CN107034261B - Method for preparing 6-O-glucose-cycloastragenol by converting astragaloside IV with aspergillus carbonarius - Google Patents

Method for preparing 6-O-glucose-cycloastragenol by converting astragaloside IV with aspergillus carbonarius Download PDF

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CN107034261B
CN107034261B CN201710311302.4A CN201710311302A CN107034261B CN 107034261 B CN107034261 B CN 107034261B CN 201710311302 A CN201710311302 A CN 201710311302A CN 107034261 B CN107034261 B CN 107034261B
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cycloastragenol
astragaloside
glucose
aspergillus carbonarius
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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 a method for preparing 6-O-glucose-cycloastragenol by converting astragaloside through Aspergillus carbonarius, and particularly relates to a method for preparing 6-O-glucose-cycloastragenol by fermenting and converting the astragaloside serving as a substrate under the premise of taking proper amount of astragalus powder as an inducer by utilizing the Aspergillus carbonarius (CICC 41254), extracting fermentation liquor obtained by fermentation through water saturated n-butanol, performing silica gel column chromatography and ethanol recrystallization, and finally obtaining a 6-O-glucose-cycloastragenol product with the purity of more than 95%. The invention utilizes a microbial conversion method to prepare 6-O-glucose-cycloastragenol, and is characterized in that the substrate astragaloside IV is almost completely converted, the conversion rate is high, the method is simple and easy to implement, the cost is low, no pollution is caused to the environment, and the method is suitable for industrial production.

Description

Method for preparing 6-O-glucose-cycloastragenol by converting astragaloside IV with aspergillus carbonarius
Technical Field
The invention belongs to the field of pharmaceutical chemicals, and particularly relates to a method for preparing 6-O-glucose-cycloastragenol by converting astragaloside IV through aspergillus carbonarius.
Background
The astragalus is a traditional Chinese medicine, has been used for more than 2000 years, and has the functions of enhancing the immunologic function of organisms, protecting the liver, promoting urination, resisting aging, resisting stress, reducing blood pressure and wider antibacterial action; the cycloastragenol saponin is the main active component in the astragalus root traditional Chinese medicine, belongs to cycloartane tetracyclic triterpene saponin, has the functions of enhancing immunity, increasing energy, resisting fatigue, protecting liver, eliminating free radicals and inhibiting osteoclast, and has high medicinal value.
6-O-glucose-Cycloastragenol (CMG) belongs to cycloastragenol saponin, and the structure of the CMG is firstly reported in 1983 by Isao Kitagawa and is obtained by hydrolyzing astragaloside by hesperidinase.
In the published patent application in China (Chinese patent application publication No.: CN1809364A), the 6-O-glucose-cycloastragenol is obtained by mild acid hydrolysis of astragaloside IV, but the yield of CMG obtained by the reaction is very low, only 21%. And the byproducts prepared by the acid method are many, the structure and the size of the byproducts are similar to those of CMG, the later separation is difficult, and the industrial production is not facilitated. Oncorhynchus chinensis et al, the university of Tianjin medical science, hydrolyzes Astragaloside to 6-O-glucose-cycloastragenol by using beta-glucosidase, the conversion rate reaches more than 90%, but compared with a microbial conversion method, the cost of enzymatic preparation is higher, and the commercialized beta-glucosidase is not Astragaloside IV specific hydrolase (Oncorhynchus chinensis, Liupeng, etc.. research on the hydrolysis of Astragaloside IV by beta-glucosidase [ J ]. Chinese herbal medicine, 2012,43(6): 1112-.
6-O-glucose-Cycloastragenol (CMG) structure
Figure BDA0001287186030000021
Disclosure of Invention
The invention aims to obtain 6-O-glucose-cycloastragenol by converting astragaloside IV by adopting a microbial conversion method, and overcomes the defects of more byproducts, low conversion rate efficiency, high cost, environmental pollution and the like in the existing 6-O-glucose-cycloastragenol preparation technology. The invention makes innovation and improvement according to the defects of the prior art and provides a high-efficiency biological method for preparing 6-O-glucose-cycloastragenol.
In order to realize the purpose of the invention, the adopted technical scheme is as follows:
A. seed liquid preparation
Transferring the aspergillus carbonarius lyophilized powder to a PDA plane culture medium after activated culture of a test tube slant, and adding sterile water into the plane culture medium to prepare the aspergillus carbonarius lyophilized powder with the spore concentration of 6 × 108Per mL Aspergillus carbonarius spore suspension.
B. Fermentation transformation
And C, inoculating the aspergillus carbonarius spore suspension in the step A into a liquid fermentation culture medium, and culturing for 6 days under the conditions that the temperature of a shaking table is 28-30 ℃ and the rotating speed of the shaking table is 180r/min to obtain fermentation liquor.
C. Separating and purifying
And C, extracting the fermentation liquor obtained in the step B for three times by using water saturated n-butanol, then carrying out vacuum concentration and evaporation to obtain an extracted product, further carrying out separation and purification by using silica gel column chromatography, and finally improving the purity of the product to more than 95% by using ethanol recrystallization to obtain a 6-O-glucose-cycloastragenol product.
The formula of the fermentation medium in the step B is as follows: the concentration of the substrate astragaloside is 1mg/mL, the concentration of the astragalus powder is 2g/L, the concentration of the yeast powder is 10g/L, the concentration of the ammonium sulfate is 2g/L, and KH is added2PO4The concentration is 4g/L, MgSO4·7H2O concentration of 1g/L, Tween80 volume percent is 0.1%, the initial pH value is 4.5, and the volume ratio of the inoculum size to the fermentation medium is 1: 100.
The astragalus powder in the step B is 80-mesh astragalus powder, is prepared by directly crushing cleaned astragalus roots and stems, and is mainly used for inducing aspergillus carbonarius to produce enzyme in the fermentation process.
The mass percentage of the substrate astragaloside in the step B is 10%.
And C, the filler of the silica gel column chromatography in the step C is silica gel powder of 100-200 meshes.
The eluent for silica gel column chromatography in the step C is a lower layer solution of methanol-chloroform-water with the volume ratio of 26:13: 4.
The invention is characterized in that the method of microbial transformation is utilized to convert the astragaloside to prepare the 6-O-glucose-cycloastragenol. Compared with the prior art, the invention has the following remarkable beneficial effects:
during the process of preparing 6-O-glucose-cycloastragenol by converting astragaloside IV by aspergillus carbonarius, no by-product is generated, and the conversion efficiency is improved.
Compared with the prior art, the conversion rate of converting the aspergillus carbonarius into the astragaloside is high, the substrate astragaloside is almost completely converted into the product 6-O-glucose-cycloastragenol, the purity of the final product is high, and the production cost is reduced.
Compared with the traditional chemical method, the microbial conversion method is clean biological conversion, has mild preparation conditions, reduces energy consumption, greatly reduces the damage to the environment in the preparation process, and is suitable for industrial production.
Drawings
FIG. 1 is a diagram of the transformation pathway of Aspergillus carbonarius.
Detailed Description
The present invention is further described with reference to specific examples, which are not intended to limit the scope of the present invention.
Example 1
Placing the aspergillus carbonarius freeze-dried powder on a test tube inclined plane containing a PDA culture medium for activation culture, then transferring the aspergillus carbonarius freeze-dried powder into a PDA plane culture medium for culture, and waiting until the aspergillus carbonarius is completely full-lengthAdding sterile water into the planar culture medium after spore emergence to make the spore concentration be 6 × 108And (4) one/mL of aspergillus carbonarius spore suspension, namely seed liquid for fermenting the strains (all operations are carried out in an ultra-clean bench in a sterile way).
The formula of the fermentation liquid culture medium is as follows: the substrate is 10% of astragaloside IV with the concentration of 1mg/mL, the concentration of astragalus powder is 2g/L, the concentration of yeast powder is 10g/L, the concentration of ammonium sulfate is 2g/L, KH2PO4The concentration is 4g/L, MgSO4·7H2The O concentration is 1g/L, the Tween 80 volume percent is 0.1 percent, and the initial pH value is 4.5. Preparing a liquid fermentation culture medium according to the formula of the culture medium.
Collecting 1mL spore with concentration of 6 × 108Inoculating the aspergillus carbonarius spore suspension into a 250mL shake flask containing 100mL liquid fermentation medium, placing the shake flask into a constant temperature shaking table at 28 ℃ for culture, adjusting the rotation speed of the shaking table to be 180r/min, and performing fermentation culture for 6 days under the condition. The resulting 95mL fermentation broth was collected.
Adding 50mL of water saturated n-butanol into the fermentation liquor for extraction, continuously extracting for three times, and then carrying out vacuum concentration and evaporation drying on the obtained extract liquor to obtain 0.16g of a crude product. Then, the crude product was completely dissolved in 10mL of methanol, and 1mL of the solution was filtered through a 0.22 μm organic filter, and the purity of the crude product was calculated to be 4.68% by HPLC-evaporative light detection.
And (3) separating and purifying 1g of the crude product with the purity by silica gel column chromatography, wherein the filler is silica gel powder with 100-200 meshes, the silica gel powder is loaded by a dry method, and the eluent is lower-layer solution of methanol-chloroform-water with the volume ratio of 26:13: 4. The fractions containing the product were collected and tested for concentration in the liquid phase, after which all fractions were collected by spin drying and weighed dry to calculate 50.32mg of product with a purity of 88.36%.
After the product obtained after silica gel column chromatography is recrystallized by ethanol, the purity of the 6-O-glucose-cycloastragenol product is finally improved to more than 95 percent.
Example 2
Placing the aspergillus carbonarius freeze-dried powder on a test tube inclined plane containing a PDA culture medium for activation culture, then transferring the aspergillus carbonarius freeze-dried powder into a PDA plane culture medium for culture, and waiting for the aspergillus carbonarius freeze-dried powder to be culturedAdding sterile water into the plane culture medium after completely growing spores to make the concentration of the spores be 6 × 108And (4) one/mL of aspergillus carbonarius spore suspension, namely seed liquid for fermenting the strains (all operations are carried out in an ultra-clean bench in a sterile way).
The formula of the fermentation liquid culture medium is as follows: the substrate is 10% of astragaloside IV with the concentration of 1mg/mL, the concentration of astragalus powder is 2g/L, the concentration of yeast powder is 10g/L, the concentration of ammonium sulfate is 2g/L, KH2PO4The concentration is 4g/L, MgSO4·7H2The O concentration is 1g/L, the volume percentage of Tween 80 is 0.1 percent, and the initial pH value is 4.5. Preparing a liquid fermentation culture medium according to the formula of the culture medium.
Collecting 1mL spore with concentration of 6 × 108Inoculating the aspergillus carbonarius spore suspension into a 250mL shake flask containing 100mL liquid fermentation medium, placing the shake flask into a constant temperature shaking table at 30 ℃ for culture, adjusting the rotation speed of the shaking table to be 180r/min, and performing fermentation culture for 6 days under the condition. 91mL of fermentation broth was collected.
Adding 50mL of water saturated n-butanol into the fermentation liquor for extraction, continuously extracting for three times, and then carrying out vacuum concentration and evaporation drying on the obtained extract liquor to obtain 0.157g of a crude product. Then, the crude product was completely dissolved in 10mL of methanol, and 1mL of the solution was filtered through a 0.22 μm organic filter, and the purity of the crude product was calculated to be 4.72% by HPLC-evaporative light detection.
And (3) separating and purifying 1g of the crude product with the purity by silica gel column chromatography, wherein the filler is silica gel powder with 100-200 meshes, and the crude product is loaded by a dry method, and the eluent is lower-layer solution of methanol-chloroform-water with the ratio of 26:13: 4. The fractions containing the product were collected and tested for concentration in the liquid phase, after which all fractions were collected by spin drying and weighed dry to calculate 52.86mg of product having a purity of 87.36%.
After the product obtained after silica gel column chromatography is recrystallized by ethanol, the purity of the 6-O-glucose-cycloastragenol product is finally improved to more than 95 percent.
Example 3
Placing the aspergillus carbonarius freeze-dried powder on a test tube inclined plane containing a PDA culture medium for activation culture, then transferring the aspergillus carbonarius freeze-dried powder into a PDA plane culture medium for culture, and waiting forAdding sterile water into the planar culture medium after the aspergillus carbonarius completely grows spores to obtain the spore concentration of 6 × 108And (4) one/mL of aspergillus carbonarius spore suspension, namely seed liquid for fermenting the strains (all operations are carried out in an ultra-clean bench in a sterile way).
The formula of the fermentation liquid culture medium is as follows: the substrate is 10% of astragaloside IV with the concentration of 1mg/mL, the concentration of astragalus powder is 2g/L, the concentration of yeast powder is 10g/L, the concentration of ammonium sulfate is 2g/L, KH2PO4The concentration is 4g/L, MgSO4·7H2The O concentration is 1g/L, the Tween 80 volume percent is 0.1 percent, and the initial pH value is 4.5. Preparing a liquid fermentation culture medium according to the formula of the culture medium.
Collecting 10mL spore with concentration of 6 × 108Inoculating the aspergillus carbonarius spore suspension into a 2500mL shake flask containing 1000mL of liquid fermentation medium, placing the shake flask in a constant temperature shaking table at 30 ℃ for culture, adjusting the rotation speed of the shaking table to be 180r/min, and performing fermentation culture for 6 days under the condition. 980mL of fermentation broth was collected.
Adding 500mL of water saturated n-butanol into the fermentation liquor for extraction, continuously extracting for three times, and then carrying out vacuum concentration and evaporation drying on the obtained extract liquor to obtain 1.82g of a crude product. 0.2g of the crude product was dissolved in 10mL of methanol, and 1mL of the solution was filtered through a 0.22 μm organic filter and the purity of the crude product was calculated to be 4.12% by HPLC-evaporative light detection.
Separating and purifying 10g of the crude product with the purity by silica gel column chromatography, and loading the crude product by a dry method, wherein the eluent is lower layer solution of methanol-chloroform-water, and the ratio is 26:13: 4. Fractions containing the product were collected and tested for concentration in the liquid phase, after which all fractions were collected by spin drying and weighed dry to calculate 464.5mg of product with a purity of 84.26%.
And recrystallizing the product after silica gel column chromatography by using ethanol, and finally improving the purity of the 6-O-glucose-cycloastragenol product to more than 95%.

Claims (5)

1. The method for preparing 6-O-glucose-cycloastragenol by converting astragaloside IV with aspergillus carbonarius is characterized by comprising the following steps: the fermentation strain is aspergillus carbonarius (A. carbonarius)Aspergillus carbonarius.) The CICC 41254 comprises the following steps in sequence:
A. seed liquid preparation
Transferring the aspergillus carbonarius lyophilized powder to a PDA plane culture medium after activated culture of a test tube slant, and adding sterile water into the plane culture medium to prepare the aspergillus carbonarius lyophilized powder with the spore concentration of 6 × 108a/mL aspergillus carbonarius spore suspension;
B. fermentation transformation
B, inoculating the aspergillus carbonarius spore suspension in the step A into a fermentation medium, placing the fermentation medium into a constant-temperature shaking table at the temperature of 28-30 ℃, and culturing for 6 days under the condition that the rotation speed of the shaking table is 180r/min to obtain fermentation liquor; the fermentation medium comprises a substrate of astragaloside IV with a concentration of 1mg/mL, radix astragali powder with a concentration of 2g/L, yeast powder with a concentration of 10g/L, ammonium sulfate with a concentration of 2g/L, KH2PO4 concentration was 4g/L, MgSO4·7H2The concentration of O is 1g/L, the volume percentage of Tween 80 is 0.1 percent, the initial pH value is 4.5, and the volume ratio of the inoculum size to the fermentation medium is 1: 100;
C. separating and purifying
And C, extracting the fermentation liquor obtained in the step B for three times by using water saturated n-butanol, then carrying out vacuum concentration and evaporation to obtain an extracted product, further carrying out separation and purification by using silica gel column chromatography, and finally improving the purity of the product to more than 95% by using ethanol recrystallization to obtain a 6-O-glucose-cycloastragenol product.
2. The method of claim 1, wherein: the radix astragali powder in the fermentation medium formula is 80 mesh radix astragali powder, and is prepared by directly pulverizing cleaned radix astragali rhizome.
3. The method of claim 1, wherein: and the mass percentage of the astragaloside in the step B is 10 percent.
4. The method of claim 1, wherein: and C, the silica gel column chromatography filler in the step C is 100-200 meshes of silica gel powder.
5. The method of claim 1, wherein: and C, eluting the silica gel column chromatography in the step C by using a lower layer solution of methanol-chloroform-water with the volume ratio of 26:13:4 respectively.
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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
CN106011213A (en) * 2016-06-03 2016-10-12 安徽大学 Method for preparing cycloastragenol by bioconversion and degradation of astragaloside

Patent Citations (2)

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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
CN106011213A (en) * 2016-06-03 2016-10-12 安徽大学 Method for preparing cycloastragenol by bioconversion and degradation of astragaloside

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
黄芪甲苷和环黄芪醇生产工艺的研究;王立媛;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20160315(第3期);B016-160 *

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