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

Abstract

This invention discloses the application of burdock root polysaccharide in the preparation of drugs for treating cognitive impairment, belonging to the field of traditional Chinese medicine technology. Experiments demonstrate that burdock root polysaccharide can improve cognitive impairment and independent exploration ability in D-galactose-induced cognitive impairment model mice, increase superoxide dismutase activity in mouse serum, reduce malondialdehyde content, decrease the content of interleukin-1β and tumor necrosis factor-α in the hippocampus of mouse brains, and alleviate the loss of neurons in the hippocampus of cognitive impairment model mice, thereby achieving a neuroprotective effect and demonstrating a good therapeutic effect on cognitive impairment. This provides a new approach for developing drugs related to the treatment of cognitive impairment.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of burdock root polysaccharide in the preparation of drugs for treating cognitive impairment. Background Technology

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

[0003] Burdock (Arctium lappa L.) is a biennial herbaceous plant belonging to the Asteraceae family and is a common plant used for both food and medicine. Burdock root polysaccharides are extracted from its roots, and studies have shown that they possess various pharmacological activities, including anti-tumor, anti-allergic, and hypoglycemic effects. However, to date, there are no reports on the use of burdock root polysaccharides in the treatment of cognitive impairment. Summary of the Invention

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

[0005] To achieve the above objectives, the technical solution of the present invention includes:

[0006] This invention provides the application of burdock root polysaccharide in the preparation of drugs for treating cognitive impairment.

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

[0008] Furthermore, the drug 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 with no toxic side effects.

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

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

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

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

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

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

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

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

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

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

[0020] This invention demonstrates through experiments that burdock root polysaccharide can improve cognitive impairment and autonomous exploration ability in D-galactose-induced cognitive impairment model mice, increase superoxide dismutase activity in mouse serum, reduce malondialdehyde content, decrease the content of interleukin-1β and tumor necrosis factor-α in the hippocampus of mouse brain, and alleviate the loss of neurons in the hippocampus of cognitive impairment model mice, thereby achieving a neuroprotective effect and showing good therapeutic effect on cognitive impairment, providing a new approach for the development of drugs related to the treatment of cognitive impairment. Attached Figure Description

[0021] Figure 1 The time when the mice in each group first reached the platform ( The results (n=10) are shown in the figure. The figure compares the results with the model control group. # P<0.05, ## P<0.01; compared with the blank control group, ** P<0.01.

[0022] Figure 2 The number of times mice in each group crossed the platform ( The results (n=10) are shown in the figure. The figure compares the results with the model control group.# P<0.05; compared with the blank control group, ** P<0.01.

[0023] Figure 3 The graph shows the percentage of time spent in the platform quadrant for 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 Escape time for each group of mice ( The result graph for n=8 is shown in the figure. * P<0.05, ** P<0.01.

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

[0026] Figure 6 The novel object recognition exploration index (NOI) of mice in each group The results (n=8) are shown in the figure. The comparison with the model control group is also shown. # P<0.05; compared with the blank control group, * P<0.05.

[0027] Figure 7 The serum SOD activity of mice in each group was measured. The results (n=8) are shown in the figure. The comparison with the model control group is also shown. # P<0.05, ## P<0.01; compared with the blank control group, ** P<0.01.

[0028] Figure 8 The serum MDA content of mice in each group ( The results (n=8) are shown in the figure. The comparison with the model control group is also shown. ## P<0.01; compared with the blank control group, ** P<0.01.

[0029] Figure 9 The content of IL-1β in the hippocampus of mice in each group of brains ( The results (n=8) are shown in the figure. The comparison with the model control group is also shown. # P<0.05, ## P<0.01; compared with the blank control group, ** P<0.01.

[0030] Figure 10The TNF-α content in the hippocampus of mice in each group of brains ( The results (n=8) are shown in the figure. The comparison with the model control group is also shown. # P<0.05; compared with the blank control group, ** P<0.01.

[0031] Figure 11 Immunofluorescence images of Neu-N, a neuronal biomarker, are shown for each group of mice. In the images, the left image is a 4× image, while CA1, CA3, and DG are 20× images. Detailed Implementation

[0032] To verify the effect of burdock root polysaccharide in treating cognitive impairment, the present invention is further illustrated through specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0033] Unless otherwise specified, all techniques or conditions used in the examples were performed in accordance with common techniques and conventional methods in the field or according to the product instructions. Instruments, reagents, and materials used without a specified manufacturer are all commercially available products from legitimate channels that meet legal standards.

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

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

[0036] (2) Add water at a ratio of 1:5 for the liquid and boil at 80°C for 3 hours. Filter the liquid through gauze and filter paper in sequence, collect the filtrate, and continue to boil the residue with water. 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) Concentrate the filtrate by rotary evaporation to a suitable volume, add 3 times the volume of anhydrous ethanol while stirring, let stand overnight, and the solution will separate into layers.

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

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

[0041] Example 2: Study on the effects of burdock root polysaccharide on cognitively impaired mice

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

[0043] Healthy male C57BL / 6 mice, weighing 18-22g, clean grade, were provided by Liaoning Changsheng Biotechnology Co., Ltd. (Certificate No.: SCXK(Liaoning)2020-0001); D-galactose (D-gal): Beijing Solarbio Science & Technology Co., Ltd.; SOD kit: Wuhan Elairit Biotechnology Co., Ltd.; MDA kit: Wuhan Elairit Biotechnology Co., Ltd.; IL-1β kit: Wuhan Elairit Biotechnology Co., Ltd.; TNF-α kit: Wuhan Elairit 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 Instruments Co., Ltd., YAN-ORM-4; Fluorescence microscope: Leica-DM4000 (Germany).

[0044] (2) Animal grouping and administration

[0045] C57BL / 6 mice were randomly divided into the following six groups: blank control group (physiological saline, ip, ultrapure water, ig), blank treatment group (physiological saline, ip, burdock root polysaccharide, 1200 mg / kg, ig), model control group (D-gal, 200 mg / kg, ip, ultrapure water, ig), low-dose model treatment group (D-gal, 200 mg / kg, ip, burdock root polysaccharide, 300 mg / kg, ig), medium-dose model treatment group (D-gal, 200 mg / kg, ip, burdock root polysaccharide, 600 mg / kg, ig), and high-dose model treatment group (D-gal, 200 mg / kg, ip, burdock root polysaccharide, 1200 mg / kg, ig). D-Gal preparation: A 20 mg / mL solution was prepared using sterile water for injection. Preparation of burdock root polysaccharide solutions: Solutions with concentrations of 30 mg / mL, 60 mg / mL, and 120 mg / mL were prepared using ultrapure water. The medication was administered once daily for 42 days. Behavioral experiments were conducted after the treatment period ended. The hippocampus and whole brain were harvested the day after the behavioral experiments to evaluate the treatment effect.

[0046] (3) Experimental methods

[0047] 1. Water Maze Experiment

[0048] The Morris water maze system mainly consists of a water maze device, an automatic water maze image acquisition system, and a software analysis system. The pool is divided into four quadrants: Quadrant 1 (Northeast), Quadrant 2 (Southeast), Quadrant 3 (Southwest), and Quadrant 4 (Northwest). Before the experiment, a certain amount of water is added to the pool, and the heating device is turned on to control the water temperature at 25±1℃. Days 1-4 are the orientation and navigation test: each day, mice are placed in the water from three different quadrants other than the one where the platform is located. If the mouse finds the platform within 60 seconds, the time is recorded as its escape latency, and the mouse stays on the platform for 5 seconds. If the mouse does not find the platform within 60 seconds, the latency is recorded as 60 seconds, and the mouse is led to the platform and stays there for 5 seconds. The indicators recorded during this stage are latency (escape time) and swimming speed. Day 5 is the spatial exploration test. The platform is removed, and the mouse is placed in the quadrant directly facing the platform, swimming freely for 60 seconds. The percentage of time the mouse spends in the original platform quadrant, the time of its first arrival at the platform, and the number of times it crosses the original platform quadrant are recorded.

[0049] 2. New Object Recognition Experiment

[0050] The new object recognition experiment was conducted in a rectangular box measuring 50cm × 50cm × 50cm. On the first day, the mouse was placed in the box to acclimatize for 1 minute. On the second day, two identical objects, A and B (cubes, 5cm × 5cm × 5cm), were placed at opposite ends of the box, equidistant from the corners. The mouse was placed with its back to the objects, its nose equidistant from the objects, and allowed to explore freely for 5 minutes while remaining quiet. After exploration, feces were cleaned and odor was removed using 75% alcohol. On the third day, object B was replaced with a completely different object, C (a cylinder, 5cm in diameter and 5cm in height). The mouse was again placed with its back to the objects, its nose equidistant from the objects. The exploration time of the mouse with the old and new objects within 5 minutes was recorded. The exploration index was calculated as: (New object exploration time / (New object exploration time + Old object exploration time)) × 100%.

[0051] 3. Measurement of serum oxidative stress markers

[0052] Perform the detection experiment according to the kit instructions, measure the protein concentration of the serum sample, and further calculate the MDA content and SOD activity of the serum sample based on the protein concentration.

[0053] 4. Measurement of inflammatory markers in the hippocampus of the brain

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

[0055] 5. Immunofluorescence

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

[0057] Statistical analysis

[0058] All measurement data are expressed as mean ± standard error (Mean ± SEM). GraphPad Prism 8.0.1 statistical software was used for data analysis, employing one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0059] (4) Experimental Results

[0060] The results of the water maze experiment are as follows Figure 1-5 As shown, compared with the blank control group, the time to first reach the platform was significantly increased, the number of times the mice crossed the platform was significantly decreased, the time spent in the target quadrant was significantly reduced, and the escape time was significantly increased in the model control group. After treatment with burdock root polysaccharide, the time to first reach the platform was significantly reduced, the number of times the mice crossed the platform was significantly increased, the time spent in the target quadrant was slightly increased, and the escape time was significantly reduced, with no significant difference compared with the blank control group. In the 4-day orientation navigation experiment, there was no significant difference in swimming speed among the groups of mice, and their motor abilities were not affected.

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

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

[0063] The effect of burdock root polysaccharide on serum MDA levels in mice is as follows: Figure 8 As shown, compared with the blank control group, the MDA content of mice in the model control group was significantly increased, while the intervention of burdock root polysaccharide administration significantly reduced the increase of MDA content in the model mice.

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

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

[0066] Results of Neu-N immunofluorescence staining for neuronal biomarkers are as follows: Figure 11 As shown, compared with the blank control group, the model control group mice lost neurons, while the intervention of burdock root polysaccharide administration 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, and not to limit them; this experiment only uses oral liquid as an example for specific illustration, and burdock root polysaccharide can also play a role in treating cognitive impairment in other dosage forms. The present invention has been described in detail with reference to the foregoing embodiments, and those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to 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; The burdock root polysaccharide mentioned above can improve cognitive impairment, increase the activity of superoxide dismutase in the serum of cognitively impaired individuals, reduce the content of malondialdehyde in the serum of cognitively impaired individuals, and improve the loss of neurons in cognitive impairment. The burdock root polysaccharide mentioned above can reduce the levels of interleukin-1β and tumor necrosis factor-α in the hippocampus of individuals with cognitive impairment. The preparation method of the burdock root polysaccharide includes the following steps: (1) Peel and wash the burdock root and slice it; (2) Add water at a ratio of 1:5 for the liquid to the material, boil at 80°C for 3 hours, filter with gauze and filter paper in sequence, collect the filtrate, add water to the residue and boil again, repeat this step 3-4 times; (3) Collect the filtrate and filter it with ordinary filter paper to initially remove insoluble impurities; (4) Concentrate the filtrate by rotary evaporation to a suitable volume, add 3 times the volume of anhydrous ethanol while stirring, let stand overnight, and the solution will separate into layers; (5) Collect the lower precipitate, dry it by rotary evaporation, and then add an appropriate amount of water to fully dissolve the burdock root polysaccharide; (6) Repeat steps (4)-(5) 3-4 times, collect the burdock root polysaccharide aqueous solution, freeze-dry, and obtain dried burdock root polysaccharide; The burdock root polysaccharide mentioned above is the only active ingredient.

2. The application according to claim 1, characterized in that, The drug comprises an effective amount of burdock root polysaccharide and pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The pharmaceutically acceptable excipients include fillers, binders, disintegrants, and emulsifiers.

4. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, granules, oral liquid preparations, injectable preparations, and capsule preparations.

5. The application according to claim 4, characterized in that, The drug is prepared in single-dose form.

6. The application according to claim 5, characterized in that, The single-dose drug contains 100-3000 mg of burdock root polysaccharide.

Images