Application of traditional Chinese medicine composition in preparation of medicine for preventing hepatic fibrosis

Abstract

The invention provides an application of a traditional Chinese medicine composition in preparing a medicine for preventing hepatic fibrosis. The traditional Chinese medicine composition is prepared from the following components in parts by weight: 128 to 137 parts of radix scutellariae, 128 to 137 parts of radix bupleuri, 103 to 120 parts of radix et rhizoma rhei, 128 to 137 parts of fructus aurantii, 128 to 137 parts of herba artemisiae scopariae, 171 to 200 parts of rhizoma polygoni cuspidati, 137 to 150 parts of fructus gardeniae, 250 to 342 parts of herba lysimachiae, 128 to 137 parts of radix paeoniae alba, 128 to 137 parts of radix aucklandiae, 90 to 103 parts of rhizoma pinelliae preparata and 34 to 40 parts of rhizoma zingiberis recens. The traditional Chinese medicine composition can obviously reduce the expression level of alpha-SMA and TGF-beta in the liver and relieve the occurrence of hepatic fibrosis, and can be used for preventing hepatic fibrosis.

Description

Technical Field

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

[0002] Liver fibrosis refers to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Under normal circumstances, the expression levels of α-SMA and TGF-β in fibrotic livers are significantly higher than those in healthy livers.

[0003] With the improvement of people's living standards, the variety of food has become more and more abundant, and the intake of high-fat and high-cholesterol foods has also increased. As a result, health problems have become more and more serious, with liver fibrosis being a common one that seriously affects people's health.

[0004] Chinese patent CN115429866A discloses a traditional Chinese medicine composition for treating cholecystitis and its preparation method. The components of this composition include: Scutellaria baicalensis, Bupleurum chinense, Rheum palmatum, Citrus aurantium, Artemisia capillaris, Polygonum cuspidatum, Gardenia jasminoides, Lysimachia christinae, Paeonia lactiflora, Aucklandia lappa, Pinellia ternata, and Zingiber officinale. The traditional Chinese medicine preparation made from this composition can be used to treat chronic cholecystitis. As an innovative traditional Chinese medicine, the composition described in this patent has been the subject of continuous and in-depth research, yielding many unexpected results. Summary of the Invention

[0005] The purpose of this invention is to provide new uses for the traditional Chinese medicine composition disclosed in Chinese Patent CN115429866A.

[0006] To achieve the above objectives, the inventors have provided the following technical solutions.

[0007] The application of a traditional Chinese medicine composition in the preparation of a drug for preventing liver fibrosis, wherein the raw materials of the traditional Chinese medicine composition are, by weight, 128-137 parts of Scutellaria baicalensis, 128-137 parts of Bupleurum chinense, 103-120 parts of Rheum palmatum, 128-137 parts of Citrus aurantium, 128-137 parts of Artemisia capillaris, 171-200 parts of Polygonum cuspidatum, 137-150 parts of Gardenia jasminoides, 250-342 parts of Lysimachia christinae, 128-137 parts of Paeonia lactiflora, 128-137 parts of Aucklandia lappa, 90-103 parts of Pinellia ternata, and 34-40 parts of Zingiber officinale.

[0008] In the above applications, the preferred raw material composition of the traditional Chinese medicine composition by weight is: 128 parts of Scutellaria baicalensis, 128 parts of Bupleurum chinense, 120 parts of Rheum palmatum, 128 parts of Citrus aurantium, 128 parts of Artemisia capillaris, 200 parts of Polygonum cuspidatum, 150 parts of Gardenia jasminoides, 250 parts of Lysimachia christinae, 128 parts of Paeonia lactiflora, 128 parts of Aucklandia lappa, 90 parts of Pinellia ternata, and 40 parts of Zingiber officinale.

[0009] In the above applications, the raw material composition of the traditional Chinese medicine composition, by weight, can preferably be: 137 parts of Scutellaria baicalensis, 137 parts of Bupleurum chinense, 103 parts of Rheum palmatum, 137 parts of Citrus aurantium, 137 parts of Artemisia capillaris, 171 parts of Polygonum cuspidatum, 137 parts of Gardenia jasminoides, 342 parts of Lysimachia christinae, 137 parts of Paeonia lactiflora, 137 parts of Aucklandia lappa, 103 parts of Pinellia ternata, and 34 parts of Zingiber officinale.

[0010] In the above applications, the preparation method of the traditional Chinese medicine composition includes the following steps:

[0011] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0012] B. Weigh out the bitter orange peel and fresh ginger, add 5-9 times the amount of water, extract the volatile oil for 8-12 hours, collect and separate the volatile oil; the distilled aqueous solution is for later use.

[0013] C. Weigh out Scutellaria baicalensis, Paeonia lactiflora, Artemisia capillaris, and Gardenia jasminoides. Add water and decoct 2-4 times. Extract for 1-3 hours for the first time, and for 1-3 hours for the second, third, and fourth times respectively. Add 7-10 times the amount of water each time. Filter the extract and combine it with the volatile oil-water extract obtained in step B. Concentrate under reduced pressure to a relative density of 1.25±0.05 at 60℃ for later use.

[0014] D. Weigh out Bupleurum, Aucklandia, Rhubarb, Polygonum cuspidatum, and Lysimachia christinae. Extract with 60-80% ethanol 2-4 times. For the first extraction, add 10-14 times the amount of ethanol and extract for 2-4 hours. For the second, third, and fourth extractions, add 8-12 times the amount of ethanol and extract for 1-3 hours each time. Filter the extract, concentrate it into a clear paste, combine it with the water extract obtained in step C, mix well, dry, and pulverize for later use.

[0015] E. The fine powder obtained in step A, the volatile oil obtained in step B, and the dried powder obtained in step D can be mixed together.

[0016] The traditional Chinese medicine composition described in this application can be prepared into various drug dosage forms as needed, including capsules, tablets, pills, oral liquids, granules, or powders.

[0017] In the above applications, the liver fibrosis is preferably caused by a high-fat, high-cholesterol diet.

[0018] In the above applications, a high-fat, high-cholesterol diet refers to a daily diet with a fat content ≥15% and a cholesterol content ≥1.25%.

[0019] In the above applications, liver fibrosis is characterized by increased expression levels of α-SMA and / or TGF-β in the liver compared to healthy livers.

[0020] Experimental studies have shown that the traditional Chinese medicine composition provided by this invention exhibits unexpected effects in preventing liver fibrosis. When mice fed a high-fat, high-cholesterol diet were given the traditional Chinese medicine composition of this invention, the fluorescence intensity (expression level) of α-SMA and TGF-β in the liver of the mice was significantly reduced, and the incidence of liver fibrosis in the high-fat, high-cholesterol diet mice was alleviated. This fully demonstrates that the traditional Chinese medicine composition of this invention is suitable for the prevention of liver fibrosis, especially for the prevention of liver fibrosis caused by a high-fat, high-cholesterol diet, with significant effects. Attached Figure Description

[0021] Figure 1 The images show Masson staining of the livers of mice in each experimental group in the experimental case.

[0022] Figure 2 The results of fluorescence experiments on α-SMA and TGF-β in the livers of mice in each experimental group in the experimental examples are shown.

[0023] Figure 3 The images show immunofluorescence staining of liver sections from mice in each group of the experimental cases. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments.

[0025] Example 1: Preparation of capsules containing traditional Chinese medicine composition

[0026] The raw material formula is as follows: Scutellaria baicalensis 128g, Bupleurum chinense 128g, Rheum palmatum 120g, Citrus aurantium 128g, Artemisia capillaris 128g, Polygonum cuspidatum 200g, Gardenia jasminoides 150g, Lysimachia christinae 250g, Paeonia lactiflora 128g, Aucklandia lappa 128g, Pinellia ternata 90g, and Zingiber officinale 40g.

[0027] Preparation process:

[0028] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0029] B. Weigh out the bitter orange peel and fresh ginger, add 7 times the amount of water, extract the volatile oil for 10 hours, collect and separate the volatile oil; the distilled aqueous solution is for later use.

[0030] C. Weigh out Scutellaria baicalensis, Paeonia lactiflora, Artemisia capillaris, and Gardenia jasminoides. Add water and decoct three times. The first extraction takes 2 hours, and the second and third extractions take 1.5 hours each time. Add 9 times the amount of water each time. Filter the extract and combine it with the volatile oil-water extract obtained in step B. Concentrate under reduced pressure to a relative density of 1.25±0.05 (60℃) to obtain an extract for later use.

[0031] D. Weigh out Bupleurum, Aucklandia, Rhubarb, Polygonum cuspidatum, and Lysimachia christinae. Extract them three times with 70% ethanol. For the first extraction, add 12 times the amount of ethanol and extract for 2.5 hours. For the second and third extractions, add 10 times the amount of ethanol and extract for 2 hours each. Filter the extract, concentrate it into a clear paste, combine it with the water extract obtained in step C, mix well, dry it, and pulverize it for later use.

[0032] E. Mix the fine powder obtained in step A and the extract powder obtained in step D. Adsorb the volatile oil obtained in step B with silica and mix well. Then, fill the mixture into capsules.

[0033] Example 2: Preparation of capsules containing traditional Chinese medicine composition

[0034] The raw material formula is as follows: Scutellaria baicalensis 137g, Bupleurum chinense 137g, Rheum palmatum 103g, Citrus aurantium 137g, Artemisia capillaris 137g, Polygonum cuspidatum 171g, Gardenia jasminoides 137g, Lysimachia christinae 342g, Paeonia lactiflora 137g, Aucklandia lappa 137g, Pinellia ternata 103g, and Zingiber officinale 34g.

[0035] Preparation process:

[0036] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0037] B. Weigh out the bitter orange peel and fresh ginger, add 9 times the amount of water, extract the volatile oil for 12 hours, collect and separate the volatile oil; the distilled aqueous solution is for later use.

[0038] C. Weigh out Scutellaria baicalensis, Paeonia lactiflora, Artemisia capillaris, and Gardenia jasminoides. Add water and decoct 14 times. The first extraction takes 13 hours, and the second and third extractions take 11 hours each. Add 110 times the amount of water each time. Filter the extract and combine it with the volatile oil-water extract obtained in step B. Concentrate under reduced pressure to obtain an extract with a relative density of 1.25±0.05 (60℃) for later use.

[0039] D. Weigh out Bupleurum, Aucklandia, Rhubarb, Polygonum cuspidatum, and Lysimachia christinae. Extract them three times with 80% ethanol. For the first extraction, add 12 times the amount of ethanol and extract for 2 hours. For the second and third extractions, add 10 times the amount of ethanol and extract for 2 hours each. Filter the extract, concentrate it into a clear paste, combine it with the water extract obtained in step C, mix well, dry it, and pulverize it for later use.

[0040] E. Mix the fine powder obtained in step A and the extract powder obtained in step D, dry them, mix them with the volatile oil obtained in step B, and then fill them into capsules.

[0041] Example 3: Preparation of the Traditional Chinese Medicine Composition

[0042] Raw material formula: Scutellaria baicalensis 150g, Bupleurum chinense 70g, Rheum palmatum 90g, Citrus aurantium 90g, Artemisia capillaris 150g, Polygonum cuspidatum 100g, Gardenia jasminoides 110g, Lysimachia christinae 500g, Paeonia lactiflora 80g, Aucklandia lappa 200g, Pinellia ternata 110g, Zingiber officinale 20g.

[0043] Preparation process:

[0044] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0045] B. Weigh out the bitter orange peel and ginger, add 5-9 times the amount of water, extract the volatile oil for 12 hours, collect and separate the volatile oil; the distilled aqueous solution is for later use.

[0046] C. Weigh out Scutellaria baicalensis, Paeonia lactiflora, Artemisia capillaris, and Gardenia jasminoides. Add water and decoct twice. Extract for 3 hours the first time, and for 3 hours the second and third times respectively. Add 7 times the amount of water each time. Filter the extract and combine it with the volatile oil-water extract obtained in step B. Concentrate under reduced pressure to a relative density of 1.25±0.05 (60℃) to obtain an extract for later use.

[0047] D. Weigh out Bupleurum, Aucklandia, Rhubarb, Polygonum cuspidatum, and Lysimachia christinae. Extract twice with 80% ethanol. For the first extraction, add 14 times the amount of ethanol and extract for 2 hours. For the second and third extractions, add 12 times the amount of ethanol and extract for 2 hours each. Filter the extract, concentrate it into a clear paste, combine it with the water extract obtained in step C, mix well, dry, and pulverize for later use.

[0048] E. The fine powder obtained in step A, the volatile oil obtained in step B, and the dried powder obtained in step D together constitute the active components of the pharmaceutical composition of the present invention.

[0049] Example 4: Preparation of Traditional Chinese Medicine Composition Tablets

[0050] Raw material formula: Scutellaria baicalensis 69g, Bupleurum chinense 205g, Rheum palmatum 52g, Citrus aurantium 205g, Artemisia capillaris 69g, Polygonum cuspidatum 255g, Gardenia jasminoides 69g, Lysimachia christinae 515g, Paeonia lactiflora 69g, Aucklandia lappa 205g, Pinellia ternata 52g, Zingiber officinale 17-52g.

[0051] Preparation process:

[0052] A. Weigh out Pinellia ternata, grind it into a fine powder, sterilize it by 60Co irradiation, and set aside for later use;

[0053] B. Weigh out the bitter orange peel and ginger, add 5 times the amount of water, extract the volatile oil for 10 hours, collect and separate the volatile oil; the distilled aqueous solution is for later use.

[0054] C. Weigh out Scutellaria baicalensis, Paeonia lactiflora, Artemisia capillaris, and Gardenia jasminoides. Add water and decoct 4 times. The first extraction takes 2 hours, and the second and third extractions take 1 hour each. Add 10 times the amount of water each time. Filter the extract and combine it with the volatile oil-water extract obtained in step B. Concentrate under reduced pressure to a relative density of 1.25±0.05 (60℃) to obtain an extract for later use.

[0055] D. Weigh out Bupleurum, Aucklandia, Rhubarb, Polygonum cuspidatum, and Lysimachia christinae. Extract them three times with 60% ethanol. For the first extraction, add 10 times the amount of ethanol and extract for 3 hours. For the second and third extractions, add 8 times the amount of ethanol and extract for 2 hours each. Filter the extract, concentrate it into a clear paste, combine it with the water extract obtained in step C, mix well, dry it, and pulverize it for later use.

[0056] E. Mix the fine powder obtained in step A and the extract powder obtained in step D, dry them, mix them with the volatile oil obtained in step B, and make them into tablets according to conventional methods.

[0057] Example 5: Preparation of Traditional Chinese Medicine Composition Granules

[0058] Raw material formula: Scutellaria baicalensis 150g, Bupleurum chinense 150g, Rheum palmatum 80g, Citrus aurantium 150g, Artemisia capillaris 90g, Polygonum cuspidatum 200g, Gardenia jasminoides 90g, Lysimachia christinae 400g, Paeonia lactiflora 90g, Aucklandia lappa 150g, Pinellia ternata 80g, Zingiber officinale 40g.

[0059] Preparation process:

[0060] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0061] B. Weigh out the bitter orange peel and ginger, add 9 times the amount of water, extract the volatile oil for 10 hours, collect and separate the volatile oil; the distilled aqueous solution is for later use.

[0062] C. Weigh out Scutellaria baicalensis, Paeonia lactiflora, Artemisia capillaris, and Gardenia jasminoides. Add water and decoct 4 times. The first extraction takes 3 hours, and the second and third extractions take 1 hour each. Add 7 times the amount of water each time. Filter the extract and combine it with the volatile oil-water extract obtained in step B. Concentrate under reduced pressure to a relative density of 1.25±0.05 (60℃) to obtain an extract for later use.

[0063] D. Weigh out Bupleurum, Aucklandia, Rhubarb, Polygonum cuspidatum, and Lysimachia christinae. Extract twice with 80% ethanol. For the first extraction, add 10 times the amount of ethanol and extract for 2 hours. For the second and third extractions, add 10 times the amount of ethanol and extract for 1 hour each. Filter the extract, concentrate it into a clear paste, combine it with the water extract obtained in step C, mix well, dry, and pulverize for later use.

[0064] E. Mix the fine powder obtained in step A and the extract powder obtained in step D, dry them, mix them with the volatile oil obtained in step B, granulate and arrange them to obtain granules.

[0065] Experimental Example

[0066] The inventors conducted an experimental investigation on the effects of the traditional Chinese medicine composition of this invention on liver fibrosis in mice fed a high-fat, high-cholesterol diet.

[0067] 1 Experimental Institution

[0068] Hainan Provincial Institute for Drug Control (Safety Evaluation Research Center)

[0069] 2 Test Substances, Reference Substances and Other Reagents

[0070] 2.1 Test Substances

[0071] 2.1.1 Name: The traditional Chinese medicine composition of the present invention (hereinafter referred to as CH) (prepared according to the formula and method provided in Example 1);

[0072] 2.1.2 Code: 22029DE-03;

[0073] 2.1.3 Source: Provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd.;

[0074] 2.1.4 Specification: Each capsule contains 0.46 g (equivalent to 1.71 g of cut crude drugs);

[0075] 2.1.5 Batch Number: A2112001;

[0076] 2.1.6 Appearance: The contents of the hard capsules are brownish-yellow granules and powders, with a fragrant smell and a slightly bitter taste;

[0077] 2.1.7 Packaging: 0.46 g * 12 capsules * 3 plates / bag;

[0078] 2.1.8 Production Date: December 27, 2021;

[0079] 2.1.9 Expiry Date: November 2024;

[0080] 2.1.10 Storage Conditions: Sealed;

[0081] 2.1.11 Quality Inspection Results: According to the finished product inspection report provided by the consignor (Report No.: BP Cheng 111 (Report) A2112001), the inspection items such as the appearance, identification, inspection, and content determination of this batch of test substances all meet the regulations.

[0082] 2.2 Reference Substances

[0083] Ursodeoxycholic acid (UDCA) capsules (250 mg / capsule; Batch Number: L19185A) were purchased from Losan Pharma GmbH, Germany.

[0084] 3 Solvents for Experiments

[0085] 3.1 Name: Sodium carboxymethylcellulose

[0086] 3.1.1 Source: Xilong Science Co., Ltd.;

[0087] 3.1.2 Specification: 250 g;

[0088] 3.1.3 Batch Number: 2101110;

[0089] 3.1.4 Appearance: White or slightly yellow cellulose-like powder, odorless and tasteless;

[0090] 3.1.5 Uses: To prepare a 0.5% sodium carboxymethyl cellulose solution as a solvent.

[0091] 4. Experimental System and Reasons for Selection

[0092] 4.1 Experimental system: Adult C57BL / 6J mice.

[0093] 4.2 Reason for selection: According to relevant literature, a diet containing 15% fat, 1.25% cholesterol and 0.5% bile acid can simulate a high-fat, high-cholesterol diet in humans.

[0094] 5. The strain, number, age, sex, weight range, origin, and grade of laboratory animals.

[0095] 5.1 Animal strain: C57BL / 6J mouse.

[0096] 5.2 Number of experimental animals: 180 animals are needed for the experiment, and 200 animals will be purchased.

[0097] 5.3 Sex of experimental animals: male.

[0098] 5.4 Age or weight of experimental animals: Purchased animals were approximately 7 weeks old and weighed 20-24g; at the start of the experiment, the animals were 8 weeks old and weighed 22-26g.

[0099] 5.5 Source of experimental animals: Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., production license number SCXK(Su)2018-0008.

[0100] 5.6 Laboratory animal grade: SPF grade.

[0101] 5.7 Animal Quality Certificate No.: 320727230100015454.

[0102] 5.8 Handling of remaining animals: After grouping, the remaining mice were used for training operations or other experimental projects.

[0103] 6. Reception and Quarantine of Laboratory Animals

[0104] The research project leader completed the "Application Form for Ethical Review of Laboratory Animal Welfare." After review by members of the Laboratory Animal Management and Use Committee (IACUC), and upon approval, the animals and feed were ordered and received according to the center's SOPs: "Ordering of Laboratory Animals and Feed," "Receiving and Placement of Laboratory Animals," and "Receiving, Storage, Issuance, and Use of Feed." The mice used in this experiment were placed in the laboratory on January 11, 2023, and underwent a 3-day quarantine period (from January 11 to January 13, 2023). During the quarantine period, the animals were observed daily, including: nutritional status, mental state, feed intake, limbs, feces, urine, fur, body surface, eyes, nose, mouth, anus, vulva, and any signs of death. These observations were recorded accurately. Animal weight was measured on the first and last days of quarantine. After passing quarantine, the mice could be used for experiments. The quarantine numbers for the mice used in this experiment ranged from 001 to 200. All mice passed quarantine and were deemed safe for use in experiments.

[0105] 7. Methods for identifying laboratory animals

[0106] According to the research center's SOP "Animal Marking and Group Numbering," the cages were marked, and labels were affixed to the cages indicating the topic code, animal species, cage number, animal number, and experiment date. After mice entered the quarantine room, their tails were marked with an oil-based marker, using numbers such as 001, 002, ..., 200 to represent the initial quarantine number. The markings should be clear and not easily confused; if blurred, they should be remarked promptly. For formal experiments, animal numbers consist of the group, sex, cage number, and animal number per cage. Groups are represented and recorded as I, II, III, etc. Each group's cage number is recorded as 01, 02, 03...36, and so on. Females are represented as F, and males as M. This experiment consisted of 6 groups: normal control group (CK), model control group (LD), low-dose test product group (LD+CH-L), medium-dose test product group (LD+CH-M), high-dose test product group (LD+CH-H), and UDCA positive control group (LD+UDCA), with 30 animals in each group. The experimental groups and animal numbers are shown in Table 1.

[0107] Table 1. Experimental Groups and Corresponding Cage Numbers and Animal Numbers

[0108]

[0109] 8. Environmental conditions for the husbandry and management of laboratory animals

[0110] 8.1 Animal housing room: This experiment was approved by the Animal Ethics Committee of Hebei Yiling Institute of Traditional Chinese Medicine (approval number: YL-IACUC-2023-004) and was carried out in accordance with the "Guide for the Care and Use of Laboratory Animals". The animal laboratory in the research center has the license number for the use of experimental animals: SYXK(Qiong)2021-0009. During the quarantine period, the animals were housed in the quarantine isolation room in the barrier area from January 11, 2023 to January 13, 2023; during the experimental period, they were housed in Laboratory 1 in the barrier area from January 17, 2023 to March 13, 2023. According to the requirements of the "National Standard of the People's Republic of China: Laboratory Animal Environment and Facilities" (GB 14925-2010): The temperature requirement is 20-26°C, which is set at 20-26°C (the actual temperature ranges in the quarantine isolation room and Laboratory 1 in the barrier area are 21.8-24.2°C and 20.3-24.5°C respectively), the daily temperature difference ≤ 4°C, the humidity is 40-70% (the actual humidity ranges in the quarantine isolation room and Laboratory 1 in the barrier area are 54.9-61.5% and 52.7-67.4% respectively), the number of air changes is required to be ≥ 15 times / h, the animal illumination is 15-20 lx, and the lighting time is 12h / 12h, with alternating light and darkness. The temperature, relative humidity, and pressure in the animal laboratory are automatically recorded, and recorded automatically once every hour. In addition, for indicators such as the temperature, relative humidity, pressure, illuminance, noise, air velocity, number of air changes, dust particles, and number of airborne bacteria in the animal laboratory, in accordance with the requirements of GB 14925-2010, a qualified unit is entrusted to monitor once a year, and the results all meet the requirements of the corresponding environmental grade.

[0111] 8.2 Environmental grade: Barrier system (SPF level).

[0112] 8.3 Cages: Composed of a cage cover and a cage feeding box. The cage cover is made of stainless steel, and the cage feeding box is a CP-3 type transparent plastic mouse cage. The cage cover is replaced once a month, the feeding box is replaced every Friday, and the bedding is replaced on Mondays, Wednesdays, and Fridays. It is carried out according to the research center's SOP "Replacement of bedding, pads, and cages". The replaced feeding boxes and cage covers are cleaned, stored, and sterilized according to the center's SOP "Cleaning, storage, and sterilization of cages".

[0113] 8.4 Bedding: Corn cob is used as bedding and is sterilized by high temperature and high pressure. The research center entrusts a qualified unit to detect heavy metals Pb, microorganisms, and aflatoxin B1 every year, and they all meet the relevant regulations.

[0114] 8.5 Feed: High-fat and high-cholesterol feed (containing 15% fat, 1.25% cholesterol, and 0.5% cholic acid).

[0115] 8.5.1 Source: Guangdong Provincial Center for Experimental Animals (Guangzhou, China).

[0116] 8.5.2 Batch number: 20221133.

[0117] 8.5.3 Shelf life: nine months.

[0118] 8.5.4 Production date: 2022.11.24.

[0119] 8.5.5 Valid until: August 23, 2023.

[0120] 8.5.6 Production License No.: Yue Feed Certificate (2019) 05073.

[0121] 8.6 Drinking Water: Sterilized tap water is provided for animals to drink freely. Water bottles are changed daily, and the bottles are cleaned and reused after each use. The process follows the research center's SOP "Bottling, Sterilization, and Testing of Drinking Water," and the water undergoes high-temperature and high-pressure sterilization before use. The research center sends drinking water samples annually to a qualified institution for physicochemical and microbiological testing according to the "Standard Examination Methods for Drinking Water" (GB / T 5750-2006). All results meet the limits set by the "Standards for Drinking Water Quality" (GB 5749-2006).

[0122] 8.7 Husbandry: Five animals are housed per cage, in accordance with the requirements of "Laboratory Animal Environment and Facilities" (GB14925-2010). During quarantine, standby observation and experimentation, animals are fed once daily according to the center's SOP "Husbandry of Mice and Rats".

[0123] 9 Experimental Methods

[0124] 9.1 Grouping and Dosage Design

[0125] 9.1.1 Animal Grouping

[0126] This experiment consisted of 6 groups: normal control group (CK), model control group (LD), low-dose test product group (LD+CH-L), medium-dose test product group (LD+CH-M), high-dose test product group (LD+CH-H), and UDCA positive control group (LD+UDCA). 180 qualified mice were randomly divided into the above 6 groups according to their body weight, with 30 mice in each group. The experimental groups and animal numbers are shown in Table 1.

[0127] Normal control group: fed standard mouse growth and reproduction diet and administered solvent by gavage for 8 weeks;

[0128] LD group: fed high-fat, high-cholesterol diet and given solvent by gavage for 8 weeks;

[0129] LD+CH-L group (0.345g powder / kg): fed with high-fat, high-cholesterol diet and administered low dose of test product by gavage for 8 weeks;

[0130] LD+CH-M group (0.69 g powder / kg): fed with high-fat, high-cholesterol diet and administered a medium dose of the test product by gavage for 8 weeks;

[0131] LD+CH-H group (1.38g powder / kg): fed with high-fat, high-cholesterol diet and administered high doses of the test product by gavage for 8 weeks;

[0132] LD+UDCA group (123.3 mg / kg): fed with high-fat, high-cholesterol diet and administered control product by gavage for 8 weeks.

[0133] 9.1.2 Dosage Design and Basis

[0134] 9.1.2.1 Test sample dosage design

[0135] Based on the clinical usage and dosage of the traditional Chinese medicine composition in this application and the previous pharmacodynamic test data, the low, medium and high dose groups in this experiment are consistent with the previous pharmacodynamic doses, with the dosages being 1.282, 2.566 and 5.132 g crude drug / kg (including the dosage of the preparation being 0.345, 0.69 and 1.38 g powder / kg, which are equivalent to 5, 10 and 20 times the clinical dose, respectively).

[0136] 9.2 Drug Preparation

[0137] 9.2.1 Preparation of the test sample

[0138] High dose of test sample: Weigh 4.14g of drug powder, add 0.5% sodium carboxymethyl cellulose solution and stir well to prepare 30ml of high dose test sample solution with a final concentration of 0.138g / ml. Place the prepared solution in a clean preparation bottle.

[0139] Medium dose of test sample: Measure 15 ml of the high dose of test sample solution (final concentration of 0.138 g / ml), and after measuring, add 15 ml of 0.5% sodium carboxymethyl cellulose solution to dilute to a final concentration of 0.069 g / ml. Place the prepared solution in a clean preparation bottle.

[0140] Low-dose test sample: Measure 15 ml of the medium-dose test sample solution (final concentration 0.069 g / ml), add 15 ml of 0.5% sodium carboxymethyl cellulose solution to dilute to a final concentration of 0.0345 g / ml, and place the prepared solution in a clean preparation bottle.

[0141] Each time, the sample is taken according to the required amount, and it is prepared and mixed in the clean bench before use.

[0142] 9.2.2 Preparation of 0.5% sodium carboxymethyl cellulose solution

[0143] Weigh 1g of sodium carboxymethyl cellulose into a 500ml clean beaker. Add 200ml of sterile water to the beaker using a graduated cylinder, and stir thoroughly to prepare a 0.5% sodium carboxymethyl cellulose solution. Then transfer the prepared solution to a clean dispensing bottle.

[0144] 9.3 Administration Method

[0145] The normal control group was fed standard mouse feed, while the model control group, the low-, medium-, and high-dose test sample groups, and the positive control group were fed a high-fat, high-cholesterol diet. All animals had normal access to water. Administration was via gavage, once daily for 8 consecutive weeks. The normal control and model control groups received 0.5% sodium carboxymethyl cellulose solution, the low-, medium-, and high-dose groups received different concentrations of the test sample solution, and the positive control group received the corresponding concentration of the control. The gavage volume per mouse was 0.1 ml / 10g (10 ml / kg).

[0146] 10. Detection items, methods, and frequencies

[0147] 10.1 Clinical Observation

[0148] During the trial, the animals were observed for general clinical symptoms at least once a day, and their health status was observed and recorded.

[0149] 10.2 Weight Record

[0150] The weight of all mice was recorded before the experiment began. The weight of all mice was measured weekly, and the trends in weight changes were observed and recorded. Finally, a trend graph was generated for analysis.

[0151] 10.3 Specimen Collection

[0152] All mice were harvested 24 hours after the last administration of the drug at the end of the eighth week of the experiment.

[0153] 10.4 Collection of liver tissue and determination of related indicators

[0154] 10.4.1 Collection of liver tissue

[0155] The liver was rapidly removed, and surrounding tissues were trimmed. The liver was washed with ice-cold 0.9% saline, blotted dry, and weighed. A portion of the tissue was kept fresh and frozen in liquid nitrogen for molecular biological assays. Another portion was fixed in 4% neutral buffered formaldehyde for histological analysis.

[0156] 10.4.2 Liver Histological Analysis

[0157] Liver tissue was fixed in 4% neutral buffered formaldehyde, embedded in paraffin, cut into 4μm thick sections, stained with Masson's trichrome, and observed under an optical microscope.

[0158] 10.4.3 Immunofluorescence analysis

[0159] Liver tissue sections were blocked with 5% bovine serum albumin in phosphate-buffered saline at room temperature for 1 hour, then incubated with α-SMA and TGF-β antibodies overnight at 4°C. The sections were then incubated with secondary antibodies at room temperature in the dark for 2 hours. The tissue sections were then placed in PBS and rinsed three times (10 min each time) on a shaker in the dark. After incubation with DAPI for 10 minutes, the sections were mounted with anti-quenching mounting medium and air-dried in the dark. The results were observed under a fluorescence microscope, photographed, and analyzed.

[0160] Transforming growth factor-β1 (TGF-β1) is a key activator of hepatic stellate cells (HSCs), and TGF-β1 activation of HSCs is an important step in the process of liver fibrosis, leading to excessive production of extracellular matrix (ECM).

[0161] α-Smooth muscle actin (α-SMA) is a marker of HSC activation.

[0162] Hepatic stellate cells (HSCs) are a key factor in the development of liver fibrosis.

[0163] 11 Experimental Materials and Methods

[0164] 11.1 Reagents and Instruments

[0165] Table 2. Main Instruments and Equipment Used in the Experiment

[0166]

[0167]

[0168] Table 3. Main reagents used in the experiment

[0169]

[0170]

[0171]

[0172] 11.2 Experimental Methods

[0173] 11.2.1 Masson staining experiment

[0174] Slicing: Place the embedded paraffin block in a -20℃ refrigerator for 30 minutes to pre-cool, and follow the steps in Table 4.

[0175] Table 4. Procedure for Masson staining experimental sections

[0176]

[0177]

[0178] Dewaxing: Follow the steps in Table 5.

[0179] Table 5 Dewaxing Operation Steps

[0180]

[0181] Staining: Use a hydrophobic pen to outline the sample distribution around the sample (the hydrophobic zone should be about 5 mm from the tissue edge). Place the slide in a humidified chamber for subsequent operations. Add an appropriate amount of the prepared Weigert iron hematoxylin staining solution (Weigert staining solution A: Weigert staining solution B = 1:1) to the slide and incubate at room temperature in the dark for 10 min. Then, add an appropriate amount of acidic ethanol differentiation solution to the slide and differentiate for 5-15 s, followed by rinsing with water. Next, add an appropriate amount of Masson's blueing solution to the slide and incubate at room temperature in the dark for 5 min, followed by rinsing with water. Then, immerse the slide in distilled water for 1 min, add an appropriate amount of Ponceau S and fuchsin staining solution to the slide and incubate at room temperature in the dark for 2 min, followed by rinsing with water. Immediately afterwards, add an appropriate amount of weak acid working solution (distilled water: weak acid solution = 2:1) to the slide and rinse for 1 min. Finally, add an appropriate amount of phosphomolybdic acid solution to the slide and wash for 1 min, followed by an appropriate amount of weak acid working solution and wash for 1 min. Add an appropriate amount of aniline blue staining solution to the slide and incubate for 1 min, then add an appropriate amount of weak acid working solution and wash for 1 min. Immediately afterwards, immerse the slide in 95% ethanol for rapid dehydration, then immerse it in anhydrous ethanol three times for 15 seconds each time. Finally, immerse the slide in xylene for 10 min to clear it, and then mount it with neutral resin.

[0182] 11.2.2 Immunofluorescence assay

[0183] Place the tissue sample in sodium citrate antigen retrieval solution (the solution should completely cover the tissue sample), microwave for 10-15 minutes, then let it sit for 30 minutes before allowing it to cool naturally at room temperature.

[0184] Then, use a hydrophobic pen to outline the sample distribution around the tissue for subsequent operations. Place the tissue sample in a humidified chamber, add 100 μl of 0.3% Triton solution to the tissue sample, evenly cover the tissue, and incubate at 37°C for 20 min.

[0185] Immerse the tissue sample in PBST and wash three times for 5 minutes each time. Then add 100 μl of 3% BSA solution to the tissue sample to evenly cover the tissue. Incubate at 37°C for 30 minutes.

[0186] Gently shake off the blocking solution on the tissue sample (do not wash), add 100 μl of the prepared primary antibody (Alpha-smooth muscle actin:PBS = 1:250, TGF-beta 1:PBS = 1:250) to the tissue sample, and incubate the tissue sample flat in a humidified chamber at 4°C overnight (add a small amount of water to the humidified chamber to prevent antibody evaporation).

[0187] Tissue samples were washed three times in PBST for 5 minutes each time. 100 μl of TRITC-labeled goat anti-rabbit IgG Rhodamine (TRITC)-conjugated Anti-Rabbit IgG (H+L):PBS = 1:100 was added to the tissue samples. The samples were incubated at 37°C in the dark for 60 minutes. The tissue samples were then washed three more times in PBST for 5 minutes each time, and then air-dried. The samples were mounted with anti-fluorescence attenuation mounting medium.

[0188] 12 Experimental Results and Analysis

[0189] 12.1 The Influence of General Animal Condition

[0190] During the experiment, mice in the normal control group, model control group, low-dose test product group, medium-dose test product group, and high-dose test product group showed good general condition in terms of appearance, signs, behavior, fur, glandular secretion, respiration, and fecal characteristics. No animals died, and no significant differences were observed.

[0191] 12.2 Effects on animal body weight

[0192] Mice body weight was recorded weekly during the experiment. C57BL / 6 mice fed with LD showed weight loss compared to CK. At week 8, mice in the test drug group showed weight gain compared to LD. The results showed that the body weight of LD-fed C57BL / 6 mice was significantly lower than that of C57BL / 6 mice fed a normal diet (CK). This LD-induced weight loss was reversed by administration of the herbal composition of this application.

[0193] 12.3 Effects on liver fibrosis in mice

[0194] Masson staining results of the liver are as follows Figure 1 As shown, after staining, collagen fibers appeared blue, muscle fibers appeared red, and cell nuclei appeared blue-black. It is evident that the CK group showed no liver fibrosis and the liver tissue structure was normal. Compared to the CK group, the LD group exhibited mild liver fibrosis, with a small amount of collagen fibers concentrated in the portal areas and around the central vein, indicating that the liver fibrosis model was successfully established. Compared to the LD group, the collagen fiber levels in the LD+CH-L, LD+CH-M, and LD+CH-H test groups did not significantly increase.

[0195] 12.4 Effects on the levels of α-SMA and TGF-β in mouse liver

[0196] like Figure 2 As shown, compared with the CK group, the expression levels and fluorescence intensity of α-SMA and TGF-β were increased in the LD group, while the fluorescence intensity of α-SMA and TGF-β in the liver was significantly reduced in the LD+CH-L, LD+CH-M, and LD+CH-H groups. Figure 3 As shown, immunofluorescence staining of liver sections (scale bar: 50 μm) reveals the expression of TGF-β1 (af) and α-SMA (gl). Cell nuclei were counterstained with DAPI (blue), and target proteins were labeled with TRITC (red). Combined images show co-localization (purple). The normal control group (CK) showed extremely weak fluorescence of TGF-β1 and α-SMA, indicating that hepatic stellate cells were in a resting state. The model group (LD) showed strong fluorescence for both markers, reflecting significant activation of hepatic stellate cells and enhanced fibrosis signaling. The fluorescence intensity decreased in the ursodeoxycholic acid (LD+UDCA) group, indicating partial inhibition of fibrosis. CH treatment showed a dose-dependent decrease in fluorescence signal: the low-dose group (LD+CH-L) moderately reduced the signal; the medium-dose group (LD+CH-M) further reduced expression; and the high-dose group (LD+CH-H) showed near-normal fluorescence levels, comparable to the LD+UDCA group.

[0197] Quantitative analysis (not shown) confirmed significant differences: compared with the CK group, the fluorescence intensity of α-SMA and TGF-β1 in the LD group increased by 6.2-fold and 4.8-fold, respectively (***p<0.001). CH treatment reduced these markers in a dose-dependent manner: CH-L reduced the fluorescence intensity of α-SMA and TGF-β1 by 18-22%, CH-M by 41-46% (p<0.01), and CH-H by 63.7% (α-SMA) and 61.9% (TGF-β1; **p<0.001). The efficacy was comparable to that of UDCA, verifying the anti-fibrotic efficacy of CH.

[0198] The results above show that the herbal composition of this application can inhibit TGF-β1 activation, reduce α-SMA expression, alleviate the occurrence of liver fibrosis, and prevent the occurrence of liver fibrosis induced by a high-fat, high-cholesterol diet.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of a drug for preventing liver fibrosis, characterized in that, The raw materials of the traditional Chinese medicine composition, by weight, are as follows: Scutellaria baicalensis 128-137 parts, Bupleurum chinense 128-137 parts, Rheum palmatum 103-120 parts, Citrus aurantium 128-137 parts, Artemisia capillaris 128-137 parts, Polygonum cuspidatum 171-200 parts, Gardenia jasminoides 137-150 parts, Lysimachia christinae 250-342 parts, Paeonia lactiflora 128-137 parts, Aucklandia lappa 128-137 parts, Pinellia ternata 90-103 parts, and Zingiber officinale 34-40 parts.

2. The application according to claim 1, characterized in that, The raw materials of the traditional Chinese medicine composition, by weight, are: 128 parts of Scutellaria baicalensis, 128 parts of Bupleurum chinense, 120 parts of Rheum palmatum, 128 parts of Citrus aurantium, 128 parts of Artemisia capillaris, 200 parts of Polygonum cuspidatum, 150 parts of Gardenia jasminoides, 250 parts of Lysimachia christinae, 128 parts of Paeonia lactiflora, 128 parts of Aucklandia lappa, 90 parts of Pinellia ternata, and 40 parts of Zingiber officinale.

3. The application according to claim 1, characterized in that, The raw materials of the traditional Chinese medicine composition, by weight, are as follows: 137 parts of Scutellaria baicalensis, 137 parts of Bupleurum chinense, 103 parts of Rheum palmatum, 137 parts of Citrus aurantium, 137 parts of Artemisia capillaris, 171 parts of Polygonum cuspidatum, 137 parts of Gardenia jasminoides, 342 parts of Lysimachia christinae, 137 parts of Paeonia lactiflora, 137 parts of Aucklandia lappa, 103 parts of Pinellia ternata, and 34 parts of Zingiber officinale.

4. The application according to claim 1, characterized in that, The preparation method of the traditional Chinese medicine composition includes the following steps: A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside; B. Weigh out the bitter orange peel and fresh ginger, add 5-9 times the amount of water, extract the volatile oil for 8-12 hours, collect and separate the volatile oil; the distilled aqueous solution is for later use. C. Weigh out Scutellaria baicalensis, Paeonia lactiflora, Artemisia capillaris, and Gardenia jasminoides. Add water and decoct 2-4 times. Extract for 1-3 hours for the first time, and for 1-3 hours for the second, third, and fourth times respectively. Add 7-10 times the amount of water each time. Filter the extract and combine it with the volatile oil-water extract obtained in step B. Concentrate under reduced pressure to a relative density of 1.25±0.05 at 60℃ for later use. D. Weigh out Bupleurum, Aucklandia, Rhubarb, Polygonum cuspidatum, and Lysimachia christinae. Extract with 60-80% ethanol 2-4 times. For the first extraction, add 10-14 times the amount of ethanol and extract for 2-4 hours. For the second, third, and fourth extractions, add 8-12 times the amount of ethanol and extract for 1-3 hours each time. Filter the extract, concentrate it into a clear paste, combine it with the water extract obtained in step C, mix well, dry, and pulverize for later use. E. The fine powder obtained in step A, the volatile oil obtained in step B, and the dried powder obtained in step D can be mixed together.

5. The application according to claim 1, characterized in that, The dosage forms of the traditional Chinese medicine composition include capsules, tablets, pills, oral liquids, granules, or powders.

6. The application according to claim 1, characterized in that, The liver fibrosis mentioned is liver fibrosis caused by a high-fat, high-cholesterol diet.

7. The application according to claim 1, characterized in that, The high-fat, high-cholesterol diet refers to a daily diet with a fat content of ≥15% and a cholesterol content of ≥1.25%.

8. The application according to claim 1, characterized in that, The liver fibrosis is characterized by increased expression levels of α-SMA and / or TGF-β in the liver.

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