Traditional Chinese medicine composition as well as preparation, pharmaceutical composition and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition and a preparation, a pharmaceutical composition and application thereof. The traditional Chinese medicine composition consists of bulbus fritillariae cirrhosae, kelp, thunberg fritillary bulb, turmeric, endothelium corneum gigeriae galli, raw hawthorn and selfheal. According to the application, the bulbus fritillariae cirrhosae and the bulbus fritillariae thunbergii are compatible to synergistically exert the effects of reducing phlegm and resolving The endothelium corneum gigeriae galli and the raw hawthorn have the effect of removing food retention, the selfheal has the effect of clearing liver and removing stasis, and all the medicines are combined to achieve the effects of reducing phlegm, removing blood stasis, softening hard mass and eliminating diseases. Experiments show that the traditional Chinese medicine composition can significantly improve the liver function of Wilson disease model mice, reduce collagen deposition of liver tissues, down-regulate fibrosis markers and inhibit activation of hepatic stellate cells.

Description

Technical Field

[0001] This application belongs to the field of traditional Chinese medicine technology, and specifically relates to a traditional Chinese medicine composition and its preparation, pharmaceutical composition and application. Background Technology

[0002] Wilson's disease (WD) is a condition caused by... ATP7B WD is an autosomal recessive inherited disorder of copper metabolism caused by gene mutations. Abnormal accumulation of copper ions in the liver leads to progressive liver damage, progressing from steatosis, inflammation, and fibrosis, ultimately developing into cirrhosis and even liver failure. Liver fibrosis is a key pathological stage in the course of WD and a major factor affecting prognosis.

[0003] Currently, standard treatment for WD primarily includes copper chelators (such as penicillamine and tricentidine) and zinc supplements. However, these therapies have significant limitations: copper chelators have significant side effects and are not tolerated by some patients; zinc supplements have a slow onset of action and limited effectiveness in reversing existing liver fibrosis. Existing Western medical treatments mainly focus on copper chelation, lacking specific interventions for the downstream pathological process of "liver fibrosis" that has already begun and is ongoing, making it difficult to completely prevent the disease from progressing to cirrhosis.

[0004] In Traditional Chinese Medicine (TCM) theory, WD liver fibrosis can be categorized under "accumulation," "hypochondriac pain," and "mass." "Phlegm-blood stasis syndrome" is a common and crucial clinical pattern. Its core pathogenesis lies in the internal accumulation of copper toxicity, leading to impaired liver function and spleen dysfunction, resulting in the failure of body fluids to be properly transformed and condensed into phlegm; stagnation of Qi and blood also hinders blood circulation, leading to blood stasis. Phlegm and blood stasis intertwine and adhere to the liver vessels, forming "mass accumulation" (i.e., liver fibrosis / early cirrhosis). Clinically, it commonly presents with a palpable mass under the ribs (hepatosplenomegaly), stabbing or distending pain in the hypochondrium, a sallow complexion, a purplish-dark tongue or ecchymosis, a greasy tongue coating, and a wiry and hesitant pulse. For this pattern, TCM treatment emphasizes "resolving phlegm and dispersing nodules, promoting blood circulation and eliminating masses."

[0005] However, existing TCM formulas for treating WD liver fibrosis mostly focus on soothing the liver, eliminating dampness, and tonifying deficiency, specifically targeting the core pathogenesis of "phlegm and blood stasis." Furthermore, there is a lack of well-formulated and relatively clearly defined compound formulas. Therefore, developing a TCM compound formula based on the treatment principle of "resolving phlegm, dispersing nodules, and eliminating masses," capable of precisely intervening in liver fibrosis caused by phlegm and blood stasis, has significant clinical importance and application prospects. Summary of the Invention

[0006] To address the aforementioned problems, in the first aspect, this application proposes a traditional Chinese medicine composition comprising the following raw materials in parts by weight: 8-12 parts of Fritillaria cirrhosa, 15-25 parts of Laminaria japonica, 25-35 parts of Fritillaria thunbergii, 8-12 parts of Curcuma longa, 15-25 parts of chicken gizzard lining, 25-35 parts of hawthorn, and 12-18 parts of Prunella vulgaris.

[0007] Furthermore, the traditional Chinese medicine composition consists of the following raw materials in parts by weight: 10 parts of Fritillaria cirrhosa, 20 parts of Laminaria japonica, 30 parts of Fritillaria thunbergii, 10 parts of Curcuma longa, 20 parts of chicken gizzard lining, 30 parts of hawthorn, and 15 parts of Prunella vulgaris.

[0008] Secondly, this application proposes a pharmaceutical preparation comprising the aforementioned traditional Chinese medicine composition and pharmaceutically acceptable excipients; the preparation method of the pharmaceutical preparation is as follows: Except for Fritillaria cirrhosa, the other raw materials (kelp, Fritillaria thunbergii, turmeric, chicken gizzard lining, hawthorn and prunella vulgaris) are decocted twice with water. For the first decoction, add 6-10 times the total weight of the other raw materials and decoct for 0.5-2 hours. For the second decoction, add 5-7 times the total weight of the other raw materials and decoct for 30-60 minutes. Combine the decoctions and filter them. Concentrate the filtrate to a thick paste with a relative density of 1.25-1.30. Fritillaria cirrhosa is ground into a fine powder, mixed evenly with a thick paste, dried at 60-70℃, and then pulverized into a fine powder. Take the fine powder, add excipients, and prepare the required dosage form according to conventional pharmaceutical processes.

[0009] Furthermore, the dosage form of the pharmaceutical preparation includes pills, powders, granules, capsules, tablets, or oral liquids.

[0010] Thirdly, this application proposes a pharmaceutical composition in which the traditional Chinese medicine composition is used as one of the active pharmaceutical ingredients.

[0011] Fourthly, this application proposes the use of the aforementioned traditional Chinese medicine composition, the aforementioned preparation, or the aforementioned pharmaceutical composition in the preparation of a drug for treating Wilson's disease with liver fibrosis.

[0012] Fifthly, this application proposes the use of the aforementioned traditional Chinese medicine composition, the aforementioned preparation, or the aforementioned pharmaceutical composition in the preparation of a drug for improving liver damage in Wilson's disease.

[0013] Sixthly, this application proposes the use of the aforementioned traditional Chinese medicine composition, the aforementioned preparation, or the aforementioned pharmaceutical composition in the preparation of a drug for downregulating the expression of liver fibrosis-related genes in Wilson's disease, wherein the liver fibrosis-related genes include... Col1a1 , Acta2 , Tgfb1 , Pdgfrb .

[0014] Seventhly, this application proposes the use of the aforementioned traditional Chinese medicine composition, the aforementioned preparation, or the aforementioned pharmaceutical composition in the preparation of a drug for regulating liver lipid metabolism in Wilson's disease.

[0015] Eighthly, this application proposes the use of the aforementioned traditional Chinese medicine composition, the aforementioned preparation, or the aforementioned pharmaceutical composition in the preparation of a drug that upregulates the expression of phosphoenolpyruvate carboxykinase 1 in the liver of Wilson's disease.

[0016] Compared with the prior art, this application has the following advantages: This application utilizes the combination of Fritillaria cirrhosa and Fritillaria thunbergii, known as the "two mothers," to synergistically resolve phlegm and dissipate nodules. Kelp softens and dissipates nodules, turmeric invigorates blood and removes blood stasis, chicken gizzard membrane and hawthorn eliminate stagnation, and prunella vulgaris clears the liver and dissipates nodules. The combined effects of these herbs resolve phlegm, remove blood stasis, soften and dissipate nodules. Experiments show that this traditional Chinese medicine composition significantly improves liver function in Wilson's disease model mice, reduces collagen deposition in liver tissue, downregulates fibrosis markers, and inhibits hepatic stellate cell activation. The composition of this application can reduce the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in WD model mice. The high-dose group can bring these indicators close to normal levels, effectively alleviating liver damage caused by WD. Simultaneously, it can significantly downregulate the expression of key serum liver fibrosis markers hyaluronic acid (HA), type IV collagen (Col IV), laminin (LN), and type III procollagen (HPC III), reducing liver collagen fiber deposition and improving liver lobular structural disorder. The composition of this application does not simply inhibit the symptoms of liver fibrosis, but intervenes in disease progression at its root by regulating key molecular pathways. On the one hand, it can significantly downregulate genes related to hepatic stellate cell activation (…). Col1a1 , Acta2 , Tgfb1 , Pdgfrb On the one hand, by upregulating the expression of phosphoenolpyruvate carboxykinase 1 (Pck1) in the liver of WD model mice, it remodels the disordered lipid metabolism network, thereby upregulating lipid synthesis genes ( Acly , Fasn , Srebf1 ) expression, and downregulation of lipid transport genes ( Cd36 , Slc27a1 ) expression, while upregulating fatty acid β-oxidation-related genes ( Acox1 , Cpt1α , Pex14 This study aims to achieve the regulatory effect of "increasing supply and reducing expenditure" by "promoting the decomposition and utilization of fatty acids and reducing the accumulation of lipids in the liver," thereby improving hepatic steatosis, alleviating liver damage from a metabolic perspective, providing core support for the reversal of liver fibrosis, and also providing experimental evidence for the connection between the TCM theory of "phlegm and blood stasis" and the modern mechanism of "metabolic liver injury."

[0017] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Representative images (×200) of Sirius Red staining in liver tissue of mice in each experimental group are shown (A), and the results of quantitative analysis of Sirius Red-stained collagen area are shown (B); all data are expressed as mean ± standard deviation (n=3); compared with the normal group, ## P <0.01; compared with the model group, P <0.05; WT is the control group, WD is the model group, EM-L is the low-dose group of Ermu Xiaozheng Wan, EM-M is the medium-dose group of Ermu Xiaozheng Wan, and EM-H is the high-dose group of Ermu Xiaozheng Wan. Figure 2 Representative images (×200) of Masson's trichrome staining in liver tissue of mice in each experimental group are shown (A), along with the quantitative analysis results of collagen area stained by Masson's trichrome (B). All data are expressed as mean ± standard deviation (n=3). Compared with the normal group, ## P <0.01; compared with the model group, P <0.001; WT is the control group, WD is the model group, EM-L is the low-dose group of Ermu Xiaozheng Wan, EM-M is the medium-dose group of Ermu Xiaozheng Wan, and EM-H is the high-dose group of Ermu Xiaozheng Wan. Figure 3The effects of each experimental group on the expression of fibrosis-related proteins are shown. A represents representative images (×200) of Collagen I immunohistochemical staining in liver tissue of each group of mice; B represents the quantitative analysis results of Collagen I protein expression; C represents representative images (×200) of α-SMA immunohistochemical staining in liver tissue of each group of mice; D represents the quantitative analysis results of α-SMA protein expression; E represents representative images (×200) of TGF-β immunohistochemical staining in liver tissue of each group of mice; F represents the quantitative analysis results of TGF-β protein expression. All data are expressed as mean ± standard deviation (n=3). Compared with the normal group... # P <0.05, ### P <0.001; compared with the model group, P <0.05, P <0.01, P <0.001; WT is the control group, WD is the model group, EM-L is the low-dose group of Ermu Xiaozheng Wan, EM-M is the medium-dose group of Ermu Xiaozheng Wan, and EM-H is the high-dose group of Ermu Xiaozheng Wan. Figure 4 This demonstrates the detection of qRT-PCR in the liver tissue of mice in each experimental group. Col1a1 (A) Acta2 (B) Tgfb1 (C) and Pdgfrb (D) mRNA expression level; all data are expressed as mean ± standard deviation (n=6); compared with the normal group, ### P <0.001; compared with the model group, P <0.01, P <0.001; WT is the control group, WD is the model group, EM-L is the low-dose group of Ermu Xiaozheng Wan, EM-M is the medium-dose group of Ermu Xiaozheng Wan, and EM-H is the high-dose group of Ermu Xiaozheng Wan. Figure 5 The results of Western blot analysis of representative bands (A) and quantitative analysis of α-SMA protein (B) and TGF-β1 protein (C) expression in the livers of mice in each experimental group are shown. All data are expressed as mean ± standard deviation (n=3). Compared with the normal group, # P <0.05, ### P <0.001; compared with the model group, P <0.05, P <0.01, P <0.001; WT is the control group, WD is the model group, EM-L is the low-dose group of Ermu Xiaozheng Wan, EM-M is the medium-dose group of Ermu Xiaozheng Wan, and EM-H is the high-dose group of Ermu Xiaozheng Wan. Figure 6 The effects of proteomics on protein expression in the liver tissues of mice in each experimental group are shown; where A represents the results of GO and KEGG enrichment analysis (n=12); B represents the expression level of Pck1 protein in the liver tissues of mice in each experimental group (n=12) as determined by proteomics analysis; and C represents the expression level of Pck1 protein in the liver tissues of mice in each experimental group as determined by qRT-PCR. Pck1 mRNA expression levels (n=6); D represents representative bands of Pck1 protein expression in the livers of mice in each experimental group detected by Western blot; E represents the quantitative analysis results of Pck1 protein expression (n=3); all data are expressed as mean ± standard deviation; compared with the normal group ### P <0.001; compared with the model group, P <0.05, P <0.01, P <0.001; WT is the control group, WD is the model group, EM-L is the low-dose group of Ermu Xiaozheng Wan, EM-M is the medium-dose group of Ermu Xiaozheng Wan, and EM-H is the high-dose group of Ermu Xiaozheng Wan. Figure 7 This demonstrates the detection of qRT-PCR in the liver tissue of mice in each experimental group. Acly (A) Fasn (B) Srebf1 (C) Cd36 (D) Slc27a1 (E) Acox1 (F) Cpt1α (G) and Pex14 (H) mRNA expression level; all data are expressed as mean ± standard deviation (n=6); compared with the normal group, # P <0.05, ### P <0.001; compared with the model group, P <0.05, P <0.01, P<0.001; WT is the control group, WD is the model group, EM-L is the low-dose group of Ermu Xiaozheng Wan, EM-M is the medium-dose group of Ermu Xiaozheng Wan, and EM-H is the high-dose group of Ermu Xiaozheng Wan. Figure 8 Representative images (×200) of Oil Red stained liver tissue from each experimental group of mice are shown (A) and the results of quantitative analysis of Oil Red stained area are shown (B); all data are expressed as mean ± standard deviation (n=3); compared with the normal group, ## P <0.01; compared with the model group, P <0.01; WT is the control group, WD is the model group, EM-L is the low-dose group of Ermu Xiaozheng Wan, EM-M is the medium-dose group of Ermu Xiaozheng Wan, and EM-H is the high-dose group of Ermu Xiaozheng Wan. Figure 9 This demonstrates the detection of different formulations of WD mice in liver tissue by qRT-PCR. Col1a1 (A) Acta2 (B) Tgfb1 (C) Pdgfrb (D) Cd36 (E) Slc27a1 (F) Cpt1α (G) Acox1 (H) and Pex14 (I) mRNA expression level; all data are expressed as mean ± standard deviation (n=6); compared with the model group, P <0.05, P <0.01, P <0.001; WT is the control group, WD is the model group, EM is the low-dose Ermu Xiaozheng Wan group, Non-C is the group without Fritillaria cirrhosa, ZB-I is the single-herb Fritillaria thunbergii increased group, EM-R is the Ermu reduced-dose group, and LL is the modified Xiaoluo Wan group. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0021] Based on the pathogenesis of phlegm-blood stasis syndrome in Wilson's disease (WD) liver fibrosis, and guided by the principle of "resolving phlegm, dispersing nodules, and eliminating masses," this application proposes a traditional Chinese medicine composition for treating WD liver fibrosis. This treatment method closely matches the pathological essence of phlegm and blood stasis congealing in the liver collaterals, forming "masses" in WD liver fibrosis. The raw materials are composed of the following parts by weight: Fritillaria cirrhosa 8-12 parts, Laminaria japonica 15-25 parts, Fritillaria thunbergii 25-35 parts, Curcuma longa 8-12 parts, Gallus gallus domesticus gizzard lining 15-25 parts, Crataegus pinnatifida 25-35 parts, and Prunella vulgaris 12-18 parts.

[0022] In the above-mentioned traditional Chinese medicine composition, the principal ingredient is "two mothers" (a traditional Chinese medicine formula) for resolving phlegm and dissipating nodules; the assistant ingredients are kelp for softening hardness and turmeric for promoting blood circulation; the adjuvant ingredients are chicken gizzard membrane and hawthorn for eliminating stagnation and promoting digestion; and the guiding ingredient is prunella vulgaris for clearing liver heat and dissipating nodules. The entire formula revolves around the three concepts of "phlegm, blood stasis, and accumulation," resolving phlegm to dissipate nodules, promoting blood circulation to eliminate blood stasis, and eliminating accumulation to aid digestion, thus achieving the effects of resolving phlegm and removing blood stasis, softening hardness and eliminating masses, precisely addressing the syndrome of phlegm and blood stasis in WD liver fibrosis. The specific formulation principles and compatibility analysis are as follows: The principal herbs (one set): Fritillaria cirrhosa (Chuan Bei Mu) and Fritillaria thunbergii (Zhe Bei Mu). Fritillaria cirrhosa is bitter and sweet in taste, slightly cold in nature, and enters the lung and heart meridians. It excels at clearing heat and moistening the lungs, resolving phlegm and dissipating nodules. It is moistening without being drying, and is especially good at resolving "stubborn phlegm" and "old phlegm". Fritillaria thunbergii is bitter in taste, cold in nature, and enters the lung and heart meridians. It excels at clearing heat and resolving phlegm, relieving stagnation and dissipating nodules, and has a strong ability to purge fire and dissipate nodules. The combination of the two herbs, one moistening and one drying, works synergistically to enhance the effect. Together, they serve as the principal herbs, targeting the core of "phlegm accumulation", and doubling the ability to resolve phlegm and dissipate nodules. This is the essence of the innovative combination of herbs in this formula.

[0023] Assistant herbs: Kelp and Turmeric. Kelp is salty and cold in nature, entering the liver, stomach, and kidney meridians. Its main functions are softening hard masses, dispersing nodules, eliminating phlegm, and promoting diuresis. The *Compendium of Materia Medica* states that it "treats goiters and breaks up accumulations," specifically targeting "lumps" formed by phlegm and blood stasis. Turmeric is pungent and bitter, warm in nature, entering the spleen and liver meridians. It excels at breaking up blood stasis, promoting qi circulation, and relieving pain, making it a key herb for invigorating blood and removing blood stasis, directly addressing the pathogenesis of "blood stasis." Together, one softens hard masses and the other invigorates blood, serving as assistant herbs to assist the principal herb in dispersing lumps.

[0024] Adjunctive herbs: Chicken gizzard lining and raw hawthorn. Chicken gizzard lining is sweet in taste and neutral in nature, entering the spleen, stomach, small intestine, and bladder meridians. It strengthens the stomach and aids digestion, astringes essence and stops seminal emission, and also promotes urination and dissolves stones. Its function of "eliminating stagnation and resolving obstructions" is utilized to assist digestion and disperse stagnation. Raw hawthorn is sour and sweet in taste and slightly warm in nature, entering the spleen, stomach, and liver meridians. It aids digestion, strengthens the stomach, promotes qi circulation and disperses blood stasis, especially effective in eliminating stagnation from meat consumption, and also invigorates blood circulation. The combination of these two herbs promotes digestion and resolves stagnation, ensuring smooth flow of qi in the middle jiao, eliminating the source of phlegm production, and assisting in invigorating blood circulation.

[0025] The guiding herb is Prunella vulgaris. It has a pungent and bitter taste, is cold in nature, and enters the liver and gallbladder meridians. It clears liver heat, improves eyesight, and disperses nodules and reduces swelling. This herb guides the medicine to the liver, clears stagnant heat in the liver meridian, and enhances the overall effect of the formula in dispersing nodules and reducing swelling; it also serves as an adjuvant.

[0026] The composition, preparation, and therapeutic effects of the traditional Chinese medicine composition are illustrated below with specific embodiments.

[0027] Example 1 1.1 Composition of the Chinese herbal medicine composition: Fritillaria cirrhosa 10g (ground into a very fine powder and taken with water), kelp 20g, Fritillaria thunbergii 30g, turmeric 10g, chicken gizzard lining 20g, hawthorn 30g, and prunella vulgaris 15g.

[0028] 1.2 Pill preparation method: a) Except for the fritillaria cirrhosa powder, the other six ingredients (kelp, fritillaria thunbergii, turmeric, chicken gizzard lining, hawthorn, and prunella vulgaris) are decocted twice with water. For the first decoction, add 8 times the amount of water (i.e., 8 times the total weight of the other six ingredients except fritillaria cirrhosa) and decoct for 1 hour. For the second decoction, add 6 times the amount of water (i.e., 6 times the total weight of the other six ingredients except fritillaria cirrhosa) and decoct for 45 minutes. Combine the decoctions and filter. Concentrate the filtrate to a thick paste with a relative density of 1.25-1.30 (60℃).

[0029] b) Mix the thick paste with the fine powder of Fritillaria cirrhosa evenly, dry at 60-70℃, and pulverize into fine powder.

[0030] c) Take the above fine powder, add an appropriate amount of refined honey (or water) to form pills, and dry them to obtain honey pills (or water pills), named Er Mu Xiao Zheng Wan. It can also be encapsulated to make capsule preparations.

[0031] It should be noted that processing Fritillaria cirrhosa alone in the above preparation process can prevent the decomposition, transformation, or volatilization of steroidal alkaloids (such as fritillary alkaloids A and B) in Fritillaria cirrhosa under high-temperature decoction conditions, thereby enhancing its expectorant and nodule-dispersing effects. Furthermore, Fritillaria cirrhosa is rich in starch and other polysaccharides. If decocted with other medicinal materials, these polysaccharides will gelatinize in hot water, making the decoction very viscous. This not only affects filtration and concentration efficiency but may also encapsulate the effective components of other medicinal materials, hindering their dissolution.

[0032] 1.3 Other dosage forms: The above-mentioned traditional Chinese medicine composition can also be prepared into other dosage forms, such as granules, tablets, powders, or oral liquids. During preparation, the medicinal materials other than Fritillaria cirrhosa can be extracted, concentrated, and dried into extract powder, and then mixed with Fritillaria cirrhosa fine powder and appropriate excipients, and prepared according to conventional pharmaceutical processes.

[0033] Example 2 Pharmacodynamic studies were conducted on the Ermu Xiaozheng Pill prepared in Example 1: 2.1 Experimental Materials: Test drug: Prepared according to the composition and preparation method of Ermu Xiaozheng Pill in Example 1. All raw materials were provided by the Traditional Chinese Medicine Pharmacy of the Affiliated Hospital of the Institute of Neurology, Anhui University of Traditional Chinese Medicine.

[0034] Laboratory animals: 100 male SPF-grade Tx-j (a classic animal model of Wilson's disease, purchased from Jackson Laboratory), weighing 20-22g, were housed at the Laboratory Animal Center of the Institute of Neurology, Anhui University of Traditional Chinese Medicine. The housing and experiments were conducted in accordance with the "Guidelines for the Housing and Use of Laboratory Animals".

[0035] 2.2 Experimental grouping and dosing regimen: Mice were randomly divided into 5 groups of 20 mice each. The specific grouping and drug administration are as follows: The low-dose group of Er Mu Xiao Zheng Wan (EM-L) was administered by gavage at a dose of 10.125 g / kg once daily. The medium-dose group of Er Mu Xiao Zheng Wan (EM-M) was administered by gavage at a dose of 20.25 g / kg once daily. High-dose group of Er Mu Xiao Zheng Wan (EM-H): Administered by gavage at a dose of 30.375 g / kg once daily; Normal group (WT): Administered an equal volume of normal saline by gavage (equal to the volume of solvent used in each dosage group of Er Mu Xiao Zheng Wan), once daily; Model group (WD): Administered an equal volume of physiological saline by gavage (the same volume of solvent used in each dosage group of Er Mu Xiao Zheng Wan), once daily.

[0036] 2.3 Experimental methods: In accordance with the experimental grouping and administration in 2.2, Tx-j mice were given the drug starting at 8 weeks of age. The dosage was calculated based on the clinical equivalent dosage of each drug and the administration ended after four weeks.

[0037] 2.4 Experimental Results: After the experiment, the mice in each group were fasted for 12 hours, sacrificed, and their liver tissue was quickly dissected and serum was collected. Serum samples were prepared by centrifugation at 3000 r / min for 15 minutes and stored at -80℃ for later use. Liver tissue was partially fixed with 4% paraformaldehyde for pathological sections and immunohistochemical detection. Some tissue was rapidly frozen in liquid nitrogen and then transferred to a -80°C freezer for storage, used for qRT-PCR, WB, and copper content determination.

[0038] The specific measurement methods for each indicator are as follows: (1) Liver function index measurement: The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in mouse serum were measured using a fully automated biochemical analyzer. The results are shown in Table 1. Table 1. Effects of Er Mu Xiao Zheng Wan on serum liver function in WD mice ( )

[0039] In Table 1, compared with the WT group, ## P <0.01; compared with the WD group, P <0.01, n=8.

[0040] It can be seen that, compared with the WT group, the serum levels of ALT, AST, and ALP in the WD group mice were significantly increased. P <0.01); Er Mu Xiao Zheng Wan can dose-dependently reduce the above indicators ( P <0.01), with the EM-H group showing ALT, AST, and ALP levels decreasing to 41.9±2.2 U / mL, 88.1±13.2 U / mL, and 116.6±9.1 U / mL, respectively, close to normal levels. This indicates that the traditional Chinese medicine composition proposed in this application can effectively alleviate liver damage in WD mice.

[0041] (2) Measurement of liver fibrosis markers: Serum fibrosis marker detection: The main components of serum extracellular matrix (ECM) include hyaluronic acid (HA), type 4 collagen (Col IV), laminin (LN), and type 3 procollagen (HPC III). As liver fibrosis progresses, the expression levels of these components change accordingly.

[0042] The level of serum fibrosis in mice was detected by ELISA, and the results are shown in Table 2. Table 2 Effects of Er Mu Xiao Zheng Wan on serum liver fibrosis markers in WD mice ( )

[0043] In Table 2, compared with the WT group, ## P <0.01; compared with the WD group, P <0.01, n=8.

[0044] The results showed that, compared with the WT group, the serum concentrations of HA, COLⅣ, LN, and HPCⅢ in Tx-j mice were significantly increased ( P <0.01), after Tx-j mice were treated with different doses of Ermu Xiaozheng Wan, the serum levels of HA, COLⅣ, LN and HPCⅢ were significantly lower than those in the model group. P <0.01).

[0045] Liver pathological changes: Liver fibrosis is one of the main pathological features of WD chronic liver disease. Due to liver inflammation and copper toxicity stimulating hepatic stellate cells, a large amount of collagen fibers are deposited in the liver. Mouse liver tissue fixed in 4% paraformaldehyde was dehydrated in a gradient, embedded in paraffin, and sectioned (4 μm thick). After sectioning, Sirius Red staining and Masson staining were performed. The deposition of collagen fibers in the liver was observed under an optical microscope, and the percentage of collagen fiber area was calculated using Image-Pro Plus 6.0 image analysis software.

[0046] The results of Sirius Red and Masson staining are as follows: Figure 1 and Figure 2 As shown, the liver structure of normal mice in the WT group was intact with no obvious collagen fiber deposition; in the WD model group, collagen fibers were deposited extensively in the liver, forming fibrous septa and causing disordered lobular structure; in the Er Mu Xiao Zheng Wan treatment group, collagen fiber deposition was significantly reduced, and the lobular structure tended to be intact, with the high-dose group showing the most significant improvement. Quantitative analysis results showed that compared with the WT group, collagen fiber deposition in the liver of Tx-j mice was significantly increased (…). P <0.01. Both Sirius Red and Masson staining showed that collagen fiber deposition was significantly reduced in the different doses of Er Mu Xiao Zheng Wan groups compared to the model group ( P <0.05).

[0047] Detection of hepatic stellate cell activation markers: To further investigate the inhibitory effect of Ermu Xiaozheng Wan on WD liver fibrosis, the effects of Ermu Xiaozheng Wan on the expression of important factors for hepatic stellate cell activation, namely collagen I, α-smooth muscle actin (α-SMA) and transforming growth factor-β (TGF-β), were examined.

[0048] ① Immunohistochemical detection: After dewaxing to water, antigen retrieval, and blocking, liver paraffin sections were incubated overnight with collagen I, α-SMA, and TGF-β primary antibodies (purchased from Servicebio). After incubation with secondary antibodies, DAB staining was performed, followed by hematoxylin counterstaining, dehydration, clearing, and mounting. Positive expression was observed under a microscope and quantitative analysis was performed.

[0049] The results are as follows Figure 3 As shown, compared with the WT group, the expression levels of collagen I, α-SMA, and TGF-β in Tx-j mice (reacted by positive area) were significantly increased. P <0.05), EM-M, EM-H group collagen I ( Figure 3 (A and B), α-SMA ( Figure 3 C and D) and TGF-β ( Figure 3The expression levels of E and F were significantly reduced. P <0.05).

[0050] ②qRT-PCR detection: Total RNA was extracted from liver tissue using the Trizol method, and cDNA was synthesized by reverse transcription. GAPDH was used as an internal reference gene, and amplification was performed according to the instructions of the real-time quantitative PCR kit (purchased from Takara). The target gene was calculated. Col1a1 , Acta2 , Tgfb1 , Pdgfrb The relative expression level.

[0051] qRT-PCR analysis results are as follows Figure 4 As shown, compared with the normal control group, the WD model group Col1a1 ( Figure 4 (A) Acta2 ( Figure 4 (B) Tgfb1 ( Figure 4 (C) Pdgfrb ( Figure 4 The level of D in the middle was significantly increased ( P <0.001). Compared to the model group, the EM-M and EM-H groups... Col1a1 , Acta2 , Tgfb1 and Pdgfrb mRNA levels were also significantly reduced. P <0.01).

[0052] ③WB detection: The protein expression levels of α-SMA and TGF-β1, markers of hepatic stellate cell (HSC) activation, in the liver tissue of WD mice were analyzed by WB experiment.

[0053] Experimental results are as follows Figure 5 As shown in the figure, A represents the representative bands of α-SMA and TGF-β1 protein expression detected by Western blot; B represents the quantitative analysis results of α-SMA protein expression; and C represents the quantitative analysis results of TGF-β1 protein expression. It can be seen that compared with the WT group mice, the levels of α-SMA and TGF-β1 proteins in the liver of Tx-j mice were significantly upregulated (…). P <0.05%, compared with the WD group mice, the levels of α-SMA and TGF-β1 proteins in the EM-M and EM-H groups were significantly downregulated ( P <0.05), suggesting that it exerts its anti-fibrotic effect by inhibiting the activation of hepatic stellate cells.

[0054] (3) Study on the mechanism of action of Ermu Xiaozheng Pill: A proteomic study was conducted on the liver of Tx-j mice after treatment with Ermu Xiaozheng Pills to explore the possible mechanism by which Ermu Xiaozheng Pills improve liver fibrosis in WD mice.

[0055] Proteomics analysis: Liver tissue samples (n=12 animals / group) from the normal group, model group, and medium-dose group of Er Mu Xiao Zheng Wan were selected for proteomics analysis.

[0056] ① Sample preparation: Extract total protein from liver tissue, and perform quantification, enzymatic digestion, and labeling (such as TMT labeling or label-free labeling).

[0057] ②LC-MS / MS analysis: High performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to separate and identify the peptides after enzymatic hydrolysis.

[0058] ③ Data Analysis: Protein identification and quantification were performed using software such as MaxQuant and Proteome Discoverer. Differentially expressed proteins (DEPs) were screened, typically with screening criteria of Fold Change > 1.5 or < 0.67, P < 0.05.

[0059] ④ Bioinformatics analysis: GO (Gene Ontology) functional enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis were performed on DEPs.

[0060] The results are as follows Figure 6 As shown, compared with the model group, the Ermu Xiaozheng Wan treatment group showed significant changes in the expression levels of multiple proteins. GO enrichment analysis of these differentially expressed proteins revealed that they were mainly enriched in biological processes such as lipid metabolism, fatty acid metabolism, immune response, and inflammatory response. Figure 6 (A). KEGG pathway enrichment analysis further indicated that the differentially expressed proteins are mainly involved in retinol metabolism, fatty acid elongation, PPAR signaling pathway, fatty acid degradation, and sphingolipid metabolism. Figure 6 (A). These results suggest that the hepatoprotective and anti-fibrotic effects of Er Mu Xiao Zheng Wan may be primarily achieved through the regulation of lipid metabolism and inflammation-related pathways.

[0061] Among the differentially expressed proteins identified in proteomics analysis, phosphoenolpyruvate carboxykinase 1 (Pck1), a key enzyme involved in lipid metabolism and gluconeogenesis pathways, was found to have significantly lower expression levels in the livers of model mice compared to the normal group. However, after treatment with Er Mu Xiao Zheng Wan (a traditional Chinese medicine), the protein expression level of Pck1 significantly increased. Figure 6 To verify this finding, the mRNA of Pck1 was detected using qRT-PCR and Western Blot techniques. Figure 6 (C) and protein ( Figure 6The relative expression levels of D and E were compared, and the results were consistent with proteomics data, confirming that Ermu Xiaozheng Pills can significantly upregulate the expression of Pck1 in the liver of WD mice.

[0062] Given the important role of Pck1 in lipid metabolism, the expression of key genes in the hepatic lipid metabolism pathway was further investigated. The results are as follows: Figure 7 The results showed that, compared with the normal group, the number of genes involved in lipid synthesis (such as...) in the liver of model group mice was significantly lower. Acly ( Figure 7 (A) Fasn ( Figure 7 (B) Srebf1 ( Figure 7 The expression of C in the middle of the body was generally downregulated. P <0.001), while genes involved in lipid transport (such as...) Cd36 ( Figure 7 (D) Slc27a1 ( Figure 7 E) expression was significantly upregulated ( P <0.001). This altered expression pattern suggests a complex reprogramming of hepatic lipid metabolism under WD conditions.

[0063] After intervention with Ermu Xiaozheng Pills, the expression level of downregulated lipid synthesis genes in the liver of WD mice was significantly upregulated. P <0.05), while upregulated lipid transport genes Cd36 and Slc27a1 The expression of [something] was significantly suppressed. P <0.01). Furthermore, it was found that Ermu Xiaozheng Wan could upregulate key genes involved in fatty acid β-oxidation (such as...). Acox1 ( Figure 7 (middle F) Cpt1α ( Figure 7 (G) Pex14 ( Figure 7 The expression of H in ( P <0.001). Er Mu Xiao Zheng Wan significantly upregulated the expression of fatty acid β-oxidation-related genes and downregulated fatty acid transport proteins. Cd36 and Slc27a1 The expression. This regulatory mode, which combines "open source" and "reduction", ultimately promotes the breakdown and utilization of fatty acids, reduces the net inflow and accumulation of lipids in the liver, and thus improves hepatic steatosis.

[0064] In addition, the results of Oil Red O staining are as follows: Figure 8 The results showed that lipid droplets accumulated extensively in the liver of mice in the WD model group, while treatment with Er Mu Xiao Zheng Wan significantly reduced the number of lipid droplets in the liver. P The result was <0.01, indicating that Er Mu Xiao Zheng Wan effectively improved hepatic steatosis in WD mice.

[0065] Based on the above experiments, it can be inferred that Er Mu Xiao Zheng Wan inhibits WD liver fibrosis by regulating Pck1 and its downstream lipid metabolism network. Er Mu Xiao Zheng Wan does not simply inhibit or activate a specific metabolic pathway, but rather exerts its therapeutic effect by reshaping the disordered metabolic network and restoring liver metabolic homeostasis.

[0066] Example 3 To verify the scientific validity of the complete formula of Er Mu Xiao Zheng Wan, five control groups were set up for the experiment: the complete Er Mu Xiao Zheng Wan group (EM), the group without Fritillaria cirrhosa (Non-C, Fritillaria cirrhosa removed), the single-herb Fritillaria thunbergii group (ZB-I, Fritillaria cirrhosa removed, simply increasing the amount of Fritillaria thunbergii to 40g), the Er Mu reduced dosage group (EM-R, reduced dosage of Fritillaria thunbergii and Fritillaria cirrhosa), and the modified Xiao Luo Wan group (LL). A Tx-j mouse model was used, and relevant indicators were measured after 4 weeks of continuous administration. The specific composition and experimental results of each control group are as follows: Experimental composition for each control group: The complete formula of Er Mu Xiao Zheng Wan (EM) is prepared according to the prescription in Example 1 of this application, namely, 10g of Fritillaria cirrhosa, 20g of Laminaria japonica, 30g of Fritillaria thunbergii, 10g of Curcuma longa, 20g of chicken gizzard lining, 30g of hawthorn, and 15g of Prunella vulgaris. Group without Fritillaria cirrhosa (Non-C): To verify the necessity of Fritillaria cirrhosa, one of the principal herbs was removed. The prescription is as follows: Fritillaria thunbergii 30g, Laminaria japonica 20g, Curcuma longa 10g, Gallus gallus domesticus gizzard lining 20g, Crataegus pinnatifida 30g, Prunella vulgaris 15g; Single-herb Fritillaria thunbergii (ZB-I): To verify the synergistic effect of the two herbs, Fritillaria cirrhosa was removed, and the dosage of Fritillaria thunbergii was simply increased to 40g. The prescription was: Fritillaria thunbergii 40g, Laminaria japonica 20g, Curcuma longa 10g, Gallus gallus domesticus gizzard lining 20g, Crataegus pinnatifida 30g, Prunella vulgaris 15g; The reduced dosage group of the two mother herbs (EM-R): To verify the importance of the dosage ratio of the two mother herbs, the ratio of the principal herbs was adjusted. The prescription was: Fritillaria cirrhosa 5g, Fritillaria thunbergii 15g, Laminaria japonica 20g, Curcuma longa 10g, Gallus gallus domesticus gizzard lining 20g, Crataegus pinnatifida 30g, and Prunella vulgaris 15g; Xiaoluo Pill Modified Group (XL): This group was compared with the classic phlegm-resolving and nodule-dispersing formula "Xiaoluo Pill" (from "Medical Insights") to evaluate the innovativeness and comprehensive efficacy advantages of this formula in resolving phlegm and dispersing nodules. The prescription is: Scrophularia ningpoensis 15g, Oyster shell 20g, Fritillaria thunbergii 30g.

[0067] Model group (WD): Administered an equal volume of physiological saline by gavage (equal to the volume of solvent used in the drug group) once daily.

[0068] (1) The improvement results of liver fibrosis indicators in each experimental group are shown in Table 3: Table 3 Comparison of the effects of different formulations on liver fibrosis markers ( )

[0069] It can be seen that, in terms of liver fibrosis indicators, the Ermu Xiaozheng Wan whole formula group showed the most significant reduction in HA, Col IV, LN, and HPC III, increasing by 17%, 23.5%, 21%, and 9.2% respectively compared to the Xiaoluo Wan modified group. The group without Fritillaria cirrhosa showed the worst effect, indicating that Fritillaria cirrhosa plays a key role in anti-liver fibrosis. The single-herb Fritillaria thunbergii group did not significantly reduce HA, Col IV, LN, and HPC III, indicating that even increasing the dosage of Fritillaria thunbergii could not compensate for the synergistic effect of lacking Fritillaria cirrhosa, highlighting the unique value of the "two mothers" combination.

[0070] (2) Effects on the expression of genes related to liver fibrosis and lipid metabolism: qRT-PCR results are as follows Figure 9 As shown.

[0071] Liver fibrosis gene: Compared with the WD group, the Er Mu Xiao Zheng Wan whole formula group was the most effective in downregulating liver fibrosis genes. Col1a1 ( Figure 9 (A) Acta2 ( Figure 9 (B) Tgfb1 ( Figure 9 (C) Pdgfrb ( Figure 9 mRNA expression of D (in the middle) P <0.001), its effect is better than the group without Fritillaria cirrhosa, the group with increased amount of Fritillaria thunbergii alone, the group with reduced amount of two mother herbs, and the modified Xiaoluo Pill group ( P <0.05).

[0072] Lipid uptake and transport genes: WD group Cd36 ( Figure 9 (E) and Slc27a1 ( Figure 9 The expression of F1 was significantly upregulated (F1) P <0.01 indicates that the model group has abnormal lipid uptake and transport. The Er Mu Xiao Zheng Wan whole formula group can significantly downregulate the expression of these two genes ( P <0.001, the effect is better than other control groups ( P <0.05). The groups with reduced Fritillaria cirrhosa, single-herb Fritillaria thunbergii, and modified Xiaoluo Pills showed effects on... Slc27a1 The downregulation effect was not significant.

[0073] Fatty acid β-oxidation genes: WD group Cpt1α ( Figure 9 (G) Acox1 ( Figure 9 (H) Pex14 ( Figure 9 Downregulation of I expression ( P<0.05 indicates impaired fatty acid β-oxidation. The complete formula of Er Mu Xiao Zheng Wan significantly reversed this trend, upregulating the expression of these three genes ( P <0.01), its promoting effect was significantly stronger than the group without Fritillaria cirrhosa, the group with increased amount of Fritillaria thunbergii alone, the group with reduced amount of both Fritillaria cirrhosa and Fritillaria thunbergii, and the modified Xiaoluowan group. Among them, the group with reduced amount of both Fritillaria cirrhosa and Fritillaria thunbergii had the best effect on promoting the growth of Fritillaria cirrhosa. Cpt1α , Acox1 , Pex14 The upregulation effect was significantly weaker in the whole formula group than in the whole formula group.

[0074] The above experiments demonstrate the synergistic necessity of the "two mothers" combination. The groups without Fritillaria cirrhosa and those with only Fritillaria thunbergii showed significantly lower performance than the complete formula group in multiple fibrosis indicators and lipid metabolism gene regulation. This indicates that the moistening and phlegm-resolving effects of Fritillaria cirrhosa and the bitter-dispersing and nodule-dissolving effects of Fritillaria thunbergii complement each other, and neither can be dispensed with. The combination of the two produced a synergistic anti-fibrotic effect of "1+1>2". The optimization of the dosage ratio of the two mothers: Although the two mothers reduction group retains the structure of the two mothers, the dosage ratio of the original formula has been changed. Its efficacy is between that of groups A and B in many aspects, which proves that the compatibility ratio of Fritillaria cirrhosa (30g) and Fritillaria thunbergii (30g) in Example 1 is an optimized ratio that can exert the best efficacy.

[0075] Advantages of the comprehensive treatment: Compared to the classic phlegm-resolving and nodule-dispersing formula E group (a modified version of Xiaoluo Pill), the comprehensive formula of this application (Group A) shows superior performance in improving liver function, inhibiting collagen deposition, regulating fibrosis genes, and correcting lipid metabolism disorders. Cd36 , Cpt1α It demonstrates comprehensive and significant advantages in gene regulation. This fully reflects the comprehensive intervention value of this formula in targeting the complex pathogenesis of "phlegm, blood stasis, and accumulation" in WD liver fibrosis, based on "resolving phlegm and dissipating nodules" and combined with the compound treatment methods of "activating blood circulation (turmeric)," "eliminating stagnation (chicken gizzard lining and hawthorn)," and "clearing the liver (prunella vulgaris)."

[0076] Potential mechanisms of action suggest that the whole-formula group regulates lipid metabolism-related genes ( Cd36 , Slc27a1 , Cpt1α , Acox1 Its outstanding performance in the area of ​​"phlegm-resolving and stasis-eliminating" suggests that its mechanism of action may be closely related to improving liver lipid metabolism, reducing lipid toxicity, and thus inhibiting the activation of hepatic stellate cells, providing experimental evidence for the connection between the TCM theory of "phlegm and blood stasis" and the modern mechanism of "metabolic liver injury".

[0077] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A traditional Chinese medicine composition, characterized in that, It is composed of the following raw materials in parts by weight: Fritillaria cirrhosa 8-12 parts, Laminaria japonica 15-25 parts, Fritillaria thunbergii 25-35 parts, Curcuma longa 8-12 parts, Chicken gizzard lining 15-25 parts, Crataegus pinnatifida 25-35 parts, and Prunella vulgaris 12-18 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, It is composed of the following raw materials in parts by weight: 10 parts of Fritillaria cirrhosa, 20 parts of Laminaria japonica, 30 parts of Fritillaria thunbergii, 10 parts of Curcuma longa, 20 parts of chicken gizzard lining, 30 parts of hawthorn, and 15 parts of Prunella vulgaris.

3. A pharmaceutical preparation, characterized in that, It comprises the traditional Chinese medicine composition as described in claim 1 or 2, and pharmaceutically acceptable excipients.

4. The pharmaceutical preparation according to claim 3, characterized in that, The dosage forms of the pharmaceutical preparations include pills, powders, granules, capsules, tablets, or oral liquids.

5. A pharmaceutical composition, characterized in that, The traditional Chinese medicine composition described in claim 1 or 2 is used as one of the active pharmaceutical ingredients.

6. The use of the traditional Chinese medicine composition according to claim 1 or 2, the preparation according to claim 3 or 4, and the pharmaceutical composition according to claim 5 in the preparation of a drug for treating Wilson's disease and liver fibrosis.

7. The use of the traditional Chinese medicine composition according to claim 1 or 2, the preparation according to claim 3 or 4, and the pharmaceutical composition according to claim 5 in the preparation of a drug for improving liver damage in Wilson's disease.

8. The use of the traditional Chinese medicine composition according to claim 1 or 2, the formulation according to claim 3 or 4, and the pharmaceutical composition according to claim 5 in the preparation of a drug for downregulating the expression of genes related to liver fibrosis in Wilson's disease, characterized in that, The liver fibrosis-related genes include Col1a1 , Acta2 , Tgfb1 , Pdgfrb .

9. The use of the traditional Chinese medicine composition according to claim 1 or 2, the preparation according to claim 3 or 4, and the pharmaceutical composition according to claim 5 in the preparation of a drug for regulating liver lipid metabolism in Wilson's disease.

10. The use of the traditional Chinese medicine composition of claim 1 or 2, the preparation of claim 3 or 4, and the pharmaceutical composition of claim 5 in the preparation of a drug that upregulates the expression of phosphoenolpyruvate carboxykinase 1 in the liver of Wilson's disease.

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