Traditional Chinese medicine composition for treating autoimmune hepatitis as well as preparation method and application thereof

Abstract

The invention relates to the field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for treating autoimmune hepatitis and a preparation method and application thereof. The traditional Chinese medicine composition provided by the invention is prepared from the following raw material medicines in parts by weight: 3-60 parts of astragalus membranaceus, 3-20 parts of bighead atractylodes rhizome, 3-20 parts of divaricate saposhnikovia root, 3-30 parts of radix paeoniae alba, 5-30 parts of hawthorn, 6-30 parts of radix curcumae, 1-10 parts of mirabilite, 3-20 parts of radix rehmanniae, 6-30 parts of poria cocos, 3-9 parts of rhizoma pinellinae praeparata, 3-20 parts of radix bupleuri, 3-10 parts of radix stephaniae tetrandrae and 3-15 parts of liquorice. The medicines are combined to achieve the functions of soothing the liver, strengthening the spleen, nourishing the liver, detoxifying, clearing heat, eliminating dampness and regulating qi and blood, and can be used for treating autoimmune hepatitis.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular to a traditional Chinese medicine composition for treating autoimmune hepatitis, a preparation method and use thereof. Background Art

[0002] Autoimmune hepatitis (AIH) is an immune-mediated hepatitis of unknown etiology, often accompanied by elevated serum aminotransferase levels, hyperimmunoglobulin G, positive serum autoantibodies, moderate to severe interface hepatitis, etc. AIH is an autoimmune-mediated chronic progressive inflammatory disease of the liver. Hepatocyte damage begins with CD4+T cells recognizing autoantigen peptides. Overexpression of inflammatory cytokines in the liver and impaired immune regulation mechanisms are important pathogenesis. The clinical manifestations of AIH are diverse, generally manifested as chronic and insidious onset, but can also manifest as acute attacks and even cause 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] Standard immunotherapy is ineffective or has incomplete biochemical response in some patients. Although hormone combined with immunosuppressants can quickly induce inflammation relief, most AIH patients face the problem of recurrence, restarting immunotherapy and lifelong maintenance treatment during the process of reducing or stopping the drug, accompanied by mild or severe clinical symptoms and adverse reactions related to immunotherapy drugs, which are more difficult to treat. Therefore, it is still clinically necessary to find an alternative treatment plan that can regulate immunity, reduce the adverse reactions of immunosuppressive drugs, and consolidate the therapeutic effect when hormones are withdrawn. In recent years, the understanding of the etiology, pathogenesis and treatment of AIH in traditional Chinese medicine has become increasingly rich, but there is still no clear guideline for treatment. Summary of the invention

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

[0005] To this end, the present invention provides the following technical solutions:

[0006] The embodiment disclosed in the present invention relates to a Chinese medicine composition, comprising the following raw materials in parts by weight:

[0007] 3-60 weight parts of Astragalus, 3-20 weight parts of Atractylodes, 3-20 weight parts of Saposhnikovia, 3-30 weight parts of White Peony, 5-30 weight parts of Crataegus, 6-30 weight parts of Curcuma, 1-10 weight parts of Sodium Sulfate, 3-20 weight parts of Rehmannia, 6-30 weight parts of Poria, 3-9 weight parts of Pinellia, 3-20 weight parts of Bupleurum, 3-10 weight parts of Stephania Tetrandrae, and 3-15 weight parts of Licorice.

[0008] In some embodiments, the method comprises the following parts by weight of the raw material:

[0009] 15-60 weight parts of raw astragalus, 3-18 weight parts of white atractylodes, 3-18 weight parts of siler, 3-28 weight parts of white peony root, 6-28 weight parts of hawthorn, 6-28 weight parts of turmeric, 3-10 weight parts of sodium dregs, 6-20 weight parts of raw rehmannia, 6-28 weight parts of poria, 6-9 weight parts of pinellia, 3-18 weight parts of bupleurum, 3-8 weight parts of tetrandra, and 6-10 weight parts of liquorice.

[0010] Optionally, 30 weight parts of raw astragalus, 12 weight parts of atractylodes, 6 weight parts of siler, 20 weight parts of white peony root, 20 weight parts of charred hawthorn, 15 weight parts of curcuma, 3 weight parts of sodium dregs, 15 weight parts of raw rehmannia, 20 weight parts of poria, 9 weight parts of pinellia, 9 weight parts of bupleurum, 6 weight parts of tetrandra, and 6 weight parts of liquorice.

[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 embodiment disclosed in the present invention also relates to a method for preparing the aforementioned Chinese medicine composition, comprising:

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

[0016] In some embodiments, the extraction is water extraction: add 1.5 to 3 times the weight of water to the mixed API, heat to 80 to 100° C., maintain for 0.5 to 0.6 hours, extract 1 to 2 times, filter, and combine the filtrate;

[0017] Optionally, the water extraction step is: adding water of 1.5 to 3 times the weight of the raw material medicine to the mixed raw material medicine, soaking for 0.4 to 0.5 hours, heating to 80 to 100° C., maintaining for 0.5 to 0.6 hours, then cooling to 60 to 80° C., filtering to obtain the first extract and the residue;

[0018] Adding 1.5 to 3 times the weight of water to the medicinal residue, then boiling the medicinal liquid to 80 to 100° C., maintaining for 0.5 to 0.6 hours, then cooling to 60 to 80° C., boiling for 0.4 to 0.5 hours, and filtering to obtain a second extract;

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

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

[0021] The embodiment disclosed in the present invention also relates to a Chinese medicine preparation, comprising the Chinese medicine composition prepared by the Chinese medicine composition or the preparation method of the Chinese medicine composition as an active ingredient.

[0022] In some embodiments, a pharmaceutically acceptable excipient is also included;

[0023] Optionally, the Chinese medicine preparation includes a liquid preparation or a solid preparation;

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

[0025] The embodiments disclosed in the present invention also relate to the use of the Chinese medicine composition or the Chinese medicine composition prepared by the method for preparing the Chinese medicine composition or the Chinese medicine preparation in any of the following items:

[0026] (1) Use in the preparation of medicines for soothing the liver and strengthening the spleen, softening the liver and detoxifying, clearing away heat and removing dampness, and / or regulating qi and blood;

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

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

[0029] Or, use in the preparation of drugs for reducing serum ALT, AST, DBIL indicators, reducing inflammatory cell infiltration in the portal area of ​​liver tissue, improving liver sinusoidal structure and LSECs microenvironment and / or reducing inflammatory damage.

[0030] The Chinese medicine composition of the present invention is an empirical prescription for treating autoimmune hepatitis after repeated clinical verification and continuous optimization by Professor Lv Wenliang. Hundreds of cases in the early clinical trials have proved that the prescription can regulate immunity, reduce the adverse reactions of immunosuppressive drugs, and consolidate the therapeutic effect when hormones are withdrawn, and can protect the liver and reduce enzymes, and can improve symptoms such as fatigue and weakness in patients, and restore the spleen and stomach function of patients. Basic experiments have proved that the Chinese medicine composition of the present invention can improve the mental state of the Con A-induced chronic immune liver injury mouse model, increase the weight of mice, reduce mortality, reduce serum ALT, AST, DBIL indicators, reduce inflammatory cell infiltration in the portal area of ​​liver tissue, improve the liver sinusoid structure and LSECs microenvironment, and effectively reduce inflammatory damage. This prescription can provide ideas and medical research evidence for the field of combining traditional Chinese and Western medicine to treat autoimmune hepatitis.

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

[0032] 1. A Chinese medicine composition provided by the present invention comprises the following raw materials in parts by weight: 3-60 parts by weight of Astragalus, 3-20 parts by weight of Atractylodes macrocephala, 3-20 parts by weight of Saposhnikovia divaricata, 3-30 parts by weight of White Peony, 5-30 parts by weight of Crataegus, 6-30 parts by weight of Curcuma, 1-10 parts by weight of Glauber's Salt, 3-20 parts by weight of Radix Rehmanniae, 6-30 parts by weight of Poria, 3-9 parts by weight of Rhizoma Pinelliae, 3-20 parts by weight of Radix Bupleuri, 3-10 parts by weight of Radix Stephaniae Tetrandrae, and 3-15 parts by weight of Radix Glycyrrhizae; in the above formula, Astragalus (main drug): sweet in taste, slightly warm in nature, and belongs to the lung and spleen meridians. It has the effects of replenishing qi and strengthening the exterior, supporting poison and promoting tissue regeneration, and promoting diuresis and reducing swelling. In this recipe, Astragalus is used to replenish qi and strengthen the spleen, enhance the body's immunity, and at the same time, it is in accordance with the nature of the liver wood ascending, which helps to dredge the liver qi.

[0033] White peony root (main drug): bitter, sour, slightly cold, enters the liver and spleen meridians. It has the effects of nourishing blood, softening the liver, relieving pain. In this prescription, white peony root is used to nourish blood, restrain yin, soften the liver and relieve pain, and relieve pain caused by liver qi stagnation.

[0034] Atractylodes macrocephala (minister drug): bitter and sweet, warm in nature, enters the spleen and stomach meridians. It has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis. In this prescription, Atractylodes macrocephala replenishes qi and strengthens the spleen, eliminates dampness, and helps Astragalus membranaceus to strengthen the spleen and strengthen the exterior.

[0035] Bupleurum (minister drug): bitter and pungent in taste, slightly cold in nature, enters the liver, gallbladder, and lung meridians. It has the effects of soothing the liver and relieving depression, and raising Yang Qi. In this prescription, Bupleurum is used to unblock liver Qi and relieve liver Qi stagnation.

[0036] Radix Rehmanniae (minister drug): sweet and bitter in taste, cold in nature, enters the heart, liver, and kidney meridians. It has the effects of clearing heat and cooling blood, nourishing yin and promoting body fluid. In this prescription, Radix Rehmanniae nourishes kidney water to contain liver wood, drains spleen earth dampness and heat, and moistens the liver.

[0037] Curcuma (adjuvant): pungent, bitter, cold in nature, enters the liver, lung, and heart meridians. It has the effects of promoting blood circulation and relieving pain, promoting qi and relieving depression, and promoting bile and relieving jaundice. In this prescription, Curcuma promotes qi and blood circulation, regulates liver qi, and helps Bupleurum to relieve liver qi and depression.

[0038] Banxia (adjuvant): pungent, warm, toxic, enters the spleen, stomach, and lung meridians. It has the effects of drying dampness and resolving phlegm, lowering adverse qi and stopping vomiting. In this prescription, Banxia lowers adverse qi, harmonizes the stomach, and clears turbidity, so as to help the spleen and stomach to transport and transform.

[0039] Poria (adjuvant): sweet, light, neutral in nature, enters the heart, lung, spleen, and kidney meridians. It has the effects of promoting diuresis and eliminating dampness, strengthening the spleen and calming the mind. In this prescription, Poria strengthens the spleen and eliminates dampness, and helps Atractylodes macrocephala to strengthen the spleen and eliminate dampness.

[0040] Stephania tetrandra (adjuvant): bitter in taste, cold in nature, enters the bladder and lung meridians. It has the effects of promoting diuresis, reducing swelling, and removing rheumatism. In this prescription, Stephania tetrandra dissolves dampness and heat in the blood, and helps Rehmannia glutinosa to clear heat and cool the blood.

[0041] Hawthorn (adjuvant): sour, sweet, slightly warm, enters the spleen, stomach, and liver meridians. It has the functions of digesting food, removing stagnation, promoting qi and dispersing blood stasis. In this prescription, hawthorn can eliminate food stagnation, dissipate blood stasis and dredge meridians, and help Poria to strengthen the spleen.

[0042] Saposhnikovia divaricata (adjuvant): pungent, sweet, slightly warm, enters the bladder, liver, and spleen meridians. It has the effects of dispelling wind and relieving exterior symptoms, removing dampness and relieving pain. In this prescription, Saposhnikovia divaricata is combined with Astragalus membranaceus and Atractylodes macrocephala to represent the meaning of a jade screen, strengthening the body and eliminating evil.

[0043] Licorice (the adjuvant drug): 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 away heat and detoxifying, and harmonizing various drugs. In this prescription, licorice harmonizes various drugs, and is combined with white peony root to sour and sweeten yin and nourish the liver.

[0044] Glauber's salt (helper drug): salty and bitter in taste, cold in nature, enters the stomach and large intestine meridians. It has the effects of purging and relieving constipation, moistening dryness and softening hard masses, clearing away heat and reducing swelling. In this prescription, Glauber's salt removes accumulation in the six internal organs, consolidates and retains addiction, dredges the meridians, and removes the old and brings in the new. The combination of the above prescription drugs has the functions of soothing the liver and strengthening the spleen, softening the liver and detoxifying, clearing away heat and dampness, and harmonizing qi and blood, and can be used to treat autoimmune hepatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 The weight change trend of mice in each group of the experimental examples of the present invention over time;

[0047] Figure 2 is the liver and spleen index of mice in each group of the experimental examples of the present invention;

[0048] Figure 3 This is the appearance of the liver of mice in each group of the experimental examples of the present invention;

[0049] Figure 4 This is the appearance of the spleen of mice in each group of the experimental examples of the present invention;

[0050] Figure 5 is the level of ALT, AST, and DBIL of mice in each group of the experimental examples of the present invention;

[0051] Figure 6 HE staining pathology of the liver of mice in each group of the experimental examples of the present invention (200×);

[0052] Figure 7 1 is a scanning electron micrograph of the liver of mice in each group of the experimental examples of the present invention. DETAILED DESCRIPTION

[0053] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0054] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0055] The raw materials in the Chinese medicine composition of the present invention are all raw materials recorded 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. of the Leguminosae family.

[0057] Atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala Koidz. of the Asteraceae family. Stir-fried Atractylodes macrocephala is the processed product of Atractylodes macrocephala.

[0058] Saposhnikovia divaricata (Turcz.) Schischk., a plant of the Umbelliferae family, is a dried root;

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

[0060] Hawthorn is the dried mature fruit of Crataegus pinnatifida Bge.var.major NEBr. or Crataegus pinnatifida Bge. of the Rosaceae family. Jiaoshanzha is a processed product of hawthorn.

[0061] Curcuma zedoaria is the dried root of Curcuma wenyujin YHChen et C.Ling, Curcuma Longa L., Curcuma kwangsiensis SGLee et CFLiang or Curcumaphaeocaulis Val. of the Zingiberaceae family;

[0062] Sodium sulfate is a crystal formed by processing and refining. It mainly contains hydrated sodium sulfate (Na2SO4·10H2O);

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

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

[0065] Pinellia ternata is the dried tuber of Pinellia ternata (Thunb.) Breit. of the Araceae family; French Pinellia ternata is the processed product of Pinellia ternata;

[0066] Bupleurum is the dried root of Bupleurum chinense DC. or Bupleurumscorzonerifolium Willd. of the Umbelliferae family.

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

[0068] Licorice is the dried root and rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. or Glycyrrhiza glabra L. of the Leguminosae family. Roasted licorice is a processed product of licorice.

[0069] The above APIs are all commercially available products.

[0070] Example

[0071] This embodiment provides a Chinese medicine composition, the formula of which is as follows:

[0072] Table 1. Formulation

[0073] API 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(g) 20 28 3 30 3 Charred hawthorn(g) 20 6 28 5 30 Turmeric(g) 15 28 6 30 6 Sodium Sulfate(g) 3 3 10 1 10 Raw land (g) 15 20 6 20 3 Poria cocos(g) 20 6 28 6 30 French Pinellia (g) 9 9 6 9 3 Bupleurum(g) 9 3 18 20 3 Stephania tetrandra(g) 6 8 3 3 10 Roasted Licorice(g) 6 6 10 15 3

[0074] The preparation method of the Chinese medicine composition of the above embodiment 1 comprises the following steps:

[0075] (1) Weigh the raw materials according to the formula, mix them evenly, then add water 3 times the weight of the raw material, heat to 100°C, maintain for 0.5 hour, and filter (200 mesh) to obtain a residue and a filtrate;

[0076] (2) Add 1.5 times the weight of the API water to the residue in step (1), heat to 100° C., maintain for 1 hour, and filter with suction (200 mesh) to obtain a residue and a filtrate;

[0077] (3) combining the filtrates from step (1) and step (2), and concentrating under reduced pressure to obtain a clear paste;

[0078] (4) Spray-dry the clear paste to collect powder, sieve to obtain dry paste powder, mix evenly, and use dry granulation to prepare 12-40 mesh granules, with a specification of 11.2 grams per granule.

[0079] The preparation method of the Chinese medicine composition of the above embodiment 2 comprises the following steps:

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

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

[0082] (3) combining the filtrates from step (1) and step (2), and concentrating under reduced pressure to obtain a clear paste;

[0083] (4) Spray-dry the clear paste to collect powder, sieve to obtain dry paste powder, mix evenly, and use dry granulation to prepare 12-40 mesh granules, with a specification of 11.2 grams per granule.

[0084] The preparation method of the Chinese medicine composition of the above embodiment 3 comprises the following steps:

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

[0086] (2) Add 2.5 times the weight of the API to the residue in step (1), heat to 80° C., maintain for 0.6 hours, and filter with suction (200 mesh) to obtain a residue and a filtrate;

[0087] (3) combining the filtrates from step (1) and step (2), and concentrating under reduced pressure to obtain a clear paste;

[0088] (4) Spray-dry the clear paste to collect powder, sieve to obtain dry paste powder, mix evenly, and use dry granulation to prepare 12-40 mesh granules, with a specification of 11.2 grams per granule.

[0089] The preparation method of the Chinese medicine composition of the above embodiment 4 comprises the following steps:

[0090] (1) Weigh the raw materials according to the formula, mix them evenly, then add water twice the weight of the raw material, heat to 100°C, maintain for 0.5 hour, and filter (200 mesh) to obtain a residue and a filtrate;

[0091] (2) Add water twice the weight of the raw material drug to the residue in step (1), heat to 80° C., maintain for 0.5 hour, and filter with suction (200 mesh) to obtain a residue and a filtrate;

[0092] (3) combining the filtrates from step (1) and step (2), and concentrating under reduced pressure to obtain a clear paste;

[0093] (4) Spray-dry the clear paste to collect powder, sieve to obtain dry paste powder, mix evenly, and use dry granulation to prepare 12-40 mesh granules, with a specification of 11.2 grams per granule.

[0094] The preparation method of the Chinese medicine composition of the above embodiment 5 comprises the following steps:

[0095] (1) Weigh the raw materials according to the formula, mix them evenly, then add water 3 times the weight of the raw material, heat to 100°C, maintain for 0.6 hours, and filter (200 mesh) to obtain a residue and a filtrate;

[0096] (2) Concentrating the filtrate in step (1) under reduced pressure to obtain a clear paste;

[0097] (3) Spray-dry the clear paste to collect powder, sieve to obtain dry paste powder, mix evenly, and use dry granulation to prepare 12-40 mesh granules, with a specification of 11.2 grams per granule.

[0098] Experimental Examples Animal Test

[0099] In this part of the experiment, a concanavalin A (Con A)-induced AIH mouse model was constructed, the 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 Chinese medicine composition of the present invention on Con A-induced AIH model mice.

[0100] 1 Experimental animals

[0101] SPF male Balb / c mice, 6-8 weeks old, weighing 18-22 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. Animal license number: SCXK (Beijing) 2021-0006. They were kept in the Experimental Animal Center of Guang'anmen Hospital, China Academy of Chinese Medical Sciences, with a constant temperature and humidity, a 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 experimental procedures strictly abided by the ethical principles of animal experiments and passed the ethical review of the Ethics Committee of Guang'anmen Hospital, China Academy of Chinese Medical Sciences.

[0103] 2 Experimental methods

[0104] 2.1 Animal grouping, modeling and drug administration

[0105] (1) Animal grouping: The mice were randomly divided into 4 groups, namely, a blank group (Control group), a ConA model group (Con A group), a prednisolone treatment group (Prednisolone group), and a Chinese medicine composition treatment group (JPYG group). In the preliminary experiments of the research group, the mortality rate of the model group was about 25%. According to the experimental needs, 16 mice were set in the Control group, and 24 mice were set in the Con A group, Prednisolone group, and ZYZL group.

[0106] (2) Experimental drug preparation:

[0107] 1. Preparation of Con A: 0.9% saline solution is placed in a 4°C refrigerator in advance. Con A (C2010, 25 mg / bottle, Sigma, USA) stored in a -20° refrigerator is added to cold saline solution to prepare a solution with a concentration of 1.25 mg / ml. The specific operation is as follows: 25 mg of ConA powder is added to 20 ml of pre-cooled saline solution and shaken repeatedly to melt. After sterilization with a sterile filter membrane, it is packaged in sterile EP tubes, 4-5 ml per tube. After packaging, it is stored in a -20° refrigerator. Prepare it on demand and take it as needed to avoid repeated freezing and thawing of the drug.

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

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

[0110] (3) Modeling and drug administration

[0111] Modeling method: Based on previous literature review and preliminary experimental results, the modeling method of chronic immune liver injury induced by tail vein injection of Con A was determined. Adaptive feeding was performed for one week, and the experiment began after the mice adapted to the environment.

[0112] Operation of mold making body: Take out the prepared ConA solution and thaw it at room temperature. After weighing the mouse, calculate the required volume of ConA solution and extract it with a 1ml insulin needle syringe. Place the mouse in the tail vein injection holder barrel, and fix the mouse in a comfortable position to avoid struggling and leaking the tail. Wipe the mouse tail repeatedly with an alcohol cotton ball to dilate the tail vein. Select one of the tail veins with more obvious expansion from both sides. Straighten the tail with the left hand to fix it. Hold the insulin needle syringe in the right hand and insert the needle parallel to the tail vein from the distal end of the tail. Push the drug gently without resistance to enter the vein. After the injection, use a sterile dry cotton ball to compress and stop bleeding. The dose of ConA injected into the mouse tail vein is 12.5mg / kg, and the injection volume is 0.1ml / 10g body weight. Once a week for 6 consecutive weeks.

[0113] Dosage: According to the "Pharmacological Experimental Methodology", the equivalent dose is converted according to the human-mouse body surface area, and the conversion factor is multiplied by 9.1 times for conversion. The safe and appropriate gavage volume for mice is 0.2ml / 10g body weight, and the concentration of the Chinese medicine composition of the present invention is 2.59g / ml. The dosage of prednisolone refers to the treatment plan and literature research of the Guidelines for the Diagnosis and Treatment of Autoimmune Hepatitis (2021). According to the adult treatment dose of 40mg / d, the mouse dosage is 0.12mg / kg body weight / d according to the above calculation method, and the concentration of prednisolone aqueous solution is 0.6mg / ml.

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

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

[0117] 3. Prednisolone positive drug control group: Con A was injected into the tail vein once a week for 6 consecutive weeks. Prednisolone solution was given from the day after the first tail vein injection, once a day for 6 consecutive weeks.

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

[0119] 2.2 Observation indicators and model verification

[0120] 2.2.1 General observations

[0121] The basic conditions of mice in each group were observed and recorded, including mental state, hair color, diet, water and excretion, and activity, and weight changes were recorded twice a week.

[0122] 2.2.2 Model Validation

[0123] According to the results of the preliminary experiment, three mice were randomly selected from each group after the fifth week of tail vein injection and gavage, and the whole blood was collected for serum liver function test after centrifugation, and the liver tissue was taken for HE pathological verification. ALT and AST in the Con A group were higher than those in the other three groups, and obvious portal inflammatory infiltration was observed in the pathology, which indicated that the model was successful. The samples were collected after the sixth week of tail vein injection and gavage cycle.

[0124] 2.3 Serum and tissue collection

[0125] Each group of mice was fasted from food and water for 12 h before sampling. After anesthesia, the eyeballs were removed to collect blood. The biochemical blood collection tubes and sterile EP tubes were marked with the corresponding groups and numbers, respectively. After the whole blood samples were collected, they were placed at room temperature for 2 h and centrifuged in a centrifuge at 4°C and 3000 rpm / min for 15 min. The supernatant was aspirated with a pipette and packaged in sterile EP tubes and stored in a -80°C refrigerator for subsequent serum liver function testing.

[0126] Immediately after blood collection, mice were killed by cervical dislocation. After alcohol disinfection, the mouse abdomen was opened, and the mouse liver and spleen were completely removed. After the residual blood was absorbed with filter paper, the liver and spleen were weighed and recorded, and the appearance, size, texture, etc. of the tissue were observed and photographed. The liver tissue was washed with PBS buffer, and part of the liver tissue was cut and fixed with 4% paraformaldehyde for 24 hours to prepare pathological sections. The remaining liver pieces were divided into sterile cryopreservation tubes, quickly placed in dry ice and transferred to a -80℃ refrigerator for freezing.

[0127] Three mice were randomly selected from each group and killed by cervical dislocation. After disinfection, the abdominal cavity was opened to expose the portal vein and inferior vena cava. Buffer was perfused through the mouse portal vein with a trocar, and the inferior vena cava was quickly cut to allow the blood and buffer in the liver to flow out. When the liver turned white, 2.5% glutaraldehyde electron microscopy fixative was perfused. After the liver hardened, the liver tissue was cut and placed on ice for cutting. The samples were then placed in 2% glutaraldehyde fixative and fixed at room temperature for 2 hours. The samples were then transferred to a 4°C refrigerator for subsequent preparation of electron microscopy samples.

[0128] 2.4 Testing indicators and methods:

[0129] 2.4.1 Serum liver function test The serum samples were tested on an automatic biochemical analyzer as soon as possible after thawing. According to the results of the previous pilot experiment, 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] The liver tissue fixed with 4% paraformaldehyde was removed and trimmed into blocks for HE staining observation. The specific steps are as follows:

[0132] ① Dehydration and embedding: Place the trimmed tissue blocks 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 melt paraffin at 65° for 3 hours.

[0133] ② Slice: Place the wax block on a paraffin slicer and slice it to a thickness of 4 μm. Bake the slices in a 60°C oven. After the moisture is dried and the paraffin is melted, take them out and store them at room temperature for later use.

[0134] ③ Dewaxing of paraffin sections: Place the sections in dewaxing solution I for 20 minutes, dewaxing solution II for 20 minutes, and

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

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

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

[0138] ⑥ Dehydration and sealing: Put the slices into anhydrous ethanol I, II, and III for 2 minutes each, n-butanol I and II for 2 minutes each, and xylene I and II for 2 minutes each to make them transparent, and then seal the slices with neutral gum.

[0139] ⑦Observe under a microscope and collect and analyze images.

[0140] 2.4.3 Preparation and observation of liver scanning electron microscopy samples

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

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

[0143] ② Alcohol gradient dehydration: Soak the liver tissue slices in 30%, 50%, 75%, 85%, 90%,

[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 samples into groups with filter paper, stack them in the critical point dryer, set the parameters, circulate CO2 12 times, and dry them.

[0146] ④ Conductive treatment of samples: Place the tissue sample tightly on the conductive carbon film double-sided tape and place it on the sample stage of the ion sputtering instrument for platinum spraying for about 30 seconds.

[0147] ⑤ Ultra-high resolution cold field emission scanning electron microscope observation: observe the ultrastructure of the liver under the cold field emission scanning electron microscope, find the hepatic portal area of ​​the specimen at low magnification, locate around the portal area, adjust the magnification until the hepatic sinusoids, intrahepatic blood vessels and capillary bile ducts can be clearly observed. Fine-tune the field of view, find the target hepatic sinusoidal structure and hepatic sinusoidal endothelial cells, adjust the magnification, and collect images.

[0148] 2.5 Statistical analysis

[0149] The measured data that conformed to normal distribution were expressed as mean ± standard deviation (x ± s), and those that did not conform to normal distribution were expressed as median and interquartile range M (P25, P75). Statistical analysis and graphing were processed using Graphpad Pism 9.5 software. One-way ANOVA was used for comparison among multiple groups, and P < 0.05 was considered statistically significant.

[0150] 3 Experimental results

[0151] 3.1 Observation of mouse survival and general condition

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

[0153] (2) General condition: The mice in the blank group were in good mental state, with shiny hair, normal food and water intake and defecation, quick activity and reaction, and gradually increased weight during the whole experimental period. The mice in the other three groups showed mental depression, hypothermia, curled up and prone position, increased defecation, reduced activity, weight loss, and dull hair from the day to 3 days after the tail vein injection of ConA each week. Some mice died, and the death mostly occurred 2h-2d after modeling. Then the mental vitality of the mice in each group gradually recovered, and the above-mentioned state recurred after the next tail vein injection of ConA. Compared with the model group, the activity and general condition of the prednisolone and the Chinese medicine composition drug intervention groups of the present invention were significantly improved, and the activity of the prednisolone intervention group increased significantly, and some mice were excited and agitated.

[0154] 3.2 Changes in mouse body weight

[0155] In the preliminary experiments, it was found that the weight of mice decreased significantly after modeling, and gradually recovered before the next modeling, showing a fluctuating increase trend. Therefore, the weight changes on the 3rd and 6th days after each modeling were recorded to compare the differences between the groups. The results showed that the weight of mice in the blank group gradually and steadily increased over time; the model group and the group treated with the Chinese medicine composition of the present invention showed a slow and fluctuating increase trend, in which the weight decreased 3 days after each modeling, and the weight gradually recovered on the 6th day; the weight of mice in the prednisolone treatment group was significantly lower than that of the other groups, and showed an alternating and fluctuating decrease after the 2nd week of modeling. See for details. Figure 1 .

[0156] 3.3 Comparison of liver and spleen index and tissue morphology

[0157] 3.3.1 Comparison of liver and spleen indexes of mice in each group

[0158] The weight, liver and spleen weight of each group of mice were recorded when the samples were collected, and the liver and spleen index was calculated (n = 6). The calculation formula is: liver index = liver weight (g) / body weight (g) × 10; spleen index = spleen weight (g) / body weight (g) × 10. For comparison among multiple groups, one-way ANOVA analysis of variance was used, and Graphpad Prism software was used for statistics and graphing.

[0159] The statistical results of liver index showed that compared with the Control group, the liver index of ConA group and JPYG group was significantly increased, and the difference was statistically significant (P<0.05). There was no difference in liver index between Prednisolone group and normal group (P>0.05). Compared with ConA group, the liver index of Prednisolone group was decreased (P<0.05), and there was no difference in ZYZL group (P>0.05), but there was a downward trend. The statistical results of spleen index showed that compared with the Control group, the spleen index of each group was significantly increased (P<0.01). Compared with ConA group, the spleen index of Prednisolone group was significantly decreased (P<0.01), and there was no statistical significance in ZYZL group (P>0.05), but there was a downward trend compared with ConA group. See Table 2 and Figure 2 .

[0160] Table 2 Comparison of liver and spleen index in each group (x±s)

[0161] Group (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 with the Control group, * P<0.05, ** P<0.01; compared with ConA group, △ P<0.05, △△P<0.01.

[0163] Note: Control group is the blank group; ConA group is the ConA model group; Prednisolone group is the prednisolone group; JPYG group is the Chinese medicine composition group of the present invention. Same below.

[0164] 3.3.2 Appearance of liver and spleen

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

[0166] Comparison of the texture and appearance of the livers of mice in each group revealed that the livers of the Control group mice had normal morphology, soft texture, and normal color; the livers of the ConA group mice were significantly enlarged, dark red in color, swollen and tough to the touch, and uneven in surface; the livers of the Prednisolone and JPYG groups were smaller in size than those of the ConA group, and had relatively smooth surfaces. The livers of the Prednisolone group were slightly yellowish in color, too soft and easy to rot; the livers of the JPYG group were larger in size, slightly dark red in color, and softer in texture than those of the ConA group. Figure 3 .

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

[0168] The spleen of mice in the Control group had normal shape and color, and was soft in texture; the spleen of mice in the ConA group was significantly swollen and enlarged, hard, and dark red in color; the volume of the spleen in the Prednisolone group and the JPYG group was smaller than that in the ConA group, the color was slightly lighter, and the texture was softer than that in the ConA group; the volume of the Prednisolone group was smaller than that in the JPYG group. Figure 4 .

[0169] 3.4 Serum liver function test results

[0170] The results of ALT, AST, and DBIL in each group of mice were statistically analyzed. The results showed that compared with the Control group, the ALT, AST, and DBIL values ​​in the ConA group were all increased, and the difference was statistically significant (P<0.01). Compared with the ConA group, the values ​​in the Prednisolone and JPYG drug intervention groups were reduced, and the difference was statistically significant (P<0.05), as shown in Table 3. Figure 5 .

[0171] Table 3 Comparison of serum ALT, AST and DBIL in each group of mice (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 with the Control group, * P<0.01; compared with ConA group, △P<0.05, △△ P<0.01.

[0174] 3.5 Pathological observation of liver HE staining

[0175] The liver lobules of mice in the Control group were normal in structure, the hepatic cords were arranged radially and neatly, the portal area structure was clear, and no obvious inflammatory cell infiltration was observed; the liver cells in the ConA group were disorderly arranged, the portal area was irregularly enlarged and congested, the liver cells were swollen and degenerated, and obvious inflammatory cell infiltration was observed; compared with the ConA group, the inflammatory cells in the portal area of ​​the Prednisolone group and the JPYG group were significantly reduced, and a small amount of fatty degeneration and extensive hepatocyte swelling and degeneration were observed in the Prednisolone group compared with the JPYG group. Figure 6 .

[0176] 3.6 Electron microscopic scanning results of liver of mice in each group

[0177] The results of electron microscopy showed that the hepatocyte cords and sinusoids of the Control group were clear and neatly arranged, and the surface of the sinusoidal endothelial cells was smooth with uniform fenestrations; the hepatocyte cords and sinusoids of the ConA group were obviously fuzzy and disordered, and the fenestrations were difficult to identify; the hepatocyte structures of the Prednisolone and JPYG groups were relatively clear and distinct, significantly better than those of the ConA group, and the sinusoidal endothelial structures were less smooth than those of the Control group, and the fenestrations of the two groups were of different sizes and densely distributed. Figure 7 .

[0178] 4 Conclusion

[0179] The Chinese medicine composition of the present invention can improve the mental state of a mouse model of chronic immune liver injury induced by Con A, increase the weight of mice, reduce mortality, reduce serum ALT, AST, and DBIL indicators, reduce inflammatory cell infiltration in the portal area of ​​liver tissue, improve the liver sinusoid structure and the microenvironment of liver sinusoidal endothelial cells (LSECs), and effectively reduce inflammatory damage.

[0180] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Chinese medicine composition, characterized in that: The composition is prepared from the following raw materials in parts by weight: 3-60 weight parts of Astragalus, 3-20 weight parts of Atractylodes, 3-20 weight parts of Saposhnikovia, 3-30 weight parts of White Peony, 5-30 weight parts of Crataegus, 6-30 weight parts of Curcuma, 1-10 weight parts of Sodium Sulfate, 3-20 weight parts of Rehmannia, 6-30 weight parts of Poria, 3-9 weight parts of Pinellia, 3-20 weight parts of Bupleurum, 3-10 weight parts of Stephania Tetrandrae, and 3-15 weight parts of Licorice.

2. The Chinese medicine composition according to claim 1, characterized in that: The composition is prepared from the following raw materials in parts by weight: 15-60 parts by weight of raw astragalus, 3-18 parts by weight of atractylodes, 3-18 parts by weight of siler, 3-28 parts by weight of white peony root, 6-28 parts by weight of hawthorn, 6-28 parts by weight of turmeric, 3-10 parts by weight of sodium sulfate, 6-20 parts by weight of raw rehmannia, 6-28 parts by weight of tuckahoe, 6-9 parts by weight of pinellia, 3-18 parts by weight of bupleurum, 3-8 parts by weight of tetrandra, and 6-10 parts by weight of liquorice; Optionally, 30 weight parts of raw astragalus, 12 weight parts of white atractylodes, 6 weight parts of siler, 20 weight parts of white peony root, 20 weight parts of charred hawthorn, 15 weight parts of curcuma, 3 weight parts of sodium dregs, 15 weight parts of raw rehmannia, 20 weight parts of poria, 9 weight parts of pinellia, 9 weight parts of bupleurum, 6 weight parts of tetrandra, and 6 weight parts of liquorice.

3. The Chinese medicine composition according to claim 1 or 2, characterized in that: The Atractylodes macrocephala is stir-fried Atractylodes macrocephala; And / or, the hawthorn is charred hawthorn; And / or, the licorice is roasted licorice.

4. A method for preparing the Chinese medicine composition according to any one of claims 1 to 3, characterized in that: include: Weigh the raw materials according to the formula. Mix and extract.

5. The method for preparing the Chinese medicine composition according to claim 4, characterized in that: The extraction is water extraction: add 1.5 to 3 times the weight of water to the mixed raw materials, heat to 80 to 100°C, keep for 0.5 to 0.6 hours, extract 1 to 2 times, filter, and combine the filtrate; Optionally, the water extraction step is: adding water of 1.5 to 3 times the weight of the raw material medicine to the mixed raw material medicine, soaking for 0.4 to 0.5 hours, heating to 80 to 100° C., maintaining for 0.5 to 0.6 hours, then cooling to 60 to 80° C., filtering to obtain the first extract and the residue; Adding 1.5 to 3 times the weight of water to the medicinal residue, then boiling the medicinal liquid to 80 to 100° C., maintaining for 0.5 to 0.6 hours, then cooling to 60 to 80° C., boiling for 0.4 to 0.5 hours, and filtering to obtain a second extract; Mix the first extract and the second extract.

6. The method for preparing the Chinese medicine composition according to claim 4 or 5, characterized in that: After the extraction step, the obtained extract is concentrated and dried.

7. A Chinese medicine preparation, characterized in that: The invention comprises a Chinese medicine composition prepared by the preparation method of the Chinese medicine composition according to any one of claims 1 to 3 or the Chinese medicine composition according to any one of claims 4 to 6 as an active ingredient.

8. The Chinese medicine preparation according to claim 7, characterized in that: Also included are pharmaceutically acceptable excipients; Optionally, the Chinese medicine preparation includes a liquid preparation or a solid preparation; Optionally, the dosage form of the traditional Chinese medicine preparation includes tablets, capsules, powders, granules or pastes.

9. Use of the Chinese medicine composition according to any one of claims 1 to 3 or the Chinese medicine composition prepared by the method for preparing the Chinese medicine composition according to any one of claims 4 to 6 or the Chinese medicine preparation according to claim 7 or 8 in any of the following items; (1) Use in the preparation of medicines for soothing the liver and strengthening the spleen, softening the liver and detoxifying, clearing away heat and removing dampness, and / or regulating qi and blood; (2) Use in the preparation of drugs for treating autoimmune liver diseases.

10. The use according to claim 9, characterized in that include: Use in the preparation of a drug for treating autoimmune hepatitis; Or, use in the preparation of drugs for reducing serum ALT, AST, DBIL indicators, reducing inflammatory cell infiltration in the portal area of ​​liver tissue, improving liver sinusoidal structure and LSECs microenvironment and / or reducing inflammatory damage.

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