Elk characteristic peptide fragment for treating Alzheimer disease and preparation method thereof

Abstract

The invention discloses an elk characteristic peptide fragment for treating Alzheimer's disease and a preparation method thereof, an elk extract with molecular weight less than 10kDa is successfully separated by optimizing an extraction and purification process and combining a membrane separation technology with Sep-Pak C18 column chromatography purification, and 13 active polypeptides with clear sequences are further obtained by screening. And a nano LC-Orbitrap-MS technology is combined with a high performance liquid chromatography method, so that the extraction efficiency and the purity of the polypeptide are remarkably improved. Experimental results show that the prepared elk feature peptide fragment shows strong effects of resisting neuroinflammation, resisting oxidation and promoting expression of neurotrophic factors in in-vivo and in-vitro experiments, and provides a more powerful drug basis for treatment of neurodegenerative diseases such as Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a cervus elaphus antler characteristic peptide segment capable of treating Alzheimer's disease and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, and its main pathological features include deposition of β-amyloid protein (Aβ), abnormal phosphorylation of tau protein to form neurofibrillary tangles, neuron loss, and inflammatory responses, etc. These pathological changes lead to the gradual decline of the cognitive function of patients and ultimately the loss of self-care ability. Although there are currently various methods for treating AD, including drugs such as cholinesterase inhibitors and NMDA receptor antagonists, these treatment means can only relieve symptoms to a certain extent and cannot cure the disease. Existing drugs have problems such as limited efficacy, large side effects, and drug resistance, so there is an urgent need to develop new and multi-target treatment means.

[0003] In this context, natural polypeptides have become a research hotspot due to their potential in multi-target effects such as antioxidant, anti-inflammatory, and neuroprotective effects. As a kind of bioactive molecule, polypeptides show great potential in drug research and development due to their low toxicity, high biocompatibility, and multifunctionality. However, problems such as poor in vivo stability, easy enzymatic degradation, and low absorption rate of polypeptide drugs limit their clinical application.

[0004] The existing technology mainly focuses on the extraction, purification, and bioactivity identification of polypeptides. In this field, the polypeptide extraction technology based on natural resources is relatively mature, and usually solvent extraction methods such as water extraction and alcohol extraction are used, combined with chromatography techniques such as ion exchange and gel filtration for purification. In recent years, the development of mass spectrometry analysis technology has provided strong support for the structure identification of polypeptides. Especially liquid chromatography-mass spectrometry tandem technology (LC-MS / MS) can accurately identify polypeptide sequences and quantitatively analyze their contents.

[0005] Insufficient multi-target treatment: Most of the existing treatment methods for Alzheimer's disease (AD) target a single target, such as inhibiting Aβ deposition or reducing abnormal phosphorylation of tau protein. This single-target treatment method often cannot effectively prevent the progression of the disease, resulting in limited efficacy. The multi-target treatment strategy for multiple neuron damage factors is insufficient and cannot comprehensively improve the cognitive function of AD patients.

[0006] Poor biological stability: Although current polypeptide drugs show certain neuroprotective effects in vitro, they are easily enzymatically degraded in vivo, resulting in unstable drug efficacy and short duration. Existing polypeptide drugs lack effective structural optimization to improve their in vivo stability, resulting in non-persistent efficacy and difficulty in achieving the ideal therapeutic effect.

[0007] Low purification and extraction efficiency: The extraction and purification process of polypeptides is complex and costly. Existing technologies mostly adopt traditional solvent extraction combined with methods such as ion exchange or gel filtration. These methods are often inefficient when dealing with complex biological samples, making it difficult to achieve efficient separation and purification, resulting in unstable product purity and quality.

[0008] Insufficient in vivo activity verification: Most existing studies mainly focus on the evaluation of polypeptide activity in in vitro cell models, lacking systematic in vivo verification. The results of in vitro experiments cannot fully reflect the effects of polypeptides under physiological conditions and are difficult to be directly applied to clinical treatment. Therefore, the verification of the in vivo efficacy and safety of polypeptides in existing technologies is still insufficient.

[0009] Side effects and toxicity: Existing AD treatment drugs (including some polypeptide drugs) have certain side effects in clinical applications, such as gastrointestinal discomfort, liver and kidney toxicity, etc., affecting patient compliance and treatment effects. Traditional polypeptide extracts may contain other impurities or non-target proteins, increasing the risk of side effects and toxicity. Summary of the Invention

[0010] The purpose of the present invention is to overcome the above-mentioned shortcomings of the existing technologies, provide a polypeptide substance extracted from elk antlers, and clarify the structure and stability of the polypeptide through innovative extraction, purification and identification technologies, so as to achieve a multi-target, highly efficient and low-toxic treatment plan for Alzheimer's disease.

[0011] Specific purposes include: Multi-target neuroprotection: The polypeptide of the present invention provides multi-target neuroprotective effects by regulating multiple signal pathways such as PI3K / AKT / Caspase 3, and can significantly improve the cognitive function of AD patients.

[0012] Efficient extraction and purification: By using nano LC-Orbitrap-MS mass spectrometry technology and efficient column chromatography, the present invention can improve the extraction and purification efficiency of polypeptides, reduce production costs and improve the purity and stability of polypeptides.

[0013] In vitro and in vivo activity verification: The present invention verifies the biological activity and safety of the polypeptide through systematic in vitro and in vivo models, ensuring its effectiveness and feasibility in clinical applications, and filling the gap in the in vivo activity verification of existing technologies.

[0014] Reduce side effects and toxicity: The present invention uses natural polypeptides and through a strict extraction and purification process, minimizes the presence of impurities and non-target proteins, reduces side effects and toxicity, and ensures the safety of the drug and the tolerance of patients.

[0015] Through the above improvements, the present invention aims to provide a safe, effective, and novel polypeptide drug with multiple neuroprotective effects for the treatment of Alzheimer's disease, overcome many deficiencies in the prior art, and provide patients with better treatment options.

[0016] The specific technical solution adopted by the present invention is: A characteristic peptide segment of elk antler for treating Alzheimer's disease, wherein the characteristic peptide segment is as follows F1-F13: Number Amino acid sequence F1 PAGERGSPGPAGPK F2 PSGASGPAGPRGPPG F3 ASGPAGPRGPPGSAG F4 GPSGPQGPSGPPGPKGN F5 PQGPSGPPGPKGN F6 SPGPAGPKGSPGEA F7 GPAGPKGSPGEAGRPG F8 PGPAGPAGPRGEVGLPGLS F9 GYDLSFLPQPPQEKAHDGGRYY F10 GHNGLQGLPGLAGHH F11 GGYDFGFDGDFYRA F12 GGGYDFGFDGDFYRA F13 FGFDGDFYR The characteristic peptide segments are the following sequences F1 to F13 as shown in sequence tables 1 to 13.

[0017] The method for preparing the characteristic peptide segment of elk antler for treating Alzheimer's disease of the present invention comprises the following steps: (1) Extraction Take the elk antlers, grind them, add water, heat them in a water bath for extraction, combine the extracts, centrifuge, and collect the supernatant; (2) Membrane separation and molecular weight fractionation The extract was separated by molecular weight cut-off membranes of 10 kDa and 100 kDa in turn to obtain an extract fraction with a molecular weight less than 10 kDa; (3) Purification Take the extraction part of step (2), add pure water to prepare a sample solution; prepare four Waters Sep-PakVac 6cc C18 columns and perform the following steps: first activate with acetonitrile; then balance with trifluoroacetic acid; load the sample and wait for natural outflow; then rinse with trifluoroacetic acid, and then elute with an aqueous solution containing 0.2% trifluoroacetic acid and 5% acetonitrile, an aqueous solution containing 0.2% trifluoroacetic acid and 20% acetonitrile, an aqueous solution containing 0.2% trifluoroacetic acid and 50% acetonitrile, and an aqueous solution containing 0.2% trifluoroacetic acid and 80% acetonitrile, respectively, collect the eluates, and obtain four components ML2-1 to ML2-4, and concentrate the collected components by centrifugation; (4) Thirteen peptides F1~F13 were identified from the ML2-1 and ML2-3 components.

[0018] As a preferred embodiment, the method for preparing the above-mentioned characteristic peptide segment of elk antler for treating Alzheimer's disease comprises the following steps: (1) Extraction Take the crude powder of elk antlers, add 8-20 times of ultrapure water, heat and reflux to extract 2-3 times, each time for 6-8 hours, filter, and combine the filtrate to obtain ML1 of total water extract of elk antlers; (2) Membrane separation and molecular weight fractionation Dilute ML1 until the liquid is clear. Prepare a membrane module with a molecular weight cut-off of 100k and 10k Da and a TNZ-1 nanofiltration separation device. After passing the total aqueous extract of elk antler ML1 sample through a nanofiltration membrane with a molecular weight of 100k Da, add pure water to rinse the residue, and collect filtrate a and retentate b respectively; filtrate a is passed through an ultrafiltration membrane with a molecular weight of 10k Da again, and filtrate c is collected; the obtained filtrate c is ML4, retentate b is ML3, and filtrate a is ML2; after the membrane separation with a molecular weight cut-off of 10k Da is completed, ML2 (MW < 10k Da), ML3 (10k < MW < 100k Da), and ML4 (MW > 100k Da) are obtained; the obtained ML1~ML4 are concentrated under reduced pressure and freeze-dried; (3)Purification Further purify the ML2 fraction by Sep-Pak column chromatography: Take the ML2 fraction, add pure water to prepare a sample solution; prepare four Waters Sep-Pak Vac 6cc C18 columns for the following steps: first activate with acetonitrile; then equilibrate with trifluoroacetic acid; load the ML2 fraction sample solution and wait for it to flow out naturally; then rinse with trifluoroacetic acid, and then elute with aqueous solutions containing 0.2% trifluoroacetic acid and 5% acetonitrile, 0.2% trifluoroacetic acid and 20% acetonitrile, 0.2% trifluoroacetic acid and 50% acetonitrile, and 0.2% trifluoroacetic acid and 80% acetonitrile respectively, and collect the eluates respectively to obtain four components ML2-1~ML2-4, and centrifuge and concentrate after collecting the components; (4)13 polypeptides F1~F13 were isolated and identified from the ML2-1 and ML2-3 components purified in step (3).

[0019] As a more preferred solution, for the preparation method of the elk antler characteristic peptide segment for treating Alzheimer's disease, in step (4), nano liquid chromatography-orbitrap mass spectrometry is used to structurally separate and identify the polypeptides in the ML2-1 and ML2-3 components; the chromatographic column is a Reprosil C18 AQ column with a specification of 150 mm×75 μm, 5 μm; the injection volume is 2 μL, and the volume flow rate is 400 nL / min; mobile phase A is acetonitrile-formic acid-water with a volume ratio of 2:0.2:98, and mobile phase B is acetonitrile-formic acid-water with a volume ratio of 80:0.2:20, and a linear gradient elution of 2% to 30% B is carried out for 120 min; the spray voltage is 2.5 kV; the temperature of the ion transfer capillary is 200 °C; the full-scan range of the mass spectrometry in the first stage is m / z 300 to 2000; the separation width is 3; tandem mass spectrometry analysis obtains a total ion chromatogram, and through collision-induced dissociation, a series of secondary tandem mass spectra are generated; the tandem mass spectrometry data is analyzed by searching a database using PEAKS8.5 software, and a self-built collagen database of Cervidae is selected; the retrieval parameters are set as follows: the precursor ion error is 1.0×10-5, and the product ion error is m / z 1; post-translational modifications: carbamidomethylation of cysteine (+57.2), oxidation of methionine (+15.99), hydroxylation of proline (+15.99), deamidation of aspartic acid and glutamine (+0.98); digestion method: trypsin digestion; allowing 2 miscleavage sites, and the false positive rate ≤1%; other parameters are default parameters, and the scores obtained under the above retrieval conditions are significant, and a P<0.05 is considered a valid identification result; Import the Nano LC-IT-Orbitrap mass spectrometry and fragment ion diagrams into the PEAKS software and analyze and identify them according to the following parameters. Search parameter settings: mass deviation of precursor ions: 10.0 ppm; mass deviation of product ions: 0.02 Da; fixed modification: carbamidomethylation, 57.02 Da; variable modifications: deamidation (NQ), 0.98 Da; oxidation (M), 15.99 Da; Phosphorylation (STY), 79.97 Da; maximum number of post-translational modifications per polypeptide: Result export parameters: de novo score (ALC%) threshold, 80; protein - 10lgP > 20; FDR (PSMs), 0.20%; The PEAKS result analysis shows that 303 peptides were identified in ML2 - 1, with molecular weights ranging from 700 to 2500 Da; derived from four proteins: alpha - 1(I) collagen chain subtype X1; alpha - 2(I) collagen chain; alpha - 1(II) collagen chain subtype X1; alpha - 1(VII) collagen chain; 330 peptides were identified in ML2 - 3, with molecular weights ranging from 600 to 3500 Da, derived from four proteins: alpha - 2(I) collagen chain; alpha - 1(I) collagen chain subtype X1; alpha - 1(II) collagen chain subtype X1; alpha - 1(XI) collagen chain; Export all polypeptide sequences in the PEAKS software, and set sorting and screening conditions in the excel table: Feature value ≥ 2 the number of times this fragment was detected; - 10lgP ≥ 30 credibility; Intensity ≥ 1×10 7 response intensity; ppm absolute value ≤ 1 deviation amount; In ML 2-1 and ML 2-3 7 and 6 potential bioactive peptide segments were respectively screened out, among which F1 - F7 are from ML 2-1 and F8 - F13 are from ML 2-3 .

[0020] Beneficial effects: Compared with the prior art, the present invention extracts and identifies polypeptides with multi - target neuroprotective effects from elk antlers, significantly overcoming many disadvantages of the prior art. The prior art mostly uses crude extraction methods (such as direct water extraction or alcohol extraction) to obtain elk antler extracts, and the resulting products have low purity of bioactive polypeptides, complex components, and it is difficult to determine specific bioactive components and their mechanisms of action, which limits further development and utilization. The present invention significantly improves the enrichment degree and therapeutic effect of bioactive polypeptides in elk antlers through optimizing extraction and purification processes and precise structure identification. By using the nano LC - Orbitrap - MS technology combined with high - performance liquid chromatography, the present invention significantly improves the extraction efficiency and purity of polypeptides, reduces the presence of impurities and non - target proteins compared with traditional methods, and improves the quality and stability of polypeptides. In addition, in the present invention, through membrane separation technology combined with Sep - Pak C18 column chromatography purification, elk antler extract (ML2) with a molecular weight less than 10 kDa was successfully separated, and 13 bioactive polypeptides with clear sequences were further screened out.

[0021] Experimental data show that in a mouse model of Alzheimer's disease, the crude extract of elk antler in the prior art has a relatively limited effect on improving learning and memory, with a latency of 38.7 ± 3.4 seconds and the number of platform crossings of 3.1 ± 0.4 times. In the high-dose group of ML2 extracted in the present invention, not only the latency was shortened to 23.4 ± 2.1 seconds, but also the number of platform crossings increased to 4.8 ± 0.5 times, significantly improving the learning and memory abilities of the mice. In addition, the ML2 extract also demonstrated strong anti-neuroinflammatory, antioxidant, and neurotrophic factor expression-promoting effects in in vivo and in vitro experiments, superior to the performance of the extract in the prior art (for example, significant reduction of TNF-α and IL-6). These data indicate that the present invention has higher extraction efficiency and stronger biological activity, providing a more powerful drug basis for the treatment of neurodegenerative diseases such as Alzheimer's disease. Brief Description of the Drawings

[0022] Figure 1 LC-MS spectra of ML2-1 (A) and ML2-3 (B); Figure 2 Results of the viability of Glu-damaged SH-SY5Y cells by F1~F13 ( Compared with the blank group, P < 0.001, * compared with the model group, P < 0.05, ** compared with the model group, P < 0.01, *** compared with the model group, P < 0.001.); Figure 3 Results of the viability of peroxide-damaged SH-SY5Y cells by F1~F13; Figure 4 Results of the in vitro mechanism study of the neuroactivity of elk antler bioactive peptide segments. Detailed Description of the Invention

[0023] The present invention will be further illustrated in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art fall within the scope defined by the appended claims of this application.

[0024] Example 1 A preparation method of elk antler characteristic peptide segments for treating Alzheimer's disease, comprising the following steps: (1) Extraction Take 100 g of crude elk antler powder, add 8 times the amount of ultrapure water, heat under reflux for 3 times, 8 h each time, filter, and combine the filtrates to obtain the total aqueous extract of elk antler (ML1). Dilute ML1 until the liquid is clear to obtain the elk antler extract sample.

[0025] (2) Membrane separation and molecular weight fractionation Prepare membrane modules with a molecular weight cut-off of 100k and 10 kDa, and a TNZ-1 nanofiltration separation device. After passing the elk antler extract sample through a nanofiltration membrane with a molecular weight of 100 kDa, add 2 L of pure water to rinse the residue, and collect filtrate a and retentate b respectively; filtrate a is passed through a ultrafiltration membrane with a molecular weight of 10 kDa again, and collect filtrate c. The obtained filtrate c above is ML4, retentate b is ML3, and filtrate a is ML2. After separation, ML2 (MW < 10 kDa), ML3 (10k < MW < 100 kDa) and ML4 (MW > 100 kDa) are obtained. Concentrate the obtained ML1 - ML4 under reduced pressure, freeze-dry, and store at -80 °C.

[0026] (3)Purification Further purify the ML2 fraction by Sep-Pak column chromatography. Take 0.05 g of the ML2 fraction, add 5 ml of pure water, and prepare a 10 mg / ml sample solution. Prepare four Waters Sep-Pak Vac 6cc C18 small columns (specification: 100 mg) for the following steps: activate with 3 ml of acetonitrile; balance with 3 ml of 0.1% TFA; load the sample (1 ml of ML2 sample solution) and wait for it to flow out naturally; wash with 6 ml of 0.1% TFA. Subsequently, elute with aqueous solutions containing 0.2% trifluoroacetic acid and 5% acetonitrile, 0.2% trifluoroacetic acid and 20% acetonitrile, 0.2% trifluoroacetic acid and 50% acetonitrile, and 0.2% trifluoroacetic acid and 80% acetonitrile respectively, 0.8 mL each time, and elute twice. Then collect the eluates, and after collecting the components, perform centrifugal concentration to obtain four components, ML2-1 to ML2-4.

[0027] (4)Structure identification (4.1)Mass spectrometry analysis Sample treatment: Re-dissolve samples ML2-1 and ML2-3 with 1% TFA. Dissolve 20 μg of protein in 200 μL of 1% TFA and vortex for 15 min. Desalt and remove impurities from the ML2-1 and ML2-3 component solutions using ZipTip. First, activate ZipTip C 18 with 80% acetonitrile (0.1% formic acid), 2 - 3 times the volume, pipette 15 - 20 times; then balance with 1 - 2 times the volume of 0.1% formic acid water, pipette 15 - 20 times; after balancing, pipette the enzymatically digested sample with ZipTip C18 15 - 20 times; rinse with 1 - 2 times the volume of 0.1% formic acid water about 10 times; first pipette with 50% acetonitrile (0.1% formic acid) 15 - 20 times for sample collection, then pipette with 80% acetonitrile (0.1% formic acid) 15 - 20 times for sample collection; combine the solutions eluted with 50% acetonitrile (0.1% formic acid) and 80% acetonitrile (0.1% formic acid), centrifuge and concentrate for standby.

[0028] The extracted polypeptides were structurally identified using nanoLC-Orbitrap-MS (nano liquid chromatography - Orbitrap mass spectrometry). The chromatographic column was a Reprosil C18 AQ column (150 mm × 75 μm, 5 μm); the injection volume was 2 μL, and the volumetric flow rate was 400 nL / min; mobile phase A was acetonitrile - formic acid - water (2:0.2:98), and mobile phase B was acetonitrile - formic acid - water (80:0.2:20), with a linear gradient elution of 2% - 30% B for 120 min. The spray voltage was 2.5 kV; the temperature of the ion transfer capillary was 200 °C; the full scan range of the mass spectrometry in the first stage was m / z 300 - 2000; the separation width was 3. The tandem mass spectrometry analysis obtained the total ion chromatogram (TIC), and through collision-induced dissociation (CID), a series of second-stage tandem mass spectra were generated. The tandem mass spectrometry data were analyzed by database searching using PEAKS 8.5 software, and the self-built deer collagen database (Home made Deer Collagen) was selected; the retrieval parameters were set as follows: the precursor ion error was 1.0×10-5, and the product ion error was m / z 1; post-translational modifications: carbamidomethylation of cysteine (+57.2), oxidation of methionine (+15.99), hydroxylation of proline (+15.99), deamidation of aspartic acid and glutamine (+0.98); digestion method: trypsin digestion; allowing 2 miscleavage sites, and the false discovery rate (FDR) ≤ 1%; other parameters were default. The scores obtained under the above retrieval conditions were considered to have significant significance (P < 0.05) as valid identification results. The mass spectrometry diagram is as Figure 1 .

[0029] The Nano LC-IT-Orbitrap mass spectrometry and fragment ion spectra were imported into the PEAKS software and analyzed and identified according to the following parameters. Search parameter settings: mass deviation of precursor ion: 10.0 ppm; mass deviation of fragment ion: 0.02 Da; fixed modification: carbamidomethylation, 57.02 Da; variable modifications: deamidation (NQ), 0.98 Da; oxidation (M), 15.99 Da; Phosphorylation (STY), 79.97 Da; maximum number of post-translational modifications per polypeptide: 3. Result export parameter settings: de novo score (ALC%) threshold, 80; protein - 10lgP > 20; FDR (PSMs), 0.20%. The PEAKS result analysis showed that 303 peptide segments with molecular weights ranging from 700 to 2500 Da were identified in ML2-1. They were derived from four proteins: alpha-1(I) collagen chain subtype X1 (Odocoileus virginianus texanus); alpha-2(I) collagen chain (Odocoileus virginianus texanus); alpha-1(II) collagen chain subtype X1 (Odocoileus virginianus texanus); alpha-1(VII) collagen chain (Odocoileus virginianus texanus). A total of 330 peptide segments with molecular weights ranging from 600 to 3500 Da were identified in ML2-3. They were derived from four proteins: alpha-2(I) collagen chain (Odocoileus virginianus texanus); alpha-1(I) collagen chain subtype X1 (Odocoileus virginianus texanus); alpha-1(II) collagen chain subtype X1 (Odocoileus virginianus texanus); alpha-1(XI) collagen chain (Odocoileus virginianus texanus).

[0030] All polypeptide sequences were exported in the PEAKS software, and sorting and filtering conditions were set in the excel table: Feature value ≥ 2 (the number of times the fragment was detected); -10lgP ≥ 30 (confidence level); Intensity ≥ 1 × 107 (response intensity); absolute value of ppm ≤ 1 (deviation amount). In ML 2-1 and ML 2-3 7 and 6 potential bioactive peptide segments were screened out respectively. The sequences are shown in Table 1, where F1 - F7 are from ML 2-1 and F8 - F13 are from ML2-3 。

[0031] Table 1 Amino acid sequences of 13 potential peptide segments Number Amino acid sequence F1 PAGERGSPGPAGPK F2 PSGASGPAGPRGPPG F3 ASGPAGPRGPPGSAG F4 GPSGPQGPSGPPGPKGN F5 PQGPSGPPGPKGN F6 SPGPAGPKGSPGEA F7 GPAGPKGSPGEAGRPG F8 PGPAGPAGPRGEVGLPGLS F9 GYDLSFLPQPPQEKAHDGGRYY F10 GHNGLQGLPGLAGHH F11 GGYDFGFDGDFYRA F12 GGGYDFGFDGDFYRA F13 FGFDGDFYR (4.2)Polypeptide biosynthesis and structure verification The identified polypeptides were synthesized and verified by solid-phase synthesis. The synthesis steps include: stepwise amino acid coupling on trityl resin, using Fmoc (9-fluorenylmethoxycarbonyl) as the amino protecting group, and the synthesis process was carried out in anhydrous dichloromethane (DCM). After each coupling step, unreacted reagents were removed by washing with tetrahydrofuran (THF). Finally, the Fmoc protecting group was removed by trifluoroacetic acid (TFA) to release the target polypeptide. The purity and structure of the synthetic product were verified by HPLC and mass spectrometry.

[0032] The peak map of the crude peptide was analyzed using a 5% - 95% acetonitrile system for 35 min. According to the detection results, the parameters of the high-performance liquid semi-preparative instrument were set to fully separate the target elution peak from other peaks, and the eluate in the determined peak region was collected. After preliminary purification, the purity of the obtained polypeptides was above 85.0%, and the mass spectrometry detection results were correct.

[0033] Example 2 Biological activity of polypeptides 1. In vitro activity verification To evaluate the activity of 13 synthetic polypeptides, the present invention used SH-SY5Y cells for in vitro activity evaluation, adopting a glutamate nerve injury model, and the results were as Figure 2 , and a nerve peroxidative injury model, and the results were as Figure 3 . Using Glu (3 mM) and H 2 O 2 (0.5 mM) for intervention to establish cell glutamate injury and peroxidative injury models, and at the same time, different concentrations of synthetic peptides F1 - F13 (100, 30, 10, 3, 1 μg / ml) were given for intervention and administration. After 24 h, the cell viability was detected by CCK8. In the Glu injury model, all peptide segments showed obvious anti-neuroexcitatory injury effects, especially F8, F9, and F11 showed a dose-dependent relationship. In the nerve peroxidative injury cell model treated with H 2 O 2 , 9 peptide segments also showed good antioxidant injury effects, among which the effects of F6 and F7 were the most significant.

[0034] 2. In vitro mechanism study 2.1 Effects of elk antler active peptides on neurotrophic factors and neurotransmitters in in vitro cell models Take out the C6 cells in the logarithmic growth phase from the incubator, digest them with trypsin, and conduct cell counting. Adjust the cell concentration according to the counting results to obtain a cell suspension with a density of approximately 3×105 cells / ml. Subsequently, inoculate the cells into a 96-well plate and place it in the incubator for culture. Administer the drug 24 h after culturing. The experimental groups are set as follows: Control: blank control group, F6: F6 peptide (100 μg / ml), F7: F7 peptide (100 μg / ml), F8: F8 peptide (100 μg / ml), F9: F9 peptide (100 μg / ml), with 6 replicates in each group. After the C6 cells transferred to the 96-well plate adhere and grow, add 100 μg / ml of F6, F7, F8, and F9 polypeptides to each replicate well. Take the supernatant of each group 24 h after drug intervention. The experimental grouping, drug administration concentration, and method for SH-SY5Y cells are the same as above. Detect the levels of neurotrophic factors and neurotransmitters in the culture medium supernatant by ELISA.

[0035] 2.2 Regulation of the PI3K / AKT / Caspase 3 pathway by antler polypeptides in vitro cell models The experimental groups are set as follows: Control: blank control group, Model group: Glu (3 mM), F6: F6 peptide (100 μg / ml) + Glu (3 mM), F7: F7 peptide (100 μg / ml) + Glu (3 mM), F8: F8 peptide (100 μg / ml) + Glu (3 mM), F9: F9 peptide (100 μg / ml) + Glu (3 mM), with 3 replicates in each group. Administer the drug for 24 h.

[0036] Extract the total protein using RIPA protein lysate, measure the total protein concentration according to the method of the BCA kit, and denature it at 100 °C for 5 min. Prepare a 12% polyacrylamide gel according to the experimental steps of the Western Blotting method. After loading the samples, perform electrophoresis at 60 V for 0.5 h, 95 V for 1 h, transfer the membrane at 300 mA for 1 h, block it with 5% BSA for 2 h, incubate with primary antibodies (PI3K, AKT, Caspase 3, antibody ratio 1:4000; GAPDH Rabbit mAb, antibody ratio 1:10000) overnight at 4 °C, incubate with secondary antibody for 1 h (HRP-anti-rabbit IgG, antibody ratio 1:5000), and expose and collect images using an imaging system. Calculate the expression levels of PI3K, AKT, and Caspase 3 proteins in the cells after processing the images.

[0037] 2.3 Regulation of Elk Antler Active Peptide on NF-κb P65 / TLR4 Pathway in In Vitro Cell Model The maturely cultured microglial cells were respectively set as Control group, LPS / ATP stimulation group, LPS / ATP+F6 group, LPS / ATP+F7 group, LPS / ATP+F8 group, and LPS / ATP+F9 group. No treatment measures were taken in the control group for blank control; in the LPS / ATP stimulation group, the cells were first stimulated with 1.0 μg / mL LPS for 4 h and then with 20.0 μM ATP for 1 h; in the LPS / ATP+F6 group, 100 μg / ml F6 active polypeptide was added on the basis of the above stimulation; in the LPS / ATP+F7 group, 100 μg / ml F7 active polypeptide was added on the basis of the above stimulation; in the LPS / ATP+F8 group, 100 μg / ml F8 active polypeptide was added on the basis of the above stimulation; in the LPS / ATP+F9 group, 100 μg / ml F9 active polypeptide was added on the basis of the above stimulation. The WB method was used to detect the expression levels of NF-κb P65 / TLR4 proteins in the cells (the method was the same as that in 2.2 before).

[0038] 2.4 Regulation of Elk Antler Active Peptide on Nrf2 / HO-1 and Caspase-3 / Bax / Bcl2 Pathways in In Vitro Cell Model HT22 cells were divided into 6 groups: Control: blank control group, Model group: H 2 O 2 (0.5 mM), F6: F6 peptide(100 μg / ml)+ H 2 O 2 (0.5 mM), F7: F7 peptide(100 μg / ml)+ H 2 O 2 (0.5 mM), F8: F8peptide(100 μg / ml)+ H 2 O 2 (0.5 mM), F9: F9 peptide(100 μg / ml)+ H 2 O 2 (0.5 mM). Each group was set with 3 replicates, and the cells were intervened with drugs for 24 h. The WB method was used to detect the expression levels of Nrf2, HO-1, Caspase-3, Bax, and Bcl2 proteins in the cells.

[0039] The polypeptide of the present invention exerts neuroprotective effects through multi-target regulation, mainly including antioxidant, anti-inflammatory, inhibiting cell apoptosis, and promoting the expression of neurotrophic factors. The results are as Figure 4 , Figure 4(A, B) show the effects of antler polypeptide of Cervus nippon Temminck on the expression levels of neurotrophic factors BDNF and NGF in C6 cells; (C, D) show the effects of antler polypeptide of Cervus nippon Temminck on the expression levels of TH and COMT in SH-SY5Y cells; (E, F, G, H) show the WB band and gray value analysis of apoptosis pathway-related proteins in Glu-damaged SH-SY5Y cells treated with antler polypeptide of Cervus nippon Temminck; (I) shows the effect of different concentrations of ATP on the viability of microglial cell line BV2. When the ATP concentration reaches 80 μM, the cell viability decreases to 50% of that of the normal group; (J) shows the effect of antler polypeptide of Cervus nippon Temminck on the expression level of TNF-α in LPS- and ATP-induced inflammatory microglial cells; (K, L, M) show the effects of antler polypeptide of Cervus nippon Temminck on the protein levels of TLR4 and NF-κB in LPS- and ATP-induced inflammatory microglial cells; The above results indicate that the characteristic peptide segments F6, 7, 8, 9 of antler of Cervus nippon Temminck provided by the present invention can significantly reduce the protein level of NF-κB, and F7 can significantly reduce the protein level of TLR4; (N, O, P, Q, R) show the effects of antler polypeptide of Cervus nippon Temminck on the expression levels of proteins related to oxidative stress and apoptosis pathways in H 2 O 2 T22 cells damaged by

[0040] The polypeptide prepared by the present invention is applicable to the treatment of Alzheimer's disease and other neurodegenerative diseases. Especially in the case of accompanied by neuroinflammation and oxidative stress, it shows obvious therapeutic effects. Based on its characteristics of low toxicity and multi-target action, it can be used as an independent drug or as part of combination therapy for AD patients.

[0041] The present invention develops a polypeptide with multi-target neuroprotective effects through innovative extraction, purification and identification techniques, providing a new, safe and effective treatment option for the treatment of Alzheimer's disease.

[0042] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A characteristic peptide segment of elk antler for treating Alzheimer's disease, characterized in that: The characteristic peptide segments are as follows F1~F13: 。 2. The method for preparing the characteristic peptide segment of elk antler for treating Alzheimer's disease according to claim 1, characterized in that: The steps include: (1) Extraction Take the elk antlers, grind them, add water, heat them in a water bath for extraction, combine the extracts, centrifuge, and collect the supernatant; (2) Membrane separation and molecular weight fractionation The extract was separated by molecular weight cut-off membranes of 10 kDa and 100 kDa in turn to obtain an extract fraction with a molecular weight less than 10 kDa; (3) Purification Take the extraction part of step (2), add pure water to prepare a sample solution; prepare four Waters Sep-Pak Vac6cc C18 columns and perform the following steps: first activate with acetonitrile; then balance with trifluoroacetic acid; load the sample and wait for natural outflow; then rinse with trifluoroacetic acid, and then elute with an aqueous solution containing 0.2% trifluoroacetic acid and 5% acetonitrile, an aqueous solution containing 0.2% trifluoroacetic acid and 20% acetonitrile, an aqueous solution containing 0.2% trifluoroacetic acid and 50% acetonitrile, and an aqueous solution containing 0.2% trifluoroacetic acid and 80% acetonitrile, respectively, collect the eluates, and obtain four components ML2-1 to ML2-4, and concentrate the collected components by centrifugation; (4) Thirteen peptides F1~F13 were identified from the ML2-1 and ML2-3 components.

3. The method for preparing the characteristic peptide segment of elk antler for treating Alzheimer's disease according to claim 2, characterized in that: The steps include: (1) Extraction Take the crude powder of elk antlers, add 8-20 times of ultrapure water, heat and reflux to extract 2-3 times, each time for 6-8 hours, filter, and combine the filtrate to obtain ML1 of total water extract of elk antlers; (2) Membrane separation and molecular weight fractionation ML1 was diluted to a clear liquid, and membrane components with molecular weight cutoffs of 100k and 10k Da and TNZ-1 nanofiltration separation equipment were prepared. After the elk antler total water extract ML1 sample was passed through a nanofiltration membrane with a molecular weight of 100k Da, pure water was added to rinse the residue, and the filtrate a and the retentate b were collected respectively; the filtrate a was passed through an ultrafiltration membrane with a molecular weight of 10k Da again, and the filtrate c was collected; the filtrate c obtained above was ML4, the retentate b was ML3, and the filtrate a was ML2; after the membrane separation with a molecular weight cutoff of 10k Da was completed, ML2 (MW <10k Da), ML3 (10k < MW < 100k Da) and ML4 (MW > 100k Da) were obtained; the obtained ML1~ML4 were concentrated under reduced pressure and freeze-dried; (3) Purification The ML2 part was further purified by Sep-Pak column chromatography: the ML2 part was taken, pure water was added, and a sample solution was prepared; four Waters Sep-Pak Vac 6cc C18 columns were prepared and the following steps were performed: first activated with acetonitrile; then balanced with trifluoroacetic acid; the ML2 part sample solution was loaded and waited for natural outflow; then rinsed with trifluoroacetic acid, and then eluted with an aqueous solution containing 0.2% trifluoroacetic acid and 5% acetonitrile, an aqueous solution containing 0.2% trifluoroacetic acid and 20% acetonitrile, an aqueous solution containing 0.2% trifluoroacetic acid and 50% acetonitrile, and an aqueous solution containing 0.2% trifluoroacetic acid and 80% acetonitrile, respectively, and the eluents were collected to obtain four components ML2-1 to ML2-4, and the collected components were centrifuged and concentrated; (4) Thirteen polypeptides F1 to F13 were separated and identified from the ML2-1 and ML2-3 fractions purified in step (3).

4. The method for preparing the characteristic peptide segment of elk antler for treating Alzheimer's disease according to claim 2 or 3, characterized in that: Step (4) separation and structural identification of peptides in components ML2-1 and ML2-3 were performed using nanoliter liquid chromatography-orbitrap mass spectrometry. The chromatographic column was a Reprosil C18 AQ column with specifications of 150 mm×75 μm, 5 μm. The injection volume was 2 μL and the volume flow rate was 400 nL / min. The mobile phase A was acetonitrile-formic acid-water with a volume ratio of 2:0.2:98, and the mobile phase B was acetonitrile-formic acid-water with a volume ratio of 80:0.2:20, with a linear gradient elution of 2% to 30% B for 120 min. The spray voltage was 2.5 kV. The ion transfer capillary temperature was 200 °C. The first-level full scan range of the mass spectrometer was m / z 300 to 2000. The separation width was 3. Tandem mass spectrometry analysis obtained a total ion chromatogram, and a series of second-level tandem mass spectra were generated by collision-induced dissociation. The tandem mass spectrometry data were analyzed using PEAKS The library search analysis was performed using 8.5 software, and the self-built Cervidae collagen database was selected; the search parameters were set as follows: precursor ion error 1.0×10-5, product ion error m / z 1; post-translational modifications: carbamoyl methylation of cysteine ​​(+57.2), oxidation of methionine (+15.99), hydroxylation of proline (+15.99), deamidation of aspartic acid and glutamine (+0.98); enzyme digestion method: trypsin digestion; 2 sites were allowed to be miscut, and the false positive rate was ≤1%; other parameters were default. The scores obtained under the above search conditions were significant, and P < 0.05 was considered a valid identification result; The Nano LC-IT-Orbitrap mass spectra and fragment ion maps were imported into PEAKS software and analyzed and identified according to the following parameters. Search parameter settings: mass deviation of parent ions: 10.0 ppm; mass deviation of fragment ions: 0.02 Da; fixed modification: carbamidomethylation, 57.02 Da; variable modification: deamidation (NQ), 0.98 Da; oxidation (M), 15.99 Da; Phosphorylation (STY), 79.97 Da; maximum number of post-translational modifications contained in each peptide: The result export parameter settings were as follows: de novo score (ALC%) threshold, 80; protein-10lgP > 20; FDR (PSMs), 0.20%; PEAKS analysis showed that 303 peptides were identified in ML2-1, with molecular weights ranging from 700 to 2500 Da; they originated from four proteins: α-1(I) collagen chain subtype X1; α-2(I) collagen chain; α-1(II) collagen chain subtype X1; α-1(VII) collagen chain; 330 peptides were identified in ML2-3, with molecular weights ranging from 600 to 3500 Da, they originated from four proteins: α-2(I) collagen chain; α-1(I) collagen chain subtype X1; α-1(II) collagen chain subtype X1; α-1(XI) collagen chain; All peptide sequences were exported in PEAKS software, and sorting and screening conditions were set in the Excel table: Feature value ≥ 2, the number of times the fragment was detected; -10lgP ≥ 30, credibility; Intensity ≥ 1×10 7 Response intensity; ppm absolute value ≤ 1 deviation; in ML 2-1 and ML 2-3 7 and 6 potential active peptides were screened out, among which F1~F7 came from ML 2-1 , F8~F13 come from ML 2-3 .

5. Use of the characteristic peptide fragment of elk antler according to claim 1 in the preparation of functional food or medicine with neuroprotective effect.

6. Use of the characteristic peptide fragment of elk antler according to claim 1 in the preparation of antioxidant or anti-inflammatory functional foods or medicines.

7. Use of the characteristic peptide segment of elk antler according to claim 1 in the preparation of anti-Alzheimer's disease drugs.

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