CN103044229A - Technology for separating acetophenone from Phellinus - Google Patents
Technology for separating acetophenone from Phellinus Download PDFInfo
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- CN103044229A CN103044229A CN2012105460708A CN201210546070A CN103044229A CN 103044229 A CN103044229 A CN 103044229A CN 2012105460708 A CN2012105460708 A CN 2012105460708A CN 201210546070 A CN201210546070 A CN 201210546070A CN 103044229 A CN103044229 A CN 103044229A
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Abstract
The invention discloses a method for separating acetophenone 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 reverse phase silica gel chromatography; and finally, properly combining eluent, and drying under reduced pressure to obtain acetophenone.
Description
Technical field
The invention belongs to the 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 be full of cracks is without cot; there is trickle fine hair at initial stage, and rear change has the concentric ring rib without hair. and the edge is blunt; dark cinnamon is to light coffee color, and downside is without thalamium. and the 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, every millimeter 4-5. and spore is subsphaeroidal; smooth, colourless, the 5-6*3-4 micron. the bristle top is sharp-pointed; base portion expands; the 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 use 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, gel commonly used has dextrane gel and sepharose.The 2nd, ion exchange chromatography.But, mainly for separating of polysaccharide, hyaluronic acid etc., the product that obtains after most the separation is mixture.The present invention uses multiple chromatographic technique to combine, and resolution is high, and using this invention can be exquisite to sterling.
Summary of the invention
The invention discloses the separation method of methyl phenyl ketone in a kind of Phellinus bacterium.At first prepare the Phellinus crude extract; then carry out the purification on normal-phase silica gel chromatography, adopt sherwood oil and acetone gradient elution, carry out TLC and detect; reuse the purification on normal-phase silica gel chromatography; then be the methanol gel chromatography, after PLC detects, carry out reversed-phase silica gel chromatography; suitably merge elutriant; drying under reduced pressure had both got 5'-methyl-3', 4'-resacetophenone.
Technical scheme of the present invention is as follows:
The Phellinus crude extract is made by following method:
(1) fermentative medium formula in/100 milliliters of grams is:
W-Gum 1-5% glucose 1-5%
Peptone 0.1-0.5% yeast extract paste 0.1-0.5%
Sal epsom 0.1-0.5% potassium primary phosphate 0.01-0.05%
(2) the Phellinus bacterial classification is inoculated into ordinary method in the Erlenmeyer flask that liquid nutrient medium is housed, with 20～35 ℃ of temperature, the shaking flask rotating speed is 80～280r/min, and under pH 3～8 conditions, vibrations were cultivated 7～15 days; In the cultivation when the pH value drops to 2.5～4, seed in the 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 rev/mins, cultivated 7～15 days, and can utilize the phellinus igniarius mycelium fermentation liquid to prepare the Phellinus crude extract;
(3) get gained phellinus igniarius mycelium fermentation liquid in the step (2), with its concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/2～1/5 of original volume;
(4) be that 60～95% the ethanol fermented liquid after concentrated to above-mentioned steps (3) extracts with volume percent, wherein, the amount that adds ethanol is 2～5 times of concentrated solution volume, can make that alcohol concn reaches 60～90% in the extracting solution;
(5) to step (4) gained extracting solution under 50～70 ℃ of conditions, heated 1～2 hour; Separate with ordinary method, and remove impurity by cascade filtration, separate obtaining ethanol extract; With 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 carries out drying with the method for frozen drying, gets the Phellinus crude extract.
Separate 5'-methyl-3', the method for 4'-resacetophenone:
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 → reversed-phase silica gel chromatography → TLC detection → evaporated under reduced pressure → 5'-methyl-3', the 4'-resacetophenone.
Concrete grammar is:
(1) at first prepares Phellinus bacterium crude extract;
(2) crude extract and the purification on normal-phase silica gel mixing with above-mentioned steps (1) gained stirs, and dry, carries out the silica gel normal phase column chromatography, uses eluent wash-out 2-5 time;
(3) the last elutriant in the collection step (2), drying under reduced pressure with the equal-volume silica gel mixed sample, carries out the purification on normal-phase silica gel chromatography again, uses the eluent wash-out;
(4) collect the elutriant that above-mentioned steps (3) obtains, concentrating under reduced pressure uses dissolve with methanol, and the methanol gel column chromatography is used the eluent wash-out, uses TLC to detect the elutriant of collecting, and suitably merges elutriant, drying under reduced pressure;
(5) product that step (4) is obtained carries out reversed-phase silica gel chromatography, and eluent is methyl alcohol: water=10%-80%;
(6) TLC detects, and appropriateness merges elutriant, and high-pressure drying is methyl phenyl ketone.
The present invention is by the significant advantage of the isolation technique of methyl phenyl ketone in the Phellinus bacterium: present method adopts multiple chromatographic technique to combine, can be exquisite clear and definite to structure, and purity is greater than 95% methyl phenyl ketone.Mature technical route is clear and definite, and is accurately efficient.
(4) description of drawings
Fig. 1 is 5'-methyl-3', the structural formula of 4'-resacetophenone;
Fig. 2 is 5'-methyl-3', the one-dimensional nuclear magnetic resonance H spectrum of 4'-resacetophenone.
(5) embodiment
Example 1:
The Phellinus crude extract is made by following method:
(1) fermentative medium formula in/100 milliliters of grams is:
W-Gum 1% glucose 1%
Peptone 0.1% yeast extract paste 0.1%
Sal epsom 0.1% potassium primary phosphate 0.01%
(2) the Phellinus bacterial classification is inoculated into ordinary method in the Erlenmeyer flask that liquid nutrient medium is housed, with 25 ℃ of temperature, the shaking flask rotating speed is 110r/min, and under pH 7 conditions, vibrations were cultivated 7 days; In the cultivation when the pH value drops to 3, seed in the 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 rev/mins, cultivated 7 days, and can utilize the phellinus igniarius mycelium fermentation liquid to prepare the Phellinus crude extract;
(3) get gained phellinus igniarius mycelium fermentation liquid in the step (2), with its concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/3 of original volume;
(4) be that 70% the ethanol fermented liquid after concentrated to above-mentioned steps (3) extracts with volume percent, wherein, the amount that adds ethanol is 5 times of concentrated solution volume, can make that alcohol concn reaches 55% in the extracting solution;
(5) to step (4) gained extracting solution under 70 ℃ of conditions, heated 1 hour; Separate with ordinary method, and remove impurity by cascade filtration, separate obtaining ethanol extract; With 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 carries out drying with the method for frozen drying, gets the Phellinus crude extract.
Above-mentioned crude extract is taken by weighing 300g and the stirring of equal-volume 100 order purification on normal-phase silica gel mixings, carry out the silica gel normal phase column chromatography.The 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, respectively 3,4,4 column volumes of wash-out.And with gained elutriant difference called after Fr-1, Fr-2, Fr-3.With Fr-3 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 sherwood oil and acetone.The concentrating under reduced pressure elutriant uses dissolve with methanol, the methanol gel column chromatography.Use TLC to detect the elutriant of collecting and suitably merge, evaporated under reduced pressure, 10% dissolve with methanol carries out reversed-phase silica gel chromatography, eluent is methyl alcohol and water, elutriant is carried out TLC detect, and suitably merges high-pressure drying, carry out the 1D-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analytical results is δ 7.34 (s, 1H), 7.28 (s, 1H), (2.47 d, J=15.2 Hz, 3H), (2.23 s, 3H). prove that it is 5'-methyl-3', the 4'-resacetophenone.
Example 2:
The Phellinus crude extract is made by following method:
(1) fermentative medium formula in/100 milliliters of grams is:
W-Gum 3% glucose 2%
Peptone 0.5% yeast extract paste 0.5%
Sal epsom 0.5% potassium primary phosphate 0.05%
(2) the Phellinus bacterial classification is inoculated into ordinary method in the Erlenmeyer flask that liquid nutrient medium is housed, with 30 ℃ of temperature, the shaking flask rotating speed is 180r/min, and under the pH6 condition, vibrations were cultivated 15 days; In the cultivation when the pH value drops to 2.5, seed in the 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 rev/mins, cultivated 15 days, and can utilize the phellinus igniarius mycelium fermentation liquid to prepare the Phellinus crude extract;
(3) get gained phellinus igniarius mycelium fermentation liquid in the step (2), with its concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/5 of original volume;
(4) be that 90% the ethanol fermented liquid after concentrated to above-mentioned steps (3) extracts with volume percent, wherein, the amount that adds ethanol is 4 times of concentrated solution volume, can make that alcohol concn reaches 70% in the extracting solution;
(5) to step (4) gained extracting solution under 55 ℃ of conditions, heated 2.5 hours; Separate with ordinary method, and remove impurity by cascade filtration, separate obtaining ethanol extract; With 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 carries out drying with the method for frozen drying, gets the Phellinus crude extract.
Above-mentioned crude extract is taken by weighing 200g and the stirring of equal-volume 100 order purification on normal-phase silica gel mixings, carry out the silica gel normal phase column chromatography.The chromatography stationary phase is 200-300 order purification on normal-phase silica gel, the high 1m of post, and diameter 15cm, eluent is for being respectively chloroform, chloroform: methyl alcohol=100:1, chloroform: methyl alcohol=50:1, respectively 2,3,3 column volumes of wash-out.And with gained elutriant difference called after Fr-1, Fr-2, Fr-3.With Fr-3 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 sherwood oil and acetone.Carry out the methanol gel column chromatography.Carry out reversed-phase silica gel chromatography, TLC detects the elutriant of collecting and suitably merges drying under reduced pressure, carry out the 1D-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analytical results is δ 7.34 (s, 1H), 7.28 (s, 1H), 2.47 (d, J=15.2 Hz, 3H), 2.23 (s, 3H). prove that it is 5'-methyl-3', the 4'-resacetophenone.
Claims (10)
1. the separation method of methyl phenyl ketone in the Phellinus bacterium, its step order is as follows:
(1) preparation Phellinus crude extract;
(2) ethanol throw out and the purification on normal-phase silica gel mixing with above-mentioned steps (1) gained stirs, and dry, carries out the silica gel normal phase column chromatography, uses eluent wash-out 2-5 time;
(3) the last elutriant in the collection step (2), drying under reduced pressure with the equal-volume silica gel mixed sample, carries out the purification on normal-phase silica gel chromatography again, uses the eluent wash-out;
(4) collect the elutriant that above-mentioned steps (3) obtains, concentrating under reduced pressure uses dissolve with methanol, and the methanol gel column chromatography is used the eluent wash-out, uses TLC to detect the elutriant of collecting, and suitably merges elutriant, drying under reduced pressure;
(5) product that step (4) is obtained carries out reversed-phase silica gel chromatography, and eluent is methyl alcohol: water=10%-80%;
(6) TLC detects, and appropriateness merges elutriant, and high-pressure drying is methyl phenyl ketone.
As claimed in claim 1 a kind of from the Phellinus bacterium method of Separation of Benzene ethanol, it is characterized in that described Phellinus crude extract, made by following method:
(1) fermentative medium formula in/100 milliliters of grams is:
W-Gum 1-5% glucose 1-5%
Peptone 0.1-0.5% yeast extract paste 0.1-0.5%
Sal epsom 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, the Phellinus bacterial classification is inoculated into ordinary method in the Erlenmeyer flask that liquid nutrient medium is housed, and with 20～35 ℃ of temperature, the shaking flask rotating speed is 80～280r/min, under pH 3～8 conditions, vibrations were cultivated 7～15 days; In the cultivation when the pH value drops to 2.5～4, seed in the 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 rev/mins, cultivated 7～15 days, and can utilize the phellinus igniarius mycelium fermentation liquid to prepare the Phellinus crude extract;
(3) get gained phellinus igniarius mycelium fermentation liquid in the step (2), with its concentrating under reduced pressure in a usual manner, make its volume be concentrated to 1/2～1/5 of original volume;
(4) be that 60～95% the ethanol fermented liquid after concentrated to above-mentioned steps (3) extracts with volume percent, wherein, the amount that adds ethanol is 2～5 times of concentrated solution volume, can make that alcohol concn reaches 60～90% in the extracting solution;
(5) to step (4) gained extracting solution under 50～70 ℃ of conditions, heated 1～2 hour; Separate with ordinary method, and remove impurity by cascade filtration, separate obtaining ethanol extract; With 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 carries out drying with the method for frozen drying, gets the Phellinus crude extract.
3. the separation method of methyl phenyl ketone in the Phellinus bacterium as claimed in claim 1 is characterized in that described compound 5'-methyl-3', the 4'-resacetophenone.
4. the separation method of methyl phenyl ketone in the Phellinus bacterium as claimed in claim 1 is characterized in that, the sample silica gel of mixing described in the step (2) is 100-200 order purification on normal-phase silica gel.
5. the separation method of methyl phenyl ketone in the Phellinus bacterium as claimed in claim 1 is characterized in that, the chromatographic silica gel described in the step (2) is positive 200-300 order, and eluent is chloroform and/or methyl alcohol.
6. the separation method of methyl phenyl ketone in the Phellinus bacterium as claimed in claim 1 is characterized in that the silica gel consumption described in the step (3) is equal-volume, and eluent is sherwood oil and acetone.
7. the separation method of methyl phenyl ketone in the Phellinus bacterium as claimed in claim 1 is characterized in that the gel described in the step (4) is Sephadex LH-20, and eluent is methyl alcohol.
8. the separation method of methyl phenyl ketone in the Phellinus bacterium as claimed in claim 1 is characterized in that, the described reversed material of step (5) is C-18, C-8.
9. the separation method of methyl phenyl ketone in the Phellinus bacterium as claimed in claim 1 is characterized in that, the described eluent of step (5) is methyl alcohol: water=10%-80%.
10. the separation method of methyl phenyl ketone in the Phellinus bacterium as claimed in claim 1 is characterized in that, the described TLC of step (6) detects, and developping agent is chloroform: methyl alcohol=12:1-15:1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001178448A (en) * | 1999-12-22 | 2001-07-03 | Yukito Akiyama | Method for culturing mycelium of phellinus linteus |
| CN1720785A (en) * | 2004-08-12 | 2006-01-18 | 陈全勇 | An artificial high-yield cultivation method of male agaric and application of fruit body thereof |
| 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 |
| CN102078339A (en) * | 2010-12-20 | 2011-06-01 | 大兴安岭林格贝有机食品有限责任公司 | Method for enriching and purifying common phellinus fungus general flavone in common phellinus fungus |
-
2012
- 2012-12-15 CN CN2012105460708A patent/CN103044229A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001178448A (en) * | 1999-12-22 | 2001-07-03 | Yukito Akiyama | Method for culturing mycelium of phellinus linteus |
| CN1720785A (en) * | 2004-08-12 | 2006-01-18 | 陈全勇 | An artificial high-yield cultivation method of male agaric and application of fruit body thereof |
| 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 |
| CN102078339A (en) * | 2010-12-20 | 2011-06-01 | 大兴安岭林格贝有机食品有限责任公司 | Method for enriching and purifying common phellinus fungus general flavone in common phellinus fungus |
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Application publication date: 20130417 |