CN103800331B - The application of Cyclic dipeptides C7 on anti-avian influenza H5N1 virus in phellinus igniarius - Google Patents
The application of Cyclic dipeptides C7 on anti-avian influenza H5N1 virus in phellinus igniarius Download PDFInfo
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- CN103800331B CN103800331B CN201310401652.1A CN201310401652A CN103800331B CN 103800331 B CN103800331 B CN 103800331B CN 201310401652 A CN201310401652 A CN 201310401652A CN 103800331 B CN103800331 B CN 103800331B
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Abstract
The invention discloses a kind of from phellinus igniarius Phellinus igniarius (L ex Fr) Quel(phellinus linteus phellinus linteus (Berk et Curt) Teng, Phellinus baumii Phellinus baumii, breathe out base of a fruit phellinus Phellinus hartigii (Allesch et Schnabl) Imaz) a kind of Cyclic dipeptides C7 with anti-avian influenza activity of being separated fermentation liquid and sporophore.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 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
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 C7 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 C7, 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 80 ~ 280r/min, pH 3 ~ 8 under condition, 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, pH 3 ~ 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 C7 from phellinus igniarius crude extract, step is as follows: the phellinus igniarius crude extract → purification on normal-phase silica gel chromatography → methanol elution gradient → anti-phase preparation of purification on normal-phase silica gel chromatography → HPLC detection → high pressure → HPLC detection → methanol gel chromatography →
1d-HNMR → Cyclic dipeptides C7.
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, with eluent 2-5 time;
(3) collect the last eluent that above-mentioned steps (2) obtains, concentrating under reduced pressure, use dissolve with methanol, methanol gel column chromatography, with eluent;
(4) product that step (3) obtains is carried out purification on normal-phase silica gel chromatography, with eluent;
(5) use HPLC to detect the eluent of the step (4) of collecting, suitably merge eluent, drying under reduced pressure;
(6) product that step (5) obtains is carried out high pressure reversed phase chromatography, eluant is methanol and water;
(7) collect eluent, evaporated under reduced pressure, carries out HPLC detection,
(8) again carry out methanol gel chromatography, collect eluent, drying under reduced pressure, is Cyclic dipeptides C7.
Preferably, the sample silica gel of mixing described in extraction step (2) is 100-200 order purification on normal-phase silica gel, and eluant is chloroform and methanol.
Eluant described in extraction step (3) is methanol.
Purification on normal-phase silica gel described in extraction step (4) is 200-300 order.
HPLC condition described in extraction step (5) is 0min:100% water, 10min:100% methanol.
Reversed material described in extraction step (6) is C-18 or C-8, and eluant is methanol: water=30%-80%, and reversed phase chromatography number of times is 2-4 time.
It is 5.20-5.58min that HPLC described in extraction step (7) goes out the cutting edge of a knife or a sword time.
Gel type described in extraction step (8) is Sephadex LH-20.
(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 110r/min, pH 7 under 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, pH 3, 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.
Then take 350g crude extract, mix with equal-volume 100 order purification on normal-phase silica gel and stir, carry out silica normal phase column chromatography.Chromatography immobile phase is 200 order purification on normal-phase silica gel, post height 1.2m, diameter 20cm, eluant for being respectively chloroform, chloroform: methanol=100:1,50:1 is eluting 3,4,4,4 column volumes respectively.And by gained eluent called after Fr-1 respectively, Fr-2, Fr-3, Fr-4.By Fr-4 dissolve with methanol, carry out methanol gel chromatography, collect eluent, suitably merge, again with 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, the silica gel used is 200 order purification on normal-phase silica gel, and carry out purification on normal-phase silica gel chromatography, eluant is chloroform and methanol.Detect through HPLC, condition is 0min:100% water, 10min:100% methanol, and appearance time is 5.25.Be associated with the eluent at target absorption peak, then, carry out the anti-phase preparation of high pressure, A phase is water, and B phase is methanol.Collect eluent, evaporated under reduced pressure, HPLC detects, and condition is 0min:100% water, 10min:100% methanol, and appearance time is 5.25.Suitable merging, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analysis result is δ 7.29 (ddd, J=31.0,18.0,7.0 Hz, 6H), 5.87 (s, 1H), 4.28 (d, J=7.7 Hz, 1H), 4.07 (t, J=7.5 Hz, 1H), 3.67 – 3.53 (m, 3H), 2.80 (dd, J=14.5,10.4 Hz, 1H), 2.36 – 2.28 (m, 2H), 2.08 – 1.80 (m, 5H). prove (L-PROLINE L-Phe).
(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 24 h, 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 48 h in incubator, abandon culture fluid supernatant, 100 μ l/ holes add 5 mg.ml
-1the maintenance medium of MTT, continue to cultivate after 1h, abandon MTT supernatant, every hole adds lysate (DMSO) 100 μ l, vibration 5-10 min, 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 C7
| Sequence number | Density (ug.mL-1) | Pathological changes rate |
| 1 | 100 | 0.93675371286 |
| 2 | 50 | 0.92633966558 |
| 3 | 25 | 0.65354242708 |
| 4 | 12.5 | 0.20421438713 |
| 5 | 6.25 | 0.025397575305 |
| Cell controls |
Known by table 1: Cyclic dipeptides C7 maximum safe concentration is 6.25ug/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, pH 3, 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.
Then take 100g crude extract, mix with equal-volume 100 order purification on normal-phase silica gel 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 10cm, and eluant is for being respectively chloroform, and chloroform: methanol=200:1,100:1,50:1, distinguishes eluting 2,3,3,3 column volumes.And by gained eluent called after Fr-1 respectively, Fr-2, Fr-3, Fr-4.。By Fr-4 dissolve with methanol, carry out methanol gel chromatography, collect eluent, suitably merge, again with equal-volume silica gel mixed sample, pentaploid amasss silica gel column chromatography, and the chromatographic silica gel of use is 200 order purification on normal-phase silica gel, carries out purification on normal-phase silica gel chromatography, and eluant is chloroform and methanol.Detect through HPLC, condition is 0min:100% water, 10min:100% methanol, and appearance time is 5.25., be associated with the eluent at target absorption peak, then, carry out the anti-phase preparation of high pressure, A phase is water, and B phase is methanol.Collect eluent, evaporated under reduced pressure, HPLC detects, and condition is 0min:100% water, 10min:100% methanol, and appearance time is 5.25.Suitable merging, carries out
1d-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analysis result is δ 7.29 (ddd, J=31.0,18.0,7.0 Hz, 6H), 5.87 (s, 1H), 4.28 (d, J=7.7 Hz, 1H), 4.07 (t, J=7.5 Hz, 1H), 3.67 – 3.53 (m, 3H), 2.80 (dd, J=14.5,10.4 Hz, 1H), 2.36 – 2.28 (m, 2H), 2.08 – 1.80 (m, 5H). prove (L-PROLINE L-Phe).
(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 2 h, 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 C7 can reach the suppression ratio of the highest 42.5% under 1ug/ml concentration to H5N1 strain, now CC
50for 20.988ug/ml, EC
50for 203.97ug/ml, SI are 0.10.
Claims (10)
1. Cyclic dipeptides C7 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that described Cyclic dipeptides C7 is L-PROLINE L-Phe.
2. Cyclic dipeptides C7 as claimed in claim 1 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that Cyclic dipeptides C7 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, with eluent 2-5 time;
(3) collect the last eluent that above-mentioned steps (2) obtains, concentrating under reduced pressure, use dissolve with methanol, methanol gel column chromatography, with eluent;
(4) product that step (3) obtains is carried out purification on normal-phase silica gel chromatography, with eluent;
(5) use HPLC to detect the eluent of the step (4) of collecting, suitably merge eluent, drying under reduced pressure;
(6) product that step (5) obtains is carried out high pressure reversed phase chromatography, eluant is methanol and water;
(7) collect eluent, evaporated under reduced pressure, carries out HPLC detection,
(8) again carry out methanol gel chromatography, collect eluent, drying under reduced pressure, is Cyclic dipeptides C7.
3. Cyclic dipeptides C7 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 80 ~ 280r/min, pH 3 ~ 8 under condition, 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, pH 3 ~ 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 C7 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 C7 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 C7 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, and 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 eluant is chloroform and methanol; Eluant described in step (3) is methanol.
7. Cyclic dipeptides C7 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, and it is characterized in that, the purification on normal-phase silica gel described in step (4) is 200-300 order.
8. Cyclic dipeptides C7 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, and it is characterized in that, the HPLC condition described in step (5) is 0min:100% water, 10min:100% methanol.
9. Cyclic dipeptides C7 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that, reversed material described in step (6) is C-18 or C-8, and eluant is the methanol aqueous solution of 30%-80%, and reversed phase chromatography number of times is 2-4 time.
10. Cyclic dipeptides C7 as claimed in claim 2 is preparing the application on anti-avian influenza H5N1 virus medicine, it is characterized in that, HPLC appearance time described in step (7) is 5.20-5.58min, and the gel type described in step (8) is Sephadex LH-20.
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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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