CN104327139A - Preparation method of cordycepin crystal - Google Patents
Preparation method of cordycepin crystal Download PDFInfo
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- CN104327139A CN104327139A CN201410507918.5A CN201410507918A CN104327139A CN 104327139 A CN104327139 A CN 104327139A CN 201410507918 A CN201410507918 A CN 201410507918A CN 104327139 A CN104327139 A CN 104327139A
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- cordycepin
- cordyceps militaris
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Abstract
The invention discloses a preparation method of a cordycepin crystal. The preparation method includes following steps: (1) with residual substrate after cultivation of cordyceps militaris stroma as a raw material, crushing the substrate, performing extraction with an organic solvent and performing centrifugation to collect a supernate; (2) performing purification to a crude liquid after the centrifugation through a macroporous resin adsorption chromatographic column; (3) performing enrichment to the cordycepin obtained through the step (2) with an aqueous two-phase system composed of ethanol and ammonium sulfate; and (4) performing elution in a chromatographic column with a methanol solution, performing reduced pressure distillation for concentrating an eluent, performing methanol elution to the concentrated eluent, and performing crystallization to the eluent to obtain the cordycepin crystal. The preparation method of the cordycepin crystal is reduced in production cost, increases the purity of the cordycepin and is free of residual organic solvent.
Description
Technical field
The present invention relates to the preparation method of cordycepin and crystal thereof, belong to medicinal chemistry arts.
Background technology
Cordyceps militaris (L.) Link. is Ascomycotina, ergot Zoopagales, the type species of Clavicipitaceae, Cordyceps, formal name used at school is Cordycepsmilitaris, has another name called Cordyceps militaris (L.) Link., Cordyceps militaris etc., and the traditional Chinese medical science is thought, Chinese caterpillar fungus enters lung kidney two warp, can tonifying lung cloudy, again can kidney-replenishing, cure mainly and suffer from a deficiency of the kidney, impotence and seminal emission, soreness of waist and knee joint, weak after being ill, chronic cough is weak, phthisical cough phlegm blood, spontaneous sweatings etc., are unique a kind of Chinese medicine that can balance simultaneously, regulate negative and positive, are widely used.
Cordycepin is the major physiological activeconstituents of Cordyceps militaris (L.) Link., it is a kind of nucleosides material, be made up of VITAMIN B4 and desoxypentose, it has very large market outlook in health care, the extraction of current cordycepin is all extracted by complicated technique from Cordyceps militaris (L.) Link., there is water-bath, alcohol bath extraction, resin adsorption method, chromatographic separation method, active carbon adsorption etc., these methods or complicated operation, extraction yield is low, need through different plastic resin treatment or must by the high performance liquid preparative chromatography technical equipment of costliness, production cost is high, be difficult to realize large-scale industrial produce.
The residue of Cordyceps militaris (L.) Link. was once attempted cultivating to obtain highly purified plate-like cordycepin crystal in this area in recent years, but this kind of method productive rate is lower, there is larger wastage of material.
Summary of the invention
For defect of the prior art, the invention discloses a kind of preparation method of cordycepin crystal, to cultivate the residue matrix after Cordyceps militaris (L.) Link. stroma for starting material, achieve the recycling of refuse, and simple to operate, mild condition, extraction yield is high, be easy to expanding production, there is not organic problem of solvent residual, and obtained cordycepin crystal purity is high.
For achieving the above object, the present invention is achieved through the following technical solutions:
The preparation method of cordycepin, comprises the steps: 1) to cultivate the remaining matrix of Cordyceps militaris (L.) Link. stroma for raw material, after matrix being pulverized, carry out lixiviate with organic solvent, collected by centrifugation supernatant liquor; 2) macroporous resin adsorption chromatographic column is utilized to carry out purifying to the thick liquid after centrifugal; 3) step 2) gained cordycepin adopt ethanol, ammonium sulfate composition double-aqueous phase system carry out enrichment and collect sample; 4) fill chromatography column, methanol solution wash-out, elutriant underpressure distillation concentrates, and concentrated eluant methanol wash-out, elutriant crystallization obtains cordycepin crystal.
In above-mentioned preparation method, with Cordyceps militaris (L.) Link. stroma cultivation residue matrix for raw material extracts the production cost that cordycepin reduces whole process, and achieve the utilization of waste material of residue matrix, improve the added value of product of Cordyceps militaris (L.) Link.; Adopt organic solvent to propose relatively traditional water-bath lixiviate to the thick immersion of cordycepin, in vat liquor, cordycepin content is higher, and impurity is less; Adopt macroporous resin adsorption chromatographic column to carry out purifying to sample, can realize continuously dynamic separation at normal temperatures, simple to operate, cost is low, and the cordycepin purity liquid of collection is high; Adopt the double-aqueous phase system of ethanol, ammonium sulfate composition to carry out enrichment to cordycepin, extraction is more complete, and product purity is higher.
Generally, method of the present invention adopts macroreticular resin absorbing method in conjunction with ethanol, ammonium sulfate aqueous two phase extraction technique, compared with traditional flooding, macroreticular resin absorbing method, simple to operate, product purity is higher, be suitable for expanding production and one does not exist organic problem of solvent residual, environmental protection.
Wherein, described cultivation Cordyceps militaris (L.) Link. stroma remaining matrix refers to that the solid medium for cultivating Cordyceps militaris (L.) Link. stroma removes the remaining matrix of Cordyceps militaris (L.) Link. stroma after cultivation completes, such matrix main component one be wheat or other compositions that Cordyceps militaris (L.) Link. stroma can be provided to grow.
In the present invention, described matrix is pulverized standard and cross 80 mesh sieves after being pulverized, and organic solvent used is ethanolic soln, is 2-10: 1 with the envelope-bulk to weight ratio of matrix, and extraction temperature is 50-80 DEG C, and extraction time is 3-8 hour.
Usually, above-mentioned ethanol solution concentration is the aqueous ethanolic solution of 40-75%, and the degree that above-mentioned matrix is pulverized and organic solvent used match, and maximizedly can improve extracting efficiency.
Wherein, chromatography parameter be cordycepin sample liquid and macroporous resin with 50ml: 0.8-1.5g amount loading, 1.5-2.0BV/h, then the distilled water removal of impurities of 3-8BV is used, flow velocity is 1.5-2.0BV/h, finally uses the ethanolic soln wash-out of 3-8BV, and flow velocity is 1.5-2.0BV/h.
Wherein, ethanol solution concentration used is the aqueous ethanolic solution of 40-75%.
Preferably, chromatography parameter be cordycepin sample liquid and macroporous resin with 50ml: 1g amount loading, (now pH is about 5), 1.8BV/h, then use the distilled water removal of impurities of 5BV, flow velocity is 1.8BV/h, finally uses the 50% ethanolic soln wash-out of 5BV, (now pH is about 3), flow velocity is 1.8BV/h.
In the present invention, it is 35-45% (w/v) that double-aqueous phase system used consists of alcohol concn, and ammonium sulfate concentrations is 15-25% (w/v), and all the other are water.
Preferably, in double-aqueous phase system, alcohol concn is 39% (w/v), and ammonium sulfate concentrations is 21% (w/v), and all the other are water.
Wherein, the methanol solution of 1.5 ~ 2BV is adopted to carry out wash-out in chromatography column.
Accompanying drawing explanation
Fig. 1 is meoh eluate thin-layer chromatography result figure;
Fig. 2 is cordycepin standard substance high performance liquid chromatography detection figure;
Fig. 3 is isolated cordycepin liquid height effect liquid phase chromatogram detection figure;
Fig. 4 is cordycepin crystal Electronic Speculum figure.
Embodiment
Embodiment 1: prepare cordycepin crystal according to following step:
(1) Cordyceps militaris (L.) Link. stroma cultivation residue matrix dried naturally, pulverize, then 80 mesh sieves are for subsequent use excessively;
(2) take 20g Cordyceps militaris (L.) Link. powder, add the ethanolic soln of 5 times of volumes 50%, alcohol bath lixiviate 5h under 60 DEG C of conditions;
(3) with supercentrifuge centrifugal 10min under 3000r/min condition, supernatant liquor is collected for subsequent use;
(4) with macroporous resin, purifying is carried out to the supernatant liquor of collected after centrifugation, sample liquid and NKAII macroporous resin with 50ml: 1g amount loading, pH 5,1.8BV/h, then use the distilled water removal of impurities of 5BV, flow velocity is 1.8BV/h, finally uses the 50% ethanolic soln wash-out of 5BV, pH 3, flow velocity is 1.8BV/h;
(5) form double-aqueous phase system with ethanol eluate and ammonium sulfate, wherein alcohol concn is 39% (w/v), and ammonium sulfate concentrations is 21% (w/v), collects supernatant liquor and carries out repeatedly enrichment;
(6) launch upper on thin layer plate, develop the color under ultraviolet 254nm, collect sample.
(7) fill chromatography column, with the methanol solution wash-out of 2BV, collect elutriant;
(8) by 1/5 of elutriant underpressure distillation simmer down to original volume;
(9) the elutriant 2BV methanol-eluted fractions after concentrating, collection elutriant, at room temperature carries out crystallization and recrystallization obtains cordycepin crystal 0.79g, and gained crystal purity reaches 98.9%.
Embodiment 2: prepare cordycepin crystal according to following step:
(1) Cordyceps militaris (L.) Link. stroma cultivation residue matrix dried naturally, pulverize, then 80 mesh sieves are for subsequent use excessively;
(2) take 20g Cordyceps militaris (L.) Link. powder, add the ethanolic soln of 6 times of volumes 60%, alcohol bath lixiviate 4h under 60 DEG C of conditions;
(3) with supercentrifuge centrifugal 15min under 3000r/min condition, supernatant liquor is collected for subsequent use;
(4) with macroporous resin, purifying is carried out to the supernatant liquor of collected after centrifugation, sample liquid and NKAII macroporous resin with 50ml: 1.2g amount loading, 1.8BV/h, then the distilled water removal of impurities of 5BV is used, flow velocity is 1.8BV/h, and finally use the 50% ethanolic soln wash-out of 5BV, flow velocity is 1.8BV/h;
(5) form double-aqueous phase system with ethanol eluate and ammonium sulfate, wherein alcohol concn is 42% (w/v), and ammonium sulfate concentrations is 25% (w/v), collects supernatant liquor and carries out repeatedly enrichment;
(6) launch upper on thin layer plate, develop the color under ultraviolet 254nm, collect sample.
(7) fill chromatography column, with the methanol solution wash-out of 2BV, collect elutriant;
(8) by 1/5 of elutriant underpressure distillation simmer down to original volume;
(9) the elutriant 1.5BV methanol-eluted fractions after concentrating, collection elutriant, at room temperature carries out crystallization and recrystallization obtains cordycepin crystal 0.82g, and gained crystal purity reaches 99.8%.
Embodiment 3: prepare cordycepin crystal according to following step:
(1) Cordyceps militaris (L.) Link. stroma cultivation residue matrix dried naturally, pulverize, then 80 mesh sieves are for subsequent use excessively;
(2) take 20g Cordyceps militaris (L.) Link. powder, add the ethanolic soln of 4 times of volumes 70%, alcohol bath lixiviate 6h under 60 DEG C of conditions;
(3) with supercentrifuge centrifugal 15min under 3000r/min condition, supernatant liquor is collected for subsequent use;
(4) with macroporous resin, purifying is carried out to the supernatant liquor of collected after centrifugation, sample liquid and NKAII macroporous resin with 50ml: 0.8g amount loading, 1.5BV/h, then the distilled water removal of impurities of 4BV is used, flow velocity is 1.6BV/h, and finally use the 40% ethanolic soln wash-out of 7BV, flow velocity is 2BV/h;
(5) form double-aqueous phase system with ethanol eluate and ammonium sulfate, wherein alcohol concn is 37% (w/v), and ammonium sulfate concentrations is 20% (w/v), collects supernatant liquor and carries out repeatedly enrichment;
(6) launch upper on thin layer plate, develop the color under ultraviolet 254nm, collect sample.
(7) fill chromatography column, with the methanol solution wash-out of 1.5BV, collect elutriant;
(8) by 1/5 of elutriant underpressure distillation simmer down to original volume;
(9) the elutriant 2BV methanol-eluted fractions after concentrating, collection elutriant, at room temperature carries out crystallization and recrystallization obtains cordycepin crystal 0.78g, and gained crystal purity reaches 98.3%.
In the above-mentioned methods, last crystallization and recrystallizing technology adopt common extract crystallization processes, such as, can adopt following technique: by elutriant crystallization at 0 ~ 4 DEG C, crystal separation gone out by vacuum filtration method, obtain cordycepin crystal crude product after drying; Ethanolic soln cordycepin crystal crude product being dissolved in 75-85% (V/V) carries out recrystallization, and then vacuum filtration separating heavy crystallized product, obtains cordycepin crystal after washing and drying.
The detection method following (crystal for embodiment 1) of above-mentioned products therefrom:
(1) thin layer detects:
Be added in by sample drop on thin layer plate, developping agent is that (chloroform: Virahol: ethyl acetate: water=8: 6: 2: 0.3), develops the color under 254nm uv irradiating, and meoh eluate thin-layer chromatography the results are shown in Figure 1.
(2) high effective liquid chromatography for measuring cordycepin content:
Stationary phase is C18 pillar; Methyl alcohol in moving phase: water=20: 80 (v/v); Flow rate, 1.0ml/min; Applied sample amount is 10 μ l, with the membrane filtration of 0.45 μm before sample uses.Finally under 260nm condition, carry out ultraviolet detection.Standard substance high performance liquid chromatography detection figure is shown in Fig. 2, and sample high performance liquid chromatography detection figure is shown in Fig. 3.
(3) by the cordycepin crystal that obtains at electric Microscopic observation, the results are shown in Figure 4.
Claims (8)
1. the preparation method of cordycepin crystal, is characterized in that comprising the steps: 1) to cultivate the remaining matrix of Cordyceps militaris (L.) Link. stroma for raw material, after matrix being pulverized, carry out lixiviate with organic solvent, collected by centrifugation supernatant liquor; 2) macroporous resin adsorption chromatographic column is utilized to carry out purifying to the thick liquid after centrifugal; 3) step 2) gained cordycepin adopt ethanol, ammonium sulfate composition double-aqueous phase system carry out enrichment and collect sample; 4) fill chromatography column, methanol solution wash-out, elutriant underpressure distillation concentrates, and concentrated eluant methanol wash-out, elutriant crystallization obtains cordycepin crystal.
2. preparation method according to claim 1, is characterized in that the remaining matrix of described cultivation Cordyceps militaris (L.) Link. stroma refers to that the solid medium for cultivating Cordyceps militaris (L.) Link. stroma removes the remaining matrix of Cordyceps militaris (L.) Link. stroma after cultivation completes.
3. preparation method according to claim 1, is characterized in that described matrix is pulverized standard and cross 80 mesh sieves after being pulverized.
4. preparation method according to claim 1, it is characterized in that organic solvent used is ethanolic soln, is 2-10: 1 with the envelope-bulk to weight ratio of matrix, and extraction temperature is 50-80 DEG C, and extraction time is 3-8 hour.
5. preparation method according to claim 1, it is characterized in that chromatography parameter is that cordycepin sample liquid and macroporous resin are with the amount loading of 50ml:0.8-1.5g, 1.5-2.0BV/h, then the distilled water removal of impurities of 3-8BV is used, flow velocity is 1.5-2.0BV/h, finally use the ethanolic soln wash-out of 3-8BV, flow velocity is 1.5-2.0BV/h.
6. preparation method according to claim 1, is characterized in that in double-aqueous phase system, and alcohol concn is 35-45% (w/v), and ammonium sulfate concentrations is 15-25% (w/v), and all the other are water.
7. preparation method according to claim 6, is characterized in that in double-aqueous phase system, and alcohol concn is 39% (w/v), and ammonium sulfate concentrations is 21% (w/v), and all the other are water.
8. preparation method according to claim 1, is characterized in that adopting the methanol solution of 1.5 ~ 2BV to carry out wash-out in chromatography column.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105111264A (en) * | 2015-08-16 | 2015-12-02 | 深圳市倍昂生物科技有限公司 | 3'-deoxyadenosine crystal form and preparation method therefor |
| CN110432258A (en) * | 2019-07-17 | 2019-11-12 | 西北农林科技大学 | Cordycepin is used to prepare the application and preparation method of goat sperm freezen protective dilution |
| CN110693898A (en) * | 2019-11-23 | 2020-01-17 | 吉林省蔚来生物科技有限公司 | A pharmaceutical composition containing ginsenoside |
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| CN101157712A (en) * | 2007-11-16 | 2008-04-09 | 上海市农业科学院 | Method for separating and purifying cordycepin |
| CN101928316A (en) * | 2010-07-23 | 2010-12-29 | 上海国宝企业发展中心 | Preparation method of high-purity plate-like cordycepin crystal |
| KR20120077691A (en) * | 2010-12-31 | 2012-07-10 | 안성구 | A cordycepin producing method using unhatched egg |
| JP2013111060A (en) * | 2011-11-30 | 2013-06-10 | Univ Of Fukui | Method for producing and refining cordycepin |
| CN103772460A (en) * | 2014-02-14 | 2014-05-07 | 江苏省中国科学院植物研究所 | Extraction process of cordycepin (active substance) from silkworm pupa cordyceps militaris |
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2014
- 2014-09-28 CN CN201410507918.5A patent/CN104327139B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101157712A (en) * | 2007-11-16 | 2008-04-09 | 上海市农业科学院 | Method for separating and purifying cordycepin |
| CN101928316A (en) * | 2010-07-23 | 2010-12-29 | 上海国宝企业发展中心 | Preparation method of high-purity plate-like cordycepin crystal |
| KR20120077691A (en) * | 2010-12-31 | 2012-07-10 | 안성구 | A cordycepin producing method using unhatched egg |
| JP2013111060A (en) * | 2011-11-30 | 2013-06-10 | Univ Of Fukui | Method for producing and refining cordycepin |
| CN103772460A (en) * | 2014-02-14 | 2014-05-07 | 江苏省中国科学院植物研究所 | Extraction process of cordycepin (active substance) from silkworm pupa cordyceps militaris |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105111264A (en) * | 2015-08-16 | 2015-12-02 | 深圳市倍昂生物科技有限公司 | 3'-deoxyadenosine crystal form and preparation method therefor |
| CN110432258A (en) * | 2019-07-17 | 2019-11-12 | 西北农林科技大学 | Cordycepin is used to prepare the application and preparation method of goat sperm freezen protective dilution |
| CN110432258B (en) * | 2019-07-17 | 2021-09-28 | 西北农林科技大学 | Application of cordycepin in preparation of diluent for cryopreservation of goat sperm and preparation method of cordycepin |
| CN110693898A (en) * | 2019-11-23 | 2020-01-17 | 吉林省蔚来生物科技有限公司 | A pharmaceutical composition containing ginsenoside |
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