CN107312054A - A kind of method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile - Google Patents
A kind of method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile Download PDFInfo
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- CN107312054A CN107312054A CN201710610814.0A CN201710610814A CN107312054A CN 107312054 A CN107312054 A CN 107312054A CN 201710610814 A CN201710610814 A CN 201710610814A CN 107312054 A CN107312054 A CN 107312054A
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- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
- C07J9/005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane containing a carboxylic function directly attached or attached by a chain containing only carbon atoms to the cyclopenta[a]hydrophenanthrene skeleton
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- C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
- C07J41/0061—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives one of the carbon atoms being part of an amide group
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Abstract
The present invention relates to a kind of method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, methods described includes extraction mixing cholic acid, separation mixing cholic acid, synthesis urso and Tauro ursodesoxy cholic acid.It is raw material using pig's bile, takes enzymolysis, ultrafiltration and the method for nanofiltration to carry out front end removal of impurities to extract mixing cholic acid, and the mixing cholic acid of extraction is separated, then synthesizes urso and Tauro ursodesoxy cholic acid.Using the method for the invention, its raw material is easy to get extensively, and Tauro ursodesoxy cholic acid purity is high; with high conversion rate; reproducible, cost is relatively low, simple to operate; preparation technology is simple; it is environment-friendly, it is more suitable for industrialized production, is conducive to the energy consumption that economizes on resources and reduce; its economic benefit and environmental benefit substantially, can promote industry sustainable development.
Description
Technical field
The invention belongs to biomedicine technical field, it is related to the preparation method of Tauro ursodesoxy cholic acid, and in particular to a kind of
The method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile.
Background technology
Bile is the product that human body or vertebrate liver cells are continuously secreted, and is a kind of coloured alkali of the viscosity with bitter taste
Property liquid, its composition has bile acid, cholate and BILE PIGMENTS than water, mainly, in addition with a small amount of cholesterol, aliphatic acid,
Lecithin, electrolyte and protein etc..Animal bile more typical now is generally bird gall, pig's bile, fel bovis, bear gall
Juice and snake bile etc..Bile acid is the main component of bile, accounts for the 50~70% of bile total amount, the physiological action of bile
Main is exactly to be played by bile acid, when as medicine, and bile acid can be for antipyretic, antispastic antibechic, regulation blood pressure, cardiac stimulant
Cholagogic, antibacterial anti-inflammatory and regulation are immune etc., and activity relatively strong, side effect is again small, cheap, therefore by numerous drug users
Welcome.In various animal biles, pig's bile is most common and yield is maximum, is just widely used in facing since the ancient times
Bed, such as with pig gall juice for treating acute icteric catarrhal jaundice, pertussis, chordapsus and gastric ulcer, can be received good
Good therapeutic effect.Mainly containing bile acids, BILE PIGMENTS, mucoprotein, lipid and inorganic matter etc. in pig's bile, bile acid is then main
Include hyodesoxycholic acid, hyocholic acid, chenodeoxycholic acid, 3-5 α of Alpha-hydroxy-6- oxygen-cholanic acid and cholelith acid etc..Wherein pig goes
Oxycholic acid accounts for 40%, and chenodeoxycholic acid accounts for 25%.
Urso (Ursodeoxycgolicacid) abbreviation UDCA, is the main component of bear gall, and its chemical name is
(3 α, 7 β)-β cholanic acids of dihydroxy -5, are the 7- hydroxyl epimers of chenodeoxycholic acid (Chenodeoxycholicacid).
Since in the 1980s, there is invention urso effective dissolving cholesterol calculus to act on, constantly there is new grind
Study carefully display urso has positive effect in cholagogic, protect liver.In recent years, foreign countries report UDCA not only to treatment original
The biliary cirrhosis of hair property, primary sclerotic cholangitis, CAH have good efficacy, also can use slow with treatment
Rejection after property hepatitis and liver transfer operation.At present, clinically urso is mainly used in the various liver and gall for the treatment of, alimentary canal
Disease.With molecular biology and the continuous progress of urso basis and clinical research, it has been found that urso
Also there is positive role in terms of immunological regulation, treatment coronary heart disease is promoted.Therefore, going deep into research, urso
Value is increasingly realized and is valued by people, and the demand to urso is also being raised year by year.But, day
Increasingly reduce in the source of right bear gall.People have been working hard seek divided by naturally bear gall obtains the alternative outside urso
Method.In early days, it by the cholic acid of the separation and Extraction from ox, sheep bile is initiation material that urso, which is, then by complicated
Chemical method production.Wherein, being made after chenodeoxycholic acid needs oxidized reduction generation urso.But, this method
Step is more, production time length, cost high.After the eighties in last century, zymetology, microbial fermentation processes grow up.With zymetology, micro-
Biofermentation method produces urso, still, this side by CDCA acid isomerization, or by lithocholic acid-hydroxylating
Method still has the high defect of many steps, production time length, cost.
Chemical entitled 2- [[(3 α, 5 β, 7 β) -3, the 7- bis- of Tauro ursodesoxy cholic acid (tauroursodeoxycholicacid)
Hydroxyl -24- oxo cholestane -24- bases] amino] ethane sulfonic acid dihydrate, found from bear gall within 1902, it is in bear gall
Primary bile acid, with the effect such as spasmolysis, anticonvulsion, anti-inflammatory and molten cholelith.Clinic be mainly used in treatment gallbladder cholesterol calculus,
Primary sclerosing cholangitis, Primary biliary cirrhosis and chronic HCV etc..Tauro ursodesoxy cholic acid can increase
The secretion of bile acid, causes the change of Bile acid, makes its content increase in bile.Tauro ursodesoxy cholic acid can also press down
The amount and the saturation index of cholesterol of the synthesis of hepatic cholesterol processed, reduction bile cholesterol and cholesteryl ester, so that favorably
Gradually dissolved in bile cholesterol.But natural bear gall juice resource is extremely precious, even if propagating drained bear bile artificially,
Market much be can't resolve for urso, the more and more substantial amounts of demand of Tauro ursodesoxy cholic acid, be that this biotechnology is closed
Technique into urso, Tauro ursodesoxy cholic acid is arisen at the historic moment.Particularly goose is extracted from extensive pig's bile of originating to go
Chemical synthesis urso again after oxycholic acid, can create obvious economic benefit and social value.
Either using the cholic acid of any animal sources, deoxycholic aicd as raw material, or with steroidal compounds（Cholesterol, sterol,
Progesterone）For raw material, it is both needed to synthesize chenodeoxycholic acid intermediate, and step is various, the time is longer, cost is higher, is this
Technology with economic implications is, using chenodeoxycholic acid as Material synthesis urso, then to synthesize Tauro ursodesoxy cholic acid.Such as
Patent CN102453071A reports direct extraction synthesis chenodeoxycholic acid and urso from porcine bile paste or leftover bits and pieces
Preparation method, the problem of it is present be：Hyodesoxycholic acid is not extracted, and its property is similar to chenodeoxycholic acid, be this not only
Added value is reduced, and adds the space steric effect of subsequent reactions；Bromine water oxidation reaction is strong, it is difficult to control, secondary anti-
Should be more, such as peroxide 3,7- diketo lithocholic acids are such as reduction of conversion ratio, add impurity；Due to metallic sodium and potassium
Then belong to activity metal, so hydrogenation reaction is strong, be not only difficult to control to, and with potential safety hazard, it is necessary to paraffin or
Preserved in kerosene；Purify link not enough, only can not remove hyodesoxycholic acid, the goose not reacted completely by a step acid precipitation deoxygenates
The impurity such as cholic acid, the 3,7- diketo lithocholic acid derivatives for crossing reaction.As patent CN102477059A reports a kind of high-purity
Tauro ursodesoxy cholic acid and preparation method thereof, the problem of it is present be：The mixed acid anhydride of synthesis is unstable, meets water and easily decomposes, meets
Hot easily racemization, thus conversion ratio is relatively low, and also low-temp reaction condition is difficult to control to；Triethylamine, chloromethane in simultaneously raw materials used
Acetoacetic ester has certain toxicity, and its residual is easily adsorbed, is embedded in coarse crystallization, because water insoluble, be difficult to water elution and removed.
The content of the invention
The technical problem to be solved in the present invention is in view of the shortcomings of the prior art, to provide a kind of using pig's bile as original for people
The method of material synthesis urso and Tauro ursodesoxy cholic acid, is raw material using pig's bile, takes enzymolysis, ultrafiltration and nanofiltration
Method carries out front end removal of impurities to extract mixing cholic acid, and the mixing cholic acid of extraction is separated, and then synthesizes urso
And Tauro ursodesoxy cholic acid.This method extraction process is simple, and raw material sources are easy to get extensively, and environmental pollution is small, and product purity is high.
In order to solve the above technical problems, the technical solution adopted by the present invention is：One kind synthesizes ursodeoxycholic from pig's bile
The method of acid and Tauro ursodesoxy cholic acid, includes extraction mixing cholic acid, separation mixing cholic acid, synthesis urso and ox
Sulphur urso, using pig's bile as raw material, takes enzymolysis, ultrafiltration and the method for nanofiltration to carry out front end removal of impurities to extract mixing courage
Acid, and the mixing cholic acid of extraction is separated, urso and Tauro ursodesoxy cholic acid are then synthesized, is specifically included following
Step：
(1) mixing cholic acid is extracted:Hyocholic acid dry powder addition pure water and compound protease are carried out into temperature control first to digest and must digest
Liquid；Enzymolysis liquid is added after precipitating reagent and carried out after centrifugal filtration, collects clarified solution；Clarified solution is being received after ultrafiltration and nanofiltration
After addition decolorising agent is decolourized in the concentrate taken, acidification is carried out to the destainer of gained, then carry out centrifugal treating receipts
Taking precipitate；Finally by sediment after esterification is handled, collect precipitate and produce the mixing cholic acid after washing；
(2) separation mixing cholic acid：The mixing cholic acid of gained in step (1) is first added into methanol and is allowed to acidifying formation mixing cholic acid first
Fat, obtains mixtures of methyl esters again by esterification and crystallization after dealcoholysis, hyodesoxycholic acid and chenodeoxycholic acid is separated, to increase pig
Deoxycholic aicd income, purifying chenodeoxycholic acid, specific requirement is in the mixing cholic acid of collection plus methanol is allowed to acidifying and forms mixed
Cholic acid formicester is closed, mixtures of methyl esters is obtained after reclaiming methanol through vacuum distillation, toluene is then added, after stirring to complete thermosol,
Centrifugal treating is carried out after crystallized again and takes crystal, the crystal collected is tied again using being recrystallized again after toluene washing
After crystalline substance is by saponification process, then dealcoholysis, water-soluble and acidification are carried out, the sediment collected is through being dried under reduced pressure to obtain pig deoxidation courage
Acid；Crystalline mother solution and cleaning solution are collected after merging, toluene is reclaimed through vacuum distillation, after taking concentrate by saponification process,
Carry out dealcoholysis, water-soluble and acidification again, the sediment finally collected filtered after hot-water soak sediment is CDCA
Acid；
(3) urso is synthesized：The chenodeoxycholic acid of gained, which first carries out hydrogenation, after separating in step (2) makes after it reduces,
Add acetone and carry out Dehydroepiandrosterone derivative, carry out acidification after resulting solution is concentrated again, filtrate passes through Subacidity cation again
Exchange resin column, crosses post liquid and adjusts its pH value with watery hydrochloric acid, then centrifugal treating collects precipitation, and with pure water to weakly acidic pH,
Methanol thermosol is added in reflux, pH value is adjusted after cooling, again with pure water to neutrality after leaching crystallization, finally
It is dried under reduced pressure and produces urso；
(4) Tauro ursodesoxy cholic acid is synthesized：Gained urso in step (3) is added into condensing agent to be activated, it is activated
Gained mixed liquor is carried out obtaining lysate after basification afterwards, and lysate is carried out after acylation reaction again, after filtering gained sediment
After pure water, then acetone thermosol is used, prompt drop Wen Houjing is put, leaching crystallization, and after being washed with ether, then washed with cold pure water
During to without ether taste, finally it is dried under reduced pressure and produces Tauro ursodesoxy cholic acid.
Further, the method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, wherein described
The method that mixing cholic acid is extracted in step (1) comprises the following steps：
(1) after pig's bile dry powder is weighed, the active compound protease of pure water, 0.5% 100,000 U/g for adding 5~6 times of amounts is carried out
Enzymolysis, regulation pH value is between 9~11, and controlled enzymatic hydrolysis temperature is stirred continuously after 2h between 50~55 DEG C, is warming up to 90 DEG C
Stop heat, 5~10% volume ratio ZTC-B 1% solution is added while hot, stirs well, adds 3~5% volume ratio ZTC-A 1% solution, pass through
After stirring well, centrifuging and taking clarified solution；
(2) clarified solution collected in step (1) is cooled to≤45 DEG C, pH value is adjusted again between 9~11, it is super by 1KD
Leaching permeate, then concentrate is taken by 300D nanofiltrations, the hydrogen peroxide for adding 1~3% volume ratio carries out decolorization, through stirring well
Afterwards, it is quiet to put after 24h, filtrate is taken through centrifugal treating, it is quiet to put layering after gained filtrate is with 10% salt acid for adjusting pH value to 2, again
Centrifugal treating collects sediment, finally by sediment pure water cyclic washing to weakly acidic pH；
(3) precipitation collected in step (2) is placed in reflux, and adds the absolute methanol of 7~8 times of amounts and carried out at esterification
Between 48~52 DEG C the concentrated sulfuric acid or concentrated hydrochloric acid of precipitation capacity 8% is added dropwise, with flow velocity 5BV/h in reason, control esterification temperature after stirring well
It is passed through the activated alumina dehydrating agent post progress post with precipitating equivalent to wash, post >=8h is crossed repeatedly, methanol is reclaimed in then vacuum distillation,
Collect precipitate after washing sediment produce mixing cholic acid.
Further, the method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, wherein institute
The method for stating separation mixing cholic acid in step (2) comprises the following steps：
(1) methanol that 5 times are first added in the mixing cholic acid of gained is allowed to acidifying and forms mixing cholic acid formicester, then through vacuum distillation
Reclaim methanol after mixtures of methyl esters, then add 3 times amount toluene be esterified, control esterification temperature 65~70 DEG C it
Between, stirring to after complete thermosol, being cooled to normal temperature, it is quiet put >=4h after, crystal is collected after centrifugal treating, gained crystal is used
After toluene is washed 3 times, then add the toluene of 2 times of amounts to be recrystallized, obtain recrystallization product, and collect the crystalline mother solution of toluene and wash
Wash after liquid merges and obtain toluene amalgamation liquid, for synthesizing urso；
(2) the recrystallization product collected in step (1) is placed in reflux, and adds 70~75% first of 3.5~4 times of amounts
Alcohol and 3% sodium hydroxide, control temperature is after 1~1.5h of stirring between 65~70 DEG C, and methanol is reclaimed in vacuum distillation, collects precipitation
Thing, and add appropriate pure water to stir molten, then pH value is adjusted to 2 with watery hydrochloric acid, quiet to put leaching sediment after layering, sediment is washed with pure water
Wash to weakly acidic pH, hyodesoxycholic acid is dried under reduced pressure to obtain under the conditions of temperature 60 C；
(3) the toluene amalgamation liquid collected in step (1) is reclaimed into toluene through vacuum distillation, the concentrate collected is placed in reflux
It is interior, and 80~85% methanol and 3% sodium hydroxide of 4~4.5 times of amounts are added, after control temperature stirs 2h between 70~75 DEG C,
Methanol is reclaimed in vacuum distillation, collects precipitate, and precipitate adds after appropriate pure water stirs evenly, then adjusts pH value to 2 with watery hydrochloric acid, quiet to put
Leaching sediment after layering, sediment pure water to weakly acidic pH, then using hot-water soak, it is soaking after refilter and collect
Sediment produces chenodeoxycholic acid.
Further, the method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, wherein institute
The method for stating synthesis urso in step (3) comprises the following steps：
(1) the CDCA acid deposit of gained is added into hydrogen source under the conditions of alkaline acid binding agent, and at reflux, makes 7
Beta-keto is converted into 7 beta-hydroxies, is then placed in again in reflux and sequentially adds the acetone of 3 times of amounts of precipitation, the first of 0.1 times of amount
Sodium alkoxide carries out Dehydroepiandrosterone derivative, is stirred continuously solution and is allowed to uniform, and under the conditions of adjusting its pH≤5, temperature≤60 DEG C, then at illumination
3% sodium hydroxide solution that condition is flowed down plus 0.2 times is measured, obtains dehydrogenation liquid；
(2) acetone is reclaimed into the dehydrogenation liquid vacuum distillation collected in step (1), concentrate regulation pH to 2, leaching sediment is with pure
Then water washing adds 3 times of amount pure water to weakly acidic pH, and adds the catalyst containing chloroethanes of weight of precipitate meter 5%, and control pH exists
Between 8~9, temperature >=80 DEG C are controlled, and are stirred continuously >=2.5h, impurity elimination is then filtered and collects filtrate；
(3) filtrate collected in step (2) is flowed through into weak-acid cation-exchange resin post with 2BV/h, crosses post liquid with watery hydrochloric acid
Adjust pH to 2, centrifuging and taking precipitation and with pure water to weakly acidic pH, then is placed in reflux and measures methanol thermosol with 4-5 times,
≤ 5 DEG C are cooled to rapidly, then watery hydrochloric acid adjusts pH to 2, quiet to put after 24h, leaching crystal pure water weakly acidic pH, in temperature
It is dried under reduced pressure under the conditions of 60 DEG C of degree and produces urso.
Further, the method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, wherein institute
The method for stating synthesis Tauro ursodesoxy cholic acid in step (4) comprises the following steps：
(1) urso of gained is placed in reflux, and adds 5 times of amount methanol, carbonic acid of 0.4mol/L quantity of methyl alcohol
After agitating solution is uniform under the conditions of the sodium taurocholate of sodium, 0.35mol/L quantity of methyl alcohol, control temperature≤60 DEG C, then flow plus condensing agent
Solution be stirred continuously in acylation reaction, course of reaction, mixing time >=2h, is then filtered impurity elimination and is collected filtrate, the contracting
The DMTMM solution that mixture adds 2mol/L by the methanol of 0.1 times of amount constitutes condensation agent solution；
(2) reaction solution of gained in step (1) is adjusted into pH to 5 with watery hydrochloric acid, is cooled to≤5 DEG C, and centrifuge removing insoluble matter,
Its filtrate adjusts pH to 2 with watery hydrochloric acid again, and leaching sediment measures 5% acetone thermosols using pure water sediment, then with 8 times,
Speed is cooled to≤5 DEG C, it is quiet put >=30h after, leaching crystallization, crystal is washed 3 times with appropriate ether, then is washed till no second with cold pure water
Ether taste, is dried under reduced pressure under the conditions of temperature 60 C and produces Tauro ursodesoxy cholic acid.
Using a kind of method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile of the present invention, with
Prior art is compared, and its advantage is：
1st, in the extraction process of mixing cholic acid, enzymolysis and the method for ultrafiltration and nanofiltration are taken, removal of impurities, its product are carried out in front end
Purity is high, is that subsequent product purifying reduces step, reduces cost.
2nd, its reaction condition is gentle in whole production extraction process, it is ensured that material molecular structure is not damaged, section
About cost and time.
In summary, the method for the invention is using pig's bile as raw material is extracted, and its raw material is easy to get extensively, by taking enzyme
Solution, ultrafiltration and the method for nanofiltration carry out front end removal of impurities to extract mixing cholic acid, and the mixing cholic acid of extraction is separated, then
Urso and Tauro ursodesoxy cholic acid are synthesized, its Tauro ursodesoxy cholic acid purity synthesized is high, with high conversion rate, repeats
Property is good, and cost is relatively low, simple to operate, environment-friendly, is more suitable for industrialized production.Be conducive to the energy consumption that economizes on resources and reduce, its
Economic benefit and environmental benefit substantially, can promote industry sustainable development.
Embodiment
In order to more fully explain the implementation of the present invention, the present invention is further illustrated below in conjunction with specific embodiment.Institute
Give an actual example and be served only for explaining the present invention, rather than limit the scope of the present invention.
Urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile using of the present invention, its design considerations includes
Herein below：
First, raw material is chosen：
Animality bile resource is many using pig, cattle and sheep, chicken and duck, but active constituent content is not quite similar, and from technique it is simple, into
This relatively low consideration, it should select the more pig of CDCA acid content, chicken and duck bile to be advisable, because swine bile acids dry powder amount great Yi
Purchase, and bilirubin has been removed, and is raw material best at present.Swine bile acids typically contain pig deoxidation courage in the market
Acid 40%, chenodeoxycholic acid 25%, cholic acid 5%, albumen, enzyme, pigment, lipid（Fat, phosphatide, cholesterol）, the impurity such as inorganic salts
20%, moisture 10% or so.
2nd, embodiment：
Selection swine bile acids are raw material, and the method to synthesize urso and Tauro ursodesoxy cholic acid includes extraction mixing
Cholic acid, separation mixing cholic acid, synthesis urso and Tauro ursodesoxy cholic acid, using pig's bile as raw material, take enzymolysis, surpass
The method of filter and nanofiltration carries out front end removal of impurities to extract mixing cholic acid, and the mixing cholic acid of extraction is separated, and then synthesizes
Urso and Tauro ursodesoxy cholic acid.
First, mixing cholic acid is extracted, because the active ingredient in hyocholic acid dry powder is mainly hyodesoxycholic acid and CDCA
Acid.Although hyodesoxycholic acid can also synthesize urso, complex steps, cost are higher, thus can as byproduct,
And using the chenodeoxycholic acid isolated as middle product, then synthesis urso, Tauro ursodesoxy cholic acid are advisable successively；Mix courage
Sour many and protein binding, many heated with saponification of conventional method is dissociated, then is isolated with calcium precipitation, displacement decalcification, acid precipitation
Cholic acid is mixed, because mixing cholic acid is 3.75 times of impurity content, is easily adsorbed in calcium precipitation, embed impurity, and repeatedly
The loss of mixing cholic acid is added in washing again；This technique is with gentle enzymatic isolation method dissociation and the combination of albumen, then with precipitating reagent
Deproteination, lipid, with ultrafiltration, nanofiltration removing sized molecules impurity, concentrate is decolourized again, and last acid precipitation must can be mixed
Cholic acid, based on the impurity that is occupied the minority with removing of whole process, the not only favourable space steric effect for reducing subsequent reactions, and mixing courage
Acid is lost in less；Substantially increase the content of material and purity of subsequent product.The property phase of hyodesoxycholic acid and chenodeoxycholic acid
Seemingly, can only be to be separated after esterification for the difference of toluene solubility；Esterification is reversible, to promote higher conversion
Rate is, it is necessary to add excessive alcohol and remove the water of generation in time, and wherein shipwreck is removed, and dehydrating agent is introduced for this, compares chlorination
Calcium, silica gel, three kinds of dehydrating agents of aluminum oxide, wherein so that aluminum oxide is water insoluble and alcohol, adsorptive dehydration is fast, easily regeneration can also be simultaneous
Turn round and look at and decolourize.
During mixing cholic acid is extracted, following steps are specifically included：
(1) after pig's bile dry powder is weighed, the active compound protease of pure water, 0.5% 100,000 U/g for adding 5~6 times of amounts is carried out
Enzymolysis, regulation pH value is between 9~11, and controlled enzymatic hydrolysis temperature is stirred continuously after 2h between 50~55 DEG C, is warming up to 90 DEG C
Stop heat, 5~10% volume ratio ZTC-B 1% solution is added while hot, stirs well, adds 3~5% volume ratio ZTC-A 1% solution, pass through
After stirring well, centrifuging and taking clarified solution；
(2) clarified solution collected in step (1) is cooled to≤45 DEG C, pH value is adjusted again between 9~11, it is super by 1KD
Leaching permeate, then concentrate is taken by 300D nanofiltrations, the hydrogen peroxide for adding 1~3% volume ratio carries out decolorization, through stirring well
Afterwards, it is quiet to put after 24h, filtrate is taken through centrifugal treating, it is quiet to put layering after gained filtrate is with 10% salt acid for adjusting pH value to 2, again
Centrifugal treating collects sediment, finally by sediment pure water cyclic washing to weakly acidic pH；
(3) precipitation collected in step (2) is placed in reflux, and adds the absolute methanol of 7~8 times of amounts and carried out at esterification
Between 48~52 DEG C the concentrated sulfuric acid or concentrated hydrochloric acid of precipitation capacity 8% is added dropwise, with flow velocity 5BV/h in reason, control esterification temperature after stirring well
It is passed through the activated alumina dehydrating agent post progress post with precipitating equivalent to wash, post >=8h is crossed repeatedly, methanol is reclaimed in then vacuum distillation,
Collect precipitate after washing sediment produce mixing cholic acid.
2nd, separation mixing cholic acid, due to hyodesoxycholic acid in esterification process and solubility of the chenodeoxycholic acid in toluene
Differ greatly, wherein hyodesoxycholic acid ester can be crystallized in toluene, and chenodeoxycholic acid ester is easily soluble in toluene, is that this can give
Separation, is further removing oil-soluble impurities, using re crystallization from toluene；And the methyl hyodeoxycholanate purified, with base catalysis ester
Solve efficiency high, then precipitation and acidified, the precipitation of precipitation pure water again removes water-solubility impurity, can obtained after being dried under reduced pressure
To yield >=288g/kg, purity >=98%, the hyodesoxycholic acid for belonging to pharmaceutical grade, you can, can also to be sold as bulk drug
It is used as the raw material of synthesis urso；And the chenodeoxycholic acid ester for being dissolved in toluene passes through precipitation, esterlysis, then it is acidified, can obtains
To chenodeoxycholic acid precipitation.
During separation mixing cholic acid, following steps are specifically included：
(1) methanol that 5 times are first added in the mixing cholic acid of gained is allowed to acidifying and forms mixing cholic acid formicester, then through vacuum distillation
Reclaim methanol after mixtures of methyl esters, then add 3 times amount toluene be esterified, control esterification temperature 65~70 DEG C it
Between, stirring to after complete thermosol, being cooled to normal temperature, it is quiet put >=4h after, crystal is collected after centrifugal treating, gained crystal is used
After toluene is washed 3 times, then add the toluene of 2 times of amounts to be recrystallized, obtain recrystallization product, and collect the crystalline mother solution of toluene and wash
Wash after liquid merges and obtain toluene amalgamation liquid, for synthesizing urso；
(2) the recrystallization product collected in step (1) is placed in reflux, and adds 70~75% first of 3.5~4 times of amounts
Alcohol and 3% sodium hydroxide, control temperature is after 1~1.5h of stirring between 65~70 DEG C, and methanol is reclaimed in vacuum distillation, collects precipitation
Thing, and add appropriate pure water to stir molten, then pH value is adjusted to 2 with watery hydrochloric acid, quiet to put leaching sediment after layering, sediment is washed with pure water
Wash to weakly acidic pH, hyodesoxycholic acid is dried under reduced pressure to obtain under the conditions of temperature 60 C；
(3) the toluene amalgamation liquid collected in step (1) is reclaimed into toluene through vacuum distillation, the concentrate collected is placed in reflux
It is interior, and 80~85% methanol and 3% sodium hydroxide of 4~4.5 times of amounts are added, after control temperature stirs 2h between 70~75 DEG C,
Methanol is reclaimed in vacuum distillation, collects precipitate, and precipitate adds after appropriate pure water stirs evenly, then adjusts pH value to 2 with watery hydrochloric acid, quiet to put
Leaching sediment after layering, sediment pure water to weakly acidic pH, then using hot-water soak, it is soaking after refilter and collect
Sediment produces chenodeoxycholic acid.
3rd, urso is synthesized, urso is synthesized using chenodeoxycholic acid, first must be turned 7 Alpha-hydroxy dehydrogenations
7 beta-ketos are turned to, and traditional method is more with bromine water dehydrogenation, but the corrosivity of molecular bromine is extremely strong, toxicity is high, easily generates bromination
Hydrogen is escaped, conversion ratio 78%, and substitutes bromine water with NBS, although is solved corrosion problems, but is the increase in amine salt impurity, and turns
Rate is down to 68%；And the method for using mixed system, the generation of impurity had both been reduced, its conversion ratio can be improved again, conversion ratio can
Up to 80% or so.In addition, 7 beta-keto repeated hydrogenations are converted into 7 beta-hydroxies, that is, synthesize urso, traditional method it is many with
Metallic potassium or sodium catalysis, it is difficult to control, side reaction is more；And the method applied in the present invention is to add to tie up in the basic conditions
Sour agent produces hydrogen source, and its hydrogen source mainly uses bromine water and the concerted reaction of hydrogen peroxide to generate hydrogen, by taking hydrogenation reaction, made
7 beta-ketos are converted into 7 beta-hydroxies, its selectivity it is good, it is easily separated, can apply mechanically, under the conditions of alkaline acid binding agent its conversion ratio >=
92%；In addition, the purity in order to ensure urso, particularly removes the similar chenodeoxycholic acid residual of property, accessory substance 3,
7- diketone based compounds, on the basis of acid precipitation, increase recrystallization method, with urso that is simple and practical, obtaining
Yield >=144g/kg, purity >=99%.
In synthesis urso process, following steps are specifically included：
(1) the CDCA acid deposit of gained is added into hydrogen source under the conditions of alkaline acid binding agent, and at reflux, makes 7
Beta-keto is converted into 7 beta-hydroxies, is then placed in again in reflux and sequentially adds the acetone of 3 times of amounts of precipitation, the first of 0.1 times of amount
Sodium alkoxide carries out Dehydroepiandrosterone derivative, is stirred continuously solution and is allowed to uniform, and under the conditions of controlling pH≤5, temperature≤60 DEG C, then at illumination bar
3% sodium hydroxide solution that part is flowed down plus 0.2 times is measured, obtains dehydrogenation liquid；
(2) acetone is reclaimed into the dehydrogenation liquid vacuum distillation collected in step (1), concentrate regulation pH to 2, leaching sediment is with pure
Then water washing adds 3 times of amount pure water to weakly acidic pH, and adds the catalyst containing chloroethanes of weight of precipitate meter 5%, and pH is 8 for control
Between~9, temperature >=80 DEG C are controlled, and are stirred continuously >=2.5h, impurity elimination is then filtered and collects filtrate；
(3) filtrate collected in step (2) is flowed through into weak-acid cation-exchange resin post with 2BV/h, crosses post liquid with watery hydrochloric acid
PH to 2 is adjusted, centrifuging and taking is precipitated, and with pure water to weakly acidic pH, then be placed in reflux with 4~5 times of amount methanol thermosols,
≤ 5 DEG C are cooled to rapidly, then watery hydrochloric acid adjusts pH to 2, quiet to put after 24h, leaching crystal uses pure water to close neutrality,
It is dried under reduced pressure under the conditions of temperature 60 C and produces urso.
4th, Tauro ursodesoxy cholic acid is synthesized, ox semi-annular jade pendant urso is the ammonia of carboxyl by urso and taurine
Base generates amido link and is condensed, and mixed anhydride method is taken current technology more, i.e., first triethylamine catalysis is lower and ethyl chloroformate is given birth to
Into the mixed acid anhydride of active ester, then generate amido link with taurine and be condensed, because mixed acid anhydride is unstable, meets water and easily divide
Solution, the easy racemization of heat, so conversion ratio is generally≤75%；Take condensing agent method, i.e., under the activation of condensing agent, conversion ratio
Height, reproducible, cost is relatively low, simple to operate, it is easy to reclaim；In addition, during this acylation reaction, to ensure higher acid amides
Key conversion ratio, one is taurine excessively addition, and two be that alcohol is high for the favourable reaction selectivity of solvent, and three be that alkaline acid binding agent prevents ester
Change reaction, four be the even adduction rapid mass transfer of condensing agent, because raw material and auxiliary material are insensitive to water, It is not necessary to remove anti-
Ying Shui；After acylation reaction, adjust pH=5 and cool, excessive taurine is separated out insoluble in subacidity methanol, can be covered under being used for
A batch of reaction, then be acidified and can separate out ox semi-annular jade pendant ursodeoxycholic acid crude, then by dilute acetone recrystallization, property can be removed similar
The residual such as urso, chenodeoxycholic acid, yield >=136g/kg, purity >=99%.
During synthesis Tauro ursodesoxy cholic acid, following steps are specifically included：
(1) urso of gained is placed in reflux, and adds 5 times of amount methanol, carbonic acid of 0.4mol/L quantity of methyl alcohol
After agitating solution is uniform under the conditions of the sodium taurocholate of sodium, 0.35mol/L quantity of methyl alcohol, control temperature≤60 DEG C, then flow plus condensing agent
Solution be stirred continuously in acylation reaction, course of reaction, mixing time >=2h, is then filtered impurity elimination and is collected filtrate, the contracting
Mixture is added 2mol/L DMTMM by the methanol of 0.1 times of amount（4- (4,6- dimethoxy-triazine -2- bases) -4- methyl morpholine hydrochloric acid
Salt）Solution constitutes condensation agent solution；
(2) reaction solution of gained in step (1) is adjusted into pH to 5 with watery hydrochloric acid, is cooled to≤5 DEG C, and centrifuge removing insoluble matter,
Its filtrate adjusts pH to 2 with watery hydrochloric acid again, and leaching sediment measures 5% acetone thermosols using pure water sediment, then with 8 times,
Speed is cooled to≤5 DEG C, it is quiet put >=30h after, leaching crystallization, crystal is washed 3 times with appropriate ether, then is washed till no second with cold pure water
Ether taste, is dried under reduced pressure under the conditions of temperature 60 C and produces Tauro ursodesoxy cholic acid.
5th, waste is handled：Organic solvent, auxiliary material in production technology of the present invention reclaim outer, and active ingredient can be recovered again
It is lost in；Neutrality, centrifugation removing precipitation are adjusted to after merging containing the waste water that plurality of impurities is not reclaimed, then passes through counter-infiltration removing
Concentrate, the reclaimed water of transmission can Reuse on Environment, clean, flush the toilet；And the thickened waste in the concentrate and production technology of counter-infiltration
Water, contamination precipitation merge, and the natural air drying in antiseepage pond merges with house refuse and is emitted into soot.
Embodiments of the invention are the foregoing is only, are not intended to limit the scope of the invention, it is every to utilize this hair
Equivalence replacement or equivalent flow conversion that bright description is made, or directly or indirectly it is used in other related technology necks
Domain, is included within the scope of the present invention.
Claims (5)
1. a kind of method that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, it is characterised in that：Methods described
Include extraction mixing cholic acid, separation mixing cholic acid, synthesis urso and Tauro ursodesoxy cholic acid, using pig's bile as original
Material, takes enzymolysis, ultrafiltration and the method for nanofiltration to carry out front end removal of impurities to extract mixing cholic acid, and carried out to the mixing cholic acid of extraction
Separation, then synthesizes urso and Tauro ursodesoxy cholic acid, specifically includes following steps：
(1) mixing cholic acid is extracted:Pig's bile dry powder addition pure water and compound protease are carried out into temperature control first to digest and must digest
Liquid；Enzymolysis liquid adds precipitating reagent and carried out after centrifugal filtration, collects clarified solution；Clarified solution is being collected after ultrafiltration and nanofiltration
Concentrate in add after decolorising agent decolourized, to the destainer progress acidification of gained, then carry out centrifugal treating and collecting
Sediment；Finally sediment is handled by esterification, precipitate is collected and produces the mixing cholic acid after washing；
(2) separation mixing cholic acid：The mixing cholic acid of gained in step (1) is first added into methanol and is allowed to acidifying formation mixing cholic acid first
Fat, obtains mixtures of methyl esters again by esterification and crystallization after dealcoholysis, hyodesoxycholic acid and chenodeoxycholic acid is separated, to increase pig
Deoxycholic aicd income, purifying chenodeoxycholic acid, specific requirement is in the mixing cholic acid of collection plus methanol is allowed to acidifying and forms mixed
Cholic acid formicester is closed, mixtures of methyl esters is obtained after reclaiming methanol through vacuum distillation, toluene is then added, after stirring to complete thermosol,
Centrifugal treating is carried out after crystallized again and takes crystal, the crystal collected is tied again using being recrystallized again after toluene washing
After crystalline substance is by saponification process, then dealcoholysis, water-soluble and acidification are carried out, the sediment collected is through being dried under reduced pressure to obtain pig deoxidation courage
Acid；Crystalline mother solution and cleaning solution are collected after merging, toluene is reclaimed through vacuum distillation, after taking concentrate by saponification process,
Carry out dealcoholysis, water-soluble and acidification again, the sediment finally collected filtered after hot-water soak sediment is CDCA
Acid；
(3) urso is synthesized：The chenodeoxycholic acid of gained, which first carries out hydrogenation, after separating in step (2) makes after it reduces,
Add acetone and carry out Dehydroepiandrosterone derivative, carry out acidification after resulting solution is concentrated again, filtrate passes through Subacidity cation again
Exchange resin column, crosses post liquid and adjusts its pH value with watery hydrochloric acid, then centrifugal treating collects precipitation, and with pure water to weakly acidic pH,
Methanol thermosol is added in reflux, pH value is adjusted after cooling, again with pure water to neutrality after leaching crystallization, finally
It is dried under reduced pressure and produces urso；
(4) Tauro ursodesoxy cholic acid is synthesized：Gained urso in step (3) is added into condensing agent to be activated, it is activated
Gained mixed liquor is carried out obtaining lysate after basification afterwards, and lysate is carried out after acylation reaction again, after filtering gained sediment
After pure water, then acetone thermosol is used, prompt drop Wen Houjing is put, leaching crystallization, and after being washed with ether, then washed with cold pure water
During to without ether taste, finally it is dried under reduced pressure and produces Tauro ursodesoxy cholic acid.
2. the method according to claim 1 that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, it is special
Levy and be, the method that mixing cholic acid is extracted in the step (1) comprises the following steps：
(1) after pig's bile dry powder is weighed, the active compound protease of pure water, 0.5% 100,000 U/g for adding 5~6 times of amounts is carried out
Enzymolysis, regulation pH value is between 9~11, and controlled enzymatic hydrolysis temperature is stirred continuously after 2h between 50~55 DEG C, is warming up to 90 DEG C
Stop heat, 5~10% volume ratio ZTC-B 1% solution is added while hot, stirs well, adds 3~5% volume ratio ZTC-A 1% solution, pass through
After stirring well, centrifuging and taking clarified solution；
(2) clarified solution collected in step (1) is cooled to≤45 DEG C, pH value is adjusted again between 9~11, it is super by 1KD
Leaching permeate, then concentrate is taken by 300D nanofiltrations, the hydrogen peroxide for adding 1~3% volume ratio carries out decolorization, through stirring well
Afterwards, it is quiet to put after 24h, filtrate is taken through centrifugal treating, it is quiet to put layering after gained filtrate is with 10% salt acid for adjusting pH value to 2, again
Centrifugal treating collects sediment, finally by sediment pure water cyclic washing to weakly acidic pH；
(3) precipitation collected in step (2) is placed in reflux, and adds the absolute methanol of 7~8 times of amounts and carried out at esterification
Between 48~52 DEG C the concentrated sulfuric acid or concentrated hydrochloric acid of precipitation capacity 8% is added dropwise, with flow velocity 5BV/ in reason, control esterification temperature after stirring well
The activated alumina dehydrating agent post that h is passed through with precipitating equivalent carries out post and washed, and post >=8h is crossed repeatedly, and first is reclaimed in then vacuum distillation
Alcohol, collect precipitate after washing sediment produce mixing cholic acid.
3. the method according to claim 1 that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, it is special
Levy and be, the method for separation mixing cholic acid comprises the following steps in the step (2)：
(1) methanol that 5 times are first added in the mixing cholic acid of gained is allowed to acidifying and forms mixing cholic acid formicester, then through vacuum distillation
Reclaim methanol after mixtures of methyl esters, then add 3 times amount toluene be esterified, control esterification temperature 65~70 DEG C it
Between, stirring to after complete thermosol, being cooled to normal temperature, it is quiet put >=4h after, crystal is collected after centrifugal treating, gained crystal is used
After toluene is washed 3 times, then add the toluene of 2 times of amounts to be recrystallized, obtain recrystallization product, and collect the crystalline mother solution of toluene and wash
Wash after liquid merges and obtain toluene amalgamation liquid, for synthesizing urso；
(2) the recrystallization product collected in step (1) is placed in reflux, and adds 70~75% first of 3.5~4 times of amounts
Alcohol and 3% sodium hydroxide, control temperature is after 1~1.5h of stirring between 65~70 DEG C, and methanol is reclaimed in vacuum distillation, collects precipitation
Thing, and add appropriate pure water to stir molten, then pH value is adjusted to 2 with watery hydrochloric acid, quiet to put leaching sediment after layering, sediment is washed with pure water
Wash to weakly acidic pH, hyodesoxycholic acid is dried under reduced pressure to obtain under the conditions of temperature 60 C；
(3) the toluene amalgamation liquid collected in step (1) is reclaimed into toluene through vacuum distillation, the concentrate collected is placed in reflux
It is interior, and 80~85% methanol and 3% sodium hydroxide of 4~4.5 times of amounts are added, after control temperature stirs 2h between 70~75 DEG C,
Methanol is reclaimed in vacuum distillation, collects precipitate, and precipitate adds after appropriate pure water stirs evenly, then adjusts pH value to 2 with watery hydrochloric acid, quiet to put
Leaching sediment after layering, sediment pure water to weakly acidic pH, then using hot-water soak, it is soaking after refilter and collect
Sediment produces chenodeoxycholic acid.
4. the method according to claim 1 that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, it is special
Levy and be, the method for synthesis urso comprises the following steps in the step (3)：
(1) the CDCA acid deposit of gained is added into hydrogen source under the conditions of alkaline acid binding agent, and at reflux, makes 7
Beta-keto is converted into 7 beta-hydroxies, is then placed in again in reflux and sequentially adds the acetone of 3 times of amounts of precipitation, the first of 0.1 times of amount
Sodium alkoxide carries out Dehydroepiandrosterone derivative, is stirred continuously solution and is allowed to uniform, and under the conditions of controlling pH≤5, temperature≤60 DEG C, then at illumination bar
3% sodium hydroxide solution that part is flowed down plus 0.2 times is measured, obtains dehydrogenation liquid；
(2) acetone is reclaimed into the dehydrogenation liquid vacuum distillation collected in step (1), concentrate regulation pH to 2, leaching sediment is with pure
Then water washing adds 3 times of amount pure water to weakly acidic pH, and adds the catalyst containing chloroethanes of weight of precipitate meter 5%, and pH is 8 for control
Between~9, temperature >=80 DEG C are controlled, and are stirred continuously >=2.5h, impurity elimination is then filtered and collects filtrate；
(3) filtrate collected in step (2) is flowed through into weak-acid cation-exchange resin post with 2BV/h, crosses post liquid with watery hydrochloric acid
PH to 2 is adjusted, centrifuging and taking is precipitated, and with pure water to weakly acidic pH, then be placed in reflux with 4~5 times of amount methanol thermosols,
≤ 5 DEG C are cooled to rapidly, then watery hydrochloric acid adjusts pH to 2, quiet to put after 24h, leaching crystal pure water weakly acidic pH, in temperature
It is dried under reduced pressure under the conditions of 60 DEG C and produces urso.
5. the method according to claim 1 that urso and Tauro ursodesoxy cholic acid are synthesized from pig's bile, it is special
Levy and be, the method for synthesis Tauro ursodesoxy cholic acid comprises the following steps in the step (4)：
(1) urso of gained is placed in reflux, and adds 5 times of amount methanol, carbonic acid of 0.4mol/L quantity of methyl alcohol
After agitating solution is uniform under the conditions of the sodium taurocholate of sodium, 0.35mol/L quantity of methyl alcohol, control temperature≤60 DEG C, then flow plus condensing agent
Solution carries out being stirred continuously >=2h in acylation reaction, course of reaction, then filters impurity elimination and collects filtrate, the condensing agent is by 0.1
The methanol of amount adds 2mol/L DMTMM solution composition condensation agent solution again；
(2) reaction solution of gained in step (1) is adjusted into pH to 5 with watery hydrochloric acid, is cooled to≤5 DEG C, and centrifuge removing insoluble matter,
Its filtrate adjusts pH to 2 with watery hydrochloric acid again, and leaching sediment measures 5% acetone thermosols using pure water sediment, then with 8 times,
Speed is cooled to≤5 DEG C, it is quiet put >=30h after, leaching crystallization, crystal is washed 3 times with appropriate ether, then is washed till no second with cold pure water
Ether taste, is dried under reduced pressure under the conditions of temperature 60 C and produces Tauro ursodesoxy cholic acid.
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