CN108096307B

CN108096307B - Application of lindley eupatorium herb ethanol extract in preparing anti-hepatitis B virus medicine

Info

Publication number: CN108096307B
Application number: CN201810045561.1A
Authority: CN
Inventors: 张健; 陈道峰; 任慧玲; 褚纯隽; 李显伦; 严彪; 吴双庆
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2014-06-04
Filing date: 2014-06-04
Publication date: 2021-02-05
Anticipated expiration: 2034-06-04
Also published as: CN104069145B; CN108113985B; CN108159099B; CN108245501A; CN108159099A; CN104069145A; CN108096307A; CN108113985A; CN108245501B

Abstract

The invention discloses an application of an ethanol extract of lindley eupatorium in preparing anti-hepatitis B virus medicines. The ethanol extract of lindley eupatorium prepared from lindley eupatorium is subjected to anti-hepatitis B virus HBV experiment, and results show that the ethanol extract of lindley eupatorium has the effects of obviously inhibiting hepatitis B surface antigen HBsAg and hepatitis B core antigen HBeAg, and has better medicinal activity compared with a clinical medicament lamivudine (3TC) for treating hepatitis B. The lindley eupatorium herb ethanol extract can be used for preparing anti-hepatitis B virus medicines.

Description

Application of lindley eupatorium herb ethanol extract in preparing anti-hepatitis B virus medicine

Technical Field

The invention relates to an application of an ethanol extract of lindley eupatorium in preparing anti-hepatitis B virus medicines.

Background

Hepatitis B Virus (HBV) infection is one of the major diseases affecting human health, and there are about 3 hundred million people as chronic hepatitis B virus carriers worldwide, and China is the country with the most hepatitis B virus carriers, and there are about 0.93 hundred million people. Acute hepatitis B is easily transformed into chronic hepatitis and even degenerates into cirrhosis and liver cancer. At present, the domestic medicines for treating chronic viral hepatitis are mainly 1, oral nucleoside (acid) chemical medicines, such as lamivudine, adefovir, entecavir, telbivudine, tenofovir and the like, but easily generate drug resistance; 2. biologics, interferons, such as: interferon alpha and pegylated interferon alpha are commonly used, but most patients have a rebound phenomenon after stopping taking the medicine, the interferon is expensive (about 2-4 ten thousand yuan is needed in each treatment course), the medicine cannot be taken through gastrointestinal tract, the use is inconvenient, and side effects are easily caused after long-term administration, such as: side effects like cold symptoms and thrombocytopenia; 3. the traditional Chinese medicine, kushenin, oleanolic acid and derivatives thereof, plant polysaccharide, chemical components in schisandra chinensis and the like, clinical medical practice and experimental research prove that the traditional Chinese medicine can inhibit hepatitis virus replication to a certain extent in preventing and treating hepatitis diseases, particularly can improve symptoms and liver functions of patients and enhance the immune function of organisms, and has the advantages of rich botanical medicine sources, low price, easy obtainment and small toxic and side effects. (see: Li Chang Gong, Qu Gui Rong. natural products research and development, 2002,14(6): 81-87; Xucong, Zen Su. pharmacokinetics research progress of anti-liver disease drugs, China pharmacy journal, 2012,47(18):1430-1433) therefore, the research and development of new traditional Chinese medicine for treating hepatitis B virus has important significance.

Herba Eupatorii Lindleyanum DC of Compositae is herba Eupatorii Lindleyanum, and is prepared from herba Eupatorii Lindleyanum DC. Has bitter taste and mild nature, enters liver and spleen meridians, has the functions of eliminating phlegm and stopping cough, clearing heat and releasing toxin, inducing diuresis and reducing edema and reducing blood pressure, and is used for treating cold, cough and excessive phlegm, headache, tonsillitis, bacillary dysentery and hypertension (see: the national traditional Chinese medicine administration, Chinese materia medica, Shanghai: Shanghai science and technology publisher, 1999:7,6876). The eupatorium extract has a certain effect of protecting rats with acute lung injury, the compound eupatorium capsule has a certain effect of inhibiting influenza viruses in vitro, the eupatorium syrup concentrated solution has a certain antibacterial effect on gram positive and negative bacteria and candida albicans, and the eupatorium total flavone extract has the effect of reducing blood viscosity (see: Jianzhou, Yanghui, Hoohiphyly, etc. the eupatorium has the effect of protecting rats from acute lung injury, Chinese pharmacy 2007, 18 (27): 4; Zhou Yu Dai, Wu Yan, etc. the eupatorium has the effects of resisting bacteria, relieving cough and relieving asthma, Chinese pharmacy 2001, 12 (12): 716 Bu Ru 718, Penn Ru, sinus, yellow fragrant, etc. the compound eupatorium capsule has the effect of resisting influenza viruses, Chinese patent medicine, 2008, 30 (5): 650 Bu Jing 654, Wang Jing, Qin, Cheng and Zhen Zhu Zhen, etc. in case the rat improvement of blood hyperlipidemia and the antioxidation effect, 2009, 25(2): 80-82. ) However, whether the lindley eupatorium has the effect of resisting the hepatitis B virus is not reported, and the invention finds that the lindley eupatorium has the activity of resisting the hepatitis B virus.

Disclosure of Invention

The invention aims to: provides an application of an ethanol extract of lindley eupatorium in preparing anti-hepatitis B virus medicaments.

The technical scheme of the invention is as follows: the application of lindley eupatorium herb in preparing the anti-hepatitis B virus medicine is that the lindley eupatorium herb is an ethanol extract of the lindley eupatorium herb, and the ethanol extract is obtained by adopting the following method: taking dry eupatorium lindleyanum whole grass, cutting the eupatorium lindleyanum into sections of 2-3 cm, adding 8-15 times of ethanol aqueous solution with volume fraction of 60-95%, refluxing and extracting for 1-3 hours, repeating for 1-3 times, and recovering the solvent under reduced pressure at 40-60 ℃ to obtain the eupatorium lindleyanum ethanol extract.

The lindley eupatorium herb is a lindley eupatorium herb flavone part containing jaceosidin and eupatorium xanthophyll, and the chemical structural formulas of the lindley eupatorium herb are respectively as follows:

the lindley eupatorium is a lindley eupatorium sesquiterpene part containing lindley eupatorium lactone F, lindley eupatorium lactone G, lindley eupatorium lactone H, lindley eupatorium lactone I and lindley eupatorium lactone K; the chemical structural formula is as follows:

the lindley eupatorium herb is an eluted part of lindley eupatorium herb with 70% ethanol water, and is prepared by the following method: taking dry eupatorium lindleyanum whole grass, cutting the eupatorium lindleyanum into sections of 2-3 cm, adding 8-15 times of ethanol aqueous solution with volume fraction of 60-95%, refluxing and extracting for 1-3 hours, repeating for 1-3 times, and recovering the solvent under reduced pressure at 40-60 ℃ to obtain an eupatorium lindleyanum ethanol extract; dissolving the extract in water to prepare a solution with the concentration of 0.5-1 g/ml, adsorbing the solution by weak-polarity macroporous adsorption resin, eluting by water to remove macromolecular compounds, eluting by 90-95% ethanol aqueous solution by volume fraction, and recovering the solvent at 40-60 ℃ under reduced pressure to obtain a macroporous resin part; adsorbing the obtained macroporous resin part by an ODS (oxide dispersion strengthened) reverse phase silica gel chromatographic column, eluting by 50% methanol aqueous solution by volume fraction, then eluting by 70% -75% methanol aqueous solution, recovering the solvent at 40-60 ℃ under reduced pressure, adsorbing by a 30-60 mesh polyamide chromatographic column, and eluting by 70% ethanol aqueous solution by volume fraction to obtain the lindley eupatorium 70% ethanol aqueous part.

Further, preferably, the weak polar macroporous absorption resin is AB-8, D101, HPD-100, HPD-200A, HPD-200B, HPD-300, HPD-700 or HPD-722 macroporous absorption resin;

further, preferably, the ODS reversed-phase silicA gel chromatographic column is A DAISOGEL ODS reversed-phase silicA gel filler with the particle size of 50-70 um, A Cosmsil ODS reversed-phase silicA gel filler with the particle size of 50-70 um or A YMCODS-A reversed-phase silicA gel filler with the particle size of 50-70 um;

furthermore, preferably, the volume ratio of the water to the low-polarity macroporous adsorption resin is 8-15: 1; the volume ratio of the 90-95% ethanol aqueous solution to the macroporous adsorption resin is 8-15: 1; the mass ratio of the volume of the ODS reversed phase silica gel chromatographic column to the obtained macroporous resin part is 20-30: 1; the volume ratio of the 50% methanol aqueous solution to the 70-75% methanol aqueous solution to the ODS reversed phase silica gel chromatographic column is 5-8: 1; the mass ratio of the volume of the polyamide chromatographic column to the adsorbed sample is 20-30: 1; the volume ratio of the 70% ethanol aqueous solution to the polyamide chromatographic column is 4-10: 1.

The lindley eupatorium is a lindley eupatorium medium-chain diterpenoid compound selected from (1)3- (hydroxymethy) -1, 14, 15-trihydroxy-7, 11, 15-trimethy-2, 6, 10-hexadecainen-13-acetate and (2)3- (hydroxymethy) -1, 13, 15-trihydroxy-7, 11, 15-trimethy-2, 6, 10-hexadecainen-14-acetate, and the structural formulas of the compounds are respectively as follows:

the four extracts and two chain diterpenoid compounds are subjected to in vitro anti-hepatitis B virus HBV experiments to obtain the extract which has obvious inhibition effect on hepatitis B surface antigen HBsAg and hepatitis B core antigen HBeAg, and the extract has obvious anti-hepatitis B virus HBV effect and can be used for preparing anti-hepatitis B virus medicines.

The invention has the advantage of providing a new source for anti-hepatitis B virus medicines.

Drawings

The invention is further described with reference to the following figures and examples:

FIG. 1 is a high performance liquid chromatogram of a tremula sesquiterpene locus of example 2;

FIG. 2 is a high performance liquid chromatogram of control Eupalinolide F in example 2;

FIG. 3 is a high performance liquid chromatogram of control Eupalinolide G of example 2;

FIG. 4 is the HPLC chromatogram of control substance Eupalinolide H in example 2;

FIG. 5 is a high performance liquid chromatogram of control Eupalinolide I in example 2;

FIG. 6 is a high performance liquid chromatogram of control Eupalinolide K in example 2;

FIG. 7 is a high performance liquid chromatogram of the lindley eupatorium herb flavone site in example 2;

FIG. 8 is the HPLC chromatogram of the control Eupatorium adenophorum xanthophyll in example 2;

FIG. 9 is a high performance liquid chromatogram of a control jaceosidin from example 2;

Detailed Description

The invention is further illustrated by the following examples.

Example 1: preparation method of herba Eupatorii Lindleyani extract and herba Eupatorii Lindleyani medium chain diterpene compound

(1) Weighing 1000g of eupatorium lindleyanum dry whole grass, cutting into sections of 2-3 cm, adding 15 times volume of 80% ethanol aqueous solution, carrying out reflux extraction for 2 hours, repeating the extraction for 2 times, recovering the solvent at 60 ℃ under reduced pressure to obtain 158g of eupatorium lindleyanum ethanol extract (EUP-EtOH), dissolving the EUP-EtOH) in about 150ml of water, adsorbing the eupatorium lindleyanum ethanol extract (EUP-EtOH) by 1800ml of AB-8 macroporous adsorption resin, eluting by 20000ml of water to remove macromolecular compounds, eluting by 20000ml of 95% ethanol aqueous solution at 60 ℃ under reduced pressure to recover the solvent to obtain 61g of macroporous resin parts; adsorbing the obtained macroporous resin part with 1500ml Cosmsil ODS reversed phase silica gel column with particle size of 70um, eluting with 10000ml methanol water solution with volume fraction of 50%, and recovering solvent under reduced pressure at 60 deg.C to obtain 21g of Eupatorium eupatorium sesquiterpene part (EUP-SQT). Then 10000ml of 70% methanol aqueous solution is used for elution, the solvent is recycled under reduced pressure at 60 ℃ to obtain 20g of extract, the extract is absorbed by 600ml of 30-60 mesh polyamide chromatographic column, 4000ml of 70% ethanol aqueous solution is used for elution, and the solvent is recycled under reduced pressure at 60 ℃ to obtain 14.7g of lindley eupatorium 70% ethanol aqueous elution part (EUP-DIT). Eluting with 4000ml 100% ethanol, and recovering solvent at 60 deg.C under reduced pressure to obtain 2.8g of herba Eupatorii Lindleyani flavone fraction (EUP-FLA).

(2) Preparation of lindley eupatorium chain diterpene compound: drying herba Eupatorii Lindleyani 2000g, extracting with 10 times of 80% ethanol-water under reflux for 2 times, each for 2 hr, mixing filtrates, and recovering ethanol under reduced pressure to obtain 250g extract; dissolving the extract in 100mL of water, extracting with 100mL of petroleum ether for degreasing, extracting with chloroform twice (100 mL each time), and concentrating the chloroform extraction layer at 30 deg.C under reduced pressure to obtain about 11g of chloroform; the chloroform fraction was subjected to 300g of silica gel column chromatography and eluted with chloroform-methanol 100:2 to give 1.08g of an eluted fraction. Performing column chromatography with 200mL Rohm and Haas resin column, eluting with 60% -80% methanol-water system, collecting 0.5g of 80% methanol eluate, performing silicA gel column chromatography, eluting with chloroform-methanol (50:1) to obtain 112mg of 2A, preparing 2A with high performance liquid chromatography, preparing chromatograph with Beijing Huideyi ILC-P3050, chromatographic column YMC-PackODS-A column (250mm £ 20mm, YMC, Kyoto, Japan), detecting wavelength of 210nm, eluting with 70% methanol-water system, separating at flow rate of 5mL/min for 38.5min to obtain 31mg of compound (A), and separating at 42.8min to obtain 26mg of compound (b).

Example 2: detection of sesquiterpene fraction, flavone fraction and active ingredient of mid-chain diterpene compound

(1) Detecting active ingredients of Eupatorium eupatorium sesquiterpene part (EUP-SQT): in this example, the components were determined by HPLC. High performance liquid chromatography (Agilent 1260Infinity, Agilent, usa) wherein mobile phase a is acetonitrile; the mobile phase B is water; the mobile phase conditions are shown in table 1. A chromatographic column: YMC-PACK ODS-A (phi 4.6 mm. times.250 mm,5 μm); column temperature: 28 ℃; detection wavelength: 210 nm; sample introduction amount: 20 mu l of the mixture; flow rate: 1 ml. min^-1。

TABLE 1 HPLC chromatographic analysis conditions

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	30	70
5	30	70
			15	32	68
25	36	64
			40	36	64

6.02mg of reference substances, namely eupalinolide F, 14.94mg of eupalinolide G, 5.08mg of eupalinolide H, 10.04mg of eupalinolide I and 13.98mg of eupalinolide K are precisely weighed respectively, and the volume is fixed to 10ml by using a mobile phase A to serve as a stock solution. Adding mobile phase A, diluting to 1:2, 1:4, 1:8, 1:16 and 1:32, filtering with 0.22 μm filter membrane, and filtering to obtain filtrate. 5 concentrations of control were chromatographed and a standard curve was plotted, with 3 replicates per concentration point. Recording peak areas of the chromatographic peaks, and performing linear regression on the concentration (x) of the chromatographic peaks by using the peak area (y) of a reference substance to obtain a standard curve of the reference substance, wherein eupalinolide F, y is 63403 x-15.408; eupalinolide G, y 24720 x-20.065; eupalinolide H, y 79063x + 6.7811; eupalinolide I, y 239018x + 53.303; eupalinolide K, y 7913.5x + 62.857.

The EUP-SQT 6mg of eupatorium sesquiterpene prepared in example 1 was precisely weighed, the volume was determined to 10ml by mobile phase a volumetric flask, and the filtrate was filtered through a 0.22 μm membrane filter and subjected to chromatography.

FIGS. 1-6 are HPLC chromatograms of sesquiterpene positions (EUP-SQT) from Eupatorium, control products eupalinolide F, eupalinolide G, eupalinolide H, eupalinolide I and eupalinolide K.

By analyzing the high performance liquid chromatography result, eupatorium pseudochinensis (EUP-SQT) part samples containing eupatorium pseudochinensis F (eupatorium pseudochinensis F), eupatorium pseudochinensis G (eupatorium pseudochinensis G), eupatorium pseudochinensis H (eupatorium pseudochinensis H), eupatorium pseudochinensis I (eupatorium pseudochinensis I) and eupatorium pseudochinensis K (eupatorium pseudochinensis K) can be obtained, the chromatographic signal values of the samples are substituted into a standard curve, and the specific content of each component is further obtained, and the specific content is shown in Table 2.

TABLE 2 Eupatorium lappacans sesquiterpene fraction (EUP-SQT) sample containing 5 Eupatorium lappacans lactone

Sample (I)

eupalinolide F

eupalinolide G

eupalinolide H

eupalinolide I

eupalinolide K

Ratio of

Content (wt.)

4.1％

31.8％

6.8％

3.4％

47.7％

93.8％

(2) Detecting active ingredients of eupatorium lindleyanum flavone part (EUP-FLA): the method is similar to the detection of active ingredients of Eupatorium eupatorium sesquiterpene part. The conditions of the high performance liquid chromatography are as follows: mobile phase a was acetonitrile (containing 0.1% formic acid); mobile phase B was water (containing 0.1% formic acid); mobile phase conditions: see table 3; a chromatographic column: YMC-PACKODS-A (Φ 4.6 mm. times.250 mm,5 μm); column temperature: 28 ℃; detection wavelength: 365 nm; sample introduction amount: 20 mu l of the mixture; flow rate: 1 ml. min^-1。

TABLE 3 HPLC chromatogram analysis conditions

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	36	64
15	36	64
			30	90	10

Precisely weighing control substances of eupatorium lucolium xanthophyll 4.67mg and jaceosidin 8.17mg respectively; and adding mobile phase A to a constant volume of 10ml to obtain stock solution, diluting with mobile phase A to obtain 1:2, 1:4, 1:8, 1:16 and 1:32, and filtering with 0.22 μm filter membrane to obtain filtrate. 5 concentrations of control were chromatographed and a standard curve was plotted, with 3 replicates per concentration point. Recording the peak area of each chromatographic peak, and performing linear regression on the concentration (x) by using the peak area (y) of a reference substance to obtain a standard curve of the reference substance, wherein eupatilin y is 52964x +19.30, and jaceosidin y is 55161x + 210.79.

6mg of the EUP-FLA sample of the eupatorium lindley EUP prepared in example 1 is precisely weighed, the volume is determined to 10ml by using the mobile phase A, the solution is filtered by a 0.22 mu m filter membrane, and the filtrate is respectively taken for chromatographic determination.

FIGS. 7-9 are high performance liquid chromatograms of a sample of Eupatorium odoratum flavone, a control of eupatilin, and a control of jaceosidin.

Analyzing by high performance liquid chromatography to obtain eupatorium adenophorum xanthophyll and jaceosidin in the sample of eupatorium adenophorum flavone part, and substituting the chromatographic signal value of the sample into a standard curve to further obtain the specific content of each component, as shown in Table 4.

TABLE 4 content of 2 flavones in Eupatorium lindleyanum flavone part

Sample (I)	Eupatorium xanthophyll	Jaceosidin	Total ratio of
				Content (wt.)	26.0％	66.1％	92.1％

(3) Detection of active ingredients of eupatorium lindleyanum medium-chain diterpenoid compounds: the compound (a) and the compound (b) prepared in example 1 were measured for spectrum, and the spectrum data were as follows:

the compound (a) is colorless oil, and the infrared spectrum IR (KBr) shows v_max：3424，2974，2930，1717，1653，1373，1258，1024cm^-1(ii) a HR-ESI-MS gives the molecular ion peak M/z 421.2568[ M + Na ]]⁺(calcd for C₂₂H₃₈O₆Na, 421.2561), indicating that its molecular formula is C₂₂H₃₈O₆. It is composed of¹H NMR and¹³c NMR spectrum ofCDCl₃The measurement in (1). The results are shown in Table 1;

the compound (b) is colorless oil, and the infrared spectrum IR (KBr) shows v_max：3402，2972，2932，1653，1576，1418，1051，1015cm^-1(ii) a HR-ESI-MS gives the molecular ion peak M/z 421.2565[ M + Na ]]⁺(calcd.for C₂₂H₃₈O₆Na, 421.2561), indicating that its molecular formula is C₂₂H₃₈O₆. It is composed of¹H NMR and¹³c NMR spectra in CDCl₃The measurement in (1). The results are shown in Table 5 below,

TABLE 5 preparation of the compounds (a), (b)¹H NMR and¹³c NMR spectrum data

Wherein the content of the first and second substances,¹h NMR and¹³c NMR was performed at 400MHz and 125MHz, respectively.

By infrared spectrum, HR-ESI-MS and,¹H NMR and¹³data analysis of C NMR spectra, in comparison with literature (see: Shuangqing Wu, Naiyu Xu, Jian Zhang, Shi Yao, Chunjun Chu.three new acrylic derivatives from Eupatorium linleyanum DC.. Journal of Asian Natural Product research.2012,14(7): 652-: compound (a) is compound (1): 3- (hydroxymethy) -1, 14, 15-trihydroxy-7, 11, 15-trimethyl-2, 6, 10-hexadecacarrien-13-acetat, and the structural formula of the compound is as follows:

compound (b) is compound (2): 3- (hydroxymethy) -1, 13, 15-trihydroxy-7, 11, 15-trimethyl-2, 6, 10-hexadecacarrien-14-acetate, the structural formula of the compound is as follows:

example 3: external anti-HBV experiment of four eupatorium lindleyanum extracts and eupatorium lindleyanum medium-chain diterpene compounds

1. Experimental drugs and reagents: the chain diterpene compounds (1) and (2) were prepared in the above example 1; dimethyl sulfoxide; MTT (thiazole blue), shanghai jianlai biotechnology limited; control drug, lamivudine tablet (3 TC); kurarin Schk pharmaceuticals (Suzhou) Inc.; a cell culture medium DMEM, a growth medium and a preparation, wherein the growth medium contains 10% fetal calf serum, 380 mug/ml G418, 0.03% glutamine, penicillin, streptomycin and the like, and each 100 mug/ml;

2. experimental equipment: ELISA kit for detecting hepatitis B surface antigen HBsAg and hepatitis B core antigen HBeAg; an incubator; 24-well plates, and the like.

3. Cell lines: 2.2.15 cell line of Hep G2, department of education/Ministry of health, molecular virology focus laboratory, Shanghai.

4. The experimental method comprises the following steps:

a2.2.15 cell line of Hep G2 was collected at 10X 10 cells per well⁵Individual cells were seeded in 24-well plates at 5% CO₂Culturing at 37 ℃ in an incubator, after 48 hours, respectively replacing the growth liquid with drug culture liquids containing the compound (1) and the compound (2) which are dissolved by dimethyl sulfoxide, setting 5 concentrations of each group of drug culture liquids to be 12.5 mu g/ml-200 mu g/ml (as shown in table 2), setting 4 parallel holes for each concentration, continuing culturing for 9 days, and replacing the liquid once every 3 days. The drug of the positive control group is lamivudine (3TC), meanwhile, the culture solution without the drug is used as a blank control group, 1 hole is respectively arranged on each of the two control groups, and the operation of the two control groups is the same as that of the drug culture solution except that the growth solution is correspondingly changed into the lamivudine drug and the culture solution without the drug. Collecting supernatant, detecting the content of HBsAg and HBeAg by using an ELISA kit, and simultaneously determining the toxicity of the two groups of compounds on cells by using MTT (methyl thiazolyl tetrazolium) at different concentrations. The results are shown in Table 6.

The above experimental procedures were repeated with ethanol extract of Eupatorium Adianum, Eupatorium Adianum flavone fraction, Eupatorium Adianum sesquiterpene fraction, and Eupatorium Adenanum 70% ethanol eluted fraction, respectively, to obtain the results shown in Table 6.

As can be seen from Table 6, both the chain diterpene compounds (1) and (2) had inhibitory effects on hepatitis B surface antigen HBsAg and hepatitis B core antigen HBeAg. The compounds (1) and (2) inhibit HBsAg and HBeAg, and the inhibition rate increases with the increase of concentration; when the compound (1) is at 50 mu g/mL, the inhibition rate of the compound on HBsAg and HBeAg is obviously higher than 3 TC; under the same concentration of the compound (2) and the 3TC, the inhibition rate of the compound (2) on HBsAg and HBeAg is obviously higher than that of the 3 TC; the compounds (1) and (2) at low concentrations had a high inhibitory rate against HBsAg and HBeAg as compared with 3 TC.

The ethanol extract, flavone part, sesquiterpene part, and 70% ethanol-eluted part of Eupatorium Adianum have effects of inhibiting HBsAg and HBeAg, and increasing the inhibition rate with increasing concentration; compared with 3TC, the low-concentration Eupatorium Adenophorum hand Mazz extract, Eupatorium Adenophorum hand Mazz fraction, and Eupatorium Adenophorum hand Mazz fraction with 70% ethanol elution fraction all have high inhibitory effect on hepatitis B surface antigen HBsAg and hepatitis B core antigen HBeAg.

TABLE 6 anti-HBV test results of four groups of Eupatorium Adenophorum extracts and two groups of chain diterpene compounds

Wherein, the ratio of +: "show toxicity" means a cell viability of < 75%; /: no inhibition rate test was performed.

The above examples can obtain that the drug prepared by extracting the lindley eupatorium ethanol extract, the lindley eupatorium flavone part, the lindley eupatorium sesquiterpene part, the lindley eupatorium 70% ethanol water elution part and the two chain diterpenoid compounds from the lindley eupatorium shows obvious inhibition effect on hepatitis B surface antigen HBsAg and hepatitis B core antigen HBeAg in an in vitro anti-hepatitis B virus HBV experiment, and has better drug activity compared with the clinical drug lamivudine (3TC) for treating hepatitis B. Therefore, the ethanol extract, the flavonoid part, the sesquiterpene part, the 70% ethanol water elution part and two chain diterpene compounds extracted from the lindley eupatorium herb can be used for preparing the anti-hepatitis B virus medicines.

Claims

1. An application of an ethanol extract of lindley eupatorium in preparing anti-hepatitis B virus medicines is characterized in that the ethanol extract is obtained by adopting the following method: taking dry eupatorium lindleyanum whole grass, cutting the eupatorium lindleyanum into sections of 2-3 cm, adding 8-15 times of ethanol aqueous solution with volume fraction of 60-95%, refluxing and extracting for 1-3 hours, repeating for 1-3 times, and recovering the solvent under reduced pressure at 40-60 ℃ to obtain the eupatorium lindleyanum ethanol extract.