CN104711312A - Method for preparing astragaloside from trichoderma viride - Google Patents
Method for preparing astragaloside from trichoderma viride Download PDFInfo
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- CN104711312A CN104711312A CN201510112855.8A CN201510112855A CN104711312A CN 104711312 A CN104711312 A CN 104711312A CN 201510112855 A CN201510112855 A CN 201510112855A CN 104711312 A CN104711312 A CN 104711312A
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- cyclosiversioside
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- 0 CC1OC1(C)[C@](CC1)O[C@@]1(C)[C@@](C(C1)O)C(C)(CC2)[C@]1(C)C(CC1)C2(C2)C22C1C(C)(C)C(*C(*1)C=IC[C@@](*)C1=O)CC2 Chemical compound CC1OC1(C)[C@](CC1)O[C@@]1(C)[C@@](C(C1)O)C(C)(CC2)[C@]1(C)C(CC1)C2(C2)C22C1C(C)(C)C(*C(*1)C=IC[C@@](*)C1=O)CC2 0.000 description 2
- CPDRBUMJBJPOKO-UHFFFAOYSA-N CCC(CCC1)=CC11C(C)C(C)C1 Chemical compound CCC(CCC1)=CC11C(C)C(C)C1 CPDRBUMJBJPOKO-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a method for preparing astragaloside from trichoderma viride. According to the method, Chinese herbal medicine astragalus is subjected to microbial conversion by utilizing the trichoderma viride, and enrichment of an effective ingredient, namely astragaloside is realized under a certain fermentation cultivation condition; then astragaloside crude extract obtained with a biological method is subjected to separation and purification by utilizing macroporous adsorption resin HPD100 and is purified with methods of silica-gel column chromatography, methanol recrystallization and the like to finally obtain astragaloside with the purity higher than 98%. The method aims to overcome shortcomings of high raw material cost, low astragaloside yield, complex process, serious pollution and the like, and is suitable for large-scale production of astragaloside with different purities.
Description
Technical field
The invention belongs to biochemical field, relating to a kind of is that prepared using viride transforms the novel method preparing Cyclosiversioside F with Chinese medicine astragalus.
Background technology
The Radix Astragali is sweet, tepor, is the conventional Chinese medicine of strengthening the body resistance to consolidate the constitution.Chinese Pharmacopoeia specifies, Chinese medicine astragalus is the dry root of leguminous plants Radix Astragali Astragalus membranaceus (Fisch.) Bge and Radix Astagali Astragalusmembranaceus (Fisch.) Bge.Var.mongholicus (Bge.) Hsiao.Its another name has continuous stilbene, the continuous Radix Astragali.Modern study finds that astragalus root comprises multiple composition.Containing Cyclosiversioside F, Isorhamnetol, Quercetin, β-sitosterol, sandlwood fruit element, linolic acid and linolenic acid etc. in the root of wherein Radix Astagali; Folic acid containing Cyclosiversioside F, trimethyl-glycine, choline, sucrose, amino acid, glucuronic acid and trace in Radix Astragali root.Radix Astragali saponin kind is very many, can from astragalus root isolation identification out just have tens of kinds, Cyclosiversioside F (AstragalosideIV, AST) is one of principle active component.It has booster immunization power, increases energy, resisting fatigue, protection liver, removes the effect of interior free yl and suppression osteoclast, has very high pharmaceutical use.
The preparation method of current Cyclosiversioside F is mainly natural product extraction, application number be 200410014786.9 Chinese invention patent application disclose and utilize Chinese medicine astragalus powder to prepare the method for Cyclosiversioside F for raw material, key step is alcohol reflux, extraction into ethyl acetate, recrystallizing methanol, traditional method is mainly extracted with alcoholic solvent, and loss of effective components is serious.Application number be 2009100681.7 Chinese invention patent application disclose a kind ofly utilize the reverse-flow extractive technique of tank group, whizzer high speed centrifugation and micro-filtration, nanofiltration produces the method for Cyclosiversioside F, the method does not consume organic solvent, but it is lower to be easy to the high product purity obtained of scale operation equipment cost.Application number be 201010615265.4 Chinese invention patent application disclose a kind of method utilizing microbial transformation Radix Astragali total saponins to prepare Cyclosiversioside F, the method is pollution-free, and specificity is strong but raw material is that Radix Astragali total saponins needs to obtain in advance.
Summary of the invention
The object of the invention is to overcome raw materials cost high, Cyclosiversioside F productive rate is low, the shortcomings such as complex process is seriously polluted, direct employing Chinese medicine astragalus is raw material, utilize viride to carry out through macroporous adsorbent resin after microbial transformation, the method such as silica gel column chromatography and recrystallizing methanol purifying finally obtains the Cyclosiversioside F of purity more than 98%.
Utilize viride to prepare the method for Cyclosiversioside F, be raw material with Chinese medicine astragalus, in turn include the following steps:
A. Chinese medicine astragalus utilizes viride to ferment
Test tube slant preservation is made after the activation of viride lyophilized powder; Get viride test tube slant to add sterilized water and make spore suspension, be inoculated in liquid nutrient medium (liquid nutrient medium is potato culture--200g potato adds water 1L, boil 30 ~ 40min, four layers of filtered through gauze removing filter residue, 20g glucose is added in filtrate, nature pH), temperature 26 DEG C, shaking speed be 120r/min cultivate 48h obtain seed liquor; Get seed liquor and join temperature 26 DEG C in fermention medium, shaking speed is that 120r/min cultivation 120h obtains fermented liquid;
B. the separation and Extraction of Cyclosiversioside F in fermented liquid
After above-mentioned fermented liquid mixing, thickening filtration obtains sample solution, and be separated through HPD100 resin column, after wash-out impurity, wash-out obtains product and obtains product purity more than 10% through n-butanol extraction; Lower floor's solution of purification by silica gel column chromatography eluent chloroform-methanol-water volume ratio 13:8:2 carries out wash-out.
It is about 85% that chloroform-methanol-water (volume ratio 13:8:2) lower floor eluant solution obtains Cyclosiversioside F product purity.Finally utilize the method for recrystallizing methanol to obtain the Cyclosiversioside F of purity more than 98%, the rate of recovery of whole technical process Cyclosiversioside F reaches more than 86%.
In steps A, fermentative medium formula is 10g/L Milkvetch Root 5g/L yeast powder, (NH4)
2sO
42.8g/L, KH
2pO
44g/L, MgSO
47H
2o 0.9g/L, anhydrous CaCl
20.9g/L, tween 80 1ml/L, regulate pH to be 5.0.
In the present invention, described bacterial classification is commercial viride; Described raw material Chinese medicine astragalus is Radix Astragali, purchased from Anguo City, Hebei province.
Feature of the present invention be with Chinese medicine astragalus be prepared using microbial transformation method obtain Cyclosiversioside F.Described microbial transformation, utilize the Cyclosiversioside F that the cellulase that produces in viride metabolic process and Glycosylase obtain in Chinese medicine astragalus, because its selectivity is strong, mild condition, with low cost, green non-pollution is suitable for industrial production.Overcome the problem that in organic reagent leaching process, loss of effective components is serious simultaneously, it also avoid in Industrial processes seriously polluted, the problem that energy consumption is high, directly prepare the Cyclosiversioside F of chromatographic purity more than 98% from Chinese medicine astragalus.
Accompanying drawing explanation
Fig. 1 is the mass spectrum of the purity astragaloside obtained.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the object of these embodiments not lying in limits the scope of the invention.
Embodiment 1:
Chinese medicine astragalus utilizes Trichoderma Viride: after commercial viride lyophilized powder activation, make the preservation of viride test tube slant.Get a viride test tube on inclined-plane, to add 10ml sterilized water make spore suspension, being inoculated in (every bottle of 1ml) is equipped with in the 250ml shaking flask of 100ml liquid nutrient medium, liquid nutrient medium is that (200g potato adds water 1L potato culture, boil 30 ~ 40min, four layers of filtered through gauze removing filter residue, 20g glucose is added, natural pH) in filtrate.26 DEG C, 120r/min cultivates 48h and obtains seed liquor.Getting 1ml seed liquor joins in 100ml fermention medium, and fermentative medium formula is 10g/L Milkvetch Root (Chinese medicine astragalus crosses 80 mesh sieves after pulverizing), 5g/L yeast powder, (NH
4)
2sO
42.8g/L, KH
2pO
44g/L, MgSO
47H
2o 0.9g/L, anhydrous CaCl
20.9g/L, tween 80 adds 1ml/L, regulates PH to be 5.0.26 DEG C, 120r/min cultivates 120h and obtains fermented liquid.Filter after fermentation and obtain 88ml fermented liquid, add 50ml n-butanol extraction, after the n-butanol layer mixing that extracting twice obtains, rotary evaporation obtains medicinal extract 0.24g.Medicinal extract is with after 10ml chromatogram dissolve with methanol constant volume, and cross 0.22 μm of organic filter membrane, then carrying out Purity with analysis mode HPLC is 0.97%.
The separation and Extraction of Cyclosiversioside F in fermented liquid: after preparing 5 bottles of above-mentioned fermented liquid mixing, thickening filtration obtains 200ml sample solution, column volume is the HPD100 resin column of 20ml, wash impurity with the ethanolic soln of the deionized water of 3 times of volumes and 20% of 3 times of volumes, obtain product with the ethanolic soln wash-out of 5 times of volumes 70%.Rosin products revolves to boil off and desolventizes rear n-butanol extraction 3 times, and being spin-dried for and obtaining product purity is 12.01%.Silica gel column layer is extracted rosin products dry method loading, silicagel column is filled with chloroform, with chloroform-methanol-water (volume ratio 13:8:2) lower floor solution as eluent product, the collection of elutriant is detected the product determining to collect in time by thin-layer chromatography, being spin-dried for and obtaining product purity is 86.5%.The product of purity 98% is obtained by recrystallizing methanol.Adopt MALDI-TOF-MS to identify product, this compound [M+Na]=807.50, the theoretical molecular of this compound is 807.95 (Fig. 1).
Embodiment 2:
Chinese medicine astragalus utilizes Trichoderma Viride: viride test tube slant adds 10ml sterilized water and makes spore suspension, being inoculated in (every bottle of 1ml) is equipped with in the 250ml shaking flask of 100ml liquid nutrient medium, liquid nutrient medium is that (200g potato adds water 1L potato culture, boil 30 ~ 40min, four layers of filtered through gauze removing filter residue, 20g glucose is added, natural pH) in filtrate.26 DEG C, 120r/min cultivates 48h and obtains seed liquor.Getting 10ml seed liquor joins in 1000ml fermention medium, and fermentative medium formula is 10g/L Milkvetch Root (Chinese medicine astragalus crosses 80 mesh sieves after pulverizing), 5g/L yeast powder, (NH
4)
2sO
42.8g/L, KH
2pO
44g/L, MgSO
47H
2o 0.9g/L, anhydrous CaCl
20.9g/L, tween 80 adds 1ml/L, regulates PH to be 5.0.26 DEG C, 120r/min cultivates 120h and obtains fermented liquid.Filter after fermentation and obtain 915ml fermented liquid, use 500ml n-butanol extraction, after the n-butanol layer mixing that extracting twice obtains, rotary evaporation obtains medicinal extract 2.81g.After getting medicinal extract 0.2g 10ml chromatogram dissolve with methanol constant volume, cross 0.22 μm of organic filter membrane, then carrying out Purity with analysis mode HPLC is 0.78%.
The separation and Extraction of Cyclosiversioside F in fermented liquid: after preparing 5 bottles of above-mentioned fermented liquid mixing, thickening filtration obtains 2L sample solution, column volume is the HPD100 resin column of 200ml, wash impurity with the ethanolic soln of the deionized water of 3 times of volumes and 20% of 3 times of volumes, obtain product with the ethanolic soln wash-out of 5 times of volumes 70%.Rosin products revolves to boil off and desolventizes rear n-butanol extraction 3 times, and being spin-dried for and obtaining product purity is 10.01%.Silica gel column layer is extracted rosin products dry method loading, silicagel column is filled with chloroform, with chloroform-methanol-water (13:8:2) lower floor solution as eluent product, the collection of elutriant is detected the product determining to collect in time by thin-layer chromatography, being spin-dried for and obtaining product purity is 83.5%.The product of purity 98% is obtained for twice by recrystallizing methanol.
Claims (2)
1. utilize viride to prepare the method for Cyclosiversioside F, be raw material with Chinese medicine astragalus, in turn include the following steps:
A. Chinese medicine astragalus utilizes viride to ferment
Make test tube slant preservation after the activation of viride lyophilized powder, get viride test tube slant and add sterilized water and make spore suspension, be inoculated in the temperature 26 DEG C of liquid nutrient medium, shaking speed is that 120r/min cultivates 48h and obtains seed liquor; Get seed liquor and join temperature 26 DEG C in fermention medium, shaking speed is that 120r/min cultivation 120h obtains fermented liquid;
B. the separation and Extraction of Cyclosiversioside F in fermented liquid
After above-mentioned fermented liquid mixing, thickening filtration obtains sample solution, and be separated through HPD100 resin column, after wash-out impurity, wash-out obtains product and obtains product purity more than 10% through n-butanol extraction; Lower floor's solution of purification by silica gel column chromatography eluent chloroform-methanol-water volume ratio 13:8:2 carries out wash-out.
2. method according to claim 1, is characterized in that: in steps A, fermentative medium formula is 10g/L Milkvetch Root 5g/L yeast powder, (NH4)
2sO
42.8g/L, KH
2pO
44g/L, MgSO
47H
2o 0.9g/L, anhydrous CaCl
20.9g/L, tween 80 1ml/L, regulate pH to be 5.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510112855.8A CN104711312B (en) | 2015-03-15 | 2015-03-15 | The method that Astragaloside IV is prepared using Trichoderma viride |
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| CN201510112855.8A CN104711312B (en) | 2015-03-15 | 2015-03-15 | The method that Astragaloside IV is prepared using Trichoderma viride |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019161719A1 (en) * | 2018-02-24 | 2019-08-29 | 苏州广奥医药开发有限公司 | Use of icarisid ii or pharmaceutically acceptable carrier thereof in erectile dysfunction |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1416843A (en) * | 2001-11-03 | 2003-05-14 | 福州济安堂菌草酸酶技术开发有限公司 | Enzyme prepn method of preparing Chinese medicine |
| CN102559828A (en) * | 2010-12-30 | 2012-07-11 | 复旦大学 | Method for preparing astragaloside IV by converting total saponins of astragalus by microorganisms |
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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1416843A (en) * | 2001-11-03 | 2003-05-14 | 福州济安堂菌草酸酶技术开发有限公司 | Enzyme prepn method of preparing Chinese medicine |
| CN102559828A (en) * | 2010-12-30 | 2012-07-11 | 复旦大学 | Method for preparing astragaloside IV by converting total saponins of astragalus by microorganisms |
Non-Patent Citations (2)
| Title |
|---|
| 孙豪栋: "黄芪总皂苷的微生物转化及其条件优化的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 焦巧芳: "中药渣微生物转化利用菌种筛选研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019161719A1 (en) * | 2018-02-24 | 2019-08-29 | 苏州广奥医药开发有限公司 | Use of icarisid ii or pharmaceutically acceptable carrier thereof in erectile dysfunction |
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