CN103800332B - In phelliuns igniarius, encircle the application of dipeptides C6 on anti-avian influenza H5N1 virus - Google Patents
In phelliuns igniarius, encircle the application of dipeptides C6 on anti-avian influenza H5N1 virus Download PDFInfo
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Abstract
The invention discloses a kind of from phelliuns igniarius<i>Phellinus</i><i>Igniarius</i>(L ex Fr) Quel(phellinus linteus <i>Phellinus</i><i>Linteus</i>(Berk et Curt) Teng, Phellinus baumii<i>Phellinus</i><i>Baumii</i>, breathe out base of a fruit phellinus <i>Phellinus</i><i>Hartigii</i>(Allesch et Schnabl) Imaz) zymotic fluid and the one ring dipeptides C6 with anti-avian influenza activity separating in fructification. The present invention discloses a 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 the new activity of this ring dipeptides, had the effect of anti-avian influenza virus.
Description
Technical field
The present invention relates to the extraction preparation method of active material in a kind of phelliuns igniarius of anti-avian influenza H5N1 virus, outstandingThe phelliuns igniarius bacterial classification that it relates to, has been deposited in that " China Committee for Culture Collection of Microorganisms is general on August 26th, 2013Logical microorganism " center ", deposit number is CGMCCNo.8108, specific name is phelliuns igniarius PhellinusIgniarius, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Background technology
Conventionally in biology, the cyclic peptide of indication refers to the compound forming with amino acid peptide bond, and in Phytochemistry, this is without exceptionThought is expanded the compounds forming with amido link, and therefore scope is enlarged to and comprises organic amine and macrocyclic alkaloidWhether class, be divided into cyclic peptide and linear peptides according to Cheng Huan. The cyclic peptide compound of having reported has many-sided biologically active, comprisesAntitumor, anti-HIV, antibacterial, antimalarial, sleep peacefully, suppress platelet aggregation, step-down, restraint of tyrosinase, inhibition Cycloxygenase, press downThe biologically actives such as lipid peroxidation enzyme processed, female hormone sample, immunosupress.
Due to varying of the ring amino acid whose number that comprises of propeptide-linear peptides and kind, cause cyclic peptideThe variation of synthetic method. Certain linear peptides is shown efficiently, and the reagent of condensation and method are to another peptide fastChain just may become poor efficiency or invalid.
Encircle dipeptides and there is the multiple important physiologically actives such as antibacterial, nowadays, do not seen anti-feelings influenza virus activityReport for work.
Summary of the invention
The invention discloses a kind of one ring dipeptides C6 with anti-avian influenza activity separating from medicinal fungus. The present inventionDisclose first this kind of newly activity of one of encircling dipeptides, proved 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 C6, hadThe effect of anti-avian influenza virus.
Technical scheme of the present invention is as follows:
First prepare phelliuns igniarius crude extract, made by following method:
(1) fermentative medium formula in gram/100 milliliters be:
Cornstarch 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) phelliuns igniarius bacterial classification is inoculated into conventional method in the conical flask that fluid nutrient medium is housed, with 20～35 DEG C of temperature, shaking flask rotating speed is 80～280r/min, under the condition of pH3～8, vibrations are cultivated 7～15 days; In cultivation, working as pH value fallsBy 2.5～4 o'clock, the seed in shaking flask is inoculated in the nutrient solution of 50L fermentation tank, with 20～35 DEG C of temperature, fermentation tank pressure0.1～0.2 kg/cm, pH3～8, throughput 0.5～1.1vvm, the condition that mixing speed is 100～280 revs/min,Cultivate 7～15 days, can utilize the full liquid of phelliuns igniarius mycelium fermentation to prepare phelliuns igniarius crude extract.
(3) get the full liquid of gained phelliuns igniarius mycelium fermentation in step (2), by its reduced pressure concentration in a usual manner, makeIts volume is concentrated to 1/2～1/5 of original volume;
(4) zymotic fluid after the ethanol that is 60～95% by percent by volume is concentrated to above-mentioned steps (3) extracts, itsIn, the amount that adds ethanol is 2～8 times of concentrate volume, can make concentration of alcohol in extract reach 60～90%, ethanol is excellentElect the edible ethanol of concentration 65-95% as, volume used is preferably 3-6 times of concentrate volume;
(5) to step (4) gained extract under 50～70 DEG C of conditions, heat 1～2 hour; Divide with conventional methodFrom, and remove impurity by cascade filtration, separate and obtain ethanol extract; Above-mentioned ethanol extract is reduced pressure dense in a usual mannerContracting, makes its volume be concentrated to 1/5～1/10 of original volume;
(6) concentrate of step (5) gained is dried with the method for frozen drying, obtains phelliuns igniarius and slightly carryThing.
Then from phelliuns igniarius crude extract, encircle dipeptides C6, step is as follows: phelliuns igniarius crude extract → purification on normal-phase silica gelThe anti-phase preparation of chromatography → methyl alcohol gradient elution → HPLC detection → high pressure → HPLC detection →1D-HNMR → ring dipeptides C6.
Concrete steps are:
(1) prepare phelliuns 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 columnChromatography, uses eluant, eluent wash-out 2-5 time;
(3) collect last eluent in above-mentioned steps (2), reduced pressure concentration, uses methyl alcohol to dissolve, methanol gel post layerAnalyse, use eluant, eluent wash-out;
(4) eluent that the step (3) that uses HPLC to detect collection obtains, suitably merges eluent, drying under reduced pressure;
(5) product step (4) being obtained carries out high pressure reversed phase chromatography, and eluant, eluent is methyl alcohol and water;
(6) product step (5) being obtained carries out HPLC detection, and drying under reduced pressure is ring dipeptides C6.
Preferably, the sample silica gel of mixing described in extraction step (2) is 100 order purification on normal-phase silica gel, and eluant, eluent is chloroform and/or firstAlcohol.
Eluant, eluent described in extraction step (3) is methyl alcohol.
HPLC condition described in extraction step (4) is 0min:100% water, 10min:100% methyl alcohol.
The described reversed material of extraction step (5) is C-18 or C-8, and eluant, eluent is methyl alcohol: water=25%-75%, heightPressing reversed phase chromatography number of times is 1-3 time.
The described HPLC appearance time of extraction step (6) is 4.95-5.47min.
(4) detailed description of the invention
Example 1:
Phelliuns igniarius crude extract, is made by following method:
(1) fermentative medium formula in gram/100 milliliters be:
Cornstarch 1% glucose 1%
Peptone 0.1% yeast extract 0.1%
Magnesium sulfate 0.1% potassium dihydrogen phosphate 0.01%
(2) phelliuns igniarius bacterial classification is inoculated into conventional method in the conical flask that fluid nutrient medium is housed, with 25 DEG CTemperature, shaking flask rotating speed is 110r/min, under pH7 condition, vibrations are cultivated 7 days; In cultivation when pH value drops to 3, by shaking flaskSeed is inoculated in the nutrient solution of 50L fermentation tank, with 25 DEG C of temperature, and fermentation tank pressure 0.1 kg/cm, pH3, ventilationAmount 0.5-1.1vvm, the condition that mixing speed is 100 revs/min, cultivates 7 days, can utilize the full liquid of phelliuns igniarius mycelium fermentationPrepare phelliuns igniarius crude extract.
(3) get the full liquid of gained phelliuns igniarius mycelium fermentation in step (2), by its reduced pressure concentration in a usual manner, makeIts volume is concentrated to 1/3 of original volume;
(4) zymotic fluid after the ethanol that is 70% by percent by volume is concentrated to above-mentioned steps (3) extracts, wherein,The amount that adds ethanol is 5 times of concentrate volume, can make concentration of alcohol in extract reach 55%;
(5) to step (4) gained extract under 70 DEG C of conditions, heat 1 hour; Separate with conventional method, and logicalCross cascade filtration and remove impurity, separate and obtain ethanol extract; By above-mentioned ethanol extract reduced pressure concentration in a usual manner, make itVolume is concentrated to 1/5 of original volume;
(6) concentrate of step (5) gained is dried with the method for frozen drying, obtains phelliuns igniarius and slightly carryThing.
Take crude extract 300g and equal-volume 100 order purification on normal-phase silica gel mix stirring, carry out silica gel normal phase column chromatography. Chromatography
Fixing is 200 order purification on normal-phase silica gel mutually, the high 1m of post, and diameter 20cm, eluant, eluent is for being respectively chloroform, chloroform: methyl alcohol=75:1 is 4,5 column volumes of wash-out respectively. And by gained eluent called after Fr-1 respectively, Fr-2. Fr-2 is dissolved with methyl alcohol, enterRow methanol gel chromatography, eluant, eluent is methyl alcohol. Then be that HPCL detects, condition is 0min:0% methyl alcohol, 10min:100% firstAlcohol, appearance time is 4.95min. Then, carry out the anti-phase preparation of high pressure, A is water mutually, and B is methyl alcohol mutually. Wash-out concentration is 25%-75% methanol aqueous solution. Collect eluent, evaporated under reduced pressure, HPLC detects, and suitably merges, and carries out 1D-HNMR nuclear magnetic resonance and dividesAnalyse, frequency is 400MHZ, and analysis result is δ 7.51 (s, 1H), 4.03 (dd, J=34.9,28.1Hz, 2H), 3.81 – 3.33 (m,2H),2.36(d,J＝8.8Hz,2H),2.15–1.68(m,5H),1.60(dd,J＝10.1,3.8Hz,2H),0.94(ddd,J=18.8,17.2,9.8Hz, 7H). prove that ring dipeptides C6 has structure shown in formula I:
(7) compound that adopts step (6) to obtain carries out the mensuration to mdck cell toxicity, determines its median toxic concentration(CC50) and maximum safe concentration.
On 96 orifice plates, 100 μ l/ holes add 4 × 105ml-1The mdck cell suspension of concentration, cultivates after 24h, thin at individual layerOn born of the same parents, add respectively variable concentrations containing sample maintenance medium, every kind of concentration repeats 3 holes, and establishes normal cell contrast. Put 37 DEG C, 5%CO2In incubator, cultivate after 48h, abandon nutrient solution supernatant, 100 μ l/ holes add 5mg.ml-1The maintenance medium of MTT, continues to cultivate 1hAfter, abandon MTT supernatant, every hole adds lysate (DMSO) 100 μ l, vibration 5-10min, dissolving completely to be crystallized, ELIASA is surveyed 492nmThe OD value at place. Calculate the median toxic concentration (CC of sample50) and maximum safe concentration. As the OD of application of sample group492Be worth significantly not lowIn cell control group OD492Time, this concentration is the maximum safe concentration of sample.
The ring dipeptides each mass concentration group of C6 (n=12)
| Sequence number | Density (ug.mL-1) | Pathology rate |
| 1 | 100 | 0.95170116583 |
| 2 | 200 | 0.93052581489 |
| 3 | 100 | 0.71068284559 |
| 4 | 50 | 0.30716155127 |
| 5 | 25 | 0.15488936474 |
| Cell contrast |
Known by table 1: ring dipeptides C6 maximum safe concentration is 25ug/mL.
Example 2:
Phelliuns igniarius crude extract, is made by following method:
(1) fermentative medium formula in gram/100 milliliters be:
Cornstarch 3% glucose 2%
Peptone 0.5% yeast extract 0.5%
Magnesium sulfate 0.5% potassium dihydrogen phosphate 0.05%
(2) phelliuns igniarius bacterial classification is inoculated into conventional method in the conical flask that fluid nutrient medium is housed, with 30 DEG CTemperature, shaking flask rotating speed is 180r/min, under pH6 condition, vibrations are cultivated 15 days; In cultivation in the time that pH value drops to 2.5, by shaking flaskSeed be inoculated in the nutrient solution of 50L fermentation tank, with 30 DEG C of temperature, fermentation tank pressure 0.2 kg/cm, pH3, logicalTolerance 0.5-1.1vvm, the condition that mixing speed is 180 revs/min, cultivates 15 days, can utilize phelliuns igniarius mycelium fermentation completeLiquid is prepared phelliuns igniarius crude extract.
(3) get the full liquid of gained phelliuns igniarius mycelium fermentation in step (2), by its reduced pressure concentration in a usual manner, makeIts volume is concentrated to 1/5 of original volume;
(4) zymotic fluid after the ethanol that is 90% by percent by volume is concentrated to above-mentioned steps (3) extracts, wherein,The amount that adds ethanol is 4 times of concentrate volume, can make concentration of alcohol in extract reach 70%;
(5) to step (4) gained extract under 55 DEG C of conditions, heat 2.5 hours; Separate with conventional method, andRemove impurity by cascade filtration, separate and obtain ethanol extract; By above-mentioned ethanol extract reduced pressure concentration in a usual manner, makeIts volume is concentrated to 1/10 of original volume;
(6) concentrate of step (5) gained is dried with the method for frozen drying, obtains phelliuns igniarius and slightly carryThing.
Take crude extract 100g and equal-volume 100 order purification on normal-phase silica gel mix stirring, carry out silica gel normal phase column chromatography. Chromatography is solidPhasing is 200-300 order purification on normal-phase silica gel, the high 0.8m of post, and diameter 10cm, eluant, eluent is for being respectively chloroform, chloroform: methyl alcohol=75:14,5 column volumes of wash-out respectively. And by gained eluent called after Fr-1 respectively, Fr-2. Fr-2 is dissolved with methyl alcohol, carry out firstAlcogel chromatography, eluant, eluent is methyl alcohol. Then be that HPCL detects, condition is 0min:0% methyl alcohol, and 10min:100% methyl alcohol, goes outPeak time is 5.10min. Then, carry out the anti-phase preparation of high pressure, A is water mutually, and B is methyl alcohol mutually. Wash-out concentration is 25%-70%'sMethanol aqueous solution. Collect eluent, evaporated under reduced pressure, HPLC detects, and suitably merges, and carries out1D-HNMR nuclear magnetic resonance spectroscopy, frequencyFor 400MHZ, analysis result is δ 7.51 (s, 1H), 4.03 (dd, J=34.9,28.1Hz, 2H), and 3.81 – 3.33 (m, 2H),2.36(d,J＝8.8Hz,2H),2.15–1.68(m,5H),1.60(dd,J＝10.1,3.8Hz,2H),0.94(ddd,J＝18.8,17.2,9.8Hz, 7H). prove that ring dipeptides C6 has structure shown in formula I.
(7) adopt within the scope of the maximum safe concentration that obtains of step (6), carry out sample in mdck cell level to H5N1The effect of strain, the half toxic concentration (CC of calculation sample50) and medium effective concentration (EC50), obtain the selection index of sample(SI)＝CC50/EC50, calculate inhibiting rate.
In test, normal cell control group (only adding cell maintenance medium) is set, virus control group (only adding virus liquid), qualityConcentration 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 μL/ hole adds 10000TCID50Virus liquid (10-4.43), 3 repetitions are set. 37 DEG C of absorption 2h, abandon virus liquid supernatant, 100 μ l/ holesAdd the sample liquid of variable concentrations. Put 37 DEG C, 5%CO2In incubator, continue to cultivate 48h. Adopt mtt assay to measure OD492Value, calculatesMedium effective concentration (EC50). As the OD of application of sample group492Value is significantly greater than virus control group OD492Time, show that this sample liquid can be remarkableSuppress virus infections MDCK, there is antiviral activity.
According to the cytopathy variability calculating and viral inhibiting rate. With the Probit recurrence of statistics software SPSS11.5Method, the half toxic concentration (CC of calculation sample50) and medium effective concentration (EC50), obtain the selection index (SI) of sample=CC50/EC50。
Calculate viral inhibiting rate according to lower formula:
Virus inhibiting rate=(A-B)/(C-B) × 100%
A: sample liquid processed group OD value, B: virus control group OD value, C: cell control group OD value
The inhibitory action experimental result of table 2 compound to H5N1 virus
Known by table 2: ring dipeptides C6 can reach the highest 53.9% inhibiting rate to H5N1 strain under 0.4ug/ml concentration, thisTime CC50For 73.15ug/ml, EC50For 1.08ug/ml, SI is 67.73.
Claims (10)
1. ring dipeptides C6, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that described ring dipeptides C6 has formulaStructure shown in I:
2. ring dipeptides C6 as claimed in claim 1, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in thatRing dipeptides C6 extracts from phelliuns igniarius, and extraction step order is as follows:
(1) prepare phelliuns 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,With eluant, eluent wash-out 2-5 time;
(3) collect last eluent in above-mentioned steps (2), reduced pressure concentration, uses methyl alcohol to dissolve, methanol gel column chromatography,Use eluant, eluent wash-out;
(4) eluent that the step (3) that uses HPLC to detect collection obtains, suitably merges eluent, drying under reduced pressure;
(5) product step (4) being obtained carries out high pressure reversed phase chromatography, and eluant, eluent is methyl alcohol and water;
(6) product step (5) being obtained carries out HPLC detection, and drying under reduced pressure is ring dipeptides C6.
3. ring dipeptides C6 as claimed in claim 2, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in thatDescribed phelliuns igniarius crude extract, is made by following method:
(1) phelliuns igniarius bacterial classification is inoculated into conventional method in the conical flask that fluid nutrient medium is housed, with 20～35 DEG CTemperature, shaking flask rotating speed is 80～280r/min, under the condition of pH3～8, vibrations are cultivated 7～15 days; In cultivation, work as pH value and drop to 2.5～4 o'clock, the seed in shaking flask is inoculated in the nutrient solution of 50L fermentation tank, with 20～35 DEG C of temperature, fermentation tank pressure 0.1～0.2 kg/cm, pH3～8, throughput 0.5～1.1vvm, the condition that mixing speed is 100～280 revs/min, cultivates 7～15 days, can obtain the full liquid of phelliuns igniarius mycelium fermentation;
(2) get the full liquid of gained phelliuns igniarius mycelium fermentation in step (1), by its reduced pressure concentration in a usual manner, make its bodyLong-pending 1/2～1/5 of the original volume that is concentrated to;
(3) zymotic fluid after the ethanol that is 60～95% by percent by volume is concentrated to above-mentioned steps (2) extracts, wherein,The amount that adds ethanol is 2～8 times of concentrate volume, can make concentration of alcohol in extract reach 60～90%;
(4) to step (3) gained extract under 50～70 DEG C of conditions, heat 1～2 hour; Separate with conventional method, andRemove impurity by cascade filtration, separate and obtain ethanol extract; By above-mentioned ethanol extract reduced pressure concentration in a usual manner, makeIts volume is concentrated to 1/5～1/10 of original volume;
(5) concentrate of step (4) gained is dried with the method for frozen drying, obtains phelliuns igniarius crude extract.
4. ring dipeptides C6 as claimed in claim 3, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in thatDescribed Liquid Culture based formulas in gram/100 milliliters be:
5. ring dipeptides C6 as claimed in claim 3, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in thatThe ethanol of phelliuns igniarius crude extract preparation process (3) is concentration 65%-95% edible ethanol, and volume used is 3-6 times of volume.
6. ring dipeptides C6 as claimed in claim 2, 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 order purification on normal-phase silica gel, and eluant, eluent is chloroform and/or methyl alcohol.
7. ring dipeptides C6 as claimed in claim 2, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that,Eluant, eluent described in step (3) is methyl alcohol.
8. ring dipeptides C6 as claimed in claim 2, in the application of preparing on anti-avian influenza H5N1 virus medicine, is characterized in that,HPLC condition described in step (4) is 0min:100% water, 10min:100% methyl alcohol.
9. ring dipeptides C6 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, eluent is methyl alcohol: water=25%-75%, high pressure reversed phase chromatographyNumber is for 1-3 time.
10. ring dipeptides C6 as claimed in claim 2 is in the application of preparing on anti-avian influenza H5N1 virus medicine, and its feature existsIn, the described HPLC appearance time of step (6) is 4.95-5.47min.
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| 桑黄粗多糖除蛋白及抗肿瘤活性;梁大勇等;《生物加工工程》;20120531;第10卷(第3期);第56-60页 * |
| 火针层孔菌（桑黄）粗多糖对荷瘤小鼠的免疫调节研究;宋爱荣等;《菌物学报》;20090315;第28卷(第2期);第295-298页 * |
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