Pharmaceutical composition containing Scrophularia or its extract and use thereof

Abstract

The present invention provides a pharmaceutical composition containing Scrophularia or its extract and its use. Compared with the prescription composed of honeysuckle, Scrophularia, angelica and liquorice, the single drug or its active component of the present invention can effectively alleviate joint damage in acute gouty arthritis (AGA) model mice, collagen antibody-induced arthritis (CAIA) model mice and collagen-induced arthritis (CIA) model mice by regulating neutrophil activity, reducing neutrophil migration, and alleviating immune damage caused by inappropriately activated neutrophils. It further confirms that the new strategy of treating diseases related to abnormal neutrophil activation by targeting neutrophils is feasible.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically to a pharmaceutical composition containing Scrophularia ningpoensis or an extract thereof and uses thereof, in particular to uses thereof for regulating neutrophil activity, and in particular to uses thereof for treating diseases related to abnormal neutrophil activation by regulating neutrophil activity. Background Art

[0002] Neutrophils are the most abundant immune cell population in the circulatory system, accounting for 40-70% of all white blood cells in the human body. They are an important component of the innate immune system, playing roles in immune surveillance, pathogen clearance and immune regulation in the body. When the body is infected with bacteria, neutrophils are recruited to the site of infection through chemotaxis; then through their phagocytosis, degranulation, respiratory burst, neutrophil extracellular traps (NETs) release and other functions, pathogens are cleared, or pathogens are restricted to the local area to prevent further spread; neutrophils can also further recruit and activate other immune cells such as monocytes / macrophages and T cells by releasing inflammatory factors and chemokines, thereby comprehensively enhancing the level of anti-infection immunity. However, once the pathogen breaks through the neutrophil barrier and develops into systemic sepsis, the excessive activation of neutrophils will release a large number of inflammatory factors, causing an inflammatory factor storm and inhibiting T cell activation, which will lead to multiple organ failure and death in patients. In recent years, studies have found that the abnormal activation of neutrophils in the local area of ​​the body also promotes the progression of related aseptic diseases such as rheumatoid arthritis (RA) and acute gouty arthritis (AGA).

[0003] Rheumatoid arthritis is a common chronic systemic autoimmune disease with clinical characteristics of persistent, symmetrical small joint lesions. Classical theory holds that rheumatoid arthritis is an autoimmune disease driven by acquired immune cells such as T cells and B cells. However, abnormal activation of neutrophils also plays a role in various stages of the development of rheumatoid arthritis: ① The cells can release NETs, ​​and the citrullinated histones carried on them are one of the main autoantigens that stimulate the formation of immune complexes; ② The cells can promote the differentiation and maturation of Th17 cells by releasing chemokines, leading to a vicious cycle of inflammatory immunity; ③ The cells act as effector cells and directly cause aseptic inflammatory damage to the bones and joints during the active stage of the disease. The first-line treatment of rheumatoid arthritis includes conventional anti-inflammatory drugs and biological disease-modifying therapies, which are mainly used to control pain and inflammation, but rarely achieve sustained relief.

[0004] Acute gouty arthritis (AGA) is a recurrent inflammatory disease caused by the deposition of monosodium urate (MSU) crystals in the synovium, bursa, cartilage and other tissues due to abnormal purine metabolism and elevated blood uric acid levels. The main manifestations are recurrent joint swelling, heat, pain and dysfunction, even joint deformity, uric acid nephropathy, etc. The deposited MSU crystals activate monocytes / macrophages, and then chemotactic neutrophils to infiltrate into the synovial fluid and synovium in large quantities, and an explosive cascade amplification reaction occurs. The infiltrated neutrophils secrete a large number of cytokines such as IL-1 and IL-8, as well as lysosomal enzymes, oxygen free radicals, arachidic acid substances, and bone marrow-related proteins S100A8 / A9, which promote the progression of acute gout inflammation. Patients with acute gouty arthritis can take allopurinol, which reduces uric acid synthesis, and probenecid, which promotes urate excretion, for a long time to prevent acute attacks of arthritis; non-steroidal anti-inflammatory drugs, glucocorticoids and colchicine are used to improve symptoms to varying degrees.

[0005] Scrophulariae is sweet, bitter and salty in taste, slightly cold in nature, and belongs to the lung, stomach and kidney meridians; it has the effects of clearing heat and cooling blood, nourishing yin and purging fire, and is mainly used to treat febrile diseases with heat entering the blood. It is clinically used to treat febrile diseases that damage yin, red tongue and thirst, fever and rashes, constipation caused by body fluid damage, bone steaming and cough, red eyes, sore throat, scrofula, diphtheria, carbuncle, sore and poisonous. Modern pharmacological studies have shown that Scrophulariae has pharmacological activities such as anti-inflammatory, antioxidant, liver protection, anti-tumor, anti-fatigue, uric acid reduction, anti-depression, and protection of myocardial cells, and has a regulatory effect on the cardiovascular system, central nervous system and immune system. It is used to treat fever, swelling, constipation, pharyngitis, laryngitis, neuritis, sore throat, rheumatism and other diseases. However, there is currently no report on Scrophulariae regulating neutrophil activity, especially treating related diseases by regulating neutrophil activity.

[0006] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0007] The inventor is committed to the research of modernization of traditional Chinese medicine. After extensive in-depth research, it is found that the prescription composed of honeysuckle, Scrophularia, Chinese angelica and liquorice can achieve effective treatment of joint inflammation related to abnormal neutrophil activation by regulating the activity of neutrophils. On this basis, the inventor further conducted in-depth research and unexpectedly found that compared with the compound prescription, when Scrophularia is used as a single drug, its effect is better, which is contrary to the general cognition of traditional theory. The present invention is completed at least in part based on this discovery. Specifically, the present invention includes the following contents.

[0008] In a first aspect of the present invention, a pharmaceutical composition is provided for regulating neutrophil activity, thereby preventing and treating diseases or illnesses, wherein the pharmaceutical composition comprises Scrophularia ningpoensis or an extract thereof.

[0009] In certain embodiments, according to the pharmaceutical composition of the present invention, the extract comprises a water extract and / or an alcohol extract of Scrophularia ningpoensis.

[0010] In certain embodiments, according to the pharmaceutical composition of the present invention, the extract comprises Scrophularia component 1: alcohol-soluble small molecules and Scrophularia component 2: Scrophularia polysaccharide.

[0011] In certain embodiments, according to the pharmaceutical composition of the present invention, the preparation of the alcohol-soluble small molecules of Scrophularia component 1 includes: adding alcohol to the water extract of Scrophularia and taking the supernatant to obtain the small molecules; the preparation of the polysaccharide of Scrophularia component 2 includes: subjecting Scrophularia to alcohol precipitation and water dissolution to obtain the small molecules.

[0012] The preparation method of the Scrophularia ningpo water extract is not limited. For example, the Scrophularia ningpo powder can be ultrasonically extracted in water to obtain a filtrate, and the supernatant can be obtained by centrifugation; or the Scrophularia ningpo can be decocted in conventional water to obtain a decoction as the water extract.

[0013] In certain embodiments, according to the pharmaceutical composition of the present invention, the temperature of the Scrophularia ningpoensis powder during ultrasound treatment in water is 40-70°C.

[0014] The second aspect of the present invention provides the use of Scrophularia ningpoensis or an extract thereof in the preparation of a medicament for regulating neutrophil activity.

[0015] In certain embodiments, according to the use of the present invention, wherein the regulating the activity of neutrophils comprises at least one of the following:

[0016] (1) Inhibit, delay, postpone, reduce, diminish or attenuate neutrophil chemotaxis;

[0017] (2) Inhibit, delay, postpone, reduce or minimize the formation of neutrophil extracellular traps (NETs).

[0018] (3) inhibit, delay, postpone, reduce, diminish or attenuate neutrophil inflammatory factors;

[0019] The third aspect of the present invention provides use of Scrophularia ningpoensis or an extract thereof in the preparation of a medicament for a disease or condition associated with abnormal neutrophil activation.

[0020] In certain embodiments, according to the use of the present invention, the diseases or conditions associated with abnormal neutrophil activation include arthritis, sepsis, etc.;

[0021] Preferably, the arthritis comprises acute gouty arthritis, chronic arthritis, persistent symmetrical polyarthritis and / or rheumatoid arthritis.

[0022] A fourth aspect of the present invention provides a method for regulating the activity of neutrophils in vitro, comprising the step of contacting the neutrophils with Scrophularia ningpoensis or an extract thereof.

[0023] In the research of the present invention, various models such as the collagen-induced arthritis (CIA) mouse model were established to observe the efficacy of the formula and single drugs. It was found that compared with the formula, the single drug Scrophularia ningpoensis was more effective in treating diseases or conditions related to abnormal neutrophil activation by regulating neutrophil activity. Such an effect was completely unexpected.

[0024] The present invention prepares a single drug and its active components capable of treating inflammatory diseases, and experiments have shown that the single drug can inhibit, reduce, decrease or weaken the chemotaxis of neutrophils, inflammatory factors, or inhibit, reduce or reduce the formation of neutrophil extracellular traps NETs, ​​thereby treating rheumatoid arthritis or acute gouty arthritis.

[0025] The single-drug Scrophularia ningpoensis obtained by the present invention or the active components obtained by extraction can effectively alleviate the damage of collagen-induced arthritis (CIA) model mice, collagen antibody-induced arthritis (CAIA) model mice and acute gouty arthritis (AGA) model mice by regulating the activity of neutrophils, reduce the migration of neutrophils to damaged joints, and alleviate the immune damage caused by inappropriately activated neutrophils. The present invention confirms that a new strategy for treating diseases or conditions related to abnormal neutrophil activation by targeting neutrophils is feasible. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of in vitro inhibition of neutrophil chemotaxis by drug serum containing the formula of the present application and Scrophularia ningpoensis, Scrophularia ningpoensis active component 1 (prepared by water extraction and alcohol dissolution process), and Scrophularia ningpoensis active component 2 (prepared by water extraction and alcohol precipitation process).

[0027] Figure 2 The results of the present application's formula and Scrophularia, Scrophularia active component 1 (prepared by water extraction and alcohol dissolution process), and Scrophularia active component 2 (prepared by water extraction and alcohol precipitation process) in alleviating the symptoms of redness and swelling of the joints of CIA mice are shown.

[0028] Figure 3 The pathological changes in the joints of CIA mice after administration of the formula of the present application and Scrophularia ningpoensis, Scrophularia ningpoensis active component 1 (prepared by water extraction and alcohol dissolution process), and Scrophularia ningpoensis active component 2 (prepared by water extraction and alcohol precipitation process) are shown.

[0029] Figure 4 Micro-CT scan reconstructed images of the joint bones of CIA mice after administration of the formula of the present application and Scrophularia ningpoensis, Scrophularia ningpoensis active component 1 (prepared by water extraction and alcohol dissolution process), and Scrophularia ningpoensis active component 2 (prepared by water extraction and alcohol precipitation process).

[0030] Figure 5 This is the mechanism of action of Scrophularia ningpoensis in diseases related to abnormal neutrophil activation. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] As used herein, the term "preventing or treating" refers to ameliorating a disease or disorder before or after it occurs, by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as compared to an untreated control group under the same conditions, as measured by any standard technique.

[0035] The drug of the present invention may optionally contain a pharmaceutically acceptable excipient, carrier or diluent. The term "pharmaceutically acceptable excipient, carrier or diluent" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, and such pharmaceutically acceptable material, composition or carrier participates in carrying or transporting the drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable", which means that it is compatible with the other ingredients of the formulation and does not harm the patient. Some examples of pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives and analogs such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl lauryl ester; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic and compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyoxyethylene-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0036] There is no particular limitation on the administration method of the drug of the present application. Representative administration methods include but are not limited to: oral administration, intramuscular injection, intravenous injection, intravenous drip, enema, spray, external application, or intraperitoneal injection.

[0037] Solid dosage forms for oral administration include tablets, pills, powders, granules, or capsules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0038] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of such drugs or compositions or active ingredients can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the composition, drug, single drug or active ingredient can also be formed into microcapsules with one or more of the above-mentioned excipients.

[0039] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0040] In the present application, the medicament for parenteral injection may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, and a sterile powder for re-dissolving into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0041] In the present application, the dosage forms of the drug for topical administration include ointments, powders, patches, sprays and inhalants. The composition, drug, single drug or active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

[0042] The drug of the present application can be administered alone or in combination with other therapeutic agents. Examples of other therapeutic agents include, but are not limited to, small molecule compounds or drugs that can be used for inflammatory diseases.

[0043] When using the drug of the present application, a safe and effective amount of the drug or its active ingredient is administered to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0044] Example 1

[0045] 1. Experimental steps

[0046] 1. Preparation of Chinese medicine

[0047] 1.1 The preparation of the decoction of the formula is as follows:

[0048] Honeysuckle 90g, Scrophularia 90g, Angelica 60g, Licorice 30g, 270g in total for ten doses. Conventional method: decoct twice, combine the liquid, filter, and concentrate to 600ml (0.45g / ml).

[0049] 1.2 The preparation of Scrophularia ningpoensis water extract is as follows:

[0050] The Scrophularia ningpoensis is crushed, and the Scrophularia ningpoensis powder is dissolved in water under ultrasound at 40-70°C, filtered, and centrifuged to obtain the supernatant to obtain the Scrophularia ningpoensis water extract; or the Scrophularia ningpoensis is soaked in water for 10-90 minutes, boiled and then simmered for 10-60 minutes, the liquid is poured out, and the residue is added with appropriate amount of water and simmered for 10-30 minutes, the two decoctions are combined, and filtered to obtain the Scrophularia ningpoensis water extract.

[0051] 1.3 The preparation of Scrophularia ningpoensis component 1 is as follows:

[0052] The Scrophularia ningpoensis is crushed, dissolved in water under ultrasound, filtered, and the supernatant is collected after alcohol precipitation, and concentrated under pressure and heating to obtain an alcohol-soluble small molecule, namely the Scrophularia ningpoensis active component 1. Or the Scrophularia ningpoensis water decoction is precipitated with alcohol, and the supernatant is collected, and concentrated under pressure and heating to obtain an alcohol-soluble small molecule, namely the Scrophularia ningpoensis active component 1.

[0053] 1.4 The preparation of Scrophularia ningpoensis component 2 is as follows:

[0054] The Scrophularia ningpoensis is crushed, dissolved in water under ultrasound, filtered, precipitated with alcohol, and precipitated, evaporated and concentrated in a water bath to obtain a crude polysaccharide, which is the active component 2 of Scrophularia ningpoensis. Alternatively, the decoction of Scrophularia ningpoensis is precipitated with alcohol, and the precipitate is collected and concentrated to obtain a crude polysaccharide, which is the active component 2 of Scrophularia ningpoensis.

[0055] 2. Preparation of drug-containing serum

[0056] The SD rats in the drug-containing serum group were intragastrically administered with a dose of 6 times the clinical equivalent dose. The doses of the Scrophularia, Scrophularia component 1, and Scrophularia component 2 drug-containing serum groups were all converted to the same Scrophularia dose in the formula. The rats in the blank serum group were intragastrically administered with normal saline. The drug was administered once every 12 hours for a total of 5 times. Blood was collected 1 hour after the last administration to separate the serum and inactivate it.

[0057] 3. Neutrophil Chemotaxis Assay

[0058] The bone marrow single cell suspension was incubated in the culture medium containing control serum and drug-containing serum for 1 h, and then incubated at 3*10 4 The cells in each well were inoculated in the chemotaxis chamber, and the lower chamber was cultured with chemokine culture medium, and cultured in a 37°C incubator for 3 hours. After the culture, the cells in the lower chamber were collected in a flow cytometer, and antibodies were added to label neutrophils. Before loading the tube, counting microspheres were added, and the number of neutrophils chemotactic to the lower chamber was analyzed by flow cytometry.

[0059] 4. Detection of NETs Formed by Activated Neutrophils

[0060] Determination of cf-DNA content in cell culture supernatant:

[0061] Neutrophils isolated from bone marrow 10 5 / well was inoculated in RPMI-1640 medium containing control serum and drug-containing serum of the formula, incubated for 1 hour; PMA was added for stimulation for 3 hours; DNase I was added to cut and interrupt NETs attached to the cells. After the reaction was completed, EDTA was added to terminate the enzyme reaction; the culture supernatant of neutrophils was collected, and the PicoGreen kit was used to detect the cf-DNA content in the culture supernatant of neutrophils.

[0062] 2. Experimental Results

[0063] 1. Inhibitory effects of the formula, Scrophularia ningpoensis, and serum containing Scrophularia ningpoensis fraction 1 and fraction 2 on neutrophil migration

[0064] According to previous experiments, 20% dose of drug-containing serum intervention is the best dosage concentration for in vitro experiments. The results of in vitro neutrophil chemotaxis experiments showed that after stimulation with CXCL1 and CXCL2, more neutrophils chemotaxis to the lower chamber compared with the control group. The drug-containing serum of the formula and the drug-containing serum of Scrophularia component 2 effectively inhibited neutrophil chemotaxis induced by CXCL1, and the drug-containing serum of Scrophularia component 1 and the drug-containing serum of Scrophularia component 1 could significantly inhibit neutrophil migration induced by CXCL1 and CXCL2 ( Figure 1 , Table 1).

[0065] Table 1. Effects of the formula, Scrophularia, Scrophularia fraction 1 and Scrophularia fraction 2 containing serum on CXCL1 and CXCL2 chemotactic granulocytes (MEAN ± SEM)

[0066]

[0067]

[0068] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0069] 2. Formula, Scrophularia, and serum containing Scrophularia fraction 1 and fraction 2 inhibit neutrophil NETs formation

[0070] After activation, neutrophils release extracellular trapping nets NETs. The release of proteases in NETs can cause tissue damage and aggravate the inflammatory response. NETs are released to the periphery, and the release of neutrophil NETs can be reflected by detecting the content of free DNA (cf-DNA). The experimental results showed that after PMA stimulation, the cf-DNA content in the neutrophil culture supernatant was significantly increased compared with the control group. The drug-containing serum of the formula, the single drug Scrophularia ningpoensis drug-containing serum, and the drug-containing serum of Scrophularia ningpoensis component 1 and component 2 all significantly reduced the release of neutrophil cf-DNA and inhibited the formation of NETs. (Table 2).

[0071] Table 2. Effects of formula, Scrophularia, and serum containing Scrophularia components on neutrophil cf-DNA (ng / mL) (MEAN±SEM)

[0072]

[0073] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0074] Example 2

[0075] 1. Experimental steps

[0076] 1. Establishment of animal model

[0077] 1.1 Establishment of collagen-induced arthritis (CIA) mouse model

[0078] Bovine type II collagen and complete Freund's adjuvant were fully emulsified in an ice-water bath at a ratio of 1:1, and the final concentration of type II collagen was 1 mg / mL, with no diffusion when added to water. Incomplete Freund's adjuvant was used for the secondary immunization. DBA / 1 mice were intradermally injected with emulsified collagen (100 μL / mouse) at the base of the tail on days 0 and 21, respectively, to induce the establishment of a CIA mouse model. Drug administration began 2 weeks after the first immunization, and the doses administered were all clinically equivalent doses. All animals were sampled after the arthritis index score began to decrease. After sampling, ELISA was used to detect plasma inflammatory factors, cf-DNA content, and joint inflammatory factor levels; real-time fluorescence quantitative PCR was used to detect the expression of neutrophil-related chemokines in the joints; flow cytometry was used to detect changes in the proportion of neutrophils; pathological section staining and Micro-CT were used to detect pathological damage and bone destruction in the mouse joints.

[0079] 1.2 Establishment of collagen antibody-induced arthritis (CAIA) mouse model

[0080] Female Balb / c mice aged 7-8 weeks were selected. Animal model establishment: Day 0: 5 clone antibody cocktail 150uL / mouse was injected into the tail vein, and day 3: LPS 70uL / mouse was injected intraperitoneally. Drug administration began after tail vein injection of antibodies, and the dosage was clinically equivalent. After modeling, the joints of mice were scored and the swelling was measured every day. The joint score and swelling began to decrease, and the samples were collected after the inflammation reached its peak.

[0081] 1.3 Establishment of acute gouty arthritis (AGA) mouse model

[0082] Grind the MSU crystals, pass them through a 40 μm sieve, and prepare an MSU crystal suspension (50 mg mL -1 ), a 0.3mL syringe with a 30-gauge needle was used to vertically puncture the ankle joint cavity along the lateral and posterior side of the right ankle joint of the mouse, and 30μL of MSU crystal suspension was injected; the control group was injected with an equal volume of saline in the same way. After modeling, a single dose was given, and the doses were all clinically equivalent. The changes in ankle joint swelling were measured within 72 hours after modeling. The AGA model was established in the same way in subsequent experiments, and samples were taken at the peak of swelling at 15 hours to detect the expression of joint inflammatory factors.

[0083] 2. Observation and testing indicators

[0084] 2.1 Symptoms, scores and swelling of mouse joints

[0085] After modeling, the joint redness and swelling symptoms of the animals were observed and recorded. Joint index score: 0 points: normal, no redness and swelling of the joints; 1 point: mild redness and swelling of the toe joints; 2 points: swelling of the toe joints and the soles of the feet; 3 points: redness and swelling of the paws below the ankles; 4 points: redness and swelling of the entire foot including the ankle joints with functional impairment; AI = the sum of the scores of the joints of the four limbs, the total score is 16 points. Scoring was done every 3 days. The diameter of the wrist and ankle joints of the mice was measured with a vernier caliper, and the swelling degree was calculated. The swelling degree = measured diameter - initial diameter, and the joint swelling degree = the sum of the swelling degrees of the joints of the four limbs.

[0086] 2.2 Joint histopathology

[0087] HE staining was used to observe the inflammatory infiltration of the joints, Safranin-O / Fast Green staining was used to observe the cartilage destruction, and Trap staining was used to observe the osteoclast formation.

[0088] 2.3 Imaging

[0089] Micro-CT scanning of the mouse ankle joint was performed and three-dimensional reconstruction imaging was performed to evaluate bone destruction.

[0090] 2.4 Cytokine expression in joint tissue and cytokine content in plasma

[0091] Real-time fluorescence quantitative PCR was used to detect the expression of cytokines in joint tissues. Total RNA was extracted using Trizol. -ΔΔ The relative expression of genes was calculated by CT method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal reference. Total joint protein was extracted, and the level of joint inflammatory factors in mice was detected by ELISA. Blood was collected from mice to separate plasma, and the levels of various cytokines in plasma were detected by CBA kit.

[0092] 2.5 Plasma cf-DNA Levels

[0093] Blood was collected from the mouse eyeballs, plasma was separated, and the cf-DNA level in plasma was detected using the PicoGreen kit.

[0094] 2.5 Neutrophil ratio in mouse spleen and bone marrow

[0095] Mouse spleen was ground to prepare single cell suspension, and femur was rinsed with PBS to prepare bone marrow single cell suspension. Neutrophils were labeled with anti-mouse CD11b-PercpCy5.5, Ly6G-BV510, and CD48-PE Cy7 antibodies, and the changes in the proportion of neutrophils in the spleen and bone marrow of CIA mice were detected by flow cytometry.

[0096] 2. Experimental Results

[0097] 1. Effects of the present application's formula, single-drug Scrophularia ningpoensis, and components isolated from Scrophularia ningpoensis on CIA mice

[0098] 1.1 The formula of this application is effective in CIA mice, and the efficacy of single-drug Scrophularia and Scrophularia component 1 is better than the formula

[0099] Tables 3 and 4 show the changes in joint scores and swelling in CIA mice. On the third day after the second immunization, the model mice began to have redness and swelling in the joints, and the joint scores continued to increase. The redness and swelling of the joints basically reached the peak on the 18th day after the second immunization. The joint swelling was maintained until the 27th day and then began to alleviate, and the joint scores decreased. The changes in joint swelling were basically consistent with the scores. Compared with the model group, the present application formula and the single-drug Scrophularia, Scrophularia component 1, and component 2 significantly reduced the degree of joint swelling in CIA mice and reduced the joint scores, and the effects of Scrophularia and Scrophularia component 1 were better than those of the formula administration group.

[0100] Table 3. Effects of the formula, Scrophularia ningpoensis decoction, Scrophularia ningpoensis fraction 1, and Scrophularia ningpoensis fraction 2 on joint scores of CIA mice at different time points after secondary immunization (MEAN±SEM)

[0101]

[0102]

[0103] Note: #There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0104] Table 4. Effects of the formula, Scrophularia ningpoensis decoction, Scrophularia ningpoensis fraction 1, and Scrophularia ningpoensis fraction 2 on joint swelling (mm) at different time points after secondary immunization of CIA mice (MEAN±SEM)

[0105]

[0106]

[0107] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0108] 1.2 The present application's formula, single-drug Scrophularia and Scrophularia components reduce joint pathological changes and bone destruction in CIA mice

[0109] The results of histopathological staining showed that the ankle joints of CIA mice were infiltrated with obvious inflammatory cells, and there was destruction of the articular cartilage structure and a large number of osteoclasts. The formula of this application, both the single drug Scrophularia and the Scrophularia components significantly improved the pathological damage of joint tissues ( Figure 2 ).

[0110] Micro-CT results showed that there was obvious bone destruction in the ankle joints of CIA mice. The degree of bone destruction in mice in the application formula, single-drug Scrophularia and Scrophularia component administration groups was significantly reduced, and the Scrophularia component improved the joint symptoms, pathology and bone damage of CIA mice more significantly than the positive drug methotrexate, the formula and single-drug Scrophularia groups ( Figure 3 ).

[0111] 1.3 The present application's formula, single-drug Scrophularia and Scrophularia components reduce systemic and joint inflammation in CIA mice

[0112] The levels of cytokines in the plasma of CIA mice were detected. The results are shown in Table 5. The levels of TNF-α, IL-6, IFN-γ and MCP-1 in the plasma of CIA model group mice were significantly increased, and the administration of Scrophularia significantly reduced the levels of IL-6 and MCP-1. The plasma IL-6 level of Scrophularia component 1 group was significantly lower than that of the model group, and the plasma MCP-1 level of Scrophularia component 2 group was significantly lower than that of the model group.

[0113] The results of the levels of inflammatory factors in joint tissues are shown in Table 6. Compared with the control group, the levels of TNF-α, IL-1β and IL-6 in the joints of the mice in the model group were significantly increased. The present application's formula and single-drug Scrophularia effectively reduce the levels of IL-1β and IL-6 in the joints. After administration of Scrophularia component 1, the levels of TNF-α and IL-6 were significantly reduced, and Scrophularia component 2 effectively reduced the level of IL-6 in the inflamed joints.

[0114] Table 5. Effects of the formula, Scrophularia ningpoensis decoction, and Scrophularia ningpoensis components on the plasma cytokine levels (pg / mL) in CIA mice (MEAN±SEM)

[0115]

[0116]

[0117] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0118] Table 6. Effects of the formula, Scrophularia ningpoensis decoction, and Scrophularia ningpoensis components on the levels of inflammatory factors (pg / ug) in joint bone tissue of CIA mice (MEAN±SEM)

[0119]

[0120] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0121] 1.4 The formula of this application, single drug Scrophularia and Scrophularia components reduce plasma cf-DNA in CIA mice

[0122] Table 7 shows the plasma cf-DNA level of CIA mice. Compared with normal mice, the plasma cf-DNA level of CIA model mice was significantly increased, and the formula of the present application and the single drug Scrophularia ningpoensis and Scrophularia ningpoensis component 2 significantly reduced the plasma cf-DNA level of CIA mice.

[0123] Table 7. Effects of the formula, Scrophularia ningpoensis decoction, and Scrophularia ningpoensis components on plasma cf-DNA (ng / mL) in CIA mice (MEAN±SEM)

[0124]

[0125] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0126] 1.5 The present application's formula, single-drug Scrophularia ningpoensis, and Scrophularia ningpoensis components reduce the spleen index of CIA mice and reduce neutrophils

[0127] After sampling, the spleen of the mice was taken, and the changes in the spleen index were analyzed. The results are shown in Table 8. The spleen of mice in the CIA model group was enlarged, and the spleen index increased significantly. The spleen index decreased after administration. The changes in the proportion of neutrophils in the spleen and bone marrow were further analyzed. As shown in Table 9, the proportion of neutrophils in the bone marrow and spleen of mice in the CIA model group increased significantly. The prescription of this application reduced the proportion of neutrophils in the spleen of CIA mice, and the proportion of neutrophils in the bone marrow and spleen of Scrophularia 1 was significantly reduced, and the proportion of neutrophils in the bone marrow was reduced by component 2. It is suggested that the prescription of this application and the separated components of Scrophularia can effectively regulate the excessive production of neutrophils in CIA mice under inflammatory conditions.

[0128] Table 8. Effects of the formula, Scrophularia ningpoensis decoction, and Scrophularia ningpoensis components on the spleen index (%) of CIA mice (MEAN±SEM)

[0129]

[0130]

[0131] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0132] Table 9. Effects of the formula, Scrophularia ningpoensis decoction, and Scrophularia ningpoensis components on the proportion of neutrophils (%) in the bone marrow and spleen of CIA mice (MEAN±SEM)

[0133]

[0134] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0135] 2. Effects of the present application's formula and single-drug Scrophularia ningpoensis on CAIA mice

[0136] 2.1 The present application's formula and single drug Scrophularia ningpoensis reduce joint swelling in CAIA mice

[0137] As shown in Table 10, after the antibody was injected into the tail vein, the mice in the CAIA model group began to have joint swelling on the 3rd day. After the intraperitoneal injection of LPS for booster immunization, the degree of joint swelling increased significantly, and the joint score increased significantly. The joint swelling basically reached the peak on the 10th day, and then the degree of joint swelling decreased, and the score decreased. Compared with the model group, the present application formula and the single drug Scrophularia significantly reduced the degree of joint swelling in CAIA mice and reduced the joint score, and the early inhibition of joint swelling by Scrophularia was more obvious than methotrexate and the formula.

[0138] Table 10. Effects of the present application formula and Scrophularia on joint scores at different time points after CAIA mouse modeling (MEAN±SEM)

[0139]

[0140] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0141] 2.2 The present application's formula and single-drug Scrophularia inhibit the expression of joint inflammatory factors and neutrophil chemokines in CAIA mice

[0142] The results of real-time quantitative PCR showed (Tables 11-12) that the local inflammatory factors TNF-α, IL-1β, and chemokines CXCL2 and IL-8 in the joints of CAIA model mice were significantly increased. The compound prescription of the present application and the single drug Scrophularia ningpoensis reduced the mRNA levels of inflammatory factors and chemokines to varying degrees.

[0143] Table 11. Effects of the present application formula and Scrophularia on the mRNA levels of joint inflammatory factors in CAIA mice (MEAN±SEM)

[0144]

[0145]

[0146] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0147] Table 12. Effects of the present application formula and Scrophularia on the mRNA levels of chemokines in the joints of CAIA mice (MEAN±SEM)

[0148]

[0149] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0150] 3. Effects of the present application's formula and single-drug Scrophularia ningpoensis on AGA mice

[0151] As shown in Table 13, compared with the control group, the ankle swelling of the mice in the AGA model group was significantly increased, and there were statistical differences between the drug-treated groups and the model group at 24h, 48h, and 72h.

[0152] According to the previous experimental results of the research group, samples were taken at the peak of swelling at 15h of AGA to detect the expression of joint inflammatory factors and chemokines, and the results are shown in Table 5. The mRNA levels of inflammatory factors TNF-α, IL-1β, IL-6 and neutrophil chemotactic factor IL-8 in the joints of the AGA model at 15h were significantly increased, and each drug-treated group reduced the expression of inflammatory factors and chemokines in the joints to varying degrees.

[0153] Table 13. Effects of the present application formula and Scrophularia ningpoensis on joint swelling (mm) at different time points after AGA mouse modeling (MEAN±SEM)

[0154]

[0155]

[0156] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0157] Table 14. Effects of the present application formula and single herb Scrophularia on the mRNA levels of joint inflammatory factors in AGA mice (MEAN±SEM)

[0158]

[0159] Note: # There was a statistically significant difference between the model group and the control group. # , p<0.05; ## , p<0.01; ### , p<0.001. * indicates that there is a statistical difference between the drug-treated group and the model group, *, p<0.05; **, p<0.01; ***, p<0.001.

[0160] From the results of neutrophil chemotaxis in vitro, the drug-containing serum of the present application formulation effectively inhibited the neutrophil chemotaxis induced by CXCL1, and the single drug Scrophularia and Scrophularia active component 1 effectively inhibited the neutrophil chemotaxis induced by CXCL1 and CXCL2.

[0161] The cf-DNA in the culture supernatant after neutrophil activation was detected. The drug-containing serum of the formulation of this application, the drug-containing serum of single-drug Scrophularia ningpoensis, and the drug-containing serum of active components of Scrophularia ningpoensis all significantly reduced the release of cf-DNA from neutrophils and inhibited the formation of NETs.

[0162] From the results of joint score and swelling, the present application formula, Scrophularia, Scrophularia active component 1 and Scrophularia active component 2 are all effective for CIA mouse arthritis, among which Scrophularia active component 1 has the best effect compared with other administration groups, and significantly inhibits the onset of CIA mouse joints in the early stage. The spleen was taken to calculate the change of spleen index. After modeling, the spleen index of CIA model mice increased significantly, and each administration group significantly reduced the spleen index.

[0163] Flow cytometry analysis was performed on the proportion of neutrophils in the bone marrow and spleen of CIA mice. The proportion of neutrophils in CIA mice increased, and the active component 1 of Scrophularia ningpoensis effectively reduced the proportion of neutrophils in the bone marrow and spleen of CIA mice.

[0164] At the same time, using two arthritis models of AGA and CAIA mice, the formula of the present application and Scrophularia ningpoensis both significantly alleviated arthritis in AGA mice; and the effect of Scrophularia ningpoensis alone in CAIA mice was better than that of the formula of the present application.

[0165] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. Use of Scrophulariaceae polysaccharide as a single drug in the preparation of a drug for preventing and treating a disease or condition, characterized in that: The disease or condition is rheumatoid arthritis associated with the formation of neutrophil NETs; wherein, the processing of the Scrophularia ningpo polysaccharide includes ultrasonically extracting Scrophularia ningpo powder in 40-70°C water to obtain a filtrate, centrifuging to obtain a supernatant to obtain a water extract, or boiling Scrophularia ningpo in conventional water to obtain a water decoction as a water extract, subjecting the water extract to alcohol precipitation, and dissolving the precipitate in water to obtain the polysaccharide.

2. The use according to claim 1, characterized in that The process further comprises the step of concentrating the supernatant by applying pressure and heating.

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