A traditional Chinese medicine composition for treating autoimmune hepatitis, its preparation method, and its uses.

Abstract

This invention relates to the field of traditional Chinese medicine, specifically to a traditional Chinese medicine composition for treating autoimmune hepatitis, its preparation method, and its uses. The traditional Chinese medicine composition provided by this invention comprises the following raw materials in parts by weight: Astragalus membranaceus 3-60 parts by weight, Atractylodes macrocephala 3-20 parts by weight, Saposhnikovia divaricata 3-20 parts by weight, Paeonia lactiflora 3-30 parts by weight, Crataegus pinnatifida 5-30 parts by weight, Curcuma longa 6-30 parts by weight, Glauber's salt 1-10 parts by weight, Rehmannia glutinosa 3-20 parts by weight, Poria cocos 6-30 parts by weight, Pinellia ternata 3-9 parts by weight, Bupleurum chinense 3-20 parts by weight, Stephania tetrandra 3-10 parts by weight, and Glycyrrhiza uralensis 3-15 parts by weight. The combined effects of these herbs are to soothe the liver and strengthen the spleen, soften the liver and detoxify, clear heat and resolve dampness, and harmonize qi and blood, and can be used to treat autoimmune hepatitis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically to a traditional Chinese medicine composition for treating autoimmune hepatitis, its preparation method, and its uses. Background Technology

[0002] Autoimmune hepatitis (AIH) is an immune-mediated hepatitis of unknown etiology, often accompanied by elevated serum aminotransferase levels, hyperimmunoglobulin G hyperemia, positive serum autoantibodies, and moderate to severe interface hepatitis. AIH is an autoimmune-mediated chronic progressive inflammatory disease of the liver. Hepatocellular damage begins with CD4+ T cells recognizing self-antigen peptides. Overexpression of inflammatory cytokines in the liver and impaired immune regulation mechanisms are important pathogenic mechanisms. The clinical manifestations of AIH are diverse, generally presenting as chronic with insidious onset, but it can also present as acute exacerbations, even leading to acute liver failure. Early diagnosis and appropriate treatment can improve the survival and quality of life of AIH patients and reduce the social medical burden.

[0003] Some patients do not respond to standard immunotherapy or have incomplete biochemical responses. Although the combination of hormones and immunosuppressants can rapidly induce inflammatory remission, most AIH patients face the challenges of disease relapse, restarting immunotherapy, and requiring lifelong maintenance therapy during dose reduction or discontinuation. Accompanied by varying degrees of clinical symptoms and adverse reactions related to immunotherapy drugs, treatment is quite challenging. Therefore, finding an alternative treatment plan that modulates immunity, reduces the adverse reactions of immunosuppressive drugs, and consolidates efficacy during hormone withdrawal remains a clinical necessity. In recent years, traditional Chinese medicine's understanding of the etiology, pathogenesis, and treatment of AIH has become increasingly rich, but currently, there is still no clear guideline system for its implementation. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a traditional Chinese medicine composition for treating autoimmune hepatitis, a preparation method thereof, and its uses.

[0005] Therefore, the present invention provides the following technical solution:

[0006] The embodiments disclosed in this invention relate to a traditional Chinese medicine composition, comprising the following raw materials in parts by weight:

[0007] Astragalus membranaceus 3-60 parts by weight, Atractylodes macrocephala 3-20 parts by weight, Saposhnikovia divaricata 3-20 parts by weight, Paeonia lactiflora 3-30 parts by weight, Crataegus pinnatifida 5-30 parts by weight, Curcuma longa 6-30 parts by weight, Glauber's salt 1-10 parts by weight, Rehmannia glutinosa 3-20 parts by weight, Poria cocos 6-30 parts by weight, Pinellia ternata 3-9 parts by weight, Bupleurum chinense 3-20 parts by weight, Stephania tetrandra 3-10 parts by weight, Glycyrrhiza uralensis 3-15 parts by weight.

[0008] In some embodiments, the drug is made from the following parts by weight of active pharmaceutical ingredient:

[0009] Astragalus membranaceus 15-60 parts by weight, Atractylodes macrocephala 3-18 parts by weight, Saposhnikovia divaricata 3-18 parts by weight, Paeonia lactiflora 3-28 parts by weight, Crataegus pinnatifida 6-28 parts by weight, Curcuma longa 6-28 parts by weight, Glauber's salt 3-10 parts by weight, Rehmannia glutinosa 6-20 parts by weight, Poria cocos 6-28 parts by weight, Pinellia ternata 6-9 parts by weight, Bupleurum chinense 3-18 parts by weight, Stephania tetrandra 3-8 parts by weight, Glycyrrhiza uralensis 6-10 parts by weight.

[0010] Optional ingredients include: 30 parts by weight of raw Astragalus membranaceus, 12 parts by weight of Atractylodes macrocephala, 6 parts by weight of Saposhnikovia divaricata, 20 parts by weight of Paeonia lactiflora, 20 parts by weight of charred Crataegus pinnatifida, 15 parts by weight of Curcuma longa, 3 parts by weight of Glauber's salt, 15 parts by weight of Rehmannia glutinosa, 20 parts by weight of Poria cocos, 9 parts by weight of processed Pinellia ternata, 9 parts by weight of Bupleurum chinense, 6 parts by weight of Stephania tetrandra, and 6 parts by weight of Glycyrrhiza uralensis.

[0011] In some embodiments, the Atractylodes macrocephala is stir-fried Atractylodes macrocephala;

[0012] And / or, the hawthorn is charred hawthorn;

[0013] And / or, the licorice is roasted licorice.

[0014] The embodiments disclosed in this invention also relate to a method for preparing the aforementioned traditional Chinese medicine composition, comprising:

[0015] Weigh the raw materials according to the formula, mix them, and then extract them.

[0016] In some embodiments, the extraction is a water extraction: 1.5 to 3 times the weight of water is added to the mixed raw materials, heated to 80 to 100°C, maintained for 0.5 to 0.6 hours, extracted 1 to 2 times, filtered, and the filtrates are combined;

[0017] Optionally, the water extraction step is as follows: add 1.5 to 3 times the weight of the raw materials to the mixed raw materials, soak for 0.4 to 0.5 hours, heat to 80 to 100°C, maintain for 0.5 to 0.6 hours, then cool to 60 to 80°C, and filter to obtain the first extract and residue;

[0018] Add 1.5 to 3 times the weight of the dregs of the medicine to the dregs, then boil the liquid at 80 to 100°C for 0.5 to 0.6 hours, then cool it down to 60 to 80°C and boil it for 0.4 to 0.5 hours. Filter to obtain the second extract.

[0019] Mix the first extract and the second extract thoroughly.

[0020] In some embodiments, after the extraction step, the obtained extract is further concentrated and dried.

[0021] The embodiments disclosed in this invention also relate to a traditional Chinese medicine preparation, comprising a traditional Chinese medicine composition prepared by the aforementioned traditional Chinese medicine composition or the preparation method of the aforementioned traditional Chinese medicine composition as an active ingredient.

[0022] In some implementations, pharmaceutically acceptable excipients are also included;

[0023] Optionally, the traditional Chinese medicine preparation may be in the form of a liquid preparation or a solid preparation;

[0024] Optionally, the dosage form of the traditional Chinese medicine preparation includes tablets, capsules, powders, granules, or ointments.

[0025] The embodiments disclosed in this invention also relate to the use of the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the method of preparing the traditional Chinese medicine composition, or the traditional Chinese medicine preparation thereof, in any of the following:

[0026] (1) Use in the preparation of drugs that soothe the liver and strengthen the spleen, soften the liver and detoxify, clear heat and resolve dampness and / or regulate qi and blood;

[0027] (2) Use in the preparation of drugs for the treatment of autoimmune liver diseases.

[0028] In some embodiments, its use in the preparation of a medicament for treating autoimmune hepatitis;

[0029] Alternatively, its use in the preparation of drugs that reduce serum ALT, AST, and DBIL levels, alleviate inflammatory cell infiltration in the portal areas of liver tissue, improve the structure of hepatic sinusoids and the microenvironment of LSECs, and / or reduce inflammatory damage.

[0030] The traditional Chinese medicine composition of this invention is an empirical formula for treating autoimmune hepatitis, repeatedly verified and continuously optimized by Professor Lü Wenliang in clinical practice. Hundreds of cases in early clinical trials have demonstrated that this formula regulates immunity, reduces the adverse reactions of immunosuppressive drugs, consolidates the therapeutic effect during hormone withdrawal, protects the liver and lowers enzymes, improves symptoms such as fatigue and weakness, and restores the patient's spleen and stomach function. Basic experiments have shown that the traditional Chinese medicine composition of this invention can improve the mental state of a Con A-induced chronic immune liver injury mouse model, increase mouse weight, reduce mortality, decrease serum ALT, AST, and DBIL levels, reduce inflammatory cell infiltration in the portal areas of liver tissue, improve hepatic sinusoidal structure and the LSECs microenvironment, and effectively reduce inflammatory damage. This formula can provide ideas and medical research evidence for the field of integrated traditional Chinese and Western medicine treatment of autoimmune hepatitis.

[0031] The technical solution of this invention has the following advantages:

[0032] 1. This invention provides a traditional Chinese medicine composition comprising the following raw materials in parts by weight: Astragalus membranaceus 3-60 parts by weight, Atractylodes macrocephala 3-20 parts by weight, Saposhnikovia divaricata 3-20 parts by weight, Paeonia lactiflora 3-30 parts by weight, Crataegus pinnatifida 5-30 parts by weight, Curcuma longa 6-30 parts by weight, Glauber's salt 1-10 parts by weight, Rehmannia glutinosa 3-20 parts by weight, Poria cocos 6-30 parts by weight, Pinellia ternata 3-9 parts by weight, Bupleurum chinense 3-20 parts by weight, Stephania tetrandra 3-10 parts by weight, and Glycyrrhiza uralensis 3-15 parts by weight; In the above formula, Astragalus membranaceus (the principal herb): sweet in taste, slightly warm in nature, and enters the lung and spleen meridians. It has the functions of tonifying qi and consolidating the exterior, promoting tissue regeneration and detoxification, and promoting diuresis and reducing swelling. In this formula, Astragalus membranaceus is used to tonify qi and strengthen the spleen, enhance the body's immunity, and at the same time, it follows the ascending nature of liver qi, which helps to regulate liver qi.

[0033] White peony root (principal ingredient): Bitter and sour in taste, slightly cold in nature, and enters the liver and spleen meridians. It has the effects of nourishing blood and softening the liver, relieving spasms and pain. In this formula, white peony root is used to nourish blood and astringe yin, soften the liver and relieve pain, and alleviate pain caused by liver qi stagnation.

[0034] Atractylodes macrocephala (assistant herb): Bitter and sweet in taste, warm in nature, and enters the spleen and stomach meridians. It has the functions of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis. In this formula, Atractylodes macrocephala replenishes qi and strengthens the spleen, and resolves dampness, thus assisting Astragalus membranaceus in strengthening the spleen and tonifying the exterior.

[0035] Bupleurum (assistant herb): Bitter and pungent in taste, slightly cold in nature, and enters the liver, gallbladder, and lung meridians. It has the functions of soothing the liver and relieving stagnation, and raising yang qi. In this formula, Bupleurum is used to promote the flow of liver qi and alleviate liver qi stagnation.

[0036] Rehmannia glutinosa (assistant herb): Sweet and bitter in taste, cold in nature, and enters the heart, liver, and kidney meridians. It has the functions of clearing heat and cooling blood, nourishing yin and generating fluids. In this formula, Rehmannia glutinosa nourishes kidney water to nourish liver wood, drains damp heat from spleen earth, and keeps the liver moist and not dry.

[0037] Turmeric (adjuvant herb): pungent and bitter in taste, cold in nature, and enters the liver, lung, and heart meridians. It has the functions of promoting blood circulation and relieving pain, regulating qi and relieving depression, and promoting bile secretion and reducing jaundice. In this formula, turmeric promotes qi and blood circulation, regulates liver qi, and assists Bupleurum in soothing the liver and relieving depression.

[0038] Pinellia ternata (adjuvant herb): pungent in taste, warm in nature, and toxic; it enters the spleen, stomach, and lung meridians. It has the functions of drying dampness and resolving phlegm, as well as suppressing nausea and vomiting. In this formula, Pinellia ternata suppresses nausea, harmonizes the stomach, and resolves turbidity to aid the spleen and stomach in their digestive functions.

[0039] Poria cocos (adjuvant herb): Sweet and bland in taste, neutral in nature, and enters the heart, lung, spleen, and kidney meridians. It has the functions of promoting diuresis and eliminating dampness, strengthening the spleen, and calming the mind. In this formula, Poria cocos strengthens the spleen and eliminates dampness, assisting Atractylodes macrocephala in strengthening the spleen and resolving dampness.

[0040] Fangji (adjuvant herb): Bitter in taste, cold in nature, and enters the bladder and lung meridians. It has the effects of promoting diuresis and reducing swelling, and dispelling wind and dampness. In this formula, Fangji transforms damp-heat in the blood and assists Rehmannia glutinosa in clearing heat and cooling the blood.

[0041] Hawthorn (auxiliary herb): Sour and sweet in taste, slightly warm in nature, and enters the spleen, stomach, and liver meridians. It has the functions of promoting digestion, resolving food stagnation, regulating qi, and dispersing blood stasis. In this formula, hawthorn promotes digestion, disperses blood stasis, and unblocks the meridians, while assisting Poria in strengthening the spleen.

[0042] Fangfeng (adjuvant herb): pungent and sweet in taste, slightly warm in nature, and enters the bladder, liver, and spleen meridians. It has the functions of dispelling wind and relieving exterior symptoms, eliminating dampness and relieving pain. In this formula, Fangfeng is used in conjunction with Huangqi and Baizhu, drawing on the meaning of "Jade Screen Wind" (a metaphor for a protective barrier), to strengthen the body's resistance and eliminate pathogenic factors.

[0043] Licorice (guide herb): Sweet in taste, neutral in nature, and enters the heart, lung, spleen, and stomach meridians. It has the functions of tonifying the spleen and replenishing qi, clearing heat and detoxifying, and harmonizing other herbs. In this formula, licorice harmonizes the other herbs and, combined with white peony root, nourishes yin and liver through its sour and sweet properties.

[0044] Glauber's salt (guide ingredient): Salty and bitter in taste, cold in nature, and enters the stomach and large intestine meridians. It has the functions of purging and relieving constipation, moistening dryness and softening hard masses, clearing heat and reducing swelling. In this formula, Glauber's salt eliminates accumulations in the six viscera, resolves lumps and stagnation, unblocks the meridians, and promotes metabolism. The combined effects of the above-mentioned ingredients have the functions of soothing the liver and strengthening the spleen, softening the liver and detoxifying, clearing heat and resolving dampness, and regulating qi and blood, and can be used to treat autoimmune hepatitis. Attached Figure Description

[0045] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0046] Figure 1 This describes the trend of mouse body weight change over time in each group of experimental examples of the present invention;

[0047] Figure 2 These are the liver and spleen indices of mice in each group of experimental cases of this invention;

[0048] Figure 3 The liver morphology of mice in each group of experimental examples of this invention;

[0049] Figure 4 The morphology of the spleen of mice in each group of experimental examples of this invention;

[0050] Figure 5 These are the ALT, AST, and DBIL levels in mice in each group of the experimental examples of this invention;

[0051] Figure 6 These are HE staining pathological images (200×) of mouse livers from each group in the experimental examples of this invention;

[0052] Figure 7 These are scanning electron microscope images of mouse livers from each group in the experimental examples of this invention. Detailed Implementation

[0053] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0054] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0055] The active pharmaceutical ingredients in the traditional Chinese medicine compositions of this invention are all listed in the 2020 edition of the Chinese Pharmacopoeia and are all commercially available products.

[0056] Astragalus (also known as raw astragalus) is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge., belonging to the legume family.

[0057] Atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala Koidz., a plant in the Asteraceae family; stir-fried Atractylodes macrocephala is a processed form of Atractylodes macrocephala.

[0058] Saposhnikovia divaricata (Turcz.) Schishk., a plant in the Apiaceae family, is a dried root.

[0059] White peony root is the dried root of Paeonia lactiflora Pall., a plant in the Ranunculaceae family.

[0060] Hawthorn is the dried, ripe fruit of Crataegus pinnatifida Bge. var. major NEBr. or Crataegus pinnatifida Bge., both belonging to the Rosaceae family; charred hawthorn is a processed form of hawthorn.

[0061] Turmeric is the dried tuberous root of Curcuma wenyujin YHChen et C.Ling, Curcuma longa L., Curcuma kwangsiensis SGLee et CFLiang, or Curcuma phaeocaulis Val., all belonging to the ginger family.

[0062] Glauber's salt is a sulfate mineral, specifically Glauber's salt (Na₂SO₄·10H₂O), which is a crystalline substance produced through processing and refining. It primarily contains hydrated sodium sulfate (Na₂SO₄·10H₂O).

[0063] Rehmannia glutinosa (also known as Dihuang) is the fresh or dried tuberous root of Rehmannia glutinosa Libosch., a plant in the Scrophulariaceae family.

[0064] Poria cocos is the dried sclerotium of the fungus Poria cocos (Schw.) Wolf, belonging to the Polyporaceae family.

[0065] Pinellia ternata is the dried tuber of Pinellia ternata (Thunb.) Breit., a plant of the Araceae family; processed Pinellia ternata is the prepared form of Pinellia ternata.

[0066] Bupleurum is the dried root of Bupleurum chinense DC. or Bupleurum corzonerifolium Willd., both belonging to the Apiaceae family.

[0067] Fangji is the dried root of Stephania tetrandra S. Moore, a plant in the Menispermaceae family.

[0068] Licorice is the dried root and rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L., all belonging to the legume family. Prepared licorice is a processed form of licorice.

[0069] All of the above raw materials are commercially available products.

[0070] Example

[0071] This embodiment provides a traditional Chinese medicine composition, the formula of which is shown in the table below:

[0072] Table 1. Formula

[0073] raw materials Example 1 Example 2 Example 3 Example 4 Example 5 Raw Astragalus (g) 30 15 60 3 60 Stir-fried Atractylodes macrocephala (g) 12 18 3 20 3 Windproof (g) 6 3 18 3 20 White peony root (g) 20 28 3 30 3 Roasted hawthorn (g) 20 6 28 5 30 Turmeric (g) 15 28 6 30 6 Glauber's salt (g) 3 3 10 1 10 Raw Rehmannia (g) 15 20 6 20 3 Poria cocos (g) 20 6 28 6 30 Pinellia ternata (g) 9 9 6 9 3 Bupleurum (g) 9 3 18 20 3 Fangji (g) 6 8 3 3 10 Prepared licorice root (g) 6 6 10 15 3

[0074] The preparation method of the traditional Chinese medicine composition in Example 1 above includes the following steps:

[0075] (1) Weigh the raw materials according to the formula, mix them evenly, then add water three times the weight of the raw materials, heat to 100°C, keep for 0.5 hours, filter (200 mesh) to obtain filter residue and filtrate;

[0076] (2) Add water at 1.5 times the weight of the raw drug to the filter residue in step (1), heat to 100°C, keep for 1 hour, filter (200 mesh) to obtain filter residue and filtrate;

[0077] (3) Combine the filtrates from steps (1) and (2) and concentrate them under reduced pressure to obtain a clear extract;

[0078] (4) The ointment is spray-dried, collected, and sieved to obtain dry ointment powder. It is mixed evenly and granulated by dry method to make 12-40 mesh granules, each granule weighing 11.2 grams.

[0079] The preparation method of the traditional Chinese medicine composition in Example 2 above includes the following steps:

[0080] (1) Weigh the raw materials according to the formula, mix them evenly, then add water at 1.5 times the weight of the raw materials, heat to 90°C, keep for 0.6 hours, filter (200 mesh) to obtain filter residue and filtrate;

[0081] (2) Add water at 3 times the weight of the raw drug to the filter residue in step (1), heat to 90°C, maintain for 0.5 hours, and filter (200 mesh) to obtain filter residue and filtrate;

[0082] (3) Combine the filtrates from steps (1) and (2) and concentrate them under reduced pressure to obtain a clear extract;

[0083] (4) The ointment is spray-dried, collected, and sieved to obtain dry ointment powder. It is mixed evenly and granulated by dry method to make 12-40 mesh granules, each granule weighing 11.2 grams.

[0084] The preparation method of the traditional Chinese medicine composition in Example 3 above includes the following steps:

[0085] (1) Weigh the raw materials according to the formula, mix them evenly, then add water at 2.5 times the weight of the raw materials, heat to 80°C, maintain for 0.6 hours, filter (200 mesh) to obtain filter residue and filtrate;

[0086] (2) Add water at 2.5 times the weight of the raw drug to the filter residue in step (1), heat to 80°C, maintain for 0.6 hours, and filter (200 mesh) to obtain filter residue and filtrate;

[0087] (3) Combine the filtrates from steps (1) and (2) and concentrate them under reduced pressure to obtain a clear extract;

[0088] (4) The ointment is spray-dried, collected, and sieved to obtain dry ointment powder. It is mixed evenly and granulated by dry method to make 12-40 mesh granules, each granule weighing 11.2 grams.

[0089] The preparation method of the traditional Chinese medicine composition in Example 4 above includes the following steps:

[0090] (1) Weigh the raw materials according to the formula, mix them evenly, then add water twice the weight of the crude drug, heat to 100 °C, hold for 0.5 hours, and filter (200 mesh) to obtain filter residue and filtrate.

[0091] (2) Add water twice the weight of the crude drug to the filter residue in step (1), heat to 80 °C, hold for 0.5 hours, and filter (200 mesh) to obtain filter residue and filtrate.

[0092] (3) Combine the filtrates in step (1) and step (2), and concentrate under reduced pressure to obtain a clear extract.

[0093] (4) Spray dry the clear extract, collect the powder, sieve to obtain dry extract powder, mix evenly, and granulate by dry granulation to make 12 - 40 mesh granules, with each granule having a specification of 11.2 grams.

[0094] The preparation method of the traditional Chinese medicine composition in Example 5 above includes the following steps:

[0095] (1) Weigh the raw materials according to the formula, mix them evenly, then add water three times the weight of the crude drug, heat to 100 °C, hold for 0.6 hours, and filter (200 mesh) to obtain filter residue and filtrate.

[0096] (2) Concentrate the filtrate in step (1) under reduced pressure to obtain a clear extract.

[0097] (3) Spray dry the clear extract, collect the powder, sieve to obtain dry extract powder, mix evenly, and granulate by dry granulation to make 12 - 40 mesh granules, with each granule having a specification of 11.2 grams.

[0098] Experimental Example Animal Experiment

[0099] In this part of the experiment, a mouse model of AIH induced by concanavalin A (Con A) was constructed. The traditional Chinese medicine composition in Example 1 was used as the experimental drug, and prednisolone was used as the positive control drug. The effects of drug intervention on the general condition, serum biochemistry, liver tissue pathology, and scanning electron microscopy of Con A - induced AIH mice were observed, aiming to evaluate the effect of the traditional Chinese medicine composition described in this invention on Con A - induced AIH model mice.

[0100] 1 Experimental Animals

[0101] SPF - grade male Balb / c mice, 6 - 8 weeks old, weighing 18 - 22 g. Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal license number: SCXK (Beijing) 2021 - 0006. Housed in the Experimental Animal Center of Guang'anmen Hospital, China Academy of Chinese Medical Sciences, with constant temperature and humidity, 12 - hour light and 12 - hour dark cycle in the room, and free access to food and water.

[0102] The feeding and experimental conditions and procedures strictly adhered to the principles of animal experimentation ethics and passed the ethical review of the Ethics Committee of Guang'anmen Hospital of China Academy of Chinese Medical Sciences.

[0103] 2 Experimental Methods

[0104] 2.1 Animal grouping, model establishment, and drug administration

[0105] (1) Animal grouping: Mice were randomly divided into four groups: a blank control group (Control group), a ConA model group (Con A group), a prednisolone treatment group (Prednisolone group), and a traditional Chinese medicine composition treatment group (JPYG group) as described in this invention. In the preliminary experiments conducted by the research group, the mortality rate of the model group was approximately 25%. Based on the experimental needs, 16 mice were set up in the Control group, and 24 mice were set up in each of the Con A group, Prednisolone group, and ZYZL group.

[0106] (2) Preparation of experimental drugs:

[0107] 1. Con A Preparation Method: Pre-store 0.9% physiological saline at 4°C. Add Con A (Sigma-Aldrich, C2010, 25mg / vial) stored at -20°C to the cold physiological saline to prepare a solution with a concentration of 1.25mg / ml. Specific procedures are as follows: Add 25mg of Con A powder to 20ml of pre-cooled physiological saline, shake repeatedly to dissolve, sterilize with a sterile filter membrane, and dispense 4-5ml into sterile EP tubes. Store at -20°C. Prepare and use immediately to avoid repeated freeze-thaw cycles that could degrade the drug.

[0108] 2. Prednisolone solution: Grind prednisolone acetate tablets (Tianjin Xinyi Jinjin Pharmaceutical Co., Ltd., 5mg / tablet, batch number: 504220410) into a fine powder using a mortar and pestle, add pure water to prepare a solution with a concentration of 0.6mg / ml, and store it in a refrigerator at 4℃.

[0109] 3. Traditional Chinese medicine composition solution: Omit the dry granulation step in step (4) and directly add the prepared dry powder of traditional Chinese medicine composition to pure water to prepare a solution with a concentration of 2.59 g / ml.

[0110] (3) Modeling and drug administration

[0111] Modeling method: Based on prior literature review and preliminary experimental results, the tail vein injection of Con A was selected as the method for inducing chronic immune liver injury. An acclimatization diet was administered for one week to allow the patient to adapt to the environment before the experiment began.

[0112] Mold creation procedure: Remove the prepared ConA solution and thaw at room temperature. Weigh the mice and calculate the required volume of ConA solution, which is then drawn using a 1ml insulin syringe. Place the mouse in the tail vein injection fixation device, ensuring it is in a comfortable position and restrained to prevent struggling, with the tail exposed. Repeatedly wipe the mouse's tail with an alcohol swab to dilate the tail vein. Select the more prominently dilated tail vein from both sides, straighten and fix the tail with your left hand, and insert the insulin syringe parallel to the tail vein from the distal end of the tail with your right hand. Gently push the drug in; if there is no resistance, it has entered the vein. After injection, apply pressure with a sterile dry cotton ball to stop bleeding. The ConA dose for tail vein injection in mice is 12.5mg / kg, with an injection volume of 0.1ml / 10g body weight. Administer once a week for 6 consecutive weeks.

[0113] Dosage: According to the "Pharmacological Experimental Methodology," the equivalent dose was calculated based on the human-to-mouse body surface area conversion, multiplied by a conversion factor of 9.1. A safe and suitable gavage volume of 0.2 ml / 10 g body weight was selected for mice, and the concentration of the traditional Chinese medicine composition described in this invention was 2.59 g / ml. The prednisolone dosage was based on the treatment guidelines for the diagnosis and treatment of autoimmune hepatitis (2021) and literature research. Following the adult treatment dose of 40 mg / day, the mouse dosage was calculated to be 0.12 mg / kg body weight / day, and the concentration of the prednisolone aqueous solution was 0.6 mg / ml.

[0114] Modeling and drug administration methods for each group:

[0115] 1. Blank control group: Each mouse was injected with an equal volume of physiological saline via the tail vein once a week for 6 consecutive weeks. Pure water was administered by gavage starting on the day of the first tail vein injection, once daily for 6 consecutive weeks.

[0116] 2. Con A model group: Con A was injected into the tail vein once a week for 6 consecutive weeks. Pure water was administered by gavage on the same day after the first tail vein injection, once daily for 6 consecutive weeks.

[0117] 3. Prednisolone positive control group: Con A was injected via tail vein once a week for 6 consecutive weeks. Prednisolone solution was started on the day of the first tail vein injection and administered once daily for 6 consecutive weeks.

[0118] 4. The traditional Chinese medicine composition group of the present invention: Con A is injected into the tail vein once a week for 6 consecutive weeks. The traditional Chinese medicine composition of the present invention is administered once daily for 6 consecutive weeks, starting on the day of the first tail vein injection.

[0119] 2.2 Observation Indicators and Model Validation

[0120] 2.2.1 General Observation

[0121] The basic conditions of each group of mice were observed and recorded, including their mental state, fur color, food and water intake and excretion, activity level, and weight changes were recorded twice a week.

[0122] 2.2.2 Model Validation

[0123] Based on the preliminary experimental results, after completing the tail vein injection and gavage cycle in week 5, three mice from each group were randomly selected. Whole blood was collected, centrifuged, and serum liver function was tested. Liver tissue was taken for HE pathological verification. The ALT and AST levels in group Con A were higher than those in the other three groups, and the pathology showed obvious inflammatory infiltration in the portal canal area, indicating successful model establishment. The tail vein injection and gavage cycle was continued for week 6 before tissue collection.

[0124] 2.3 Serum and tissue collection

[0125] Mice in each group were fasted for 12 hours before sampling. After anesthesia, blood was collected by enucleation. Biochemical blood collection tubes and sterile EP tubes were labeled with the corresponding group and number. Whole blood samples were placed at room temperature for 2 hours after collection, and then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was aspirated by pipette and dispensed into sterile EP tubes. The samples were stored at -80°C for subsequent serum liver function testing.

[0126] Immediately after blood collection, mice were euthanized by cervical dislocation. After disinfection with alcohol, the abdominal cavity was opened, and the liver and spleen were completely removed. Residual blood was blotted dry with filter paper, and the liver and spleen were weighed and recorded. The appearance, size, and texture of the tissues were observed and photographed. The liver tissue was washed with PBS buffer, and a portion of the liver tissue was cut and fixed in 4% paraformaldehyde for 24 hours to prepare pathological sections. The remaining liver sections were aliquoted into sterile cryovials and quickly transferred to dry ice for freezing at -80°C.

[0127] Three mice were randomly selected from each group and euthanized by cervical dislocation. After disinfection, the abdominal cavity was opened to expose the portal vein and inferior vena cava. Buffer was infused through the mouse's portal vein using a cannula. The inferior vena cava was quickly cut to allow the blood and buffer solution to drain from the liver. When the liver turned white, 2.5% glutaraldehyde electron microscopy fixative was started. After the liver hardened, liver tissue was excised, placed on ice, cut into pieces, and then placed in 2% glutaraldehyde fixative. The pieces were fixed at room temperature for 2 hours and then transferred to a 4°C refrigerator for storage for subsequent electron microscopy sample preparation.

[0128] 2.4 Detection Indicators and Methods:

[0129] 2.4.1 Serum Liver Function Tests: Serum samples were thawed and tested as soon as possible using a fully automated biochemical analyzer. Based on the preliminary experimental results, the main test items were alanine aminotransferase (ALT), aspartate aminotransferase (AST), and direct bilirubin (DBIL).

[0130] 2.4.2 Liver tissue pathological sections and HE staining

[0131] Liver tissue fixed with 4% paraformaldehyde was removed, trimmed into blocks, and subjected to HE staining for observation. The specific procedures are as follows:

[0132] ① Dehydration and embedding: The trimmed tissue blocks were sequentially immersed in 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I and II for 30 minutes each, benzene for 5-10 minutes, xylene I and II for 5-10 minutes each, and paraffin was melted at 65°C for 3 hours.

[0133] ② Sectioning: Place the wax block in a paraffin microtome and section it to a thickness of 4μm. Bake the sections in a 60℃ oven. After the moisture is dried and the paraffin is melted, remove the sections and store them at room temperature for later use.

[0134] ③ Dewaxing paraffin sections to water: Immerse the sections in dewaxing solution I for 20 minutes, then in dewaxing solution II.

[0135] After 20 minutes, immerse in anhydrous ethanol I and II for 5 minutes each, and 75% alcohol for 5 minutes, then rinse with tap water.

[0136] ④ Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, and rinse with running water after blueing with blueing solution.

[0137] ⑤ Eosin staining: Dehydrate the sections in 95% alcohol for 1 minute, then stain them in eosin staining solution for 15 seconds.

[0138] ⑥ Dehydration and mounting: The sections are placed in anhydrous ethanol I, II, and III for 2 min each, n-butanol I and II for 2 min each, and xylene I and II for 2 min each for clearing, and then mounted with neutral resin.

[0139] ⑦ Observe under a microscope and perform image acquisition and analysis.

[0140] 2.4.3 Liver Scanning Electron Microscopy Sample Preparation and Observation

[0141] Remove liver tissue from glutaraldehyde fixative in a 4°C refrigerator and prepare electron microscopy samples according to the following steps.

[0142] ① Post-fixation: Rinse to remove glutaraldehyde from the sample, fix in 2% glutaraldehyde for 3 hours, then rinse repeatedly several times at 4℃ with 0.1 mol / L sodium dimethylarsinate buffer (pH 7.4), and place the tissue in the buffer overnight. On the second day, remove the buffer, add 1% osmium tetroxide for 1 hour, discard the fixative, and rinse repeatedly with buffer 5 times, 15 minutes each time.

[0143] ② Alcohol gradient dehydration: Liver tissue slices were sequentially immersed in 30%, 50%, 75%, 85%, 90% alcohol solutions, and...

[0144] Dehydrate in 95% alcohol for 15 minutes each time; then dehydrate in two groups of anhydrous ethanol for 15 minutes each time.

[0145] ③ Drying: Separate the sample groups with filter paper, stack them in a critical point dryer, set the parameters, and circulate CO2 12 times to dry.

[0146] ④ Sample conductivity treatment: Place the tissue sample tightly onto the conductive carbon film double-sided adhesive and put it on the sample stage of the ion sputtering instrument for platinum sputtering treatment for about 30 seconds.

[0147] ⑤ High-resolution cold field emission scanning electron microscopy observation: Observe the ultrastructure of the liver under a cold field emission scanning electron microscope. Locate the portal area of ​​the specimen at low magnification, position the area around the portal area, and adjust the magnification until the structure of the sinusoids, intrahepatic vessels, and capillary bile ducts can be observed clearly. Fine-tune the field of view to find the target sinusoidal structures and sinusoidal endothelial cells, adjust the magnification, and acquire images.

[0148] 2.5 Statistical Analysis

[0149] For measurement data that conform to a normal distribution, the mean ± standard deviation (x ± s) is used; for those that do not conform to a normal distribution, the median and interquartile range M (P25, P75) are used. Statistical analysis and graphing were performed using Graphpad Pism 9.5 software. One-way ANOVA was used for comparisons among multiple groups, with P < 0.05 considered statistically significant.

[0150] 3 Experimental Results

[0151] 3.1 Observation of mouse survival and general condition

[0152] (1) Survival status: During the entire experimental period, no mice died in the blank group; 6 mice died in the model group; 2 mice died in the prednisolone treatment group; and 3 mice died in the traditional Chinese medicine composition group described in this invention.

[0153] (2) General condition: Mice in the blank group were in good spirits, with shiny fur, normal food and water intake and bowel movements, and were agile. Their weight gradually increased throughout the experimental period. Mice in the other three groups showed lethargy, hypothermia, a tendency to curl up and lie prone, increased bowel movements, decreased activity, weight loss, and dull fur within 3 days of weekly tail vein injection of ConA. Some mice died, with most deaths occurring 2 hours to 2 days after model establishment. Subsequently, the mice in each group gradually recovered their mental and physical condition, only to experience the same condition again after the next tail vein injection of ConA. Compared to the model group, the activity and general condition of the prednisolone and the traditional Chinese medicine composition groups described in this invention were significantly improved. The activity of the prednisolone intervention group was significantly increased, and some mice were hyperactive.

[0154] 3.2 Changes in mouse body weight

[0155] In preliminary experiments, it was observed that the mice's body weight significantly decreased after modeling, gradually recovering before the next modeling attempt, showing a fluctuating upward trend. Therefore, the body weight changes on the 3rd and 6th days after each modeling attempt were recorded to compare the differences between the groups. The results showed that the body weight of the control group mice gradually and steadily increased over time; the model group and the treatment group using the traditional Chinese medicine composition described in this invention showed a slow, fluctuating upward trend, with a decrease in body weight 3 days after each modeling attempt, gradually recovering by the 6th day; the body weight of the mice in the prednisolone treatment group was significantly lower than that of the other groups, showing an alternating fluctuating decrease after the second week of modeling. See details... Figure 1 .

[0156] 3.3 Comparison of liver and spleen indices and histological morphology

[0157] 3.3.1 Comparison of liver and spleen indices among different groups of mice

[0158] At the time of sampling, the body weight, liver and spleen weights of mice in each group were recorded, and the liver and spleen indices were calculated (n=6). The calculation formulas were: Liver index = Liver weight (g) / Body weight (g) × 10; Spleen index = Spleen weight (g) / Body weight (g) × 10. One-way ANOVA was used for comparisons among multiple groups, and Graphpad Prism software was used for statistical analysis and graphing.

[0159] Liver index statistics showed that compared with the Control group, the liver indexes in the ConA and JPYG groups were significantly increased (P<0.05), while the liver index in the Prednisolone group was not significantly different from that in the normal group (P>0.05). Compared with the ConA group, the liver index in the Prednisolone group was decreased (P<0.05), while there was no significant difference in the ZYZL group (P>0.05), but a decreasing trend was observed. Spleen index statistics showed that compared with the Control group, the spleen indexes in all groups were significantly increased (P<0.01). Compared with the ConA group, the spleen index in the Prednisolone group was significantly decreased (P<0.01), while there was no statistically significant difference in the ZYZL group (P>0.05), but a decreasing trend was observed compared to the ConA group. See Table 2 for details. Figure 2 .

[0160] Table 2 Comparison of liver and spleen indices among groups (x±s)

[0161] Groups (n=6) Liver index Spleen Index Control group 0.3917±0.0289 0.0388±0.0026 ConA Group <![CDATA[0.4683±0.0290 ** ]]> <![CDATA[0.0957±0.0147 ** ]]> Prednisolone group <![CDATA[0.4155±0.0048 △ ]]> <![CDATA[0.0612±0.0052 **△△ ]]> JPYG Group <![CDATA[0.4437±0.0058 * ]]> <![CDATA[0.0823±0.0096 ** ]]>

[0162] Note: Compared to the Control group, * P<0.05, ** P<0.01; compared with group ConA, △ P<0.05, △△P<0.01.

[0163] Note: The Control group is the blank group; the ConA group is the concanavalin A model group; the Prednisolone group is the prednisolone group; and the JPYG group is the traditional Chinese medicine composition group described in this invention. The same applies below.

[0164] 3.3.2 External morphology of the liver and spleen

[0165] (1) Appearance of liver in mice of each group

[0166] Comparison of liver texture and appearance among the groups of mice revealed the following: The Control group mice had normal liver morphology, soft texture, and normal color; the ConA group mice had significantly enlarged livers, a dark red color, a distended and firm texture, and an uneven surface; the Prednisolone and JPYG groups had smaller livers than the ConA group, with relatively smooth surfaces. Specifically, the Prednisolone group livers were slightly yellow, excessively soft, and prone to decay; the JPYG group livers were increased in volume, slightly dark red in color, and softer than the ConA group. (See...) Figure 3 .

[0167] (2) Appearance of spleen in mice of each group

[0168] The spleens of mice in the control group were normal in morphology and color, and soft in texture; the spleens of mice in the ConA group were significantly swollen and enlarged, hard in texture, and dark red in color; the spleens of mice in the Prednisolone and JPYG groups were smaller in volume than those in the ConA group, slightly lighter in color, and softer in texture; the spleens of mice in the Prednisolone group were smaller in volume than those in the JPYG group. (See...) Figure 4 .

[0169] 3.4 Serum liver function test results

[0170] Statistical analysis was performed on the ALT, AST, and DBIL levels of mice in each group. The results showed that compared with the Control group, the ALT, AST, and DBIL levels in the ConA group were all significantly increased (P<0.01). Compared with the ConA group, the Prednisolone and JPYG intervention groups showed significantly decreased levels (P<0.05), as shown in Table 3. Figure 5 .

[0171] Table 3 Comparison of serum ALT, AST, and DBIL levels in mice across different groups (x±s)

[0172] Group ALT(U / L) AST(U / L) DBIL(umol / l) Control group 29.98±1.627 92.35±13.670 6.76±0.690 ConA Group <![CDATA[40.58±4.504 * ]]> <![CDATA[122.4±7.784 * ]]> <![CDATA[10.21±2.194 * ]]> Prednisolone group <![CDATA[31.40±4.468 △ ]]> <![CDATA[101.1±7.391 △ ]]> <![CDATA[7.419±1.284 △ ]]> JPYG Group <![CDATA[29.18±3.667 △△ ]]> <![CDATA[97.28±7.394 △ ]]> <![CDATA[7.30±1.31 △ ]]> F 9.122 7.849 5.434 P 0.001 0.004 0.007

[0173] Note: Compared to the Control group, * P<0.01; compared with group ConA, △P<0.05, △△ P<0.01.

[0174] 3.5 Liver HE staining pathological observation

[0175] In the Control group, the liver lobule structure of mice was normal, the hepatic cords were arranged radially and neatly, the portal area structure was clear, and no obvious inflammatory cell infiltration was observed. In the ConA group, hepatocytes were disordered, the portal area was irregularly enlarged and congested, hepatocytes were swollen and degenerated, and obvious inflammatory cell infiltration was visible. Compared with the ConA group, the number of inflammatory cells in the portal area was significantly reduced in both the Prednisolone group and the JPYG group. Compared with the JPYG group, the Prednisolone group showed a small amount of fatty degeneration and extensive hepatocyte swelling and degeneration. See Figure 6 .

[0176] 3.6 Electron microscopy results of mouse livers in each group

[0177] Electron microscopy results showed that in the Control group, the hepatocyte cords and sinusoidal structures were clear and neatly arranged, with smooth sinusoidal endothelial cells exhibiting a uniform network of fenestrated foci. In the ConA group, the hepatocyte cords and sinusoidal structures were significantly blurred and disordered, with the fenestrated foci difficult to discern. The hepatocyte structures in the Prednisolone and JPYG groups were relatively clear and distinct, significantly better than those in the ConA group. The sinusoidal endothelial structures in these groups were less smooth than those in the Control group, and the fenestrated foci varied in size and were more densely distributed. (See...) Figure 7 .

[0178] 4. Conclusion

[0179] The traditional Chinese medicine composition described in this invention can improve the mental state of mice with Con A-induced chronic immune liver injury, increase mouse weight, reduce mortality, reduce serum ALT, AST, and DBIL levels, alleviate inflammatory cell infiltration in the portal area of ​​liver tissue, improve the structure of hepatic sinusoids and the microenvironment of hepatic sinusoidal endothelial cells (LSECs), and effectively reduce inflammatory damage.

[0180] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A traditional Chinese medicine composition for treating autoimmune hepatitis, characterized in that, is prepared from the following raw medicinal materials by weight: Radix Astragali 3-60 parts by weight, fried Atractylodes 3-20 parts by weight, Ledebouriella 3-20 parts by weight, Radix Paeoniae Alba 3-30 parts by weight, Fructus Crataegi 5-30 parts by weight, Radix Notoginseng 6-30 parts by weight, Mirabilite 1-10 parts by weight, Radix Rehmanniae 3-20 parts by weight, Poria 6-30 parts by weight, Rhizoma Pinelliae 3-9 parts by weight, Radix Bupleuri 3-20 parts by weight, Radix Stephaniae Tetrandrae 3-10 parts by weight, Radix Glycyrrhizae 3-15 parts by weight.

2. The traditional Chinese medicine composition for treating autoimmune hepatitis according to claim 1, characterized in that, is prepared from the following raw medicinal materials by weight: Radix Astragali 3-60 parts by weight, fried Atractylodes 3-20 parts by weight, Ledebouriella 3-20 parts by weight, Radix Paeoniae Alba 3-30 parts by weight, Fructus Crataegi 5-30 parts by weight, Radix Notoginseng 6-30 parts by weight, Mirabilite 1-10 parts by weight, Radix Rehmanniae 3-20 parts by weight, Poria 6-30 parts by weight, Rhizoma Pinelliae 3-9 parts by weight, Radix Bupleuri 3-20 parts by weight, Radix Stephaniae Tetrandrae 3-10 parts by weight, Radix Glycyrrhizae 3-15 parts by weight.

3. The traditional Chinese medicine composition for treating autoimmune hepatitis according to claim 1, characterized in that, is prepared from the following raw medicinal materials by weight: Radix Astragali 3-60 parts by weight, fried Atractylodes 3-20 parts by weight, Ledebouriella 3-20 parts by weight, Radix Paeoniae Alba 3-30 parts by weight, Fructus Crataegi 5-30 parts by weight, Radix Notoginseng 6-30 parts by weight, Mirabilite 1-10 parts by weight, Radix Rehmanniae 3-20 parts by weight, Poria 6-30 parts by weight, Rhizoma Pinelliae 3-9 parts by weight, Radix Bupleuri 3-20 parts by weight, Radix Stephaniae Tetrandrae 3-10 parts by weight, Radix Glycyrrhizae 3-15 parts by weight.

4. A method for preparing the traditional Chinese medicine composition for treating autoimmune hepatitis according to any one of claims 1-3, characterized in that, comprises: The raw medicinal materials are weighed according to the prescription, mixed, and extracted; the extraction is water extraction: 1.5-3 times the weight of water is added to the mixed raw medicinal materials, heated to 80-100℃, maintained for 0.5-0.6 hours, extracted 1-2 times, filtered, and the filtrates are combined; after the extraction step, the obtained extract is concentrated and dried.

5. The method for preparing the traditional Chinese medicine composition for treating autoimmune hepatitis according to claim 4, characterized in that, The water extraction step is: 1.5-3 times the weight of water is added to the mixed raw medicinal materials, soaked for 0.4-0.5 hours, heated to 80-100℃, maintained for 0.5-0.6 hours, then cooled to 60-80℃, filtered to obtain a first extract and a residue; 1.5-3 times the weight of water is added to the residue, then the liquid is boiled to 80-100℃, maintained for 0.5-0.6 hours, then cooled to 60-80℃ and boiled for 0.4-0.5 hours, filtered to obtain a second extract; The first extract and the second extract are mixed.

6. A traditional Chinese medicine preparation for treating autoimmune hepatitis, characterized in that, Prepared by the preparation method of the traditional Chinese medicine composition of any one of claims 1-3 or the preparation method of any one of claims 4-5.

7. The traditional Chinese medicine preparation according to claim 6, characterized in that, Further comprises a pharmaceutically acceptable excipient.

8. The traditional Chinese medicine preparation of claim 7, wherein The form of the traditional Chinese medicine preparation comprises a liquid preparation or a solid preparation.

9. The traditional Chinese medicine preparation of claim 8, wherein The dosage form of the traditional Chinese medicine preparation comprises a tablet, a capsule, a powder, a granule, or a paste.

10. Use of the traditional Chinese medicine composition of any one of claims 1-3 or the traditional Chinese medicine composition prepared by the preparation method of any one of claims 4-5 or the traditional Chinese medicine preparation of any one of claims 6-9 in the preparation of a drug for treating autoimmune hepatitis.

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