Traditional Chinese medicine composition for treating chronic viral hepatitis B

Abstract

The invention discloses a traditional Chinese medicine composition for treating chronic viral hepatitis B. The traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 16-20 parts of herba artemisiae scopariae, 10-15 parts of fructus gardeniae, 4-8 parts of radix et rhizoma rhei, 13-17 parts of rhizoma atractylodis macrocephalae and 8-12 parts of pericarpium citri reticulatae. On the basis of the artemisia capillaris decoction, the atractylodes macrocephala koidz and the pericarpium citri reticulatae are combined, and the dosage proportion of all the medicines is precisely matched, so that the syndrome performance, liver function and liver pathological change of the CHB liver and gallbladder damp-heat syndrome are remarkably improved, FBG and TG are reduced, and the Lep and EPI levels are improved; the traditional Chinese medicine composition disclosed by the invention has incomparable advantages in the aspects of improving the typical clinical manifestation fatigue and weakness of the CHB liver and gallbladder damp-heat syndrome and highly active HBV replication.

Description

Technical Field

[0001] The present invention specifically relates to a Chinese medicine composition for treating chronic hepatitis B. Background Art

[0002] Chronic hepatitis B (CHB) is a chronic inflammatory disease of the liver caused by infection with the hepatitis B virus (HBV), with clinical manifestations such as fatigue, anorexia, nausea, abdominal distension, and pain in the liver area. CHB can be complicated by complications of multiple systems such as hepatic diabetes, which can lead to liver fibrosis, cirrhosis, and even liver cancer, seriously affecting the quality of life of patients and threatening people's health. According to data from the World Health Organization, there are approximately 257 million chronic HBV carriers worldwide, and approximately 887,000 people die each year from CHB-related diseases such as cirrhosis and liver cancer. Although Western antiviral treatment can effectively inhibit viral replication and control disease progression, there are still problems such as long medication cycles, drug side effects, and HBV drug-resistant mutations. Therefore, in-depth research on the prevention and treatment of CHB remains an important issue in my country's public health.

[0003] A large number of clinical practices have shown that the combined use of traditional Chinese medicine to treat CHB can make antiviral Western medicine "more effective and less toxic", alleviate patient symptoms, improve liver fibrosis, and reduce HBV drug-resistant mutations, which is superior to Western medicine alone. Traditional Chinese medicine treatment of chronic hepatitis B emphasizes syndrome differentiation and treatment, and formulates personalized treatment plans based on the patient's specific symptoms. The main syndromes include liver and gallbladder damp-heat syndrome, liver depression and spleen deficiency syndrome, etc. Studies have found that HBV replication is highly active in CHB with liver and gallbladder damp-heat syndrome, the degree of liver tissue inflammation is higher than other syndromes, and the degree of liver damage is more severe, which is a key link in the treatment of CHB. Therefore, it is of great significance to carry out research on the prevention and treatment of CHB liver and gallbladder damp-heat syndrome with traditional Chinese medicine to maximize the therapeutic advantages of traditional Chinese medicine, which is conducive to controlling the progression of CHB and reducing the incidence of CHB-related cirrhosis and even liver cancer.

[0004] Yinchenhao Decoction was created by Zhang Zhongjing, a famous doctor in the Eastern Han Dynasty. It comes from the Chinese medicine classic "Treatise on Febrile Diseases" "If the head sweats but the body does not, the neck is still, urination is not smooth, and the patient is thirsty and wants to drink water, this is due to blood stasis and heat in the body, and the body will turn yellow. Yinchenhao Decoction is the main treatment." Modern studies have found that Yinchenhao Decoction has a good effect in treating CHB. However, in the previous study of the present invention, it was found that for CHB with liver and gallbladder damp-heat syndrome, Yinchenhao Decoction did not have a positive effect in improving the feeling of fatigue and weakness caused by liver and gallbladder damp-heat, as well as HBeAg replication. Summary of the invention

[0005] To solve the above problems, the purpose of the present invention is to provide a method having a good therapeutic effect on CHB with liver and gallbladder damp-heat syndrome, which can improve the symptoms of CHB with liver and gallbladder damp-heat syndrome and improve the physiological and biochemical indicators of CHB, thereby controlling the occurrence and development of CHB.

[0006] The present invention provides a Chinese medicine composition for treating chronic hepatitis B, which is prepared from the following raw materials in weight proportion:

[0007] 16-20 parts of Artemisia capillaris, 10-15 parts of Gardenia jasminoides, 4-8 parts of Rhubarb, 13-17 parts of Atractylodes macrocephala, and 8-12 parts of dried tangerine peel.

[0008] Furthermore, it is prepared from the following raw materials in the following weight ratio:

[0009] 18 parts of Herba Artemisiae Scopariae, 12 parts of Fructus Gardeniae, 6 parts of Radix Rhei, 15 parts of Atractylodes Macrocephalae, 10 parts of Pericarpium Citri Reticulatae.

[0010] Furthermore, the Atractylodes macrocephala is stir-fried Atractylodes macrocephala.

[0011] Furthermore, it is a preparation prepared from the powder of the raw material drug, or the water or organic solvent extract of the raw material drug as the active ingredient, and pharmaceutically acceptable excipients.

[0012] Furthermore, the preparation is an oral preparation.

[0013] Furthermore, the oral preparation is a granule, paste, powder, pill or solution.

[0014] The present invention also provides a method for preparing the aforementioned pharmaceutical composition, which comprises the following steps:

[0015] (1) Weigh the API according to the above ratio;

[0016] (2) The raw material drug is ground into powder, or the water or organic solvent extract of the raw material drug is added with excipients or auxiliary ingredients commonly used in pharmaceuticals to obtain the powder.

[0017] Finally, the present invention provides a use of the above-mentioned composition in preparing a medicine for treating chronic hepatitis B.

[0018] Furthermore, the medicine is a medicine for treating chronic hepatitis B of liver and gallbladder damp-heat syndrome.

[0019] Furthermore, the drug has the effects of improving liver function and liver pathological changes, reducing FBG and TG, increasing Lep and EPI levels, improving fatigue, increasing activity, and / or reducing HBeAg levels.

[0020] The present invention combines Atractylodes macrocephala and dried orange peel on the basis of Yinchenhao decoction, which can not only clear away damp-heat in liver and gallbladder, but also invigorate the spleen and remove dampness, regulate qi and harmonize the middle, restrict the bitter and cold nature of the medicine, and prevent spleen and stomach damage. Yinchenhao decoction clears away damp-heat in liver and gallbladder and treats liver, Atractylodes macrocephala invigorates spleen and replenishes qi to invigorate spleen, and dried orange peel regulates qi and invigorates spleen to regulate spleen, which conforms to the purpose of liver disease strengthening spleen method. In the prescription, Yinchenhao clears away heat and removes dampness, soothes liver and promotes gallbladder as the main ingredient, Gardenia jaundices heat and removes dampness and relieves jaundice, Atractylodes macrocephala invigorates spleen and removes dampness, dried orange peel regulates qi and harmonizes the middle ingredient to regulate spleen as the minister, and rhubarb has laxative and heat-conducting properties as the adjuvant. Yinchenhao decoction combines Atractylodes macrocephala and dried orange peel to clear away damp-heat and invigorate qi and invigorate spleen, and purges damp-heat without causing bitter and cold damage to spleen and stomach.

[0021] The present invention proves through animal experiments that, on the basis of Yinchenhao decoction, white atractylodes and dried orange peel are used in combination, and the dosage ratio of each drug is properly matched, which significantly improves the symptom manifestations of CHB liver and gallbladder damp-heat syndrome, liver function and liver pathological changes, reduces FBG and TG, and improves Lep and EPI levels. Compared with Yinchenhao decoction, the Chinese medicine composition of the present invention has incomparable advantages in improving fatigue, a typical clinical manifestation of CHB liver and gallbladder damp-heat syndrome, and highly active HBV replication.

[0022] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0023] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 HBsAg levels in mice 3 days after tail vein injection NC, normal control group; MODEL, model group. Compared with the normal control group, *p<0.05, **p<0.01;

[0025] Figure 2 Changes of serum bilirubin in mice NC, normal control group; MODEL, model group; compared with the normal control group, *p<0.05;

[0026] Figure 3 Dermoscopic images; NC, normal control group; MODEL, model group;

[0027] Figure 4 HE staining of mouse skin; NC, normal control group; MODEL, model group; 10× scale bar is 100μm, 20× scale bar is 50μm;

[0028] Figure 5 Changes of rectal temperature in mice during moist heat modeling NC, normal control group; Model, model group; compared with the normal control group, *p<0.05, **p<0.01;

[0029] Figure 6 Changes in water intake of mice during damp-heat modeling A: Line graph of water intake, B: Bar graph of water intake; NC, normal control group; MODEL, model group; compared with the normal control group, *p<0.05, **p<0.01;

[0030] Figure 7 The salivary flow rate of mice at different time points of moist heat modeling; A: comparison between different groups; B: comparison between the same group; W5, 9, 13, 5, 9, 13 weeks of moist heat modeling; NC, normal control group; MODEL, model group; compared with the normal control group, *p<0.05, **p<0.01, ***p<0.001, ns, no statistical difference;

[0031] Figure 8 HE staining of mouse submandibular gland; NC, normal control group; MODEL, model group; 10× scale bar is 100μm, 20× scale bar is 50μm;

[0032] Fig. 9 Changes of food intake and serum leptin in mice during damp-heat modeling A: food intake; B: LEP; NC, normal control group; Model, model group; compared with the normal control group, *p<0.05, **p<0.01, ns, no statistical difference;

[0033] Fig.10 Weighted swimming time of mice A: Comparison between different groups; B: Comparison within the same group; NC, normal control group; MODEL, model group; Comparison between the two groups, *p<0.05, **p<0.01, ns, no statistical difference

[0034] Fig.11 Blood glucose levels in mice A: fasting blood glucose on the 5th day, B: fasting blood glucose on the 13th day; NC, normal control group; MODEL, model group; compared with the normal control group, *p<0.05, **p<0.01, ns, no statistical difference;

[0035] Fig.12 Serum adrenaline levels in mice NC, normal control group; MODEL, model group; compared with the normal control group, **p<0.01;

[0036] Fig.13 Gastrointestinal motility of mice; A. Gastric emptying rate B. Small intestinal propulsion rate C. Serum gastrin D. Serum motilin NC, normal control group; Model, model group; compared with the normal control group, *p<0.05, ns, no statistical difference;

[0037] Fig.14 Mouse behavior at different times A: total distance, B: central area visit time; NC, normal control group; Model, model group; comparison between the two groups, *p<0.05, **p<0.01, ns, no statistical difference;

[0038] Fig.15 Mouse trajectory diagram and time heat map; 1_1_1 represents the first time of the first mouse in group 1, and so on. In W0 and W5, 1, normal control group; 2, model group; in W11 and W13, C, normal control group; M, model group;

[0039] Fig.16 Bilirubin content of mice in each group NC, normal control group; Model, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. Compared with the normal control group, *p<0.05; compared with the model group, #p<0.05, ##p<0.01, ###p<0.001;

[0040] Fig.17 HE staining of skin; NC, normal control group; MODEL, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. 10× scale bar is 100μm, 20× scale bar is 50μm;

[0041] Fig.18 Changes in rectal temperature of mice during treatment (℃) A: Rectal temperature line graph.; B: Rectal temperature bar graph; NC, blank control group; MODEL, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. Compared with the normal control group, *p<0.05; compared with the model group, #p<0.05, ns, no statistical difference;

[0042] Fig.19 Changes in water intake of mice during treatment A: Line graph of water intake.; B: Bar graph of water intake; NC, normal control group; MODEL, experimental group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. Compared with the normal control group, *p<0.05, **p<0.01; compared with the model group, #p<0.05;

[0043] Fig. 20 Mouse salivary flow rate NC, normal control group; MODEL, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. ns, no statistical difference;

[0044] Fig.21 Changes of food intake and serum leptin in mice during treatment A: Food intake; B: LEP; NC, blank control group; MODEL, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. Compared with the normal control group, *p<0.05, **p<0.01, ***p<0.001; compared with the model group, #p<0.05, ##p<0.01; compared with the modified Yinchenhao decoction group, △p<0.05, △△p<0.01;

[0045] Fig. 22 A. Weight-bearing swimming time B. Fasting blood glucose C. Serum adrenaline content of mice in each group NC, normal control group; Model, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. Compared with the normal control group, *p<0.05; compared with the model group, #p<0.05, ##p<0.01; compared with the modified Yinchenhao decoction group, △p<0.05, △△p<0.01;

[0046] Fig.23 Gastrointestinal motility of mice; A. Gastric emptying rate B. Small intestinal propulsion rate C. Serum gastrin D. Serum motilin NC, normal control group; MODEL, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. Compared with the normal control group, *p<0.05, **p<0.01, ***p<0.001; compared with the model group, #p<0.05;

[0047] Fig.24 The distance traveled and average speed in the central area of ​​the open field of mice after 4 weeks of drug administration A, C: total distance, B, D: central area visit time; NC, normal control group; MODEL, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. Compared with the normal control group, *p<0.05, **p<0.01, ***p<0.001; compared with the model group, #p<0.05; ns, no statistically significant difference;

[0048] Fig.25 Representative heat maps of open field activity trajectories and residence time of mice; C, normal control group; M, model group; Y, Yinchenhao decoction group; B, modified Yinchenhao decoction group; U, ursodeoxycholic acid group;

[0049] Fig.26 Changes of biochemical indexes in mice after 4 weeks of drug administration A: ALT, B: AST, C: TC, D: TG; NC, normal control group; MODEL, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. Compared with the normal control group, *p<0.05, **p<0.01; compared with the model group, #p<0.05, ##p<0.01;

[0050] Fig. 27 Pathological changes in mouse liver; NC, normal control group; MODEL, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group;

[0051] Fig.28 Pathological changes of colon tissue; NC, normal control group; MODEL, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group;

[0052] Fig.29 Changes of serum HBsAg (A), HBeAg (B), and HBV DNA (C) in mice after 4 weeks of administration NC, normal control group; Model, model group; YCHT, Yinchenhao decoction group; JWYCH, modified Yinchenhao decoction group; UDCA, ursodeoxycholic acid group. Compared with the normal control group, *p<0.05, **p<0.01, ***p<0.01; compared with the model group, #p<0.05, ##p<0.01. DETAILED DESCRIPTION

[0053] The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products. Among them, the preparation process of modern Chinese medicine preparations made from the Chinese medicine composition of the present invention is mature and can be made by referring to the conventional methods of the prior art.

[0054] Example 1. Preparation of the composition of the present invention

[0055] Recipe: Artemisia capillaris 18g, Gardenia 12g, Rhubarb 6g, Stir-fried Atractylodes macrocephala 15g, Tangerine peel 10g.

[0056] Preparation method: Weigh the above raw materials according to the ratio, add 10 times the amount of water and boil twice, each time for 30 minutes, filter, combine the filtrate, decompress the filtrate and concentrate it into a paste, add the commonly used auxiliary materials in medicines to make granules.

[0057] Example 2. Preparation of the composition of the present invention

[0058] Recipe: Artemisia capillaris 16g, Gardenia 10g, Rhubarb 4g, Stir-fried Atractylodes macrocephala 13g, Tangerine peel 8g

[0059] Preparation method: Weigh the above raw materials according to the ratio, add 8 times the amount of water and boil twice, each time for 20 minutes, filter, and combine the filtrate to obtain the medicinal solution.

[0060] Example 3. Preparation of the composition of the present invention

[0061] Recipe: Artemisia capillaris 20g, Gardenia 15g, Rhubarb 8g, Stir-fried Atractylodes macrocephala 17g, Tangerine peel 112g.

[0062] Preparation method: Weigh the above raw materials according to the ratio, add 10 times the amount of water and boil twice, each time for 30 minutes, filter, combine the filtrate, decompress the filtrate into a thick paste, add the commonly used auxiliary materials in medicines to make tablets.

[0063] The beneficial effects of the present invention are described below through test examples.

[0064] Experimental Example 1 Study on the Treatment of Chronic Hepatitis B Virus Hepatitis by the Chinese Medicine Composition of the Present Invention

[0065] In the previous research, based on the good efficacy of Yinchenhao Decoction in the treatment of CHB, it was applied to CHB with liver-gallbladder damp-heat syndrome, and it was found that the efficacy was poor. Therefore, based on the syndrome differentiation and prescription formulation of CHB liver-gallbladder damp-heat syndrome, Atractylodes macrocephala was added to Yinchenhao Decoction, and it was found that it could reduce the HBeAg level in CHB. However, it could not effectively reduce the HBeAg level for the highly active HBV in CHB with liver-gallbladder damp-heat syndrome, and it had no effective improvement on the fatigue caused by liver-gallbladder damp-heat. Based on this, the syndromes composed of syndrome elements such as the location and nature of the disease in the process of CHB with liver-gallbladder damp-heat syndrome were identified and treated in a simple and concise manner, and the prescription was accurately differentiated. Atractylodes macrocephala and dried tangerine peel were added to Yinchenhao Decoction, and the dosage of each drug was adjusted to obtain a further improved Yinchenhao Decoction, in order to study its therapeutic effect on CHB with liver-gallbladder damp-heat syndrome.

[0066] 1. Experimental Materials

[0067] 1.1 Experimental animals

[0068] 90 4-6 week old SPF male wild-type C57BL / 6J mice were purchased from Beijing Huafukang Company, license number: SCXK (Beijing) 2019-0008, batch number: 1103222301023714. The normal control group and the experimental group were fed with ordinary feed and high-fat feed, respectively, and had free access to water. Ordinary maintenance feed was purchased from Chengdu Dashuo Company, and 60% high-fat feed (TP 23400) was purchased from Nantong Trofi Feed Technology Co., Ltd. The ingredients of high-fat feed were: casein, dextrin, sucrose, soybean oil, lard, cellulose, minerals, vitamins, L-cystine, choline bitartrate, TBHQ. Calories: 5.0 kcal / g, protein accounted for 14.1%, carbohydrates accounted for 25.9%, and fat accounted for 60.0%. Cholesterol content: 217 mg / kg.

[0069] 1.2 Main reagents and instruments

[0070] Table 1 Experimental reagents

[0071]

[0072] Table 2 ELISA and PCR kits

[0073]

[0074]

[0075] Table 3 Main instruments

[0076]

[0077] 2 Experimental methods

[0078] 2.1 Animal model preparation and group administration

[0079] 2.1.1 Model preparation method

[0080] Preparation method of HBV infection mouse model: After one week of adaptive feeding, rAAV-1.3HBV was injected into the tail vein and diluted with PBS buffer to a titer of 1×10 10 GC, injection volume 200 μL / mouse, 3 days after modeling, blood was collected from the orbits to detect HBsAg and evaluate whether the disease modeling was successful.

[0081] Preparation method of liver-gallbladder damp-heat syndrome model: After the mouse HBV infection model is successfully replicated, it is fed with high-fat feed and placed in a damp heat box for 9 weeks (temperature 30°C, relative humidity 95%, 6 hours a day), and high-fat diet for 13 weeks. When the high-fat diet is fed for the 10th week, the mice are gavaged with 50 mg / kg ANIT (5 mg / mL dissolved in olive oil) to establish the model once a week until the 13th week. The success of the model is evaluated by serum bilirubin, rectal temperature, water intake, salivary flow rate, food intake, blood sugar, motilin, gastrin, open field test, etc. When bilirubin increases, rectal temperature increases, and food intake decreases, it indicates that the liver-gallbladder damp-heat syndrome model is successfully established.

[0082] 2.1.2 Grouped drug administration method

[0083] In this study, 90 mice were divided into a normal control group (NC, n = 18) and a model group (Model, n = 18), a Yinchenhao decoction group (YCHT, n = 18), a modified Yinchenhao decoction group (JWYCH, n = 18), and an ursodeoxycholic acid capsule group (UDCA, n = 18). Except for the normal control group and the model group, each treatment group was treated with Yinchenhao decoction, modified Yinchenhao decoction, and ursodeoxycholic acid aqueous solution by gavage. The dosage of mice was calculated according to the clinical adult dosage and the equivalent dose coefficient was 9.1. The Yinchenhao decoction group (4.6 mg / g), the modified Yinchenhao decoction group (6.6 mg / g), and the ursodeoxycholic acid capsule group (60 mg / kg) were gavaged with aqueous solution, and the gavage volume was 100 μL / 10g, and the administration was continued for 4 weeks.

[0084] After intraperitoneal injection of pilocarpine (used to measure salivary flow rate, not used in subsequent experiments) in the model group, the mice secreted too much saliva, and one of them died of suffocation; electrocardiogram examination of the mice in the Yinchenhao decoction group (used to evaluate heart rate, not used in subsequent experiments) showed that one mouse died of anesthesia, and two mice died after ANIT modeling; three mice in the ursodeoxycholic acid group died after ANIT modeling; there was no unexpected death in the mice in the modified Yinchenhao decoction group.

[0085] Yinchenhao decoction composition: 18g of Yinchenhao, 12g of Gardenia, 6g of Rhubarb. Jiawei Yinchenhao decoction composition: 18g of Yinchenhao, 12g of Gardenia, 6g of Rhubarb, 15g of stir-fried Atractylodes macrocephala, 10g of dried orange peel. The granules prepared according to Example 1 were used, and the aqueous solution preparation method was: the granules were dissolved in boiling water and shaken to mix. Positive control drug: ursodeoxycholic acid capsules, manufacturer: Losan Pharma GmbH, specification 250mg / capsule, and the aqueous solution preparation method for intragastric administration was: the capsules were dissolved in boiling water and shaken to mix.

[0086] 3 Experimental results

[0087] 3.1 Disease model evaluation

[0088] Three days after AAV-HBV was injected into the tail vein of mice, the HBsAg level in the model group increased compared with that in the normal control group (P<0.01), indicating that the HBV infection model in mice was successfully established ( Figure 1 ).

[0089] 3.2 Evaluation of the liver-gallbladder damp-heat syndrome model

[0090] 3.2.1 Bilirubin, dermatoscopy and skin HE staining

[0091] Compared with the normal control group, the serum Bb level of mice in the model group was significantly increased after damp-heat syndrome modeling (p<0.05) ( Figure 2 ). Dermoscopic observation showed that the hair in the model group was greasy and slightly sparse compared with the normal control group, and the skin color of the two groups did not show obvious yellowing ( Figure 3 ).

[0092] The epidermis, dermis, subcutaneous tissue and muscle fiber structures of mice in the normal control group were clear, with no bleeding or obvious inflammatory cell infiltration. The subcutaneous tissue and muscle tissue of the model group were homogeneously stained light red, with thickened spinous processes and no obvious inflammatory cell infiltration ( Figure 4 ).

[0093] 3.2.2 Rectal temperature measurement

[0094] Before the modeling, there was no statistical difference in rectal temperature between the normal control group and the model group (p>0.05). Starting from the first week of damp-heat modeling, the rectal temperature of mice in the model group and the normal control group gradually increased, reaching a peak of 36°C in the third week of modeling. By the fourth week of modeling, the rectal temperature of both groups dropped back to around 34.5°C. From then on until the end of modeling, the rectal temperature of mice in the model group was always higher than that of the normal control group, and the difference between the two groups was statistically significant at the 6th, 7th, 8th, 12th, and 13th weeks of modeling (p<0.05) ( Figure 5 ).

[0095] 3.2.3 Measurement of water intake and saliva flow rate

[0096] Before modeling, there was no statistical difference in water intake between the two groups (p>0.05). During the modeling process, the water intake of the normal control group gradually increased with the extension of modeling time, and the overall fluctuation of water intake of the model group mice was large, and the water intake of the model group was lower than that of the normal control group during the entire modeling period. Among them, the difference in water intake from the 2nd week to the 6th week and from the 8th week to the 13th week was statistically significant (p<0.05).

[0097] The water intake of mice in the normal control group was compared every 4 weeks. The water intake in the 5th, 9th and 13th weeks was significantly increased compared with that in the 1st week (p<0.01), while there was no significant change between the 5th, 9th and 13th weeks (p>0.05). The water intake of mice in the model group was compared every 4 weeks. The water intake in the 13th week of modeling was significantly increased compared with that in the 1st week (p<0.05), while there was no significant change in water intake between other time points of modeling (p>0.05) ( Figure 6 ).

[0098] Compared with the normal control group, there was no statistical difference in the salivary flow rate of mice in the model group (p>0.05). The salivary flow rate of mice showed an increasing trend over time. The salivary flow rate of the normal control group at the 13th week of modeling was higher than that at the 5th week (p<0.05). The salivary flow rate of the model group at the 13th week of modeling was significantly higher than that at the 5th week (p<0.001) and higher than that at the 9th week (p<0.05) ( Figure 7 ).

[0099] At 6 weeks of damp-heat modeling, the submandibular glands of mice were taken for HE staining. The submandibular glands of the normal control group and the model group were intact in structure, with obvious lobes, tightly arranged glandular ducts, dense ducts and glandular cells, no obvious acinar atrophy, connective tissue hyperplasia, etc., and no obvious inflammatory granule infiltration, indicating that damp-heat syndrome did not affect the morphological structure of the submandibular glands ( Figure 8 ).

[0100] 3.2.4 Food intake and leptin measurement

[0101] During the damp-heat syndrome modeling period, the food intake of the model group was always less than that of the normal control group mice, and the difference in food intake in the 6th week and the 9th to 13th week was statistically significant (p<0.05) ( Fig. 9 A).

[0102] Compared with the normal control group, the serum leptin level in the model group increased, but the difference was not statistically significant (p>0.05)( Fig. 9 B).

[0103] 3.2.5 Weighted swimming time measurement

[0104] At the 5th, 9th and 13th weeks of wet heat modeling, the weighted swimming time of mice in the model group was shortened compared with that in the normal control group, but the difference was not statistically significant (p>0.05). In the longitudinal comparison between the two groups, compared with the 5th week of modeling, the weighted swimming time of the normal control group and the model group at the 9th week of modeling was significantly increased (p<0.05), while the weighted swimming time of the two groups at the 13th week of modeling was significantly shortened compared with the 9th week (p<0.05), and the change trend was consistent ( Fig.10 ).

[0105] 3.2.6 Blood glucose and adrenaline determination

[0106] At the 5th week of damp-heat modeling, the FBG of mice in the model group increased compared with that in the normal control group (p<0.05). At the 13th week of modeling, the FBG of the model group was significantly higher than that in the normal control group (p<0.01). Fig.11 ), which indicates that the ability to regulate sugar metabolism is impaired in damp-heat syndrome, and pain may occur.

[0107] Compared with the normal control group, the serum adrenaline content of mice in the model group decreased (p<0.01) ( Fig.12 ).

[0108] 3.2.7 Gastric emptying rate, small intestinal propulsion rate, GAS, and MTL determination

[0109] Compared with the normal control group, there was no statistical difference in gastric emptying rate and intestinal propulsion rate in the model group (p>0.05) ( Fig.13 AB).

[0110] Compared with the normal control group, the mean serum GAS level of mice in the model group was slightly increased, but there was no statistical difference (p>0.05); compared with the normal control group, the serum MTL level of mice in the model group was significantly increased (p<0.05), indicating that the mice in the model group had gastrointestinal motility abnormalities ( Fig.13 CD).

[0111] 3.2.8 Routine examination of bowel movements

[0112] According to the naked eye observation, during the modeling process, the urine of the mice in the model group was yellower than that of the mice in the normal control group. Urinalysis showed that there was no difference in urobilinogen, urine ketones, bilirubin, glucose, vitamin C, urine protein, urine occult blood, urine leukocytes, nitrite, and urine specific gravity between the model group and the normal control group (Table 4). According to the naked eye observation, the stool of the model group was softer than that of the normal control group, and no significant difference was found in the routine stool test of red blood cells, white blood cells, epithelial cells, fat globules, etc. (Table 5).

[0113] Table 4 Routine urine test results of mice

[0114]

[0115] Table 5 Routine results of mouse stool

[0116]

[0117]

[0118] Note: C1-C5 are normal control groups, M1-M5 are model groups; RBC, red blood cells; HP, high power microscope field; WBC, white blood cells; +, small amount; ++, medium amount.

[0119] 3.2.9 Open field test

[0120] Before modeling, there was no statistically significant difference in the total distance traveled in the open field test between the model group and the normal control group (p>0.05). At the 5th and 11th weeks of wet heat modeling, the total distance traveled in the model group was significantly reduced (p<0.05), and there was no statistically significant difference in the time spent in the central area between the two groups (p>0.05) ( Figures 14-15 ).

[0121] 3.3 Results of modified Yinchenhao decoction in the treatment of CHB with liver-gallbladder damp-heat syndrome

[0122] 3.3.1 Modified Yinchenhao Decoction improves CHB liver and gallbladder damp-heat syndrome

[0123] 3.3.1.1 Bilirubin and skin HE staining

[0124] Compared with the normal control group, the serum bilirubin in the model group was significantly increased (p<0.05). Compared with the model group, the bilirubin concentration in each treatment group was significantly decreased (p<0.05). There was no statistical difference in the change of bilirubin between the modified Yinchenhao decoction group and the Yinchenhao decoction group (p>0.05). Fig.16 The epidermis, dermis, subcutaneous tissue and muscle fiber structures of mice in each drug-treated group were clear, with no bleeding or obvious inflammatory cell infiltration. There was no significant difference between the drug-treated groups ( Fig.17 ). 3.3.1.2 Rectal temperature

[0125] At the first and second weeks of administration, the rectal temperature in the modified Yinchenhao decoction group and the ursodeoxycholic acid group was significantly lower than that in the model group (p<0.05). At the third week of administration, there was no significant difference in rectal temperature between the treatment groups and the normal control group (p>0.05). At the fourth week of administration, the rectal temperature in the ursodeoxycholic acid group was lower than that in the model group (p<0.05), and there was no statistical difference in rectal temperature between the treatment groups (p>0.05). Fig.18 A).

[0126] Compared with before drug intervention, the rectal temperature of the normal control group, model group, Yinchenhao decoction group and ursodeoxycholic acid group increased (p<0.05), while there was no significant difference in the rectal temperature of the modified Yinchenhao decoction group (p>0.05) ( Fig.18 B).

[0127] 3.3.1.3 Water intake and saliva flow rate

[0128] Before the drug intervention, there was no statistical difference in water intake between the drug groups and the model group (p>0.05). From the first to the second week of drug intervention, compared with the model group, the water intake of the modified Yinchenhao decoction group was significantly reduced (p<0.05). At the third week, the water intake increased. Compared with the model group, the water intake of the Yinchenhao decoction group and the ursodeoxycholic acid group was significantly increased (p<0.05). At the fourth week, the water intake of each drug group increased compared with the model group, but there was no statistical difference (p>0.05). Fig.19 A).

[0129] Compared with before administration, there was no significant difference in water intake among the groups after 4 weeks of administration (p>0.05) ( Fig.19 B).

[0130] Compared with the model group, there was no significant difference in salivary flow rate among the drug-treated groups (p>0.05)( Fig. 20 ).

[0131] 3.3.1.4 Food intake and leptin

[0132] During the administration period, the food intake of each drug group was less than that of the normal control group (p<0.05). In the first week of administration, compared with the model group, the food intake of the modified Yinchenhao decoction group and the ursodeoxycholic acid group was significantly reduced (p<0.05). From the third week of administration, the food intake of each drug group gradually increased, and there was no statistical difference with the model group (p>0.05). There was no statistical difference in food intake between the drug groups (p>0.05) ( Fig.21 A).

[0133] After drug intervention, the serum leptin level in the modified Yinchenhao decoction group was higher than that in the model group, Yinchenhao decoction group and ursodeoxycholic acid group (p<0.01), and there was no significant difference between the other drug groups and the model group (p>0.05) ( Fig.21 B).

[0134] 3.3.1.5 Weighted swimming time

[0135] After drug intervention, the weight-bearing swimming time of mice in the modified Yinchenhao decoction group was significantly longer than that in the model group, Yinchenhao decoction group, and ursodeoxycholic acid group (p<0.05), while there was no statistical difference between the Yinchenhao decoction group and the ursodeoxycholic acid group and the model group (p>0.05) ( Fig. 22 A).

[0136] 3.3.1.6 Blood sugar and adrenaline

[0137] Compared with the normal control group, the blood glucose level of mice in the model group was significantly increased (p<0.05). Compared with the model group, the fasting blood glucose level of each drug-treated group decreased, and there was no statistical difference with the normal control group (p>0.05). There was no statistically significant difference in fasting blood glucose between the drug-treated groups (p>0.05). Fig. 22 B).

[0138] Compared with the normal control group, the EPI of the model group was significantly decreased (p<0.05), and the serum EPI of the modified Yinchenhao decoction group was significantly increased compared with the model group (p<0.05). There was no significant difference between the Yinchenhao decoction group and the ursodeoxycholic acid group and the model group (p>0.05). Fig. 22 C).

[0139] 3.3.1.7 Gastric emptying rate, small intestinal propulsion rate, GAS, and MTL

[0140] Compared with the model group, the gastric emptying rate of mice in the ursodeoxycholic acid group was increased (p<0.05), and there was no significant difference between the other drug groups and the model group (p>0.05); the gastric emptying rate of the modified Yinchenhao decoction group was higher than that of the normal control group (p<0.01); there was no significant difference in gastric emptying rate among the drug groups (p>0.05). There was no significant difference in small intestinal propulsion rate among the groups (p>0.05) Fig.23 AB).

[0141] There was no statistical difference in the serum GAS content of mice in each group (p>0.05). Compared with the normal control group, the MTL level in the model group was significantly increased (p>0.05); compared with the model group, the MTL level in the Yinchenhao decoction group, the modified Yinchenhao decoction group and the ursodeoxycholic acid group decreased, and the difference was not statistically significant (p>0.05) ( Fig.23 CD).

[0142] 3.3.1.8 Characteristics of stool and feces

[0143] Macroscopic observation showed that the urine color of the mice in the Yinchenhao Decoction group and the modified Yinchenhao Decoction group was lighter than that of the model group, and the stool of the Yinchenhao Decoction group and the modified Yinchenhao Decoction group was brown and looser than that of the model group. There was no significant difference in the stool between the ursodeoxycholic acid group and the model group.

[0144] 3.3.1.9 Open field test

[0145] At the second week of drug intervention, compared with the normal control group, the total distance traveled by mice in the model group was significantly reduced (p<0.001), and compared with the model group, there was no significant difference in the total distance traveled by mice in each drug-treated group (p>0.05). At the fourth week of drug administration, compared with the model group, the total distance traveled by mice in the modified Yinchenhao decoction group was significantly increased (p<0.05), and there was no statistical difference between the other drug-treated groups and the model group (p>0.05). Compared with the second week of drug intervention, the total distance traveled by mice in each drug-treated group increased in the fourth week of intervention (p<0.05). There was no statistically significant difference in the central area visit time and different intervention times among the groups (p>0.05) ( Figures 24-25 ).

[0146] 3.3.2 Improvement of serum biochemistry in CHB patients with modified Yinchenhao decoction

[0147] After 4 weeks of drug intervention, compared with the normal control group mice, the serum ALT, AST, and TC in the model group were increased (p<0.05), indicating that in addition to liver function damage, lipid metabolism regulation function was also damaged.

[0148] Compared with the model group, ALT levels in the modified Yinchenhao decoction group and the ursodeoxycholic acid group were significantly reduced (p<0.05), and there was no significant difference among the medication groups (p>0.05).

[0149] Compared with the model group, AST in the Yinchenhao decoction group, modified Yinchenhao decoction group, and ursodeoxycholic acid group was significantly reduced (p<0.05), and there was no statistical difference among the medication groups (p>0.05).

[0150] Compared with the model group, there was no statistical difference in TC levels among the drug-treated groups (p>0.05).

[0151] Compared with the normal control group, the TG levels of each drug-treated group were significantly reduced (p<0.05); compared with the model group, the TG levels of the ursodeoxycholic acid group were significantly reduced (p<0.01), and there was no significant difference in TG levels in the other drug-treated groups (p>0.05) ( Fig.26 ).

[0152] 3.3.3 Modified Yinchenhao Decoction relieves CHB liver and gallbladder damp-heat syndrome liver and colon histopathology

[0153] The hepatocytes of mice in the normal control group had normal morphology, intact hepatic lobule structure, neatly arranged hepatic cords, and no hepatocyte necrosis or inflammatory cell infiltration was observed; the hepatocytes of the model mice showed alveolar hepatocyte fatty degeneration, with a small amount of collagen fiber production. Oil red O staining showed that no red fat droplets were observed in the normal control group, while obvious fat infiltration appeared in the model group. The fatty degeneration of the liver in each drug-treated group was alleviated compared with the model group, and the liver pathological damage was improved. The fat droplets in the modified Yinchenhao decoction group were reduced compared with the Yinchenhao decoction group and the ursodeoxycholic acid group ( Fig. 27 ).

[0154] Colon HE staining showed that the mucosal layer, submucosal layer and muscular layer of the control group mice were arranged normally, the absorptive columnar cells at the top of the mucosal layer were arranged neatly, and the lamina propria contained a small amount of lymphocytes. The muscular layer of the model group became thinner, and the lamina propria was infiltrated with inflammatory cells. The inflammatory cells in the drug-treated group were reduced compared with the model group, and there was no significant difference between the drug-treated groups ( Fig.28 ).

[0155] 3.3.4 Modified Yinchenhao Decoction regulates CHB viral indexes

[0156] Compared with the normal control group, the HBsAg and HBeAg levels of mice in the model group were significantly increased (p<0.05).

[0157] Compared with the model group, the mean HBsAg levels in the Yinchenhao decoction group, modified Yinchenhao decoction group, and ursodeoxycholic acid group were reduced, but there was no statistically significant difference (p>0.05).

[0158] Compared with the model group, the HBeAg level in the modified Yinchenhao decoction group was significantly reduced (p<0.05), while there was no significant difference between the Yinchenhao decoction group and the ursodeoxycholic acid group (p>0.05).

[0159] There was no significant difference in HBsAg, HBeAg, and HBV DNA between the treatment groups (p>0.05) ( Fig.29 ).

[0160] 4. Discussion

[0161] 4.1 Discussion on the evaluation of the CHB liver-gallbladder damp-heat syndrome model

[0162] The animal model of disease and syndrome combination is an important basis for exploring the essence of TCM syndrome and the development of clinical new drugs. The present invention uses tail vein injection of rAAV-HBV1.3 to construct a CHB immune tolerance mouse model, and combines the use of a hot and humid box + high-fat diet feeding + ANIT gavage to model liver and gallbladder damp-heat syndrome. After modeling, the mice in the model group showed jaundice, fever, reduced food intake, reduced weight-bearing swimming time, increased blood sugar, decreased adrenaline, and increased motilin.

[0163] After successfully obtaining the CHB liver-gallbladder damp-heat syndrome model, Yinchenhao decoction and modified Yinchenhao decoction were used to intervene to evaluate whether the syndrome model was successful. The results showed that after the intervention of the prescription and medicine, the serum bilirubin of the model mice was significantly reduced, the rectal temperature was reduced, the diet and water intake were increased, the weighted swimming time was prolonged, the blood sugar was reduced, the gastric emptying rate was increased, the total activity distance was increased, the serum ALT and AST levels were significantly reduced, the liver function was improved, and the HBeAg level was reduced. The prescription test showed that the CHB liver-gallbladder damp-heat syndrome model was established.

[0164] 4.2 Discussion on the effect of modified Yinchenhao decoction in treating CHB with liver-gallbladder damp-heat syndrome

[0165] The present invention combines stir-fried Atractylodes macrocephala and dried tangerine peel with Yinchenhao decoction to treat CHB liver and gallbladder damp-heat syndrome. The results show that compared with Yinchenhao decoction alone, it has more advantages in improving fatigue and weakness of model mice, increasing weight-bearing swimming time, increasing total activity distance, increasing LEP and EPI levels, and reducing HBeAg levels of model mice, but its mechanism has not yet been revealed.

[0166] 5. Summary

[0167] This study established a mouse model of CHB liver and gallbladder damp-heat syndrome through AAV-HBV+high-fat diet+humid heat chamber+ANIT combined modeling, which was manifested by jaundice, fever, reduced diet, fatigue, reduced activity, etc. Pathological changes were manifested by alveolar fatty degeneration of hepatocytes and a small amount of inflammatory cell infiltration, and serology was characterized by increased ALT, AST, HBsAg, and HBeAg levels. At the same time, FPG and TC in mice in the model group increased, indicating that abnormal glucose and lipid metabolism may have occurred.

[0168] The intervention of the Chinese medicine composition of the present invention can improve the symptoms of liver and gallbladder damp-heat syndrome, improve liver function and liver pathological changes, reduce FBG and TG, and increase Lep and EPI levels. Compared with Yinchenhao decoction, the Chinese medicine composition of the present invention has the advantages of improving fatigue of model group mice, increasing weight-bearing swimming time and activity, increasing LEP levels, and reducing HBeAg levels.

Claims

1. A Chinese medicine composition for treating chronic hepatitis B, characterized in that: It is prepared from the following raw materials in the following weight ratio: 16-20 parts of Artemisia capillaris, 10-15 parts of Gardenia jasminoides, 4-8 parts of Rhubarb, 13-17 parts of Atractylodes macrocephala, and 8-12 parts of Tangerine peel.

2. The Chinese medicine composition according to claim 1, characterized in that: It is prepared from the following raw materials in the following weight ratio: 18 parts of Artemisia capillaris, 12 parts of Gardenia jasminoides, 6 parts of Rhubarb, 15 parts of Atractylodes macrocephala, and 10 parts of dried orange peel.

3. The Chinese medicine composition according to claim 2, characterized in that: The Atractylodes macrocephala is stir-fried Atractylodes macrocephala.

4. The composition according to claim 1, characterized in that: It is a preparation made from the powder of the raw material drug, or the water or organic solvent extract of the raw material drug as the active ingredient, and pharmaceutically acceptable excipients.

5. The composition according to any one of claims 1 to 4, characterized in that: The preparation is an oral preparation.

6. The composition according to claim 5, characterized in that: The oral preparation is a granule, paste, powder, pill or solution.

7. The method for preparing the pharmaceutical composition according to any one of claims 1 to 6, characterized in that: It includes the following steps: (1) Weighing the raw material according to the ratio described in claim 1; (2) The raw material drug is ground into powder, or the water or organic solvent extract of the raw material drug is added with excipients or auxiliary ingredients commonly used in pharmaceuticals to obtain the powder.

8. Use of the composition according to any one of claims 1 to 6 in the preparation of a medicament for treating chronic hepatitis B.

9. The composition according to claim 8, characterized in that: The medicine is a medicine for treating chronic hepatitis B of liver and gallbladder damp-heat syndrome type.

10. The Chinese medicine composition according to claim 9, characterized in that: The drug has the effects of improving liver function and liver pathological changes, reducing FBG and TG, increasing Lep and EPI levels, improving fatigue, increasing activity, and / or reducing HBeAg levels.

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