CN114796233A

CN114796233A - Application of asiatic acid in preparing medicine for treating hepatitis B

Info

Publication number: CN114796233A
Application number: CN202210692906.9A
Authority: CN
Inventors: 陈娟; 杨祯; 程胜桃
Original assignee: International Institute Of In Vitro Diagnostics Chongqing Medical University
Current assignee: International Institute Of In Vitro Diagnostics Chongqing Medical University
Priority date: 2022-06-17
Filing date: 2022-06-17
Publication date: 2022-07-29
Also published as: WO2023241179A1

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of asiatic acid in preparation of a medicine for treating hepatitis B. The invention discovers that asiatic acid can reduce the level of HBx protein, inhibit cccDNA transcription activity so as to reduce the level of total HBV RNAs and HBV3.5-kb RNA in cells and the level of HBsAg in supernatant, and can effectively reduce the level of HBsAg and HBV DNA in serum and the level of HBV RNA and HBV DNA in liver tissues in a recombinant cccDNA mouse model, so that the asiatic acid is a potential novel medicament targeting HBx and can be used for treating hepatitis B. The invention provides a new direction and theoretical basis for reducing the infection rate of HBV and exploring a novel medicament for effectively treating hepatitis B.

Description

Application of asiatic acid in preparing medicine for treating hepatitis B

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of asiatic acid in preparing a medicament for treating hepatitis B.

Background

Hepatitis B Virus (HBV) infection is a major public health problem worldwide. According to WHO, about 20 million people worldwide have been infected with HBV, and about 2.57 million patients with chronic hepatitis B infection. China belongs to the HBV high-traffic area. About 9700 ten thousand hepatitis B virus carriers account for 8 to 10 percent of the total population in China, wherein about 2000 ten thousand patients with chronic hepatitis B are treated, and the number of the patients with the complications such as cirrhosis, liver cancer and the like caused by the chronic hepatitis B is more than 26.3 ten thousand patients with the complications of death each year. Therefore, the task of preventing and treating hepatitis B is very severe.

Antiviral drugs commonly used in the treatment of chronic hepatitis B at present include Interferon (IFN) and Nucleoside Analogues (NAs). However, both of these drugs have certain disadvantages, such as long administration time of nucleoside analogs, poor patient compliance, easy rebound after drug withdrawal, significant effect of interferon on less than 30% of patients, and more adverse reactions. More importantly, neither of the two drugs can effectively cure hepatitis B, so that a novel anti-HBV drug is urgently developed.

HBV belongs to the family Hepadnaviridae (hepadnaviridae), and its genome is a partially double-stranded, relaxed circular DNA (rcDNA) of about 3.2kb in length. After the HBV virus enters the host hepatocyte, rcDNA is transferred to the nucleus and forms Covalently closed circular DNA (cccDNA) with a supercoiled structure. cccDNA persists in the hepatocyte nucleus in the form of minichromosomes, is structurally stable, and is a transcription template for viral RNA. cccDNA is therefore the leading cause of HBV persistent infection, drug resistance, and HBV reactivation after inactivation of antiviral drugs.

In infected HBV cells, the virus may encode a transactivator, HBx, which activates cccDNA transcription by hijacking DDB1, thereby relieving E3 ubiquitin ligase CUL4B from ubiquitination degradation of Smc5/6, and also maintains cccDNA transcription by binding to cccDNA minichromosomes to inhibit the recruitment of transcription repressors to cccDNA. These studies all indicate that HBx plays a key role in the transcription of cccDNA. Inhibition of HBx function may inhibit cccDNA transcription. Therefore, HBx is an important target of a new therapy for treating chronic HBV infection, and a novel medicament for targeted inhibition of HBx is developed, so that a new direction and a theoretical basis can be provided for reducing the infection rate of HBV and exploring a novel medicament for effectively treating hepatitis B.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide the use of asiatic acid in preparing a medicament for treating hepatitis B, wherein the asiatic acid can inhibit HBx in a targeted manner, and provides a new medicament and technical means for the effective treatment of hepatitis B.

To achieve the above and other related objects, the present invention provides, in a first aspect, use of asiatic acid as an active ingredient in the manufacture of a medicament for the treatment of hepatitis b.

Further, the hepatitis B therapeutic drug has at least one of the following functions:

reduce the level of HBsAg, reduce the level of intracellular total HBV RNAs, reduce the level of intracellular HBV3.5-kb RNA, and reduce the level of HBV DNA.

Further, the therapeutic agent for hepatitis B necessarily includes asiatic acid, and asiatic acid is used as an effective ingredient for the aforementioned functions.

Furthermore, in the hepatitis B therapeutic drug, the effective component exerting the functions can be only asiatic acid, and other molecules playing similar functions can also be contained.

Further, the asiatic acid is one of the effective components of the hepatitis B therapeutic drug or the only effective component.

Furthermore, the hepatitis B therapeutic drug can be a single-component substance or a multi-component substance.

Further, the form of the therapeutic agent for hepatitis b is not particularly limited, and may be in the form of various substances such as solid, liquid, gel, semifluid, aerosol, and the like.

Furthermore, the hepatitis B therapeutic drug is mainly directed to mammals such as rodents, primates and the like.

In a second aspect, the invention provides a pharmaceutical formulation for the treatment of hepatitis B comprising a safe and effective amount of asiatic acid.

Further, the medicinal preparation for treating hepatitis B comprises 10-30 mu M of asiatic acid.

Furthermore, the dosage of the medicinal preparation for treating hepatitis B is 15-30 mg/kg of asiatic acid.

Furthermore, the pharmaceutical preparation for treating hepatitis B also comprises a pharmaceutically acceptable carrier and/or an auxiliary material.

Furthermore, the pharmaceutical preparation for treating hepatitis B necessarily comprises asiatic acid, and the asiatic acid is taken as an effective component of the functions.

Furthermore, in the pharmaceutical preparation for treating hepatitis B, the effective component exerting the functions can be asiatic acid only, and other molecules capable of playing similar functions can also be contained.

Further, the asiatic acid is used as one of the effective components or the only effective component of the medicinal preparation for treating hepatitis B.

Furthermore, the medicinal preparation for treating hepatitis B can be a single-component substance or a multi-component substance.

Furthermore, the form of the pharmaceutical preparation for treating hepatitis b is not particularly limited, and may be in the form of various substances such as solid, liquid, gel, semifluid, aerosol, and the like.

Furthermore, the pharmaceutical preparation for treating hepatitis B is mainly aimed at mammals, such as rodents, primates and the like.

In a third aspect, the invention provides a method of treating hepatitis B by administering to a subject asiatic acid.

Further, the subject may be a mammal or a mammalian hepatitis b cell. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. The primate is preferably a monkey, ape or human. The hepatitis b cell may be an isolated hepatitis b cell.

Further, the subject may be a patient suffering from hepatitis b or an individual in whom treatment for hepatitis b is desired. Alternatively, the subject is a hepatitis b cell of a hepatitis b patient or an individual expected to treat hepatitis b.

Further, the asiatic acid may be administered to the subject before, during, or after receiving treatment for hepatitis B.

The invention provides a hepatitis B combined treatment medicine composition, which comprises safe and effective dose of asiatic acid and at least one other hepatitis B treatment medicine, and the balance is pharmaceutically acceptable carriers and/or auxiliary materials.

Further, the hepatitis b combination therapy drug combination may be in any one of the following forms:

the asiatic acid and other hepatitis B treating medicine are prepared into different preparation forms, which may be the same or different and may be administered through the same or different routes.

When the other therapeutic agent for hepatitis B is an antibody, parenteral administration is generally employed. When other hepatitis B treatment drugs are chemical drugs, the administration forms can be rich, and the drug can be administered in the gastrointestinal tract or can be administered in the parenteral tract. Known routes of administration for each chemical are generally recommended.

And (II) the asiatic acid and the other hepatitis B therapeutic drugs are prepared into a compound preparation, and when the asiatic acid and the other hepatitis B therapeutic drugs are administered by the same administration route and are applied simultaneously, the asiatic acid and the other hepatitis B therapeutic drugs can be prepared into the compound preparation.

Further, the other hepatitis b treatment drugs are drugs known to be useful for treating hepatitis b before the filing date of this application, for example, antiviral drugs: interferon (IFN), nucleoside analogs, and the like.

In a fifth aspect, the invention provides a method of treating hepatitis b by administering to a subject an effective amount of asiatic acid and administering to the subject an effective amount of another hepatitis b treatment agent and/or administering to the subject another hepatitis b treatment modality.

Further, an effective dose of asiatic acid and at least one other hepatitis B treating agent may be administered simultaneously or sequentially.

The medicine for treating hepatitis B, which is discovered for the first time based on asiatic acid, can at least play a role in adding curative effects when being used together with other medicines for treating hepatitis B except asiatic acid, and further enhances the treatment effect on hepatitis B.

Further, other therapeutic agents for hepatitis b include, but are not limited to: antibody drugs, chemical drugs or targeted drugs, etc.

Further, the asiatic acid may be administered gastrointestinal or parenteral, and the other therapeutic agents for hepatitis B may be administered gastrointestinal or parenteral. For antibody drugs, parenteral administration is generally employed.

In a sixth aspect, the invention provides the use of asiatic acid in the preparation of a substance having any one or more of the following actions: use for the preparation of a substance inhibiting HBx protein; use for the preparation of a substance that inhibits HBsAg levels; use for the preparation of a substance that inhibits the level of intracellular total HBV RNAs; use for the preparation of a substance that inhibits the intracellular HBV3.5-kb RNA level; the use for preparing a substance for inhibiting the level of HBV DNA.

As mentioned above, the use of asiatic acid in preparing the medicament for treating hepatitis B has the following beneficial effects:

the invention discovers for the first time that asiatic acid can reduce the HBx protein level, inhibit cccDNA transcription activity so as to reduce the intracellular total HBV RNAs and HBV3.5-kb RNA level and the supernatant HBsAg level, can effectively reduce the HBsAg and HBV DNA level in serum and the HBV RNA and HBV DNA level in liver tissue in a recombinant cccDNA mouse model, and is a potential novel targeted HBx medicine. The invention provides a new direction and a theoretical basis for reducing the infection rate of HBV and exploring a novel medicament for effectively treating hepatitis B, and has great application prospect in the aspect of treating hepatitis B.

Drawings

FIG. 1 shows the analysis of the cytotoxicity of asiatic acid in HepG2-NTCP, HepG2.2.15 cells for MTT experiments.

FIG. 2 shows the effect of asiatic acid on the levels of 3 Xflag-HBx and 2 XHA-HBx protein measured by Western blot.

FIG. 3 shows the effect of asiatic acid on total HBV RNAs and HBV3.5-kb RNA detected by fluorescence quantitative PCR method and Northern blot experiment.

FIG. 4 shows the fluorescent quantitative PCR method and Southern blot experiment for detecting the effect of asiatic acid on HBV DNA.

FIG. 5 shows the effect of asiatic acid on HBsAg secretion measured by ELISA.

FIG. 6 shows the effect of the Taq Man probe PCR experiment on the level and transcriptional activity of the cccDNA.

FIG. 7 shows the effect of asiatic acid on total HBV RNAs and HBV3.5-kb RNA in HepG2.2.15 cells detected by fluorescence quantitative PCR method and Northern blot experiment.

FIG. 8 shows the effect of asiatic acid on HBV DNA in HepG2.2.15 cells detected by fluorescence quantitative PCR and Southern blot.

FIG. 9 shows the effect of asiatic acid on cccDNA in HepG2.2.15 cells for Taq Man probe PCR experiments.

Figure 10 shows a mouse treatment flow chart.

FIG. 11 shows the effect of ELISA and fluorescent quantitative PCR on the HBsAg and HBV DNA levels in the serum of mice.

FIG. 12 shows the effect of fluorescent quantitative PCR method for detecting the level of HBV RNA in liver tissue of mice by asiatic acid.

FIG. 13 shows the effect of fluorescent quantitative PCR on the level of HBV DNA in liver tissue of mice by detection of asiatic acid.

Detailed Description

Asiatic acid, often abbreviated as AA, is known in Chinese as Asiatic acid. It shows antihypertensive, neuroprotective, cardioprotective, antibacterial and antitumor activity in preclinical studies. The research of the invention finds that AA can reduce the protein level of HBx, inhibit the transcription activity of cccDNA so as to reduce the level of total HBV RNAs and HBV3.5-kb RNAs in cells and the level of HBsAg in supernatant, and can effectively reduce the level of HBsAg and HBV DNA in serum and the level of HBV RNA and HBV DNA in liver tissues in a recombinant cccDNA mouse model, thus being a potential novel medicament targeting HBx.

Based on the above, the invention provides the application of asiatic acid as an effective component in preparing a medicament for treating hepatitis B.

Generally, the therapeutic agent for hepatitis B comprises, in addition to a safe and effective amount of asiatic acid, one or more pharmaceutically acceptable carriers or excipients, as required by the dosage form of the respective agent.

By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

A "pharmaceutically acceptable carrier or adjuvant" should be compatible with, i.e., capable of being blended with, asiatic acid without substantially diminishing the effectiveness of the pharmaceutical composition under normal circumstances. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as glycerol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and may be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by a conventional method. The choice of the pharmaceutical dosage form should be matched to the mode of administration.

In addition, the invention also provides a hepatitis B combined treatment medicine composition and an application method. The hepatitis B combination therapy drug combination can be any one of the following forms:

the asiatic acid and other hepatitis B treating medicine are prepared into different preparation forms, which may be the same or different and may be administered through the same or different routes. When in use, several medicines can be used simultaneously or sequentially. When administered sequentially, the other drugs should be administered to the body during the period that the first drug is still effective in the body.

When the other therapeutic agent for hepatitis B is an antibody, a parenteral administration type such as intravenous injection, intravenous drip or arterial infusion is generally used. The usage and the dosage can refer to the prior art.

When other hepatitis B treatment drugs are chemical drugs, the administration forms can be rich, and the drug can be administered in the gastrointestinal tract or can be administered in the parenteral tract. Known routes of administration for each chemical are generally recommended.

It should be noted that the combination in the present invention refers to a reasonable combination, and should be based on the principle of improving the therapeutic effect and/or reducing the adverse reaction. When used in combination, drug interactions shall include those that affect pharmacokinetics and those that affect pharmacodynamics. When the medicines are used in combination, the medicine types are reduced as much as possible, adverse reaction of the medicines caused by the interaction of the medicines is reduced, the curative effect of the medicines is prevented from being influenced or the toxicity is increased, and the adverse effect is prevented from being generated.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Example 1

First, experiment method

1. Cell culture

HepG2-NTCP cells were cultured in DMEM medium containing 10% fetal calf serum, 1% penicillin-streptomycin and 2ug/mL doxycycline, and HepG2.2.15 cells were cultured in DMEM medium containing 10% fetal calf serum, 1% penicillin-streptomycin and 400ug/mL G418, and cultured in a 5% CO2 incubator at 37 ℃. Asiatic acid (cas number 464-92-6) was purchased from MCE.

2. Plasmids and drugs

prcccDNA and pCMV-KRAB-Cre were given by professor dung (university of cowden, shanghai, china). 3 XFlag-HBx was constructed by inserting full-length HBx into p3 XFlag-CMV 7.1. 3 XFlag-HBx was constructed by inserting full-length HBx into p3 XFlag-CMV 7.1. The 2 × HA HBx was given by professor chenjieliang (university of fudan, shanghai, china). Asiatic acid (cas number 464-92-6) was purchased from MCE, dissolved in DMSO, prepared as a 20mM stock solution, dispensed and stored at-80 ℃ until use.

3. MTT assay

HepG2-NTCP cells, HepG2.2.15 cells at 15X 10 ⁴ The cells were inoculated in 96-well plates per ml. After 24h of incubation, asiatic acid was diluted in DMEM medium to 200, 100, 50, 25, 12.5, 6.25, 3.125, 0uM 8 concentration gradients, 3 more wells per concentration and normal cell controls. After 72 hours, 10ul MTT reagent was added, incubated at 37 ℃ for 4h, 100ul DMSO was added, and shakenAfter oscillating for 10min, detecting the OD value at 490nm by using an enzyme labeling instrument, and calculating and drawing a cell activity curve.

4. Detection of the Effect of Asiatic acid on HBx protein level in HBV-infected cell model HepG2-NTCP

HepG2-NTCP cells at 20X 10 ⁴ Perml were inoculated in twelve well plates. After culturing for 24h, respectively transfecting 3 XFlag-HBx and 2 XHA-HBx; 24h after transfection, HepG2-NTCP cells were treated with DMEM medium by diluting the asiatic acid to 30, 20, 10, 0uM 4 concentration gradients. And extracting total protein after 48 hours and detecting the HBx protein level by using Western blot.

5. Detection of influence of asiatic acid on HBsAg, total HBV RNAs, HBV3.5-kb RNA and HBV DNA levels in HBV infected cell model HepG2-NTCP

HepG2-NTCP cells at 20X 10 ⁴ Perml were inoculated in twelve well plates. After 24h of culture, diluting the asiatic acid to 30, 20, 10 and 0uM 4 concentration gradients by using a DMEM culture medium, respectively treating HepG2-NTCP cells 3d, 6d and 9d, collecting culture medium supernatant and cells, detecting HBsAg level in the supernatant by ELISA, detecting total HBV RNAs and HBV3.5-kb RNA levels in the cells by Real-time PCR and Northern blot, and detecting HBV DNA level in the cells by Real-time PCR and Southern blot.

6. Detection of the Effect of Asiatic acid on the levels of Total HBV RNAs, HBV3.5-kb RNA and HBV DNA in the HBV Stable replication model HepG2.2.15

HepG2.2.15 cells at 20X 10 ⁴ Perml were inoculated in twelve well plates. After 24h of culture, the asiatic acid is diluted to 30, 20, 10 and 0uM 4 concentration gradients by using a DMEM culture medium to process HepG2.2.15 cells, the cells are collected after 4d, the total HBV RNAs and HBV3.5-kb RNA levels in the cells are detected by Real-time PCR and Northern blot, and the HBV DNA level in the cells is detected by Real-time PCR and Southern blot.

7. HBV recombinant cccDNA mouse model verifies the influence of asiatic acid on HBV replication

50 male C57BL/6 mice, aged 6-8 weeks and weighing 18-20g, were selected. 4. mu.g of plasmid prcccDNA and 4. mu.g of plasmid pCMV-KRAB-Cre were injected into the tail vein, respectively. After one week, the serum HBV DNA copy number is detected by fluorescent quantitative PCR to judge whether the model is successfully established.

Successfully modeled mice were randomly divided into four groups: negative control, positive control, low-concentration experimental group and high-concentration experimental group, wherein 6 of each group are orally administered with 0.9% physiological saline, 0.02mg/kg entecavir, 15mg/kg asiatic acid and 30mg/kg asiatic acid respectively, the treatment is carried out on the groups, the groups are administered every other day, blood is collected once every four days, serum is separated, and the HBsAg and HBV DNA levels in the serum are detected. After 24 days of drug treatment, liver tissues are ground to detect the HBV RNAs and HBV DNA levels.

8、Real-time PCR

Detection of HBV DNA: after extracting HBV DNA, a reaction system was prepared and reaction conditions were set according to SYBR Green (Roche, Germany) instructions, HBV DNA primers, F: CCTAGTAGTCAGTTATGTCAAC (SEQ ID NO.1), R: TCTATAAGCTGGAGGAGTGCGA (SEQ ID NO. 2). Each sample was provided with 3 replicate wells, and each set of experiments was replicated 3 times.

Detecting HBV RNA, namely reverse transcription of cDNA synthesized by RNA, preparing a reaction system and setting reaction conditions according to SYBR Green (Bio Rad) specifications, wherein HBV3.5 kb RNA primers are F: CTCTTCCAGCCTTCCTTCCT (SEQ ID NO.3), R: AGCACTGTGTTGGCGTACAG (SEQ ID NO.4), total HBV RNAs primers are F: ACCGACCTTGAGGCATACTT (SEQ ID NO.5) and R: GCCTACAGCCTCCTAGTACA (SEQ ID NO. 6). Each sample was provided with 3 replicate wells, and each set of experiments was replicated 3 times.

9. Statistical method

Statistics is carried out by adopting GraphPad Prism statistical software, a non-pairing t test is adopted for comparison between two groups, and the difference with P <0.05 has statistical significance.

II, experimental results:

1. MTT assay

FIG. 1 shows the analysis of the cytotoxicity of asiatic acid in HepG2-NTCP, HepG2.2.15 cells for MTT experiments. The results shown in FIG. 1 indicate that asiatic acid has CC50 ═ 74.82. mu.M in the HBV-infected cell line HepG2-NTCP and CC50 ═ 67.6. mu.M in the HBV stable replication model HepG2.2.15.

2. Detection of the Effect of Asiatic acid on HBx protein level in HBV-infected cell model HepG2-NTCP

FIG. 2 shows the effect of asiatic acid on the levels of 3 Xflag-HBx and 2 XHA-HBx protein measured by Western blot. Western blot results shown in FIG. 2 indicate that exogenous 3 XFlag-HBx and 2 XHA-HBx protein levels in HepG2-NTCP cells are reduced in a concentration gradient dependence after asiatic acid treatment, suggesting that asiatic acid can inhibit HBx expression.

3. Detection of influence of asiatic acid on levels of HBsAg, intracellular total HBV RNAs, HBV3.5-kb RNA, HBV DNA and cccDNA in supernatant in HBV infected cell model HepG2-NTCP

FIG. 3 shows the effect of asiatic acid on total HBV RNAs and HBV3.5-kb RNA detected by fluorescence quantitative PCR method and Northern blot experiment. The Real-time PCR result shown in FIG. 3 shows that asiatic acid is time-dependent and concentration gradient-dependent, so that total HBV RNAs and HBV3.5-kb RNA levels are significantly reduced, and Northern blot verifies the PCR result. FIG. 4 shows the effect of asiatic acid on HBV DNA levels as detected by fluorescence quantitative PCR and Southern blot. The Real-time PCR results shown in FIG. 4 show that asiatic acid is time-dependent and has concentration gradient to reduce the intracellular HBV DNA level significantly compared with the negative control group, and Southern blot verifies the PCR results.

FIG. 5 shows the effect of asiatic acid on HBsAg secretion measured by ELISA. The ELISA results shown in FIG. 5 show that asiatic acid was effective in reducing HBsAg levels in HepG2-NTCP cells, and was time-and concentration-gradient-dependent.

FIG. 6 shows the effect of the Taq Man probe PCR experiment on the level and transcriptional activity of the cccDNA. Since HBx plays a key role in the transcription process of cccDNA. Inhibition of HBx function may inhibit cccDNA transcription. The effect of asiatic acid on cccDNA transcription was therefore further determined. The results of the Taq Man probe PCR experiments shown in fig. 6 indicate that asiatic acid has no significant effect on cccDNA levels. Further analysis of cccDNA transcriptional activity (total RNAs/cccDNA and pgRNA/cccDNA ratios) revealed that asiatic acid significantly inhibited cccDNA transcriptional activity.

4. Detection of the Effect of Asiatic acid on the levels of Total HBV RNAs, HBV3.5-kb RNA and HBV DNA in the HBV Stable replication model HepG2.2.15

FIG. 7 shows the effect of detecting asiatic acid on total HBV RNAs, HBV3.5-kb RNA in HepG2.2.15 cells. FIG. 8 shows the effect of detecting asiatic acid on HBV DNA in HepG2.2.15 cells. The results show that asiatic acid can significantly inhibit intracellular HBV RNA (fig. 7), HBV DNA levels (fig. 8), but does not affect cccDNA levels (fig. 9).

5. HBV recombinant cccDNA mouse model verifies the influence of asiatic acid on HBV replication

Figure 10 shows a mouse treatment flow chart. FIG. 11 shows the effect of ELISA and fluorescent quantitative PCR on the HBsAg and HBV DNA levels in the serum of mice. FIG. 12 shows the effect of fluorescent quantitative PCR method for detecting the level of HBV RNA in liver tissue of mice by asiatic acid. FIG. 13 shows the effect of fluorescent quantitative PCR on the level of HBV DNA in liver tissue of mice by detection of asiatic acid.

Separating mouse serum to detect HBsAg and HBV DNA. The results showed that asiatic acid treated group significantly inhibited serum HBsAg and HBV DNA levels compared to control group (fig. 11). After 24 days of drug treatment, the liver tissues were ground to further detect the levels of HBV RNA and HBV DNA in the liver tissues, and experimental results showed that the asiatic acid treated group could significantly inhibit the levels of HBV RNA (FIG. 12) and HBV DNA (FIG. 13) in the liver tissues.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

SEQUENCE LISTING

<110> Chongqing university of medicine international institute of in vitro diagnostics

Application of asiatic acid in preparing medicine for treating hepatitis B

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Claims

1. Use of asiatic acid as effective component in preparing medicine for treating hepatitis B is provided.

2. Use according to claim 1, characterized in that: the hepatitis B treatment drug has at least one of the following functions:

reducing the level of HBx protein, reducing the level of HBsAg, reducing the level of total HBV RNAs in cells, reducing the level of HBV3.5-kb RNA in cells, and reducing the level of HBV DNA.

3. Use according to claim 2, characterized in that: the hepatitis B therapeutic agent necessarily comprises asiatic acid, and asiatic acid is used as an effective ingredient for the aforementioned functions.

4. Use according to claim 1, characterized in that: the asiatic acid is used as one of the effective components of the medicine for treating hepatitis B or the only effective component.

5. Use according to claim 1, characterized in that: the hepatitis B therapeutic drug is in the form selected from the group consisting of solid, liquid, gel, semifluid, and aerosol.

6. A pharmaceutical preparation for treating hepatitis B is characterized in that: comprises a safe and effective dose of asiatic acid.

7. The pharmaceutical formulation for the treatment of hepatitis b according to claim 6, wherein: the asiatic acid is used as one of the effective components or the only effective component of the medicinal preparation for treating hepatitis B.

8. A hepatitis B combined treatment medicine combination is characterized in that: comprises safe and effective dose of asiatic acid and at least one other medicament for treating hepatitis B, and the balance is pharmaceutically acceptable carriers and/or auxiliary materials.

9. The hepatitis b combination therapeutic drug combination according to claim 8, characterized in that: the hepatitis B combined treatment medicine combination can be any one of the following forms:

firstly, asiatic acid and other hepatitis B treatment medicines are respectively prepared into independent preparations, the preparation formulations can be the same or different, and the administration routes can be the same or different;

10. Use of asiatic acid in the preparation of a substance having any one or more of the following effects:

use for the preparation of a substance inhibiting HBx protein;

use for the preparation of a substance that inhibits HBsAg levels;

use for the preparation of a substance that inhibits the level of intracellular total HBV RNAs;

use for the preparation of a substance that inhibits the intracellular HBV3.5-kb RNA level;

the use for preparing a substance for inhibiting the level of HBV DNA.