Application of radix polygoni multiflori preparata polysaccharide in preparation of medicine for treating and / or preventing ischemia-reperfusion injury
By preparing and applying Polygonum multiflorum polysaccharide, the problem of insufficient neuroprotection in acute ischemic stroke was solved, and the neurological dysfunction and cerebral infarction of cerebral ischemia and reperfusion injury was significantly alleviated, providing safe and efficient treatment and prevention solutions. .
Patent Information
- Application Number
- CN202411749162.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2025-01-10
AI Technical Summary
The prior art cannot effectively protect patients from ischemic injury and dysfunction, especially in the treatment of acute ischemic stroke, and there is a lack of effective neuroprotective measures.
The prepared polysaccharide polysaccharide is prepared through extraction and purification processes to reduce I/R-induced neuronal damage and reduce activation of microglia in the brain, providing oral or injection dosage forms for prevention and Treatment of ischemia-reperfusion injury.
It significantly alleviates neurological dysfunction caused by cerebral ischemia and reperfusion injury, reduces the area of cerebral infarction, improves the survival rate of rats, and has high safety and no toxic side effects. It is suitable for long-term use, providing ischemic Effective prevention and treatment plan for stroke.
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Figure CN119280267A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical technology, and particularly relates to application of Radix Polygoni Multiflori Preparata polysaccharide in preparing medicine for treating and / or preventing ischemia-reperfusion injury. Background Art
[0002] Current status of clinical treatment of stroke: Stroke is the second leading cause of death worldwide, of which 70% are ischemic strokes. Currently, the main strategies for clinical treatment of acute ischemic stroke include intravenous thrombolysis and mechanical thrombectomy, but these methods cannot protect patients from ischemic damage and functional impairment.
[0003] Therefore, it is particularly important to find neuroprotective treatments to improve ischemic injury and ischemia / reperfusion injury. Summary of the invention
[0004] In view of the problems existing in the prior art, the present invention provides a method for mildly preventing and treating ischemic stroke.
[0005] In order to solve the above problems, the technical solution adopted by the present invention is as follows:
[0006] The invention provides an application of Radix Polygoni Multiflori Preparata polysaccharide in preparing a medicine for treating and / or preventing ischemia-reperfusion injury.
[0007] The Radix Polygoni Multiflori Preparata polysaccharide of the present invention is used for alleviating neuronal damage caused by I / R and reducing the number of activated microglial cells in the brain.
[0008] The cerebral ischemia-reperfusion injury described in the present invention is the ischemia-reperfusion injury generally known in the art, and its triggering factors include but are not limited to traumatic shock, surgery, organ transplantation, burns, frostbite, thrombosis and other blood circulation disorders that cause cerebral ischemia-reperfusion injury.
[0009] The dosage form of the drug of the present invention can be an oral dosage form, such as tablets, capsules, pills, powders, granules, suspensions, syrups, etc.; it can also be an injectable dosage form, such as injections, powder injections, etc., through intravenous, intraperitoneal, subcutaneous or intramuscular routes. All dosage forms used are well known to ordinary technicians in the pharmaceutical field.
[0010] The drug of the present invention can be applied to any animal that will or has suffered cerebral ischemia-reperfusion injury, including humans and non-human animals, such as pets or livestock.
[0011] The drugs described in the present invention can be administered to subjects by routes known in the art, including but not limited to oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal, buccal, sublingual, intranasal, transdermal, etc.
[0012] The dosage of the drug administered according to the present invention depends on the age, health and weight of the recipient, the frequency of treatment, the route of administration, etc.
[0013] In a specific embodiment of the present invention, the dosage of the drug administered is 20 mg / kg to 50 mg / kg.
[0014] The preparation method of the prepared Polygonum multiflorum polysaccharide of the present invention comprises: adding distilled water to the prepared Polygonum multiflorum at a solid-liquid ratio of 1:20, extracting for 2 hours by means of reflux heating, and pouring out the filtrate; then repeating the extraction process, combining the filtrate and concentrating under reduced pressure to a density of 1.12-1.18 to obtain a combined liquid; then adding anhydrous ethanol to the combined liquid until the ethanol concentration is 80%, precipitating overnight at a temperature of 4°C, collecting the precipitate, and repeatedly washing with ethanol and acetone to obtain an alcohol precipitate; re-dissolving the alcohol precipitate and deproteinizing by a Sevage method, dialyzing with distilled water for 48 hours, and then concentrating and freeze-drying to obtain the prepared Polygonum multiflorum polysaccharide.
[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:
[0016] The present invention establishes an in vitro model of cerebral ischemia-reperfusion by middle cerebral artery occlusion (MCAO), observes the effect of prepared Polygonum multiflorum polysaccharide on SD rats, and monitors the changes in body weight and neurological function of prepared Polygonum multiflorum polysaccharide on SD rats after modeling. The 2,3,5-triphenyltetrazolium chloride (TTC) method is used to detect the changes in brain ischemia in rats, and a high dose of prepared Polygonum multiflorum polysaccharide is used as a positive drug. The results show that prepared Polygonum multiflorum polysaccharide has a significant effect on resisting cerebral ischemia-reperfusion injury, and compared with the control group, its drug efficacy is significant, the raw material source is wide, and the safety is high, and it is suitable for preparing drugs for treating and / or preventing ischemia-reperfusion injury.
[0017] The prepared Polygonum multiflorum polysaccharide in the present invention can significantly alleviate the neurological dysfunction caused by MCAO modeling, reduce the area of cerebral infarction, and increase the survival rate of rats, providing a reference for the prevention and treatment of ischemic stroke. The prepared Polygonum multiflorum polysaccharide has the characteristics of high safety, no toxic side effects, and is suitable for long-term use. As a natural plant polysaccharide, the prepared Polygonum multiflorum polysaccharide may have better biocompatibility and bioavailability, and may have better absorption and distribution in the body compared with traditional synthetic drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 Total sugar content and its curve, protein content and its curve;
[0019] Figure 2 This is the infrared image of Polygonum multiflorum polysaccharide.
[0020] Figure 3This is a graph showing the weight changes and neurological function scores of rats;
[0021] Figure 4 This is the rat brain ischemic slice diagram and ischemic area statistics;
[0022] Figure 5 HE staining image of rat brain and statistical diagram of microglia (Iba1). DETAILED DESCRIPTION
[0023] In recent years, TCM has made great progress in the treatment of ischemic stroke, including etiology and pathogenesis, syndrome differentiation and treatment, special prescription treatment, Chinese medicine injection treatment, external treatment, integrated Chinese and Western medicine therapy, etc. Prepared Polygonum multiflorum is a famous traditional Chinese herbal medicine, which is now widely used as a popular functional food; it is one of the fourth batch of Chinese herbal medicines that can be used for dietary supplements announced by the Ministry of Health of the People's Republic of China. It is now widely used in compound medicines for the treatment of "cerebral thrombosis" and "stroke", such as Peiyuantongnao capsules, Kangshuan Zaizao pills, Xingnao Zaizao capsules, etc., with the main effects of "invigorating qi" and "nourishing blood". Polysaccharides have been verified to provide new strategies and directions for the treatment of stroke through multiple aspects such as neuroprotection, anti-inflammation, and promotion of angiogenesis. The inventors found that the anti-ischemic stroke effect of prepared Polygonum multiflorum polysaccharide was evaluated by using the MCAO model through indicators such as weight change, neurological function score, cerebral infarction rate, and microglia number. The results showed that different concentrations of Radix Polygoni Multiflori (20mg / kg, 50mg / kg) had a protective effect on acute cerebral ischemia caused by MCAO, showing concentration dependence, and could restore the area of cerebral ischemia and significantly reduce the number of microglia, indicating that Radix Polygoni Multiflori has good activity in preventing and treating ischemic stroke. These studies provide a scientific basis for Radix Polygoni Multiflori as a potential drug for the treatment of stroke, which can be further applied to the development of health products and medicines, and has a good market prospect.
[0024] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.
[0025] The product performance tests used in the following examples are as follows:
[0026] 1. Extraction and purification of Polygonum multiflorum polysaccharide: 100g of Polygonum multiflorum polysaccharide was added with 2L of distilled water (solid-liquid ratio 1:20) and extracted with an electric heating jacket for 2h. The filtrate was poured out and the previous operation was repeated. The combined solution was concentrated under reduced pressure to a density of 1.12-1.18. Anhydrous ethanol was added to an ethanol concentration of 80%, and the mixture was precipitated in a refrigerator at 4°C overnight. The precipitate was collected and washed repeatedly with ethanol and acetone. The alcohol precipitate was redissolved and deproteinized by the Sevage method. The solution was dialyzed with distilled water for 48h, and the solution was concentrated and freeze-dried to obtain Polygonum multiflorum polysaccharide. The total sugar content of the obtained Polygonum multiflorum polysaccharide was determined by the sulfuric acid phenol method, and the protein content was determined by the biuret (Bicinchoninic Acid Assay, BCA) method.
[0027] 2. Perform MCAO surgery on rats. Fast for 12 hours, then inject 2% sodium pentobarbital (0.2mL / 100g) intraperitoneally.
[0028] Anesthetized, the common carotid artery (CCA) was ligated, and then a monofilament was inserted from the external carotid artery (ECA) and gently advanced to occlude the ipsilateral middle cerebral artery. After 1 hour of occlusion, reperfusion was achieved by removing the monofilament, and reperfusion was then ensured for 24 hours.
[0029] 3. High and low dose interventions were performed. After SD rats were acclimated for 1 week, they were randomly divided into 5 groups, with 10 rats in each group. The control group (normal saline), MCAO group (normal saline), LM group (20 mg / kg PPM), HM group (50 mg / kg PPM), and HCK group-PMP high-dose positive control (50 mg / kg PPM) were gavaged for 21 days. Except for the control group rats and the high-dose positive control group, the rats in the other groups underwent MCAO surgery, ischemia for 1 hour and then reperfusion, and all intervention measures lasted for 7 days after modeling.
[0030] 4. Neurological score adopts Longa scoring method. In short, 3 points means severe neurological damage, 2 points means moderate neurological damage, and 1 point means mild neurological damage.
[0031] 5. After euthanasia, the whole brain of the rat was taken and cut into 2 mm tissue slices. The slices were stained with 2% TTC (2,3,5-triphenyltetrazolyl ammonium chloride solution) for 20 min to show the extent of ischemic infarction. The stained brain slices were fixed in 10% formalin at pH 7.4. Coronal slices were taken. The area of cerebral infarction was calculated using Imagej1.41 software. The percentage of cerebral infarction = infarction area / area of non-ischemic hemisphere × 100%.
[0032] 6. Brain tissues were collected, fixed in 4% formaldehyde solution, and then embedded in paraffin after dehydration in a graded ethanol series (70% to 100%). The embedded samples were sectioned with a rotary microtome (5 μm thick), H&E stained according to standard protocols, and images were captured using an Olympus fluorescence microscope (Olympus, Tokyo, Japan).
[0033] 7. Statistical methods: Graphpadprism 9.0 software was used to analyze and compare all data as "mean ± SD". Statistical significance: #p<0.05, ##p<0.01, ###p<0.001 and ####p<0.0001 vs. model group; *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 vs. control group.
[0034] 8. Experimental results:
[0035] (1) Total sugar content, protein content and infrared fingerprint of Radix Polygoni Multiflori polysaccharide: The total sugar content of Radix Polygoni Multiflori polysaccharide is about 85%, containing a small amount of protein (see Figure 1 ), the infrared fingerprint of Radix Polygoni Multiflori polysaccharide showed: 3405cm -1 The band represents the stretching of hydroxyl groups. -1 The small bands nearby are caused by carbon-hydrogen bond stretching and bending vibrations. -1 、1419cm -1 、1327cm -1 The broad absorption band at 1200 cm-1 can be attributed to the deformation vibration of carbon-hydrogen bonds. -1 ~1000cm -1 The region has a specific energy band. This region is dominated by ring vibration, which overlaps with the stretching vibration of the (C-OH) side group and the (COC) glycosidic bond. -1 、1106cm -1 、1025cm -1 The absorption at 934 cm indicates the presence of a pyranose unit. -1 There is a characteristic peak nearby, indicating the β configuration of carbon-hydrogen deformation vibration in the pyran ring. -1 The absorption at Figure 2 ).
[0036] (2) Effect of Radix Polygoni Multiflori Polysaccharide on Body Weight Change and Neurological Function Scores of Rats: Different doses of Radix Polygoni Multiflori Polysaccharide improved the body weight changes and neurological function of rats in a dose-dependent manner. The dose of 50 mg / kg improved the body weight of rats towards the control group (see Figure 3 ).
[0037] (3) Analysis of the effect of processed Polygonum multiflorum polysaccharide on cerebral infarction rate in rats: After MCAO modeling, rats had obvious cerebral congestion and swelling. The prevention of processed Polygonum multiflorum polysaccharide significantly improved the cerebral ischemia area and brain swelling (see Figure 4 ).
[0038] Analysis of the effect of Preparata multiflora polysaccharide on HE sections and the number of microglia in the brains of MCAO rats: Histological examination of HE-stained ischemic brain sections showed that MCAO caused deep staining and concentration of neuronal nuclei, atrophy of cell bodies, and peripheral cavitation. In contrast, in the high-dose group, Preparata multiflora polysaccharide intervention alleviated I / R-induced neuronal damage. Representative images and quantification showed a significant increase in iba1 positivity in MCAO rats on the 7th day after surgery. It was observed in the low-dose and high-dose groups that Preparata multiflora polysaccharide administration reduced the number of activated microglia in the mouse brain, and the improvement was dose-dependent (see Figure 5 ).
[0039] In summary, Radix Polygoni Multiflori polysaccharides can significantly improve the neurobehavioral dysfunction of rats with cerebral ischemia-reperfusion, reduce the rate of cerebral infarction, and promote the recovery of cognitive behavioral ability in the late stage of stroke. Radix Polygoni Multiflori polysaccharides can be used as a drug for the prevention of ischemic stroke and post-stroke repair, providing an effective solution for the prevention and treatment of stroke.
[0040] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.
Claims
1. Application of prepared Polygonum multiflorum polysaccharide in the preparation of drugs for treating and / or preventing ischemia-reperfusion injury.
2. The use according to claim 1, characterized in that: The prepared Polygonum multiflorum polysaccharide is used for alleviating neuronal damage caused by I / R and reducing the number of activated microglial cells in the brain.
3. The use according to claim 1, characterized in that: The ischemia-reperfusion injury includes ischemia-reperfusion injury caused by blood circulation disorder caused by traumatic shock, surgical operation, organ transplantation, burns, frostbite or thrombosis.
4. The use according to claim 1, characterized in that: The dosage form of the medicine is tablet, capsule, pill, powder, granule, suspension, syrup, injection or powder injection.
5. The use according to claim 1, characterized in that: The drug is administered to humans or non-human animals.
6. The use according to claim 1, characterized in that: The drug can be administered orally, parenterally, subcutaneously, intramuscularly, intravenously, intraperitoneally, intrahepatically, intramyocardially, intrarenally, vaginally, rectally, buccally, sublingually, intranasally or transdermally.
7. The use according to claim 1, characterized in that: The dosage of the prepared Polygonum multiflorum polysaccharide in the medicine is 20mg / kg to 50mg / kg.
8. The use according to claim 1, characterized in that: The preparation method of Radix Polygoni Multiflori Polysaccharide comprises: Add distilled water to the prepared Polygonum multiflorum at a solid-liquid ratio of 1:20, extract for 2 hours by reflux heating, and pour out the filtrate; then repeat the extraction process, combine the filtrates, and concentrate under reduced pressure to a density of 1.12-1.18 to obtain a combined liquid; then add anhydrous ethanol to the combined liquid until the ethanol concentration is 80%, precipitate overnight at 4°C, collect the precipitate, and wash it repeatedly with ethanol and acetone to obtain an alcohol precipitate; re-dissolve the alcohol precipitate and deproteinize it by Sevage method, dialyze it with distilled water for 48 hours, and then concentrate and freeze-dry to obtain the prepared Polygonum multiflorum polysaccharide.