Wheat peptide with antioxidant activity and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a wheat peptide with antioxidant stress activation, a preparation method thereof, and an application thereof. The present application provides a wheat peptide with antioxidant stress activation, the amino acid sequence of which is shown in SEQ ID NO: 1. The present invention discloses a wheat peptide FGWPGPK for protecting oxidative stress neurons, and a screening method and application thereof. Through enzymatic hydrolysis and multidimensional screening strategies, the target peptide segment was identified from wheat germ, and it was confirmed that it can significantly increase the survival rate of PC-12 cells damaged by H2O2 (72.88%) and reduce the ROS level (125.54%). The peptide segment has both strong antioxidant activity and neural targeting, and is suitable for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease.

Description

Technical Field

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

[0002] Oxidative stress is a core pathogenesis of neurodegenerative diseases such as Alzheimer's and Parkinson's. Currently, there is a lack of effective and low-toxic neuroprotectants in clinical practice. Natural antioxidant peptides have become a research hotspot due to their safety and well-established activity. Existing research on the antioxidant activity of wheat peptides has primarily focused on in vitro chemical models, lacking systematic screening and mechanistic validation of oxidative stress damage in neurons.

[0003] This invention uses an enzymatic hydrolysis process combined with a multi-dimensional screening strategy to identify the specific peptide FGWPGPK from wheat germ for the first time, 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] In order to solve the above problems, the present application provides a wheat peptide with antioxidant activity.

[0005] A wheat peptide with antioxidant activity, the amino acid sequence of which is 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 method for preparing wheat peptides with antioxidant activity.

[0008] The preparation method of wheat peptide with antioxidant activity comprises the following steps:

[0009] (1) Defatted wheat germ powder was hydrolyzed with pepsin and trypsin, and crude wheat peptide extract was obtained by ultrafiltration and freeze-drying;

[0010] (2) Screening process:

[0011] Mass spectrometry identification: peptide sequence identification based on LC-MS / MS technology;

[0012] Activity prediction: combining PeptideRanker, free radical scavenging scoring, molecular docking, non-toxicity and non-allergenicity to screen candidate peptides;

[0013] ORAC assay: The target peptide FGWPGPK was finally identified.

[0014] 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.

[0015] Step (1) specifically includes the following steps:

[0016] 1) Defatted wheat germ powder was added with 0.9% NaCl solution at a solid-liquid ratio of 1:10, shaken at 30°C for 1.5 h, centrifuged, and the supernatant was freeze-dried to obtain wheat germ protein powder;

[0017] 2) Take the wheat germ protein powder obtained above and incubate it at 37℃ for 30min at a material-liquid ratio of 1:20 (w / v). -1 The pH of the HCl solution was adjusted to 2.5, and then pepsin (2000 U / mL) was added and shaken at 37 °C and 150 rpm for 2 h; then 1 mol·L -1 The pH of the NaOH solution was adjusted to 7.5, and trypsin (100 U / mL) was added, and enzymatic hydrolysis was carried out at 37°C and 150 rpm for 2 h;

[0018] 3) Ultrafiltration (molecular weight cut-off 3 kDa) followed by freeze-drying to obtain a crude wheat peptide extract;

[0019] In step (2):

[0020] Mass spectrometry identification: based on LC-MS / MS technology (mass spectrometry parameters: scanning range 100-1500 m / z, primary resolution 60,000, secondary resolution 15,000);

[0021] Activity prediction: Candidate peptides were screened based on PeptideRanker > 0.5, free radical scavenging score > 0.5, molecular docking binding energy ≥ -9.5 kcal / mol, and non-toxicity and non-allergenicity;

[0022] ORAC assay: The peptide with the highest activity was selected, and the target peptide FGWPGPK (sequence: Phe-Gly-Trp-Pro-Gly-Pro-Lys) was finally identified.

[0023] In addition, the present application also provides the use of wheat peptides with antioxidant activity.

[0024] The invention relates to the use of a wheat peptide with antioxidant activity in the preparation of neuroprotective drugs or health products, wherein the amino acid sequence is shown in SEQ ID NO: 1.

[0025] Preferably, the drug is a drug that protects nerve cells from oxidative stress damage.

[0026] Preferably, the drug is used for the prevention and treatment of neurodegenerative diseases, wherein the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

[0027] The effective dose of the drug is 50-200 μM, preferably 100 μM or 200 μM.

[0028] Preferably, the medicine further comprises pharmaceutically acceptable excipients.

[0029] Verification of the neuroprotective effect of wheat peptide FGWPGPK with antioxidant activity:

[0030] Cell model: H2O2-induced oxidative stress injury in rat adrenal pheochromocytoma cells (PC-12);

[0031] Evaluation indicators:

[0032] Cell viability (CKK-8 assay): FGWPGPK (200 μM) increased the cell viability from 53.04% in the injury group to 72.88%;

[0033] ROS level: FGWPGPK (200 μM) reduced the ROS level from 190.8% to 125.54% in the injury group;

[0034] Fluorescence inverted microscope observation: significantly reduced cell oxidative damage.

[0035] This invention discloses a wheat peptide, FGWPGPK, that protects neurons from oxidative stress, as well as its screening method and application. Through enzymatic hydrolysis and a multidimensional screening strategy, the target peptide was identified from wheat germ and demonstrated to significantly increase the survival rate of PC-12 cells damaged by H₂O₂ (72.88%) and reduce ROS levels (125.54%). This peptide possesses both strong antioxidant activity and neural targeting, making it suitable for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease.

[0036] Innovations of the present invention:

[0037] 1. Innovation in screening methods: For the first time, enzymatic hydrolysis, mass spectrometry identification, bioactivity prediction, molecular docking, and ORAC analysis are combined to achieve multi-dimensional targeted screening;

[0038] 2. Peptide characteristics: FGWPGPK has both strong antioxidant activity and neuronal cell targeting;

[0039] 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%, which is not significantly different from the control GSH and has no cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 : Total ion current chromatogram (TIC) of wheat germ peptide.

[0041] Figure 2 : Infographic of wheat germ peptide PeptideRanker, free radical scavenging score, toxicity and allergenicity.

[0042] Figure 3 : ORAC activity diagram of candidate peptides.

[0043] Figure 4 : MS / MS spectrum of the target peptide (FGWPGPK).

[0044] Figure 5 : HPLC purity analysis of the target peptide (FGWPGPK).

[0045] Figure 6 : Effects of peptide treatment on H2O2-induced neuronal activity.

[0046] Figure 7 : Effects of peptide treatment on H2O2-induced ROS levels in neural cells.

[0047] Figure 8 : Cell morphology was observed under an inverted fluorescence microscope, where arrows indicate oxidative damage. DETAILED DESCRIPTION

[0048] Example 1: Preparation and screening of wheat peptides

[0049] 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 hours, centrifuge, and freeze-dry the supernatant to obtain wheat germ protein powder;

[0050] 2. Take the wheat germ protein powder obtained above and incubate it at 37℃ for 30min at a material-liquid ratio of 1:20 (w / v). -1 The pH of the HCl solution was adjusted to 2.5, and then pepsin (2000 U / mL) was added and shaken at 37 °C and 150 rpm for 2 h; then 1 mol·L -1 The pH of the NaOH solution was adjusted to 7.5, and trypsin (100 U / mL) was added and enzymatic hydrolysis was performed at 37 °C for 2 h.

[0051] 3. Ultrafiltration (molecular weight cut-off 3 kDa) followed by freeze-drying to obtain crude wheat peptide extract;

[0052] 4. LC-MS / MS analysis: The peptide sample is dissolved in water, followed by reductive alkylation and desalting, and then mass spectrometry identification;

[0053] Mass spectrometry identification: Based on LC-MS / MS technology (mass spectrometry parameters: scan range 100-1500 m / z, primary resolution 60,000, secondary resolution 15,000), followed by searching the target protein database;

[0054] Figure 1 This is the total ion current chromatogram (TIC) of wheat germ peptides. The total ion current chromatogram of enzymatically hydrolyzed wheat germ is a graph of the fractions eluted from the chromatographic separation, which continuously enter the mass spectrometer. The mass spectrometer continuously scans for data acquisition, generating a mass spectrum with each scan. The total ion current intensity is obtained by summing all ion intensities at each time point in each mass spectrum. The total ion current intensity is then plotted with ion intensity as the vertical axis and time as the horizontal axis as the total ion chromatogram (TIC). The full mass spectrometer scan range was 100-1500 m / z, with the primary mass spectrometer resolution set to 60,000, AGC to Custom, MaximumIT: Custom; the secondary mass spectrometer resolution was set to Resolution: 15,000, AGC to Custom, MaximumIT: Custom, Cycle time: 2s, and the peptide fragmentation collision energy set to 32. Raw mass spectrometry data (.raw) were generated. The raw mass spectrometry file was searched against the target protein database using software with the following search parameters:

[0055] 1) Fixed modifications: Carbamidomethyl (C).

[0056] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term).

[0057] 3) Enzyme: Non specific.

[0058] 4) Database: uniprotkb_taxonomy_id_4565.

[0059] 5) Peptide Mass Tolerance: 20 ppm

[0060] 6) Fragment Mass Tolerance: 0.02 Da.

[0061] The raw files collected by mass spectrometry were searched through the software database to obtain the identification results, and 302 peptides were identified in the sample.

[0062] Figure 2 The PeptideRanker, free radical scavenging score, toxicity and allergenicity information of wheat germ peptides were obtained, and wheat germ peptides with PeptideRanker and free radical scavenging scores greater than 0.5 and no toxicity or allergenicity were selected as candidates.

[0063] Table 1 PeptideRanker, free radical scavenging score, binding energy, toxicity and allergenicity information of candidate wheat germ peptides

[0064]

[0065] Note: The smaller the binding energy, the higher the potential activity. Peptides with a binding energy of -9.5 or less were selected for activity verification.

[0066] Figure 3 The ORAC activity of the candidate peptides was verified. Among them, FGWPGPK had the highest ORAC activity, which was significantly higher than that of the control group GSH (p < 0.01).

[0067] Figure 4 This is the secondary mass spectrum of the target peptide FGWPGPK: During the mass spectrometry scanning process, each cycle will generate a primary spectrum (i.e., MS1). In the primary spectrum, the device will select the top 20 in abundance / response (i.e., TOP 20 in the mass spectrometry conditions) for fragmentation. The fragments of each peptide will be recorded by the mass spectrometer and generate a secondary mass spectrum (i.e., MS2 or MS / MS).

[0068] Figure 5 HPLC purity assessment of the target peptide (FGWPGPK): The main peak retention time is 9.65 min, with a high peak response (significant mV value). Other peaks are at 10.350 min (possibly a minor component or systemic peak) and 13.668 min (possibly an impurity or residual solvent). If the main peak area accounts for >95% and the sum of the minor peak areas is <5%, the purity is high.

[0069] Activity prediction: Candidate peptides were screened based on PeptideRanker > 0.5, free radical scavenging score > 0.5, molecular docking binding energy ≥ -9.5 kcal / mol, and non-toxicity and non-allergenicity;

[0070] 5. ORAC activity was verified and FGWPGPK (sequence: Phe-Gly-Trp-Pro-Gly-Pro-Lys) was identified.

[0071] Example 2: Neuronal cell protection experiment

[0072] 1. PC-12 cells were divided into 8 groups: normal control group, H2O2 injury group, FGWPGPK low / medium / high dose groups (50 / 100 / 200 μM), and GSH positive control group (50 / 100 / 200 μM).

[0073] 2. The experimental group (FGWPGPK) and the positive control group (GSH) were treated with culture medium containing the peptide for 12 hours, followed by H2O2 (100 μM) for 6 hours. The H2O2-damaged group was treated with culture medium without the peptide for 12 hours, followed by H2O2 (100 μM) for 6 hours. The normal control group was treated with culture medium without the peptide for 12 hours, followed by culture medium without H2O2 (100 μM) for 6 hours.

[0074] 3. After treatment, PC-12 cells were added with CKK-8 solution and DCFH-DA solution to measure cell viability and ROS content, and cell morphology was observed under an inverted fluorescence microscope.

[0075] Figure 6 The effects of peptide treatment on H2O2-induced neuronal activity were analyzed. These included control, H2O2 (injury model), FGWPGPK (intervention group, 50 μM, 100 μM, 200 μM), and GSH (positive control group, 50 μM, 100 μM, 200 μM). Cell viability significance analysis showed that at 50 μM, FGWPGPK (62.72%) showed no significant difference from GSH (64.67%) (p > 0.05). At 100 μM, FGWPGPK (70.13%) was significantly superior to GSH (66.55%) (p < 0.05). At 200 μM, FGWPGPK (72.88%) showed no significant difference from GSH (75.38%) (p > 0.05). Advantage analysis: Concentration dependence: FGWPGPK has a weak effect at low concentration (50 μM), but the effect increases significantly with increasing concentration, especially at a concentration of 100 μM, where the effect is higher than GSH.

[0076] Potential advantages: FGWPGPK is derived from natural wheat, with lower costs or fewer side effects. Even if its activity is slightly lower, it may still have application value.

[0077] Figure 7 The effects of peptide treatment on H2O2-induced ROS levels in neuronal cells were shown. These included control, H2O2 (injury model), FGWPGPK (intervention group, 50μM, 100μM, 200μM), and GSH (positive control group, 50μM, 100μM, 200μM). At a concentration of 200μM, FGWPGPK reduced ROS levels from 190.80% to 125.54% of the injury group, significantly reducing oxidative damage to the cells.

[0078] Figure 8 The cell morphology was observed under an inverted fluorescence microscope (arrows indicate cell oxidative damage); the protective effect of the peptide (FGWPGPK) on oxidative stress damage of neurons was observed under an inverted fluorescence microscope. FGWPGPK intervention reduced H2O2-induced cell damage of neurons.

[0079] Conclusion of optimal dose: FGWPGPK has the best effect at 200 μM, which is equivalent to the GSH positive control group, suggesting that it has potential application value as a substitute for GSH.

[0080] 4. Results: FGWPGPK (100 μM) increased the survival rate from 53.04% in the injury group to 70.13% (significantly better than the GSH group at the same dose, p < 0.05). FGWPGPK (200 μM) increased the survival rate from 53.04% in the injury group to 72.88% (equivalent to the GSH group at the same dose, p > 0.05). FGWPGPK intervention reduced ROS levels from 190.80% in the injury group to 125.54%, significantly reducing oxidative damage to neurons.

Claims

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

1.

2. The method for preparing the wheat peptide having antioxidant activity according to claim 1, comprising the following steps: (1) Defatted wheat germ powder was hydrolyzed with pepsin and trypsin, and crude wheat peptide extract was obtained by ultrafiltration and freeze-drying; (2) Screening process: Mass spectrometry identification: peptide sequence identification based on LC-MS / MS technology; Activity prediction: combining PeptideRanker, free radical scavenging scoring, molecular docking, non-toxicity and non-allergenicity to screen candidate peptides; ORAC verification: Select the peptide with the highest ORAC activity and finally lock in the target peptide FGWPGPK.

3. The preparation method according to claim 2, characterized in that Step (1) specifically includes the following steps: 1) Defatted wheat germ powder was added with 0.9% NaCl solution at a solid-liquid ratio of 1:10, shaken at 30°C for 1.5 h, centrifuged, and the supernatant was freeze-dried to obtain wheat germ protein powder; 2) Take the wheat germ protein powder obtained in step 1) and incubate it at 37°C for 30 min at a material-liquid ratio of 1:20 w / v. -1 The pH of the HCl solution was adjusted to 2.5, and then 2000 U / mL of pepsin was added and shaken at 37 °C and 150 rpm for 2 h; then 1 mol·L - 1 The pH of the NaOH solution was adjusted to 7.5, and 100 U / mL of trypsin was added. The cells were enzymatically digested at 37°C and 150 rpm for 2 h. 3) Ultrafiltration with a molecular weight cut-off of 3 kDa and freeze-drying to obtain a crude wheat peptide extract.

4. The preparation method according to claim 2, characterized in that Mass spectrometry parameters for the mass spectrometry identification in step (2): scanning range 100-1500 m / z, primary resolution 60,000, secondary resolution 15,000; activity prediction: screening candidate peptides based on PeptideRanker>0.5, free radical scavenging score>0.5, molecular docking binding energy ≥-9.5 kcal / mol, non-toxicity and non-allergic properties; ORAC verification: selecting the peptide with the highest ORAC activity.

Images