CN103044207A - Technology for separating methyl benzenediol from Phellinus - Google Patents
Technology for separating methyl benzenediol from Phellinus Download PDFInfo
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- CN103044207A CN103044207A CN201210546221XA CN201210546221A CN103044207A CN 103044207 A CN103044207 A CN 103044207A CN 201210546221X A CN201210546221X A CN 201210546221XA CN 201210546221 A CN201210546221 A CN 201210546221A CN 103044207 A CN103044207 A CN 103044207A
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- phellinus
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Abstract
The invention discloses a method for separating methyl benzenediol 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 methyl benzenediol.
Description
Technical field
The invention belongs to the 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, 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; 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, and old tube layer is full of white hypha. the mouth of pipe becomes rusty brown to dark reddish brown, circle; every millimeter 4-5. 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 methylbenzene diphenol 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 TLC detects, again carry out the purification on normal-phase silica gel chromatography; suitably merge elutriant; drying under reduced pressure namely gets the methyl isophthalic acid into 3-, the 2-dihydroxy-benzene.
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.
The method of separating the methylbenzene diphenol:
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 → purification on normal-phase silica gel chromatography → TLC detection → evaporated under reduced pressure → be the 3-methyl isophthalic acid, the 2-dihydroxy-benzene.
Concrete grammar is:
(1) preparation Phellinus bacterium crude extract;
(2) crude extract and the purification on normal-phase silica gel mixing with step (1) gained stirs, and carries out the silica gel normal phase column chromatography after the drying, uses the eluent wash-out 2-5 time, obtains elutriant;
(3) with last elutriant evaporated under reduced pressure in the step (2), the equal-volume silica gel mixed sample carries out the purification on normal-phase silica gel chromatography, uses the eluent wash-out;
(4) collect the elutriant that step (3) obtains, concentrating under reduced pressure uses dissolve with methanol, carries out the methanol gel column chromatography, uses the eluent wash-out, uses TLC to detect the elutriant of collecting, and suitably merges elutriant, drying under reduced pressure;
(5) product that obtains with step 4 carries out the purification on normal-phase silica gel chromatography again, and eluent is chloroform and methyl alcohol;
(6) TLC detects, and appropriateness merges elutriant, and high-pressure drying is the methylbenzene diphenol.
The present invention is by the significant advantage of the isolation technique of methylbenzene diphenol 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% methylbenzene diphenol.Mature technical route is clear and definite, and is accurately efficient.
(4) description of drawings
Fig. 1 is the 3-methyl isophthalic acid, the structural formula of 2-dihydroxy-benzene;
Fig. 2 is the 3-methyl isophthalic acid, the one-dimensional nuclear magnetic resonance H spectrum of 2-dihydroxy-benzene.
(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 100g 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 0.8m of post, and diameter 10cm, eluent is for being respectively chloroform, chloroform: methyl alcohol=100:1, respectively 3,4 column volumes of wash-out.And with gained elutriant difference called after Fr-1, Fr-2.With Fr-2 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: acetone=20:1.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 is carried out the purification on normal-phase silica gel chromatography again, eluent is chloroform and methyl alcohol, 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 6.71 (s, 1H), (2.26 s, 1H). prove the methyl isophthalic acid into 3-, the 2-dihydroxy-benzene.
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 100g 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 0.8m of post, and diameter 10cm, eluent is for being respectively chloroform, chloroform: methyl alcohol=100:1, respectively 3,4 column volumes of wash-out.And with gained elutriant difference called after Fr-1, Fr-2.With Fr-2 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: acetone=40:1.Then carry out the methanol gel column chromatography.Again carry out the purification on normal-phase silica gel chromatography, TLC detects the elutriant of collecting and suitably merges, and drying under reduced pressure carries out the 1D-HNMR nuclear magnetic resonance spectroscopy, frequency is 400MHZ, and analytical results is δ 6.71 (s, 1H), (2.26 s, 1H). prove the methyl isophthalic acid into 3-, the 2-dihydroxy-benzene.
Claims (9)
1. the separation method of methylbenzene diphenol 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 step (1) gained stirs, and carries out the silica gel normal phase column chromatography after the drying, uses the eluent wash-out 2-5 time, obtains elutriant;
(3) with last elutriant evaporated under reduced pressure in the step (2), the equal-volume silica gel mixed sample carries out the purification on normal-phase silica gel chromatography, uses the eluent wash-out;
(4) collect the elutriant that step (3) obtains, concentrating under reduced pressure uses dissolve with methanol, carries out the methanol gel column chromatography, uses the eluent wash-out, uses TLC to detect the elutriant of collecting, and suitably merges elutriant, drying under reduced pressure;
(5) product that obtains with step 4 carries out the purification on normal-phase silica gel chromatography again, and eluent is chloroform and methyl alcohol;
(6) TLC detects, and appropriateness merges elutriant, and high-pressure drying is the methylbenzene diphenol.
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 methylbenzene diphenol in the Phellinus bacterium as claimed in claim 1 is characterized in that described methylbenzene diphenol is the 3-methyl isophthalic acid, the 2-dihydroxy-benzene.
4. the separation method of methylbenzene diphenol 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 methylbenzene diphenol 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 methylbenzene diphenol 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: acetone=20:1-40:1.
7. the separation method of methylbenzene diphenol 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 methylbenzene diphenol in the Phellinus bacterium as claimed in claim 1 is characterized in that, the described eluent of step (5) is chloroform: methyl alcohol=100:1-150:1.
9. the separation method of methylbenzene diphenol 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=25:1-40:1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 吴秀丽等: "火木层孔菌液体培养物的化学成分研究", 《中国中药杂志》, vol. 36, no. 7, 30 April 2011 (2011-04-30), pages 874 - 880 * |
| 钟石等: "优化培养基对桑黄菌丝体生长的影响", 《浙江农业科学》, no. 1, 31 December 2011 (2011-12-31), pages 173 - 175 * |
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Application publication date: 20130417 |