Method for separating and purifying glyceroglycolipid from seaweed

Abstract

The invention provides a method for separating and purifying glyceroglycolipid from seaweed, which comprises the following steps: mixing dry seaweed powder with a methanol solution according to a certain proportion to obtain a crude glyceroglycolipid extract; mixing the glyceroglycolipid crude extract with an extraction solution, and extracting to obtain an extract a; adding a chloroform solution into the extract a, uniformly mixing, adding a methanol solution, and adding a sodium chloride solution into the solution to obtain an extract b; and separating and purifying the extract b by using column chromatography to obtain the glyceroglycolipid. The method disclosed by the invention is simple and convenient to operate and high in purification efficiency, fills the blank of the separation and purification technology of the seaweed glycerol glycolipid, and provides a technical support for the application of the glycerol glycolipid in the fields of medicines, foods, cosmetics and the like.

Description

Technical Field

[0001] The present invention belongs to the field of marine biochemical engineering, and particularly relates to a method for separating and purifying glyceroglycolipids from Durvillaea antarctica. Glyceroglycolipids are natural compounds with important biological activities and are widely used in fields such as pharmaceuticals, foods, and cosmetics. Background Art

[0002] Durvillaea antarctica, also known as noodle algae and Antarctic bull kelp, belongs to the phylum Phaeophyta, specifically classified as class Phaeophyceae, order Gigartinales, family Durvillaeaceae, and is an alga. Durvillaea antarctica is a precious blue food that prefers to grow in cold and nutrient-rich waters, has strong toughness, and can withstand strong winds and waves. Durvillaea antarctica contains various trace elements and bioactive substances beneficial to the human body. Extracting active substances from Durvillaea antarctica will not cause serious environmental impacts and helps promote the development of green chemistry and biotechnology. Durvillaea antarctica is a renewable resource, and through reasonable management and utilization, sustainable development can be achieved, which greatly improves the high-value utilization and development of Durvillaea antarctica.

[0003] Glyceroglycolipids have glycerol as the basic backbone, and glyceroglycolipids are compounds in which sugars are linked to glycerolipids by glycosidic bonds. Due to differences in polar head groups, glyceroglycolipids exhibit different types, such as monogalactosyl diacylglycerol (MGDG), monogalactosylmonoacylglycerol (MGMG), digalactosyl diacylglycerol (DGDG), digalactosylmonoacylglycerol (DGMG), sulfoquinovosyldiacylglycerol (SQDG), and sulfoquinovosylmonoacylglycerol (SQMG), etc. According to the different fatty acyl chains linked to the sn-1 and sn-2 positions of the glycerol backbone, glyceroglycolipids can be further divided into different subclasses.

[0004] Glycolipids mainly exist in plants and microorganisms and are the main components of the thylakoid membranes of plant chloroplasts and the plasma membranes of bacteria, participating in the recognition activities of cell membranes. Glycolipids naturally exist in various seaweeds, including planktonic algae and benthic algae. In these seaweeds, glycolipids are not only components of cell membranes but also show great potential in clinical treatment. Specifically, glycolipids have been found in the following types of seaweeds: brown algae (such as Undaria pinnatifida, Laminaria japonica, etc.), red algae (such as Corallina officinalis, Porphyra yezoensis, etc.), green algae (such as Chlorella vulgaris, Spirulina platensis, etc.), and planktonic algae (such as single-celled algae). The glycolipids in these seaweeds are not only important in biological functions but also have broad application prospects in fields such as scientific research, drug preparation, food and cosmetic R & D due to their unique biological activities, such as antioxidant, antibacterial, antitumor, anti-HIV (human immunodeficiency virus), enhancing the immune function of the body, and other biological activities.

[0005] Currently, the extraction of glycolipids mainly relies on plants and microorganisms, but its low yield and complex purification process limit its wide application. As a renewable resource, the research on the separation and purification of glycolipids from Eisenia bicyclis has not been fully developed. Therefore, it is of great significance to develop an efficient and environmentally friendly method for the separation and purification of glycolipids from Eisenia bicyclis. Based on this, this invention patent uses Eisenia bicyclis as the raw material to establish a method for the separation and purification of glycolipids from Eisenia bicyclis. Summary of the Invention

[0006] The first aspect of the present invention aims to provide a method for separating and purifying glycolipids from Eisenia bicyclis.

[0007] The second aspect of the present invention aims to provide a glycolipid.

[0008] The third aspect of the present invention aims to provide an application of the glycolipid.

[0009] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0010] The first aspect of the present invention provides a method for separating and purifying glycolipids from Eisenia bicyclis, which is characterized by including the following steps:

[0011] (1) Mix the dry powder of Eisenia bicyclis with a methanol solution in a certain proportion to obtain a crude extract of glycolipids;

[0012] (2) Mix the crude extract of glycolipids with an extraction solution for extraction to obtain extract a;

[0013] (3) Add a chloroform solution to extract a, mix well, then add a methanol solution, and then add a sodium chloride solution to the solution to obtain extract b;

[0014] (4) The extract b was separated and purified using column chromatography silica gel to obtain glyceroglycolipid.

[0015] In some embodiments of the present invention, the dried seaweed powder described in step (1) is a powder passed through a 40-mesh sieve.

[0016] In some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 80 - 90%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 80%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 81%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 82%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 83%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 84%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 85%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 86%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 87%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 88%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 89%; in some embodiments of the present invention, the concentration of the methanol solution described in step (1) is 90%.

[0017] In some embodiments of the present invention, the certain ratio is that the solid-liquid ratio of the dried seaweed powder to the methanol aqueous solution is 1:25 - 1:30 g / mL. In some embodiments of the present invention, the certain ratio is that the solid-liquid ratio of the dried seaweed powder to the methanol aqueous solution is 1:26 g / mL; in some embodiments of the present invention, the certain ratio is that the solid-liquid ratio of the dried seaweed powder to the methanol aqueous solution is 1:27 g / mL; in some embodiments of the present invention, the certain ratio is that the solid-liquid ratio of the dried seaweed powder to the methanol aqueous solution is 1:28 g / mL; in some embodiments of the present invention, the certain ratio is that the solid-liquid ratio of the dried seaweed powder to the methanol aqueous solution is 1:29 g / mL; in some embodiments of the present invention, the certain ratio is that the solid-liquid ratio of the dried seaweed powder to the methanol aqueous solution is 1:30 g / mL.

[0018] In some embodiments of the present invention, the dried seaweed powder described in step (1) is added to an 87% methanol aqueous solution at a solid-liquid ratio of 1:30 g / mL, and extracted by shaking in a water bath at 50 °C for 150 min. The extract is subjected to freeze centrifugation at a rotation speed of 8000 r / min for 10 min, and the supernatant is taken and dried after reduced pressure evaporation to obtain a crude extract of glyceroglycolipid;

[0019] In some embodiments of the present invention, the extraction solution described in step (2) is chloroform:methanol (2:1, v / v).

[0020] In some embodiments of the present invention, the solid-liquid ratio of the crude glyceroglycolipid extract to the extraction solution in step (2) is 1:10 g / mL.

[0021] In some embodiments of the present invention, the volume ratio of the chloroform solution to the methanol solution in step (3) is 1.5:1 - 2:1. In some embodiments of the present invention, the volume ratio of the chloroform solution to the methanol solution in step (3) is 1.5:1; in some embodiments of the present invention, the volume ratio of the chloroform solution to the methanol solution in step (3) is 1.6:1; in some embodiments of the present invention, the volume ratio of the chloroform solution to the methanol solution in step (3) is 1.7:1; in some embodiments of the present invention, the volume ratio of the chloroform solution to the methanol solution in step (3) is 1.8:1; in some embodiments of the present invention, the volume ratio of the chloroform solution to the methanol solution in step (3) is 1.9:1; in some embodiments of the present invention, the volume ratio of the chloroform solution to the methanol solution in step (3) is 2.0:1.

[0022] In some embodiments of the present invention, the volume ratio of the extract a to the chloroform solution in step (3) is 1:15; the concentration of the sodium chloride solution is 0.9%.

[0023] In some embodiments of the present invention, the column chromatography silica gel described in step (4) is 200 - 300 mesh.

[0024] In some embodiments of the present invention, the extract b described in step (4) is dissolved in a chloroform solution and passed through column chromatography silica gel; dichloromethane solution and methanol solution are used as eluents in turn in a mixed solvent with ratios of 96:4, 90:10, 70:30, 55:45, and 30:70 to separate the glyceroglycolipid solution. Each tube of elution fraction is analyzed for glycolipid by high performance liquid chromatography, and samples with the same retention time are enriched and dried under reduced pressure to obtain glyceroglycolipid.

[0025] An object of the second aspect of the present invention is to provide a glyceroglycolipid.

[0026] In some embodiments of the present invention, the glycoglycerolipid is isolated and purified from Eisenia bicyclis.

[0027] In some embodiments of the present invention, the method for isolating and purifying the glycoglycerolipid from Eisenia bicyclis is as described above.

[0028] The object of the third aspect of the present invention is to provide an application of a glycoglycerolipid.

[0029] In some embodiments of the present invention, the glycoglycerolipid can be used in pharmaceuticals, foods or cosmetics.

[0030] In some embodiments of the present invention, the obtained glycoglycerolipid is analyzed by high performance liquid chromatography, and the retention times of the elution peaks obtained are 6.617 min and 10.963 min, and the maximum absorption peak wavelengths are 202.15 nm and 266.65 nm respectively, and the absorption peak of the standard product is at 204.12 nm. The absorption peak of the glycoglycerolipid is between 201 nm and 210 nm, so the glycoglycerolipid meets the ultraviolet absorption spectral characteristics of the glycoglycerolipid.

[0031] In some embodiments of the present invention, the obtained glycoglycerolipid is analyzed by infrared spectroscopy, and it is found that there are absorption peaks in the ranges of 3600 - 3200 cm -1 、2925 - 2850 cm -1 、1740 cm -1 、1400 - 1000 cm -1 、1240 - 1020 cm -1 which can prove the existence of the glycoglycerolipid.

[0032] The dried and pre-treated seaweed velvet powder (passed through a 40-mesh sieve) of the present invention was added to an 87% methanol aqueous solution at a solid-liquid ratio of 1:30 g / mL, and extracted at 50 °C for 150 min. The extraction was repeated twice, and the extracts were combined. After centrifugation at 8000 rmp for 10 minutes at room temperature, the supernatant was taken. The supernatant was evaporated under reduced pressure at 45 °C and then dried in an oven at 45 °C to prepare a crude extract of glyceroglycolipid. The crude extract was mixed with a chloroform / methanol mixture (2:1, v / v), and the solid-liquid ratio was 1:10. The mixture was placed in a magnetic stirrer and magnetically stirred for 8 h at a stirring temperature of 45 °C. The mixture was collected and evaporated to dryness under reduced pressure, and then dried in an oven at 45 °C to obtain extract a. Extract a was added to chloroform at a solid-liquid ratio of 1:15, mixed well, and then methanol was added. The chloroform / methanol mixture was (2:1, v / v). Then, 0.9% NaCl solution was added to the solution. After shaking well and standing for 30 min, extraction was carried out using a separating funnel. After centrifugation, it was concentrated by a rotary evaporator under vacuum to obtain extract b. Extract b was dissolved in chloroform to prepare a concentration of 400 mg / mL, and passed through a 0.22-μm organic membrane. Using refined column chromatography silica gel of 200-300 mesh (Silica gel for C.C) as the packing material, a mixed solvent of dichloromethane and methanol in the ratios of 96:4, 90:10, 70:30, 55:45, and 30:70 in turn was used as the eluent to separate the glyceroglycolipid solution. After each eluent eluted 2 column volumes, the next eluent was used. The elution fractions of each tube were analyzed for glycolipids by high-performance liquid chromatography. Samples with the same retention time were enriched and evaporated to dryness under reduced pressure to obtain glyceroglycolipid. The glyceroglycolipid was redissolved in ethanol. By high-performance liquid chromatography, the retention times of the main elution peaks of glyceroglycolipid were 6.617 min and 10.963 min, and the maximum absorption peak wavelengths were 202.15 nm and 266.65 nm respectively. The absorption peak of the standard product was at 204.12 nm. The absorption peak of glyceroglycolipid was between 201 nm and 210 nm. Therefore, glyceroglycolipid meets the ultraviolet absorption spectral characteristics of glyceroglycolipid. Thin-layer chromatography and infrared spectroscopy detection showed that the sample presented the characteristics of glyceroglycolipid.

[0033] The present invention discloses a method for extracting glyceroglycolipid from Durvillaea antarctica for the first time, filling the blank in the separation and purification technology of glyceroglycolipid from Durvillaea antarctica. As a renewable resource, the extraction of glyceroglycolipid from Durvillaea antarctica not only provides a new raw material source for the research of glyceroglycolipid, but also promotes the high-value utilization and development of Durvillaea antarctica. A method for separating and purifying glyceroglycolipid from Durvillaea antarctica is provided, which has the advantages of simple operation and high purification efficiency. Through high-performance liquid chromatography, thin-layer chromatography and infrared spectroscopy analysis, the characteristic absorption peaks and spectral characteristics of glyceroglycolipid were confirmed. The present invention fills the blank in the separation and purification technology of glyceroglycolipid from Durvillaea antarctica, providing technical support for the application of glyceroglycolipid in the fields of medicine, food and cosmetics, etc. Description of the Drawings

[0034] Figure 1 : Figure 1 a is the high performance liquid chromatography (HPLC) chromatogram of glyceroglycolipid; Figure 1 b is the integration result of the HPLC chromatogram of glyceroglycolipid

[0035] Figure 2 : Figure 2 a is the HPLC chromatogram of the reference standard; Figure 2 b is the integration result of the HPLC chromatogram of the reference standard

[0036] Figure 3 : TLC detection result of glyceroglycolipid

[0037] Figure 4 : IR spectrum detection result of glyceroglycolipid

[0038] Figure 5 : Figure 5 a is the HPLC chromatogram of the glyceroglycolipid in Example 2; Figure 5 b is the integration result of the HPLC chromatogram of the glyceroglycolipid in Example 2

[0039] Figure 6 : Figure 6 a is the HPLC chromatogram of the reference standard; Figure 6 b is the integration result of the HPLC chromatogram of the reference standard {same as Figure 2}

[0040] Figure 7 : TLC detection result of the glyceroglycolipid in Example 2

[0041] Figure 8 : IR spectrum detection result of the glyceroglycolipid in Example 2

[0042] Figure 9 : Figure 9 a is the HPLC chromatogram of the glyceroglycolipid in Example 3; Figure 9 b is the integration result of the HPLC chromatogram of the glyceroglycolipid in Example 3

[0043] Figure 10 : Figure 10 a is the HPLC chromatogram of the reference standard; Figure 10 b is the integration result of the HPLC chromatogram of the reference standard {same as Figure 2}

[0044] Figure 11 : TLC detection result of the glyceroglycolipid in Example 3

[0045] Figure 12 : IR spectrum detection result of the glyceroglycolipid in Example 3

[0046] Detailed Implementation Modes (Examples)

[0047] To enable those skilled in the art to better understand the solutions of this application, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application.

[0048] It should be noted that the terms "include" and "have" in the specification and claims of this application and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units does not necessarily have to be limited to those clearly listed steps or units, but may include other steps or units that are not clearly listed or are inherent to these processes, methods, products, or devices.

[0049] The following are some definitions in the present invention:

[0050] Extract: The extract refers to a mixture containing glyceroglycolipids separated from dried seaweed powder through the extraction process.

[0051] Extraction solution: The extraction solution refers to the solvent or solvent mixture used in the extraction process. In the present invention, chloroform and methanol solution are used as the extraction solution, which can effectively extract glyceroglycolipids from seaweed.

[0052] Column chromatography: Column chromatography is a technique for separating and purifying glyceroglycolipids. In the present invention, silica gel is used as the stationary phase, and a mixed solvent of dichloromethane and methanol is used as the mobile phase to separate glyceroglycolipids.

[0053] Column chromatography silica gel: Column chromatography silica gel is a porous silica particle with a particle size range of 200 - 300 mesh. Its surface characteristics can effectively separate glyceroglycolipids from other impurities.

[0054] Thin layer chromatography: Thin layer chromatography is used to detect the separation effect of glyceroglycolipids. In the present invention, the sample is spotted on a silica gel plate, and the solvent is used as the mobile phase to observe the separation of glyceroglycolipids.

[0055] Mesh number: The mesh number refers to the number of holes per inch length of the sieve mesh, which is used to represent the aperture size of the sieve mesh. The larger the mesh number, the finer the particles screened.

[0056] 40 mesh: 40 mesh refers to the aperture size of the sieve mesh, approximately 0.425 mm. In the present invention, the dried seaweed powder needs to pass through a 40-mesh sieve to ensure that the particle size meets the extraction requirements.

[0057] Solid-liquid ratio: The solid-liquid ratio refers to the mass or volume ratio of the raw material to the solvent. In the present invention, a solid-liquid ratio of 1:30 g / mL means that 30 mL of an 87% methanol aqueous solution is used per 1 gram of dried seaweed powder.

[0058] Standard substance: A standard substance refers to a substance with known composition, purity, and characteristics, which is used as a qualitative or quantitative reference in experimental analysis. Standard substances are usually used as reference substances to verify the characteristics of the target substance (such as retention time, absorption wavelength, spectral characteristics, etc.) or to calibrate instruments.

[0059] Dried seaweed powder: The dried seaweed powder described in the present invention refers to a powdery substance obtained by processing seaweed (Durvillaea antarctica) as raw material through washing, drying, and pulverizing. The dried seaweed powder is not limited to any specific method or approach for obtaining, including but not limited to the following methods:

[0060] Source of raw material: Seaweed can be sourced from natural collection or artificial cultivation and grows in cold and nutrient-rich waters.

[0061] Drying method: Drying can be carried out by natural air drying, hot air drying, freeze drying, or other drying techniques.

[0062] Pulverizing method: Pulverizing can be achieved by mechanical pulverization, grinding, or other pulverization techniques, and the particle size range of the powder is not limited to a specific mesh size (such as 40 mesh).

[0063] Component characteristics: The dried seaweed powder contains various bioactive components such as glyceroglycolipids, polysaccharides, proteins, and trace elements.

[0064] The following further analyzes the present invention in conjunction with specific examples.

[0065] Example 1

[0066] Place 1000 g of dried seaweed powder that has passed through a 40-mesh sieve into 30 L of an 87% methanol aqueous solution (at a solid-liquid ratio of 1:30 g / mL), and perform water bath shaking extraction at 50 °C for 180 min. Repeat this step twice and collect all the extracts. Subsequently, centrifuge the extract at 8000 r / min for 10 minutes, take the supernatant, filter the supernatant through a vacuum pump, concentrate the filtered supernatant by reduced pressure evaporation at 45 °C, pour the concentrated solution into a petri dish and dry it in an oven at 45 °C to prepare 187.6 g of a crude glyceroglycolipid extract. Mix 187.6 g of the crude extract with a chloroform / methanol mixture (2:1, v / v) in a 1000 mL Erlenmeyer flask, with a solid-liquid ratio of 1:10 g / mL. Place the Erlenmeyer flask on a magnetic stirrer and stir for 8 h at a stirring temperature of 45 °C. Subsequently, filter the mixture and collect it, evaporate it to dryness under reduced pressure, and dry it in an oven at 45 °C to obtain extract a. Add chloroform to extract a at a solid-liquid ratio of 1:15 g / mL, mix well and then add methanol, where the chloroform / methanol mixture is (2:1, v / v). Then add 0.9% NaCl solution to the solution, shake it well and let it stand for 30 min, perform extraction with a separatory funnel, and concentrate it with a rotary evaporator under vacuum after centrifugation to obtain extract b. Take 2.5 g of extract b, dissolve it in chloroform, prepare a concentration of 400 mg / mL, filter it through a 0.22 μm organic membrane, and load it onto a column chromatography using refined column chromatography silica gel 200 - 300 mesh as the packing (Silica gel for C.C) for purification. Use a mixed solvent of dichloromethane and methanol in the ratios of 96:4, 90:10, 70:30, 55:45, and 30:70 in turn as the eluent to separate the glyceroglycolipid solution. After each eluent elutes 2 column volumes, change to the next eluent. Analyze the glycolipids in each elution fraction by high performance liquid chromatography, enrich the samples with the same retention time and evaporate them to dryness under reduced pressure to obtain glyceroglycolipids. Redissolve the glyceroglycolipids in ethanol, and by high performance liquid chromatography, the retention time of the main elution peak of the glyceroglycolipids is 6.617 min, the relative peak area of the maximum absorption wavelength reaches 94.32%, and the maximum absorption peak wavelength is 203.69 nm( Figure 1 ), and the absorption peak of the standard product is at 204.12 nm( Figure 2 ). The absorption peak of the glyceroglycolipids is between 201 nm and 210 nm, so the glyceroglycolipids meet the ultraviolet absorption spectral characteristics of glyceroglycolipids. After redissolving the glyceroglycolipids in ethanol, perform thin layer chromatography development, and it is found that the target substance appears on the chromatography plate( Figure 3 ). At the same time, perform infrared spectroscopy analysis on the dried glyceroglycolipids, and it is found that it has absorption at 722.56 cm -1 , 1077.62 cm -1 , 1377.7 cm -1 , 1734 cm -1, 2951.45 cm -1 , 2923.99 cm -1 , 3420.13 cm -1 There are absorption peaks at these positions, which can prove the existence of glyceroglycolipids ( Figure 4 ).

[0067] Example 2

[0068] Place 300 g of dried and 40-mesh sieved seaweed powder in 15 L of 87% methanol aqueous solution (at a solid-liquid ratio of 1:30 g / mL), and extract it by shaking in a water bath at 50 °C for 180 min. Repeat this step twice and collect all the extracts. Subsequently, centrifuge the extract at 8000 r / min for 10 minutes, take the supernatant, filter the supernatant through a vacuum pump, concentrate the filtered supernatant by vacuum evaporation at 45 °C, pour the concentrated solution into a petri dish and dry it in an oven at 45 °C to prepare 56.1 g of crude glyceroglycolipid extract. Mix 56.1 g of the crude extract with a chloroform / methanol mixture (2:1, v / v) in a 1000 mL Erlenmeyer flask, and the solid-liquid ratio is 1:10 g / mL. Place the Erlenmeyer flask on a magnetic stirrer and stir for 8 h at a stirring temperature of 45 °C. Then filter and collect the mixed solution and evaporate it to dryness under reduced pressure, and dry it in an oven at 45 °C to obtain extract a. Add chloroform to extract a according to a solid-liquid ratio of 1:15 g / mL, mix well and then add methanol, where the chloroform / methanol mixture is (2:1, v / v). Then add 0.9% NaCl solution to the solution, shake it well and let it stand for 30 min, extract it with a separating funnel, and concentrate it with a rotary evaporator under vacuum after centrifugation to obtain extract b. Take 5 g of extract b and dissolve it in chloroform to prepare a concentration of 400 mg / mL, filter it through a 0.22 μm organic membrane, and load it onto a column chromatography using refined column chromatography silica gel 200 - 300 mesh as the packing (Silicagel for C.C) for purification. Use a mixed solvent of dichloromethane and methanol in the ratios of 96:4, 90:10, 70:30, 55:45, and 30:70 in turn as the eluent to separate the glyceroglycolipid solution. After each eluent elutes 2 column volumes, change to the next eluent. Analyze the glycolipids in each eluted fraction by high performance liquid chromatography, enrich and evaporate to dryness under reduced pressure the samples with the same retention time to obtain glyceroglycolipids. Redissolve the glyceroglycolipids in ethanol, and by high performance liquid chromatography, the retention time of the main elution peak of the glyceroglycolipids is 6.617 min, the relative peak area of the maximum absorption wavelength reaches 95.04%, and the maximum absorption peak wavelength is 204.09 nm ( Figure 5 ), and the absorption peak of the standard product is at 204.12 nm ( Figure 6)。The absorption peak of glyceroglycolipid is between 201 nm and 210 nm, so glyceroglycolipid meets the ultraviolet absorption spectral characteristics of glyceroglycolipid. After redissolving glyceroglycolipid with ethanol, thin-layer chromatography was carried out, and it was found that the target substance appeared on the chromatographic plate ( Figure 7 )。At the same time, infrared spectroscopy analysis was carried out on the dried glyceroglycolipid, and it was found that there were absorption peaks at 6672.61 cm -1 , 1079.21 cm -1 , 1377.60 cm -1 , 1735.53 cm -1 , 2854.44 cm -1 , 2922.65 cm -1 , 3425.44 cm -1 , which can prove the existence of glyceroglycolipid ( Figure 8 )。

[0069] Example 3

[0070] Place 1000 g of dried seaweed powder that has passed through a 40-mesh sieve into 30 L of an 87% methanol aqueous solution (at a solid-liquid ratio of 1:30 g / mL), and perform water bath shaking extraction at 50 °C for 180 min. Repeat this step twice and collect all the extracts. Subsequently, centrifuge the extract at 8000 r / min for 10 minutes, take the supernatant, filter the supernatant through a vacuum pump, concentrate the filtered supernatant by reduced pressure evaporation at 45 °C, pour the concentrated solution into a petri dish and dry it in an oven at 45 °C to prepare 187.6 g of crude glyceroglycolipid extract. Mix 187.6 g of the crude extract with a chloroform / methanol mixture (2:1, v / v) in a 1000 mL Erlenmeyer flask, with a solid-liquid ratio of 1:10 g / mL. Place the Erlenmeyer flask on a magnetic stirrer and stir for 8 h at a stirring temperature of 45 °C. Subsequently, filter and collect the mixed solution, evaporate it to dryness under reduced pressure, and dry it in an oven at 45 °C to obtain extract a. Add chloroform to extract a at a material-liquid ratio of 1:15 g / mL, mix well and then add methanol, where the chloroform / methanol mixture is (2:1, v / v). Then add 0.9% NaCl solution to the solution, shake it well and let it stand for 30 min, perform extraction with a separatory funnel, and concentrate it with a rotary evaporator under vacuum after centrifugation to obtain extract b. Take 7.5 g of extract b, dissolve it in chloroform, prepare a concentration of 400 mg / mL, filter it through a 0.22 μm organic membrane, and load it onto a column chromatography using refined column chromatography silica gel 200 - 300 mesh as the packing (Silicagel for C.C) for purification. Use a mixed solvent of dichloromethane and methanol in the ratios of 96:4, 90:10, 70:30, 55:45, and 30:70 in turn as the eluent to separate the glyceroglycolipid solution. After each eluent elutes 2 column volumes, change to the next eluent. Analyze the eluted fractions of each tube for glycolipids using analytical high-performance liquid chromatography, enrich the samples with the same retention time and wavelength, evaporate them to dryness under reduced pressure to obtain glyceroglycolipids. Redissolve the glyceroglycolipids in ethanol, and detect them by high-performance liquid chromatography. The retention time of the main elution peak of glyceroglycolipids is 6.61 min, the relative peak area of the maximum absorption wavelength reaches 96.04%, and the maximum absorption peak wavelength is 203.89 nm( Figure 9 ), and the absorption peak of the standard product is at 204.12 nm( Figure 10 ). The absorption peak of glyceroglycolipids is between 201 nm and 210 nm, so glyceroglycolipids meet the ultraviolet absorption spectral characteristics of glyceroglycolipids. After redissolving the glyceroglycolipids in ethanol, perform thin-layer chromatography development, and it is found that the target substance appears on the chromatography plate( Figure 11 ). At the same time, perform infrared spectroscopy analysis on the dried glyceroglycolipids, and it is found that it is at 619.87 cm -1 , 1075.72 cm -1 , 1377.62 cm -1 , 1737.67 cm-1 、2852.83 cm -1 、2924.76 cm -1 、3414.93 cm -1 There are absorption peaks at the positions, which can prove the existence of glyceroglycolipids( Figure 12 ).

Claims

1. A method for separating and purifying glyceroglycolipids from Undaria pinnatifida, characterized in that, It includes the following steps: (1) Mix the dried seaweed velvet powder with a methanol solution in a certain proportion to obtain a crude extract of glyceroglycolipid; (2) Mix the crude extract of glyceroglycolipid with an extraction solution for extraction to obtain extract a; (3) Add a chloroform solution to extract a, mix well and then add a methanol solution, and then add a sodium chloride solution to the solution to obtain extract b; (4) Use column chromatography to separate and purify extract b to obtain glyceroglycolipid.

2. The method according to claim 1, wherein The dried seaweed velvet powder described in step (1) is a powder passed through a 40-mesh sieve.

3. The method according to claim 1, wherein The concentration of the methanol solution described in step (1) is 80-90%, and the certain proportion is that the solid-liquid ratio of the dried seaweed velvet powder to the methanol aqueous solution is 1:25-1:30 g / mL.

4. The method according to claim 1, wherein The extraction solution described in step (2) is chloroform:methanol (2:1, v / v); the solid-liquid ratio of the crude glyceroglycolipid extract to the extraction solution is 1:10 g / mL.

5. The method according to claim 1, characterized in that, The volume ratio of the chloroform solution to the methanol solution described in step (3) is 1.5:1-2:

1.

6. The method according to claim 1, characterized in that, The column chromatography silica gel for column chromatography described in step (4) is 200-300 mesh.

7. A glyceroglycolipid, characterized in that, The glyceroglycolipid is separated and purified from seaweed.

8. The glyceroglycolipid according to claim 7, wherein The method for separating and purifying glyceroglycolipid from seaweed is as shown in the method described in any one of claims 1-7.

9. Use of a glyceroglycolipid, characterized in that, The glyceroglycolipid is prepared by the method described in any one of claims 1-6 and is used in pharmaceuticals, foods or cosmetics.

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