CN104983787B - A kind of composition of anti-hepatitis B virus and the preparation method and application thereof - Google Patents
A kind of composition of anti-hepatitis B virus and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses compositions of a kind of anti-hepatitis B virus and the preparation method and application thereof, and the medicinal material for preparing the composition is Gentianaceae Swertia plant；The preparation method comprises the following steps: ethyl alcohol extracts, filtrate concentration；Medicinal extract adds water to mix；Successively use petroleum ether, ethyl acetate, extracting n-butyl alcohol；Petroleum ether is mutually concentrated to get the component A；Ethyl acetate phase is first eluted through macroreticular resin, is carried out column chromatography with silicagel column after eluent concentration, is collected eluent, be concentrated to get the component B；N-butanol is concentrated to get the component C after mutually being eluted with macroreticular resin.The composition include component A be 1-50 parts, component B is 1-50 parts, component C is 1-50 parts；The composition toxicity is low, and composition inhibits hepatitis B ability stronger than one-component or two-component, remains to prevent HBV-DNA level from rebounding after drug withdrawal, therefore can be widely used in the treatment of hepatitis B.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of composition of anti-hepatitis B virus and preparation method thereof
With application.
Background technique
Hepatitis type B virus (HBV) infection is a global health problem.Global 2,000,000,000 populations have received HBV infection,
Chronic patients are up to 3.6 hundred million, and annual Died Patients are up to 520,000 (50,000 die of acute HBV patient, and 470,000 die of cirrhosis and liver cancer).
China HBV crowd infection rate 57.6%, hepatitis B surface antigen (HBsAg) positive 9.75%, the existing disease chronic hepatitis B in the whole nation
About 3000-4000 ten thousand.Hepatitis type B virus and cirrhosis and primary carcinoma of liver it is in close relations, hepatitis B virus infection has become shadow
Ring one of the nine big diseases of human longevity.Virus B hepatitis is caused by hepatitis B (HBV), with liver inflammation lesion
Based on, a kind of disease of multiple organ injury can be caused, such as treat bad, or even cirrhosis and liver cancer can be changed into.
HBV-DNA is the DNA (i.e. hepatitis B virus gene) of hepatitis B, it be HBV infection most directly,
High specificity and the high index of sensitivity.HBV-DNA is positive, prompts hbv replication and is infectious.The higher expression disease of HBV-DNA
Poison duplication is more severe, and infectiousness is strong.The drug for the treatment of hepatitis B mainly includes interferon, nucleotide analog and Chinese medicine at present
Chinese medicine.Using interferon is big as the biological species drug therapy hepatitis B toxicity of representative, adverse reaction is serious and expensive, Huan Zhejing
Ji burden is big, and clinic is difficult to be widely used；Nucleotide analog (Lamivudine, adefovirdipivoxil, Entecavir, Sebivo etc.)
There is good inhibiting effect to hepatitis type B virus, but the structure type of these synthesis antiviral agents is single, exists multiple after being discontinued
Hair rate is high, unobvious to HBeAg effect, and long-term administration is needed to cause a disease malicious the problems such as being resistant to；Traditional Chinese medicine advantage is that the mechanism of action is complete
Face has the multiple actions such as antiviral, adjusting is immune, anti-inflammatory anti-hepatic fibrosis, but it is slow to work, and antiviral speed is difficult to
It is compared with Western medicine.Therefore, novel Anti-HBV drugs are researched and developed to treat hepatitis B, the lapsing to for control and disease to the infection sources has
Extremely important social effect and economic significance.
Summary of the invention
It is low the purpose of the present invention is developing a kind of toxicity, it still can inhibit the drug of hepatitis B after drug withdrawal.To realize
Object above, the present invention can realize by the following technical solutions:
A kind of composition of anti-hepatitis B virus prepares the raw medicinal material of the composition as the plant of Gentianaceae Swertia
Object；Prepare the composition the preparation method comprises the following steps: takes the raw medicinal material first, adds ethanol solution to extract, filters, merging filtrate
Afterwards, it is concentrated to get medicinal extract；Gained medicinal extract is taken, is added water and mixed；Successively use petroleum ether, ethyl acetate, extracting n-butyl alcohol；Gained stone
Oily ether is mutually directly concentrated to get the component A；Gained ethyl acetate phase is first eluted through macroreticular resin, uses silica gel after eluent concentration
Column carries out column chromatography, collects eluent, is concentrated to get the component B；Gained n-butanol is concentrated to give after mutually being eluted with macroreticular resin
To the component C；By three of the above component according to component A be 1-50 parts, component B is 1-50 parts, component C be 1-50 parts of mixing i.e.
Obtain the composition.
The Gentianaceae Swertia plant is preferably mussot swertia herb (Swertia mussotii Franch.), embraces stem
Swertia patens (Swertia franchetiana H.Smith), Tibet Herba Swertiae bimaculatae (Swertia racemosa (Griseb.)
Wall.ex C.B.Clarke), Swertia przewalskii Pissjauk (Swertia przewalskii Pissjauk.), Swertia erythrosticta Maxim
(Swertia erythrosticta Maxim.)；
When composition described in preparation, 1 part of the raw medicinal material agents area is taken first, is crushed, and is added 70%~95%
5-12 parts of ethanol solution mixing, refluxing extraction, filtering after merging filtrate, is concentrated to get medicinal extract；Medicinal extract obtained by taking, addition 2~
The water of 3 times of volume ratios mixes；Successively with 3~4 times of volume ratio petroleum ethers, 4~5 times of volume ratio ethyl acetate, 4~6 times of volume ratios
Extracting n-butyl alcohol.
The processing method of the ethyl acetate phase is to dissolve the ethyl acetate phase, then with methanol with 1-2 parts of macropore trees
Rouge/polyamide, 1-3 parts of macroreticular resin/microcrystalline celluloses are that the mixed fillers of ratio fill column, and successively use 10%-98% methanol
Aqueous solution gradient elution, according to wash water color by no discoloration it is coloured collect, until brown become it is light yellow collection terminate, will
Use 200-300 mesh silicagel column again after eluent concentration, petroleum ether: ethyl acetate=1:1 elution is collected eluent, is concentrated to get
The component B.
The processing method of the n-butanol phase is mutually to dissolve the n-butanol with methanol, with 1-3 parts of macroreticular resins/poly-
Amide, 1-2 parts of macroreticular resin/microcrystalline celluloses are that the mixed fillers of ratio fill column, and successively use 12%-98% methanol aqueous solution
Gradient elution, according to wash water color by no discoloration it is coloured collect, until brown become it is light yellow collection terminate, by eluent
Concentration, obtains the component C.
The method of the preparation composition, takes the raw medicinal material first, ethanol solution is added to extract, and filters, and merges filter
After liquid, it is concentrated to get medicinal extract；Gained medicinal extract is taken, is added water and mixed；Successively use petroleum ether, ethyl acetate, extracting n-butyl alcohol；Gained
Petroleum ether is mutually directly concentrated to get the component A；Gained ethyl acetate phase is first eluted through macroreticular resin, uses silicon after eluent concentration
Rubber column gel column carries out column chromatography, collects eluent, is concentrated to get the component B；Gained n-butanol is concentrated after mutually being eluted with macroreticular resin
Obtain the component C；By three of the above component according to component A be 1-50 parts, component B is 1-50 parts, component C is 1-50 parts of mixing
Obtain the composition.
Preferably, 1 part of the raw medicinal material agents area is taken first, is crushed, and 70%~95% ethanol solution 5- is added
12 parts of mixing, refluxing extraction filter, and after merging filtrate, are concentrated to get medicinal extract；Gained medicinal extract is taken, 2~3 times of volume ratios are added
Water mixes；Successively with 3~4 times of volume ratio petroleum ethers, 4~5 times of volume ratio ethyl acetate, 4~6 times of volume ratio extracting n-butyl alcohols.
Preferably, the processing method of the ethyl acetate phase is to dissolve the ethyl acetate phase, then with methanol with 1-2
Part macroreticular resin/polyamide, the mixed fillers that 1-3 parts of macroreticular resin/microcrystalline celluloses are ratio fill column, and successively use 10%-
98% aqueous methanol gradient elution, according to wash water color by no discoloration it is coloured collect, until brown becomes light yellow receipts
Collection terminates, and uses 200-300 mesh silicagel column after eluent is concentrated again, petroleum ether: ethyl acetate=1:1 elution collects eluent,
It is concentrated to get the component B.
Preferably, the processing method of the n-butanol phase is mutually to dissolve the n-butanol with methanol, with 1-3 parts of macropores
Resin/polyamide, 1-2 parts of macroreticular resin/microcrystalline celluloses are that the mixed fillers of ratio fill column, and successively use 12%-98% first
Alcohol solution gradient elution, according to wash water color by no discoloration it is coloured collect, until brown become it is light yellow collection terminate,
Eluent is concentrated, the component C is obtained.
The composition is preparing the application in anti-hepatic-B virus medicine.
The invention has the benefit that toxicity is low, composition inhibits hepatitis B ability strong than one-component and two-component, stops
It remains to reduce HBV-DNA level after medicine, the treatment of hepatitis B can be widely used in.
Specific embodiment
The present invention is done below with reference to embodiment and is further explained.The following example is merely to illustrate the present invention, but
It is not used to limit practical range of the invention.
Embodiment 1:
Taking Gentianaceae Swertia plant --- the drying herb 500g of mussot swertia herb is crushed, and the ethyl alcohol with 70% is molten
Liquid is mixed by solid-liquid mass ratio 1:5, and refluxing extraction 4 times, 3 hours every time, filtering, merging filtrate simultaneously recycled ethanol reagent.It will filter
Liquid, which is placed in be concentrated in Rotary Evaporators, is made medicinal extract, and the water that 2 times of volume ratios of medicinal extract are added is uniformly mixed, successively with 3 times of volumes
Petroleum ether, the ethyl acetate of 4 times of volumes, extracting n-butyl alcohol 5 times of 4 times of volumes.Petroleum ether is mutually directly concentrated as component A；With
The medicinal extract of ethyl acetate phase is raw material, is mixed with methanol by 1g:6mL, and for heating stirring to being completely dissolved, heat filtering obtains filtrate, will
Filtrate is using macroreticular resin/polyamide, macroreticular resin/microcrystalline cellulose as mixed fillers (the two mass ratio is 1:3), organic solvent
Wet method dress post is successively eluted with 10%-90% aqueous methanol gradient, elution flow rate 100mL/min.According to wash water color
By no discoloration it is coloured collect, until brown become it is light yellow collection terminate, after eluent is concentrated again use 200-300 mesh column layer
Chromatographic silica gel carries out column chromatography, and with petroleum ether: ethyl acetate=1:1 rushes column, collects eluent, obtains the component B；Finally will
N-butanol phase medicinal extract is mixed with methanol by 1g:8mL, and for heating stirring to being completely dissolved, heat filtering obtains filtrate, by filtrate with macropore tree
Rouge/polyamide, macroreticular resin/microcrystalline cellulose are mixed fillers (the two mass ratio is 1:2), organic solvent wet method dress post, according to
It is secondary to be eluted with 12%-92% aqueous methanol gradient, elution flow rate 150mL/min.Had according to wash water color by no discoloration
Color starts to collect, until brown, which becomes light yellow collection, to be terminated, it is component C after eluent is concentrated.
Embodiment 2
Gentianaceae Swertia plant --- the extracting method of Swertia franchetiana H.Smith is the same as embodiment 1.
Embodiment 3:
Taking Gentianaceae Swertia plant --- the dry herb 500g of Tibet Herba Swertiae bimaculatae is crushed, the ethanol solution with 85% is pressed
Solid-liquid mass ratio 1:10 mixing, refluxing extraction 5 times, 3 hours every time, filtering, merging filtrate simultaneously recycled ethanol reagent.Filtrate is set
It carries out that obtained medicinal extract is concentrated in Rotary Evaporators, the water that 3 times of volume ratios of medicinal extract are added is uniformly mixed；Successively with 4 times of volumes
Petroleum ether, the ethyl acetate of 5 times of volumes, extracting n-butyl alcohol 5 times of 6 times of volumes；Petroleum ether is mutually directly concentrated as component A；
Using the medicinal extract of ethyl acetate phase as raw material, being mixed with methanol by 1g:8mL, for heating stirring to being completely dissolved, heat filtering obtains filtrate,
By filtrate using macroreticular resin/polyamide, macroreticular resin/microcrystalline cellulose as mixed fillers (the two mass ratio is 1:2), You Jirong
Agent wet method dress post is successively the elution of 16%-96% aqueous methanol gradient, elution flow rate 100mL/min with percent by volume.
According to wash water color by no discoloration it is coloured for the first time collect, until brown become it is light yellow collection terminate, eluent is dense
Column chromatography is carried out with 200-300 mesh column layer chromatography silicone rubber again after contracting, with petroleum ether: ethyl acetate 1:1 rushes column, eluent is collected,
It is concentrated to get the component B；Finally n-butanol phase medicinal extract is mixed with methanol by 1g:9mL, heating stirring is to being completely dissolved, heat
It filters to get filtrate, by filtrate, using macroreticular resin/polyamide, macroreticular resin/microcrystalline cellulose, as mixed fillers, (the two mass ratio is
1:2), organic solvent wet method dress post is successively the elution of 12%-92% aqueous methanol gradient, elution flow rate with percent by volume
For 150mL/min.According to wash water color by no discoloration it is coloured collect, until brown become it is light yellow collection terminate, will wash
It is component C after de- liquid concentration.
Embodiment 4
Gentianaceae Swertia plant --- the extracting method of Swertia przewalskii Pissjauk is the same as embodiment 3.
Embodiment 5
Taking Gentianaceae Swertia plant --- the drying herb 500g of Swertia erythrosticta Maxim is crushed, and the ethyl alcohol with 95% is molten
Liquid is mixed by solid-liquid mass ratio 1:12, and refluxing extraction 7 times, 4 hours every time, filtering, merging filtrate simultaneously recycled ethanol reagent.It will filter
Liquid, which is placed in Rotary Evaporators, be concentrated obtained medicinal extract, and the water that 3 times of volume ratios of medicinal extract are added is uniformly mixed；Successively with 4 times of bodies
Long-pending petroleum ether, the ethyl acetate of 5 times of volumes, extracting n-butyl alcohol 5 times of 6 times of volumes；Petroleum ether is mutually directly concentrated as component
A；It using the medicinal extract of ethyl acetate phase as raw material, is mixed with methanol by 1g:10mL, to being completely dissolved, heat filtering must be filtered heating stirring
Liquid has by filtrate using macroreticular resin/polyamide, macroreticular resin/microcrystalline cellulose as mixed fillers (the two mass ratio is 2:1)
Solvent wet method dress post is successively eluted with 18%-98% aqueous methanol gradient, elution flow rate 100mL/min.According to elution
Liquid color by no discoloration it is coloured collect, until brown become it is light yellow collection terminate, 200-300 is used after eluent is concentrated again
Mesh column layer chromatography silicone rubber carries out column chromatography, and with petroleum ether: ethyl acetate 1:1 rushes column, collects eluent, is concentrated to get the component
B；N-butanol phase medicinal extract will finally be mixed with methanol by 1g:10mL, for heating stirring to being completely dissolved, heat filtering obtains filtrate, will
Filtrate is using macroreticular resin/polyamide, macroreticular resin/microcrystalline cellulose as mixed fillers (the two mass ratio is 3:1), organic solvent
Wet method dress post is successively eluted with 18%-98% aqueous methanol gradient, elution flow rate 150mL/min.According to wash water color
By no discoloration it is coloured collect, until brown become it is light yellow collection terminate, after eluent is concentrated be component C.
The quality list of three kinds of components obtained by 1. 5 kinds of Gentianaceae Swertia plant extracts of table
| Mussot swertia herb | Swertia franchetiana H.Smith | Tibet Herba Swertiae bimaculatae | Swertia przewalskii Pissjauk | Swertia erythrosticta Maxim | |
| Component A | 185mg | 156mg | 125mg | 130mg | 116mg |
| Component B | 90mg | 76mg | 68mg | 72mg | 55mg |
| Component C | 220mg | 209mg | 185mg | 196mg | 167mg |
Embodiment 6: inhibiting effect of the composition and Lamivudine of various ratios to hepatitis type B virus
1.HepG2.2.15 cell culture
Cell culture fluid is MEM culture solution, and every 100mL contains fetal calf serum 10%, glutamine 0.03%, G418380 μ g/
ML, 50 μ g/mL of kanamycins.Cell culture processes: add 0.25% pancreatin in the culture bottle for covering with 2.2.15 cell, 37 DEG C disappear
Change 10 minutes, culture solution is added to dispel, 1:3 passage is covered with for 10 days.
2. the cell toxicity test of drug
With the cytotoxicity of mtt assay measurement drug, experiment is divided into the composition group (shown in table 2) and positive drug of various ratios
Lamivudine group.Cell dissociation, is configured to every milliliter of 200,000 cells, inoculated and cultured plate, 100 μ L of the every hole of 96 orifice plates, and 37 DEG C 5%
CO2 is cultivated 24 hours, and cell is tested after growing up to single layer.Experiment repeats 2 batches.The composition of various ratios is prepared with culture solution
2 times of medical fluid dilutions.First experiment be made into 1000 μ g/mL, 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 62.5 μ g/mL,
31.25 μ g/mL and 15.61 μ g/mL.Second batch experiment increases 1 times, since 2000 μ g/mL, is made into 2000 μ g/mL, 1000 μ
G/mL, 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 62.5 μ g/mL and 31.25 μ g/mL, control drug Lamivudine is from 2000 μ
G/mL starts, totally 7 dilutions, every 4 hole of concentration, and 96 porocyte culture plates are added, change same concentration liquid within every 4 days, is falling after 8 days
Set microscopically observation cytopathy.Median toxic concentration (CC is calculated by Reed-Muench method50) and maximal non-toxic concentration
(CC0)。CC50=Antilog [A+ (50-B/C-B) × D], wherein A is the drug concentration of the cumulative inhibiting rate of log < 50%, and B is
The cumulative inhibiting rate of log<50%, the cumulative inhibiting rate of C log>50%, D are log extension rate.
Table 3 shows that the composition for the various ratios that 5 kinds of plant extracts obtain is lower than sun to the toxicity of HepG2.2.15 cell
Property comparison medicine Lamivudine.In table 3, CC50And CC0For the mean value of two batches test.
The composition laboratory sample list of the various ratios of table 2
| Sample number | The composition of various ratios |
| Sample 1 | The resulting mussot swertia herb component A of embodiment 1 is 50 parts, component B is 1 part, component C is 1 part |
| Sample 2 | The resulting mussot swertia herb component A of embodiment 1 is 25 parts, component B is 1 part, component C is 1 part |
| Sample 3 | The resulting Swertia franchetiana H.Smith component A of embodiment 2 is 1 part, component B is 50 parts, component C is 1 part |
| Sample 4 | The resulting Tibet Herba Swertiae bimaculatae component A of embodiment 3 is 1 part, component B is 25 parts, component C is 1 part |
| Sample 5 | The resulting Swertia przewalskii Pissjauk component A of embodiment 4 is 1 part, component B is 1 part, component C is 50 parts |
| Sample 6 | The resulting Swertia erythrosticta Maxim component A of embodiment 5 is 1 part, component B is 1 part, component C is 25 parts |
| Sample 7 | The resulting mussot swertia herb component A of embodiment 1 is 1 part, component B is 1 part, component C is 1 part |
| Sample 8 | The mussot swertia herb component A of the resulting one-component of embodiment 1 |
| Sample 9 | The Swertia franchetiana H.Smith component B of the resulting one-component of embodiment 2 |
| Sample 10 | The Tibet Herba Swertiae bimaculatae component C of the resulting one-component of embodiment 3 |
| Sample 11 | The resulting mussot swertia herb component B of embodiment 1 is 1 part, and component C is 1 part |
| Sample 12 | The resulting mussot swertia herb component A of embodiment 1 is 1 part, and component C is 1 part |
| Sample 13 | The resulting mussot swertia herb component A of embodiment 1 is 1 part, and component B is 1 part |
Cytotoxic effect of the composition and Lamivudine of the various ratios of table 3 to HepG2.2.15
| Sample number | CC50(μg/mL) | CC0(μg/mL) |
| Sample 1 | 1100±7.21 | 503±6.32 |
| Sample 2 | 1098±6.25 | 505±5.58 |
| Sample 3 | 1143±5.56 | 501±4.76 |
| Sample 4 | 1123±6.43 | 506±5.02 |
| Sample 5 | 1100±4.29 | 502±3.98 |
| Sample 6 | 1104±6.73 | 500±4.72 |
| Sample 7 | 1108±7.01 | 503±5.01 |
| Sample 8 | 1000±7.05 | 510±5.24 |
| Sample 9 | 1004±6.86 | 508±4.65 |
| Sample 10 | 1001±5.43 | 505±3.97 |
| Sample 11 | 1000±5.78 | 500±4.56 |
| Sample 12 | 1004±6.69 | 495±3.33 |
| Sample 13 | 1005±6.50 | 490±3.79 |
| Lamivudine | 1000±7.55 | 487±4.72 |
Embodiment 7: effect of the various compositions to 2.2.15 cell culture HBsAg and HBeAg
The composition group and positive drug lamivudine group of the various ratios of experiment point.2.2.15 cells/ml 200,000 inoculations
24 porocyte culture plates, every hole 1mL, 37 DEG C of 5%CO2 are cultivated 24 hours, 2 below the composition maximal non-toxic concentration of various ratios
It dilutes again, 5 dilutions, test repeats three batches.Comparison medicine Lamivudine dilutes for 3 times below 1/5 maximal non-toxic concentration, and 5
Dilution: 100 μ g/mL, 33.33 μ g/mL, 11.11 μ g/m, 3.70 μ g/mL and 1.23 μ g/mL, test repeat two batches.Every concentration
Two holes.37 DEG C of 5%CO2Culture.Change within every 4 days original content medical fluid culture, the 8th day collection culture solution, -20 DEG C of stored frozens.Simultaneously
HBsAg and HBeAg OD value is measured by enzyme linked immunological kit method microplate reader.Effect of drugs calculates: it is effectively dense to calculate half
Spend (IC50) and selection index.
(1) Drug inhibition medium effective concentration (IC is calculated50): IC50=Antilog [A+ (50-B/C-B) × D],
Middle A is the drug concentration of the cumulative inhibiting rate of log<50%, and B is the cumulative inhibiting rate of log<50%, the cumulative suppression of C log>50%
Rate processed, D are log extension rate.
(2) the selection index (SI) of HBsAg and HBeAg is calculated according to the following formula in 2.2.15 cell culture.
SI=CC 50/IC50, the results are shown in Table 4.Table 4 shows the anti-hepatitis B activity of composition than one-component and double groups
Part is all good.In table 4, IC50It is mean value with SI.
The inhibiting effect of the various compositions of table 4 and Lamivudine to HBeAg, HBsAg
Embodiment 8: the effect of the anti-duck hepatitis B virus of the composition of various ratios
Nanjing sheldrake DHBV-DNA positive duck blood is injected intravenously through leg shin into an age in days according to the dosage of 0.2mL/ only clearly
In Beijing duck body, infection took blood after 7 days, the content of DHBV-DNA in Detection and Extraction serum.Serum detected it is positive in DHBV after,
Duckling is randomly divided into 5 groups, i.e., virus control group, Lamivudine (50mg/kg) and treatment group 1-3 (100mg/kg, 50mg/kg,
25mg/kg), every group 6.Oral administration, two times a day；Virus control group gives the physiological saline of same volume.Successive administration or life
Reason salt water 10 days upon administration the 5th day, the 10th day and the 15th day after being discontinued respectively, through duck shin venous blood sampling, and separates on the 30th day
Serum does the dot hybridization of serum according to the method for specification in kit, measures OD value, and it is close to calculate serum DHBV-DNA light
Degree.It the results are shown in Table 5.The results show that being all positive after virus control group duckling infection serum DHBV-DNA, test in overall process
The level of serum DHBV-DNA significantly rises.Compared with before self administration, treatment group 1-3 duck the 10th day upon administration, after drug withdrawal
15th day, it can reduce within the 30th day the level of duckling infection serum DHBV-DNA.Positive drug can be shown on the the 5th, 10 day upon administration
Landing reduces the level of DHBV-DNA in serum, but when being discontinued 15 days, 30 days, the level of DHBV-DNA has highly significant in serum
Rising, have " rebound phenomenon ".The result shows that the composition that the extract of 5 kinds of plants is prepared can inhibit after drug withdrawal
The knock-on of DHBV-DNA level.
The effect of the various compositions of table 5 and the anti-duck hepatitis B virus of Lamivudine
Claims (4)
1. a kind of composition of anti-hepatitis B virus, which is characterized in that the raw medicinal material for preparing the composition is Gentianaceae
Swertia plant；Prepare the composition the preparation method comprises the following steps: takes the raw medicinal material first, adds ethanol solution to extract, mistake
It filters, after merging filtrate, is concentrated to get medicinal extract；Gained medicinal extract is taken, is added water and mixed；Successively use petroleum ether, ethyl acetate, n-butanol
Extraction；Gained petroleum ether is mutually directly concentrated to get component A；Gained ethyl acetate phase is first eluted through macroreticular resin, eluent concentration
Column chromatography is carried out with silicagel column afterwards, eluent is collected, is concentrated to get component B；Gained n-butanol is dense after mutually being eluted with macroreticular resin
Contracting obtains component C；By three of the above component according to component A be 1-50 parts, component B is 1-50 parts, component C is 1-50 parts
Mixing obtains the composition；
The method that the raw medicinal material is concentrated to get medicinal extract is: taking 1 part of the raw medicinal material agents area, crushes, is added 70%
~ 95% 5-12 parts of ethanol solution mixing, refluxing extraction filter, and after merging filtrate, are concentrated to get medicinal extract；
It is to the method that medicinal extract is extracted is concentrated to get: takes gained medicinal extract, the water that 2 ~ 3 times of volume ratios are added mixes；Successively use
3 ~ 4 times of volume ratio petroleum ethers, 4 ~ 5 times of volume ratio ethyl acetate, 4 ~ 6 times of volume ratio extracting n-butyl alcohols；
The processing method of the ethyl acetate phase be the ethyl acetate phase is dissolved with methanol, then with 1-2 parts of macroreticular resins/
Polyamide, 1-3 parts of macroreticular resin/microcrystalline celluloses are that the mixed fillers of ratio fill column, and successively use 10%-98% methanol
Aqueous solution gradient elution, according to wash water color by no discoloration it is coloured collect, until brown become it is light yellow collection terminate, will
Use 200-300 mesh silicagel column again after eluent concentration, petroleum ether: ethyl acetate=1:1 elution is collected eluent, is concentrated to give
To the component B；
The processing method of the n-butanol phase is mutually to dissolve the n-butanol with methanol, with 1-3 parts of macroreticular resin/polyamides
Amine, 1-2 parts of macroreticular resin/microcrystalline celluloses are that the mixed fillers of ratio fill column, and successively use 12%-98% methanol aqueous solution
Gradient elution, according to wash water color by no discoloration it is coloured collect, until brown become it is light yellow collection terminate, by eluent
Concentration, obtains the component C.
2. the composition as described in claim 1, which is characterized in that the Gentianaceae Swertia plant is mussot swertia herb
(Swertia mussotii Franch.), Swertia franchetiana H.Smith (Swertia franchetiana H. Smith), Tibet Herba Swertiae bimaculatae
(Swertia racemosa (Griseb.) Wall.ex C.B.Clarke), Swertia przewalskii Pissjauk (Swertia
Przewalskii Pissjauk.), one of Swertia erythrosticta Maxim (Swertia erythrosticta Maxim.).
3. a kind of method for preparing the composition as described in claim 1 or 2, which is characterized in that take the raw material first
Medicinal material adds ethanol solution to extract, filtering, after merging filtrate, is concentrated to get medicinal extract；Gained medicinal extract is taken, is added water and mixed；Successively use
Petroleum ether, ethyl acetate, extracting n-butyl alcohol；Gained petroleum ether is mutually directly concentrated to get the component A；Gained ethyl acetate phase
It is first eluted through macroreticular resin, carries out column chromatography with silicagel column after eluent concentration, collect eluent, be concentrated to get the component B
；Gained n-butanol is concentrated to get the component C after mutually being eluted with macroreticular resin；It is according to component A by three of the above component
1-50 parts, component B be 1-50 parts, component C is that 1-50 parts of mixing obtain the composition.
4. a kind of composition as described in any in claim 1-2 is in the application prepared in anti-hepatic-B virus medicine.
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| CN104370871A (en) * | 2013-08-12 | 2015-02-25 | 北京大学 | Xanthine separated from swertia punicea and application thereof in inhibiting hepatitis B virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104370871A (en) * | 2013-08-12 | 2015-02-25 | 北京大学 | Xanthine separated from swertia punicea and application thereof in inhibiting hepatitis B virus |
Non-Patent Citations (1)
| Title |
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| 紫红獐牙菜醋酸乙酯部位体外抗乙型肝炎病毒作用的研究;田峦鸢等;《湖北中医学院学报》;20060331;第8卷(第1期);第5-7页，尤其是第6页左栏第1段和4讨论 * |
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