Use of polygala root powder in preparation of a medicine for preventing and / or treating central nervous system inflammatory acute demyelinating diseases

Abstract

The application discloses application of a polygala root powder in preparation of a medicine for preventing and / or treating central nervous inflammatory acute demyelination diseases and belongs to the technical field of medicines.The polygala root powder is composed of the following components in mass fractions: 12 parts of polygalae radix, 15 parts of poriae cocos, 18 parts of aconiti tuber, 12 parts of coptidis rhizoma and 9 parts of ginseng.The application has an in-depth analysis on the treatment and prevention effect of the polygala root powder on central nervous inflammatory acute demyelination diseases, finds that the polygala root powder can effectively improve the clinical symptoms of CPZ demyelination mice and relieve the demyelination of the CPZ mice, and the mechanism is related to inhibition of abnormal activation of astrocytes and reduction of inflammatory reaction in the brain of the CPZ mice.The application provides a new use of the polygala root powder, widens the application range of the polygala root powder and provides more possibilities for application of the polygala root powder.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of Polygala tenuifolia powder in the preparation of medicaments for the prevention and / or treatment of acute inflammatory demyelinating diseases of the central nervous system. Background Technology

[0002] Central nervous system inflammatory demyelinating diseases are a class of diseases characterized by inflammatory responses and widespread primary demyelination. Among them, relapsing-remitting multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD) are the most common demyelinating diseases. Their course typically involves alternating relapses and remissions, with repeated attacks causing irreversible damage to neurological function, making them a leading cause of non-traumatic disability in young and middle-aged adults. The pathogenesis of these diseases is not yet fully understood, but pathologically, they are characterized by neuronal damage and demyelination. The pathogenesis of CNS degeneration is related to oxidative stress, inflammatory responses, and neuronal damage. In clinical treatment, the combination of traditional Chinese medicine and Western medicine is more effective than either alone. Traditional Chinese medicine often employs methods to invigorate qi and promote blood circulation, which can effectively improve patient symptoms and reduce the probability of relapse.

[0003] Astrocytes (AS) are the most widely distributed cells in the central nervous system (CNS), playing a crucial role in maintaining the stability of the CNS microenvironment and regulating neuroinflammation. Studies have shown that in CNS degenerative diseases, after demyelination, AS is activated through the combined action of multiple signaling pathways, including the JAK-STAT3 signaling pathway, nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK). On the one hand, autoimmune atrophy (AS) can produce various pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-18 (IL-18), IL-1α / β, IL-10, and TGF-β1, which promote demyelination. On the other hand, reactive AS can secrete nerve growth factor (NGF) and basic fibroblast growth factor (BFGF) to promote myelin repair. Targeting AS to inhibit the inflammatory response is of great significance for the prevention and treatment of inflammatory demyelinating diseases of the central nervous system.

[0004] Yuanzhi Powder is composed of Polygala tenuifolia, Acorus tatarinowii, Ginseng, Poria cocos, and Coptis chinensis. Polygala tenuifolia is bitter, pungent, and slightly warm, entering the heart, kidney, and lung meridians; it can calm the mind, improve intelligence, resolve phlegm, and open the orifices. Acorus tatarinowii is pungent, bitter, and warm, entering the heart and stomach meridians; it can resolve dampness, stimulate appetite, awaken the mind, open the orifices, resolve phlegm, and improve intelligence. Ginseng is sweet, slightly bitter, and slightly warm, entering the spleen, lung, and heart meridians; it has the effects of greatly replenishing vital energy, restoring pulse and consolidating the body, tonifying the spleen and lungs, calming the mind, and improving intelligence. Poria cocos is sweet, bland, and neutral, entering the heart, spleen, and kidney meridians; it has the effects of strengthening the spleen and replenishing qi, calming the mind, and soothing the nerves. Coptis chinensis is slightly cold and bitter, entering the heart, spleen, stomach, liver, and gallbladder meridians; it can clear heat and purge fire, restraining the warming properties of Polygala tenuifolia and Acorus tatarinowii, thus achieving the effects of tonifying kidney essence, replenishing qi and nourishing blood, resolving phlegm, and dispersing blood stasis. However, there are currently no reports on the use of Yuanzhi Powder in the treatment of degenerative diseases of the central nervous system. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Yuanzhi San in the preparation of medicaments for the prevention and / or treatment of acute inflammatory demyelinating diseases of the central nervous system, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of Yuanzhi powder in the preparation of a medicament for the prevention and / or treatment of acute inflammatory demyelinating diseases of the central nervous system, wherein the Yuanzhi powder is composed of the following components in parts by weight: 12 parts of Yuanzhi, 15 parts of Poria cocos, 18 parts of Acorus tatarinowii, 12 parts of Coptis chinensis and 9 parts of ginseng.

[0008] Preferably, the central nervous system inflammatory demyelinating disease includes multiple sclerosis, neuromyelitis optica, or acute disseminated encephalomyelitis.

[0009] The present invention also provides a pharmaceutical composition for treating acute demyelinating diseases of the central nervous system, comprising Polygala tenuifolia powder;

[0010] The central nervous system inflammatory acute demyelinating diseases mentioned are multiple sclerosis, neuromyelitis optica, or acute disseminated encephalomyelitis.

[0011] The present invention also provides a pharmaceutical preparation for treating acute demyelinating diseases of the central nervous system, comprising Polygala tenuifolia powder or the pharmaceutical composition thereof and a pharmaceutically acceptable carrier.

[0012] Preferably, the pharmaceutically acceptable carrier is suitable for tablets, capsules, powders, pills, granules, oral liquids, injections, or emulsions.

[0013] This invention also provides the use of Polygala tenuifolia powder in the preparation of drugs that inhibit demyelination.

[0014] This invention also provides the use of Polygala tenuifolia powder in the preparation of drugs that inhibit astrocyte activation.

[0015] This invention also provides the use of Yuanzhi powder in the preparation of drugs that inhibit inflammatory factors and protect myelin.

[0016] Based on the above technical solution, the present invention has the following technical effects:

[0017] This invention provides an in-depth analysis of the therapeutic and preventive effects of Polygala tenuifolia powder in demyelinating diseases. It reveals that Polygala tenuifolia powder can effectively improve the clinical symptoms of Cuprizone (CPZ)-induced demyelinating mice and alleviate demyelination in CPZ mice. The mechanism is related to its inhibition of abnormal activation of astrocytes and reduction of inflammatory responses in the brains of CPZ mice. This invention provides a new use for Polygala tenuifolia powder, broadens its application scope, and offers more possibilities for its application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This application presents the effects of Polygala tenuifolia powder on the behavior and demyelination of CPZ mice in the experimental examples. Specifically, A and B compare the total distance traveled by mice in the closed arms and the number of times they entered the open arms of the cross maze; C and D compare the number of times mice entered the central area of ​​the maze and the total distance traveled; E and F show the effects of Polygala tenuifolia powder on demyelination of the corpus callosum region of CPZ mice, as detected by the black gold staining method in the experimental examples of this application.

[0020] Figure 2 The effect of Polygala tenuifolia powder on MBP expression in the corpus callosum region of CPZ mice is shown in Figure A, where A represents the effect of immunofluorescence staining on MBP protein expression in the corpus callosum region of CPZ mice in the experimental examples of this application; B is a schematic diagram of the staining location in the mouse brain; and C is the data analysis of the fluorescence intensity of MBP in the corpus callosum region of CPZ mice by Polygala tenuifolia powder in the experimental examples of this application.

[0021] Figure 3 The effect of Polygala tenuifolia powder on dMBP expression in the corpus callosum region of CPZ mice is shown in Figure A, where A represents the effect of immunofluorescence staining on dMBP expression in the corpus callosum region of CPZ mice in the experimental examples of this application; B is a schematic diagram of the staining location in the mouse brain; and C is the data analysis of the fluorescence intensity of dMBP in the corpus callosum region of CPZ mice by Polygala tenuifolia powder in the experimental examples of this application.

[0022] Figure 4 The effect of Polygala tenuifolia powder on GFAP expression in the corpus callosum region of CPZ mice is shown in Figure A, where A represents the effect of immunofluorescence staining on GFAP expression in the corpus callosum region of CPZ mice in the experimental examples of this application; B is a schematic diagram of the staining location in the mouse brain; and C is the data analysis of the fluorescence intensity of GFAP in the corpus callosum region of CPZ mice by Polygala tenuifolia powder in the experimental examples of this application.

[0023] Figure 5 The experimental examples in this application show the effects of Polygala tenuifolia powder on inflammatory factors in the brains of CPZ mice. Among them, A represents the effect of Polygala tenuifolia powder on the expression of IL-1β inflammatory factor in the brains of CPZ mice, B represents the effect of Polygala tenuifolia powder on the expression of TNF-α inflammatory factor in the brains of CPZ mice, C represents the effect of Polygala tenuifolia powder on the expression of IL-6 inflammatory factor in the brains of CPZ mice, and D represents the effect of Polygala tenuifolia powder on the expression of IL-10 inflammatory factor in the brains of CPZ mice. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0030] This invention provides the application of Yuanzhi powder in the preparation of a medicament for the prevention and / or treatment of acute inflammatory demyelinating diseases of the central nervous system. The Yuanzhi powder is composed of the following components in parts by weight: 12 parts of Yuanzhi, 15 parts of Poria cocos, 18 parts of Acorus tatarinowii, 12 parts of Coptis chinensis, and 9 parts of ginseng.

[0031] In some specific embodiments, the central nervous system inflammatory acute demyelinating disease is a dicyclohexanone oxaloyl dihydrazone-mediated central nervous system inflammatory acute demyelinating disease.

[0032] It should be noted that the aforementioned demyelination refers to inflammatory demyelinating diseases of the central nervous system related to abnormal autoimmune responses, genetics, environment, etc., such as multiple sclerosis, neuromyelitis optica, or acute disseminated encephalomyelitis.

[0033] This invention provides a pharmaceutical composition for treating acute demyelinating inflammatory diseases of the central nervous system, comprising Polygala tenuifolia powder.

[0034] This invention provides a pharmaceutical preparation for treating acute demyelinating inflammatory diseases of the central nervous system, comprising Polygala tenuifolia powder or the pharmaceutical composition thereof and a pharmaceutically acceptable carrier.

[0035] In some specific embodiments, the pharmaceutically acceptable carrier is suitable for tablets, capsules, powders, pills, granules, oral liquids, injections, or emulsions.

[0036] It should be noted that, by examining the effect of Polygala tenuifolia on improving the inflammatory microenvironment of the central nervous system in demyelinating model mice, the inventors found that Polygala tenuifolia significantly inhibited the inflammatory response in the central nervous system. The mechanism may be related to the fact that Polygala tenuifolia inhibited the abnormal activation of AS, thereby suppressing the inflammatory response in acute CPZ mice.

[0037] It should be noted that Polygala tenuifolia powder can inhibit the activation of astrocytes and improve the inflammatory microenvironment of the central nervous system. This improvement mainly includes inhibiting the expression of inflammatory factors such as IL-6, IL-1β, and TNF-α, and promoting the expression of the anti-inflammatory factor IL-10. The inventors found through in vivo verification that Polygala tenuifolia powder significantly inhibits the activation of astrocytes and improves the inflammatory microenvironment of the central nervous system.

[0038] In some specific embodiments, the Yuanzhi powder is a compound composed of 12g of Yuanzhi, 15g of Poria cocos, 18g of Acorus tatarinowii, 12g of Coptis chinensis and 9g of ginseng.

[0039] It should be noted that the formulation can be prepared according to conventional methods in the art, including tablets, capsules, granules, suspensions, emulsions, solutions, syrups or injections.

[0040] It should be noted that the Yuanzhi San recorded in the *Shengji Zonglu* has the effects of invigorating qi and nourishing the heart, calming the mind and improving intelligence, resolving phlegm and opening the orifices, and is mainly used to treat forgetfulness. However, it is unclear whether Yuanzhi San can treat diseases by inhibiting myelin demyelination. Therefore, this invention established a CPZ mouse model and used Yuanzhi San for intervention to verify whether Yuanzhi San can inhibit myelin demyelination and to explore the specific mechanism of inhibiting myelin demyelination.

[0041] It should be noted that Traditional Chinese Medicine (TCM) believes that the main pathogenesis of demyelinating diseases is deficiency of kidney essence and insufficiency of qi and blood, leading to phlegm and blood stasis obstruction, malnourishment of the brain, and dysfunction of the mind, ultimately resulting in sensory and motor dysfunction. In the formula Yuanzhi San, Yuanzhi (Radix Polygalae) is bitter, pungent, and slightly warm, entering the heart, kidney, and lung meridians; it can calm the mind, improve intelligence, and clear phlegm and open the orifices. Shichangpu (Radix Acoruse Tatarinowii) is pungent, bitter, and warm, entering the heart and stomach meridians; it can resolve dampness, stimulate appetite, awaken the mind, clear phlegm, and improve intelligence. Renshen (Radix Ginseng) is sweet, slightly bitter, and slightly warm, entering the spleen, lung, and heart meridians; it has the effects of greatly replenishing vital energy, restoring pulse and consolidating the body, tonifying the spleen and lungs, and calming the mind and improving intelligence. Fuling (Poria) is sweet, bland, and neutral, entering the heart, spleen, and kidney meridians; it has the effects of strengthening the spleen and replenishing qi, calming the mind and soothing the nerves. Huanglian (Rhizoma Coptise) is slightly cold and bitter, entering the heart, spleen, stomach, liver, and gallbladder; it can clear heat and purge fire, restraining the warming properties of Yuanzhi and Shichangpu, thus achieving the effects of replenishing kidney essence, nourishing qi and blood, and resolving phlegm and dispersing blood stasis.

[0042] It should be noted that the aforementioned Polygala tenuifolia powder can be used alone or in combination with other drugs. It can be used together with Polygala tenuifolia powder to treat inflammatory demyelinating diseases of the central nervous system, or it can have other functions, such as alleviating the side effects of Polygala tenuifolia powder during treatment.

[0043] It should be noted that the pharmaceutical products of Yuanzhi San can be manufactured into various dosage forms using conventional production methods known in the art. These dosage forms include one or more of the following: tablets, capsules, granules, suspensions, emulsions, solutions, syrups, or injections, administered via one or more routes of administration, such as oral or injection (including one or more of intravenous injection, intravenous drip, intramuscular injection, or subcutaneous injection).

[0044] The dosage or administration of the Polygala tenuifolia powder described in this invention is generally determined based on the patient's or user's age, weight, physical condition, or the severity of the patient's symptoms. Furthermore, it should be understood that for any particular patient, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges set forth herein are merely illustrative and do not limit the scope or practice of the claimed compositions.

[0045] The mechanism of action of Yuanzhi Powder will be verified below with specific experimental examples.

[0046] Main reagents in the embodiments of this invention:

[0047] Myelin basic protein (MBP, Abcam, ab40390);

[0048] Glial fibrillary acidic protein (GFAP, Oasis Biofarm, OB-PGP055);

[0049] Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) was purchased from Invitrogen, catalog number A-11001;

[0050] The TNF-α, IL-1β, IL-6, and IL-10 kits were purchased from Invitrogen, with catalog numbers 88-7324-88, 88-7013A-88, 88-7064-22, and 88-7105-22, respectively.

[0051] The TrueGold myelin staining kit was purchased from Osasi Biotechnology Co., Ltd., catalog number BK-AC001.

[0052] Main instruments in this embodiment of the invention:

[0053] Cryostat slicer (Leica GmbH, Germany, model: CM1950);

[0054] Elevated cross-maze and open field experimental analysis system (Reward Life Technology Co., Ltd.);

[0055] Electronic analytical balance (Mettler Toledo, Switzerland, model: PL303);

[0056] High-speed refrigerated centrifuge (Eppendorf GmbH, Germany, model: 5840);

[0057] Microplate reader (Berten Instruments, USA, model: SYNERGY / H1);

[0058] Upright fluorescence microscope (Olympus, Japan, model: BX51).

[0059] Example 1

[0060] 1. Experimental Methods

[0061] ① Drug preparation:

[0062] The formula for Yuanzhi Powder is prepared according to the original dosage ratio: Yuanzhi 12g, Poria 15g, Acorus tatarinowii 18g, Coptis chinensis 12g, and Ginseng 9g. The extracts are coarsely filtered three times and concentrated at 80℃ to 1.2g / mL.

[0063] ② Grouping and administration of experimental mice:

[0064] Male C57BL / 6 mice (8-10 weeks old) were purchased from Vital River Pharmaceuticals in Beijing and randomly divided into three groups (n=10 per group) according to body weight, with three parallel control groups in each group:

[0065] Normal group (Nor): fed with regular feed for 6 weeks;

[0066] CPZ treatment group (CPZ): The model was established by feeding the diet containing 0.2% CPZ for 6 consecutive weeks, followed by gavage with an equal volume of physiological saline from the end of week 4.

[0067] Polygala tenuifolia treatment group (CPZ + Polygala tenuifolia): The model was established by feeding the diet containing 0.2% CPZ for 6 consecutive weeks, and Polygala tenuifolia (12g / kg / d) was administered by gavage starting from the end of the 4th week.

[0068] ③ Behavioral testing

[0069] (1) Elevated Plus Maze (EPM) Experiment: Mice explore new environments out of curiosity, but they are naturally afraid of heights. Mice with normal cognitive abilities and stable emotions avoid heights. The EPM consists of two arms of equal length crossing each other. One arm, the closed arm, is surrounded by a barrier; the other arm is an open arm without a barrier. The unbarriered area at the intersection is the central area. The EPM is about 50 cm above the ground. During the experiment, mice are placed in the central area, and their movement trajectory data is recorded for 5 minutes in a quiet environment using a camera. Between experiments, the maze is cleaned with 70% alcohol to eliminate the influence of odor.

[0070] (2) Open field test: The open field test is a method to evaluate the autonomous behavior, exploratory behavior, and stress level of experimental animals in a new environment, and to detect their movement trajectory. Before the test, the mice are placed in an opaque square open field box and allowed to adapt to the environment for 1 minute. The test is then conducted for 5 minutes, and the movement trajectory data of the mice are recorded. After each mouse is tested, the odor is eliminated with 70% alcohol.

[0071] ④ Specimen preparation

[0072] Five mice were randomly selected from each group and anesthetized with 1% sodium pentobarbital (50 mg / kg). The heart was perfused with physiological saline until the liver turned white. Then, the heart was perfused with 4% paraformaldehyde. The brain tissue was dissected and removed. After fixation with paraformaldehyde, the tissue was dehydrated in a gradient of 10%, 20%, and 30% sucrose solutions. After OTC embedding, the tissue was flash-frozen in liquid nitrogen and stored at -80°C for later use. The brain tissue was cut into 10 μm sections using a cryostat for immunofluorescence staining.

[0073] The remaining 5 mice in each group underwent cardiac perfusion with physiological saline. Brain tissue was harvested, weighed, and placed in sterile EP tubes. Lysis buffer was added by weight, and the mixture was homogenized until a paste was formed. Lysis was performed on ice for 30 minutes, with shaking every 5 minutes. The mixture was then transferred to a centrifuge at 12,000 rpm for 15 minutes at 4°C. The supernatant was transferred to new sterile EP tubes, which contained the brain tissue protein solution. To minimize protein degradation, the solution was aliquoted and stored at -80°C for subsequent ELISA assays.

[0074] ⑤ TrueGold staining

[0075] After the prepared frozen sections of brain tissue were brought to room temperature, they were dried at 37°C for 30 minutes. The required amount of dye and staining agent were diluted with double-distilled water. The dye was added, and the sections were placed in a humidified chamber and stained at 45°C in the dark for 30 minutes. The sections were then rinsed twice with PBS for 1 minute each time, and the staining agent was added. The sections were incubated at 45°C for 2-3 minutes. After that, the sections were rinsed three times with PBS for 1 minute each time, until the gray and white matter could be clearly distinguished. The sections were then blotted dry with absorbent paper, mounted, and observed and photographed under a microscope.

[0076] ⑥ Immunofluorescence staining to detect the expression of MBP, dMBP, and GFAP

[0077] Prepared frozen sections of brain tissue were washed five times with PBS for 5 min each time. Primary antibody was added to PBS solution containing 0.3% Triton X-100 and 1% BSA, and the suspension was dropped onto the sections. The sections were incubated overnight at 4°C in a humidified chamber. The next day, the sections were washed five times with PBS for 5 min each time, and the corresponding species' secondary antibody was added and incubated at room temperature for 1 h. The sections were washed five times with PBS again. Cell nuclei were stained with DAPI for 10 min, and the sections were mounted with fluorescent anti-quenching mounting medium. The results were observed under a fluorescence microscope. Three parallel controls were set up for each group. The fluorescence density was semi-quantitatively analyzed using ImageJ fiji 2.14.0.

[0078] ⑦ Detection of TNF-α, IL-1β, IL-6, and IL-10 in brain tissue protein solution using ELISA and microenzyme labeling methods

[0079] The brain tissue protein solutions from each group were processed according to the instructions of the ELISA and micro-enzyme labeling kits. The optical density (OD) values ​​were read using an enzyme-linked immunosorbent assay (ELISA) reader to detect the levels of TNF-α, IL-1β, IL-6, and IL-10 in the mouse brain tissue protein solutions.

[0080] 2. Statistical methods

[0081] Statistical analysis was performed using GraphPad 8.0 software. Normally distributed continuous data were expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for pairwise comparisons between groups. P < 0.05 was considered statistically significant.

[0082] 3. Results

[0083] 3.1 Behavioral test results

[0084] In EPM (Extended Perimeter Movement), mice are sensitive to changes in the external environment, prefer dim lighting, are nocturnal, and are naturally timid and afraid of heights, but are also very curious and enjoy exploring. Therefore, this invention statistically analyzes the total distance mice travel within the closed arm and the number of times they enter the open arm. For example... Figure 1 As shown in Figures A and B, the EPM results showed that, compared with the normal group, the number of times mice entered the open arm in the CPZ group decreased (P<0.01), and the total distance of movement in the closed arm decreased (P<0.001); compared with the CPZ group, the number of times mice entered the open arm in the CPZ+Yuanzhisan group increased (P<0.05), and the total distance of movement in the closed arm increased (P<0.01).

[0085] In open field experiments, mice are naturally afraid of open spaces. Therefore, this invention records the number of times a mouse enters the central area and the total distance traveled. Figure 1 As shown in Tables C and D, the open field experiment results showed that, compared with the normal group, the CPZ group mice had an increased total distance to enter the central region in the open field (P<0.01) and an increased number of times they entered the central region (P<0.001); compared with the CPZ group, the CPZ+Yuanzhisan group mice had a decreased total distance to enter the central region in the open field (P<0.01) and a decreased number of times they entered the central region (P<0.0001). The results are shown in Table 1.

[0086] The results showed that Yuanzhi powder effectively improved the behavior of CPZ-demyelinated mice and significantly improved anxiety and memory deficits in the demyelinating model mice.

[0087] Table 1. Behavioral Detection of Mice

[0088]

[0089] Note: * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 vs CPZ.

[0090] 3.2 The protective effect of Polygala tenuifolia powder on myelin sheath in CPZ mice

[0091] Brain sections of mice from the normal group, CPZ treatment group, and Polygala tenuifolia treatment group were selected and pathologically stained. Three parallel controls were set up in each group to observe the staining of each section in each group and to evaluate the effect of Polygala tenuifolia on demyelination morphology.

[0092] 1) TrueGold staining

[0093] Brain slices containing the corpus callosum were stained with True Gold, and myelin morphology was assessed by color difference and percentage of stained area. Figure 1As shown in Figures E and F, the black-gold staining of the normal group mice was evenly distributed and deeply stained, with a large stained area ratio. Compared with the normal group, the corpus callosum of the CPZ-treated group mice was lightly stained, and the stained area ratio was reduced (P<0.0001). Compared with the CPZ group, the black-gold staining of the corpus callosum in the CPZ + Polygala tenuifolia group was deeper, and the stained area ratio was increased (P<0.0001). The results are shown in Table 2.

[0094] Analysis results showed that the black gold staining of the Yuanzhi powder treatment group was darker and the myelin sheath was thicker. Based on the judgment criteria of the area occupied by the staining of the demyelinated area, it can be seen that Yuanzhi powder has a protective effect on the myelin sheath of the corpus callosum.

[0095] Table 2. Polygala tenuifolia powder inhibits demyelination in CPZ mice.

[0096]

[0097] Note: * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 vs CPZ.

[0098] 2) Immunofluorescence staining

[0099] Immunofluorescence staining was used to stain myelin basic protein (MBP) and degraded myelin basic protein (dMBP) in the corpus callosum region of demyelinating mouse models. Figure 2 , 3 As shown in Table 3, compared with the normal group, the fluorescence intensity of MBP staining in the corpus callosum was significantly decreased in the CPZ group (P<0.0001), while the fluorescence intensity of dMBP staining was significantly increased (P<0.0001). Compared with the CPZ group, the fluorescence intensity of MBP staining in the corpus callosum was increased in the CPZ+Yuanzhisan group (P<0.0001), while the fluorescence intensity of dMBP staining was significantly decreased (P<0.0001).

[0100] The results showed that Polygala tenuifolia powder enhanced the expression of MBP in the corpus callosum region of demyelinating mouse model mice and reduced the expression of dMBP. Staining analysis indicated that Polygala tenuifolia powder could inhibit demyelination in demyelinating mouse model mice.

[0101] Table 3. Effects of Yuanzhi powder on myelin expression in CPZ mice.

[0102]

[0103] Note: * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 vs CPZ.

[0104] 3.3 Effects of Polygala tenuifolia powder on GFAP expression in the brain of CPZ mice

[0105] The atherosclerotic cells (ASTs) are the most widely distributed cells in the central nervous system (CNS), playing a crucial role in maintaining the stability of the CNS microenvironment and regulating neuroinflammation. GFAP is a marker of AST activation; its expression increases during demyelination, and it secretes inflammatory factors such as TNF-α, IL-1β, and IL-17, exacerbating demyelination. Therefore, detecting the status of the AST is essential. Figure 4 As shown in Table 4, compared with the normal group, the fluorescence intensity of GFAP in the corpus callosum of mice in the CPZ group was significantly increased (P<0.001), and compared with the CPZ group, the immunofluorescence intensity of GFAP in the CPZ+Yuanzhisan group was significantly decreased (P<0.001).

[0106] The results showed that Yuanzhi powder could inhibit the activation of astrocytes and reduce the inflammatory response.

[0107] Table 4. Polygala tenuifolia powder inhibits abnormal activation of astrocytes in the brain of CPZ mice.

[0108]

[0109] Note: * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 vs CPZ.

[0110] 3.4 Effects of Polygala tenuifolia powder on inflammatory factors in CPZ mice

[0111] Slow myelin repair in a CPZ-mediated chronic mouse model is closely related to central nervous system inflammation. Therefore, the applicant used ELISA to detect the levels of IL-1β, TNF-α, IL-6, and IL-10 in brain tissue protein solution. Three parallel controls were set up in each group.

[0112] like Figure 5 As shown, compared with the normal group, the CPZ group showed significantly increased secretion levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 (P<0.0001, P<0.0001, and P<0.0001, respectively), and significantly decreased secretion level of the anti-inflammatory factor IL-10 (P<0.0001). Compared with the CPZ group, the CPZ + Polygala tenuifolia powder group downregulated the secretion levels of IL-1β, TNF-α, and IL-6 (P<0.01, P<0.01, and P<0.001, respectively), and upregulated the secretion level of IL-10 (P<0.001). Polygala tenuifolia powder can inhibit the inflammatory response in CPZ mice. The results are as follows. Figure 5 As shown.

[0113] The results showed that Yuanzhi powder could effectively inhibit the secretion of inflammatory factors in CPZ mice.

[0114] Table 5. Polygala tenuifolia powder inhibits inflammatory responses in the brains of CPZ mice.

[0115] Normal CPZ CPZ+Yuanzhi Powder TNF-α (pg / ml) 3.943±0.199**** 35.78±0.995 11.05±1.599** IL-6 (pg / ml) 1.717±0.209**** 25.74±0.418 5.370±1.990*** IL-1β (pg / ml) 82.86±0.787**** 777.9±2.981 83.32±8.344** IL-10 (pg / ml) 1601±84.88**** 521.8±8.598 1063±82.66**

[0116] Note: * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 vs CPZ.

[0117] In summary, this invention provides an in-depth analysis of the therapeutic and preventive effects of Polygala tenuifolia powder in demyelinating diseases. A CPZ-induced acute demyelinating mouse model was established, and the anxiety levels of the mice were evaluated using the elevated cruciate maze test and the open field test. Subsequently, mouse brain tissue was collected for black-gold staining to compare myelin changes in each group. Immunofluorescence staining was used to detect the average fluorescence intensity of MBP, dMBP, and GFAP in the brains of each group of mice. The levels of IL-1β, TNF-α, IL-6, and IL-10 in mouse brain tissue were detected using ELISA. The results show that Polygala tenuifolia powder effectively improves the behavior of CPZ-induced demyelinating mice, inhibits demyelination in CPZ-induced mice, inhibits astrocyte activation, and suppresses the inflammatory response in CPZ-induced mice. This invention provides a new use for Polygala tenuifolia powder, broadens its application scope, and offers more possibilities for its application.

[0118] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The use of Yuanzhi powder in the preparation of drugs for the prevention and / or treatment of acute inflammatory demyelinating diseases of the central nervous system, characterized in that, The Yuanzhi powder is composed of the following components in parts by weight: 12 parts of Yuanzhi, 15 parts of Poria cocos, 18 parts of Acorus tatarinowii, 12 parts of Coptis chinensis, and 9 parts of ginseng.

2. The application according to claim 1, characterized in that, The central nervous system inflammatory demyelinating diseases mentioned are multiple sclerosis, neuromyelitis optica, or acute disseminated encephalomyelitis.

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