Peony flower peptide for inhibiting MMP activity and preparation method and application thereof

Abstract

The invention discloses peony flower peptide for inhibiting MMP activity and a preparation method and application thereof, and relates to the technical field of cosmetics. The peony flower peptide is prepared by the following steps: adding dried peony flower powder into deionized water according to a solid-liquid ratio of (1: 10)-(1: 50) (g / mL), adding 1-10% (g / g) of protease, adjusting the pH value to 7.5-9.0, carrying out enzymolysis at 50-70 DEG C for 2-6 hours, heating the reaction solution in boiling water to remove volatile grease, centrifuging, and taking supernatant, so as to obtain primarily extracted peony flower crude peptide; performing ultrafiltration on the peony flower crude peptide to obtain molecular weight lt; the composition is prepared from the components of 3kDa and 3kDa. According to the present invention, the peony flower peptide can simultaneously remove a variety of free radicals, has good antioxidant effect, can simultaneously inhibit the activity of a variety of MMP, inhibits the degradation of collagen, and has good anti-aging effect, the preparation method of the peony flower peptide is simple, the complex complex enzyme configuration is not required, and the obtained peony flower peptide has characteristics of small molecular weight, easy absorption, and no toxic-side effect. Wide application prospects are realized in the field of cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular to a peony flower peptide for inhibiting MMP activity, a preparation method and application thereof. BACKGROUND

[0002] Peony is a traditional Chinese flower, belonging to Paeonia Linn, which is divided into ornamental peony and oil type peony. It not only has high ornamental value, but also is rich in various bioactive substances and has been widely used in the field of traditional Chinese medicine for a long time. In recent years, peony flowers and peony seeds as by-products of the production of Danpi have also attracted more and more attention. Peony peptide is the product of peony flower or peony seed protein enzymolysis, which has biological activities such as antioxidant, blood pressure lowering and blood sugar lowering, and has been widely used. However, the existing technology is mostly the research on the preparation method of peony peptide, and the resource opening of peony peptide is not comprehensive enough.

[0003] Especially, the antioxidant effect of modern peony flower peptide is mostly focused on free radical scavenging, and the research on matrix metalloproteinase (MMP) is still relatively less. MMP plays a key role in promoting the degradation of old collagen and promoting wound repair in the normal metabolism of skin, and is an important factor to maintain skin health. However, when it is over-activated, it becomes the core driver of skin damage. The trigger factors of MMP activity imbalance are diverse, and the damage path is also relatively complex. Under normal conditions, the MMP activity of the skin is strictly regulated, which can maintain the normal structure and function of the skin, such as maintaining the tightness, elasticity and good barrier function of the skin. When MMP is over-activated, the skin will show obvious changes, such as increased wrinkles, sagging, sensitive and fragile, etc. Mechanistically, MMP activity imbalance will break the original homeostasis of the skin and cause a series of damage to the structure and function of the skin.

[0004] CN117051069A discloses a peony antioxidant peptide extraction method and its application, which is an antioxidant peptide obtained by twice enzymolysis of peony seeds. However, peony seeds and Danfeng peony petals do not belong to the same part, and the oil contained in Danfeng peony petals is mainly volatile oil, and the oil content is less than that of peony seeds. Therefore, the extraction method of peony seeds cannot be directly used for the extraction of peony petals, and the antioxidant effect is only reflected in the free radical scavenging aspect, and the effect of inhibiting MMP activity is unknown.

[0005] CN117363678A discloses a method for preparing peony fresh flower extract. In particular, the method for preparing peony fresh flower extract by enzymolysis develops various peony active peptides, which are pure natural and free of additives, and have effects of antioxidant, whitening and moisturizing, lightening of color spots, delaying of aging, and antibacterial and anti-inflammatory. Then, the method also needs to be subjected to preliminary enzymolysis by lipase, cellulase and glycosidase, and then subjected to enzymolysis by a complex enzyme of 1:1:1:1:1:1 papain, trypsin, neutral protease, alkaline protease, subtilisin and chymotrypsin to obtain small molecular peptides less than 10 kDa. Although the obtained polypeptides have many effects, the types and ratio of the complex enzyme are strictly required, and the effects of MMP activity inhibition are not involved.

[0006] In summary, there is no related research on peony flower peptides with the effect of inhibiting MMP activity. SUMMARY

[0007] In view of the important role of MMP in maintaining metabolic balance in the skin, in view of the blank of the prior art, the present application discloses a peony flower peptide for inhibiting MMP activity, the molecular weight of the peony flower peptide is <3 kDa, preferably, the molecular weight of the peony flower peptide is 160 Da-2100 Da, the peony flower peptide not only has good free radical scavenging effect, but also can inhibit the activity of various MMPs, and has wide application prospect in the preparation of anti-oxidation and anti-aging products, the content of the present application is as follows:

[0008] In the first aspect of the present application, a method for preparing a peony flower peptide for inhibiting MMP activity is provided, the method comprises the following steps:

[0009] S1. The peony flower powder is added to deionized water at a ratio of 1:10-1:50 (g / mL), 1%-10% (g / g) of protease is added, and the pH is adjusted to 7.5-9.0, and then the reaction solution is heated in boiling water to remove volatile oil and fat after enzymolysis at 50-70℃ for 2-6h, and the supernatant is obtained by centrifugation to obtain the preliminary extracted peony flower crude peptide;

[0010] S2. The peony flower crude peptide obtained in S1 is subjected to ultrafiltration to obtain a component with a molecular weight of <3 kDa, i.e. the peony flower peptide for inhibiting MMP activity.

[0011] Further, the protease in step S1 is selected from one of alkaline protease, neutral protease, papain, trypsin and pepsin.

[0012] Further, the centrifugation condition in step S1 is 4000 rpm for 10 min.

[0013] Further, the step S2 is specifically operated as follows: using 3kDa ultrafiltration centrifuge tube to centrifuge at 4000-6000rpm for 20-30min to obtain the component with molecular weight <3kDa.

[0014] In a specific embodiment of the present application, the step S1 is specifically operated as follows: drying and crushing the peony flower to obtain peony flower powder; adding the peony flower powder into deionized water at a solid-liquid ratio of 1:22(g / mL), adding alkaline protease at an enzyme amount of 7%(g / g), stirring uniformly at low speed, adjusting pH to 8.0, placing in a water bath pot for constant temperature heating reaction at 60℃, and heating the reaction solution in boiling water for 10min to obtain the product.

[0015] In a specific embodiment of the present application, the step S2 is specifically operated as follows: ultrafiltration of the peony flower crude peptide: using ultrafiltration centrifuge tube to centrifuge the peony flower crude peptide, first using 10kDa ultrafiltration centrifuge tube to centrifuge at 5000rpm for 20min to obtain the component with molecular weight >10kDa, <10kDa part is taken out and centrifuged at 5000rpm for 20min using 3kDa ultrafiltration membrane to obtain the component with molecular weight <3kDa; measuring the antioxidant activity of each component, and taking the component with the highest antioxidant activity in the subsequent experiment, i.e. the component with molecular weight <3kDa.

[0016] Further, the method further comprises step S3: gel filtration chromatography of the peony flower peptide obtained in step S2 to obtain the peony flower peptide with molecular weight 160Da-2100Da.

[0017] The step of gel filtration chromatography is specifically operated as follows: eluting the peony flower peptide obtained in step S2 at a rotation speed of 50-60rpm and a flow rate of 2.5-3.5mL / min to obtain the peony flower peptide with molecular weight 160Da-2100Da.

[0018] In a specific embodiment of the present application, the step S3 is specifically operated as follows:

[0019] The chromatography column was equilibrated with 0.025 mol / L Tris-HCl phosphate buffer (pH 7.2) at a flow rate of 1.0 mL / min. The peristaltic pump speed was adjusted so that the inlet and outlet flow rates were the same. 1 mL of the fraction with a molecular weight <3 kDa was added to the gel column. Deionized water was used as the eluent. The peristaltic pump speed was 54 rpm and the flow rate was 3 mL / min. The eluent was collected in 3 mL tubes. After collection, the sample was scanned at 245 nm to obtain seven fractions: G1, G2, G3, G4, G5, G6, and G7. The antioxidant and MMP inhibition effects of the seven fractions were verified, and the fractions with the highest activity, G2 and G3, were found. The molecular weights of G2 and G3 were calculated to be 160 Da-2100 Da based on the elution volume.

[0020] In a third aspect, the present invention provides peony flower peptides prepared by applying the method described above.

[0021] In a fourth aspect, the present invention provides the application of the peony flower peptide in any of the following aspects:

[0022] A1: Applications in the preparation of free radical scavenging products;

[0023] A2: Applications in the preparation of products that inhibit MMP activity.

[0024] In a fifth aspect, the present invention provides the application of the peony flower peptide in any of the following aspects:

[0025] B1: Applications in the preparation of antioxidant products;

[0026] B2: Applications in the preparation of products that inhibit collagen degradation.

[0027] In a sixth aspect, the present invention provides the application of the aforementioned peony flower peptide in the preparation of anti-aging products. Furthermore, the anti-aging products can achieve antioxidant effects by scavenging free radical activity; they can also inhibit collagen degradation by suppressing MMP activity, thereby achieving anti-wrinkle effects, improving skin elasticity, and maintaining skin homeostasis.

[0028] Furthermore, the products include at least cosmetics.

[0029] Furthermore, the product also includes additives acceptable in the cosmetics field.

[0030] The beneficial effects of the present invention include, but are not limited to:

[0031] The peony flower peptide disclosed in this invention can not only simultaneously scavenge multiple free radicals and have a good antioxidant effect, but also simultaneously inhibit the activity of multiple MMPs and inhibit the degradation of collagen, thus having a good anti-aging effect. Moreover, the preparation method of the peony flower peptide of this invention is simple, does not require the preparation of complex compound enzymes, and the obtained peony flower peptide has a small molecular weight, making it easier to absorb, and has broad application prospects in the cosmetics field. Attached Figure Description

[0032] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0033] Figure 1 The DPPH free radical scavenging rate of crude peony peptides at different concentrations is shown.

[0034] Figure 2 The ABTS free radical scavenging rate of crude peony peptides at different concentrations is shown.

[0035] Figure 3 The DPPH radical scavenging rate and IC50 of components F1, F2, and F3 at different concentrations were compared. 50 In the figure, A: DPPH radical scavenging rate of component F1 at different concentrations; B: DPPH radical scavenging rate of component F2 at different concentrations; C: DPPH radical scavenging rate of component F3 at different concentrations; D: IC50 values ​​for each component. 50 Value comparison.

[0036] Figure 4 The ABTS radical scavenging rate and IC50 of components F1, F2, and F3 at different concentrations were compared. 50 In the figure, A: ABTS radical scavenging rate of component F1 at different concentrations; B: ABTS radical scavenging rate of component F2 at different concentrations; C: ABTS radical scavenging rate of component F3 at different concentrations; D: IC50 values ​​for each component. 50 Value comparison.

[0037] Figure 5 This is the UV-Vis full-wavelength absorption scan of peony flower peptides in fraction F3.

[0038] Figure 6 This is a chromatographic elution result of the separation and purification of component F3 by gel filtration chromatography.

[0039] Figure 7 The DPPH free radical scavenging rate of each component obtained by Sephadex G-25 gel filtration chromatography is given for component F3.

[0040] Figure 8ABTS radical scavenging rate of each fraction of F3 component separated by Sephadex G-25 gel filtration chromatography.

[0041] Figure 9 Total activity and non-specific activity fluorescence difference of each fraction of different molecular weight separated by ultrafiltration.

[0042] Figure 10 MMP-1 activity inhibition rate of each fraction of different molecular weight separated by ultrafiltration.

[0043] The same letter represents no significant difference (P>0.05) and different letters represent significant difference (P<0.05); in the analysis of difference marked by asterisk, *** represents extremely significant difference (P<0.001).

[0044] Figure 11 Total activity and non-specific activity fluorescence difference of each fraction of F3 component separated by Sephadex G-25 gel filtration chromatography.

[0045] Figure 12 MMP-1 activity inhibition rate of each fraction of F3 component separated by Sephadex G-25 gel filtration chromatography; in the analysis of difference marked by asterisk, ns represents no significant difference (P>0.05) and *** represents extremely significant difference (P<0.001).

[0046] Figure 13 MMP-3 activity inhibition fluorescence value of each fraction of different molecular weight separated by ultrafiltration.

[0047] Figure 14 MMP-3 activity inhibition rate of each fraction of different molecular weight separated by ultrafiltration; the same letter represents no significant difference (P>0.05) and different letters represent significant difference (P<0.05); in the analysis of difference marked by asterisk, *** represents extremely significant difference (P<0.001).

[0048] Figure 15 MMP-3 activity inhibition fluorescence value of each fraction of F3 component separated by Sephadex G-25 gel filtration chromatography.

[0049] Figure 16The inhibitory rate of each component of the F3 component separated by Sephadex G-25 gel filtration chromatography on the activity of MMP-3; the same letter indicates no significant difference (P>0.05), and different letters indicate significant difference (P<0.05). In the difference analysis marked by an asterisk, *** represents extremely significant difference (P<0.001). DETAILED DESCRIPTION

[0050] The present application will be described in detail below in conjunction with examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are all purchased through commercial channels.

[0051] Matrix metalloproteinase-1: purchased from Taizhou Mornai Biological Material Sales Department;

[0052] Matrix metalloproteinase-3: purchased from Wuhan Elabscience Biotechnology Co., Ltd.

[0053] Example 1:

[0054] A preparation method of a peony flower peptide having a collagen protection effect and inhibiting MMP activity, comprising the following steps:

[0055] S1, dry peony flowers are ground and sieved to obtain peony flower powder. The peony flower powder is added to deionized water at a solid-liquid ratio of 1:22 (g / mL), and alkaline protease is added at an enzyme amount of 7% (g / g). After uniform stirring at low speed, the pH is adjusted to 8.0, and the reaction is placed in a water bath pot for constant temperature heating at 60°C. After enzymolysis for 4h, the reaction solution is heated in boiling water for 10min to obtain the preliminary extracted peony flower crude peptide;

[0056] S2, ultrafiltration of the peony flower crude peptide: the peony flower crude peptide is subjected to ultrafiltration centrifugation using an ultrafiltration centrifuge tube. First, a 10kDa ultrafiltration centrifuge tube is used for centrifugation at 5000rpm for 20min for interception. The <10kDa part is taken out and subjected to ultrafiltration centrifugation using a 3kDa ultrafiltration membrane centrifuge tube at 5000rpm for 20min for interception to obtain a component with a molecular weight >10kDa (F1), a component with a molecular weight 3-10kDa (F2), and a component with a molecular weight <3kDa (F3);

[0057] S3, gel filtration chromatography:

[0058] S3-1, gel swelling: 8.0g of Sephadex G-25 gel dry powder is weighed, then 100mL of deionized water is added for swelling at room temperature for 36h. After the swelling is completed, the upper impurities are removed and washed with deionized water for 3 times;

[0059] S3-2, Column loading: Fix the column on the iron stand, add a certain amount of deionized water, then start the peristaltic pump, continuously add the gel, and the gel particles are 5 cm away from the water inlet. The column loading is completed; the column size is 2.6 cm x 70 cm, and the column is installed on the Biotage rapid preparation liquid phase system. The column is equilibrated with 0.025 mol / L Tris-HCl phosphate buffer (pH 7.2) at a flow rate of 1.0 mL / min;

[0060] S3-3, Sample loading and elution: Adjust the speed of the peristaltic pump to the appropriate speed, that is, the flow rates of the water inlet and outlet are the same. Add 1 mL of components with a molecular weight of <3 kDa to the gel column, and deionized water is used as the eluent. The speed of the peristaltic pump (Chongqing Jieheng BT-600EA) is 54 rpm, and the flow rate is 3 mL / min. Start collecting the eluent, and collect one tube every 3 mL. After the collection is completed, scan the sample at 245 nm to obtain seven components G1, G2, G3, G4, G5, G6, and G7. The elution volume of the G2 component preparation is 33-43 mL.

[0061] The elution volume of the G3 component preparation is 51-61 mL.

[0062] The gel column volume is about 31.4 mL

[0063] That is, for the general case (including the case of column volume amplification), the elution volume of the G2 component preparation is 1.05-1.37 gel column volumes; the elution volume of the G3 component preparation is 1.62-1.94 column volumes. Therefore, the molecular weight range of G2 and G3 is 160.18-2066.53 Da.

[0064] The desalination step of the peptide segment is as follows:

[0065] (1) Activate the column with about 1 column volume of methanol, and add 1 column volume of 80% ACN / 0.1% TFA when the dripping is almost complete;

[0066] (2) Equilibrate the column with 2 column volumes of 0.1% TFA;

[0067] (3) Add the G2 or G3 component to the desalination column;

[0068] (4) After the sample is loaded, wash the column with 2 column volumes of 1% acetic acid;

[0069] (5) Elute with 1.3 column volumes of 80% ACN / 0.1% acetic acid, collect the eluent, and blow off the solution remaining in the packing after the dripping is complete. The column can be used again by starting with methanol;

[0070] (6) The eluate containing the peptide segment is aliquoted and placed in a rotary vacuum dryer to evaporate the organic solvent. The peptide segment is stored at -80°C.

[0071] The capillary high performance liquid chromatography separation step is specifically as follows:

[0072] The desalted peptide segment is separated by a nanoflow rate HPLC liquid phase system. Buffer A is 0.1% formic acid aqueous solution, and buffer B is 0.08% formic acid acetonitrile aqueous solution (80% acetonitrile). The chromatographic column is equilibrated with 100% A solution, and the sample is loaded onto the mass spectrometry pre-column by an automatic sampler, and then separated by an analysis column to obtain the sample, and the flow rate and related liquid phase gradient are shown in Table 1.

[0073] Table 1

[0074]

[0075] Example 2: Determination of the antioxidant property of the peony flower peptide prepared in Example 1:

[0076] I. Determination of DPPH free radical scavenging rate

[0077] 1. Preparation of DPPH solution

[0078] 0.0079 g of DPPH solid was accurately weighed using an analytical balance and dissolved in an appropriate amount of anhydrous methanol to prepare a DPPH stock solution with a concentration of 200 μmol / L, which was placed in the dark for standby.

[0079] 15 mL of the above solution was measured and diluted to obtain a DPPH solution with a concentration gradient of 60 μmol / L.

[0080] 2. Determination of DPPH scavenging capacity of sample solution

[0081] The experimental group was 0.1 mL of sample solution with different concentration gradients of 70, 80, 90, 100, 110, 120, 130, 150, and 180 μg / mL, and 0.9 mL of diluted DPPH solution was added. In the experimental process, the well-mixed sample to be tested was placed in the dark for 30 min. The blank control group: 0.1 mL of solvent (anhydrous methanol) + 0.9 mL of DPPH working solution; background control: 0.1 mL of sample solution + 0.9 mL of anhydrous methanol. The absorbance of each group was measured at 518 nm using a UV spectrophotometer. The DPPH free radical scavenging rate was calculated, and the calculation formula is as follows:

[0082]

[0083] Wherein A is the absorbance of the experimental group; B is the absorbance of the background control group; and C is the absorbance of the blank control group.

[0084] II. Determination of ABTS free radical scavenging rate

[0085] 1. Preparation of ABTS free radical mother liquor

[0086] Accurately weigh 0.0384 g of ABTS and add 5 mL of ultrapure water to obtain an ABTS solution with a concentration of 14 mmol / L, designated as solution A. Weigh 0.0066 g of potassium persulfate and add 5 mL of ultrapure water to obtain a K₂S₂O₈ solution with a concentration of 4.9 mmol / L, designated as solution B. Mix solutions A and B in a 1:1 ratio and react at room temperature in the dark for 12–16 hours.

[0087] 2. Preparation of ABTS free radical working solution

[0088] Prepare a 70% ethanol solution by mixing 350 mL of 100% anhydrous ethanol and 150 mL of deionized water. Mix 1 mL of ABTS radical stock solution with 80 mL of 70% ethanol solution and measure the absorbance at 734 nm. Adjust the absorbance to 0.70 ± 0.02 with 70% ethanol solution to obtain the ABTS radical working solution.

[0089] 3. Determination of ABTS free radical scavenging rate in sample solution

[0090] In the experimental group, sample solutions and ABTS free radical working solution with different concentration gradients (100, 150, 200, 250, 300, 350, 400, 450, and 500 μg / mL) were added at a volume ratio of 1:5. The prepared samples were mixed thoroughly and placed in the dark for 6 min, and the absorbance was measured at a wavelength of 734 nm. The blank control group consisted of 0.2 mL of 70% ethanol and 1.0 mL of ABTS free radical working solution; the background control group consisted of 0.2 mL of sample solution and 1.0 mL of 70% ethanol. The formula for calculating the ABTS free radical removal rate is as follows:

[0091]

[0092] A represents the absorbance of the experimental group; B represents the absorbance of the background control group; and C represents the absorbance of the blank control group.

[0093] III. Measurement Results

[0094] 1. DPPH and ABTS free radical scavenging rates of crude peony peptides at different concentrations

[0095] The experiments in Example 2 were conducted using crude peony peptides at different concentrations, and the results are as follows: Figure 1 , 2 As shown;

[0096] Figure 1 The fitted equation is Y = -0.002227X. 2-0.06801X+8.478, correlation coefficient R 2 =0.9903.

[0097] Figure 2 The fitted equation is Y = -0.0001336X 2 +0.05586X - 3.139, correlation coefficient R 2 =0.9919.

[0098] 2. Determination of DPPH radical scavenging rate, ABTS radical scavenging rate and FRAP value of components with different molecular weights obtained by ultrafiltration separation. The experiments in Example 2 were performed using different concentrations of component F1 (molecular weight > 10 kDa), component F2 (molecular weight 3-10 kDa), and component F3 (molecular weight < 3 kDa). The results are as follows:

[0099] 2.1. DPPH free radical scavenging rate

[0100] like Figure 3 As shown, Figure 3 A shows the DPPH radical scavenging rate of component F1 at different concentrations. The curves were fitted and the IC50 was calculated. 50 The fitted equation was obtained as Y = 0.008307X. 2 +1.592X+17.74, correlation coefficient R 2 =0.9958. Calculate IC. 50 =19.92μg / mL.

[0101] Figure 3 B shows the DPPH radical scavenging rate of component F2 at different concentrations. The curves were fitted, and IC50 was calculated. 50 The fitted equation is obtained as Y = -0.003152X. 2 +1.013X+25.65, correlation coefficient R 2 =0.9888. Calculate IC. 50 =13.93μg / mL.

[0102] Figure 3 C shows the DPPH radical scavenging rate of component F3 at different concentrations. The curves were fitted, and IC50 was calculated. 50 The fitted equation is obtained as Y = -0.04135X. 2 +3.768X+25.49, correlation coefficient R 2 =0.9910. Calculate IC. 50 =5.079 μg / mL.

[0103] In summary and Figure 3 As shown in D, component F3 (molecular weight < 3kDa) has a stronger ability to scavenge DPPH.

[0104] 2.2. ABTS free radical scavenging rate

[0105] Figure 4 A shows the ABTS radical scavenging rate of component F1 at different concentrations. The curves were fitted and the IC50 was calculated. 50 The fitted equation obtained is Y = -0.0006299X. 2 +0.4640X+6.229, correlation coefficient R 2 =0.9919. Calculate IC. 50 = 97.39 μg / mL.

[0106] Figure 4 B shows the ABTS radical scavenging rate of component F2 at different concentrations. The curves were fitted, and IC50 was calculated. 50 The fitted equation obtained is Y = -0.002979X. 2 +0.9186X+21.33, correlation coefficient R 2 =0.9828. Calculate IC. 50 =29.35μg / mL.

[0107] Figure 4 C shows the ABTS radical scavenging rate of component F3 at different concentrations. The curves were fitted, and IC50 was calculated. 50 The fitted equation is obtained as Y = 0.03133X. 2 +3.168X+26.09, correlation coefficient R 2 =0.9965. Calculate IC. 50 = 6.861 μg / mL.

[0108] In summary and Figure 4 As shown in Figure D, component F3 (<3KD) exhibited a more pronounced ABTS radical scavenging effect. This is likely because the smaller molecular weight peptide chains allow for greater exposure of antioxidant amino acid residues, enabling them to fully exert their antioxidant activity. Therefore, component F3 was collected for further separation and purification.

[0109] 3. DPPH radical scavenging rate and ABTS radical scavenging rate of each component obtained by Sephadex G-25 gel filtration chromatography.

[0110] Analysis revealed that fraction F3 exhibited stronger anti-aging activity. Further gel filtration chromatography was performed on fraction F3 as the primary research subject. Full-wavelength scanning was conducted on fraction F3, such as... Figure 5The F3 component was determined to have an optical absorption wavelength of 245 nm by full wavelength scanning, thus determining the detection wavelength of the sample after gel filtration chromatography. The F3 component was separated into seven peaks by the elution process and named G1, G2, G3, G4, G5, G6, and G7, respectively, as shown in Figure 6 The seven components were collected, their protein concentrations were determined, and they were then diluted to the same concentration (2 μg / mL) and their DPPH and ABTS were determined, as shown in Figure 7 、 8 It was shown that the G3 component had the highest scavenging capacity.

[0111] Example 3: Peptide from peony flower for determination of inhibition of matrix metalloproteinase 1 (MMP-1) activity:

[0112] 1. Total activity determination of the sample to be tested

[0113] The detection was performed according to the experimental procedure of the matrix metalloproteinase-1 assay kit (purchased from the Mohnai Biological Material Sales Department in Xiaodian District, Taiyuan City) instructions, specifically: all samples to be tested were diluted to a concentration of 0.5 mg / mL, the wells of a 96-well plate were labeled (model group, Bose factor control group, and sample solution), 78 μL of buffer was transferred to the corresponding wells, 10 μL of deionized water and 10 μL of matrix metalloproteinase-1 solution at a concentration of 28.4 μg / mL were added to the model group, hydroxypropyl tetrahydro pyranetriol (C8H16O5) was added to the Bose factor control group, and 10 μL of sample solution and 10 μL of matrix metalloproteinase-1 solution were added to the other wells. After the addition was completed, the plate was incubated in a 37°C incubator for 10 min, then 2 μL of MCA peptide substrate solution (MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2) was added, and detection was performed after 5 min (excitation wavelength 330 nm, emission wavelength 400 nm), obtaining the corresponding fluorescence value of the total activity of the sample.

[0114] 2. Non-specific activity determination of the sample to be tested

[0115] Dilute all samples to be tested to a concentration of 0.5 mg / mL, label the model group, the Bose factor control group and the sample solution on a 96-well plate, respectively, and then transfer 68 μL of buffer to the corresponding wells, and then add 10 μL of the specific solution, add 10 μL of deionized water and 10 μL of matrix metalloproteinase-1 solution to the model group, add hydroxypropyl tetrahydro pyranetriol (C8H16O5) to the Bose factor control group, and add the crude peptide of peony, the components F1-F3 and F3 obtained by ultrafiltration separation, and the components G1-G3 obtained by Sephadex G-25 gel filtration chromatography separation to the sample solution, and then place the 96-well plate in a 37°C incubator for 10 min, and then respectively add 2 μL of the substrate solution (MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2), gently shake the 96-well plate, and then place it in a fluorescence enzyme label instrument for detection at an excitation wavelength of 330 nm and an emission wavelength of 400 nm after 5 min, and then obtain the non-specific activity fluorescence reading of the sample.

[0116] 3. Calculation of the inhibition rate of MMP-1 activity

[0117] The actual fluorescence reading of the specific activity of the sample to be tested is equal to the total activity fluorescence reading at 5 min minus the non-specific activity fluorescence reading at 5 min, and then the specific activity fluorescence reading of each group is calculated, and then the inhibition rate of MMP-1 activity of the sample is calculated.

[0118] 4. Determination results

[0119] 4.1 Determination of the inhibition of MMP-1 activity by the components obtained by ultrafiltration separation with different molecular weights

[0120] Figure 9 The difference between the total activity and the non-specific activity fluorescence of each component is Figure 10 The MMP-1 activity inhibition rate of each component is Figure 10 It can be seen that the inhibition rate of F3 on MMP-1 activity is the highest, and when the concentration is 0.5 mg / mL, the inhibition rate of F3 on MMP-1 activity can reach 85.24%.

[0121] 4.2 Determination of the inhibition of MMP-1 activity by the components obtained by Sephadex G-25 gel filtration chromatography separation of the F3 component

[0122] Figure 11 The difference between the total activity and the non-specific activity fluorescence of each component is Figure 12 The MMP-1 activity inhibition rate of each component is Figure 11 and 12 It can be seen that the inhibition rate of G2 on MMP-1 activity is the highest, and when the concentration is 0.5 mg / mL, the inhibition rate of G2 on MMP-1 activity is the highest, reaching 93.11%.

[0123] Example 4: Peony flower peptide inhibits matrix metalloproteinase 3 (MMP-3) activity assay

[0124] According to the experimental steps of the matrix metalloproteinase 3 assay kit (purchased from Wuhan Elyra Biotech Co., Ltd.), the specific steps are as follows:

[0125] 1. Preparation of reagent four (substrate) working solution

[0126] Mix reagent four (substrate) and deionized water according to the volume ratio of 3:20, centrifuge and store on ice in the dark. Note that reagent four working solution needs to be prepared immediately, and the prepared working solution is effective on the same day.

[0127] 2. Preparation of control working solution

[0128] Estimate the amount of working solution needed according to the number of samples to be tested, mix reagent one (buffer) and reagent two (activator) according to the volume ratio of 21:0.2, incubate in a 37°C constant temperature incubator for 10 min in the dark, and then store on ice for standby. Mainly prepare immediately, and store the prepared control working solution at 2-8°C in the dark for 2 days.

[0129] 3. Preparation of reagent six (matrix metalloproteinase-3) working solution

[0130] Mix reagent six, one and two according to the ratio of 1:20:0.2, and incubate at 37°C for 10 min. Take out and store on ice for standby. Prepare immediately, and store the prepared reagent six working solution at 2-8°C in the dark for 2 days.

[0131] 4. Determination of MMP-3 activity inhibition rate

[0132] Dilute all samples to be tested to a concentration of 0.1 μg / mL, and label the model group, blank control and sample wells (including Bose factor control and positive control) on the 96-well plate. First, add 10 μL of control working solution to the blank control well, and add 10 μL of reagent six working solution to the remaining wells. Then add 90 μL of reagent one (buffer) and 10 μL of reagent three (stabilizer) to all wells, shake the plate for 3 s and let it stand at room temperature for 3 min. Then add 10 μL of double distilled water to the model group and blank control wells, and add 10 μL of sample to the sample wells. Add 10 μL of GM6001 (Iloperidone) to the positive control well, shake the plate again for 3 s and let it stand at room temperature for 5 min. Finally, add 10 μL of reagent four working solution to all wells, shake the plate for 3 s and incubate in a 37°C constant temperature incubator for 10 min. Put it into the fluorescence plate reader for detection at an excitation wavelength of 325 nm and an emission wavelength of 393 nm. The fluorescence values of each well are obtained. The MMP-3 activity inhibition rate of the sample is calculated.

[0133] The sample inhibition rate was calculated by the following formula:

[0134]

[0135] 5. Assay results

[0136] 5.1 Assay of the inhibitory effect of the components of different molecular weights obtained by ultrafiltration on MMP-3 activity

[0137] Figure 13 is the fluorescence value of the inhibitory effect of each component on MMP-3 activity, Figure 14 is the inhibitory rate of each component on MMP-3 activity. As can be seen from the figure, the inhibitory rate of component F3 on MMP-3 activity is the highest, and when the concentration is 0.1 μg / mL, the inhibitory rate of F3 on MMP-3 activity can reach 30.07%. This can be due to the small volume of the small molecule peptide, which is more easily diffused and entered into the narrow space of the active center, and combined with the metal ions or key amino acid residues required for catalysis.

[0138] 5.2 Assay of the inhibitory effect of each component obtained by Sephadex G-25 gel filtration chromatography separation of component F3 on MMP-3 activity

[0139] Figure 15 is the fluorescence value of the inhibitory effect of each component on MMP-3 activity, Figure 16 is the inhibitory rate of each component on MMP-3 activity. As can be seen from the figure, the inhibitory rate of component F3 on MMP-3 activity is the highest, and when the concentration is 0.1 μg / mL, the inhibitory rate of F3 on MMP-3 activity can reach 30.07%. This can be due to the small volume of the small molecule peptide, which is more easily diffused and entered into the narrow space of the active center, and combined with the metal ions or key amino acid residues required for catalysis. Figure 15 16

[0140] In summary, G3 has the highest antioxidant activity, and G2 has the highest inhibitory rate.

[0141] In the description in the specification, the description of the terms "one embodiment", "an example", "a specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0142] The above description is only an embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principles of the present application shall be included in the protection scope of the present application.​​

Claims

1. A method for preparing peony flower peptide for inhibiting MMP activity, characterized in that, The method comprises the following steps: S1. Dry peony pollen powder is added to deionized water at a ratio of 1:10-1:50 (g / mL), 1%-10% (g / g) protease is added, and the pH is adjusted to 7.5-9.

0. After enzymolysis at 50-70°C for 2-6h, the reaction solution is heated in boiling water to remove volatile oil and fat, and the supernatant is obtained by centrifugation to obtain the preliminary extracted peony flower crude peptide; S2. The peony flower crude peptide obtained in S1 is subjected to ultrafiltration to obtain a component with a molecular weight of <3kDa, i.e. the peony flower peptide for inhibiting MMP activity.

2. The method of claim 1, wherein, The protease in step S1 is selected from one of alkaline protease, neutral protease, papain, trypsin, and pepsin.

3. The method of claim 1, wherein, The operation in step S2 is specifically as follows: a 3kDa ultrafiltration centrifuge tube is used to centrifuge at 4000-6000rpm for 20-30min to obtain a component with a molecular weight of <3kDa.

4. The method of claim 1, wherein The method further comprises step S3: the peony flower peptide obtained in step S2 is subjected to gel filtration chromatography to obtain a peony flower peptide with a molecular weight of 160Da-2100Da.

5. The method of claim 4, wherein, The gel filtration chromatography step is specifically as follows: the peony flower peptide obtained in step S2 is eluted at a rotation speed of 50-60rpm and a flow rate of 2.5-3.5mL / min to obtain a peony flower peptide with a molecular weight of 160Da-2100Da.

6. The peony flower peptide prepared by the method of any one of claims 1-5.

7. The peony flower peptide of claim 6 for use in any one of the following aspects: A1: for use in preparing a free radical scavenging product; A2: for use in preparing a product for inhibiting MMP activity.

8. The peony flower peptide of claim 6 for use in any one of the following aspects: B1: for use in preparing an antioxidant product; B2: for use in preparing a product for inhibiting collagen degradation.

9. The peony flower peptide of claim 6 for use in preparing an anti-aging product.

10. Use according to any one of claims 7 to 9, characterized in that, The product at least comprises a cosmetic product.

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