EP0583468A1 - Process for making pure phosphatidylethanolamine and phosphatidylmyoinositol - Google Patents
Process for making pure phosphatidylethanolamine and phosphatidylmyoinositolInfo
- Publication number
- EP0583468A1 EP0583468A1 EP19930917399 EP93917399A EP0583468A1 EP 0583468 A1 EP0583468 A1 EP 0583468A1 EP 19930917399 EP19930917399 EP 19930917399 EP 93917399 A EP93917399 A EP 93917399A EP 0583468 A1 EP0583468 A1 EP 0583468A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- filtration
- phosphatidylmyoinositol
- silica
- precipitate
- filtrate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 14
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 title claims abstract description 10
- 150000008104 phosphatidylethanolamines Chemical class 0.000 title claims abstract description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 13
- 239000002904 solvent Substances 0.000 claims abstract description 8
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 5
- 238000011282 treatment Methods 0.000 claims abstract 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 13
- 239000000706 filtrate Substances 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 9
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 229940083466 soybean lecithin Drugs 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 238000001704 evaporation Methods 0.000 claims description 4
- 230000008020 evaporation Effects 0.000 claims description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 239000011877 solvent mixture Substances 0.000 claims description 3
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 claims description 2
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 claims description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 2
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical group ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 claims description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-O azanium;hydron;hydroxide Chemical compound [NH4+].O VHUUQVKOLVNVRT-UHFFFAOYSA-O 0.000 claims description 2
- DLNWMWYCSOQYSQ-UHFFFAOYSA-M benzyl-hexadecyl-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 DLNWMWYCSOQYSQ-UHFFFAOYSA-M 0.000 claims description 2
- 238000002425 crystallisation Methods 0.000 claims description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 claims description 2
- UBOXGVDOUJQMTN-UHFFFAOYSA-N trichloroethylene Natural products ClCC(Cl)Cl UBOXGVDOUJQMTN-UHFFFAOYSA-N 0.000 claims description 2
- 238000001291 vacuum drying Methods 0.000 claims description 2
- 230000008025 crystallization Effects 0.000 claims 1
- 238000001033 granulometry Methods 0.000 claims 1
- 230000007928 solubilization Effects 0.000 claims 1
- 238000005063 solubilization Methods 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- PZNPLUBHRSSFHT-RRHRGVEJSA-N 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[C@@H](COP([O-])(=O)OCC[N+](C)(C)C)COC(=O)CCCCCCCCCCCCCCC PZNPLUBHRSSFHT-RRHRGVEJSA-N 0.000 abstract 1
- 238000001179 sorption measurement Methods 0.000 abstract 1
- 239000008347 soybean phospholipid Substances 0.000 abstract 1
- 239000000047 product Substances 0.000 description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 4
- 229960000367 inositol Drugs 0.000 description 4
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 4
- 150000001414 amino alcohols Chemical class 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 235000019198 oils Nutrition 0.000 description 3
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 2
- 230000001476 alcoholic effect Effects 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 150000008103 phosphatidic acids Chemical class 0.000 description 2
- 235000012424 soybean oil Nutrition 0.000 description 2
- 239000003549 soybean oil Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N glycerol 1-phosphate Chemical compound OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940042880 natural phospholipid Drugs 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 125000001095 phosphatidyl group Chemical group 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical group 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
- C07F9/103—Extraction or purification by physical or chemical treatment of natural phosphatides; Preparation of compositions containing phosphatides of unknown structure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/117—Esters of phosphoric acids with cycloaliphatic alcohols
Definitions
- the present invention refers to a novel industrial process for the production, in a form suitable for pharmaceutical use, of pure phosphatidylethanolamine and phosphatidylmyoinositol from soybean lecithin.
- the whole phospholipides mixture from soybean oil is already widely used as such together with soybean oil, glucosides and various pigments, which are precipitated by adding water to the oil itself, centrifuged, dried and directly used.
- Phospholipides naturally occur, in various amounts, in all the cells of living animal as well vegetal organisms, always as complex mixtures of terms which differ from each other by the structure of the polyvalent alcoholic function, of the amino alcohol and, mainly, of the fatty acids they come from.
- phospholipides Of all types of phospholipides, the most abundant in vegetal species are the glycerophosphatides, their structure deriving from alpha-glycerophosphoric acid.
- the glycerophosphatides present in rather high quantities are found especially as esters of phosphatidic acids with amino alcohols, such as ethanolamine, choline and with the myoinositol polyalcohol.
- R and R which may be the same or different, represent a saturated or unsaturated fatty acid residue, with a chain length between 15 and 20 carbon atoms.
- R and R represent a saturated or unsaturated fatty acid residue, with a chain length between 15 and 20 carbon atoms.
- Purpose and object of this invention is to provide a process for the production of phosphatidylethanolamine and phosphatylmyoinositol by separation from soybean lecithin, such process being industrially advantageous from the standpoint of the yields, of purity of the compounds resulting therefrom and of the process reproducibility.
- a more specific purpose of the invention is to obtain the above mentioned products with a purity degree suitable for therapeutic use.
- step (h) heat treatment of the precipitate (P) of step (c) with a mixture of a chlorinated solvent as above defined and of an aliphatic alcohol selected from methanol, ethanol, n-propanol, isopropanol and mixtures thereof, and filtration;
- step (i) heat treatment of step(c) residual silica with a like solvent mixture and filtration as per step (h);
- a relevant and remarkable aspect of the process of this invention is that it is based in a number of operations not involving chemical reactions that are likely to alter or anyhow cause losses of the desired compounds as by products thereof.
- the following example illustrates a preferred embodiment of the process of the present invention, while not limiting it.
- Dissolving is accomplished in a glass flask equipped with stirrer, thermometer, bubble heating and cooling bath and to adjust boiling of the liquid.
- a solvent refluxing is carried out for 1 hour untill the solubilisation is almost completed.
- the solution contained in the flask is cooled to " 20°C and, still under stirring. 1 kg chromatography grade silica is added, and the material is maintained under stirring and again heated for additional 2 hours.
- the resulting suspension is cooled and filtered on a Buckner filter, equipped with porous septum, or by centrifuging, then washing repeatedly the silica portion with methylene chloride.
- the filtrate contains phosphatidylinositol, not pure due to the presence of phosphatidic acids, whereas the precipitate and silica portion are set apart and further treated for phosphatidylethanolamine recovery.
- the filtrate i.e. the solution as above obtained, is poured again in a flask and added with 50 g of a long chain quaternary ammonium base, such as cetyltrimethyl ammonium bromide or benzyldimethylhexadecylammonium bromide, which dissolve in the solution and additional 100 g chromatography grade silica are added.
- a long chain quaternary ammonium base such as cetyltrimethyl ammonium bromide or benzyldimethylhexadecylammonium bromide
- the solution is refluxed for 1 hour, cooled and filtered on a porous septum.
- the filtrate is treated with 1% of its total volume of 32% NH 4 OH, followed by stirring at room temperature for 30 minutes and filtering thereafter on a porous septum.
- the product phosphatidylmyoinositol, crystallizes and is separated b filtration as a powder and vacuum dried with a mechanical pump.
- the so obtained product has a purity higher than 98% if compared with Sigma standard.
- the previously set apart precipitate is treated with 5000 ml of 85/15 V/V solution of methylene chloride and methanol and refluxed for 1 hour. After filtering on porous septum, phosphatidylethanolamine remains in solution together with impurities of other phospatidic-like material, anyhow with an about neutral pH.
- silica freed from phosphatidylmyoinositol, is also treated with 1000 ml of a methylene chloride/methanol 85/15 (V/V) solution and refluxed for 15 minutes. After that time it is filtered on glass porous septum. A further warm washing of the silica, for 15 minutes with additional 500 ml of the previous solution, follows.
- the precipitate is washed with three subsequent 200 ml pure acetone portions and then vacuum dried.
- the obtained phosphatidylethanolamine is ground and has a purity of about 95%.
- Phosphatidylmyoinositol (2-3%) is the main impurity.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
Abstract
Procédé consistant à soumettre la lécithine de soja à une succession d'opérations qui n'impliquent aucune réaction chimique mais impliquent des traitements par solvant à adsorptions alternatives sur de la silice pour chromatographie. On obtient ainsi de la phosphatidyléthanolamine et du phosphatidylmyoïnositol dont la pureté en permet une utilisation pharmaceutique.Process consisting in subjecting the soy lecithin to a succession of operations which do not involve any chemical reaction but involve solvent treatments with alternative adsorption on silica for chromatography. Phosphatidylethanolamine and phosphatidylmyoïnositol are thus obtained, the purity of which allows pharmaceutical use.
Description
PROCESS FOR MAKING PURE PHOSPHATIDYLETHANOLAMINE AND
PHOSPHATIDYLMYOINOSITOL
The present invention refers to a novel industrial process for the production, in a form suitable for pharmaceutical use, of pure phosphatidylethanolamine and phosphatidylmyoinositol from soybean lecithin.
The whole phospholipides mixture from soybean oil is already widely used as such together with soybean oil, glucosides and various pigments, which are precipitated by adding water to the oil itself, centrifuged, dried and directly used.
Phospholipides naturally occur, in various amounts, in all the cells of living animal as well vegetal organisms, always as complex mixtures of terms which differ from each other by the structure of the polyvalent alcoholic function, of the amino alcohol and, mainly, of the fatty acids they come from.
The chemical investigation is further complicated since, besides the complexity of their natural mixture, also hydrolysis and transesterification phenomena come into play, which occur also during the extractive operation from raw materials, as well as because of the interfering by enzymes therein contained, and, in addition, for those derived from insaturated fatty acids, the oxidation reactions they easily undergo brought about by the atmospheric oxygen.
Of all types of phospholipides, the most abundant in vegetal species are the glycerophosphatides, their structure deriving from alpha-glycerophosphoric acid.
In soybean lecithin, the glycerophosphatides, present in rather high quantities are found especially as esters of phosphatidic acids with amino alcohols, such as ethanolamine, choline and with the myoinositol polyalcohol.
An alcoholic group and, also, an amino group, being simultaneously present in ethanolamine and choline, it would lead to envisage the formation of a saline bond between the phosphatidic acid and the amino group itself.
As a matter of fact, if a chemical reaction would take place, the forming of a salt or of an amide would be mainly possible, depending on whether the reaction is carried out at low or high temperature, whereas an ester is formed instead owing to an enzymatic type of reaction.
It is also worth to mention that the peculiar final chemical nature of these esters with amino alcohols might result in products showing a betaine-like structure as below indicated:
R -COO-CH
CH -O-P-O-CH -CH -NH 2 / 2 2 3
/
0 where R and R , which may be the same or different, represent a saturated or unsaturated fatty acid residue, with a chain length between 15 and 20 carbon atoms. As regards the phosphatidic ester with inositol, only myoinositol has been until now perfectly characterized in the natural phospholipids: the phosphatidyl myoinositols have the following general formula:
where R and R are as previously indicated.
The complete characterization of the isolated product confirmed that the hydroxyl group at the number 1 position of inositol is that esterified with the phosphatidic acid grouping.
Purpose and object of this invention is to provide a process for the production of phosphatidylethanolamine and phosphatylmyoinositol by separation from soybean lecithin, such process being industrially advantageous from the standpoint of the yields, of purity of the compounds resulting therefrom and of the process
reproducibility.
A more specific purpose of the invention is to obtain the above mentioned products with a purity degree suitable for therapeutic use.
The purpose and main object of the invention is achieved by a process which is characterized by the following steps:
(a) heat solubilisation of the starting soybean lecithin with a chlorinated solvent, selected from chloroform, dichloromethane, dichloroethane and trichloroethylene;
(b) addition under stirring to the solution, coooled to room temperature, of a chromatography grade silica, with a granule size between 60 and 500 Angstrom, and heating of the resulting suspension.
(c) filtration of the suspension to obtain a filtrate (F) and a precipitate (P);
(d) heat treatment of the filtrate with a long alkyl chain quaternary ammonium base and silica of the above indicated grade;
(e) filtration of the solution after cooling and treating the filtrate with ammonium hydrate under stirring;
(f) evaporation under reduced pressure of the solution to yield an oil and refluxing with ethanol;
(g) separation of phosphatidylmyoinositol by crystallisation;
(h) heat treatment of the precipitate (P) of step (c) with a mixture of a chlorinated solvent as above defined and of an aliphatic alcohol selected from methanol, ethanol, n-propanol, isopropanol and mixtures thereof, and filtration;
(i) heat treatment of step(c) residual silica with a like solvent mixture and filtration as per step (h);
(j) evaporation to dryness under reduced pressure of the combinated filtrates of the steps (h) and (i), followed by suspension of the resulting residue in heated, absolute ethanol;
(k) filtration of the resulting suspension and vacuum drying of the precipitate, yielding phosphadidylethanolamine.
A relevant and remarkable aspect of the process of this invention is that it is based in a number of operations not involving chemical reactions that are likely to alter or anyhow cause losses of the desired compounds as by products thereof. The following example illustrates a preferred embodiment of the process of the present invention, while not limiting it.
A detailed description of the process of the invention will be found hereinafter, scheduled for the following purificative steps utilizing soybean lecithin.
EXAMPLE
1 kg raw, moist, ground soybean lecithin is dissolved as such by contact in 10 1 methylene chloride or other chlorinated solvent.
Dissolving is accomplished in a glass flask equipped with stirrer, thermometer, bubble heating and cooling bath and to adjust boiling of the liquid.
A solvent refluxing is carried out for 1 hour untill the solubilisation is almost completed.
The solution contained in the flask is cooled to" 20°C and, still under stirring. 1 kg chromatography grade silica is added, and the material is maintained under stirring and again heated for additional 2 hours.
The resulting suspension is cooled and filtered on a Buckner filter, equipped with porous septum, or by centrifuging, then washing repeatedly the silica portion with methylene chloride.
The filtrate contains phosphatidylinositol, not pure due to the presence of phosphatidic acids, whereas the precipitate and silica portion are set apart and further treated for phosphatidylethanolamine recovery.
The filtrate, i.e. the solution as above obtained, is poured again in a flask and added with 50 g of a long chain quaternary ammonium base, such as cetyltrimethyl ammonium bromide or benzyldimethylhexadecylammonium bromide, which dissolve in the solution and additional 100 g chromatography grade silica are added.
The solution is refluxed for 1 hour, cooled and filtered on a porous septum.
The filtrate is treated with 1% of its total volume of 32% NH4OH, followed by stirring at room temperature for 30 minutes and filtering thereafter on a porous septum.
The resulting solution is evaporated under reduced pressure until an oil is obtained;
1000 ml of anhydrous ethyl alcohol are then added and refluxed for 30 minutes.
After that time the product, phosphatidylmyoinositol, crystallizes and is separated b filtration as a powder and vacuum dried with a mechanical pump. The so obtained product has a purity higher than 98% if compared with Sigma standard.
The previously set apart precipitate is treated with 5000 ml of 85/15 V/V solution of methylene chloride and methanol and refluxed for 1 hour. After filtering on porous septum, phosphatidylethanolamine remains in solution together with impurities of other phospatidic-like material, anyhow with an about neutral pH.
The previously set apart silica, freed from phosphatidylmyoinositol, is also treated with 1000 ml of a methylene chloride/methanol 85/15 (V/V) solution and refluxed for 15 minutes. After that time it is filtered on glass porous septum. A further warm washing of the silica, for 15 minutes with additional 500 ml of the previous
solution, follows.
The filtrates are combined and evaporated to dryness under reduced pressure.
The residue is then suspended in 1 1 of absolute ethanol and refluxed under heating for 30 minutes, and the suspension filtered thereafter.
The precipitate is washed with three subsequent 200 ml pure acetone portions and then vacuum dried.
The obtained phosphatidylethanolamine is ground and has a purity of about 95%.
Phosphatidylmyoinositol (2-3%) is the main impurity.
Claims
CLAIMS 1 ) Process for making phosphatidylethanolamine and phosphatidylmyoinositol by separation from soybean lecithin, characterized by the following steps:
(a) solubilization under heat of the starting soybean lecithin with a chlorinated solvent selected from chloroform, dichloromethane, dichloroethane and trichloroethylene;
(b) addition under stirring, to the room temperature cooled solution, of chromatography grade silica, having a granulometry from 20 to 100 micron and porosity from 60 to 500 Angstrom, and heating the resulting suspension;
(c) filtration of the suspension, thereby obtaining a filtrate (F) and a precipitate
(P);
(d) heat treatment of the filtrate with a long , alkyl chain quaternary ammonium base and silica of same above defined grade;
(e) filtration of the solution after cooling and treatment of the filtrate under stirring with ammonium hydrate;
(f) evaporation under reduced pressure of the solution until an oil is obtained and refluxing with ethanol;
(g) separation of phosphatidylmyoinositol by crystallization.
(h) heat treatment of precipitate (P) of step (c) with a mixture of a chlorinated solvent as above defined and an aliphatic alcohol, selected from methanol, ethanol, n-propanol, isopropanol and mixtures thereof, and filtration;
(i) heat treatment of step (c) residual silica with a solvent mixture and filtration as that of step (h);
(j) evaporation to dryness under reduced pressure of steps (h) and (i) combined filtrates and suspension in absolute and warm ethanol of the resulting residue;
(k) filtration of the resulting suspension and vacuum drying of precipitate to yield phosphatidylethanolamine.
2. Process according to claim 1 , characterized in that the chlorinated solvent is methylene chloride.
3. Process according to claim 1 , characterized in that said quaternary ammonium base of said step (d) is selected between cetyltrimethylammonium bromide and benzyldimethylhexadecyl ammonium bromide.
4. Process according to claim 1 , characterized in that said solvent mixture of said step (i) is a methylene chloride and methanol mixture.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH34692A CH682489A5 (en) | 1992-02-06 | 1992-02-06 | Procedure for obtaining phosphatidylethanolamine and fosfatidilmioinositolo pure. |
| CH346/92 | 1992-02-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0583468A1 true EP0583468A1 (en) | 1994-02-23 |
Family
ID=4184922
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP19930917399 Withdrawn EP0583468A1 (en) | 1992-02-06 | 1993-02-05 | Process for making pure phosphatidylethanolamine and phosphatidylmyoinositol |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0583468A1 (en) |
| CA (1) | CA2107912A1 (en) |
| CH (1) | CH682489A5 (en) |
| WO (1) | WO1993016086A1 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1617679A1 (en) * | 1967-08-21 | 1971-03-18 | Nattermann A & Cie | Process for the production of highly purified phosphatidylcholine |
| US4847015A (en) * | 1986-02-10 | 1989-07-11 | Kewpie Kabushiki Kaisha | Process for producing egg yolk lecithin having reduced PE content and/or containing substantially no impurities |
-
1992
- 1992-02-06 CH CH34692A patent/CH682489A5/en unknown
-
1993
- 1993-02-05 WO PCT/EP1993/000275 patent/WO1993016086A1/en not_active Application Discontinuation
- 1993-02-05 EP EP19930917399 patent/EP0583468A1/en not_active Withdrawn
- 1993-02-05 CA CA 2107912 patent/CA2107912A1/en not_active Abandoned
Non-Patent Citations (1)
| Title |
|---|
| See references of WO9316086A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1993016086A1 (en) | 1993-08-19 |
| CA2107912A1 (en) | 1993-08-07 |
| CH682489A5 (en) | 1993-09-30 |
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