CN105316384A - Synthetic method for prostaglandin E1 - Google Patents
Synthetic method for prostaglandin E1 Download PDFInfo
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- CN105316384A CN105316384A CN201510823186.5A CN201510823186A CN105316384A CN 105316384 A CN105316384 A CN 105316384A CN 201510823186 A CN201510823186 A CN 201510823186A CN 105316384 A CN105316384 A CN 105316384A
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Abstract
The invention relates to a synthetic method for prostaglandin E1. The synthetic method includes the following steps that 1, prostaglandin wet enzymes are extracted from a sheep seminal vesicle; 2, a prostaglandin E1 crude product is obtained by carrying out an enzymatic reaction on the prostaglandin wet enzymes; 3, the crude product passes through a column to be purified; 4, the prostaglandin E1 is refined in a recrystallization mode.
Description
Technical field
The invention belongs to the preparation method of medical compounds, be specifically related to a kind of preparation method of prostaglandin E1.
Technical background
Prostaglandin E1, structure is as follows:
Have another name called: Prostaglandin E1, chemical name: (1R, 2R, 3R)-3-hydroxyl-2-[(E)-(3S)-3-hydroxyl-1-octenyl]-5-oxo-cyclopentane enanthic acid.
Prostaglandin E1 comes from 20 carbon fatty acids, is the broad spectrum material existed in human body.It directly acts on vascular smooth muscle, and vasodilation and raising volume of blood flow, improve microcirculatory perfusion; And there is anticoagulant and thrombus A2 generates, suppress atherosclerosis Lipid Plaque to be formed and the formation of immunocomplex; Peripheral blood vessel and coronary vasodilator can be expanded, reduce peripheral vascular resistance and blood pressure, prevent thrombosis and protection platelet cell, protection ischemic myocardium, reducing myocardial infarction area, heart failure resistance; And energy nephrectasia blood vessel, increase renal blood flow, remove non-protein nitrogen(NPN), regulate water sodium balance, there is diuresis and the effect protecting the organ functions such as liver, kidney, lungs.There is expansion arteria penis, lax corpus cavernosum smooth muscle simultaneously, accelerate the effect of arteria penis Hemodynamic environment.Nineteen eighty-two, the model Enbrel scholar from Britain and doctor Samuelson from Sweden and Burger Strong doctor are because finding and studying Prostaglandin E1 and obtain Nobel Prize in Physiology or Medicine.
In recent years, due to the extensive clinical application range of prostaglandin E1, market increases year by year to its demand.Now, the national large-scale industrial production realizing prostaglandin E 1 as raw material of medicines such as existing such as day, moral, U.S. in the world, but in product purity and market value, transportation cost etc., be difficult to the production demand meeting domestic drug manufacturing enterprise.Meanwhile, the domestic production to prostaglandin E 1 as raw material of medicines rests on the laboratory scale production phase mostly, small part is only had to realize suitability for industrialized production, wherein a part is then the pure chemistry synthesizing mean used, the bulk drug product purity that they produce is high, but complex manufacturing, the production cycle is long; Have is exactly to relate to the environment problems such as the use of a large amount of organic solvents and the process of waste liquid in process of production again.Remaining a part of manufacturing enterprise carries out continuous seepage under utilizing the Bio-enzyme Combined Pre-treatment catalysis of extracting in the seminal vesicle of macro-organism reactor in metallic nickel and mammalian body, this production model greatly reduces the complex process degree of this product, also reduce the usage quantity of organic reagent simultaneously, but the use of metallic nickels a large amount of in its production process, add the sensitization risk of human body to this bulk drug, also considerably increase production cost simultaneously.
Summary of the invention
The object of the invention is, provide a kind of effectively simple, and more practical process for preparing prostaglandin E 1 as raw material of medicines, to reach reduction raw materials cost, simplify production technique, shorten the production cycle, reduce the risk that external source allergenic substance occurs, reduce the object of organic reagent discharge.
The invention provides the preparation method that a kind of sheep spermary extracts prostaglandin E1, described method steps is as follows:
Step 1, prostaglandin(PG) wet the extraction of enzyme:
(1) fresh sheep spermary is got, except degrease, muscle and reticular tissue;
(2) add potassium chloride solution, soak;
(3) homogenate is ground to form;
(4) centrifugally obtain clear liquid, filter, filtrate adjusts pH to be 4-6;
(5) the centrifugal precipitation obtained homogenate again, centrifugally obtains clear liquid, filters, and filtrate adjusts pH to be 4-6.
(6) product that mixing step (4) and (5) obtain is prostaglandin(PG) and wets enzyme;
Step 2, enzymatic reaction:
(1) prostaglandin(PG) wets in enzyme and adds dispersion liquid, adjusts pH to 7-8, adds reduced glutathion, Resorcinol, dihomo-gamma-linolenic acid mixing solutions, reaction;
(2) reaction solution adjusts pH to 4-5, is centrifugally precipitated, and precipitation adds organic solvent;
(3) filter, obtain filtrate, filtrate is reclaimed organic solvent and is obtained concentrated solution, adds damping fluid, adjusts pH to 8-9, adds petroleum ether extraction, obtain water layer, adjusts pH to 3-4, and by extracted with diethyl ether, ether layer is dry;
(4) dried ether layer after filtering, removes solvent under reduced pressure, obtains Prostaglandin E1 crude product.
Step 3, purifying:
(1) wet method dress post after 200-300 order silica gel activating.
(2) add in silicagel column after being dissolved by Prostaglandin E1 crude product trichloromethane, add trichloromethane balance, washings washs, elution, collects effluent liquid;
(3) steaming desolventizes, and obtains purifying product;
(4) obtain purifying product is repeated the purification process walking (1) ~ (3) step, obtain secondarily purified product.Step 4, refining:
(1) secondarily purified for Prostaglandin E1 product are dissolved in ethyl acetate, after filtration, filtrate crystallization, filtration drying obtains a recrystallization product;
(2) secondary recrystallization product is obtained at this by re-crystallizing in ethyl acetate;
(3) Prostaglandin E1 purified product is obtained after secondary recrystallization product drying.
Preferably, the preparation method of prostaglandin E1 of the present invention, step is as follows:
Step 1, prostaglandin(PG) wet the extraction of enzyme:
(1) get fresh sheep spermary, removing superabundant fat, muscle and reticular tissue, quick freezing and cold preserving is preserved.
(2) get freezing sheep spermary and be placed in the potassium chloride solution (being as the criterion not have sheep spermary completely) that concentration is 0.154mol/L, soak 30 ~ 60min.
(3) add in colloidal mill be ground into homogenate by soaking the sheep spermary after thawing.Grinding particle size is in the homogenate of about 0.8mm.
(4) homogenate is in 4000-4500r/min, temperature 2-8 DEG C, centrifugal 20min.By the supernatant liquid filtering after centrifugal, filtrate adjusts pH=5.0 ± 1.0 with 2mol/L citric acid soln, centrifugal once wet enzyme.
(5) precipitation after homogenate is centrifugal again homogenate obtains secondary and to wet enzyme.
(7) twice gained is wet enzyme mixing, weigh, the wet enzyme quality of record.
Step 2, enzymatic reaction:
(1) the enzyme phosphoric acid salt/EDTA-2Na mixed solution that will wet disperses, pH to 7.5 ± 0.5 is adjusted with 1mol/L potassium hydroxide solution, add reduced glutathion, Resorcinol, dihomo-gamma-linolenic acid mixing solutions that phosphate buffered saline buffer dissolves, logical oxygen 35-40 DEG C of reaction 0.5-1 hour, reacts complete and is cooled to room temperature.
(2) adjust pH to 4.0 ± 0.5 with dilute hydrochloric acid solution, after centrifugal, filtering supernatant liquor, precipitation is disperseed with the acetone of 2 times of volumes, places 12h.
(3) suction filtration acetone dispersion liquor, filter cake washing with acetone, merging filtrate and washings.Remove solvent (recyclable recycling) under reduced pressure, in concentrated solution, add the phosphate buffered saline buffer of pH8.2, and adjust pH to 8.2 ± 0.5 with potassium hydroxide solution, then use petroleum ether degreasing.After water layer adjusts pH to 3.2 ± 0.5 with citric acid soln, anhydrous diethyl ether extracts.Merge ether layer, with purified water washing, anhydrous sodium sulfate drying ether layer more than 8 hours.
(4) dried ether layer after filtering, removes solvent under reduced pressure, obtains Prostaglandin E1 crude product.
Step 3, purifying:
(1) wet method dress post after 200-300 order silica gel activating.
(2) add in silicagel column after Prostaglandin E1 crude product trichloromethane being dissolved, add the trichloromethane balance of 1 times of column volume successively, the washings washing of 3 times of column volumes and 5 times of column volume elution, cover after 1 column volume until elutriant and start to collect effluent liquid.
(3) remove collection liquid under reduced pressure solvent, obtain purifying product.
(4) a purifying product repeating step (1) ~ step (3) operation will obtained, obtains secondarily purified product.
Step 4, refining:
(1) be dissolved in ethyl acetate by secondarily purified for Prostaglandin E1 product in 1g: 20ml ratio, sealing after filtering, ambient temperatare puts 2 ~ 4h crystallization, filters after gained crystallization is drained and obtains a recrystallization product.
(2) be dissolved in ethyl acetate by a recrystallization product in 1g: 30ml ratio, sealing after filtering, room temperature places 2 ~ 4h crystallization.Drain after filtering the washing of gained crystallized from ethyl acetate, obtain secondary recrystallization product.
(3) secondary recrystallization product is put into vacuum drying oven, vacuum-drying 4h under room temperature ,-0.09MPa, obtains Prostaglandin E1 purified product.
In aforesaid method of the present invention,
Wherein, in step 2, enzymatic reaction, each composition weight percentage composition is as follows:
Wherein being prepared as follows of pH8.2 phosphate buffered saline buffer: take dipotassium hydrogen phosphate 44.0g respectively, potassium primary phosphate 1.5g, is settled to 1000ml.
Being prepared as follows of wherein phosphoric acid salt/EDTA-2Na mixed solution: pH8.2 phosphate buffered saline buffer: 0.125mol/L disodium ethylene diamine tetra-acetic acid solution (volume ratio)=9:1.
Wherein, in the purifying of step 3, each component composed as follows:
Washings: trichloromethane: methyl alcohol (volume ratio)=98:2
Elutriant: trichloromethane: methyl alcohol (volume ratio)=96:4
Method of the present invention is through screening acquisition, and screening process is as follows:
1, in the crushing process of sheep spermary, we analyze the relation of grinding particle size and total yield of products.Keeping on the basis that other processing parameters are constant, the grinding particle size of setting sheep spermary is 1.5mm, 1.0mm, 0.8mm, the finished product yield sheep spermary of equal in quality being carried out pulverize gained by three groups of parallel tests compares, and optimizes the degree of grinding parameter of sheep spermary.Its analytical results is as follows:
Table one, sheep spermary grinding particle size experimental result
| Grinding particle size 1.5mm | Grinding particle size 1.0mm | Grinding particle size 0.8mm | |
| Experimental group 1 | 18.1％ | 20.2％ | 20.9％ |
| Experimental group 2 | 17.5％ | 19.5％ | 20.2％ |
| Experimental group 3 | 18.4％ | 20.0％ | 20.5％ |
From upper table result, along with the raising of sheep spermary degree of grinding, product yield is corresponding raising also, selects sheep spermary to be crushed to 0.8mm so final.
2, in wet enzyme extraction process, we have studied the impact of different centrifugal condition on the extract yield of wet enzyme.
Table two, centrifugal condition experimental result
| Centrifugal rotational speed (r/min) | Centrifugation time (min) | Wet enzyme yield | |
| 1 | 2000 | 10 | 8.6％ |
| 2 | 2000 | 20 | 12.9％ |
| 3 | 2000 | 30 | 14.2％ |
| 4 | 3000 | 10 | 17.8％ |
| 5 | 3000 | 20 | 19.0％ |
| 6 | 3000 | 30 | 19.7％ |
| 7 | 4000 | 10 | 20.1％ |
| 8 | 4000 | 20 | 20.6％ |
| 9 | 4000 | 30 | 20.7％ |
The result that upper table orthogonal test draws shows, when rotating speed is 2000r/min and 3000r/min, the suspended substance in sheep spermary homogenate can not reach effective separating effect; When rotating speed is 4000r/min, during centrifugal 20min and 30min time wet enzyme yield difference be only 0.1%, within tolerance interval.So finally determine that centrifugal condition is more than rotating speed 4000r/min, centrifugal 20min.
3, when purifying products, the purifying elute effect of washings when we have also investigated column chromatography and the different configuration proportion of elutriant.By adjusting the configuration proportion of trichloromethane and methyl alcohol in washings and elutriant in twice purge process, detect yield and the content of products obtained therefrom after twice purifying, finally compare obtain a result as follows:
Table three, purge process washings and elutriant configuration proportion experimental result
| Washings | Elutriant | Purification yield | Content | |
| 1 | 97:3 | 95:5 | 48.9％ | 93.1％ |
| 2 | 97:3 | 96:4 | 47.5％ | 93.6％ |
| 3 | 97:3 | 97:3 | 40.3％ | 94.2％ |
| 4 | 98:2 | 95:5 | 76.0％ | 90.2％ |
| 5 | 98:2 | 96:4 | 75.7％ | 94.4％ |
| 6 | 98:2 | 97:3 | 72.1％ | 94.7％ |
| 7 | 99:1 | 95:5 | 85.8％ | 72.9％ |
| 8 | 99:1 | 96:4 | 82.5％ | 79.4％ |
| 9 | 99:1 | 97:3 | 77.4％ | 80.7％ |
By above the selection result, to the product yield after twice purifying and content carry out comprehensive analyze consider after, select washings configuration proportion to be 98:2; Elutriant configuration proportion is 96:4.
Below data declaration beneficial effect of the present invention by experiment.
Compared to the prior art, relevant data of the present invention is listed as follows:
| Yield a | Content | Color | Total assorted | Maximum single impurity | |
| The embodiment of the present invention 1 method | 18.4％ | 99.7％ | White crystals | 0.3％ | 0.1％ |
| The embodiment of the present invention 2 method | 18.9％ | 99.8％ | White crystals | 0.2％ | 0.1％ |
| Prior art 1 document | 15.7％ | 96.0％ | White crystal | 0.8％ | 0.4％ |
| Prior art 2 document | 16.2％ | 97.5％ | White crystal | 0.7％ | 0.2％ |
| Prior art 3 document | 16.4％ | 99.0％ | White crystal | 0.5％ | 0.3％ |
The yield related in a, example of the present invention is all with dihomo-gamma-linolenic acid Dosage calculation
As can be known from the above table, method of the present invention is better than prior art.
The present invention be advantageous in that:
1, the present invention is by optimum synthesis reactions steps, reduces the technical difficulty of synthetic operation, utilizes biological enzyme synthesis of prostaglandins E1 bulk drug, improves product yield.
2, the production technique that the present invention relates to completes by simple device instrument, and whole process does not relate to heavy mechanical equipment; Working method is simple, and a small amount of personnel can complete all operations, reduce further the early investment of product.
3, in production process of the present invention, do not relate to heavy metal substance catalysis, and chemical reagent usage quantity is less, reduces the risk that external source allergenic substance occurs, reduce the discharge of organic reagent.
Embodiment
Further illustrate the present invention by the following examples.
Embodiment 1:
1, the extraction of prostaglandin(PG) enzyme:
(1) fresh food frozen sheep spermary 0.154mol/L Klorvess Liquid soaks 60min.
(2) sheep spermary colloidal mill is crushed to about 0.8mm.
(3) homogenate centrifugal after, get supernatant liquor and adjust pH=5.4, centrifugal once wet enzyme.
(4) precipitation after homogenate is centrifugal again homogenate obtains secondary and to wet enzyme.
(5) twice gained is wet enzyme mixing, weigh, the wet enzyme quality of record.
2, enzymatic reaction:
The composition of raw materials of prostaglandin E1, this formula forms by weight:
(1) the enzyme phosphoric acid salt/EDTA-2Na mixed solution that will wet disperses, and adjusts pH to 7.8, adds the reduced glutathion of phosphate buffered saline buffer dissolving, Resorcinol, dihomo-gamma-linolenic acid mixing solutions participation reaction.
(2) adjust pH to 4.3, centrifugal rear filtering supernatant liquor, precipitation acetone disperses.
(3) suction filtration dispersion liquid, filter cake washing with acetone, merging filtrate and washings.Remove solvent under reduced pressure, water layer adjusts pH to 8.2, after petroleum ether degreasing, adjusts pH to 3.2, extracts with anhydrous diethyl ether.Merge ether layer, with purified water washing, and dry more than 8 hours.
(4) dried ether layer after filtering, removes solvent under reduced pressure, obtains Prostaglandin E1 crude product.
3, purifying:
(1) Prostaglandin E1 crude product is added in silicagel column, cover after 1 column volume until elutriant and start to collect effluent liquid.
(2) remove collection liquid under reduced pressure solvent, obtain purifying product.
(3) the purifying product obtained are repeated above-mentioned steps, obtain secondarily purified product.
4, refining:
(1) be dissolved in ethyl acetate by secondarily purified for Prostaglandin E1 product, after filtering, ambient temperatare is put, and filters after crystallization, and gained crystallization obtains a recrystallization product after draining.
(2) be dissolved in ethyl acetate by a recrystallization product, after filtering, room temperature is placed, and filter after crystallization, gained crystallized from ethyl acetate washs and drains, and obtains secondary recrystallization product.
(3) secondary recrystallization product is put into vacuum drying oven, vacuum-drying obtains Prostaglandin E1 purified product.
Embodiment 2:
1, the extraction of prostaglandin(PG) enzyme:
(1) sheep spermary 0.154mol/L Klorvess Liquid is gone to soak 50min.
(2) sheep spermary colloidal mill is crushed to the homogenate of about 0.8mm.
(3) homogenate centrifugal after, get supernatant liquor and adjust pH=5.7, centrifugal once wet enzyme.
(4) precipitation after homogenate is centrifugal again homogenate obtains secondary and to wet enzyme.
(5) twice gained is wet enzyme mixing, weigh, the wet enzyme quality of record.
2, enzymatic reaction:
The composition of raw materials of prostaglandin E1, this formula forms by weight:
(1) wet enzyme phosphate buffered saline buffer is disperseed, adjust pH to 7.4, add the reduced glutathion of phosphate buffered saline buffer dissolving, Resorcinol, dihomo-gamma-linolenic acid mixing solutions participation reaction.
(2) adjust pH to 4.2, centrifugal rear filtering supernatant liquor, precipitation acetone disperses.
(3) suction filtration dispersion liquid, filter cake washing with acetone, merging filtrate and washings.Remove solvent under reduced pressure, water layer adjusts pH to 8.2, after petroleum ether degreasing, adjusts pH to 3.5, extracts with anhydrous diethyl ether.Merge ether layer, with purified water washing, and dry more than 8 hours.
(4) dried ether layer after filtering, removes solvent under reduced pressure, obtains Prostaglandin E1 crude product.
3, purifying:
(1) Prostaglandin E1 crude product is added in silicagel column, cover after 1 column volume until elutriant and start to collect effluent liquid.
(2) remove collection liquid under reduced pressure solvent, obtain purifying product.
(3) the purifying product obtained are repeated above-mentioned steps, obtain secondarily purified product.
4, refining:
(1) be dissolved in ethyl acetate by secondarily purified for Prostaglandin E1 product, after filtering, ambient temperatare is put, and filters after crystallization, and gained crystallization obtains a recrystallization product after draining.
(2) be dissolved in ethyl acetate by a recrystallization product, after filtering, room temperature is placed, and filter after crystallization, gained crystallized from ethyl acetate washs and drains, and obtains secondary recrystallization product.
(3) secondary recrystallization product is put into vacuum drying oven, vacuum-drying obtains Prostaglandin E1 purified product.
Claims (6)
1. extract a preparation method for prostaglandin E1 with sheep spermary, it is characterized in that, described method steps is as follows:
Step 1, prostaglandin(PG) wet the extraction of enzyme:
(1) fresh sheep spermary is got, except degrease, muscle and reticular tissue;
(2) add potassium chloride solution, soak;
(3) homogenate is ground to form;
(4) centrifugally obtain clear liquid, filter, filtrate adjusts pH to be 4-6;
(5) the centrifugal precipitation obtained homogenate again, centrifugally obtains clear liquid, filters, and filtrate adjusts pH to be 4-6,
(6) product that mixing step (4) and (5) obtain is prostaglandin(PG) and wets enzyme;
Step 2, enzymatic reaction:
(1) prostaglandin(PG) wets in enzyme and adds dispersion liquid, adjusts pH to 7-8, adds reduced glutathion, Resorcinol, dihomo-gamma-linolenic acid mixing solutions, reaction;
(2) reaction solution adjusts pH to 4-5, is centrifugally precipitated, and precipitation adds organic solvent;
(3) filter, obtain filtrate, filtrate is reclaimed organic solvent and is obtained concentrated solution, adds damping fluid, adjusts pH to 8-9, adds petroleum ether extraction, obtain water layer, adjusts pH to 3-4, and by extracted with diethyl ether, ether layer is dry;
(4) dried ether layer after filtering, removes solvent under reduced pressure, obtains Prostaglandin E1 crude product;
Step 3, purifying:
(1) wet method dress post after 200-300 order silica gel activating;
(2) add in silicagel column after being dissolved by Prostaglandin E1 crude product trichloromethane, add trichloromethane balance, washings washs, elution, collects effluent liquid;
(3) steaming desolventizes, and obtains purifying product;
(4) obtain purifying product are repeated the purification process walking (1) ~ (3) step, obtain secondarily purified product, step 4, refining:
(1) secondarily purified for Prostaglandin E1 product are dissolved in ethyl acetate, after filtration, filtrate crystallization, filtration drying obtains a recrystallization product;
(2) secondary recrystallization product is obtained at this by re-crystallizing in ethyl acetate;
(3) Prostaglandin E1 purified product is obtained after secondary recrystallization product drying.
2. preparation method according to claim 1, is characterized in that, step is as follows:
Step 1, prostaglandin(PG) wet the extraction of enzyme:
(1) get fresh sheep spermary, removing superabundant fat, muscle and reticular tissue, quick freezing and cold preserving is preserved,
(2) get freezing sheep spermary and be placed in potassium chloride solution immersion 30 ~ 60min that concentration is 0.154mol/L,
(3) add in colloidal mill be ground into homogenate by soaking the sheep spermary after thawing, grinding particle size in the homogenate of about 0.8mm,
(4) homogenate is in 4000-4500r/min, temperature 2-8 DEG C, centrifugal 20min, and by the supernatant liquid filtering after centrifugal, filtrate adjusts pH=5.0 ± 1.0 with 2mol/L citric acid soln, centrifugal once wet enzyme,
(5) precipitation after homogenate is centrifugal again homogenate obtains secondary and to wet enzyme,
(7) twice gained is wet enzyme mixing, weigh, the wet enzyme quality of record,
Step 2, enzymatic reaction:
(1) the enzyme phosphoric acid salt/EDTA-2Na mixed solution that will wet disperses, pH to 7.5 ± 0.5 is adjusted with 1mol/L potassium hydroxide solution, add reduced glutathion, Resorcinol, dihomo-gamma-linolenic acid mixing solutions that phosphate buffered saline buffer dissolves, logical oxygen 35-40 DEG C of reaction 0.5-1 hour, react complete and be cooled to room temperature
(2) pH to 4.0 ± 0.5 is adjusted with dilute hydrochloric acid solution, after centrifugal, filtering supernatant liquor, precipitation is disperseed with the acetone of 2 times of volumes, places 12h,
(3) suction filtration acetone dispersion liquor, filter cake washing with acetone, merging filtrate and washings, remove solvent under reduced pressure, in concentrated solution, add the phosphate buffered saline buffer of pH8.2, and adjust pH to 8.2 ± 0.5 with potassium hydroxide solution, then use petroleum ether degreasing, after water layer adjusts pH to 3.2 ± 0.5 with citric acid soln, anhydrous diethyl ether extracts, and merges ether layer, washs by purified water, anhydrous sodium sulfate drying ether layer more than 8 hours
(4) dried ether layer after filtering, removes solvent under reduced pressure, obtains Prostaglandin E1 crude product,
Step 3, purifying:
(1) wet method dress post after 200-300 order silica gel activating,
(2) add in silicagel column after Prostaglandin E1 crude product trichloromethane being dissolved, add the trichloromethane balance of 1 times of column volume successively, the washings washing of 3 times of column volumes and 5 times of column volume elution, cover after 1 column volume until elutriant and start to collect effluent liquid
(3) remove collection liquid under reduced pressure solvent, obtain purifying product,
(4) a purifying product repeating step (1) ~ step (3) operation will obtained, obtains secondarily purified product,
Step 4, refining:
(1) be dissolved in ethyl acetate by secondarily purified for Prostaglandin E1 product in 1g: 20ml ratio, sealing after filtering, ambient temperatare puts 2 ~ 4h crystallization, filters after gained crystallization is drained and obtains a recrystallization product,
(2) be dissolved in ethyl acetate by a recrystallization product in 1g: 30ml ratio, sealing after filtering, room temperature places 2 ~ 4h crystallization, drains, obtain secondary recrystallization product after filtering the washing of gained crystallized from ethyl acetate,
(3) secondary recrystallization product is put into vacuum drying oven, vacuum-drying 4h under room temperature ,-0.09MPa, obtains Prostaglandin E1 purified product.
3. preparation method according to claim 1, is characterized in that, wherein, in step 2, enzymatic reaction, each composition weight is composed as follows:
Wherein being prepared as follows of pH8.2 phosphate buffered saline buffer: take dipotassium hydrogen phosphate 44.0g respectively, potassium primary phosphate 1.5g, is settled to 1000ml,
Being prepared as follows of wherein phosphoric acid salt/EDTA-2Na mixed solution: pH8.2 phosphate buffered saline buffer: 0.125mol/L disodium ethylene diamine tetra-acetic acid solution (volume ratio)=9:1.
4. preparation method according to claim 1, is characterized in that, wherein, in the purifying of step 3, and each component composed as follows:
Washings: trichloromethane: methyl alcohol (volume ratio)=98:2
Elutriant: trichloromethane: methyl alcohol (volume ratio)=96:4.
5. preparation method according to claim 1, is characterized in that, step is as follows:
A, get the sheep spermary that 0.154mol/L Klorvess Liquid soaked, utilize colloidal mill to be crushed to the homogenate of about 0.8mm, after homogenate is centrifugal, gets supernatant liquor and adjust pH=5.0 ± 1.0, the centrifugal enzyme that must wet,
B, by the wet enzyme that phosphate buffered saline buffer disperses, adjust pH to 7.5 ± 0.5, add the reduced glutathion that phosphate buffered saline buffer dissolves, Resorcinol, dihomo-gamma-linolenic acid mixing solutions, 35-40 DEG C of reaction 0.5-1 hour, adjust pH to 4.0 ± 0.5, centrifugal rear filtering supernatant liquor, precipitation acetone disperses, suction filtration dispersion liquid, filter cake washing with acetone, merging filtrate and washings, remove solvent under reduced pressure, water layer adjusts pH to 8.2 ± 0.5, after petroleum ether degreasing, adjust pH to 3.2 ± 0.5, extract with anhydrous diethyl ether, merge ether layer, wash by purified water, and dry more than 8 hours, dried ether layer after filtering, remove solvent under reduced pressure, obtain Prostaglandin E1 crude product,
C, Prostaglandin E1 crude product is added in silicagel column, covers after 1 column volume until elutriant and start to collect effluent liquid, remove collection liquid under reduced pressure solvent, obtain purifying product, the purifying product obtained are repeated above-mentioned steps, obtains secondarily purified product,
D, secondarily purified for Prostaglandin E1 product are dissolved in ethyl acetate, after filtering, ambient temperatare is put, filter after crystallization, gained crystallization obtains a recrystallization product after draining, and is dissolved in ethyl acetate by a recrystallization product, after filtering, room temperature is placed, filter after crystallization, gained crystallized from ethyl acetate washs and drains, and obtains secondary recrystallization product, secondary recrystallization product is put into vacuum drying oven, and vacuum-drying obtains Prostaglandin E1 purified product.
6. preparation method according to claim 1, is characterized in that, step is as follows:
The extraction of step 1, prostaglandin(PG) enzyme:
(1) fresh food frozen sheep spermary 0.154mol/L Klorvess Liquid soaks 60min,
(2) sheep spermary colloidal mill is crushed to about 0.8mm,
(3) homogenate centrifugal after, get supernatant liquor and adjust pH=5.4, centrifugal once wet enzyme,
(4) precipitation after homogenate is centrifugal again homogenate obtains secondary and to wet enzyme,
(5) twice gained is wet enzyme mixing, weigh, the wet enzyme quality of record,
Step 2, enzymatic reaction:
Each compositions in weight percentage composed as follows:
(1) the enzyme phosphoric acid salt/EDTA-2Na mixed solution that will wet disperses, and adjusts pH to 7.8, adds the reduced glutathion of phosphate buffered saline buffer dissolving, Resorcinol, dihomo-gamma-linolenic acid mixing solutions participation reaction,
(2) adjust pH to 4.3, centrifugal rear filtering supernatant liquor, precipitation acetone disperses,
(3) suction filtration dispersion liquid, filter cake washing with acetone, merging filtrate and washings, remove solvent under reduced pressure, and water layer adjusts pH to 8.2, after petroleum ether degreasing, adjusts pH to 3.2, with anhydrous diethyl ether extraction, merges ether layer, with purified water washing, and dry more than 8 hours,
(4) dried ether layer after filtering, removes solvent under reduced pressure, obtains Prostaglandin E1 crude product,
Step 3, purifying:
Each component composed as follows:
Washings: trichloromethane: methyl alcohol (volume ratio)=98:2
Elutriant: trichloromethane: methyl alcohol (volume ratio)=96:4
(1) Prostaglandin E1 crude product is added in silicagel column, covers after 1 column volume until elutriant and start to collect effluent liquid,
(2) remove collection liquid under reduced pressure solvent, obtain purifying product,
(3) the purifying product obtained are repeated above-mentioned steps, obtain secondarily purified product,
Step 4, refining:
(1) be dissolved in ethyl acetate by secondarily purified for Prostaglandin E1 product, after filtering, ambient temperatare is put, and filters after crystallization, and gained crystallization obtains a recrystallization product after draining,
(2) be dissolved in ethyl acetate by a recrystallization product, after filtering, room temperature is placed, and filter after crystallization, gained crystallized from ethyl acetate washs and drains, and obtains secondary recrystallization product,
(3) secondary recrystallization product is put into vacuum drying oven, vacuum-drying obtains Prostaglandin E1 purified product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN110951814A (en) * | 2019-12-30 | 2020-04-03 | 长春理工大学 | Method for preparing prostaglandin E1 by using genetically engineered cyclooxygenase-1 and genetically engineered prostaglandin E synthetase-1 |
| CN114599791A (en) * | 2019-12-26 | 2022-06-07 | 协和医药化工股份有限公司 | Method for producing prostaglandin |
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| CN114599791A (en) * | 2019-12-26 | 2022-06-07 | 协和医药化工股份有限公司 | Method for producing prostaglandin |
| CN110951814A (en) * | 2019-12-30 | 2020-04-03 | 长春理工大学 | Method for preparing prostaglandin E1 by using genetically engineered cyclooxygenase-1 and genetically engineered prostaglandin E synthetase-1 |
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