CN103450217B - Thick hair Boreostereum vibrans thalline is preparing the application in Ginkgolide B - Google Patents
Thick hair Boreostereum vibrans thalline is preparing the application in Ginkgolide B Download PDFInfo
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- CN103450217B CN103450217B CN201310393185.2A CN201310393185A CN103450217B CN 103450217 B CN103450217 B CN 103450217B CN 201310393185 A CN201310393185 A CN 201310393185A CN 103450217 B CN103450217 B CN 103450217B
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- SQOJOAFXDQDRGF-WJHVHIKBSA-N ginkgolide B Natural products O=C1[C@@H](C)[C@@]2(O)[C@@H]([C@H](O)[C@]34[C@@H]5OC(=O)[C@]23O[C@H]2OC(=O)[C@H](O)[C@@]42[C@H](C(C)(C)C)C5)O1 SQOJOAFXDQDRGF-WJHVHIKBSA-N 0.000 title claims abstract description 61
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 241001624913 Boreostereum vibrans Species 0.000 title claims abstract description 22
- SQOJOAFXDQDRGF-MMQTXUMRSA-N ginkgolide-b Chemical compound O[C@H]([C@]12[C@H](C(C)(C)C)C[C@H]3OC4=O)C(=O)O[C@H]2O[C@]24[C@@]13[C@@H](O)[C@@H]1OC(=O)[C@@H](C)[C@]21O SQOJOAFXDQDRGF-MMQTXUMRSA-N 0.000 title claims abstract 10
- MOLPUWBMSBJXER-YDGSQGCISA-N bilobalide Chemical compound O([C@H]1OC2=O)C(=O)[C@H](O)[C@@]11[C@@](C(C)(C)C)(O)C[C@H]3[C@@]21CC(=O)O3 MOLPUWBMSBJXER-YDGSQGCISA-N 0.000 claims abstract description 54
- 238000006243 chemical reaction Methods 0.000 claims abstract description 36
- 239000007788 liquid Substances 0.000 claims abstract description 29
- 230000001580 bacterial effect Effects 0.000 claims abstract description 28
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000000605 extraction Methods 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000002425 crystallisation Methods 0.000 claims abstract description 9
- 230000008025 crystallization Effects 0.000 claims abstract description 9
- 230000009466 transformation Effects 0.000 claims abstract description 8
- 238000011218 seed culture Methods 0.000 claims abstract description 6
- 239000012530 fluid Substances 0.000 claims abstract description 5
- 238000000926 separation method Methods 0.000 claims abstract 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- 239000000706 filtrate Substances 0.000 claims description 15
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- 238000010899 nucleation Methods 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 11
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 210000000582 semen Anatomy 0.000 claims description 10
- 238000009423 ventilation Methods 0.000 claims description 10
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 210000005069 ears Anatomy 0.000 claims description 7
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 206010013786 Dry skin Diseases 0.000 claims description 5
- 239000013530 defoamer Substances 0.000 claims description 5
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- 238000011081 inoculation Methods 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- FPUXKXIZEIDQKW-MFJLLLFKSA-N ginkgolide A Natural products O=C1[C@H](C)[C@@]2(O)[C@@H](O1)C[C@]13[C@@H]4OC(=O)[C@]21O[C@@H]1OC(=O)[C@H](O)[C@]31[C@@H](C(C)(C)C)C4 FPUXKXIZEIDQKW-MFJLLLFKSA-N 0.000 claims description 4
- FPUXKXIZEIDQKW-VKMVSBOZSA-N ginkgolide-a Chemical compound O[C@H]([C@]12[C@H](C(C)(C)C)C[C@H]3OC4=O)C(=O)O[C@H]2O[C@]24[C@@]13C[C@@H]1OC(=O)[C@@H](C)[C@]21O FPUXKXIZEIDQKW-VKMVSBOZSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 238000012807 shake-flask culturing Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 2
- 244000061456 Solanum tuberosum Species 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 239000012153 distilled water Substances 0.000 claims description 2
- 239000008121 dextrose Substances 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 17
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- SQOJOAFXDQDRGF-ZMVGXLHTSA-N ginkgolide b Chemical compound O[C@H]([C@]12[C@H](C(C)(C)C)C[C@H]3OC4=O)C(=O)O[C@H]2O[C@]24[C@@]13[C@H](O)[C@@H]1OC(=O)[C@@H](C)[C@]21O SQOJOAFXDQDRGF-ZMVGXLHTSA-N 0.000 description 51
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000001816 cooling Methods 0.000 description 5
- 238000012856 packing Methods 0.000 description 5
- 229930063422 Bilobalide A Natural products 0.000 description 4
- 241000218628 Ginkgo Species 0.000 description 4
- 235000011201 Ginkgo Nutrition 0.000 description 4
- 235000008100 Ginkgo biloba Nutrition 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- 239000001965 potato dextrose agar Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000002464 receptor antagonist Substances 0.000 description 3
- 229940044551 receptor antagonist Drugs 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 108700023400 Platelet-activating factor receptors Proteins 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
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- 102000030769 platelet activating factor receptor Human genes 0.000 description 2
- 230000010118 platelet activation Effects 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
- AMOGMTLMADGEOQ-FNZROXQESA-N Ginkgolide C Chemical compound O([C@H]1O2)C(=O)[C@H](O)C31[C@]14[C@@H](O)[C@@H]5OC(=O)[C@@H](C)[C@]5(O)[C@@]12C(=O)O[C@@H]4[C@@H](O)[C@H]3C(C)(C)C AMOGMTLMADGEOQ-FNZROXQESA-N 0.000 description 1
- CBAUUWCEZZNYTD-OOWJTCQTSA-N Ginkgolide M Natural products O=C1[C@@H](C)[C@@H]2[C@@H]([C@@H](O)[C@@]34[C@H]5[C@@H](O)[C@@H](CC(C)C)[C@@]63[C@@H](O)C(=O)O[C@@H]6O[C@@]24C(=O)O5)O1 CBAUUWCEZZNYTD-OOWJTCQTSA-N 0.000 description 1
- KDKROYXEHCYLJQ-DYXVGVPESA-N Ginkgolide M Chemical compound C[C@H]1[C@H]2[C@H]([C@@H](C34[C@]25C(=O)O[C@@H]3[C@@H]([C@H](C46[C@H](C(=O)O[C@H]6O5)O)C(C)(C)C)O)O)OC1=O KDKROYXEHCYLJQ-DYXVGVPESA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000123055 Stereum hirsutum Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
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- AMOGMTLMADGEOQ-DPFZUGDXSA-N ginkgolide C Natural products O=C1[C@@H](C)[C@]2(O)[C@H]([C@H](O)[C@@]34[C@H]5[C@H](O)[C@@H](C(C)(C)C)[C@]63[C@H](O)C(=O)O[C@H]6O[C@@]24C(=O)O5)O1 AMOGMTLMADGEOQ-DPFZUGDXSA-N 0.000 description 1
- LMEHVEUFNRJAAV-HOSIAMDISA-N ginkgolide J Natural products O=C1[C@H](C)[C@@]2(O)[C@H](O1)C[C@@]13[C@H]4[C@@H](O)[C@@H](C(C)(C)C)[C@@]51[C@@H](O)C(=O)O[C@@H]5O[C@@]23C(=O)O4 LMEHVEUFNRJAAV-HOSIAMDISA-N 0.000 description 1
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- Medicines Containing Plant Substances (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
The invention discloses thick hair Boreostereum vibrans thalline and prepare the application in Ginkgolide B, the present invention is by carrying out enlarged culturing to thick hair Boreostereum vibrans bacterial classification, from test tube slant, liquid shaking bottle, seed culture be separated with thalline, utilize thalline as catalyzer, transform bilobalide synthesis Ginkgolide B, conversion fluid through centrifugal, extraction into ethyl acetate, raw spirit crystallization Ginkgolide B from separation of fermentative broth out.Purity >=99% of the Ginkgolide B utilizing the method to obtain, bilobalide transformation efficiency & gt; 90%.
Description
Technical field
The present invention relates to technical field of bioengineering, prepare the application in Ginkgolide B in particular to thick hair Boreostereum vibrans thalline.
Background technology
Research proves that bilobalide is one of activeconstituents main in Ginkgo Leaf, is a class special effective platelet activation factor (PlateletactivatingfactorPAF) receptor antagonist.Platelet activation factor is a kind of endogenous phosphide produced by thrombocyte and inflammation tissue secretion, is the most effective platelet aggregation found at present, closely related with the Emergence and Development of numerous disease.Bilobalide, as paf receptor specific antagonists, has pharmacological action widely.Research finds that Ginkgolide B has provide protection to central nervous system and ischemic injuries, antishock, antianaphylaxis, antimicrobial antiphlogistic effect, and the rejection in anti-organ transplantation.Also find that Ginkgolide B can reduce hepatic vein pressure and improve system vascular tolerance simultaneously, illustrate that it has certain curative effect to liver cirrhosis.Bilobalide has therapeutic action for injury of the kidney.These effects are all relevant as paf receptor antagonists with Ginkgolide B, and it is considered to act on native platelets activation factor (PAF) receptor antagonist that is the strongest, that have potential applicability in clinical practice most at present.
Produce the method for Ginkgolide B at present both at home and abroad mainly from Folium Ginkgo extract, the patent of the Ginkgolide B of leading of having appeared in the newspapers has 51 sections, wherein a part is preparation (as: a kind of ginkgolide B solid dispersion and preparation method thereof of the preparation about Ginkgolide B, application number: CN200910204198.4), three sections of physiologic function (a kind of novelty teabag of Ginkgolide B about Ginkgolide B and derivative thereof; Application number: CN200910092750.5; The novelty teabag of bilobalide B derivates in pharmacy, application number: CN200910234319.X; Bilobalide B hydrolyzed derivatives and uses thereof, application number: CN201010122039.2).And only have nine sections of (a kind of novel method extracting Ginkgolide B from Ginkgo Leaf or Folium Ginkgo extract, application number 200510063407.X about the preparation of Ginkgolide B; A kind of method of separating bilobalide B from bilobalide mixture, application number: CN201010608847.X, etc.), containing the homologue such as Ginkgolide A. B. C, M in bilobalide, these patents adopt isolation technique separating bilobalide B from bilobalide mixture, is difficult to industrially produce Ginkgolide B in enormous quantities.
Summary of the invention
The present invention is to provide thick hair Boreostereum vibrans thalline and prepare the application in Ginkgolide B, should with thick hair Boreostereum vibrans (Stereumhirsutum) for bacterial classification obtains thalline by liquid culture technology, thalline is as catalyzer, conversion Ginkgolide A, C and M generate Ginkgolide B, and obtain Ginkgolide B goods by extraction step.
One aspect of the present invention relates to thick hair Boreostereum vibrans thalline and is preparing the application in Ginkgolide B, it is characterized in that comprising the steps:
Distilled water and acid is added at bilobalide (comprising Ginkgolide A. B. C and M), regulate pH6.0-7.0, be made into 20-30 gram of bilobalide/liter reaction solution, thick hair Boreostereum vibrans and reaction solution are mixed according to weight/volume 1:20-30, at temperature 20-30 DEG C, stir speed (S.S.) 100-150 rev/min, reacts more than 10 hours;
Reaction solution after reaction removed thalline according to 4000-6000 rev/min of centrifugal 10-20 minute, filtrate hydrochloric acid or sulfuric acid regulate pH2.0-3.0, be extracted with ethyl acetate 2-4 time continuously, combined ethyl acetate extraction liquid, vacuum concentration is to dry, residue anhydrous alcohol solution, filter, filtrate, as less than-20 DEG C crystallizations more than 70 hours, is filtered, filter residue is washed, and drying obtains Ginkgolide B.
In a preferred embodiment of the present invention, described thick hair Boreostereum vibrans obtains in the following way: with thick hair Boreostereum vibrans for starting strain, carry out the steps such as test tube strains goes down to posterity, liquid shaking bottle bacterial classification, seeding tank bacterial classification, catalyzed conversion and obtain conversion fluid containing Ginkgolide B, conversion fluid containing Ginkgolide B is through centrifugal, extraction into ethyl acetate, concentrated, alcohol redissolution, and the extraction processes such as-20 DEG C of crystallizations, filtration, oven dry obtain Ginkgolide B crystal.The substratum of wherein said test tube strains is potato dextrose broth (Ge Jian, Wang Zhengxiang, industrial microorganism experimental technique handbook, 1994:P367); Wherein said test tube slant spawn culture technique is that slant strains is cut into 4 × 4mm fritter bacterial classification, the switching of picking one fritter is containing in the test tube of potato dextrose agar, cultivate 5 ~ 20 days for 20 ~ 32 DEG C, obtained test tube slant bacterial classification, 4 DEG C, this test tube slant saves backup; Wherein said liquid submerged culture base consists of (unit is grams per liter): glucose 5 ~ 40, Semen Maydis powder 5 ~ 25, peptone 1 ~ 20, KH
2pO
41 ~ 6, MgSO
41 ~ 4, add water to proper volume, initial ph value is 5.0 ~ 8.0,100 ~ 140 DEG C of sterilizings 20 ~ 60 minutes; The culture condition of wherein said shake-flask culture is: 250mL triangular flask liquid amount is 60 ~ 150mL, inoculates 3 ~ 4 pieces of 4 × 4mm fritter bacterial classifications, is placed in temperature 22 ~ 30 DEG C, rotating speed 120 ~ 160 revs/min, incubation time 24 ~ 48 hours; The substratum of wherein said seed tank culture consists of (unit is grams per liter): glucose 5 ~ 40, Semen Maydis powder 5 ~ 25, peptone 1 ~ 10, KH
2pO
41 ~ 6, MgSO
41 ~ 4, defoamer 0.1 ~ 0.8, adds water to proper volume, and initial ph value is 5.0 ~ 8.0,100 ~ 140 DEG C of sterilizings 20 ~ 60 minutes; The culture condition of wherein said seeding tank is: inoculum size 5 ~ 10% (after seed liquor volume/inoculation volume), culture temperature 22 ~ 30 DEG C, stir speed (S.S.) 90 ~ 150 revs/min, ventilation 1:0.3 ~ 1v/v/m, incubation time 80 ~ 90 hours; Wherein said conversion reaction liquid is (unit is grams per liter): bilobalide 10 ~ 50, and initial ph value is 5.0 ~ 8.0; Wherein said conversion process condition is: seed liquor 4000 ~ 6000 revs/min, centrifugal 5 ~ 20 minutes, collect thalline, thalline and reaction solution are pressed 1:5 ~ 50 (weight: volume) and are mixed, temperature 22 ~ 30 DEG C, stir speed (S.S.) 90 ~ 150 revs/min, ventilation 1:0.3 ~ 1v/v/m, reacts 6 ~ 18 hours; Wherein said Ginkgolide B extraction process is, centrifugal 10 ~ 20 minutes of conversion fluid 4000 ~ 6000 revs/min removes thalline, and filtrate hydrochloric acid or sulfuric acid regulate pH1.0 ~ 4.0, uses the extraction into ethyl acetate 2 ~ 4 times of 0.3 ~ 3 times of fermentating liquid volume continuously, combined ethyl acetate extraction liquid, vacuum concentration is to dry, and residue anhydrous alcohol solution, filters, filtrate was as-20 DEG C of crystallizations 24 ~ 120 hours, filter, filter residue is washed, and 80 DEG C of dryings obtain Ginkgolide B sterling.
Thick hair Boreostereum vibrans bacterial classification used in the present invention is the concomitance bacterium of golden ear, any one golden ears or side handles of a utensil entity optional, such as the golden ears or side handles of a utensil entity in Yunnan, Tibet or Shanxi, can obtain wild thick hair Boreostereum vibrans bacterial classification by separate tissue.This bacterium is not formed or is difficult to form sporophore, and mycelium meets the dimmed or blackening of potassium hydroxide solution.Generative hyphae is weave in loosely, branch, has clamp connexion, closely colourless.
Technique Ginkgolide B white of the present invention, purity >=99%, bilobalide transformation efficiency >90%.
According to the bilobalide measuring method that this technique adopts be: precision takes Ginkgolide B reference substance 10mg, is dissolved in 1mL hplc grade methanol, is mixed with the reference liquid of 10mg/mL.0.2 μm of filtering with microporous membrane, controlling sample size is 1,2,3,4,5,6,7 and 8 μ l, utilizes high performance liquid chromatograph (HIMADZU (Shimadzu) LC-20AT) to measure Ginkgolide B content.Chromatographic column is silicagel column (4.6mm × 250mm, 5 μm).Condition determination is moving phase: methyl alcohol: 0.1% phosphoric acid solution=10:90; Column temperature: 30 DEG C, flow velocity: 0.8mL/min, determined wavelength: 270nm.Take sample size as X-coordinate (X), carry out linear regression with corresponding peak area for ordinate zou (Y), try to achieve the equation of linear regression of bilobalide concentration and peak area.The supernatant liquor of pipette samples liquid (in fermenting process and after fermentation) is a small amount of, with 0.45 μm of filtering with microporous membrane, measure peak area respectively by above-mentioned chromatographic condition, adopt external standard peak area according to the regression equation calculation Ginkgolide B content of above-mentioned Ginkgolide B concentration of trying to achieve and peak area.
Ginkgolide B transformation efficiency calculates by following formula (1):
In formula, R
tfor the transformation efficiency of t time Ginkgolide B; C
0(mg/L), V
0(mL) be the bilobalide concentration added for 0 hour and reaction solution volume; C
t(mg/L) and Vt (mL) be the reaction concentration of t hour Ginkgolide B and reaction solution volume.
Embodiment
In order to set forth the material and progress involved by technical scheme of the present invention further, give following examples.The scope that these embodiments do not limit the present invention in any way.
Embodiment 1
The preparation of test tube strains
The preparation of potato dextrose agar: take potato 200g, is cut into sheet, adds 1000mL water, boil 30 minutes, filter, filtrate is settled to 1000mL, adds agar 20g, and heating is after agar melts completely, the test tube of packing 18 × 200mm, often pipe 15mL substratum, 120 DEG C of sterilizings 30 minutes, are cooled to 50 DEG C, be put into inclined-plane, chamfer length is the half of test tube length; The thick hair Boreostereum vibrans bacterial classification be separated from golden ears or side handles of a utensil entity of preservation is cut into 4 × 4mm fritter bacterial classification, the switching of picking one fritter is containing in the test tube of potato dextrose agar, cultivate 20 days for 20 DEG C, obtained test tube slant bacterial classification, 4 DEG C, this test tube slant saves backup;
The preparation of the fermented liquid containing Ginkgolide B
1, the preparation of liquid shaking bottle bacterial classification
Liquid shaking bottle spawn culture based formulas is (unit is grams per liter) glucose 5, Semen Maydis powder 5, peptone 1, KH
2pO
41, MgSO
41, initial ph value is 5.0.Prepare 5500mL altogether, packing 250mL triangular flask, every bottle of 60mL, totally 84 bottles, 100 DEG C of sterilizings 60 minutes.Prepare 5500mL altogether, packing 250mL triangular flask, every bottle of 60mL, totally 84 bottles, the thick hair Boreostereum vibrans test tube slant bacterial classification be separated from the gold ears or side handles of a utensil entity of Yunnan preserved is cut into the fritter of 4 × 4mm size, picking 3 pieces is transferred and is equipped with in the 250mL triangular flask of Shake flask medium, is placed in 22 DEG C, 90 revs/min shaking tables and cultivates 24 hours.
2, the preparation of seeding tank bacterial classification
75L seeding tank is equipped with 45L seed culture medium, and wherein the formula of seed culture medium is (unit is grams per liter): glucose 5, Semen Maydis powder 5, peptone 1, KH
2pO
41, MgSO
41, defoamer 0.5, initial ph value is 5.0,100 DEG C of sterilizings 60 minutes, after cooling, accessed by the bacterial classification 5000mL of above-mentioned cultivation in seeding tank, namely inoculum size 10% (after seed liquor volume/inoculation volume), maintains tank temperature 22 DEG C, tank pressure 0.05MPa, stirring velocity 90 revs/min, ventilation 1:0.3v/v/m, fermentation time 90 hours.
3, Ginkgolide B transforms
Seed liquor 4000 revs/min collects thalline in centrifugal 20 minutes, obtains fresh thalli 2kg.Accurately take bilobalide 100 grams, adding distil water 10L, regulates pH5.0 with hydrochloric acid, is made into the reaction solution of 10 grams per liters, 2kg and 10L reaction solution (1:5, weight/volume) mixes, temperature 22 DEG C, stir speed (S.S.) 90 revs/min, ventilation 1:0.3v/v/m, reacts 18 hours.
4, the extraction of Ginkgolide B
Above-mentioned reaction solution 4000 revs/min removes thalline in centrifugal 20 minutes, filtrate hydrochloric acid or sulfuric acid regulate pH4.0, the extraction into ethyl acetate of continuous use 3 times of fermentating liquid volumes 2 times, combined ethyl acetate extraction liquid, vacuum concentration is to dry, residue anhydrous alcohol solution, filter, filtrate, as-20 DEG C of crystallizations 24 hours, is filtered, filter residue is washed, and 80 DEG C of dryings obtain Ginkgolide B sterling.Ginkgolide B content 99.1% by analysis, transformation efficiency 90.8%.
The preparation of the fermented liquid containing Ginkgolide B
1, the making of shaking flask bacterial classification
The formula of Shake flask medium is (unit is grams per liter): glucose 20, Semen Maydis powder 15, peptone 6, KH
2pO
43, MgSO
42, initial ph value is 6.5, prepares 4500mL altogether, packing 250mL triangular flask (totally 50 bottles), every bottle of 90mL, 120 DEG C of sterilizings 40 minutes.After cooling, the thick hair Boreostereum vibrans test tube slant bacterial classification be separated from the gold ears or side handles of a utensil entity of Shanxi preserved is cut into the fritter of 4 × 4mm size, test tube slant bacterial classification is cut into the fritter of 4 × 4mm size, picking 4 pieces is transferred and is equipped with in the triangular flask of Shake flask medium, be placed on 25 DEG C, 120 revs/min shaking tables, cultivate 36 hours.
2, the preparation of seeding tank bacterial classification
100L seeding tank is equipped with 50L seed culture medium, and wherein the formula of seed tank culture base is (unit is grams per liter): glucose 20, Semen Maydis powder 15, peptone 6, KH
2pO
43, MgSO
42, defoamer 0.1, initial ph value is 6.5,120 DEG C of sterilizings 40 minutes.After cooling, accessed by the bacterial classification 4000mL of above-mentioned cultivation in 100L seeding tank, i.e. inoculum size 7.4% (after seed liquor volume/inoculation volume), in seeding tank, the overall of fermented liquid is 54L; Maintain tank temperature 25 DEG C, tank pressure 0.05MPa, stirring velocity 120 rpms, ventilation 1:0.5v/v/m, incubation time 90 hours.
3, Ginkgolide B transforms
Seed liquor 5000 revs/min collects thalline in centrifugal 12 minutes, obtains fresh thalli 3.5kg.Accurately take bilobalide 1250 grams, adding distil water 50L, pH6.5 is regulated with hydrochloric acid, be made into the reaction solution of 25 grams per liters, 2kg and 50L reaction solution (1:25, weight/volume) mixes, temperature 25 DEG C, stir speed (S.S.) 120 revs/min, ventilation 1:0.8v/v/m, reacts 12 hours.
4, the extraction of Ginkgolide B
Centrifugal 15 minutes of above-mentioned reaction solution 5000 revs/min removes thalline, and filtrate hydrochloric acid or sulfuric acid regulate pH2.5, continuously with the extraction into ethyl acetate 3 times of 2 times of fermentating liquid volumes, combined ethyl acetate extraction liquid, vacuum concentration is to dry, and residue anhydrous alcohol solution, filters, filtrate was as-20 DEG C of crystallizations 72 hours, filter, filter residue is washed, and 80 DEG C of dryings obtain Ginkgolide B sterling, Ginkgolide B content 99.5% by analysis, transformation efficiency 95.8%.
The preparation of the fermented liquid of embodiment 4 containing Ginkgolide B
1, the making of shaking flask bacterial classification
The formula of Shake flask medium is (unit is grams per liter): glucose 40, Semen Maydis powder 25, peptone 10, KH
2pO
46, MgSO
44, initial ph value is 8.0.Prepare 2000mL altogether, packing 250mL triangular flask (totally 10 bottles), every bottle of 150mL, 140 DEG C of sterilizings 20 minutes.After cooling, the thick hair Boreostereum vibrans test tube slant bacterial classification be separated from the gold ears or side handles of a utensil entity of Tibet preserved is cut into the fritter of 4 × 4mm size, picking 4 pieces is transferred and is equipped with in the triangular flask of Shake flask medium, is placed in 30 DEG C, 150 rev/min shaking tables are cultivated 46 hours.
2, the preparation of seeding tank bacterial classification
50L seeding tank is equipped with 30L seed culture medium, and wherein the formula of seed tank culture base is (unit is grams per liter): glucose 40, Semen Maydis powder 25, peptone 10, KH
2pO
46, MgSO
44, defoamer 0.8, initial ph value is 8.0,140 DEG C of sterilizings 20 minutes.After cooling, the bacterial classification 1600mL of aforesaid liquid shake-flask culture is accessed in 50L seeding tank, i.e. inoculum size 5.1% (after seed liquor volume/inoculation volume); Maintain tank temperature 30 DEG C, tank pressure 0.05MPa, stirring velocity 150 revs/min, ventilation 1:1v/v/m, fermentation time 90 hours.
3, Ginkgolide B transforms
Seed liquor 6000 revs/min collects thalline in centrifugal 5 minutes, obtains fresh thalli 1.2kg.Accurately take bilobalide 3000 grams, adding distil water 60L, pH8.0 is regulated with sodium hydroxide, be made into the reaction solution of 50 grams per liters, 1.2kg fresh thalli and 60L reaction solution (1:50, weight/volume) mix, temperature 30 DEG C, stir speed (S.S.) 150 revs/min, ventilation 1:1v/v/m, reacts 6 hours.
4, the extraction of Ginkgolide B
Above-mentioned reaction solution 6000 revs/min removes thalline in centrifugal 10 minutes, filtrate hydrochloric acid or sulfuric acid regulate pH1.0, the extraction into ethyl acetate of continuous use 0.3 times of fermentating liquid volume 4 times, combined ethyl acetate extraction liquid, vacuum concentration is to dry, residue anhydrous alcohol solution, filter, filtrate, as-20 DEG C of crystallizations 120 hours, is filtered, filter residue is washed, and 80 DEG C of dryings obtain Ginkgolide B sterling.Ginkgolide B content 99.9% by analysis, transformation efficiency 98.5.8%.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any change of expecting without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, the protection domain that protection scope of the present invention should limit with claims is as the criterion.
Claims (5)
1. thick hair Boreostereum vibrans thalline is preparing the application in Ginkgolide B, and it is characterized in that thick hair Boreostereum vibrans transforms bilobalide by liquid submerged culture, seed tank culture and thalline, separation and Extraction obtains Ginkgolide B; Wherein said liquid shaking bottle seed culture medium consists of, and unit is grams per liter: glucose 5 ~ 40, Semen Maydis powder 5 ~ 25, peptone 1 ~ 20, KH
2pO
41 ~ 6, MgSO
41 ~ 4, add water to proper volume, initial ph value is 5.0 ~ 8.0,100 ~ 140 DEG C of sterilizings 20 ~ 60 minutes; Wherein said seed tank culture base consists of, and unit is grams per liter: glucose 5 ~ 40, Semen Maydis powder 5 ~ 25, peptone 1 ~ 10, KH
2pO
41 ~ 6, MgSO
41 ~ 4, defoamer 0.1 ~ 0.8, adds water to proper volume, and initial ph value is 5.0 ~ 8.0,100 ~ 140 DEG C of sterilizings 20 ~ 60 minutes; Wherein said conversion bilobalide reaction solution consists of, and unit is grams per liter: bilobalide 10 ~ 50, and initial ph value is 5.0 ~ 8.0; The culture condition of wherein said shake-flask culture is: 250mL triangular flask liquid amount is 60 ~ 150mL, inoculates 3 ~ 4 pieces of 4 × 4mm fritter bacterial classifications, is placed in temperature 22 ~ 30 DEG C, rotating speed 120 ~ 160 revs/min, incubation time 24 ~ 48 hours; The culture condition of wherein said seeding tank is: volume % after the volume/inoculation of inoculum size 5 ~ 10 seed liquor, culture temperature 22 ~ 30 DEG C, stir speed (S.S.) 90 ~ 150 revs/min, ventilation 1: 0.3 ~ 1v/v/m, incubation time 80 ~ 90 hours; Wherein said conversion process condition is: seed liquor 4000 ~ 6000 revs/min, centrifugal 5 ~ 20 minutes, collect thalline, thalline and conversion bilobalide reaction solution by weight volume ratio 1: 5 ~ 50 mix, temperature 22 ~ 30 DEG C, stir speed (S.S.) 90 ~ 150 revs/min, ventilation 1: 0.3 ~ 1v/v/m, transforms 6 ~ 18 hours; According to this conversion process, wherein before carrying out shake-flask culture, first carry out Secondary Culture, culture temperature 22 ~ 30 DEG C, incubation time 24 ~ 248 hours by the potato dextrose medium of new for the access of the slant strains of thick hair Boreostereum vibrans preparation.
2. application according to claim 1, wherein said thick hair Boreostereum vibrans bacterial classification is isolated golden ear concomitance bacterium bacterial classification from any one golden ears or side handles of a utensil entity in Yunnan, Shanxi and Tibet.
3. application according to claim 1, conversion fluid 4000 ~ 6000 revs/min is centrifugal, and filtrate hydrochloric acid or sulfuric acid regulate pH1.0 ~ 4.0, uses the extraction into ethyl acetate 2 ~ 4 times of 0.3 ~ 3 times of fermentating liquid volume continuously, combined ethyl acetate extraction liquid, vacuum concentration is to dry, and residue anhydrous alcohol solution, filters, filtrate is placed in-20 DEG C of crystallizations 24 ~ 120 hours, filter, filter residue is washed, and 80 DEG C of dryings obtain Ginkgolide B sterling.
4. application according to claim 3, obtained Ginkgolide B white, purity >=99%, bilobalide transformation efficiency > 90%.
5. application according to claim 1, is characterized in that comprising the steps:
Distilled water and acid is added at bilobalide, described bilobalide comprises Ginkgolide A. B. C and M, regulate pH6.0-7.0, be made into 20-30 gram of bilobalide/liter reaction solution, thick hair Boreostereum vibrans and conversion bilobalide reaction solution are mixed according to weight/volume 1: 20-30, at temperature 22-30 DEG C, stir speed (S.S.) 100-150 rev/min, reacts more than 10 hours;
Reaction solution after reaction removed thalline according to 4000-6000 rev/min of centrifugal 10-20 minute, filtrate hydrochloric acid or sulfuric acid regulate pH2.0-3.0, be extracted with ethyl acetate 2-4 time continuously, combined ethyl acetate extraction liquid, vacuum concentration is to dry, residue anhydrous alcohol solution, filter, filtrate, as less than-20 DEG C crystallizations more than 70 hours, is filtered, filter residue is washed, and drying obtains Ginkgolide B.
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| CN105907810A (en) * | 2016-06-06 | 2016-08-31 | 江苏大学 | Method for simultaneously preparing ginkgolide B and quercetin from stereum hirsutum |
| CN113718000A (en) * | 2021-09-10 | 2021-11-30 | 陈开云 | Industrial preparation method for producing ginkgolide B by fermenting endophytic fungi of ginkgo |
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