CN103816156B - The application of Cyclic dipeptides C2 on anti-avian influenza H5N1 virus in phellinus igniarius - Google Patents
The application of Cyclic dipeptides C2 on anti-avian influenza H5N1 virus in phellinus igniarius Download PDFInfo
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Abstract
The invention discloses a kind of wood from the fire layer of & lt;I>Phellinus</ i>& lt;I> Igniarius</ i>(L The ex Fr.) Quel (split hoof wood layer hole bacteria & lt; i> phellinus< / i> & lt; i> Linteus< / i> (Berk et Curt) Teng, bowman's layer hole bacteria & lt; i> phellinus< / i> & lt; i> Baumii< / i> bacteria, harty needle hole & lt; i> phellinus< / i> & lt; i> Hartigii< / i> (Allesch et Schnabl) Imaz) fermented liquid and fruiting body of separation with avian flu activity in a ring dipeptide C2.The one that the present invention makes public for the first time a kind of Cyclic dipeptides 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 Cyclic dipeptides, 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 " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; deposit number is CGMCCNo.8108; specific name is phellinus igniarius Phellinusigniarius; 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
In usual biology, the cyclic peptide of indication refers to the compound formed with aminoacid peptide bond, in phytochemistry, this concept is expanded the compounds formed 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 reported has many-sided biological activity, comprises the biological activitys such as antitumor, AntiHIV1 RT activity, antibacterial, malaria, sleeping, anticoagulant, blood pressure lowering, restraint of tyrosinase, suppression Cycloxygenase, anti-lipid peroxidation enzyme, estrogen sample, immunosuppressant.
Varying of the amino acid whose number comprised due to ring propeptide-linear peptides and kind, causes the variation of cyclic peptide synthetic method.Show efficiently to certain linear peptides, the reagent of rapid condensation effect and method just may become poor efficiency or invalid to another peptide chain.
Cyclic dipeptides has the multiple important physiologically actives such as antibacterial, nowadays, does not see reporting for work of anti-feelings influenza activity.
Summary of the invention
The invention discloses a kind of a kind of Cyclic dipeptides C2 with anti-avian influenza activity be separated from medicinal fungus.The one that the present invention makes public for the first time this kind of Cyclic dipeptides 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 a kind of Cyclic dipeptides C2, 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 Cyclic dipeptides C2 from phellinus igniarius crude extract, 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 → methanol gel chromatography → TLC detects → and reversed-phase silica gel chromatography → methanol gel chromatography HPLC detects → evaporated under reduced pressure → Cyclic dipeptides C2.
Concrete grammar is:
(1) phellinus igniarius crude extract is prepared;
(2) crude extract of above-mentioned steps (1) gained and purification on normal-phase silica gel are mixed stir, and dry, carry out silica normal phase column chromatography, use eluent 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 eluent;
(4) collect the eluent obtained in above-mentioned steps (3), concentrating under reduced pressure, use dissolve with methanol, methanol gel column chromatography, with eluent, use TLC to detect the eluent collected, suitably merge eluent, drying under reduced pressure;
(5) carry out reversed-phase silica gel chromatography to the product that step (4) obtains, 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 Cyclic 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 SephadexLH-20 or SephadexLH-25, and eluant is methanol.
Reversed material described in extraction step (5) is C-18 or C-8, and eluant is methanol: water=15%-70%.
Gel described in extraction step (6) is methanol gel.
HPLC condition described in extraction step (7) is, 0min, 100%A water → 10min, 100% methanol, and it is 5.3-5.9min that HPLC goes out the cutting edge of a knife or a sword time.
(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 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 order purification on normal-phase silica gel, post height 1m, diameter 20cm, eluant for being respectively chloroform, chloroform: methanol=100:1,50:1,10:1,5:1 is eluting 3 column volumes 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, 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 order purification on normal-phase silica gel.Eluant is chloroform and methanol.Collect eluent, concentrating under reduced pressure, use dissolve with methanol, carry out methanol gel column chromatography, eluant is methanol.Use TLC to detect the eluent collected, suitably merge eluent, drying under reduced pressure, carry out reversed-phase silica gel chromatography, eluant is methanol: water=15%, and TLC detects the eluent collected 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 Cyclic dipeptides C2, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analysis result is δ 7.35 – 7.13 (m, 5H), 4.45 (t, J=4.1Hz, 1H), 4.32 (dd, J=10.9,5.2Hz, 1H), 4.24 (d, J=4.7Hz, 1H), 3.66 (dd, J=13.0,5.0Hz, 1H), 3.12 (d, J=5.0Hz, 2H), 2.02 (dd, J=13.0,5.9Hz, 1H), 1.36 – 1.26 (m, 1H). prove six hydrogen-7-hydroxyl-3-(phenyl methyls) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone.
(7) compound 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
5ml
-1the mdck cell suspension of 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 5mg.ml
-1the maintenance medium of MTT, continue to cultivate after 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 each mass concentration group (n=12) of Cyclic dipeptides C2
| 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 controls |
Known by table 1: Cyclic dipeptides C2 maximum safe concentration is 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 150g and equal-volume purification on normal-phase silica gel to mix and stir, carry out silica normal phase column chromatography.Chromatography immobile phase is 200 order purification on normal-phase silica gel, post height 0.8m, diameter 14cm, eluant for being respectively chloroform, chloroform: methanol=100:1,50:1,10:1,5:1 is eluting 2 column volumes 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, use dissolve with methanol, carry out methanol gel column chromatography, eluant is methanol.Use TLC to detect the eluent collected, suitably merge eluent, drying under reduced pressure, carry out reversed-phase silica gel chromatography, eluant is methanol: water=15%, and TLC detects the eluent collected 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 Cyclic dipeptides C2, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analysis result is δ 7.35 – 7.13 (m, 5H), 4.45 (t, J=4.1Hz, 1H), 4.32 (dd, J=10.9,5.2Hz, 1H), 4.24 (d, J=4.7Hz, 1H), 3.66 (dd, J=13.0,5.0Hz, 1H), 3.12 (d, J=5.0Hz, 2H), 2.02 (dd, J=13.0,5.9Hz, 1H), 1.36 – 1.26 (m, 1H). prove six hydrogen-7-hydroxyl-3-(phenyl methyls) pyrrolo-[1,2-a) pyrazine-Isosorbide-5-Nitrae-diketone.
(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), 3 repetitions are set.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: Cyclic dipeptides C2 can reach the suppression ratio of the highest 69.6% under 2ug/ml concentration to H5N1 strain, now CC
50for 42.296ug/ml, EC
50for 26.08ug/ml, SI are 1.62.
Claims (9)
1. Cyclic dipeptides C2 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that described Cyclic dipeptides C2 is six hydrogen-7-hydroxyl-3-(phenyl methyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone.
2. Cyclic dipeptides C2 as claimed in claim 1 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that Cyclic dipeptides C2 extracts from phellinus igniarius, and extraction step order is as follows:
(1) phellinus igniarius crude extract is prepared;
(2) crude extract of above-mentioned steps (1) gained and purification on normal-phase silica gel are mixed stir, and dry, carry out silica normal phase column chromatography, use eluent 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 eluent;
(4) collect the eluent obtained in above-mentioned steps (3), concentrating under reduced pressure, use dissolve with methanol, methanol gel column chromatography, with eluent, use TLC to detect the eluent collected, suitably merge eluent, drying under reduced pressure;
(5) carry out reversed-phase silica gel chromatography to the product that step (4) obtains, 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 Cyclic dipeptides C2.
3. Cyclic dipeptides C2 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that described phellinus igniarius crude extract, is 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. Cyclic dipeptides C2 as claimed in claim 3 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that described liquid culture based formulas in gram/100 milliliters be:
5. Cyclic dipeptides C2 as claimed in claim 3 is preparing the application on anti-avian influenza H5N1 virus medicine, and 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. Cyclic dipeptides C2 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that, 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. Cyclic dipeptides C2 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, and it is characterized in that, the silica gel consumption described in step (3) is equal-volume, and eluant is chloroform and methanol.
8. Cyclic dipeptides C2 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, and it is characterized in that, the gel described in step (4) is SephadexLH-20 or SephadexLH-25, and eluant is methanol.
9. Cyclic dipeptides C2 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, and it is characterized in that, the reversed material described in step (5) is C-18 or C-8, and eluant is methanol: water=15%-70%.
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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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| 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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