Hippocampus-derived neuroprotective and anti-oxidative stress polypeptide and preparation method thereof

Abstract

The invention relates to the technical field of hippocampus extract, in particular to a hippocampus-sourced neuroprotection and anti-oxidative stress polypeptide and a preparation method thereof.The amino acid sequence of the polypeptide is shown as SEQ ID No.1. The preparation method comprises the following steps that 1, 500 g of fresh hippocampus is weighed, surface silt is removed, freeze-drying is conducted at the temperature of-60 DEG C, then the hippocampus is smashed through a smashing machine and sieved through a 20-mesh sieve, and the hippocampus extract is obtained; dialyzing with distilled water for 24 hours, and freeze-drying to obtain hippocampus powder; (2) weighing 200g of hippocampus powder, adding 1000ml of 95% ethanol, extracting for three times, removing fat-soluble substances, and extracting and freeze-drying precipitates by using a 1 mol / L NaCl solution under the condition of 4 DEG C in combination with ultrasonic assistance to obtain crude hippocampus protein; (3) the hippocampus crude protein is taken, the hippocampus polypeptide is prepared through an ultrasonic-assisted bionic digestion method, and the hippocampus polypeptide has the advantages of being specific in molecular structure, clear in action mechanism, stable in gastrointestinal tracts, capable of protecting nerves, resistant to oxidative stress and the like.

Description

Technical Field

[0001] The invention relates to the technical field of hippocampus extracts, in particular to a hippocampus-derived neuroprotective and anti-oxidative stress polypeptide and a preparation method thereof. Background Art

[0002] As we all know, neurodegenerative diseases are a series of serious diseases characterized by persistent functional decline of the nervous system. Their impact has become increasingly prominent with the intensification of the global aging population trend, and has become a public health issue of great concern to all sectors of society. Such diseases not only seriously affect the quality of life of patients, but also bring heavy burdens to the socioeconomic system and individual families. Its characteristics are the chronicity, irreversibility and high difficulty of cure, mainly including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, motor neuron disease, cerebellar lesions and multiple system atrophy. Traditional neurodegenerative disease drugs, cholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists, etc. may cause side effects such as liver damage, headache, diarrhea, insomnia, muscle spasms, etc. Finding safe and effective bioactive substances against neurodegenerative diseases has become the key to alleviating the progression of such diseases.

[0003] The ocean is rich in biological resources. These organisms survive in extreme environments and evolve many compounds with potential medicinal value. These compounds are known for their diverse biological activities, such as antioxidant, anti-inflammatory and anti-tumor properties, which bring new dawn and hope to the treatment of neurodegenerative diseases. Their unique biological properties and broad therapeutic potential are gradually becoming an important breakthrough in overcoming the challenges of these complex diseases. Marine bioactive peptides, as a class of biological molecules with unique physiological activities, are increasingly attracting attention from the medical community. These peptides exist naturally in the bodies of marine organisms or are extracted from marine organisms through advanced proteolysis technology. With their significant advantages such as high efficiency, versatility and biodegradability, they show great potential in medical applications. Seahorse is a traditional Chinese medicinal material with a high protein content, with a total protein content of up to 60%. Considering the therapeutic effect of seahorse on neurological diseases, it is of great significance to convert seahorse protein into specific peptides through biological fermentation and further examine its neuroprotective activity. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the above-mentioned prior art and to provide a hippocampal-derived neuroprotective and anti-oxidative stress polypeptide with a specific molecular structure, a clear mechanism of action, and stability in the gastrointestinal tract, and a preparation method thereof.

[0005] The technical solution adopted by the present invention to solve its technical problem is: A neuroprotective and anti-oxidative stress polypeptide derived from hippocampus, characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID No. 1.

[0006] A method for preparing a hippocampus-derived neuroprotective and anti-oxidative stress polypeptide, characterized in that the preparation steps are as follows: (1) Weigh 500 g of fresh seahorse, clean the surface sand, freeze-dry at -60°C, crush the seahorse with a grinder, pass through a 20-mesh sieve, dialyze with distilled water for 24 hours, and freeze-dry to obtain seahorse powder; (2) Weigh 200 g of hippocampus powder, add 1000 ml of 95% ethanol and extract three times to remove fat-soluble substances. Use 1 mol / L NaCl solution to extract the precipitate at 4°C with ultrasound assistance, and freeze-dry to obtain hippocampus crude protein. (3) Taking crude hippocampal protein, using ultrasound-assisted biomimetic digestion method to prepare hippocampal peptides.

[0007] The specific method for preparing hippocampal polypeptides by using ultrasound-assisted bionic digestion in step (3) of the present invention is as follows: using simulated gastric juice, dissolving hippocampal crude protein in simulated gastric juice, adjusting the pH to 3.0, using pepsin (2000 U / mL) for enzymatic hydrolysis under the assistance of ultrasound, the ultrasonic power is 400 W, the reaction temperature is 37°C, the reaction time is 2 h, after the reaction is completed, the pH is adjusted to 7, the enzyme is inactivated in a 100°C water bath for 20 min, the supernatant is collected by centrifugation at 5000 rpm, and the hippocampal gastric hydrolysate is dissolved in simulated intestinal juice after freeze-drying, and enzymatic hydrolyzate is performed using trypsin (100 U / mL) and α-chymotrypsin (25 U / mL) under the assistance of ultrasound, the pH is 7.4, the ultrasonic power is 400 W, the reaction temperature is 37°C, the reaction time is 2 h, the final enzymatic hydrolyzate is steamed at 100°C for 20 minutes, and then heated at 4 The mixture was centrifuged at 4 °C for 10 minutes, and 95% ethanol was added to the collected supernatant at a ratio of 1:3 to precipitate the sugar components in the hippocampus. The mixture was centrifuged at 4 °C for 10 minutes, and the collected supernatant was passed through a 3000 Da membrane, freeze-dried, and purified using RP-HPLC to obtain the hippocampal polypeptide PAAGSPIR.

[0008] The components of the simulated gastric fluid described in the present invention include KCl, KH2PO4, NaHCO3, NaCl, and MgCl2, and the ratio is: KCl 0.51 mg / mL, KH2PO4 0.12 mg / mL, NaHCO3 2.1 mg / mL, NaCl 2.758 mg / mL, (NH4)2CO3 0.048 mg / mL, MgCl2 2.375 μg / mL, and CaCl2 4.15 μg / mL.

[0009] The components of the simulated intestinal fluid described in the present invention include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and pig bile salt, and the ratio is: KCl 0.5 mg / mL, KH2PO4 0.1 mg / mL, NaHCO3 7.14 mg / mL, NaCl 2.24 mg / mL, MgCl2 0.03 mg / mL, pig bile salt 0.974 mg / mL, CaCl2 0.066 mg / mL.

[0010] The hippocampal polypeptide PAAGSPIR obtained by the method has the advantages of specific molecular structure, clear mechanism of action, stability in the gastrointestinal tract, neuroprotection, and anti-oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the liquid chromatography spectrum of the purified hippocampal neuroprotective peptide PAAGSPIR.

[0012] Figure 2 This is a high-resolution mass spectrum of the hippocampal neuroprotective peptide PAAGSPIR.

[0013] Figure 3 It is a structural diagram of hippocampal collagen α-1(X) chain protein and polypeptide PAAGSPIR.

[0014] Figure 4 This is a diagram showing the protective effect of hippocampal neuroprotective peptide PAAGSPIR on different cells.

[0015] Figure 5 This is a molecular docking diagram of the interaction between hippocampal neuroprotective peptide PAAGSPIR and tripeptidyl peptidase II. DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with the accompanying drawings: As shown in the accompanying drawings, a neuroprotective and anti-oxidative stress polypeptide derived from hippocampus is characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID No. 1, and the amino acid sequence shown in SEQ ID No. 1 is proline-alanine-alanine-glycine-serine-proline-isoleucine-arginine (Pro-Ala-Ala-Gly-Ser-Pro-Ile-Arg).

[0017] A method for preparing a hippocampus-derived neuroprotective and anti-oxidative stress polypeptide, characterized in that the preparation steps are as follows: (1) Weigh 500 g of fresh seahorse, clean the surface sand, freeze-dry at -60°C, crush the seahorse with a grinder, pass through a 20-mesh sieve, dialyze with distilled water for 24 hours, and freeze-dry to obtain seahorse powder; (2) Weigh 200 g of hippocampus powder, add 1000 ml of 95% ethanol and extract three times to remove fat-soluble substances. Use 1 mol / L NaCl solution to extract the precipitate at 4°C with ultrasound assistance, and freeze-dry to obtain hippocampus crude protein. (3) Taking hippocampal crude protein, using ultrasound-assisted biomimetic digestion method to prepare hippocampal peptides.

[0018] The specific method for preparing hippocampal polypeptides by using ultrasound-assisted bionic digestion in step (3) of the present invention is as follows: using simulated gastric juice, dissolving hippocampal crude protein in simulated gastric juice, adjusting the pH to 3.0, using pepsin (2000 U / mL) for enzymatic hydrolysis under the assistance of ultrasound, the ultrasonic power is 400 W, the reaction temperature is 37°C, the reaction time is 2 h, after the reaction is completed, the pH is adjusted to 7, the enzyme is inactivated in a 100°C water bath for 20 min, the supernatant is collected by centrifugation at 5000 rpm, and the hippocampal gastric hydrolysate is dissolved in simulated intestinal juice after freeze-drying, and enzymatic hydrolyzate is performed using trypsin (100 U / mL) and α-chymotrypsin (25 U / mL) under the assistance of ultrasound, the pH is 7.4, the ultrasonic power is 400 W, the reaction temperature is 37°C, the reaction time is 2 h, the final enzymatic hydrolyzate is steamed at 100°C for 20 minutes, and then heated at 4 The mixture was centrifuged at 4 °C for 10 minutes, and 95% ethanol was added to the collected supernatant at a ratio of 1:3 to precipitate the sugar components in the hippocampus. The mixture was centrifuged at 4 °C for 10 minutes, and the collected supernatant was passed through a 3000 Da membrane, freeze-dried, and purified using RP-HPLC to obtain the hippocampal polypeptide PAAGSPIR.

[0019] The components of the simulated gastric fluid described in the present invention include KCl, KH2PO4, NaHCO3, NaCl, and MgCl2, and the ratio is: KCl 0.51 mg / mL, KH2PO4 0.12 mg / mL, NaHCO3 2.1 mg / mL, NaCl 2.758 mg / mL, (NH4)2CO3 0.048 mg / mL, MgCl2 2.375 μg / mL, and CaCl2 4.15 μg / mL.

[0020] The components of the simulated intestinal fluid described in the present invention include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and pig bile salt, and the ratio is: KCl 0.5 mg / mL, KH2PO4 0.1 mg / mL, NaHCO3 7.14 mg / mL, NaCl 2.24 mg / mL, MgCl2 0.03 mg / mL, pig bile salt 0.974 mg / mL, CaCl2 0.066 mg / mL.

[0021] The above-mentioned hippocampal enzymatic hydrolysate is obtained by ultrasound-assisted bionic digestion, and is further purified and separated by ultrafiltration centrifugation and RP-HPLC. A new hippocampal polypeptide sequence is obtained by high-resolution mass spectrometry analysis. The sequence is retrieved by the Byonic database software. The polypeptide is derived from the collagen α-1(X) chain protein in the hippocampus. The above-mentioned hippocampus uses the trispot hippocampus.

[0022] PAAGSPIR, a new neuroprotective and anti-oxidative stress peptide derived from hippocampus, possesses the following elements: a) The hippocampal neuroprotective peptide PAAGSPIR is prepared by ultrasound-assisted biomimetic enzymatic hydrolysis, which has a specific enzymatic hydrolysis sequence and uses ultrasound to improve the extraction efficiency; in addition, this peptide has gastrointestinal stability.

[0023] b) PAAGSPIR, a peptide derived from Hippocampus trisaccharids, has an amino acid sequence of Pro-Ala-Ala-Gly-Ser-Pro-Ile-Arg; c) It has neuroprotective activity, and its mechanism of action is a tripeptidyl peptidase II inhibitor; it has antioxidant stress ability and can alleviate cell damage caused by alcohol.

[0024] The hippocampal polypeptide PAAGSPIR obtained by the method has the advantages of specific molecular structure, clear mechanism of action, stability in the gastrointestinal tract, neuroprotection, and anti-oxidative stress.

[0025] Example 1 The preparation method of hippocampal neuroprotective and anti-oxidative stress peptide PAAGSPIR, the specific process parameters are as follows: (1) Weigh 500 g of fresh seahorse, clean the surface sand, freeze-dry at -60°C, crush the seahorse with a grinder, and pass through a 20-mesh sieve. Dialyze with distilled water for 24 hours, freeze-dry, and obtain the coarse fraction of seahorse powder; (2) Weigh 200 g of hippocampus powder, add 1000 ml of 95% ethanol and extract three times to remove fat-soluble substances. Use 1 mol / L NaCl solution to extract the precipitate at 4 °C with ultrasound assistance, and freeze-dry to obtain hippocampus crude protein. (3) Take the crude protein from the hippocampus and use the ultrasound-assisted biomimetic digestion method to prepare the hippocampal peptides. The specific method is as follows: using simulated gastric fluid, the crude protein from the hippocampus is dissolved in the simulated gastric fluid (material-liquid ratio of 1:40), the pH is adjusted to 3.0, and pepsin (2000 U / mL) is used for enzymatic hydrolysis under the assistance of ultrasound, the ultrasonic power is 400 W, the reaction temperature is 37 °C, and the reaction time is 2 h. After the reaction, the pH is adjusted to 7, the enzyme is inactivated in a 100 °C water bath for 20 min, and the supernatant is obtained by centrifugation at 5000 rpm. After freeze-drying, the hippocampal gastric hydrolysate is dissolved in simulated intestinal fluid (material-liquid ratio of 1:25), and trypsin (100 U / mL) and α-chymotrypsin (25 U / mL) are used for enzymatic hydrolysis under the assistance of ultrasound, the pH is 7.4, the ultrasonic power is 400 W, the reaction temperature is 37 °C, and the reaction time is 2 h. The final enzymatic hydrolysate is steamed at 100 °C for 20 min and then heated at 4 The mixture was centrifuged at 4 °C for 10 minutes, and 95% ethanol was added to the collected supernatant at a ratio of 1:3 to precipitate the sugar components in the hippocampus. The mixture was centrifuged at 4 °C for 10 minutes, and the collected supernatant was passed through a 3000 Da membrane, freeze-dried, and purified using RP-HPLC to obtain the hippocampal polypeptide PAAGSPIR, whose liquid phase and mass spectrum are shown in the attached figure. The polypeptide was found to be derived from collagen α-1(X) chain protein in the hippocampus by searching the Byonic database.

[0026] The components of the simulated gastric fluid described above include KCl, KH2PO4, NaHCO3, NaCl, and MgCl2, and the ratio is: KCl 0.51 mg / mL, KH2PO4 0.12 mg / mL, NaHCO3 2.1 mg / mL, NaCl 2.758 mg / mL, (NH4)2CO3 0.048 mg / mL, MgCl2 2.375 μg / mL, and CaCl2 4.15 μg / mL.

[0027] The components of the simulated intestinal fluid described above include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and pig bile salt, and the ratio is: KCl 0.5 mg / mL, KH2PO4 0.1 mg / mL, NaHCO3 7.14 mg / mL, NaCl 2.24 mg / mL, MgCl20.03 mg / mL, pig bile salt 0.974 mg / mL, and CaCl2 0.066 mg / mL.

[0028] The protein sequence is attached in the sequence listing.

[0029] The genomic position of the nucleic acid sequence of this protein sequence is KV880001.1: 1,220,905 - 1,224,937.

[0030] The hippocampal peptide PAAGSPIR is located at positions 537-544 of the protein and is obtained from an organism for the first time. Its basic properties are as follows: Molecular mass: 767.4278 Isoelectric point (pI): 11.56 Net charge: +1 Hydrophobicity: +11.48Kcal·mol -1 Figure 1 This is the liquid chromatography spectrum of the purified hippocampal neuroprotective peptide PAAGSPIR. Figure 2 This is the high-resolution mass spectrum of the hippocampal neuroprotective peptide PAAGSPIR. The ion fragments in the primary and secondary mass spectra can correspond to the peptide information. Figure 3 It is a structural diagram of hippocampal collagen α-1(X) chain protein and polypeptide PAAGSPIR.

[0031] Example 2 In vitro cell protection activity and mechanism of action of hippocampal neuroprotective and anti-oxidative stress peptide PAAGSPIR: (1) The hippocampal neuroprotective and anti-oxidative stress peptide PAAGSPIR was dissolved in phosphate buffered saline (PBS) and diluted to 200 μg / mL using dulbecco's modified eagle medium (DMEM). Logarithmically growing human hepatoma HepG2 cells and human neuroblastoma SH-SY5Y cells were digested and plated in 96-well plates. Alcohol oxidative damage and nerve damage models were established using 500 mM alcohol and 30 mM L-glutamate, respectively. Normal cells and damaged cells were treated with hippocampal neuropeptides, respectively. After 24 h, the absorbance was measured at 490 nm using the MTT method and a microplate reader. Cell viability was calculated as follows: Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100%, where As, Ac and Ab are the absorbances of the experimental group, control group and blank well, respectively. At a concentration of 200 μg / mL, the hippocampal neuroprotective and anti-oxidative stress peptide PAAGSPIR had significant protective activity against damaged cells. Molecular docking was used to explore the possible sites of inhibition of tripeptidyl peptidase II by the hippocampal neuroprotective and anti-oxidative stress peptide PAAGSPIR. The cell viability and molecular docking results are shown in the attached figure. Figure 4 and 5 As shown, Figure 4This is a diagram showing the protective effect of hippocampal neuroprotective peptide PAAGSPIR on different cells. After hippocampal neuroprotective peptide PAAGSPIR acts on HepG2 cells and SH-SY5Y cells damaged by alcohol, the cell viability changes. Figure 5 This is a molecular docking diagram of the interaction between hippocampal neuroprotective peptide PAAGSPIR and tripeptidyl peptidase II. It is the molecular docking result of hippocampal neuroprotective peptide PAAGSPIR and tripeptidyl peptidase II. The peptide can combine well with tripeptidyl peptidase II and easily form hydrogen bonds.

[0032] The above specific implementation examples are only preferred examples of the present invention and are not intended to limit the present invention in other forms.

Claims

1. A neuroprotective and anti-oxidative stress polypeptide derived from hippocampus, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

2. A method for preparing a hippocampus-derived neuroprotective and anti-oxidative stress polypeptide, characterized in that The preparation steps are as follows: (1) Weigh 500 g of fresh seahorse, clean the surface sand, freeze-dry at -60°C, crush the seahorse with a grinder, pass through a 20-mesh sieve, dialyze with distilled water for 24 hours, and freeze-dry to obtain seahorse powder; (2) Weigh 200 g of hippocampus powder, add 1000 ml of 95% ethanol and extract three times to remove fat-soluble substances. Use 1 mol / L NaCl solution to extract the precipitate at 4°C with ultrasound assistance, and freeze-dry to obtain hippocampus crude protein. (3) Taking hippocampal crude protein, using ultrasound-assisted biomimetic digestion method to prepare hippocampal peptides.

3. The method for preparing a hippocampus-derived neuroprotective and anti-oxidative stress polypeptide according to claim 1, characterized in that The specific method for preparing hippocampal polypeptides using ultrasound-assisted bionic digestion in step (3) is as follows: using simulated gastric fluid, dissolving hippocampal crude protein in simulated gastric fluid, adjusting the pH to 3.0, using pepsin for enzymatic hydrolysis under ultrasound, the ultrasonic power is 400 W, the reaction temperature is 37 ° C, the reaction time is 2 h, after the reaction is completed, adjusting the pH to 7, inactivating the enzyme in a 100 ° C water bath for 20 min, centrifuging at 5000 rpm to obtain the supernatant, and dissolving the hippocampal gastric hydrolysate in simulated intestinal fluid after freeze-drying, using trypsin and α-chymotrypsin for enzymatic hydrolysis under ultrasound, pH 7.4, ultrasonic power is 400 W, reaction temperature is 37 ° C, reaction time is 2 h, the final enzymatic hydrolysate is steamed at 100 ° C for 20 minutes, centrifuged at 4 ° C for 10 minutes, the collected supernatant is added with 95% ethanol at a ratio of 1:3 to precipitate the sugar components in the hippocampus, centrifuged at 4 ° C for 10 minutes, and the collected supernatant is filtered through 3000 rpm. Da membrane, lyophilized and purified by RP-HPLC to obtain the hippocampal peptide PAAGSPIR.

4. The method for preparing a hippocampus-derived neuroprotective and anti-oxidative stress polypeptide according to claim 3, characterized in that The components of simulated gastric fluid include KCl, KH2PO4, NaHCO3, NaCl, and MgCl2, and the ratios are: KCl 0.51 mg / mL, KH2PO4 0.12 mg / mL, NaHCO3 2.1 mg / mL, NaCl 2.758 mg / mL, (NH4)2CO3 0.048 mg / mL, MgCl2 2.375 μg / mL, and CaCl2 4.15 μg / mL.

5. The method for preparing a hippocampus-derived neuroprotective and anti-oxidative stress polypeptide according to claim 3, characterized in that The components of simulated intestinal fluid include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and pig bile salt, and the ratio is: KCl 0.5 mg / mL, KH2PO4 0.1 mg / mL, NaHCO3 7.14 mg / mL, NaCl 2.24 mg / mL, MgCl2 0.03 mg / mL, pig bile salt 0.974 mg / mL, and CaCl2 0.066 mg / mL.

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