Active peptide with improved memory and antioxidant efficacy, preparation and application

Abstract

The application discloses active peptides with memory improving and antioxidant efficacy, a preparation and application, and belongs to the technical field of bioactive peptides. The amino acid sequence of the active peptide with memory improving and antioxidant efficacy is one of SEQ ID NO:1-SEQ ID NO:3. The application takes defatted walnut powder as raw material, and through directional enzymolysis, ultrafiltration grading and liquid chromatography-mass spectrometry (LC-MS / MS) technology, three specific active peptide sequences with significant acetylcholinesterase inhibitory activity and antioxidant activity are accurately identified. The preparation process of the application is simple, green, mild, the raw material source is wide, the cost is low, the obtained peptide segment is natural, safe, biocompatible, has high acetylcholinesterase inhibitory activity and antioxidant activity, and therefore, has important application prospect in development of products with high activity, small side effects, memory improving and antioxidant functions.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to bioactive peptides, formulations, and applications that have the effects of improving memory and anti-oxidation. Background Technology

[0002] Walnuts (Juglans regia L.) are a type of nut rich in high-quality protein and various bioactive components. The small molecule peptides produced by the enzymatic hydrolysis of their protein have been reported to have various biological activities such as antioxidant, anti-inflammatory and neuroprotective effects. However, the specific active ingredients and mechanisms of action of walnut peptides in improving memory are still unclear in the current technology.

[0003] Currently, there is considerable research on enzymatic hydrolysis techniques for walnut protein, such as using alkaline proteases, neutral proteases, or flavor proteases to hydrolyze walnut protein in order to obtain enzymatic hydrolysates with specific functions. However, due to the complex structure of walnut protein and the specificity of the enzymatic hydrolysis process, different proteases exhibit significant differences in the hydrolysis sites of walnut protein and the sequence composition of the resulting peptides, which directly affects the bioactivity of the hydrolysates. Existing technologies lack systematic research on the inhibitory activity of specific amino acid sequence peptides derived from walnut protein on acetylcholinesterase (AChE), and also lack in-depth exploration of synergistic combinations of active peptides in walnut protein enzymatic hydrolysates.

[0004] Acetylcholinesterase inhibitors are currently an important strategy for improving memory function. By inhibiting acetylcholinesterase activity, they can increase the level of acetylcholine in the brain, thereby improving learning and memory abilities. Although some chemically synthesized acetylcholinesterase inhibitors have been used clinically, they often come with certain side effects. Therefore, isolating and identifying specific peptides with significant acetylcholinesterase inhibitory activity from naturally sourced walnut protein and clarifying their sequence structure is of great significance for developing products with high safety and few side effects to improve memory function.

[0005] Current research on walnut peptides largely focuses on evaluating the activity of crude enzymatic hydrolysates, failing to delve into the sequences of the core peptides and their interrelationships. Therefore, isolating and screening small molecule peptides with memory-improving effects from defatted walnut powder holds significant promise for developing memory-enhancing products with high activity and minimal side effects. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of this invention is to provide active peptides, preparations and applications with memory-improving and antioxidant effects.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: An active peptide with memory-improving and antioxidant effects, wherein the amino acid sequence of the active peptide with memory-improving and antioxidant effects is one of SEQ ID NO:1 to SEQ ID NO:3.

[0008] The aforementioned active peptides with memory-improving and antioxidant effects are used to prepare products with these effects.

[0009] Based on the above scheme, the product is a pharmaceutical product.

[0010] Based on the above scheme, the dosage form of the product is one of tablets, capsules, powders, granules, or oral liquids.

[0011] Based on the above scheme, pharmaceutically acceptable excipients are further included.

[0012] Based on the above scheme, the effect of improving memory is that it has acetylcholinesterase inhibitory activity.

[0013] Based on the above scheme, the antioxidant effect is that it has ABTS and DPPH free radical scavenging activity.

[0014] A preparation with memory-improving and antioxidant effects, the active ingredient being walnut peptide with an amino acid sequence as shown in at least one of SEQ ID NO:1~SEQ ID NO:3.

[0015] Based on the above scheme, the percentage of active ingredient in the preparation mass is 0.1-99%.

[0016] Advantages of the technical solution of this invention This invention uses defatted walnut powder as raw material and employs targeted enzymatic hydrolysis, ultrafiltration fractionation, and liquid chromatography-tandem mass spectrometry (LC-MS / MS) to precisely identify three specific walnut peptide sequences with significant acetylcholinesterase inhibitory and antioxidant activities, which are then chemically synthesized. In vitro activity verification showed that all three synthesized peptides exhibit significant acetylcholinesterase inhibitory and antioxidant activities.

[0017] The preparation process of this invention is simple, green, and mild, with widely available and inexpensive raw materials. The resulting peptides are natural, safe, and biocompatible, and have high acetylcholinesterase inhibitory and antioxidant activities. Therefore, they have important application prospects in the development of products with high activity, few side effects, and memory-improving and antioxidant functions. Attached Figure Description

[0018] Figure 1 This is the molecular weight spectrum of the VEDELRVL peptide mass spectrometry. Figure 2This is the molecular weight diagram of the LDDELRVL mass spectrometry of the peptide. Figure 3 This is a molecular weight diagram of the peptide LPSFSNAPR mass spectrometry. Figure 4 The image shows the reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram of the peptide VEDELRVL. Figure 5 The image shows the reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram of the peptide LDDELRVL. Figure 6 The image shows the reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram of the peptide LPSFSNAPR. Figure 7 The effect of different proteases on the hydrolysis of crude and fine walnut powder is shown in the figure (where different lowercase letters indicate significant differences between groups, P<0.05). Figure 8 The AChE inhibition rate of flavor protease <3KDa enzymatic hydrolysate at different dilution factors is shown in the figure (where different lowercase letters indicate significant differences between groups, P<0.05). Figure 9 The graph shows the ABTS and DPPH free radical scavenging rates of the flavor protease hydrolysates of coarse and fine walnut powder (where different lowercase letters indicate significant differences between groups, P<0.05). Figure 10 The graph shows the inhibitory activity of each single peptide and complex peptide on acetylcholinesterase (AChE) (where different lowercase letters indicate significant differences between groups, P<0.05). Figure 11 The graph shows the antioxidant activity of each single peptide and complex peptide (where different lowercase letters indicate significant differences between groups, P<0.05). Detailed Implementation

[0019] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0020] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0021] In the following embodiments, 1. Determination of degree of hydrolysis The degree of hydrolysis was determined using the o-phthalaldehyde (OPA) method. 80 mg of OPA was dissolved in 2 mL of β-mercaptoethanol, 5 mL of SDS (10%, mass fraction), and 92.8 mL of sodium tetraborate (0.1 mol / L) to prepare a 100 mL OPA reagent solution. 4 mL of walnut protein hydrolysate was mixed with 4 mL of OPA reagent and incubated at room temperature for 2 min. The absorbance was measured at 340 nm. Defatted walnut powder samples were hydrolyzed with 6 mol / L HCl at 110 °C for 24 h to achieve complete hydrolysis. The content of free amino acids in the hydrolysate was determined using a serine standard curve. The degree of hydrolysis (H, %) of the walnut protein hydrolysate was calculated using the following formula:

[0022] In the formula: H is the degree of hydrolysis, %; Nt is the number of free amino acids in the enzymatic hydrolysis product at time t; No is the number of free amino acids that have not undergone enzymatic hydrolysis; NT is the number of free amino acids after complete hydrolysis.

[0023] 2. Determination of acetylcholinesterase (AChE) inhibitory activity Acetylcholinesterase (AChE) activity assay kit from Beijing Solarbio Science & Technology Co., Ltd. was used for the determination, based on the Ellman colorimetric method. Test tubes and control tubes were prepared, and samples were added according to the instructions: 15 μL of the test sample was added to the test tube, and 50 μL of reagent II (substrate) was added to the control tube. After reacting in a 37°C water bath for 5 min, 50 μL of reagent IV was added to both tubes to stop the reaction. Then, 50 μL of reagent II was added to the control tube. After mixing, 10 μL of the supernatant was taken from each tube, and 170 μL of reagent I and 20 μL of reagent III colorimetric solution were added to each tube. After mixing and incubation for 2 min, the absorbance was measured at 412 nm. A blank control group (equal volume of distilled water instead of the sample) was set up, with three replicates per group. The AChE inhibition rate (J,%) was calculated as follows:

[0024] In the formula: A 样 A represents the absorbance of the sample group. 空 Absorbance of the blank control group; A 对 The absorbance is for the control group.

[0025] 3. Determination of ABTS free radical scavenging rate Prepare a 7 mmol / L ABTS solution and a 2 mmol / L potassium persulfate solution, mix them in a 1:1 ratio, and react at room temperature in the dark for 16 h. Dilute the resulting ABTS radical solution with distilled water to achieve an absorbance of 0.700 ± 0.005 at 734 nm. This solution should be prepared fresh before use. Add 4 mL of the ABTS working solution to a colorimetric tube, then add 1 mL of the enzymatic hydrolysate sample, allow to stand for 6 min, and measure the absorbance using a UV spectrophotometer. Calculate the ABTS radical scavenging rate using the following formula.

[0026]

[0027] In the formula: A0 is the absorbance of distilled water and ABTS working solution at a wavelength of 734 nm after the reaction; A j The absorbance value at a wavelength of 734 nm is the result of the reaction between the walnut enzymatic hydrolysate sample and the ABTS working solution.

[0028] 4. DPPH free radical scavenging rate determination Weigh 0.00528 g of DPPH, dissolve it in anhydrous ethanol, and dilute to a volumetric flask of 100 mL to obtain a 0.1339 mmol / L DPPH ethanol solution. Store at 4 °C for later use. Transfer 0.2 mL of the test sample solution to a 10 mL centrifuge tube, add 4 mL of the 0.1339 mmol / L DPPH ethanol solution, mix well, and incubate at 37 °C for 20 min. Measure the absorbance at 510 nm using a spectrophotometer. Use a mixture of 4 mL DPPH and 0.2 mL deionized water as a blank control. The DPPH free radical scavenging rate is expressed as the inhibition rate. The higher the inhibition rate (IR value), the stronger the antioxidant activity of the test subject; conversely, the lower the IR value, the weaker the antioxidant activity.

[0029]

[0030] In the formula: A x The absorbance is the result of the reaction between walnut enzymatic hydrolysate and DPPH free radical solution. A0 is the absorbance of the enzymatic hydrolysate and deionized water; A0 is the absorbance of the mixture of deionized water and DPPH solution.

[0031] The walnut powder raw materials in the following examples all come from the same batch of defatted walnut kernel meal: using commercially available walnut kernels from Shandong Province produced in the current year as raw materials, after removing most of the oil through a cold pressing and degreasing process, walnut meal with a moisture content of ≤5% and a crude protein content of ≥40% is obtained. The meal is free from mold and rancidity and meets the food safety standards of GB 19300 "Nuts and Seeds".

[0032] The specific preparation methods for the two types of walnut powder are as follows: Preparation method of walnut coarse powder: Take the walnut cake residue after cold pressing and deoiling, remove impurities and crush it into small pieces with a diameter of ≤1cm; put it into a stainless steel food-grade hammer mill and set the speed to 1500~2000r / min for preliminary crushing; then transfer it to a low temperature pulverizer, control the chamber temperature to ≤20℃, and crush for 3~5min; pass it through a 40-mesh standard sieve, and the material passing through the sieve is the raw material of walnut coarse powder, which is sealed for later use.

[0033] Walnut powder preparation method (obtained from coarse powder): Walnut coarse powder is fed into the feed hopper of a food-grade airflow ultrafine pulverizer (with nitrogen protection system). The nitrogen protection system is activated to create an inert gas atmosphere in the chamber. The classifying wheel speed is set to 3000~4000 r / min, and the chamber temperature is ≤30℃. Pulverization is performed for 10~15 min. The material is crushed by collision under high-speed airflow. After screening by the classifying wheel, fine powder with a particle size ≤50μm enters the collection hopper. A small amount of agglomerated particles are removed by passing through a 300-mesh standard sieve, finally obtaining walnut powder, which is then vacuum-packed with nitrogen for later use.

[0034] Example 1

[0035] The method for extracting bioactive peptides with memory-improving and antioxidant effects from walnuts involves the following steps: Step 1: Enzymatic hydrolysis Take 1.5g of defatted walnut powder (coarse powder), add 50mL of distilled water, adjust the pH to 6.5, add 0.3g of flavor protease (enzyme activity 20U / mg), the amount of enzyme added is 2.0% of the substrate mass, and enzymatically hydrolyze at 50℃ for 6h, using 0.1mol / L NaOH to maintain a constant pH. After enzymatic hydrolysis, heat at 100℃ for 10min to inactivate the enzyme, cool, and centrifuge at 4℃ and 8000r / min for 15min. Collect the supernatant to obtain the walnut protease hydrolysate.

[0036] Step 2: Ultrafiltration Fractionation The walnut protein hydrolysate obtained in step one above was subjected to ultrafiltration fractionation through an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa. The fraction was centrifuged at 4°C and 4000 r / min for 15 min. The filtered fraction with a molecular weight <3 kDa was collected and freeze-dried to obtain crude walnut peptide.

[0037] Step 3: Sequence identification of active peptides The <3kDa fraction obtained by ultrafiltration was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS), with specific analysis performed by Sangon Biotech (Shanghai) Co., Ltd. The sample was reconstituted in mobile phase A (2% acetonitrile, 0.1% formic acid) containing 0.1% formic acid. Using a nano-liquid chromatography system, the sample was first desalted on a C18 pre-column (nanoViper™, 3μm, 100Å), followed by separation on a C18 reversed-phase column (Acclaim™ PepMap™ 100). The gradient conditions were as follows: mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 30 min at a flow rate of 200 nL / min. Mass spectrometry was performed using a high-resolution mass spectrometer (Q Exactive™) equipped with a nano-spray ionization source (Nanospray Flex™), a spray voltage of 1.9 kV, and an ion transfer tube temperature of 275 °C. The primary mass spectrometry scan resolution was 70,000 m / z, with a scan range of 350–2,000 m / z. A data-dependent acquisition mode was used, selecting the most intense 2+ to 5+ charged ions for high-energy collision dissociation and fragmentation in each cycle (collision energy 28 eV). The secondary scan resolution was 17,500 m / z, with a dynamic exclusion time of 25 s. Data were analyzed using PEAKS Online software, identifying multiple peptide sequences from this fraction.

[0038] Step 4: Use bioinformatics techniques to screen the peptides obtained above. The screening criteria are as follows: (1) Molecular weight less than 1000 Da; (2) Remove peptides with phosphorylation, glycosylation, or other modifications; (3) Sequence confidence (ALC) > 50%; (4) Remove low-abundance peptides (peak area <10) 7 ); (5) Peptide Ranker score > 0.8; (6) Toxicity prediction threshold < 0.2.

[0039] Based on the above screening criteria, three peptides were finally selected from the identified multiple peptides, as shown in Table 1. The mass spectrometry molecular weight spectra of the three peptides are shown below. Figures 1-3 As shown.

[0040] Table 1. Information on the three peptides obtained through screening.

[0041] Step 5: Chemical synthesis of the target peptide Based on the LC-MS / MS results, the three peptides were chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method. The synthesized products were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic conditions were as follows: column: SHIMADZU Inertsil ODS-SP (4.6 × 250 mm, 5 μm); mobile phase A: aqueous solution containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile solution containing 0.1% trifluoroacetic acid; gradient increase of phase B from 21% to 41% within 0–20 min; flow rate: 1.0 mL / min; detection wavelength: 214 nm. The purities of the three peptides, as determined by RP-HPLC, were: VEDELRVL 99.0%, LDDELRVL 98.0%, and LPSFSNAPR 96.8%. The RP-HPLC chromatograms of the three peptides are shown below. Figures 4-6 As shown.

[0042] Example 2

[0043] Effects of protease type on the degree of hydrolysis and AChE inhibition rate of walnut powder enzymatic hydrolysates Defatted walnut powder (coarse powder) and defatted walnut powder (fine powder) were used as raw materials. Five proteases, namely alkaline protease, acidic protease, neutral protease, flavor protease and papain, were used to enzymatically hydrolyze the coarse and fine walnut powders under their respective optimal conditions (Table 2) according to the method of Example 1. The degree of hydrolysis and the acetylcholinesterase (AChE) inhibitory activity of each enzymatic hydrolysis product were measured.

[0044] Table 2. Optimal temperature and optimal pH for different hydrolytic enzymes

[0045] The effect of protease type on the degree of hydrolysis of walnut powder enzymatic hydrolysates, such as Figure 7 As shown, the five proteases exhibited significant differences in their hydrolytic capacity for walnut protein. Among them, the flavor protease showed the highest degree of hydrolysis for both coarse and fine walnut powder, significantly higher than the other four proteases (P<0.05). This indicates that the flavor protease can efficiently degrade large walnut protein molecules into smaller peptides, which is beneficial for the full exposure of active functional groups.

[0046] The AChE inhibition rate of walnut powder hydrolysate obtained by flavor protease hydrolysis at different dilution ratios is as follows: Figure 8 As shown, at the same dilution factor, the AChE inhibition rate of the enzymatic hydrolysate of crude walnut powder was higher than that of fine walnut powder, and the inhibition rate reached 38.5% under the condition of 6-fold dilution, showing good potential for improving memory in vitro.

[0047] The ABTS and DPPH free radical scavenging rates of the enzymatic hydrolysates of walnut powder obtained by enzymatic degreasing of walnut powder (coarse powder) and defatted walnut powder (fine powder) using flavor protease were determined. The results are as follows: Figure 9 As shown, the flavor protease hydrolysates of both types of walnut powder exhibit good free radical scavenging capabilities, demonstrating excellent in vitro antioxidant properties. This confirms that walnut protein, after being hydrolyzed by flavor protease, can generate a large number of small molecule peptides and amino acid components with antioxidant activity. The study confirms that antioxidant active peptides can protect the nervous system by scavenging excess free radicals in the body and alleviating oxidative stress damage, providing a good active basis for the memory-improving effects of walnut active peptides.

[0048] Example 3

[0049] An active peptide with memory-improving and antioxidant effects, wherein the amino acid sequence of the active peptide is any one of SEQ ID NO:1 to SEQ ID NO:3.

[0050] SEQ ID NO:1: VEDELRVL; SEQ ID NO:2: LDDELRVL; SEQ ID NO:3: LPSFSNAPR.

[0051] Example 4

[0052] A preparation with memory-improving and antioxidant effects, the active ingredient being at least one of the following active peptides; SEQ ID NO:1: VEDELRVL; SEQ ID NO:2: LDDELRVL; SEQ ID NO:3: LPSFSNAPR.

[0053] Antioxidant and AChE inhibitory activities of single peptide solutions and complex peptide solutions Three synthetic peptides were separately prepared into single peptide solutions with a concentration of 1 mg / mL using deionized water (where 1: VEDELRVL; 2: LDDELRVL; 3: LPSFSNAPR). At the same time, a compound peptide solution was prepared (where 1+2 is peptide 1 + peptide 2; 1+3 is peptide 1 + peptide 3; 2+3 is peptide 2 + peptide 3; 1+2+3 is peptide 1 + peptide 2 + peptide 3; the compound peptide solution contains equal mass ratios of each peptide, with a total concentration of 1 mg / mL).

[0054] The AChE inhibitory activity of single peptide solutions and complex peptide solutions is as follows: Figure 10As shown, in single peptide solutions, all three single peptides exhibited AChE inhibitory activity, with peptide 2 (LDDELRVL) showing the highest AChE inhibition rate at 58%, significantly higher than peptide 1 (33%) and peptide 3 (48%) (P<0.05). In complex peptide solutions, the inhibition rate increased to 72.56% for the peptide 1 + peptide 2 (1+2) group, 56.23% for the peptide 1 + peptide 3 (1+3) group, and 75.51% for the peptide 2 + peptide 3 (2+3) group; the inhibition rate was 63.81% for the peptide 1 + peptide 2 + peptide 3 (1+2+3) group. These results indicate a significant synergistic effect among the peptides, with peptide 2 (LDDELRVL) + peptide 3 (LPSFSNAPR) (2+3) exhibiting the best AChE inhibitory activity.

[0055] The results of ABTS and DPPH free radical scavenging rates for single peptide solutions and complex peptide solutions are as follows: Figure 11 As shown, all three individual peptides exhibited certain ABTS and DPPH free radical scavenging abilities. Peptide 2, LDDELRVL, showed the highest scavenging rate (ABTS: 67%, DPPH: 79%), significantly higher than peptides 1 (57% / 69%) and 3 (61% / 72%) (P<0.05). In the complex peptide solution, the scavenging rates were significantly higher than their corresponding single active peptides, with the 2+3 group showing the highest increase (ABTS: 94.51%, DPPH: 86.37%), consistent with the trend of AChE inhibitory activity. These results indicate a significant synergistic effect among the peptides in terms of antioxidant activity, further confirming that the combination of peptides 2 and 3 is the most crucial peptide for exerting multiple biological activities.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An active peptide with memory-improving and antioxidant effects, characterized in that, The amino acid sequence of the active peptide that has the effects of improving memory and anti-oxidation is shown in SEQ ID NO:

2.

2. The application of the active peptide with memory-improving and antioxidant effects as described in claim 1, characterized in that, Used to prepare products with memory-improving and antioxidant effects.

3. The application of the active peptide with memory-improving and antioxidant effects according to claim 2, characterized in that, The product in question is a pharmaceutical product.

4. The application of the active peptide with memory-improving and antioxidant effects according to claim 3, characterized in that, The product is available in one of the following dosage forms: tablets, capsules, powders, granules, or oral liquids.

5. The application of the active peptide with memory-improving and antioxidant effects according to claim 4, characterized in that, It further includes pharmaceutically acceptable excipients.

6. The application of the active peptide with memory-improving and antioxidant effects according to claim 2, characterized in that, The effect of improving memory is due to its acetylcholinesterase inhibitory activity.

7. The application of the active peptide with memory-improving and antioxidant effects according to claim 6, characterized in that, The antioxidant effect is described as having ABTS and DPPH free radical scavenging activity.

8. A formulation with memory-improving and antioxidant effects, characterized in that, The active ingredient is an active peptide with an amino acid sequence as shown in SEQ ID NO:2, or a combination of active peptides with an amino acid sequence as shown in SEQ ID NO:2 and SEQ ID NO:

3.

9. The preparation with memory-improving and antioxidant effects according to claim 8, characterized in that, The percentage of active ingredient in the formulation is 0.1% to 99% of the total mass.

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