CN114796220A

CN114796220A - Application of neferine in preparation of anti-hepatitis B virus medicine

Info

Publication number: CN114796220A
Application number: CN202210531337.XA
Authority: CN
Inventors: 胡康洪
Original assignee: Kangluo Biotechnology Wuhan Co ltd
Current assignee: Kangluo Biotechnology Wuhan Co ltd
Priority date: 2022-05-16
Filing date: 2022-05-16
Publication date: 2022-07-29

Abstract

The invention provides an application of neferine in preparing anti-hepatitis B virus drugs, wherein the neferine targets hepatitis B virus RNA and interferes the synthesis of the hepatitis B virus RNA, so that the transcription of pgRNA, preS/S RNA and X RNA of virus is reduced, the reverse transcription synthesis of cDNA in virus nucleocapsid in cells and the synthesis of virus protein are reduced, and the synthesis and secretion of antigen HBsAg and HBeAg are reduced. The invention uses an in vitro cell model to discover that neferine has an inhibitory effect on HBV for the first time, and provides a new clue for the subsequent deep research and clinical medication of anti-HBV.

Description

Application of neferine in preparation of anti-hepatitis B virus medicine

Technical Field

The invention relates to the technical field of medical biology, in particular to application of neferine in preparation of a hepatitis B virus resistant medicine.

Background

Hepatitis B Virus (HBV) infection and its resulting chronic Hepatitis B constitute a worldwide public health threat, with HBV infection in 30% of the world, approximately 20 million people, and with chronic infections and carriers exceeding 3.5 million, these patients with Hepatitis B are also more likely to progress further to cirrhosis and even liver cancer, with about 80 million people worldwide each year being killed. China is a region with high incidence of hepatitis B virus infection, about 6 hundred million people have been infected with hepatitis B virus, about 8600 million virus carriers and 2800 million chronic hepatitis B patients exist, and the number of the people is high in the first place of the world. In order to control the development of hepatitis B epidemic, the traditional Chinese medicine mainly starts from two aspects of prevention and treatment. Due to the large-scale inoculation of the vaccine, the prevention means is greatly enhanced, and the possibility of the disease of the juveniles is effectively reduced; however, the existing clinical treatment scheme and effect are not ideal for the patient population with huge amount of developed chronic hepatitis B.

At present, the medicines for treating hepatitis B are mainly divided into two categories, one category is an immunomodulator, such as alpha-interferon, the course of treatment of the medicines is short, generally 6 months to 1 year, the curative effect is obvious, but the response rate is low, generally about 30 percent, and the response rate of the medicines in Asian patients is lower due to factors such as genetic background and the like; in addition, its clinical use is limited by strong side effects and expensive price; the other class of drugs is nucleoside/nucleotide analogs, such as lamivudine (3-TC), Adefovir (ADV), Entecavir (ETV) and the like, which are specially targeted to viral polymerase, control the disease condition by inhibiting the replication of viruses, have low toxicity, but cannot be eliminated for viral genome-covalent closed circular DNA (cccDNA) in a cell nucleus, and have the potential risks of drug resistance, drug withdrawal rebound and the like after long-term administration. Undoubtedly, the discovery of drugs with novel targets has become an imminent challenge for the treatment of hepatitis b.

The research in 2007 discovers that neferine has anti-HIV activity, and the action mechanism of neferine mainly relates to two metabolic pathways of N-demethylation and O-demethylation. In 2021, neferine was reported to inhibit infection by coronavirus (SARS) and new coronavirus (SARS-CoV 2). However, it has not been known whether or not it can inhibit hepatitis B virus.

Disclosure of Invention

The invention aims to provide application of neferine in preparation of a hepatitis B virus resistant medicament. The hepatoma cell line HepG2.2.15 supporting the replication of hepatitis B virus and the plasmid containing complete HBV genome are used to transiently transfect Huh7 cell line, so that HBV is replicated in Huh7 cells, and the cells are further incubated by using different concentrations of neferine for a certain time. Tests show that neferine has inhibitory effect on HBV. The specific analysis steps are as follows: HepG2.2.15 or Huh7 cell line transiently transfected with 1.1/1.3x HBV plasmid is treated with neferine with specific concentration for 1-4 days, then cell culture supernatant, intracellular total RNA and newly synthesized HBV DNA in virus nucleocapsid are respectively collected, and enzyme linked immunosorbent assay (ELISA), Northern Blotting and Southern Blotting are used for detection in sequence, and the result proves that neferine has inhibitory effect on HBV.

The neferine can react with appropriate organic acid or inorganic acid to form pharmaceutically acceptable salt, and can be used in medicine for resisting hepatitis B virus. Specifically, the salt can be hydrochloride, perchlorate, methanesulfonate, phosphate, citrate and sulfate of neferine.

In another aspect, the present invention also provides a pharmaceutical composition for resisting hepatitis b virus, which comprises neferine and/or a pharmaceutically acceptable salt of neferine.

On the basis of the technical scheme, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers include, but are not limited to: microcapsules, microspheres, polymer nanocapsules, polymer nanospheres, pharmacosomes, solid lipid nanoparticles, nanoemulsions, polymer micelles and the like.

On the basis of the technical scheme, preferably, the dosage form of the pharmaceutical composition is tablets, pills, dropping pills, capsules, granules, powder, suppositories, powder, paste, patches, injection, solution, suspension, spray, lotion, drops, liniment or emulsion.

The specific scheme of the invention is as follows:

firstly, adding neferine into a culture solution of a HepG2.2.15 cell line containing HBV genome and a cell line Huh7 transiently transfected with 1.1/1.3xHBV plasmid for incubation treatment for 3 days, and respectively collecting cell culture supernatant, intracellular total RNA and virus DNA in intracellular HBV nucleocapsid; subsequently, the secretion level changes of virus antigen HBsAg and HBeAg in the supernatant are detected by using an enzyme-linked immunosorbent assay (ELISA), the transcription change of virus mRNA in cells is detected by a Northern blotting method, and the level change of newly synthesized virus DNA in HBV nucleocapsids in cells is detected by a Southern blotting method.

Compared with the prior art, the invention has the following beneficial effects:

(1) the neferine is derived from the plant lotus plumule, is easy to obtain, has low cost, is nontoxic to human bodies, has homology of medicine and food, is suitable for low-income patients, and is easy to popularize;

(2) the neferine can reduce the expression of various mRNAs of the hepatitis B virus from the transcription level, which is different from other currently known anti-HBV medicines. Because transcription is the initial step of virus, it is the template of reverse transcription synthesis of virus cDNA, and is the template of translation of virus protein, inhibit virus from this level and can interfere virus replication and assembly greatly, the medicament effect is apparent;

(3) because the action mechanisms are different, the neferine can be used together with other existing clinical medicines, so that the drug resistance of patients to the existing chemotherapeutic medicines is solved, and the clinical symptoms are improved.

Drawings

In order to more clearly illustrate the embodiments or prior art solutions of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious that the drawings in the description are only some embodiments of the present invention, and other similar results can be obtained by those skilled in the art without inventive efforts.

FIG. 1 is a graph showing the effect of neferine on HBsAg secretion in cell supernatants;

FIG. 2 is a graph showing the effect of neferine on HBeAg secretion in cell supernatants;

FIG. 3 is a graph of the effect of neferine on the level of viral mRNA transcription in cells;

FIG. 4 is a graph of the effect of neferine on the level of cDNA synthesis in viral nucleocapsids in cells.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Embodiment 1:

detection of changes in HBsAg and HBeAg levels in supernatants of neferine-treated cells by enzyme-linked immunosorbent assay (ELISA)

Step 1, inoculating liver cancer cells into a six-hole plate, and starting to add medicine stimulation after 1 day of HepG2.2.15 inoculation or 1 day of transfection of 1.1/1.3x HBV plasmid by Huh 7. Adding 0.5 μ M, 1.5 μ M, 2.0 μ M and 2.5 μ M neferine into the culture medium in sequence, adding 30nM ETV into the positive control culture medium, treating for 3 days, changing the solution every day, collecting the cell supernatant after 3 days, and standing at-20 deg.C.

Wherein, the 1.1x HBV plasmid is pCH9-3091, which contains HBV genome of 1.1-fold adw genotype, the upstream of which is started by a Human Cytomegalovirus (HCMV)1 type early-medium protein (IE1) promoter, and the strong promoter can make the plasmid transfect into mammal eukaryotic cells and then efficiently transcribe HBV RNA transcript. The plasmid was gifted by doctor Michael Nassal, university Hospital, Frieberg, Germany.

The 1.3X HBV plasmid is p1.3HBV, which inserts 1.3 times adw gene type HBV genome into pGEM-3Z plasmid skeleton, and the virus genome initiation site is I-X enhancer initiation region. The plasmid was gifted by doctor Josef Shaul, the science institute of wilhelmy, israel.

And 2, adding 2.5 mu M of neferine into the culture medium of the cells under the same conditions as the step 1 and adding 30nM ETV into the culture medium under the same conditions and serving as a positive control group, treating for 4 days, changing the liquid every day, collecting cell supernatants on each day from the 1 st day to the 4 th day of treatment, and standing at-20 ℃ for later use.

And 3, after the cell supernatant collected in the step 1 and the step 2 is unfrozen, 10000g of cell supernatant is subjected to centrifugation for 5min, and then the cell supernatant is preheated for 30min in a 37 ℃ incubator.

And 4, taking out 500 mu L of each sample, and detecting the content of the HBsAg in the supernatant by using an enzyme-linked immunosorbent assay (ELISA) kit of the HBsAg in Shanghai science, wherein the detection process is carried out according to the product operation instruction.

And 5, taking out 200 mu L of each sample, and detecting the content of the HBeAg in the supernatant by using an enzyme-linked immunosorbent assay (ELISA) kit, wherein the detection process is carried out according to the product operation instruction.

And 6, repeating the steps 1 to 5 twice to obtain 3 groups of experimental data, importing the experimental data into GraphPad Prism 6 software, drawing a histogram and calculating a P value to determine whether the difference exists between the medicine group and the non-medicine control group. The specific experimental results are shown in fig. 1 and 2.

As can be seen from FIG. 1A, after incubation for 3 days with different concentrations of neferine, the level of HBsAg secreted from HepG2.2.15 cells into the supernatant gradually decreased, and the decreased level of HBsAg increased with increasing neferine concentration. Wherein after 3 days of 2.5. mu.M neferine treatment, the average HBsAg antigen level in the supernatant is only about 40% of that in the control group. As can be seen in FIG. 1B, HBsAg levels in the HepG2.2.15 supernatant gradually decreased with increasing treatment time and showed a clear time dependence under 2.5. mu.M neferine stimulation. In conclusion, the stimulation with neferine can cause the reduction of HBsAg secretion level of HepG2.2.15 cells, and shows dose-and time-dependence.

From FIG. 2A, it can be found that the secretion level of HBeAg in the supernatant decreased after 3 days of incubation of 1.5. mu.M, 2.0. mu.M, 2.5. mu.M neferine-treated HepG2.2.15 cells, and the tendency of HBeAg decreasing increased with increasing neferine concentration. After 3 days of 2.5. mu.M neferine stimulation, the average HBeAg level in the supernatant had decreased to about 50% of that in the control group. As shown in fig. 2B, after 2.5 μ M of neferine-stimulated hepg2.2.15 cells for 1 to 4 days, the level of HBeAg in the supernatant gradually decreased, with the degree of decrease increasing with the stimulation time. Taken together, as with the change in HBsAg in hepg2.2.15 cells caused by neferine stimulation, neferine stimulation of the cells resulted in a decrease in the level of HBeAg secretion in the supernatant and showed dose-and time-dependence.

As can be seen from FIG. 1C, after Huh7 cells were transfected with 1.1xHBV plasmid, the HBsAg secretion level in the supernatant was significantly reduced after 3 days of stimulation at neferine concentrations greater than 2.0. mu.M, whereas the stimulation with other concentrations of neferine did not cause significant change in HBsAg secretion. As shown in FIG. 1D, 2.5. mu.M neferine stimulated Huh7 cells transfected with 1.1xHBV plasmid resulted in a gradual decrease in HBsAg secretion levels in the supernatant over time. In conclusion, 2.5 μ M neferine was able to inhibit HBsAg secretion from Huh7 cells transfected with 1.1xHBV plasmid.

As can be seen from FIG. 2C, the secretion level of HBeAg in the cell supernatant decreased after 3 days of neferine stimulation at 1.5. mu.M, 2.0. mu.M, 2.5. mu.M, and when the neferine concentration reached 2.0. mu.M or more, the HBeAg decreased significantly, and the secretion level decreased to less than 50% of the control group. In FIG. 2D, the HBeAg secretion level in the supernatant of Huh7 cells transfected with 1.1XHBV plasmid was gradually decreased with increasing stimulation time under the action of 2.5. mu.M neferine. In conclusion, three concentrations of neferine, namely 1.5. mu.M, 2.0. mu.M and 2.5. mu.M, can inhibit HBeAg secretion of Huh7 cells transfected by 1.1xHBV plasmid, and have certain dose dependence and time dependence.

From the results in FIG. 1E, it can be seen that HBsAg secretion in the supernatant of Huh7 cells transfected with 1.3xHBV plasmid was reduced 3 days after the neferine concentration was greater than 2.0. mu.M, while HBsAg secretion was not affected by neferine stimulation at other concentrations. As can be seen from the results in fig. 1F, when 2.5 μ M neferine stimulates 1.3xHBV plasmid to transfect Huh7 cells, the level of HBsAg secretion in the supernatant decreases from day 2, and the results on days 3 and 4 are significantly different from those of the control group. From the results, it can be seen that the HBsAg level in the supernatant of Huh7 cells transiently transfected with 1.3xHBV was significantly inhibited only when the neferine concentration was greater than 2.0. mu.M for more than 3 days.

From FIG. 2E, it can be seen that the HBeAg level in the supernatant decreased significantly and tended to increase with increasing concentration after the 1.3xHBV plasmid transfected Huh7 cells were stimulated with 2.0. mu.M and 2.5. mu.M neferine for 3 days. FIG. 2F shows that a decrease in HBeAg levels in the supernatant occurred 2 days after 2.5. mu.M neferine stimulation of Huh7 cells transfected with 1.3xHBV plasmid, and the longer the stimulation time, the more significant the decrease in HBeAg. Taken together, the reduction of HBeAg secretion after transfection of Huh7 with 1.3xHBV plasmid requires stimulation with neferine at a concentration of 2.0. mu.M or more and takes at least 2 days.

Embodiment 2:

detection of intracellular viral mRNA levels by Northern blotting

Step 1, washing each group of cells for detecting the HBsAg and the HBeAg in the supernatant for 3 times by using PBS, then adding 1mL of Trizol solution into each hole of a six-hole plate, digesting for 5min at room temperature, slightly shaking, transferring the Trizol and the cells to a new centrifuge tube, and repeatedly blowing and sucking until no obvious precipitate exists.

And 2, adding 1/5 volumes of chloroform, violently shaking until the solution is milky white, and standing for 5min at room temperature.

And 3, centrifuging at 12000g at 4 ℃ for 15min, and dividing the mixed solution into 3 layers: the colorless supernatant containing RNA, the middle genomic DNA and the colored lower organic phase were carefully transferred to another new centrifuge tube.

And step 4, adding isopropanol with the same volume, fully and uniformly mixing, and standing at room temperature for 10 min.

And 5, centrifuging at 12000g at 4 ℃ for 10min, wherein RNA precipitates can be seen at the bottom of the centrifuge tube.

Step 6, carefully discard the supernatant, add 1mL of 70% ethanol, turn the tube upside down and wash the pellet.

And 7, centrifuging 12000g at 4 ℃ for 5min, removing ethanol, standing at room temperature for 5min, and adding 43 mu L of RNase-free deionized water for dissolution.

Step 8, preparing each sample according to the following formula: total RNA 43. mu.L, 10 XDNase I Buffer 5. mu.L, recombined DNase I (RNase-free) 1. mu.L, RNase Inhibitor 1. mu.L, reacted at 37 ℃ for 30 min.

Step 9, after the volume of the solution was increased to 100. mu.L by treating with 50. mu.L of DEPC water, an equal volume of a mixture of phenol/chloroform/isoamyl alcohol (25:24:1) was added thereto, and the mixture was shaken by inversion. Centrifugation was carried out at 12000g for 5min at room temperature and the supernatant was transferred to a fresh centrifuge tube.

Step 10, adding a chloroform/isoamyl alcohol (24:1) mixture with the same volume, reversing, uniformly mixing, centrifuging at 12000g at room temperature for 5min, and transferring the supernatant to a new centrifuge tube.

Step 11, add 1/10 volumes of NaAc (pH5.2) with 3M and 2.5 times of absolute ethanol, mix well and let stand overnight at-20 ℃.

And step 12, centrifuging 12000g at 4 ℃ for 10min, discarding supernatant, washing twice with 70% ethanol, drying at room temperature, and adding 20 mu L of RNase-free water for dissolving.

And step 13, taking a 20 mu gRNA sample, adding an equal amount of RNA loading buffer solution containing ethidium bromide, treating at 5 ℃ for 10min, and quickly placing on ice.

Step 14, preparing 1.2% agarose gel containing 18% formaldehyde according to molecular cloning guidelines. RNA samples are subjected to denaturation treatment, loading electrophoresis and electrophoresis at 105V and 4 ℃ for 3 h.

And step 15, after electrophoresis is finished, washing the surface of the gel by DEPC treatment water, soaking in Northern blotting gel treatment solution (0.4 g of NaOH, 175g of NaCl, dissolved in 1000mL of RNase-free water) for 20min, and then transferring to 20 XSSC (175 g of NaCl, 88g of trisodium citrate dihydrate, pH of 7.0, dissolved in 1000mL of RNase-free water) to soak for 10 min.

Step 16, transferring the treated gel to an acetate fiber membrane according to the molecular cloning experimental instruction, and carrying out alpha- ³² And (3) carrying out development observation after the P isotope labeled HBV probe is hybridized. The results are shown in FIG. 3.

From the Northern Blot results in FIG. 3, it can be seen that neferine stimulation can reduce the transcription of HBV RNA in cells, and can simultaneously reduce the levels of pgRNA, preS/S RNA and X RNA. pgRNA, in addition to serving as a template for the synthesis of (-) -DNA, is also capable of forming, by translation, viral polymerase (Pol protein) and Core protein (Core protein); translation of preS RNA produces LHBs, translation of S RNA produces MHBs and SHBs, and these products are all components constituting HBV HBsAg. The reduction in the levels of preS and S RNA leads to a reduction in HBsAg synthesis, resulting in a concomitant reduction in HBsAg secretion into the supernatant.

Transcription of X RNA forms HBx protein, and a decrease in X RNA transcript reduces intracellular viral HBx protein synthesis. It is known that the reduction of HBV RNA level has various negative effects on HBV.

In FIG. 3, Entecavir (ETV) has no inhibitory effect on HBVRNA, because the drug acts on the target of inhibiting reverse transcription synthesis of viral cDNA and has no influence on viral RNA, and the experimental result is as expected.

FIG. 3Northern blotting results the agarose gel electrophoresis of 28SRNA and 18SRNA as internal references is shown below, and the bands of the samples are not different from each other, indicating that the total nucleic acid amount is the same between the samples.

Embodiment 3:

detection of viral DNA levels in intracellular HBV nucleocapsids by Southern blotting

Step 1, HepG2.2.15 was inoculated in a 10cm dish, Huh7 was inoculated in a 6cm dish, and drug stimulation was started 1 day after HepG2.2.15 inoculation or 1 day after Huh7 transfection of 1.1/1.3XHBV plasmid. Sequentially adding 0.5 μ M, 1.5 μ M, 2.0 μ M and 2.5 μ M neferine and 30nM ETV as positive control into the culture medium, treating for 3 days, changing liquid every day, and freezing the cells at-80 deg.C after 3 days;

step 2, adding 2.5 mu M of neferine into the culture medium of the cells under the same conditions in the step 1, treating the cells for 4 days by using 30nM ETV as a positive control, changing the liquid every day, collecting the cells on each day from the 1 st day to the 4 th day of treatment, and freezing the cells at-80 ℃ for later use;

and 3, adding cell lysate into each sample, adding 1mL of lysate into cells on a 10cm plate, adding 500 mu L of lysate into a 60mm plate, shaking and cracking at 4 ℃ for 30min, centrifuging at 12000g for 20min after cracking is finished, and collecting the supernatant into a new precooled 1.5mL centrifuge tube.

Step 4, taking an equal amount of sample, and adding 1M MgCl into the sample respectively ₂ (final concentration 8mM), 1. mu.L of DNase I (70U/. mu.L), 5. mu.L of RNase A, digested at 37 ℃ for 1h to remove intracellular host DNA.

And 5, adding 10 mu L of 0.5M EDTA into each sample, fully and uniformly mixing, and treating at 60 ℃ for 10 min.

Step 6, 1/10 volumes of 10% SDS (final concentration of 1%), 20mg/mL protease K (final concentration of 50. mu.g/mL) were added and treated at 55 ℃ for 2h to lyse HBV nucleocapsids.

And 7, adding equal volume of Tris-saturated phenol, shaking, uniformly mixing, then 12000g, centrifuging for 10min, and transferring the supernatant into a new centrifuge tube.

Step 8, 1/10 volumes of 3M NaAc (pH5.2) were added, and after mixing by inversion an appropriate amount of glycogen (final concentration: 0.1. mu.g/. mu.L) was added thereto, 2.5 volumes of ethanol were added, and the mixture was allowed to stand at-20 ℃ overnight.

And 9, centrifuging at 4 ℃ and 12000g/min for 15min, removing supernatant, and washing precipitates once by using 70% ethanol.

And step 10, drying at room temperature for 10min, and adding 20 mu L of deionized water to dissolve the DNA precipitate.

Step 11, adding the purified sample into a DNA loading buffer solution, and then carrying out electrophoresis in 1% agarose gel at 105V and 4 ℃ for 3 h. Note that when the staining agent bromophenol blue migrates to the bottom of the gel, the electrophoresis can be stopped, so as to avoid the phenomenon that the voltage is too high, otherwise the generated heat can deform the gel.

Step 12, after electrophoresis is finished, washing the gel with double distilled water, treating the gel with freshly prepared hydrolysate (2 mL of concentrated HCl is added into 100mL of deionized water) for 5min, and washing with double distilled water; soaking the denatured liquid (NaOH 20g, NaCl 87.6g, dissolved in 1000mL deionized water) for 15min, treating for 2 times, and washing with double distilled water; neutralizing solution (Tris 60g, NaCl 87.6g, dissolved by adding about 800mL of deionized water, adjusting pH to 7.5 by concentrated HCl, and fixing volume to 1000mL) for 15min, treating for 2 times, and washing by double distilled water; 20 XSSC buffer (NaCl 175g, trisodium citrate dihydrate 88g, pH 7.0, dissolved in 1000mL deionized water) for 10 min.

Step 13, transferring the treated gel to an acetate fiber membrane according to the molecular cloning experimental instruction, and performing alpha- ³² P isotope labeled HBV probe is observed by autoradiography after hybridization. The results are shown in FIG. 4.

As can be seen from FIG. 4A, with the increase of neferine concentration, the replication level of HBV DNA in viral nucleocapsid in HepG2.2.15 cell gradually decreased, and after 3 days of 2.5. mu.M neferine stimulation, HBV SS DNA had been significantly weakened and RC DNA was also weakened. As shown in FIG. 4B, HepG2.2.15 cells were stimulated with 2.5. mu.M neferine for 1 to 4 days, respectively, and it can be seen that the HBV DNA level in the intracellular viral nucleocapsid is sequentially decreased as the stimulation time is increased. From the above results, it was found that neferine can inhibit replication of HBV in hepg2.2.15 cells.

As can be seen from fig. 4C, in Huh7 cells transfected with 1.1 × HBV plasmid, the replication level of HBV was decreased in the neferine-treated group compared to the control group, but the decrease tendency did not show dose dependence. FIG. 4D shows that 2.5. mu.M neferine treated cells sequentially decreased the level of HBV replication in the cells with increasing treatment time. As shown in fig. 4E, in Huh7 cells transfected with 1.3 xlhbv plasmid, after 3 days of 2.0 μ M, 2.5 μ M neferine stimulation, the replication level of HBV in the cells was reduced to some extent; while the HBV replication is not inhibited when the 0.5 mu M and 1.5 mu M neferine are stimulated; FIG. 4F shows that 2.5. mu.M neferine-stimulated for 1 to 4 days, the replication of HBV in cells was inhibited to some extent compared to the control group. In conclusion, neferine can inhibit HBV replication in Huh7 cells transiently transfected with HBV; among them, the inhibitory effect on HBV replication in 1.1xHBV transfected Huh7 cell was slightly stronger than that of 1.3xHBV transfected Huh7 HBV.

In FIG. 4, the positive control Entecavir (ETV) acts on the target to inhibit reverse transcription synthesis of viral cDNA, and the result shows that no DNA band exists, and the experimental result is in line with the expectation.

FIG. 4 Southern blotting results Western blotting showing β -actin as an internal reference, with no band difference between samples, suggesting that there is no absolute amount error between samples in preparation.

In conclusion, the invention firstly proves that neferine can inhibit the synthesis of HBV RNA from the transcription level, and further influences the reverse transcription of downstream virus cDNA and the synthesis and secretion of virus antigen. The discovery not only widens the idea of HBV treatment and increases the possibility of drug combination selection, but also provides new understanding for the traditional botanical drug neferine and provides new clues for HBV clinical treatment.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. Application of neferine and its pharmaceutically acceptable salt in preparing medicine for resisting hepatitis B virus is provided.

2. The use of neferine and its pharmaceutically acceptable salts in the preparation of anti-hepatitis B virus drugs according to claim 1, wherein the pharmaceutically acceptable salts are neferine hydrochloride, perchlorate, methanesulfonate, phosphate, citrate and sulfate.

3. A pharmaceutical composition for use against hepatitis b virus, comprising: one or two of the pharmaceutically acceptable salts of neferine and neferine are mixed and pharmaceutically acceptable carrier.

4. The pharmaceutical composition for resisting hepatitis b virus according to claim 3, wherein the pharmaceutical composition is in the form of a tablet, a pill, a drop pill, a capsule, a granule, a powder, a suppository, a powder, an ointment, a patch, an injection, a solution, a suspension, a spray, a lotion, a drop, a liniment or an emulsion.