CN103044206A - Technology for separating benzenediol from Phellinus - Google Patents
Technology for separating benzenediol from Phellinus Download PDFInfo
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- CN103044206A CN103044206A CN2012105462008A CN201210546200A CN103044206A CN 103044206 A CN103044206 A CN 103044206A CN 2012105462008 A CN2012105462008 A CN 2012105462008A CN 201210546200 A CN201210546200 A CN 201210546200A CN 103044206 A CN103044206 A CN 103044206A
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Abstract
The invention discloses a technology for separating benzenediol from Phellinus (Phellinusigniarius (LexFr) Quel, Phellinuslinteus (BerketCurt) Teng, Phellinus baumii and Phellinushartigii (AlleschetSchnabl) Imaz). The technology comprises the following steps of: firstly, preparing a crude Phellinus extract; secondly, performing normal phase silica gel chromatography, performing gradient elution by using chloroform and methanol, and performing normal phase silica gel chromatography again; thirdly, performing chloroform and methanol gel chromatography; fourthly, performing normal phase silica gel chromatography again; and finally, drying by distillation under reduced pressure to obtain benzenediol.
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 a kind of Phellinus bacterium (phelliuns igniarius
Phellinus igniarius(L ex Fr) Quel, phellinus linteus
Phellinus linteus(Berk et Curt) Teng, Phellinus baumii, Kazakhstan base of a fruit phellinus
Phellinus hartigii(Allesch et Schnabl) Imaz) in 1, the isolation technique of 2-dihydroxy-benzene.At first preparing the Phellinus crude extract, then is the purification on normal-phase silica gel chromatography, uses chloroform and methyl alcohol gradient elution, repeats one time the purification on normal-phase silica gel chromatography, then carries out the chloroform methanol gel chromatography, again carries out the purification on normal-phase silica gel chromatography, and last evaporated under reduced pressure obtains 1,2-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 Separation of Benzene diphenol: Phellinus bacterium crude extract → ethanol precipitation → purification on normal-phase silica gel chromatography → chloroform and methyl alcohol gradient elution → TLC detection → purification on normal-phase silica gel chromatography → chloroform methanol gel chromatography → TLC detection → purification on normal-phase silica gel chromatography → TLC detection → evaporated under reduced pressure → colorless oil (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 the last elutriant evaporate to dryness 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) elutriant in the collection step (3), concentrating under reduced pressure, use chloroform: methyl alcohol=1:1 dissolving, carry out the methanol gel column chromatography, use the eluent wash-out, the elutriant that uses the TLC detection to collect suitably merges elutriant, drying under reduced pressure;
(5) again carry out the purification on normal-phase silica gel chromatography with the product that obtains in the step (4), use the eluent wash-out;
(6) collect the elutriant that obtains in the step (5), use TLC to detect each elutriant, appropriateness merges elutriant, and drying under reduced pressure is 1,2-dihydroxy-benzene.
(4) description of drawings
Fig. 1 is the structural formula of 1,2-dihydroxy-benzene;
Fig. 2 is the one-dimensional nuclear magnetic resonance H spectrum of 1,2-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 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 0.8m of post, and diameter 15cm, eluent is for being respectively chloroform, methyl alcohol, chloroform: methyl alcohol=100:1, respectively 2,3 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 and acetone.The concentrating under reduced pressure elutriant uses methyl alcohol: chloroform=1:1 dissolving, chloroform methanol gel filtration chromatography.Use TLC to detect the elutriant of collecting and suitably merge, evaporated under reduced pressure reuses the purification on normal-phase silica gel chromatography, elutriant is chloroform methanol, collects elutriant, uses TLC to detect each elutriant, developping agent is sherwood oil: acetone=20:1, merges to collect the elutriant that spot occurs.Drying under reduced pressure carries out the 1D-HNMR nuclear magnetic resonance spectroscopy, and frequency is 400MHZ, and analytical results is δ 8.78 (s, 1H), (6.68 dt, J=40.7,20.4 Hz, 1H), 6.58 (dd, J=5.7,3.5 Hz, 1H), proving is 1,2-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 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, 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 and acetone.The concentrating under reduced pressure elutriant uses methyl alcohol: chloroform=1:1 dissolving, chloroform methanol gel filtration chromatography.Use TLC to detect the elutriant of collecting and suitably merge, evaporated under reduced pressure reuses the purification on normal-phase silica gel chromatography, elutriant is chloroform methanol, collects elutriant, uses TLC to detect each elutriant, developping agent is sherwood oil: acetone=35:1, merges to collect the elutriant that spot occurs.Drying under reduced pressure carries out the 1D-HNMR nuclear magnetic resonance spectroscopy, and frequency is 400MHZ, and analytical results is δ 8.78 (s, 1H), (6.68 dt, J=40.7,20.4 Hz, 1H), 6.58 (dd, J=5.7,3.5 Hz, 1H), proving is 1,2-dihydroxy-benzene.
Claims (9)
1. the separation method of dihydroxy-benzene 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 the last elutriant evaporate to dryness 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) elutriant in the collection step (3), concentrating under reduced pressure, use chloroform: methyl alcohol=1:1 dissolving, carry out the methanol gel column chromatography, use the eluent wash-out, the elutriant that uses the TLC detection to collect suitably merges elutriant, drying under reduced pressure;
(5) again carry out the purification on normal-phase silica gel chromatography with the product that obtains in the step (4), use the eluent wash-out;
(6) collect the elutriant that obtains in the step (5), use TLC to detect each elutriant, appropriateness merges elutriant, and drying under reduced pressure is 1,2-dihydroxy-benzene.
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 dihydroxy-benzene in the Phellinus bacterium as claimed in claim 1 is characterized in that described dihydroxy-benzene is 1,2-dihydroxy-benzene.
4. the separation method of dihydroxy-benzene 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 dihydroxy-benzene 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 or methyl alcohol.
6. the separation method of dihydroxy-benzene 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=20:1-35:1.
7. the separation method of dihydroxy-benzene 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 chloroform: methyl alcohol=1:1.
8. the separation method of dihydroxy-benzene in the Phellinus bacterium as claimed in claim 1 is characterized in that, the described chromatographic silica gel of step (5) is the 200-300 order, and eluent is chloroform: methyl alcohol=200:1-50:1.
9. the separation method of dihydroxy-benzene in the Phellinus bacterium as claimed in claim 1 is characterized in that, the described detection point of step (6) is spot to occur, and developping agent is sherwood oil: acetone=20:1-35:1.
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| CN104988192A (en) * | 2015-06-01 | 2015-10-21 | 宁夏医科大学 | Preparation method for morin D |
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| 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 |
| 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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| 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 |
| 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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104988192A (en) * | 2015-06-01 | 2015-10-21 | 宁夏医科大学 | Preparation method for morin D |
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