Application of burdock root oligosaccharide in preparation of medicine for treating cerebral arterial thrombosis

Abstract

The invention discloses application of burdock root oligosaccharide in preparation of a medicine for treating cerebral arterial thrombosis, and belongs to the technical field of medicines. Firstly, the burdock root oligosaccharide is obtained through water extraction and alcohol precipitation, membrane separation and preparation chromatography, and experiments prove that the burdock root oligosaccharide can improve the cerebral infarction area of ischemic stroke mice induced by a bilateral common carotid artery occlusion model, improve neuroinflammation of the ischemic stroke mice and improve the neurological function; according to the application, the content of tumor necrosis factor-alpha in a mouse body is reduced, the content of interleukin-10 in the mouse body is increased, a very good treatment effect on the cerebral arterial thrombosis is achieved, and a new way is provided for developing the medicine for treating the cerebral arterial thrombosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of burdock root oligosaccharides in the preparation of drugs for treating ischemic stroke. Background Technology

[0002] Stroke is one of the most serious neurological diseases threatening human health, causing long-term disability and even death in a large number of patients worldwide. Hemorrhagic stroke and ischemic stroke are the main types of stroke, with ischemic stroke being more common in clinical practice, accounting for more than 80% of cases. Therefore, prevention, active treatment, and disease prognosis recovery for patients with ischemic stroke are of great significance.

[0003] Burdock (Arctium lappa L.) is a biennial herbaceous plant belonging to the Asteraceae family. It is a common plant used for both food and medicine. Burdock root oligosaccharides are low-polymer polysaccharide components isolated from burdock roots. To date, there have been no reports on the use of burdock root oligosaccharides in the treatment of ischemic stroke. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide the application of burdock root oligosaccharides in the preparation of drugs for treating ischemic stroke. The burdock root oligosaccharides prepared by this invention can improve ischemic stroke induced by a bilateral common carotid artery occlusion model, reduce the infarct area in mice with ischemic stroke, improve the neurological function in mice with ischemic stroke, reduce the content of tumor necrosis factor-α (TNF-α) in mice, and increase the content of interleukin-10 (IL-10) in mice, thereby achieving a neuroprotective effect. It has a good therapeutic effect on ischemic stroke and provides a new approach for developing drugs for treating ischemic stroke.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of burdock root oligosaccharides in the preparation of drugs for treating ischemic stroke.

[0006] Based on the above technical solution, the burdock root oligosaccharide is further described as the sole active ingredient.

[0007] Based on the above technical solution, the preparation method of the burdock root oligosaccharide further includes the following steps: (1) Peel and wash the burdock root, slice it, dry it, and crush it; (2) Add water at a mass ratio of 1:5 to 1:25, boil at 70 to 90°C for 2 to 5 hours, filter with gauze and filter paper in sequence, collect the filtrate, add water to the filter residue and boil, repeat this step 3 to 4 times; (3) The filtrate obtained by vacuum filtration is used to initially remove insoluble impurities; (4) Concentrate the filtrate obtained in step (3), add 2 to 4 times the volume of anhydrous ethanol while stirring, let stand overnight, and the solution will separate into layers; (5) Collect the precipitate, dry it, and dissolve it in water to obtain burdock root polysaccharide; (6) Pass the obtained burdock root polysaccharide aqueous solution through a 5kDa hollow fiber membrane to collect the filtrate with a concentration lower than 5kDa, then pass it through a 3kDa hollow fiber membrane to collect the low-polymerization degree burdock polysaccharide solution with a concentration lower than 3kDa, freeze-dry it to obtain low-polymerization degree burdock polysaccharide. (7) The low-polymerization degree burdock polysaccharide obtained by redissolving in an ethanol-water solution with a volume ratio of 1:1 to 3:1 was centrifuged and the supernatant was filtered through a 0.45 μm filter membrane and loaded onto a preparative chromatography column for purification. The packing material was a packing material bonded with cysteine. The mobile phase A was water and the mobile phase B was ethanol. Gradient elution was performed: 0 min 20-30% A, 45 min 50-60% A, 45.1 min 65-75% A. The eluent was collected after 10-25 min and freeze-dried.

[0008] Based on the above technical solution, the chromatographic column is a dynamic axial compression column with an inner diameter of 100 mm, a packing amount of 2000 g, and a bed volume of 2 liters; the flow rate is 200~400 mL / min; and the UV detection wavelength is 190~200 nm.

[0009] Based on the above technical solution, the drug further includes an effective amount of burdock root oligosaccharide and pharmaceutically acceptable excipients.

[0010] Based on the above technical solution, the pharmaceutically acceptable excipients further include fillers, diluents, binders, disintegrants, and emulsifiers.

[0011] Based on the above technical solution, the dosage form of the drug further includes tablets, granules, oral liquid preparations, drops, injectable preparations, and capsule preparations.

[0012] Based on the above technical solution, the drug is further prepared in the form of a single-dose drug.

[0013] Based on the above technical solution, the single-dose drug further comprises 100~3000 mg of burdock root oligosaccharides.

[0014] Based on the above technical solution, the burdock root oligosaccharide can further improve ischemic stroke.

[0015] Based on the above technical solution, the burdock root oligosaccharide can further reduce the infarct area in patients with ischemic stroke.

[0016] Based on the above technical solution, the burdock root oligosaccharide can further improve the neurological function of patients with ischemic stroke.

[0017] Based on the above technical solution, the burdock root oligosaccharide can further reduce the TNF-α content in patients with ischemic stroke.

[0018] Based on the above technical solution, the burdock root oligosaccharide can further increase the IL-10 content in patients with ischemic stroke.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention demonstrates through systematic experiments that burdock root oligosaccharides can improve ischemic stroke induced by a bilateral common carotid artery occlusion model, reduce the infarct area in mice with ischemic stroke, improve the neurological function of mice with ischemic stroke, reduce the content of tumor necrosis factor-α (TNF-α) in mice, and increase the content of interleukin-10 (IL-10) in mice, thereby achieving a neuroprotective effect. It has a good therapeutic effect on ischemic stroke and provides a new approach for developing drugs to treat ischemic stroke. Attached Figure Description

[0020] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0021] Figure 1 The graph shows the degree of polymerization of the burdock root oligosaccharide prepared in Example 1.

[0022] Figure 2 The image shows the TTC staining results of mice in each group in Example 2.

[0023] Figure 3 The neurological function scores of mice in each group in Example 2 ( The results (±S, n=10) are shown in the figure. The figure compares the results with the model control group. ## P <0.01; compared with the sham surgery group, ** P <0.01.

[0024] Figure 4 The latency period of movement during treatment in Example 2 ( The results (±S, n=10) are shown in the figure. The figure compares the results with the model control group. # P <0.05; compared with the sham surgery group, ** P <0.01.

[0025] Figure 5The kinetic latency period (3 weeks) of mice in each group after treatment in Example 2. The results (±S, n=10) are shown in the figure. The figure compares the results with the model control group. # P <0.05; compared with the sham surgery group, ** P <0.01.

[0026] Figure 6 The TNF-α content in the plasma of mice in each group in Example 2 ( The results (±S, n=7) are shown in the figure. The figure compares the results with the model control group. # P <0.05, ## P <0.01; compared with the sham surgery group, ** P <0.01.

[0027] Figure 7 The IL-10 content in the plasma of mice in each group in Example 2 ( The results (±S, n=7) are shown in the figure. The figure compares the results with the model control group. # P <0.05; compared with the sham surgery group, * P <0.05. Detailed Implementation

[0028] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0029] Specific techniques or conditions not specified in the examples were performed in accordance with common techniques and conventional methods in the field or according to the product instructions. Instruments, reagents, materials, etc., used in the examples without specified manufacturers are all commercially available products from legitimate channels that meet legal standards.

[0030] Example 1: This embodiment provides a method for preparing burdock root oligosaccharide (ALR-OS), comprising the following steps: (1) Peel and wash the burdock root, slice it, dry it in an oven at 60°C, and then pulverize it; (2) Add water at a mass ratio of 1:15 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. (3) Collect the filtrate and filter it with ordinary filter paper to initially remove insoluble impurities; (4) Concentrate the filtrate to a suitable volume by low-temperature rotary evaporation, 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) Collect burdock root polysaccharide aqueous solution and separate burdock root polysaccharides with different degrees of polymerization using membrane separation technology: first pass the burdock root polysaccharide aqueous solution through a 5kDa hollow fiber membrane and collect the filtrate on both sides of the membrane, then pass the filtrate in the section below 5kDa through a 3kDa hollow fiber membrane and collect the filtrate on both sides of the membrane, and then freeze-dry the low-polymerization-degree burdock polysaccharide solution below 3kDa to obtain low-polymerization-degree burdock polysaccharide; (7) The low-polymerization degree burdock polysaccharide was redissolved in an ethanol-water solution with a volume ratio of 2:1 at a concentration of 50 mg / mL. The supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane to obtain a clear solution. The solution was then purified in fractions using preparative chromatography under the following conditions: Packing material: A preparative column (DAC 100) with an inner diameter of 100 mm, packed with cysteine-bonded packing material of 40 μm particle size (CYS 40 μm), with a packing volume of 2000 g and a bed volume (BV) of 2 liters (1 BV = 2 L).

[0031] Mobile phase A: water, mobile phase B: food ethanol. After loading the sample onto the pump, gradient elution is performed, and the gradient settings are shown in Table 1.

[0032] Table 1 Elution gradient

[0033] Flow rate: 300 mL / min; UV detection wavelength: 195 nm.

[0034] Sample: After loading the sample, collect the eluent for 10-25 minutes, freeze-dry it to obtain burdock root oligosaccharide powder. Polymerization analysis showed that the degree of polymerization of this burdock root oligosaccharide was ≤8. Figure 1 ).

[0035] Example 2: This embodiment examines the therapeutic effect of burdock root oligosaccharide prepared in Example 1 on mice with ischemic stroke. (1) Laboratory animals, reagents and equipment Healthy male C57BL / 6 mice, weighing 18-22g, SPF grade, were provided by Liaoning Changsheng Biotechnology Co., Ltd. (Certificate No.: SCXK(Liaoning)2020-0001); 2% TTC staining solution: Beijing Solarbio Science & Technology Co., Ltd.; TNF-α reagent kit: Quanzhou Ruixin Biotechnology Co., Ltd.; IL-10 reagent kit: Quanzhou Ruixin Biotechnology Co., Ltd.; Rotary fatigue tester: Shanghai Yuyan Scientific Instruments Co., Ltd.

[0036] (2) Animal model preparation A bilateral common carotid artery occlusion (BCCAO) model was established using male C57BL / 6 mice. The specific procedure was as follows: Mice were anesthetized with 2.5% aflutole, placed with their limbs facing upwards, and secured to a warming pad with adhesive tape. Strict aseptic technique was maintained throughout the procedure. The neck was carefully incised, and the left common carotid artery was first separated from the vagus nerve. The clean carotid artery was then dissected, and the same steps were repeated to dissect the right common carotid artery. A 4-0 suture was inserted under each of the two common carotid arteries, and both arteries were blocked to induce ischemia for 15 minutes. The sutures were then released for 10 minutes of rest; this was repeated twice, with ligation for 10 minutes followed by another 10 minutes of rest. The sutures were then removed, and the mice's vital signs were observed for approximately 10 minutes. Once the vital signs stabilized, the incision was carefully sutured, and the mice were returned to their cages. In the sham surgery group, only bilateral common carotid artery dissection was required; bilateral carotid artery ligation was not necessary.

[0037] (3) Animal grouping and administration C57BL / 6 mice were randomly divided into six groups: sham-operated group (saline, ig), sham-operated drug administration group (burdock root oligosaccharide, 1500 mg / kg, ig), model control group (saline, ig), low-dose model drug administration group (burdock root oligosaccharide, 500 mg / kg, ig), and high-dose model drug administration group (burdock root oligosaccharide, 1500 mg / kg, ig). Burdock root oligosaccharide solutions were prepared at concentrations of 50 mg / ml and 150 mg / ml using ultrapure water. Administration was once daily for 21 days. Behavioral experiments were conducted after the drug administration period. Whole brain tissue was harvested the day after the behavioral experiments to evaluate the therapeutic effect of burdock root oligosaccharide.

[0038] (3) Experimental methods 1. TTC staining Brain tissue was collected from mice in each group and dissected on ice. The tissue was cut into 2mm coronal sections and placed in 24-well plates. The sections were incubated with 2% TTC (2,3,5-triphenyltetrazolium chloride) phosphate buffer at 37°C in the dark for 15 minutes. The sections were then flipped and incubated for another 15 minutes. Normal brain tissue appeared red, while infarcted areas appeared white.

[0039] 2. Neurological function score of mice After the drug intervention ended, the neurological function of mice in each group was scored using the LONGA method: 0 points: No signs of neurological damage; 1 point: The mouse's right forepaw cannot be fully extended when the tail is lifted; 2 points: The mouse turns to the right when walking; 3 points: The mouse leans to the right when walking; 4 points: The mouse cannot walk and loses consciousness. The higher the score, the more severe the neurological damage in the mouse.

[0040] 3. Rotating bar experiment The fatigue rotundimeter has 6 channels. Before the experiment, the instrument parameters were set as follows: the rotation speed was uniformly increased from 1 rpm / min to 40 rpm / min within 90 seconds, with a total rotation time of 600 seconds. Mice were placed in the experimental environment for 1 hour to acclimatize before the formal experiment. During the formal experiment, each mouse was tested once in the morning and once in the afternoon, on the day after surgery, and at 1 week, 2 weeks, and 3 weeks post-surgery. The time it took for the mouse to drop the rotundimeter was recorded, and the average of the two tests was used as the experimental data. After each measurement, the feces of each mouse were cleaned and the odor was removed using 75% alcohol.

[0041] 4. Measurement of inflammatory markers in plasma The levels of TNF-α and IL-10 in the plasma of mice in each group were measured according to the kit instructions.

[0042] 5. Statistical Analysis All measurement data are expressed as mean ± standard error (Mean ± SEM). Data analysis was performed using GraphPad Prism 10.1.2 statistical software, employing one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0043] (4) Experimental results TTC experimental results are as follows Figure 2 As shown, compared with the sham-operated group, the infarct area of ​​the brain in mice in the model control group was significantly increased. Administration of burdock root oligosaccharides (especially the high-dose group) could significantly improve the increase in the infarct area of ​​the brain in mice in the model group.

[0044] Neurological function score results as follows Figure 3 As shown, compared with the sham-operated group, the neurological function scores of mice in the model control group were significantly increased, while the neurological function scores of mice were significantly reduced after administration of burdock root oligosaccharides (especially the high-dose group).

[0045] The results of the rotator experiment are as follows Figure 4-5As shown, compared with the sham-operated group (CON), the motor latency of the model control group (BCCAO) mice was significantly reduced, showing slight recovery during the three-week experiment, but still significantly lower than that of the sham-operated group. Treatment with burdock root oligosaccharides accelerated the recovery of mice during the three-week treatment period, and significantly increased the motor latency after the end of treatment, with no significant difference compared to the sham-operated group.

[0046] The effects of burdock root oligosaccharides on mouse plasma TNF-α were as follows: Figure 6 As shown, compared with the sham-operated group, the plasma TNF-α content of mice in the model control group was significantly increased, and administration of burdock root oligosaccharide could significantly improve the increase in plasma TNF-α content in model mice.

[0047] The effect of burdock root oligosaccharides on mouse plasma IL-10 results are as follows: Figure 7 As shown, compared with the sham-operated group, the plasma IL-10 content of mice in the model control group was significantly reduced, and administration of burdock root oligosaccharide could significantly improve the reduction of plasma IL-10 content in model mice.

[0048] 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; although the present invention has been described in detail with reference to the foregoing embodiments, 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. Use of arctin in the preparation of a drug for treating ischemic stroke.

2. The use according to claim 1, wherein the arctin is the only active ingredient.

3. The use according to claim 1, wherein the arctin is prepared by the following steps: (1) peeling and washing burdock root, slicing, drying, and crushing; (2) adding water in a mass ratio of 1:5-1:25 and a liquid ratio of 1:5-1:25, boiling at 70-90°C for 2-5 hours, filtering successively with gauze and filter paper, collecting the filtrate, and continuing to add water to the residue for boiling, repeating this step 3-4 times; (3) filtering the obtained filtrate to preliminarily remove insoluble impurities; (4) concentrating the filtrate obtained in step (3), adding 2-4 times the volume of anhydrous ethanol under stirring, standing overnight, and separating the solution into layers; (5) collecting the precipitate, drying, and dissolving the obtained burdock root polysaccharide in water; (6) passing the obtained burdock root polysaccharide solution through a 5-kDa hollow fiber membrane to collect the filtrate below 5 kDa, then passing the filtrate through a 3-kDa hollow fiber membrane to collect the oligomerization burdock polysaccharide solution below 3 kDa, and freeze-drying to obtain the oligomerization burdock polysaccharide; (7) resuspending the obtained oligomerization burdock polysaccharide with an ethanol-water solution in a volume ratio of 1:1-3:1, centrifuging to take the supernatant, passing through a 0.45-μm filter membrane, and loading onto a preparative chromatography column for purification, with a filler bonded with cysteine, a mobile phase A of water, a mobile phase B of ethanol, gradient elution, 0 min 20-30% A, 45 min 50-60% A, 45.1 min 65-75% A, collecting the eluate at 10-25 min, and freeze-drying. The drug comprises an effective amount of arctin and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient comprises a filler, a diluent, a binder, a disintegrant, and an emulsifier. The dosage form of the drug comprises tablets, granules, oral liquid preparations, drops, injection preparations, and capsule preparations. The drug is prepared in the form of a single-dose drug. The single-dose drug contains 100-3000 mg of arctin. The arctin can improve ischemic stroke, reduce the infarction area of patients with ischemic stroke, and improve the neurological function of patients with ischemic stroke. The arctin can reduce the content of TNF-α in the body of patients with ischemic stroke and increase the content of IL-10 in the body of patients with ischemic stroke.

4. Use according to claim 1, characterized in that, ​ 5. Use according to claim 4, characterized in that, ​ 6. Use according to claim 1, characterized in that, ​ 7. The use according to claim 1, characterized in that, ​ 8. Use according to claim 7, characterized in that, ​ 9. The use according to claim 1, characterized in that, ​ 10. The use according to claim 1, characterized in that, ​

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