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

Abstract

The invention belongs to the technical field of traditional Chinese medicine composition application, and particularly relates to application of a traditional Chinese medicine composition in preparation of a medicine for preventing and treating gall-stone and a product. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 90 to 110 parts of herba artemisiae scopariae, 90 to 110 parts of radix bupleuri, 65 to 85 parts of folium isatidis, 30 to 50 parts of radix et rhizoma rhei and 22 to 42 parts of radix sophorae flavescentis. The traditional Chinese medicine composition prepared by the invention has the effects of remarkably reducing the gall-stone lithogenesis rate, inhibiting cholesterol synthesis, promoting bile excretion, improving blood lipid metabolism and the like, and has excellent multiple treatment effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of the application of traditional Chinese medicine compositions, and particularly relates to the application of a traditional Chinese medicine composition in the preparation of a drug for preventing and treating gallstones and a product thereof. Background Art

[0002] Cholelithiasis refers to a disease in which stones occur in the biliary tract system, including the gallbladder and bile ducts. Its components are composed of cholesterol, bilirubin, calcium salts, and mixed stones, etc. Its clinical manifestations depend on factors such as the location of the gallstones, whether biliary tract obstruction and infection are caused, etc. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme that catalyzes the decomposition of cholesterol into bile acids in the liver, and HMG-CoA reductase HMGR is a key enzyme in the cholesterol synthesis pathway in vivo. The activities of both are regulated by various factors and are crucial for maintaining cholesterol metabolism balance. The bile salt export pump (BSEP) is located on the cell membrane of the canalicular surface of hepatocytes and is the most important transporter for bile acid excretion, responsible for transporting bile salts from hepatocytes to bile ducts, which can maintain the balance of bile salts in the body and reduce the possibility of gallstones. The blood lipid levels (total cholesterol (TC), triglyceride (TG)) are significantly higher in patients with gallstones than in non-gallstone populations, reflecting abnormal lipid metabolism. Therefore, indicators such as CYP7A1, HMGR, BSEP, TC, and TG are closely related to gallstones.

[0003] For example, Chinese Patent CN1994378A discloses a compound preparation for treating jaundice viral hepatitis and its preparation method. The preparation is made from Artemisia capillaris, Bupleurum falcatum, Sophora flavescens, Rheum palmatum, and Isatis indigotica in a weight ratio. The preparation method is as follows: Artemisia capillaris and Bupleurum falcatum are subjected to steam distillation to extract volatile oils for standby; the aqueous solution and medicinal residues after distillation are combined with Sophora flavescens and Isatis indigotica, and then decocted with water. The decoction is concentrated, ethanol is added to make the ethanol content reach 70%, and after refrigeration, it is filtered. The filtrate is recovered from ethanol, water is added, and after refrigeration, it is filtered to obtain a filtrate. Rheum palmatum is decocted with water, the decoction is concentrated, ethanol is added, refrigerated and filtered, the filtrate is recovered from ethanol, water is added, and after refrigeration, it is filtered to obtain a filtrate. The filtrate is combined with the above-mentioned filtrates of Sophora flavescens, etc., concentrated to obtain an extract, and volatile oils and excipients of different dosage forms are added, and any dosage form in pharmacy can be prepared by a conventional preparation method; the clinical trial results of the preparation of the invention show that the cure rate of treating jaundice viral hepatitis reaches 48.76%, and the total effective rate reaches 88.43%. However, its application in cholesterol stones is not yet known.

[0004] As disclosed in Chinese Patent CN105688039A, a Yinchen Lidan Granule includes the following components by weight: 5-15 parts by weight of Artemisia capillaris Thunb., 1-10 parts by weight of Lysimachia christinae Hance, 5-15 parts by weight of Paeonia lactiflora Pall., 2-6 parts by weight of Rheum officinale Baill. (processed with wine), 5-15 parts by weight of Isatis indigotica Fortune, 1-10 parts by weight of Curcuma aromatica Salisb., 2-6 parts by weight of Atractylodes macrocephala Koidz., 2-6 parts by weight of Poria cocos (Schw.) Wolf, 2-6 parts by weight of Bupleurum chinense DC., and 3-11 parts by weight of Salvia miltiorrhiza Bunge. This preparation is used to treat cholestatic hepatitis, which is not only economical and convenient to take, but also can shorten the course of disease, improve the curative effect, and fully develop green medicine. However, its composition is complex.

[0005] Therefore, there is an urgent need to study a traditional Chinese medicine composition that has outstanding curative effects in preventing and treating gallstones. Summary of the Invention

[0006] Based on the deficiencies of the prior art, the present invention provides the use of a traditional Chinese medicine composition in the preparation of a drug for preventing and treating gallstones and the product. The traditional Chinese medicine composition prepared by the present invention can significantly reduce the stone formation rate of gallbladder stones, inhibit cholesterol synthesis, promote bile excretion, and improve blood lipid metabolism, and has excellent multiple therapeutic effects.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides the use of a traditional Chinese medicine composition in preventing and treating gallstones. The traditional Chinese medicine composition is composed of the following raw materials by weight: 90-110 parts of Artemisia capillaris Thunb., 90-110 parts of Bupleurum chinense DC., 65-85 parts of Isatis indigotica Fortune, 30-50 parts of Rheum officinale Baill., and 22-42 parts of Sophora flavescens Ait.

[0009] Preferably, the traditional Chinese medicine composition is composed of the following raw materials by weight:

[0010] 100-110 parts of Artemisia capillaris Thunb., 90-100 parts of Bupleurum chinense DC., 75-85 parts of Isatis indigotica Fortune, 30-40 parts of Rheum officinale Baill., and 32-42 parts of Sophora flavescens Ait.

[0011] More preferably, the traditional Chinese medicine composition is composed of the following raw materials by weight: 100 parts of Artemisia capillaris Thunb., 100 parts of Bupleurum chinense DC., 75 parts of Isatis indigotica Fortune, 40 parts of Rheum officinale Baill., and 32 parts of Sophora flavescens Ait.

[0012] On the other hand, the present invention also provides a preparation method of the above traditional Chinese medicine composition for preventing and treating gallstones, including the following steps:

[0013] (1) Take the formula amount of Artemisia capillaris Thunb. and Bupleurum chinense DC., add water, distill, and collect the volatile oil for standby;

[0014] (2) Add water to the aqueous solution after distillation in step (1), the medicinal residues, Sophora flavescens Ait., and Isatis indigotica Fortune, and decoct for 2-3 times, 1-2 hours each time. Combine the decoction liquids and concentrate to obtain a concentrated liquid;

[0015] (3) Add ethanol to the concentrated solution obtained in step (2) to make the alcohol content reach 70%-80%, refrigerate at 0-4°C for 48-60 hours, filter, recover ethanol, add water again, refrigerate, and filter to obtain refined concentrated solution 1;

[0016] (4) Take the formula amount of Rheum officinale, add water and decoct for 2-3 times, 1-2 hours each time. Combine the decoction liquids and concentrate. Add ethanol to make the alcohol content reach 70%-80%, filter, recover ethanol, add water again, refrigerate at 0-4°C for 48-60 hours, and filter to obtain refined concentrated solution 2;

[0017] (5) Mix refined concentrated solution 1 and refined concentrated solution 2, dry, and add the volatile oil obtained in step (1) to obtain the described traditional Chinese medicine composition.

[0018] As a preferred embodiment, the preparation method of the traditional Chinese medicine composition for preventing and treating gallstones includes the following steps:

[0019] (1) Take the formula amount of Artemisia capillaris and Bupleurum chinense, add water, distill, and collect the volatile oil for standby;

[0020] (2) Decoct the aqueous solution after distillation in step (1), the medicinal residues, Sophora flavescens and Isatis indigotica with water for 2 times, 1.5 hours each time. Combine the decoction liquids and concentrate to obtain a concentrated solution;

[0021] (3) Add ethanol to the concentrated solution obtained in step (2) to make the alcohol content reach 70%, refrigerate at 0-4°C for 48 hours, filter, recover ethanol, add water again, refrigerate, and filter to obtain refined concentrated solution 1;

[0022] (4) Take the formula amount of Rheum officinale, add water and decoct for 2 times, 1.5 hours each time. Combine the decoction liquids and concentrate. Add ethanol to make the alcohol content reach 70%, filter, recover ethanol, add water again, refrigerate at 0-4°C for 48 hours, and filter to obtain refined concentrated solution 2;

[0023] (5) Mix refined concentrated solution 1 and refined concentrated solution 2, dry, add the volatile oil obtained in step (1) to obtain the described traditional Chinese medicine composition.

[0024] On the other hand, the present invention provides the application of the above traditional Chinese medicine composition in preventing and treating gallstones, and the gallstones include cholesterol stones, pigment stones, and mixed stones.

[0025] Furthermore, the present invention mainly studies the effect of the traditional Chinese medicine composition on cholesterol stones.

[0026] Furthermore, the related indexes of the cholesterol stones include gallbladder stone formation rate, CYP7A1, HMGR, BSEP mRNA level, serum triglyceride (TG), and total cholesterol (TC) level.

[0027] The present invention also provides the use of the above traditional Chinese medicine composition for treating gallstones in the preparation of a drug for preventing and treating gallstones.

[0028] Preferably, the dosage form of the drug is pill, powder, granule, capsule, injection or tablet.

[0029] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0030] The present invention also provides the use of Kuhuang Granules in the preparation of a drug for preventing and treating gallstones, comprising the traditional Chinese medicine composition and pharmaceutically acceptable excipients in the above use.

[0031] The present invention also provides the use of Kuhuang Injection in the preparation of a drug for preventing and treating gallstones, comprising the traditional Chinese medicine composition and pharmaceutically acceptable excipients in the above use.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The composition of the present invention is composed of five Chinese herbal medicines, namely Sophora flavescens, Rheum palmatum, Artemisia capillaris, Bupleurum chinense var. sutchuenense and Isatis indigotica Fortune, and has the effects of clearing heat and promoting diuresis, soothing the liver and reducing jaundice. In the formula, Artemisia capillaris is bitter, pungent and slightly cold in taste, and belongs to the liver, gallbladder, spleen and stomach meridians. It has a good effect of clearing damp-heat and reducing jaundice, and can promote the excretion of bile. It is a commonly used monarch drug in traditional Chinese medicine for treating jaundice; Bupleurum has the functions of relieving exterior syndrome and subsiding fever, soothing the liver and relieving depression, and raising yang. It enters the liver and gallbladder meridians and can remove the excess fire of the liver and gallbladder, so it is used as an assistant; Isatis indigotica Fortune has the functions of clearing heat and cooling blood, and detoxifying. Clinically, it can be used to treat viral infections. For hepatitis patients with high fever and coma, and subcutaneous hemorrhage, this drug is commonly used as an adjuvant in traditional Chinese medicine; Rheum palmatum has the functions of purging heat and dredging the intestines, cooling blood and detoxifying, treating damp-heat jaundice, and promoting blood circulation and removing stasis. In the formula, Rheum palmatum is used to have the effect of clearing heat and purging fire as an assistant; Sophora flavescens has the functions of clearing heat and drying dampness, diuresis and killing insects, and is mainly used to treat hot dysentery, jaundice with oliguria and other syndromes. In the formula, its strong dampness-drying effect is mainly used to promote diuresis and reduce jaundice.

[0034] (2) The composition prepared by the present invention shows significant effects in preventing and treating gallstones, such as significantly reducing the stone formation rate of gallbladder stones, inhibiting cholesterol cooperation, promoting bile excretion and improving blood lipid metabolism, and has excellent multiple treatment effects. Detailed Embodiments

[0035] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are usually in accordance with conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0036] Example 1 A traditional Chinese medicine composition for treating gallstones

[0037] It is composed of the following raw materials:

[0038] 100 parts of Artemisia capillaris Thunb., 100 parts of Bupleurum chinense DC., 75 parts of Isatis indigotica Fortune, 40 parts of Rheum officinale Baill., 32 parts of Sophora flavescens Ait.

[0039] Example 2 A traditional Chinese medicine composition for treating gallstones

[0040] It is composed of the following raw materials:

[0041] 90 parts of Artemisia capillaris Thunb., 110 parts of Bupleurum chinense DC., 65 parts of Isatis indigotica Fortune, 50 parts of Rheum officinale Baill., 32 parts of Sophora flavescens Ait.

[0042] Example 3 A traditional Chinese medicine composition for treating gallstones

[0043] It is composed of the following raw materials:

[0044] 100 parts of Artemisia capillaris Thunb., 100 parts of Bupleurum chinense DC., 75 parts of Isatis indigotica Fortune, 30 parts of Rheum officinale Baill., 42 parts of Sophora flavescens Ait.

[0045] Example 4 A traditional Chinese medicine composition for treating gallstones

[0046] It is composed of the following raw materials:

[0047] 110 parts of Artemisia capillaris Thunb., 90 parts of Bupleurum chinense DC., 85 parts of Isatis indigotica Fortune, 40 parts of Rheum officinale Baill., 22 parts of Sophora flavescens Ait.

[0048] Comparative Example 1 A traditional Chinese medicine composition for treating gallstones

[0049] It is composed of the following raw materials:

[0050] 115 parts of Artemisia capillaris Thunb., 75 parts of Bupleurum chinense DC., 90 parts of Isatis indigotica Fortune, 55 parts of Rheum officinale Baill., 12 parts of Sophora flavescens Ait.

[0051] Comparative Example 2 A traditional Chinese medicine composition for treating gallstones

[0052] It is composed of the following raw materials:

[0053] 100 parts of Artemisia capillaris Thunb., 100 parts of Bupleurum chinense DC., 40 parts of Rheum officinale Baill.

[0054] It is prepared according to the following method:

[0055] (1) Take the formula amount of Artemisia capillaris Thunb. and Bupleurum chinense DC., add water, distill, and collect the volatile oil for standby;

[0056] (2) Decoct the aqueous solution and the medicinal residues obtained in step (1) twice with water, each time for 1.5 hours, combine the decoctions and concentrate to obtain a concentrated solution;

[0057] (3) Add ethanol to the concentrated solution obtained in step (2) to make the alcohol content reach 70%, refrigerate at 0 - 4 °C for 48 hours, filter, recover the ethanol, add water again, refrigerate, and filter to obtain refined concentrated solution 1;

[0058] (4) Take the formula amount of Rheum palmatum, add water and decoct twice, each time for 1.5 hours, combine the decoction liquids and concentrate, add ethanol to make the alcohol content reach 70%, filter, recover the ethanol, add water again, refrigerate at 0 - 4 °C for 48 hours, and filter to obtain concentrated solution 2;

[0059] (5) Mix concentrated solution 1 and concentrated solution 2, dry, and add the volatile oil obtained in step (1) to obtain the described traditional Chinese medicine composition.

[0060] Comparative Example 3 A traditional Chinese medicine composition for treating gallstones

[0061] It consists of the following raw materials:

[0062] 75 parts of Isatis indigotica Fort. and 32 parts of Sophora flavescens Ait.

[0063] It is prepared according to the following method:

[0064] (1) Take the formula amount of Sophora flavescens Ait. and Isatis indigotica Fort., add water and decoct twice, each time for 1.5 hours, combine the decoction liquids and concentrate to obtain a concentrated solution;

[0065] (2) Add ethanol to the concentrated solution obtained in step (1) to make the alcohol content reach 70%, refrigerate at 0 - 4 °C for 48 hours, filter, recover the ethanol, add water again, refrigerate, and filter to obtain refined concentrated solution 1;

[0066] (3) Dry the refined concentrated solution 1 to obtain the described traditional Chinese medicine composition.

[0067] The above-mentioned Examples 1 - 4 and Comparative Example 1 were prepared into traditional Chinese medicine compositions according to the following method:

[0068] (1) Take the formula amount of Artemisia capillaris Thunb. and Bupleurum chinense DC., add water and distill, collect the volatile oil for later use;

[0069] (2) Decoct the aqueous solution after distillation in step (1) together with the medicinal residues, Sophora flavescens Ait. and Isatis indigotica Fort. twice with water, each time for 1.5 hours, combine the decoction liquids and concentrate to obtain a concentrated solution;

[0070] (3) Add ethanol to the concentrated solution obtained in step (2) to make the alcohol content reach 70%, refrigerate at 0 - 4 °C for 48 hours, filter, recover the ethanol, add water again, refrigerate, and filter to obtain refined concentrated solution 1;

[0071] (4) Add water to the formulated amount of rhubarb and boil it twice, each time for 1.5 hours. Combine the decoctions and concentrate them. Add ethanol to make the alcohol content reach 70%. Filter and recover the ethanol. Add water again and refrigerate at 0-4°C for 48 hours. Filter to obtain the refined concentrated solution 2.

[0072] (5) The refined concentrated solution 1 and the refined concentrated solution 2 are mixed, dried, and the volatile oil obtained in step (1) is added to obtain the traditional Chinese medicine composition.

[0073] Effect Experiment

[0074] 1.1 Experimental animals and groups

[0075] 120 SPF-grade healthy C57BL / 6 mice (6 weeks old, body weight 21-23 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021-0011) were selected and kept in a clean animal room at 25°C, maintaining relative humidity (40% ± 5%), and given free access to food and water. After 1 week, they were weighed, numbered, and randomly divided into 10 groups, namely: blank group, model group, Example 1 group, Example 2 group, Example 3 group, Example 4 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, and ursodeoxycholic acid group.

[0076] Ursodeoxycholic acid group: Ursodeoxycholic acid capsules, specification: 250 mg / capsule, batch number: National Medicine Standard H20213697, purchased from Anshi Pharmaceutical (Zhongshan) Co., Ltd.

[0077] 1.2 Construction of gallstone model

[0078] After the animals were grouped, except for the blank group which was given normal feed, the other groups were fed with high-fat feed to construct a gallstone mouse model: they were fed with high-fat, high-calorie, high-cholesterol (15% fat, 1% cholesterol, 0.5% bile acid) feed for 8 consecutive weeks.

[0079] 1.3 Administration

[0080] The Chinese medicine compositions prepared in Examples 1-4 and Comparative Examples 1-4 were dissolved in an appropriate amount of physiological saline for convenient gavage. Animals were grouped and then gavage administered once daily for 8 weeks. The blank and model groups were given physiological saline. The dosages for each group are shown in Table 1.

[0081]

[0082]

[0083] Test Example 1: Application of a Chinese medicine composition in the treatment and prevention of gallstones

[0084] General observation

[0085] Before the observation experiment and after the last administration, observe the general conditions of the animals in each group, including food intake, activity, mental state, and dorsal hair.

[0086] Before the experiment, the animals in each group drank water normally, had a good mental state, moved freely, reacted flexibly, were quick in movement, had shiny dorsal hair, had normal food intake, defecation, and urination, and no abnormal conditions occurred; after the experiment, the body weights of the rats in the blank group increased naturally, the condition was good, and the food intake, defecation, and urination were normal; the animals in the model group had reduced food intake, slow movement, arched backs, dull reactions, loose and dull dorsal hair, indicating successful modeling. Compared with the model group, the animals in the groups of Examples 1-4 and the ursodeoxycholic acid group had better improvements in aspects such as food intake, body weight, mental state, arched back condition, and dorsal hair state, and no animal death occurred during the experiment.

[0087] Test Example 2 Application of a traditional Chinese medicine composition in the treatment and prevention of gallstones

[0088] Gallstone formation rate in the gallbladder

[0089] After the last administration, the animals in each group were fasted but allowed to drink water for 12 h. Before sampling, the animals were anesthetized with 5% chloral hydrate (0.1 mL / 10 g) and fixed supine on the surgical board. The abdominal cavity was opened, the gallbladder was found, and the gallstone formation situation in the gallbladder was observed. Calculate the gallstone formation rate of the animals in each group: Gallstone formation rate (%) = Number of animals with gallbladder stones in each group / Number of animals in each group × 100%.

[0090] After dissection, gross observation showed that the bile of the rats in the blank group was clear and transparent; 91.67% of the animals in the model group had flocculent precipitates. The stone formation rates of Comparative Examples 1, 2, and 3 were higher than those of Examples 1, 2, 3, 4 groups and the ursodeoxycholic acid group, and flocculent precipitates were visible. Compared with the model group, the groups of Examples 1-4, Comparative Example 1 group, and the ursodeoxycholic acid group significantly reduced the turbidity of bile and inhibited the formation of cholesterol stones (p < 0.05). The effect of Example 1 group was equivalent to that of the ursodeoxycholic acid group. The data are shown in Table 2.

[0091] Table 2 Gallstone formation rate in the gallbladder of animals in each group (n, %)

[0092]

[0093]

[0094] Note: Chi-square test was used. Compared with the blank group, *p < 0.05, **p < 0.01; compared with the model group, #p < 0.05, ##p < 0.01; compared with Example 1 group, &p < 0.05, &&p < 0.01.

[0095] Test Example 3 Application of a traditional Chinese medicine composition in the treatment and prevention of gallstones

[0096] Detection of CYP7A1, HMGR, and BSEP mRNA Levels

[0097] After the last administration, the animals in each group were fasted but allowed water for 12 h. Before sampling, the animals were anesthetized with 5% chloral hydrate (0.1 mL / 10 g) and fixed supine on the operating board. The abdominal cavity was opened, and the liver was taken to detect the transcriptional levels of CYP7A1, HMGR, and BSEP mRNA using RT-PCR technology. The operation was as follows:

[0098] Total RNA was extracted from mouse liver tissues using Trizol (Invitrogen, USA, catalog number: 15596-026), and reverse transcription was completed using HiFiScriptT (Beijing Kangwei Reagent Company, catalog numbers: CW2582M, CW0957H). The experimental operations were carried out strictly according to the product instructions. Amplification was performed using the internal reference gene primers and target gene primers respectively, as shown in Table 3. The total volume of the reaction system was 20 μL. The program was as follows: The reaction solution was pre-denatured at 95 °C for 15 min, 1 cycle; denatured at 95 °C for 10 s, annealed at 58 °C for 30 s, and extended at 72 °C for 30 s, for 40 cycles. At the same time, melting curve analysis was performed at 60 - 90 °C. Finally, a fluorescence quantitative PCR instrument (Shanghai Fengling Biotechnology Co., Ltd., model: FTC-3000P) was used, and the relative quantitative analysis of the data was carried out using the 2 –△△Ct method, and the results obtained are shown in Table 4.

[0099] Table 3 Gene Primer Sequences

[0100]

[0101]

[0102] Table 4 Comparison of CYP7A1, HMGR, and BSEP mRNA Levels in Animals of Each Group

[0103] Group n CYP7A1 HMGR BSEP Blank group 12 0.98±0.05 0.62±0.29 0.98±0.11 Model group 12 <![CDATA[0.12±0.02 ** > <![CDATA[3.82±0.42 ** > <![CDATA[0.21±0.01 ** > Comparative example 1 group 12 <![CDATA[0.30±0.01 ##&& > <![CDATA[2.40±0.91 ##& > <![CDATA[0.49±0.09 ##&& > Comparative example 2 group 12 <![CDATA[0.28±0.08 ##&& > <![CDATA[2.79±0.49 ##&& > <![CDATA[0.37±0.08 ##&& > Comparative example 3 group 12 <![CDATA[0.13±0.04 && > <![CDATA[3.25±1.09 && > <![CDATA[0.28±0.14 && > Example 1 group 12 <![CDATA[0.46±0.05 ## > <![CDATA[1.47±1.27 ## > <![CDATA[0.61±0.09 ## > Example 2 group 12 <![CDATA[0.45±0.07 ## > <![CDATA[1.62±0.68 ## > <![CDATA[0.62±0.13 ## > Example 3 group 12 <![CDATA[0.48±0.08 ## > <![CDATA[1.51±0.11 ## > <![CDATA[0.61±0.02 ## > Example 4 group 12 <![CDATA[0.45±0.10 ## > <![CDATA[1.44±0.53 ## > <![CDATA[0.65±0.04 ## > Ursodeoxycholic acid group 12 <![CDATA[0.49±0.07 ## > <![CDATA[1.22±0.37 ## > <![CDATA[0.64±0.08 ## >

[0104] Note: T-test was used. Compared with the blank group, *p < 0.05, **p < 0.01; compared with the model group, #p < 0.05, ##p < 0.01; compared with the Example 1 group, &p < 0.05, &&p < 0.01.

[0105] Cholelithiasis refers to the disease of the biliary tract system, including the gallbladder and bile ducts, where stones occur. Its components consist of cholesterol, bilirubin, calcium salts, and mixed stones, etc. Its clinical manifestations depend on factors such as the location of the gallstones, whether it causes biliary tract obstruction and infection. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme that catalyzes the decomposition of cholesterol into bile acids in the liver, and HMG-CoA reductase (HMGR) is the key enzyme in the cholesterol synthesis pathway in the body. The activities of both are regulated by various factors and are crucial for maintaining cholesterol metabolism balance. The bile salt export pump (BSEP) is located on the cell membrane of the hepatocyte canalicular surface and is the most important transport protein for bile acid excretion. It is responsible for transporting bile salts from hepatocytes to the bile ductules, can maintain the balance of bile salts in the body, and reduce the possibility of gallstones.

[0106] Compared with the blank group, the mRNA expression levels of CYP7A1 and BSEP in the model group were significantly decreased, and the mRNA expression level of HMGR was significantly increased, with statistically significant differences (P<0.05); compared with the model group, the mRNA expression levels of CYP7A1 and BSEP in the comparative example 1-2 groups, the example 1-4 groups, and the ursodeoxycholic acid group were significantly increased, and the mRNA expression level of HMGR was significantly decreased, with statistically significant differences (P<0.05); compared with the example 1 group, the mRNA expression levels of CYP7A1 and BSEP in the comparative example 1-3 groups were significantly decreased, and the mRNA expression level of HMGR was significantly increased, with statistically significant differences (P<0.05); there was little difference in the effects between the example 2-4 groups and the example 1 group, and there were significant differences from the comparative example 3 group; the effects of the example 1 group were comparable to those of the ursodeoxycholic acid group.

[0107] Experimental example 4 Application of a traditional Chinese medicine composition in the treatment and prevention of gallstones

[0108] Detection of serum TG and TC levels

[0109] After the last administration, the animals in each group were fasted but allowed to drink water for 12 h. Before sampling, the animals were anesthetized with 5% chloral hydrate (0.1 mL / 10 g) and fixed supine on the operating board. The tail was wiped with warm water, and the tail tip of the mouse was quickly cut off by 0.5 - 1 cm with a sterile scalpel and directly dropped into the collection tube. The levels of triglyceride (TG) and total cholesterol (TC) in the serum of each group of mice were detected using an automatic biochemical analyzer (Beckman Coulter Biotechnology Co., Ltd., USA, model: AU-480). All reagent kits were purchased from Beckman Coulter Biotechnology Co., Ltd., USA, and the batch numbers were 695874 and 741854 respectively. The results obtained are shown in Table 5.

[0110] Table 5 Comparison of TG and TC levels in each group of animals ( n = 12, μmol / L)

[0111] Group TG TC Blank group 1.66±0.22 2.40±0.67 Model group <![CDATA[4.42±0.36 ** > <![CDATA[5.52±0.60 ** > Comparative example 1 group <![CDATA[3.29±0.32 ##&& > <![CDATA[4.73±0.39 ##&& > Comparative example 2 group <![CDATA[3.54±0.35 ##&& > <![CDATA[4.94±0.76 #&& > Comparative example 3 group <![CDATA[4.37±0.37 && > <![CDATA[5.26±0.53 && > Example 1 group <![CDATA[2.36±0.34 ## > <![CDATA[3.98±0.73 ## > Example 2 group <![CDATA[2.52±0.32 ## > <![CDATA[4.03±0.51 ## > Example 3 group <![CDATA[2.54±0.38 ## > <![CDATA[4.10±0.50 ## > Example 4 group <![CDATA[2.41±0.33 ## > <![CDATA[4.13±0.64 ## > Ursodeoxycholic acid group <![CDATA[2.40±0.35 ## > <![CDATA[4.10±0.67 ## >

[0112] Note: T - test was used. Compared with the blank group, *p < 0.05, **p < 0.01; compared with the model group, #p < 0.05, ##p < 0.01; compared with Example 1 group, &p < 0.05, &&p < 0.01.

[0113] Compared with the blank group, the levels of TG and TC in the serum of animals in the model group were significantly increased (P < 0.05); compared with the model group, the levels of TG and TC in the serum of animals in Comparative Example 1 - 2 groups, Example 1 - 4 groups and Ursodeoxycholic acid group were significantly decreased (P < 0.05); compared with Example 1 group, the levels of TG and TC in the serum of animals in Comparative Example 1 - 3 groups were significantly increased (P < 0.05). The effects of Example 2 - 4 groups were not much different from those of Example 1 group, and there were significant differences from Comparative Example 3 group; the effects of Example 1 group and Ursodeoxycholic acid group were comparable.

[0114] The present invention replicated a gallstone mouse model by the high - fat diet feeding method and systematically evaluated the effect of the composition prepared by the present invention on gallstones. The research results show that this composition exhibits significant pharmacological effects in improving the survival state of gallstone mice, reducing the gallstone formation rate in the gallbladder, inhibiting cholesterol cooperation, promoting bile excretion, and improving blood lipid metabolism; especially the compositions within a specific combination ratio range (Examples 1 - 4), whose effects are comparable to those of the positive control drug Ursodeoxycholic acid and even better in some indicators; in addition, although the combination outside the range (Comparative Example 1) shows certain therapeutic effects in some indicators, its overall effect is significantly lower than that of the compositions within the protected scope of this application, indicating that the multi - component synergistic effect of the composition may be an important mechanism for its efficacy.

[0115] In summary, the composition prepared by the present invention shows significant effects in reducing the gallstone formation rate in the gallbladder, inhibiting cholesterol cooperation, promoting bile excretion, and improving blood lipid metabolism during the prevention and treatment of gallstones, and has excellent multiple therapeutic effects.

[0116] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a traditional Chinese medicine composition in the preparation of a drug for preventing and treating gallstones, characterized in that, The described traditional Chinese medicine composition is composed of the following raw materials by weight: 90-110 parts of Artemisia capillaris, 90-110 parts of Bupleurum chinense, 65-85 parts of Isatis indigotica, 30-50 parts of Rheum palmatum, and 22-42 parts of Sophora flavescens.

2. The application according to claim 1, characterized in that, The described traditional Chinese medicine composition is composed of the following raw materials by weight: 100-110 parts of Artemisia capillaris, 90-100 parts of Bupleurum chinense, 75-85 parts of Isatis indigotica, 30-40 parts of Rheum palmatum, and 32-42 parts of Sophora flavescens.

3. The application according to claim 1, characterized in that The described traditional Chinese medicine composition is composed of the following raw materials by weight: 100 parts of Artemisia capillaris, 100 parts of Bupleurum chinense, 75 parts of Isatis indigotica, 40 parts of Rheum palmatum, and 32 parts of Sophora flavescens.

4. The application according to claim 1, characterized in that The preparation method of the described traditional Chinese medicine composition includes the following steps: (1) Take the formula amount of Artemisia capillaris and Bupleurum chinense, add water, distill, and collect the volatile oil for standby; (2) Add water to decoct the aqueous solution and medicinal residues after distillation in step (1), Sophora flavescens, and Isatis indigotica 2-3 times, 1-2 hours each time, combine the decoction liquids and concentrate to obtain a concentrated liquid; (3) Add ethanol to the concentrated liquid obtained in step (2) to make the alcohol content reach 70%-80%, refrigerate at 0-4°C for 48-60 hours, filter, recover ethanol, add water again, refrigerate, and filter to obtain refined concentrated liquid 1; (4) Take the formula amount of Rheum palmatum, add water to decoct 2-3 times, 1-2 hours each time, combine the decoction liquids and concentrate, add ethanol to make the alcohol content reach 70%-80%, filter, recover ethanol, add water again, refrigerate at 0-4°C for 48-60 hours, filter to obtain refined concentrated liquid 2; (5) Mix refined concentrated liquid 1 and refined concentrated liquid 2, dry, and add the volatile oil obtained in step (1) to obtain the described traditional Chinese medicine composition.

5. The application according to claim 1, characterized in that The preparation method of the described traditional Chinese medicine composition includes the following steps: (1) Take the formula amount of Artemisia capillaris and Bupleurum chinense, add water, distill, and collect the volatile oil for standby; (2) Add water to decoct the aqueous solution and medicinal residues after distillation in step (1), Sophora flavescens, and Isatis indigotica 2 times, 1.5 hours each time, combine the decoction liquids and concentrate to obtain a concentrated liquid; (3) Add ethanol to the concentrated liquid obtained in step (2) to make the alcohol content reach 70%, refrigerate at 0-4°C for 48 hours, filter, recover ethanol, add water again, refrigerate, and filter to obtain refined concentrated liquid 1; (4) Take the formula amount of Rheum palmatum, add water to decoct 2 times, 1.5 hours each time, combine the decoction liquids and concentrate, add ethanol to make the alcohol content reach 70%, filter, recover ethanol, add water again, refrigerate at 0-4°C for 48 hours, filter to obtain refined concentrated liquid 2; (5) Mix refined concentrated liquid 1 and refined concentrated liquid 2, dry, add the volatile oil obtained in step (1) to obtain the described traditional Chinese medicine composition.

6. The application according to claim 1, characterized in that, The described types of gallstones include cholesterol stones, pigment stones, or mixed stones.

7. The application according to claim 6, characterized in that, The described type of gallstone is cholesterol stone.

8. The application according to claim 1, characterized in that The dosage form of the described drug includes pills, powders, granules, capsules, injections, or tablets prepared by adding pharmaceutically acceptable excipients.

9. Use of a Kuhuang Granule in the preparation of a drug for preventing and treating gallstones, characterized in that, Containing the traditional Chinese medicine composition in the application described in claim 1 and pharmaceutically acceptable excipients.

10. Use of a Kuhuang injection in the preparation of a drug for preventing and treating gallstones, characterized in that, Containing the traditional Chinese medicine composition in the application described in claim 1 and pharmaceutically acceptable excipients.

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