Application of burdock root polysaccharide in preparation of medicine for treating cognitive impairment

Abstract

The invention discloses application of burdock root polysaccharide in preparation of a medicine for treating cognitive impairment, and belongs to the technical field of traditional Chinese medicines. Experiments prove that the burdock root polysaccharide can improve cognitive impairment and autonomous exploration ability of D-galactose induced cognitive impairment model mice, increase the activity of superoxide dismutase in serum of the mice, reduce the content of malondialdehyde, reduce the content of hippocampus interleukin-1beta and tumor necrosis factor-alpha in brains of the mice, and improve the cognitive impairment activity of the mice. According to the present invention, with the composition, the loss of the neuron in the hippocampal region of the cognitive impairment model mouse can be reduced so as to achieve the neuron protection effect, such that the good treatment effect on the cognitive impairment can be provided, and the new approach is provided for the development of the cognitive impairment-related drug.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to the application of burdock root polysaccharide in preparing a medicine for treating cognitive impairment. Background Art

[0002] Cognition is a high-level neurophysiological activity of the brain and one of the main functions of the brain of higher animals. Cognitive impairment refers to abnormalities in the high-level intelligent processing of the brain related to learning, memory, thinking and judgment, which seriously affects the quality of life and greatly increases the burden on society. Cognitive impairment is also a common clinical symptom of neurological diseases. With the rapid development of society, how to effectively prevent and treat cognitive impairment has become one of the hot issues in the field of life and health in my country.

[0003] Burdock (Arctium lappa L.) is a biennial herbaceous plant of the Asteraceae family and a common edible plant. Burdock root polysaccharide is extracted from its root. Studies have shown that burdock root polysaccharide has multiple pharmacological activities such as anti-tumor, anti-allergy, and hypoglycemic. However, there has been no report on the use of burdock root polysaccharide in the treatment of cognitive impairment. Summary of the invention

[0004] The present invention aims to provide the use of burdock root polysaccharide in the preparation of a drug for treating cognitive impairment. The burdock root polysaccharide can improve the cognitive impairment and independent exploration ability of D-galactose-induced cognitive impairment model mice, increase the superoxide dismutase (SOD) activity of mouse serum, reduce the malondialdehyde (MDA) content, reduce the content of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the hippocampal tissue of the mouse brain, and reduce the loss of neurons in the hippocampus of the cognitive impairment model mice, thereby achieving the effect of protecting neurons, having a good therapeutic effect on cognitive impairment, and providing a new way for the development of drugs related to the treatment of cognitive impairment.

[0005] In order to achieve the above object, the technical solution of the present invention includes:

[0006] The invention provides application of burdock root polysaccharide in preparing medicine for treating cognitive impairment.

[0007] Furthermore, the burdock root polysaccharide is the only active ingredient.

[0008] Furthermore, the medicine comprises an effective amount of burdock root polysaccharide and pharmaceutically acceptable excipients.

[0009] Furthermore, the pharmaceutically acceptable excipients include fillers, diluents, binders, disintegrants, emulsifiers and drug carriers without toxic side effects.

[0010] Furthermore, the dosage forms of the drug include tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

[0011] Furthermore, the drug is prepared in the form of a single-dose drug.

[0012] Furthermore, the single-dose medicine contains 100 to 3000 mg of burdock root polysaccharide.

[0013] Furthermore, the burdock root polysaccharide can improve cognitive impairment.

[0014] Furthermore, the burdock root polysaccharide can increase the SOD enzyme activity in the serum of people with cognitive impairment.

[0015] Furthermore, the burdock root polysaccharide can reduce the MDA content in the serum of people with cognitive impairment.

[0016] Furthermore, the burdock root polysaccharide can reduce the IL-1β content in the hippocampal tissue of people with cognitive impairment.

[0017] Furthermore, the burdock root polysaccharide can reduce the TNF-α content in the hippocampus tissue of people with cognitive impairment.

[0018] Furthermore, the burdock root polysaccharide can improve neuronal loss in cognitive impairment.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention proves through experiments that burdock root polysaccharide can improve cognitive impairment and independent exploration ability of D-galactose-induced cognitive impairment model mice, increase superoxide dismutase activity in mouse serum, reduce malondialdehyde content, reduce interleukin-1β and tumor necrosis factor-α content in hippocampal tissue of mouse brain, and reduce the loss of neurons in the hippocampus of cognitive impairment model mice, thereby achieving the effect of protecting neurons, having a good therapeutic effect on cognitive impairment, and providing a new way for developing drugs related to the treatment of cognitive impairment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the time when each group of mice first reached the platform ( n=10) Result graph, in which, compared with the model control group, # P<0.05, ## P<0.01; compared with the blank control group, ** P<0.01.

[0022] Figure 2 is the number of times each group of mice crossed the platform ( n=10) Result graph, in which, compared with the model control group,# P<0.05; compared with the blank control group, ** P<0.01.

[0023] Figure 3 The results of the percentage of platform quadrant residence time of each group of mice ( n=10), in the figure, compared with the model control group, # P<0.05; compared with the blank control group, * P<0.05.

[0024] Figure 4 is the escape time of mice in each group ( n=8) Result graph, in the figure, * P<0.05, ** P<0.01.

[0025] Figure 5 is the swimming speed of mice in each group ( n=8)Result graph.

[0026] Figure 6 The novel object recognition exploration index of each group of mice ( n=8) Result graph, in which, compared with the model control group, # P<0.05; compared with the blank control group, * P<0.05.

[0027] Figure 7 is the SOD activity in the serum of each group of mice ( n=8) Result graph, in which, compared with the model control group, # P<0.05, ## P<0.01; compared with the blank control group, ** P<0.01.

[0028] Figure 8 is the MDA content in the serum of each group of mice ( n=8) Result graph, in which, compared with the model control group, ## P<0.01; compared with the blank control group, ** P<0.01.

[0029] Fig. 9 is the IL-1β content in the hippocampus of each group of mice ( n=8) Result graph, in which, compared with the model control group, # P<0.05, ## P<0.01; compared with the blank control group, ** P<0.01.

[0030] Fig.10is the TNF-α content in the hippocampus of each group of mice ( n=8) Result graph, in which, compared with the model control group, # P<0.05; compared with the blank control group, ** P<0.01.

[0031] Fig.11 Immunofluorescence images of the neuronal biomarker Neu-N of each group of mice. In the figure, the left image is a 4× image, and CA1, CA3 and DG are 20× images. DETAILED DESCRIPTION

[0032] In order to verify the effect of burdock root polysaccharide in treating cognitive impairment, the present invention is further described by specific examples and in combination with the accompanying drawings. It should be understood that the specific examples described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0033] If no specific technology or conditions are specified in the examples, they are all carried out according to the general technology, conventional methods or product instructions in the art. If no manufacturer is specified for the instruments, reagents, materials, etc. used, they are all commercially available products from regular channels and meet the statutory standards.

[0034] Example 1: Preparation of burdock root polysaccharide, comprising the following steps:

[0035] (1) Peel and wash the burdock root, then slice it.

[0036] (2) Add water at a solid-liquid ratio of 1:5, boil at 80°C for 3 h, filter with gauze and filter paper in turn, collect the filtrate, continue to add water to boil the residue, and repeat this step 3-4 times.

[0037] (3) Collect the filtrate and filter it with ordinary filter paper to initially remove insoluble impurities.

[0038] (4) The filtrate was concentrated by rotary evaporation to an appropriate volume, and 3 volumes of anhydrous ethanol were added while stirring. The mixture was allowed to stand overnight to separate the layers.

[0039] (5) Collect the lower precipitate, dry it by rotary evaporation, and then add appropriate amount of water to fully dissolve the burdock root polysaccharide.

[0040] (6) Repeat steps (4) to (5) 3-4 times, collect the burdock root polysaccharide aqueous solution, and freeze-dry to obtain a burdock root polysaccharide dry product.

[0041] Example 2: Study on the effect of burdock root polysaccharide on cognitive impairment mice

[0042] (1) Experimental animals, reagents and equipment

[0043] Healthy male C57BL / 6 mice, weighing 18-22 g, clean grade, were provided by Liaoning Changsheng Biotechnology Co., Ltd. (certificate number: SCXK(Liao)2020-0001); D-galactose (D-gal): Beijing Solebao Technology Co., Ltd.; SOD kit: Wuhan Elerite Biotechnology Co., Ltd.; MDA kit: Wuhan Elerite Biotechnology Co., Ltd.; IL-1β kit: Wuhan Elerite Biotechnology Co., Ltd.; TNF-α kit: Wuhan Elerite Biotechnology Co., Ltd.; Neu-N antibody: Abcam Limited, USA; Morris water maze: Shanghai Xinruan Information Technology Co., Ltd., XR-XM101; Shanghai new object recognition box: Shanghai Yuyan Scientific Instrument Co., Ltd., YAN-ORM-4; fluorescence microscope: Leica-DM4000, Germany.

[0044] (2) Animal grouping and drug administration

[0045] C57BL / 6 mice were randomly divided into the following six groups: blank control group (normal saline, ip, ultrapure water, ig), blank administration group (normal saline, ip, burdock root polysaccharide, 1200 mg / kg, ig), model control group (D-gal, 200 mg / kg, ip, ultrapure water, ig), model administration low-dose group (D-gal, 200 mg / kg, ip, burdock root polysaccharide, 300 mg / kg, ig), model administration medium-dose group (D-gal, 200 mg / kg, ip, burdock root polysaccharide, 600 mg / kg, ig), model administration high-dose group (D-gal, 200 mg / kg, ip, burdock root polysaccharide, 1200 mg / kg, ig). D-Gal preparation: Prepare a 20 mg / mL solution using sterile water for injection. Preparation of burdock root polysaccharide solution: Use ultrapure water to prepare 30mg / mL, 60mg / mL, and 120mg / mL solutions. Administer once a day for 42 days. After administration, conduct behavioral experiments. The hippocampus and whole brain are removed the next day after the behavioral experiment to evaluate the therapeutic effect.

[0046] (3) Experimental methods

[0047] 1. Water maze experiment

[0048] The Morris water maze system mainly includes a water maze device, a water maze image automatic acquisition system, and a software analysis system. The pool is divided into four areas, namely the first quadrant (northeast), the second quadrant (southeast), the third quadrant (southwest), and the fourth quadrant (northwest). Before the experiment, a certain amount of water was injected into the pool, and the heating device was turned on to control the water temperature at 25±1℃. Days 1-4 were for positioning navigation tests: mice were placed in the water from the other three quadrants of the quadrant where the platform was located every day. If the platform was found within 60s, the time at this time was recorded as its escape latency, and the mice were allowed to stay on the platform for 5s. If the platform was not found within 60s, the latency was recorded as 60s, and the mice were led to the platform to stay for 5s. The indicators recorded at this stage were latency (escape time) and swimming speed. Day 5 was a spatial exploration test. The platform was removed and the mice were placed in the quadrant facing the platform. They swam freely for 60s, and the percentage of time the mice stayed in the quadrant of the original platform, the time to reach the platform for the first time, and the number of crossings of the original platform quadrant were recorded.

[0049] 2. New object recognition experiment

[0050] The new object recognition experiment was conducted in a rectangular box of 50cm×50cm×50cm. On the first day, the mice were placed in the box to adapt to the environment for 1 minute. On the second day, two identical objects A and B (cubes, 5cm×5cm×5cm) were placed at the left and right ends of the box. The objects were placed at the same distance from the corners on both sides. The mice were placed in the venue with their backs facing the two objects, and the length of the nose tip from the two objects was equal. They were allowed to explore freely in the box for 5 minutes and kept quiet. After the exploration, the feces were cleaned up and 75% alcohol was used to remove the odor. On the third day, the B object in the box was replaced with a completely different object C (cylinder, 5cm in diameter and 5cm in height). The mice were still placed in the box with their backs facing the two objects, and the length of the nose tip from the two objects was equal. The exploration time of the mice with the new and old objects was recorded within 5 minutes. The exploration index = new object exploration time / (new object exploration time + old object exploration time)×100%.

[0051] 3. Determination of oxidative stress indicators in serum

[0052] The detection experiment was carried out according to the steps of the kit to measure the protein concentration of the serum sample, and the MDA content and SOD activity of the serum sample were further calculated based on the protein concentration.

[0053] 4. Determination of inflammatory indicators in hippocampal tissue

[0054] After measuring the protein concentration of the hippocampal tissue in the brain, the detection experiment was carried out according to the steps of the kit, and the content of IL-1β and TNF-α in the sample was further calculated according to the protein concentration.

[0055] 5. Immunofluorescence

[0056] Coronal brain sections of 30 μm thickness were taken and stored at 4°C. The brain sections were washed with PBS, repaired with antigen repair solution, washed with PBS, and pre-incubated in 0.4% Triton-100X, 1% BSA and 4% sheep serum in 0.01M PBS at room temperature for 1 hour. Then, they were incubated with primary antibody Neu-N (1:500) at 4°C overnight. After washing with PBS, the fluorescent secondary antibody Alexa Fluor TM 594 (room temperature for 1 h). After washing with PBS, the slides were mounted with a mounting medium containing DAPI, and images were taken using a Leica fluorescence microscope.

[0057] Statistical analysis

[0058] The measured data were expressed as mean ± standard error (Mean ± SEM). GraphPad Prism 8.0.1 statistical software was used for data analysis, and one-way analysis of variance was used. P < 0.05 indicated a significant difference, which was statistically significant.

[0059] (4) Experimental results

[0060] The results of the water maze test were as follows Figure 1-5 As shown in the figure, compared with the blank control group, the time for the mice in the model control group to first reach the platform was significantly increased, the number of times they crossed the platform was significantly reduced, the time they stayed in the target quadrant was significantly reduced, and the escape time was significantly increased. After treatment with burdock root polysaccharides, the time for the mice to first reach the platform was significantly reduced, the number of times they crossed the platform was significantly increased, the time they stayed in the target quadrant was increased, and the escape time was significantly reduced, which was not significantly different from the blank control group. In the 4-day positioning navigation test, there was no significant difference in the swimming speed of the mice in each group, and their motor ability was not affected.

[0061] The experimental results of new object recognition are as follows Figure 6 As shown in the figure, the exploration index of mice in the model control group was significantly reduced compared with the blank control group. After treatment with burdock root polysaccharides, the exploration index of mice increased significantly, with no significant difference compared with the blank control group.

[0062] The results of the effect of burdock root polysaccharide on the activity of SOD enzyme in mouse serum are as follows Figure 7 As shown, compared with the blank control group, the SOD enzyme activity of the mice in the model control group was significantly reduced, while the intervention with burdock root polysaccharide significantly improved the reduction of SOD enzyme activity in the model mice.

[0063] Effect of burdock root polysaccharide on serum MDA content in mice Figure 8 As shown, compared with the blank control group, the MDA content of the mice in the model control group was significantly increased, while the intervention with burdock root polysaccharides significantly attenuated the increase in MDA content in the model mice.

[0064] The results of the effects of burdock root polysaccharides on IL-1β in the hippocampus of mice are as follows Fig. 9 As shown, compared with the blank control group, the content of IL-1β in the hippocampus tissue of the mice in the model control group was significantly increased, while intervention with burdock root polysaccharide significantly improved the increase in the content of IL-1β in the hippocampus tissue of the model mice.

[0065] The results of the effects of burdock root polysaccharides on TNF-α in the hippocampus of mice are as follows Fig.10 As shown, compared with the blank control group, the content of TNF-α in the hippocampus of the mice in the model control group was significantly increased, while the intervention of burdock root polysaccharide significantly improved the increase of TNF-α content in the hippocampus of the model mice.

[0066] The results of immunofluorescence staining of neuronal biomarker Neu-N are shown in Fig.11 As shown, compared with the blank control group, the model control group mice lost neurons, while intervention with burdock root polysaccharides significantly improved the neuronal loss in the model mice.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; this experiment only takes oral liquid as an example for specific description, and burdock root polysaccharides in other dosage forms can also play a role in treating cognitive impairment. The present invention is described in detail with reference to the above embodiments, and ordinary technicians in the field should understand that it is still possible to modify the technical solutions recorded in the above embodiments, or to replace some or all of the technical features therein; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of burdock root polysaccharide in the preparation of drugs for treating cognitive impairment.

2. The use according to claim 1, characterized in that: The burdock root polysaccharide is the only active ingredient.

3. The use according to claim 1, characterized in that: The medicine comprises an effective amount of burdock root polysaccharide and pharmaceutically acceptable auxiliary materials.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable excipients include fillers, diluents, binders, disintegrants, emulsifiers and drug carriers without toxic or side effects.

5. The use according to claim 1, characterized in that: The dosage forms of the medicine include tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

6. The use according to claim 5, characterized in that: The medicine is prepared in the form of a single-dose medicine.

7. The use according to claim 6, characterized in that: The single-dose medicine contains 100-3000 mg of burdock root polysaccharide.

8. The use according to claim 1, characterized in that: The burdock root polysaccharide can improve cognitive impairment.

9. The use according to claim 1, characterized in that: The burdock root polysaccharide can increase the activity of superoxide dismutase in the serum of people with cognitive impairment and reduce the content of malondialdehyde in the serum of people with cognitive impairment; The burdock root polysaccharide can reduce the content of interleukin-1β (Interleukin-1beta) and tumor necrosis factor-α (Tumor necrosis factor-α) in the hippocampus tissue of people with cognitive impairment.

10. The use according to claim 1, characterized in that: The burdock root polysaccharide can improve neuronal loss in cognitive impairment.

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