with an enzyme-linked immunosorbent assay (ELISA) reader.

[0106] The results are as Figure 1 shown. 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 a DOXO-stimulated mouse skin fibroblast (MSF) collagen loss model

[0108] The mechanisms of DOXO-induced cell senescence mainly include DNA damage and oxidative stress. After DOXO enters the cells, it will embed in the DNA, leading to DNA damage, and then activating the DNA damage response mechanism in the cells. At the same time, DOXO will also trigger oxidative stress, resulting in an increase in the level of reactive oxygen species (ROS) in the cells, further promoting cell senescence and leading to a decrease in the collagen content in the cells.

[0109] 1. Cell seeding: When the MSF cells are cultured to the appropriate passage number and the confluence reaches 90%, aspirate the original culture medium, wash twice with PBS, digest with 0.25% trypsin, let it stand in the cell culture incubator for 3 min until the cells shrink, become round, and gradually detach from the culture flask. Then add complete medium with twice the volume of trypsin to stop digestion. Collect the cell suspension, centrifuge, resuspend with an appropriate amount of complete medium, and count. After counting, seed the cells into a black 96-well plate at a density of 11,000 cells / well and place it in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity.

[0110] 2. Cell drug treatment: When the cells reach the logarithmic growth phase, drug treatment is carried out to establish a model. Specifically, the following 3 drugs are involved: collagen peptide (750 μg / mL), EGCG (3.2 μM), and Garcinone E (0.5 μM). The experimental groups include a background well group, a blank group (Control), a 1 μM DOXO model group, and a group with drugs added after modeling (single drugs: collagen peptide 750 μg / mL group, EGCG 3.2 μM group, Garcinone E 0.5 μM group; combined formulation: collagen peptide 750 μg / mL + EGCG 3.2 μM + Garcinone E 0.5 μM group). In the model group and the group with drugs added after modeling, 100 μL of culture medium containing the corresponding concentration of drugs is added to each well, and 100 μL of complete DMEM medium is added to each well in the background well group and the blank group. Each group is set with 3 replicate wells. After drug administration, the black 96-well plate is placed in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity for 24 h.

[0111] 3. ICW experiment: After aspirating the complete medium from the MSF cells cultured overnight, 150 μL of fixative is added to each well using a multi-channel pipette and left standing at room temperature for 60 min. Then, the fixative is aspirated, and 150 μL of permeabilization solution (0.1% Triton X-100 solution) is added to each well to make holes, and the plate is incubated on a shaker (at about 50 revolutions amplitude) at room temperature for 20 min. After permeabilization, the permeabilization solution is aspirated, and 150 μL of blocking buffer (purchased from a gene company) is added to each well, and the plate is incubated on a shaker (at about 50 revolutions amplitude) at room temperature for 1.5 h. Then, the blocking buffer in each well is aspirated, and 50 μL of primary antibody working solution (anti-COL3A1 primary antibody, 1:500, prepared with 1% TBS BSA, and 1% TBS BSA is added to the background well group) is added, and the plate is incubated on a shaker at 4 °C overnight. The next day, the plate is washed 3 times with TBST for 5 min each time, and then incubated with a mixture of secondary antibody (anti-rabbit 800) and Cell Tag (purchased from a gene company) (1:500, prepared with TBST, and only the secondary antibody is added to the background well group) for 1 h at room temperature in the dark. After that, the plate is washed 3 times with TBST for 5 min each time, and then the liquid is aspirated, and data is detected and analyzed using an Odyssey fluorescence imaging instrument.

[0112] The results are as Figure 2 shown. Compared with the blank control group, the content of COL3A1 in cells after DOXO stimulation decreased significantly, indicating that the DOXO-stimulated MSF cell collagen loss model was successfully established. After repairing the damage with the collagen peptide + EGCG + Garcinone E combination, the results showed that compared with the DOXO model group, the expression of COL3A1 increased significantly and was significantly better than that of each group treated with drugs alone.

[0113] Example 3 Effect of the Composition on the Expression of P21 in a Doxorubicin (DOXO)-stimulated Senescence Model of Mouse Skin Fibroblasts (MSF)

[0114] 1. Cell seeding: When the MSF cells were cultured and grew to an appropriate passage number and the confluence reached 90%, the original culture medium was aspirated, and the cells were washed twice with PBS, digested with 0.25% trypsin, and left standing in the cell culture incubator for 3 min until the cells shrank, became round, and gradually detached from the culture flask. Then, a complete medium with a volume twice that of trypsin was added to stop the digestion. The cell suspension was collected, centrifuged, and resuspended with an appropriate amount of complete medium for counting. After counting, the cells were seeded into 6-cm dishes at a density of 7.5×10⁵ cells / dish and placed in a cell culture incubator at 37 °C, 5% CO₂, and saturated humidity.

[0115] 2. Cell drug treatment: When the cells were observed to reach the logarithmic growth phase, drug treatment was performed to establish a model. Specifically, the following three drugs were involved: collagen peptide (750 μg / mL), EGCG (3.2 μM), and Garcinone E (0.5 μM). The experimental groups included a blank group (Control), a 1-μM DOXO model group, and a group treated with drugs (combination) after model establishment (combination formulation: 750 μg / mL collagen peptide + 3.2 μM EGCG + 0.5 μM Garcinone E). In the model group and the group treated with drugs after model establishment, 5 mL of a culture medium containing the corresponding concentration of drugs was added to each well, and 5 mL of DMEM complete medium was added to each well in the blank group. After drug administration, the 6-cm dishes were placed in a constant temperature incubator at 37 °C, 5% CO₂, and saturated humidity and cultured for 24 h.

[0116] 3. qPCR experiment: The next day, the cells were scraped from the culture dish with a cell scraper and collected into a centrifuge tube together with the culture medium. After centrifugation at 3000 rpm at room temperature for 5 min, the supernatant was discarded. RNA was extracted using an RNA extraction kit (purchased from Novoprotein), and then reverse-transcribed into cDNA using a reverse transcription kit (purchased from TIANGEN). Subsequently, a qPCR experiment was performed using a qPCR kit (purchased from Novoprotein).

[0117] The results are as Figure 3 shown. Compared with the blank control group, the expression level of P21 in the cells was significantly increased after DOXO stimulation, indicating that the DOXO-stimulated MSF cell senescence model was successfully established. After treating with the collagen peptide + EGCG + Garcinone E composition for damage repair, the results showed that compared with the DOXO model group, the expression of P21 was significantly decreased, proving that the composition has anti-aging effects.

[0118] Example 4 Effect of the Composition on the Recovery of Collagen Content in a Doxorubicin (DOXO)-stimulated Collagen Loss Model of Mouse Skin Fibroblasts (MSF)

[0119] 1. Cell seeding: When the MSF cells are cultured and grown to an appropriate passage number and the confluence reaches 90%, aspirate the original culture medium, wash twice with PBS, digest with 0.25% trypsin, let it stand in the cell culture incubator for 3 min until the cells shrink and become round and gradually detach from the culture flask, then add complete medium with twice the volume of trypsin to stop digestion. Collect the cell suspension, centrifuge, and resuspend with an appropriate amount of complete medium, and count. After counting, seed the cells into 6-cm dishes at a density of 750,000 cells / dish and place them in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity.

[0120] 2. Cell drug treatment: When it is observed that the cells grow to the logarithmic growth phase, perform drug-induced modeling. Specifically, the following 5 drugs are involved: collagen peptide (750 μg / mL), EGCG (3.2 μM), Garcinone E (0.5 μM), and two drugs similar in structure to Garcinone E, Salidroside (0.5 μM) and CR-1-31-B (0.5 μM). Set up a blank group (Control), a 1 μM DOXO modeling group, and a group with drugs added after modeling (single drugs: collagen peptide 750 μg / mL group, EGCG 3.2 μM group, Garcinone E 0.5 μM group; combined formulations: collagen peptide 750 μg / mL + EGCG 3.2 μM group, collagen peptide 750 μg / mL + Garcinone E 0.5 μM group, EGCG 3.2 μM + Garcinone E 0.5 μM group, collagen peptide 750 μg / mL + EGCG 3.2 μM + Salidroside 0.5 μM group, collagen peptide 750 μg / mL + EGCG 3.2 μM + CR-1-31-B 0.5 μM group, collagen peptide 750 μg / mL + EGCG 3.2 μM + Garcinone E 0.5 μM group). Add 5 mL of culture medium containing the corresponding concentration of drugs to each well in the modeling and sample groups, and add 5 mL of DMEM complete medium to each well in the blank group; after drug administration, place the 6-cm dishes in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity and culture for 24 h.

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

[0122] The results are as Figures 4 - 5As shown, compared with the blank control group, the contents of COL1A1 and COL3A1 in cells were significantly decreased after DOXO stimulation, indicating that the collagen loss model of MSF cells was successfully established by DOXO stimulation. After the collagen peptide + EGCG + Jiashengwo E composition was used for post-injury repair, the results showed that compared with the DOXO-induced model group, the expressions of COL1A1 and COL3A1 were significantly increased, which was better than the individual administration of each group and better than the combination of two drugs similar in structure to Jiashengwo E.

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

[0124] 1. Cell seeding: When HACAT cells were cultured and grown to an appropriate passage number and the confluence reached 90%, the original culture medium was aspirated, washed twice with PBS, digested with 0.25% trypsin, and left standing in the cell culture incubator for 7 min until the cells shrank, became round, and gradually detached from the culture flask. Then, a complete medium with twice the volume of trypsin was added to stop digestion. The cell suspension was collected, centrifuged, and resuspended with an appropriate amount of complete medium for counting. After counting, the cells were seeded into 6-cm dishes at a density of 2.5×10^6 cells / dish and placed in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity.

[0125] 2. Cell drug treatment: When it was observed that the cells grew to the logarithmic growth phase, drug-induced modeling treatment was carried out. Specifically, the following 5 drugs were involved: collagen peptide (750 μg / mL), EGCG (3.2 μM), Jiashengwo E (0.5 μM), and two drugs similar in structure to Jiashengwo E, salidroside (0.5 μM) and CR-1-31-B (0.5 μM). The experimental groups included a blank group (Control), a 3-μM DOXO-induced model group, and a group with drugs added after modeling (see Example 4). In the model group and the group with drugs added after modeling, 5 mL of a culture medium containing the corresponding concentration of drugs was added to each well, and 5 mL of DMEM complete medium was added to each well in the blank group. After drug administration, the 6-cm dishes were placed in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity and cultured for 24 h.

[0126] 3. WB experiment: The next day, the cells were scraped off the culture dish with a cell scraper and collected into a centrifuge tube with the culture medium. Centrifuged at 3000 rpm at room temperature for 5 min, and the supernatant was discarded. The precipitate was lysed with RIPA, sonicated for 1 min after 10 min, and the supernatant was collected by centrifugation to obtain a protein sample. After measuring the protein concentration with a BCA kit (purchased from Thermo), the WB experiment was carried out.

[0127] The results are as Figure 6As shown, compared with the blank control group, the content of COL17A1 in cells significantly decreased after DOXO stimulation, indicating that the collagen loss model of HACAT cells was successfully established by DOXO stimulation. After the damage was repaired with the collagen peptide + EGCG + Garcinone E composition, the results showed that compared with the DOXO model group, the expression of COL17A1 was significantly increased, which was better than that of each group administered alone and better than the combination of two drugs with a structure similar to Garcinone 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 seeding: When the MSF cells were cultured to the appropriate passage number and the confluence reached 90%, the original culture medium was aspirated, washed twice with PBS, digested with 0.25% trypsin, and left standing in the cell culture incubator for 3 min until the cells shrank, became round, and gradually detached from the culture flask. Then, a complete medium with twice the volume of trypsin was added to stop digestion. The cell suspension was collected, centrifuged, and resuspended with an appropriate amount of complete medium for counting. After counting, the cells were seeded into a black 96-well plate at a density of 11,000 cells / well and placed in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity.

[0130] 2. Cell drug treatment: When the cells were observed to reach the logarithmic growth phase, drug-induced modeling treatment was performed. Specifically, the following three drugs were involved: collagen peptide (750 μg / mL), Aronia melanocarpa fruit concentrate (100 μg / mL), and Garcinone E (0.5 μM). The experimental groups included a background well group, a blank group (Control), a 1 μM DOXO model group, and a group with drugs added after modeling (single drug: collagen peptide 750 μg / mL group, Aronia melanocarpa fruit concentrate 100 μg / mL group, Garcinone E 0.5 μM group; combined formulation: collagen peptide 750 μg / mL + Aronia melanocarpa fruit concentrate 100 μg / mL + Garcinone E 0.5 μM group). 100 μL of the culture medium containing the corresponding concentration of the drug was added to each well in the model group and the group with drugs added after modeling, and 100 μL of DMEM complete medium was added to each well in the background well group and the blank group. Each group was set with 3 replicate wells. After the drug administration was completed, the black 96-well plate was placed in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity and cultured for 24 h.

[0131] 3. ICW experiment: After aspirating and discarding the complete medium in the overnight-cultured MSF cells, add 150 μL of fixative to each well using a multi-channel pipette, and let it stand at room temperature for 60 min. Then aspirate and discard the fixative, add 150 μL of permeabilization solution (0.1% Triton X-100 solution) to each well to create pores, and incubate on a shaker (at about 50 revolutions amplitude) at room temperature for 20 min. After permeabilization, aspirate and discard the permeabilization solution, add 150 μL of blocking buffer (purchased from a gene company) to each well, and incubate on a shaker (at about 50 revolutions amplitude) at room temperature for 1.5 h. Then aspirate and discard the blocking buffer in each well, add 50 μL of the primary antibody working solution (anti-COL3A1 primary antibody, 1:500, prepared with 1% TBS BSA, add 1% TBS BSA to the background well group), and incubate on a shaker at 4 °C overnight. The next day, wash the plate 3 times with TBST, 5 min each time, then incubate with the secondary antibody (anti-rabbit 800) and Cell Tag (purchased from a gene company) mixed solution (1:500, prepared with TBST, only add the secondary antibody to the background well group), incubate at room temperature in the dark for 1 h, then wash the plate 3 times with TBST, 5 min each time, then aspirate the liquid, and use an Odyssey fluorescence imaging instrument to detect and analyze the data.

[0132] The results are as Figure 7 shown. Compared with the blank control group, the content of COL3A1 in cells was significantly decreased after DOXO stimulation, indicating that the collagen loss model of DOXO-stimulated MSF cells was successfully established. After performing injury repair with the collagen peptide + aronia melanocarpa fruit + Galsenwo E composition, the results showed that compared with the DOXO model group, the expression of COL3A1 was significantly increased, and it was significantly better than the individual administration of each group.

[0133] Example 7 Effect of the composition on the expression of P21 in the senescence model of doxorubicin-stimulated mouse skin fibroblasts (MSF)

[0134] 1. Cell seeding: When the MSF cells are cultured and grown to an appropriate passage and the confluence reaches 90%, aspirate the original culture medium, wash 2 times with PBS, digest with 0.25% trypsin, let it stand in the cell culture incubator for 3 min until the cells shrink, become round, and gradually detach from the culture flask, then add a complete medium with 2 times the volume of trypsin to stop digestion, collect the cell suspension, centrifuge, and resuspend with an appropriate amount of complete medium, and count. After counting, seed the cells into 6-cm dishes at a density of 750,000 cells / dish, and place them in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity.

[0135] 2. Cell drug treatment: When it is observed that the cells reach the logarithmic growth phase, drug treatment is performed to establish a model. Specifically, the following 3 drugs are involved: collagen peptide (750 μg / mL), concentrated fruit juice of Aronia melanocarpa (100 μg / mL), and Galsenwo E (0.5 μM). The experiment sets up a blank group (Control), a doxorubicin (DOXO) model group at 1 μM, and a group with drugs added after model establishment (combination). 5 mL of culture medium containing the corresponding concentration of drugs is added to each well in the model group and the group with drugs added after model establishment, and 5 mL of complete DMEM medium is added to each well in the blank group. After drug administration, the 6-cm dishes are placed in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity for 24 h.

[0136] 3. qPCR experiment: The next day, the cells are scraped off the culture dish with a cell scraper and collected into a centrifuge tube with the culture medium. Centrifuge at 3000 rpm at room temperature for 5 min and discard the supernatant. After extracting RNA using an RNA extraction kit (purchased from Novoprotein), reverse transcribe the RNA into cDNA using a reverse transcription kit (purchased from TIANGEN). Then perform qPCR experiments using a qPCR kit (purchased from Novoprotein).

[0137] The results are as Figure 8 shown. Compared with the blank control group, the expression level of P21 in cells was significantly increased after DOXO stimulation, indicating that the establishment of the DOXO-stimulated MSF cell senescence model was successful. After repairing the damage with the combination of collagen peptide + concentrated fruit juice of Aronia melanocarpa + Galsenwo E, the results showed that compared with the DOXO model group, the expression of P21 was significantly reduced, proving that this combination has anti-aging effects.

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

[0139] 1. Cell seeding: When the MSF cells are cultured to the appropriate passage and the confluence reaches 90%, aspirate the original culture medium, wash twice with PBS, digest with 0.25% trypsin, and let it stand in the cell culture incubator for 3 min until the cells shrink, become round, and gradually detach from the culture flask. Then add complete medium with twice the volume of trypsin to stop digestion. Collect the cell suspension, centrifuge, and resuspend with an appropriate amount of complete medium for counting. After counting, seed the cells into 6-cm dishes at a density of 750,000 cells / dish and place them in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity.

[0140] 2. Cell drug treatment: When the cells reach the logarithmic growth phase, drug treatment is performed to establish a model. Specifically, the following 5 drugs are involved: collagen peptide (750 μg / mL), Aronia melanocarpa fruit concentrate (100 μg / mL), Garcinone E (0.5 μM), and two drugs similar in structure to Garcinone E, Salidroside (0.5 μM) and CR-1-31-B (0.5 μM). The experimental groups include a blank group (Control), a 1 μM DOXO model group, and a group with drugs added after model establishment (single drugs: collagen peptide 750 μg / mL group, Aronia melanocarpa fruit concentrate 100 μg / mL group, Garcinone E 0.5 μM group; combined drugs: collagen peptide 750 μg / mL + Aronia melanocarpa fruit concentrate 100 μg / mL group, collagen peptide 750 μg / mL + Garcinone E 0.5 μM group, Aronia melanocarpa fruit concentrate 100 μg / mL + Garcinone E 0.5 μM group, collagen peptide 750 μg / mL + Aronia melanocarpa fruit concentrate 100 μg / mL + Salidroside 0.5 μM group, collagen peptide 750 μg / mL + Aronia melanocarpa fruit concentrate 100 μg / mL + CR-1-31-B 0.5 μM group, collagen peptide 750 μg / mL + Aronia melanocarpa fruit concentrate 100 μg / mL + Garcinone E 0.5 μM group). 5 mL of culture medium containing the corresponding concentration of drugs is added to each well in the model group and the group with drugs added after model establishment, and 5 mL of complete DMEM medium is added to each well in the blank group. After drug administration, the 6-cm dishes are placed in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity for 24 h.

[0141] 3. WB experiment: The next day, the cells are scraped off the culture dish with a cell scraper and collected into a centrifuge tube with the culture medium. Centrifuge at 3000 rpm at room temperature for 5 min and discard the supernatant. The precipitate is lysed with RIPA, sonicated for 1 min after 10 min, and the supernatant is collected by centrifugation to obtain the protein sample. After measuring the protein concentration with a BCA kit (purchased from Thermo), the WB experiment is performed.

[0142] The results are as Figures 9 - 10 shown. Compared with the blank control group, the contents of COL1A1 and COL3A1 in the cells were significantly decreased after DOXO stimulation, indicating that the model of collagen loss in MSF cells induced by DOXO stimulation was successfully established. After repairing the damage with the combination of collagen peptide + Aronia melanocarpa + Garcinone E, the results showed that compared with the DOXO model group, the expressions of COL1A1 and COL3A1 were significantly increased, which was better than that of each group administered alone and better than the combination of two drugs similar in structure to Garcinone E.

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

[0144] 1. Cell seeding: When the HACAT cells were cultured and grown to an appropriate passage number and the confluence reached 90%, the original culture medium was aspirated, washed twice with PBS, digested with 0.25% trypsin, and left standing in the cell culture incubator for 7 min until the cells shrank, became round, and gradually detached from the culture flask. Then, a complete medium with twice the volume of trypsin was added to stop the digestion. The cell suspension was collected, centrifuged, and resuspended with an appropriate amount of complete medium for counting. After counting, the cells were seeded into 6-cm dishes at a density of 2.5×10^6 cells / dish and placed in a cell culture incubator at 37 °C, 5% CO2, and saturated humidity.

[0145] 2. Cell drug treatment: When it was observed that the cells grew to the logarithmic growth phase, drug treatment was performed to establish a model. Specifically, the following 5 drugs were involved: collagen peptide (750 μg / mL); Aronia melanocarpa fruit concentrate (100 μg / mL), Gashengwo E (0.5 μM), and two drugs similar in structure to Gashengwo E, salidroside (0.5 μM) and CR-1-31-B (0.5 μM). The experimental groups included a blank group (Control), a 3 μM DOXO model group, and a group treated with drugs after model establishment (see Example 8). In the model group and the group treated with drugs after model establishment, 5 mL of a culture medium containing the corresponding concentration of drugs was added to each well, and 5 mL of DMEM complete medium was added to each well in the blank group. After drug administration, the 6-cm dishes were placed in a constant temperature incubator at 37 °C, 5% CO2, and saturated humidity and cultured for 24 h.

[0146] 3. WB experiment: The next day, the cells were scraped off the culture dish with a cell scraper and collected into a centrifuge tube with the culture medium. Centrifuged at 3000 rpm at room temperature for 5 min, and the supernatant was discarded. The precipitate was lysed with RIPA, ultrasonicated for 1 min after 10 min, and the supernatant was obtained by centrifugation to obtain a protein sample. After measuring the protein concentration with a BCA kit (purchased from Thermo), the WB experiment was performed.

[0147] The results are as Figure 11 shown. Compared with the blank control group, the content of COL17A1 in the cells was significantly decreased after DOXO stimulation, indicating that the DOXO-stimulated HACAT cell collagen loss model was successfully established. After repairing the damage with the collagen peptide + Aronia melanocarpa fruit concentrate + Gashengwo E composition, the results showed that compared with the DOXO model group, the expression of COL17A1 was significantly increased, which was better than the individual administration of each group and better than the combination of two drugs similar in structure to Gashengwo E.

[0148] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, 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, it should be considered as falling within the scope described in this specification.

[0149] The above-described embodiments merely represent several implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, 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 appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A collagen peptide composition, characterized in that: The collagen peptide composition comprises Gastrointestinal E and collagen peptides, and the collagen peptide composition further comprises Aronia nigra fruit concentrate or EGCG, wherein the EGCG is epigallocatechin gallate, and the concentration of the collagen peptides in the collagen peptide composition is 750 μg / mL; The concentration of the Gastrointestinal tract E in the collagen peptide composition is 0.5 μM; The concentration of the Aronia melanocarpa fruit concentrate in the collagen peptide composition is 100 μg / mL; The concentration of EGCG in the collagen peptide composition is 3.2 μM; The black fruit glandular rib aronia fruit concentrate was purchased from Microstructure Sp. z oo, with the product number R695335; The collagen peptide was purchased from Hainan Huayan Collagen Technology Co., Ltd. with batch number JDA240618.

2. The collagen peptide composition according to claim 1, 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 collagen peptide composition is a pharmaceutical composition.

3. The method for preparing the collagen peptide composition according to claim 1 or 2, characterized in that: The method comprises mixing relevant components with a solvent to prepare a collagen peptide mixture.

4. Use of the collagen peptide composition according to claim 1 or 2 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 claim 1 or 2 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 claim 1 or 2.

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 claim 1 or 2, wherein the method is not a diagnostic or therapeutic method.

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