CN103194502B - Separating and purifying method of taxol synthesized by biological fermentation of nodulisporium sylviforme entophytic fungi as well as precursors of taxol - Google Patents
Separating and purifying method of taxol synthesized by biological fermentation of nodulisporium sylviforme entophytic fungi as well as precursors of taxol Download PDFInfo
- Publication number
- CN103194502B CN103194502B CN201310146249.9A CN201310146249A CN103194502B CN 103194502 B CN103194502 B CN 103194502B CN 201310146249 A CN201310146249 A CN 201310146249A CN 103194502 B CN103194502 B CN 103194502B
- Authority
- CN
- China
- Prior art keywords
- taxol
- methyl alcohol
- acetone
- fermentation
- ethyl acetate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a separating and purifying method of taxol synthesized by biological fermentation of nodulisporium sylviforme entophytic fungi as well as precursors of the taxol, relates to a separating and purifying method and aims at solving the problems that taxol in fermentation liquor is low in content and the taxol precursors are low in the recovery rate and low in purity in the traditional method for synthesizing by biological fermentation of entophytic fungi. The separating and purifying method disclosed by the invention comprises the following steps of: activating a bacterial strain and culturing seeds; 2, carrying out fermentation culture; 3, separating and recovering fermentation liquor and mycelium, decoloring the fermentation liquor, and obtaining a sample through extracting and concentrating; 4, carrying out primary chromatography; 5, carrying out secondary chromatography; and 6, separating by using a reversed phase chromatography method to finish separation and purification. According to the separating and purifying method, the content of taxol in the fermentation liquor is 556.80mug/L, the taxol purity is 98 percent, the precursor baccatin purity is 97 percent, and the precursor cephalomannine purity is 98 percent; the separating and purifying method has the characteristics of simple operation process, high product purity and low use amount of a solvent; and meanwhile, the production cost of the taxol is indirectly reduced.
Description
Technical field
The present invention relates to the isolation and purification method of treelike menu endogenetic fungus HDFS4-26 biological fermentation taxol biosynthesis and precursor thereof.
Background technology
Malignant tumour seriously threatens the health of the mankind, and cancer is the second largest cause of the death being only second to cardio-cerebrovascular disorder in current human diseases.
Taxol is the diterpene alkaloid class medicine with multiple antitumor curative effect extracted from Chinese yew genus plants.Because Ramulus et folium taxi cuspidatae (having another name called Japanese yew) is rare tree species, the speed of growth is slow, and its biological characteristics requires higher to ecotope, natural regeneration ability, so population quantity is very limited, belong to imminent extinction protected plants, resource is very limited.Therefore, Chinese scholars has made many-sided effort in the problem of research and development taxol source new drugs.
The antitumour activity of taxol uniqueness is approved by the world, and its natural resource Chinese yew is deficient and the execution of restriction collection measure, certainly will cause studying the new resources of taxol and analogue thereof and alternate resources.Production by Microorganism Fermentation taxol reduces one of production cost, the most promising method obtaining taxol in a large number.
Taxol Producion by Microbe Fermentation is utilized to have following advantages:
(1) microorganism is as industrial source, can carry out in fermentor tank, can carrying out not to the utmost produce in a steady stream;
(2) method of fermentable is easily magnified, and is beneficial to suitability for industrialized production;
(3) microbial growth only needs general culture technique, collection taxol before, can by improving culture environment, improvement opportunity improves output;
(4) microorganism is easily through method screening superior strains such as genetically engineereds, improves the output of taxol;
(5) endogenetic fungus growth rapidly, and be easy to cultivate, the substratum of application is relatively cheap;
(6) be expected the demand meeting market, reduce the price of taxol.Plant endogenesis epiphyte is the resource microorganism that a class has a extensive future, and produces taxol by the method for fermentable, is the effective way solving medicine source problem.
But it is low to there is content of taxol in fermented liquid in the method for current endogenetic fungus biological fermentation taxol biosynthesis, the problem that the paclitaxel precursor thing rate of recovery is low and purity is low.
Summary of the invention
It is low to there is content of taxol in fermented liquid in the method that the present invention seeks to solve existing endogenetic fungus biological fermentation taxol biosynthesis, the problem that the paclitaxel precursor thing rate of recovery is low and purity is low, and the isolation and purification method of treelike menu endogenetic fungus HDFS4-26 biological fermentation taxol biosynthesis and precursor thereof is provided.
The isolation and purification method of treelike menu endogenetic fungus HDFS4-26 biological fermentation taxol biosynthesis and precursor thereof, realizes according to the following steps:
One, bacterial strain activation and seed culture: treelike menu HDFS4-26 is inoculated on PDA slant medium, 2 ~ 3d is activated under the condition of 28 DEG C, then the treelike menu HDFS4-26 after activation is connected in PDA liquid nutrient medium, at 28 DEG C, 120r/min shaking table cultivation 3d, obtain seed culture fluid;
Two, fermentation culture: seed culture fluid is proceeded in the S-7 liquid nutrient medium of improvement with the inoculum size of volume ratio 5%, at 28 DEG C, 120r/min fermentation culture 12d, wherein add the sucrose of 4g/L and the VITAMIN concentrated solution of 3mL/L at the 4d of fermentation culture, the peptone of 0.4g/L and the soya-bean cake hydrolyzed solution of 0.2mL/L is added at the 6d of fermentation culture, add the sucrose of 2g/L and the styracin of 20mg/L at the 7d of fermentation culture, add the amphotericin B of 28 μ g/L at the 9d of fermentation culture;
Three, after step 2 fermentation ends, separate fermentation liquid and mycelium also reclaim, and with sherwood oil to fermented liquid decolouring twice, then add isopyknic ethyl acetate and methyl alcohol mixed liquor extracted overnight, separate organic phase, again add isopyknic ethyl acetate and methyl alcohol mixed liquor extracted overnight, separate organic phase, then merge organic phase, again with Rotary Evaporators removing ethyl acetate and methyl alcohol, to remaining 5mL solution, solution is poured in plate and dries, obtain sample;
Four, a chromatography: sample is dissolved in chloroform by mass volume ratio 1:10, add the diatomite of 5 times amount, mix rear natural air drying thoroughly, after dress post, use chloroform, chloroform successively: methyl alcohol=98:2, chloroform: methyl alcohol=87:3, chloroform: methyl alcohol=90:10 and chloroform: methyl alcohol=60:40 carries out wash-out, TLC detects, merge the fraction containing target product taxol, after evaporate to dryness, be dissolved in methyl alcohol in 8mL/g ratio, heating in water bath is to 50 DEG C, simultaneously, the distilled water of instillation 1/3rd methyl alcohol volumes when stirring, cooling 48 ~ 72h, obtains primary crystallization sample;
Five, secondary chromatography: the length-to-diameter ratio of chromatographic column is at more than 8:1, dry column-packing, the mass ratio of stationary phase silica gel and primary crystallization sample is 30:l, elution mode is hexane/ethyl acetate 60/40(3BV)-55/45(2BV)-50/50(5BV), TLC detects, merge the fraction containing target product taxol, after evaporate to dryness, be dissolved in methyl alcohol in 8mL/g ratio, heating in water bath, to 50 DEG C, meanwhile, instills the distilled water of 1/3rd methyl alcohol volumes when stirring, cooling 48 ~ 72h, obtains secondary crystal sample;
Six, reversed phase chromatography separation: with PRP-6 resin (polystyrene-divinylbenzene porous microsphere) for stationary phase, cylinder is shaping uses 2BV acetone respectively afterwards, ethyl acetate, acetone, 20% acetone-water balances, after adding secondary crystal sample, be the acetone-water system of 20% with volume content successively, volume content is the acetone-water system of 40%, volume content is the acetone-water system of 45%, volume content is the acetone-water system of 50%, volume content is the acetone-water system of 55%, volume content be 60% acetone-water system and acetone system carry out gradient elution, TLC detects, merge identical cut, concentrated, crystallization, namely the isolation and purification method of treelike menu endogenetic fungus HDFS4-26 biological fermentation taxol biosynthesis and precursor thereof is completed,
Wherein in step 3, ethyl acetate and methyl alcohol mixed liquor mix according to volume ratio 1:1.
The method of treelike menu endogenetic fungus HDFS4-26 biological fermentation taxol biosynthesis in the present invention, in fermented liquid, content of taxol is 556.80 μ g/L, and taxol purity is 98%, and precursor Bakating III purity is 97%, Cephalomannine purity is 98%.
Taxol of the present invention and precursor isolation and purification method thereof have the advantages that operating process is simple, product purity is high, solvent usage quantity is little; Meanwhile, for utilizing chemosynthesis taxol to provide precursor substance, indirectly can reduce the production cost of taxol, realizing the suitability for industrialized production of taxol as early as possible.
Accompanying drawing explanation
Fig. 1 is the chemical structural drawing of taxol;
Fig. 2 is the ESI-MS collection of illustrative plates of taxol;
Fig. 3 is taxol
1h-NMR collection of illustrative plates;
Fig. 4 is taxol
13c-NMR collection of illustrative plates;
Fig. 5 is the chemical structural drawing of Bakating III;
Fig. 6 is the ESI-MS collection of illustrative plates of Bakating III;
Fig. 7 is Bakating III
1h-NMR collection of illustrative plates;
Fig. 8 is Bakating III
13c-NMR collection of illustrative plates;
Fig. 9 is the chemical structural drawing of Cephalomannine;
Figure 10 is the ESI-MS collection of illustrative plates of Cephalomannine;
Figure 11 is Cephalomannine
1h-NMR collection of illustrative plates;
Figure 12 is Cephalomannine
13c-NMR collection of illustrative plates.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the isolation and purification method of present embodiment treelike menu endogenetic fungus HDFS4-26 biological fermentation taxol biosynthesis and precursor thereof, realizes according to the following steps:
One, bacterial strain activation and seed culture: treelike menu HDFS4-26 is inoculated on PDA slant medium, 2 ~ 3d is activated under the condition of 28 DEG C, then the treelike menu HDFS4-26 after activation is connected in PDA liquid nutrient medium, at 28 DEG C, 120r/min shaking table cultivation 3d, obtain seed culture fluid;
Two, fermentation culture: seed culture fluid is proceeded in the S-7 liquid nutrient medium of improvement with the inoculum size of volume ratio 5%, at 28 DEG C, 120r/min fermentation culture 12d, wherein add the sucrose of 4g/L and the VITAMIN concentrated solution of 3mL/L at the 4d of fermentation culture, the peptone of 0.4g/L and the soya-bean cake hydrolyzed solution of 0.2mL/L is added at the 6d of fermentation culture, add the sucrose of 2g/L and the styracin of 20mg/L at the 7d of fermentation culture, add the amphotericin B of 28 μ g/L at the 9d of fermentation culture;
Three, after step 2 fermentation ends, separate fermentation liquid and mycelium also reclaim, and with sherwood oil to fermented liquid decolouring twice, then add isopyknic ethyl acetate and methyl alcohol mixed liquor extracted overnight, separate organic phase, again add isopyknic ethyl acetate and methyl alcohol mixed liquor extracted overnight, separate organic phase, then merge organic phase, again with Rotary Evaporators removing ethyl acetate and methyl alcohol, to remaining 5mL solution, solution is poured in plate and dries, obtain sample;
Four, a chromatography: sample is dissolved in chloroform by mass volume ratio 1:10, add the diatomite of 5 times amount, mix rear natural air drying thoroughly, after dress post, use chloroform, chloroform successively: methyl alcohol=98:2, chloroform: methyl alcohol=87:3, chloroform: methyl alcohol=90:10 and chloroform: methyl alcohol=60:40 carries out wash-out, TLC detects, merge the fraction containing target product taxol, after evaporate to dryness, be dissolved in methyl alcohol in 8mL/g ratio, heating in water bath is to 50 DEG C, simultaneously, the distilled water of instillation 1/3rd methyl alcohol volumes when stirring, cooling 48 ~ 72h, obtains primary crystallization sample;
Five, secondary chromatography: the length-to-diameter ratio of chromatographic column is at more than 8:1, dry column-packing, the mass ratio of stationary phase silica gel and primary crystallization sample is 30:l, elution mode is hexane/ethyl acetate 60/40(3BV)-55/45(2BV)-50/50(5BV), TLC detects, merge the fraction containing target product taxol, after evaporate to dryness, be dissolved in methyl alcohol in 8mL/g ratio, heating in water bath, to 50 DEG C, meanwhile, instills the distilled water of 1/3rd methyl alcohol volumes when stirring, cooling 48 ~ 72h, obtains secondary crystal sample;
Six, reversed phase chromatography separation: with PRP-6 resin (polystyrene-divinylbenzene porous microsphere) for stationary phase, cylinder is shaping uses 2BV acetone respectively afterwards, ethyl acetate, acetone, 20% acetone-water balances, after adding secondary crystal sample, be the acetone-water system of 20% with volume content successively, volume content is the acetone-water system of 40%, volume content is the acetone-water system of 45%, volume content is the acetone-water system of 50%, volume content is the acetone-water system of 55%, volume content be 60% acetone-water system and acetone system carry out gradient elution, TLC detects, merge identical cut, concentrated, crystallization, namely the isolation and purification method of treelike menu endogenetic fungus HDFS4-26 biological fermentation taxol biosynthesis and precursor thereof is completed,
Wherein in step 3, ethyl acetate and methyl alcohol mixed liquor mix according to volume ratio 1:1.
Treelike menu (Nodulisporium sylviforme) HDFS4-26 in present embodiment step one, open in Chinese patent " paclitaxel genome rearrangement strain HD FS4-26(patent No. ZL200810064048.3; applying date 2008.2.29) ", its bacterial strain is preserved in China typical culture collection center, preservation date is 2008.2.28, and preserving number is CCTCC M:208026.
The S-7 liquid nutrient medium improved in present embodiment step 2 in 1993, phenylalanine, tyrosine, linolic acid is increased to final concentration 1.5 ~ 5.0mg/L and make by the former S-7 medium base of Strobel.
The diatomite adding 5 times amount in present embodiment step 4 is for benchmark with medicinal extract sample (i.e. the total mass of sample and chloroform) quality.
In present embodiment, primary crystallization sample is needle-like crystal, and secondary crystal system is methanol/water.
In present embodiment step 4, gained taxol purity can reach 50% ~ 70%, the rate of recovery more than 95%.Mother liquor ethyl acetate after primary crystallization is extracted, dehydration evaporate to dryness, and with next batch accumulation of material circular treatment, can further improve total recovery.
In present embodiment step 5, secondary crystal can make taxol purity reach more than 98.5%, the rate of recovery more than 95%, if need to improve purity further, available methanol/water system recrystallize once.
Gained crystallization in present embodiment step 6, by HPLC, MS,
1h-NMR and
13c-NMR determines structure: HPLC detects, chromatographic column: Dikma Diamonsil C
18(250 × 4.6mm i.d., 5 μm), moving phase: acetonitrile: water=45: 55(v: v); Column temperature: 30 DEG C; Determined wavelength: 227nm; Sample size: 20 μ L; Flow velocity: 10mL/min.Taxol Standard concentration is 100 μ g/mL(available from Sigma).NMR analyzes: sample trichloromethane dissolves, and measures
1h NMR and
13c NMR spectrogram.
Taxol, white powdery solids (chemical structure is shown in Fig. 1), fusing point 213-216 DEG C, ESI-MS m/z:854 place visible [M+H]
+quasi-molecular ions, 876 places visible [M+Na]
+, molecular formula is C
47h
51nO
14, molecular weight is 853; ESI-MS,
1h-NMR and
13c-NMR collection of illustrative plates is shown in Fig. 2-4 respectively.
Bakating III (Baccatin III), white, needle-shaped crystals (chemical structure is shown in Fig. 5), fusing point 223-225 DEG C, ESI-MSm/z:587 place visible [M+H]
+quasi-molecular ions, 609 places visible [M+Na]
+, molecular formula is C
31h
38o
11, molecular weight is 586.ESI-MS,
1h-NMR and
13c-NMR collection of illustrative plates is shown in Fig. 6-8 respectively.
Cephalomannine (Cephalomannine), white powder (chemical structure is shown in Fig. 9), fusing point 240-242 DEG C, ESI-Msm/z:832 place visible [M+H]
+, 609 places visible [M+Na]
+, molecular formula is C
45h
53nO
14, molecular weight is 831.ESI-MS,
1h-NMR and
13c-NMR collection of illustrative plates is shown in Figure 10-12 respectively.
Embodiment two: present embodiment and embodiment one unlike, in step 2, the every 100mL of VITAMIN concentrated solution is by 15mg tyrosine, 50mg phenylalanine, 10mg VB
6, 10mg VB
1, 10mg V
h, 10mg calcium pantothenate, 10mg linolic acid and surplus distilled water composition.Other is identical with embodiment one.
Claims (1)
1. the isolation and purification method of treelike menu HDFS4-26 biological fermentation taxol biosynthesis and precursor Bakating III and Cephalomannine, wherein treelike menu (Nodulisporium sylviforme) HDFS4-26, in Chinese patent " paclitaxel genome rearrangement strain HD FS4-26 ", oneself is open, its bacterial strain is preserved in China typical culture collection center, preservation date is 2008.2.28, and preserving number is CCTCC M:208026; It is characterized in that it realizes according to the following steps:
One, bacterial strain activation and seed culture: treelike menu HDFS4-26 is inoculated on PDA slant medium, 2 ~ 3d is activated under the condition of 28 DEG C, then the treelike menu HDFS4-26 after activation is connected in PDA liquid nutrient medium, at 28 DEG C, 120r/min shaking table cultivation 3d, obtain seed culture fluid;
Two, fermentation culture: seed culture fluid is proceeded in the S-7 liquid nutrient medium of improvement with the inoculum size of volume ratio 5%, at 28 DEG C, 120r/min fermentation culture 12d, wherein add the sucrose of 4g/L and the VITAMIN concentrated solution of 3mL/L at the 4d of fermentation culture, the peptone of 0.4g/L and the soya-bean cake hydrolyzed solution of 0.2mL/L is added at the 6d of fermentation culture, add the sucrose of 2g/L and the styracin of 20mg/L at the 7d of fermentation culture, add the amphotericin B of 28ug/L at the 9d of fermentation culture;
Three, after step 2 fermentation ends, separate fermentation liquid and mycelium also reclaim, and with sherwood oil to fermented liquid decolouring twice, then add isopyknic ethyl acetate and methyl alcohol mixed liquor extracted overnight, separate organic phase, again add isopyknic ethyl acetate and methyl alcohol mixed liquor extracted overnight, separate organic phase, then merge organic phase, again with Rotary Evaporators removing ethyl acetate and methyl alcohol, to remaining 5mL solution, solution is poured in plate and dries, obtain sample;
Four, a chromatography: sample is dissolved in chloroform by mass volume ratio 1:10, add the diatomite of 5 times amount, mix rear natural air drying thoroughly, after dress post, use chloroform successively, chloroform: methyl alcohol=98:2, chloroform: methyl alcohol=87:3, chloroform: methyl alcohol=90:10 and chloroform: methyl alcohol=60:40 carries out wash-out, TLC detects, merge the fraction containing target product taxol, after evaporate to dryness, be dissolved in methyl alcohol in 8mL/g ratio, heating in water bath is to 50 DEG C, simultaneously, the distilled water of instillation 1/3rd methyl alcohol volumes when stirring, cooling 48 ~ 72h, obtain primary crystallization sample,
Five, secondary chromatography: the length-to-diameter ratio of chromatographic column is at more than 8:1, dry column-packing, the mass ratio of stationary phase silica gel and primary crystallization sample is 30:1, and elution mode is first use the hexane/ethyl acetate solution of 3 times of column volumes, and hexane/ethyl acetate liquor capacity carries out wash-out than 60:40; Then, with the hexane/ethyl acetate solution of 2 times of column volumes, hexane/ethyl acetate liquor capacity carries out wash-out than 55:45; Finally, with the hexane/ethyl acetate solution of 5 times of column volumes, hexane/ethyl acetate liquor capacity carries out wash-out than 50:50; TLC detects, and merges the fraction containing target product taxol, after evaporate to dryness, is dissolved in methyl alcohol in 8mL/g ratio, and heating in water bath, to 50 DEG C, meanwhile, instills the distilled water of 1/3rd methyl alcohol volumes when stirring, cooling 48 ~ 72h, obtains secondary crystal sample;
Six, reversed phase chromatography separation: with PRP-6 resin for stationary phase, cylinder is shaping uses 2BV acetone respectively afterwards, ethyl acetate, acetone, 20% acetone-water balances, after adding secondary crystal sample, be the acetone-water system of 20% with volume content successively, volume content is the acetone-water system of 40%, volume content is the acetone-water system of 45%, volume content is the acetone-water system of 50%, volume content is the acetone-water system of 55%, volume content be 60% acetone-water system and acetone system carry out gradient elution, TLC detects, merge identical cut, concentrated, crystallization, namely complete treelike menu HDFS4-26 to ferment the isolation and purification of paclitaxel produced and precursor Bakating III and Cephalomannine, in step 2, the every 100mL of VITAMIN concentrated solution is by 15mg tyrosine, 50mg phenylalanine, 10mg VB
6, 10mg VB
1, 10mg V
h, 10mg calcium pantothenate, 10mg linolic acid and surplus distilled water composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310146249.9A CN103194502B (en) | 2013-04-24 | 2013-04-24 | Separating and purifying method of taxol synthesized by biological fermentation of nodulisporium sylviforme entophytic fungi as well as precursors of taxol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310146249.9A CN103194502B (en) | 2013-04-24 | 2013-04-24 | Separating and purifying method of taxol synthesized by biological fermentation of nodulisporium sylviforme entophytic fungi as well as precursors of taxol |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103194502A CN103194502A (en) | 2013-07-10 |
| CN103194502B true CN103194502B (en) | 2015-05-27 |
Family
ID=48717317
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310146249.9A Expired - Fee Related CN103194502B (en) | 2013-04-24 | 2013-04-24 | Separating and purifying method of taxol synthesized by biological fermentation of nodulisporium sylviforme entophytic fungi as well as precursors of taxol |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103194502B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107674890A (en) * | 2016-08-02 | 2018-02-09 | 武汉臻智生物科技有限公司 | The method for preparing taxol |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280281A (en) * | 2008-02-29 | 2008-10-08 | 黑龙江大学 | Paclitaxel genome rearrangement bacterial strain HDFS4-26 and breeding method of high-yield bacterial strain |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2001255821A1 (en) * | 2000-03-28 | 2001-10-08 | Cubist Pharmaceuticals, Inc. | Enhanced production of secondary metabolites in the presence of organic additives |
-
2013
- 2013-04-24 CN CN201310146249.9A patent/CN103194502B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280281A (en) * | 2008-02-29 | 2008-10-08 | 黑龙江大学 | Paclitaxel genome rearrangement bacterial strain HDFS4-26 and breeding method of high-yield bacterial strain |
Non-Patent Citations (2)
| Title |
|---|
| Biotransformation of taxol;Tom S Chen et al;《Tetrahedron Letters》;20011231;第42卷;3787-3789 * |
| 抗癌药物紫杉醇的提取与分离纯化技术;赵凯等;《生物技术通讯》;20040531;第15卷(第3期);309-312 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103194502A (en) | 2013-07-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103910708B (en) | The Azaphilones compound in a kind of thalassiomycetes source and its preparation method and application | |
| CN103910746B (en) | The Berkeleyones compound in one class marine fungi source and its preparation method and application | |
| CN103655215B (en) | There is Paecilomyces varioti extract of restraint of tyrosinase activity and Free-radical scavenging activity and uses thereof | |
| CN102337240A (en) | Serratia marcescen capable of producing red pigment through fermentation and method for producing red pigment by fermentation | |
| CN109134574B (en) | Steroid compound, preparation method and application thereof, and anti-tumor drug | |
| CN102234677B (en) | A kind of method of Fusarium moniliforme microbe conversion panaxdiols saponin | |
| CN103911407B (en) | The preparation method of the Azaphilone class dimer compound in a kind of marine fungi source and application | |
| CN103194502B (en) | Separating and purifying method of taxol synthesized by biological fermentation of nodulisporium sylviforme entophytic fungi as well as precursors of taxol | |
| CN103910701B (en) | The naphthoquinone compound in a kind of thalassiomycetes source and its preparation method and application | |
| CN112300243A (en) | Cyclopeptide compound and preparation method and application thereof | |
| CN101319199B (en) | Method for cell abduction generation of taxone with Chinese yew | |
| CN103667387A (en) | Method for separation and extraction of epothilone B from sorangium cellulosum fermentation liquid | |
| CN102757443B (en) | Sulfur-substituted podophyllum derivative and bioconversion, separation and purification method thereof | |
| CN101684451B (en) | Microorganism for hydrolyzing 7-xylose group and 13-side chain of taxane | |
| CN102234669B (en) | Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin | |
| CN103145722B (en) | A kind of method of high speed adverse current chromatogram separating-purifying ebormycine | |
| CN102382866B (en) | Preparation, purification and content detection methods for cerebroside | |
| CN107954839A (en) | A kind of antiinflammatory active compound peniroquesine A and its preparation method and application | |
| CN101492704B (en) | Method for converting podophyllinic acid lactone into podophyllic acid and picropodophyllin | |
| CN108774120B (en) | Lapatiquinone compound and preparation method thereof | |
| CN102051394A (en) | Preparation method and application of sulfo-diketopiperazine compounds | |
| CN101386874B (en) | Two-liquid phase fermentation method for improving paclitaxel yield from fungi | |
| CN103484505B (en) | Method for generating resveratrol by converting glucose by using intracellular enzyme of alternaria microorganism | |
| CN110041401B (en) | Kentanson namycin, preparation method and application thereof | |
| CN103819430A (en) | Paclitaxel purifying method of paclitaxel crude product produced by Chinese yew cell culture |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150527 Termination date: 20180424 |