CN112618584A

CN112618584A - Application of four-tile extract in preparing medicine for resisting hepatitis B virus and/or preventing and treating hepatitis B

Info

Publication number: CN112618584A
Application number: CN202011477717.7A
Authority: CN
Inventors: 郭弘妍; 王辉; 郝艳同; 刘京京; 邢婉丽; 程京
Original assignee: Boao Biological Group Co ltd
Current assignee: Boao Biological Group Co ltd; CapitalBio Corp
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2021-04-09

Abstract

The invention relates to the technical field of natural medicines, in particular to a preparation method and application of a four-tile extract. The invention provides application of a chloranthus japonicus extractive, and CCK8 cell proliferation detection experiments show that the chloranthus japonicus extractive has no cytotoxicity on HepG2.2.15, has obvious inhibition on HBsAg and HBeAg antigen secretion, and shows good anti-hepatitis B virus application prospect. Can reduce the AST content in the supernatant of HepG2.2.15 cells and has better effect of resisting liver injury. Analysis of transcriptome data of the Huvec cell model shows that the gene expression level of inflammatory pathways such as TNF signal pathways, IL-17 signal pathways and the like can be effectively regulated, and the gene expression level has a better liver inflammation regulation function.

Description

Application of four-tile extract in preparing medicine for resisting hepatitis B virus and/or preventing and treating hepatitis B

Technical Field

The invention relates to the technical field of natural medicines, in particular to application of a chloranthus japonicus extractive in preparing a medicine for resisting hepatitis B virus and/or preventing and treating hepatitis B.

Background

The liver is the largest parenchymal organ in the abdominal cavity and is responsible for important physiological functions of the human body. Its main function is to regulate the metabolism and energy metabolism of the whole body and at the same time it is also an important immune organ. Liver damage may be caused by liver irritation or infection, such as infectious diseases, immunodeficiency diseases, and the like, and chemical poisons and the like. When the liver is damaged, inflammation, such as hepatocyte necrosis, degeneration, swelling, inflammatory infiltration of various degrees, fibroplasia, etc., may occur in the liver, which in turn may affect liver function. Persistent inflammation can disrupt tissue integrity, chromosomal stability, promote apoptosis, proliferation, and carcinogenesis. Inflammation of the liver can be caused by a number of infectious (hepatitis virus, etc.) and non-infectious (alcohol, obesity, etc.) factors. If the pathogen (hepatitis A or E virus) or other harmful factors (drugs, alcohol) are cleared or eliminated in time, the inflammation will subside. In China, viral hepatitis, especially chronic hepatitis B, is the most common cause of liver damage and ultimately cirrhosis.

The anti-hepatitis B virus treatment is the key to prevent further development of liver injury, and the research and development of new drugs with good antiviral effect and low side effect are imperative. At present, the anti-hepatitis B virus drugs mainly comprise interferon and antiviral drugs. These drugs have quick curative effect, but are easy to recur, and have certain side effects. There is no specific drug in the treatment of preventing liver injury and relieving liver inflammation. China is one of the countries with higher incidence of hepatitis B, and the search and research of natural medicines with both antiviral and liver-protecting effects are always important directions for the research of traditional Chinese medicines. The traditional Chinese medicine utilizes holism concept and characteristics of treatment based on dialectical theory, exerts multiple ways, multiple levels and multiple targets of the traditional Chinese medicine, has low price and less side effect, and has great advantages in the aspects of hepatitis B virus resistance and liver injury resistance.

The herba Chloranthi Henryi is whole herb of herba Lysimachiae Christinae or herba Lysimachiae Christinae of Paris polyphylla or Primulaceae. Modern scientific research shows that the four-piece shingles contains chloranthus japonicus lactone, eurasia glechoma lactone, chloranthus japonicus lactone A, atractyloide, chloranthus japonicus spirodiclol, isofraxidin and the like. Has effects in dispelling cold, relieving cough, promoting blood circulation, relieving pain, removing blood stasis, and removing toxic substances. Can be used for treating cough due to wind-cold evil, rheumatic osteodynia, and amenorrhea; it is used externally to treat traumatic injury, swelling and pain due to blood stasis, and snake bite. However, there is no report on anti-hepatitis B virus, anti-inflammation and liver protection.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide an application of a chloranthus japonicus extractive in preparation of a medicament for resisting hepatitis b virus and/or preventing and treating hepatitis b, and experiments of the present invention prove that the chloranthus japonicus extractive has obvious hepatitis b virus resisting effect, good liver injury resisting effect and good liver inflammation reaction regulating effect.

The invention also provides application of the four-tile extract in preparing a medicament for inhibiting the level of inflammatory-related pathways. In some embodiments, the inflammatory-related pathway is an inflammatory-related pathway in a Huvec cell. Studies have shown that four-tile alcohol extracts are effective in modulating inflammatory pathways such as impaired inflammatory-related pathways including: the gene expression level of TNF signal channel, IL-17 signal channel, FoxO signal channel, NOD-like receptor signal channel, viral protein and cytokine receptor interaction, epithelial cell signal channel in helicobacter pylori infection, rheumatoid arthritis and other channels has better liver inflammation regulation function.

The invention provides application of a chloranthus japonicus extract in preparing a medicament for inhibiting the level of hepatitis B surface antigen and/or hepatitis B core antigen.

In the present invention, the hepatitis B surface antigen and/or hepatitis B core antigen level is the hepatitis B surface antigen and/or hepatitis B core antigen level in a hepatitis B virus-infected cell. Research shows that the four-tile extract has obvious inhibition effect on HBsAg and HBeAg antigen secretion of HepG2.2.15 cells, and shows good anti-hepatitis B virus application prospect.

In the invention, the hepatitis B virus infected cell is HepG2.2.15. CCK8 cell proliferation assay showed that the four tile extract had no cytotoxic effect on HepG2.2.15. Research shows that the four-tile extract has a good effect of resisting liver injury on reducing the AST content in the supernatant of HepG2.2.15 cells. Therefore, the invention also provides the application of the four-tile extract in preparing the medicine for protecting the hepatitis B virus infected cells. In the present invention, the protection includes suppression of AST levels. The hepatitis B virus infected cell is HepG2.2.15.

The invention also provides application of the four-tile extract in preparing a medicament for treating hepatitis B.

In the present invention, the four-tile extract is a four-tile ethanol extract.

In some embodiments, the method of preparing the four-tile ethanol extract comprises: extracting the four tiles by 80-95% ethanol solution to obtain four tile ethanol extracts.

In the preparation method of the extract, the volume fraction of ethanol in the ethanol solution is 90%. The extraction mode is Soxhlet extraction or ultrasonic-assisted extraction.

In the Soxhlet extraction method, the mass-volume ratio of the four tiles to the ethanol solution is 10g:150 mL. The parameters of the soxhlet extraction include: reflux extraction for 3 h. Concentrating the obtained extract, and drying to obtain ethanol extract of four tiles.

In the ultrasound-assisted extraction method, the mass-to-volume ratio of the four tiles to the ethanol solution is 10g:120 mL. The parameters of the ultrasound-assisted extraction include: soaking for 1-2h, and carrying out ultrasonic extraction for 10-30 min. Concentrating the obtained extract, and drying to obtain ethanol extract of four tiles.

The preparation method of the four-tile extract also comprises the step of dissolving the four-tile ethanol extract and then extracting with an organic solvent, wherein the organic solvent comprises at least one of petroleum ether, dichloromethane, ethyl acetate or n-butanol. In the invention, the solvent used for dissolving the ethanol extract of the four tiles is water. Dissolving into four tiles, and dissolving the ethanol extract into DMSO to obtain ethanol extract solution. In the embodiment of the invention, the organic solvent is: ethyl acetate, petroleum ether, dichloromethane or n-butanol. Preferably, the organic solvent is petroleum ether or dichloromethane. The volume ratio of the alcohol extract solution to the organic solvent is 20: 15.

According to the experimental result of the invention, the ethanol extract of four tiles, the ethyl acetate part, the petroleum ether part, the dichloromethane part or the n-butanol part have certain anti-hepatitis B virus effect, and the effect is obviously superior to that of lamivudine, catechin or epicatechin. Through detection, the four tile ethanol extracts and the four organic solvent parts contain no or only trace catechin or epicatechin, which suggests that other components in the extracts have the effect of inhibiting the hepatitis B virus.

The invention also provides a medicament for resisting hepatitis B virus and/or preventing and treating hepatitis B, which comprises the four-tile extract. The medicine also comprises pharmaceutically acceptable auxiliary materials.

The pharmaceutically acceptable adjuvants are one or more of fruit powder, edible essence, sweetener, sour agent, bulking agent, lubricant, antiseptic, suspending agent, edible pigment, diluent, emulsifier, disintegrating agent or plasticizer.

The medicament of the invention is in the dosage form of tablets, pills, oral liquid, capsules, syrups, dripping pills or granules. The capsule is hard capsule or soft capsule. The tablet is oral tablet or buccal tablet. Oral tablets refer to tablets intended for oral administration, most of which act by absorption through the gastrointestinal tract, and some of which act locally in the gastrointestinal tract. In some embodiments provided herein, the oral tablet is a compressed tablet, a dispersible tablet, an effervescent tablet, a chewable tablet, a coated tablet, or a sustained release tablet.

The invention also provides a method for resisting hepatitis B virus and/or preventing and treating hepatitis B, which is to administer the medicament. The administration is oral and/or injection.

The invention provides a four-tile extract and application of an organic solvent extraction part of the extract, and CCK8 cell proliferation detection experiments show that the four-tile extract and the organic solvent extraction part of the extract have no cytotoxicity to HepG2.2.15, have obvious inhibition to secretion of HBsAg and HBeAg antigens, and show good anti-hepatitis B virus application prospects. Can reduce the AST content in the supernatant of HepG2.2.15 cells and has better effect of resisting liver injury. Analysis of transcriptome data of the Huvec cell model shows that the gene expression level of inflammatory pathways such as TNF signal pathways, IL-17 signal pathways and the like can be effectively regulated, and the gene expression level has a better liver inflammation regulation function.

Drawings

FIG. 1 shows the effect of four-tile ethanol extracts on HepG2.2.15 cell proliferation;

FIG. 2 shows the results of ELISA detection of hepatitis B surface antigen HBeAg;

FIG. 3 shows the results of ELISA detection of hepatitis B core antigen HBsAg;

FIG. 4 shows the AST assay results for four tile extracts (100. mu.g/mL) treated HepG2.2.15 cell supernatant;

FIG. 5 shows the differential gene KEGG pathway enrichment results for the four tile extract treatment group;

FIG. 6 shows HPLC chromatogram (50min) of each active site of catechin standard and Tetrastigma croci Sativi extract;

FIG. 7 shows HPLC chromatogram (40min) of each active site of catechin standard and Tetrastigma croci extract;

FIG. 8 shows an HPLC chromatogram (33min) of the catechin content in each active fraction of a four-piece Tile extract;

FIG. 9 shows the results of the anti-HBV experiment of each active site of the four-tile alcohol extract;

FIG. 10 shows the results of lamivudine, catechin and epicatechin anti-HBV tests.

Detailed Description

The invention provides the application of the four-tile extract, and the technical personnel can use the content for reference and appropriately improve the technological parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The preparation method of the four-tile extract comprises the following steps: the four tiles adopt a Soxhlet extraction method, ethanol with the concentration of 90 percent is used as a solvent, and the four tiles are heated, refluxed, extracted, filtered, concentrated and freeze-dried to obtain four tile alcohol extracts.

The four-tile alcohol extract disclosed by the invention has the following effects:

(1) has obvious hepatitis B virus resisting effect. The invention adopts a Soxhlet extraction method, uses 90 percent ethanol as a solvent for four tiles, and obtains the four tiles extract by heating, refluxing, extracting, filtering, concentrating, freezing and drying. Based on a HepG2.2.15 cell hepatitis B virus model, the four-tile extract is subjected to anti-hepatitis B drug screening, and CCK8 cell proliferation detection experiments show that the four-tile extract has no cytotoxicity on HepG2.2.15 and has obvious inhibition on HBsAg and HBeAg antigen secretion.

(2) Has good effect of resisting liver injury. The supernatant after HepG2.2.15 cell treatment is detected by detecting the four-tile extract, and the AST in the HepG2.2.15 cell supernatant is reduced by the four-tile extract, so that the four-tile extract has potential functions of protecting liver cells and reducing liver injury.

(3) Has good effect of regulating the inflammatory reaction of the liver.

The four tile alcohol extracts are used for treating a vascular endothelial cell inflammation model, then transcriptome sequencing analysis is carried out, and the KEGG channel analysis result shows that the four tile alcohol extracts can effectively reduce the expression level of inflammatory channels related to the vascular endothelial cell inflammation model. In addition, transcriptome Disease analysis shows that related differential genes can be enriched on pathways of atherosclerosis and Hepatitis C related diseases, and pathway gene expression is reduced. The four-tile alcohol extract has relevant regulation effect on disease pathways such as atherosclerosis, Hepatitis C and the like.

The invention is further illustrated by the following examples:

example 1: soxhlet preparation method of ethanol extract of four-piece tile

1. Taking 10g of four tiles, and crushing the four tiles into powder by a crusher.

2. The four tiles of powder were wrapped in filter paper and placed in a soxhlet extractor and 150mL 90% ethanol was added.

3. Heating and refluxing for 3h by using a Soxhlet extractor.

4. Filtering the extractive solution, and concentrating at 45 deg.C to obtain extract.

5. The four-tile ethanol extract was obtained after 24h by freeze-drying.

Collecting lyophilized ethanol extract of four tiles, and placing in a refrigerator at-80 deg.C for use

Example 2: ultrasonic preparation method of four-tile extract

2. Adding 90% ethanol solution according to the material ratio of 1:12, soaking for 1-2h, ultrasonic extracting for 10-30min, standing, and concentrating the supernatant at 70 deg.C.

3. Freezing the concentrated solution at-80 deg.C for more than 6h, and vacuum drying at low temperature to obtain dry powder.

Example 3: experiment of anti-hepatitis B virus of four-tile extract.

1. Materials and methods

(1) Preparation of ethanol extract of four tiles: four tile alcohol extracts prepared according to the method of the invention in example 1 or example 2 (research shows that the extracts obtained by the two methods have similar activity and show similar activity at different parts of organic extraction), 50mg of freeze-dried powder is taken and added with 1mLDMSO to prepare 50mg/mL of concentrated solution.

(2) Preparation of formula experimental working solution: according to the experimental requirements, the concentrated solution will be diluted with culture medium to 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL working solution.

(3) Hepg2.2.15 cell plating: for the hepatotoxicity test, hepg2.2.15 cells were used. Taking culture bottles of two types of cells from a culture box, adding 1mL of pancreatin into the culture bottles respectively, slightly shaking to cover the whole cell surface with the pancreatin, putting the culture bottles into the culture box for digestion until the cells partially float, adding 2mL of complete culture medium to stop digestion, blowing and beating the culture medium by using a Pasteur pipette to enable the cells to fall off, centrifuging, removing supernatant, adding 2mL of culture medium to lightly blow and evenly mix the cells, and counting by using a cell counting plate. 96 well cell plates were plated at 10000 cells/well. Placing in an incubator for 24 h.

(4) Cell dosing: the cell culture plate was removed from the incubator and the medium was slowly discarded. Washing with PBS for 1 time, setting experimental holes and control holes, taking DMSO as the control holes for alcohol extraction, and adding 100 μ L of prescription experiment working solution with different concentration gradients into the experimental holes. Culturing in an incubator for 24 h.

(5) And (3) CCK-8 detection: the cell culture plate was removed from the incubator, the medium aspirated and transferred to another 96 well cell culture plate for detection of hepatitis b surface antigen HBsAg and hepatitis b core antigen HBeAg. Then, 110. mu.L of the prepared cck8 complete medium was added to a 96-well plate in which cells were cultured. After incubation in an incubator for 3h, the measurement was carried out with a microplate reader (wavelength 450 nm).

(6) Detecting hepatitis B surface antigen HBsAg and hepatitis B core antigen HBeAg: and (3) performing ELISA detection on the cell culture solution collected in the previous step by adopting a hepatitis B surface antigen detection kit and a hepatitis B core antigen detection kit.

2. Results of the experiment

(1) HepG2.2.15 cell CCK8 cell proliferation assay

This example uses the CCK8(Cell Counting Kit-8) Kit to examine the effect of four-tile extract on HepG2.2.15 Cell proliferation. Four tile extracts were diluted to different concentrations (400, 200, 100, 50, 25 μ g/mL), and hepg2.2.15 cells in log phase of growth were treated with DMSO control, and 24h later, cell viability was determined using CCK8 kit and cell proliferation rate was calculated. As can be seen from FIG. 1, the four-tile ethanol extract has small proliferation inhibition effect on HepG2.2.15 cells under various concentrations, and the inhibition rate is less than 10%.

(2) ELISA detection result of hepatitis B surface antigen HBeAg

Diluting four tile extracts into different concentrations (400, 200, 100, 50 and 25 mu g/mL), treating HepG2.2.15 cells in a growth log phase, setting DMSO control, detecting by adopting an ELISA detection kit of HBeAg after 24 hours, and calculating the ratio of the control to the drug-added group ELISA detection. As can be seen from FIG. 2, the four-tile ethanol extract had a greater inhibitory effect on the secretion of HBeAg by HepG2.2.15 cells at each concentration.

(3) ELISA detection result of hepatitis B core antigen HBsAg

Diluting four tile extracts into different concentrations (400, 200, 100, 50 and 25 mu g/mL), treating HepG2.2.15 cells in a growth log phase, setting DMSO control, detecting by adopting an ELISA detection kit of HBsAg after 24 hours, and calculating the ratio of the control to the ELISA detection of a dosing group. As can be seen from FIG. 3, the four-tile ethanol extract had a greater inhibitory effect on the secretion of HBsAg from HepG2.2.15 cells at each concentration.

Example 3: experiment for reducing hepatitis B cell model HepG2.2.15 cell damage by using four-tile extract

1. Materials and methods

(1) Preparation of ethanol extract of four tiles: the four-tile alcohol extract prepared by the method of the

embodiment

1 or 2 is prepared by taking 50mg of freeze-dried powder, adding 1mL of DMSO and preparing into 50mg/mL of concentrated solution.

(2) Preparation of formula experimental working solution: the concentrate will be diluted with medium to 100. mu.g/mL depending on the experimental requirements.

(4) Cell dosing: the cell culture plate was removed from the incubator and the medium was slowly discarded. Washing with PBS for 1 time, setting experimental holes and control holes, taking DMSO as the control holes for alcohol extraction, and adding 200 μ L of prescription experiment working solution with different concentration gradients into the experimental holes. Culturing in an incubator for 24 h.

(5) And (3) CCK-8 detection: the cell culture plate was removed from the incubator, the medium aspirated and transferred to another 96 well cell culture plate for detection of hepatitis b surface antigen HBsAg and hepatitis b core antigen HBeAg. Then, 110. mu.L of the prepared CCK8 complete medium was added to a 96-well plate in which the cells were cultured. After incubation in an incubator for 3h, the measurement was carried out with a microplate reader (wavelength 450 nm).

(7) Hepg2.2.15 cells: the AST value of HepG2.2.15 cell culture supernatant was determined on the collected supernatant sample using an AST detection kit.

2. Results of the experiment

The results show that the treatment of hepg2.2.15 cells with the four-tile extract has potential anti-hepatocyte damage function at 100 μ g/mL, and specifically the four-tile extract reduces AST in hepg2.2.15 cell supernatant (fig. 4). Shows that the four-piece alcohol extract has potential functions of protecting liver cells and reducing liver damage.

Example 4: transcriptome experiment of four-tile extract processing Huvec inflammation model

1. Materials and methods

(1) Preparing a four-tile ethanol extract: the four-tile alcohol extract prepared by the method of the

embodiment

(2) Huvec cell plating: taking culture bottles of the Huvec cells from the culture box, adding 1mL of pancreatin into the culture bottles respectively, slightly shaking to enable the whole cell surface to be covered by the pancreatin, putting the culture bottles into the culture box for digestion until the cells partially float, adding 2mL of complete culture medium to stop digestion, using a Pasteur pipette to blow and beat the culture medium to enable the cells to fall off, centrifuging, removing supernatant, adding 2mL of culture medium to lightly blow and beat the cells uniformly, and counting by using a cell counting plate. 96 well cell plates were plated at 10000 cells/well. Placing in an incubator for 12 h.

(3) ECM complete medium (LPS inducer containing): 50 μ L of 1mg/mL LPS was added to 50mL of complete ECM medium.

(4) Preparation of formula experimental working solution: four portions of the aqueous ethanolic extract concentrate were diluted to 200. mu.g/mL using ECM complete medium (containing LPS inducer) according to the experimental requirements.

(5) The cell culture plate was removed from the incubator, the medium was slowly discarded (to prevent discarding of cells), 100. mu.L/well of complete medium (containing LPS inducer) containing four tiles of ethanol extract was sequentially added according to the arrangement of the well plates, and the cells were incubated for 24 hours in the incubator. Control wells were filled with 100. mu.L/well of complete medium (LPS inducer) only.

(6) The 96-well cell culture plate was removed and approximately 95. mu.L of medium was discarded. Add 100. mu.L PBS to each sample well and wash once. Add 18.0. mu.L of lysate to each sample well, gently blow and suck 5 times and mix (on ice) and lyse on ice for 5 min.

(7) The cleaved RNA is subjected to reverse transcription to produce cDNA. After fragmentation, end repair filling, tail addition, linker ligation, library enrichment.

(8) The library was subjected to illumina high throughput sequencing.

2. Results of the experiment

And performing transcriptome sequencing data analysis on the off-line data of the library, performing differential gene analysis on the transcriptome data of the medicated Huvec cells and the non-medicated Huvec cells, and performing KEGG gene path enrichment analysis. The results show that the four-piece kavalol extract can effectively reduce the gene expression level of inflammatory pathways related to vascular endothelial cell inflammation models, such as TNF signaling pathway, IL-17 signaling pathway and the like (figure 5).

Example 5: and (3) identifying active ingredients of the four tile extracts and analyzing the ingredients:

1. materials and methods

(1) The four tile extracts obtained in example 1 or example 2 were suspended with the appropriate amount of water. Extracting with organic solvent such as petroleum ether, dichloromethane, ethyl acetate, and n-butanol for three times, mixing the three extractive solutions, vacuum concentrating with rotary evaporator (petroleum ether concentration temperature 40 deg.C, dichloromethane concentration temperature 30 deg.C, ethyl acetate concentration temperature 55 deg.C, and n-butanol concentration temperature 90 deg.C) until the extractive agents are removed, respectively adding water for suspension, freezing at-80 deg.C for more than 6 hr, and vacuum drying at low temperature to obtain dried powder of organic solvent extraction part.

(2) Precisely weighing 5.0mg of each sample obtained in the step (1), fully dissolving the sample with methanol to prepare a sample with a final concentration of 1mg/mL, filtering the sample with a 0.22 mu m filter membrane, and analyzing the filtrate by high performance liquid chromatography. The catechin standard substance is prepared to have a concentration of 25 μ g/mL, and is filtered through a 0.22 μm filter membrane, and the filtrate is analyzed by high performance liquid chromatography. The sample information was analyzed as follows:

TABLE 1 analysis of samples

Sample name	Sample numbering	Solvent(s)	Detecting the concentration
				Catechin	C	Methanol	25μg/mL
Epigallocatechin	EGC	Methanol	25μg/mL
				Epicatechin gallate	ECG	Methanol	25μg/mL
Epimetechin	EC	Methanol	25μg/mL
				Epigallocatechin gallate	EGCG	Methanol	25μg/mL
Mixed label (5 kinds)	SD	Methanol	25μg/mL
				Four-tile total butanol extract	Total	Methanol	1mg/mL
Petroleum ether fraction of four tiles	Pe	Methanol	1mg/mL
				Four tiles of methylene chloride fraction	Di	Methanol	1mg/mL
Four tile ethyl acetate fraction	Et	Methanol	1mg/mL
				N-butanol fraction of four tiles	Bu	Methanol	1mg/mL

(3) Performing chromatographic analysis on the sample, and performing ultraviolet detection by using an Agilent 1260-type high performance liquid chromatograph, wherein a chromatographic column is Waters, Symmetry 300TM C18, (4.6 multiplied by 250mm,5 mu m), a mobile phase is a water (A) -chromatographic methanol (B) binary system, a gradient elution procedure is 0-10min, and the concentration of B is 35-40%; 10-30min, 40% -58% B; 30-35min, 58% -70% B; 35-40min, 70% -95% B; 40-50min, 95% B. The flow rate was 1mL/min, the amount of sample was 20. mu.L, and the detection wavelength was 232 nm.

2. Results of the experiment

(1) Chromatographic analysis is carried out on each part of the four-tile extract, and a chromatogram shows that the active site components of petroleum ether, dichloromethane, ethyl acetate and n-butanol of the four-tile extract are obviously different (figures 6-8).

(2) The chromatogram of each of the catechin standard substance and the four tiles is analyzed by software of a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), as shown in fig. 6, the components of catechins: concentrated distribution in retention time Rt 2-5min, Et: the difference from other four tile parts is large, and the enrichment effect on chemical components with Rt being 41.2min is very obvious. Other chromatographic peaks were not evident by the effect of the peak in Et. Processed by software of traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), as shown in fig. 7, and compared with chromatographic peaks of standard catechin products, chromatographic peaks of 5 catechins cannot be detected at the Pe and Di parts under the separation effect of the extracting agent. Et shows a weak peak, Bu shows a clear chromatographic peak, and catechin-containing components are more likely to be contained.

(2) Other chromatographic conditions were unchanged, the elution procedure was optimized to be: 0-5min, 5% -9% B; 5-8min, 9% B; 8-10min, 9-15%, 10-13min, 15-20% B, 13-25min, 20-40% B; 25-40min, 40% B. Under the chromatographic conditions, 5 kinds of catechin compounds were injected and the peak positions (SD) of the chromatographic peaks were determined, and it was confirmed from the HPLC chromatogram of the catechin compounds in each part of the four tiles in FIG. 8: pe (petroleum ether fraction) and Di (methylene chloride fraction) have no catechin component, and Bu (n-butanol fraction), Total (ethanol extract), Et (ethyl acetate fraction) may contain a certain amount of catechin component. The contents of n-butanol-containing fractions of catechin (C) and Epicatechin (EC) were both < 0.05% by chromatographic peak calculation.

Example 6: anti-hepatitis B virus experiment of organic solvent extraction part of four-tile extract and comparison with lamivudine, catechin and epicatechin

1. Materials and methods

(1) Preparation of petroleum ether, dichloromethane, ethyl acetate, n-butanol fractions of the four tile extract prepared in example 5: the petroleum ether, dichloromethane, ethyl acetate, n-butanol fractions of the four-tile extract prepared according to the method of example 3 of the present invention. 50mg of the freeze-dried powder is taken and added with 1mLDMSO to prepare a concentrated solution of 50 mg/mL.

(2) Preparation of formula experimental working solution: according to the experimental requirements, petroleum ether, dichloromethane, ethyl acetate and n-butanol of the four-tile extract are diluted into 400, 200, 100, 50 and 25 mu g/mL working solution by using a culture medium. The control drugs, lamivudine, catechin and epicatechin, were diluted in a medium to give a working solution of 12.5. mu.g/mL, 25. mu.g/m, 50. mu.g/mL, 100. mu.g/mL and 200. mu.g/mL.

2. Results of the experiment

The results of experiments on the proliferation of HepG2.2.15 cell CCK8 cells of four tile extracts containing petroleum ether, dichloromethane, ethyl acetate and n-butanol parts and control drugs of lamivudine, catechin and epicatechin and ELISA detection results of hepatitis B surface antigen HBeAg and HBsAg show that the petroleum ether and dichloromethane active parts of the four tile extracts have small proliferation inhibition effect and small cytotoxicity on HepG2.2.15 cells at the concentration of 25-200 [ mu ] g/mL, and remarkably inhibit the secretion of hepatitis B surface antigen HBsAg and hepatitis B core antigen HBeAg of HepG2.2.15 cells. The ethyl acetate and n-butanol parts of the four-tile extract have small proliferation inhibition effect on HepG2.2.15 cells and small cytotoxicity at low concentration (25 mu g/mL) and high concentration (400 mu g/mu L). And can obviously inhibit the secretion of hepatitis B surface antigen HBsAg and hepatitis B core antigen HBeAg of HepG2.2.15 cells (figure 9). In addition, as a comparison, the present invention also used the reference drugs lamivudine and catechin, epicatechin, etc. to perform anti-hepatitis b virus experiments, and as a result, the anti-hepatitis b virus effects of the four-tile extract and the active site (petroleum ether, dichloromethane, ethyl acetate, n-butanol extract site) were superior to those of lamivudine, catechin, epicatechin (fig. 10).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims

1. Use of a chloranthus japonicus extract for the manufacture of a medicament for inhibiting the level of an inflammatory-related pathway.

2. The use of claim 1, wherein the impairment of an inflammatory-related pathway comprises: TNF signaling pathways, IL-17 signaling pathways, FoxO signaling pathways, NOD-like receptor signaling pathways, interactions of viral proteins with cytokines and cytokine receptors, epithelial cell signaling pathways in H.pylori infection, and/or rheumatoid arthritis.

3. Use of extract of herba Chloranthi Henryi in preparing medicine for inhibiting hepatitis B surface antigen and/or hepatitis B core antigen level is provided.

4. Application of herba Chloranthi Henryi extract in preparing medicine for protecting hepatitis B virus infected cell is provided.

5. The use of claim 4, wherein said protection comprises suppression of AST levels.

6. The application of the four-tile extract in preparing the medicine for resisting hepatitis B virus and/or preventing and treating hepatitis B.

7. The use according to any one of claims 1 to 6, wherein the preparation method of the four-tile extract comprises extracting four tiles with 80% to 95% ethanol solution to obtain the four-tile ethanol extract.

8. The use of claim 7, wherein the preparation method further comprises the step of dissolving the ethanol extract of the four tiles and then extracting with an organic solvent, wherein the organic solvent comprises at least one of petroleum ether, dichloromethane, ethyl acetate or n-butanol.

9. A medicine for resisting hepatitis B virus and/or preventing and treating hepatitis B is characterized by comprising four tile extracts; the preparation method of the four-tile extract comprises the step of extracting the four tiles by using 80-95% ethanol solution to obtain the four-tile ethanol extract.

10. The pharmaceutical of claim 9, wherein the preparation method further comprises the step of dissolving the ethanol extract of the four-piece tile and then extracting with an organic solvent, wherein the organic solvent comprises at least one of petroleum ether, dichloromethane, ethyl acetate or n-butanol.