Application of traditional Chinese medicine composition in preparation of medicine for preventing gall-stone

Abstract

The invention provides an application of a traditional Chinese medicine composition in preparing a medicine for preventing gall-stone. 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 disclosed by the invention can be used for remarkably reducing the formation of gall-stone and preventing the occurrence of gall-stone diseases.

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] Gallstones, also known as cholelithiasis, are a disease in which stones form in the biliary system. Clinically, they mainly manifest as gallbladder stones and hepatobiliary duct stones, severely impacting patients' quality of life. Treatment for gallstones primarily involves medication and surgery. For patients with small stones and no obvious symptoms, litholytic drugs can be used. For patients with large stones and severe symptoms, surgery is usually performed to remove the stones. In cases of severe complications, the entire gallbladder may need to be removed.

[0003] Previous studies have found a close correlation between the occurrence of gallstones and abnormal bile acid metabolism and blood lipid levels. There are significant differences in the bile acid composition of bile between gallstone patients and non-gallstone patients, and the incidence of gallstones is significantly higher in people with dyslipidemia than in those with normal blood lipid levels. In Xu Xia's article entitled "Analysis of Bile Acid Composition in Gallstone Patients," the differences in the types and contents of bile acid components in the bile of gallstone patients and non-gallstone patients, as well as patients with gallstones in different locations, were investigated. The contents of free bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and lithocholic acid (LCA) differed significantly among the three groups of patients with common bile duct stones, gallbladder stones, and non-gallstone disease. Both common bile duct stone and gallbladder stone patients had LCA and DCA in their bile, while these two free bile acids were not detected in non-gallstone patients. In Liu Siyu's article entitled "Correlation Study between TCM Syndrome Differentiation and Clinical Objective Indicators of Gallstones", the experiment found that dyslipidemia accounted for 44.55% of the four pathogenic factors of gallstones (dyslipidemia, obesity, diabetes and hypertension) in the study.

[0004] On the one hand, the prognosis for gallstones is not ideal, whether treated with medication or surgery, and the high recurrence rate and serious complications are difficult to effectively address. On the other hand, skipping breakfast, lack of exercise, and a preference for high-fat, high-cholesterol foods have been proven to be important causes of gallstones. Therefore, preventing gallstone formation at its source is receiving increasing attention. In addition to changing dietary habits and increasing exercise, medication is also an effective means of preventing gallstones.

[0005] Studies have found that trace components in the traditional Chinese medicine Yin Chen Hao Tang can lower calcium ion levels in bile, which is beneficial in preventing gallstone formation. It can also improve liver function and prevent bile reflux, thus avoiding bile stasis. Long Zhaoming et al. used Danfu Shu capsules to observe the effect of preventing experimental cholesterol stones in rabbits. The results showed that Danfu Shu capsules could reduce the concentrations of serum cholesterol, triglycerides, low-density lipoprotein cholesterol, and cholesterol and mucoprotein in bile, while increasing the content of bile acids and lecithin in bile, thus having an anti-gallstone effect. Ma Shiping et al., through their research on the prevention and treatment of gallstones using Dan Dao Pai Shi capsules, found that Dan Dao Pai Shi capsules could inhibit the increase of free bilirubin in bile caused by feeding stone-inducing foods.

[0006] Chinese patent CN115429866A discloses a traditional Chinese medicine composition for treating cholecystitis and its preparation method. The composition comprises: 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 clinically for the treatment of chronic cholecystitis. As an innovative traditional Chinese medicine, research on this composition has been continuously deepening, and it has also achieved unexpected results in the prevention of gallstones. Summary of the Invention

[0007] The purpose of this invention is to provide new uses for the traditional Chinese medicine composition disclosed in Chinese Patent CN115429866A.

[0008] To achieve the above objectives, the inventors have provided the following technical solutions.

[0009] The application of a traditional Chinese medicine composition in the preparation of a drug for the prevention or treatment of gallstones, 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.

[0010] 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.

[0011] 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.

[0012] In the above applications, the preparation method of the traditional Chinese medicine composition includes the following steps:

[0013] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] In the application of the traditional Chinese medicine composition provided by this invention, the gallstones are preferably gallstones caused by a high-fat, high-cholesterol diet. A high-fat, high-cholesterol diet refers to a daily diet with a fat content ≥15% and a cholesterol content ≥1.25%, meeting the conditions for dietary-induced gallstones.

[0020] This invention has shown that when mice fed a high-fat, high-cholesterol diet were given the herbal composition of this invention, the number of gallstones in the gallbladder of the mice was significantly reduced compared to the model group that was not given the herbal composition of this invention. Furthermore, the number of large crystals and stacked crystal masses was significantly reduced, and the bile was clearer. The herbal composition of this invention can be used for the prevention or treatment of gallstones, and is particularly suitable for gallstones caused by a high-fat, high-cholesterol diet.

[0021] Simultaneously, the herbal composition of this invention can also improve bile acid metabolism abnormalities in mice fed a high-fat, high-cholesterol diet. Abnormal bile acid metabolism refers to abnormalities in bile acid synthesis and / or transport in the liver, specifically manifested as decreased CYP7A1 mRNA expression levels, increased ABCG5 mRNA expression levels, and / or increased ABCG8 mRNA expression levels, and / or increased SCP2 mRNA expression levels in the liver, as well as increased ABCG5 protein expression levels and / or increased ABCG8 protein expression levels. Experimental studies of this invention show that in mice administered the herbal composition of this invention, the protein levels of ABCG5 and ABCG8 in the liver were significantly lower than in the model group, the CYP7A1 mRNA expression level was higher than in the model group, the ABCG5 mRNA expression level was lower than in the model group, the ABCG8 mRNA expression level was lower than in the model group, and the SCP2 mRNA expression level was lower than in the model group. The herbal composition of this invention has a regulatory effect on abnormal bile acid metabolism and can be used for the prevention of abnormal bile acid metabolism, especially suitable for the prevention of bile acid metabolism abnormalities caused by a high-fat, high-cholesterol diet.

[0022] In addition, the herbal composition of this invention can improve dyslipidemia in mice fed a high-fat, high-cholesterol diet, specifically, serum total cholesterol levels higher than normal, and / or serum triglyceride levels lower than normal, and / or serum low-density lipoprotein levels higher than normal, and / or serum high-density lipoprotein levels lower than normal. Experimental studies of this invention show that mice given the herbal composition of this invention exhibit significantly reduced total cholesterol levels and a decreasing trend in low-density lipoprotein levels, while triglyceride and high-density lipoprotein levels showed varying degrees of increase, fully demonstrating that the herbal composition of this invention has a regulatory effect on lipid metabolism disorders. Attached Figure Description

[0023] Figure 1 These are photographs showing the anatomical structure of the gallbladders of mice in each experimental group in the experimental case.

[0024] Figure 2 These are polarized light microscope images of gallstones in mice from each experimental group in the experimental examples.

[0025] Figure 3The images show representative photographs of the gallbladders of mice in each experimental group, quantitative analysis diagrams of bile volume, polarized microscope images of bile crystals, and HE-stained images of the gallbladders.

[0026] Figure 4 The expression levels of ABCG5, ABCG8, CYP7A1, and SCP2 mRNA in the livers of mice in each experimental group (n=8) are given.

[0027] Figure 5 The image shows the protein bands of ABCG5 and ABCG8 in the livers of mice in each experimental group in the experimental example.

[0028] Figure 6 This is a comparison of the grayscale values ​​of ABCG5 and ABCG8 proteins in the livers of mice in each experimental group (n=3).

[0029] Figure 7 The results show the expression of ABCG5 and ABCG8 proteins in the liver of mice in each experimental group, as well as the liver mRNA expression of genes related to cholesterol / bile acid metabolism.

[0030] Figure 8 The values ​​represent the serum lipid levels of mice in each experimental group in the experimental case. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments.

[0032] Example 1: Preparation of capsules containing traditional Chinese medicine composition

[0033] 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.

[0034] Preparation process:

[0035] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0036] B. Weigh out the bitter orange peel and 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] Example 2: Preparation of capsules containing traditional Chinese medicine composition

[0041] 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.

[0042] Preparation process:

[0043] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0044] B. Weigh out the bitter orange peel and 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Example 3: Preparation of the Traditional Chinese Medicine Composition

[0049] 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.

[0050] Preparation process:

[0051] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] Example 4: Preparation of Traditional Chinese Medicine Composition Tablets

[0057] 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.

[0058] Preparation process:

[0059] A. Weigh out Pinellia ternata, grind it into a fine powder, sterilize it by 60Co irradiation, and set it aside for later use;

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Example 5: Preparation of Traditional Chinese Medicine Composition Granules

[0065] 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.

[0066] Preparation process:

[0067] A. Weigh out ginger and Pinellia ternata, grind them into fine powder, sterilize by 60Co irradiation, and set aside;

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Experimental Example

[0073] The inventors conducted an experimental investigation into the effects of the herbal composition of this invention on gallstone formation in mice fed a high-fat, high-cholesterol diet.

[0074] 1 Experimental Institution

[0075] Hainan Provincial Institute for Drug Control (Safety Evaluation Research Center)

[0076] 2 Test samples, reference substances and other reagents

[0077] 2.1 Test samples

[0078] 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);

[0079] 2.1.2 Code: 22029DE-03;

[0080] 2.1.3 Source: Provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd.;

[0081] 2.1.4 Specification: Each capsule contains 0.46 g (equivalent to 1.71 g of cut crude drugs);

[0082] 2.1.5 Batch number: A2112001;

[0083] 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;

[0084] 2.1.7 Packaging: 0.46 g * 12 capsules * 3 plates / bag;

[0085] 2.1.8 Production date: December 27, 2021;

[0086] 2.1.9 Expiry date: November 2024;

[0087] 2.1.10 Storage conditions: Sealed;

[0088] 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 samples all meet the regulations.

[0089] 2.2 Reference substances

[0090] Ursodeoxycholic acid (UDCA) capsules (250 mg / capsule; Batch number: L19185A) were purchased from Losan Pharma GmbH, Germany.

[0091] 3 Solvents for experiments

[0092] 3.1 Name: Sodium carboxymethylcellulose

[0093] 3.1.1 Source: Xilong Science Co., Ltd.;

[0094] 3.1.2 Specification: 250 g;[[ID=-56]]

[0095] 3.1.3 Batch number: 2101110;

[0096] 3.1.4 Appearance: White or slightly yellow cellulose-like powder, odorless and tasteless;

[0097] 3.1.5 Uses: To prepare a 0.5% sodium carboxymethyl cellulose solution as a solvent.

[0098] 4. Experimental System and Reasons for Selection

[0099] 4.1 Experimental system: Adult C57BL / 6J mice.

[0100] 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.

[0101] 5. The strain, number, age, sex, weight range, origin, and grade of laboratory animals.

[0102] 5.1 Animal strain: C57BL / 6J mouse.

[0103] 5.2 Number of experimental animals: 180 animals are needed for the experiment, and 200 animals will be purchased.

[0104] 5.3 Sex of experimental animals: male.

[0105] 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.

[0106] 5.5 Source of experimental animals: Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., production license number SCXK(Su)2018-0008.

[0107] 5.6 Laboratory animal grade: SPF grade.

[0108] 5.7 Animal Quality Certificate No.: 320727230100015454.

[0109] 5.8 Handling of remaining animals: After grouping, the remaining mice were used for training operations or other experimental projects.

[0110] 6. Reception and Quarantine of Laboratory Animals

[0111] 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.

[0112] 7. Methods for identifying laboratory animals

[0113] 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), and high-dose test product group (LD+CH-H), with 30 animals in each group. The experimental groups and animal numbers are shown in Table 1.

[0114] Table 1. Experimental Groups and Corresponding Cage Numbers and Animal Numbers

[0115]

[0116]

[0117] 8. Environmental conditions for the husbandry and management of laboratory animals

[0118] 8.1 Breeding Room: This experiment was approved by the Animal Ethics Committee of Hebei Yiling Institute of Traditional Chinese Medicine (Approval No.: YL-IACUC-2023-004) and was carried out in accordance with the Guide for the Care and Use of Laboratory Animals. The animal laboratory of the research center has the license number for the use of experimental animals: SYXK(Qiong)2021-0009. During the quarantine period, the animals were raised in the quarantine isolation room in the barrier area from January 11, 2023 to January 13, 2023; during the experimental period, they were raised 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 was set at 20-26°C (the actual temperature ranges in the quarantine isolation room and Laboratory 1 in the barrier area were 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 were 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 light and dark alternating. The temperature, relative humidity, and pressure in the animal laboratory are automatically recorded, once every hour. In addition, 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 are monitored once a year by a qualified unit according to the requirements of GB 14925-2010, and the results all meet the requirements of the corresponding environmental grade.

[0119] 8.2 Environmental Grade: Barrier system (SPF level).

[0120] 8.3 Cages: Consist of a cage cover and a cage breeding box. The cage cover is made of stainless steel, and the cage breeding box is a CP-3 type transparent plastic mouse cage. The cage cover is replaced once a month, the breeding box is replaced once a week on Friday, and the bedding is replaced once a week on Monday, Wednesday, and Friday, following the research center's SOP "Replacement of bedding, bedding trays, and cages". The replaced breeding boxes and cage covers are cleaned, stored, and sterilized according to the center's SOP "Cleaning, storage, and sterilization of cages".

[0121] 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 every year to detect heavy metals Pb, microorganisms, and aflatoxin B1, and all meet the relevant regulations.

[0122] 8.5 Feed: High-fat and high-cholesterol feed (containing 15% fat, 1.25% cholesterol, and 0.5% cholic acid).

[0123] 8.5.1 Source: Guangdong Provincial Center for Laboratory Animals (Guangzhou, China).

[0124] 8.5.2 Batch number: 20221133.

[0125] 8.5.3 Shelf life: nine months.

[0126] 8.5.4 Production date: 2022.11.24.

[0127] 8.5.5 Valid until: August 23, 2023.

[0128] 8.5.6 Production License No.: Yue Feed Certificate (2019) 05073.

[0129] 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).

[0130] 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".

[0131] 9 Experimental Methods

[0132] 9.1 Grouping and Dosage Design

[0133] 9.1.1 Animal Grouping

[0134] 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.

[0135] Normal control group: fed standard mouse growth and reproduction diet and administered solvent by gavage for 8 weeks;

[0136] LD group: fed high-fat, high-cholesterol diet and given solvent by gavage for 8 weeks;

[0137] 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;

[0138] 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;

[0139] 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;

[0140] LD+UDCA group (123.3 mg / kg): fed with high-fat, high-cholesterol diet and administered control product by gavage for 8 weeks.

[0141] 9.1.2 Dosage Design and Basis

[0142] 9.1.2.1 Test sample dosage design

[0143] 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).

[0144] 9.2 Drug Preparation

[0145] 9.2.1 Preparation of the test sample

[0146] 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.

[0147] 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.

[0148] 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.

[0149] Each time, the sample is taken according to the required amount, and it is prepared and mixed in the clean bench before use.

[0150] 9.2.2 Preparation of 0.5% sodium carboxymethyl cellulose solution

[0151] 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.

[0152] 9.3 Administration Method

[0153] 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).

[0154] 10. Detection items, methods, frequency, and result interpretation

[0155] 10.1 Clinical Observation

[0156] During the trial, the animals were observed for general clinical symptoms at least once a day, and their health status was observed and recorded.

[0157] 10.2 Weight Record

[0158] 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.

[0159] 10.3 Specimen Collection

[0160] All mice were harvested 24 hours after the last administration of the drug at the end of the eighth week of the experiment.

[0161] 10.4 Observation of stones

[0162] All mice were dissected 24 hours after the last administration of the drug at the end of week 8 of the experiment to observe the gallstones. Bile was collected, and the crystallization of gallstones in the bile was observed using a polarizing microscope.

[0163] 10.5 Collection of liver tissue and determination of related indicators

[0164] 10.5.1 Collection of liver tissue

[0165] The liver was quickly removed, the surrounding tissues were trimmed, washed with ice-cold 0.9% saline, dried, and weighed. A portion of the tissue was kept fresh and frozen in liquid nitrogen for the determination of molecular biological indicators.

[0166] 10.5.2 Immunoblotting assay

[0167] Liver X receptor (LXR) pathway related markers: ABCG5, ABCG8

[0168] After thorough grinding with lysis buffer, the tissue was centrifuged at 10000g for 10 minutes at 4°C, and the protein content of the supernatant was detected using a BCA protein assay kit. Equal volumes of protein were separated using a 10% SDS-PAGE gel. The protein was transferred from the gel to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour, followed by incubation with primary and secondary antibodies. After development and exposure, images were taken and analyzed using ImageJ.

[0169] 10.5.3 Real-time quantitative PCR

[0170] (1) Detection of target genes in the liver that affect bile acid synthesis and transport. These are: CYP7A1, ABCG5, ABCG8, and SCP2.

[0171] (2) Target gene sequences were searched in NCBI (National Library of Medicine) using the screening criteria of "Mus musculus," and suitable sequences were further screened in PrimerBank, considering primer conditions such as Tm, GC%, Amplicon Size, and Primer length. PCR primers for detecting the fragments were designed to span exons. Sequence validation was performed in Primer Blast, considering predicted primer dimers and possible secondary structures within the primers themselves or between primer pairs. Finally, relatively suitable primer sequences were selected for detection, with one spare sequence reserved for each gene, procured in the same batch.

[0172] Total RNA was extracted from liver tissue using TRIzol reagent. cDNA was extracted using PrimeScript. TM RT Master Mix was synthesized, and the reverse transcription system was performed according to the manufacturer's instructions. RT-PCR was performed using SYBR Green for real-time quantitative PCR, with a final reaction volume of 20 μl.

[0173] 10.6 Collection of mouse serum

[0174] Blood was collected from each mouse via the orbital venous plexus, and serum was separated by centrifugation (3000g, 15min, 4℃) for later use. The following indicators were measured according to the kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China): serum total cholesterol (TCH-O), total bile acids (TBA), triglycerides (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) levels.

[0175] 10.7 Histological Analysis

[0176] The gallbladder was dissected, bile was aspirated, and the tissue was rinsed with 0.9% saline, fixed in 4% neutral buffered formaldehyde (pH 7.4) for 24 hours, dehydrated with graded ethanol (70%-100%), cleared with xylene, and embedded in paraffin. Serial 4μm sections were stained with hematoxylin and eosin (H&E) using standard methods. Stained sections were observed under an optical microscope (BX53, Olympus, Japan), and photographed at 100× and 400× magnification. The degree of gallbladder hyperplasia and inflammatory infiltration was assessed by a pathologist unaware of the grouping.

[0177] 11 Data Statistical Processing Methods

[0178] Data are expressed as mean ± standard deviation (SD). Data processing was performed using GraphPad Prism (version 8.0). One-way ANOVA was used to analyze the data. Duncan's multiple comparison test was used to determine significance; P < 0.05 was considered significant, and P < 0.01 was considered highly significant.

[0179] 12 Experimental Materials and Methods

[0180] 12.1 Reagents and Instruments

[0181] Table 2. Main Instruments and Equipment Used in the Experiment

[0182]

[0183]

[0184] Table 3. Main reagents used in the experiment

[0185]

[0186]

[0187]

[0188] 12.2 Experimental Methods

[0189] 12.2.1 Observational Experiment of Stones

[0190] A gallstone model was established in experimental animals by feeding them a high-fat, high-cholesterol diet. Mice in each experimental group were dissected 24 hours after the last administration of the drug at the end of the eighth week of the experiment. Bile was collected, and the crystallization of gallstones in the bile was observed using a polarizing microscope.

[0191] 12.2.2 Immunoblotting assay

[0192] Total protein extraction from tissue: Wash tissue blocks 2-3 times with pre-cooled PBS to remove blood contamination, cut into small pieces and place in a homogenization tube. Add two 4mm homogenization beads and 10 times the tissue volume of lysis buffer (add various protease inhibitors a few minutes before use). Set the homogenization program to homogenize (if a higher protein concentration is needed, the volume of lysis buffer can be reduced appropriately). Remove the homogenized tube and place it on ice with lysis buffer for 30 minutes, shaking every 5 minutes to ensure complete tissue lysis. Centrifuge at 12000 rpm, 4℃ for 10 minutes, and collect the supernatant, which is the total protein solution. Take the undenatured protein solution and determine the protein concentration using a BCA protein concentration assay kit. Then, add 5× reducing protein loading buffer to the protein solution at a ratio of 4:1, denature in a boiling water bath for 15 minutes, and store at -20℃ for later use.

[0193] SDS-PAGE electrophoresis: Prepare separating gels of different concentrations according to experimental requirements (Table 4), add TEMED, mix thoroughly, and pour into the gel. After 30 minutes, wait for the separating gel to solidify, then prepare a 5% stacking gel (Table 5), add TEMED, mix thoroughly immediately, pour into the gel, and insert the comb into the stacking gel. Once the stacking gel has solidified, remove the gel casting apparatus, carefully remove the comb, and you are ready to begin electrophoresis. Place the gel casting apparatus in the electrophoresis tank, add electrophoresis buffer, and perform protein loading and electrophoresis. Use 90V for the stacking gel and 150V for the separating gel. Stop electrophoresis when the gel reaches approximately 1cm from the bottom of the bromophenol blue layer, and then proceed with the transfer.

[0194] Table 4. Proportions of separating gels at different concentrations

[0195]

[0196] Table 5. Proportion of 5% Concentrated Gum

[0197]

[0198]

[0199] Transfer: Prepare 7×9cm filter paper and a PVDF (0.45µm) membrane of appropriate size. The PVDF membrane should be activated with methanol for 2 minutes before use. Place the transfer clamp in a bowl containing the transfer buffer. Open the clamp; the left side is white, and the right side is black. Place a sponge and three layers of filter paper on each side. Carefully peel off the separating gel and place it on the filter paper. Place the PVDF membrane on the gel, ensuring there are no air bubbles between the membrane and the gel. Cover the membrane with three layers of filter paper and remove any air bubbles. Finally, cover with another layer of sponge. Incubate at a constant current of 300mA for 30 minutes. During the transfer process, place the transfer equipment in ice water to cool it down.

[0200] Immunological reaction: Place the transferred membrane into an incubator containing TBST, rinse quickly once, then add 5% milk and block at room temperature for 30 min. Prepare the primary antibody dilution according to the antibody instructions and incubate overnight at 4°C on a shaker (slow shaking). Wash the membrane quickly with TBST for 5 min each time, 3 times. Dilute the secondary antibody with TBST at a ratio of 1:5000, incubate at room temperature for 30 min, then rinse the membrane quickly with TBST for 5 min each time, 3 times.

[0201] Chemiluminescence: Take out the eluted PVDF membrane and place it on absorbent paper to slightly dry the liquid on the membrane. Place the membrane on the shelf of the chemiluminescence instrument and add the ECL luminescent solution mixed in a 1:1 ratio, so that the liquid completely submerges the membrane. After reacting for 1 minute, use absorbent paper to dry the excess liquid on the membrane and place it in the chemiluminescence instrument. Start chemiluminescence according to the preset program. After exposure is complete, save the original image as a TIFF file.

[0202] WB Results and Analysis: The saved TIFF format original image was analyzed using AIWBwell. TM The analysis software performs data analysis, and the raw grayscale values ​​it reads are the raw data. Then, the ratio of the indicator grayscale value to the internal reference grayscale value is calculated, which represents the relative content of the sample.

[0203] 12.2.3 Real-time PCR Experiment

[0204] qRT-PCR was used to detect target genes in the liver that affect bile acid synthesis and transport, including CYP7A1, ABCG5, ABCG8, and SCP2. CYP7A1 is the rate-limiting enzyme in the classic bile acid biosynthesis pathway.

[0205] Total RNA extraction: Take a grinding tube, add 1 ml of RNA extraction buffer and 3 3 mm grinding beads, and pre-chill on ice. Then, take 5-20 mg of tissue and add it to the grinding tube, grinding thoroughly until no visible tissue fragments remain. Centrifuge at 12000 rpm for 10 min at 4°C and collect the supernatant. Add 100 μl of chloroform substitute, invert the centrifuge tube for 15 seconds to mix thoroughly, let stand for 3 min, and centrifuge at 12000 rpm for 10 min at 4°C. Transfer 400 μl of supernatant to a new centrifuge tube, add 550 μl of isopropanol, invert to mix thoroughly, and incubate at -20°C for 15 min. Centrifuge at 12000 rpm for 10 min at 4°C; the white precipitate at the bottom of the tube is RNA. Further, remove the liquid, add 1 ml of 75% ethanol, invert and mix well to wash the precipitate, centrifuge at 12000 rpm for 5 min at 4°C, remove the liquid again, add 1 ml of 75% ethanol, invert and mix well to wash the precipitate, remove all liquid, place the centrifuge tube on a clean bench and blow for 3-5 min, add 15 μl of RNA dissolving buffer to dissolve the RNA. Use Nanodrop 2000 to detect RNA concentration and purity. After zeroing the instrument with a blank, take 2.5 μl of the RNA solution to be tested onto the detection base, lower the sample arm, and start the absorbance detection using the software on the computer. Dilute the RNA with excessively high concentrations appropriately to achieve a final concentration of 200 ng / μl.

[0206] Reverse transcription: Prepare the reverse transcription reaction system according to the instructions (4 μl 5×SweScript All-in-One SuperMix for qPCR, 1 μl gDNARemover, 10 μl Total RNA, and add Nuclease-Free Water to a total volume of 20 μl), mix gently, and centrifuge. Then, perform reverse transcription at 25°C for 5 min, 42°C for 30 min, and 85°C for 5 s on a standard PCR instrument.

[0207] Quantitative PCR: Take 0.1 ml of PCR reaction plate and prepare the reaction mixture (7.5 μl 2×Universal Blue SYBR Green qPCR Master Mix, 1.5 μl 2.5 μM mouse gene primers (Table 6), 2.0 μl reverse transcription product, and 4.0 μl Water Nuclease-Free). Prepare 3 tubes for each reverse transcription product. After loading the samples, seal the PCR plate with a PCR sealing film using a sealing device, and centrifuge using a microplate centrifuge. Then perform PCR amplification: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 15 s, 60℃ annealing / extension for 30 s, and 65℃→95℃ melting curve. All these processes were completed on a real-time PCR instrument. Data were analyzed using Method 2. -△△Ct conduct.

[0208] Table 6. Primer sequences for each mouse gene.

[0209]

[0210] 12.2.4 Serum Biochemical Index Determination Experiment

[0211] Blood was collected from each mouse via the orbital venous plexus, and the serum was separated by centrifugation (3000g, 15min, 4℃) and stored at -80℃. Relevant indicators were measured directly; if the levels exceeded the linear range, the serum was diluted with physiological saline before measurement. Relevant biochemical indicators in mouse serum were measured according to the instructions of the respective kits, including the total cholesterol test kit (Nanjing Jiancheng Bioengineering Institute, catalog number: A111-1-1), the total bile acid test kit (Nanjing Jiancheng Bioengineering Institute, catalog number: E003-2-1), the low-density lipoprotein cholesterol test kit (Nanjing Jiancheng Bioengineering Institute, catalog number: A113-1-1), and the high-density lipoprotein cholesterol test kit (Nanjing Jiancheng Bioengineering Institute, catalog number: A112-1-1).

[0212] 13 Experimental Results and Analysis

[0213] 13.1 The Influence of General Animal Condition

[0214] 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.

[0215] 13.2 Effects on animal body weight

[0216] 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.

[0217] 13.3 Effects on gallstone formation in mice induced by a high-fat, high-cholesterol diet

[0218] like Figure 1 and Figure 3As shown in figure a, the gallbladders of mice in the CK group were smaller, and the bile was clear and yellow, with no obvious crystals observed. Compared with the CK group, the gallbladders of mice in the LD group were larger, with granular or silt-like sediments visible in the gallbladder, yellowish-white in color (indicated by the red arrows in the figure), and the bile was slightly cloudy and thick, indicating successful model establishment. The bile in the UDCA group (LD+UDCA) was clear and yellow, and the gallbladder was smaller. A small number of gallstones were visible in the gallbladders of mice in the LD+CH-L group (indicated by the red arrows), and the number of large crystals and stacked crystal blocks was significantly reduced compared with the LD group. Compared with the LD group, no yellowish-white granular gallstones were observed in the gallbladders of mice in the LD+CH-M and LD+CH-H groups, or the number of gallstones was significantly reduced, and the bile was clear.

[0219] Quantitative analysis of gallbladder bile volume ( Figure 3 b) shows that, compared with CK, the LD group had a significantly increased bile volume, indicating cholestasis. LD+CH-L, LD+CH-M, and LD+CH-H significantly reduced bile volume compared with LD, with the LD+CHLD-M group showing the most significant reduction.

[0220] To further observe gallstones, researchers used a polarizing microscope to examine the crystallization of gallstones in the bile of mice in each experimental group. Figure 2 and Figure 3 As shown in Figure c, the bile of mice in the CK group contained virtually no crystals, while the bile of mice in the LD group contained large crystal particles and stacked crystal blocks, which were polygonal and quadrilateral in shape. Compared with the LD group, the bile of mice in the LD+UDCA group contained sparse microcrystals, with few large particles and amorphous quadrilaterals; the number of crystals in the bile of mice in the LD+CH-L, LD+CH-M, and LD+CH-H groups was significantly reduced, and the number of large crystal particles and stacked crystal blocks was significantly reduced, with single-layer solid quadrilaterals visible.

[0221] HE staining showed ( Figure 3 d) In the CK group, the gallbladder wall (mucosa, muscular layer, serosa) structure was intact, with normal mucosal folds, a single layer of columnar epithelium, oval nuclei located at the base, and a clear basement membrane, without hyperplasia or inflammation. In the LD group, epithelial thickening, structural destruction, and inflammatory infiltration occurred under litholytic conditions. The histopathology of the LD+CH-L / M / H treatment group showed improvement, with reduced epithelial thickening and inflammation.

[0222] The results above show that the traditional Chinese medicine composition provided by the present invention can significantly inhibit the formation of gallstones in the gallbladder, improve bile quality, improve gallbladder pathology, and is suitable for the prevention of gallstones caused by a high-fat, high-cholesterol diet.

[0223] 13.4 Results of Real-Time PCR

[0224] like Figure 4As shown in Table 7, compared with the CK group, the CYP7A1 mRNA level in the LD group showed a decreasing trend, suggesting an abnormality in the bile acid synthesis process in the LD group mice. After administration of the herbal composition of the present invention, the CYP7A1 mRNA levels in the LD+CH-L, LD+CH-M, and LD+CH-H groups were restored to varying degrees. In addition, compared with the CK group, the ABCG5, ABCG8, and SCP2 mRNA levels in the LD group mice were significantly increased. After administration of the test product, the ABCG5, ABCG8, and SCP2 mRNA levels in the LD+CH-L, LD+CH-M, and LD+CH-H groups were reduced to varying degrees.

[0225] Table 7. mRNA expression level of the target gene

[0226]

[0227] Note: ***P<0.001, **P<0.01, *P<0.05

[0228] Based on the significant (greater than 6-fold) difference in ABCG5 / ABCG8 mRNA expression between model establishment and drug administration, researchers further investigated the expression levels of ABCG5 and ABCG8 proteins using Western blotting. The results are as follows... Figure 5 , Figure 6 and Figure 7 As shown in Table 8, compared with the CK group, the levels of ABCG5 and ABCG8 proteins in the liver of mice in the LD group were significantly increased. After administration of the traditional Chinese medicine composition of the present invention, the protein levels of ABCG5 and ABCG8 in the LD+CH-L, LD+CH-M and LD+CH-H groups were significantly decreased.

[0229] Table 8. Gray value analysis of target protein expression

[0230]

[0231]

[0232] Note: ***P<0.001, **P<0.01, *P<0.05

[0233] 13.5 Serum biochemical markers (T-CHO, TBA) results

[0234] As shown in Table 9, compared with the CK group, the serum levels of total cholesterol (T-CHO) and total bile acids (TBA) in the LD group mice were significantly increased. After administration of the herbal composition of the present invention, compared with the LD group, the serum T-CHO levels of mice in the LD+CH-L, LD+CH-M, and LD+CH-H experimental groups were significantly decreased, and the TBA levels all showed a decreasing trend.

[0235] Table 9

[0236]

[0237] Note: ***P<0.001, **P<0.01, *P<0.05

[0238] The above experimental results show that the herbal composition of this invention can regulate the levels of CYP7A1 mRNA and ABCG5, ABCG8, and SCP2 mRNA in the liver of mice fed a high-fat, high-cholesterol diet (e.g., Figure 7 It can also regulate the expression levels of ABCG5 and ABCG8 proteins in the liver of mice on a high-fat, high-cholesterol diet, effectively affecting the synthesis and transport of bile acids.

[0239] 13.6 Effects on serum lipid levels in mice induced by a high-fat, high-cholesterol diet

[0240] like Figure 8 As shown in Table 10, compared with the control group (CK), the serum T-CHO and LDL-C levels in the LD group mice were significantly increased, while the TG level was significantly decreased, indicating that the model was successfully established. After administration of the test product, compared with the LD group, the serum T-CHO levels in the LD+CH-L, LD+CH-M, and LD+CH-H experimental groups were significantly decreased, and the high-dose group (CH-H) showed efficacy comparable to UDCA. LDL-C levels also showed a decreasing trend, while TG and HDL-C levels showed varying degrees of increase. Therefore, the herbal composition of this application has a regulatory effect on lipid metabolism disorders in mice fed a high-fat, high-cholesterol diet, and has the effect of improving cholesterol metabolism disorders. Its high-dose efficacy is comparable to that of UDCA.

[0241] Table 10 Serum lipid index determination

[0242]

[0243]

[0244] Note: ***P<0.001, **P<0.01, *P<0.05.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of a drug for the prevention or treatment of gallstones, 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 any one of claims 1-4, characterized in that, The gallstones mentioned are gallstones caused by a high-fat, high-cholesterol diet.

7. The application according to claim 6, characterized in that, The high-fat, high-cholesterol diet is defined as a diet with a fat content of ≥15% and a cholesterol content of ≥1.25%.

8. The application according to any one of claims 1-4, characterized in that, The application of the drug in the preparation of drugs to improve bile acid metabolism disorders.

9. The application according to claim 8, characterized in that, The drug improves abnormal bile acid metabolism by regulating the levels of CYP7A1 mRNA, ABCG5 mRNA, ABCG8 mRNA, and SCP2 mRNA, and / or regulating the expression levels of ABCG5 and ABCG8 proteins.

10. The application according to any one of claims 1-4, characterized in that, The application of the drug in the preparation of drugs for regulating lipid metabolism disorders.

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