CN109825547A - A method of passing through half enzymatic clarification ursodesoxycholic acid by raw material of cholic acid - Google Patents
A method of passing through half enzymatic clarification ursodesoxycholic acid by raw material of cholic acid Download PDFInfo
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Abstract
The invention discloses a kind of methods for passing through half enzymatic clarification ursodesoxycholic acid using cholic acid as raw material, ketone group is first converted for the trihydroxy of cholic acid using chemical method, recycle distinctive 3 α hydroxyl dehydrogenase, 7 β hydroxyl dehydrogenases and some cofactors, efficiently reduction converts hydroxyl to orientation, 12 ketone groups finally are removed using huang-Minlon reaction, obtain ursodeoxycholic acid crude.Synthetic method of the present invention is environmentally friendly, safe and efficient, and the conversion ratio of every step process is 95% or more.
Description
Technical field
The present invention relates to technical field of bioengineering, pass through half enzymatic clarification bear by raw material of cholic acid more particularly to one kind
The method of deoxycholic aicd.
Background technique
Ursodesoxycholic acid (- 5 β of 3 α, 7 β-dihydroxy-cholestane -24- acid) is a kind of bile acid, for treating gallbladder knot
Stone, cholestatic liver disease, fatty liver, various hepatitis, Poisoning hepatopathy, cholecystitis, cholangitis and bilious dyspepsia,
Bile gastritis, eye disease etc..Ursodesoxycholic acid is primarily present in bear gall, since bear is national protection animal,
It cannot arbitrarily catch and kill, therefore ursodesoxycholic acid on the market is mainly derived from synthesis at present.
Cholic acid (3 α, 7 α, -5 β of 12 α-trihydroxy-cholestane -24- acid) has the benefit that clears liver in bovine and sheep bile
Gallbladder, removing toxic substances and other effects, is that the current country is mainly used in the raw material for preparing calculus bovis factitius and Qingkailing preparation.As the country cultivates
The gradually development of industry, cholic acid raw material also substantially increase therewith, and existing cholic acid has begun the original as synthesis ursodesoxycholic acid
Material.However, existing synthesis technology is all to protect 3 α using acetylation in industry, 7 α hydroxyls, then huang-Minlon reaction removes 12 α
Position hydroxyl, hydrolysis production chenodeoxycholic acid, then restored through chemical method oxidation, chemical method, finally obtain ursodeoxycholic acid crude.Cause
This, which has following defects that first is that process route is long, and the production cycle is long, and processing step is more, and cause cost to occupy high,
Process stabilizing controlling is poor；Second is that being related to strong oxidizer and strong reductant in production process, cause security risk huge；And
A large amount of solvents can be also related to as carrier, generate a large amount of waste water, cause environmental protection very risky；Third is that chemical oxidation and reduction can
A large amount of by-products are generated, to influence conversion ratio and recovery rate, cause cost high.
Summary of the invention
In view of this, it is an object of the invention to propose that one kind passes through half enzymatic clarification ursodesoxycholic acid by raw material of cholic acid
Method, the process of the process is: first by the trihydroxy of cholic acid converting ketone group, i.e., 3,7,12-triketones using chemical method
- 5 β of base-cholestane -24- is sour (neocholan)；Recycle distinctive 3 α hydroxyl dehydrogenase, 7 β hydroxyl dehydrogenases and some coenzyme
The factor orients efficiently reduction conversion hydroxyl, i.e. 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid；Finally utilize Huang Minglong
12 ketone groups are removed in reaction, obtain ursodesoxycholic acid (- 5 β of 3 α, 7 β-dihydroxy-cholestane -24- acid) crude product.Above-mentioned synthesis side
Method is environmentally friendly, safe and efficient, and the conversion ratio of every step process is 95% or more.
To achieve the above object, the present invention adopts the following technical scheme:
A method of passing through half enzymatic clarification ursodesoxycholic acid by raw material of cholic acid, which is characterized in that including aoxidizing cholic acid
The step of for neocholan, enzymatic conversion method are 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid step, and utilization
Huang-Minlon reaction is converted into the step of ursodesoxycholic acid, and concrete technology method is as follows:
One, cholic acid is oxidized to neocholan, i.e., 3,7, the preparation of tri- -5 β of ketone group of 12--cholestane -24- acid；
(1.1) it takes cholic acid and the solvent A of 8 times of cholic acid deals to be pumped into oxidation tank, under room temperature after stirring and dissolving, 0.2 times of gallbladder is added
The glacial acetic acid of sour deal stirs 30 minutes, and adjusting pH value is 2-4；It takes a sample to check until completely dissolved, freezing liquid refrigeration is added, makes
Solution temperature is down to 0-5 DEG C, for use.
(1.2) oxidant of cholic acid deal 25-30% is added in three times in oxidation tank, controlled at 5-10 DEG C, continues
Heat preservation 1 hour, HPLC monitors the extent of reaction, until cholic acid content is less than 0.2%.
(1.3) 20% sodium hydrate aqueous solution is added in oxidation tank, adjusting pH value is 9-10, continue stirring 30 minutes,
It opens oxidation bottom valve and solution is pumped into concentration kettle.
(1.4) to concentration kettle steam heating recovery solvent A, until solution is in thick paste in kettle, tap water is added, continues to steam
Evaporate remaining solvent A, gas-chromatography inspection into fraction solvent A content to 1% hereinafter, solution is cooled to 35-40 DEG C, for use.
(1.5) solution stand-by in step (1.4) is pumped into acidification kettle, 10% aqueous hydrochloric acid solution is added while stirring, adjusts
Section pH value is 2-3, continues stirring 30 minutes.Centrifugation, mother liquor enter sewage plant processing；Collection material, obtain neocholan wet product (3,
7, tri- -5 β of ketone group of 12--cholestane -24- acid), for use.
Two, the preparation of 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid；
(2.1) purified water is added in enzymatic hydrolysis kettle, stand-by neocholan wet product is added while stirring, the deal of water is dehydrogenation gallbladder
Then the sodium dihydrogen phosphate of cholic acid deal 3-3.5% is added in 5 times of sour wet product, adjust PH using 20% sodium hydrate aqueous solution
Value is 7-8, and temperature control is 30-35 DEG C, after stirring and dissolving, for use.
(2.2) glucose that cholic acid deal 50-60% is added in enzymatic hydrolysis kettle continuously adds cholic acid part after stirring 15 minutes
Measure the Portugal of the 3 α dehydrogenation bacterial enzymes of 13-14%MU, the 7 β dehydrogenation bacterial enzymes of cholic acid deal 13-14%MU, cholic acid deal 50-60%MU
Grape sugar dehydrogenation bacterial enzyme, the coenzyme one of ten thousand/1.4-1.5 of cholic acid deal, ten thousand/2.3-2.5 of cholic acid deal coenzyme two.Drop
Add 5% sodium hydrate aqueous solution stablize pH value be 7-7.5, react 3 hours, HPLC monitor neocholan content < 0.2%, 3,
- 7 β hydroxycholan acid content < 0.2% of 12- diketo, 7, -3 α hydroxycholan acid content < 0.2% of 12- diketo.Using
Liquid temperature is steam heated to up to 50 DEG C, keeps the temperature 30 minutes, for use.
(2.3) enzymolysis liquid in step (2.2) is separated into enzyme slag by diaphragm filter press, purifying is added in kettle after the completion
Water cleaning of enzyme slag, merging filtrate enter souring tank, and enzyme slag is discarded, can be used as bio-feritlizer regeneration.
(2.4) cooling water is opened in souring tank, so that solution temperature is down to 35-40 DEG C, the hydrochloric acid of agitation and dropping 10% is water-soluble
It is 2-3 that liquid, which adjusts pH value, insulated and stirred 10 minutes, puts into filter press, and filters pressing separates bile acid, and sour water enters sewage plant processing,
Collect to obtain wet product material (3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid).
(2.5) the wet product material collected in step (2.4) is put into vacuum rake dryer, 80 DEG C of temperature control, be dried in vacuo
To moisture < 2.0%, collection material is stand-by.
Three, huang-Minlon reaction prepares ursodesoxycholic acid
(3.1) by material investment pyroreaction kettle stand-by in step (2.5), the water of 2 times of material deals is added, is added 4 times
The sodium hydroxide of 0.15 times of material deal, the hydrazine hydrate of 0.11 times of material deal, oil bath heating is added in the solvent B of material deal
It is warming up to 140 DEG C, waste water is collected in recycling (into sewage plant).Temperature control opens reflux after reaching 140 DEG C, keeps the temperature 2 hours and reacts.
(3.2) recycling is opened, is continuously heating to 200 DEG C, recycles extra hydrazine hydrate around here (after this hydrazine hydrate detects purity
Can apply), 200 DEG C of reflux 2-4 hours, sampling Detection are kept the temperature, HPLC monitors 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-
The content < 0.2% of 24- acid.
(3.3) vacuum degree -0.07Mpa is controlled, at 120-140 DEG C, vacuum recycles solvent B for temperature control, collects solvent B set
With until paste, stops concentration.
(3.4) water of 8 times of material deals is added, stirring dissolves paste sufficiently, is transferred in acidification kettle, cooling is added
Water is cooled to 40-50 DEG C, and it is 2-3 that the aqueous hydrochloric acid solution that stirring is added 10%, which adjusts pH value, keeps the temperature 10-30 minutes, and bear deoxidation is precipitated
Cholic acid crude product, filters pressing separation, waste water enter sewage plant, and collection material enters boiled bed drying machine, the drying 4 hours of 100 DEG C of temperature control,
Ursodeoxycholic acid crude is obtained, detecting its purity is 95% or more.
Further, the solvent A in the step (1.1) is one of acetone, methanol, ethyl alcohol, ethyl acetate.
Further, the freezing liquid in the step (1.1) is freezing ethylene glycol.
Further, the oxidant in the step (1.2) is calcium hypochlorite.
Further, the solvent B in the step (3.1) is one of ethylene glycol, diethylene glycol (DEG), glycerol.
The present invention has the advantages that
1, entire technical process is divided into three-step reaction, cholic acid is converted into ursodesoxycholic acid, conversion process is not required to excessively arrive
Chenodeoxycholic acid, process cycle reduce 2/3 compared with traditional handicraft, and conversion yields are increased to 90% or more.Through the detection present invention
The conversion yields of method are 96%, and the conversion yields of conventional chemical methods technique are 80% or so.
2, process cycle is reduced, then significantly reduces production cost, improves the competitiveness of product in market.
3, the introducing of biological enzyme technology reduces traditional handicraft using strong oxidizer, strong metal reducing agent and carries out chiral turn
The high safety risk and high-environmental risk changed.
4, feature efficient using biological enzyme, single-minded targets group and reacts, and no coupling product generates, and greatly reduces
Impurity improves conversion ratio and recovery rate.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this hair
Bright further description.
Embodiment 1
A method of using cholic acid it is raw material by half enzymatic clarification ursodesoxycholic acid, concrete technology method is as follows:
One, cholic acid is oxidized to neocholan, i.e., 3,7, the preparation of tri- -5 β of ketone group of 12--cholestane -24- acid；
(1.1) it is pumped into 400 kilograms of acetone in the enamel oxidation tank of 2000L, the cholic acid 50 that content is 95.1% is put into acetone
Kilogram, under room temperature after stirring and dissolving, 10 kilograms of glacial acetic acids are added, stir 30 minutes, adjusting pH value is 2.5；It takes a sample to check to complete
After fully dissolved, enamel aoxidizes the freezing ethylene glycol refrigeration that tank interlayer is added -10 DEG C, so that solution temperature is down to 3 DEG C, for use.
(1.2) 13.5 kilograms of calcium hypochlorite is added in three times in enamel oxidation tank, wherein the deal being added three times point
It Wei not be 5 kilograms, 5 kilograms, 3.5 kilograms.Controlled at 6.8 DEG C, the extent of reaction is monitored by HPLC after continuing heat preservation 1 hour,
Sample detection cholic acid content is 0.08%.
(1.3) 20% sodium hydrate aqueous solution is added in enamel oxidation tank, adjusting pH value is 9.5, continues 30 points of stirring
Clock opens oxidation bottom valve for solution and is pumped into oxidation concentration kettle.
(1.4) to oxidation concentration kettle steam heating recovery acetone, until solution is in thick paste in kettle, 400 kilograms are added certainly
Water, continues to distill residual acetone, gas-chromatography inspection into fraction content of acetone to 1% hereinafter, solution is cooled to 36 DEG C,
For use.
(1.5) solution stand-by in step (1.4) is pumped into enamel acidification kettle, the hydrochloric acid of addition 10% is water-soluble while stirring
Liquid, adjusting pH value is 2.7, continues stirring 30 minutes.Centrifugation, mother liquor enter sewage plant processing；It is wet to obtain neocholan for collection material
107 kilograms of net weight of product (3,7, tri- -5 β of ketone group of 12--cholestane -24- acid), for use.
Two, the preparation of 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid；
(2.1) 535 kilograms of purified waters are added in stainless steel enzymatic hydrolysis kettle, 107 public affairs stand-by in step (1.5) is added while stirring
Jin neocholan wet product, is then added 1.56 kilograms of sodium dihydrogen phosphate, use 20% sodium hydrate aqueous solution adjust pH value for
7.37, temperature control is 31.5 DEG C, after stirring and dissolving, for use.
(2.2) 26.7 kilograms of glucose is added in stainless steel enzymatic hydrolysis kettle and continuously adds 6.7MU after stirring 15 minutes
3 α dehydrogenation bacterial enzymes, the 7 β dehydrogenation bacterial enzymes of 6.7MU, the glucose dehydrogenation bacterial enzyme of 26.7MU, 7.2 grams one, 12 gram of coenzyme
Coenzyme two.The sodium hydrate aqueous solution for being added dropwise 5% stablizes pH value as 7-7.5, reacts 3 hours, and HPLC monitoring neocholan content is
0.11%, 3, -7 β hydroxycholan acid content of 12- diketo be 0.05%, 7, -3 α hydroxycholan acid content of 12- diketo be
0.09%.Using liquid temperature is steam heated to up to 50 DEG C, 30 minutes are kept the temperature, for use.
(2.3) enzymolysis liquid in step (2.2) is separated into enzyme slag by diaphragm filter press, is added 100 in kettle after the completion
Kilogram purified water cleaning of enzyme slag, merging filtrate enter souring tank, and enzyme slag is discarded, can be used as bio-feritlizer regeneration.
(2.4) cooling water is opened in souring tank, so that solution temperature is down to 39 DEG C, the aqueous hydrochloric acid solution of agitation and dropping 10%
Adjusting pH value is 2.4, insulated and stirred 10 minutes, puts into filter press, filters pressing separates bile acid, and sour water enters sewage plant processing, receives
Collect to obtain 110.2 kilograms of wet product material (3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid).
(2.5) the wet product material collected in step (2.4) is put into vacuum rake dryer, 80 DEG C of temperature control, dry 4 is small
When, being dried under vacuum to moisture is 1.8%, and collection material 47.6 is stand-by.
Three, huang-Minlon reaction prepares ursodesoxycholic acid
(3.1) by material investment pyroreaction kettle stand-by in step (2.5), 95.2 kg of water is added, are added 190.4 kilograms
Ethylene glycol, 7.14 kilograms of sodium hydroxide, 5.2 kilograms of hydrazine hydrate is added, oil bath heating is warming up to 140 DEG C, and recycling is collected
Waste water (into sewage plant).Temperature control opens reflux after reaching 140 DEG C, keeps the temperature 2 hours and reacts.
(3.2) recycling is opened, is continuously heating to 200 DEG C, recycles extra hydrazine hydrate around here (after this hydrazine hydrate detects purity
Can apply), it keeps the temperature 200 DEG C and flows back 3.5 hours, sampling Detection, HPLC monitors 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-
The content of 24- acid is 0.06%, ursodesoxycholic acid 96.1%.
(3.3) vacuum degree -0.07Mpa is controlled, at 120-140 DEG C, vacuum recycles ethylene glycol for temperature control, collects ethylene glycol
It applies, until paste, stops concentration.
(3.4) 380 kilograms of tap water is added, stirring dissolves paste sufficiently, is transferred in enamel acidification kettle, is added
For cooling water temperature to 45 DEG C, it is 2.2 that the aqueous hydrochloric acid solution that stirring is added 10%, which adjusts pH value, keeps the temperature 10 minutes, and ursodeoxycholic is precipitated
Acid crude, filters pressing separation, waste water enter sewage plant, and collection material enters boiled bed drying machine, and the drying 4 hours of 100 DEG C of temperature control obtains
46.1 kilograms of ursodeoxycholic acid crude, detecting its purity is 96.6%.
Embodiment 2
A method of using cholic acid it is raw material by half enzymatic clarification ursodesoxycholic acid, concrete technology method is as follows:
One, cholic acid is oxidized to neocholan, i.e., 3,7, the preparation of tri- -5 β of ketone group of 12--cholestane -24- acid；
(1.1) it is pumped into 500 kilograms of methanol in the enamel oxidation tank of 2000L, the cholic acid that content is 92.9% is put into methanol
62.5 kilograms, under room temperature after stirring and dissolving, 12.5 kilograms of glacial acetic acids are added, stir 30 minutes, adjusting pH value is 3；It takes a sample to check
Until completely dissolved, -10 DEG C of freezing ethylene glycol refrigeration is added in enamel oxidation tank interlayer, and solution temperature is made to be down to 2.5 DEG C, to
With.
(1.2) 16.9 kilograms of calcium hypochlorite is added in three times in enamel oxidation tank, wherein the deal being added three times point
It Wei not be 5 kilograms, 5 kilograms, 6.9 kilograms.Controlled at 8.8 DEG C, the extent of reaction is monitored by HPLC after continuing heat preservation 1 hour,
Sample detection cholic acid content is 0.12%.
(1.3) 20% sodium hydrate aqueous solution is added in enamel oxidation tank, adjusting pH value is 9.7, continues 30 points of stirring
Clock opens oxidation bottom valve for solution and is pumped into oxidation concentration kettle.
(1.4) to oxidation concentration kettle steam heating recovery methanol, until solution is in thick paste in kettle, 400 kilograms are added certainly
Water, continues to distill residual methanol, gas-chromatography inspection into fraction content of acetone to 1% hereinafter, solution is cooled to 39 DEG C,
For use.
(1.5) solution stand-by in step (1.4) is pumped into enamel acidification kettle, the hydrochloric acid of addition 10% is water-soluble while stirring
Liquid, adjusting pH value is 2.1, continues stirring 30 minutes.Centrifugation, mother liquor enter sewage plant processing；It is wet to obtain neocholan for collection material
130.8 kilograms of net weight of product (3,7, tri- -5 β of ketone group of 12--cholestane -24- acid), for use.
Two, the preparation of 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid；
(2.1) 654 kilograms of purified waters are added in stainless steel enzymatic hydrolysis kettle, 130.8 in step (1.5) for use is added while stirring
Then 1.95 kilograms of sodium dihydrogen phosphate is added in kilogram neocholan wet product, adjust pH value using 20% sodium hydrate aqueous solution
It is 7.27, temperature control is 34.5 DEG C, after stirring and dissolving, for use.
(2.2) 33.4 kilograms of glucose is added in stainless steel enzymatic hydrolysis kettle and continuously adds 8.4MU after stirring 15 minutes
3 α dehydrogenation bacterial enzymes, the 7 β dehydrogenation bacterial enzymes of 8.4MU, the glucose dehydrogenation bacterial enzyme of 26.7MU, 15 grams one, 15 gram of coenzyme it is auxiliary
Enzyme two.The sodium hydrate aqueous solution for being added dropwise 5% stablizes pH value as 7-7.5, reacts 3 hours, and HPLC monitoring neocholan content is
0.11%, 3, -7 β hydroxycholan acid content of 12- diketo be 0.09%, 7, -3 α hydroxycholan acid content of 12- diketo be
0.03%.Using liquid temperature is steam heated to up to 50 DEG C, 30 minutes are kept the temperature, for use.
(2.3) enzymolysis liquid in step (2.2) is separated into enzyme slag by diaphragm filter press, is added 125 in kettle after the completion
Kilogram purified water cleaning of enzyme slag, merging filtrate enter souring tank, and enzyme slag is discarded, can be used as bio-feritlizer regeneration.
(2.4) cooling water is opened in souring tank, so that solution temperature is down to 35 DEG C, the aqueous hydrochloric acid solution of agitation and dropping 10%
Adjusting pH value is 2.2, insulated and stirred 10 minutes, puts into filter press, filters pressing separates bile acid, and sour water enters sewage plant processing, receives
Collect to obtain 130.1 kilograms of wet product material (3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid).
(2.5) the wet product material collected in step (2.4) is put into vacuum rake dryer, 80 DEG C of temperature control, dry 4 is small
When, being dried under vacuum to moisture is 1.8%, and collection material 59.8 is stand-by.
Three, huang-Minlon reaction prepares ursodesoxycholic acid
(3.1) by material investment pyroreaction kettle stand-by in step (2.5), 119.6 kg of water is added, it is public to be added 239.2
8.97 kilograms of sodium hydroxide, 6.58 kilograms of hydrazine hydrate is added in the diethylene glycol (DEG) of jin, and oil bath heating is warming up to 140 DEG C, and recycling is received
Collect waste water (into sewage plant).Temperature control opens reflux after reaching 140 DEG C, keeps the temperature 2 hours and reacts.
(3.2) recycling is opened, is continuously heating to 200 DEG C, recycles extra hydrazine hydrate around here (after this hydrazine hydrate detects purity
Can apply), it keeps the temperature 200 DEG C and flows back 3 hours, sampling Detection, HPLC monitors 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24-
The content of acid is 0.04%, ursodesoxycholic acid 95.7%.
(3.3) vacuum degree -0.07Mpa is controlled, at 120-140 DEG C, vacuum recycles diethylene glycol (DEG) for temperature control, collects diethylene glycol (DEG)
It applies, until paste, stops concentration.
(3.4) 478.4 kilograms of tap water is added, stirring dissolves paste sufficiently, is transferred in enamel acidification kettle, adds
Enter cooling water temperature to 42 DEG C, it is 2.6 that the aqueous hydrochloric acid solution that stirring is added 10%, which adjusts pH value, keeps the temperature 10 minutes, and bear deoxidation is precipitated
Cholic acid crude product, filters pressing separation, waste water enter sewage plant, and collection material enters boiled bed drying machine, the drying 4 hours of 100 DEG C of temperature control,
57.5 kilograms of ursodeoxycholic acid crude are obtained, detecting its purity is 93.4%.
Embodiment 3
A method of using cholic acid it is raw material by half enzymatic clarification ursodesoxycholic acid, concrete technology method is as follows:
One, cholic acid is oxidized to neocholan, i.e., 3,7, the preparation of tri- -5 β of ketone group of 12--cholestane -24- acid；
(1.1) 480 kg ethanols are pumped into the enamel oxidation tank of 2000L, the cholic acid 60 that content is 93.8% is put into ethyl alcohol
Kilogram, under room temperature after stirring and dissolving, 12 kilograms of glacial acetic acids are added, stir 30 minutes, adjusting pH value is 2.5；It takes a sample to check to complete
After fully dissolved, enamel aoxidizes the freezing ethylene glycol refrigeration that tank interlayer is added -10 DEG C, so that solution temperature is down to 2.5 DEG C, for use.
(1.2) 16.2 kilograms of calcium hypochlorite is added in three times in enamel oxidation tank, wherein the deal being added three times point
It Wei not be 5 kilograms, 5 kilograms, 6.2 kilograms.Controlled at 8.8 DEG C, the extent of reaction is monitored by HPLC after continuing heat preservation 1 hour,
Sample detection cholic acid content is 0.07%.
(1.3) 20% sodium hydrate aqueous solution is added in enamel oxidation tank, adjusting pH value is 9.9, continues 30 points of stirring
Clock opens oxidation bottom valve for solution and is pumped into oxidation concentration kettle.
(1.4) to oxidation concentration kettle steam heating recovery ethyl alcohol, until solution is in thick paste in kettle, 400 kilograms are added certainly
Water, continues to distill residual ethanol, gas-chromatography inspection into fraction content of acetone to 1% hereinafter, solution is cooled to 39 DEG C,
For use.
(1.5) solution stand-by in step (1.4) is pumped into enamel acidification kettle, the hydrochloric acid of addition 10% is water-soluble while stirring
Liquid, adjusting pH value is 2.7, continues stirring 30 minutes.Centrifugation, mother liquor enter sewage plant processing；It is wet to obtain neocholan for collection material
128 kilograms of net weight of product (3,7, tri- -5 β of ketone group of 12--cholestane -24- acid), for use.
Two, the preparation of 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid；
(2.1) 640 kilograms of purified waters are added in stainless steel enzymatic hydrolysis kettle, 128 public affairs stand-by in step (1.5) is added while stirring
Jin neocholan wet product, is then added 1.87 kilograms of sodium dihydrogen phosphate, use 20% sodium hydrate aqueous solution adjust pH value for
7.24, temperature control is 32.7 DEG C, after stirring and dissolving, for use.
(2.2) 32 kilograms of glucose is added in stainless steel enzymatic hydrolysis kettle and continuously adds 3 α of 8MU after stirring 15 minutes
Dehydrogenation bacterial enzyme, the 7 β dehydrogenation bacterial enzymes of 8MU, the glucose dehydrogenation bacterial enzyme of 32MU, 14.4 grams of coenzyme, one, 14.4 gram of coenzyme
Two.The sodium hydrate aqueous solution for being added dropwise 5% stablizes pH value as 7-7.5, reacts 3 hours, and HPLC monitoring neocholan content is
0.10%, 3, -7 β hydroxycholan acid content of 12- diketo be 0.01%, 7, -3 α hydroxycholan acid content of 12- diketo be
0.05%.Using liquid temperature is steam heated to up to 50 DEG C, 30 minutes are kept the temperature, for use.
(2.3) enzymolysis liquid in step (2.2) is separated into enzyme slag by diaphragm filter press, is added 120 in kettle after the completion
Kilogram purified water cleaning of enzyme slag, merging filtrate enter souring tank, and enzyme slag is discarded, can be used as bio-feritlizer regeneration.
(2.4) cooling water is opened in souring tank, so that solution temperature is down to 35 DEG C, the aqueous hydrochloric acid solution of agitation and dropping 10%
Adjusting pH value is 2.1, insulated and stirred 10 minutes, puts into filter press, filters pressing separates bile acid, and sour water enters sewage plant processing, receives
Collect to obtain 119.5 kilograms of wet product material (3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid).
(2.5) the wet product material collected in step (2.4) is put into vacuum rake dryer, 80 DEG C of temperature control, dry 4 is small
When, being dried under vacuum to moisture is 1.4%, and collection material 57.5 is stand-by.
Three, huang-Minlon reaction prepares ursodesoxycholic acid
(3.1) by material investment pyroreaction kettle stand-by in step (2.5), 115 kg of water is added, are added 230 kilograms
8.63 kilograms of sodium hydroxide, 6.33 kilograms of hydrazine hydrate is added in glycerol, and oil bath heating is warming up to 140 DEG C, and waste water is collected in recycling
(into sewage plant).Temperature control opens reflux after reaching 140 DEG C, keeps the temperature 2 hours and reacts.
(3.2) recycling is opened, is continuously heating to 200 DEG C, recycles extra hydrazine hydrate around here (after this hydrazine hydrate detects purity
Can apply), it keeps the temperature 200 DEG C and flows back 3 hours, sampling Detection, HPLC monitors 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24-
The content of acid is 0.11%, ursodesoxycholic acid 96.9%.
(3.3) vacuum degree -0.07Mpa is controlled, at 120-140 DEG C, vacuum recycles glycerol for temperature control, collects glycerol set
With until paste, stops concentration.
(3.4) 460 kilograms of tap water is added, stirring dissolves paste sufficiently, is transferred in enamel acidification kettle, is added
For cooling water temperature to 48 DEG C, it is 2.5 that the aqueous hydrochloric acid solution that stirring is added 10%, which adjusts pH value, keeps the temperature 10 minutes, and ursodeoxycholic is precipitated
Acid crude, filters pressing separation, waste water enter sewage plant, and collection material enters boiled bed drying machine, and the drying 4 hours of 100 DEG C of temperature control obtains
55.5 kilograms of ursodeoxycholic acid crude, detecting its purity is 94.4%.
Cholic acid raw material, ursodeoxycholic acid crude in above three embodiments are examined, correlated results is as follows:
Subordinate list one: investment, output yield and conversion ratio
It is known that the method for the present invention passes through half enzymatic clarification ursodesoxycholic acid, conversion stream by raw material of cholic acid from subordinate list one
Cheng Buxu excessively arrives chenodeoxycholic acid, cholic acid directly can be converted into ursodesoxycholic acid, conversion yields be increased to 90% with
On, it is significantly better than the conversion yields (80% or so) of conventional chemical methods technique.
Claims (5)
1. a kind of pass through the method for half enzymatic clarification ursodesoxycholic acid by raw material of cholic acid, which is characterized in that including by cholic acid oxygen
The step of turning to neocholan, enzymatic conversion method are 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid step, Yi Jili
The step of being converted into ursodesoxycholic acid with huang-Minlon reaction, concrete technology method are as follows:
One, cholic acid is oxidized to neocholan, i.e., 3,7, the preparation of tri- -5 β of ketone group of 12--cholestane -24- acid
(1.1) it takes cholic acid and the solvent A of 8 times of cholic acid deals to be pumped into oxidation tank, under room temperature after stirring and dissolving, 0.2 times of gallbladder is added
The glacial acetic acid of sour deal stirs 30 minutes, and adjusting pH value is 2-4；It takes a sample to check until completely dissolved, freezing liquid refrigeration is added, makes
Solution temperature is down to 0-5 DEG C, for use；
(1.2) oxidant of cholic acid deal 25-30% is added in three times in oxidation tank, controlled at 5-10 DEG C, continues to keep the temperature
1 hour, HPLC monitored the extent of reaction, until cholic acid content is less than 0.2%；
(1.3) 20% sodium hydrate aqueous solution is added in oxidation tank, adjusting pH value is 9-10, continues stirring 30 minutes, opens
It aoxidizes bottom valve and solution is pumped into concentration kettle；
(1.4) to concentration kettle steam heating recovery solvent A, until solution is in thick paste in kettle, tap water is added, continues to distill residual
Remaining solvent A, gas-chromatography inspection into fraction solvent A content to 1% hereinafter, solution is cooled to 35-40 DEG C, for use；
(1.5) solution stand-by in step (1.4) is pumped into acidification kettle, 10% aqueous hydrochloric acid solution is added while stirring, adjusts PH
Value is 2-3, continues stirring 30 minutes；Centrifugation, mother liquor enter sewage plant processing；Collection material, obtain neocholan wet product (3,7,
Tri- -5 β of ketone group of 12--cholestane -24- acid), for use；
Two, the preparation of 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid
(2.1) purified water is added in enzymatic hydrolysis kettle, stand-by neocholan wet product is added while stirring, the deal of water is dehydrogenation gallbladder
Then the sodium dihydrogen phosphate of cholic acid deal 3-3.5% is added in 5 times of sour wet product, adjust PH using 20% sodium hydrate aqueous solution
Value is 7-8, and temperature control is 30-35 DEG C, after stirring and dissolving, for use；
(2.2) glucose that cholic acid deal 50-60% is added in enzymatic hydrolysis kettle continuously adds cholic acid deal after stirring 15 minutes
The 3 α dehydrogenation bacterial enzymes of 13-14%MU, the 7 β dehydrogenation bacterial enzymes of cholic acid deal 13-14%MU, cholic acid deal 50-60%MU grape
Sugared dehydrogenation bacterial enzyme, the coenzyme one of ten thousand/1.4-1.5 of cholic acid deal, ten thousand/2.3-2.5 of cholic acid deal coenzyme two；It is added dropwise
It is 7-7.5 that 5% sodium hydrate aqueous solution, which stablizes pH value, is reacted 3 hours, and HPLC monitors neocholan content < 0.2%, 3,12-
- 7 β hydroxycholan acid content < 0.2% of diketo, 7, -3 α hydroxycholan acid content < 0.2% of 12- diketo；Using steaming
Vapour is heated to liquid temperature up to 50 DEG C, keeps the temperature 30 minutes, for use；
(2.3) enzymolysis liquid in step (2.2) is separated into enzyme slag by diaphragm filter press, it is clear that purified water is added in kettle after the completion
Enzyme slag is washed, merging filtrate enters souring tank, and enzyme slag is discarded；
(2.4) cooling water is opened in souring tank, so that solution temperature is down to 35-40 DEG C, the aqueous hydrochloric acid solution tune of agitation and dropping 10%
Section pH value is 2-3, insulated and stirred 10 minutes, puts into filter press, filters pressing separates bile acid, and sour water enters sewage plant processing, collects
Obtain wet product material (3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid)；
(2.5) the wet product material collected in step (2.4) is put into vacuum rake dryer, 80 DEG C of temperature control, is dried under vacuum to water
Divide < 2.0%, collection material is stand-by；
Three, huang-Minlon reaction prepares ursodesoxycholic acid
(3.1) by material investment pyroreaction kettle stand-by in step (2.5), the water of 2 times of material deals is added, is added 4 times
The sodium hydroxide of 0.15 times of material deal, the hydrazine hydrate of 0.11 times of material deal, oil bath heating is added in the solvent B of material deal
140 DEG C are warming up to, waste water is collected in recycling；Temperature control opens reflux after reaching 140 DEG C, keeps the temperature 2 hours and reacts；
(3.2) recycling is opened, 200 DEG C is continuously heating to, recycles extra hydrazine hydrate around here, keep the temperature 200 DEG C of reflux 2-4 hours,
Sampling Detection, HPLC monitor 3 α ,-12-5 β of ketone of 7 beta-dihydroxy-cholestane-24- acid content < 0.2%；
(3.3) vacuum degree -0.07Mpa is controlled, at 120-140 DEG C, vacuum recycles solvent B for temperature control, and it collects solvent B and applies,
To paste, stop concentration；
(3.4) water of 8 times of material deals is added, stirring dissolves paste sufficiently, is transferred in acidification kettle, and cooling water drop is added
For temperature to 40-50 DEG C, it is 2-3 that the aqueous hydrochloric acid solution that stirring is added 10%, which adjusts pH value, keeps the temperature 10-30 minutes, and ursodesoxycholic acid is precipitated
Crude product, filters pressing separation, waste water enter sewage plant, and collection material enters boiled bed drying machine, and the drying 4 hours of 100 DEG C of temperature control obtains bear
Deoxycholic aicd crude product, detecting its purity is 95% or more.
2. a kind of method for passing through half enzymatic clarification ursodesoxycholic acid using cholic acid as raw material as described in claim 1, feature
It is, the solvent A in the step (1.1) is one of acetone, methanol, ethyl alcohol, ethyl acetate.
3. a kind of method for passing through half enzymatic clarification ursodesoxycholic acid using cholic acid as raw material as described in claim 1, feature
It is, the freezing liquid in the step (1.1) is freezing ethylene glycol.
4. a kind of method for passing through half enzymatic clarification ursodesoxycholic acid using cholic acid as raw material as described in claim 1, feature
It is, the oxidant in the step (1.2) is calcium hypochlorite.
5. a kind of method for passing through half enzymatic clarification ursodesoxycholic acid using cholic acid as raw material as described in claim 1, feature
It is, the solvent B in the step (3.1) is one of ethylene glycol, diethylene glycol (DEG), glycerol.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115536720A (en) * | 2022-10-18 | 2022-12-30 | 湖南科瑞生物制药股份有限公司 | Cholic acid intermediate A8 and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012532A (en) * | 2012-12-10 | 2013-04-03 | 辽宁百凤生物药业有限公司 | Method for producing ursodesoxycholic acid with 98.5 percent sodium cholate |
| CN105418716A (en) * | 2015-12-25 | 2016-03-23 | 成都市新功生物科技有限公司 | Method for synthesizing chenodeoxycholic acid by using duck cholic acid extracted from duck bile |
| CN108218943A (en) * | 2018-03-05 | 2018-06-29 | 常德云港生物科技有限公司 | By the chenodeoxycholic acid in chicken courage and the method for cholic acid synthesis ursodesoxycholic acid |
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- 2019-03-18 CN CN201910201308.5A patent/CN109825547A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012532A (en) * | 2012-12-10 | 2013-04-03 | 辽宁百凤生物药业有限公司 | Method for producing ursodesoxycholic acid with 98.5 percent sodium cholate |
| CN105418716A (en) * | 2015-12-25 | 2016-03-23 | 成都市新功生物科技有限公司 | Method for synthesizing chenodeoxycholic acid by using duck cholic acid extracted from duck bile |
| CN108218943A (en) * | 2018-03-05 | 2018-06-29 | 常德云港生物科技有限公司 | By the chenodeoxycholic acid in chicken courage and the method for cholic acid synthesis ursodesoxycholic acid |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115536720A (en) * | 2022-10-18 | 2022-12-30 | 湖南科瑞生物制药股份有限公司 | Cholic acid intermediate A8 and preparation method thereof |
| CN115536720B (en) * | 2022-10-18 | 2024-05-14 | 湖南科瑞生物制药股份有限公司 | Cholic acid intermediate A8 and preparation method thereof |
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