Xanthoceras sorbifolia kernel active peptide with antioxidant activity and separation and purification method and application thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to an active peptide from Xanthoceras sorbifolia seed kernel with antioxidant activity, and a separation and purification method and application thereof. The Xanthoceras sorbifolia seed kernel antioxidant peptide provided by the present invention is selected from AKPAFFAPR, PSPSFELPK, TKPFAWARR, VKKPFMPPRE, PRESDNTMLF, KKPSPSFELP, AGIPPLAGFC, GVTASFGMLG, PWVFEPEYPG, LKPPPYQLN, GGLPPLAGFF, EEYGPWVPF, and KKPSPSFELP. The screening method provided by the present invention has high enzymatic hydrolysis efficiency and effectively avoids amino acid deficiency by optimizing process conditions; the active peptide provided by the present invention has good antioxidant effect, can effectively remove free radicals such as DPPH and ABTS, has the characteristics of low price, strong antioxidant activity and high safety, can be used as an excellent substitute for chemically synthesized antioxidants, and provides a theoretical basis and practical reference for the research and development of new natural additives in the pharmaceutical and cosmetic industries.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an active peptide from Xanthoceras sorbifolia seed kernel with antioxidant activity, and a separation and purification method and application thereof. Background Art

[0002] Xanthoceras sorbifolia belongs to the Sapindaceae family and the Xanthoceras genus. It is cultivated in Northeast my country, Northwest China, and the northern part of North China, and has a history of being eaten in Inner Mongolia, Gansu, Shaanxi, Shandong and other regions. Xanthoceras sorbifolia has rich bioactive ingredients. Among the 278 compounds isolated and identified from Xanthoceras sorbifolia, triterpenoids and flavonoids are considered to be the main bioactive substances due to their wide variety, high content, and high pharmacological activity. At present, there is no record of screening and isolating peptides from Xanthoceras sorbifolia kernels.

[0003] There are many methods for the preparation and production of peptides. Generally speaking, they are roughly divided into acid-base hydrolysis, enzyme hydrolysis, directional synthesis and microbial fermentation. Among them, acid-base hydrolysis is the simplest process, but it cannot avoid the problems of amino acid deficiency, hydrolysis degree and amino acid sequence of product peptides, which are difficult to control. It has been gradually abandoned by people. Relatively speaking, the process conditions of enzyme hydrolysis and microbial fermentation are relatively mild, and no harmful chemicals are mixed into the product, so it has been widely used.

[0004] As a biologically active peptide that has been studied in depth, antioxidant peptides have been proven by a large number of studies to have good free radical scavenging ability and the role of maintaining the body's redox balance, and have broad development prospects. However, due to the characteristics of their structural composition, there are many types of peptides, and factors such as the amino acid composition and arrangement order of the peptide sequence can affect the biological activity of the peptide, which brings certain difficulties to the study and analysis of the structure-activity of antioxidant peptides, resulting in the current unclear relationship between the sequence structure of antioxidant peptides and their biological activity. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides an active peptide from Xanthoceras sorbifolia seed kernel with antioxidant activity.

[0006] The present invention further provides a method for separating and screening the Xanthoceras sorbifolia seed kernel active peptides having antioxidant activity.

[0007] Another object of the present invention is to provide the use of Xanthoceras sorbifolia seed kernel active peptides in the preparation of antioxidant drugs.

[0008] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0009] The present invention provides a Xanthoceras sorbifolia seed kernel active peptide having antioxidant activity, wherein the Xanthoceras sorbifolia seed kernel antioxidant peptide is selected from SEQ ID NO.1- SEQ ID NO.13;

[0010] The sequences of the antioxidant peptides are specifically: AKPAFFAPR (SEQ ID NO.1), PSPSFELPK (SEQ ID NO.2), TKPFAWARR (SEQ ID NO.3), VKKPFMPPRE (SEQ ID NO.4), PRESDNTMLF (SEQ ID NO.5), KKPSPSFELP (SEQ ID NO.6), AGIPPLAGFC (SEQ ID NO.7), GVTASFGMLG (SEQ ID NO.8), PWVFEPEYPG (SEQ ID NO.9), LKPPPYQLN (SEQ ID NO.10), GGLPPLAGFF (SEQ ID NO.11), EEYGPWVPF (SEQ ID NO.12), and KKPSPSFELP (SEQ ID NO.13).

[0011] Most preferably, the Xanthoceras sorbifolia seed kernel antioxidant peptides are selected from AKPAFFAPR, TKPFAWARR, PRESDNTMLF, and GVTASFGMLG.

[0012] The present invention also provides a method for separating and screening the active peptides from the kernels of Xanthoceras sorbifolia, comprising the following steps:

[0013] (1) The kernels of Xanthoceras sorbifolia seeds are shelled and crushed, and then defatted to obtain defatted Xanthoceras sorbifolia powder.

[0014] (2) Adding defatted Xanthoceras sorbifolia powder to deionized water, adjusting the pH value, ultrasonically extracting, centrifuging, taking the supernatant, adjusting the pH value of the supernatant, centrifuging to obtain protein precipitate, and washing to obtain protein powder;

[0015] (3) Add distilled water to the protein powder, shake it ultrasonically, cool it to room temperature, adjust the pH, perform enzymatic hydrolysis by constant temperature ultrasonic shaking, inactivate the enzyme, immediately cool it, perform refrigerated centrifugation, take the supernatant, and freeze-dry it to obtain the polypeptide powder;

[0016] (4) The polypeptide powder was separated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa and 10 kDa to obtain an XSP-Ⅰ component <3 kDa; then the screened sequence was combined with Keap1 as a receptor protein, and active peptides with good antioxidant effects were screened based on the "-CDOCKEREnergy" value and the "LibDock" value.

[0017] Preferably, in step (1), the degreasing is carried out using n-hexane.

[0018] Preferably, in step (2), the solid-liquid ratio of the defatted Xanthoceras sorbifolia powder to deionized water is 1:10; after the defatted Xanthoceras sorbifolia powder is added to deionized water, the pH value is adjusted to 11.0 with a 1 mol / L NaOH solution; the extraction is ultrasonic extraction at 40°C for 30 min; and the pH value of the supernatant is adjusted to 4.6 with a 1 mol / L HCl solution.

[0019] Preferably, in step (3), the mass concentration of the protein powder in the aqueous solution is 2-4%; preferably, the mass fraction of the substrate is 3.244%; the ultrasonic oscillation is constant temperature oscillation at 50°C for 10 min; the pH is adjusted to 8.0 using 1 mol / L NaOH solution; the amount of the enzyme added is 6-10% of the mass of the protein powder; and the enzymatic hydrolysis is performed at 50-60°C for 90-150 min.

[0020] Preferably, in step (3), the enzyme is alkaline protease.

[0021] The present invention also provides the use of the active peptide in the preparation of antioxidant drugs.

[0022] The present invention is used to indicate the exact binding site between the active peptide and keap1 through molecular docking. The active peptide provided by the present invention contains more active groups, has higher affinity to the target, and exhibits excellent antioxidant activity.

[0023] The present invention uses Design-Expert 13.0 software to analyze the regression model and obtain that the optimal process parameters for the enzymatic hydrolysis of Xanthoceras sorbifolia antioxidant peptide are: substrate mass fraction 3.244%, enzyme addition amount 8.05%, enzymatic hydrolysis temperature 50.79°C, pH8, and enzymatic hydrolysis time 99.11min. Under these conditions, the scavenging rate of the enzymatic hydrolyzate for DPPH free radicals reaches 71.25%. Taking into account the actual operation situation, the process parameters are adjusted to substrate mass fraction 3%, enzyme addition amount 8%, enzymatic hydrolysis temperature 50°C, pH8, and enzymatic hydrolysis time 100min. In order to verify the feasibility of the experiment, a verification experiment was carried out under the optimal conditions. The result showed that the DPPH free radical scavenging rate was 70.4%, which was only 0.85% different from the predicted value, indicating that the regression equation fits the actual situation well. The constructed model can perform regression analysis and parameter optimization on the preparation process of Xanthoceras sorbifolia antioxidant peptide, indicating the feasibility of the response surface model.

[0024] The beneficial effects of the present invention are:

[0025] (1) The screening method provided by the present invention optimizes the process conditions, has high enzymatic hydrolysis efficiency, and effectively avoids amino acid deficiency;

[0026] (2) The active peptides provided by the present invention have good antioxidant effects and can effectively remove free radicals such as DPPH and ABTS. They are low-priced, have strong antioxidant activity, and are highly safe. They can be used as an excellent substitute for chemically synthesized antioxidants and provide a theoretical basis and practical reference for the research and development of new natural additives in the pharmaceutical and cosmetics industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 bovine serum albumin standard curve;

[0028] Figure 2 Effects of different protease enzymes on the hydrolysis degree of Xanthoceras sorbifolia protein;

[0029] Figure 3 Effects of different protease hydrolysis on DPPH free radical scavenging rate of Xanthoceras sorbifolia protein hydrolysate;

[0030] Figure 4 Effect of pH on the degree of protein hydrolysis and DPPH free radical scavenging ability;

[0031] Figure 5 Effect of enzyme dosage on protein hydrolysis degree and DPPH free radical scavenging ability;

[0032] Figure 6 Effect of enzymatic hydrolysis time on protein hydrolysis degree and DPPH free radical scavenging ability;

[0033] Figure 7 Effects of substrate mass fraction on protein hydrolysis degree and DPPH free radical scavenging ability;

[0034] Figure 8 Effect of enzymatic hydrolysis temperature on protein hydrolysis degree and DPPH free radical scavenging ability;

[0035] Fig. 9 is the interaction diagram of substrate mass fraction and enzyme addition amount;

[0036] Fig.10 Interaction plot of substrate mass fraction and temperature;

[0037] Fig.11 Interaction plot of substrate mass fraction and time;

[0038] Fig.12 Interaction diagram of enzyme addition amount and temperature;

[0039] Fig. 13 is the interaction diagram of enzyme addition amount and time;

[0040] Fig.14 Interaction plot of temperature and time;

[0041] Fig.15DPPH free radical scavenging rate of each component after ultrafiltration;

[0042] Fig.16 ABTS free radical scavenging rate of each component after ultrafiltration;

[0043] Fig.17 The amino acid composition ratio of peptides of each sample, where: Note: the X-axis is the sample name, and the Y-axis is the percentage of amino acid quantity;

[0044] Fig.18 XSP-Ⅰ mass spectrum base peak diagram;

[0045] Fig.19 ABTS free radical scavenging rate of synthetic peptide;

[0046] Fig. 20 DPPH free radical scavenging rate of synthetic peptide;

[0047] Fig.21 3D and 2D docking diagrams of AR-9 and Keap1 active site;

[0048] Fig. 22 3D and 2D docking diagrams of TR-9 and Keap1 active site;

[0049] Fig.23 3D and 2D docking maps of PR-10 and Keap1 active site;

[0050] Fig.24 3D and 2D docking images of GR-10 and Keap1 active site. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0052] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available.

[0053] In some embodiments, the degree of hydrolysis is determined as:

[0054] Referring to the Adler-Nissen method, the pH-stat method was used to calculate the degree of hydrolysis. During the hydrolysis process, 1 mol / L NaOH was added to maintain the pH value stable at 9 and the volume of the added NaOH solution was recorded. The formula for calculating the degree of hydrolysis is as follows:

[0055]

[0056]

[0057]

[0058] DH—degree of protein hydrolysis; C—concentration of NaOH solution (mol / L); V—volume of NaOH solution consumed (mL)

[0059] m—sample protein mass (g); h—millimoles of peptide bonds per gram of almond protein raw material (h=7.84mmol / g); T—test temperature (K); α—average dissociation degree of amino group; pKa—negative logarithm of the dissociation degree of α-amino group.

[0060] In some embodiments, the DPPH free radical scavenging rate is determined by:

[0061] The DPPH free radical scavenging activity was determined according to the method reported by Liu (2011). DPPH was dissolved in 95% ethanol to prepare a 0.1 mmol / L DPPH solution. 100 μL of DPPH solution was placed in a 96-well plate, and then 100 μL of sample solutions of different concentrations were added. A sample color control and a sample control group were set up. The control group used ethanol instead of the sample solution. After oscillation and mixing, the mixture was reacted in a shaker in the dark for 30 minutes. The absorbance value was measured at 517 nm using an enzyme reader. The calculation formula for DPPH free radical scavenging activity is as follows:

[0062]

[0063] Ac—absorbance of the control group; As—absorbance of the experimental group.

[0064] In some embodiments, the protein concentration is determined by:

[0065] According to the Coomassie Brilliant Blue G-250 method, the protein content of the alkali-dissolved protein solution was determined. Using the Bio-Tech BCA protein concentration determination kit, the standard protein was diluted in a gradient manner (0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1ug / ul) according to the instructions, 20μL of protein solution was taken, 200μL of BCA solution was added, and the absorbance value was measured at 565nm using an enzyme reader after reacting in a shaker for 30 minutes, thereby obtaining a standard protein curve, as shown in FIG. Figure 1 As shown, the formula is:

[0066] y= 1.1177x + 0.1603 (R² = 0.999).

[0067] The moisture, ash, carbohydrate, protein and fat contents in the kernel of Xanthoceras sorbifolia used in the present invention are analyzed as shown in Table 1, wherein the fat and protein have the highest mass fractions, which are 58.4% and 28.7% respectively.

[0068] Table 1 Chemical components of Xanthoceras sorbifolia kernels

[0069]

[0070] Example 1 Extraction of Xanthoceras sorbifolia Kernel Protein

[0071] (1) The kernels of Xanthoceras sorbifolia seeds were shelled and crushed, and defatted with n-hexane at a solid-liquid ratio of 1:10, and the process was repeated three times until the n-hexane became clear. After defatting and filtration, the kernels were naturally air-dried to obtain defatted Xanthoceras sorbifolia powder;

[0072] (2) The protein was extracted by alkali dissolution and acid precipitation. First, the defatted powder of Xanthoceras sorbifolia was dissolved in deionized water at a solid-liquid ratio of 1:10, and the pH value was adjusted to 11.0 with 1 mol / L NaOH solution. The mixture was extracted in an ultrasonic pot at 40°C for 30 min, centrifuged (4000 r / min, 15 min), and the supernatant was collected (part of the supernatant was collected for protein concentration determination). The pH value was adjusted to 4.6 with 1 mol / L HCl solution, and centrifuged (4000 r / min, 10 min) to obtain protein precipitate. Finally, deionized water was added to the precipitate for washing three times, and the precipitate was stored at low temperature (-80°C).

[0073] Example 2 Preparation of Xanthoceras sorbifolia polypeptide

[0074] The protein powder was dissolved in a certain amount of distilled water, and then cooled to room temperature after constant temperature ultrasonic oscillation at 50°C for 10 minutes. The pH was adjusted to 9.0 with 1 mol / L NaOH solution, and a certain amount of enzyme was added to constant temperature oscillation for a period of time (pH 9.0 was maintained by dropping 1 mol / L NaOH), and the enzyme was added for enzymatic hydrolysis for a period of time, and then the enzyme was inactivated at 90°C for 10 minutes, and the mixture was immediately cooled, centrifuged at 4000 r / min for 15 minutes, and the supernatant was taken and freeze-dried to obtain the polypeptide powder.

[0075] Example 3 Single Factor Experiment

[0076] During the enzymatic hydrolysis process, the hydrolysis degree of Xanthoceras sorbifolia protein and the DPPH radical scavenging rate were used as evaluation indicators, and the effects of different enzyme types, enzymatic hydrolysis pH, liquid-to-solid ratio, extraction temperature, extraction time, and extraction temperature on the extraction rate of Xanthoceras sorbifolia polypeptides and the free radical scavenging rate were explored.

[0077] Table 2 Factors of single factor experiment

[0078]

[0079] (1) Effect of enzyme type

[0080] In order to screen the protease with high enzymatic hydrolysis efficiency on Xanthoceras sorbifolia seed kernels, neutral protease, alkaline protease, pepsin, composite protease and papain were selected to hydrolyze Xanthoceras sorbifolia protein in this experiment.

[0081] The hydrolysis degree of Xanthoceras sorbifolia proteases and the scavenging rate of DPPH free radicals of the products are shown in Figures 2 and Figure 3 As shown. The antioxidant capacity of the peptides obtained by the five protease hydrolysis products is not the same. Among them, the alkaline protease hydrolysis product has the strongest antioxidant capacity, and the DPPH free radical scavenging rate reaches 84.3%. The relationship between the 5 protease hydrolysis products' DPPH free radical scavenging ability is: alkaline protease>neutral protease>complex protease>pepsin>papain. Therefore, considering the hydrolysis degree and the antioxidant capacity of the hydrolysis products, alkaline protease will be selected in subsequent studies.

[0082] (2) Effect of pH on hydrolysis degree and DPPH free radical scavenging rate

[0083] like Figure 4 As shown in the figure, as the pH value increases, the degree of hydrolysis of the reaction system gradually increases, but the free radical scavenging rate gradually decreases. When the pH value reaches 8, the degree of hydrolysis and the ability of the hydrolyzate to scavenge DPPH free radicals are relatively high. This pH is consistent with the optimal pH of alkaline protease, so the optimal pH selected for the preparation of Xanthoceras sorbifolia antioxidant peptides is 8.0, and pH is not selected as an independent variable factor for response surface experiments.

[0084] (3) Effect of enzyme addition amount

[0085] like Figure 5 As shown in the figure, with the increase of enzyme dosage, the probability of protein-enzyme binding increases, and the degree of hydrolysis of the reaction system gradually increases. With the increase of enzyme dosage, the DPPH free radical scavenging activity of the enzymatic hydrolyzate first shows a rising trend. When the enzyme dosage is 8%, the DPPH free radical scavenging activity reaches the maximum value (65.9.67%). Considering comprehensively, the enzyme dosages of 6%, 8% and 10% were selected for response surface experiments.

[0086] (4) Effect of enzymatic hydrolysis time on hydrolysis degree and DPPH free radical scavenging rate

[0087] Depend on Figure 6 It can be seen that the hydrolysis degree of Xanthoceras sorbifolia protein showed a significant upward trend with the increase of enzymatic hydrolysis time, reaching a peak at 180 min, and then slightly decreased. Alkaline protease mainly affects the peptide bond connected to serine. When the enzymatic hydrolysis reaches a certain degree, the alkaline protease cleavage site reaches saturation, which may be the main reason why the hydrolysis degree of Xanthoceras sorbifolia protein no longer increases with the extension of enzymatic hydrolysis time. The scavenging ability of the enzymatic hydrolyzate for DPPH free radicals showed a trend of first increasing and then decreasing, and the scavenging rate reached a maximum value of 39% at 120 min. Taking comprehensive considerations, the enzymatic hydrolysis time of 90 min, 120 min h and 150 min was selected for response surface experiment.

[0088] (5) Effect of substrate mass fraction on hydrolysis degree and DPPH free radical scavenging rate

[0089] like Figure 7 As shown in the figure, with the increase of substrate mass fraction, the hydrolysis degree showed a trend of first increasing and then decreasing. When the substrate mass fraction was 4%, the hydrolysis degree reached a maximum value of 40.253%. At the same time, when the substrate mass fraction was 3%, the DPPH free radical scavenging ability of the enzymatic hydrolyzate reached the highest value of 60.2%. After comprehensive consideration, substrate mass fractions of 2%, 3% and 4% were selected for response surface experiments.

[0090] (6) Effect of enzymatic hydrolysis temperature on hydrolysis degree and DPPH free radical scavenging rate

[0091] Depend on Figure 8 It can be seen that the hydrolysis degree of Xanthoceras sorbifolia protein shows a trend of first increasing and then decreasing with the increase of enzymatic hydrolysis temperature. At 55 ℃, the hydrolysis degree is the highest, and the hydrolysis degree decreases significantly after 55 ℃. This may be due to the fact that the temperature destroys the internal structure of the enzyme, the enzyme activity decreases, and the enzymatic hydrolysis effect of the enzyme on Xanthoceras sorbifolia protein decreases. When the enzymatic hydrolysis temperature is 50 ℃, the DPPH free radical scavenging rate of the enzymatic hydrolysis product reaches a maximum value of 34.3%. Since the purpose of this study is to study the antioxidant activity of the enzymatic hydrolysis product, the enzymatic hydrolysis temperatures of 50 ℃, 55 ℃, and 60 ℃ were selected for response surface experiments after comprehensive consideration.

[0092] The single factor analysis showed that there was no linear relationship between the hydrolysis degree of Xanthoceras sorbifolia kernel protein and the antioxidant capacity of the enzymatic hydrolysis products, so the DPPH free radical scavenging rate was selected as the response value in the response surface experiment.

[0093] Example 4 Response surface optimization experiment

[0094] Taking the free radical scavenging rate of Xanthoceras sorbifolia antioxidant peptides as an indicator, a four-factor three-level experimental design was conducted on substrate concentration, enzyme addition amount, enzymatic hydrolysis temperature, and enzymatic hydrolysis time to determine the optimal treatment conditions. The factor levels of the response surface experiment are shown in Table 3. Design-Expert 13.0 software was used for analysis to determine the optimal process conditions for enzymatic preparation of antioxidant peptides.

[0095] Table 3 Box-Behnken test factor level table

[0096]

[0097] According to the above experimental design, four factors, namely, substrate mass fraction (A), enzyme addition amount (B), enzymatic hydrolysis temperature (C), and enzymatic hydrolysis time (D), were selected as the evaluation indicators, and the DPPH free radical scavenging rate was used as the response value. The four-factor three-level Box-Behnken experimental design was performed using Design Expert 13.0 software. The experimental scheme and results are shown in the figure below. Taking the DPPH free radical scavenging rate Y as the response value, the data obtained from the experiment were subjected to multivariate regression fitting analysis, and the regression equation of the DPPH free radical scavenging rate of Xanthoceras sorbifolia polypeptide hydrolysate for each variable was obtained as follows: Y(%)=70.53+0.258A+0.549B-1.50C+0.594D-1.83AB-1.9506AC-1.786AD-0.501BC-0.198BD+2.555CD-3.063A²-1.322B²-2.051C²-1.579D²

[0098] Table 4 Response surface methodology experimental design and results

[0099]

[0100] (1) Box-Behnken test analysis of variance

[0101] The regression equation was subjected to a significance test of variance analysis, and the results are shown in the table. Model P = 0.0001 < 0.05, and the R 2 =0.9627, R 2 adj=0.9253, CV%=1.06%, indicating that the results are reliable, indicating that the fitting correlation between the actual test value and the model prediction value is good, indicating that the selected model is significant and can be used to predict the DPPH free radical scavenging ability of Xanthoceras sorbifolia polypeptide hydrolysate, and the experimental design is correct. The F value of the regression model is 25.79, which is significant (P<0.05), indicating that the difference is not significant, indicating that there is no significant influence of other factors in this experiment. The first-order terms B and D are significant, C is extremely significant, and the second-order term A is 2 , B 2 , C 2 , D 2 It can be seen from the F value that among the four factors, in order of their influence on the results, C>B>D>A, that is, temperature>enzyme addition>time>substrate mass fraction.

[0102] Table 5 ANOVA results of quadratic regression model

[0103]

[0104] Note: ** indicates extremely significant difference (P<0.01), * indicates significant difference (P<0.05)

[0105] (2) Response surface analysis of the interaction of various factors

[0106] According to the obtained regression equation, response surface graphs and contour plots were made. Two of the four factors, substrate mass fraction (A), enzyme addition amount (B), enzymatic hydrolysis temperature (C), and enzymatic hydrolysis time (D), were fixed at the origin, and the response surface graphs and contour plots of the other two factors were obtained in turn. The three-dimensional response surface graph and contour plot of the interaction of each factor on DPPH clearance rate are shown in Figure 2. Figure 9-14 As shown, it can be seen that there is a certain interaction between the factors.

[0107] The interactions between substrate mass fraction and enzymatic hydrolysis time, and between enzymatic hydrolysis temperature and enzyme dosage were quite significant, and the contour plots showed ellipses, which were consistent with the results of variance analysis.

[0108] Example 5

[0109] 1. Ultrafiltration separation of active peptides

[0110] The Xanthoceras sorbifolia polypeptides were separated by tangential cross-flow membranes with molecular weight cutoffs of 3 kDa and 10 kDa, and MW < 3 kDa and 3 kDa were obtained. <MW<10 kDa , MW> The three components were collected and freeze-dried, and recorded as XSP-Ⅰ, XSP-Ⅱ, XSP-Ⅲ, and the unfiltered component XSP-0 were all stored at -20℃ for future use. The in vitro antioxidant capacity of each component was compared.

[0111] 2. Determination of antioxidant capacity of active peptides

[0112] 1. DPPH free radical scavenging ability

[0113] The DPPH free radical scavenging activity was determined according to the method reported by Liu (2011). DPPH was dissolved in 95% ethanol to prepare a 0.1 mmol / L DPPH solution. 100 μL of DPPH solution was placed in a 96-well plate, and then 100 μL of sample solutions of different concentrations were added. A sample color control and a sample control group were set up. The control group used ethanol instead of the sample solution. After oscillation and mixing, the mixture was reacted in a shaker in the dark for 30 minutes. The absorbance value was measured at 517 nm using an enzyme reader. The calculation formula for DPPH free radical scavenging activity is as follows:

[0114]

[0115] Ac—absorbance of the control group; As—absorbance of the experimental group.

[0116] 2. Determination of ABTS free radical scavenging ability

[0117] ABTS free radical scavenging activity assay. The antioxidant solution and ABTS solution were mixed in equal volumes and reacted at room temperature for 16 h to prepare the ABTS working solution. The ABTS working solution was diluted with PBS buffer before use so that its absorbance at 734 nm was 0.70±0.02. 180 μL of the diluted ABTS working solution was placed in a 96-well plate, and then 20 μL of sample solutions of different concentrations were added. After oscillation and mixing, the mixture was reacted at room temperature in the dark for 10 min. The absorbance of the reaction solution at 734 nm was measured. The control group used PBS buffer instead of the sample solution. The calculation formula for ABTS free radical scavenging activity is as follows:

[0118]

[0119] Ac—absorbance of the control group; As—absorbance of the experimental group.

[0120] 3. Determination of amino acid sequence of active peptides

[0121] The LC-MS / MS method was used for determination. The specific method is as follows:

[0122] 1. Pretreatment method

[0123] 1) Sample preparation: Use an analytical balance to accurately measure 1 mg of sample, use a pipette to accurately transfer 100 µL of 50 mM NH4HCO3 into the sample and shake to re-dissolve.

[0124] 2) Reductive alkylation: Take 50 μg according to the peptide concentration and add 50mM NH4HCO3 to 100μL. Use a pipette to accurately transfer 1 μL of 1M DTT solution to the sample, so that the final concentration of DTT is 10 mmol / L, and reduce it in a 56℃ water bath for 1 h; use a pipette to accurately transfer 10 μL of 0.5M IAM solution to the sample, so that the final concentration of IAM is 50 mmol / L, and react at room temperature in the dark for 40 min; use a pipette to accurately transfer 2 μL of 1M DTT solution to the sample, so that the final concentration of DTT is 20 mmol / L, to neutralize the unreacted IAM.

[0125] 3) C18 desalting (Stage-Tip): Activation: Activate the desalting column twice with 300 μL 100% ACN, Equilibrate the desalting column twice with 200 μL 0.1% TFA, Loading: Load twice, Desalting: Desalt twice with 200 μL 0.1% TFA, Elution: Elute once with 300 μL 80% ACN-20% 0.1% TFA, and dry in vacuo at 45°C.

[0126] 4) Peptide quantification: Use a pipette to accurately transfer 25 µL of sample into a new 1.5 mL EP tube, use a pipette to accurately transfer 20 µL of standard and sample into a microplate, and add 180 µL of working reagent to each well. Mix on a microplate shaker for 30 s, and incubate at 37°C for 15 min. After cooling to room temperature, place the 96-well plate into a full-wavelength microplate reader and measure the absorbance at 480 nm. Prepare a standard curve and calculate the sample concentration.

[0127] 2. Machine conditions

[0128] (1) Liquid chromatography conditions

[0129] 1) Analytical column: 75μm id×250 mm, packing: Reprosil-Pur 120 C18-AQ 1.9μm

[0130] 2) Mobile phase A: 0.1% FA;

[0131] 3) Mobile phase B: 0.1% FA, 80% ACN;

[0132] 4) Flow rate: 600 nL / min;

[0133] 5) Analysis time for each component: 66 min;

[0134] 6) Specific chromatographic conditions are shown in Table 6;

[0135] Table 6

[0136]

[0137] (2) Mass spectrometry conditions

[0138] The full mass spectrometry scan range was 300-1800 m / z, the primary mass spectrometry resolution was set to 120000, AGC was Standard, MaximumIT: 20 ms; the secondary mass spectrometry resolution was set to 15000, AGC was Standard, MaximumIT: 22 ms, and the peptide fragmentation collision energy was set to 30 to generate mass spectrometry detection raw data (.raw).

[0139] (3) Database search conditions

[0140] The mass spectrometry raw file uses software to search the target protein database, and the search parameters are as follows:

[0141] 1) Fixed modifications: Carbamidomethyl (C).

[0142] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term).

[0143] 3) Enzyme: Non specific.

[0144] 4) Database: Xanthoceras sorbifolium.fasta.

[0145] 5) Peptide Mass Tolerance: 20 ppm

[0146] 6) Fragment Mass Tolerance: 0.02 Da

[0147] 4. Molecular docking simulation

[0148] The binding ability of Xanthoceras sorbifolia peptides to Keap1 was predicted by molecular docking. First, the X-ray crystal structure of Keap1 (kelch-like ECH-associated protein 1) was obtained from the RCSB protein database and defined as a receptor. The active peptide was defined as a ligand, and the energy of the small molecule ligand was optimized by Discovery Studio.2019 software, and the structural formula and 3D structure of the active peptide were drawn. After removing water molecules and adding hydrogen atoms to the receptor target, its active center was defined in the "Receptor-Ligand Interactions" module (coordinates: x=5, y=9, z=1; radius: 15 Å). The structure of the selected Xanthoceras sorbifolia peptide was drawn by ChemDraw 20.0 client software, and its energy was minimized by the CHARMm force field. The docking was performed using the "-CDOCKER" and "LibDock" protocols. The Keap1-peptide complex with a high "-CDOCKER" energy value was considered to be the most stable conformation, and the higher the "LibdockScore", the higher the activity of the small molecule binding to the receptor. In addition, the interaction mechanism between Xanthoceras sorbifolia peptide and Keap1 was obtained through the receptor-ligand interaction map.

[0149] 5. Chemical synthesis of active peptides

[0150] The active peptide was synthesized by Shanghai Jier Chemical using the Fmoc solid phase synthesis method. The synthesis method is briefly described as follows:

[0151] (1) Resin swelling

[0152] Select the starting resin and add it to a reaction column with a volume of 500 mL. Add DCM to soak it and drain it to complete the swelling of the resin.

[0153] (2) Deprotection

[0154] Add deprotection reagent, stir under N2 for 30 min, filter out the solvent; then wash the resin with DMF 6 times, drain and complete the deprotection of the resin.

[0155] (3) Amino acid coupling reaction

[0156] The reaction process was monitored by the ninhydrin method. The specific operation process is as follows:

[0157] Weigh the corresponding amount of TBTU and protected amino acid in a beaker, add DMF to dissolve; then add the reaction solution to the resin, add DIEA, pass N2 for 90 min, and detect by ninhydrin reaction. After the reaction is completed, remove the solvent and wash the resin with DMF 3 times. Add 2 deprotection reagents to the resin, pass N2 for 30 minutes, then remove the solvent and wash the resin with DMF 6 times to complete the coupling of this amino acid. Repeat the above reaction procedure until the condensation reaction of all protected amino acids is completed.

[0158] (4) Cleavage and separation of synthetic peptides

[0159] The synthetic peptide was cleaved with 95% TFA solution, the solution was concentrated in vacuo, washed with diethyl ether, and lyophilized to obtain the product.

[0160] 6. Results

[0161] In this experiment, ultrafiltration membranes with a molecular weight cutoff of 3 kDa and 10 kDa were used to separate the Xanthoceras sorbifolia kernel polypeptides, and three components, XSP-Ⅰ (<3 kDa), XSP-Ⅱ (3 kDa~10 kDa), and XSP-Ⅲ (>10 kDa) were obtained. In order to screen polypeptide components with strong antioxidant capacity, the antioxidant properties of XSP-Ⅰ, XSP-Ⅱ, XSP-Ⅲ, and XSP-0 were determined by DPPH free radical scavenging rate and ABTS free radical scavenging rate.

[0162] 1. DPPH free radical scavenging rate of each component after ultrafiltration

[0163] As shown in Figure 15, within the concentration range of 0.1~1 mg / mL, each group showed DPPH free radical scavenging ability in a dose-dependent manner. Among them, XSP-Ⅰ with the smallest molecular weight showed the strongest DPPH free radical scavenging ability.

[0164] 2. ABTS free radical scavenging rate of each component after ultrafiltration

[0165] As shown in Figure 16, within the concentration range of 0.1-1 mg / mL, each group showed ABTS free radical scavenging ability in a dose-dependent manner. Among them, XSP-Ⅰ showed the strongest ABTS free radical scavenging activity at all concentrations.

[0166] By comparing the DPPH free radical scavenging rate and ABTS free radical scavenging rate of each component after ultrafiltration, it can be found that XSP-Ⅰ has the strongest antioxidant capacity. Therefore, XSP-Ⅰ was selected for subsequent amino acid sequence sequencing and computer simulation screening.

[0167] 3. Analysis of amino acid composition of each component after ultrafiltration

[0168] The amino acid compositions of XSP-Ⅰ and its different molecular weight components were determined, such as Fig.17 As shown in Table 7, in XSP-Ⅰ, the level of Leu was the highest, and peptides containing aromatic or hydrophobic amino acids (i.e., Ile, Ala, Val, Phe, Asp, Tyr, Pro, Trp, Gly, and Leu) promoted free radical scavenging activity by enhancing their interaction with lipids or acting as proton or hydrogen donors. In addition, peptides with Arg and Ser residues also contributed to the antioxidant activity.

[0169] Table 7 Amino acid composition analysis

[0170]

[0171] Note: The X-axis is the sample name, and the Y-axis is the percentage of amino acid quantity.

[0172] 4. Structural identification of XSP-Ⅰ and screening of active peptides based on molecular docking assay

[0173] (1) Structural identification and virtual screening of XSP-Ⅰ

[0174] In order to further study the structure-activity relationship between the structure and antioxidant activity of XSP-Ⅰ, the structure of XSP-Ⅰ was first identified. The mass spectrometry Basepeak results of XSP-Ⅰ are shown in Fig.18 .

[0175] The results of qualitative analysis of the database search are shown in Table 8. A total of 980 Xanthoceras sorbifolia kernel-derived peptides were identified. The number of amino acids in the detected peptides ranged from 7 to 32, and the molecular weight distribution ranged from 625.31 to 3356.69 Da.

[0176] The sequences after simulation screening were used with Keap1 as receptor protein, and active peptides with good potential antioxidant effects were screened according to the "-CDOCKER Energy" value and "LibDock" value. The results are shown in Table 18. Among the 17 Xanthoceras sorbifolia peptides, 13 active peptides can bind to the target protein keap1 to varying degrees. The four peptides with the highest "-CDOCKER Energy" value and "LibDock" value are AKPAFFAPR, TKPFAWARR, PRESDNTMLF, and GVTASFGMLG, which are recorded as AR-9, TR-9, PR-10, and GR-10, respectively.

[0177] Table 8 XSP-Ⅰ amino acid sequence identification results

[0178]

[0179] Effect Example Antioxidant Activity Verification of Synthetic Peptides

[0180] (1) To verify the antioxidant activity of the four peptides AKPAFFAPR, TKPFAWARR, PRESDNTMLF, and GVTASFGMLG, the DPPH free radical scavenging ability and ABTS activity of the chemically synthesized peptides were measured. The results are shown in Figures 19-20. The antioxidant activity of PR-10 was higher than that of the other three peptides. Its DPPH free radical scavenging ability reached 54.249%, and its ABTS free radical scavenging rate reached 89.041% (close to the activity of GSH).

[0181] (2) Molecular docking experiment

[0182] Molecular docking was used to reveal the exact binding sites of the active peptides and keap1. The docking results of the four Xanthoceras sorbifolia peptides binding to keap1 are shown in Figure 2. Figure 21-24 As shown. TR-9 and PR-10 contain more active groups compared to the other two peptides, among which PR-10 has the highest "-CDOCKER energy" value indicating that the peptide may have the highest affinity for the target. TR-9 forms van der Waals interactions with some residues of Keap1, including Asn414, Tyr334, and Ser602, which are important residues in the Keap1-Nrf2 interaction. The contact of TR-9 with the above residues indicates that TR-9 has the potential to competitively bind to Keap1 and release Nrf2, and exhibits antioxidant activity in vivo by activating the Keap1-Nrf2 pathway.

Claims

1. An active peptide from Xanthoceras sorbifolia kernels with antioxidant activity, characterized in that: The Xanthoceras sorbifolia seed kernel antioxidant peptide is PRESDNTMLF.

2. A method for separating and screening active peptides from Xanthoceras sorbifolia kernels as claimed in claim 1, characterized in that: The following steps are involved: (1) Shelling and crushing the kernels of Xanthoceras sorbifolia and defatting them to obtain defatted Xanthoceras sorbifolia powder; (2) Adding defatted Xanthoceras sorbifolia powder to deionized water, adjusting the pH value, ultrasonically extracting, centrifuging, taking the supernatant, adjusting the pH value of the supernatant, centrifuging to obtain protein precipitate, and washing to obtain protein powder; (3) Add distilled water to the protein powder, shake it ultrasonically, cool it to room temperature, adjust the pH, perform enzymatic hydrolysis by constant temperature ultrasonic shaking, inactivate the enzyme, immediately cool it, perform refrigerated centrifugation, take the supernatant, and freeze-dry it to obtain the polypeptide powder; The amount of the enzyme added is 6-10% of the mass of the protein powder; the enzymolysis is performed at 50-60° C. for 90-150 minutes; The enzyme is alkaline protease; (4) The polypeptide powder was separated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa and 10 kDa to obtain an XSP-Ⅰ component <3 kDa; then the screened sequence was combined with Keap1 as a receptor protein, and active peptides with good antioxidant effects were screened based on the "-CDOCKEREnergy" value and the "LibDock" value.

3. The separation and screening method according to claim 2, characterized in that In step (1), the degreasing is carried out using n-hexane.

4. The separation and screening method according to claim 2 or 3, characterized in that: In step (2), the solid-liquid ratio of the defatted Xanthoceras sorbifolia powder to deionized water is 1:10; after the defatted Xanthoceras sorbifolia powder is added to deionized water, the pH value is adjusted to 11.0 with a 1 mol / L NaOH solution; the extraction is ultrasonic extraction at 40° C. for 30 min; and the pH value of the supernatant is adjusted to 4.6 with a 1 mol / L HCl solution.

5. The separation and screening method according to claim 2, characterized in that: In step (3), the mass concentration of the protein powder in the aqueous solution is 2-4%; the ultrasonic oscillation is performed at a constant temperature of 50° C. for 10 min; and the pH is adjusted to 8.0 using a 1 mol / L NaOH solution.

6. Use of the active peptide from Xanthoceras sorbifolia seed kernel as claimed in claim 1 in the preparation of antioxidant drugs.

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