Brain peptide enzymolysis active peptide as well as preparation method and application thereof

Abstract

The invention discloses a brain peptide enzymolysis active peptide as well as a preparation method and application thereof. Belongs to the technical field of active peptide preparation. The specific preparation method is adopted, the pig brain extract is subjected to enzymolysis and purification, the brain peptide enzymolysis active peptide VVAVP is successfully prepared, and the enzymolysis yield and the enzymolysis purity are remarkably improved by adopting the preparation method. In addition, efficacy verification is carried out on the prepared oligopeptide, and experimental results show that the oligopeptide VVAVP can play a certain anti-anxiety role on an anxiety mouse model, the treatment effect of the oligopeptide VVAVP is equivalent to that of a positive control group, and it is indicated that the active peptide VVAVP has a certain emotion regulation ability.

Description

Technical Field

[0001] The present invention relates to the technical field of active peptide preparation, and more particularly to a brain peptide hydrolysate active peptide, its preparation method and application. Background Art

[0002] Brain Peptides generally refer to a class of active peptide substances that exist in the brain or nervous system and participate in regulating nerve functions. They include naturally occurring neuropeptides (such as endorphins, enkephalins, substance P, etc.) or synthetic peptide compounds. Common natural brain peptides usually have the following functions: For example, endorphins have analgesic, stress-relieving, and pleasure-inducing effects and are known as "natural painkillers"; enkephalins can regulate pain signal transmission and are related to memory. Substance P is involved in pain signal transmission and inflammatory responses and is related to diseases such as migraine and arthritis. Adrenocorticotropic hormone (ACTH) regulates stress responses and cortisol secretion and affects learning and memory. Ghrelin, in addition to regulating appetite, may also affect cognitive function and neuroprotection.

[0003] However, the above-mentioned natural brain peptides are easily degraded rapidly by enzymes in the body (such as proteases), have a short half-life (for example, enkephalins only survive in the blood for a few minutes), resulting in a short acting time and 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, which limits their direct action on the central nervous system. In addition, natural brain peptides have non-specific effects and may simultaneously activate multiple receptor subtypes (such as substance P acting on NK1, NK2, etc. receptors simultaneously), causing side effects (such as inflammation or abnormal pain sensitivity). Therefore, the defects of natural brain peptides have promoted researchers to carry out related research on synthetic brain peptides, but there are few applications of synthetic brain peptides in mood regulation at present.

[0004] Therefore, how to provide a brain peptide hydrolysate active peptide, its preparation method and related applications is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0005] In view of this, the present invention provides a brain peptide hydrolysate active peptide, its preparation method and application, successfully prepares the brain peptide hydrolysate active peptide short VVAVP, and significantly improves the enzymatic hydrolysis yield and enzymatic hydrolysis purity of the product. Moreover, the short peptide prepared by the present invention has an obvious anti-anxiety effect and has a mood regulation effect.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

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

[0008] Another object of the present invention is to provide a nucleic acid molecule that encodes the above-mentioned brain peptidase-cleaved active peptide or is reverse complementary to the nucleotide sequence encoding the above-mentioned brain peptidase-cleaved active peptide.

[0009] Another object of the present invention is to provide a biological material that contains the above nucleic acid molecule and is capable of expressing the above brain peptidase-cleaved active peptide;

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

[0011] Another object of the present invention is to provide the use of the above brain peptidase-cleaved active peptide, the above nucleic acid molecule or the above biological material in the preparation of a product for improving mood.

[0012] As a preferred technical solution, the product includes food or medicine.

[0013] Another object of the present invention is to provide a drug for improving mood, which includes the above brain peptidase-cleaved active peptide.

[0014] Another object of the present invention is to provide a food for improving mood, which includes the above brain peptidase-cleaved active peptide.

[0015] Through the above technical solutions, compared with the prior art, the present invention has the following beneficial effects: The present invention uses a specific preparation method to enzymatically hydrolyze and purify the porcine brain extract, and successfully prepares the brain peptidase-cleaved active peptide VVAVP. Moreover, the use of the preparation method of the present invention significantly improves the enzymatic hydrolysis yield and enzymatic hydrolysis purity. In addition, the present invention verifies the efficacy of the prepared short peptide. The experimental results show that the short peptide VVAVP of the present invention can play a certain anti-anxiety role in the anxiety mouse model, and the treatment effect is equivalent to that of the positive control group, having the potential for mood regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0017] Figure 1 It is: the mass spectrum of the protease-cleaved new active peptide VVAVP. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the protection scope of the present invention.

[0019] The carboxypeptidase B used in the embodiments of the present invention was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0020] Example 1

[0021] A preparation method of brain peptide hydrolysate active peptide includes the following steps:

[0022] (1) Pretreatment of raw materials: Using pig brain extract as the raw material, stir and degrease it twice with 2 volumes of acetone-ether solution (1:0.5 (V:V)) for 0.5 h each time. After filtration, vacuum dry it to obtain defatted powder for standby.

[0023] (2) Water extraction and preliminary purification: Add the defatted powder to 0.1 M phosphate buffer solution (pH 7.3) at a weight-to-volume ratio of 1:8, stir and extract at 3°C for 4 hours, centrifuge at 6000 g for 15 min, and take the supernatant. The supernatant is fractionally precipitated with ammonium sulfate at 30% - 60% saturation, the precipitate is collected, dialyzed (2 kDa) to remove salts, and freeze-dried to obtain crude brain peptide powder.

[0024] (3) Multi-enzyme - stepwise enzymatic hydrolysis:

[0025] First enzymatic hydrolysis: First, adjust the pH of the substrate crude brain peptide powder to 8.5, add alkaline protease according to the weight ratio of alkaline protease to substrate of 0.5:100, react at 45°C for 2.5 h, inactivate the enzyme by boiling water bath treatment for 4 min, centrifuge at 8000 g for 10 min, and take the supernatant.

[0026] Second enzymatic hydrolysis: Adjust the pH of the supernatant to 7.5, add trypsin according to the weight ratio of trypsin to substrate of 0.5:200, react at 36°C for 1.5 h, add acetic acid to adjust the pH to 3.5 to inactivate the enzyme, centrifuge at 8000 g for 10 min, and take the supernatant.

[0027] Third enzymatic hydrolysis: Adjust the pH of the supernatant to 7.0, add carboxypeptidase B according to the weight ratio of carboxypeptidase B to substrate of 0.5:500, react at 36°C for 0.5 h, adjust the pH to 1.5 with hydrochloric acid to inactivate the enzyme, centrifuge at 8000 g for 10 min, and take the supernatant to obtain the enzymatic hydrolysate.

[0028] (4) Precipitation and purification of proteins: Heat the enzymatic hydrolysate to 70°C and maintain for 5 min, cool and centrifuge at 8000 g for 10 min, and take the supernatant.

[0029] (5) Ethanol fractional precipitation:

[0030] 20% ethanol precipitation: Slowly add cold ethanol (-20 °C) to the supernatant of step (4) at 4 °C at a rate of ≤1 mL / min, control the final concentration of ethanol to be 20%, stir for 5 min at 4 °C, centrifuge at 8000 g for 10 min, and take the supernatant;

[0031] 40% ethanol precipitation: Take the supernatant, slowly add cold ethanol at 4 °C, control the final concentration of ethanol to be 40%, stir and centrifuge at 4 °C, take the precipitate, ultrasonically dissolve the precipitate with deionized water at 0 °C, ultrasonic power 20 W, 5 s pulse mode, treat for 20 s, dialyze to remove alcohol, and freeze-dry to obtain crude peptides;

[0032] (6) Chromatographic separation and extraction: Pass the crude peptides through a chromatographic column, collect the main peak of VVAVP, ultrafiltrate and concentrate, and freeze-dry to obtain enzymatically hydrolyzed active peptides of brain peptides;

[0033] The chromatographic conditions are as follows:

[0034] Chromatographic column: Sephadex G-15 (2.5×100 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 method for preparing enzymatically hydrolyzed active peptides of brain peptides, comprising the following steps:

[0040] (1) Pretreatment of raw materials: Using pig brain extract as raw material, stir and defat with 3 times the volume of acetone-ether solution (2:0.5 (V:V)) 3 times, 1 h each time, filter and vacuum dry to prepare defatted powder for later use;

[0041] (2) Water extraction and preliminary purification: Add defatted powder to 0.1 M phosphate buffer (pH 7.5) at a weight-to-volume ratio of 1:12, stir and extract at 3-4 °C for 4 hours, centrifuge at 10000 g for 25 min, and take the supernatant; The supernatant is fractionally precipitated with ammonium sulfate at 30% - 60% saturation, collect the precipitate, dialyze (4 kDa) to remove salts, and freeze-dry to obtain crude brain peptide powder.

[0042] (3) Multi-enzyme - stepwise enzymatic hydrolysis:

[0043] First enzymatic hydrolysis: First, adjust the pH of the crude cerebropeptide powder substrate to 9.5. Add alkaline protease according to the weight ratio of alkaline protease to substrate of 1.5:100, react at 55 °C for 3.5 h, inactivate the enzyme by boiling water bath treatment for 5 min, centrifuge at 12000 g for 20 min, and take the supernatant;

[0044] Second enzymatic hydrolysis: Adjust the pH of the supernatant to 8.5. Add trypsin according to the weight ratio of trypsin to substrate of 1.5:200, react at 38 °C for 3 h, add acetic acid to adjust the pH to 4.5 to inactivate the enzyme, centrifuge at 10000 g for 20 min, and take the supernatant;

[0045] Third enzymatic hydrolysis: Adjust the pH of the supernatant to 8.0. Add carboxypeptidase B according to the weight ratio of carboxypeptidase B to substrate of 1.5:500, react at 38 °C for 1 h, adjust the pH to 2.5 with hydrochloric acid to inactivate the enzyme, centrifuge at 10000 g for 20 min, take the supernatant, and prepare the enzymatic hydrolysate;

[0046] (4) Protein precipitation and purification: Heat the enzymatic hydrolysate to 80 °C and maintain for 10 min, cool and centrifuge at 10000 g for 15 min, and take the supernatant;

[0047] (5) Ethanol fractional precipitation:

[0048] 20% ethanol precipitation: Place the supernatant from step (4) at 4 °C and slowly add cold ethanol (-20 °C) at a rate of ≤ 1 mL / min, control the final concentration of ethanol to 20%, stir for 10 min at 4 °C, centrifuge at 10000 g for 15 min, and take the supernatant;

[0049] 40% ethanol precipitation: Take the supernatant, place it at 4 °C and slowly add cold ethanol, control the final concentration of ethanol to 40%, stir and centrifuge at 4 °C, take the precipitate, dissolve the precipitate in deionized water by ultrasonic wave at 4 °C, ultrasonic power 50 W, 5 s pulse mode, treat for 30 s, dialyze to remove alcohol and freeze-dry to obtain crude peptide;

[0050] (6) Chromatographic separation and extraction: Pass the crude peptide through a chromatographic column, collect the main peak of VVAVP, ultrafiltrate and concentrate, and freeze-dry to obtain the cerebropeptide enzymatic active peptide;

[0051] The chromatographic conditions are as follows:

[0052] Chromatographic column: Sephadex G-15 (2.5×100 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 peptidase hydrolyzed active peptide, comprising the following steps:

[0058] (1) Pretreatment of raw materials: Using porcine brain extract as the raw material, stir and degrease it 3 times with 3 volumes of acetone-ether solution (1:1 (V:V)) for 1 hour each time. After filtration, vacuum dry it to obtain defatted powder for standby;

[0059] (2) Water extraction and preliminary purification: Add the defatted powder to 0.1M phosphate buffer (pH 7.4) at a weight-to-volume ratio of 1:10, stir and extract at 4°C for 4 hours, centrifuge at 8000g for 20 minutes, and take the supernatant; The supernatant is fractionated and precipitated with ammonium sulfate at 30% - 60% saturation, collect the precipitate, dialyze (3 kDa) to remove salts, and freeze-dry to obtain crude brain peptide powder.

[0060] (3) Multi-enzyme - stepwise enzymatic hydrolysis:

[0061] First enzymatic hydrolysis: First, adjust the pH of the substrate crude brain peptide powder to 9.0, add alkaline protease according to a weight ratio of alkaline protease to substrate of 1:100, react at 50°C for 3.0 hours, treat it in a boiling water bath for 5 minutes to inactivate the enzyme, centrifuge at 10000g for 15 minutes, and take the supernatant;

[0062] Second enzymatic hydrolysis: Adjust the pH of the supernatant to 8.0, add trypsin according to a weight ratio of trypsin to substrate of 1:200, react at 37°C for 2 hours, add acetic acid to adjust the pH to 4.0 to inactivate the enzyme, centrifuge at 10000g for 15 minutes, and take the supernatant;

[0063] Third enzymatic hydrolysis: Adjust the pH of the supernatant to 7.5, add carboxypeptidase B according to a weight ratio of carboxypeptidase B to substrate of 1:500, react at 37°C for 1 hour, adjust the pH to 2.0 with hydrochloric acid to inactivate the enzyme, centrifuge at 10000g for 15 minutes, and take the supernatant to obtain the enzymatic hydrolysate;

[0064] (4) Precipitation and purification of proteins: Heat the enzymatic hydrolysate to 75°C and maintain for 10 minutes, cool it, centrifuge at 8000g for 10 minutes, and take the supernatant;

[0065] (5) Ethanol fractional precipitation:

[0066] 20% ethanol precipitation: Place the supernatant from step (4) at 4°C and slowly add cold ethanol (-20°C) at a rate of ≤1 mL / min, control the final concentration of ethanol to be 20%, stir for 10 minutes at 4°C, centrifuge at 8000g for 15 minutes, and take the supernatant;

[0067] 40% Ethanol Precipitation: Take the supernatant and slowly add cold ethanol at 4°C, controlling the final concentration of ethanol to be 40%. Stir and centrifuge at 4°C. Take the precipitate and dissolve it in deionized water by ultrasonic treatment at 3°C. The ultrasonic power is 20W, the pulse mode is 5s, and the treatment time is 30s. Then dialyze to remove ethanol and lyophilize to obtain crude peptides.

[0068] (6)Chromatographic Separation and Extraction: Pass the crude peptides through a chromatographic column, collect the main peak of VVAVP, ultrafiltrate and concentrate, and lyophilize to obtain the enzymatically hydrolyzed active peptide of brain peptide.

[0069] The chromatographic conditions are as follows:

[0070] Chromatographic column: Sephadex G-15 (2.5×100 cm);

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

[0072] Flow rate: 0.5 mL / min;

[0073] Detection: UV 220 nm.

[0074] Perform mass spectrometry detection on the extracted enzymatically hydrolyzed active peptide of brain peptide. The detection results are as Figure 1 shown, and the specific results are shown in Table 1.

[0075] Table 1 Mass Spectrometry Fragment Ion Detection Results Ion type Measured m / z Sequence position y1 116.0706 P y2 215.139 VP y3 286.1761 AVP b2 199.1441 VV b3 270.1812 VVA

[0076] Parent ion: m / z 485.73 ([M+2H] 2+ , theoretical calculated value 485.23).

[0077] In summary, by matching the b / y ion series, the detected peptide segment is confirmed to be VVAVP.

[0078] Comparative Example 1

[0079] A method for preparing an enzymatically hydrolyzed active peptide of brain peptide, comprising the following steps:

[0080] 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 in Example 3.

[0081] Comparative Example 2

[0082] A method for preparing an enzymatically hydrolyzed active peptide of brain peptide, comprising the following steps:

[0083] It is basically the same as Example 3, except that the first enzymatic hydrolysis process is not included in step (3), and the remaining operations are the same as in Example 3.

[0084] Comparative Example 3

[0085] A method for preparing a brain peptide hydrolysate active peptide, comprising the following steps:

[0086] It is basically the same as Example 3, except that the ethanol used in step (5) is at room temperature, and the rest of the operations are the same as in Example 3.

[0087] Comparative Example 4

[0088] A method for preparing a brain peptide hydrolysate active peptide, comprising the following steps:

[0089] It is basically the same as Example 3, except that the ethanol fractional precipitation process in step (5) is not involved, and the rest of the operations are the same as in Example 3.

[0090] Comparative Example 5

[0091] A method for preparing a brain peptide hydrolysate active peptide, comprising the following steps:

[0092] It is basically the same as Example 3, except that in step (5), 30% ethanol precipitation and 50% ethanol precipitation are used for ethanol fractional precipitation, and the rest of the operations are the same as in Example 3.

[0093] In order to verify the effects of the preparation methods of different examples, the enzymatic hydrolysis yields and the purities of the enzymatic hydrolysis fragments of different examples and comparative examples were measured, and the experimental results are shown in Table 2:

[0094] Table 2 Enzymatic hydrolysis yields and enzymatic hydrolysis purities of the preparation methods of different groups Group Enzymatic hydrolysis yield (%) Enzymatic hydrolysis 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

[0095] Result analysis: According to the content of Table 2, it can be seen that the preparation method of the present invention can significantly improve the enzymatic hydrolysis yield of the brain peptide hydrolysate active peptide VVAVP and the purity of the product compared with Comparative Examples 1-5.

[0096] Meanwhile, in order to further verify the efficacy of the peptide segments of the present invention, the following mouse experiment is now carried out.

[0097] Construction of an anxiety mouse model and detection of anxiety-like behaviors

[0098] Select several male C57BL / 6J mice at 8 - 12 weeks of age. One week before the start of model construction, after adapting the experimental mice to a strange environment for one week, one day before the start of model construction, perform the elevated plus maze test on the mice participating in the experiment, screen out the mice with normal mental and emotional states, and exclude the mice with abnormal indicators for the next experiment. Feeding conditions: 12 h light - dark cycle, free access to food and water. Divide the above - screened mice into a control group, an experimental group, and a positive control group, with no less than 3 mice in each group. Through restraint treatment of the mice, make them produce anxiety under stress, and verify the mechanism of action of the brain peptidase - hydrolyzed active peptide VVAVP in alleviating this process. The model was constructed for 7 days.

[0099] Control group mice: Without restraint treatment, and only give them normal saline.

[0100] Experimental group mice: Perform restraint treatment, and respectively give them low, medium, and high - dose peptide segments (0.1, 1, and 10 mg / kg) prepared in Example 3.

[0101] Positive control group mice: Perform restraint treatment, and give them diazepam at 1 mg / kg.

[0102] Perform anxiety - like behavior detection on the mice in the above different groups using the elevated plus maze (EPM). Place the mice in the central area, facing the open arms, and record the number of times the mice enter the open arms within 5 minutes (when all four limbs enter completely is counted as one time); the proportion of time spent in the open arms (the lower the anxiety, the longer the time), and the total movement distance (excluding the interference of motor ability). The experimental results are shown in Table 3.

[0103] Table 3 Anxiety - like behavior results of different treatment groups Group Control group Experimental group (low) Experimental group (medium) Experimental group (high) Positive control group Number of entries into the open arm (times) 3 8 9 10 11 Percentage of residence time in the open arm (%) 22.6 36.4 39.8 43.3 44.1 Total movement distance (cm) 1400 1406 1396 1405 1410

[0104] Result analysis: As can be seen from Table 3, compared with the control group, giving VVAVP prepared in Example 3 can, to a certain extent, increase the number of times the experimental group mice enter the open arms. As the number of entries increases, it indicates that the anxiety behavior of the mice decreases. At the same time, the time spent by the mice in the high - dose group of the experimental group in the open arms is significantly closer to that of the positive control group and is significantly different from the control group, which also indicates that the anxiety level of the mice decreases. Observing the total movement distance of the mice in different groups, there is no obvious difference, indicating that the peptide segment does not affect the motor ability of the mice, excluding the sedative or muscle toxicity effects of the peptide segment.

[0105] In this specification, each example is described in a progressive manner. The key point of each example is to illustrate the differences from other examples. For the same and similar parts between each example, reference can be made to each other.

[0106] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not 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 hydrolyzed active peptide, characterized in that, The amino acid sequence of the active peptide is VVAVP, SEQ ID NO.

1.

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

3. A biological material, characterized in that, The biological material contains the nucleic acid molecule described in claim 2 and is capable of expressing the brain peptide hydrolytic active peptide described in claim 1; The biological material includes a recombinant plasmid, a recombinant vector or a recombinant host cell.

4. Use of the brain peptide hydrolytic active peptide described in claim 1, or the nucleic acid molecule described in claim 2, or the biological material described in claim 3 in the preparation of a product for improving mood.

5. The application according to claim 4, wherein The product includes a food or a drug.

6. A drug for improving mood, characterized in that, It includes the brain peptide hydrolytic active peptide described in claim 1.

7. A food for improving mood, characterized in that, It includes the brain peptide hydrolytic active peptide described in claim 1.

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