CN103816156A - Application of cyclic dipeptide C2 in phellinus igniarius to resisting H5N1 avian influenza virus - Google Patents
Application of cyclic dipeptide C2 in phellinus igniarius to resisting H5N1 avian influenza virus Download PDFInfo
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Abstract
The invention discloses a cyclic dipeptide C2 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 the cyclic dipeptide and proves that the cyclic dipeptide 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 the cyclic dipeptide is found, and the cyclic dipeptide 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 " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; deposit number is CGMCC No.8108; specific name is phellinus igniarius Phellinus igniarius; preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica on August 26th, 2013.
background technology
Conventionally in biology, the cyclic peptide of indication refers to the compound forming with aminoacid peptide bond, in phytochemistry, this concept is expanded the compounds forming with amido link, therefore scope is enlarged to and comprises organic amine and macrocyclic alkaloid class, whether is divided into cyclic peptide and linear peptides according to ring formation.The cyclic peptide compound of having reported has many-sided biological activity, comprises antitumor, anti-HIV, antibacterial, malaria, sleeps peacefully, anticoagulant, blood pressure lowering, restraint of tyrosinase, inhibition Cycloxygenase, suppresses the biological activitys such as lipid peroxidation enzyme, estrogen sample, immunosuppressant.
Due to varying of the ring amino acid whose number that comprises of propeptide-linear peptides and kind, cause the variation of cyclic peptide synthetic method.Certain linear peptides is shown efficiently, and reagent and the method for condensation just may become poor efficiency or invalid to another peptide chain fast.
Encircle dipeptides and there is the multiple important physiologically actives such as antibacterial, nowadays, do not see reporting for work of anti-feelings influenza virus activity.
Summary of the invention
The invention discloses a kind of one ring dipeptides C2 with anti-avian influenza activity separating from medicinal fungus.The present invention discloses this kind of newly activity of one of encircling dipeptides first, proves that it has anti-avian influenza effect.
This method is compared with the advantage of vague generalization compound application: found this kind of a kind of new activity of encircling dipeptides C2, 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 medium ring dipeptides C2, 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 → methanol gel chromatography → TLC detection → reversed-phase silica gel chromatography → methanol gel chromatography HPLC detection → evaporated under reduced pressure → ring dipeptides C2.
Concrete grammar is:
(1) prepare phellinus igniarius crude extract;
(2) crude extract and the purification on normal-phase silica gel of above-mentioned steps (1) gained are mixed to stirring, and dry, carry out silica gel normal phase column chromatography, use eluant eluting 3-5 time;
(3) collect the last eluent in step (2), drying under reduced pressure, with equal-volume silica gel mixed sample, carries out purification on normal-phase silica gel chromatography again, uses eluant eluting;
(4) collect the eluent obtaining in above-mentioned steps (3), concentrating under reduced pressure, uses dissolve with methanol, and 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 step (4) being obtained carries out reversed-phase silica gel chromatography, and eluant is first alcohol and water;
(6) collect eluent, evaporated under reduced pressure, carries out methanol gel chromatography.
(7) HPLC detects, and appropriateness merges eluent, and drying under reduced pressure is ring dipeptides C2.
Preferably, the sample silica gel of mixing 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 or methanol.
Silica gel consumption described in extraction step (3) is equal-volume, and eluant is chloroform and methanol.
Gel described in extraction step (4) is Sephadex LH-20 or Sephadex LH-25, and eluant is methanol.
The described reversed material of extraction step (5) is C-18 or C-8, and eluant is methanol: water=15%-70%.
The described gel of extraction step (6) is methanol gel.
The described HPLC condition of extraction step (7) is, 0min, and 100%A water → 10min, 100% methanol, it is 5.3-5.9min that HPLC goes out the cutting edge of a knife or a sword time.
(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 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 order purification on normal-phase silica gel, the high 1m of post, and diameter 20cm, eluant is for being respectively chloroform, chloroform: methanol=100:1,50:1,10:1,5:1 is 3 column volumes of eluting respectively.And by gained eluent called after Fr-1 respectively, Fr-2, Fr-3, Fr-4, Fr-5.。By Fr-5 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 order purification on normal-phase silica gel.Eluant is chloroform and methanol.Collect eluent, concentrating under reduced pressure, uses dissolve with methanol, carries out methanol gel column chromatography, and eluant is methanol.Use TLC to detect the eluent of collecting, suitably merge eluent, drying under reduced pressure, carry out reversed-phase silica gel chromatography, eluant is methanol: water=15%, and TLC detects the eluent of collecting and suitably merges, drying under reduced pressure, carries out methanol gel chromatography again, by methanol-eluted fractions, by eluent evaporated under reduced pressure, carry out HPLC detection, 0min, 100%A water → 10min, 100% methanol, suitably merges eluent, drying under reduced pressure, obtains encircling dipeptides C2, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, analysis result is δ 7.35 – 7.13 (m, 5H), 4.45 (t, J=4.1 Hz, 1H), 4.32 (dd, J=10.9, 5.2 Hz, 1H), 4.24 (d, J=4.7 Hz, 1H), 3.66 (dd, J=13.0, 5.0 Hz, 1H), 3.12 (d, J=5.0 Hz, 2H), 2.02 (dd, J=13.0, 5.9 Hz, 1H), 1.36 – 1.26 (m, 1H). prove six hydrogen-7-hydroxyl-3-(phenyl methyl) pyrrolo-[1, 2-a] pyrazine-1, 4-diketone.
(7) compound 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
5ml
-1the mdck cell suspension of 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 5 mg.ml
-1the maintenance medium of MTT, continues to cultivate after 1h, abandons MTT supernatant, and every hole adds lysate (DMSO) 100 μ l, vibration 5-10 min, and 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 ring dipeptides each mass concentration group of C2 (n=12)
Sequence number | Density (ug.mL -1) | Pathological changes rate |
1 | 200 | 0.94513575092 |
2 | 100 | 0.92862567596 |
3 | 50 | 0.67233851242 |
4 | 25 | 0.15087414497 |
5 | 12.5 | -0.056898798321 |
? | Cell contrast | ? |
Known by table 1: ring dipeptides C2 maximum safe concentration is 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 150g and equal-volume purification on normal-phase silica gel mixes stirring, carry out silica gel normal phase column chromatography.Chromatography immobile phase is 200 order purification on normal-phase silica gel, the high 0.8m of post, and diameter 14cm, eluant is for being respectively chloroform, chloroform: methanol=100:1,50:1,10:1,5:1 is 2 column volumes of eluting respectively.And by gained eluent called after Fr-1 respectively, Fr-2, Fr-3, Fr-4, Fr-5.。By Fr-5 equal-volume silica gel mixed sample, pentaploid amasss silica gel column chromatography, and the silica gel of use is 200 order purification on normal-phase silica gel.Eluant is chloroform and methanol.Collect eluent, concentrating under reduced pressure, uses dissolve with methanol, carries out methanol gel column chromatography, and eluant is methanol.Use TLC to detect the eluent of collecting, suitably merge eluent, drying under reduced pressure, carries out reversed-phase silica gel chromatography, eluant is methanol: water=15%, and TLC detects the eluent of collecting and suitably merges, drying under reduced pressure, again carry out methanol gel chromatography, by methanol-eluted fractions, by eluent evaporated under reduced pressure, carry out HPLC detection, 0min, 100%A water → 10min, 100% methanol, appearance time is 5.45min, suitably merges eluent, drying under reduced pressure, obtains encircling dipeptides C2, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, analysis result is δ 7.35 – 7.13 (m, 5H), 4.45 (t, J=4.1 Hz, 1H), 4.32 (dd, J=10.9, 5.2 Hz, 1H), 4.24 (d, J=4.7 Hz, 1H), 3.66 (dd, J=13.0, 5.0 Hz, 1H), 3.12 (d, J=5.0 Hz, 2H), 2.02 (dd, J=13.0, 5.9 Hz, 1H), 1.36 – 1.26 (m, 1H). prove six hydrogen-7-hydroxyl-3-(phenyl methyl) pyrrolo-[1, 2-a) pyrazine-1, 4-diketone.
(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), 3 repetitions are set.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: ring dipeptides C2 can reach the highest 69.6% suppression ratio, now CC to H5N1 strain under 2ug/ml concentration
50for 42.296ug/ml, EC
50for 26.08ug/ml, SI is 1.62.
Claims (10)
1. ring dipeptides C2, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that described ring dipeptides C2 is six hydrogen-7-hydroxyl-3-(phenyl methyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone.
2. ring dipeptides C2 as claimed in claim 1, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that encircling dipeptides C2 and extracts from phellinus igniarius, and extraction step order is as follows:
(1) prepare phellinus igniarius crude extract;
(2) crude extract and the purification on normal-phase silica gel of above-mentioned steps (1) gained are mixed to stirring, and dry, carry out silica gel normal phase column chromatography, use eluant eluting 3-5 time;
(3) collect the last eluent in step (2), drying under reduced pressure, with equal-volume silica gel mixed sample, carries out purification on normal-phase silica gel chromatography again, uses eluant eluting;
(4) collect the eluent obtaining in above-mentioned steps (3), concentrating under reduced pressure, uses dissolve with methanol, and 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 step (4) being obtained carries out reversed-phase silica gel chromatography, and eluant is first alcohol and water;
(6) collect eluent, evaporated under reduced pressure, carries out methanol gel chromatography.
(7) HPLC detects, and appropriateness merges eluent, and drying under reduced pressure is ring dipeptides C2.
3. ring dipeptides C2 as claimed in claim 2, 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. ring dipeptides C2 as claimed in claim 3 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. ring dipeptides C2 as claimed in claim 3 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. ring dipeptides C2 as claimed in claim 2 is in the application of preparing on anti-avian influenza H5N1 virus medicine, it is characterized in that, the sample silica gel of mixing described in 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 or methanol.
7. ring dipeptides C2 as claimed in claim 2, 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 chloroform and methanol.
8. ring dipeptides C2 as claimed in claim 2, 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 or Sephadex LH-25, and eluant is methanol.
9. ring dipeptides C2 as claimed in claim 2, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that, the described reversed material of step (5) is C-18 or C-8, and eluant is methanol: water=15%-70%.
10. ring dipeptides C2 as claimed in claim 2, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that, the described gel of step (6) is methanol gel.
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CN114805364A (en) * | 2022-04-12 | 2022-07-29 | 常州工程职业技术学院 | Fused ring compound of pyrrolidine and piperazine dione, preparation and pharmaceutical use thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001178448A (en) * | 1999-12-22 | 2001-07-03 | Yukito Akiyama | Method for culturing mycelium of phellinus linteus |
CN102603577A (en) * | 2011-01-19 | 2012-07-25 | 董慧珍 | Derivatives of anti-influenza and anti-avian influenza medicament and application thereof |
CN103073551A (en) * | 2013-01-30 | 2013-05-01 | 青岛农业大学 | Separation technology for cyclic dipeptide C2 in phellinus igniarius |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001178448A (en) * | 1999-12-22 | 2001-07-03 | Yukito Akiyama | Method for culturing mycelium of phellinus linteus |
CN102603577A (en) * | 2011-01-19 | 2012-07-25 | 董慧珍 | Derivatives of anti-influenza and anti-avian influenza medicament and application thereof |
CN103073551A (en) * | 2013-01-30 | 2013-05-01 | 青岛农业大学 | Separation technology for cyclic dipeptide C2 in phellinus igniarius |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN114805364A (en) * | 2022-04-12 | 2022-07-29 | 常州工程职业技术学院 | Fused ring compound of pyrrolidine and piperazine dione, preparation and pharmaceutical use thereof |
CN114805364B (en) * | 2022-04-12 | 2023-04-07 | 常州工程职业技术学院 | Fused ring compound of pyrrolidine and piperazine dione, preparation and pharmaceutical use thereof |
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