CN105017307A - Method for preparing high-purity natural L-alpha-glycerylphosphorylcholine - Google Patents
Method for preparing high-purity natural L-alpha-glycerylphosphorylcholine Download PDFInfo
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- CN105017307A CN105017307A CN201510443689.XA CN201510443689A CN105017307A CN 105017307 A CN105017307 A CN 105017307A CN 201510443689 A CN201510443689 A CN 201510443689A CN 105017307 A CN105017307 A CN 105017307A
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Abstract
The invention discloses a method for preparing high-purity natural L-alpha-glycerylphosphorylcholine (L-alpha-GPC), and belongs to the technical field of phosphatides products. The method comprises the following preparation steps: taking an alkaline hydrolysis reaction solution as a raw material, directly performing alumina column chromatography, then using cation exchange resin column chromatography to remove residual choline, salts and other impurities, and performing active carbon adsorption and concentration on the obtained chromatographical liquid, so as to finally obtain the product. The obtained product has the purity of 99.0% or more through detection by high performance liquid chromatography-evaporative light scattering detector, and accords with purity demands of medicines and health products. According to the technical scheme, a new production technological method is provided for separation purification of L-alpha-glycerylphosphorylcholine, also application of alumina column chromatography in phosphatides deep processing field is increased, and also safety of L-alpha-glycerylphosphorylcholine as a bulk drug and a health product is guaranteed.
Description
Technical field
The present invention relates to pharmaceutical chemistry, separation and purification, technical field of health care food, particularly relate to the method that one prepares high-purity natural L-α-glycerolphosphocholine (L-α-GPC).
Background technology
L-α-glycerolphosphocholine is the deacylated tRNA based products that the aliphatic chain gained on 1 and 2 carbon removed by the phosphatidylcholine in natural phospholipid; it is naturally occurring active substance in animal and plant body; there is important physiologically active, can be applicable to the fields such as protective foods, medicine, makeup.In field of medicaments, glycerolphosphocholine, to neural function disease, has obvious therapeutic action to patients of senile dementia especially.
At present, domestic have the separation and purification of several scholar to L-α-glycerolphosphocholine to be studied, and its separation purification method comprises extraction, precipitated crystal method, ion exchange resin column chromatography, silica gel column chromatography etc.As in patent CN102516292 and CN102964377 the separation purification method that adopts, GPC sample to be separated first dissolves by low-carbon alcohol, and then in this low-carbon alcohol solution, add non-polar organic solvent precipitation GPC, the purity applying the GPC product that this kind of method obtains is low, cannot arrive the requirement of market to this product purity.The separation purification method adopted in patent CN102875592 and CN103172659, make GPC sample to be separated cross Zeo-karb and anionite-exchange resin successively, this kind of method organic solvent is few, but it is strict to the purity requirement of phosphatidylcholine in raw material, can only be raw material with highly purified phosphatidylcholine, if the powdered soybean phospholipid adopting low-purity is raw material, obtained glycerolphosphocholine often purity can not reach more than 98%.Patent CN102093410 adopts and makes alkali solution liquid first carry out silica gel column chromatography separation, and then carry out cation exchange resin column chromatography separation, this method can obtain the L-α-glycerolphosphocholine product of high-content, but when carrying out silica gel column chromatography, eluent can dissolve part silicone gel and enter in GPC sample, and subsequent operations is difficult to remove this silica gel impurity.
Summary of the invention
In order to overcome above the deficiencies in the prior art, the invention provides a kind of method preparing high-purity natural L-α-glycerolphosphocholine, solving the shortcomings such as inadequate at separation and purification process moderate purity, ingredient requirement is high, new introduction impurity cannot be removed.
The technical solution adopted in the present invention is: this method adopts powdered soybean phospholipid to be initial feed, through sodium methylate alkaline degradation, neutral alumina column chromatography removes the impurity such as L-ALPHA-GPE, thanomin, glyceryl phosphatide acyl inositol, inositol, glyceryl phosphatide acid in alkali solution liquid, apply Zeo-karb again and remove other impurity such as neutral grease, lipid acid, choline, finally decolour with charcoal absorption, concentrated, obtain the finished product.
Comprise the steps:
Basic fluxing raction: powdered soybean phospholipid (or concentrated phosphatide and phosphatidylcholine PC purity contained by other be greater than the soybean phospholipid product of 20%mg/mg) adds methyl alcohol in 40 ± 2 DEG C of stirring and dissolving 25 ± 5min, add the sodium methylate of lecithin materials 5 ± 0.5% (mg/mg) in 40 ± 2 DEG C of stirring reaction 4 ± 0.2h, liquid pH value is regulated to be 6.0 ± 0.5 with glacial acetic acid after reaction, cross 0.45 μm of filter membrane, it is 40 ± 3% (mg/mg) that filtrate is concentrated into butt ratio in 50 ± 2 DEG C.
Alumina column chromatography: neutral alumina adds low carbon alcohol solution and is stirred to bubble-free, pours in chromatography column, hold over night, loading, carries out chromatographic separation with low carbon alcohol solution, and TLC detects, and merges GPC cut, concentrated.The calculating of aluminum oxide consumption calculates with dry biomass contained in Basic fluxing raction liquid, Basic fluxing raction liquid dry biomass: neutral alumina quality=1:10 to 1:15 (g/g).
Described neutral alumina order number is 80-160 order or 200-300 order.
Described low-carbon alcohol is methyl alcohol, ethanol.
The volume of water in described low carbon alcohol solution: low carbon alcohol solution cumulative volume=1% to 5% (ml/ml).
Ion-exchange chromatography: the Zeo-karb methyl alcohol soaked overnight that pre-treatment is good, pours in chromatography column, loading, first uses low-carbon alcohol washing oil lipid impurities, then use purified water wash-out GPC, collects GPC cut.
Described Zeo-karb is sulfuric acid H
+type ion exchange resin, preferred D001 macroporous ion exchange resin.
Described low-carbon alcohol is methyl alcohol, ethanol.
Activated carbon decolorizing: exchange resin elution gained GPC cut is warming up to 58-62 DEG C, adds gac whip attachment, crosses 0.22 μm of filter membrane and removes gac, and filtrate is concentrated into butt than being 85-90% (mg/mg) in 50 ± 2 DEG C, obtains finished product.
Described gac is wood source gac, mineral origin gac.
The present invention utilizes the Basic fluxing raction liquid of neutral alumina column chromatography separation and purification powdered soybean phospholipid to prepare L-α-glycerolphosphocholine, and cheaper starting materials, technique are simple, Impurity removal is complete, without newly-increased impurity, purity is high, and is easy to realize suitability for industrialized production.
Embodiment
Below by embodiment, the present invention is further described.Should be noted that the preparation method described by embodiments of the invention is only for illustration of the present invention, instead of limitation of the present invention, under thinking described in the invention, the scope that the present invention comprises all is belonged to the simple modifications of preparation method of the present invention.
Embodiment 1
Get powdered soybean phospholipid 400g, add 2000ml anhydrous methanol and stir 25min in 40 DEG C, add 20g sodium methylate, in 40 DEG C of stirring reaction 4h.Adjust liquid pH value to be 6.0 with glacial acetic acid after reaction, be cooled to 25 DEG C, cross 0.45 μm of filter membrane, it is 40% that filtrate is concentrated into butt ratio.
Get 80-160 order neutral alumina 960g, add the methyl alcohol 1440ml of moisture 1%, be stirred to bubble-free, dress post, hold over night.Loading, the methyl alcohol with 1%, as elution, collects the 0.5 to 2.0 times of column volume GPC cut, concentrated.
Getting D001 macroporous ion exchange resin volume is 800ml, and use anhydrous methanol soaked overnight, next day fills post, loading, washs 4 times of column volumes, then use purified water wash-out with anhydrous methanol, collects the 0.5 to 1.5 times of column volume GPC cut of purified water wash-out, merges mixing.
The GPC aqueous solution crossing ion exchange resin gained is warming up to 60 DEG C, adds 40g injection active carbon 767, in 60 DEG C of whip attachment 2h, cross 0.22 μm of filter membrane and remove gac, it is 87% that filtrate is concentrated into butt ratio, obtains product 16.1g.
The GPC content that high performance liquid chromatography evaporative light external standard method detects this sample is 99.2%, and yield is 4.0%.
Embodiment 2
Get powdered soybean phospholipid 400g, add 2000ml anhydrous methanol and stir 30min in 40 DEG C, add 20g sodium methylate, in 40 DEG C of stirring reaction 6h.Adjust liquid pH value to be 5.2 with glacial acetic acid after reaction, be cooled to 25 DEG C, cross 0.45 μm of filter membrane, it is 41% that filtrate is concentrated into butt ratio in 45-55 DEG C.
Get 80-160 order neutral alumina 920g, add the ethanol 1380ml of moisture 5%, be stirred to bubble-free, dress post, hold over night.Loading, the ethanol with 5%, as elution, collects the 0.5 to 2.1 times of column volume GPC cut, concentrated.
Getting D001 macroporous ion exchange resin volume is 1000ml, and use anhydrous methanol soaked overnight, next day fills post, loading, washs 4 times of column volumes, then use purified water wash-out with anhydrous methanol, collects the 0.5 to 1.5 times of column volume GPC cut of purified water wash-out, merges mixing.
The GPC aqueous solution crossing ion exchange resin gained is warming up to 60 DEG C, adds 40g injection active carbon 767, in 60 DEG C of whip attachment 2h, cross 0.22 μm of filter membrane and remove gac, it is 89% that filtrate is concentrated into butt ratio, obtains product 15.5g.
The GPC content that high performance liquid chromatography evaporative light external standard method detects this sample is 99.5%, and yield is 3.9%.
Embodiment 3
Get powdered soybean phospholipid 400g, add 2000ml anhydrous methanol and stir 30min in 40 DEG C, add 20g sodium methylate, in 40 DEG C of stirring reaction 6h.Adjust liquid pH value to be 5.0 after reaction, be cooled to 25 DEG C, cross 0.45 μm of filter membrane, it is 38% that filtrate is concentrated into butt ratio in 45-55 DEG C.
Get 200-300 order neutral alumina 640g, add the methyl alcohol 960ml of moisture 2%, be stirred to bubble-free, dress post, hold over night.Loading, the methyl alcohol with 2%, as elution, collects the 0.6 to 2.1 times of column volume GPC cut, concentrated.
Getting D001 macroporous ion exchange resin volume is 800ml, and use anhydrous methanol soaked overnight, next day fills post, loading, washs 4 times of column volumes, then use purified water wash-out with anhydrous methanol, collects the 0.5 to 1.5 times of column volume GPC cut of purified water wash-out, merges mixing.
The GPC aqueous solution crossing ion exchange resin gained is warming up to 60 DEG C, adds 40g injection active carbon 767, in 60 DEG C of whip attachment 2h, cross 0.22 μm of filter membrane and remove gac, it is 86% that filtrate is concentrated into butt ratio, obtains product 16.5g.
The GPC content that high performance liquid chromatography evaporative light external standard method detects this sample is 99.4%, and yield is 4.1%.
Embodiment 4
Get powdered soybean phospholipid 400g, add 2000ml anhydrous methanol and stir 30min in 40 DEG C, add 20g sodium methylate, in 40 DEG C of stirring reaction 6h.Adjust liquid pH value to be 5.0 after reaction, be cooled to 25 DEG C, cross 0.45 μm of filter membrane, it is 36% that filtrate is concentrated into butt ratio in 45-55 DEG C.
Get 200-300 order neutral alumina 620g, add the ethanol 930ml of moisture 4%, be stirred to bubble-free, dress post, hold over night.Loading, the ethanol with 4%, as elution, collects the 0.7 to 2.3 times of column volume GPC cut, concentrated.
Getting D001 macroporous ion exchange resin volume is 1000ml, and use anhydrous methanol soaked overnight, next day fills post, loading, washs 4 times of column volumes, then use purified water wash-out with anhydrous methanol, collects the 0.5 to 1.5 times of column volume GPC cut of purified water wash-out, merges mixing.
The GPC aqueous solution crossing ion exchange resin gained is warming up to 60 DEG C, adds 40g injection active carbon 767, in 60 DEG C of whip attachment 2h, cross 0.22 μm of filter membrane and remove gac, it is 86% that filtrate is concentrated into butt ratio, obtains product 16.9g.
The GPC content that high performance liquid chromatography evaporative light external standard method detects this sample is 99.6%, and yield is 4.2%.
Claims (8)
1. prepare a method for high-purity natural L-α-glycerolphosphocholine, it is characterized in that, comprise following processing step:
Be initial feed with powdered soybean phospholipid, utilize sodium methylate to prepare Basic fluxing raction liquid for reaction reagent, cross 0.45 μm of filter membrane, concentrated; Weigh neutral alumina, add low carbon alcohol solution and be stirred to bubble-free, dress post, loading, merges GPC cut, concentrated; Measure Zeo-karb, add low-carbon alcohol and be stirred to bubble-free, dress post, loading, first uses low-carbon alcohol wash-out, then uses purified water wash-out, merges GPC cut; Charcoal absorption is decoloured, and crosses 0.22 μm of filter membrane, concentrates, to obtain final product.
2. the method preparing high-purity natural L-α-glycerolphosphocholine according to claim 1, it is characterized in that, the calculating of aluminum oxide consumption calculates with dry biomass in Basic fluxing raction liquid, Basic fluxing raction liquid dry biomass: neutral alumina quality=1:10 to 1:15(g/g).
3. the method preparing high-purity natural L-α-glycerolphosphocholine according to claim 1, is characterized in that, described low-carbon alcohol is methyl alcohol, ethanol.
4., according to the method for preparing high-purity natural L-α-glycerolphosphocholine of claims 1 to 3 described in any one, it is characterized in that, neutral alumina chromatographic stuffing is 80-160 order, 200-300 order.
5. according to the method for preparing high-purity natural L-α-glycerolphosphocholine of Claims 1-4 described in any one, it is characterized in that, the volume of water in described low carbon alcohol solution: low carbon alcohol solution cumulative volume=1% to 5%(ml/ml).
6., according to the method for preparing high-purity natural L-α-glycerolphosphocholine of claim 1 to 5 described in any one, it is characterized in that, described Zeo-karb is sulfuric acid H
+type ion exchange resin.
7., according to the method for preparing high-purity natural L-α-glycerolphosphocholine of claim 1 to 6 described in any one, it is characterized in that, described gac is wood source gac, mineral origin gac.
8. according to the method for preparing high-purity natural L-α-glycerolphosphocholine of claim 1-7 described in any one, it is characterized in that, described powdered soybean phospholipid can also be greater than the soybean phospholipid product of 20% mg/mg for concentrated phosphatide or phosphatidylcholine PC purity contained by other.
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Cited By (8)
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|---|---|---|---|---|
| CN108516987A (en) * | 2018-04-19 | 2018-09-11 | 厦门大学 | A kind of glycerolphosphocholine and its aqueous synthesis method |
| CN108745324A (en) * | 2018-05-15 | 2018-11-06 | 天津工业大学 | A kind of intelligent silica gel of chirality for the purification of L-a- glycerolphosphocholines |
| CN108997411A (en) * | 2018-07-19 | 2018-12-14 | 芜湖福民生物药业股份有限公司 | The purification process of glycerolphosphocholine |
| CN108997412A (en) * | 2018-07-19 | 2018-12-14 | 芜湖福民生物药业股份有限公司 | The method of purification of crude glycerol phosphatidyl choline |
| CN109134531A (en) * | 2018-09-05 | 2019-01-04 | 湖南中茂生物科技有限公司 | A kind of preparation method of α-GPC |
| CN109965103A (en) * | 2017-12-28 | 2019-07-05 | 丰益(上海)生物技术研发中心有限公司 | A kind of feed addictive and preparation method thereof substituting fish oil |
| CN111116642A (en) * | 2020-02-18 | 2020-05-08 | 苏州富士莱医药股份有限公司 | Method for preparing natural L- α -glycerophosphatidylcholine |
| CN114106036A (en) * | 2021-11-08 | 2022-03-01 | 云南开放大学(云南国防工业职业技术学院) | Separation and extraction method of L-alpha-glycerophosphatidylcholine |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109965103A (en) * | 2017-12-28 | 2019-07-05 | 丰益(上海)生物技术研发中心有限公司 | A kind of feed addictive and preparation method thereof substituting fish oil |
| CN108516987A (en) * | 2018-04-19 | 2018-09-11 | 厦门大学 | A kind of glycerolphosphocholine and its aqueous synthesis method |
| CN108745324A (en) * | 2018-05-15 | 2018-11-06 | 天津工业大学 | A kind of intelligent silica gel of chirality for the purification of L-a- glycerolphosphocholines |
| CN108997411A (en) * | 2018-07-19 | 2018-12-14 | 芜湖福民生物药业股份有限公司 | The purification process of glycerolphosphocholine |
| CN108997412A (en) * | 2018-07-19 | 2018-12-14 | 芜湖福民生物药业股份有限公司 | The method of purification of crude glycerol phosphatidyl choline |
| CN109134531A (en) * | 2018-09-05 | 2019-01-04 | 湖南中茂生物科技有限公司 | A kind of preparation method of α-GPC |
| CN111116642A (en) * | 2020-02-18 | 2020-05-08 | 苏州富士莱医药股份有限公司 | Method for preparing natural L- α -glycerophosphatidylcholine |
| CN114106036A (en) * | 2021-11-08 | 2022-03-01 | 云南开放大学(云南国防工业职业技术学院) | Separation and extraction method of L-alpha-glycerophosphatidylcholine |
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Application publication date: 20151104 |