Preparation method of phosphatidylserine

Abstract

The invention belongs to the technical field of medicines, and provides a preparation method of phosphatidylserine, which comprises the following steps: S1, raw material pretreatment; s2, absolute ethyl alcohol extraction; s3, precipitating zinc acetate; s4, extracting with normal hexane; according to the method, a zinc acetate precipitation method is used for the separation and purification process of phosphatidylserine for the first time, and main impurities such as PC and PE in a phospholipid mixture are effectively removed; by adopting an ethanol and n-hexane dual extraction system, PC and PE are thoroughly removed, and impurities such as inositol, phospholipid and the like are removed through a salting-out effect, so that the separation and purification of phosphatidylserine are efficiently realized; repeatedly washing the zinc acetate precipitate with acetone to obtain PC and PE mixed phospholipid with relatively high purity; the method has the advantages of simple operation in the whole preparation process, mild conditions, easy industrial large-scale production, and production cost reduction.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically provides a method for preparing phosphatidylserine. The method effectively achieves high-purity separation and purification of phosphatidylserine through ethanol extraction combined with zinc acetate precipitation technology. Background Art

[0002] Phosphatidylserine (PS), as an important functional phospholipid, has been widely used in health products, food additives and drugs for the treatment of neurodegenerative diseases.

[0003] However, traditional methods for preparing phosphatidylserine mostly use large amounts of organic solvents, such as chloroform and methanol, which pose potential threats to human health and the environment. Preparation often relies on complex, multi-step extraction and purification processes, which are subject to low efficiency, high cost, and the potential introduction of impurities. Especially when extracting from natural raw materials such as animal lung tissue, how to effectively remove other major components in the phospholipid mixture, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), becomes the key to improving PS purity.

[0004] Therefore, those skilled in the art have proposed a method for preparing phosphatidylserine to solve the problems raised in the background art. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing phosphatidylserine, so as to solve the problems of traditional phosphatidylserine preparation methods in the prior art, which mostly rely on complex multi-step extraction and purification processes, have low efficiency, high cost and may introduce impurities.

[0006] A method for preparing phosphatidylserine comprises the following steps:

[0007] S1. Raw material pretreatment: First, select animal tissues rich in phosphatidylserine, then thoroughly wash them to remove surface impurities and blood, then freeze-dry them to remove moisture. The dried freeze-dried tissues are pulverized into powder, sealed in a nitrogen-filled plastic bag, and stored in a -20°C refrigerator for later use.

[0008] S2. Anhydrous ethanol extraction: The pretreated animal tissue powder is mixed with an appropriate amount of anhydrous ethanol, stirred and soaked thoroughly for 30 minutes each time, and the extraction is repeated three times. The good solubility of ethanol in phospholipids is utilized to effectively extract phospholipid compounds from the raw material. The solid-liquid separation is then performed by centrifugation, and the ethanol solution containing phospholipids is collected;

[0009] S3. Zinc acetate precipitation: A 100 mM zinc acetate aqueous solution is added dropwise to the phospholipid ethanol solution and stirred thoroughly. Zinc acetate reacts specifically with impurities such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to form a precipitate that is insoluble in the solvent. The precipitate containing a large amount of impurities is then separated by centrifugation again to obtain a relatively pure phosphatidylserine ethanol solution. The precipitate can then be repeatedly washed with acetone to obtain a high-purity mixed phospholipid of PC and PE.

[0010] S4, n-hexane extraction: After concentrating the phosphatidylserine ethanol solution purified in step S3, n-hexane is added for extraction, followed by adding a certain concentration of metal salt solution. After stirring for 1 minute, the mixture is allowed to stand for separation. The light yellow n-hexane layer is removed and subjected to reduced pressure distillation using a rotary evaporator. The n-hexane is vacuum evaporated to dryness to obtain a high-purity phosphatidylserine product.

[0011] Preferably, in step S1, the animal tissue includes brain, lung, placenta, etc.

[0012] Preferably, in step S2, the material-liquid ratio of the animal tissue powder to an appropriate amount of anhydrous ethanol is 1:10 (weight:volume).

[0013] Preferably, in step S3, the amount of the zinc acetate aqueous solution added is 1 / 10 to 1 / 5 of the total volume of the extract.

[0014] Preferably, in step S4, during the extraction, the volume ratio of n-hexane to the phosphatidylserine ethanol solution is 2:1, and the extraction time is 30-60 minutes.

[0015] Preferably, in step S4, the reduced pressure distillation refers to evaporating n-hexane to dryness in a vacuum using a rotary evaporator.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The present invention is the first to use zinc acetate precipitation method in the separation and purification process of phosphatidylserine, effectively removing the main impurities such as PC and PE in the phospholipid mixture.

[0018] 2. The present invention adopts a dual extraction system of ethanol and n-hexane, which not only completely removes PC and PE, but also removes impurities such as inositol phospholipids through the salting-out effect, thereby efficiently achieving the separation and purification of phosphatidylserine.

[0019] 3. The zinc acetate precipitate of the present invention is repeatedly washed with acetone to obtain a high-purity PC and PE mixed phospholipid.

[0020] 4. The entire preparation process of the present invention is simple to operate, with mild conditions, and is easy to industrialize and scale up, thereby reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flow chart of the preparation method of phosphatidylserine of the present invention; Figure 2 Schematic diagram of the phosphatidylserine passivation treatment of the present invention. DETAILED DESCRIPTION

[0022] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0023] As attached Figure 1 As shown:

[0024] The present invention provides a method for preparing phosphatidylserine, comprising the following steps:

[0025] S1. Raw material pretreatment: First, select animal tissues rich in phosphatidylserine, such as brain, lung, and placenta. Then, thoroughly wash them to remove surface impurities and blood. Then, freeze-dry them to remove moisture. The dried freeze-dried tissues are pulverized into powder, sealed in a nitrogen-filled plastic bag, and stored in a -20°C refrigerator for later use.

[0026] S2. Anhydrous ethanol extraction: The pretreated animal tissue powder is mixed with an appropriate amount of anhydrous ethanol at a solid-liquid ratio of 1:10 (weight:volume). The mixture is thoroughly stirred and soaked for 30 minutes each time. The extraction is repeated three times. The good solubility of ethanol in phospholipids is utilized to effectively extract phospholipid compounds from the raw material. The solid-liquid separation is then performed by centrifugation, and the ethanol solution containing phospholipids is collected.

[0027] S3. Zinc acetate precipitation: A 100 mM zinc acetate aqueous solution is added dropwise to the phospholipid ethanol solution in an amount of 1 / 10-1 / 5 of the total volume of the extract, and the mixture is stirred thoroughly. Zinc acetate reacts specifically with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) impurities to form a precipitate insoluble in the solvent. The precipitate containing a large amount of impurities is then separated by centrifugation again to obtain a relatively pure phosphatidylserine ethanol solution. The precipitate can then be repeatedly washed with acetone to obtain a high-purity mixed phospholipid of PC and PE.

[0028] S4, n-hexane extraction: After concentrating the phosphatidylserine ethanol solution purified in step S3, n-hexane is added for extraction. The volume ratio of n-hexane to the phosphatidylserine ethanol solution is 2:1, and the extraction time is 30-60 minutes. Subsequently, a certain concentration of metal salt solution is added, and the mixture is stirred for 1 minute and allowed to stand for separation. The light yellow n-hexane layer is removed and subjected to reduced pressure distillation using a rotary evaporator. The n-hexane is vacuum evaporated to dryness to obtain a high-purity phosphatidylserine product.

[0029] As can be seen from the above, the present invention has the following beneficial effects:

[0030] 1) The present invention is the first to use zinc acetate precipitation method in the separation and purification process of phosphatidylserine, effectively removing the main impurities such as PC and PE in the phospholipid mixture.

[0031] 2) The dual extraction system of ethanol and n-hexane not only completely removes PC and PE, but also removes impurities such as inositol phospholipids through the salting-out effect, thereby efficiently achieving the separation and purification of phosphatidylserine.

[0032] 3) The zinc acetate precipitate is repeatedly washed with acetone to obtain a high-purity mixed phospholipid of PC and PE.

[0033] 4) The entire preparation process is simple to operate, with mild conditions, and is easy to scale up for industrial production, thereby reducing production costs.

[0034] like Figure 2 As shown, in order to verify the treatment effect of phosphatidylserine purity:

[0035] Samples before purification: Prepare rabbit lungs and extract them with anhydrous ethanol. Centrifuge and collect the supernatant to obtain an anhydrous ethanol extract. Take a portion of the extract and blow dry it with nitrogen, dissolve it in n-hexane, and analyze it by silica gel G thin layer chromatography.

[0036] After purification, the ethanol extract was precipitated with zinc acetate, the supernatant was concentrated appropriately using a rotary evaporator, and finally extracted with n-hexane and evaporated to dryness to obtain the phosphatidylserine sample; a small amount of the sample was dissolved in n-hexane and analyzed by silica gel G thin layer chromatography;

[0037] As can be seen from the above, the intuitive silica gel G thin layer chromatography analysis results show that the purity of phosphatidylserine in the sample before and after purification is significantly improved, thereby verifying the feasibility and effectiveness of the preparation method.

[0038] Example 1: The present invention provides a method for preparing phosphatidylserine, comprising the following steps:

[0039] S1. Raw material pretreatment: First, select animal tissues rich in phosphatidylserine (brain, lung, placenta), then thoroughly wash them to remove surface impurities and blood, then freeze-dry them to remove moisture. The dried freeze-dried tissues are pulverized into powder, sealed in a nitrogen-filled plastic bag, and stored in a -20°C refrigerator for later use.

[0040] S2. Anhydrous ethanol extraction: The pretreated animal tissue powder is mixed with an appropriate amount of anhydrous ethanol at a solid-liquid ratio of 1:10 (weight:volume). The mixture is thoroughly stirred and soaked for 30 minutes each time. The extraction is repeated three times. The good solubility of ethanol in phospholipids is utilized to effectively extract phospholipid compounds from the raw material. The solid-liquid separation is then performed by centrifugation, and the ethanol solution containing phospholipids is collected.

[0041] S3. Zinc acetate precipitation: A 100 mM zinc acetate aqueous solution is added dropwise to the phospholipid ethanol solution in an amount of 1 / 10-1 / 5 of the total volume of the extract, and the mixture is stirred thoroughly. Zinc acetate reacts specifically with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) impurities to form a precipitate insoluble in the solvent. The precipitate containing a large amount of impurities is then separated by centrifugation again to obtain a relatively pure phosphatidylserine ethanol solution. The precipitate can then be repeatedly washed with acetone to obtain a high-purity mixed phospholipid of PC and PE.

[0042] S4, n-hexane extraction: After concentrating the phosphatidylserine ethanol solution purified in step S3, n-hexane is added for extraction. The volume ratio of n-hexane to the phosphatidylserine ethanol solution is 2:1, and the extraction time is 30-60 minutes. Subsequently, a certain concentration of metal salt solution is added, and the mixture is stirred for 1 minute and allowed to stand for separation. The light yellow n-hexane layer is removed and subjected to reduced pressure distillation using a rotary evaporator. The n-hexane is vacuum evaporated to dryness to obtain a high-purity phosphatidylserine product.

[0043] Example 2: The present invention provides a method for preparing phosphatidylserine, comprising the following steps:

[0044] S1. Raw material pretreatment: First, select animal tissues rich in phosphatidylserine (brain, lung, placenta), then thoroughly wash them to remove surface impurities and blood, then freeze-dry them to remove moisture. The dried freeze-dried tissues are pulverized into powder, sealed in a nitrogen-filled plastic bag, and stored in a -20°C refrigerator for later use.

[0045] S2. Anhydrous ethanol extraction: The pretreated animal tissue powder is mixed with an appropriate amount of anhydrous ethanol at a solid-liquid ratio of 1:10 (weight:volume). The mixture is thoroughly stirred and soaked for 30 minutes each time. The extraction is repeated three times. The good solubility of ethanol in phospholipids is utilized to effectively extract phospholipid compounds from the raw material. The solid-liquid separation is then performed by centrifugation, and the ethanol solution containing phospholipids is collected.

[0046] S3. Zinc acetate precipitation: A 100 mM zinc acetate aqueous solution is added dropwise to the phospholipid ethanol solution in an amount of 1 / 10-1 / 5 of the total volume of the extract, and the mixture is stirred thoroughly. Zinc acetate reacts specifically with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) impurities to form a precipitate insoluble in the solvent. The precipitate containing a large amount of impurities is then separated by centrifugation again to obtain a relatively pure phosphatidylserine ethanol solution. The precipitate can then be repeatedly washed with acetone to obtain a high-purity mixed phospholipid of PC and PE.

[0047] S4, n-hexane extraction: After concentrating the phosphatidylserine ethanol solution purified in step S3, n-hexane is added for extraction. The volume ratio of n-hexane to the phosphatidylserine ethanol solution is 2:1, and the extraction time is 30-60 minutes. Subsequently, a certain concentration of metal salt solution is added, and the mixture is stirred for 1 minute and allowed to stand for separation. The light yellow n-hexane layer is removed and subjected to reduced pressure distillation using a rotary evaporator. The n-hexane is vacuum evaporated to dryness to obtain a high-purity phosphatidylserine product.

[0048] Example 3: The present invention provides a method for preparing phosphatidylserine, comprising the following steps:

[0049] S1. Raw material pretreatment: First, select animal tissues rich in phosphatidylserine (brain, lung, placenta), then thoroughly wash them to remove surface impurities and blood, then freeze-dry them to remove moisture. The dried freeze-dried tissues are pulverized into powder, sealed in a nitrogen-filled plastic bag, and stored in a -20°C refrigerator for later use.

[0050] S2. Anhydrous ethanol extraction: The pretreated animal tissue powder is mixed with an appropriate amount of anhydrous ethanol at a solid-liquid ratio of 1:10 (weight:volume). The mixture is thoroughly stirred and soaked for 30 minutes each time. The extraction is repeated three times. The good solubility of ethanol in phospholipids is utilized to effectively extract phospholipid compounds from the raw material. The solid-liquid separation is then performed by centrifugation, and the ethanol solution containing phospholipids is collected.

[0051] S3. Zinc acetate precipitation: A 100 mM zinc acetate aqueous solution is added dropwise to the phospholipid ethanol solution in an amount of 1 / 10-1 / 5 of the total volume of the extract, and the mixture is stirred thoroughly. Zinc acetate reacts specifically with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) impurities to form a precipitate insoluble in the solvent. The precipitate containing a large amount of impurities is then separated by centrifugation again to obtain a relatively pure phosphatidylserine ethanol solution. The precipitate can then be repeatedly washed with acetone to obtain a high-purity mixed phospholipid of PC and PE.

[0052] S4, n-hexane extraction: After concentrating the phosphatidylserine ethanol solution purified in step S3, n-hexane is added for extraction. The volume ratio of n-hexane to the phosphatidylserine ethanol solution is 2:1, and the extraction time is 30-60 minutes. Subsequently, a certain concentration of metal salt solution is added, and the mixture is stirred for 1 minute and allowed to stand for separation. The light yellow n-hexane layer is removed and subjected to reduced pressure distillation using a rotary evaporator. The n-hexane is vacuum evaporated to dryness to obtain a high-purity phosphatidylserine product.

[0053] The beneficial effects of the preparation methods of phosphatidylserine in Example 1, Example 2 and Example 3 are compared with the traditional preparation method of phosphatidylserine, as shown in the following table:

[0054]

[0055] It can be seen from the above table:

[0056] 1) Extraction and Purification Steps: Conventional methods for preparing phosphatidylserine typically rely on complex, multi-step extraction and purification processes, which are cumbersome and prone to the introduction of impurities. However, Examples 1, 2, and 3 of the present invention all employ a preparation method that combines ethanol extraction, zinc acetate precipitation, and n-hexane extraction. This method employs a simple and straightforward process that effectively avoids the introduction of impurities.

[0057] 2) Purity: The phosphatidylserine prepared by conventional methods has a medium purity, which is difficult to meet the demand of the high-end market for high-purity products. However, Examples 1, 2, and 3 of the present invention effectively remove the main impurities such as PC and PE in the phospholipid mixture through zinc acetate precipitation. The resulting phosphatidylserine product has a high purity, meeting market demand.

[0058] 3) Efficiency: Traditional preparation methods are inefficient and require a lot of time and manpower. However, Examples 1, 2, and 3 of the present invention improve preparation efficiency and shorten preparation cycles by optimizing preparation steps and conditions.

[0059] 4) Cost: Traditional preparation methods are expensive, primarily due to complex extraction and purification processes and high raw material costs. However, Examples 1, 2, and 3 of the present invention reduce production costs and improve economic benefits by simplifying the preparation steps and optimizing raw material selection.

[0060] 5) Environmental friendliness: Conventional preparation methods can generate large amounts of waste and pollutants during the preparation process, impacting the environment. However, Examples 1, 2, and 3 of the present invention utilize environmentally friendly solvents and reagents during the preparation process, and the preparation steps are concise and clear, effectively reducing waste generation and pollutant emissions, resulting in a highly environmentally friendly approach.

[0061] 6) Difficulty of industrial production: Traditional preparation methods are difficult to industrialize, mainly due to the complex preparation steps and harsh production conditions. However, the preparation steps of Examples 1, 2, and 3 of the present invention are simple and clear, the conditions are mild and easy to control, and they are easy to scale up for industrial production, reducing the production difficulty and cost.

[0062] In summary, the phosphatidylserine preparation method of the present invention exhibits significant advantages over conventional methods, including high purity, high efficiency, low cost, environmental friendliness, and ease of industrial production. These advantages give the preparation method of the present invention broad application prospects and market value in the fields of medicine, health products, and food.

[0063] The embodiments of the present invention are provided for the purpose of illustration and description. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations of the present invention. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing phosphatidylserine, characterized in that: The following steps are involved: S1. Raw material pretreatment: First, select animal tissues rich in phosphatidylserine, then wash them thoroughly to remove surface impurities and blood, and then freeze-dry them to remove moisture. The dried freeze-dried tissues are crushed into powder, sealed in a plastic bag filled with nitrogen, and stored in a -20°C refrigerator for later use; S2. Anhydrous ethanol extraction: The pretreated animal tissue powder is mixed with an appropriate amount of anhydrous ethanol, and the mixture is fully stirred and soaked for 30 minutes each time, and the extraction is repeated 3 times. The good solubility of ethanol in phospholipids is used to effectively extract phospholipid compounds from the raw materials, and then solid-liquid separation is performed by centrifugation to collect the ethanol solution containing phospholipids; S3, zinc acetate precipitation: adding a 100 mM zinc acetate aqueous solution to the phospholipid ethanol solution and stirring the solution thoroughly; zinc acetate reacts specifically with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) impurities to form a precipitate insoluble in the solvent, and then centrifuging again to separate the precipitate containing a large amount of impurities to obtain a relatively pure phosphatidylserine ethanol solution, and then the precipitate can be further washed repeatedly with acetone to obtain a PC and PE mixed phospholipid with a higher purity; S4, n-hexane extraction: After the phosphatidylserine ethanol solution purified in step S3 is concentrated, n-hexane is added for extraction, and then a certain concentration of metal salt solution is added. After stirring for 1 minute, the layer is allowed to stand for stratification, and the light yellow n-hexane layer is taken out. A rotary evaporator is used for reduced pressure distillation, and the n-hexane is vacuum evaporated to dryness to finally obtain a high-purity phosphatidylserine product.

2. A method for preparing phosphatidylserine as claimed in claim 1, characterized in that: In step S1, the animal tissues include brain, lung, and placenta.

3. A method for preparing phosphatidylserine as claimed in claim 1, characterized in that: In step S2, the material-liquid ratio of the animal tissue powder to an appropriate amount of anhydrous ethanol is 1:

10.

4. A method for preparing phosphatidylserine as claimed in claim 1, characterized in that: In step S3, the amount of the zinc acetate aqueous solution added is 1 / 10 to 1 / 5 of the total volume of the extract.

5. The method for preparing phosphatidylserine according to claim 1, characterized in that: In step S4, during the extraction, the volume ratio of n-hexane to the phosphatidylserine ethanol solution is 2:1, and the extraction time is 30-60 minutes.

6. A method for preparing phosphatidylserine as claimed in claim 1, characterized in that: In step S4, the reduced pressure distillation refers to evaporating n-hexane to dryness under vacuum using a rotary evaporator.

Images