Application of arabinoxylan and composition of arabinoxylan

Abstract

The invention provides an application of arabinoxylan, and the application is selected from any one of the following: an application of arabinoxylan in preparation of a medicine for improving dyskinesia, the dyskinesia including dyskinesia, and an application of arabinoxylan in preparation of a medicine for improving dyskinesia. The application is used for preparing medicines for improving the expression quantity of TH and BDNF in the brain nigra and reducing the expression quantity of alpha-syn, IFN-gamma and IL1-beta proteins; the application is used for preparing a medicine for improving brain tissue nigra neuron damage; the application is used for preparing medicines for improving the expression quantity of BDNF in serum and reducing the expression quantity of TNF-alpha, IL-1beta and NF-kappa B proteins; the compound is used for preparing a medicine for treating Parkinson's disease. According to the technical scheme, a new thought is provided for clinical medication selection of the Parkinson's disease.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the use of arabinoxylan and a composition thereof. Background Art

[0002] Arabinoxylan (AX) is a non-starch polysaccharide found in many cereals. It has immunomodulatory and probiotic properties, and its functional activity is closely related to the structural composition. As a dietary fiber, AX can increase the beneficial microbial flora such as bifidobacteria and lactic acid bacteria. When bifidobacteria ferment dietary fiber in the colon, the short-chain fatty acids produced can reduce the pH value of the intestine and inhibit the growth of potential pathogenic bacteria. As a functional oligosaccharide, its probiotic effect can reduce the risk of many chronic diseases. A large number of studies have found that the aggregation of α-synuclein in the intestine can cause intestinal flora imbalance, thereby increasing intestinal permeability, which may cause the accumulation of α-synuclein in the enteric nervous system and affect the brain, promoting the occurrence and development of the disease. At present, the research on the medicinal value of AX mainly focuses on regulating intestinal flora, improving the symptoms of Parkinson's disease rats, and enhancing immunity.

[0003] Parkinson's disease is one of the common diseases in the degenerative nervous system, with a constantly evolving level of complexity. It has been characterized by the classic motor features of Parkinson's disease associated with Lewy bodies and the loss of dopaminergic neurons in the substantia nigra. The incidence rate is generally increased in the middle-aged and elderly population, seriously affecting the quality of life of patients. The clinical manifestations of Parkinson's disease include a variety of motor symptoms and non-motor symptoms. The clinical manifestations of gastrointestinal dysfunction in Parkinson's patients include malnutrition, oral and oral diseases, salivation, dysphagia, gastroparesis, constipation, and defecation dysfunction.

[0004] Currently, the use of dopamine in the clinical treatment of Parkinson's disease can temporarily control the patient's motor symptoms, but non-motor symptoms such as the continued loss of dopaminergic neurons and gastrointestinal dysfunction have not yet been resolved. Therefore, more treatments and drugs are needed clinically to slow the progression of Parkinson's disease. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a use of arabinoxylan and a composition thereof to solve the problem that the treatment methods in the prior art cannot control the non-motor symptoms of Parkinson's disease, so as to delay the progression of Parkinson's disease.

[0006] To achieve the above-mentioned purpose and other related purposes, the present invention is implemented through the following technical solutions.

[0007] The first aspect of the present invention provides the use of arabinoxylan, wherein the use is selected from any one of the following:

[0008] Use for preparing a drug for improving movement dysfunction, wherein the movement dysfunction includes dystonia;

[0009] Use for preparing a drug for increasing the expression of TH and BDNF in the substantia nigra and reducing the expression of α-syn and IFN-γ;

[0010] Use for preparing a drug for improving damage to substantia nigra neurons in brain tissue;

[0011] Use for preparing a drug for reducing the expression of IL-1β;

[0012] Used for preparing drugs for treating Parkinson's disease.

[0013] According to the above-mentioned use, the polysaccharide content in the arabinoxylan is greater than 60%.

[0014] According to the above-mentioned use, the arabinoxylan is prepared by pre-treating plant raw materials to remove impurities, then extracting with alkali solution, and purifying with dealkalization.

[0015] According to the above-mentioned use, the plant raw material includes one or more of straw, corn cobs, bran or wood, etc., whose hemicellulose components are mainly arabinoxylan.

[0016] According to the above-mentioned use, the specific preparation method of the arabinoxylan is as follows:

[0017] 1) After the above-mentioned plant raw materials are crushed, 8 to 12 times the weight of NaOH solution with a mass concentration of 0.2 to 1.0% is added, and the mixture is heat-treated at 40 to 90° C. for 1.5 to 2.5 hours, and then replaced and washed with clean water and filtered and dehydrated to obtain clean fibers.

[0018] 2) Add 6 to 10 times the weight of a NaOH solution with a mass concentration of 8 to 12% to the clean fiber obtained in step 1), extract at 55 to 65° C. for 5.5 to 6.5 hours, collect a primary supernatant by sedimentation and centrifugation, and continuously countercurrent replace the residue with 8 to 12 times the weight of a NaOH solution with a mass concentration of 3 to 5%. Collect a secondary supernatant after sedimentation and centrifugation of the replacement liquid, and combine the primary and secondary supernatants to obtain an arabinoxylan alkali solution.

[0019] 3) The arabinoxylan alkali solution obtained in step 2) is subjected to de-alkali treatment, then acid is added to neutralize it to a pH of 6.0 to 8.0, concentrated and dehydrated, and then clean water is added to perform continuous displacement desalination treatment, and dried to obtain the product.

[0020] According to the above-mentioned use, in the above step 2), the first sedimentation centrifugation adopts a horizontal spiral sedimentation centrifuge, and the second sedimentation centrifugation adopts a butterfly centrifuge with a higher separation factor.

[0021] According to the above-mentioned use, the above-mentioned step 3) can be carried out by the dealkalization method using an ultrafiltration membrane or an electrodialysis membrane replacement method, or both. Preferably, the ultrafiltration membrane has a molecular weight cutoff of ≤10 kDa.

[0022] According to the above-mentioned use, the acid used in the above step 3) is any one of hydrochloric acid, citric acid or acetic acid, and the mass concentration is 4-6%.

[0023] According to the above-mentioned use, the above-mentioned step 3) concentration and dehydration is concentrated by ultrafiltration membrane filtration; preferably, the ultrafiltration membrane has a molecular weight cutoff of ≤10kDa.

[0024] The second aspect of the present invention provides a composition for treating Parkinson's disease, wherein the composition comprises arabinoxylan and a pharmaceutically acceptable adjuvant.

[0025] According to the above-mentioned composition, the composition further comprises an anti-Parkinson's disease drug.

[0026] According to the above-mentioned composition, the anti-Parkinson's disease drug is Madopar.

[0027] According to the above-mentioned composition, the mass ratio of arabinoxylan to Madopar is 15-20:1. Preferably, the mass ratio may be 15-17:1, 17-19:1, or 19-20:1. In a specific embodiment, the mass ratio is 16:1.

[0028] According to the above-mentioned composition, the composition is in the form of tablets, powders, granules, suspensions, capsules or emulsions. The composition of the present invention can be administered parenterally in the form of sterile injectable aqueous or oily suspensions, and the parenteral administration is subcutaneous administration or intravenous administration or intramuscular administration or intrasternal administration or administration by infusion technology. The suspending agent can be prepared according to known technology using the above-mentioned appropriate dispersants, wetting agents or suspending agents or other acceptable substances.

[0029] The third aspect of the present invention provides a use of the above-mentioned composition for preparing a drug for treating Parkinson's disease.

[0030] According to the above-mentioned use, it is used to prepare a drug for treating one or more of the following: motor dysfunction caused by Parkinson's disease, damage to substantia nigra neurons in brain tissue, changes in intestinal flora, and low levels of neurotrophic factors in serum and substantia nigra.

[0031] The arabinoxylan may be commercially available arabinoxylan or arabinoxylan prepared according to the preparation method of the present invention.

[0032] The present application provides the use of arabinoxylan and its composition. According to the data results of multiple groups of animal experiments, the technical solution of the present application has the following beneficial effects:

[0033] 1. The present application has discovered the uses of arabinoxylan, including the use of arabinoxylan for preparing drugs for improving motor dysfunction, wherein the motor dysfunction includes dystonia; the use of arabinoxylan for preparing drugs for increasing the expression of TH and BDNF in the substantia nigra and reducing the expression of α-syn and IFN-γ; the use of arabinoxylan for preparing drugs for improving neuronal damage in the substantia nigra of brain tissue; the use of arabinoxylan for preparing drugs for reducing the expression of IL-1β; and the use of arabinoxylan for preparing drugs for treating Parkinson's disease.

[0034] 2. At the same time, the present application also provides an arabinoxylan composition for treating Parkinson's disease, the composition comprising arabinoxylan and Madopar. The composition can not only relieve the motor symptoms of Parkinson's disease, but also increase the expression of TH and BDNF in the substantia nigra of Parkinson's rats, reduce the expression of α-syn and IFN-γ proteins; increase the content of BDNF in serum, and reduce the content of TNF-α and IL-1β in serum; regulate the diversity of intestinal microbial flora, and improve the changes in intestinal flora caused by Parkinson's disease; alleviate the damage of substantia nigra neurons in the brain tissue of Parkinson's rats, and improve the non-motor symptoms of Parkinson's disease.

[0035] 3. The technical solution of this application provides new ideas for the clinical drug selection for Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Shown are the immunohistochemical staining pictures of rats in each group in Example 3.

[0037] Figure 2 The figure shows the inter-group difference analysis of the α diversity of the intestinal flora of each group of rats in Example 4. Among them, Figures 2 to 6 In the figure, K represents the blank group, R represents the sham operation group, D represents the model group, M represents the Madopar group, X represents the arabinoxylan group, and MX represents the Madopar + arabinoxylan group.

[0038] Figure 3 Shown is a bar graph of the LDA value distribution in the LEfSe species difference analysis results in Example 4.

[0039] Figure 4 Shown is a graph showing the results of the analysis of the differential species among the intestinal flora groups of each group of rats in Example 4.

[0040] Figure 5 The relative expression levels of α-syn, TH, NF-κBP65, BDNF and INF-γ in the substantia nigra of each group of rats in Example 5 are shown.

[0041] Figure 6 Shown are electron microscopic images of the ultrastructural changes in substantia nigra neurons of rats in each group in Example 6. DETAILED DESCRIPTION

[0042] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0043] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0044] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0045] The arabinoxylan in the following embodiments of the present application is prepared by a specific method, comprising the following steps:

[0046] 1) 10 kg of dry, mold-free cereal bran was selected, 100 kg of a 0.2% NaOH solution was added, and the mixture was kept warm at 40° C. for 2 h, and then washed with clean water and filtered to obtain clean fiber.

[0047] 2) Add 80 kg of 8% NaOH solution to the clean fiber obtained in step 1), extract at 60°C for 6 hours, collect the primary supernatant by sedimentation centrifugation using a horizontal spiral sedimentation centrifuge, and continuously countercurrent replace the residue with 100 kg of 4% NaOH solution, collect the secondary supernatant by sedimentation centrifugation using a butterfly centrifuge, combine the primary and secondary supernatants to obtain 165 kg of arabinoxylan alkali solution.

[0048] 3) After the arabinoxylan alkali solution obtained in step 2) is de-alkalized by ultrafiltration membrane, hydrochloric acid with a mass concentration of 5% is slowly added while stirring to neutralize to pH 6.0-8.0, concentrated and dehydrated by ultrafiltration membrane, and then added with clean water for continuous displacement desalination treatment, spray dried, and 1.1 kg of arabinoxylan is obtained. The ultrafiltration membrane has a molecular weight cutoff of ≤10kDa. The hydrolyzable polysaccharide content in the finally obtained arabinoxylan is 82%, and the various pollutant indicators are within the national standard range for cereal foods.

[0049] Example 1 Establishment of rat Parkinson's disease model and group intervention

[0050] Forty-eight SPF healthy male rats were selected and randomly divided into blank group, sham operation group, model group, Madobar group, arabinoxylan group, and arabinoxylan+Madobar group, with 8 rats in each group.

[0051] 1) Establishment of Parkinson's disease model in rats

[0052] SPF healthy male rats were selected and injected subcutaneously with rotenone sunflower oil emulsion at the back of the rat's neck. The injection volume was 1.5 mg / (kg·d). The concentration of rotenone in sunflower oil was 1.5 mg / mL. The injection was continued for 6 consecutive days a week, once a day, and then stopped for 1 day for a total of four weeks, 28 days. During the injection period of rotenone sunflower oil emulsion, when the animal showed quadriplegia, inability to stand and walk, difficulty turning over, and inability to eat, the injection was immediately terminated. The criterion for the successful establishment of the screening model is: rotenone administration is terminated after the occurrence of behavioral scoring (3 to 5 weeks). Behavioral scoring criteria, score according to Table 1: Behavioral scores of 2-8 points can be considered as models.

[0053] Sham operation group: rats were injected subcutaneously with sunflower oil emulsion without rotenone at the back of the neck, with an injection volume of 1.5 mg / (kg·d), for 6 consecutive days a week, once a day, and then stopped for 1 day for a total of four weeks, 28 days.

[0054] Table 1 Parkinson model scoring criteria

[0055]

[0056] 2) Grouping by intervention method

[0057] Blank group: SPF healthy male rats, raised normally, without any treatment.

[0058] Sham operation group: The mice were raised normally without any treatment.

[0059] Model group: The selected Parkinson's disease model rats were given distilled water 10 ml / kg by gavage once a day for 14 consecutive days.

[0060] Madopar group: The selected Parkinson's disease model rats were given Madopar (50 mg / kg / d) suspension by gavage once a day for 14 consecutive days.

[0061] Arabinoxylan group: The selected Parkinson's disease model rats were given arabinoxylan (800 mg / kg / d) suspension by oral gavage once a day for 14 consecutive days.

[0062] Madopar + arabinoxylan group: The selected Parkinson's disease model rats were given Madopar (50 mg / kg / d) suspension + arabinoxylan (800 mg / kg / d) suspension by oral gavage once a day for 14 consecutive days. The solvent was distilled water.

[0063] 3) Changes in body weight of rats in each group

[0064] There was no significant difference in the initial body weight of rats in each group before administration. As shown in Table 2, compared with the blank group and the sham operation group, the body weight of the model group decreased significantly (P<0.05). After 14 days of intervention, the body weight of the rats in the Madobar group, the arabinoxylan group and the Madobar+arabinoxylan group increased significantly compared with the model group (P<0.05). Compared with the Madobar group, there was no statistically significant difference in the body weight of the rats in the arabinoxylan group (P>0.05). Compared with the arabinoxylan group, the body weight of the rats in the Madobar+arabinoxylan group increased significantly, and the difference was statistically significant (P<0.05). The drug itself did not cause a significant decrease in the body weight of the rats, indicating that the safety of each composition itself was good.

[0065] Table 2 Body weight changes of rats in each group after 14 days of intervention (x - ±s)

[0066]

[0067] Note: ① The measurement data in the table are expressed as (x - ±s) indicates② compared with the sham operation group, a P<0.05, aa P<0.01; compared with the model group, b P<0.05, bb P<0.01; compared with the arabinoxylan group, c P<0.05, cc P<0.01.

[0068] Example 2

[0069] Used to prove that the combined use of arabinoxylan and Madopar has a significant improvement effect on the behavior of rats in the Parkinson's model group.

[0070] 1) Behavioral testing methods

[0071] A) Suspension test: used to test rat muscle tension. Before the test, let the rats adapt to the environment for 30 minutes. Prepare a wire with a diameter of 2 mm and a length of about 20 cm in advance at a height of 1 meter from the ground. Hang the rats' two front paws on the horizontal wire and record the time before the rats fall to the ground. If the rats fall within 3 seconds or only one paw grabs the wire, they are considered failures. Each rat is measured 3 times, with an interval of 5 minutes each time. The final suspension time is the average of the 3 times.

[0072] B) Pole climbing test: The pole climbing test device is a 50cm high and 1cm diameter wooden stick fixed on a wooden base, with a 2.5cm diameter sphere on the top. The outer layer of the wooden stick is wrapped with two layers of gauze to prevent the rat from slipping during the crawling process. The pole climbing test was carried out at the end of the first and second weekends of the experiment. First, the rats were adapted to the training, and then placed on the top of the ball. The total time for the rats to climb from the top of the pole to the bottom of the pole and land on their hind legs was recorded. The test was repeated 3 times, with an interval of at least 10 minutes each time. If the rat paused or climbed in the opposite direction, it was retested. The average of the 3 times was taken. At the same time, the rats were scored according to the scoring criteria for the pole climbing test, see Table 3 for details.

[0073] Table 3 Pole climbing experiment scoring table

[0074]

[0075]

[0076] 2) Test results

[0077] A) Suspension test results

[0078] See Table 4 for details. As can be seen from Table 3, compared with the sham operation group, the hanging time of rats in the model group was shorter (P<0.05), and there was no significant difference between the sham operation group and the normal group (P>0.05); after 14 days of intervention, compared with the model group, the hanging time of rats in the arabinoxylan group, the Madobar group and the arabinoxylan+Madobar group increased significantly (P<0.05), and compared with the arabinoxylan group, the hanging time of the arabinoxylan+Madobar group increased more significantly (P<0.05), and the difference was statistically significant. It shows that the pharmaceutical composition provided by the present invention has a significant improvement effect on the behavior of rats.

[0079] Table 4 Hanging time of rats in each group after 14 days of intervention

[0080]

[0081] Note: ① The measurement data in the table are expressed as (x - ±s) indicates② compared with the sham operation group, a P<0.05,aa P<0.01; compared with the model group, b P<0.05, bb P<0.01; compared with the arabinoxylan group, c P<0.05, cc P<0.01.

[0082] B) Pole climbing experiment results

[0083] According to the scores in Table 3, as shown in Table 5, it can be seen from Table 5 that compared with the blank group and the sham operation group, the model group has significantly weakened the coordination ability of the rats' limbs, accelerated the decline, and even failed to hold the pole and fell directly. The difference is significant (P < 0.001). During the climbing process of the rats in the arabinoxylan group, the Madopar group, and the arabinoxylan + Madopar group, the rats used all four limbs and coordinated their movements, and were able to climb to the bottom of the pole at one time. Compared with the model group, the coordination of the limbs was significantly improved, and the difference was statistically significant (P < 0.05). The arabinoxylan group was compared with the arabinoxylan + Madopar group, and the difference was statistically significant (P < 0.05). This shows that the pharmaceutical composition provided by the present invention can significantly improve the coordination ability of the limbs of Parkinson's rats, and the effect is better than using Madopar alone.

[0084] Table 5 Changes of the pole climbing test in each group of rats after 14 days of intervention

[0085]

[0086] Note: ① The measurement data in the table are expressed as (x - ±s) indicates② compared with the sham operation group, a P<0.05, aa P<0.01; compared with the model group, b P<0.05, bb P<0.01; compared with the arabinoxylan group, c P<0.05, cc P<0.01.

[0087] Example 3

[0088] It is used to illustrate that the combination of arabinoxylan and Madopar in the technical solution of the present application increases the expression levels of TH and BDNF in the substantia nigra of rats in the Parkinson's model group, and reduces the expression levels of α-syn, IFN-γ, and IL1-β proteins.

[0089] 1) Immunohistochemical staining method

[0090] After anesthesia, rats in each group were decapitated and brains were removed. The whole brain was fixed in 4% paraformaldehyde. After fixation for 24 hours, the midbrain was cut off and embedded in paraffin. The slices were routinely dewaxed and placed in 3% citric acid repair solution for high pressure repair. After the valve was opened, the repair was performed for 10 minutes. The pressure cooker was naturally cooled to room temperature and washed with distilled water for 5 minutes. 3% methanol hydrogen peroxide was added for 20 minutes, washed with distilled water for 5 minutes, and soaked in PBS for 1 minute. 5% BSA was added dropwise, incubated at room temperature for 30 minutes and discarded. The primary antibody working solution (dilution multiple of 1:120) was added dropwise, incubated at 4°C overnight, and washed with PBS buffer 3 times, each time for 5 minutes. The secondary antibody working solution was added dropwise and incubated at 37°C for 20 minutes. Washed with PBS buffer 3 times, each time for 5 minutes. The cells were stained with DAB, washed with tap water, counterstained with hematoxylin, differentiated, and blued, dehydrated, made transparent, and sealed. The expressions of α-syn and TH proteins in the substantia nigra tissue of the midbrain of each group of rats were observed under a microscope, and pictures were collected at 100 times the magnification.

[0091] 2) Immunohistochemical staining results

[0092] like Figure 1 As shown, the cell nucleus of a negative result is blue, and that of a positive result is brown or tan.

[0093] Compared with the sham operation group and the blank control group, the expression levels of α-syn, IFN-γ, and IL1-β in the model group increased; compared with the model group, the expression levels of α-syn, IFN-γ, and IL1-β in the Madopar group, the arabinoxylan group, and the Madopar + arabinoxylan group decreased. Compared with the blank group and the sham operation group, the expression levels of TH and BDNF in the model group decreased; compared with the model group, the expression levels of TH and BDNF in the Madopar group, the arabinoxylan group, and the Madopar + arabinoxylan group increased.

[0094] These results indicate that the pharmaceutical composition provided by the present invention can increase the expression levels of TH and BDNF in the substantia nigra of Parkinson's rats, and reduce the expression levels of α-syn, IFN-γ, and IL1-β proteins.

[0095] Example 4

[0096] Used to illustrate that arabinoxylan combined with Madopar enriches the diversity of intestinal flora in rats in the Parkinson's model group.

[0097] 1) Intestinal flora detection method

[0098] The colonic feces of rats in each group were collected using a 1.5 ml sterile EP tube, and the feces were immediately stored in a -80 ° C refrigerator after collection. After quality inspection, the 16SrDNA gene high-throughput sequencing technology was used to detect the intestinal flora of rats. The V3-V4 region sequence of bacterial 16SrDNA was selected for gene amplification and gene sequencing. The diversity was compared by using the α diversity index Chao1 (species richness), Shannon and Simpson (used to evaluate species diversity), and the difference in flora between groups was found out by analyzing the difference in flora between groups.

[0099] 2) Intestinal flora test results

[0100] A) Analysis of bacterial alpha diversity

[0101] The colonic feces of rats were collected and 16SrDNA high-throughput gene sequencing was performed to detect the intestinal flora of rats. The α diversity analysis of the intestinal flora of rats was performed, and the results are detailed in Figure 2 , Figure 2 This is the analysis of the differences between the Alpha diversity groups; the box plot can intuitively reflect the median, dispersion, maximum, minimum, and outliers of the species diversity within the group. At the same time, the T-test and Wilcoxon rank sum test (T-test when there are only 2 groups, Wilcoxon rank sum test when there are more than 2 groups) are used to analyze whether the differences in species diversity between the groups are significant. Figure 2 It can be seen that compared with the blank group and sham operation, the Chao1, Shannon, and Simpson indexes of the model group were reduced, and the diversity of the flora was less, and the differences were statistically significant (all P<0.05). Compared with the model group, the α diversity index of the arabinoxylan group was increased, and the flora diversity was richer, and the differences were statistically significant (all P<0.05). The results show that arabinoxylan can increase the α diversity index of intestinal flora and effectively improve the richness of intestinal flora.

[0102] B) LEfSe species difference analysis

[0103] LEfSe (LDA Effect Size) is an analytical tool for discovering and interpreting high-dimensional biomarkers (genes, pathways, and taxa). It can be used to compare two or more groups. It emphasizes statistical significance and biological relevance, and can find biomarkers with statistical differences between groups. It allows researchers to identify features of different abundances and associated categories. The LDA value distribution bar chart is a statistical result of LEfSe. The LDA value distribution bar chart shows species with significant abundance differences in different groups, and the length of the bar chart represents the effect size of the different species. The vertical axis is the species with significant differences between groups, and the horizontal axis uses a bar chart to intuitively display the LDA analysis logarithmic score value of each species. Species are sorted according to the size of the score value. The longer the length, the more significant the difference of the species. The color of the bar graph is the sample group corresponding to the species, that is, red (counting from top to bottom, the 33rd to 40th bars) represents the blank group, blue (counting from top to bottom, the 18th to 24th bars) represents the model group, green (counting from top to bottom, the 25th to 32nd bars) represents the sham operation group, cyan (counting from top to bottom, the 4th to 6th bars) represents the Madopar group, purple (counting from top to bottom, the 7th to 17th bars) represents the arabinoxylan group, and yellow (counting from top to bottom, the 1st to 3rd bars) represents the Madopar + arabinoxylan group. An LDA threshold greater than 2 indicates that the microorganisms have significant differences. The specific results are as follows Figure 3 shown.

[0104] LEfSe species difference analysis was performed on the intestinal flora of rats, and an LDA threshold greater than 2 represented significant differences in microorganisms. Figure 3 It can be seen that there are 8 dominant bacteria in the blank group, 8 dominant bacteria in the sham operation group, 7 dominant bacteria in the model group, 11 dominant bacteria in the arabinoxylan group, 3 dominant bacteria in the Madopar group, and 3 dominant bacteria in the Madopar + arabinoxylan group. The results of LEfSe species difference analysis showed that the intestinal flora of rats in each group was significantly different.

[0105] C) Analysis of species differences among groups

[0106] The species differences between groups of rat intestinal flora were analyzed. Through statistical analysis, species with significant differences in abundance between groups can be found out in a targeted manner, and the enrichment of differential species between different groups can be obtained. At the same time, the size of intra-group differences and inter-group differences can be compared to determine whether the differences in community structure between different groups are significant. Figure 4 The middle left figure shows the species abundance differences among the intestinal bacteria of each group of rats. Each bar in the figure represents the mean value of the species (bacteria) in each group with significant abundance differences among the groups. Figure 4The middle right figure is a display of the confidence level of the differences between the intestinal bacteria of each group of rats. The leftmost end point of each circle in the figure represents the lower limit of the 95% confidence interval of the mean difference, and the rightmost end point of the circle represents the upper limit of the 95% confidence interval of the mean difference. The center of the circle represents the difference in the mean. The group represented by the color of the circle is the group with a high mean. Figure 4 The far right end of the middle right figure is the p value of the inter-group significance test corresponding to the different species.

[0107] This study found that at the genus level, Figure 4 As shown in the data, for the abundance of Prevotellaceae UCG-001, Bacteroides, Blautia, Parabacteroides, Alistipes, Ruminococcaceae UCG-005, and Prevotellaceae NK3B31_groupPrevotellaceae NK3B31group, the abundance of the above genera in the model group was significantly decreased compared with that in the blank group, and their abundance in the arabinoxylan group, the Madobar group, and the Madobar + arabinoxylan group was significantly increased after intervention compared with the model group. As for the abundance of Vagococcus, Kurthia, Desulfovibrio, and Romboutsia, the abundance of the above genera in the model group was significantly higher than that in the blank group.

[0108] The above analysis results comprehensively show that the pharmaceutical composition provided by the present invention can enrich the diversity of the intestinal flora of Parkinson's rats, regulate the disorder of the intestinal flora of Parkinson's rats, and improve non-motor symptoms.

[0109] Example 5

[0110] It is used to prove that arabinoxylan combined with Madopar increases the expression of TH and BDNF in the substantia nigra of rats in the Parkinson's model group, and reduces the expression of α-synuclein and IFN-γ.

[0111] 1) Western blotting assay

[0112] 10 mg of the substantia nigra tissue of each group of rats was weighed and cut into small pieces, added with 100-200 μl of the prepared RIPA lysis solution, and fully crushed with a glass homogenizer. After sufficient lysis, centrifuged at 10000-12000 r / min for 10 minutes, took the supernatant, and transferred the supernatant to a new EP tube to obtain the total protein product of the tissue; BCA protein quantification, calculated the concentration of the protein to be tested according to the absorbance, and unified the protein loading amount; according to the relative molecular weight of the measured protein, appropriate concentrated gel and separation gel were selected for vertical electrophoresis, and then the protein was transferred to the PVDF membrane, blocked with 5% skim milk for 1 hour, washed 3 times, added mouse anti-TH antibody (1:1000), and incubated at 4°C overnight. After TBST washing, HRP-labeled goat anti-mouse IgG (1:5000) was added, incubated at room temperature for 2 hours, and the membrane was washed; according to every 10 cm 2 Prepare 1 ml of ECL working solution, and finally use a fluorescent imager for exposure and development. Image J software calculates the gray value, and statistical analysis is performed based on the ratio of target protein / internal reference.

[0113] 2) Western blotting results

[0114] See Table 6 and Figure 5 Figures A to E are the protein immunoblotting results of α-syn, TH, NF-κBP65, INF-γ and BDNF, respectively; Figures F to J are the quantitative bar graphs of the protein immunoblotting results of α-syn, TH, NF-κBP65, INF-γ and BDNF, respectively.

[0115] Compared with the sham operation group, the expression levels of α-synuclein and IFN-γ in the model group increased; compared with the model group, the expression levels of α-synuclein and IFN-γ in the Madobar group, arabinoxylan group and Madobar + arabinoxylan group were significantly decreased, and compared with the arabinoxylan group, the expression level of α-synuclein in the Madobar + arabinoxylan group decreased more significantly, and the difference was statistically significant. Compared with the blank group and the sham operation group, the expression levels of TH and BDNF in the model group decreased; compared with the model group, the expression levels of TH and BDNF in the Madobar group, arabinoxylan group and Madobar + arabinoxylan group were significantly increased, and compared with the Madobar group, the expression levels of TH and BDNF in the Madobar + arabinoxylan group increased more significantly, and the difference was statistically significant.

[0116] Table 6 Relative expression of α-syn, TH, NF-κBP65, BDNF and INF-γ in the substantia nigra of rats in each group (x - ±s)

[0117]

[0118] Note: Compared with the sham operation group, a p<0.05,aa p<0.01; compared with the model group, b p<0.05, bb p<0.01; compared with the arabinoxylan group, cc p<0.05, cc p<0.01.

[0119] Therefore, the above data show that the present application found that arabinoxylan can increase the expression levels of TH and BDNF in the substantia nigra of Parkinson's model rats, and reduce the expression levels of α-synuclein and IFN-γ. At the same time, the arabinoxylan composition provided by the present invention can also increase the expression levels of TH and BDNF in the substantia nigra of Parkinson's model rats, and reduce the expression levels of α-synuclein and IFN-γ, and the effect is obvious.

[0120] Example 6

[0121] Used to illustrate that arabinoxylan combined with Madopar alleviates the damage of substantia nigra neurons in the brain tissue of Parkinson's rats.

[0122] 1) Transmission electron microscopy to observe the ultrastructure of substantia nigra neurons

[0123] The substantia nigra neuron specimens of rat brain tissue in each group were fixed with 2% paraformaldehyde-2.5% glutaraldehyde at 4℃ for 2h; rinsed with sodium cacodylate buffer (pH 7.2) for 10min×3; fixed with 1% osmium acid (OsO4) for 2h; rinsed with double distilled water for 10min×3; dehydrated with gradient alcohol, 50% alcohol for 10min, 70% alcohol overnight; 90% alcohol for 10min, 100% alcohol for 15min×2; replaced with propylene oxide for 15min; propylene oxide: resin 1:1, 1h, propylene oxide: resin 1:4, 1h at room temperature, pure resin soaking for 2h, embedded with epoxy resin Epon812; polymerization: 35℃ for 16h; 45℃ for 8h, 55℃ for 14h; 65℃ for 48h; embedded block trimming; 1μm semi-thin section, azure-methylene blue staining, optical microscope positioning; uranyl acetate / lead citrate staining, Hitachi Observation by H-7650 transmission electron microscope.

[0124] 2) Transmission electron microscopy observation of the ultrastructure of substantia nigra neurons

[0125] Ultrastructural changes of substantia nigra neurons were observed by transmission electron microscopy. Figure 6As shown, in the blank group, the inner and outer membranes of mitochondria were intact, the cristae structure was intact without edema or dissolution, the endoplasmic reticulum structure was normal without expansion, the nuclear membrane structure was intact without rupture, the nuclear chromatin distribution was normal, the myelin sheath was normal without rupture and stratification, and the synaptic structure was normal; in the sham operation group, the mitochondria were basically normal, with a small amount of slight edema, the endoplasmic reticulum structure was normal without rupture, the nuclear membrane structure was intact without rupture, the nuclear chromatin distribution was normal, the myelin sheath was normal without rupture and stratification, and the synaptic structure was not widened; in the model group, severe mitochondrial edema, the inner and outer membranes and cristae structure were dissolved, the cavity was vacuolated, and only residual images could be seen to distinguish, most of the endoplasmic reticulum degranulated, half of them expanded, the cell membrane was not expanded, the nuclear membrane was broken in many places, the nucleus was edematous, the myelin synaptic structure was normal, and after the cell edema, the cell membrane was broken and dissolved in many places, the cytoplasm overflowed, and some membrane components dissolved to form large vesicle-like substances. Compared with the model group, the damage in the Madopar group, arabinoxylan group, and arabinoxylan+Madopar group was improved, with mild to moderate mitochondrial edema, dissolution and rupture of some mitochondrial inner and outer membranes, mild dilation of the endoplasmic reticulum, relatively normal distribution of nuclear chromatin, occasional local ruptures in the nucleus, sparse chromatin, and normal myelin and synaptic structures.

[0126] The above results show that the pharmaceutical composition provided by the present invention, namely, arabinoxylan combined with Madopar treatment, can alleviate the damage of substantia nigra neurons in the brain tissue of Parkinson's rats caused by injection of rotenone sunflower oil emulsion, and improve the non-motor symptoms of Parkinson's rats.

[0127] Example 7

[0128] It is used to illustrate that arabinoxylan combined with Madopar reduces the serum TNF-α and IL-1β levels and increases the serum BDNF level in Parkinson's rats.

[0129] 1) Rat serum ELISA detection method

[0130] Take out the rat serum from each group from the -80℃ refrigerator and let it stand for 20 minutes. Dilute the samples and standards according to the instructions, add 100ul to each well, add sample diluent, standards with different concentration gradients, and samples to be tested, seal the plate with a sealing film, incubate at 37°, add enzyme, add stop dye after color development, and use an enzyme reader to detect the absorbance value at a wavelength of 450nm.

[0131] 2) Rat serum ELISA test results

[0132] The test results show that arabinoxylan combined with Madopar can improve the symptoms of Parkinson's rats. See Table 6 for details.

[0133] A) Serum TNF-α levels in rats of each group

[0134] Compared with the blank group and the sham operation group, the serum TNF-α content of the model group increased, and the difference was significant (P < 0.001); compared with the model group, the serum TNF-α content of the arabinoxylan, Madobar group, and Madobar + arabinoxylan group decreased; compared with the model group, the arabinoxylan group was P > 0.05, which was not statistically significant; compared with the model group, the difference between the Madobar group and the model group was statistically significant (P < 0.05); compared with the model group, the difference between the Madobar + arabinoxylan group and the model group was significant (P < 0.001). There was no statistically significant difference between the Madobar + arabinoxylan group and the Madobar group (P > 0.05); compared with the arabinoxylan group, the difference between the Madobar + arabinoxylan group and the arabinoxylan group was statistically significant (P < 0.05). It shows that the pharmaceutical composition provided by the present invention can significantly reduce the TNF-α content in serum.

[0135] B) Serum IL-1β levels in rats of each group

[0136] Compared with the blank group and the sham operation group, the serum IL-1β content of the model group increased, and the difference was significant (P < 0.001); compared with the model group, the serum IL-1β content of the arabinoxylan, Madobar group, and Madobar + arabinoxylan group decreased, and the difference between the arabinoxylan group and the model group was statistically significant (P < 0.05); compared with the model group, the difference between the Madobar group and the Madobar + arabinoxylan group was statistically significant (P < 0.001). There was no statistically significant difference between the Madobar + arabinoxylan group and the Madobar group (P > 0.05); compared with the arabinoxylan group, the Madobar group and the Madobar + arabinoxylan group had a statistically significant difference (P < 0.05). It shows that the pharmaceutical composition provided by the present invention can significantly reduce the IL-1β content in serum, and the effect is better than that of arabinoxylan alone.

[0137] C) Serum BDNF levels of rats in each group

[0138] Serum BDNF content of rats in each group: compared with the blank group, the serum BDNF content of the model group decreased, and the difference was significant (P < 0.01); compared with the model group, the serum BDNF content of the arabinoxylan, Madopar group, and Madopar + arabinoxylan group increased, and compared with the model group, the arabinoxylan and Madopar group had no statistical significance (P > 0.05); compared with the model group, the difference between the Madopar + arabinoxylan group and the model group was statistically significant (P < 0.05). It shows that the pharmaceutical composition provided by the present invention can significantly increase the BDNF content in serum, and the effect is better than that of using arabinoxylan or Madopar alone.

[0139] Table 7 Results of ELISA test of TNF-α, IL-1β and BDNF in rats of each group Unit: ug / ul

[0140]

[0141] Note: ① The measurement data in the table are =PD group is the model group; AX group is the arabinoxylan group; M group is the Madopar group; MX group is the arabinoxylan + Madopar group②Compared with the sham operation group, a p<0.05, aa p<0.01; compared with the model group, b p<0.05, bb p<0.01; compared with the arabinoxylan group, c p<0.05, cc p<0.01.

[0142] In summary, the present application found that arabinoxylan can reduce the serum IL-1β content of Parkinson's rats. At the same time, the arabinoxylan composition provided by the present application can also reduce the serum TNF-α and IL-1β content of Parkinson's rats and increase the serum BDNF content. The effect is better than using arabinoxylan or Madopar alone, and it has an improvement effect on the non-motor symptoms of Parkinson's rats.

Claims

1. The use of arabinoxylan, characterized in that: The use is selected from any of the following: Use for preparing a drug for improving movement dysfunction, wherein the movement dysfunction includes dystonia; Use for preparing a drug for increasing the expression of TH and BDNF in the substantia nigra and reducing the expression of α-syn and IFN-γ; Use for preparing a drug for improving damage to substantia nigra neurons in brain tissue; Use for preparing a drug for reducing the expression of IL-1β; Use for preparing medicine for treating Parkinson's disease.

2. The use according to claim 1, characterized in that The polysaccharide content in the arabinoxylan is greater than 60%.

3. The use according to claim 1, wherein the arabinoxylan is obtained by pre-treating plant raw materials to remove impurities, then extracting with alkali solution, and purifying by dealkalization; the plant raw materials include one or more of straw, corn cobs, bran or wood, whose hemicellulose components are mainly arabinoxylan.

4. The use according to claim 3, characterized in that The specific preparation method of the arabinoxylan is as follows: 1) After the plant raw material is crushed, 8 to 12 times the weight of a NaOH solution with a mass concentration of 0.2 to 1.0% is added, and the mixture is heat-treated at 40 to 90° C. for 1.5 to 2.5 hours, and then the mixture is replaced and washed with clean water and then filtered and dehydrated to obtain clean fibers; 2) adding 6 to 10 times the weight of a NaOH solution with a mass concentration of 8 to 12% to the clean fiber obtained in step 1), extracting at 55 to 65° C. for 5.5 to 6.5 hours, collecting a primary supernatant by sedimentation and centrifugation, and continuously countercurrently replacing the residue with 8 to 12 times the weight of a NaOH solution with a mass concentration of 3 to 5%, collecting a secondary supernatant after sedimentation and centrifugation of the replacement liquid, and combining the primary and secondary supernatants to obtain an arabinoxylan alkali solution; 3) The arabinoxylan alkali solution obtained in step 2) is de-alkalized, then acid is added to neutralize it to pH 6.0-8.0, concentrated and dehydrated, and then clean water is added to perform continuous displacement desalination treatment, and dried to obtain arabinoxylan.

5. A composition for treating Parkinson's disease, characterized in that: The composition comprises arabinoxylan and pharmaceutically acceptable adjuvants.

6. The composition according to claim 5, characterized in that The composition also includes an anti-Parkinson's disease drug.

7. The composition according to claim 6, characterized in that The anti-Parkinson's disease drug is Madopar.

8. The composition according to claim 7, characterized in that The mass ratio of the arabinoxylan to Madopar is (15-20):

1.

9. The composition according to claim 5, characterized in that The pharmaceutical composition may be in the form of one or more of tablets, powders, granules, suspensions, capsules or emulsions.

10. Use of the composition according to any one of claims 5 to 9 for preparing a drug for treating Parkinson's disease.

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