CN104711300A - Preparation method of icaritin - Google Patents
Preparation method of icaritin Download PDFInfo
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- CN104711300A CN104711300A CN201310674836.5A CN201310674836A CN104711300A CN 104711300 A CN104711300 A CN 104711300A CN 201310674836 A CN201310674836 A CN 201310674836A CN 104711300 A CN104711300 A CN 104711300A
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- herba epimedii
- epimedii extract
- polygalacturonase
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Abstract
The invention provides a preparation method of icaritin. In the preparation method, epimedium extract carries out enzymatic hydrolysis reactions in the presence of pectase to produce icaritin. The yield of icaritin relative to general flavone can reach 20% or more, and the icaritin purity exceeds 99%.
Description
Technical field
The present invention relates to the preparation method of Icaritin, belong to field of medicaments.
Background technology
A Kelading, has another name called Icaritin, epimedium aglucone, and be the new effective monomer that extraction and isolation obtains from Chinese medicinal materials Herba Epimedii main active ingredient Herba Epimedii extract obtains through enzymatic conversion, its structural formula is as shown in the formula shown in (I):
The preparation method of this compound is disclosed in CN101302548B.The method is raw material with icarin, is hydrolyzed with beta-glucosidase, the centrifugal precipitation acetone solution obtained of hydrolysate, filters and obtains supernatant liquor.Again this supernatant liquor water is carried out recrystallization, obtain Icaritin sterling.
Be disclose a kind of method adopting naringinase to obtain epimedium aglucone from icarin in the Chinese patent of 201010517793.6 at application number, icarin standard substance are dissolved in alcohol solvent by the method, add naringinase when 40-70 DEG C and carry out enzymolysis, thus obtain epimedium aglucone.
Be the preparation method disclosing a kind of epimedium aglucone in the Chinese patent of 200910184282.4 at application number, the method is raw material with Herba Epimedii, and by methanol extraction, the steps such as helicase hydrolysis obtain epimedium aglucone crude product.But all there is enzymolysis low conversion rate in the enzymolysis process of above Icaritin preparation method, the defect that enzymolysis product impurity is many.
Summary of the invention
An object of the present invention is to provide a kind of preparation method of Icaritin, the Icaritin purity prepared by the method is high.
One aspect of the present invention provides a kind of preparation method of Icaritin, and the method comprises carries out enzyme digestion reaction by Herba Epimedii extract under the effect of polygalacturonase, obtains Icaritin.
Preferably, described polygalacturonase comprises polygalacturonase and a-N-arabinofuranosidase.
Preferably, the icarin mass content in described Herba Epimedii extract is 5%-100%.
Preferably, described Herba Epimedii extract prepares by the following method: take Herba Epimedii as raw material, extracted by ethanolic soln, obtain Herba Epimedii extract.
Preferably, the enzymolysis product obtained by enzyme digestion reaction, is dissolved in weakly polar organic solvent, filters, and collects filtrate, adds polar solvent, crystallization in filtrate.
Preferably, described weakly polar organic solvent is one or more in ethyl acetate, acetone, methylene dichloride and methyl tertiary butyl ether, and described polar solvent is one or more in methyl alcohol, second alcohol and water.
Most preferably, described weakly polar organic solvent is acetone, and described polar solvent is water.
Preferably, described enzyme digestion reaction comprises the following steps:
A. the enzymolysis product obtained by enzyme digestion reaction is centrifugal;
B. acetone is added by the centrifugal precipitation obtained;
C. again acetone is filtered in the filtrate obtained and add distilled water, reflux;
D. by the liquid at room temperature crystallization obtained that refluxes.
Preferably, the filtrate in step c and the volume ratio of distilled water are 1-5:1, and described reflux temperature is 50-100 DEG C.
Preferably, Herba Epimedii extract liquid concentration, is then adsorbed by macroporous resin column, then carries out wash-out with the aqueous ethanolic solution that volumetric concentration is 30%-100%, obtains the Herba Epimedii extract for enzyme digestion reaction.
Preferably, be 20%-100% for the mass content of flavones in the Herba Epimedii extract of enzyme digestion reaction.
Preferably, the quality of described Herba Epimedii and the volume ratio of ethanolic soln are 1 kilogram: 2-50 liter, and Extracting temperature is 30-100 DEG C.
Preferably, the aqueous ethanolic solution of described ethanolic soln to be volume ratio be 20-90%.
Preferably, the condition of described enzyme digestion reaction is: Herba Epimedii extract and phosphoric acid buffer are made the substrate solution that pH value is 4.0-6.0, in substrate solution, add polygalacturonase, the quality of polygalacturonase is 5-30 times of Herba Epimedii extract quality, and hydrolysis temperature is 45-55 DEG C.
More preferably, the quality of polygalacturonase is 10-20 times of Herba Epimedii extract quality, and the pH value of substrate solution is 4.5-5.5, and hydrolysis temperature is 48-50 DEG C.
Most preferably, the quality of described polygalacturonase is 15 times of Herba Epimedii extract quality, the ethanol of the 10-15% containing its cumulative volume in substrate solution.
Preferably, the pH value of described substrate solution is regulated by phosphoric acid buffer.
More preferably, described phosphoric acid buffer is phosphoric acid-citrate buffer solution or Sodium phosphate dibasic-potassium phosphate buffer.
Beneficial effect of the present invention is: the Icaritin purity that the inventive method prepares is very high, and content is greater than 99%, the Icaritin transformed by polygalacturonase, relative to Herba Epimedii extract, the yield of Icaritin can reach 20%, and the by product after conversion is few, easily removes.
Accompanying drawing explanation
Fig. 1 represents the total flavones in medicinal raw material, and by step 1,2 and 3 total flavoness obtained of embodiment 1.
Fig. 2 represents the high-efficient liquid phase chromatogram of the Icaritin after purifying.
Fig. 3 represents that enzyme digestion reaction carries out the high-efficient liquid phase chromatogram of 48 hours.
Fig. 4 represents that enzyme digestion reaction carries out the high-efficient liquid phase chromatogram of 70 hours.
Embodiment
Unless otherwise indicated, term " enzyme digestion reaction " herein refers to the hydrolysis reaction under enzyme effect.
Unless otherwise indicated, term " icarin " herein refers to the extract in the dry cauline leaf of Herba Epimedii, is single component, has following molecular structure:
Unless otherwise indicated, term " total flavones " herein refers to the main component in the Herba Epimedii extract obtained by ethanol extraction method, and total flavones comprises icarin, Epimedin A, Epimedin B, epimedin, icariside, and treasured is glycosides etc. suddenly.Have recorded the method for calculation of total flavones at Chinese Pharmacopoeia version in 2010 first 306-308 page, according to ultraviolet visible spectrophotometry, the content of total flavones can be calculated in the absorbancy at 270nm wavelength place.
Unless otherwise indicated, term " polygalacturonase " herein purchased from the trade mark of DSM company be RAPIDASE polygalacturonase as enzyme digestion reaction enzyme, production code member is 984.
Embodiment 1
the preparation of Herba Epimedii extract
Step 1: with the Folium Epimedii 300g except stem for raw material, rubbed broken, extract with the 600L aqueous ethanolic solution that volumetric concentration is 70%, extract 3 times altogether at 75 DEG C.Reclaim ethanol extract extremely without alcohol taste, then by centrifugal for this concentrated solution, gained filtrate is adsorbed by macroporous resin column.
Step 2: be the macroporous resin column in the aqueous ethanolic solution washing step 1 of 20% by volumetric concentration, elutriant discards.
Step 3: carry out the macroporous resin column in washing step 2 with the aqueous ethanolic solution that volumetric concentration is 80%, collects elutriant now.
The elutriant that above three steps obtain is concentrated, obtains Herba Epimedii extract and amount to 46g.The Herba Epimedii extract that above three-step approach obtains detects flavone component wherein by high performance liquid chromatography, sees second figure of Fig. 1.
Macroporous resin in the present embodiment is purchased from Hebei Bao En sorbing material company limited, and model is D101.
Detect the flavone component in medicinal material by high performance liquid chromatography, see first figure of Fig. 1.
High performance liquid chromatography (HPLC) condition: chromatographic column: Agela-C18 post; Moving phase: acetonitrile-water (wherein containing volume fraction is the trifluoroacetic acid of 0.0125%), gradient sees the following form 1, elution time 60min, flow velocity: 1.0mL/min, column temperature: 35 DEG C.UV-detector determined wavelength is respectively 254,272,320nm.
Table 1 solvent gradient elution table
Result shows: except the peak of appearance 3 minutes time is except solvent peak, the peak that peak is flavone component is gone out 22 minutes, 24 minutes, 46 minutes and 52 minutes, the total flavones collection of illustrative plates of the medicinal material detected by above method, contrast can find out with the collection of illustrative plates (HPLC-UV) of total flavones in Herba Epimedii extract, after macroporous resin purification, in Herba Epimedii extract, the content of total flavones significantly increases, and wherein total flavones reaches 5-15% relative to the raw-material yield of Herba Epimedii.
enzyme digestion reaction prepares Icaritin
According to above step, repeatedly extract epimedium herb, obtain Herba Epimedii extract 55 grams, Sodium phosphate dibasic-potassium phosphate buffer 0.15L, the ethanol 0.22L of pH5.2 of 1mol/L, 660 grams of enzymes are dissolved in the water the 0.7L made, pH value is that the enzyme solution of 5.2 is in 5L reactor, temperature of reaction carries out enzymolysis under the condition of 48 DEG C, and concrete condition is as table 2:
Table 2 enzymatic hydrolysis condition
| Herba Epimedii extract | Enzyme | Ethanol | Water | Damping fluid |
| 55g | 660g | 0.22L | 1.1L | 0.15L(1M) |
Select the trade mark of DSM company be RAPIDASE polygalacturonase as enzyme digestion reaction enzyme, production code member is 984.
the purifying of Icaritin
After reaction in 70 hours terminates, reaction solution carries out centrifugal, removes supernatant liquor, and precipitation uses 300mL acetone solution, filters, obtains filtrate 160mL, then in filtrate, add about 150mL distilled water, and 75 DEG C of backflows are dissolved, and room temperature places crystallization.Crystallization, after 24 hours, is filtered and is obtained crystallization, and dry, weigh to obtain 11.5g.Crystallize out is Icaritin.Icaritin is 20.9% relative to the yield of Herba Epimedii extract, reaches 99.5%, see Fig. 2 by the purity of high-efficient liquid phase color spectral test Icaritin.
Embodiment 2
Be purchased Herba Epimedii extract enzymolysis process and prepare Icaritin
herba Epimedii extract in the present embodiment is purchased from Jiahe, Shaanxi plant Chemical Co., Ltd., and trade name is " Herba Epimedii extract ", and wherein containing massfraction is the icarin of 90%.
step one: enzymolysis process prepares Icaritin
By the 80g Herba Epimedii extract be purchased, wherein containing massfraction is the icarin of 90%, be scattered in Sodium phosphate dibasic-potassium phosphate buffer 2.0L that concentration is the pH5.2 of 1mol/L, add ethanol 0.6L, RAPIDASE polygalacturonase 1400g, amount to 1.4L in 5L reactor, temperature of reaction carries out enzymolysis under the condition of 50 DEG C, and concrete condition is as table 3:
Table 3 enzymatic hydrolysis condition
Detected by HPLC, reaction system transforms 70 hours, and the transformation efficiency that icarin is converted into Icaritin reaches 90%.
Step 2: the purifying of Icaritin
After reaction in 70 hours terminates, reaction solution carries out centrifugal, removes supernatant, and precipitation uses 2L acetone solution, filters, then in filtrate, adds about 1L distilled water, and 75 DEG C of backflows are dissolved, and room temperature places crystallization.Crystallization, after 24 hours, is filtered and is obtained crystallization, and dry, weigh to obtain 32.9g.Crystallize out is Icaritin.Icaritin is 41.1% relative to the yield of 90% Herba Epimedii extract, reaches 99.0% by the purity of high-efficient liquid phase color spectral test Icaritin.
Embodiment 3
Enzymatic conversion total flavones prepares the condition optimizing of Icaritin
1, the investigation of pH value, temperature of reaction, concentration of substrate
The Herba Epimedii extract selecting 1mg to be purchased Herba Epimedii extract or to obtain according to embodiment 1 method is as substrate, at 200 μ L damping fluids, under the condition of the 7 μ L polygalacturonases that 15mg enzyme is mixed with and 250 μ L water, change pH value, temperature and concentration of substrate respectively, react 24 hours, after reaction terminates, water-saturated n-butanol extracts, and finds optimal reaction conditions.By thin-layer chromatographic analysis, determine optimal reaction conditions.(developping agent is trichloromethane to thin-layer chromatographic analysis condition: methyl alcohol: lower floor's solution of water=76:26:6; Developer is 10% ethanol solution of sulfuric acid).
The variation range of table 4pH value
The variation range of table 5 temperature of reaction
The further investigation of reaction conditions
In order to determine substrate Herba Epimedii extract concentration and alcohol concn further, with 10mg/mL and 20mg/mL of the 15mg/ml concentration of substrate left and right sides, ethanol contend concentration (10%, 15%) and dissimilar ion damping fluid, carry out following experiment:
Table 6 concentration of substrate, damping fluid are on the impact of transformation efficiency
Result shows: when ethanol contend concentration is 15%, enzyme is part inactivation, and therefore ethanol contend concentration is 10% most suitable; When substrate Herba Epimedii extract concentration is 20mg/mL, transform not exclusively, and when concentration of substrate is 15mg/mL, transform comparatively complete, and the changing effect of acetate buffer solution is slightly poorer than phosphoric acid buffer and phosphoric acid-citrate buffer solution.
2. sum up
From above result, this polygalacturonase is utilized to carry out Herba Epimedii total flavones when transforming in a large number, the concentration of substrate chosen can be 15mg/mL, alcohol concn is 10%, damping fluid is Sodium phosphate dibasic-potassium phosphate buffer or phosphoric acid-citrate buffer solution, pH4.5, and temperature controls between 50-55 DEG C, yield can reach more than 20%, obtains the purity of Icaritin more than 99%.
Claims (10)
1. a preparation method for Icaritin, the method comprises carries out enzyme digestion reaction by Herba Epimedii extract under the effect of polygalacturonase, obtains Icaritin, and preferably, described polygalacturonase comprises polygalacturonase and a-N-arabinofuranosidase.
2. method according to claim 1, is characterized in that, the icarin mass content in described Herba Epimedii extract is 5%-100%.
3. method according to claim 1 and 2, is characterized in that, described Herba Epimedii extract prepares by the following method: take Herba Epimedii as raw material, extracted by ethanolic soln, obtain Herba Epimedii extract.
4. method according to claim 1 and 2, is characterized in that, the enzymolysis product obtained by enzyme digestion reaction, is dissolved in weakly polar organic solvent, filters, and collects filtrate, adds polar solvent, crystallization in filtrate.
5. method according to claim 4, it is characterized in that, described weakly polar organic solvent is one or more in ethyl acetate, acetone, methylene dichloride and methyl tertiary butyl ether, and described polar solvent is one or more in methyl alcohol, second alcohol and water; Most preferably, described weakly polar organic solvent is acetone, and described polar solvent is water.
6. method according to claim 4, is characterized in that, the method comprises the following steps:
A. the enzymolysis product obtained by enzyme digestion reaction is centrifugal;
B. acetone is added by the centrifugal precipitation obtained;
C. again acetone is filtered in the filtrate obtained and add distilled water, reflux;
D. by the liquid at room temperature crystallization obtained that refluxes.
Preferably, the filtrate in step c and the volume ratio of distilled water are 1-5:1, and described reflux temperature is 50-100 DEG C.
7. method according to claim 3, is characterized in that, Herba Epimedii extract liquid concentration, is then adsorbed by macroporous resin column, then carries out wash-out with the aqueous ethanolic solution that volumetric concentration is 30%-100%, obtains the Herba Epimedii extract for enzyme digestion reaction; Preferably, be 20%-100% for the mass content of total flavones in the Herba Epimedii extract of enzyme digestion reaction.
8. method according to claim 3, is characterized in that, the volume ratio of described Herba Epimedii quality and ethanolic soln is 1 kilogram: 2-50 liter, and Extracting temperature is 30-100 DEG C; Preferably, the aqueous ethanolic solution of described ethanolic soln to be volume ratio be 20-90%.
9. method according to claim 1, it is characterized in that, the condition of described enzyme digestion reaction is: Herba Epimedii extract and phosphoric acid buffer are made the substrate solution that pH value is 4.0-6.0, polygalacturonase is added in substrate solution, the quality of polygalacturonase is 5-30 times of Herba Epimedii extract quality, and hydrolysis temperature is 45-55 DEG C; Preferably, the quality of polygalacturonase is 10-20 times of Herba Epimedii extract quality, and the pH value of substrate solution is 4.5-5.5, and hydrolysis temperature is 48-50 DEG C; Most preferably, the quality of described polygalacturonase is 15 times of Herba Epimedii extract quality, the ethanol of the 10-15% containing its cumulative volume in substrate solution.
10. method according to claim 9, is characterized in that, the pH value of substrate solution is regulated by phosphoric acid buffer, and preferably, described phosphoric acid buffer is phosphoric acid-citrate buffer solution or Sodium phosphate dibasic-potassium phosphate buffer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106148454A (en) * | 2015-03-24 | 2016-11-23 | 北京珅奥基医药科技有限公司 | A kind of preparation method of baohuoside Ⅰ |
| CN109459515A (en) * | 2018-07-05 | 2019-03-12 | 广州卡马生物科技有限公司 | A kind of Herba Epimedii reference extract (arrow leaf) and its application |
| CN110699263A (en) * | 2019-10-29 | 2020-01-17 | 浙江工业大学 | Aspergillus niger YH-6 and application thereof in improving content of icaritin in epimedium |
| CN113355373A (en) * | 2021-06-29 | 2021-09-07 | 劲牌持正堂药业有限公司 | Preparation method of low polycyclic aromatic hydrocarbon icaritin |
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| CN101302548A (en) * | 2007-05-09 | 2008-11-12 | 北京珅奥基医药科技有限公司 | Preparation of icaritin |
| CN101316573A (en) * | 2005-11-30 | 2008-12-03 | 株式会社太平洋 | Cosmetic composition containing hydrolysates of icariin |
| CN103305564A (en) * | 2013-07-05 | 2013-09-18 | 西安纽赛生物科技有限公司 | Method for converting icariin to herba epimedii |
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2013
- 2013-12-13 CN CN201310674836.5A patent/CN104711300B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101316573A (en) * | 2005-11-30 | 2008-12-03 | 株式会社太平洋 | Cosmetic composition containing hydrolysates of icariin |
| CN101302548A (en) * | 2007-05-09 | 2008-11-12 | 北京珅奥基医药科技有限公司 | Preparation of icaritin |
| CN103305564A (en) * | 2013-07-05 | 2013-09-18 | 西安纽赛生物科技有限公司 | Method for converting icariin to herba epimedii |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148454A (en) * | 2015-03-24 | 2016-11-23 | 北京珅奥基医药科技有限公司 | A kind of preparation method of baohuoside Ⅰ |
| CN106148454B (en) * | 2015-03-24 | 2021-01-26 | 北京珅奥基医药科技有限公司 | Preparation method of baohuoside I |
| CN109459515A (en) * | 2018-07-05 | 2019-03-12 | 广州卡马生物科技有限公司 | A kind of Herba Epimedii reference extract (arrow leaf) and its application |
| CN110699263A (en) * | 2019-10-29 | 2020-01-17 | 浙江工业大学 | Aspergillus niger YH-6 and application thereof in improving content of icaritin in epimedium |
| CN113355373A (en) * | 2021-06-29 | 2021-09-07 | 劲牌持正堂药业有限公司 | Preparation method of low polycyclic aromatic hydrocarbon icaritin |
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Application publication date: 20150617 Assignee: Beijing Shengnuoji Medicine Technology Co., Ltd. Assignor: Beijing Kun'aoji Medical Sci-tech Co., Ltd. Contract record no.: 2017110000017 Denomination of invention: Preparation of icaritin License type: Exclusive License Record date: 20170919 |
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Assignee: BEIJING SHENOGEN PHARMA Group Ltd. Assignor: BEIJING SHENOGEN BIOMEDICAL Co.,Ltd. Contract record no.: 2017110000017 Date of cancellation: 20200714 |