CN1325048C - Application of wogonin for preparing medicine to treat or prevent hepatitis B - Google Patents

Abstract

The present invention belongs to the field of pharmacy, which relates to the application of wogonin to the preparation of medicine for treating or preventing hepatitis B. The medicine prepared from wogonin provided by the present invention for treating or preventing hepatitis B can inhibit the duplication of HBV DNA, and has stronger inhibiting effect on the secretion of s antigens and e antigens of hepatitis B. The medicine can obviously reduce the content of blood serum HBsAg, and has smaller toxicity to cells.

Description

The application of wogonin in preparation treatment or prevention hepatitis B medicine

Technical field

The invention belongs to pharmaceutical field, relate to the application of wogonin in preparation treatment or prevention hepatitis B medicine.

Background technology

Hepatitis B (hepatitis B) is caused, is served as main and can cause a kind of infectious disease of multiple organ injury with the liver inflammatory lesion by hepatitis B virus (HBV), be common clinical, frequently-occurring disease, be widely current in countries in the world, main infringement child and person between twenty and fifty, small number of patients can be converted into liver cirrhosis or hepatocarcinoma.Hepatitis B has become the worldwide disease of serious threat human health, also be that China's current popular is the most extensive, the most serious a kind of infectious disease of hazardness, it is reported, China has 1.2 hundred million people to carry hepatitis B virus approximately, wherein the chronic viral hepatitis B patient more than 3,000 ten thousand, have every year 350000 people to die from the chronic viral hepatitis B relevant disease.The medicine of research and development treatment and prevention hepatitis B is the emphasis of drug development, and the medicine for the treatment of hepatitis B at present is more, and curative effect differs, and is the hepatitis B resisting medicated of main component but seldom have with the wogonin.

Wogonin is Labiatae (Labiatase) plant Radix Scutellariae (Scutellaria baicalensis Georgi) and belongs to flavone compound contained in the root of various plants together, has the structure of formula (1).

Formula (1)

Studies show that wogonin has multiple pharmacological effect, comprise antioxidation, blood coagulation resisting function, antitumor action, spasmolysis, antibiotic in addition in addition, antivirus action and diuresis etc.The patent No. is 200310110337.X, the patent documentation that name is called " extraction process of wogonin, Pharmaceutical composition and preparation process thereof " points out that highly purified wogonin and pharmaceutical composition thereof " can be used for various diseases of viral infection, preferably use " in the medicine of acute respiratory syndrome that preparation treatment hepatitis, viral influenza, viral infection cause etc.But the document openly wogonin the pharmacological effect of hepatitis is not pointed out the particular type of the hepatitis that can treat yet, the present invention has carried out further research on this basis.

Summary of the invention

The purpose of this invention is to provide the new purposes of wogonin in pharmacy, i.e. the application of wogonin in preparation treatment or prevention hepatitis B medicine.

Technical scheme of the present invention is that application, the especially wogonin of wogonin in preparing treatment or prevention hepatitis B medicine preparing the application that reduces in the hepatitis B virus surface antigen medicine at application and the wogonin that preparation suppresses in the hepatitis B virus duplication medicine.

The wogonin of treatment effective dose and any one adjuvant of pharmaceutically permission can be made pharmaceutical composition according to the present invention, also can add the other treatment hepatitis B medicine that other and wogonin do not have antagonism.Its preparation can be any one dosage form that pharmaceutically allows, and includes but not limited to tablet, granule, capsule, pill, oral liquid, injection, liposome etc.The consumption of wogonin can be according to variations such as route of administration, patient age, body weight, body surface area, the disease type of being treated and the orders of severity, and its daily dose can be 80-3600mg, can use by one or many.

Beneficial effect of the present invention:

Prepared in accordance with the present invention is that the medicine of main component can obviously suppress duplicating of HBV DNA with the wogonin, antigenic secretion has stronger inhibitory action to hepatitis B s, e, can significantly reduce the content of HBsAg in serum, and pair cell toxicity less (seeing effect embodiment part experimental data for details), to hepatitis B have good curative effect and side effect little.

Description of drawings

Fig. 1: HH effect different time is secreted the inhibitory action of HBsAg, HBeAg to HepG2215.Data are expressed as the antigen suppression ratio of each drug treating group number with respect to matched group.

Fig. 2: the HH effect is to the inhibitory action of HepG2215 secretion HBV DNA.

Fig. 3: HH is to the inhibitory action of duck hepatitis-B DNA polymerase.

Fig. 4: transgenic mice serum HBsAg detection by quantitative.

Fig. 5: carry out southern hybridization with digoxin labelled probe behind the total DNA agarose gel electrophoresis of the dirty extraction of duck liver.

The specific embodiment

The invention will be further elaborated by the following examples, but do not limit the present invention.

Used test material in following examples:

1.1 be subjected to the reagent thing: injection wogonin (HH), Medicinal College, China Medical Univ. provides, batch: 20040702, content: 25mg/ props up.

1.2 cell strain source: the HepG2215 cell strain, draw the molecule virus laboratory from university of Fudan University.

1.3 laboratory animal:

Transgenic mouse: the BALB/C strain, the ayw hypotype, body weight 18-22 gram is available from Guangzhou air hospital.Mice adopts toeclipping to carry out labelling.Ad lib, drinking-water (Co during the administration ⁶⁰Irradiation feedstuff and aquesterilisa), illumination 12 hours every days, 12 hours dark, 22 ± 2 ℃ of temperature, humidity 55-70%.

Dhbv dna (DHBV) infected duck model: choose the duck blood sampling about 3 ages in week, fluorescence quantitative PCR method detects serum DHBV DNA.The DNA titre is 10 in the serum ⁵-10 ⁷Duck in the scope is judged as the positive duck of DHBV, can be used as model and experimentizes.Duck is raised at 18-24 ℃, under the environment of relative humidity 70%, and ad lib, drinking-water; Illumination 12 hours every days, 12 hours dark.

1.4 positive control:

Lamivudine, GlaxoSmithKline PLC production, 100mg/ sheet, lot number: 04020040

Foscarnet sodium, the honest day fine Pharma Inc. in Jiangsu produces 12mg/ml, lot number: 0403301

1.5 detection kit:

Antigenic reagent box, section China hepatitis B s antigen, e antigen ELISA test kit, lot number: 20030922

Virus is extracted test kit, goes up the biological company limited of marine Ke Kairui, lot number: 81101B

HBV nucleic acid amplification fluorescent quantitative detection kit, the basic biological engineering in Shenzhen limited company produces lot number: 20041001

The digoxigenin labeled test kit is available from Luo Shi diagnosis, lot number: 10271420

Effect embodiment 1:HH detects the toxicity of HepG 2215 cells.

Experimental technique: get one bottle in HepG 2215 cells, with being prepared into single cell suspension after the trypsinization, cell concentration to 2 * 10 are adjusted in the counting back ⁴Cell/ml adds in 96 well culture plates.Place cell culture incubator, 37 ℃, 5% CO ₂Overnight incubation.Suction goes to add the DMEM culture medium (5% hyclone) that contains the variable concentrations medicine behind the supernatant.Drug level is respectively: 200 μ g/ml, 50 μ g/ml, 20 μ g/ml, 2 μ g/ml, 0.2 μ g/ml, 0.02 μ g/ml.After the drug effect 9 days, add the MTT (tetrazole indigo plant) of 10 μ l in each hole and continue cultivation 4 hours.The careful suction goes supernatant, every hole to add 150 μ l DMSO, and the Shi Jia Za that vibrates gently dissolving is with the OD value at microplate reader detection 570nm place.Calculate growth inhibition ratio, carry out the t check.While observation of cell paramophia and cell breakage situation.

Experimental result:

Under the HH effect, medication group and not medication group there is no tangible cellular morphology and reach toxicity variations such as cell breakage unusually.But when high concentration, the metabolic activity of HepG 2215 there is certain inhibitory action.Try to achieve the TD of HH by the Logit method ₅₀Be about 600 μ g/ml, TD ₀Be about 20 μ g/ml; And the TD of HH ₉₀＞＞1mg/ml.The results are shown in Table 1.

Table 1:HH to the growth inhibited effect of HepG 2215 (Means ± SD, n=4)

Concentration (HH)	OD 570	The P value	TC	Growth inhibition ratio (IC)
					200μg/ml 50μg/ml 20μg/ml 2μg/ml 0.2μg/ml 0.02μg/ml	1.009±0.168 ** 1.381±0.150 ** 2.022±0.219 2.015±0.068 2.073±0.152 2.079±0.146	＜0.01 ＜0.01 ＞0.05 ＞0.05 ＞0.05 ＞0.05	0.519 0.710 1.040 1.036 1.066 1.069	48.1％ 29.0％ 0 0 0 0

^*P＜0.01 vs. contrast.

IC (%)=1-(OD _{Processed group}/ OD _{Matched group}) * 100%, down together.

Conclusion: HH pair cell toxicity a little less than, do not see cellular morphology in the experiment and reach toxicity such as cell breakage unusually and change.When concentration 200 μ g/ml, cell growth has inhibitory action.

Effect embodiment 2:HH is to HepG 2215 emiocytosis s, the antigenic influence of e

Experimental technique:

HepG 2215 cells are prepared into single cell suspension after with trypsinization, regulate cell concentration to 2 * 10 ⁴Cell/ml adds in the 24 porocyte culture plates (1ml/well) 37 ℃, 5% CO ₂Overnight incubation.(concentration is respectively: 50,20,10,5,2 μ g/ml) to add the HH of different extension rates, establish cell matched group and medicine matched group (3TC 10 μ g/ml) simultaneously, cultivate and got supernatant mensuration HBsAg and HBeAg content on the the 3rd, 6,9 day respectively, and compare, calculate suppression ratio, IC with matched group ₅₀And TI (therapeutic index), and carry out the t check.

Experimental result:

The secretion of HH pair cell HBsAg has inhibitory action, drug effect the 9th day, and its IC ₅₀Be about 7.7 μ g/ml, IC ₉₀Be about 13.2 μ g/ml, therapeutic index (TI) is 77.9; HH also has inhibitory action to HBeAg, drug effect the 9th day, and IC ₅₀Be about 3.9 μ g/ml, IC ₉₀Be about 15.1 μ g/ml, therapeutic index (TI) is 153.8.The results are shown in Table 2,3 and Fig. 1.

Table 2:HH to the inhibitory action of HepG2215 secretion HBsAg (Means ± SD, n=3)

Medicine to be measured (dosage)	The 3rd day		The 6th day		The 9th day
							OD 450	IC (％)	OD 450	IC (％)	OD 450	IC (％)
	HH 50μg/ml 20μg/ml	0.173±0.026 ** 0.140±0.004 **	81.2 84.8	0.070±0.050 ** 0.040±0.006	92.6 95.6	0.038±0.038 ** 0.026±0.002 **							97.7 98.4

10 μ g/ml, 5 μ g/ml, 2 μ g/ml 3TC, 10 μ g/ml contrast

0.288±0.020 ** 0.413±0.013 ** 0.498±0.033 ** 0.487±0.065 ** 0.866±0.002

68.7 55.1 45.9 43.8 /

0.371±0.055 0.654±0.123 0.723±0.074 0.625±0.125 0.886±0.664

60.5 30.5 23.1 29.5 /

0.592±0.159 * 1.333±0.170 0.995±0.631 1.081±0.155 1.577±0.132

63.8 18.3 39.1 31.4 /

^*P＜0.05, ^*P＜0.01, the vs. contrast.

Table 3:HH to the inhibitory action of HepG2215 secretion HBeAg (Means ± SD, n=3)

Medicine to be measured (dosage)	The 3rd day		The 6th day		The 9th day
							OD 450	IC (％)	OD 450	IC (％)	OD 450	IC (％)
	HH 50 μ g/ml 20 μ g/ml 10 μ g/ml 5 μ g/ml 2 μ g/ml 3TC 10 μ g/ml contrast	0.736±0.084 0.626±0.083 0.652±0.127 0.574±0.109 0.544±0.113 0.636±0.113 0.696±0.052	0 10.0 6.3 17.5 21.8 8.6 /	0.139±0.019 ** 0.215±0.031 ** 0.479±0.126 * 0.591±0.131 * 0.528±0.098 * 0.742±0.161 0.953±0.126	85.4 77.4 49.7 38.0 44.6 22.1 /	0.018±0.015 ** 0.084±0.010 ** 0.510±0.141 ** 0.880±0.235 ** 1.053±0.178 * 0.823±0.189 ** 1.721±0.183							98.9 95.0 70.3 48.8 38.8 52.2 /

^*P＜0.05, ^*P＜0.01, the vs. contrast.

Conclusion: HH (50,20,10 μ g/ml) has strong inhibitory action (p＜0.01, p＜0.01, p＜0.05), IC to HepG 2215 emiocytosis hepatitis B s antigens ₅₀Be 7.7 μ g/ml; HH (50,20,10 μ g/ml) has strong inhibitory action (p＜0.01, p＜0.01, p＜0.01), IC to HepG 2215 emiocytosis hepatitis B virus e antigens ₅₀Be 3.9 μ g/ml.

Effect embodiment 3:HH produces the influence of viral nucleic acid to HepG 2215 cells

Experimental technique:

Drug effect is collected culture supernatant and is used post adsorption-type extraction agent box (going up the biological company limited of marine Ke Kairui) extracting HBV DNA in cell 9 days; And use HBV nucleic acid amplification fluorescent quantitative detection kit (Shenzhen basic biological engineering limited company) to carry out fluorescent quantitation and detect (Bio-Rad iCycler).Pcr amplification carries out according to operation instruction: 37 ℃, and 5min; 94 ℃ of 1min; 95 ℃ of 1sec; 60 ℃ of 30sec, 42 circulations of increasing.PCR finishes back 60 ℃ and collects Fam fluorescence, and the result carries out the t check.

Reaction system:

Reagent

Volume (μ l)

10 * R-PCR buffer Mg 2+(250mmol/L) dNTP (10mmol/L) SYBR Green I Calibration (10-3x) primer COL13 S (20 μ M) primer COL 13 R (20 μ M) Taq-Ex dna profiling ddH2O

2.5 0.3 0.75 2.5 1.0 0.75 0.75 0.25 5 to add to cumulative volume be 40

Experimental result:

HH has certain inhibitory action to HBV DNA, and when concentration was 20 μ g/ml, HH was 35.3% to the suppression ratio of HBV DNA; When concentration was 50 μ g/ml, suppression ratio was 66.1%, and positive control medicine 3TC 10 μ g/ml are 62.8% to the suppression ratio of HBV DNA.The results are shown in Table 4 and Fig. 2.

Table 4:HH to the inhibitory action of HepG2215 secretion HBV DNA (Means ± SD, n=3)

Medicine	HBV copy number (* 10 3Copies/ml)
			Mean	SD
	HH
50 μ g/ml 20 μ g/ml 10 μ g/ml 5 μ g/ml 2 μ g/m 3TC 10 μ g/ml contrast 1		2.64 ** 5.04 4.95 5.32 6.08 2.90 ** 7.79	0.45 0.62 1.03 0.89 1.23 0.74 0.88

^*P＜0.05, ^*P＜0.01, the vs. contrast

Conclusion: HH (50 μ g/ml) pair cell secretion HBV DNA has inhibitory action (p＜0.01).

Effect embodiment 4:HH is to the influence of duck hepatitis-B DNA polymerase

Experimental technique:

Duck hepatitis-B DNA polymerase in the suitableeest enzyme reaction condition and reaction system, can with ³The H-dTTP substrate is added in the system, in measurement of enzymatic reaction products ³The H incorporation detects the activity of enzyme.Get positive Sanguis Anas domestica, 180, centrifugal 4 hours separating viral particles of 000g.Add behind the drug dilution and contain (Tris-HCl, β-ME, NP-40,3H-dTTP, MgCl2 among reaction Buffer KCl), and adds an amount of dhbv dna granule.37 ℃, reacted 1.5 hours.Radioactive intensity is surveyed in sampling.The positive control medicine adopts foscarnet sodium.

Experimental result:

By ultracentrifugal method separating viral particles from the male Sanguis Anas domestica of DHBV, and detect the inhibitory action of HH to duck hepatitis B DNA polymerase with this system.The result shows that HH has inhibitory action to duck hepatitis-B DNA polymerase, its IC ₅₀Be about 0.57 μ g/ml; The IC of positive control medicine foscarnet sodium (PFA) ₅₀For generally between 0.36-0.72 μ g/ml.The results are shown in Table 5 and Fig. 3.

Table 5:HH is to the inhibitory action of duck hepatitis-B DNA polymerase

Medicine	Initial concentration (μ g/ml)	Suppression ratio (%) (n=3)	IC 50(μg/ml)
				HH	3.2 1.6 0.8 0.4 0.2	75.4±4.3 68.3±3.2 58.3±4.1 42.2±2.8 32.5±1.9	0.57
PFA	0.76	83.0±3.8

Conclusion: HH has inhibitory action, IC to duck hepatitis-B DNA polymerase ₅₀Be about 0.57 μ g/ml.

Effect embodiment 5:HH is to the influence of transgenic mouse serum HBsAg

Get blood examination before the transgenic mouse experiment and survey HBsAg in serum (Shanghai section China hepatitis B s antigen ELISA test kit, lot number: 20030922), choose positive transgenic mouse as experimental model (table 6).

Experimental technique:

40 of HBV transgenic mices just are divided into 5 groups according to serum HBsAg concentration, are respectively HH high dose group 28mg/kg, dosage group 14mg/kg among the HH, HH low dose group 7mg/kg, positive controls (lamivudine 100mg/kg), blank group (normal saline).Every group of eight mices, administration ten days; High, medium and low dosage group and matched group passed through the tail vein injection administration in per two days; Lamivudine group gastric infusion every day.Before serum sample picks up from administration respectively, administration 5 days, administration 10 days and drug withdrawal 5 days.Respectively put to death 3 mices at random for every group after 24 hours of last administration, get liver, kidney and other organs specimen; All the other mices are put to death after getting blood the last time, get liver, kidney.

The transgenic mice serum HBsAg detects and uses hepatitis B surface antigen detection by quantitative test kit (Shanghai Fudan-Yueda Bio-Tech Co., Ltd.), and operation reference reagent box description is carried out.Standard curve uses recombination hepatitis B surface antigen to make up.(Victor 1420, PE) with detecting after 20 times of the normal saline dilutions for serum sample.

Table 6

(Cov＞0.138 is judged as the positive for feminine gender 0.066, the positive 2.242.)

Experimental result:

Treated the tenth day, HH high dose group mice serum HBsAg content obviously descends, and has descended approximately 25.5% with comparing before the treatment, and compares before normal saline matched group and the administration that there were significant differences; Compare before dosage group mice serum HBsAg content and the treatment among the HH and descended approximately 24.6%, have remarkable meaning; Lamivudine group mice serum HBsAg content has descended about 36.1%.After the drug withdrawal 5 days, it is stable that HH high dose group and middle dosage group mice serum HBsAg content keep, and rebound phenomenon does not take place, and on the contrary, lamivudine group mice serum HBsAg content raises in the time of ten days to some extent than administration.The results are shown in Table 7 and Fig. 4.

Table 7:HH is to the influence of transgenic mice serum HBsAg.

Chemical compound	Dosage (mg/kg)	Serum HBsAg content (ng/ml)
						Day0	Day5	Day10 (n＝8)	Day15 (n＝5)
		The HH lamivudine	28 14 7 100	17.24±3.51 21.23±4.74 20.62±2.99 18.84±5.59	15.71±6.61 16.13±4.41 17.43±5.55 13.29±2.75 *					12.85±3.76 *#14.91±3.64 *16.78±5.26 12.04±1.69 **##	13.39±1.28 *# 14.22±3.23 * 17.76±4.93 13.35±8.88

Normal saline

-

17.08±6.02

18.80±8.91

19.17±5.91

18.26±4.33

^*P＜0.05, ^*P＜0.01, vs. is on the same group before the administration, ^#P＜0.05, ^##P＜0.01, vs. matched group on the same day.

(28,14mg/kg) treatment hepatitis B transgenic mice is 10 days, can obviously reduce the amount of serum hepatitis B surface antigen, compares before with treatment with matched group to have remarkable meaning (p＜0.05, p＜0.05) for conclusion: HH; After drug withdrawal 5 days, (28, the serum hepatitis B surface antigen of 14mg/kg) treatment group still has obvious decline (p＜0.05, p＜0.05) to HH.

Effect embodiment 6:HH is to the influence of the clear DHBV DNA of Sanguis Anas domestica

Experimental technique:

72 of the positive ducks of DHBV, being divided into 6 groups at random, is respectively positive controls (the DHBV DNA positive, not administration), negative control group (DHBV DNA feminine gender, not administration), positive drug group (the DHBV DNA positive, lamivudine 50mg/kg/day treatment), drug study group (the DHBV DNA positive, the HH treatment) comprising: high dose group (20mg/kg/day), middle dosage group (10mg/kg/day), low dose group (5mg/kg/day).Animal intravenous administration every day; Positive drug group gastric infusion every day, totally ten days.Before the medication, after the medication, drug withdrawal is in the time of 10 days, 3 venous blood collections ,-20 ℃ of preservations.

Fluorescence quantitative PCR method carries out detection by quantitative (Bio-Rad ICycler) to serum DHBV DNA.Every group of sample comprises the clear extract of 2.5 μ l Sanguis Anas domestica, 7.5 μ l deionized waters, 10 μ l reaction mixtures.Reaction mixture comprises 2 μ l SYBR Green1 fluorescent dyes, 2.4 μ l MgCl ₂(4mM), the primer of 0.5 μ l, 10 μ M, and the water of 5.1 μ l.Primer sequence is: 5 '-AGC TGG CCT AAT CGG ATTAC-3 ' and 5 '-TGT CCG TCA GAT ACA GCAAG-3 '.

The DNA copy number calculates by standard curve, and carries out the t check.

Reaction is provided with as follows:

Be provided with	Period
		95℃ 10min 95℃ 5s，55℃ 10s，72℃ 15s 4℃	1 40 ∞

Experimental result:

The serum DHBV DNA copy number of lamivudine group, HH high dose group obviously descends, and compares before normal saline matched group of the same period and the administration that there were significant differences (P＜0.01); Comparing before dosage group and low dose group serum DHBVDNA content and the treatment among the HH all has decline, also has statistical significance (P＜0.05) with comparing before normal saline matched group of the same period and the administration.The results are shown in Table 8.

Table 8:HH is to the influence of the clear DHBV DNA of Sanguis Anas domestica.

Chemical compound	Dosage (mg/kg/day)	Serum DHBV dna content (* 10 7copies/ml)
					Before the administration (Day0)	After the administration (Day10)	Drug withdrawal 10 days (Day20)
		HH lamivudine normal saline	20 10 5 50 /	1.67±1.20 1.66±1.52 1.82±1.81 1.32±1.17 1.36±1.56				0.76±0.62 **## 0.83±0.55 *# 0.97±0.74 *# 0.35±0.51 **## 2.05±1.24	1.04±0.74 1.51±1.46 0.76±1.07 1.30±1.10 2.05±1.54

^*P＜0.05, ^*P＜0.01, vs. is on the same group before the administration, #P＜0.05, ##P＜0.01, vs. matched group on the same day.

Each dosage group of conclusion: HH was treated the positive duck of DHBV 10 days, can reduce the copy number (having remarkable meaning with matched group with treating to compare before, wherein high dose group p＜0.01) of DHBV DNA in the serum; After drug withdrawal, the serum DHBV DNA copy number of each group increases to some extent.

Effect embodiment 7:HH is to the influence of the dirty DHBV DNA of duck liver

Experimental technique:

The dirty total DNA of duck liver adopts following method extracting: the duck liver of getting liquid nitrogen cryopreservation organizes 400mg to grind; Add 50 ℃ of cracking of dna cleavage liquid 4ml (containing E.C. 3.4.21.64 0.5mg/ml) 3 hours; The centrifugal 10min of 13,000 * g gets supernatant; The extracting of phenol chloroform; 2 times of volume ethanol add 1/10 sodium acetate precipitation; The washing of 70% ethanol, the TER buffer of sample dissolution 800 μ l (100 μ g/ml RNase A, 0.01 M pH7.5 Tris-Hcl, EDTA 0.002 M).Total DNA carries out Southern hybridization at the DHBV dna probe with digoxigenin labeled behind 1% agarose gel electrophoresis.Each dosage group is got 2-3 specimen and is illustrated as the typical case, and shows the duck numbering of each group.Picture is quantitative through gray scale scanning, and carries out the t check.

Experimental result:

Liver extracts total DNA, the situation of duplicating of the dirty middle DHBV DNA of duck liver after the effect of southern blot method detection of drugs.Lamivudine group, the dirty middle DHBV amount (RC of HH high dose group duck liver, Relaxed circular and RI, Replicativeintermediates) than matched group obvious decline is arranged, suppression ratio is 65.6% and 53.6% (p＜0.01, p＜0.01), and dosage group and low dose group also have decline among the HH with respect to matched group, and suppression ratio is 37.4% and 25.2% (p＜0.01, p＜0.05).The results are shown in Table 9 and Fig. 5.

Table 9:HH is to the influence of the dirty DHBV DNA of duck liver.

Medicine (mg/kg)

Inhibition rate(％control)

	Mean	SD
			HH
20 10 5 lamivudines 100	53.6 37.4 25.2 65.6	4.3 6.7 2.5 5.3

Gray scale scanning carries out quantitatively (n=3), Inhibition rate=1-(gray value _Medicine/ gray value _Contrast) * 100%

Example of formulations 1

Get wogonin 10g, add suitably adjuvant of injection (comprising lyophilized injectable powder and aseptic subpackaged dry powder injection), become hepatitis B resisting medicated injection by injection (comprising lyophilized injectable powder and aseptic subpackaged dry powder injection) prepared.

Example of formulations 2

Get wogonin 10g, add suitably adjuvant of tablet (comprising slow-release tablet, matrix tablet, coated tablet, dispersible tablet etc.), become hepatitis B resisting medicated tablet by tablet (comprising slow-release tablet, matrix tablet, coated tablet, dispersible tablet etc.) prepared.

Example of formulations 3

Get wogonin 10g, add the suitable adjuvant of capsule, become hepatitis B resisting medicated capsule by the capsule prepared.

Example of formulations 4

Get wogonin 10g, add suitably adjuvant of Emulsion (comprising microemulsion, nano-emulsion etc.), become hepatitis B resisting medicated Emulsion by Emulsion (comprising microemulsion, nano-emulsion etc.) prepared.

Example of formulations 5

Get wogonin 10g, add the suitable adjuvant of granule, become hepatitis B resisting medicated granule by the granule prepared.

Example of formulations 6

Get wogonin 10g, add the suitable adjuvant of sustained-release and controlled release agent, become hepatitis B resisting medicated sustained-release and controlled release agent by sustained-release and controlled release agent agent prepared.

Example of formulations 7

Get wogonin 10g, add the suitable adjuvant of oral liquid, become hepatitis B resisting medicated oral liquid by the oral liquid prepared.

Example of formulations 8

Get wogonin 10g, add the suitable adjuvant of liposome dosage form, become hepatitis B resisting medicated liposome dosage form by the liposome prepared.

Claims (3)

1, the application of wogonin in preparation treatment or prevention hepatitis B medicine.

2, application according to claim 1 is characterized in that the application of wogonin in preparation inhibition hepatitis B virus duplication medicine.

3, application according to claim 1 is characterized in that the application of wogonin in preparation reduction hepatitis B virus surface antigen medicine.

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