Sea cucumber peptide against pdl, computer simulation screening and synthesis method and application in treating parkinson's disease

Abstract

The sea cucumber peptide for anti-PD effect and its computer simulation screening and synthesis method and application in treating Parkinson's disease belong to the field of active peptide screening and application, solve the problem of screening and synthesizing sea cucumber peptide for anti-PD effect, the technical points are to prepare sea cucumber peptide, and screen and synthesize sea cucumber active peptide from the sea cucumber peptide, the amino acid sequence of the sea cucumber active peptide is Gln-Trp-Phe-Asp-Trp, the effect is that the prepared sea cucumber peptide mixture can improve the Rot-induced mouse behavior level, reduce the loss of substantia nigra dopaminergic neurons, and improve the survival rate of Rot-induced SH-SY5Y cells, the active sea cucumber peptide screened based on peptidomics analysis through computer simulation analysis and in-vitro activity experiment can improve the Rot-induced 7d zebra fish larvae behavior level, and reduce the pathological process of PD by reducing the endogenous ROS content of Rot-induced SH-SY5Y cells and reducing the mitochondrial membrane potential damage, and has good anti-PD effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of active peptide screening and application, and relates to sea cucumber peptide with anti-PD effect and a computer simulation screening and synthesis method thereof and application of the sea cucumber peptide in treatment of Parkinson's disease. BACKGROUND

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. Due to the gradual loss of dopaminergic (DAergic) neurons in the substantia nigra (SN) pars compacta (SNpc) and the aggregation of alpha-synuclein (a-syn) in the structure of Lewy bodies (LB), it leads 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 Parkinson's disease. The main feature of oxidative stress is the increase in the level of reactive oxygen species (ROS) and the decrease or dysfunction of the antioxidant system to counteract free radicals. Rotenone (ROT) can easily cross the blood-brain barrier and selectively inhibit the activity of mitochondrial complex I, which can cause mitochondrial dysfunction, lead to a decrease in ATP production and produce a large amount of ROS, thereby inducing oxidative stress to cause damage to the substantia nigra striatal 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 relieving Parkinson's disease, but long-term use of levodopa can exacerbate the movement disorders of PD patients. Due to the limitations of PD treatment, there is an urgent need to develop new drugs to combat this disease.

[0003] Bioactive peptides (BPs) are small fragments containing two or more amino acid residues, which have important nutritional value and various bioactivities such as antioxidant, neuroprotective and immunomodulatory activities. Among them, marine peptides have antioxidant, anti-inflammatory, anti-fatigue, neuroprotective and other bioactive functions, and are an important source of bioactive peptides. Sea cucumber is an important traditional tonic, which has long been favored by people in Asian countries. The function of sea cucumber is often related to the active compounds contained therein. The content of protein in sea cucumber is as high as 40.7%-63.3%, and these abundant proteins are an important source of high-quality active peptides. Sea cucumber peptides (SCP) are hydrolysis or enzymatic products of sea cucumber protein, which have neuroprotective, antioxidant, ACE inhibitory, immunomodulatory, anti-fatigue, anti-aging, trace mineral chelation and many other biological activities. Among them, sea cucumber peptides may have important bioactive functions in neuroprotective activity. Studies have shown that various hydrolysis components of sea cucumber body wall can effectively alleviate PD, and various active sea cucumber peptides may be involved. In addition, sea cucumber peptide extract can alleviate scopolamine-induced cognitive impairment in mice by reducing cholinergic dysfunction, increasing histone acetylation levels, up-regulating LTP pathways and unsaturated lipid levels. The unique amino acid sequence of sea cucumber peptide often determines its neuroprotective bioactivity. The peptides identified from sea cucumber peptide hydrolysate may be candidates for alleviating Alzheimer's disease by resisting Aβ aggregation. Sea cucumber egg peptide NDEELNK can alleviate scopolamine-induced PC12 cell damage by improving the cholinergic system, enhancing cell energy metabolism, up-regulating 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 can alleviate scopolamine-induced PC12 cell damage by resisting oxidative stress and regulating the cholinergic system. And it 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 peptides (SCP) have many bioactive functions, among which they play a very important role in neuroprotection, but there are few clear reports of SCP on PD. Therefore, the purpose of the present invention is to study the therapeutic potential of SCP on ROT-induced PD. The ability of SCP hydrolysis extract to alleviate PD was studied through in vivo and in vitro experiments. Then, the exact SCP molecules with potential PD treatment activity were further screened and synthesized by using peptidomics analysis combined with computer simulation analysis. Finally, the biological activity and mechanism of action of SCP in treating PD were studied through in vivo and in vitro experiments. SUMMARY

[0005] To solve the problem of sea cucumber peptide for anti-PD, in a first aspect, an 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, a method for preparing the active peptide according to the first aspect of some embodiments of the present application is synthesized by Fmoc / tBu solid phase method.

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

[0008] S10. Mix the pulp sea cucumber and distilled water in the reaction vessel to obtain a first mixture;

[0009] S20. Add 4% (w / w) pepsin to the first mixture and stir to obtain a second mixture, the enzyme hydrolysis temperature is 37℃, the enzyme hydrolysis pH is 2.0, and the enzyme hydrolysis time is 3h;

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

[0011] S40. Heat the third mixture to 65-70℃ and stir for 30min to inactivate the enzyme;

[0012] S50. Add 4% (w / w) alkaline protease to the third mixture after inactivation of the enzyme and stir to obtain a fourth mixture, the enzyme hydrolysis temperature is 50℃, the enzyme hydrolysis pH is 9.0, and the enzyme hydrolysis time is 3h;

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

[0014] S70. Centrifuge the fifth mixture after boiling water bath, take the supernatant and freeze-dry to obtain the sea cucumber peptide.

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

[0016] Wherein, boiling water bath for 10min;

[0017] Wherein, the pH is adjusted with 0.1mol / L HCl and / or NaOH.

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

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

[0020] Desalting the sea cucumber peptide according to the fourth aspect;

[0021] Performing liquid chromatography-mass spectrometry analysis on the desalted sea cucumber peptide;

[0022] Identifying peptide segments of the sea cucumber peptide according to the mass spectrometry data;

[0023] Analyzing the biological activity, toxicity, physicochemical properties, absorption, distribution and metabolism characteristics, excretion characteristics, and anti-oxidation, anti-inflammatory and neuroprotective activity of each peptide segment of the sea cucumber peptide;

[0024] Screening a potential sea cucumber peptide with anti-PD activity according to the analysis.

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

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

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

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

[0029] Advantages of the present application: The SCP mixture is used to treat Rot-induced C57BL / 6J mice and SH-SY5Y cells, and the behavior level, histopathology level, cell survival rate, and potential biological activity in vitro of the mice are evaluated. Peptidomics analysis combined with computer simulation analysis is used to construct a sea cucumber peptide spectrum, analyze and screen new sea cucumber peptides with potential anti-PD function. Rotenone (Rot) is used to induce 7-day-old zebrafish larvae and SH-SY5Y cells, and the anti-PD effect and mechanism of the candidate peptides are studied.

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

[0031] Figure 1 Figure S1. Experimental data graphs showing that SCP improves the behavioral deficits of rotenone-induced mice, wherein (A) is a schematic flow chart showing the experimental workflow; (B) is the time for mice to stay on the rotarod device; (C) is the length of the left forelimb stride of mice; (D) is the length of the right forelimb stride of mice; (E) is the length of the left hindlimb stride of mice; (F) is the length of the right hindlimb stride of mice; (G) is the forelimb stride distance of mice; (H) is the hindlimb stride distance of mice; n = 10 in each group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0032] Figure 2 Figure S2. Experimental data graphs showing that SCP reduces the dopaminergic neuronal damage of rotenone-induced mice, wherein (A) is a representative immunohistochemical micrograph of TH in the substantia nigra (SN) of mice; (B) is a quantitative evaluation of TH-positive neurons in the substantia nigra of mice; (C) is a representative immunohistochemical micrograph of TH in the striatum of mice; (D) is a quantitative analysis of the optical density of TH-positive neurons in the striatum of mice; (E) is a representative immunoblotting band of TH protein level in the substantia nigra of mice; (F) is a quantitative analysis of the ratio of TH to β-actin in the blot; n = 3 in each group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; scale bar = 100 μM.

[0033] Figure 3In 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 without 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 1 μM rotenone-induced SH-SY5Y cells; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; data are expressed as mean ± standard deviation (n=3).

[0034] Figure 4 Characterization of peptide identification and quantification, wherein (A) peptide segment count of different lengths; (B) distribution of peptide segment molecular weight; (C) abundance of peptide segments with precise amino acid composition; (D) peptide segment statistics with multiple unique amino acids; n=3 per group.

[0035] Figure 5 Computer strategy for screening potential new peptide segments with biological activity and bioavailability;

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

[0037] Figure 7 Sea cucumber peptide QWFDW improves the behavioral level of 7-day-old zebrafish larvae induced by rotenone; (A) Typical free swimming trajectory of zebrafish in each group, green path and red line represent the movement trajectory recorded by Viewpoint Zebrabox system; (B) Total swimming distance of zebrafish in 5 minutes; (C) 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 8Figure legends for experimental data graphs showing that QWFDW reduced intracellular ROS levels and alleviated mitochondrial membrane dysfunction, where (A) ROS expression was detected using a ROS fluorescent probe; (B) ROS fluorescence intensity was evaluated; (C) mitochondrial membrane potential was detected using a TMRE fluorescent probe; (D) TMRE fluorescence intensity was evaluated; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; data are presented as mean ± standard deviation (n = 3).

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

[0040] Figure 10 Chromatography 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 attached drawing figures, wherein the examples of embodiments are shown in the drawings and like or similar designations refer to like or to functionally similar elements throughout the whole drawing.

[0042] A. Explanation of terms:

[0043] Parkinson's disease (PD): the second most common neurodegenerative disease. It leads to motor impairment, including bradykinesia, resting tremor, and muscle rigidity, due to the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra (SN) pars compacta (SNpc) and the aggregation of alpha-synuclein (a-syn) in the structure of Lewy bodies (LB).

[0044] Bioactive peptides (BPs): a small fragment containing two or more amino acid residues, which has important nutritional value and various biological activities such as antioxidant, neuroprotective, and immunomodulatory activities.

[0045] Sea cucumber peptides (SCP): the hydrolysis or enzymatic products of sea cucumber proteins, which have numerous biological activities such as neuroprotection, antioxidant, ACE inhibition, immunomodulation, anti-fatigue, anti-aging, and trace mineral chelation.

[0046] B. Explanation of experimental materials:

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

[0050] The antibody against β-actin (1:1000, ab8227) was from Abeam (Cambridge, UK).

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

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

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

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

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

[0056] Cell Counting Kit-8 (CCK-8, C0041), reactive oxygen species assay kit with CM-H2DCFDA (S0035S), mitochondrial membrane potential assay kit with tetramethylrhodamine ethyl ester (TMRE, C2001S), BCA protein quantification kit and protein lysis buffer (P0009) were from Biyun Tian (Shanghai, China).

[0057] C. Examples (1-3):

[0058] Example 1: This example provides a sea cucumber peptide preparation method, which uses fresh Stichopus japonicus with an average size of about 20 centimeters long, obtained from the sea area of Dalian, China. The preparation specifically includes the following steps:

[0059] First, the sea cucumber body wall is broken into a slurry, and then the slurry sea cucumber and distilled water are mixed (10%, w / v) and placed in a reaction container.

[0060] At 37°C, the pH is adjusted to 2.0 with 0.1 mol / L HC1, and the enzyme is digested with pepsin (4% w / w, protein basis) for 3 h.

[0061] Adjust pH to 8 with 0.1 mol / L NaOH, and then stir the enzyme with trypsin (4% w / w, protein basis) for 3 h.

[0062] Warm up to 65-70℃, and then stir for 30 min.

[0063] Adjust temperature to 50℃, and then adjust pH to 9, and then stir the enzyme with alkaline protease (4% w / w, protein basis) for 3 h.

[0064] Then adjust pH to 6, and then stir the enzyme with papain (4% w / w, protein basis) for 3 h.

[0065] Finally, terminate the reaction in a boiling water bath for 10 min, and then centrifuge the mixture (10000 g, 20 min, 4℃), and then discard the precipitate.

[0066] Freeze-dry the supernatant, and then store it at -20℃ to obtain sea cucumber peptide powder X1.

[0067] Embodiment 2: The embodiment provides a method for constructing and analyzing a sea cucumber peptide mixture spectrum, which is used for preliminarily discovering a sea cucumber active peptide that may have a potential treatment effect on PD, and the method comprises the following steps:

[0068] S210. Extraction and desalination of the peptide;

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

[0070] S230. Peptide fragment identification and screening;

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

[0072] In the embodiment of the present application, step S210. Extraction and desalination of the peptide, comprises

[0073] According to the volume, 5 times of 50% (v / v) ethanol-water mixed extraction solution is used to fully stir the sea cucumber peptide powder X1, and then the mixture is oscillated overnight for extraction. After completion, the supernatant is freeze-dried by centrifugation at a speed of 5000-6000 rpm for 10-30 min.

[0074] After re-dissolving in pure water, 3 times of dichloromethane:n-butanol=4:1 (v / v) organic mixed reagent is added, and then the mixture is fully oscillated, centrifuged at a speed of 12000 rpm for 30 min, and then the polypeptide layer (aqueous phase) is taken.

[0075] To the MonoSpin C18 (SHIMADZU, Japan) desalting column, 200 μL of acetonitrile was added to activate the desalting column, centrifuged at 5000-6000 rpm for 2 min, 200 μL of 0.1% trifluoroacetic acid was added, centrifuged for 1 min x 2 times, 800 μL of polypeptide extract was continuously added, centrifuged at 5000-6000 rpm for 2 min, 200 μL of 0.1% trifluoroacetic acid was added, centrifuged at 5000-6000 rpm for 1 min, 200 μL of 60% acetonitrile was added, centrifuged at 5000-6000 rpm for 2 min, and the purified sample was obtained, and the sample solution was combined and freeze-dried for use.

[0076] wherein, in the embodiments of the present application, step S220. Liquid chromatography-mass spectrometry (LC-MS) analysis, comprising

[0077] The desalted polypeptide sample was dissolved in a 0.1% formic acid solution and filtered with a 0.22 μm membrane for testing. LCMS analysis was performed using an EASY-nLC 1200-Orbitrap Fusion Lumos LCMS system (Thermo Fisher, USA), a laboratory self-filled Nano column (C18, 75 μm ID, 3 um particle size) was used as the chromatographic column, the mobile phase A was a 0.1% (v / v) formic acid aqueous solution, the mobile phase B was a 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 spectrometry was detected in a DDA (Data Dependent Acquisition) mode, the ion mode was a positive ion mode, the full scan range of the mass spectrometry was m / z 350-1500, the resolution was 60000 FWHM. The charge amount (z) of the precursor ion was 1-7, MS2 scanning was performed on 20 most abundant ions, the activation type was HCD, the collision energy was set to 30%, the mass resolution of the tandem mass spectrometry 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 equally divided into three sections according to the ion distribution, and the secondary spectrum of single-charge and multi-charge precursor ions was collected by multiple injections, and the corresponding intervals were as follows: when z = 1, mz = 350-530, 520-695, and 685-1500; when mz = 2-7, mz = 350-540, 530-780, and 770-1500.

[0079] In the embodiments of the present application, step 230. peptide fragment identification and screening, includes

[0080] The mass spectrometry data was imported into PEAKS Studio 7.0 software (BSI, Canada) to identify the peptide fragments by using two ways of peptide-spectrum matches (PSM) and De Novo, so as to make the identification more comprehensive.

[0081] The PSM database was UniProt Sea Cucumber (30032 entries). The peptide sequence search parameters were set as follows: the precursor ion mass error was 10 ppm, the daughter ion mass error was 0.05 Da, the precursor ion mass search type was single isotope ion. No enzyme hydrolysis type was specified. The carbamidomethylation on cysteine (Cys) was specified as a fixed modification, the acetylation (N-term) at the N-terminus of the protein and the oxidation (M) of methionine were specified as variable modifications. The maximum number of peptide fragment 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 application adopts a label-free peptide group strategy to identify and quantify the peptide segment. In the PSM result, there are 2192 polypeptides with FDR (false discovery rate) <1%, i.e. -10lgP>20. In the De Novo result, there are 1201 polypeptides with ALC (average local confidence) ≥90%.

[0083] After removing the duplicate amino acid sequences from the two identification results, there are 3267 peptides left. According to the de-duplicated amino acid sequence of the peptide segment, the count and proportion of the peptide segment with different amino acid numbers are counted. Most of the identified peptides are short sequence peptides, and the count (proportion) of the peptides containing 4-9 amino acids is 143 (4.4%), 1101 (33.7%), 612 (18.7%), 446 (13.7%), 300 (9.2%), and 204 (6.2%). The proportion of the peptides with 5-7 amino acid numbers is more than 10%, and the number of the peptides with 5 amino acid numbers is the largest.

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

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

[0086] In the embodiments of the present application, step S240, computer simulation analysis of potential functional sea cucumber peptide sequences, includes

[0087] The identified peptides in the sea cucumber peptide spectrum were predicted for biological activity 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 that can have, the higher the score.

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

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

[0090] The excretion characteristics of the peptides, including "clearance" and "t1 / 2", were analyzed using ADMETlab2.0 (https: / / admetmesh.scbdd.com / service / screening / cal).

[0091] The activities of antioxidant, anti-inflammatory, neuroprotective, etc. were searched using the online databases BIOPEP-UWM (https: / / biochema.UWM.edu.pl / ) and Neuropeptide Database (isyslab.info / Neuropep / basic_search.jsp). The structural sequence novelty of the polypeptides was searched by SciFinder.

[0092] In step S240, in order to further screen new sea cucumber peptides in the sea cucumber peptide spectrum that can have therapeutic PD biological activity, computer simulation analysis methods were used to predict the potential biological activity and various physicochemical properties of the sea cucumber peptides. The active peptide screening strategy is as follows: Figure 5The PeptideRanker program was used to predict the biological activity of the 3267 polypeptide sequences with high reliability, with a threshold of 0.5, and 325 polypeptides with PeptideRanker values≥0.8, and 310 remaining after deduplication. Then, the potential toxicity, hydrophobicity, hydrophilicity, and relative molecular weight of the sea cucumber peptides were predicted according to the ToxinPred software. The human intestinal absorption (HIA), blood-brain barrier (BBB) penetration, and cytochrome P450 (CYP450) interaction of the peptides were analyzed by admetSAR2 to determine the absorption, distribution, and metabolism characteristics, respectively. The excretion characteristics of the peptides, including "clearance" and "t1 / 2", were analyzed by ADMETlab2.0. The activities of antioxidant, anti-inflammatory, neuroprotective, etc. were searched using the online databases BIOPEP-UWM and Neuropeptide Database. Finally, the novelty of the sea cucumber peptides was searched by SciFinder, and the biological activity prediction values PeptideRanker values, potential antioxidant, anti-inflammatory, neuroprotective activity, blood-brain barrier (BBB) penetration, toxicity, etc. of each peptide segment were comprehensively considered to screen out new polypeptide segments with good potential biological activity (Table 1). Among them, the new sea cucumber peptide shown as SEQ ID No. 1 scored the highest, which contains the typical acidic amino acid residue Asp, and its Trp and Phe are also important hydrophobic amino acid residues for providing antioxidant capacity. In addition, it has potential human intestinal absorption and blood-brain barrier penetration characteristics in computer simulation analysis, which also means that it can play a role in the brain. The new sea cucumber peptide shown as SEQ ID No. 1 can have potential therapeutic effects on PD. Among them, SEQ ID No. 1: Gln-Trp-Phe-Asp-Trp. (SEQ ID No. 1: QWFDW).

[0093] Table 1 One optimal new sea cucumber peptide sequence with potential treatment of 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 by Fmoc / tBu solid phase method.

[0096]

[0097] Firstly, 100-200 m of Wang resin is subjected to the operation of 1, which is 20% Piperidine (Remove the n-terminal Fmoc group with piperidine) reaction for 20 minutes, and the color detected by the detection reagent is blue. The operation of 2 is performed. The operation of 2 is HBTU+DIEA+Fmoc-Asp(otbu)-OH reaction for 1 hour, and the color detected by the detection reagent is colorless. Then, the operation of 1 is performed again, which is 20% Piperidine (Remove the n-terminal Fmoc group with piperidine) reaction for 20 minutes, and the color detected by the detection reagent is blue. The operation of 2 is performed. HBTU+DIEA+Fmoc-Phe-OH reaction for 1 hour, and the color detected by the detection reagent is colorless. Then, the operation of 1 is performed again, and so on until the last amino acid Gln is removed from the Fmoc. After that, the resin is dried and cut to obtain the crude polypeptide. The crude polypeptide is purified to obtain the desired polypeptide. Figures 9-10 The chromatographic and mass spectrometric analysis report of the sea cucumber peptide (SEQ ID No. 1: QWFDW).

[0098] D. Experimental explanation:

[0099] I. Mouse experiment: All animal experiments strictly follow the principles of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and have obtained the formal approval of the Institutional Animal Ethics Committee of Dalian Medical University. In addition, the animal experiment report of the present application also fully meets the requirements of the ARRIVE guidelines. 60 C57BL / 6J mice (male, 8 weeks old), weighing between 20-25 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. These mice were placed in standard experimental conditions and could freely access food and water. They were randomly divided into 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 high-dose SCP (1000 mg / kg / day) alone group (Con+L) (n=10). Rotenone was freshly prepared in an equal volume of DMSO: polyethylene glycol (PEG, 10%) solution every day, and was given to mice intraperitoneally (1.5 mg / kg / d) every day for 21 consecutive days, and the blank group was injected with an equal amount of solvent. SCP was dissolved in normal saline, and 30 minutes after injection, the mice were given intragastrically according to the group for 21 consecutive days, and the blank group was given intragastrically with an equal amount of normal saline.

[0100] 1.1. Rotarod test: The rotarod test was used to evaluate the behavioral ability of mice. Briefly, mice were trained on the rotarod for 3 consecutive days before the formal test. During the training, each mouse was allowed to move freely at a constant speed of 22 rpm, and if the mouse fell, it was placed on the device again until the end of 300 s. In the formal test, the speed of the rotarod instrument was accelerated from 4 rpm to 50 rpm within 300 s. If the mouse fell, the falling time and the current speed of the mouse were recorded. Each mouse was tested 3 times with an interval of 2 hours. The average time of 3 repeated tests was statistically analyzed.

[0101] 1.2. Gait analysis: Gait analysis was performed as follows. Briefly, mice were placed in a runway that was 50 cm long and 10 cm wide, and all mice were trained to walk along the runway. Then the forelimbs and hindlimbs of the mice were dyed with black ink and red ink, respectively, and the mice were allowed to freely leave footprints on white paper during exercise. In the formal test, 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 steps of each mouse were recorded. Then the step length and stride distance of the four limbs were analyzed.

[0102] 1.3. Immunohistochemistry: Mouse brain tissue samples were obtained after cardiac perfusion of mice with pre-chilled PBS and 4% formaldehyde. Then, mouse brain tissues were re-fixed with 4% paraformaldehyde for 48 h at 4 °C, and then the brain tissues were transferred into 30% sucrose solution until they sank to the bottom. The coronal brain sections were cut about 30 pm thick with a Leica cryostat microtome (CM 1950, Leica, Wetzlar, Germany) free-floating. For immunohistochemistry experiments, briefly, after washing the sections with PBS, and inactivating peroxidase by incubating with 3% H2O2 for 10 min at room temperature. 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, overnight at 4 °C, the sections were incubated with TH (1 : 1000) antibody. The next day, after washing the sections with PBS, the sections were incubated with HRP-conjugated secondary antibody (ZK1030, Vector, Shenzhen, China) for 1 h at room temperature. After washing the sections with PBS, the sections were incubated with ABC kit (ZK1030, Vector, Shenzhen, China) for 1 h at room temperature. Then, after washing the sections with PBS, the sections were incubated with 3-3’ diaminobenzidine (34002, Thermo Fisher Scientific, Waltham, MA, USA) to visualize the binding signal. Finally, the sections were scanned by digital slide scanner KF-PRO-020 (Kfbio, Ningbo, China). The brain sections were analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

[0103] 1.4. Western Blot Analysis: Western blot experiment mouse brain tissue material was obtained from PBS only perfused mice. The samples were mixed homogenate with 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) and protein was extracted. Then the resulting protein concentration was measured with BCA kit (P0009, Beyotime, Shanghai, China). Each sample was loaded according to equal amount of protein and sample protein was separated by electrophoresis in 10% SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF) membrane (IPVH 07850, Waltham, MA, USA). Next, the membrane was blocked with Superblocking Buffer (37515, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. After that, the membrane was incubated with primary antibody at 4°C overnight. The next day, the membrane was washed with Tris-buffered saline with Tween 20 and incubated with HRP-labeled secondary antibody (ab 6721, Abeam, Cambridge, UK) for 2 h at room temperature. Finally, the membrane was evenly covered with ECL reagent (P0018 S, Beyotime, Shanghai, China) and imaged with an imaging system (Bio-Rad, Hercules, CA, USA). Finally, ImageLab (4.0) software was used for quantitative analysis.

[0104] II. In vitro evaluation of potential antioxidant capacity and biological activity, including ABTS, DPPH, and hydroxyl radical scavenging capacity assays, using ABTS radical scavenging capacity assay kit, DPPH radical scavenging capacity assay kit, and hydroxyl radical scavenging capacity assay kit purchased from Solarbio, and following the manufacturer's instructions.

[0105] III. Cell experiments: Human neuroblastoma cell line (SH-SY5Y) cells were cultured in Dulbecco's Modified Eagle Medium (11965118, Gibco, Waltham, MA, USA) mixed homogeneously with 10% fetal bovine serum (A5669701, Gibco, Waltham, MA, USA) and 1% penicillin-streptomycin solution (15140122, Thermo, Waltham, MA, USA) at 37°C in a humidified atmosphere with 5% CO2.

[0106] 3.1. Cell viability and toxicity detection (CCK-8): The enhanced cell counting kit-8 (CCK-8, C0041) was purchased from Biyun Tian Company (Shanghai, China). Briefly, SH-SY5Y cells were seeded in 96-well plates and incubated until reaching 90% confluence. Subsequently, 100 μL of DMEM medium supplemented with 10% CCK-8 reagent was added to each well and incubated at 37°C in the dark for 1 hour. Then, the absorbance was measured at 450 nm using a microplate reader (Infinite 200 PRO, USA).

[0107] 3.2. ROS generation detection: In a 24-well plate, 1000 μL of fresh medium without serum was added. SH-SY5Y cells were divided into four groups: Con (Rot, QWFDW concentration is 0), only 1 μM Rot, 1 μM Rot with 10 μM QWFDW, only 10 μM QWFDW, cultured for 24 h, and allowed to grow and stabilize. Then according to the DCFH-DA kit experimental method, the probe was added, and observed and photographed under an inverted fluorescence microscope

[0108] 3.3. Mitochondrial membrane potential detection: TMRE fluorescent probe was from Biyun Tian. According to the TMRE kit method, it was processed and observed and photographed under an inverted fluorescence microscope.

[0109] IV. Zebrafish experiments: Zebrafish wild-type AB were provided by the National Zebrafish Resource Center. Fish were maintained in water (200 mg instant sea salt / 1 L reverse osmosis water; conductivity, 480-510 μ8 / cm; pH 6.9-7.2; hardness, 53.7-71.6 mg / L CaCO3) at 28 °C with a 14 h light / 10 h dark cycle, fed once daily with dry flakes and twice daily with live saltwater brine shrimp. Feeding management was in accordance with the requirements of the Association for Assessment and Accreditation of Laboratory Animal Care International. Embryos were collected by natural spawning and incubated in E3 embryo medium (5 mM NaCl, 0.17 mM KC1, 0.33 mM CaCl2, 0.33 mM MgSO4). The experiment was divided into four groups: Con group (0.01% DMSO only), Rot group only, Rot and 10 μΜ QWFDW co-treatment group (Rot + QWFDW), and Con group with only 10 μΜ QWFDW added (Con + QWFDW). At 3 dpf, 20 larvae were placed in each well of a 6-well plate for each group, with a solution volume of 3 mL. Larvae were treated from 3 dpf to 7 dpf, with normal replacement of the solution every day. Rot was prepared at a concentration of 100 mM in 100% dimethyl sulfoxide (DMSO), then serially diluted in 10% water diluted DMSO to 1,000,000 times to the working concentration of 50 nM. The rotenone control was exposed to a 0.01% DMSO solution, and all treatments were performed in the dark.

[0110] Behavioral assessment: The behavioral level of swimming activity of 7 d old zebrafish larvae was detected after the end of administration. Individual larvae were transferred to E3 embryo medium in a 24-well plate and acclimated for 15 min under ambient light before recording. Activity was recorded using Zebralab software and a Zebrabox tracking system (ViewPoint Life Science, Lyon, France). Larval swimming activity was performed for 3 times, 5 min each time. The total distance traveled each time and the average speed of 5 min test were counted, and the speed was set to high active state at 5 mm / s, and low active state at 0-5 mm / s. Ten samples were selected from each group for behavioral assessment. The data integrity of statistical analysis was ensured by strict experimental design, and each measurement was repeated three times, both technically and biologically. The results were presented as mean ± standard error (SEM). Statistical analysis was performed using GraphPad Prism version 8.0.1, and one-way or two-way ANOVA was used to identify significant differences. A threshold of p < 0.05 was set to represent statistical significance, ensuring that both reliable and robust findings were discovered.

[0111] E. Experimental results analysis:

[0112] E1. Sea cucumber peptide (X1) SCP result analysis:

[0113] I. Mouse experiment:

[0114] SCP alleviates Rot-induced behavioral impairment in mice: To investigate whether SCP can play an anti-Parkinson's disease role in vivo, the present application uses 8-month-old C57BL / 6J mice to construct an animal PD model by intraperitoneal injection of Rot (1.5 mg / kg / day) to the model group mice every day, and to treat the treatment group mice by intragastrically administering different concentrations of SCP every day. After 21 days of continuous treatment, the mice are subjected to behavioral tests Figure 1 A). The rotarod test and gait test of mice are classic experiments for studying the motor behavior of mice and important methods for studying the behavior of PD.

[0115] 1.1. The rotarod test performed on mice is to evaluate their motor coordination and balance. Compared with the control group, the Con+H group shows no significant difference, indicating that a high concentration of SCP does not impair the motor function of normal mice. On the contrary, compared with the control group, the Rot group reduces the time spent on the rotating rod. After treatment with 250, 500, and 1000 mg / kg of SCP, Rot-induced motor impairment is significantly alleviated, and the higher the concentration of SCP, the better the protective effect, thereby proving the protective effect of SCP on the motor ability of mice subjected to Rot challenge Figure 1 B).

[0116] 1.2. The gait test of mice can often directly reflect the motor behavior impairment of mice. Compared with the Con group, the Rot group significantly reduces the stride of the forelimbs and hindlimbs of mice, and after treatment with SCP, the reduction in stride is significantly inhibited. In addition, the C+H group shows no significant change compared with the Con group Figure 1 C-F). In addition, for the hindlimb spacing of mice, the Rot group has a certain extension compared with the Con, and after treatment with SCP, the spacing is dose-dependently shortened. However, the forelimb spacing of mice shows no significant change Figure 1 G-H). The results show that SCP can effectively alleviate Rot-induced behavioral impairment in mice.

[0117] SCP prevents the loss of dopaminergic neurons in the substantia nigra: The pathological features of PD include the degeneration of dopaminergic neurons in the substantia nigra compacta of the midbrain. To investigate whether SCP can alleviate Rot-induced PD model in mice, the present application studies 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.

[0118] 1.3. Immunohistochemistry results showed that there was a significant loss of TH+ neurons in the SNpc of mice in the Rot group compared with the Con group, while the administration of SCP alleviated the loss of TH+ neurons in the SNpc in a dose-dependent manner Figure 2 A). 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. Compared with the Rot group, the number of TH+ neurons in the SCP administration groups was significantly increased Figure 2 B). Secondly, the present application found that in the striatum, compared with the Con group, there was no significant difference in the C+H group, while the TH immunostaining of the Rot group was significantly reduced. After administration of SCP treatment, the treatment group alleviated this phenomenon in a dose-dependent manner Figure 2 C). The TH immunostaining density statistics of the mouse striatum were consistent with the histological observation results Figure 2 D). The present application further confirmed the neuroprotective effect of SCP by detecting the expression level of TH protein in the mouse midbrain.

[0119] 1.4. Western blot results were consistent with immunohistochemistry, showing that Rot induced a significant reduction in the expression level of TH in the mouse midbrain, while this damage was alleviated in a dose-dependent manner after administration of SCP Figure 2 E). Compared with the Con group, there was no significant difference between the Con group and the C+H group. Compared with the Rot group, the TH / β-Actin of the R+L, R+M, R+H groups administered with SCP was 1.342 times, 1.534 times, and 1.859 times, respectively Figure 2 F). The experiment confirmed that SCP can play a role in treating PD by preventing the loss of dopaminergic neurons in the substantia nigra.

[0120] II. In vitro evaluation of potential antioxidant capacity and biological activity:

[0121] Evaluation of the in vitro biological activity of SCP and its effect on the survival rate of rotenone (Rot)-induced SH-SY5Y cells: The function of bioactive peptides is closely related to their antioxidant capacity, and the potential biological activity of SCP mixtures can be better analyzed by in vitro antioxidant activity. 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 is easily reacted with antioxidants in aqueous systems. ·OH scavenging rate 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 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 might have good antioxidant capacity, which was consistent with previous reports.

[0122] III. Cell experiment:

[0123] 3.1. To determine the toxicity of sea cucumber peptide extract on SH-SY5Y cells, the cell viability of different concentrations of sea cucumber peptide mixture was determined using CCK-8 kit. Between the Con group and the drug administration group, it was observed that the cells in the drug administration group had a certain cell proliferation phenomenon, and when the concentration of sea cucumber peptide extract was 50 μg / mL, the promotion of cell proliferation was significant Figure 3 D). This showed that the SCP mixture had no significant toxic effect on cells, but had a certain promoting effect on cell proliferation. To evaluate the potential neuroprotective effect of sea cucumber peptide mixture, SH-SY5Y cells were exposed to 1 μM Rot, then treated with 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, after being given sea cucumber peptide mixture with a concentration of 10, 25 and 50 μg / mL, the survival rate of cells was significantly improved Figure 3 E). Therefore, the sea cucumber peptide mixture had a potential neuroprotective effect on Rot-induced SH-SY5Y cells. This showed that in the sea cucumber peptide mixture, there might be a certain sea cucumber polypeptide that had a protective effect on Rot-induced PD.

[0124] E2. Analysis of sea cucumber peptide (SEQ ID No. 1) results:

[0125] II. In vitro evaluation of potential antioxidant capacity and biological activity:

[0126] The sea cucumber peptide (SEQ ID No. 1) in vitro activity experiment was evaluated to further screen out the peptide segment with potential anti-PD activity. In the ABTS radical scavenging rate, DPPH radical scavenging rate and hydroxyl radical scavenging rate experiments, the sea cucumber peptide (SEQ ID No. 1) with a concentration of 10 mM was evaluated. The results showed that the ABTS radical scavenging rate Figure 6 A) of sea cucumber peptide (SEQ ID No. 1) was high, and it might have better in vitro antioxidant capacity. Figure 6 B) and hydroxyl radical scavenging rate Figure 6 C) were high.

[0127] III. Cell experiment:

[0128] 3.1. In order to determine the toxicity of sea cucumber peptide (SEQ ID No. 1) to SH-SY5Y cells, the present application administered sea cucumber peptide (SEQ ID No. 1) to the cells at a concentration of 10 μM for 24 h of co-culture. The results showed that compared with the Con group, no obvious cell proliferation and cell death phenomenon was 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 application found that 10 μM sea cucumber peptide (SEQ ID No. 1) can significantly improve the survival rate of cells in Rot-induced SH-SY5Y cells compared with the Rot group Figure 6 D), the protection activity is obvious, therefore sea cucumber peptide (SEQ ID No. 1) has good potential for treating PD.

[0129] Effect of sea cucumber peptide (SEQ ID No. 1) on rotenone (Rot) induced SH-SY5Y cells: a large increase in intracellular ROS levels and damage to mitochondria are often important causes of PD.

[0130] In order to further study how sea cucumber peptide (SEQ ID No. 1) reduces Rot-induced damage to SH-SY5Y cells and plays a role in relieving PD, the intracellular ROS level and the mitochondrial membrane potential level were detected.

[0131] 3.2. The results of ROS generation detection showed that Rot induced a significant increase in intracellular ROS levels, and after administration of sea cucumber peptide (SEQ ID No. 1), the intracellular ROS level was down-regulated, as shown in Figure 8 A-8B.

[0132] 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 administration of sea cucumber peptide (SEQ ID No. 1), compared with the Rot group, the fluorescence intensity of mitochondrial membrane potential was significantly increased, effectively reducing the damage of Rot to the mitochondrial membrane potential of the cells Figure 8 C-8D).

[0133] The results of cell experiments showed that sea cucumber peptide (SEQ ID No. 1) may play an anti-PD biological activity by reducing intracellular ROS levels and reducing mitochondrial membrane potential damage.

[0134] Four. Zebrafish experiments:

[0135] Zebrafish has been proved to be a powerful in vivo vertebrate model for the research and high-throughput drug screening of various neurodegenerative diseases. The present application further studies whether sea cucumber peptide (SEQ ID No. 1) has neuroprotective activity in vivo by the changes of behavioral level of 7d-old zebrafish larvae induced by Rot. As shown in Figure 7 A, 7B, 7C, the results show that there is no significant change in Con+QWFDW group compared with Con, while the zebrafish in Rot group shows changes of reduced motor level and decreased active state. Rot+QWFDW group can effectively improve this Rot-induced motor level decline. The analysis of 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 significantly decrease compared with Con group. After the administration of sea cucumber peptide (SEQ ID No. 1), the total swimming distance and average swimming speed of zebrafish are significantly increased. Therefore, the present application finds that the novel sea cucumber peptide (SEQ ID No. 1) can improve the behavioral level of 7d-old zebrafish larvae induced by Rot.

[0136] The above is only a preferred embodiment of the present application, but the protection scope of the present application is not limited thereto, any skilled person in the art can make equivalent replacement or change according to the technical scheme and inventive concept of the present application within the disclosed technical scope, which should be covered in the protection scope of the present application.

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, It was synthesized by the Fmoc / tBu solid-state method.

3. A method for preparing sea cucumber peptides, characterized in that, Includes the following steps: S10. A mixture of slurry seaweed and distilled water is prepared in a reaction vessel to obtain a first mixture; S20. Add 4% (w / w) pepsin to the first mixture and stir to hydrolyze it to obtain the second mixture. The hydrolysis temperature is 37℃, the hydrolysis pH is 2.0, and the hydrolysis time is 3h. S30. Add 4% (w / w) trypsin to the second mixture and stir to hydrolyze to obtain the third mixture. The hydrolysis temperature is 37℃, the hydrolysis pH is 8.0, and the hydrolysis time is 3h. S40. Heat the third mixture to 65-70℃ and stir for 30 minutes to inactivate the enzyme; S50. Add 4% (w / w) alkaline protease to the third mixture after enzyme inactivation and stir to obtain the fourth mixture. The enzymatic hydrolysis temperature is 50℃, the enzymatic hydrolysis pH is 9.0, and the enzymatic hydrolysis time is 3h. S60. Add 4% (w / w) papain of the mixture to the fourth mixture and stir to obtain the fifth mixture. The enzymatic hydrolysis temperature is 50℃, the enzymatic hydrolysis pH is 6.0, and the enzymatic hydrolysis time is 3h. S70. After centrifuging the fifth mixture in a boiling water bath, the supernatant is collected and freeze-dried to obtain the sea cucumber peptide.

4. The preparation method according to claim 3, characterized in that, in, The slurry seaweed was mixed with 10% (w / v) distilled water and placed in the reaction vessel; Among them, boiling water bath for 10 minutes; The pH is adjusted using 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 anti-PD active sea cucumber peptides, characterized in that, Includes the following steps: Desalting the sea cucumber peptides described in claim 5; The desalted sea cucumber peptides were analyzed by liquid chromatography-mass spectrometry. Identification of sea cucumber peptide fragments based on mass spectrometry data; The bioactivity, toxicity, physicochemical properties, absorption, distribution and metabolic characteristics, excretion properties, and antioxidant, anti-inflammatory and neuroprotective activities of each peptide fragment of sea cucumber peptide were analyzed. Based on the analysis, potential anti-PD active sea cucumber peptides were screened.

7. Use of the peptide of claim 1 or 5 in the preparation of a medicament for treating Parkinson's disease.

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