CN104711312B - The method that Astragaloside IV is prepared using Trichoderma viride - Google Patents
The method that Astragaloside IV is prepared using Trichoderma viride Download PDFInfo
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- CN104711312B CN104711312B CN201510112855.8A CN201510112855A CN104711312B CN 104711312 B CN104711312 B CN 104711312B CN 201510112855 A CN201510112855 A CN 201510112855A CN 104711312 B CN104711312 B CN 104711312B
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Abstract
It the present invention relates to the use of the method that Trichoderma viride prepares Astragaloside IV.The method of the present invention is carried out carrying out microorganism conversion to Chinese herbal medicine astragalus using Trichoderma viride, and the enrichment of active ingredient Astragaloside IV is realized under certain fermentation culture conditions.Then Astragaloside IV crude extract bioanalysis obtained, is isolated and purified using macroporous absorbent resin HPD100, and the Astragaloside IV of purity more than 98% is further finally given using purifying the methods of silica gel column chromatography and recrystallizing methanol.The shortcomings of this method aims to overcome that cost of material is high, Astragaloside IV low yield, and complex process is seriously polluted, it is adapted to the Astragaloside IV of large-scale production different purity.
Description
Technical field
The invention belongs to biochemical field, is related to a kind of yellow using Trichoderma viride conversion preparation by raw material of Chinese herbal medicine astragalus
The new method of stilbene first glycosides.
Background technology
The Radix Astragali is sweet, tepor, is the conventional Chinese medicine strengthened the body resistance to consolidate the constitution.Chinese Pharmacopoeia provides that Chinese herbal medicine astragalus is legume film
The pod Radix Astragali Astragalus membranaceus (Fisch.) Bge and astragalus mongolicus Astragalus membranaceus
(Fisch.) Bge.Var.mongholicus (Bge.) Hsiao dry root.Its alias has continuous stilbene, the continuous Radix Astragali.Modern study
It was found that astragalus root uses composition comprising a variety of.Wherein in the root of astragalus mongolicus containing Astragaloside IV, Isorhamnetin, Quercetin, β-
Sitosterol, sandlwood fruit element, linoleic acid and leukotrienes etc.；Contain Astragaloside IV, glycine betaine, choline, sucrose, ammonia in Astragalus membranacus root
Base acid, glucuronic acid and micro folic acid.Astragaloside species is very more, can separate to identify from astragalus root and come just
There are tens of kinds, Astragaloside IV (AstragalosideIV, AST) is one of principle active component.It has booster immunization power, increases
Add energy, resisting fatigue, protect liver, remove interior free yl and suppress the effect of osteoclast, there is very high medicinal valency
Value.
The preparation method of Astragaloside IV is mainly natural product extraction at present, the China of Application No. 200410014786.9
Application for a patent for invention discloses the method for preparing Astragaloside IV using Chinese herbal medicine astragalus powder for raw material, mainly comprises the following steps ethanol and returns
Stream, ethyl acetate extraction, recrystallizing methanol, conventional method are mainly extracted with alcohols solvent, and loss of effective components is serious.Application number
For 2009100681.7 Chinese invention patent application disclose it is a kind of using the reverse-flow extractive technique of tank group, centrifuge at a high speed from
The heart and micro-filtration, the method for nanofiltration production Astragaloside IV, this method do not consume organic solvent, are easy to large-scale production but set
The product purity that standby cost height obtains is relatively low.The Chinese invention patent application of Application No. 201010615265.4 discloses one kind
The method that Astragaloside IV is prepared using microorganism conversion astragalus root total saponin, this method is pollution-free, and selectivity is strong but raw material is Huang
Stilbene total saposins need to be made in advance.
The content of the invention
It is an object of the invention to overcome cost of material high, Astragaloside IV low yield, the shortcomings of complex process is seriously polluted,
Chinese herbal medicine astragalus is directly used as raw material, using Trichoderma viride carry out microorganism conversion after by macroporous absorbent resin, silica gel column layer
The purifying of the methods of analysis and recrystallizing methanol finally gives the Astragaloside IV of purity more than 98%.
The method that Astragaloside IV is prepared using Trichoderma viride, using Chinese herbal medicine astragalus as raw material, in turn include the following steps：
A. Chinese herbal medicine astragalus is fermented using Trichoderma viride
Test tube slant preservation is made after the activation of Trichoderma viride freeze-dried powder；Trichoderma viride test tube slant is taken to add sterilized water that spore is made
Sub- suspension, be inoculated in fluid nutrient medium (fluid nutrient medium is potato culture -- 200g potatos add water 1L, boil 30~
40min, four layers of filtered through gauze remove filter residues, 20g glucose, natural pH are added in filtrate), 26 DEG C of temperature, shaking speed is
120r/min cultures 48h obtains seed liquor；Seed liquor is taken to be added to 26 DEG C of temperature in fermentation medium, shaking speed 120r/
Min cultures 120h obtains zymotic fluid；
B. in zymotic fluid Astragaloside IV separation and Extraction
Thickening filtration obtains sample solution after above-mentioned zymotic fluid mixing, through HPD100 resin post separations, is eluted after eluting impurity
Obtain product and obtain product purity more than 10% through extracting n-butyl alcohol；Eluent chloroform-first is used in silica gel column chromatography purifying
Alcohol-water accumulates ratio 13:8:2 lower floor's solution is eluted.
Chloroform-methanol-water (volume ratio 13:8:2) lower floor's solution affords Astragaloside IV product purity as 85% or so.
It is final to obtain the Astragaloside IV of purity more than 98%, the recovery of whole technological process Astragaloside IV using the method for recrystallizing methanol
Rate reaches more than 86%.
Fermentative medium formula is 10g/L Milkvetch Root 5g/L dusty yeasts in step A, (NH4)2SO42.8g/L, KH2PO4
4g/L, MgSO4·7H2O 0.9g/L, anhydrous CaCl20.9g/L, Tween 80 1ml/L, regulation pH are 5.0.
In the present invention, the strain is Trichoderma viride purchased in market；The raw material Chinese herbal medicine astragalus is Astragalus membranacus, purchased from Hebei province
Anguo City.
It is characteristic of the invention that obtain Astragaloside IV using the method for microorganism conversion using Chinese herbal medicine astragalus as raw material.It is described micro-
Bioconversion, the Radix Astragali first in Chinese herbal medicine astragalus is obtained using caused cellulase and glycosidase in Trichoderma viride metabolic process
Glycosides, because its selectivity is strong, mild condition, cost is cheap, and green non-pollution is suitable to industrial production.Simultaneously organic reagent is overcome to carry
The problem of loss of effective components is serious during taking, it also avoid it is seriously polluted in industrial processes, the problem of high energy consumption, directly
Connect the Astragaloside IV that chromatographic purity more than 98% is prepared from Chinese herbal medicine astragalus.
Brief description of the drawings
Fig. 1 is the mass spectrogram of the purity astragaloside obtained.
Embodiment
With reference to specific embodiment, the present 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：
Chinese herbal medicine astragalus utilizes Trichoderma Viride：After Trichoderma viride freeze-dried powder activation purchased in market, it is oblique that Trichoderma viride test tube is made
Face preservation.Take a Trichoderma viride test tube on inclined-plane plus spore suspension is made in 10ml sterilized waters, be inoculated in (every bottle of 1ml) and be equipped with
In the 250ml shaking flasks of 100ml fluid nutrient mediums, fluid nutrient medium is that (200g potatos add water 1L to potato culture, boil 30
~40min, four layers of filtered through gauze remove filter residue, 20g glucose, natural pH are added in filtrate).26 DEG C, 120r/min cultures 48h
Obtain seed liquor.1ml seed liquors are taken to be added in 100ml fermentation mediums, fermentative medium formula is 10g/L Milkvetch Roots
(Chinese herbal medicine astragalus crosses 80 mesh sieves after crushing), 5g/L dusty yeasts, (NH4)2SO42.8g/L, KH2PO44g/L, MgSO4·7H2O
0.9g/L, anhydrous CaCl20.9g/L, Tween 80 add 1ml/L, and regulation PH is 5.0.26 DEG C, 120r/min cultures 120h is obtained
Zymotic fluid.88ml zymotic fluids are filtrated to get after fermentation, add 50ml extracting n-butyl alcohols, are extracted twice obtained n-butanol layer mixing
Rotary evaporation obtains medicinal extract 0.24g afterwards.After medicinal extract dissolves constant volume with 10ml chromatograms methanol, 0.22 μm of organic filter membrane, Ran Houyong are crossed
It is 0.97% that analytic type HPLC, which carries out Purity,.
The separation and Extraction of Astragaloside IV in zymotic fluid：Thickening filtration is obtained on 200ml after preparing 5 bottles of above-mentioned zymotic fluid mixing
Sample liquid, column volume are 20ml HPD100 resin columns, with the deionized water of 3 times of volumes and 20% ethanol solution of 3 times of volumes
Impurity is washed, product is afforded with the ethanol solution of 5 times of volumes 70%.Naval stores revolving uses extracting n-butyl alcohol after removing solvent
3 times, it is spin-dried for obtaining product purity as 12.01%.Silica gel column layer is extracted naval stores dry method loading, is filled silicagel column with chloroform, is used
Chloroform-methanol-water (volume ratio 13:8:2) lower floor's solution is as eluent product, the collection of eluent by thin-layer chromatography and
When detection determine the product collected, be spin-dried for obtaining product purity as 86.5%.The product of purity 98% is obtained with recrystallizing methanol.
Product is identified using MALDI-TOF-MS, the compound [M+Na]=807.50, the theoretical molecular of the compound is
(807.95 Fig. 1).
Embodiment 2：
Chinese herbal medicine astragalus utilizes Trichoderma Viride：Trichoderma viride test tube slant adds 10ml sterilized waters that spore suspension is made,
It is inoculated in the 250ml shaking flasks of (every bottle of 1ml) equipped with 100ml fluid nutrient mediums, fluid nutrient medium is potato culture (200g
Potato adds water 1L, boils 30~40min, and four layers of filtered through gauze remove filter residue, 20g glucose, natural pH are added in filtrate).
26 DEG C, 120r/min cultures 48h obtains seed liquor.10ml seed liquors are taken to be added in 1000ml fermentation mediums, fermented and cultured
Based formulas is 10g/L Milkvetch Roots (Chinese herbal medicine astragalus crush then cross 80 mesh sieves), 5g/L dusty yeasts, (NH4)2SO42.8g/L
KH2PO44g/L, MgSO4·7H2O 0.9g/L, anhydrous CaCl20.9g/L, Tween 80 add 1ml/L, and regulation PH is 5.0.26
DEG C, 120r/min cultures 120h obtains zymotic fluid.915ml zymotic fluids are filtrated to get after fermentation, with 500ml extracting n-butyl alcohols, extraction
Rotary evaporation obtains medicinal extract 2.81g after taking the n-butanol layer obtained twice mixing.Medicinal extract 0.2g 10ml chromatograms methanol is taken to dissolve
After constant volume, 0.22 μm of organic filter membrane is crossed, then carries out Purity with analytic type HPLC as 0.78%.
The separation and Extraction of Astragaloside IV in zymotic fluid：Thickening filtration obtains 2L loadings after preparing 5 bottles of above-mentioned zymotic fluid mixing
Liquid, column volume are 200ml HPD100 resin columns, are washed with the deionized water of 3 times of volumes and 20% ethanol solution of 3 times of volumes
Impurity, product is afforded with the ethanol solution of 5 times of volumes 70%.Naval stores revolving uses extracting n-butyl alcohol 3 after removing solvent
It is secondary, it is spin-dried for obtaining product purity as 10.01%.Silica gel column layer is extracted naval stores dry method loading, is filled silicagel column with chloroform, is used chlorine
Imitation-carbinol-water (13:8:2) lower floor's solution is detected really in time as eluent product, the collection of eluent by thin-layer chromatography
Surely the product collected, it is spin-dried for obtaining product purity as 83.5%.Obtain the product of purity 98% twice with recrystallizing methanol.
Claims (1)
1. preparing the method for Astragaloside IV using Trichoderma viride, using Chinese herbal medicine astragalus as raw material, in turn include the following steps：
A. Chinese herbal medicine astragalus is fermented using Trichoderma viride
Test tube slant preservation is made after the activation of Trichoderma viride freeze-dried powder, takes Trichoderma viride test tube slant to add sterilized water that spore is made and hangs
Supernatant liquid, is inoculated in 26 DEG C of the temperature of fluid nutrient medium, and shaking speed is that 120r/min cultures 48h obtains seed liquor；Take seed liquor
26 DEG C of temperature in fermentation medium are added to, shaking speed is that 120r/min cultures 120h obtains zymotic fluid；
B. in zymotic fluid Astragaloside IV separation and Extraction
Thickening filtration obtains sample solution after above-mentioned zymotic fluid mixing, through HPD100 resin post separations, is afforded after eluting impurity
Product obtains product purity more than 10% through extracting n-butyl alcohol；Eluent chloroform-methanol-water is used in silica gel column chromatography purifying
Volume ratio 13:8:2 lower floor's solution is eluted；
Fermentative medium formula is 10g/L Milkvetch Roots in step A, 5g/L dusty yeasts,（NH4)2SO42.8g/L, KH2PO4 4g/
L, MgSO4•7H2O 0.9g/L, anhydrous CaCl20.9g/L, Tween 80 1ml/L, regulation pH are 5.0.
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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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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 (1)
| Title |
|---|
| 黄芪总皂苷的微生物转化及其条件优化的研究;孙豪栋;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20110815(第8期);第E057-20页 * |
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