Wheat peptide with antioxidant stress activity as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a wheat peptide with antioxidant stress activity and a preparation method and application thereof. The invention provides a wheat peptide with antioxidant stress activity. The amino acid sequence of the wheat peptide is as shown in SEQ ID NO: 1. The invention discloses a wheat peptide FGWPGPK for protecting oxidative stress nerve cells as well as a screening method and application of the wheat peptide FGWPGPK. A target peptide fragment is identified from wheat germs through enzymolysis and multi-dimensional screening strategies, and it is proved that the target peptide fragment can significantly improve the survival rate of PC-12 cells damaged by H2O2 (72.88%) and reduce the ROS level (125.54%). The peptide fragment has strong antioxidant activity and nerve targeting property, and is suitable for preventing and treating neurodegenerative diseases such as Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a wheat peptide with antioxidant stress activity, a preparation method thereof, and an application thereof. Background Art

[0002] Oxidative stress is one of the core pathogenic mechanisms of neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease). Currently, there is a lack of highly effective and low-toxic neuroprotective agents in clinical practice. Natural antioxidant peptides have become a research hotspot due to their high safety and clear activity. In the prior art, the research on the antioxidant activity of wheat peptides mostly focuses on in vitro chemical models, lacking systematic screening and mechanism verification for oxidative stress damage of nerve cells.

[0003] Through an enzymatic hydrolysis process combined with a multi-dimensional screening strategy, the present invention first identifies a specific peptide segment FGWPGPK from wheat germ, and confirms through cell experiments that it significantly reduces H2O2-induced nerve cell damage, providing a new candidate molecule for the development of neuroprotective drugs. Summary of the Invention

[0004] To solve the above problems, the present application provides a wheat peptide with antioxidant stress activity.

[0005] The wheat peptide with antioxidant stress activity has an amino acid sequence as shown in SEQ ID NO:1.

[0006] The sequence is: Phe-Gly-Trp-Pro-Gly-Pro-Lys (FGWPGPK).

[0007] The present application also provides a preparation method of the wheat peptide with antioxidant stress activity.

[0008] The preparation method of the wheat peptide with antioxidant stress activity includes the following steps: (1) Subject the defatted wheat germ powder to combined enzymatic hydrolysis with pepsin and trypsin, and obtain a crude extract of wheat peptide through ultrafiltration and freeze-drying; (2) Screening process: Mass spectrometry identification: Identify the peptide segment sequence based on LC-MS / MS technology; Activity prediction: Combine PeptideRanker, free radical scavenging score, molecular docking, non-toxicity and non-allergenicity to screen candidate peptides; ORAC determination: Finally lock in the target peptide segment FGWPGPK.

[0009] Preferably, the pepsin is used at 2000 U / mL, and the enzymatic hydrolysis conditions are 37 °C, 150 rpm, and 2 h; the trypsin is used at 100 U / mL, and the enzymatic hydrolysis conditions are 37 °C, 150 rpm, and 2 h.

[0010] Step (1) specifically includes the following steps: 1) Take defatted wheat germ powder, add 0.9% NaCl solution at a material-liquid ratio of 1:10, shake at 30 °C for 1.5 h, centrifuge, and freeze-dry the supernatant to obtain wheat germ protein powder; 2) Take the wheat germ protein powder obtained above, incubate at a material-liquid ratio of 1:20 (w / v) at 37 °C for 30 min, adjust the pH to 2.5 with 1 mol·L -1 HCl solution, then add pepsin (2000 U / mL) and shake at 37 °C and 150 rpm for 2 h; then adjust the pH to 7.5 with 1 mol·L -1 NaOH solution, add trypsin (100 U / mL), and enzymatically hydrolyze at 37 °C and 150 rpm for 2 hours; 3) After ultrafiltration (cut-off molecular weight 3 kDa), freeze-dry to obtain a crude extract of wheat peptides; In step (2): Mass spectrometry identification: Based on LC-MS / MS technology (mass spectrometry parameters: scanning range 100 - 1500 m / z, first-order resolution 60,000, second-order resolution 15,000); Activity prediction: Combine PeptideRanker > 0.5, free radical scavenging score > 0.5, binding energy of molecular docking ≥ -9.5 kcal / mol, non-toxic, and non-allergenic to screen candidate peptides; ORAC determination: Select the peptide segment with the highest activity, and finally lock in the target peptide segment FGWPGPK (sequence: Phe-Gly-Trp-Pro-Gly-Pro-Lys).

[0011] In addition, the present application also provides the use of wheat peptides with antioxidant stress activity.

[0012] Use of wheat peptides with antioxidant stress activity in the preparation of neuroprotective drugs or health products, and the amino acid sequence is as shown in SEQ ID NO:1.

[0013] Preferably, the drug is a drug for protecting nerve cells from oxidative stress damage.

[0014] Preferably, the drug is used for the prevention and treatment of neurodegenerative diseases. The neurodegenerative diseases are Alzheimer's disease or Parkinson's disease.

[0015] The effective dose of the drug is 50 - 200 μM, preferably 100 μM or 200 μM.

[0016] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0017] Verification of the neuroprotective effect of wheat peptide FGWPGPK with antioxidant stress activity: Cell model: Oxidative stress injury of rat adrenal pheochromocytoma cells (PC-12) induced by H2O2; Evaluation indicators: Cell survival rate (CKK-8 method): FGWPGPK (200 μM) increased the survival rate from 53.04% in the injury group to 72.88%; ROS level: FGWPGPK (200 μM) decreased the ROS level from 190.8% in the injury group to 125.54%; Observation by fluorescence inverted microscope: Significantly reduced cell oxidative damage.

[0018] The present invention discloses a wheat peptide FGWPGPK for protecting oxidative stress nerve cells, its screening method and application. Through enzymatic hydrolysis and multi-dimensional screening strategies, the target peptide segment was identified from wheat germ, and it was confirmed that it could significantly increase the survival rate of PC-12 cells damaged by H2O2 (72.88%) and reduce the ROS level (125.54%). This peptide segment has both strong antioxidant activity and nerve targeting, and is suitable for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease.

[0019] Innovative points of the present invention: 1. Innovative screening method: For the first time, enzymatic hydrolysis, mass spectrometry identification, bioactivity prediction, molecular docking and ORAC analysis were combined to achieve multi-dimensional targeted screening; 2. Peptide segment characteristics: FGWPGPK has both strong antioxidant activity and nerve cell targeting; 3. Application advantages: The comprehensive protection rate of the medium dose (100 μM) is as high as 70%, which is better than the positive control GSH; the comprehensive protection rate of the high dose (200 μM) is as high as 72.88%, showing no significant difference from the control GSH, and there is no cytotoxicity. Description of the drawings

[0020] Figure 1 : Total ion current chromatogram (TIC) of wheat germ peptides.

[0021] Figure 2 : Information diagram of PeptideRanker, free radical scavenging score, toxicity and allergenicity of wheat germ peptides.

[0022] Figure 3 : ORAC activity diagram of candidate peptides.

[0023] Figure 4 : Secondary mass spectrum diagram of the target peptide segment (FGWPGPK).

[0024] Figure 5: HPLC purity analysis of the target peptide segment (FGWPGPK).

[0025] Figure 6 : Effects of peptide treatment on the activity of H2O2-induced nerve cells.

[0026] Figure 7 : Effects of peptide treatment on the ROS level of H2O2-induced nerve cells.

[0027] Figure 8 : Observation of cell morphology by inverted fluorescence microscope, where the arrows indicate cell oxidative damage. Detailed implementation mode

[0028] Example 1: Preparation and screening of wheat peptides 1. Take defatted wheat germ powder, add 0.9% Nacl solution at a material-liquid ratio of 1:10, shake at 30 °C for 1.5 h, centrifuge, and freeze-dry the supernatant to obtain wheat germ protein powder; 2. Take the wheat germ protein powder obtained above, incubate at a material-liquid ratio of 1:20 (w / v) at 37 °C for 30 min, adjust the pH to 2.5 with 1 mol·L -1 HCl solution, then add pepsin (2000 U / mL), shake at 37 °C and 150 rpm for 2 h; then adjust the pH to 7.5 with 1 mol·L -1 NaOH solution, add trypsin (100 U / mL), and enzymatically hydrolyze at 37 °C for 2 hours; 3. After ultrafiltration (molecular weight cut-off 3 kDa), freeze-dry to obtain a crude extract of wheat peptides; 4. LC-MS / MS analysis: Dissolve the polypeptide sample in water, then perform reduction alkylation and desalting, and then perform mass spectrometry identification; Mass spectrometry identification: Based on LC-MS / MS technology (mass spectrometry parameters: scanning range 100-1500 m / z, first-level resolution 60,000, second-level resolution 15,000), then retrieve the target protein Database database; Figure 1Figure 0 is the total ion chromatogram (TIC) of wheat germ peptides. The total ion chromatogram of enzymatically hydrolyzed wheat germ is obtained by continuously introducing the components eluted from chromatographic separation into the mass spectrometer, and the mass spectrometer continuously scans for data acquisition. Each scan generates a mass spectrum. The ion intensities at all time points in each mass spectrum are added together to obtain a total ion current intensity. Then, a graph with the ion intensity as the vertical axis and time as the horizontal axis is the total ion chromatogram (TIC, Total Ion Chromatogram). The full scan range of the mass spectrometer is 100 - 1500 m / z, the resolution of the first-order mass spectrum is set to 60000, AGC is Custom, MaximumIT: Custom; the resolution of the second-order mass spectrum is set to Resolution: 15000, AGC is Custom, MaximumIT: Custom, Cycle time: 2 s, and the peptide fragmentation collision energy is set to 32 to generate the original mass spectrometry detection data (.raw). The original mass spectrometry file is used to search the target protein database with the following search parameters: 1) Fixed modifications: Carbamidomethyl (C).

[0029] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term).

[0030] 3) Enzyme: Non specific.

[0031] 4) Database: uniprotkb_taxonomy_id_4565.

[0032] 5) Peptide Mass Tolerance of the first-order mass spectrum: 20 ppm 6) Fragment Mass Tolerance of the second-order mass spectrum: 0.02 Da.

[0033] The raw file collected by mass spectrometry is searched against the software database to obtain the identification results. 302 peptide segments are identified in the sample.

[0034] Figure 2 For the PeptideRanker, free radical scavenging score, toxicity, and allergenicity information of wheat germ peptides, wheat germ peptide segments with both PeptideRanker and free radical scavenging scores greater than 0.5 and no toxicity and no allergenicity are selected as candidates.

[0035] Table 1 PeptideRanker, free radical scavenging score, binding energy, toxicity and allergenicity information of candidate wheat germ peptides

[0036] Note: The smaller the binding energy, the higher the possible activity. Peptides with a binding energy less than or equal to -9.5 were selected for activity verification.

[0037] Figure 3 For the ORAC activity verification of candidate peptides, among which the ORAC activity of FGWPGPK was the highest, significantly higher than that of the control group GSH (p < 0.01).

[0038] Figure 4 The secondary mass spectrum of the target peptide FGWPGPK: During the mass spectrometry scan, a primary spectrum (i.e., MS1) is generated in each cycle. In the primary spectrum, the instrument selects the top 20 in terms of abundance / response (i.e., TOP 20 in the mass spectrometry conditions) for fragmentation. The fragments fragmented from each peptide are recorded by the mass spectrometer and a secondary mass spectrum (i.e., MS2 or MS / MS) is generated.

[0039] Figure 5 The HPLC purity evaluation of the target peptide (FGWPGPK): The retention time of the main peak is 9.65 min, and the peak response value is relatively high (the mV value is significant). Other peaks: 10.350 min (possibly minor components or system peaks), 13.668 min (possibly impurities or solvent residues); the proportion of the main peak area > 95%, and the total area of the minor peaks < 5%, then the purity is relatively high.

[0040] Activity prediction: Candidate peptides were screened by combining PeptideRanker > 0.5, free radical scavenging score > 0.5, binding energy of molecular docking ≥ -9.5 kcal / mol, non-toxicity and non-allergenicity. 5. Through ORAC activity verification, FGWPGPK (sequence: Phe - Gly - Trp - Pro - Gly - Pro - Lys) was locked.

[0041] Example 2: Nerve cell protection experiment 1. PC-12 cells were divided into 8 groups: normal control group, H2O2 damage group, low / middle / high dose groups of FGWPGPK (50 / 100 / 200 μM), and GSH positive control group (50 / 100 / 200 μM). 2. The experimental group (FGWPGPK) and the positive control group (GSH) were added to the culture medium containing peptide segments and intervened for 12 hours, and then treated with H2O2 (100 μM) for 6 hours; the H2O2 injury group was added to the culture medium without peptide segments and intervened for 12 hours, and then treated with H2O2 (100 μM) for 6 hours; the normal control group was added to the culture medium without peptide segments and intervened for 12 hours, and then treated with the culture medium without H2O2 (100 μM) for 6 hours; 3. After treatment, CCK-8 solution and DCFH-DA solution were added to the PC-12 cells respectively to detect the cell viability and ROS content, and the cell morphology was observed under an inverted fluorescence microscope; Figure 6 Shows the effect of peptide treatment on the activity of H2O2-induced nerve cells. It includes Control (control group), H2O2 (injury model group), FGWPGPK (intervention group, 50 μM, 100 μM, 200 μM), GSH (positive control group, 50 μM, 100 μM, 200 μM). Through significant analysis of cell activity: 50 μM: There was no significant difference between FGWPGPK (62.72%) and GSH (64.67%) (p > 0.05). 100 μM: FGWPGPK (70.13%) was significantly better than GSH (66.55%) (p < 0.05). 200 μM: There was no significant difference between FGWPGPK (72.88%) and GSH (75.38%) (p > 0.05). Advantage analysis: Concentration dependence: FGWPGPK had a weak effect at low concentration (50 μM), but the effect was significantly improved with the increase of concentration. Especially at the concentration of 100 μM, the effect was higher than that of GSH.

[0042] Potential advantages: FGWPGPK is derived from natural wheat, with lower cost or fewer side effects. Even if the activity is slightly lower, it may still have application value.

[0043] Figure 7 Shows the effect of peptide treatment on the ROS level of H2O2-induced nerve cells. It includes Control (control group), H2O2 (injury model group), FGWPGPK (intervention group, 50 μM, 100 μM, 200 μM), GSH (positive control group, 50 μM, 100 μM, 200 μM). When the concentration of FGWPGPK was 200 μM, the ROS level decreased from 190.80% in the injury group to 125.54%, significantly reducing the oxidative damage of cells.

[0044] Figure 8 Shows the cell morphology observed under an inverted fluorescence microscope (the arrow indicates cell oxidative damage); through the inverted fluorescence microscope images, the protective effect of peptide (FGWPGPK) on oxidative stress injury of nerve cells was observed, and FGWPGPK intervention reduced the cell damage of H2O2-induced nerve cells.

[0045] Optimal dose conclusion: FGWPGPK has the best effect at 200 μM, which is comparable to the GSH positive control group, indicating its potential application value as a substitute for GSH.

[0046] 4. Results: FGWPGPK (100 μM) increased the survival rate from 53.04% in the injury group to 70.13% (significantly better than the same-dose GSH group, p < 0.05), and FGWPGPK (200 μM) increased the survival rate from 53.04% in the injury group to 72.88% (comparable to the same-dose GSH group, p > 0.05); the intervention of FGWPGPK decreased the ROS level from 190.80% in the injury group to 125.54%, significantly reducing the oxidative damage of nerve cells.

Claims

1. A wheat peptide with antioxidant stress activity, characterized in that, The amino acid sequence is shown as SEQ ID NO:

1.

2. The preparation method of the wheat peptide with antioxidant stress activity according to claim 1, comprising the following steps: (1) Subject the defatted wheat germ powder to combined enzymatic hydrolysis with pepsin and trypsin, and obtain the crude extract of wheat peptide by ultrafiltration and freeze-drying; (2) Screening process: Mass spectrometry identification: Identify the peptide sequence based on LC-MS / MS technology; Activity prediction: Combine PeptideRanker, free radical scavenging score, molecular docking, non-toxicity and non-allergy to screen candidate peptides; ORAC verification: Select the peptide segment with the highest ORAC activity, and finally lock the target peptide segment FGWPGPK.

3. The preparation method according to claim 2, characterized in that, Step (1) specifically includes the following steps: 1) Take the defatted wheat germ powder, add 0.9% Nacl solution at a material-liquid ratio of 1:10, shake at 30 °C for 1.5 h, centrifuge, and freeze-dry the supernatant to obtain wheat germ protein powder; 2) Take the obtained wheat germ protein powder above, incubate it at 37 °C for 30 min according to the material-liquid ratio of 1:20 w / v, adjust the pH to 2.5 with 1 mol·L -1 HCl solution, then add pepsin at 2000 U / mL and shake at 37 °C and 150 rpm for 2 h; then use 1 mol·L - 1 NaOH solution to adjust the pH to 7.5, add trypsin at 100 U / mL, and enzymatically hydrolyze at 37 °C and 150 rpm for 2 hours; 3) Ultrafiltration, with a molecular weight cut-off of 3 kDa, and then freeze-dry to obtain the crude extract of wheat peptide.

4. The preparation method according to claim 2, wherein Mass spectrometry parameters in step (2): Scanning range 100 - 1500 m / z, primary resolution 60,000, secondary resolution 15,000; Activity prediction: Combine PeptideRanker > 0.5, free radical scavenging score > 0.5, binding energy of molecular docking ≥ -9.5 kcal / mol, non-toxicity, non-allergy, and the highest ORAC activity to screen candidate peptides.

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