Low-temperature freeze-dried fresh ginseng polypeptide, and preparation method and application thereof

Abstract

The application discloses a kind of low-temperature freeze-dried fresh ginseng polypeptide and its preparation method and application, belong to medical field.Low-temperature freeze-dried fresh ginseng polypeptide has no significant cytotoxicity effect, and the antioxidant activity fraction has good protective effect on H2O2 induced PC12 cell damage.The low-temperature freeze-dried fresh ginseng polypeptide can significantly reduce oxidative stress, inhibit brain cell apoptosis, and increase the expression of synaptic markers PSD95 and SYN1 related to memory capacity, improve D-galactose induced aging damage.Low-temperature freeze-dried fresh ginseng polypeptide can reduce the content of SOD, MDA and NO in brain tissue, activate Bcl-2 and Bax to play anti-apoptotic activity, while inhibit apoptosis proteins such as Caspase-3 and GSK-3β, and then improve aging damage.In addition, the application of optimized SNOTRAP proteomics technology shows that the pathways related to synapse and apoptosis are significantly enriched, indicating that the freeze-dried ginseng antioxidant peptide regulates biological processes through the above signaling pathways and plays a role in improving aging model.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a low-temperature freeze-dried fresh ginseng polypeptide, its preparation method, and its application. Background Technology

[0002] Aging is a long process characterized by the continuous accumulation of biological changes. This complex process is closely related to oxidative stress, mitochondrial dysfunction, and inflammatory responses. As time passes, aging gradually leads to a decline in the body's structure, metabolic function, and adaptability. More seriously, aging may ultimately trigger a series of disabling and fatal diseases, such as Parkinson's disease, Alzheimer's disease, cancer, and diabetes. This aging process not only seriously harms the physical and mental health of the elderly but may even accelerate death.

[0003] Oxidative stress caused by excessive free radicals produced in the body is a major cause of aging. Based on this theory, it is believed that supplementing with antioxidants can eliminate excess free radicals in the body, which is an important means of delaying aging. Polypeptides are compounds composed of multiple amino acids linked by peptide bonds, typically consisting of 10 to 100 amino acid molecules linked in the same way as proteins. They have a relative molecular mass of less than 10 kDa and generally lack complex spatial structures. Polypeptides that can regulate the life activities of organisms or have certain physiological activities are called bioactive polypeptides. Bioactive polypeptides play a variety of biological roles, one of the most important of which is antioxidant activity.

[0004] Ginseng ( Panaxginseng C. A. Mey Ginseng, a plant of the Araliaceae family, is a traditional and precious medicinal herb with a long history of application in Chinese medicine. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) records that ginseng "nourishes the five internal organs, calms the spirit, stabilizes the soul, stops palpitations, eliminates evil influences, brightens the eyes, opens the heart and improves intelligence; long-term use lightens the body and prolongs life." Modern pharmacology shows that ginseng also has anti-aging, anti-tumor, and immune-enhancing effects. Therefore, scholars both domestically and internationally have conducted in-depth and systematic research on the chemical composition and pharmacological effects of ginseng.

[0005] To date, there have been no reports of freeze-dried fresh ginseng peptides being used in the preparation of antioxidant or anti-aging drugs. Summary of the Invention

[0006] In view of this, the purpose of this invention is to overcome the shortcomings of the prior art, realize the development and utilization of natural products using modern drug research methods, and provide the application of low-temperature freeze-dried fresh ginseng polypeptides in the preparation of antioxidant or anti-aging drugs by combining a large number of pharmacodynamic experiments.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a polypeptide with antioxidant and anti-aging effects, wherein the polypeptide is a freeze-dried fresh ginseng polypeptide with a molecular weight of less than 3000 Da.

[0008] Furthermore, the polypeptide comprises four oligopeptides, the amino acid sequences of which are Gly-Trp-Gly-Pro-Gln, Gly-Trp, Leu-Trp, and Gly-Glu, respectively.

[0009] In a second aspect, the present invention provides a method for preparing any of the aforementioned polypeptides, wherein the low-temperature freeze-dried fresh ginseng polypeptide is prepared by the following method: pulverizing ginseng obtained after low-temperature freeze-drying, extracting the powder by alkaline dissolution and acid precipitation, obtaining endogenous fractions with a molecular weight greater than 3000 Da by ultrafiltration fractionation, and obtaining low-temperature freeze-dried fresh ginseng polypeptides with a molecular weight less than 3000 Da by enzymatic hydrolysis.

[0010] Furthermore, the specific steps include: (1) Take dried ginseng that has been frozen at low temperature, crush it to obtain powder, soak the powder in 5 to 7 times the amount of n-hexane, stir magnetically at room temperature for 6 to 10 hours, pour off the supernatant, and evaporate the powder to dryness; (2) Weigh the powder obtained after treatment in step (1), mix it with double-distilled water at a weight ratio of 1:13~18, adjust the pH to 8~10, extract at 2~6 ℃ for 10~14 h, centrifuge, take the supernatant, repeat 2~3 times, combine the supernatants, add HCl to adjust the pH to 4~5, let stand, incubate at 2~6 ℃ overnight, centrifuge, collect the precipitate, freeze dry to obtain protein; (3) Take the protein, add double-distilled water to completely dissolve it, and obtain a protein solution with a concentration of 4~6 mg / mL. Use an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to perform ultrafiltration fractionation to obtain endogenous fractions with molecular weights less than 3000 Da and greater than 3000 Da respectively. Freeze-dry for later use. (4) Take the endogenous fraction with a concentration greater than 3000 Da after freeze-drying, prepare it with double-distilled water to a concentration of 25~35 mg / mL, and use pepsin to enzymatically hydrolyze it for 3~5 h to obtain the hydrolysate; (5) Dissolve the enzymatic hydrolysate in double-distilled water at a concentration of 4-6 mg / mL and a pH of 6-8. Use an ultrafiltration membrane for ultrafiltration fractionation to obtain low-temperature freeze-dried fresh ginseng polypeptide with a molecular weight of less than 3000 Da.

[0011] Furthermore, after step (5), the method further includes: (6) Weigh the freeze-dried fresh ginseng polypeptide with a molecular weight of less than 3000 Da obtained in step (5), dissolve it in ultrapure water at a mass-to-volume ratio of 0.8 mg:10 μL, filter the sample solution through a 0.22 μm filter membrane for later use, and then connect the Superdex30 increase pre-packed column with ÄKTA. TM The pure protein purification system was used in conjunction with ultrapure water as the elution solution. Continuous elution was performed at a flow rate of 0.1 mL / min, 0.5 mL / tube. The absorbance of the elution peak was detected at 280 nm, and fractions F1-F6 were enriched.

[0012] In a third aspect, the present invention provides the application of the aforementioned polypeptide in the preparation of drugs with antioxidant and anti-aging effects.

[0013] Furthermore, the drug comprises low-temperature freeze-dried fresh ginseng polypeptides and pharmaceutically acceptable excipients, wherein the low-temperature freeze-dried fresh ginseng polypeptides are the active ingredients in the drug with antioxidant and anti-aging effects.

[0014] Furthermore, the drug is at least one of the following: (1) Drugs that activate Bcl-2 and Bax to exert anti-apoptotic activity, while inhibiting apoptotic proteins and promoting neuronal survival. Apoptotic proteins include Caspase-3 and GSK-3β. (2) Drugs that increase the expression of synaptic markers PSD95 and SYN1, which are related to memory ability; (3) Drugs that reduce the content of SOD, MDA and NO in the cerebral cortex of mice and alleviate oxidative stress.

[0015] Furthermore, the dosage form of the drug includes capsules, tablets, granules, or oral liquids.

[0016] The present invention also provides a drug comprising the polypeptide described above.

[0017] In this invention, the drug did not exhibit significant cytotoxic effects, and the antioxidant active peptide fraction had a good protective effect against H2O2-induced PC12 cell damage.

[0018] The drug activates Bcl-2 and Bax to exert anti-apoptotic activity, while inhibiting apoptotic proteins such as Caspase-3 and GSK-3β, thus promoting neuronal survival.

[0019] The drug increased the expression of synaptic markers PSD95 and SYN1, which are associated with memory ability, thereby improving D-galactose-induced aging damage.

[0020] The drug reduces the levels of SOD, MDA, and NO in the cerebral cortex of mice, thereby alleviating oxidative stress and improving D-galactose-induced aging damage.

[0021] The pharmaceutically acceptable excipients refer to conventional drug carriers in the field of pharmaceutical formulations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0022] The fillers of this invention include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binders include, but are not limited to, water, ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, etc.; the disintegrants include, but are not limited to, starch effervescent mixtures, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the solvents include, but are not limited to, water, ethanol, salt solutions, etc.

[0023] The present invention also provides an optimized SNOTRAP proteomics technology, which can detect more proteins and peptides and has higher specificity.

[0024] In the pharmaceutical applications described above, the timing, frequency, and duration of administration of freeze-dried fresh ginseng polypeptides should be determined based on the specific diagnostic results of the patient's condition, which is within the technical scope of those skilled in the art.

[0025] The beneficial effects of this invention include at least the following: This invention, leveraging my country's strengths in natural product research, is the first to verify the antioxidant and anti-aging effects of low-temperature freeze-dried fresh ginseng peptides. The low-temperature freeze-dried fresh ginseng peptides did not exhibit significant cytotoxicity, and the antioxidant active peptide fraction showed good protective effects against H2O2-induced PC12 cell damage. Simultaneously, it significantly activated Bcl-2 and Bax to exert anti-apoptotic activity and promote neuronal survival. It also increased the expression of synaptic markers PSD95 and SYN1, which are related to memory ability, thereby improving D-galactose-induced aging damage. Low-temperature freeze-dried fresh ginseng peptides can reduce the levels of SOD, MDA, and NO in the mouse cerebral cortex, thereby alleviating oxidative stress and improving D-galactose-induced aging damage. Therefore, low-temperature freeze-dried fresh ginseng peptides have the advantages of significant therapeutic effects and low toxicity. Attached Figure Description

[0026] Figure 1A bar chart is presented to illustrate the separation and purification of peptides from freeze-dried fresh ginseng using a protein purification system, and the analysis of the antioxidant activity of different fractions. A: Chromatogram of peptide separation and purification from freeze-dried fresh ginseng; B: DPPH radical scavenging activity of different fractions; C: •OH radical scavenging activity of different fractions. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 are considered statistically significant. Any insignificant difference is expressed in ns.

[0027] Figure 2 Mass spectrum for polypeptide identification in fraction F3. A: m / z 566.4450 primary and secondary mass spectra; B: m / z Primary and secondary mass spectra at 284.7285; C: m / z Primary and secondary mass spectra at 340.2726; D: m / z The primary and secondary mass spectra of 205.1673.

[0028] Figure 3 The effects of antioxidant active fraction F3 on cell viability and its protective effect. A: Effect of antioxidant active fraction F3 on PC12 cell viability; * indicates P<0.05, significant difference; ** indicates P<0.01, relatively significant difference; *** indicates P<0.001, highly significant difference; any insignificant difference is expressed as ns. B: Protective effect of antioxidant active fraction F3 on H2O2-induced cell damage model. # Compared to the model group, # A p-value < 0.05 indicates a significant difference. ## This indicates that P < 0.01, meaning the difference is statistically significant. ### P < 0.001 indicates a highly significant difference. * indicates a difference compared to the lowest effective concentration, * indicates a significant difference (P < 0.05), ** indicates a moderately significant difference (P < 0.01), and *** indicates a highly significant difference (P < 0.001). Any insignificant difference is expressed in ns.

[0029] Figure 4 The effect of different concentrations of fraction F3 on H2O2-induced apoptosis in PC12 cells was investigated. Representative histograms show: A: Untreated control group; B: PC12 cells treated with 200 µM H2O2 for 24 h; C: PC12 cells pretreated with 15 µg / mL fraction F3 for 24 h, followed by induction of apoptosis with 200 µM H2O2 for 24 h; D: PC12 cells pretreated with 25 µg / mL, 50 µg / mL, and 100 µg / mL fraction F3 for 24 h, followed by induction of apoptosis with 200 µM H2O2 for 24 h.

[0030] Figure 5 Representative images of PC12 cells stained with TUNEL. Scale bar: 50 μm. The control group, H2O2-only treatment group, 15 µg / mL fraction F3 + H2O2 treatment group, 25 µg / mL fraction F3 + H2O2 treatment group, 50 µg / mL fraction F3 + H2O2 treatment group, and 100 µg / mL fraction F3 + H2O2 treatment group were detected using a TUNEL assay kit. Photographs of randomly selected fields of view were taken, and at least three fields of view were selected from each photograph.

[0031] Figure 6 This study investigates the effects of low-temperature freeze-dried fresh ginseng peptides on a D-galactose-induced aging model using optimized SNOTRAP proteomics technology. A: Bar chart of SNO protein levels in four mouse groups; B: Venn plot of the treatment group, control group, and model group; C: Volcano plot of the control and model groups; D: Volcano plot of the model and L group; E: Volcano plot of the model and H group.

[0032] Figure 7 Biological process (GO) and signaling pathway (KEGG pathway) analysis of SNO protein data. A: KEGG pathway and GO biological process analysis of SNO protein in the Control group. B: KEGG pathway and GO biological process analysis of upregulated SNO protein in the Model group. C: KEGG pathway and GO biological process analysis of downregulated SNO protein after treatment with low-dose LG antioxidant peptide compared with the Model group. D: KEGG pathway and GO biological process analysis of downregulated SNO protein after treatment with high-dose LG antioxidant peptide compared with the Model group.

[0033] Figure 8 The effect of freeze-dried fresh ginseng antioxidant peptides on nNOS in the mouse cerebral cortex. The expression level of nNOS protein in the mouse cerebral cortex was investigated. ## P<0.01, *P<0.05, **P<0.01.

[0034] Figure 9 This study investigated the effects of freeze-dried fresh ginseng antioxidant peptides on the expression of apoptosis proteins in the mouse cerebral cortex. A: Expression levels of p-AKT, AKT, Bcl-2, Bax, Caspase-8, and Caspase-3 proteins in the cerebral cortex of four groups of mice. B and C: Quantitative analysis of apoptosis proteins Bax, Caspase-8, and Caspase-3 in the cerebral cortex of four groups of mice. D: Quantitative analysis of anti-apoptotic proteins p-AKT and Bcl-2 in the cerebral cortex of four groups of mice.

[0035] Figure 10This study investigated the effects of freeze-dried fresh ginseng antioxidant peptides on the expression of PSD-95 and SYN-1, synaptic markers related to memory, in the mouse cerebral cortex. A: Western blot analysis of PSD-95 and SYN-1 synaptic proteins in the cerebral cortex of four groups of mice. B: Quantitative analysis of PSD-95 and SYN-1 proteins. # P<0.05, **P<0.01.

[0036] Figure 11 The effects of low-temperature freeze-dried fresh ginseng antioxidant peptides on the levels of NO, MDA, and SOD in the cerebral cortex of mice. A: NO content in the cerebral cortex of four groups of mice; B: MDA content in the cerebral cortex of four groups of mice; C: SOD content in the cerebral cortex of four groups of mice. # P<0.05, **P<0.01. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0039] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Preparation of fresh ginseng polypeptides by low-temperature freeze-drying The dried ginseng obtained through low-temperature freeze-drying was pulverized and sieved. The powder was soaked in 6 times its volume of n-hexane and magnetically stirred at room temperature for 8 hours. The supernatant was then discarded, and the powder was evaporated to dryness. An appropriate amount of defatted, low-temperature freeze-dried fresh ginseng powder was accurately weighed and mixed with double-distilled water at a ratio of 1:15. NaOH was added to adjust the pH of the solution to 9, and the mixture was extracted at 4 °C for 12 hours. After centrifugation at 8000×g for 20 minutes, the supernatant was collected. This process was repeated twice, and the supernatants were combined. HCl was added to adjust the pH to 4.5, and the mixture was allowed to stand overnight at 4 °C. After centrifugation at 8000×g for 20 minutes, the precipitate was collected and freeze-dried. An appropriate amount of protein was dissolved completely in double-distilled water to a concentration of 5 mg / mL. Ultrafiltration fractionation was performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain endogenous fractions of <3000 Da and >3000 Da, which were then freeze-dried for later use. An endogenous fraction of freeze-dried fresh ginseng with a substrate concentration of 30 mg / mL and a molecular weight cutoff of >3000 Da was prepared. This fraction was then enzymatically hydrolyzed with pepsin for 4 h. An appropriate amount of the freeze-dried fresh ginseng pepsin hydrolysate was dissolved in double-distilled water at a concentration of 5 mg / mL and a pH of 7.0. Ultrafiltration fractionation was performed using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain freeze-dried fresh ginseng peptides with a molecular weight cutoff of less than 3000 Da after 4 h of pepsin hydrolysis. These peptides were then freeze-dried for later use.

[0040] Example 2: Establishment of a purification method and structural analysis of freeze-dried fresh ginseng polypeptides 1. Establishment of a purification method for fresh ginseng polypeptides obtained by low-temperature freeze-drying Accurately weigh 40 mg of the above-mentioned low-temperature freeze-dried fresh ginseng polypeptide sample and dissolve it in 500 μL of ultrapure water. Filter the sample solution through a 0.22 μm filter membrane for later use. Then, connect the Superdex 30 increase pre-packed column with ÄKTA... TM The pure protein purification system was used in conjunction with ultrapure water as the elution solution. Continuous elution was performed at a flow rate of 0.1 mL / min, 0.5 mL / tube. The absorbance of the elution peak was detected at 280 nm. Different fractions were enriched and freeze-dried for later use.

[0041] 2. Antioxidant Activity Test of Freeze-Dried Fresh Ginseng Peptide Fraction 2.1 Detection of antioxidant activity of different fractions 2.1.1 Determination of DPPH free radical scavenging activity Add 100 μL of 3 mg / mL sample solution and 100 μL of 0.1 mmol / L 99.7% DPPH ethanol solution to a 96-well plate, mix thoroughly, and repeat three times. Incubate the mixture in the dark at room temperature for 30 min, then measure the absorbance at 515 nm using a microplate reader. The calculation formula is as follows: DPPH free radical scavenging rate (%) = [1-(A sample - A blank ) / A control ]×100% In the formula: A sample The absorbance of the sample group; A blank The absorbance of deionized water is used instead of the sample solution. A control The absorbance is measured using anhydrous ethanol instead of DPPH solution.

[0042] 2.1.2 • OH Scavenging Activity Assay Method In a 96-well plate, 50 μL of 6 mmol / L FeSO4 was added to 50 μL of 1 mg / mL sample solution and 1 mg / mL glutathione (GSH) positive control solution, followed by 50 μL of 6 mmol / L H2O2. The mixture was shaken thoroughly, repeated three times, and allowed to stand at room temperature for 10 min. Then, 50 μL of 6 mmol / L salicylic acid ethanol solution was added, and the mixture was reacted in the dark for 30 min. The absorbance of the reaction system was measured at 510 nm using a microplate reader. The calculation formula is as follows: •OH scavenging rate (%) = [1-( A 1- A 2) / A 0]×100% In the formula: A o For the absorbance of the control, 50 μL of distilled water was used instead of the sample solution. A 1. To measure the absorbance of the sample, A 2 represents the absorbance without salicylic acid (95% ethanol is used instead of salicylic acid).

[0043] 3. Structural analysis of the antioxidant active fraction of peptides from freeze-dried fresh ginseng at low temperature 3.1 UPLC-MS Conditions Mass spectrometry analysis was performed using a Waters Xevo G2-XS QTof MS mass spectrometer equipped with an electrospray ionization (ESI) source and electrospray ionization positive ion mode (ESI). + The scanning range is as follows: m / z The range is 100-1000, the ion source temperature is 320 ℃, the cone voltage is 2.5 kV, the collision gas is nitrogen, and the collision energy is set to 80 V. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. The gradient elution program was as follows: 0–5 min, 10% B; 5–25 min, 100% B; 25–30 min, 100% B. The flow rate was 0.3 mL / min. -1 The injection volume was 2 μL. Instrument control and all data processing were performed using the Waters MassLynx workstation software.

[0044] 4. Experimental Results 4.1 Isolation, purification, and antioxidant activity detection of ginseng peptides obtained by low-temperature freeze-drying The freeze-dried fresh ginseng polypeptide prepared in Example 1 was separated and purified using a protein purification system. The spectrum showed six fraction peaks with significant UV absorption (e.g., Figure 1 A), which were enriched and freeze-dried separately. The DPPH free radical scavenging activity test results showed (as shown in Table 1) that the antioxidant activities of fractions F1-F6 were, in descending order: fraction 3 > fraction 2 > fraction 1 > fraction 4 > fraction 6 > fraction 5 (e.g., ...). Figure 1 B), the results of the •OH free radical scavenging activity test (as shown in Table 1) show that the antioxidant activity is in the order of fraction 3 > fraction 1 > fraction 2 > fraction 5 > fraction 4 > fraction 6 (e.g. Figure 1 (C) Since fraction 3 has the highest yield after separation and purification and its antioxidant activity is significantly superior to other fractions, fraction F3 with strong antioxidant activity was selected for further investigation.

[0045] Table 1: DPPH and •OH free radical scavenging activities (%) of fractions F1-F6 ± S, n = 3)

[0046] 4.2 Structural analysis of fraction F3 Tandem mass spectrometry analysis identified four polypeptides in fraction F3, namely... m / z 566.4450, 284.7285, 340.2726, and 205.1673. Mass spectrometry data and identification results of amino acids in fraction F3 are as follows: Figure 2 As shown.

[0047] Secondary mass spectrometry analysis was performed on peptides with strong primary mass spectrometry signals in fraction F3. m / z The 566.4450 polypeptide yielded a secondary mass spectrum with uniformly distributed fragments and a highly reliable amino acid sequence, such as... Figure 2 A. The mass-to-charge ratio of the analyte and fragment ions was obtained through primary and secondary mass spectra, and their structures were identified, yielding a pentapeptide. Fragment ions were detected in the secondary mass spectrum. m / z114.0947 [M+K] + , m / z 227.1841 [M+Na] + and m / z 265.1352 [M+Na] + Their relative molecular masses are 75.0947 and 204.1841, respectively, while m / z 265.1352 [M+Na] + The ion is presumed to be formed by the dehydration condensation of one molecule of Pro and one molecule of Gln, therefore it is presumed that... m / z 566.4450[M+Na] + It is formed by the dehydration condensation of 2 molecules of Gly with 1 molecule of Trp, 1 molecule of Pro, and 1 molecule of Gln. The amino acid sequence is Gly-Trp-Gly-Pro-Gln, and the molecular weight is 543.4450.

[0048] For peptides with strong first-order mass spectrometry signals in fraction F3 m / z Secondary mass spectrometry analysis was performed at 284.7285, yielding a secondary mass spectrum showing uniform fragment distribution and highly reliable amino acid sequences, such as... m / z B. The mass-to-charge ratio of the analyte and fragment ions was obtained through primary and secondary mass spectra, and their structures were identified, yielding a dipeptide. Fragment ions were detected in the secondary mass spectrum. Figure 2 114.0947 [M+K] + , m / z 205.0673 [M+H] + Their relative molecular masses are 75.0947 and 204.0673, respectively, therefore it is inferred that... m / z 284.7285[M+Na] + It is formed by the dehydration condensation of one molecule of Gly and one molecule of Trp, with the amino acid sequence Gly-Trp and a molecular weight of 261.7285.

[0049] For peptides with strong first-order mass spectrometry signals in fraction F3 m / z Secondary mass spectrometry analysis was performed on 340.2726, yielding a secondary mass spectrum showing uniform fragment distribution and highly reliable amino acid sequences, such as... m / z C. The mass-to-charge ratio of the analyte and fragment ions was obtained through primary and secondary mass spectra, and their structures were identified, yielding a dipeptide. Fragment ions were detected in the secondary mass spectrum. Figure 2 132.1072[M+H] + , m / z 205.0673 [M+H] + Their relative molecular masses are 131.1072 and 204.0673, respectively, therefore it is inferred that... m / z340.2726 [M+Na] + It is formed by the dehydration condensation of one molecule of Leu and one molecule of Trp, with the amino acid sequence Leu-Trp and a molecular weight of 317.2726.

[0050] For peptides with strong first-order mass spectrometry signals in fraction F3 m / z Secondary mass spectrometry analysis was performed at 205.1673, yielding a secondary mass spectrum showing uniform fragment distribution and highly reliable amino acid sequences, such as... m / z D. The mass-to-charge ratio of the analyte and fragment ions was obtained through primary and secondary mass spectra, and their structures were identified, yielding a dipeptide. Fragment ions were detected in the secondary mass spectrum. Figure 2 114.0985 [M+K] + , m / z 148.9755 [M+H] + Their relative molecular masses are 75.0985 and 147.9755, respectively, therefore it is inferred that... m / z 205.1673 [M+H] + It is formed by the dehydration condensation of one molecule of Gly and one molecule of Glu, with the amino acid sequence Gly-Glu and a molecular weight of 204.1673.

[0051] Example 3: Pharmacodynamic study of the anti-aging effects of freeze-dried fresh ginseng peptides 3.1 Study on the effect of antioxidant active fraction on improving in vitro cell aging model 3.1.1 Effect of antioxidant active fraction on PC12 cell survival PC12 cells in logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / well in sterile 96-well plates and cultured for 24 h. After confirming good cell adhesion and growth under microscopic examination, the culture medium in each well was discarded. Complete culture medium containing a series of concentrations (5, 10, 15, 20, 25, 50, 100, 200, 400 μg / mL) of antioxidant active fraction was added, and the cells were cultured for another 24 h. The culture medium was then discarded, and 100 μL of CCK-8 working solution was added to each well. The cells were cultured for another 1 h, and the absorbance of the test wells was measured at 450 nm. Following the kit instructions, with the control group (no drug treatment group) having a survival rate of 100%, the relative cell viability of the experimental groups was calculated as follows: Relative cell survival rate (%) = [( A 测定 - A 空白 ) / ( A 对照 - A 空白 )]×100% 3.1.2 Protective effect of antioxidant active fractions on H2O2-induced cell damage model PC12 cells in logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 1×10⁻⁶. 5 Cells were inoculated at a rate of 100 μL / well into sterile 96-well plates and cultured for 24 h. The culture medium in each well was discarded, and complete culture medium containing a series of concentrations (10, 15, 20, 25, 50, 100 μg / mL) of antioxidant active fractions was added. After 24 h of culture, the culture medium was discarded, and H2O2 (200 μM) was added and cultured for another 24 h. The relative cell viability was calculated according to the method in 3.1.1. The following groups were used in this experiment: the control group (only complete culture medium was added), the model group (H2O2 (200 μM) treatment group), and the drug treatment group (pretreated with different concentrations of fractions before being treated with H2O2 (200 μM)). The groupings for the following experiments were the same as above.

[0052] 3.1.3 Annexin V-FITC double staining method for detecting cell apoptosis level PC12 cells in logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 2 mL / well in sterile 6-well plates and cultured for 24 h. The culture medium was discarded, and complete culture medium containing different concentrations (15, 25, 50, 100 μg / mL) of antioxidant active fractions was added to each well. After 24 h of culture, the culture medium was discarded, and 500 μL of trypsin was added to each well for 2 min of digestion. The cell suspension was collected and centrifuged at 1000 rpm and 4 °C for 5 min. The supernatant was discarded, and 5 μL of Annexin V-FITC staining solution and 5 μL of PI staining solution were added to each cell tube. After incubation at room temperature for 10 min, the apoptosis level was detected by flow cytometry.

[0053] 3.1.4 DAPI staining observation 4',6-diamidino-2-phenylindole (DAPI) is a fluorescent dye that binds strongly to DNA. Observing the fluorescence intensity of cells through DAPI staining can indicate the state of cell apoptosis. PC12 cells in the logarithmic growth phase were taken, and the cell suspension concentration was adjusted to 1×10⁻⁶. 5Cells were inoculated at 1 mL / well in sterile 12-well plates and cultured for 24 h. The culture medium in the wells was discarded, and complete culture medium containing fractions 3 at a series concentrations (15, 25, 50, 100 μg / mL) was added. The cells were cultured for 24 h, the culture medium was discarded, and H2O2 (200 μM) was added and cultured for 24 h. The culture medium was discarded, and 4% paraformaldehyde was added to fix the cells for 20 min. After washing three times with PBS, 10 μg / mL DAPI solution was added and incubated for 20 min. The fluorescence intensity was observed using a fluorescence inverted microscope.

[0054] 3.1.5 Experimental Results 3.1.5.1 Effect of fraction F3 on PC12 cell viability The effect of fraction F3 on PC12 cell survival rate is as follows: m / z As shown in Figure A, the effect of fraction F3 in the concentration range of 5–400 μg / mL on the survival rate of PC12 cells was investigated. The cell survival rate of the 400 μg / mL fraction F3 administration group was slightly reduced, but no significant cytotoxic effect was observed. Based on the pharmacodynamic effects of the preliminary experiment, the protective effect of fraction F3 on H2O2-induced PC12 cell damage was explored at administration concentrations of 25, 50, and 100 μg / mL in the concentration range of 5–400 μg / mL.

[0055] 3.1.5.2 Protective effect of fraction F3 on H2O2-induced cell damage model like Figure 3 As shown in Figure B, fraction F3 reversed the H2O2-induced decrease in cell viability. Compared with the control group, PC12 cell viability was significantly reduced after treatment with 200 μM H2O2. Compared with the model group, PC12 cell survival rate was significantly increased after 24 h of pretreatment with fraction F3 in a dose-dependent manner, indicating that fraction F3 has a good protective effect against H2O2-induced PC12 cell damage.

[0056] 3.1.5.3 Results of Annexin V-FITC double staining method for detecting cell apoptosis level The effect of different concentrations of distillate F3 on H2O2-induced apoptosis in PC12 cells was investigated using flow cytometry. In this study, PC12 cells were first treated with 200 µM H2O2. The results showed that, compared with the control group, 200 µM H2O2 significantly induced apoptosis, with the apoptosis level increasing to 12.22% (e.g., ...). Figure 3 (AB). PC12 cells undergoing apoptosis were treated with different concentrations of fraction F3. The results showed that 15 µg / mL fraction F3 had no significant inhibitory effect on H2O2-induced apoptosis (e.g., ...). Figure 4 C). However, as Figure 4As shown in Figure D, treatment with fractions F3 at concentrations of 25, 50, and 100 µg / mL significantly inhibited apoptosis in the cell lines, reducing apoptosis levels to 7.92%, 8.31%, and 6.61%, respectively. These results indicate that fractions F3 at concentrations of 25, 50, and 100 µg / mL can significantly inhibit the apoptotic effects of hydrogen peroxide on PC12 cells.

[0057] 3.1.5.4 Observation results of DAPI staining The effect of different concentrations of distillate F3 on H2O2-induced apoptosis in PC12 cells was detected using TUNEL staining. Results are as follows: Figure 4 As shown, compared with the control group, the number of TUNEL-positive cells in PC12 cells treated with 200 µM H2O2 increased significantly. However, the number of TUNEL-positive cells in cells treated simultaneously with fractions F3 and H2O2 at concentrations of 25 µg / mL, 50 µg / mL, and 100 µg / mL, respectively, was lower than that in the group treated with 200 µM H2O2 alone.

[0058] 3.2 Study on the effects of optimized SNOTRAP proteomics technology on a D-galactose-induced aging model of freeze-dried fresh ginseng peptides 3.2.1 Animals Twenty-four healthy C57BL / 6 mice, clean-grade, 2 months old, weighing 20-22 g, were provided by Liaoning Changsheng Biotechnology Co., Ltd. (Shenyang, China), Animal Qualification Certificate No.: SCXK(Liaoning)2020-0001. The animals were housed at the Experimental Animal Center of Changchun University of Traditional Chinese Medicine, with strict temperature (25 ± 2 ℃), relative humidity (55~60%), and a 12-hour light-dark cycle. Prior to the experiment, the animals were acclimatized for one week in a local animal facility. All experimental procedures were approved by the Experimental Animal Management Committee of Changchun University of Traditional Chinese Medicine.

[0059] 3.2.2 Drugs and Experimental Protocol Animals were randomly divided into four groups of six per group: a control group, a model group, a low-dose treatment group (L, 12.5 mg / kg / day of freeze-dried fresh ginseng antioxidant peptides), and a high-dose treatment group (H, 25 mg / kg / day of freeze-dried fresh ginseng antioxidant peptides). Animals in the model and treatment groups received intraperitoneal injections of D-galactose (150 mg / kg) daily, while the control group received saline. Simultaneously, the low-dose group (12.5 mg / kg) and the high-dose group (25 mg / kg) received intraperitoneal injections of freeze-dried fresh ginseng antioxidant peptides for eight consecutive weeks.

[0060] 3.2.3 Preparation of SNO brain tissue samples Brain tissue from mice euthanized by cervical dislocation was placed on ice and washed with physiological saline. The cerebral cortex was rapidly dissected, and striatal, hippocampal, and cortical samples were collected separately to form biological replicates. Appropriate amounts of lysis buffer (100 mmol / L HEPES-NaOH, 1 mmol / L EDTA, 20 mmol / L IAM, 0.1 mmol / L neoocuproine, 1% Triton X-100, 0.1% SDS, and 1% protease inhibitor, pH 7.7) were added to each replicate, and the tissues were homogenized on ice and lysed at low temperature for 30 min. The tissues were centrifuged at 16000×g for 15 min, and the supernatant was collected. Protein concentration was determined using the BCA method.

[0061] The supernatant was washed with 1 mL of 50 mmol / L HEPES (pH 7.7) and centrifuged at 5500×g for 30 min using spin filters. 1.5 mmol / L SNOTRAP probe was added to each sample to bind 10 mg of protein, and the mixture was incubated at room temperature for 2 h. The samples were then washed three times with 50 mmol / L HEPES buffer (pH 7.7) and 10 kD MWCO centrifuge tubes to remove excess chemicals, and then digested overnight with trypsin at 37°C.

[0062] High-capacity streptavidin agarose beads were gently incubated with each digested sample at room temperature for 2 h. The beads were then washed twice sequentially with five volumes of the following five buffers to remove non-specific binding: Buffer I: 100 mmol / L ammonium bicarbonate, 150 mmol / L NaCl, 1 mmol / L EDTA, 0.05% SDS, and 0.1% Triton X-100 (pH 7.4); Buffer ⅠⅠ: 100 mmol / L ammonium bicarbonate, 50 mmol / L NaCl, 1 mmol / L EDTA, and 0.1% SDS (pH 7.4); Buffer ⅠⅢⅠ: 100 mmol / L ammonium bicarbonate, 0.05% SDS, and 150 mmol / L NaCl; Buffer ⅠⅢⅠ: 100 mmol / L ammonium bicarbonate; Buffer ⅠⅢⅢⅠ: 50 mmol / L HEPES (pH 7.7). The bound peptide was eluted with 100 mmol / L TCEP, followed by alkylation with 100 mmol / L NEM. After alkylation, the sample was desalted and stored at -80°C for analysis.

[0063] 3.2.3.1 Nano LC-MS Analysis Peptides were analyzed using an Orbitrap Exploris 480 mass spectrometer coupled with an Easy-nLC1000 liquid chromatograph (ThermoFisher Scientific, Waltham, MA) and a nanoSpray Flex ion source interface. Each biological sample was analyzed in triplicate. Chromatographic conditions: Samples were separated using a gradient mobile phase of 0.1% formic acid aqueous solution (A) and acetonitrile (B). 3 μL of sample was injected into a C18 capillary column (75 μm × 20 mm, 3 μm, ThermoScientific), and eluted using a C18 capillary column (75 μm × 250 mm, 2 μm, Thermo Scientific) at a flow rate of 300 nL / min with a gradual gradient elution: 0–10 min, 1% B; 10–110 min, 60% B; 110–120 min, 60–100% B. Mass spectrometry conditions: spray voltage 2.2 kV; capillary temperature 250 °C; S-lens voltage 60%; resolution 70,000; scan range 350–1800. Figure 5 Ten of the most abundant multi-charged ions were selected for fragmentation, with an isolation window of 2 Th, a resolution of 35,000, and a collision energy of 28%. Data acquisition and analysis were performed using Xcalibur 2.0 software.

[0064] 3.2.3.2 Data Processing Peak list generation, database search, label-free semi-quantitative assessment, and FDR estimation were performed using Thermo Scientific Proteome Discoverer proteomics software. The parameters extracted from the data were: precursor ion MH... + 300-8000 Da, sequence tag length >1. Mass spectrometry / mass spectrometry with consistent ion tolerance was searched based on the mouse SwissProt protein database. The scope of the study included asparagine deamidation, N-terminal acetylation of proteins, methionine oxidation, and immobilization modifications of N-ethylmaleimide, with an FDR cutoff of 1%. The minimum peptide length was specified as 5 amino acids, allowing a maximum of two deletions.

[0065] 3.2.3.3 Experimental Results Using SNOTRAP proteomics technology, a database of SNO proteins and SNO modification sites in the cerebral cortex of aging model mice treated with low-temperature freeze-dried fresh ginseng antioxidant peptides was successfully constructed. 589 SNO proteins were identified in the brains of control mice, 621 SNO proteins in the brains of aging mice, 622 SNO proteins in the brains of mice treated with low-dose antioxidant peptides, and 623 SNO proteins in the brains of mice treated with high-dose antioxidant peptides. There was no significant difference in the number of SNO proteins between the model group and the treatment group. m / z A). Venn diagrams were used to further analyze the effects of lyophilized ginseng antioxidant peptides on SNO protein in D-galactose model mice. Approximately 76% of SNO protein was identical between the low-dose treatment group, the control group, and the model group, while 2.7% was different. Similarly, approximately 76.1% of SNO protein was identical between the high-dose treatment group, the control group, and the model group, while 2.6% was different. Figure 6 B). The Volcano plot shows a scatter plot of Fold change (FC) versus p-value for each protein to interpret the results of this quantitative analysis. Red and blue circles represent significantly upregulated (FC≥2) and downregulated (FC≤2) proteins, respectively (P<0.05). Black circles represent significant changes (P<0.05) but with a fold change <2. A black horizontal dashed line represents P=0.05, and gray circles below this line represent no significant change. Fold change (FC; log 2 ) and signal strength P-value (-log 10 The Volcano plot, a pie chart showing the percentage of protein (e.g.) Figure 6 These results demonstrate that the SNOTRAP method has the advantages of high reproducibility and high sensitivity.

[0066] To assess the differences in molecular processes in the cerebral cortex among the mouse groups, GO and KEGG pathway analyses were performed on the SNO protein data from the four groups. The results are as follows: Figure 6 As shown. Bioinformatics analysis revealed that, compared to the model group, the downregulated SNO protein in the brain tissue of mice in both the low-dose and high-dose treatment groups was mainly enriched in signaling pathways related to synaptic function regulation and apoptosis inhibition, such as "positive regulation of the ERK1 and ERK2 cascade" and "positive regulation of synaptic maturation." Low-temperature freeze-dried fresh ginseng antioxidant peptides regulate biological processes through these signaling pathways and play a role in improving aging models.

[0067] 3.3 Effects of low-temperature freeze-dried fresh ginseng antioxidant peptides on nNOS, apoptosis proteins, and synaptic expression in mouse cerebral cortex 3.3.1 Brain tissue preparation Mice were euthanized by cervical dislocation and their brains were quickly decapitated. Hippocampal and cortical tissues were harvested from both cerebral hemispheres, rapidly frozen, and stored at -80°C for later analysis.

[0068] 3.3.2 Western blot analysis Cortical tissue was homogenized in RIPA lysis buffer (1:5, w / v) containing PMSF and protease inhibitor cocktail (100:1:1, v / v / v) at ice temperature. The mixture was then centrifuged at 12000 r / min for 15 min at 4 °C. Protein concentration was determined using the Bradford method with BSA as a standard. 10% SDS-PAGE gels were prepared using an SDS-PAGE gel kit, with 25 μg sample loaded per well for subsequent electrophoresis and transfer. Primary antibody Keap-1 (1:2000), Nrf2 (1:1000), and internal control GAPDH (1:1000) were diluted with primary antibody dilution buffer. The transferred samples were blocked in TBST with 5% (w / v) skim milk at room temperature for 1 hour, then incubated overnight with primary antibody at 4 °C. After washing the membrane 5 times with TBST, horseradish peroxidase-conjugated secondary antibody was added and incubated at room temperature for 1 hour. Protein bands were treated with ECL solution, and each protein band was exposed using a ChemiDoc MP imaging system (BIO-RAD, USA). Quantification was performed using ImageJ software.

[0069] 3.3.3 Enzyme-linked immunosorbent assay (ELISA) For biochemical analysis, tissue samples were homogenized with ice-cold physiological saline solution (0.9%, v / w) at a ratio of 1:9 (w / v). The homogenate was centrifuged at 3000 rpm for 10 minutes at 4 °C, and the supernatant was collected. The supernatant was diluted with an appropriate buffer solution before measuring relevant biochemical parameters. SOD, MDA, and NO levels were measured using an ELISA kit (Hubei, China, mouse-specific) provided by Huangshi Yansheng Biotechnology Co., Ltd., according to the manufacturer's instructions.

[0070] 3.3.4 Experimental Results 3.3.4.1 Effects of low-temperature freeze-dried fresh ginseng antioxidant peptides on nNOS in mouse cerebral cortex This invention investigated the effect of low-temperature freeze-dried fresh ginseng antioxidant peptides on nNOS expression in the mouse cerebral cortex. The results are as follows: Figure 7 As shown, compared with the control group, the expression of nNOS in the cerebral cortex of the model group was significantly increased ( ## P<0.01. Compared with the model group, nNOS expression was reduced to varying degrees in the treatment group, and the low-dose group significantly reduced nNOS expression (P<0.01). ** P<0.01), high doses can also significantly reduce nNOS expression ( *P<0.05), no dose-dependent effect.

[0071] 3.3.4.2 Effects of low-temperature freeze-dried fresh ginseng antioxidant peptides on the expression of apoptosis proteins in mouse cerebral cortex GO and pathway analysis revealed enrichment of apoptosis and synaptic pathways, therefore the expression of proteins related to apoptosis and synapse was measured. Results are as follows: Figure 8 The results showed that, compared with the control group, the expression of Bax, Caspase-8, and Caspase-3 in the cerebral cortex of the model group mice was significantly increased, while the expression levels of p-AKT and Bcl-2 were significantly decreased, and the differences were statistically significant. ## P<0.01).

[0072] Compared with the model group, the low-dose treatment group showed a significant increase in p-AKT expression in the cerebral cortex. ** P<0.01, high-dose treatment group showed increased p-AKT expression in the cerebral cortex (P<0.01). * P<0.05, no dose-dependent effect; compared with the model group, the low-dose treatment group showed increased Bcl-2 expression in the cerebral cortex (P<0.05). * P<0.05, high-dose treatment group showed increased Bcl-2 expression in the cerebral cortex (P<0.05). * P<0.05, no dose-dependent effect; compared with the model group, the low-dose treatment group showed decreased Bax expression in the cerebral cortex (P<0.05). ** P<0.01, the high-dose treatment group showed decreased Bax expression in the cerebral cortex (P<0.01). ** P<0.01, no dose-dependent effect; compared with the model group, the low-dose treatment group showed decreased Caspase-8 expression in the cerebral cortex (P<0.01). * P<0.05, Caspase-8 expression in the cerebral cortex was decreased in the high-dose treatment group (P<0.05). * P<0.05, no dose-dependent effect; compared with the model group, the low-dose treatment group showed decreased Caspase-3 expression in the cerebral cortex (P<0.05). * P<0.05, Caspase-3 expression in the cerebral cortex was decreased in the high-dose treatment group (P<0.05). * P<0.05), no dose-dependent effect.

[0073] 3.3.4.3 Effects of low-temperature freeze-dried fresh ginseng antioxidant peptides on the expression of synaptic proteins in the mouse cerebral cortex To further investigate the effects of low-temperature freeze-dried fresh ginseng antioxidant peptides on the mouse cerebral cortex, this invention detected the expression of SYN1 and PSD95 proteins in the mouse cerebral cortex, such as... Figure 9 As shown in the figure. Compared with the control group, the expression of SYN1 and PSD95 in the cerebral cortex of the model group was significantly reduced, and the differences were statistically significant. ##P<0.01. Compared with the model group, the low-dose treatment group showed increased SYN1 expression in the cerebral cortex (P<0.01). ** P<0.01, high-dose treatment group showed increased SYN1 expression in the cerebral cortex (P<0.01). ** P<0.01, no dose-dependent effect; compared with the model group, the low-dose treatment group showed increased PSD95 expression in the cerebral cortex (P<0.01). ** P<0.01, the expression of PSD95 in the cerebral cortex increased in the high-dose treatment group (P<0.01). * P<0.05), no dose-dependent effect.

[0074] 3.3.4.4 Effects of low-temperature freeze-dried fresh ginseng antioxidant peptides on the content of SOD, MDA, and NO in the cerebral cortex of mice To determine whether low-dose and high-dose treatment groups of freeze-dried fresh ginseng antioxidant peptides could inhibit oxidative damage in D-galactose-treated mice, this invention detected the levels of the major antioxidant enzyme SOD, lipid-oxygen free radical reaction products MDA, and NO in the cerebral cortex. The results showed that compared with the control group ( Figure 10 Figure 11 The SOD level in the cerebral cortex of the model group was significantly downregulated. # P<0.05, after low-dose treatment, SOD levels were significantly upregulated ( # P<0.05, the high-dose group did not upregulate SOD levels. In the model group, MDA and NO levels in the cerebral cortex were significantly upregulated (P<0.05). # P<0.05. Low-dose treatment significantly downregulated MDA levels in the mouse cerebral cortex. ** P<0.01, and MDA levels were also significantly downregulated after high-dose treatment ( * P<0.05, no dose-dependent effect. After low-dose treatment, NO levels in the mouse cerebral cortex were significantly downregulated (P<0.05). ** P<0.01, and NO levels were also significantly downregulated after high-dose treatment ( * (P<0.05), with no dose-dependent effect. These data collectively indicate that low-temperature freeze-dried fresh ginseng antioxidant peptides can provide protection against D-galactose-induced oxidative stress damage.

[0075] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0076] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0077] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A low-temperature freeze-dried fresh ginseng polypeptide, characterized in that, The polypeptide was prepared by the following method: (1) Take dried ginseng that has been frozen at low temperature, crush it to obtain powder, soak the powder in 5 to 7 times the amount of n-hexane, stir magnetically at room temperature for 6 to 10 hours, pour off the supernatant, and evaporate the powder to dryness; (2) Weigh the powder obtained after treatment in step (1), mix it with double-distilled water at a weight ratio of 1:13~18, adjust the pH to 8~10, extract at 2~6 ℃ for 10~14 h, centrifuge, take the supernatant, repeat 2~3 times, combine the supernatants, add HCl to adjust the pH to 4~5, let stand, incubate at 2~6 ℃ overnight, centrifuge, collect the precipitate, freeze dry to obtain protein; (3) Take the protein, add double-distilled water to completely dissolve it, and obtain a protein solution with a concentration of 4~6 mg / mL. Use an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to perform ultrafiltration fractionation to obtain endogenous fractions with molecular weights less than 3000 Da and greater than 3000 Da respectively. Freeze-dry for later use. (4) Take the endogenous fraction with a concentration greater than 3000 Da after freeze-drying, prepare it with double-distilled water to a concentration of 25~35 mg / mL, and use pepsin to enzymatically hydrolyze it for 3~5 h to obtain the hydrolysate; (5) Dissolve the enzymatic hydrolysate in double-distilled water at a concentration of 4-6 mg / mL and a pH of 6-8. Use an ultrafiltration membrane for ultrafiltration fractionation to obtain low-temperature freeze-dried fresh ginseng polypeptide with a molecular weight of less than 3000 Da.

2. The use of the polypeptide of claim 1 in the preparation of antioxidant drugs.

3. The application according to claim 2, characterized in that, The drug comprises freeze-dried fresh ginseng polypeptides and pharmaceutically acceptable excipients, wherein the freeze-dried fresh ginseng polypeptides are the active ingredient in the antioxidant drug.

4. The application according to claim 2, characterized in that, The dosage forms of the drug include capsules, tablets, granules, or oral liquids.

Images