Preparation method and effect research of peony flower polypeptide

Abstract

The invention provides a preparation method and efficacy research of peony flower polypeptide. The preparation method of the peony flower polypeptide comprises the following steps: S1, performing ultrahigh-pressure auxiliary pretreatment: crushing selected and cleaned peony flowers into powder, putting the powder into a pressure-resistant preparation container, adding a proper amount of deionized water to prepare uniform slurry, controlling the material-liquid ratio to be (1: 6)-(1: 8), pressurizing the outer surface of the pressure-resistant preparation container, and treating for a corresponding time under the pressure of 100-600MPa, so as to obtain peony flower polypeptide powder; ultrahigh pressure can destroy cell structures and denature macromolecules such as protein, polypeptide is easier to release in subsequent enzymolysis, after the treatment time is up, pressure is released, pulp is taken out, and a compound enzyme solution is prepared: a proper amount of beta-glucosidase can be added except 537 acid proteinase, pectinase and cellulase; according to the preparation method of the peony flower polypeptide, through the design, the cost of preparation equipment of the peony flower polypeptide is reduced, the preparation efficiency of the peony flower polypeptide is improved, side reactions in the preparation process are reduced, and the preparation effect of the peony flower polypeptide is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of peony flower polypeptide preparation, and in particular to a peony flower polypeptide preparation method and efficacy research. Background Art

[0002] The peony, a perennial deciduous shrub belonging to the Paeoniaceae family, has stems up to 2 meters tall and short, thick branches. Its leaves are typically bi-ternate, hairless green on the outside and light green on the back. The most eye-catching feature is the solitary flower at the top of the branch.

[0003] Peony polypeptide is a bioactive peptide extracted from peony flowers. It is obtained from peony flower extracts through a specific process and has a variety of ingredients and functions that are beneficial to the human body. Peony polypeptide is rich in a variety of ingredients that are beneficial to the human body, including 17 amino acids, VC, VE, proanthocyanidins, paeonol, astragaloside, gallic acid, paeoniflorin and polypeptides. Peony polypeptide has multiple biological activities, mainly including antioxidant, anti-inflammatory, immune regulation, and promotion of collagen synthesis.

[0004] However, the traditional preparation process of peony flower peptides has high equipment costs and a long time, low preparation efficiency, and many side reactions during the preparation process will affect the quality and activity of the peptides.

[0005] Therefore, it is necessary to provide a method for preparing peony flower polypeptide and study its efficacy to solve the above technical problems. Summary of the Invention

[0006] The present invention provides a method for preparing a peony polypeptide and research on its efficacy, which solves the problems of high cost, long time, low preparation efficiency and many side reactions in the preparation process that may affect the preparation quality and activity of the peony polypeptide.

[0007] To solve the above technical problems, the present invention provides a method for preparing peony polypeptide, which includes the following preparation methods:

[0008] S1: Ultrahigh pressure assisted pretreatment: After the selected and cleaned peony flowers are crushed into powder, they are placed in a pressure-resistant preparation container, and an appropriate amount of deionized water is added to form a uniform slurry. The material-liquid ratio is controlled at 1:6-1:8. The outer surface of the pressure-resistant preparation container is then pressurized and treated at a pressure of 100-600 MPa for a corresponding time. Ultrahigh pressure can destroy cell structure and denature macromolecules such as proteins, making it easier to release polypeptides in subsequent enzymatic hydrolysis. After the treatment time is up, the pressure is released and the slurry is removed;

[0009] S2: Prepare complex enzyme solution: In addition to 537 acid protease, pectinase and cellulase, add appropriate amount of β-glucosidase. Calculated based on the protein mass of the slurry, 537 acid protease 8000u / g, pectinase and cellulase 3g each, β-glucosidase 1-2g, mix and dissolve;

[0010] S3: Membrane separation coupled reaction: The pretreated peony pollen slurry is transferred to an enzymatic hydrolysis-membrane separation coupled reaction vessel, the pH is adjusted to 4.5-5.0, the temperature is 50-55°C, and the enzyme solution is added to allow the peony pollen slurry and the enzyme solution to fully react. After the reaction is completed, the reaction system after enzymatic hydrolysis is rapidly heated to 90-95°C and maintained for 5-10 minutes. The high temperature denatures the protein structure of the enzyme, thereby losing its activity, terminating the enzymatic hydrolysis reaction and performing enzyme inactivation treatment;

[0011] S4: Separation and purification: Separation and purification is performed using a magnetic nanoparticle adsorption separation method. Magnetic nanoparticles are added to the crude polypeptide extract and incubated with shaking at a certain temperature of 25-35°C for 30-60 minutes to allow the nanoparticles to fully bind to the polypeptide. The nanoparticles adsorbed with the polypeptide are separated using an external magnetic field, and eluted with a suitable eluent. The eluate is collected to obtain a purified peony polypeptide solution;

[0012] S5: Drying: Drying is performed using a microwave vacuum drying method. The polypeptide solution is placed on a tray of a microwave vacuum drying device with a spreading thickness not exceeding 1 cm. The microwave power is set to 200-500W and the vacuum degree is set to 0.08-0.09 MPa. The material is continuously stirred during the drying process to prevent local overheating. Dry until the moisture content is less than 5%. Remove the solution, cool it, and seal it for packaging.

[0013] During the reaction, an ultrafiltration membrane with a molecular weight cutoff of 500-1000Da is used to separate the small molecule polypeptides and hydrolysis products produced by enzymatic hydrolysis in real time, continuously replenish the substrate, maintain the forward progress of the reaction, and shorten the enzymatic hydrolysis time to 2-3 hours.

[0014] Preferably, the pressure-resistant preparation container used in the peony polypeptide preparation method includes: a supporting frame;

[0015] A booster tank, which is fixedly connected to the top of the support base frame through a fixing frame, a preparation tank is installed inside the booster tank through a mounting frame, and a discharge pipe is installed at the bottom of the preparation tank;

[0016] A valve is provided at one end of the discharge pipe, and there is a certain space between the boosting tank and the preparation tank, which is convenient for pressurizing the preparation tank.

[0017] Preferably, a second sealing cover is installed on the top of the boosting tank, a pressure gauge is installed on the top of the second sealing cover, a first sealing cover is installed on the top of the preparation tank, a heating device is installed on the top of the first sealing cover, and a feeding structure is installed on the top of the first sealing cover;

[0018] The heating equipment can heat the liquid inside the preparation tank, the pressure gauge can monitor the pressure value between the preparation tank and the boosting tank, and the feeding structure includes pipes and valves.

[0019] Preferably, the support chassis comprises a bottom plate, a mounting structure and an adjustment structure, the mounting structure being used to mount the adjustment structure on the bottom of the bottom plate, a boosting device being mounted on the top of the support chassis, a pressure supply pipe being mounted at the outlet of the boosting device, a connecting pipe being mounted at the other end of the pressure supply pipe via a first valve, and a filter assembly with an air intake pipe being mounted on the top of the support chassis;

[0020] The adjusting structure and the mounting structure are threadedly connected.

[0021] Preferably, a control box is installed on the top of the supporting chassis, a box door is rotatably connected to the front of the control box, and an operation screen is installed on one side of the control box;

[0022] The operating screen can be used to set the equipment operating parameters. The box door is equipped with a lock. The control box is equipped with a power switch and a controller for controlling the operation of the equipment.

[0023] Preferably, a driving assembly is installed on the top of the second sealing cover, and a stirring frame is installed on the output end of the driving assembly through a rotating shaft, and the driving assembly includes a protective shell, an angle sensor and a driving component;

[0024] The driving component is an electric motor.

[0025] Preferably, the filter assembly comprises a filter box, an intercepting component and a mounting pipe, wherein the mounting pipe is used to connect the filter box and the inlet of the boosting device;

[0026] The intercepting component is a filter.

[0027] Preferably, a fixing ring is installed near the bottom of the preparation tank, and a conical intercepting structure is installed on the top of the fixing ring;

[0028] The conical interception structure is located below the stirring frame.

[0029] Preferably, a slag discharge pipe is installed at the bottom of the conical intercepting structure, and a second valve is installed at the other end of the slag discharge pipe;

[0030] After the second valve is opened, the residue can be discharged. If necessary, a scraper can be installed at the bottom of the mixing frame.

[0031] A study on the efficacy of a peony polypeptide, wherein the study requires the use of a peony polypeptide, comprises the following steps:

[0032] S1: Peonies of different varieties and growth stages were selected, and high-purity peony peptide samples were obtained using innovative preparation methods. A blank control group was also prepared. The peony peptide samples were analyzed using proteomics and metabolomics techniques. Liquid chromatography-mass spectrometry (LC-MS) was used to identify the amino acid sequence, molecular weight, and other information of the peptides. Metabolomics analysis was used to detect metabolites related to skin care efficacy. With the help of bioinformatics tools, data from different samples were compared and analyzed to screen out key peptide components and their metabolites with potential skin care activity. Their targets and pathways were preliminarily inferred, thereby completing the exploration of components and mechanisms of action.

[0033] S2: Through cell culture: culture human epidermal keratinocytes and dermal fibroblasts separately, and wait for the cells to grow to the logarithmic growth phase for use; model construction: use bioprinting technology or layer-by-layer assembly method to construct a 3D skin model, inoculate keratinocytes on the upper layer to form the epidermis, and mix fibroblasts with extracellular matrix components and inoculate them on the lower layer to form the dermis. Culture for several days to mature the model; efficacy evaluation: apply different concentrations of peony peptide solution to the 3D skin model, set up blank control group and positive control group, and evaluate the antioxidant, anti-wrinkle, moisturizing, anti-inflammatory and other skin care effects of peony peptide by detecting indicators such as cell viability, collagen secretion, hyaluronic acid content, and inflammatory factor expression, thereby completing the in vitro test;

[0034] S3: Model construction: Using CRISPR-Cas9 gene editing technology, construct animal models with gene defects or overexpression related to skin aging, inflammation, etc., making them closer to human skin diseases or aging conditions; Experimental treatment: Prepare peony flower peptides into suitable dosage forms such as creams and gels, apply them to the skin surface of animal models, set up different dosage groups, and set up blank control groups and positive drug control groups; Index detection: Regularly observe changes in the appearance of animal skin, and detect collagen content, inflammatory cell infiltration, oxidative stress indicators, etc. through skin tissue section staining such as HE staining, Masson staining, and immunohistochemical analysis to evaluate the skin care efficacy of peony flower peptides, thereby completing in vivo experiments;

[0035] S3: Through subject recruitment and grouping: Recruit subjects with different skin types (dry, oily, mixed, sensitive), and different age groups, and randomly divide them into different experimental and control groups; Skin testing: Use skin testers such as VISIA skin testers and Corneometer moisture testers to conduct comprehensive skin tests on subjects to obtain baseline data, including skin moisture content, pigmentation, wrinkle depth, sebum secretion, etc.; Personalized intervention: Based on the subject's skin test results, develop a personalized peony peptide skin care plan, adjust the product dosage form, frequency of use, and dosage. The control group uses a placebo product; Long-term follow-up evaluation: At different time points after using the product, such as 2 weeks, 4 weeks, and 8 weeks, the subject's skin will be tested again. Combined with the subject's self-assessment questionnaire, the effectiveness and safety of the personalized peony peptide skin care plan will be comprehensively evaluated, thus completing the human clinical trial.

[0036] Compared with related technologies, the preparation method of peony flower polypeptide provided by the present invention and its efficacy research have the following beneficial effects:

[0037] The invention provides a method for preparing a peony flower polypeptide. In order to improve the efficiency and effect of the peony flower polypeptide preparation, the selected and cleaned peony flowers are first crushed into powder, and then placed in a pressure-resistant preparation container. An appropriate amount of deionized water is added to form a uniform slurry, and the material-liquid ratio is controlled at 1:6-1:8. The pressure-resistant preparation container is located in an ultra-high pressure device. The outer surface of the pressure-resistant preparation container is pressurized to 100-600 MPa and treated for a corresponding time. The ultra-high pressure can destroy the cell structure, denature macromolecules such as proteins, and make it easier to release polypeptides in subsequent enzymatic hydrolysis, which is beneficial to improving the pretreatment time. After the treatment time is reached, the pressure is released and the slurry is taken out. In this process, in addition to 537 acid protease, pectinase and cellulase, an appropriate amount of beta-glucosidase can be added. Calculated based on the protein mass of the slurry, 8000 u / g of 537 acid protease, 3 g of pectinase and cellulase, and 1-2 g of beta-glucosidase are added to prepare a composite enzyme solution. After the preparation of the slurry is completed, the pretreated peony pollen slurry is transferred to an enzymatic hydrolysis-membrane separation coupling reaction container, the pH is adjusted to 4.5-5.0 and the temperature is 50-55° C., an enzyme solution is added, and the peony pollen slurry and the enzyme solution are fully reacted. After the reaction is completed, the reaction system after the enzymatic hydrolysis is rapidly heated to 90-95° C. and maintained for 5-10 minutes. The high temperature denatures the protein structure of the enzyme, thereby losing its activity, terminating the enzymatic hydrolysis reaction, and performing an enzyme inactivation treatment. After the treatment is completed, a magnetic nanoparticle adsorption separation method is used for separation and purification. After the purification is completed, a microwave vacuum drying method is used for drying. During the drying process, the material is continuously stirred to prevent local overheating. The material is dried until the moisture content is less than 5%, and the material is taken out, cooled, and sealed for packaging. The design reduces the cost of peony polypeptide preparation equipment, improves the peony polypeptide preparation efficiency, reduces side reactions during the preparation process, and is conducive to improving the peony polypeptide preparation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the first embodiment of the method for preparing peony polypeptide provided by the present invention;

[0039] Figure 2 A schematic structural diagram of a pressurized tank is provided for the present invention;

[0040] Figure 3 Provides a structural schematic diagram of a filter assembly for the present invention;

[0041] Figure 4 Provides a structural schematic diagram of a preparation tank for the present invention;

[0042] Figure 5 Provides a structural schematic diagram of the interception component of the present invention;

[0043] Figure 6 Provides a structural schematic diagram of a driving component for the present invention;

[0044] Figure 7 A schematic diagram of the present invention is provided for studying the efficacy of peony polypeptides;

[0045] Figure 8 This is a schematic structural diagram of a second embodiment of the method for preparing peony polypeptides provided by the present invention;

[0046] Figure 9 A structural schematic diagram of a conical intercepting structure is provided for the present invention.

[0047] Numbers in the figure: 1. Support base frame, 101. Bottom plate, 102. Mounting structure, 103. Adjustment structure, 2. Fixed frame, 3. Control box, 4. Box door, 5. Operation screen, 6. Booster equipment, 7. Air intake pipe, 8. Filter assembly, 801. Filter box, 802. Mounting pipe, 803. Interception component, 9. Pressure supply pipe, 10. First valve, 11. Feed structure, 12. Drive assembly, 121. Protective shell, 122. Angle sensor, 123. Drive component, 13. First sealing cover, 14. Second sealing cover, 15. Connecting pipe, 16. Booster tank, 17. Discharge pipe, 18. Pressure gauge, 19. Heating equipment, 20. Preparation tank, 21. Mounting frame, 22. Rotating shaft, 23. Stirring frame, 24. Slag discharge pipe, 25. Fixed ring, 26. Conical interception structure, 27. Second valve. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0049] Please refer to Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 and Figure 7 ,in, Figure 1 This is a schematic diagram of the first embodiment of the method for preparing peony polypeptide provided by the present invention; Figure 2 A schematic structural diagram of a pressurized tank is provided for the present invention; Figure 3 Provides a structural schematic diagram of a filter assembly for the present invention; Figure 4 Provides a structural schematic diagram of a preparation tank for the present invention; Figure 5 Provides a structural schematic diagram of the interception component of the present invention; Figure 6 Provides a structural schematic diagram of a driving component for the present invention; Figure 7 The present invention provides a schematic diagram of the research on the efficacy of peony flower polypeptide. The preparation method of peony flower polypeptide includes the following preparation methods:

[0050] S1: Ultrahigh pressure assisted pretreatment: After the selected and cleaned peony flowers are crushed into powder, they are placed in a pressure-resistant preparation container, and an appropriate amount of deionized water is added to form a uniform slurry. The material-liquid ratio is controlled at 1:6-1:8. The outer surface of the pressure-resistant preparation container is then pressurized and treated at a pressure of 100-600 MPa for a corresponding time. Ultrahigh pressure can destroy cell structure and denature macromolecules such as proteins, making it easier to release polypeptides in subsequent enzymatic hydrolysis. After the treatment time is up, the pressure is released and the slurry is removed;

[0051] S2: Prepare complex enzyme solution: In addition to 537 acid protease, pectinase and cellulase, add appropriate amount of β-glucosidase. Calculated based on the protein mass of the slurry, 537 acid protease 8000u / g, pectinase and cellulase 3g each, β-glucosidase 1-2g, mix and dissolve;

[0052] S3: Membrane separation coupled reaction: The pretreated peony pollen slurry is transferred to an enzymatic hydrolysis-membrane separation coupled reaction vessel, the pH is adjusted to 4.5-5.0, the temperature is 50-55°C, and the enzyme solution is added to allow the peony pollen slurry and the enzyme solution to fully react. After the reaction is completed, the reaction system after enzymatic hydrolysis is rapidly heated to 90-95°C and maintained for 5-10 minutes. The high temperature denatures the protein structure of the enzyme, thereby losing its activity, terminating the enzymatic hydrolysis reaction and performing enzyme inactivation treatment;

[0053] S4: Separation and purification: Separation and purification is performed using a magnetic nanoparticle adsorption separation method. Magnetic nanoparticles are added to the crude polypeptide extract and incubated with shaking at a certain temperature of 25-35°C for 30-60 minutes to allow the nanoparticles to fully bind to the polypeptide. The nanoparticles adsorbed with the polypeptide are separated using an external magnetic field, and eluted with a suitable eluent. The eluate is collected to obtain a purified peony polypeptide solution;

[0054] S5: Drying: Drying is performed using a microwave vacuum drying method. The polypeptide solution is placed on a tray of a microwave vacuum drying device with a spreading thickness not exceeding 1 cm. The microwave power is set to 200-500W and the vacuum degree is set to 0.08-0.09 MPa. The material is continuously stirred during the drying process to prevent local overheating. Dry until the moisture content is less than 5%. Remove the solution, cool it, and seal it for packaging.

[0055] During the reaction, an ultrafiltration membrane with a molecular weight cutoff of 500-1000Da is used to separate the small molecule polypeptides and hydrolysis products produced by enzymatic hydrolysis in real time, continuously replenish the substrate, maintain the forward progress of the reaction, and shorten the enzymatic hydrolysis time to 2-3 hours.

[0056] The pressure-resistant preparation container used in the method for preparing peony polypeptide comprises: a supporting frame 1;

[0057] A boost tank 16 is fixedly connected to the top of the support frame 1 via a fixing frame 2. A preparation tank 20 is installed inside the boost tank 16 via a mounting frame 21. A discharge pipe 17 is installed at the bottom of the preparation tank 20.

[0058] A valve is provided at one end of the discharge pipe 17 , and there is a certain space between the pressurizing tank 16 and the preparation tank 20 , which facilitates pressurizing the preparation tank 20 .

[0059] A second sealing cover 14 is installed on the top of the boosting tank 16, a pressure gauge 18 is installed on the top of the second sealing cover 14, a first sealing cover 13 is installed on the top of the preparation tank 20, a heating device 19 is installed on the top of the first sealing cover 13, and a feeding structure 11 is installed on the top of the first sealing cover 13;

[0060] The heating device 19 can heat the liquid inside the preparation tank 20, the pressure gauge 18 can monitor the pressure value between the preparation tank 20 and the boosting tank 16, and the feeding structure 11 includes a pipe and a valve. After the feeding is completed, the valve is closed to seal the preparation tank 20.

[0061] The support frame 1 includes a base plate 101, a mounting structure 102, and an adjustment structure 103. The mounting structure 102 is used to mount the adjustment structure 103 on the bottom of the base plate 101. A booster device 6 is mounted on the top of the support frame 1. A pressure supply pipe 9 is mounted at the outlet of the booster device 6. A connecting pipe 15 is mounted on the other end of the pressure supply pipe 9 through a first valve 10. A filter assembly 8 with an air intake pipe 7 is mounted on the top of the support frame 1.

[0062] The adjusting structure 103 and the mounting structure 102 are threadedly connected to each other, which can increase the stability of the base plate 101 . The other end of the connecting pipe 15 is connected to the second sealing cover 14 , and the boosting device 6 boosts the pressure in the boosting tank 16 .

[0063] A control box 3 is installed on the top of the supporting chassis 1, a box door 4 is rotatably connected to the front of the control box 3, and an operation screen 5 is installed on one side of the control box 3;

[0064] The operation screen 5 can be used to set the equipment operating parameters, the box door 4 is provided with a lock, and the control box 3 is internally installed with a power switch and a controller for controlling the operation of the equipment.

[0065] A driving assembly 12 is installed on the top of the second sealing cover 14. A stirring frame 23 is installed on the output end of the driving assembly 12 via a rotating shaft 22. The driving assembly 12 includes a protective shell 121, an angle sensor 122 and a driving component 123.

[0066] The driving component 123 is an electric motor, and the angle sensor 122 cooperates with the driving component 123 controller to accurately control the speed and rotation angle.

[0067] The filter assembly 8 comprises a filter box 801, an intercepting component 803 and a mounting pipe 802, wherein the mounting pipe 802 is used to connect the filter box 801 and the inlet of the boosting device 6;

[0068] The intercepting component 803 is a filter screen, and a cover is installed on the top of the filter box 801.

[0069] A study on the efficacy of a peony polypeptide, wherein the study requires the use of a peony polypeptide, comprises the following steps:

[0070] S1: Peonies of different varieties and growth stages were selected, and high-purity peony peptide samples were obtained using innovative preparation methods. A blank control group was also prepared. The peony peptide samples were analyzed using proteomics and metabolomics techniques. Liquid chromatography-mass spectrometry (LC-MS) was used to identify the amino acid sequence, molecular weight, and other information of the peptides. Metabolomics analysis was used to detect metabolites related to skin care efficacy. With the help of bioinformatics tools, data from different samples were compared and analyzed to screen out key peptide components and their metabolites with potential skin care activity. Their targets and pathways were preliminarily inferred, thereby completing the exploration of components and mechanisms of action.

[0071] S2: Through cell culture: culture human epidermal keratinocytes and dermal fibroblasts separately, and wait for the cells to grow to the logarithmic growth phase for use; model construction: use bioprinting technology or layer-by-layer assembly method to construct a 3D skin model, inoculate keratinocytes on the upper layer to form the epidermis, and mix fibroblasts with extracellular matrix components and inoculate them on the lower layer to form the dermis. Culture for several days to mature the model; efficacy evaluation: apply different concentrations of peony peptide solution to the 3D skin model, set up blank control group and positive control group, and evaluate the antioxidant, anti-wrinkle, moisturizing, anti-inflammatory and other skin care effects of peony peptide by detecting indicators such as cell viability, collagen secretion, hyaluronic acid content, and inflammatory factor expression, thereby completing the in vitro test;

[0072] S3: Model construction: Using CRISPR-Cas9 gene editing technology, construct animal models with gene defects or overexpression related to skin aging, inflammation, etc., making them closer to human skin diseases or aging conditions; Experimental treatment: Prepare peony flower peptides into suitable dosage forms such as creams and gels, apply them to the skin surface of animal models, set up different dosage groups, and set up blank control groups and positive drug control groups; Index detection: Regularly observe changes in the appearance of animal skin, and detect collagen content, inflammatory cell infiltration, oxidative stress indicators, etc. through skin tissue section staining such as HE staining, Masson staining, and immunohistochemical analysis to evaluate the skin care efficacy of peony flower peptides, thereby completing in vivo experiments;

[0073] S3: Through subject recruitment and grouping: Recruit subjects with different skin types (dry, oily, mixed, sensitive), and different age groups, and randomly divide them into different experimental and control groups; Skin testing: Use skin testers such as VISIA skin testers and Corneometer moisture testers to conduct comprehensive skin tests on subjects to obtain baseline data, including skin moisture content, pigmentation, wrinkle depth, sebum secretion, etc.; Personalized intervention: Based on the subject's skin test results, develop a personalized peony peptide skin care plan, adjust the product dosage form, frequency of use, and dosage. The control group uses a placebo product; Long-term follow-up evaluation: At different time points after using the product, such as 2 weeks, 4 weeks, and 8 weeks, the subject's skin will be tested again. Combined with the subject's self-assessment questionnaire, the effectiveness and safety of the personalized peony peptide skin care plan will be comprehensively evaluated, thus completing the human clinical trial.

[0074] The working principles of the method for preparing peony polypeptides provided by the present invention and the research on their efficacy are as follows:

[0075] First, the selected and cleaned peony flowers are crushed into powder, and then placed in a pressure-resistant preparation container. An appropriate amount of deionized water is added to form a uniform slurry, and the material-liquid ratio is controlled at 1:6-1:8. The pressure-resistant preparation container is located in an ultra-high pressure device. By pressurizing the outer surface of the pressure-resistant preparation container to 100-600MPa and treating it for a corresponding time, ultra-high pressure can destroy the cell structure, denature macromolecules such as proteins, and make it easier to release polypeptides in subsequent enzymatic hydrolysis, which is beneficial to increase the pretreatment time. After the treatment time is reached, the pressure is released and the slurry is taken out. In this process, in addition to 537 acid protease, pectinase and cellulase, an appropriate amount of β-glucosidase can be added. According to the slurry protein mass, 537 acid protease 8000u / g, pectinase and cellulase 3g each, β-glucosidase 1 -2g, prepare a composite enzyme solution, after the preparation of the peony pollen slurry is completed, transfer the pretreated peony pollen slurry to an enzymatic hydrolysis-membrane separation coupling reaction vessel, adjust the pH to 4.5-5.0, the temperature to 50-55°C, add the enzyme solution, and allow the peony pollen slurry and the enzyme solution to fully react. After the reaction is completed, the reaction system after enzymatic hydrolysis is quickly heated to 90-95°C and maintained for 5-10 minutes. The high temperature denatures the protein structure of the enzyme, thereby losing its activity, terminating the enzymatic hydrolysis reaction, and performing enzyme inactivation treatment. After the treatment is completed, separation and purification are performed using a magnetic nanoparticle adsorption separation method. After purification is completed, drying is performed using a microwave vacuum drying method. During the drying process, the material is continuously stirred to prevent local overheating. Drying is performed until the moisture content is less than 5%, and the product is taken out, cooled, and sealed and packaged.

[0076] After the peony flowers and the corresponding amount of liquid are transported to the interior of the preparation tank 20 through the feeding structure 11, the valve inside the feeding structure 11 is closed, and then the boosting device 6 is started to increase the pressure inside the boosting tank 16, so that the preparation tank 20 is in a corresponding pressure environment. During this process, the driving component 12 is started to stir the peony flowers and liquid inside the preparation tank 20 through the stirring frame 23 to improve the mixing efficiency. At the same time, the pressure value is monitored by the pressure gauge 18, and the heating device 19 is used to heat the slurry inside the preparation tank 20. After the preparation is completed, the valve on the discharge pipe 17 is opened to discharge the prepared slurry.

[0077] Compared with related technologies, the preparation method of peony flower polypeptide provided by the present invention and its efficacy research have the following beneficial effects:

[0078] In order to improve the efficiency and effect of peony flower polypeptide preparation, the selected and cleaned peony flowers are first crushed into powder, placed in a pressure-resistant preparation container, and an appropriate amount of deionized water is added to make a uniform slurry. The material-liquid ratio is controlled at 1:6-1:8, and the pressure-resistant preparation container is located in an ultra-high pressure device. By pressurizing the outer surface of the pressure-resistant preparation container to 100-600MPa and treating it for a corresponding time, ultra-high pressure can destroy the cell structure, denature macromolecules such as proteins, and make it easier to release polypeptides in subsequent enzymatic hydrolysis, which is beneficial to increase the pretreatment time. After the treatment time is reached, the pressure is released and the slurry is taken out. In this process, in addition to 537 acid protease, pectinase and cellulase, an appropriate amount of β-glucosidase can be added. According to the slurry protein mass, 537 acid protease 8000u / g, pectinase and cellulase 3g each, β-glucosidase 1-2g, and a composite enzyme solution are prepared. After the peony pollen slurry is prepared, The pretreated peony pollen slurry is transferred to an enzymatic hydrolysis-membrane separation coupled reaction vessel, the pH is adjusted to 4.5-5.0 and the temperature is 50-55° C., an enzyme solution is added, and the peony pollen slurry and the enzyme solution are allowed to fully react. After the reaction is completed, the reaction system after enzymatic hydrolysis is rapidly heated to 90-95° C. and maintained for 5-10 minutes. The high temperature denatures the protein structure of the enzyme, thereby losing its activity, terminating the enzymatic hydrolysis reaction, and performing enzyme inactivation treatment. After the treatment is completed, separation and purification are performed using a magnetic nanoparticle adsorption separation method. After purification, drying is performed using a microwave vacuum drying method. During the drying process, the material is continuously stirred to prevent local overheating. The material is dried until the moisture content is less than 5%. The material is taken out, cooled, and sealed and packaged. This design reduces the cost of peony polypeptide preparation equipment, improves the efficiency of peony polypeptide preparation, reduces side reactions during the preparation process, and is conducive to improving the effect of peony polypeptide preparation.

[0079] Second embodiment

[0080] Please refer to Figure 8-Figure 9 , Figure 8 This is a schematic structural diagram of a second embodiment of the method for preparing peony polypeptides provided by the present invention; Figure 9 The present invention provides a schematic diagram of a conical interception structure. Based on the method for preparing a peony flower polypeptide and its efficacy research provided in the first embodiment of this application, the second embodiment of this application provides another method for preparing a peony flower polypeptide and its efficacy research. The second embodiment is merely a preferred embodiment of the first embodiment, and its implementation will not affect the independent implementation of the first embodiment.

[0081] Specifically, the method for preparing peony polypeptide and its efficacy research provided in the second embodiment of the present application are different in that a fixing ring 25 is installed near the bottom of the preparation tank 20, and a conical intercepting structure 26 is installed on the top of the fixing ring 25;

[0082] The conical intercepting structure 26 is located below the stirring frame 23 and does not affect the rotation of the stirring frame 23 .

[0083] A slag discharge pipe 24 is installed at the bottom of the conical interception structure 26, and a second valve 27 is installed at the other end of the slag discharge pipe 24;

[0084] After the second valve 27 is opened, the residue can be discharged. If necessary, a scraper can be installed at the bottom of the stirring frame 23 to clean the conical intercepting structure 26 during the rotation of the stirring frame 23.

[0085] Compared with related technologies, the preparation method of peony flower polypeptide provided by the present invention and its efficacy research have the following beneficial effects:

[0086] To facilitate the discharge of residue during the peony flower pretreatment process, a conical interception structure 26 is installed inside the preparation tank 20 through a fixing ring 25. Then, a residue discharge pipe 24 with a second valve 27 is installed at the bottom of the conical interception structure 26. During actual use, when the peony flower fragments are liquefied inside the preparation tank 20, the residue will be intercepted and filtered by the conical interception structure 26. After the peony pollen slurry is prepared and discharged, the second valve 27 can be opened to discharge the residue from the inside of the conical interception structure 26. This design can preliminarily filter the peony pollen slurry, which is beneficial to improving the efficiency of peony flower polypeptide preparation.

[0087] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing peony polypeptide, characterized in that: The invention comprises the following preparation methods: S1: Ultrahigh pressure assisted pretreatment: After the selected and cleaned peony flowers are crushed into powder, they are placed in a pressure-resistant preparation container, and an appropriate amount of deionized water is added to form a uniform slurry. The material-liquid ratio is controlled at 1:6-1:

8. The outer surface of the pressure-resistant preparation container is then pressurized and treated at a pressure of 100-600 MPa for a corresponding time. Ultrahigh pressure can destroy cell structure and denature macromolecules such as proteins, making it easier to release polypeptides in subsequent enzymatic hydrolysis. After the treatment time is up, the pressure is released and the slurry is removed; S2: Prepare complex enzyme solution: In addition to 537 acid protease, pectinase and cellulase, add appropriate amount of β-glucosidase. Calculated based on the protein mass of the slurry, 537 acid protease 8000u / g, pectinase and cellulase 3g each, β-glucosidase 1-2g, mix and dissolve; S3: Membrane separation coupled reaction: The pretreated peony pollen slurry is transferred to an enzymatic hydrolysis-membrane separation coupled reaction vessel, the pH is adjusted to 4.5-5.0, the temperature is 50-55°C, and the enzyme solution is added to allow the peony pollen slurry and the enzyme solution to fully react. After the reaction is completed, the reaction system after enzymatic hydrolysis is rapidly heated to 90-95°C and maintained for 5-10 minutes. The high temperature denatures the protein structure of the enzyme, thereby losing its activity, terminating the enzymatic hydrolysis reaction and performing enzyme inactivation treatment; S4: Separation and purification: Separation and purification is performed using a magnetic nanoparticle adsorption separation method. Magnetic nanoparticles are added to the crude polypeptide extract and incubated with shaking at a certain temperature of 25-35°C for 30-60 minutes to allow the nanoparticles to fully bind to the polypeptide. The nanoparticles adsorbed with the polypeptide are separated using an external magnetic field, and eluted with a suitable eluent. The eluate is collected to obtain a purified peony polypeptide solution; S5: Drying: Use microwave vacuum drying to dry the peptide solution. Place the peptide solution on the tray of the microwave vacuum drying equipment with a spreading thickness of no more than 1 cm. Set the microwave power to 200-500W and the vacuum degree to 0.08-0.09 MPa. Stir the material continuously during the drying process to prevent local overheating. Dry until the moisture content is less than 5%. Remove the solution and seal it after cooling.

2. The pressure-resistant preparation container used in the method for preparing peony polypeptide according to claim 1, characterized in that: include: Support chassis; The boosting tank is fixedly connected to the top of the supporting base frame through a fixing frame, a preparation tank is installed inside the boosting tank through a mounting frame, and a discharge pipe is installed at the bottom of the preparation tank.

3. The method for preparing peony polypeptide according to claim 2, characterized in that: A second sealing cover is installed on the top of the boosting tank, a pressure gauge is installed on the top of the second sealing cover, a first sealing cover is installed on the top of the preparation tank, a heating device is installed on the top of the first sealing cover, and a feeding structure is installed on the top of the first sealing cover.

4. The method for preparing peony polypeptide according to claim 2, characterized in that: The supporting base frame includes a base plate, a mounting structure and an adjustment structure. The mounting structure is used to install the adjustment structure on the bottom of the base plate. A boosting device is installed on the top of the supporting base frame. A pressure supply pipe is installed at the outlet of the boosting device. A connecting pipe is installed at the other end of the pressure supply pipe through a first valve. A filter assembly with an air intake pipe is installed on the top of the supporting base frame.

5. The method for preparing peony polypeptide according to claim 2, characterized in that: A control box is installed on the top of the supporting chassis, a box door is rotatably connected to the front of the control box, and an operation screen is installed on one side of the control box.

6. The method for preparing peony polypeptide according to claim 3, characterized in that: A driving assembly is installed on the top of the second sealing cover, and a stirring frame is installed on the output end of the driving assembly through a rotating shaft. The driving assembly includes a protective shell, an angle sensor and a driving component.

7. The method for preparing peony polypeptide according to claim 4, characterized in that: The filter assembly comprises a filter box, an intercepting component and a mounting pipe, wherein the mounting pipe is used to connect the filter box and the inlet of the boosting device.

8. The method for preparing peony polypeptide according to claim 2, characterized in that: A fixing ring is installed near the bottom of the preparation tank, and a conical intercepting structure is installed on the top of the fixing ring.

9. The method for preparing peony polypeptide according to claim 8, characterized in that: A slag discharge pipe is installed at the bottom of the conical intercepting structure, and a second valve is installed at the other end of the slag discharge pipe.

10. A study on the efficacy of peony polypeptide, characterized in that: The method for preparing a peony flower polypeptide according to any one of claims 1 to 9, wherein the peony flower polypeptide is required for efficacy research, comprising the following steps: S1: Peonies of different varieties and growth stages were selected, and high-purity peony peptide samples were obtained using innovative preparation methods. A blank control group was also prepared. The peony peptide samples were analyzed using proteomics and metabolomics techniques. Liquid chromatography-mass spectrometry (LC-MS) was used to identify the amino acid sequence, molecular weight, and other information of the peptides. Metabolomics analysis was used to detect metabolites related to skin care efficacy. With the help of bioinformatics tools, data from different samples were compared and analyzed to screen out key peptide components and their metabolites with potential skin care activity. Their targets and pathways were preliminarily inferred, thereby completing the exploration of components and mechanisms of action. S2: Through cell culture: culture human epidermal keratinocytes and dermal fibroblasts separately, and wait for the cells to grow to the logarithmic growth phase for use; model construction: use bioprinting technology or layer-by-layer assembly method to construct a 3D skin model, inoculate keratinocytes on the upper layer to form the epidermis, and mix fibroblasts with extracellular matrix components and inoculate them on the lower layer to form the dermis. Culture for several days to mature the model; efficacy evaluation: apply different concentrations of peony peptide solution to the 3D skin model, set up blank control group and positive control group, and evaluate the antioxidant, anti-wrinkle, moisturizing, anti-inflammatory and other skin care effects of peony peptide by detecting indicators such as cell viability, collagen secretion, hyaluronic acid content, and inflammatory factor expression, thereby completing the in vitro test; S3: Model construction: Using CRISPR-Cas9 gene editing technology, construct animal models with gene defects or overexpression related to skin aging, inflammation, etc., making them closer to human skin diseases or aging conditions; Experimental treatment: Prepare peony flower peptides into suitable dosage forms such as creams and gels, apply them to the skin surface of animal models, set up different dosage groups, and set up blank control groups and positive drug control groups; Index detection: Regularly observe changes in the appearance of animal skin, and detect collagen content, inflammatory cell infiltration, oxidative stress indicators, etc. through skin tissue section staining such as HE staining, Masson staining, and immunohistochemical analysis to evaluate the skin care efficacy of peony flower peptides, thereby completing in vivo experiments; S3: Through subject recruitment and grouping: Recruit subjects with different skin types (dry, oily, mixed, sensitive), and different age groups, and randomly divide them into different experimental and control groups; Skin testing: Use skin testers such as VISIA skin testers and Corneometer moisture testers to conduct comprehensive skin tests on subjects to obtain baseline data, including skin moisture content, pigmentation, wrinkle depth, sebum secretion, etc.; Personalized intervention: Based on the subject's skin test results, develop a personalized peony peptide skin care plan, adjust the product dosage form, frequency of use, and dosage. The control group uses a placebo product; Long-term follow-up evaluation: At different time points after using the product, such as 2 weeks, 4 weeks, and 8 weeks, the subject's skin will be tested again. Combined with the subject's self-assessment questionnaire, the effectiveness and safety of the personalized peony peptide skin care plan will be comprehensively evaluated, thus completing the human clinical trial.

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