A hippocampus-derived neuroprotective and antioxidant stress polypeptide and a preparation method thereof

Abstract

The present application relates to hippocampus extract technical field, specifically speaking, a hippocampus derived neuroprotective and antioxidant stress polypeptide and its preparation method, the amino acid sequence of the polypeptide is as shown in SEQ ID No. 1, the preparation steps are as follows: (1) weigh the fresh hippocampus 500g, clean the surface silt, freeze-dry at-60 DEG C, then use the crusher to break the hippocampus, pass through 20 mesh screen, dialysis for 24 hours with distilled water, freeze-dry, obtain hippocampus powder;(2) weigh 200g hippocampus powder, add 1000ml 95% ethanol to extract three times, remove fat-soluble substances, under the condition of 4 DEG C, use 1 mol / L NaCl solution to extract the precipitate with the aid of ultrasonic, freeze-dry, obtain hippocampus crude protein;(3) take hippocampus crude protein, use the method of ultrasonic-assisted biomimetic digestion to prepare hippocampus polypeptide, which has the advantages of specific molecular structure, clear mechanism, stability in gastrointestinal tract, neuroprotection, antioxidant stress and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to hippocampus extract technical field, specifically a hippocampus-derived neuroprotective and antioxidant stress polypeptide and a preparation method thereof. BACKGROUND

[0002] It is well known that neurodegenerative diseases are a series of serious conditions characterized by persistent functional decline of the nervous system, and their impact is increasingly prominent as the global population ages, becoming a public health issue of great concern to all sectors of society. This type of disease not only seriously affects the quality of life of patients, but also places a heavy burden on the social and economic system and individual families. Its characteristics are chronicity, irreversibility and high cure difficulty, and it mainly includes Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, motor neuron disease, cerebellar disease and multiple system atrophy, and other disease forms. Traditional neurodegenerative disease drugs, such as cholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists, can cause liver damage, headaches, diarrhea, insomnia, muscle spasms and other side effects. Finding safe and effective anti-neurodegenerative disease bioactive substances has become the key to alleviating the progression of this type of disease.

[0003] The ocean is rich in biological resources, and these organisms survive in extreme environments and evolve a large number of compounds containing potential medicinal value. These compounds are known for their diverse biological activities, such as antioxidant, anti-inflammatory and anti-tumor properties, bringing new hope and light to the field of neurodegenerative disease treatment. Their unique biological characteristics 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 activity, are increasingly attracting attention in the medical community. These polypeptides naturally exist in marine organisms or are extracted from marine organisms through advanced proteolysis techniques, and exhibit great potential in medical applications due to their high efficiency, multifunctionality, biodegradability and other significant advantages. Hippocampus is a traditional Chinese medicinal material with high protein content, with a total protein content of up to 60%. Considering the therapeutic effect of hippocampus on neurological diseases, it is of great significance to convert hippocampus protein into specific polypeptides through biological fermentation and further investigate their neuroprotective activity. SUMMARY

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

[0005] The technical solution adopted by the present application to solve its technical problems is:

[0006] A hippocampus-derived neuroprotective and antioxidant stress polypeptide, characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID No. 1.

[0007] A preparation method of a hippocampus-derived neuroprotective and antioxidant stress polypeptide, characterized in that the preparation steps are as follows:

[0008] (1) 500 g of fresh hippocampus is weighed, the surface silt is cleaned, and after freeze-drying at -60 DEG C, the hippocampus is crushed by a pulverizer, passed through a 20-mesh sieve, dialyzed in distilled water for 24 hours, freeze-dried, and hippocampus powder is obtained;

[0009] (2) 200 g of the hippocampus powder is weighed, extracted by 1000 ml of 95% ethanol for three times, the fat-soluble substances are removed, and the precipitate is extracted by using a 1 mol / L NaCl solution under the condition of 4 DEG C combined with ultrasonic assistance, freeze-dried, and hippocampus crude protein is obtained;

[0010] (3) the hippocampus crude protein is taken, and the hippocampus polypeptide is prepared by using an ultrasonic-assisted biomimetic digestion method.

[0011] The specific method for preparing the hippocampus polypeptide by using the ultrasonic-assisted biomimetic digestion method in step (3) is as follows: the hippocampus crude protein is dissolved in simulated gastric juice, the pH is adjusted to 3.0, pepsin (2000 U / mL) is used for enzymolysis under ultrasonic assistance, the ultrasonic power is 400 W, the reaction temperature is 37 DEG C, the reaction time is 2 h, after the reaction is completed, the pH is adjusted to 7, the enzyme is inactivated by water bath at 100 DEG C for 20 min, the supernatant is obtained by centrifugation at 5000 rpm, the hippocampus gastric enzymolysis product is dissolved in simulated intestinal juice after freeze-drying, trypsin (100 U / mL) and alpha-chymotrypsin (25 U / mL) are used for enzymolysis under ultrasonic assistance, the pH is 7.4, the ultrasonic power is 400 W, the reaction temperature is 37 DEG C, the reaction time is 2 h, the final enzymolysis product is cooked at 100 DEG C for 20 min, centrifuged at 4 DEG C for 10 min, 95% ethanol is added to the collected supernatant in a proportion of 1:3, the sugar components in the hippocampus are precipitated, centrifuged at 4 DEG C for 10 min, the supernatant is passed through a 3000 Da membrane, freeze-dried, and purified by RP-HPLC to obtain the hippocampus polypeptide PAAGSPIR.

[0012] The components of the simulated gastric juice include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and the ratio is as follows: 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, CaCl2 4.15 μg / mL.

[0013] The composition of the simulated intestinal fluid according to the present application comprises KCl, KH2PO4, NaHCO3, NaCl, MgCl2, 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.

[0014] The hippocampus polypeptide PAAGSPIR obtained by the above method has the advantages of specific molecular structure, clear action mechanism, stability in the gastrointestinal tract, neuroprotection, antioxidant stress, etc. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is the liquid chromatogram of the purified hippocampus neuroprotective polypeptide PAAGSPIR.

[0016] Figure 2 is the high-resolution mass spectrum of the hippocampus neuroprotective polypeptide PAAGSPIR.

[0017] Figure 3 is the structure diagram of the hippocampus collagen alpha-1 (X) chain protein and the polypeptide PAAGSPIR.

[0018] Figure 4 is the protection effect diagram of the hippocampus neuroprotective polypeptide PAAGSPIR on different cells.

[0019] Figure 5 is the molecular docking diagram of the interaction between the hippocampus neuroprotective polypeptide PAAGSPIR and tripeptidyl peptidase II. DETAILED DESCRIPTION

[0020] The present application will be further described below in conjunction with the accompanying drawings:

[0021] As shown in the drawings, a hippocampus-derived neuroprotective and antioxidant stress polypeptide is characterized in that the amino acid sequence of the polypeptide is 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).

[0022] A preparation method of a hippocampus-derived neuroprotective and antioxidant stress polypeptide is characterized by the following preparation steps:

[0023] (1) Take 500 grams of fresh sea horse, clean the surface silt, freeze-dry at -60℃, then crush the sea horse using a pulverizer, pass through a 20-mesh sieve, dialyze for 24 hours using distilled water, freeze-dry, and obtain sea horse powder;

[0024] (2) Take 200 grams of sea horse powder, add 1000 milliliters of 95% ethanol to extract three times, remove the fat-soluble substances, and extract the precipitate using 1 mol / L NaCl solution under the condition of 4°C, freeze-dry, and obtain sea horse crude protein;

[0025] (3) Take the sea horse crude protein, and prepare sea horse polypeptides using the method of ultrasonic-assisted biomimetic digestion.

[0026] The specific method for preparing sea horse polypeptides using the method of ultrasonic-assisted biomimetic digestion in step (3) is as follows: using simulated gastric juice, dissolving the sea horse crude protein in the simulated gastric juice, adjusting the pH to 3.0, and performing enzymolysis using pepsin (2000 U / mL) under ultrasonic assistance, the ultrasonic power is 400 W, the reaction temperature is 37℃, the reaction time is 2 hours, after the reaction is completed, the pH is adjusted to 7, the enzyme is inactivated in a 100℃ water bath for 20 minutes, centrifuged at 5000 rpm to take the supernatant, freeze-dried, and then the sea horse gastric enzymolysis product is dissolved in simulated intestinal juice, and enzymolysis is performed using trypsin (100 U / mL) and α-chymotrypsin (25 U / mL) under ultrasonic assistance, the pH is 7.4, the ultrasonic power is 400 W, the reaction temperature is 37℃, and the reaction time is 2 hours, the final enzymolysis product is cooked at 100℃ for 20 minutes, centrifuged at 4℃ for 10 minutes, the collected supernatant is added with 95% ethanol at a ratio of 1:3, the sugar components in the sea horse are precipitated, centrifuged at 4℃ for 10 minutes, the collected supernatant is passed through a 3000 Da membrane, freeze-dried, and then purified using RP-HPLC to obtain sea horse polypeptides PAAGSPIR.

[0027] The components of the simulated gastric juice include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and the ratio is as follows: 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.

[0028] The components of the simulated intestinal fluid according to the application include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, 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.

[0029] The hippocampus enzymatic hydrolysate obtained by the above-mentioned ultrasonic-assisted biomimetic digestion is further purified and separated by ultrafiltration centrifugation and RP-HPLC, and a new hippocampus polypeptide sequence is obtained by using high-resolution mass spectrometry analysis. The sequence is searched by the software Byonic database, and the polypeptide is derived from the collagen alpha-1 (X) chain protein in the hippocampus. The hippocampus adopts three-spotted hippocampus.

[0030] The new hippocampus-derived neuroprotective and antioxidant stress polypeptide PAAGSPIR has the following elements:

[0031] a) The hippocampus neuroprotective polypeptide PAAGSPIR is prepared by an ultrasonic-assisted biomimetic enzymatic hydrolysis method, has a specific enzymatic hydrolysis sequence, and uses ultrasonic to improve the extraction efficiency. In addition, the polypeptide has gastrointestinal stability.

[0032] b) The polypeptide PAAGSPIR derived from the three-spotted hippocampus has the amino acid sequence Pro-Ala-Ala-Gly-Ser-Pro-Ile-Arg.

[0033] c) It has neuroprotective activity and the mechanism is a tripeptidyl peptidase II inhibitor. It has antioxidant stress capacity and can alleviate alcohol-induced cell damage.

[0034] The hippocampus polypeptide PAAGSPIR obtained by the above-mentioned method has the advantages of specific molecular structure, clear mechanism, stability in the gastrointestinal tract, neuroprotection, antioxidant stress, etc.

[0035] Example 1

[0036] The preparation method of the hippocampus neuroprotective and antioxidant stress polypeptide PAAGSPIR is as follows:

[0037] (1) Take 500 grams of fresh hippocampus, clean the surface sand, freeze-dry at -60℃, and then use a pulverizer to crush the hippocampus, and pass through a 20-mesh sieve. Distilled water dialysis for 24 hours, freeze-drying, to obtain a hippocampus powder component;

[0038] (2) Take 200 grams of hippocampus powder, extract three times with 1000 milliliters of 95% ethanol to remove lipid-soluble substances, and use 1 mol / L NaCl solution to extract the precipitate under the condition of 4 °C combined with ultrasonic assistance, freeze-dry to obtain hippocampus crude protein;

[0039] (3) Take the hippocampus crude protein, and use the method of ultrasonic-assisted biomimetic digestion to prepare hippocampus polypeptide. The specific method is as follows: use simulated gastric juice to dissolve the hippocampus crude protein in the simulated gastric juice (solid-liquid ratio 1:40), adjust the pH to 3.0, and use pepsin (2000 U / mL) for enzymolysis under ultrasonic assistance, ultrasonic power 400 W, reaction temperature 37 °C, reaction time 2 h, adjust the pH to 7 after the reaction is completed, 100 °C water bath for 20 min to inactivate the enzyme, 5000 rpm centrifugation to take the supernatant, freeze-drying, and then dissolve the hippocampus gastric enzymolysis product in simulated intestinal juice (solid-liquid ratio 1:25), and use trypsin (100 U / mL) and α-chymotrypsin (25 U / mL) for enzymolysis under ultrasonic assistance, pH 7.4, ultrasonic power 400 W, reaction temperature 37 °C, reaction time 2 h, and then cook the final enzymolysis product at 100 °C for 20 minutes, centrifuge at 4 °C for 10 minutes, collect the supernatant, add 95% ethanol to the supernatant at a ratio of 1:3, precipitate the sugar components in the hippocampus, centrifuge at 4 °C for 10 minutes, collect the supernatant, pass it through a 3000 Da membrane, freeze-dry, and then purify using RP-HPLC to obtain hippocampus polypeptide PAAGSPIR, and the liquid chromatogram and mass spectrum are shown in the attached figure. After database retrieval by software Byonic, it is found that the polypeptide is derived from the collagen alpha-1 (X) chain protein in the hippocampus.

[0040] The components of the above-mentioned simulated gastric juice include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and the ratio is as follows: 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, CaCl2 4.15 μg / mL.

[0041] The components of the above-mentioned simulated intestinal juice include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and pig bile salt, and the ratio is as follows: 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.

[0042] The protein sequence is attached in the sequence listing.

[0043] The nucleic acid sequence of the protein sequence is located at KV880001.1: 1,220,905 - 1,224,937.

[0044] The hippocampus polypeptide PAAGSPIR is located at 537-544 of the protein, which is obtained from the organism for the first time. Its basic properties are as follows:

[0045] Molecular mass: 767.4278

[0046] Isoelectric point (pI): 11.56

[0047] Net charge: +1

[0048] Hydrophobicity: +11.48 Kcal·mol -1

[0049] Figure 1 is the liquid chromatogram of the purified hippocampus neuroprotective polypeptide PAAGSPIR,

[0050] Figure 2 is the high-resolution mass spectrum of the hippocampus neuroprotective polypeptide PAAGSPIR, and the ion fragments in the primary and secondary mass spectrometry can be corresponded to the polypeptide information;

[0051] Figure 3 is the structural diagram of the hippocampus collagen α-1 (X) chain protein and the polypeptide PAAGSPIR.

[0052] Example 2

[0053] In vitro cell protection activity and mechanism of hippocampal neuroprotective and antioxidant stress polypeptide PAAGSPIR: (1) Dissolve hippocampal neuroprotective and antioxidant stress polypeptide PAAGSPIR in phosphate buffered saline (PBS), and dilute to 200 μg / mL with dulbecco's modified eagle medium (DMEM) medium. After digestion, logarithmically growing human hepatoma HepG2 cells and human neuroblastoma SH-SY5Y cells were plated in 96-well plates. 500 mM alcohol and 30 mM L-glutamic acid were used to establish alcohol oxidative damage and nerve damage models, respectively. Hippocampal peptides were used to treat normal cells and damaged cells, respectively. After 24 h, the absorbance was measured by MTT method and using a microplate reader at 490 nm wavelength. Cell viability was calculated as follows: Cell viability (%) = [(As - Ab) / (Ac - Ab)] x 100%, where As, Ac and Ab are the absorbance of the experimental group, the control group and the blank well, respectively. At a concentration of 200 μg / mL, hippocampal neuroprotective and antioxidant stress polypeptide PAAGSPIR has obvious protective activity on damaged cells. The possible site of hippocampal neuroprotective and antioxidant stress polypeptide PAAGSPIR tripeptidyl peptidase II inhibition was explored by molecular docking. Cell viability and molecular docking results are shown in FIGS. Figure 4 and 5 Figure 4 is a graph of the protective effect of hippocampal neuroprotective polypeptide PAAGSPIR on different cells. After hippocampal neuroprotective polypeptide PAAGSPIR was applied to HepG2 cells, SH-SY5Y cells and the above-mentioned alcohol-damaged cells, the cell viability changed, Figure 5 is a molecular docking diagram of hippocampal neuroprotective polypeptide PAAGSPIR and tripeptidyl peptidase II. The molecular docking results of hippocampal neuroprotective polypeptide PAAGSPIR and tripeptidyl peptidase II show that the polypeptide can bind well with tripeptidyl peptidase II and easily form hydrogen bonds.

[0054] The above specific embodiments are only preferred examples of the present application and are not intended to limit the present application in other forms.​

Claims

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

1.

2. The method for preparing a hippocampal-derived neuroprotective and antioxidant stress-relieving polypeptide according to claim 1, characterized in that... The preparation steps are as follows: (1) Take 500 grams of fresh sea horse, clean the surface sand, freeze-dry at -60°C, and then crush the sea horse using a pulverizer, pass through a 20-mesh sieve, dialyze in distilled water for 24 hours, freeze-dry, and obtain sea horse powder; (2) Take 200 grams of sea horse powder, extract three times with 1000 milliliters of 95% ethanol to remove lipid-soluble substances, and extract the precipitate using 1 mol / L NaCl solution under the condition of 4°C combined with ultrasonic assistance, and freeze-dry to obtain sea horse crude protein; (3) Take the sea horse crude protein, and prepare sea horse polypeptides using the ultrasonic-assisted biomimetic digestion method, wherein the specific method for preparing sea horse polypeptides using the ultrasonic-assisted biomimetic digestion method in step (3) is as follows: dissolve the sea horse crude protein in simulated gastric juice, adjust the pH to 3.0, and perform enzymolysis using pepsin under ultrasonic assistance, with an ultrasonic power of 400 W, a reaction temperature of 37°C, and a reaction time of 2 hours; after the reaction is completed, adjust the pH to 7, perform enzyme inactivation in a 100°C water bath for 20 minutes, centrifuge at 5000 rpm to take the supernatant, freeze-dry, dissolve the sea horse gastric enzymolysis product in simulated intestinal juice, perform enzymolysis using trypsin and α-chymotrypsin under ultrasonic assistance, with a pH of 7.4, an ultrasonic power of 400 W, a reaction temperature of 37°C, and a reaction time of 2 hours; cook the final enzymolysis product in a 100°C water bath for 20 minutes, centrifuge at 4°C for 10 minutes, collect the supernatant, add 95% ethanol to the supernatant at a ratio of 1:3 to precipitate the sugar components in the sea horse, centrifuge at 4°C for 10 minutes, collect the supernatant, pass the supernatant through a 3000 Da membrane, freeze-dry, purify using RP-HPLC, and obtain sea horse polypeptides PAAGSPIR; the components of the simulated gastric juice include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and the ratio is as follows: 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; the components of the simulated intestinal juice include KCl, KH2PO4, NaHCO3, NaCl, MgCl2, and pig bile salt, and the ratio is as follows: 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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