CN103044206B - A kind of isolation technics of benzenediol in Phellinus bacterium - Google Patents
A kind of isolation technics of benzenediol in Phellinus bacterium Download PDFInfo
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- CN103044206B CN103044206B CN201210546200.8A CN201210546200A CN103044206B CN 103044206 B CN103044206 B CN 103044206B CN 201210546200 A CN201210546200 A CN 201210546200A CN 103044206 B CN103044206 B CN 103044206B
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Abstract
The invention discloses a kind of Phellinus bacterium (phelliuns igniarius<i>Phellinus igniarius</i>(L ex Fr) Quel, phellinus linteus <i>Phellinus linteus</i>(Berk et Curt) Teng , Phellinus baumii, Kazakhstan base of a fruit phellinus <i>Phellinus hartigii</i>(Allesch et Schnabl) Imaz) isolation technics of benzenediol in. First preparing Phellinus bacterium crude extract, is then purification on normal-phase silica gel chromatography, uses chloroform and methyl alcohol gradient elution, repeats one time purification on normal-phase silica gel chromatography, then carries out chloroform methanol gel chromatography, again carries out purification on normal-phase silica gel chromatography, and last evaporated under reduced pressure obtains benzenediol.
Description
The invention belongs to biotech medicine product field.
Phellinus, fructification stockless, the flat hemispherical of cap or the shape of a hoof, 212*321 centimetre, thick 1.510 centimetres, 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, without hair, has concentric ring rib.Edge is blunt, and dark cinnamon is to light coffee color, and downside is without hymenium. and bacterial context dark brown is hard, wooden. tube and bacterial context are closely homochromy,Multilayer, but level is not obvious, old tube layer is full of white hypha. and the mouth of pipe becomes rusty brown to dark reddish brown, circle, 45 every millimeter.Spore is subsphaeroidal, smooth, colourless, 56*34 micron. bristle top is sharp-pointed, and base portion expands, 1025*57 micron. mycelia regardless ofBranch, without tabula, 35 microns of diameters.
At present, the purification process of epiphyte product is more, mainly contains precipitation classification, preparation property high performance liquid chroma-tography, post layerAnalyse method, ultrafiltration, centrifugation chromatography, etc. several method. And wherein apply maximum column chromatographies that surely belongs to, be divided into two classes:The one, only have the gel filtration chromatography of molecular sieve effect, conventional gel has sephadex and Ago-Gel. The 2nd, ion is handed overChange chromatography. But, mainly for separating of polysaccharide, hyaluronic acid etc., the product obtaining after most separation is mixture. The present invention usesMultiple chromatographic technique combines, and separating degree is high, and applying this invention can be exquisite to sterling.
The invention discloses a kind of Phellinus bacterium (phelliuns igniarius Phellinusigniarius (LexFr) Quel, cracked feetPhellinus phellinuslinteus (BerketCurt) Teng, Phellinus baumii, Kazakhstan base of a fruit phellinusPhellinushartigii (AlleschetSchnabl) Imaz) in the isolation technics of 1,2 benzenediol. First prepare PhellinusCrude extract, is then purification on normal-phase silica gel chromatography, uses chloroform and methyl alcohol gradient elution, repeats one time purification on normal-phase silica gel chromatography, follow intoRow chloroform methanol gel chromatography, carries out purification on normal-phase silica gel chromatography again, and last evaporated under reduced pressure obtains 1,2 benzenediol.
Technical scheme of the present invention is as follows:
Phellinus crude extract, is made by following method:
(1) fermentative medium formula in gram/100 milliliters be:
Cornstarch 15% glucose 15%
Peptone 0.10.5% yeast extract 0.10.5%
Magnesium sulfate 0.10.5% potassium dihydrogen phosphate 0.010.05%
(2) Phellinus bacterial classification is inoculated into conventional method in the conical flask that fluid nutrient medium is housed, with 20～35 DEG C of temperatureDegree, shaking flask rotating speed is 80～280r/min, under the condition of pH3～8, vibrations are cultivated 7～15 days; In cultivation when pH value 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 0.1～0.2Kg/cm, pH3～8, throughput 0.5～1.1vvm, the condition that mixing speed is 100～280 revs/min, cultivates 7～15My god, can utilize phellinus igniarius mycelium fermentation liquid to prepare Phellinus crude extract;
(3) get gained phellinus igniarius mycelium fermentation liquid in step (2), by its reduced pressure concentration in a usual manner, make its volumeBe 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 2～5 times of concentrate volume, can make concentration of alcohol in extract reach 60～90%;
(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 Phellinus crude extract.
The method of Separation of Benzene diphenol: Phellinus bacterium crude extract → ethanol precipitation → purification on normal-phase silica gel chromatography → chloroform and methyl alcohol gradientWash-out → TLC detection → purification on normal-phase silica gel chromatography → chloroform methanol gel chromatography → TLC detection → purification on normal-phase silica gel chromatography → TLC detects→ evaporated under reduced pressure → colorless oil (benzenediol).
Concrete grammar is:
1. prepare Phellinus bacterium crude extract;
2. by step 1. crude extract and the purification on normal-phase silica gel of gained mix stirring, carry out silica gel normal phase column chromatography after dry, makeWith eluant, eluent wash-out 25 times, obtain eluent;
3. the last eluent evaporate to dryness in 2. by step, equal-volume silica gel mixed sample, carries out purification on normal-phase silica gel chromatography, usesEluant, eluent wash-out;
4. collect the eluent of step in 3., reduced pressure concentration, uses chloroform: methyl alcohol=1:1 dissolves, and carries out methanol gel postChromatography, uses eluant, eluent wash-out, uses TLC to detect the eluent of collecting, and merges eluent, drying under reduced pressure;
5. the product obtaining in 4. by step carries out purification on normal-phase silica gel chromatography again, uses eluant, eluent wash-out;
6. collect the eluent that step obtains in 5., use TLC to detect each eluent, merge eluent, drying under reduced pressure,It is 1,2 benzenediol.
(4) brief description of the drawings
Fig. 1 is the structural formula of 1,2 benzenediol;
Fig. 2 is the one-dimensional nuclear magnetic resonance H spectrum of 1,2 benzenediol.
(5) detailed description of the invention
Example 1:
Phellinus 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) Phellinus bacterial classification is inoculated into conventional method in the conical flask that fluid nutrient medium is housed, with 25 DEG C of temperature, shakesBottle rotating speed is 110r/min, and 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, throughput 0.51.1vvm, the condition that mixing speed is 100 revs/min, cultivates 7 days, can utilize phellinus igniarius mycelium fermentation liquid to prepare Phellinus and slightly carryThing;
(3) get gained phellinus igniarius mycelium fermentation liquid in step (2), by its reduced pressure concentration in a usual manner, make its volumeBe 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 Phellinus crude extract.
Above-mentioned crude extract is taken to 200g and equal-volume 100 order purification on normal-phase silica gel mix stirring, carry out silica gel normal phase column chromatography.Chromatography is fixing is 200300 order purification on normal-phase silica gel mutually, the high 0.8m of post, and diameter 15cm, eluant, eluent is for being respectively chloroform, methyl alcohol, chloroform:Methyl alcohol=100:1, respectively 2,3 column volumes of wash-out. And by gained eluent called after Fr1 respectively, Fr2. By bodies such as Fr2Long-pending silica gel mixed sample, using silica gel is 100 order purification on normal-phase silica gel, pentaploid amasss silica gel column chromatography, the silica gel of use be 200-300 orders justPhase silica gel. Eluant, eluent is benzinum and acetone. Reduced pressure concentration eluent, uses methyl alcohol: chloroform=1:1 dissolves, and chloroform methanol is solidifyingPlastic column chromatography. The eluent that uses TLC to detect collection merges, and evaporated under reduced pressure, reuses purification on normal-phase silica gel chromatography, and eluent is chlorineImitative methyl alcohol, collects eluent, uses TLC to detect each eluent, and solvent is benzinum: acetone=20:1, merges to collect to occurThe eluent of spot. Drying under reduced pressure, carries out 1DHNMR nuclear magnetic resonance spectroscopy, and frequency is 400MHZ, analysis result be δ 8.78 (s,1H), 6.68 (dt, J=40.7,20.4Hz, 1H), 6.58 (dd, J=5.7,3.5Hz, 1H), proving is 1,2 benzenediol.
Example 2:
Phellinus 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) Phellinus bacterial classification is inoculated into conventional method in the conical flask that fluid nutrient medium is housed, with 30 DEG C of temperature, shakesBottle rotating speed is 180r/min, and 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, throughput0.51.1vvm, the condition that mixing speed is 180 revs/min, cultivates 15 days, can utilize phellinus igniarius mycelium fermentation liquid to prepare PhellinusCrude extract;
(3) get gained phellinus igniarius mycelium fermentation liquid in step (2), by its reduced pressure concentration in a usual manner, make its volumeBe 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) just the concentrate of step (5) gained is dried with the method for frozen drying, obtains Phellinus crude extract.
Above-mentioned crude extract is taken to 300g and equal-volume 100 order purification on normal-phase silica gel mix stirring, carry out silica gel normal phase column chromatography.Chromatography is fixing is 200300 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=100:1, respectively 3,4 column volumes of wash-out. And by gained eluent called after Fr1 respectively, Fr2. By Fr2 equal-volume silica gelMix sample, using silica gel is 100 order purification on normal-phase silica gel, and pentaploid amasss silica gel column chromatography, and the silica gel of use is 200-300 order purification on normal-phase silica gel.Eluant, eluent is benzinum and acetone. Reduced pressure concentration eluent, uses methyl alcohol: chloroform=1:1 dissolves, chloroform methanol gel column layerAnalyse. The eluent that uses TLC to detect collection merges, and evaporated under reduced pressure, reuses purification on normal-phase silica gel chromatography, and eluent is chloroform firstAlcohol, collects eluent, uses TLC to detect each eluent, and solvent is benzinum: acetone=35:1, merges to collect to occur spotEluent. Drying under reduced pressure, carries out 1DHNMR nuclear magnetic resonance spectroscopy, and frequency is 400MHZ, and analysis result is δ 8.78 (s, 1H),6.68 (dt, J=40.7,20.4Hz, 1H), 6.58 (dd, J=5.7,3.5Hz, 1H), proving is 1,2 benzenediol.
Claims (7)
1. the separation method of benzenediol in Phellinus bacterium, its sequence of steps is as follows:
1. prepare Phellinus crude extract;
2. by step 1. Phellinus crude extract and the purification on normal-phase silica gel of gained mix stirring, carry out silica gel normal phase column chromatography after dry, makeWith eluant, eluent wash-out 2-5 time, obtain eluent;
3. the last eluent evaporate to dryness in 2. by step, equal-volume silica gel mixed sample, carries out purification on normal-phase silica gel chromatography, uses wash-outAgent wash-out;
4. collect the eluent of step in 3., reduced pressure concentration, uses chloroform: methyl alcohol=1:1 dissolves, and carries out methanol gel post layerAnalyse, use eluant, eluent wash-out, use TLC to detect the eluent of collecting, merge eluent, drying under reduced pressure;
5. the product obtaining in 4. by step carries out purification on normal-phase silica gel chromatography again, uses eluant, eluent wash-out;
6. collect the eluent that step obtains in 5., use TLC to detect each eluent, merge eluent, drying under reduced pressure, is 1,2-benzenediol,
Wherein said Phellinus crude extract, is 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) fermentation culture method of described Phellinus crude extract is, Phellinus bacterial classification is inoculated into Liquid Culture is housed with conventional methodIn the conical flask of base, with 20～35 DEG C of temperature, shaking flask rotating speed is 80～280r/min, and under the condition of pH3～8, vibrations cultivate 7～15 days; In cultivation, in the time that pH value drops to 2.5～4, the seed in shaking flask is inoculated in the nutrient solution of 50L fermentation tank, with temperatureSpend 20～35 DEG C, fermentation tank pressure 0.1～0.2 kg/cm, pH3～8, throughput 0.5～1.1vvm, mixing speedThe condition of 100～280 revs/min, cultivates 7～15 days, can utilize phellinus igniarius mycelium fermentation liquid to prepare Phellinus crude extract;
(3) get gained phellinus igniarius mycelium fermentation liquid in step (2), by its reduced pressure concentration in a usual manner, make its volume concentratedTo 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, wherein,The amount that adds ethanol is 2～5 times of concentrate volume, can make concentration of alcohol in extract reach 60～90%;
(5) to step (4) 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;
(6) concentrate of step (5) gained is dried with the method for frozen drying, obtains Phellinus crude extract.
2. the separation method of benzenediol in Phellinus bacterium as claimed in claim 1, is characterized in that, step is mixed sample described in 2.Silica gel is 100-200 order purification on normal-phase silica gel.
3. the separation method of benzenediol in Phellinus bacterium as claimed in claim 1, is characterized in that, the chromatography of step described in 2.Silica gel is positive 200-300 order, and eluant, eluent is chloroform or methyl alcohol.
4. the separation method of benzenediol in Phellinus bacterium as claimed in claim 1, is characterized in that, the silica gel of step described in 3.Consumption is equal-volume, and eluant, eluent is benzinum: acetone=20:1-35:1.
5. the separation method of benzenediol in Phellinus bacterium as claimed in claim 1, is characterized in that, the gel of step described in 4.For SephadexLH-20, eluant, eluent is chloroform: methyl alcohol=1:1.
6. the separation method of benzenediol in Phellinus bacterium as claimed in claim 1, is characterized in that, 5. described chromatography silicon of stepGlue is 200-300 order, and eluant, eluent is chloroform: methyl alcohol=200:1-50:1.
7. the separation method of benzenediol in Phellinus bacterium as claimed in claim 1, is characterized in that, step 6. described detection is wantedPoint is to occur spot, and solvent is benzinum: acetone=20:1-35:1.
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| CN101182550A (en) * | 2007-11-16 | 2008-05-21 | 上海市农业科学院 | Flavonoids from phellinus, method of producing the same and use |
| CN102491827A (en) * | 2011-11-14 | 2012-06-13 | 江苏省农业科学院 | Solid fermentation culture medium for phellinus igniarius mycelium and solid fermentation cultural method for phellinus igniarius mycelium |
| CN102766663A (en) * | 2012-05-30 | 2012-11-07 | 陕西师范大学 | Preparation method of active polysaccharides from phellinus linteus |
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| CN101182550A (en) * | 2007-11-16 | 2008-05-21 | 上海市农业科学院 | Flavonoids from phellinus, method of producing the same and use |
| CN102491827A (en) * | 2011-11-14 | 2012-06-13 | 江苏省农业科学院 | Solid fermentation culture medium for phellinus igniarius mycelium and solid fermentation cultural method for phellinus igniarius mycelium |
| CN102766663A (en) * | 2012-05-30 | 2012-11-07 | 陕西师范大学 | Preparation method of active polysaccharides from phellinus linteus |
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