Pharmaceutical composition containing polypeptide and application of pharmaceutical composition in preparation of medicine for preventing and treating Parkinson's disease

Abstract

The invention provides a pharmaceutical composition containing polypeptide and application of the pharmaceutical composition to preparation of a medicine for preventing and treating Parkinson's disease, and belongs to the technical field of polypeptide and biology. The amino acid sequence of the polypeptide is PCVLYDLREKM, the damage of 6-OHDA induced BZ555 nematode dopaminergic neurons can be obviously relieved, mouse dyskinesia caused by a Parkinson's disease model can be obviously improved, the spontaneous standing frequency can be improved, the movement speed in an open field can be increased, and meanwhile the IL-1beta and IL-18 levels caused by the mouse Parkinson's disease model can be reduced; the polypeptide provided by the invention can significantly reduce MAO-B level while significantly increasing DA level in nigra of brain tissues of rats with Parkinson's disease, which prompts that the polypeptide provided by the invention has a significant therapeutic effect on Parkinson's disease, and has a wide market prospect in many fields such as health food and medicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polypeptides and biotechnology, and particularly relates to a pharmaceutical composition comprising a polypeptide and application thereof in preparing a medicine for preventing and treating Parkinson's disease. BACKGROUND

[0002] Parkinson's Disease (PD) is a common central nervous system degenerative disease with complex pathogenesis, which is closely related to the progressive degeneration and necrosis of dopaminergic neurons in the substantia nigra of the midbrain, insufficient secretion of dopamine neurotransmitters, and other pathological characteristics such as abnormal aggregation of alpha-synuclein to form Lewy bodies. Clinically, patients often show symptoms such as static tremor, muscle rigidity, bradykinesia, and postural abnormalities, which seriously affect the quality of life, and often lead to death due to complications in the late stage. With the intensification of global population aging, the incidence of Parkinson's disease is increasing year by year, and it has become the second most common neurodegenerative disease after Alzheimer's disease, causing heavy medical burden and economic pressure to society and families. At present, the treatment of Parkinson's disease in the clinic is still mainly symptomatic treatment, and the core goal is to supplement dopamine neurotransmitters or regulate the activity of dopaminergic neural pathways. There is no specific drug that can delay disease progression, prevent neuronal degeneration, or achieve a cure.

[0003] The existing clinical drugs mainly include dopamine replacement therapy drugs (such as levodopa), dopamine receptor agonists (such as pramipexole), and monoamine oxidase B inhibitors (such as selegiline). Among them, levodopa is the "gold standard" for treating Parkinson's disease, which can quickly relieve motor symptoms, but long-term use can easily cause problems such as efficacy decay, motor complications (such as dyskinesia, on-off phenomenon), and inability to improve non-motor symptoms (such as cognitive impairment, depression, sleep disorders, etc.); dopamine receptor agonists can reduce the risk of motor complications, but have adverse reactions such as hallucinations and impulse control disorders, especially poor tolerance in elderly patients; other adjunctive therapy drugs also have limitations such as limited efficacy, obvious adverse reactions, or narrow application population. In addition, recent studies have found that the occurrence and development of Parkinson's disease are also related to oxidative stress, inflammatory response, mitochondrial dysfunction, and cell apoptosis. Single-target treatment drugs are difficult to comprehensively cover the complex pathological aspects of the disease, resulting in poor treatment effect. Therefore, developing new anti-Parkinson's disease drugs with multi-target regulation, accurate efficacy, high safety, and the ability to delay disease progression has become a research hotspot and urgent need in the field of biomedicine.

[0004] The living environment of marine organisms is unique and contains rich bioactive substances. Marine polypeptides have great potential in the field of drug research and development due to their diverse structures, extensive biological activities, low toxicity, and good biocompatibility. Studies have shown that some marine polypeptides have biological activities such as antioxidant, anti-inflammatory, neuroprotective, and inhibition of abnormal protein aggregation, which can regulate the physiological functions of the nervous system through various pathways and provide a new idea for the treatment of neurodegenerative diseases.

[0005] However, there is no related report on the application of natural polypeptides extracted from marine organisms, especially sea cucumbers, in the prevention and treatment of Parkinson's disease. There is also a lack of drug compositions constructed for such polypeptides that have synergistic effects. Based on the shortcomings of existing Parkinson's disease treatment drugs and the unique advantages of marine active polypeptides, developing a drug composition with sea cucumber natural polypeptides as the core component for preventing and treating Parkinson's disease is expected to break through the existing treatment bottleneck, provide patients with safer and more effective treatment options, and has important clinical value and social significance. SUMMARY

[0006] In order to understand the problems in the prior art, the present application provides a drug composition containing polypeptides and its application in the preparation of drugs for preventing and treating Parkinson's disease. Specifically, polypeptides with the ability to prevent or treat Parkinson's symptoms are extracted from sea cucumbers, and then used to prepare corresponding drug compositions, which can provide important support for the development of high-quality protein functional products and food-derived polypeptides from sea cucumbers, and expand the application of sea cucumbers in the fields of drugs, health products and food.

[0007] To this end, the present application adopts the following technical solutions: The present application provides a polypeptide for preventing or treating Parkinson's disease, characterized in that the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1. Further, the amino acid sequence is PCVLYDLREKM, i.e. Pro-Cys-Val-Leu-Tyr-Asp-Leu-Arg-Glu-Lys-Met.

[0008] Further, the theoretical isoelectric point (pI) of the polypeptide is about 6.8, and the solubility in the in vivo environment is good.

[0009] Further, the polypeptide is derived from sea cucumber or artificially synthesized. The present application also provides a preparation method of a polypeptide for preventing or treating Parkinson's disease, wherein the polypeptide is extracted from sea cucumber.

[0010] The present application also provides a drug composition for preventing or treating Parkinson's disease.

[0011] Further, the pharmaceutical composition contains the polypeptide for preventing or treating Parkinson's disease.

[0012] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0013] Further, the pharmaceutically acceptable excipient comprises at least one of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a flavoring agent, a solvent, a solubilizer, a co-solvent, an emulsifier, an absorption enhancer, a surfactant, an antioxidant, an excipient, a stabilizer, a flavoring agent, a sustained release agent, a metal complexing agent, an inert gas, a preservative, a filler, a local analgesic, a pH adjusting agent, and an isotonic or tonicity adjusting agent.

[0014] Further, the diluent includes, but is not limited to, at least one of starches, sugars, celluloses, inorganic salts. The wetting agent includes, but is not limited to, at least one of water, ethanol, propanol. The binder includes, but is not limited to, at least one of starch paste, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol. The disintegrant includes, but is not limited to, at least one of microcrystalline cellulose, cross-linked polyvinylpyrrolidone, croscarmellose sodium starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, cross-linked povidone, surfactants (such as sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate, etc.), effervescent disintegrants. The lubricant includes, but is not limited to, at least one of talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, microfine silica, calcium carbonate, sodium bicarbonate, polyethylene glycol. The color, flavor, and odor modifier includes, but is not limited to, at least one of pigments, flavors, sweeteners, gum base, odor correctors. The solvent includes, but is not limited to, at least one of water, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, ethyl acetate, vegetable oil (such as soybean oil, castor oil, peanut oil, blended oil, etc.). The solubilizer includes, but is not limited to, at least one of Tween, Brij, polyoxyethylene fatty alcohol ether, soap, sulfate, sulfonate. The co-solvent includes, but is not limited to, at least one of organic acids and their salts, amide and amine compounds, inorganic salts, polyethylene glycol, povidone, glycerol. The emulsifier includes, but is not limited to, at least one of Span, Tween, Brij, benzyl zein, glycerol fatty acid ester, higher fatty acid salt, sulfate, sulfonate, gum arabic, tragacanth gum, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silicon dioxide, bentonite. The suspending agent includes, but is not limited to, at least one of glycerol, sugar syrup, gum arabic, tragacanth gum, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol, thixotropic gel. The antioxidant includes, but is not limited to, at least one of sulfite, pyrosulfite, bisulfite, ascorbic acid, gallic acid and its esters. The metal complexing agent includes, but is not limited to, at least one of disodium ethylenediaminetetraacetate, polycarboxylic acid compound; the inert gas includes, but is not limited to, at least one of nitrogen, carbon dioxide. The preservative includes, but is not limited to, at least one of nipagin, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, volatile oils. The filler includes at least one of starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose. The flavoring agent includes at least one of aspartame, sucralose, essence, steviol, acesulfame potassium, citric acid, or sodium saccharin. The local analgesic agent includes, but is not limited to, at least one of benzyl alcohol, trichlorobenzyl alcohol, lidocaine, procaine.The pH adjuster includes, but is not limited to, at least one of the following: hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, and citrate. The isotonic or isotropic adjuster includes, but is not limited to, at least one of the following: glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

[0015] Furthermore, the dosage form of the pharmaceutical composition is selected from oral formulations, injectable formulations, transdermal absorption formulations, or mucosal delivery formulations.

[0016] Furthermore, the dosage form of the oral preparation is selected from any one of pills, powders, tablets, ointments, capsules, granules, oral liquids, and decoctions.

[0017] Furthermore, the pharmaceutical composition may also contain combination drugs.

[0018] Furthermore, the combination drugs include medications for treating or preventing Parkinson's-related diseases.

[0019] The present invention also provides the use of the described polypeptide or pharmaceutical composition in the preparation of medicaments for the prevention or treatment of Parkinson's disease.

[0020] The present invention also provides food, health products or medicines containing the aforementioned polypeptides or pharmaceutical compositions.

[0021] The present invention also provides a product for the prevention or treatment of Parkinson's disease, wherein the active ingredient of the product includes the aforementioned polypeptide.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention extracts polypeptides from sea cucumbers using a bio-enzymatic hydrolysis method, which have the effect of preventing or treating Parkinson's disease symptoms. The amino acid sequence of the polypeptide is PCVLYDLREKM. It can significantly reduce 6-OHDA-induced damage to dopaminergic neurons in BZ555 nematodes. At the same time, it can significantly improve motor function abnormalities in mice induced by Parkinson's disease, improve the number of spontaneous standing up, increase the speed of movement in the open field, and reduce the levels of IL-1β and IL-18 induced by Parkinson's disease in mice. While significantly increasing the level of DA in the substantia nigra of the brain tissue of Parkinson's disease rats, it can also significantly reduce the level of MAO-B, suggesting that the polypeptides of this invention have significant therapeutic effects on Parkinson's disease and have broad market prospects in many fields such as health foods and medicines. Attached Figure Description

[0023] Figure 1 This is a molecular structure diagram of the polypeptide of the present invention; Figure 2 This is a three-dimensional structural diagram of the polypeptide of the present invention; Figure 3This is the HPLC detection chromatogram of the polypeptide of the present invention. Detailed Implementation

[0024] To illustrate the possible application scenarios, technical principles, implementable specific solutions, and achievable objectives and effects of this application in detail, the following description, in conjunction with the listed specific embodiments and accompanying drawings, provides a detailed explanation. The embodiments described herein are merely illustrative of the technical solutions of this application and are therefore intended to limit the scope of protection of this application.

[0025] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0026] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0027] Unless otherwise specified, the use of terms such as “comprising,” “including,” “having,” or other similar expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.

[0028] Similar to the understanding in the Examination Guidelines, in this application, expressions such as "greater than," "less than," and "exceeding" are understood to exclude the stated number; expressions such as "above," "below," and "within" are understood to include the stated number. Furthermore, in the description of the embodiments in this application, "multiple" means two or more (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups" and "multiple times," unless otherwise explicitly specified.

[0029] The term "Parkinsonian syndrome" defines a class of progressive neurodegenerative diseases characterized by resting tremor and / or bradykinesia, associated with rigidity, postural instability, loss of postural reflexes, flexed posture, and / or freezing phenomena (when the feet are briefly "glued" to the ground). Examples of Parkinsonian syndrome symptoms include Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, diffuse Lewy body disease, Parkinson-dementia, X-linked dystonia-Parkinsonian syndrome, and secondary Parkinsonian syndrome. Parkinson's disease is the most common form of Parkinsonian syndrome. Parkinson's disease causes motor and nonmotor manifestations and is characterized by widespread degeneration of dopaminergic neurons in the substantia nigra-striatal system. The motor manifestations of Parkinson's disease can be attributed to the degeneration of dopaminergic neurons in the substantia nigra. These include tremor, decreased motor function, postural instability, gait abnormalities, and dysphagia.

[0030] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods. The sea cucumbers used in this invention were purchased from the Changbai Road Aquatic Products Market in Changchun City, Jilin Province (address: No. 5, Changbai Road, Kuancheng District, Changchun City, Jilin Province).

[0031] Example 1: Extraction and preparation of polypeptides A method for preparing a polypeptide for the prevention or treatment of Parkinson's disease includes the following steps: (1) Raw material pretreatment Select fresh sea cucumbers (150-200g / each), rinse them with clean water to remove mud and impurities, cut open the body wall with sterile scissors, remove the internal organs and digestive tract, and retain the body wall tissue; rinse the body wall tissue three times with pre-cooled deionized water, drain the water and cut it into small pieces of 1cm×1cm; place the small pieces in an ultra-low temperature freezer at -80℃ overnight, then transfer them to a freeze dryer to dry to constant weight, and obtain dried sea cucumber body wall products; put the dried products into a high-speed freeze grinder to grind them, pass them through a 100-mesh stainless steel sieve, collect the dried sea cucumber powder, place it in a desiccator and seal it for storage to prevent moisture absorption and deterioration.

[0032] (2) Enzymatic hydrolysis reaction Add Tris-HCl buffer solution at pH 7.2 to the dried sea cucumber powder at a material-to-liquid ratio of 1:25 (g / mL), and stir at room temperature for 30 min on a magnetic stirrer to obtain a sea cucumber homogenate. Add a compound enzyme (trypsin and papain mixed at a mass ratio of 2:1, with trypsin activity of 8000 U / g and papain activity of 6000 U / g) to the sea cucumber homogenate, with the enzyme addition amount being 3.0% of the mass of the dried sea cucumber powder. Transfer the above system to a constant temperature water bath shaker, set the enzymatic hydrolysis temperature to 55℃, the shaker speed to 180 r / min, and the enzymatic hydrolysis time to 5 h. After the enzymatic hydrolysis reaction is completed, place the reaction system in a 95℃ constant temperature water bath and heat for 20 min to inactivate the enzyme.

[0033] (3) Preparation of crude extract The enzymatic hydrolysis system was rapidly cooled to room temperature in an ice bath and then transferred to a high-speed refrigerated centrifuge. It was centrifuged at 10,000 rpm for 25 min, and the supernatant was collected. The centrifuged precipitate was reconstituted with an equal volume of pH 7.2 Tris-HCl buffer, and the two supernatants were combined by centrifugation to obtain a crude extract of sea cucumber polypeptides. The crude extract was filtered through a 0.22 μm organic filter membrane to remove suspended impurities and microorganisms, and a clear crude extract was obtained for later use.

[0034] (4) Purification and refining Ultrafiltration fractionation: The clarified crude extract is injected into the ultrafiltration device, and a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 2000 Da is selected. Ultrafiltration is carried out under the conditions of 0.1 MPa pressure and room temperature. The permeate is collected, and the retained large molecular weight proteins and impurities are discarded to obtain the ultrafiltration permeate. Preliminary purification by RP-HPLC: A Symmetry Shield™ RP C18 column (4.6 mm × 250 mm, 5 μm) was used with acetonitrile as mobile phase A and deionized water (containing 0.1% trifluoroacetic acid) as mobile phase B, and gradient elution was performed. The elution program was as follows: 0-10 min, mobile phase A volume fraction increased from 5% to 20%; 10-30 min, mobile phase A volume fraction increased from 20% to 45%; 30-40 min, mobile phase A volume fraction increased from 45% to 80%; flow rate 1.0 mL / min, detection wavelength 220 nm, column temperature 30 °C. 20 μL of the ultrafiltration permeate was loaded, and the eluted components were collected according to the chromatographic peaks. The peak components corresponding to the target peptide were preliminarily screened by HPLC chromatogram, and the components with the same retention time were combined. The acetonitrile was removed by concentration under reduced pressure to obtain the preliminarily purified peptide solution. RP-HPLC secondary purification: The preliminarily purified peptide solution was loaded again onto a C18 column. Elution conditions: gradient elution of mobile phase A (acetonitrile) and mobile phase B (deionized water containing 0.1% trifluoroacetic acid). From 0 to 20 min, the volume fraction of mobile phase A increased from 10% to 35%; from 20 to 35 min, the volume fraction of mobile phase A increased from 35% to 50%. Flow rate: 0.8 mL / min; detection wavelength: 220 nm; column temperature: 28 °C. The eluent corresponding to a single symmetrical peak was collected, concentrated under reduced pressure, and dialyzed against deionized water for 24 h, with the deionized water replaced every 6 h to remove trifluoroacetic acid and residual acetonitrile. Freeze-drying: The dialyzed peptide solution was placed in a freeze dryer (vacuum degree 5 Pa, condensation temperature -55℃) and dried to constant weight to obtain a white powder of pure peptide product, which was then sealed and stored in a -20℃ refrigerator for later use.

[0035] (5) Validation of peptide structure Molecular weight verification: Molecular weight was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Sample preparation: 1 mg of the pure peptide was dissolved in 0.1% trifluoroacetic acid solution and mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) at a volume ratio of 1:1. The mixture was spotted onto the target and allowed to air dry. Detection conditions: positive ion mode, accelerating voltage 20 kV, detection range 500-5000 Da. Results showed that the molecular weight corresponding to the molecular ion peak of the pure peptide was 1366.66 Da, consistent with the theoretical molecular weight (1366.64 Da). The structural diagram is shown below. Figure 1 As shown.

[0036] Amino acid sequencing: N-terminal sequencing was performed using the Edman degradation method with an automated protein sequencer (Applied Biosystems 491). 200 pmol of the purified peptide was sequenced. The sequencing steps were as follows: the peptide reacted with phenyl isothiocyanate (PITC) to generate a phenylaminothiocarbamate (PTC) derivative, which was then cyclized to obtain a thiazolinone aniline (ATZ) derivative containing the first N-terminal amino acid. This ATZ derivative was then converted into a stable phenylhydantoin (PTH) amino acid, which was separated and identified by high-performance liquid chromatography (HPLC). The above steps were repeated to sequentially determine the N-terminal amino acids from position 2 to 11. The sequencing results showed that the amino acid sequence of the peptide was Pro-Cys-Val-Leu-Tyr-Asp-Leu-Arg-Glu-Lys-Met, which is the target sequence PCVLYDLREKM.

[0037] Purity verification: The purity of the peptide was verified by RP-HPLC, and the detection conditions were the same as those for the secondary purification in step (4); the results are as follows. Figure 3 As shown, the chromatogram of the pure polypeptide exhibits a single symmetrical peak with a purity >98% and no other interfering peaks, indicating that the target polypeptide with high purity has been obtained.

[0038] Example 2 Artificial Synthesis of Peptides The polypeptide shown in SEQ ID NO: 1 (sequence: PCVLYDLREKM) was synthesized by Sangon Biotech (Shanghai) Co., Ltd. After mass spectrometry and high performance liquid chromatography analysis, the product was confirmed to be the target polypeptide compound. The polypeptide compound is a white powder with a purity of ≥98% and is stored at low temperature for later use.

[0039] Example 3: Protective effect of peptides against 6-OHDA-induced dopaminergic neuronal damage in BZ555 nematodes BZ555 nematodes (purchased from Caenorhabditis Genetics Center (CGC)) were inoculated onto NMG plates coated with Escherichia coli (purchased from Beijing Bio-Bio Biotechnology Co., Ltd., catalog number: bio-67405) and cultured in an incubator at 20°C. When the nematodes reached adulthood, synchronization treatment was performed. Plates with a large number of adults and some hatched nematode eggs were selected, and the nematodes were flushed off the culture medium with M9 buffer and transferred to centrifuge tubes. The plates were centrifuged at 3600 rpm, the supernatant was discarded, lysis buffer was added to the centrifuge tubes, and the plates were vortexed for 2 min until all nematodes were lysed. The plates were then aliquoted into 1.5 mL centrifuge tubes, and the nematode eggs were washed three times with M9 buffer.

[0040] Detection of dopaminergic neurons in BZ555 nematodes: The experiment included a blank group, a model group (6-OHDA group, purchased from MedChemExpress, HY-B1081A), a positive control group (2mM L-DOPA, purchased from MedChemExpress, HY-N0304R), and peptide groups (high, medium, and low concentrations: 50, 100, and 200 μg / mL). Synchronized L3-stage BZ555 nematodes were incubated for 1 h in a modeling solution containing 50 mM 6-OHDA and 10 mM ascorbic acid. The nematodes were washed three times with M9 buffer and then transferred to NMG plates of different treatment groups. After 48 h of culture, the nematodes were washed three times with M9 buffer and then transferred to 1% agarose pads on glass slides. A few drops of 100 mM levamisole were added to fix the nematodes. The nematodes were observed and photographed under a fluorescence microscope, and the fluorescence intensity of dopaminergic neurons was analyzed using ImageJ software. Each experiment was repeated three times. Data are expressed as mean ± SEM. One-way ANOVA was performed using Graphpad Prism. T-test was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant.

[0041] Table 1. Relative fluorescence intensity of the polypeptide of the present invention on dopaminergic neurons of nematodes (n=3)

[0042] Note: Compared with the blank group, # P<0.05, ## P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0043] The detection results are shown in Table 1. The relative fluorescence intensity of the model group was extremely significant compared with that of the blank group (P<0.01), indicating that the nematode model was successful. The polypeptide prepared by the present invention has a significant protective effect on the damage of dopaminergic neurons induced by 6-OHDA in BZ555 nematodes, and shows a positive correlation with the concentration of the polypeptide; the high-concentration polypeptide of 200 μg / mL can restore the viability of dopaminergic neurons to 80.64% at most, which is equivalent to the effect of the positive control group (P<0.01).

[0044] Example 4 Therapeutic effect of polypeptide on MPTP-induced mouse Parkinson's disease model Experimental animals: 48 male C57BL / 6 mice, weighing 18-22 g, purchased from Liaoning Changsheng Biotechnology Co., Ltd. Animal grade: SPF. Animal license number of the production unit: SCXK(Liao)2020-0001.

[0045] Animal laboratory: Laboratory Animal Room of Changchun University of Chinese Medicine. Experimental animal use license number: SYXK(Ji)2018-0014. Room temperature: 22±2°C, relative humidity 60-80%.

[0046] Drugs and kits: MPTP (Beyotime, ST1020); DMF (InvivoChem, V13644); Mouse IL-1β enzyme-linked immunosorbent assay kit (Thermo Fisher, BMS6002-2); Mouse IL-18 enzyme-linked immunosorbent assay kit (Thermo Fisher, KMC0181); Dopamine (DA) adsorption assay kit (novus, NBP2-67270); Mouse monoamine oxidase B (MAO-B) enzyme-linked immunosorbent assay kit (novus, NBP2-81161).

[0047] Dose design: The daily oral dose of the polypeptide is 200 mg crude drug / day. According to the body surface area conversion method, the equivalent dose for mice is 85 g / 60 kg * 12.3 = 41 mg crude drug / kg. According to the 1 / 4:1 / 2:1 dosing method, the low dose is 10.25 mg crude drug / kg, the medium dose is 20.5 mg crude drug / kg, and the drug is administered by gavage at 10 mL / kg once a day. Dimethyl fumarate (DMF), the dosing dose is 50 mg / kg / day.

[0048] Experimental Methods: C57 / BL6 mice were acclimatized in a quarantine room for 7 days with free access to feed and water. They were randomly divided into four groups (n=8 per group): a normal control group, a model control group, a DMF control group, and high-, medium-, and low-dose polypeptide groups. Mice in each group were administered the corresponding dose via gavage once daily for 15 consecutive days. The normal and model control groups received the same volume of pure water. Starting on day 8, except for the normal control group, all mice were intraperitoneally injected with MPTP at a dose of 30 mg / (kg·d) to establish a Parkinson's disease model. The control group received the same volume of physiological saline for 7 consecutive days. On day 15, 10 minutes after MPTP injection, the presence of tremors, arched backs, erect tails, and piloerection indicated successful model establishment. One hour after MPTP injection on the last day, the mice underwent a behavioral assessment, namely the open field test: mice were placed in the experimental environment for at least 30 minutes to acclimatize before being placed in an open field. The mice's movement distance, speed, and number of spontaneous standing movements were recorded and analyzed using a behavioral analysis system over a period of 5 minutes. After the behavioral experiment, blood was collected from the mice's eyes and brain tissue. The mouse blood was then subjected to treatment at 4°C and 3000 rpm. -1 Centrifuge for 10 min to separate serum. Perform ELISA kit instructions to measure serum inflammatory factors IL-1β and IL-18 levels. Take substantia nigra from brain tissue, homogenize it with physiological saline at a ratio of 1:9, centrifuge at 12000 r / min for 15 min at 4℃, collect the supernatant, and perform enzyme-linked immunosorbent assay (ELISA) to measure DA and MAO-B levels.

[0049] Results: Seven days after intraperitoneal injection of MPTP to induce modeling in mice, the number of spontaneous standing up and the speed of movement in the open field were significantly reduced in the model control group compared with the normal control group (P < 0.001), indicating successful modeling. Compared with the model group, the number of spontaneous standing up in the DMF control group, the high-dose peptide group, and the medium-dose peptide group all showed significant increases to varying degrees, and the speed of movement in the open field in the DMF control group, the high-dose peptide group, the medium-dose peptide group, and the low-dose peptide group all showed significant increases. The results are shown in Table 2.

[0050] Serum analysis of mice revealed that, compared with the normal control group, the levels of inflammatory factors IL-1β and IL-18 in the model group were significantly increased (P < 0.001), indicating successful modeling. Compared with the model group, the serum levels of inflammatory factors IL-1β and IL-18 in the DMF control group and the high, medium, and low dose peptide groups were significantly decreased. Analysis of mouse brain homogenate showed that, compared with the normal control group, the DA level in the model control group was significantly decreased (P < 0.001), and the MAO-B level was significantly increased (P < 0.001). Compared with the model control group, the DA level in the DMF control group and the high, medium, and low dose peptide groups showed varying degrees of significant recovery, while the MAO-B level in the DMF control group and the high, medium, and low dose peptide groups was significantly decreased, with the high-dose group showing the best results. The results are shown in Table 3.

[0051] Table 2. The effect of the peptides of this invention on behavioral indicators in a mouse model of Parkinson's disease (n=8)

[0052] Note: Compared with the normal control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the model control group, *P<0.05, **P<0.01, ***P<0.001. The same applies to the following table.

[0053] Table 3. The ameliorative effect of the peptides of this invention on molecular indicators in a mouse model of Parkinson's disease (n=8)

[0054] The sea cucumber polypeptide of this invention can improve motor dysfunction in mice induced by Parkinson's disease, improve the number of spontaneous standing up, increase movement speed in an open field, reduce the elevated serum IL-1β, IL-18 and MAO-B levels in the substantia nigra of the brain induced by Parkinson's disease in mice, and simultaneously increase brain dopamine (DA) levels. The sea cucumber polypeptide of this invention has a significant therapeutic effect on MPTP-induced Parkinson's disease in mice.

[0055] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A polypeptide for the prevention or treatment of Parkinson's disease, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide is derived from sea cucumber or is artificially synthesized.

3. A pharmaceutical composition for the prevention or treatment of Parkinson's disease, characterized in that, The pharmaceutical composition contains the polypeptide according to any one of claims 1-2.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition also contains pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: diluents, wetting agents, binders, disintegrants, lubricants, flavor and color modifiers, solvents, solubilizers, co-solvents, emulsifiers, absorption enhancers, surfactants, antioxidants, excipients, stabilizers, flavoring agents, sustained-release agents, metal complexing agents, inert gases, preservatives, fillers, local analgesics, pH adjusters, and isotonic or isotropic modifiers.

6. The pharmaceutical composition according to any one of claims 3-5, characterized in that, The dosage form of the pharmaceutical composition is selected from oral preparations, injectable preparations, transdermal absorption preparations, or mucosal delivery preparations.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the oral preparation is selected from any one of pills, powders, tablets, ointments, capsules, granules, oral liquids, and decoctions.

8. The use of the polypeptide according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 7 in the preparation of a medicament for the prevention or treatment of Parkinson's disease.

9. A food, health product, or drug containing the polypeptide according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 7.

10. A product for the prevention or treatment of Parkinson's disease, characterized in that, The active ingredient of the product includes the polypeptide as described in any one of claims 1 to 2.

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