Walnut peptide capable of penetrating blood-brain barrier and its application in preparing intelligence-enhancing products

Abstract

The present invention relates to a walnut peptide that can penetrate the blood-brain barrier and its application in the preparation of intelligence-enhancing products. Existing studies have confirmed that walnut protein has a variety of beneficial effects on the human body, especially its brain-boosting and intelligence-enhancing effects. At present, there are many walnut polypeptides under development, but there are few related studies proving that they can penetrate the blood-brain barrier. The present invention uses walnut meal as a raw material, prepares and screens a walnut peptide product with blood-brain barrier penetration activity, and it has been verified that the walnut peptide product can not only penetrate the blood-brain barrier and disperse in the animal brain, but also has the effects of improving the memory of mice, and is expected to be applied to the development of intelligence-enhancing foods or medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of walnut active peptide development, and specifically relates to a walnut peptide that can penetrate the blood-brain barrier and a preparation method thereof, a walnut peptide obtained by the preparation method, a pharmaceutical composition containing the walnut peptide and an application thereof in the preparation of intelligence-enhancing products. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Walnut (Juglans regia L.) is a plant of the Juglandaceae family and the genus Juglans. It is known as the "four major dried fruits" along with almonds, cashews, and hazelnuts. At present, my country is the largest producer and consumer of walnuts. Walnuts are rich in nutrients, including protein, lipids, and carbohydrates. At the same time, they contain a variety of trace elements and minerals such as calcium, phosphorus, and iron, as well as a variety of vitamins such as carotene and riboflavin. Among them, walnut protein peptides have antioxidant, anti-inflammatory, memory-enhancing, blood pressure-lowering, cholesterol-lowering, and anti-cancer effects.

[0004] At present, walnuts are mainly eaten raw or pressed for oil. The waste product of oil pressing, walnut meal, is rich in walnut protein peptides, which has good brain-boosting and intelligence-enhancing value, and conforms to the theory of "like cures like" in traditional Chinese medicine. At present, the development and utilization of active ingredients in walnut meal are not sufficient. In addition, the protective effect of the blood-brain barrier on the brain also prevents some active ingredients from reaching the brain tissue to exert their active effects.

[0005] The main methods for preparing bioactive peptides are: protease hydrolysis, chemical synthesis, gene recombination, separation and extraction, and chemical hydrolysis. The walnut peptides prepared by these methods are of various types, complex activities, and poor purpose. It is not possible to guide the formulation of peptide preparation methods and screen the corresponding peptide fragments based on whether they can pass through the blood-brain barrier. At this stage, a large number of studies have shown that walnut peptides have antioxidant activity, anti-tumor activity, blood pressure lowering, anticoagulant and immunomodulatory activity, etc. However, there are no reports on whether walnut peptides can pass through the blood-brain barrier to play a role in brain health and intelligence.

[0006] The inventor believes that, at present, the preparation methods of walnut polypeptides are mainly based on protease hydrolysis, chemical hydrolysis, separation and extraction, etc., and have not been combined with animal experiments to produce walnut polypeptides that can pass through the blood-brain barrier. Therefore, the development of a preparation method for walnut polypeptides that can pass through the blood-brain barrier is of great significance to the diversification of walnut products and the development of healthy foods. Summary of the invention

[0007] Based on the above technical background, the purpose of the present invention is to provide a walnut peptide component that can pass through the blood-brain barrier and exert intelligence-enhancing activity. The present invention selects walnut meal, a by-product of walnut processing, as the raw material for screening active peptides. Walnut meal, as a by-product of walnut oil pressing, contains a large amount of protein and dietary fiber. It is currently mainly used as animal feed or directly discarded, and has low economic value. The present invention takes into account that walnut meal retains most of the protein components in walnuts and is an ideal raw material for walnut peptide extraction. Furthermore, the present invention screened out active polypeptide products that can pass through the blood-brain barrier and improve memory through biological enzymatic hydrolysis. Therefore, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a walnut peptide that can penetrate the blood-brain barrier and a preparation method thereof. The preparation method comprises the following steps: crushing the walnuts and dispersing them in water, adding a complex enzyme for enzymolysis, obtaining the clarified solution after enzymolysis and drying it to obtain an extract. The walnut peptide that can penetrate the blood-brain barrier is detected by the detection method for the proportion of collagen peptides with a relative molecular mass of less than 10,000 in Appendix A of "GB31645-2018 National Food Safety Standard Collagen Peptide" (high performance size exclusion chromatography) (GPC), and has a set of 5 characteristic peaks with specific retention time and peak area ratio.

[0009] In the above-mentioned preparation method provided by the present invention, the walnut meal is hydrolyzed by two steps, and the non-protein components are degraded by polysaccharidase and lipase enzymolysis in the first step, and then the filter residue is filtered; in the second step, water and protease are added to the filter residue for enzymolysis, the filtrate is filtered and dried to obtain solid walnut peptides. Specifically, the combination of trypsin and xylanase, the combination of papain and pectinase, the combination of trypsin, cellulase and lipase, the combination of papain, glucanase and lipase, and the combination of alkaline protease, xylanase and lipase can obtain active walnut peptides with barrier penetration. It is generally believed in the art that drugs with high fat solubility, relatively small molecular weight and relatively simple chemical structure are more likely to penetrate the blood-brain barrier. It has been verified that the active walnut peptide prepared by the present invention, its ability to penetrate the blood-brain barrier is not simply related to the molecular weight, and the enzymolysis products of smaller molecular weight segments do not show similar penetration ability. This means that the walnut peptide provided by the present invention has special permeability, or contains active peptides with strong membrane penetration ability.

[0010] Therefore, the present invention secondly provides the use of the above-mentioned walnut peptide that can penetrate the blood-brain barrier in the preparation of intelligence-enhancing products.

[0011] It has been verified by the present invention that the walnut peptide separated by the above-mentioned first preparation method has the ability to pass through the blood-brain barrier and has the effect of improving memory, and can be used in the preparation of corresponding foods, nutritional supplements or medicines. Therefore, the present invention also provides a pharmaceutical composition with intelligence-enhancing effect, and the use of the walnut peptide and / or pharmaceutical composition in the preparation of intelligence-enhancing products. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0013] Figure 1 The mouse imaging results of walnut polypeptides in Example 1 and Example 2;

[0014] The left figure is the result of Example 1, and the right figure is the result of Example 2;

[0015] Figure 2 The mouse imaging results of walnut polypeptides in comparative examples 1 and 2 are shown;

[0016] The left figure is the result of comparative example 1, and the right figure is the result of example 2;

[0017] Figure 3 The imaging results of mice in the blank control group and the Cy7 control group;

[0018] Figure 4 Characteristic GPC spectrum of Example 1 walnut peptide that can pass through the blood-brain barrier;

[0019] Figure 5 Characteristic GPC spectrum of Example 2 walnut peptide that can pass through the blood-brain barrier;

[0020] Figure 6 GPC spectrum of walnut peptide in comparative example 1 that cannot pass the blood-brain barrier;

[0021] Figure 7 GPC spectrum of walnut peptide of comparative example 2 that cannot pass the blood-brain barrier. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0023] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0024] As introduced in the background technology, it is generally recognized in the art that the bioactive peptides in walnuts have high brain-boosting and intelligence-enhancing value. Due to the existence of the blood-brain barrier, whether the existing walnut polypeptide products can really penetrate the blood-brain barrier to play the effect of brain-boosting and intelligence-enhancing is still lacking. In order to solve the above technical problems, the present invention provides a walnut peptide product that can penetrate the blood-brain barrier and has intelligence-enhancing effects, using walnut meal, a byproduct of walnut processing, as a raw material, from which the above-mentioned active parts are screened.

[0025] In a first aspect, the present invention provides a walnut peptide that can penetrate the blood-brain barrier, wherein the molecular weight distribution characteristics of the walnut peptide are as follows:

[0026] The walnut peptide is analyzed by liquid chromatography, and the liquid chromatography uses a hydrophilic vinyl resin matrix protein separation column, and the mobile phase is as follows: the organic phase is acetonitrile, and the aqueous phase is trifluoroacetic acid aqueous solution; the detection wavelength is 220nm, the flow rate is 0.3-0.7ml / min, and the column temperature is 28-32°C;

[0027] In the GPC analysis of the walnut peptide, the peak area at 17.6-18.7 min accounts for the largest proportion, and it is taken as the main peak; the GPC characteristic peaks 1-5 of the walnut peptide are: peak 1 is 3.8-2.5 min before the main peak, peak 2 is 2.5-1.3 min before the main peak, peak 3 is the main peak, peak 4 is 0.3-1.5 min after the main peak, and peak 5 is 2.7-3.8 min after the main peak.

[0028] Peak area ratio characteristic 1 is: the ratio of peak 3 area to peak 1 is 1:0.07-0.24;

[0029] Peak area ratio feature 2 is: the area ratio of peak 3 to peak 2 is 1:0.24-0.56;

[0030] Peak area ratio feature 3 is: the area ratio of peak 3 to peak 4 is 1:0.33-1.01;

[0031] Peak area ratio characteristic 4 is: the area ratio of peak 3 to peak 5 is 1:0.09-0.50.

[0032] Furthermore, the main peak of the GPC characteristic peak is 17.8min to 18.5min, and the retention times of characteristic peaks 1 to 5 are 3.6min to 2.7min before the main peak of peak 1, 2.3min to 1.5min before the main peak of peak 2, 0.5min to 1.3min after the main peak of peak 3, and 2.9min to 3.6min after the main peak of peak 4;

[0033] Peak area ratio characteristic 1 is: the ratio of peak 3 area to peak 1 is 1:0.10-0.21;

[0034] Peak area ratio feature 2 is: the area ratio of peak 3 to peak 2 is 1:0.30-0.48;

[0035] Peak area ratio feature 3 is: the area ratio of peak 3 to peak 4 is 1:0.43-0.86;

[0036] Peak area ratio characteristic 4 is: the area ratio of peak 3 to peak 5 is 1:0.11-0.43.

[0037] The liquid chromatography analysis detection conditions provided in the first aspect above are from Appendix A of "GB 31645-2018 National Food Safety Standard Collagen Peptide"; according to the detection method for the proportion of collagen peptides with a relative molecular mass of less than 10,000 in Appendix A (high performance size exclusion chromatography) (GPC), the walnut peptide that can penetrate the blood-brain barrier has a set of 5 characteristic peaks with specific retention times and peak area ratios.

[0038] The second aspect of the present invention provides a method for preparing the walnut peptide that can penetrate the blood-brain barrier as described in the first aspect, the method comprising the following steps: crushing walnuts and dispersing them in water to obtain a suspension, adding a composite enzyme for enzymolysis, obtaining a clear solution after enzymolysis and drying it to obtain an extract, and performing chromatographic analysis on the extract to obtain a liquid chromatography characteristic peak retention time of the walnut peptide that can penetrate the blood-brain barrier is 14.3min-15.6min, 15.6min-16.8min, 17.6min-18.7min, 18.4min-19.6min, 20.8min-21.9min;

[0039] The enzymatic hydrolysis method is as follows:

[0040] Add polysaccharidase and lipase to the suspension, and retain the filter residue after enzymolysis; add protease and water to the filter residue, and retain the filtrate after enzymolysis, which is the clarified solution.

[0041] In the preparation method of the second aspect of the present invention, a two-step enzymatic hydrolysis method is adopted, which is beneficial to the degradation of impurities, the exposure of walnut protein, the reduction of oil content, and the improvement of walnut peptide yield.

[0042] In the above preparation method, the walnut is a powder after mechanical crushing of walnuts, the walnut includes unprocessed walnut kernels after removing the shell, and also includes walnut processing byproducts with a high protein content. In one embodiment provided by the present invention, walnut meal is used as a raw material for extracting the above walnut peptide. The preparation method provided by the present invention has no restrictions on the source of walnut meal, and any byproduct produced by the walnut oil pressing process can be used.

[0043] Furthermore, the walnut meal is crushed and then passed through a 200-mesh sieve.

[0044] Furthermore, in the suspension, the ratio of walnut powder to water is 4-15:100g.

[0045] In the above preparation method, the polysaccharidase is selected from 1-3 of cellulase, pectinase, lysozyme, hemicellulase, xylanase, glucanase, ligninase and amylase, and lipase is added in a ratio of total polysaccharidase activity: lipase activity = 20-100:1, and the amount of enzyme added is 50,000-100,000 U of polysaccharidase per kg of walnuts;

[0046] The protease is a combination of 2-3 kinds of trypsin, papain, bromelain, neutral protease and alkaline protease; the proteases are mixed according to equal enzyme activity, and the amount of protease added is 200,000-600,000 U of protease per kg of walnuts; during the enzymatic hydrolysis of the protease, the pH of the reaction is adjusted within the range of 7.5-10.5 according to the type of protease.

[0047] The enzymatic hydrolysis process is carried out in a water bath with a temperature of 20 to 75° C. and a water bath time of 3 to 36 hours.

[0048] In the above preparation method, the clear solution is obtained as follows: the reaction system after enzymatic hydrolysis is subjected to solid-liquid separation to obtain a supernatant, and the supernatant is sterilized and filtered to obtain the clear solution.

[0049] Furthermore, the solid-liquid separation may be centrifugation, dead-end filtration and cross-flow filtration.

[0050] Furthermore, the sterilization filtration is performed using a 0.45 μm water filter membrane.

[0051] Furthermore, the clear solution is dried by concentrated distillation to remove most of the solvent, and then spray-dried or freeze-dried to obtain the extract.

[0052] The third aspect of the present invention provides a pharmaceutical composition with intelligence-enhancing effect, in which the walnut peptide that can penetrate the blood-brain barrier as described in the first aspect is used as an active ingredient.

[0053] Preferably, in the pharmaceutical composition, the dosage of the walnut peptide can be adjusted according to the purpose of use of the pharmaceutical composition, which includes but is not limited to repairing brain damage, improving intelligence and memory, etc.

[0054] Preferably, the pharmaceutical composition further comprises other active ingredients having intelligence-enhancing effects, and the walnut peptide and other active ingredients may be mixed physically, or may be connected by inclusion or chemical bonds.

[0055] Preferably, the pharmaceutical composition also contains a pharmaceutically necessary carrier; further, the pharmaceutical composition is preferably used orally, and is one of the following: tablets, powders, granules, decoctions, suspensions, and emulsions.

[0056] The fourth aspect of the present invention provides the use of the blood-brain barrier permeable walnut peptide described in the first aspect and / or the pharmaceutical composition described in the third aspect in the preparation of intelligence-enhancing products.

[0057] The above-mentioned intelligence-enhancing products mainly refer to foods or medicines that can repair brain damage, improve memory or promote the development of the nervous system. Preferably, the intelligence-enhancing products include but are not limited to one of medical foods, nutritional supplements and medicines.

[0058] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0059] Example 1

[0060] 1. Separation of walnut peptide samples

[0061] Small pieces of walnut meal were crushed into powder using a grinder. 40 g of walnut meal powder was added into a dispersion flask with 1000 g of distilled water. 500 U of cellulase and 5 U of lipase were added. The mixture was placed in a shaking water bath and incubated at 40 °C for 8 h.

[0062] Filter, collect the residue, add distilled water, the solid-liquid ratio is 1:20. Add 2000U papain, shake in a 60℃ water bath for 5h.

[0063] The enzymatic hydrolysate was poured into a centrifuge cup for centrifugation at a speed of 8000r / min for 10min. The clarified liquid was concentrated and distilled at a temperature of 65°C and a speed of 85r / min. After the concentrated liquid was obtained, it was spray-dried to obtain a walnut peptide sample. The yield of walnut peptide preparation was 56%, and there was no special odor after being placed at room temperature for one month.

[0064] The above samples were subjected to GPC testing in accordance with the collagen peptide operating procedures of the national food safety standard GB 31645-2018.

[0065] 2. HPLC Conditions

[0066] Reagent A

[0067] A.1 Acetonitrile: chromatographic grade, trifluoroacetic acid: analytical grade, water: deionized

[0068] A.2 Standards used for relative molecular mass calibration curve: cytochrome c (MW12384), aprotinin (MW6512), bacillus enzyme (MW1423), tyrosine-tyrosine-arginine (MW451), tyrosine-tyrosine-tyrosine (MW189)

[0069] B. Chromatographic conditions

[0070] B.1 Chromatographic column: TSKgel G2000 SWXL 300mm×7.8mm (GEL LOT 502R).

[0071] B.2 Mobile phase: acetonitrile: water: trifluoroacetic acid = 40:60:0.5.

[0072] B.3 Detection wavelength: 220nm.

[0073] B.4 Flow rate: 0.5 ml / min.

[0074] B.5 Column temperature: 30℃.

[0075] B.6 Injection volume: 10 μl.

[0076] CSample Preparation

[0077] Weigh 125.0 mg of walnut peptide powder, transfer it to a 25 ml volumetric flask, dilute to the mark with mobile phase, sonicate for 10 min, filter with a 0.22 μm organic filter membrane, and use the filtrate to determine its molecular weight distribution.

[0078] Table 1

[0079] Characteristic peak Retention time (min) area% The ratio of peak 3 to its area 1 14.878 5.06 1：0.13 2 16.157 16.525 1：0.43 3 18.161 38.398 1 4 19.053 18.516 1：0.48 5 21.321 5 1：0.13

[0080] Example 2

[0081] Small pieces of walnut meal were crushed into powder using a grinder. 15 g of walnut meal was taken, 100 g of distilled water was added to a dispersion flask, 100 U of hemicellulase and 5 U of lipase were added, and the mixture was placed in a shaking water bath at 40 °C for 3 h.

[0082] Filter, collect the residue, add distilled water, the solid-liquid ratio is 1:15, adjust the pH to 8.5, add 500U trypsin and 100U alkaline protease, and shake in a 37℃ water bath for 5h.

[0083] The enzymatic hydrolyzate was filtered through a Buchner funnel through filter paper and a 0.45 μm filter membrane to obtain a clarified solution. The clarified solution was concentrated and distilled at a temperature of 90°C and a speed of 100 r / min. After the concentrated solution was obtained, it was placed in a freeze dryer to obtain a walnut peptide sample. The yield of walnut peptide preparation was 40%, and there was no special odor when placed at room temperature for one month.

[0084] The above samples were subjected to GPC testing in accordance with the collagen peptide operating procedures of the national food safety standard GB 31645-2018.

[0085] Table 2

[0086] Characteristic peak Retention time (min) area% The ratio of peak 3 to its area 1 15.039 4.158 1：0.15 2 16.213 9.187 1：0.339 3 18.176 27.091 1 4 18.987 21.02 1：0.776 5 21.356 10.251 1：0.378

[0087] Example 3

[0088] Small pieces of walnut meal were crushed into powder using a grinder. 10g of walnut meal was taken, 100g of distilled water was added into a dispersion conical flask, 40U of pectinase, 20U of lysozyme, 10U of ligninase and 3U of lipase were added, and the mixture was placed in a shaking water bath at 70℃ for 10h.

[0089] Filter, collect the residue, add distilled water, the solid-liquid ratio is 1:10, adjust the pH to 8.0, add 100U of ficin, 150U of bromelain and 100U of alkaline protease, and shake in a 60℃ water bath for 20h.

[0090] The enzymatic hydrolysate was filtered through a 10000Da ultrafiltration membrane and a 0.45μm filter membrane to obtain a clarified liquid. The clarified liquid was concentrated and distilled at a temperature of 75°C and a rotation speed of 120r / min. After the concentrated liquid was obtained, it was placed in a freeze dryer to obtain a walnut peptide sample. The yield of walnut peptide preparation was 55%, and there was no special odor when placed at room temperature for one month.

[0091] The above samples were subjected to GPC testing in accordance with the collagen peptide operating procedures of the national food safety standard GB 31645-2018.

[0092] Characteristic peak Retention time (min) area% The ratio of peak 3 to its area 1 13.800 1.751 1：0.07 2 15.097 6.005 1：0.24 3 17.600 25.019 1 4 17.904 8.256 1：0.33 5 20.301 2.252 1：0.09

[0093] Example 4

[0094] Small pieces of walnut meal were crushed into powder using a grinder. 8 g of walnut meal was taken and added with 100 g of distilled water in a dispersion flask. 80 U of pectinase, 20 U of lysozyme and 3.5 U of lipase were added. The mixture was placed in a shaking water bath and placed in a water bath at 35°C for 3 h.

[0095] Filter, collect the residue, add distilled water, the solid-liquid ratio is 1:20. Add 400U neutral protease and 100U papain, and shake in a 60℃ water bath for 3h.

[0096] The enzymatic hydrolysate was poured into a centrifuge tube for centrifugation at a speed of 8000r / min for 10min. The clarified liquid was concentrated and distilled at a temperature of 65°C and a speed of 85r / min. After the concentrated liquid was obtained, it was placed in a freeze dryer to obtain a walnut peptide sample. The yield of walnut peptide preparation was 52%, and there was no special odor after being placed at room temperature for one month.

[0097] The above samples were subjected to GPC testing in accordance with the collagen peptide operating procedures of the national food safety standard GB 31645-2018.

[0098] Characteristic peak Retention time (min) area% The ratio of peak 3 to its area 1 16.202 7.215 1：0.24 2 17.398 16.835 1：0.56 3 18.700 30.063 1 4 20.201 30.364 1：1.01 5 22.500 15.032 1：0.5

[0099] Example 5

[0100] Small pieces of walnut meal were crushed into powder using a grinder. 14 g of walnut meal was taken, 100 g of distilled water was added to a dispersion flask, 85 U of xylanase and 2 U of lipase were added, and the mixture was placed in a shaking water bath at 50°C for 3 h.

[0101] Filter, collect the residue, add distilled water, the solid-liquid ratio is 1:20. Add 450U bromelain, shake in a 60℃ water bath for 3h.

[0102] The enzymatic hydrolysate was poured into a centrifuge tube for centrifugation at a speed of 8000r / min for 10min. The clarified liquid was concentrated and distilled at a temperature of 65°C and a speed of 85r / min. After the concentrated liquid was obtained, it was placed in a freeze dryer to obtain a walnut peptide sample. The yield of walnut peptide preparation was 45%, and there was no special odor after being placed at room temperature for one month.

[0103] The above samples were subjected to GPC testing in accordance with the collagen peptide operating procedures of the national food safety standard GB 31645-2018.

[0104] Characteristic peak Retention time (min) area% The ratio of peak 3 to its area 1 14.703 5.434 1：0.16 2 15.948 15.223 1：0.40 3 17.850 35.058 1 4 18.752 25.499 1：0.67 5 21.100 10.342 1：0.30

[0105] Comparative Example 1

[0106] Small pieces of walnut meal were crushed into powder using a grinder. 5 g of the crushed walnut meal was added to 100 g of distilled water and dispersed in a conical flask. 600 U of papain was added. The pH was adjusted to 6.0 with 1 mol / L hydrochloric acid, and then placed in a shaking water bath at 60°C for 3 h.

[0107] The enzymatic hydrolysate was poured into a centrifuge tube and centrifuged at a speed of 8000 r / min for 10 min. The supernatant was filtered using a 0.45 μm water filter membrane to obtain a clear solution.

[0108] The clarified liquid was concentrated and distilled at a temperature of 65°C and a rotation speed of 85 r / min. After the concentrated liquid was obtained, it was placed in a freeze dryer to obtain a walnut peptide sample. The yield of walnut peptide preparation was 10%. After being placed at room temperature for one month, it had a distinct rancid smell.

[0109] GPC Testing

[0110] HPLC conditions (same as above)

[0111] Table 3

[0112] Characteristic peak Retention time (min) area% The ratio of peak 3 to its area 1 14.76 10.83 1：0.383 2 15.967 21.587 1：0.763 3 18.083 28.275 1 4 18.976 9.896 1：0.35 5 21.328 6.134 1：0.217

[0113] By spectrum Figure 6 It can be seen that the comparative ratio does not meet the patent peak-to-peak ratio characteristics.

[0114] Comparative Example 2

[0115] Small pieces of walnut meal were crushed into powder using a grinder. 15 g of the crushed walnut meal was taken and dispersed in a conical flask with 100 g of distilled water. 100 U of alkaline protease, 300 U of papain and 2 U of esterase were added. The pH was adjusted to 9.5 with 1 mol / L of sodium hydroxide, and then placed in a shaking water bath at 55 °C for 3 h.

[0116] The enzymatic hydrolysate was poured into a centrifuge tube and centrifuged at a speed of 8000 r / min for 10 min. The supernatant was filtered using a 0.45 μm water filter membrane to obtain a clear solution.

[0117] The clarified liquid was concentrated and distilled at a temperature of 65°C and a rotation speed of 85 r / min. After the concentrated liquid was obtained, it was placed in a freeze dryer to obtain a walnut peptide sample. The yield of walnut peptide preparation was 15%. After being placed at room temperature for one month, it had a distinct rancid smell.

[0118] GPC Testing

[0119] HPLC conditions (same as above)

[0120] Table 4

[0121] Characteristic peak Retention time (min) area% The ratio of peak 3 to its area 1 14.878 10.063 1：0.496 2 16.025 12.138 1：0.598 3 18.068 20.295 1 4 18.841 6.443 1：0.317 5 21.324 12.785 1：0.63

[0122] By spectrum Figure 7 It can be seen that the comparative ratio does not meet the patent peak-to-peak ratio characteristics.

[0123] Comparative Example 3

[0124] Small pieces of walnut meal were crushed into powder using a grinder. 10 g of the crushed walnut meal was taken and dispersed in a conical flask with 100 g of distilled water. 500 U of trypsin and 1 mol / L of sodium hydroxide were added to adjust the pH to 8. The mixture was then placed in a shaking water bath and incubated at 37 °C for 3 h.

[0125] The enzymatic hydrolysate was poured into a centrifuge tube and centrifuged at a speed of 8000 r / min for 10 min. The supernatant was filtered using a 0.45 μm water filter membrane to obtain a clear solution.

[0126] The clarified liquid was concentrated and distilled at a temperature of 65°C and a rotation speed of 85 r / min. After the concentrated liquid was obtained, it was placed in a freeze dryer to obtain a walnut peptide sample. The yield of walnut peptide preparation was 20%. After being placed at room temperature for one month, it had a distinct rancid smell.

[0127] Activity determination of walnut peptides in Examples 1-5 and Comparative Example 1

[0128] 1. Blood-brain barrier permeability test

[0129] Walnut polypeptide was prepared into a 13.3mg / ml polypeptide aqueous solution, pH was adjusted to about 8.0, centrifuged at 13000rpm at 4℃ for 5min, and the supernatant was taken for use. Cy7 1mg was weighed and DMSO 50ul was added to dissolve. Then 150ul of polypeptide supernatant was added and slowly mixed for about 2h. Injected into the caudal vein, fluorescence imaging was performed to observe its blood-brain barrier crossing.

[0130] The results are as follows Figure 1-3 As shown, Figure 1 The results of mouse imaging of walnut polypeptides in Example 1 and Example 2 are shown below. Walnut polypeptides were labeled with fluorescent dye Cy7 and injected into the tail edge of mice for in vivo imaging experiments. Figure 1 As shown, there is a large area of ​​strong fluorescence distribution on the mouse head, indicating that the walnut polypeptide in the example group can pass through the blood-brain barrier.

[0131] Figure 2 The mouse imaging results of walnut polypeptides in comparative examples 1 and 2 were as follows. Walnut polypeptides were labeled with fluorescent dye Cy7, injected into the tail edge of mice, and subjected to in vivo imaging experiments. As shown in the figure, no fluorescent signal appeared in the head of the mouse, indicating that the walnut polypeptides prepared by this method cannot penetrate the blood-brain barrier.

[0132] Figure 3 The following are the imaging results of walnut peptides in the blank control group and the Cy7 control group. The mice were injected with saline and Cy7 at the tail edge and subjected to in vivo imaging experiments. As shown in the figure, a very small amount of fluorescent signal appeared in the abdomen of the blank group, a strong fluorescent signal appeared in the tail of the Cy7 control group, and no fluorescent signal distribution appeared in the head of the two groups.

[0133] 2. Research on improving memory function

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0135] (1) Animal grouping and dosage design

[0136] SPF grade Kunming female mice were selected and raised in a barrier grade animal room with a temperature range of (22±2)°C and a relative humidity of 50%-60%. Day: night = 12h: 12h. Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3 and a blank control group were set up respectively. The gavage dose was 4.5g / kg·bw. The blank control group was treated with gavage of distilled water. There were 10 test animals in each group, and gavage was continued for 30d. The test was started 24h after the last gavage.

[0137] (2) Water maze test

[0138] The water depth of the maze lane was 9 cm and the water temperature was 20°C. In the first training, the mice were made to climb the stairs automatically 3 times. In each subsequent training, the mice were made to face the stairs with their backs and climb the stairs automatically once. The distance was gradually lengthened according to the learning performance of the mice. The first training started from point A, and the time it took for the mice to reach the end point from point A was recorded; the second training started from point B, and the distance was lengthened. After training 3 times until more than 80% of the animals reached the end point within 2 minutes, the distance was further lengthened, and the time required for each mouse to reach the end point from point B and the number of errors occurred were recorded respectively (entering any blind end once was counted as an error); the last test was carried out from the starting point, and the time required to reach the end point from the starting point and the number of errors occurred were recorded. In order to achieve the training purpose, mice that did not complete the training within 2 minutes should be guided to reach the end point. The training time of the test animals was limited to 120 seconds, and the test animals that did not reach the end point within 120 seconds were all recorded as 120 seconds. During each training or test, each mouse headed towards the starting point, and finally the total time to reach the end point, the total number of errors, and the total number of mice that reached the end point within 2 minutes for each group of mice in 5 tests were calculated.

[0139] (3) Dark avoidance test

[0140] The training started 24 hours after the last oral gavage. The mice were placed in the bright room with their backs facing the hole. The timer was started at the same time. The mice passed through the hole and entered the dark room to receive electric shock. The timer stopped automatically and the mice were kept in the dark room for 5 minutes. This was the training process. The test started 24 hours later. The latency of each mouse entering the dark room and the number of electric shocks within 5 minutes were recorded. The percentage of mice entering the dark room (wrong response) within 5 minutes was calculated (the percentage of mice entering the dark room to the total number of mice in the group).

[0141] (4) Platform test

[0142] The training started 24 hours after the last oral gavage. The mice were placed on the copper grid in the reaction box and immediately connected to 36V AC. The mice were shocked and jumped back to the platform to avoid injury. The mice stayed on the platform in the reaction box for 5 minutes. This was the training process. The test started 24 hours later. The latency of each mouse jumping off the platform for the first time and the number of errors within 3 minutes were recorded. At the same time, the percentage of mice with incorrect reactions was calculated (the number of mice shocked was the percentage of the total number of mice in the group).

[0143] (5) Statistical methods

[0144] SPSS 20.0 software was used to analyze the data, and the results were expressed as mean ± standard deviation. One-way analysis of variance was used for each group of data, and the least significant difference method was used for pairwise comparison between groups. P < 0.05 was considered statistically significant.

[0145] (6) Experimental results

[0146] As can be seen from Table 1, there was no significant change in the body weight of each group of mice compared with the blank control group. In the water maze test, the time for the mice in Example 1 to reach the end point was less than that in the control group, and there was a significant difference (P<0.05); the number of errors of the mice was significantly lower than that in the control group (P<0.01), indicating that the result of the water maze test in Example 1 was positive. The time for the mice in Example 2 to reach the end point was significantly less than that in the control group (P<0.05); the number of animals reaching the end point was significantly higher than that in the control group (P<0.05), indicating that the result of the water maze test in Example 2 was positive. The time for the mice in Example 3 to reach the end point was significantly less than that in the control group (P<0.05), indicating that the result of the water maze test in Example 3 was positive. There were no significant differences in the time for the mice in Comparative Example 1, Comparative Example 2, and Comparative Example 3 to reach the end point, the number of errors, and the number of animals reaching the end point compared with the control group, and the water maze experiments of Comparative Example 1, Comparative Example 2, and Comparative Example 3 were all negative.

[0147] In the dark avoidance test, the latency of the mice in Example 1 was significantly higher than that in the control group (P<0.01), indicating that the dark avoidance test in Example 1 was positive. The latency of the mice in Example 2 was significantly higher than that in the control group (P<0.05), indicating that the dark avoidance test in Example 2 was positive. The number of errors of the mice in Example 3 was significantly less than that in the control group (P<0.05), indicating that the dark avoidance test in Example 3 was positive. There were no significant differences in the latency, number of errors, and error reaction rate of the mice in Comparative Example 1, Comparative Example 2, and Comparative Example 3 compared with those in the control group, and the dark avoidance tests in Comparative Example 1, Comparative Example 2, and Comparative Example 3 were all negative.

[0148] In the platform jumping test, the latency of the mice in Example 1 was significantly higher than that in the control group (P<0.05), indicating that the platform jumping test in Example 1 was positive. There were no significant differences in the latency, number of errors, and error response rate of the mice in Example 2 compared with the control group, so the platform jumping test in Example 2 was negative. The latency of Example 3 was significantly higher than that in the control group (P<0.05), and the error response rate was significantly lower than that in the control group (P<0.01), indicating that the platform jumping test in Example 3 was positive. There were no significant differences in the latency, number of errors, and error response rate of the mice in Comparative Examples 1, Comparative Example 2, and Comparative Example 3 compared with the control group, and the platform jumping tests in Comparative Examples 1, Comparative Example 2, and Comparative Example 3 were all negative.

[0149] The above results show that the walnut peptides in the example group can promote the active avoidance ability and passive avoidance ability of the test animals, while the walnut peptides in the comparative example group cannot promote the active avoidance ability and passive avoidance ability of the test animals, that is, the walnut peptides in the example group have the effect of improving memory, while the walnut peptides in the comparative example group do not have the effect of improving memory, and the chromatographic peaks and area ratio characteristics of the GPC spectrum of the example group have a guiding role in the evaluation of brain function.

[0150] Table 5 Effect of walnut peptide on body weight of mice

[0151]

[0152]

[0153] Table 6 Effects of walnut peptide on the mouse water maze test

[0154]

[0155] Table 7 Effect of walnut peptide on dark avoidance test in mice

[0156]

[0157] Table 8 Walnut peptides on mice jumping platform experiment

[0158]

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A walnut peptide for improving memory, characterized in that: The preparation method of the walnut peptide comprises the following steps: crushing walnuts and dispersing them in water to obtain a suspension, adding a composite enzyme for enzymolysis, obtaining a clear solution after enzymolysis and drying it to obtain an extract, wherein the extract is walnut peptide; The enzymatic hydrolysis method is as follows: Add pectinase, lysozyme, ligninase and lipase to the suspension, and put it into a shaking water bath; retain the filter residue after enzymolysis; add ficin, bromelain, alkaline protease and water to the filter residue, and retain the filtrate after enzymolysis, which is the clarified solution; During the protease hydrolysis process, the pH of the reaction is adjusted within the range of 7.5 to 10.5 according to the type of protease; the temperature of the water bath is 20 to 75°C, and the water bath time is 3 to 36 hours.

2. The walnut peptide for improving memory according to claim 1, characterized in that: The walnuts are powders obtained by mechanically crushing walnuts. The walnuts include unprocessed walnut kernels after removing the shells, and also include walnut processing byproducts.

3. The walnut peptide for improving memory according to claim 1, characterized in that: The walnut is walnut meal.

4. The walnut peptide for improving memory according to claim 3, characterized in that: The walnut meal is crushed and passed through a 200-mesh sieve.

5. The walnut peptide for improving memory according to claim 1, characterized in that: In the suspension, the ratio of walnut powder to water is 4-15g:100g.

6. The walnut peptide for improving memory according to claim 1, characterized in that: In the preparation method, the clear solution is obtained by performing solid-liquid separation on the reaction system after enzymatic hydrolysis to obtain a supernatant, and sterilizing and filtering the supernatant to obtain the clear solution.

7. The walnut peptide for improving memory according to claim 6, characterized in that: The solid-liquid separation is centrifugation, dead-end filtration and cross-flow filtration.

8. The walnut peptide for improving memory according to claim 6, characterized in that: The sterilization filtration is performed using a 0.45 μm water filter membrane.

9. The walnut peptide for improving memory according to claim 1, characterized in that: The clear solution is dried by concentrating and distilling to remove the solvent, and then spray drying or freeze drying to obtain the extract.

10. A pharmaceutical composition for improving memory, characterized in that: In the pharmaceutical composition, the walnut peptide for improving memory according to claim 1 is used as an active ingredient.

11. The pharmaceutical composition for improving memory according to claim 10, characterized in that: The pharmaceutical composition also contains a pharmaceutically necessary carrier.

12. The pharmaceutical composition for improving memory according to claim 10, characterized in that: The pharmaceutical composition is used orally and is in the form of, but not limited to, tablets, powders, granules, decoctions, suspensions, and emulsions.

13. Use of the memory-improving walnut peptide according to claim 1 and / or the pharmaceutical composition according to claim 10 in the preparation of a product for improving memory; The product described is a drug.

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