Purified egg yolk lecithin and method for preparing purified egg yolk lecithin containing DMC

Abstract

The invention provides a purified egg yolk lecithin and a preparation method comprising DMC purification, and environment-friendly solvents and conditions are used, so that the influence on the environment is reduced. The invention also provides a lipid emulsion comprising the purified egg yolk lecithin obtained by the method of the invention.

Description

Technical Field

[0001] The present invention relates to a purified egg yolk lecithin and a preparation method thereof. In addition, the present invention also relates to a lipid emulsion comprising the purified egg yolk lecithin obtained by the method of the present invention, and a propofol composition comprising the lipid emulsion. Background Art

[0002] Egg yolk lecithin is a natural phospholipid mixture extracted and refined from egg yolk. Historically, it has been used as an emulsifier in the food and pharmaceutical industries. Rich in phosphatidylcholine, egg yolk lecithin is used as a pharmaceutical additive, particularly as an emulsifier in fat emulsions.

[0003] During the extraction of phospholipids from egg yolk, impurities such as cholesterol and neutral lipids are also extracted. There are several methods for purifying egg yolk phosphatidylcholine, including solvent extraction, chromatography, and low-temperature solvent precipitation. The choice of purification method depends on the intended use of the isolated phosphatidylcholine.

[0004] One method for separating egg yolk lecithin from egg yolk is based on the use of acetone. Since egg yolk lecithin is poorly soluble in acetone, it precipitates, while impurities such as neutral lipids dissolve in the acetone and are separated.

[0005] EP2502928B1 discloses a method for producing purified egg yolk phospholipids by treating the egg yolk with water and an acetone-containing alcohol. The acetone content is 80 vol%, and the alcohol content is 0.3-3 vol%. However, this relatively inefficient method does not produce a high yield of phosphatidylcholine-rich phospholipids, making it insufficient for industrial application.

[0006] Luz Palacios and Tong Wang (Journal of American Oil Chemists Society (2005) 82, 565–569) describe a method for extracting de-oiled and non-de-oiled egg yolk lecithin using ethanol. The purity of the phosphatidylcholine-rich egg yolk lecithin was low (35.7–53.3%). This method also uses a variety of organic solvents, including chlorinated solvents such as chloroform, making it an environmentally unsuitable method.

[0007] Current extraction methods involving the use of acetone often result in egg yolk lecithin containing high levels of residual impurities.

[0008] Even if certain neutral lipids can be sufficiently reduced, obtaining purified egg yolk lecithin that meets the requirements for producing pharmaceutical-grade emulsion ingredients may require repeated treatment with other solvents, thereby increasing time and cost while also reducing the yield of egg yolk lecithin.

[0009] These traditional extraction methods involve the use of organic solvents, which, if not managed properly, can have negative impacts on the environment, leading to environmental and safety issues.

[0010] Egg yolk lecithin is commercially available from egg yolks and is used for certain specialized pharmaceutical and food applications.

[0011] The presence of certain impurities is believed to be the main cause of the color of egg yolk lecithin.

[0012] As a starting material for egg yolk lecithin isolation or purification methods, eggs are low in certain pigments, which is considered important in the industry. Therefore, the inventors have been searching for an efficient purification method that can use a wider variety of eggs, and therefore a wider variety of egg yolk powders (such as SFE-EYP) as a source or starting material for extraction and separation methods. This would eliminate the need to select only those eggs with low pigment content.

[0013] Because egg yolk lecithin is used as an emulsifier in pharmaceutical or nutritional emulsions, the presence of these impurities can also affect the appearance of such emulsions. The inventors of the present invention have discovered that the method for preparing purified egg yolk lecithin from SFE-EYP, using activated carbon decolorization alone, can only remove colored impurities to a certain extent. However, due to the presence of these residual impurities in the egg yolk lecithin, the final egg yolk lecithin still exhibits significant nonspecific absorption.

[0014] Propofol is an alkylphenol derivative (2,6-diisopropylphenol) sold in the form of an oil-in-water emulsion using soybean oil (10%) and egg lecithin (1.2%) as emulsifiers.

[0015] Therefore, there is a need in the art to develop a method for isolating egg yolk lecithin to a purity sufficient to prepare pharmaceutical grade emulsions, such as propofol emulsions, which provides an adequate yield of the desired product, results in a white product appearance, and allows for the reduction or avoidance of the use of environmentally harmful solvents.

[0016] Purpose of the Invention

[0017] The main purpose of the present invention is to solve and overcome one or more deficiencies of the prior art methods for separating egg yolk lecithin, thereby producing egg yolk lecithin with reduced impurities.

[0018] Another object of the present invention is to provide a purified egg yolk lecithin.

[0019] Another object of the present invention is to provide a method for preparing purified egg yolk lecithin, which can obtain a sufficient amount of purified egg yolk lecithin suitable for industrial production.

[0020] Yet another object of the present invention is to provide a method for preparing purified egg yolk lecithin using environmentally friendly solvents and conditions, thereby reducing the impact on the environment and ensuring the safety of the purified egg yolk lecithin production process.

[0021] Another object of the present invention is to provide a lipid emulsion comprising purified egg yolk lecithin.

[0022] Another object of the present invention is to provide a method for preparing a pharmaceutical emulsion, which uses the egg yolk lecithin obtained by the new method as an emulsifier to obtain a white and stable pharmaceutical emulsion. Summary of the Invention

[0023] The present invention relates to purified egg yolk lecithin and a preparation method thereof. The present invention also relates to a lipid emulsion comprising the purified egg yolk lecithin obtained by the method of the present invention and a method for preparing the lipid emulsion.

[0024] In a first aspect, the present invention provides a method for preparing purified egg yolk lecithin, comprising the following steps:

[0025] a. providing supercritical fluid extracted egg yolk powder as a starting material,

[0026] b. treating the supercritical fluid extracted egg yolk powder with a solvent,

[0027] c. separating the product obtained in step b) from the solvent used in step b),

[0028] d. extracting the egg yolk lecithin fraction from the product obtained in step c),

[0029] e. Optionally, treating the egg yolk lecithin fraction obtained in step d) with charcoal,

[0030] f. precipitating impurities from the egg yolk lecithin fraction obtained in step d) or step e),

[0031] g. removing precipitated impurities from the product obtained in step f),

[0032] h. separating the purified egg yolk lecithin from the product obtained in step g), and

[0033] i. Optionally, drying purified egg yolk lecithin;

[0034] Wherein, in step b), the solvent is dimethyl carbonate.

[0035] In a second aspect, the present invention provides purified egg yolk lecithin obtained by the above method.

[0036] In a third aspect, the present invention provides a nutritional product comprising the purified egg yolk lecithin obtained by the method of the present invention.

[0037] In a fourth aspect, the present invention provides a lipid emulsion comprising the purified egg yolk lecithin obtained by the method of the present invention. The lipid emulsion may contain a pharmaceutically active ingredient and may be used as a pharmaceutical composition.

[0038] In a fifth aspect, the present invention provides a method for preparing a lipid emulsion, comprising the following steps:

[0039] a. providing an oil phase comprising soybean oil, a lipophilic pharmaceutically active ingredient, preferably propofol, and optionally a pharmaceutically

[0040] Acceptable antioxidants;

[0041] b. providing an aqueous phase comprising water for injection, and optionally a pharmaceutically acceptable tonicity agent, a pH adjusting agent, and

[0042] optionally a pharmaceutically acceptable co-emulsifier, preferably a free long-chain fatty acid, more preferably oleic acid;

[0043] c. by mixing the oil phase provided in step a) with the aqueous phase provided in step b) to form a pre-emulsion;

[0044] d. forming an emulsion by high pressure homogenization of the pre-emulsion obtained in step c); and

[0045] e. sterilizing the emulsion obtained in step d), wherein the emulsion is optionally filled into suitable containers before or after sterilization;

[0046] Wherein, the purified egg yolk lecithin of the present invention is added in step a) or step b).

[0047] In a sixth aspect, the present invention provides an oil-in-water emulsion for parenteral administration, comprising:

[0048] 10-20 mg / mL propofol;

[0049] 50-100 mg / mL soybean oil;

[0050] 22.5 mg / mL glycerol;

[0051] 12 mg / mL of purified egg yolk lecithin according to the present invention;

[0052] Oleic acid;

[0053] Optionally, 50 mg / mL medium-chain triglycerides;

[0054] Optionally, 0.05 mg / mL anhydrous EDTA disodium (equivalent to 0.055 mg EDTA disodium); sodium hydroxide to adjust pH, and water for injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 is an HPLC chromatogram analyzed by the HPLC-UV analysis method described in Example 5, in which the solid line represents the UV absorbance at 447 nm of the purified egg yolk lecithin not treated with dimethyl carbonate, and the dotted line represents the UV absorbance at 447 nm of the purified egg yolk lecithin treated with dimethyl carbonate in step b) of the method according to the present invention. DETAILED DESCRIPTION

[0056] The present invention provides a method for preparing purified egg yolk lecithin and its use in compositions. This method has several advantages, such as using a green solvent and obtaining highly pure egg yolk lecithin. Various methods for purifying egg yolk lecithin are known in the art. However, these methods have several disadvantages. Therefore, the present invention focuses on the use of an environmentally friendly solvent, namely dimethyl carbonate, for purifying egg yolk lecithin.

[0057] definition

[0058] Unless the context indicates otherwise, the following definitions apply in this application.

[0059] As used herein, the terms "comprises," "includes," "comprising," "including," "having," "having" or any other variation thereof, are intended to cover a non-exclusive inclusion.

[0060] Dimethyl carbonate (DMC), also known as DMC, is a carbonate ester, in which both hydrogen atoms in carbonic acid are replaced by methyl groups.

[0061] As used herein, the term "treating" refers to the process of contacting a substance (e.g., supercritical fluid extracted egg yolk powder) with one or more solvents or other agents. Such treatment may involve various methods, including but not limited to mixing, stirring, agitating, soaking, or exposure, to allow physical interaction between the substance and the solvent or agent. The results of the treatment may include purification, extraction, reaction, or other modification of the substance to achieve a desired result.

[0062] The term "separating" as used herein refers to the process of separating insoluble matter from a solvent or solution. This process can be accomplished by various techniques, including but not limited to filtration, decantation, extraction, distillation, evaporation, centrifugation, drying, or a combination thereof.

[0063] As used herein, the term "purified egg-yolk lecithin" refers to egg-yolk lecithin that is free of certain impurities and significantly reduced in others. Purified egg-yolk lecithin is further characterized by being rich in phosphatidylcholine. Its purity and stability make it suitable for further application in nutritional and pharmaceutical products.

[0064] The term "phosphatidylcholine" refers to a class of phospholipids with choline as the head group. It therefore refers to a variety of compounds rather than one unique compound, as several different fatty acid chain types may be present.

[0065] The term "supercritical fluid extracted egg-yolk powder" or "SFE-EYP" refers to egg-yolk powder that has been extracted for neutral lipids (such as cholesterol) using the "supercritical fluid extraction" technique. This technique typically uses non-hazardous carbon dioxide as a fluid. Above its critical point, carbon dioxide behaves both like a gas and a liquid, allowing for selective extraction based on solubility. Under high pressure and high temperature conditions, the resulting powder has a potentially reduced fat content while retaining valuable components such as phospholipids, depending on the extraction conditions used. Supercritical fluid can selectively extract certain lipids, such as phospholipids (primarily triglycerides and cholesterol), from egg yolk under mild conditions. Supercritical fluid-extracted egg-yolk powder is used as the starting material in the method of the present invention. It may also be referred to as defatted or de-oiled egg-yolk powder. Hietaniemi (2009) Eur Food Res Technol 228: pp. 857-863 describes this method in detail.

[0066] Therefore, the supercritical fluid extracted egg yolk powder (SFE-EYP) referred to herein, as the starting material for separating egg yolk lecithin, is a composition obtained by subjecting egg yolk to supercritical fluid extraction with carbon dioxide. Preferably, the composition comprises phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol, sphingomyelin (SHP), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC), as well as various impurities. More preferably, the composition has a reduced neutral lipid content, more preferably, a reduced triglyceride and cholesterol content, compared to dry egg yolk powder.

[0067] The exact composition of SFE-EYP depends on the type of egg yolk used and the conditions used in the extraction method.

[0068] Preferably, the SFE-EYP used as starting material for the process of the present invention contains less than 2% w / w cholesterol, 1-2% w / w residual triglycerides and more than 70% w / w phosphatidylcholine.

[0069] As used herein, the term "extraction" refers to the process of separating a specific substance from a mixture of components using a solvent. This method typically involves dissolving the desired substance in a solvent or solvent mixture to separate it from other components of the mixture that are insoluble in the solvent or solvent mixture. The solvent can then be removed to yield a purified substance. In the methods of the present invention, the term extraction, as used in step d, refers to this understanding.

[0070] As used herein, the term "precipitating" or "precipitation" refers to the process by which a substance solidifies. It may crystallize or otherwise change from a liquid to a solid state. Precipitation can be induced by changes in temperature, pH, concentration, or the addition of another reagent or solvent.

[0071] As used herein, the term "drying" refers to the process of removing residual liquid (such as solvent) from a substance, typically using heat, air flow, vacuum, or a combination of these methods.

[0072] As used herein, "medium chain triglyceride" refers to a triglyceride containing two or three fatty acids with an aliphatic tail of 6-12 carbon atoms.

[0073] A UV-Vis spectrophotometer is an analytical instrument used to measure the amount of ultraviolet (UV) and visible light absorbed by a sample. The UV-Vis spectrophotometer works by passing a beam of light through the sample and measuring the amount of light absorbed at each wavelength. The amount of light absorbed is proportional to the concentration of the absorbing compound in the sample. It measures the intensity of the light after it passes through the sample (I) and compares it to the intensity of the light before it passes through the sample (I o ) for comparison. I / I o The ratio of absorbance to transmittance is called transmittance and is usually expressed as a percentage (T%). Absorbance A is based on transmittance:

[0074] A = -log(T% / 100%)

[0075] It is a technique widely used in chemistry, biochemistry, and other fields to identify and quantify compounds in various samples. Sometimes, a UV spectrometer is a built-in component of a high-performance liquid chromatograph.

[0076] High-performance liquid chromatography (HPLC) is a technique for separating molecules based on properties such as size and surface charge. Ultraviolet (UV) spectroscopy combined with HPLC allows the determination of the concentration of identified and separated molecules, or simply the comparison of relative concentrations of the same eluent in different runs. In this application, references to detection at a wavelength of 447 nm should be understood to include detection at wavelengths between 445 and 450 nm.

[0077] Combining a charged aerosol detector (CAD) with HPLC allows the mass of compounds present in a sample to be determined by generating charged aerosol particles, which are detected using an electrometer. The process employed produces a signal that is proportional to the mass of the analyte present. It is often used to analyze compounds that cannot be detected using traditional UV / Vis methods due to the lack of a chromophore.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although similar or equivalent methods and materials as described herein can be used in the practice or testing of the embodiments of the present invention, suitable methods and materials are described below. In the event of any conflict, this specification (including definitions) shall prevail. In addition, the materials, methods and examples are illustrative only and are not restrictive.

[0079] When an amount, concentration or other value or parameter is given as a range, preferred range or preferred upper value and / or preferred lower value list, this should be understood as specifically disclosing all ranges formed by any pair of any upper range limit or preferred value and any lower range limit or preferred value, whether or not a range is disclosed separately. Unless otherwise stated, the numerical ranges recited herein should include their endpoints and all integers and fractions within the range.

[0080] Egg yolk is composed of various nutrients, including protein, fat, and phospholipids. According to research by Aro et al. (Eur Food Res Technol (2009) 228:857-863), approximately 50% of egg yolk is water. After spray drying, the resulting powder contains approximately 60% lipids, including neutral lipids (65%), phospholipids (31%), and cholesterol (4%). It is described that dried egg yolk powder also contains approximately 15% phospholipids, mainly composed of approximately 70% phosphatidylcholine and 20% phosphatidylethanolamine (Burley RW, Vadehra DV, The Avian Egg Chemistry and Biology, John Wiley & Sons (1989)). It seems to be an indisputable fact that the main phospholipids in egg yolk are phosphatidylcholine (PC), which accounts for approximately 70% of the total egg yolk phospholipids, and phosphatidylethanolamine (PE), which accounts for approximately 15% of the total egg yolk phospholipids. Yolk proteins include lipoproteins and phosphoproteins, which together account for approximately 16% of the yolk weight. However, as reported in the literature, the composition of egg yolk varies greatly, which may be due to different breeds of hens and the diet they are fed.

[0081] Even though SFE-EYP primarily consists of organic solvent-soluble phospholipids, organic solvent-soluble proteins, and water-insoluble proteins, it may also contain various impurities, such as natural pigments such as carotenes and xanthophylls (lutein, canthaxanthin, and zeaxanthin). These impurities may be responsible for the dark yellow to orange color of egg yolk lecithin, which indeed affects the usability of the composition in pharmaceuticals and nutritional products that rely on a clear white appearance. These impurities can affect the quality and appearance of lipid emulsion compositions. Studies have found that washing with a solvent such as dimethyl carbonate before extracting egg yolk lecithin from SFE-EYP can help remove these impurities.

[0082] In the process of separating and purifying egg yolk lecithin from SFE-EYP, the inventors of the present application tried different strategies to control or remove these impurities. They treated SFE-EYP with several different organic solvents and tested them in combination with activated carbon treatment.

[0083] When searching for a more environmentally friendly solvent (green solvent) that is beneficial to workplace safety, health, and the environment (e.g., reducing hazardous waste), various factors need to be considered, such as the solubility of egg yolk lecithin in the solvent, the solvent's low boiling point, its lack of reactivity toward phospholipids, and good recyclability. Based on these characteristics, dimethyl carbonate, acetone, methyl acetate, and diisopropyl ether were identified as the most suitable candidates for the purification of SFE-EYP.

[0084] Surprisingly, while testing these different solvents, the inventors of the present application discovered that treating supercritical fluid extracted egg yolk powder with dimethyl carbonate was not only effective in purifying SFE-EYP, thereby providing a more environmentally friendly alternative to existing methods, but also improved the impurity profile of egg yolk lecithin without significantly reducing yield or generating additional unwanted byproducts. The formation of such byproducts, which can be observed when SFE-EYP is treated with acetone, creates new impurities, likely due to the reaction of the amine groups in phosphatidylethanolamine (PE) with acetone.

[0085] The effects of using different solvents to purify SFE-EYP on the purity of the final egg yolk lecithin (i.e., the purified egg yolk lecithin after completing all the above steps) are shown in Table 1. The table lists the yield (in w / w) obtained by HPLC-CAD, the amount of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (in % w / w), and the absorbance of the color-causing impurities measured by UV-visible spectrophotometer at 447 nm:

[0086] Table 1:

[0087]

[0088]

[0089] The samples analyzed in this UV absorption assay all contained the same concentration of purified egg yolk lecithin (final concentration).

[0090] The inventors of the present application have discovered that the use of the environmentally friendly solvent dimethyl carbonate in step b) of the method of the present invention can very effectively reduce impurities in supercritical fluid extracted egg yolk powder, preferably those impurities that are believed to cause yellow to brown coloration, which are believed to be detrimental to the suitability of egg yolk lecithin for preparing white pharmaceutical emulsions (such as propofol emulsions).

[0091] The dimethyl carbonate (DMC) treatment method not only provides high yields but also excellent purity. UV absorbance at 447 nm is significantly reduced. This method also yields a product enriched in phosphatidylcholine (PC) and depleted in phosphatidylethanolamine (PE) within the purified egg yolk phosphatidylcholine.

[0092] The UV absorbance value measured at 447 nm corresponds to the content of carotenoids (mainly lutein and zeaxanthin). Therefore, treatment of SFE-EYP with dimethyl carbonate reduces the carotenoids in egg yolk lecithin, mainly lutein and zeaxanthin.

[0093] Liquid chromatography analysis (the results are shown in Figure 1) confirmed this effect. It also showed that when dimethyl carbonate was used to purify SFE-EYP, impurities could be further effectively removed.

[0094] In a first aspect, the present invention relates to a method for preparing purified egg yolk lecithin, comprising the following steps:

[0095] a. providing supercritical fluid extracted egg yolk powder as a starting material,

[0096] b. Treating the supercritical fluid extracted egg yolk powder with a solvent,

[0097] c. separating the product obtained in step b) from the solvent used in step b),

[0098] d. extracting the egg yolk lecithin fraction from the product obtained in step c),

[0099] e. Optionally, treating the egg yolk lecithin fraction obtained in step d) with charcoal,

[0100] f. precipitating impurities from the egg yolk lecithin fraction obtained in step d) or step e),

[0101] g. removing precipitated impurities from the product obtained in step f),

[0102] h. separating the purified egg yolk lecithin from the product obtained in step g), and

[0103] i. optionally, drying the purified egg yolk lecithin,

[0104] Wherein, in step b), the solvent is dimethyl carbonate.

[0105] Therefore, the present invention provides a multi-step preparation method for purified egg yolk lecithin using SFE-EYP as a starting material and dimethyl carbonate as a solvent, wherein the first step is to provide SFE-EYP and the second step is to treat SFE-EYP with dimethyl carbonate (DMC). The treatment may involve one or more of mixing, stirring, agitating, soaking or exposing the SFE-EYP powder to dimethyl carbonate. Preferably, the treatment comprises adding SFE-EYP to a dimethyl carbonate solution and stirring the resulting mixture at an elevated temperature. Preferably, the temperature range is 30-60°C, preferably 35-55°C, and most preferably 40-45°C. Further preferably, the stirring lasts for 50 to 70 minutes, preferably 50 to 65 minutes, and most preferably 55 to 65 minutes. In a preferred embodiment, this treatment of SFE-EYP powder with dimethyl carbonate produces a slurry.

[0106] The addition of a free radical scavenger, particularly when oxidation protection is required, can improve the efficiency of the process. Preferably, steps b), d), e), and / or g) further include the addition of a free radical scavenger. Preferably, the free radical scavenger is DL-α-tocopherol. This free radical scavenger can be added during step b), during step d), during and after the charcoal treatment in step e), and / or during step g).

[0107] The desired amount of dimethyl carbonate added to SFE-EYP in step b) may be 2 mL to 10 mL per gram of SFE-EYP, preferably 3 mL to 8 mL per gram of SFE-EYP, more preferably 4 mL to 8 mL per gram of SFE-EYP.

[0108] Step b) may further comprise adding a free radical scavenger. Preferably, the free radical scavenger is DL-α-tocopherol.

[0109] In step c), the product obtained in step b) is separated from the solvent.

[0110] In a preferred embodiment, the desired product obtained in step b) is a solid.

[0111] The solid product can be isolated by conventional methods such as filtration, decantation, centrifugation or a combination thereof.

[0112] In some embodiments, isolation of the product is accomplished by filtration to remove soluble impurities dissolved by the solvent.

[0113] The resulting product can be washed with dimethyl carbonate.

[0114] In order to remove excess dimethyl carbonate, the product of step c) may be further dried, whether or not washed, before adding the extraction solvent in step d). Drying may be carried out under vacuum conditions at a pressure of 200 to 300 mbar, at a temperature in the range of 15° C. to 35° C., preferably in the range of 20° C. to 30° C., for a period of 4 to 6 hours.

[0115] In step d), the egg yolk lecithin fraction is extracted from the product obtained in step c) using a solvent.

[0116] The extraction solvent in step d) can be selected from C 1- C3 alcohols, toluene, C5-C7 aliphatic alkanes, mixtures of alcohols and nitriles, mixtures of alcohols and dimethyl carbonate, and mixtures of alcohols and diethyl carbonate.

[0117] Preferably, the solvent in step d) is selected from methanol, ethanol, propanol, toluene, n-heptane, a mixture of ethanol and acetonitrile, a mixture of ethanol and dimethyl carbonate, and a mixture of ethanol and diethyl carbonate.

[0118] The temperature range for extracting the egg yolk lecithin fraction with the solvent is 15°C to 45°C, preferably 20°C to 30°C.

[0119] In a preferred embodiment, step d) may further comprise adding a free radical scavenger. The free radical scavenger is preferably DL-α-tocopherol.

[0120] In another preferred embodiment, as described above, the free radical scavenger may be dissolved in the solvent of step d) before the extraction process. The mixture is heated at a temperature range of 55° C. to 60° C., preferably 55° C. to 60° C., and then stirred for 30 to 40 minutes.

[0121] The mixture can be filtered to remove insoluble particles. The insoluble material can optionally be rinsed with a solvent mixture. The rinsed solution is then added to the filtrate.

[0122] Step e) is optional. It involves treating the obtained egg yolk lecithin fraction with charcoal, wherein the treatment comprises stirring a liquid solvent containing the extracted egg yolk lecithin fraction with the added charcoal. The amount of charcoal added in the process can vary from 1% w / w to 5% w / w, preferably 2% w / w to 4% w / w. A preferred embodiment is one in which the charcoal treatment is carried out at a temperature in the range of 30°C to 50°C for a time period of 30 minutes to 3 hours, more preferably at a temperature in the range of 40°C to 45°C for a time period of 1 hour to 2 hours.

[0123] During step e) of the method, different types of charcoal (strongly acidic or basic) can be used.

[0124] Step e) may comprise adding a free radical scavenger after separating the charcoal from the solution. Preferably, the free radical scavenger is DL-α-tocopherol.

[0125] The method further comprises step f), which is the step of precipitating impurities such as PE from the egg yolk lecithin fraction obtained in the previous step. Preferred embodiments involve cooling the egg yolk lecithin fraction obtained in step d) or step e) to achieve precipitation. Preferably, cooling is performed at a temperature in the range of -12°C to -8°C, more preferably -11°C to -10°C, followed by stirring at the reduced temperature for 1 to 1.5 hours to further precipitate impurities.

[0126] Step g) involves separating the precipitated impurities from the egg yolk lecithin-containing solution. If optional step e) is performed before step f), charcoal adsorbed with other impurities can be removed simultaneously. The precipitated impurities can be removed from the egg yolk lecithin fraction obtained in step d) or step e) by conventional methods such as filtration or centrifugation, preferably by filtration.

[0127] In step g), a free radical scavenger may be added after removing the precipitated impurities in the solution. Preferably, the free radical scavenger is DL-α-tocopherol.

[0128] After removal of the precipitate (cooling and filtration), the product obtained in step g) is a solution.

[0129] In step h) of the method, the dissolved purified egg yolk lecithin is separated from the product obtained in step g). This step can be carried out by conventional methods such as concentration, distillation, or evaporation. Preferably, the purified egg yolk lecithin is separated by evaporation. In this step, a viscous product is obtained.

[0130] The isolated purified egg yolk lecithin can optionally be dried. The product can be converted into a fluffy solid by an optional drying step i), for example using a belt freeze dryer. Drying is preferably carried out at a temperature in the range of 25°C to 35°C for 1 to 4 hours.

[0131] In a second aspect, the present invention provides purified egg yolk lecithin obtained by the above-described method. This purified egg yolk lecithin is significantly whiter than egg yolk lecithin prepared by the method that does not involve step b) of treating SFE-EYP with dimethyl carbonate. The purified egg yolk lecithin exhibits significantly reduced UV absorbance during the retention time of lutein and zeaxanthin elution, as measured at a wavelength of 447 nm, indicating a significant reduction in the amount of zeaxanthin and / or lutein compared to egg yolk lecithin prepared by the method that does not involve step b) of treating SFE-EYP with dimethyl carbonate. The UV absorbance, expressed as AUC, measured at 447 nm using a UV-visible spectrophotometer, is lower than that of egg yolk lecithin prepared by the method that differs only in that step b) is not involved. Preferably, the UV absorbance value detected at 447 nm at the retention time at which lutein and zeaxanthin elute is 10 times lower, more preferably 25 times lower, and most preferably 50 times lower than the UV absorbance value of egg yolk lecithin prepared by a method that differs only in that it does not involve step b). In a most preferred embodiment, the UV absorbance value is 68 times lower than the UV absorbance value of egg yolk lecithin prepared by a method that differs only in that it does not involve step b).

[0132] Preferably, the purified egg yolk lecithin obtained according to the method of the present invention does not contain impurities A, B, and C. In the HPLC-UV analysis described in Example 5, these impurities elute at RT of 18.66 minutes, RT of 19.0 minutes, and RT of 19.5 minutes, respectively. The relative retention time of lutein is 16.2 minutes, as shown in Table 2.

[0133] The following table lists the effect of treating SFE-EYP with dimethyl carbonate or without solvent on the final egg yolk phosphatidylcholine, i.e., the impurities eluting at 16.2 minutes, 18.66 minutes, 19.0 minutes and 19.5 minutes, respectively, in HPLC-UV analysis (see Example 5).

[0134] Table 2:

[0135]

[0136] Compared with the commercially available products tested, the purified egg yolk lecithin obtained by the method of the present invention has better lipid properties, which are manifested in that it is rich in phosphatidylcholine (PC) and has reduced amounts of phosphatidylethanolamine (PE), cholesterol, sphingomyelin (SHP), phosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), and lysophosphatidylcholine (LPC).

[0137] In another preferred embodiment, the purified egg yolk lecithin obtained by this method has a better yield than when using a different solvent, and has an improved purity profile compared to products obtained using a different solvent or compared to alternative methods.

[0138] The enhanced purity is characterized by reduced levels of phosphatidylethanolamine, cholesterol, sphingomyelin, phosphatidylinositol, lysophosphatidylethanolamine, and lysophosphatidylcholine.

[0139] The data in Table 3 show the effects of treating SFE-EYP with different solvents in step b) of the method on the lipid properties and yield of the final egg yolk lecithin, and are compared with commercial egg yolk lecithin. The first row lists the different compounds measured. The second to fourth rows list the masses of these compounds in commercial egg yolk lecithin (Batch 1); the masses of these compounds in egg yolk lecithin from which SFE-EYP was purified using acetone in step b) of the method (Batch 2); and the masses of these compounds in egg yolk lecithin from which SFE-EYP was purified using DMC in step b) of the method (Batch 3).

[0140] Table 3:

[0141]

[0142] When DMC is used as solvent in step b) of method (3) according to the first aspect of the present invention, the PE content in the obtained product is significantly reduced to less than 6% w / w, preferably 5.6% w / w, and the PC content is significantly increased to more than 78% w / w, preferably 79.38% w / w, compared to when acetone is used as solvent in step b) of method (2) or compared to commercially available egg yolk lecithin (1).

[0143] According to a second aspect of the present invention, purified egg yolk lecithin may also contain trace amounts of dimethyl carbonate. In pilot-scale batches, this amount was as low as 50 ppm to 110 ppm, as determined by GC-MS. This is clearly well below the values ​​required for pharmaceutical use.

[0144] Another advantage of the purified egg yolk lecithin according to the second aspect of the present invention is that the residual amount of ketone organic solvents or chlorinated solvents is less than 10 ppm. Preferably, the ketone organic solvent is selected from acetone, butanone (also known as methyl ethyl ketone (MEK)), and methyl isobutyl ketone (MIBK). Preferably, the chlorinated solvent is selected from chloroform, dichloromethane, and tetrachloromethane. Preferably, the purified egg yolk lecithin does not contain any such solvents.

[0145] In a third aspect, the present invention provides a lipid emulsion comprising purified egg yolk lecithin obtained by the method of the present invention. The lipid emulsion may contain egg yolk lecithin as an emulsifier. This lipid emulsion can be used to provide a pharmaceutically active ingredient that is considered substantially water-insoluble for intravenous injection.

[0146] The egg yolk lecithin of the present invention is beneficial for these lipid emulsions because it does not contain the aforementioned impurities. Consequently, it appears whiter, and so does the lipid emulsion. Drug products that appear yellowish or even have a slight orange tint are not as easily accepted by patients as bright white drugs. Patients tend to associate lower quality with a less-than-pure white color, which can affect compliance. More importantly, if patients even subconsciously mistrust their medication, the significant therapeutic effects based on personal confidence in the efficacy of the drug they are taking may be diminished.

[0147] Propofol is a very lipophilic compound that is essentially insoluble in water or other aqueous media. Therefore, it is formulated as an intravenous emulsion containing 10% lipid (1 mL contains 1.1 kcal; 0.1 g fat) containing soybean oil and egg yolk lecithin, so that the lipid component can serve as a drug carrier.

[0148] In a fourth aspect, the present invention provides a method for preparing a lipid emulsion containing a lipophilic pharmaceutical active ingredient, preferably propofol, comprising the following steps:

[0149] a) providing an oil phase comprising soybean oil, a lipophilic pharmaceutically active ingredient, preferably propofol, and optionally a pharmaceutically acceptable antioxidant,

[0150] b) providing an aqueous phase comprising water for injection, and optionally a pharmaceutically acceptable tonicity agent, optionally a pharmaceutically acceptable co-emulsifier, preferably the co-emulsifier is a free long-chain fatty acid, more preferably oleic acid, and a pH adjuster and optionally a chelating agent;

[0151] c) forming a pre-emulsion by mixing the oil phase provided in step a) with the water phase provided in step b);

[0152] d) forming an emulsion by high pressure homogenizing the pre-emulsion obtained in step c); and

[0153] e) sterilizing the emulsion obtained in step d), wherein the emulsion is optionally filled into suitable containers before or after sterilization,

[0154] Wherein, the purified egg yolk lecithin of the present invention is added in step a) or step b).

[0155] The purified egg yolk lecithin added is the egg yolk lecithin described above.

[0156] The oil phase comprises soybean oil and a lipophilic pharmaceutically active ingredient. Preferably, the pharmaceutically active ingredient is propofol. The oil phase may also comprise a pharmaceutically acceptable antioxidant. Preferably, the oil phase does not comprise a pharmaceutically acceptable antioxidant.

[0157] The oil component (preferably soybean oil) and the lipophilic active ingredient propofol can be mixed.

[0158] In a preferred embodiment, the oil phase in step a) further comprises medium chain triglycerides.

[0159] The aqueous phase may contain a pharmaceutically acceptable tonicity agent and, optionally, a pharmaceutically acceptable co-emulsifier and / or a pH adjuster and / or a chelating agent. Preferred embodiments are those in which the aqueous phase comprises a pharmaceutically acceptable tonicity agent, a pharmaceutically acceptable co-emulsifier, and a pH adjuster. Even more preferably, the pharmaceutically acceptable tonicity agent is glycerol and the co-emulsifier is a free long-chain fatty acid, more preferably oleic acid.

[0160] Oleic acid is a fatty acid also known as cis-9-octadecenoic acid. Fatty acids (or their salts) do not typically occur as fatty acids in biological systems. Instead, fatty acids such as oleic acid exist as esters (usually triglycerides). However, as a component of this emulsion, it is used as a free fatty acid.

[0161] The components of the aqueous phase can be dispersed in water to form a suspension. This suspension can be achieved by high-intensity mixing. During this process, pH adjustment can be performed. Sodium hydroxide can be used for pH adjustment.

[0162] The oil phase can be transferred to the water phase through a membrane filter. The water phase ingredients and the oil phase ingredients can be mixed together to obtain a pre-emulsion.

[0163] The emulsion can be obtained by high pressure homogenization of a pre-emulsion.

[0164] The homogenization process can be carried out at a temperature ranging from 50°C to 80°C and a pressure of 350 to 400 bar. It is preferably carried out at a temperature ranging from 55°C to 70°C. This process can be followed by sterilization. The emulsion can be filled into suitable containers before or after sterilization. The emulsion can be further diluted with an appropriate amount of water under stirring and then filtered to obtain the final emulsion.

[0165] The propofol emulsion prepared by the above method using the purified egg yolk lecithin according to the present invention has a surprisingly stable state and appears whiter than the emulsion prepared using the egg yolk lecithin which has not been purified according to the present invention.

[0166] In a fifth aspect, the present invention provides an oil-in-water emulsion composition for parenteral administration, comprising 10 to 20 mg / mL propofol, 50 to 100 mg / mL soybean oil, 22.5 mg / mL glycerol, 12 mg / mL purified egg yolk lecithin, optionally 0.05 mg / mL anhydrous disodium edetate (equivalent to 0.055 mg disodium edetate), sodium hydroxide for adjusting the pH, and water for injection.

[0167] In a preferred embodiment, the oil-in-water emulsion composition further comprises medium chain triglycerides in an amount ranging from 50 to 100 mg / mL.

[0168] In a further preferred embodiment, the oil-in-water emulsion composition further comprises oleic acid.

[0169] In a preferred embodiment, the present application provides an oil-in-water emulsion for parenteral administration, comprising:

[0170] 10.0 mg / mL propofol,

[0171] 100.0mg / mL soybean oil,

[0172] 22.5mg / mL glycerol,

[0173] 12.0 mg / mL of the purified egg yolk lecithin of the present invention,

[0174] 0.055 mg / mL anhydrous EDTA disodium (equivalent to 0.05 mg EDTA disodium) as a microbial inhibitor, and

[0175] Sodium hydroxide and water for injection to adjust pH.

[0176] In another preferred embodiment, the present application provides an oil-in-water emulsion for parenteral administration, comprising:

[0177] 10.0 mg / mL propofol,

[0178] 100.0mg / mL soybean oil,

[0179] 22.5mg / mL glycerol,

[0180] 12.0 mg / mL of the purified egg yolk lecithin of the present invention,

[0181] Sodium hydroxide and water for injection to adjust pH.

[0182] In another preferred embodiment, the present invention provides an oil-in-water emulsion for parenteral administration, comprising:

[0183] 20.0 mg / mL propofol,

[0184] 100.0mg / mL soybean oil,

[0185] 22.5mg / mL glycerol,

[0186] 12 mg / mL of the purified egg yolk lecithin of the present invention,

[0187] Sodium hydroxide and water for injection to adjust pH.

[0188] In another preferred embodiment, the present invention provides an oil-in-water emulsion for parenteral administration, comprising:

[0189] 10.0 mg / mL propofol,

[0190] 50.0mg / mL soybean oil,

[0191] 50mg / mL medium chain triglycerides,

[0192] 22.5mg / mL glycerol,

[0193] 12.0 mg / mL of the purified egg yolk lecithin of the present invention,

[0194] Sodium hydroxide and water for injection to adjust pH.

[0195] In another preferred embodiment, the present invention provides an oil-in-water emulsion for parenteral administration, comprising:

[0196] 20mg / mL propofol,

[0197] 50mg / mL soybean oil,

[0198] 50.0mg / mL medium chain triglycerides,

[0199] 22.5mg / mL glycerol,

[0200] 12 mg / mL of the purified egg yolk lecithin of the present invention,

[0201] Sodium hydroxide and water for injection to adjust pH.

[0202] Preferably, all of these compositions also contain oleic acid.

[0203] abbreviation

[0204] SFE-EYP: Supercritical Fluid Extracted Egg Yolk Powder

[0205] PC: Phosphatidylcholine

[0206] PE: Phosphatidylethanolamine

[0207] LPE: Lysophosphatidylethanolamine

[0208] LPC: Lysophosphatidylcholine

[0209] SPH: sphingomyelin

[0210] PI: Phosphatidylinositol

[0211] HPLC: High Performance Liquid Chromatography

[0212] LC: Liquid chromatography

[0213] UV: Ultraviolet

[0214] w / w: weight ratio

[0215] DMC: dimethyl carbonate

[0216] DL: right-handed and left-handed

[0217] Mbar: millibar

[0218] CAD: Charged Aerosol Detector

[0219] MTBE: 2-methoxy-2-methylpropane (also known as methyl tert-butyl ether)

[0220] experiment

[0221] The following examples provide detailed experimental parameters according to the present invention. These examples are intended to illustrate the present invention, rather than to limit any possible implementation of the present invention.

[0222] Example

[0223] Example 1: Preparation of Purified Egg Yolk Lecithin by DMC Washing and Charcoal Treatment

[0224] 3540 mL of dimethyl carbonate was injected into a 5 L reactor. A solution of DL-α-tocopherol (0.15 g) dissolved in dimethyl carbonate (60 mL) was prepared and added to the reactor. 600 g of supercritical fluid extracted egg yolk powder was added and mixed with 3600 mL of the solution under stirring. The mixture was heated to 40-45° C. and stirred at this temperature for 55-65 minutes. The hot mixture was filtered using a Buchner funnel equipped with 10 μ Whatman filter paper and 10 μ filter cloth. The filtered solid was washed with 1200 mL (600 mL×2) of dimethyl carbonate. The solid was dried under vacuum at 200-300 mbar (610-535 mmHg) and 20-30° C. for 4-6 hours.

[0225] The DMC-washed SFE-EYP was extracted with 3540 mL of an ethanol and acetonitrile solvent mixture (4800 mL of a solvent mixture made up of 2736 mL of anhydrous ethanol and 2064 mL of acetonitrile) at a temperature in the range of 20-25°C, followed by the addition of the DL-α-tocopherol solution. The mixture was heated to a temperature of 55-60°C and then stirred for 30-40 minutes. The hot slurry was filtered, and the resulting solid was washed with 1200 mL (600 mL x 2) of the solvent mixture. The filtrate was treated with charcoal at a temperature in the range of 40-45°C and then stirred for 55-65 minutes.

[0226] Under the stirring condition of 100RPM, the obtained solution is cooled to -12℃ to -8℃ for 90 minutes to further precipitate impurities. The obtained mixture is filtered through a jacketed funnel while the jacket temperature is maintained in the range of -12℃ to -8℃, and then DL-α-tocopherol (prepared by dissolving 0.048g DL-α-tocopherol in 30mL of the solvent mixture prepared before) is added to the obtained filtered solution. The obtained solution is filtered again through 5μm and 0.22μm polyvinylidene fluoride filters, and then concentrated under vacuum conditions below 35℃ until the remaining residue is viscous. The remaining viscous material is dried to obtain 161.8g (0.27w / w) of purified egg yolk lecithin.

[0227] Example 2: Comparison of different amounts of DMC in the preparation of purified egg yolk lecithin

[0228] The method of Example 1 using dimethyl carbonate as the purification solvent was repeated using different amounts of dimethyl carbonate and different process parameters (such as temperature and time). The amounts in column 2 are expressed as coefficients, where, for example, 3+1 means that 3 times the amount of SFE-EYP in grams (in mL) was used for stirring, and 1 times the amount was used to wash the filtered SFE-EYP. For example, in row 1, if 150 g of SFE-EYP was processed, it was placed in 450 mL of DMC and stirred, and then washed with 150 mL of DMC. The resulting yields and UV absorbance values ​​are shown in Table 4 below:

[0229] Table 4:

[0230]

[0231] It can be seen that the method is robust under various circumstances, but it appears that the purification process can be optimized without compromising yield if six times the amount of SFE-EYP is used in the initial treatment and then twice the amount of SFE-EYP is used in further washing steps.

[0232] Example 3: Comparative Experiment of Preparing Egg Yolk Lecithin with and without DMC

[0233] Example 3.1: In step e) charcoal treatment is used , Preparation of egg yolk lecithin without DMC (no step b)

[0234] 240.0 mL of mixed solvent was prepared with anhydrous ethanol (136.8 mL) and acetonitrile (103.2 mL) at a temperature in the range of 20-25 °C for use in the process.

[0235] Under stirring, a solution of DL-α-tocopherol (prepared by dissolving 2.4 mg of DL-α-tocopherol in 5 mL of mixed solvent) and SFE-EYP (30 g) was added to 170 mL of the solvent mixture prepared above. The mixture was heated to 55-60° C. and then stirred for 30-40 minutes. The hot mixture was filtered and the resulting solid was washed twice with 60 mL (30 mL×2) of the solvent mixture. The combined filtrate was treated with charcoal at a temperature in the range of 40-45° C. and then stirred for 55-65 minutes.

[0236] The resulting solution was cooled to -12°C to -8°C and stirred for 80-100 minutes. The resulting mixture was filtered at a temperature in the range of -12°C to -8°C, and DL-α-tocopherol (prepared by dissolving 2.4 mg of DL-α-tocopherol in 5 mL of a solvent mixture) was added to the filtered solution. The resulting solution was concentrated, and the remaining viscous material was dried under vacuum to obtain 7.9 g (0.26 w / w) of egg yolk lecithin.

[0237] Example 3.2: Preparation of egg yolk lecithin without DMC (without step b) and without charcoal treatment (without step e)

[0238] Anhydrous ethanol (136.8 mL) and acetonitrile (103.2 mL) were used to prepare 240.0 mL of a solvent mixture at a temperature in the range of 20-25 °C for use in the process.

[0239] Under stirring, a solution of DL-α-tocopherol (prepared by dissolving 2.4 mg of DL-α-tocopherol in 5 mL of mixed solvent) and SFE-EYP (30 g) was added to 170 mL of the solvent mixture prepared above. The mixture was heated to 55-60° C. and then stirred for 30-40 minutes. The hot mixture was filtered and the resulting solid was washed twice with 60 mL (30 mL×2) of the solvent mixture.

[0240] The resulting solution was cooled to -12°C to -8°C and stirred for 80-100 minutes. The resulting mixture was filtered at a temperature in the range of -12°C to -8°C. The resulting solution was concentrated and the remaining viscous material was dried under vacuum to obtain 8.3 g (0.28 w / w) of egg yolk lecithin.

[0241] Example 3.3: Use of DMC in step b) Preparation of egg yolk lecithin without charcoal treatment (no step e): Stir a solution of DL-α-tocopherol (2.4 mg) in dimethyl carbonate (10 mL), SFE-EYP (30 g), and dimethyl carbonate (170 mL). Heat the mixture to 40-45°C and stir for 55-65 minutes. Filter the mixture and wash the resulting solid with 60 mL (30 mL x 2) of dimethyl carbonate. Dry the solid in vacuo for 4-6 hours.

[0242] Anhydrous ethanol (136.8 mL) and acetonitrile (103.2 mL) were used to prepare 240.0 mL of a solvent mixture at a temperature in the range of 20-25 °C for use in the process.

[0243] 170 mL of the solvent mixture, DL-α-tocopherol solution (prepared by dissolving 2.4 mg of DL-α-tocopherol in 10 mL of the solvent mixture) and the resulting solid were heated to 55-60° C. and then stirred for 30-40 minutes. The hot slurry was filtered and the resulting solid was washed with 60 mL (30 mL×2) of the solvent mixture.

[0244] The resulting solution was cooled to -12°C to -8°C and stirred for 80-100 minutes. The resulting mixture was filtered at a temperature in the range of -12°C to -8°C. The resulting solution was concentrated and the remaining viscous material was dried under vacuum to obtain 8.2 g (0.27 w / w) of purified egg yolk lecithin.

[0245] Example 3.4: Treatment with DMC in step b) and charcoal in step e) Preparation of egg yolk lecithin

[0246] A solution of DL-α-tocopherol (2.4 mg) in dimethyl carbonate (10 mL), SFE-EYP (30 g), and dimethyl carbonate (170 mL) was stirred. The mixture was heated to 40-45°C and then stirred for 55-65 minutes. The mixture was filtered and the resulting solid was washed with 60 mL (30 mL x 2) of dimethyl carbonate. The solid was dried in vacuo for 4-6 hours.

[0247] Anhydrous ethanol (136.8 mL) and acetonitrile (103.2 mL) were used to prepare 240.0 mL of a solvent mixture at a temperature in the range of 20-25 °C for use in the process.

[0248] 170 mL of the solvent mixture, the DL-α-tocopherol solution (prepared by dissolving 2.4 mg of DL-α-tocopherol in 5 mL of the solvent mixture) and the resulting solid were heated to 55-60° C. and then stirred for 30-40 minutes. The hot slurry was filtered and the resulting solid was washed with 60 mL (30 mL×2) of the solvent mixture. The combined filtrates were treated with activated carbon at a temperature in the range of 40-45° C. and stirred for 55-65 minutes.

[0249] The resulting solution was cooled to -12°C to -8°C while stirring. The resulting mixture was filtered at a temperature in the range of -12°C to -8°C, and then a DL-α-tocopherol solution (prepared by dissolving 2.4 mg of DL-α-tocopherol in 5 mL of a solvent mixture) was added to the filtered solution. The resulting solution was concentrated, and the remaining viscous material was dried under vacuum to obtain 8.0 g (0.26 w / w) of purified egg yolk lecithin.

[0250] The effects of these different methods on the purity of the final egg yolk lecithin (i.e., the purified egg yolk lecithin after all the above steps) were analyzed by high performance liquid chromatography coupled with a charged aerosol detector (also known as HPLA-CAD). Details of the analysis are shown in Example 6.

[0251] The results are shown in Table 5. The table lists the contents (% w / w) of phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SPH), and lysophosphatidylcholine (LPC). The achieved yield (in w / w) is also shown, along with the total amount of color-causing impurities absorbed by the UV-visible spectrophotometer at 447 nm. The sample preparation procedure for HPLC-UV analysis is as follows: 40 mg of purified egg yolk lecithin sample was added to a 20 mL volumetric flask, approximately 15 mL of ethanol was added, and the material was vortexed to completely dissolve the material. The resulting solution was cooled to a temperature in the range of 20-25°C. The solution was then diluted to a certain volume with ethanol and mixed thoroughly to obtain a purified egg yolk lecithin sample solution with a concentration of 2 mg / mL (0.2% w / v).

[0252] Table 5:

[0253]

[0254] Treatment with dimethyl carbonate alone or in combination with charcoal significantly reduced UV absorbance at 447 nm, thereby increasing the purity of egg yolk lecithin. Combining DMC with charcoal further improved this result, but at the cost of a 1% reduction in yield.

[0255] Example 4: Comparative Example of Preparing Egg Yolk Lecithin Using Two Different Solvents (DMC, Acetone)

[0256] The procedure described in Example 3.3 was repeated using acetone instead of dimethyl carbonate.

[0257] The yield (w / w ratio of purified egg yolk lecithin to each starting material), PC, and PE (% w / w) obtained from Example 3.2 (without any solvent), Example 3.3 (using DMC), and the same method in step b) using acetone as the solvent were analyzed by HPLC-CAD and UV absorbance at 447 nm. The data are shown in Table 6.

[0258] Sample preparation for HPLC-UV analysis was as follows: 200 mg of purified egg yolk lecithin sample was added to a 10 mL volumetric flask. Approximately 5 mL of ethanol was added and vortexed to completely dissolve the material. The resulting solution was cooled to a temperature between 20 and 25°C. The solution was then diluted to volume with ethanol and mixed to obtain a 20 mg / mL (2% w / v) purified egg yolk lecithin sample solution.

[0259] Table 6:

[0260]

[0261] Results: Compared to egg yolk lecithin obtained with acetone, the egg yolk lecithin obtained in the dimethyl carbonate experiments showed an increase in PC content and a decrease in PE content. Furthermore, the amount of compounds detectable under UV light at 447 nm was significantly reduced when the SFE-EYP was washed prior to the extraction step compared to when it was not washed with any solvent. This reduction was even more pronounced when dimethyl carbonate was used as the solvent.

[0262] Example 5: HPLC-UV analysis of purified egg yolk lecithin

[0263] Purified egg yolk lecithin samples obtained from different methods and commercially available samples were analyzed by HPLC-UV analysis (High Pressure Liquid Chromatography (HPLC) equipped with a UV-Visible Diode Array Detector, wavelength range 200-600 nm, 1 cm sample cell) at an absorbance wavelength of 447 nm to better understand the reduction of impurities eluted from the column. The column type used was a core-shell-(SPP-)-fused core-C18-HPLC column, also known as Express C18 (2.7 μm) HPLC column. Commercially available from, for example, Sigma Aldrich. 250 mg of sample was diluted in 3 mL of ethanol with vigorous shaking and then injected into the column.

[0264] The mobile phase used was mobile phase A, which consisted of a degassed mixture of 700 mL of acetonitrile and 300 mL of water in a 1-L bottle. Mobile phase B consisted of a degassed mixture of 500 mL of MTBE and 500 mL of methanol in a 1-L bottle. Ethanol was used as the diluent. The blank was also ethanol. The chromatographic conditions used were as follows:

[0265]

[0266] Table 2 provides the area under the curve (AUC) values ​​for the UV chromatograms of impurities A, B, and C eluting at retention times of approximately 16.2 (i.e., the retention time of zeaxanthin and lutein in this setup), and 18.66, 19.0, and 19.5, respectively, and is presented again here:

[0267]

[0268]

[0269] Example 6: Determination of purified egg yolk lecithin by HPLC-CAD analysis

[0270] The PC, PE, SPH, LPC, PI, LPE, and cholesterol contents shown in Table 5 were determined by high-performance liquid chromatography (HPLC) equipped with CAD. CAD is used to detect nonvolatile analytes that do not absorb UV light by detecting charged aerosol particles generated upon evaporation of the mobile phase and analytes under the following conditions.

[0271] The mobile phase used was as follows:

[0272] Mobile phase A: Contains acetonitrile.

[0273] Mobile phase B: Prepare by accurately weighing 1.3 g of ammonium formate into a 1 L bottle containing 1000 mL of milli-q water. Ultrasonicate the mixture until homogenized. Adjust the pH to 3.30 by adding formic acid. Filter the solution through 0.22 mm filter paper.

[0274] Mobile phase C: Contains a mixture prepared by accurately transferring 500 mL of methanol and 500 mL of milli-q water into a 1 L bottle, then ultrasonically mixing and degassing.

[0275] Chloroform:methanol at a ratio of 2:1 was used as diluent and blank.

[0276] The chromatographic conditions used were as follows:

[0277] Table 7:

[0278] left pump

[0279]

[0280] Table 8: Right Pump

[0281]

Claims

1. A method for preparing purified egg yolk lecithin, comprising the following steps: a. providing supercritical fluid extracted egg yolk powder as a starting material, b. treating the supercritical fluid extracted egg yolk powder with a solvent, c. separating the product obtained in step b) from the solvent used in step b), d. extracting the egg yolk lecithin fraction from the product obtained in step c), e. Optionally, treating the egg yolk lecithin fraction obtained in step d) with charcoal, f. precipitating impurities from the egg yolk lecithin fraction obtained in step d) or step e), g. removing precipitated impurities from the product obtained in step f), h. separating the purified egg yolk lecithin from the product obtained in step g), and i. optionally, drying the purified egg yolk lecithin, Wherein, in step b), the solvent is dimethyl carbonate.

2. The method according to claim 1, wherein step b), step d), step e) and / or step g) further comprises adding a free radical scavenger.

3. The method according to claim 1, wherein step b), step d), step e) and / or step g) further comprises adding DL-α-tocopherol.

4. The method according to any one of claims 1 to 3, wherein step b) comprises adding the supercritical fluid extracted egg yolk powder to a dimethyl carbonate solution and stirring the resulting mixture at an elevated temperature.

5. The method according to any one of claims 1 to 3, wherein the treatment in step b) comprises adding the supercritical fluid extracted egg yolk powder to a dimethyl carbonate solution and stirring the resulting mixture at a temperature in the range of 30°C to 60°C for a period of 50 to 65 minutes.

6. The method according to any one of claims 1 to 3, wherein in step b), the amount of dimethyl carbonate used is 2 mL to 10 mL per gram of supercritical fluid extracted egg yolk powder.

7. The process according to any one of claims 1 to 3, wherein in step d) the egg yolk lecithin fraction is extracted with one solvent or a mixture of several solvents.

8. The method according to any one of claims 1 to 3, wherein in step e), the charcoal treatment is performed at a temperature in the range of 30°C to 50°C for a time interval of 30 minutes to 3 hours.

9. The process according to any one of claims 1 to 3, wherein in step f), precipitation is achieved by cooling.

10. The process according to any one of claims 1 to 3, wherein in step f), precipitation is achieved by cooling at a temperature in the range of -12°C to -8°C.

11. Purified egg yolk lecithin obtained by the method according to any one of claims 1 to 10.

12. The purified egg yolk lecithin according to claim 11, wherein The UV absorbance values ​​of lutein and zeaxanthin expressed as AUC at 447 nm detected by UV-visible spectrophotometer are lower than the UV absorbance value of egg yolk lecithin prepared by the method according to claim 1 but not involving step b).

13. The purified egg yolk lecithin according to claim 11 or 12, wherein the UV absorbance values ​​of lutein and zeaxanthin are 10 times lower than the UV absorbance values ​​of the egg yolk lecithin prepared by the method according to claim 1 but not involving step b) but adopting step e).

14. The purified egg yolk lecithin according to claim 11 or 12, which does not contain impurities A, B and C, whose elution retention times in HPLC-UV analysis performed at an absorption wavelength of 447 nm are 18.66 minutes, 19.0 minutes and 19.5 minutes, respectively, wherein the relative retention time of lutein is 16.2 minutes.

15. The purified egg yolk lecithin according to claim 11 or 12, wherein the residual amount of dimethyl carbonate is 50 ppm to 100 ppm as detected by GC-MS.

16. The purified egg yolk lecithin according to claim 11 or 12, wherein the residual amount of ketone organic solvents or chlorinated solvents is less than 10 ppm as determined by GC-MS.

17. A lipid emulsion comprising the purified egg yolk lecithin according to any one of claims 11 to 16.

18. A method for preparing a lipid emulsion containing propofol, comprising: a) providing an oil phase comprising soybean oil and propofol; b) providing an aqueous phase comprising water for injection and a pharmaceutically acceptable tonicity agent, oleic acid and / or a pH adjuster; c) forming a pre-emulsion by mixing the oil phase provided in step a) with the water phase provided in step b); d) forming an emulsion by high pressure homogenizing the pre-emulsion obtained in step c); and e) sterilizing the emulsion obtained in step d), wherein the emulsion is optionally filled into suitable containers before or after sterilization, Wherein, the purified egg yolk lecithin according to any one of claims 11 to 16 is added in step a) or step b).

19. The method according to claim 18, wherein the oil phase in step a) further comprises medium chain triglycerides.

20. An oil-in-water emulsion for parenteral administration comprising: 10mg / mL to 20mg / mL propofol, 50mg / mL to 100mg / mL soybean oil, 22.5mg / mL glycerol, 12 mg / mL of the purified egg yolk lecithin according to any one of claims 11 to 16, Optionally, 0.05 mg / mL anhydrous EDTA disodium (equivalent to 0.055 mg EDTA disodium), Sodium hydroxide to adjust pH, and water for injection.

21. The oil-in-water emulsion of claim 20, wherein the composition further comprises medium chain triglycerides in the range of 50 mg / mL to 100 mg / mL.

22. An oil-in-water emulsion for parenteral administration comprising: 10mg / mL to 20mg / mL propofol, 50mg / mL to 100mg / mL soybean oil, 22.5mg / mL glycerol, 12 mg / mL of the purified egg yolk lecithin according to any one of claims 11 to 16, Sodium hydroxide to adjust pH, and water for injection.

23. The oil-in-water emulsion of claim 22, wherein the composition further comprises medium chain triglycerides in the range of 50 mg / mL to 100 mg / mL.

24. An oil-in-water emulsion for parenteral administration comprising: 10.0 mg / mL propofol, 100.0mg / mL soybean oil, 22.5mg / mL glycerol, 12.0 mg / mL of the purified egg yolk lecithin according to any one of claims 11 to 16, oleic acid, Sodium hydroxide to adjust pH, and water for injection.

25. An oil-in-water emulsion for parenteral administration comprising: 20.0 mg / mL propofol, 100.0mg / mL soybean oil, 22.5mg / mL glycerol, 12 mg / mL of the purified egg yolk lecithin according to any one of claims 11 to 16, oleic acid, Sodium hydroxide to adjust pH, and water for injection.

26. An oil-in-water emulsion for parenteral administration comprising: 10.0 mg / mL propofol, 50.0mg / mL soybean oil, 50mg / mL of medium-chain triglycerides, 22.5mg / mL glycerol, 12.0 mg / mL of the purified egg yolk lecithin according to any one of claims 11 to 16, oleic acid, Sodium hydroxide to adjust pH, and water for injection.

27. An oil-in-water emulsion for parenteral administration comprising: 20 mg / mL propofol in water for injection, 50.0mg / mL soybean oil, 50.0mg / mL medium chain triglycerides, 22.5mg / mL glycerol, 12 mg / mL of the purified egg yolk lecithin of any one of claims 11-16, oleic acid, and Sodium hydroxide to adjust pH.

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