CN103059032B - The isolation technique of trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone in Phellinus bacterium - Google Patents
The isolation technique of trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone in Phellinus bacterium Download PDFInfo
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Abstract
Do you the invention discloses a kind of Phellinus bacterium (phelliuns igniarius Phellinus? igniarius (L? ex? Fr) Quel, phellinus linteus phellinus? linteus (Berk? et? Curt) Teng, Phellinus baumii, Kazakhstan base of a fruit phellinus Phellinus? hartigii (Allesch? et? Schnabl) Imaz) in trans-six hydrogen-3-(1-benzyl) pyrrolo-[1; 2-a] separation method of pyrazine-Isosorbide-5-Nitrae-diketone.First Phellinus bacterium crude extract is prepared; then purification on normal-phase silica gel chromatography is carried out; then be methanol elution gradient; again carrying out purification on normal-phase silica gel chromatography, is then anti-phase preparation, methanol gel chromatography again; then HPLC detects is finally that 1D-HNMR detects; finally obtain trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone.
Description
Technical field
The invention belongs to biotech medicine product field.
Background technology
Phellinus, sporophore stockless, the flat semisphere of cap or the shape of a hoof, 2-12*3-21 centimetre, thick 1.5-10 centimetre, wooden, shallow liver brown to lead or black, always often chap, without cot, there is trickle fine hair at initial stage, rear change is without hair, there is concentric ring rib. edge is blunt, dark cinnamon is to light coffee color, downside is without thalamium. bacterial context dark brown, firmly, wooden. tube and bacterial context are closely homochromy, multilayer, but level is not obvious, old tube layer is full of white hypha. and mouth of pipe rust brown is to dark reddish brown, circular, every millimeter 4-5. spore is subsphaeroidal, smooth, colourless, 5-6*3-4 micron. bristle top is sharp-pointed, base portion expands, 10-25*5-7 micron. mycelia is branch not, without tabula, diameter 3-5 micron.
At present, the purification process of epiphyte product is more, mainly contain precipitation classification, preparation property high performance liquid chromatography, column chromatography, ultrafiltration process, centrifugation chromatography, etc. several method.And wherein apply maximum column chromatographies that surely belongs to, be divided into two classes: one is the gel filtration chromatography only having molecular sieve effect, conventional gel has dextrane gel and sepharose.Two is ion exchange chromatographies.But mainly for separating of polysaccharide, hyaluronic acid etc., the product obtained after most separation is mixture.The present invention uses multiple chromatographic technique to combine, and resolution is high, and applying this invention can be exquisite to sterling.
Summary of the invention
The invention discloses trans-six hydrogen-3-(1-benzyl) pyrrolo-[1 in a kind of Phellinus bacterium (phelliuns igniarius Phellinusigniarius (LexFr) Quel, phellinus linteus phellinuslinteus (BerketCurt) Teng, Phellinus baumii, Kazakhstan base of a fruit phellinus Phellinushartigii (AlleschetSchnabl) Imaz); 2-a] separation method of pyrazine-Isosorbide-5-Nitrae-diketone.First Phellinus bacterium crude extract is prepared; then purification on normal-phase silica gel chromatography is carried out; then be methanol elution gradient; again carrying out purification on normal-phase silica gel chromatography, is then anti-phase preparation, methanol gel chromatography again; then HPLC detects is finally that 1D-HNMR detects; obtain trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone.This compound has the effect suppressing phytotoxin, broad-spectrum antimicrobial and raising intestinal cell maturation.
Technical scheme of the present invention is as follows:
Phellinus crude extract, is obtained by following method:
(a) fermentative medium formula in gram/100 milliliters be:
B Phellinus bacterial classification is inoculated into and is equipped with in the Erlenmeyer flask of liquid nutrient medium by () in conventional manner, 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 nutrient solution of 50L fermentor tank, with temperature 20 ~ 35 DEG C, fermentor tank pressure 0.1 ~ 0.2 kg/cm, pH3 ~ 8, air flow 0.5 ~ 1.1vvm, the condition that stirring velocity is 100 ~ 280 revs/min, cultivate 7 ~ 15 days, phellinus igniarius mycelium fermentation liquid can be utilized to prepare Phellinus crude extract;
C () gets gained phellinus igniarius mycelium fermentation liquid in step (b), by its concentrating under reduced pressure in a usual manner, make its volume concentration to 1/2 ~ 1/5 of original volume;
(d) by volume percent be 60 ~ 95% ethanol concentrated to above-mentioned steps (c) after fermented liquid extract, wherein, the amount adding ethanol is 2 ~ 5 times of concentrated solution volume, and alcohol concn in extracting solution can be made to reach 60 ~ 90%;
E (), heats 1 ~ 2 hour step (d) gained extracting solution under 50 ~ 70 DEG C of conditions; 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;
F the concentrated solution of () just step (e) gained carries out drying with the method for frozen drying, obtain Phellinus crude extract.
Be separated the method for trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone:
Phellinus bacterium crude extract → purification on normal-phase silica gel chromatography → chloroform and methanol elution gradient → purification on normal-phase silica gel chromatography → methanol gel chromatography → TLC detect → and reversed-phase silica gel chromatography → HPLC detects → methanol gel chromatography → evaporated under reduced pressure → trans-six hydrogen-3-(1-benzyl) pyrrolo-[1; 2-a] pyrazine-Isosorbide-5-Nitrae-diketone.
Concrete grammar is:
(1) Phellinus bacterium 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 3-4 time;
(3) collect last elutriant in above-mentioned steps (2), concentrating under reduced pressure, uses dissolve with methanol, methanol gel column chromatography, with eluent, uses TLC to detect the elutriant collected, suitably merges elutriant, drying under reduced pressure;
(4) product that step (3) obtains is carried out methanol gel column chromatography, again with eluent;
(5) product that step (4) obtains is carried out reversed-phase silica gel chromatography, eluent is methyl alcohol: water=30%-70%;
(6) appropriateness merges elutriant, drying under reduced pressure, collects elutriant, and HPLC detects, and drying under reduced pressure, is trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone.
The present invention is by-six hydrogen-3-(1-benzyl) pyrrolo-[1 trans in Phellinus bacterium; 2-a] pyrazine-1; the significant advantage of the isolation technique of 4-diketone: present method adopts multiple chromatographic technique to combine; can be exquisite clear and definite to structure; trans-six hydrogen-3-(1-benzyl) pyrrolo-[1 that purity is greater than 95%; 2-a] pyrazine-Isosorbide-5-Nitrae-diketone.Mature technical route is clear and definite, efficiently accurately.
Accompanying drawing explanation
Fig. 1 is the structural formula of trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone;
Fig. 2 is that the one-dimensional nuclear magnetic resonance H of trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone composes.
Embodiment
Example 1:
Phellinus crude extract, is obtained by following method:
(a) fermentative medium formula in gram/100 milliliters be:
B Phellinus bacterial classification is inoculated into and is equipped with in the Erlenmeyer flask of liquid nutrient medium by () in conventional manner, 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 nutrient solution of 50L fermentor tank, with temperature 25 DEG C, fermentor tank pressure 0.1 kg/cm, pH3, air flow 0.5-1.1vvm, the condition that stirring velocity is 100 revs/min, cultivate 7 days, phellinus igniarius mycelium fermentation liquid can be utilized to prepare Phellinus crude extract;
C () gets gained phellinus igniarius mycelium fermentation liquid in step (b), by its concentrating under reduced pressure in a usual manner, make its volume concentration to 1/3 of original volume;
(d) by volume percent be 70% ethanol concentrated to above-mentioned steps (c) after fermented liquid extract, wherein, the amount adding ethanol is 5 times of concentrated solution volume, and alcohol concn in extracting solution can be made to reach 55%;
E (), heats 1 hour step (d) gained extracting solution under 70 DEG C of conditions; 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;
F the concentrated solution of () just step (e) gained carries out drying with the method for frozen drying, obtain Phellinus crude extract.
Take crude extract 250g and equal-volume 100 order purification on normal-phase silica gel to mix and stir, carry out silica normal phase column chromatography.Chromatography stationary phase is 300 order purification on normal-phase silica gel, post height 1.0m, diameter 14cm, and eluent is respectively chloroform, chloroform: methyl alcohol=300:1 is wash-out 3,4,4,4 column volumes respectively.Elutriant is called after Fr-1, Fr-2, Fr-3 and Fr-4 respectively.By Fr-4 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 300 order purification on normal-phase silica gel.Eluent is chloroform and methyl alcohol.Carry out methanol gel column chromatography.Then, carry out reversed-phase silica gel chromatography, HPLC detects the elutriant collected, condition is 0min, 100%A water → 10min, 100% methyl alcohol, appearance time is 4.38min, suitable merging, drying under reduced pressure, again carry out methanol gel chromatography, elutriant is subtracted evaporate to dryness, carry out 1D-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, analytical results is δ 7.39 – 7.08 (m, 5H), 4.20 (t, J=4.4Hz, 1H), 3.53 (dt, J=16.8, 8.3Hz, 1H), 3.19 (dd, J=13.6, 4.7Hz, 1H), 2.99 (dd, J=13.6, 4.6Hz, 1H), 2.59 (dd, J=10.3, 6.3Hz, 1H), 2.03 (dd, J=11.9, 6.1Hz, 1H), 1.90 (d, J=5.4Hz, 1H), 1.73 – 1.48 (m, 2H). prove trans-six hydrogen-3-(1-benzyl) pyrrolo-[1, 2-a) pyrazine-1, 4-diketone.
Example 2:
Phellinus crude extract, is obtained by following method:
(a) fermentative medium formula in gram/100 milliliters be:
B Phellinus bacterial classification is inoculated into and is equipped with in the Erlenmeyer flask of liquid nutrient medium by () in conventional manner, 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 nutrient solution of 50L fermentor tank, with temperature 30 DEG C, fermentor tank pressure 0.2 kg/cm, pH3, air flow 0.5-1.1vvm, the condition that stirring velocity is 180 revs/min, cultivate 15 days, phellinus igniarius mycelium fermentation liquid can be utilized to prepare Phellinus crude extract;
C () gets gained phellinus igniarius mycelium fermentation liquid in step (b), by its concentrating under reduced pressure in a usual manner, make its volume concentration to 1/5 of original volume;
(d) by volume percent be 90% ethanol concentrated to above-mentioned steps (c) after fermented liquid extract, wherein, the amount adding ethanol is 4 times of concentrated solution volume, and alcohol concn in extracting solution can be made to reach 70%;
E (), heats 2.5 hours step (d) gained extracting solution under 55 DEG C of conditions; 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;
F the concentrated solution of () just step (e) gained carries out drying with the method for frozen drying, obtain Phellinus crude extract.
Take crude extract 100g and equal-volume 100 order purification on normal-phase silica gel to mix and stir, carry out silica normal phase column chromatography.Chromatography stationary phase is 300 order purification on normal-phase silica gel, post height 0.8m, diameter 14cm, and eluent is respectively chloroform, chloroform: methyl alcohol=300:1-50:1 is wash-out 2,3,3,3 column volumes respectively.Elutriant is called after Fr-1, Fr-2, Fr-3 and Fr-4 respectively.By Fr-4 equal-volume silica gel mixed sample, use silica gel is 100-200 order purification on normal-phase silica gel, and pentaploid amasss silica gel column chromatography, and the silica gel of use is 300 order purification on normal-phase silica gel.Eluent is chloroform and methyl alcohol.Carry out methanol gel column chromatography.Then, carry out reversed-phase silica gel chromatography, HPLC detects the elutriant collected, condition is 0min, 100%A water → 10min, 100% methyl alcohol, appearance time is 4.60min, suitable merging, drying under reduced pressure, again carry out methanol gel chromatography, elutriant is subtracted evaporate to dryness, carry out 1D-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, analytical results is δ 7.39 – 7.08 (m, 5H), 4.20 (t, J=4.4Hz, 1H), 3.53 (dt, J=16.8, 8.3Hz, 1H), 3.19 (dd, J=13.6, 4.7Hz, 1H), 2.99 (dd, J=13.6, 4.6Hz, 1H), 2.59 (dd, J=10.3, 6.3Hz, 1H), 2.03 (dd, J=11.9, 6.1Hz, 1H), 1.90 (d, J=5.4Hz, 1H), 1.73 – 1.48 (m, 2H). prove trans-six hydrogen-3-(1-benzyl) pyrrolo-[1, 2-a) pyrazine-1, 4-diketone.
Claims (6)
1. the separation method of trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone in Phellinus bacterium, its sequence of steps is as follows:
(1) Phellinus bacterium 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 3-4 time;
(3) collect last elutriant in above-mentioned steps (2), concentrating under reduced pressure, uses dissolve with methanol, methanol gel column chromatography, with eluent, uses TLC to detect the elutriant collected, suitably merges elutriant, drying under reduced pressure;
(4) product that step (3) obtains is carried out methanol gel column chromatography, again with eluent;
(5) product that step (4) obtains is carried out reversed-phase silica gel chromatography, eluent is methyl alcohol: water=30%-70%;
(6) TLC detects, and appropriateness merges elutriant, drying under reduced pressure, collects elutriant, HPLC detects, and HPLC condition is 0min, 100% water → 10min, 100% methyl alcohol, drying under reduced pressure, is trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone;
Wherein said Phellinus crude extract, is obtained by following method:
(a) fermentative medium formula in gram/100 milliliters be:
B the fermentation culture method of () described Phellinus crude extract is, by Phellinus strain inoculation in the Erlenmeyer flask that liquid nutrient medium is housed, 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 nutrient solution of 50L fermentor tank, with temperature 20 ~ 35 DEG C, fermentor tank pressure 0.1 ~ 0.2 kg/cm, pH3 ~ 8, air flow 0.5 ~ 1.1vvm, the condition that stirring velocity is 100 ~ 280 revs/min, cultivate 7 ~ 15 days, phellinus igniarius mycelium fermentation liquid can be utilized to prepare Phellinus crude extract;
C () gets gained phellinus igniarius mycelium fermentation liquid in step (b), by its concentrating under reduced pressure, make its volume concentration to 1/2 ~ 1/5 of original volume;
(d) by volume percent be 60 ~ 95% ethanol concentrated to above-mentioned steps (c) after fermented liquid extract, wherein, the amount adding ethanol is 2 ~ 5 times of concentrated solution volume, and alcohol concn in extracting solution can be made to reach 60 ~ 90%;
E (), heats 1 ~ 2 hour step (d) gained extracting solution under 50 ~ 70 DEG C of conditions; Be separated, and remove impurity by cascade filtration, be separated and obtain ethanol extract; By above-mentioned ethanol extract concentrating under reduced pressure, make its volume concentration to 1/5 ~ 1/10 of original volume;
F the concentrated solution of step (e) gained is carried out drying with the method for frozen drying by (), obtain Phellinus crude extract.
2. trans-six hydrogen-3-(1-benzyl) pyrrolo-[1 in Phellinus bacterium as claimed in claim 1; 2-a] pyrazine-1; the separation method of 4-diketone, is characterized in that, the sample silica gel of mixing described in step (2) is 100-200 order purification on normal-phase silica gel.
3. the separation method of trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone in Phellinus bacterium as claimed in claim 1, it is characterized in that, the chromatographic silica gel described in step (2) is positive 200-300 order.
4. trans-six hydrogen-3-(1-benzyl) pyrrolo-[1 in Phellinus bacterium as claimed in claim 1; 2-a] pyrazine-1; the separation method of 4-diketone; it is characterized in that; gel described in step (4) is SephadexLH-20 or SephadexLH-25, and eluent is methyl alcohol.
5. the separation method of trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone in Phellinus bacterium as claimed in claim 1, it is characterized in that, the reversed material described in step (5) is C-18 or C-8.
6. the separation method of trans-six hydrogen-3-(1-benzyl) pyrrolo-[1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone in Phellinus bacterium as claimed in claim 1, it is characterized in that, the HPLC appearance time described in step (6) is 4.3-5.6min.
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| CN1557961A (en) * | 2004-01-13 | 2004-12-29 | 浙江大学 | Biologic preparation method of cyclic dipeptide analogue compound and uses thereof |
| US20060147410A1 (en) * | 2004-12-16 | 2006-07-06 | Pei-Yuan Qian | Use of marine fungus originated compounds as antifouling agents |
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| KR100474111B1 (en) * | 2001-12-26 | 2005-03-10 | 한국생명공학연구원 | Streptomyces sp. AMLK-335 strain and use of compound produced therefrom |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1557961A (en) * | 2004-01-13 | 2004-12-29 | 浙江大学 | Biologic preparation method of cyclic dipeptide analogue compound and uses thereof |
| US20060147410A1 (en) * | 2004-12-16 | 2006-07-06 | Pei-Yuan Qian | Use of marine fungus originated compounds as antifouling agents |
Non-Patent Citations (3)
| Title |
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| DD-Diketopiperazines: Antibiotics Active against Vibrio anguillarum Isolated from Marine Bacteria Associated with Cultures of Pecten maximus;Faouzi Fdhila，等;《Journal of Natural Products》;20030917;第66卷(第10期);第1299-1301页，尤其第1300页左栏 * |
| Diketopiperazines and Nucleosides from Mycelial Cultures of Dichomitus squalens;XIE Hai-Hui，等;《Chin. J. Nat. Med.》;20080930;第6卷(第5期);第395-398页 * |
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