CN107034261A - The method that Aspergillus carbonerius conversion Astragaloside IV prepares 6 O glucose ring Astragenols - Google Patents

The method that Aspergillus carbonerius conversion Astragaloside IV prepares 6 O glucose ring Astragenols Download PDF

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CN107034261A
CN107034261A CN201710311302.4A CN201710311302A CN107034261A CN 107034261 A CN107034261 A CN 107034261A CN 201710311302 A CN201710311302 A CN 201710311302A CN 107034261 A CN107034261 A CN 107034261A
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astragaloside
aspergillus
carbonerius
aspergillus carbonerius
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CN107034261B (en
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袁其朋
程磊雨
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Beijing University of Chemical Technology
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Abstract

The invention discloses the method that Aspergillus carbonerius conversion Astragaloside IV prepares 6 O glucose ring Astragenols, it is specifically related to a kind of to utilize Aspergillus carbonerius (Aspergillus carbonarius.CICC 41254) on the premise of appropriate astragalus membranaceus powder is inducer, converted by fermenting substrate of Astragaloside IV and 6 O glucose ring Astragenols are made, and will fermentation gained zymotic fluid extracted through water-saturated n-butanol, silica gel column chromatography and ethyl alcohol recrystallization, finally give the 6 O glucose ring Astragenol products that purity reaches more than 95%.The present invention is to prepare 6 O glucose ring Astragenols using microbe transformation method, and its feature is that substrate Astragaloside IV almost all is converted, and high conversion rate, method is simple and easy to apply, and cost is low, and environmentally safe, is adapted to industrialized production.

Description

The method that Aspergillus carbonerius conversion Astragaloside IV prepares 6-O- glucose-cycloastragenol
Technical field
The invention belongs to field of medicine and chemical technology, and in particular to one kind prepares 6-O- Portugals using Aspergillus carbonerius conversion Astragaloside IV The method of grape sugar-cycloastragenol.
Background technology
The Radix Astragali is traditional Chinese medicine simply, its medicinal history for having more than 2000 years so far, with enhancing body's immunity, Liver protection, diuresis, anti-aging, resisting stress, decompression and wide antibacterial action；Cycloastragenol saponins are the masters in Radix Astragali Chinese medicine Active component is wanted, belongs to cyclic-ahltin alkane tetracyclic triterpene saponins, with booster immunization power, increase energy, resisting fatigue, protection Liver, the effect for removing free radical and suppression osteoclast, there is very high medical value.
6-O- glucose-cycloastragenol (CMG) belongs to cycloastragenol saponins, its structure by Isao Kitagawa in Nineteen eighty-three is reported first, is obtained by hesperidinase hydrolysis for astragalus first glycosides.
(Chinese patent application publication number in the published patent application of China：CN1809364A it is) yellow with mild acid hydrolysis Stilbene first glycosides obtains 6-O- glucose-cycloastragenol, but the yield for reacting obtained CMG is very low, and only 21%.And prepared by acid system Accessory substance it is many, its structure and size are similar with CMG, and later stage separation is difficult, is unfavorable for industrialized production.Medical University Of Tianjin Summer wide duckweed et al. arrives 6-O- glucose-cycloastragenol using beta-glucosidase hydrolysis for astragalus first glycosides, and conversion ratio reaches 90% More than, but compared to microbe transformation method, cost prepared by enzyme process is higher, and the beta-glucosidase of commercialization is also not yellow Stilbene first glycosides specific hydrolase enzyme (Xia Guangping, Liu Peng, wait research [J] Chinese herbal medicines of beta-glucosidase hydrolysis for astragalus first glycosides, 2012,43(6):1112-1114.
6-O- glucose-cycloastragenol (CMG) structure
The content of the invention
It is an object of the invention to select to obtain 6-O- Portugals using a kind of method of microorganism conversion to convert Astragaloside IV Grape sugar-cycloastragenol, overcomes in existing 6-O- glucose-cycloastragenol technology of preparing that accessory substance is more, and conversion ratio efficiency is low, cost The shortcomings of height, pollution environment.The present invention according to the deficiencies in the prior art make innovation and improve there is provided a kind of efficient bioanalysis The method for preparing 6-O- glucose-cycloastragenol.
For achieving the above object, the technical scheme used is：
A. prepared by seed liquor
Aspergillus carbonerius freeze-dried powder is transferred to after test tube slant activation culture in PDA plating mediums, then is trained to plane It is 6 × 10 to support addition sterilized water in base and spore concentration is made8Individual/mL Aspergillus carbonerius spore suspensions.
B. microbe conversion
Aspergillus carbonerius spore suspension in step A is seeded in liquid fermentation medium, it is 28 to be placed in shaking table temperature ~30 DEG C, shaking speed be 180r/min under conditions of cultivate 6 days, obtain zymotic fluid.
C. isolate and purify
It is concentrated in vacuo to be evaporated after zymotic fluid in step B is extracted three times via water-saturated n-butanol and obtains extracted products, Silica gel column chromatography separating purification is further utilized, the purity of product is brought up to more than 95% eventually through ethyl alcohol recrystallization, obtained To 6-O- glucose-cycloastragenol product.
Fermentative medium formula in above-mentioned steps B is：The concentration of substrate Astragaloside IV is 1mg/mL, and astragalus membranaceus powder concentration is 2g/L, dusty yeast concentration is 10g/L, and ammonium sulfate concentrations are 2g/L, KH2PO4Concentration is 4g/L, MgSO4·7H2O concentration is 1g/ L, Tween 80 percent by volume is 0.1%, and initial pH value is 4.5, and the volume ratio of inoculum concentration and fermentation medium is 1:100.
Astragalus membranaceus powder in above-mentioned steps B is the astragalus membranaceus powder of 80 mesh, is directly to crush clean Radix Astragali rhizome to be made, in hair It is mainly used to induce Aspergillus carbonerius producing enzyme during ferment.
The mass percent of substrate Astragaloside IV in above-mentioned steps B is 10%.
The filler of silica gel column chromatography in above-mentioned steps C is the silica white of 100~200 mesh.
The eluant, eluent of silica gel column chromatography is that volume ratio is 26 in above-mentioned steps C:13:The lower floor of 4 methanol-chloroform-water Solution.
It is characteristic of the invention that preparing 6-O- glucose-ring Radix Astragali using the method conversion Astragaloside IV of microorganism conversion Alcohol.Compared with prior art, the significant beneficial effect of the present invention is：
Aspergillus carbonerius conversion Astragaloside IV will not produce any by-product during preparing 6-O- glucose-cycloastragenol Thing, improves transformation efficiency.
Compared with prior art, Aspergillus carbonerius converts the high conversion rate of Astragaloside IV, and substrate Astragaloside IV almost all turns Product 6-O- glucose-cycloastragenol is turned to, and the purity of finished product is high, reduces production cost.
Compared with conventional chemical methods, microbe transformation method is a kind of bioanalysis conversion of cleaning, and its preparation condition is gentle, energy The injury of environment is substantially reduced in consumption reduction, preparation process, is adapted to industrialized production.
Brief description of the drawings
Fig. 1 is Aspergillus carbonerius conversion pathway figure.
Embodiment
With reference to specific embodiment, the invention will be further described, but the purpose of these embodiments and does not lie in limit Protection scope of the present invention processed.
Embodiment 1
Take Aspergillus carbonerius freeze-dried powder to be placed in activation culture on the test tube slant containing PDA culture medium, after transfer to PDA put down Cultivated in the culture medium of face, adding sterilized water into plating medium after Aspergillus carbonerius grows spore completely is made spore concentration and is 6×108(all operations are sterile in super-clean bench for the seed liquor of individual/mL Aspergillus carbonerius spore suspension, as strain fermentation Operation).
Fermentation broth is formulated：Substrate is the Astragaloside IV of 10% mass percent, and concentration is 1mg/mL, astragalus membranaceus powder Concentration is 2g/L, and dusty yeast concentration is 10g/L, and ammonium sulfate concentrations are 2g/L, KH2PO4Concentration is 4g/L, MgSO4·7H2O concentration For 1g/L, Tween 80 percent by volume is 0.1%, and initial pH value is 4.5.Liquid fermentation and culture is prepared according to the formula of culture medium Base.
It is 6 × 10 to take 1mL spore concentrations8Individual/mL Aspergillus carbonerius spore suspensions are seeded to containing the training of 100mL liquid fermentations In the shaking flask for the 250mL for supporting base, and shaking flask is placed in culture in 28 DEG C of constant-temperature table, regulation shaking speed is 180r/min, Fermented and cultured 6 days on this condition.Collect the zymotic fluid for obtaining 95mL.
Add the extraction of 50mL water-saturated n-butanols into zymotic fluid, it is dense through vacuum to obtain extract after continuous extraction three times Contracting, which is evaporated, obtains crude product 0.16g.Crude product is completely dissolved with 10mL methanol afterwards, taking 1mL solution to cross 0.22 μm has machine filter Film, in high performance liquid chromatography-lower measure of evaporative light detection, the purity for being computed crude product is 4.68%.
Take the crude product 1g of above-mentioned purity in silica gel column chromatography separating purification, filler is the silica white of 100~200 mesh, is adopted Dry method loading is used, eluant, eluent is that volume ratio is 26:13:Lower floor's solution of 4 methanol-chloroform-water.Collection wherein contains product Cut and in liquid phase detectable concentration, after all fraction collections be spin-dried for and weigh dry weight, calculate 50.32mg purity For 88.36% product.
By the product after silica gel column chromatography by ethyl alcohol recrystallization after, most at last 6-O- glucose-cycloastragenol product Purity has brought up to more than 95%.
Embodiment 2
Take Aspergillus carbonerius freeze-dried powder to be placed in activation culture on the test tube slant containing PDA culture medium, after transfer to PDA put down Cultivated in the culture medium of face, adding sterilized water into plating medium after Aspergillus carbonerius grows spore completely is made spore concentration and is 6×108(all operations are sterile in super-clean bench for the seed liquor of individual/mL Aspergillus carbonerius spore suspension, as strain fermentation Operation).
Fermentation broth is formulated：Substrate is the Astragaloside IV of 10% mass percent, and concentration is 1mg/mL, astragalus membranaceus powder Concentration is 2g/L, and dusty yeast concentration is 10g/L, and ammonium sulfate concentrations are 2g/L, KH2PO4Concentration is 4g/L, MgSO4·7H2O concentration For 1g/L, the percent by volume of Tween 80 is 0.1%, and initial pH value is 4.5.Formula according to culture medium prepares liquid fermentation training Support base.
It is 6 × 10 to take 1mL spore concentrations8Individual/mL Aspergillus carbonerius spore suspensions are seeded to containing the training of 100mL liquid fermentations In the shaking flask for the 250mL for supporting base, and shaking flask is placed in culture in 30 DEG C of constant-temperature table, regulation shaking speed is 180r/min, Fermented and cultured 6 days on this condition.Collect the zymotic fluid for obtaining 91mL.
Add the extraction of 50mL water-saturated n-butanols into zymotic fluid, it is dense through vacuum to obtain extract after continuous extraction three times Contracting, which is evaporated, obtains crude product 0.157g.Crude product is completely dissolved with 10mL methanol afterwards, taking 1mL solution to cross 0.22 μm has machine filter Film, in high performance liquid chromatography-lower measure of evaporative light detection, the purity for being computed crude product is 4.72%.
Take the crude product 1g of above-mentioned purity in silica gel column chromatography separating purification, filler is the silica white of 100~200 mesh, is adopted Dry method loading is used, eluant, eluent is lower floor's solution of methanol-chloroform-water, and its ratio is 26:13:4.Collect wherein containing product Cut and in liquid phase detectable concentration, after all fraction collections be spin-dried for and weigh dry weight, calculating 52.86mg purity is 87.36% product.
By the product after silica gel column chromatography by ethyl alcohol recrystallization after, most at last 6-O- glucose-cycloastragenol product Purity has brought up to more than 95%.
Embodiment 3
Take Aspergillus carbonerius freeze-dried powder to be placed in activation culture on the test tube slant containing PDA culture medium, after transfer to PDA put down Cultivated in the culture medium of face, adding sterilized water into plating medium after Aspergillus carbonerius grows spore completely is made spore concentration and is 6×108(all operations are sterile in super-clean bench for the seed liquor of individual/mL Aspergillus carbonerius spore suspension, as strain fermentation Operation).
Fermentation broth is formulated：Substrate is the Astragaloside IV of 10% mass percent, and concentration is 1mg/mL, astragalus membranaceus powder Concentration is 2g/L, and dusty yeast concentration is 10g/L, and ammonium sulfate concentrations are 2g/L, KH2PO4Concentration is 4g/L, MgSO4·7H2O concentration For 1g/L, Tween 80 percent by volume is 0.1%, and initial pH value is 4.5.Liquid fermentation and culture is prepared according to the formula of culture medium Base.
It is 6 × 10 to take 10mL spore concentrations8Individual/mL Aspergillus carbonerius spore suspensions are seeded to containing 1000mL liquid fermentations In the 2500mL of culture medium shaking flask, and shaking flask is placed in culture in 30 DEG C of constant-temperature table, regulation shaking speed is 180r/ Min, on this condition fermented and cultured 6 days.Collect the zymotic fluid for obtaining 980mL.
Add the extraction of 500mL water-saturated n-butanols into zymotic fluid, extract will be obtained through vacuum after continuous extraction three times Concentration, which is evaporated, obtains crude product 1.82g.Take 0.2g crude product fully to be dissolved in 10mL methanol, take 1mL solution to cross 0.22 μ The organic filter membranes of m, in high performance liquid chromatography-lower measure of evaporative light detection, the purity for being computed crude product is 4.12%.
Take the crude product 10g of above-mentioned purity in silica gel column chromatography separating purification, use a dry method on a sample, eluant, eluent be methanol- Lower floor's solution of chloroform-water, its ratio is 26:13:4.Collect the wherein cut containing product and in liquid phase detectable concentration, after will All fraction collections are spin-dried for and weigh dry weight, calculate 464.5mg purity for 84.26% product.
Afterwards by the product after silica gel column chromatography by ethyl alcohol recrystallization after, most at last 6-O- glucose-cycloastragenol product Purity brought up to more than 95%.

Claims (5)

1. the method that Aspergillus carbonerius conversion Astragaloside IV prepares 6-O- glucose-cycloastragenol, it is characterised in that：Fermented bacterium is Aspergillus carbonerius (Aspergillus carbonarius.CICC 41254), comprises the following steps successively：
A. prepared by seed liquor
Aspergillus carbonerius freeze-dried powder is transferred to after test tube slant activation culture in PDA plating mediums, then into plating medium It is 6 × 10 to add sterilized water and spore concentration is made8Individual/mL Aspergillus carbonerius spore suspension；
B. microbe conversion
Aspergillus carbonerius spore suspension in step A is seeded in fermentation medium, the perseverance that temperature is 28~30 DEG C is placed in In warm shaking table, cultivated 6 days under conditions of shaking speed is 180r/min, obtain zymotic fluid.Fermentative medium formula is substrate Astragaloside IV concentration is 1mg/mL, and astragalus membranaceus powder concentration is 2g/L, and dusty yeast concentration is 10g/L, and ammonium sulfate concentrations are 2g/L, KH2PO4 concentration is 4g/L, MgSO4·7H2O concentration is 1g/L, and Tween 80 percent by volume is 0.1%, and initial pH value is 4.5, Inoculum concentration and the volume ratio of fermentation medium are 1:100；
C. isolate and purify
It is concentrated in vacuo to be evaporated after zymotic fluid in step B is extracted three times via water-saturated n-butanol and obtains extracted products, enters one Step utilizes silica gel column chromatography separating purification, and the purity of product is brought up into more than 95% eventually through ethyl alcohol recrystallization, 6- is obtained O- glucose-cycloastragenol product.
2. according to the method described in claim 1, it is characterised in that：Astragalus membranaceus powder in fermentation based formulas is the astragalus membranaceus powder of 80 mesh, It is directly to crush clean Radix Astragali rhizome to be made.
3. according to the method described in claim 1, it is characterised in that：The quality percentage of Astragaloside IV in step B is 10%.
4. according to the method described in claim 1, it is characterised in that：Silica gel column chromatography filler in step C is 100~200 mesh Silica white.
5. according to the method described in claim 1, it is characterised in that：The eluant, eluent of silica gel column chromatography is volume ratio in step C Respectively 26:13:Lower floor's solution of 4 methanol-chloroform-water.
CN201710311302.4A 2017-05-05 2017-05-05 Method for preparing 6-O-glucose-cycloastragenol by converting astragaloside IV with aspergillus carbonarius Active CN107034261B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109609581A (en) * 2018-12-28 2019-04-12 北京化工大学 A kind of method that microorganism mixed fermentation conversion degradation Astragaloside IV prepares cycloastragenol

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CN101225424A (en) * 2007-09-13 2008-07-23 天津药物研究院 Single-glucopyranoside of cyclomembranousol, preparation method, medicament combination and uses thereof
CN106011213A (en) * 2016-06-03 2016-10-12 安徽大学 Method for preparing cycloastragenol by bioconversion and degradation of astragaloside

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Publication number Priority date Publication date Assignee Title
CN101225424A (en) * 2007-09-13 2008-07-23 天津药物研究院 Single-glucopyranoside of cyclomembranousol, preparation method, medicament combination and uses thereof
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109609581A (en) * 2018-12-28 2019-04-12 北京化工大学 A kind of method that microorganism mixed fermentation conversion degradation Astragaloside IV prepares cycloastragenol

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