CN103030613B - Method for purifying lipstatin - Google Patents
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- CN103030613B CN103030613B CN201110297723.9A CN201110297723A CN103030613B CN 103030613 B CN103030613 B CN 103030613B CN 201110297723 A CN201110297723 A CN 201110297723A CN 103030613 B CN103030613 B CN 103030613B
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Abstract
The invention provides a method for purifying lipstatin. The method comprises the following steps: 1) providing a solid mixture containing the lipstatin; 2) extracting the solid mixture by utilizing a low-polarity first organic solvent or a non-polarity organic solvent, so as to obtain an extract liquor containing the lipstatin; 3) adsorbing the extract liquor by a molecular sieve; eluting the extract liquor by a first eluent so as to obtain the first extract liquor containing the lipstatin; and 4) purifying the first eluent containing the lipstatin obtained in the step 3) through chromatography. The method provided by the invention has the following beneficial effects: the solvent containing the lipstatin is selectively adsorbed by adopting the organic molecular sieve; the method is low in cost and more stable at high temperature; the use amount of silica gel is reduced, the utilization ratio of the silica gel is improved, and the separation effect is improved, so that the production running cost is greatly reduced.
Description
Technical field
The present invention relates to pharmacy field, in particular to a kind of method of purification of Lipstatin.
Background technology
Lipstatin (lipstatin) is the meta-bolites of Streptomyces toxytricini (Streptomyces toxytricini), and energy selectivity suppresses the activity of steapsase in gi tract, reduces fatty decomposition and absorption, and its structural formula is suc as formula shown in I.Its tetrahydrochysene derivative orlistat (orlistat) Yi Bei Roche Holding Ag (Roche lnc.) is successfully developed as diet pill---orlistat, is the medicine of unique treatment of obesity of going on the market as non-central nervous system effect at present.
Formula I
The Lipstatin technological process of production synthetic by microorganism biological mainly comprises the following steps: microbial fermentation solution directly adopts ultrasonication cell, with organic solvent extraction, afterwards that extract is concentrated, thus coarse crystal obtained, then through chromatogram purification, obtain the finished product.
The open No.CN1266058A of Chinese patent application discloses a kind of method of purification of orlistat, and the Lipstatin that is about to produce after streptomyces fermentation carries out double fluid extraction by heptane and aqueous acetic acid, and Lipstatin hydrogenation post crystallization is obtained to orlistat.But double-current abstraction technique requires very high to the Lipstatin weight content in fermented liquid, for example in above-mentioned purifying process, the Lipstatin weight content in fermented liquid need be up to 90%, otherwise cannot purify at all, obtain other orlistat of pharmaceutical grade, and extraction equipment costliness once drops into larger.
U.S. Patent No. US4598089 discloses a kind of method of and purifying Lipstatin concentrated by chromatographic process, wherein will be containing chromatographic column wash-out, concentrated on the fermented product of Lipstatin.A kind of purification process of Lipstatin is disclosed in the open No.CN101775416A of Chinese patent application, wherein the fermented liquid containing Lipstatin is extracted with 60-85% aqueous ethanolic solution, phase inversion enters in normal heptane again, after concentrating under reduced pressure again with acetonitrile extraction, oil removing, washing, upper silica gel column chromatography after concentrating under reduced pressure, elutriant concentrating under reduced pressure obtains Lipstatin crude product.
It is mainly to adopt preparative chromatography post, resin column and silicagel column that chromatographic column is separated in industrial.Preparative chromatography post is expensive, is only suitable for carrying out the production of the high and product that yields poorly of added value; Although resin column is widely used, resin price is still comparatively expensive compared with silica gel.
Therefore, for Lipstatin, still generally adopt at present silica gel separated, use organic solvent (being generally methyl alcohol, ethanol) to zymocyte liquid extract, separation, the concentrated rear upper silica gel column chromatography of extraction liquid obtaining, with eluent, carry out wash-out, the elutriant containing target product part is concentrated, crystallization obtains product.For example, in the open No.CN 101775416A of Chinese patent application, disclose employing silica gel separation method and extracted Lipstatin.
But the Lipstatin content extracting in actual production is very low, and particularly weight content is very low, had a strong impact on separating effect and the efficiency of silica gel from bacterium slag.In addition, in extract, have part material and silica gel adsorption seriously and can not or be difficult to again solution, " the extremely absorption " being commonly called as, this not only can strengthen silica regeneration difficulty, increase production cost, and this part material can affect the separating effect of silica gel.
To this, be mostly to allow Lipstatin extract reach purifying object by multiple organic solvent phase inversion at present.By removing this impurity of part in extract after multi-solvents phase inversion, reach the object of purifying Lipstatin extract.But the use of a large amount of organic solvents has not only increased recovery operation amount, also increased production safety risk, and often contained large, the volatile organic solvent of this class toxicity of acetonitrile in this process, this increases the invisible cost of all respects.
Summary of the invention
For solving the above-mentioned problems in the prior art, the invention provides a kind of method of purification of Lipstatin.
Particularly, the invention provides:
(1) method of purification for Lipstatin, the method comprises:
1) provide the solid mixt that contains Lipstatin;
2) using step 1) solid mixt that contains Lipstatin that obtains extracts as extraction agent with low polarity or non-polar the first organic solvent, thus obtain the extraction liquid that contains Lipstatin;
3) by step 2) extraction liquid that contains Lipstatin that obtains adsorbs with molecular sieve, and carries out wash-out with the first eluent, thus obtain the first elutriant of containing Lipstatin; And
4) by step 3) the first elutriant that contains Lipstatin that obtains is further purified by chromatography.
(2), according to the method for purification (1) described, wherein, the described solid mixt that contains Lipstatin is the dry bacterium powder of Lipstatin fermented liquid.
(3) according to the method for purification (1) described, wherein, described the first organic solvent is one or more that are selected from heptane, normal hexane, hexanaphthene and sherwood oil.
(4) according to the method for purification (1) described, wherein, described molecular sieve is inorganic zeolite molecular sieve; Preferably, described inorganic zeolite molecular sieve is to be selected from a kind of in 5A type, 10X type, 13X type or Y zeolite; More preferably, described inorganic zeolite molecular sieve is 13X type molecular sieve.
(5) according to the method for purification (1) described, wherein, described the first eluent is the mixed organic solvents of the second organic solvent of polarity or middle polarity and the 3rd organic solvent of low polarity; Preferably, described the second organic solvent is one or more that are selected from methyl alcohol, ethanol, acetone, ethyl acetate, butylacetate; Preferably, described the 3rd organic solvent is one or more that are selected from heptane, normal hexane, sherwood oil; Preferably, the volume ratio of described the second organic solvent and described the 3rd organic solvent is 1: (5-20).
(6) method of purification according to (1) or (5), wherein, in described step 3) in, before carrying out described wash-out, adopt described step 2) in the described molecular sieve of described extraction agent after to absorption wash.
(7) according to the method for purification (1) described, wherein, described step 4) comprising:
A) by step 3) the first elutriant that contains Lipstatin that obtains carries out chromatography, and carries out wash-out with the second eluent, thus obtain the second elutriant of containing Lipstatin;
B) the second elutriant that contains Lipstatin step a) being obtained carries out crystallization, thereby obtains Lipstatin.
(8) according to the method for purification (7) described, wherein, described chromatography is silica gel column chromatography; The mixed organic solvents of the 4th organic solvent that preferably, described the second eluent is middle polarity and the 5th organic solvent of low polarity; Preferably, described the 4th organic solvent is selected from ethyl acetate and/or butylacetate; Preferably, described the 5th organic solvent is selected from heptane and/or sherwood oil; Preferably, the volume ratio of described the 4th organic solvent and described the 5th organic solvent is 1: (5-15).
(9) according to the method for purification described in any one in (1)-(8), wherein, in described step 4) in, before carrying out described chromatography, described the first elutriant that contains Lipstatin is concentrated.
(10) according to the method for purification (1) described, wherein, in described step 5) in, described crystallization comprises: described the second elutriant that contains Lipstatin is concentrated, and at the temperature of-10 ℃ to 5 ℃, carry out crystallization, thereby obtain Lipstatin.
Method of the present invention has following beneficial effect:
The present invention adopts inorganic molecule sieve to carry out selective adsorption to the solvent containing Lipstatin, its cost ratio silica gel is slightly cheap, under high temperature, the more stable regeneration life-span is long, and molecular weight and molecular structure are had compared with strong selectivity, reached the effect of purifying Lipstatin, and to silica gel, there is the material of " extremely absorption " effect to greatly reduce in the Lipstatin condensed cream after selective adsorption, improved the weight content of Lipstatin, reduced the usage quantity of silica gel, improved the utilization ratio of silica gel, separating effect, efficiency, and use silica gel later just renewable by polar solvent drip washing, reduced the number of times that needs high temperature sintering regeneration, improved the work-ing life of silica gel, thereby greatly reduce production run cost.
Embodiment
Below the invention will be further described for the description by embodiment, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modifications or improvement, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
" molecular sieve " as herein described comprises the silico-aluminate of crystal type, has uniform pore texture.In this molecular sieve, contain a large amount of crystal water, during heating, can vaporize and remove, therefore claim again zeolite.Its chemical constitution can be expressed as M
x/n[(AlO
2)
x(SiO
2)
y] ZH
2o, in its Chinese style, M is metallic cation, and n is its valence mumber, and x is AlO
2molecule number, y is SiO
2molecule number, Z is water molecule number.Because AlO
2electronegative, the existence of metallic cation can make molecular sieve keep electric neutrality.
Conventional molecular sieve mainly contains: square na-pretreated zeolite, as A type molecular sieve; Faujasite, as X-type, Y-shaped molecular sieve; Mercerising type zeolite; High-silicon type zeolite, as ZSM-5 etc.The constitutional features of molecular sieve can be divided into different layer of structure.First layer of structure is the most basic structural unit silicon-oxy tetrahedron (SiO namely
4) and aluminum-oxygen tetrahedron (AlO
4), they form the skeleton of molecular sieve.Adjacent tetrahedron connects to ring by oxo bridge.Ring is second level of molecular sieve structure, and proportionately the oxygen atomicity of ring is divided, and has quaternary oxygen ring, five yuan of oxygen rings, hexa-atomic oxygen ring, eight yuan of oxygen rings, ten yuan of oxygen rings and ten binary oxygen rings etc.Ring is the passage aperture of molecular sieve, to playing sieving action by molecule.Oxygen ring interconnects by oxo bridge, forms and has three-dimensional polyhedron.Various polyhedrons are the 3rd levels of molecular sieve structure.Polyhedron has the cage of hollow, and cage is the key character of molecular sieve structure.Cage is divided into α cage, octahedral zeolite cage, β cage and γ cage etc.Wherein octahedral zeolite cage is the main hole that forms X-type and Y-shaped molecular sieve skeleton, also claims supercage.Inorganic zeolite molecular sieve in the present invention is selected from a kind of in 5A type, 10X type, 13X type or Y zeolite, preferably has the X-type of supercage structure and the type molecular sieve of Y.
" nonpolarity " as herein described (or title " nonpolar ") organic solvent generally refers to that its molecule is symmetrical organic solvent; " rudimentary property " (or title " low-pole ") organic solvent generally refers to the slightly asymmetric organic solvent of its molecule.Nonpolarity or rudimentary property organic solvent generally comprises: hydro carbons, sherwood oil, hydro carbons comprise (such as): chloralkane, nitration alkane etc.
" middle polarity " as herein described organic solvent generally refers to the comparatively asymmetric organic solvent of its molecule." polarity " as herein described organic solvent generally refers to the higher organic solvent of the asymmetric degree of its molecule.Middle polarity organic solvent generally comprises: ketone, ester class, ethers and alcohol ethers.Polar organic solvent generally comprises: alcohols.
The method of purification that the object of this invention is to provide the Lipstatin that a kind of production cost is low.
Lipstatin is product in spore, and its fermented liquid is a kind of dense thick, dense high oily waterborne liquid of bacterium, and without obvious solid, fermented liquid is not easy to solid-liquid separation; Lipstatin is the material of a kind of polyene hydrocarbon and ester structure, and it is subject to various factors and destroyed and degrade, and produces impurity.For example, extraction temperature has important impact for extracting Lipstatin.When extraction temperature is too low, extraction not exclusively, can cause waste to raw material; Along with the rising of extraction temperature, extraction efficiency improves thereupon, but excess Temperature can make Lipstatin that degraded occurs, increases the impurity in product.In technique, conventionally adopt at present ethanol as extraction agent, and the solvability of ethanol is strong, the material that can dissolve is extensive, but the poor selectivity to solute, impurity in extraction liquid is too much, to after silica gel have the content of material of " extremely absorption " effect high, so in actual production process the work-ing life of silica gel short, production cost is high.
The inventor found through experiments: first adopt the organic solvent of low-pole to extract the solid matter containing Lipstatin, with inorganic molecule sieve, the extraction liquid containing Lipstatin is carried out to selective adsorption more afterwards, can reach the effect of purifying Lipstatin, and to silica gel, there is the material of " extremely absorption " effect to greatly reduce in the Lipstatin condensed cream after selective adsorption, improved the weight content of Lipstatin, reduced the usage quantity of silica gel, improve the separating effect of silica gel, efficiency, and use silica gel later just renewable by polar solvent drip washing, reduced the number of times that needs high temperature sintering, improved the work-ing life of silica gel, thereby greatly reduce production run cost.
Embodiment of the present invention are:
A method of purification for Lipstatin, it comprises:
1) provide the solid mixt that contains Lipstatin;
2) using step 1) solid mixt that contains Lipstatin that obtains extracts as extraction agent with low polarity or non-polar organic solvent, thus obtain the extraction liquid that contains Lipstatin;
3) by step 2) extraction liquid that contains Lipstatin that obtains adsorbs with molecular sieve, and carries out wash-out with the first eluent, thus obtain the first elutriant of containing Lipstatin; And
4) by step 3) the first elutriant that contains Lipstatin that obtains is further purified by chromatography.
Wherein, above-mentioned step 1) solid mixt that contain Lipstatin of the solid mixt that contains Lipstatin in for being obtained by fermented liquid or alternate manner, does not limit especially to the content of Lipstatin in this solid mixt.Preferably by the fermented liquid of producing the Streptomyces toxytricini (Streptomyces toxytricini) of Lipstatin is carried out to the solid mixt that contains Lipstatin that dry method obtains.More preferably, described dry be that mode by spray dry or freeze-drying realizes.
Preferably, the solid mixt that contains Lipstatin is the dry bacterium powder of Lipstatin fermented liquid, comprises by the fermented liquid of producing the Streptomyces toxytricini of Lipstatin is dried to the dry bacterium powder obtaining, as Lipstatin fermented liquid sprays dry slag.
Fermented liquid as herein described refers to the fermented liquid of the Streptomyces toxytricini of producing Lipstatin, its can by (such as) method described in following scientific and technical literature ferments and obtains: " Streptomyces toxytricini is produced fermentation and the purifying technique of Lipstatin " < < Chinese Journal of Pharmaceuticals > > of the people such as the people that act like a bully, 2007,38 (10), 705-708 page.Streptomyces toxytricini can be any Streptomyces toxytricini that can mainly produce Lipstatin, comprises the high-yield strains obtaining by mutagenesis.
The process that is obtained solid state powder by fermented liquid can be adopted with the following method: Direct spraying is dried or freeze-drying.The dry resistance to transportation of bacterium powder of solid state fermentation, the easily preservation that obtain thus.But the invention is not restricted to this.For example, the employing spray drying device of mentioning in the open No.CN101885713A of Chinese patent application is to carrying out spray-dired method containing Lipstatin fermented liquid.For example, the dry step of Direct spraying can be carried out according to following manner: fermented liquid is directly sprayed when dry, the gas feed temperature of spray-drying tower can be controlled to 150-180 ℃, gas outlet temperature is controlled at 50-70 ℃, in tower, temperature of charge remains on 60-80 ℃, centrifugal atomizer rotating speed is controlled at 9000-12000rpm, obtains the dry bacterium powder of solid fermentation.For example, freeze-drying can be carried out according to following manner: the adoptable device of step of freeze drying is LG-0.2 series small System of Dry-out Test Machine (deriving from Shenyang Aero Space Xinyang Quick Freezing Equip. Manuf. Co., Ltd.), but is not limited to this; The temperature of eutectic point of Lipstatin fermented liquid is about-20 ℃, and the pre-freeze temperature of product is-38 ℃; The pre-freeze time is 3-4h, and the dry heating-up time of trunk is 4h, and main drying temperature is-8 ℃; Pressure is 0.1mbar, is no more than 0.25mbar judges that trunk is dry and complete with 1min internal pressure liter; Redrying temperature is 30 ℃-60 ℃, and pressure 0.03mbar is no more than 0.06mbar with 1 minute internal pressure liter and judges that freeze-drying finishes, and obtains faint yellow lyophilized powder.
Preferably, the low polarity above-mentioned step 2) or non-polar organic solvent are one or more in heptane, normal hexane, hexanaphthene and sherwood oil.Extraction temperature is 20-35 ℃.Extraction can also repeat 1-4 time again, fully to extract Lipstatin.
Preferably, the molecular screening above-mentioned step 3) sieves from inorganic molecule.More preferably, inorganic molecule sieve is inorganic zeolite molecular sieve.Further preferably, inorganic zeolite molecular sieve is selected from a kind of in 5A type, 10X type, 13X type or Y zeolite, is most preferably 13X type molecular sieve.
Preferably, above-mentioned step 3) eluent in can be the mixed solvent of polarity or medium polar solvent and weak polar solvent, and described polarity or medium solvent comprise one or more in methyl alcohol, ethanol, acetone, ethyl acetate, butylacetate; Weak polar solvent comprises one or more in heptane, normal hexane, sherwood oil; The volume ratio of described polarity or medium solvent and weak polar solvent is preferably 1: (5-20).
Preferably, in above-mentioned step 3) in, before carrying out described wash-out, adopt above-mentioned step 2) in the extraction agent that the adopts molecular sieve after to absorption wash.Further preferably, molecule being sieved and washed to elutant with extraction agent, be near when colourless, then start to carry out wash-out with eluent.
Preferably, using described molecular sieve in sorbent material is filled in adsorption column, and make above-mentioned step 2) in extract flow through this adsorption column, to adsorb.Absorption can be carried out under 10-45 ℃ and flow are the condition of 1-10BV/h (column volume is per hour).Wash-out can carry out under the condition that be 1-4BV/h at 10-45 ℃ and flow.
Molecular sieve can carry out wash-out by one or more in methyl alcohol, ethanol, acetone after use, to recover its absorption property.
Preferably, above-mentioned step 4) comprising:
A) by step 3) the first elutriant that contains Lipstatin that obtains carries out chromatography, and carries out wash-out with the second eluent, thus obtain the second elutriant of containing Lipstatin;
B) the second elutriant that contains Lipstatin step a) being obtained carries out crystallization, thereby obtains Lipstatin.
Preferably, in above-mentioned step 4) in, described chromatography can be any conventional chromatography method known in the art, is preferably silica gel column chromatography, for example silica gel column chromatography.Preferably, the mixed organic solvents that described the second eluent is middle polarity organic solvent and weakly polar organic solvent; Preferably, described middle polarity organic solvent is selected from ethyl acetate and/or butylacetate; Preferably, described weakly polar organic solvent is selected from heptane and/or sherwood oil; Preferably, the volume ratio of described middle polarity organic solvent and described weakly polar organic solvent is 1: (5-15).Preferably, before carrying out chromatography, the first elutriant that contains Lipstatin is concentrated, concentrated can be any conventional concentration method known in the art, and for example reduction vaporization is concentrated.
Preferably, at above-mentioned step b) in, described the second elutriant that contains Lipstatin is concentrated, concentrated can be any conventional concentration method known in the art, for example reduction vaporization is concentrated, and reduction vaporization is concentrated can carry out at 20-50 ℃; Then carry out crystallization, low temperature crystallization (for example carrying out crystallization at the temperature of-10 ℃ to 5 ℃) for example, thus obtain Lipstatin.
Wherein, silica gel can be reused 5-8 time through method of the present invention, each only need carry out wash-out by one or more in methyl alcohol, ethanol, acetone, just can recover its absorption property.
A kind of specific embodiment of the present invention can be:
1. will spray low polarity or non-polar organic solvent extraction for dry slag containing the fermented liquid of Lipstatin, this organic solvent is a kind of in heptane, normal hexane, hexanaphthene, sherwood oil.
2. extraction temperature is at 20-35 ℃, extracting twice, and single extraction solvent is 4-6 times of envelope-bulk to weight ratio of the dry bacterium slag of spray; Reextraction solvent is 3-5 times of envelope-bulk to weight ratio of the dry bacterium slag of spray.Extraction liquid adopts 3-foot automatic scraper conveyer centrifugal to carry out separation.
3. extraction liquid is crossed after 100-200 order deep bed filter, the filling post that inorganic zeolite molecular sieve is housed of flowing through adsorbs, and adsorption column is in-built that inorganic zeolite molecular sieve is selected from any one of 5A, 10X, 13X or Y zeolite.
4. the adsorption liquid of the inorganic zeolite molecular sieve of flowing through carries out under 10-45 ℃ and flow 1-10BV/h; Eluent is the mixed solution of polarity or medium polar solvent and weak polar solvent, and polarity or medium polar solvent comprise methyl alcohol, ethanol, acetone, ethyl acetate, butylacetate; Weak polar solvent comprises heptane, normal hexane, sherwood oil.The ratio of mixed solvent is that polar solvent is 1 than weak polar solvent: 5-20.Eluent carries out wash-out under 10-45 ℃ and 1-4BV/h.
5. the inorganic zeolite molecular sieve of wash-out after completing used eluant solution by 10-100% methyl alcohol or ethanol, acetone to recover its absorption property.When inorganic zeolite molecular sieve is adsorbed Lipstatin Efficiency Decreasing after reusing for a long time, zeolite molecular sieve is taken out in adsorption column, at 400-800 ℃, calcination 3-5 hour, puts in back adsorption column, and adsorption efficiency recovers.
6. on the condensed cream obtaining after elutriant reduced vacuum being concentrated, silicagel column carries out chromatography.In silicagel column after desorb completes, for silica gel, polar solvent (comprising methyl alcohol, ethanol, acetone) can recover absorption property, and this silica gel repeated regeneration can recover its absorption property through high temperature sintering after using 5-8 time.
7. silicagel column elutriant is at 20-50 ℃ after vacuum-concentrcted, low temperature crystallization.
By object lesson, further explain and describe content of the present invention below, but these examples are not to be construed as limiting the scope of the invention.
In following examples, the used solid mixt that contains Lipstatin is that Lipstatin fermented liquid sprays dry slag, is to obtain according to the mode of test example 1, but the invention is not restricted to this.
In following examples, according to following method, measure content the calculate recovery rate (percentage recovery) of Lipstatin: adopt HPLC method, the condition of HPLC method is: high performance liquid chromatograph (purchased from the LC-10AD of Shimadzu Corporation); Chromatographic column XDB-C18 (250mm * 4.6mm); Moving phase is 90% acetonitrile (30: 70); Flow 1.0ml/min; 30 ℃ of column temperatures; Detect wavelength 195nm; Sampling volume 10 μ l.
13X type, 10X type and 5A type molecular sieve used in following examples can be purchased from Shanghai zeolite molecular sieve company limiteds; Heptane, sherwood oil, ethyl acetate can be purchased from Chongqing Chuan Dong Chemical Co., Ltd..
Test example 1
Lipstatin fermented liquid is directly sprayed dry, the gas feed temperature of spray-drier is controlled to 160 ℃, and gas outlet temperature is controlled at 70 ℃, and in tower, temperature of charge remains on 80 ℃, centrifugal atomizer rotating speed is controlled at 10000rpm, obtains Lipstatin fermented liquid and sprays dry slag.
Embodiment 1
Lipstatin fermented liquid sprays dry slag 1000g heptane extracting twice, and extraction temperature is 30 ℃, and it is 5L and 3L that single extraction and reextraction are used heptane amount.HPLC method detects Lipstatin in heptane extraction mixed solution, and recording its purity is 20.59%.The heptane that takes a morsel extraction mixed solution reduced vacuum at 40 ℃ is concentrated obtains condensed cream, and measuring weight content is 8%; In residue extraction mixed solution, containing Lipstatin product volume, be 6.5g.
Molecular sieve column blade diameter length ratio is 1: 7, and 13X type molecular sieve for molecular screening, takes 13X type molecular sieve 50g, and before use molecular sieve is soaked more than 8 hours with heptane.Heptane extraction mixed solution is by molecular sieve column under 25 ℃ and flow 4BV/h, and after completing, continuation is flowed through molecular sieve column until become colourless with fresh heptane, and upper prop adsorption rate is 95%.Preparation ethyl acetate: heptane is the eluent of 1: 10 (volume ratio) is carried out wash-out under 35 ℃ and flow 1BV/h.Gained elutriant reduced vacuum at 40 ℃ is concentrated obtains oily concentrated solution, and the purity of its Lipstatin HPLC is 68.1%, and weight content is 56.3%, and product volume is 6.11g, and desorption efficiency is 99%.By silica gel column chromatography on oily concentrated solution, silicagel column filling silica gel 110g, blade diameter length ratio approximately 1: 6.After upper prop completes by ethyl acetate: heptane=1: 9 carry out wash-out, flow 1BV/h, Eluant temperature is 25 ℃, collect the HPLC purity of Lipstatin in silicagel column at more than 90% elutriant, concentrated through reduced vacuum at 40 ℃ again, and at-5 ℃, carry out crystallization, thus obtaining Lipstatin finished product 4.88g, it is 95.3% that HPLC detects purity.
Embodiment 2
Lipstatin fermented liquid sprays dry petroleum ether extraction twice for slag 1000g, and extraction temperature is 30 ℃, and it is 5L and 3L that single extraction and reextraction are used sherwood oil amount.The Lipstatin purity that HPLC method detects petroleum ether extraction mixed solution is 22.7%.The concentrated condensed cream that obtains of the petroleum ether extraction mixed solution reduced vacuum at 40 ℃ that takes a morsel, measuring weight content is 7.6%; In residue extraction mixed solution, Lipstatin product content is 6g.
Molecular sieve column blade diameter length ratio is 1: 8, and molecular sieve is 10X type molecular sieve, takes 10X type molecular sieve 63g, more than first molecular sieve being soaked to 8h with sherwood oil before use.Petroleum ether extraction mixed solution is by molecular sieve column under 25 ℃ and flow 3BV/h, and after completing, continuation is flowed through molecular sieve column until become colourless with live oil ether, and upper prop adsorption rate is 85%.Preparation ethyl acetate is the eluent of 1: 13 (volume ratio) than heptane, under 35 ℃ and flow 1BV/h, carries out wash-out.The concentrated oily concentrated solution that obtains of gained elutriant reduced vacuum at 40 ℃, the purity that HPLC method detects Lipstatin is 54.3%, and weight content is 45.6%, and product volume is 5.06g, and desorption efficiency is 99.2%.By silica gel column chromatography on oily concentrated solution, silicagel column filling silica gel 170g, blade diameter length ratio approximately 1: 7.After upper prop completes by ethyl acetate: heptane=1: 9 carry out wash-out, flow 1BV/h, Eluant temperature is 27 ℃, collect the HPLC purity of Lipstatin in silicagel column at more than 90% elutriant, concentrated through reduced vacuum at 40 ℃ again, low temperature crystallization at-5 ℃, obtains Lipstatin finished product 3.54g, and it is 92% that HPLC detects purity.
Embodiment 3
Lipstatin fermented liquid sprays dry petroleum ether extraction twice for slag 1000g, and extraction temperature is 30 ℃, and it is 5L and 3L that single extraction and reextraction are used sherwood oil amount.The Lipstatin purity that HPLC method detects in petroleum ether extraction mixed solution is 22.56%.The concentrated condensed cream that obtains of the petroleum ether extraction mixed solution reduced vacuum at 40 ℃ that takes a morsel, measuring weight content is 8.2%; In residue extraction mixed solution, Lipstatin product content is 6.7g.
Molecular sieve column blade diameter length ratio is 1: 7, and molecular sieve is 5A type molecular sieve, takes 5A type molecular sieve 60g, more than first molecular sieve being soaked to 8h with sherwood oil before use.Petroleum ether extraction mixed solution is by molecular sieve column under 20 ℃ and flow 3BV/h, and after completing, continuation is flowed through molecular sieve column until become colourless with live oil ether, and upper prop adsorption rate is 78%.Preparation ethyl acetate is the eluent of 1: 13 (volume ratio) than heptane, under 35 ℃ and flow 1BV/h, carries out wash-out.The concentrated oily concentrated solution that obtains of gained elutriant reduced vacuum at 40 ℃, the purity that HPLC detects Lipstatin is 48%, and weight content is 20.7%, and product volume is 5.17g, and desorption efficiency is 99%.By silica gel column chromatography on oily concentrated solution, silicagel column filling silica gel 500g, blade diameter length ratio approximately 1: 10.After upper prop completes by ethyl acetate: heptane=1: 12 carry out wash-out, flow 1BV/h, Eluant temperature is 26 ℃, collect the HPLC purity of Lipstatin in silicagel column at more than 90% elutriant, concentrated through reduced vacuum at 40 ℃ again, and at-5 ℃ low temperature crystallization, obtain Lipstatin finished product 2.58g, it is 91.6% that HPLC detects purity.
Result by embodiment 1-3 can be found out, by method of the present invention, can obtain highly purified Lipstatin finished product, and adopts the result of 13X type molecular sieve to be better than the result that adopts other types of molecules sieve.
Embodiment 4
Adopt processing step purification Lipstatin substantially the same manner as Example 1, wherein see table 1 from parameter and result different in embodiment 1.
Embodiment 5
Adopt processing step purification Lipstatin substantially the same manner as Example 2, wherein see table 1 from parameter and result different in embodiment 2.
Comparative example 1
Adopt the processing step purification Lipstatin roughly the same with embodiment 1, but do not pass through the step of inorganic zeolite molecular sieve, its processing parameter and result see table 1.
Comparative example 2
Adopt the processing step purification Lipstatin roughly the same with embodiment 1, but do not pass through the step of inorganic zeolite molecular sieve, each parameter of its technique and result see table 1.
The experimental result of table 1 comparative example 1-2 and embodiment 4-5
By above-mentioned contrast, can find: the Lipstatin enriched material after molecular sieve is processed, due to the significantly lifting of weight content and HPLC content, silica gel consumption in the time of silica gel column chromatography is greatly reduced, and chromatography yield significantly improves; The repeating utilization factor again of silica gel promotes and also greatly reduces production cost.
Claims (14)
1. a method of purification for Lipstatin, the method comprises:
1) provide the solid mixt that contains Lipstatin, wherein, the described solid mixt that contains Lipstatin is the dry bacterium powder of Lipstatin fermented liquid;
2) using step 1) solid mixt that contains Lipstatin that obtains extracts as extraction agent with low polarity or non-polar the first organic solvent, thus obtain the extraction liquid that contains Lipstatin;
3) by step 2) extraction liquid that contains Lipstatin that obtains adsorbs with molecular sieve, and carry out wash-out with the first eluent, thereby obtain the first elutriant that contains Lipstatin, wherein, described molecular sieve is inorganic zeolite molecular sieve, and described inorganic zeolite molecular sieve is to be selected from a kind of in 5A type, 10X type, 13X type or Y zeolite; And
4) by step 3) the first elutriant that contains Lipstatin that obtains is further purified by chromatography, wherein, described chromatography is silica gel column chromatography, and described step 4) comprising:
A) by step 3) the first elutriant that contains Lipstatin that obtains carries out chromatography, and carries out wash-out with the second eluent, thus obtain the second elutriant of containing Lipstatin;
B) the second elutriant that contains Lipstatin step a) being obtained carries out crystallization, thereby obtains Lipstatin.
2. method of purification according to claim 1, wherein, described the first organic solvent is one or more that are selected from heptane, normal hexane, hexanaphthene and sherwood oil.
3. method of purification according to claim 1, wherein, described inorganic zeolite molecular sieve is 13X type molecular sieve.
4. method of purification according to claim 1, wherein, described the first eluent is the mixed organic solvents of the second organic solvent of polarity or middle polarity and the 3rd organic solvent of low polarity.
5. method of purification according to claim 4, wherein, described the second organic solvent is one or more that are selected from methyl alcohol, ethanol, acetone, ethyl acetate, butylacetate.
6. method of purification according to claim 4, wherein, described the 3rd organic solvent is one or more that are selected from heptane, normal hexane, sherwood oil.
7. method of purification according to claim 4, wherein, the volume ratio of described the second organic solvent and described the 3rd organic solvent is 1:(5-20).
8. method of purification according to claim 1, wherein, in described step 3) in, before carrying out described wash-out, adopt described step 2) in the described molecular sieve of described extraction agent after to absorption wash.
9. method of purification according to claim 1, wherein, the mixed organic solvents of the 4th organic solvent that described the second eluent is middle polarity and the 5th organic solvent of low polarity.
10. method of purification according to claim 9, wherein, described the 4th organic solvent is selected from ethyl acetate and/or butylacetate.
11. methods of purification according to claim 9, wherein, described the 5th organic solvent is selected from heptane and/or sherwood oil.
12. methods of purification according to claim 9, wherein, the volume ratio of described the 4th organic solvent and described the 5th organic solvent is 1:(5-15).
13. according to the method for purification described in any one in claim 1-12, wherein, and in described step 4) in, before carrying out described chromatography, described the first elutriant that contains Lipstatin is concentrated.
14. methods of purification according to claim 1, wherein, at described step b) in, described crystallization comprises: described the second elutriant that contains Lipstatin is concentrated, and at the temperature of-10 ℃ to 5 ℃, carry out crystallization, thereby obtain Lipstatin.
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|---|---|---|---|---|
| US4598089A (en) * | 1983-06-22 | 1986-07-01 | Hoffmann-La Roche Inc. | Leucine derivatives |
| CN1266058A (en) * | 1999-01-29 | 2000-09-13 | 弗·哈夫曼-拉罗切有限公司 | Process for refining lipstatin |
| WO2003048335A2 (en) * | 2001-12-04 | 2003-06-12 | Biogal Gyogyszergyar Rt | A fermentation process for lipstatin and method of extracting lipstatin from a fermentation broth |
| CN101885713A (en) * | 2010-07-19 | 2010-11-17 | 大邦(湖南)生物制药有限公司 | New process for separating and extracting lipstatin from stretomyces toxytricini fermentation liquor |
| CN101948450A (en) * | 2010-10-13 | 2011-01-19 | 鲁南制药集团股份有限公司 | Method for preparing orlistat |
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2011
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4598089A (en) * | 1983-06-22 | 1986-07-01 | Hoffmann-La Roche Inc. | Leucine derivatives |
| CN1266058A (en) * | 1999-01-29 | 2000-09-13 | 弗·哈夫曼-拉罗切有限公司 | Process for refining lipstatin |
| WO2003048335A2 (en) * | 2001-12-04 | 2003-06-12 | Biogal Gyogyszergyar Rt | A fermentation process for lipstatin and method of extracting lipstatin from a fermentation broth |
| CN101885713A (en) * | 2010-07-19 | 2010-11-17 | 大邦(湖南)生物制药有限公司 | New process for separating and extracting lipstatin from stretomyces toxytricini fermentation liquor |
| CN101948450A (en) * | 2010-10-13 | 2011-01-19 | 鲁南制药集团股份有限公司 | Method for preparing orlistat |
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