Application of walnut dietary fiber in the preparation of drugs for treating Parkinson's disease

Abstract

The invention discloses the application of walnut dietary fiber in the preparation of a drug for treating Parkinson's disease. The walnut dietary fiber is insoluble walnut dietary fiber. The insoluble dietary fiber is rich in galacturonic acid, glucose and xylose, and its monosaccharide composition also includes: arabinose, galactose, guluronic acid, rhamnose, glucuronic acid, mannuronic acid and fucose. Experimental results show that after oral administration of low-dose, medium-dose and high-dose insoluble walnut dietary fiber, the motor dysfunction of rotenone-induced PD model mice is restored, and the PD mice intervened by walnut fiber diet also show significantly improved limb strength and motor coordination, which indicates that walnut dietary fiber also has an improving effect on the motor dysfunction of PD.

Description

Technical Field

[0001] The invention relates to the field of drugs for treating diseases, and in particular to application of walnut dietary fiber in preparing drugs for treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is one of the most common neurodegenerative diseases, second only to Alzheimer's disease. The clinical features of PD are typical motor symptoms, including slow movement, rigidity, static tremor and postural instability, and may also be accompanied by a large number of non-motor disorders, such as constipation, hyposmia, dysphagia, nausea, vomiting, weight loss, depression and sleep disorders.

[0003] The clinical pathological characteristics of PD are mainly manifested as a large number of apoptosis of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc), a specific brain region of the midbrain, and the presence of Lewy bodies (LB) formed by the misfolding and aggregation of α-synuclein (α-Syn) in the surviving neurons, and a decrease in the expression level of DA in the striatum. It is currently believed that the onset of PD is jointly determined by aging, genetics, behavior and environmental factors. The exact cause of this pathological change is still unclear. Oxidative stress, damage to the intracellular protein degradation pathway, abnormal protein aggregation, mitochondrial dysfunction, neuronal apoptosis and neuroinflammation may all be involved in the degeneration and death of PD dopaminergic neurons. At present, the mainstream theories on the pathogenic mechanism of PD include the neuroinflammation theory, the misfolding and overexpression theory of α-Syn protein, the oxidative stress theory, the mitochondrial dysfunction theory, etc. There is currently no targeted cure for PD. Some drugs currently used in clinical practice to treat Parkinson's disease can only slow down movement disorders, but cannot cure them, and long-term use has serious side effects and may even endanger life.

[0004] People continue to find that exposure to environmental toxins may increase the risk of PD through gene-environment interactions. Among them, widely used pesticides contain rotenone. Rotenone belongs to the ketone chemical agent. It is naturally present in the seeds and rhizomes of plants of the genus Derris in the Leguminosae family. It causes Parkinson's disease by inducing mitochondrial damage and oxidative stress, inducing cell apoptosis, producing neuroinflammatory reactions and abnormal protein aggregation. Rotenone is often easily absorbed by the body through the digestive tract and skin. It has extremely strong lipophilicity and can penetrate the blood-brain barrier and cell membranes, causing excessive reactive oxygen to induce mitochondrial damage and oxidative stress, and highly selectively causes degeneration of dopaminergic neurons in the substantia nigra and striatum, leading to cell apoptosis. Studies have shown that the pathogenesis of the rotenone-induced chronic PD mouse model is closely related to intestinal flora imbalance. In addition, rotenone can be successfully administered in different ways to prepare rat or mouse PD models. Rotenone can simulate some of the movement defects and pathological characteristics of Parkinson's disease, and the induced Parkinson's disease is more similar to the anatomical and neuropathological characteristics of PD patients. Therefore, it is widely used to establish a rodent Parkinson's disease model.

[0005] Walnuts have high nutritional value, with an oil content of more than 60%, a protein content of more than 20%, dietary fiber of about 9%, and high content of trace elements such as manganese, zinc, and copper. Walnut meal is a by-product left over from oil extraction and other production and processing. It is rich in dietary fiber and can be used as an important source of dietary fiber extraction. At present, the research on walnut dietary fiber mainly focuses on the physical and chemical comparison of water holding capacity and oil holding capacity of soluble and insoluble dietary fiber prepared by enzymatic and alkaline methods, as well as the preparation of high-protein and high-dietary fiber chewable tablets. According to research reports, the crude fiber content of pecans prepared by alkaline method is 5%-8%. There are also studies that use the high-quality protein and dietary fiber resources in walnut meal to develop walnut nutritious breakfast and walnut dietary fiber biscuits. In terms of efficacy research, it is mainly reflected in the study of water-soluble fiber improving metabolic syndrome through intestinal flora. There is no literature reporting that walnut dietary fiber can be used to treat Parkinson's disease. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides an application of walnut dietary fiber in the preparation of a drug for preventing, improving or treating Parkinson's disease. The walnut dietary fiber can effectively alleviate the symptoms of Parkinson's patients; relieve Parkinson's symptoms mainly characterized by motor dysfunction; and be beneficial to inhibiting the development of Parkinson's disease and promoting the reversal or recovery of Parkinson's disease.

[0007] In order to solve the above technical problems, the technical solution of the present invention is as follows: application of walnut dietary fiber in preparing drugs for treating Parkinson's disease.

[0008] As a further description of the above scheme: the walnut dietary fiber is preferably insoluble walnut dietary fiber.

[0009] As a further description of the above scheme: the preparation method of the insoluble walnut dietary fiber is as follows:

[0010] (1) Grease removal

[0011] After grinding the walnuts, defatting was performed three times with a ratio of walnuts to petroleum ether of 1:10-1:15 (w / v), each time for 100-150 min, stirring once every 15-20 min, and then drying at room temperature to obtain a defatted sample, which was sieved before use;

[0012] (2) Removal of starch

[0013] The defatted sample is dispersed in deionized water at a ratio of 1:10-1:15 (w / v), and when heated in a water bath to 83-88°C, 0.2-0.3 mL / g of heat-resistant α-amylase is added, and the reaction is continued with stirring at 88-92°C for 20-40 min; after cooling to 55-65°C, 0.10-0.15 mL / g of amyloglucosidase is added, and the reaction is continued with stirring at 58-62°C for 2.5-3.5 h; after the enzymolysis is completed, the enzyme is inactivated in a boiling water bath for 15-20 min, 3-5 times the volume of 95% ethanol preheated to 58-62°C is added to the enzymolysis solution, and the solution is cooled and refrigerated overnight, and then centrifuged and freeze-dried to obtain a de-starched sample;

[0014] (3) Protein removal

[0015] The destarch sample was dispersed in deionized water at a ratio of 1:20-30 (w / v) and hydrolyzed by alkaline protease, flavor protease and acid protease in sequence to remove protein;

[0016] (4) Inactivation

[0017] After the enzymatic hydrolysis is completed, the enzyme is inactivated by boiling for 15-20 minutes; cooled to room temperature, the enzymatic hydrolyzate is centrifuged to obtain the residue, and then the residue is washed with water, neutralized, washed with alcohol, and freeze-dried to obtain walnut insoluble dietary fiber.

[0018] As a further description of the above scheme: the alkaline protease enzymolysis reaction conditions are: pH 10.5-11.5, enzyme addition amount 1500-1800U / mL, reaction temperature 40-48°C, reaction time 4-6h; the flavor protease enzymolysis reaction conditions are: pH 7.5-8.5, enzyme addition amount 400-600U / mL, reaction temperature 48-53°C, reaction time 4-6h; the acidic protease enzymolysis reaction conditions are pH 2.8-3.2, enzyme addition amount 400-600U / mL, reaction temperature 48-53°C, reaction time 4-6h.

[0019] Preferably, the daily dosage range of the walnut dietary fiber is 50-900 mg / kg.

[0020] As a further description of the above scheme: the monosaccharide composition of the insoluble dietary fiber is: galacturonic acid, glucose, xylose, arabinose, galactose, guluronic acid, rhamnose, glucuronic acid, mannuronic acid and fucose.

[0021] The characteristics of the present invention are as follows: motor dysfunction is the main clinical symptom of PD, and observing the improvement of the motor coordination ability of PD model animals by drugs is an important indicator for evaluating the neuroprotective effect of drugs. Climbing pole test, grip test, open field test, adhesive removal test and rotating rod test are classic experiments in behavioral experiments of PD model animals, so these behavioral experiments are also selected in the present invention to evaluate the motor function of PD model mice. Behavioral results show that compared with the control group, the motor coordination ability of rotenone mice is significantly reduced. After other drug interventions, the changes in the body's motor symptoms will be affected by drug toxicity, the body itself, the environment and other aspects, and the motor symptoms of PD are generally later than the pathological symptoms. When the dopaminergic neurons in the substantia nigra are damaged by more than 50%, or the content of dopamine transmitters in the striatum exceeds 80%, the symptoms of motor dysfunction will appear. Studies have reported that low-dose rotenone gavage for 1 month will show obvious motor dysfunction, and the dopamine neurons in the substantia nigra and striatum of mice will show degenerative changes and motor coordination will be significantly reduced when the rotenone dose of 30 mg / kg is continuously gavaged for 28 days. The experimental results showed that after oral administration of Madopar, low-dose, medium-dose and high-dose insoluble walnut dietary fiber, the motor dysfunction of rotenone-induced PD model mice was improved. Some studies have found that Madopar can improve Parkinson's disease by supplementing dopamine to make up for the lack of motor ability. PD mice intervened with insoluble walnut dietary fiber also showed significantly improved limb strength and motor coordination, which indicates that insoluble walnut dietary fiber has an improving effect on the movement disorders of PD.

[0022] The present invention further studies the pathological mechanism, and the experimental results show that insoluble walnut dietary fiber significantly increases the number of TH-positive neurons and the number of Nissl-positive neurons, which means that the administration of insoluble walnut dietary fiber can protect dopaminergic neurons.

[0023] For achieving the purpose of medication, enhancing the therapeutic effect, the medicine of the present invention can be administered with any known method of administration. The dosage of walnut dietary fiber in the present invention can vary widely according to the nature and severity of the disease to be prevented or treated, the individual conditions of the patient or animal, the route of administration and the dosage form, etc. Generally speaking, the suitable dosage range of walnut dietary fiber every day of the present invention is 50-900mg / kg body weight. The above dosage can be administered in one dosage unit or divided into several dosage units, which depends on the doctor's clinical experience and includes the dosage regimen of utilizing other therapeutic means.

[0024] The walnut dietary fiber of the present invention can be taken alone or used in combination with other therapeutic drugs or symptomatic drugs. When there is a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to actual conditions.

[0025] Compared with the prior art, the present invention has the following beneficial effects: the present invention constructs a rotenone-induced mouse Parkinson's disease animal model, and walnut dietary fiber has a significant improvement effect in anti-Parkinson's disease, especially in improving behavioral performance. Experimental results show that after oral administration of low-dose, medium-dose and high-dose insoluble walnut dietary fiber, the motor dysfunction of rotenone-induced PD model mice is improved, which provides a safe, effective and economical solution for the prevention, improvement and treatment of Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Structural characteristics and monosaccharide composition of WIDF; (A) SEM analysis of defatted walnut powder (a, b, c) and WIDF (d, e, f) at 500, 1000, and 2000 times, respectively; (B) AFM analysis of WIDF; (C) XRD pattern of WIDF; (D) TG and DTG curves of WIDF; (E) FT-IR spectrum of WIDF; (F) Monosaccharide composition of WIDF; in the figure, Fuc, Rha, Ara, Gal, Glc, Xyl, Gal-UA, Gul-UA, Glc-UA, and Man-UA represent fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid, and mannuronic acid, respectively;

[0027] Figure 2 Animal grouping and treatment process;

[0028] Figure 3 Effects of insoluble dietary fiber administration for 1, 5, and 9 weeks on the climbing time of PD mice (n=12); Note: Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the PD model group, *P<0.05, **P<0.01, ***P<0.001;

[0029] Figure 4 Effects of insoluble dietary fiber on the grip strength of PD mice for 1, 5, and 9 weeks (n=12); Note: Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the PD model group, *P<0.05, **P<0.01, ***P<0.001;

[0030] Figure 5 Effects of insoluble dietary fiber administration for 1, 5, and 9 weeks on the rod time of PD mice (n=12); Note: Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; Compared with the PD model group, *P<0.05, **P<0.01, ***P<0.001;

[0031] Figure 6 Effects of insoluble dietary fiber administration for 1, 5, and 9 weeks on adhesive removal time in PD mice (n=12); Note: Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; Compared with the PD model group, *P<0.05, **P<0.01, ***P<0.001;

[0032] Figure 7 Effects of insoluble dietary fiber administration for 1, 5, and 9 weeks on the movement distance, movement speed, and movement trajectory of PD mice (n=12)A: Movement distance; B: Movement speed; C: Movement trajectory at week 9; Note: Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; Compared with the PD model group, *P<0.05, **P<0.01, ***P<0.001;

[0033] Figure 8 Effects of insoluble dietary fiber on dopaminergic neurons in PD mice; A: TH+ positive neurons (immunohistochemical staining); B: Nissl staining results. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

[0035] The experimental reagents used in the following examples were purchased from the market. Alkaline protease, flavor protease, acid protease, etc. were purchased from Beijing Solebow Technology Co., Ltd. (Beijing, China); rotenone was purchased from China Macklin Company; Madopar was purchased from Roche Company, China; carboxymethylcellulose sodium salt, α-amylase, and amyloglucosidase were purchased from Sigma Company, USA; petroleum ether, etc. were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0036] The experimental results of the following examples are expressed as mean ± standard deviation (mean SEM), and the two groups were compared using independent t-test using GraphPadPrism statistical software. P values ​​< 0.05 were considered statistically significant.

[0037] Example 1 Preparation and structural characterization of insoluble walnut dietary fiber

[0038] 1. Preparation of Insoluble Walnut Dietary Fiber

[0039] After grinding the walnuts, defatting was performed three times with a ratio of walnuts to petroleum ether of 1:15 (w / v), each time for 120 min, stirring every 20 min, and then drying at room temperature to obtain defatted samples, which were sieved before use;

[0040] Disperse defatted walnut powder in deionized water at a ratio of 1:10 (w / v), add thermostable α-amylase (0.2mL / g), and react at 90℃ with continuous stirring for 30min; after cooling to 60℃, add amyloglucosidase (0.1mL / g), and react at 60℃ for 3h. After enzymolysis, inactivate the enzyme in a boiling water bath for 15min. Add 4 times the volume of 95% ethanol preheated to 60℃ to the above enzymolysis solution, cool, transfer to 4℃ overnight, and then centrifuge and freeze-dry to obtain the de-starch sample.

[0041] The de-starch sample was dispersed in deionized water at a ratio of 1:25 (w / v), and hydrolyzed by alkaline protease (pH11.0, 1500U / mL, 45℃, 5h), flavor protease (pH 8.0, 500U / mL, 50℃, 5h) and acid protease (pH3.0, 500U / mL, 45℃, 5h) to remove protein. After the enzymatic hydrolysis, it was inactivated in a boiling water bath for 15min. The enzymatic hydrolyzate was centrifuged at 4000r / min for 15min to obtain the residue, which was then washed with water (10-20 times the mass of the de-starch sample with deionized water), neutralized (adding 10 times the mass of the de-starch sample with deionized water, and then adjusting the pH to 7.0), washed with alcohol (washed twice with 75%-85% ethanol and twice with 90%-95% ethanol), and freeze-dried to obtain insoluble walnut dietary fiber.

[0042] 2. Composition and structure determination of walnut insoluble dietary fiber (WIDF)

[0043] 2.1 Scanning electron microscopy (SEM) analysis of WIDF

[0044] A small amount of dry insoluble walnut dietary fiber powder was fixed on a sample holder of double-sided carbon conductive tape and coated with a 10 nm gold layer. The surface and microstructure of the sample were observed using a scanning electron microscope (FlexSEM1000) at a voltage of 5 kV, and photographed at 500× (scale bar 100 μm), 1000× (scale bar 50 μm), and 2000× (scale bar 20 μm), respectively.

[0045] 2.2 Atomic force microscopy (AFM) analysis of WIDF

[0046] Part of the insoluble walnut dietary fiber powder was dispersed in an ethanol solution for ultrasonic treatment, and then a few drops of the dispersed liquid were dripped dropwise onto a mica sheet. After drying at room temperature, an atomic force microscope (Bruker DimensionIcon, Germany) was used to observe the ultrastructure of the insoluble walnut dietary fiber.

[0047] 2.3 X-ray diffractometer (XRD) analysis of WIDF

[0048] The XRD pattern of insoluble walnut dietary fiber was obtained using an X-ray diffractometer (Rigaku Ultima IV, Japan). The test conditions were: pipeline voltage 40 kV, Cu-Kα radiation The diffraction angle scanning range (2θ) was 5° to 70°, and the scanning speed was 2° / min.

[0049] 2.4 TG-DTG analysis of WIDF

[0050] The thermal stability of insoluble walnut dietary fiber was measured by thermogravimetric-differential thermal analysis (HITACHISTA200, Japan) at a heating rate of 10°C / min from 30°C to 800°C.

[0051] 2.5 FT-IR analysis of WIDF

[0052] A small amount of insoluble walnut dietary fiber sample was weighed and mixed with 200 mg potassium bromide, and then pressed into a 1 mm thick sheet. The sheet was then scanned and analyzed using a Fourier transform infrared spectrometer (Nicolet iZ-10, Thermo Nicolet, USA) with an instrument resolution of 4.00 cm -1 , scanning range is 4000-400cm -1 , the number of scans is 32 times.

[0053] 2.6 Analysis of monosaccharide composition of WIDF

[0054] Weigh 5 mg (± 0.05 mg) of polysaccharide sample accurately, add 1 mL of 2M TFA acid solution, and heat at 105 ° C for 6 h. Pass nitrogen and blow dry. Add methanol to wash, blow dry again, and repeat the methanol washing 3 times. The residue was redissolved in deionized water and filtered through a 0.22 μm microporous filter membrane. The sample (5 μL) and the monosaccharide standard were analyzed by high-performance anion exchange chromatography (HPAEC) on a CarboPac PA-10 anion exchange column (250*4.0 mm, 10 um; Dionex) using a pulsed current detector (PAD; Dionex ICS 5000+system). Data were collected on an ICS 5000+ (Thermo Scientific) and processed using chromeleon 7.2CDS (Thermo Science).

[0055] Structural characteristics and monosaccharide composition of insoluble walnut dietary fiber Figure 1 The results showed that defatted walnut powder showed a spherical or flaky and spherical composite structure, while insoluble walnut dietary fiber showed a single-layer flaky network structure or a multi-layer, loose skeleton network structure, similar to the network composition of several layers of fiber cell walls ( Figure 1 A). Defatted walnut flour contains a large number of spherical structures, which are mainly formed by the combination of protein and dietary fiber. However, the spherical structures disappear in WIDF, which is mainly attributed to the degradation of the bound proteins by various protease treatments. In addition, the planar and 3D AFM images of WIDF are shown in Figure 2. Figure 1 B. It is mainly composed of a cellulose network structure formed by many microfibrils cross-linked with each other and some irregular particles, which is consistent with the SEM results. The thickness of WIDF varies significantly, indicating that the single-chain molecules may form polymers through van der Waals forces or hydrogen bonds between WIDF molecules.

[0056] WIDF has only one strong diffraction peak at 21.34° ( Figure 1 C), attributed to cellulose and hemicellulose, because 15°-25° is dominated by the diffraction peaks of cellulose and hemicellulose. At the same time, the peak near 21°-22° may also come from cellulose crystals, indicating that WIDF may contain some cellulose crystal structures. TG and DTG were used to evaluate the thermal stability and structural characteristics of WIDF. Figure 1As shown in Figure D, the DTG curve of WIDF shows three peaks. The first peak at 92°C between 33 and 180°C may be caused by water evaporation, the second peak at 254°C between 180 and 276°C may be attributed to the degradation of hemicellulose, and the third peak at 326°C between 280 and 380°C may be due to the degradation of cellulose. After 380°C, the TG curve shows a slow downward trend, which may be related to the continuous decomposition of lignin, because lignin degradation starts at about 200°C and continues to 520°C.

[0057] In the FT-IR spectrum, the absorption peaks of WIDF and commercial cellulose show more similarities ( Figure 1 E). 3412-3392cm -1 The absorption peak is caused by the stretching vibration of OH, 2937-2902cm -1 The absorption peaks at 1651, 1657 cm -1 The absorption peak of is asymmetric C=O stretching vibration, indicating the presence of amide groups in WIDF. -1 The absorption peak may come from the typical fiber structure, 900-1200cm -1 The spectral region within the range corresponds to the backbone CO and CC vibration bands of the glycosidic bond and pyranose ring. In addition, a WIDF at 896 cm -1 The typical absorption peak of glucose β configuration.

[0058] After determining the structural characteristics of WIDF, the present invention conducted a monosaccharide composition analysis. A total of 6 monosaccharides and 4 uronic acids were detected in WIDF, of which glucose was the most abundant monosaccharide and galacturonic acid was the most abundant uronic acid, accounting for 25.63% and 34.76%, respectively. Figure 1 F and Table 1).

[0059] Table 1 Monosaccharide composition of WIDF

[0060]

[0061] In conclusion, the present invention obtains an insoluble dietary fiber having typical dietary fiber structural characteristics such as β-linkage, cellulose crystals, high galacturonic acid and glucose through separation and purification by a composite enzyme method.

[0062] Example 2 Study on the improvement of Parkinson's disease behavior by walnut dietary fiber

[0063] Experimental animal preparation

[0064] Eight-week-old C57BL / 6J male mice (n=84, 20-22 g) were housed in the animal laboratory of Yunnan Agricultural University. The animals were kept in a constant temperature environment (22±2°C, 12 h light, 12 h dark, lights off at 20:00 every day) and had free access to food (basic feed) and drinking water.

[0065] Animal grouping and establishment of Parkinson's disease animal model

[0066] Animal groups and treatment procedures Figure 2 As shown, the mice were randomly divided into 7 groups, with 12 mice in each group: blank group (Con group), rotenone (Rot group), Madopar group (Rot+Madopar group), low-dose insoluble walnut dietary fiber group (Rot+L-IDF group), medium-dose insoluble walnut dietary fiber group (Rot+M-IDF group), and high-dose insoluble walnut dietary fiber group (Rot+H-IDF group).

[0067] The drugs were administered by gavage with a 12-gauge straight-tip gavage needle. The rotenone group was gavaged with 20 mg / kg of rotenone per day, and rotenone was dissolved in 0.25% carboxymethyl cellulose sodium salt (CMC) saline solution, followed by gavage with normal saline; the blank control group was gavaged with 0.25% CMC solution per day, followed by gavage with normal saline; the Madopar group was gavaged with 20 mg / kg of rotenone first and then 50 mg / kg of Madopar per day; the low-dose insoluble walnut dietary fiber group was gavaged with 20 mg / kg of rotenone first and then 300 mg / kg of insoluble fiber every day; the medium-dose insoluble walnut dietary fiber group was gavaged with 20 mg / kg of rotenone first and then 600 mg / kg of insoluble fiber every day; the high-dose insoluble walnut dietary fiber group was gavaged with 20 mg / kg of rotenone first and then 900 mg / kg of insoluble fiber every day. One week before the experiment, the Madopar group, the low-dose insoluble walnut dietary fiber group, the medium-dose insoluble walnut dietary fiber group, and the high-dose insoluble walnut dietary fiber group were pre-administered with 50 mg / Kg of Madopar solution, 200 mg / kg, 600 mg / Kg, and 900 mg / Kg of insoluble walnut dietary fiber solution, respectively. At the same time, the blank control group and the rotenone group were gavaged with normal saline for 1 week. One week later, rotenone was gavaged together with each drug. The gavage volume was determined according to the weight of the mice. The gavage of the two drugs was separated by more than 4 hours and the total gavage volume per day did not exceed 1 ml. The experimental period was 9 weeks in total. At the same time, gastrointestinal function tests and feces collection were performed every week, and behavioral tests were performed at the 1st, 3rd, 5th, 7th, and 9th weeks, respectively. The entire process of mouse treatment is as follows. Figure 1 shown.

[0068] Solution preparation

[0069] 0.25% CMC solution: Dissolve 0.25g CMC in every 100ml saline and stir with a magnetic stirrer;

[0070] 20mg / kg rotenone solution: add 20ml 0.25% CMC solution to every 40mg rotenone and stir with a magnetic stirrer;

[0071] 50mg / kg / day Madopar solution: add 10ml PBS solution for every 16.7mg Madopar and stir with a magnetic stirrer;

[0072] 300mg / kg / day low-dose insoluble walnut dietary fiber solution: add 10ml PBS solution to every 100mg insoluble walnut dietary fiber solution and mix well;

[0073] 600mg / kg / day medium dose insoluble walnut dietary fiber solution: add 10ml PBS solution to every 200mg insoluble walnut dietary fiber solution and mix well;

[0074] 900mg / kg / day high-dose insoluble walnut dietary fiber solution: add 10ml PBS solution to every 300mg insoluble walnut dietary fiber solution and mix well;

[0075] Experimental methods

[0076] Behavioral testing

[0077] Five behavioral tests were performed to assess motor function. All mice were tested three times with 1 hour between each test. The training was performed 3 days in advance to prevent the mice from developing anxiety or panic.

[0078] (1) Rotarod test

[0079] The rotating rod test is used to evaluate the motor coordination ability of mice. The mice were trained continuously for 3 days before modeling. The speed of each mouse was 5-20rpm for the first training and 10-20rpm for the second training, and they were trained 3 times. Each training time did not exceed 180s. After each training, they rested for 5 minutes to relieve the tension and fatigue of the mice. At the beginning of the formal experiment, the speed was set to 35r / min and the test time was 180s. Each mouse was placed on the rotating rod to adapt for 5 seconds each time and prevented from slipping. After the mouse stood firmly, the instrument was started. The time from the beginning of the rotation of the roller to the falling of the roller within 180s was recorded. This time was used as the movement latency of the mouse. Each mouse was measured 3 times and the average value was taken. The interval between the tests of each group of mice was at least 20min. After each experiment, the instrument was cleaned with 75% alcohol before the next group of experiments could be carried out.

[0080] (2) Pole climbing experiment

[0081] The pole climbing test is a behavioral test used to evaluate the limb coordination ability of rodents. The pole is 50cm high, 1cm in diameter, and has a 35mm diameter ball on the top. During the test, the mouse is placed on the ball and the time it takes for the mouse to climb to the bottom of the pole is recorded. Each mouse begins adaptive training 3 days before the test. On the day of the test, each mouse repeats the pole climbing 3 times and the average value is recorded. After each experiment, the instrument must be cleaned with 75% alcohol before the next set of experiments can be carried out.

[0082] (3) Degumming experiment

[0083] The purpose of the adhesive removal test is to evaluate the limb coordination and balance of mice by testing the time it takes to remove adhesive. Each mouse was assigned to a clean cage and allowed 3 minutes of free movement to adapt to the environment. To perform the removal test, a circular adhesive label was first gently pressed on the front paw. Then, each mouse was returned to the cage, and the time it took for each mouse to completely remove the label was recorded. Three consecutive trials were performed for each mouse, and a 20-minute interval was allowed between each trial.

[0084] (4) Grip test

[0085] The grip strength test can be used to evaluate limb strength and to identify aging, nerve damage, and muscle damage in animals. Place the laboratory-specific rat grip strength meter (Grip Strength Meter) on a stable operating table, and place the mouse on the dynamometer rack. Be gentle and adjust the position of the mouse. After its limbs grasp the rack, grab the mouse's tail and pull the mouse evenly in the horizontal direction until the mouse lets go of the rack. When the mouse lets go of the rack, the tester will record the mouse's maximum gripping force during this process. Each mouse is measured 3 times and the average is taken. It should be noted that in order to reduce experimental errors, the grip strength measurement experiment should be performed by the same experimenter, and the force should be balanced.

[0086] (5) Open field test

[0087] The purpose of the open field experiment is to reflect the autonomous activity ability of mice through the movement distance and movement speed of mice in the open field. The instrument used is a program-controlled autonomous activity box. During the experiment, 4 mice were randomly selected from the same group and placed in four square chambers (50cm×50cm) of the autonomous activity box at the same time to adapt to it for 3 minutes. After 3 minutes, the program was turned on for video collection, and the collection time was 5 minutes. Note that the test environment should be kept dark and quiet as much as possible. After every 4 mice are tested, the autonomous activity box should be cleaned to remove the remaining smell and excrement to prevent affecting other mice. The experiment was tested in parallel 3 times, with an interval of 30 minutes each time. After all the mouse behavior information was collected, the collected data was processed and analyzed using Mouse Doctor. The total distance of the mouse movement was short and the average speed was slow, indicating that the experimental animals had motor dysfunction.

[0088] Insoluble walnut dietary fiber treatment alleviates motor dysfunction in rotenone-induced PD mice

[0089] (1) Effect of walnut insoluble dietary fiber on pole climbing time in rotenone-induced PD mice

[0090] Movement disorders are the main clinical symptoms of PD, so the detection of motor function in PD animal models is the basis for judging whether the model is successful. The pole climbing test is a commonly used method for judging motor function in PD mouse models, and some studies have shown that the time it takes for mice to climb the pole may be related to the degree of loss of dopamine neurons. Therefore, the pole climbing test was performed on mice to evaluate their body coordination and balance. Figure 3 As shown: In the first week of experimental animal administration, there was no significant change in the pole climbing time of mice in the model group compared with the control group (P>0.05); compared with the model group, there was no significant change in the pole climbing time of mice in the other four groups (Medopar group, walnut insoluble dietary fiber low dose / medium dose / high dose group) (P>0.05). In the fifth and ninth weeks of experimental animal administration, compared with the control group, the pole climbing time of mice in the model group increased significantly (P<0.05); compared with the model group, the pole climbing time of mice in the other four groups (Medopar group, walnut insoluble dietary fiber low dose / medium dose / high dose group) decreased significantly (P<0.05). The results show that insoluble walnut dietary fiber can improve the body coordination and balance of Parkinson's mice.

[0091] (2) Effect of walnut insoluble dietary fiber on the grip strength of rotenone-induced PD mice

[0092] The purpose of the grip test is to evaluate the muscle strength of mice. Figure 4 As shown in the results, in the first week of drug administration, there was no significant difference in the grasping strength of mice in the model group compared with the control group (P>0.05); compared with the model group, there was no significant difference in the grasping strength of mice in the other four groups (Medobar group, walnut insoluble dietary fiber group, medium dose, and high dose walnut group) (P>0.05). In the fifth and ninth weeks of drug administration, compared with the control group, the grasping strength of mice in the model group was significantly reduced (P<0.05); compared with the model group, the grasping strength of mice in the other four groups (Medobar group, walnut insoluble dietary fiber low dose, medium dose, and high dose group) was significantly increased (P<0.05). This indicates that walnut insoluble dietary fiber can improve the muscle strength of mice, and high-dose walnut insoluble dietary fiber has the best effect.

[0093] (3) Effect of walnut insoluble dietary fiber on the time spent on the rod in rotenone-induced PD mice

[0094] The rotarod test was used to record the duration of continuous movement of mice on the rotarod to evaluate the motor coordination and balance of mice. Figure 5As shown. In the first week of drug administration, the model group mice maintained no significant difference in exercise time on the rotarod compared with the control group (P>0.05); compared with the model group, the other four groups (Medopar group, walnut insoluble dietary fiber low dose / medium dose / high dose group) mice maintained no significant difference in exercise time on the rotarod (P>0.05). In the fifth week of drug administration, the model group mice maintained a significantly shorter exercise time on the rotarod compared with the control group (P<0.05); compared with the model group, the other four groups (Medopar group, walnut insoluble dietary fiber low dose / medium dose / high dose group) mice maintained no significant difference in exercise time on the rotarod (P>0.05). At the 9th week of drug administration, the model group mice maintained a significantly shorter exercise time on the rotarod than the control group (P<0.05); compared with the model group, the Madopar group mice maintained a significantly longer exercise time on the rotarod (P<0.05); the other three groups (low-dose / medium-dose / high-dose walnut insoluble dietary fiber groups) maintained an increasing exercise time on the rotarod but without statistical significance (P>0.05). This indicates that walnut insoluble dietary fiber can improve the motor coordination and balance of mice to a certain extent, and the high-dose walnut insoluble dietary fiber has the best effect.

[0095] (4) Effect of walnut insoluble dietary fiber on adhesive removal time in rotenone-induced PD mice

[0096] The purpose of the glue removal experiment is to evaluate the coordination and balance of mice by testing the time it takes them to remove glue. Figure 6 As shown. After 1 week of drug administration, there was no significant difference in the adhesive removal time of mice in the model group compared with the control group (P>0.05); compared with the model group, there was no significant difference in the adhesive removal time of mice in the other four groups (Medopar group, walnut insoluble dietary fiber low dose / medium dose / high dose group) (P>0.05). At the 5th week of drug administration, compared with the control group, the adhesive removal time of mice in the model group was significantly increased (P<0.05); compared with the model group, the adhesive removal time of mice in the Medopar group was significantly reduced (P<0.05); there was no significant difference in the adhesive removal time of mice in the other three groups (walnut insoluble dietary fiber low dose / medium dose / high dose group) (P>0.05). At the 9th week of drug administration, the model group mice had a significantly longer adhesive removal time than the control group (P<0.05); compared with the model group, the adhesive removal time of the other four groups (Medopar group, walnut insoluble dietary fiber low dose / medium dose / high dose group) was significantly reduced (P<0.05). This indicates that walnut insoluble dietary fiber can improve the limb coordination and balance of mice, with the best effect at medium and high doses.

[0097] (5) Effect of walnut insoluble dietary fiber on the spontaneous activity of rotenone-induced PD mice

[0098] The distance and speed of movement of mice in the open field reflect the autonomous activity ability of mice. Figure 7 As shown, after 1 and 5 weeks of drug administration, the movement distance of mice in the model group was significantly shortened compared with that in the control group (P<0.05), while the movement distance of mice in the other four groups (Medopar group, low-dose / medium-dose / high-dose walnut insoluble dietary fiber groups) did not change significantly (P>0.05). After 9 weeks of drug administration, compared with that in the control group, the movement distance of mice in the model group was significantly shortened (P<0.05), while the movement distance of mice in the other four groups (Medopar group, low-dose / medium-dose / high-dose walnut insoluble dietary fiber groups) increased significantly (P<0.05).

[0099] In the first week of experimental animal administration, the movement speed of mice in the model group was significantly reduced compared with the control group (P<0.05); compared with the model group, there was no significant difference in the movement speed of mice in the other four groups (Madopar group, walnut insoluble dietary fiber low dose / medium dose / high dose group) (P>0.05). In the fifth week of experimental animal administration, the movement speed of mice in the model group was significantly reduced compared with the control group (P<0.05); compared with the model group, the movement speed of mice in the Madopar group was significantly increased (P<0.05), and there was no significant difference in the movement speed of mice in the other three groups (walnut insoluble dietary fiber low dose / medium dose / high dose group) (P>0.05). In the ninth week of experimental animal administration, the movement speed of mice in the model group was significantly reduced compared with the control group (P<0.05); compared with the model group, the movement speed of mice in the other four groups (Madopar group, walnut insoluble dietary fiber low dose / medium dose / high dose group) was significantly increased (P>0.05). The movement trajectory in the 9th week also showed that walnut insoluble dietary fiber can significantly improve the autonomous activity ability of mice, and high-dose walnut insoluble dietary fiber has the best effect.

[0100] Example 3 Walnut dietary fiber can improve Parkinson's disease symptoms by protecting dopaminergic neurons

[0101] PD is characterized by the loss of dopaminergic neurons in the substantianigra (SN). Tyrosine hydroxylase (TH) is a key enzyme in the synthesis of DA in dopaminergic neurons and their terminals. During the pathological progression of PD, the level of TH decreases. In order to explore whether walnut insoluble dietary fiber can protect dopaminergic neurons from the toxicity of rotenone, the present invention performed immunohistochemistry and Nissl staining on the SN region.

[0102] Immunohistochemistry

[0103] Mouse brain tissue was dissected, fixed, dried, paraffin-embedded and cut into 4 μm thick slices. After inhibiting endogenous peroxidase activity with 3% H2O2 and repairing antigens with citrate buffer, the slices were blocked with 5% goat serum and treated with anti-TH primary antibody (ab137869, Abcam, 1:500) overnight at 4°C. The next day, the slices were incubated with biotin-conjugated goat anti-rabbit IgG antibody and stained using the avidin-biotin complex (ABC) system and diaminobenzidine (DAB) incubation.

[0104] Nissl stain

[0105] Coronal sections of mouse brain were attached to slides, dried naturally, and then soaked in xylene for 10 minutes, fresh xylene for 10 minutes, ethanol for 5 minutes, fresh ethanol for 5 minutes, and then treated with 75% ethanol for 5 minutes. The sections were washed with distilled water for 2 minutes and stained with Nissl staining solution for 4 minutes. The slides were gently washed in deionized water until all the washing mixture was removed. Subsequently, the excess water on the sections was drained and the sections were placed in an oven at 65°C to dry. They were then infiltrated with xylene and sealed with neutral gum.

[0106] The experimental results are as follows Figure 8 As shown in Figure 2, compared with the model group, walnut insoluble dietary fiber significantly increased the number of TH-positive neurons ( Figure 8 A) and the number of Nissl-positive neurons ( Figure 8 B), which means that administration of walnut insoluble dietary fiber protects dopaminergic neurons.

[0107] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. An application of walnut dietary fiber in preparing a drug for treating Parkinson's disease, characterized in that: The walnut dietary fiber is insoluble walnut dietary fiber; The monosaccharide composition of the insoluble walnut dietary fiber is: galacturonic acid, glucose, xylose, arabinose, galactose, guluronic acid, rhamnose, glucuronic acid, mannuronic acid and fucose; The preparation method of the insoluble walnut dietary fiber is as follows: (1) Grease removal After grinding the walnuts, defatting was performed three times with a ratio of walnuts to petroleum ether of 1:10-1:15 (w / v), each time for 100-150 min, stirring once every 15-20 min, and then drying at room temperature to obtain defatted samples, which were sieved before use; (2) Removal of starch The defatted sample was dispersed in deionized water at a ratio of 1:10-1:15 (w / v), and when heated in a water bath to 83-88°C, 0.2-0.3 mL / g of heat-resistant α-amylase was added, and the mixture was stirred and reacted at 88-92°C for 20-40 min; after cooling to 55-65°C, 0.10-0.15 mL / g of amyloglucosidase was added, and the mixture was stirred and reacted at 58-62°C for 2.5-3.5 h; after the enzymolysis was completed, the enzyme was inactivated in a boiling water bath for 15-20 min, 3-5 times the volume of 95% ethanol preheated to 58-62°C was added to the enzymolysis solution, and the solution was cooled and refrigerated overnight, and then the de-starched sample was obtained by centrifugation and freeze-drying; (3) Protein removal The destarch samples were dispersed in deionized water at a ratio of 1:20-30 (w / v) and hydrolyzed by alkaline protease, flavor protease, and acid protease in sequence to remove proteins; (4) Inactivation After the enzymatic hydrolysis is completed, the enzyme is inactivated by boiling for 15-20 min; the enzyme is cooled to room temperature, and the enzymatic hydrolyzate is centrifuged to obtain the residue, which is then washed with water, neutralized, washed with alcohol, and freeze-dried to obtain insoluble walnut dietary fiber.

2. The use of walnut dietary fiber according to claim 1 in preparing a drug for treating Parkinson's disease, characterized in that: The enzymatic hydrolysis reaction conditions of the alkaline protease are as follows: pH 10.5-11.5, enzyme addition amount 1500-1800 U / mL, reaction temperature 40-48°C, and reaction time 4-6 h; the enzymatic hydrolysis reaction conditions of the flavor protease are as follows: pH 7.5-8.5, enzyme addition amount 400-600 U / mL, reaction temperature 48-53°C, and reaction time 4-6 h; the enzymatic hydrolysis reaction conditions of the acidic protease are as follows: pH 2.8-3.2, enzyme addition amount 400-600 U / mL, reaction temperature 48-53°C, and reaction time 4-6 h.

3. The use of walnut dietary fiber according to claim 1 in preparing a drug for treating Parkinson's disease, characterized in that: The daily dosage range of the insoluble walnut dietary fiber is 50-900 mg / kg.

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