CN101584702B - Method of preparing extract of Mallotus apelta and uses for fighting hepatitis B virus - Google Patents
Method of preparing extract of Mallotus apelta and uses for fighting hepatitis B virus Download PDFInfo
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Abstract
The invention relates to anti-HBV activity of Mallotus apelta extract and uses in prepartion of medicine for treating related diseases caused by hepatitis B. The extract of Mallotus apelta provided by the invention has basic components of apiolin, apiin-7-O-belta-D-glucoside, and flavonoids compounds of 5,7-dihydroxy-6-isopentene group-4'-methoxyl flavonone, flavonoids compounds Mallotus apelta have obvious inhibiting effect on hepatitis B virus surface antigen and hepatitis B virus e antigen ono surface of HepG 2215, and cabaple of inhibiting duplication of Duck Hepatitis B Virus deoxyribonucleic acid, withdraw rebound is weaker than positive reference medicine lamivudine, and hepatitis B is provided with a subsequent inhibiting effect after withdraw. The medicine according to the invention has strong function for fighting hepatitis B virus, clear active ingredient, which is suitable for industrial production, predictablly used for preparing medicine of preparing diseases infected by hepatitis B virus.
Description
Technical field
The present invention relates to Chinese medicine extract of a kind of anti-hepatitis virus and preparation method thereof, the anti-hepatitis B activity agent preparation of particularly extracting the gained effective component group in the Folium Malloti Apeltae has the purposes of the various disease drugs with function that treatment causes by hepatitis B virus infection.
Background technology
Hepatitis B virus (HBV) is the pathogen of Type B viral hepatitis, and the persistence hbv replication causes the liver inflammatory disease to cause hepatitis B.This disease is commonly encountered diseases, the frequently-occurring disease of China, has that infectiousness is strong, the route of transmission is complicated, popular wide general, and sickness rate is than characteristics such as height.According to the Ministry of Public Health statistics, only had the hepatitis B case of record just to reach 910,000 people in 2004, and chronic asymptomatic HBV carrier may account for 9.09% of the common size of population, reaches 1.2 hundred million people.Long-term HBV is duplicated the liver inflammation that causes can cause serious consequences such as patient's hepatic fibrosis, liver cirrhosis, canceration of hepatic cell.According to statistics, 1,000,000 patients that have an appointment global every year die from hepatitis b virus infected relevant liver cirrhosis or hepatocarcinoma, are higher than 200 times of normal persons in China by the relative risk that chronic hepatitis patient develops into primary hepatocarcinoma.
Anti-hepatitis virus treatment can suppress or remove HBV, reduce and prevent that the liver function mistake is compensatory, the generation of liver cirrhosis, hepatocarcinoma and complication thereof, improve patients ' life quality and prolong its time-to-live.Present clinical antiviral therapy often adopts interferon and nucleoside medicine.Interferon is that a class has the active protein of broad-spectrum antiviral, can produce antiviral protein by inducing cell.But interferon therapy is realizing that curative effect is limited in cloudy commentaries on classics of hepatitis B virus e antigen (HBeAg) and the HBV removing, the HBeAg positive patients only is respectively 33% and 37% through the HBeAg negative conversion rate and hepatitis B virus deoxyribonucleotide (HBV-DNA) clearance rate of Interferon Alpha-2b treatment, and the interferon side effect is more, comprises influenza-like symptom, fatigue, irritability depression and bone marrow depression etc.Nucleoside medicine comprises medicines such as lamivudine (3TC), adefovirdipivoxil, Entecavir, and such drug main will suppress the HBV archaeal dna polymerase, and causing preceding genome rna (RNA) can't reverse transcription be DNA; Or synthetic with certain triphosphoric acid dezyribonucleoside competition participation HBV-DNA, integrate the extension that the back stops viral DNA.But nucleoside medicine can only suppress virus replication, can not fundamentally remove virus, and long-time medication easily causes virus mutation, the generation drug resistance.In addition, interferon and nucleoside medicine all have tangible drug withdrawal knock-on, are prone to viral massive duplication, the liver function symptom of deterioration rapidly after the drug withdrawal.And involving great expense of medicine, the financial burden of patient treatment is very heavy.
Folium Malloti Apeltae (whitebackleaf) is Euphorbiaceae Mallotus plant Mallotus apelta (Lour.) MuelL.-Arg., [another name]: yeast for brewing rice wine subtree, wild paulownia, Folium Malloti Apeltae, hang chestnut, and [meridian distribution of property and flavor] little hardship, puckery, flat.[function cures mainly] root: easing the affected liver is invigorated blood circulation, invigorating the spleen for eliminating dampness, and convergence is taken off admittedly.Be used for chronic hepatitis, hepatosplenomegaly, uterine prolapse, proctoptosis, leucorrhea, edema during pregnancy.Leaf: anti-inflammation hemostasia.Otitis media is controlled in external, furuncle and phyma, traumatic injury, traumatic hemorrhage.Document record, Folium Malloti Apeltae have the merit of clearing away heat-damp and promoting diuresis, detoxifcation hemostasis, pain relieving, are usually used in enteritis, diarrhea, chronic hepatitis, spleen enlargement and traumatic injury etc.The Xu Yixin of Second Military Medical University, PLA, Chen Haisheng etc. adopt alcohol extraction, silicon chamber TLC, and low pressure column chromatography separates, spectroscopic identification.Result: get 4 chemical compounds, their structure is: Acetyl aleuritolic acid (I), erythrodiol-3-acetate (erythordiol-3-acetate) (II), scopoletin (acopoletin) (III), cupreol-3-O-β-D-glucopyanoside (β-sitosterol-3-O-β-D-glucopyranoside) (IV), think that this is the main active Folium Malloti Apeltae composition of Folium Malloti Apeltae, " Radix Malloti Apelitae chemical constitution study " " PLA's Acta Pharmaceutica Sinica " .1999,15 (5) .-7-10.The Wu Guifan of Colleges Of Traditional Chinese Medicine Of Guangxi, Wei Song etc. separate the ethanol soluble extraction of Folium Malloti Apeltae and purification with various column chromatographies, and the gained chemical compound is identified with physicochemical property and spectral data.The result gets 5 chemical compounds, is accredited as taraxerol (I), Sitosterolum (II), 5 respectively, 7-dihydroxy-6-isopentene group-4 '-melonia flavone (III), apiolin (IV), apiolin-7-O-β-D-glucoside (V)." new isopentene group flavanone in the Folium Malloti Apeltae " " Chinese herbal medicine " .2006,37 (8) .-1126-1128.But do not carry out the pharmacodynamics test of these chemical compounds.
Discovery Radix Malloti Apelitae decocting liquid such as Zhang Xiaogang have obvious inhibition to the secretion of HepG 2215 cell hbs antigenes (HBsAg) and HBeAg, its half toxic concentration (TC50) to 2213 cells is 48.25mg/ml, and all obviously suppressing 2215 emiocytosis HBsAg and HBeAg at this decocting liquid below concentration, medium effective concentration (IC50) is respectively 3.64 and 1.12mg/ml.But Radix Malloti Apelitae decocting liquid to the inhibitory action of 2215 cell HBV dna replication dnas not significantly [Zhang Xiaogang, etc., the time precious traditional Chinese medical science traditional Chinese medicines, 2006,17 (8)), 1437-1438].Xu Shu etc. find that by the dhbv dna experiment Radix Malloti Apelitae decocting liquid has the effect of inhibition dhbv dna (DHBV) dna replication dna, and the effect [Xu Shu that still has follow-up inhibition DHBV to duplicate after the drug withdrawal, Deng, combination of Chinese and Western medicine journal, 2006,4 (3), 285-288].
The Zhao Jinjun of No.1 Military Medical Univ. etc. study the effect and the Radix Malloti Apelitae Study on anti-oxidative ability of Radix Malloti Apelitae control hepatic fibrosis by the rat liver fibrosis animal model.Method: use 40%CCl
4Peanut oil solution prepares the rat liver fibrosis model, observe the influence of Radix Malloti Apelitae to multiple index in hepatic fibrosis rats serum and the hepatic tissue, the index of observing comprises: alanine aminotransferase (ALT), Tianmen propylhomoserin transaminase (AST), MDA, the content of hydroxyproline and liver tissue injury degree (pathology section examination) in NO and the hepatic tissue, the result: Radix Malloti Apelitae can significantly reduce the level of many index in the rat blood serum, and can alleviate liver collagen fiber hyperplasia degree, think that Radix Malloti Apelitae has effect of anti hepatic fibrosis and oxidation resistance preferably, antioxidation may be one of mechanism of Radix Malloti Apelitae blocking-up hepatic fibrosis formation." Chinese crude drug .2002,25 (3) .-185-187, " experimentation of Radix Malloti Apelitae antioxidation in the hepatic fibrosis animal model " "
Recognize that from above-mentioned open source literature Folium Malloti Apeltae and root have the supression effect to hepatitis virus, also can prevent and treat hepatic fibrosis and have oxidation resistance.Have, prevent and treat hepatic fibrosis but that part in the Folium Malloti Apeltae extract works to restrain hepatitis virus, open source literature does not disclose.
Summary of the invention
The purpose of this invention is to provide the preparation method that has anti-hepatitis virus in the anti-Folium Malloti Apeltae plant extract, be specifically related to flavone compound anti-hepatitis B activity that extracts in the Folium Malloti Apeltae plant and the purposes for the treatment of disease that hepatitis B virus infection causes.
We see from above-mentioned background technology, and many kinds of chemical compounds are arranged in the Folium Malloti Apeltae, and different extracting method obtains different materials, their structure and also different as the disease of Drug therapy.
The inventor analyzes according to the flavone compound that extracts from the Folium Malloti Apeltae plant in the scientific experimentation in the past few years, through pharmacodynamics test, has determined the medicine of anti-hepatitis B activity and preparation treatment hepatitis B virus infection disease.
Technical scheme of the present invention is as follows:
One, the chemical compound in the Folium Malloti Apeltae plant extracts and purifies:
Get the dried plant leaf of Folium Malloti Apeltae, remove impurity, smash or shred the back and extract in boiling water, per kilogram dried plant leaf adds water 1～20L, heating extraction 1～8 hour, and filtered while hot is collected filtrate.Filtering residue adopts boiling water to repeat to extract once more 1～3 time, merges gained filtrate after the filtered while hot.
Gained filtrate temperature control is by macroporous adsorptive resins, and 20 ℃～80 ℃ of temperature adopt water elution, adopt 10%～80% ethanol to carry out eluting again, collect and the ethanol elution that hydrochloric acid-magnesium powder solution is positive, and drying under reduced pressure, recrystallization obtains end-product.
Described macroporous adsorbent resin can be selected D101 macroporous resin (domestic have a lot of manufacturer production) for use, also can select X5, AB-8 resin for use, (Chemical Plant of Nankai Univ.'s production); Perhaps XAD-4, XAD-7 resin, (production of U.S. Rohm.Hass company).
Gained Folium Malloti Apeltae flavone compound is the crystallization of pale brown toner powder after the extract drying, and through liquid-phase chromatographic analysis, its solvent is an apiolin, apiin-7-O-β-D-glucoside, 5,7-dihydroxy-6-isopentene group-4 '-the melonia flavone, structure is as follows:
The apiolin structural formula:
Apiin-7-O-β-D-glucoside structural formula:
5,7-dihydroxy-6-isopentene group-4 '-melonia flavone structural formula:
The solvent that the present invention extracts can be above-mentioned two or three compound compositions, best ratio is: apiolin 5%～70%, apiin-7-O-β-D-glucoside 30%～90%, 5,7-dihydroxy-6-isopentene group-4 '-melonia flavone 0%～20%.The invention provides the expression that above-mentioned Folium Malloti Apeltae flavone compound suppresses HBsAg and HBeAg, and suppress the activity that hepatitis B virus DNA duplicates; And be used to prepare the new purposes for the treatment of medicine that hepatitis B virus infection gives rise to diseases.
Two, the anti-HBV effect of Folium Malloti Apeltae flavone compound
The inventor's pharmacodynamic experiment shows that the Folium Malloti Apeltae flavone compound is not having under the Cytotoxic concentration, and HepG2215 cell HBsAg and HBeAg expression are had remarkable inhibitory action.Folium Malloti Apeltae flavone compound maximal non-toxic concentration is 37.5 μ g/ml, after cultivating three days under this concentration, the suppression ratio of 2215 emiocytosis HBsAg and HBeAg is reached 86.6% and 74.79% respectively, to the IC of 2215 emiocytosis HBsAg and HBeAg
50Be respectively 8.61 μ g/ml and 20.87 μ g/ml.In addition, the inventor finds that through the dhbv dna experiment Folium Malloti Apeltae flavone compound can significantly suppress duplicating of DHBV-DNA.Infected the duckling of dhbv dna, continuous 10 days oral 50～200mg/kg Folium Malloti Apeltae flavone compounds have significantly suppressed serum DHBV-DNA concentration, take medicine 5～10 days, its suppression ratio is 14～20%, and after the drug withdrawal serum DHBV DNA is had follow-up inhibition effect.
According to inventor's above-mentioned research, the invention provides the Folium Malloti Apeltae flavone compound and have the HBsAg of inhibition and HBeAg expression, suppress the activity that hepatitis B virus DNA duplicates, be expected to be used for the treatment of the disease that hepatitis B virus infection causes.This invention has the following advantages: Folium Malloti Apeltae flavone compound preparation method is simple, suitability for industrialized production; Our experiments show that Folium Malloti Apeltae flavone compound anti-HBV effect is remarkable, be better than other thick extractions of Folium Malloti Apeltae; Folium Malloti Apeltae is abundant in china natural resources, and widely distributed, cost of material is cheap.
The specific embodiment
For characteristics of the present invention and essence are described better, form with embodiment provides Folium Malloti Apeltae flavone compound (apiolin 5%～70% below, apiin-7-O-β-D-glucoside 30%～90%, 5,7-dihydroxy-6-isopentene group-4 '-melonia flavone 0%～20%) extraction process and the The pharmacological results of anti-hepatitis virus, its purposes in pharmaceutical field is described.
Embodiment 1: the flavone compound in the Folium Malloti Apeltae plant extracts and purifies:
Get the dried plant leaf of Folium Malloti Apeltae, remove impurity, smash or shred the back and extract in boiling water, per kilogram dried plant leaf adds water 1～20L, heating extraction 1～8 hour, and filtered while hot is collected filtrate.Filtering residue adopts boiling water to repeat to extract once more 1～3 time, merges gained filtrate after the filtered while hot.
Gained filtrate temperature control is by macroporous adsorptive resins, and 20 ℃～80 ℃ of temperature adopt water elution, adopt 10%～80% ethanol to carry out eluting again, collect and the ethanol elution that hydrochloric acid-magnesium powder solution is positive, and drying under reduced pressure, recrystallization obtains end-product.
Described macroporous resin adopts D101 macroporous resin or AB-8 resin.To use the Cotton Gossypii jam-pack at the bottom of the chromatographic column, the ethanol that adds one times of dried resin volume fully stirs back wet method dress post with Glass rod, and chromatographic column post blade diameter length ratio is generally 1: 5～10.The water flushing eliminates the ethanol in the post behind the alcohol flushing.Load onto and hold the liquid ball, open the post lower piston, gained filtrate heating back impouring is held in the liquid ball, cross post filtrate control temperature at 20～80 ℃, to improve adsorption efficiency.Adopt water elution interchangeable ethanol elution after eluent is colourless, and note the control eluent flow rate.The drying under reduced pressure ethanol elution is concentrated into 5%～50% of original volume with effluent volume, leaves standstill recrystallization afterwards, and the concentrating degree of eluent influences the speed and the purity of recrystallization to a great extent, so should strict control.
Gained Folium Malloti Apeltae flavone compound is the crystallization of pale brown toner powder after the extract drying, through liquid-phase chromatographic analysis, its solvent is an apiolin 5%～70%, apiin-7-O-β-D-glucoside 30%～90%, 5,7-dihydroxy-6-isopentene group-4 '-melonia flavone 0%～20%.
Embodiment 2: the Folium Malloti Apeltae flavone compound is to the inhibitory action of HepG 2215 cell HBsAg and HBeAg expression.2.1 reagent: Eagles MEM dry powder (U.S. Gibco product); Hyclone (U.S. Hyclone Lab product); G-418 (Genticin product); L-glutaminate (Amresco packing); Kanamycin (Amresco packing); DMSO (vast Imtech product); 0.25% trypsin U.S. Hyclone Lab product); Hepatitis B surface antigen testing cassete and hepatitis B virus e antigen testing cassete (euzymelinked immunosorbent assay (ELISA), Shanghai Rongsheng Bioisystech Co., Ltd's product).
2.2 cell in vitro is cultivated: 2215 cells are made up by U.S. Mount Sinai medical center, and Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences is so kind as to give.Cell culture contains hyclone 10% with the MEM culture fluid, glutamine 300 μ g/ml, and G-418380 μ g/ml, kanamycin 750 μ g/ml use 5%NaHCO
3Adjust pH to 7.2～7.4.Cell is put 37 ℃, 5%CO in the MEM culture fluid that contains 10% hyclone
2Incubator is cultivated, and changes culture fluid once in 2～3 days, covers with culture bottle in about 7～10 days.
2.3 experimental technique: with the morphocytology pathological changes is the toxicity of index evaluation Folium Malloti Apeltae flavone compound to 2215 cells.If (solvent is an apiolin 5%～70% for cell matched group and 7 Folium Malloti Apeltae flavone compound medicine groups, apiin-7-O-β-D-glucoside 30%～90%, 5,7-dihydroxy-6-isopentene group-4 '-melonia flavone 0%～20%), the medicine final concentration is respectively 150,75,37.5,18.75,9.38,4.69,2.34 μ g/ml.Establish 3 multiple holes for every group.After cell covers with,, adjust cell concentration to 10 through 0.25% trypsinization
5Cells/ml is inoculated in 96 orifice plates, every hole 0.2ml.Abandon original fluid after cultivating 24h, the cell matched group adds normal culture fluid, and the medication group adds the pastille culture fluid, cultivates 3 days.To cultivating the 4th day, each group is all changed normal culture fluid, continues to cultivate 4 days.To cultivating observation of cell morphological change under inverted microscope the 8th day.With the morphocytology pathological changes is the toxicity of index evaluation WF to 2215 cells, according to the Reed-Muench method the impaired percentage rate of cell is higher than 25% the medicine of thinking cytotoxicity is arranged, be lower than 25% and think that promptly cell is not damaged by medicine source property, determine maximal non-toxic concentration thus.Collect maximal non-toxic concentration and be lower than each hole supernatant that this concentration WF cultivates, measure usefulness for HBsAg and HBeAg.
Get above-mentioned cell culture supernatant, press HBsAg and HBeAg enzyme linked immunological (ELISA) test kit description and measure HBsAg and HBeAg OD value.Antigen suppression ratio, IC
50, be calculated as follows respectively:
Wherein: A=1g (suppression ratio greater than 50% drug level) B=1g (suppression ratio less than 50% drug level)
C＝A-B
2.4 statistical analysis: experimental result is all represented with mean+SD.Adopt each group difference of t check analysis.
2.5 experimental result: cytotoxicity experiment was rescued demonstration, and the Folium Malloti Apeltae flavone compound of variable concentrations is cultivated and caused cell death in various degree to occur, wherein about 98% death of 150 μ g/ml group cell; About 40% death of 75 μ g/ml group cell, cell shrinkage; 37.5 about 15% death of μ g/ml group cell, cell shrinkage; 18.75 μ g/ml group and this concentration following group cell growth state and cell matched group comparison cellular morphology do not have obvious change, cell quantity does not obviously reduce yet.Determine that thus 37.5 μ g/ml are maximal non-toxic concentration.
Adopt the ELISA method to detect of the influence of the following Folium Malloti Apeltae flavone compound of maximal non-toxic concentration to 2215 cell HBsAg and HBeAg expression, experimental result shows, the Folium Malloti Apeltae flavone compound all has stronger inhibitory action at 2.34～37.5 μ g/ml to 2215 cellular expression HBsAg and HBeAg, and its inhibition degree be proportionate with concentration, the two suppression ratio is reached 84.6% and 74.8% respectively.The Folium Malloti Apeltae flavone compound suppresses the IC of 2215 cellular expression HBsAg
50Be 8.61 μ g/ml, to the IC of HBeAg expression
50Be 20.87 μ g/ml.Experimental data sees Table one and table two.
Suppression ratio is up to:
2.6 conclusion: HBsAg and HBeAg are the important indicators of hepatitis B virus active degree, and inventor's experiment shows that the Folium Malloti Apeltae flavone compound has powerful inhibitory action to 2215 cellular expression HBsAg and HBeAg under avirulent concentration.
Table one. the Folium Malloti Apeltae flavone compound to the inhibitory action of 2215 cellular expression HBsAg (x ± s, n=3)
Annotate:
*Expression is compared P＜0.01 with the cell matched group.
Table two. the Folium Malloti Apeltae flavone compound to the inhibitory action of 2215 cellular expression HBeAg (x ± s, n=3)
Annotate:
*Expression is compared P＜0.01 with the cell matched group.
Embodiment 3: the inhibitory action experiment that the Folium Malloti Apeltae flavone compound duplicates DHBV-DNA
3.1 reagent: positive control medicine lamivudine (lamivudine, 3TC, GlaxoSmithKline PLC drugmaker); DMSO (Guangzhou New Port chemical industry company limited); α-32P-Dctp (the auspicious biotechnology of Beijing good fortune engineering company); Nick translation test kit (Promega company); SephadexG-50 (Ficoll company); PVP (Pharmacia company); SDS (Merck company); 0.45 μ m nitrocellulose filter (Amersham company); Milt DNA and bovine serum albumin are all available from Instite of Biophysics, Chinese Academy of Sciences.
3.2 laboratory animal and viral source: 1 age in days Beijing sheldrake, body weight 60～100g is available from progressive species duck animal feeding field, Beijing.DHBV-DNA strong positive serum picks up from the Nanjing sheldrake ,-70 ℃ of preservations.
3.3 experimental technique: 1 age in days Beijing duck, through lower limb shin intravenous injection DHBV-DNA positive serum 0.2ml/ only.Duckling infects through DHBV and was divided into 4 groups at random on the 7th day, be respectively virus control group (normal saline, p.o.), Folium Malloti Apeltae flavone compound low dose group (75mg/kg, p.o.), Folium Malloti Apeltae flavone compound high dose group (150mg/kg, p.o.), every group of (3TC 50mg/kg of positive controls, p.o.), 6 every group.The 7th day (being T0) begins administration, 2 times/day, successive administration 10 days after infecting DHBV.(Folium Malloti Apeltae flavone compound solvent is an apiolin 5%～70%, apiin-7-O-β-D-glucoside 30%～90%, 5, and 7-dihydroxy-6-isopentene group-4 '-melonia flavone 0%～20%)
Respectively before DHBV infects back (T0) medication in the 7th day, medication the 5th day (T5) after medication the 10th day (T10) and the drug withdrawal the 3rd day (P3), is got blood from duck lower limb shin vein, centrifugalize serum, and-70 ℃ of preservations are to be checked.Measure serum DHBV-DNA level in strict accordance with nick translation test kit description.With
32P labelling DHBV-DNA probe, and do the clear dot blot hybridization of Sanguis Anas domestica, autoradiography diaphragm speckle, enzyme mark detector is measured the 490nm OD of place value, calculating serum DHBV-DNA density, with hybridization spot OD value as specimen DHBV-DNA level value.Calculate medicine as follows at the suppression ratio of each time point to animal serum DHBV-DNA:
3.4 statistical analysis: experimental result is all represented with mean+SD.Adopt the animal serum DHBV-DNA level variation on the same group of paired sample t check analysis Drug therapy each time point of front and back; Adopt the suppression ratio of the medicine of each time point of burst data t check analysis to serum DHBV-DNA.
3.5 experimental result: DHBV infects back T0, T5, T10 and the clear DHBV-DNA of P3 time point Sanguis Anas domestica and measures through spot hybridization, and behind all 4 groups of experimental animal infection DHBV, serum DHBV-DNA all is strong positive.Microplate reader detects each experiment sample and shows in the OD of 490nm place value, and 5-10 days animal serum DHBV-DNA concentration is extremely significantly decline behind the oral Folium Malloti Apeltae flavone compound, and drug withdrawal still significantly suppresses DHBV-DNA after 3 days.And also significantly reduced animal serum DHBV-DNA concentration in oral positive drug 3TC 5-10 days, but drug withdrawal obviously knock-on property rise of animal serum DHBV-DNA concentration after 3 days.Experimental result sees Table three and accompanying drawing.
Calculating medicine finds the suppression ratio of DHBV-DNA, the Folium Malloti Apeltae flavone compound is to suppression ratio and the administration time positive correlation of animal serum DHBV-DNA, and drug withdrawal after 3 days low dosage Folium Malloti Apeltae flavone compound the suppression ratio of DHBV-DNA is still risen to some extent, and high dose to the suppression ratio of DHBV-DNA only a little less than medication the 10th day.And positive drug 3TC has obvious inhibition to animal serum DHBV-DNA during medication, but drug withdrawal after 3 days its suppression ratio descend rapidly.Experimental data sees Table three.
Table three. the influence that the Folium Malloti Apeltae flavone compound duplicates animal serum DHBV-DNA)
Annotate: n=6, serum DHBV-DNA represents that at 490nm place light absorption value data are listed with x ± s to measure sample, adopts paired data t check,
*P＜0.05,
*P＜0.01 is with T0 comparison on the same group.The DNA suppression ratio is represented with meansigma methods, adopts burst data t check,
#P＜0.05, and relatively with time point virus control group.
3.6 conclusion: the duckling that infects DHBV childhood can develop into persistence DHBV and infect.DHB and viruses of human hepatitis B have its similarity at aspects such as gene structure and copy modes, so often with the model of DHBV infection animal, study the curative effect of anti-HBV medicine, kinetics, the disinfectant observation on effect of inhibition virus replication.The inventor's experiment shows that the Folium Malloti Apeltae flavone compound is to the remarkable inhibition of having duplicated of duckling serum DHBV-DNA, and knock-on is significantly less than lamivudine after its drug withdrawal, and after the drug withdrawal DHBV-DNA is still had follow-up inhibition effect.
Description of drawings
Fig. 1-Fig. 4 is the dynamic change situation that .DHBV infects back duckling serum DHBV-DNA, and wherein Fig. 1 is the virus control group; Fig. 2 is the 3TC group; Fig. 3 is a Folium Malloti Apeltae flavone compound low dose group; Fig. 4 is a Folium Malloti Apeltae flavone compound high dose group.
See among the figure that duckling serum DHBV-DNA was with different medicines after DHBV infected, the result: the Whitebackleaf Mallotus Root flavone compound is to the remarkable inhibition that copied of duckling serum DHBV-DNA.
Claims (2)
1. Folium Malloti Apeltae method for preparing extractive, it is characterized in that: the dried plant leaf of getting Folium Malloti Apeltae, remove impurity, smashing or shred the back extracts in boiling water, per kilogram dried plant leaf adds 1～20 liter in water, heating extraction 1～8 hour, filtered while hot, collect filtrate, filtering residue adopts boiling water to repeat to extract once more 1～3 time, merges gained filtrate after the filtered while hot, and gained filtrate temperature control passes through macroporous adsorptive resins, 20 ℃～80 ℃ of temperature, adopt water elution, adopt 10%～80% ethanol to carry out eluting again, collection and the ethanol elution that hydrochloric acid-magnesium powder solution is positive, drying under reduced pressure, recrystallization obtains end-product.
2. the purposes of the described Folium Malloti Apeltae extract of claim 1 in the medicine of the disease that preparation treatment hepatitis B virus infection causes, the main chemical compositions of described extract is:
Apiolin:
Apiin-7-O-β-D-glucoside:
5,7-dihydroxy-6-isopentene group-4 '-the melonia flavone:
The mass content of each composition in the described extract: apiolin 5%～70%; Apiin-7-O-β-D-glucoside 30%～90% and 5,7-dihydroxy-6-isopentene group-4 '-melonia flavone 0%～20%.
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