Application of total flavonoids of artemisia scoparia in preparation of medicine for preventing or treating autoimmune demyelination disease

Abstract

The invention belongs to the technical field of medicines, and discloses application of artemisia scoparia total flavonoids in preparation of medicines for preventing, relieving and / or treating autoimmune demyelination diseases. In a mouse experimental autoimmune encephalomyelitis model, the total flavonoids of artemisia scoparia show a good treatment effect, and can effectively treat pathological changes and disease progression of diseases. The treatment effect is realized by relieving spinal cord inflammatory cell infiltration and reducing spinal cord demyelination.

Description

Technical Field

[0001] This invention relates to the application of total flavonoids from Artemisia scoparia in the preparation of drugs for the prevention, relief and / or treatment of autoimmune demyelinating diseases, and belongs to the field of pharmaceutical technology. Background Technology

[0002] Multiple sclerosis (MS) is an immune-mediated, chronic, progressive inflammatory demyelinating disease of the central nervous system (CNS). It affects more than 2 million people worldwide, posing a serious threat to human health and imposing a heavy economic burden on society. The pathogenesis of MS is complex, the cause is unknown, and there is currently no cure; patients require long-term treatment. MS is characterized by inflammation and demyelinating lesions in the brain, spinal cord, and optic nerve. Its clinical manifestations are widespread, including muscle weakness, sensory disturbances, cognitive impairment, and fatigue. The etiology of MS is unclear, but it may be related to multiple factors such as genetics, environment, and infection. Based on the patient's clinical presentation, MS can be classified into four types: relapsing-remitting (relapsing and remittent), characterized by alternating periods of relapse and remission with no significant disease progression; primary progressive (primarily progressive), characterized by continuous deterioration after onset; secondary progressive (initially relapsing and remittent), characterized by a relapsing-remitting pattern followed by continuous deterioration; and progressive-relapsing (gradually progressing with relapses). Currently, most clinical treatments target patients with relapsed or remission-type illnesses.

[0003] Multiple sclerosis is considered an autoimmune disease, primarily caused by autoreactive immune cells crossing the blood-brain barrier into the central nervous system (CNS). Early lesions manifest as peripheral immune cell infiltration and blood-brain barrier (BBB) ​​leakage. Cellular infiltration is predominantly dominated by macrophages and CD8+ cells. + T cells followed by CD4 + The number of T cells, B cells, and plasma cells is relatively low. The composition of T cells does not change with disease progression, but the relative proportion of B cells and plasma cells increases. Microglia and macrophages remain chronically activated throughout the disease, forming plaques of myelin and oligodendrocyte loss. As the disease progresses, focal white matter lesions appear in the brain. Brain and spinal cord damage is not obvious, but brain atrophy is widespread, accompanied by ventricular enlargement. Astrocytes form multiple sclerotic glial scars in the white matter lesions. Demyelination also occurs in the gray matter of the cerebral cortex, nuclei, and spinal cord, but the demyelinated areas of white matter can be partially repaired through myelin regeneration. In addition, the disease process not only affects myelin but also leads to degenerative changes in axons and neurons, resulting in irreversible disability in patients.

[0004] Over the past two decades, as our understanding of the pathogenesis of multiple sclerosis (MS) has deepened, researchers have developed a variety of drugs targeting the disease's unique physiological pathways. Currently, drugs used to treat MS are mainly divided into five categories: immunomodulators, hormones, monoclonal antibodies, interferons, and neurorepair agents. These drugs can alleviate disease progression and control patient symptoms, but they are ineffective in repairing damaged neurons, do not improve functional disabilities, and have serious toxic side effects with long-term use.

[0005] Experimentally allergic encephalomyelitis (EAE) is a classic animal model of multiple sclerosis. It involves the specific activation of brain helper T cells by myelin autoantigens, leading to inflammatory infiltration and demyelination of the central nervous system. Its biochemical, immunological, and pathological characteristics are very similar to those of multiple sclerosis. Furthermore, rat and mouse EAEs are ideal animal models for studying other autoimmune demyelinating diseases such as experimentally allergic encephalomyelitis, neuroencephalomyelitis optica, and acute disseminated encephalomyelitis. Their clinical, pathological, immunological, and biochemical changes are quite similar to those of human demyelinating diseases, thus making them widely used.

[0006] Artemisia scoparia Waldst. et Kit. is a traditional Uyghur medicinal plant. It is slightly cold in nature and has a bitter and pungent taste. It is used to clear heat and dry dampness, cool the liver, and stop bleeding. The main chemical components of Artemisia scoparia include volatile oils, flavonoids, coumarins, and acids.

[0007] The total flavonoids from Artemisia scoparia Waldst. et Kit. (ASTF) described in this invention are flavonoid extracts from Artemisia scoparia. To date, Chinese patent CN 101669979A (publication number) describes "Artemisia scoparia extract and its production method and application," wherein the Artemisia scoparia extract has significant anti-influenza virus, hepatitis B virus, and HIV effects; Chinese patent CN 105832793A (publication number) describes "Artemisia scoparia extract as a preparation of a drug for treating pneumonia caused by Streptococcus pneumoniae and / or beta-hemolytic streptococci," wherein the Artemisia scoparia extract has a therapeutic effect on pneumonia caused by Streptococcus pneumoniae and / or beta-hemolytic streptococci. The above-mentioned Artemisia scoparia extracts are characterized by containing 25% to 75% flavonoid components. However, there are no reports, domestically or internationally, of this extract exerting direct or indirect effects on autoimmune demyelinating diseases. Therefore, this invention mainly relates to total flavonoids from Artemisia scoparia and drug combinations, for clinical application in the preparation of drugs for the prevention, relief and / or treatment of autoimmune demyelinating diseases. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide the use of total flavonoids from Artemisia scoparia Waldst. et Kit. in the preparation of drugs for the prevention, relief and / or treatment of autoimmune demyelinating diseases.

[0009] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0010] The first aspect of the present invention is to provide the use of total flavonoids from Artemisia scoparia in the preparation of drugs for the prevention, relief and / or treatment of autoimmune demyelinating diseases.

[0011] The autoimmune demyelinating diseases mentioned include, but are not limited to, demyelinating autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis, neuromyelitis optical spectrum disorder (NMOSD), acute disseminated encephalomyelitis, leukoencephalitis, and transverse myelitis; and demyelinating autoimmune diseases affecting the peripheral nervous system such as acute inflammatory demyelinating polyneuropathy (AIDP; Guillain-Barré syndrome), chronic inflammatory demyelinating polyneuropathy, anti-MAG peripheral neuropathy, motor and sensory neuropathy (HMSN), hereditary sensorimotor neuropathy (HSMN), and peroneal muscular atrophy. Atrophy, progressive neurogenic peroneal muscular atrophy (Charcot-Marie-Tooth Disease), etc.

[0012] The multiple sclerosis includes relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, and progressive-relapsing multiple sclerosis.

[0013] An EAE model was established using female C57BL / 6 mice. The effects of total flavonoids from *Artemisia scoparia* on animal body weight and disease scores were examined. Limb strength was observed using a suspension test to assess treatment progress. H&E and LFB staining were used to detect the ameliorative effect of total flavonoids on inflammatory cell infiltration and demyelination in the spinal cord of the experimental animals. An LPS-induced microglia BV2 cell activation model was used to detect the effect of total flavonoids on LPS-induced BV2 cell inflammatory responses. Based on these findings, the role of total flavonoids from *Artemisia scoparia* in the preparation of drugs for the prevention, alleviation, and / or treatment of autoimmune demyelinating diseases was determined.

[0014] The second aspect of the present invention is to provide the use of a pharmaceutical composition in the preparation of drugs for the prevention, relief and / or treatment of autoimmune demyelinating diseases, characterized in that the pharmaceutical composition contains an effective dose of total flavonoids from Artemisia scoparia and pharmaceutical excipients.

[0015] The pharmaceutical composition, in addition to containing total flavonoids from Artemisia scoparia as the active pharmaceutical ingredient, also contains other active ingredients. The pharmaceutical composition includes the following dosage forms: solution, suspension, lyophilized powder for injection, emulsion, pills, capsules, powder, controlled release, sustained release formulations, and microparticle delivery systems. The pharmaceutical excipients include starch, dextrin, sodium polymethyl cellulose, magnesium stearate, and talc. The product is selected from pharmaceuticals and health products.

[0016] The present invention therefore also relates to the use of pharmaceutical compositions using total flavonoids from Artemisia scoparia of the present invention as the active ingredient in the prevention, relief, and / or treatment of autoimmune demyelinating diseases. These pharmaceutical compositions can be prepared according to methods known in the art. They can be formulated into any dosage form suitable for human or animal use by combining the extracts of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0017] The extracts or pharmaceutical compositions containing them of this invention can be administered in unit doses via enteral or non-enteric routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc. The dosage form can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, droplets, suppositories, films, patches, aerosols, sprays, etc.; semi-solid dosage forms can include ointments, gels, pastes, etc. The extract of this invention can be formulated into ordinary preparations, as well as sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle delivery systems.

[0018] To formulate the extract of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. To formulate the drug delivery unit into capsules, the active ingredient, the extract of this invention, can be mixed with a diluent and a disintegrant, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, the extract of this invention, can be first formulated into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The various diluents, binders, wetting agents, disintegrants, and disintegrants used to prepare tablets of the extract of this invention can also be used to prepare capsules of the extract of this invention. To formulate the extract of this invention into an injection, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, cosolvents, pH adjusters, and osmotic pressure adjusters can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure adjusters can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. When preparing lyophilized powder injections, mannitol, glucose, etc., can also be added as supporting agents. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, or other additives can be added to the pharmaceutical preparation. To achieve the intended therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered using any known method of administration.

[0019] Beneficial technical effects

[0020] 1. The total flavonoids of Artemisia scoparia of the present invention can prevent, alleviate and / or treat autoimmune demyelinating diseases, providing a drug option for the clinical treatment of autoimmune demyelinating diseases.

[0021] 2. The total flavonoids from Artemisia annua of this invention can achieve therapeutic and preventative effects with oral administration and appropriate dosage. The drug is safe and reliable. Its development as a medicine has significant advantages. Attached Figure Description

[0022] Figure 1 Effects of total flavonoids from Artemisia annua on body weight in EAE mice. In this experiment, the body weight of mice in the EAE model group was significantly lower than that of the normal control group after the onset of the disease. However, after drug administration, compared with the model group, the body weight of mice in the Artemisia annua total flavonoid treatment group increased in the later stage of drug administration.

[0023] Figure 2 Effects of total flavonoids from Artemisia annua on disease scores in EAE mice. In this experiment, the disease scores of mice in the EAE model group were significantly higher than those in the normal control group after the onset of the disease. However, after drug administration, the disease scores of mice in the Artemisia annua total flavonoid treatment group were significantly lower than those in the EAE model group in the later stages of drug administration.

[0024] Figure 3 Effects of total flavonoids from Artemisia annua on suspension time in EAE mice. In this experiment, compared with the normal control group, the suspension time of mice in the EAE model group was significantly reduced, while total flavonoids from Artemisia annua could prolong the suspension time of the model mice. ### P < 0.001 vs. normal control group, ***P < 0.001 vs. EAE model group.

[0025] Figure 4 Effects of total flavonoids from Artemisia annua on suspension grading in EAE mice. In this experiment, compared with the normal control group, the suspension scores of mice in the EAE model group were significantly reduced after the onset of the disease, while total flavonoids from Artemisia annua could increase the suspension scores of EAE mice. ### P < 0.001 vs. normal control group, *P < 0.1, ***P < 0.001 vs. EAE model group.

[0026] Figure 5 Effects of total flavonoids from Artemisia annua on inflammatory cell infiltration in the spinal cord of EAE mice. In this experiment, compared with the normal control group, the EAE model group mice showed significant inflammatory cell infiltration in the spinal cord, while total flavonoids from Artemisia annua significantly reduced the infiltration area of ​​inflammatory cells.

[0027] Figure 6 Effects of total flavonoids from Artemisia scoparia on demyelination of the spinal cord in EAE mice. In this experiment, after LFB staining, compared with the normal control group, the spinal cord of mice in the EAE model group showed demyelination lesions with larger areas. Total flavonoids from Artemisia scoparia could reduce the area of ​​white matter whitening in the spinal cord, effectively improving the demyelination situation. Detailed Implementation

[0028] The pharmacological effects of total flavonoids from Artemisia scoparia in the prevention, relief and / or treatment of autoimmune demyelinating diseases are further explained below in conjunction with the present invention.

[0029] The following embodiments illustrate the invention in more detail, but are not intended to limit the invention in any way.

[0030] Example 1: Preparation process of total flavonoids from Artemisia annua

[0031] 1.1 Preparation of Artemisia annua extract

[0032] The first step involves crude extraction of the above-ground parts or whole plant of *Artemisia scoparia* using water and / or ethanol. The water extraction is performed by adding 8 to 10 times the amount of water to the above-ground parts or whole plant, extracting twice at 80°C to 100°C for 2 to 3 hours each time, filtering, and combining the filtrates. The ethanol extraction is performed by adding 8 to 10 times the amount of 30% to 90% ethanol to the above-ground parts or whole plant of *Artemisia scoparia*, extracting twice at 60°C to 80°C for 2 to 3 hours each time, filtering, and combining the filtrates.

[0033] The second step is to concentrate the above filtrate to one-third of its original volume, add anhydrous ethanol equivalent to 1 to 2 times the volume of the concentrated filtrate, let it precipitate for 12 to 24 hours, and filter out the precipitate to obtain an ethanol solution.

[0034] The third step involves concentrating the above ethanol solution and adjusting the pH to 3-6 with dilute hydrochloric acid, then adsorbing it onto a macroporous resin and / or polyamide resin chromatography column. The macroporous resin used is a weakly polar macroporous resin, specifically one of HPD400, D-101, or AB-8 type weakly polar macroporous resins. When using a mixture of macroporous resin and polyamide resin, they should be mixed in a 1:1 weight ratio.

[0035] Fourth step: Rinse the above chromatography column with deionized water equivalent to 3 to 8 column volumes, and then perform gradient elution with 10% to 90% ethanol as the eluent. The amount of each concentration of eluent used is equivalent to 3 to 8 column volumes, and the flow rate is 2 to 5 column volumes / hour. Collect the ethanol eluent and combine them.

[0036] Fifth, the combined ethanol eluent is concentrated at 60°C to 90°C to recover the ethanol, and then dried under reduced pressure at 50°C to 80°C to obtain the desired Artemisia annua extract. The Artemisia annua extract is the total Artemisia annua flavonoids described in the invention.

[0037] 1.2 Determination of total flavonoid content in Artemisia annua extract

[0038] Preparation of the reference solution: Accurately weigh 50 mg of dried rutin reference standard to constant weight and place it in a 25 mL volumetric flask. Add an appropriate amount of methanol, heat in a water bath to dissolve, cool, add methanol to the mark, and shake well. Accurately measure 10 mL and place it in a 100 mL volumetric flask, add water to the mark, and shake well to obtain a reference solution containing 0.2 mg of anhydrous rutin per mL.

[0039] Preparation of the standard curve: Accurately measure 1, 2, 3, 4, 5, and 6 mL of the reference solution and place them into 25 mL volumetric flasks. Add water to each flask to a final volume of 6 mL. Add 1 mL of 5% sodium nitrite solution, mix well, and let stand for 6 minutes. Then add 1 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes. Next, add 10 mL of sodium hydroxide solution, add distilled water to the mark, shake well, and let stand for 15 minutes. Using the corresponding reagent as a blank, measure the absorbance at 500 nm. Plot the standard curve with absorbance as the ordinate and concentration as the abscissa.

[0040] Determination of total flavonoid content in Artemisia annua extract: Accurately weigh 1.0 g of the obtained Artemisia annua extract sample and place it in a 100 mL volumetric flask. Add methanol to the mark and shake well. Accurately measure 10 mL and place it in a 100 mL volumetric flask. Add water to the mark and shake well. Accurately measure 3 mL and place it in a 25 mL volumetric flask. Add water to 6 mL. Add 1 mL of 5% sodium nitrite solution and mix well. Let stand for 6 minutes. Add 1 mL of 10% aluminum nitrate solution and shake well. Let stand for 6 minutes. Add 10 mL of sodium hydroxide solution and distilled water to the mark. Shake well and let stand for 15 minutes. Using the corresponding reagent as a blank, measure the absorbance at 500 nm. Read the amount of anhydrous rutin in the sample solution from the above standard curve. Calculate the total flavonoid content (calculated as anhydrous rutin) in the Artemisia annua extract sample. It should be in the range of 25% to 75%.

[0041] Example 2: The effect of total flavonoids from Artemisia scoparia on the behavioral improvement of mice with experimental autoimmune encephalomyelitis 2.1 Establishment of the mouse model of experimental autoimmune encephalomyelitis and drug administration

[0042] Experimental Principle

[0043] C57BL / 6 mice were treated with MOG 35-55 Induced EAE model.

[0044] Experimental methods

[0045] Female C57BL / 6 mice, 6-8 weeks old, weighing 16-18g, were subcutaneously injected with MOG after 3-5 days of acclimatization feeding. 35-55 An experimental autoimmune encephalomyelitis (EAE) animal model was established by intravenous injection of 300 μg of pertussis toxin and 400 ng of pertussis toxin. A normal control group was also set up. Eleven days after modeling, the animals began to show symptoms, exhibiting varying degrees of limb paralysis, confirming the successful establishment of the mouse model of experimental autoimmune encephalomyelitis.

[0046] Female C57BL / 6 mice were randomly divided into two groups after immunization: an EAE control group and a group treated with 600 mg / kg total flavonoids from Artemisia scoparia. After grouping, the mice were administered the total flavonoids once daily by gavage. The normal control group and the EAE model group received the same volume of solvent control (0.5% sodium carboxymethyl cellulose solution). Total flavonoids from Artemisia scoparia were administered continuously from the date of immunization until day 28 of modeling. Disease scores were measured daily, and animal mortality was recorded. Suspension grading was measured on days 7, 14, 21, and 28 post-immunization, and the suspension time on the wire mesh was measured on day 28.

[0047] Experimental results

[0048] The animals began to show symptoms on day 11 after immunization, including weight loss and decreased neurological function scores.

[0049] 2.2 Effects of total flavonoids from Artemisia annua on body weight in EAE mice

[0050] Experimental methods

[0051] Body weight is an important indicator of an animal's energy balance and growth. In this experiment, we observed and recorded the animals' activity level, coat condition, and other general characteristics daily, and monitored their body weight daily.

[0052] Experimental results

[0053] In this experiment, the body weight of mice in the EAE model group was significantly lower than that of the normal group after the onset of the disease. However, after drug administration, compared with the model group, the body weight of mice in the Artemisia scoparia total flavonoid treatment group increased in the later stage of drug administration. Results are shown below. Figure 1 .

[0054] 2.3 Effects of total flavonoids from Artemisia annua on disease scores in EAE mice

[0055] Experimental methods

[0056] After modeling, the experimental mice were scored daily using the following criteria: 0 points: normal mouse; 0.5 points: tail weakness; 1 point: complete tail paralysis; 1.5 points: weakness in one hind limb; 2 points: weakness in both hind limbs; 2.5 points: paralysis in one hind limb and weakness in the other hind limb; 3 points: paralysis in both hind limbs; 3.5 points: partial weakness in the forelimbs; 4 points: partial paralysis in the forelimbs; 4.5 points: complete paralysis in the forelimbs; 5 points: death.

[0057] Experimental results

[0058] In this experiment, the disease score of mice in the EAE model group was significantly higher than that of the normal control group after the onset of the disease. However, after drug administration, the disease score of mice in the Artemisia scoparia total flavonoid treatment group was significantly lower than that of the EAE model group in the later stage of drug administration. Results are shown below. Figure 2 .

[0059] 2.4 Effect of total flavonoids from Artemisia annua on suspension time in EAE mice

[0060] Experimental methods

[0061] On day 28 after modeling, the time it took for mice to fall from a 180° wire mesh was measured; a time greater than 120 seconds was counted as 120 seconds.

[0062] Experimental results

[0063] In this experiment, compared with the normal control group, the suspension time of mice in the EAE model group was significantly reduced, while total flavonoids from Artemisia annua could prolong the suspension time of the model mice. Results are shown below. Figure 3 And Table 1.

[0064] Table 1. Effects of total flavonoids from Artemisia annua on suspension time in MOG-induced EAE mice.

[0065]

[0066] Mean±SEM (n=6)

[0067] ### P < 0.001 vs. normal control group, ***P < 0.001 vs. EAE model group.

[0068] 2.5 Effects of total flavonoids from Artemisia annua on suspension grading in EAE mice

[0069] Experimental methods

[0070] On days 7, 14, 21, and 28 after modeling, the mice's forepaws were suspended from a balance rope 30 cm above the ground. The suspension status of the mice's limbs on the balance rope was observed and scored. The grading criteria were as follows: 5 points: Grasping the rope and able to pull with the hind limbs, with the tail tightly wrapped around the rope; 4 points: Grasping the rope and able to pull with the hind limbs, with the tail raised but not wrapped around the rope; 3 points: Grasping the rope and able to pull with the hind limbs, with the tail drooping; 2 points: Raising the hind limbs and grasping the rope but unable to pull; 1 point: Raising the hind limbs but unable to grasp the rope; 0 points: Unable to raise the hind limbs.

[0071] Experimental results

[0072] In this experiment, compared with the normal control group, the suspension scores of mice in the EAE model group were significantly lower after the onset of the disease. After drug administration, the suspension scores of the drug treatment group were significantly increased compared with the EAE model group, and this increase was time-dependent. Results are shown below. Figure 4 And Table 2.

[0073] Table 2. Effects of total flavonoids from Artemisia annua on suspension grading in MOG-induced EAE mice.

[0074]

[0075] Mean±SEM (n=6)

[0076] ### P < 0.001 vs. normal control group; *P < 0.05, ***P < 0.001 vs. EAE model group

[0077] Example 3: Effects of total flavonoids from Artemisia annua on inflammatory cell infiltration and demyelination in the spinal cord of EAE model mice 3.1 Establishment of EAE mouse model and drug administration

[0078] The experimental principle, experimental method, and experimental results are the same as in Example 2.1.

[0079] 3.2 Effects of total flavonoids from Artemisia annua on inflammatory cell infiltration in the spinal cord of EAE mice

[0080] Experimental methods

[0081] On day 28 after modeling, three mice from each group were anesthetized with 4% tribromoethanol. The mice were first perfused with physiological saline until the liver turned white, then perfused with 4% paraformaldehyde until the limbs stiffened. The mice were then decapitated, and the lumbar enlarged portion of the spinal cord was fixed in 4% paraformaldehyde. Paraffin sections of the lumbar enlarged portion of the spinal cord were prepared and stained with hematoxylin and eosin (H&E) to observe the infiltration of inflammatory cells in the spinal cord.

[0082] Experimental results

[0083] In this experiment, compared with the normal control group, the EAE model group mice showed significant infiltration of inflammatory cells in the spinal cord. Treatment with 600 mg / kg Artemisia scoparia total flavonoids significantly inhibited the infiltration of inflammatory cells in the spinal cord of EAE mice, and the pathological changes such as fibrosis and vacuolation caused by inflammatory infiltration were significantly improved, confirming that Artemisia scoparia total flavonoids can significantly improve inflammatory cell infiltration in the spinal cord of EAE mice. Results are shown below. Figure 5 .

[0084] 3.3 Effects of total flavonoids from Artemisia annua on demyelination in the spinal cord of EAE mice

[0085] Experimental methods

[0086] On day 28 after modeling, three mice from each group were anesthetized with 4% tribromoethanol. The mice were first perfused with physiological saline until the liver turned white, then perfused with 4% paraformaldehyde until the limbs stiffened. The mice were then decapitated, and the lumbar enlargement of the spinal cord was fixed in 4% paraformaldehyde. Paraffin sections of the lumbar enlargement of the spinal cord were prepared and stained with Fast Blue (LFB) to observe the demyelination within the spinal cord.

[0087] Experimental results

[0088] In this experiment, after LFB staining, compared with the normal control group, the spinal cord of EAE model mice showed demyelinating lesions with a larger area. 600 mg / kg total flavonoids from Artemisia annua could reduce the area of ​​white matter in the spinal cord of EAE mice, effectively improving the demyelination condition. Results are shown below. Figure 6 .

[0089] Example 4: The effect of total flavonoids from Artemisia annua on improving inflammation in LPS-induced microglia BV2 cells

[0090] 4.1 Establishment and drug administration of the LPS-induced BV2 cell inflammation model

[0091] Experimental methods

[0092] BV2 cells were cultured at 37°C, 5% CO2, and saturated humidity in DMEM high-glucose complete medium (Gibco) containing 10% fetal bovine serum (FBS). When the cells showed logarithmic growth, they were cultured at a rate of 2.5 × 10⁻⁶ cells / year. 4 Cells were seeded per well in 96-well plates, with 100 μL of cell suspension per well, and cultured overnight at 37°C in a 5% CO2 incubator. A normal control group, a model group, and a drug-treated group were set up. The supernatant was discarded and replaced with serum-free medium, 80 μL per well. Physiological saline containing DMSO was added to the normal control and model groups to bring the final DMSO concentration to 0.1%. The drug-treated group was added to a final concentration of 100 μg / mL of Artemisia scoparia total flavonoids. After incubation for 2 h, LPS (Yuan Ye, S11060) was added to each well of the model and drug-treated groups to bring the final concentration to 1 μg / mL. After 24 h of culture, 50 μL of the supernatant was collected to detect NO production, following the instructions of the NO detection kit (Pulley, E1030). (If the detection exceeds the upper limit of the standard curve or the microplate reader, the supernatant can be appropriately diluted before detection). After discarding the supernatant, cell viability was detected using the MTS method.

[0093] NO inhibition rate (%) = (NO 模型组 -NO 给药组 ) / (NO 模型组 -NO 对照组 )×100%

[0094] Experimental results:

[0095] The results are shown in Table 3.

[0096] Table 3. Inhibition rate of total flavonoids from Artemisia annua on NO production and cell viability in LPS-stimulated BV2 cells (n=4)

[0097]

Claims

1. Use of total flavonoids of Artemisia sphaerocephala Krasn. in the preparation of a medicament for preventing, alleviating and / or treating an autoimmune demyelinating disease.

2. Use according to claim 1, characterized in that, The autoimmune demyelinating disease includes a demyelinating autoimmune disease of the central nervous system and a demyelinating autoimmune disease affecting the peripheral nervous system.

3. Use according to claim 2, characterised in that, The demyelinating autoimmune disease of the central nervous system includes multiple sclerosis, neuromyelitis optica spectrum disease, acute disseminated encephalomyelitis, leukoencephalitis and transverse myelitis. The demyelinating autoimmune disease affecting the peripheral nervous system includes acute inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyneuropathy, anti-MAG peripheral neuropathy, motor and sensory neuropathy, hereditary sensorimotor neuropathy, Charcot-Marie-Tooth disease, progressive neural Charcot-Marie-Tooth disease.

4. Use according to claim 3, characterised in that, The multiple sclerosis includes relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis and progressive-relapsing multiple sclerosis.

5. The use according to claim 1, characterized in that, The flavonoid content in the total flavonoids of Artemisia sphaerocephala Krasn. is 25% to 75% by mass.

6. Use of a pharmaceutical composition in the manufacture of a medicament for preventing, alleviating and / or treating an autoimmune demyelinating disease, characterized in that, The pharmaceutical composition contains an effective dose of the total flavonoids of Artemisia sphaerocephala Krasn. and optionally a pharmaceutically acceptable pharmaceutical excipient.

7. Use according to claim 6, characterised in that, The pharmaceutical composition contains, in addition to the total flavonoids of Artemisia sphaerocephala Krasn. as the pharmaceutically active ingredient, other active ingredients.

8. Use according to any one of claims 6-7, characterised in that, The pharmaceutical composition includes the following dosage forms: solution, suspension, lyophilized powder injection, emulsion, pill, capsule, powder, controlled release, sustained release preparation and microsomal drug delivery system.

9. Use according to claim 8, characterised in that, The pharmaceutical excipient includes starch, dextrin, sodium polymethylcellulose, magnesium stearate, talc.

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