Use of a traditional Chinese medicine composition in the preparation of a drug for treating chronic obstructive pulmonary disease

Abstract

The application relates to an application of a traditional Chinese medicine composition in a drug for chronic obstructive pulmonary disease, characterized in that the traditional Chinese medicine composition comprises the following components in parts: Magnolia officinalis 1-100 parts, Strychnos ignatii 1-100 parts, Toddalia asiatica 1-100 parts, Ephedra 1-100 parts, Amygdalus 1-100 parts, Notopterygium 1-100 parts, Zingiber officinale 1-100 parts, Pogostemonis 1-100 parts, Perilla 1-100 parts, Atractylodes 1-100 parts, Poria cocos 1-160 parts, Atractylodes macrocephala 1-120 parts, Gypsum fibrosum 1-100 parts, Fructus Crataegi 1-100 parts, Fructus Junci 1-100 parts, Fructus Hordei 1-100 parts, Lumbricus 1-100 parts, Radix et Rhizoma Veratri 1-100 parts, Herba Eupatorii Fortunei 1-100 parts, Semen Lepidii 1-100 parts and Semen Platycladi 1-100 parts.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology and relates to a new use of a traditional Chinese medicine composition, specifically the use of a traditional Chinese medicine composition in the preparation of drugs for treating chronic obstructive pulmonary disease, upper respiratory tract infection, autoimmune hepatitis, ulcerative colitis, chronic nephritis, rheumatoid arthritis, Sjögren's syndrome, vasculitis, or allergic purpura. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a destructive lung disease characterized by incompletely reversible airflow limitation. This airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to harmful particles or gases. COPD is a preventable and treatable chronic inflammatory airway disease. Although COPD is an airway disease, its systemic effects should not be ignored. The main symptoms include chronic cough, sputum production, shortness of breath or dyspnea, wheezing, and chest tightness. Incompletely reversible airflow limitation is a prerequisite for the diagnosis of COPD. An FEV1 / FVC ratio <70% and an FEV1 <80% of predicted value after inhalation of bronchodilators confirm incompletely reversible airflow limitation. A small number of patients may not have cough or sputum production symptoms, but only have an FEV1 / FVC ratio <70% and an FEV1 ≥80% of predicted value on pulmonary function tests; in such cases, after ruling out other diseases, COPD can also be diagnosed. Emphysema refers to a pathological state characterized by decreased elasticity of the airways distal to the terminal bronchioles, excessive inflation, increased lung volume, or simultaneous airway wall destruction. Based on its etiology, emphysema can be classified into several types: senile emphysema, compensatory emphysema, interstitial emphysema, focal emphysema, paraseptal emphysema, and obstructive emphysema. In Traditional Chinese Medicine (TCM), it is called "lung distension." External wind-cold and wind-heat are important factors inducing acute exacerbations of this disease. External wind-heat and wind-dryness can cause impaired lung function, leading to phlegm-heat accumulation in the lungs. Its pathogenesis involves qi stagnation, phlegm obstructing the airways, and phlegm and blood stasis, representing a deficiency in the root and an excess in the branch. Acute exacerbations often present as phlegm-heat accumulation in the lungs.

[0003] Asthma, also known as bronchial asthma, is a heterogeneous disease characterized by chronic airway inflammation involving multiple cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, and airway epithelial cells) and cellular components. This chronic inflammation is associated with airway hyperresponsiveness, typically presenting as widespread and variable reversible expiratory airflow limitation, leading to recurrent episodes of wheezing, shortness of breath, chest tightness, and / or cough, with intensity varying over time. Attacks often occur and worsen at night and / or in the early morning, and most patients experience spontaneous remission or relief with treatment. If bronchial asthma is not diagnosed and treated promptly, irreversible airway narrowing and remodeling can occur with prolonged disease progression. Symptoms include paroxysmal expiratory dyspnea with wheezing or paroxysmal cough and chest tightness. In severe cases, patients are forced to sit upright or adopt an orthopneic posture, experiencing a dry cough or coughing up large amounts of white frothy sputum, and even cyanosis. Sometimes, cough is the only symptom (cough-variant asthma). Some adolescent patients present with chest tightness, cough, and shortness of breath as their sole clinical manifestation during exercise (exercise-induced asthma). Asthma symptoms can occur within minutes and last for hours to days, relieved by bronchodilators or spontaneously. Some patients may experience a recurrence several hours after remission. Nighttime and early morning attacks and exacerbations are often characteristic of asthma. Traditional Chinese medicine (TCM) considers the lungs to be the master of qi and the kidneys to be the root of qi. When asthma occurs, lung deficiency prevents the lungs from governing qi, and kidney deficiency prevents the kidneys from receiving qi, causing qi to rise upwards and manifest as shortness of breath. The spleen is the source of qi production; spleen deficiency leads to phlegm production, which obstructs the airways, causing chest tightness and shortness of breath. Therefore, asthma is considered a condition of kidney and spleen deficiency with excessive dampness.

[0004] Upper respiratory tract infection (URTI) falls under the categories of "common cold" and "cough" in Traditional Chinese Medicine (TCM). Clinically, it is characterized by nasal congestion, runny nose, sneezing, cough, headache, chills, fever, general malaise, and a floating pulse. Clinical observations reveal that the primary cause of URTI is wind-heat. Wind-heat is a yang pathogenic factor, easily damaging the body's yin fluids. It is characterized by its tendency to disperse and drain, often resulting in sweating after infection. The opening of the pores (the skin and pores) serves to expel pathogenic factors, but it also acts as a channel for external pathogens to enter the body. Especially after improper medication or care, the body's vital energy (qi) is damaged, leading to recurrent infections. Upper respiratory tract infections are predominantly wind-heat type, corresponding to the Wei-level syndrome of febrile diseases. Regarding the pathogenesis of the Wei-level syndrome, Ye Tianshi believed that "when warm pathogens invade the body, they first attack the lungs." The lungs govern qi and belong to the wei (defensive) system, externally connecting with the skin and hair. When warm pathogens invade the lungs, they cause lung qi stagnation, preventing the wei yang from dispersing and spreading, resulting in a lack of warmth on the body surface, hence the symptom of "aversion to wind and cold." This aversion to cold is not the same as the aversion to cold in superficial syndromes. Yang Lishan explicitly stated that "in febrile diseases, when pathogenic heat attacks internally, all external symptoms are due to internal heat stagnation that floats to the surface. Although there are external symptoms, there is actually no external pathogen." In other words, the appearance of external symptoms in the Wei level syndrome does not mean the disease is located on the surface, but rather that internal heat stagnation has floated to the surface. Because Ye Tianshi once said, "The lung governs qi, and it is associated with the skin and hair, hence it is said to be on the surface," the Wei level syndrome is still conventionally referred to as an external syndrome. The *Neijing* states, "For those on the skin, induce sweating to expel the pathogen," indicating that sweating is the treatment principle for external syndromes. However, the location of the Wei level syndrome in febrile diseases is in the lungs, not on the surface; therefore, there is a saying that sweating is contraindicated in febrile diseases. Ye Tianshi's statement, "Sweating is permissible in the Wei level," means using pungent and cooling herbs to clear heat and clear the pores, harmonize the Ying and Wei (nutritive and defensive qi), and naturally induce sweating to resolve the illness. This is not a method of inducing sweating. He also stated in *Essentials of Pediatrics* that "Sweating is contraindicated in cases of wind-heat or spring-heat." Wu Jutong further emphasized that "...merely inducing sweating on the surface is of no benefit." Therefore, the formation mechanism of Wei level syndrome can be summarized as "warm pathogens attacking the lungs, causing lung qi stagnation and internal heat accumulation." Treatment for Wei level syndrome should focus on clearing lung heat and expelling pathogens externally.

[0005] Bronchitis is an acute or chronic specific inflammation of the bronchial mucosa and surrounding tissues caused by biological or non-biological factors, manifesting as symptoms such as cough, sputum production, and discomfort. Acute bronchitis is an acute inflammation of the bronchial mucosa; in traditional Chinese medicine, it is classified as "cough," with the pathogenesis mainly involving impaired lung function and upward reversal of lung qi. Treatment focuses on clearing heat and removing blood stasis, and promoting lung function and resolving phlegm. Chronic bronchitis is a chronic inflammation of the trachea, bronchi, and surrounding tissues caused by infectious or non-infectious factors. Clinically, it is characterized by a chronic course of recurrent cough, sputum production, or wheezing. Pathological features include goblet cell proliferation in the bronchial walls, massive airway obstruction by mucus, and infiltration of inflammatory cells such as macrophages. Current treatment primarily involves antibacterial, anti-inflammatory, and antiviral therapies, with commonly used Western medicines including amoxicillin, cephalexin, and methimazole. Traditional Chinese medicine preparations are widely used in the treatment of different types of bronchitis. However, based on the current clinical situation, there is still a need for better drugs to play a role in treating bronchitis and to solve the physical and economic problems caused by the disease for patients.

[0006] Pharyngitis is a nonspecific inflammation of the pharynx, a general term for inflammation caused by various microbial infections of the pharynx, and can exist alone. Acute pharyngitis is one of the common and frequently occurring diseases in clinical otolaryngology, and is called "acute throat obstruction" in traditional Chinese medicine. Acute pharyngitis is an acute inflammation of the pharyngeal mucosa, submucosal tissue, and lymphoid tissue caused by viral, bacterial, or mycoplasma infections. It has a rapid onset and progression. The inflammation can affect the entire pharynx. Clinically, the initial symptoms are dryness, burning, roughness, and mild pain in the throat, with the sore throat gradually worsening, followed by pain when swallowing; or it may be limited to only part of the nasopharynx, oropharynx, with symptoms such as cough and hoarseness; systemic symptoms such as malaise, headache, loss of appetite, dry mouth, thirst, chills, and limb aches may also occur, often accompanied by fever. In addition, some cases of acute pharyngitis present with edema or secondary to laryngeal angioedema; it can also occur alone, but this is less common and easily spreads to the larynx, causing suffocation. Traditional Chinese medicine believes that acute pharyngitis is caused by the invasion of the body by external pathogens and toxins. The causes are mostly related to external wind-heat, excessive heat in the lungs and stomach, etc. Redness, swelling, heat and pain are common clinical symptoms. The main treatment is to dispel wind and clear heat, detoxify and soothe the throat.

[0007] Autoimmune hepatitis (AIH) presents with complex and diverse clinical manifestations, lacking specificity. It is more common in women (70%), but can occur at any age and in any population. Type 2 AIH is common in children, with approximately 20% of adult patients developing the disease after age 60, resulting in an incidence rate of 0.1‰–0.2‰. It is often accompanied by other autoimmune diseases. A small number of cases may develop fulminant hepatitis and liver failure, and some patients, especially the elderly, may be asymptomatic. Acute AIH presents with jaundice, joint pain, loss of appetite, and fatigue; liver biopsy may reveal signs of acute hepatitis. Some cases present with symptoms of acute hepatitis, but liver biopsy reveals signs of chronic liver disease such as fibrosis or cirrhosis. AIH can also have an insidious onset, only manifesting clinically after progression to decompensated cirrhosis, and is more common in the elderly. Increasingly, AIH is diagnosed during routine checkups or when abnormal liver biochemical markers are found during consultations for other medical conditions. Physical examination may be normal, or it may present with signs of chronic liver disease such as hepatomegaly, splenomegaly, and jaundice. Clinical features of autoimmune hepatitis (AIH) include elevated serum aminotransferase levels, hyperimmunoglobulin G (HG)emia, positive serum autoantibodies, and moderate to severe interface hepatitis on liver histology. AIH is classified into three types based on serum autoantibody testing: ① Type 1, characterized by positive anti-smooth muscle antibody (SMA) and / or antinuclear antibody (ANA), is the most common; ② Type 2, characterized by positive anti-hepatic and kidney microsomal antibody (LKM-1), is rare in adults; ③ Type 3, characterized by positive anti-soluble liver antigen antibody / anti-hepatopancreatic antibody (SLA / LP). The diagnosis of AIH integrates clinical manifestations, biochemical indicators, serological indicators, histological features, and exclusion of other etiologies.

[0008] Ulcerative colitis (UC) is a common digestive system disease characterized by abdominal pain, diarrhea, mucus and bloody stools, and tenesmus. The disease primarily affects the colon and rectum. Modern medicine believes that a combination of genetic, environmental, and psychological factors leads to damage to the intestinal mucosal barrier, neuroendocrine dysfunction, and immune imbalance, causing localized ulceration of the intestinal mucosa and ultimately resulting in the disease. The severity of the condition varies, and it often presents as a recurring condition. This disease can occur at any age, but is most common between 20 and 40 years old, although it can also occur in children and the elderly; there is no significant difference in incidence between men and women. Western medicine often treats it with aminosalicylic acid drugs or hormones, which frequently cause various adverse reactions. Traditional Chinese medicine believes that the occurrence of this disease is related to external wind-cold, internal injury from diet, damp-heat accumulation, and spleen and stomach dysfunction. Traditional Chinese medicine (TCM) classifications of ulcerative colitis (UC) primarily include damp-heat in the large intestine, intense heat and toxicity, spleen deficiency with dampness, mixed cold and heat patterns, liver stagnation and spleen deficiency, spleen and kidney yang deficiency, and yin and blood deficiency. The primary disease location is in the spleen, stomach, and large intestine; prolonged illness can affect the kidneys. TCM treatment of UC mainly relies on syndrome differentiation and treatment, utilizing mechanisms such as immune regulation, antioxidant activity, scavenging of oxygen free radicals, regulation of gastrointestinal hormones and intestinal flora, and regulation of intestinal motility disorders. This approach offers advantages such as definite efficacy, multi-target regulation, and minimal side effects.

[0009] Chronic glomerulonephritis (CGN) is a glomerular disease, also known as chronic nephritis, and is an autoimmune disease. It progresses slowly, has a complex pathogenesis, varied onset, and is protracted and difficult to cure. Clinically, it manifests as proteinuria, hematuria, hypertension, edema, and varying degrees of renal function impairment. Severe cases can lead to glomerular sclerosis and eventually chronic renal failure. Currently, Western medicine treatments are limited, and treatment often involves glucocorticoids and immunosuppressants. While these have good clinical efficacy, long-term use can cause significant side effects. For example, glucocorticoids can easily cause skin, respiratory, and urinary tract infections, and may also cause adverse reactions in the skin and soft tissues, such as moon face and buffalo hump. Ocular adverse reactions such as cataracts, glaucoma, and exophthalmos may also occur. Cardiovascular effects include high cholesterol and high triglycerides, increasing the risk of atherosclerosis and osteoporosis in elderly patients. Immunosuppressants suppress the immune system, reducing the body's ability to fight off pathogens and making it more susceptible to infectious diseases. They also affect the blood system, potentially causing bone marrow suppression symptoms such as decreased white blood cell, hemoglobin, and platelet counts, and may damage liver or kidney function. Traditional Chinese medicine (TCM) considers chronic glomerulonephritis to be primarily determined by both internal and external factors. External factors include invasion of wind, cold, and dampness, damaging the spleen and stomach; internal factors include spleen and stomach deficiency, imbalance of yin and yang, qi and blood leading to dampness obstruction, qi stagnation, and blood stasis. TCM treatment for chronic nephritis follows the principle of dialectical treatment, addressing the root cause of the disease by comprehensively understanding its development and progression, resulting in fewer side effects and allowing for long-term use. Traditional Chinese medicine (TCM) also possesses strong anti-inflammatory and de-inflammatory effects in the treatment of chronic nephritis, and can assist Western medicine treatment by reducing the side effects of immunosuppressants. Because TCM has significant characteristics and advantages in alleviating patients' clinical symptoms, reducing the toxic side effects and dosage of Western medicine, and improving patients' quality of life, it achieves better therapeutic effects.

[0010] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease of unknown etiology, characterized by erosive, symmetrical, and polyarticular arthritis. It can affect various organs and tissues throughout the body, including the skin, muscles, blood vessels, nerves, pleura, myocardium, and lymph nodes. The basic pathological changes include synovitis, pannus formation, and gradual destruction of articular cartilage and bone, ultimately leading to joint deformities and loss of function. Clinically, anti-inflammatory and analgesic drugs are commonly used. However, nonsteroidal anti-inflammatory drugs (NSAIDs), slow-acting antirheumatic drugs (SRAs), and glucocorticoids may cause side effects such as gastritis, gastric ulcers, liver damage, skin rashes, and osteoporosis, and therefore cannot be taken long-term. Currently, the scientific community has conducted in-depth and extensive research on the key roles of a class of cytokines with broad biological activity, synthesized or secreted by immune or non-immune cells upon stimulation, such as IL-1, IL-6, IL-12, IL-18, and TNF-α, in the development of this disease. Numerous studies have reported that IL-1β, IL-6, and TNF-α are inflammatory mediators that play a major role in the pathogenesis of rheumatoid arthritis (RA). They stimulate chondrocytes and synovial cells to secrete collagenase and other substances, disrupting the chondrocyte environment and promoting chondrocyte apoptosis. These mediators are closely related to changes in the pathogenesis, treatment, and prognosis of RA. Therefore, cytokine antagonists, such as monoclonal antibodies, recombinant soluble receptor antagonists, and cytokine receptor antagonists, can be used to delay the progression of joint or cartilage destruction in patients. However, clinical studies have found that cytokine antagonists can cause certain adverse reactions. There are reports that TNF-α antagonists theoretically increase the risk of tumor development, and patients using the IL-1 receptor antagonist Anakinra may experience mild, transient injection site reactions.

[0011] Sjogren's syndrome (SS) is an autoimmune disease that commonly affects the lacrimal and salivary glands, manifesting as dry mouth and tongue, and dry conjunctivitis. In severe cases, it can lead to internal organ damage. The disease has an extremely high incidence rate, and its pathogenesis remains to be explored. There is currently no cure, and treatment primarily focuses on symptomatic relief, using medications such as corticosteroids, hydroxychloroquine, immunosuppressants, and biologics. However, the treatment effects are not always satisfactory, and some medications can cause significant side effects and adverse reactions, such as liver and kidney damage, leukopenia, osteoporosis, obesity, and elevated blood sugar and blood pressure, making long-term use unsuitable. Currently, the development of drugs with good efficacy and fewer side effects is a key research focus in this field. Traditional Chinese medicine (TCM) literature does not use the term "Sjögren's syndrome." Based on its onset and clinical manifestations, it is categorized under "dryness syndrome," "dryness-related arthralgia," and "dryness-related toxins." TCM believes that this disease is mostly caused by external invasion of dryness pathogens, wind-cold-heat pathogens transforming into dryness and damaging Yin, or by inherent Yin deficiency, insufficient endowment, or loss of body fluids after sweating, vomiting, or purging, leading to insufficient Yin fluids, Qi and blood, and blood stasis and obstruction of the collaterals. The pathological mechanism is most closely related to the imbalance of Yin and Yang in the liver, spleen, and kidneys, with Yin deficiency and Yang excess. TCM excels at grasping the treatment method from a "holistic perspective." While having the effect of "causal treatment," it also has the ability to adjust and improve the function of the human body's organs, Qi and blood, and overall function, achieving multi-target treatment, thereby improving the symptoms of Sjögren's syndrome, delaying the progression of the disease, and without side effects.

[0012] Vasculitis is a type of vascular disease, referring to a group of diseases caused by inflammation of the blood vessel wall and perivascular area, involving inflammatory cell infiltration and vascular damage, including fibrin deposition, collagen fiber degeneration, and necrosis of endothelial and myocyte cells. It is also known as vasculitis. The common pathophysiological basis is the inflammatory lesions such as degeneration and necrosis of the walls and surrounding tissues of arteries, veins, and microvessels of varying sizes caused by various factors, leading to endothelial necrosis, thrombosis, and luminal occlusion, resulting in hemodynamic dysfunction of the affected organs. The pathogenesis is complex, involving multiple components such as inflammatory cells and endothelial cells. Some types of vasculitis are mild and self-limiting, primarily affecting one organ. Other types, involving medium-sized blood vessels, can affect different organs, causing functional failure, severely impacting the patient's quality of life and ability to work, and even leading to disability or death due to heart, brain, or kidney failure, such as Kawasaki disease (KD). Kawasaki disease is a systemic vasculitis of unknown etiology in children, clinically manifested as persistent fever, rash, cervical lymphadenopathy, conjunctival congestion, and changes in the extremities and oral mucosa. Without timely treatment, one-quarter of affected children will develop coronary artery disease; these children can be classified as high-risk Kawasaki disease coronary artery disease patients. Kawasaki disease-induced coronary arteritis can lead to vascular remodeling and reconstruction, resulting in endothelial hyperplasia and vascular stenosis, with significant thickening of the vessel wall. Furthermore, persistent coronary lesions caused by Kawasaki disease have a high probability of cardiac complications, including myocardial infarction due to thrombosis or stenosis. Therefore, treatment of Kawasaki disease is crucial for preventing its associated coronary artery disease. Retinal vasculitis (RV) is another common vasculitis, an intraocular inflammatory disease affecting the retinal vessels, prevalent in young adults aged 20-40. Its etiology is complex and often related to systemic diseases. Typical manifestations include grayish-white vascular sheaths in the fundus, exudation, hemorrhage, and retinal edema. Clinically, retinal vasculitis (RV) can be divided into three types: arteritis, phlebitis, and capillary vasculitis. The pathogenesis of RV is currently unclear, but most scholars believe it is related to pattern recognition receptors and gene products involved in immune and inflammatory responses. Laser treatment is necessary if the patient presents with significant retinal ischemia and capillary non-perfusion. Vitreous hemorrhage or retinal detachment requires vitrectomy. Currently, Western medicine primarily uses corticosteroids and various novel immunosuppressants, but these often involve long treatment courses and significant side effects, which are detrimental to patient treatment. From a Traditional Chinese Medicine (TCM) perspective, vasculitis is considered a form of blood stasis. The treatment principle is to clear heat and remove blood stasis, invigorate blood circulation, warm the yang and remove blood stasis, clear heat and detoxify, and promote tissue regeneration. This improves blood circulation in the limbs, reducing fever, pain, and swelling, and even restoring skin color. It also cleans the wound, promoting local anti-inflammatory effects and wound healing. Traditional Chinese medicine treats both internal and external ailments by regulating yin and yang, regulating qi and blood, and eliminating wind, heat, and dampness.

[0013] Henoch-Schonlein Purpura (HSP) is a spontaneous immune system disorder primarily characterized by small vessel inflammation mediated by IgA immune complex deposition. It typically presents as non-thrombocytopenic purpura, with an incidence of approximately 6.21 per 100,000 (0-15 years old), making it the most common vasculitis in children. Although its etiology is not fully understood, exposure to many antigenic substances and drugs is known to potentially trigger immunological changes. Clinical symptoms mainly include skin purpura, joint pain, abdominal pain, and gastrointestinal bleeding, and the disease is characterized by a long course and frequent recurrence. Ancient Chinese medicine literature does not record a disease called "allergic purpura." However, based on clinical symptoms, such as the appearance of skin purpura in batches, fluctuating and unpredictable, joint swelling and pain appearing in no fixed location, and occasional skin itching, it aligns with the characteristics of wind ("wind is characterized by its rapid movement and frequent changes") and the saying "no wind, no itching" in traditional Chinese medicine. In Traditional Chinese Medicine (TCM), this condition is called purpura. Purpura falls under the categories of "blood disorders," "grape epidemic," and "rashes," caused by heat toxins congesting the blood, forcing blood to flow erratically, burning the blood vessels, causing blood seepage, and the inability of Qi to control the blood. It can also be caused by a prolonged illness leading to Qi deficiency and an inability to control the blood. Some scholars use the theory of latent pathogens to explain the etiology and pathogenesis of allergic purpura, believing it is due to congenital deficiencies combined with external pathogenic factors such as the six evils and epidemic diseases. Other scholars believe that a weak constitution combined with external wind-dampness and heat toxins can lead to the disease. Excessive heat in the body forces blood to flow erratically, or a weak constitution with prolonged illness leads to deficiency of both Qi and blood, resulting in loss of blood control, and is easily exacerbated by external wind-heat. Still other scholars believe that children's internal organs are delicate, and contact with hot and dry substances can easily cause internal heat to clash with the blood, burning the blood vessels, forcing blood to flow erratically, and overflowing to the skin surface as purpura. Some scholars believe that the pathogenesis of allergic purpura is essentially a combination of dampness and heat, leading to blood depletion and blood stasis, with the causes attributed to five aspects: wind, heat, dampness (toxin), blood stasis, and deficiency. Currently, Western medicine's treatment methods for allergic purpura depend on the severity of the condition. For simple cutaneous purpura, antihistamines or compound glycyrrhizin, i.e., non-hormonal drugs, are used. If allergic purpura affects the kidneys, resulting in protein in the urine, appropriate use of hormones is necessary. However, hormone use can cause osteoporosis, disordered glucose and lipid metabolism, and blood system disorders, while histamines can cause drowsiness and dry mouth; therefore, they cannot be used long-term. Traditional Chinese medicine combines internal and external treatments for allergic purpura, while strengthening dietary and lifestyle care to prevent complications, demonstrating significant efficacy and advantages. Summary of the Invention

[0014] In view of this, the present invention provides the application of a traditional Chinese medicine composition in the preparation of drugs for chronic obstructive pulmonary disease, upper respiratory tract infection, autoimmune hepatitis, ulcerative colitis, chronic nephritis, rheumatoid arthritis, Sjögren's syndrome, and vasculitis. By weight, the traditional Chinese medicine composition comprises: 1-100 parts of Magnolia officinalis, 1-100 parts of Areca catechu (roasted), 1-100 parts of Amomum villosum (roasted), 1-100 parts of Ephedra sinica, and 1-100 parts of Prunus armeniaca (bitter). The ingredients are: 1-100 parts Notopterygium root, 1-100 parts fresh ginger, 1-100 parts Pogostemon cablin, 1-100 parts Eupatorium fortunei, 1-100 parts Atractylodes lancea, 1-160 parts Poria cocos, 1-120 parts Atractylodes macrocephala, 1-100 parts gypsum, 1-100 parts charred hawthorn, 1-100 parts charred Shenqu (medicated leaven), 1-100 parts charred malt, 1-100 parts earthworm, 1-100 parts Cynanchum paniculatum, 1-100 parts Dryopteris crassirhizoma, and 1-100 parts Lepidium apetalum. The ingredients can be used in any way that is beneficial to improving the corresponding symptoms of the patient, including treatment or prevention. The composition can be made by directly grinding the raw materials into powder, or by extracting or other forms obtained through conventional methods. The raw materials used can also be used by directly grinding into powder, extracting, or other processed forms.

[0015] Furthermore, the traditional Chinese medicine composition includes: 10-80 parts of Magnolia officinalis, 10-80 parts of Areca catechu (roasted), 10-80 parts of Amomum villosum (roasted), 10-60 parts of Ephedra sinica, 10-60 parts of Prunus armeniaca (bitter), 10-80 parts of Notopterygium incisum, 10-60 parts of Zingiber officinale (fresh), 10-80 parts of Pogostemon cablin, 10-60 parts of Eupatorium fortunei, 10-80 parts of Atractylodes lancea, 10-160 parts of Poria cocos, 10-120 parts of Atractylodes macrocephala, 10-80 parts of Gypsum fibrosum, 10-50 parts of Crataegus pinnatifida (roasted), 10-80 parts of Massa fermentata (roasted), 10-60 parts of Hordeum vulgare (roasted), 10-80 parts of Pheretima aspergillum, 10-80 parts of Cynanchum paniculatum, 10-60 parts of Dryopteris crassirhizoma, and 10-80 parts of Lepidium apetalum.

[0016] Preferably, the traditional Chinese medicine composition may be: 30-50 parts of Magnolia officinalis, 20-30 parts of Areca catechu (roasted), 30-50 parts of Amomum villosum (roasted), 20-30 parts of Ephedra sinica, 20-30 parts of Prunus armeniaca (bitter), 30-50 parts of Notopterygium incisum, 30-50 parts of Zingiber officinale (fresh), 30-50 parts of Pogostemon cablin, 20-30 parts of Eupatorium fortunei, 30-50 parts of Atractylodes lancea, 120-150 parts of Poria cocos, 80-100 parts of Atractylodes macrocephala, 30-50 parts of Gypsum fibrosum, 20-30 parts of Crataegus pinnatifida (roasted), 30-50 parts of Massa fermentata (roasted), 20-30 parts of Hordeum vulgare (roasted), 30-50 parts of Pheretima aspergillum, 30-50 parts of Cynanchum paniculatum, 20-30 parts of Dryopteris crassirhizoma, and 30-50 parts of Lepidium apetalum.

[0017] Preferably, the traditional Chinese medicine composition includes: 50 parts Magnolia officinalis, 30 parts Areca catechu (roasted), 30 parts Amomum villosum (roasted), 20 parts Ephedra sinica, 30 parts Prunus armeniaca (bitter), 50 parts Notopterygium incisum, 50 parts fresh ginger, 50 parts Pogostemon cablin, 30 parts Eupatorium fortunei, 50 parts Atractylodes lancea, 150 parts Poria cocos, 100 parts Atractylodes macrocephala, 50 parts gypsum, 30 parts Crataegus pinnatifida (roasted), 30 parts Massa fermentata (roasted), 30 parts Hordeum vulgare (roasted), 50 parts Pheretima aspergillum, 50 parts Cynanchum paniculatum, 30 parts Dryopteris crassirhizoma, and 50 parts Lepidium apetalum.

[0018] Specifically, the aforementioned drugs include oral, injectable, or topical dosage forms. The upper respiratory tract infection can be viral or bacterial; more specifically, it can be an upper respiratory tract infection caused by influenza A virus or methicillin-resistant Staphylococcus aureus. The autoimmune hepatitis is selected from Con A-mediated autoimmune hepatitis or autoimmune liver injury caused by immunosuppressants. The aforementioned autoimmune liver injury caused by immunosuppressants includes, but is not limited to, hepatitis S100 antigen-induced autoimmune hepatitis.

[0019] In some specific embodiments of the present invention, the treatment for ulcerative colitis includes any of the following:

[0020] (I) Improve diarrhea; and / or

[0021] (II) Improve abdominal pain; and / or

[0022] (III) Improves mucus and bloody stools; and / or

[0023] (IV) Reduce colon damage; and / or

[0024] (V) Improve inflammation.

[0025] In some specific embodiments of the present invention, the reduction of colonic damage includes any of the following:

[0026] (I) Increase colon length; and / or

[0027] (II) Improvement of colonic ulcers; and / or

[0028] (III) Improve colonic mucosal damage.

[0029] In some specific embodiments of the present invention, the improvement of inflammation includes any of the following:

[0030] (I) Improves inflammatory cell infiltration; and / or

[0031] (II) Reduce and / or suppress serum inflammatory factors.

[0032] In some specific embodiments of the present invention, the serum inflammatory factors include, but are not limited to, IL-6.

[0033] In some specific embodiments of the present invention, the serum inflammatory factor is IL-6.

[0034] The dosage form of the drug of the present invention is prepared by weighing the raw material in proportion, and then adding pharmaceutically acceptable excipients such as fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrices during the preparation process, and preparing it according to conventional production methods in the pharmaceutical field to produce a pharmaceutically acceptable conventional dosage form, including but not limited to decoctions, granules, capsules, tablets, oral liquids, pills, tinctures, syrups, suppositories, gels, sprays, and injections.

[0035] The present invention also provides a method for preparing any of the aforementioned traditional Chinese medicine compositions, characterized in that the preparation method includes:

[0036] Weigh out the following herbs: Magnolia officinalis, Areca catechu (roasted), Amomum villosum (roasted), Ephedra sinica, Prunus armeniaca (bitter), Notopterygium incisum, Zingiber officinale, Pogostemon cablin, Eupatorium fortunei, Atractylodes lancea, Poria cocos, Atractylodes macrocephala, Gypsum fibrosum, Crataegus pinnatifida (roasted), Massa fermentata (roasted), Hordeum vulgare (roasted), Pheretima aspergillum, Cynanchum paniculatum, Dryopteris crassirhizoma, and Lepidium apetalum. Extract each herb twice with water. For the first extraction, add 6 times the amount of water and extract for 1.5 hours. For the second extraction, add 4 times the amount of water and extract for 1.0 hour. Combine the extracts, filter, concentrate the filtrate to a relative density of 1.10–1.15, centrifuge, filter, and dry the filtrate.

[0037] Furthermore, the preparation method of the aforementioned drug involves the following steps:

[0038] Take 50 parts of Magnolia officinalis, 30 parts of Areca catechu (roasted), 30 parts of Amomum villosum (roasted), 20 parts of Ephedra sinica, 30 parts of Prunus armeniaca (bitter), 50 parts of Notopterygium incisum, 50 parts of fresh ginger, 50 parts of Pogostemon cablin, 30 parts of Eupatorium fortunei, 50 parts of Atractylodes lancea, 150 parts of Poria cocos, 100 parts of Atractylodes macrocephala, 50 parts of gypsum, 30 parts of Crataegus pinnatifida (roasted), 30 parts of Massa fermentata (roasted), 30 parts of malt (roasted), 50 parts of Pheretima aspergillum, 50 parts of Cynanchum paniculatum, 30 parts of Dryopteris crassirhizoma, and 50 parts of Lepidium apetalum. Add water and reflux twice. For the first extraction, add 6 times the amount of water and extract for 1.5 hours. For the second extraction, add 4 times the amount of water and extract for 1.0 hour. Combine the extracts, filter, concentrate the filtrate to a relative density of 1.10-1.15, centrifuge, filter, vacuum dry the filtrate, spray dry and pulverize to obtain an intermediate composition. Add sucralose and dextrin, mix well, and obtain granules.

[0039] This invention also proposes the application of a traditional Chinese medicine composition in the preparation of drugs for emphysema or asthma, improving immunity, treating or preventing bronchitis or pharyngitis, or allergic purpura. The traditional Chinese medicine composition, by weight, comprises: 1-100 parts Magnolia officinalis, 1-100 parts Areca catechu (roasted), 1-100 parts Amomum villosum (roasted), 1-100 parts Ephedra sinica, 1-100 parts Prunus armeniaca (bitter), 1-100 parts Notopterygium incisum, and ginger. 1-100 parts of the following herbs: Patchouli 1-100 parts, Eupatorium fortunei 1-100 parts, Atractylodes lancea 1-100 parts, Poria cocos 1-160 parts, Atractylodes macrocephala 1-120 parts, Gypsum 1-100 parts, Roasted Crataegus pinnatifida 1-100 parts, Roasted Shenqu 1-100 parts, Roasted Malt 1-100 parts, Earthworm 1-100 parts, Cynanchum paniculatum 1-100 parts, Dryopteris crassirhizoma 1-100 parts, and Lepidium apetalum 1-100 parts. The bronchitis described can be acute or chronic bronchitis, and the pharyngitis can be acute pharyngitis.

[0040] Furthermore, the traditional Chinese medicine composition includes: 30-50 parts of Magnolia officinalis, 20-30 parts of Areca catechu (roasted), 30-50 parts of Amomum villosum (roasted), 20-30 parts of Ephedra sinica, 20-30 parts of Prunus armeniaca (bitter), 30-50 parts of Notopterygium incisum, 30-50 parts of Zingiber officinale (fresh), 30-50 parts of Pogostemon cablin, 20-30 parts of Eupatorium fortunei, 30-50 parts of Atractylodes lancea, 120-150 parts of Poria cocos, 80-100 parts of Atractylodes macrocephala, 30-50 parts of Gypsum fibrosum, 20-30 parts of Crataegus pinnatifida (roasted), 30-50 parts of Massa fermentata (roasted), 20-30 parts of Hordeum vulgare (roasted), 30-50 parts of Pheretima aspergillum, 30-50 parts of Cynanchum paniculatum, 20-30 parts of Dryopteris crassirhizoma, and 30-50 parts of Lepidium apetalum.

[0041] The composition of this invention can significantly improve lung function in rats with chronic obstructive pulmonary disease (COPD), reduce lung inflammation and emphysema; increase serum α1-AT content, prevent progressive destruction of alveolar walls, reduce HA and PCIII content, and alleviate pulmonary interstitial fibrosis; significantly reduce serum MMP-9 content and increase TIMP-1 content in rats with elastase-induced emphysema; significantly improve the levels of inflammatory cytokines and various cell counts in bronchoalveolar lavage fluid of an asthmatic mouse model; significantly reduce the levels of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid of an OVA-induced asthma mouse model; and significantly inhibit the increase in total cell count, total eosinophil count, total macrophage count, and total lymphocyte count. These findings suggest that the traditional Chinese medicine composition can significantly inhibit airway remodeling, alleviate pulmonary interstitial fibrosis, inhibit lung inflammation, reduce inflammatory cell recruitment, and delay the progression of COPD and asthma, demonstrating good therapeutic effects on both COPD and asthma.

[0042] The composition of this invention can significantly regulate the lung index in mice with upper respiratory tract infection, reduce the level of the inflammatory factor TNF-α in serum, and also has a certain preventive and therapeutic effect on mice with acute upper respiratory tract infection induced by methicillin-resistant Staphylococcus aureus, thus having a good therapeutic effect on upper respiratory tract infection. It can significantly reduce the content of IL-6 and IL-8 in bronchoalveolar lavage fluid and serum of rats, increase the content of IL-10, significantly reduce the content of MDA, reduce lipid peroxidation, and alleviate oxygen free radical damage. It can significantly improve the lesions such as congestion and swelling of pharyngeal mucosa tissue in rats caused by group A beta-hemolytic streptococcal infection. It can significantly reduce the content of IL-1β, IL-6, and TNF-α in serum of rats with acute pharyngitis induced by ammonia water, suggesting that the traditional Chinese medicine composition can promote the reduction or elimination of local inflammation of pharyngeal mucosa tissue, and that the traditional Chinese medicine composition can significantly improve the inflammatory response and pharyngitis treatment effect.

[0043] The composition of this invention can alleviate clinical symptoms, restore IgG to normal levels, and eliminate significant active inflammation histologically. For example, ALP (alkaline phosphatase), AST (aspartate aminotransferase), ALT (alanine aminotransferase), and autoantibodies such as ANA, ASMA, anti-SLA / LP, anti-LKM-1, and anti-LC-1 return to normal, while serum immunoglobulin IgG and / or gamma globulin levels decrease. Liver histology (interface hepatitis, lymphoplasmacytic infiltration, hepatocyte rosette and penetration phenomena, etc.) is alleviated, demonstrating a good therapeutic effect on autoimmune hepatitis.

[0044] The composition of this invention can effectively improve the symptoms of acute and chronic ulcerative colitis in mice induced by DSS and inhibit disease progression.

[0045] The composition of this invention can significantly reduce 24-hour urinary protein, serum urea nitrogen (BUN), and creatinine (Scr) levels in rats with chronic glomerulonephritis, decrease serum levels of inflammatory factors tumor necrosis factor (TNF-α) and interferon (IFN-γ), and increase interleukin-4 (IL-4) levels. It also increases the levels of CD3, CD4, and CD4 / CD8 in rat peripheral blood and decreases CD8 expression. Furthermore, it reduces the levels of TNF-α, interleukin-6 (IL-6), and interleukin-1β (IL-1β) in rat kidney tissue. This suggests that the traditional Chinese medicine composition of this application can protect the kidney tissue of rats with chronic glomerulonephritis, thereby improving renal function, reducing inflammatory response, stabilizing the proportion of CD3, CD4, and CD8 subsets in peripheral blood T lymphocytes, and has a good therapeutic effect on chronic nephritis.

[0046] The composition of this invention significantly reduces joint swelling in rats with rheumatoid arthritis and significantly decreases serum levels of immunomodulatory factors and pro-inflammatory cytokines such as IL1, IL6, and TNF-α, suggesting that the composition can alleviate joint swelling in rats with rheumatoid arthritis, inhibit the production of cytokines mediating inflammation and tissue damage, and regulate abnormal cellular immune function in rats with rheumatoid arthritis. The composition of this application also significantly improves histopathological changes such as synovial inflammatory infiltration, synovial hyperplasia, pannus formation, and cartilage and bone erosion, demonstrating that the composition can delay the progression of synovial and cartilage damage in rheumatoid arthritis.

[0047] The composition of this invention can significantly improve salivary flow rate and water intake in mice with primary Sjögren's syndrome, reduce serum levels of related immunological markers IgG and IgM, reduce whole blood viscosity, plasma viscosity and serum levels of IL-6, IL-17 and IgG in rats with antigen-induced Sjögren's syndrome, and alleviate inflammatory response. This demonstrates that the traditional Chinese medicine composition can significantly regulate immunity, inhibit inflammatory response, and delay disease progression, thus having a good therapeutic effect on Sjögren's syndrome.

[0048] The composition of this invention can significantly reduce vascular damage caused by inflammatory stimulation, thereby alleviating vasculitis in Kawasaki disease mice; it can significantly reduce the inflammatory response of retinal vasculitis in mice, and can reduce inflammatory cell adhesion by reducing the expression of intercellular adhesion molecule-1 (ICAM-1) in the retinal vascular region of mice, indicating that the composition has a good therapeutic effect on vasculitis.

[0049] The composition of this invention can significantly reduce the serum levels of immunoglobulin A (IgA), circulating immune complexes (CIC), and interleukin-6 (IL-6) in ovalbumin-induced allergic purpura in rats, while increasing the levels of interferon-γ (IFN-γ). It can also significantly reduce the serum levels of circulating immune complexes (CIC), blood urea nitrogen (BUN), and creatinine (Cr) in gliadin-induced allergic purpura in mice, indicating that the traditional Chinese medicine composition can significantly delay the course of allergic purpura, improve inflammatory responses and renal function, and has a good therapeutic effect on allergic purpura. Attached Figure Description

[0050] Figure 1 This refers to the cytokine levels in mouse bronchoalveolar lavage fluid.

[0051] Figure 2 It is a cell typing count in mouse bronchoalveolar lavage fluid;

[0052] Figure 3 It is the incubation period of guinea pig panting seizures;

[0053] Figure 4These are HE staining images of the morphological changes of the ankle joint in rats with rheumatoid arthritis caused by the composition (A: normal control group; B: model group; C: low-dose group; D: medium-dose group; E: high-dose group; F: positive control group; arrows: synovial proliferation, pannus, inflammatory cell infiltration, bone erosion; in group AF, the first image is the ankle joint, the second is the synovial tissue, and the third is the cartilage tissue).

[0054] Figure 5 The effect on the pathological morphology of liver tissue in mice with autoimmune hepatitis (HE staining, ×200).

[0055] Figure 6 The effect on the pathological morphology of liver tissue in mice with autoimmune hepatitis (HE staining, ×200).

[0056] Figure 7 The effect of this on the histopathological morphology of colon length in mice with acute ulcerative colitis (HE staining, ×200).

[0057] Figure 8 The effect of this on the histopathological morphology of colon length in mice with chronic ulcerative colitis (HE staining, ×200). Detailed Implementation

[0058] As previously stated, this invention aims to provide the application of a traditional Chinese medicine composition in the preparation of drugs for chronic obstructive pulmonary disease, upper respiratory tract infection, autoimmune hepatitis, ulcerative colitis, chronic nephritis, rheumatoid arthritis, Sjögren's syndrome, vasculitis, or allergic purpura. The following will describe this in detail with reference to specific experiments.

[0059] Unless otherwise specified, all experiments in the following experiments were conducted under standard conditions or conditions recommended by the manufacturer. Active pharmaceutical ingredients (APIs) or excipients, as well as reagents or instruments whose manufacturers are not specified, are all commercially available products. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention.

[0061] Example 1: Preparation of the composition granules

[0062] The traditional Chinese medicine composition in this embodiment is made from the following raw materials: 50g Magnolia officinalis, 30g Areca catechu (roasted), 30g Amomum villosum (roasted), 20g Ephedra sinica, 30g Prunus armeniaca (bitter), 50g Notopterygium incisum, 50g Zingiber officinale, 50g Pogostemon cablin, 30g Eupatorium fortunei, 50g Atractylodes lancea, 150g Poria cocos, 100g Atractylodes macrocephala, 50g Gypsum fibrosum, 30g Crataegus pinnatifida (roasted), 30g Massa fermentata (roasted), 30g Hordeum vulgare (roasted), 50g Pheretima aspergillum, 50g Cynanchum paniculatum, 30g Dryopteris crassirhizoma, and 50g Lepidium apetalum.

[0063] The traditional Chinese medicine composition is prepared according to the following method:

[0064] Take the above-mentioned medicinal materials, add water and reflux extract twice. The first time, add 6 times the amount of water and extract for 1.5 hours. The second time, add 4 times the amount of water and extract for 1.0 hour. Combine the extracts, filter, concentrate the filtrate to a relative density of 1.10-1.15, centrifuge and filter, vacuum dry the filtrate, spray dry and pulverize to obtain an intermediate composition. Add sucralose and dextrin, mix well, and obtain granules.

[0065] Pharmacological test

[0066] Test drug: Particles from Example 1, provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0067] Experiment Example 1: Effects of combined smoke and LPS on COPD

[0068] Experimental materials

[0069] Animals: SD rats, SPF grade, male, 60 in total, weighing 130-150g.

[0070] Drug: Aminophylline tablets, product of Tianjin Lisheng Pharmaceutical Co., Ltd., batch number: 2020009; specification: 0.1g / tablet, 100 tablets / bottle.

[0071] Reagents: LPS: SIGMA, extracted from E. coli O55: B5, batch number 039M4004V; 0.9% sodium chloride solution, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., specification: 500mL / bottle, batch number: 2012192103; formaldehyde, Nanjing Chemical Reagent Co., Ltd., specification: 500mL / bottle, batch number: 200517018E; chloral hydrate, Shanghai Guoyao Group Chemical Reagent Co., Ltd., specification: 250g / bottle, batch number: 20201129; sodium penicillin for injection, Harbin Pharmaceutical Group Pharmaceutical Factory, batch number: P2101719;

[0072] Instruments: Ultra-low temperature freezer (Haier, model: DW-86L728); Electronic balance (Sartorius Scientific Instruments, model: BSA224S-CW); DHG-9076A electric thermostatic drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.); PowerLab8 / 35 biosignal acquisition system (AD Instruments, Australia); CENTRFUGE5840 refrigerated centrifuge (Eppendorf); MD FLEX-STION3 microplate reader.

[0073] Experimental methods

[0074] Male SD rats, approximately 200g, were injected intratracheally with 200μg / 200μL endotoxin on days 1 and 14. From days 2 to 30 (except day 14), the rats were exposed to 5% cigarette smoke (Qianmen brand) for 1 hour / day, twice a day, to establish a COPD model. The normal control group received intratracheal instillation of physiological saline on days 1 and 14 without smoke exposure. Different dosage groups of the herbal composition granules (6.48g crude drug / kg, 12.96g crude drug / kg, 25.92g crude drug / kg) and the aminophylline tablet positive control group were administered the herbal composition granules at a dose of 10ml / kg once by gavage starting on day 1, for 30 consecutive days. Thirty minutes after the last administration, rats were anesthetized and tested by intraperitoneal injection of 0.35g / kg of 10% chloral hydrate.

[0075] Detection indicators: Rats were anesthetized with 0.35 g / kg of 10% chloral hydrate. The skin of the neck was longitudinally incised to expose the trachea. An inverted "T"-shaped incision was made under the cricoid cartilage, and an endotracheal tube with a three-way valve was inserted. One end was connected to the animal ventilator, and the other end was connected to a signal acquisition system to measure respiratory parameters. The forced expiratory volume in 0.3 seconds (FEV0.3), forced vital capacity (FVC), FEV0.3 / FVC, and peak expiratory flow (PEF) were measured.

[0076] The results showed that FVC, FEV0.3, FEV0.3 / FVC, and PEF in the model group were significantly lower than those in the normal group (P<0.01), indicating successful model establishment. The high-dose group and the positive control group of the traditional Chinese medicine composition showed significant improvements in FVC, FEV0.3, FEV0.3 / FVC, and PEF compared to the model group (P<0.01, P<0.05). The medium-dose group of the traditional Chinese medicine composition showed significant improvements in FEV0.3, FEV0.3 / FVC, and PEF compared to the model group (P<0.01, P<0.05). The results are shown in Table 1.

[0077] After modeling, the levels of IL1β, IL6, and TNFα in rat lung tissue increased (P<0.01, P<0.05), and administration of the traditional Chinese medicine composition significantly reduced lung tissue inflammation. Compared with the model group, the high-dose, medium-dose, and positive control groups of the traditional Chinese medicine composition significantly reduced the levels of inflammatory factors (P<0.01, P<0.05), and the low-dose group showed a significant decrease in IL-1β compared with the model group (P<0.05). The results are shown in Table 2.

[0078] Table 1 Effects on pulmonary function in rats with chronic obstructive pulmonary disease

[0079]

[0080] Note: Compared with the model group, *P<0.05, **P<0.01.

[0081] Table 2 Effects of cytokines on rats with chronic obstructive pulmonary disease.

[0082]

[0083] Note: Compared with the model group, *P<0.05, **P<0.01.

[0084] Experiment Example 2: Effect of elastase-induced emphysema

[0085] Animals: SD rats, SPF grade, male, 60 in total, weighing 140-160g.

[0086] Drug: Dexamethasone acetate tablets, Chenxin Pharmaceutical Co., Ltd., batch number: 210915201.

[0087] Reagents: Elastase, Nanjing Zelang, batch number 20220218; 0.9% sodium chloride solution, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., specification: 500mL / bottle, batch number: 2012192103; Formaldehyde, Nanjing Chemical Reagent Co., Ltd., specification: 500mL / bottle, batch number: 200517018E; Chloral hydrate, Shanghai Sinopharm Group Chemical Reagent Co., Ltd., specification: 250g / bottle, batch number: 20201129;

[0088] Instruments: Electronic balance (Sartorius Scientific Instruments, model: BSA224S-CW); Centurf UGE 5840 refrigerated centrifuge, Eppendorf; Ultra-low temperature freezer (Haier, model: DW-86L728); MD FLEX-STION3 microplate reader

[0089] Experimental Methods: Sixty male SD rats, weighing 180-220g, were randomly divided into 6 groups (n=10 per group). The 6 groups were: normal control group, model group, low-, medium-, and high-dose groups of the traditional Chinese medicine composition (6.48g crude drug / kg, 12.96g crude drug / kg, and 25.92g crude drug / kg, respectively), and positive control group (dexamethasone 4mg / kg). An experimental rat emphysema model was established using the elastase intratracheal instillation method. Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, exposing the trachea. 0.5ml of elastase solution (200U / kg) was slowly instilled into the trachea using a 1ml syringe. The rat was immediately held upright and gently shaken from side to side to ensure even distribution. The incision was sutured and disinfected after the surgery. The normal control group underwent the same procedure, with 0.4ml / kg of physiological saline instilled intratracheally. Drug administration began on the second day post-surgery, once daily. The positive control group received the drug every other day for 4 weeks.

[0090] Detection indicators: Measurement of serum α1-antitrypsin (α1-AT), hyaluronic acid (HA), laminin (LN), and rat type III procollagen PCⅢ. Measurement of lung tissue matrix metalloproteinases (MMP-9) and TIMP-1.

[0091] The results showed that after elastase modeling, the serum α1-AT content in rats was significantly lower than that in the normal group, while the serum HA and PCIII content was significantly higher (P<0.01), and the LN content showed no significant difference. After administration of the traditional Chinese medicine composition, the serum α1-AT content in the low, medium, and high dose groups was significantly higher than that in the model group (P<0.01); the serum HA and PCIII content in the medium and high dose groups was significantly lower than that in the model group (P<0.01). These results suggest that the traditional Chinese medicine composition can increase the serum α1-AT content in rats after modeling, prevent progressive alveolar wall destruction, reduce the serum HA and PCIII content, and alleviate pulmonary interstitial fibrosis. The results are shown in Table 3.

[0092] After elastase administration to induce a model, the levels of MMP-9 in the lung tissue of rats increased and the levels of TIMP-1 decreased, indicating that airway inflammation was deepened and airway mucosal damage was observed in rats with emphysema. Compared with the model group, after administration of the traditional Chinese medicine composition, the levels of MMP-9 decreased and the levels of TIMP-1 increased in both the high-dose and low-dose groups (P<0.01), suggesting that the traditional Chinese medicine composition could inhibit airway remodeling. The results are shown in Table 4.

[0093] Table 3. Effects of elastase-induced emphysema on α1-antitrypsin (α1-AT) and the levels of HA, LN, and PCⅢ in rats.

[0094]

[0095] Note: Compared with the model group, *P<0.05, **P<0.01.

[0096] Table 4. Effects of elastase on lung tissue factor in rats with elastase-induced emphysema.

[0097]

[0098] Note: Compared with the model group, *P<0.05, **P<0.01.

[0099] The composition proposed in this invention can significantly delay disease progression, improve respiratory function, and reduce pulmonary interstitial fibrosis and airway remodeling. Example 3: Effects on an OVA-induced mouse asthma model.

[0100] Experimental materials

[0101] Animals: 60 BABL / c mice, SPF grade, female, weighing 18-20g.

[0102] Drug: Salbutamol sulfate, Jiangsu Yabang Aipusen Pharmaceutical Co., Ltd., batch number: 201108; specification: 2mg / tablet.

[0103] Reagents: OVA, MCE, catalog number: HY-P1489A, specification: 100mg / vial, batch number: 119448; OVA, Sigma, catalog number: A5503, specification: 10g / bottle, batch number: SLCB8249; Aluminum hydroxide (Al(OH)3), Aladdin Company, catalog number: A110525, specification: 500g / bottle, batch number: H2106415; Physiological saline, Chenxin Pharmaceutical Co., Ltd., national drug approval number H20013310, specification: 100ml, batch number: 2101282721

[0104] Instruments: Sartorius BS224S electronic balance (Beijing Sartorius Instrument Systems Co., Ltd.); ACX-SC-DA animal weight scale (Shanghai Xiangchuan Electronic Weighing Instrument Co., Ltd.); Flexstation 3 multi-functional microplate reader (MD Instruments, USA); multi-functional cough and asthma induction device (Shanghai Xinruan Information Technology Co., Ltd., model: XR-YLS-8A).

[0105] Experimental methods

[0106] After the acclimatization period, 60 mice of suitable weight were selected and randomly divided into a blank control group, a model group, a low-dose group, a medium-dose group, a high-dose group, and a positive control group, with 10 mice in each group, all of which were female.

[0107] Asthma model mice were sensitized by intraperitoneal injection of an antigen mixture (OVA 100 μg / mouse, aluminum hydroxide 2.25 mg / mouse) on days 1 and 14. Challenge was initiated on day 21 by ultrasonic nebulization of 1% OVA once daily for 30 minutes for one week. Normal control mice were intraperitoneally injected with an equal volume of physiological saline on days 1 and 14, and then ultrasonically nebulized with physiological saline once daily for 30 minutes for one week, starting on day 21. Drug-treated mice were administered the drug 30 minutes before nebulization. From day 21, mice were administered the corresponding low, medium, and high doses of the drug via gavage at doses of 9.36, 18.72, and 37.44 g crude drug / kg, respectively, for one week. Both the normal control and asthma model mice were administered an equal volume of physiological saline via gavage starting on day 21 for one week.

[0108] Testing indicators:

[0109] 1. Bronchoalveolar lavage fluid contains IL-4, IL-5, and IL-13, etc.

[0110] 2. Bronchoalveolar lavage fluid lymphocyte count.

[0111] The results showed that the levels of IL-4, IL-5, IL-13, total cell count, total eosinophil count, total macrophage count, and total lymphocyte count in the bronchoalveolar lavage fluid of the model group mice were significantly higher than those in the normal group (P<0.05), indicating successful model establishment. In the medium-dose group of the traditional Chinese medicine composition and the positive control group, the levels of IL-4, IL-5, IL-13, total cell count, total eosinophil count, total macrophage count, and total lymphocyte count were significantly improved compared to the model group (P<0.05). In the high-dose group of the traditional Chinese medicine composition, the levels of IL-4, IL-5, IL-13, total cell count, total macrophage count, and total lymphocyte count were significantly improved compared to the model group (P<0.05). See Figure 1 and Figure 2 Tables 5 and 6.

[0112] Table 5. Effects of OVA on cytokines in bronchoalveolar lavage fluid of a mouse asthma model.

[0113]

[0114] Compared with the blank control group, P<0.001, standard ### Compared with the model group, P<0.001, standard *** .

[0115] Table 6. Effects on cell typing of bronchoalveolar lavage fluid in an OVA-induced mouse asthma model.

[0116]

[0117]

[0118] Compared with the blank control group, P<0.001, standard ### Compared with the model group, P<0.05, standard * P<0.01, standard ** P < 0.001, standard *** .

[0119] Experiment Example 4: Effects of acetylcholine and histamine-induced asthma in guinea pigs

[0120] Animal: Guinea pig, standard grade, weight 180-200g

[0121] Drug: Aminophylline tablets, product of Tianjin Lisheng Pharmaceutical Co., Ltd., specification: 0.1g / tablet, batch number: 1905006.

[0122] Reagent: Acetylcholine, Sigma, Specification: 25g / bottle, Batch No.: BCBR3675.

[0123] Histamine, Sigma, Specification: 5g / bottle, Batch No.: BCBQ3927V.

[0124] Sodium chloride solution, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., specification: 500mL / bottle, batch number: 1803193205.

[0125] Instrument: Electronic balance, METTLER TOLEOD, model: MS1003S101.

[0126] Animal weight scale, Shanghai Xiangchuan Electronic Weighing Instrument Co., Ltd., Model: ACX-SC-DA.

[0127] Cough and wheezing induction device, Shanghai Xinruan, model: XR-YLS-8A.

[0128] Experimental methods: Animal screening: After pre-feeding, healthy guinea pigs were placed in a cough and wheezing induction device. 20 mL of a mixture of equal volumes of 2% acetylcholine and 0.1% histamine was placed in the nebulizer cup. The mixture was sprayed for 10 seconds, and the latency period for wheezing convulsions in the guinea pigs was observed. Those with a latency period of less than 150 seconds were used for the experiment.

[0129] Formal experiment: Qualified animals were randomly divided into 6 groups according to body weight, with 10 animals in each group, half male and half female. Each group was administered the drug by gavage. The model control group was administered the same volume of physiological saline (5 mL / kg body weight) by gavage. The test drug was administered at low, medium and high doses, with doses of 5.57, 11.15 and 22.30 g crude drug / kg, respectively, once a day for 7 consecutive days. One hour after the last administration, the same method was used to observe the latency period of panting convulsions in guinea pigs.

[0130] Detection indicator: the incubation period of panting convulsions in guinea pigs.

[0131] The results showed that, compared with the model group, the low-dose, medium-dose, and high-dose traditional Chinese medicine combination and the positive control drug all significantly prolonged the latency of wheezing seizures. (Results are as follows...) Figure 3 Table 7.

[0132] Table 7 Effects of acetylcholine and histamine-induced latency of panting in guinea pigs

[0133]

[0134]

[0135] Compared with the model group, P<0.001, standard *** .

[0136] Experiment 5: Effects of Experiment 5 on Lung Index and Serum TNF-α in Mice with Viral Upper Respiratory Tract Infection

[0137] Experimental materials

[0138] Animals: BALB / c mice, SPF grade, both male and female, a total of 72 mice, weighing 18-22g.

[0139] Drug: Ribavirin tablets, Sichuan Meidakang Pharmaceutical Co., Ltd., batch number: H20213197; specification: 0.1g / tablet.

[0140] Drug: Composition granules, provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.

[0141] Reagents:

[0142] Chloral hydrate, Shanghai Sinopharm Chemical Reagent Co., Ltd., Specification: 250g / bottle, Batch No.: 30037517;

[0143] Influenza A virus A / PR8 / 34 (H1N1): provided by the Institute of Virology, Chinese Academy of Preventive Medicine;

[0144] TNF-α Reagent Kit: Thermo Fisher Scientific, Catalog No.: 88-50450-86

[0145] instrument:

[0146] Electronic balance, Sartorius, Germany, model: BS224S;

[0147] Weight scale, G&G brand, China, Model: TC3K;

[0148] Centrifuge, Eppendorf, Germany, Model: 5804R;

[0149] Microplate reader, Meigu Molecular, USA, Model: Flexstation3.

[0150] Experimental methods

[0151] Seventy-two healthy SPF-grade mice, half male and half female, weighing 18–22 g, were randomly divided into six groups of 12 mice each using a random number lookup table. The groups were then labeled: blank control group, model control group, positive control group, low-dose composition group, medium-dose composition group, and high-dose composition group. On the day of administration, mice in the normal control group were lightly anesthetized with 0.35 g / kg of 10% chloral hydrate and administered 30 μL of physiological saline intranasally. The other groups were administered the drug at 4 times the LD50. 50 (LD50) 50 =10 -1.71 Influenza A virus (A / PR8 / 34) was administered via nasal drops to mice at a dose of 30 μL per mouse, representing a 1-fold LD50 infection. 50 Half an hour after infection, the normal group (equal volume of normal saline), the model data group (equal volume of normal saline), and the ribavirin group (150 mg / kg) were divided into three groups. -1 The composition was divided into three groups: a high-dose group (37.44 g crude drug / kg), a medium-dose group (18.72 g crude drug / kg), and a low-dose group (9.36 g crude drug / kg). The volume of medication administered to each group was 0.2 mL / 10 g. -1 The medication was administered via gavage once daily for 7 consecutive days.

[0152] Testing indicators:

[0153] ① Observe the growth and behavioral changes of mice in each group using routine observation.

[0154] ② After the last administration of the drug, the animals in each group were sacrificed, and the intact lung tissue of the mice was harvested. The tissue was dried with clean gauze, weighed, and the lung index and lung index inhibition rate were calculated.

[0155] Lung Index = Total Lung Weight (g) / Body Weight (g) * 10

[0156] Inhibition rate = (mean lung index) 模型组 - Average Lung Index 实验组 )*100%

[0157] ③ After the last administration of the drug, blood was collected from the eyes of mice in each group by enucleation, centrifugation, and serum collection. The TNF-α level in mice was measured using ELISA.

[0158] Experimental results

[0159] Effects on lung index in mice with upper respiratory tract infection

[0160] As shown in Table 8, the lung index of the model group mice was significantly different from that of the normal group (P<0.01). After drug treatment, the ribavirin group, as well as the high-dose, medium-dose, and low-dose groups of the composition, all reduced the lung index of mice with upper respiratory tract infection, showing significant differences compared with the model group (P<0.05). This indicates that the composition can inhibit upper respiratory tract infection in mice caused by influenza A virus.

[0161] Table 8 Effects of the composition on lung index in mice with upper respiratory tract infection

[0162]

[0163] Note: Compared with the blank control group, ▲P<0.05; compared with the model control group, △P<0.05; compared with the positive drug group, ■P<0.05.

[0164] As shown in Table 9, serum TNF-α levels were significantly higher in the upper respiratory tract infection model group compared to the normal group (P<0.05). After drug treatment, serum TNF-α levels decreased in the ribavirin group and the high-dose combination group, showing a significant difference compared to the model group (P<0.05). This indicates that the inhibitory effect of the combination on upper respiratory tract infection caused by influenza A virus is related to the reduction of serum TNF-α levels.

[0165] Table 9. Effects of the composition on TNF-α in mice with upper respiratory tract infection.

[0166]

[0167] Note: Compared with the blank control group, ▲P<0.05; compared with the model control group, △P<0.05; compared with the positive drug group, ■P<0.05.

[0168] Effects of Experiment 6 on Mice with Acute Bacterial Upper Respiratory Tract Infection

[0169] Experimental materials

[0170] Animals: SPF-grade ICR mice, weighing (20±2.0)g, half male and half female, a total of 72 mice, weighing 18-20g.

[0171] drug:

[0172] Methicillin-resistant Staphylococcus aureus (MRSA), ATCC (American Technical Control Center), Catalog No.: ATCC43300.

[0173] Xylozoline hydrochloride nasal spray, Grand Pharmaceutical (China) Co., Ltd., batch number: 210821.

[0174] instrument:

[0175] Electronic balance, Sartorius, Germany, model: BS224S;

[0176] Weight scale, G&G brand, China, Model: TC3K;

[0177] Centrifuge, Eppendorf, Germany, Model: 5804R;

[0178] Microplate reader, Meigu Molecular, USA, Model: Flexstation3;

[0179] Cell counter: Thermo Fisher Scientific, USA, Model: Invitrogen Countess;

[0180] Upright fluorescence microscope: Leica GmbH, Germany, model: DM4000B.

[0181] Experimental methods

[0182] Seventy-two ICR mice (half male and half female, weighing 20-22g) were randomly divided into six groups of 12 mice each. The six groups were: a blank control group, a model control group, low-, medium-, and high-dose groups of the traditional Chinese medicine composition (9.36g crude drug / kg, 18.72g crude drug / kg, and 37.44g crude drug / kg, respectively), and a positive control group (0.05g / kg xylometazoline hydrochloride nasal spray). Before the experiment, methicillin-resistant Staphylococcus aureus (MRSA) was passaged twice in live mice and cultured at 37℃ in a CO2 environment for 24 hours. The cultured bacteria were then eluted with sterile physiological saline at a concentration of 1.2 × 10⁻⁶. 9 1 colony forming unit (CFU) / ml bacterial suspension. The external nasal cavity, oral cavity and head of mice were disinfected with 75% alcohol. After the alcohol evaporated, the mice were lightly anesthetized with ether and placed in a supine position. The control group was treated with sterile saline solution, while the other groups were treated with methicillin-resistant Staphylococcus aureus (MRSA) bacterial suspension for nasal instillation, 20 μl per mouse.

[0183] Starting from the day of model establishment, the following intranasal administration methods were used: blank group and model group, 0.5% Tween-80; positive group, xylometazoline hydrochloride nasal spray; low, medium, and high dose groups of the traditional Chinese medicine composition (9.36g crude drug / kg, 18.72g crude drug / kg, 37.44g crude drug / kg). 10μl per mouse, twice a day for two consecutive days. Mice in each group were sacrificed by cervical dislocation on the 4th day after administration.

[0184] Before euthanizing the mice, the external nasal cavity, oral cavity, and head were disinfected with 75% alcohol. After the alcohol evaporated, the mice were euthanized, and the nasopharynx was irrigated with sterile physiological saline twice, 50 μl each time. 5 μl of the irrigation fluid was spread on an agar plate and incubated at 37°C in a CO2 environment for 48 h. The number of colonies was then counted and expressed as CFU / mouse.

[0185] The results show:

[0186] The number of methicillin-resistant Staphylococcus aureus (MRSA) colonies cultured from the nasopharyngeal lavage fluid of the model group mice was significantly higher than that of the control group mice, showing a statistically significant difference. The number of MRSA colonies in the nasopharyngeal lavage fluid of the positive group and each dose group of the composition was significantly lower than that of the model group mice, showing a statistically significant difference. The results are shown in Table 10.

[0187] Table 10. Methicillin-resistant Staphylococcus aureus colony count in nasopharyngeal lavage fluid (CFU / animal, ×10⁻¹⁰) 3 )

[0188]

[0189] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0190] The composition proposed in this invention can significantly regulate the lung index of mice with virus-induced upper respiratory tract infection, reduce the level of the inflammatory factor TNF-α in serum, and also has a certain preventive and therapeutic effect on mice with acute upper respiratory tract infection induced by methicillin-resistant Staphylococcus aureus.

[0191] Experiment 7 Effects of Bacterial Combined with Smoke-Induced Acute Bronchitis in Rats

[0192] Experimental materials

[0193] Animals: SD rats, SPF grade, male, 60 in total, weighing 200-220g.

[0194] Drug: Yinhuang Qingfei Capsules (Hunan Anbang Pharmaceutical Co., Ltd., Specification: 0.15g / capsule, Batch No.: 191209).

[0195] Reagents: Tobacco leaves (self-made), IL-6 (batch number: 201912), IL-8 (batch number: 201912), IL-10 (batch number: 201912) ELISA detection kit (Bio-Swamp);

[0196] Instruments: Ultra-low temperature freezer (Haier, model: DW-861286); Electronic balance (Beijing Sartorius Instrument Systems Co., Ltd., model: BS2244); Electric thermostatic drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd., model: DHG-9076A); Microplate reader (Molecular Devices, model: FlexStation 3).

[0197] Experimental methods

[0198] Sixty SD rats were randomly divided into a normal control group, a model control group, a Yinhuang Qingfei Capsule control drug group (0.18 g·kg-1), and low-, medium-, and high-dose traditional Chinese medicine composition granule groups (6.48 g crude drug / kg, 12.96 g crude drug / kg, 25.92 g crude drug / kg), with 10 animals in each group. Except for the normal control group which was not treated, an acute bronchitis model in rats was replicated by the smoking method (50 g of sawdust and tobacco shreds were placed in a smoking furnace and ignited in each group to avoid open flames, and the rats in the smoking chamber were stimulated by smoking, 30 minutes each time, twice a day) for 2 consecutive weeks. After the model was established, the rats in each group were continuously administered for 7 days, once a day. The normal control group and the model control group were respectively intragastrically administered an equal volume of pure water.

[0199] Detection indexes: The rats were anesthetized with 10% chloral hydrate at 0.35 g / kg, and the left lung lobe was lavaged with 1 mL of normal saline, repeated 3 times, and the alveolar lavage fluid was collected. The contents of IL-6, IL-8, and IL-10 in the alveolar lavage fluid were detected by ELISA according to the steps in the kit instruction manual.

[0200] The results showed that compared with the normal group, the rats in the model group showed symptoms such as rapid breathing, louder breathing sounds, and coughing. The contents of IL-6 and IL-8 increased, and the content of IL-10 decreased (P<0.01), indicating that the model was successfully established. Compared with the model control group, the content of IL-6 in the alveolar lavage fluid in the medium- and high-dose groups of the traditional Chinese medicine composition, and the contents of IL-8 in the low-, medium-, and high-dose groups were significantly reduced (P<0.05), and the content of IL-10 in the alveolar lavage fluid in the medium- and high-dose groups was significantly increased (P<0.05). See Table 11.

[0201] Table 11 Effects on acute bronchitis in rats caused by bacteria combined with smoking

[0202]

[0203] Note: Compared with the model group, *P<0.05, **P<0.01.

[0204] Experimental Example 8 Effects on chronic bronchitis in rats caused by LPS

[0205] Animals: SD rats, SPF grade, male, a total of 60, with a body weight of 180 - 220 g. Purchased from Beijing Spebio Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0010.

[0206] Drugs: Ke Te Ling Capsules, produced by the General Pharmaceutical Factory of Guangzhou Baiyunshan Pharmaceutical Holdings Co., Ltd., batch number 4560062.

[0207] Reagents: Tobacco leaves (self-made); LPS (Beijing Dingguo Changsheng Biotechnology Co., Ltd., batch number 156M4131V); Interleukin (IL)-6, IL-10 ELISA kit (Shanghai Jijin Chemical Technology Co., Ltd., batch numbers 20201127); Chloral hydrate, Shanghai Guoyao Chemical Reagent Co., Ltd., specification: 250g / bottle, batch number: 20200905;

[0208] Instruments: Ultra-low temperature freezer (Haier, model: DW-861286); Electronic balance (Beijing Sartorius Instrument Systems Co., Ltd., model: BS2244); Electric thermostatic drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd., model: DHG-9076A); Microplate reader (Molecular Devices, model: FlexStation 3).

[0209] Experimental Methods: Sixty SD rats were randomly divided into three groups according to body weight: a normal control group, a model control group, a cough suppressant capsule control group (0.11 g·kg⁻¹), and low, medium, and high dose traditional Chinese medicine granule groups (6.48 g crude drug / kg, 12.96 g crude drug / kg, and 25.92 g crude drug / kg), with 10 animals in each group. A rat model of chronic bronchitis was established using a combination of fumigation and intratracheal injection of LPS. The five rat groups in the model group were placed in a fumigation chamber and fumigated with 150 g of tobacco leaves twice daily for 30 minutes each time, for 30 days. The normal control group was kept in a standard environment. On days 1 and 15, the rats in the model group were injected intratracheally with 200 μg (1 mg / mL) of LPS per rat, while the normal control group received an equal volume of physiological saline intratracheally. Drug administration began on day 2 of model establishment, with an administration volume of 10 mL / kg, and continued by gavage for 30 days.

[0210] Detection indicators: ① Malondialdehyde (MDA) content in bronchoalveolar lavage fluid was detected by colorimetric method according to the kit instructions. ② Serum inflammatory factors IL-6 and IL-10 were detected by ELISA.

[0211] The results showed that the rat model replicated using LPS exhibited significantly increased cellular oxygen consumption due to hypoxia and chronic inflammation, resulting in the production of a large number of free radicals. The MDA content in the lung tissue of this rat model was significantly higher than that of the control group. Administration of the traditional Chinese medicine composition significantly reduced MDA content, significantly decreased serum levels of the pro-inflammatory factor IL-6, and significantly increased levels of the anti-inflammatory factor IL-10. This traditional Chinese medicine composition can reduce lipid peroxidation, alleviate oxygen free radical damage, and regulate the inflammatory response, thereby reducing airway damage from chronic bronchitis and protecting lung tissue. The results are shown in Table 12.

[0212] Table 12 Effects of LPS on LPS-induced chronic bronchitis in rats

[0213]

[0214] Note: Compared with the model group, *P<0.05, **P<0.01.

[0215] Experimental Example 9: Group A beta-hemolytic streptococcus-induced acute pharyngitis model in rats

[0216] Experimental materials

[0217] Animals: SD rats, SPF grade, half male and half female, a total of 60 rats, weighing 220-250g.

[0218] Drug: Amoxicillin capsules, product of Harbin Pharmaceutical Group General Factory, batch number: A1406029; specification: 0.25g / capsule.

[0219] Strains: Group A beta-hemolytic streptococci were purchased from the National Institutes for Food and Drug Control.

[0220] Reagents: 0.9% sodium chloride solution, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., specification: 500mL / bottle, batch number: 1803193205; chloral hydrate, Shanghai Guoyao Group Chemical Reagent Co., Ltd., specification: 250g / bottle, batch number: 20180629.

[0221] Instruments: Electronic balance, METTLER TOLEOD, model: MS1003S 101; Animal weight scale, Kagawa, model: ACX-SC-DA.

[0222] Experimental methods

[0223] Sixty SD rats, weighing 220–250 g (half male and half female), were randomly divided into six groups according to body weight: a normal control group, a model group, low-, medium-, and high-dose groups of the traditional Chinese medicine composition (6.48 g crude drug / kg, 12.96 g crude drug / kg, and 25.92 g crude drug / kg, respectively), and a positive control group (amoxicillin 0.275 g / kg), with 10 rats in each group. Except for the normal control group, the oral cavity of each rat in the other groups was opened with sterile forceps to expose the maxillary mucosa, and 1×10⁻⁶ aspirated aspirate of the traditional Chinese medicine composition was injected into the rats. 9A CFU / mL solution of beta-hemolytic streptococci was used to horizontally puncture the nasopharyngeal mucosa, followed by injection of the bacterial solution. The procedure was performed to the extent that slight punctate bleeding was expected. 0.1 mL was injected per animal, and the infection was repeated for 2 days. The normal control group received physiological saline under the same conditions. One hour after infection on day 1, animals in each group were administered the drug by gavage at a dose of 10 mL / kg, once daily for 3 consecutive days. The normal control and model control groups received distilled water under the same conditions. On day 4, the pharyngeal tissue lesions of the rats were observed, and pharyngeal signs were scored. The scoring criteria for pharyngeal signs by visual observation were as follows: 0 points: no redness or swelling of the hard palate; 1 point: redness and swelling of the soft palate; 2 points: congestion and redness and swelling of the soft palate, soft palatal arch, or posterior pharyngeal wall; 3 points: red petechiae on the soft palate, soft palatal arch, and posterior pharyngeal wall; 4 points: red petechiae on the soft palate, soft palatal arch, and posterior pharyngeal wall, with white purulent spots visible.

[0224] The results showed that, upon visual observation, the rats in the model control group exhibited red ecchymoses with white purulent spots in their pharynx, and their pharyngeal signs scores were significantly increased, showing a significant difference compared to the normal control group (P < 0.01), indicating successful model establishment. The pharyngeal lesions in rats in the high, medium, and low dose groups of the traditional Chinese medicine composition were significantly reduced, and the pharyngeal signs scores decreased, showing significant differences compared to the model control group (P < 0.01, P < 0.01, P < 0.05). The results are shown in Table 13.

[0225] Table 13 Effects on the rat model of acute pharyngitis induced by group A beta-hemolytic streptococcus.

[0226]

[0227] Note: Compared with the model control group, **P<0.01, *P<0.05.

[0228] Experimental Example 10: Ammonia-induced acute pharyngitis model in rats

[0229] Animals: SD rats, SPF grade, half male and half female, a total of 60 rats, weighing 150-170g.

[0230] Drug: Amoxicillin capsules, product of Harbin Pharmaceutical Group General Factory, batch number: A1406029; specification: 0.25g / capsule.

[0231] Reagents: 0.9% sodium chloride solution, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., specification: 500mL / bottle, batch number: 1803193205; chloral hydrate, Shanghai Guoyao Group Chemical Reagent Co., Ltd., specification: 250g / bottle, batch number: 20180629; ammonia water, Nanjing Chemical Reagent Co., Ltd., specification: 500mL / bottle, batch number: 13041230175; interleukin-1β (IL-1β, catalog number: SEKR-0002) kit, interleukin-6 (IL-6, catalog number: SEKR-0005) kit, tumor necrosis factor-α (TNF-α, catalog number: SEKR-0009) kit, Beijing Solarbio Science & Technology Co., Ltd.

[0232] Instruments: Electronic balance, METTLER TOLEOD, model: MS1003S101; Animal weight scale, Kagawa, model: ACX-SC-DA; CENTRFUGE5840 refrigerated centrifuge, eppendorf; Laryngeal nebulizer, Jiangsu Ping An Medical Instrument Co., Ltd.; Microplate reader, FlexStation 3, American Microplate Reader.

[0233] Experimental Methods: Sixty SD rats, weighing 150–170 g (half male and half female), were randomly divided into six groups according to body weight: a normal control group, a model group, low-, medium-, and high-dose groups of the traditional Chinese medicine composition (6.48 g crude drug / kg, 12.96 g crude drug / kg, and 25.92 g crude drug / kg, respectively), and a positive control group (amoxicillin 0.36 g / kg), with ten rats in each group. Except for the normal control group, all other groups were treated with a laryngeal spray containing 15% ammonia water, three puffs each time, twice daily for three consecutive days, to establish an acute pharyngitis model in rats. The normal control group was sprayed with an equal volume of physiological saline. On the fourth day after modeling, all groups were administered the drug by gavage at 10 mL / kg once daily for five consecutive days. The normal control group and the model group were given an equal volume of distilled water. The general condition of the rats in each group was observed and recorded daily after modeling. One hour after the last administration, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, blood was collected from the abdominal aorta, and serum was obtained by centrifugation. The levels of IL-1β, IL-6, and TNF-α in the serum were then determined by enzyme-linked immunosorbent assay (ELISA).

[0234] The results showed that: compared with the normal control group, the levels of IL-1β, IL-6, and TNF-α in the serum of rats in the model group were significantly increased (P < 0.01). Compared with the model group, the high, medium, and low dose groups of the traditional Chinese medicine composition could significantly reduce the content of IL-1β in the serum of rats (P < 0.01, P < 0.01, P < 0.05); the high, medium, and low dose groups of the traditional Chinese medicine composition could significantly reduce the content of IL-6 in the serum of rats (P < 0.01, P < 0.01, P < 0.05); the high and medium dose groups of the traditional Chinese medicine composition could significantly reduce the content of TNF-α in the serum of rats (P < 0.01, P < 0.05). The results are shown in Table 14.

[0235] Table 14 Effects of the traditional Chinese medicine composition on IL-1β, IL-6, and TNF-α in the serum of rats with acute pharyngitis induced by ammonia water

[0236]

[0237]

[0238] Note: Compared with the model group, *P < 0.05, **P < 0.01.

[0239] The composition proposed by the present invention can significantly improve the pathological changes of the pharyngeal mucosal tissue in acute pharyngitis and promote the reduction or elimination of pharyngeal inflammation.

[0240] Experimental Example 11 Effects on the mouse model of Con A-mediated autoimmune hepatitis (AIH)

[0241] Experimental materials

[0242] Animals: C57BL / 6 mice, SPF grade, male, a total of 60 mice, with a body weight of about 22 g. Purchased from Beijing Sino-British SIPPR / BK Lab Animal Co., Ltd., license number: SCXK (Beijing) 2019-0010, certificate number: No. 110324221105351178.

[0243] Positive control drug: Azathioprine, produced by Shanghai Xinyi Pharmaceutical Factory Co., Ltd., specification: 50 mg / tablet, batch number 20200109.

[0244] Test drug: The granules of Example 1, provided by Jiangsu Kanion Pharmaceutical Co., Ltd.

[0245] Reagents: Concanavalin A (Con A), Sigma-Aldrich, USA, batch number: 11028-71-0; 0.9% sodium chloride solution, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., specification: 500mL / bottle, batch number: 1803193205; formaldehyde, Nanjing Chemical Reagent Co., Ltd., specification: 500mL / bottle, batch number: 160517018E; alanine aminotransferase (ALT) and aspartate aminotransferase (AST) detection kits, Shanghai Renjie Biotechnology Co., Ltd., batch numbers: 080120, 080122; IL-4, Mouse IL-4Valukine ELISA Kit, batch number: 600803, specification: VAL803; 96-well plate; TNF-α, Mouse TNF-AValukine ELISA Kit, batch number: 600609, specification: VAL609; 96-well plate.

[0246] Instruments: Ultra-low temperature freezer, Haier, model: DW-86L728; Electronic balance, Sartorius Scientific Instruments, model: BSA224S-CW; Electronic balance, Sartorius, Germany, model: BS224S; Weight scale, G&G, China, model: TC3K; Centrifuge, Eppendorf, Germany, model: 5804R; Microplate reader, Flexstation 3, USA.

[0247] Experimental methods

[0248] Sixty mice were randomly divided into a normal control group, a model group, different dosage groups of the herbal composition granules of this invention (9.36 g crude drug / kg, 18.72 g crude drug / kg, 37.44 g crude drug / kg), and a positive control group (2 mg / kg), with 10 mice in each group. The model group, the low, medium, and high dosage groups of the herbal composition granules, and the positive control group were injected with ConA (12 mg / kg) via tail vein once to establish an AIH model. The normal control group and the model group were given an equal volume of physiological saline by gavage. From day 1, the mice were administered the drug once daily by gavage at a dose of 10 mL / kg for 5 consecutive days at fixed times. Twenty-four hours after the last administration, the mice were enucleated, blood was collected by centrifugation, serum was obtained, and the abdominal cavity was opened to harvest liver tissue for later use.

[0249] Detection indicators: ① Serum ALT and AST level determination: Centrifuge whole blood (4500 r / min) for 10 min to separate serum, and use kits to detect serum ALT and AST levels. ② HE staining observation of mouse liver pathological changes: Separate liver tissue, fix the left lobe of the liver with formaldehyde, and after 24 h of formaldehyde fixation, dehydrate and embed it, section with a thickness of 1 μm, routinely stain with HE, and observe the pathological changes of liver tissue. ③ ELISA method for detecting IL-4 and TNF-α levels in liver tissue: Take frozen liver tissue, prepare homogenate, add 100 μL of liver homogenate to the reaction wells, and incubate at 37℃ for 2 h; add 100 μL of the corresponding antibody, incubate for 2 h; add 100 μL of chromogenic solution, incubate in the dark for 10-15 min; add stop solution to terminate the reaction, and measure the absorbance (OD) value of each well at 450 nm.

[0250] Liver function tests in mice showed that the levels of ALT and AST in the model group were significantly higher than those in the normal group (P<0.001), indicating successful model establishment. The levels of ALT and AST in the medium-dose and high-dose groups of the traditional Chinese medicine composition and the positive control group were significantly improved compared with the model group (P<0.01, P<0.001). The results are shown in Table 15.

[0251] Results of TNF-α and IL-4 levels in mouse liver tissue showed that compared with the normal group, the IL-4 level in the model group was significantly decreased (P < 0.001), while the TNF-α level was significantly increased (P < 0.001). After treatment with the traditional Chinese medicine composition and positive control drug, the IL-4 level rebounded significantly (P < 0.001), and the expression level of TNF-α was significantly inhibited (P < 0.001). This indicates that the traditional Chinese medicine composition can effectively promote the release of anti-inflammatory factors and inhibit the secretion of pro-inflammatory factors. The results are shown in Table 16.

[0252] Pathological results showed that the model group mice exhibited extensive hepatocyte death, disordered liver lobule structure, abundant inflammatory cell infiltration in the portal area, and localized focal necrosis, indicating successful replication of the mouse immune-mediated liver injury model (P<0.001). The traditional Chinese medicine composition reduced liver inflammation and hepatocyte apoptosis, with the high and medium dose groups showing better effects and statistically significant differences compared to the model group (P<0.01, P<0.001). No obvious abnormalities were observed in the liver structure of the normal control group mice; the model group mice showed extensive hepatocyte necrosis, disordered hepatic cord arrangement, and inflammatory cell infiltration; the pathological changes in each dose group of the traditional Chinese medicine composition and the positive control group were less severe than those in the model group, and the high-dose group of the traditional Chinese medicine composition and the positive control group only showed mild hepatocyte necrosis (P<0.001). Figure 5 The scoring results are shown in Table 17.

[0253] Table 15 Effects of ALT and AST levels in liver tissue of mice with autoimmune hepatitis

[0254]

[0255] Note: Compared with the normal group, ### P < 0.001; compared with the model group, **P < 0.01, ***P < 0.001

[0256] Table 16 Effects on TNF-α and IL-4 levels in liver tissues of mice with autoimmune hepatitis

[0257]

[0258] Note: Compared with the normal group, ### P < 0.001; compared with the model group, ***P < 0.001.

[0259] Table 17 Liver pathological scores of mice with autoimmune hepatitis

[0260]

[0261] Note: Compared with the normal group, ### P < 0.001; compared with the model group, *P < 0.05, **P < 0.01, ***P <0.001.

[0262] Effect of Experimental Example 12 on the mouse model of autoimmune liver injury induced by immunizing agents

[0263] Experimental materials

[0264] Animals: C57BL / 6 mice, SPF grade, male, a total of 70 mice, weighing 22 - 24 g. Purchased from Beijing Spepharm Biotechnology Co., Ltd., license number: SCXK(Beijing)2019 - 0010, certificate number: No.110324221106451 (175).

[0265] Positive control drug: Azathioprine, Shanghai Xinyi Pharmaceutical Factory Co., Ltd., specification: 50 mg / tablet, batch number 20200109.

[0266] Test drug: Granules of Example 1, provided by Jiangsu Kanion Pharmaceutical Co., Ltd.

[0267] Reagents: Complete Freund's adjuvant, Sigma-Aldrich, USA, batch number: 9007-81-2; 0.9% sodium chloride solution, Shijiazhuang No. 4 Pharmaceutical Co., Ltd., specification: 500mL / bottle, batch number: 1803193205; formaldehyde, Nanjing Chemical Reagent Co., Ltd., specification: 500mL / bottle, batch number: 160517018E; alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assay kit, Shanghai Renjie Biotechnology Co., Ltd., batch numbers: 080120, 080122; IL-1β, Mouse IL-1beta Valukine ELISA Kit, batch number: 6006403, specification: VAL601; 96-well plate; IL-6, Mouse IL-6 Valukine ELISA Kit, batch number: 600504, specification: VAL604; 96-well plate; TNF-α, Mouse TNF-A Valukine ELISA Kit, lot number: 600609, specification: VAL609; 96-well plate.

[0268] Instruments: Ultra-low temperature freezer, Haier, model: DW-86L728; Electronic balance, Sartorius Scientific Instruments, model: BSA224S-CW; Electronic balance, Sartorius (Germany), model: BS224S; Weighing scale, G&G (China), model: TC3K; Centrifuge, Eppendorf (Germany), model: 5804R; Microplate reader, Amicon Ultra-15, model: Flexstation 3; Amicon Ultra-15 filter membrane, Millipore (USA), model: UFC910096; Sepharose column, Pharmacia (USA), model: Sepharose CL-2B.

[0269] Experimental methods

[0270] An autoimmune hepatitis (AIH) model was induced using liver S100 antigen. Ten mice were sacrificed by intraperitoneal injection of an excessive amount of sodium pentobarbital (2%). The liver was removed by dissection, minced, and homogenized with PBS under ice bath conditions. The homogenate was centrifuged at 150g for 10 min. The supernatant was further centrifuged at 100,000g for 60 min. The resulting supernatant was reduced to 5 mL using an Amicon Ultra-15 filter membrane and then purified using a 90cm-6B Sepharose column to collect the S100 antigen. The S100 antigen was emulsified with full Freund's adjuvant to a concentration of 1 g / L. Sixty mice were randomly divided into a normal control group (n=10) and a model group (n=50). Mice in the model group received an intraperitoneal injection of the S100 emulsifier on day 1 of modeling, and the injection was repeated on day 7, with each injection being 1 mL. Mice that successfully modeled the disease were randomly divided into three groups: a model group, a azathioprine positive control group (2 mg / kg), and different dosage groups of traditional Chinese medicine granules (9.36 g crude drug / kg, 18.72 g crude drug / kg, and 37.44 g crude drug / kg), with 10 mice in each group. The normal control group and the model group were given an equal volume of physiological saline by gavage, while the other groups were given the drug by gavage once a day at a dose of 10 mL / kg for two consecutive weeks at fixed times. Twenty-four hours after the last administration, the mice were enucleated, blood was collected by centrifugation, serum was obtained, and the liver tissue was harvested from the abdominal cavity for later use.

[0271] Detection indicators: ① Serum indicators: ELISA was used to detect serum inflammatory factors. Whole blood was collected and allowed to stand at room temperature for 30 minutes, then centrifuged for 10 minutes (3000 r / min) to separate serum. One portion of the serum sample was treated with antibodies and chromogenic agents according to the kit instructions, and the absorbance at 450 nm was measured using an ELISA reader. The concentrations of IL-1β, IL-6, and tumor necrosis factor (TNF-α) were calculated based on the standard curve. The other portion of the serum sample was used to detect liver injury indicators, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. ② HE staining for observation of mouse liver pathological changes: Liver tissue was separated, and the left lobe of the liver was fixed in formaldehyde. After 24 hours of formaldehyde fixation, the tissue was dehydrated, embedded, and sectioned to a thickness of 1 μm. Routine HE staining was performed to observe the pathological changes in the liver tissue.

[0272] Liver function tests in mice showed that the levels of ALT and AST in the model group were significantly higher than those in the normal group (P < 0.001), indicating successful model establishment. ALT and AST levels in all dosage groups of the traditional Chinese medicine composition and the positive control group were significantly improved compared to the model group (P < 0.05, P < 0.001). The results are shown in Table 18.

[0273] The results of serum inflammatory factors IL-1β, IL-6, and TNF-α in mice showed that, compared with the normal group, the levels of IL-1β, IL-6, and TNF-α in the liver tissue of the model group mice were significantly higher than those in the normal group (P < 0.001). After treatment with the traditional Chinese medicine composition and positive control drug, the levels of IL-1β, IL-6, and TNF-α were significantly reduced (P < 0.05, P < 0.01, P < 0.001). This indicates that the traditional Chinese medicine composition effectively alleviated the inflammatory response in mice with autoimmune hepatitis. The results are shown in Table 19.

[0274] Pathological results showed that the liver tissue of the normal control group mice had normal structure with no cell necrosis or obvious lymphocyte infiltration. In the model group mice, inflammatory cell infiltration was observed in the portal areas and central lobules, with significant hepatocyte swelling and necrosis. The total pathological score was significantly different from that of the normal control group (P < 0.001). Both the positive control group and the traditional Chinese medicine combination group inhibited lymphocyte infiltration and hepatocyte necrosis, with the positive control group and the high-dose traditional Chinese medicine combination group showing the most significant effects (P < 0.001). Figure 6 The scoring results are shown in Table 20.

[0275] Table 18 Effects of Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of mice with autoimmune hepatitis

[0276]

[0277] Note: Compared with the normal group ### P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0278] Table 19 Effects of IL-1β, IL-6, and TNF-α levels in the serum of mice with autoimmune hepatitis

[0279]

[0280] Note: Compared with the normal group ### P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0281] Table 20 shows the liver pathological scores of mice with autoimmune hepatitis.

[0282]

[0283] Note: Compared with the normal group ### P<0.001; compared with the model group, *P<0.05, ***P<0.001.

[0284] The composition proposed in this invention can significantly reduce liver inflammation in mice with autoimmune hepatitis, inhibit hepatocyte apoptosis, and exert a liver-protective effect. It may be a potential therapeutic drug for treating autoimmune hepatitis and has high application value and broad development prospects.

[0285] Experimental Example 13: Effects on a 3% DSS-induced mouse model of acute ulcerative colitis

[0286] 1. Experimental materials

[0287] Animals: 60 male C57BL / 6J mice, SPF grade, weighing 18-20g, purchased from the Comparative Medicine Center of Yangzhou University.

[0288] Drug: Ciprofloxacin Hydrochloride Capsules, Zhejiang CR Sanjiu Zhongyi Pharmaceutical Co., Ltd., 0.25g / capsule, batch number: 211205.

[0289] Reagents: Dextran sulfate (DSS), MP Biomedicals, USA, 100g, batch number: S7102. Mouse IL-6 ELISA kit: Beyotime, 96T, catalog number: PI326. Sodium chloride, Sinopharm Chemical Reagent Co., Ltd. (Shanghai), 500g / bottle, batch number: 20171221. Formaldehyde, Sinopharm Chemical Reagent Co., Ltd. (Shanghai), 500mL / bottle, batch number: 20180313.

[0290] Instruments: Electronic balance, Sartorius (Germany), model: BS224S. Weighing scale, G&G (China), model: TC3K. Centrifuge, Eppendorf (Germany), model: 5804R. Microplate reader, Flexstation 3 (USA).

[0291] Test drug: The traditional Chinese medicine composition granules prepared in the embodiments of this invention were provided by Jiangsu Kangyuan Pharmaceutical Co., Ltd.; Drug dosage: The daily dose of raw medicinal materials for humans is 288g of raw medicinal materials. The clinical dose for adults (based on 70kg) is 4.1g of raw medicinal materials / kg. The equivalent dose for mice was calculated to be 37.44g of raw medicinal materials / kg. This dose was set as the high dose. Low, medium and high dose groups were set according to a dose ratio of 0.25:0.5:1, with doses of 9.36, 18.72 and 37.44g of raw medicinal materials / kg, respectively, which are equivalent to 2.3, 4.6 and 9.1 times the clinical dose, respectively.

[0292] 2. Experimental Methods

[0293] C57BL6 / J mice were randomly divided into 6 groups: a normal control group, a model control group, a positive control group (ciprofloxacin, 615 mg / kg), a low-dose group (9.36 g crude drug / kg), a medium-dose group (18.72 g crude drug / kg), and a high-dose group (37.44 g crude drug / kg). Each group consisted of 10 mice. Except for the normal control group, which had free access to purified water, the other groups were given freshly prepared 3% DSS solution daily for 7 days. From day 1 of model establishment, the positive control group (ciprofloxacin) and the low, medium, and high-dose groups of the present invention were administered the corresponding drugs by gavage at 20 mL / kg, while the normal control and model control groups were administered purified water by gavage once daily for 10 consecutive days.

[0294] 3. Detection indicators

[0295] (1) Disease Activity Index (DAI) score: Starting from day 1 of the experiment, the body weight of each group of mice was weighed and recorded at the same time every day. The characteristics of the stool of each group of mice were observed. The anus of the mice was wiped with a moist cotton ball to observe the blood in the stool. The DAI score was calculated according to the standards shown in Table 21. DAI score = body weight change score + stool characteristics score + bloody stool score.

[0296] Table 21 DAI Scoring Criteria

[0297]

[0298] (2) Colon length and pathological score: One hour after the last administration, mice were anesthetized and blood was collected. The colon from the cecum to the anus was retrieved and its length was measured. A 1 cm section of the distal colon was taken, fixed with 10% formaldehyde solution, and then subjected to dehydration, paraffin embedding, sectioning, and HE staining. The pathological scoring criteria are shown in Table 22.

[0299] Table 22. Colonic Histopathological Scoring Criteria

[0300]

[0301]

[0302] (3) ELISA method for determining serum inflammatory factor IL-6 activity level: Take serum, thaw and shake well. Perform the determination according to the method shown in the ELISA kit instructions.

[0303] 4. Experimental Results

[0304] (1) Effect of the traditional Chinese medicine composition of the present invention on the DAI score of mice with acute ulcerative colitis

[0305] Compared with the normal control group, the model control group had higher DAI scores on days 7-9 (p<0.01), and mice experienced weight loss and severe rectal bleeding, indicating successful model establishment. Compared with the model control group, the positive drug ciprofloxacin group had significantly lower DAI scores on days 7-9 (p<0.05, p<0.01), less weight loss, milder or no rectal bleeding, and better overall activity. The medium dose of the herbal composition of this invention significantly reduced DAI scores on days 8 and 9 (p<0.05); the high dose of the herbal composition of this invention significantly reduced DAI scores on days 7-9 (p<0.05, p<0.01), and mouse weight loss and rectal bleeding were improved to varying degrees. The results are shown in Table 23.

[0306] Table 23 Effects of the herbal composition of the present invention on the DAI score of mice

[0307]

[0308] Note: Compared with the normal control group, ## p<0.01; compared with the model control group, * p<0.05, ** p<0.01.

[0309] (2) Effects of the herbal composition of the present invention on colon length and pathological score in mice with acute ulcerative colitis

[0310] Compared with the normal control group, the colon length of mice in the model control group was significantly shortened, and large-scale mucosal damage, ulceration, and inflammatory cell infiltration were observed in the colon, with a significantly increased pathological score, indicating that the mouse model of acute ulcerative colitis was successfully established (p<0.01). Compared with the model control group, the colon length of the ciprofloxacin group was significantly increased, and pathological examination showed that only a few mice had mild ulceration or inflammation, with a significantly lower score, indicating better drug efficacy (p<0.01). The colon length and colon pathological score of mice in the medium and high dose groups of the herbal composition of this invention were between those of the model control group and the ciprofloxacin group, with a significantly increased colon length compared with the model control group (p<0.01), a significantly lower pathological score compared with the model control group, and significant improvement in pathological conditions such as colonic ulceration and inflammatory cell infiltration (p<0.05, p<0.01), suggesting that the herbal composition of this invention can alleviate colonic damage in mice. Figure 7 The results are shown in Table 24.

[0311] Table 24 Effects of the herbal composition of the present invention on colon length in mice

[0312]

[0313] Note: Compared with the normal control group, ## p<0.01; compared with the model control group, * p<0.05,** p<0.01.

[0314] (3) Effect of the herbal composition of the present invention on the activity of serum inflammatory factor IL-6 in mice with acute ulcerative colitis

[0315] Compared with the normal control group, the serum inflammatory factor IL-6 level in the model control group mice was significantly increased (p<0.01). Compared with the model control group, the serum IL-6 level in the ciprofloxacin group was significantly decreased (p<0.01). Medium and high doses of the herbal composition of this invention also significantly reduced the serum IL-6 level (p<0.01), indicating that the herbal composition of this invention has the effect of slowing down the release of the inflammatory factor IL-6. The results are shown in Table 25.

[0316] Table 25 Effects of the herbal composition of the present invention on serum IL-6 levels in mice with colitis

[0317]

[0318] Note: Compared with the normal control group, ## p<0.01; compared with the model control group, ** p<0.01.

[0319] Experimental Example 14: Effects on a 2% DSS-induced mouse model of chronic ulcerative colitis

[0320] 1. Experimental materials

[0321] Animals: 60 male C57BL / 6J mice, SPF grade, weighing 18-20g, purchased from the Comparative Medicine Center of Yangzhou University.

[0322] Drug: Mesalazine enteric-coated tablets, Jiamusi Luling Pharmaceutical Co., Ltd., Sunflower Pharmaceutical Group, Specification: 0.25g / tablet, Batch No.: 2005015;

[0323] Reagents: Same as in Experiment 13.

[0324] Instruments: Same as in Experiment 13.

[0325] Test drug: Same as in Experiment 13.

[0326] 2. Experimental Methods

[0327] C57BL6 / J mice were randomly divided into 6 groups: a normal control group, a model control group, a positive control group (mesalazine, 300 mg / kg), a low-dose group (9.36 g crude drug / kg), a medium-dose group (18.72 g crude drug / kg), and a high-dose group (37.44 g crude drug / kg). Each group consisted of 10 mice. Except for the normal control group, which received pure water, the other groups were given 2% DSS solution for free drinking for 5 days, followed by 14 days of free drinking pure water. This cycle was repeated once, and then 2% DSS solution was given for another 5 days of free drinking. The experimental period was 43 days. The 2% DSS solution was prepared every 2 days, and the bedding was changed daily. From day 1 of model establishment, the positive control group (mesalazine) and the low, medium, and high-dose groups of the present invention were administered the corresponding drugs by gavage at 20 mL / kg, while the normal control group and the model control group were given pure water by gavage. Administration was once daily for 43 consecutive days.

[0328] 3. Detection indicators

[0329] (1) Colon length and pathological score: Same as experimental case 13.

[0330] (2) ELISA method to determine the activity level of serum inflammatory factor IL-6: same as experimental example 13.

[0331] 4. Experimental Results

[0332] The results showed that by repeatedly administering 2% DSS to the drinking water to establish a mouse model of chronic ulcerative colitis, the colon length of the mice was significantly shorter than that of the normal control group, and the colon pathological score and serum IL-6 level were significantly increased (p<0.01), indicating that the mouse model of chronic ulcerative colitis was successfully established. The positive control group (mesalazine) and the low, medium, and high dose groups of the traditional Chinese medicine composition of this invention all significantly increased colon length, improved the pathological state of colonic ulceration, inflammatory cell infiltration, and mucosal damage, and inhibited the release of the inflammatory factor IL-6 (p<0.05, p<0.01). Figure 8 The results indicate that the herbal composition of this invention can effectively improve chronic ulcerative colitis in mice. The results are shown in Table 26.

[0333] Table 26 Effects of the herbal composition of the present invention on colon length, pathological score, and serum IL-6 level in mice with chronic ulcerative colitis.

[0334]

[0335] Note: Compared with the normal control group, ## p<0.01; compared with the model control group, * p<0.05, ** p<0.01.

[0336] The traditional Chinese medicine composition of this invention can effectively improve the symptoms of acute and chronic ulcerative colitis in mice induced by DSS and inhibit the development of the disease.

[0337] Experimental Example 15: Effects of Traditional Chinese Medicine Composition on Calf Serum Albumin C-BSA-Induced Chronic Glomerulonephritis

[0338] Preparation of cationized bovine serum albumin (C-BSA)

[0339] Following the Border method, add 67 ml of anhydrous ethylenediamine (EDA, Tianjin Damao Chemical Reagent Factory, batch number: 20210103) to 500 ml of double-distilled water, then slowly add 350 ml of 6 mol / L hydrochloric acid (Beijing Solarbio Science & Technology Co., Ltd., batch number: P1022) to adjust the pH to 4.75, maintaining the solution temperature at 25°C. Dissolve 5 g of bovine serum albumin (BSA, Sigma-Aldrich, batch number: A1933) in 25 ml of double-distilled water, then slowly add this solution to the anhydrous ethylenediamine solution while stirring constantly, maintaining the solution temperature at a constant 25°C. Finally, add 1.8 g of carbodiimide (EDC, Maclean's, batch number: N808856). Two hours later, the reaction was terminated with 30 ml of acetate buffer solution with pH 4.75 to obtain the C-BSA solution with the isoelectric point. The C-BSA solution was dialyzed with double-distilled water at 4℃ for 72 hours (with water changed every 3-5 hours), and then freeze-dried to obtain C-BSA powder with an isoelectric point (PI) of 8.4 or higher, which was stored at -20℃ for later use.

[0340] Establishing an animal model of chronic nephritis

[0341] Eighty male SPF-grade SD rats, weighing approximately 200g, were selected and acclimatized for one week, with fasting but free access to water for 12 hours. In week 1, an appropriate amount of cationic bovine serum albumin was dissolved in phosphate buffer (pH 7.2–7.4) to a concentration of 2 mg / mL, and mixed with an equal volume of incomplete Freund's adjuvant (Sigma-Aldrich, batch number: SLBT5128) to prepare a milky white suspension with a final concentration of 1 mg / mL. This suspension was injected subcutaneously at multiple points in the bilateral axillae and groin of each rat, 2 mL per rat. In week 2, PBS containing cationic bovine serum albumin was mixed with an equal volume of complete Freund's adjuvant (Sigma-Aldrich, batch number: SLBV6895) to prepare a milky white suspension with a final concentration of 1 mg / mL, and injected subcutaneously in the same manner, 2 mL per rat. In week 3, an appropriate amount of cationic bovine serum albumin was dissolved in PBS to a concentration of 2.5 mg / mL, and injected into the tail vein of each rat every other day, 1 mL per rat, for three consecutive weeks. Three weeks after administering cationic bovine serum albumin via tail vein injection, all urine samples were collected from each rat over 24 hours. The 24-hour urinary protein content was measured, and a 24-hour urinary protein level >10 mg was considered a successful model replication. Rats in the normal control group were injected with an equal volume of physiological saline at the same site on the same day.

[0342] The traditional Chinese medicine composition granules prepared in Example 1 of this application were administered to rats by gavage at different dosage groups (6.48 g crude drug / kg, 12.96 g crude drug / kg, 25.92 g crude drug / kg) and a positive control group of prednisolone acetate tablets (Guangdong Huanan Pharmaceutical Group Co., Ltd., batch number: 140502). Starting from day 1, rats were administered the drug once by gavage at a dose of 10 ml / kg for four consecutive weeks. The normal control group and the model group were administered an equal volume of physiological saline by gavage. One hour after the last administration, all urine samples were collected within 24 hours. Twenty-four hours after the last administration, rats were anesthetized by intraperitoneal injection of 0.35 g / kg of 10% chloral hydrate, and serum samples were collected for testing.

[0343] Detection indicators: ① The total urine of each rat within 24 hours after 4 weeks of drug administration was collected and the 24-hour urinary protein content of the rats was determined using the Coomassie Brilliant Blue reagent kit (Nanjing Jiancheng Technology Co., Ltd., catalog number: A045-2).

[0344] ② Rats were anesthetized with chloral hydrate. Blood was collected after exposing the abdominal aorta and centrifuged at 4°C (using a benchtop high-speed refrigerated centrifuge, Shanghai Luxiangyi Laboratory Instruments Co., Ltd., model: TGL-16M). Renal function indicators creatinine (Scr) and blood urea nitrogen (BUN) were measured using a blood urea nitrogen (BUN) kit (Nanjing Jiancheng Bioengineering Institute, batch number: 20220517) and a creatinine (Scr) kit (Nanjing Jiancheng Bioengineering Institute, batch number: 20220630). The levels of inflammatory factors TNF-α, IL-4, and IFN-γ in rat serum were detected using a TNF-α, IFN-γ, and IL-4 kit (Invitrogen, batch numbers 305271-004, 282909-006, and 271790-002, respectively). Values ​​were read using a multi-functional microplate reader (Molecular Devices, model: FlexStation3).

[0345] ③ Collect orbital venous blood from each group of rats into flow cytometry tubes. Add 10 μL of CD3, CD4, and CD8 monoclonal antibodies and 50 μL of EDTA anticoagulant (EDTA disodium salt, Sinopharm Chemical Reagent Co., Ltd., batch number: 20130528) to each tube, mix well, and incubate in the dark for 20 min. Add 2 mL of hemolysin (BD Biosciences, catalog number: 349202) to each tube, incubate in the dark for 20 min, centrifuge at 2000 rpm for 10 min, discard the supernatant, mix with 2 mL of PBS in each tube, centrifuge and discard the supernatant, repeat washing twice, discard the supernatant, resuspend in 2 mL of PBS, mix well, and then analyze the proportions of peripheral blood T lymphocyte subsets CD3, CD4, and CD8 by flow cytometry (NoveCyte, Eisen Biosciences, USA). (eBioscience, batch numbers D160312, D160328, D160407)

[0346] The results showed that compared with the normal control group, the 24-hour urinary protein and serum Scr and BUN levels in the model group rats were significantly increased, and the differences were statistically significant (P<0.01). Compared with the model group, the 24-hour urinary protein and serum Scr and BUN levels in rats in different dose groups of the traditional Chinese medicine granules and the prednisone acetate group were significantly decreased, and the differences were statistically significant (P<0.01, P<0.05). See Table 27.

[0347] Table 27 Effects of traditional Chinese medicine composition on 24-hour urinary protein, serum urea nitrogen, and creatinine in rats with chronic glomerulonephritis (x±s, n=10)

[0348]

[0349]

[0350] Note: *P<0.01, compared with the normal group; ▲ P<0.05, ▲▲ P<0.01, compared with the model group.

[0351] Compared with the normal control group, the serum levels of TNF-α and IFN-γ in the model group rats were significantly increased, while the IL-4 level was significantly decreased (P < 0.01). Compared with the model group, except for the low-dose group of the traditional Chinese medicine composition granules where there was no statistically significant difference in serum IL-4 and IFN-γ levels, the serum levels of TNF-α and IFN-γ in the other dose groups of the traditional Chinese medicine composition granules were significantly decreased, while the IL-4 level was significantly increased (P < 0.01, P < 0.05). See Table 28.

[0352] Table 28 Effects of traditional Chinese medicine composition on serum inflammatory factors in rats with chronic glomerulonephritis (x±s, n=10)

[0353]

[0354] Note: *P<0.01, compared with the normal group; ▲ P<0.05, ▲▲ P<0.01, compared with the model group.

[0355] Compared with the normal control group, the levels of CD3, CD4, and CD4 / CD8 in the peripheral blood of rats in the model group were significantly decreased (P<0.01, P<0.05), while CD8 was significantly increased (P<0.01, P<0.05). Compared with the model group, the levels of CD3, CD4, and CD4 / CD8 in the peripheral blood of rats in the low, medium, and high dose groups of the traditional Chinese medicine composition granules were significantly increased (P<0.01, P<0.05), while CD8 was significantly decreased (P<0.01, P<0.05). See Table 29.

[0356] Table 29 Effects of traditional Chinese medicine composition on peripheral blood T lymphocyte subsets in rats with chronic glomerulonephritis (%, x±s, n=10)

[0357]

[0358] Note: *P<0.01, compared with the normal group; ▲ P<0.05, ▲▲ P<0.01, compared with the model group;

[0359] Experimental Example 16: Effects of a Traditional Chinese Medicine Composition on Doxorubicin-Induced Chronic Glomerulonephritis in Rats

[0360] Eighty-five male SPF-grade SD rats, weighing approximately 200g, were injected twice via tail vein on days 1 and 14 with doxorubicin (epirarubicin hydrochloride, Xinghai Zhengfang Pfizer Pharmaceutical Co., Ltd., batch number: 15029611). The normal control group received an equivalent volume of physiological saline via tail vein injection. On day 21 post-injection, all urine samples were collected from the rats within 24 hours. A 24-hour urinary protein level >50mg / kg indicated successful model establishment. The normal control group rats received an equal volume of physiological saline injected at the same site on the same day.

[0361] Different dosage groups of the traditional Chinese medicine composition granules prepared in Example 1 of this application (6.48 g crude drug / kg, 12.96 g crude drug / kg, 25.92 g crude drug / kg) and the prednisolone acetate tablet positive control group were administered by gavage at a dose of 10 ml / kg once a day for 30 consecutive days. The blank control group and model group rats were administered an equal volume of physiological saline by gavage. One hour after the last administration, all urine was collected within 24 hours. Twenty-four hours after the last administration, rats were anesthetized by intraperitoneal injection of 0.35 g / kg of 10% chloral hydrate, and serum samples were collected and kidneys were harvested for testing.

[0362] Test various indicators: ① Collect all urine samples from each rat within 24 hours after 30 days of drug administration, and use the urine protein quantification kit (Nanjing Jiancheng Biotechnology Co., Ltd., batch number: 202009123) to determine the 24-hour urine protein content of each group of rats using the endpoint method.

[0363] ② Rats were anesthetized with chloral hydrate, and blood was collected after exposing the abdominal aorta. The serum was obtained by centrifugation, and the levels of Scr, BUN, TNF-α, IL-6, and IL-1β in the serum and kidney tissue were measured (Invitrogen, batch numbers 305271-004, 324017-001, and 1102060222, respectively). The values ​​were read using a multi-functional microplate reader.

[0364] ③ Collect orbital venous blood from each group of rats into flow cytometry tubes, add 10 μL of CD3, CD4 and CD8 monoclonal antibodies and 50 μL of EDTA anticoagulated blood respectively, mix well, incubate in the dark for 20 min, add 2 mL of hemolysin to each tube, incubate in the dark for 20 min, centrifuge at 2000 r / min for 10 min, discard the supernatant, mix with 2 mL of PBS in each tube, centrifuge and discard the supernatant, repeat washing twice, discard the supernatant, add 2 mL of PBS to resuspend and mix well, and then analyze the proportion of peripheral blood T lymphocyte subsets CD3, CD4 and CD8 by flow cytometry.

[0365] The results showed that compared with the normal control group, the 24-hour urinary protein, serum Scr, and BUN levels in the model group rats were significantly increased, and the differences were statistically significant (P < 0.01). Compared with the model group, except for the low-dose group of the traditional Chinese medicine composition granules which showed no statistically significant difference in serum BUN levels, the 24-hour urinary protein, serum Scr, and BUN levels in the other dose groups of the traditional Chinese medicine composition granules and the prednisolone acetate group were significantly decreased, and the differences were statistically significant (P < 0.01). See Table 30.

[0366] Table 30 Effects of 24-hour urinary protein, serum urea nitrogen, and creatinine on rats with chronic glomerulonephritis (x±s, n=10)

[0367]

[0368] *P<0.01, compared with the normal group; ▲ P<0.01, compared with the model group.

[0369] Compared with the normal control group, the levels of TNF-α, IL-6, and IL-1β in the kidney tissue of rats in the model group were significantly increased (P < 0.05). Compared with the model group, after 30 days of drug intervention, the different dose groups of the traditional Chinese medicine composition granules and the positive drug group could significantly reduce the levels of TNF-α, IL-6, and IL-1β in the kidney tissue of rats, thereby alleviating the doxorubicin-induced renal inflammatory response (P < 0.05). See Table 31.

[0370] Table 31 Effects of inflammatory factors on renal tissue of rats with chronic glomerulonephritis (x±s, n=10)

[0371]

[0372] Note: *P<0.01, compared with the normal group; ▲ P<0.01, compared with the model group.

[0373] Compared with the normal control group, the levels of CD3, CD4, and CD4 / CD8 in the peripheral blood of rats in the model group were significantly decreased (P<0.01, P<0.05), while CD8 was significantly increased (P<0.01, P<0.05). Compared with the model group, the levels of CD3, CD4, and CD4 / CD8 in the peripheral blood of rats in the low, medium, and high dose groups of the traditional Chinese medicine composition granules were significantly increased (P<0.01, P<0.05), while CD8 was significantly decreased (P<0.01, P<0.05). See Table 32.

[0374] Table 32 Effects of traditional Chinese medicine composition on peripheral blood T lymphocyte subsets in rats with chronic glomerulonephritis (%, x±s, n=10)

[0375]

[0376] Note: *P<0.01, compared with the normal group; ▲ P<0.05, ▲▲ P<0.01, compared with the model group.

[0377] The composition proposed in this application can protect the kidney tissue of rats with chronic glomerulonephritis, improve kidney function, and reduce inflammatory response.

[0378] Experimental Example 17: Effects of the Composition on Type II Collagen-Induced Rheumatoid Arthritis in Rats

[0379] One hundred and fifty healthy SPF-grade SD rats weighing 150-160g were divided into equal groups of males and females. After one week of acclimatization, collagen-induced arthritis (CIA) was induced. Multiple subcutaneous injections were administered at the base of the tail using a microsyringe. The initial immunization consisted of 0.2 mL of an equal volume of CII-CFA emulsion (bovine type II collagen (CII), Chondrex, catalog number: 220176; complete Freund's adjuvant (CFA), Sigma, catalog number: SLCL4383) at the same site on day 14. A booster immunization was administered at the same site on day 14, with an injection of 0.15 mL of CII-IFA (incomplete Freund's adjuvant (IFA), Sigma, catalog number: SLCL0729) emulsion. The normal control group rats were injected with an equal volume of physiological saline (Shandong Kelun Pharmaceutical Co., Ltd., catalog number: B21050102B) using the same method. Fourteen days after injection, the swelling of the rats' joints and toes was observed daily, and arthritis scores were calculated.

[0380] Specifically, rats with successfully established collagen arthritis models were selected (arthritis score sum ≥4; 0 points: normal paws or no inflammation; 1 point: swollen or slightly red toe joints; 2 points: red and swollen toe joints and soles; 3 points: red and swollen paws below the ankle; 4 points: severely red and swollen ankle joints with signs of joint deformity). These rats were then randomly assigned to groups.

[0381] The different dosage groups of the traditional Chinese medicine composition granules prepared in Example 1 (6.48g crude drug / kg, 12.96g crude drug / kg, 25.92g crude drug / kg) and the methotrexate positive drug group (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: SN1125RA18) were administered by gavage at a dose of 10ml / kg once a day for 28 consecutive days. 30 minutes after the last administration, anesthetized rats were injected intraperitoneally with 3ml / kg of 10% chloral hydrate solution (Shanghai Chuangsai Technology Co., Ltd., catalog number: PM22473) for sample collection and testing.

[0382] Detection indicators: ① Before and weekly during drug administration, the volume of the rat's hind paws was measured using a paw volume analyzer (Chengdu Taimeng Software Co., Ltd., PV-200 model). The average volume of the hind paws was taken as the final rat paw swelling volume. Calculation formula: Swelling degree = (Paw volume on day i - Pre-inflammatory paw volume) ÷ Pre-inflammatory paw volume × 100% ② Experimental endpoint: After anesthetizing the rats with 0.35g / kg of 10% chloral hydrate, blood was collected from the abdominal aorta. The serum IL1β, IL6, and TNF-α levels were detected using an enzyme-linked immunosorbent assay (ELISA) reader (SpectraMax M2e model) according to the instructions of the IL1β, IL6, and TNF-α ELISA kits (TNF-α detection kit, IL-1β ELISA kit, IL6 ELISA kit, Wuhan Saipei Biotechnology Co., Ltd., catalog numbers: 202205, 202203, 202203). ③Tissue from one side of the rat's ankle joint was fixed in 10% formalin, and routine paraffin sections were prepared and stained with hematoxylin and eosin (HE) for microscopic examination. The pathological scoring criteria are as follows: (1) Synovial cell proliferation score: 0 = no proliferation; 1 = slight proliferation, 2-4 layers of synovial cells; 2 = moderate proliferation, more than 4 layers of synovial cells; 3 = excessive proliferation of synovial cells, erosion of cartilage and bone, and disappearance of joint space. (2) Cell erosion score: 0 = no erosion; 1 = a few 1 to 2 cell erosion foci; 2 = 2 to 5 local cell erosion foci; 3 = more than 5 cells extensively eroding the joint capsule. (3) Pannus score: 0 = no change; 1 = pannus appears in two locations; 2 = pannus appears in four locations, accompanied by erosion of the cartilage surface; 3 = pannus appears in more than four locations or extensive pannus appears in two locations. (4) Inflammation score: 0 = normal; 1 = mild inflammation, with one aggregate or a small number of scattered inflammatory cells (T cells, B cells, macrophages and plasma cells) infiltrating; 2 = moderate inflammation, with two or more white blood cell aggregates; 3 = severe inflammation, with a large number of inflammatory cells infiltrating, a large number of white blood cells aggregated, and obvious scattered infiltration. (5) Bone erosion score: 0 = normal; 1 = trace erosion, 1 to 2 small superficial sites; 2 = small erosion, 1 to 4 sites of medium size and depth; 3 = moderate erosion, more than 5 sites, with local erosion to the bone cortex; 4 = severe erosion, multiple injuries, with local or complete erosion to the bone cortex; 5 = extensive injury, with the cortex penetrating more than 25% of the bone length.

[0383] The results show

[0384] After establishing the CIA rat model, paw swelling was significantly increased, and serum levels of inflammatory factors IL1β, IL6, and TNF-α were significantly elevated. Compared with the model group, administration of the traditional Chinese medicine composition significantly reduced the degree of ankle and paw swelling in CIA rats and significantly reduced serum IL1β, IL6, and TNF-α levels, suggesting that the traditional Chinese medicine composition can significantly alleviate joint swelling, reduce the expression of inflammatory cytokines, and inhibit inflammatory responses and tissue damage in rheumatoid arthritis rats. The results are shown in Tables 33 and 34.

[0385] Table 33 Effect of the composition on joint swelling in rats with rheumatoid arthritis (%, X±SD)

[0386]

[0387] Note: Compared with the model group, *P<0.05, **P<0.01.

[0388] Table 34 Effects of the composition on serum inflammatory cytokines in rheumatoid arthritis rats (n = 6, X ± SD)

[0389]

[0390] Note: Compared with the model group, *P<0.05, **P<0.01.

[0391] Pathological results showed

[0392] The main pathological manifestations of the ankle joint in the model group rats were: significant thickening of the synovial lining cells, villous or finger-like hypertrophy of the synovial tissue, and infiltration of inflammatory cells such as lymphocytes, plasma cells, macrophages, and neutrophils within the synovium, tending to form lymphoid nodules or lymphoid follicles; increased synovial blood vessels with significant inflammatory cell infiltration in the vessel walls and surrounding tissues; and detachment and defects on the articular cartilage surface, especially in the junction area between the synovium and cartilage and subchondral bone. Proliferating synovial cells, aggregated inflammatory cells, and vasculitis formed pannus tissue, eroding and destroying adjacent articular cartilage, subchondral bone, and surrounding tendons, and even invading bone tissue, resulting in the disappearance of the joint space. Different doses of the drug significantly reduced inflammatory cell infiltration and vasculitis in the synovium, and reduced damage to the articular cartilage surface and erosion of cartilage and subchondral bone. The reduction achieved with the high dose was significantly different from that in the model group, and statistically significant. Results are shown in […]. Figure 4 Table 35.

[0393] Table 35 Effects of the composition on the histopathological score of the ankle joint in rats with rheumatoid arthritis

[0394]

[0395] Note: Compared with the model group, *P<0.05, **P<0.01.

[0396] Effect of Traditional Chinese Medicine Composition on Adjuvant-induced Rheumatoid Arthritis Rats in Experimental Example 18

[0397] After 72 male Wistar rats weighing 60 - 80 g (Spebefu Biotechnology Co., Ltd., certificate number: 110324221104520838, animal production license number: SCXK(Beijing)2019 - 0010) were adaptively fed for 1 week, Freund's complete adjuvant (CFA) (Sigma Company, USA, product number: SLCL4383) was used to induce the model. First, the same batch of Bacillus Calmette - Guérin (Beijing Institute of Biological Products, batch number 2022 - 1 company, product number: 202208) was inactivated in an 80℃ water bath (DK - 8D type electrothermal constant temperature water bath, Shanghai Jinghong Experimental Equipment Co., Ltd.) for 1 hour, and then formulated into an emulsion with a concentration of 15 g / L with Freund's complete adjuvant and fully emulsified. 0.1 ml / rat of the CFA emulsion was injected intradermally into the toe of the rat to cause inflammation. After 14 days, rats with obvious toe swelling were randomly grouped.

[0398] According to the different dose groups of the traditional Chinese medicine composition granules (6.48 g crude drug / kg, 12.96 g crude drug / kg, 25.92 g crude drug / kg) and the positive drug group of methotrexate, gavage administration was carried out once a day at a dose of 10 ml / kg from the first day for 28 consecutive days. 30 minutes after the last administration, the rats were anesthetized with 10% chloral hydrate at a dose of 3 ml / kg, and then sacrificed for detection.

[0399] Detection indexes: ① Before administration and every week during the administration process, the volume of the hind feet of the rats was measured with a toe volume measuring instrument, and the average value of the swelling of the hind feet was used as the final foot swelling volume of the rats. ② At the end of the experiment, the rats were anesthetized with 10% chloral hydrate at a dose of 3 ml / kg, and blood was taken from the abdominal aorta. The content of IL1β, IL6, and TNF - ɑ in the rat serum was detected using an enzyme - labeled instrument (Infinite M200Pro enzyme - labeled instrument, TECAN Company) according to the instructions of the IL1β, IL6, TNF - ɑ Elisa kit. ③ One side of the ankle joint tissue of the rats was fixed in 10% formalin, and routine paraffin sections were made and stained with HE. Professional pathologists examined and scored them under a microscope.

[0400] The results showed

[0401] After the establishment of the adjuvant arthritis rat model, the toe swelling degree increased significantly, and the content of serum inflammatory factors IL1β, IL6, and TNF - ɑ increased significantly. Administration of the traditional Chinese medicine composition could significantly reduce the ankle toe swelling degree of CIA rats and significantly reduce the levels of serum IL1β, IL6, and TNF - ɑ, suggesting that the traditional Chinese medicine composition could significantly reduce the joint swelling degree of rheumatoid arthritis rats, reduce the expression of inflammatory cytokines, and inhibit the inflammatory reaction and tissue damage. The results are shown in Tables 36 and 37.

[0402] Table 36 Effect of the composition on joint swelling in adjuvant-induced rheumatoid arthritis rats (%, X±SD)

[0403]

[0404]

[0405] Note: Compared with the model group, *P<0.05, **P<0.01.

[0406] Table 37 Effects of the composition on serum inflammatory factors in adjuvant-induced rheumatoid arthritis rats

[0407]

[0408] Note: Compared with the model group, *P<0.05, **P<0.01.

[0409] Pathological results showed

[0410] The main pathological manifestations of the ankle joint in the model group rats were: significant thickening of the synovial lining cells, villous or finger-like hypertrophy of the synovial tissue, and infiltration of inflammatory cells such as lymphocytes, plasma cells, macrophages, and neutrophils within the synovium, tending to form lymphoid nodules or lymphoid follicles; increased synovial blood vessels with significant inflammatory cell infiltration in the vessel walls and surrounding tissues; and detachment and defects on the articular cartilage surface, especially in the junction area between the synovium and cartilage and subchondral bone. Proliferating synovial cells, aggregated inflammatory cells, and vasculitis formed pannus tissue, eroding and destroying adjacent articular cartilage, subchondral bone, and surrounding tendons, and even invading bone tissue, resulting in the disappearance of the joint space. Different doses of the drug significantly reduced inflammatory cell infiltration and vasculitis in the synovium, and reduced damage to the articular cartilage surface and erosion of cartilage and subchondral bone. The reduction achieved with the high dose showed a statistically significant difference compared to the model group. The results are shown in Table 38.

[0411] Table 38 Effects of Traditional Chinese Medicine Compositions on the Histopathological Scores of Ankle Joints in Rats with Adjuvant-Induced Rheumatoid Arthritis

[0412]

[0413] Note: Compared with the model group, *P<0.05, **P<0.01.

[0414] The composition of this application can reduce joint swelling in rats with rheumatoid arthritis, inhibit the production of cytokines that mediate inflammatory responses and tissue damage, improve the histopathological changes of synovial hyperplasia, inflammatory infiltration, pannus formation and cartilage damage in rheumatoid arthritis, and significantly delay the disease progression of rheumatoid arthritis.

[0415] Effects of the traditional Chinese medicine composition on N49 peptide-induced Sjögren's syndrome model mice in Experiment Example 19

[0416] Antigen preparation: M3 peptide N49 (Shanghai Yingjun Biotechnology Co., Ltd.) was diluted with 0.01 mol / L PBS solution (Nanjing Senbeijia Biotechnology Co., Ltd.) at pH 7.2 to a concentration of 200 μg / mL. The solution was then fully emulsified with an equal volume of complete Freund's adjuvant (Sigma-Aldrich, USA) in an ice bath to prepare a 100 μg / mL M3 peptide N49 inducing agent.

[0417] Modeling: Sixty female BALB / c mice, aged 5 weeks and weighing (16±2)g, were selected and randomly divided into a blank control group, a model group, different dosage groups of traditional Chinese medicine granules (prepared in Example 1) (9.36g crude drug / kg, 18.72g crude drug / kg, 37.44g crude drug / kg), and a hydroxychloroquine tablet positive drug group (5.23mg / kg) (Shanghai Zhongxi Pharmaceutical Co., Ltd., 0.1g / tablet).

[0418] A Sjögren's syndrome animal model was established by repeatedly immunizing BALB / c mice with the prepared antigens described above. Mice in each group were administered the corresponding drug or an equal volume of physiological saline (Shanghai Beyotime Biotechnology Co., Ltd.) by gavage starting on day 14 post-immunization, once daily for 28 consecutive days.

[0419] detection indicators

[0420] ① Saliva flow rate and water intake detection: 24 hours after the last administration, the saliva flow rate and water intake of each mouse were measured for 24 hours. Saliva flow rate = saliva volume / 10min. ② 1mL of blood was collected from the eyeballs and centrifuged using a high-speed refrigerated centrifuge (Eppendorf, model: BY-20) to separate the serum. The supernatant serum was collected. The concentrations of IgG and IgM in the serum were detected using an enzyme-linked immunosorbent assay (ELISA) kit for mouse immunoglobulins IgG and IgM (Lianke Biotechnology). The values ​​were read using a microplate reader (PerkinElmer P, model: Enspire).

[0421] The results show

[0422] Compared with the blank control group, the model group showed a decrease in saliva flow rate and an increase in water intake (P < 0.001); compared with the high-dose and hydroxychloroquine groups of the traditional Chinese medicine composition, the model group showed an increase in saliva flow rate and a decrease in water intake (P < 0.001, P < 0.01); compared with the medium-dose group of the traditional Chinese medicine composition, the model group showed an increase in saliva flow rate and a decrease in water intake (P < 0.001, P < 0.01). The results are shown in Table 39.

[0423] Table 39 Effects on salivary flow rate and water intake in Sjögren's syndrome mice ( n=10)

[0424]

[0425] Note: Compared with the model group, *P<0.05, **P<0.01, **P<0.001

[0426] ELISA results showed that the levels of immunoglobulins IgG and IgM in the serum of model mice were elevated compared with normal mice (P < 0.001). The levels of IgG and IgM in the high-dose group of the traditional Chinese medicine composition were significantly lower in the model group compared with the hydroxychloroquine group (P < 0.01). The results are shown in Table 40.

[0427] Table 40 Effects on serum IgG and IgM levels in mice with Sjögren's syndrome n=10)

[0428]

[0429]

[0430] Note: Compared with the model group, *P<0.05, **P<0.01, **P<0.001

[0431] Experiment Example 20: Effects of Traditional Chinese Medicine Composition on a Rats with Freund's Complete Adjuvant and Antigen Combined Induction of Sjögren's Syndrome

[0432] A Sjögren's syndrome animal model was induced using Freund's complete adjuvant (Shanghai Beyotime Biotechnology Co., Ltd.) and SD rat submandibular gland antigen.

[0433] First, the submandibular glands of SD rats were dissected on a clean bench, and the glandular capsule and connective tissue were removed. After weighing, the glands were minced, homogenized, and repeatedly centrifuged. The supernatant was collected, and the protein antigen content was determined using a BCA assay kit. The protein antigen was thoroughly emulsified with Freund's complete adjuvant, and the final concentration of the protein antigen (Shanghai Beyotime Biotechnology Co., Ltd.) was detected using a BCA microprotein assay kit. Then, 60 SPF-grade male SD rats weighing 180-200g were randomly divided into 6 groups of 10 each. The 6 groups were: a normal control group, a model group, low, medium, and high dose groups of the traditional Chinese medicine composition (6.48g crude drug / kg, 12.96g crude drug / kg, and 25.92g crude drug / kg), and a positive control hydroxychloroquine tablet group (3.62mg / kg) (Shanghai Zhongxi Pharmaceutical Co., Ltd., 0.1g / tablet). Except for the normal control group, the above-mentioned protein antigen was subcutaneously injected into the plantar region of both hind paws of each rat, with 0.2 mL injected per rat. The normal control group was injected with an equal volume of physiological saline at the same site. The model was considered successful when the rats showed increased water intake and decreased salivation. The low, medium, and high dose groups of the traditional Chinese medicine composition and the positive control group were administered the medication by gavage once a day for 30 consecutive days. The normal control group and the model group were administered the medication by gavage with an equal volume of physiological saline.

[0434] Blood rheological parameters were measured: Rats were dissected after 30 days, and 5 mL of blood was collected from the femoral artery. 3 mL of this blood was used to determine the whole blood high-shear viscosity, low-shear viscosity, and plasma viscosity of each group of rats using a blood viscosity analyzer (LG-R-80B, Beijing Shidi Scientific Instruments Co., Ltd.). The remaining blood from the rats was collected, allowed to stand for 15 min, and then centrifuged at 3000 r / min for 15 min to separate serum (Eppendorf, model: BY-20). The levels of IL-6 and IL-17 in the serum were measured using an interleukin-17 (IL-17) and interleukin-6 (IL-6) ELISA kit (Nanjing Chuanbo Biotechnology Co., Ltd.), and the values ​​were read using a multifunctional enzyme-linked immunosorbent assay (ELISA) reader.

[0435] The results show

[0436] Compared with the normal control group, the model group showed increased whole blood viscosity and plasma viscosity (P<0.001, P<0.01); compared with the model group, the high-dose traditional Chinese medicine composition and hydroxychloroquine group showed decreased whole blood viscosity (P<0.001, P<0.01); compared with the model group, the medium-dose traditional Chinese medicine composition group showed decreased whole blood viscosity (high shear) and plasma viscosity (P<0.01).

[0437] Table 41 Effects of Traditional Chinese Medicine Compositions on Hemorheology in Rats n=10)

[0438]

[0439] Note: Compared with the model group, *P<0.05, **P<0.01, **P<0.001

[0440] ELISA results showed that the expression of IL-6 and IL-17 in the model group rats was increased compared with that in normal mice (P<0.001). The expression of IL-6 and IL-17 in the high- and medium-dose Chinese medicine combination and the hydroxychloroquine group was significantly decreased compared with that in the model group rats (P<0.01).

[0441] Table 42 Effects of traditional Chinese medicine composition on serum IL-6 and IL-17 levels in rats n=10)

[0442]

[0443] Note: Compared with the model group, *P<0.05, **P<0.01, **P<0.001

[0444] The composition proposed in this invention can significantly improve the levels of inflammatory cytokines in the serum of rats with Sjögren's syndrome and reduce blood viscosity. It can delay the progression of Sjögren's syndrome, regulate immunity, and inhibit inflammatory responses.

[0445] Experiment 21 Effects on a mouse model of Kawasaki disease (KD) vasculitis induced by intraperitoneal injection of Lactobacillus casei cell wall

[0446] Take an appropriate amount of Lactobacillus casei (LCWE) powder (Shanghai Chunshi Biotechnology Co., Ltd., batch number: 20210307002) to prepare an LCWE solution with a concentration of 1 mg / ml. Select 60 SPF-grade male BALB / c mice weighing 18-22g and establish a KD model by a single intraperitoneal injection of 0.5ml of the freshly prepared LCWE solution. Observe the behavior of the mice within 0-4 days after injection. If the mice show reduced eating and drinking activity, dull fur, slight redness and swelling in the nose and mouth area, and trembling, it indicates that the model has been successfully established.

[0447] Fifty mice that successfully developed the model were randomly divided into a model group, low-, medium-, and high-dose groups (9.36, 18.72, and 37.44 g crude drug / kg) of the traditional Chinese medicine composition prepared in Example 1, and an aspirin group (250 mg / kg). The low-, medium-, and high-dose traditional Chinese medicine composition and aspirin (Shanghai Shifeng Biotechnology Co., Ltd., batch number: 202103004001, specification: 0.5 g / tablet) were prepared into suspensions of 0.936 g / ml, 1.872 g / ml, 3.744 g / ml, and 0.025 g / ml, respectively, using physiological saline (Shijiazhuang No. 4 Pharmaceutical Co., Ltd., batch number: 2103193205). Both the traditional Chinese medicine composition and aspirin were administered by gavage at a volume of 0.1 ml / 10 g. All groups were administered the drug once a day for 7 consecutive days. The other 10 mice served as the normal control group and the model group and were given physiological saline by gavage at a volume of 0.1 ml / 10 g.

[0448] Detection indicators: ① During the drug administration period, the body weight of mice in each group was measured, and basic conditions such as fur and food intake were observed. ② Twelve hours after the last drug administration, blood was collected by enucleation of the eyeballs and centrifuged at 3000 rpm for 10 min (BY-220A medical centrifuge, Beijing Baiyang Medical Instrument Co., Ltd.). After separation, serum was collected and analyzed using a Cayman enzyme-linked immunosorbent assay (ELISA) kit for endothelin (ET). Chemical, batch number: 583151-480; Vascular endothelial growth factor (VEGF) ELISA kit (Wuhan Feien Biotechnology Co., Ltd., batch number: EM0205); Interleukin-6 (IL-6) ELISA kit and Tumor necrosis factor-α (TNF-α) ELISA kit (Invitrogen, batch number: 225266-025, batch number: 234889-005); Interleukin-1β (IL-1β) ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd., batch number: ml301814). Serum TNF-α, IL-1β, IL-6, ET, and VEGF levels were measured according to the kit instructions, and the values ​​were read using an Enspire microplate reader (PerkinElmer).

[0449] The general observation results of the mice showed that, compared with the normal control group, the model group mice exhibited tremors, sparse hair, reduced eating and activity, and a significant decrease in body weight (P<0.01). Compared with the model group, the mice in the low, medium, and high dose groups of the traditional Chinese medicine composition and the aspirin group showed varying degrees of improvement in sparse hair, reduced eating and activity, and a significant increase in body weight (P<0.05, P<0.01). The above indicators of each dose group of the traditional Chinese medicine composition showed a dose-dependent relationship. The results are shown in Table 43.

[0450] Table 43 Effects of traditional Chinese medicine compositions on body weight (g) in Kawasaki disease mice. n=10)

[0451]

[0452]

[0453] Note: Compared with the model group, *P<0.05, **P<0.01.

[0454] The results of serum inflammatory cytokines TNF-α, IL-1β, and IL-6 in mice of each group showed that, compared with the normal control group, the serum TNF-α, IL-1β, and IL-6 levels in the model group were significantly increased (P<0.01). Compared with the model group, the serum TNF-α, IL-1β, and IL-6 levels in the low, medium, and high dose groups of the traditional Chinese medicine composition and the aspirin group were significantly decreased (P<0.01), and all dose groups of the traditional Chinese medicine composition showed a dose-dependent effect. The results are shown in Table 44.

[0455] Table 44. Effects of traditional Chinese medicine composition on serum TNF-α, IL-1β, and IL-6 levels in Kawasaki disease mice (pg / ml). n=10)

[0456]

[0457] Note: **P<0.01 compared to the model group.

[0458] The results of serum endothelin (ET) and vascular endothelial growth factor (VEGF) levels in mice from each group showed that, compared with the normal control group, the serum ET and VEGF levels in the model group were significantly increased (P<0.01); compared with the model group, the serum ET and VEGF levels in the low, medium, and high dose groups of the traditional Chinese medicine composition and the aspirin group were significantly decreased (P<0.01), and all dose groups of the traditional Chinese medicine composition showed a dose-dependent effect. See Table 45.

[0459] Table 45. Effects of traditional Chinese medicine composition on serum vascular endothelial injury markers ET and VEGF levels in Kawasaki disease mice (pg / ml). n=10)

[0460]

[0461] Note: **P<0.01 compared to the model group.

[0462] Experiment Example 22: Effects of a Traditional Chinese Medicine Composition on a Mouse Autoimmune Uveoretinal Vasculitis Model

[0463] Sixty SPF-grade male C57BL / 6 mice, aged 8–10 weeks and weighing (20±5) g, were selected. After acclimatization for one week, the mice were randomly divided into 6 groups of 10 mice each: normal control group, model group, positive control group, low-dose group, medium-dose group, and high-dose group of traditional Chinese medicine composition.

[0464] After the mice's activity stabilized, except for the normal control group, the other groups of mice were anesthetized by injection of xerazine hydrochloride (Changsha Byte Biotechnology Research Institute Co., Ltd., batch number: 20210102). 100 μL of IRBP solution (5 mg / mL) (Med ChemExpress, USA, batch number: 298202-25-2) and 100 μL of complete Freund's adjuvant containing 5 mg / mL Mycobacterium tuberculosis (Sigma, USA, batch number: 9007812) were thoroughly mixed to form a white emulsion. This emulsion was then subcutaneously injected into the head and neck, bilateral groin, and tail base of the mice, respectively. After active immunization, the mice were intraperitoneally injected with 100 μL of pertussis toxin (Beijing Bolede Technology Development Co., Ltd., batch number: 211242A1) as an immune adjuvant to enhance the immune effect. The control group mice were given an equal volume of physiological saline (Shijiazhuang No. 4 Pharmaceutical Co., Ltd., batch number: 2103193205) via subcutaneous injection and an equal volume of physiological saline via intraperitoneal injection.

[0465] Twenty-four hours after modeling, mice in each group were administered the drug via gavage once daily, with a gavage volume of 0.02 mL / g. The normal control group and model group were administered 0.02 mL / g physiological saline via gavage; the low, medium, and high dose groups of the traditional Chinese medicine composition were administered 9.36, 18.72, and 37.44 g crude drug / kg via gavage, respectively; the positive control group was given an intraperitoneal injection of dexamethasone sodium phosphate injection (Tianjin Jinyao Amino Acid Co., Ltd., batch number 0820221) at a dose of 1.3 mg / kg once daily. After 21 days, blood was collected from the eyeballs of mice in each group. The blood was centrifuged at 3000 rpm for 10 min (BY-220A medical centrifuge, Beijing Baiyang Medical Instrument Co., Ltd.). The supernatant was collected after separation and stored at -20℃ for later use. The procedures were performed according to the instructions of the mouse interleukin (IL-1β) (Shanghai Enzyme-Linked Biotechnology Co., Ltd., batch number: ml301814) and the mouse intercellular adhesion molecule-1 (ICAM-1) enzyme-linked immunosorbent assay kit (Wuhan Boster Biological Engineering Co., Ltd., batch number: EK0371). The absorbance was measured at 450 nm using an Enspire microplate reader (PerkinElmer, USA). A standard curve was plotted to determine the levels of IL-1β and ICAM-1 in mouse serum.

[0466] The results of serum IL-1β and ICAM-1 levels in mice of each group showed that, compared with the normal control group, the serum IL-1β and ICAM-1 levels in the model group were significantly increased (P<0.01); compared with the model group, the serum IL-1β and ICAM-1 levels in the low, medium, and high dose groups of the traditional Chinese medicine composition and the positive control group were significantly decreased (P<0.01), and the levels of the traditional Chinese medicine composition showed a dose-dependent effect. See Table 46.

[0467] Table 46. Effects of traditional Chinese medicine composition on serum IL-1β and ICAM-1 levels in mice with retinal vasculitis (pg / ml). n=10)

[0468]

[0469] Note: **P<0.01 compared to the model group.

[0470] The composition significantly reduces vascular damage caused by inflammatory stimulation, thereby alleviating vasculitis in Kawasaki disease mice. It also significantly reduces the inflammatory response in rabbit retinal vasculitis by decreasing the expression of ICAM-1 in the retinal vascular region, thereby reducing inflammatory cell adhesion.

[0471] Experiment Example 23: Effects of Traditional Chinese Medicine Composition on Serum Indicator Factors in Ovalbumin-Induced Allergic Purpura Rats

[0472] Drugs required for preparing an allergic purpura rat model

[0473] Take dried ginger (from Bozhou Wanzhen Chinese Medicine Pieces Factory), long pepper, and black pepper (from Bozhou Wanzhen Chinese Medicine Pieces Factory) in a 1:1:1 ratio and add 16 times the amount of water. Bring to a boil over high heat, then simmer over low heat for 2 hours. Filter the mixture, and boil the dregs again with 16 times the amount of water for 2 hours. Combine the filtrates, concentrate to 0.125 g / mL, and store at 4°C for later use.

[0474] Preparation of ovalbumin and Freund's complete adjuvant emulsion

[0475] To prepare 100mL of emulsion, weigh 1g of ovalbumin (Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and dissolve it completely in 50mL of physiological saline (Chenxin Pharmaceutical Co., Ltd.). Then add 50mL of Freund's complete adjuvant (Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and stir until the emulsion does not disperse when dropped into water for 5 minutes. The concentration of ovalbumin emulsion is 10mg / mL.

[0476] To prepare a 0.3% ovalbumin saline solution: Weigh 0.21g of ovalbumin and dissolve it completely in 70mL of saline solution. Stir well to obtain a 0.3% ovalbumin saline solution.

[0477] Preparation of test drug and positive control drug

[0478] Weigh 504.06g of the composition particles prepared in Example 1 and dissolve them in 400mL of distilled water to prepare a high-dose solution of composition particles with a concentration of 1.260g / mL; weigh 252.04g of the composition particles and dissolve them in 400mL of distilled water to prepare a medium-dose solution of composition particles with a concentration of 0.630g / mL; weigh 126.03g of the composition particles and dissolve them in 400mL of distilled water to prepare a low-dose solution of composition particles with a concentration of 0.315g / mL.

[0479] Weigh 0.0220g of dexamethasone acetate tablets (Zhejiang Xianju Pharmaceutical Co., Ltd., specification: 0.75mg / tablet), grind them thoroughly, and dissolve them in 400mL of distilled water to prepare a dexamethasone aqueous solution of 0.055mg / mL.

[0480] Modeling, drug administration, and biological sample collection of allergic purpura rats

[0481] Sixty SPF-grade 3-week-old male rats weighing 60-80g were acclimatized for one week and then randomly divided into 6 groups according to body weight: a normal control group, a model control group, low-, medium-, and high-dose groups of the traditional Chinese medicine composition (3.15g granules / kg, 6.30g granules / kg, and 12.60g granules / kg), and a positive control group (dexamethasone 0.55mg / kg). Each group consisted of 10 rats. The model rats were administered a decoction of dried ginger, long pepper, and black pepper (0.15g / kg) by gavage once daily, while the normal control rats were administered the same volume of physiological saline (Chenxin Pharmaceutical Co., Ltd.) by gavage for 3 consecutive weeks. From day 1 of week 4, the model rats were intraperitoneally injected with a mixture of ovalbumin and Freund's complete adjuvant emulsion (10mg / kg), while the normal control rats were intraperitoneally injected with the same volume of physiological saline, once weekly for 3 consecutive weeks. Three days after the third intraperitoneal injection of the aforementioned ovalbumin emulsion, the skin on the back of the model rats was shaved, and 1 mL of 0.3% ovalbumin saline solution was injected intradermally at 5 points. The normal control group was injected with saline. Starting from week 6, each treatment group was administered the corresponding dose of the drug by gavage for 9 consecutive days, while the normal control and model control groups were administered the same volume of saline by gavage. On the first day of week 7 at 5 pm, the model group rats were injected with 0.25 mL of ovalbumin saline solution (10 mg / kg) via the tail vein to establish the model. After the last administration, the rats were fasted but allowed free access to water for 12 hours. They were then anesthetized by intraperitoneal injection of 10% chloral hydrate (Shanghai Yuanye Biotechnology Co., Ltd.), and blood was collected from the abdominal aorta. After standing for more than 0.5 hours, the blood was centrifuged at 3500 rpm for 10 min using a medical centrifuge (Beijing Baiyang Medical Instrument Co., Ltd., model: BY-220A). The serum was then collected and stored at -80℃ for later use.

[0482] The levels of IgA, CIC, IFN-γ, and IL-6 in serum were detected using rat IgA and IFN-γ ELISA kits (abcam product numbers: ab157735, ab239425) and rat CIC and IL-6 ELISA kits (Wuhan Huamei Biotechnology Co., Ltd. product numbers: CSB-E17938r, CSB-E04640r). The values ​​were read using an ELISA reader (PerkinElmer Inc., model: Enspire).

[0483] The results showed that the levels of immunoglobulin A (IgA), circulating immune complexes (CIC), and interleukin-6 (IL-6) in the model control group were significantly higher than those in the normal group (P<0.001), while the level of interferon-γ was significantly lower (P<0.01), indicating successful model establishment. Administration of the traditional Chinese medicine composition significantly delayed the course of allergic purpura. Compared with the model control group, the low, medium, and high dose groups of the traditional Chinese medicine composition showed significant improvements in IgA, CIC, IFN-γ, and IL-6 compared to the model group (P<0.05, P<0.01, P<0.001). The dexamethasone group also showed some improvement in IgA, CIC, IFN-γ, and IL-6 compared to the model control group (P<0.05, P<0.01, P<0.001). The results are shown in Table 47.

[0484] Table 47 Effects of the composition on serum marker factors in allergic purpuric rats

[0485]

[0486]

[0487] Note: Compared with the blank control group ### P < 0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0488] Experiment 24: Effects of serum cytokines and other indicators on a mouse model of gliadin-induced allergic purpura.

[0489] The drugs required for preparing a mouse model of allergic purpura.

[0490] Accurately measure 360 ​​μL of Indian ink (Beijing Benoway Biotechnology Co., Ltd.) with a pipette and dissolve it in 90 mL of physiological saline (Chenxin Pharmaceutical Co., Ltd.) to prepare a 4 mg / mL Indian ink physiological saline solution.

[0491] Measure 2 mL of HCl solution (6 mol / L) (Nanjing Chemical Reagent Co., Ltd.) using a pipette, dissolve it in 2000 mL of distilled water to prepare 6 mmol / L acidified water; accurately weigh 1.5 g of gliadin (Beijing Benoway Biotechnology Co., Ltd.), grind it thoroughly, dissolve it in 1500 mL of acidified water to prepare an acidified aqueous solution containing gliadin, allow it to dissolve at room temperature for 10 days, and then store it at 4℃.

[0492] Measure 50 μL of HCl solution (6 mol / L) (Nanjing Chemical Reagent Co., Ltd.) using a pipette, dissolve it in 60 ml of distilled water to prepare 5 mmol / L acidified water, and store it at 4℃.

[0493] Preparation of test drug and positive control drug

[0494] Weigh 546.04 g of the composition granules prepared in Example 1 and dissolve them in 300 ml of distilled water to prepare a high-dose solution of composition granules with a concentration of 1.820 g / mL; weigh 273.01 g of the composition granules and dissolve them in 300 ml of distilled water to prepare a medium-dose solution of composition granules with a concentration of 0.910 g / mL; weigh 136.54 g of the composition granules and dissolve them in 300 ml of distilled water to prepare a low-dose solution of composition granules with a concentration of 0.455 g / mL; weigh 0.0189 g of dexamethasone tablets, grind them thoroughly, and dissolve them in 300 ml of distilled water to prepare a dexamethasone aqueous solution with a concentration of 0.063 mg / mL.

[0495] Modeling, drug administration, and biological sample collection of a mouse model of allergic purpura:

[0496] An experimental mouse model of allergic purpura was constructed using Indian ink and gliadin (Beijing Benoway Biotechnology Co., Ltd.). Sixty SPF-grade ICR mice weighing 18-22g (half male, half female) were randomly divided into six groups based on weight: a normal control group, a model control group, low-, medium-, and high-dose groups of the herbal composition (4.55g granules / kg, 9.10g granules / kg, and 18.2g granules / kg), and a positive control group (dexamethasone 0.63mg / kg), with ten mice in each group. Specifically, for the first three weeks, except for the normal group, each group received a tail vein injection of 0.2ml / mouse of 4mg / ml Indian ink saline solution. This was repeated weekly for three weeks, while the normal group received an equal volume of saline solution. From the fourth week onwards, except for the normal group, each group received a gavage injection of 0.5ml / mouse of acidified saline solution containing gliadin every other day until the end of the 14th week. The normal group received an equal volume of acidified saline solution. During the last 3 days of gavage, except for the control group, all other groups received a tail vein injection of PBS solution containing gliadin (1 mg gliadin added to 0.01 mmol / L pH 7.4 PBS acidified with 5 mmol / L HCl), 0.2 ml / 20 g, once daily for 3 consecutive days. The control group received an equal volume of 0.01 mmol / L PBS solution acidified with 5 mmol / L HCl (Beijing Solarbio Science & Technology Co., Ltd.) via tail vein injection. After the last administration, patients were kept NPO for 12 hours but allowed free access to water. Blood was collected from the eyeballs, allowed to stand for at least 0.5 hours, then centrifuged at 3500 rpm for 10 min. The serum was collected and stored at -80°C.

[0497] The levels of CIC, BUN, and Cr in serum were detected using a mouse CIC ELISA kit (Wuhan Huamei Biotechnology Co., Ltd., catalog number: CSB-E16553m) and mouse BUN and Cr kits (Nanjing Jiancheng Biotechnology Research Institute, catalog numbers: C013-2-1, C011-2-1).

[0498] The results showed that after gliadin modeling, the levels of CIC, BUN, and Cr in the serum of mice increased, indicating that the inflammation in HSP mice was more severe and that kidney function was impaired. Administration of the traditional Chinese medicine composition significantly delayed the course of HSP. Compared with the model group, the levels of CIC, BUN, and Cr in HSP mice decreased, suggesting that the traditional Chinese medicine composition has a certain therapeutic effect on HSP. The results are shown in Table 48.

[0499] Table 48 Effects of the composition on serum factors in mice with gliadin-induced allergic purpura.

[0500]

[0501] Note: Compared with the blank control group ### P<0.001; compared with the model group, * P<0.05,** P<0.01, *** P<0.001.

[0502] The composition proposed in this application can significantly delay the course of allergic purpura, improve inflammatory response and kidney function.

[0503] The above embodiments of the present invention are merely examples to clearly illustrate the present invention and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications 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 application of a traditional Chinese medicine composition in the preparation of a medicament for treating chronic obstructive pulmonary disease, characterized in that, The raw materials of this traditional Chinese medicine composition, by weight, are: Magnolia officinalis 30-50 parts, Areca catechu 20-30 parts, Amomum villosum 30-50 parts, Ephedra sinica 20-30 parts, Prunus armeniaca 20-30 parts, Notopterygium incisum 30-50 parts, Zingiber officinale 30-50 parts, Pogostemon cablin 30-50 parts, Eupatorium fortunei 20-30 parts, Atractylodes lancea 30-50 parts, Poria cocos 120-150 parts, Atractylodes macrocephala 80-100 parts, Gypsum 30-50 parts, Crataegus pinnatifida 20-30 parts, Massa fermentata 30-50 parts, Hordeum vulgare 20-30 parts, Pheretima aspergillum 30-50 parts, Cynanchum paniculatum 30-50 parts, Dryopteris crassirhizoma 20-30 parts, and Lepidium apetalum 30-50 parts.

2. The application according to claim 1, characterized in that, The raw materials of the traditional Chinese medicine composition are: 50 parts Magnolia officinalis, 30 parts Areca catechu (roasted), 30 parts Amomum villosum (roasted), 20 parts Ephedra sinica, 30 parts Prunus armeniaca (bitter), 50 parts Notopterygium incisum, 50 parts fresh ginger, 50 parts Pogostemon cablin, 30 parts Eupatorium fortunei, 50 parts Atractylodes lancea, 150 parts Poria cocos, 100 parts Atractylodes macrocephala, 50 parts Gypsum fibrosum, 30 parts Crataegus pinnatifida (roasted), 30 parts Massa fermentata (roasted), 30 parts Hordeum vulgare (roasted), 50 parts Pheretima aspergillum, 50 parts Cynanchum paniculatum, 30 parts Dryopteris crassirhizoma, and 50 parts Lepidium apetalum.

3. The application as described in any one of claims 1 to 2, characterized in that, The drug is in the form of an oral administration.

4. The application according to claim 3, characterized in that, The medications for chronic obstructive pulmonary disease are in the form of decoctions, granules, capsules, tablets, oral liquids, pills, tinctures, or syrups.

5. The application according to any one of claims 1 to 2, characterized in that, The method for preparing the traditional Chinese medicine composition includes: Take 50 parts of Magnolia officinalis, 30 parts of Areca catechu (roasted), 30 parts of Amomum villosum (roasted), 20 parts of Ephedra sinica, 30 parts of Prunus armeniaca (bitter), 50 parts of Notopterygium incisum, 50 parts of fresh ginger, 50 parts of Pogostemon cablin, 30 parts of Eupatorium fortunei, 50 parts of Atractylodes lancea, 150 parts of Poria cocos, 100 parts of Atractylodes macrocephala, 50 parts of gypsum, 30 parts of Crataegus pinnatifida (roasted), 30 parts of Massa fermentata (roasted), 30 parts of malt (roasted), 50 parts of Pheretima aspergillum, 50 parts of Cynanchum paniculatum, 30 parts of Dryopteris crassirhizoma, and 50 parts of Lepidium apetalum. Add water and reflux twice. For the first extraction, add 6 times the amount of water and extract for 1.5 hours. For the second extraction, add 4 times the amount of water and extract for 1.0 hour. Combine the extracts, filter, concentrate the filtrate to a relative density of 1.10~1.15, centrifuge and filter, vacuum dry the filtrate, spray dry and pulverize to obtain the intermediate composition.

6. The application according to any one of claims 1 to 2, characterized in that, The medication for treating chronic obstructive pulmonary disease is selected from granules, which further include sucralose and dextrin.

7. The use of a traditional Chinese medicine composition in the preparation of a medicament for treating emphysema or asthma, characterized in that, The raw materials of the traditional Chinese medicine composition, by weight ratio, are: Magnolia officinalis 30-50 parts, Areca catechu 20-30 parts, Amomum villosum 30-50 parts, Ephedra sinica 20-30 parts, Prunus armeniaca 20-30 parts, Notopterygium incisum 30-50 parts, Zingiber officinale 30-50 parts, Pogostemon cablin 30-50 parts, Eupatorium fortunei 20-30 parts, Atractylodes lancea 30-50 parts, Poria cocos 120-150 parts, Atractylodes macrocephala 80-100 parts, Gypsum 30-50 parts, Crataegus pinnatifida 20-30 parts, Massa fermentata 30-50 parts, Hordeum vulgare 20-30 parts, Pheretima aspergillum 30-50 parts, Cynanchum paniculatum 30-50 parts, Dryopteris crassirhizoma 20-30 parts, Lepidium apetalum 30-50 parts.

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