CN103816138B - The application of 1,2-Benzodiazepines on anti-avian influenza H5N1 virus in phellinus igniarius - Google Patents
The application of 1,2-Benzodiazepines on anti-avian influenza H5N1 virus in phellinus igniarius Download PDFInfo
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- CN103816138B CN103816138B CN201310404207.0A CN201310404207A CN103816138B CN 103816138 B CN103816138 B CN 103816138B CN 201310404207 A CN201310404207 A CN 201310404207A CN 103816138 B CN103816138 B CN 103816138B
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Abstract
The invention discloses a kind of from phellinus igniarius <i>Phellinus</iG reatT.GreaT.GT<i>? igniarius</i> (Lex? Fr) Quel(phellinus linteus? <i>phellinus</iG reatT.GreaT.GT<i>? linteus</i> (Berket? Curt) Teng, Phellinus baumii <i>Phellinus</iG reatT.GreaT.GT<i>? baumii</i>, breathe out base of a fruit phellinus? <i>Phellinus</iG reatT.GreaT.GT<i>? hartigii</i> (Alleschet? Schnabl) Imaz) fermentation liquid be separated in sporophore there is 1 of anti-avian influenza activity, 2-Benzodiazepines.The one that the present invention makes public for the first time 1,2-Benzodiazepines is newly active, proves that it has anti-avian influenza effect.The advantage that this method is applied compared with vague generalization compound is: found the new activity of this 1,2-special Benzodiazepines, had the effect of anti-avian influenza virus.
Description
Technical field
The present invention relates to the extraction preparation method of active substance in a kind of phellinus igniarius of anti-avian influenza H5N1 virus, the phellinus igniarius strain particularly related to, be deposited in No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on August 26th, 2013, Institute of Microorganism, Academia Sinica, " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; specific name: phellinus igniarius Phellinusigniarius, deposit number is CGMCCNo.8108.
Background technology
Benzodiazepines can volatilize together with steam, it is strong reductant, meet naked light can burn, by high heat give out poisonous, be corrosive abnormal smells from the patient, can cause through skin poisoning, can distil, have stimulation to nervus centralis, respiratory system, rat oral LD50 is 3890mg/kg, different from resorcinol and hydroquinone.
Benzodiazepines has the multiple important physiologically actives such as antibacterial, nowadays, does not see reporting for work of anti-avian influenza virus activity.
Summary of the invention
The invention discloses a kind of Benzodiazepines with anti-avian influenza activity be separated from medicinal fungus.The one that the present invention makes public for the first time this Benzodiazepines is newly active, proves that it has anti-avian influenza effect.
The advantage that this method is applied compared with vague generalization compound is: the new activity having found this kind of Benzodiazepines, has the effect of anti-avian influenza virus.
Technical scheme of the present invention is as follows:
First prepare phellinus igniarius crude extract, obtained by following method:
(1) fermentative medium formula in gram/100 milliliters be:
Corn starch 1-5% glucose 1-5%
Peptone 0.1-0.5% yeast extract 0.1-0.5%
Magnesium sulfate 0.1-0.5% potassium dihydrogen phosphate 0.01-0.05%
(2) be inoculated in conventional manner by phellinus igniarius strain and be equipped with in the conical flask of fluid medium, with 20 ~ 35 DEG C of temperature, shaking flask rotating speed is under 80 ~ 280r/min, pH3 ~ 8 conditions, vibrations cultivation 7 ~ 15 days; In cultivation when pH value drops to 2.5 ~ 4, seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, with temperature 20 ~ 35 DEG C, fermentation tank pressure 0.1 ~ 0.2 kg/cm, pH3 ~ 8, ventilation 0.5 ~ 1.1vvm, the condition that mixing speed is 100 ~ 280 revs/min, cultivate 7 ~ 15 days, the full liquid of phellinus igniarius mycelium fermentation can be utilized to prepare phellinus igniarius crude extract.
(3) get the full liquid of gained phellinus igniarius mycelium fermentation in step (2), by its concentrating under reduced pressure in a usual manner, make its volume concentration to 1/2 ~ 1/5 of original volume;
(4) by percent by volume be 60 ~ 95% ethanol concentrated to above-mentioned steps (3) after fermentation liquid extract, wherein, the amount adding ethanol is 2 ~ 8 times of concentrated solution volume, concentration of alcohol in extracting solution can be made to reach 60 ~ 90%, ethanol is preferably the edible ethanol of concentration 65-95%, and volume used is preferably the 3-6 of concentrated solution volume doubly;
(5) to step (4) gained extracting solution under 50 ~ 70 DEG C of conditions, heat 1 ~ 2 hour; Be separated in conventional manner, and remove impurity by cascade filtration, be separated and obtain ethanol extract; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume concentration to 1/5 ~ 1/10 of original volume;
(6) just the concentrated solution of step (5) gained carries out drying with the method for frozen drying, obtains phellinus igniarius crude extract.
Then from phellinus igniarius crude extract, 1 is prepared, 2-Benzodiazepines, step is as follows: and phellinus igniarius crude extract → purification on normal-phase silica gel chromatography → chloroform and methanol elution gradient → TLC detect → purification on normal-phase silica gel chromatography → chloroform methanol gel chromatography → TLC detects → and purification on normal-phase silica gel chromatography → TLC detects → evaporated under reduced pressure → colorless oil (1,2-Benzodiazepines).
Concrete grammar is:
(1) phellinus igniarius crude extract is prepared;
(2) precipitate of step (1) gained and purification on normal-phase silica gel are mixed stir, after drying, carry out silica normal phase column chromatography, use eluent 2-5 time, obtain eluent;
(3) by eluent evaporate to dryness last in step (2), equal-volume silica gel mixed sample, carries out purification on normal-phase silica gel chromatography, uses eluent;
(4) collect the eluent in step (3), concentrating under reduced pressure, use chloroform: methanol=1:1 dissolves, and carries out methanol gel column chromatography, use eluent, use TLC to detect the eluent collected, suitably merge eluent, drying under reduced pressure;
(5) product obtained in step (4) is carried out purification on normal-phase silica gel chromatography again, use eluent;
(6) collect the eluent that step (5) obtains, use TLC to detect each eluent, appropriateness merges eluent, and drying under reduced pressure, is 1,2-Benzodiazepines.
Preferably, the silica gel described in extraction step (2) is 100-200 order purification on normal-phase silica gel, and chromatographic silica gel is positive 200-300 order, and eluant is chloroform and/or methanol;
Silica gel consumption described in extraction step (3) is 1-3 times of volume, and eluant is petroleum ether and acetone;
Gel described in extraction step (4) is SephadexLH-20, and eluant is chloroform: methanol=1:1 ~ 1:3;
Chromatographic silica gel described in extraction step (5) is 200-300 order, and eluant is chloroform: methanol=100:1-50:1;
In extraction step (6), TLC detection point occurs speckle, and developing solvent is petroleum ether: acetone=30:1-35:1.
Then use the mensuration that 1, the 2-Benzodiazepines obtained carries out mdck cell toxicity, determine its median toxic concentration (CC
50) and maximum safe concentration;
Finally within the scope of maximum safe concentration, carry out sample effect to H5N1 strain in mdck cell level, the half toxic concentration (CC of calculation sample
50) and medium effective concentration (EC
50), obtain selection index (the SI)=CC of sample
50/ EC
50, calculate suppression ratio.
Significant advantage of the present invention: this method adopts multiple chromatographic technique to combine, can be exquisite clear and definite to structure, 1, the 2-Benzodiazepines that purity is greater than 95%.Mature technical route clearly, efficiently accurately, and demonstrates the activity that it has anti-avian influenza H5N1 virus first.
(4) detailed description of the invention
Example 1:
Phellinus igniarius crude extract, is obtained by following method:
(1) fermentative medium formula in gram/100 milliliters be:
Corn starch 1% glucose 1%
Peptone 0.1% yeast extract 0.1%
Magnesium sulfate 0.1% potassium dihydrogen phosphate 0.01%
(2) be inoculated in conventional manner by phellinus igniarius strain and be equipped with in the conical flask of fluid medium, with 25 DEG C of temperature, shaking flask rotating speed is under 110r/min, pH7 condition, vibrations cultivation 7 days; In cultivation when pH value drops to 3, seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, with temperature 25 DEG C, fermentation tank pressure 0.1 kg/cm, pH3, ventilation 0.5-1.1vvm, the condition that mixing speed is 100 revs/min, cultivate 7 days, the full liquid of phellinus igniarius mycelium fermentation can be utilized to prepare phellinus igniarius crude extract.
(3) get the full liquid of gained phellinus igniarius mycelium fermentation in step (2), by its concentrating under reduced pressure in a usual manner, make its volume concentration to 1/3 of original volume;
(4) by percent by volume be 70% ethanol concentrated to above-mentioned steps (3) after fermentation liquid extract, wherein, the amount adding ethanol is 5 times of concentrated solution volume, and concentration of alcohol in extracting solution can be made to reach 55%;
(5) to step (4) gained extracting solution under 70 DEG C of conditions, heat 1 hour; Be separated in conventional manner, and remove impurity by cascade filtration, be separated and obtain ethanol extract; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume concentration to 1/5 of original volume;
(6) just the concentrated solution of step (5) gained carries out drying with the method for frozen drying, obtains phellinus igniarius crude extract.
Above-mentioned crude extract is taken 200g and equal-volume 100 order purification on normal-phase silica gel to mix and stir, carry out silica normal phase column chromatography.Chromatography immobile phase is 200-300 order purification on normal-phase silica gel, post height 0.8m, diameter 15cm, and eluant is for being respectively chloroform, methanol, chloroform: methanol=100:1, respectively eluting 2,3 column volumes.And by gained eluent called after Fr-1, Fr-2 respectively.By Fr-2 equal-volume silica gel mixed sample, use silica gel to be 100 order purification on normal-phase silica gel, pentaploid amasss silica gel column chromatography, and the silica gel of use is 200-300 order purification on normal-phase silica gel.Eluant is petroleum ether and acetone.Concentrating under reduced pressure eluent, uses methanol: chloroform=1:1 dissolves, chloroform methanol gel filtration chromatography.Use TLC to detect the eluent collected suitably to merge, evaporated under reduced pressure, reuses purification on normal-phase silica gel chromatography, eluent is chloroform methanol, collects eluent, uses TLC to detect each eluent, developing solvent is petroleum ether: acetone=20:1, merges the eluent collected and occur speckle.Drying under reduced pressure, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analysis result is δ 8.78 (s, 1H), 6.68 (dt, J=40.7,20.4Hz, 1H), 6.58 (dd, J=5.7,3.5Hz, 1H), and proving is 1,2-Benzodiazepines.
(7) ergot sterone adopting step (6) to obtain carries out the mensuration to mdck cell toxicity, determines its median toxic concentration (CC
50) and maximum safe concentration.
On 96 orifice plates, 100 μ l/ holes add 4 × 10
5the mdck cell suspension of ml-1 concentration, after cultivating 24h, what cell monolayer added variable concentrations respectively contains sample maintenance medium, and often kind of concentration repeats 3 holes, and establishes normal cell controls.Put 37 DEG C, 5%CO
2after cultivating 48h in incubator, abandon culture fluid supernatant, 100 μ l/ holes add the maintenance medium of 5mg.ml-1MTT, after continuing to cultivate 1h, abandon MTT supernatant, every hole adds lysate (DMSO) 100 μ l, vibration 5-10min, to be crystallizedly dissolves completely, and microplate reader surveys the OD value at 492nm place.Calculate the median toxic concentration (CC of sample
50) and maximum safe concentration.As the OD of application of sample group
492value is not significantly lower than cell controls group OD
492time, this concentration is the maximum safe concentration of sample.
The impact of each concentration versus cell growth of table 1 compound
Known by table 1: the maximum safe concentration of 1,2-Benzodiazepines is and 25ug/mL.
Example 2:
Phellinus igniarius crude extract, is obtained by following method:
(1) fermentative medium formula in gram/100 milliliters be:
Corn starch 3% glucose 2%
Peptone 0.5% yeast extract 0.5%
Magnesium sulfate 0.5% potassium dihydrogen phosphate 0.05%
(2) be inoculated in conventional manner by phellinus igniarius strain and be equipped with in the conical flask of fluid medium, with 30 DEG C of temperature, shaking flask rotating speed is under 180r/min, pH6 condition, vibrations cultivation 15 days; In cultivation when pH value drops to 2.5, seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, with temperature 30 DEG C, fermentation tank pressure 0.2 kg/cm, pH3, ventilation 0.5-1.1vvm, the condition that mixing speed is 180 revs/min, cultivate 15 days, the full liquid of phellinus igniarius mycelium fermentation can be utilized to prepare phellinus igniarius crude extract.
(3) get the full liquid of gained phellinus igniarius mycelium fermentation in step (2), by its concentrating under reduced pressure in a usual manner, make its volume concentration to 1/5 of original volume;
(4) by percent by volume be 90% ethanol concentrated to above-mentioned steps (3) after fermentation liquid extract, wherein, the amount adding ethanol is 4 times of concentrated solution volume, and concentration of alcohol in extracting solution can be made to reach 70%;
(5) to step (4) gained extracting solution under 55 DEG C of conditions, heat 2.5 hours; Be separated in conventional manner, and remove impurity by cascade filtration, be separated and obtain ethanol extract; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume concentration to 1/10 of original volume;
(6) just the concentrated solution of step (5) gained carries out drying with the method for frozen drying, obtains phellinus igniarius crude extract.
Above-mentioned crude extract is taken 300g and equal-volume 100 order purification on normal-phase silica gel to mix and stir, carry out silica normal phase column chromatography.Chromatography immobile phase is 200-300 order purification on normal-phase silica gel, post height 1m, diameter 20cm, and eluant is for being respectively chloroform, and chloroform: methanol=100:1, distinguishes eluting 3,4 column volumes.And by gained eluent called after Fr-1, Fr-2 respectively.By Fr-2 equal-volume silica gel mixed sample, use silica gel to be 100 order purification on normal-phase silica gel, pentaploid amasss silica gel column chromatography, and the silica gel of use is 200-300 order purification on normal-phase silica gel.Eluant is petroleum ether and acetone.Concentrating under reduced pressure eluent, uses methanol: chloroform=1:1 dissolves, chloroform methanol gel filtration chromatography.Use TLC to detect the eluent collected suitably to merge, evaporated under reduced pressure, reuses purification on normal-phase silica gel chromatography, eluent is chloroform methanol, collects eluent, uses TLC to detect each eluent, developing solvent is petroleum ether: acetone=35:1, merges the eluent collected and occur speckle.Drying under reduced pressure, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analysis result is δ 8.78 (s, 1H), 6.68 (dt, J=40.7,20.4Hz, 1H), 6.58 (dd, J=5.7,3.5Hz, 1H), and proving is 1,2-Benzodiazepines.
(7) sample effect to H5N1 strain in mdck cell level is carried out, the half toxic concentration (CC of calculation sample within the scope of the maximum safe concentration adopting step (6) to obtain
50) and medium effective concentration (EC
50), obtain selection index (the SI)=CC of sample
50/ EC
50, calculate suppression ratio.
In test, arrange normal cell controls group (only adding cell maintenance medium), virus control group (only adding virus liquid), mass concentration is 2 times of dilutions, 4 mass concentrations in nontoxic scope, arrange 3 holes and repeat.Abandon 96 porocytes and cultivate supernatant in plate hole, 100 μ l/ holes add 10000TCID
50virus liquid (10-4.43), arranges 3 repetitions.37 DEG C of absorption 2h, abandon virus liquid supernatant, 100 μ l/ holes add the sample liquid of variable concentrations.Put 37 DEG C, 5%CO
2continue in incubator to cultivate 48h.Mtt assay is adopted to measure OD
492value, calculates medium effective concentration (EC
50).As the OD of application of sample group
492value is significantly greater than virus control group OD
492time, show that this sample liquid can significantly suppress viral infection MDCK, there is antiviral activity.
According to the cytopathy variability calculated and viral suppression.By the Probit Return Law of statistics software SPSS11.5, the half toxic concentration (CC of calculation sample
50) and medium effective concentration (EC
50), obtain selection index (the SI)=CC of sample
50/ EC
50.
According to lower formulae discovery viral suppression:
Viral suppression=(A-B)/(C-B) × 100%
A: sample liquid processed group OD value, B: virus control group OD value, C: cell controls group OD value
Table 2 compound is to the inhibitory action experimental result of H5N1 virus
Known by table 2: 1,2-Benzodiazepines can reach the suppression ratio of the highest 52.4% under 6.25ug/ml concentration to H5N1 strain, now CC
50for 66.239ug/ml, EC
50for 3.3767ug/ml, SI are 19.62.
Claims (10)
- The application on anti-avian influenza H5N1 virus medicine prepared by 1.1,2-Benzodiazepines.
- 2. the application on anti-avian influenza H5N1 virus medicine prepared by 1,2-Benzodiazepines as claimed in claim 1, and it is characterized in that Benzodiazepines is is extract the phellinus igniarius of CGMCCNo.8108 from deposit number, and extraction step order is as follows:(1) phellinus igniarius crude extract is prepared;(2) the phellinus igniarius crude extract of step (1) gained and purification on normal-phase silica gel are mixed stir, after drying, carry out silica normal phase column chromatography, use eluent 2-5 time, obtain eluent;(3) by the last eluent evaporate to dryness in step (2), equal-volume silica gel mixed sample, carries out purification on normal-phase silica gel chromatography, uses eluent;(4) collect the eluent in step (3), concentrating under reduced pressure, use chloroform: methanol=1:1 dissolves, and carries out methanol gel column chromatography, use eluent, use TLC to detect the eluent collected, suitably merge eluent, drying under reduced pressure;(5) again carry out purification on normal-phase silica gel chromatography with the product obtained in step (4), use eluent;(6) collect the eluent obtained in step (5), use TLC to detect each eluent, appropriateness merges eluent, and drying under reduced pressure, is 1,2-Benzodiazepines.
- 3. the application on anti-avian influenza H5N1 virus medicine prepared by 1,2-Benzodiazepines as claimed in claim 2, it is characterized in that described phellinus igniarius crude extract, obtained by following method:(1) be inoculated in conventional manner by phellinus igniarius strain and be equipped with in the conical flask of fluid medium, with 20 ~ 35 DEG C of temperature, shaking flask rotating speed is under 80 ~ 280r/min, pH3 ~ 8 conditions, vibrations cultivation 7 ~ 15 days; In cultivation when pH value drops to 2.5 ~ 4, seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, with temperature 20 ~ 35 DEG C, fermentation tank pressure 0.1 ~ 0.2 kg/cm, pH3 ~ 8, ventilation 0.5 ~ 1.1vvm, the condition that mixing speed is 100 ~ 280 revs/min, cultivate 7 ~ 15 days, the full liquid of phellinus igniarius mycelium fermentation can be obtained;(2) get the full liquid of gained phellinus igniarius mycelium fermentation in step (1), by its concentrating under reduced pressure in a usual manner, make its volume concentration to 1/2 ~ 1/5 of original volume;(3) by percent by volume be 60 ~ 95% ethanol concentrated to above-mentioned steps (2) after fermentation liquid extract, wherein, the amount adding ethanol is 2 ~ 8 times of concentrated solution volume, and concentration of alcohol in extracting solution can be made to reach 60 ~ 90%;(4) to step (3) gained extracting solution under 50 ~ 70 DEG C of conditions, heat 1 ~ 2 hour; Be separated in conventional manner, and remove impurity by cascade filtration, be separated and obtain ethanol extract; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume concentration to 1/5 ~ 1/10 of original volume;(5) concentrated solution of step (4) gained is carried out drying with the method for frozen drying, obtain phellinus igniarius crude extract.
- 4. the application on anti-avian influenza H5N1 virus medicine prepared by 1,2-Benzodiazepines as claimed in claim 3, it is characterized in that described liquid culture based formulas in gram/100 milliliters be:
- 5. as claimed in claim 31, the application on anti-avian influenza H5N1 virus medicine prepared by 2-Benzodiazepines, it is characterized in that the ethanol of phellinus igniarius crude extract preparation process (3) is concentration 65%-95% edible ethanol, volume used is 3-6 times of volume.
- 6. as claimed in claim 21,2-Benzodiazepines, preparing the application on anti-avian influenza H5N1 virus medicine, is characterized in that, the sample silica gel of mixing described in step (2) is 100-200 order purification on normal-phase silica gel, chromatographic silica gel is positive 200-300 order, and eluant is chloroform or methanol.
- 7. 1,2-Benzodiazepines as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that, the silica gel consumption described in step (3) is equal-volume, and eluant is petroleum ether: acetone=20:1-35:1.
- 8. 1,2-Benzodiazepines as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that, the gel described in step (4) is SephadexLH-20, and eluant is chloroform: methanol=1:1.
- 9. 1,2-Benzodiazepines as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that, the chromatographic silica gel described in step (5) is 200-300 order, and eluant is chloroform: methanol=200:1-50:1.
- 10. 1,2-Benzodiazepines as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that, the detection point described in step (6) occurs speckle, and developing solvent is petroleum ether: acetone=20:1-35:1.
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| CN101726597A (en) * | 2008-10-27 | 2010-06-09 | 中国科学院动物研究所 | Test paper for detecting avian influenza virus and preparation method thereof |
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| Title |
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| 板蓝根水提取物的化学成分研究;王晓良等;《中国中药杂志》;20130430;第38卷(第8期);1172-1182 * |
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