Clearing Aβ 1-42 Walnut-derived peptides with high antioxidant activity in protein deposition improve learning and memory and their applications

Abstract

The present invention discloses a method for removing Aβ 1‑42 The walnut-derived peptide with high antioxidant activity for protein deposition improves learning and memory, and its amino acid sequence is Tyr‑Val‑Tyr‑Tyr‑Pro‑Ser‑Pro‑Lys. The present invention also discloses its use in the preparation of drugs for neuroprotection and improvement of learning and memory, and its use in the preparation of drugs for delaying the occurrence and development of AD. The present invention replaces the leucine at the 3rd and 4th positions of the C-terminus of the original peptide YVLLPSPK with tyrosine to form the derivative peptide YVYYPSPK, which significantly improves its antioxidant activity, thereby having higher neuroprotective activity and improving learning and memory. The derivative peptide YVYYPSPK of the present invention can effectively eliminate Aβ, the early clinical symptom of AD 1‑42 Protein deposition can be used to prevent or delay nerve cell damage, thereby delaying the occurrence and development of AD. It can be used to prepare drugs that delay the occurrence and development of AD, and has huge market potential.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for removing Aβ 1-42 Walnut-derived peptides with high antioxidant activity for protein deposition improve learning and memory and their applications. Background Art

[0002] According to the results of the eighth national census in 2022, my country currently has 280.04 million people aged 60 and above, accounting for 19.8% of the total population, of which 209.78 million are aged 65 and above, accounting for 14.9%, officially entering a stage of deep population aging. The incidence, prevalence and mortality of diseases related to deep aging have also increased significantly, bringing heavy pressure to society. Among them, the number of people with Alzheimer's disease (AD) and other dementias characterized by learning and memory disorders continues to increase, and the socioeconomic burden is becoming increasingly obvious.

[0003] Oxidative stress refers to the imbalance of the body's oxidative and antioxidant systems due to the inability of the cell's endogenous antioxidant system to effectively remove accumulated free radicals. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are free radicals produced by endogenous redox reactions in eukaryotic organisms. ROS include hydroxyl radicals, superoxide anions, hydrogen peroxide, and peroxyl radicals; RNS include nitric oxide and peroxynitrite ions. At normal physiological levels in the body, ROS / RNS, as important signaling molecules, help promote a series of biological processes, such as gene expression, cell proliferation, angiogenesis, programmed cell death, and aging. When the accumulated ROS / RNS exceeds the body's threshold, oxidative and nitrosative stress will occur due to a series of external factors, such as radiation (X-rays and ultraviolet rays, etc.), chemical reagents (metal ions, ozone, hypochlorous acid and hypobromous acid, etc.), environmental pollutants, exogenous substances such as drugs and their metabolites, and smoking, or internal factors. Excessive ROS and RNS can damage biological macromolecules in the body, causing a decline in learning and memory, cognitive impairment, and further induce neurodegenerative diseases such as AD. Amyloid β (Aβ 1-42 ) is a proven biomarker for detecting early AD.

[0004] The World Health Organization reported that dietary nutrition is one of the important factors in regulating human health. Its impact on human health is far greater than that of medical factors. It plays an important role in enhancing and promoting the health of the body and preventing or controlling pathological processes. Active peptides have a wide range of sources, low production input, easy production, high affinity, and strong physiological activity in the body. They have advantages in dietary nutrition that cannot be matched by drug interventions, such as easy absorption, early prevention, and green safety. Active peptides have unique physiological functions that original proteins or amino acid molecules do not have at all. At the same time, the amino acids that make up active peptides are not necessarily essential amino acids, which provides opportunities for humans to make full use of protein resources.

[0005] Our team's previous in-depth research found that walnut active peptide YVLLPSPK can significantly improve nerve cell damage caused by oxidative stress and learning and memory disorders in model mice 1 In order to enhance the neuroprotective activity of active peptides, the present invention intends to obtain derivative peptides with high antioxidant activity by replacing the amino acid composition of active peptides, so as to have higher neuroprotective activity, prevent or delay nerve cell damage, and thus delay the occurrence and development of AD.

[0006] References

[0007] 1 Zhao F, Liu C, Fang L, Lu H, Wang J, Gao Y, Gabbianelli R, Min W.Walnut-Derived Peptide Activates PINK1 via the NRF2 / KEAP1 / HO-1 Pathway,Promotes Mitophagy, and Alleviates Learning and Memory Impairments in a MiceModel. J Agric Food Chem. 2021 Mar 10;69(9):2758-2772. doi: 10.1021 / acs.jafc.0c07546. Epub 2021 Feb 16. PMID: 33591165. Summary of the invention

[0008] The present invention aims to provide a method for removing Aβ 1-42 Walnut-derived peptides with high antioxidant activity for protein deposition improve learning and memory and their applications to address the deficiencies of the prior art.

[0009] The present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for removing Aβ 1-42 High antioxidant activity of protein deposition improves learning and memory Walnut-derived peptide with the amino acid sequence of Tyr-Val-Tyr-Tyr-Pro-Ser-Pro-Lys (YVYYPSPK).

[0011] The second aspect of the present invention provides the use of the above walnut-derived peptide in the preparation of medicines for neuroprotection and improving learning and memory.

[0012] Furthermore, neuroprotection involves the removal of Aβ from neurons 1-42 Protein deposition.

[0013] The third aspect of the present invention provides the use of the above-mentioned walnut-derived peptide in the preparation of a drug for delaying the occurrence and development of Alzheimer's disease.

[0014] Beneficial effects of the present invention:

[0015] The present invention replaces the leucine (L) at the 3rd and 4th positions of the C-terminus of the original peptide YVLLPSPK, which has the ability to significantly improve the nerve cell damage caused by oxidative stress and the learning and memory impairment of model mice, with tyrosine (Y), thereby forming a derivative peptide YVYYPSPK, which significantly improves its antioxidant activity, thereby having higher neuroprotective activity and improving learning and memory. The derivative peptide YVYYPSPK of the present invention can effectively eliminate Aβ, a preclinical symptom of AD 1-42 Protein deposition can be used to prevent or delay nerve cell damage, thereby delaying the occurrence and development of AD, and can be used to prepare drugs for delaying the occurrence and development of AD, with huge market potential. The derivative peptide YVYYPSPK of the present invention is a natural derivative peptide, which has the characteristics of high activity, high bioavailability and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the ABTS free radical scavenging rate of the peptide derived from Example 1.

[0017] Figure 2 This is a graph showing the hydroxyl radical scavenging rate of the peptide derived from Example 2.

[0018] Figure 3 This is a graph showing the DPPH radical scavenging rate of the peptide derived from Example 3.

[0019] Figure 4 This is the ORAC graph of the peptide derived from Example 4.

[0020] Figure 5 This is a map of the navigation path for mice in the Morris water maze test in Example 5.

[0021] Figure 6 This is a bar graph of the escape latency of mice in the Morris water maze test in Example 5.

[0022] Figure 7 This is a bar graph of the effective movement distance of mice in the Morris water maze test in Example 5.

[0023] Figure 8 Aβ derived from the peptide of Example 6 1-42 Protein deposition clearance effect diagram Aβ 1-42 Western blot image. The depth of the band color in the image represents the intracellular Aβ 1-42 The darker the band, the higher the Aβ content. 1-42 The higher the content, the higher the Aβ 1-42The more protein deposits there are, the greater the damage to learning and memory. β-actin is used as an internal reference.

[0024] Fig. 9 Aβ derived from the peptide of Example 6 1-42 Protein deposition clearance effect diagram Aβ 1-42 Relative protein expression bar graph.

[0025] The different letters above the bar graphs above indicate significant differences between different treatments ( p <0.05). DETAILED DESCRIPTION

[0026] The present invention is further explained below in conjunction with the embodiments and the accompanying drawings, but the embodiments do not limit the present invention in any way.

[0027] In order to further improve the antioxidant activity, the following amino acids were replaced in the walnut active peptide YVLLPSPK (hereinafter referred to as the original peptide YVLLPSPK):

[0028] 1) The third leucine (L) at the C-terminus of the original peptide YVLLPSPK was replaced with tyrosine (Y) to form the walnut-derived peptide YVYLPSPK (hereinafter referred to as the derived peptide YVYLPSPK);

[0029] 2) The 3rd and 4th leucine (L) at the C-terminus of the original peptide YVLLPSPK were replaced with tyrosine (Y) to form the walnut-derived peptide YVYYPSPK (hereinafter referred to as the derived peptide YVYYPSPK).

[0030] The original peptide YVLLPSPK, the derived peptide YVYLPSPK, and the derived peptide YVYYPSPK involved in the following examples were directly synthesized by a commercial company. The specific method of synthesis can be found in the reference (Huang Bei. Research Progress in Solid Phase Synthesis of Peptides [J]. Henan Chemical Industry, 2013, 30(01): 28-30+58.).

[0031] Example 1 Study on the scavenging rate of ABTS free radicals by derivative peptides

[0032] Dissolve 0.0045 g ABTS in 1.1025 mL distilled water to prepare a 7.4 mmol / L ABTS aqueous solution. Dissolve 0.0025 g potassium persulfate in 3.575 mL distilled water to prepare a 2.6 mmol / L potassium persulfate aqueous solution. Mix the two in a volume ratio of 1:1, and let stand at 4 °C for 12 h-16 h in the dark to prepare the ABTS free radical stock solution. When using, use 5 mmol / L pH7.4 PBS to adjust the concentration so that the absorbance of the ABTS free radical stock solution at a wavelength of 734 nm is 0.700±0.002 to obtain the ABTS free radical working solution. Take 10 μL of 0.1 mg / mL original peptide YVLLPSPK aqueous solution, 10 μL of 0.1 mg / mL derivative peptide YVYLPSPK aqueous solution, 10 μL of 0.1 mg / mL derivative peptide YVYYPSPK aqueous solution and 10 μL of 0.1 mg / mL GSH aqueous solution, add them into a black 96-well plate respectively, and then add 190 μL A BTS free radical working solution respectively. After accurately reacting for 6 min, use a microplate reader to measure the absorbance value (A1) at a wavelength of 734 nm, and set three replicates.

[0033] The calculation formula of ABTS free radical scavenging rate is as follows:

[0034]

[0035] Wherein: A0 is the absorbance value measured by replacing the original peptide YVLLPSPK aqueous solution, the derivative peptide YVYLPSPK aqueous solution, the YVYYPSPK aqueous solution or the GSH aqueous solution with an equal volume of distilled water in the ABTS free radical working solution (without adding the original peptide YVLLPSPK, the derivative peptide YVYLPSPK, the derivative peptide YVYYPSPK or GSH); A1 is the absorbance value measured by adding the original peptide YVLLPSPK aqueous solution, the derivative peptide YVYLPSPK aqueous solution, the derivative peptide YVYYPSPK aqueous solution or the GSH aqueous solution to the ABTS free radical working solution; A2 is the absorbance value measured by replacing the ABTS free radical working solution with an equal volume of 5 mmol / L pH 7.4 PBS solution (with adding the original peptide YVLLPSPK, the derivative peptide YVYLPSPK, the derivative peptide YVYYPSPK or GSH).

[0036] like Figure 1 As shown in Figure 2, at a concentration of 0.1 mg / mL, the ABTS free radical scavenging rate of the positive control GSH was 44.17±0.0023%, and the ABTS free radical scavenging rate of the original peptide YVLLPSPK was 27.66±0.0084%. The ABTS free radical scavenging rates of the derived peptides were significantly higher than those of the original peptide YVLLPSPK and the positive control GSH ( p<0.05), which were 67.76±0.0089% for YVYLPSPK and 89.22±0.0070% for YVYYPSPK. The ABTS free radical scavenging rate of the derivative peptide YVYYPSPK was significantly higher than that of the derivative peptide YVYLPSPK.

[0037] Example 2 Study on the scavenging rate of hydroxyl radicals by derivative peptides

[0038] 200 μL of 6 mmol / L ferrous sulfate aqueous solution, 200 μL of 6 mmol / L salicylic acid-ethanol solution, 200 μL of 6 mmol / L hydrogen peroxide aqueous solution, and 200 μL of sample solution (sample aqueous solution: 1 mg / mL original peptide YVLLPSPK aqueous solution, 1 mg / mL derivative peptide YVYLPSPK aqueous solution, 1 mg / mL derivative peptide YVYYPSPK aqueous solution, or 1 mg / mL GSH aqueous solution) were added to a centrifuge tube, reacted in a 37°C water bath for 30 min, adjusted to zero with distilled water, and the absorbance was measured at 510 nm (A X ), set up three repetitions.

[0039] The calculation formula of hydroxyl radical scavenging rate is as follows:

[0040] Hydroxyl free radical scavenging rate%=[A0-(A X -A X0 ) / A0]×100%

[0041] Where: A0 is the absorbance value when the sample solution is replaced by an equal volume of distilled water; A X A is the absorbance value when the sample solution is added; X0 It is the absorbance value when an equal volume of ethanol replaces the salicylic acid-ethanol solution.

[0042] like Figure 2 As shown in Figure 2, at a concentration of 1 mg / mL, the hydroxyl radical scavenging rate of the positive control GSH was 32.46±0.0021%, and the hydroxyl radical scavenging rate of the original peptide YVLLPSPK was 14.21±0.00014%, while the hydroxyl radical scavenging rates of the derived peptides were significantly higher than those of the original peptides ( p <0.05), respectively, YVYLPSPK was 22.91±0.0086%, YVYYPSPK was 36.96±0.0019%, among which, the hydroxyl radical scavenging rate of the derivative peptide YVYYPSPK was also significantly higher than that of the positive control GSH ( p <0.05).

[0043] Example 3 Study on the scavenging rate of DPPH free radicals by the derivative peptides

[0044] Dissolve 0.0023 g DPPH in 100 mL 95 v / v% ethanol to prepare a 60 μmol / L DPPH free radical working solution and store it in the dark. Mix the sample solution (sample aqueous solution: 1 mg / mL original peptide YVLLPSPK aqueous solution, 1 mg / mL derivative peptide YVYLPSPK aqueous solution, 1 mg / mL derivative peptide YVYYPSPK aqueous solution or 1 mg / mL GSH aqueous solution) with 60 μmol / L DPPH free radical working solution in a volume ratio of 1:1 and let it stand in the dark at room temperature for 30 min. After centrifugation at 4500 rpm for 10 min, take the supernatant and test the absorbance (A1) at a wavelength of 517 nm, setting three replicates.

[0045] The calculation formula of DPPH free radical scavenging rate is as follows:

[0046]

[0047] Wherein: A0 is the absorbance value measured by replacing the sample solution with an equal volume of distilled water in the DPPH free radical working solution (without adding the original peptide YVLLPSPK, derivative peptide YVYLPSPK, derivative peptide YVYYPSPK or GSH); A1 is the absorbance value measured by adding the sample solution to the DPPH free radical working solution; A2 is the absorbance value measured by replacing the DPPH free radical working solution with an equal volume of 95 v / v% ethanol (with adding the original peptide YVLLPSPK, derivative peptide YVYLPSPK, derivative peptide YVYYPSPK or GSH).

[0048] like Figure 3 As shown in Figure 2, at a concentration of 1 mg / mL, the DPPH free radical scavenging rate of the positive control GSH was 96.03±0.0029%, and there was no significant difference in the DPPH free radical scavenging rate between the two derivative peptides and the original peptide ( p >0.05), which were 33.52±0.0084% for original peptide YVLLPSPK, 31.91±0.0055% for derivative peptide YVYLPSPK, and 35.10±0.0126% for derivative peptide YVYYPSPK. The DPPH free radical scavenging rate of derivative peptide YVYYPSPK was significantly higher than that of derivative peptide YVYLPSPK.

[0049] Example 4 Study on the Oxidative Radical Absorption Capacity (ORAC) of Derivatized Peptides

[0050] Fluorescein sodium salt and azobisisobutylamidine hydrochloride (AAPH) were diluted with 75 mmol / L pH7.4 PBS buffer to 63 mmol / L fluorescein sodium salt working solution and 6 mmol / L AAPH working solution, respectively, and stored in the dark. 25 μL 75 mmol / L pH7.4 PBS buffer and 25 μL Trolox standard solution (concentrations of 6.25 μM, 12.5 μM, 25 μM and 50 μM, prepared with 75 mmol / L pH7.4 PBS buffer) or 25 μL sample solution (sample solution: 1 mg / mL original peptide YVLLPSPK in PBS, 1 mg / mL derivative peptide YVYLPSPK in PBS, 1 mg / mL derivative peptide YVYYPSPK in PBS or 1 mg / mL GSH in PBS, prepared with 75 mmol / L pH7.4 PBS) were added to a black 96-well plate, and three parallel wells were set for each Trolox standard solution or sample solution. After shaking the black 96-well plate for 2 min, incubate at 37 °C for 10 min. Use a multi-channel pipette to add 150 μL of 63 mmol / L sodium fluorescein working solution to each well at the same time, shake for 5 s, and incubate at 37 °C for 20 min. Use a multi-channel pipette to quickly add 25 μL of 6 mmol / L AAPH working solution to each well to start the reaction. At the same time, two control groups were set up, one of which was a black 96-well plate with free radicals (+AAPH) added to the plate. This group replaced the Trolox standard solution or sample solution with an equal volume of 75 mmol / L pH7.4 PBS buffer, and the other group was a black 96-well plate without free radicals (-AAPH). This group replaced the Trolox standard solution or sample solution with an equal volume of 75 mmol / L pH7.4 PBS buffer, and the AAPH working solution was also replaced with an equal volume of 75 mmol / L pH7.4 PBS buffer; three parallel wells were set up in each group. The excitation wavelength was set to 485 nm and the emission wavelength to 535 nm. The whole system was maintained at 37°C. The fluorescence intensity was measured every 2 min until it reached 5% of the initial fluorescence intensity (60 cycles were set in this experiment). The fluorescence intensity of each well at different time points measured in the experiment was compared with the fluorescence intensity data at the initial time to obtain the relative fluorescence intensity (the initial fluorescence intensity value was set to 1), and the area under the fluorescence decay curve (AUC) was calculated using the approximate integration method:

[0051] AUC=0.5×[2×(ƒ0+ƒ 1… +ƒ n-1 +ƒ n )-ƒ0-ƒ n ]×△t

[0052] Where, nis the relative fluorescence intensity of the nth measurement point; △t is the interval between adjacent time points, 2 min.

[0053] The above formula can be simplified to

[0054] AUC=2×(ƒ0+ƒ 1… +ƒ n-1 +ƒ n )-ƒ0-ƒ n

[0055] With Trolox concentration as the horizontal axis, AUC Trolox -AUC 空白 The value is the vertical axis, and the Trolox standard curve is drawn.

[0056] The results are expressed as ORAC values. The ORAC values ​​of the samples are expressed as μmol Trolox Equivalent g -1 The specific calculation formula is as follows:

[0057] ORAC value = [(AUC 样品 -AUC 空白 ) / (AUC Trolox -AUC 空白 )]×(C Trolox / C 样品 ).

[0058] Among them, AUC 样品 The result of adding sample solution; AUC 空白 The results are for the +AAPH control group; AUC Trolox -AUC 空白 is the value obtained by substituting 100 μM into the Trolox standard curve; C Trolox AUC 样品 -AUC 空白 Substitute the Trolox concentration obtained by Trolox standard curve; C 样品 is the sample molar concentration.

[0059] like Figure 4 As shown, at a concentration of 1 mg / mL, the ORAC value of the positive control GSH was 5533.64±155.99 μmolTrolox Equivalent g -1 The ORAC value of the original peptide YVLLPSPK was 4401.32±129.03 μmol TroloxEquivalent g -1 , while the ORAC values ​​of the two derived peptides were significantly higher than those of the original peptide and the positive control GSH ( p<0.05), respectively, YVYLPSPK was 6086.65±144.33 μmol Trolox Equivalent g -1 , YVYYPSPK is 7950.54±174.25μmol Trolox Equivalent g -1 The ORAC value of the derivative peptide YVYYPSPK was significantly higher than that of the derivative peptide YVYLPSPK.

[0060] Example 5 Study on the effect of derivative peptides on learning and memory in cognitively impaired mice

[0061] Mouse studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the European Commission. Male C57BL / 6J mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Benxi, China) and weighed 18–20 g. Mice were housed in a stable temperature (22 ± 2 °C) with a 12 h / 12 ​​h light / dark cycle. Mice were fed standard chow and water ad libitum. The mice were divided into six groups (n = 10 / group): (i) blank control group: mice were gavaged with normal saline for 37 days; (ii) learning and memory impairment group: mice were gavaged with normal saline for 37 days, and scopolamine (1 mg / kg.bw / d, scopolamine concentration was 0.05 mg / mL) was intraperitoneally injected for 7 days starting from the 31st day; (iii) positive control group: mice were gavaged with normal saline for 30 days, and piracetam (400 mg / kg.bw / d, piracetam concentration was 40 mg / mL) was gavaged and scopolamine (1 mg / kg.bw / d, scopolamine concentration was 0.05 mg / mL) was intraperitoneally injected for 7 days starting from the 31st day; (iv) original peptide YVLLPSPK treatment group: mice were treated with original peptide YVLLPSPK (60 mg / kg.bw / d, original peptide YVLLPSPK concentration was 6 The mice were gavaged with 100 mg / mL of scopolamine for 37 days, and scopolamine (1 mg / kg.bw / d, 0.05 mg / mL) was injected intraperitoneally on the 31st day for 7 days; (v) derivative peptide YVYLPSPK treatment group: the mice were gavaged with 100 mg / mL of scopolamine for 37 days, and scopolamine (1 mg / kg.bw / d, 0.05 mg / mL) was injected intraperitoneally on the 31st day for 7 days; (vi) derivative peptide YVYYPSPK treatment group: the mice were gavaged with 100 mg / mL of scopolamine for 37 days, and scopolamine (1 mg / kg.bw / d, 0.05 mg / mL) was injected intraperitoneally on the 31st day for 7 days. mg / kg.bw / d, scopolamine concentration was 0.05 mg / mL) by intraperitoneal injection for 7 days.

[0062] The physiological saline involved in this embodiment is 0.9 w / v% physiological saline, and scopolamine, piracetam, original peptide YVLLPSPK, derivative peptide YVYLPSPK, and derivative peptide YVYYPSPK are all prepared with physiological saline.

[0063] The Morris water maze test started on the 31st day and lasted for 7 days, with the first 6 days being training and the last day being the formal test (each time the test was started again after the intraperitoneal injection wound had healed).

[0064] Warm water at 23±2℃ was injected into the circular pool of the water maze with a diameter of 120 cm. At the same time, a circular platform with a diameter of 8 cm and a height of 35 cm was placed 1 cm under the water and fixed in the NW quadrant. Each mouse was tested once a day. The mouse was gently placed in the pool. The image acquisition system recorded the swimming trajectory of the mouse and timed for 120 s. The time taken by the mouse to find the circular platform within 120 s was recorded as the escape latency. The mouse that reached the circular platform stayed on the platform for 10 s and then was put back into the cage. The mouse that failed to find the circular platform within 120 s was guided to the circular platform and was also allowed to stay on the platform for 10 s. The escape latency was recorded as 120 s. After each training session, the mouse was wiped dry with a dry towel to prevent stress caused by low temperature. Subsequently, the time from the mouse being placed in the water to finding the circular platform was analyzed and calculated according to the data acquisition system. The experimental results are as follows: Figure 5-7As shown. In the Morris water maze test, the exploration paths of animals are generally divided into four types: "random, linear, trend, and edge". The exploration paths of mice in the scopolamine-induced learning and memory impairment group were not regular, mainly edge-based, and required a long time to find the circular platform (the escape latency of mice in the learning and memory impairment group was 113.5 s, and the escape latency of mice in the blank control group was 39.6 s). This shows that the model was successfully established by intraperitoneal injection of scopolamine, and the learning ability of mice in the learning and memory impairment group was significantly reduced. The escape latency of mice in the positive control group was 49.7 s; the escape latency of mice in the original peptide YVLLPSPK treatment group was 65 s; the escape latency of mice in the two derivative peptide treatment groups was significantly shorter than that in the original peptide YVLLPSPK treatment group, namely 56.1 s in the derivative peptide YVYLPSPK treatment group and 26 s in the derivative peptide YVYYPSPK treatment group; among them, the escape latency of mice in the derivative peptide YVYYPSPK treatment group was also significantly shorter than that in the positive control group. The effective movement distance of mice in the learning and memory impairment group was 5.0 cm, which was significantly lower than the effective movement distance of 33.5 cm in the blank control group. The effective movement distance of mice in the original peptide YVLLPSPK treatment group was 15.2 cm, the effective movement distance of mice in the derivative peptide YVYLPSPK treatment group was 16.9 cm, and the effective movement distance of mice in the derivative peptide YVYYPSPK treatment group was 36.0 cm. The effective movement distance of mice in the derivative peptide YVYYPSPK treatment group was significantly higher than the effective movement distance of 21.0 cm in the positive control group.

[0065] Example 6 Effect of Derivative Peptides on Aβ in Hippocampal Neurons Induced by Scopolamine in Mice 1-42 Research

[0066] Original peptide / derivative peptide group: Mouse hippocampal neuronal cells HT-22 were inoculated into T25 culture flasks and cultured at 37°C until the cells were completely attached to the bottom of the flask. Then, original peptide YVLLPSPK / derivative peptide YVYLPSPK / derivative peptide YVYYPSPK (original peptide / derivative peptide were prepared with DMEM complete culture medium, and the stock concentration was 1000 μM) were added at a final concentration of 100 μM. After culturing at 37°C for 24 hours, the supernatant was removed, and scopolamine (scopolamine was prepared with DMEM cell culture medium, and the stock concentration was 40 mg / mL) was added at a final concentration of 4 mg / mL. After induction at 37°C for 4 hours, the cells were collected, resuspended and washed three times with PBS to remove the residual culture medium in the cells. Blank control group (blank): The original peptide / derivative peptide was replaced with an equal volume of DMEM complete culture medium, and scopolamine was replaced with an equal volume of DMEM complete culture medium. Model injury group (model): the original peptide / derivative peptide was replaced with an equal volume of DMEM complete culture medium. Lysis buffer (cell and lysate mass volume ratio of 1 mg: 10 µL, lysate: Shanghai Biotech Biotechnology Co., Ltd., P0013) was added to the washed cells of each group. After lysis on ice for 30 min, the cells were sonicated at 300 w for 2 min and centrifuged at 12000 rpm for 10 min. The supernatant was the protein sample. After the protein concentration of the protein sample was determined using the BCA protein concentration assay kit (Shanghai Biotech Biotechnology Co., Ltd.), the protein sample was mixed with 2xSDS-PAGE protein loading buffer at a volume ratio of 1:1 and the protein was fully denatured in a 100 ℃ metal bath for 10 min. The proteins in the protein sample were separated on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene fluoride derivative membrane (PVDF) for transfer for 40 min. The membrane was washed three times with TSBT buffer (10X) (5 min each time), immersed in 5 w / v% skim milk powder (5 w / v% skim milk powder prepared with TSBT buffer (10X)) for blocking at room temperature for 1 h, and then washed again three times with TBST buffer (10X) (5 min each time). 10 mL of primary antibody, i.e., Aβ 1-42 Antibody (Cat#14974, Cellsignaling, prepared with 3 w / v% skim milk powder in TSBT buffer (10X)) was diluted 1:1000 against Aβ 1-42Antibody), incubated overnight at 4 ℃. The next day, after recovering the primary antibody, the membrane was washed 3 times with TBST buffer (10X) (5 min each time). Add 10 mL of goat anti-rabbit IgG secondary antibody (GB23303, Servicebio, diluted with 3 w / v% skim milk powder (3 w / v% skim milk powder with TSBT buffer (10X)) at a dilution ratio of 1:15000), incubate at room temperature for 1 h, recover the secondary antibody, and wash the membrane 3 times with TBST buffer (10X) (5 min each time). Apply ECL luminescent solution and use ImageQuant LAS 500 software (GE Healthcare) to observe protein bands.

[0067] The test results are as follows Figure 8 and 9 As shown in the figure, compared with the blank control group, the Aβ 1-42 The color of the band was significantly enhanced. After adding the original peptide YVLLPSPK, Aβ 1-42 The color of the band weakened, and after adding the derivative peptide YVYLPSPK and derivative peptide YVYYPSPK, Aβ 1-42 The color of the bands is weaker than that of the original peptide. 1-42 The color of the band is the weakest. Compared with the model injury group, the Aβ 1-42 The protein expression levels were significantly reduced, indicating that both the original peptide and the derived peptide have the ability to clear Aβ 1-42 The ability of the derived peptide to deposit Aβ 1-42 The protein expression level was significantly reduced, indicating that the derived peptide has a stronger ability to clear Aβ 1-42 Among them, the derivative peptide YVYYPSPK had the strongest ability to clear Aβ 1-42 Protein deposition capacity.

[0068] The above results show that the derivative peptides YVYLPSPK and YVYYPSPK have higher antioxidant capacity than the original peptide YVLLPSPK. Among them, the antioxidant activity of YVYYPSPK is significantly higher than that of glutathione, and it significantly improves the learning and memory of mice with cognitive impairment induced by scopolamine. In addition, the derivative peptide YVYYPSPK has a higher antioxidant capacity than the original peptide YVLLPSPK. 1-42 These results indicate that the derivative peptide YVYYPSPK has the ability to clear Aβ 1-42 A derived peptide with high antioxidant activity and protein deposit-clearing ability that improves learning and memory.

Claims

1. Clear Aβ 1-42 High antioxidant activity of protein deposition improves learning and memory Walnut-derived peptide with the amino acid sequence of Tyr-Val-Tyr-Tyr-Pro-Ser-Pro-Lys.

2. Use of the walnut-derived peptide according to claim 1 in the preparation of medicines for improving learning and memory.

3. Use of the walnut-derived peptide according to claim 1 in the preparation of a drug for delaying the occurrence and development of Alzheimer's disease.

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