A brain peptide hydrolysis active peptide and a preparation method and application thereof

Abstract

The application discloses a brain peptide enzymolysis active peptide and a preparation method and application thereof, and belongs to the technical field of active peptide preparation. The brain peptide enzymolysis active peptide VVAVP is successfully prepared by adopting a specific preparation method to perform enzymolysis and purification on a pig brain extract, and the enzymolysis yield and enzymolysis purity are significantly improved by adopting the preparation method. In addition, the efficacy of the prepared short peptide is verified, and the experimental results show that the short peptide VVAVP can play an anti-anxiety role on an anxiety mouse model, and the treatment effect is equivalent to that of a positive control group, so that it is indicated that the active peptide VVAVP has certain emotion regulation ability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of active peptide preparation, and more particularly to a brain peptide enzymatic active peptide, a preparation method and application thereof. BACKGROUND

[0002] Brain peptides generally refer to a class of active peptide substances existing in the brain or nervous system and participating in the regulation of neural function. They include naturally occurring neuropeptides (such as endorphins, enkephalins, substance P, etc.) or artificially synthesized peptide compounds. Common natural brain peptides usually have the following functions: endorphins have the effects of analgesia, stress relief, and generation of pleasant feelings, and are called "natural analgesics"; enkephalins can regulate pain transmission and are related to memory. Substance P is involved in pain signal transmission and inflammatory response and is related to diseases such as migraine and arthritis. Adrenocorticotropic hormone (ACTH) regulates stress response and cortisol secretion and affects learning and memory. Ghrelin regulates appetite and may also affect cognitive function and neuroprotection.

[0003] However, the above-mentioned natural brain peptides are easily degraded by enzymes (such as proteases) in the body, have a short half-life (such as enkephalins only exist for a few minutes in the blood), have a short-acting time, and have relatively poor stability; and most natural brain peptides have a large molecular weight and high polarity, and are difficult to freely pass through the blood-brain barrier, limiting their direct effect on the central nervous system. In addition, natural brain peptides have non-specific effects and can simultaneously activate multiple receptor subtypes (such as substance P acting on NK1, NK2, etc.), causing side effects (such as inflammation or abnormal pain sensitivity). Therefore, the defects of natural brain peptides have driven researchers to carry out related research on artificially synthesized brain peptides, but there are few applications of artificially synthesized brain peptides in emotion regulation.

[0004] Therefore, how to provide a brain peptide enzymatic active peptide, a preparation method and related applications thereof is a technical problem that needs to be solved by those skilled in the art. SUMMARY

[0005] Therefore, the present application provides a brain peptide enzymatic active peptide, a preparation method and application thereof, successfully prepares the brain peptide enzymatic active peptide short VVAVP, significantly improves the enzymatic yield and enzymatic purity of the product, and the short peptide prepared by the present application has obvious anxiolytic effect and emotion regulation effect.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] A brain peptide enzymatic active peptide, wherein the amino acid sequence of the active peptide is VVAVP, SEQ ID NO. 1.

[0008] Still another object of the present application is to provide a nucleic acid molecule encoding the above-mentioned brain peptide enzymatic active peptide, or reverse complement of the nucleotide sequence encoding the above-mentioned brain peptide enzymatic active peptide.

[0009] Still another object of the present application is to provide a biological material containing the above-mentioned nucleic acid molecule and capable of expressing the above-mentioned brain peptide enzymatic active peptide.

[0010] The biological material includes a recombinant plasmid, a recombinant vector or a recombinant host cell.

[0011] Still another object of the present application is to provide an application of the above-mentioned brain peptide enzymatic active peptide, the above-mentioned nucleic acid molecule or the above-mentioned biological material in the preparation of a mood improvement product.

[0012] As a preferred technical solution, the product is a pharmaceutical product.

[0013] Still another object of the present application is to provide a drug for improving mood, comprising the above-mentioned brain peptide enzymatic active peptide.

[0014] Still another object of the present application is to provide a food product comprising the above-mentioned brain peptide enzymatic active peptide.

[0015] As can be known from the above technical solution, compared with the prior art, the present application has the following beneficial effects: the present application uses a specific preparation method to enzymatically hydrolyze and purify pig brain extract, successfully preparing brain peptide enzymatic active peptide VVAVP, and the preparation method of the present application significantly improves the enzymatic hydrolysis yield and enzymatic hydrolysis purity. In addition, the short peptide prepared by the present application is verified for efficacy, and the experimental results show that the short peptide VVAVP of the present application can have an anti-anxiety effect on an anxiety mouse model, and has a mood regulation potential comparable to the therapeutic effect of the positive control group. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

[0017] Figure 1 is a mass spectrum of the protein enzymatic active peptide VVAVP. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0019] The carboxypeptidase B used in the embodiments of the present application is purchased from Beijing Solabio Science and Technology Co., Ltd.

[0020] Embodiment 1

[0021] A preparation method of a brain peptide hydrolyzed active peptide, comprising the following steps:

[0022] (1) Pretreatment of raw materials: taking pig brain extract as raw material, defatting twice with 2 times volume of acetone-ether solution (1:0.5 (V:V)) stirring, each time for 0.5 h, filtering and vacuum drying to prepare defatted powder for use;

[0023] (2) Water extraction and preliminary purification: defatted powder is added into 0.1M phosphate buffer (pH 7.3) at a ratio of 1:8 by weight and volume, stirred at 3℃ for 4 hours, centrifuged at 6000g for 15 min, and the supernatant is taken; the supernatant is fractionally precipitated with 30%-60% saturated ammonium sulfate, the precipitate is collected, dialyzed (2 kDa) to remove salt, and freeze-dried to obtain crude brain peptide powder.

[0024] (3) Multi-enzyme-stepwise enzymolysis:

[0025] First enzymolysis: the pH of the substrate crude brain peptide powder is adjusted to 8.5, alkaline protease is added according to the weight ratio of 0.5:100 of alkaline protease and substrate, and the reaction is carried out at 45℃ for 2.5 h, then the enzyme is inactivated by boiling water bath treatment for 4 min, centrifuged at 8000g for 10 min, and the supernatant is taken;

[0026] Second enzymolysis: the pH of the supernatant is adjusted to 7.5, trypsin is added according to the weight ratio of 0.5:200 of trypsin and substrate, and the reaction is carried out at 36℃ for 1.5 h, then the pH is adjusted to 3.5 by adding acetic acid to inactivate the enzyme, centrifuged at 8000g for 10 min, and the supernatant is taken;

[0027] Third enzymolysis: the pH of the supernatant is adjusted to 7.0, carboxypeptidase B is added according to the weight ratio of 0.5:500 of carboxypeptidase B and substrate, and the reaction is carried out at 36℃ for 0.5 h, then the pH is adjusted to 1.5 by using hydrochloric acid to inactivate the enzyme, centrifuged at 8000g for 10 min, and the supernatant is taken to prepare an enzymolysis solution;

[0028] (4) Protein precipitation and purification: the enzymolysis solution is heated to 70℃ for 5 min, and then centrifuged at 8000g for 10 min after cooling, and the supernatant is taken;

[0029] (5) Ethanol fractionation precipitation:

[0030] 20% ethanol precipitation: the supernatant of step (4) was slowly added with cold ethanol (-20℃) at a speed of ≤1 mL / min at 4℃, and the final concentration of ethanol was controlled at 20%. After stirring for 5 min at 4℃, the supernatant was centrifuged at 8000g for 10 min, and the supernatant was taken;

[0031] 40% ethanol precipitation: the supernatant was slowly added with cold ethanol at 4℃, and the final concentration of ethanol was controlled at 40%. After stirring and centrifugation at 4℃, the precipitate was taken, and the precipitate was dissolved with deionized water at 0℃. The ultrasonic power was 20W, and the pulse mode was 5s. The treatment time was 20s. After dialysis and alcohol removal, the crude peptide was freeze-dried;

[0032] (6) Chromatographic separation and extraction: the crude peptide was passed through a chromatographic column, and the main peak of WAWP was collected. After ultrafiltration and concentration, the brain peptide enzymatic active peptide was obtained by freeze-drying;

[0033] The chromatographic conditions are as follows:

[0034] Chromatographic column: Sephadex G-15 (2.5x100 cm);

[0035] Mobile phase: 0.1 M NH4HCO3 (pH 7.8);

[0036] Flow rate: 0.5 mL / min;

[0037] Detection: UV 220 nm.

[0038] Example 2

[0039] A preparation method of a brain peptide enzymatic active peptide, comprising the following steps:

[0040] (1) Pretreatment of raw materials: the pig brain extract was used as raw material, and 3 volumes of acetone-ether solution (2:0.5 (V:V)) was stirred for 3 times, each for 1 h. After filtration, vacuum drying was performed to prepare defatted powder for use;

[0041] (2) Water extraction and preliminary purification: the defatted powder was added with 0.1 M phosphate buffer (pH 7.5) at a weight to volume ratio of 1:12 at 3-4℃, and stirred for 4 hours. After centrifugation at 10000g for 25 min, the supernatant was taken. The supernatant was fractionally precipitated with 30%-60% saturated ammonium sulfate, and the precipitate was collected. After dialysis (4 kDa) to remove salt, the crude brain peptide powder was freeze-dried.

[0042] (3) Multi-enzyme stepwise enzymolysis:

[0043] First enzymolysis: the pH of the substrate crude encephalin powder was adjusted to 9.5, alkaline protease was added according to the weight ratio of 1.5:100 of the enzyme and the substrate, and the reaction was carried out at 55°C for 3.5h, and then the enzyme was inactivated by boiling water treatment for 5min, centrifugation was carried out at 12000g for 20min, and the supernatant was taken;

[0044] Second enzymolysis: the pH of the supernatant was adjusted to 8.5, trypsin was added according to the weight ratio of 1.5:200 of the enzyme and the substrate, and the reaction was carried out at 38°C for 3h, and then the enzyme was inactivated by adjusting the pH to 4.5 with acetic acid, centrifugation was carried out at 10000g for 20min, and the supernatant was taken;

[0045] Third enzymolysis: the pH of the supernatant was adjusted to 8.0, carboxypeptidase B was added according to the weight ratio of 1.5:500 of the enzyme and the substrate, and the reaction was carried out at 38°C for 1h, and then the enzyme was inactivated by adjusting the pH to 2.5 with hydrochloric acid, centrifugation was carried out at 10000g for 20min, and the supernatant was taken to prepare an enzymolysis solution;

[0046] (4) Precipitation purification of the protein: the enzymolysis solution was heated to 80°C and maintained for 10min, and then cooled, centrifugation was carried out at 10000g for 15min after cooling, and the supernatant was taken;

[0047] (5) Ethanol fractionation precipitation:

[0048] 20% ethanol precipitation: the supernatant of step (4) was slowly added with cold ethanol (-20°C) at a speed of ≤1 mL / min at 4°C, the final concentration of ethanol was controlled to be 20%, stirring was carried out for 10min at 4°C, centrifugation was carried out at 10000g for 15min at 4°C, and the supernatant was taken;

[0049] 40% ethanol precipitation: the supernatant was slowly added with cold ethanol at 4°C, the final concentration of ethanol was controlled to be 40%, stirring and centrifugation were carried out at 4°C, the precipitate was taken, the precipitate was ultrasonically dissolved with deionized water at 4°C, the ultrasonic power was 50W, the 5s pulse mode was used, the treatment time was 30s, and the crude peptide was obtained by dialysis to remove ethanol and freeze-drying;

[0050] (6) Chromatographic separation and extraction: the crude peptide was passed through a chromatographic column, the WWA VP main peak was collected, ultrafiltration and concentration were carried out, and freeze-drying was carried out to obtain the brain peptide enzymolysis active peptide;

[0051] The chromatographic conditions are as follows:

[0052] Chromatographic column: Sephadex G-15 (2.5x100 cm);

[0053] Mobile phase: 0.1 M NH4HCO3 (pH 7.8);

[0054] Flow rate: 0.5 mL / min;

[0055] Detection: UV 220 nm.

[0056] Example 3

[0057] A preparation method of a brain peptide hydrolysis active peptide, comprising the following steps:

[0058] (1) Pretreatment of raw materials: taking pig brain extract as raw material, defatting 3 times with 3 times volume of acetone-ether solution (1:1 (V:V)) stirring, filtering and vacuum drying after 1 h each time to prepare defatted powder for standby;

[0059] (2) Water extraction and preliminary purification: defatted powder is added into 0.1M phosphate buffer (pH 7.4) at a ratio of 1:10 by weight and volume, stirred at 4°C for 4 hours, centrifuged at 8000g for 20 min, and the supernatant was taken; the supernatant is fractionally precipitated with 30%-60% saturated ammonium sulfate, the precipitate is collected, dialyzed (3 kDa) to remove salt, and freeze-dried to obtain crude brain peptide powder.

[0060] (3) Multi-enzyme-stepwise enzymolysis:

[0061] First enzymolysis: the pH of the substrate crude brain peptide powder is adjusted to 9.0, alkaline protease is added according to the weight ratio of 1:100 of alkaline protease and substrate, 50°C reaction for 3.0h, boiling water bath treatment for 5min to inactivate the enzyme, 10000g, centrifugal 15min to take the supernatant;

[0062] Second enzymolysis: adjust the pH of the supernatant to 8.0, add trypsin according to the weight ratio of 1:200 of trypsin and substrate, 37°C reaction for 2h, add acetic acid to adjust the pH to 4.0 to inactivate the enzyme, 10000g centrifugal 15min, take the supernatant;

[0063] Third enzymolysis: adjust the pH of the supernatant to 7.5, add carboxypeptidase B according to the weight ratio of 1:500 of carboxypeptidase B and substrate, 37°C reaction for 1h, use hydrochloric acid to adjust the pH to 2.0 to inactivate the enzyme, 10000g, centrifugal 15min, take the supernatant, to prepare the enzymolysis solution;

[0064] (4) Protein precipitation purification: heat the enzymolysis solution to 75°C for 10 min, cool it, centrifugal 10min at 8000g, and take the supernatant;

[0065] (5) Ethanol fractionation precipitation:

[0066] 20% ethanol precipitation: the supernatant of step (4) is slowly added with cold ethanol (-20°C) at a speed of ≤1 mL / min at 4°C, the final concentration of ethanol is controlled at 20%, stirred for 10 min at 4°C, centrifugal 15min at 8000g, and the supernatant is taken;

[0067] 40% ethanol precipitation: the supernatant was slowly added with cold ethanol at 4°C to control the final concentration of ethanol at 40%, stirring, centrifugation at 4°C, taking the precipitate, dissolving the precipitate with deionized water at 3°C, ultrasonic power 20W, 5s pulse mode, processing 30s, dialysis to remove alcohol, freeze-drying to obtain crude peptide;

[0068] (6) Chromatographic separation and extraction: the crude peptide was passed through a chromatographic column, the main peak of WWA VP was collected, ultrafiltration was concentrated, and freeze-drying was performed to obtain brain peptide enzymolysis active peptide;

[0069] The chromatographic conditions are as follows:

[0070] Chromatographic column: Sephadex G-15 (2.5x100 cm);

[0071] Mobile phase: 0.1 M NH4HCO3 (pH 7.8);

[0072] Flow rate: 0.5 mL / min;

[0073] Detection: UV 220 nm.

[0074] The brain peptide enzymolysis active peptide extracted was subjected to mass spectrometry detection, and the detection results are shown in Table 1. Figure 1

[0075] Table 1 Mass spectrometry fragment ion detection results

[0076] Ion type Observed m / z Sequence position y1 116.0706 P y2 215.139 VP y3 286.1761 AVP b2 199.1441 VV b3 270.1812 VVA

[0077] Parent ion: m / z 485.73 ([M+2H] 2+ , theoretical calculation value 485.23).

[0078] In summary, by matching the b / y ion series, it is confirmed that the detected peptide segment is WWA VP.

[0079] Comparative Example 1

[0080] A preparation method of a brain peptide enzymolysis active peptide, comprising the following steps:

[0081] It is basically the same as Example 3, except that steps (1) and (2) are not included, and the remaining operations are the same as Example 3.

[0082] Comparative Example 2

[0083] A preparation method of a brain peptide enzymolysis active peptide, comprising the following steps:

[0084] It is basically the same as Example 3, except that the first enzymolysis process is not included in step (3), and the remaining operations are the same as Example 3.

[0085] Comparative Example 3​

[0086] A method for preparing a brain peptide hydrolysis active peptide, comprising the following steps:

[0087] The method is basically the same as that in Example 3, except that the ethanol used in step (5) is at room temperature, and the rest of the operations are the same as those in Example 3.

[0088] Comparative Example 4

[0089] A method for preparing a brain peptide hydrolysis active peptide, comprising the following steps:

[0090] The method is basically the same as that in Example 3, except that the ethanol used in step (5) is at room temperature, and the rest of the operations are the same as those in Example 3.

[0091] Comparative Example 5

[0092] A method for preparing a brain peptide hydrolysis active peptide, comprising the following steps:

[0093] The method is basically the same as that in Example 3, except that the ethanol used in step (5) is at room temperature, and the rest of the operations are the same as those in Example 3.

[0094] In order to verify the effects of the preparation methods of different examples, the enzymatic hydrolysis yield and the purity of the enzymatic hydrolysis fragments of different examples and comparative examples were determined, and the experimental results are shown in Table 2:

[0095] Table 2 Enzymatic hydrolysis yield and purity of different preparation methods

[0096] Group Enzymolysis yield (%) Enzymolysis purity (%) Example 1 87.4 98.5 Example 2 86.5 98.7 Example 3 88.2 99.2 Comparative Example 1 54.2 75.6 Comparative Example 2 56.8 76.9 Comparative Example 3 43.5 60.5 Comparative Example 4 30.8 55.3 Comparative Example 5 35.6 58.9

[0097] According to the content of Table 2, the method of the present application can significantly improve the enzymatic hydrolysis yield of the brain peptide hydrolysis active peptide VVAVP and the purity of the product compared with Comparative Examples 1-5.

[0098] At the same time, in order to further verify the efficacy of the peptide segment of the present application, the following mouse experiments are carried out,

[0099] Construction of anxiolytic mouse model and detection of anxiolytic behavior

[0100] Select 8-12 weeks old, male C57BL / 6J mice, one week before the model is built, the experimental mice are adapted to the unfamiliar environment for one week, and the day before the model is built, the mice participating in the experiment are subjected to the elevated plus maze test, and the mice with normal mental and emotional state are selected, and the mice with abnormal indicators are excluded, and the next experiment is performed. Raising conditions: 12 h light-dark cycle, free drinking and drinking water. The above selected mice are divided into a control group, an experimental group and a positive control group, each group not less than 3. By binding the mouse, it produces anxiety under stress, and verifies the mechanism of brain peptide enzyme active peptide VVAVP in relieving this process. The model is built for 7 days.

[0101] Control group mice: no binding treatment, and only physiological saline is given to them;

[0102] Experimental group mice: binding treatment, and low, medium and high dose peptide segments prepared in Example 3 (0.1, 1 and 10 mg / kg) are respectively given to them;

[0103] Positive control group mice: binding treatment, and 1 mg / kg of diazepam is given to them.

[0104] The above different groups of mice are subjected to anxiety-like behavior detection by using the elevated plus maze (EPM), the mice are placed in the central area, facing the open arm, and the number of times the mice enter the open arm within 5 minutes (four limbs completely enter once); The proportion of open arm stay time (the lower the anxiety, the longer the time), and the total movement distance (excluding the interference of movement ability), and the experimental results are shown in Table 3.

[0105] Table 3 Anxiety-like behavior results of different treatment groups

[0106] Group Control group Experimental group (low) Experimental group (medium) Experimental group (high) Positive control group Number of times of entering open arm (times) 3 8 9 10 11 Percentage of open arm residence time (%) 22.6 36.4 39.8 43.3 44.1 Total movement distance (cm) 1400 1406 1396 1405 1410

[0107] Result analysis: from the content of Table 3, it can be seen that, compared with the control group, the number of times the experimental group mice enter the open arm can be increased to a certain extent by giving VVAVP prepared in Example 3, and with the increase of the number of times, it shows that the anxiety behavior of the mice is reduced. At the same time, the open arm stay time of the mice in the experimental group (high) dose group tends to be significantly different from the stay time of the positive control group, and is significantly different from the control group, also indicating that the anxiety degree of the mice is reduced. The total movement distance of mice in different groups is not significantly different, indicating that the peptide segment does not affect the movement ability of the mice, and the sedative or muscle toxicity effect of the peptide segment is excluded.

[0108] Each embodiment in the specification is described in a progressive manner, and each embodiment focuses on the difference from other embodiments. The same or similar parts of each embodiment can be referred to each other.

[0109] The foregoing description of the disclosed embodiments enables one skilled in the art to make or use the application. Numerous modifications of those embodiments can be apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without the use of the innovation falling outside the spirit and scope of the application. Therefore, the application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A brain peptide with enzymatic hydrolysis activity, characterized in that, The amino acid sequence of the active peptide is VVAVP.

2. A nucleic acid molecule, characterized in that, It encodes the brain peptide hydrolysis active peptide of claim 1, or is reverse complementary to the nucleotide sequence encoding the brain peptide hydrolysis active peptide of claim 1.

3. A biomaterial, characterized in that, The biomaterial contains the nucleic acid molecule of claim 2 and is able to express the brain peptide enzymatic hydrolysis active peptide of claim 1; The biomaterials include recombinant vectors or recombinant host cells.

4. The biomaterial according to claim 3, characterized in that, The recombinant vector is a recombinant plasmid.

5. The application of the brain peptide enzymatic hydrolysis active peptide of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of anti-anxiety products.

6. The application according to claim 5, characterized in that, The product in question is a pharmaceutical product.

7. A drug for treating anxiety, characterized in that, Includes the brain peptide enzymatic hydrolysis active peptides as described in claim 1.

8. A food product, characterized in that, Includes the brain peptide enzymatic hydrolysis active peptides as described in claim 1.

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