CN106011213B - A kind of method that bioconversion degradation Astragaloside IV prepares cycloastragenol - Google Patents
A kind of method that bioconversion degradation Astragaloside IV prepares cycloastragenol Download PDFInfo
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- CN106011213B CN106011213B CN201610409748.6A CN201610409748A CN106011213B CN 106011213 B CN106011213 B CN 106011213B CN 201610409748 A CN201610409748 A CN 201610409748A CN 106011213 B CN106011213 B CN 106011213B
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- cycloastragenol
- astragaloside
- culture medium
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- WENNXORDXYGDTP-UOUCMYEWSA-N cycloastragenol Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O)C4(C)C)[C@H]4[C@@H](O)C[C@H]3[C@]2(C)C[C@@H]1O WENNXORDXYGDTP-UOUCMYEWSA-N 0.000 title claims abstract description 32
- WENNXORDXYGDTP-UHFFFAOYSA-N cyclosiversigenin Natural products O1C(C(C)(O)C)CCC1(C)C1C2(C)CCC34CC4(CCC(O)C4(C)C)C4C(O)CC3C2(C)CC1O WENNXORDXYGDTP-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 22
- QMNWISYXSJWHRY-YLNUDOOFSA-N astragaloside IV Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)[C@H]4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)C[C@H]3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-YLNUDOOFSA-N 0.000 title claims abstract description 21
- QMNWISYXSJWHRY-BCBPIKMJSA-N astragaloside IV Natural products CC(C)(O)[C@@H]1CC[C@@](C)(O1)[C@H]2[C@@H](O)C[C@@]3(C)[C@@H]4C[C@H](O[C@@H]5O[C@H](CO)[C@H](O)[C@@H](O)[C@H]5O)[C@H]6C(C)(C)[C@H](CC[C@@]67C[C@@]47CC[C@]23C)O[C@@H]8OC[C@@H](O)[C@H](O)[C@H]8O QMNWISYXSJWHRY-BCBPIKMJSA-N 0.000 title claims abstract description 21
- PFKIBRPYVNVMRU-UHFFFAOYSA-N cyclosieversioside F Natural products CC(C)(O)C1COC(C)(C1)C2C(O)CC3(C)C4CC(OC5OC(CO)C(O)C(O)C5O)C6C(C)(C)C(CCC67CC47CCC23C)OC8OCC(O)C(O)C8O PFKIBRPYVNVMRU-UHFFFAOYSA-N 0.000 title claims abstract description 21
- 230000015556 catabolic process Effects 0.000 title claims abstract description 10
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims abstract description 17
- 230000004151 fermentation Effects 0.000 claims abstract description 17
- 239000000758 substrate Substances 0.000 claims abstract description 11
- 241000170282 Absidia sp. (in: Fungi) Species 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- 239000000284 extract Substances 0.000 claims description 24
- 239000001963 growth medium Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 13
- 239000006143 cell culture medium Substances 0.000 claims description 11
- 238000000746 purification Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000003638 chemical reducing agent Substances 0.000 claims description 6
- 229930182470 glycoside Natural products 0.000 claims description 6
- 150000002338 glycosides Chemical class 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 6
- 239000012074 organic phase Substances 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 238000010898 silica gel chromatography Methods 0.000 claims description 6
- 239000009636 Huang Qi Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 4
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 229910052928 kieserite Inorganic materials 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 7
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 239000000047 product Substances 0.000 description 13
- PJANXHGTPQOBST-VAWYXSNFSA-N Stilbene Natural products C=1C=CC=CC=1/C=C/C1=CC=CC=C1 PJANXHGTPQOBST-VAWYXSNFSA-N 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- PJANXHGTPQOBST-UHFFFAOYSA-N stilbene Chemical compound C=1C=CC=CC=1C=CC1=CC=CC=C1 PJANXHGTPQOBST-UHFFFAOYSA-N 0.000 description 8
- 235000021286 stilbenes Nutrition 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 208000012839 conversion disease Diseases 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 235000006533 astragalus Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 241000235389 Absidia Species 0.000 description 2
- 241000045403 Astragalus propinquus Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- -1 Saponins compound Chemical class 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000003712 anti-aging effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000000105 evaporative light scattering detection Methods 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229930182490 saponin Natural products 0.000 description 2
- 235000017709 saponins Nutrition 0.000 description 2
- BSLYZLYLUUIFGZ-JRUDBKCSSA-N 4,4,14-trimethyl-9,19-cyclo-5alpha,9beta-cholestane Chemical compound C1CCC(C)(C)[C@H]2[C@@]31C[C@@]13CC[C@]3(C)[C@@H]([C@H](C)CCCC(C)C)CC[C@@]3(C)[C@@H]1CC2 BSLYZLYLUUIFGZ-JRUDBKCSSA-N 0.000 description 1
- KNESISUQBYQIIU-UHFFFAOYSA-N 6-Angeloyl-1,6-Dihydroxyfuranoeremophilan-9-one Natural products O1C(C(C)(O)C)CCC1(C)C1C2(C)CC=C3C4(C)CCC(O)C(C)(C)C4C(O)CC3C2(C)CC1O KNESISUQBYQIIU-UHFFFAOYSA-N 0.000 description 1
- SMDOOINVMJSDPS-UHFFFAOYSA-N Astragaloside Natural products C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)=C1 SMDOOINVMJSDPS-UHFFFAOYSA-N 0.000 description 1
- 241001061264 Astragalus Species 0.000 description 1
- KZGXEJKMJNLRSF-UHFFFAOYSA-N Astragenol Natural products CC(C)(O)C1CCC(C)(O1)C2C(O)CC3(C)C4CC(O)C5(C)C(C)(C)C(O)CCC5(C)C4=CCC23C KZGXEJKMJNLRSF-UHFFFAOYSA-N 0.000 description 1
- RRTBTJPVUGMUNR-UHFFFAOYSA-N Cycloartanol Natural products C12CCC(C(C(O)CC3)(C)C)C3C2(CC)CCC2(C)C1(C)CCC2C(C)CCCC(C)C RRTBTJPVUGMUNR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241001480490 Mucoraceae Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- QMNWISYXSJWHRY-XWJCTJPOSA-N astragaloside Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)C4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)CC3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-XWJCTJPOSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- YNBJLDSWFGUFRT-UHFFFAOYSA-N cycloartenol Natural products CC(CCC=C(C)C)C1CCC2(C)C1(C)CCC34CC35CCC(O)C(C)(C)C5CCC24C YNBJLDSWFGUFRT-UHFFFAOYSA-N 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 210000004233 talus Anatomy 0.000 description 1
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical group N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 1
- 229930182493 triterpene saponin Natural products 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/20—Preparation of steroids containing heterocyclic rings
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the methods that a kind of bioconversion degradation Astragaloside IV prepares cycloastragenol, it is characterised in that: using Astragaloside IV as substrate, is fermented by the mould Absidia sp.CGMCC 3.2834 of strain colter, cycloastragenol is made.The present invention obtains the cycloastragenol of high-purity with microbial fermentation technology degradation Astragaloside IV, simple for process, at low cost, and the conversion ratio of final cycloastragenol reaches 60% or more, purity and is up to 95% or more, and conversion specificity is higher；Entire technical process carries out under room temperature, normal pressure, and required appointed condition is low, is suitable for producing in enormous quantities.
Description
Technical field
The invention discloses the methods that a kind of bioconversion degradation Astragaloside IV prepares cycloastragenol, belong to medication chemistry neck
Domain.
Background technique
Radix Astragali is the dry root of astragalus mongholicus or astragalus membranaceus of leguminous plant.It is first recorded in Shennong's Herbal, so far
There are more than 400 years medicinal histories, still treats one of most common drug at present for tcm clinical practice.Saponins compound is Astragalus
Most important a kind of active constituent in plant, saponins compound contained therein are mainly cycloartane type from structure
Tetracyclic triterpene saponins.Cycloastragenol is the aglycon part of astragaloside, molecular formula C30H50O5, relative molecular weight be
490.71.Its content in plant is few, accounts for about the 0.1% of dry weight, is primary product of the Astragaloside IV in enteron aisle intracellular metabolite, though
Right content is very low but has good biological activity: promoting nerve cell increment, promotes wound healing, anti-aging etc..Especially
It is cycloastragenol as known unique natural Activation of Telomerase agent in anti-aging, and enhancing immunity of organisms etc. has not
Alternative effect, has a vast market foreground.
Traditional method is cycloastragenol directly to be extracted from Radix Astragali, but be limited by lower content in Radix Astragali, yield compared with
Low and higher cost, therefore it is a kind of feasible for selecting the higher Astragaloside IV conversion production of and content similar to cycloastragenol structure
Production technology.Since the difference of the two is only with the presence or absence of glycosyl modified, domestic production technology is using each at present
Kind method opens glycosidic bond conversion production cycloastragenol.Due to the unstability of structure, general hydrolysis can be generated largely
By-product Astragenol, destroy original ring structure, cause isolated difficulty.
Domestic at present seldom about cycloastragenol related methods of production report, Chinese invention patent CN104817610A is in
State patent of invention CN103880910A destroys saccharide ring knot according to the directly sour hydrolyzing glucosidic bonds of the principles of chemistry or through oxidization-reduction
Structure hydrolyzing glucosidic bonds again, by the available cycloastragenol of series of steps, but reaction process requirement condition is higher, technique compared with
To be cumbersome, which has limited large-scale production and application.
The mould Absidia sp.CGMCC 3.2834 of colter, belong to Eumycota, Zygomycotina, Zygomycetes, Mucoales,
Mucoraceae.It is distributed widely in soil, distiller's yeast and excrement, often with the presence of their spore, other microorganisms easy to pollute in air
Pure culture.Absidia is widely used in the research of bioconversion, for example, the mould Absdia orchidis of Absidia by
In with C11β-hydroxylation acts on the production for having been used for hydrocortisone.
The method of cycloastragenol is prepared there is not yet report with microbial fermentation technology.
Summary of the invention
The present invention be to avoid above-mentioned existing deficiencies in the technology, provide it is a kind of efficiently, low cost, it is simple and
The high cycloastragenol preparation method of product purity, the technical problem to be solved is that pass through bioconversion degradation Astragaloside IV preparation
Cycloastragenol.
The present invention solves technical problem, adopts the following technical scheme that
The method that bioconversion degradation Astragaloside IV of the present invention prepares cycloastragenol, it is characterized in that: it is with Astragaloside IV
Substrate is fermented by the mould Absidia sp.CGMCC 3.2834 of strain colter, cycloastragenol is made.Specifically comprise the following steps:
(1) seed culture
PDB culture medium is prepared in conical flask, is then accessed the mould Absidia sp.CGMCC 3.2834 of strain colter and is sent out
Ferment is cultivated 3 days under conditions of 25~35 DEG C of shaking table temperature, 150~200r/min of shaking speed, obtains the culture of seed level-one
Base；
(2) conversion culture
Step (1) seed first cell culture medium obtained is improved into PDB culture medium by 10% inoculum concentration access 150mL
In, it is cultivated under conditions of 25~35 DEG C of shaking table temperature, 150~200r/min of shaking speed 3 days, it is yellow that 25~50mg is then added
Stilbene first glycosides substrate continues culture 6 days, obtains fermentation liquid；
(3) it purifies
It is extracted at room temperature three times, every time after fermentation liquid obtained by step (2) is mixed in equal volume with extractant ethyl acetate
10min, gained extract liquor are concentrated in vacuo to dry, acquisition extract；
Extract is dissolved in methanol, reducing agent NaBH is added according still further to the dosage of extract mass ratio 1:14, often
Temperature reaction 10min, the separation of gained reaction solution, purification, i.e. acquisition target product cycloastragenol.
Wherein, the composition of the improvement PDB culture medium are as follows: potato leachate 300g/L, KH2PO43g/L, MgSO4·
H2O1.5g/L, Vb1mg/L.The composition of PDB culture medium are as follows: potato leachate 200g/L, glucose 20g/L, KH2PO43g/L,
MgSO4·H2O 1.5g/L, Vb 1mg/L。
The purity of the Astragaloside IV substrate is 50%~100%.
In step (3) the step of separation, purification are as follows: it is added the water that volume is no less than reaction solution volume in reaction solution, 40
Methanol is removed in DEG C reduced pressure, and isometric ethyl acetate, stratification is then added；It is molten with methanol after taking organic phase to be concentrated to dryness
Solution, it is dry by silica gel column chromatography column purification, that is, obtain target product.Column chromatography uses silica normal phase column.
The reagents and materials used in the present invention are commercially available.
This method obtains the cycloastragenol of high-purity with microbial fermentation technology degradation Astragaloside IV.The preparation process is simple
Easy, at low cost, whole operation process carries out under room temperature, normal pressure, is suitable for producing in enormous quantities.
Compared with the prior art, the beneficial effects of the present invention are embodied in:
1, the present invention obtains the cycloastragenol of high-purity with microbial fermentation technology degradation Astragaloside IV, and simple process is easy
Capable, at low cost, the conversion ratio of final cycloastragenol reaches 60% or more, purity and is up to 95% or more, and conversion specificity is higher；
2, entire technical process of the invention carries out under room temperature, normal pressure, and required appointed condition is low, is suitable for large quantities of
Amount production efficiently solves the problems such as production chinese raw materials limits, recovery rate is low at present.
Detailed description of the invention
Fig. 1 is the chromatogram of 1 products therefrom of embodiment.
Specific embodiment
Embodiment 1
The present embodiment prepares cycloastragenol as follows:
(1) seed culture
PDB culture medium is prepared in conical flask, then accesses the mould Absidia sp.CGMCC 3.2834, In of strain colter
It is cultivated 3 days under conditions of 30 DEG C of shaking table temperature, shaking speed 180r/min, obtains seed first cell culture medium；
(2) conversion culture
Step (1) seed first cell culture medium obtained is improved into PDB culture medium by 10% inoculum concentration access 150mL
In, it is cultivated under conditions of 30 DEG C of shaking table temperature, shaking speed 180r/min 3 days, it is (yellow that 50mg Astragaloside IV substrate is then added
Stilbene first glycosides contains 34.5mg), continue culture 6 days, obtains fermentation liquid；
It is extracted at room temperature three times, every time after fermentation liquid obtained by step (2) is mixed in equal volume with extractant ethyl acetate
10min, gained extract liquor are concentrated in vacuo to dry, acquisition extract；
Extract is dissolved in methanol by the mass volume ratio of 10mg/mL, according still further to the use with extract mass ratio 1:1
Reducing agent NaBH is added in amount4, normal-temperature reaction 10min, acquisition reaction solution；
Isometric water is added in reaction solution, 40 DEG C of concentrations remove methanol, isometric ethyl acetate is then added, and stand
Layering；It is dissolved after taking organic phase to be concentrated to dryness with methanol, dry by silica gel column chromatography column purification, i.e. acquisition target product ring is yellow
Stilbene alcohol.
Through detecting, product purity 95.5%, reaction conversion ratio 61.5%.
Purity: the cycloastragenol sample high performance liquid chromatograph that the present embodiment is obtained detects purity, chromatostrip
Part: chromatographic column is analytical column Hypersil GOLD column (4.6mm × 150mm, 5 μm, Thermo Scientific)；Column
30 DEG C of temperature；Mobile phase: 0min~13min methanol: water=75:25,13min~20min methanol: water=85:15,20min~
30min methanol 100%；Flow velocity: 1mL/min；Applied sample amount is 120 DEG C of drift tube temperature of 10 μ L, ELSD, ELSD throughput 2.1L/
min.The cycloastragenol purity for detecting this sample is 95.5%, and chromatogram is as shown in Figure 1.
Embodiment 2
The present embodiment prepares cycloastragenol as follows:
(1) seed culture
PDB culture medium is prepared in conical flask, is then accessed the mould Absidia sp.CGMCC 3.2834 of strain colter and is sent out
Ferment is cultivated 3 days under conditions of 30 DEG C of shaking table temperature, shaking speed 180r/min, obtains seed first cell culture medium；
(2) conversion culture
Step (1) seed first cell culture medium obtained is improved in PDB culture medium by 5% inoculum concentration access 150mL,
It is cultivated under conditions of 30 DEG C of shaking table temperature, shaking speed 180r/min 3 days, 50mg Astragaloside IV substrate (Radix Astragali is then added
First glycosides contains 34.5mg), continue culture 6 days, obtains fermentation liquid；
(3) it purifies
It is extracted at room temperature three times, every time after fermentation liquid obtained by step (2) is mixed in equal volume with extractant ethyl acetate
10min, gained extract liquor are concentrated in vacuo to dry, acquisition extract；
Extract is dissolved in methanol by the mass volume ratio of 10mg/mL, according still further to the dosage with extract quality 1:1
Reducing agent NaBH is added4, normal-temperature reaction 10min, acquisition reaction solution；
Isometric water is added in reaction solution, 40 DEG C of concentrations remove methanol, isometric ethyl acetate is then added, and stand
Layering；It is dissolved after taking organic phase to be concentrated to dryness with methanol, dry by silica gel column chromatography column purification, i.e. acquisition target product ring is yellow
Stilbene alcohol.
Through detecting, product purity 95.1%, reaction conversion ratio 27.7%.
Embodiment 3
The present embodiment prepares cycloastragenol as follows:
(1) seed culture
PDB culture medium is prepared in conical flask, then accesses the mould Absidia sp.CGMCC 3.2834, In of strain colter
It is cultivated 3 days under conditions of 30 DEG C of shaking table temperature, shaking speed 180r/min, obtains seed first cell culture medium；
(2) conversion culture
Step (1) seed first cell culture medium obtained is accessed into the common PDB culture medium of 150mL by 10% inoculum concentration
In, it is cultivated under conditions of 30 DEG C of shaking table temperature, shaking speed 180r/min 3 days, it is (yellow that 50mg Astragaloside IV substrate is then added
Stilbene first glycosides contains 34.5mg), continue culture 6 days, obtains fermentation liquid；
(3) it purifies
It is extracted at room temperature three times, every time after fermentation liquid obtained by step (2) is mixed in equal volume with extractant ethyl acetate
10min, gained extract liquor are concentrated in vacuo to dry, acquisition extract；
Extract is dissolved in methanol by the mass volume ratio of 10mg/mL, according still further to the use with extract mass ratio 1:1
Reducing agent NaBH is added in amount4, normal-temperature reaction 10min, acquisition reaction solution；
Isometric water is added in reaction solution, 40 DEG C of concentrations remove methanol, isometric ethyl acetate is then added, and stand
Layering；It is dissolved after taking organic phase to be concentrated to dryness with methanol, dry by silica gel column chromatography column purification, i.e. acquisition target product ring is yellow
Stilbene alcohol.
Through detecting, product purity 95.1%, reaction conversion ratio 44.8%.
Embodiment 4
The present embodiment prepares cycloastragenol as follows:
(1) seed culture
PDB culture medium is prepared in conical flask, then accesses the mould Absidia sp.CGMCC 3.2834, In of strain colter
It is cultivated 3 days under conditions of 30 DEG C of shaking table temperature, shaking speed 180r/min, obtains seed first cell culture medium；
(2) conversion culture
Step (1) seed first cell culture medium obtained is improved into PDB culture medium by 10% inoculum concentration access 150mL
In, it is cultivated under conditions of 30 DEG C of shaking table temperature, shaking speed 180r/min 3 days, it is (yellow that 50mg Astragaloside IV substrate is then added
Stilbene first glycosides contains 34.5mg), continue culture 2 days, obtains fermentation liquid；
(3) it purifies
It is extracted at room temperature three times, every time after fermentation liquid obtained by step (2) is mixed in equal volume with extractant ethyl acetate
10min, gained extract liquor are concentrated in vacuo to dry, acquisition extract；
Extract is dissolved in methanol by the mass volume ratio of 10mg/mL, according still further to the use with extract mass ratio 1:1
Reducing agent NaBH is added in amount4, normal-temperature reaction 10min, acquisition reaction solution；
Isometric water is added in reaction solution, 40 DEG C of concentrations remove methanol, isometric ethyl acetate is then added, and stand
Layering；It is dissolved after taking organic phase to be concentrated to dryness with methanol, dry by silica gel column chromatography column purification, i.e. acquisition target product ring is yellow
Stilbene alcohol.
Through detecting, product purity 96.4%, reaction conversion ratio 34.0%.
Claims (5)
1. a kind of method that bioconversion degradation Astragaloside IV prepares cycloastragenol, it is characterised in that: using Astragaloside IV as substrate,
By mould (Absidia sp.) CGMCC 3.2834 fermentation of strain colter, cycloastragenol is made.
2. according to the method described in claim 1, it is characterized by comprising following steps:
(1) seed culture
PDB culture medium is prepared in conical flask, is then accessed mould (Absidia sp.) CGMCC 3.2834 of strain colter, is being shaken
It is cultivated 3 days under conditions of 25~35 DEG C of bed tempertaure, 150~200r/min of shaking speed, obtains seed first cell culture medium；
(2) conversion culture
Step (1) seed first cell culture medium obtained is improved in PDB culture medium by 10% inoculum concentration access 150mL, In
It is cultivated under conditions of 25~35 DEG C of shaking table temperature, 150~200r/min of shaking speed 3 days, 25~50mg Radix Astragali first is then added
Glycosides substrate continues culture 6 days, obtains fermentation liquid；
(3) it purifies
It is extracted at room temperature three times, every time after fermentation liquid obtained by step (2) is mixed in equal volume with extractant ethyl acetate
10min, gained extract liquor are concentrated in vacuo to dry, acquisition extract；
Extract is dissolved in methanol, reducing agent NaBH is added according still further to the dosage of extract mass ratio 1:14, normal-temperature reaction
10min, the separation of gained reaction solution, purification, i.e. acquisition target product cycloastragenol.
3. according to the method described in claim 2, it is characterized by: the composition of the improvement PDB culture medium are as follows: potato leachate
300g/L, KH2PO43g/L, MgSO4·H2O 1.5g/L, Vb 1mg/L。
4. according to the method described in claim 2, it is characterized by: the purity of the Astragaloside IV substrate is 50%~100%.
5. the method according to weighing and require 2, it is characterised in that: in step (3) the step of separation, purification are as follows: in reaction solution
The water that volume is no less than reaction solution volume is added, 40 DEG C of reduced pressures remove methanol, isometric ethyl acetate is then added, and stand
Layering；It is dissolved after taking organic phase to be concentrated to dryness with methanol, it is dry by silica gel column chromatography column purification, that is, obtain target product.
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| 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 |
| CN111728194A (en) * | 2020-02-21 | 2020-10-02 | 香港科技大学深圳研究院 | Method for fermenting astragalus membranaceus by using probiotics and probiotic astragalus membranaceus fermentation liquor |
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| Smith degradation ,an efficient method for the preparation of cycloastragenol from astragaloside IV;FENG LM,ET AL;《FITOTERAPIA》;20140305;全文 * |
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