CN107964555B - Method for biological enrichment and production of icariside II - Google Patents
Method for biological enrichment and production of icariside II Download PDFInfo
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- CN107964555B CN107964555B CN201810012075.XA CN201810012075A CN107964555B CN 107964555 B CN107964555 B CN 107964555B CN 201810012075 A CN201810012075 A CN 201810012075A CN 107964555 B CN107964555 B CN 107964555B
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention provides a method for producing icariside II by biological enrichment, which comprises the following steps: firstly preparing a PDA solid culture medium to culture trichoderma viride, then inoculating the cultured trichoderma viride to a liquid culture medium containing icariin for fermentation culture, and obtaining icariside II enriched in the trichoderma viride. The Trichoderma viride used in the method has convenient source, is easy to propagate, and does not need to extract hydrolase; the fermentation equipment is common, and the subsequent purification method is simple. Extracting with low boiling point solvent, concentrating easily, extracting, soaking, filtering, adsorbing, chromatography, and recrystallizing to obtain high purity product. Is suitable for industrial production and has low production cost.
Description
Technical Field
The invention belongs to the field of medicines, and relates to a method for enriching icariside II by using Trichoderma viride organisms.
Background
Herba Epimedii(Epimedii Folium) Is berberidaceae (Berbridaceae) Epimedium genus (Epimedium) The plant is a common Chinese traditional medicine, and has the medicinal value of nourishing yin, tonifying yang, strengthening yang and strengthening body. Icariin (Icariin, ICA; molecular formula: C)33H40O15(ii) a Molecular weight: 676.77) is the main component, and the content can reach 1%. Recent studies have shown that ICA is hydrolyzed to Icariside II (Icariside II, ICA II; formula: C) under intestinal cellulase catalysis after oral administration27H32O10(ii) a Molecular weight: 514.54) and absorbed into the blood. Pharmacokinetic studies suggest that ICAII is the major active agent of ICAThe product is obtained by the process of production. Therefore, the method for preparing icariside II with low cost and high efficiency has medical value.
The prior art uses glycosyl hydrolase extracted or purified (Quan Xia 2009 Fitotterapia 81 (2010) 437-442; Zhang Zhenhai 2011 China pharmacy Vol 22, No 43) to enzymolyze icariin to obtain icariside II. The chinese patent application No. 201610289040.1 discloses a process for producing icariside II, which comprises extracting icariin monomer from herba Epimedii, and hydrolyzing the icariin monomer with glycosyl hydrolase with high catalytic activity to obtain icariside II. However, because icariin is hardly dissolved in water, and the solvent is obtained on the premise of hydrolyzing the icariin, the existing hydrolysis technology has large reaction volume and low concentration of raw materials and products, and cannot realize industrial application.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a method for enriching icariside II by using Trichoderma viride organisms.
The technical scheme adopted by the invention is as follows: the invention provides a method for biotransformation of icariin into icariside II by trichoderma viride, which comprises the following steps: firstly, trichoderma viride is cultured in a trichoderma culture medium, then the cultured trichoderma viride is inoculated into a liquid culture medium containing icariin for fermentation culture, the icariin is hydrolyzed and converted to obtain icariside II, and finally, the icariside II is separated, extracted and purified to obtain a pure product of the icariside II. The Trichoderma viride used in the method has convenient source, is easy to propagate, and does not need to extract hydrolase; the fermentation equipment is common, the subsequent purification method is simple, the concentration is easy due to the extraction of a low-boiling-point solvent, a high-purity product is obtained only by extraction soaking, filtration, adsorption chromatography and recrystallization, and the method is suitable for industrial production and low in production cost.
Further, the method for enriching icariside II by using Trichoderma viride organisms comprises the steps of firstly preparing a PDA solid culture medium to culture Trichoderma viride, and then inoculating the cultured Trichoderma viride to a liquid culture medium containing icariin for fermentation culture. The trichoderma viride can transport icariin into cells, complete hydrolysis by hydrolase in the cells, and enrich hydrolysate, so that industrial preparation of icariside II becomes possible.
The method specifically comprises the following steps: a. culturing Trichoderma viride in a small test tube culture medium in an inclined plane; b. inoculating the cultured trichoderma viride into a liquid culture medium containing epimedium for fermentation culture; c. when icariin is completely converted and hydrolyzed, filtering the fermentation liquor to obtain filter residue containing trichoderma viride and icariside II (and a small amount of icariin); d. soaking the residue in methanol, filtering again to obtain filtrate containing icariside II (and small amount of icariin), and repeating the process until all icariside II and small amount of icariin are dissolved in methanol solution; e. detecting with high performance liquid chromatography, and purifying by silica gel adsorption column chromatography if the conversion rate of icariside II is more than 50% and less than 85%; f. and e, further purifying the hypo-glycoside II which is subjected to silica gel column in the step e and has the conversion rate of more than 85% by using a recrystallization method to obtain a pure icariside II product.
Further, the steps of preparing the culture medium and culturing the trichoderma viride are as follows: firstly, preparing a PDA solid culture medium by using potatoes and glucose, and culturing trichoderma viride in the PDA solid culture medium for 3-5 days under the culture condition of 26-29 ℃ to obtain the trichoderma viride which is high-activity thallus.
Further, the method for culturing trichoderma viride by using the PDA solid culture medium is characterized by specifically adopting slant culture in a small test tube culture medium. The preparation method of the PDA solid culture medium comprises the following steps: weighing 200g of potato and 1L of distilled water, adding distilled water into the potato, boiling the potato until the potato is soft and rotten, filtering, heating the filtrate, adding 20g of glucose and 15-20g of agar, subpackaging the dissolved potato in small test tubes, sterilizing at the temperature of 121 ℃ for 15-20 minutes, taking out the potato, shaking uniformly, placing the potato in an inclined manner at an angle of 45 ℃, and cooling to obtain the PDA solid culture medium.
Further, the preparation method of the icariin-containing liquid culture medium comprises the following steps: adding 0.5-10g of crushed potato and 0.8-16g of icariin into a 0.1L-3L triangular conical flask, adding distilled water to 0.05-1L, fastening the bottle mouth with a sealing film, placing in a sterilizing box at 121 deg.C, sterilizing for 15-20 min, cooling to 20-30 deg.C to obtain liquid culture medium containing icariin.
Preferably, the icariin-containing liquid culture medium is prepared by mashing and sterilizing fresh potatoes in a ratio of 10g of fresh potatoes, 1L of distilled water and 16g of epimedium herb.
Further, the fermentation culture process comprises the following steps: inoculating the cultured trichoderma viride to a liquid culture medium containing icariin on an ultra-clean workbench, culturing for 9 days in a shaking table at the temperature of 28 ℃ and the rotating speed of 165r/min, filtering the fermentation liquor, soaking the filtered filter residue in methanol, filtering again, and repeating the process for 2-5 times until the trichoderma viride is soaked to be white and icariside II is completely dissolved in the methanol; and concentrating the methanol soak solution to obtain powder containing icariside II, and purifying the powder to obtain a pure product of the icariside II. Icariside II and icariin have the best solubility in methanol, and Trichoderma viride does not dissolve in methanol and Trichoderma viride can be separated from the icariside II and icariin.
Further, the inoculation, i.e., 16g of icariin, required 4 small test tubes (1.2 cm in diameter) of Trichoderma for 9 days, at which the conversion rate was the highest.
Further, the method for purifying the powder containing icariside II comprises the following steps: firstly, detecting the conversion rate of icariside II by liquid phase detection of powder containing icariside II, mixing the mixed powder with the conversion rate of icariside II being more than 50 percent and less than 85 percent by a dry method through macroporous resin DM301 of 1.5-2 times, filling 200-mesh and 300-mesh industrial chromatographic silica gel of 6-8 times into an upper column, and using dichloromethane: the volume ratio of methanol is 15: 1, eluting with a mixed eluent of pure methanol, monitoring effluent liquid by thin-layer chromatography, mixing single-component solutions containing icariside II, concentrating the mixed solution into powder at 60 ℃ under reduced pressure, dissolving the powder with the conversion rate of more than 85% by using methanol at 65-70 ℃ by using a heating reflux method, removing the methanol solution containing the icariside II after dissolving, standing at normal temperature, separating out crystals, filtering the crystals, and drying under vacuum at the temperature of below 45 ℃ to constant weight to obtain the pure product of the icariside II with the content of more than 99%. When the conversion rate is between 50% and 85%, the icariside II is purified by recrystallization after being purified by silica gel adsorption column chromatography, and when the conversion rate is more than 85%, the icariside II is directly purified by recrystallization, so that the quality of the prepared icariside II can be effectively ensured.
The invention has the beneficial effects that: according to the method for producing icariside II by utilizing trichoderma viride biological enrichment, trichoderma viride is used, icariin can be transferred into cells, hydrolysis is completed by hydrolase in the cells, hydrolysis products are enriched, industrial preparation of icariside II is possible, the content of the prepared icariside II is more than 99%, and the yield is more than 91%. The method has the advantages of simple operation, easy implementation, low cost, high yield, no pollution, suitability for industrial large-scale production and extremely high application prospect.
Drawings
FIG. 1 shows the detection result of high performance liquid chromatography in step e of example 1 according to the present invention;
FIG. 2 shows the detection result of high performance liquid chromatography in step f of example 1;
FIG. 3 shows the detection result of high performance liquid chromatography in step e of example 2 according to the present invention;
FIG. 4 shows the detection result of high performance liquid chromatography in step f of example 2 according to the present invention.
Detailed Description
The invention will be further elucidated by means of several specific examples, which are intended to be illustrative only and not limiting.
Example 1:
a method for obtaining icariside II by biotransformation and enrichment of 8g of icariin comprises the following steps:
a. propagating the trichoderma viride which is frozen and stored at 4 ℃ in a potato culture medium for 4 days at the propagation culture temperature of 28 ℃, and freezing and storing the trichoderma viride in a refrigerator at 4 ℃ until the slant of the test tube is full of thalli; the potato culture medium is prepared by adding distilled water to fresh potatoes, anhydrous glucose and agar, namely 200g of fresh potatoes is firstly boiled to be soft and rotten by an iron pot with the distilled water, the mixture is filtered, filtrate is heated again, 20g of the anhydrous glucose and 18g of the agar are added, the mixture is subpackaged into small test tubes with the diameter of 1.2cm after being dissolved, the height of the culture medium does not exceed 1/3 of the total height of the test tubes, the test tubes are plugged, each 7 test tubes are bundled, the test tubes are wrapped by kraft paper, the mixture is sterilized for 20 minutes at the temperature of 121 ℃, taken out and shaken uniformly, placed at an inclined angle of 45 degrees, cooled and stored for later use;
b. adding 0.5g of potatoes (broken) and 0.8g of icariin into a 100mL conical flask, adding distilled water to 50mL, fastening the mouth of the conical flask by using a sealing film, putting the conical flask into a 10 ℃ sterilizing box, sterilizing for 20 minutes, cooling to about 25 ℃, inoculating the trichoderma viride of 1 test tube cultured in the step a into a liquid culture medium of the conical flask on an ultra-clean workbench, and culturing for 9 days by a shaking table at the temperature of 28 ℃ and the rotating speed of 165 r/min;
c. after culturing for 9 days, filtering out the mixture containing trichoderma viride, water-insoluble icariside II and icariin (a small amount) by using a suction filtration device;
d. dissolving the icariside II and the icariin filtered out in the step c by using pure methanol until the trichoderma viride is soaked to be white, and dissolving the icariside II and the icariin in the methanol;
soaking in methanol for 3 times, and filtering out Trichoderma viride in the amount of 1000ml, 500ml and 500ml respectively.
e. Concentrating the methanol soak solution in the step d to obtain mixture powder mainly containing icariside II, and weighing 6.2430 g; the conversion was measured by passing through the liquid phase, and the measured result was calculated to be 91.56% (FIG. 1);
f. and e, dissolving the powder in the step e by using a heating reflux method at the temperature of 65-70 ℃ with minimum methanol, removing a methanol solution containing icariside II after dissolution, standing at normal temperature, filtering to obtain crystals, continuously standing at normal temperature, filtering to obtain crystals after the crystals are separated out for the second time, filtering to obtain the crystals, combining the crystals obtained by the first filtering, and drying in vacuum at the temperature of below 45 ℃ to constant weight to obtain 5.5371g of the icariside II pure product with the content of 99%, wherein the yield is 91.07% (figure 2).
Example 2:
a method for obtaining icariside II by biotransformation and enrichment of 192g icariin comprises the following steps:
a. propagating the trichoderma viride which is frozen and stored at 4 ℃ in a potato culture medium for 4 days at the propagation culture temperature of 28 ℃, and freezing and storing the trichoderma viride in a refrigerator at 4 ℃ until the slant of the test tube is full of thalli; the potato culture medium is prepared by adding distilled water to fresh potatoes, anhydrous glucose and agar, namely 200g of fresh potatoes is firstly boiled to be soft and rotten by an iron pot with the distilled water, the mixture is filtered, filtrate is heated again, 20g of the anhydrous glucose and 18g of the agar are added, the mixture is subpackaged into small test tubes with the diameter of 1.2cm after being dissolved, the height of the culture medium does not exceed 1/3 of the total height of the test tubes, the test tubes are plugged, each 7 test tubes are bundled, the test tubes are wrapped by kraft paper, the mixture is sterilized for 20 minutes at the temperature of 121 ℃, taken out and shaken uniformly, placed at an inclined angle of 45 degrees, cooled and stored for later use;
b. adding 10g of potatoes (broken) and 16g of icariin into a 3L triangular conical flask, adding distilled water to 1L, fastening the bottle mouth with a sealing film, putting the bottle into a sterilization box at 121 ℃, sterilizing for 20 minutes, cooling to about 25 ℃, inoculating the trichoderma viride of 6 test tubes cultured in the step a into a liquid culture medium of the triangular conical flask on an ultra-clean workbench, and culturing for 9 days in a shaking table at 28 ℃ and the rotating speed of 165 r/min;
c. after culturing for 9 days, filtering out the mixture containing trichoderma viride, water-insoluble icariside II and icariin (a small amount) by using a suction filtration device;
d. dissolving the icariside II and the icariin filtered out in the step c by using pure methanol until the trichoderma viride is soaked to be white, and dissolving the icariside II and the icariin in the methanol;
soaking in methanol for 3 times, respectively in the amount of 36L, 18L, and 18L to filter out Trichoderma viride.
e. Concentrating the methanol soak solution in the step d to obtain mixture powder mainly containing icariside II, and weighing 143.5265 g; the conversion was measured by passing through the liquid phase, and the measured result was calculated to be 93.12% (FIG. 3);
f. and e, dissolving the powder in the step e by using a heating reflux method at the temperature of 65-70 ℃ with minimum methanol, removing a methanol solution containing icariside II after dissolution, standing at normal temperature, filtering to obtain crystals, continuously standing at normal temperature, filtering to obtain crystals after the crystals are separated out for the second time, filtering to obtain the crystals, combining the crystals obtained by the first filtering, and drying in vacuum at the temperature of below 45 ℃ to constant weight to obtain 133.4286g of icariside II pure product with the product content of 99.12 percent and the yield of 91.44 percent (figure 4).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.
Claims (1)
1. A method for enriching and producing icariside II by using Trichoderma viride organisms is characterized by comprising the following steps:
(a) weighing 200g of potato and 1L of distilled water, adding distilled water into the potato, boiling the potato until the potato is soft and rotten, filtering, heating the filtrate, adding 20g of glucose and 15-20g of agar, subpackaging the dissolved potato into small test tubes, sterilizing at the temperature of 121 ℃ for 15-20 minutes, taking out the potato, shaking uniformly, placing the potato in an inclined manner at an angle of 45 ℃, and cooling to obtain a PDA solid culture medium; culturing Trichoderma viride in PDA solid culture medium at 26-29 deg.C for 3-5 days;
(b) adding 10g of crushed potato and 16g of icariin into 1L of distilled water, sealing, placing in a sterilization box at 121 ℃, sterilizing for 15-20 minutes, and cooling to obtain a liquid culture medium; adding the trichoderma viride cultured in the step (a) into a liquid culture medium in a sterile environment, and culturing for 9 days in a shaking table at the temperature of 28 ℃ and the rotating speed of 165 r/min;
(c) filtering the fermentation culture solution: filtering the filter residue containing trichoderma viride, water-insoluble icariside II and icariin by using a suction filtration device to obtain trichoderma viride thallus enriched with icariside II;
(d) and (3) detecting the enrichment degree: soaking the residue in methanol, filtering again, repeating for 2-5 times, collecting filtrate, concentrating to obtain concentrated powder, and detecting icariside II content in the powder;
(e) when the conversion rate of icariside II in the concentrated powder is detected to be more than 50 percent and less than 85 percent, the concentrated powder is purified by silica gel adsorption column chromatography to obtain purified powder; the silica gel adsorption column chromatography purification comprises the following steps: mixing the concentrated powder with 1.5-2 times of macroporous resin by a dry method, filling the concentrated powder into an upper column by 6-8 times of 200-mesh 300-mesh chromatographic silica gel, eluting by using an eluent, and firstly, using a solvent with a volume ratio of 15: 1 dichloromethane: eluting icariside II with methanol, eluting icariside with methanol after icariside II is completely eluted, monitoring the effluent by thin layer chromatography, mixing the effluent, and concentrating under reduced pressure at 60 deg.C to obtain the purified powder;
(f) recrystallizing and purifying the purified powder to obtain a pure product of icariside II; the recrystallization purification comprises the following steps: dissolving with methanol at 65-70 deg.C by heating and refluxing, removing methanol solution containing icariside II, standing at room temperature, filtering to obtain crystal, standing at room temperature again, filtering to obtain crystal, mixing the crystals obtained by multiple filtering, vacuum drying at 45 deg.C below to constant weight to obtain icariside II pure product with content of more than 99%;
(g) when the conversion rate of the icariside II in the concentrated powder is detected to be more than 85 percent, the concentrated powder is recrystallized and purified to obtain a pure product of the icariside II; the recrystallization purification comprises the following steps: dissolving in methanol at 65-70 deg.C by heating and refluxing, removing methanol solution containing icariside II, standing at room temperature, filtering to obtain crystal, standing at room temperature again, filtering to obtain crystal, mixing the crystals obtained by multiple filtering, vacuum drying at 45 deg.C or below to constant weight to obtain icariside II pure product with content of 99% or above.
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| WO2007064085A1 (en) * | 2005-11-30 | 2007-06-07 | Amorepacific Corporation | Cosmetic composition containing hydrolysates of icariin |
| CN101516325A (en) * | 2006-09-19 | 2009-08-26 | 株式会社太平洋 | Method for preparing icariside II, cosmetic composition containing the same and the use thereof for skin whitening |
| CN103305572A (en) * | 2013-05-31 | 2013-09-18 | 西昌丹阳生物科技有限责任公司 | Method for producing baohuoside I through herba epimdii |
| CN105925635A (en) * | 2016-05-04 | 2016-09-07 | 宁波旋光医药科技有限公司 | Production process of icariside II |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007064085A1 (en) * | 2005-11-30 | 2007-06-07 | Amorepacific Corporation | Cosmetic composition containing hydrolysates of icariin |
| CN101516325A (en) * | 2006-09-19 | 2009-08-26 | 株式会社太平洋 | Method for preparing icariside II, cosmetic composition containing the same and the use thereof for skin whitening |
| CN103305572A (en) * | 2013-05-31 | 2013-09-18 | 西昌丹阳生物科技有限责任公司 | Method for producing baohuoside I through herba epimdii |
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Effective date of registration: 20221222 Address after: 102600 Beijing city Daxing District Huangcun emperor Road No. 12 Patentee after: BEIJING DONGFANG BAIAO MEDICAL DEVELOPMENT Co.,Ltd. Address before: 215400 Biological Medicine Industrial Park, Shaxi Town, Taicang City, Suzhou City, Jiangsu Province Patentee before: SUZHOU GUANG'AO HEALTHCARE Co.,Ltd. |