New active peptide composition for regulating emotion and application thereof

Abstract

The invention discloses a novel active peptide composition for regulating emotion and application thereof. Belongs to the technical field of active peptides. In the process of extracting new active peptides from brain peptide by taking brain peptide as a raw material, various new active peptides, including VVAVP and VVAVPGN, are found, the efficacy of the new active peptides is further studied, and experimental results show that VVAVP and VVAVPGN have a certain anti-depression effect, but when VVAVP and VVAVPGN synergistically act on mice at the same time according to a ratio of 1: 1-1: 3, the anti-depression effect of VVAVP and VVAVPGN is remarkably improved, and the anti-depression effect of VVAVP and VVAVPGN is remarkably improved. It is accidentally found that the anti-depression effect is multiplied, it is indicated that when fluoxetine and fluoxetine are mixed in a specific proportion, the synergistic effect is achieved in the anti-depression aspect, the anti-depression effect can be remarkably improved, the effect is equivalent to that of fluoxetine in a positive control group, and a new research direction is provided for research and development of anti-depression drugs.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and more specifically to a novel bioactive peptide composition for regulating mood and its applications. Background Technology

[0002] Brain peptides generally refer to a class of bioactive peptides found in the brain or nervous system that participate in regulating nerve function. These include naturally occurring neuropeptides (such as endorphins, enkephalins, and substance P) or synthetic peptide compounds. Common natural brain peptides typically have the following functions: endorphins have analgesic, stress-relieving, and pleasure-inducing effects, and are known as "natural painkillers"; enkephalins can regulate pain transmission and are associated with memory; substance P participates in pain signal transmission and inflammatory responses, and is associated with diseases such as migraines and arthritis; adrenocorticotropic hormone (ACTH) regulates stress responses and cortisol secretion, affecting learning and memory; and ghrelin, in addition to regulating appetite, may also affect cognitive function and neuroprotection.

[0003] However, these natural brain peptides are easily degraded rapidly by enzymes in the body (such as proteases), have short half-lives (e.g., enkephalins only exist in the blood for a few minutes), resulting in short duration of action and relatively poor stability. Furthermore, most natural brain peptides have large molecular weights and high polarity, making it difficult for them to freely cross the blood-brain barrier, limiting their direct effects on the central nervous system. In addition, natural brain peptides exhibit non-specificity, potentially activating multiple receptor subtypes simultaneously (e.g., substance P acts on NK1 and NK2 receptors simultaneously), leading to side effects (such as inflammation or abnormal pain sensitivity). Therefore, the limitations of natural brain peptides have driven researchers to develop artificial brain peptides. However, current research on the application of artificial brain peptides in mood regulation is relatively limited, resulting in a small number of artificial brain peptides suitable for mood regulation, poor selectivity, and unreliable mood-regulating effects from a single artificial brain peptide.

[0004] Therefore, how to provide a new active peptide composition for regulating emotions and its application, enrich the quantity of artificial brain peptides for emotion regulation, and improve the regulatory effect are technical problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a novel active peptide composition for regulating mood and its application. Two novel active peptides were discovered during the enzymatic hydrolysis of brain peptides. It was also found that the two have a synergistic effect, and compared with a single active peptide, the anti-anxiety effect is more obvious, and the mood regulation effect is significant.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A novel active peptide composition for regulating mood includes novel active peptide 1 and novel active peptide 2; the amino acid sequence of novel active peptide 1 is VVAVP; and the amino acid sequence of novel active peptide 2 is VVAVPGN.

[0008] As a preferred technical solution, the molar ratio of novel active peptide 1 to novel active peptide 2 in the novel active peptide composition is 1:1 to 1:3.

[0009] As a preferred technical solution, the novel active peptide composition for regulating mood also includes other excipients or active ingredients.

[0010] As a preferred technical solution, the novel active peptide 1 is prepared by the following method:

[0011] S1: Pretreatment of raw materials: Using pig brain extract as raw material, the extract is defatted by stirring with 2-3 times the volume of acetone-ether solution, filtered and vacuum dried to prepare defatted powder for later use.

[0012] S2: Water extraction and preliminary purification: Add defatted powder to phosphate buffer at a weight-to-volume ratio of 1:8-12 and stir for 4 hours at 3-4℃. Centrifuge and collect the supernatant. Use 30%~60% saturated ammonium sulfate to precipitate the supernatant in fractional fractions. Collect the precipitate, dialyze, and freeze-dry to obtain crude brain peptide powder.

[0013] S3: Multi-enzyme stepwise enzymatic hydrolysis:

[0014] First enzymatic hydrolysis: Adjust the pH of the crude brain peptide substrate to 8.5-9.5, add alkaline protease, react at 45-55℃ for 2.5-3.5h, inactivate the enzyme in a water bath, and centrifuge to collect the supernatant;

[0015] Second enzymatic hydrolysis: Adjust the pH of the supernatant to 7.5-8.5, add trypsin, react at 36-38℃ for 1.5-3 hours, adjust the pH to 3.5-4.5 to inactivate the enzyme, centrifuge and collect the supernatant;

[0016] Third enzymatic hydrolysis: Adjust the pH of the supernatant to 7.0-8.0, add carboxypeptidase B, react at 36-38℃ for 0.5-1h, adjust the pH to 1.5-2.5 to inactivate the enzyme, centrifuge and collect the supernatant to prepare the enzymatic hydrolysate;

[0017] S4: Protein precipitation and purification: Heat the enzymatic hydrolysate to 70-80℃ and maintain for 5-10 min, then cool and centrifuge to collect the supernatant;

[0018] S5: Ethanol fractionation precipitation:

[0019] 20% ethanol precipitation: Place the supernatant from step (4) at 4°C and slowly add cold ethanol to control the final concentration of ethanol to 20%. Stir, centrifuge at 4°C, and take the supernatant.

[0020] 40% ethanol precipitation: Take the supernatant and place it at 4℃, slowly add cold ethanol, control the final concentration of ethanol to 40%, stir, centrifuge at 4℃, take the precipitate, dissolve the precipitate with deionized water by sonication, remove alcohol, freeze dry to obtain crude peptide.

[0021] S6: Chromatographic separation and extraction: Pass the crude peptide through a chromatographic column, collect the VVAVP main peak, and freeze-dry to obtain the new active peptide 1.

[0022] As a preferred technical solution, the volume ratio of acetone to diethyl ether in S1 is 1-2:0.5-2; the stirring and degreasing time is 0.5-1 hour, and the stirring and degreasing is performed 2-3 times;

[0023] The phosphate buffer solution in S2 has a concentration of 0.1M and a pH of 7.3-7.5; the centrifugation speed is 6000-10000×g and the time is 15-25min; the molecular weight cutoff for dialysis is 2-4 kDa.

[0024] In the first enzymatic hydrolysis of S3, the enzyme inactivation is performed by boiling water bath for 4-5 min; the centrifugation speed is 8000-12000×g and the time is 10-20 min; the weight ratio of alkaline protease to substrate is 0.5-1.5:100.

[0025] In the second enzymatic hydrolysis in S3, acetic acid is used to adjust the pH to 3.5-4.5; the centrifugation speed is 8000-10000×g, and the time is 10-20min; the weight ratio of trypsin to substrate is 0.5-1.5:200.

[0026] In the third enzymatic hydrolysis described in S3, the pH is adjusted to 1.5-2.5 using hydrochloric acid; the centrifugation speed is 8000-10000×g, and the time is 10-20min; the weight ratio of carboxypeptidase B to substrate is 0.5-1.5:500.

[0027] The centrifugation speed described in S4 is 8000-10000×g, and the time is 10-15 min;

[0028] The addition rate of cold ethanol in S5 is ≤1 mL / min, and the temperature is -20℃; the stirring time is 5-10 min; the centrifugation speed is 8000-10000×g, and the time is 10-15 min.

[0029] The conditions for ultrasonic dissolution of the precipitate are as follows: 0-4℃, ultrasonic power 20-50W, 5s pulse mode, treatment time 20-30s; the alcohol removal is performed by dialysis.

[0030] The chromatographic conditions described in S5 are as follows:

[0031] Chromatographic column: Sephadex G-15 (2.5 × 100 cm);

[0032] Mobile phase: 0.1 M NH4HCO3 (pH 7.8);

[0033] Flow rate: 0.5 mL / min;

[0034] Detection: UV 220 nm.

[0035] As a preferred technical solution, the novel active peptide 2 is prepared by the following method:

[0036] (1) Pretreatment of raw materials: Take fresh pig brain, remove blood vessels and meninges, wash, add 3-4 times the volume of pre-cooled PBS buffer, homogenize at high speed, centrifuge, take the supernatant, and prepare pig brain extract.

[0037] (2) Multi-enzyme stepwise enzymatic hydrolysis:

[0038] First enzymatic hydrolysis: Adjust the pH of the pig brain extract to 1.5-2.5, add pepsin, and react at 36-38℃ for 1.5-2.5 h; adjust the pH to 7.0 to terminate the enzymatic hydrolysis reaction and prepare the pepsin hydrolysate;

[0039] Second enzymatic hydrolysis: Tris-HCl and CaCl2 were added to the pepsin hydrolysate to adjust the pH to 7.5-7.8, thermophilic protease was added, and the reaction was carried out at 50°C for 4 hours. The enzyme was then inactivated by boiling water bath to prepare the thermophilic protease hydrolysate.

[0040] Third enzymatic hydrolysis: NH4HCO3 was added to the thermophilic bacteria protease hydrolysate to adjust the pH to 8.0-8.2, trypsin was added, and the reaction was carried out at 36-38℃ for 0.5-1h to prepare the trypsin hydrolysate;

[0041] (3) Precipitation to remove macromolecules: Centrifuge the trypsin hydrolysate, collect the supernatant, concentrate by ultrafiltration, and collect the permeate;

[0042] (4) Ethanol precipitation and concentration: Add 3-4 times the volume of cold ethanol to the permeate, let stand, centrifuge, and redissolve the precipitate with a small amount of water to obtain a crude peptide mixture;

[0043] (5) HPLC separation:

[0044] Column: C18 reversed-phase column;

[0045] Mobile phase: 0.1% TFA in water / acetonitrile;

[0046] Flow rate: 1.0 mL / min;

[0047] Detection: 220 nm.

[0048] As a preferred technical solution, in the pretreatment of the raw materials in step (1), 0.9% cold physiological saline is used for washing; the PBS buffer contains 1mM PMSF protease inhibitor; the high-speed homogenization process is as follows: 10000rpm, 30 seconds × 3 times, with ice bath intervals;

[0049] In step (2), during the first enzymatic hydrolysis, the pH was adjusted to 1.5-2.5 using 1M HCl; the final concentration of pepsin was 0.1 mg / mL; and the pH was adjusted to 7.0 using 1M NaOH.

[0050] In step (2), during the second enzymatic hydrolysis, the final concentration of CaCl2 in the pepsin hydrolysate is 2 mM; the amount of thermophilic protease added is 0.4-0.6 U / mg protein; and the time for inactivating the enzyme in the boiling water bath is 5-10 min.

[0051] In step (2), during the third enzymatic hydrolysis, the amount of trypsin added is 0.04-0.06 U / mg protein;

[0052] In step (3), the centrifugation speed is 10,000 × g and the time is 5-10 min; the molecular weight cutoff of the ultrafiltration is 3-3.5 kDa.

[0053] The temperature of the cold ethanol in step (4) is -20℃; the standing time is 0.5-1h; the centrifugation speed is 8000-10000×g; and the time is 10-15min.

[0054] Another object of the present invention is to provide the use of the above-described novel active peptide composition for regulating mood in the preparation of mood-regulating drugs.

[0055] Another object of the present invention is to provide a medicine for regulating mood, comprising the above-described novel active peptide composition for regulating mood.

[0056] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: In the process of extracting new active peptides from brain peptides using brain peptides as raw materials, the present invention discovered a variety of new active peptides, including the new active peptides VVAVP and VVAVPGN. Further research was conducted on the efficacy of the above-mentioned new active peptides. The experimental results showed that VVAVP and VVAVPGN have certain antidepressant effects. However, when the two were synergistically acted on mice in a ratio of 1:1 to 1:3, an unexpected increase in the antidepressant-like effect was found. This indicates that when the two are mixed in a specific ratio, they have a synergistic effect in antidepressant treatment, which can significantly enhance the antidepressant effect. Its effect is comparable to that of fluoxetine in the positive control group, providing a new research direction for the development of antidepressant drugs. Attached Figure Description

[0057] Appendix Figure 1 The image shows the results of mass spectrometry detection of the extracted novel active peptide 1.

[0058] Appendix Figure 2 The image shows the results of mass spectrometry detection of the extracted new active peptide 2. Detailed Implementation

[0059] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0060] The carboxypeptidase B used in the embodiments of this invention was purchased from Beijing Solarbio Technology Co., Ltd.

[0061] Example 1

[0062] Preparation of New Active Peptide 1:

[0063] (1) Pretreatment of raw materials: Using pig brain extract as raw material, the degreased powder was prepared by stirring with 3 times the volume of acetone-ether solution (1:1 (V:V)) for 3 times, 1 hour each time, filtered and vacuum dried.

[0064] (2) Water extraction and preliminary purification: The defatted powder was added to 0.1M phosphate buffer (pH 7.4) at a weight-to-volume ratio of 1:10 and extracted at 4℃ with stirring for 4 hours. After centrifugation at 8000×g for 20 min, the supernatant was collected. The supernatant was fractionally precipitated with 30%~60% saturated ammonium sulfate. The precipitate was collected, dialyzed (3 kDa) to remove salt, and freeze-dried to obtain crude brain peptide powder.

[0065] (3) Multi-enzyme stepwise enzymatic hydrolysis:

[0066] First enzymatic hydrolysis: First, adjust the pH of the crude brain peptide substrate to 9.0. Add alkaline protease according to the weight ratio of alkaline protease to substrate of 1:100. React at 50℃ for 3.0h, inactivate the enzyme by boiling water bath for 5min, centrifuge at 10000×g for 15min and collect the supernatant.

[0067] Second enzymatic hydrolysis: Adjust the pH of the supernatant to 8.0, add trypsin at a weight ratio of 1:200 to substrate, react at 37℃ for 2 hours, add acetic acid to adjust the pH to 4.0 to inactivate the enzyme, centrifuge at 10000×g for 15 minutes, and collect the supernatant.

[0068] Third enzymatic hydrolysis: Adjust the pH of the supernatant to 7.5, add carboxypeptidase B at a weight ratio of 1:500 to substrate, react at 37℃ for 1 h, adjust the pH to 2.0 with hydrochloric acid to inactivate the enzyme, centrifuge at 10000×g for 15 min, take the supernatant, and prepare the enzymatic hydrolysate.

[0069] (4) Protein precipitation and purification: Heat the enzymatic hydrolysate to 75°C and maintain for 10 min, cool and centrifuge at 8000×g for 10 min, and take the supernatant;

[0070] (5) Ethanol fractionation and precipitation:

[0071] 20% ethanol precipitation: Place the supernatant from step (4) at 4℃ and slowly add cold ethanol (-20℃) at a rate of ≤1 mL / min, control the final concentration of ethanol to 20%, stir for 10 min, centrifuge at 4℃ for 8000×g for 15 min, and take the supernatant.

[0072] 40% ethanol precipitation: Take the supernatant and place it at 4℃, slowly add cold ethanol, control the final concentration of ethanol to 40%, stir, centrifuge at 4℃, take the precipitate, and sonicate the precipitate with deionized water at 3℃, ultrasonic power 20W, 5s pulse mode, process for 30s, dialyze to remove alcohol, freeze dry to obtain crude peptide.

[0073] (6) Chromatographic separation and extraction: Pass the crude peptide through a chromatographic column, collect the VVAVP main peak, and freeze-dry to obtain the new active peptide 1;

[0074] The chromatographic conditions are as follows:

[0075] Chromatographic column: Sephadex G-15 (2.5 × 100 cm);

[0076] Mobile phase: 0.1 M NH4HCO3 (pH 7.8);

[0077] Flow rate: 0.5 mL / min;

[0078] Detection: UV 220 nm.

[0079] The extracted novel active peptide 1 was analyzed by mass spectrometry, and the results are as follows: Figure 1 As shown in Table 1, the specific results are as follows.

[0080] Table 1. Mass spectrometry fragment ion detection results Ion type Actual m / z Sequence position y1 116.0706 P y2 215.139 VP y3 286.1761 AVP b2 199.1441 VV b3 270.1812 VVA

[0081] Precipitating ion: m / z 485.73 ([M+2H]²⁺, theoretically calculated value 485.23).

[0082] In summary, by matching the b / y ion series, the detected peptide was confirmed to be VVAVP.

[0083] Example 2

[0084] Preparation and extraction of new active peptide 2:

[0085] (1) Pretreatment of raw materials: Take fresh pig brain, remove blood vessels and meninges, wash with 0.9% cold physiological saline, add 4 times the volume of pre-cooled PBS buffer (containing 1mM PMSF protease inhibitor), 10000rpm, 30 seconds × 3 times, ice bath intermittently, high speed homogenize, centrifuge, take the supernatant to prepare pig brain extract.

[0086] (2) Multi-enzyme stepwise enzymatic hydrolysis:

[0087] First enzymatic hydrolysis: Add 1M HCl to adjust the pH of the pig brain extract to 1.5-2.5, add pepsin (final concentration 0.1mg / mL), and react at 36-38℃ for 1.5-2.5h; add 1M NaOH to adjust the pH to 7.0 to terminate the enzymatic hydrolysis reaction and prepare the pepsin hydrolysate.

[0088] Second enzymatic hydrolysis: Tris-HCl and CaCl2 (final concentration 2mM) were added to the pepsin hydrolysate, the pH was adjusted to 7.5-7.8, thermophilic protease was added at a concentration of 0.4-0.6 U / mg protein, the reaction was carried out at 50℃ for 4 hours, and the enzyme was inactivated by boiling water bath for 10 min to prepare thermophilic protease hydrolysate.

[0089] Third enzymatic hydrolysis: Add NH4HCO3 to the thermophilic bacteria protease hydrolysate, adjust the pH to 8.0-8.2, add trypsin at a rate of 0.04-0.06 U / mg protein, and react at 36-38℃ for 0.5-1 h to prepare the trypsin hydrolysate;

[0090] (3) Precipitation to remove macromolecules: Take 10,000×g of trypsin hydrolysate, centrifuge for 10 min, take the supernatant, concentrate by ultrafiltration (molecular weight cutoff is 3kDa), and collect the permeate;

[0091] (4) Ethanol precipitation and concentration: Add 3-4 times the volume of cold ethanol (-20℃) to the permeate, let stand for 1 hour, centrifuge at 8000×g for 10 minutes, and redissolve the precipitate with a small amount of water to obtain a crude peptide mixture.

[0092] (5) HPLC separation:

[0093] Column: C18 reversed-phase column;

[0094] Mobile phase A: Ultrapure water + 0.1% trifluoroacetic acid (TFA, v / v)

[0095] Flow phase B: acetonitrile + 0.1% TFA (v / v); gradient (10%-40% acetonitrile, 30 min);

[0096] Flow rate: 1.0 mL / min;

[0097] Detection: 220 nm.

[0098] The specific elution procedure is as follows: 0 min: 10% B; 1-30 min: 10-40% B; 30-31 min: 40-90% B; 31-35 min: 90% B; 35-36 min: 90-10% B; 36-40 min: 10% B.

[0099] The extracted novel active peptide 2 was analyzed by mass spectrometry, and the results are as follows: Figure 2 As shown, the precursor ion is m / z 219.13. By matching the b / y ion series, the detected peptide was confirmed to be VVAVPGN.

[0100] Example 3

[0101] A novel active peptide composition for regulating mood, comprising VVAVP 5mg + VVAVPGN 10mg + mannitol (carrier).

[0102] Example 4

[0103] A novel active peptide composition for regulating mood, comprising VVAVP 5mg + VVAVPGN 5mg + mannitol (carrier).

[0104] Example 5

[0105] A novel active peptide composition for regulating mood, comprising VVAVP 5mg + VVAVPGN 15mg + mannitol (carrier).

[0106] Comparative Example 1

[0107] A novel active peptide composition for regulating mood, comprising VVAVP 5mg + VVAVPGN 4mg + mannitol (carrier).

[0108] Comparative Example 2

[0109] A novel active peptide composition for regulating mood, comprising VVAVP 5mg + VVAVPGN 18mg + mannitol (carrier).

[0110] Comparative Example 3

[0111] A novel active peptide composition for regulating mood, comprising VVAVPGN 15 mg + mannitol (carrier).

[0112] Comparative Example 4

[0113] A novel active peptide composition for regulating mood, comprising VVAVP 5mg + mannitol (carrier).

[0114] To verify the mood-regulating effects of the novel active peptide composition in different groups, the following mouse experiments were conducted:

[0115] Construction of a mouse model of depression and detection of depression-like behaviors

[0116] A social frustration stress (SDS) model was constructed to simulate human social stress by inducing depressive-like behaviors through failure in social competition.

[0117] Procedure: The experimental mice (C57BL / 6J) were placed in cages with aggressive CD1 mice and subjected to 10 minutes of physical attack each day (monitoring was required to avoid serious injury). After 10 consecutive days, the escape behavior was assessed by social interaction tests. When CD1 was present, the experimental mice were considered to have successfully modeled the behavior if their stay time was less than 50% of the baseline value.

[0118] Blank control group mice: Depressed model mice, which were given only saline solution;

[0119] Experimental group mice: Depressed model mice were administered the novel active peptide compositions prepared in Examples 3-5 and Comparative Examples 1-2, respectively, at a dose of 5 mg / kg;

[0120] Positive control mice: Depressed model mice that were given fluoxetine 5 mg / kg.

[0121] The different groups were given the drug continuously for 14 days, and the depressive-like behaviors were detected by the Forced Swim Test (FST).

[0122] Apparatus: Cylindrical water tank (20 cm in diameter, 15 cm in water depth, 25±1℃). The mice were placed in the water and allowed to adapt for 1 minute. The time they remained still for 5 minutes after that was recorded (they only maintained a passive state with their heads floating).

[0123] The experimental results are shown in Table 2.

[0124] Table 2. Depression-like behavior outcomes in different treatment groups Group Example 3 Example 4 Example 5 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Blank control group Positive control group Stationary time (s) 91 90 88 124 119 130 133 142 90

[0125] Results analysis: As shown in Table 2, the immobility time of Examples 3-5 of the present invention is significantly lower than that of the blank control group. In addition, the immobility time of Examples 3-5 is comparable to or slightly better than that of the positive control group, indicating that the novel active peptide composition of the present invention has a good antidepressant effect.

[0126] Furthermore, a comparison of Examples 3-5 and Comparative Examples 1 and 2 shows that when New Active Peptide 1 and New Active Peptide 2 are mixed in a specific ratio (1:1-1:3), the antidepressant effect is good. When the ratio is outside this range (such as in Examples 1 and 2), although there is a certain antidepressant effect, it is not as good as in Examples 3-5.

[0127] Furthermore, by comparing Example 5 with Comparative Examples 3 and 4, it can be concluded that the new active peptide 1 and the new active peptide 2 have a synergistic effect, which can make the antidepressant effect better.

[0128] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0129] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not 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 novel active peptide composition for regulating mood, characterized in that, It includes novel active peptide 1 and novel active peptide 2; the amino acid sequence of novel active peptide 1 is VVAVP; the amino acid sequence of novel active peptide 2 is VVAVPGN.

2. The novel active peptide composition for regulating mood according to claim 1, characterized in that, The molar ratio of novel active peptide 1 to novel active peptide 2 in the novel active peptide composition is 1:1 to 1:

3.

3. The novel active peptide composition for regulating mood according to claim 1, characterized in that, It also includes other excipients or active ingredients.

4. The novel active peptide composition for regulating mood according to any one of claims 1-3, characterized in that, The novel active peptide 1 was prepared using the following method: S1: Pretreatment of raw materials: Using pig brain extract as raw material, the extract is defatted by stirring with 2-3 times the volume of acetone-ether solution, filtered and vacuum dried to prepare defatted powder for later use. S2: Water extraction and preliminary purification: Add defatted powder to phosphate buffer at a weight-to-volume ratio of 1:8-12 and stir for 4 hours at 3-4℃. Centrifuge and collect the supernatant. Use 30%~60% saturated ammonium sulfate to precipitate the supernatant in fractional fractions. Collect the precipitate, dialyze, and freeze dry to obtain crude brain peptide powder. S3: Multi-enzyme stepwise enzymatic hydrolysis: First enzymatic hydrolysis: Adjust the pH of the crude brain peptide substrate to 8.5-9.5, add alkaline protease, react at 45-55℃ for 2.5-3.5h, inactivate the enzyme in a water bath, and centrifuge to collect the supernatant; Second enzymatic hydrolysis: Adjust the pH of the supernatant to 7.5-8.5, add trypsin, react at 36-38℃ for 1.5-3 hours, adjust the pH to 3.5-4.5 to inactivate the enzyme, centrifuge and collect the supernatant; Third enzymatic hydrolysis: Adjust the pH of the supernatant to 7.0-8.0, add carboxypeptidase B, react at 36-38℃ for 0.5-1h, adjust the pH to 1.5-2.5 to inactivate the enzyme, centrifuge and collect the supernatant to prepare the enzymatic hydrolysate; S4: Protein precipitation and purification: Heat the enzymatic hydrolysate to 70-80℃ and maintain for 5-10 min, then cool and centrifuge to collect the supernatant; S5: Ethanol fractionation precipitation: 20% ethanol precipitation: Place the supernatant from step (4) at 4°C and slowly add cold ethanol to control the final concentration of ethanol to 20%. Stir, centrifuge at 4°C, and take the supernatant. 40% ethanol precipitation: Take the supernatant and place it at 4℃, slowly add cold ethanol, control the final concentration of ethanol to 40%, stir, centrifuge at 4℃, take the precipitate, dissolve the precipitate with deionized water by sonication, remove alcohol, freeze dry to obtain crude peptide. S6: Chromatographic separation and extraction: Pass the crude peptide through a chromatographic column, collect the VVAVP main peak, and freeze-dry to obtain the new active peptide 1.

5. The novel active peptide composition for regulating mood according to claim 4, characterized in that, The volume ratio of acetone to diethyl ether in S1 is 1-2:0.5-2; the stirring and degreasing time is 0.5-1 hour, and the stirring and degreasing is performed 2-3 times. The phosphate buffer solution in S2 has a concentration of 0.1M and a pH of 7.3-7.5; the centrifugation speed is 6000-10000×g and the time is 15-25min; the molecular weight cutoff for dialysis is 2-4 kDa. In the first enzymatic hydrolysis of S3, the enzyme inactivation is performed by boiling water bath for 4-5 min; the centrifugation speed is 8000-12000×g and the time is 10-20 min; the weight ratio of alkaline protease to substrate is 0.5-1.5:

100. In the second enzymatic hydrolysis in S3, acetic acid is used to adjust the pH to 3.5-4.5; the centrifugation speed is 8000-10000×g, and the time is 10-20min; the weight ratio of trypsin to substrate is 0.5-1.5:

200. In the third enzymatic hydrolysis described in S3, the pH is adjusted to 1.5-2.5 using hydrochloric acid; the centrifugation speed is 8000-10000×g, and the time is 10-20min; the weight ratio of carboxypeptidase B to substrate is 0.5-1.5:

500. The centrifugation speed described in S4 is 8000-10000×g, and the time is 10-15 min; The addition rate of cold ethanol in S5 is ≤1 mL / min, and the temperature is -20℃; the stirring time is 5-10 min; the centrifugation speed is 8000-10000×g, and the time is 10-15 min. The conditions for ultrasonic dissolution of the precipitate are as follows: 0-4℃, ultrasonic power 20-50W, 5s pulse mode, treatment time 20-30s; the alcohol removal is performed by dialysis. The chromatographic conditions described in S5 are as follows: Chromatographic column: Sephadex G-15 (2.5 × 100 cm); Mobile phase: 0.1 M NH4HCO3 (pH 7.8); Flow rate: 0.5 mL / min; Detection: UV 220 nm.

6. The novel active peptide composition for regulating mood according to any one of claims 1-3, characterized in that, The novel active peptide 2 was prepared using the following method: (1) Pretreatment of raw materials: Take fresh pig brain, remove blood vessels and meninges, wash, add 3-4 times the volume of pre-cooled PBS buffer, homogenize at high speed, centrifuge, take the supernatant, and prepare pig brain extract. (2) Multi-enzyme stepwise enzymatic hydrolysis: First enzymatic hydrolysis: Adjust the pH of the pig brain extract to 1.5-2.5, add pepsin, and react at 36-38℃ for 1.5-2.5 h; adjust the pH to 7.0 to terminate the enzymatic hydrolysis reaction and prepare the pepsin hydrolysate; Second enzymatic hydrolysis: Tris-HCl and CaCl2 were added to the pepsin hydrolysate to adjust the pH to 7.5-7.8, thermophilic protease was added, and the reaction was carried out at 50°C for 4 hours. The enzyme was then inactivated by boiling water bath to prepare the thermophilic protease hydrolysate. Third enzymatic hydrolysis: Add NH4HCO3 to the thermophilic bacteria protease hydrolysate, adjust the pH to 8.0-8.2, add trypsin, and react at 36-38℃ for 0.5-1h to prepare the trypsin hydrolysate; (3) Precipitation to remove macromolecules: Centrifuge the trypsin hydrolysate, collect the supernatant, concentrate by ultrafiltration, and collect the permeate; (4) Ethanol precipitation and concentration: Add 3-4 times the volume of cold ethanol to the permeate, let stand, centrifuge, and redissolve the precipitate with a small amount of water to obtain a crude peptide mixture; (5) HPLC separation: Column: C18 reversed-phase column; Mobile phase: 0.1% TFA in water / acetonitrile; Flow rate: 1.0 mL / min; Detection: 220 nm.

7. The novel active peptide composition for regulating mood according to claim 6, characterized in that, In step (1), the raw materials are pretreated by washing with 0.9% cold physiological saline; the PBS buffer contains 1mM PMSF protease inhibitor; the high-speed homogenization process is as follows: 10000rpm, 30 seconds × 3 times, with ice bath intervals; In step (2), during the first enzymatic hydrolysis, the pH was adjusted to 1.5-2.5 using 1M HCl; the final concentration of pepsin was 0.1 mg / mL; and the pH was adjusted to 7.0 using 1M NaOH. In step (2), during the second enzymatic hydrolysis, the final concentration of CaCl2 in the pepsin hydrolysate is 2 mM; the amount of thermophilic protease added is 0.4-0.6 U / mg protein; and the time for inactivating the enzyme in the boiling water bath is 5-10 min. In step (2), during the third enzymatic hydrolysis, the amount of trypsin added is 0.04-0.06 U / mg protein; In step (3), the centrifugation speed is 10,000 × g and the time is 5-10 min; the molecular weight cutoff of the ultrafiltration is 3-3.5 kDa. The temperature of the cold ethanol in step (4) is -20℃; the standing time is 0.5-1h; the centrifugation speed is 8000-10000×g; and the time is 10-15min.

8. Use of the novel active peptide composition for regulating mood according to any one of claims 1-7 in the preparation of a mood-regulating drug.

9. A drug for regulating mood, characterized in that, The novel active peptide composition for regulating mood includes any of the claims 1-7.

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