Traditional Chinese medicine compound for treating metabolism-related fatty liver diseases and hepatic fibrosis thereof

Abstract

The invention belongs to the field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine compound for treating metabolism-related fatty liver diseases and hepatic fibrosis thereof. The phlegm-eliminating and blood-activating composition disclosed by the invention can be used for treating both symptoms and root causes from pathogenesis and comprehensively regulating the body. On the basis of the traditional Chinese medicine compatibility theories of monarch, minister, assistant and guide and seven-estrus combination, the traditional Chinese medicine composition has the effects of treating lipid deposition, resisting inflammation and apoptosis, improving fibrosis and the like.

Description

Technical Field

[0001] This application belongs to the field of traditional Chinese medicine, specifically relating to a traditional Chinese medicine compound for treating metabolic-related fatty liver disease and liver fibrosis. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD), formerly known as nonalcoholic fatty liver disease (NAFLD), has a new definition. In early 2020, an international expert panel of 30 experts from 22 countries released an international expert consensus statement on the new definition of MAFLD. The panel detailed the clinical significance of renaming NAFLD to MAFLD and recommended that MAFLD be a more appropriate disease name to describe liver diseases associated with metabolic dysfunction. MAFLD is the most common chronic liver disease worldwide, encompassing metabolic dysfunction-associated fatty liver (MAFL), metabolic dysfunction-associated steatohepatitis (MASH), metabolic dysfunction-associated fatty liver fibrosis, and metabolic dysfunction-associated fatty liver cirrhosis. Currently, approximately 38% of the world's population has MAFLD, and its incidence is rapidly increasing, with an estimated 4,613 new cases per 100,000 people annually. Furthermore, hepatocellular carcinoma associated with MAFLD is also rapidly increasing, posing a significant economic and medical burden globally. In my country, due to changes in lifestyle and dietary structure, the incidence of MAFLD is rising rapidly. Currently, the national prevalence rate is about 29.2%, making it the most common chronic liver disease in my country. Moreover, the end-stage liver diseases caused by MAFLD, such as cirrhosis and liver cancer, are increasing.

[0003] Liver fibrosis is a key pathological stage in the progression of MAFLD to end-stage liver disease, and it is the core pathological link in this progression. Although the main causes of death in MAFLD patients are cardiovascular disease and extrahepatic malignancies, the degree of liver fibrosis, especially advanced fibrosis (F3 / F4 stage), is a key indicator for predicting liver-related outcomes and overall mortality. MAFLD patients who have progressed to F3 / F4 stage fibrosis have a significantly increased risk of liver complications such as hepatic encephalopathy, esophageal varices, and hepatocellular carcinoma, as well as an increased risk of type 2 diabetes and kidney damage, leading to a substantial increase in related mortality. Even early fibrosis, progressing from hepatic steatosis to fibrosis, significantly increases the risk of end-stage liver disease. Therefore, liver fibrosis is a core liver disease closely related to poor long-term prognosis in MAFLD patients. Finding key targets and therapeutic drugs that can effectively prevent, especially reverse, liver fibrosis is of urgent clinical need and significant scientific importance.

[0004] However, the pathogenesis of MAFLD, especially its associated liver fibrosis, is extremely complex, involving multiple aspects such as insulin resistance, oxidative stress, inflammatory response, and lipid metabolism disorders. Many new drug developments have failed in clinical trials, and highly effective treatments are still lacking. Some guidelines recommend the use of vitamin E, as it can improve lipid peroxidation in MAFLD and shows potential to improve liver fibrosis, but its long-term efficacy and safety still require more evidence. While the farnesoid X receptor (FXR) agonist obeticholic acid can improve liver fibrosis in some patients, its exact therapeutic benefits and safety need further evaluation. The thyroxine beta receptor agonist resmetirom is the first drug for treating non-alcoholic steatohepatitis (NASH) with liver fibrosis. It can promote the breakdown of liver fat and control the level of normal mitochondria by regulating mitochondrial activity. Although it achieved a 29.9% NASH remission rate in a phase III trial, the improvement rate of liver fibrosis in the high-dose group (100 mg / day) was only 11.7%, and the high annual treatment cost for patients severely limits its clinical application. Bariatric and metabolic surgery (MBS) is the most effective treatment for obesity. Lifestyle interventions (such as weight loss and dietary adjustments) are important measures for the treatment of MAFLD, but patient compliance is poor and lifestyle interventions alone cannot reverse fibrosis, so drug therapy is still required.

[0005] Therefore, there is an urgent need to develop a drug that can treat MAFLD and its liver fibrosis.

[0006] Application content

[0007] In order to solve the technical problems in the background art mentioned above, this application provides a phlegm-reducing and blood-activating formula composition.

[0008] The technical solution adopted in this application is as follows:

[0009] This application provides a formula composition for relieving phlegm and promoting blood circulation, the composition comprising Gynostemma pentaphyllum and hawthorn as principal ingredients, and Atractylodes macrocephala, Rhodiola rosea and turmeric as assistant ingredients.

[0010] Based on the above technical solution, the following improvements can be made to this application.

[0011] Furthermore, by weight, the composition comprises 10-30 parts of Gynostemma pentaphyllum and 10-30 parts of hawthorn as principal ingredients, and 4-10 parts of Rhodiola rosea, 8-16 parts of Atractylodes macrocephala and 4-12 parts of turmeric as adjuvant ingredients.

[0012] Furthermore, by weight, the composition comprises 20 parts of Gynostemma pentaphyllum and 15 parts of hawthorn as principal ingredients, and 6 parts of Rhodiola rosea, 12 parts of Atractylodes macrocephala and 9 parts of turmeric as adjuvant ingredients.

[0013] This application also provides the use of the expectorant and blood-activating composition as described above in the preparation of a medicament for treating lipid deposition, inflammation, or improving fibrosis.

[0014] This application also provides the use of the expectorant and blood-activating formula composition as described above in the preparation of a medicament for treating metabolic-related fatty liver disease and its liver fibrosis.

[0015] This application also provides a method for screening therapeutic targets of the expectorant and blood-activating formula composition as described above, comprising the following steps:

[0016] S1. Screen the active ingredients in the expectorant and blood-activating formula composition through the TCMSP database, and summarize the target points of the active ingredients in the expectorant and blood-activating formula.

[0017] S2. Based on the active ingredients obtained in step S1, set screening conditions and search for disease-related genes in the GeneCards, OMIM and TTD databases using disease keywords as targets to be identified.

[0018] S3. After integrating the target points to be determined in step S2, remove duplicates and take the intersection to obtain the disease target points.

[0019] S4. Combine the target of the active ingredient of the expectorant and blood-activating formula in step S1 with the disease target in step S3 and take the intersection to obtain the therapeutic target of the expectorant and blood-activating formula.

[0020] Based on the above technical solution, the following improvements can be made to this application.

[0021] Furthermore, in step S2, the disease keywords are non-alcoholic fatty liver disease or metabolic-associated fatty liver disease.

[0022] Furthermore, in step S3, an R language script is used to remove duplicates and find the intersection.

[0023] This application also provides a method for enriching rutin using the expectorant and blood-activating formula composition described above.

[0024] The beneficial effects of this application are: the phlegm-removing and blood-activating formula composition of this application can address both the root cause and symptoms of the disease, and comprehensively regulate the body. Based on the traditional Chinese medicine compatibility theory of "principal, assistant, adjuvant, and guide" and "seven emotions and harmony", this application has studied Chinese medicines with anti-inflammatory, anti-apoptotic, and fibrosis-reversing effects. The composition of this application can exert the synergistic effect of multiple drug components, namely rutin. Rutin has rich pharmacological effects, mainly manifested in the following aspects: (1) achieving anti-inflammatory effect by regulating inflammatory response through multiple targets; (2) achieving anti-apoptotic effect by protecting cell survival; (3) achieving lipid deposition effect by improving metabolic homeostasis; (4) having a therapeutic effect on fibrosis. Moreover, the composition of this application can play a huge role in the role of hawthorn. The phlegm-removing and blood-activating formula with hawthorn is more effective than that without hawthorn. The effect is reflected in improving fatty degeneration and inflammation, and also has a therapeutic effect on fibrosis. Attached Figure Description

[0025] Figure 1 The liver weight and liver body weight ratio of mice in each group are shown in this application. Figure 1 A neutralization Figure 1 In group B, CON represents the control group, CDAHFD represents the model group, PIO represents the pioglitazone group, ZY represents the first group of the expectorant and blood-activating formula, QTHXF-L represents the low-dose group of the second group of the expectorant and blood-activating formula, and QTHXF-H represents the high-dose group of the second group of the expectorant and blood-activating formula. Compared with the CON group, aP<0.05; compared with the CDAHFD group, bP<0.05; compared with the ZY group, cP<0.05.

[0026] Figure 2 This paper shows schematic diagrams of HE staining and NAS scores for each group of mice in this application. Figure 2 A shows a schematic diagram of HE staining of liver tissue (Bar: 100μm). Figure 2 B shows the NAS score of liver tissue; CON represents the control group, CDAHFD represents the model group, PIO represents the pioglitazone group, ZY represents the first group of the expectorant and blood-activating formula, QTHXF-L represents the low-dose group of the second group of the expectorant and blood-activating formula, and QTHXF-H represents the high-dose group of the second group of the expectorant and blood-activating formula. Compared with the CON group, aP<0.05; compared with the CDAHFD group, bP<0.05; compared with the ZY group, cP<0.05.

[0027] Figure 3 The serum ALT and AST activities of mice in each group presented in this application were shown. Figure 3 A showed serum ALT activity. Figure 3 B represents serum AST activity; CON represents the control group, CDAHFD represents the model group, PIO represents the pioglitazone group, ZY represents the first group of the expectorant and blood-activating formula, QTHXF-L represents the low-dose group of the second group of the expectorant and blood-activating formula, QTHXF-H represents the high-dose group of the second group of the expectorant and blood-activating formula, and CDAHFD+ST represents the low methionine and choline deficiency high-lipid group + STING inhibitor group. Compared with the CON group, aP<0.05; compared with the CDAHFD group, bP<0.05; compared with the ZY group, cP<0.05.

[0028] Figure 4 The study showed the TG and HYP content in the liver tissue of mice in each group. Figure 4 A shows the TG content in liver tissue. Figure 4 B shows the HYP content in liver tissue; CON represents the control group, CDAHFD represents the model group, PIO represents the pioglitazone group, ZY represents the first group of the expectorant and blood-activating formula, QTHXF-L represents the low-dose group of the second group of the expectorant and blood-activating formula, QTHXF-H represents the high-dose group of the second group of the expectorant and blood-activating formula, and CDAHFD+ST represents the low methionine and choline deficiency high-lipid group + STING inhibitor group. Compared with the CON group, aP<0.05; compared with the CDAHFD group, bP<0.05; compared with the ZY group, cP<0.05.

[0029] Figure 5 The diagram shows the core target protein interaction network of QTHX in this application.

[0030] Figure 6 The pathway enrichment analysis of QTHX in this application is shown.

[0031] Figure 7 The diagram shows HE staining of mouse liver tissue in each group of this application. CON represents the control group, CDAHFD represents the model group, QTHXF-L represents the low-dose group of the expectorant and blood-activating formula, QTHXF-H represents the high-dose group of the expectorant and blood-activating formula, and CDAHFD+ST represents the low methionine and choline deficiency high-lipid group + STING inhibitor group.

[0032] Figure 8 The graphs showing TG content and NAS score of liver tissue from each group of mice in this application are displayed. Figure 8 Figure A shows the TG content in liver tissue. Figure 8 B shows the liver tissue pathological NAS score. CON represents the control group, CDAHFD represents the model group, QTHXF-L represents the low-dose group of the expectorant and blood-activating formula, QTHXF-H represents the high-dose group of the expectorant and blood-activating formula, and CDAHFD+ST represents the low methionine and choline deficiency high-lipid group + STING inhibitor group. *P<0.05, **P<0.01, ***P<0.001, n=8.

[0033] Figure 9 The graph showing the changes in serum ALT activity in mice of each group in this application is shown. Figure 9 Table A shows the serum ALT levels. Figure 9 B shows the serum AST level. Figure 9 C represents the liver HYP content, CON represents the control group, CDAHFD represents the model group, QTHXF-L represents the low-dose group of the expectorant and blood-activating formula, QTHXF-H represents the high-dose group of the expectorant and blood-activating formula, and CDAHFD+ST represents the low methionine and choline deficiency high-lipid group + STING inhibitor group. *P<0.05, **P<0.01, ***P<0.001, n=8.

[0034] Figure 10 The diagram shows the Sirius red staining of the liver tissue of mice in each group of this application.

[0035] Figure 11 The diagram shows the traditional Chinese medicine-component-target action network of rutin in this application. Detailed Implementation

[0036] The principles and features of this application are described below with reference to the accompanying drawings. The examples given are only for explaining this application and are not intended to limit the scope of this application.

[0037] Terminology definition:

[0038] Drugs or pharmaceutical compositions: The expectorant and blood-activating formula compositions disclosed herein, together with one or more excipients such as adjuvants, carriers, or diluents, can be incorporated into pharmaceutical compositions, unit dosages, or dosage forms. The pharmaceutical compositions may be in solid dosage forms (e.g., powders, granules, pills, coated or uncoated tablets or filled capsules), liquid dosage forms (e.g., solutions, suspensions, emulsions, or filled capsules), or semi-solid dosage forms (e.g., gels, creams, and ointments). The solubility and release characteristics of one or more active ingredients in the pharmaceutical dosage form can vary from seconds to months.

[0039] The “medicine” or “medicinal composition” is designed for use in animals and humans and can be administered via all routes of administration. Preferred routes of administration are injection, oral, pulmonary, nasal, rectal, and parenteral. Such pharmaceutical compositions and their unit dosage forms may conventionally or specifically contain conventional or novel ingredients, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient to be used, commensurate with the target daily dose range.

[0040] Treatment methods and pharmaceutical formulations: The active ingredient of Formula I of this disclosure, or with one or more pharmaceutical-acceptable excipients, carriers or diluents, particularly and preferably in the form of pharmaceutical compositions thereof, may be administered in an effective amount to a subject in need, such as an active animal (including a human), for the treatment, relief or improvement, alleviation or elimination of an indication or condition to which the subject is sensitive, or an indication or condition otherwise described in this disclosure.

[0041] As used herein, the term “treatment” means to reduce or alleviate at least one symptom of the disease in the subject, and within the scope of the meaning of this disclosure, the term “treatment” also means to suppress, delay the onset (i.e., the early stage of clinical manifestations of the disease) and / or reduce the risk of developing or worsening the disease.

[0042] As used herein, the term "recovery" means that under the influence of certain endogenous or exogenous stimuli, such as endotoxins, cellular function and / or structure may change, resulting in structural and / or functional abnormalities, and "recovery" refers to restoring this abnormal state to a normal state. "Protection" means reducing or avoiding further alterations or damage to cellular structure and / or function caused by the aforementioned endogenous or exogenous stimuli.

[0043] The pharmaceutical products or pharmaceutical compositions disclosed herein can be administered orally, topically, parenterally, or via mucosal routes (e.g., sublingually, by inhalation, or rectally) in dosage units comprising a conventional, non-toxic, pharmaceutically acceptable carrier. Oral administration is generally preferred. The active agent can be administered orally in capsule, tablet, or other similar forms (see Remington: The Science and Practice of Pharmacy, 20th Edition).

[0044] Example 1: The efficacy of the expectorant and blood-activating formula II in treating metabolic-associated fatty liver disease (MASH).

[0045] 1.1 Animal grouping

[0046] Forty-eight 5-week-old SPF male C57BL / 6J mice, weighing 16 - 20 g, were purchased from Shanghai Slack Experimental Animal Co., Ltd. The animal production license number is SCXK(Shanghai)2022 - 0004. They were fed in the Experimental Animal Center of Ningbo Institute of Life and Health Industry, Chinese Academy of Sciences, given a 12-hour light-dark cycle, free access to food and water, and adaptively fed for one week. The mice were randomly divided into a normal group (CON), a low-methionine and choline-deficient high-fat group (CDAHFD), a CDAHFD + pioglitazone group (PIO), a CDAHFD + Qutan Huoxue Formula 1 (ZY), a CDAHFD + Qutan Huoxue Formula 2 low-dose group (QTHXF-L), and a CDAHFD + Qutan Huoxue Formula 2 high-dose group (QTHXF-H), with 8 mice in each group.

[0047] The ingredients of Qutan Huoxue Formula 1 (ZY) include: 20 g of Gynostemma pentaphyllum, 6 g of Rhodiola rosea, 12 g of raw Atractylodes macrocephala, and 9 g of Curcuma longa.

[0048] The ingredients of Qutan Huoxue Formula 2 (QTHXF) include: 20 g of Gynostemma pentaphyllum, 6 g of Rhodiola rosea, 12 g of raw Atractylodes macrocephala, 9 g of Curcuma longa, and 15 g of raw Hawthorn.

[0049] 1.2 Model establishment

[0050] The CON group was fed with a control diet (XTMRCD10-C, with 10% of energy from fat, normal methionine and choline content, Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.), and the remaining 5 groups were fed with a CDAHFD diet (CDAHF60, with 60% of energy from fat, 0.17% methionine, choline-deficient, Wuxi Dietz Biotechnology Co., Ltd.). After 2 weeks, the PIO group was given intragastric administration of 10 mg·kg -1 ·d -1 of pioglitazone; the ZY group was given intragastric administration of traditional Chinese medicine at a dose of 2275 mg·kg -1 ·d -1 of raw Atractylodes macrocephala, -1 ·d -1 1820 mg·kg -1 ·d -1 of Gynostemma pentaphyllum, -1 ·d -1 910 mg·kg -1 ·d -1 of Rhodiola rosea, -1 ·d -1 1365 mg·kg

[0051] 1.3 Experimental results

[0052] 1.3.1 Effects on liver quality and liver index in MASH mice

[0053] like Figure 1 As shown, compared with the CON group mice, the liver weight and liver-to-body ratio of the CDAHFD group mice were significantly increased (P<0.05); compared with the CDAHFD group mice, the liver weight and liver-to-body ratio of the PIO, ZY, QTHXF-L and QTHXF-H groups mice were significantly decreased (P<0.05); compared with the ZY group mice, the liver weight and liver-to-body ratio of the QTHXF-H group mice were significantly decreased (P<0.05).

[0054] 1.3.2 Effects on liver lesions in MASH mice

[0055] like Figure 2 As shown in Figure A, HE staining results revealed that the liver tissue structure of mice in the CON group was normal, with no hepatocyte steatosis or inflammatory cell infiltration. In contrast, the liver tissue of mice in the CDAHFD group showed significant steatosis, with numerous fat droplets in the cytoplasm, scattered inflammatory cell infiltration, and ballooning degeneration. However, the aforementioned pathological changes were significantly reduced in the QTHXF-L, QTHXF-H, and PIO groups. Compared to the ZY group, the QTHXF-H group showed reduced steatosis, inflammatory cell infiltration, and ballooning degeneration in its liver tissue.

[0056] like Figure 2 As shown in Figure B, compared with the CON group mice, the NAS score of liver tissue in the CDAHFD group mice was significantly increased (P<0.05); compared with the CDAHFD group mice, the NAS score of liver tissue in the PIO, ZY, QTHXF-L and QTHXF-H groups mice was significantly decreased (P<0.05); compared with the ZY group mice, the NAS score of liver tissue in the QTHXF-H group mice was significantly decreased (P<0.05).

[0057] 1.3.3 Effects on serum transaminase levels in MASH mice

[0058] like Figure 3 As shown, compared with the CON group mice, the serum ALT and AST activities of the CDAHFD group mice were significantly increased (P<0.05); compared with the CDAHFD group mice, the serum ALT and AST activities of the PIO, ZY, QTHXF-L and QTHXF-H groups mice were significantly decreased (P<0.05); compared with the ZY group mice, the serum ALT activity of the QTHXF-H group mice was significantly decreased (P<0.05).

[0059] 1.3.4 Effects on TG and HYP in liver tissue of MASH mice

[0060] like Figure 4As shown, compared with the CON group mice, the liver tissue TG and HYP content of the CDAHFD group mice was significantly increased (P<0.05); compared with the CDAHFD group mice, the liver tissue TG and HYP content of the PIO, ZY, QTHXF-L and QTHXF-H groups mice was significantly decreased (P<0.05); compared with the ZY group mice, the liver tissue TG and HYP content of the QTHXF-H group mice was significantly decreased (P<0.05).

[0061] Example 2: Key components and target points of the expectorant and blood-activating formula II in the treatment of MASH

[0062] 2.1 Network Pharmacology-Based Approach

[0063] The ingredients of the expectorant and blood-activating formula 2 (QTHXF) include: 20g of Gynostemma pentaphyllum, 6g of Rhodiola rosea, 12g of raw Atractylodes macrocephala, 9g of Curcuma longa, and 15g of raw Crataegus pinnatifida.

[0064] (1) First, the active ingredients in QTHXF were screened using the TCMSP (Traditional Chinese Medicine Systems Pharmacology Database). According to the general standards of network pharmacology, the screening criteria were set as oral bioavailability (OB>20%) and drug-likeness (DL>0.15). Disease-related genes were retrieved from the GeneCards, OMIM, and TTD databases using the keywords "NAFLD" (Non-alcoholic fatty liver disease) and "MAFLD" (Metabolic-dysfunction-associated fatty liver disease). After integrating targets from multiple databases, a disease target set was constructed using R scripts to remove duplicates and take the intersection. Finally, the intersection of the QTHX active ingredient targets and the disease targets was taken to obtain the potential targets for QTHX treatment of MAFLD, as shown in Table 1.

[0065] Table 1. Main active ingredients and number of targets in QTHX formula

[0066]

[0067]

[0068]

[0069] (2) Protein-protein interaction (PPI) analysis was performed on the QTHX target proteins using the STRING database (Search Tool for Retrieval of Interacting Genes / Proteins). A confidence threshold greater than 0.7 was set to exclude isolated nodes, generating a network diagram containing key interaction relationships, as shown below. Figure 5 As shown. The STRING results were imported into Cytoscape software, and the MCODE plugin was used to screen for core genes. The top 10 genes with the highest degree values ​​were selected as the core target proteins for QTHX treatment of MAFLD.

[0070] (3) Based on the above important targets, conduct pathway enrichment analysis, such as... Figure 6 As shown, the above targets were found to be mainly related to the IL-4 and IL-13 inflammatory signaling pathways and the intrinsic pathway for apoptosis. The IL-4 and IL-13 inflammatory signaling pathways play crucial roles in immune regulation, allergic reactions, and inflammation-related diseases, exhibiting both synergistic and specific functions. The IL-4 and IL-13 signaling pathways form a complex regulatory network in allergy, fibrosis, and immune homeostasis through shared receptors and downstream pathways (such as JAK-STAT6). The intrinsic apoptosis pathway responds to cellular stress through mitochondrial signaling, playing a central role in maintaining homeostasis and participating in disease pathology. These results suggest that the QTHX compound has anti-inflammatory and anti-apoptotic biological effects.

[0071] Example 3: The effects and efficacy of the expectorant and blood-activating formula in treating MASH and its liver fibrosis, and its ability to significantly reduce the degree of liver fibrosis and decrease the content of hydroxyproline in liver tissue in MASH mice.

[0072] Single-target drugs are insufficient to simultaneously improve problems such as steatosis, inflammation, and fibrosis. However, the expectorant and blood-activating formula in this application can address both non-alcoholic steatohepatitis (MASH) and related liver fibrosis issues, achieving better therapeutic effects. Specific experiments are as follows:

[0073] 3.1 Animal grouping and model establishment:

[0074] Forty 5-week-old SPF male C57BL / 6J mice, weighing 16 - 20 g, were purchased from Shanghai Slake Experimental Animal Co., Ltd. The animal production license number is SCXK(Shanghai)2022 - 0004. They were fed in the Experimental Animal Center of Ningbo Institute of Life and Health Industry, Chinese Academy of Sciences, given a 12-hour light-dark cycle, free access to food and water, and adaptively fed for one week. The mice were randomly divided into a normal group (CON), a low-methionine and choline-deficient high-fat group (CDAHFD), a CDAHFD + low-dose Qutan Huoxue formula group (QTHXF-L), a CDAHFD + high-dose Qutan Huoxue formula group (QTHXF-H), and a CDAHFD + STING inhibitor group (CDAHFD + ST), with 8 mice in each group. The CON group was fed a normal diet (rodent maintenance diet, product number: 0205SHO228A, Speywood Biotechnology Co., Ltd.), and the other 4 groups were fed a CDAHFD diet (CDAHF60, 60% of energy from fat, 0.17% methionine, choline-deficient, Wuxi Dietz Biotechnology Co., Ltd.). After 3 weeks, the QTHXF-L group was given intragastric administration of 1895.83 mg·kg -1 ·d -1 of the Qutan Huoxue formula; the QTHXF-H group was given intragastric administration of 3791.66 mg·kg -1 ·d -1 of the Qutan Huoxue formula; the STING inhibitor group was intraperitoneally injected with C-176 (15 mg / kg); the other groups were given intragastric administration of an equal amount of normal saline, and the intervention period was 6 weeks.

[0075] The Qutan Huoxue formula group (QTHXF) used Qutan Huoxue formula II, whose ingredients include: 20 g of Gynostemma pentaphyllum, 6 g of Rhodiola rosea, 12 g of Atractylodes macrocephala, 9 g of Curcuma longa, and 15 g of Crataegus pinnatifida.

[0076] 3.1 Experimental results

[0077] As Figure 7 shown, the HE staining results showed that the hepatic tissue structure of the mice in the normal group was normal, without hepatocyte steatosis and inflammatory cell infiltration. The hepatic tissue of the mice in the model group showed obvious steatosis, with a large number of lipid droplets in the cytoplasm, scattered inflammatory cell infiltration and ballooning degeneration. The above pathological changes in the mice of the Qutan Huoxue formula group were significantly alleviated.

[0078] As Figure 8 shown, compared with the mice in the normal group, the NAS score and TG content of the hepatic tissue of the mice in the model group were significantly increased (P < 0.01); compared with the mice in the model group, the NAS score and TG content of the hepatic tissue of the mice in the high- and low-dose Qutan Huoxue formula groups were significantly decreased (P < 0.01).

[0079] Therefore, the results show that the expectorant and blood-activating formula composition of this application can improve liver lipid deposition and inflammation in MASH liver fibrosis mice.

[0080] like Figure 9 As shown, compared with the normal group mice, the liver HYP content, serum AST and ALT activities of the model group mice were significantly increased (P<0.001); compared with the model group mice, the liver HYP content, serum ALT and AST activities of the phlegm-removing and blood-activating formula group mice were significantly decreased (P<0.05).

[0081] like Figure 10 As shown, the Sirius red staining results indicated that collagen fibers were evenly distributed and few in number in the liver tissue of normal mice, with lighter Sirius red staining and less collagen deposition. In the model group, collagen fibers were significantly increased and disordered, with darker Sirius red staining and significantly increased collagen deposition. Compared with the model group, the expectorant and blood-activating formula group had fewer collagen fibers, a more normal distribution, lighter staining, reduced collagen deposition, and a lessened degree of fibrosis.

[0082] The above results show that the expectorant and blood-activating formula composition of this application can improve liver inflammation and liver fibrosis in MASH liver fibrosis mice.

[0083] Example 4: The effect and potential mechanism of the expectorant and blood-activating formula in inhibiting colorectal cancer liver metastasis by improving metabolic-associated fatty liver disease (MAFLD). The therapeutic effect of the expectorant and blood-activating formula has been confirmed in MAFLD mouse experiments.

[0084] 4.1 Animal Experiments

[0085] Fifty SPF-grade C57BL / 6J mice were randomly divided into a control group and a model group. The control group was fed a diet with 10% fat content, while the model group was fed a diet with 60% high fat content to establish a MAFLD model. At week 10, the model group mice were randomly divided into a placebo group, a low-dose group of a traditional Chinese medicine formula for phlegm removal and blood circulation, a high-dose group of a traditional Chinese medicine formula for phlegm removal and blood circulation, and a metformin group for intervention. The intervention was performed by gavage for 2 weeks. At week 12, 5 mice from each group were sacrificed, and the livers were isolated. The liver pathology was observed by HE, Masson, and Oil Red O staining. The distribution of immune cells in the liver was detected by flow cytometry, and the expression of T cells and dendritic cells-related cytokines was detected by ELISA and qPCR. The remaining mice were used to establish a MAFLD-colorectal cancer liver metastasis (MAFLD-CRLM) model by injecting MC38 cells into the spleen. The pathological condition of colorectal tumor liver metastasis was observed after week 15.

[0086] The expectorant and blood-activating formula uses Expectorant and Blood-Activating Formula Two, whose components include: 20g of Gynostemma pentaphyllum, 6g of Rhodiola rosea, 12g of raw Atractylodes macrocephala, 9g of Curcuma longa, and 15g of raw Crataegus pinnatifida.

[0087] 4.2 Results

[0088] Compared with the control group, MAFLD model mice showed increased colorectal cancer liver metastasis (P < 0.01); the immune cell pattern in the liver of MAFLD mice was significantly remodeled, including CD8... + T cells and dendritic cells were significantly reduced (P < 0.01), and the secretion of related cytokines IFN-γ, MHC II, CCR7, and CXCL9 was also reduced (P < 0.05). The expectorant and blood-activating formula significantly inhibited liver metastasis of colorectal cancer in MAFLD mice, reduced the expression levels of fasting blood glucose, serum and liver TC, TG, AST, and ALT in MAFLD mice (P < 0.05), promoted the expression of IFN-γ, MHC II, CCR7, and CXCL9 (P < 0.05), and significantly increased liver CD8+. + Infiltration and maturation of T cells and dendritic cells (P < 0.05).

[0089] The expectorant and blood-activating formula can inhibit liver metastasis of colorectal cancer in MAFLD mice, and its mechanism may be related to increasing CD8. + T cell and dendritic cell infiltration and maturation are related to the remodeling of the liver's immune microenvironment.

[0090] Example 5: Key components and target points of the expectorant and blood-activating formula II

[0091] The expectorant and blood-activating formula II (QTHXF) developed in this application includes the following ingredients: 20g of Gynostemma pentaphyllum, 6g of Rhodiola rosea, 12g of raw Atractylodes macrocephala, 9g of Curcuma longa, and 15g of raw Crataegus pinnatifida. Based on the traditional Chinese medicine compatibility theories of "principal, assistant, adjuvant, and guide" and "harmony of the seven emotions," this application studied Chinese herbs with anti-inflammatory, anti-apoptotic, and fibrosis-reversing effects. Furthermore, through the TCMSP database, it identified rutin as an effective ingredient that can exert synergistic effects with multiple drug components (including Gynostemma pentaphyllum, Crataegus pinnatifida, and Rhodiola rosea) (see details). Figure 11 Rutin has a wide range of pharmacological effects, mainly in the following aspects: (1) it achieves anti-inflammatory effects by regulating inflammatory responses through multiple targets; (2) it achieves anti-apoptotic effects by protecting cell survival; and (3) it achieves the effect of preventing lipid deposition by improving metabolic homeostasis.

[0092] In the description of this specification, the terms "one embodiment," "some embodiments," "specific embodiment," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0093] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A formula composition for relieving phlegm and promoting blood circulation, characterized in that, The composition includes Gynostemma pentaphyllum and hawthorn as principal ingredients, and Atractylodes macrocephala, Rhodiola rosea and turmeric as adjuvant ingredients.

2. The expectorant and blood-activating formula composition according to claim 1, characterized in that, By weight, the composition comprises 10-30 parts of Gynostemma pentaphyllum and 10-30 parts of hawthorn as principal ingredients, and 4-10 parts of Rhodiola rosea, 8-16 parts of Atractylodes macrocephala and 4-12 parts of turmeric as adjuvant ingredients.

3. The expectorant and blood-activating formula composition according to any one of claims 1 or 2, characterized in that, By weight, the composition comprises 20 parts of Gynostemma pentaphyllum and 15 parts of hawthorn as principal ingredients, and 6 parts of Rhodiola rosea, 12 parts of Atractylodes macrocephala and 9 parts of turmeric as adjuvant ingredients.

4. The use of the expectorant and blood-activating formula composition according to any one of claims 1 to 3 in the preparation of a medicament for treating lipid deposition and inflammation, or for improving fibrosis.

5. The use of the expectorant and blood-activating formula composition according to any one of claims 1 to 3 in the preparation of a medicament for treating metabolic-related fatty liver disease and liver fibrosis.

6. The method for screening therapeutic targets of the expectorant and blood-activating formula composition according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Screen the active ingredients in the expectorant and blood-activating formula composition through the TCMSP database, and summarize the target points of the active ingredients in the expectorant and blood-activating formula. S2. Based on the active ingredients obtained in step S1, set screening conditions and search for disease-related genes in the GeneCards, OMIM and TTD databases using disease keywords as targets to be identified. S3. After integrating the target points to be determined in step S2, remove duplicates and take the intersection to obtain the disease target points. S4. Combine the target of the active ingredient of the expectorant and blood-activating formula in step S1 with the disease target in step S3 and take the intersection to obtain the therapeutic target of the expectorant and blood-activating formula.

7. The method for screening therapeutic targets of the expectorant and blood-activating formula composition according to claim 6, characterized in that, In step S2, the disease keywords are non-alcoholic fatty liver disease or metabolic-associated fatty liver disease.

8. The method for screening therapeutic targets of the expectorant and blood-activating formula composition according to claim 6, characterized in that, In step S3, an R language script is used to remove duplicates and find the intersection.

9. A method for enriching rutin, characterized in that, The phlegm-reducing and blood-activating formula composition as described in any one of claims 1 to 3 is used.

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