Active peptide capable of improving cognitive impairment, preparation method and application thereof

Abstract

The present invention discloses an active peptide capable of improving cognitive impairment, a preparation method thereof, and applications thereof. The amino acid sequence of the active peptide is shown in SEQ ID NO: 1, 2, 3, 4, 5, or 6. The present invention aims to address the problems of limited means and unstable effects of existing methods for improving cognitive impairment by providing a new active peptide, a preparation method thereof, and applications thereof. The active peptide prepared by the present invention can, to a certain extent, improve problems of impaired learning and memory and decreased cognitive function, enriching the technical approaches for improving cognitive impairment and providing new application options for related fields.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and more particularly to an active peptide capable of improving cognitive impairment, and a preparation method and application thereof. Background Art

[0002] Cognitive impairment is an increasingly prominent health issue in modern society. Its pathological mechanisms are closely related to neuroinflammation, oxidative stress, and synaptic plasticity damage, with clinical manifestations including decreased attention, decreased memory function, and impaired executive function. Currently, interventions mainly fall into two categories: one is sedative-hypnotic drugs such as benzodiazepines and melatonin receptor agonists, but long-term use can easily lead to tolerance, dependence, and further cognitive decline; the other is non-drug interventions such as cognitive behavioral therapy, but these have limitations such as poor compliance and slow onset of effect. Therefore, the development of new, safe, efficient, and well-targeted active substances has become an urgent need in this field.

[0003] In recent years, naturally derived bioactive peptides have garnered significant attention for their neuroprotective effects. For example, the milk-derived peptide OPA-1 can improve spatial memory in mice by regulating the BDNF / TrkB signaling pathway, and the marine collagen peptide MCP-3 has been shown to inhibit oxidative stress in the hippocampus. However, the existing technology has the following key problems: high raw material costs: active peptides mostly rely on extraction from dairy products or deep-sea fish, and although meat duck processing by-products (such as duck bones) are rich in collagen (accounting for about 30% of the dry weight of duck bones), they have not been fully developed as a raw material source for cognitive improvement peptides, resulting in resource waste and rising costs; low process efficiency: traditional duck bone utilization technology mostly focuses on bone polysaccharide extraction (such as defatting, acid hydrolysis, alcohol precipitation and other multi-step operations), but the process is complicated and it is difficult to achieve targeted enzymatic hydrolysis of collagen and precise screening of active peptides, and the utilization rate of functional components is insufficient; insufficient targeting: existing active peptides mostly act through the pan-antioxidant pathway, lack of molecular design targeting pathological mechanisms (such as circadian rhythm disorders, abnormal synaptic protein phosphorylation), and related studies mostly focus on Alzheimer's disease models, and there is a significant lack of systematic verification of sleep deprivation models; incomplete functional verification chain: the reported active peptides have not yet been systematically evaluated in animal models for their effects on key indicators such as spatial memory consolidation and prefrontal cortex metabolism, which restricts their clinical application transformation.

[0004] In summary, how to develop an efficient preparation process based on low-cost raw materials, design targeted active peptides based on pathological characteristics, and build a complete functional verification system is a bottleneck that urgently needs to be broken through in the current technology field. Summary of the Invention

[0005] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide an active peptide, a preparation method and application thereof, which innovatively screens active peptides with specific sequences from duck bone collagen, and solves the problems of high raw material costs and lack of functional verification in the existing technology by optimizing the enzymatic hydrolysis process and active function verification, and ultimately achieves specific intervention in cognitive impairment.

[0007] In order to achieve these objects and other advantages according to the present invention, an active peptide is provided, the amino acid sequence of which is shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0008] The present invention also provides a method for preparing an active peptide, which comprises the following steps:

[0009] S1, taking duck bones and crushing them after pretreatment;

[0010] S2, soaking the crushed duck bones in anhydrous ethanol to defatted, and freeze-drying to obtain defatted bone residue;

[0011] S3, suspending the defatted bone residue in phosphate buffer, adding protease for enzymatic hydrolysis, and inactivating the enzyme at 100° C. for 15 minutes to obtain an enzymatic hydrolyzate;

[0012] S4. The enzymatic hydrolysate is centrifuged to obtain the supernatant, which is filtered through an ultrafiltration membrane to fractionate the solution with a molecular weight cutoff of less than 3 kDa, and then freeze-dried to obtain the active peptide.

[0013] Preferably, in the method for preparing the active peptide, the enzymatic hydrolysis conditions in S1 are: pH 7.6, temperature 30° C., and enzymatic hydrolysis time 2.3 h.

[0014] Preferably, in the method for preparing the active peptide, in S1, the duck bones of fresh meat duck are taken, the residual muscles and connective tissues are removed, and then the bones are crushed, and then rinsed with deionized water three times to obtain the crushed duck bones.

[0015] Preferably, the preparation method of the active peptide, S2 is specifically: soaking the crushed duck bones in anhydrous ethanol for 3 hours, collecting bone residues, repeating the soaking and collecting twice, combining the bone residues and freeze-drying them to obtain defatted bone residues.

[0016] Preferably, in the method for preparing the active peptide, the protease in S3 is collagenase, and the ratio of protease to defatted bone residue is 0.013 IU / mg.

[0017] The present invention also provides a use of the active peptide or the active peptide prepared above in preparing a product for improving cognitive impairment.

[0018] Preferably, in the application, cognitive impairment includes spatial memory impairment, decreased ability to recognize new objects, and increased levels of inflammatory factors IL-1β and TNF-α in the hippocampus.

[0019] Preferably, in the application, the active peptide improves cognitive impairment by increasing superoxide dismutase activity and reducing malondialdehyde content.

[0020] The present invention has at least the following beneficial effects:

[0021] The present invention provides an active peptide, its preparation method, and its application. The active peptide has a clear source, a controllable preparation process, and is easy to use, effectively enhancing cognitive performance. Furthermore, the active peptide exhibits excellent biosafety and stability, with no significant adverse reactions observed during application, demonstrating its promising potential for application.

[0022] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the mass spectrum of the active peptide prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments so that those skilled in the art can implement the invention with reference to the description.

[0025] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0026] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0027] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0028] Example 1

[0029] The present invention provides an active peptide, and its preparation method is as follows:

[0030] S1, taking duck bones and crushing them after pretreatment;

[0031] S2, soaking the crushed duck bones in anhydrous ethanol to defatted, and freeze-drying to obtain defatted bone residue;

[0032] S3, suspending the defatted bone residue in phosphate buffer, adding protease for enzymatic hydrolysis, and inactivating the enzyme at 100° C. for 15 minutes to obtain an enzymatic hydrolyzate;

[0033] S4. The enzymatic hydrolysate is centrifuged to obtain the supernatant, which is filtered through an ultrafiltration membrane to fractionate the solution with a molecular weight cutoff of less than 3 kDa, and then freeze-dried to obtain the active peptide.

[0034] Optimization of the above extraction process parameters:

[0035] Single-factor experiments: Using the proliferation activity of SH-SY5Y cells (human neuroblastoma cells) as an indicator, we used single-factor experiments to investigate the effects of enzyme addition, pH, hydrolysis time, and hydrolysis temperature on SH-SY5Y cell viability. Four sets of experiments were conducted. The enzyme addition levels were 0.0025, 0.005, 0.01, 0.02, and 0.04 IU / mg; the pH values ​​were 6.0, 6.5, 7.0, 7.5, and 8.0; the hydrolysis times were 0.5, 1, 2, 4, and 8 h; and the hydrolysis temperatures were 20, 30, 40, 50, and 60°C. Each experiment was repeated three times, and the average results are shown in Table 1.

[0036] Table 1 Single factor experimental results

[0037]

[0038] Orthogonal experiment: As shown in Table 2, based on the single-factor experiment, a 4-factor 3-level L9(34) orthogonal experiment was designed with enzyme addition amount, pH value, enzymatic hydrolysis time, and enzymatic hydrolysis temperature as factors and cell viability as an indicator. The levels of each factor are shown in the table. IBM SPSS Statistics. V20 was used to design the orthogonal experiment and perform variance analysis to determine the optimal conditions for the preparation process. The results are shown in Table 3.

[0039] Table 2 Orthogonal experimental design

[0040]

[0041] result:

[0042] Table 3 Orthogonal experiment results

[0043]

[0044] The optimal group is A1B2C2D2, and the order of influence is: D>B>C>A.

[0045] Table 3 finally determined the optimal experimental parameters for the preparation process of the active peptide of the present invention. The enzymatic hydrolysis conditions were as follows: temperature of 30°C, pH of 7.6, enzymatic hydrolysis time of 2.3 h, a ratio of protease to sample of 0.013 IU / mg, and the protease used was collagenase, Sigma-Aldric, CAS number: 9001-12-1.

[0046] The active peptides of the enzymatic hydrolysis products prepared under optimized conditions were detected by mass spectrometry, such as Figure 1 Mass spectrometry data were acquired using a Q Exactive HF mass spectrometer coupled to an UltiMate 3000 RSLCnano liquid chromatography-mass spectrometry system. Active peptide samples were dissolved in loading buffer, drawn up by an autosampler, and separated on an analytical column (75 μm × 25 cm, C18, 1.9 μm, 120 Å). An analytical gradient was established using two mobile phases (mobile phase A: 0.1% formic acid, 3% DMSO and mobile phase B: 0.1% formic acid, 3% DMSO, 80% ACN). The flow rate was set at 300 nL / min. Mass spectrometry data were acquired in DDA mode. Each scan cycle consisted of a full MS scan (R = 60K, AGC = 3e6, max IT = 25 ms, scan range = 350–1500 m / z) followed by 20 MS / MS scans (R = 15K, AGC = 1e5, max IT = 50 ms). The HCD collision energy was set to 27. The quadrupole screening window was set to 1.4 Da. The dynamic exclusion time for repeated ion acquisition was set to 24 s.

[0047] Mass spectrometry data were retrieved using MaxQuant (V1.6.6) software, using the Andromeda database search algorithm. The following search parameters were used: LFQ was selected for the project type; Oxidation (M), Acetyl (Protein N-term) was selected for the variable modification; Carbamidomethyl (C) was selected for the fixed modification; and Unspecific was selected for the enzyme digestion. The search results were screened using a 1% FDR at both the protein and peptide levels. Six peptides with pro-proliferative effects on SH-SY5Y cells were identified, with amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. These six amino acid sequences were synthesized to generate individual active peptides, which are numbered as follows:

[0048] Peptide 1: SEQ ID NO: 1 is KQYVPLRSTW;

[0049] Peptide 2: SEQ ID NO: 2 is GFNHCQDM;

[0050] Peptide 3: SEQ ID NO: 3 is MLKICFTRGP;

[0051] Peptide 4: SEQ ID NO: 4 is CRVPTQYWGE;

[0052] Peptide 5: SEQ ID NO: 5 is VPLGQTRKFS;

[0053] Peptide 6: SEQ ID NO: 6 is YWDHCNQKLP.

[0054] Example 2 Active Peptide Stability Test

[0055] The stability of the six synthesized active peptides was tested. The active peptides (dissolved in sterile PBS, pH 7.4) were aliquoted into sealed, light-proof EP tubes and stored at 40°C, 25°C, 4°C, and -20°C, with three replicates per group. Samples were collected at 0, 7, 14, and 28 days and immediately frozen at -80°C until further use. SH-SY5Y cell viability was then assayed. The results are shown in Table 4.

[0056] Table 4 Active peptide stability test results

[0057]

[0058] The results in Table 4 show that the active peptides were relatively stable during storage, with no significant change in activity. The active peptides provided by the present invention exhibited excellent stability in solution, as shown in the following:

[0059] Wide temperature adaptability: After the active peptide solution was stored at high temperature (40°C), room temperature (25°C), refrigerated (4°C) and frozen (-20°C) for 28 days, the cell proliferation rate still maintained 96.5%, 98.3%, 99.0% and 99.2% of the initial activity, respectively, indicating that its molecular structure is stable and not easily degraded;

[0060] Outstanding long-term stability: Even in an accelerated aging environment at 40°C, the activity retention rate after 28 days is still as high as 94.1% (e.g., peptide 1: 47.17% vs. initial 48.88%), far exceeding that of conventional active peptides (usually <80%), meeting the requirements of industrial storage and transportation;

[0061] Durable biological activity: After 28 days of storage at low temperature (4°C / -20°C), the active peptide's proliferation-promoting effect on SH-SY5Y cells showed no significant fluctuation (e.g., peptide 5: 43.08% vs. initial 43.39%), demonstrating that its functional activity can be maintained over the long term, providing a reliable basis for formulation development.

[0062] In summary, the active peptides obtained by optimizing the preparation process in the present invention have both high efficiency, stability and functional durability, breaking through the technical bottlenecks of easy inactivation and harsh storage conditions of traditional active peptides, and providing ideal candidate molecules for the development of related products to improve cognitive impairment.

[0063] Example 3 Gastrointestinal digestibility test of active peptides

[0064] 3.1 Experimental Materials

[0065] 3.1.1. Active peptide samples: six active peptides synthesized in Example 1 (dissolved in sterile PBS, pH 7.4, concentration 1 mg / mL).

[0066] 3.1.2 Digestive enzymes: pepsin (≥250 U / mg, dissolved in 0.1 M HCl, pH 2.0), trypsin (≥1000 U / mg, dissolved in 0.1 M Tris-HCl, pH 7.5).

[0067] 3.1.3 Reagents: hydrochloric acid (HCl), tris (hydroxymethyl)aminomethane (Tris), phosphate buffered saline (PBS, pH 7.4).

[0068] 3.1.4 Equipment: Constant temperature water bath shaker (37°C), pH meter, centrifuge, ultrafiltration centrifuge tubes (3 kDa molecular weight cut-off).

[0069] 3.2 Experimental Procedure

[0070] 3.2.1. Simulated gastric digestion stage

[0071] Solution preparation: Take 1 mL of active peptide solution and add 9 mL of gastric digestion buffer (0.1 M HCl, pH 2.0).

[0072] Enzymatic hydrolysis reaction: pepsin was added at a ratio of pepsin to substrate of 1:20 (w / w), and the reaction was carried out at 37°C with constant temperature shaking (200 rpm) for 2 hours.

[0073] Stop the reaction: adjust the pH to 7.0 (using 1 M NaOH), immediately place on ice to stop enzyme activity, centrifuge (10,000 × g, 10 min), collect the supernatant, and label it as "gastric digestion sample."

[0074] 3.2.2 Simulated intestinal digestion stage

[0075] Solution preparation: Take 1 mL of the above gastric digestion sample and add 9 mL of intestinal digestion buffer (0.1 M Tris-HCl, pH 7.5).

[0076] Enzymatic hydrolysis reaction: Trypsin was added at a trypsin to substrate ratio of 1:50 (w / w), and the reaction was carried out at 37°C with constant shaking (200 rpm) for 4 hours.

[0077] Termination of the reaction: Heat the sample in a water bath at 100°C for 5 min to inactivate the enzyme, centrifuge (10,000 × g, 10 min), and collect the supernatant, which is labeled as “gastrointestinal digestion sample.”

[0078] 3.2.3. Active peptide stability test

[0079] Ultrafiltration purification: The digested sample was filtered through an ultrafiltration centrifuge tube (3 kDa) to remove large molecular enzymes and undigested products, and the filtrate was collected.

[0080] Cell viability assay:

[0081] Cell culture: SH-SY5Y cells were seeded in 96-well plates (density 5 × 10³ cells / well) and cultured at 37°C, 5% CO2 for 24 h.

[0082] Sample treatment: Initial active peptide, gastric digestion sample, and gastrointestinal digestion sample (final concentration 50 μg / mL) were added respectively, with 6 replicate wells in each group.

[0083] Detection method: CCK-8 assay was used. After incubation at 37°C for 2 hours, the absorbance at 450 nm was measured with a microplate reader, and the cell proliferation rate was calculated (the untreated group was set as 100% activity benchmark).

[0084] 3.3 Data Processing

[0085] Activity retention rate calculation: Activity retention rate (%) = (proliferation rate of digested sample / proliferation rate of initial sample) × 100

[0086] Statistical analysis: One-way analysis of variance (ANOVA) was performed using GraphPad Prism 8.0. Data are expressed as mean values ​​(n=3). P<0.05 was considered significant.

[0087] 3.4. Key Parameter Description

[0088] pH control: pH 2.0 in the gastric phase (simulating gastric acid environment), pH 7.5 in the intestinal phase (simulating neutral intestinal environment).

[0089] Enzyme activity ratio: pepsin to substrate ratio 1:20 (w / w), trypsin to substrate ratio 1:50 (w / w), to ensure that the digestion conditions match the physiological concentration.

[0090] Ultrafiltration cut-off: 3 kDa ultrafiltration membrane accurately separates active peptides (<3 kDa).

[0091] According to the above method, simulated gastrointestinal digestion (pepsin 2h + trypsin 4h) was carried out to test the gastrointestinal digestibility of the six synthesized active peptides. The results are shown in Table 5.

[0092] Table 5 Gastrointestinal digestion performance (proliferation-promoting activity on SH-SY5Y cells) test results

[0093]

[0094] As can be seen from Table 5, after simulated gastrointestinal digestion (pepsin 2h + trypsin 4h), the in vitro cell proliferation activity did not decrease significantly. After simulated gastrointestinal digestion (pepsin + trypsin), the active peptides (such as peptide 1 and peptide 5) still retained more than 97.1% of their initial activity, which is much higher than similar products (usually <70%), indicating that the active peptides obtained by the present invention have a good oral bioavailability advantage.

[0095] The active peptide provided by the present invention exhibits excellent stability and function retention in a simulated gastrointestinal digestive environment, specifically:

[0096] High resistance to digestion and degradation: After digestion with pepsin for 2 hours and trypsin for 4 hours, the core active peptides (such as peptide 1 and peptide 5) still retained more than 97.1% of their initial activity (peptide 1: 47.24% vs. initial 48.82%; peptide 5: 40.85% vs. initial 43.07%), which is significantly better than conventional active peptides (usually losing more than 30%), indicating that their structure is stable and can resist gastrointestinal enzymatic damage;

[0097] Key functional peptides have significant advantages: Peptide 1 loses only 3.2% of its activity during gastrointestinal digestion (48.82% → 47.24%), and Peptide 5 retains 94.8% of its activity, highlighting its potential as an oral candidate molecule. Comparable products (such as milk-derived peptides) generally have activity retention rates below 70% under the same conditions.

[0098] Broad-spectrum tolerance: Although the activity of some peptides (such as peptide 3 and peptide 6) decreased significantly, the overall active peptide population still maintained functionality after digestion (for example, peptide 4 retained 86.1% activity and peptide 2 retained 87.8%), proving that the process of the present invention can screen out advantageous peptides with both high activity and digestion stability.

[0099] In summary, the active peptides obtained by the directed enzymatic hydrolysis and ultrafiltration fractionation technology of the present invention not only have significant in vitro cell proliferation activity, but also break through the technical bottleneck of traditional active peptides that are easily inactivated after oral administration, laying a solid foundation for the development of oral cognitive improvement preparations.

[0100] The present invention also provides specific applications of the six active peptides synthesized in the laboratory, and the relevant applications are described below.

[0101] Example 4

[0102] Verification of active peptides improving cognitive function

[0103] 4.1 Experimental Design Method

[0104] 4.1.1 Animal Model Construction

[0105] Animal selection: Healthy male C57BL / 6 mice (8 weeks old, weighing 20-25 g) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and kept in an SPF-grade environment.

[0106] Group design: Randomly divided into the following groups (n=10 / group):

[0107] Control group (Control): Normal feeding, no sleep deprivation, gavage with equal volume of normal saline;

[0108] Sleep deprivation group (SD): sleep deprivation treatment + gavage with normal saline;

[0109] Low-dose peptide intervention group (LP): sleep deprivation treatment + 50 mg / kg / d active peptide solution gavage (divided into 6 groups: LP-peptide 1 to LP-peptide 6);

[0110] High-dose peptide intervention group (HP): sleep deprivation treatment + oral administration of 100 mg / kg / d active peptide solution (divided into 6 groups: HP-peptide 1 to HP-peptide 6).

[0111] Sleep deprivation method: Using a modified multi-platform water environment method, mice were placed in a water tank (45 cm × 30 cm × 20 cm) containing multiple small platforms (3 cm diameter), with a water depth of 15 cm and a temperature of 25 ± 1°C. Each platform housed only one mouse, and the sleep deprivation period was 20 hours per day (8:00 AM to 4:00 AM the following day) for 7 consecutive days.

[0112] 4.1.2 Dosage Regimen

[0113] Preparation of active peptide solutions: The six active peptides (peptide 1 to peptide 6) prepared in Example 1 were dissolved in physiological saline (pH 7.4) to prepare 50 mg / mL (LP group) and 100 mg / mL (HP group) solutions, respectively.

[0114] Gavage time: After the end of sleep deprivation every day (4:00-5:00), gavage at 10 mL / kg body weight.

[0115] 4.1.3 Sleep State

[0116] The actual sleep state of mice during sleep deprivation was tested, including the detection duration (min) and sleep ratio (%), and the results are shown in Table 6;

[0117] After modeling: record the sleep data of mice after deprivation;

[0118] Post-drug intervention: Monitor sleep recovery.

[0119] The mouse's activities are monitored in real time through a camera, automatically distinguishing between resting (sleeping) and moving (awake) states.

[0120] Set the inactivity threshold (e.g., continuous inactivity for ≥ 40 seconds is considered sleep).

[0121] Monitoring time points: After model establishment, mice were returned to their normal cages and their sleep status was monitored continuously for 24 hours. After drug intervention, sleep recovery was monitored continuously for 24 hours.

[0122] Each mouse was monitored individually to avoid group interference;

[0123] Environmental parameters (light, temperature) were recorded simultaneously to ensure consistency.

[0124] Detection duration: 24 hours / time;

[0125] Awake time: the total time the mouse was active during the monitoring period (min);

[0126] Sleep time: the total duration of inactivity (sleep) during the monitoring period (min);

[0127] Sleep ratio: sleep time / test duration × 100%.

[0128] 4.1.4 Behavioral testing

[0129] Morris water maze test:

[0130] Positioning navigation: Training was continued for 4 days (4 times per day). On the fifth day, the test was conducted. Mice were placed from the quadrant opposite to the platform quadrant, and the time it took for the mice to find the hidden platform (escape latency, seconds) was recorded. The results are shown in Table 7.

[0131] Spatial exploration test: On the 6th day, the platform was removed and the time (in seconds) the mice stayed in the target quadrant and the number of times they crossed the platform were recorded. The results are shown in Table 8.

[0132] New object recognition experiment:

[0133] Adaptation period: mice were placed in an open field containing two identical objects and allowed to explore freely for 10 min;

[0134] Test period (24 hours later): One of the objects was replaced with a new object, and the time the mouse spent exploring the new and old objects was recorded. The recognition index (new object exploration time / total exploration time × 100%) was calculated. The results are shown in Table 9.

[0135] 4.1.5 Biochemical index detection

[0136] Sample collection: After behavioral testing, mice were anesthetized and hippocampal tissue was obtained. The tissue was aliquoted and stored at -80°C.

[0137] Inflammatory factor detection: ELISA kits were used to measure the levels of IL-1β and TNF-α (unit: pg / mg protein).

[0138] Oxidative stress indicators: superoxide dismutase (SOD) activity: determined by the xanthine oxidase method (unit: U / mg protein); malondialdehyde (MDA) content: determined by the thiobarbituric acid method (unit: nmol / mg protein); the specific results are shown in Table 10.

[0139] 4.2 Test data and analysis

[0140] Table 6 Sleep status results

[0141]

[0142] Table 7 Results of the concealed station search test (mean ± SD, n = 10)

[0143]

[0144] Table 8. Spatial exploration test results (mean ± SD, n = 10)

[0145]

[0146] Table 9 Experimental results of novel object recognition (mean ± SD, n = 10)

[0147]

[0148] Table 10 Results of biochemical indexes in hippocampus tissue (mean ± SD, n = 10)

[0149]

[0150] As shown in Table 6, the sleep state data show that the sleep deprivation group (SD) had a sleep percentage of only 10% and wake time of 90%, indicating successful model construction. High-dose peptide 1 (HP-peptide 1) restored the sleep percentage to 30%, significantly higher than the SD group (p < 0.001), and the sleep duration (900 seconds) was close to that of the control group (1200 seconds), indicating a direct improvement in the effects of sleep deprivation. Peptide 5 (HP-peptide 5) achieved the highest sleep percentage (31.7%) of the intervention group, suggesting that it may enhance cognitive recovery by regulating sleep rhythms.

[0151] As shown in Table 7, the escape latency of the sleep-deprived group (SD) (80.8 seconds) was significantly longer than that of the control group (26.4 seconds), and the platform area was not found, indicating that sleep deprivation severely impairs spatial memory. The escape latency of high-dose peptide 1 (HP-peptide 1) (31.2 seconds) was close to that of the control group and significantly better than that of the SD group (p < 0.001), indicating that peptide 1 effectively restored spatial memory. The escape latency of high-dose peptide 5 (HP-peptide 5) (50.9 seconds) was significantly shorter than that of the SD group, suggesting that its improvement effect was superior to that of other peptides. The effect of the low-dose group was limited (e.g., LP-peptide 4 and LP-peptide 6 still failed to find the platform), indicating a dose-dependent effect.

[0152] As shown in Table 8, the spatial exploration test results show that the sleep deprivation group (SD) had significantly lower platform quadrant dwell time (19.5 seconds) and shuttle frequency (0.8 times) than the control group (48.14 seconds, 5.4 times). High-dose peptide 1 (HP-peptide 1) restored dwell time (42.1 seconds) and shuttle frequency (4 times) to near-normal levels, indicating a significant improvement in spatial memory consolidation. Peptide 5 (HP-peptide 5) achieved superior dwell time (34.0 seconds) and shuttle frequency (3 times) compared to most intervention groups, confirming its stable therapeutic effect.

[0153] As shown in Table 9, the novel object recognition test results show that the sleep deprivation group (SD) achieved a significantly lower recognition index (31%) than the control group (65%), indicating impaired novel object recognition ability. The recognition index of high-dose peptide 1 (HP-peptide 1) (56%) was significantly higher than that of the SD group (p < 0.01), approaching normal levels, suggesting that it rapidly improves cognitive flexibility. Peptide 2 (HP-peptide 2) achieved a similar recognition index (54%), indicating its potential application.

[0154] Table 10 shows the results of hippocampal biochemical indices. The sleep deprivation group (SD) showed significant increases in IL-1β (28.7 pg / mg), TNF-α (22.1 pg / mg), and MDA (9.1 nmol / mg), while SOD activity (18.9 U / mg) decreased, indicating that neuroinflammation and oxidative stress are core mechanisms of cognitive impairment. High-dose peptide 1 (HP-peptide 1) significantly decreased IL-1β (14.8 pg / mg) and TNF-α (10.9 pg / mg), while SOD activity (32.3 U / mg) was elevated to near-normal levels, confirming its dual anti-inflammatory and antioxidant effects. Peptide 5 (HP-peptide 5) showed significantly lower MDA (5.1 nmol / mg) than the SD group, demonstrating its superiority in alleviating lipid peroxidation.

[0155] In summary, active peptides significantly improved cognitive impairment. A high dose of peptide 1 shortened escape latency by 61.4% (80.8 seconds to 31.2 seconds), restoring it to near-normal levels. Peptides 1 and 2 increased the recognition index by 80.6% and 74.2%, respectively, significantly outperforming existing drugs (such as melatonin receptor agonists). Active peptides achieved neuroprotection by inhibiting IL-1β (48.4% reduction) and TNF-α (50.7% reduction) and increasing SOD activity (70.9%).

[0156] The high-dose group (100 mg / kg) showed significantly better efficacy than the low-dose group (50 mg / kg), as evidenced by the sleep rate of peptide 1 (30% vs 20%), indicating that the active peptide of the present invention has the advantages of dose-dependency and targeting.

[0157] Peptide 1 and peptide 5 performed outstandingly in inflammation inhibition, oxidative stress relief and sleep recovery, verifying the molecular design advantages of the present invention for sleep deprivation-specific pathologies.

[0158] The cost of duck bone-derived active peptides was 60% lower than that of milk-derived peptides (with raw material utilization increased to 95%). Optimization of the enzymatic hydrolysis process (pH 7.6, 30°C) increased the yield of active peptides by 32%. Ultrafiltration fractionation (<3 kDa) precisely selected highly stable peptides, resulting in >94% activity retention after gastrointestinal digestion (Table 5), breaking through the technical bottleneck of traditional oral inactivation of peptides.

[0159] The active peptide combines safety with long-lasting efficacy (Table 4: 99.2% activity retention after 28 days at -20°C), making it an ideal candidate for development in oral formulations (such as capsules and tablets). Its multi-target mechanisms of action (anti-inflammatory, antioxidant, and sleep-promoting) offer broad application prospects in sleep-related cognitive disorders, such as Alzheimer's disease.

[0160] The active peptides (SEQ ID NOs: 1-6) obtained through targeted enzymatic hydrolysis of duck bone collagen in this invention demonstrate significant cognitive improvement in a sleep deprivation model. Key advantages include: high doses of peptide 1 restore cognitive indicators to 80%-90% of normal levels; simultaneous inhibition of neuroinflammation, alleviation of oxidative stress, and promotion of sleep recovery; low-cost raw materials, stable process, and excellent oral bioavailability. This technical solution provides a novel solution for the intervention of cognitive impairment and possesses significant clinical translational value.

[0161] The number of devices and processing scales described herein are intended to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be readily apparent to those skilled in the art.

[0162] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Active peptide, characterized in that The amino acid sequence of the active peptide is shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6.

2. Use of the active peptide according to claim 1 in the preparation of a product for improving cognitive impairment.

3. The use according to claim 2, characterized in that Cognitive impairment included decreased spatial memory, reduced ability to recognize new objects, and increased levels of inflammatory factors IL-1β and TNF-α in the hippocampus.

4. The use according to claim 3, characterized in that Active peptides improve cognitive impairment by increasing superoxide dismutase activity and reducing malondialdehyde content.

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