Novel active peptide composition for improving concentration degree as well as preparation method and application of novel active peptide composition

Abstract

The invention discloses a novel active peptide composition for improving concentration and a preparation method and application thereof. Belongs to the technical field of new active peptide composition preparation. The novel active peptide composition is prepared through synergism of the novel active peptide PAVP, the blood-brain barrier penetrating peptide and the metabolism regulator, when the novel active peptide PAVP, the blood-brain barrier penetrating peptide and the metabolism regulator act together, the novel active peptide composition has a synergistic effect, and the concentration improving effect of the novel active peptide composition is better than that of each independent component; the time for the mouse to explore new things can be prolonged to a certain extent, the relative expression quantity of synaptic plasticity markers PSD-95, BDNF and p-CREB can be increased to a certain extent, and technical support is provided for research and development of concentration-related products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of new bioactive peptide composition preparation, more particularly to a new bioactive peptide composition for improving concentration and a preparation method and application thereof. BACKGROUND

[0002] Brain peptides generally refer to a class of new bioactive 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 pleasure generation, and are called "natural painkillers"; 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 prone to degradation, have short half-lives, and poor stability; and most natural brain peptides have large molecular weights and high polarity, making it difficult for them to freely pass through the blood-brain barrier, thereby 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.), which can cause 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 brain protease-degraded short peptides with small molecular weights. During the related research, the present application found a brain protease-degraded new bioactive peptide PAVP, which has a certain effect on improving concentration. However, PAVP alone is prone to degradation, has poor stability, and the effect of improving concentration needs to be further improved.

[0004] Therefore, how to provide a new bioactive peptide composition for improving concentration and a preparation method and application thereof is a technical problem that those skilled in the art need to solve. SUMMARY

[0005] Therefore, the present application provides a new bioactive peptide composition for improving concentration and a preparation method and application thereof. The present application takes the new bioactive peptide PAVP prepared by extraction as the main active ingredient, and is combined with a blood-brain barrier penetrating peptide and a metabolic regulator. The three components interact with each other and synergistically improve the stability of the new bioactive peptide composition, and further improve the concentration.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: The new peptide composition for improving concentration comprises new peptide PAVP, blood-brain barrier penetrating peptide and metabolic regulator in a weight ratio of 10:3-5:1.

[0007] As a preferred technical solution, the blood-brain barrier penetrating peptide can be a blood-brain barrier penetrating peptide with an amino acid sequence TWLPYPR; and the metabolic regulator can be Noopept.

[0008] Another purpose of the present application is to provide a preparation method of the new peptide composition for improving concentration, which is prepared according to the above-mentioned ratio and specifically comprises the following steps: (1) Preparation of new peptide composition mixture: new peptide PAVP, blood-brain barrier penetrating peptide and metabolic regulator are weighed according to the ratio, dissolved in PBS solution to prepare a new peptide composition mixture; (2) Preparation of lipid film: DSPC, cholesterol and DSPE-PEG are weighed according to a weight ratio of 45-60:35-45:3-8 to prepare a lipid mixture material, which is dissolved in a mixed solvent, rotary evaporated, vacuum dried overnight to remove organic solvent, and dried lipid film is prepared; (3) Hydration of lipid film: the dried lipid film is added to the new peptide composition mixture prepared in step (1), incubated at 55-65°C for 20-40 min, and vortexed to prepare a multi-compartment liposome suspension; (4) Preparation of nanoparticles by microfluidization: ethanol is added as an organic phase and PBS solution is used as an aqueous phase to prepare nanoparticles by microfluidization under a pressure of 50 psi, and the nanoparticles are stored in pre-cooled PBS solution for standby; (5) Ultrafiltration, freeze-drying protection and freeze-drying: the nanoparticle solution is ultrafiltrated and centrifuged to remove free drugs and organic solvents, and a purified nanoparticle suspension is obtained, trehalose is added and vortexed, and the mixture is equilibrated at 4°C for 20-30 min; after pre-freezing, the mixture is freeze-dried to prepare the new peptide composition.

[0009] As a preferred technical solution, the pH of the PBS solution in step (1) is 7.3-7.5; and the amount of the PBS solution added is 70-80% of the volume of the new peptide composition mixture.

[0010] As a preferred technical solution, the mixed solvent in step (2) is prepared by mixing chloroform and methanol in a volume ratio of 2:1; the mass-volume ratio of the lipid mixture material to the mixed solvent is 8-12 mg:1 mL; and the rotary evaporation temperature is 38-42°C. As a preferred technical solution, the volume-weight ratio of the new peptide composition mixture and the dried lipid film in step (3) is 5-20 mL:10-50 mg.

[0011] As a preferred technical scheme, the flow rate ratio of the water phase and the organic phase in step (4) is 3:1; the temperature of the pre-cooled PBS solution is 4 DEG C; the amount of the added ethanol is 20-25% of the total volume of the organic phase; and the volume ratio of the organic phase to the water phase is 1:1-1:3. As a preferred technical scheme, the molecular cut-off of the ultrafiltration in step (5) is 80-100 kDa; and the centrifugation is performed at 4 DEG C, 3000-4000g for 10-20 min. As a preferred technical scheme, the final concentration of the trehalose in the nanoparticle suspension in step (5) is 5-7% w / v; and the pre-freezing treatment is performed by rapid freezing in liquid nitrogen for 5-8 min. As a preferred technical scheme, the freeze-drying in step (5) is performed as follows: Primary drying: -40 DEG C, 0.1 mbar, 24 h; Secondary drying: 25 DEG C, 0.01 mbar, 12 h.

[0012] Still another purpose of the present application is to provide the application of the new bioactive peptide composition or the new bioactive peptide composition prepared by the method in the preparation of a product for improving concentration.

[0013] As a preferred technical scheme, the product comprises food or medicine.

[0014] Still another purpose of the present application is to provide a medicine for improving concentration, which comprises the new bioactive peptide composition or the new bioactive peptide composition prepared by the method.

[0015] Still another purpose of the present application is to provide a food for improving concentration, which comprises the new bioactive peptide composition or the new bioactive peptide composition prepared by the method.

[0016] According to the above technical scheme, compared with the prior art, the present application has the following beneficial effects: The new bioactive peptide composition of the present application is prepared by the synergistic effect of the new bioactive peptide PAVP, the blood-brain barrier penetrating peptide and the metabolic regulator, and has a synergistic effect when the three components act together, and the effect of improving concentration is better than that of each component alone; in addition, when the composition of the present application is prepared into nanoparticles by being wrapped with a lipid film, the time of exploring new things of mice and the relative expression amounts of synaptic plasticity markers PSD-95, BDNF and p-CREB can be improved to a certain extent. BRIEF DESCRIPTION OF DRAWINGS

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 Here is the mass spectrum of the newly active peptide PAVP obtained by proteolytic digestion.

[0019] The blood-brain barrier penetrating peptide used in this invention is the TWLPYPR peptide disclosed in the patent document with publication number CN 117229351 A. Detailed Implementation

[0020] 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.

[0021] DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine) CAS 816-94-4; Noopept (N-phenylacetyl-L-prolyl glycine ethyl ester) CAS 157115-85-0; DSPE-PEG2000, CAS 119446-29-4.

[0022] Example 1 The specific preparation process of PAVP, a novel active peptide derived from brain protein hydrolysis, is as follows: (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. (2) Multi-enzyme stepwise enzymatic hydrolysis: First enzymatic hydrolysis: Add 1M HCl to adjust the pH of the pig brain extract to 2, add pepsin (final concentration 0.1mg / mL), and react at 37℃ for 2h; add 1M NaOH to adjust the pH to 7.0 to terminate the enzymatic hydrolysis reaction and prepare the pepsin hydrolysate. Second enzymatic hydrolysis: Tris-HCl and CaCl2 (final concentration of 2mM) were added to the pepsin hydrolysate, the pH was adjusted to 7.5, thermophilic protease was added at a rate of 0.5 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. Third enzymatic hydrolysis: NH4HCO3 was added to the thermophilic bacteria protease hydrolysate to adjust the pH to 8.0, and trypsin was added at a rate of 0.04 U / mg protein. The mixture was reacted at 37°C for 0.5 h to prepare the trypsin hydrolysate. (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 3 kDa), and collect the permeate; (4) Ethanol precipitation and concentration: Add 3 times the volume of cold ethanol (-20℃) to the permeate, let stand for 1 hour, centrifuge at 8000g for 10 minutes, 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 A: Ultrapure water + 0.1% trifluoroacetic acid (TFA, v / v) Flow phase B: acetonitrile + 0.1% TFA (v / v); gradient (10%-40% acetonitrile, 30 min); Flow rate: 1.0 mL / min; Detection: 220 nm.

[0023] 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.

[0024] The elution peaks of PAVP were collected and analyzed by mass spectrometry. The results are as follows: Figure 1 As shown, the precursor ion is m / z 382.73. By matching the b / y ion series, the detected peptide was confirmed to be PAVP.

[0025] Example 2 A novel active peptide composition for enhancing focus comprises PAVP, TWLPYPR blood-brain barrier penetrating peptide, and Noopept in a weight ratio of 10:3:1. The specific preparation method is as follows: (1) Preparation of the new active peptide composition mixture: Weigh the new active peptide PAVP, TWLPYPR blood-brain barrier penetrating peptide and Noopept according to the ratio, dissolve them in PBS (pH 7.3) solution, and add PBS solution at 70% of the volume of the new active peptide composition mixture to prepare the new active peptide composition mixture; (2) Preparation of lipid film: DSPC, cholesterol and DSPE-PEG were weighed in a weight ratio of 45:35:3 to prepare lipid mixture material. It was dissolved in a mixed solvent (chloroform:methanol = 2:1, v / v). The mass-volume ratio of lipid mixture material to mixed solvent was 8 mg:1 mL. The mixture was rotary evaporated at 38 °C and vacuum dried overnight to remove organic solvent, thus preparing a dry lipid film. (3) Lipid film hydration: Add the dried lipid film to the mixture of the new active peptide composition prepared in step (1) at a volume-to-weight ratio of 5 mL: 10 mg, incubate at 55 °C for 20 min, vortex, and prepare a multi-compartment liposome suspension. (4) Microfluidic preparation of nanoparticles: Ethanol was added to the multi-chamber liposome suspension as the organic phase, and the amount of ethanol added was 20% of the total volume of the organic phase. PBS solution was used as the aqueous phase. The mixture was placed under a pressure of 50 psi and the flow rate of organic phase to aqueous phase was 1:1 and 1:3. The nanoparticles were prepared by microfluidic control and stored in pre-cooled PBS solution (4°C) for later use. (5) Ultrafiltration, lyophilization and freeze-drying: The nanoparticle solution was ultrafiltered (molecular cutoff of 80 kDa), centrifuged (4℃, 3000g, 10min) to remove free drug and organic solvent, and purified nanoparticle suspension was obtained. Trehalose (final concentration of 5% w / v) was added to it, vortexed and mixed, and equilibrated at 4℃ for 20min. After being rapidly frozen in liquid nitrogen for 5min, it was placed at -40℃, 0.1 mbar for 24h for primary drying. Then it was placed at 25℃, 0.01 mbar for 12h for secondary drying to prepare the new active peptide composition.

[0026] Example 3 A novel active peptide composition for enhancing focus comprises PAVP, TWLPYPR blood-brain barrier penetrating peptide, and Noopept in a weight ratio of 10:5:1. The specific preparation method is as follows: (1) Preparation of the new active peptide composition mixture: Weigh the new active peptide PAVP, TWLPYPR blood-brain barrier penetrating peptide and Noopept according to the ratio, dissolve them in PBS (pH 7.5) solution, and add 80% of the volume of the new active peptide composition mixture to prepare the new active peptide composition mixture; (2) Preparation of lipid film: DSPC, cholesterol and DSPE-PEG were weighed in a weight ratio of 60:45:8 to prepare lipid mixture material, which was dissolved in a mixed solvent (chloroform:methanol = 2:1, v / v). The mass-volume ratio of lipid mixture material to mixed solvent was 12mg:1mL. The mixture was rotary evaporated at 42℃ and vacuum dried overnight to remove organic solvent, thus preparing a dry lipid film. (3) Lipid film hydration: Add the dried lipid film to the mixture of the new active peptide composition prepared in step (1) at a volume-to-weight ratio of 20 mL: 50 mg, incubate at 65 °C for 40 min, vortex, and prepare a multi-compartment liposome suspension. (4) Microfluidic preparation of nanoparticles: Ethanol was added to the multi-chamber liposome suspension as the organic phase, and the amount of ethanol added was 25% of the total volume of the organic phase. PBS solution was used as the aqueous phase. The nanoparticles were prepared by microfluidic control under a pressure of 50 psi and a flow rate of 1:3 between the organic phase and the aqueous phase. The nanoparticles were stored in pre-cooled PBS solution (4°C) for later use. (5) Ultrafiltration, lyophilization and freeze-drying: The nanoparticle solution was ultrafiltered (molecular cutoff of 100 kDa), centrifuged (4℃, 4000g, 20min) to remove free drug and organic solvent, and purified nanoparticle suspension was obtained. Trehalose (final concentration of 7% w / v) was added to it, vortexed and mixed, and equilibrated at 4℃ for 30min. After being rapidly frozen in liquid nitrogen for 8min, it was placed at -40℃, 0.1 mbar for 24h for primary drying. Then it was placed at 25℃, 0.01 mbar for 12h for secondary drying to prepare the new active peptide composition.

[0027] Example 4 A novel active peptide composition for enhancing focus comprises PAVP, TWLPYPR blood-brain barrier penetrating peptide, and Noopept in a weight ratio of 10:4:1. The specific preparation method is as follows: (1) Preparation of the new active peptide composition mixture: Weigh the new active peptide PAVP, TWLPYPR blood-brain barrier penetrating peptide and Noopept according to the ratio, dissolve them in PBS (pH 7.4) solution, and add PBS solution at 75% of the volume of the new active peptide composition mixture to prepare the new active peptide composition mixture; (2) Preparation of lipid film: DSPC, cholesterol and DSPE-PEG were weighed in a weight ratio of 55:40:5 to prepare lipid mixture material, which was dissolved in a mixed solvent (chloroform:methanol = 2:1, v / v). The mass-volume ratio of lipid mixture material to mixed solvent was 10mg:1mL. The mixture was rotary evaporated at 40℃ and vacuum dried overnight to remove organic solvent, thus preparing a dry lipid film. (3) Lipid film hydration: Add the dried lipid film to the mixture of the new active peptide composition prepared in step (1) at a volume-to-weight ratio of 10 mL: 20 mg, incubate at 60 °C for 30 min, vortex, and prepare a multi-compartment liposome suspension. (4) Microfluidic preparation of nanoparticles: Ethanol was added to the multi-chamber liposome suspension as the organic phase, and the amount of ethanol added was 25% of the total volume of the organic phase. PBS solution was used as the aqueous phase. The nanoparticles were prepared by microfluidic control under a pressure of 50 psi and a flow rate of 1:3 between the organic phase and the aqueous phase. The nanoparticles were stored in pre-cooled PBS solution (4°C) for later use. (5) Ultrafiltration, lyophilization and freeze-drying: The nanoparticle solution was ultrafiltered (molecular cutoff of 100 kDa), centrifuged (4℃, 3000g, 15min) to remove free drug and organic solvent, and purified nanoparticle suspension was obtained. Trehalose (final concentration of 6% w / v) was added to it, vortexed and mixed, and equilibrated at 4℃ for 25min. After being rapidly frozen in liquid nitrogen for 5min, it was placed at -40℃, 0.1 mbar for 24h for primary drying. Then it was placed at 25℃, 0.01 mbar for 12h for secondary drying to prepare the new active peptide composition.

[0028] Comparative Example 1 The operation is basically the same as in Example 4, except that TWLPYPR blood-brain barrier penetrating peptide and Noopept are removed from the new active peptide composition. The remaining steps and operations are the same as in Example 4.

[0029] Comparative Example 2 The operation is basically the same as in Example 4, except that the new active peptides PAVP and Noopept are removed from the new active peptide composition. The remaining steps and operations are the same as in Example 4.

[0030] Comparative Example 3 The operation is basically the same as in Example 4, except that the new active peptide PAVP and TWLPYPR blood-brain barrier penetrating peptide are removed from the new active peptide composition. The remaining steps and operations are the same as in Example 4.

[0031] Comparative Example 4 The operation is basically the same as in Example 4, except that the lipid film is not encapsulated.

[0032] Efficacy verification To verify the efficacy of the novel active peptide compositions prepared in Examples 2-4 and Comparative Examples 1-4, the following mouse experiments were conducted. (1) Establishment of a mouse model of chronic unpredictable mild stress Healthy 6-week-old SPF-grade male C57BL / 6J mice, weighing 18-22g, were selected and housed in a constant temperature and humidity environment (temperature 22±1℃, humidity 55±10%) with a 12h:12h light-dark cycle. They were provided with free access to food and water. After a one-week acclimatization period, the mice were divided into groups as follows: Normal group: 5 mg / kg physiological saline; New active peptide composition group: CUMS modeling, and respectively administered 5 mg / kg of the new active peptide composition prepared in Examples 2-4 and Comparative Examples 1-3 (with an equal weight of mannitol added as a carrier when administering the new active peptide composition); Placebo group: CUMS modeling, 5 mg / kg normal saline; Positive control group: CUMS modeling, methylphenidate (2 mg / kg, standard treatment for ADHD).

[0033] Based on the above grouping, the mice were stimulated daily using 1-3 different methods: restraint for 4 hours, tail clamping for 1 minute, swimming in ice water for 5 minutes, all-night lighting for 24 hours, continuous darkness for 24 hours, no bedding for 24 hours, fasting for 24 hours, water deprivation for 24 hours, tilting of the cage for 24 hours, noise stimulation for 20 minutes, and foreign object stimulation for 2 hours. The stimulation was performed at irregular intervals each day for 4 weeks. The mice were weighed once a week, and the mice that successfully developed the model were selected for subsequent experiments.

[0034] (2) Evaluation of the focus-enhancing efficacy of the new active peptide PAVP in the novel object recognition experiment The Novel Object Recognition (NOR) experiment focuses on the differences in animals' exploration behaviors towards new and old objects, thereby assessing their attention span.

[0035] Before the formal test, the mice were trained for 3 days. On the first day, the mice were allowed to freely explore and adapt in an open field for 15 minutes. On the second day, two identical objects A were placed diagonally opposite each other, and the mice were allowed to explore freely for 10 minutes. One hour later, one of the objects A was replaced with a new object B, and the mice were allowed to explore freely for 10 minutes. In the formal experiment, object A was kept in the open field, and object B was replaced with object C. The mice were placed in the open field in the same direction as before and allowed to explore freely for 10 minutes. The percentage of time the mice spent exploring the new object C was recorded. The experimental results are shown in Table 1.

[0036] Table 1. Time taken for mice in different groups to explore new things Group Percentage of time exploring new things Fold increase in time exploring new things compared to placebo group Duration of action (h) Example 2 27.1% 10.84 4.5 Example 3 27.6% 11.04 4.7 Example 4 25.4% 10.16 4.6 Comparative Example 1 12.4% 4.96 1.6 Comparative Example 2 5.6% 2.24 0.6 Comparative Example 3 3.2% 1.28 0.5 Comparative Example 4 21.3% 8.52 1.2 Placebo group 2.5% 1 - Positive control group 11.8% 4.72 2.4 Normal group 8.0% 3.2 - Results analysis: As shown in Table 1, there was a significant difference in the percentage of time spent exploring new things between the placebo group and the normal group, indicating that the mouse chronic unpredictable mild stress model of the present invention was successfully constructed. By comparing Examples 2-4 with Comparative Examples 1-4, it is shown that the active peptide composition prepared in the present invention can significantly improve the time mice spend exploring new things, and the three components have a synergistic effect when used together.

[0037] Furthermore, by exploring the duration of drug action in different groups, it was found that the three drugs worked synergistically, significantly enhancing the duration of action.

[0038] (3) Detection of synaptic plasticity markers Western blot was used to extract total protein from the prefrontal cortex (PFC) tissue of mice in different groups. The relative expression levels of synaptic plasticity markers PSD-95, BDNF, and p-CREB in mice in different groups were detected compared with the placebo group. The experimental results are shown in Table 2.

[0039] Table 2. Relative expression levels of PSD-95, BDNF, and p-CREB, markers of synaptic plasticity, in mice from different groups. Group PSD-95 BDNF p-CREB Example 2 2.83±0.06 2.87±0.04 2.98±0.06 Example 3 2.86±0.04 2.89±0.06 2.99±0.07 Example 4 2.79±0.07 2.86±0.04 2.96±0.04 Comparative Example 1 1.84±0.04 1.76±0.04 2.09±0.03 Comparative Example 2 1.35±0.11 1.46±0.05 1.42±0.03 Comparative Example 3 1.28 ± 0.06 1.35 ± 0.11 1.31 ± 0.06 Comparative Example 4 1.67±0.05 1.58±0.10 1.69±0.10 Placebo group 1.00 ± 0.04 1.00 ± 0.03 1.00 ± 0.05 Positive control group 1.65 ± 0.14 1.61 ± 0.08 1.91 ± 0.08 Normal group 1.13±0.04 1.25±0.04 1.24±0.08 Results analysis: As shown in Table 2, Examples 2-4 showed a synergistic effect with Comparative Examples 1-3 and the placebo group in terms of PSD-95, BDNF, and p-CREB expression levels. This indicates that the three components of the composition of the present invention work synergistically to significantly enhance the synaptic plasticity damage in mouse models and improve synaptic function. This is the core molecular mechanism by which the composition enhances focus.

[0040] 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.

[0041] 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 enhancing focus, characterized in that, It contains a novel active peptide PAVP, a blood-brain barrier penetrating peptide, and a metabolic regulator in a weight ratio of 10:3-5:

1.

2. The novel active peptide composition for enhancing focus according to claim 1, characterized in that, The blood-brain barrier penetrating peptide can be a blood-brain barrier penetrating peptide with the amino acid sequence TWLPYPR; the metabolic regulator can be Noopept.

3. A method for preparing the novel active peptide composition for enhancing focus as described in any one of claims 1-2, characterized in that, The preparation according to the proportions described in claim 1 or 2 specifically includes the following steps: (1) Preparation of the new active peptide composition mixture: Weigh the new active peptide PAVP, blood-brain barrier penetrating peptide and metabolic regulator according to the ratio, dissolve them in PBS solution to prepare the new active peptide composition mixture; (2) Preparation of lipid film: DSPC, cholesterol and DSPE-PEG were weighed in a weight ratio of 45-60:35-45:3-8 to prepare a lipid mixture. The mixture was dissolved in a mixed solvent, rotary evaporated, and vacuum dried overnight to remove the organic solvent, thus preparing a dry lipid film. (3) Lipid film hydration: Add the dried lipid film to the mixture of the new active peptide composition prepared in step (1), incubate at 55-65℃ for 20-40 min, vortex, and prepare a multi-compartment liposome suspension. (4) Microfluidic preparation of nanoparticles: Ethanol was added to the multi-chamber liposome suspension as the organic phase and PBS solution was used as the aqueous phase. The nanoparticles were prepared by microfluidic control under a pressure of 50 psi and stored in pre-cooled PBS solution for later use. (5) Ultrafiltration, freeze-drying protection and freeze-drying: The nanoparticle solution was ultrafiltered and centrifuged to remove free drugs and organic solvents, and purified nanoparticle suspension was obtained. Trehalose was added to it, vortexed and mixed, and equilibrated at 4℃ for 20-30 min. After pre-freezing treatment, freeze-drying was performed to prepare the new active peptide composition.

4. The preparation method according to claim 3, characterized in that, The pH of the PBS solution in step (1) is 7.3-7.5; the amount of PBS solution added is 70-80% of the volume of the new active peptide composition mixture.

5. The preparation method according to claim 3, characterized in that, The mixed solvent in step (2) is prepared by mixing chloroform and methanol in a volume ratio of 2:1; the mass-volume ratio of the lipid mixture to the mixed solvent is 8-12 mg: 1 mL; the rotary evaporation temperature is 38-42 °C. In step (3), the volume-to-weight ratio of the new active peptide composition mixture and the dried lipid film is 5-20 mL: 10-50 mg.

6. The preparation method according to claim 3, characterized in that, In step (4), the flow rate ratio of the aqueous phase to the organic phase is 3:1; the temperature of the pre-cooled PBS solution is 4°C; the amount of ethanol added is 20-25% of the total volume of the organic phase; and the volume ratio of the organic phase to the aqueous phase is 1:1-1:

3. The molecular cutoff of the ultrafiltration in step (5) is 80-100 kDa; the centrifugation conditions are as follows: 4℃, 3000-4000g, 10-20min; The final concentration of trehalose in the nanoparticle suspension in step (5) is 5-7% w / v; the pre-freezing process is as follows: quick-freezing with liquid nitrogen for 5-8 min; The freeze-drying process described in step (5) is as follows: Primary drying: -40℃, 0.1 mbar, 24h; Secondary drying: 25℃, 0.01 mbar, 12h.

7. The use of the novel active peptide composition for enhancing focus as described in any one of claims 1-2 or the novel active peptide composition prepared by any one of claims 3-6 in the preparation of products for enhancing focus.

8. The application according to claim 7, characterized in that, The products include food or medicine.

9. A drug for improving concentration, characterized in that, This includes the novel active peptide composition according to any one of claims 1-2 or the novel active peptide composition prepared by any one of claims 3-6.

10. A food product for improving concentration, characterized in that, This includes the novel active peptide composition according to any one of claims 1-2 or the novel active peptide composition prepared by any one of claims 3-6.

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