Active peptide for regulating stress adaptation of nerve cells, and preparation method and application thereof

Abstract

This invention discloses an active peptide that regulates the stress adaptation ability of nerve cells, its preparation method, and its application. It belongs to the field of bioengineering technology. The amino acid sequence of the active peptide is shown in SEQ ID NO: 1, 2, 3, 4, 5, or 6. The preparation method of the active peptide of this invention is as follows: Step 1: Duck liver is collected and extracted to obtain a duck liver protein extract; Step 2: The duck liver protein extract is enzymatically hydrolyzed, and the supernatant is collected by centrifugation to obtain an enzymatic hydrolysate; Step 3: The enzymatic hydrolysate is subjected to ultrafiltration fractionation to collect low molecular weight peptide components, which are then freeze-dried to obtain a crude active peptide powder; Step 4: Peptides exhibiting excellent performance in the indicators are screened from the crude active peptide powder to obtain the target active peptide. The active peptide of this invention can significantly improve the tolerance of nerve cells under continuous stress, maintain mitochondrial functional homeostasis and redox balance, and reduce cell apoptosis damage. It can be used to prepare products that improve corticosterone-induced nerve cell stress adaptation disorders.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology. More specifically, this invention relates to an active peptide that regulates the stress adaptation ability of nerve cells, its preparation method, and its applications. Background Technology

[0002] Under sustained or excessive stress, nerve cells often exhibit changes such as the accumulation of reactive oxygen species, decreased mitochondrial membrane potential, increased apoptosis levels, and weakened neurotrophic signals, ultimately leading to decreased cell viability and impaired function. The corticosterone-induced PC12 cell injury model has been widely used to simulate nerve cell stress damage induced by glucocorticoid overdose and to evaluate the intervention effects of candidate active substances.

[0003] Currently, considerable research has been conducted on the neuroprotective effects of peptides, with related patents and literature primarily focusing on neuroprotection, antioxidation, anti-apoptosis, memory improvement, and intervention in neurodegenerative diseases. Some existing patents disclose polypeptides or short peptides with neuroprotective effects, typically emphasizing the acquisition of specific peptide sequences and their effects on improving nerve damage, neurodegenerative diseases, or oxidative stress. For example, existing technologies disclose the use of neuroprotective peptides to treat nerve damage, and there are reports on peptides reducing amyloid-β protein toxicity, lowering Tau protein levels, and improving neurodegenerative pathological changes. There are also technical solutions for using bioactive peptides to protect nerve cells from oxidative stress damage and improve memory function. In addition to patent literature, non-patent literature also indicates that peptides derived from natural protein hydrolysates have certain protective effects in corticosterone-induced PC12 cell injury models or SH-SY5Y cell stress models. These effects typically manifest as increased cell viability, reduced reactive oxygen species levels, reduced mitochondrial damage, inhibition of apoptosis, and regulation of the expression of some neurotrophic factors.

[0004] However, existing technologies still have significant limitations. Current descriptions of peptide functions are mostly limited to broad areas such as neuroprotection, antioxidation, anti-inflammation, and memory improvement, lacking targeted research and technical solutions for the more specific functional phenotype of nerve cell stress adaptation. Existing technologies typically focus on improving end-stage damage outcomes, such as increasing cell survival, reducing oxidative damage levels, or inhibiting apoptosis, while lacking clear and systematic technical disclosures on how nerve cells maintain stress tolerance, stabilize neurotrophic signals, and enhance adaptive responses under sustained stress. Some existing technologies primarily target specific pathological or broadly defined damage scenarios such as Alzheimer's disease, brain injury, or general oxidative stress damage, and their technical objectives are not entirely aligned with the stress-induced imbalance of nerve cell function. Other existing technologies employ artificially designed peptides, peptides rich in specific amino acid residues, or peptides targeting specific disease sites; their applicable scenarios, key functions, and technical implementation pathways differ significantly from the technical problems they aim to solve. There is still a lack of specific disclosures regarding bioactive peptides that can directly regulate the stress adaptation ability of nerve cells, improve the tolerance of nerve cells to stress stimuli, and are applicable to the intervention of stress-induced nerve cell damage.

[0005] Therefore, developing a bioactive peptide that can effectively regulate the stress adaptation ability of nerve cells and alleviate stress-induced cell damage, and that has a clear source and structure, has important scientific research value and clinical application prospects. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] Another objective of this invention is to provide an active peptide that regulates the stress adaptation ability of nerve cells, its preparation method, and its application, which solves the technical problem in the prior art of lacking bioactive peptides that can directly regulate the stress adaptation ability of nerve cells, improve the tolerance of nerve cells to continuous stress stimulation, and maintain mitochondrial functional homeostasis.

[0008] To achieve these objectives and other advantages according to the present invention, an active peptide is provided that regulates the stress adaptation ability of nerve cells, the amino acid sequence of which is shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0009] This invention also provides a method for preparing the aforementioned active peptide that regulates the stress adaptation ability of nerve cells, comprising the following steps: Step 1: After pretreatment, duck liver is homogenized, heat-treated, and subjected to ultrasonic-assisted extraction to obtain duck liver protein extract. Step 2: After adjusting the duck liver protein extract to the conditions for the first step of enzymatic hydrolysis, add the first protease to carry out the first step of enzymatic hydrolysis, then adjust to the conditions for the second step of enzymatic hydrolysis and add the second protease to continue enzymatic hydrolysis; after the enzymatic hydrolysis is completed, heat to inactivate the enzyme, centrifuge and collect the supernatant to obtain the enzymatic hydrolysate; Step 3: The enzymatic hydrolysate is subjected to ultrafiltration fractionation to collect the low molecular weight peptide components, which are then freeze-dried to obtain crude active peptide powder; the crude active peptide powder contains multiple candidate active peptides; Step 4: Multiple candidate active peptides were applied to a corticosterone-induced neuronal stress injury system. The effects of each candidate active peptide on neuronal viability, lactate dehydrogenase release, reactive oxygen species level, mitochondrial membrane potential, and apoptosis rate were detected and compared. Peptides that performed well in the indicators were selected and purified to obtain the target active peptide. The amino acid sequence of the target active peptide is shown in SEQ ID NO: 1, 2, 3, 4, 5, or 6.

[0010] Preferably, in the preparation method of the active peptide, in step two, the enzymatic hydrolysis conditions are: pH value of 7.8–8.5, preferably 8.0; temperature of 42–48℃, preferably 45℃; and hydrolysis time of 1.5–3 h, preferably 2 h. Preferably, the enzymatic hydrolysis conditions in the second step are: pH value of 6.8–7.4, preferably 7.2; temperature of 35–40℃, preferably 37℃; and hydrolysis time of 2–4 h, preferably 3 h.

[0011] Preferably, in the preparation method of the active peptide, the ultrafiltration fractionation in step three uses a molecular weight cutoff of 1-10 kDa, preferably 2-8 kDa, and further separates the positively charged peptide components by cation exchange chromatography.

[0012] Preferably, in the method for preparing the active peptide, the first protease is trypsin and the second protease is chymotrypsin; the enzyme activity ratio of the first protease to the second protease is 1:1 to 2:1, preferably 1:1; the amount of trypsin added in the first enzymatic hydrolysis is 0.015 IU / mg based on protein, the amount of chymotrypsin added in the second enzymatic hydrolysis is 0.015 IU / mg based on protein, and the total amount of enzyme added is 0.03 IU / mg based on protein.

[0013] The present invention also provides the application of the above-mentioned active peptide in a product for improving corticosterone-induced neuronal stress adaptation disorder.

[0014] Preferably, the application described herein includes stress adaptation disorder, which includes decreased nerve cell vitality, increased lactate dehydrogenase release, elevated reactive oxygen species levels, decreased mitochondrial membrane potential, and increased cell apoptosis rate.

[0015] Preferably, in the aforementioned application, the product alleviates nerve cell stress damage through one or more of the following mechanisms: improving nerve cell stress tolerance, reducing intracellular reactive oxygen species levels, maintaining mitochondrial membrane potential stability, regulating redox balance, inhibiting the activation of apoptosis signaling pathways, and maintaining cellular energy metabolism homeostasis.

[0016] The present invention has at least the following beneficial effects: This invention provides an active peptide that regulates the stress adaptation ability of nerve cells, its preparation method, and its application. Using duck liver as raw material, the invention involves extraction, two-step enzymatic hydrolysis, ultrafiltration fractionation, cation exchange separation, and functional screening to obtain an active peptide with a defined amino acid sequence. Applying this active peptide to a corticosterone-induced nerve cell stress injury system significantly improves the tolerance of nerve cells under continuous stress, maintains mitochondrial homeostasis and redox balance, and reduces apoptotic damage.

[0017] This invention is the first to use a positively charged short peptide with a specific sequence to directly regulate the stress adaptation ability of nerve cells. Unlike existing technologies that only improve the pan-neuroprotective effect of terminal damage (such as increasing survival rate and reducing oxidative damage), this invention achieves targeted intervention on the tolerance and adaptation ability of nerve cells under continuous stress.

[0018] The mechanism of action of the active peptides in this invention is clearly directed towards the maintenance of mitochondrial function and redox homeostasis. By stabilizing mitochondrial membrane potential, reducing the accumulation of reactive oxygen species, and regulating apoptosis-related signals, it alleviates stress-induced neuronal damage at its source, which is different from the technical solutions of general antioxidant peptides that only exert their effects by non-specifically scavenging free radicals.

[0019] This invention employs a two-step enzymatic hydrolysis combined with ultrafiltration fractionation and cation exchange chromatography, which effectively enriches target peptides with specific charge properties and functional characteristics, improving the targeting and efficiency of active peptide screening. Compared with traditional single-step enzymatic hydrolysis or simple ultrafiltration separation processes, the technical approach of this invention is more suitable for obtaining neurostimulation-regulating active peptides with clearly defined functional orientations.

[0020] The active peptides described in this invention have a clear source, well-defined structure, and controllable preparation process, and have good development potential. They can be applied to the intervention of nerve cell stress injury, the development of neurohealth-related preparations and functional products.

[0021] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0022] Figure 1 This is the mass spectrum of the active peptide prepared in Example 1 of the present invention. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it based on the description.

[0024] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0025] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0027] Example 1 This invention provides an active peptide that regulates the stress adaptation ability of nerve cells, and its preparation method includes the following steps: Step 1: Take fresh duck liver, remove the surface fascia and blood, rinse thoroughly with pre-cooled physiological saline, drain, and chop. Add phosphate buffer (PBS, pH 7.4) at a material-to-liquid ratio of 1:3 (g / mL) for homogenization. Heat-treat the homogenate at 80℃ for 10 min to inactivate endogenous enzymes, followed by ultrasonic-assisted extraction. The ultrasonic conditions are: power 350W, on / off time 3s / 2s, total processing time 15 min. After extraction, centrifuge at 10000 rpm for 15 min, collect the supernatant to obtain duck liver protein extract. Step 2: The obtained duck liver protein extract is subjected to two-step enzymatic hydrolysis: First, the pH of the system is adjusted to 8.0, and the first protease is added for pre-enzymatic hydrolysis at a temperature of 45℃ for 2 hours; Second, the pH of the system is adjusted to 7.2, and the second protease is added for further enzymatic hydrolysis at a temperature of 37℃ for 3 hours.

[0028] The first protease is trypsin, and the second protease is chymotrypsin. The enzyme activity ratio of the first protease to the second protease is 1:1 to 2:1, preferably 1:1. The amount of trypsin added in the first enzymatic hydrolysis step is 0.015 IU / mg based on protein, the amount of chymotrypsin added in the second enzymatic hydrolysis step is 0.015 IU / mg based on protein, and the total amount of enzyme added is 0.03 IU / mg based on protein. After enzymatic hydrolysis, the enzyme is inactivated by heating in a 95°C water bath for 10 min, cooled, and centrifuged at 10,000 rpm for 10 min. The supernatant is collected to obtain the enzymatic hydrolysate.

[0029] Step 3: The enzymatic hydrolysate is fractionated by ultrafiltration membrane to collect low molecular weight peptide fractions of 1–10 kDa, preferably fractions in the range of 2–8 kDa. These fractions are then further separated by cation exchange chromatography to collect positively charged peptide fractions, which are then freeze-dried to obtain crude active peptide powder; the crude active peptide powder contains multiple candidate active peptides. Step 4: Multiple candidate active peptides were applied to a corticosterone-induced neuronal stress injury system. The effects of each candidate active peptide on neuronal viability, lactate dehydrogenase release, reactive oxygen species level, mitochondrial membrane potential and apoptosis rate were detected and compared. Peptides that performed well in the above indicators were selected and purified to obtain active peptides.

[0030] Optimization of the above extraction process parameters: With the second-step enzymatic hydrolysis conditions fixed at pH 7.2, hydrolysis temperature 37℃, and hydrolysis time 3h, and the ratio of the first protease to the second protease activity fixed at 1:1, the first-step pre-enzymatic hydrolysis conditions and total enzyme addition were optimized using the protection rate of the hydrolysis product on PC12 cells (rat adrenal pheochromocytoma cells) as the evaluation index.

[0031] Single-factor experiment: Single-factor experiments were conducted to investigate the effects of pH, time, temperature, and total enzyme dosage in the first-step enzymatic hydrolysis on the activity of the enzymatic hydrolysis products. When investigating one factor, the other factors were fixed as follows: pH 8.0, time 2.0 h, temperature 45 °C, and total enzyme dosage 0.03 IU / mg.

[0032] The total enzyme dosage, calculated based on protein content, was set at 0.01, 0.02, 0.03, and 0.04 IU / mg, respectively. Under the condition that the ratio of the first protease to the second protease was 1:1, the enzymes were allocated to the two-step enzymatic hydrolysis system. The pH values ​​for the first-step hydrolysis were set to 7.6, 8.0, 8.4, and 8.8, respectively; the hydrolysis times were set to 1.5, 2.0, 2.5, and 3.0 h, respectively; and the hydrolysis temperatures were set to 40, 45, 50, and 55 °C, respectively. Each experiment was repeated three times in the single-factor experiments, and the average value was taken. The results are shown in Table 1.

[0033] Table 1 Results of the single-factor experiment Table 1 shows that, under the condition of fixed second-step enzymatic hydrolysis, the first-step pre-hydrolysis conditions and the total enzyme addition amount significantly affected the cell protection activity of the obtained hydrolysate. Specifically, the hydrolysate obtained at a pH of 8.0, a hydrolysis time of 2.0 h, and a hydrolysis temperature of 45℃ showed a high protection rate for PC12 cells; the cell protection rate reached its highest when the total enzyme addition amount was 0.03 IU / mg. Based on the results of the single-factor experiments, pH, hydrolysis time, hydrolysis temperature, and total enzyme addition amount were selected as orthogonal optimization factors.

[0034] Orthogonal experiment: Based on the single-factor experiment, a 4-factor, 3-level L9(34) orthogonal experiment was designed with pH value, enzymatic hydrolysis time, enzymatic hydrolysis temperature and enzyme addition amount as factors and cell protection rate as the indicator. The levels of each factor are shown in Table 2. The orthogonal experiment was designed and variance analysis was performed using IBM SPSS Statistics V20 to determine the optimal conditions for the preparation process. The results are shown in Table 3.

[0035] Table 2 Orthogonal Experimental Design result: Table 3 Results of the orthogonal experiment The optimal group is A2B2C2D2, and the order of influence is: hydrolysis time (B) > pH (A) > hydrolysis temperature (C) > enzyme addition amount (D).

[0036] Table 3 shows the optimal experimental parameters for the preparation process of the active peptides of this invention: First-step enzymatic hydrolysis at pH 8.0, hydrolysis time 2.0 h, and hydrolysis temperature 45℃; second-step enzymatic hydrolysis at pH 7.2, 37℃, and 3 h. The sequential enzymatic hydrolysis system used is: first protease is trypsin, and second protease is chymotrypsin. The total enzyme addition is 0.03 IU / mg, and the enzyme activity ratio of the first protease to the second protease is preferably 1:1. Correspondingly, the first-step trypsin addition is 0.015 IU / mg based on protein content, and the second-step chymotrypsin addition is 0.015 IU / mg based on protein content.

[0037] The enzymatic hydrolysate prepared under optimized conditions was first fractionated by ultrafiltration membrane to remove large molecular weight proteins and excessively small molecular weight impurities, collecting peptide fractions with a molecular weight cutoff of 1–10 kDa, preferably those in the range of 2–8 kDa. The obtained ultrafiltration fractions were desalted by dialyzing with deionized water and then lyophilized for later use. The activity of different ultrafiltration molecular weight fractions was evaluated, and the results are shown in Table 4. The results showed that the 1–10 kDa fraction had the best protective effect against corticosterone-induced PC12 cell damage; therefore, this fraction was selected for subsequent cation exchange chromatography separation.

[0038] Table 4. Protective effects of different ultrafiltration molecular weight fractions on corticosterone-induced PC12 cell injury model. Table 4 shows that different molecular weight fractions exhibited significantly different protective effects on the corticosterone-induced PC12 cell injury model. The 2–10 kDa fraction showed the highest cell protection rate, reaching 78.4%, significantly higher than other molecular weight fractions (P<0.05), indicating that this molecular weight range is enriched with bioactive peptides possessing strong neuronal stress regulation capabilities. Therefore, the 2–10 kDa fraction was selected as the target fraction for subsequent cation exchange chromatography separation.

[0039] The lyophilized samples were dissolved in a low ionic strength buffer, preferably 20 mmol / L phosphate buffer (pH 6.0). After centrifugation to remove insoluble matter, the samples were loaded onto a pre-equilibrated cation exchange chromatography column. The cation exchange chromatography column was preferably a strong cation exchange column with sulfonic acid groups as ligands. After loading, the samples were eluted with the equilibration buffer to remove unadsorbed components; subsequently, elution was performed using a linear salt concentration gradient containing 0–1.0 mol / L NaCl, and each peptide fraction was collected according to the elution peak. The protective effect of each component on the corticosterone-induced PC12 cell injury model was evaluated, and components with higher activity were screened for subsequent purification and sequence identification steps. The results are shown in Table 5.

[0040] Table 5. Protective effects of different cation exchange chromatography eluents on a corticosterone-induced PC12 cell injury model. Table 5 shows that different cation exchange chromatography eluents exhibited significantly different protective effects against corticosterone-induced PC12 cell damage. Among them, fraction F3, obtained under elution conditions of 0.3–0.5 mol / L NaCl, showed the highest cell protection rate, reaching 84.6%, significantly higher than the other fractions (P<0.05), indicating that this fraction was enriched with bioactive peptides with strong neuronal stress regulation effects. Therefore, fraction F3 was selected as the target fraction for subsequent reversed-phase high-performance liquid chromatography (RP-HPLC) separation, purification, and sequence identification. The crude bioactive peptide powder obtained from fraction F3 was analyzed by mass spectrometry, as shown in the figure. Figure 1As shown; the mass spectrometry method is as follows: mass spectrometry data are acquired using a QExactive HF mass spectrometer connected in series with an UltiMate 3000 RSLC nano liquid chromatography system. The active peptide sample is dissolved in the loading buffer, aspirated by the autosampler, and separated by the analytical column. The analytical column specifications are:

[75] μm ×

[25] cm, C18, [2] μm,

[100] Å. An analytical gradient is established using two mobile phases: mobile phase A is a 0.1% formic acid aqueous solution (containing 2% dimethyl sulfoxide), and mobile phase B is a 0.1% formic acid-80% acetonitrile aqueous solution (containing 2% dimethyl sulfoxide). The flow rate of the liquid phase is set to

[300] nL / min.

[0041] Mass spectrometry data were acquired in DDA mode, with each scan cycle consisting of one MS full scan and 20 subsequent MS / MS scans (Top20). Specific parameters were as follows: MS full scan resolution of 60,000, and automatic gain control target value of 3e. 6 The maximum ion implantation time was 50 ms, and the scan range was 300–1800 m / z; the MS / MS scan resolution was 15000, and the automatic gain control target value was 1e. 5 The maximum ion implantation time was 45 ms. The high-energy collision dissociation collision energy was set to 28 kDa, the quadrupole screening window was set to 1.6 Da, and the dynamic exclusion time was set to 30 s.

[0042] Mass spectrometry data were retrieved using MaxQuant (V1.6.17.0) software with the Andromeda algorithm. The main search parameters were as follows: label-free quantification was selected for the item type; methionine oxidation and protein N-terminal acetylation were selected for the variable modifications; cysteine ​​carboxymethylation was selected for the fixed modifications; and non-specific enzyme digestion was selected for the enzyme digestion method. Search results were screened based on a 1% false discovery rate at both the protein and peptide levels. Several candidate peptides with protective effects against corticosterone-induced PC12 cells were ultimately identified. The amino acid sequences of the superior peptides are SEQ ID NO: 1, 2, 3, 4, 5, and 6. These six amino acid sequences were used to synthesize a single active peptide, which is numbered as follows: Peptide 1: SEQ ID NO: 1 is QRSWFSLGAVQSAA; Peptide 2: SEQ ID NO: 2 is DHYYETQDALK; Peptide 3: SEQ ID NO: 3 is VFQATGLYKAHAS; Peptide 4: SEQ ID NO: 4 is AYTVAFTQGYSAA; Peptide 5: SEQ ID NO: 5 is TAFYYLESWKLVA; Peptide 6: SEQ ID NO: 6 is QFVATSAQYTWKQ.

[0043] Example 2: Stability Test of Active Peptides The stability of the six synthesized target active peptides was tested: the active peptides (dissolved in sterile PBS, pH 7.4) were aliquoted into sealed, light-protected EP tubes and stored at constant temperatures of 40℃, 25℃, 4℃, and -20℃, with three replicates for each group. Samples were taken at 0, 7, 14, and 28 days and immediately frozen at -80℃ for later use. Subsequently, the samples at each time point were applied to a corticosterone-induced PC12 cell injury model. The cell protection rate was used as the evaluation index to detect the functional stability of the six active peptides under different storage conditions. The experimental results are shown in Table 6.

[0044] Table 6 Results of Stability Tests for Active Peptides As shown in Table 6, the PC12 cell protection rates of the six active peptides showed certain differences under different storage temperatures and storage times. Overall, the higher the temperature and the longer the storage time, the lower the activity retention rate, indicating that storage conditions have a significant impact on the structural stability and functional retention of active peptides.

[0045] At 40℃, the cell protection rates of all six bioactive peptides gradually decreased with prolonged storage time. After 7 days of storage, each bioactive peptide maintained high activity; however, the decline in activity accelerated further with extended storage times of 14 and 28 days. After 28 days of storage, the cell protection rates of peptides 1–6 were 86.9%, 82.8%, 79.8%, 75.2%, 70.6%, and 66.1%, respectively. These results indicate that under higher temperature conditions, all bioactive peptides experienced varying degrees of functional decline, with peptides 1 and 2 exhibiting relatively stronger activity retention and better thermostability. At 25℃, all six bioactive peptides maintained a relatively high level of cell protection activity overall, but a gradual decline was observed with prolonged storage time. After 28 days of storage, the cell protection rates of peptides 1–6 were 90.7%, 87.9%, 85.1%, 81.8%, 78.5%, and 75.0%, respectively. Compared to 40℃, the activity loss of each bioactive peptide was significantly reduced at 25℃, indicating that the bioactive peptides of this invention still have good storage adaptability under room temperature conditions. At 4℃, the cell protection rate of each bioactive peptide showed little change, demonstrating good cold storage stability. After 28 days of storage, the cell protection rates of peptides 1–6 were 96.2%, 94.8%, 92.8%, 90.6%, 88.2%, and 85.7%, respectively. These results indicate that cold storage conditions can effectively slow down the decline in bioactive peptide activity, which is more conducive to maintaining their structural stability and functional integrity. At -20℃, all six bioactive peptides exhibited optimal storage stability, with the smallest decrease in activity throughout the storage period. After 28 days of storage, the cell protection rates of peptides 1–6 were 98.1%, 96.9%, 97.2%, 96.1%, 94.9%, and 93.6%, respectively. This indicates that freezing conditions can further inhibit the inactivation of bioactive peptides during storage, thereby more effectively maintaining their biological activity.

[0046] The experimental results under various temperature conditions show that the six bioactive peptides prepared in this invention exhibit good storage stability in solution, especially maintaining a high level of PC12 cell protection activity over a relatively long storage period at 4℃ and below. Even after storage at 40℃ for 28 days, each bioactive peptide still retains a certain degree of cell protection activity, indicating good environmental tolerance and application adaptability. Among them, peptides 1 and 2 show superior activity retention under most conditions, and have higher development and application value.

[0047] In summary, the active peptides obtained by this invention through optimized preparation process exhibit good storage stability in solution, especially maintaining high activity for extended periods at 4°C and below. Even after 28 days of storage at 40°C, they still maintain strong PC12 cell protection effects, overcoming the shortcomings of some traditional active peptides that are prone to inactivation and require stringent storage conditions. This provides experimental evidence and technical support for their application and development as ideal candidate molecules in interventions for nerve cell stress injury, functional foods, special medical foods, and related bioactive preparations.

[0048] Example 3: Protective effect of active peptides against corticosterone-induced neuronal stress damage. 3.1 Experimental Materials 3.1.1 Active peptide samples: Target active peptides (SEQ ID NO: 1, 2, 3, 4, 5, 6) prepared in Example 1.

[0049] 3.1.2 Cell line: PC12 cells (rat adrenal pheochromocytoma cells).

[0050] 3.1.3. Main reagents: corticosterone, DMEM medium, fetal bovine serum, CCK-8 kit, lactate dehydrogenase (LDH) detection kit, reactive oxygen species (ROS) detection kit, mitochondrial membrane potential detection kit (JC-1), apoptosis detection kit (Annexin V-FITC / PI), ATP content detection kit.

[0051] 3.2 Experimental Procedure 3.2.1 Cell Culture: PC12 cells were routinely cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator.

[0052] 3.2.2 Model Construction and Grouping: PC12 cells in the logarithmic growth phase were seeded into 6-well culture plates. A blank control group, a model group (corticosterone treatment group), and groups treated with different concentrations of active peptides were set up. Except for the blank control group, all other groups were treated with 200 μM corticosterone to induce cell stress damage. Simultaneously, the active peptide treatment groups were treated with different concentrations (e.g., 10, 20, 40 μM) of the target active peptide for 24 h.

[0053] 3.2.3 Cell viability assay: After treatment, CCK-8 solution was added to each well and incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0054] 3.2.4 Lactate dehydrogenase (LDH) release detection: Collect cell culture supernatant, follow the LDH detection kit instructions, measure the absorbance at 490 nm, and calculate the LDH release rate.

[0055] 3.2.5 Detection of reactive oxygen species (ROS) level: Cells were incubated with the DCFH-DA probe, and the fluorescence intensity was detected by a fluorescence microplate reader or flow cytometer to represent the ROS level.

[0056] 3.2.6 Mitochondrial membrane potential detection: Cells were incubated with the JC-1 probe, and the red / green fluorescence ratio was detected by a fluorescence microplate reader or flow cytometer to reflect changes in mitochondrial membrane potential.

[0057] 3.2.7 Apoptosis rate detection: The apoptosis rate was detected by flow cytometry using Annexin V-FITC / PI double staining.

[0058] 3.2.8 ATP Content Detection: Intracellular ATP content was measured using an ATP assay kit to reflect the energy metabolism status. The results are shown in Table 7.

[0059] Table 7 Effects of bioactive peptides on various parameters of corticosterone-induced PC12 cells (mean ± SD, n=3) Group Cell viability (%) LDH release rate (%) ROS level (%) Mitochondrial membrane potential (red / green ratio) Apoptosis rate (%) ATP content (%) Blank control group 100.0±3.2 100.0±4.1 100.0±3.8 3.12±0.21 5.3±0.8 100.0±4.0 Model group 64.7±4.5 158.3±5.2 182.6±6.1 1.21±0.18 24.6±2.1 58.9±4.3 Low-dose group of active peptide 1 75.8±3.9 139.7±4.8 152.3±5.7 1.89±0.19 17.8±1.9 72.5±3.9 Medium dose group of active peptide 1 86.4±4.1 121.5±4.3 130.6±5.2 2.42±0.20 12.6±1.6 84.3±4.1 High-dose group of active peptide 1 94.1±3.6 109.2±3.9 112.8±4.7 2.89±0.22 8.7±1.2 93.6±3.8 Low-dose group of active peptide 2 73.2±3.8 142.6±4.6 158.9±5.5 1.78±0.18 18.9±1.8 70.3±3.7 Medium dose group of active peptide 2 83.5±4.0 125.8±4.2 136.4±5.0 2.31±0.19 13.8±1.5 81.2±3.9 High-dose group of active peptide 2 91.6±3.5 112.7±3.8 118.6±4.6 2.75±0.21 9.6±1.3 90.8±3.6 Low-dose group of active peptide 3 71.5±3.7 146.2±4.7 162.7±5.6 1.72±0.17 19.6±1.9 68.9±3.6 Medium dose group of active peptide 3 80.8±3.9 130.4±4.3 140.9±5.1 2.24±0.18 14.9±1.6 79.6±3.8 High-dose group of active peptide 3 89.3±3.4 116.8±3.9 122.5±4.7 2.68±0.20 10.8±1.3 88.7±3.5 Low-dose group of active peptide 4 69.8±3.6 149.5±4.9 166.3±5.8 1.66±0.16 20.4±2.0 67.2±3.5 Medium dose group of active peptide 4 78.6±3.8 134.2±4.4 145.8±5.3 2.18±0.18 15.8±1.6 77.8±3.7 High-dose group of active peptide 4 87.5±3.3 120.6±4.0 126.9±4.9 2.61±0.20 11.7±1.4 86.9±3.4 Low-dose group of active peptide 5 68.1±3.5 152.8±5.0 170.5±6.0 1.60±0.15 21.2±2.1 65.6±3.4 Medium dose group of active peptide 5 76.9±3.7 137.6±4.5 149.7±5.4 2.12±0.17 16.7±1.7 75.9±3.6 High-dose group of active peptide 5 85.8±3.2 124.3±4.1 130.8±5.0 2.54±0.19 12.6±1.4 84.8±3.3 Low-dose group of active peptide 6 66.5±3.4 156.3±5.2 174.8±6.2 1.54±0.32 22.1±2.2 63.9±3.3 Medium dose group of active peptide 6 75.2±3.6 141.5±4.6 153.6±5.6 2.05±0.17 17.6±1.8 74.1±3.5 High-dose group of active peptide 6 84.2±3.1 128.7±4.2 134.6±5.3 2.47±0.18 13.5±1.5 82.9±3.2 Experimental results showed that, compared with the blank control group, PC12 cell viability was significantly decreased, LDH release rate, ROS level, and apoptosis rate were significantly increased, and mitochondrial membrane potential and ATP content were significantly decreased after treatment with corticosterone in the model group. This indicates that corticosterone can induce significant neuronal stress damage, accompanied by enhanced oxidative stress, mitochondrial dysfunction, and energy metabolism imbalance. Compared with the model group, intervention with the six active peptides could improve PC12 cell viability, reduce LDH release rate, ROS level, and apoptosis rate to varying degrees, and restore mitochondrial membrane potential and ATP content, indicating that each active peptide has a certain neuroprotective effect. Overall, the protective effect of each active peptide showed a trend of increasing with increasing dose, with the high-dose group showing the most significant improvement. Further comparison of the effects of different active peptides showed that all six active peptides could alleviate corticosterone-induced cell damage, but there were certain differences in their overall protective capabilities. Overall, peptides 1 and 2 showed better effects, while the other active peptides also showed good intervention effects. The above results indicate that the active peptides provided by this invention can not only alleviate corticosterone-induced neuronal damage, but also improve the adaptability and tolerance of neuronal cells under continuous stress conditions by reducing oxidative stress levels, maintaining mitochondrial functional homeostasis, and improving cellular energy metabolism, thereby effectively alleviating corticosterone-induced stress damage.

[0060] Explanation of the structural characteristics and functional relationship of active peptides The amino acid sequences of the active peptides described in this invention are shown in SEQ ID NO: 1-6, specifically as follows: Peptide 1: SEQ ID NO: 1 is QRSWFSLGAVQSAA; Peptide 2: SEQ ID NO: 2 is DHYYETQDALK; Peptide 3: SEQ ID NO: 3 is VFQATGLYKAHAS; Peptide 4: SEQ ID NO: 4 is AYTVAFTQGYSAA; Peptide 5: SEQ ID NO: 5 is TAFYYLESWKLVA; Peptide 6: SEQ ID NO: 6 is QFVATSAQYTWKQ.

[0061] The six bioactive peptides obtained in this invention are all short-chain peptides with a length of 12 to 15 amino acids. Their common structural features include a relatively high isoelectric point, the presence of aromatic amino acid residues (tryptophan, tyrosine, and phenylalanine), and an amphiphilic distribution. These features endow the bioactive peptides with the ability to bind efficiently to nerve cell membranes and mitochondrial membranes, enabling them to penetrate the cell barrier and exert their effects intracellularly. Specifically, the positive charge carried by the bioactive peptides (mainly derived from arginine, lysine, and histidine) promotes electrostatic interactions with negatively charged cell membrane phospholipids and mitochondrial cardiolipin at physiological pH, thereby targeting and enriching them on the mitochondrial membrane surface, stabilizing the mitochondrial membrane potential, inhibiting cytochrome c release, and thus blocking the activation of apoptosis signaling pathways. The aromatic amino acid residues directly scavenge reactive oxygen species through their conjugated system, while their hydrophobic side chains can insert into the membrane lipid bilayer, repairing stress-induced membrane fluidity abnormalities and maintaining the normal function of the mitochondrial electron transport chain complex. The amphiphilic helical or turn conformation helps the bioactive peptides resist protease degradation and prolong their half-life in vivo.

[0062] Based on the unique sequences of each bioactive peptide, peptide 1 (QRSWFSLGAVQSAA) exhibits a positive charge at its N-terminus (arginine), while the aromatic rings of tryptophan and phenylalanine can embed into the inner mitochondrial membrane. Its C-terminal hydrophobic alanine cluster further enhances membrane anchoring ability. Experiments have confirmed that this peptide can restore mitochondrial membrane potential to near-normal levels at high doses. Peptide 2 (DHYYETQDALK) contains two tyrosine and histidine residues. The phenolic hydroxyl group of tyrosine can directly donate electrons to scavenge hydroxyl radicals, while the imidazole ring of histidine can chelate transition metal ions to inhibit the Fenton reaction. Therefore, this peptide demonstrates outstanding performance in reducing intracellular reactive oxygen species levels. The GLYK motif in peptide 3 (VFQATGLYKAHAS) is homologous to the binding epitope of the TrkB receptor in brain-derived neurotrophic factor (BDNF), potentially competitively antagonizing corticosterone-induced receptor desensitization. Simultaneously, the positive charges provided by histidine and lysine help the peptide maintain conformational activity under acidic stress. Although peptide 4 (AYTVAFTQGYSAA) does not contain basic amino acids, its two tyrosine residues endow it with strong antioxidant capacity. Furthermore, the YTV fragment in its sequence is homologous to mitochondrial targeting signal peptides, allowing it to be recognized by the mitochondrial delivery system and enriched in the mitochondrial matrix. Peptide 5 (TAFYYLESWKLVA) possesses a continuous phenylalanine-tyrosine-tyrosine triaromatic structure, forming a hydrophobic "sandwich" that can efficiently embed into the phospholipid-rich region of the mitochondrial membrane core, repairing membrane fluidity. Experiments show that this peptide has the most significant effect on maintaining mitochondrial membrane potential. The YTW motif in peptide 6 (QFVATSAQYTWKQ) is similar to the activating peptide of some G protein-coupled receptors, potentially alleviating stress damage by regulating the cAMP-PKA signaling pathway. Simultaneously, multiple glutamine residues form a hydrogen bond network, ensuring water solubility while promoting interaction with polar head groups on the membrane surface. In summary, the active peptides of this invention, through a multi-target structure-function synergistic mechanism, comprehensively improve the adaptability and tolerance of nerve cells under continuous stress environments from multiple levels, such as scavenging free radicals, stabilizing mitochondrial membranes, blocking apoptosis, and regulating signaling pathways.

[0063] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0064] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. An active peptide that regulates the stress adaptation ability of nerve cells, characterized in that, The amino acid sequences of the active peptides are shown in SEQ ID NO: 1, 2, 3, 4, 5 or 6.

2. The method for preparing the active peptide for regulating the stress adaptation ability of nerve cells as described in claim 1, characterized in that, Includes the following steps: Step 1: After pretreatment, duck liver is homogenized, heat-treated, and subjected to ultrasonic-assisted extraction to obtain duck liver protein extract. Step 2: After adjusting the duck liver protein extract to the conditions for the first step of enzymatic hydrolysis, add the first protease to carry out the first step of enzymatic hydrolysis, then adjust to the conditions for the second step of enzymatic hydrolysis and add the second protease to continue enzymatic hydrolysis; after the enzymatic hydrolysis is completed, heat to inactivate the enzyme, centrifuge and collect the supernatant to obtain the enzymatic hydrolysate; Step 3: The enzymatic hydrolysate is subjected to ultrafiltration fractionation to collect the low molecular weight peptide components, which are then freeze-dried to obtain crude active peptide powder; the crude active peptide powder contains multiple candidate active peptides; Step 4: Multiple candidate active peptides were applied to a corticosterone-induced neuronal stress injury system. The effects of each candidate active peptide on neuronal viability, lactate dehydrogenase release, reactive oxygen species level, mitochondrial membrane potential, and apoptosis rate were detected and compared. Peptides that performed well in the indicators were selected and purified to obtain the target active peptide. The amino acid sequence of the target active peptide is shown in SEQ ID NO: 1, 2, 3, 4, 5, or 6.

3. The method for preparing the active peptide as described in claim 2, characterized in that, In step two, the first step of enzymatic hydrolysis conditions are: pH value of 7.8-8.5, temperature of 42-48℃, and enzymatic hydrolysis time of 1.5-3h; the second step of enzymatic hydrolysis conditions are: pH value of 6.8-7.4, temperature of 35-40℃, and enzymatic hydrolysis time of 2-4h.

4. The method for preparing the active peptide as described in claim 3, characterized in that, In step three, ultrafiltration fractionation is performed using components with a molecular weight cutoff of 1–10 kDa, and positively charged peptide components are further separated by cation exchange chromatography.

5. The method for preparing the active peptide as described in claim 2, characterized in that, The first protease is trypsin, and the second protease is chymotrypsin; the enzyme activity ratio of the first protease to the second protease is 1:1 to 2:1; the amount of trypsin added in the first enzymatic hydrolysis step is 0.015 IU / mg based on protein, the amount of chymotrypsin added in the second enzymatic hydrolysis step is 0.015 IU / mg based on protein, and the total amount of enzyme added is 0.03 IU / mg based on protein.

6. The use of the active peptide as described in claim 1 or the active peptide prepared according to any one of claims 2 to 5 in the preparation of a product for improving corticosterone-induced neuronal stress adaptation disorder.

7. The application as described in claim 6, characterized in that, Stress adaptation disorders include decreased nerve cell vitality, increased release of lactate dehydrogenase, elevated levels of reactive oxygen species, decreased mitochondrial membrane potential, and increased apoptosis rate.

8. The application as described in claim 6, characterized in that, The product reduces apoptotic damage by improving nerve cell stress tolerance, maintaining mitochondrial functional homeostasis, and redox balance.

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