A Pueraria lobata polypeptide with antioxidant activity and its application

Abstract

The present invention belongs to the field of bioactive peptides, and particularly relates to a Pueraria polypeptide with antioxidant activity, its preparation method and application. The amino acid sequence of the Pueraria polypeptide is YLWVGA, that is, Tyr-Leu-Trp-Val-Gly-Ala. The polypeptide YLWVGA of the present invention has a significant protective effect on ethanol-induced oxidative damage of HepG2 cells, and inhibits ethanol-induced oxidative stress by reducing the release amounts of ALT, AST and LDH and increasing the activities of antioxidant enzymes, thereby exerting the activity of protecting HepG2 cells from oxidative damage. The natural polypeptide YLWVGA provided by the present invention has the advantages of strong antioxidant activity, small molecular weight, easy digestion and absorption, etc., and can be applied to functional foods or health products for relieving oxidative stress damage.

Description

Technical Field:

[0002] The present invention belongs to the field of bioactive peptides, and particularly relates to a Pueraria polypeptide with antioxidant activity, its preparation method and application. Background Art:

[0004] Pueraria, the dried root of Pueraria lobata (Willd.) Ohwi of the Leguminosae family, was first recorded in "Shennong Ben Cao Jing" in the Han Dynasty of China, and has the effects of lowering blood pressure, blood sugar, anti-tumor, relieving alcoholism and protecting the liver. In 2002, Pueraria was officially approved by the Ministry of Health of China as one of the first batch of plants that can be used both as medicine and food. A large number of health foods containing Pueraria have emerged on the market, with high economic value and application value. At present, researchers mainly study the efficacy of components such as flavonoids, triterpenoids, and coumarin compounds in Pueraria, but there are relatively few research reports on the preparation and antioxidant activity of Pueraria protein and Pueraria polypeptide. In particular, there is no report on polypeptides with antioxidant activity.

[0005] Pueraria protein, as a food-derived protein belonging to leguminous plant protein, is rich in essential amino acids that cannot be synthesized by the human body. However, due to the complex structure and large molecular weight of proteins, it is difficult to exert its nutritional value after being ingested by the human body. Therefore, taking Pueraria as the raw material, preparing Pueraria protein into polypeptides with antioxidant activity that are easy to digest and absorb through directional enzymatic hydrolysis and separation and purification is of great significance for in-depth research and product development of Pueraria protein, and at the same time has practical significance for improving the added value of Pueraria protein and expanding the application fields of products. Summary of the Invention:

[0007] The primary object of the present invention is to provide a Pueraria polypeptide with antioxidant activity, the amino acid sequence of the Pueraria polypeptide is YLWVGA, that is, Tyr-Leu-Trp-Val-Gly-Ala, and its molecular weight is 708.37 Da.

[0008] Another object of the present invention is to provide the application of the above YLWVGA polypeptide, especially its application as an antioxidant or in the preparation of antioxidant products, more particularly in the preparation of health products with antioxidant efficacy.

[0009] The Pueraria polypeptide YLWVGA provided by the present invention can be obtained by solid-phase chemical synthesis technology. It can also be obtained by enzymatic hydrolysis and separation and purification using Pueraria as the raw material. Specifically, it includes the following steps:

[0010] (1)Pueraria lobata protein extraction: The enzyme hydrolysis method was used to extract Pueraria lobata protein. The specific process was as follows: The Pueraria lobata powder was sieved through a 60-mesh sieve to remove impurities, deionized water was added according to the mass-volume ratio of 1:20, 2% (w / w) of cellulase based on the mass of Pueraria lobata powder was added, the pH value was adjusted to 5.0, the enzymatic hydrolysis temperature was 50 °C, the enzymatic hydrolysis time was 2 h, and the enzyme was inactivated at 100 °C for 10 min after the enzymatic hydrolysis was completed. Then the pH value was adjusted to 8.0 - 9.0, and the extraction was carried out by shaking in a water bath at 50 °C for 1 - 2 h to obtain the extract. The extract was centrifuged at 4000 r / min for 10 min, the pH value of the obtained supernatant was adjusted to 3.5 - 4.5, the precipitate was taken after standing, and the Pueraria lobata protein was obtained by freeze-drying.

[0011] (2)Pueraria lobata protein enzymatic hydrolysis: The Pueraria lobata protein was dissolved in deionized water, the concentration of Pueraria lobata protein was 2 - 4%, 4 - 6% (w / w) of protease based on the mass of Pueraria lobata protein was added, the enzymatic hydrolysis time was 4 - 6 h, the enzymatic hydrolysis temperature was 55 °C, and the pH value was 8.0; after the enzymatic hydrolysis was completed, the enzyme was inactivated at 100 °C for 10 min, and the supernatant was taken after centrifugation as the Pueraria lobata protein enzymatic hydrolysate.

[0012] (3)Separation and purification of Pueraria lobata polypeptides: The Pueraria lobata protein enzymatic hydrolysate was ultrafiltered and separated, and the fraction with a molecular weight <3 kDa after ultrafiltration was further separated and purified by Sephadex G-15 dextran gel column and DEAE-52 anion exchange chromatography column in sequence. The components of multiple elution peaks were collected and combined, and the fraction with stronger antioxidant activity was selected for reverse high performance liquid chromatography purification. The components of each elution peak were collected and stored at -20 °C after freeze-drying for standby.

[0013] (4)Identification of Pueraria lobata polypeptide sequence: The fraction of the elution peak with the best antioxidant activity separated by reverse high performance liquid chromatography was collected, and the amino acid sequence was determined by liquid chromatography-tandem mass spectrometry to obtain peptide segments with relatively high credibility. The amino acid sequences were YLWVGA, LLVYY, DVLPLA, and VLSALP respectively. Using solid-phase chemical synthesis technology, the peptide segments were synthesized in sequence, and the antioxidant activities of each synthesized peptide segment were compared.

[0014] Preferably, the polypeptide with the amino acid sequence of YLWVGA has the optimal antioxidant activity. Its DPPH free radical scavenging rate at a concentration of 2 mg / mL is 74.57 ± 0.72%, the ABTS free radical scavenging rate is 68.53 ± 0.61%, and the hydroxyl free radical scavenging rate is 75.73 ± 0.79%; and the intervention of YLWVGA has a good protective effect on ethanol-induced oxidative damage of HepG2 cells, especially in terms of improving cell survival rate, reducing the release of ALT, AST, and LDH, and significantly increasing the activities of CAT, GSH-Px, and SOD.

[0015] Further, the protease in step (2) includes one or more of neutral protease, alkaline protease, compound protease, trypsin, and papain. Preferably, the protease is a compound protease composed of alkaline protease and neutral protease in a mass ratio of 3:1.

[0016] Preferably, the enzymatic hydrolysis conditions in step (2) are as follows: the substrate concentration is 3%, the enzyme dosage is 5%, the pH value is 8.0, the enzymatic hydrolysis temperature is 55 °C, and the enzymatic hydrolysis time is 5 h.

[0017] Further, for the separation by Sephadex G-15 dextran gel column in step (3), the eluent is deionized water, the flow rate is 1 mL / min, the detection wavelength is 220 nm, and 5 mL is collected in each tube; the antioxidant activities of each elution fraction are collected and measured.

[0018] Further, for the separation by DEAE-52 anion exchange chromatography column in step (3), gradient elution is carried out using sodium chloride solutions with concentrations of 0 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L, the flow rate is 2 mL / min, the detection wavelength is 220 nm, and 5 mL is collected in each tube; the antioxidant activities of each elution fraction are collected and measured.

[0019] The beneficial effects of the present invention are as follows:

[0020] (1) The present invention uses cellulase to assist in the extraction of puerarin protein, improving the protein extraction rate, and the extraction rate reaches 42.05%.

[0021] (2) The present invention uses ultrafiltration separation, gel column chromatography, and reverse high-performance liquid chromatography techniques in combination to separate and purify puerarin polypeptides, and the obtained polypeptides have higher purity. Moreover, taking the survival rate of HepG2 cells as a discrimination index to screen the components with stronger activity, it can be accurately used for the preparation of puerarin polypeptides with antioxidant activity, and the method is simple and highly practical.

[0022] (3) The polypeptide YLWVGA of the present invention has a significant protective effect on ethanol-induced oxidative damage of HepG2 cells, and inhibits ethanol-induced oxidative stress by reducing the release amounts of ALT, AST, and LDH and increasing the activities of antioxidant enzymes, thereby exerting the activity of protecting HepG2 cells from oxidative damage.

[0023] (4) The natural polypeptide YLWVGA provided by the present invention has the advantages of strong antioxidant activity, small molecular weight, and easy digestion and absorption, and can be applied to health products for relieving oxidative stress damage. Description of the drawings:

[0025] Figure 1 It is a schematic diagram of the hydrolysis degree of puerarin protein by different proteases;

[0026] Figure 2 Schematic diagram of DPPH radical scavenging ability of each ultrafiltration fraction of puerarin protease hydrolysate;

[0027] Figure 3 Elution curve of Sephadex G-15 dextran gel column for <3k ultrafiltration fraction;

[0028] Figure 4 Effect diagram of different fractions of Sephadex G-15 dextran gel column on the survival rate of HepG2 cells;

[0029] Figure 5 Elution curve of DEAE-52 anion exchange chromatography column for G1 fraction;

[0030] Figure 6 Effect diagram of different fractions of DEAE-52 anion exchange chromatography column on the survival rate of HepG2 cells;

[0031] Figure 7 Reverse high performance liquid chromatography diagram of G1D1 fraction;

[0032] Figure 8 Effect diagram of different fractions of reverse high performance liquid chromatography on the survival rate of HepG2 cells;

[0033] Figure 9 Total ion chromatogram of F5 fraction of reverse high performance liquid chromatography;

[0034] Figure 10 Secondary mass spectrum diagram of peptide YLWVGA;

[0035] Figure 11 Effect diagram of peptide YLWVGA on the survival rate of HepG2 cells. Specific implementation method:

[0037] The technical solutions of the present invention will be further explained and illustrated below through specific examples, but the implementation manners of the present invention are not limited thereto.

[0038] Example 1 Extraction of puerarin protein

[0039] The kudzu root powder was passed through a 60-mesh sieve to remove impurities, and deionized water was added at a mass-to-volume ratio of 1:20. Cellulase at 2% (w / w) of the mass of the kudzu root powder was added, the pH value was adjusted to 5.0, the enzymatic hydrolysis temperature was 50 °C, the enzymatic hydrolysis time was 2 h, and after the enzymatic hydrolysis was completed, the enzyme was inactivated at 100 °C for 10 min. Then the pH value was adjusted to 8.0, and the mixture was extracted by shaking in a water bath at 50 °C for 1 h. The extract was centrifuged at 4000 r / min for 10 min to obtain the supernatant. The pH value of the obtained supernatant was adjusted to 3.5, allowed to stand for 30 min, and then centrifuged at 4000 r / min for 15 min. The precipitate was collected and freeze-dried to obtain kudzu root protein. It was determined that the extraction rate of kudzu root protein under this condition was 42.05%, indicating that this method could effectively extract kudzu root protein.

[0040] Example 2: Enzymatic Hydrolysis of Kudzu Root Protein

[0041] The kudzu root protein was dissolved in deionized water at a mass-to-volume ratio of 1:40, and proteases at 4% (w / w) of the mass of the kudzu root protein were added (respectively (1) neutral protease, (2) alkaline protease, (3) a composite protease composed of alkaline protease and neutral protease in a mass ratio of 3:1, (4) trypsin, (5) papain). The enzymatic hydrolysis was carried out at 55 °C and pH 8.0 for 4 h, and the types of proteases were screened with the degree of hydrolysis as the index.

[0042] Compare the effects of different proteases on the degree of hydrolysis of the enzymatic hydrolysis solution of kudzu root protein. The results are as Figure 1 shown. The degrees of hydrolysis of the enzymatic hydrolysis solutions of the 5 proteases were different. Among them, the composite protease composed of alkaline protease and neutral protease in a mass ratio of 3:1 had the best hydrolysis effect on kudzu root protein. Considering industrial production and economic benefits, the composite protease was selected to hydrolyze kudzu root protein.

[0043] The kudzu root protein was dissolved in deionized water, and a protease (a composite protease composed of alkaline protease and neutral protease in a mass ratio of 3:1) was added. The substrate concentration of kudzu root protein was 2 - 4%, the enzyme addition amount was 4 - 6%, and the enzymatic hydrolysis time was 4 - 6 h. The enzymatic hydrolysis temperature was 55 °C and the pH value was 8.0. Response surface experiments were used to analyze the effects of substrate concentration, enzyme addition amount, and enzymatic hydrolysis time on the degree of hydrolysis to obtain the optimal enzymatic hydrolysis parameters. The factor levels of the experimental design are shown in Table 1.

[0044] Table 1: Factor and Level Coding of Response Surface Experiments

[0045]

[0046] The results of the response surface experiments of kudzu root protein enzymatic hydrolysis are shown in Table 2. The experimental results were fitted by multiple regression using DesignExpert.V8.0.6.1 software, and the obtained regression equation was:

[0047] Degree of hydrolysis = 30.71 - 0.34A - 0.025B + 0.23C + 0.76AB - 0.36AC - 0.23BC - 1.37A 2 - 1.55B 2 - 1.5C 2 , the variance analysis of the regression model is shown in Table 3. The R of this model 2 is 0.9796, indicating that the experimental results are in good agreement with the model. From the F value shown in Table 3, the influencing factors of pueraria protease hydrolysis are as follows: substrate concentration > hydrolysis time > enzyme dosage. The optimal technological conditions for pueraria protease hydrolysis are determined as follows: substrate concentration is 3%, enzyme dosage is 5%, pH value is 8.0, hydrolysis temperature is 55 °C, and hydrolysis time is 5 h. The predicted value of the degree of hydrolysis under these optimal conditions is 30.75%. Using the optimized conditions for verification tests, the obtained degree of hydrolysis is 31.14%, which is close to the predicted value, proving that the data of the optimal technological parameters have practical value.

[0048] Table 2 Response surface test design and results

[0049]

[0050] Table 3 Results of variance analysis of the regression model

[0051]

[0052] Example 3 Separation and purification of pueraria polypeptides

[0053] Ultrafiltration separation After the enzymatic hydrolysis under the optimal conditions in Example 1 was completed, the enzyme was inactivated at 100 °C for 10 min, and the supernatant was taken as the pueraria protease hydrolysate after centrifugation. The pueraria protease hydrolysate was successively passed through ultrafiltration membranes with a molecular weight cut-off of 3 and 10 kDa to obtain three ultrafiltration fractions with molecular weights < 3 kDa, 3 - 10 kDa, and > 10 kDa. Each fraction was adjusted to 0.5, 1, 2, 4, 6, 8, 10 mg / mL, and then the antioxidant capacity (DPPH free radical scavenging ability) of each ultrafiltration fraction was measured and compared. The results are as Figure 2 shown. In the concentration range of 0.5 - 10 mg / mL, the < 3 kDa ultrafiltration fraction has the strongest DPPH free radical scavenging ability. Among them, when the concentration is 8.0 mg / mL, the DPPH free radical scavenging rate is as high as 89.19%, showing good antioxidant activity. Therefore, the < 3 kDa ultrafiltration fraction was selected for the next purification step.

[0054] Sephadex G-15 dextran gel column separation After purification by Sephadex G-15 dextran gel column, the eluent was deionized water, the flow rate was 1 mL / min, the detection wavelength was 220 nm, and 5 mL was collected in each tube. The elution curve is as Figure 3As shown, the ultrafiltration fraction <3 kDa was separated into two peak fractions, named G1 and G2, respectively.

[0055] An ethanol-induced oxidative damage model of HepG2 cells was established to detect the protective effects of different concentrations of G1 and G2 fractions on HepG2 cells. The specific experiments were as follows:

[0056] Take 100 μL of the HepG2 cell suspension in the logarithmic growth phase and inoculate it into a 96-well plate at a density of 1×10 4 / well, incubate in a 37 °C incubator for 24 h, and then set up a blank group, a model group, and a sample group, with 3 replicates in each group. Use fresh medium without cells as the blank well. The grouping is as follows:

[0057] Aspirate and discard the original medium. For the blank group (add 100 μL of fresh medium and culture for 30 h), the model group (add 100 μL of medium and culture for 24 h, then aspirate and discard the medium, and add 100 μL of fresh medium with an ethanol final concentration of 500 mM and culture for 6 h), and the sample group (add 100 μL of medium containing different concentrations of polypeptides and intervene for 24 h, then aspirate and discard the medium, and add 100 μL of fresh medium with an ethanol final concentration of 500 mM and culture for 6 h). The CCK8 method was used to measure the cell viability: After the culture was completed, take out the plate, aspirate and discard the medium, add 90 μL of fresh medium and 10 μL of CCK8 reagent to each well, incubate in a 37 °C incubator for 2 - 3 h, and then measure the absorbance at 450 nm with an enzyme-labeled instrument. The absorbance values of the model group and the sample group are A; the absorbance value of the blank well is A0; the absorbance value of the blank group is A1. The cell viability is calculated according to the following formula:

[0058] Cell viability (%) = (A - A0) / (A1 - A0) × 100

[0059] The protective effects of different concentrations of G1 and G2 fractions on HepG2 cells are as Figure 4 shown. The cell viability of the model group was 55.29%, about half of that of the blank group, indicating that the model was successfully established. Compared with the model group, when the concentration was 25 - 100 μg / mL, the cell viability of the G1 experimental group was significantly higher than that of the model group ( P <0.01), showing a dose-dependent manner. When the concentration was 100 μg / mL, the highest cell viability was 82.52%, indicating that the G1 fraction could significantly protect HepG2 cells from ethanol-induced oxidative damage.

[0060] Separation using DEAE-52 anion exchange chromatography column: The G1 fraction was further separated using a DEAE-52 anion exchange chromatography column. Gradient elution was carried out with sodium chloride solutions at 0 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L. The flow rate was 2 mL / min, the detection wavelength was 220 nm, and 5 mL was collected in each tube. The elution curve is as Figure 5 shown. Four distinct elution peaks were detected at 220 nm and were named G1D1, G1D2, G1D3, and G1D4, respectively. The protective effects of different elution peak fractions on the ethanol-induced oxidative damage model of HepG2 cells were determined using the same method as in Step 2 above. The results are shown in Figure 6 It can be seen that with the increase in concentration, the cell survival rates of each fraction increased to varying degrees. At the same concentration, the cell survival rate of the G1D1 fraction was higher than that of the other three fractions and showed a dose-dependence, indicating that the G1D1 fraction had a stronger protective effect on ethanol-induced oxidative damage of HepG2 cells.

[0061] Reverse high performance liquid chromatography separation: The G1D1 fraction was selected and purified using a C-18 chromatographic column. Mobile phase A: 0.1% trifluoroacetic acid - water; Mobile phase B: 0.1% trifluoroacetic acid - acetonitrile. Gradient elution conditions: 0 - 5 min, 95% A; 5 - 15 min, 95% - 80% A; 15 - 20 min, 80% - 95% A. The flow rate was 21 mL / min, the elution time was 24 min, the detection wavelength was 280 nm, and the target components were collected according to the elution time and freeze-dried.

[0062] The obtained reverse high performance liquid chromatography is as Figure 7 shown. Eight main absorption peaks were detected and named F1, F2, F3, F4, F5, F6, F7, and F8, respectively. The HepG2 cells were intervened with the eight fractions using the same method as in Step 2 above, and the protective effects of different separation peak fractions on the ethanol-induced oxidative damage model of HepG2 cells were determined. The results are as Figure 8 shown. When the concentration of each fraction was 100 μg / mL, the cell survival rate of the F5 fraction was higher than that of the other fractions, reaching up to 86.45%. Therefore, the F5 fraction was selected for amino acid sequence identification.

[0063] Example 4 Identification of the polypeptide sequence of Pueraria lobata

[0064] The specific method of liquid chromatography-tandem mass spectrometry is as follows: The liquid phase is an Easy nLC 1200 nanoliter liquid system. After the sample is desalted and retained on the pre-column, it is separated by the analytical column. The specification of the analytical column is a C18 reversed-phase chromatography column. The gradient used in the experiment is that the mobile phase B (80% acetonitrile, 0.1% formic acid) is increased from 5% to 38% within 60 minutes. The mass spectrometry uses a ThermoFisher Q Exactive system combined with a Nano Flex nano-spray ion source. The spray voltage is 1.9 kV, and the heating temperature of the ion transfer tube is 275 °C. The mass spectrometry scanning mode is in the information-dependent acquisition working mode (DDA, Data Dependent Analysis). The resolution of the first-level mass spectrometry scan is 70000, the scanning range is 100 - 1500 m / z, and the maximum injection time is 100 ms. In each DDA cycle, at most 20 secondary spectra with charges from 1+ to 3+ are collected. The maximum injection time of the secondary mass spectrometry ions is 50 ms. The collision cell energy (high-energy collision-induced dissociation, HCD) is set to 28 eV, which is applicable to all precursor ions, and the dynamic exclusion is set to 6 seconds. For the original raw spectrum files collected by the mass spectrometry, data processing and retrieval analysis are carried out using PEAKS Studio 8.5 software. The database is the Pueraria lobata species protein database downloaded from NCBI. The retrieval parameters are set as follows: the mass tolerance of the first-level mass spectrometry is 10 ppm, and that of the secondary mass spectrometry is 0.05 Da.

[0065] Amino acid sequence determination was performed by liquid chromatography-tandem mass spectrometry to obtain peptide segments with relatively high credibility. The amino acid sequences are YLWVGA, LLVYY, DVLPLA, and VLSALP, respectively. Figure 9 It is the total ion chromatogram of the reverse high-performance liquid chromatography F5 fraction.

[0066] Solid-phase chemical synthesis technology was used to synthesize the above 4 polypeptides ( Figure 10 is the secondary mass spectrum of the peptide segment YLWVGA). By measuring the scavenging ability of DPPH radicals, ABTS radicals, and hydroxyl radicals, the antioxidant activities of the above synthetic peptide segments were compared. The specific method is as follows:

[0067] (1) Determination of DPPH radical scavenging ability: Mix 2.0 mL of a 2 mg / mL polypeptide sample with 2.0 mL of a 0.1 mmol / L DPPH radical solution, shake well, incubate in the dark for 30 min, and measure the absorbance value Ai of the mixed solution at 517 nm; for the control group, measure the absorbance value A0 by replacing the polypeptide sample solution with 2.0 mL of distilled water; for the blank group, measure the absorbance value Aj by replacing the DPPH solution with 2.0 mL of absolute ethanol. The DPPH radical scavenging rate is calculated according to the following formula:

[0068] DPPH radical scavenging rate (%) = [1 - (Ai - Aj) / A0] × 100

[0069] (2) Determination of ABTS radical scavenging ability: Prepare ABTS reagent containing 7 mmol / L ABTS and 2.45 mmol / L potassium persulfate, and incubate at room temperature for 12 - 16 hours. Dilute with absolute ethanol to an absorbance value of 0.700 ± 0.005 at 734 nm to obtain the ABTS radical cation working solution. Take 3.9 mL of the ABTS radical cation working solution and mix it with 0.1 mL of the 2 mg / mL polypeptide sample solution, shake well, incubate in the dark for 6 min, measure the absorbance value Ai at 734 nm, and use absolute ethanol instead of the polypeptide sample solution as a blank control to measure the absorbance value A0. The ABTS radical scavenging rate is calculated according to the following formula:

[0070] ABTS radical scavenging rate (%) = (A0 - Ai) / A0 × 100

[0071] (3) Determination of hydroxyl radical scavenging ability: Place 2 mL of the 2 mg / mL polypeptide sample solution in a 10 mL stoppered test tube, then add 2 mL of hydrogen peroxide solution (6 mmol / L) and 2 mL of ferrous sulfate (6 mmol / L) solution, shake well and let stand in the dark for 30 min, then add 2 mL of freshly prepared salicylic acid solution (6 mmol / L) to the tube, shake well and let stand for 10 min, measure the absorbance value of the reaction mixture at 510 nm as Ai, use distilled water instead of the polypeptide sample solution for the blank control to measure the absorbance value A0, and use distilled water instead of the salicylic acid solution to measure the absorbance value as Aj. The hydroxyl radical scavenging rate is calculated according to the following formula:

[0072] Hydroxyl radical scavenging rate (%) = (A0 + Aj - Ai) / A0 × 100

[0073] As shown in Table 4, the antioxidant activity of the peptide segment YLWVGA is higher than that of other amino acid sequences, with its DPPH radical scavenging rate being 74.57 ± 0.72%, ABTS radical scavenging rate being 68.53 ± 0.61%, and hydroxyl radical scavenging rate being 75.73 ± 0.79%.

[0074] Table 4 Comparison of antioxidant activities of synthetic peptide segments

[0075]

[0076] Example 5 Protective effect of YLWVGA on ethanol-induced oxidative damage in HepG2 cells

[0077] Take 1 mL of the HepG2 cell suspension in the logarithmic growth phase and inoculate it into a 12-well plate at a density of 2×105 / wells. After incubation in a 37 °C incubator for 24 h, a blank group, a model group, and a sample group were set up, with 3 replicate wells in each group: Discard the original culture medium. The blank group (add only 1 mL of fresh culture medium and culture for 30 h), the model group (add 1 mL of fresh culture medium and culture for 24 h, then discard the culture medium and add 1 mL of fresh culture medium with an ethanol final concentration of 500 mM and culture for 6 h), and the sample group (add 1 mL of fresh culture medium containing different concentrations of YLWVGA and intervene for 24 h, then discard the culture medium and add 1 mL of fresh culture medium with an ethanol final concentration of 500 mM and culture for 6 h). After the culture is completed, measure the cell survival rate. At the same time, lyse the cells with cell lysate and measure the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) according to the kit instructions; measure the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px).

[0078] As Figure 11 shown, compared with the model group, the cell survival rate was significantly increased after YLWVGA intervention ( P <0.01). Among them, the protective effect was the greatest at a concentration of 100 μg / mL, and the cell survival rate was 88.68%. It shows that within the concentration range of 25 - 100 μg / mL, YLWVGA can protect cells from oxidative damage.

[0079] As can be seen from Table 5, the release amounts of ALT, AST, and LDH in the model group increased, indicating cell damage. After YLWVGA intervention, the release amounts of the three enzymes were significantly decreased ( P <0.05), indicating that YLWVGA can repair the integrity of the cell membrane to inhibit the release of cytoplasmic transaminases and LDH into the extracellular space.

[0080] Table 5 Effects of YLWVGA on the release amounts of ALT, AST, and LDH in ethanol-induced HepG2 cell damage

[0081]

[0082] As can be seen from Table 6, after YLWVGA intervention, compared with the model group, the activities of CAT, GSH-Px, and SOD were significantly increased ( P <0.05), indicating that YLWVGA can inhibit ethanol-induced oxidative stress by increasing the activities of antioxidant enzymes, and thus play a role in protecting the oxidative damage activity of HepG2 cells.

[0083] Table 6 Effects of YLWVGA on oxidative stress-related indexes in ethanol-induced HepG2 cell damage

[0084]

[0085] In summary, the present invention effectively hydrolyzes Pueraria protein with compound protease, separates the Pueraria protease hydrolysate by ultrafiltration, and obtains an ultrafiltration fraction with a molecular weight of <3 kDa that has a strong free radical scavenging ability; further separation is carried out using Sephadex G-15 dextran gel column, DEAE-52 anion exchange chromatography column and reverse high performance liquid chromatography, and the elution peak fractions are collected and combined. After activity screening and sequence identification, 4 peptide segments with strong antioxidant activity (YLWVGA, LLVYY, DVLPLA and VLSALP) are synthesized. Among them, the molecular weight of YLWVGA is 708.37 Da, and the scavenging rate of free radicals is higher than that of the other 3 peptide segments. At the same time, the intervention of YLWVGA has a good protective effect on ethanol-induced oxidative damage of HepG2 cells, especially in terms of increasing cell survival rate, reducing the release of ALT, AST and LDH, and increasing the activities of CAT, GSH-Px and SOD. It shows that the polypeptide YLWVGA provided by the present invention has strong antioxidant activity and can be applied to the preparation of health products with antioxidant efficacy.

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A Pueraria lobata polypeptide with antioxidant activity, Characterized in that, The amino acid sequence of the Pueraria lobata polypeptide is YLWVGA, i.e., Tyr-Leu-Trp-Val-Gly-Ala.

2. Application of the Pueraria lobata polypeptide according to claim 1, Characterized in that, It is the application of the Pueraria lobata polypeptide as an antioxidant or in the preparation of antioxidant products; the antioxidant products are health products with antioxidant effects.

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