CN103816138A - Application of 1,2-benzenediol in phellinus igniarius to resisting H5N1 avian influenza virus - Google Patents
Application of 1,2-benzenediol in phellinus igniarius to resisting H5N1 avian influenza virus Download PDFInfo
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- CN103816138A CN103816138A CN201310404207.0A CN201310404207A CN103816138A CN 103816138 A CN103816138 A CN 103816138A CN 201310404207 A CN201310404207 A CN 201310404207A CN 103816138 A CN103816138 A CN 103816138A
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Abstract
The invention discloses 1,2-benzenediol with an avian influenza-resisting activity, which is separated from fermentation broth and fruiting bodies of phellinus igniarius (LexFr) Quel (phellinus linteus (BerketCurt) Teng, phellinus baumii and phellinus hartigii (AlleschetSchnabl) Imaz). The invention firstly discloses a new activity of 1,2-benzenediol and proves that the 1,2-benzenediol has an avian influenza-resisting action. Compared with the application of a general compound, the method has the advantages that the special new activity of 1,2-benzenediol is found, and the 1,2-benzenediol has the action of resisting the 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 relating in particular to, be deposited in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 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 Phellinus igniarius, deposit number is CGMCC No.8108.
background technology
Benzodiazepines can volatilize together with steam, it is strong reductant, meeting naked light can burn, be subject to high heat give out poisonous, abnormal smells from the patient is corrosive, can cause through skin poisoningly, can distil, nervus centralis, respiratory system are had to stimulation, 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 separating from medicinal fungus.The present invention discloses the newly activity of one of this Benzodiazepines first, proves that it has anti-avian influenza effect.
This method is compared with the advantage of vague generalization compound application: found the new activity of this kind of Benzodiazepines, had the effect of anti-avian influenza virus.
Technical scheme of the present invention is as follows:
First prepare phellinus igniarius crude extract, made 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) phellinus igniarius strain is inoculated into conventional method in the conical flask that fluid medium is housed, with 20～35 ℃ of temperature, shaking flask rotating speed is 80～280r/min, and under pH 3～8 conditions, vibrations are cultivated 7～15 days; In cultivation in the time that pH value drops to 2.5～4, seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, with 20～35 ℃ of temperature, fermentation tank pressure 0.1～0.2 kg/cm, pH 3～8, ventilation 0.5～1.1vvm, the condition that mixing speed is 100～280 revs/min, cultivate 7～15 days, can utilize the full liquid of phellinus igniarius mycelium fermentation 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 be concentrated to 1/2～1/5 of original volume;
(4) fermentation liquid after the ethanol that is 60～95% by percent by volume is concentrated to above-mentioned steps (3) extracts, wherein, the amount that adds ethanol is 2～8 times of concentrated solution volume, can make concentration of alcohol in extracting solution reach 60～90%, ethanol is preferably the edible ethanol of concentration 65-95%, and volume used is preferably 3-6 times of concentrated solution volume;
(5) to step (4) gained extracting solution under 50～70 ℃ of conditions, heat 1～2 hour; Separate with conventional method, and remove impurity by cascade filtration, separate and obtain ethanol extract; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5～1/10 of original volume;
(6) just the concentrated solution of step (5) gained is dried with the method for frozen drying, obtains phellinus igniarius crude extract.
Then from phellinus igniarius crude extract, prepare 1,2-Benzodiazepines, step is as follows: phellinus igniarius crude extract → purification on normal-phase silica gel chromatography → chloroform and methanol gradient elution → TLC detection → purification on normal-phase silica gel chromatography → chloroform methanol gel chromatography → TLC detection → purification on normal-phase silica gel chromatography → TLC detection → evaporated under reduced pressure → colorless oil (1,2-Benzodiazepines).
Concrete grammar is:
(1) prepare phellinus igniarius crude extract;
(2) precipitate and the purification on normal-phase silica gel of step (1) gained are mixed to stirring, after being dried, carry out silica gel normal phase column chromatography, use eluant eluting 2-5 time, obtain eluent;
(3) by last eluent evaporate to dryness in step (2), equal-volume silica gel mixed sample, carries out purification on normal-phase silica gel chromatography, uses eluant eluting;
(4) collect the eluent in step (3), concentrating under reduced pressure, uses chloroform: methanol=1:1 dissolves, and carries out methanol gel column chromatography, uses eluant eluting, uses TLC to detect the eluent of collecting, and suitably merges eluent, drying under reduced pressure;
(5) product obtaining in step (4) is carried out to purification on normal-phase silica gel chromatography again, use eluant eluting;
(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 Sephadex LH-20, and eluant is chloroform: methanol=1:1～1:3;
The described chromatographic silica gel of extraction step (5) is 200-300 order, and eluant is chloroform: methanol=100:1-50:1;
In extraction step (6), TLC detection point is to occur speckle, and developing solvent is petroleum ether: acetone=30:1-35:1.
Then use obtain 1,2-Benzodiazepines carries out the mensuration to mdck cell toxicity, determines 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, purity be greater than 95% 1,2-Benzodiazepines.Mature technical route clearly, efficiently accurate, and proved that first it has the activity of anti-avian influenza H5N1 virus.
(4) specific embodiment
Example 1:
Phellinus igniarius crude extract, is made 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) phellinus igniarius strain is inoculated into conventional method in the conical flask that fluid medium is housed, with 25 ℃ of temperature, shaking flask rotating speed is 110r/min, and under pH 7 conditions, vibrations are cultivated 7 days; In cultivation in the time that pH value drops to 3, seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, with 25 ℃ of temperature, fermentation tank pressure 0.1 kg/cm, pH 3, ventilation 0.5-1.1vvm, the condition that mixing speed is 100 revs/min, cultivate 7 days, can utilize the full liquid of phellinus igniarius mycelium fermentation 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 be concentrated to 1/3 of original volume;
(4) fermentation liquid after the ethanol that is 70% by percent by volume is concentrated to above-mentioned steps (3) extracts, and wherein, the amount that adds ethanol is 5 times of concentrated solution volume, can make concentration of alcohol in extracting solution reach 55%;
(5) to step (4) gained extracting solution under 70 ℃ of conditions, heat 1 hour; Separate with conventional method, and remove impurity by cascade filtration, separate and obtain ethanol extract; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5 of original volume;
(6) just the concentrated solution of step (5) gained is dried with the method for frozen drying, obtains phellinus igniarius crude extract.
Above-mentioned crude extract is taken to 200g and equal-volume 100 order purification on normal-phase silica gel mix stirring, carry out silica gel normal phase column chromatography.Chromatography immobile phase is 200-300 order purification on normal-phase silica gel, the high 0.8m of post, and diameter 15cm, eluant is for being respectively chloroform, methanol, and chloroform: methanol=100:1, distinguishes 2,3 column volumes of eluting.And by gained eluent called after Fr-1 respectively, Fr-2.By Fr-2 equal-volume silica gel mixed sample, using silica gel is 100 order purification on normal-phase silica gel, and 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 of collecting and suitably 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 and collects the eluent that occurs speckle.Drying under reduced pressure, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, analysis result is δ 8.78 (s, 1H), 6.68 (dt, J=40.7,20.4 Hz, 1H), 6.58 (dd, J=5.7,3.5 Hz, 1H), proving is 1,2-Benzodiazepines.
(7) ergot sterone that adopts 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, cultivates after 24 h, on cell monolayer, add respectively variable concentrations containing sample maintenance medium, every kind of concentration repeats 3 holes, and establishes normal cell contrast.Put 37 ℃, 5%CO
2in incubator, cultivate after 48 h, abandon culture fluid supernatant, 100 μ l/ holes add the maintenance medium of 5 mg.ml-1MTT, continue to cultivate after 1h, abandon MTT supernatant, every hole adds lysate (DMSO) 100 μ l, vibration 5-10 min, dissolving completely to be crystallized, microplate reader is surveyed 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 matched group OD
492time, this concentration is the maximum safe concentration of sample.
The impact of the each concentration 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 made 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) phellinus igniarius strain is inoculated into conventional method in the conical flask that fluid medium is housed, with 30 ℃ of temperature, shaking flask rotating speed is 180r/min, and under pH6 condition, vibrations are cultivated 15 days; In cultivation in the time that pH value drops to 2.5, seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, with 30 ℃ of temperature, fermentation tank pressure 0.2 kg/cm, pH 3, ventilation 0.5-1.1vvm, the condition that mixing speed is 180 revs/min, cultivate 15 days, can utilize the full liquid of phellinus igniarius mycelium fermentation 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 be concentrated to 1/5 of original volume;
(4) fermentation liquid after the ethanol that is 90% by percent by volume is concentrated to above-mentioned steps (3) extracts, and wherein, the amount that adds ethanol is 4 times of concentrated solution volume, can make concentration of alcohol in extracting solution reach 70%;
(5) to step (4) gained extracting solution under 55 ℃ of conditions, heat 2.5 hours; Separate with conventional method, and remove impurity by cascade filtration, separate and obtain ethanol extract; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/10 of original volume;
(6) just the concentrated solution of step (5) gained is dried with the method for frozen drying, obtains phellinus igniarius crude extract.
Above-mentioned crude extract is taken to 300g and equal-volume 100 order purification on normal-phase silica gel mix stirring, carry out silica gel normal phase column chromatography.Chromatography immobile phase is 200-300 order purification on normal-phase silica gel, the high 1m of post, and diameter 20cm, eluant is for being respectively chloroform, chloroform: methanol=100:1, respectively 3,4 column volumes of eluting.And by gained eluent called after Fr-1 respectively, Fr-2.By Fr-2 equal-volume silica gel mixed sample, using silica gel is 100 order purification on normal-phase silica gel, and 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 of collecting and suitably 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 and collects the eluent that occurs speckle.Drying under reduced pressure, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, analysis result is δ 8.78 (s, 1H), 6.68 (dt, J=40.7,20.4 Hz, 1H), 6.58 (dd, J=5.7,3.5 Hz, 1H), proving is 1,2-Benzodiazepines.
(7) within the scope of the maximum safe concentration that adopts step (6) to obtain, 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.
In test, normal cell matched group (only adding cell maintenance medium) is set, virus control group (only adding virus liquid), mass concentration is 4 mass concentrations of 2 times of dilutions in nontoxic scope, 3 holes are set 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 ℃ of absorption 2 h, abandon virus liquid supernatant, and 100 μ l/ holes add the sample liquid of variable concentrations.Put 37 ℃, 5% CO
2in incubator, continue to cultivate 48h.Adopt mtt assay 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, has antiviral activity.
According to the cytopathy variability calculating and viral suppression ratio.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.
Calculate viral suppression ratio according to lower formula:
Virus suppression ratio=(A-B)/(C-B) × 100%
A: sample liquid processed group OD value, B: virus control group OD value, C: cell matched group OD value
The inhibitory action experimental result of table 2 compound to H5N1 virus
Known by table 2: 1,2-Benzodiazepines can reach the highest 52.4% suppression ratio, now CC to H5N1 strain under 6.25ug/ml concentration
50for 66.239ug/ml, EC
50for 3.3767ug/ml, SI is 19.62.
Claims (10)
1.1,2-Benzodiazepines is in the application of preparing on anti-avian influenza H5N1 virus medicine.
2. as claimed in claim 11,2-Benzodiazepines, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that Benzodiazepines extracts from phellinus igniarius, and extraction step order is as follows:
(1) prepare phellinus igniarius crude extract;
(2) ethanol precipitate and the purification on normal-phase silica gel of step (1) gained are mixed to stirring, after being dried, carry out silica gel normal phase column chromatography, use eluant eluting 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 eluant eluting;
(4) collect the eluent in step (3), concentrating under reduced pressure, uses chloroform: methanol=1:1 dissolves, and carries out methanol gel column chromatography, uses eluant eluting, uses TLC to detect the eluent of collecting, and suitably merges eluent, drying under reduced pressure;
(5) again carry out purification on normal-phase silica gel chromatography with the product obtaining in step (4), use eluant eluting;
(6) collect the eluent obtaining in step (5), use TLC to detect each eluent, appropriateness merges eluent, and drying under reduced pressure, is 1,2-Benzodiazepines.
3. as claimed in claim 21,2-Benzodiazepines, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that described phellinus igniarius crude extract, is made by following method:
(1) phellinus igniarius strain is inoculated into conventional method in the conical flask that fluid medium is housed, with 20～35 ℃ of temperature, shaking flask rotating speed is 80～280r/min, and under pH 3～8 conditions, vibrations are cultivated 7～15 days; In cultivation in the time that pH value drops to 2.5～4, seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, with 20～35 ℃ of temperature, fermentation tank pressure 0.1～0.2 kg/cm, pH 3～8, ventilation 0.5～1.1vvm, the condition that mixing speed is 100～280 revs/min, cultivate 7～15 days, can obtain the full liquid of phellinus igniarius mycelium fermentation;
(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 be concentrated to 1/2～1/5 of original volume;
(3) fermentation liquid after the ethanol that is 60～95% by percent by volume is concentrated to above-mentioned steps (2) extracts, and wherein, the amount that adds ethanol is 2～8 times of concentrated solution volume, can make concentration of alcohol in extracting solution reach 60～90%;
(4) to step (3) gained extracting solution under 50～70 ℃ of conditions, heat 1～2 hour; Separate with conventional method, and remove impurity by cascade filtration, separate and obtain ethanol extract; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5～1/10 of original volume;
(5) just the concentrated solution of step (4) gained is dried with the method for frozen drying, obtains phellinus igniarius crude extract.
4. as claimed in claim 31,2-Benzodiazepines is in the application of preparing on anti-avian influenza H5N1 virus medicine, it is characterized in that described liquid culture based formulas 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%.
5. as claimed in claim 31,2-Benzodiazepines is in the application of preparing on anti-avian influenza H5N1 virus medicine, and the ethanol that it is characterized in that phellinus igniarius crude extract preparation process (3) is concentration 65%-95% edible ethanol, and volume used is 3-6 times of volume.
6. as claimed in claim 21,2-Benzodiazepines, in the application of preparing 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. as claimed in claim 21,2-Benzodiazepines, in the application of preparing on anti-avian influenza H5N1 virus medicine, 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. as claimed in claim 21,2-Benzodiazepines, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that, the gel described in step (4) is Sephadex LH-20, and eluant is chloroform: methanol=1:1.
9. as claimed in claim 21,2-Benzodiazepines, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that, the described chromatographic silica gel of step (5) is 200-300 order, and eluant is chloroform: methanol=200:1-50:1.
10. as claimed in claim 21,2-Benzodiazepines, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that, the described detection point of step (6) is to occur 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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| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 王晓良等: "板蓝根水提取物的化学成分研究", 《中国中药杂志》, vol. 38, no. 8, 30 April 2013 (2013-04-30), pages 1172 - 1182 * |
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