阅读说明：本技术 一种用于预防阿尔兹海默症的核桃多肽及其应用 (Walnut polypeptide for preventing Alzheimer's disease and application thereof ) 是由 申烨华 林立钶 李聪 李婷婷 陈邦 于 2021-11-08 设计创作，主要内容包括：本发明公开了一种用于预防阿尔兹海默症的核桃多肽及其应用,所述核桃多肽自C端到N端的氨基酸序列为Glu-Pro-Glu-Val-Leu-Arg。本发明从核桃粕提取核桃蛋白,用胰蛋白酶对核桃蛋白进行酶解得到核桃多肽,1kDa超滤膜分离后用LC-MS/MS鉴定多肽的组成。根据液质结果及分子对接模拟多肽与乙酰胆碱酯酶的相互作用,筛选出1条具备改善记忆功效的多肽。通过小鼠实验证明了其可有效改善D型半乳糖带来的脑力损伤,证实其具有预防阿尔兹海默症(AD)的潜力。脑组织HE染色、体外抗氧化实验验和分子对接模拟证实其可减少海马区蛋白斑块、降低体内氧化应激和抑制乙酰胆碱酯酶活性、预防AD。另外,本发明从核桃粕中酶解制备核桃多肽,不仅步骤简便,而且有利于提升核桃价值。(The invention discloses a walnut polypeptide for preventing Alzheimer's disease and application thereof, wherein the amino acid sequence of the walnut polypeptide from the C end to the N end is Glu-Pro-Glu-Val-Leu-Arg. The invention extracts walnut protein from walnut meal, uses trypsin to carry out enzymolysis on the walnut protein to obtain walnut polypeptide, and uses LC-MS/MS to identify the composition of the polypeptide after the 1kDa ultrafiltration membrane is separated. And screening 1 polypeptide with memory improving effect according to the liquid quality result and the interaction between the molecular docking simulation polypeptide and the acetylcholinesterase. Proved by experiments on mice, the D-type galactose has the effect of effectively improving the mental injury caused by the D-type galactose, and the potential of preventing Alzheimer's Disease (AD) is proved. Brain tissue HE staining, in vitro antioxidant experiment and molecular docking simulation prove that the composition can reduce hippocampal protein plaques, reduce in vivo oxidative stress, inhibit acetylcholinesterase activity and prevent AD. In addition, the method for preparing the walnut polypeptide from the walnut meal through enzymolysis has simple and convenient steps and is beneficial to improving the walnut value.)

1. A walnut polypeptide for preventing Alzheimer's disease, which is characterized in that: the amino acid sequence of the walnut polypeptide from the C end to the N end is Glu-Pro-Glu-Val-Leu-Arg.

2. The use of the walnut polypeptide of claim 1 in the preparation of a medicament for preventing alzheimer's disease.

Technical Field

The invention belongs to the technical field of polypeptides, and particularly relates to a polypeptide which is derived from walnut meal and can prevent Alzheimer's disease.

Background field of the invention

The learning and memory functions are the high-level functions of the brain, one of the essential abilities of human survival, and have been highly valued. Learning and memory dysfunction is often the symptom of various diseases, such as brain trauma, mental retardation, dysmnesia caused by stress trauma, coxsackie syndrome, dementia, carbon monoxide poisoning, etc. As the population ages, the number of dementia patients increases rapidly. Alzheimer's Disease (AD) is a major form of dementia, and is widely appreciated by researchers as it places a heavy mental and economic burden on family members and society. The world alzheimer's disease report of 2018 states that there is one case of dementia every three seconds worldwide. In 2018, 5000 million patients with AD worldwide are expected to increase by a factor of two by 2050. To date, a number of drugs have been approved for mild to moderate AD, including: donepezil and galantamine, and the like. They were found to have modest overall therapeutic efficacy but no effect on long-term disease progression. Furthermore, the existing data do not support the use of these drugs in very mild AD, i.e. not in patients with early AD. In addition, the pathology of AD patients has already accumulated in their brains 15-20 years before clinical symptoms appear, and AD is really an advanced stage of biological disease. Therefore, early prevention may be one of the good AD treatment strategies.

Oxidative stress is reported to be one of the early changes in AD pathogenesis, disease progression and progressive accumulation. Oxidative stress is a state of imbalance between oxidation and antioxidation in the body, and is characterized by a significant increase in free radicals such as Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) in the body. With the occurrence of oxidative stress, a large amount of ROS and RNS accumulate in the body, resulting in oxidation of proteins and peroxidation of lipids, causing structural and functional disorders of proteins and lipids. The brain, as an organ with high oxygen consumption, has weak oxidation resistance and is easily damaged by free radicals. The increase in free radicals in the brain directly leads to neuronal apoptosis and loss of synapses. Reducing the level of oxidative stress in vivo is a good strategy for the treatment and prevention of AD. At present, chemically synthesized antioxidants such as: butylated Hydroxyanisole (BHA) and Butylated Hydroxytoluene (BHT) are not good for human health due to their own toxicity and side effects. Therefore, it is important to develop new products which are beneficial to human health and possess the functions of resisting oxidation and improving memory.

In addition, it is also manifested as a massive loss of acetylcholine (ACh) during AD, which plays an important role in the conduction of neural signals. ACh is synthesized from choline and acetyl-coa under the catalysis of choline acetyltransferase (ChAT) and is rapidly hydrolyzed to choline and acetate by the action of acetylcholinesterase (AChE). During AD, the balance of ACh synthesis and hydrolysis is disrupted, resulting in a significant drop in ACh levels. AChE is therefore also a major target for the treatment and prevention of AD.

Walnut (Juglans regia L.) is a plant of the genus Juglans of the family Juglandaceae, with the fruits of Prunus amygdalus, Anacardium occidentalis, and Corylus heterophylla, and is known as the world's famous "four-dry fruit". The walnut kernel contains rich oil (65.8%) and various unsaturated fatty acids, so that the walnut kernel can be used as one of the sources of vegetable oil. After the walnut kernel is pressed, most of grease is pressed, and the rest walnut dregs are byproducts. The walnut meal contains a large amount of protein and complete amino acid types, has biological activities of resisting oxidation, improving immunity, improving intelligence and the like, and is an excellent source of active polypeptide. However, most of walnut meal is used as feed or directly discarded, which not only causes waste of walnut protein resources, but also seriously hinders the development of walnut industry.

Disclosure of Invention

In order to solve the problems, the method takes walnut pulp as a raw material, after degreasing, protein in the degreased walnut pulp is extracted by an alkali extraction and acid precipitation method, a walnut polypeptide enzymolysis product is obtained by enzymolysis with trypsin, after ultrafiltration is carried out by using a 1kDa ultrafiltration membrane, a sequence of a walnut polypeptide is identified by a liquid chromatography-mass spectrometry combined technology (LC-MS/MS) on filtrate, a fragment with the effect of preventing the polypeptide of Alzheimer's disease is screened by using a molecular docking technology, and the polypeptide with the lowest docking score is artificially synthesized and verified.

The walnut polypeptide for preventing Alzheimer's disease provided by the invention has the following amino acid sequence from C end to N end: Glu-Pro-Glu-Val-Leu-Arg, abbreviated EPEVLR.

The walnut polypeptide can be used for preparing a medicament for preventing Alzheimer's disease. When the walnut polypeptide is used, the walnut polypeptide can be obtained by artificial synthesis or separation and purification after enzymolysis of walnut meal.

The invention has the following beneficial effects:

the walnut polypeptide is obtained by taking walnut meal as a raw material and carrying out enzymolysis on walnut protein by adopting trypsin. The polypeptide sequence is determined by LC-MS/MS, the molecular docking technology is utilized to screen fragments with the effect of preventing Alzheimer's disease, 1 walnut polypeptide with the best score is screened, and then the walnut polypeptide is artificially synthesized by a solid phase to carry out animal in vivo experiments, so that the capability of improving memory is verified. According to the result of HE staining of the hippocampal region of the brain, EPEVLR is proved to prevent AD from reducing protein plaques of the hippocampal region; according to the in vitro antioxidant test results (IC)₅₀1.44mg/mL), validating the potential of EPEVLR in preventing AD from reducing oxidative stress. Furthermore, the EPEVLR and the AChE are subjected to molecular docking through a molecular docking technology, and key amino acids and acting force of interaction of the EPEVLR and the AChE are analyzed, so that a theoretical basis is laid for deep development and utilization of the walnut pulp. The molecular docking result shows that EPEVLR and AChE are connected through pi-sigma, pi-alkyl bonds and hydrogen bonds. In addition, the method for preparing the walnut polypeptide from the walnut meal through enzymolysis has simple and convenient steps and is beneficial to improving the walnut value.

Drawings

FIG. 1 is the number of times a mouse crosses a platform in a water maze experiment.

FIG. 2 is the quadrant distance of the platform of the mouse in the water maze experiment.

FIG. 3 shows the effective residence time of the mouse in the quadrant of the platform in the water maze experiment.

FIG. 4 shows the number of errors in the mouse in the jump bench test.

FIG. 5 is the first latency of the mice in the jump bench experiment.

Fig. 6 is a HE stained section of hippocampal regions of brain tissue, model group (left) and EPEVLR group (right).

FIG. 7 is a diagram of molecular docking 2D of EPEVLR with AChE.

FIG. 8 is a 3D map of molecular docking of EPEVLR with AChE.

Detailed Description

The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.

Example 1

(1) Walnut meal pretreatment

Crushing walnut cakes into walnut cake powder by using a high-speed crusher, adding the walnut cake powder into n-hexane according to the feed-liquid ratio of 1g to 5mL, stirring for 40min at room temperature, and performing suction filtration, wherein the operation is repeated twice; air drying at room temperature overnight to remove excessive n-hexane to obtain defatted walnut cake, and storing at 4 deg.C.

(2) Extracting walnut protein

Adding degreased walnut pulp into distilled water according to the material-liquid ratio of 1g:10mL, uniformly stirring, adjusting the pH to 9.0 by using 1mol/L NaOH aqueous solution, carrying out ultrasonic treatment for 10min, carrying out stirring extraction in a water bath at 45 ℃ for 30min, standing, cooling, centrifuging at 8000rpm for 15min, collecting filtrate, adjusting the pH to 4.5 by using 1mol/L HCl aqueous solution, precipitating protein from the filtrate, standing at 4 ℃ for 4h, centrifuging at 4 ℃ and 8000rpm for 15min, collecting precipitate, adjusting the pH to 7.0 by using 1mol/L NaOH aqueous solution or HCl aqueous solution, freezing and drying to obtain walnut pulp protein powder, and storing at-80 ℃.

(3) Enzymolysis of walnut protein

Adding walnut meal protein powder into deionized water according to the feed-liquid ratio of 1g to 25mL, and heating at 85 DEG CPretreating for 20min, cooling, adjusting pH to 9 with 1mol/L NaOH water solution or HCl water solution, adding trypsin with a mass of 4% of walnut cake protein powder, and performing enzymolysis for 4h in a water bath kettle at 55 deg.C. In the enzymolysis process, 0.5mol/L NaOH aqueous solution is used for controlling the pH value to be 9.0 +/-0.1. After enzymolysis is finished, enzyme is inactivated in a water bath kettle at 90 ℃ for 10min, centrifugation is carried out at 10000rpm for 10min, supernatant is collected, ultrafiltration is carried out on the supernatant by using an ultrafiltration membrane with the molecular weight cutoff of 1kDa, LC-MS/MS analysis is carried out on filtrate with the molecular weight range of less than 1kDa, 3080 polypeptides with different sequences are identified, and scoring is carried out according to software (ALC% > 95%), and relative content (Area > 1 multiplied by 10)⁸) 144 polypeptides are screened out. Then, 10 walnut polypeptides with determined sequences, which have high content of glutamic acid (Glu, E), arginine (Arg, R), tryptophan (Trp, W) and tyrosine (Tyr, Y), in the polypeptide sequences, are screened out. Finally, the molecular docking technology is utilized to simulate the interaction between the polypeptide and acetylcholinesterase (the result is shown in table 1, the lower the docking score is, the stronger the interaction between the polypeptide and acetylcholinesterase is), and a polypeptide sequence with the best docking effect is screened, wherein the amino acid sequence from the C end to the N end is as follows: Glu-Pro-Glu-Val-Leu-Arg, amino acid abbreviated as EPEVLR.

TABLE 1 docking results of polypeptides with acetylcholinesterase

Polypeptide sequence	Fractional docking (kcal/mol)
		ELEWER	-7.8
ETASELPR	-7.8
		LADSFR	-7.7
LNQLPR	-7.5
		EELEELFR	-8.3
EPEVLR	-8.6
		FVPTER	-8.2
VEDELR	-7.3
		LLTHDSR	-8.5
SGFDEEFLR	-8.1

(4) Solid phase synthesis of polypeptides

The synthesis of polypeptide EPEVLR is carried out by adopting Fmoc solid-phase polypeptide synthesis strategy, Fmoc-AA-Wang resin is used as initial solid-phase carrier, anhydrous DMF is used as reaction solvent, ethanolamine is used for removing Fmoc protecting group at the tail end of amino, HBTU, HoBt and N-methylmorpholine are used as activating agents, carboxyl of Fmoc protecting amino acid is activated, and the Fmoc protecting amino acid is coupled with free amino on the resin. After coupling, the side chain protecting groups are removed by trifluoroacetic acid, and the free polypeptide is obtained by cleavage from the resin. Purifying a solid phase by using a semi-preparative reverse phase liquid chromatography to synthesize the polypeptide, wherein the mobile phase of the reverse phase liquid chromatography is as follows: a: 0.1% TFA/water, B: 0.1% TFA/acetonitrile, mobile phase conditions: 0-90% B/30 min-90% B/10min, and the chromatographic column comprises: eilide 5 μm 10.0mm X250 mm C18, sample size 100 μ L, flow rate 1mL/min, detection wavelength 220nm, and identification of component amino acid sequence by LC-MS/MS.

(5) Test of AD prevention activity of walnut polypeptide

The activity of walnut polypeptide in preventing AD is verified through a mouse experiment. The mice are quarantined for one week and are subjected to adaptive feeding, and are randomly grouped, wherein the groups comprise 3 groups: normal control group, model group, and polypeptide group. The model group and walnut polypeptide group mice are firstly subjected to D-type galactose (D-gal) molding treatment, the two groups of mice receive 500mg/kg/D continuous subcutaneous injection for 8 weeks, from the 5 th week, the model group is simultaneously perfused with stomach physiological saline, and the polypeptide group is simultaneously perfused with stomach walnut polypeptide EPEVLR. Normal groups had free access to food. After feeding, the effect of preventing AD of the walnut polypeptide is verified through a behavioral experiment.

Behavioral experiments: the behavioral experiments of the mice are mainly divided into a diving platform experiment and a water maze experiment. Four days after training, the test was started. And starting the water maze experiment 30min after the bench jump experiment is finished.

Water maze experiment: the diameter of the water maze is 1.2m, the height is 0.5m, the diameter of the platform is 0.09m, the height is 0.3m, and the platform is hidden under the water surface by about 1 cm. Running water is injected into the water pool, and the temperature of the water pool needs to be maintained at about 23 ℃. The parameters are set as follows: swimming time (60s), stay time on the platform (10s), selecting a mouse experiment mode, and adjusting a red line circle to accurately frame the ranges of the pool and the platform respectively. And (3) selecting the alignment quadrant of the quadrant where the platform is located as a research quadrant, putting the mouse into water, and immediately recording the time required by the mouse to stably climb up the platform after the mouse enters the water and is searched by software. If the mouse can climb up the platform within 60s and stably stand for 3s, recording the use time; and if the platform cannot be found within 60s after the mouse enters water, recording the latency period as 60s, guiding the mouse to stably stand on the platform for 15s, then taking down, resting for 30-60 s, and then performing next training, wherein the training is finished and the mouse is wiped to dry hair.

A jump bench experiment: the diving platform is a rectangular reflecting box, eight areas are totally arranged, and a copper grid capable of being electrified with proper current is laid at the bottom. Each small compartment has a small platform with a height and diameter of 4.5 cm. The setting parameters are as follows: time (5min), stimulation intensity (0.6 mA). The mice were placed on a copper grid, powered on after their mood had stabilized, and the number of shocks received by each mouse (number of errors), time to first jump (latency) was recorded.

(6) HE staining of mouse brain tissue hippocampus

After the mice were subjected to the behavioral activity test, the mice were fasted and water-free for 12h before sacrifice. The mouse is performed with inhalation type anesthesia by isoflurane with the concentration of 2% -3%. After anesthesia, the mice were sacrificed by cervical spondylolysis. The brain tissue of the mice was carefully removed, washed with physiological saline, and post-fixed by placing it in a 4% paraformaldehyde solution. After fixation, it was subjected to embedding treatment using paraffin to obtain a tissue paraffin-embedded wax block. The paraffin block was then sliced to a slice thickness of 4 μm. Finally, the tissue sections were stained using Hematoxylin-Eosin staining and viewed under a microscope in the bright field.

(7) Test for antioxidant Activity

Mixing polypeptides with different concentrations with DPPH solution (5mg/100mL, prepared with absolute ethanol) at equal volume, standing at room temperature in dark place for 30min, and measuring light absorption value at 517 nm; 2mL of absolute ethyl alcohol solution is used as a sample reference group to replace a DPPH solution; blank set was 2mL DPPH solution with 2mL deionized water. DPPH radical scavenging activity of the samples was calculated according to the following formula:

in the formula A₀Blank absorbance values; a is the absorbance value of the sample group; b is the absorbance value of the reference group.

(8) Molecular docking

AChE (PDB ID: 1gqr) was downloaded from the PDB protein database, and acceptor AChE was hydrogenated, removed of water molecules, etc. using Autodock tools1.5.6 software. The identified small molecule polypeptide structure is mapped by using ChemDraw3D 18.1.1, and the small molecule structure energy is minimized and then converted into a pdbqt format file.

Autodock Vina as docking software, docking center coordinates were set to (x:3.302, y:65.556, z:63.181), and box size was (x:40, y:40, z: 40). The software can respectively butt-joint the polypeptide with the AChE receptor protein, calculate the degree of fit between different conformations of the ligand and the crystal structure of the AChE and sequentially score, wherein the butt-joint energy is generally negative, and the smaller the value, the higher the score of the butt-joint result is, namely the tighter the combination between the ligand and the AChE receptor protein is, the strongest the interaction is, and the more possible the AChE inhibitory activity is. Subsequently, the interaction between the single small molecule and the ligand was visually analyzed using Discovery studio software.

(9) Results of the experiment

The water behavioural results of the mice are shown in figures 1-5, after the model group is subjected to D-gal modeling, the times of platform crossing, the path occupation ratio of a quadrant where the platform is located and the effective residence time of the quadrant where the platform is located in a water maze experiment are all obviously reduced, the error times in a jump platform experiment are obviously increased, the first latency is also obviously lower than that of the blank group, and the success of the model building of the mouse aging model is indicated. However, the behavioral experiment related indexes of the mice after the intragastric administration of the polypeptide EPEVLR are very close to those of a blank control group, which indicates that the EPEVLR has the effect of preventing AD.

After the mouse behaviourology is finished, the brain tissue of the mouse is taken to carry out HE staining, the number of protein plaques in the hippocampal region is observed, the result is shown in figure 6, the number of protein plaques in the hippocampal region of the brain of the mouse perfused with the EPEVLR polypeptide is obviously lower than that of the model group, and the EPEVLR can prevent AD from the aspect of reducing the brain protein plaques. In addition, IC was determined from the in vitro antioxidant assay of EPEVLR₅₀1.44mg/mL, indicating that EPEVLR has better antioxidant property and can prevent AD from reducing the oxidative stress of mice. The molecular docking results of EPEVLR and acetylcholinesterase are shown in FIGS. 7 and 8, the molecular docking fraction is-8.6, and the low docking fraction indicates that EPEVLR can effectively inhibit the activity of acetylcholinesterase in mouse brain, and is helpful for increasing the content of acetylcholine. Wherein, Asn-85, Asp-72, Aer-81, Tyr-121, Ser-286, Trp-279 and Leu-282 of AChE can form hydrogen bond with H atom of side chain on EPEVLR and N and O atoms of N terminal and C terminal; phr-330 forms pi-sigma conjugation with the pyrrole ring on EPEVLR; phe-331, Ile-287, His-440 and Ttrp-279 are linked to EPEVLR by a pi-alkyl linkage.