Non-sulfated monosialic acid ganglioside as well as preparation method and application thereof

Abstract

The invention provides non-sulfated monosialic acid ganglioside as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The structural formula of the ganglioside is Gal-3GalNAc-4 (NeuAc [alpha] 2-3) Gal [beta] 1-4Glc-2C < er >. The non-sulfated monosialic acid ganglioside SJ-2 prepared by the invention has good activity, can be used for preparing a medicine for promoting nerve regeneration or nourishing brain cells, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a non-sulfated monosialoganglioside and a preparation method and application thereof. Background Art

[0002] Ganglioside (GLS) is a class of glycosphingolipids with important biological functions. They play a key role in cell signaling, cell recognition, and neural development. GLS can be recognized by platelets and secrete proinflammatory factors and neurotransmitters, thereby regulating the functions of neurons and immune cells.

[0003] GLS has a complex structure and consists of three main components: sphingosine, long-chain fatty acids and oligosaccharide chains. The base of sphingosine is connected to ceramide through a glycosidic bond. Researchers have found that GLS is present in high levels in ganglia, and more than 180 different GLS structures have been identified in vertebrates. In addition, gangliosides have also been found in echinoderms such as sea cucumbers, starfish and echinoids. Their structures are significantly different from those of GLS in vertebrates: (1) The sugar group that connects GLS to ceramide in vertebrates is galactose, while the sugar group that connects GLS to ceramide in most echinoderms is glucose; (2) The gangliosides of echinoderms contain a variety of sialic acids including N-acetylneuraminic acid, N-hydroxyacetylneuraminic acid and 8-sulfate-N-hydroxyacetylneuraminic acid, while GSLs in vertebrates usually only contain one sialic acid, Neu5Ac; (3) In vertebrates, the sialic acid of GLS is connected to galactose through an α2-3 bond, while in echinoderms, the sialic acid of GLS is connected to glucose through an α2-6 bond.

[0004] Since GLS is present in high levels in the nervous system, the brain tissue of vertebrates has become the preferred biological material for obtaining GLS. However, as the study of GLS deepens, researchers have discovered that echinoderms such as sea urchins, sea cucumbers and starfish also contain GLS, and have proved through in vitro experiments that GLS in echinoderms also has good activity in promoting the growth of neurites, and that GLS in echinoderms has better intervention effects on the development of the nervous system and neurodegenerative diseases than vertebrates. However, due to the complex lipid composition of echinoderms and the large amount of impurities contained in the extracted total lipids, the initial extraction mixture containing glycolipids, phospholipids and trace proteins must be separated and purified. The purified GLS contains a variety of different fatty acid chains, sugar chains and sialic acid, making it relatively difficult to conduct accurate structural analysis of GLS.

[0005] Electrospray ionization-quadrupole-time of flight mass spectrometry (ESI-Q-TOF-MS) is an advanced mass spectrometry technology that integrates the high-efficiency ionization of ESI, the mass screening of quadrupoles, and the high-resolution function of time-of-flight mass spectrometry. It has become a powerful tool for analyzing complex mixtures and identifying unknown compounds. Although there are reports on the structural identification of gangliosides by chromatography-mass spectrometry, there is still a lack of detailed mass spectrometry fragmentation rules for specific categories of GLS.

[0006] Therefore, it is necessary to provide a new GLS isolated from echinoderms and to use spectroscopic techniques to conduct detailed structural identification of the isolated GLS, which not only provides a scientific theoretical basis for the structural analysis of echinoderm GLS, but also lays an important reference value for future exploration of the biological activity of echinoderm GLS. In addition, by studying the mass spectrometry fragmentation rules of GLS, it can provide guidance for the development of new analytical methods and strategies, thereby promoting the application of GLS in the biomedical field, which is of great significance for understanding the pathogenesis of nervous system diseases, developing new therapeutic drugs, and exploring other biological functions of GLS. Summary of the invention

[0007] The purpose of the present invention is to provide a non-sulfated monosialoganglioside and a preparation method and application thereof, in order to lay a foundation for the development of new GLS resources and realize the high-value comprehensive utilization of sea cucumber gonads.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a non-sulfated monosialoganglioside, wherein the structural formula of the ganglioside is Gal-3GalNAc-4(NeuAcα2-3)Galβ1-4Glc-2Cer.

[0010] The present invention provides a method for preparing the non-sulfated monosialoganglioside, comprising the steps of extracting total lipids from sea cucumber gonads by chloroform-methanol filtration, separating the extracted total lipids by silica gel column chromatography to obtain crude gangliosides, and purifying the crude gangliosides to obtain the non-sulfated monosialogangliosides.

[0011] Preferably, the mixing mass ratio of chloroform to methanol is 1.6-2.4:0.8-1.2.

[0012] Preferably, the sea cucumber gonad is sea cucumber gonad freeze-dried powder, and the amount of chloroform-methanol used is 8 to 12 times the volume of the sea cucumber gonad freeze-dried powder.

[0013] Preferably, during silica gel column chromatography, glycolipids and pigments are first eluted with acetone solution, and then phospholipids are eluted with methanol solution.

[0014] Preferably, the purification method is weak anion exchange chromatography and gel exclusion chromatography.

[0015] The present invention also provides a preparation containing the non-sulfated monosialoganglioside or the non-sulfated monosialoganglioside prepared according to the method.

[0016] The present invention further provides a use of the non-sulfated monosialoganglioside or the non-sulfated monosialoganglioside prepared according to the method or the preparation in the preparation of a drug for promoting nerve regeneration or nourishing brain cells.

[0017] The present invention uses chloroform-methanol filtration and silica gel column chromatography to extract and separate crude gangliosides, with a yield of 6.52%, and then uses weak anion exchange chromatography and gel exclusion chromatography separation technology to purify the crude gangliosides, with a yield of 24 mg / 50 g. Compared with the yield of 0.502 mg / 50 g of gangliosides separated from pig brain as a raw material, the yield of the present invention is higher, about 47 times that of the pig brain raw material, proving that the ganglioside content in the gonad of sea cucumber is relatively high, and it is expected to replace pig brain as the main source of gangliosides.

[0018] The present invention also infers that the sugar chain portion of the prepared non-sulfated monosialoganglioside SJ-2 contains GalNAc, GalNAc+Gal, Cer·H respectively through the ion fragment peaks of m / z=205 and m / z=367. 2 O and Glc+Cer·H 2 O; HPLC analysis showed that SJ-2 contained galactose and glucose; the fatty acid composition of SJ-2 was mainly C16:1 and C18:1, with contents of 16.7% and 70.1% respectively; the long-chain bases in SJ-2 were mainly d18:0 and d18:1, with contents of 47.52% and 31.26% respectively; combined with the methylation analysis results and the above experimental results, it can be inferred that SJ-2 in the sea cucumber gonad is a non-sulfated monosialoganglioside with the structural formula of Gal-3GalNAc-4(NeuAcα2-3)Galβ1-4Glc-2Cer.

[0019] The non-sulfated monosialoganglioside SJ-2 prepared by the invention has good activity, can be used for preparing drugs for promoting nerve regeneration or nourishing brain cells, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 The DEAE Sepharose FastFlow separation chromatogram in Example 2;

[0022] Figure 2 is the HPLC analysis spectrum of the ganglioside substance after weak anion exchange chromatography in Example 2;

[0023] Figure 3 The Sephacryl S-100HR separation chromatogram in Example 2;

[0024] Figure 4 is the HPLC analysis spectrum of ganglioside substances after gel exclusion chromatography in Example 2;

[0025] Figure 5 is the fragment peak obtained by SJ-2 in positive ion mode in Example 3;

[0026] Figure 6 is the mass spectrum of the sugar chain of SJ-2 in Example 3;

[0027] Figure 7 is the lipid chain mass spectrum of SJ-2 in Example 3;

[0028] Figure 8 This is the mass spectrum of the sphingosine chain on the fatty chain in SJ-2 in Example 3. DETAILED DESCRIPTION

[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] The extraction and separation method of sea cucumber gangliosides comprises the following steps:

[0032] (1) Total lipid extraction

[0033] Accurately weigh 5 g of Dalian sea cucumber gonad freeze-dried powder and add 10 times the volume of sea cucumber gonad freeze-dried powder in CHCl 3 -CH 3OH mixed solution (2:1, v / v), stirred evenly, let stand for 10 minutes, filtered using a vacuum filtration device, collected the filtrate, and repeated the above operation twice for the remaining filter residue. The filtrate collected three times was placed in a separatory funnel, and 1 / 5 of the volume of the filtrate was added with a 0.9% NaCl solution, and allowed to stand overnight. The lower layer of solution was collected in a round-bottomed flask and placed in a rotary evaporator, and evaporated to dryness. The residue in the bottle was the total lipid in the sea cucumber gonad, and the weight was recorded. 3 Dissolve it and place it in a -20℃ refrigerator for later use.

[0034] (2) Glycolipid Separation

[0035] Accurately weigh 180 g silica gel (250 mesh) and dissolve it in 400 mL CHCl 3 Slowly pour the above solution into the glass column and continuously add CHCl 3 Accurately weigh 600 mg of the total lipid prepared in step (1) and add 1 mL of CHCl 3 After the solution is dissolved, the sample is loaded and eluted with organic solvents of different polarities. First, 3 column volumes of acetone solution are used to elute glycolipids and pigments, and then 2.5 column volumes of methanol solution are used to elute phospholipids. The eluate is collected and evaporated to dryness. The residue in the bottle is crude ganglioside (glycolipids and phospholipids in the total lipid of sea cucumber gonads), and the weight is recorded. Use 2mL CHCl 3 The solution was dissolved and placed in a -20°C refrigerator for later use.

[0036] Table 1 shows the separation results of crude gangliosides. It can be seen that the present invention uses chloroform-methanol filtration and silica gel column chromatography to extract and separate crude gangliosides, with a yield of 6.52%.

[0037] Table 1 Separation results of crude gangliosides

[0038] Total fat (%) Crude ganglioside (%) Sea cucumber gonad (freeze-dried powder) 17.3％ 6.52％

[0039] Example 2

[0040] The purification method of sea cucumber gonadal gangliosides comprises the following steps:

[0041] (1) Weak anion exchange chromatography of gangliosides

[0042] Weigh 200 mL of DEAE Sepharose Fast Flow gel, wash the filler with pure water, and then rinse the filler with Tris-HCl solution until the pH value is close to 7.4, adjust the flow rate to 1 mL / min, add equilibrium buffer A solution at the upper end (accurately measure 84 mL of 0.2 mol / L hydrochloric acid solution, dilute to 1000 mL with the prepared Tris solution, and then adjust the pH to 7.4). After the column is balanced, the separation and purification of GLS can be carried out. The crude ganglioside prepared in Example 1 is washed with CHCl 3 After dissolving (m:v=0.6), add to the top of the column. The amount of sample should not exceed 20% of the column volume. After the sample is loaded, elute with equilibrium buffer A. First elute with 100% A, gradually change the ratio of buffer A to buffer B (A containing 1 mol / L NaCl), and the final ratio is 30% A + 70% B (100% A + 0% B → 95% A + 5% B → 90% A + 10% B → ... → 30% A + 70% B). Store the material in the collection tube in a 4°C refrigerator.

[0043] DEAE Sepharose Fast Flow separation chromatogram is shown below: Figure 1 As shown, peak 1 (F1): 30min-36min, peak 2 (F2): 66min-108min, peak 3 (F3): 132min-144min, peak 4 (F4): 192min-198min, peak 5 (F5): 252min-288min, peak 6 (F6): 369min-444min. It can be seen that the crude ganglioside prepared in Example 1 was subjected to DEAE Sepharose Fast Flow weak anion chromatography to obtain 6 components.

[0044] The gangliosides after weak anion exchange chromatography were further analyzed and detected by HPLC. The HPLC conditions were as follows: chromatographic column Hypersil BDS C18 (4.6 mm × 250 mm, 5 μm); mobile phase acetonitrile-0.03% triethylamine (72:28, v / v), pH adjusted to 7.5 with phosphoric acid; flow rate 1.0 mL / min; UV detection wavelength 205 nm; injection volume 20 μL. The analysis results are shown in Figure 2 As shown, it can be seen that F1, F2 and F3 do not contain the target substance GLS; F4, F5 and F6 contain the target substance GLS.

[0045] (2) Gel Exclusion Chromatography of Gangliosides

[0046] Weigh 200mL of propylene dextran gel Sephacryl S-100HR filler, wash it with pure water, and then rinse the filler with equilibrium buffer until the pH value is close to 7.4. Slowly add the washed propylene dextran gel Sephacryl S-100HR into the column along the column wall, and gently tap the column to help the filler settle evenly. After installing the column, rinse the column with PBS buffer, and the mobile phase volume is 18 column volumes. Fix the column on an iron stand, and after the column is balanced, enrich the F4, F5 and F6 components collected in step (1) and add them to the upper end of the column. The sample loading amount cannot exceed 20% of the column volume. Use 0.01mol / L PBS buffer with a pH of 7.4 for isocratic elution, and use the resorcinol-HCl method to qualitatively detect the collected samples, collect the samples at the peak time, and freeze-dry until the samples are white powder.

[0047] The separation results are as follows Figure 3 As shown, peak 1 (S1): 72min~44min, peak 2 (S2): 132min~177min, peak 3 (S3): 246min~315min, peak 4 (S4): 384min~426min. It can be seen that after enriching the F4, F5 and F6 components, four components were obtained by gel exclusion chromatography.

[0048] HPLC was further used to analyze and detect the ganglioside substances after gel exclusion chromatography. The HPLC conditions were as follows: chromatographic column Hypersil BDS C18 (4.6 mm × 250 mm, 5 μm); mobile phase acetonitrile-0.03% triethylamine (72:28, v / v), pH adjusted to 7.5 with phosphoric acid; flow rate 1.0 mL / min; UV detection wavelength 205 nm; injection volume 20 μL. The analysis results are shown in Figure 4 As shown in the figure, by comparing the peak times of the four components and the ganglioside standard, it can be seen that S1, S2, S3 and S4 all contain the target substance ganglioside. At the same time, the above four components were analyzed by ESI-Q-TOF. The results showed that the response values ​​of S1 and S3 components were close, so these two components were enriched and named SJ-1; the response values ​​of S2 and S4 components were close, so these two components were enriched and named SJ-2.

[0049] Example 3

[0050] (1) Structural identification of sea cucumber gonadal ganglioside SJ-2

[0051] The sea cucumber gonadal ganglioside SJ-2 obtained in Example 2 was passed through an organic filter membrane and then subjected to LC-MS analysis. LC-MS conditions: mobile phase A was pure water, mobile phase B was acetonitrile (1:1, v / v), flow rate was 0.2 mL / min, and injection volume was 0.5 μL. The electrospray ion source adopted positive ion mode, the primary mass spectrometry scanning range was m / z 300-2000, the secondary mass spectrometry scanning range was m / z100-2000, spray voltage 5500 V, declustering potential 80 V, and ion source temperature was 500 ° C. A two-way connection was made between the liquid chromatography and the mass spectrometry.

[0052] The fragment peaks obtained by SJ-2 in positive ion mode are as follows Figure 5 As shown, it can be seen that SJ-2 contains fragment peaks such as m / z=205, m / z=367, m / z=547 and m / z=701, from which the sugar chain structure and lipid chain structure of SJ-2 can be inferred.

[0053] (2) Sugar chain structure analysis

[0054] When SJ-2 was cleaved by high collision energy 80eV, its fragment structure was found to be similar to that of ganglioside standards. Figure 6 As shown in the figure, by comparing the fragment peaks with the NIST 02 database, it was found that the fragments m / z = 205, m / z = 367, m / z = 547, and m / z = 701 in the figure can be inferred that the sugar chain part of SJ-2 contains acetylgalactosamine (GalNAc), GalNAc + galactose (Gal), ceramide (Cer·H 2 O) and glucose (Glc) + Cer·H 2 O.

[0055] (3) Lipid chain structure analysis

[0056] Figure 7 This is the mass spectrum of the partial structure of the lipid chain of SJ-2. By comparing the fragment peaks with the NIST 02 database, it can be found that the characteristic fragment peak of m / z=264 can be seen, indicating that the sphingosine chain of SJ-2 has not changed.

[0057] (4) Analysis of fatty acid and long-chain base composition

[0058] Sample derivatization: Accurately weigh 1.0 mg of sea cucumber gonadal ganglioside SJ-2 and place it in an ampoule tube. Add 1 mL of 5% HCl-CH 3OH solution, seal the tube mouth under nitrogen protection to avoid sample oxidation, place the sealed ampoule tube in a 70°C water bath for 8 hours to heat the reaction to achieve fatty acid methyl esterification. After the reaction, cool the ampoule tube to room temperature, add 1 mL of n-hexane for extraction, repeat the extraction 3 times to fully extract fatty acid methyl esters, combine all n-hexane layers, use a rotary evaporator to concentrate the n-hexane layer to 1 mL, and refrigerate the filtered extract at 4°C for fatty acid composition analysis.

[0059] The remaining layer was neutralized with aqueous ammonia and CH 3 The OH layer was concentrated to 1 mL, and the residue was dried in a vacuum oven at 50°C overnight. 200 μL of pyridine was added to dissolve it, and then 200 μL of TMS (trimethylsilane) was added for silylation reaction at 70°C for 30 minutes. After silylation, 200 μL of Ac 2 O / pyridine mixed solution (1:1, v / v) was heated at 90°C for 10 min to promote dissolution, and then the reaction was continued at 100°C for 1 hour to complete the derivatization. Finally, 1 mL of water was added to terminate the reaction and refrigerated at 4°C until the long-chain base composition analysis was performed.

[0060] GC-MS conditions: HP-5ms column (30m×0.32mm, 0.25μm, J&W); injection port temperature was set to 250℃, splitless injection; fatty acid temperature program: starting from 140℃, heating to 280℃ at a rate of 5℃ / min, and maintaining for 10min; long-chain base temperature program: starting from 180℃, heating to 250℃ at a rate of 4℃ / min, and maintaining for 10min; carrier gas flow rate was 1.2mL / min, and the transfer line temperature was 280℃; mass spectrometry scanning range: m / z 45-550.

[0061] The total ion current (TIC) and mass spectrum obtained were compared and analyzed with the NIST 02 database, and the peak area was normalized to obtain the relative content ratio of fatty acids and long-chain bases. The fatty acid composition of SJ-2 is shown in Table 2, and the long-chain base composition is shown in Table 3.

[0062] Table 2 SJ-2 fatty acid composition

[0063]

[0064] Table 3 SJ-2 long chain base composition

[0065]

[0066] As can be seen from Table 2, compared with gangliosides from the spine, gangliosides in the gonads of sea cucumbers showed a significant extension of their fatty acid chains, and the content of unsaturated fatty acids also increased, and obvious hydroxylation was observed. These characteristics may be one of the reasons for the improvement of the biological activity of GLS in the gonads of sea cucumbers. As can be seen from Table 3, the long-chain bases in SJ-2 are mainly d 18:0 and d 18:1, with contents of 47.52% and 31.26%, respectively, of which d18:1 has a higher content in SJ-2. Compared with other echinoderms, the types of long-chain bases of gangliosides in the gonads of sea cucumbers are relatively rich, including t-series long-chain bases and d-series long-chain bases.

[0067] (5) Analysis of neutral monosaccharide composition

[0068] Sample derivatization: Accurately weigh 1.0 mg of sea cucumber gonadal ganglioside SJ-2, add 1 mL of 2 mol / L trifluoroacetic acid to degrade for 8 hours, derivatize with 1 mL of PMP and dilute to 5 mL, pass through the membrane, and wait for HPLC analysis. The monosaccharide composition and content ratio of the sample are determined by the retention time and peak area of ​​the PMP derivatives of glucose and galactose standards.

[0069] HPLC conditions: Hypersil BDS C18 column (4.6 mm × 250 mm, 5 μm); UV detector, detection wavelength 250 nm; mobile phase: solvent A: 15% (v / v) acetonitrile and 0.05 mol / L phosphate buffer (KH 2 PO 4 -NaOH, pH 6.9), solvent B: 40% (v / v) acetonitrile + 0.05 mol / L phosphate buffer (KH 2 PO 4 -NaOH, pH 6.9); gradient mode: time gradient, 0→10→30min; corresponding solvent B concentration gradient, 0→8%→20%; flow rate was 1.0mL / min; injection volume was 20μL.

[0070] The results of the determination of the composition and content ratio of neutral monosaccharides in SJ-2 are shown in Table 4. It can be seen that SJ-2 contains glucose and galactose. The prior art shows that gangliosides in echinoderms such as sea urchins and sea cucumbers usually only contain glucose, but the inventors unexpectedly found galactose in the gangliosides extracted from the gonads of sea cucumbers.

[0071] Table 4 Neutral monosaccharide composition and content ratio determination results in SJ-2

[0072] sample glucose / % Galactose / % SJ-2 38 72

[0073] (6) Structural identification of gangliosides

[0074] Refer to the improved Hakomori methylation method. First, accurately weigh 1.0 mg of sea cucumber gonadal ganglioside SJ-2, add 1 mL DMSO, transfer to a 50 mL flask and stir at 90 ° C for 10 min, then add 100 mg NaH and continue to react for 30 min until the solution turns orange-yellow; then slowly add 750 μL iodomethane under light-proof conditions and continue to react for 1 h, then add 1 mL water to terminate the reaction, extract with 2 mL dichloromethane 3 times, combine the dichloromethane layers, and wash with water 3 times; then place in a 50 ° C oven to dry, add 1 mL 2 mol / L trifluoroacetic acid and transfer to an ampoule and seal the tube, degrade at 105 ° C for 6 h, dry under reduced pressure rotary evaporation, repeatedly add 2 mL methanol and rotary evaporation to remove trifluoroacetic acid (until the attachment on the bottle wall turns white); add 5 mg NaBH 4 Add 1mL 2mmol / LNaOH and react at 70℃ for 1h, transfer to a rotary evaporator and dry, repeatedly add 1mL methanol and rotary evaporate to remove boric acid (until the attachment on the bottle wall turns white), evaporate to dryness and dry under reduced pressure overnight; add 2mLAc 2 After heating O / pyridine (1:1, v / v) at 90℃ for 10min to promote dissolution, react at 100℃ for 1h, add 1mL of pure water to terminate the reaction, extract with dichloromethane 3 times and wash with water 3 times, and then perform GC-MS analysis on the neutral monosaccharide connection mode after vacuum concentration. Sialic acid is unstable in strong acid solution and its structure is easily destroyed. Weaker acidic conditions must be used for hydrolysis. Methanolysis is performed at 70℃ with anhydrous methanol solution containing 8% hydrochloric acid. After the hydrolysis is completed, acetylation reaction is directly carried out at 80℃. After vacuum concentration, GC-MS analysis is performed on the sialic acid substitution position.

[0075] Structural Elucidation by Gas Chromatography-Mass Spectrometry

[0076] GC-MS analysis conditions: Neutral monosaccharide derivatives were analyzed using a DB-35ms column (30m×0.25mm, 0.25μm), using a program temperature rise method, with a starting temperature of 170°C, held for 1min, then heated to 230°C at a rate of 2°C / min, held for 20min, and the carrier gas was helium at a flow rate of 1mL / min. The mass scan range was m / z 40-450amu. Acidic sialic acid analysis used a DB-1701 column (30m×0.25mm, 0.32μm), with a temperature rise program of: starting temperature of 200°C, heated to 250°C at a rate of 2.5°C / min, held for 10min, carrier gas flow rate of 1.2mL / min, and mass scan range of m / z 40-450amu. The mass spectrometer inlet temperature was set to 250°C, the transfer line temperature was 280°C, and the EI ion source was 230°C.

[0077] The sphingosine chain on the fatty acid chain of the SJ-2 sample has not changed, and its mass spectrum is as follows Figure 8 Based on the results of methylation analysis, it was inferred that SJ-2 in the gonad of sea cucumber is a non-sulfated monosialoganglioside with a structural formula of Gal-3GalNAc-4(NeuAcα2-3)Galβ1-4Glc-2Cer.

[0078] Studies have shown that sialic acid in sea urchins is linked to glucose through an α2-6 bond, sialic acid in starfish is linked to galactose through an α2-3 bond, and sialic acid in vertebrates is linked to galactose through an α2-3 bond. The differences in sugar chains and connection methods will affect the activity of gangliosides.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A non-sulfated monosialoganglioside, characterized in that: The structural formula of the ganglioside is Gal-3GalNAc-4(NeuAcα2-3)Galβ1-4Glc-2Cer.

2. A method for preparing the non-sulfated monosialoganglioside according to claim 1, characterized in that: The total lipids in the gonads of sea cucumbers were extracted by chloroform-methanol filtration method, and then the extracted total lipids were separated by silica gel column chromatography to obtain crude gangliosides. After the crude gangliosides were purified, non-sulfated monosialogangliosides were obtained.

3. The method according to claim 2, characterized in that The mixing mass ratio of the chloroform to the methanol is 1.6-2.4:0.8-1.

2.

4. The method according to claim 3, characterized in that The sea cucumber gonad is sea cucumber gonad freeze-dried powder, and the amount of chloroform-methanol used is 8 to 12 times the volume of the sea cucumber gonad freeze-dried powder.

5. The method according to claim 4, characterized in that During silica gel column chromatography, glycolipids and pigments are first eluted with acetone solution, and then phospholipids are eluted with methanol solution.

6. The method according to any one of claims 2 to 5, characterized in that: The purification method is weak anion exchange chromatography and gel exclusion chromatography.

7. A preparation comprising the non-sulfated monosialoganglioside according to claim 1 or the non-sulfated monosialoganglioside prepared by the method according to any one of claims 2 to 6.

8. Use of the non-sulfated monosialoganglioside according to claim 1 or the non-sulfated monosialoganglioside prepared by the method according to any one of claims 2 to 6 or the preparation according to claim 7 in the preparation of a drug for promoting nerve regeneration or nourishing brain cells.

Images