CN105566381A - Preparation method for phosphatidylcholine for injection - Google Patents
Preparation method for phosphatidylcholine for injection Download PDFInfo
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- CN105566381A CN105566381A CN201511005969.9A CN201511005969A CN105566381A CN 105566381 A CN105566381 A CN 105566381A CN 201511005969 A CN201511005969 A CN 201511005969A CN 105566381 A CN105566381 A CN 105566381A
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- chromatographic column
- phosphatidylcholine
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- injection phosphorus
- injection
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- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 title claims abstract description 61
- 238000002347 injection Methods 0.000 title claims abstract description 31
- 239000007924 injection Substances 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 239000007789 gas Substances 0.000 claims abstract description 27
- 239000000243 solution Substances 0.000 claims abstract description 26
- 239000003960 organic solvent Substances 0.000 claims abstract description 13
- 239000011261 inert gas Substances 0.000 claims abstract description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 35
- 239000000787 lecithin Substances 0.000 claims description 35
- 229940067606 lecithin Drugs 0.000 claims description 35
- 235000010445 lecithin Nutrition 0.000 claims description 35
- 239000000463 material Substances 0.000 claims description 33
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 22
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 22
- 239000000945 filler Substances 0.000 claims description 22
- 229910052698 phosphorus Inorganic materials 0.000 claims description 22
- 239000011574 phosphorus Substances 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 16
- 238000006073 displacement reaction Methods 0.000 claims description 14
- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
- 229910052786 argon Inorganic materials 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 238000004090 dissolution Methods 0.000 claims description 7
- 229910052754 neon Inorganic materials 0.000 claims description 7
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 5
- 229920005989 resin Polymers 0.000 claims description 5
- 230000008929 regeneration Effects 0.000 claims description 4
- 238000011069 regeneration method Methods 0.000 claims description 4
- 238000002604 ultrasonography Methods 0.000 claims description 4
- 150000001408 amides Chemical class 0.000 claims description 3
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 238000002203 pretreatment Methods 0.000 claims description 2
- 238000004806 packaging method and process Methods 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 19
- 238000011049 filling Methods 0.000 abstract description 8
- 238000001914 filtration Methods 0.000 abstract description 8
- 239000002994 raw material Substances 0.000 abstract description 7
- 150000003904 phospholipids Chemical class 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 6
- 238000012856 packing Methods 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 210000002969 egg yolk Anatomy 0.000 description 9
- 238000011068 loading method Methods 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 239000002904 solvent Substances 0.000 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 description 8
- 239000000843 powder Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000007791 liquid phase Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 150000008105 phosphatidylcholines Chemical class 0.000 description 6
- 229940083466 soybean lecithin Drugs 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 238000010606 normalization Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 4
- 230000006837 decompression Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000003995 emulsifying agent Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000004952 Polyamide Substances 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 229920002647 polyamide Polymers 0.000 description 3
- 239000008347 soybean phospholipid Substances 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 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 description 1
- GGRLKHMFMUXIOG-UHFFFAOYSA-M 2-acetyloxyethyl(trimethyl)azanium;hydroxide Chemical compound [OH-].CC(=O)OCC[N+](C)(C)C GGRLKHMFMUXIOG-UHFFFAOYSA-M 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 208000004930 Fatty Liver Diseases 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 239000004902 Softening Agent Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007177 brain activity Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004134 energy conservation Methods 0.000 description 1
- 208000010706 fatty liver disease Diseases 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229940042880 natural phospholipid Drugs 0.000 description 1
- 230000007830 nerve conduction Effects 0.000 description 1
- 230000001473 noxious effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 238000010525 oxidative degradation reaction Methods 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
- 239000002699 waste material Substances 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
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
The invention provides a preparation method for phosphatidylcholine for injection, wherein the preparation method comprises the following steps: (1) phospholipid raw material pretreatment: adding an organic solvent into a phospholipid raw material, carrying out ultrasonic dissolving, and then filtering, to obtain a phospholipid raw material solution; (2) adding the phospholipid raw material solution obtained in the step (1) to a chromatographic column having gas replaced with inert gas for protection; (3) after the phospholipid raw material solution is completely adsorbed to a chromatographic column packing, adding an organic solvent, eluting, and at the same time, filling the chromatographic column with inert gas to make the pressure in the chromatographic column to be 0.1-2 MPa; and (4) collecting the eluates of the step (3), detecting the purities of phosphatidylcholine in the collected flow fraction eluates, merging the flow fraction eluates with the purity of more than 99%, carrying out reduced pressure concentration in 30-50 DEG C water bath to be dried, and thus obtaining the phosphatidylcholine for injection. The phosphatidylcholine prepared by the method has high purity and high yield; and the preparation method has the advantages of simple process, environmental protection and low cost, and can be used for mass production.
Description
Technical field
The present invention relates to medical auxiliary materials production technical field, more properly, the present invention relates to a kind of preparation method of injection phosphorus phosphatidylcholine.
Background technology
Phosphatidylcholine (phosphatidylcholine is called for short PC), popular name Yelkin TTS (Lecithin), be the main component of natural phospholipid, many physiologically actives are all associated, always the favor of extremely people.The absorption of phosphatidylcholine can significantly improve the concentration of vagusstoff in body, and thus it can strengthen brain activity, promotes nerve conduction, delay body aging and arteriosclerosis; The effect that lipid is changed can be promoted, can effectively prevent and treat the effect such as liver cirrhosis and fatty liver.Phosphatidylcholine can be used as the foodstuff additive such as good emulsifying agent, antioxidant, softening agent, moistening agent in the food industry in addition.
Lecithin in high purity is a kind of natural, safe, efficient emulsifying agent, and can be used for transfusion or used for intravenous injection emulsifying agent, is the most frequently used, the safest emulsifying agent in injection field.Meanwhile, phosphatidylcholine, due to the feature of its liposome, can be used as the carrier of cancer therapy drug and other slow releasing pharmaceuticals, significantly improves the activity of medicine, reduces toxic side effect, have the efficient advantage of low toxicity compared with other medicines carrier.When being used for the purposes such as injection, liposome when phosphatidylcholine, all strict demand is had to the purity of phosphatidylcholine, foreign matter content, water-content, usually require that phosphatidylcholine purity is greater than 95-98%, the content of phosphatidylethanolamine and Lysophosphatidylcholone is less than 1% simultaneously, water-content lower than 2%, simultaneously to pyrogen, the equal finite quantity requirement of heavy metal content.Therefore, the difficulty of preparation technology of these injection stage lecithin in high purities is very large.Existing method can only obtain the phosphatidylcholine of high level usually, is difficult to obtain the phosphatidylcholine meeting injection requirement.
Phosphatidylcholine is extensively present in the biological tissue such as soybean, yolk, adopts suitable extraction means can obtain mixed phosphatide from these biological tissues.Except containing except phosphatidylcholine in mixed phosphatide, also containing, for example other phosphatide impurity such as phosphatidylethanolamine, Lysophosphatidylcholone, suitable means need be adopted to give separation and purification.
Up to the present, the separation method of phosphatide mainly contains TLC separation technology, column chromatography for separation technology, high performance liquid chromatography isolation technique, separated from solvent technology etc.The supercritical CO just risen in recent years in addition
2extraction process, semi-transparent embrane method etc.
Supercritical fluid extraction good separating effect, quality product is high, effectively can avoid the use of noxious solvent, and solvent consumption is few simultaneously.But because overcritical facility investment is expensive and process cost is high, heavy industrialization application is limited.If publication number is CN103288870A, name is called a kind of preparation method of injection stage lecithin in high purity; Membrane technique, owing to not using chemical reagent, energy-conservation, has attracted the research of a lot of chemical engineer.At present, although the resistant to pollution ability of film has obtained good improvement, treatment capacity is little, the life-span etc. of infiltration capacity and film still governs the industrialization of membrane process.To combine with column chromatography the method for separating phospholipids phatidylcholine as Chinese patent ZL201010105688.1 discloses a kind of membrane sepn, obtain the product phosphatidylcholines that purity is more than 95%.Chinese patent ZL200510086600.5 discloses a kind of method utilizing inorganic ceramic membrane sepn to prepare food grade soy lecithin; Pillar layer separation method equipment is simple, and production cost is low, is the method that uniquely can be used in the high-purity phosphatide of scale operation at present, thus becomes study hotspot.But major part research rests in AG preparation, and preparation process multiselect toxic reagent or mix reagent carry out gradient elution, and this causes very large difficulty to solvent recuperation, and easily causes environmental pollution.As Chinese patent ZL200710052284.9, disclose the phosphatidylcholine purification process that a kind of ethanol leaches and column chromatography combines; Organic solvent extractionprocess one is that extraction purity is lower, cannot meet injection requirement, and two is that to use solvent species many, and solvent recuperation cost is high or even be difficult to reclaim, and easily cause waste and environmental pollution, its industrial application is also subject to a definite limitation.Chinese patent ZL02121550.2, ZL02149601.3 and disclose and a kind ofly adopt the mixed solvent of acetonitrile and the unitary such as methyl alcohol, ethanol low-carbon alcohol to carry out multi-stage countercurrent leaching to powdered soybean phospholipid to prepare the method for phosphatidylcholine.
Summary of the invention
The present invention is intended to solve prior art preparing to be existed in phosphatidylcholine process: phosphatidylcholine purity is low, can not meet injection requirement; Product yield is low, adsorbent reactivation is difficult and toxic solvent, solvent are difficult to recycle, stationary phase is expensive, sample easily the problems such as oxidative degradation occurs, a kind of preparation method of injection phosphorus phosphatidylcholine is provided, the phosphatidylcholine purity that the method prepares is high, yield is high, technique is simple, environmental protection, with low cost, can be mass-produced.
For solving the problems of the technologies described above, the present invention is achieved through the following technical solutions.
A preparation method for injection phosphorus phosphatidylcholine, comprises the following steps:
(1) lecithin materials pre-treatment: lecithin materials is dissolved in organic solvent, filters after ultrasonic dissolution, obtains lecithin materials solution; The millipore filtration of 0.45um was adopted during filtration;
(2) by the lecithin materials dissolution homogeneity of step (1) gained join with inert gas replacement protection chromatographic column in; Lecithin materials solution adopts wet method loading; Chromatographic column filler filling adopts dry column-packing, and chromatographic column installs in backward chromatographic column the air be filled with in inert gas replacement chromatographic column;
(3) treat that lecithin materials solution is adsorbed onto on chromatographic column filler completely, add organic solvent and carry out wash-out, in chromatographic column, be filled with rare gas element simultaneously and make the pressure in chromatographic column be 0.1 ~ 2MPa; The take-off rate being filled with control liquid that can be effective and reasonable of rare gas element is at 10ml/min ~ 50ml/min;
(4) collect the elutriant of step (3), detect the purity of phosphatidylcholine in each stream part elutriant collected, be that the stream part elutriant being greater than 99% merges by purity, be evaporated under 30 ~ 50 DEG C of water-baths and do and obtain injection phosphorus phosphatidylcholine.Detecting elutriant purity adopts high performance liquid chromatography to detect; Concentrating under reduced pressure, pressure-controlling is at-0.05 ~-0.1MPa.
In step (1), when adding organic solvent dissolution lecithin materials, the weightmeasurement ratio of described lecithin materials and machine solvent is 1:1 ~ 1:10.
In step (1), the ultrasound works frequency of described ultrasonic dissolution is 15 ~ 80KHz, and under ultrasound condition, sample can be dissolved in organic phase in the short period at room temperature, reduces sample and air duration of contact, the degradation speed of reduction sample.
In step (1), step (3), described organic solvent is any one in raw spirit, edible ethanol and industrial spirit, and from cost and reagent, the content of the objectionable impurities such as methyl alcohol is considered, prioritizing selection edible ethanol.
In step (2), described inert gas replacement protection is filled into by rare gas element in chromatographic column to carry out displacement protection, and described displacement protection carries out 2 ~ 5 times, and the volume being at every turn filled with rare gas element is identical with the volume of chromatographic column filler.
The weight of described chromatographic column filler is 20 ~ 100 times of lecithin materials weight.
Described rare gas element is any one in nitrogen, argon gas and neon.
Described chromatographic column filler is any one in silica gel, aluminum oxide, polymeric amide and macroporous resin.
In order to protect the purity of the phosphatidylcholine prepared, also comprising in the injection phosphorus phosphatidylcholine prepared in above-mentioned steps (4) and being filled with rare gas element protection.Rare gas element is any one in nitrogen, argon gas and neon, and charge is 1 container volume.
Also comprise and used chromatographic column filler methyl alcohol is carried out manipulation of regeneration, the envelope-bulk to weight ratio of described methyl alcohol and chromatographic column filler is 1:1 ~ 10:1, and after methyl alcohol volatilization is dry, chromatographic column filler is reused.
Beneficial effect:
Compared with prior art, the present invention has advantage.
1, the present invention is first by yolk powder organic solvent dissolution, crosses millipore filtration, avoids insolubles wherein to block up pillar, improves separating effect.In elution process, keep the pressure in pillar with rare gas element, can elution speed be controlled on the one hand, improve elution efficiency, reduce costs; The effect of protection can be played on the other hand, avoid sample oxidized decomposition in elution process.Concentrating sample solution at a certain temperature, both can keep the recovery speed of reagent, and sample also can be avoided to degrade under the high temperature conditions.
2, in concentrate drying sample, rare gas element has been filled, can starvation and sample contacts, avoid sample to be oxidized, finally obtain the phosphatidylcholine that purity is greater than 99%.
3, technique is simple, and with low cost, product purity is high, and wherein organic solvent can recovery, and filler is reused by manipulation of regeneration.Do not have particular requirement to equipment, suitability for industrialized is produced.The preparation method of phosphatidylcholine of the present invention is simple to operate, have higher yield and purity simultaneously, is convenient to industrial amplification production, meets the demand of China to lecithin in high purity.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of embodiment 1;
Fig. 2 is the high-efficient liquid phase chromatogram of embodiment 2;
Fig. 3 is the high-efficient liquid phase chromatogram of embodiment 4.
In Fig. 1,2,3, X-coordinate is time (min), and ordinate zou is absorbancy (AU).
Embodiment
Specifically the present invention is described below in conjunction with specific embodiment.
Embodiment 1
A preparation method for injection phosphorus phosphatidylcholine, comprises the following steps:
(1) dissolve: take lecithin materials 100g, add edible ethanol 100ml, regulate ultrasound works frequency to be 30KHz, after lecithin materials dissolves completely, cross 0.45 μm of millipore filtration, obtain lecithin materials solution.
(2) post, the displacement protection of chromatographic column and the loading of lecithin materials solution is filled:
Dress post: chromatographic column internal diameter × height (8cm × 40cm) used, takes alumina packing 5kg, pour in chromatographic column equably, make filling surface smooth; Filling with inert gas displacement protection: chromatographic column installs in backward chromatographic column has filled nitrogen, opens valve under chromatographic column, replaces the air in chromatographic column, and repeat 3 times, the volume of the nitrogen be at every turn filled with is identical with the volume of alumina packing; Loading: the surface lecithin materials solution obtained in step (1) slowly being joined aluminum oxide;
(3) wash-out: be all adsorbed on after on filling alumina until lecithin materials solution, in chromatographic column, add edible ethanol wash-out, and be filled with nitrogen in chromatographic column, the pressure 1MPa that chromatographic column is obtained, thus making the flow velocity of elutriant be 30ml/min, the every 500ml of elutriant is gathered into a stream part;
(4) concentrated: each stream part of being collected by step (3) wash-out is detected with high performance liquid phase HPLC, merge the product section that purity is greater than 99%, in the present embodiment, the elutriant collected in order, 5-12 stream part purity is greater than 99%, therefore 5-12 stream part is merged, regulate bath temperature 45 DEG C, vacuum decompression (pressure-0.09MPa) is concentrated into dry product phosphatidylcholines, weigh to obtain 75.3g, HPLC testing product, its color atlas as shown in Figure 1, employing area normalization method calculates, and obtaining phosphatidylcholine purity is 99.3%.
Embodiment 2
The lecithin materials of the present embodiment adopts yolk powder, take phosphatidylcholine content as being advisable of 50%-80%.
A preparation method for injection phosphorus phosphatidylcholine, comprises the following steps:
(1) dissolve: take yolk powder 100g, add edible ethanol 300ml, regulate ultrasonic merit operating frequency to be 80KHz, after yolk powder dissolves completely, cross 0.45 μm of millipore filtration, obtain yolk powder solution;
(2) post, the displacement protection of chromatographic column and the loading of yolk powder material solution is filled: chromatographic column internal diameter × height (8cm × 30cm) used takes polyamide filler 2kg, pours in chromatographic column equably, makes filling surface smooth; Displacement: chromatographic column installs in backward chromatographic column has filled argon gas, opens valve under chromatographic column, replaces the air in chromatographic column, and repeat 3 times, the volume of the argon gas be at every turn filled with is identical with the volume of polyamide filler; Loading: the yolk powder solution obtained in step (1) is slowly joined polyamide surface;
(3) wash-out: treat that yolk powder solution is all absorbed by polymeric amide, raw spirit wash-out is added in chromatographic column, and the pressure be filled with in chromatographic column in argon gas maintenance chromatographic column is 0.1MPa, make the flow velocity of elutriant be 10ml/min, the every 500ml of elutriant is gathered into a stream part.
(4) concentrated: each eluent stream part of collecting after step (3) wash-out is detected with high performance liquid phase HPLC, merge the product section that purity is greater than 99%, after testing, in the present embodiment, in 3-7 stream part, the purity of phosphatidylcholine is greater than 99%, therefore low 3-7 stream part is merged, regulate bath temperature 40 DEG C, vacuum decompression (pressure-0.1MPa) is concentrated into dry phosphatidylcholine, weigh to obtain 68.6g, HPLC testing product, as shown in Figure 2, adopt area normalization method to calculate, obtain phosphatidylcholine purity is 99.1% to its color atlas.
Embodiment 3
The lecithin materials of the present embodiment adopts soybean lecithin, take phosphatidylcholine content as being advisable of 20%-70%.
A preparation method for injection phosphorus phosphatidylcholine, comprises the following steps:
(1) dissolve: take soybean lecithin 100g, add industrial spirit 1000ml, regulate ultrasonic merit operating frequency to be 15KHz, after soybean lecithin dissolves completely, cross 0.45 μm of millipore filtration, obtain soybean lecithin solution;
(2) post, the displacement protection of chromatographic column and the loading of soybean lecithin material solution is filled: internal diameter × height (12cm × 100cm), takes macroporous resin filler 10kg, pour into equably in chromatographic column, make filling surface smooth; Displacement: chromatographic column installs in backward chromatographic column has filled nitrogen, opens valve under chromatographic column, replaces the air in chromatographic column, and repeat 5 times, the nitrogen volume be at every turn filled with is identical; Loading: after displacement protection, the soybean lecithin solution obtained in step (1) is slowly joined macroporous resin surface;
(3) wash-out: treat that soybean phospholipid solution is all absorbed by macroporous resin, industrial spirit wash-out is added in chromatographic column, and the pressure be filled with in chromatographic column in nitrogen maintenance chromatographic column is 0.5MPa, make the flow velocity of elutriant be 50ml/min, the every 500ml of elutriant is gathered into a stream part;
(4) concentrated: each eluent stream part of collecting after step (3) wash-out is detected with high performance liquid phase HPLC, merge the product section that purity is greater than 99%, after testing, in the present embodiment, in 12-20 stream part, the purity of phosphatidylcholine is greater than 99%, therefore 12-20 stream part is merged, regulate bath temperature 50 DEG C, vacuum decompression (pressure-0.09MPa) is concentrated into dry phosphatidylcholine, weigh to obtain 56.3g, HPLC testing product, employing area normalization method calculates, and obtaining phosphatidylcholine purity is 99.2%.
Embodiment 4
A preparation method for injection phosphorus phosphatidylcholine, comprises the following steps:
(1) dissolve: take lecithin materials 100g, add raw spirit 200ml, regulate ultrasonic merit operating frequency to be 50KHz, after lecithin materials dissolves completely, cross 0.45 μm of millipore filtration, obtain lecithin materials solution;
(2) post, the displacement protection of chromatographic column and the loading of lecithin materials solution is filled: internal diameter × height (8cm × 30cm), takes silica filler 2kg, pours in chromatographic column equably, makes filling surface smooth; Displacement: chromatographic column installs in backward chromatographic column has filled neon, opens valve under chromatographic column, replaces the air in chromatographic column, repeats 4 times; Loading: after displacement protection, the lecithin materials solution obtained in step (1) is slowly joined Silica Surface;
(3) wash-out: treat that lecithin materials solution is all by absorbed on silica gel, raw spirit wash-out is added in chromatographic column, and the pressure be filled with in chromatographic column in neon maintenance chromatographic column is 0.2MPa, make the flow velocity of elutriant be 20ml/min, the every 500ml of elutriant is gathered into a stream part;
(4) concentrated: each eluent stream part of collecting after step (3) wash-out is detected with high performance liquid phase HPLC, merge the product section that purity is greater than 99%, after testing, in the present embodiment, in 2-6 stream part, the purity of phosphatidylcholine is greater than 99%, therefore low 12-20 stream part is merged, regulate bath temperature 50 DEG C, very
Empty decompression (pressure-0.1MPa) is concentrated into dry phosphatidylcholine, and 53.1g, HPLC testing product of weighing to obtain, as shown in Figure 3, adopt area normalization method to calculate, obtain phosphatidylcholine purity is 99.5% to its color atlas.
Embodiment 5
The difference of the present embodiment and embodiment 1 is: in the present embodiment, and one be the edible ethanol that dissolved phosphorus fat raw material is used in step (1) is 500ml, and two is internal diameter × height (10cm × 120cm) of chromatographic column used, chromatographic column alumina packing 7kg.In the present embodiment, 5th ~ 9 stream parts are merged by the elutriant collected in order, by the operation of embodiment 1, the elutriant of merging is carried out concentrating to obtain product phosphatidylcholines, 50.8g, HPLC testing product of weighing to obtain, employing area normalization method calculates, and obtaining phosphatidylcholine purity is 99.2%.
By embodiment 6
The difference of the present embodiment and embodiment 4 is; the product phosphatidylcholines that step (4) obtains is protected by the present embodiment further; its concrete operations are filled with rare gas element protection in product phosphatidylcholines; and sealing is preserved; rare gas element be selected from nitrogen, argon gas, neon any one, be advisable for 0.1 ~ 10 times that the charge (by volume/L) of rare gas element is product phosphatidylcholines weight (g).
In embodiment 1-5, manipulation of regeneration is carried out to the chromatographic column raw material after using, particularly with pure methyl alcohol, the filler in chromatographic column is rinsed, then pour out, it is reusable after pure methyl alcohol volatilization is dry, during flushing, the volumetric usage of pure methyl alcohol is 1 ~ 10 times of filler weight, as in embodiment 1, and the pure washed with methanol of available 15L, in embodiment 2, the pure washed with methanol of available 10L, in embodiment 3, the pure washed with methanol of available 20L, the pure washed with methanol of available 20L in embodiment 4, the embodiment 5 pure washed with methanol of 7L.
Claims (10)
1. a preparation method for injection phosphorus phosphatidylcholine, is characterized in that: comprise the following steps:
(1) lecithin materials pre-treatment: add organic solvent in lecithin materials, filters after ultrasonic dissolution, obtains lecithin materials solution;
(2) the lecithin materials solution of step (1) gained is joined in the chromatographic column with inert gas replacement protection;
(3) treat that lecithin materials solution is adsorbed onto on chromatographic column filler completely, add organic solvent and carry out wash-out, in chromatographic column, be filled with rare gas element simultaneously and make the pressure in chromatographic column be 0.1 ~ 2MPa;
(4) collect the elutriant of step (3), detect the purity of phosphatidylcholine in each stream part elutriant collected, be that the stream part elutriant being greater than 99% merges by purity, be evaporated under 30 ~ 50 DEG C of water-baths and do and obtain injection phosphorus phosphatidylcholine.
2. preparation method as claimed in claim 1, is characterized in that: in step (1), described lecithin materials and organic solvent weightmeasurement ratio be 1:1 ~ 1:10.
3. the preparation method of a kind of injection phosphorus phosphatidylcholine as claimed in claim 1, is characterized in that: in step (1), and the ultrasound works frequency of described ultrasonic dissolution is 15 ~ 80KHz.
4. the preparation method of a kind of injection phosphorus phosphatidylcholine as claimed in claim 1, is characterized in that: in step (1), step (3), described organic solvent is any one in raw spirit, edible ethanol and industrial spirit.
5. the preparation method of a kind of injection phosphorus phosphatidylcholine as claimed in claim 1; it is characterized in that: in step (2); described inert gas replacement protection is filled into by rare gas element in chromatographic column to carry out displacement protection; described displacement protection carries out 2 ~ 5 times, and the volume being at every turn filled with rare gas element is identical with the volume of chromatographic column filler.
6. the preparation method of a kind of injection phosphorus phosphatidylcholine as claimed in claim 1, is characterized in that: the weight of described chromatographic column filler is 20 ~ 100 times of lecithin materials weight.
7. the preparation method of a kind of injection phosphorus phosphatidylcholine as described in claim 1 or 5, is characterized in that: described rare gas element is any one in nitrogen, argon gas and neon.
8. the preparation method of a kind of injection phosphorus phosphatidylcholine as described in claim 1 or 6, is characterized in that: described chromatographic column filler is any one in silica gel, aluminum oxide, polymeric amide and macroporous resin.
9. the preparation method of a kind of injection phosphorus phosphatidylcholine as described in any one of claim 1-6; it is characterized in that: also comprise and be filled with rare gas element protection in the packaging vessel of the injection phosphorus phosphatidylcholine described in step (4), described rare gas element is any one in nitrogen, argon gas and neon.
10. the preparation method of a kind of injection phosphorus phosphatidylcholine as described in any one of claim 1-6, it is characterized in that: also comprise and used chromatographic column filler methyl alcohol is carried out manipulation of regeneration, the envelope-bulk to weight ratio of described methyl alcohol and chromatographic column filler is 1:1 ~ 10:1, and after methyl alcohol volatilization is dry, chromatographic column filler is reused.
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