Active peptide composition with effect of promoting cell repair as well as preparation method and application of active peptide composition

Abstract

The invention discloses an active peptide composition with an effect of promoting cell repair as well as a preparation method and application thereof, and belongs to the technical field of biological active peptides. The technical problem to be solved is to provide a pure natural and more effective bioactive peptide composition for promoting cell repair, and the bioactive peptide composition can significantly promote cell proliferation and repair oxidative damage and dry damage. According to the technical scheme, the active peptide composition is characterized in that the active peptide composition composed of sea cucumber peptide, casein peptide, rice peptide and fish skin collagen peptide is provided, and the dosage ratio of the sea cucumber peptide to the casein peptide to the rice peptide to the fish skin collagen peptide is (6-19): 15: (52-65): 15 by mass.

Description

Technical Field

[0001] This invention belongs to the field of active peptide composition technology, specifically relating to an active peptide composition that promotes cell repair, its preparation method, and its application. Background Technology

[0002] For understanding the technical content of this invention:

[0003] Bioactive peptides are peptides that perform specific functions within living organisms. They range in size from containing only two amino acids to being composed of hundreds, representing a substance intermediate between amino acids and proteins. Widely distributed in the biological world, bioactive peptides often act as messengers, transmitting chemical signals within or between cells, and regulating intercellular or organ-related behaviors. Scientific research has found that small-molecule bioactive peptides are involved in various functions, including those related to the nervous system, immunity, and aging process.

[0004] Food-derived bioactive peptides, due to their natural, safe, and highly effective properties, have shown great potential in delaying skin aging. Over the past decade, research on bioactive peptides (including collagen peptides, sea cucumber peptides, and bird's nest peptides) in promoting cell proliferation, migration, and matrix synthesis has increased rapidly both domestically and internationally. Numerous experiments have shown that small molecule peptides can penetrate cell membranes or enter cells via endocytosis, activating signaling pathways such as ERK1 / 2, PI3K-Akt, and TGF-β / Smad, thereby upregulating the expression of collagen types I, III, and IV, as well as laminin, and accelerating the re-epithelialization and barrier reconstruction of damaged tissues. For example, sea cucumber peptides have been shown to reduce oxidative stress damage to fibroblasts by inhibiting excessive ROS production and downregulating inflammatory factors such as TNF-α and IL-6.

[0005] However, single active peptides often have limited targets, and their repair efficiency is greatly affected by peptide sequence, dosage, and stability.

[0006] Relevant patent documents retrieved: The patent, published in China (CN115517369A) on December 27, 2022, discloses a composite protein polypeptide powder comprising: soybean peptide powder, wheat oligopeptide, corn oligopeptide powder, rice oligopeptide powder, hydrolyzed collagen, black tea powder, inulin, sea cucumber peptide, xylitol, water-soluble starch, and steviol glycosides. However, this patent only provides the technical solution and claimed technical effects; its actual effectiveness still requires further verification.

[0007] Relevant non-patent literature retrieved: The journal or book title is "Journal of Food Safety and Quality Testing," and the article title is "Comparison of the Protective Effects of Three Peptides on Cell Oxidative Damage and Their Cell Proliferation-Promoting Ability," volume number 15(09):332-338, publication date 2024.5.15. This article discloses that the oxidative damage protection ability of sea cucumber peptide is superior to that of soybean peptide and wheat peptide. The proliferation index of fibroblasts treated with 200 μg / mL soybean peptide and sea cucumber peptide was significantly increased compared with the blank control. That is, soybean peptide and sea cucumber peptide can promote fibroblast proliferation by affecting the cell cycle and increasing the proliferation index. Moreover, the ability of sea cucumber peptide to promote fibroblast proliferation at the same concentration is better than that of soybean peptide at the same concentration, while the effect of wheat peptide on the cell cycle is not significantly different. Sea cucumber peptide has the strongest inhibitory effect on fibroblast apoptosis. After treatment with 200 μg / mL sea cucumber peptide, the apoptosis rate of cells decreased from (3.59±0.11)% in the blank control to (1.89±0.09)%.

[0008] The existing technologies represented by the aforementioned literature still lack an active peptide composition that can effectively promote cell repair. Summary of the Invention

[0009] The purpose of this invention is to provide: An active peptide composition with cell repair-promoting effects, its preparation method and application, and related technologies, to solve the technical problems such as the lack of purely natural and highly efficient cell repair-promoting products in the prior art, or combinations thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definition of the standard chemical term can be found in the reference "Bioactive Peptide Functions and Preparation", China Light Industry Press, Luo Yongkang.

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as cell culture, shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The term "active peptide composition" as used in this article refers to a mixture of sea cucumber peptides, casein peptides, rice peptides, and fish skin collagen peptides in a specific mass ratio, which has the function of promoting cell repair, reducing the production of cell free radicals, alleviating oxidative damage, reducing cell cycle arrest, increasing cell proliferation rate, and reducing tyrosinase activity.

[0016] The term "cell repair" used in this article refers to the process by which cells affected by external factors such as oxidative damage and desiccation damage restore their normal growth state and improve their activity and proliferation capacity by enhancing their own metabolic capacity and antioxidant capacity. In this invention, HSF fibroblasts are mainly used as the research object to demonstrate this function.

[0017] The term "simulated digestion treatment" used in this article refers to the treatment of bioactive peptide compositions under laboratory conditions by adding pepsin and pancreatic enzymes and adjusting parameters such as pH and temperature to simulate the digestive environment of the human stomach and intestines, in order to study their stability and post-digestion activity during human digestion.

[0018] The term "cell proliferation rate" used in this article refers to the growth ratio of experimental group cells relative to blank control group cells, calculated by detecting the growth activity of cells under specific conditions using the CCK-8 assay. It is an important indicator for evaluating cell growth status and repair capacity.

[0019] In a first aspect, the present invention provides: an active peptide composition having a cell repair promoting effect.

[0020] This includes technical features such as the composition of the active peptide composition and the amount of each component.

[0021] The active peptide composition with technical characteristics is selected from sea cucumber peptides, casein peptides, rice peptides and fish skin collagen peptides.

[0022] The mass ratio of the technical features sea cucumber peptide, casein peptide, rice peptide, and fish skin collagen peptide is selected from 6-19:15:52-65:15.

[0023] The preferred ratio of technical feature quality is 6:15:65:15.

[0024] The preferred ratio of technical feature quality is 19:15:52:15.

[0025] Secondly, the present invention provides a method for preparing an active peptide composition, comprising the following steps: A method for preparing an active peptide composition, characterized by comprising the following steps: (1) Take 3-8g of rice protein and add it to 15-25 times its weight of water, heat it to 45-55℃, and adjust the pH to 7.0±0.2; (2) Add 1.5-2.5‰ w / w of papain and 1.5-2.5‰ w / w of alkaline protease, and hydrolyze for 3.5-4.5h. Then add 0.5-1.5‰ w / w of neutral protease and continue hydrolysis for 0.5-1.5h. Adjust the pH to 7.0±0.2. (3) After the enzymatic hydrolysis is completed, centrifuge at 9000-11000r / min for 10-20min and take the supernatant. Measure the soluble solids content of the supernatant and calculate the weight of the rice peptides. (4) Weigh out sea cucumber peptide, casein peptide, rice peptide and fish skin collagen peptide, stir and mix evenly, and then freeze-dry under vacuum to obtain the final product.

[0026] Thirdly, the present invention provides the use of the above-mentioned active peptide composition in the preparation of products that promote cell repair.

[0027] Fourthly, the present invention provides a product comprising the above-described active peptide composition.

[0028] The products mentioned include, but are not limited to, cosmetics, food, or pharmaceuticals.

[0029] The cosmetics mentioned include, but are not limited to, at least one of the following: body lotion, hand cream, serum, essential oil, emulsion, toner, face cream, BB cream, shower gel, face mask, foundation, eye cream, facial cleanser, or sunscreen.

[0030] The food includes at least one of health products or beverages.

[0031] The drug also includes pharmaceutically acceptable excipients.

[0032] The pharmaceutically acceptable excipients include at least one of binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0033] The present invention has at least the following beneficial effects: 1. Compared with existing technologies, the present invention has better technical effects in terms of safety and cell repair.

[0034] The active peptide composition of the present invention consists of only four naturally sourced active peptides: sea cucumber peptide, casein peptide, rice peptide, and fish skin collagen peptide, and has no cytotoxicity to fibroblasts.

[0035] The active peptide composition of the present invention can more significantly promote cell proliferation, repair oxidative damage and desiccation damage than a single peptide, and in particular, it can maintain good technical effects even after digestion. Attached Figure Description

[0036] Figure 1 To investigate the effect of different concentrations of samples in Example 1 on the proliferation rate of HSF fibroblasts, A, E, and D were determined to be concentrations of 100, 200, 400, 800, and 1200 µg / mL, respectively; the letters a, b, c, and d represent significant differences between groups. p <0.05).

[0037] Figure 2 To investigate the effect of different concentrations of the sample in Example 1 on the proliferation rate of HSF fibroblasts after digestion, A and D were concentrations of 100, 200, 400, and 800 µg / mL, respectively; different letters a, b, c, and d represent significant differences between groups ( p <0.05).

[0038] Figure 3 To investigate the effect of different H2O2 concentrations on the proliferation rate of HSF cells in Example 2.

[0039] Figure 4 To test the ability of different concentrations of the compound peptide in Example 2 to repair oxidative damage in HSF fibroblasts, A, E were concentrations of 100, 200, 400, 800, and 1200 µg / mL, respectively; the letters a, b, c, d, and e represent significant differences between groups ( p <0.05).

[0040] Figure 5 To investigate the effect of different concentrations of the compound active peptides in Example 2 on the proliferation rate of HSF fibroblasts after digestion, A and D were concentrations of 100, 200, 400, and 800 µg / mL, respectively; different letters a, b, c, d, e, f, and g represented significant differences between groups ( p <0.05).

[0041] Figure 6 To investigate the effect of different air-drying times on HSF fibroblasts in Example 3.

[0042] Figure 7 To demonstrate the protective effect of different concentrations of the compound active peptides on desiccation-damaged HSF fibroblasts, A, E, and E were concentrations of 100, 200, 400, 800, and 1200 µg / mL, respectively; the letters a, b, c, d, e, and f represent significant differences between groups.p <0.05).

[0043] Figure 8 The protective effects of different concentrations of the compound active peptides on desiccation-damaged HSF fibroblasts were evaluated. A and D were concentrations of 100, 200, 400, and 800 µg / mL, respectively; letters a, b, c, d, and e represented significant differences between groups. p <0.05). Detailed Implementation

[0044] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0046] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0047] Example 1 a. Take 5g of rice protein (purchased from Xi'an Weizhen Biotechnology Co., Ltd.) and add it to 100g of water. While stirring, heat the mixture to 50℃, and then adjust the pH of the solution to 7.0±0.2 with 7.5% sodium hydroxide aqueous solution. b. After the liquid is stirred evenly and the temperature reaches 50℃, add 2.0‰ (mass ratio) of papain and 2.0‰ (mass ratio) of alkaline protease per gram of rice protein substrate. After 4 hours of enzymatic hydrolysis, add 1.0‰ of neutral protease and continue enzymatic hydrolysis for 1 hour. During the enzymatic hydrolysis process, use 7.5% sodium hydroxide aqueous solution to maintain the pH of the liquid between 6.8 and 7.2. c. After enzymatic hydrolysis, the supernatant is collected after centrifuging at 10000r / min for 15 minutes. d. Determine the soluble solids content of the supernatant and calculate the weight of rice peptides; e. Add sea cucumber peptide, casein peptide and collagen peptide in sequence at a ratio of 6:15:65:15 (mass ratio) of sea cucumber peptide: casein peptide: rice peptide: fish skin collagen peptide, and stir to mix evenly to obtain an active peptide composition solution. f. An active peptide composition was prepared by freeze-drying (temperature -35℃, pressure <100Pa).

[0048] g. Dissolve the active peptide composition obtained after freeze-drying in sterile water to prepare a high-concentration sample solution. Sterilize the solution by passing it through a 0.22 µM sterile membrane and then dilute it to the experimental concentration with culture medium (DMEM high-glucose medium).

[0049] Example 2 a. Dissolve 1g of the mixed active peptide obtained in Example 1 in 20mL of hydrochloric acid solution with pH 2.0, add 4% pepsin (w / v), and hydrolyze in a constant temperature shaker at 37℃ for 60min to simulate gastric digestion. Adjust the pH to 7.5 with sodium hydroxide solution, bring the volume to 40mL, add 3% pancreatin (w / v), and hydrolyze again in a constant temperature shaker at 37℃ for 60min to simulate intestinal digestion. After the simulated digestion is complete, freeze-dry the digestive fluid for 72h to obtain the digestive product.

[0050] b. Dissolve the digested product of the active peptide composition obtained after freeze-drying in sterile water to prepare a high-concentration sample solution. Sterilize the solution by passing it through a 0.22 µM sterile membrane and then dilute it to the experimental concentration with culture medium (DMEM high-glucose medium).

[0051] Example 3 a. Take 5g of rice protein (purchased from Xi'an Weizhen Biotechnology Co., Ltd.) and add it to 100g of water. While stirring, heat the mixture to 50℃, and then adjust the pH of the solution to 7.0±0.2 with 7.5% sodium hydroxide aqueous solution. b. After the liquid is stirred evenly and the temperature reaches 50℃, add 2.0‰ (mass ratio) of papain and 2.0‰ (mass ratio) of alkaline protease per gram of rice protein substrate. After 4 hours of enzymatic hydrolysis, add 1.0‰ of neutral protease and continue enzymatic hydrolysis for 1 hour. During the enzymatic hydrolysis process, use 7.5% sodium hydroxide aqueous solution to maintain the pH of the liquid between 6.8 and 7.2. c. After enzymatic hydrolysis, the supernatant is collected after centrifuging at 10000r / min for 15 minutes. d. Determine the soluble solids content of the supernatant and calculate the weight of rice peptides; e. Add sea cucumber peptide, casein peptide and collagen peptide in sequence at a ratio of 19:15:52:15 (mass ratio) of sea cucumber peptide: casein peptide: rice peptide: fish skin collagen peptide, and stir to mix evenly to obtain an active peptide composition solution. f. An active peptide composition was prepared by freeze-drying (temperature -35℃, pressure <100Pa).

[0052] g. Dissolve the active peptide composition obtained after freeze-drying in sterile water to prepare a high-concentration sample solution. Sterilize the solution by passing it through a 0.22 µM sterile membrane and then dilute it to the experimental concentration with culture medium (DMEM high-glucose medium).

[0053] Example 4 a. Dissolve 1g of the mixed active peptide obtained in Example 3 in 20mL of hydrochloric acid solution with pH 2.0, add 4% pepsin (w / v), and hydrolyze in a constant temperature shaker at 37℃ for 60min to simulate gastric digestion. Adjust the pH to 7.5 with sodium hydroxide solution, bring the volume to 40mL, add 3% pancreatin (w / v), and hydrolyze again in a constant temperature shaker at 37℃ for 60min to simulate intestinal digestion. After the simulated digestion is complete, freeze-dry the digestive fluid for 72h to obtain the digestive product.

[0054] b. Dissolve the digested product of the active peptide composition obtained after freeze-drying in sterile water to prepare a high-concentration sample solution. Sterilize the solution by passing it through a 0.22 µM sterile membrane and then dilute it to the experimental concentration with culture medium (DMEM high-glucose medium).

[0055] Comparative Example 1 Sea cucumber peptides (patent number: ZL 20201005698.3, sample provided by Hangzhou Kangyuan Food Technology Co., Ltd.) were dissolved in sterile water to prepare a high-concentration sample solution. The solution was then sterilized by passing it through a 0.22 µM sterile membrane and diluted to the experimental concentration with culture medium (DMEM high-glucose medium). Since the market price of sea cucumber peptides is relatively high, and 75% of the protein structure in sea cucumber protein is collagen, 25% sea cucumber peptides were added by weight, and the remaining 75% was supplemented with collagen peptides to prepare a sea cucumber peptide composition.

[0056] Comparative Example 2 Casein peptide (ZL202411215502.6, sample provided by Hangzhou Kangyuan Food Technology Co., Ltd.) was dissolved in sterile water to prepare a high-concentration sample solution. The solution was then sterilized by passing it through a 0.22 µM sterile membrane and diluted to the experimental concentration with culture medium (DMEM high-glucose medium).

[0057] Comparative Example 3 a. Take 5g of rice protein (purchased from Xi'an Weizhen Biotechnology Co., Ltd.) and add it to 100g of water. While stirring, heat the mixture to 50℃, and then adjust the pH of the solution to 7.0±0.2 with 7.5% sodium hydroxide aqueous solution. b. After the liquid is stirred evenly and the temperature reaches 50℃, add 2.0‰ (mass ratio) of papain and 2.0‰ (mass ratio) of alkaline protease per gram of rice protein substrate. After 4 hours of enzymatic hydrolysis, add 1.0‰ of neutral protease and continue enzymatic hydrolysis for 1 hour. During the enzymatic hydrolysis process, use 7.5% sodium hydroxide aqueous solution to maintain the pH of the liquid between 6.8 and 7.2. c. After enzymatic hydrolysis, the supernatant is collected after centrifuging at 10000r / min for 15 minutes. d. Rice peptides were obtained by freeze-drying the supernatant (temperature -35℃, pressure <100Pa).

[0058] e. Dissolve the rice peptides obtained after freeze-drying in sterile water to prepare a high-concentration sample solution. Sterilize the solution by passing it through a 0.22 µM sterile membrane and then dilute it to the experimental concentration with culture medium (DMEM high-glucose medium).

[0059] Comparative Example 4 Collagen peptides (sample provided by Hubei Ruibang Biotechnology Co., Ltd.) were dissolved in sterile water to prepare a high-concentration sample solution. The solution was then sterilized by passing it through a 0.22 µM sterile membrane and diluted to the experimental concentration with culture medium (DMEM high-glucose medium).

[0060] Comparative Example 5 a. Dissolve 1g of sea cucumber peptide composition (same as Comparative Example 1, composed of 25% sea cucumber peptide and 75% collagen peptide by mass) in 20mL of hydrochloric acid solution with pH 2.0, add 4% pepsin (w / v), and hydrolyze in a constant temperature shaker at 37℃ for 60min to simulate gastric digestion. Adjust the pH to 7.5 with sodium hydroxide solution, bring the volume to 40mL, add 3% pancreatin (w / v), and hydrolyze again in a constant temperature shaker at 37℃ for 60min to simulate intestinal digestion. After simulated digestion, freeze-dry the digestive fluid for 72h to obtain the digestive product.

[0061] b. Dissolve the sea cucumber peptide digestion product obtained after freeze-drying in sterile water to prepare a high-concentration sample solution. Sterilize the solution by passing it through a 0.22 µM sterile membrane and then dilute it to the experimental concentration with culture medium (DMEM high-glucose medium).

[0062] Comparative Example 6 a) Dissolve 1g of casein peptide in 20mL of hydrochloric acid solution with pH 2.0, add 4% pepsin (w / v), and hydrolyze in a constant temperature shaker at 37℃ for 60min to simulate gastric digestion. Adjust the pH to 7.5 with sodium hydroxide solution, bring the volume to 40mL, add 3% pancreatin (w / v), and hydrolyze again in a constant temperature shaker at 37℃ for 60min to simulate intestinal digestion. After the simulated digestion is complete, freeze-dry the digestive fluid for 72h to obtain the digestive product.

[0063] b. Dissolve the casein peptide digestion product obtained after freeze-drying in sterile water to prepare a high-concentration sample solution. Sterilize the solution by passing it through a 0.22 µM sterile membrane and then dilute it to the experimental concentration with culture medium (DMEM high-glucose medium).

[0064] Comparative Example 7 a) Dissolve 1g of rice peptide (Comparative Example 3) in 20mL of hydrochloric acid solution with pH 2.0, add 4% pepsin (w / v), and hydrolyze in a constant temperature shaker at 37℃ for 60min to simulate gastric digestion. Adjust the pH to 7.5 with sodium hydroxide solution, bring the volume to 40mL, add 3% pancreatin (w / v), and hydrolyze again in a constant temperature shaker at 37℃ for 60min to simulate intestinal digestion. After the simulated digestion is complete, freeze-dry the digestive fluid for 72h to obtain the digestive product.

[0065] b. Dissolve the rice peptide digestion product obtained after freeze-drying in sterile water to prepare a high-concentration sample solution. Sterilize the solution by passing it through a 0.22 µM sterile membrane and then dilute it to the experimental concentration with culture medium (DMEM high-glucose medium).

[0066] Comparative Example 8 a) Dissolve 1g of collagen peptides in 20mL of hydrochloric acid solution with pH 2.0, add 4% pepsin (w / v), and hydrolyze in a constant temperature shaker at 37℃ for 60min to simulate gastric digestion. Adjust the pH to 7.5 with sodium hydroxide solution, bring the volume to 40mL, add 3% pancreatin (w / v), and hydrolyze again in a constant temperature shaker at 37℃ for 60min to simulate intestinal digestion. After the simulated digestion is complete, freeze-dry the digestive fluid for 72h to obtain the digestive product.

[0067] b. Dissolve the digested product of the active peptide composition obtained after freeze-drying in sterile water to prepare a high-concentration sample solution. Sterilize the solution by passing it through a 0.22 µM sterile membrane and then dilute it to the experimental concentration with culture medium (DMEM high-glucose medium).

[0068] Detection Example 1 The cell line used in the experiment was HSF cells, which were cultured in DMEM high-glucose medium supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were passaged every 2 to 3 days in an incubator maintained at 37°C and 5% CO2.

[0069] HSF fibroblasts in logarithmic growth phase were selected and digested with 0.25% trypsin. Cell suspensions of the same concentration were seeded into 96-well plates at a volume of 100 µL per well. The plates were incubated at 37°C with 5% CO2 for 24 hours. Cells were observed using an inverted microscope to ensure complete adhesion and good growth. The old culture medium was carefully aspirated using a vacuum pump, and culture medium containing different samples was added, with six replicates for each concentration. A blank control group without sample-containing medium was used. The plates were then incubated at 37°C with 5% CO2 for another 24 hours. The old culture medium was aspirated, and after rinsing with PBS, culture medium containing 10% CCK-8 solution was quickly added. The plates were returned to the 37°C, 5% CO2 incubator and reacted for 2 hours. After the reaction, the absorbance of each well was measured at 450 nm using a microplate reader. The cell proliferation rate was calculated using the following formula:

[0070] In the formula, A1 refers to the OD value of the well containing CCK-8, sample and cells; A2 refers to the OD value of the well containing only CCK-8; A3 refers to the OD value of the well containing CCK-8 and cells.

[0071] The effects of different concentrations of samples prepared in Examples 1, 3, and Comparative Examples 1-4 on the proliferation rate of HSF fibroblasts are shown in the figures. Figure 1 It can be seen that within the experimental concentration range, the mixed peptides at each concentration have no cytotoxicity to fibroblasts. Compared with the blank group (under normal conditions), at a concentration of 200 μg / mL, Examples 1 and 3 can significantly increase the proliferation rate of HSF fibroblasts, by 29.09% and 10.45%, respectively.

[0072] The effects of different concentrations of samples prepared in Examples 2, 4, and Comparative Examples 5-8 on the proliferation rate of HSF fibroblasts are shown in the figures. Figure 2 It can be seen that within the experimental concentration range, the mixed peptides at each concentration have no cytotoxicity to fibroblasts. Compared with the blank group (under normal conditions), Example 4 significantly increased the proliferation rate of HSF fibroblasts at a concentration of 800 μg / mL, by 27.56%, and the effect on the proliferation rate of HSF fibroblasts was more significant than that of the control group.

[0073] Detection Example 2 The cell line used in the experiment was HSF cells, which were cultured in DMEM high-glucose medium supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were passaged every 2 to 3 days in an incubator maintained at 37°C and 5% CO2.

[0074] To determine the effective dose of H2O2 in HSF cells, 100 µL of H2O2 was seeded into each well of a 96-well plate and cultured at 37°C and 5% CO2 for 24 h. The cultured cells were then treated with different concentrations of H2O2 (200 µM, 400 µM, 600 µM, 800 µM, 1000 µM, 1200 µM, and 1400 µM). After 24 h of treatment, the 96-well plates were removed from the incubator, the H2O2-containing culture medium was aspirated using a vacuum pump, and the cells were washed twice with PBS. Then, 200 µL of culture medium containing 10% CCK-8 reagent was quickly added. The cell proliferation rate was calculated using the same method as in Example 1.

[0075] like Figure 3 As shown, HSF cells were treated with H2O2 at concentrations of 0, 200, 400, 600, 800, 1000, 1200, and 1400 µM for 24 h. The results showed that the proliferation rate of HSF cells decreased with increasing H2O2 concentration, exhibiting a dose-dependent effect, indicating that H2O2 induces apoptosis. The optimal concentration for modeling was 50%-75% cell proliferation, with 800 µM resulting in a HSF cell proliferation rate of (68.03±3.13)%. Considering all factors, 800 µM H2O2 was selected as the modeling concentration for the oxidative damage model.

[0076] Select healthy HSF cells and seed 100 µL per well in a 96-well plate. Incubate for 24 h. Observe under an inverted microscope that the cells in each well are fully adherent and the confluence is greater than 80%. Remove the 96-well plate, discard the culture medium, wash with PBS, add complete culture medium (containing 10% FBS and 1% penicillin antibody) to the blank group, and add 800 µM hydrogen peroxide medium to the sample group. Incubate for 24 h, discard the culture medium, add sample culture medium containing different concentrations, and add complete culture medium to the blank group. Continue culturing for another 24 h, and measure cell proliferation rate according to the method in Example 1.

[0077] The ability of different concentrations of compound peptides prepared in Examples 1, 3, and Comparative Examples 1-4 to repair oxidative damage in HSF fibroblasts is shown in the figures. Figure 4 .Depend on Figure 4 It can be seen that, compared with the blank group, the cell proliferation rate of the model group was 56.27%, significantly lower than that of the blank group, indicating that the modeling was successful. Compared with the model group (damaged group), at a concentration of 100 μg / mL, Examples 1 and 3 significantly repaired oxidative damage to cells and improved cell survival. Example 1 showed the highest cell proliferation rate, higher than other single peptide groups, and at the same level as the blank group (normal state), indicating that Example 1 had the best effect on repairing oxidative damage to cells, restoring cellular oxidative damage to the level of the blank group (normal). Compared with the model group (damaged state), the cell proliferation rate of Example 1 increased by 73.80%.

[0078] The effects of different concentrations of compound active peptides prepared in Examples 2, 4, and Comparative Examples 5-8 on the proliferation rate of HSF fibroblasts are shown in the figures below. Figure 5 .Depend on Figure 5 It can be seen that, compared with the blank group, the cell proliferation rate of the model group was 53.87%, significantly lower than that of the blank (normal) group, indicating that the modeling was successful. Compared with the model group (damaged group), at a concentration of 100 μg / mL, Examples 2 and 4 significantly repaired oxidative damage to cells and improved cell survival. Example 2 showed the highest cell proliferation rate, higher than other single peptide groups and significantly higher than the blank group (normal state), indicating that Example 2 had the best effect on repairing oxidative damage to cells, not only repairing oxidative damage to the level of the blank group (normal state) but also significantly better than the blank group (normal state). Compared with the model group (damaged state), the cell proliferation rate of Example 2 increased by 117.83%.

[0079] Detection Example 3 Select healthy HSF cells and seed 100 µL per well in a 96-well plate. Incubate for 24 h in a cell culture incubator. Observe under an inverted microscope that the cells in each well are completely adherent and the confluence is greater than 80%. Remove the 96-well plate and, under a drying air velocity of 0.4 m / s, remove all culture medium from each well. Place the plates in a clean bench for 5, 10, 15, 20, 25, 30, 35, and 40 min respectively. After adding complete culture medium, continue culturing for another 24 h. Measure the absorbance at 570 nm using the CCK-8 assay. The cell drying mortality rate formula is as follows:

[0080] In the formula, A1 is the OD value of the blank group, A2 is the OD value of the experimental group, and A3 is the OD value of the CCK-8 culture medium solution.

[0081] Effects of different air-drying times on HSF fibroblasts, such as Figure 6 As shown, after 5 min of drying stimulation, the cell viability was (52.09±3.02)%. When the drying time was 10 min, the cell viability decreased to (30.25±5.07)%. When the cell proliferation rate was less than 50%, the cell viability was poor, which was not conducive to subsequent research. Furthermore, the cell drying damage experiment results were more stable at 5 min, so 5 min was chosen as the cell drying damage time.

[0082] Select healthy HSF cells and seed 100 µL per well in a 96-well plate. Incubate for 24 h in a cell culture incubator. Observe under an inverted microscope to ensure complete cell adhesion and good growth in each well. Remove the 96-well plate and, except for the control group, remove the culture medium from each well using a pipette. Place the plate in a clean bench with a dry air velocity of 0.4 m / s for 5 min, then add 100 µL of sample culture medium solution of different concentrations and incubate for 24 h. Remove the 96-well plate, discard the culture medium, wash with PBS, and measure the cell proliferation rate according to the method in Example 1.

[0083] The protective effects of different concentrations of compound active peptides prepared in Examples 1, 3, and Comparative Examples 1-4 on desiccation-damaged HSF fibroblasts are as follows: Figure 7 As shown. By Figure 7 As can be seen, compared with the blank group, the proliferation rate of the model group was 73.6%, significantly lower than that of the blank group, indicating that the modeling was successful. Compared with the model group, the compound active peptides increased the proliferation rate of fibroblasts to varying degrees. At a concentration of 1200 μg / mL, Examples 1 and 3 significantly increased the proliferation rate of HSF fibroblasts, reaching the same level as the blank group (normal state), with increases of 30.86% and 26.30%, respectively. This indicates that the active peptide composition can significantly repair cell desiccation damage and improve cell survival.

[0084] The protective effects of different concentrations of compound active peptides prepared in Examples 2, 4, and Comparative Examples 5-8 on desiccation-damaged HSF fibroblasts are as follows: Figure 8 As shown, by Figure 8 It can be seen that, compared with the blank group, the proliferation rate of the model group was 69.44%, significantly lower than that of the blank group, indicating that the modeling was successful. Compared with the model group, at a concentration of 100 μg / mL, the compound active peptides increased the proliferation rate of fibroblasts to varying degrees, with Example 4 showing the best effect, significantly superior to other single peptide groups and the blank group. When the concentration reached 800 μg / mL, Examples 2 and 4 significantly increased the proliferation rate of HSF fibroblasts, and were significantly superior to other single peptide groups. Compared with the model group (dry damage state), Examples 2 and 4 increased the proliferation rate of HSF fibroblasts by 70.72% and 59.68%, respectively.

[0085] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An active peptide composition with cell repair promoting effects, characterized in that, The active peptide composition is composed of sea cucumber peptide, casein peptide, rice peptide and fish skin collagen peptide; the mass ratio of the sea cucumber peptide, casein peptide, rice peptide and fish skin collagen peptide is 6-19:15:52-65:

15.

2. The active peptide composition according to claim 1, characterized by, The mass ratio of the sea cucumber peptide, casein peptide, rice peptide and fish skin collagen peptide is 19:15:52:

15.

3. A method for preparing the active peptide composition according to claim 1 or 2, characterized by, The method comprises the following steps: (1) 3-8 g of rice protein is taken and added into 15-25 times mass of water, the temperature is raised to 45-55 ℃, and the pH is adjusted to 7.0±0.2; (2) 1.5-2.5 ‰ w / w of papain and 1.5-2.5 ‰ w / w of alkaline protease are added, after enzymolysis for 3.5-4.5 h, 0.5-1.5 ‰ w / w of neutral protease is added for continuous enzymolysis for 0.5-1.5 h, and the pH is adjusted to 7.0±0.2; (3) after the enzymolysis is completed, the supernatant is obtained by high-speed centrifugation at 9000-11000 r / min for 10-20 min, the soluble solid content of the supernatant is determined, and the weight of the rice peptide is converted; (4) sea cucumber peptide, casein peptide, rice peptide and fish skin collagen peptide are weighed, stirred and uniformly mixed, and then frozen and vacuum dried to obtain the product.

4. The use of the active peptide composition of claim 1 or 2 in the preparation of a product for promoting cell repair.

5. A product having a cell repair-promoting effect, characterized by, The product comprises the active peptide composition of claim 1 or 2.

6. The product of claim 5, wherein, The product comprises a cosmetic product, a food product or a pharmaceutical product.

7. The product of claim 6, wherein, The cosmetic product comprises at least one of body milk, hand cream, essence, essence oil, emulsion, lotion, face cream, BB cream, shower gel, facial mask, foundation, eye cream, facial cleanser or sunscreen.

8. The product of claim 6, wherein, The food product comprises at least one of health care product or beverage.

9. The product of claim 6, wherein, The pharmaceutical product further comprises a pharmaceutically acceptable excipient.

10. The product of claim 9, wherein, The pharmaceutically acceptable excipient comprises at least one of binding agent, filling agent, disintegrating agent, lubricant, preservative, antioxidant, flavoring agent, aromatic agent, solubilizing agent, emulsifying agent, solubilizing agent or osmotic pressure adjusting agent.

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