Cordyceps sinensis polypeptide and application thereof

Abstract

The invention relates to the field of bioactive peptides, and discloses a cordyceps sinensis polypeptide. The cordyceps sinensis polypeptide is selected from at least one of a peptide with an amino acid sequence as shown in SEQ ID NO: 1, a peptide with an amino acid sequence as shown in SEQ ID NO: 2 and a peptide with an amino acid sequence as shown in SEQ ID NO: 3. Besides, the invention also provides application of the cordyceps sinensis polypeptide as an antioxidant in preparation of a product for improving cell viability, a product for improving the survival rate of cells damaged by oxidation, a product for reducing ROS (reactive oxygen species) in cells, a product for reducing malonaldehyde in cells and a product for improving the activity of antioxidant enzyme in cells. The invention also relates to the application of the Keap1-Nrf2-ARE in preparation of a product for improving the activity of antioxidant enzyme in cells and in preparation of a product for activating a Keap1-Nrf2-ARE signal channel. The cordyceps sinensis polypeptide has relatively high antioxidant activity, and has good effects on resisting oxidation and delaying senescence.

Description

Technical Field

[0001] The present invention relates to the field of bioactive peptides, and in particular to a Cordyceps peptide. In addition, the present invention also relates to the application of the Cordyceps peptide. Background Art

[0002] Aging is a common problem faced by all animals, including humans. As living standards continue to improve, people, beyond material needs, are increasingly pursuing spiritual fulfillment. Slowing down aging has garnered widespread attention, and antioxidants are a useful approach.

[0003] Bioactive peptides are short polymers composed of 2-20 amino acids derived from natural sources. These peptides possess potential biological activity and can provide a variety of health benefits without side effects, such as antimicrobial, anti-inflammatory, antioxidant, and antihypertensive peptides. These physicochemical effects of bioactive peptides stem from their unique amino acid composition, sequence, and molecular structure. Because most bodily functions result from the interactions of amino acids (either as small peptide fragments or small protein chains), bioactive peptides have broad potential for application in nutritional supplements, functional foods, and dietary supplements.

[0004] However, the antioxidant properties of some current antioxidant peptides need to be further improved. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem that the antioxidant activity of some antioxidant peptides in the prior art needs to be further improved, and to provide a Cordyceps peptide and its application. The Cordyceps peptide has high antioxidant activity and has a good effect on anti-oxidation and anti-aging.

[0006] To achieve the above object, the present invention provides a Cordyceps polypeptide, which is selected from at least one of a peptide with an amino acid sequence such as SEQ ID NO: 1, a peptide with an amino acid sequence such as SEQ ID NO: 2, and a peptide with an amino acid sequence such as SEQ ID NO: 3.

[0007] A second aspect of the present invention provides a use of a Cordyceps polypeptide as an antioxidant, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0008] A third aspect of the present invention provides a use of a Cordyceps polypeptide in the preparation of a product for improving cell viability, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0009] A fourth aspect of the present invention provides a use of a Cordyceps polypeptide in the preparation of a product for improving the survival rate of oxidatively damaged cells, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0010] A fifth aspect of the present invention provides a use of a Cordyceps polypeptide in the preparation of a product for reducing intracellular reactive oxygen species (ROS), wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0011] In a sixth aspect, the present invention provides a use of a Cordyceps polypeptide in preparing a product for reducing intracellular malondialdehyde (MDA), wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0012] In a seventh aspect, the present invention provides a use of a Cordyceps polypeptide in preparing a product for improving the activity of intracellular antioxidant enzymes, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0013] Preferably, the antioxidant enzyme is at least one selected from catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX).

[0014] In an eighth aspect, the present invention provides a use of a Cordyceps polypeptide in preparing a product for reducing intracellular β-galactosidase, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0015] In a ninth aspect, the present invention provides a use of a Cordyceps polypeptide in preparing a product that activates the Keap1-Nrf2-ARE signaling pathway, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0016] Preferably, the product is a medicine and / or a functional food.

[0017] Through the above technical solution, the amino acid sequence of the Cordyceps peptide is shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and has high antioxidant activity. The Cordyceps peptide can significantly increase cell viability, improve the survival rate of cells damaged by oxidative damage, enhance the activity of intracellular antioxidant enzymes, activate the Keap1-Nrf2-ARE signaling pathway, reduce intracellular ROS content, reduce intracellular malondialdehyde content, and reduce intracellular β-galactosidase content. Therefore, it has high application in pharmaceuticals and functional foods with the above-mentioned functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The primary mass spectrum (A) and secondary mass spectrum (B) of the peptide having the sequence of SEQ ID NO: 1; Figure 2 The primary mass spectrum (A) and secondary mass spectrum (B) of the peptide having the sequence of SEQ ID NO: 2; Figure 3 The primary mass spectrum (A) and secondary mass spectrum (B) of the peptide having the sequence of SEQ ID NO: 3; Figure 4 The vertical axis represents the cell survival rate, and the vertical axis represents the cell survival rate. Different letters indicate significant differences (p < 0.05). Figure 5 The figure shows the protective effects of three peptides at different concentrations on HepG2 cells under H2O2-induced oxidative stress (+ represents addition, - represents no addition). The vertical axis represents the cell survival rate. Different letters indicate significant differences (p<0.05). Figure 6 Effects of three peptides and boletus polypeptide (BBP) on ROS and MDA in HepG2 cells induced by H2O2 oxidative damage, fluorescence intensity (A) and MDA content (B), different letters indicate significant differences (p < 0.05); Figure 7The effects of the three peptides on the antioxidant enzymes of HepG2 cells induced by H2O2, including SOD activity (A), CAT activity (B) and GPX activity (C). Different letters indicate significant differences (p < 0.05). Figure 8 This is an optical microscopic image of cellular senescence-associated β-galactosidase staining (×20). DETAILED DESCRIPTION

[0019] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0020] As mentioned above, the first aspect of the present invention provides a Cordyceps polypeptide, which is selected from at least one of a peptide with an amino acid sequence such as SEQ ID NO: 1, a peptide with an amino acid sequence such as SEQ ID NO: 2, and a peptide with an amino acid sequence such as SEQ ID NO: 3.

[0021] Among them, SEQ ID NO: 1: LFNHF.

[0022] SEQ ID NO: 2: PLDRHPF.

[0023] SEQ ID NO: 3: YPFLRPL.

[0024] During their research, the inventors discovered that the amino acid sequences of Cordyceps peptides, as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, exhibit high antioxidant activity. These peptides can significantly enhance cell viability, increase the survival rate of oxidatively damaged cells, increase the activity of intracellular antioxidant enzymes, activate the Keap1-Nrf2-ARE signaling pathway, and reduce intracellular ROS, malondialdehyde, and β-galactosidase levels. These peptides are therefore promising for use as pharmaceuticals and functional foods with these properties.

[0025] The peptides with amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 can be obtained by enzymatic hydrolysis of the fermentation product of Xinjiang Cordyceps.

[0026] Preferably, the preparation method comprises the following steps: (1) Drying, dissolving, and separating the Xinjiang Cordyceps mycelium obtained by liquid fermentation to obtain a crude Xinjiang Cordyceps protein extract; (2) Mixing the crude extract of Xinjiang Cordyceps protein with water, performing enzymatic hydrolysis, and separating to obtain Xinjiang Cordyceps peptide; (3) Xinjiang Cordyceps peptide was mixed with water and then subjected to membrane separation to collect four fractions: OGP-1 (>10 kDa), OGP-2 (5-10 kDa), OGP-3 (3-5 kDa), and OGP-4 (<3 kDa); (4) Purification of the OGP-4 fraction using a Q Sepharose High Performance anion exchange chromatography column to obtain three main fractions (OGP-4-1, OGP-4-2, and OGP-4-3); (5) Sephadex G-25 column was used to further separate and purify the OGP-4-3 fraction with the strongest free radical scavenging activity, and two main peaks (OGP-4-3-1 and OGP-4-3-2) were eluted. (6) De novo LC-MS / MS analysis was performed to isolate peptides with amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0027] Preferably, in step (1), the drying is freeze-drying. The freeze-drying conditions can be determined by the experimenter according to actual conditions.

[0028] Preferably, step (1) further comprises grinding the dried Xinjiang Cordyceps mycelium into powder before dissolving. Further preferably, the Xinjiang Cordyceps mycelium powder obtained by grinding has a particle size greater than 120 mesh. The powder can be obtained by sifting through a 120 mesh sieve.

[0029] Preferably, in step (1), the dissolution can be performed using a buffer solution. Further preferably, the pH of the buffer solution can be 7.2-7.8, more preferably 7.4. More preferably, the buffer solution is a phosphate buffer solution. The amount of the buffer solution used is 15-25 mL relative to 1 g of Xinjiang Cordyceps mycelium powder.

[0030] Preferably, in step (1), the separation process includes: homogenizing the solution obtained after dissolution and then separating the solid and liquid, then using ammonium sulfate solution for graded precipitation and then using ammonium sulfate solution for gradient dissolution, and finally dialyzing and drying.

[0031] Preferably, in step (2), the amount of water used is 8-12 mL relative to 1 g of Xinjiang Cordyceps protein crude extraction.

[0032] Preferably, the enzyme used for the enzymatic hydrolysis is selected from at least one of papain, pepsin, alkaline protease, and trypsin, more preferably papain and / or pepsin. More preferably, papain and pepsin are used. Further preferably, the amount of papain used is 1500-2500 mg, and the amount of pepsin used is 1500-2500 mg, per 1 g of the crude extract of Xinjiang Cordyceps protein.

[0033] Preferably, the enzymatic hydrolysis step comprises first hydrolyzing the product with papain and then hydrolyzing the product with pepsin. Papain and pepsin are both hydrolyzed under suitable conditions, including papain (pH 6.5, 60°C), pepsin (pH 2, 37°C), alkaline protease (pH 10, 50°C), and trypsin (pH 8, 37°C).

[0034] Preferably, in step (3), the conditions for membrane separation include at least: 5500-6500 g, 12-18 min, 2-6°C.

[0035] As a specific embodiment of the present invention, the preparation method of Cordyceps polypeptide includes: (1) The Xinjiang Cordyceps mycelium obtained by liquid fermentation was freeze-dried, ground into powder, sieved, and mixed with phosphate buffered saline (PBS, pH 7.4) at a ratio of 1:20 (w / v), homogenized on ice, centrifuged (6000g, 15min, 4℃), and the supernatant was collected. The supernatant was filtered through a 0.45μm filter membrane and precipitated with (NH4)2SO4 at 30%, 70%, and 100% saturation at 4℃ for 1 day. The precipitate was gradiently dissolved with (NH4)2SO4 at 80%, 40%, 20%, 10%, and 5% saturation, and dialyzed in ultrapure water at 4℃ for 2 days using a 400Da dialysis membrane. The water was changed 12 times and the crude Xinjiang Cordyceps protein extract was obtained by freeze-drying.

[0036] (2) Xinjiang Cordyceps protein was dissolved in ultrapure water at a material-water ratio of 1:10. The pH and temperature were adjusted to the optimal conditions for each enzyme and hydrolyzed for 4 hours. The reaction was terminated by heating at 95°C for 10 minutes. After cooling to room temperature, the hydrolyzate was centrifuged (6000g, 15min, 4°C) and the supernatant was collected, filtered through a 0.22μm filter, freeze-dried, and stored at -20°C until subsequent analysis. The optimal conditions for each enzyme were: papain (pH 6.5, 60°C) and pepsin (pH 2, 37°C) were purchased from Solebao Biotechnology Co., Ltd., with an enzyme: substrate ratio (E:S) of 2000U / g.

[0037] (3) Xinjiang Cordyceps peptide was reconstituted with ultrapure water and ultrafiltration was performed using 3 kDa, 5 kDa and 10 kDa molecular weight cutoff (MWCO) membranes (6000 g, 15 min, 4 °C) to collect four fractions: OGP-1 (>10 kDa), OGP-2 (5-10 kDa), OGP-3 (3-5 kDa) and OGP-4 (<3 kDa).

[0038] (4) OGP-4 was reconstituted with ultrapure water and initially purified using a Q Sepharose High Performance anion exchange chromatography column. The sample was dissolved in 20 mM Tris-HCl buffer containing 1 M NaCl (pH 7.5) and placed on a HiTrap Q Sepharose High Performance chromatography column (5 mL, Cytiva) equilibrated with the same buffer. The column was then eluted using a linear gradient of NaCl (0-1 M) at a flow rate of 1 mL / min and monitored at 280 nm to obtain three main fractions (OGP-4-1, OGP-4-2, and OGP-4-3).

[0039] (5) Weigh an appropriate amount of Sephadex G-25 dry powder, add excess ultrapure water and fully swell at room temperature for 24 hours, remove the floating particles on the upper layer, change the water, and degas. Use a glass rod to drain the treated filler and slowly pour it into the column at once (no stratification and no bubbles), allowing the gel to settle naturally in the column under gravity. After the gel is deposited for about 3 cm, open the water outlet at the bottom of the column to let the water flow out. When 5 cm of water column remains in the upper layer of the column, close the water outlet. Equilibrate the gel chromatography column with 3 times the column volume of ultrapure water. Dissolve the sample in ultrapure water at a concentration of 10 mg, load 2 mL, elute at a flow rate of 1 mL / min, and monitor at 280 nm to obtain fractions OGP-4-3-1 and OGP-4-3-2.

[0040] (6) An Easy-nLC 1200 system was connected to a Q Exactive™ hybrid quadrupole-orbitrap™ mass spectrometer (Thermo Fisher Scientific, USA) with an ESI nanospray source. The OGP-4-3-1 fraction was reduced with 10 mM dithiothreitol (DTT) at 56°C for 1 hour, then alkylated with 50 mM iodoacetamide (IAA) at room temperature for 40 minutes in the dark, desalted, and the solvent evaporated in a vacuum centrifuge concentrator at 45 =°C. Prior to LC-MS / MS analysis, the peptide was resuspended in 2-20 μL of 0.1% formic acid. The reversed-phase column was an Acclaim PepMap RPLC C18 nano column (150 μm × 15 cm) (1.9 μm, 100 Å, Dr. Maisch GmbH, Germany), and the sample load was 5 μL. The mobile phase consisted of 0.1% formic acid in water (A) and 20% 0.1% formic acid in water-80% acetonitrile (B), with a flow rate of 600 nL / min. The analysis time for each component was 66 minutes. The gradient elution was from 4% to 8% B in 2 minutes; from 8% to 28% B in 43 minutes; from 28% to 40% B in 10 minutes; from 40% to 95% B in 1 minute; and from 95% to 95% B in 10 minutes. The capillary temperature was set at 270°C, and the spray voltage was set at 2.2 kV.

[0041] Primary mass spectrometry parameters: resolution 70,000, AGC target 3e6, maximum dwell time (IT) 100 ms, scan range 300–1800 m / z; secondary mass spectrometry parameters: resolution 17,500, AGC target 1e5, maximum IT 50 ms, TopN 20, and NCE / steppedNCE 28. De novo sequencing analysis of raw MS files was performed using PEAKS Studio 8.5. The peptide mass tolerance (PMT) was 20 ppm, and the fragment mass tolerance (FMT) was 0.02 Da.

[0042] The fermentation strain of Cordyceps sinensis (P. dubia) HL-119 (CGMCC No. 20731) was obtained from the China Center for Microbiological Culture Collection (CGMCC) and stored at -80°C in 20% (v / v) glycerol. Cordyceps sinensis mycelium was obtained from liquid fermentation in the laboratory of Academician Huang He, School of Food and Pharmaceutical Engineering, Nanjing Normal University. After freeze-drying, the powder was ground through a 120-mesh sieve and stored at 4°C until use. Papain and pepsin used in the enzymatic hydrolysis were purchased from Solebao Biotechnology Co., Ltd.

[0043] In the product, the ALC value of the amino acid sequence of SEQ ID NO: 1 is 98%, the ALC value of the amino acid sequence of SEQ ID NO: 2 is 99%, and the ALC value of the amino acid sequence of SEQ ID NO: 1 is 97%. The molecular weights of the peptides of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are 676.33, 880.46, and 904.52 Da, respectively. For the gas chromatography-mass spectrometry, see Figure 1-Figure 3 .

[0044] In a second aspect, the present invention also provides a use of a Cordyceps polypeptide as an antioxidant, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0045] During the research process, the inventors found that the Cordyceps peptide provided above has higher antioxidant activity and has better application as a Cordyceps peptide.

[0046] In a third aspect, the present invention provides a use of a Cordyceps polypeptide in preparing a product for improving cell viability, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0047] During the research process, the inventors found that the Cordyceps peptide provided above can significantly improve cell viability and has better application in the preparation of products that improve cell viability.

[0048] In a fourth aspect, the present invention provides a use of a Cordyceps polypeptide in preparing a product for improving the survival rate of oxidatively damaged cells, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0049] During the research process, the inventors found that the Cordyceps peptide provided above can improve the survival rate of cells damaged by oxidation, and has better application in the preparation of products that improve the survival rate of cells damaged by oxidation, and can reduce aging caused by oxidation.

[0050] In a fifth aspect, the present invention provides a use of a Cordyceps polypeptide in preparing a product for reducing intracellular ROS, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0051] ROS produced during cellular metabolism can bind to polyunsaturated fatty acid-associated phospholipids, enzymes, and membrane receptors, triggering lipid peroxidation to form malondialdehyde (MDA). Excessive MDA ultimately disrupts cellular metabolism. Therefore, increased MDA levels are a hallmark of aging, indirectly reflecting the extent of ROS-mediated cellular oxidative damage.

[0052] During the research process, the inventors found that the Cordyceps peptide provided above can reduce the content of ROS in cells, thereby reducing oxidative damage and slowing down aging.

[0053] In a sixth aspect, the present invention provides a use of a Cordyceps polypeptide in preparing a product for reducing intracellular malondialdehyde, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0054] During the research process, the inventors found that the Cordyceps peptide provided above can effectively reduce the content of malondialdehyde in cells, thereby reducing oxidative damage and slowing down aging.

[0055] In a seventh aspect, the present invention provides a use of a Cordyceps polypeptide in the preparation of a product for improving the activity of intracellular antioxidant enzymes, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0056] During the research process, the inventors found that the Cordyceps peptide provided above can effectively increase the activity of antioxidant enzymes in cells, thereby delaying aging.

[0057] Preferably, the antioxidant enzyme is selected from at least one of CAT, SOD and GPX. Cordyceps peptides can significantly improve the activity of the above antioxidant enzymes.

[0058] In an eighth aspect, the present invention provides a use of a Cordyceps polypeptide in preparing a product for reducing intracellular β-galactosidase, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0059] During the research process, the inventors found that the Cordyceps peptide provided above can effectively reduce the content of β-galactosidase in cells, protect cells from oxidative damage, and effectively inhibit premature cell aging.

[0060] In a ninth aspect, the present invention provides a use of a Cordyceps polypeptide in the preparation of a product for activating the Keap1-Nrf2-ARE signaling pathway, wherein the Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO: 3.

[0061] Studies have found that the above-mentioned Cordyceps peptides can inhibit the binding between Nrf2 and Keap1, thereby activating the Keap1-Nrf2-ARE signaling pathway, increasing the activity of CAT, SOD and GPX, and thereby enhancing the body's ability to scavenge free radicals and having a better antioxidant effect.

[0062] According to the present invention, in the above application, the product is a medicine and / or a functional food.

[0063] The present invention will be described in detail below through examples.

[0064] Human hepatocellular carcinoma cells (HepG2) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Penicillin-streptomycin solution, fetal bovine serum (FBS), and modified Dulbecco's Modified Eagle Medium (DMEM) were purchased from Jiangsu KeyGen Biotech Co., Ltd. (Nanjing, China). CAT, GPX, SOD, MDA, ROS, and BCA protein concentration assay kits were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Boletus peptides were purchased commercially with an active ingredient content of 90%.

[0065] Cell culture: Remove the cryovial containing HepG2 cells from liquid nitrogen and quickly place the cells in a 37°C water bath. Shake by hand to thaw as quickly as possible, disinfect with 75% alcohol, and transfer to a clean bench. Gently pipette to mix the cells. HepG2 cells were cultured in DMEM high-glucose medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin solution in a 37°C, 5% CO2 incubator. The cell culture medium was changed every other day. When the cells reached 90% confluence, the medium was removed, and the cells were gently washed with PBS. The cells were then digested and passaged using 0.05% trypsin-EDTA solution for further processing. All HepG2 cells used had been passaged fewer than 10 times.

[0066] Example 1 HepG2 cell viability was determined using the MTT assay with minor modifications. HepG2 cells were seeded at a density of 1×10⁴ cells / well in 96-well plates and cultured for 24 hours to determine the cytotoxic effects of the peptides and establish a cell model of oxidative stress-induced senescence. The cells were then divided into treatment and control groups. The culture medium was removed. 100 μL of culture medium was added to each well of the control group, while 100 μL of peptides (dissolved in DMEM) at different concentrations (100, 200, 300, 400, 600, 800, 1000, 2000, and 3000 μg / mL) and 100 μL of H₂O₂ (diluted in DMEM) at different concentrations (0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, and 2 mM) were added to each well of the treatment group. The blank group was treated with culture medium without HepG2 cells. The H₂O₂ group was cultured for 4 hours, while the other groups were cultured for 24 hours. 10 μL of MTT (5 mg / mL) was added to each well for 4 hours. After removing the MTT reagent, add 150 μL of dimethyl sulfoxide (DMSO) and mix. Vortex at low speed for 10 minutes. Measure the absorbance at 490 nm. Calculate cell viability using the following formula: HepG2 cell survival rate (%) = (A1- A0) / ( A2- A0) × 100; Where: A0 is the absorbance of blank; A1 is the absorbance of treatment group; A2 is the absorbance of control group.

[0067] HepG2 cells were treated with the three peptides at different concentrations for 24 h and then treated with H2O2 for 4 h ( Figure 5Compared with the blank control group, the cell survival rate in the H2O2 injury model group was 53.13%, demonstrating a significant damage effect (p<0.05). Compared with the H2O2 model group, when treated with the three peptides at different concentrations, HepG2 cell survival initially increased with increasing peptide concentration, but then decreased with increasing peptide dosage. This indicates that the protective effect against oxidative damage increases in a dose-dependent manner within a certain concentration range, and all peptide concentrations can reverse H2O2-induced cell damage. LFNHF (SEQ ID NO: 1) treatment exhibited significant protective effects against oxidative damage at low, medium, and high concentrations (p<0.05). HepG2 cells were virtually unaffected by the damage, and at the medium concentration (600 μg / mL), cell proliferation was even enhanced after recovery to the same level as the control group. The PLDRHPF (SEQ ID NO: 2) treatment group also demonstrated substantial protection against oxidative damage at low, medium, and high concentrations (p < 0.05). However, the high concentration (1000 μg / mL) did not restore the effect to the control group. The low and medium concentration groups, especially the medium concentration group, promoted cell proliferation. YPFLRPL (SEQ ID NO: 3) treatment group demonstrated significant protection against oxidative damage at low and medium concentrations (p < 0.05), but showed no protective effect against HepG2 cell damage at the high concentration (55.14%). Overall, the three peptides demonstrated a strong protective effect against H2O2-induced oxidative damage in HepG2 cells.

[0068] Example 2 HepG2 cells were collected at 1×10 4 Cells were seeded at a density of 100 cells / well in a 96-well plate and cultured for 2 hours. The cells were divided into control, protection, and injury groups. The culture medium was removed, and 200 μL of culture medium was added to each well of the control group and cultured for 28 hours. For the protection group, 100 μL of 200, 600, and 1000 μg / mL peptides (dissolved in DMEM) were added to each well. After 24 hours of culture, 100 μL of 0.6 mM H₂O₂ (diluted in DMEM) was added to each well and cultured for 4 hours. For the injury group, 100 μL of culture medium was added to each well for 24 hours, followed by 100 μL of 0.6 mM H₂O₂ (diluted in DMEM) and cultured for 4 hours. For the blank group, culture medium without HepG2 cells was added for 28 hours. HepG2 cell viability was determined using the MTT assay.

[0069] Human hepatocellular carcinoma cells (HepG2) are found in the liver, which plays an important role in the body's antioxidant processes. Because HepG2 cells are highly representative and easy to culture, they have been widely used to evaluate the antioxidant defense system [. Figure 4As shown in Figures 4A, 4B, and 4C, the peptide of SEQ ID NO: 1 significantly increased the survival rate of HepG2 cells within the concentration range of 0-1000 μg / mL (p<0.05), while the cell survival rate remained almost unchanged within the concentration range of 1000-3000 μg / mL ( Figure 4 A). The survival rate of HepG2 cells was greater than 90% when the peptide sequence of SEQ ID NO: 2 was in the concentration range of 0-1000 μg / mL. When the concentration exceeded 1000 μg / mL, the cell survival rate showed a downward trend with increasing concentration ( Figure 4 B). Within the concentration range of 0-1000 μg / mL, the peptide with the sequence of SEQ ID NO: 3 significantly improved the survival rate of HepG2 cells (p<0.05). Within the concentration range of 1000-3000 μg / mL, the cell survival rate was greater than 90% ( Figure 4 C). The results showed that the peptides with sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 had no toxic side effects on HepG2 cells and could promote the proliferation of HepG2 cells.

[0070] Example 3 HepG2 cells were collected at 1×10 4 Cells were seeded at a density of 100 cells / well in a 96-well plate and cultured for 2 hours. The cells were divided into a control group, a protection group, a damaged group, and a porcini peptide group. The culture medium was removed, and 200 μL of culture medium was added to each well of the control group and cultured for 28 hours. In the protection group, 100 μL of 600 μg / mL peptide (dissolved in DMEM) was added to each well. After 24 hours of culture, 100 μL of 0.6 mM H₂O₂ (diluted in DMEM) was added to each well and cultured for 4 hours. In the damaged group, 100 μL of culture medium was added to each well for 24 hours, followed by 100 μL of 0.6 mM H₂O₂ (diluted in DMEM) and cultured for 4 hours. In the porcini peptide group, 100 μL of 600 μg / mL peptide was added to each well. The blank group was cultured with culture medium without HepG2 cells for 28 hours.

[0071] HepG2 cells were collected from the control, protection, injury, and boletus peptide groups. The cells were centrifuged at 1000 g for 10 minutes, and the cell pellet was collected. ROS levels in HepG2 cells were measured according to the instructions of the ROS detection kit, using the fluorescence value of the control group as a reference. Fluorescence images were observed using an inverted fluorescence microscope. Malondialdehyde (MDA) content in HepG2 cells was measured according to the instructions of the MDA detection kit.

[0072] The "free radical theory of aging" points out that intracellular ROS plays a vital role in the aging process. When there are too many ROS in the cells, the intracellular antioxidant system cannot remove the excessive ROS, which leads to oxidative damage to the cells and premature aging. The production of ROS is the most direct indicator for evaluating antioxidant capacity. The present invention uses DCFH-DA fluorescent dye to measure the ROS levels in different treatment groups. Figure 6 As shown in A, the fluorescence signal of the H2O2 model group was the brightest, indicating that H2O2 induced oxidative stress in HepG2 cells by accumulating ROS. Compared with the H2O2 model group, the fluorescence level of HepG2 cells decreased after treatment with the three peptides. The fluorescence intensity of all groups was also quantified ( Figure 6 B) Compared with the control group, ROS levels in the model group increased significantly to 161.13% (p < 0.05), indicating enhanced cellular oxidative stress after H2O2 treatment. The ROS fluorescence intensities in the three peptide-treated groups were significantly lower than those in the H2O2 model group and the boletus peptide group. Therefore, the cytoprotective effects of the three peptides may be related to their ability to scaveng intracellular ROS production, thereby reducing oxidative damage. Furthermore, the fluorescence intensities of the PLDRHPF (SEQ ID NO: 2) and YPFLRPL (SEQ ID NO: 3)-treated groups were lower than those in the boletus peptide group, indicating that their antioxidant properties are superior to those of the boletus peptide.

[0073] ROS produced by cells during metabolism can bind to phospholipids, enzymes, and membrane receptors related to polyunsaturated fatty acids, triggering lipid peroxidation to form malondialdehyde (MDA). Excessive MDA will eventually lead to the destruction of cell metabolism. Therefore, an increase in MDA content is a sign of aging, indirectly reflecting the degree of ROS-mediated cellular oxidative damage. Figure 6 As shown in Figure B, the MDA content in the model group was significantly increased compared to the control group (p < 0.05), reaching 7.25 nmol / mg pro. Compared to the model group, the MDA content in the groups treated with the three peptides LFNHF (SEQ ID NO: 1), PLDRHPF (SEQ ID NO: 2), and YPFLRPL (SEQ ID NO: 3) was significantly decreased (p < 0.05), reaching 4.66, 1.93, and 3.55 nmol / mg pro, respectively. This indicates that all three peptides can effectively inhibit lipid peroxidation and reduce MDA production. The MDA content of the boletus peptide was 5.48 nmol / mg pro, indicating that these three peptides have superior antioxidant properties to the boletus peptide.

[0074] Example 4 HepG2 cells from the control, protection, and injury groups described in Example 2 were collected. The cells were centrifuged at 1000 g for 10 minutes, and the cell pellet was collected. CAT, SOD, and GPX activities of the HepG2 cells were measured according to the instructions of the assay kit. Protein concentration was determined using a BCA assay kit.

[0075] Endogenous antioxidants are products of human metabolism and play a crucial role as the first line of defense against ROS. SOD converts superoxide anions into H2O2 and O2. CAT binds to mitochondria and peroxidases within cells to rapidly remove the toxic H2O2 produced by cellular metabolism, breaking it down into water and oxygen. GPX catalyzes the conversion of GSH to GSSG, reducing toxic peroxides to non-toxic hydroxyl compounds, protecting the integrity of cell membrane structure and function.

[0076] Compared with the control group, when treated with 600 μg / mL H2O2 for 4 h, SOD ( Figure 7 A), CAT ( Figure 7 B) and GPX ( Figure 7 C) activity was significantly decreased (p<0.05), reaching 66.68U / mg pro, 2.41U / mg pro and 7.79mU / mg pro, respectively. Compared with the model group, the SOD activity was significantly increased in the LFNHF (SEQ ID NO: 1) and PLDRHPF (SEQ ID NO: 2) peptide treatment groups (p<0.05), reaching 93.72U / mg pro and 98.97U / mg pro, respectively, while the SOD activity was not significantly increased in the YPFLRPL (SEQ ID NO: 3) peptide treatment group (71.39U / mg pro). Figure 7 As shown in Figure B, the CAT activity in the group treated with the peptide PLDRHPF (SEQ ID NO: 2) was high (6.15 U / mg pro), while the peptides LFNHF (SEQ ID NO: 1) and YPFLRPL (SEQ ID NO: 3) had minimal effects on CAT activity, reaching 2.91 U / mg pro and 3.31 U / mg pro, respectively. Regarding GPX activity, all three peptides significantly enhanced GPX activity in HepG2 cells. These results indicate that all three peptides positively impact CAT, SOD, and GPX activity in HepG2 cells, thereby alleviating H₂O₂-induced cellular oxidative damage and exhibiting cytoprotective effects, particularly the peptide PLDRHPF (SEQ ID NO: 2).

[0077] Example 5 HepG2 cells were collected from the control, protection, and injury groups described in Example 2. The cells were centrifuged at 1000 g for 10 minutes to collect the cell pellet. The HepG2 cells were stained according to the kit instructions, observed under an optical microscope, and photographed.

[0078] The cell senescence-related β-galactosidase staining kit uses X-Gal as a substrate to produce a dark blue substance. The stained senescent cells or tissues can be observed under an ordinary optical microscope. The test results of senescence-related β-galactosidase are as follows: Figure 8 As shown, dark blue cells are senescent cells expressing SA-β-gal. The figure demonstrates that, compared to the control group, after H2O2-induced oxidative damage, the number of senescent cells increased significantly, their volume dramatically increased, their morphology became irregular, and their surface showed bulges or collapses. This indicates that the addition of 600 μg / mL H2O2 successfully induced premature cellular senescence. In the three peptide-treated groups, although some damaged cells with increased volume and collapsed surfaces remained, the number of SA-β-gal-positive cells decreased significantly, indicating a reduction in the number of senescent cells. Therefore, these images provide visual experimental evidence that the three peptides protect HepG2 cells from H2O2-induced oxidative damage and can effectively inhibit premature cellular senescence.

[0079] Example 6 Molecular docking was used to estimate the interactions between peptides and target proteins. The X-ray crystal structure of the receptor Keap1 protein (PDB ID: 4IQK) was obtained from the Protein Data Bank (RCSB PDB: Homepage). Two-dimensional structures of the three novel peptides were drawn using MarvinSketch software (Chemaxon), and ligand energy minimization was performed. The protonation state of the small molecules was set to pH 7.4, and the compounds were expanded into three-dimensional structures using Open Babel. A series of ligand and receptor protein preparations were performed using AutoDockTools 1.5.6 software. The docking box was generated using the AutoGrid program, and molecular docking was performed using AutoDock Vina 1.2.0. The optimal binding conformation was selected for interaction analysis. The association between the peptides and the Keap1-Nrf2 signaling pathway was evaluated based on the binding free energies of the ligand and receptor. Low binding free energies suggested that the peptides might activate the Keap1-Nrf2 signaling pathway and exert antioxidant effects. Finally, the molecular docking results were visualized and analyzed using PyMOL software.

[0080] The Keap1-Nrf2 signaling pathway is a classic signaling pathway for the body's resistance to oxidative damage. Activation of the pathway begins with the dissociation of Keap1 and Nrf2. Competitive inhibition is the predominant dissociation mode, whereby exogenous antioxidant molecules occupy the Keap1-Nrf2 binding site, directly inhibiting the Keap1-Nrf2 interaction and enhancing Nrf2 dissociation, thereby activating the pathway. Therefore, if a peptide can occupy the binding site of Keap1 and Nrf2, it can increase the ubiquitination level of Nrf2, thereby enhancing the body's antioxidant capacity.

[0081] Molecular docking simulations revealed that LFNHF (SEQ ID NO: 1), PLDRHPF (SEQ ID NO: 2), and YPFLRPL (SEQ ID NO: 3) had affinities for Keap1 of -9.0, -7.5, and -7.9 kcal / mol, respectively. All three peptides spontaneously bound to Keap1. The results indicated that LFNHF (SEQ ID NO: 1) formed hydrogen and carbon-hydrogen bonds with amino acid residues Ser363, Gly462, Val463, Asn414, Ala510, and Ser602 of Keap1, and formed π-cation and π-anion interactions with Gly364, Arg415, Ser508, Tyr572, and Gly603. PLDRHPF (SEQ ID NO: 2) forms carbon-hydrogen and hydrogen bonds with Gly527, Gln530, Ser508, Gly364, and Ser363, and π-alkyl interactions with Tyr572, Ala556, Arg380, Phe577, and Tyr334. Furthermore, PLDRHPF (SEQ ID NO: 2) forms electrostatic interactions with Arg415 and Arg483. YPFLRPL (SEQ ID NO: 3) forms carbon-hydrogen and hydrogen bonds with Ser363, Arg415, Tyr525, Gln528, Gln530, and Ser555, and π-π, π-σ, and π-alkyl hydrophobic interactions with Ala556, Tyr572, and Phe577. In addition, YPFLRPL (SEQ ID NO: 3) also forms electrostatic interactions with Arg415 and Arg415.

[0082] In summary, LFNHF (SEQ ID NO: 1), PLDRHPF (SEQ ID NO: 2), and YPFLRPL (SEQ ID NO: 3) inhibit the binding of Nrf2 to Keap1 by forming steric hindrance. Combined with the results of cell-based experiments, these three peptides have the potential to activate the Keap1-Nrf2-ARE signaling pathway by occupying the binding site between Nrf2 and Keap1, ultimately increasing the activity of CAT, SOD, and GPX, thereby enhancing the body's ability to scavenge free radicals.

[0083] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A Cordyceps polypeptide, characterized in that: The Cordyceps polypeptide is selected from at least one of a peptide having an amino acid sequence such as SEQ ID NO: 1, a peptide having an amino acid sequence such as SEQ ID NO: 2, and a peptide having an amino acid sequence such as SEQ ID NO:

3.

2. Use of the Cordyceps polypeptide according to claim 1 as an antioxidant.

3. Use of the Cordyceps polypeptide according to claim 1 in preparing a product for improving cell viability.

4. Use of the Cordyceps polypeptide according to claim 1 in the preparation of a product for improving the survival rate of cells damaged by oxidative damage.

5. Use of the Cordyceps polypeptide according to claim 1 in preparing a product for reducing intracellular ROS.

6. Use of the Cordyceps polypeptide according to claim 1 in preparing a product for reducing intracellular malondialdehyde.

7. Use of the Cordyceps polypeptide according to claim 1 in the preparation of a product for increasing the activity of intracellular antioxidant enzymes; Preferably, the antioxidant enzyme is at least one selected from catalase, superoxide dismutase and glutathione peroxidase.

8. Use of the Cordyceps polypeptide according to claim 1 in the preparation of a product for increasing the activity of intracellular antioxidant enzymes.

9. Use of the Cordyceps polypeptide according to claim 1 in the preparation of a product for activating the Keap1-Nrf2-ARE signaling pathway.

10. The use according to any one of claims 3 to 9, characterized in that The products are medicines and / or functional foods.

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