CN103044340B - Technology for separating uracil from Phellinus - Google Patents
Technology for separating uracil from Phellinus Download PDFInfo
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- CN103044340B CN103044340B CN201210546456.9A CN201210546456A CN103044340B CN 103044340 B CN103044340 B CN 103044340B CN 201210546456 A CN201210546456 A CN 201210546456A CN 103044340 B CN103044340 B CN 103044340B
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- phellinus
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Abstract
The invention discloses a method for separating uracil from Phellinus (Phellinusigniarius (LexFr) Quel, Phellinuslinteus (BerketCurt) Teng, Phellinus baumii and Phellinushartigii (AlleschetSchnabl) Imaz). The method comprises the following steps of: firstly, preparing a crude Phellinus extract; secondly, performing normal phase silica gel chromatography, performing gradient elution by using petroleum ether and acetone, performing TLC (thin-layer chromatography) detection, and performing normal phase silica gel chromatography again; thirdly, performing methanol gel chromatography; fourthly, after the TLC detection, performing normal phase silica gel chromatography again; and finally, properly combining eluent, and drying under reduced pressure to obtain uracil.
Description
Technical field
The invention belongs to biotech medicine product field.
Background technology
Phellinus (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, often be full of cracks always, 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, and be divided into two classes: the one, only have the gel filtration chromatography of molecular sieve effect, conventional gel has dextrane gel and sepharose.The 2nd, ion exchange chromatography.But, mainly for separating of polysaccharide, hyaluronic acid etc., the product obtaining 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 the separation method of uridylic in a kind of Phellinus bacterium.First prepare Phellinus crude extract, then carry out purification on normal-phase silica gel chromatography, adopt sherwood oil and acetone gradient elution; carrying out TLC detection, reuse purification on normal-phase silica gel chromatography, is then methanol gel chromatography; after PLC detects, suitably merge elutriant, drying under reduced pressure, had both obtained uridylic.
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:
W-Gum 1-5% glucose 1-5%
Peptone 0.1-0.5% yeast extract paste 0.1-0.5%
Magnesium sulfate 0.1-0.5% potassium primary phosphate 0.01-0.05%
(2) Phellinus bacterial classification is inoculated into ordinary method in the Erlenmeyer flask that liquid nutrient medium is housed, with 20～35 ℃ of temperature, shaking flask rotating speed is 80～280r/min, and under pH 3～8 conditions, vibrations are cultivated 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 20～35 ℃ of temperature, fermentor tank pressure 0.1～0.2 kg/cm, pH 3～8, air flow 0.5～1.1vvm, the condition that stirring velocity is 100～280 revs/min, cultivate 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 concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/2～1/5 of original volume;
(4) fermented liquid after the ethanol that is 60～95% by volume percent is concentrated to above-mentioned steps (3) extracts, and wherein, the amount that adds ethanol is 2～5 times of concentrated solution volume, can make alcohol concn in extracting solution reach 60～90%;
(5) to step (4) gained extracting solution under 50～70 ℃ of conditions, heat 1～2 hour; With ordinary method, carry out separation, and remove impurity by cascade filtration, the separated ethanol extract that obtains; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5～1/10 of original volume;
(6) just the concentrated solution of step (5) gained is dried with the method for frozen drying, obtains Phellinus crude extract.
The method of separated uridylic:
Phellinus bacterium crude extract → purification on normal-phase silica gel chromatography → chloroform and methyl alcohol gradient elution → TLC detection → purification on normal-phase silica gel chromatography → methanol gel chromatography → TLC detection → TLC detection → evaporated under reduced pressure → white powder (uridylic).
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, use eluent wash-out 2-7 time;
(3) collect last elutriant in step (2), drying under reduced pressure, with equal-volume silica gel mixed sample, carries out purification on normal-phase silica gel chromatography again, uses eluent wash-out;
(4) collect the elutriant in above-mentioned steps (3), concentrating under reduced pressure, is used dissolve with methanol, and methanol gel column chromatography, uses eluent wash-out;
(5) elutriant of TLC detecting step (4), appropriateness merges elutriant, and high-pressure drying, is uridylic.
The significant advantage of the present invention's isolation technique of uridylic in Phellinus bacterium: present method adopts multiple chromatographic technique to combine, can be exquisite clear and definite to structure, the uridylic that purity is greater than 95%.Mature technical route is clear and definite, efficiently accurate.
(4) accompanying drawing explanation
Fig. 1 is the structural formula of uridylic of the present invention;
Fig. 2 is the one-dimensional nuclear magnetic resonance H spectrum of uridylic
(5) embodiment
Example 1:
Phellinus crude extract, is made by following method:
(1) fermentative medium formula in gram/100 milliliters be:
W-Gum 1% glucose 1%
Peptone 0.1% yeast extract paste 0.1%
Magnesium sulfate 0.1% potassium primary phosphate 0.01%
(2) Phellinus bacterial classification is inoculated into ordinary method in the Erlenmeyer flask that liquid nutrient medium is housed, with 25 ℃ of temperature, shaking flask rotating speed is 110r/min, and under pH 7 conditions, vibrations are cultivated 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 25 ℃ of temperature, fermentor tank pressure 0.1 kg/cm, pH 3, air flow 0.5-1.1vvm, the condition that stirring velocity is 100 revs/min, cultivate 7 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 concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/3 of original volume;
(4) fermented liquid after the ethanol that is 70% by volume percent is concentrated to above-mentioned steps (3) extracts, and wherein, the amount that adds ethanol is 5 times of concentrated solution volume, can make alcohol concn in extracting solution reach 55%;
(5) to step (4) gained extracting solution under 70 ℃ of conditions, heat 1 hour; With ordinary method, carry out separation, and remove impurity by cascade filtration, the separated ethanol extract that obtains; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5 of original volume;
(6) just the concentrated solution 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 stationary phase is 200-300 order purification on normal-phase silica gel, the high 1m of post, and diameter 20cm, eluent is for being respectively chloroform, chloroform: methyl alcohol=100:1, chloroform: methyl alcohol=50:1, chloroform: methyl alcohol=10:1, chloroform: methyl alcohol=5:1, difference wash-out 3,4,4,4,4 column volumes.And by gained elutriant difference called after Fr-1, Fr-2, Fr-3, Fr-4, Fr-5.By Fr-5 equal-volume silica gel mixed 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.Eluent is chloroform and methyl alcohol.Concentrating under reduced pressure elutriant, is used dissolve with methanol, and methanol gel column chromatography, uses methanol-eluted fractions.Use TLC to detect the elutriant of collecting and suitably merge, obtain white powder, carry out 1D-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analytical results is δ 7.41,7.39,5.62, and 5.61. proves that it is uridylic.
Example 2:
Phellinus crude extract, is made by following method:
(1) fermentative medium formula in gram/100 milliliters be:
W-Gum 3% glucose 2%
Peptone 0.5% yeast extract paste 0.5%
Magnesium sulfate 0.5% potassium primary phosphate 0.05%
(2) Phellinus bacterial classification is inoculated into ordinary method in the Erlenmeyer flask that liquid nutrient medium is housed, with 30 ℃ of temperature, shaking flask rotating speed is 180r/min, and under pH6 condition, vibrations are cultivated 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 30 ℃ of temperature, fermentor tank pressure 0.2 kg/cm, pH 3, air flow 0.5-1.1vvm, the condition that stirring velocity is 180 revs/min, cultivate 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 concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5 of original volume;
(4) fermented liquid after the ethanol that is 90% by volume percent is concentrated to above-mentioned steps (3) extracts, and wherein, the amount that adds ethanol is 4 times of concentrated solution volume, can make alcohol concn in extracting solution reach 70%;
(5) to step (4) gained extracting solution under 55 ℃ of conditions, heat 2.5 hours; With ordinary method, carry out separation, and remove impurity by cascade filtration, the separated ethanol extract that obtains; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/10 of original volume;
(6) just the concentrated solution of step (5) gained is dried with the method for frozen drying, obtains Phellinus crude extract.
Above-mentioned crude extract is taken to 100g and equal-volume 100 order purification on normal-phase silica gel mix stirring, carry out silica gel normal phase column chromatography.Chromatography stationary phase is 200-300 order purification on normal-phase silica gel, the high 0.7m of post, and diameter 10cm, eluent is for being respectively chloroform, chloroform: methyl alcohol=100:1, chloroform: methyl alcohol=50:1, chloroform: methyl alcohol=10:1, chloroform: methyl alcohol=5:1.By Fr-5 equal-volume silica gel mixed 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.Eluent is chloroform and methyl alcohol.Carry out methanol gel column chromatography.TLC detects the elutriant of collecting and suitably merges, and drying under reduced pressure, carries out 1D-HNMR nuclear magnetic resonance spectroscopy, and frequency is 400MHZ, and analytical results is δ 7.41,7.39,5.62, and 5.61. proves that it is uridylic.
Claims (7)
1. the separation method of uridylic in Phellinus bacterium, its step order is as follows:
(1) prepare Phellinus 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 eluent wash-out 2-7 time;
(3) collect last elutriant in step (2), drying under reduced pressure, with equal-volume silica gel mixed sample, carries out purification on normal-phase silica gel chromatography again, uses eluent wash-out;
(4) collect the elutriant in above-mentioned steps (3), concentrating under reduced pressure, is used dissolve with methanol, and methanol gel column chromatography, uses eluent wash-out;
(5) elutriant of TLC detecting step (4), appropriateness merges elutriant, and high-pressure drying, is uridylic.
As claimed in claim 1 a kind of from Phellinus bacterium the method for separated uridylic, it is characterized in that described Phellinus crude extract, by following method, made:
(1) fermentative medium formula in gram/100 milliliters be:
W-Gum 1-5% glucose 1-5%
Peptone 0.1-0.5% yeast extract paste 0.1-0.5%
Magnesium sulfate 0.1-0.5% potassium primary phosphate 0.01-0.05%
(2) fermentation culture method of Phellinus crude extract is as claimed in claim 1, Phellinus bacterial classification is inoculated into ordinary method in the Erlenmeyer flask that liquid nutrient medium is housed, with 20～35 ℃ of temperature, 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 drops to 2.5～4; seed in shaking flask is inoculated in the nutrient solution of 50L fermentor tank; with 20～35 ℃ of temperature; 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, 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 concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/2～1/5 of original volume;
(4) fermented liquid after the ethanol that is 60～95% by volume percent is concentrated to above-mentioned steps (3) extracts, and wherein, the amount that adds ethanol is 2～5 times of concentrated solution volume, can make alcohol concn in extracting solution reach 60～90%;
(5) to step (4) gained extracting solution under 50～70 ℃ of conditions, heat 1～2 hour; With ordinary method, carry out separation, and remove impurity by cascade filtration, the separated ethanol extract that obtains; By above-mentioned ethanol extract concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5～1/10 of original volume;
(6) just the concentrated solution of step (5) gained is dried with the method for frozen drying, obtains Phellinus crude extract;
3. the separation method of uridylic in Phellinus bacterium as claimed in claim 1, is characterized in that, the chromatographic silica gel described in step (2) is positive 200-300 order, and eluent is chloroform and/or methyl alcohol.
4. the separation method of uridylic in Phellinus bacterium as claimed in claim 1, is characterized in that, the silica gel consumption described in step (3) is equal-volume, and eluent is chloroform and methyl alcohol.
5. the separation method of uridylic in Phellinus bacterium as claimed in claim 1, is characterized in that, the gel described in step (4) is Sephadex LH-20, Sephadex LH-15, and eluent is methyl alcohol.
6. the separation method of uridylic in Phellinus bacterium as claimed in claim 1, is characterized in that, the described TLC of step (5) detects, and developping agent is chloroform: methyl alcohol=4:1-5:1.
7. the separation method of uridylic in Phellinus bacterium as claimed in claim 2, is characterized in that, the alcohol concn described in step (4) is 95% edible ethanol, and volume used is 5 times of volumes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102363749B (en) * | 2011-10-09 | 2012-11-28 | 东北林业大学 | Preparation method of Phellinus linteus mycelium |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102363749B (en) * | 2011-10-09 | 2012-11-28 | 东北林业大学 | Preparation method of Phellinus linteus mycelium |
Non-Patent Citations (4)
| Title |
|---|
| 李翠翠等.桑黄液体发酵培养基优化的初步研究.《中国食用菌》.2009,第28卷(第2期),46-48. |
| 桑黄液体发酵培养基优化的初步研究;李翠翠等;《中国食用菌》;20091231;第28卷(第2期);46-48 * |
| 桑黄液体发酵培养液初步研究;郭忠军;《黑龙江医药》;20051231;第18卷(第3期);198-199 * |
| 郭忠军.桑黄液体发酵培养液初步研究.《黑龙江医药》.2005,第18卷(第3期),198-199. |
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