CN103468760B - Utilize Tremella thalline as the method for catalyst preparing ginkalide B - Google Patents
Utilize Tremella thalline as the method for catalyst preparing ginkalide B Download PDFInfo
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- 241001506047 Tremella Species 0.000 title claims abstract description 26
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- Medicines Containing Plant Substances (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses the method utilizing Tremella (Stereum hirsutum) thalline to convert bilobalide mixture synthesis ginkalide B as catalyst, belong to biological technical field.The present invention by being enlarged cultivation to Tremella strain, from test tube slant, liquid shaking bottle, seed culture separate with thalline, utilize thalline as catalyst, converting bilobalide synthesis ginkalide B, conversional solution is through centrifugal, extraction into ethyl acetate, anhydrous alcohol crystallization ginkalide B from separation of fermentative broth out.Utilize purity >=99% of the obtained ginkalide B of the method, bilobalide conversion ratio > 90%.
Description
Technical field
The present invention relates to technical field of bioengineering, cultivated by Tremella thalline strain liquid, convert Folium Ginkgo extract simultaneously and prepare a kind of new technology of ginkalide B.
Background technology
Research proves that bilobalide is one of active component main in Folium Ginkgo, is a class special effective platelet activating factor (PlateletactivatingfactorPAF) receptor antagonist.Platelet activating factor is a kind of endogenous phosphide produced by platelet and inflammation tissue secretion, is the maximally effective platelet aggregation having now been found that, closely related with the Emergence and Development of numerous disease.Bilobalide, as paf receptor specific antagonists, has pharmacological action widely.Research finds that central nervous system is had protective effect, shock, antiallergic, antimicrobial antiphlogistic effect, and the rejection in anti-organ transplantation with ischemic injuries by ginkalide B.Also find that ginkalide B can reduce hepatic portal vein pressure and improve system vascular toleration simultaneously, illustrate that liver cirrhosis is had certain curative effect by it.Bilobalide has therapeutical effect for injury of kidney.These effects are all relevant as paf receptor antagonists with ginkalide B, and it is presently believed to be effect native platelets activation factor (PAF) receptor antagonist the strongest, that have potential applicability in clinical practice most.
Produce the method for ginkalide B at present both at home and abroad mainly from Folium Ginkgo extract, the patent of the ginkalide B led of having appeared in the newspapers has 51 sections, a portion is that the preparation of the preparation about ginkalide B is (such as a kind of ginkgolide B solid dispersion and preparation method thereof, application number: CN200910204198.4), three sections of (a kind of new application of ginkalide B of the physiologic function about ginkalide B and derivant thereof；Application number: CN200910092750.5；Bilobalide B derivates new application in pharmacy, application number: CN200910234319.X；Bilobalide B hydrolyzed derivatives and application thereof, application number: CN201010122039.2).And the preparation about ginkalide B only has nine sections of (a kind of new method extracting ginkalide B from Folium Ginkgo or Folium Ginkgo extract, application number 200510063407.X；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 are to adopt isolation technics separating bilobalide B from bilobalide mixture, it is difficult to industrially produce ginkalide B in enormous quantities.
Tremella strain used in the present invention, any one Tremella fructification optional, for instance the Tremella in Yunnan, Tibet or Shanxi, can be obtained by separate tissue.This bacterium is formed without or is hardly formed sporophore, branch, has clamp connection, closely colourless.
Summary of the invention
The present invention is to provide the production technology of a kind of ginkalide B, the process for separation and purification that utilizes that this technology is not simple obtains substantial amounts of ginkalide B, but obtain thalline for strain by liquid culture technology with Tremella (Tremellafuciformis), thalline is as catalyst, convert ginkalide A in bilobalide mixture, C and M generates ginkalide B, and obtains ginkalide B goods by extraction step.
The present invention is with Tremella for starting strain, carry out that test tube strains goes down to posterity, the step such as liquid shaking bottle strain, seed tank strain, catalyzed conversion prepares the reactant liquor containing ginkalide B, reactant liquor containing ginkalide B redissolves through centrifugal, extraction into ethyl acetate, concentration, ethanol, and the extraction process such as-20 DEG C of crystallizations, filtration, drying obtains ginkalide B crystal.The culture medium 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 strain, the switching of picking one fritter is containing in the test tube of potato dextrose agar, cultivating 5～20 days for 20～32 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；Wherein said liquid submerged culture is basis set to be become (unit for g/l): glucose 5～40, Semen Maydis powder 5～25, peptone 1～20, KH2PO41～6, MgSO41～4, add water to proper volume, initial ph value is 5.0～8.0,100～140 DEG C of sterilizings 20～60 minutes；The condition of culture of wherein said shake-flask culture is: 250mL triangular flask liquid amount is 60～150mL, inoculates 3～4 pieces of 4 × 4mm fritter strains, is placed in temperature 22～30 DEG C, rotating speed 120～160 revs/min, incubation time 24～48 hours；The culture medium of wherein said seed tank culture consists of (unit for g/l): glucose 5～40, Semen Maydis powder 5～25, peptone 1～10, KH2PO41～6, MgSO41～4, defoamer 0.1～0.8, add water to proper volume, initial ph value is 5.0～8.0,100～140 DEG C of sterilizings 20～60 minutes；The condition of culture of wherein said seed tank is: inoculum concentration 5～10% (after seed liquor volume/inoculation volume), cultivation 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 for g/l): bilobalide 10～50, 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 reactant liquor press 1:5～50 (weight: volume) mixing, 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 ginkalide B extraction process is, conversional solution 4000～6000 revs/min is removed thalline, filtrate hydrochloric acid or sulphuric acid for centrifugal 10～20 minutes and is regulated pH1.0～4.0, continuously with the extraction into ethyl acetate 2～4 times of 0.3～3 times of fermentating liquid volume, combined ethyl acetate extract, vacuum is concentrated into dry, residue anhydrous alcohol solution, filters, filtrate was as-20 DEG C of crystallizations 24～120 hours, filtering, filtering residue is washed, and 80 DEG C dry obtains ginkalide B sterling.
Tremella strain used in the present invention, any one Tremella fructification optional, for instance the Tremella in Yunnan, Tibet or Shanxi, can be obtained by separate tissue.This bacterium is formed without or is hardly formed sporophore, branch, has clamp connection, closely colourless.
Technique ginkalide B of the present invention white, purity >=99%, bilobalide conversion ratio > 90%.
According to the bilobalide assay method that this technique adopts it is: precision weighs ginkalide B reference substance 10mg, is dissolved in 1mL hplc grade methanol, is configured to the titer 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 ginkalide B content.Chromatographic column is silicagel column (4.6mm × 250mm, 10 μm).Condition determination is mobile phase: methanol: 0.1% phosphoric acid solution=10:90；Column temperature: 30 DEG C, flow velocity: 0.8mL/min, detects wavelength: 270nm.With sample size for abscissa (X), carry out linear regression with corresponding peak area for vertical coordinate (Y), try to achieve the equation of linear regression of bilobalide concentration and peak area.The supernatant of pipette samples liquid (in sweat 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 the external standard peak area regression equation calculation ginkalide B content according to the above-mentioned ginkalide B concentration tried to achieve Yu peak area.
Ginkalide B conversion ratio calculates by following equation (1):
In formula, RtConversion ratio for t time ginkalide B；C0(mg/L), V0(mL) it is the bilobalide concentration added for 0 hour and culture volume；Ct(mg/L) and Vt (mL) be the reaction concentration of t hour ginkalide B and reactant liquor volume.
Detailed description of the invention
In order to material technical scheme involved by and technique are expanded on further, give following example.The scope that these embodiments do not limit the present invention in any way.
The preparation of embodiment 1 test tube strains
The preparation of potato dextrose agar: weigh Rhizoma Solani tuber osi 200g, is cut into lamellar, 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 subpackage 18 × 200mm, often pipe 15mL culture medium, 120 DEG C of sterilizings 30 minutes, it is cooled to 50 DEG C, being put into inclined-plane, chamfer length is the half of test tube length；The Tremella strain separated from Tremella fructification of preservation is cut into 4 × 4mm fritter strain, and the switching of picking one fritter is containing, in the test tube of potato dextrose agar, cultivating 20 days for 20 DEG C, prepare test tube slant strain, and 4 DEG C, this test tube slant saves backup:
The preparation of the embodiment 2 fermentation liquid containing ginkalide B
1, the preparation of liquid shaking bottle strain
Liquid shaking bottle spawn culture based formulas is (unit for g/l) glucose 5, Semen Maydis powder 5, peptone 1, KH2PO41, MgSO41, initial ph value is 5.0.Prepare 5500mL altogether, subpackage 250mL triangular flask, every bottle of 60mL, totally 84 bottles, 100 DEG C of sterilizings 60 minutes.Prepare 5500mL altogether, subpackage 250mL triangular flask, every bottle of 60mL, totally 84 bottles, the Tremella test tube slant strain separated from the Tremella fructification of Yunnan preserved is cut into the fritter of 4 × 4mm size, picking 3 pieces is transferred equipped with, in the 250mL triangular flask of Shake flask medium, being placed in 22 DEG C, 90 revs/min shaking tables and cultivate 24 hours.
2, the preparation of seed tank strain
75L seed tank is equipped with 45L seed culture medium, and wherein the formula of seed culture medium is (unit for g/l): glucose 5, Semen Maydis powder 5, peptone 1, KH2PO41, MgSO41, defoamer 0.5, initial ph value is 5.0,100 DEG C of sterilizings 60 minutes, after cooling, the strain 5000mL of above-mentioned cultivation is accessed in seed tank, i.e. inoculum concentration 10% (after seed liquor volume/inoculation volume), maintain tank temperature 22 DEG C, tank pressure 0.05MPa, mixing speed 90 revs/min, ventilation 1:0.3v/v/m, fermentation time 90 hours.
3, ginkalide B converts
Seed liquor 4000 revs/min collects thalline in centrifugal 20 minutes, obtains fresh thalli 2kg.Accurately weigh bilobalide 100 grams, add distilled water 10L, regulate pH5.0 with hydrochloric acid, be made into the reactant liquor of 10 g/l, 2kg and 10L reactant liquor (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 ginkalide B
Above-mentioned reactant liquor 4000 revs/min removes thalline in centrifugal 20 minutes, filtrate hydrochloric acid or sulphuric acid regulate pH4.0, continuously with the extraction into ethyl acetate 2 times of 3 times of fermentating liquid volumes, combined ethyl acetate extract, vacuum is concentrated into dry, residue anhydrous alcohol solution, filtering, filtrate, as-20 DEG C of crystallizations 24 hours, is filtered, filtering residue is washed, and 80 DEG C dry obtains ginkalide B sterling.Ginkalide B content 99.1% by analysis, conversion ratio 90.8%.
The preparation of the embodiment 3 fermentation liquid containing ginkalide B
1, the making of shaking flask strain
The formula of Shake flask medium is (unit for g/l): glucose 20, Semen Maydis powder 15, peptone 6, KH2PO43, MgSO42, initial ph value is 6.5, prepares 4500mL altogether, subpackage 250mL triangular flask (totally 50 bottles), every bottle of 90mL, 120 DEG C of sterilizings 40 minutes.After cooling, the Tremella test tube slant strain separated from the Tremella fructification of Shanxi preserved is cut into the fritter of 4 × 4mm size, test tube slant strain is cut into the fritter of 4 × 4mm size, picking 4 pieces is transferred equipped with in the triangular flask of Shake flask medium, it is placed on 25 DEG C, 120 revs/min shaking tables, cultivates 36 hours.
2, the preparation of seed tank strain
100L seed tank is equipped with 50L seed culture medium, and wherein the formula of seed tank culture base is (unit for g/l): glucose 20, Semen Maydis powder 15, peptone 6, KH2PO43, MgSO42, defoamer 0.1, initial ph value is 6.5,120 DEG C of sterilizings 40 minutes.After cooling, the strain 4000mL of above-mentioned cultivation is accessed in 100L seed tank, i.e. inoculum concentration 7.4% (after seed liquor volume/inoculation volume), the generally 54L of fermentation liquid in seed tank；Maintain tank temperature 25 DEG C, tank pressure 0.05MPa, mixing speed 120 rpms, ventilation 1:0.5v/v/m, incubation time 90 hours.
3, ginkalide B converts
Seed liquor 5000 revs/min collects thalline in centrifugal 12 minutes, obtains fresh thalli 3.5kg.Accurately weigh bilobalide 1250 grams, add distilled water 50L, regulate pH6.5 with hydrochloric acid, be made into the reactant liquor of 25 g/l, 2kg and 50L reactant liquor (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 ginkalide B
Above-mentioned reactant liquor 5000 revs/min is removed thalline, filtrate hydrochloric acid or sulphuric acid for centrifugal 15 minutes and is regulated pH2.5, continuously with the extraction into ethyl acetate 3 times of 2 times of fermentating liquid volumes, combined ethyl acetate extract, vacuum is concentrated into dry, residue anhydrous alcohol solution, filters, filtrate was as-20 DEG C of crystallizations 72 hours, filtering, filtering residue is washed, and 80 DEG C dry obtains ginkalide B sterling, ginkalide B content 99.5% by analysis, conversion ratio 95.8%.
The preparation of the embodiment 4 fermentation liquid containing ginkalide B
1, the making of shaking flask strain
The formula of Shake flask medium is (unit for g/l): glucose 40, Semen Maydis powder 25, peptone 10, KH2PO46, MgSO44, initial ph value is 8.0.Prepare 2000mL altogether, subpackage 250mL triangular flask (totally 10 bottles), every bottle of 150mL, 140 DEG C of sterilizings 20 minutes.After cooling, the Tremella test tube slant strain separated from the Tremella fructification of Tibet preserved being cut into the fritter of 4 × 4mm size, picking 4 pieces is transferred equipped with in the triangular flask of Shake flask medium, is placed in 30 DEG C, cultivates 46 hours on 150 rev/min shaking tables.
2, the preparation of seed tank strain
50L seed tank is equipped with 30L seed culture medium, and wherein the formula of seed tank culture base is (unit for g/l): glucose 40, Semen Maydis powder 25, peptone 10, KH2PO46, MgSO44, defoamer 0.8, initial ph value is 8.0,140 DEG C of sterilizings 20 minutes.After cooling, the strain 1600mL of aforesaid liquid shake-flask culture is accessed in 50L seed tank, i.e. inoculum concentration 5.1% (after seed liquor volume/inoculation volume)；Maintain tank temperature 30 DEG C, tank pressure 0.05MPa, mixing speed 150 revs/min, ventilation 1:1v/v/m, fermentation time 90 hours.
3, ginkalide B converts
Seed liquor 6000 revs/min collects thalline in centrifugal 5 minutes, obtains fresh thalli 1.2kg.Accurately weigh bilobalide 3000 grams, add distilled water 60L, pH8.0 is regulated with sodium hydroxide, it is made into the reactant liquor of 50 g/l, 1.2kg fresh thalli and 60L reactant liquor (1:50, weight/volume) mixing, temperature 30 DEG C, stir speed (S.S.) 150 revs/min, ventilation 1:1v/v/m, reacts 6 hours.
4, the extraction of ginkalide B
Above-mentioned reactant liquor 6000 revs/min removes thalline in centrifugal 10 minutes, filtrate hydrochloric acid or sulphuric acid regulate pH1.0, continuously with the extraction into ethyl acetate 4 times of 0.3 times of fermentating liquid volume, combined ethyl acetate extract, vacuum is concentrated into dry, residue anhydrous alcohol solution, filtering, filtrate, as-20 DEG C of crystallizations 120 hours, is filtered, filtering residue is washed, and 80 DEG C dry obtains ginkalide B sterling.Ginkalide B content 99.9% by analysis, conversion ratio 98.5%.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention be not limited thereto, any change expected without creative work or replacement, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with claims protection defined.
Claims (2)
1. utilize Tremella thalline as the method for catalyst preparing ginkalide B, it is characterized in that with Tremella for starting strain, by test tube slant strain, liquid shaking bottle strain, seed tank strain, centrifugal acquisition thalline, the thalline catalysis bilobalide synthesis reactant liquor containing ginkalide B, reactant liquor containing ginkalide B redissolves through centrifugal, extraction into ethyl acetate, concentration, ethanol, and the extraction process such as-20 DEG C of crystallizations, filtration, drying obtains ginkalide B crystal；Tremella strain converts bilobalide by liquid shaking bottle, seed tank and thalline, and separation and Extraction obtains ginkalide B；Wherein said liquid shaking bottle seed culture medium consists of: glucose 5～40, Semen Maydis powder 5～25, peptone 1～20, KH2PO41～6, MgSO41～4, unit is g/l, 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 seed tank culture is basis set to be become: glucose 5～40, Semen Maydis powder 5～25, peptone 1～10, KH2PO41～6, MgSO41～4, defoamer 0.1～0.8, unit is g/l, 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 reactant liquor consists of bilobalide 10 g/l～50 g/l, and described bilobalide includes Ginkgolide A. B. C and M, and initial ph value is 5.0～8.0；The condition of culture of wherein said shake-flask culture is: 250mL triangular flask liquid amount is 60～150mL, inoculates 3～4 pieces of 4 × 4mm fritter strains, is placed in temperature 22～30 DEG C, rotating speed 120～160 revs/min, incubation time 24～48 hours；The condition of culture of wherein said seed tank is: inoculum concentration 5%～10%, described inoculum concentration is the percent by volume of volume, cultivation temperature 22～30 DEG C, stir speed (S.S.) 90～150 revs/min after seed liquor volume and inoculation, 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 reactant liquor are by 1 weight: 5～50 volumes, mixing, in temperature 22～30 DEG C, stir speed (S.S.) 90～150 revs/min, ventilation 1:0.3～1v/v/m, converts 6～18 hours；According to this conversion process, wherein before carrying out shake-flask culture, first the slant strains of Tremella thalline is accessed in the potato dextrose medium of new preparation and carry out Secondary Culture, cultivation temperature 22～30 DEG C, incubation time 24～248 hours；Wherein, described Tremella thalline strain is isolated Tremella aurantialba Bandoni et Zang concomitance bacterium strain from any one Tremella aurantialba Bandoni et Zang sporophore in Yunnan, Shanxi and Tibet.
2. the method preparing ginkalide B according to claim 1, conversional solution 4000～6000 revs/min is centrifuged, and filtrate hydrochloric acid or sulphuric acid regulate pH1.0～4.0, continuously with the extraction into ethyl acetate 2～4 times of 0.3～3 times of fermentating liquid volume, combined ethyl acetate extract, vacuum is concentrated into dry, residue anhydrous alcohol solution, filters, filtrate is placed in-20 DEG C of crystallizations 24～120 hours, filtering, filtering residue is washed, and 80 DEG C dry obtains ginkalide B sterling.
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