Sea cucumber peptide with anti-PD (Parkinson's Disease) effect, computer simulation screening and synthesis method of sea cucumber peptide and application of sea cucumber peptide in treating Parkinson's

Abstract

The invention relates to a sea cucumber peptide with an anti-PD (Parkinson's Disease) effect, a computer simulation screening method, a synthesis method and application thereof in treatment of Parkinson's Disease, belongs to the field of active peptide screening and application, and solves the problem that the screened and synthesized sea cucumber peptide has the anti-PD effect, and has the technical key points that the sea cucumber peptide is prepared, and the sea cucumber active peptide is screened and synthesized from the sea cucumber peptide. The amino acid sequence of the sea cucumber active peptide is Gln-Trp-Phe-Asp-Trp, and the sea cucumber active peptide mixture has the effects that the prepared sea cucumber active peptide mixture can improve the behavioral level of a mouse induced by Rot, reduce the loss of dopaminergic neurons of nigra, improve the survival rate of SH-SY5Y cells induced by the Rot and improve the survival rate of the SH-SY5Y cells induced by the Rot. The active sea cucumber peptide screened by computer simulation analysis and in-vitro activity experiments on the basis of peptidomics analysis can improve the behavioral level of Rot-induced juvenile zebrafish of 7-day-old zebrafish, alleviates the pathological process of PD by reducing the endogenous ROS content of Rot-induced SH-SY5Y cells and alleviating mitochondrial membrane potential damage, and has a relatively good anti-PD effect.

Description

Technical Field

[0001] The invention belongs to the field of active peptide screening and application, and relates to sea cucumber peptides with anti-PD effect and computer simulation screening and synthesis methods thereof, and applications in treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. The progressive loss of dopaminergic (DAergic) neurons in the substantia nigra (SN) pars compacta (SNpc) and the aggregation of α-synuclein (α-syn) in the structure of Lewy bodies (LB) lead to movement disorders, including bradykinesia, resting tremor, and muscle rigidity. Although the pathogenesis and progression of PD are not fully understood, a large number of studies have shown that oxidative stress and mitochondrial dysfunction play an important role in the pathogenesis of PD. The main characteristics of oxidative stress are increased levels of reactive oxygen species (ROS) and reduced or dysfunctional antioxidant systems to combat free radicals. Rotenone (ROT) easily crosses the blood-brain barrier and can selectively inhibit the activity of mitochondrial complex I, causing mitochondrial dysfunction, reduced ATP production, and the generation of a large amount of ROS, thereby inducing oxidative stress and causing damage to the nigrostriatal dopaminergic system. At present, most of the drugs used clinically to treat PD are difficult to prevent or slow down the progression of the disease. Levodopa is an effective drug for the treatment of Parkinson's disease, but long-term use of levodopa may aggravate movement disorders in PD patients. Due to the limitations of PD treatment, there is an urgent need to develop new drugs to fight this disease.

[0003] Bioactive peptides (BPs) are small fragments containing more than two amino acid residues. They have important nutritional value and multiple biological activities such as antioxidant, neuroprotective and immunomodulatory. Among them, marine biological peptides have biological activities such as antioxidant, anti-inflammatory, anti-fatigue, and neuroprotective functions, and are an important source of bioactive peptides. Sea cucumber is an important traditional tonic and has long been loved by people in Asian countries. The functions of sea cucumbers are often related to the active compounds they contain. The content of its protein is as high as 40.7%-63.3%, and these rich proteins are often an important source of high-quality active peptides. Sea cucumber peptides (SCP) are hydrolyzed or enzymatic products of sea cucumber protein, which have many biological activities such as neuroprotection, antioxidant, ACE inhibition, immunomodulatory, anti-fatigue, anti-aging, and trace mineral chelation. Among them, sea cucumber peptides may have important biological active functions in neuroprotective activity. Studies have shown that various hydrolyzed components of the sea cucumber body wall can effectively alleviate PD, among which various active sea cucumber peptides may be involved. In addition, sea cucumber peptide extracts can alleviate cognitive impairment in scopolamine-induced mice by alleviating cholinergic dysfunction, increasing histone acetylation levels, and upregulating the LTP pathway and unsaturated lipid levels. The unique amino acid sequence of sea cucumber peptides often determines their biological activity in exerting neuroprotective functions. Peptides identified from sea cucumber peptide hydrolysates may become candidates for anti-Aβ aggregation to alleviate Alzheimer's disease. Sea cucumber egg peptide NDEELNK can alleviate scopolamine-induced PC12 cell damage by improving the cholinergic system, enhancing cellular energy metabolism, and upregulating the expression of phosphorylated protein kinase A (p-PKA), brain-derived neurotrophic factor (BNDF), and nerve growth factor (NGF) signaling proteins. Sea cucumber peptide SFGDI alleviates scopolamine-induced PC12 cell damage by protecting against oxidative stress and regulating the cholinergic system. It also plays a neuroprotective role by regulating the Sirt3 / SOD / ROS pathway, regulating lipid metabolism, and enhancing the energy metabolism of BV2 cells.

[0004] Sea cucumber peptide (SCP) has many biologically active functions, among which it plays a very important role in neuroprotection, but there are few clear reports on SCP for PD. Therefore, the purpose of the present invention is to study the therapeutic potential of SCP for ROT-induced PD. The ability of SCP hydrolyzed extract to alleviate PD was studied by in vivo and in vitro experiments. Then, the exact SCP molecules with potential therapeutic activity for PD were further screened and synthesized by using peptide omics analysis combined with computer simulation analysis. Finally, the biological activity and mechanism of action of SCP in treating PD were studied by in vivo and in vitro experiments. Summary of the invention

[0005] In order to solve the problem of using sea cucumber peptides for anti-PD effects, in the first aspect, the active peptide according to some embodiments of the present application has an amino acid sequence of Gln-Trp-Phe-Asp-Trp.

[0006] In a second aspect, the method for preparing the active peptide according to the first aspect of some embodiments of the present application is synthesized by the Fmoc / tBu solid phase method.

[0007] In a third aspect, the method for preparing sea cucumber peptide according to some embodiments of the present application is characterized by comprising the following steps:

[0008] S10. Mixing slurry of sea cucumber with distilled water in a reaction container to obtain a first mixture;

[0009] S20. adding 4% (w / w) pepsin to the first mixture and stirring for enzymolysis to obtain a second mixture, wherein the enzymolysis temperature is 37° C., the enzymolysis pH is 2.0, and the enzymolysis time is 3 h;

[0010] S30. Add 4% (w / w) trypsin to the second mixture and stir to obtain a third mixture by enzymolysis. The enzymolysis temperature is 37° C., the enzymolysis pH is 8.0, and the enzymolysis time is 3 h;

[0011] S40. The third mixture is heated to 65-70° C. and stirred for 30 min to inactivate the enzyme;

[0012] S50. 4% (w / w) alkaline protease is added to the third mixture after the enzyme is inactivated, and stirred for enzymolysis to obtain a fourth mixture. The enzymolysis temperature is 50° C., the enzymolysis pH is 9.0, and the enzymolysis time is 3 h;

[0013] S60. Add 4% (w / w) papain to the fourth mixture and stir to obtain a fifth mixture by enzymolysis. The enzymolysis temperature is 50° C., the enzymolysis pH is 6.0, and the enzymolysis time is 3 h;

[0014] S70. The fifth mixture is placed in a boiling water bath and then centrifuged, and the supernatant is freeze-dried to obtain the sea cucumber peptide.

[0015] According to the preparation method of some embodiments of the present application, slurry sea cucumber is mixed with 10% (w / v) distilled water and placed in a reaction container;

[0016] Among them, boiling water bath for 10min;

[0017] The pH was adjusted with 0.1 mol / L HCl and / or NaOH.

[0018] In a fourth aspect, a sea cucumber peptide is prepared according to the method described in the third aspect of some embodiments of the present application.

[0019] In a fifth aspect, the method for screening sea cucumber peptides with anti-PD activity according to some embodiments of the present application comprises the following steps:

[0020] Desalting the sea cucumber peptide described in the fourth aspect;

[0021] The desalted sea cucumber peptides were subjected to liquid chromatography-mass spectrometry analysis;

[0022] Identify the peptide segments of sea cucumber peptides based on mass spectrometry data;

[0023] The biological activity, toxicity, physicochemical properties, absorption, distribution and metabolic characteristics, excretion characteristics, and antioxidant, anti-inflammatory, and neuroprotective activities of each peptide segment of sea cucumber peptide were analyzed;

[0024] Screening of sea cucumber peptides with potential anti-PD activity based on analysis.

[0025] In a sixth aspect, a sea cucumber peptide is screened according to the method described in the fifth aspect of some embodiments of the present application.

[0026] According to some embodiments of the present application, the sea cucumber peptide has an amino acid sequence of Gln-Trp-Phe-Asp-Trp.

[0027] In the seventh aspect, the peptide (active peptide) according to any aspect of some embodiments of the present application is used in the preparation of a drug for treating degenerative neurological diseases, or in the preparation of a drug for treating Parkinson's disease, or in the preparation of a drug for treating epilepsy, or in the preparation of a drug for reducing the endogenous ROS content in Rot-induced SH-SY5Y cells, or in the preparation of a drug for reducing mitochondrial membrane potential damage.

[0028] In the eighth aspect, the computer simulation analysis according to some embodiments of the present application is used to screen sea cucumber peptides with anti-PD activity.

[0029] Beneficial effects of the present invention: The present invention treats Rot-induced C57BL / 6J mice and SH-SY5Y cells with an SCP mixture, and evaluates the behavioral level, histopathological level, cell survival rate and its potential biological activity in vitro. Peptidomics analysis is combined with computer simulation analysis to construct a sea cucumber peptide spectrum, and analyze and screen new sea cucumber peptides with potential anti-PD function. Rotenone (Rot)-induced 7-day-old zebrafish larvae and SH-SY5Y cells are used to study the anti-PD effect and mechanism of candidate peptides.

[0030] The sea cucumber peptide mixture can improve the behavior level of Rot-induced mice, reduce the loss of substantia nigra dopaminergic neurons, and increase the survival rate of Rot-induced SH-SY5Y cells. Peptidomics analysis combined with computer simulation analysis and in vitro activity screening initially screened sea cucumber peptide (SEQ ID No.1: Gln-Trp-Phe-Asp-Trp) may have good anti-PD activity. Finally, it was found that sea cucumber peptide (SEQ ID No.1: Gln-Trp-Phe-Asp-Trp) can improve the behavior level of 7d-old zebrafish larvae induced by Rot, and reduce the pathological process of PD by reducing the endogenous ROS content of Rot-induced SH-SY5Y cells and reducing mitochondrial membrane potential damage. Therefore, the findings of the present invention indicate that sea cucumber peptide (SEQ ID No.1: Gln-Trp-Phe-Asp-Trp) is a potential candidate for the treatment of PD. Additional aspects and advantages of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 .Figure of experimental data of SCP improving the behavioral defects induced by rotenone in mice, including: (A) schematic flow chart showing the experimental workflow; (B) the time the mouse stayed on the rotating rod device; (C) measurement of the stride length of the left forelimb of the mouse, (D) measurement of the stride length of the right forelimb of the mouse, (E) measurement of the stride length of the left hindlimb of the mouse, (F) measurement of the stride length of the right hindlimb of the mouse, (G) measurement of the stride distance of the forelimb of the mouse, (H) measurement of the stride distance of the hindlimb of the mouse; n=10 per group; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0032] Figure 2 . SCP alleviated rotenone-induced dopaminergic neuron damage in mice, including: (A) Representative immunohistochemical micrographs of TH in mouse substantia nigra (SN); (B) Quantitative evaluation of TH-positive neurons in mouse substantia nigra; (C) Representative immunohistochemical micrographs of TH in mouse striatum; (D) Quantitative analysis of the optical density of TH-positive neurons in mouse striatum; (E) Representative immunoblot bands of TH protein levels in mouse substantia nigra; (F) Quantitative analysis of the ratio of TH to β-actin in the blot; n = 3 per group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Scale bar = 100 μM.

[0033] Figure 3.In vitro experimental data of SCP; (A) ABTS experiment of SCP (20, 40, 60, 80, 100 mg / mL); (B) DPPH experiment of SCP (10, 20, 30, 40, 50 mg / mL); (C) Hydroxyl radical scavenging activity of SCP (4, 8, 12, 16, 20 mg / mL); (D) Effect of sea cucumber peptide extract (Con group does not contain sea cucumber peptide, 1, 5, 10, 25, 50 μg / mL SCP) on SH-SY5Y cell viability; (E) Protective effect of sea cucumber peptide extract on SH-SY5Y cells induced by 1 μM rotenone; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Data are expressed as mean ± standard deviation (n=3).

[0034] Figure 4 .Feature analysis of peptide identification and quantification, including: (A) counts of peptides of different lengths; (B) distribution of peptide molecular weights; (C) abundance of peptides with precise amino acid composition; (D) statistics of peptides with multiple unique amino acids; n = 3 per group.

[0035] Figure 5 .In silico strategies for screening potential new peptides with biological activity and bioavailability;

[0036] Figure 6 .In vitro experiments of sea cucumber peptide QWFDW; (A) ABTS experiment of SCP (10μM QWFDW); (B) DPPH experiment of SCP; (C) Hydroxyl radical scavenging activity of SCP; (D) Effect of SCP (Con, 10μ, MQWFDW) on SH-SY5Y cell viability; (E) Protective effect on SH-SY5Y cells induced by 1μM rotenone; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Data are expressed as mean±SD (n=3).

[0037] Figure 7 .Sea cucumber peptide QWFDW improved the behavioral level of 7-day-old zebrafish larvae induced by rotenone. Experimental data, where (A) the typical free swimming trajectory of each group of zebrafish, the green path and red line represent the movement trajectory recorded by the ViewpointZebrabox system; (B) the total swimming distance of zebrafish in 5 minutes; (C) the average swimming speed of zebrafish; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; data are expressed as mean±standard deviation (n=10).

[0038] Figure 8.QWFDW reduces intracellular ROS levels and alleviates mitochondrial membrane dysfunction experimental data, where (A) ROS expression is detected using ROS fluorescent probe; (B) ROS fluorescence intensity is evaluated; (C) mitochondrial membrane potential is detected using TMRE fluorescent probe; (D) TMRE fluorescence intensity is evaluated; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; data are expressed as mean±standard deviation (n=3).

[0039] Fig. 9 .Mass spectrometry analysis report of sea cucumber peptide (SEQ ID No.1: QWFDW).

[0040] Fig.10 .Chromatographic analysis report of sea cucumber peptide (SEQ ID No.1: QWFDW). DETAILED DESCRIPTION

[0041] Embodiments of the present application are described in detail below with reference to the accompanying drawings, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions.

[0042] A. Terminology:

[0043] Parkinson's disease (PD) is the second most common neurodegenerative disease. It is caused by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra (SN) pars compacta (SNpc) and the aggregation of α-synuclein (α-syn) in the structure of Lewy bodies (LB), leading to movement disorders including bradykinesia, resting tremor and muscle rigidity.

[0044] Bioactive peptides (BPs) are small fragments containing two or more amino acid residues, which have important nutritional value and multiple biological activities such as antioxidant, neuroprotective and immunomodulatory.

[0045] Sea Cucumber Peptides (SCP): It is the hydrolysis or enzymatic hydrolysis product of sea cucumber protein, which has many biological activities such as neuroprotection, anti-oxidation, ACE inhibition, immunomodulation, anti-fatigue, anti-aging, trace mineral chelation, etc.

[0046] B. Experimental Materials Description:

[0047] Trifluoroacetic acid was purchased from Maclean (Shanghai, China).

[0048] Rotenone (Rot, R8875) was purchased from Sigma-Aldrich (Shanghai, China).

[0049] Ethanol, acetonitrile, formic acid, dichloromethane, and n-butanol were purchased from Aladdin Company (Shanghai, China).

[0050] Antibody against β-actin (1:1000, ab8227) was from Arbor (Cambridge, UK).

[0051] Antibodies against tyrosine hydroxylase (TH, 1:1000, 25859-1-AP) were from Proteintech (Wuhan, China).

[0052] The amino acid sequence of the sea cucumber peptide was Gln-Trp-Phe-Asp-Trp (QWFDW) with a purity of 98%, and it was synthesized by GLBiochem Ltd (Shanghai, China).

[0053] Goat anti-mouse (1:1000, 31430) or goat anti-rabbit (1:1000, 31460) IgG-HRP antibodies were from Thermo Fisher Scientific (Waltham, MA, USA).

[0054] ABTS (BC4770), DPPH (BC4750), and hydroxyl radical scavenging kit (BC1320) were purchased from Solebol (Beijing, China).

[0055] Pepsin (P6322), trypsin (T6325), alkaline protease (P750258), and papain (P6321) were from MacLean (Shanghai, China).

[0056] Enhanced cell counting kit-8 (CCK-8, C0041), reactive oxygen species detection kit with CM-H2DCFDA (S0035S), mitochondrial membrane potential detection kit with tetramethylrhodamine ethyl ester (TMRE, C2001S), BCA protein quantification kit and protein lysis buffer (P0009) were all from Beyotime (Shanghai, China).

[0057] C. Embodiment (1~3):

[0058] Example 1: This example provides a method for preparing sea cucumber peptides. Fresh sea cucumbers (Stichopus japonicus) with an average length of about 20 cm are used for preparation. The preparation method is obtained from the waters of Dalian, China. The preparation method specifically includes the following steps:

[0059] First, the body wall of the sea cucumber is broken into pulp, and then the pulped sea cucumber is mixed with distilled water (10%, w / v) and put into a reaction container.

[0060] The pH was adjusted to 2.0 with 0.1 mol / L HCl at 37°C, and pepsin (4% w / w, protein basis) was used for enzymatic hydrolysis with stirring for 3 h.

[0061] The pH was adjusted to 8 with 0.1 mol / L NaOH, and trypsin (4% w / w, protein basis) was used for enzymatic hydrolysis with stirring for 3 h.

[0062] Heat to 65-70°C and stir for 30 minutes.

[0063] The temperature was adjusted to 50°C, the pH was adjusted to 9, and alkaline protease (4% w / w, protein basis) was used for enzymatic hydrolysis for 3 h with stirring.

[0064] The pH was then adjusted to 6, and papain (4% w / w, protein basis) was used for enzymatic hydrolysis with stirring for 3 h.

[0065] Finally, the reaction was terminated in a boiling water bath for 10 min, the mixture was centrifuged (10000 g, 20 min, 4°C), and the precipitate was discarded.

[0066] The supernatant was freeze-dried and stored at -20°C to obtain sea cucumber peptide powder X1.

[0067] Example 2: This example provides a method for constructing and analyzing a spectrum of a sea cucumber peptide mixture, which is used to preliminarily discover sea cucumber active peptides that may have potential for treating PD. The method comprises the following steps:

[0068] S210. Peptide extraction and desalting;

[0069] S220. Liquid chromatography-mass spectrometry (LC-MS) analysis;

[0070] S230. Peptide fragment identification and screening;

[0071] S240. Computer simulation analysis of potential functional sea cucumber peptide sequences.

[0072] Among them, in an embodiment of the present invention, step S210. Extraction and desalting of peptides includes

[0073] By volume, 5 times the amount of 50% (v / v) ethanol-water mixed extract was used to fully stir the sea cucumber peptide powder X1, and the mixture was shaken and then extracted overnight. After completion, the mixture was centrifuged at 5000-6000 rpm for 10-30 minutes, and the supernatant was freeze-dried.

[0074] After re-dissolving in pure water, add 3 times the amount of dichloromethane: n-butanol = 4:1 (v / v) organic mixed reagent, shake thoroughly, centrifuge at 12000 rpm for 30 min and take the polypeptide layer (aqueous phase).

[0075] Add 200 μL of acetonitrile to a MonoSpin C18 (SHIMADZU, Japan) desalting column to activate the desalting column, centrifuge at 5000-6000 rpm for 2 min, add 200 μL of 0.1% trifluoroacetic acid, centrifuge for 1 min×2 times, continue to add 800 μL of polypeptide extract, centrifuge at 5000-6000 rpm for 2 min, add 200 μL of 0.1% trifluoroacetic acid, centrifuge at 5000-6000 rpm for 1 min, add 200 μL of 60% acetonitrile, centrifuge at 5000-6000 rpm for 2 min to obtain a purified sample, combine the sample solutions, and lyophilize for later use.

[0076] Wherein, in an embodiment of the present invention, step S220. Liquid chromatography-mass spectrometry (LC-MS) analysis includes

[0077] The desalted peptide sample was fully dissolved in a 0.1% formic acid solution and filtered through a 0.22 μm membrane for testing. LCMS analysis was performed using an EASY-nLC 1200-Orbitrap Fusion Lumos LCMS system (Thermo Fisher, USA), the chromatographic column used was a laboratory self-filled Nano column (C18, 75 μm ID, 3 um particle size), the mobile phase A was an aqueous solution containing 0.1% (v / v) formic acid, the mobile phase B was an 80% acetonitrile aqueous solution containing 0.1% (v / v) formic acid, the flow rate was 500 nL / min, and the injection volume was 2 μL. The separation gradient for 120 min was as follows: 0-5 min, 5% B; 5-95 min, 5-15% B; 95-105 min, 15-25% B; 105-110 min, 25-80% B; 110-115 min, 80% B; 115-120 min, 5% B.

[0078] The mass spectrometer was detected in DDA (Data Dependent Acquisition) mode, the ion mode was positive, the full scan range was m / z350-1500, and the resolution was 60000 FWHM. The precursor ion charge (z) range was 1-7, the 20 most abundant ions were scanned by MS2, the activation type was HCD, the collision energy was set to 30%, the mass resolution of the tandem mass spectrometer was 15000 FWHM, and the quadrupole isolation window of the precursor ion was 2 m / z. In order to improve the signal coverage, the number of precursor ions was divided into three sections according to the ion distribution, and multiple injections were performed to collect the secondary spectra of singly charged and multiply charged precursor ions, respectively. The corresponding intervals were mz = 350-530, 520-695, 685-1500 when z = 1; mz = 350-540, 530-780, 770-1500 when mz = 2-7.

[0079] Among them, in an embodiment of the present invention, step 230. peptide fragment identification and screening includes

[0080] The mass spectrometry data were imported into PEAKS Studio 7.0 software (BSI, Canada) and peptides were identified by peptide-spectrum matches (PSM) and de novo sequencing (De Novo) to make the identification more comprehensive.

[0081] The PSM database is UniProt Sea Cucumber (30032 entries). The peptide sequence search parameters were set to 10 ppm for the precursor ion mass error, 0.05 Da for the daughter ion mass error, and monoisotopic ion for the precursor ion mass search type. No enzymatic hydrolysis type was specified. Carbamidomethylation on cysteine ​​(Cys) was specified as a fixed modification, and acetylation (N-term) at the N-terminus of the protein and oxidation (M) of methionine were specified as variable modifications. The maximum number of peptide modifications was set to 4.

[0082] In step 230, in order to study the potential biological activity of the exact peptide segment of sea cucumber peptide, the present invention adopts a label-free peptide group strategy to identify and quantify the peptide segment. In the PSM results, there are 2192 peptides with FDR (false discovery rate) <1%, that is, -10lgP> 20. In the De Novo results, there are 1201 peptides with ALC (average local confidence) ≥ 90%.

[0083] After removing duplicate amino acid sequences from the two identification results, 3267 peptides remained. After removing duplicates from the peptide amino acid sequences, the counts and proportions of peptides with different numbers of amino acids were counted. Most of the identified peptides are short sequence peptides. The counts (proportions) of peptides containing 4-9 amino acids are 143 (4.4%), 1101 (33.7%), 612 (18.7%), 446 (13.7%), 300 (9.2%), and 204 (6.2%), respectively. Peptides with 5-7 amino acids account for more than 10%, with the largest number of peptides being 5 amino acids.

[0084] like Figure 4 As shown in Figure A, in the sea cucumber peptide spectrum constructed by the present invention, the size of the peptide segments is mainly small molecule peptides. The number of peptide segments with a molecular weight distribution of 500-600Da is the largest, and the proportion of peptide segments of 600-700Da and 700-800Da also exceeds 10% of the total, which are 1064 (32.57%), 680 (20.81%), and 407 (12.46%) respectively. The proportion of peptides with a molecular weight <800Da is 73.33% ( Figure 4 B).

[0085] In the sea cucumber peptide spectrum constructed by the present invention, the size of the peptide segments is mainly small molecule peptides. The number of peptide segments with a molecular weight distribution of 500-600Da is the largest, and the proportion of peptide segments of 600-700Da and 700-800Da also exceeds 10% of the total, which are 1064 (32.57%), 680 (20.81%), and 407 (12.46%) respectively. The proportion of peptides with a molecular weight <800Da is 73.33% ( Figure 4 B). Most of these peptides have hydrophobic amino acids, acidic amino acids and basic amino acids. The content of peptides with hydrophobic amino acids is as high as 99.76% ( Figure 4 C-4D). According to previous reports, amino acid residues including glycine, proline, tryptophan, leucine and methionine are considered to be the hydrophobic amino acids most closely related to antioxidant activity. The biological activity of peptides often depends on their specific amino acid composition and sequence. Therefore, these peptides may have potential biological activity for the treatment of Parkinson's disease.

[0086] Wherein, in an embodiment of the present invention, step S240. Computer simulation analysis of potential functional sea cucumber peptide sequences includes

[0087] The biological activity of the peptides in the identified sea cucumber peptide spectrum was predicted by the PeptideRanker program (http: / / distilldeep.ucd.ie / PeptideRanker / ), and the peptides were scored ranging from 0 to 1. The greater the potential biological activity, the higher the score.

[0088] The potential toxicity prediction and related physicochemical properties (hydrophobicity, hydrophilicity, molecular weight, etc.) of the peptides were predicted online using ToxinPred software (http: / / crdd.osdd.net / raghava / toxinpred / ).

[0089] Human intestinal absorption (HIA), blood-brain barrier (BBB) ​​penetration, and cytochrome P450 (CYP450) interactions of the peptides were analyzed using admetSAR2 (http: / / lmmd.ecust.edu.cn / admetsar2) to determine the absorption, distribution, and metabolic characteristics, respectively.

[0090] ADMETlab2.0 (https: / / admetmesh.scbdd.com / service / screening / cal) was used to analyze the excretion properties of the peptides, including “clearance” and “t1 / 2”.

[0091] The online databases BIOPEP-UWM (https: / / biochema.UWM.edu.pl / ) and Neuropeptide Database (isyslab.info / Neuropep / basic_search.jsp) were used to search for antioxidant, anti-inflammatory, neuroprotective and other activities. The novelty of the peptide structure sequence was searched through SciFinder.

[0092] In step S240, in order to further screen the new sea cucumber peptides that may have biological activity in treating PD in the sea cucumber peptide map, a computer simulation analysis method is used to predict the potential biological activity and various physicochemical properties of the sea cucumber peptides. Figure 5As shown. The PeptideRanker program was used to predict the biological activity of 3267 peptide sequences with high credibility, with a threshold of 0.5. There were 325 peptides with PeptideRanker values ​​≥ 0.8, and 310 peptides remained after deduplication. Afterwards, the potential toxicity, hydrophobicity, hydrophilicity and relative molecular weight of sea cucumber peptides were predicted according to the ToxinPred software. The human intestinal absorption (HIA), blood-brain barrier (BBB) ​​penetration rate and cytochrome P450 (CYP450) interaction of the peptides were analyzed by admetSAR2 to determine the absorption, distribution and metabolic characteristics, respectively. The excretion characteristics of the peptides, including "clearance" and "t1 / 2", were analyzed by ADMETlab2.0. Antioxidant, anti-inflammatory, neuroprotective and other activities were searched using the online databases BIOPEP-UWM and Neuropeptide Database. Finally, SciFinder was used to search for the novelty of sea cucumber peptides, and the PeptideRanker values ​​of the biological activity prediction of each peptide segment, potential antioxidant, anti-inflammatory, neuroprotective activity, blood-brain barrier (BBB) ​​penetration rate, toxicity and other properties were comprehensively considered to screen out more credible new polypeptide fragments that may have good potential biological activity (Table 1). Among them, the new sea cucumber peptide shown in SEQ ID No.1 has the highest score. It contains a typical acidic amino acid residue Asp, and its Trp and Phe are also extremely important hydrophobic amino acid residues that provide antioxidant capacity. In addition, in computer simulation analysis, it has the potential for human intestinal absorption and passing through the blood-brain barrier, which also means that it may play a role in the brain. The new sea cucumber peptide shown in SEQ ID No.1 may have a potential role in treating PD. Among them, SEQ ID No.1: Gln-Trp-Phe-Asp-Trp. (SEQ ID No.1: QWFDW).

[0093] Table 1 The best new sea cucumber peptide sequence with potential for treating Parkinson's disease predicted by computer simulation

[0094]

[0095] Example 3: Synthesis of sea cucumber peptide (SEQ ID No. 1: QWFDW), SEQ ID No. 1: QWFDW, synthesized using Fmoc / tBu solid phase method.

[0096]

[0098] First, 100-200m Wang resin is subjected to operation 1, where operation 1 is 20% Piperidine (Remove the n-terminal Fmoc group with piperidine) for 20 minutes, and the color is blue when detected by a detection reagent. Then, operation 2 is performed. Operation 2 is HBTU+DIEA plus Fmoc-Asp(otbu)-OH for 1 hour, and the color is colorless when detected by a detection reagent, and then operation 1 is performed, 20% Piperidine (Remove the n-terminal Fmoc group with piperidine) for 20 minutes, and the color is blue when detected by a detection reagent. Operation 2 is performed. HBTU+DIEA plus Fmoc-Phe-OH for 1 hour, and the color is colorless when detected by a detection reagent, and then operation 1 is performed, and so on until the last amino acid Gln is de-Fmoced, the resin is drained and cut to obtain a crude polypeptide, and the crude polypeptide is purified to obtain the desired polypeptide. Figure 9-10 This is a report on the chromatography and mass spectrometry analysis of sea cucumber peptide (SEQ ID No.1: QWFDW).

[0099] D. Experimental description:

[0100] 1. Mouse experiment: All animal experiments strictly followed the guidelines of the National Institutes of Health on the care and use of experimental animals and have been formally approved by the Institutional Ethics Committee of Dalian Medical University. In addition, the animal experiment report of the present invention also fully complies with the requirements of the ARRIVE guidelines. 60 C57BL / 6J mice (male, 8 weeks old), weighing between 20-25g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. These mice were housed under standard experimental conditions and had free access to food and water. They were randomly divided into a control group (Con), rotenone (Rot), rotenone + low-dose SCP (250 mg / kg / day) (Rot+L), rotenone + medium-dose SCP (500 mg / kg / day) (Rot+M), rotenone + high-dose SCP (1000 mg / kg / day) (Rot+H) and a high-dose SCP (1000 mg / kg / day) group alone (Con+L) (n=10). Rotenone was freshly prepared in an equal volume solution of DMSO:polyethylene glycol (PEG, 10%) and intraperitoneally injected into mice (1.5 mg / kg / d) for 21 consecutive days. The blank group was injected with the same amount of solvent. SCP was dissolved in saline and administered to mice by gavage according to the group 30 minutes after injection for 21 consecutive days. The blank group was gavaged with the same amount of saline.

[0101] 1.1. Rotarod test: The rotarod test of mice is to evaluate the behavioral ability of mice. In short, before the formal experiment of mice, the mice were trained in the rotarod for 3 consecutive days. During the training, each mouse was allowed to move freely at a constant speed of 22rpm. If the mouse fell, the mouse would be placed on the device again until the end of 300s. In the formal experiment, the acceleration mode was adopted to accelerate the speed of the rotarod instrument from 4rpm to 50rpm within 300s. If the mouse falls, the falling time and current speed of the mouse are recorded. Each mouse was subjected to 3 experiments, with an interval of 2 hours each time. The average time of the 3 repeated experiments was statistically analyzed.

[0102] 1.2. Gait analysis: In brief, the gait analysis experiment was conducted by placing mice on a 50 cm long and 10 cm wide runway and training all mice to walk along the runway. The forelimbs and hindlimbs of the mice were then dyed with black ink and red ink, respectively, and they were free to leave footprints on white paper during movement. In the formal experiment, white paper of appropriate length and width was placed on the runway, and the mice were allowed to walk freely along the runway. Three to four consecutive strides of each mouse were recorded. The stride length and stride distance of the limbs were then analyzed.

[0103] 1.3. Immunohistochemistry: Mouse brain tissue samples were obtained by transcardial perfusion of mouse brains with pre-cooled PBS and 4% formaldehyde. Then, the mouse brain tissue was fixed with 4% paraformaldehyde at 4°C for another 48 h, and then the brain tissue was transferred to a 30% gradient sucrose solution until it sank to the bottom. Free-floating coronal brain sections of approximately 30 μm thickness were cut using a Leica cryostat microtome (CM 1950, Leica, Wetzlar, Germany). For immunohistochemistry experiments, briefly, the sections were washed with PBS and incubated with 3% H2O2 at room temperature for 10 minutes to inactivate peroxidase. Then, after washing the sections with PBS, the selected mouse brain sections were incubated with 1% BSA, 0.4% Triton X-100 and 4% normal goat serum for 1 h. Then, the sections were incubated with TH (1:1000) antibody at 4°C overnight. The next day, the sections were washed with PBS and then incubated with HRP-conjugated secondary antibody (ZK1030, Vector, Shenzhen, China) for 1 h at room temperature. After washing the sections with PBS, they were incubated with ABC kit (ZK1030, Vector, Shenzhen, China) for 1 h at room temperature. Then, after washing the sections in PBS, they were incubated with 3-3′ diaminobenzidine (34002, Thermo Fisher Scientific, Waltham, MA, USA) to visualize the binding signal. Finally, the sections were scanned by a digital slide scanner KF-PRO-020 (Kfbio, Ningbo, China). Brain slices were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

[0104] 1.4. Western Blot Analysis: Western blot experimental mouse brain tissue material was obtained from mice perfused with PBS only. RIPA buffer (P0013 B, Beyotime, Shanghai, China) mixed with 1% protease inhibitor (78429, Thermo Fisher Scientific, Waltham, MA, USA) and phosphatase inhibitor (78440, Thermo Fisher Scientific, Waltham, MA, USA) was mixed and homogenized with the sample, and protein was extracted. The protein concentration was then measured using a BCA kit (P0009, Beyotime, Shanghai, China). Each sample was loaded with equal amounts of protein, and the sample protein was separated by electrophoresis in 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (IPVH 07850, Waltham, MA, USA). Next, the membrane was blocked with Superblocking Buffer (37515, Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 30 min. The membrane was then incubated with the primary antibody at 4°C overnight. The next day, the membrane was washed with Tris-buffered saline with Tween 20 and incubated with a secondary antibody labeled with HRP (ab 6721, Abcam, Cambridge, UK) for 2 h at room temperature. Finally, the membrane was evenly covered with ECL reagent (P0018 S, Beyotime, Shanghai, China) and images were taken with an imaging system (Bio-Rad, Hercules, CA, USA). Finally, quantitative analysis was performed using ImageLab (4.0) software.

[0105] 2. In vitro evaluation of potential antioxidant capacity and biological activity, including ABTS, DPPH and hydroxyl radical scavenging ability tests. The experimental materials were ABTS free radical scavenging ability test kit, DPPH free radical scavenging ability test kit, hydroxyl free radical scavenging ability test kit purchased from Solebao, and the test method reference kit.

[0106] 3. Cell experiment: Human neuroblastoma cell line (SH-SY5Y) cells were cultured in a humid environment of 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (11965118, Gibco, Waltham, MA, USA) mixed with 10% fetal bovine serum (A5669701, Gibco, Waltham, MA, USA) and 1% penicillin-streptomycin solution (15140122, Thermo, Waltham, MA, USA).

[0107] 3.1. Cell viability and toxicity assay (CCK-8): Enhanced cell counting kit-8 (CCK-8, C0041) was purchased from Bio-Tech (Shanghai, China). Briefly, SH-SY5Y cells were seeded in 96-well plates and incubated until 90% confluence was achieved. Subsequently, 100 μL of DMEM medium supplemented with 10% CCK-8 reagent was added to each well and incubated for 1 hour in the dark at 37 ° C. Then, the absorbance was measured at 450 nm using a microplate reader (Infinite 200PRO, USA).

[0108] 3.2. ROS generation detection: In a 24-well plate, add 1000 μL of fresh medium without serum. Divide SH-SY5Y cells into four groups: Con (the concentration of Rot and QWFDW is 0), 1 μM Rot only, 1 μM Rot with 10 μM QWFDW, and 10 μM QWFDW only, culture for 24 hours, and allow them to grow and stabilize. Then add the probe according to the DCFH-DA kit experimental method, and observe and take pictures under an inverted fluorescence microscope.

[0109] 3.3. Mitochondrial membrane potential detection: TMRE fluorescent probe was from Bio-Tech, processed according to the TMRE kit method, and observed and photographed under an inverted fluorescence microscope.

[0110] 4. Zebrafish experiments: Wild-type zebrafish AB were provided by the National Zebrafish Resource Center. They were raised in water (200 mg instant sea salt / 1 L reverse osmosis water; conductivity, 480-510 μs / cm; pH 6.9-7.2; hardness, 53.7-71.6 mg / LCaCO3) at 28 °C with a 14-h light / 10-h dark cycle, and fed with dry slices once a day and live brine shrimp twice a day. Feeding management complies with the requirements of the International Association for Accreditation, Assessment and Accreditation of Laboratory Animal Care. Embryos were collected by natural spawning and incubated in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). The experiment was divided into 4 groups: Con group (0.01% DMSO only), Rot group only, Rot and 10μM QWFDW co-treatment group (Rot+QWFDW) and Con group with only 10μM QWFDW added (Con+QWFDW). At 3dpf, 20 larvae were placed in each well of a 6-well plate in a solution volume of 3mL. Larvae were treated at 3dpf to 7dpf, and the solution was changed every day as normal. Rot was prepared at a concentration of 100mM in 100% dimethyl sulfoxide DMSO and then serially diluted to 1,000,000 times in 10% water-diluted DMSO to a working concentration of 50nM. Rotenone controls were exposed to 0.01% DMSO solution, and all treatments were performed in the dark.

[0111] Behavioral assessment: After the end of drug administration, the behavioral level of swimming activity of 7-day-old zebrafish larvae was tested. Individual larvae were transferred to E3 embryo culture medium, placed in a 24-well plate, and adapted to ambient light for 15 minutes before recording. Activity was recorded using Zebralab software and Zebrabox tracking system (ViewPoint Life Science, Lyon, France). Larval swimming activities were performed 3 times, 5 minutes each time. The total travel distance and the average speed of the 5-minute test were counted each time, and the speed was set to high activity at 5mm / s and low activity at 0-5mm / s. A sample size of 10 was selected for each group for behavioral evaluation. Among them, the data integrity of statistical analysis was ensured by rigorous experimental design, and each measurement was repeated three times. Both technically and biologically, the results were presented in the form of mean ± standard error (SEM). Statistical analysis was performed using GraphPad Prism version 8.0.1, and significant differences were identified using one-way or two-way ANOVA. A threshold of p < 0.05 was set to indicate statistical significance, ensuring that the findings were both reliable and robust.

[0112] E. Experimental results analysis:

[0113] E1. Analysis of SCP results of sea cucumber peptide (X1):

[0114] 1. Mouse Experiment:

[0115] SCP alleviates behavioral disorders induced by Rot in mice: In order to study whether SCP can play an anti-Parkinson's disease role in vivo, the present invention uses 8-month-old C57BL / 6J mice to construct an animal PD model by intraperitoneally injecting Rot (1.5 mg / kg / day) into the model group mice every day, and then gavages different concentrations of SCP into the treatment group mice for treatment. This was continued for 21 consecutive days, and then the mice were subjected to behavioral tests ( Figure 1 A). The mouse rotarod test and gait test are classic experiments to study the motor behavior of mice, and are also important methods to study the behavioral ability of PD.

[0116] 1.1. Rotarod test was performed on mice to evaluate their motor coordination and balance ability. The Con+H group showed no significant difference compared with the control group, indicating that high concentrations of SCP did not impair the motor function of normal mice. In contrast, the Rot group showed a reduced time remaining on the rotating rod compared with the control group. Treatment with 250, 500, and 1000 mg / kg of SCP significantly alleviated the Rot-induced motor deficits, and the higher the concentration of SCP, the better the protective effect, thus demonstrating the protective effect of SCP on the motor ability of mice subjected to Rot challenge ( Figure 1 B).

[0117] 1.2. The gait test of mice can often directly reflect the motor behavior disorder of mice. Compared with the Con group, the Rot group significantly reduced the movement stride of the front and hind limbs of mice, and after SCP treatment, the reduction of stride was significantly curbed. In addition, there was no significant change in the C+H group compared with the Con group ( Figure 1 CF). In addition, the distance between the hind limbs of mice in the Rot group was extended compared with that in the Con group, but it was shortened after administration of SCP. However, there was no significant change in the distance between the forelimbs of mice ( Figure 1 GH). The results showed that SCP could effectively alleviate the behavioral disorders induced by Rot in mice.

[0118] SCP prevents loss of dopaminergic neurons in the substantia nigra: The pathological features of PD include degeneration of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. In order to study whether SCP can alleviate the degeneration of dopaminergic neurons in the Rot-induced mouse PD model, the present invention studied the degeneration of dopaminergic neurons in the brain. TH is the rate-limiting enzyme for dopamine synthesis, and its immunostaining is used to detect dopaminergic neurons.

[0119] 1.3. Immunohistochemistry results showed that compared with the Con group, there was a significant loss of TH+ neurons in the SNpc of mice in the Rot group, and the administration of SCP dose-dependently alleviated the loss of TH+ neurons in the SNpc ( Figure 2 A). The cell counting results were consistent with the above results. Compared with the Con group, there was no significant difference in the C+H group, while the number of TH+ neurons in the Rot group was significantly reduced. The number of TH+ neurons in the SCP group was significantly increased compared with the Rot group ( Figure 2 B). Secondly, the present invention found that in the striatum, there was no significant difference in the C+H group compared with the Con group, while the TH immunostaining in the Rot group was significantly reduced. After SCP treatment, the treatment group reduced this phenomenon in a dose-dependent manner ( Figure 2 C). The density statistics of TH immunostaining in the mouse striatum are consistent with the histochemical observation results ( Figure 2 D). The present invention further determines the neuroprotective effect of SCP by detecting the expression level of TH protein in the mouse midbrain.

[0120] 1.4. The results of immunoblotting were consistent with those of immunohistochemistry, showing that Rot induction significantly reduced the expression level of TH in the midbrain of mice, while administration of SCP alleviated this damage in a dose-dependent manner ( Figure 2 E). Compared with the Con group, there was no significant difference between the Con group and the C+H group. However, the TH / β-Actin of the mice in the R+L, R+M, and R+H groups given sea cucumber peptides was 1.342 times, 1.534 times, and 1.859 times, respectively, compared with the Rot group ( Figure 2 F) Experiments have confirmed that SCP can play a role in treating PD by preventing the loss of dopaminergic neurons in the substantia nigra.

[0121] 2. In vitro evaluation of potential antioxidant capacity and biological activity:

[0122] In vitro bioactivity evaluation of SCP and its effect on the survival rate of SH-SY5Y cells induced by rotenone (Rot): The function of bioactive peptides is closely related to their antioxidant capacity. The potential bioactivity of SCP mixtures can be better analyzed through in vitro antioxidant activity. The antioxidant activity was evaluated using ABTS radical scavenging, DPPH radical scavenging and hydroxyl radical scavenging assays. DPPH is a relatively stable free radical, and DPPH radical scavenging is a common method for evaluating free radical scavenging activity. ABTS radical scavenging rate ABTS is a water-soluble free radical that easily reacts with antioxidants in aqueous systems. ·OH scavenging rate The hydroxyl radical (·OH) has the strongest reaction among ROS and can be captured by DMPO. The results showed that as the concentration of sea cucumber peptide extract increased, its ABTS radical scavenging rate ( Figure 3A) DPPH free radical scavenging rate ( Figure 3 B) and hydroxyl radical scavenging rate ( Figure 3 C) gradually increased, and the results showed that the sea cucumber peptide mixture may have good antioxidant capacity, which is consistent with previous reports.

[0123] 3. Cell experiment:

[0124] 3.1. In order to determine the toxicity of sea cucumber peptide extract on SH-SY5Y cells, the cell viability of sea cucumber peptide mixtures of different concentrations was determined using a CCK-8 kit. Between the Con group and the drug group, it was observed that the cells in the drug group had a certain cell proliferation phenomenon. When the concentration of sea cucumber peptide extract was 50 μg / mL, this promotion of cell proliferation was significant ( Figure 3 D). This indicates that the SCP mixture has no significant toxic effect on cells, but has a certain effect of promoting cell proliferation. To evaluate the potential neuroprotective effect of the sea cucumber peptide mixture, SH-SY5Y cells were exposed to 1 μM Rot and then treated with the sea cucumber peptide mixture, and then the cell survival rate was evaluated. Compared with the Con group, the survival rate of SH-SY5Y cells incubated with Rot was significantly reduced. Compared with the Rot group, the administration of the sea cucumber peptide mixture at concentrations of 10, 25, and 50 μg / mL significantly increased the cell survival rate ( Figure 3 E). Therefore, the sea cucumber peptide mixture has a potential neuroprotective effect on Rot-induced SH-SY5Y cells. This suggests that there may be a certain sea cucumber peptide in the sea cucumber peptide mixture that has a protective effect on Rot-induced PD.

[0125] E2. Sea cucumber peptide (SEQ ID No.1) result analysis:

[0126] 2. In vitro evaluation of potential antioxidant capacity and biological activity:

[0127] The in vitro activity of sea cucumber peptide (SEQ ID No.1) was evaluated to further screen out peptides with potential anti-PD activity. In the ABTS free radical scavenging rate, DPPH free radical scavenging rate and hydroxyl free radical scavenging rate experiments, the present invention evaluated the sea cucumber peptide (SEQ ID No.1) at a concentration of 10mM. The results showed that the ABTS free radical scavenging rate of sea cucumber peptide (SEQ ID No.1) Figure 6 A) DPPH free radical scavenging rate ( Figure 6 B) and hydroxyl radical scavenging rate ( Figure 6 C) high, and may have better in vitro antioxidant capacity.

[0128] 3. Cell experiment:

[0129] 3.1. In order to determine the toxicity of sea cucumber peptide (SEQ ID No.1) to SH-SY5Y cells, the present invention administered sea cucumber peptide (SEQID No.1) to cells at a concentration of 10 μM and co-cultured for 24 hours. The results showed that compared with the Con group, no obvious cell proliferation and cell death were found in the administration group. Therefore, at a concentration of 10 μM, sea cucumber peptide (SEQ ID No.1) had no significant toxic effect on SH-SY5Y cells ( Figure 6 D). In addition, the present invention found that in Rot-induced SH-SY5Y cells, 10 μM sea cucumber peptide (SEQ ID No. 1) can significantly improve the cell survival rate compared with the Rot group ( Figure 6 D), the protective activity is obvious, so the sea cucumber peptide (SEQ ID No.1) has a good potential for treating PD.

[0130] Effect of sea cucumber peptide (SEQ ID No. 1) on SH-SY5Y cells induced by rotenone (Rot): A large increase in intracellular ROS levels and mitochondrial damage are often important causes of PD.

[0131] In order to further study how sea cucumber peptide (SEQ ID No. 1) alleviates the damage of SH-SY5Y cells induced by Rot and plays a role in alleviating PD, the intracellular ROS level and mitochondrial membrane potential level were detected.

[0132] 3.2. The results of ROS generation detection showed that the intracellular ROS level induced by Rot increased significantly, while the intracellular ROS level was downregulated after the administration of sea cucumber peptide (SEQ ID No. 1), such as Figure 8 A-8B.

[0133] 3.3. The results of mitochondrial membrane potential detection showed that in TMRE detection, Rot can destroy the mitochondrial membrane potential, causing mitochondrial damage, which may lead to cell apoptosis. After the administration of sea cucumber peptide (SEQ ID No.1), the fluorescence intensity of mitochondrial membrane potential increased significantly compared with the Rot group, effectively reducing the damage of Rot to the cell mitochondrial membrane potential ( Figure 8 C-8D).

[0134] The results of cell experiments showed that sea cucumber peptide (SEQ ID No. 1) may exert its anti-PD biological activity by reducing the level of intracellular ROS and alleviating the damage of mitochondrial membrane potential.

[0135] 4. Zebrafish experiment:

[0136] Zebrafish has been shown to be a powerful in vivo vertebrate model for the study of various neurodegenerative diseases and high-throughput drug screening. The present invention further studies whether sea cucumber peptide (SEQ ID No. 1) can have neuroprotective activity in vivo by observing the behavioral changes of 7-day-old zebrafish larvae induced by Rot. Figure 7 A, 7B, 7C, the results showed that compared with Con, there was no significant change in the Con+QWFDW group, while the zebrafish in the Rot group showed a weakened movement level and a reduced active state. The Rot+QWFDW group can effectively improve this Rot-induced decline in movement level. The analysis of the total swimming distance and average swimming speed of zebrafish is consistent with the above results. Under the induction of Rot, the total swimming distance and average swimming speed of zebrafish decreased significantly compared with the Con group. After administering sea cucumber peptide (SEQ ID No.1), the total swimming distance and average swimming speed of zebrafish were significantly increased. Therefore, the present invention found that the new sea cucumber peptide (SEQ ID No.1) can improve the behavioral level of 7d-old zebrafish fry induced by Rot.

[0137] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An active peptide, characterized in that Its amino acid sequence is Gln-Trp-Phe-Asp-Trp.

2. The method for preparing the active peptide according to claim 1, characterized in that: Synthesized by Fmoc / tBu solid phase method.

3. A method for preparing sea cucumber peptide, characterized in that: The steps include: S10. Mixing slurry of sea cucumber with distilled water in a reaction container to obtain a first mixture; S20. adding 4% (w / w) pepsin to the first mixture and stirring for enzymolysis to obtain a second mixture, wherein the enzymolysis temperature is 37° C., the enzymolysis pH is 2.0, and the enzymolysis time is 3 h; S30. Add 4% (w / w) trypsin to the second mixture and stir to obtain a third mixture by enzymolysis. The enzymolysis temperature is 37° C., the enzymolysis pH is 8.0, and the enzymolysis time is 3 h; S40. The third mixture is heated to 65-70° C. and stirred for 30 min to inactivate the enzyme; S50. 4% (w / w) alkaline protease is added to the third mixture after the enzyme is inactivated, and stirred for enzymolysis to obtain a fourth mixture. The enzymolysis temperature is 50° C., the enzymolysis pH is 9.0, and the enzymolysis time is 3 h; S60. Add 4% (w / w) papain to the fourth mixture and stir to obtain a fifth mixture by enzymolysis. The enzymolysis temperature is 50° C., the enzymolysis pH is 6.0, and the enzymolysis time is 3 h; S70. The fifth mixture is placed in a boiling water bath and then centrifuged, and the supernatant is freeze-dried to obtain the sea cucumber peptide.

4. The preparation method according to claim 3, characterized in that: in, The slurry of sea cucumber is mixed with 10% (w / v) distilled water and placed in a reaction container; Among them, boiling water bath for 10min; The pH was adjusted with 0.1 mol / L HCl and / or NaOH.

5. Sea cucumber peptide prepared by the method of claim 3 or 4.

6. A method for screening sea cucumber peptides with anti-PD activity, characterized in that: The steps include: Desalting the sea cucumber peptide according to claim 5; The desalted sea cucumber peptides were subjected to liquid chromatography-mass spectrometry analysis; Identify the peptides of sea cucumber peptides based on mass spectrometry data; The biological activity, toxicity, physicochemical properties, absorption, distribution and metabolic characteristics, excretion characteristics, and antioxidant, anti-inflammatory, and neuroprotective activities of each peptide segment of sea cucumber peptide were analyzed; Screening of sea cucumber peptides with potential anti-PD activity based on analysis.

7. Sea cucumber peptide screened by the method of claim 6.

8. The sea cucumber peptide according to claim 7, characterized in that: Its amino acid sequence is Gln-Trp-Phe-Asp-Trp.

9. Use of the peptide according to any one of claims 1, 5, 7, and 8 in the preparation of a drug for treating degenerative neurological diseases, or in the preparation of a drug for treating Parkinson's disease, or in the preparation of a drug for treating epilepsy, or in the preparation of a drug for reducing the endogenous ROS content in Rot-induced SH-SY5Y cells, or in the preparation of a drug for reducing mitochondrial membrane potential damage.

10. Application of computer simulation analysis in screening sea cucumber peptides with anti-PD activity.

Images