CN103304396B - Method for separating and purifying 1403C from fermentation liquid of 1403C - Google Patents
Method for separating and purifying 1403C from fermentation liquid of 1403C Download PDFInfo
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Abstract
The invention discloses a method for separating and purifying 1403C from fermentation liquid of 1403C. The method comprises the following steps of: (1) adding polar organic solvent or mixture of the polar organic solvent and water into the fermentation liquid of 1403C, adjusting the pH to 1-4, stirring for 0.5-2 hours at the temperature of 50-75 DEG C, then performing solid-liquid separation and taking the liquid phase to obtain extraction liquid; (2) concentrating the extraction liquid obtained by the step (1), adsorbing by using a non-polar adsorbent, detecting the adsorbed liquid by using high performance liquid chromatography and collecting the part with peak purity of being over 90 percent of the 1403C to obtain purified liquid; and (3) concentrating the purified liquid obtained by the step (2) and then crystallizing under the environment of 20 DEG C below zero to 25 DEG C to obtain the crystal which is the 1403C. According to the method, the 1403C with high purity is obtained at last, and the weight purity can reach over 95 percent. The method has the advantages of easiness in operation and control, high process stability, less pollution and high safety in the production process and contribution to industrialization.
Description
Technical field
The invention belongs to separation and purification field, particularly a kind of method of separation and purification 1403C from the fermented liquid of 1403C.
Background technology
From special Biological resources, find that lead compound is the important directions of current International Pharmaceutical research.Ocean is grand strategy space and the resources bank of human kind sustainable development.Marine microorganism is due to the singularity of environment and the function in Marine ecosystems, develop the mode of some unique metabolic, thus the meta-bolites of extremely abundant, various structures and novelty can be produced, the research and development for original new drug provide a large amount of newtype drug lead compound mode configurations.
Along with the development of marine microorganism cultivation technology and Protocols in Molecular Biology in recent years, from ocean, be separated to a large amount of new microorganism, and therefrom isolated and many there is bioactive secondary metabolite.Wherein, in South China Sea mangrove forest leaf, the endogenetic fungus Halorosellinia sp.1403 obtained is separated by people such as E.B.G.Jones and L.L.P.Vrijmoed, after cultivating, various active product can be isolated from its fermented liquid, comprise the material such as grisovin and multiple anthraquinone, naphthoquinones and xanthone class, these compounds mostly have antitumor, anti-microbial activity (Xiao Nana. the secondary metabolite research of solid fermentation two strain South Sea mangrove endophytic fungus. Zhongshan University master thesis .2010).
1403C is the anthraquinone analog compound (Jiang Guangce of isolated novel structure from the nutrient solution of Halorosellinia sp.1403, Lin Yongcheng etc. the research of South China Sea mangrove endophytic fungus No.1403 secondary metabolites. Zhongshan University's journal, 2000,39 (6): 68-72).Through the active testing that Zhongshan University carries out it, determine its have good anti-tumor activity (She Zhigang, Lin Yongcheng etc. quinone compounds and preparation method thereof applies .ZL200610035637.X with antitumor).Therefore this compound has a good application prospect.
The preparation method of the 1403C reported at present is biological fermentation process, comprises fermentation culture and link is prepared in separation and purification two.The fermentation culture method of 1403C has more report, comprises the formula of substratum, culture condition, inoculation method, feed process etc.
What the separation purification method of 1403C adopted at present is extraction--silica gel/polyamide chromatography--method of recrystallization, as patent ZL200610035637.X report, thalline is removed by after fermented liquid solid-liquid separation, heating is concentrated into the 1/3-1/5 of original volume, repeatedly extract by ethyl acetate, concentrate, chromatographic separation is carried out in silicagel column, with petrol ether/ethyl acetate/methanol elution gradient, collect 50%--100% ethyl acetate/petroleum ether part, again through polyamide column chromatography after concentrated, 70% ethyl acetate/petroleum ether wash-out, recrystallization purifying, obtains red 1403C crystal.
Adopt known method to carry out 1403C separation and purification, complex steps, operational difficulty, technique is unstable; Organic solvent volatility used and toxicity are all comparatively strong, large usage quantity, and the pollution of production process is large, poor stability, is unfavorable for industrialized production operation.
Summary of the invention
The invention provides a kind of method of separating and purifying high-purity 1403C from fermented liquid, the method operation is more simple and easy to control, technology stability is strong; The pollution of production process is little, security is high, is more beneficial to industrialized production operation.
The technical solution used in the present invention is as follows: a kind of method of separation and purification 1403C from the fermented liquid of 1403C, comprises the following steps:
1) in the fermented liquid of 1403C, add the mixture of polar organic solvent or polar organic solvent and water, regulate pH to pH 1-4,50 DEG C-75 DEG C are stirred 0.5-2 hour, and then solid-liquid separation gets liquid phase, obtains extract;
2) by step 1) after the extract of gained is concentrated with non-polar adsorbent absorption, the liquid high performance liquid chromatography through adsorb detects, and the part of collection 1403C peak purity more than 90%, obtains refined solution;
3) by step 2) refined solution of gained concentrate after in the environment of-20 DEG C to 25 DEG C crystallization, get crystal and namely obtain 1403C.
In the present invention, step 1) be: the mixture adding polar organic solvent or polar organic solvent and water in the fermented liquid of 1403C, regulates pH to pH 1-4, and 50 DEG C-75 DEG C are stirred 0.5-2 hour, and then solid-liquid separation gets liquid phase, obtains extract.
The fermented liquid of 1403C of the present invention can be any fermented liquid that can produce the thalline of 1403C, as long as this thalline can produce 1403C.As obtained after cultivating by mangrove endophytic fungus Halorosellinia sp.1403, the preparation method of this fermented liquid has been disclosed in patent 201010179396.2,200910199117.6 and document " research of South China Sea mangrove endogenetic fungus No.1403 secondary metabolites ", " research of seven kinds of South Sea thalassiomycetess and actinomycetes meta-bolites ".The bacterial classification of Halorosellinia sp.1403 has preservation, preserving number in China typical culture collection center (CCTCC): M201018.
Polar organic solvent of the present invention is known any polar organic solvent.Comprise alcohols, ketone, aldehydes etc.Be preferably alcohols, preferred lower alcohol.Lower alcohol of the present invention refers to C1-C4 alcohol, can be methyl alcohol, ethanol, Virahol, preferred alcohol.Described polar organic solvent and the mixture of water refer to the mixture of polar organic solvent of the present invention volume percent in the mixture in 0%-100% (not comprising endpoint value) scope.Preferably polar organic solvent volume percent is in the mixture 30%-98%, more preferably 50%-95%, best as 75%.The addition of the mixture of described polar organic solvent or polar organic solvent and water be make organic solvent account in whole mixed solution volume content be 30%-90%, more preferably 50%-90%, best as 75%.
Step 1) object be all be dissolved in liquid phase with the 1403C that thalline produces by above-mentioned organic solvent.In research process, we find, 1403C is at polar organic solvent and higher with solubleness in the mixed solvent of water, and solubleness in water is very little.The 1403C major part that thalline produces is accumulated in thalline inside or sticks on mycelium in solid form, and can not all be dissolved in aqueous phase.Therefore, in fermented liquid, add polar organic solvent can improve the solubleness of liquid phase to 1403C, the 1403C that thalline is accumulated is discharged in liquid phase.Regulate pH thalline can be made to break to acidity and discharge 1403C.Therefore, the 1403C extracting in fermented liquid enters in the mixed solution of organic phase or organic phase and water by the present invention, and the method for the extracting adopted comprises stirring, is separated the equal step of each extracting.Preferably after adding the mixture of polar organic solvent or polar organic solvent and water in fermented liquid, regulate pH of mixed to 1-4,50 DEG C-75 DEG C are stirred 0.5-2 hour, and better for regulated pH of mixed to 2.5,70 DEG C of constant temperature stir 1 hour.
Step 1) also comprise the fermented liquid solid-liquid separation after being stirred by the mixture adding polar organic solvent or polar organic solvent and water, after solid-liquid separation, liquid phase contains 1403C.The solid-liquid separating method adopted in the present invention is the solid-liquid separating method of this area routine, as centrifugal, suction filtration, micro-filtration etc.
In the present invention, step 2) be: by step 1) after the extract of gained is concentrated with non-polar adsorbent absorption, the liquid high performance liquid chromatography through adsorb detects, and the effluent liquid of collection 1403C peak purity more than 90%, obtains refined solution.
Wherein, first by step 1) extract of gained concentrates.The concentration method adopted is this area ordinary method, and as normal pressure evaporation concentration, reduced vacuum is concentrated.Extract, after concentrated, not only can improve the concentration of 1403C, can also adjust the content of organic solvent, thus affect the adsorbed state in non-polar adsorbent of 1403C and impurity.After concentrated, the volume content of organic solvent is 15% ~ 85% time, and yield is higher, and preferably 30%.
Step 2) object be with a part of impurity in non-polar adsorbent Adsorption extract.Non-polar adsorbent can adsorb nonpolar material from polar solvent, and polarity is more weak, adsorbs stronger.In research process, we find, the impurity major part polarity in extract is more weak compared with 1403C, is more easily adsorbed by non-polar adsorbent.Therefore the present invention adopts non-polar adsorbent by more weak for extract Semi-polarity or segment polarity is comparatively strong but the impurity absorption removing that content is lower, reaches the object of purifying.Described non-polar adsorbent can be aromatic series macroporous adsorbent resin as Amberlite XAD-1600, Amberchrom CG-161 etc., or halogen-containing aromatic series macroporous adsorbent resin is as SP-207 etc., also can be gac or carbide resin.Optimization aromatic macroporous adsorbent resin and halogen-containing aromatic series macroporous adsorbent resin, more preferably aromatic series macroporous adsorbent resin Amberchrom CG161.The method that the present invention preferably adopts percolation to adsorb carries out adsorption-edulcoration.More preferably non-polar adsorbent fluidized-bed is crossed.
Step 2) in described " peak purity " refer in the high-efficient liquid phase chromatogram of 1403C solution, the percentage composition that the peak area of 1403C component is obtained by normalization method.The high performance liquid phase peak purity that the present invention preferably collects 1403C is greater than 90%, more preferably greater than the effluent liquid of 95%.
The HPLC analytical procedure of the present invention one preferably 1403C is as follows:
Chromatographic column: anti-phase C
18chromatographic column, 260mm × 4.6mm
Flow velocity: 1mL/min
Sample size: 15 μ L
Column temperature: 26 DEG C
Determined wavelength: 280nm
Solution A: 1% acetic acid: the aqueous solution (V: V)
B solution: methyl alcohol
Elution process:
In the present invention, step 3) be: by step 2) refined solution of gained concentrate after in the environment of-20 DEG C to 25 DEG C crystallization, get crystal and namely obtain 1403C.
Step 3) object be make 1403C crystallization from refined solution, impurity stronger for polarity a part of in refined solution is stayed in mother liquor simultaneously, reaches refining effect.Refined solution, by concentrated, can improve concentration, and can adjust the content of organic solvent.In refined solution after concentrated, the volume content of organic solvent is 25% ~ 100% time, and yield is higher, and preferably 75%.In order to improve purity and yield further, refined solution can also be concentrated into dry, and then in 50 DEG C-75 DEG C, preferably at 70 DEG C, dissolve 1403C completely with the water mixed solution of the polar organic solvent of 25% ~ 100%, and excessively filter insoluble impurity, obtain clear liquid and carry out crystallization again.Described polar solvent is selected from alcohols, ketone and aldehydes, is preferably alcohols, preferred lower alcohol, described lower alcohol particular methanol, ethanol, Virahol, preferred alcohol.
Step 3) in crystallization carry out in the environment of-20 DEG C to 25 DEG C, preferably place 4-8 hour, namely obtain 1403C crystal.For improving yield, refined solution can be placed in the environment being equal to or less than 4 DEG C, but above freezing in crystal solution of envrionment temperature will be ensured, in order to avoid solvent solidifies.
Step 3) middle crystal of separating out can as the process washing of routine and drying and other steps.The object of crystal washing is to remove the residual mother liquor on filter cake.Washing methods is by dissolving each other with mother liquor (solution remaining after magma removes crystal after the crystallization that mother liquor refers to) but to the poor solvent wash crystal of 1403C solvability 2 ~ 3 times with a kind of, and suction filtration.The mixture of the preferred polar organic solvent of cleaning solvent used or polar organic solvent and water, more preferably as methyl alcohol, ethanol, Virahol and the mixed solution etc. with water thereof.Dry method is conventional crystal drying means, as reduced vacuum is dry, preferably as 20-40 DEG C, and-0.08 ~-0.1MPa air pressure, 2-4 hour or be dried to constant weight.Step 3) the red tabular crystal of separating out, after washing, drying, obtain the 1403C that purity by weight is greater than 95%.
Concentration method described in the present invention is this area ordinary method, and as normal pressure evaporation concentration, reduced vacuum is concentrated.
The volumn concentration of organic solvent after concentrated, can regulate by controlling solvent evaporation amount; Or add water/organic solvent after concentrated to regulate; Or be concentrated into dry, again 1403C all dissolved with the mixed solvent of certain volume content in a heated condition and regulate.
In addition, produce in the present invention containing the waste liquid of 1403C, as non-polar adsorbent absorption terminate after stripping liquid, mother liquor etc. after crystallization, can recovery in other batches, to improve yield further.
A preferred embodiments of the present invention is, a kind of method of separation and purification 1403C from the fermented liquid of 1403C, comprises the following steps:
1) add the mixture of polar organic solvent or polar organic solvent and water in fermented liquid, regulate pH of mixed to 1-4, stir 0.5-2 hour at 50 DEG C-75 DEG C, then solid-liquid separation obtains extract;
2) volume content extract being concentrated into organic solvent is 15% ~ 80%, and non-polar adsorbent fluidized-bed crossed by concentrated solution, the effluent liquid that the high performance liquid phase peak purity collecting 1403C is greater than 90%, obtains refined solution;
3) refined solution is concentrated into dry, at 50 DEG C-75 DEG C, dissolves 1403C completely with the polar organic solvent of 25% ~ 100% and the mixing solutions of water, cross the environment that leaching clear liquid is placed in-20 DEG C to 25 DEG C, place 4-8 hour, make 1403C crystallization;
4) the mixture washing of the crystal polar organic solvent of separating out or polar organic solvent and water, 20-40 DEG C, under-0.08 ~-0.1MPa, vacuum-drying 2-4 hour.
The raw material that the present invention is used or reagent except special instruction, all commercially.
On the basis meeting this area general knowledge, each technical characteristic optimum condition above-mentioned in the present invention arbitrary combination can obtain preferred embodiments.
Positive progressive effect of the present invention is: the present invention finally obtains highly purified 1403C, and purity by weight can reach more than 95%.The method that the inventive method is reported more at present, operate more simple and easy to control, technology stability is strong; The polar organic solvent adopted is as ethanol, Virahol etc., and than former methodical organic solvent, volatility is low, toxicity is little, and the pollution of production process is little, security is high, is more beneficial to industrialized production operation.1403C prepared by the present invention has antitumor action, can be applied to the preparation of antitumor drug, in the open and patent CN 200610035637.X of its anti-tumor activity.
Embodiment
The invention provides a kind of method of separating and purifying high-purity 1403C from fermented liquid, the feature of the method is to utilize non-polar adsorbent to the more weak impurity of polarity in the absorption removing fermentation extract of impurity, removed other impurity again by the method for crystallization, finally obtain highly purified 1403C.
Further illustrate the present invention by embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.Room temperature described in embodiment generally refers to 25 DEG C.
In the present embodiment, the acquisition of fermented liquid is carried out as follows.
Bacterial classification: Halorosellinia sp.1403, CCTCC NO:M201018.
Slant medium: glucose 10g; Yeast powder 1g; Peptone 2g; PH 7; Artificial seawater (ASW) 1000mL; Agar 15 ~ 20g.
Seed culture medium: glucose 10g; Yeast powder 1g; Peptone 2g; PH 7; Artificial seawater (ASW) 1000mL.
Fermention medium: glucose 10g; Yeast powder 1g; Peptone 2g; PH 7; Artificial seawater (ASW) 200mL; Deionized water 800mL.
Cultural method:
The well-grown thalline of picking from slant medium, access is through 121 DEG C of (0.1MPa) high-temperature sterilizations seed culture medium of 25 minutes, cultivate 3 days same fermention mediums through high-temperature sterilization of access afterwards, inoculum size 5% (volume percent relative to substratum), culture temperature 28 DEG C, incubation time 96 hours, and to add mol ratio for 24 hours in fermentation in fermented liquid be the sodium acetate of 1: 4 and the mixed solution of propanedioic acid, makes its final concentration in fermented liquid be 6mM.
Embodiment 1
Fermented liquid 1.2L is altogether collected after fermentation ends, 1403C concentration is that (1403C method for measurement of concentration is 1.1g/L: get 10ml fermented liquid, add 30ml acetic acid, stir 1 hour at 70 DEG C, centrifugal, supernatant liquor high performance liquid phase detects), add ethanol 3.0L, regulate pH of mixed to 1.0 with hydrochloric acid, 75 DEG C of constant temperature stir 0.5 hour, obtain the extract 4.0L clarified after suction filtration.The solution of 2.8L alcohol volume content 85% is mixed with after concentrating extract with Rotary Evaporators, after filtering, the resin bed of 50mL carbide resin crossed by clear liquid, effluent liquid HPLC detects, and detection method, as described in summary of the invention, collects the effluent liquid 0.7L altogether of 1403C peak purity more than 90%.After effluent liquid Rotary Evaporators is concentrated into 150mL (alcohol volume content is 25%), be placed in room temperature lower 5 hours, suction filtration magma, crystal 20mL deionized water wash 2 times, crystal vacuum-drying 4 hours at 30 DEG C after suction filtration, obtain the 1403C crystal 0.26g of purity by weight 96.2%, yield 19%.
Embodiment 2
The concentration of collecting fermented liquid altogether 1.4L, 1403C is 0.9g/L, centrifugally obtains thalline 80g, add the aqueous ethanolic solution 1.0L of 75%, regulate pH to 2.6, stir 1 hour at 70 DEG C, suction filtration mixed solution, bacterium slag repeats aforesaid operations once, and twice extract merges 1.74L altogether.Be evaporated to 1.1L, filter after adding suitable quantity of water, be made into the solution of 2L alcohol volume content 30%.This solution is through 80mLAmberchrom CG161 resin bed, and effluent liquid HPLC detects, and collects the effluent liquid 1.4L altogether of 1403C peak purity more than 95%.This solution decompression is concentrated into after doing, adds 250mL, the aqueous ethanolic solution of 75%, at 70 DEG C after stirring and dissolving, heat filtering obtains clear soln, under slowly stirring, is placed in 4 DEG C of conditions lower 8 hours, suction filtration magma, crystal 50mL, 30% aqueous ethanolic solution washs 2 times, crystal vacuum-drying 4 hours at 30 DEG C after suction filtration, obtain the 1403C crystal 0.58g of purity by weight 99.7%, yield 46%.
Embodiment 3
Collecting common 1.2L, the 1403C concentration of fermented liquid after fermentation ends is 1.0g/L, and add ethanol 3.0L, in mixed solution, alcohol volume content is 75%, and regulate pH of mixed to 2.5 with hydrochloric acid, 70 DEG C of constant temperature stir 2 hours, obtains the extract 4.0L clarified after suction filtration.Be mixed with the solution of 3.0L alcohol volume content 30% after concentrating under reduced pressure extract, after filtering, clear liquid is through the resin bed of 80mL Amberchrom CG161, and effluent liquid HPLC detects, and collects the effluent liquid 2.5L altogether of 1403C peak purity more than 95%.Be evaporated to dry, with 75% aqueous ethanolic solution 320mL, 1403C is dissolved completely at 70 DEG C, filter insolubles, clear liquid slowly stirs 5 hours under being placed in room temperature, then is placed in 4 DEG C of environment lower 3 hours, suction filtration magma, crystal 50mL, 20% aqueous ethanolic solution washs 2 times, crystal vacuum-drying after suction filtration, obtain the 1403C crystal 0.65g of purity by weight 99.4%, yield 54%.
Embodiment 4
Collecting common 1.2L, the 1403C concentration of fermented liquid after fermentation ends is 0.9g/L, and add ethanol 3.0L, regulate pH of mixed to 4.0 with hydrochloric acid, 50 DEG C of constant temperature stir 2 hours, obtain the extract 4.0L clarified after suction filtration.Be mixed with the solution of 3.0L alcohol volume content 30% after concentrating under reduced pressure extract, after filtering, clear liquid is through the resin bed of 80mL SP-207, and effluent liquid HPLC detects, and collects the effluent liquid 1.7L altogether of 1403C peak purity more than 90%.Effluent liquid concentrating under reduced pressure also regulates alcohol volume content 65%, is settled to 250mL, leaves standstill 4 hours at being placed in-20 DEG C, suction filtration magma, crystal 20mL, 30% aqueous ethanolic solution washs 2 times, crystal vacuum-drying after suction filtration, obtains the 1403C crystal 0.31g of purity by weight 96.7%, yield 28%.
Embodiment 5
Collecting common 1.0L, the 1403C concentration of fermented liquid after fermentation ends is 1.0g/L, and add ethanol 2.5L, regulate pH of mixed to 2.5 with hydrochloric acid, 70 DEG C of constant temperature stir 1 hour, obtain the extract 3.3L clarified after suction filtration.Be mixed with the solution of 3.0L alcohol volume content 30% after concentrating under reduced pressure extract, after filtering, the resin bed of 80mL Amberlite XAD-1600 crossed by clear liquid, and effluent liquid HPLC detects, and collects the effluent liquid 1.2L altogether of 1403C peak purity more than 90%.Effluent liquid atmospheric evaporation is concentrated into 150mL, and be placed in left at room temperature 5 hours, suction filtration magma, crystal 20mL, 30% aqueous ethanolic solution washs 2 times, crystal vacuum-drying after suction filtration, obtains the 1403C crystal 0.22g of purity by weight 95.8%, yield 21%.
Embodiment 6
Collecting common 1.2L, the 1403C concentration of fermented liquid after fermentation ends is 1.1g/L, and add methyl alcohol 3.0L, regulate pH of mixed to 2.5 with hydrochloric acid, 70 DEG C of constant temperature stir 1 hour, obtain the extract 4.0L clarified after suction filtration.Be mixed with the solution of 3.0L methyl alcohol volume content 30% after concentrating under reduced pressure extract, after filtering, the resin bed of 80mL Amberchrom CG161 crossed by clear liquid, and effluent liquid HPLC detects, and collects the effluent liquid 2.2L altogether of 1403C peak purity more than 90%.Effluent liquid is evaporated to 200mL, filter insolubles, clear liquid slowly stirs 5 hours under being placed in room temperature, suction filtration magma, crystal 50mL, 20% methanol aqueous solution washs 2 times, crystal vacuum-drying 4 hours at 30 DEG C after suction filtration, obtain the 1403C crystal 0.58g of purity by weight 97.7%, yield 43%.
Embodiment 7
Other operations are with embodiment 6, and the lower alcohol dissolved each other with water only used is Virahol, obtains the 1403C crystal 0.48g of purity by weight 97.2%, yield 35%.
Embodiment 11
Other operations are with embodiment 3, and only the concentrated rear adjustment alcohol volume content of extract is 15%, obtains the 1403C crystal 0.29g of purity by weight 99.2%, yield 24%.
Embodiment 12
Other operations are with embodiment 3, and only the concentrated rear adjustment alcohol volume content of extract is 80%, obtains the 1403C crystal 0.42g of purity by weight 98.9%, yield 35%.
Embodiment 13
Other operation with embodiment 3, only after refined solution concentrating under reduced pressure by 25% aqueous ethanolic solution stirring and dissolving at 75 DEG C, obtain the 1403C crystal 0.36g of purity by weight 99.2%, yield 30%.
Embodiment 14
Other operations, with embodiment 3, are only used dehydrated alcohol stirring and dissolving at 50 DEG C after refined solution concentrating under reduced pressure, are obtained the 1403C crystal 0.51g of purity by weight 99.4%, yield 42%.
Embodiment 15
Other operations are with embodiment 3, and only crystallization clear liquid slowly stirs after 5 hours under being placed in room temperature, then is placed in 0 DEG C of environment lower 3 hours, obtains the 1403C crystal 0.66g of purity by weight 99.4%, yield 54%.
Embodiment 16
Other operations are with embodiment 3, and only add the aqueous ethanolic solution 3.0L of 70% to fermented liquid, in mixed solution, alcohol volume content is 50%.Obtain the 1403C crystal 0.41g of purity by weight 99.0%, yield 43%.
Embodiment 17
Other operations are with embodiment 3, and only add ethanol 11.0L to fermented liquid, in mixed solution, alcohol volume content is 90%.Obtain the 1403C crystal 0.64g of purity by weight 99.4%, yield 53%.
Embodiment 18
Other operations are with embodiment 3, and only add the aqueous ethanolic solution 3.0L of 45% to fermented liquid, in mixed solution, alcohol volume content is 30%.Obtain the 1403C crystal 0.41g of purity by weight 98.6%, yield 34%.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (9)
1. a method of separation and purification 1403C from the fermented liquid of 1403C, is characterized in that, comprise the following steps:
1) in the fermented liquid of 1403C, add the mixture of polar organic solvent or polar organic solvent and water, regulate pH to pH 1-4,50 DEG C-75 DEG C are stirred 0.5-2 hour, then solid-liquid separation gets liquid phase, obtain extract, described polar organic solvent be selected from methyl alcohol, ethanol and Virahol one or more;
2) by step 1) adsorb with non-polar adsorbent after the extract of gained is concentrated, liquid high performance liquid chromatography through adsorbing detects, collect the part of 1403C peak purity more than 90%, obtain refined solution, described non-polar adsorbent be selected from aromatic series macroporous adsorbent resin, gac and carbide resin one or more;
3) by step 2) refined solution of gained concentrate after in the environment of-20 DEG C to 25 DEG C crystallization, get crystal and namely obtain 1403C.
2. the method for claim 1, is characterized in that, step 1) described in polar organic solvent be ethanol.
3. the method for claim 1, is characterized in that, step 1) described in polar organic solvent or the addition of mixture of polar organic solvent and water be the 30%-90% making organic solvent account for the volume percent in whole mixed solution.
4. the method for claim 1, is characterized in that, step 2) described in non-polar adsorbent be carbide resin, Amberlite XAD-1600, Amberchrom CG-161 or SP-207.
5. the method for claim 1, is characterized in that, step 2) described in extract concentrate after the volume content of wherein organic solvent be 15% ~ 85%.
6. the method for claim 1, is characterized in that, step 3) in, after described refined solution concentrates, wherein the volume content of organic solvent is 25% ~ 100%.
7. the method for claim 1, is characterized in that, step 3) in, described refined solution is concentrated into dry, then in 50 DEG C-75 DEG C, dissolve 1403C completely with the water mixed solution of the polar organic solvent of 25% ~ 100%, and leaching clear liquid carries out described crystallization excessively.
8. the method for claim 1, is characterized in that, step 3) in, described crystallization is by concentrated solution in the environment of-20 DEG C to 25 DEG C, places 4-8 hour.
9. the method for claim 1, is characterized in that, the method also comprises step 3) middle crystal of separating out washs, drying.
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| CN1850765A (en) * | 2006-05-26 | 2006-10-25 | 中山大学 | Quinone compounds, and their preparing method and antitumour use |
| CN101544556A (en) * | 2008-06-06 | 2009-09-30 | 中山大学 | Quinone compound Bostrycin, preparation method thereof and anti-tumor application thereof |
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| CN1850765A (en) * | 2006-05-26 | 2006-10-25 | 中山大学 | Quinone compounds, and their preparing method and antitumour use |
| CN101544556A (en) * | 2008-06-06 | 2009-09-30 | 中山大学 | Quinone compound Bostrycin, preparation method thereof and anti-tumor application thereof |
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