CN103834706A - Method of converting ginkgo biloba extract product by tremella aurantialba bacterial strain to obtain bilobalide B - Google Patents
Method of converting ginkgo biloba extract product by tremella aurantialba bacterial strain to obtain bilobalide B Download PDFInfo
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- CN103834706A CN103834706A CN201410041574.3A CN201410041574A CN103834706A CN 103834706 A CN103834706 A CN 103834706A CN 201410041574 A CN201410041574 A CN 201410041574A CN 103834706 A CN103834706 A CN 103834706A
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Abstract
The invention discloses a method of converting a ginkgo biloba extract product by a tremella aurantialba bacterial strain to obtain bilobalide B, and belongs to the field of biotechnology. The method includes subjecting the tremella aurantialba bacterial strain to amplification culture by utilization of a potato glucose culture medium, converting the ginkgo biloba extract product to obtain the bilobalide B through steps of test-tube slant culturing, liquid flask shaking, primary seed culture and fermentation culture, and subjecting the fermentation liquid to extraction with ethyl acetate and crystallization with absolute alcohol so as to separate the bilobalide B from the fermentation liquid. The purity of the bilobalide B prepared by the method is not less than 90%.
Description
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Technical field
The present invention relates to technical field of bioengineering, by tremella kind liquid culture, transform a kind of novel process that Folium Ginkgo extract is prepared Ginkgolide B simultaneously.
Background technology
Studies have shown that bilobalide is one of activeconstituents main in Ginkgo Leaf, is the special effective platelet activation factor of a class (Platelet activating factor) receptor antagonist.Platelet activation factor (PAF) is a kind of endogenous phosphide being produced by thrombocyte and inflammation tissue secretion, is the most effective platelet aggregation of finding 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 rejection in anti-organ transplantation.Also find that Ginkgolide B can reduce hepatic vein and press raising whole body blood vessel 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 natural platelet activation factor (PAF) receptor antagonist the strongest, that have potential applicability in clinical practice most at present.
The method of producing at present Ginkgolide B is both at home and abroad mainly from Folium Ginkgo extract, the patent of the Ginkgolide B of leading of having appeared in the newspapers at present has 5 pieces, wherein there is one piece of (a kind of ginkgolide B solid dispersion and preparation method thereof about the preparation of Ginkgolide B, application number: CN200910204198.4), three pieces of (new purposes of one of Ginkgolide B of the physiologic function about Ginkgolide B and derivative thereof; Application number: CN200910092750.5; The new purposes of bilobalide B derivates in pharmacy, application number: CN200910234319.X; Bilobalide B hydrolyzed derivatives and uses thereof, application number: CN201010122039.2).And about the preparation of Ginkgolide B only have one piece (a kind of from bilobalide mixture the method for separating bilobalide B, application number: a kind of from bilobalide mixture the method for separating bilobalide B, application number: CN201010608847.X), in this bilobalide, contain the homologue such as Ginkgolide A. B. C, M, this patent is to adopt isolation technique separating bilobalide B from bilobalide mixture, is different from the present invention and utilizes fungi golden fungus liquid to transform bilobalide generation Ginkgolide B technology.
There is in recent years scholar to propose herb fermenting pharmaceutical technology.So-called herb fermenting pharmaceutical technology is to inherit on the basis of science of Chinese drug processing fermentation method, draw Microecology achievement, the high-tech herbal pharmaceutical new technology forming in conjunction with the fermentation technique of modern biological project is the new curative effect of finding medicine from Chinese medicine (natural drug) pharmacy aspect.Mostly traditional herb fermenting be to carry out under natural condition, and present herb fermenting pharmaceutical technology forms gradually fully having absorbed microecology in modern age, bionic achievement in research.The present invention, drawing on the basis of herb fermenting pharmaceutical technology, adopts submerged fermentation technology, adds appropriate Folium Ginkgo extract in liquid medium within, gold ear growth metabolism in substratum, transform the activeconstituents of Ginkgo Leaf simultaneously, as bilobalide mixture, generate Ginkgolide B.
Summary of the invention
The present invention is to provide a kind of production technology of Ginkgolide B, this technology is not that the simple separating and purifying method that utilizes obtains a large amount of Ginkgolide Bs, but in substratum, add Folium Ginkgo extract, pass through liquid culture technical transform Folium Ginkgo extract take golden ear as bacterial classification, generation contains Ginkgolide B, and obtains Ginkgolide B goods by extraction step.
Optional any one tremella kind of tremella kind used in the present invention, the wild golden ear in for example Yunnan, Tibet or Shanxi, the bacterial classification obtaining through conventional separate tissue.It is take golden ear as starting strain, carry out the steps such as test tube strains, liquid shaking bottle bacterial classification, seeding tank bacterial classification, fermentation and make the fermented liquid that contains Ginkgolide B, the fermented liquid that contains Ginkgolide B redissolves through ethyl acetate extraction, concentrated, alcohol, and the extraction processes such as-20 ℃ of crystallizations, filtration, oven dry obtain Ginkgolide B crystal.Wherein said potato glucose liquid nutrient medium is potato 200g, is cut into sheet, adds 1000mL water, boils 30 minutes, filters, and filtrate is settled to 1000mL, need to carry out packing according to test, 120 ℃ of sterilizings 30 minutes; Wherein said potato dextrose agar is that potato liquid nutrient medium 1000mL adds agar 20g, boils thawing agar, packing test tube, and 120 ℃ of sterilizings, after 30 minutes, are put into inclined-plane; Wherein said test tube slant bacterial classification is that preservation of bacteria strain is cut into 4 × 4mm fritter bacterial classification, picking one fritter is transferred in the test tube that contains potato dextrose agar, cultivate 5～20 days for 20～32 ℃, make test tube slant bacterial classification, 4 ℃ of this test tube slants save backup; Wherein said liquid shaking bottle bacterial classification is the fritter that test tube slant bacterial classification is cut into 4 × 4mm size, 3～8 of pickings are transferred and are equipped with in the 250mL triangular flask of 60～150mL potato glucose liquid nutrient medium, triangular flask is placed in constant-temperature shaking incubator, 20～32 ℃, 80～150 revs/min of shaking culture make golden fungus liquid shaking flask bacterial classification for 3～8 days; Wherein said seeding tank bacterial classification is that 10～50 bottles of golden fungus liquid shaking flask bacterial classification accesses are equipped with in the potato glucose liquid nutrient medium of 30～100L and the seeding tank of 105～350mL soya-bean oil, 20～32 ℃ of temperature, air flow 0.3～1:1/ minute (ventilation gas volume/medium liquid volume), 80～150 revs/min of cultivations of mixing speed 1～5 day; Wherein said zymotechnique is equipped with containing in 0.1～3% the potato glucose liquid nutrient medium of Folium Ginkgo extract and the fermentor tank of 630～6300mL soya-bean oil of 180～1800L for 60L seeding tank bacterial classification access, 20～32 ℃ of temperature, air flow 0.3～1:1/ minute (ventilation gas volume/medium liquid volume), 80～150 revs/min of cultivations of mixing speed make the fermented liquid that contains Ginkgolide B for 3～10 days; Wherein said Ginkgolide B extraction process is fermented liquid hydrochloric acid or sulphur acid for adjusting pH 1.0～4.0, extract 2～4 times by the ethyl acetate 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 for-20 ℃ of crystallizations 24～120 hours, filter filter residue washing, 80 ℃ of dry Ginkgolide B sterlings that obtain.
Technique of the present invention is applicable to 1000～10000L fermentation scale, and the Folium Ginkgo extract of process using of the present invention is domestic any Folium Ginkgo extract, but general flavone content ﹥ 24%, total lactone content ﹥ 6%.Utilize this technique to obtain Ginkgolide B Chun Du≤90%.
Accompanying drawing explanation
Fig. 1 is process flow sheet of the present invention.
Embodiment
In order further to set forth related material and the technique of technical scheme of the present invention, following examples are provided.The scope that these embodiment do not limit the present invention in any way.
The tremella strain using in following embodiment was to screen by contriver the golden ear B101 bacterial strain obtaining, and golden ear B101 bacterial strain is submitted the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms on April 21st, 2011.The Classification And Nomenclature of gold ear B101 be golden ear (
tremella aurantialba), preserving number is CGMCC No.4785.Depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
In technique of the present invention, the preparation method of substratum is as follows:
The preparation of potato glucose liquid nutrient medium: the potato that takes required weight, be cut into sheet, add the water of 5 times of potato weight, boil 30 minutes, filter, filtrate is settled to added water volume, need in packing 250mL triangular flask, seeding tank or fermentor tank according to test, 120 ℃ of sterilizings 30 minutes, cooling for subsequent use.
The preparation of potato dextrose agar: take potato 200g, be cut into sheet, add 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, every pipe 15mL substratum, 120 ℃ of sterilizings 30 minutes, are cooled to 50 ℃, be put into inclined-plane, chamfer length is the half of test tube length.
Embodiment 1
Preservation of bacteria strain is cut into 4 × 4mm fritter bacterial classification, and picking one fritter is transferred in the test tube that contains potato dextrose agar, cultivates 20 days for 20 ℃, makes test tube slant bacterial classification, and 4 ℃ of this test tube slants save backup; Test tube slant bacterial classification is cut into the fritter of 4 × 4mm size, 3 of pickings are transferred and are equipped with in the 250mL triangular flask of 60～150mL potato glucose liquid nutrient medium, triangular flask is placed in constant-temperature shaking incubator, and 20～32 ℃, 80 revs/min of shaking culture make golden fungus liquid shaking flask bacterial classification for 8 days; Wherein said seeding tank bacterial classification is that 10 bottles of golden fungus liquid shaking flask bacterial classification accesses are equipped with in the potato glucose liquid nutrient medium of 30L and the seeding tank of 105mL soya-bean oil, 20 ℃ of temperature, air flow 0.3:1/ minute (ventilation gas volume/medium liquid volume), 80 revs/min of cultivations of mixing speed 5 days; Containing in the potato glucose liquid nutrient medium of Folium Ginkgo extract and the fermentor tank of 630mL soya-bean oil that 0.1% sky, Xuzhou Lik-Sang Products Co., Ltd produces of 180L is equipped with in 60L seeding tank bacterial classification access, 20 ℃ of temperature, air flow 0.3:1/ minute (ventilation gas volume/medium liquid volume), 80 revs/min of cultivations of mixing speed make the fermented liquid that contains Ginkgolide B for 10 days.
By salt acid for adjusting pH 4.0 for fermented liquid, add the ethyl acetate extraction of 0.3 times of fermentating liquid volume, water extracts once by the ethyl acetate of 0.3 times of fermentating liquid volume again, combined ethyl acetate extraction liquid, and vacuum concentration is to dry, residue anhydrous alcohol solution, filter, filtrate, as for-20 ℃ of crystallizations 120 hours, is filtered, filter residue washing, 80 ℃ of dry Ginkgolide B sterlings that obtain.
Accurately take sample 5mg and be dissolved in 1mL dehydrated alcohol, 0.2 μ m membrane filtration, filtrate is for analyzing; Accurately take 1,2,3,4,5,6,7 and 8mg Ginkgolide B be dissolved in 1 mL dehydrated alcohol, 0.2 μ m membrane filtration, filtrate being divided into analysed use; Standard substance Ginkgolide B purity check adopt high-efficient liquid phase chromatogram technology, chromatography column is C-18 post, moving phase be second fine-water, adopt continuous gradient wash-out, compare with standard substance, according to peak area drawing standard curve, try to achieve calculation formula, per sample middle calculated by peak area purity.The content of measuring thus Ginkgolide B is respectively 90.5%.
Embodiment 2
Preservation of bacteria strain is cut into 4 × 4mm fritter bacterial classification, and picking one fritter is transferred in the test tube that contains potato dextrose agar, cultivates 10 days for 25 ℃, makes test tube slant bacterial classification, and 4 ℃ of this test tube slants save backup; Wherein said liquid shaking bottle bacterial classification is the fritter that test tube slant bacterial classification is cut into 4 × 4mm size, 5 of pickings are transferred and are equipped with in the 250mL triangular flask of 100mL potato glucose liquid nutrient medium, triangular flask is placed in constant-temperature shaking incubator, 26 ℃, 120 revs/min of shaking culture make golden fungus liquid shaking flask bacterial classification for 5 days; 30 bottles of golden fungus liquid shaking flask bacterial classification accesses are equipped with in the potato glucose liquid nutrient medium of 50L and the seeding tank of 250mL soya-bean oil, 26 ℃ of temperature, air flow 0.6:1/ minute (ventilation gas volume/medium liquid volume), 100 revs/min of cultivations of mixing speed 3 days; Containing in the potato glucose liquid nutrient medium of Folium Ginkgo extract and the fermentor tank of 2500mL soya-bean oil that 1.5% Ningbo herbal pharmaceutical company limited produces of 600L is equipped with in 60L seeding tank bacterial classification access, 26 ℃ of temperature, air flow 0.6:1/ minute (ventilation gas volume/medium liquid volume), 120 revs/min of cultivations of mixing speed make the fermented liquid that contains Ginkgolide B for 6 days.
By salt acid for adjusting pH 2.5 for fermented liquid, extract 3 times by the ethyl acetate of 1.5 times of fermentating liquid volumes continuously, combined ethyl acetate extraction liquid, vacuum concentration is to dry, and residue anhydrous alcohol solution, filters, filtrate was as for-20 ℃ of crystallizations 60 hours, filter filter residue washing, 80 ℃ of dry Ginkgolide B sterlings that obtain.
Accurately take sample 5mg and be dissolved in 1mL dehydrated alcohol, 0.2 μ m membrane filtration, filtrate is for analyzing; Accurately take 1,2,3,4,5,6,7 and 8mg Ginkgolide B be dissolved in 1 mL dehydrated alcohol, 0.2 μ m membrane filtration, filtrate being divided into analysed use; Standard substance Ginkgolide B purity check adopt high-efficient liquid phase chromatogram technology, chromatography column is C-18 post, moving phase be second fine-water, adopt continuous gradient wash-out, compare with standard substance, according to peak area drawing standard curve, try to achieve calculation formula, per sample middle calculated by peak area purity.The content of measuring thus Ginkgolide B is respectively 91.2%.
Embodiment 3
Preservation of bacteria strain is cut into 4 × 4mm fritter bacterial classification, and picking one fritter is transferred in the test tube that contains potato dextrose agar, cultivates 5 days for 32 ℃, makes test tube slant bacterial classification, and 4 ℃ of this test tube slants save backup; Test tube slant bacterial classification is cut into the fritter of 4 × 4mm size, 8 of pickings are transferred and are equipped with in the 250mL triangular flask of 150mL potato glucose liquid nutrient medium, triangular flask is placed in constant-temperature shaking incubator, and 32 ℃, 150 revs/min of shaking culture make golden fungus liquid shaking flask bacterial classification for 3 days; 50 bottles of golden fungus liquid shaking flask bacterial classification accesses are equipped with in the potato glucose liquid nutrient medium of 100L and the seeding tank of 350mL soya-bean oil, 32 ℃ of temperature, air flow 1:1/ minute (ventilation gas volume/medium liquid volume), 150 revs/min of cultivations of mixing speed 1 day; Containing in the potato glucose liquid nutrient medium of Folium Ginkgo extract and the fermentor tank of 630mL soya-bean oil that 3% Xi'an Chang Yue plant Chemical Co., Ltd. produces of 1800L is equipped with in 60L seeding tank bacterial classification access, 32 ℃ of temperature, air flow 1:1/ minute (ventilation gas volume/medium liquid volume), 150 revs/min of cultivations of mixing speed make the fermented liquid that contains Ginkgolide B for 3 days.
By sulphur acid for adjusting pH 1.0 for fermented liquid, use continuously the ethyl acetate of 3 times of volumes of fermentating liquid volume to extract 4 times, combined ethyl acetate extraction liquid, vacuum concentration is to dry, and residue anhydrous alcohol solution, filters, filtrate is as for-20 ℃ of crystallization 24-120 hour, filter filter residue washing, 80 ℃ of dry Ginkgolide B sterlings that obtain.
Accurately take sample 5mg and be dissolved in 1mL dehydrated alcohol, 0.2 μ m membrane filtration, filtrate is for analyzing; Accurately take 1,2,3,4,5,6,7 and 8mg Ginkgolide B be dissolved in 1 mL dehydrated alcohol, 0.2 μ m membrane filtration, filtrate being divided into analysed use; Standard substance Ginkgolide B purity check adopt high-efficient liquid phase chromatogram technology, chromatography column is C-18 post, moving phase be second fine-water, adopt continuous gradient wash-out, compare with standard substance, according to peak area drawing standard curve, try to achieve calculation formula, per sample middle calculated by peak area purity.The content of measuring thus Ginkgolide B is respectively 92.1%.
Claims (5)
1. a tremella kind transforms Folium Ginkgo extract and obtains the method for Ginkgolide B, it is characterized in that take golden ear as starting strain, make by steps such as test tube slant bacterial classification, liquid shaking bottle bacterial classification, seeding tank bacterial classification, fermentations the fermented liquid that contains Ginkgolide B, the fermented liquid that contains Ginkgolide B redissolves through ethyl acetate extraction, concentrated, alcohol, and the extraction processes such as-20 ℃ of crystallizations, filtration, oven dry obtain Ginkgolide B crystal.
2. obtain the method for Ginkgolide B according to a kind of tremella kind conversion Folium Ginkgo extract described in claims 1, tremella kind is cut into 4 × 4mm fritter bacterial classification, picking one fritter is transferred in the test tube that contains potato dextrose agar, cultivate 5～20 days for 20～32 ℃, make test tube slant bacterial classification, 4 ℃ of this test tube slants save backup; Gold ear test tube slant bacterial classification is cut into the fritter of 4 × 4mm size, 3～8 of pickings are transferred in the triangular flask of 250mL that 60～150mL potato glucose liquid nutrient medium is housed, triangular flask is placed in constant-temperature shaking incubator, 20～32 ℃, 80～150 revs/min of shaking culture make golden fungus liquid shaking flask bacterial classification for 3～8 days; 10～50 bottles of golden fungus liquid shaking flask bacterial classification accesses are equipped with in the potato glucose liquid nutrient medium of 30～100L and the seeding tank of 105～350mL soya-bean oil, 20～32 ℃ of temperature, air flow 0.3～1:1/ minute (ventilation gas volume: medium liquid volume), 80～150 revs/min of cultivations of mixing speed make for 1～5 day; Containing in 0.1～3% the potato glucose liquid nutrient medium of Folium Ginkgo extract and the fermentor tank of 630～6300L soya-bean oil of 180～1800L is equipped with in 60L seeding tank bacterial classification access, 20～32 ℃ of temperature, air flow 0.3～1:1/ minute (ventilation gas volume: medium liquid volume), 80～150 revs/min of cultivations of mixing speed make the fermented liquid that contains Ginkgolide B for 3～10 days; Wherein said potato glucose liquid nutrient medium is potato 200g, is cut into sheet, adds 1000mL water, boils 30 minutes, filters, and filtrate is settled to 1000mL, need to carry out packing according to test, 120 ℃ of sterilizings 30 minutes; Wherein said potato dextrose agar is that potato liquid nutrient medium 1000mL adds agar 20g, boils thawing agar, packing test tube, and 120 ℃ of sterilizings, after 30 minutes, are put into inclined-plane.
3. a kind of tremella kind conversion Folium Ginkgo extract according to claim 1 obtains the method for Ginkgolide B, hydrochloric acid or sulphur acid for adjusting pH 1.0～4.0 for fermented liquid, extract 2～4 times by the ethyl acetate of 0.3～3 times of fermentating liquid volume continuously, combined ethyl acetate extraction liquid, vacuum concentration is to dry, residue anhydrous alcohol solution, filter, filtrate, as for-20 ℃ of crystallizations 24～120 hours, is filtered, filter residue washing, 80 ℃ of dry Ginkgolide B sterlings that obtain.
4. obtain the method for Ginkgolide B according to a kind of tremella kind conversion Folium Ginkgo extract described in claims 1, Folium Ginkgo extract is domestic any Folium Ginkgo extract with manufacturer production, but in Folium Ginkgo extract, general flavone content ﹥ 24%, total lactone content ﹥ 6%.
5. a kind of tremella kind conversion Folium Ginkgo extract according to claim 1 obtains the method for Ginkgolide B, the prepared Ginkgolide B white of fermenting extraction process, interior ester content≤90%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103451251A (en) * | 2013-09-02 | 2013-12-18 | 张志才 | Method for preparing ginkgolide B by using stereum hirsutum thalli as catalyst |
| CN103468760A (en) * | 2013-09-02 | 2013-12-25 | 张志才 | Method for preparing bilobalide B by using Stereum hirsutum thalli as catalyst |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451251A (en) * | 2013-09-02 | 2013-12-18 | 张志才 | Method for preparing ginkgolide B by using stereum hirsutum thalli as catalyst |
| CN103468760A (en) * | 2013-09-02 | 2013-12-25 | 张志才 | Method for preparing bilobalide B by using Stereum hirsutum thalli as catalyst |
Non-Patent Citations (2)
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| 刘春卉，等: "金耳与其近似种的rDNA-ITS序列分析", 《云南植物研究》 * |
| 刘春卉，等: "金耳与其近似种的rDNA-ITS序列分析", 《云南植物研究》, vol. 29, no. 2, 31 December 2007 (2007-12-31), pages 237 - 242 * |
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