A method for preparing a salty taste enhancing peptide-quercetin complex and its use in improving cognitive decline

Abstract

The application belongs to the field of food processing and biomedical technology, and discloses a preparation method of a salty taste enhancing peptide-naringenin compound and application of the compound in improving cognitive decline. The compound is prepared by compounding salty taste enhancing peptides from soft-shelled turtle eggs and naringenin at a ratio of 20-150:1 (w:w). The preparation process comprises the following steps: soft-shelled turtle egg enzymolysis, ultrafiltration, freeze-drying to obtain salty taste enhancing peptides, mixing the salty taste enhancing peptides with a naringenin solution, concentration, and freeze-drying. The compound has good stability, salty taste enhancing and antioxidant activities, can reduce the use of salt and maintain salty taste. The compound can improve cognitive decline and anxiety caused by aging and diabetes, improve autonomous activity, memory and social skills, strengthen physical fitness, protect nerve cells and skeletal muscles, maintain brain tissue health, and can be applied to the preparation of related preparations for improving cognitive decline.

Description

Technical Field

[0001] This invention belongs to the fields of food processing and biomedical technology, and relates to a method for preparing a salty-enhancing peptide-naringenin complex and its use in improving cognitive decline. Background Technology

[0002] Salt-enhancing peptides, a class of specific peptides that synergistically interact with NaCl to enhance salty taste perception without tasting salty themselves, meet the needs of people pursuing healthy diets and reducing salt intake, and have become a research hotspot in the food processing field. Existing technology CN120081895A has achieved the preparation of salt-enhancing peptides with a molecular weight of less than 3kDa from natural raw materials such as turtle eggs through papain enzymatic hydrolysis, ultrafiltration, and freeze-drying. These peptides not only retain the nutritional value and antioxidant activity of the raw materials, but also effectively reduce the amount of salt used without diminishing the salty taste experience. Among them, 16 specific amino acid sequences of salt-enhancing peptides, including dipeptides, tripeptides, and tetrapeptides, have been identified, exhibiting excellent binding ability to salty taste receptors and demonstrating application potential in the condiment and food industries.

[0003] Naringenin, a common flavonoid found in citrus fruits, possesses a wealth of biological functions, including antioxidant and anti-inflammatory effects, blood sugar regulation, and improvement of memory impairment in Alzheimer's disease rats and diabetes, making it valuable for health applications. However, current research clearly indicates that naringenin suffers from low bioavailability and poor water solubility, significantly limiting its efficient function. Therefore, improving its bioavailability is a pressing issue that needs to be addressed.

[0004] With the increasing aging of the population, the co-occurrence of aging and diabetes is becoming more and more common. Age-related diabetes is often accompanied by a series of health problems such as cognitive and memory decline, anxiety, skeletal muscle damage, and decreased motor coordination, seriously affecting patients' quality of life. In existing technologies, salt-enhancing peptides are mainly used in food seasoning and salt reduction applications. While naringenin has the potential to improve memory impairment, its bioavailability is limited. There is currently no technology to combine the two, leveraging their respective advantages and compensating for the application limitations of naringenin, to specifically address the cognitive decline and related health problems caused by age-related diabetes. Therefore, developing a complex that combines salt-enhancing properties with high bioavailability and can effectively improve age-related diabetes-related health problems has significant practical significance and application value. Summary of the Invention

[0005] This invention aims to solve two core technical problems: First, the low bioavailability and poor water solubility of naringenin in existing technologies make it difficult to effectively exert its biological functions such as anti-oxidation and improvement of memory impairment; Second, existing turtle egg-derived salt-enhancing peptides are limited to the scenario of reducing salt in food seasoning and have not yet been combined with naringenin to form a formulation with multiple functions, which cannot meet the needs of coping with health problems such as cognitive decline, anxiety, and skeletal muscle dysfunction caused by aging and diabetes.

[0006] To address the above problems, this invention provides a method for preparing a salty-enhancing peptide-naringenin complex and its application in improving cognitive decline. The specific technical solution is as follows:

[0007] In a first aspect, the present invention provides a method for preparing a salty-enhancing peptide-naringenin complex (NP), comprising: preparing a salty-enhancing peptide solution, preparing a naringenin solution, and mixing the salty-enhancing peptide solution with the naringenin solution; Preparation of salty-enhanced peptide solution: Dissolve lyophilized turtle egg powder in water to prepare turtle egg solution; add papain for enzymatic hydrolysis, collect the liquid by solid-liquid separation of the hydrolysis product, and filter and ultrafilter the liquid in sequence to obtain salty-enhanced peptide (Pep) with a molecular weight of less than 3kDa; dissolve the salty-enhanced peptide in a solvent to prepare a salty-enhanced peptide solution of 20~150mg / mL. Preparation of naringenin solution: Dissolve naringenin in a solvent to prepare a 1 mg / mL naringenin solution; Salty flavor-enhancing peptide solution and naringenin solution were mixed: the salty flavor-enhancing peptide solution and naringenin solution were mixed evenly at a volume ratio of 1:1, concentrated, and freeze-dried to obtain the salty flavor-enhancing peptide-naringenin complex.

[0008] Furthermore, the concentration of the turtle egg solution is 1-10%.

[0009] Furthermore, in the above preparation method, the papain activity is 4500~5500 U / g, and the enzymatic hydrolysis temperature is 45~55℃.

[0010] Furthermore, in the above preparation method, the solvent for dissolving the salty flavor-enhancing peptide is water, and the solvent for dissolving naringenin is 70-80% ethanol.

[0011] Secondly, the present invention seeks to protect the use of the salty-enhancing peptide-naringenin complex obtained by the above-described preparation method in the preparation of formulations that improve cognitive decline.

[0012] Furthermore, in the above applications, the cognitive decline includes one or more of the following: new object recognition memory decline, social memory decline, and spatial learning memory decline.

[0013] Thirdly, the present invention seeks protection for the use of the salty-enhancing peptide-naringenin complex obtained by the above-described preparation method in the preparation of formulations that improve anxiety states.

[0014] Fourthly, the present invention seeks protection for the use of the salty-enhancing peptide-naringenin complex obtained by the above-described preparation method in the preparation of formulations that improve the body's motor coordination ability.

[0015] Furthermore, the formulation can also improve skeletal muscle injury, enhance the brain tissue's antioxidant stress capacity, and alleviate brain nerve cell damage.

[0016] Furthermore, the formulation is an oral formulation.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention effectively improves the application limitations of naringenin by forming a stable nanoscale complex with a salty-enhancing peptide derived from turtle egg. This significantly enhances the water solubility and bioavailability of naringenin, allowing its antioxidant and memory function improvement functions to be effectively performed, thus solving the problem of limited naringenin function in existing technologies.

[0018] This complex achieves functional synergy and expansion. It retains the high binding capacity of the salt-enhancing peptides and salt-taste receptors, increasing the saltiness of 0.05 mol / L NaCl solution by 27.01–159.12%, meeting the need for a healthy diet that reduces salt intake without sacrificing overall saltiness. Furthermore, it incorporates the antioxidant activity of naringenin, which inhibits DPPH and ABTS. + The free radical scavenging rate is no less than 80%, breaking through the single application scenario of existing salty flavor-enhancing peptides, which are only used for food seasoning.

[0019] With targeted health-improving effects, compared to existing technologies that lack related compound formulations to address health problems caused by aging and diabetes, the compound of this invention can significantly improve cognitive decline and anxiety caused by aging and diabetes by increasing the activity of SOD and GSH-Px in brain tissue, reducing MDA content, protecting nerve cells from damage, and enhancing autonomous activity, memory, and social abilities. At the same time, it can alleviate muscle fiber atrophy, improve skeletal muscle function, enhance motor coordination and fatigue resistance, and provide a new solution for health problems related to aging and diabetes.

[0020] The preparation process is mild and controllable, employing mature processes such as papain enzymatic hydrolysis, ultrafiltration, and freeze drying. The reaction conditions are mild, making it easy to scale up production. Furthermore, the raw materials are derived from natural turtle eggs and citrus extracts, ensuring high safety and broad application prospects. Attached Figure Description

[0021] Figure 1The figures show the particle size, potential, and polydispersity index (PDI) for different proportions of NP; the horizontal axis represents the mass ratio of salty-enhancing peptides to naringenin, and the vertical axis represents the particle size ("Size" in the figure), zeta potential ("Zeta-potential" in the figure), and PDI value, respectively.

[0022] Figure 2 The image shows UV full-wavelength scans of different proportions of NP and salty-enhancing peptide (Pep); the horizontal axis represents wavelength (“Wavelength” in the image) and the vertical axis represents absorbance (“Absorbance” in the image).

[0023] Figure 3 The graphs show fluorescence spectra of NP and Pep at different ratios; the horizontal axis represents the emission wavelength (“Wavelength” in the graph), and the vertical axis represents the fluorescence intensity (“Fluorescence intensity” in the graph).

[0024] Figure 4 Fourier transform infrared spectra of naringenin (Nar), NP, and Pep; the horizontal axis represents wavenumber (“Wavenumber” in the figure), and the vertical axis represents transmittance (“Transmittance” in the figure).

[0025] Figure 5 Microscopic images of the salty-enhancing peptide ("Pep" in the figure), naringenin ("Nar" in the figure), and complex ("NP" in the figure) under a laser confocal microscope; 5μm, 1μm, and 500nm represent different observation scales.

[0026] Figure 6 The original glutathione (GSH), Pep and NP are the inhibitors of DPPH and ABTS. + Scavenging capacity statistics chart; the horizontal axis represents the concentration of each sample (“concentration” in the chart), and the vertical axis represents the scavenging activity of different free radicals (“radicalscavenging activity” in the chart).

[0027] Figure 7 This is a taste analysis graph of the complex NP; the horizontal axis represents the NP concentration (in %), and the vertical axis represents the saltiness value (“Salinity” in the graph). NaCl indicates that 0.05 mol / L NaCl was used as a control.

[0028] Figure 8Figure 1 shows the open field test results for mice in the control group (CON), model group (Model), metformin group (MET), low-dose NP group (NPL), and high-dose NP group (NPH). In the figure, (A) is the total distance (“Total Distance”), (B) is the average speed (“Average speed”), (C) is the number of hind limbs held upright (“Number of areas”), (D) is the number of times the mice entered the central zone (“Number of central zone entries”), (E) is the time spent in the central zone (“Time spent in the central zone”), and (F) is the movement trajectory.

[0029] Figure 9 The figures above show the results of the elevated cross maze test for each group of mice; where (A) is the total distance traveled (“Total distance traveled” in the figure), (B) is the total number of arm entries (“Total arm entries” in the figure), (C) is the number of open arm entries (“Open arm entries” in the figure), (D) is the time spent in open arms (“Timespent in open arms” in the figure), (E) is the percentage of time spent in open arms (“Open arm times” in the figure), (F) is the percentage of open arm entries (“Open arm entry” in the figure), and (G) is the movement trajectory.

[0030] Figure 10 The results of the novel object recognition test for each group of mice are shown in the figure; where (A) is the recognition coefficient ("Discrimination index" in the figure) and B is the movement trajectory.

[0031] Figure 11 The figures show the results of the three-chamber socialization test for each group of mice; where (A) is the time spent in the chamber during the socialization test (“Chamber times” in the figure), (B) is the time spent sniffing during the socialization test (“Sniffing” in the figure), (C) is the time spent in the chamber during the socialization test (“Chamber times” in the figure), (D) is the time spent sniffing during the socialization test (“Sniffing” in the figure), (E) is the movement trajectory during the socialization test, and F is the movement trajectory during the socialization test.

[0032] Figure 12Figure 1 shows the results of the Morris water maze test for each group of mice; where (A) is the escape latency during the positioning and cruising phase (“Escape latency” in the figure), (B) is the number of platforms crossed during the spatial exploration phase (“Number of crossing platform” in the figure), (C) is the target quadrant time ratio (“Target quadrant time ratio” in the figure), (D) is the target quadrant path ratio (“Target quadrant path ratio” in the figure), and (E) is the swimming trajectory.

[0033] Figure 13 The results of the rotarod test for each group of mice are shown in the figure; (A) is the fall latency (“Latency of fall” in the figure), and (B) is the actual movement figure.

[0034] Figure 14 Images of H&E staining results of skeletal muscle sections from each group of mice (magnified 100x); green circles indicate areas of muscle fiber necrosis or breakage, black arrows indicate infiltrated cells, and green arrows indicate the distribution of cell nuclei.

[0035] Figure 15 The graph shows the brain coefficient and brain tissue oxidative stress level of mice in each group; where (A) is the brain ("Brain") index (%), (B) is the SOD activity (U / mgprot), (C) is the GSH-Px level (U / mgprot), and (D) is the MDA content (nmol / mgprot).

[0036] Figure 16 Images of H&E staining results of mouse brain tissue sections from each group; the hippocampus (“Hippocampus” in the image) and the CA1, CA3, and DG regions are labeled respectively. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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.

[0038] Example 1 Preparation of salty-enhancing peptide-naringenin complex (NP).

[0039] I. Experimental Materials and Equipment 1. Experimental materials: The freeze-dried powder of turtle egg obtained by mixing egg white and egg yolk and then freeze-drying under vacuum, papain (activity 5000U / g, Shanghai Yuanye), naringenin (Aladdin, ≥97%), ultrapure water, and 75% ethanol were all commercially available analytical grade or food grade.

[0040] 2. Experimental equipment: magnetic stirrer, high-speed refrigerated centrifuge, vacuum freeze dryer, high-throughput vacuum parallel concentrator, Malvern Master Sizer 3000 nanoparticle size analyzer, 0.45μm filter membrane, 4 layers of 200-mesh gauze, 5kDa and 3kDa ultrafiltration plates, electronic balance, constant temperature water bath.

[0041] II. Test Methods 1. Preparation of salty-enhancing peptide: Take the freeze-dried turtle egg powder and add ultrapure water to prepare a 5% freeze-dried turtle egg powder solution; add papain to the solution and enzymatically hydrolyze at 50℃ for 4h; after the enzymatic hydrolysis, centrifuge the hydrolysate at 4℃ and 10000g for 20min, collect the supernatant, filter it through 4 layers of 200-mesh gauze, and then filter it through a 0.45μm filter membrane; then ultrafilter it through 5kDa and 3kDa ultrafiltration plates in sequence, collect the fraction with a molecular weight less than 3kDa, freeze-dry it under vacuum and store it in a -80℃ freezer for later use to obtain salty-enhancing peptide (Pep).

[0042] 2. Preparation of composite solutions: The salty taste-enhancing peptides prepared above were fully dissolved in ultrapure water to prepare salty taste-enhancing peptide solutions with concentrations of 20 mg / mL, 50 mg / mL, 100 mg / mL, 125 mg / mL, and 150 mg / mL, respectively; at the same time, naringenin was fully dissolved in 75% ethanol to prepare a naringenin solution with a concentration of 1 mg / mL.

[0043] 3. Preparation of the complex: Under magnetic stirring, naringenin solution was slowly added dropwise to salty taste-enhancing peptide solutions of different concentrations at a volume ratio of 1:1. The mixture was stirred continuously at room temperature for 2 hours to obtain complex systems with mass ratios of salty taste-enhancing peptide to naringenin of 20:1, 50:1, 100:1, 125:1, and 150:1, respectively. Each complex system was concentrated using a high-throughput vacuum parallel concentrator at 37°C and 170 rpm, and then freeze-dried under vacuum to obtain freeze-dried powders of salty taste-enhancing peptide-naringenin complex (NP) with different proportions.

[0044] 4. Stability testing of composite systems: Before freeze-drying, the particle size, zeta potential and polydispersity index (PDI) of each composite system were determined using a Malvern nanoparticle size analyzer. The refractive index of water was set to 1.33 and the test temperature was 25℃. Each sample was measured in parallel three times and the average value was taken as the test result.

[0045] III. Experimental Results and Analysis Test results as follows Figure 1 As shown, the composite systems with different mass ratios exhibit differences in particle size, zeta potential, and PDI value. When the mass ratio of salty-enhancing peptide to naringenin is 100:1, the composite system has the smallest particle size (452.97 nm), corresponding to a zeta potential of -21.53 mV and a PDI value of 0.49. This parameter combination indicates that the composite system has the best stability at this ratio. As the mass ratio of salty-enhancing peptide to naringenin deviates from 100:1, the particle size of the composite gradually increases, the PDI value increases, the absolute value of the zeta potential decreases, and the system stability declines. Therefore, the preferred mass ratio of salty-enhancing peptide to naringenin is 100:1.

[0046] Example 2 Spectroscopic characterization and verification of the salty-enhancing peptide-naringenin complex (NP).

[0047] I. Experimental Materials and Equipment Experimental materials: lyophilized compound (NP) powders prepared in Example 1 at different mass ratios (20:1, 50:1, 100:1, 125:1, 150:1), lyophilized single salty-enhancing peptide (Pep) powder prepared in Example 1, naringenin (Nar) powder (Aladdin, ≥97%), ultrapure water, and potassium bromide (spectrally pure).

[0048] Experimental equipment: ultraviolet spectrophotometer, fluorescence spectrophotometer, Fourier transform infrared spectrometer, electronic balance, agate mortar and pestle, tablet press.

[0049] II. Test Methods Ultraviolet full-wavelength scanning: Composite systems with different mass ratios were prepared using the method in Example 1. Salty-enhancing peptide (Pep) was dissolved in ultrapure water to prepare a sample solution with a concentration of 100 mg / mL. Ultrapure water was used as a blank control. The absorbance was scanned at a full wavelength of 200-800 nm on an ultraviolet spectrophotometer and the absorbance change curve with wavelength was recorded.

[0050] Fluorescence spectroscopy determination: The above sample solutions were placed in a fluorescence spectrophotometer, the excitation wavelength was set to 260 nm, the emission wavelength scanning range was 260~450 nm, the excitation slit width was 10 nm, and the emission slit width was 5 nm. The fluorescence spectra of each sample were measured at room temperature, and the fluorescence intensity and characteristic peak positions were recorded.

[0051] Fourier transform infrared spectroscopy determination: The preferred ratio (100:1) lyophilized complex (NP), lyophilized salty-enhancing peptide (Pep), and naringenin (Nar) powder selected in Example 1 were mixed with potassium bromide at a mass ratio of 1:150. After thorough grinding in an agate mortar, the mixture was pressed into transparent sheets using a tablet press. The sheets were then placed in a Fourier transform infrared spectrometer and analyzed at 4000~400 cm⁻¹. -1 Scanning was performed within the wavenumber range to record the infrared spectrum and the positions of characteristic peaks.

[0052] III. Experimental Results and Analysis Results of ultraviolet full-wavelength scan: such as Figure 2 As shown, the salty-enhancing peptide (Pep) alone exhibits only a single absorption peak at 275 nm; however, the composite systems with different mass ratios all show a distinct bimodal characteristic at 275 nm and 323 nm, and the intensity of the UV absorption peak gradually changes as the proportion of naringenin decreases. This bimodal characteristic reflects the inherent absorption characteristics of both the salty-enhancing peptide and naringenin, confirming that they interact and form a complex.

[0053] Fluorescence spectroscopy results: such as Figure 3 As shown, the salty flavor-enhancing peptide (Pep) alone exhibits strong fluorescence emission intensity at an excitation wavelength of 260 nm. However, the fluorescence intensity of all composite systems is significantly lower than that of Pep. Furthermore, the fluorescence intensity gradually increases with the decrease of the proportion of naringenin in the composite system, indicating that naringenin quenches the fluorescence of the salty flavor-enhancing peptide, and the degree of quenching is positively correlated with the relative content of naringenin. Simultaneously, the characteristic fluorescence emission peak positions of all composite systems did not shift significantly, indicating that the binding of naringenin to the salty flavor-enhancing peptide does not change the central fluorescence emission wavelength of the salty flavor-enhancing peptide, but only reduces its fluorescence intensity through interaction.

[0054] Fourier transform infrared spectroscopy results: as follows Figure 4 As shown, naringenin (Nar) at 3290 cm⁻¹ -1 Phenolic hydroxyl groups (OH) appear at the location - The characteristic peak of stretching vibration, with the salt-enhancing peptide (Pep) alone at 3294 cm⁻¹. -1 The characteristic peak of the amino (NH) stretching vibration in the peptide chain appears at 3286 cm⁻¹, while the characteristic peak of the complex (NP) shifts to 3286 cm⁻¹. -1 The peak shape is wider at 1647 cm⁻¹, suggesting that hydrogen bonds form between the amino group of the salty-enhancing peptide and the phenolic hydroxyl group of naringenin, and this hydrogen bonding is one of the main forces binding them. Furthermore, the salty-enhancing peptide (Pep) alone shows a peak shape at 1647 cm⁻¹. -1 An amide I band appears at 1600-1700 cm⁻¹ (characteristic peak of peptide secondary structure). -1The peak of the complex (NP) shifted to 1643 cm⁻¹. -1 At this wavenumber range, naringenin (Nar) showed no obvious characteristic peak, indicating that the combination of the two led to a change in the secondary structure of the salty-enhancing peptide, further confirming the successful formation of the complex.

[0055] Example 3 Microstructural observation of the salty-enhancing peptide-naringenin complex (NP).

[0056] I. Experimental Materials and Equipment Experimental materials: the optimal ratio (100:1) complex (NP) lyophilized powder screened in Example 1, the salty taste-enhancing peptide (Pep) lyophilized powder alone, naringenin (Nar) powder (Aladdin, ≥97%), FITC solution (concentration 1 mg / mL), and ultrapure water, all of which were commercially available analytical grade.

[0057] Experimental equipment: laser confocal microscope, electronic balance, centrifuge tubes, glass slides, pipettes.

[0058] II. Test Methods Sample staining: Take 1 mL of NP solution (100:1 mass ratio), 1 mL of Pep solution (100 mg / mL concentration), and 1 mL of Nar solution (1 mg / mL concentration) and place them in three centrifuge tubes respectively. Add 10 μL of FITC solution to each tube, mix gently, and then incubate in a 4°C refrigerator in the dark for 4 hours to allow the fluorescent dye to fully bind to the sample.

[0059] Slide preparation: After appropriate dilution, take 10 μL of the above-stained NP solution, Pep solution, and Nar solution respectively, and add them to a clean glass slide. Let it stand at room temperature in the dark for 10 minutes, and let it air dry naturally before use. Avoid strong light exposure throughout the process.

[0060] Microscopic observation: The prepared slides were placed under a laser confocal microscope, and 488nm and 405nm lasers were used as excitation sources. The samples were observed with a 20x objective lens, and microscopic morphological images at the scales of 5μm, 1μm, and 500nm were acquired. The morphological characteristics and fluorescence distribution of the samples were recorded.

[0061] III. Experimental Results and Analysis Observation results as follows Figure 5As shown, the Pep and Nar groups exhibited dispersed, single morphologies at different scales, without obvious composite structure formation, and the fluorescence signal was uniformly and uniformly distributed. In contrast, the NP group displayed morphological characteristics different from the single component at the 5μm, 1μm, and 500nm scales, forming aggregated structures of varying sizes, with the fluorescence signal concentrated in these aggregated structures, confirming the successful binding of the salty-enhancing peptide and naringenin through interaction. Furthermore, the image scale indicates that the NP particle size is at the nanometer level, consistent with the 452.97nm particle size detected by the Malvern nanoparticle size analyzer in Example 1, directly verifying the successful preparation of the nanoscale complex.

[0062] Example 4 Determination of the antioxidant activity of salty-enhancing peptide-naringenin complex (NP).

[0063] I. Experimental Materials and Equipment Experimental materials: NP lyophilized powder in the optimal ratio (100:1) in Example 1, Pep lyophilized powder prepared in Example 1, reduced glutathione (GSH), DPPH reagent, ABTS reagent, potassium persulfate, anhydrous ethanol, 95% ethanol, and ultrapure water, all of which were commercially available analytical grade.

[0064] Experimental equipment: UV spectrophotometer, vortex mixer, constant temperature incubator, electronic balance, centrifuge tubes, pipettes.

[0065] II. Test Methods Sample solution preparation: Take NP lyophilized powder and dissolve it in ultrapure water to prepare NP solutions of 20 mg / mL and 30 mg / mL respectively; take Pep lyophilized powder and prepare a Pep solution of 30 mg / mL (Pep); take GSH and prepare control solutions of different concentration gradients in ultrapure water.

[0066] DPPH free radical scavenging rate determination: (1) Take 2 mL of sample solutions of various concentrations (NP solution, Pep solution, GSH control solution), add 2 mL of 0.2 mmol / L DPPH ethanol solution, mix well with a vortex mixer, react in the dark at 25℃ for 30 min, and measure the absorbance value A1 at a wavelength of 517 nm; (2) Replace the DPPH ethanol solution with anhydrous ethanol and measure the absorbance value A2 according to the above steps; (3) Replace the sample solution with 2 mL of ultrapure water and measure the absorbance value A3 according to the above steps; (4) Calculate the free radical scavenging rate according to formula I: DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A3] × 100%.

[0067] ABTS +Determination of free radical scavenging rate: (1) Prepare 7.0 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution, mix them at a volume ratio of 1:1, react in the dark for 12-16 h, and then dilute with 95% ethanol until the absorbance at 734 nm is 0.7 ± 0.05 to obtain ABTS. + (2) Take 50 μL of each concentration sample solution (NP solution, Pep solution, GSH control solution), add 3 mL of ABTS + After vortexing the reaction solution, react it in the dark for 6 minutes. Using 95% ethanol as a blank control, measure the absorbance value A at 734 nm. t (3) Take 50 μL of ultrapure water and 3 mL of ABTS + Mix the reaction solution and measure the absorbance value A0 according to the above steps; (4) Calculate the free radical scavenging rate according to formula II: ABTS + Free radical scavenging rate (%) = (A0 - A) t ) / A0×100%.

[0068] Parallel experiments: Three parallel experiments were set up for each sample and control, and the average value was taken as the final measurement result.

[0069] III. Experimental Results and Analysis The test results are as follows Figure 6 As shown, the NP complex exhibits excellent antioxidant activity, particularly against DPPH and ABTS. + The free radical scavenging rate reached over 80%, and the antioxidant effect was superior to that of the salty taste-enhancing peptide alone. Specifically, the DPPH and ABTS of a 30 mg / mL Pep solution alone were significantly better. + The scavenging rate was close to that of high-concentration GSH, while the DPPH scavenging rate of 20 mg / mL NP solution reached the level of equivalent concentrations of Pep and high-concentration GSH; when the NP concentration was 30 mg / mL, its ABTS... + The scavenging rate is close to that of high-concentration GSH. These results indicate that the antioxidant activity is further enhanced after Pep is combined with Nar, a characteristic that provides a foundation for the subsequent application of NP.

[0070] Example 5 Detection of the saltiness-enhancing effect of saltiness-enhancing peptide-naringenin complex (NP).

[0071] I. Experimental Materials and Equipment Experimental materials: NP lyophilized powder in the optimal ratio (100:1) of Example 1, sodium chloride (analytical grade), tartaric acid (analytical grade), potassium chloride (analytical grade), and ultrapure water, all of which were commercially available analytical grade.

[0072] Experimental equipment: electronic tongue (taste sensing system SA402B, Beijing Yingsheng Hengtai Technology Co., Ltd.), electronic balance, volumetric flask, pipette, beaker. All containers used in the experiment were cleaned.

[0073] II. Test Methods Solution preparation: (1) Preparation of reference solution: Weigh 0.3 mmol / L tartaric acid and 30 mmol / L potassium chloride, dissolve them in ultrapure water and bring the volume to 1L, shake well and set aside; (2) Preparation of control solution: Weigh an appropriate amount of sodium chloride, dissolve it in ultrapure water to prepare a 0.05 mol / L NaCl solution as a salty control; (3) Preparation of sample solution: Take NP lyophilized powder, add it to 0.05 mol / L NaCl solution, mix well and prepare a series of sample solutions with NP concentrations of 0.1%, 0.3%, 0.5%, 0.7% and 0.9% (w / v).

[0074] Electronic tongue detection: (1) The electronic tongue is calibrated according to the operating procedure. The reference solution is used as the standard. The collection time is set to 120s and the sample detection time is 30s. Each sample is tested 4 times. (2) The control solution and sample solutions of each concentration are tested in sequence. After removing the unstable data from the first test, the average value of the last three test data is taken as the electronic tongue analysis result. The focus is on analyzing the change in the saltiness value of the sample.

[0075] III. Experimental Results and Analysis Test results as follows Figure 7 As shown, compared with the 0.05 mol / L NaCl control solution, the saltiness values ​​of sample solutions with different concentrations of NP were significantly increased, with the enhancement ranging from 27.01% to 159.12%. Specifically, the 0.1% NP sample solution showed a 27.01% increase in saltiness, and as the NP concentration gradually increased to 0.9%, the increase in saltiness continued to rise to 159.12%, exhibiting a clear dose-dependent effect. Specific taste value data from electronic tongue detection showed that NP only specifically enhances the perception of saltiness, without significantly interfering with other tastes such as sourness, bitterness, and astringency, thus retaining the advantage of reducing salt intake without reducing saltiness. These results confirm that NP retains the synergistic effect of saltiness-enhancing peptides derived from turtle eggs, maintaining the salty experience of food while reducing salt consumption, thus meeting the needs of a healthy diet.

[0076] Example 6 The open field test verified the effect of NP on improving spontaneous activity and anti-anxiety ability in aging diabetic mice.

[0077] I. Experimental Materials and Equipment Experimental animals: 8-week-old male C57BL / 6 mice, purchased from Zhuhai Bestong Biotechnology Co., Ltd., weighing 18.0 - 20.0 g (License number: SCXK (Guangdong) 2020 - 0051), Ethical review number: GOU-LAE-2025-013. The mice were adaptively fed for 1 week under the conditions of a breeding temperature of 22 ± 2°C, a humidity of 50 ± 10%, and a 12h light / dark cycle before being used in the experiment.

[0078] Experimental reagents: The NP freeze-dried powder prepared in Example 1, metformin (analytical pure), and normal saline, all of which are commercially available qualified products.

[0079] Experimental equipment: A white cube open-field box (length × width × height = 50 cm × 50 cm × 50 cm, with an open upper end), a high-definition camera, Shanghai Xinruan behavior analysis software, and a 75% alcohol sprayer.

[0080] II. Experimental methods Animal modeling: The mice that had been adaptively fed for 1 week were randomly divided into 5 groups, with 12 mice in each group. One group served as the blank control group (CON), and the remaining 4 groups were the aging model groups (Model): Aging model group (Model): Intraperitoneally injected with 500 mg / kg bw D-galactose every morning at 9:00 for 9 consecutive weeks; in the 6th week of modeling, after the mice were fasted but allowed water for 12 h, they were intraperitoneally injected with 70 mg / kg bw streptozotocin (STZ) for 5 consecutive days; STZ was freshly prepared with 0.1 M citrate buffer (pH 4.5), and the whole operation was carried out under light avoidance. 20% glucose aqueous solution was given 4 h after injection to avoid death due to hypoglycemia.

[0081] Blank control group (CON): Intraperitoneally injected with an equal volume of normal saline and sterile citrate buffer.

[0082] 5 days after injecting STZ, the mice were fasted but allowed water for 12 h, and their fasting blood glucose (FBG) was measured. Mice with FBG > 11.1 mmol / L were determined to be aging diabetic mice. Mice that did not meet the standard were supplemented with an injection of STZ until their blood glucose reached the standard. 4 mice that did not meet the standard and 2 mice that died accidentally during the modeling period were excluded. Finally, 54 aging diabetic mice were obtained. During the feeding period, the young and healthy control group was fed a basal maintenance diet, and the aging diabetic model group was fed a high-fat diet. The mice were allowed to eat and drink freely.

[0083] Animal grouping and intervention: The 54 aging diabetic mice were randomly divided into 4 groups, and together with the young and healthy control group, a total of 5 groups were set up. The number of mice in each group and the intervention methods are as follows: Young and healthy control group (CON): 12 mice, gavaged with an equal volume of normal saline for 8 consecutive weeks; The aging diabetes model group (Model): 11 animals were administered an equal volume of physiological saline by gavage for 8 weeks. Metformin-positive drug group (MET): 10 animals were administered metformin by gavage at a dose of 150 mg / kg. bw, lasting 8 weeks; Low-dose NP group (NPL): 10 animals were administered NP by gavage at a dose of 0.5 g / kg. bw, lasting 8 weeks; High-dose NP group (NPH): 11 animals were administered NP by gavage at a dose of 1 g / kg. bw, lasting 8 weeks.

[0084] The gavage volume for each group was 0.1 mL / 10 g. After 8 weeks of NP intervention, the open field trial was conducted in week 16.

[0085] Open field test procedure: (1) Before the test, all mice were transferred to the test site and allowed to adapt to the environment for 2 hours; (2) The bottom of the open field box was divided into 9 grids, with the middle grid defined as the central area and the surrounding 8 grids as the surrounding area; (3) The inner wall and bottom of the box were sprayed with 75% alcohol, and after drying to remove residual odor interference, the mice were gently placed in the center of the box. At the same time, the camera and behavioral analysis software were activated to record the movement of the mice within 5 minutes; (4) After each mouse was tested, the excrement in the box was cleaned immediately, and the box was wiped with 75% alcohol again. After drying, the next mouse was tested. The environment was kept quiet throughout the test.

[0086] Data collection: The total distance, average speed, number of rears, number of central zone entries, and time spent in the central zone of mice were analyzed using Shanghai Xinruan software. All data were the average of three parallel tests.

[0087] III. Experimental Results and Analysis The test results are as follows Figure 8 As shown, compared with the young healthy control group (CON), the total movement distance of the aging diabetic model mice (Model) was significantly greater. Figure 8 A) Average speed of motion ( Figure 8 (B) Number of hind limbs standing upright ( Figure 8 The number of times C in the middle region decreased significantly, and the number of times it entered the central region ( Figure 8 D in the text) and the length of stay ( Figure 8The significant reduction in E in the mice indicates that aging-related diabetes leads to decreased autonomous activity, reduced exploratory drive, and increased anxiety levels in mice.

[0088] Compared with the aging diabetic model group (Model), the above indicators were significantly improved after NP intervention: the total movement distance of mice in the NPL group reached 29.02m, the average movement speed reached 9.68cm / s, the number of hindlimb upright positions reached 25.67, the number of times they entered the central area reached 13.83, and the activity time reached 16.27s. All indicators were significantly higher than those in the Model group (p<0.001). The NPH group also showed significant improvement, with some indicators approaching the level of the CON group. The mouse movement trajectory diagram (F) visually showed that NP can effectively improve the motor ability, exploration ability, and anti-anxiety ability of aging diabetic mice. These results confirm that NP can effectively improve the autonomous activity and exploration ability of aging diabetic mice, while reducing their anxiety behavior, providing behavioral support for subsequent improvement of cognitive decline.

[0089] Example 7 The elevated cross maze test verified the effect of NP on improving anxiety in aging diabetic mice.

[0090] I. Experimental Materials and Equipment Experimental animals: Same as in Example 6, including young healthy control group (CON, n=12), aging diabetes model group (Model, n=11), metformin positive drug group (MET, n=10), low-dose NP group (NPL, n=10), and high-dose NP group (NPH, n=11). All mice were modeled and treated for 9 weeks.

[0091] Test reagents: The NP lyophilized powder, metformin (analytical grade), and physiological saline prepared in Example 1 were all commercially available qualified products.

[0092] Experimental equipment: elevated cross maze (consisting of two open arms and a closed arm, each 30cm long and 5cm wide, with a total platform height of 74cm), high-definition camera, Shanghai Xinruan behavioral analysis software, and 75% alcohol sprayer.

[0093] II. Test Methods Preparation before the experiment: 30 minutes before the experiment, all mice were transferred to the experimental room to acclimatize to the environment and reduce stress response; each arm of the maze and the central platform were wiped with 75% alcohol and dried to remove residual odor interference.

[0094] Experimental procedure: The mouse was placed with its back to the experimenter and its head facing the open arm, and gently placed on the central platform area. At the same time, the camera and behavioral analysis software were activated to record the mouse's activities over 10 minutes. During the experiment, the experimenter should observe from at least 1 meter away from the center of the maze to avoid disturbing the mouse.

[0095] Sample preparation: After each mouse test, immediately clean up the excrement inside and outside the maze, and wipe each arm and platform thoroughly with 75% alcohol again. After it is completely dry, proceed to test the next mouse. Keep the experimental environment quiet and undisturbed throughout the process.

[0096] Data collection: The total distance traveled, total arm entries, open arm entries, and time spent in open arms were extracted using Shanghai Xinruan Behavioral Analysis Software. The percentage of open arm times and the percentage of open arm entries were calculated. All data were the average of three parallel tests.

[0097] III. Experimental Results and Analysis The test results are as follows Figure 9 As shown, compared with the young healthy control group (CON), the total distance traveled by mice in the aging diabetes model group (Model) was significantly greater. Figure 9 A) Total number of times the arm enters ( Figure 9 B) Number of times the open arm enters ( Figure 9 (C) and open arm dwell time ( Figure 9 The levels of D in the mice were significantly reduced, indicating that aging-related diabetes led to a decline in the mice's motor abilities, and that they avoided open-arm environments due to anxiety.

[0098] After NP intervention, anxiety-related indicators in mice were significantly improved: the NPH group showed the most significant effect, with a total movement distance of 20.09 m, a total number of arm entry attempts of 29.5, an open arm entry attempt of 7.83, and an open arm dwell time of 303.46 s, all of which were significantly higher than those in the Model group (p<0.05). Furthermore, compared with the Model group, the percentage of open arm dwell time in the NPH group was significantly prolonged by 115.91% (…). Figure 9 In E), the percentage of open arm entry times increased significantly by 96.79% ( Figure 9 (F in the text); the movement trajectories of the mice in each group are as follows: Figure 9 As shown in G in the figure. In the elevated cross maze test, the more times mice entered the open arms and the longer they stayed, the lower their anxiety level. This result confirms that NP can effectively improve the anxiety state caused by the disease in aging diabetic mice, and the improvement effect is more prominent in the high-dose group.

[0099] Example 8 A novel object recognition experiment validated the effect of NP on improving cognitive and memory abilities in aging diabetic mice.

[0100] I. Experimental Materials and Equipment Experimental animals: Same as in Example 6, including young healthy control group (CON, n=12), aging diabetes model group (Model, n=11), metformin positive drug group (MET, n=10), low-dose NP group (NPL, n=10), and high-dose NP group (NPH, n=11). All mice were modeled and treated for 9 weeks.

[0101] Test reagents: The NP lyophilized powder, metformin (analytical grade), and physiological saline prepared in Example 1 were all commercially available qualified products.

[0102] Experimental equipment: white cube test box (length × width × height = 40cm × 40cm × 40cm), two old triangular prism objects (A1, A2) that are identical in size, shape and color, a new object (B) that is the same size as the old objects but different in shape, high-definition camera, Shanghai Xinruan behavioral analysis software, 75% alcohol sprayer.

[0103] II. Test Methods The experiment was conducted over three days, with the experimental environment kept quiet and the light uniform throughout: (1) Adaptation period (Day 1): The test box was empty of any objects. The mouse was placed in the box and allowed to explore freely for 5 minutes to adapt to the environment. After the experiment, the inside of the box was wiped with 75% alcohol and dried to remove odor interference. (2) Familiarization period (Day 2): Two old objects, A1 and A2, were placed at a fixed distance on the diagonal of the box. The mouse faced the box wall and was placed in the box from the perpendicular bisector of the two objects. It was allowed to explore the old objects freely and become familiar with them for 5 minutes. The box was cleaned after the experiment using the same method. (3) New object recognition period (Day 3): The old object A1 was replaced with the new object B. The position of A2 remained unchanged. The mouse was placed in the box in the same way as during the familiarization period. The exploration time of the mouse for the new object B and the old object A2 within 5 minutes was recorded (exploration was defined as the mouse's nose approaching the object ≤2cm or touching the object with its paw).

[0104] Data collection and calculation: The exploration time of mice for new and old objects was extracted using Shanghai Xinruan Behavioral Analysis Software, and the recognition coefficient was calculated according to the formula: Recognition coefficient = (exploration time of new object - exploration time of old object) / (exploration time of new object + exploration time of old object); The test chamber was cleaned immediately after each mouse was tested, and all data were the average of 3 parallel tests.

[0105] III. Experimental Results and Analysis The test results are as follows Figure 10As shown, the discrimination index of young healthy control group (CON) mice was significantly higher than that of other groups, indicating that they had good cognitive memory ability. Compared with the CON group, the discrimination index of aging diabetic model group (Model) mice was significantly lower (p<0.01), and their willingness to explore new objects was significantly weakened, suggesting that aging-related diabetes leads to a significant decline in cognitive memory ability in mice. After NP intervention, the discrimination index of mice in NPL and NPH groups was significantly increased (p<0.05), and the exploration time of new objects was significantly prolonged, with the discrimination index of NPH group approaching that of CON group. The movement trajectories of mice in each group are shown in the figure. Figure 10 As shown in B in the figure. The higher the recognition coefficient, the clearer the mouse's memory of familiar objects and the stronger its ability to distinguish new objects. This result confirms that NP can effectively improve cognitive memory impairment in aging diabetic mice and enhance their ability to recognize new objects.

[0106] Example 9 Three-box social experiment verified the effect of NP on enhancing social preference and social memory in aging diabetic mice.

[0107] I. Experimental Materials and Equipment Experimental animals: Same as in Example 6, including young healthy control group (CON, n=12), aging diabetes model group (Model, n=11), metformin positive drug group (MET, n=10), low-dose NP group (NPL, n=10), and high-dose NP group (NPH, n=11). All mice were stable after 9 weeks of intervention. In addition, healthy male C57BL / 6 mice were used as stranger mice (M1, M2), with similar weight to the experimental group mice and no prior contact with the experimental group mice.

[0108] Test reagents: The NP lyophilized powder, metformin (analytical grade), and physiological saline prepared in Example 1 were all commercially available qualified products.

[0109] Experimental equipment: a transparent three-box social interaction box (length × width × height = 600mm × 400mm × 220mm), with partition walls inside the box (with a door for free entry and exit), dividing it into three spaces: left, middle, and right; two wire cages of the same size, shape, and color; a high-definition camera; Shanghai Xinruan behavioral analysis software; and a 75% alcohol sprayer.

[0110] II. Test Methods Preparation before the experiment: 30 minutes before the experiment, the mice in the experimental group were transferred to the experimental room to adapt to the environment and reduce stress; the inner wall of the social box and the wire cage were wiped with 75% alcohol and dried to remove residual odor interference.

[0111] The experiment was conducted in three phases, with the environment kept quiet and the light soft throughout: (1) Adaptation period: The mice were placed in the middle box facing the experimenters, the door of the partition wall was opened, and the mice were allowed to explore freely in the three boxes for 10 minutes. The activity status during the adaptation period was recorded. The social box was cleaned after the experiment. (2) Social test phase: The unfamiliar mouse M1 was placed in the wire cage in the left box, the wire cage in the right box was empty (O), and there was no object in the middle box. The experimental group mice were placed in the middle box, the door was opened, and the time the mice stayed in the left, middle, and right boxes within 10 minutes was recorded, as well as the sniffing time of the wire cage where M1 was located (the sniffing time was defined as the effective contact time when the mouse approached the wire cage less than 2 cm). (3) Social memory test phase: The empty cage in the right box was removed, and a new unfamiliar mouse M2 (unrelated to M1) was placed in it. The left box was still M1. The experimental group mice were placed in the same way as above, and the time the mice stayed in the left and right boxes within 10 minutes, as well as the sniffing time of M1 and M2, was recorded.

[0112] Data collection: Chambertimes and sniffing time of mice at each stage were extracted using Shanghai Xinruan Behavioral Analysis Software. All data were the average of three parallel tests. After each mouse was tested, the social box and wire cage were cleaned immediately to avoid odor residue affecting the next test.

[0113] III. Experimental Results and Analysis The test results are as follows Figure 11 As shown, during the social testing phase, except for the NPH group, mice in other groups did not show a significant preference for the unfamiliar mouse M1. The NPH group mice spent 281.05 seconds in the box containing M1. Figure 11 (A) The sniffing time reached 397.14s. Figure 11 The B in the model was significantly longer than the 192.09s and 203.98s in the empty cage (p<0.05), indicating that NPH can significantly improve the social preference of aging diabetic mice.

[0114] In the social memory test phase, such as Figure 11As shown in C and D, the aging diabetic model group (Model) and the metformin group (MET) mice showed no significant differences in dwell time and sniffing time with the unfamiliar mouse M2 and the familiar mouse M1, suggesting impaired social memory. However, the CON, NPL, and NPH groups showed significantly longer dwell times in the box containing M2 (324.0s, 312.45s, and 354.36s, respectively) and sniffing times (351.78s, 333.54s, and 351.54s, respectively) and sniffing times (191.08s, 165.44s, and 144.77s, respectively, p<0.05) with the box containing the familiar mouse M1. These results confirm that NP can effectively enhance the social initiative of aging diabetic mice and improve their social memory, helping them distinguish between familiar and unfamiliar individuals.

[0115] Example 10 Morris water maze test verified the effect of NP on enhancing spatial learning and memory in aging diabetic mice.

[0116] I. Experimental Materials and Equipment Experimental animals: Same as in Example 6, including young healthy control group (CON, n=12), aging diabetes model group (Model, n=11), metformin positive drug group (MET, n=10), low-dose NP group (NPL, n=10), and high-dose NP group (NPH, n=11). All mice were stable after 9 weeks of intervention and showed no abnormal behavior.

[0117] Test reagents: The NP lyophilized powder, metformin (analytical grade), physiological saline, and titanium dioxide (analytical grade) prepared in Example 1 were all commercially available qualified products.

[0118] Experimental equipment: Morris water maze (circular pool, 1200cm in diameter and 50cm in height; escape platform, 10cm in diameter), high-definition camera, Shanghai Xinruan behavioral analysis software, towel.

[0119] II. Test Methods Preparation before the experiment: Add ultrapure water to the pool and add titanium dioxide to make the water white (to facilitate the observation of the black mouse trajectory). Adjust the water temperature to 23±2℃. Divide the pool into four quadrants. Fix the escape platform in the second quadrant, with the upper surface of the platform 1cm above the water surface. Keep the environment quiet throughout the experiment and avoid external interference such as light and sound.

[0120] The experiment was conducted in two phases: (1) Positioning and cruising phase (first 4 days): Each mouse was placed on the platform for 15 seconds before the experiment. Then, it was placed in the water from the central placement point of each of the four quadrants, facing the pool wall. At the same time, the camera and analysis software were activated to record the time it took for the mouse to find the platform within 60 seconds, which is the escape latency. If the mouse did not find the platform within 60 seconds, it was guided to climb onto the platform and stay for 15 seconds. The escape latency was recorded as 60 seconds. After completing one set of tests each day, the mouse was dried with a towel and put back into the cage. The same method was used to complete the tests for 4 days. (2) Spatial exploration phase (day 5): The platform was removed, and the mouse was placed in the water from the central placement point of the fourth quadrant (opposite to the quadrant where the platform is located), facing the pool wall. The swimming trajectory of the mouse, the number of times it crossed the original platform position, the target quadrant time ratio, and the target quadrant pathratio were recorded within 60 seconds.

[0121] Data collection: Experimental data for each stage were extracted using Shanghai Xinruan Behavioral Analysis Software. All data were the average of three parallel tests, and the differences between groups were statistically analyzed.

[0122] III. Experimental Results and Analysis The test results are as follows Figure 12 As shown, during the navigation phase, there was no significant difference in escape latency among the groups of mice on day 1. From day 2 onwards, the escape latency of the aging diabetic model group (Model) mice was significantly longer than that of the other groups, indicating that their spatial learning ability was significantly impaired. From day 4 onwards, the escape latency of mice in both the MET and NP groups was significantly shortened, with the NPH group having the shortest escape latency, even better than the young healthy control group (CON). Compared with the Model group, the escape latency of the NP group was shortened by 30.98%–51.62%. Figure 12 (A in the middle).

[0123] like Figure 12 As shown in Figures B-D, during the spatial exploration phase, the Model group mice traversed the platform an average of only 0.33 times, with a target quadrant dwell time ratio of 7.80% and a distance traveled of 8.60%, both significantly lower than the CON group (p<0.001). After NP intervention, the mice's spatial memory ability was significantly improved. The NPL group traversed the platform an average of 1.83 times, with a target quadrant dwell time ratio of 21.82% and a distance traveled of 23.42%. The NPH group further improved these indicators to 3.17 times, 34.31%, and 34.21% (p<0.001), and some indicators of the NPH group were better than those of the CON group. Figure 12E in the figure represents the swimming trajectory of each group of mice in the space exploration experiment. It can be seen that the Model group mice could hardly find the target platform, while the NP group mice were able to cross the platform location more often and had more swimming trajectories in the quadrant where the platform was located.

[0124] The results confirm that NP can effectively improve the spatial learning and memory abilities of aging diabetic mice, helping them to quickly establish spatial cognition and accurately locate target positions.

[0125] Example 11 Rotary bar test verified the effect of NP on improving motor coordination and fatigue resistance in aging diabetic mice.

[0126] I. Experimental Materials and Equipment Experimental animals: Same as in Example 6, including young healthy control group (CON, n=12), aging diabetes model group (Model, n=11), metformin positive drug group (MET, n=10), low-dose NP group (NPL, n=10), and high-dose NP group (NPH, n=11). All mice were stable after 9 weeks of intervention.

[0127] Test reagents: The lyophilized powder of the NP complex prepared in Example 1, metformin (analytical grade), and physiological saline were all commercially available qualified products.

[0128] Experimental equipment: Rotary bar fatigue tester (model YLS-4C), high-definition camera, Shanghai Xinruan behavioral analysis software.

[0129] II. Test Methods Pre-experiment training: All mice underwent two days of acclimatization training before the formal experiment, with one training session per day, each lasting 10 minutes. After the rotundus was started, it was accelerated at a constant speed of 2 seconds per revolution, eventually stabilizing at 30 rpm, to help the mice overcome fear and become familiar with the rotundus movement pattern.

[0130] Formal experiment: On the 3rd day, the mice were placed on the rotundus of the rotundus fatigue tester. After the instrument was started, it was accelerated at a constant speed of 2 seconds per rotation to 30 r / min. The formal experiment duration was set to 10 minutes. At the same time, the camera and analysis software were started to record the time from the start of the rotundus movement to the fall of each mouse, which was recorded as the fall latency.

[0131] Repeated testing: Each mouse was tested 3 times, with a 60-minute interval between each test. During this period, the mice were placed in a quiet environment to rest. After each round of testing, the surface of the rotatine rod was cleaned of any remaining hair and excrement to avoid interfering with the results of subsequent tests.

[0132] Data collection: The dwell time for each test was extracted using Shanghai Xinruan Behavioral Analysis Software. The average dwell time for each mouse across three tests was calculated as the final statistical data.

[0133] III. Experimental Results and Analysis The test results are as follows Figure 13 As shown, the fall latency of young healthy control group (CON) mice on the rotundus was 9.07 min, while that of aging diabetic model group (Model) mice was only 1.92 min, with a highly significant difference (p<0.001). This indicates that aging-related diabetes severely impairs the motor coordination and muscle function of mice, leading to a significant decrease in their fatigue tolerance. After NP intervention, the motor function of mice was significantly improved: the fall latency of NPL group mice reached 8.47 min, and that of NPH group mice reached 10 min, both of which were significantly higher than those of Model group and MET group (p<0.001), and the fall latency of NPH group was slightly higher than that of CON group (…). Figure 13 (A in the middle). Figure 13 B in the figure represents the actual movement of the rotarod test. In the rotarod test, the longer the fall latency of the mice, the better their motor coordination, muscle endurance and fatigue resistance. This result confirms that NP can effectively protect the motor ability of aging diabetic mice from damage, alleviate muscle function decline, and restore their motor coordination and fatigue resistance to the level of young healthy mice.

[0134] Example 12 HE staining of skeletal muscle verified the alleviating effect of NP on muscle fiber atrophy in aging diabetic mice.

[0135] I. Experimental Materials and Equipment Experimental animals: Same as in Example 6, including young healthy control group (CON, n=12), aging diabetes model group (Model, n=11), metformin positive drug group (MET, n=10), low-dose NP group (NPL, n=10), and high-dose NP group (NPH, n=11). All mice were euthanized after 9 weeks of intervention, and skeletal muscle tissue was collected for later use.

[0136] Test reagents: 4% paraformaldehyde solution, graded concentrations of alcohol (70%, 80%, 90%, 95%, 100%), paraffin, hematoxylin staining solution, eosin staining solution, and xylene, all of which were commercially available analytical grade.

[0137] Experimental equipment: pathological sectioner, paraffin embedding machine, inverted microscope, constant temperature oven, dehydrator, staining tank, glass slides, coverslips, forceps, and blades.

[0138] II. Test Methods Tissue processing: Skeletal muscle tissue from the ipsilateral hind limb of a mouse was taken and quickly fixed in 4% paraformaldehyde solution for no less than 24 hours; then the tissue was sequentially placed in alcohol of varying concentrations for dehydration, with each concentration gradient soaking for 1 hour; after dehydration, the tissue was immersed in paraffin solution and placed in an embedding machine for paraffin embedding to prepare paraffin blocks.

[0139] Sectioning and staining: Using a pathological microtome, continuous sections with a thickness of 5-8 μm were cut from the paraffin block. The sections were then attached to clean glass slides and baked in a constant temperature oven at 60°C for 2 hours to ensure firm adhesion. The standard HE staining procedure was followed: after dewaxing with xylene and rehydration with a gradient of alcohols, the sections were stained with hematoxylin for 5 minutes, rinsed with running water to remove excess stain, stained with eosin for 3 minutes, and finally dehydrated with a gradient of alcohols and cleared with xylene.

[0140] Observation and Imaging: After staining, the sections were dried and observed under an inverted microscope at 100x magnification. The focus was on observing the tightness of muscle bundle arrangement, the integrity of muscle fiber morphology, cell infiltration, and the distribution of cell nuclei. Typical fields of view were selected for photographing and recording.

[0141] III. Experimental Results and Analysis Observation results as follows Figure 14 As shown, HE-stained sections of skeletal muscle from young, healthy control (CON) mice revealed tightly packed muscle bundles, uniform and regular muscle fiber morphology, clear cell boundaries, and evenly distributed nuclei, with no obvious cell infiltration or muscle fiber damage. In contrast, skeletal muscle from aging diabetic model (Model) mice exhibited significant pathological damage: significantly enlarged interfascicular spaces and a loose structure; muscle fibers showed atrophy, irregular morphology, and disordered arrangement; some muscle fibers underwent focal necrosis, breakage, thinning, and dissolution; numerous deeply stained cells infiltrated and fibrotic were observed; and the distribution of nuclei was uneven and their number significantly reduced. In the metformin-positive drug (MET) group, the interfascicular spaces in skeletal muscle remained relatively large, with only a slight reduction in inflammatory infiltration and limited improvement in muscle fiber damage. In the low-dose NP group (NPL), muscle fiber arrangement was significantly more regular than in the Model group, with a morphology similar to the CON group; muscle bundles were tightly packed, with no obvious cell infiltration and evenly distributed nuclei. In the high-dose NP group (NPH), muscle fibers were fuller, the interstitial space was more compact, and the density of the muscle bundle structure was superior to the NPL group, with almost complete remission of pathological damage. The results confirm that NP can effectively alleviate myofibrous atrophy caused by age-related diabetes by improving insulin resistance and promoting myoprotein synthesis. Moreover, this improvement effect is dose-dependent, which is consistent with the results of the NPH group mice having the best motor ability in the rotarod test.

[0142] Example 13 The effects of NP on reducing antioxidant and lipid oxidative damage were verified by detecting brain index and brain tissue oxidative stress indicators.

[0143] I. Experimental Materials and Equipment Experimental animals: Same as in Example 6, including young healthy control group (CON, n=12), aging diabetes model group (Model, n=11), metformin positive drug group (MET, n=10), low-dose NP group (NPL, n=10), and high-dose NP group (NPH, n=11). After 9 weeks of intervention, all mice were euthanized, and their brain tissue was quickly separated, weighed, and frozen at -80°C for later use.

[0144] Test reagents: physiological saline, SOD detection kit (Nanjing Jiancheng, catalog number A001-3), GSH-Px detection kit (Nanjing Jiancheng, catalog number A005-1), and MDA detection kit (Nanjing Jiancheng, catalog number A003-1), all of which are commercially available qualified products.

[0145] Experimental equipment: electronic balance, tissue homogenizer, high-speed refrigerated centrifuge, enzyme-linked immunosorbent assay (ELISA) reader. All containers involved in the experiment were pretreated at low temperature.

[0146] II. Test Methods Brain index calculation: Immediately after sacrifice, weigh the mouse body and the weight of intact brain tissue, and calculate the brain index according to the formula: Brain index (%) = (brain tissue weight / mouse body weight) × 100%.

[0147] Preparation of brain tissue homogenate: Weigh 0.1g of frozen brain tissue and add pre-cooled physiological saline at a ratio of weight (g):volume (mL) = 1:9. Grind the homogenate for 1 min at 60 Hz using a tissue homogenizer under ice bath conditions to prepare a 10% brain tissue homogenate. Centrifuge the homogenate at 3500 rpm for 10 min and collect the supernatant. Maintain low temperature operation throughout the process to avoid loss of enzyme activity.

[0148] Indicator detection: Strictly follow the instructions of each kit to measure the SOD activity, GSH-Px activity and MDA content in the supernatant of brain tissue homogenate. Set up 3 sets of parallel replicates for each sample, read the corresponding absorbance value with a microplate reader, and calculate the actual concentration or activity value according to the standard curve of the kit.

[0149] III. Experimental Results and Analysis The test results are as follows Figure 15 As shown, compared with the young healthy control group (CON), the brain index of the aging diabetic model group (Model) mice was significantly increased (p<0.001), suggesting that the dual effects of aging and diabetes induce pathological changes in the brain, leading to an increase in relative brain weight. Figure 15 (A in the middle).

[0150] like Figure 15As shown in Figures B-D, the SOD activity (73.12 U / mgprot) and GSH-Px activity (2752.19 U / mgprot) in the brain tissue of the Model group mice were significantly decreased, while the MDA content was significantly increased by 26.10%, indicating that the antioxidant capacity of their brain tissue was weakened and lipid oxidation damage was severe. After NP intervention, the brain indices of mice in both the MET group and the NP group were significantly reduced (p<0.001), and approached the level of the CON group, indicating that NP can effectively improve the pathological state of brain tissue. Regarding antioxidant indicators, the SOD activity in the NPH group mice reached 124.16 U / mgprot, significantly higher than that in the Model group (p<0.01); the GSH-Px activities in the NPL and NPH groups were 3796.96 U / mgprot and 3975.97 U / mgprot, respectively, which were extremely significantly higher than those in the Model group (p<0.01); at the same time, the MDA content in the NPL and NPH groups decreased by 26.74% and 33.40%, respectively (p<0.01), both returning to the levels of the CON group. SOD and GSH-Px are core antioxidant enzymes in brain tissue, and their activities reflect the ability to scavenge oxygen free radicals. MDA is a lipid oxidation product, and its content represents the degree of oxidative damage. These results confirm that NP can reduce the production of oxidative stress products by increasing the activity of antioxidant enzymes in brain tissue, thereby alleviating lipid oxidation damage in brain tissue caused by aging and diabetes and maintaining the healthy state of brain tissue.

[0151] Example 14 HE staining of brain tissue verified the protective effect of NP on nerve cells in aging diabetic mice.

[0152] I. Experimental Materials and Equipment Experimental animals: Same as in Example 6, including young healthy control group (CON, n=12), aging diabetes model group (Model, n=11), metformin positive drug group (MET, n=10), low-dose NP group (NPL, n=10), and high-dose NP group (NPH, n=11). After 9 weeks of intervention, all mice were euthanized, and their intact brain tissue was rapidly separated and fixed in 4% paraformaldehyde solution for later use.

[0153] Test reagents: 4% paraformaldehyde solution, graded concentrations of alcohol (70%, 80%, 90%, 95%, 100%), paraffin, hematoxylin staining solution, eosin staining solution, xylene, and phosphate buffered saline (PBS), all of which were commercially available analytical grade.

[0154] Experimental equipment: pathological sectioner, paraffin embedding machine, optical microscope, constant temperature oven, dehydrator, staining tank, glass slides, coverslips, forceps, and blades.

[0155] II. Test Methods Tissue fixation and embedding: Brain tissue was fixed in 4% paraformaldehyde solution for 48 h and then rinsed 3 times with PBS to remove residual fixative. Subsequently, it was dehydrated in alcohol of varying concentrations, soaking for 1 h at each concentration. After dehydration, it was cleared twice with xylene (30 min each time) and then immersed in melted paraffin (held in a 60℃ oven for 3 h). Finally, brain tissue was embedded in paraffin to form paraffin blocks.

[0156] Sectioning and staining: Using a pathological microtome, continuous sections with a thickness of 5 μm were cut from the paraffin block along the coronal plane. Sections containing the complete hippocampus were selected and attached to poly-L-lysine-treated slides. The slides were baked in a constant temperature oven at 60°C for 2 hours to ensure firm adhesion. The standard HE staining procedure was followed: see Example 12 for the staining procedure.

[0157] Observation and Imaging: The mounted slides were placed under an optical microscope and observed at 100x magnification, focusing on the three subregions of the hippocampus: CA1, CA3, and DG. The arrangement density, morphological regularity, cell body integrity, and nucleus clarity of neurons were recorded. Typical fields of view of each subregion were selected for photographing and archiving.

[0158] III. Experimental Results and Analysis Observation results as follows Figure 16 As shown, neurons in the CA1, CA3, and DG subregions of the hippocampus in the brain tissue of young healthy control (CON) mice were tightly and neatly arranged, with plump, round or oval cell bodies, regular morphology, and uniformly stained nuclei located in the center of the cell body. There was no neuronal loss, degeneration, or necrosis, and the nerve fibers were arranged in an orderly manner. In contrast, the hippocampal subregions of the aging diabetic model (Model) mice showed obvious neurodegenerative changes: neurons were arranged in a disordered and loose manner, with significantly reduced density. Many neuronal cell bodies were shrunken and deformed, and some neurons showed cytoplasmic dissolution, nuclear pyknosis, or nuclear fragmentation. Significant neuronal loss and interstitial edema were observed, with the CA1 region showing the most severe damage. The metformin-positive drug group (MET) showed slightly less neuronal damage in the hippocampus than the Model group, but disordered neuronal arrangement and some cell body deformation still existed, indicating limited protective effect. After NP intervention, neuronal damage was significantly improved: in the NPL group, neurons in various subregions of the hippocampus were arranged much more densely than in the Model group, cell body morphology was more regular, nuclear pyknosis was reduced, and the number of lost neurons was significantly decreased; in the NPH group, the density of neurons in the hippocampus was close to that in the CON group, cell bodies were full and intact, cell nuclei were clear, and only a few neurons had slight morphological abnormalities, indicating that neuronal damage was almost completely repaired. These results confirm that NP can significantly protect hippocampal neurons in aging diabetic mice by reducing oxidative stress damage to brain tissue and inhibiting neuronal apoptosis, which is also an important histological basis for its improvement of cognitive and memory abilities in mice.

[0159] The above embodiments can well illustrate the technical solution of the present invention, but they are only describing preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all kinds of changes and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A method for preparing a salty-enhancing peptide-naringenin complex, characterized in that, include: Preparation of salty-enhancing peptide solution, preparation of naringenin solution, and mixing of salty-enhancing peptide solution and naringenin solution; Preparation of salty-enhancing peptide solution: Dissolve the freeze-dried turtle egg powder in water to prepare a turtle egg solution; Papain was added for enzymatic hydrolysis. The hydrolysis product was collected by solid-liquid separation. The liquid was then subjected to filtration and ultrafiltration to obtain a salty flavor-enhancing peptide with a molecular weight of less than 3 kDa. The salty flavor-enhancing peptide was dissolved in a solvent to prepare a salty flavor-enhancing peptide solution of 20-150 mg / mL. Preparation of naringenin solution: Dissolve naringenin in a solvent to prepare a 1 mg / mL naringenin solution; Salty flavor-enhancing peptide solution and naringenin solution were mixed: the salty flavor-enhancing peptide solution and naringenin solution were mixed evenly at a volume ratio of 1:1, concentrated, and freeze-dried to obtain the salty flavor-enhancing peptide-naringenin complex.

2. The preparation method according to claim 1, characterized in that, The concentration of the turtle egg solution is 1-10%.

3. The preparation method according to claim 1, characterized in that, The papain activity is 4500~5500 U / g, and the enzymatic hydrolysis temperature is 45~55℃.

4. The preparation method according to claim 1, characterized in that, Water is used to dissolve the salty-enhancing peptides, and 70-80% ethanol is used to dissolve the naringenin.

5. The use of the salty-enhancing peptide-naringenin complex obtained by the preparation method of claim 1 in the preparation of formulations that improve cognitive decline.

6. The application according to claim 5, characterized in that, The cognitive decline includes one or more of the following: new object recognition memory decline, social memory decline, and spatial learning memory decline.

7. The use of the salty-enhancing peptide-naringenin complex obtained by the preparation method according to claim 1 in the preparation of formulations that improve anxiety.

8. The application of the salty-enhancing peptide-naringenin complex obtained by the preparation method according to claim 1 in the preparation of formulations that enhance the body's motor coordination ability.

9. The application according to claim 5, 6, 7 or 8, characterized in that, The formulation can also improve skeletal muscle damage, enhance the brain tissue's antioxidant stress capacity, and alleviate brain nerve cell damage.

10. The application according to claim 5, 6, 7 or 8, characterized in that, The preparation is an oral preparation.

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