CN103130808B - The also anti-phase preparation isolation technics of normal pressure of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium - Google Patents
The also anti-phase preparation isolation technics of normal pressure of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium Download PDFInfo
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Abstract
The invention discloses a kind of Phellinus bacterium (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, Kazakhstan base of a fruit phellinus <i>Phellinus</i><i>Hartigii</i>(Allesch et Schnabl) Imaz) the also separation method of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in. First prepare Phellinus bacterium crude extract; then carry out purification on normal-phase silica gel chromatography; then methanol gel chromatography, carries out purification on normal-phase silica gel chromatography again, follows methanol gel chromatography again; next the anti-phase preparation of normal pressure; then carry out HPLC detection, last 1D-HNMR detects, and obtains (8aS)-hexahydropyrrolo also [1; 2-a] pyrazine-Isosorbide-5-Nitrae-diketone.
Description
Technical field
The invention belongs to biotech medicine product field.
Background technology
Phellinus, fructification stockless, the flat hemispherical 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, be full of cracks often always, without cot, there is trickle fine hair at the initial stage, and rear change is without hair,There is concentric ring rib. edge is blunt, and dark cinnamon is to light coffee color, and downside is without hymenium. bacterial context dark brown is hard, wooden. tubeClosely homochromy with bacterial context, multilayer, but level is not obvious, and old tube layer is full of white hypha. and the mouth of pipe becomes rusty brown to dark reddish brown,Circle, every millimeter 4-5. spore is subsphaeroidal, smooth, colourless, 5-6*3-4 micron. and bristle top is sharp-pointed, and 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 contains precipitation classification, preparation property high performance liquid chroma-tography, postChromatography, ultrafiltration, centrifugation chromatography, etc. several method. And wherein apply maximum column chromatographies that surely belongs to, divideBe two classes: the one, only have the gel filtration chromatography of molecular sieve effect, conventional gel has sephadex and Ago-Gel.The 2nd, ion-exchange chromatography. But, mainly for separating of polysaccharide, hyaluronic acid etc., the product obtaining after most separation is for mixedCompound. The present invention uses multiple chromatographic technique to combine, and separating degree is high, and applying this invention can be exquisite to sterling.
Summary of the invention
The invention discloses a kind of Phellinus bacterium (phelliuns igniarius Phellinusigniarius (LexFr) Quel, cracked feet wood layerPore fungi phellinuslinteus (BerketCurt) Teng, Phellinus baumii, Kazakhstan base of a fruit phellinus Phellinushartigii(AlleschetSchnabl) Imaz) in the also separation method of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo. First systemStandby Phellinus bacterium crude extract, then carries out purification on normal-phase silica gel chromatography, and then methanol gel chromatography, carries out purification on normal-phase silica gel chromatography again,Follow methanol gel chromatography again, the next anti-phase preparation of normal pressure, then carries out HPLC detection, last 1D-HNMRDetect, obtain also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo. The effect of this compound not yet has relevant report at present.
Technical scheme of the present invention is as follows:
Phellinus crude extract, is made by following method:
(a) 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%
(b) Phellinus 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 flaskRotating speed is 80～280r/min, and under the condition of pH3～8, vibrations are cultivated 7～15 days; In cultivation, work as pH value and drop to 2.5～4Time, 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, trainingSupport 7～15 days, can utilize phellinus igniarius mycelium fermentation liquid to prepare Phellinus crude extract;
(c) get gained phellinus igniarius mycelium fermentation liquid in step (b), by its reduced pressure concentration in a usual manner, its volume is concentrated to former1/2～1/5 of volume;
(d) zymotic fluid after the ethanol that is 60～95% by percent by volume is concentrated to above-mentioned steps (c) extracts, and wherein, addsThe amount of ethanol is 2～5 times of concentrate volume, can make concentration of alcohol in extract reach 60～90%;
(e) to step (d) 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,Make its volume be concentrated to 1/5～1/10 of original volume;
(f) just the concentrate of step (e) gained is dried with the method for frozen drying, obtains Phellinus crude extract.
Separate the also method of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo:
The anti-phase system of Phellinus bacterium crude extract → purification on normal-phase silica gel chromatography → methyl alcohol gradient elution → purification on normal-phase silica gel chromatography → methanol gel → normal pressureStandby → HPLC detection → 1D-HNMR → (8aS)-hexahydropyrrolo is [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone also.
Concrete grammar is:
(1) prepare Phellinus bacterium 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 the last eluent that above-mentioned steps (2) obtains, reduced pressure concentration, uses methyl alcohol to dissolve, methanol gel post layerAnalyse, use eluant, eluent wash-out;
(4) product step (3) being obtained carries out purification on normal-phase silica gel chromatography, uses eluant, eluent wash-out;
(5) collect eluent, suitably merge, evaporated under reduced pressure, carries out methanol gel chromatography;
(6) product step (5) being obtained carries out normal pressure reversed phase chromatography, and eluant, eluent is methyl alcohol and water;
(7) HPLC detects, and drying under reduced pressure, is also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo.
The present invention is by the also significant advantage of the isolation technics of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium:This method adopts multiple chromatographic technique to combine, can be exquisite clear and definite to structure, and (8aS)-hexahydropyrrolo that purity is greater than 95%And [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone. Mature technical route is clear and definite, efficiently accurate.
Brief description of the drawings
Fig. 1 is the also structural formula of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo;
Fig. 2 is the also one-dimensional nuclear magnetic resonance H spectrum of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo.
Detailed description of the invention
Example 1:
Phellinus crude extract, is made by following method:
(a) 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%
(b) Phellinus bacterial classification is inoculated into conventional method in the conical flask that fluid nutrient medium is housed, with 25 DEG C of temperature, shaking flask rotating speedFor 110r/min, under pH7 condition, vibrations are cultivated 7 days; In cultivation, in the time that pH value drops to 3, the seed in shaking flask is connectPlant in the nutrient solution of 50L fermentation tank, with 25 DEG C of temperature, 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 phellinus igniarius mycelium fermentation liquid systemStandby Phellinus crude extract;
(c) get gained phellinus igniarius mycelium fermentation liquid in step (b), by its reduced pressure concentration in a usual manner, its volume is concentrated to former1/3 of volume;
(d) zymotic fluid after the ethanol that is 70% by percent by volume is concentrated to above-mentioned steps (c) extracts, and wherein, adds ethanolAmount be 5 times of concentrate volume, can make concentration of alcohol in extract reach 55%;
(e) to step (d) gained extract under 70 DEG C of conditions, heat 1 hour; Separate with conventional method, and pass through secondaryRemove by filter impurity, separate and obtain ethanol extract; By above-mentioned ethanol extract reduced pressure concentration in a usual manner, make its bodyLong-pending 1/5 of the original volume that is concentrated to;
(f) just the concentrate of step (e) gained is dried with the method for frozen drying, obtains Phellinus crude extract.
Take 300g crude extract and isopyknic 100 order purification on normal-phase silica gel mix, silica gel normal phase column chromatography. Chromatography is fixed phaseFor 200-300 order purification on normal-phase silica gel, the high 1.5m of post, diameter 20cm, eluant, eluent is for being respectively chloroform, chloroform: methyl alcohol=200:1,100:1,40:1 is 4,5,5,5 column volumes of wash-out respectively. Last eluent is suitably merged, evaporated under reduced pressure,Carry out methanol gel chromatography. Collect eluent, suitably merge, with equal-volume silica gel mixed sample, the fixing silicon using mutually of chromatographyGlue is 200-300 order purification on normal-phase silica gel. Eluant, eluent is chloroform and methyl alcohol. Collect eluent, carry out methanol gel column chromatography.Then, carry out the anti-phase preparation of normal pressure, A is water mutually, and B is methyl alcohol mutually. Collect eluent, evaporated under reduced pressure, HPLC detects,Condition is 0min:95% water, 10min:100% methyl alcohol, and going out the cutting edge of a knife or a sword time is that 5.95min suitably merges, and carries out 1D-HNMRNuclear magnetic resonance spectroscopy, frequency is 400MHZ, analysis result is δ 7.30 (s, 1H), 4.06 (dd, J=15.8,10.2Hz, 2H),3.87(dd,J＝16.5,4.4Hz,1H),3.65–3.48(m,2H),2.42–2.26(m,1H),2.03(dddd,J＝16.1,13.3,8.0,5.2Hz, 3H), 1.95 – 1.81 (m, 1H). prove also [1,2-a] pyrazine-Isosorbide-5-Nitrae of (8aS)-hexahydropyrrolo-Diketone.
Example 2:
Phellinus crude extract, is made by following method:
(a) 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%
(b) Phellinus bacterial classification is inoculated into conventional method in the conical flask that fluid nutrient medium is housed, with 30 DEG C of temperature, shaking flask rotating speedFor 180r/min, under pH6 condition, vibrations are cultivated 15 days; In cultivation in the time that pH value drops to 2.5, by the seed in shaking flaskBe inoculated in the nutrient solution of 50L fermentation tank, with 30 DEG C of temperature, fermentation tank pressure 0.2 kg/cm, pH3 is logicalTolerance 0.5-1.1vvm, the condition that mixing speed is 180 revs/min, cultivates 15 days, can utilize phellinus igniarius mycelium fermentation liquidPreparation Phellinus crude extract;
(c) get gained phellinus igniarius mycelium fermentation liquid in step (b), by its reduced pressure concentration in a usual manner, its volume is concentrated to former1/5 of volume;
(d) zymotic fluid after the ethanol that is 90% by percent by volume is concentrated to above-mentioned steps (c) extracts, and wherein, adds ethanolAmount be 4 times of concentrate volume, can make concentration of alcohol in extract reach 70%;
(e) to step (d) gained extract under 55 DEG C of conditions, heat 2.5 hours; Separate with conventional method, and by twoLevel removes by filter impurity, separates and obtains ethanol extract; By above-mentioned ethanol extract reduced pressure concentration in a usual manner, make itVolume is concentrated to 1/10 of original volume;
(f) just the concentrate of step (e) gained is dried with the method for frozen drying, obtains Phellinus crude extract.
Take 100g crude extract and isopyknic 100 order purification on normal-phase silica gel mix, silica gel normal phase column chromatography. Chromatography is fixed phaseFor 200-300 order purification on normal-phase silica gel, the high 0.8m of post, diameter 14cm, eluant, eluent is for being respectively chloroform, chloroform: methyl alcohol=200:1,100:1,40:1 is 3,4,4,4 column volumes of wash-out respectively. Last eluent is suitably merged, evaporated under reduced pressure,Carry out methanol gel chromatography. Collect eluent, suitably merge, with equal-volume silica gel mixed sample, the fixing silicon using mutually of chromatographyGlue is 200-300 order purification on normal-phase silica gel. Eluant, eluent is chloroform and methyl alcohol. Collect eluent, carry out methanol gel column chromatography.Then, carry out the anti-phase preparation of normal pressure, A is water mutually, and B is methyl alcohol mutually. Collect eluent, evaporated under reduced pressure, HPLC detects,Condition is 0min:95% water, 10min:100% methyl alcohol, and going out the cutting edge of a knife or a sword time is that 5.93min suitably merges, and carries out 1D-HNMRNuclear magnetic resonance spectroscopy, frequency is 400MHZ, analysis result is δ 7.30 (s, 1H), 4.06 (dd, J=15.8,10.2Hz, 2H),3.87(dd,J＝16.5,4.4Hz,1H),3.65–3.48(m,2H),2.42–2.26(m,1H),2.03(dddd,J＝16.1,13.3,8.0,5.2Hz, 3H), 1.95 – 1.81 (m, 1H). prove also [1,2-a] pyrazine-Isosorbide-5-Nitrae of (8aS)-hexahydropyrrolo-Diketone.
Claims (8)
1. the also anti-phase separation method of preparing of normal pressure of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium, its sequence of steps is as follows:
(1) prepare Phellinus bacterium crude extract;
(2) crude extract and the purification on normal-phase silica gel of above-mentioned steps (1) gained are mixed to stirring, and dry, carry out silica gel normal phase column chromatography, use eluant, eluent wash-out 2～5 times;
(3) collect the last eluent that above-mentioned steps (2) obtains, reduced pressure concentration, uses methyl alcohol to dissolve, and methanol gel column chromatography, uses eluant, eluent wash-out;
(4) product step (3) being obtained carries out purification on normal-phase silica gel chromatography, uses eluant, eluent wash-out;
(5) collect eluent, suitably merge, evaporated under reduced pressure, carries out methanol gel chromatography;
(6) product step (5) being obtained carries out normal pressure reversed phase chromatography, and eluant, eluent is methyl alcohol and water;
(7) HPLC detects, and HPLC condition is 0min:95% water, 10min:100% methyl alcohol, and appearance time is 5.85～6.54min; Drying under reduced pressure, is also [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo; It is characterized in that described Phellinus crude extract, made by following method:
(a) 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%
(b) fermentation culture method of described Phellinus crude extract is, Phellinus bacterial classification is inoculated in the conical flask that fluid nutrient medium is housed, and with 20～35 DEG C of temperature, shaking flask rotating speed is 80～280r/min, and under the condition of pH3～8, vibrations are cultivated 7～15 days; In cultivation in the time that pH value drops to 2.5～4, seed in shaking flask is inoculated in the 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, cultivate 7～15 days, can utilize phellinus igniarius mycelium fermentation liquid to prepare Phellinus crude extract;
(c) get gained phellinus igniarius mycelium fermentation liquid in step (b), by its reduced pressure concentration, make its volume be concentrated to 1/2～1/5 of original volume;
(d) zymotic fluid after the ethanol that is 60～95% by percent by volume is concentrated to above-mentioned steps (c) extracts, and wherein, the amount that adds ethanol is 2～5 times of concentrate volume, can make concentration of alcohol in extract reach 60～90%;
(e) to step (d) gained extract under 50～70 DEG C of conditions, heat 1～2 hour; Separate, and remove impurity by cascade filtration, separate and obtain ethanol extract; By above-mentioned ethanol extract reduced pressure concentration, make its volume be concentrated to 1/5～1/10 of original volume;
(f) concentrate of step (e) gained is dried with the method for frozen drying, obtains Phellinus crude extract.
2. (8aS)-hexahydropyrrolo also [1 in Phellinus bacterium as claimed in claim 1; 2-a] separation method of pyrazine-Isosorbide-5-Nitrae-diketone, 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, eluent is chloroform and/or methyl alcohol.
3. the also separation method of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium as claimed in claim 1, is characterized in that, the eluant, eluent described in step (3) is methyl alcohol.
4. the also separation method of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium as claimed in claim 1, is characterized in that, the purification on normal-phase silica gel described in step (4) is 200～300 orders, and eluant, eluent is chloroform and methyl alcohol.
5. the also separation method of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium as claimed in claim 1, is characterized in that, the gel type described in step (5) is LH-20.
6. the also separation method of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium as claimed in claim 1, is characterized in that, the described reversed material of step (6) is that C-18 or C-8 use the anti-phase preparation of normal pressure.
7. the also separation method of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium as claimed in claim 1, is characterized in that, the described eluant, eluent of step (6) is methyl alcohol: water=40%～85%.
8. the also separation method of [1,2-a] pyrazine-Isosorbide-5-Nitrae-diketone of (8aS)-hexahydropyrrolo in Phellinus bacterium as claimed in claim 1, is characterized in that, the described reversed phase chromatography number of times of step (6) is 1～3 time.
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| CN101724305B (en) * | 2009-12-08 | 2012-09-05 | 中国科学院南海海洋研究所 | Application of cycle (proline-alanine) in preventing and removing marine fouling organism |
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