Method for extracting and purifying tetraacetyl phytosphingosine

Abstract

The invention relates to the technical field of chemical purification, and particularly discloses an extraction and purification method of tetraacetyl phytosphingosine. The invention provides an extraction and purification method of tetraacetyl phytosphingosine. The extraction and purification method comprises the following steps: sample treatment, ethanol extraction, C8 chromatography, PA308 chromatography and PS-5 chromatography. Wherein an eluent used in the steps of C8 chromatography and PA308 chromatography is a 50-60v / v% acetonitrile-water solution; an eluent used in the PS-5 chromatography step is an acetonitrile-water solution with the concentration of 65-85v / v%. According to the extraction and purification method of the tetraacetyl phytosphingosine provided by the invention, the tetraacetyl phytosphingosine in thalli can be fully extracted, and the purity of the tetraacetyl phytosphingosine in the obtained tetraacetyl phytosphingosine extract is greater than or equal to 95%.

Description

Technical Field

[0001] The present application relates to the technical field of chemical purification, and in particular to a method for extracting and purifying tetraacetyl phytosphingosine. Background Art

[0002] Tetraacetyl phytosphingosine is a non-ionic surfactant-ester compound with good emulsification, dispersing and lubricating abilities. It also has antiviral and immunity-enhancing functions. It can effectively improve skin aging, wrinkles, pigmentation and other problems, enhance the skin's moisturizing ability and overall health, and is currently widely used in the field of personal care products.

[0003] In order to obtain high-purity tetraacetyl phytosphingosine, a method for extracting tetraacetyl phytosphingosine is reported in the related art. The method first acidifies the microbial fermentation liquid containing tetraacetyl phytosphingosine and then adds a cationic resin for adsorption to achieve preliminary extraction of tetraacetyl phytosphingosine; then the cationic resin is eluted with water and acetic acid solution in turn; finally, the eluate is concentrated and washed under reduced pressure to obtain tetraacetyl phytosphingosine; however, although the above method can achieve the extraction of tetraacetyl phytosphingosine to a certain extent, impurities are easily introduced during the extraction process, resulting in low purity of the final product, and the purity of the final product is only about 90-95%.

[0004] Therefore, how to further improve the purity of tetraacetyl phytosphingosine while ensuring efficient extraction has become a technical problem that needs to be solved urgently. Summary of the invention

[0005] In order to overcome the problem of low product purity in the existing method for extracting and purifying tetraacetyl phytosphingosine, the present application provides a method for extracting and purifying tetraacetyl phytosphingosine.

[0006] In a first aspect, the present application provides a method for extracting and purifying tetraacetyl phytosphingosine, using the following technical solution: A method for extracting and purifying tetraacetyl phytosphingosine, comprising the following steps: sample treatment, ethanol extraction, C8 chromatography, PA308 chromatography and PS-5 chromatography; C8 chromatography: The crude extract I obtained by ethanol extraction is concentrated by rotary evaporation and filtered; then, a chromatographic column filled with C8 is used for column chromatography, and the eluent is 50-60 v / v% acetonitrile-water solution; ultraviolet 200nm monitoring is performed, and the target peak of tetraacetyl phytosphingosine is collected to obtain a crude extract II; PA308 chromatography: the crude extract II is subjected to rotary evaporation; then column chromatography is performed using a chromatographic column filled with PA308, and the eluent is a 50-60 v / v% acetonitrile-water solution; ultraviolet 200 nm monitoring is performed, and the target peak of tetraacetyl phytosphingosine is collected to obtain a crude extract III; PS-5 chromatography: the crude extract III is subjected to rotary evaporation to remove the acetonitrile, ethyl acetate is added for extraction, the ethyl acetate phase is collected and concentrated by rotary evaporation; then column chromatography is performed using a PS-5 chromatographic column, and the eluent is a 65-85 v / v% acetonitrile-water solution; ultraviolet 200 nm monitoring is performed, and the target peak of tetraacetylphytosphingosine is collected to obtain a crude extract IV.

[0007] The present application provides a method for extracting and purifying tetraacetyl phytosphingosine. The method firstly breaks the cell wall by crushing the bacteria and then freeze-drying them in the sample processing stage, thereby fully releasing the tetraacetyl phytosphingosine and laying the foundation for subsequent thorough extraction; then anhydrous ethanol is used for extraction, which can maximize the transfer of tetraacetyl phytosphingosine and dissolve it in anhydrous ethanol; finally, through three chromatography and post-treatment, tetraacetyl phytosphingosine with a purity of ≥98% is obtained. The extraction and purification method provided by the present application comprehensively uses a variety of technical means to achieve efficient and accurate enrichment and purification of tetraacetyl phytosphingosine, and has important application value and development prospects.

[0008] In the chromatography step of the present application, firstly, C8 chromatography is used to accurately separate the relevant active peak distillate of tetraacetyl phytosphingosine with a purity of more than 75% using acetonitrile-water solution as the elution medium; then PA308 chromatography is used to further remove part of the alkaline impurities, thereby reducing the pressure of subsequent purification; finally, PS-5 chromatography is used to use acetonitrile-water solution as the elution medium, so that the purity of tetraacetyl phytosphingosine reaches more than 95%.

[0009] In some embodiments, the eluent in the C8 chromatography and PA308 chromatography steps can be 50-55 v / v% acetonitrile-water solution or 55-60 v / v% acetonitrile-water solution.

[0010] In a specific embodiment, the eluent in the C8 chromatography and PA308 chromatography steps can also be 50 v / v% acetonitrile-water solution, 55 v / v% acetonitrile-water solution or 60 v / v% acetonitrile-water solution.

[0011] In some embodiments, the eluent in the PS-5 chromatography step can be 65-75 v / v% acetonitrile-water solution, 65-80 v / v% acetonitrile-water solution, 65-85 v / v% acetonitrile-water solution, 75-80 v / v% acetonitrile-water solution, 75-85 v / v% acetonitrile-water solution or 80-85 v / v% acetonitrile-water solution.

[0012] In a specific embodiment, the eluent in the PS-5 chromatography step can be a 65 v / v% acetonitrile-water solution, a 75 v / v% acetonitrile-water solution, an 80 v / v% acetonitrile-water solution or an 85 v / v% acetonitrile-water solution.

[0013] Optionally, the flow rate of the eluent in the C8 chromatography step is 8-10 mL / min, and the flow rate of the eluent in the PA308 chromatography step is 3-5 mL / min.

[0014] Optionally, in the PS-5 chromatography step, the flow rate of the eluent in the period from 0 to 38 min is 15 to 25 mL / min, and the flow rate after 38 min is adjusted to 25 to 35 mL / min.

[0015] In the PS-5 chromatography step of the present application, by reducing the flow rate in the early stage of sample loading, it is possible to effectively prevent the high-pressure chromatography column from being over-pressurized and avoid damage to the chromatography column.

[0016] Optionally, the rotary evaporation temperature in the chromatography step is 50±5°C.

[0017] Optionally, the sample processing is to wash, crush and freeze-dry the bacteria to obtain freeze-dried bacterial powder; the pressure during the crushing process is 1000-1500psi, and the temperature for freeze-drying is -80±5℃.

[0018] In some embodiments, the pressure during the crushing process can be 1000-1100 psi, 1000-1200 psi, 1000-1300 psi, 1000-1400 psi, 1000-1500 psi, 1100-1200 psi, 1100-1300 psi, 1100-1400 psi, 1100-1500 psi, 1200-1300 psi, 1200-1400 psi, 1200-1500 psi, 1300-1400 psi, 1300-1500 psi, or 1400-1500 psi.

[0019] In a specific embodiment, the pressure during the crushing process may also be 1000 psi, 1100 psi, 1200 psi, 1300 psi, 1400 psi or 1500 psi.

[0020] Optionally, the pressure during the crushing process is 1200-1400 psi.

[0021] In the present application, the pressure during the crushing process will affect the recovery amount of tetraacetyl phytosphingosine extract. Through experimental exploration, the present application found that further controlling the pressure during the crushing process within the above-mentioned range can reduce energy consumption on the one hand, and on the other hand, can fully extract tetraacetyl phytosphingosine in the bacteria, thereby increasing the yield of tetraacetyl phytosphingosine extract.

[0022] Optionally, the specific steps of the ethanol extraction are: adding anhydrous ethanol to the freeze-dried bacterial powder after sample treatment, the mass volume ratio of the freeze-dried bacterial powder to the anhydrous ethanol is 1: (8-12); then ultrasonic extraction and filtration to obtain a crude extract I.

[0023] In the ethanol extraction step of the present application, by controlling the appropriate mass-to-volume ratio and performing the extraction under ultrasonic conditions, not only the penetration rate of anhydrous ethanol into the bacteria is accelerated, but also the transfer of effective ingredients in the bacteria to anhydrous ethanol is promoted, so that the tetraacetyl phytosphingosine in the bacteria can be completely dissolved; at the same time, the filtration step can also effectively remove insoluble impurities to ensure the quality of the crude extract.

[0024] Optionally, the extraction and purification method also includes post-treatment, the specific steps of which are: subjecting the crude extract IV to rotary evaporation at a temperature of 50±5°C to remove acetonitrile, then adding petroleum ether for extraction, collecting the petroleum ether phase and concentrating it by rotary evaporation at a temperature of 50±5°C; then blowing nitrogen and freezing it at -20±5°C for 2h, and then placing it at 25±5°C for 0.5h to obtain purified tetraacetyl phytosphingosine.

[0025] In a second aspect, the present application provides a tetraacetyl phytosphingosine extract, wherein the purity of tetraacetyl phytosphingosine in the tetraacetyl phytosphingosine extract is ≥95%.

[0026] In summary, this application has the following beneficial effects: The present application provides a method for extracting and purifying tetraacetyl phytosphingosine. The method first obtains a crude tetraacetyl phytosphingosine extract by crushing and freeze-drying the bacterial cells and then extracting them through ethanol; then, by adopting three chromatographic methods, namely, C8 chromatography, PA308 chromatography and PS-5 chromatography, the tetraacetyl phytosphingosine in the bacterial cells is fully extracted, and tetraacetyl phytosphingosine with a purity of ≥95% is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a chromatogram of the effluent after C8 chromatography in Example 1 of the present application; Figure 2is a chromatogram of the effluent after chromatography of PA308 in Example 1 of the present application; Figure 3 is a chromatogram of the effluent after PS-5 chromatography in Example 1 of the present application; Figure 4 This is a high performance liquid chromatogram of the tetraacetyl phytosphingosine obtained in Example 1 of the present application. DETAILED DESCRIPTION

[0028] The present application provides a method for extracting and purifying tetraacetyl phytosphingosine, comprising the following steps: (1) Sample processing: first, add purified water to the frozen bacteria, stir evenly, and then centrifuge at a speed of 7000-10000 r / min for 10-30 min; pour out the supernatant after centrifugation, add purified water again, stir evenly, and then centrifuge at a speed of 7000-10000 r / min for 10-30 min; pour out the supernatant after centrifugation and harvest the washed bacteria; add purified water to the washed bacteria, stir evenly, pour into a high-pressure homogenizer for crushing, the pressure is 1000-1500 psi, repeat three times, add water to wash the bacteria during the process, collect the crushed bacteria, freeze them at -80±5℃ overnight, and then freeze-dry them to obtain freeze-dried bacterial powder; (2) Ethanol extraction: add most of anhydrous ethanol to the freeze-dried bacterial powder, extract by water bath ultrasonication for 3-4 hours, filter with a 0.22 μm filter, and take filtrate 1; wash the residue with a small amount of anhydrous ethanol, and take filtrate 2; combine filtrate 1 and filtrate 2 to obtain crude extract I; wherein the mass volume ratio of the freeze-dried bacterial powder to anhydrous ethanol is 1:(8-12); (3) C8 chromatography: The crude extract I was concentrated by rotary evaporation and filtered using a 0.22 μm filter; then, column chromatography was performed using a chromatographic column filled with C8, with a sample volume of 10 mL, an eluent of 50-60 v / v% acetonitrile-water solution, and a flow rate of 8-10 mL / min; ultraviolet monitoring at 200 nm, and the tetraacetyl phytosphingosine target peak was collected by an automatic collector to obtain a crude extract II; (4) PA308 chromatography: The crude extract II was subjected to rotary evaporation at a temperature of 50±5°C until a white precipitate was about to appear but no white precipitate appeared. The rotary evaporation was then stopped. Then, a chromatographic column filled with PA308 was used for column chromatography. The sample volume was 10 mL. The eluent was 50-60 v / v% acetonitrile-water solution. The flow rate was 3-5 mL / min. The target peak of tetraacetyl phytosphingosine was collected by an automatic collector under ultraviolet light at 200 nm to obtain a crude extract III. (5) PS-5 chromatography: subject the crude extract III to rotary evaporation at a temperature of 50±5°C until the acetonitrile is removed, then add ethyl acetate for extraction, collect the ethyl acetate phase and concentrate it by rotary evaporation; then use a PS-5 chromatographic column for column chromatography, with a sample volume of 10 mL, an eluent of 65-85 v / v% acetonitrile-water solution, a flow rate of 15-25 mL / min from 0 to 38 min, and a flow rate of 25-35 mL / min after 38 min; monitor at 200 nm ultraviolet, and collect the target peak of tetraacetyl phytosphingosine through an automatic collector to obtain a crude extract IV; (6) Post-treatment: The crude extract IV is subjected to rotary evaporation at a temperature of 50±5°C until the acetonitrile is removed, and then petroleum ether is added for extraction. The petroleum ether phase is collected and concentrated by rotary evaporation at a temperature of 50±5°C; then nitrogen is blown and placed in a freezer at -20±5°C for 2-3 hours, and then placed at 25±5°C for 0.5-1 hour to obtain a tetraacetyl phytosphingosine extract.

[0029] In the embodiments of the present application, the frozen cells are obtained by freezing the yeast cells after fermentation and centrifugation; the specifications of the C8 chromatography column are 21.2×250 mm, the filler is UniHybrid8-200 C8, and the particle size is 8 μm; the specifications of the PA308 chromatography column are 16×400 mm, the filler is DIAION PA308, and the particle size is 100 μm; the specifications of the PS-5 chromatography column are 30×250 mm, the filler is UniPS5-300, and the particle size is 5 μm, which is purchased from Suzhou Nanotech; the raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.

[0030] The present application is further described in detail below in combination with embodiments, performance testing and accompanying drawings. Example 1

[0031] Embodiment 1 provides a method for extracting and purifying tetraacetyl phytosphingosine, comprising the following steps: (1) Sample processing: First, add 800 mL of purified water to 510 g of frozen bacteria, stir evenly, and then centrifuge at a speed of 8000 r / min for 20 min; pour out the supernatant after centrifugation, add 800 mL of purified water again, stir evenly, and then centrifuge at a speed of 8000 r / min for 20 min; pour out the supernatant after centrifugation, and harvest 985 g of washed bacteria; add 400 mL of purified water to the washed bacteria, stir evenly, pour into a high-pressure homogenizer for crushing, the pressure is 1300 psi, repeat three times, add water to wash the bacteria during the process, collect 1917 g of crushed bacteria, freeze them at -80±5°C overnight, and then freeze-dry them to obtain 170 g of freeze-dried bacterial powder; (2) Ethanol extraction: add 1500 mL of anhydrous ethanol to 170 g of freeze-dried bacterial powder, perform water bath ultrasonic extraction for 3 h, filter using a 0.22 μm filter, and take filtrate 1; wash the residue with 200 mL of anhydrous ethanol, and take filtrate 2; combine filtrate 1 and filtrate 2 to obtain 1540 mL of crude extract I, containing 2.605 g of tetraacetyl phytosphingosine; (3) C8 chromatography: The crude extract I was concentrated by rotary evaporation and filtered using a 0.22 μm filter; then, column chromatography was performed using a C8-filled column, with a sample volume of 10 mL, an eluent of 55 v / v% acetonitrile-water solution, and a flow rate of 10 mL / min; UV monitoring at 200 nm, and the target peak of tetraacetylphytosphingosine ( Figure 1 66-150min), obtaining 840mL of crude extract II, containing 2.2g of tetraacetyl phytosphingosine; (4) PA308 chromatography: The crude extract II was subjected to rotary evaporation at a temperature of 50°C until a white precipitate was about to appear but no white precipitate appeared. The rotary evaporation was then stopped. Then, a chromatographic column filled with PA308 was used for column chromatography. The sample volume was 10 mL, the eluent was 55 v / v% acetonitrile-water solution, and the flow rate was 4 mL / min. The UV was monitored at 200 nm, and the tetraacetyl phytosphingosine target peak ( Figure 2 6-232min), obtaining 904mL of crude extract III, containing 2.092g of tetraacetyl phytosphingosine; (5) PS-5 chromatography: The crude extract III was subjected to rotary evaporation at a temperature of 50°C until the acetonitrile was removed, and then ethyl acetate was added for extraction. The ethyl acetate phase was collected and concentrated by rotary evaporation; then column chromatography was performed using a PS-5 column, with a sample volume of 10 mL, an eluent of 80 v / v% acetonitrile-water solution, a flow rate of 20 mL / min from 0 to 38 min, and a flow rate of 30 mL / min after 38 min; ultraviolet monitoring at 200 nm, and the tetraacetyl phytosphingosine target peak ( Figure 3 49-66min), obtaining a crude extract IV of 510mL, containing 1.82g of tetraacetylphytosphingosine; (6) Post-treatment: The crude extract IV was subjected to rotary evaporation at 50°C until the acetonitrile was removed, and then petroleum ether was added for extraction. The petroleum ether phase was collected and concentrated by rotary evaporation at 50°C; then nitrogen was blown and frozen at -20°C for 2 h, and then placed at 25°C for 0.5 h to obtain 1.7 g of a waxy semi-solid tetraacetyl phytosphingosine extract. Embodiment 2-8

[0032] Examples 2-8 respectively provide a method for extracting and purifying tetraacetyl phytosphingosine.

[0033] The difference between the above embodiment and embodiment 1 is that the eluents in the C8 chromatography, PA308 chromatography and PS-5 chromatography steps are specifically shown in Table 1 below. Comparative Examples 1-3

[0034] Comparative Examples 1-3 respectively provide a method for extracting and purifying tetraacetyl phytosphingosine.

[0035] The difference between the comparative example and Example 1 is that the eluents in the C8 chromatography, PA308 chromatography and PS-5 chromatography steps are specifically shown in Table 1 below.

[0036] Table 1 Eluents used in the chromatography steps of Examples 2-8 and Comparative Examples 1-3 Examples 9-13

[0037] Examples 9-13 respectively provide a method for extracting and purifying tetraacetyl phytosphingosine.

[0038] The difference between the above embodiment and embodiment 1 is that in the sample processing step, the pressure of the high-pressure homogenizer during the bacterial cell crushing process is specifically shown in Table 2 below.

[0039] Table 2 Pressure of high pressure homogenizer during bacterial cell crushing in the sample processing steps of Examples 9-13 Comparative Example 4

[0040] Comparative Example 4 provides a method for extracting and purifying tetraacetyl phytosphingosine.

[0041] The difference between the above comparative example and Example 1 is that the chromatography step does not include C8 chromatography. Comparative Example 5

[0042] Comparative Example 5 provides a method for extracting and purifying tetraacetyl phytosphingosine.

[0043] The difference between the comparative example and Example 1 is that the chromatography step does not include PA308 chromatography. Comparative Example 6

[0044] Comparative Example 6 provides a method for extracting and purifying tetraacetyl phytosphingosine.

[0045] The difference between the above comparative example and Example 1 is that the chromatography step does not include PS-5 chromatography. Performance testing

[0046] 510 g of frozen bacterial cells were used as raw materials, and extraction and purification were performed according to the methods of Examples 1-13 and Comparative Examples 1-6, respectively. The recovery amount of tetraacetyl phytosphingosine extract obtained by each method was counted, and then the purity of tetraacetyl phytosphingosine in each tetraacetyl phytosphingosine extract was detected by high performance liquid chromatography. The results are shown in Table 3 below. Among them, the high performance liquid chromatogram of the tetraacetyl phytosphingosine extract obtained in Example 1 is as follows Figure 4 shown.

[0047] Table 3 Purity test results of tetraacetyl phytosphingosine extracts obtained by extraction and purification in Examples and Comparative Examples

[0048] According to the test results in Table 3, 510 g of frozen bacterial cells were extracted and purified by the extraction and purification method of tetraacetyl phytosphingosine provided in Examples 1-13, and the recovery amount of the obtained tetraacetyl phytosphingosine extract was 1.56-1.71 g, and the purity of tetraacetyl phytosphingosine in the tetraacetyl phytosphingosine extract was 95.4-99.6%. Therefore, it is explained that C8 chromatography, PA308 chromatography and PS-5 chromatography are used in the chromatography step of the extraction and purification method of the present application, and 50-60 v / v% acetonitrile-water solution is used as the eluent in the C8 chromatography and PA308 chromatography steps, and 65-85 v / v% acetonitrile-water solution is used as the eluent in the PS-5 chromatography step, so that tetraacetyl phytosphingosine with a purity of ≥95% can be obtained.

[0049] In Comparative Example 1, 55 v / v% acetonitrile-water solution was used as the eluent in the C8 chromatography, PA308 chromatography and PS-5 chromatography steps, and the purity of the tetraacetyl phytosphingosine obtained by extraction and purification was only 84.5%.

[0050] In Comparative Example 2, 40 v / v% acetonitrile-water solution was used as the eluent in the C8 chromatography and PA308 chromatography steps, and 80 v / v% acetonitrile-water solution was used as the eluent in the PS-5 chromatography step. The purity of the tetraacetylphytosphingosine obtained by extraction and purification was only 81.3%.

[0051] In Comparative Example 3, 40 v / v% acetonitrile-water solution was used as the eluent in the C8 chromatography and PA308 chromatography steps, and 80 v / v% acetonitrile-water solution was used as the eluent in the PS-5 chromatography step. The purity of the tetraacetylphytosphingosine obtained by extraction and purification was only 86.8%.

[0052] In the chromatography step of Comparative Example 4, only PA308 chromatography and PS-5 chromatography were used, and the purity of tetraacetyl phytosphingosine obtained by extraction and purification was only 81.7%.

[0053] In the chromatography step of Comparative Example 5, only C8 chromatography and PS-5 chromatography were used, and the purity of tetraacetyl phytosphingosine obtained by extraction and purification was only 92.4%.

[0054] In the chromatography step of Comparative Example 6, only C8 chromatography and PA308 chromatography were used, and the purity of tetraacetyl phytosphingosine obtained by extraction and purification was only 86.3%.

[0055] Further comparison of the test results of Example 1 and Examples 9-13 shows that the purity of tetraacetyl phytosphingosine in the tetraacetyl phytosphingosine extract obtained in Examples 1 and 9-13 is 99.4-99.6%; however, the recovery amount of the tetraacetyl phytosphingosine extract obtained in Examples 9-10 is only 1.56-1.59 (<1.6 g), while the recovery amount of the tetraacetyl phytosphingosine extract obtained in Examples 1 and 11-12 is 1.64-1.70 (≥1.6 g). Therefore, it is explained that the crushing pressure during the sample processing process will affect the recovery amount of the tetraacetyl phytosphingosine extract. The present application further controls the pressure of the high-pressure homogenizer in the cell crushing process in the sample processing step within the range of 1200-1400 psi, which can fully crush the cell with less energy consumption, and the tetraacetyl phytosphingosine can be more fully exposed, and the yield of the obtained tetraacetyl phytosphingosine extract is higher.

[0056] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for extracting and purifying tetraacetyl phytosphingosine, characterized in that: The following steps are involved: Sample processing, ethanol extraction, C8 chromatography, PA308 chromatography, and PS-5 chromatography; C8 chromatography: The crude extract I obtained by ethanol extraction is concentrated by rotary evaporation and filtered; then, a chromatographic column filled with C8 is used for column chromatography, and the eluent is 50-60 v / v% acetonitrile-water solution; ultraviolet 200nm monitoring is performed, and the target peak of tetraacetyl phytosphingosine is collected to obtain a crude extract II; PA308 chromatography: the crude extract II is subjected to rotary evaporation; then column chromatography is performed using a chromatographic column filled with PA308, and the eluent is a 50-60 v / v% acetonitrile-water solution; ultraviolet 200 nm monitoring is performed, and the target peak of tetraacetyl phytosphingosine is collected to obtain a crude extract III; PS-5 chromatography: the crude extract III is subjected to rotary evaporation to remove the acetonitrile, ethyl acetate is added for extraction, the ethyl acetate phase is collected and concentrated by rotary evaporation; then column chromatography is performed using a PS-5 chromatographic column, and the eluent is a 65-85 v / v% acetonitrile-water solution; ultraviolet 200 nm monitoring is performed, and the target peak of tetraacetylphytosphingosine is collected to obtain a crude extract IV.

2. The extraction and purification method according to claim 1, characterized in that: The flow rate of the eluent in the C8 chromatography step is 8-10 mL / min, and the flow rate of the eluent in the PA308 chromatography step is 3-5 mL / min.

3. The extraction and purification method according to claim 1, characterized in that: In the PS-5 chromatography step, the flow rate of the eluent in the period from 0 to 38 min was 15 to 25 mL / min, and the flow rate after 38 min was adjusted to 25 to 35 mL / min.

4. The extraction and purification method according to claim 1, characterized in that: The rotary evaporation temperature in the chromatography step is 50±5°C.

5. The extraction and purification method according to claim 1, characterized in that: The sample processing includes washing, crushing and freeze-drying the bacteria to obtain freeze-dried bacterial powder; the pressure during the crushing process is 1000-1500psi, and the freeze-drying temperature is -80±5°C.

6. The extraction and purification method according to claim 5, characterized in that: The pressure during the crushing process is 1200-1400 psi.

7. The extraction and purification method according to claim 1, characterized in that: The specific steps of the ethanol extraction are: adding anhydrous ethanol to the freeze-dried bacterial powder after sample treatment, with the mass volume ratio of the freeze-dried bacterial powder to the anhydrous ethanol being 1:(8-12); then ultrasonic extraction and filtration to obtain a crude extract I.

8. The extraction and purification method according to claim 1, characterized in that: The extraction and purification method also includes post-treatment, which specifically comprises the following steps: subjecting the crude extract IV to rotary evaporation at a temperature of 50±5°C until the acetonitrile is removed, then adding petroleum ether for extraction, collecting the petroleum ether phase and subjecting it to rotary evaporation concentration at a temperature of 50±5°C; then blowing nitrogen and freezing it at -20±5°C for 2h, and then placing it at 25±5°C for 0.5h to obtain purified tetraacetyl phytosphingosine.

9. A tetraacetyl phytosphingosine extract obtained by the method for extracting and purifying tetraacetyl phytosphingosine according to any one of claims 1 to 8, characterized in that: The purity of tetraacetyl phytosphingosine in the tetraacetyl phytosphingosine extract is ≥95%.

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