Paeonia ostii flower peptide capable of whitening, moisturizing, tightening and promoting collagen synthesis as well as preparation method and application of paeonia ostii flower peptide

Abstract

The invention relates to the technical field of deep processing of paeonia ostii, in particular to paeonia ostii peptide capable of whitening, moisturizing, tightening and promoting collagen synthesis as well as a preparation method and application of the paeonia ostii peptide. The paeonia ostii flower peptide capable of whitening, moisturizing, tightening and promoting collagen synthesis is prepared through water bath, homogenization, alkali extraction and acid precipitation pretreatment, three times of enzymolysis through compound enzyme of acid protease, cellulase and pectinase and alkaline protease and solid-liquid separation, operation is easy, the requirement for equipment is low, and the paeonia ostii flower peptide is suitable for industrial large-scale production. The prepared paeonia ostii flower peptide has good inhibition capability on tyrosinase, hyaluronidase and elastinase, and also has excellent anti-saccharification and anti-inflammatory effects. Besides, five paeonia ostii active peptide fragments are screened out, are high in activity and non-toxic, can be combined with tyrosinase, hyaluronidase, MMP-1, MMP-3 and COX-2 receptor protein, and have the effects of whitening, moisturizing, tightening and promoting collagen synthesis.

Description

Technical Field

[0001] The invention relates to the technical field of deep processing of peony flowers, and in particular to a peony flower peptide having the functions of whitening, moisturizing, firming and promoting collagen synthesis, and a preparation method and application thereof. Background Art

[0002] Peony is a plant of the genus Paeonia in the family Ranunculaceae. It is dignified and gorgeous and is known as the "king of flowers". China is the birthplace of peonies in the world. Peonies are distributed in most parts of China, including Luoyang, Henan and Heze, Shandong. The flowers, roots, skins, seeds and other parts of the peony have good edible and medicinal value. In 2013, Danfeng peony was listed as a new resource food raw material. It is rich in nutrients. It not only contains rich protein, fat, amino acids, trace elements, vitamins and other nutrients that are beneficial to the human body, but also contains astragalus glycosides, paeoniflorin, gallic acid, paeonol and other active ingredients that can lower blood pressure, relieve cough and fight tumors. It has been developed and used by many researchers.

[0003] At present, the research on the effective ingredients in peony flowers mainly focuses on the development of peony flavonoids and polyphenol active substances. Chinese patent CN106074747B disclosed a method and application of extracting tyrosinase inhibitors from peony flowers on November 9, 2016. Peony petals are used as raw materials, and the effective components containing tyrosinase inhibitors in peony petals are extracted by heating and reflux. This method can obtain tyrosinase inhibitors with a high extraction rate, which can reach more than 6%; Chinese patent CN104152521B disclosed peony pollen protein polypeptides, preparation methods and applications on November 19, 2014. Peony pollen protein is obtained by breaking the wall, extracting crude protein and vacuum drying with dried peony pollen as raw materials; Peony pollen protein is subjected to proteolysis, debittering and decolorization, centrifugation or filtration. Peony pollen polypeptide is obtained, and the product can be in the form of powder, granules, tablets, capsules, oral liquid, etc. The peony protein polypeptide has the advantages of lowering cholesterol, lowering blood pressure, promoting fat metabolism, anti-fatigue, enhancing human immunity, and regulating human physiological functions; Chinese patent CN114410724A disclosed on April 29, 2022 a Danfeng peony flower peptide with the ability to inhibit tyrosinase activity and its preparation method and application. Dried Danfeng peony flowers are mixed with water and ground and homogenized to form a slurry; the slurry is pretreated with alkali extraction and acid precipitation, and after pretreatment, it is successively enzymatically hydrolyzed, centrifugally filtered, concentrated, and dried to obtain Danfeng peony flower peptide. The Danfeng peony flower peptide can only inhibit tyrosinase activity.

[0004] However, for women's skin health, pure whitening is not attractive enough. Moisturizing, anti-wrinkle and firming effects are also of great importance to skin health. In addition, there are few reports on the characterization of specific active peptide fragments with specific functions. Therefore, it is of great significance to develop specific active peptide fragments with tyrosinase inhibition activity, hyaluronidase inhibition activity, anti-glycation and anti-inflammatory effects. Summary of the invention

[0005] In order to develop a specific active peptide segment with tyrosinase inhibition activity, hyaluronidase inhibition activity, anti-glycation and anti-inflammatory effects, the present invention provides a preparation method of Danfeng peony flower peptide, wherein the Danfeng peony flower peptide has the functions of whitening, moisturizing, firming, and promoting collagen synthesis, and the preparation method comprises the following steps: Mixing dried red peony flowers with pure water, and then grinding and homogenizing the mixture to obtain red peony flower slurry; Performing alkali extraction on the peony flower slurry, centrifuging to obtain the supernatant, then performing acid precipitation and standing overnight, centrifuging to retain the precipitate, and adding pure water to prepare the peony flower suspension; Acidic protease, a complex enzyme of cellulase and pectinase and alkaline protease are sequentially added to the suspension of Danfeng peony flowers for three-step hydrolysis, followed by enzyme inactivation treatment to obtain Danfeng peony flower hydrolyzate; The Danfeng Peony Flower hydrolyzate is subjected to solid-liquid separation to obtain a Danfeng Peony Flower peptide solution.

[0006] In one embodiment, the dried Danfeng peony flower is mixed with pure water in a mass percentage ratio of 1: (15-30), and the grinding and homogenization treatment temperature is 55-65°C, and the treatment time is 1-2 h; during the alkali extraction operation, the treatment temperature is 55±1°C, the treatment pH is 9.5-10, and the treatment time is 1-3 h; during the acid precipitation operation, the treatment temperature is 37±1°C, the treatment pH is 4.2, and it is left to stand overnight at 4°C.

[0007] In one embodiment, the added amount of the acidic protease is 0.5%~1.5% of the mass of the dried Danfeng peony flower; the added amount of the cellulase and pectinase is 0.5%~2% of the mass of the dried Danfeng peony flower; and the added amount of the alkaline protease is 1%~2% of the mass of the dried Danfeng peony flower.

[0008] In one embodiment, when acidic protease is added for primary hydrolysis, the hydrolysis temperature is 40-42°C, the pH is 2.5-3, and the hydrolysis time is 1-2 h; when a composite enzyme of cellulase and pectinase is added for secondary hydrolysis, the hydrolysis temperature is 49-52°C, the pH is 4.7-5.0, and the hydrolysis time is 1-2 h; when alkaline protease is added for tertiary hydrolysis, the hydrolysis temperature is 55-57°C, the pH is 8.4-8.5, and the hydrolysis time is 2-3 h, and then the pH is adjusted to 4.5 and the enzyme is inactivated at 85-90°C for 15-20 min.

[0009] In one embodiment, after the three-step hydrolysis and enzyme inactivation, activated carbon is added for decolorization, the amount of activated carbon added is 4% to 5% of the mass of the dried Danfeng peony flower, the treatment time is 1 to 1.5 h, and after the treatment is completed, centrifuge at 4000 to 6000 rpm for 5 to 10 min, take the supernatant, and then filter with diatomaceous earth to obtain a Danfeng peony flower peptide solution.

[0010] In one embodiment, the Danfeng Peony Flower Peptide Solution is subjected to ultrafiltration to obtain a solution containing Danfeng Peony Flower Peptide with a molecular weight of less than 5000 Da.

[0011] In one embodiment, the Danfeng Peony Flower Peptide Solution is spray-dried to obtain Danfeng Peony Flower Peptide Powder.

[0012] The present invention also provides a Danfeng peony flower peptide prepared according to the preparation method of Danfeng peony flower peptide as described in any one of the above items, comprising at least one of the following Danfeng peony flower peptide active peptide segments: the amino acid sequence of which is Phe-Ser-Pro-Leu or Phe-Gly-Pro-Leu or Phe-Gly-Pro-Ile or Ser-Leu-Gly-Phe or Leu-Phe-Leu.

[0013] The present invention also provides a functional product, the components of which include the Danfeng peony flower peptide as described above.

[0014] Furthermore, the functional product includes at least one of the following functions: (1) Tyrosinase inhibitory activity; (2) Hyaluronidase inhibitory activity; (3) Anti-glycation activity; (4) Anti-inflammatory activity; (5) Anti-wrinkle activity.

[0015] The preparation method of the Danfeng peony flower peptide provided by the present invention comprises the following steps: pretreatment by water bath, homogenization, alkali extraction and acid precipitation, and then enzymolysis by acid protease, cellulase and pectinase complex enzyme and alkaline protease for three times, and then solid-liquid separation to obtain the Danfeng peony flower peptide with whitening, moisturizing, firming and collagen synthesis promotion effects; in addition, activated carbon can be used for decolorization before solid-liquid separation, ultrafiltration can be used for interception during solid-liquid separation to obtain a Danfeng peony flower peptide solution with a molecular weight of less than 5000 Da, and spray drying can be performed after solid-liquid separation to obtain Danfeng peony flower peptide powder; the process steps are simple and suitable for industrial large-scale production.

[0016] In particular, the process scheme of the present invention uses a specific combination of acidic protease, cellulase, pectinase, and alkaline protease for enzymatic hydrolysis to achieve the desired effect, while the double enzyme combination hydrolysis or other arbitrary protease hydrolysis combination enzymatic hydrolysis treatment cannot achieve the Danfeng peony flower peptide obtained by the preparation method of the present invention, which has the advantages of tyrosinase inhibition activity, hyaluronidase inhibition activity, anti-glycation and anti-inflammatory effects.

[0017] The present invention also provides a Danfeng peony flower peptide, which has good tyrosinase inhibitory activity, can inhibit the production of melanin and whiten the skin; it also has good hyaluronidase inhibitory activity, can moisturize and tighten the skin; it also has high AGEs inhibitory ability, and has significant efficacy in resisting glycation and aging of the skin; it also has good elastase inhibitory activity, can tighten the skin and resist wrinkle formation; it also has the ability to inhibit protein denaturation, and can play an anti-inflammatory and stabilizing role.

[0018] Based on the preparation method of the Danfeng peony flower peptide, the present invention combines separation and purification-bioinformatics combined technology to screen out 4 Danfeng peony flower active peptide segments that have not been reported and have the functions of whitening, moisturizing, firming and promoting collagen synthesis, and the amino acid sequences thereof are: Phe-Ser-Pro-Leu (sequence abbreviation: FSPL), Phe-Gly-Pro-Leu (sequence abbreviation: FGPL), Phe-Gly-Pro-Ile (sequence abbreviation: FGPI), Ser-Leu-Gly-Phe (sequence abbreviation: SLGF), Leu-Phe-Leu (sequence abbreviation: LFL); the 5 peptide segments are highly active and non-toxic, and can bind to tyrosinase, hyaluronidase, MMP-1, MMP-3, and COX-2 receptor proteins, so as to play the role of whitening, moisturizing, firming and promoting collagen synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a graph showing the cell viability data analysis in Experiment 6 of the present invention; Figure 2 This is a graph showing the qRT-PCR analysis results of the Danfeng peony flower peptide sample on the Col Ⅰ gene and Col Ⅲ gene in HSF cells in Experiment 7 of the present invention; Figure 3 This is a graph showing the qRT-PCR analysis results of the Danfeng peony flower peptide sample on the HAS2 gene in HSF cells in Experiment 7 of the present invention; Figure 4 This is a 2D diagram of the docking of the peptide segment FSPL with the tyrosinase receptor protein molecule in Experiment 8 of the present invention; Figure 5 This is a 2D diagram of the docking of the peptide segment FSPL with the hyaluronidase receptor protein molecule in Experiment 8 of the present invention; Figure 6 This is a 2D diagram of the docking of the peptide FSPL with the MMP-1 receptor protein molecule in Experiment 8 of the present invention; Figure 7 This is a 2D diagram of the docking of the peptide FSPL with the MMP-3 receptor protein molecule in Experiment 8 of the present invention; Figure 8 This is a 2D diagram of the docking of the peptide FSPL with the COX-2 receptor protein molecule in Experiment 8 of the present invention; Fig. 9 This is a 2D diagram of the docking of the peptide segment FGPL and the tyrosinase receptor protein molecule in Experiment 8 of the present invention; Fig.10 This is a 2D diagram of the docking of the peptide segment FGPL and the hyaluronidase receptor protein molecule in Experiment 8 of the present invention; Fig.11 This is a 2D diagram of the docking of the peptide segment FGPL and the MMP-1 receptor protein molecule in Experiment 8 of the present invention; Fig.12 This is a 2D diagram of the docking of the peptide segment FGPL and the MMP-3 receptor protein molecule in Experiment 8 of the present invention; Fig.13 This is a 2D diagram of the docking of the peptide segment FGPL and the COX-2 receptor protein molecule in Experiment 8 of the present invention; Fig.14 This is a 2D diagram of the docking of the peptide segment FGPI and the tyrosinase receptor protein molecule in Experiment 8 of the present invention; Fig.15 This is a 2D diagram of the docking of the peptide segment FGPI and the hyaluronidase receptor protein molecule in Experiment 8 of the present invention; Fig.16 This is a 2D diagram of the docking of the peptide FGPI and the MMP-1 receptor protein molecule in Experiment 8 of the present invention; Fig.17 This is a 2D diagram of the docking of peptide FGPI and MMP-3 receptor protein molecule in Experiment 8 of the present invention; Fig.18 This is a 2D diagram of the docking of the peptide FGPI and the COX-2 receptor protein molecule in Experiment 8 of the present invention; Fig.19 It is a 2D diagram of the docking of the peptide segment SLGF and the tyrosinase receptor protein molecule in Experiment 8 of the present invention; Fig. 20 This is a 2D diagram of the docking of the peptide segment SLGF and the hyaluronidase receptor protein molecule in Experiment 8 of the present invention; Fig.21 This is a 2D diagram of the docking of the peptide segment SLGF and the MMP-1 receptor protein molecule in Experiment 8 of the present invention; Fig. 22 This is a 2D diagram of the docking of the peptide SLGF and the MMP-3 receptor protein molecule in Experiment 8 of the present invention; Fig.23 This is a 2D diagram of the docking of the peptide segment SLGF and the COX-2 receptor protein molecule in Experiment 8 of the present invention; Fig.24 This is a 2D diagram of the docking of the peptide LFL with the tyrosinase receptor protein molecule in Experiment 8 of the present invention; Fig.25 This is a 2D diagram of the docking of the peptide LFL with the hyaluronidase receptor protein molecule in Experiment 8 of the present invention; Fig.26 This is a 2D diagram of the docking of the peptide LFL with the MMP-1 receptor protein molecule in Experiment 8 of the present invention; Fig. 27 This is a 2D diagram of the docking of the peptide LFL with the MMP-3 receptor protein molecule in Experiment 8 of the present invention; Fig.28 This is a 2D diagram of the docking of the peptide LFL and the COX-2 receptor protein molecule in Experiment 8 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and the technical features designed in different implementations of the present invention described below can be combined with each other as long as they do not conflict with each other. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The present invention provides the following examples 1 to 3 for preparing a Danfeng peony flower peptide having whitening, moisturizing, firming, and promoting collagen synthesis, and the specific steps are as follows: S100, mixing dried Danfeng peony flowers and pure water in a mass percentage ratio of 1: (15-30), heating to 55-65° C. and incubating for 1-2 h, and grinding and mixing after the incubation to obtain Danfeng peony flower slurry; S101, raise the temperature to 55±1℃, adjust the pH of the peony slurry to 9.5-10 with NaOH solution, perform alkali extraction for 1-2 h, centrifuge at 5000-6000 rpm for 10-20 min, and take the supernatant; S102, cooling to 37±1°C, adjusting the pH to 4.2 with HCl solution, standing at 4°C overnight, centrifuging at 5000-6000 rpm for 10-20 min, collecting the precipitate, adding an equal amount of pure water to resuspend, and obtaining a Danfeng peony flower suspension; S103, raising the temperature to 40-42° C., adjusting the pH of the Danfeng peony flower suspension to 2.5-3.0 with HCl solution, adding acidic protease to hydrolyze for 1-2 h to obtain a primary hydrolyzate, wherein the amount of the acidic protease added is 0.5%-1.5% of the mass of the dried Danfeng peony flower; S104, heating to 49-52°C, adjusting the pH of the primary hydrolyzate to 4.7-5.0 with a NaOH solution, adding cellulase and pectinase for co-hydrolysis for 1-2 h to obtain a secondary hydrolyzate, wherein the addition amount of the cellulase and pectinase is 0.5%-2% of the mass of the dried Danfeng peony flower, and the addition ratio is 1:1; S105, raising the temperature to 55-57° C., adjusting the pH of the secondary hydrolyzate to 8.4-8.5 with a NaOH solution, and adding alkaline protease to hydrolyze for 2-3 h, wherein the amount of alkaline protease added is 1%-2% of the mass of the dried Danfeng peony flower; S106, adjust the pH to 4.5 with HCl solution, and heat to 85-90℃ to inactivate the enzyme for 15-20 min; S107, cooling to 57° C., adding activated carbon, stirring for decolorization for 1-1.5 h, centrifuging at 4000-6000 rpm for 5-10 min, taking the supernatant and filtering it with diatomaceous earth to obtain a Danfeng peony peptide solution, wherein the amount of activated carbon added is 4%-5% of the mass of the dried Danfeng peony flower.

[0023] Example 1 S100, evenly mix 500 g of dried Danfeng peony flowers with 10000 g of pure water, raise the temperature to 60°C and incubate for 1 h, grind and homogenize after the incubation to obtain Danfeng peony flower slurry; S101, heating to 55°C, adjusting the pH of the peony slurry to 9.5 with NaOH solution, alkali extraction for 2 h, centrifuging at 5000 rpm for 10 min, and taking the supernatant; S102, cooling to 37°C, adjusting the pH to 4.2 with HCl solution, standing at 4°C overnight, centrifuging at 5000 rpm for 10 min, collecting the precipitate, adding an equal amount of pure water to resuspend, and obtaining a Danfeng peony flower suspension; S103, raising the temperature to 42°C, adjusting the pH of the Danfeng peony flower suspension to 2.5 with HCl solution, adding 5 g of acidic protease to hydrolyze for 2 h, and obtaining a primary hydrolyzate; S104, raising the temperature to 50°C, adjusting the pH of the primary hydrolyzate to 5.0 with a NaOH solution, adding 10 g of cellulase and 10 g of pectinase to hydrolyze for 1 h to obtain a secondary hydrolyzate; S105, raise the temperature to 57°C, adjust the pH of the secondary hydrolyzate to 8.5 with NaOH solution, add 5 g of alkaline protease and hydrolyze for 2 h; S106, adjust the pH to 4.5 with HCl solution, and heat to 85°C to inactivate the enzyme for 20 min; S107, cool to 57°C, add 25 g activated carbon, stir to decolorize for 1 h, centrifuge at 5000 rpm for 10 min, take the supernatant and filter it with diatomaceous earth to obtain Danfeng peony peptide solution.

[0024] Example 2 S100, mixing 500 g of dried Danfeng peony flowers with 15000 g of pure water, heating to 60°C and incubating for 2 h, grinding and homogenizing after the incubation to obtain Danfeng peony flower slurry; S101, heating to 55°C, adjusting the pH of the peony slurry to 9.5 with NaOH solution, alkali extraction for 2 h, centrifuging at 5000 rpm for 10 min, and taking the supernatant; S102, cooling to 37°C, adjusting the pH to 4.2 with HCl solution, standing at 4°C overnight, centrifuging at 5000 rpm for 10 min, collecting the precipitate, adding an equal amount of pure water to resuspend, and obtaining a Danfeng peony flower suspension; S103, raising the temperature to 42°C, adjusting the pH of the Danfeng peony flower suspension to 2.5 with HCl solution, adding 2.5 g of acidic protease to hydrolyze for 2 h to obtain a primary hydrolyzate; S104, raising the temperature to 50°C, adjusting the pH of the primary hydrolyzate to 5.0 with a NaOH solution, adding 2.5 g of cellulase and 2.5 g of pectinase to hydrolyze for 1 h to obtain a secondary hydrolyzate; S105, raise the temperature to 57°C, adjust the pH of the secondary hydrolyzate to 8.5 with NaOH solution, add 10 g alkaline protease and hydrolyze for 2 h; S106, adjust the pH to 4.3 with HCl solution, and heat to 85°C to inactivate the enzyme for 20 min; S107, cool to 57°C, add 25 g activated carbon, stir to decolorize for 1 h, centrifuge at 5000 rpm for 10 min, take the supernatant and filter it with diatomaceous earth to obtain Danfeng peony peptide solution.

[0025] Example 3 S100, evenly mix 500 g of dried Danfeng peony flowers with 7500 g of pure water, heat to 60°C and incubate for 1 h, grind and homogenize after the incubation to obtain Danfeng peony flower slurry; S101, heating to 55°C, adjusting the pH of the peony slurry to 9.5 with NaOH solution, alkali extraction for 2 h, centrifuging at 5000 rpm for 10 min, and taking the supernatant; S102, cooling to 37°C, adjusting the pH to 4.2 with HCl solution, standing at 4°C overnight, centrifuging at 5000 rpm for 10 min, collecting the precipitate, adding an equal amount of pure water to resuspend, and obtaining a Danfeng peony flower suspension; S103, raising the temperature to 42°C, adjusting the pH of the peony flower suspension to 2.5 with HCl solution, adding 7.5 g of acidic protease to hydrolyze for 2 h, and obtaining a primary hydrolyzate; S104, raising the temperature to 50°C, adjusting the pH of the primary hydrolyzate to 5.0 with a NaOH solution, adding 5 g of cellulase and 5 g of pectinase to hydrolyze for 1 h to obtain a secondary hydrolyzate; S105, raise the temperature to 57°C, adjust the pH of the secondary hydrolyzate to 8.5 with NaOH solution, add 5 g of alkaline protease and hydrolyze for 2 h; S106, adjust the pH to 4.3 with HCl solution, and heat to 85°C to inactivate the enzyme for 20 min; S107, cool to 57°C, add 25 g activated carbon, stir to decolorize for 1 h, centrifuge at 5000 rpm for 10 min, take the supernatant and filter it with diatomaceous earth to obtain Danfeng peony peptide solution.

[0026] In Examples 1 to 3 of the present invention, preferably, the protein content in the dried Danfeng peony flowers is 30%.

[0027] In Examples 1 to 3 of the present invention, preferably, the enzyme activity of the acidic protease is 150,000 U / g.

[0028] In Examples 1 to 3 of the present invention, preferably, the enzymatic activity of the cellulase is 20,000 U / g.

[0029] In Examples 1 to 3 of the present invention, preferably, the enzyme activity of the pectinase is 30,000 U / g.

[0030] In Examples 1 to 3 of the present invention, preferably, the enzyme activity of the alkaline protease is 200,000 U / g.

[0031] The present invention also provides the following comparative examples 1 to 9 for preparing a Danfeng peony flower peptide: Comparative Example 1 This comparative example prepares a Danfeng peony flower peptide, which differs from Example 1 in that: the amount of acid protease added in step S103 is changed to 10 g; step S104 is changed to heating to 47°C, adjusting the pH to 5.0, adding 15 g of cellulase for hydrolysis for 1 h, then heating to 50°C, adjusting the pH to 4.7, adding 15 g of pectinase for hydrolysis for 1 h; and step S105 is deleted. The remaining components, proportions, operating steps and process parameters are consistent with those in Example 1.

[0032] It should be noted that, based on the protein quality of Danfeng peony flower, the total amount of acidic protease, cellulase and pectinase in this comparative example is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1, and the specific calculation process is as follows: In Example 1, the amount of dried Danfeng peony flower added is 500 g, and its protein content is 30%. It is calculated that the protein mass of Danfeng peony flower in 500 g of dried Danfeng peony flower is 150 g; the amount of acid protease added is 5 g, and its enzyme activity is 150,000 U / g. The enzyme activity of 5 g of acid protease is 150,000 U / g×5 g=750,000 U, which is converted to the enzyme activity of the added enzyme per unit gram weight of protein in the dried Danfeng peony flower, that is, 750,000 U / 150 g=5000 U / g; similarly, the amount of cellulase added is 10 g, and its enzyme activity is 20,000 U / g. It is calculated that, based on the protein mass of Danfeng peony flower, an enzyme with an enzyme activity of 1300 U / g is added; the amount of pectinase added is 10 g, and its enzyme activity is 30,000 U / g. It is calculated that, based on the protein mass of Danfeng peony flower, an enzyme with an enzyme activity of 2000 U / g is added. U / g enzyme activity; the added amount of alkaline protease is 5 g, and its enzyme activity is 200,000 U / g. It can be calculated that, based on the quality of Danfeng peony flower protein, an enzyme with an enzyme activity of 6700 U / g is added; then in Example 1, based on the quality of Danfeng peony flower protein, the total amount of acidic protease, cellulase, pectinase and alkaline protease is 15000 U / g.

[0033] In Comparative Example 1, the amount of acid protease added is 10 g, and calculated based on the quality of Danfeng peony flower protein, the amount of acid protease used is 150,000 U / g×10 g / 150 g=10,000 U / g; the amount of cellulase added is 15 g, and calculated based on the quality of Danfeng peony flower protein, the amount of cellulase used is 20,000 U / g×15 g / 150 g=2,000 U / g; the amount of pectinase added is 15 g, and calculated based on the quality of Danfeng peony flower protein, the amount of pectinase used is 30,000 U / g×15 g / 150 g=3,000 U / g; then in Comparative Example 1, calculated based on the quality of Danfeng peony flower protein, the total amount of acid protease, cellulase and pectinase used is 15,000 U / g.

[0034] Therefore, based on the protein quality of Danfeng peony flowers, the total amount of acidic protease, cellulase and pectinase in Comparative Example 1 is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1.

[0035] Comparative Example 2 This comparative example prepares a Danfeng peony flower peptide, which differs from Example 1 in that: Step S103 is deleted; the addition amount of cellulase and pectinase in Step S104 is changed to 5 g; the addition amount of alkaline protease in Step S105 is changed to 10 g. The remaining components, proportions, operating steps and process parameters are consistent with those in Example 1.

[0036] It should be noted that, based on the quality of Danfeng peony flower protein, the total amount of cellulase, pectinase and alkaline protease in this comparative example is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1, and the specific calculation process is as follows: In Example 1, based on the protein quality of the peony peony, the total amount of acidic protease, cellulase, pectinase and alkaline protease used is 15000 U / g.

[0037] In Comparative Example 2, the amount of cellulase added is 5 g, and the amount of cellulase used is 20,000 U / g×5 g / 150 g=700 U / g based on the quality of Danfeng peony flower protein; the amount of pectinase added is 5 g, and the amount of pectinase used is 30,000 U / g×5 g / 150 g=1000 U / g based on the quality of Danfeng peony flower protein; the amount of alkaline protease added is 10 g, and the amount of alkaline protease used is 200,000 U / g×10 g / 150 g=13300 U / g based on the quality of Danfeng peony flower protein; then in Comparative Example 2, based on the quality of Danfeng peony flower protein, the total amount of cellulase, pectinase and alkaline protease used is 15000 U / g, which is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease used in Example 1.

[0038] Comparative Example 3 This comparative example prepared a Danfeng peony flower peptide, which differed from Example 1 in that: step S104 was deleted; and a step was added after step S105, specifically, heating to 57°C, adjusting the pH to 7.0, adding 2.75 g of Bacillus subtilis neutral protease and 2.75 g of Bacillus subtilis flavor protease for co-hydrolysis for 2 h. The remaining components, proportions, operating steps and process parameters were consistent with those of Example 1.

[0039] In the comparative example of the present invention, the enzyme activity of the Bacillus subtilis neutral protease is 80,000 U / g, and the enzyme activity of the Bacillus subtilis flavor protease is 100,000 U / g.

[0040] It should be noted that, based on the quality of Danfeng peony flower protein, the total amount of acidic protease, alkaline protease, Bacillus subtilis neutral protease and Bacillus subtilis flavor protease in this comparative example is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1, and the specific calculation process is as follows: In Example 1, based on the protein quality of the peony peony, the total amount of acidic protease, cellulase, pectinase and alkaline protease used is 15000 U / g.

[0041] In Comparative Example 3, the amount of acidic protease added is 5 g, and the amount of acidic protease used is 150,000 U / g×5 g / 150 g=5000 U / g based on the quality of Danfeng peony flower protein; the amount of alkaline protease added is 5 g, and the amount of alkaline protease used is 200,000 U / g×5 g / 150 g=6700 U / g based on the quality of Danfeng peony flower protein; the amount of Bacillus subtilis neutral protease added is 2.5 g, and the amount of Bacillus subtilis neutral protease used is 80,000 U / g×2.75 g / 150 g=1500 U / g based on the quality of Danfeng peony flower protein; the amount of Bacillus subtilis flavor protease added is 2.5 g, and the amount of Bacillus subtilis flavor protease used is 100,000 U / g×2.75 g / 150 g=1800 U / g; in Comparative Example 3, based on the quality of Danfeng peony flower protein, the total amount of acidic protease, alkaline protease, Bacillus subtilis neutral protease and Bacillus subtilis flavor protease is 15000 U / g, which is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1.

[0042] Comparative Example 4 This comparative example prepared a Danfeng peony flower peptide, which differed from Example 1 in that: step S103 was deleted; and a step was added after step S106, specifically, heating to 57°C, adjusting the pH to 6.5, adding 1.86 g of papain to hydrolyze for 2h, and then inactivating the enzyme at 85°C for 20 min. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0043] In the comparative example of the present invention, the enzyme activity of the papain is 400,000 U / g.

[0044] It should be noted that, based on the quality of Danfeng peony flower protein, the total amount of cellulase, pectinase, alkaline protease and papain in this comparative example is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1, and the specific calculation process is as follows: In Example 1, based on the protein quality of the peony peony, the total amount of acidic protease, cellulase, pectinase and alkaline protease used is 15000 U / g.

[0045] In Comparative Example 4, the amount of cellulase added is 10 g, and the amount of cellulase used is 20,000 U / g×10 g / 150 g=1300 U / g according to the quality of Danfeng peony flower protein; the amount of pectinase added is 10 g, and the amount of cellulase used is 30,000 U / g×10 g / 150 g=2000 U / g according to the quality of Danfeng peony flower protein; the amount of alkaline protease added is 5 g, and the amount of alkaline protease used is 200,000 U / g×5 g / 150 g=6700 U / g according to the quality of Danfeng peony flower protein; the amount of papain added is 1.86 g, and the amount of papain used is 400,000 U / g×1.86 g / 150 g=5000 U / g according to the quality of Danfeng peony flower protein. U / g; in Comparative Example 4, based on the protein quality of Danfeng Peony, the total amount of cellulase, pectinase, alkaline protease and papain is 15000 U / g, which is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1.

[0046] Comparative Example 5 This comparative example prepared a Danfeng peony flower peptide, which differed from Example 1 in that: step S103 was deleted; and a step was added after step S106, specifically, heating to 57°C, adjusting the pH to 7.0, adding 4.2 g of Bacillus subtilis neutral protease and 4.2 g of Bacillus subtilis flavor protease for co-hydrolysis for 2 h, and then inactivating the enzyme at 85°C for 20 min. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0047] It should be noted that, based on the quality of Danfeng peony flower protein, the total amount of cellulase, pectinase, alkaline protease, Bacillus subtilis neutral protease and Bacillus subtilis flavor protease in this comparative example is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1, and the specific calculation process is as follows: In Example 1, based on the protein quality of the peony peony, the total amount of acidic protease, cellulase, pectinase and alkaline protease used is 15000 U / g.

[0048] In Comparative Example 5, the amount of cellulase added is 10 g, and the amount of cellulase used is 20,000 U / g×10 g / 150 g=1300 U / g based on the quality of Danfeng peony flower protein; the amount of pectinase added is 10 g, and the amount of cellulase used is 30,000 U / g×10 g / 150 g=2000 U / g based on the quality of Danfeng peony flower protein; the amount of alkaline protease added is 5 g, and the amount of alkaline protease used is 200,000 U / g×5 g / 150 g=6700 U / g based on the quality of Danfeng peony flower protein; the amount of Bacillus subtilis neutral protease added is 4.2 g, and the amount of Bacillus subtilis neutral protease used is 80,000 U / g×4.2 g / 150 g=2200 U / g based on the quality of Danfeng peony flower protein; the amount of Bacillus subtilis flavor protease added is 4.2 g, based on the quality of Danfeng peony flower protein, the dosage of Bacillus subtilis flavor protease is 100,000 U / g×4.2 g / 150 g=2800 U / g; in Comparative Example 5, based on the quality of Danfeng peony flower protein, the total dosage of cellulase, pectinase, alkaline protease, Bacillus subtilis neutral protease and Bacillus subtilis flavor protease is 15,000 U / g, which is equal to the total dosage of acidic protease, cellulase, pectinase and alkaline protease in Example 1.

[0049] Comparative Example 6 This comparative example prepared a Danfeng peony flower peptide, which differed from Example 1 in that: the amount of acid protease added in step S103 was changed to 2.5 g; the amount of cellulase and pectinase added in step S104 were both changed to 7.5 g; and a step was added after step 105, specifically, heating to 57°C, adjusting the pH to 7.0, adding 2.75 g of Bacillus subtilis neutral protease and 2.75 g of Bacillus subtilis flavor protease for co-hydrolysis for 2 h. The remaining components, proportions, operating steps and process parameters were consistent with those in Example 1.

[0050] It should be noted that, based on the quality of Danfeng peony flower protein, the total amount of acidic protease, cellulase, pectinase, alkaline protease, Bacillus subtilis neutral protease and Bacillus subtilis flavor protease in this comparative example is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1, and the specific calculation process is as follows: In Example 1, based on the protein quality of the peony peony, the total amount of acidic protease, cellulase, pectinase and alkaline protease used is 15000 U / g.

[0051] In Comparative Example 6, the amount of acid protease added is 2.5 g, and the amount of acid protease used is 150,000 U / g×2.5 g / 150 g=2500 U / g according to the quality of Danfeng peony flower protein; the amount of cellulase added is 7.5 g, and the amount of cellulase used is 20,000 U / g×7.5 g / 150 g=1000 U / g according to the quality of Danfeng peony flower protein; the amount of pectinase added is 7.5 g, and the amount of cellulase used is 30,000 U / g×7.5 g / 150 g=1500 U / g according to the quality of Danfeng peony flower protein; the amount of alkaline protease added is 5 g, and the amount of alkaline protease used is 200,000 U / g×5 g / 150 g=6700 U / g according to the quality of Danfeng peony flower protein; the amount of Bacillus subtilis neutral protease added is 2.75 g, based on the quality of Danfeng peony flower protein, the amount of Bacillus subtilis neutral protease is 80,000 U / g×2.75 g / 150 g=1500 U / g; the added amount of Bacillus subtilis flavor protease is 2.75 g, and based on the quality of Danfeng peony flower protein, the amount of Bacillus subtilis flavor protease is 100,000 U / g×2.75 g / 150 g=1800 U / g; in Comparative Example 6, based on the quality of Danfeng peony flower protein, the total amount of acid protease, cellulase, pectinase, alkaline protease, Bacillus subtilis neutral protease and Bacillus subtilis flavor protease is 15,000 U / g, which is equal to the total amount of acid protease, cellulase, pectinase and alkaline protease in Example 1.

[0052] Comparative Example 7 This comparative example prepares a Danfeng peony flower peptide, which differs from Example 1 in that the amount of acidic protease added in step S103 is changed to 15 g, and steps S104 and S105 are deleted. The remaining components, proportions, operating steps and process parameters are consistent with those in Example 1.

[0053] It should be noted that, based on the quality of Danfeng peony flower protein, the amount of acidic protease in this comparative example is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1, and the specific calculation process is as follows: In Example 1, based on the protein quality of the peony peony, the total amount of acidic protease, cellulase, pectinase and alkaline protease used is 15000 U / g.

[0054] In Comparative Example 7, the amount of acid protease added is 15 g. Calculated based on the quality of Danfeng peony flower protein, the amount of acid protease used is 150,000 U / g×15 g / 150 g=15,000 U / g, which is equal to the total amount of acid protease, cellulase, pectinase and alkaline protease used in Example 1.

[0055] Comparative Example 8 This comparative example prepares a Danfeng peony flower peptide, which differs from Example 1 in that the addition amount of cellulase and pectinase in step S104 is changed to 45 g, and steps S103 and S105 are deleted. The remaining components, proportions, operating steps and process parameters are consistent with those in Example 1.

[0056] It should be noted that, based on the protein quality of Danfeng peony flower, the amount of cellulase and pectinase used in this comparative example is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease used in Example 1, and the specific calculation process is as follows: In Example 1, based on the protein quality of the peony peony, the total amount of acidic protease, cellulase, pectinase and alkaline protease used is 15000 U / g.

[0057] In Comparative Example 8, the amount of cellulase added is 45 g, and the amount of cellulase used is 20,000 U / g×45 g / 150 g=6000 U / g based on the quality of Danfeng peony flower protein; the amount of pectinase added is 45 g, and the amount of pectinase used is 30,000 U / g×45 g / 150 g=9000 U / g based on the quality of Danfeng peony flower protein; then in Comparative Example 8, based on the quality of Danfeng peony flower protein, the total amount of cellulase and pectinase used is 15,000 U / g, which is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease used in Example 1.

[0058] Comparative Example 9 This comparative example prepares a Danfeng peony flower peptide, which differs from Example 1 in that: Steps S103 and S104 are deleted; the amount of alkaline protease added in step S105 is changed to 11.2 g. The remaining components, proportions, operating steps and process parameters are consistent with those in Example 1.

[0059] It should be noted that, based on the quality of Danfeng peony flower protein, the amount of alkaline protease in this comparative example is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease in Example 1, and the specific calculation process is as follows: In Example 1, based on the protein quality of the peony peony, the total amount of acidic protease, cellulase, pectinase and alkaline protease used is 15000 U / g.

[0060] In Comparative Example 9, the amount of alkaline protease added is 11.2 g. Calculated based on the quality of Danfeng peony flower protein, the amount of acidic protease used is 200,000 U / g×11.2 g / 150 g=15,000 U / g, which is equal to the total amount of acidic protease, cellulase, pectinase and alkaline protease used in Example 1.

[0061] The present invention also provides the following test to perform performance testing and peptide segment screening on the Danfeng Peony Flower Peptide prepared by the present invention: It should be noted that in step S107, the solid-liquid separation operation after centrifugation of Comparative Examples 7, 8, and 9 was difficult, probably because the single enzyme hydrolysis retained a large amount of large molecular proteins that were difficult to filter. Therefore, subsequent tests no longer studied Comparative Examples 7, 8, and 9.

[0062] Experiment 1 Determination of the inhibition rate of tyrosinase by Danfeng peony flower peptide Tyrosinase is a key enzyme in the synthesis of melanin, which can catalyze the substrate to generate L-dopa, and further convert it into L-dopaquinone, and finally form melanin. Therefore, by measuring the inhibitory effect of Danfeng Peony Flower Peptide on tyrosinase activity, it can be used to evaluate its ability to inhibit melanin production, that is, to evaluate whether it has whitening effect.

[0063] The Danfeng Peony Flower Peptide Solutions prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were concentrated to a solid content of 3 and used as the test sample. 1.0 mL of sample solution and 0.5 mL of tyrosinase solution were added to the sample tube, 1.0 mL of sample solution and 0.5 mL of buffer were added to the sample background tube, 1.0 mL of buffer and 0.5 mL of tyrosinase solution were added to the enzyme reaction tube, and 1.5 mL of buffer was added to the solvent background tube; all were placed in a water bath at 37°C for incubation for 10 min, 2.0 mL of levodopa solution was added to each, and after reacting at room temperature for 5 min, the absorbance at 475 nm was measured and recorded as T, T0, C, and C0, respectively.

[0064] The tyrosinase inhibition rate was calculated according to formula (1) and the results are summarized in Table 1.

[0065]

[0066] Table 1 Tyrosinase inhibition rate of Danfeng peony flower peptides prepared in Examples 1 to 3 and Comparative Examples 1 to 6

[0067] According to the data in Table 1, the Danfeng peony flower peptides prepared in Examples 1 to 3 and Comparative Examples 1 to 6 all have tyrosinase inhibition ability, but the tyrosinase inhibition ability of Examples 1 to 3 is significantly higher than that of Comparative Examples 1 to 6; among them, the Danfeng peony flower peptide prepared in Example 1 has the highest tyrosinase inhibition rate, reaching 60.2%.

[0068] Comparing Comparative Examples 1 and 2 with the Example, it is shown that the addition of acidic protease and alkaline protease can further hydrolyze active polypeptide components; and comparing Comparative Example 6 with the Example, it is shown that excessive addition of neutral and flavor protease complex enzymes may cause excessive hydrolysis, which in turn affects the ability of its tyrosinase activity.

[0069] Experiment 2 Determination of the inhibition rate of hyaluronidase by Danfeng peony flower peptide Hyaluronic acid has strong water-retaining properties in the skin, and hyaluronidase is an enzyme used to degrade hyaluronic acid. Therefore, the presence of hyaluronidase will reduce skin moisture, leading to aging phenomena such as reduced skin elasticity and increased wrinkles.

[0070] In the experiment, after the complexation of CTAB with hyaluronic acid, the turbidimetric method was used to determine the content of HA-CTAB at 400 nm, which could quickly calculate the change in hyaluronic acid content before and after enzymatic hydrolysis, thereby characterizing the inhibitory effect of Danfeng peony flower peptide on hyaluronidase.

[0071] Experimental group: Add 50 μL of the sample solution to be tested, 4.85 mL of sodium acetate buffer and 50 μL of hyaluronidase solution to a 10 mL centrifuge tube, shake well, add 50 μL of sodium hyaluronate solution, react at 37°C for 15 min, add 2 mL of 2.5% CTAB (dissolved in 2% sodium hydroxide solution), let stand at room temperature for 15 min, and measure the absorbance of the solution at 400 nm after the reaction, which is recorded as A1; Sample control group: 50 μL of hyaluronidase solution was replaced with 50 μL of sodium acetate buffer, and the absorbance was recorded as A c1 ; Blank control group: 50 μL of the sample solution and 50 μL of hyaluronidase solution were replaced with 100 μL of sodium acetate buffer, and the absorbance was recorded as A c2 ; Enzyme solution control group: 50 μL of the sample solution to be tested was replaced with 50 μL of sodium acetate buffer, and the absorbance was recorded as A0.

[0072] The hyaluronidase inhibition rate was calculated according to formula (2), and the results are summarized in Table 2.

[0073]

[0074] Table 2 Inhibition rate of hyaluronidase by Danfeng peony flower peptides prepared in Examples 1 to 3 and Comparative Examples 1 to 6

[0075] According to the data in Table 2, the hyaluronidase inhibition rate of the Danfeng peony flower peptides prepared in Examples 1 to 3 is relatively high, with the highest being 63.3% of Example 1; and comparing Comparative Examples 1 and 2 with the Examples, respectively, it can be shown that acidic protease or alkaline protease has a significant effect on the hyaluronidase inhibition activity of the Danfeng peony flower peptides.

[0076] Experiment 3 Determination of the inhibition rate of Danfeng peony flower peptide on AGEs As people age, their sugar intake accumulates and their metabolism slows down. The sugar ingested in the body is very easy to accumulate. The accumulated sugar combines with proteins and oxidizes, eventually forming advanced glycation end products, or AGEs. By measuring the inhibition rate of Danfeng Peony Flower Peptide on AGEs, it can be used to evaluate its anti-glycation effect.

[0077] A phosphate buffer solution with a pH of 7.4, a 4.0 mg / mL bovine serum albumin (BSA) solution, a 0.5 mol / L glucose / D-fructose solution, and a 2.0 mmol / L aminoguanidine solution were prepared and then subjected to membrane treatment.

[0078] 100 μL BSA, 100 μL glucose / D-fructose solution and 100 μL peptide sample were added to sample group F1, 100 μL PBS, 100 μL glucose / D-fructose solution and 100 μL peptide sample were added to sample control group F2, 100 μL BSA, 100 μL glucose / D-fructose solution and 100 μL sample solvent solution were added to blank group F3, and 100 μL PBS, 100 μL glucose / D-fructose solution and 100 μL sample solvent solution were added to blank control group F4; 2.0 mmol / L aminoguanidine (AG) solution was used as a positive control.

[0079] The cells were incubated at 37°C for 7 days and then excited with 360 nm excitation light. The fluorescence intensity at 453 nm was measured, where the excitation and emission slit widths were 5 nm.

[0080] The AGEs inhibition rate was calculated according to formula (3), and the results are summarized in Table 3.

[0081]

[0082] Table 3 Inhibition rate of AGEs by Danfeng peony flower peptides prepared in Examples 1 to 3 and Comparative Examples 1 to 6

[0083] According to the data in Table 3, the AGEs inhibition rate of comparative example 1 is significantly lower than that of other comparative examples and examples, indicating that alkaline protease has an important influence on the AGEs inhibition rate of Danfeng peony flower peptide; the AGEs inhibition rates of Examples 1 to 3 are higher than those of comparative examples 3 and 5, indicating that the enzymatic hydrolysis combination of acidic protease, cellulose pectin complex enzyme and alkaline protease can play a synergistic role in the AGEs inhibition activity of Danfeng peony flower peptide.

[0084] Experiment 4 Determination of the inhibition rate of Danfeng peony flower peptide on elastase Elastase (MMP-12) is a metal matrix oxidase that can degrade elastin. Therefore, by inhibiting elastase and protecting elastin, it can maintain skin elasticity.

[0085] In the experiment, after elastase specifically binds to the substrate N-succinyl-Ala-Ala-Ala-pNa, it will replace the p-nitroaniline in the substrate, and p-nitroaniline will have a characteristic peak at 410 nm. Therefore, the activity of elastase can be characterized by measuring the concentration of p-nitroaniline.

[0086] Add 200 μL of the sample to be tested to the 96-well plate, and the blank control is Tris-HCl buffer; quickly add 50 μL of elastase solution and 50 μL of color substrate, and measure the absorbance of the mixed solution at 410 nm when it is not reacted, which is recorded as A s0 The blank control group was recorded as A c0 ; After incubation in the microplate reader for 5 min, the absorbance at 410 nm was recorded as A s , A c .

[0087] The results were summarized in Table 4 according to the calculation formula (4) for the inhibition rate of elastase.

[0088]

[0089] Table 4 Inhibition rate of Danfeng peony flower peptides prepared in Examples 1 to 3 and Comparative Examples 1 to 6 on elastase

[0090] According to the data in Table 4, the inhibition rate of elastase by the Danfeng peony flower peptides prepared in Examples 1 to 3 is significantly higher than that of all comparative examples, reaching a maximum of 54.7%, indicating that the enzymatic hydrolysis combination of acidic protease, cellulose pectin complex enzyme and alkaline protease can improve the elastase inhibition rate of the Danfeng peony flower peptide compared with other enzymatic hydrolysis combinations with equal enzyme activity; according to the elastase inhibition rate data of comparative examples 2, 4, and 5, it can be shown that acidic protease plays an important role in the enzymatic hydrolysis process; and by comparing comparative examples 5 and 6, the necessity of acidic protease can be further illustrated.

[0091] Experiment 5 Determination of the inhibition rate of Danfeng peony flower peptide on albumin denaturation Certain substances released by the body during inflammation can cause protein denaturation, so inhibiting protein denaturation is an effective indicator of anti-inflammatory activity. The reaction of pH=7.2 phosphate buffer with ovalbumin will destroy the protein structure, and after a 70℃ water bath, the protein will also denature, and the solution will eventually change from clear and transparent to white and turbid.

[0092] 25 μL ovalbumin, 350 μL phosphate buffer solution (0.1M, pH=7.2) and 250 μL sample were mixed and incubated in a water bath at 37°C for 15 min, and then incubated in a water bath at 70°C for 5 min. After cooling in an ice water bath for 5 min, the absorbance value measured at 660 nm was recorded as A sample, and the absorbance value measured by replacing ovalbumin with phosphate buffer solution was recorded as A 对照 , the absorbance value measured by ultrapure water instead of the sample is recorded as A 空白 .

[0093] The results were summarized in Table 5 according to the albumin denaturation inhibition rate calculation formula (5).

[0094]

[0095] Table 4 Albumin denaturation inhibition rate of Danfeng peony flower peptides prepared in Examples 1 to 3 and Comparative Examples 1 to 6

[0096] According to the data in Table 5, the inhibition rate of albumin denaturation of the Danfeng Peony Flower Peptide prepared in Examples 1 to 3 is significantly higher than that of the comparative example, with the highest being 89.4% of Example 1, indicating that the Danfeng Peony Flower Peptide solution prepared in Examples 1 to 3 of the present invention can effectively inhibit albumin denaturation, thereby achieving an anti-inflammatory effect.

[0097] Experiment 6: Cytotoxicity test of Danfeng peony peptide Since Comparative Examples 1, 3, and 5 performed poorly in the results of in vitro tyrosinase inhibition activity, hyaluronidase inhibition activity, AGEs inhibition activity, elastase inhibition activity, and albumin denaturation inhibition activity, only Examples 1 to 3 and Comparative Examples 2, 4, and 6 were subjected to cytotoxicity experiments.

[0098] HSF cells growing in the logarithmic phase were collected, and a cell suspension was prepared with DMEM complete culture medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at a density of 1.0×105 cells / mL, and inoculated into a 96-well plate, and 100 μL was added to each well; the plate was placed in a carbon dioxide incubator and cultured at 37°C for 24 h; the 96-well plate was taken out, the culture medium was aspirated, and different concentrations of Danfeng Peony Flower Peptide Solutions were added, among which the blank group did not need to be treated, and the concentrations of the Danfeng Peony Flower Peptide Solutions were 2%, 4%, 6%, 8%, and 10%, respectively; the plate was placed in a carbon dioxide incubator and cultured at 37°C for 24 h, the culture medium was aspirated again, and 10 μL of CCK8 solution was added to each well in the dark; the absorbance value of each well at 450 nm was measured with an enzyme marker, and the average value of 3 wells in each group was taken, the sample group was recorded as A1, and the blank group was recorded as A0.

[0099] Calculate according to the cell viability calculation formula (6).

[0100]

[0101] Figure 1 This is a cell viability data analysis chart. Figure 1 It can be seen that in Comparative Example 6, when the concentration of the Danfeng Peony Flower Peptide Solution reached 6%, the cell viability decreased significantly, indicating that the peptide solution of Comparative Example 6 at this concentration was toxic to cells to a certain extent. Therefore, Comparative Example 6 was no longer subjected to subsequent experiments; Figure 1 As a result of data analysis, it was finally determined that the concentration of 4% which was non-toxic to cells in Examples 1 to 3 and Comparative Examples 2 and 4 was used as the cell administration concentration.

[0102] Experiment 7 Effect of Danfeng Peony Flower Peptide on mRNA Expression Levels of Related Genes HSF cells were collected and seeded in a 24-well plate at a density of 2×104 cells / mL and cultured in an incubator for 24 h. When the cells grew to 70%, a 4% concentration of Danfeng peony peptide solution was added. A blank control group was set up, and each group had 3 parallel wells and was cultured in a carbon dioxide incubator at 37°C for 24 h.

[0103] Total cellular RNA was extracted according to the Simply P total RNA extraction kit (BioFlux). 2 μL of sterile enzyme-free water was dropped on a concentration measurement plate (Nanodrop one) to detect the RNA concentration and purity of each group (the purity was between 1.8 and 2.1).

[0104] A 20 μL qRT-PCR reaction system was established, and the PCR amplification conditions were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 54°C for 30 s, extension at 72°C for 45 s, and reaction cycles for 40 times.

[0105] The reaction conditions of the melting curve were as follows: 95°C, 30 s, 60°C, 1 min, and the fluorescence signal was collected every 5°C rise.

[0106] The primers were designed and synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., with GAPDH as the internal reference. The primer sequence information is listed in Table 6: Table 6 Primer information

[0107] It should be noted that the above primers are all existing primers.

[0108] Under the premise of ensuring the stability of cell growth and not affecting the proliferation ability, the qRT-PCR analysis results of the Danfeng Peony Flower Peptide samples prepared in Examples 1 to 3 and Comparative Examples 2, 4, and 6 on the Col Ⅰ gene, Col Ⅲ gene, and HAS2 gene in HSF cells were as follows: Figure 2 and Figure 3 shown.

[0109] Collagen is the most abundant organic protein in the skin, mainly composed of Col I (85%~90%) and a small amount of Col Ⅲ (10%~15%). As we age, skin cells gradually age, resulting in a decrease in collagen synthesis, and ultimately the cessation of collagen production and secretion.

[0110] refer to Figure 2 It can be seen that compared with Comparative Examples 2, 4, and 6, Examples 1 to 3 significantly promoted the expression of Col I and Col III genes in HSF cells, and can promote the synthesis of Col I and Col III, and have firming and anti-wrinkle effects, which indicates that the Danfeng peony flower peptide solution prepared in Examples 1 to 3 can maintain firming and anti-wrinkle effects by promoting the synthesis of collagen in the dermis of the skin.

[0111] Hyaluronic acid plays an important role in retaining skin moisture and maintaining skin structure. It also has the function of promoting skin regeneration, enhancing skin elasticity, and degrading free radicals in the skin.

[0112] according to Figure 3 It can be seen that Examples 1 to 3 can significantly increase the mRNA expression level of HAS2 in HSF cells, promote the synthesis of hyaluronic acid in the skin, increase the hyaluronic acid content, and thus delay cell or tissue aging, which indicates that the Danfeng peony flower peptide solution prepared in Examples 1 to 3 can increase skin hydration, increase skin elasticity, and reduce wrinkle formation.

[0113] Experiment 8: Screening of Danfeng Peony Flower Peptide In this experiment, the peptide segments of the Danfeng peony flower peptide prepared in Example 1 were screened and analyzed.

[0114] 8.1 Mass spectrometry experimental methods (1) Pretreatment In this experiment, the Danfeng peony peptide was pre-treated by the reductive alkylation method, and the specific steps are as follows: S200, use a pipette to accurately transfer 50 μL of sample into the EP tube, add 50 μL of water and mix well; S201. Use a pipette to accurately transfer 1 μL of 1M DTT solution to the sample to make the final concentration of DTT 10 mmol / L. Reduce in a water bath at 56°C for 1 h. S202. Use a pipette to accurately transfer 2 μL of 1M IAM solution to the sample to make the final IAM concentration 20 mmol / L. Incubate in dark at room temperature for 40 min. S203. Use a pipette to accurately transfer 1 μL of 1M DTT solution to the sample to make the final concentration of DTT 10 mmol / L to neutralize the unreacted IAM.

[0115] (2) C18 desalting (Stage-Tip) Desalt using a C18 stage-tip and dry under vacuum at 45°C.

[0116] 8.2 Computer Requirements (1) Liquid chromatography conditions 1) Pre-column: 150 μm id × 50 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm; Analytical column: 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm; 2) Mobile phase A: 0.1% FA; 3) Mobile phase B: 0.1% FA, 80% ACN; 4) Flow rate: 600 nL / min; 5) Analysis time for each component: 66 min; 6) Specific chromatographic conditions are listed in Table 7: Table 7 Specific chromatographic conditions of liquid chromatography

[0117] (2) Mass spectrometry conditions The primary mass spectrometry parameters were: 1) Resolution: 70,000; 2) AGCtarget: 3e6; 3) MaximumIT: 100 ms; 4) Scanrange: 100 to 1500 m / z.

[0118] The secondary mass spectrometry parameters were: 1) Resolution: 17,500; 2) AGCtarget: 1e5; 3) MaximumIT: 50 ms; 4) TopN: 20; 5) NCE / steppedNCE: 28.

[0119] The original data was obtained through mass spectrometry acquisition.

[0120] 8.3 Search conditions The mass spectrometry raw file uses software to search the target protein database, and the search parameters are as follows: 1) Fixed modifications: Carbamidomethyl (C); 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term); 3) Enzyme: Non specific; 4) Database: uniprotkb_Acipenser (genus)_2024_11_13; uniprotkb_Oreochromis (genus)_2024_12_05; 5) Peptide Mass Tolerance: 20 ppm; 6) Fragment Mass Tolerance: 0.02 Da.

[0121] 8.4 Results Analysis (1) Peptide screening The raw files collected by mass spectrometry contain more than 5900 peptides ranging from 2 to 13. Since 2 peptides have been studied more, they are not included in this screening. After searching the Byonic database, 104 peptides with an abundance greater than 100,000,000 were screened out, and the fragments were scored for activity prediction (http: / / distilldeep.ucd.ie / PeptideRanker / ), and 7 peptides with results > 0.9 were selected. After searching the peptide database BIOPEP (http: / / bis.zju.edu.cn / biopepdbr / index.php), 5 peptides were new peptides that had not been reported, and the toxicity prediction of the 5 peptides was non-toxic (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php); the relevant information of the 5 peptides is listed in Table 8: Table 8 Peptide information

[0122] (2) Molecular docking Molecular docking software was used to perform molecular docking on the above five peptides with tyrosinase, hyaluronidase, MMP-1, MMP-3, and COX-2 receptors. The three-dimensional structures of tyrosinase (PDB code: 966c), hyaluronidase (PDB code: 2Pe4), MMP-1 (PDB code: 966c), MMP-3 (PDB code: 2JT6), and cyclooxygenase-2 (COX-2) (PDB code: 5IKV) were downloaded from the PDB database (http: / / www.rcsb.org / ). The docking binding energy data of the five peptides with different receptors are shown in Table 9 Table 9 Docking binding energy of peptides and receptors

[0123] Hyaluronidase is a type of enzyme that can break down hyaluronic acid. Hyaluronic acid plays an important role in retaining skin moisture and maintaining skin structure. Therefore, peptides that can inhibit hyaluronidase are believed to have moisturizing effects.

[0124] The main components of the extracellular matrix include collagen, elastin, etc. The degradation of the extracellular matrix mainly relies on protease, and matrix metalloproteinases (MMPs) are the most important group of protease, which can degrade collagen and elastin in the dermis. Therefore, inhibiting the expression of MMP-1 / 3 and blocking the binding of MMP1 / 3 to the substrate can inhibit its activity and reduce the degradation of matrix collagen.

[0125] Cyclooxygenase-2 (COX-2) has always been considered to play an important role in the pathological process of inflammation. Inhibiting the binding of COX-2 active sites and preventing its catalytic activity can inhibit the inflammatory response.

[0126] According to the data in Table 9, the five peptides have a certain binding energy with the five receptor proteins of tyrosinase, hyaluronidase, MMP-1, MMP-3 and COX-2, which indicates that the five peptides of FSPL, FGPL, FGPI, SLGF and LFL are active fragments of Danfeng peony flower peptide with the functions of whitening, moisturizing, firming and promoting collagen synthesis.

[0127] Figures 4 to 8 These are the 2D images of FSPL docking with five receptor protein molecules: tyrosinase, hyaluronidase, MMP-1, MMP-3, and COX-2; Figures 9 to 13 They are represented as 2D images of the docking of FGPL with five protein molecules: tyrosinase, hyaluronidase, MMP-1, MMP-3, and COX-2; Figures 14 to 18 They are represented as 2D images of the docking of FGPI with five protein molecules: tyrosinase, hyaluronidase, MMP-1, MMP-3, and COX-2; Figures 19 to 23They are represented as 2D images of SLGF docking with five protein molecules: tyrosinase, hyaluronidase, MMP-1, MMP-3, and COX-2; Figures 24 to 28 They are represented as 2D images of the docking of LFL with five receptor protein molecules: tyrosinase, hyaluronidase, MMP-1, MMP-3, and COX-2.

[0128] like Figure 4 As shown, there are donor hydrogen bonds, van der Waals forces, and π-π interactions between FSPL and tyrosinase; Figure 5 As shown, the interactions between FSPL and hyaluronidase are mainly carbon-hydrogen bonds, conventional hydrogen bonds, van der Waals forces, attractive charges, and salt bridges; Figure 6 As shown, there are conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, salt bridges, and van der Waals forces between FSPL and MMP-1; Figure 7 As shown, the main interactions between FSPL and MMP-3 are carbon-hydrogen bonds, conventional hydrogen bonds, attractive charges, van der Waals forces, and alkyl forces; Figure 8 As shown, the interactions between FSPL and COX-2 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, salt bridges and van der Waals forces.

[0129] like Fig. 9 As shown, the main forces between FGPL and tyrosine are carbon-hydrogen bonds, conventional hydrogen bonds, alkyl groups, π-anion interactions, and van der Waals forces; Fig.10 As shown, there are carbon-hydrogen bonds, van der Waals forces, alkyl interactions and salt bridge interactions between FGPL and hyaluronidase; Fig.11 As shown, the interactions between FGPL and MMP-1 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, π-alkyl, van der Waals forces, and salt bridges; Fig.12 As shown, the interactions between FGPL and MMP-3 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, alkyl groups, and van der Waals forces; Fig.13 As shown, the interactions between FGPL and COX-2 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, van der Waals forces, alkyl effects and attractive charge interactions.

[0130] like Fig.14 As shown, the main forces between FGPI and tyrosine are van der Waals forces, conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, π-π interactions, alkyl groups, and salt bridges; Fig.15 As shown, the interactions between FGPI and hyaluronidase are mainly conventional hydrogen bonds, carbon-hydrogen bonds, van der Waals forces, attractive charges, and salt bridges; Fig.16 As shown, the interactions between FGPI and MMP-1 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, van der Waals forces, and alkyl forces; Fig.17As shown, the interactions between FGPI and MMP-3 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, alkyl groups, and van der Waals forces; Fig.18 As shown, the interactions between FGPI and COX-2 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, van der Waals forces, attractive charges, π-π interactions, alkyl groups and salt bridges.

[0131] like Fig.19 As shown, the interactions between SLGF and tyrosine are mainly conventional hydrogen bonds, carbon-hydrogen bonds and van der Waals forces, and there are also metal receptor and π-alkyl interactions; Fig. 20 As shown, the interactions between SLGF and hyaluronidase are mainly conventional hydrogen bonds, carbon-hydrogen bonds, van der Waals forces, attractive charges, and salt bridges; Fig.21 As shown, the interactions between SLGF and MMP-1 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, van der Waals forces, and π-π interactions; Fig. 22 As shown, the interactions between SLGF and MMP-3 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, van der Waals forces, and π-alkyl forces; Fig.23 As shown, the interactions between SLGF and COX-2 are mainly conventional hydrogen bonds, carbon-hydrogen bonds and van der Waals forces, and there are also π-cation and π-alkyl forces.

[0132] like Fig.24 As shown, the forces between LFL and tyrosine are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges and van der Waals forces, and there are also π-π interactions, π-alkyl and π-anion interactions; Fig.25 As shown, LFL interacts with hyaluronidase via carbon-hydrogen bonds, van der Waals forces, and covalent bonds; Fig.26 As shown, the interactions between LFL and MMP-1 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, van der Waals forces, alkyl groups and salt bridges, and there is also the influence of metal receptors; Fig. 27 As shown, the interactions between LFL and MMP-3 are mainly conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, donor hydrogen bonds, alkyl groups, and van der Waals forces; Fig.28 As shown, the forces between LFL and COX-2 are mainly conventional hydrogen bonds, attractive charges, salt bridges and van der Waals forces, and there are also alkyl and π-cation interactions.

[0133] It should be noted that the terms used in the present invention, such as ultrafiltration, centrifugation, spray drying, mass spectrometry experiment, molecular docking, RNA extraction, etc., are the names of processing steps commonly used by those skilled in the art, and their names can accurately describe the processing process and will not be repeated in the present invention.

[0134] The raw materials used in the present invention, such as acid protease, cellulase, pectinase, alkaline protease, papain, Bacillus subtilis flavor protease, Bacillus subtilis neutral protease and the like, are all existing commercially available enzymes and can be purchased by those skilled in the art; in addition, unless otherwise specified, other raw materials used in the present invention may also be conventional commercial products in the art or prepared by conventional methods in the art, that is, the reagents, instruments, and consumables used in the present invention, without specifying the manufacturer and other information, can also be purchased from the market.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a Danfeng peony peptide, characterized in that: The following steps are involved: Mixing dried red peony flowers with pure water, and then grinding and homogenizing the mixture to obtain red peony flower slurry; Performing alkali extraction on the peony flower slurry, centrifuging to obtain the supernatant, then performing acid precipitation and standing overnight, centrifuging to retain the precipitate, and adding pure water to prepare the peony flower suspension; Acidic protease, a complex enzyme of cellulase and pectinase and alkaline protease are sequentially added to the suspension of Danfeng peony flowers for three-step hydrolysis, followed by enzyme inactivation treatment to obtain a Danfeng peony flower hydrolyzate; The Danfeng Peony Flower hydrolyzate is subjected to solid-liquid separation to obtain a Danfeng Peony Flower peptide solution.

2. The method for preparing the Danfeng Peony Flower Peptide according to claim 1, characterized in that: The dried Danfeng peony flowers are mixed with pure water in a mass percentage ratio of 1: (15-30), and the temperature is raised to 55-65° C. and incubated for 1-2 h; During the alkaline extraction operation, the treatment temperature is 55±1℃, the treatment pH is 9.5~10, and the treatment time is 1~3 h; During the acid precipitation operation, the processing temperature is 37±1℃, the processing pH is 4.2, and it is allowed to stand at 4℃ overnight. The precipitate is retained by centrifugation and an equal amount of pure water is added to resuspend it.

3. The method for preparing the Danfeng Peony Flower Peptide according to claim 1, characterized in that: The amount of the acidic protease added is 0.5% to 1.5% of the mass of the dried Danfeng peony flowers; The added amount of the cellulase and pectinase is 0.5% to 2% of the mass of the dried Danfeng peony flowers; The added amount of the alkaline protease is 1% to 2% of the mass of the dried Danfeng peony flowers.

4. The method for preparing the phoenix peony flower peptide according to claim 1, characterized in that: When acid protease is added for primary hydrolysis, the hydrolysis temperature is 40-42°C, the pH is 2.5-3, and the hydrolysis time is 1-2 h; When the complex enzyme of cellulase and pectinase is added for secondary hydrolysis, the hydrolysis temperature is 49-52°C, the pH is 4.7-5.0, and the hydrolysis time is 1-2 h; When alkaline protease is added for three hydrolysis, the hydrolysis temperature is 55-57°C, the pH is 8.4-8.5, the hydrolysis time is 2-3h, and then the pH is adjusted to 4.5 and the enzyme is inactivated at 85-90°C for 15-20 min.

5. The method for preparing the Danfeng Peony Flower Peptide according to claim 1, characterized in that: After three-step hydrolysis to inactivate the enzyme, activated carbon was added for decolorization. The amount of activated carbon added was 4% to 5% of the mass of the dried Danfeng peony flower. The treatment time was 1 to 1.5 h. After the treatment was completed, it was centrifuged at 4000 to 6000 rpm for 5 to 10 min, the supernatant was taken, and then filtered with diatomaceous earth to obtain the Danfeng peony flower peptide solution.

6. The method for preparing the Danfeng Peony Flower Peptide according to claim 1, characterized in that: The Danfeng Peony Flower Peptide Solution is subjected to ultrafiltration interception to obtain a solution containing Danfeng Peony Flower Peptide with a molecular weight of less than 5000 Da.

7. The method for preparing the Danfeng Peony Flower Peptide according to claim 1, characterized in that: The Danfeng Peony Flower Peptide solution is spray-dried to obtain Danfeng Peony Flower Peptide powder.

8. A Danfeng Peony Flower Peptide prepared according to the method for preparing Danfeng Peony Flower Peptide according to any one of claims 1 to 7, characterized in that: The active peptide segments of Danfeng peony flower peptides are included: the amino acid sequence is Phe-Ser-Pro-Leu or Phe-Gly-Pro-Leu or Phe-Gly-Pro-Ile or Ser-Leu-Gly-Phe or Leu-Phe-Leu.

9. A functional product, characterized in that: Its components include the Danfeng peony flower peptide described in claim 8.

10. The functional product according to claim 9, characterized in that: Include at least one of the following functions: (1) Tyrosinase inhibitory activity; (2) Hyaluronidase inhibitory activity; (3) Anti-glycation activity; (4) Anti-inflammatory activity; (5) Anti-wrinkle activity.

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