RU2698715C1 - Method of producing unsaturated alkyl-glycerine esters of marine fats - Google Patents

Abstract

FIELD: pharmaceuticals; food industry.

SUBSTANCE: invention relates to food and pharmaceutical industry and can be used for production of alkyl-glycerol esters from marine fats with high biological effect. Proposed method comprises hydrolysis of sea fat, neutralization of obtained fatty mixture with diluted sulfuric acid, washing with aqueous solution of NaCl, sequential double crystallisation from acetone at minus 15 °C for 18 hours, separating precipitate of saturated alkyl-glycerine ethers and drying it, evaporation of liquid fraction containing unsaturated alkyl-glycerine ethers, separation of unsaturated alkyl-glycerine ethers by column chromatography with eluating system benzene:chloroform = 10:0 → 0:10, vol./vol.; after which the purified alkyl-glycerol ester concentrate is acetylated with acetyl chloride, then separated into individual acetylated alkyl-glycerol esters by high-performance liquid chromatography on a reversed phase column (C18) and elution with 85 % aqueous ethanol, fractions with individual acetylated alkyl-glycerol ethers are collected and evaporated, acetyl groups are removed by alkaline hydrolysis, alkyl-glycerine ethers are extracted with hexane and evaporated. Method provides obtaining high-purity preparations of unsaturated AGE in laboratory conditions suitable for use both in biochemical and medical experiments.

EFFECT: preparations produced by the method reduce dependence on imported products and enable rapid scientific research.

1 cl, 2 dwg, 1 tbl, 1 ex

Description

The invention relates to the food and pharmaceutical industries and can be used to produce alkyl-glycerol esters with high biological effect from sea fats.

Ether-linked lipids, including 1-O-alkyl-glycerol ethers (AGE), attract the attention of physicians, nutritionists, and biochemists of high biological activity. In FIG. 1 shows the structural formulas of the main unsaturated natural alkyl-glycerol esters, provided that: R ₁ = C ₁₆ H ₃₃ is chymyl alcohol, R ₁ = C ₁₈ H ₃₇ is butyl alcohol, R ₁ = C ₁₈ H ₃₅ is selachyl alcohol.

In the body, AGEs are part of membranes in the form of plasmalogen structures, especially significant amounts of them are present in the nervous tissue, primarily in the brain. Being in lipid rafts regulate the transport of cholesterol, proteins, the activity of secretions responsible for the breakdown of amyloid protein β42, preventing the development of neurodegenerative diseases. The first to take on the effects of oxidative stress, protecting other vital molecules from oxidation and preventing more severe effects on the body (Wood P., Khan A., Mankidy R., Goodenowe D. “Plasmalogen deficit: a new and testable hypothesis for the etiology of Alzheimer's disease. "In:" Alzheimer's disease pathogenesis - Core Concepts, Shifting Paradigms and Therapeutic Targets ". 2011. P. 561-588).

The diet of modern man is such that these compounds are always in short supply. Given that with age, peroxisomes lose their activity on the synthesis of a simple ether bond, it is necessary to have additional sources of these important lipids. Terrestrial plants and animals are poor in them, in contrast to the inhabitants of the deep sea - sharks, rays, squids. Abroad and in Russia, technologies for producing AGE have been implemented. However, the radical in the first position of the glycerol may be saturated or unsaturated. In the case of saturated AGEs, their isolation is well developed, not to say about unsaturated AGEs.

, чем у насыщенных АГЭ (A.-L. Deniau, P. Mosset, D.Le Bot, A.B. Legrand Which alkylglycerols from shark liver oil have anti-tumour activities? / Biochimie. 2010. Р. 1-3). Поэтому поиск методов выделения таких соединений и создания препаратов с их участием является важным в свете профилактики и лечения многих недугов человека. Recent studies have revealed particular biological activity in unsaturated selachyl alcohol,

than in saturated AGEs (A.-L. Deniau, P. Mosset, D.Le Bot, AB Legrand Which alkylglycerols from shark liver oil have anti-tumor activities? / Biochimie. 2010. P. 1-3). Therefore, the search for methods for isolating such compounds and creating drugs with their participation is important in the light of the prevention and treatment of many human ailments.

It is especially worthwhile to dwell on the possibility of obtaining these compounds in laboratory conditions, since the further promotion of the created drugs depends on biochemical and medical studies. The absence of some of them in the market should not limit the researchers in their experiments.

At the moment, there is no way to obtain unsaturated AGE in one process with a high concentration and yield, simplification of the entire technological scheme, allowing the method to be carried out by affordable means in any laboratory.

A known method of producing concentrates of unsaturated AGE from the liver of a prickly shark (Katran), a Greenland shark and a chimera (Hallgren B., Larsson SO "Separation and identification of alkoxyglycerols" // Acta Chem. Scand. 1959. Vol. 13, N 7. P. 2147-2148). The method is as follows: unsaponifiable substances extracted from hydrolyzed liver oil of fish are chromatographed on an activated alumina column, a mixture of 10% methanol in methylene chloride is used to elute AGE. The following concentrates of unsaturated AGE were obtained (by selachyl alcohol) - 47.8% for Katran, 59.4% for the Greenland shark and 53.6% for the chimera. Other specifying data on the release of AGE are absent.

The disadvantages of the method:

1) the implementation of this method rather illustrates the very presence of AGE in the liver of aquatic organisms than actually offers a rational way to isolate them;

2) chromatographic isolation of AGE, without preliminary concentration, for obtaining large quantities of drugs is not acceptable - low efficiency of the method, problems with sorbent regeneration, and, most importantly, high cost of the product obtained;

3) the use of methyl alcohol reduces interest in the method, as it uses an extremely toxic solvent, not acceptable in food production.

A known method for the isolation of alkyl glycerol esters from the fat of Commander squid Berryteuthis magister (RF Patent No. 2415125 IPC C07C 43/13, C07C 41/16, C07C 41/40, publ. 03/27/2011). The method includes hydrolysis of sea fat to isolate free fatty acids and release an unsaponifiable fraction containing AGE, neutralizing the resulting fat mixture with acid, washing with water and sequential double crystallization from an organic solvent, filtering and drying the precipitate formed after each crystallization. For the first and second crystallization, the fat mixture in an organic solvent is kept first for 10-12 hours at room temperature, and then for 12-15 hours at 0-4 ° C.

Acetone or hexane is used in the process, and only one solvent can be used in the whole process, or a combination thereof.

When using hexane as an organic solvent, the ratio of fat mixture to hexane in the first crystallization is 1:10, kg / l, and in the second crystallization of the intermediate product 1:50, kg / l.

When using acetone as an organic solvent, the ratio of fat mixture to acetone is 1:10, kg / l, both in the first and second crystallization of the intermediate product.

The method allows to obtain saturated AGE with a yield of 50-59.0% and a purity of more than 99%.

Disadvantages:

1) the method uses significant volumes of solvents, which requires the use of large containers, appropriate filtering systems and continuous regeneration of solvents;

2) the two crystallizations of the method are separated by drying the precipitate so that when changing the solvent there is no contamination of one solvent with another; drying really interrupts the process and postpones the subsequent second crystallization for several hours;

3) the duration of the entire process - from the initial fat to the target product (AGE) - is at least 59 hours, an average of 65 hours, despite the fact that the process between the stages is inextricable. Such a long process time has an extremely negative effect on the performance of the AGE production line and does not allow for quick and timely processing of incoming raw materials.

4) AGE released by this method is 98-99% composed of saturated compounds, all unsaturated AGEs with the highest biological activity remain in an unused residue and are lost due to the undeveloped technology.

A known method of producing alkyl-glycerol esters from sea fats (RF Patent No. 2476611, IPC C22B 7/04, C22B 15/00, C22B 23/00, C22B 4/06, publ. 02/27/2013), including hydrolysis of sea fat to highlight free fatty acids and the release of unsaponifiable fraction containing AGE, neutralization of the resulting fat mixture with acid, washing with water and sequential double crystallization from an organic solvent, filtering and drying the precipitate formed at the last stage of crystallization. For the first crystallization, the room temperature fat mixture is mixed with an organic solvent of temperature -15 ° -5 ° C with vigorous stirring and the precipitate formed is immediately filtered off, washed with a small amount of organic solvent at the same temperature and this amorphous precipitate is mixed again with an organic temperature solvent - 15 ° -5 ° C (2nd crystallization) with vigorous stirring and immediately filter the precipitate and dry it from the solvent at room temperature, obtaining intact howling product - AGE. The ratio of fat mixture: organic solvent during the first crystallization is 1: 1-5, kg / l, in the second it is also 1: 1-5, kg / l, calculated on the initial amount of the fat mixture. Acetone and hexane can be taken as organic solvents, and the entire process should use only one solvent to avoid contamination of one solvent with another.

The total yield of AGE is 50-59%.

Disadvantages:

1) the product contains almost only saturated AGE;

2) the method does not provide complete extraction of AGE from lipids, therefore, all unsaturated AGE with high biological potential remain in the filtrate and, in the future, get into waste.

Closest to the claimed invention is a method for producing a concentrate of unsaturated alkyl glycerol esters from marine lipids (RF Patent No. 2649014, MKP 11/11, published on March 29, 2018). The method includes (example 1) hydrolysis of sea fat to isolate free fatty acids and release an unsaponifiable fraction containing AGE, neutralizing the resulting fat mixture with diluted sulfuric acid, washing with water and sequential double crystallization from acetone at minus 15 ° C for 18 hours, filtering and drying the precipitate. The sediment contains saturated AGE (more than 99% in total), which are used as dietary supplement Lipidomarin or in dietetics (RF Patent No. 2658431 “Dry nutrient mixture for diet food”, MCP A23J 1/04, A23L 33/00, A23L 17 / 50, publ. 06/21/2018).

The liquid fraction containing free fatty acids and unsaturated AGE is evaporated from the solvent, esterified with a solution of sulfuric acid in anhydrous ethanol, water is added, lipids are separated and the unsaturated AGE is extracted from the lipid mixture with aqueous ethanol solutions and evaporated.

The resulting unsaturated AGE product is purified (Example 3, Part B) by column chromatography on silica gel with an elution system of petroleum ether - ethyl acetate in a gradient.

The method has the following disadvantages:

1. Etherification involves the use of anhydrous ethanol, which means serious preparation for this stage - either benzene (azeatropic distillation), or molecular sieves, or alkali metals, etc., will be used as a desiccant.

2. Extraction of AGE from lipids by aqueous solutions of ethanol is a long process, since in each case it is necessary to achieve equilibrium of the system in which AGE is transferred to the water-alcohol layer to the maximum, and ethyl esters of fatty acids, on the contrary, are removed from it. The process is accelerated by the use of a centrifuge, but this does not reduce the time spent on it.

3. Column chromatography used in the process allows the production of a combined concentrate of unsaturated AGE (a total of 96.8% with a yield of 91.1% per stage).

However, it does not suggest the production of individual unsaturated AGEs, primarily selachyl alcohol. Therefore, the method can be considered as one of the possible ways to implement the task of isolating the net total unsaturated AGE, but not the method of obtaining valuable individual compounds.

The technical problem posed by the invention is the development of an effective and economical method for obtaining unsaturated alkyl-glycerol esters from sea fats, with the aim of extracting them from raw materials with maximum quality of the target product (high purity) as promising means of preventing socially significant diseases .

The technical problem posed is solved by the fact that in the known method for the separation of unsaturated and saturated alkyl-glycerol esters from sea fats, including hydrolysis of sea fat to isolate free fatty acids and release the unsaponifiable fraction containing AGE, neutralizing the resulting fat mixture with diluted sulfuric acid, washing 1% NaCl solution, sequential double crystallization from acetone at minus 15 ° C for 18 hours, filtering and drying the resulting precipitate containing saturated AGE. The resulting liquid fraction containing unsaturated AGE is evaporated, the total unsaturated AGE is isolated from the evaporated product by column chromatography on silica gel with an eluting system, according to the invention, benzene is used as an eluting system: chloroform = 10: 0 → 0: 10, v / v ., the purified AGE concentrate is acetylated with acetyl chloride, and then separated into individual acetylated AGE by high-performance liquid chromatography on a column with a reversed phase (C18) and elution with 85% aqueous ethanol, fractions with ind vidual acetylated AGE collected and evaporated, acetyl groups removed by alkaline hydrolysis, AGE was extracted with hexane and evaporated.

When developing the method, solutions were found that made it possible to implement it in the most economical, and most importantly, simple way.

The use of a new eluting system for the isolation of pure unsaturated AGEs: the benzene-chloroform system, 10: 0 → 0: 10, vol./about., With an increase in the gradient, made it possible not only to purify the unsaturated AGE concentrate by column chromatography on silica gel to the content of the main substance in it more than 98%, but also apply cleaning immediately after evaporation of the liquid fraction. Thus, in contrast to the prototype, there is no need for the esterification of free fatty acids of the lipid mixture with a solution of sulfuric acid in anhydrous ethanol, separation of lipids from the reaction mixture and repeated extraction of unsaturated AGE from the lipid mixture with aqueous solutions of ethanol, combining the extracts and their evaporation.

 Deviation from the parameters of the benzene-chloroform system, 10: 0 → 0: 10, v / v, in one direction or another reduced the yield or purity of the product.

Acetylation of the purified AGE concentrate with acetyl chloride allowed us to separate unsaturated AGE fractions in the form of their acetylated derivatives by high performance liquid chromatography into individual unsaturated AGEs. In this case, the preparation applied to the column is liquid, unlike the starting materials, it is easily applied to the column and is easily separated, since the polar hydroxyl groups are esterified into individual compounds with the elution system ethanol-water 85:15, vol./about.

Description of the General scheme of the claimed method.

Hydrolysis of fat from the digestive gland of the commander squid (according to the prototype)

The initial stage of AGE isolation is the alkaline hydrolysis of squid digestive gland lipids, which results in cleavage of the FA from the alkyl-diacyl-glycerol (ADAG) molecule; the hydrolysis scheme is shown in FIG. 2.

To 2 kg of fat add 1.5 l of 96% ethanol and 1 l of a 40% solution of KOH. The reaction is carried out at 60 ° C for 1 hour. The end of the hydrolysis reaction is controlled by thin layer chromatography.

At the end of the reaction, the mixture was transferred to a separatory funnel, 4 L of water was added and neutralized with an aqueous solution of H ₂ SO ₄ to pH = 4 using indicator paper. Hydrolyzed lipids are washed twice with 3 L of a 1% NaCl solution.

Precipitation of saturated alkyl glycerol esters

1 kg of hydrolyzed lipids was taken, 2.8 L of cold (-15 ° С) acetone was poured, stirred vigorously and placed in a freezer (-15 ° С) for 18 hours to precipitate (crystallized) saturated AGE. The process of crystallization from acetone is carried out twice.

The precipitate after the second crystallization is filtered through filtered paper at -15 ° C. The resulting filtrate containing fatty acids and unsaturated alkyl glycerol esters was evaporated in vacuo.

Column chromatography

The resulting unsaturated AGE concentrate is purified by column chromatography on silica gel 230-400 mesh, 60A (Merck) using an elution system benzene: chloroform 10: 0 → 0: 10, vol./about. with increasing gradient. Fractions containing total net AGE were combined and evaporated.

High Performance Liquid Chromatography

To obtain individual unsaturated AGEs, the purified AGE concentrate is acetylated with acetyl chloride in a known manner in which the hydroxyl groups of AGE form an ester bond with acetic acid (Lipid analysis: a practical approach / Ed. By RJ Hamilton and Sh. Hamilton, Oxford University Press, 1992 Protocol 37. P. 55), adding acetyl chloride to the AGE dropwise and keeping in the bath for 1 minute, add a 3-fold volume of water and extract the acetyl derivatives of AGE with chloroform, and the solvent is evaporated.

HPLC of acetylated AGE is carried out on an LC-8A chromatograph (Shimadzu, Japan) with a RID-10A refractometric detector. Preparative separation is carried out on a Discovery HS C-18 column, 10 μm 25 cm × 50 mm (Supelco, USA) in isocratic mode with an ethanol-water solvent system (85:15, v / v). The elution rate is 50 ml / min. The eluates of individual acetylated unsaturated AGE are collected and evaporated in vacuo.

The removal of acetyl groups is carried out by alkaline hydrolysis, treating individual acetylated AGE with a 5% aqueous-alcoholic solution of NaOH, add an equal volume of water and extract individual unsaturated AGE with hexane, and evaporate.

The invention is illustrated by the following FIG. and table

Figure 1. shows the structural formula of alkyl glycerol esters,

* - if R ₁ = C ₁₆ H ₃₃ is a chemical alcohol,

R ₁ = C ₁₈ H ₃₇ - butyl alcohol,

R ₁ = C ₁₈ H ₃₅ - selachyl alcohol;

in FIG. 2 is a diagram of alkaline hydrolysis of alkyl diacyl glycerides (ADAG);

in table Figure 1 shows the composition of lipids in products (and intermediates) at different stages of the isolation of unsaturated AGE.

The method is as follows.

EXAMPLE 1. Isolation of saturated and unsaturated AGE from the digestive fat of the Commander squid Berryteuthis magister.

The main classes of digestive lipids are alkyl-diacyl-glycerols (ADAG) and free fatty acids, 37.5% and 29.7%, respectively. In addition to these components, the composition of lipids also includes triacyl glycerols (18.5%), cholesterol (7.0%). Polar lipids, sterol esters and waxes are present in small quantities.

The qualitative and quantitative composition of lipid mixtures at all stages of the process is evaluated using thin layer chromatography (TLC) (Sorbfil plates, hexane-diethyl ether-acetic acid system 50: 50: 1, v / v / v) using a flatbed scanner and evaluating the density (intensity) of spots shown on TLC chromatograms using the TLC manager version 3.1 2005 @PinSoft program.

The composition of AGE isolated from hydrolyzed lipids, as well as the obtained fractions, was determined by GC-MS.

Take 100 kg of the digestive gland (frozen squid guts) are heated in a screw with a water jacket and separated. Get 48.5 kg of fat.

Hydrolysis of squid digestive lipids

To 2 kg of fat add 1.5 l of 96% ethanol and 1 l of a 40% solution of KOH. The reaction is carried out at 60 ° C for 1 hour. The end of the hydrolysis reaction is controlled by thin layer chromatography.

At the end of the reaction, the mixture was transferred to a separatory funnel, 4 L of water was added and neutralized with an aqueous solution of H ₂ SO ₄ to pH = 4 using indicator paper. The separated lipids are washed twice with 3 L of a 1% aqueous NaCl solution.

Received 1.76 kg of hydrolyzed fat.

The yield (to the initial mass of lipids) is 88.0%.

The composition of lipids after hydrolysis is shown in the summary table.

Isolation of saturated alkyl glycerol esters

After alkaline hydrolysis, crystallization of saturated AGE from acetone is carried out according to a previously developed method (RF Patent No. 2476611 "Method for the production of alkyl glycerol esters from sea fats", IPC C22B 7/04, C22B 15/00, C22B 23/00, C22B 4 / 06, publ. 02.27.2013). In the first stage of crystallization at -15 ° C, a precipitate formed, which was re-precipitated from acetone at the same temperature.

1 kg of hydrolyzed lipids was taken, 2.8 L of cold (-15 ° С) acetone was poured, stirred vigorously and placed in the freezer (-15 ° С) for 18 hours to precipitate saturated AGE. The process is repeated under the same conditions. The precipitate formed is filtered through filter paper at -15 ° C. The resulting product is a mixture of saturated AGE with a purity of 99.2%, the main component of which was chymyl alcohol (content 93.4%).

The yield of saturated AGE was 11.0% of the lipids taken for crystallization, or 42.0% of the AGE content in hydrolyzed lipids.

The resulting filtrate containing fatty acids and unsaturated alkyl glycerol esters is evaporated in vacuo and unsaturated alkyl glycerol esters are isolated from it.

Isolation of total unsaturated alkyl glycerol esters

Column chromatography

2 g of the resulting total unsaturated AGE concentrate was purified by column chromatography on silica gel 230-400 mesh, 60A (Merck) using an elution system benzene: chloroform 10: 0 → 0: 10, vol./about. with increasing gradient. Fractions containing total net AGE were combined and evaporated.

Received: 320 mg of unsaturated AGE with the content of AGE-18: 1 (selachyl alcohol) 74.5% and AGE-20: 1 (radical in the 1st position - 20: 1) 11.8%.

The output at the stage: 100%% (for selachyl alcohol, AGE-18: 1)

100% for AGE-20: 1.

High Performance Liquid Chromatography

To obtain individual unsaturated AGEs (selachyl alcohol), 320 mg purified AGE concentrate is acetylated with acetyl chloride in a known manner (Lipid analysis: a practical approach / Ed. By RJ Hamilton and Sh. Hamilton, Oxford University Press, 1992. Protocol 37. P. 55), adding acetyl chloride to the AGE dropwise and keeping in the bath for 1 minute, add a 3-fold volume of water and extract the acetyl derivatives of AGE with chloroform, and the solvent is evaporated.

HPLC of acetylated AGE is carried out on an LC-8A chromatograph (Shimadzu, Japan) with a RID-10A refractometric detector. Preparative separation is carried out on a Discovery HS C-18 column, 10 μm 25 cm × 50 mm (Supelco, USA) in isocratic mode with an ethanol-water solvent system (85:15, v / v). The elution rate is 50 ml / min. The eluates of individual acetylated unsaturated AGE are combined and evaporated in vacuo.

The removal of acetyl groups is carried out by alkaline hydrolysis, treating individual acetylated AGE with a 5% aqueous-alcoholic NaOH solution. An equal volume of water is added and the individual unsaturated AGE is extracted with a hexane hydrocarbon solvent and evaporated.

Received: 224.0 mg AGE-18: 1 (selachyl alcohol) with a concentration of 98.1%

The output at the stage: 91.4%.

Received: 35.7 mg AGE-20: 1 (radical in the 1st position - 20: 1) with a concentration of 97.8%.

Yield: 89.5%.

The proposed methodological approach made it possible to obtain both saturated AGE with a purity of 99.2% (the content of chemical alcohol, AGE-16: 0 - 93.4%), and individual unsaturated AGE-18: 1 (selachyl alcohol) with a purity of 98.1% and AGE -20: 1 with a purity of 97.8%. At the same time, a feature is the use of simple isolation methods available in modern laboratories.

We carried out the isolation of individual unsaturated AGEs from marine raw materials using a combination of column chromatography on silica gel using an eluting benzene - chloroform (10: 0 → 0: 10, v / v) elution system with increasing polarity and high performance liquid chromatography with an ethanol eluting system - water (85:15, v / v), moreover, to increase the mobility, it was not the AGE themselves that separated them, but their acetylated derivatives.

The advantage of this method is the ability to isolate pure individual unsaturated AGE.

The method provides in laboratory conditions obtaining preparations of unsaturated AGE with a high degree of purification, suitable for use in both biochemical and medical experiments.

Obtained by the method of drugs reduce dependence on imported products and allow you to quickly carry out the necessary scientific research.

Table AGE Source Sediment
(saturated. AGE) Filtrate
(combined) Column chromatography HPLC
(AGE-18: 1) HPLC
(AGE-20: 1) 14: 0 1.21 ¹ 1.04 1.93 1.88 - - 15: 0 0.33 0.22 next next - - 16: 0
(chemical alcohol) 73.52 93.43 6.39 6.21 - - 17: 0 0.50 0.47 0.16 0.14 - - 18: 0
(butyl alcohol) 5.62 3.84 next next - 1.95 16: 1 0.62 - 3.42 3.49 0.41 - 18: 1
(selachyl alcohol) 06/14 - 74.50 74.79 98.10 - 20: 1 2.18 - 11.82 11.95 1.39 97.81 22: 1 0.49 - 1.47 1.54 0.10 0.24 ∑ us. 82.65 100 8.79 8.09 - 1.95 ∑ nenas. 17.35 - 91.21 91.91 100 98.05 AGE content
products
% 19.14 99.22 7.35 100 100 100 The output at the stage,% - 42.0 100 100 91.4 89.5

¹ us. - saturated alkyl moieties, nenas. - unsaturated alkyl fragments, cl. - trace amounts (less than 0.1%).

Claims (1)

A method of producing unsaturated alkyl glycerol esters from sea fats, including hydrolysis of sea fat, neutralizing the resulting fat mixture with dilute sulfuric acid, washing with an aqueous NaCl solution, sequential double crystallization from acetone at minus 15 ° C for 18 hours, separating the precipitate of saturated alkyl glycerol esters and its drying, evaporation of the liquid fraction containing unsaturated alkyl glycerol esters, their isolation by column chromatography with an eluting system, characterized in that as eluting benzene: chloroform = 10: 0 → 0:10, vol./about .; The purified concentrate of alkyl glycerol ethers is acetylated with acetyl chloride, and then it is separated into individual acetylated alkyl glycerol ethers by high performance liquid chromatography on a reverse phase column (C18) and elution with 85% aqueous ethanol, fractions with individual acetylated alkyl glycerol ethers are collected evaporated, the acetyl groups are removed by alkaline hydrolysis, the alkyl glycerol esters are extracted with hexane and evaporated.

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