Use of a traditional Chinese medicine composition in the preparation of a medicine for preventing or treating hepatitis B virus

Abstract

The present application belongs to the technical field of medicine, and particularly relates to a traditional Chinese medicine composition with the effect of preventing and / or treating hepatitis B. The traditional Chinese medicine composition is mainly prepared from Notopterygium incisum, Angelica pubescens, Poria cocos, Saposhnikovia divaricata, Schizonepeta tenuifolia, Chuanxiong, Platycodon grandiflorum, Bupleurum chinense, Peucedanum praeruptorum, Citrus aurantium, and Glycyrrhiza uralensis. The traditional Chinese medicine composition can not only significantly inhibit the activity of hepatitis B virus, but also significantly inhibit the secretion of HBSAg and HBeAg of hepatitis B virus, inhibit rebound after drug withdrawal, has a significant preventive and / or therapeutic effect on liver damage caused by hepatitis B virus infection, and can promote the production of hepatitis B surface antibody, thus having a broad clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the pharmaceutical use of a traditional Chinese medicine composition in the preparation of drugs for the prevention and / or treatment of hepatitis B. Background Technology

[0002] Jingfang Granules are a traditional Chinese medicine preparation of Jingfang Baidu Powder, made from 11 Chinese herbs: Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Bupleurum, Peucedanum, Poria, Ligusticum chuanxiong, Platycodon grandiflorus, Citrus aurantium, and Glycyrrhiza uralensis.

[0003] Jingfang Baidu San is a modified version of Renshen Baidu San. Renshen Baidu San first appeared in the Song Dynasty official medical text, *Taiping Huimin Heji Jufang*, and its ingredients include Bupleurum, Licorice, Platycodon, Ginseng, Ligusticum, Poria, Aurantium, Peucedanum, Notopterygium, and Angelica pubescens. It is effective in treating epidemic diseases. The book states that it "treats typhoid fever and seasonal illnesses." *Leizheng Huoren Shu* and *Xiao'er Yaozheng Zhijue* state: "It treats colds, epidemic diseases, rheumatism… during epidemics… this medicine is indispensable." In the Ming Dynasty, Yu Chang's *Yimen Falu·Sanqimen Fang* points out that this formula "treats typhoid fever, epidemic diseases, rheumatism, wind-phlegm, headache, dizziness, limb pain, aversion to cold, high fever, stiff neck, eye pain, and can be taken by the elderly and children. It is indispensable in places with miasma, during epidemics, where people have a lot of wind-phlegm, or in low-lying, damp places with weak legs." This shows that Ginseng Detoxifying Powder was an effective remedy for treating epidemics in ancient times and was considered the "number one prescription for treating epidemics".

[0004] Jingfang Baidu San is a modified version of Renshen Baidu San (Ginseng Baidu San). It was first recorded in Zhang Shiche's *Shesheng Zhongmiao Fang* (Collection of Wonderful Prescriptions for Nourishing Life) from the Ming Dynasty. It is essentially Renshen Baidu San without ginseng, with the addition of Jingjie (Schizonepeta) and Fangfeng (Saposhnikovia). It is primarily used to treat "all kinds of sores, seasonal infections, swelling and pain, and fever" and "the initial stage of sores and swellings." Jingfang Baidu San, with the addition of Jingjie and Fangfeng, has a stronger dispersing and penetrating effect than Renshen Baidu San, making it suitable for expelling pathogens in the early stages of epidemics. It has been widely used in the treatment of epidemics in later generations.

[0005] Jingfang Baidu San and Renshen Baidu San are of the same lineage, both capable of clearing the exterior and rescuing the interior, raising the sunken pathogenic qi, and reversing the downward flow of clear yang, allowing the pathogenic qi to dissipate from the skin surface, making them suitable for the "early stage of epidemic disease". In modern clinical practice, they are often used to treat epidemic and infectious diseases, such as acute viral upper respiratory tract infections, dengue fever, H1N1 influenza A, chickenpox, and mumps.

[0006] Hepatitis B (HBV type B) is an infectious disease primarily affecting the liver, caused by the hepatitis B virus. Clinically, it is mainly characterized by loss of appetite, nausea, upper abdominal discomfort, liver area pain, and fatigue. It is primarily transmitted through blood, mother-to-child transmission, and sexual contact. HBV infection can cause acute hepatitis, chronic hepatitis, and asymptomatic carriers. Hepatitis B is a serious public health problem not only in my country but also globally, and it is also a social issue that deserves attention.

[0007] Currently, antiviral treatment for hepatitis B primarily relies on nucleoside (acid) analogs and interferon. Nucleoside analogs act directly on viral reverse transcriptase, blocking hepatitis B virus replication. However, they have drawbacks such as a long treatment course requiring lifelong therapy, the potential for drug resistance development with increased side effects, and a high risk of viral rebound upon discontinuation. Interferon, an immunomodulator, activates the JAK-STAT signaling pathway to inhibit viral replication. While interferon treatment offers long-lasting efficacy and a high viral surface antigen clearance rate, it suffers from low response rates and significant side effects, including flu-like symptoms, fatigue, neutropenia, thrombocytopenia, and lethargy. Therefore, identifying new drug targets and developing novel antiviral drugs for hepatitis B is extremely urgent. Summary of the Invention

[0008] This invention provides a traditional Chinese medicine composition for the prevention or treatment of hepatitis B. The traditional Chinese medicine composition is mainly prepared from Notopterygium incisum, Angelica pubescens, Poria cocos, Saposhnikovia divaricata, Schizonepeta tenuifolia, Ligusticum chuanxiong, Platycodon grandiflorus, Bupleurum chinense, Peucedanum praeruptorum, Citrus aurantium, and Glycyrrhiza uralensis.

[0009] Schizonepeta and Saposhnikovia, being pungent and warm, are the principal herbs for dispelling wind and cold, while Notopterygium and Angelica pubescens dispel wind, eliminate dampness, and relieve pain, thus aiding Schizonepeta and Saposhnikovia in their ability to dispel wind. Ligusticum chuanxiong dispels wind from the blood and relieves pain, serving as the assistant herbs. Bupleurum, being pungent and cool, disperses heat on the skin's surface and, together with Peucedanum praeruptorum, Platycodon grandiflorus, and Citrus aurantium, has both ascending and descending effects, collectively promoting lung function, resolving phlegm, and opening the chest. Poria cocos, being sweet, bland, and neutral, strengthens the spleen and eliminates dampness; these four herbs serve as adjuvant herbs. Licorice is the guiding herb, harmonizing the other herbs. Together, these herbs work to induce sweating, dispel wind, eliminate cold, and remove dampness. It has the functions of inducing sweating, dispelling wind, and removing dampness. Clinically, it is used to treat colds due to wind-cold, headache, body aches, chills without sweating, nasal congestion, runny nose, and cough. It is a commonly used medicine on the market for treating colds due to wind-cold, headache, body aches, chills without sweating, nasal congestion, runny nose, and cough.

[0010] One objective of this invention is to provide the composition of the above-mentioned traditional Chinese medicine composition, namely, the traditional Chinese medicine composition is mainly prepared from the following raw materials:

[0011]

[0012] Furthermore, the traditional Chinese medicine composition is mainly prepared from the following raw materials:

[0013]

[0014] Furthermore, the traditional Chinese medicine composition is mainly prepared from the following raw materials:

[0015]

[0016] A second objective of this invention is to provide the use of the above-mentioned traditional Chinese medicine composition in the preparation of drugs for the prevention or treatment of hepatitis B. Example 9 of this invention shows that Jingfang granules have a significant inhibitory effect on HepG2.2.15 cells and inhibit the secretion of HB by HepG2.2.15 cells. S Jingfang granules showed significant inhibitory effects on HBsAg and HBeAg. At a concentration of 50 μg / ml, the inhibition rates of Jingfang granules against HBsAg and HBeAg were 82.9% and 56.7%, respectively. This suggests that Jingfang granules have an anti-HBV cell secretion HBc inhibitory effect in vitro. S The effects of Ag and HBeAg. Example 10: Jingfang granules exhibit better anti-duck hepatitis B virus activity, with a more significant inhibitory effect on DHBV-DNA levels in infected duck serum, showing better efficacy than modified Jingfang Baidu powder. Furthermore, it still showed inhibitory effects on hepatitis B virus 3 days after discontinuation of administration, demonstrating the characteristic of inhibiting rebound after drug withdrawal. Example 11: Jingfang granules significantly reduced serum ALT and AST levels in DHBV-infected rat models, showing better efficacy than modified Jingfang Baidu powder in treating liver damage caused by hepatitis B virus. Clinical cases show that the Jingfang preparation of this invention can be used to treat hepatitis B with reliable efficacy and promotes the production of hepatitis B surface antibodies.

[0017] The third objective of this invention is to provide a method for preparing the above-mentioned traditional Chinese medicine composition, the method mainly comprising the following steps:

[0018] Step A: Distill and extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, and Citrus aurantium for later use. Prepare the distilled residue and the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium for later use.

[0019] Step B: Prepare a 10-30% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use;

[0020] Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved.

[0021] Step D: Add water to decoct the Bupleurum, Platycodon, Licorice, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, and Peucedanum praeruptorum residue obtained in Step A. Concentrate the decoction for later use.

[0022] Step E: Mix the percolate obtained in step C and the decoction obtained in step D, concentrate them, and add the volatile oil obtained in step A to obtain the final product.

[0023] In one embodiment of the present invention, the preparation method mainly includes the following steps:

[0024] Step A: Distill and extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, and Citrus aurantium for later use. Prepare the distilled residue and the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium for later use.

[0025] Step B: Prepare a 20% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use;

[0026] Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved.

[0027] Step D: Add water to decoct the Bupleurum, Platycodon, Licorice, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, and Peucedanum praeruptorum residue obtained in Step A. Concentrate the decoction into a thick paste for later use.

[0028] Step E: Mix the percolate obtained in Step C and the thick paste obtained in Step D, concentrate them into a clear paste, and add the volatile oil obtained in Step A to obtain the final product.

[0029] The fourth objective of this invention is to provide a traditional Chinese medicine preparation containing the above-mentioned traditional Chinese medicine composition, wherein the traditional Chinese medicine preparation is a clinically acceptable oral preparation;

[0030] In one embodiment of the present invention, the clinically acceptable oral formulation is one or more of pills, capsules, tablets, granules, or liquid oral formulations;

[0031] Furthermore, the oral formulation is in the form of granules.

[0032] Compared with the prior art, the advantages of the present invention are:

[0033] 1. The herbal composition of this invention has a significant inhibitory effect on HepG2.2.15 cells and inhibits the secretion of HB by HepG2.2.15 cells. S Ag and HBeAg showed significant inhibitory effects, with inhibition rates of 82.9% and 56.7% against HBsAg and HBeAg, respectively, at a concentration of 50 μg / ml. This suggests that the herbal composition of the present invention has the ability to inhibit HBV secretion by cells in vitro. S The interaction between Ag and HBeAg.

[0034] 2. The traditional Chinese medicine composition of this invention is safe for clinical use, has a definite curative effect against hepatitis B virus in vivo, and has a significant inhibitory effect on the DHBV-DNA level in infected duck serum. Compared with the modified Jingfang Baidu San, the difference is significant, indicating that the anti-hepatitis B virus effect is better than that of the modified Jingfang Baidu San, and it can be used to prevent and treat hepatitis B.

[0035] 3. The traditional Chinese medicine composition of the present invention has the characteristic of inhibiting rebound after drug withdrawal; compared with acyclovir, it still has an inhibitory effect on hepatitis B virus 3 days after drug withdrawal, and the traditional Chinese medicine composition of the present invention does not rebound after drug withdrawal.

[0036] 4. The herbal composition of this invention can repair liver damage caused by hepatitis B virus infection and significantly reduce the levels of serum transaminase ALT and AST in rats infected with DHBV. Compared with the modified Jingfang Baidu San, the difference is significant, indicating that its effect in treating liver damage is better than that of the modified Jingfang Baidu San.

[0037] 5. The traditional Chinese medicine composition of the present invention can be used to treat hepatitis B with reliable efficacy and can promote the production of hepatitis B surface antibodies. Detailed Implementation Specific Implementation

[0039] Example 1: Preparation of Granules

[0040] prescription:

[0041]

[0042] Preparation method:

[0043] Step A: Extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, and Citrus aurantium separately by distillation and set aside. Set aside the distilled residue and the distilled aqueous solutions of Ligusticum chuanxiong and Citrus aurantium.

[0044] Step B: Prepare a 20% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use;

[0045] Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved.

[0046] Step D: Add water to the Bupleurum, Platycodon, Licorice, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens and Peucedanum praeruptorum residue obtained in Step A and decoct twice, 2 hours each time. Combine the two decoctions, filter and concentrate into a thick paste for later use.

[0047] Step E: Mix the percolate obtained in Step C and the thick paste obtained in Step D, let stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, make into granules, dry, add the volatile oil obtained in Step A, mix well, and the product is ready.

[0048] Example 2: Granule Preparation

[0049] prescription:

[0050]

[0051] The preparation method is the same as in Example 1.

[0052] Example 3: Preparation of Granules

[0053] prescription:

[0054]

[0055] The preparation method is the same as in Example 1.

[0056] Example 4: Capsule Preparation

[0057] prescription:

[0058]

[0059] Step A: Extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, and Citrus aurantium separately by distillation and set aside. Set aside the distilled residue and the distilled aqueous solutions of Ligusticum chuanxiong and Citrus aurantium.

[0060] Step B: Prepare a 30% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use;

[0061] Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved.

[0062] Step D: Add water to the Bupleurum, Platycodon, Licorice, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens and Peucedanum praeruptorum residue obtained in Step A and decoct twice, 2 hours each time. Combine the two decoctions, filter and concentrate into a thick paste for later use.

[0063] Step E: Mix the percolate obtained in Step C and the thick paste obtained in Step D, let stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, make into granules, dry, add the volatile oil obtained in Step A, mix well, make into granules, dry, pulverize, and fill into capsules to obtain the final product.

[0064] Example 5: Syrup Preparation

[0065] prescription:

[0066]

[0067] Preparation method:

[0068] Step A: Extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, and Citrus aurantium separately by distillation and set aside. Set aside the distilled residue and the distilled aqueous solutions of Ligusticum chuanxiong and Citrus aurantium.

[0069] Step B: Prepare a 30% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use;

[0070] Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved.

[0071] Step D: Add water to the Bupleurum, Platycodon, Licorice, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens and Peucedanum praeruptorum residue obtained in Step A and decoct twice, 2 hours each time. Combine the two decoctions, filter and concentrate into a thick paste for later use.

[0072] Step E: Mix the percolate obtained in Step C and the thick paste obtained in Step D, let stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, add 500ml of volatile oil and simple sugar syrup obtained in Step A, mix well, let stand, filter, add water to 1000ml, and you have the final product.

[0073] Example 6: Preparation of Oral Liquid

[0074] prescription:

[0075]

[0076] Preparation method:

[0077] Step A: Extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, and Citrus aurantium separately by distillation and set aside. Set aside the distilled residue and the distilled aqueous solutions of Ligusticum chuanxiong and Citrus aurantium.

[0078] Step B: Prepare a 10% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use;

[0079] Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved.

[0080] Step D: Add water to the Bupleurum, Platycodon, Licorice, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens and Peucedanum praeruptorum residue obtained in Step A and decoct twice, 2 hours each time. Combine the two decoctions, filter and concentrate into a thick paste for later use.

[0081] Step E: Mix the percolate obtained in Step C and the thick paste obtained in Step D, let stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, add the volatile oil obtained in Step A, mix well, add water to 1000ml, and you have the final product.

[0082] Example 7 Tablet Preparation

[0083] prescription:

[0084]

[0085] Preparation method:

[0086] Step A: Extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, and Citrus aurantium separately by distillation and set aside. Set aside the distilled residue and the distilled aqueous solutions of Ligusticum chuanxiong and Citrus aurantium.

[0087] Step B: Prepare a 15% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use;

[0088] Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved.

[0089] Step D: Add water to the Bupleurum, Platycodon, Licorice, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens and Peucedanum praeruptorum residue obtained in Step A and decoct twice, 2 hours each time. Combine the two decoctions, filter and concentrate into a thick paste for later use.

[0090] Step E: Mix the percolate obtained in Step C and the thick paste obtained in Step D, let stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, make into granules, dry, add the volatile oil obtained in Step A, mix well, make into granules, add an appropriate amount of excipients, mix well, compress into tablets, and the product is obtained.

[0091] Example 8: Preparation of Pills

[0092] prescription:

[0093]

[0094] Preparation method:

[0095] Step A: Extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, and Citrus aurantium separately by distillation and set aside. Set aside the distilled residue and the distilled aqueous solutions of Ligusticum chuanxiong and Citrus aurantium.

[0096] Step B: Prepare a 20% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use;

[0097] Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved.

[0098] Step D: Add water to the Bupleurum, Platycodon, Licorice, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens and Peucedanum praeruptorum residue obtained in Step A and decoct twice, 2 hours each time. Combine the two decoctions, filter and concentrate into a thick paste for later use.

[0099] Step E: Mix the percolate obtained in Step C and the thick paste obtained in Step D, let stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, make into granules, dry, add the volatile oil obtained in Step A, mix well, dry, pulverize, sieve, add 30-50g of refined honey, add an appropriate amount of water to make pills, dry, and the product is obtained.

[0100] Comparative Example: Preparation of Jingfang Baidu Powder Granules (with modifications)

[0101] prescription:

[0102]

[0103] Preparation method:

[0104] Step A: Extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, Citrus aurantium, and Mentha haplocalyx separately by distillation and set aside. Set aside the distilled residue and the distilled aqueous solutions of Ligusticum chuanxiong and Citrus aurantium.

[0105] Step B: Prepare a 20% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use;

[0106] Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved.

[0107] Step D: Add water to the Bupleurum, Platycodon, Licorice, Forsythia, Hawthorn, Six-Fermented Soybean Curd, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, and Peucedanum praeruptorum residue obtained in Step A and decoct twice, 2 hours each time. Combine the two decoctions, filter, and concentrate into a thick paste for later use.

[0108] Step E: Mix the percolate obtained in Step C and the thick paste obtained in Step D, let stand, filter and concentrate into a clear paste, add an appropriate amount of sucrose, mix well, make into granules, dry, add the volatile oil obtained in Step A, mix well, and the product is ready.

[0109] Example 9: In vitro efficacy determination of Jingfang granules against HBV

[0110] 1. HepG2.2.15 cell culture

[0111] This experiment used the HepG2.2.15 cell, a currently recognized cell model for studying HBV replication, as the research subject. The HepG2.2.15 cells used in the experiment were purchased from the China Center for Type Microbiology.

[0112] The cell culture medium was MEN medium, containing 10 ml of fetal bovine serum, 1 ml of 3% glutamine solution, 380 μg / ml of G418, and 50 U / ml of gentamicin per 100 ml. The cell digestion solution was prepared with Hanks' solution containing 0.25% trypsin.

[0113] Cell culture method: After thawing HepG 2.2.15 cells, they were seeded into 25 cm² cells. 2 Culture flask, concentration 1×10 5 Cells were cultured at a density of 10 cells / ml. Once the cells reached confluence, 0.25% trypsin was added and the cells were digested at 37°C for 3-5 minutes. Culture medium was added and the cells were pipetted. The cells were passaged at a ratio of 1:3, and the supernatant was collected daily for two consecutive weeks. The cells were then refrigerated for testing.

[0114] 2. Inhibitory effect of Jingfang granules on the proliferation of HepG2.2.15 cells

[0115] The cytotoxicity of the drug was determined using the MTT assay. The granules prepared in Example 1 were diluted with DMEM culture medium to concentrations of 50 μg / ml, 25 μg / ml, 12.5 μg / ml, and 6.25 μg / ml. 100 μl of HepG2.2.15 cells (cell concentration of 3 × 10⁻⁶ cells) were added to each well of a 96-well plate. 5 Cells / ml were cultured in a 37℃, 5% CO2 incubator. After 24 hours of culture, the medium was replaced with drug-containing medium, 4 wells per concentration. The medium was replaced with the same concentration of drug-containing medium every 3 days, and the cells were cultured for another 8 days in a 37℃, 5% CO2 incubator. After the culture was completed, 10 μl of 5 μg / ml MTT was added to each well, and 100 μl of supernatant was retained in each well. After 4 hours of culture in a 37℃, 5% CO2 incubator, the OD value was measured at 570 / 630 nm using a microplate reader. Four wells each were used for the blank control and cell control. The percentage of cell inhibition was calculated using the following formula.

[0116] Cell inhibition percentage = [(Cell control OD value - Drug treatment group OD value) / (Cell control OD value - Blank control OD value)] × 100%.

[0117] Table 1. Inhibitory effect of Jingfang granules on the proliferation of HepG2.2.15 cells.

[0118]

[0119]

[0120] Cellular experiments showed that Jingfang granules had a significant inhibitory effect on HepG2.2.15 cells, suggesting that Jingfang granules may have an inhibitory effect on hepatitis B virus.

[0121] 3. Jingfang granules affect the secretion of HB by HepG2.2.15 cells. S Effects of Ag and HBeAg

[0122] 96-well plate, 3×10 per well 5 Cells / ml 2.2.15 100 μl of cells were cultured in a 37℃ 5% CO2 incubator. After one day, the culture medium was replaced with drug-containing medium. For each concentration, 4 wells were prepared. The granules prepared in Example 1 were diluted with DMEM culture medium to (50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml). The culture medium was replaced with the same concentration every 3 days. On day 9, the culture supernatant was collected and stored at -20℃. HBsAg and HBeAg were measured using an ELISA kit, and OD values ​​were measured at 540 / 630 nm wavelength using a microplate reader. Four wells each were used for the blank control and cell control.

[0123] Inhibition percentage = [(OD value of cell control - OD value of drug treatment group) / (OD value of cell control - OD value of blank control)] × 100%. The results are shown in Table 2.

[0124] Table 2. Effects of Jingfang Granules on HB secretion in HepG2.2.15 cells. S Effects of Ag and HBeAg

[0125]

[0126] HBsAg and HBeAg are the main markers in the serum of patients with hepatitis B. The results of this experiment show that Jingfang granules inhibit the secretion of HBsAg by HepG2.2.15 cells. S Jingfang granules showed significant inhibitory effects on HBsAg and HBeAg. At a concentration of 50 μg / ml, the inhibition rates of Jingfang granules against HBsAg and HBeAg were 82.9% and 56.7%, respectively. This suggests that Jingfang granules have an anti-HBV cell secretion HBc inhibitory effect in vitro. S The interaction between Ag and HBeAg.

[0127] Example 10: The therapeutic effect of Jingfang preparation on hepatitis B virus infection in ducks.

[0128] This invention uses an internationally recognized experimental model to conduct experiments in ducks infected with hepatitis B virus.

[0129] 1. DHBV infection: 48 ducklings were injected intravenously with DHBV-DNA positive duck serum, 0.2 ml per duck. Blood was collected 7 days post-infection, and serum was separated. It was stored at -70℃ for testing.

[0130] 2. Drug Treatment Trial: One-day-old Beijing ducks infected with DHBV were randomly divided into three groups. The treatment group was administered Jingfang granules prepared in Example 1 by gavage at three doses: a low-dose group (1.4 g / kg), a medium-dose group (2.8 g / kg), and a high-dose group (5.6 g / kg). The model control group received physiological saline instead of the drug. The positive control group received acyclovir (35 mg / kg), and the control group received the granules prepared in the control example (5.6 g / kg). All treatments were administered three times daily for 7 consecutive days. Blood samples were collected from the tibial vein of the ducks before administration, on days 3 and 7 of administration, and 3 days after discontinuation. Serum was separated and stored at -70°C for testing.

[0131] 3. Detection Method: Take the above-mentioned duck serum samples and spot them onto a membrane simultaneously for each batch. Measure the dynamic level of DHBV-DNA in the duck serum. Following the instructions of the gap translation kit, use a 32p-labeled DHBV-DNA probe to perform dot hybridization of the duck serum. Measure the OD value (490nm) of the autoradiographic membrane spots and calculate the serum DHBV-DNA density. Use the OD value of the hybridization spots as the DHBV-DNA level value of the sample.

[0132] 4. Efficacy Calculation: Serum DHBV-DNA levels were calculated at different time points for each group. Paired t-tests were used to compare the DHBV-DNA inhibition rate before and after drug administration in each group. The dynamic changes in the DHBV-DNA inhibition rate of duck serum were compared among the groups. Independent t-tests were used to compare the drug-treated group with the virus control group. DHBV-DNA inhibition rate = (OD value before administration - OD value after administration) / OD value before administration × 100%. The results are shown in Table 1.

[0133] Table 1. Effects of Jingfang Granules on Serum DHBV-DNA Levels in DHBV-Infected Ducks ( n=8)

[0134]

[0135] Compared with the model control group, *p<0.05, **p<0.01; compared with the comparative example group, # p < 0.05 ## p < 0.01; compared with the acyclovir group, Δp < 0.05, ΔΔp < 0.01

[0136] Table 1 shows that, compared with the model control group, all dosage groups of Jingfang granules significantly inhibited the DHBV-DNA level in infected duck serum (p < 0.01). Compared with the comparative example group, the inhibitory effects of each dosage group of Jingfang granules on the DHBV-DNA level in infected duck serum were significantly or extremely significantly different (p < 0.05, p < 0.01); compared with the acyclovir group, all dosage groups of Jingfang granules significantly inhibited the DHBV-DNA level in infected duck serum (p < 0.01). These findings indicate that Jingfang granules have better anti-duck hepatitis B virus activity, a more significant inhibitory effect on the DHBV-DNA level in infected duck serum, and are superior to the comparative example. Furthermore, the inhibitory effect on hepatitis B virus persists 3 days after discontinuation of administration, demonstrating the characteristic of inhibiting rebound after drug withdrawal.

[0137] Example 11 Effect of Jingfang preparation on serum transaminase in rats with hepatitis B virus model

[0138] Experimental animals: 48 SPF-grade SD rats, half male and half female, weighing (200±20) g. The rats were fed routinely and then subjected to intervention experiments after one week of acclimatization.

[0139] Grouping and Model Establishment: Rats were randomly divided into 6 groups according to body weight: normal control group, model group, comparative example group, low-dose Jingfang granules group, medium-dose Jingfang granules group, and high-dose Jingfang granules group. Except for the normal control group, hepatitis B infection models were established in the other groups. The method of model establishment and the determination of model success were based on the research of Guo Chunxia (Guo Chunxia, ​​He Yongwen, Peng Cheng, et al. Establishment of mouse model of acute hepatitis B virus infection [J]. Chinese Journal of Zoonoses, 2009, 25(12): 1170-1173.). The specific operation is as follows: HBV plasmid was injected into the tail vein of rats to transfect liver cancer cell lines, which can achieve complete transcription, replication and expression of HBV gene.

[0140] Drug intervention: After the hepatitis B infection model was established, rats in each group were given drugs three times a day for 7 consecutive days. The drugs and their dosages are as follows:

[0141] Normal control group: an equal volume of physiological saline

[0142] Model group: Equal volume of physiological saline

[0143] Comparative Example Group: Jingfang Baidu San modified granules prepared in the comparative example, 5.6 g / kg

[0144] Low-dose group of Jingfang granules: Jingfang granules preparation prepared in Example 2, 1.4 g / kg

[0145] Medium-dose group of Jingfang granules: Jingfang granules preparation prepared in Example 2, 2.8 g / kg

[0146] High-dose group of Jingfang granules: Jingfang granules preparation prepared in Example 2, 5.6 g / kg

[0147] Twenty-four hours after the last administration, 0.2 ml of blood was collected from the abdominal aorta, and the changes in serum ALT and AST levels in rats were measured strictly according to the instructions of the fully automated biochemical analyzer. The results are shown in Table 2.

[0148] Table 2 Effects of Jingfang Granules on Serum Transaminases ALT and AST in HBV-Infected Rats ( n=8)

[0149]

[0150] Compared with the normal control group, *p<0.05, **p<0.01; compared with the model group, #p<0.05, ##p<0.01; compared with the comparative example group, Δ p < 0.05 ΔΔ p < 0.01

[0151] Table 2 shows that compared with the normal control group, the serum ALT and AST levels in the model group rats were significantly increased (p < 0.01), indicating successful model establishment. Compared with the model group, the serum ALT and AST levels in rats in each dose group of Jingfang granules were significantly decreased (p < 0.01); compared with the comparative example group, the serum ALT and AST levels in the high-dose group of Jingfang granules were significantly different (p < 0.05). These findings indicate that Jingfang granules can significantly reduce the levels of serum ALT and AST in DHBV-infected rats, and its effect in treating liver damage caused by hepatitis B virus is superior to that of modified Jingfang Baidu San.

[0152] Typical cases

[0153] Mr. Zhang, male, 30 years old, was diagnosed with chronic hepatitis B with high viral load. His liver function was abnormal in multiple tests. Three days ago, a test showed ALT 662, AST 355, and total bilirubin 60. After taking Jingfang granules of this invention for 2 weeks, his liver function returned to normal. He continued to take the granule preparation of this invention for another 2 months. His surface antigen turned negative, and his surface antibody became positive, achieving clinical cure.

[0154] Ms. Zhao, 17 years old, was found to have chronic hepatitis B with high viral load during a physical examination. Her ALT was 215 and AST was 185. After taking the Jingfang granules of this invention for one week, her liver function returned to normal. She continued to take the granules of this invention for another month, and her hepatitis B surface antigen turned negative while her hepatitis B surface antibody became positive, achieving clinical cure.

[0155] The present invention has been further described above through specific embodiments. Any equivalent substitutions will be obvious to those skilled in the art and are included in the present invention.

Claims

1. The use of a traditional Chinese medicine composition in the preparation of drugs for the prevention and / or treatment of hepatitis B, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials: 10-30 parts by weight of Schizonepeta tenuifolia, 10-30 parts by weight of Saposhnikovia divaricata, and 10-30 parts by weight of Notopterygium incisum. Angelica pubescens 10-30 parts by weight, Bupleurum chinense 10-30 parts by weight, Peucedanum praeruptorum 10-30 parts by weight Chuanxiong 10-30 parts by weight, Zhi Ke 10-30 parts by weight, Fu Ling 10-30 parts by weight Platycodon grandiflorus 10-30 parts by weight, licorice 3-10 parts by weight.

2. The use as described in claim 1, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials: 20 parts by weight of Schizonepeta tenuifolia, 20 parts by weight of Saposhnikovia divaricata, and 20 parts by weight of Notopterygium incisum. Angelica pubescens 20 parts by weight, Bupleurum chinense 20 parts by weight, Peucedanum praeruptorum 20 parts by weight Chuanxiong 20 parts by weight, Zhi Ke 20 parts by weight, Fu Ling 20 parts by weight Platycodon grandiflorus 20 parts by weight, licorice 2 parts by weight.

3. The use as described in claim 1, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials: 15 parts by weight of Schizonepeta tenuifolia, 15 parts by weight of Saposhnikovia divaricata, and 15 parts by weight of Notopterygium incisum. Angelica pubescens 15 parts by weight, Bupleurum chinense 15 parts by weight, Peucedanum praeruptorum 15 parts by weight Chuanxiong 15 parts by weight, Zhi Ke 15 parts by weight, Fu Ling 15 parts by weight Platycodon grandiflorus 15 parts by weight, licorice 5 parts by weight.

4. The use as described in claim 1, characterized in that, The preparation method of the traditional Chinese medicine composition includes the following steps: Step A: Distill and extract volatile oils from Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, Peucedanum praeruptorum, Ligusticum chuanxiong, and Citrus aurantium for later use. Prepare the distilled residue and the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium for later use. Step B: Prepare a 10-30% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in Step A for later use; Step C: Mix Poria cocos, the distilled Ligusticum chuanxiong and Citrus aurantium residue obtained in Step A, and extract by percolation with the ethanol solution obtained in Step B. The percolate is reserved. Step D: Add water to decoct the Bupleurum, Platycodon, Licorice, and the distilled Schizonepeta, Saposhnikovia, Notopterygium, Angelica pubescens, and Peucedanum praeruptorum residue obtained in Step A. Concentrate the decoction for later use. Step E: Mix the percolate obtained in step C and the decoction obtained in step D, concentrate them, and add the volatile oil obtained in step A to obtain the final product.

5. The use as described in claim 4, characterized in that, Step B involves preparing a 20% ethanol solution from the distilled aqueous solution of Ligusticum chuanxiong and Citrus aurantium obtained in step A for later use.

6. The use as described in any one of claims 1-5, characterized in that, The traditional Chinese medicine composition can be prepared into a clinically acceptable oral formulation.

7. The use as described in claim 6, characterized in that, The clinically acceptable oral formulation is one of the following: pills, capsules, tablets, granules, or liquid oral formulations.

8. The use as described in claim 7, characterized in that, The oral preparation is in granule form.

9. The use of a traditional Chinese medicine composition in the preparation of a drug for inhibiting the activity of hepatitis B virus, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials: 10-30 parts by weight of Schizonepeta tenuifolia, 10-30 parts by weight of Saposhnikovia divaricata, and 10-30 parts by weight of Notopterygium incisum. Angelica pubescens 10-30 parts by weight, Bupleurum chinense 10-30 parts by weight, Peucedanum praeruptorum 10-30 parts by weight Chuanxiong 10-30 parts by weight, Zhi Ke 10-30 parts by weight, Fu Ling 10-30 parts by weight Platycodon grandiflorus 10-30 parts by weight, licorice 3-10 parts by weight.

10. The use of a traditional Chinese medicine composition in the preparation of a drug for the prevention and / or treatment of liver damage caused by hepatitis B virus, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials: 10-30 parts by weight of Schizonepeta tenuifolia, 10-30 parts by weight of Saposhnikovia divaricata, and 10-30 parts by weight of Notopterygium incisum. Angelica pubescens 10-30 parts by weight, Bupleurum chinense 10-30 parts by weight, Peucedanum praeruptorum 10-30 parts by weight Chuanxiong 10-30 parts by weight, Zhi Ke 10-30 parts by weight, Fu Ling 10-30 parts by weight Platycodon grandiflorus 10-30 parts by weight, licorice 3-10 parts by weight.

Images