CN105734109A - Producing and refining method for high-purity cycloastragenol - Google Patents
Producing and refining method for high-purity cycloastragenol Download PDFInfo
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- CN105734109A CN105734109A CN201610071864.1A CN201610071864A CN105734109A CN 105734109 A CN105734109 A CN 105734109A CN 201610071864 A CN201610071864 A CN 201610071864A CN 105734109 A CN105734109 A CN 105734109A
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- cycloastragenol
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- astragaloside
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J53/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by condensation with a carbocyclic rings or by formation of an additional ring by means of a direct link between two ring carbon atoms, including carboxyclic rings fused to the cyclopenta(a)hydrophenanthrene skeleton are included in this class
- C07J53/001—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by condensation with a carbocyclic rings or by formation of an additional ring by means of a direct link between two ring carbon atoms, including carboxyclic rings fused to the cyclopenta(a)hydrophenanthrene skeleton are included in this class spiro-linked
Abstract
The invention relates to a producing and refining method for cycloastragenol with the purity being 95% or more.The method includes the steps that astragaloside is used as a raw material, the cycloastragenol with the purity being 95% or more is obtained through two-phase enzyme hydrolysis, extraction and recrystallization.Compared with an existing producing method for cycloastragenol, the producing and refining method is simple in production technology, mild in reaction condition, high in reaction conversion rate and more suitable for mass production; the problem that when cycloastragenol is hydrolyzed on the acid-alkali condition, the ninth and tenth bits are prone to ring opening to generate astragenol is solved.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to a kind of high-purity Cycloastragenol and produce refining new method.
Background technology
Effective ingredient in the Radix Astragali includes flavonoid, saponins, polysaccharide and amino acids.Wherein Radix Astragali saponin has heart tonifying, blood pressure lowering, antitumor, reduction blood glucose in diabetic rats and promotes the effects such as insulin secretion.Separated from astragalus root identifying tens of kinds of Radix Astragali saponins, astragaloside is the principle active component of saponin.Cycloastragenol (Cycloastragenol), belongs to triterpene saponin componds, is mainly obtained by the hydrolysis of astragaloside (Astragaloside IV), and physical property is white powder tasteless, unformed.Cycloastragenol is the Activation of Telomerase agent found from the Radix Astragali, by increasing telomerase activation, delays telomere to shorten, thus being considered to have the effect of slow down aging.Chinese patent CN1809364B proposes with acid hydrolysis astragaloside, through the method that purification by silica gel column chromatography obtains Cycloastragenol, but facts have proved that it generates that the ratio of ring-opened byproducts-(+)-Astragenol is higher and complex process, it is difficult to the problem of industrialized production.Chinese patent CN103880910B adopts redox method to prepare Cycloastragenol, reactions steps is more, employ strong oxidizer periodic acid, sodium metaperiodate or Potassium metaperiodate., strong reductant sodium borohydride, potassium borohydride etc., and produce a large amount of formaldehyde after course of reaction adds ethylene glycol, operator are produced certain injury, does not meet the requirement of environmental protection.
Summary of the invention
The purpose of the present invention is to propose to a kind of high-purity Cycloastragenol and produce refining method, the method reaction condition is gentle, easy to operate, is suitable for large-scale industrial production.This method uses more than 80% astragaloside to be raw material, adopts the method for biphase enzyme hydrolysis that astragaloside is hydrolyzed, then adopts extraction again, and recrystallization method obtains the Cycloastragenol of high-load.
Cycloastragenol chemical structural formula is as follows:
(+)-Astragenol (Cycloastragenol ring-opened byproducts) structure is as follows:
A kind of high-purity Cycloastragenol produces refining method, comprises the following steps:
A, biphase enzymatic hydrolysis reaction
Use astragaloside is raw material, is added in the buffer solution that PH is 5-9, and the solid-liquid mass ratio example of astragaloside and buffer solution is 1:50-1:100, is sufficiently stirred for formation suspension；Adding hydrolytic enzyme in suspension, the mass ratio of astragaloside and hydrolytic enzyme is 1:1-9:1；Adding water-insoluble organic solvent, so as to become biphase, organic solvent and volume of buffer solution ratio is for 0.5:1-3:1；Enzyme hydrolysis 20-60h under 25-45 DEG C of condition；
B, extraction
After enzymatic hydrolysis reaction terminates, stand after aqueous phase and organic facies are kept completely separate, take upper organic phase solution；Being subsequently adding a certain amount of water-insoluble organic solvent to extract in buffer solution, the volume ratio of organic solvent and buffer solution is 0.5:1-3:1；Standing, after aqueous phase and organic facies are kept completely separate, takes upper organic phase solution；Merging twice organic phase solution, filter, recovered under reduced pressure organic phase solution obtains the crude product of Cycloastragenol；
C, recrystallization
Taking above-mentioned Cycloastragenol crude product and join in recrystallization solvent, Cycloastragenol crude product and solvent quality ratio for 1:30-1:80, adds the activated carbon of solvent quality 0.05%-0.2%, and temperature 25-90 DEG C is sufficiently stirred for 10-120min；Filter, put it in the refrigerator-freezer that temperature is 10 to-25 DEG C and place, precipitate out Cycloastragenol crystal, filter, be drying to obtain the Cycloastragenol more than or equal to 95% purity.
In above-mentioned biphase enzymatic hydrolysis reaction, the material content of astragaloside should be not less than 80%.
Above-mentioned water-insoluble organic solvent is n-butyl alcohol, ethyl acetate, chloroform, dichloromethane, petroleum ether or its mixture.
Said hydrolyzed enzyme is beta-glucosidase, Snailase, naringinase, the combination of cellulase or above several enzyme.
Above-mentioned recrystallization solvent is ethyl acetate, petroleum ether, methanol, ethanol, chloroform, dichloromethane, acetone or its mixture.
The present invention compares with existing technology, and advantage is in that:
One, astragaloside raw material adopts the method for biphase enzyme hydrolysis to be hydrolyzed, and reactions steps is few, and conversion ratio is higher, and effectively avoids the patent CN1809364B problem adopting acid hydrolysis to be easily caused generation ring-opened byproducts-(+)-Astragenol in actual mechanical process.Enzyme hydrolysis temperature is 25-45 DEG C, and reaction dissolvent is water, a small amount of organic solvent, and organic solvent all reclaims, repeatable
Two, in the present invention, Cycloastragenol is refined the method that adopts crystallization and uses silicagel column loading eluting to be purified and compare in other patents, has easy to operate, and operating efficiency is high, is more suitable for carrying out industrialization promotion.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further described, but not in this, as limitation of the present invention.
Embodiment 1
A, biphase enzyme hydrolysis
1000ml0.02MKH2PO4In-NaOHPH5.0 buffer solution, add 20g90% astragaloside and 4.0g beta-glucosidase stirring suspendible, add 1000ml ethyl acetate, be slowly stirred, react 48h at 37 DEG C；
B, extraction
After enzymatic hydrolysis reaction terminates, stand 1h, take the ethyl acetate solution on upper strata；Aqueous phase adds the ethyl acetate of 500ml be sufficiently stirred for and then stand 1h, after aqueous phase and organic facies are kept completely separate, take upper strata ethyl acetate solution；Merging twice ethyl acetate solution, filter, reclaim ethyl acetate and obtain the crude product 10.68g of Cycloastragenol, the purity detecting Cycloastragenol crude product through HPLC is 83.20%, conversion ratio 78.98%；
C, recrystallization
Taking in the above-mentioned Cycloastragenol crude product 5g mixed solvent joining 250ml ethyl acetate and petroleum ether, the ratio of ethyl acetate and petroleum ether is 1:1, adds 0.25g activated carbon, 70 DEG C of heated and stirred 30min；Filter, treat that recrystallization solvent temperature puts it into standing 24h in the refrigerator-freezer that temperature is 0 to-5 DEG C after dropping to room temperature, precipitate out Cycloastragenol crystal, filter, be drying to obtain Cycloastragenol 3.14g；
Detecting through HPLC, Cycloastragenol purity is 99.08%.Conversion ratio 73.83%.
Embodiment 2
A, biphase enzyme hydrolysis
800ml0.04MNaH2PO4In-NaOHPH5.5 buffer solution, add 10g80% astragaloside and 2.0g Snailase stirring suspendible, add 800ml dichloromethane, be slowly stirred, react 58h at 40 DEG C；
B, extraction
After enzymatic hydrolysis reaction terminates, stand 1.5h, take off the dichloromethane solution of layer；Aqueous phase adds the dichloromethane of 500ml be sufficiently stirred for and then stand 1h, after aqueous phase and organic facies are kept completely separate, take off a layer dichloromethane solution；Merging twice dichloromethane solution, filter, reclaim dichloromethane and obtain the crude product 4.25g of Cycloastragenol, the purity detecting Cycloastragenol crude product through HPLC is 78.32%, and conversion ratio is 66.57%；
C, recrystallization
Take above-mentioned Cycloastragenol crude product 2g and join in 100ml ethyl acetate, add 0.15g activated carbon, 60 DEG C of heated and stirred 60min；Filter, treat that recrystallization solvent temperature puts it into standing 12h in the refrigerator-freezer that temperature is-5 to-10 DEG C after dropping to room temperature, precipitate out Cycloastragenol crystal, filter, be drying to obtain Cycloastragenol 1.29g.
Embodiment 3
A, biphase enzyme hydrolysis
1500ml0.04MNaH2PO4In-NaOHPH6.0 buffer solution, add 20g85% astragaloside and 3.0g naringinase, 1.0g Snailase stirring suspendible, add 1000ml chloroform, be slowly stirred, react 60h at 30 DEG C；
B, extraction
After enzymatic hydrolysis reaction terminates, stand 2h, take off the chloroformic solution of layer；Aqueous phase adds the chloroform of 1000ml be sufficiently stirred for and then stand 1h, after aqueous phase and organic facies are kept completely separate, take off a layer chloroformic solution；Merging twice chloroformic solution, filter, reclaim chloroform and obtain the crude product 11.29g of Cycloastragenol, the purity detecting Cycloastragenol crude product through HPLC is 79.41%, and conversion ratio is 84.38%；
C, recrystallization
Taking in the above-mentioned Cycloastragenol crude product 5g mixed solvent joining 400ml acetone and petroleum ether, the ratio of acetone and petroleum ether is 2:1, adds 0.8g activated carbon, 40 DEG C of heated and stirred 120min；Filter, treat that recrystallization solvent temperature puts it into standing 24h in the refrigerator-freezer that temperature is-25 to-20 DEG C after dropping to room temperature, precipitate out Cycloastragenol crystal, filter, be drying to obtain Cycloastragenol 3.24g；
Detecting through HPLC, Cycloastragenol purity is 99.05%, and conversion ratio is 80.60%.
Embodiment 4
A, biphase enzyme hydrolysis
2200ml0.03MKH2PO4In-NaOHPH8.0 buffer solution, add 40g98% astragaloside and 4.0g beta-glucosidase, 20.0g Snailase and 2.0g cellulase stirring suspendible, add 4000ml ethyl acetate and 1000ml petroleum ether, be slowly stirred, react 48h at 37 DEG C；
B, extraction
After enzymatic hydrolysis reaction terminates, stand 1h, take ethyl acetate and the petroleum ether solution on upper strata；Aqueous phase adds the ethyl acetate of 1000ml be sufficiently stirred for and then stand 1h, after aqueous phase and organic facies are kept completely separate, take upper strata ethyl acetate solution；Merging twice solution, filter, recycling design obtains the crude product 37.85g of Cycloastragenol, and the purity detecting Cycloastragenol crude product through HPLC is 83.48%, and conversion ratio is 80.60%；
C, recrystallization
Taking above-mentioned Cycloastragenol crude product 10g and join in 450ml ethanol, add 0.5g activated carbon, 90 DEG C are heated to reflux 100min；Filter, treat that recrystallization solvent temperature puts it into temperature and is after dropping to room temperature；
Detecting through HPLC, Cycloastragenol purity is 99.85%, and conversion ratio is 87.84%.
Claims (5)
1. high-purity Cycloastragenol produces a refining method, in turn includes the following steps:
A. biphase enzyme hydrolysis
Use astragaloside is raw material, is added in the buffer solution that PH is 5-9, and the solid-liquid mass ratio example of astragaloside and buffer solution is 1:50-1:100, is sufficiently stirred for formation suspension；Adding hydrolytic enzyme in suspension, the mass ratio of astragaloside and hydrolytic enzyme is 1:1-9:1；Adding water-insoluble organic solvent, so as to become biphase, organic solvent and volume of buffer solution ratio is for 0.5:1-3:1；Enzyme hydrolysis 20-60h under 25-45 DEG C of condition；
B. extract
After enzymatic hydrolysis reaction terminates, stand after aqueous phase and organic facies are kept completely separate, take upper organic phase solution；Being subsequently adding a certain amount of water-insoluble organic solvent to extract in buffer solution, the volume ratio of organic solvent and buffer solution is 0.5:1-3:1；Standing, after aqueous phase and organic facies are kept completely separate, takes upper organic phase solution；Merging twice organic phase solution, filter, recovered under reduced pressure organic phase solution obtains the crude product of Cycloastragenol；
C. recrystallization
Taking above-mentioned Cycloastragenol crude product and join in recrystallization solvent, Cycloastragenol crude product and solvent quality ratio for 1:30-1:80, adds the activated carbon of solvent quality 0.05%-0.2%, and temperature 25-80 DEG C is sufficiently stirred for 10-120min；Filter, put it in the refrigerator-freezer that temperature is 10 to-25 DEG C and place, precipitate out Cycloastragenol crystal, filter, be drying to obtain the Cycloastragenol more than or equal to 95% purity.
2. method according to claim 1, it is characterised in that the material content of described astragaloside should be not less than 80%.
3. method according to claim 1, it is characterised in that described water-insoluble organic solvent is n-butyl alcohol, ethyl acetate, chloroform, dichloromethane, petroleum ether or its mixture.
4. method according to claim 1, it is characterised in that described hydrolytic enzyme is beta-glucosidase, Snailase, naringinase, the combination of cellulase or above several enzyme.
5. method according to claim 1, it is characterised in that described recrystallization solvent is ethyl acetate, petroleum ether, methanol, ethanol, chloroform, dichloromethane, acetone or its mixed solvent.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058445A (en) * | 2017-05-09 | 2017-08-18 | 北京化工大学 | It is a kind of to convert the method that Astragaloside IV prepares cycloastragenol using two step enzymatic isolation methods |
| CN109942663A (en) * | 2019-04-22 | 2019-06-28 | 中国人民解放军联勤保障部队第九八九医院 | The method for preparing cycloastragenol using two-phase sour water solution |
| CN111378002A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Novel cycloastragenol crystal form A and preparation method thereof |
| CN111378000A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form F and preparation method thereof |
| CN111378004A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form D and preparation method thereof |
| CN111377998A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form C and preparation method thereof |
| CN111377999A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form B and preparation method thereof |
| CN111378001A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form E and preparation method thereof |
| CN111378003A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form G and preparation method thereof |
| CN114369636A (en) * | 2021-12-27 | 2022-04-19 | 泰州丹鼎生物科技有限公司 | Biocatalytic preparation method of cycloastragenol |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058445A (en) * | 2017-05-09 | 2017-08-18 | 北京化工大学 | It is a kind of to convert the method that Astragaloside IV prepares cycloastragenol using two step enzymatic isolation methods |
| CN107058445B (en) * | 2017-05-09 | 2020-11-20 | 北京化工大学 | Method for preparing cycloastragenol by converting astragaloside IV by two-step enzymolysis method |
| CN111378000A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form F and preparation method thereof |
| CN111378002A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Novel cycloastragenol crystal form A and preparation method thereof |
| CN111378004A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form D and preparation method thereof |
| CN111377998A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form C and preparation method thereof |
| CN111377999A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form B and preparation method thereof |
| CN111378001A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form E and preparation method thereof |
| CN111378003A (en) * | 2018-12-30 | 2020-07-07 | 山东新时代药业有限公司 | Cycloastragenol crystal form G and preparation method thereof |
| CN111378003B (en) * | 2018-12-30 | 2022-06-14 | 鲁南制药集团股份有限公司 | Cycloastragenol crystal form G and preparation method thereof |
| CN109942663A (en) * | 2019-04-22 | 2019-06-28 | 中国人民解放军联勤保障部队第九八九医院 | The method for preparing cycloastragenol using two-phase sour water solution |
| CN109942663B (en) * | 2019-04-22 | 2021-10-26 | 中国人民解放军联勤保障部队第九八九医院 | Method for preparing cycloastragenol by using diphasic acid hydrolysis |
| CN114369636A (en) * | 2021-12-27 | 2022-04-19 | 泰州丹鼎生物科技有限公司 | Biocatalytic preparation method of cycloastragenol |
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