CN1515277A - Method for extractings ubstance resisting respiratory syncytial virus from patrinia - Google Patents
Method for extractings ubstance resisting respiratory syncytial virus from patrinia Download PDFInfo
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- CN1515277A CN1515277A CNA031001823A CN03100182A CN1515277A CN 1515277 A CN1515277 A CN 1515277A CN A031001823 A CNA031001823 A CN A031001823A CN 03100182 A CN03100182 A CN 03100182A CN 1515277 A CN1515277 A CN 1515277A
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- respiratory syncytial
- herba patriniae
- syncytial virus
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Abstract
The present invention relates to a method for extracting substance for resisting respiratory syncytial virus from Chinese herbal medicine patrinia. The method includes the following steps: using ethyl alcohol method to preparing ethyl alcohol precipitate, slowly adding 95% ethyl alcohol into concentrated extract (1g/ml) and continuously stirring until the ethyl alcohol volume is reached to 20%-90%, then placing it for 12 hr. at 4 deg.C, then centrifugalizing for 10 min, in 3500 rpm, filtering and cottecting precipitate, using macroporous adsorption resin column chromatography to prepare patrinia eluent for resisting respiratory syncytial cirus, adding water and dissolving ethyl alcohol precipitate, adding it on the pretreated macroporous adsorption resin column top end, using water to make elution, decompression concentrating eluent, drying so as to obtain the invented product.
Description
Technical field: the extracting method that the present invention relates to Chinese herbal medicine Herba Patriniae antiviral substance.
Background technology: (respiratory syncytial virus RSV) as one of important cause of disease of global range children virus respiratory tract infection, accounts for critical positions to respiratory syncytial virus in respiratory tract infection.Rsv infection easily develops into serious lower respiratory infection in infant below 1 years old, also can bring out asthma, even causes death, and is bigger to infant harm.In recent years find that RSV is the important cause of disease that adult immunosuppressant person infects, also can be popular in the old people, still there are not gratifying prevention and treatment way at present.The respiratory syncytial virus vaccines development just began as far back as the sixties in last century, but did not still have safe and effective person at present.Reason is the 1. intravital female RSV of the biography antibody of baby, can reduce the immunogenicity of vaccine; 2. the immunity of organism that excites owing to the RSV natural infection is incomplete immunity, so the RSV vaccine needs repeatedly inoculation; 3. RSV exists two hypotypes of A, B, and two hypotype cross-neutralization activity are low, so the RSV of two hypotypes of RSV vaccine reply has protective effect.Virazole (ribavirin) obtains Food and Drug Administration (FDA) approval the earliest and is used for the treatment of the serious symptom respiratory syncytial virus infection.But because its toxicity is bigger, the medical expense costliness is used inconvenience, and the clinical practice of virazole is very restricted.The new drug research of anti respiratory syncytial virus is a lot, comprises monoclonal antibody, interferon, antisense oligonucleotide etc.Although prospect is relatively more optimistic, also do not have in the short time to enter clinical medicine.
Summary of the invention: for solving the problem that prior art exists, the invention provides the extracting method of anti respiratory syncytial virus material in a kind of Herba Patriniae, it is simple that it should have extraction process, the high characteristics of anti respiratory syncytial virus material purity of extraction.It also should have the effect that utilizes cell culture processes to detect and confirm Herba Patriniae antiviral active substance inhibition respiratory syncytial virus, for developing new antiviral two class Chinese medicines and further studying Herba Patriniae antiviral effective ingredient, the characteristics of scientific basis are provided, the present invention at first prepares the water extract of Herba Patriniae, with Herba Patriniae (Herba patriniae) 1kg, clean five times with tap water, distilled water cleans three times, 7000ml distilled water immersion 12 hours, filter with absorbent cotton after boiling 90 minutes in 100 ℃, medicinal residues add the 5000ml distilled water and boiled 90 minutes in 100 ℃, filter with absorbent cotton, twice filtrate is mixed, mix filtrate decompression and concentrate, being concentrated to concentration is 1g/ml (every ml contains crude drug 1 gram).Water alcohol legal system is equipped with the ethanol precipitation thing then, slowly adds 95% ethanol in spissated water extract (1g/ml), and constantly stirs, after the ethanol volume reaches 20%-90%, under 4 ℃ of temperature, placed 12 hours, in 3500 rev/mins centrifugal 10 minutes, filter collecting precipitation.To precipitate with the dissolving of an amount of distilled water after filter paper filtering, filtrate concentrates through rotary evaporator, drying, powder.Also available macroporous adsorbent resin column chromatography legal system is equipped with the eluate of Herba Patriniae anti respiratory syncytial virus, after the ethanol precipitation thing is dissolved in water, is added in the good macroporous adsorptive resins top of pretreatment, the water eluting, and the eluent concentrating under reduced pressure, drying gets eluate A.
Check Herba Patriniae ethanol precipitation thing adopts the medicine contrast experiment: Herba Patriniae ethanol precipitation thing to the inhibitory action of respiratory syncytial virus (RSV).The positive control medicine: virazole (Ribavirin) injection, content is 100mg/ml, lot number 000302, Yantai No.2 Pharmaceutical Factory produces.HeLa cell Hela is provided by Virology Inst., Chinese Academy of Preventive Medical Science.The conventional method cultivation of going down to posterity.Respiratory syncytial virus (RSV) Long strain is provided by Virology Inst., Chinese Academy of Preventive Medical Science.In the Hela cell, organize median infective dose (TCID
50) be 10
-7.7
Toxicity trial:, add cell respectively by constant and grown up in the % well culture plate of monolayer the continuous 0.8 times of dilution of medicinal liquid with cell maintenance medium, place 37 ℃, 5%CO
2Incubator in cultivate, observation of cell form under the every day inverted microscope was observed 3 days continuously.Each dosage adds four porocytes, sets up four hole normal cell contrasts simultaneously.Record medicine maximal non-toxic dosage (TC
0).
Medicine is to the inhibiting test of respiratory syncytial virus: adopt cytopathy inhibition test (CPEI) that Herba Patriniae ethanol precipitation thing is carried out pharmacodynamics and detect.Continuous ten times of dilutions of respiratory syncytial virus are added the cell that has grown up to monolayer, place 37 ℃, 5%CO
2Incubator in hatch and add medicine (drug level according to maximal non-toxic dosage determine) after 2 hours, place 37 ℃, 5%CO again
2Incubator in cultivate.Every day, the observation of cell form was observed 3 days continuously, record cytopathy (CPE) degree.The CPE degree: the no obvious CPE of 0 expression, 1 expression CPE are that 0%～25%, 2 expression CPE are that 25%～50%, 3 expression CPE are that 50%～75%, 4 expression CPE are 75%～100%.Set up normal cell contrast, virus control and each four hole of positive drug contrast simultaneously.
Statistical method: Kruskal-Wallis method of inspection comparative drug is to the inhibitory action and the virus control cytopathy degree of different titers respiratory syncytial virus.
The drug toxicity measurement result: according to the toxicity trial result, Herba Patriniae ethanol precipitation thing TC
0Be 0.3125mg/ml; Virazole TC
0Be 11.52ug/ml.
Herba Patriniae ethanol precipitation thing sees Table 1 to the inhibiting result of the test of respiratory syncytial virus.
The effect of table 1 Herba Patriniae ethanol precipitation thing anti respiratory syncytial virus
| Virus titer ethanol precipitation thing virazole virus control (0.3125mg/ml) (10 μ g/ml) |
| ??10 -6???????3231?????????4444???????4444 |
| ??10 -7??????1222??????????2343???????4343 |
| ??10 -8??????1112??????????1211???????4333 |
Compare inhibitory action and the virus control cytopathy degree of Herba Patriniae precipitate with the Kruskal-Wallis method of inspection to the different titers respiratory syncytial virus.The result shows that virus titer is 10
-6, 10
-7, 10
-8The time, the Herba Patriniae precipitate all has obvious suppression effect (P＜0.01) to respiratory syncytial virus.Illustrate that the Herba Patriniae precipitate has the effect of obvious suppression respiratory syncytial virus.Check the inhibitory action of Herba Patriniae antiviral active substance (eluate A): experiment medicine: eluate A (Herba Patriniae anti respiratory syncytial virus active substance) to respiratory syncytial virus.The positive control medicine: virazole (Ribavirin) injection, content is 100mg/ml, lot number 000302 Yantai No.2 Pharmaceutical Factory produces.HeLa cell Hela is provided by Virology Inst., Chinese Academy of Preventive Medical Science.The conventional method cultivation of going down to posterity.Respiratory syncytial virus (RSV) Long strain is provided by Virology Inst., Chinese Academy of Preventive Medical Science.In the Hela cell, organize median infective dose (TCID
50) be 10
-7.7
Toxicity trial:, add cell respectively by constant and grown up in the % well culture plate of monolayer the continuous 0.8 times of dilution of medicinal liquid with cell maintenance medium, place 37 ℃, 5%CO
2Incubator in cultivate, observation of cell form under the every day inverted microscope was observed 3 days continuously.Each dosage adds four porocytes, sets up four hole normal cell contrasts simultaneously.Record medicine maximal non-toxic dosage (TC
0), calculate medicine median toxic concentration (TC
50).
Medicine is tested the inhibitory action of respiratory syncytial virus: adopt cytopathy inhibition test (CPEI) that medicine is carried out pharmacodynamics and detect.The cell inoculation dosage that has grown up to monolayer is 50TCID
50Respiratory syncytial virus, place 37 ℃, 5%CO
2Incubator in hatched 2 hours.Discard then and contain virus and keep liquid, add and contain the liquid of keeping that variable concentrations contains medicine, place 37 ℃, 5%CO
2Incubator in cultivate.Every day, the observation of cell form was observed 3 days continuously, record CPE degree.The CPE degree: the no obvious CPE of 0 expression, 1 expression CPE are that 0%～25%, 2 expression CPE are that 25%～50%, 3 expression CPE are that 50%～75%, 4 expression CPE are 75%～100%.Set up normal cell contrast, virus control and each four hole of positive drug contrast simultaneously.Calculate medicine median effective dose (EC
50) and therapeutic index (TI).
The influence that the different dosing time suppresses virus: grown up in the cell of monolayer and added respiratory syncytial virus, infective dose is 50 TCID
50, put 4 ℃ 1 hour, discard viral liquid, in 0 hour, 2 hours, 4 hours, 6 hours, added respectively with concentration eluate A and virazole in 8 hours, cultivate to observe record CPE degree.
Statistical method: the Reed-Muench method is calculated medicine median toxic concentration (TC
50) and medium effective concentration (EC
50), therapeutic index (TI)=median toxic concentration (TC
50)/medium effective concentration (EC
50); The population distribution of each dosage group of Kruskal-Wallis method of inspection comparative drug and virus control group cytopathy degree and different dosing time are respectively organized the population distribution of medicine and virus control group cytopathy degree.Date processing utilization SAS software is finished.
The Herba Patriniae antiviral substance sees Table 2 to the inhibiting result of the test of respiratory syncytial virus.
Each dosage group of Kruskal-Wallis method of inspection comparative drug and virus control group cytopathy degree.The result shows, Herba Patriniae water extract 5,2.5,1.25,0.625mg/ml dosage group, Herba Patriniae eluate A 2,1,0.5,0.25mg/ml dosage group, virazole 10,5,2.5ug/ml dosage group all have obvious inhibitory action (P＜0.01) to respiratory syncytial virus.
Adopt the Reed-Muench method to calculate Herba Patriniae water extract median toxic concentration (TC
50) be 32mg/ml, the EC of Herba Patriniae water extract
50For 125mg/ml, therapeutic index (TI) are 25.6; Herba Patriniae eluate A median toxic concentration (TC
50) be 11.52mg/ml, medium effective concentration (EC
50) be 0.0986mg/ml, therapeutic index (TI) is 116.84; Virazole median toxic concentration (TC
50) be 76.43ug/ml, medium effective concentration (EC
50) be 1.43 μ g/ml, therapeutic index (TI) is 53.45.
Table 2 Herba Patriniae water extract and eluate A are to the inhibitory action of respiratory syncytial virus
The average CPE inhibition of every hole CPE accumulation CPE suppression ratio divides into groups
CPE degree CPE suppresses CPE (%)
Herba Patriniae 5 1,011 0.75 3.25 14.50 3.25 81.69
Water extract 2.5 1,121 1.25 2.75 11.25 6.00 65.22
mg/ml?????1.25????1212????1.50????2.50???????8.50??????8.50????50.00
0.625???2112????1.50????2.50???????6.00??????14.50???29.27
0.3125???3223???2.50????1.50????3.50????18.00???16.28
0.156????2332???2.50????1.50????2.00????20.00???9.09
0.078????3434???3.50????0.50????0.50????20.50???2.38
Herba Patriniae 2 0,000 0.00 4.00 19.25 0.00 100.00
Eluate 1 1,010 0.50 3.50 15.25 0.50 96.82
A????????0.5??????1100???0.50????3.50????11.75???1.00????92.16
mg/ml??????0.25?????1110???0.75????3.25????8.25????1.75????82.50
0.125????3211???1.75????2.25????5.00????3.50????58.82
0.0625???1322???2.00????2.00????2.75????5.50????33.33
0.0312???4343???3.50????0.50????0.75????9.00????7.69
0.0162???4344???3.75????0.25????0.25????12.75???1.92
10???????0000???0.00????4.00????13.50???0.00????100.00
Sick 5 1,010 0.50 3.50 9.50 0.50 95.00
Poison 2.5 1,221 1.50 2.50 6.00 2.00 75.00
Azoles 1.25 1,323 2.25 1.75 3.50 4.25 45.16
μg/ml?????0.625????3233???2.75????1.25????1.75????7.00????20.00
0.3125???4433???3.50????0.50????0.50????10.50???4.54
Normal cell 0,000 0 4.00
Virus control 4,434 3.75 0.25
The influence that the different dosing time suppresses virus:
The result of the test of Herba Patriniae eluate A different dosing time to viral inhibition sees Table 3.
The Kruskal-Wallis method of inspection comparison different dosing time is respectively organized medicine and virus control group cytopathy degree.The result shows, infects after the respiratory syncytial virus 0 hour, 2 hours, 4 hours, adds eluate A in 6 hours, and the effect (P＜0.01) that suppresses respiratory syncytial virus is arranged; Infect back 0 hour, 2 hours, 4 hours adding virazoles, the effect (P＜0.01) that suppresses respiratory syncytial virus is all arranged.
The table 3 Herba Patriniae eluate A different dosing time influences medicine grouping CPE pathological changes medicine grouping CPE pathological changes to viral inhibition
The degree degree
0h???????0000??????????????????????0h??????????0000
Eluate A 2h 0000 malicious azoles 2h 1101
(2mg/ml)????4h???????1111???????(10ug/ml)??????4h??????????1321
6h???????1223??????????????????????6h??????????2334
8h???????4334??????????????????????8h??????????4443
Virus control 4444
It is the chemical extraction method of guiding that the present invention adopts biological activity, extracts anti respiratory syncytial virus material (eluate A) from the Chinese herbal medicine Herba Patriniae.Eluate A, the Molish reacting positive is confirmed as polysaccharose substance.
Use the cell in vitro culture method, surveyed the inhibitory action of Herba Patriniae antiviral substance respiratory syncytial virus with the positive contrast medicine inspection of virazole.Result of the test shows that the Herba Patriniae antiviral substance has the effect of tangible anti respiratory syncytial virus.
Median toxic concentration (the TC of Herba Patriniae antiviral substance
50) be 11.52mg/ml, medium effective concentration (EC
50) be 0.0986mg/ml, therapeutic index (TI) is 116.84.
Virazole (positive control drug) median toxic concentration (TC
50) be 76.43ug/ml, medium effective concentration (EC
50) be 1.43 μ g/ml, therapeutic index (TI) is 53.45.
The present invention also learns, add eluate A in infective virus 0-6 hour, all can obviously suppress the respiratory syncytial virus increment, and delay the appearance of CPE, according to the supposition of virus replication cycle, eluate A can act on the rna replicon process of respiratory syncytial virus and bring into play its antiviral activity.
Simultaneously, also detected Herba Patriniae water extract, Herba Patriniae ethanol precipitation thing inhibitory action to respiratory syncytial virus.Result of the test shows that Herba Patriniae water extract, Herba Patriniae ethanol precipitation thing also have the effect of tangible anti respiratory syncytial virus.
Chemical extraction method of the present invention has adopted water extraction method, has used water alcohol method and macroporous adsorbent resin column chromatography method.In the extract drugs process, avoid using, but the method for employing ethanol precipitation has reduced the pollution of noxious substance in the course of processing, improves the safety of drug use to the deleterious chemical reagent of human body.The eluate antivirus test result of macroporous adsorbent resin column chromatography shows that water elution partly has the effect of tangible anti respiratory syncytial virus.Macroporous adsorbent resin column chromatography separates the Herba Patriniae antiviral substance, and as eluant, this just makes production process simple with water, and production cost descends significantly.It is simple to have extraction process, the high advantage of extracting of anti respiratory syncytial virus material purity, it also has the effect that utilizes cell culture processes to detect and confirm Herba Patriniae antiviral active substance inhibition respiratory syncytial virus, provides the advantage of scientific basis for developing new antiviral two class Chinese medicines and further research Herba Patriniae antiviral effective ingredient.
The specific embodiment one: present embodiment at first prepares the water extract of Herba Patriniae, with Herba Patriniae (Herba patriniae) 1kg, clean five times with tap water, distilled water cleans three times, 7000ml distilled water immersion 12 hours, filter with absorbent cotton after boiling 90 minutes in 100 ℃, medicinal residues add the 5000ml distilled water and boiled 90 minutes in 100C, filter with absorbent cotton, and twice filtrate is mixed, mix filtrate decompression and concentrate, being concentrated to concentration is 1g/ml (every ml contains crude drug 1 gram).Water alcohol legal system is equipped with the ethanol precipitation thing then, slowly adds 95% ethanol in spissated water extract (1g/ml), and constantly stirs, after the ethanol volume reaches 20%～90%, under 4 ℃ of temperature, placed 12 hours, in 3500 rev/mins centrifugal 10 minutes, filter collecting precipitation.To precipitate with the dissolving of an amount of distilled water after filter paper filtering, filtrate concentrates through rotary evaporator, drying, powder.Also available macroporous adsorbent resin column chromatography legal system is equipped with the eluate of Herba Patriniae anti respiratory syncytial virus, after the ethanol precipitation thing is dissolved in water, is added in the good macroporous adsorptive resins top of pretreatment, the water eluting, and the eluent concentrating under reduced pressure, drying gets eluate A.
The specific embodiment two: in the present embodiment, alcoholic acid volume is 20%～34%.
The specific embodiment three: in the present embodiment, alcoholic acid volume is 35%～48%.
The specific embodiment four: in the present embodiment, alcoholic acid volume is 49%～62%.
The specific embodiment five: in the present embodiment, alcoholic acid volume is 63%～76%.
The specific embodiment six: in the present embodiment, alcoholic acid volume is 77%～90%.
Claims (7)
1, the extracting method of anti respiratory syncytial virus material in the Herba Patriniae, it at first prepares the water extract of Herba Patriniae, with Herba Patriniae (Herba patriniae) 1kg, clean five times with tap water, distilled water cleans three times, 7000ml distilled water immersion 12 hours, filter with absorbent cotton after boiling 90 minutes in 100 ℃, medicinal residues add the 5000ml distilled water and boiled 90 minutes in 100 ℃, filter with absorbent cotton, twice filtrate is mixed, mix filtrate decompression and concentrate, being concentrated to concentration is 1g/ml, it is characterized in that preparing the ethanol precipitation thing with Herba Patriniae water extract, the ethanol of slow adding 95% in spissated water extract (1g/ml), and constantly stir, after the ethanol volume reaches 20%～90%, under 4 ℃ of temperature, placed 12 hours, in 3500 rev/mins centrifugal 10 minutes, filter collecting precipitation.To precipitate with distilled water dissolving after filter paper filtering, filtrate concentrates through rotary evaporator, drying, powder.
2, the extracting method of anti respiratory syncytial virus material in the Herba Patriniae according to claim 1, it is characterized in that being equipped with the eluate of Herba Patriniae anti respiratory syncytial virus with the macroporous adsorbent resin column chromatography legal system, after the ethanol precipitation thing is dissolved in water, be added in the good macroporous adsorptive resins top of pretreatment, the water eluting, the eluent concentrating under reduced pressure, drying gets eluate A.
3, the extracting method of anti respiratory syncytial virus material in the Herba Patriniae according to claim 1 is characterized in that alcoholic acid volume is 20%～34%.
4, the extracting method of anti respiratory syncytial virus material in the Herba Patriniae according to claim 1 is characterized in that alcoholic acid volume is 35%～48%.
5, the extracting method of anti respiratory syncytial virus material in the Herba Patriniae according to claim 1 is characterized in that alcoholic acid volume is 49%～62%.
6, the extracting method of anti respiratory syncytial virus material in the Herba Patriniae according to claim 1 is characterized in that alcoholic acid volume is 63%～76%.
7, the extracting method of anti respiratory syncytial virus material in the Herba Patriniae according to claim 1 is characterized in that alcoholic acid volume is 77%～90%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03100182 CN1233358C (en) | 2003-01-09 | 2003-01-09 | Method for extractings ubstance resisting respiratory syncytial virus from patrinia |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03100182 CN1233358C (en) | 2003-01-09 | 2003-01-09 | Method for extractings ubstance resisting respiratory syncytial virus from patrinia |
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| Publication Number | Publication Date |
|---|---|
| CN1515277A true CN1515277A (en) | 2004-07-28 |
| CN1233358C CN1233358C (en) | 2005-12-28 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168571B (en) * | 2007-11-28 | 2010-06-09 | 李鑫 | Method for exactly extracting antiviral field pennycress polysaccharide from field pennycress |
| CN103965368A (en) * | 2013-02-04 | 2014-08-06 | 陈朋 | Bitter herbs polysaccharide extraction method |
| CN115919920A (en) * | 2023-02-13 | 2023-04-07 | 四川大学 | A composition with antiviral effect |
-
2003
- 2003-01-09 CN CN 03100182 patent/CN1233358C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168571B (en) * | 2007-11-28 | 2010-06-09 | 李鑫 | Method for exactly extracting antiviral field pennycress polysaccharide from field pennycress |
| CN103965368A (en) * | 2013-02-04 | 2014-08-06 | 陈朋 | Bitter herbs polysaccharide extraction method |
| CN115919920A (en) * | 2023-02-13 | 2023-04-07 | 四川大学 | A composition with antiviral effect |
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| Publication number | Publication date |
|---|---|
| CN1233358C (en) | 2005-12-28 |
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