Extraction method of plant glycosphingolipid

Abstract

The invention provides a plant glycosphingolipid extraction method, which comprises: taking a plant material, grinding liquid nitrogen, adding a hot ethanol solution (containing beta-mercaptoethanol), carrying out oscillation extraction at a high temperature, carrying out high-temperature high-speed centrifugation, taking the supernatant, carrying out low-temperature precipitation, and carrying out high-speed centrifugation to obtain a crude extract; washing the crude extract with acetone and diethyl ether, removing the organic solvent through high-speed centrifugation, adding a dissolving solution (tetrahydrofuran, methanol and water), carrying out vortex uniform mixing, carrying out high-temperature dissolution, filtering, and carrying out vacuum centrifugal drying on the filtrate to obtain a crude product; adding n-butyl alcohol and water into the crude product, dissolving, precipitating and centrifuging at high temperature, taking an upper layer n-butyl alcohol phase, and performing vacuum centrifugal drying to obtain a target product. The extraction method provided by the invention has the advantages of high extraction efficiency and time saving, and more importantly, small-weight plant material extraction can be effectively realized by virtue of simple equipment, and the sample size required by mass spectrum identification of GIPCs composition and ion chromatography identification of GIPCs glycosyl composition can be obtained.

Description

Technical Field

[0001] The invention relates to the technical field of lipid extraction, and in particular to a method for extracting plant glycosphingolipids. Background Art

[0002] The plant plasma membrane (PM) is an important barrier between cells and the external environment, controlling signal perception and transmission. It is composed of an asymmetric lipid bilayer consisting of three different lipid classes: sphingolipids, sterols, and phospholipids. Glycosyl inositol phosphoryl ceramide (GIPCs) accounts for about 1 / 4 of the total lipids and is the most abundant sphingolipid, showing great structural diversity and playing an important role in signal transduction in the biological responses of plant cells. As GIPCs have attracted more and more attention from molecular biologists in recent years, a small amount and simple extraction method for GIPCs has been put on the agenda. The glycosyl head of GIPCs, unlike ordinary lipids, has stronger amphiphilicity and the extraction process is more complicated. In the past, the extraction of GIPCs required dozens of grams of plant materials and required some extraction equipment and heating devices equipped in chemical laboratories. Not only were the operation steps cumbersome, but it was also easy to reduce the extraction purity of GIPCs. Summary of the invention

[0003] In view of this, in order to facilitate small-scale extraction and identification by biological researchers, the purpose of the present invention is to propose a method for extracting plant glycosphingolipids. The present invention can extract GIPCs for subsequent identification from 1 to 3 g of plant materials. In the extraction method provided by the present invention, liquid nitrogen grinding, oscillation extraction, high-speed centrifugation and vacuum drying are used to replace traditional stirring, gravity extraction, vacuum filtration and nitrogen drying, so that the extraction efficiency is higher and it is convenient for molecular biology laboratory operations and saves time; and in this extraction method, β-mercaptoethanol is added for the first time when 60% to 70% ethanol is used for the initial extraction of GIPCs to prevent lipid oxidation, thereby improving the extraction efficiency of GIPCs; after the ethanol extraction is completed for the first time, high-temperature centrifugation is performed to remove cell debris and insoluble matter, and after the crude GIPCs precipitate is redissolved, it is filtered in a high-temperature environment using a 0.22μm or 0.45μm organic phase filter, thereby greatly improving the extraction purity of GIPCs.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A method for extracting plant glycosphingolipids comprises the following steps:

[0006] S1: Take plant materials, grind them with liquid nitrogen, put them into 50mL centrifuge tubes, add hot ethanol solution with a concentration of 60% to 70% for oscillation extraction, centrifuge at 8000 to 14000rpm, take the supernatant, place it in a -20℃ or -40℃ low-temperature refrigerator for more than 1 hour, and then centrifuge at high speed to obtain a crude extract; the mass volume ratio of the plant material and the ethanol solution is 1 to 3g: 10 to 30mL; if necessary, the extraction can be scaled up in equal proportions.

[0007] S2: washing the crude extract with an organic solvent, discarding the organic solvent after high-speed low-temperature centrifugation, retaining the precipitate, and then standing in a fume hood to volatilize the residual organic solvent, adding a dissolving solution, dissolving the precipitate with high-temperature oscillation, and immediately filtering, taking the filtrate and performing vacuum centrifugation to dry, to obtain a crude product; the organic solvent includes acetone and ether; the dissolving solution includes tetrahydrofuran, methanol and water;

[0008] S3: adding n-butanol and water to the crude product, dissolving the precipitate at high temperature, centrifuging at high speed and high temperature, taking the upper n-butanol phase, and performing vacuum centrifugal drying to obtain the target product.

[0009] The plant glycosphingolipid extraction method provided by the present invention abandons the stirring, filtration, reflux extraction and other equipment used in the previous GIPCs extraction method in terms of the use of instruments and equipment, because these equipment are not suitable for extracting small samples and are often not equipped in molecular biology laboratories. Instead, high-speed centrifuges, ovens, shakers, vortex shakers and small vacuum centrifuges commonly available in molecular biology laboratories are used. In the extraction step, the present invention uses liquid nitrogen grinding instead of crushing plant tissues with a crusher, which is more complete and has a higher extraction efficiency ( Figure 1 A); The previous steps of adding 0.1% acetic acid to break up the plant tissue and then low-temperature vacuum filtration to obtain the precipitate are omitted. The ground plant material powder is put into a centrifuge tube and 60% to 70% hot ethanol (0.8 to 1.2% β-mercaptoethanol is added to prevent oxidation) is directly added, which saves time and is more convenient ( Figure 1 B); extracting GIPCs from plant materials by high temperature oscillation with 60% to 70% ethanol in a sealed centrifuge tube and combining it with high temperature centrifugation to obtain an extract instead of using gravity to extract and precipitate multiple times in a high temperature environment. This not only prevents ethanol evaporation and saves time, but also makes the extraction more complete and avoids insoluble impurities from penetrating the filter cloth into the filtrate ( Figure 1 B, C); using high temperature oscillation of the lysis solution to dissolve the GIPCs crude extract precipitate instead of ultrasonic heating to dissolve the precipitate, the dissolution is more complete and the extraction efficiency is further improved ( Figure 1 H); using a vacuum centrifuge instead of the previous nitrogen dryer to obtain the precipitate, saving time, making the operation easier and the precipitate less likely to be lost ( Figure 1IK), using the extraction method of the present invention, the sample amount required for mass spectrometry identification of GIPCs and ion chromatography identification of GIPCs glycosylation can be extracted from a few grams of sample. In addition, using a large 500mL centrifuge bottle, this method is also suitable for GIPCs extraction of 30 to 40g of a large amount of plant tissue, and the extraction method is flexible and has a wide range of applications.

[0010] A further solution is that in step S1, the plant material includes at least one of leaves, stems or roots, and the plant material includes herbaceous plants, grasses or solanaceae plants.

[0011] A further solution is that in step S1, 0.8% v / v to 1.2% v / v of β-mercaptoethanol is also added to the 60-70% volume concentration ethanol solution; the conditions for the oscillation extraction are: 100-2000 rpm, 50-70°C, 20min to 3h.

[0012] A further solution is that in steps S1 and S3, the high-speed high-temperature centrifugation refers to centrifugation at 50-70°C for 4-10 min; in steps S1 and S2, the high-speed low-temperature centrifugation refers to centrifugation at 2-6°C, 8000-14000 rpm, for 8-20 min.

[0013] A further solution is that in step S2, the acetone or ether is pre-cooled to -20 or -40°C before use, the centrifugation method is: 8000-14000rpm, 2-6°C, 8-20min; the blowing and washing is repeated 1-2 times.

[0014] A further scheme is that in step S2, the dissolving solution includes tetrahydrofuran, methanol and water in a mass ratio of 3 to 4:3 to 4:1, the dissolving solution also includes 0.1% v / v formic acid, and 4 to 12 mL of the dissolving solution is added to each 50 mL centrifuge tube; the dissolving is oscillating dissolving, and the conditions are: 100 to 300 rpm, 50 to 70°C, 1 to 3 hours. The filtering is that after the high-temperature dissolution is completed, the solution is immediately passed through a 0.22 μm or 0.45 μm organic phase filter, and the filtrate 1 mL / tube is obtained and divided into 2 mL centrifuge tubes for subsequent vacuum centrifugation and drying.

[0015] A further solution is that in step S3, 600-800 μL of n-butanol and 600-800 μL of water are added to each 2 mL centrifuge tube; after the precipitate is dissolved at 50-70° C., centrifuge at 50-70° C. and 10,000-14,000 rpm for 4-10 min, and the supernatant is aspirated for vacuum centrifugal drying.

[0016] A further solution is that the vacuum centrifugal drying in steps S2 and S3 refers to placing the 2 mL centrifuge tube containing the GIPCs solution in a vacuum centrifuge with the tube open, maintaining a vacuum degree of 0.05 to 0.1 MPa, and centrifuging at a low speed for 2 to 6 hours to obtain a GIPCs precipitate.

[0017] The present invention provides the glycosphingolipid extracted by the above-mentioned method for extracting plant glycosphingolipids, specifically glycosyl inositol phosphoceramide.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention uses liquid nitrogen to fully grind and combine with an ethanol solution with beta-mercaptoethanol to perform high-temperature oscillation extraction to improve the yield of GIPCs, and removes impurities in the extract through high-temperature centrifugation and high-temperature microporous membrane filtration to improve the purity of GIPCs. The extraction method provided by the present invention is not only simple to operate and has high extraction efficiency, but also can effectively achieve the extraction of a small amount of plant material to obtain the sample amount required for mass spectrometry identification of GIPCs and ion chromatography identification of GIPCs glycosylation composition.

[0020] The present invention develops a simple, easy-to-operate and efficient GIPCs extraction method for a small amount of plant samples. The extraction process is stable. By using the extraction method provided by the present invention, the sample amount required for mass spectrometry, chromatography and the like to identify GIPCs can be obtained in one extraction. The extraction efficiency is high and the extraction time is short. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a flow chart of the extraction steps of plant glycosphingolipids GIPCs of the present invention;

[0022] Figure 2 The primary mass spectrometry identification results of extracted tobacco, wheat and banana GIPCs, m / z, mass-to-charge ratio;

[0023] Figure 3 The secondary mass spectrometry identification results of the main peaks (1261 and 1423) of banana GIPCs extracted in Example 1 of the present invention, m / z, mass-to-charge ratio;

[0024] Figure 4 The ion chromatography identification results of monosaccharides after chemical hydrolysis of banana GIPCs extracted in Example 1 of the present invention. Time, time; Response, response value;

[0025] Figure 5 The traditional method for extracting GIPCs and the steps and primary mass spectrometry identification results, m / z, mass-to-nuclear ratio. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0028] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0029] Example 1

[0030] A method for extracting plant glycosphingolipids, comprising the following steps:

[0031] (1) Take 3 g of healthy banana leaves, grind them into powder with liquid nitrogen, and put them into a 50 mL centrifuge tube.

[0032] (2) Add 30 mL (70% ethanol, 1% β-mercaptoethanol) solution and place in a shaker for 2 hours (conditions: 200 rpm, 60°C)

[0033] (3) Preheat the centrifuge rotor to 60°C and centrifuge (conditions: 12,000 rpm, 5 minutes. If there is a high-temperature centrifuge, it can be set to 60 degrees.)

[0034] (4) Transfer the supernatant into a new 50 mL centrifuge tube and place at -20°C for 2 hours.

[0035] (5) Precool the centrifuge to 4°C, centrifuge (conditions: 12,000 rpm, 4°C, 10 min), and discard the supernatant.

[0036] (6) Wash the precipitate with 15 mL of -20°C precooled acetone by aspiration. Centrifuge at 4°C (12,000 rpm, 4°C, 10 min), discard the supernatant, and repeat the wash once.

[0037] (7) Wash the precipitate by pipetting with 15 mL of -20°C precooled ether, centrifuge at 4°C (12,000 rpm, 4°C, 10 min), discard the supernatant, and repeat the wash process once.

[0038] (8) Let stand in a fume hood for 5 minutes to evaporate the residual ether. Add 12 mL of solvent (tetrahydrofuran: methanol: water = v4: v4: v1 and 0.1% formic acid), vortex to mix. Place in a shaker for 2 hours (conditions: 200 rpm, 60°C).

[0039] (9) After dissolution is complete, filter the mixture immediately using an organic phase filter. Dispense 1 mL of the filtrate into 12 2 mL centrifuge tubes and vacuum centrifuge to obtain the precipitate.

[0040] (10) The GIPCs crude product was precipitated. 700 μL of n-butanol and 700 μL of water were added to each tube and the precipitate was dissolved at 60°C. The centrifuge rotor was preheated at 60°C and centrifuged (conditions: 12,000 rpm, 5 min).

[0041] (11) Immediately transfer the upper n-butanol phase into a new 1.5 mL centrifuge tube while it is still hot, and vacuum centrifuge to dry to obtain approximately 0.6 mg of GIPCs precipitate with a purity of 50%.

[0042] Example 2

[0043] A method for extracting plant glycosphingolipids, comprising the following steps:

[0044] (1) Take 1 gram of tobacco leaves, grind them into powder with liquid nitrogen, and put them into a 50 mL centrifuge tube.

[0045] (2) Add 10 mL of (70% ethanol, 1% β-mercaptoethanol) solution and place in a shaker for 2 hours (conditions: 200 rpm, 60°C).

[0046] (3) Preheat the centrifuge rotor to 60°C and centrifuge (conditions: 12,000 rpm, 5 minutes).

[0047] (4) Transfer the supernatant into a new 50 mL centrifuge tube and place at -20°C for 2 hours.

[0048] (5) Precool the centrifuge to 4°C, centrifuge (conditions: 12,000 rpm, 4°C, 10 min), and discard the supernatant.

[0049] (6) Wash the precipitate with 5 mL of -20°C precooled acetone by aspiration. Centrifuge at 4°C (12,000 rpm, 4°C, 10 min), discard the supernatant, and repeat the wash once.

[0050] (7) Wash the precipitate by pipetting with 5 mL of -20°C precooled ether, centrifuge at 4°C (12,000 rpm, 4°C, 10 min), discard the supernatant, and repeat the wash process once.

[0051] (8) Let the precipitation stand in a fume hood for 5 minutes to evaporate the residual ether. Add 4 mL of dissolving solution (tetrahydrofuran: methanol: water = v4: v4: v1 add 0.1% formic acid), vortex to mix. Place in a shaker for 2 hours (conditions: 200 rpm, 60°C).

[0052] (9) After dissolution is complete, filter the mixture immediately using an organic phase filter. Dispense 1 mL of the filtrate into four 2 mL centrifuge tubes and vacuum centrifuge to obtain the precipitate.

[0053] (10) The GIPCs crude product was precipitated. 700 μL of n-butanol and 700 μL of water were added to each tube and the precipitate was dissolved at 60°C. The centrifuge rotor was preheated at 60°C and centrifuged (conditions: 12,000 rpm, 5 min).

[0054] (11) Immediately transfer the upper n-butanol phase into a new 1.5 mL centrifuge tube while it is still hot and vacuum centrifuge to dry to obtain approximately 0.2 mg of GIPCs precipitate.

[0055] Example 3

[0056] A method for extracting plant glycosphingolipids, comprising the following steps:

[0057] (1) Take 1 gram of wheat leaves, grind them into powder with liquid nitrogen, and put them into a 50 mL centrifuge tube.

[0058] (2) Add 10 mL (65% ethanol, 1% β-mercaptoethanol) solution and place in a shaker for 2 hours (conditions: 200 rpm, 60°C).

[0059] (3) Preheat the centrifuge rotor to 60°C and centrifuge (conditions: 12,000 rpm, 5 minutes).

[0060] (4) Transfer the supernatant into a new 50 mL centrifuge tube and store at -20°C overnight.

[0061] (5) Precool the centrifuge to 4°C, centrifuge (conditions: 12,000 rpm, 4°C, 10 min), and discard the supernatant.

[0062] (6) Wash the precipitate by aspiration with 5 mL of -20°C precooled acetone. Precool the centrifuge rotor to 4°C, centrifuge (conditions: 12,000 rpm, 4°C, 10 minutes), discard the supernatant, and repeat the wash once.

[0063] (7) Wash the precipitate by pipetting with 5 mL of -20°C precooled ether, precool the centrifuge rotor to 4°C, centrifuge (conditions: 12,000 rpm, 4°C, 10 min), discard the supernatant, and repeat the wash once.

[0064] (8) The precipitation was allowed to stand in a fume hood for 5 minutes to evaporate the residual ether. 4 mL of dissolving solution (tetrahydrofuran: methanol: water = v4: v4: v1 plus 0.1% formic acid) was added, and the mixture was vortexed on a multi-hole vortex oscillator at 60°C and 1500 rpm for 30 minutes.

[0065] (9) After dissolution is complete, filter the mixture immediately using an organic phase filter. Dispense 1 mL of the filtrate into four 2 mL centrifuge tubes and vacuum centrifuge to obtain the precipitate.

[0066] (10) The GIPCs crude product was precipitated. 700 μL of n-butanol and 700 μL of water were added to each tube and the precipitate was dissolved at 60°C. The centrifuge rotor was preheated at 60°C and centrifuged (conditions: 12,000 rpm, 5 min).

[0067] (11) Immediately transfer the upper n-butanol phase into a new 1.5 mL centrifuge tube while it is still hot and vacuum centrifuge to dry to obtain 0.2 mg of GIPCs precipitate.

[0068] Comparative Example 1

[0069] The traditional method for extracting GIPCs is:

[0070] Tobacco or Arabidopsis (about 100-200 g fresh weight) was mixed with 400 mL of cold 0.1 N aqueous acetic acid in a refrigerated Waring blender at maximum speed for 2 minutes. The slurry was filtered under vacuum through 16 layers of acid-washed miracloth on a large funnel. The residue was extracted twice again in the same manner. The acetic acid aqueous filtrate was discarded, and the residue was then back-extracted with hot 70% ethanol (70°C) containing 0.1 N HCl. The slurry was hot filtered through miracloth and washed with acidic 70% ethanol. The residue was back-extracted twice more with acidic 70% ethanol. The combined filtrate was immediately cooled and left overnight at -20°C. Precipitation was achieved by centrifugation at 2000g for 15 minutes at 4°C. The GIPC-containing precipitate was washed with cold acetone until the wash solution was colorless and finally washed with cold ether to obtain a white precipitate. GIPCs were then dissolved in tetrahydrofuran / methanol / water (4:4:1, v / v / v) containing 0.1% formic acid by heating at 60°C followed by gentle sonication. The GIPC extract was further dried and subjected to n-butanol / water (1:1, v / v) phase partitioning. This process leaves proteins and cell walls in the lower aqueous phase, while GIPCs are recovered in the upper butanol phase. Finally, the latter phase was dried and the residue was dissolved in tetrahydrofuran / methanol / water (4:4:1, v / v / v) containing 0.1% formic acid. The solution was further used for mass spectrometry analysis. (Buré et al., 2011; Fast screening of highly glycosylated plantphingolipids by tandemmass spectrometry).

[0071] The GIPCs identification results extracted from Comparative Example 1 are as follows Figure 5As shown in A. The extraction methods of GIPCs published in the literature (such as Liu et al., 2020; Lipidomic Analysis Reveals the Importance of GIPCs in Arabidopsis Leaf Extracellular Vesicles; Cassim et al., 2021; Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation) have similar operation processes as those in Comparative Example 1, but a small amount of operation details are missing. After complementing each other, the extraction process is as follows Figure 5 B. The extraction of tobacco GIPCs was performed several times according to the extraction steps of Comparative Example 1, and only a small part of the samples obtained mass spectrometry identification results, such as Figure 5 C shows similar results. Figure 5 The results obtained in A showed a high baseline and a low signal value. In the process of extraction using traditional methods, many problems were also found: the blender is only suitable for a large number of samples, and the crushing effect is not good; during the vacuum filtration process using acid-washed filter cloth, the residue blocks the filter cloth, and the filtration time can be as long as ten hours. The long-term high temperature also causes a large amount of hot alcohol to evaporate, the volume is seriously reduced, and the concentration is even more uncontrollable; the filtrate obtained by filtration is still relatively turbid, resulting in a large amount of impurities in the precipitate obtained after centrifugation, which seriously interferes with the dissolution of GIPCs in the precipitate and easily brings impurities into the further purification process. The identification success rate of GIPCs obtained by the final extraction is not high, the baseline of the successful case is not flat, and the signal response value is relatively low.

[0072] Comparative Example 2

[0073] The main difference between this comparative example and Example 2 is that no β-mercaptoethanol is added and no filtration is performed. The specific steps are as follows:

[0074] (1) Take 3 g of healthy tobacco leaves, grind them into powder with liquid nitrogen, and put them into a 50 mL centrifuge tube.

[0075] (2) Add 30 mL (70% ethanol) solution and place in a shaker for 2 hours (conditions: 200 rpm, 60°C)

[0076] (3) Preheat the centrifuge rotor to 60°C and centrifuge (conditions: 12,000 rpm, 5 minutes.)

[0077] (4) Transfer the supernatant into a new 50 mL centrifuge tube and place at -20°C for 2 hours.

[0078] (5) Precool the centrifuge to 4°C, centrifuge (conditions: 12,000 rpm, 4°C, 10 min), and discard the supernatant.

[0079] (6) Wash the precipitate with 15 mL of -20°C precooled acetone by aspiration. Centrifuge at 4°C (12,000 rpm, 4°C, 10 min), discard the supernatant, and repeat the wash once.

[0080] (7) Wash the precipitate by pipetting with 15 mL of -20°C precooled ether, centrifuge at 4°C (12,000 rpm, 4°C, 10 min), discard the supernatant, and repeat the wash process once.

[0081] (8) Let stand in a fume hood for 5 minutes to evaporate the residual ether. Add 12 mL of solvent (tetrahydrofuran: methanol: water = v4: v4: v1 and 0.1% formic acid), vortex to mix. Place in a shaker for 2 hours (conditions: 200 rpm, 60°C).

[0082] (9) After dissolution, 1 mL / tube was divided into 12 2 mL centrifuge tubes and vacuum centrifuged to obtain the precipitate.

[0083] (10) The GIPCs crude product was precipitated. 700 μL of n-butanol and 700 μL of water were added to each tube and the precipitate was dissolved at 60°C. The centrifuge rotor was preheated at 60°C and centrifuged (conditions: 12,000 rpm, 5 min).

[0084] (11) Immediately transfer the upper n-butanol phase into a new 1.5 mL centrifuge tube while it is still hot and vacuum centrifuge to dry it to obtain GIPCs.

[0085] The GIPCs precipitate obtained by this method is darker in color, and the GIPCs precipitate contains impurities or is oxidized.

[0086] The above is a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for extracting plant glycosphingolipids, characterized in that: The steps include: S1: Take plant materials, grind them with liquid nitrogen, put them into centrifuge tubes, add hot ethanol solution with β-mercaptoethanol, perform high-temperature oscillation extraction, and centrifuge at 8000-14000 rpm to obtain supernatant; place the supernatant at low temperature and then centrifuge at high speed at low temperature to obtain crude extract precipitate; the mass volume ratio of the plant material and the ethanol solution is 1-3 g: 10-30 mL; S2: washing the crude extract with an organic solvent, discarding the organic solvent by high-speed low-temperature centrifugation, adding a dissolving solution and vortexing to mix and precipitate, filtering immediately after high-temperature dissolution, and dispensing the filtrate into centrifuge tubes for vacuum centrifugation and drying to obtain a crude product; the organic solvent includes acetone or ether; the dissolving solution includes tetrahydrofuran, methanol and water; S3: adding n-butanol and water to the crude product, dissolving the precipitate at high temperature, centrifuging at high speed and high temperature, taking the upper n-butanol phase, and performing vacuum centrifugation and drying to obtain the target product.

2. The method for extracting plant glycosphingolipids according to claim 1, characterized in that: In step S1, the plant material includes at least one of leaves, stems or roots, and the plant raw material includes herbaceous plants, grass plants or solanaceae plants.

3. The method for extracting plant glycosphingolipids according to claim 1, characterized in that: In step S1, the concentration of the ethanol solution is 60% v / v to 70% v / v, 0.8% v / v to 1.2% v / v of β-mercaptoethanol is added, and the solution is preheated to 50 to 70° C. before use; the conditions for the oscillation extraction are: 100 to 2000 rpm, 50 to 70° C., and 20 min to 3 h.

4. The method for extracting plant glycosphingolipids according to claim 1, characterized in that: In steps S1 and S3, the high-speed high-temperature centrifugation refers to centrifugation at 50-70° C. and 8000-14000 rpm for 4-10 min.

5. The method for extracting plant glycosphingolipids according to claim 1, characterized in that: In steps S1 and S2, the low-temperature placement refers to placement in a -20 or -40°C refrigerator for at least 1 hour, and the high-speed low-temperature centrifugation refers to centrifugation at 2-6°C and 8000-14000 rpm for 8-20 minutes.

6. The method for extracting plant glycosphingolipids according to claim 1, characterized in that: In step S2, the acetone or ether is pre-cooled to -20 or -40°C before use, and the centrifugation method is: 10000-14000rpm, 2-6°C, 8-20min; each solution is repeatedly blown and washed 1-2 times; the dissolving solution includes tetrahydrofuran, methanol and water in a volume ratio of 3-4:3-4:1, and the dissolving solution also includes 0.1% v / v formic acid.

7. The method for extracting plant glycosphingolipids according to claim 1, characterized in that: In step S2, the high temperature dissolution is high temperature oscillating dissolution, 4 to 12 mL of dissolving solution is added to each 50 mL centrifuge tube, and the dissolution conditions are: 100 to 300 rpm, 50 to 70° C., 1 to 3 h; the filtration is that after the high temperature dissolution is completed, the solution is immediately passed through a 0.22 μm or 0.45 μm organic phase filter to obtain 1 mL / tube of filtrate, which is then divided into 2 mL centrifuge tubes.

8. The method for extracting plant glycosphingolipids according to claim 1, characterized in that: In step S3, 600-800 μL of n-butanol and 600-800 μL of water are added to each 2 mL centrifuge tube; after the precipitate is dissolved at 50-70° C., the upper butanol phase is obtained by centrifugation at 50-70° C. and 10,000-14,000 rpm for 4-10 min.

9. The method for extracting plant glycosphingolipids according to claim 1, characterized in that: In steps S2 and S3, vacuum centrifugal drying means that a 2 mL centrifuge tube containing the GIPCs solution is placed open in a vacuum centrifuge, maintained at a vacuum degree of 0.05 to 0.1 MPa, and centrifuged at a low speed for 2 to 6 hours to obtain a GIPCs precipitate.

10. The glycosphingolipid extracted by the method for extracting plant glycosphingolipids according to any one of claims 1 to 9.

Images