Method for preparing high-purity phosphatidylinositol

Abstract

The invention belongs to the field of phospholipid processing, and discloses a method for preparing high-purity phosphatidylinositol. The method comprises the following steps: mixing and stirring soybean concentrated phospholipid, phospholipase C and water, carrying out hydrolysis reaction to obtain an enzymatically modified phospholipid mixture, and sequentially carrying out rotary evaporation, vacuum drying and acetone washing treatment to obtain an acetone insoluble substance; the method comprises the following steps: extracting an acetone insoluble substance in a methanol solution of 1-butyl-3-methylimidazolium bromide to obtain phospholipid powder rich in phosphatidylinositol; and carrying out separation, purification and elution on the phospholipid powder rich in phosphatidylinositol by adopting a column chromatography to obtain a high-purity phosphatidylinositol product. The method disclosed by the invention is simple in process, mild in condition, efficient, green and environment-friendly, low in cost, easy to realize industrial production, and capable of preparing the phosphatidylinositol with the purity of more than 95%, and the yield of the phosphatidylinositol is more than 70%.

Description

Technical Field

[0001] The invention belongs to the field of phospholipid processing, and more specifically, relates to a method for preparing high-purity phosphatidylinositol. Background Art

[0002] Phosphatidylinositol (PI) is a glycerol phospholipid compound with an inositol group. The 3rd, 4th, and 5th hydroxyl groups on the PI inositol ring are phosphorylated under the catalysis of corresponding kinases to form a variety of PI derivatives. These substances can be used as signal precursors to produce second messengers. PI can also directly participate in many cell activities, such as transmembrane transport, ion transport, membrane vesicle transport, cell framework, and cell apoptosis. Due to its unique physiological functions and emulsifying properties, phosphatidylinositol (PI) has a wide range of applications in the medical field, food industry, cosmetics industry, etc.

[0003] There are many methods for preparing PI. Currently, the preparation of PI mainly utilizes the difference in polarity between its components to separate and prepare PI, which includes solvent extraction, column chromatography, thin layer chromatography, etc.

[0004] Wang Qiang et al. used soybean powder lecithin as raw material and alkaline ethanol as extraction solvent to prepare high-purity PI. Since the solubility of phosphatidylethanolamine (PE) in alkaline ethanol is significantly higher than that of PI, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in soybean lecithin can be effectively removed by using alkaline ethanol extraction. After filtration, the alcohol insoluble matter was obtained as crude PI, which was then vacuum dried to obtain PI with a purity of 58.86% and a yield of 70.14%. Zhu Meijin et al. used alkaline ethanol as extraction solvent, combined with freezing purification, and determined the optimal extraction conditions of PI by response surface optimization experiment based on single factor experiment: freezing time 17h, freezing temperature -20℃, extraction temperature 40℃, solid-liquid ratio 1:27, 98% ethanol and 25% ammonia volume ratio 100:4, extraction time 40min, extraction times 3 times. Under the optimal conditions, the product PI content was 82.62% and the yield was 14.29%. Although this method can achieve good separation and purification effects, it is time-consuming, consumes a lot of solvent, and is expensive.

[0005] Column chromatography is a commonly used method for separating and purifying high-purity phospholipid products, generally using adsorption column chromatography and ion exchange column chromatography. In order to achieve the best separation effect, column chromatography conditions such as column geometry, filling method, filler particle size, temperature, sample dosage, flow rate, eluent composition and other factors must be selected and controlled. Currently, the methods used to separate PI include silica gel column chromatography, alumina column chromatography, ion exchange column chromatography, etc. The adsorbents selected are generally silica gel, alumina, silicon dioxide, etc., and the eluent is often a mixture of several solvents such as chloroform and lower alcohols. However, the separation time required for column chromatography is relatively long, and the mixed solvent used is consumed a lot and difficult to recover.

[0006] Therefore, the invention of an efficient, green and environmentally friendly method for separating, purifying and preparing phosphatidylinositol has good economic benefits and social value. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing high-purity phosphatidylinositol in view of the deficiencies of the prior art. The method of the present invention has simple process, mild conditions, high efficiency, green environmental protection, low cost, and is easy to realize industrial production. It can prepare phosphatidylinositol with a purity greater than 95%, and its yield is greater than 70%.

[0008] In order to achieve the above object, the present invention provides a method for preparing high-purity phosphatidylinositol, the method comprising the following steps:

[0009] S1: mixing concentrated soybean lecithin, phospholipase C and water, stirring and performing a hydrolysis reaction to obtain an enzyme-modified lecithin mixture;

[0010] S2: subjecting the enzymatically modified phospholipid mixture to rotary evaporation, vacuum drying and acetone washing in sequence to obtain an acetone-insoluble material rich in phosphatidylinositol;

[0011] S3: extracting the acetone insoluble matter rich in phosphatidylinositol in a methanol solution of 1-butyl-3-methylimidazolium bromide ([Emim]Br), centrifuging and vacuum drying to obtain a phosphatidylinositol-rich phospholipid powder;

[0012] S4: Separating, purifying and eluting the phospholipid powder rich in phosphatidylinositol by column chromatography, detecting the eluate by an ultraviolet online detector, collecting the high-purity phosphatidylinositol eluate, and performing low-temperature desolventizing and drying to obtain a high-purity phosphatidylinositol product, wherein:

[0013] The stationary phase of the chromatographic column of the column chromatography is silica gel;

[0014] The mobile phase system of the column chromatography is chloroform-methanol-1-butyl-3-methylimidazole hydroxide ([Bmim]OH) ionic liquid.

[0015] In the present invention, a soybean concentrated phospholipid and phospholipase C mixed system is subjected to a hydrolysis reaction under the condition of adding a large amount of water (mechanical stirring), and phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the soybean concentrated phospholipid are hydrolyzed under the catalytic action of phospholipase C to generate diglycerides and triglycerides, thereby obtaining an enzyme-modified phospholipid mixture (in which phosphatidylinositol is enriched); the enzyme-modified phospholipid mixture is subjected to rotary evaporation and vacuum drying treatment to remove water therein, and acetone washing treatment is used to remove diglycerides and triglycerides in the hydrolyzed phospholipid mixture to obtain acetone-insoluble matter rich in phosphatidylinositol.

[0016] According to the present invention, preferably, the ratio of the concentrated soybean lecithin to water is (1-2): (1-3) g / mL;

[0017] The added mass of the phospholipase C accounts for 1-3% of the mass of the concentrated soybean lecithin.

[0018] According to the present invention, preferably, the temperature of the hydrolysis reaction is 30-60° C. (as a preferred embodiment, the hydrolysis reaction is carried out in a water bath) and the time is 1-5 h.

[0019] According to the present invention, preferably, the concentration of 1-butyl-3-methylimidazolium bromide in the 1-butyl-3-methylimidazolium bromide methanol solution is 0.5-1.5%; in the present invention, the 1-butyl-3-methylimidazolium bromide ([Emim]Br) methanol solution is also an ionic liquid;

[0020] The solid-liquid ratio of the acetone-insoluble matter rich in phosphatidylinositol to the 1-butyl-3-methylimidazolium bromide methanol solution is 1:(4.5-5.5).

[0021] According to the present invention, preferably, the extraction time is 25-35 min, and the extraction temperature is 35-45°C.

[0022] According to the present invention, preferably, in the mobile phase system, the volume ratio of chloroform, methanol and 1-butyl-3-methylimidazolium hydroxide ionic liquid is 1.8:1:(0.05-0.35), more preferably 1.8:1:0.2; the flow rate of the mobile phase system is 8-20 mL / min.

[0023] According to the present invention, preferably, the pore size of the silica gel is The particle size of silica gel is 20-35 μm.

[0024] According to the present invention, preferably, the ratio of the mass of the phosphatidylinositol-rich phospholipid powder loaded onto the chromatographic column to the mass of the stationary phase filled in the chromatographic column is (0.1-10):100.

[0025] According to the present invention, preferably, the detection wavelength of the ultraviolet online detector is 200-205 nm.

[0026] According to the present invention, preferably, the purity of phosphatidylinositol in the high-purity phosphatidylinositol product is greater than 95%, and the yield is greater than 70%.

[0027] The beneficial effects of the technical solution of the present invention are as follows: the method of the present invention has simple process, mild conditions, high efficiency, green environmental protection, low cost, easy to realize industrial production, and can prepare phosphatidylinositol with a purity greater than 95%, and its yield is greater than 70%. The present invention solves the problems of complex production process, long reaction time, low product purity, high cost and the like in the prior art. Specifically:

[0028] (1) The present invention adds a large amount of water to phospholipase C and soybean concentrated phospholipids, which significantly improves the catalytic effect of phospholipase C, fully hydrolyzes phosphatidylcholine (PC) and phosphatidylethanolamine (PE), reduces the difficulty of subsequent purification of PI, and is conducive to obtaining high-purity and high-recovery phosphatidylinositol, which can become an important source of high-purity phosphatidylinositol in the pharmaceutical and health care industry.

[0029] (2) The purity and yield of PI in the present invention are significantly improved, with the purity increasing from 26% to more than 95%, and the yield being higher than 70%.

[0030] (3) The present invention adopts column chromatography for separation and purification, which is a green and environmentally friendly process with strong separation ability, good repeatability, simple and controllable operation, and easy to realize industrial production.

[0031] (4) The present invention uses 1-butyl-3-methylimidazole hydroxide ([Bmim]OH) ionic liquid as a chromatographic column mobile phase additive, which significantly shortens the separation time, has a good peak shape, high column efficiency, and a better separation effect. Ionic liquids are ionic compounds composed of anions and cations, which are liquid at room temperature or near room temperature, and are therefore also called room temperature molten salts. Compared with traditional organic solvents, ionic liquids not only have excellent properties such as low vapor pressure, wide liquid range, high thermal stability, and good solubility, but are also widely used in the fields of organic synthesis, catalysis, and extraction. As an extraction medium, ionic liquids can replace traditional organic solvents, improve the efficiency and selectivity of extraction, and have great advantages and potential in terms of environmental friendliness and recycling.

[0032] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0034] Figure 1 A schematic flow chart of a method for preparing high-purity phosphatidylinositol provided in Example 1 of the present invention is shown. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0036] In the following embodiments:

[0037] Soybean concentrated lecithin was purchased from Shenzhen Siwei Tongchuang Trading Co., Ltd.;

[0038] Phospholipase C was purchased from DSM Biotechnology Co., Ltd.;

[0039] 1-Butyl-3-methylimidazolium bromide ([Emim]Br) methanol solution was purchased from Shanghai Chengjie Chemical Co., Ltd.;

[0040] 1-Butyl-3-methylimidazolium hydroxide ([Bmim]OH) ionic liquid was purchased from Shanghai Chengjie Chemical Co., Ltd.

[0041] Example 1

[0042] This embodiment provides a method for preparing high-purity phosphatidylinositol, such as Figure 1 As shown, the method comprises the following steps:

[0043] S1: 2 g of concentrated soybean phospholipids, phospholipase C and 4 mL of deionized water were mixed and mechanically stirred and hydrolyzed in a water bath at 40° C. for 4 h to obtain an enzyme-modified phospholipid mixture (the enzyme-modified phospholipid mixture includes the following components: phosphatidylinositol, diglyceride and triglyceride);

[0044] The added mass of the phospholipase C accounts for 3% of the mass of the soybean concentrated phospholipids.

[0045] S2: subjecting the enzymatically modified phospholipid mixture to rotary evaporation, vacuum drying and acetone washing in sequence to obtain an acetone-insoluble material rich in phosphatidylinositol;

[0046] The acetone washing treatment was performed three times.

[0047] S3: extracting the acetone insoluble matter rich in phosphatidylinositol in a methanol solution of 1-butyl-3-methylimidazolium bromide at 40° C. for 30 min, centrifuging and vacuum drying to obtain a phosphatidylinositol-rich phospholipid powder (PI content of 51.3 g / 100 g);

[0048] The concentration of 1-butyl-3-methylimidazolium bromide in the 1-butyl-3-methylimidazolium bromide methanol solution is 1.0%; the solid-liquid ratio of the acetone-insoluble matter rich in phosphatidylinositol to the 1-butyl-3-methylimidazolium bromide methanol solution is 1:5.

[0049] S4: Separate, purify and elute the phospholipid powder rich in phosphatidylinositol by column chromatography, detect the eluent by ultraviolet online detector at a wavelength of 203nm, receive the eluent from the time when the target peak emerges until the target peak returns to the baseline position, receive the eluent once every 40mL, and analyze the eluent by liquid phase, combine the eluents to obtain a high-purity phosphatidylinositol eluent, perform low-temperature desolventization and drying, and obtain a high-purity phosphatidylinositol product. Detection by evaporative light-high performance liquid chromatography shows that the purity of phosphatidylinositol in the product is 95.3%, and the yield is 71.6%; wherein:

[0050] The stationary phase of the chromatographic column of the column chromatography is silica gel, and the pore size of the silica gel is The particle size of silica gel is 20-35 μm, and the mass of silica gel is 40 g;

[0051] The mobile phase system (ie, eluent) of the column chromatography is chloroform-methanol-1-butyl-3-methylimidazolium hydroxide ionic liquid, with a volume ratio of 1.8:1:0.2; the flow rate of the mobile phase system is 12 mL / min.

[0052] The ratio of the mass of the phosphatidylinositol-rich phospholipid powder loaded onto the chromatographic column to the mass of the stationary phase filled in the chromatographic column was 1:40.

[0053] Example 2

[0054] This embodiment provides a method for preparing high-purity phosphatidylinositol. The difference between this embodiment and embodiment 1 is only that:

[0055] The mobile phase system (i.e., eluent) of the column chromatography is chloroform-methanol-1-butyl-3-methylimidazolium hydroxide ionic liquid, with a volume ratio of 1.8:1:0.1

[0056] The results of evaporative light-high performance liquid chromatography (ELHPLC) showed that the purity of the phosphatidylinositol in the product was 89.6% and the yield was 68.2%.

[0057] Example 3

[0058] This embodiment provides a method for preparing high-purity phosphatidylinositol. The difference between this embodiment and embodiment 1 is only that:

[0059] The mobile phase system (ie, eluent) of the column chromatography is chloroform-methanol-1-butyl-3-methylimidazolium hydroxide ionic liquid, with a volume ratio of 1.8:1:0.3.

[0060] The results of evaporative light-high performance liquid chromatography (ELHPLC) showed that the purity of the phosphatidylinositol in the product was 87.5% and the yield was 66.9%.

[0061] Comparative Example 1

[0062] This comparative example provides a method for preparing phosphatidylinositol. The difference between this comparative example and Example 1 is that this comparative example only performs steps S1-S3 of Example 1.

[0063] The phosphatidylinositol product obtained in this comparative example was detected by evaporative light-high performance liquid chromatography, and it was found that the purity of the phosphatidylinositol in the phosphatidylinositol product obtained in this comparative example was 52.3%, and the yield was 62%.

[0064] Comparative Example 2

[0065] The only difference from Comparative Example 1 is that after the acetone washing treatment, pure methanol solution is used for extraction. After extraction, a phosphatidylinositol product is obtained. Evaporative light-high performance liquid chromatography is used for detection. It can be seen that the purity of phosphatidylinositol in the phosphatidylinositol product obtained in this comparative example is 29.1%, and the yield is 48.7%.

[0066] Comparative Example 3

[0067] The only difference from Example 1 is that after the acetone washing treatment, pure methanol solution is used for extraction, and a phospholipid powder containing phosphatidylinositol is obtained after extraction, and the PI content in the powder is 27.8g / 100g; then, according to step S4 of Example 1, the phospholipid powder rich in phosphatidylinositol is separated, purified and eluted by column chromatography to obtain a high-purity phosphatidylinositol product, and evaporative light-high performance liquid chromatography is used for detection to show that the purity of phosphatidylinositol in the product is 53.7%, and the yield is 51.4%.

[0068] Comparative Example 4

[0069] The only difference from Example 1 is that chloroform-methanol solution (v / v, 1.8:1) is used as eluent for column chromatography separation, and [Bmim]OH ionic liquid is not added, and the purity and yield of phosphatidylinositol obtained are 56.1% and 54.8%, respectively.

[0070] Table 1

[0071]

[0072]

[0073] In the present invention, phospholipase C is used to catalyze the hydrolysis of PE and PC in concentrated soybean phospholipids, so that PI is enriched and the separation difficulty is reduced. In addition, 1-butyl-3-methylimidazolium bromide ([Emim]Br) methanol solution is used to extract acetone insoluble matter, which significantly improves the separation efficiency.

[0074] In Comparative Example 1, the purity after extraction with 1-butyl-3-methylimidazolium bromide ([Emim]Br) methanol solution was 52.3%, and the yield was 62%;

[0075] On the basis of Comparative Example 1, chloroform-methanol-1-butyl-3-methylimidazolium hydroxide ([Bmim]OH) ionic liquid (v / v, 1.8:1:0.2) was used as the eluent for column chromatography separation (Example 1), and the purity and yield were 95.3% and 71.6%, respectively.

[0076] Compared with Example 1, Comparative Example 2 neither used 1-butyl-3-methylimidazolium bromide ([Emim]Br) methanol solution extraction nor performed column chromatography separation. In Comparative Example 3, 1-butyl-3-methylimidazolium bromide ([Emim]Br) methanol solution extraction was not performed. In Comparative Example 4, 1-butyl-3-methylimidazolium hydroxide ([Bmim]OH) ionic liquid was not added to the eluent. After column chromatography separation, the purity and yield of phosphatidylinositol were significantly lower than those in Experimental Example 1.

[0077] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for preparing high-purity phosphatidylinositol, characterized in that: The method comprises the following steps: S1: mixing concentrated soybean lecithin, phospholipase C and water, stirring and performing a hydrolysis reaction to obtain an enzyme-modified lecithin mixture; S2: subjecting the enzymatically modified phospholipid mixture to rotary evaporation, vacuum drying and acetone washing in sequence to obtain an acetone-insoluble material rich in phosphatidylinositol; S3: extracting the acetone insoluble matter rich in phosphatidylinositol in a methanol solution of 1-butyl-3-methylimidazolium bromide, centrifuging and vacuum drying to obtain a phosphatidylinositol-rich phospholipid powder; S4: Separating, purifying and eluting the phospholipid powder rich in phosphatidylinositol by column chromatography, detecting the eluate by an ultraviolet online detector, collecting the high-purity phosphatidylinositol eluate, and performing low-temperature desolventizing and drying to obtain a high-purity phosphatidylinositol product, wherein: The stationary phase of the chromatographic column of the column chromatography is silica gel; The mobile phase system of the column chromatography is chloroform-methanol-1-butyl-3-methylimidazolium hydroxide ionic liquid.

2. The method for preparing high-purity phosphatidylinositol according to claim 1, wherein: The ratio of the concentrated soybean lecithin to water is (1-2): (1-3) g / mL; The added mass of the phospholipase C accounts for 1-3% of the mass of the concentrated soybean lecithin.

3. The method for preparing high-purity phosphatidylinositol according to claim 1, wherein: The temperature of the hydrolysis reaction is 30-60° C. and the time is 1-5 h.

4. The method for preparing high-purity phosphatidylinositol according to claim 1, wherein: The concentration of 1-butyl-3-methylimidazolium bromide in the 1-butyl-3-methylimidazolium bromide methanol solution is 0.5-1.5%; The solid-liquid ratio of the acetone-insoluble matter rich in phosphatidylinositol to the 1-butyl-3-methylimidazolium bromide methanol solution is 1:(4.5-5.5).

5. The method for preparing high-purity phosphatidylinositol according to claim 1, wherein: The extraction time is 25-35 minutes, and the extraction temperature is 35-45°C.

6. The method for preparing high-purity phosphatidylinositol according to claim 1, wherein: In the mobile phase system, the volume ratio of chloroform, methanol and 1-butyl-3-methylimidazolium hydroxide ionic liquid is 1.8:1:(0.05-0.35); the flow rate of the mobile phase system is 8-20 mL / min.

7. The method for preparing high-purity phosphatidylinositol according to claim 1, wherein: The pore size of the silica gel is The particle size of silica gel is 20-35 μm.

8. The method for preparing high-purity phosphatidylinositol according to claim 1, wherein: The ratio of the mass of the phosphatidylinositol-rich phospholipid powder loaded on the chromatographic column to the mass of the stationary phase filled in the chromatographic column is (0.1-10):

100.

9. The method for preparing high-purity phosphatidylinositol according to claim 1, wherein: The detection wavelength of the ultraviolet online detector is 200-205nm.

10. The method for preparing high-purity phosphatidylinositol according to claim 1, wherein: The purity of the phosphatidylinositol in the high-purity phosphatidylinositol product is greater than 95%, and the yield is greater than 70%.

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