CN107502630B - A kind of method that bioanalysis prepares Dehydro and drographolide - Google Patents
A kind of method that bioanalysis prepares Dehydro and drographolide Download PDFInfo
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- CN107502630B CN107502630B CN201710964407.XA CN201710964407A CN107502630B CN 107502630 B CN107502630 B CN 107502630B CN 201710964407 A CN201710964407 A CN 201710964407A CN 107502630 B CN107502630 B CN 107502630B
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Abstract
The invention discloses a kind of methods that bioanalysis prepares Dehydro and drographolide, include the following steps: step S1, Spawn incubation；Step S2, microorganism conversion；Step S3, converted product extract；Step S4, high-speed counter-current purifying；Wherein, the ionic liquid of effective concentration is added in the fermentation medium in step S1.Preparation method provided by the invention can be conversion bacterial strain with the common cunninghamella blakesleana easily cultivated, and add the functional ion liquid of specific structure in the fermentation medium, convert Dehydro and drographolide for andrographolide；Meanwhile if adding the valine of effective concentration in the fermentation medium, the selectivity of the conversion reaction can be improved.
Description
Technical field
The invention belongs to microorganism conversion fields, and in particular to a kind of method that bioanalysis prepares Dehydro and drographolide.
Background technique
Andrographolide is one of main active of Herba Andrographitis, has significant clearing heat and detoxicating, cool blood detumescence, disease-resistant
The effects of malicious.Modern pharmacology research shows the dehydration product-Dehydro and drographolide and its derivative tool of andrographolide
There is better bioactivity, therefore people's is caused to the research of andrographolide, Dehydro and drographolide and its derivative
Greatly concern.
Since the market demand of Dehydro and drographolide is larger, seek to be suitble to industrial method very heavy
It wants.Preparing Dehydro and drographolide mostly uses chemical method (bibliography: Synthsis of andrographolide at present
cyclophosphate derivatives and their antitumor activities.SynthComm,2006,36:
407-414), this method toxicity is big, seriously polluted, and has dissolvent residual.Lee close equality from the soil of acquisition separation screening to
Andrographolide, can be converted into Dehydro and drographolide by one plant of strain for having higher activity of conversion to andrographolide, should
Bacterial strain is accredited as Serratia proteamaculans (bibliography: microorganism conversion legal system through U.S.'s microbial identification and DNA identification experiment room
Standby Dehydro and drographolide, China Medicine University's journal, 2008,39:479-480), although this method mild condition and pollution compared with
It is few, but the bacterial strain used is the bacterial strain of new screening discovery, availability and popularization are poor, are not easy large-scale promotion.
Applicants have found that product is complex when andrographolide bioconversion prepares Dehydro and drographolide, until
There is the following two kinds dewatering type less:
But dehydration product 1 is only the active high Dehydro and drographolide of document record, therefore, bioconversion legal system
The selectivity that standby Dehydro and drographolide also needs to improve strain to conversion reaction.
Summary of the invention
The purpose of the present invention is to provide a kind of methods that bioanalysis prepares Dehydro and drographolide, it is intended to use routine
Bacterial strain carries out bioconversion as raw material using andrographolide and prepares Dehydro and drographolide.
The present invention is achieved by following technical solution:
A kind of preparation method of Dehydro and drographolide, includes the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, becomes seed liquor after shaken cultivation 2d；Draw seed liquor switching
Into the triangular flask equipped with fermentation medium, become culture solution after shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
The fermentation medium be added again in aforesaid liquid culture medium 40-60 μM season phosphine ionic liquid, cation knot
Structure is as follows:
Wherein, n is natural number；
Or 20-40 μM of benzotriazole ionic liquid of addition, cationic structural are as follows:
Wherein, n is natural number；
Or 30-50 μM of glyoxaline ion liquid of addition, cationic structural are as follows:
Wherein, n is natural number；
Step S2, microorganism conversion:
Andrographolide 0.5-1.5g/L is put into above-mentioned fermentation culture, shaken cultivation 3d is converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；
Step S4, high-speed counter-current purifying:
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
Preferably, the fermentation medium also contains 4-8 μM of valine.
Preferably, the anion of the ionic liquid is bromine or chloride ion.
Preferably, the shaken cultivation condition is 25 DEG C, 135r/min.
Preferably, the solvent system is ethyl acetate, alcohol and water-formic acid, and four volume ratios are 4:1:5:0.05.
Preferably, the separating pipe of the instrument of high speed adverse current chromatogram is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8,
Spiral pipe diameter 2.3mm, working speed 750r/min.
Preferably, the flow rate of mobile phase of the high speed adverse current chromatogram is 1.3mL/min, and separation temperature is 35 DEG C.
Season, phosphine ionic liquid converted andrographolide in Dehydro and drographolide in induction cunninghamella blakesleana
Using the cationic structural of the season phosphine ionic liquid is as follows:
Wherein, n is natural number.
Benzotriazole ionic liquid converts dehydration Herba Andrographitis for andrographolide in induction cunninghamella blakesleana
The cationic structural of application in lactone, the benzotriazole ionic liquid is as follows:
Wherein, n is natural number.
Glyoxaline ion liquid converts andrographolide in Dehydro and drographolide in induction cunninghamella blakesleana
Application, the cationic structural of the glyoxaline ion liquid is as follows:
Wherein, n is natural number.
Preferably, the anion of the ionic liquid is bromine or chloride ion.
Advantages of the present invention:
Preparation method provided by the invention can be conversion bacterial strain with the common cunninghamella blakesleana easily cultivated, and ferment
The functional ion liquid that specific structure is added in culture medium, converts Dehydro and drographolide for andrographolide；Meanwhile if
The valine that effective concentration is added in fermentation medium, can be improved the selectivity of the conversion reaction.
Detailed description of the invention
Fig. 1 is the liquid chromatogram for the converted product for adding ionic liquid and not adding ionic liquid；
Fig. 2 is that the high-speed counter-current of converted product purifies chromatogram.
Specific embodiment
Substantial technical scheme of the invention is discussed in detail below with reference to embodiment.
Every a kind of ionic liquid only enumerates 3-4 ionic liquid as space is limited, their chemical structure and number is such as
Under:
Outsourcing company can be entrusted to customize for above-mentioned ionic liquid or independently synthesis, those skilled in the art can obtain.
In embodiments of the present invention, these ionic liquids are synthesized by applicant according to literature method.
Cunninghamella blakesleana Cunninghamella blakesleeana Lendner CGMCC 3.970, is purchased from
Institute of microbiology of the academy of sciences of state China General Microbiological culture presevation administrative center.
The preparation (season phosphine -1) of 1 Dehydro and drographolide of embodiment
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
The fermentation medium is to add the season phosphine ionic liquid that 50 μM of numbers are season phosphine -1 again in aforesaid liquid culture medium
Body；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2, chromatogram are as shown in Figure 1 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component is collected according to chromatogram, chromatogram is as shown in Fig. 2, purity is greater than 98%.
The preparation (season phosphine -2) of 2 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that, substitutes season phosphine -1 using season phosphine -2.Specifically comprise the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
The fermentation medium is to add the season phosphine ionic liquid that 40 μM of numbers are season phosphine -2 again in aforesaid liquid culture medium
Body；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (season phosphine -3) of 3 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that, substitutes season phosphine -1 using season phosphine -3.Specifically comprise the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
The fermentation medium is to add the season phosphine ionic liquid that 60 μM of numbers are season phosphine -3 again in aforesaid liquid culture medium
Body；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (season phosphine -4) of 4 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that, substitutes season phosphine -1 using season phosphine -4.Specifically comprise the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
The fermentation medium is to add the season phosphine ionic liquid that 50 μM of numbers are season phosphine -4 again in aforesaid liquid culture medium
Body；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (phendioxin) of 5 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that, substitutes season phosphine -1 using phendioxin.Specifically comprise the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium be added again in aforesaid liquid culture medium 30 μM number be phendioxin benzotriazole from
Sub- liquid；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (benzo -2) of 6 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that, substitutes season phosphine -1 using benzo -2.Specifically comprise the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium be added again in aforesaid liquid culture medium 20 μM number be benzo -2 benzotriazole from
Sub- liquid；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (benzo -3) of 7 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that, substitutes season phosphine -1 using benzo -3.Specifically comprise the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium be added again in aforesaid liquid culture medium 40 μM number be benzo -3 benzotriazole from
Sub- liquid；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (imidazoles -1) of 8 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that, substitutes season phosphine -1 using imidazoles -1.Specifically comprise the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium is to add the glyoxaline ion liquid that 40 μM of numbers are imidazoles -1 again in aforesaid liquid culture medium；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (imidazoles -2) of 9 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that, substitutes season phosphine -1 using imidazoles -2.Specifically comprise the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium is to add the glyoxaline ion liquid that 30 μM of numbers are imidazoles -2 again in aforesaid liquid culture medium；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (imidazoles -3) of 10 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that, substitutes season phosphine -1 using imidazoles -3.Specifically comprise the following steps:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium is to add the glyoxaline ion liquid that 50 μM of numbers are imidazoles -3 again in aforesaid liquid culture medium；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (season phosphine -1+ valine) of 11 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 1 is only that fermentation medium also adds valine.Specifically include following step
It is rapid:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium be added again in aforesaid liquid culture medium 50 μM season phosphine -1 and 6 μM of valines；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (phendioxin+valine) of 12 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 5 is only that fermentation medium also adds valine.Specifically include following step
It is rapid:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium is to add 30 μM of phendioxins and 6 μM of valines again in aforesaid liquid culture medium；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
The preparation (imidazoles -1+ valine) of 13 Dehydro and drographolide of embodiment
The difference of the embodiment and embodiment 8 is only that fermentation medium also adds valine.Specifically include following step
It is rapid:
Step S1, Spawn incubation:
By cunninghamella blakesleana strain inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By thallus from oblique
Face culture medium is forwarded in the triangular flask equipped with fluid nutrient medium, and 25 DEG C, become seed liquor after 135r/min shaken cultivation 2d；It inhales
Seed liquor is taken to be forwarded in the triangular flask equipped with fermentation medium, 25 DEG C, become culture solution after 135r/min shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；
Magnesium sulfate, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium is to add 40 μM of imidazoles -1 and 6 μM of valines again in aforesaid liquid culture medium；
Step S2, microorganism conversion:
Put into andrographolide 1g/L into above-mentioned fermentation culture, 25 DEG C, 135r/min shaken cultivation 3d converted.
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate, filtrate acetic acid second are collected
Ester extracts repeatedly, combined ethyl acetate extract liquor, and evaporation recycling ethyl acetate is up to converted product；It is measured and is converted using HPLC method
The content of dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in product；
Step S4, high-speed counter-current purifying:
The separating pipe of high-speed counter-current chromatograph is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min；It is equipped with 8823B- UV detector；
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-
Alcohol-water-formic acid, stratification after which is acutely shaken separate upper and lower two-phase, and upper phase is stationary phase, lower phase
For mobile phase, Dehydro and drographolide is collected according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product.
First 12 hours preparation ethyl acetate, alcohol and water-formic acid (volume ratio 4:1:5:0.05) solvent systems are separated, specifically
Preparation method are as follows: mix four kinds of solvents in proportion, acutely after concussion, standing 12 hours is layered it completely, will in funnel
Upper and lower mutually to separate, upper phase is used as stationary phase, and lower phase is used as mobile phase, with preceding ultrasonic half an hour.Phase 5mL and lower phase are taken respectively
5mL, the sample solution by above-mentioned converted product ultrasonic dissolution in the in the mixed solvent after mixing, as high-speed counter-current.
By in two phase solvent system the upper phase (stationary phase) of ultrasonic degassing with the flow velocity of 20mL/min be pumped into HSCCC separation
In pipe (35 DEG C of separation temperature), after upper phase is full of entire separating pipe, adjusts engine speed and reaches 750r/min, rotate clockwise,
After stabilization of speed, lower phase (mobile phase), Detection wavelength 225nm are pumped into 1.3mL/min flow velocity.When mobile phase is from host mouth
When outflow, illustrate that system has reached fluid dynamic equilibrium, the 10mL sample solution being ready for injected into HSCCC instrument at this time,
Acquisition data are started simultaneously at, target component are collected according to chromatogram, purity is greater than 98%.
14 comparative example of embodiment
The difference of the embodiment and embodiment 1 is only that fermentation medium does not add any ionic liquid.
Containing for dehydration product 1 (Dehydro and drographolide) and dehydration product 2 in embodiment 1-14 converted product is measured respectively
After amount, conversion ratio (%) of the andrographolide to dehydration product 1 and dehydration product 2 is calculated, the result is as follows:
To sum up, preparation method provided by the invention can be using the common cunninghamella blakesleana easily cultivated as transformed bacteria
Strain adds the functional ion liquid of specific structure in the fermentation medium, converts Dehydro and drographolide for andrographolide；
Meanwhile if adding the valine of effective concentration in the fermentation medium, the selectivity of the conversion reaction can be improved.
Claims (4)
1. a kind of preparation method of Dehydro and drographolide, which comprises the steps of:
Step S1, Spawn incubation:
Cunninghamella blakesleana bacterial strain CGMCC 3.970 is inoculated on fresh slant medium, 25 DEG C of constant temperature incubation 3d；By bacterium
Body is forwarded in the triangular flask equipped with fluid nutrient medium from slant medium, becomes seed liquor after shaken cultivation 2d；Draw seed
Liquid is forwarded in the triangular flask equipped with fermentation medium, becomes culture solution after shaken cultivation 2d；
Wherein, the formula of the fluid nutrient medium are as follows: remove the peel the mashed potatoes boiled, 200g/L；Potassium dihydrogen phosphate, 3g/L；Sulfuric acid
Magnesium, 0.75g/L；Glucose, 20g/L；Vitamin B1,0.15g/L；PH value is adjusted to 6.0；
Fermentation medium is to add 30-50 μM of glyoxaline ion liquid again in aforesaid liquid culture medium, and cationic structural is as follows:
；Wherein, n=1 or 3, anion Cl-；Or n=5, anion Br-；
Step S2, microorganism conversion:
Andrographolide 0.5-1.5g/L is put into above-mentioned fermentation culture, shaken cultivation 3d is converted；
Step S3, converted product extract:
The pH value of conversion fluid is adjusted to 5.2 after conversion termination, is stood overnight, is filtered, filtrate is collected, filtrate is anti-with ethyl acetate
Multiple extraction, combined ethyl acetate extract liquor, evaporation recycling ethyl acetate is up to converted product；
Step S4, high-speed counter-current purifying:
The Dehydro and drographolide in converted product is isolated and purified with high speed adverse current chromatogram, solvent system is ethyl acetate-ethanol-
Water-formic acid, four volume ratios are 4:1:5:0.05, and stratification after which is acutely shaken separates upper and lower two-phase,
Upper phase is stationary phase, and lower phase is mobile phase, collects Dehydro and drographolide according to chromatogram and corresponds to fraction, is concentrated and dried to obtain the final product；
Wherein, the separating pipe of the instrument of high speed adverse current chromatogram is the multilayer polytetrafluoroethylarticles helix tube of β value 0.5-0.8, spiral pipe diameter
2.3mm, working speed 750r/min, the flow rate of mobile phase of the high speed adverse current chromatogram are 1.3mL/min, separation temperature 35
℃。
2. preparation method according to claim 1, it is characterised in that: the fermentation medium also contains 6 μM of valines.
3. preparation method according to claim 1 or 2, it is characterised in that: shaken cultivation condition is 25 DEG C, 135r/min.
4. glyoxaline ion liquid converts dehydration punching for andrographolide in induction cunninghamella blakesleana CGMCC 3.970
The cationic structural of application in lotus lactone, the glyoxaline ion liquid is as follows:
；Wherein, n=1 or 3, anion Cl-；Or n=5, anion Br-。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710964407.XA CN107502630B (en) | 2017-10-17 | 2017-10-17 | A kind of method that bioanalysis prepares Dehydro and drographolide |
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