Polypeptide with neuroprotective function and preparation method and application thereof

Abstract

The present invention relates to a polypeptide with neuroprotective function and a preparation method and application thereof, and relates to the technical field of functional polypeptides. A polypeptide with neuroprotective function, the amino acid sequence of which is shown in SEQ ID NO.1. The present invention also discloses the use of the above-mentioned polypeptide as a pharmaceutical ingredient in the preparation of a drug for treating Parkinson's disease (PD). The present invention discloses for the first time three polypeptide compounds containing 6-7 amino acid residues extracted from Spirulina C-phycocyanin (C-PC). Through detection, it is found that the polypeptide compound can alleviate the vascular damage and PD behavior of the zebrafish brain caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methy1-4-pheny1-1,2,3,6-terahydro-pyridine, MPTP), protect dopamine neurons, regulate the abnormal expression of genes related to PD, have very significant anti-PD activity, can be used for the subsequent development of drugs for the prevention or treatment of PD, and has broad market prospects.

Description

Technical Field

[0001] The present invention relates to a polypeptide with neuroprotective function and a preparation method and application thereof, belonging to the technical field of functional polypeptides. Background Art

[0002] Parkinson's disease (PD) is a common age-related neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra (SNpc). The clinical symptoms of the disease are mainly movement disorders including body rigidity, static tremor, postural instability, and non-motor symptoms including cognitive impairment, sleep disorders and autonomic dysfunction. PD is a multifactorial disease involving multiple molecular and cellular mechanisms, such as oxidative stress, apoptosis, neuroinflammation, autophagy and dysbiosis. Currently, the most common PD drug treatments focus on the replacement of substantia nigra dopamine (DA) neurons, but there are adverse effects that may cause severe movement disorders or abnormal involuntary movements. Therefore, low-efficiency natural dietary antioxidants become the best choice.

[0003] The ocean covers about 70% of the Earth's surface and is one of the richest components of the Earth's biosphere, containing more than 80% of the world's plant and animal species. Due to its vastness, the marine environment is extremely rich in biodiversity and chemical diversity compared to terrestrial plants and animals, so the ocean is considered to be a natural treasure trove of new bioactive molecules. Many studies have shown that bioactive peptides from the ocean show efficacy as potential drug candidates, with multiple functions such as antioxidant, anti-inflammatory, anticancer, antibacterial, and treatment of neurodegenerative diseases. Compared with proteins or antibodies, marine peptides have some significant advantages, such as small size, high safety and low side effects, simple synthesis, easy modification, cell membrane cross-capability, and low drug-drug interaction. Recent studies have increasingly focused on bioactive peptides isolated from marine organisms.

[0004] Spirulina ( Spirulina ) is a blue-green cyanobacteria that has attracted much attention in recent years for its unique medicinal value. Studies have shown that the phycobiliprotein C-phycocyanin (C-PC) and other phenolic phytochemicals from Spirulina have strong antioxidant, anti-inflammatory and neuroprotective effects. The C-PC content in Spirulina is as high as 60%. It is a light-harvesting pigment protein composed of α and β subunits. So far, most studies on C-PC have focused on biology, such as the genome, nutrition, nutritional components and biological functions of C-PC macromolecules. There are currently few reports on C-PC bioactive peptides, and only early studies have found that α and β subunits have anti-cancer and reproductive functions. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a polypeptide with neuroprotective function and a preparation method and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] The amino acid sequences of three polypeptides with neuroprotective function are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0008] SEQ ID NO.1: Met-Ala-Ala-Ala-His-Arg (MAAAHR);

[0009] SEQ ID NO.2: Met-Pro-Gln-Pro-Pro-Ala-Lys (MPQPPAK);

[0010] SEQ ID NO. 3: Met-Thr-Ala-Ala-Ala-Arg (MTAAAR).

[0011] A method for extracting the above polypeptide, comprising the following steps:

[0012] S1. Preparation of Phycocyanin (C-PC) Hydrolysate

[0013] Weigh 1g C-PC powder and dissolve it in 60.64 mL pure water. Use trypsin to hydrolyze C-PC. The conditions for enzymatic hydrolysis are: enzyme addition amount 55.18 mg, temperature 49.4 ℃, pH 8, time 120 min. After the hydrolysis is completed, inactivate the enzyme in a 95 ℃ water bath for 15 min. The obtained hydrolyzate was centrifuged at 4 ℃ and 12000r / min for 20 min, and the supernatant was collected and filtered with a 0.45 μm regenerated cellulose filter membrane, rotary evaporated, and concentrated to obtain the C-PC enzymatic hydrolyzate (C-PCH);

[0014] S2. Preparation of peptide mixed stock solution

[0015] The C-PCH prepared in step S1 was separated by Sephadex G-100. 500 mg of freeze-dried C-PCH was dissolved in 2 mL of deionized water, filtered through a 0.45 μm microporous membrane and loaded onto a Sephadex column (2.0x40 cm). The eluent was deionized water at a flow rate of 0.5 mL / min. One portion was collected every 20 min. The fractions were combined and concentrated according to the results of thin layer chromatography analysis to obtain a peptide mixed stock solution.

[0016] S3. Preparation of crude extracts of active peptide segments

[0017] The polypeptide mixed stock solution prepared in step S2 was dissolved in 50% methanol-water, filtered through a 0.45 μm microporous membrane, and then separated through Sephadex LH-20, using 50% methanol-water as the eluent, collecting samples at a rate of 0.25 mL / min, collecting one portion every 40 minutes, combining the same active segment eluates, and concentrating to obtain a crude extract of the polypeptide active segment;

[0018] S4. Final preparation

[0019] The crude extract of the active segment of the polypeptide obtained in step S3 was dissolved in ultrapure water to prepare a 10 mg / mL solution, filtered through a 0.45 μm microporous membrane, and the filtrate was passed through a preparative C 18 RP-HPLC(ZORBAX, 300Extent-C 18 ) was further separated and purified, with mobile phase A being water and mobile phase B being acetonitrile. Gradient elution was used, with mobile phase B ranging from 1% to 100% in six column volumes at a flow rate of 0.8 mL / min. The eluate with an absorption peak at 214 nm was collected to screen for multifunctional active polypeptides. After concentration and drying, polypeptides with neuroprotective function were obtained.

[0020] Preferably, in step S1, the concentration is performed by rotary evaporation at 38-40°C to remove water from the solution.

[0021] Preferably, in step S2, the vacuum concentration conditions are as follows: the vacuum degree is 0.08-0.1 MPa and the water bath temperature is 40-45°C.

[0022] Preferably, in step S4, ZORBAX, 300Extent-C 18 The column specifications are 4.6×250mm, 5 μm.

[0023] Preferably, according to step S4, the zebrafish dopamine neurons, brain blood vessels and behavioral evaluation models are used to screen out the fractions with the highest activity in the three evaluation models.

[0024] Preferably, in step S4, the drying condition is freeze-drying at -20°C.

[0025] The above-mentioned polypeptide with neuroprotective function is used as a medicinal ingredient in the preparation of drugs for treating Parkinson's disease (PD).

[0026] The present invention provides a polypeptide having a neuroprotective function and a preparation method and application thereof. It has the following beneficial effects:

[0027] The present invention discloses for the first time three polypeptide compounds containing 6-7 amino acid residues extracted from C-PC. Through detection, it is found that the polypeptide compounds can alleviate the vascular damage and PD behavior in the zebrafish brain caused by MPTP, protect dopamine neurons, regulate the abnormal expression of PD-related genes, have very significant anti-PD activity, can be used for the subsequent development of PD prevention and / or treatment drugs, and have broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Peak diagram of the product after separation and purification by Sephadex G-100;

[0029] Figure 2 The results of scavenging ability of DPPH and ABTS free radicals of each fraction eluted by Sephadex G-100;

[0030] Figure 3 Peak diagram of the product after separation and purification by Sephadex LH-20;

[0031] Figure 4 The results of scavenging ability of DPPH and ABTS free radicals of each fraction eluted by Sephadex LH-20;

[0032] Figure 5 Peak diagram of the product after separation and purification by RP-HPLC;

[0033] Figure 6 Results of dopamine neurons, brain blood vessels and PD behavioral tests in each fraction eluted by RP-HPLC;

[0034] Figure 7 The effect of peptide samples on the repair of zebrafish dopamine neurons and brain blood vessels;

[0035] Figure 8 The effect of peptide samples on relieving zebrafish PD behavior;

[0036] Fig. 9 The results of the peptide samples' effects on the production of reactive oxygen species (ROS) in zebrafish induced by MPTP;

[0037] Fig.10 The results of the peptide samples' effects on the number of apoptotic cells in the zebrafish brain induced by MPTP;

[0038] Fig.11 Results of the effects of peptide samples on the expression of PD-related genes;

[0039] Fig.12 This is a graph showing the effects of the polypeptide sample in Comparative Example 1 on zebrafish dopamine neurons, brain blood vessels and PD behavior. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Sources of biological materials

[0042] The C-phycocyanin (C-PC) powder described in the examples was purchased from Zhejiang Binmei Biotechnology Co., Ltd. Example

[0043] A method for extracting a polypeptide having a neuroprotective function, comprising the following steps:

[0044] S1. Preparation of Phycocyanin (C-PC) Hydrolysate

[0045] Weigh 1g C-PC powder and dissolve it in 60.64 mL pure water. Use trypsin to hydrolyze C-PC. The conditions for enzymatic hydrolysis are: enzyme addition amount 55.18 mg, temperature 49.4 ℃, pH 8, time 120 min. After the hydrolysis is completed, inactivate the enzyme in a 95 ℃ water bath for 15 min. The obtained hydrolyzate was centrifuged at 4 ℃ and 12000 r / min for 20 min, and the supernatant was collected and filtered with a 0.45 μm regenerated cellulose filter membrane, rotary evaporated, and concentrated to obtain the C-PC enzymatic hydrolyzate (C-PCH);

[0046] S2. Preparation of peptide mixed stock solution

[0047] The C-PCH prepared in step S1 was separated by Sephadex G-100. 500 mg of freeze-dried C-PCH was dissolved in 2 mL of deionized water, filtered through a 0.45 μm microporous membrane and loaded onto a Sephadex column (2.0x40 cm). The eluent was deionized water at a flow rate of 0.5 mL / min. One portion was collected every 20 min. The fractions were combined and concentrated according to the results of thin layer chromatography analysis to obtain a peptide mixed stock solution.

[0048] S3. Preparation of crude extracts of active peptide segments

[0049] The polypeptide mixed stock solution prepared in step S2 was dissolved in 50% methanol-water, filtered through a 0.45 μm microporous membrane, and then separated through Sephadex LH-20, using 50% methanol-water as the eluent, collecting samples at a rate of 0.25 mL / min, collecting one portion every 40 minutes, combining the same active segment eluates, and concentrating to obtain a crude extract of the polypeptide active segment;

[0050] S4. Final preparation

[0051] The crude extract of the active segment of the polypeptide obtained in step S3 was dissolved in ultrapure water to prepare a 10 mg / mL solution, filtered through a 0.45 μm microporous membrane, and the filtrate was passed through a preparative C 18 RP-HPLC(ZORBAX, 300Extent-C 18 ) was further separated and purified, with mobile phase A being water and mobile phase B being acetonitrile. Gradient elution was used, with mobile phase B ranging from 1% to 100% in six column volumes at a flow rate of 0.8 mL / min. The eluate with an absorption peak at 214 nm was collected to screen for multifunctional active polypeptides. After concentration and drying, polypeptides with neuroprotective function were obtained.

[0052] After testing, the amino acid sequence of the polypeptide with neuroprotective function is shown as SEQ ID NO.1. Example

[0053] The polypeptide with the amino acid sequence shown in SEQ ID NO.1 was artificially synthesized by the Fmoc solid phase synthesis method described by Liu Zhennan et al. (Liu Zhennan, Huang Qiang. Fmoc solid phase synthesis method. Journal of Guangxi University for Nationalities, 1999, 5(2): 110-112).

[0054] Comparative Example 1:

[0055] As described in Example 1, except that the fraction A was separated and purified in step S4 and then subjected to mass spectrometry to obtain four polypeptide sequences (P4: MNIGQK; P5: MAAATR; P6: MSAGER; P7: MVTVDR). No significant anti-PD activity was observed in zebrafish dopamine neurons, brain vessels, and behavioral tests ( Fig.12 ). This indicates that the active antioxidant polypeptide is only present in the Fraction B fraction. Therefore, the sample prepared in step S4 is used as the test sample for the next neuroprotection evaluation experiment.

[0056] Experimental example

[0057] DPPH free radical scavenging ability

[0058] The DPPH free radical scavenging ability of the sample peptides was tested according to the method described by Lee et al. with appropriate modifications.

[0059] 1 ml of sample was added to 1 ml of 0.15 mmol·L -1 The mixture was centrifuged and mixed with DPPH ethanol solution. Then, the mixture was reacted for 30 min in the dark. Ultrapure water was used as a control. The absorbance of each sample was measured at 517 nm and the determination was performed in triplicate. The DPPH free radical scavenging rate was calculated as follows:

[0060] DPPH free radical scavenging rate (%) = (1-As / Ac) × 100

[0061] Where As is the absorbance of the sample and Ac is the absorbance of the reference

[0062] ABTS free radical scavenging ability

[0063] According to the method described by Ji Chenxi et al., with appropriate modifications, the ABTS free radical scavenging ability of the sample peptides was detected.

[0064] Take 10 ml 7.4 mmol·L -1 ABTS solution and 10 ml of 2.6 mmol·L -1 After mixing well, react at room temperature in the dark for 12 h~16 h to obtain ABTS + Stock solution. + The stock solution was diluted with anhydrous ethanol to an absorbance of 0.70 ± 0.002, i.e. ABTS + Working solution. Mix 600 μL of sample with 5.0 mL ABTS + Mix the working solution, shake well and react for 15 min, and measure the absorbance of each sample at 734 nm as A. Take 5.0 mL of ethanol solution and add 600 μL of sample solution, shake well and react for 15 min, and measure the absorbance of each sample at 734 nm as A. j . Take 5.0 mL ABTS + Add 600 μL of ultrapure water to the working solution and measure its absorbance value A0 at 734 nm. Perform the measurement in triplicate. The ABTS free radical scavenging rate is calculated as follows:

[0065] ABTS free radical scavenging rate (%) = {1-(AA j ) / A0}×100

[0066] Detection of dopamine neurons and brain blood vessels

[0067] The transgenic zebrafish larvae Vmat:GFP and Fli1:GFP in each group at 96 hpf were placed under a Zeiss stereofluorescence microscope to observe the development of dopamine neurons and brain blood vessels, respectively.

[0068] Behavioral testing

[0069] The 144 h old zebrafish larvae from the control group, model group, and different concentrations of peptide sample groups were collected, washed, and aspirated into a 48-well plate, one per well, and 500 μl of fish water was added to each well. The zebrafish were placed in a Zebra-box zebrafish behavior analyzer dark box suitable environment. After 15 min, the movement trajectory of each larva was recorded for 20 minutes using an automatic computer video tracking system (Viewpoint, Lyon, France). Zeblab software (Viewpoint, Lyon, France) was used to analyze the digital trajectory, and the average speed was analyzed every 60 seconds.

[0070] Determination of reactive oxygen species (ROS) levels

[0071] By using wild-type zebrafish strain AB, the level of ROS production in zebrafish induced by MPTP of each polypeptide sample was detected.

[0072] ROS levels in zebrafish larvae were measured using 2,7-dichlorofluorescein diacetate (DCF-DA) probe. Zebrafish at 72 hpf were placed in 24-well plates supplemented with DCF-DA (10 mg / mL) and purified water to a final concentration of 10 μg / mL and incubated at 28°C in the dark for 1 h. Afterwards, each group was washed with PBS and photographed in a fluorescence microscope (Olympus, Tokyo, Japan). Fluorescence intensity was quantified by Image Pro-Plus (IPP).

[0073] Acridine orange staining

[0074] The AB zebrafish treated with MPTP and peptide samples for 72 hours were drained with a pipette, washed three times with PBS, and then incubated in a 37 ℃ incubator in the dark for 30 minutes. After 30 minutes, the zebrafish were washed three times with PBS, each time for 10 minutes. Finally, the zebrafish were anesthetized and placed on a glass slide with the abdomen facing down, and images of zebrafish brain cell apoptosis were collected using a fluorescence microscope.

[0075] Real-time quantitative polymerase chain reaction (RT-PCR)

[0076] Wild-type AB zebrafish at 144 hpf were collected from each group for RT-PCR detection, with 30 zebrafish larvae in each group. Reverse transcription and real-time quantitative PCR were performed according to the kit instructions.

[0077] Statistical analysis

[0078] All tests were repeated three times, and the results are expressed as mean ± SD. Prism 9.3.0 (Prism, SanDiego, CA) was used for data preparation and statistical analysis. One-way analysis of variance (ANOVA) was used to analyze the differences. P values ​​less than 0.05 were considered statistically significant. Pearson's correlation coefficient was used to assess the correlation between content and activities. The linear regression equation was calculated by linear regression analysis.

[0079] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A polypeptide having neuroprotective function, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A polypeptide having neuroprotective function, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

2.

3. A polypeptide having neuroprotective function, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

3.

4. A method for preparing the polypeptide having neuroprotective function according to any one of claims 1 to 3, characterized in that: Here are the steps: S1. Preparation of phycocyanin hydrolysate 1 g of C-PC powder was weighed and dissolved in 60.64 mL of pure water. Trypsin was used to hydrolyze C-PC. The conditions for enzymatic hydrolysis were as follows: enzyme addition amount 55.18 mg, temperature 49.4 °C, pH 8, time 120 min. After the hydrolysis was completed, the enzyme was inactivated in a 95 °C water bath for 15 min. The obtained hydrolyzate was centrifuged at 4 °C and 12000 r / min for 20 min. The supernatant was collected and filtered with a 0.45 μm regenerated cellulose filter membrane, rotary evaporated, and concentrated to obtain the C-PC enzymatic hydrolyzate. S2. Preparation of peptide mixed stock solution The C-PCH prepared in step S1 was separated by Sephadex G-100. 500 mg of freeze-dried C-PCH was dissolved in 2 mL of deionized water, filtered through a 0.45 μm microporous membrane and loaded onto a Sephadex column. The eluent was deionized water at a flow rate of 0.5 mL / min. One portion was collected every 20 min. The fractions were combined and concentrated according to the results of thin layer chromatography analysis to obtain a polypeptide mixed stock solution. S3. Preparation of crude extracts of active peptide segments The polypeptide mixed stock solution prepared in step S2 was dissolved in 50% methanol-water, filtered through a 0.45 μm microporous membrane, and then separated through Sephadex LH-20, using 50% methanol-water as the eluent, collecting samples at a rate of 0.25 mL / min, collecting one portion every 40 minutes, combining the same active segment eluates, and concentrating to obtain a crude extract of the polypeptide active segment; S4. Final preparation The crude extract of the active segment of the polypeptide obtained in step S3 was dissolved in ultrapure water to prepare a 10 mg / mL solution, filtered through a 0.45 μm microporous membrane, and the filtrate was filtered through a ZORBAX-300Extent-C 18 Preparative Column C 18 RP-HPLC was used for further separation and purification, with mobile phase A being water and mobile phase B being acetonitrile. Gradient elution was used, with mobile phase B ranging from 1% to 100% in six column volumes at a flow rate of 0.8 mL / min. The eluate with an absorption peak at 214 nm was collected to screen for multifunctional active peptides, which were concentrated and dried to obtain peptides with neuroprotective function. In the S4 step, the multifunctional polypeptide is screened using activity-guided technology, and the specific steps are as follows: Zebrafish dopamine neurons, brain blood vessels and behavioral evaluation models were used to screen out the fractions with the highest activity in the three evaluation models.

5. The method for preparing a polypeptide having neuroprotective function according to claim 4, characterized in that: According to step S1, the concentration is to remove water in the solution by rotary evaporation at 38-40°C.

6. The method for preparing a polypeptide having neuroprotective function according to claim 4, characterized in that: In the step S2, the vacuum concentration conditions are as follows: the vacuum degree is 0.08-0.1 MPa and the water bath temperature is 40-45°C.

7. The method for preparing a polypeptide having neuroprotective function according to claim 4, characterized in that: According to step S4, ZORBAX, 300Extent-C 18 The column specifications are 4.6×250mm, 5μm.

8. The method for preparing a polypeptide having neuroprotective function according to claim 4, characterized in that: In step S4, the drying condition is freeze drying at -20°C.

9. Use of the polypeptide with neuroprotective function according to any one of claims 1 to 3 as a pharmaceutical ingredient in the preparation of a drug for treating Parkinson's disease.

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