Marine galactooligosaccharide lipid as well as preparation method and application thereof

Abstract

The invention discloses a marine galactooligosaccharide lipid as well as a preparation method and application thereof, and belongs to the field of marine biological resource utilization and drug development. According to the invention, a marine red algae polysaccharide degradation technology and high efficiency of reductive amination aliphatic chain modification are combined together for the first time, and modification of saturated aliphatic chains with different lengths of marine galactooligosaccharides with different polymerization degrees is realized. Cellular level pharmacodynamic evaluation proves that the marine galactooligosaccharide lipid can significantly inhibit release of nitric oxide in microglial cells and expression of inflammatory factors such as interleukin-6 and tumor necrosis factor alpha, and has neuroinflammation inhibition activity; meanwhile, proliferation of tumor cells U87MG and GL261 can be remarkably inhibited, and the compound has certain anti-tumor potential and can be used for preparing drugs for preventing and / or treating neuroinflammation and anti-tumor related diseases. Therefore, the marine galactooligosaccharide lipid prepared by the invention has relatively high clinical application value and development prospect.

Description

Technical Field

[0001] The present invention belongs to the field of utilization of marine biological resources and drug development, and particularly relates to the preparation of marine galactooligosaccharide lipids and their applications in anti-neuroinflammatory and anti-tumor drugs. Background Art

[0002] The ocean covers more than 70% of the total global area, and the vast ocean environment has given rise to a huge marine ecosystem. In the marine environment, polysaccharides are one of the most abundant components of organic carbon. However, marine polysaccharides have large molecular weights and complex structures, resulting in low solubility and high solution viscosity of marine polysaccharides, which limits their applications. By degrading marine polysaccharides, marine oligosaccharides can be obtained. They have good solubility and are easily absorbed and utilized by the body. They have been widely used in the fields of anti-tumor, immunomodulation, anti-inflammatory, etc., and are one of the hotspots in marine drug research.

[0003] However, due to problems such as high hydrophilicity, insufficient structural rigidity, and poor metabolic stability of natural oligosaccharides, their applications in drug development, biomaterials, and functional preparations are limited. Summary of the Invention

[0004] The purpose of the present invention is to provide a marine galactooligosaccharide lipid, a preparation method of the marine galactooligosaccharide lipid, and specific applications of the marine galactooligosaccharide lipid to make up for the deficiencies of the prior art.

[0005] To achieve the above invention purpose, the present invention adopts the following technical solutions: A marine galactooligosaccharide lipid, the chemical formula structure of the marine galactooligosaccharide lipid is shown as Formula I or Formula II: Formula I; Formula II; Wherein, in Formula I, n = 0 - 4, m = 4 - 16; in Formula II, n = 0 - 4, m = 4 - 16.

[0006] A preparation method of a marine galactooligosaccharide lipid, using agar as a raw material, carrying out controllable degradation by chemical method and bio-enzymatic method to prepare galactooligosaccharides with different degrees of polymerization, and then carrying out fatty acid chain modifications with different lengths on the galactooligosaccharides to obtain the marine galactooligosaccharide lipid; specifically including the following steps: (1) Adopting the synergistic effect of β-agarase-mediated site-specific enzymatic hydrolysis process and controllable acid degradation process, and realizing the selective preparation of the target oligosaccharide chain length by precisely regulating the reaction pH, temperature, enzyme / substrate ratio, and hydrolysis time; (2) Based on the graphitized carbon column chromatography separation technology, high-purity galactooligosaccharide monomer components are purified by ethanol gradient elution, providing a highly homogeneous substrate for subsequent fatty acid chain modification research.

[0007] (3) Using agar-derived galacto-oligosaccharides with a degree of polymerization of 2 - 10 as the substrate for the fatty chain modification reaction, the reaction system is based on methanol. Galacto-oligosaccharides (1.0 equivalent), amino-modified saturated fatty chains (1.0 - 5.0 equivalents), and NaBH3CN (1.0 - 5.0 equivalents) are dissolved in the methanol system. The pH value of the reaction system is adjusted to 4.0 - 7.0, the reaction temperature is 10 - 60 °C, the rotation speed is 10 - 200 r / min, and the reaction is carried out for 10 - 24 h under nitrogen protection. The progress of the reaction is tracked by thin-layer chromatography, and the developing agent prepared by mixing n-butanol: formic acid: water = 4:6:1 by volume ratio is used for detection; (4) After obtaining galacto-oligosaccharide lipids through the above synthesis method, dilute hydrochloric acid (HCl) is added dropwise to the reaction solution to adjust the pH to 3.0 - 4.0 (operation in a fume hood), and the solvent is evaporated to dryness. Finally, the target product is separated and purified using a silica gel column and a C18 column to obtain marine galacto-oligosaccharide lipids. Among them, the mobile phase for silica gel column purification is dichloromethane: methanol = 8:1 - 1:1, and each gradient is eluted for 2 - 5 column volumes, and the fractions containing the target product are collected. Further, the C18 column is used for fine separation of the target product, and the mobile phase for C18 column purification is 20% - 100% methanol, and each gradient is eluted for 3 column volumes. The separation of the product is monitored by TLC to achieve the precise preparation of the target product. The application of the marine galacto-oligosaccharide lipids in the preparation of anti-neuroinflammatory products; the products are drugs or health products.

[0008] The application of the marine galacto-oligosaccharide lipids in the preparation of anti-tumor drugs.

[0009] Among them, the anti-neuroinflammatory products include: neuroinflammatory inhibitors, products for treating and / or preventing nervous system diseases caused by neuroinflammation.

[0010] The nervous system diseases include: Parkinson's disease, Alzheimer's disease, multiple sclerosis, Huntington's chorea, vascular dementia, stroke, depression, schizophrenia, meningitis, etc.; the tumors include: human glioma, liver cancer, lung cancer, skin cancer, prostate cancer, gastric cancer, breast cancer, colorectal cancer, melanoma, etc.

[0011] Drugs with the compounds shown in Formula I - II of the present invention as active ingredients can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, and mucosal tissues by injection, spraying, nasal dropping, eye dropping, penetration, absorption, physical or chemical mediated methods; or be introduced into the body after being mixed or encapsulated with other substances.

[0012] When needed, one or more pharmaceutically acceptable carriers can also be added to the above-mentioned drug. The carriers include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0013] The above-mentioned drug can be made into various forms such as injection solutions, tablets, powders, granules, capsules, oral liquids, ointments, creams, etc. The drugs in the above various dosage forms can all be prepared according to the conventional methods in the pharmaceutical field.

[0014] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: The present invention combines the technology of degrading marine red algal polysaccharides with the high efficiency of reductive amination and fatty chain modification for the first time, realizing the modification of different lengths of saturated fatty chains on marine galactooligosaccharides with different degrees of polymerization. And through the evaluation of pharmacodynamics at the cellular level, it is proved that marine galactooligosaccharide lipids can significantly inhibit the release of nitric oxide (NO) and the expression of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in microglia, and have the activity of inhibiting neuroinflammation; at the same time, it can significantly inhibit the proliferation of tumor cells U87MG and GL261, and has certain anti-tumor potential, and can be used to prepare drugs for preventing and / or treating neuroinflammation and anti-tumor related diseases. Therefore, it has high clinical application value and development prospects. Description of the Drawings

[0015] Figure 1 1H NMR spectrum of marine galactooligosaccharide lipid in Example 1 1 H spectrum.

[0016] Figure 2 13C NMR spectrum of marine galactooligosaccharide lipid in Example 1 13 C spectrum.

[0017] Figure 3 Mass spectrum of marine galactooligosaccharide lipid in Example 1, where A is the mass spectrum of compound 1, B is the mass spectrum of compound 2, C is the mass spectrum of compound 3, and D is the mass spectrum of compound 4.

[0018] Figure 4 Effects of marine galactooligosaccharide lipid in Example 2 on cytotoxicity (A) and the expression of inflammatory mediators such as NO (B), TNF-α (C), and IL-6 (D) in LPS-induced BV2 microglia; where, * indicates the difference between the LPS group and the Control group (* P < 0.05, ** P < 0.01, **** P < 0.0001); # indicates the difference between the experimental group and the LPS group (# P < 0.05, ##P < 0.01, P < 0.001, # P < 0.0001); ns indicates no statistical difference ( P > 0.05).

[0019] Figure 5 Effects of marine galactooligosaccharide lipids with different concentrations in Example 3 on the viability of tumor cells; where A is the microscopic observation image of the morphology of U87MG cells after treatment with Compound 3, and B is the effect of Compound 3 on the viability of U87MG cells; * indicates the difference between the experimental group and the Control group (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

[0020] Figure 6 Effects of marine galactooligosaccharide lipids with different concentrations in Example 3 on the viability of tumor cells; where A is the microscopic observation image of the morphology of GL261 cells after treatment with Compound 3, and B is the effect of Compound 3 on the viability of GL261 cells; * indicates the difference between the experimental group and the Control group (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Detailed implementation manners

[0021] Next, the technical solutions of the present invention will be further described in conjunction with specific embodiments, but the present invention is not limited thereto.

[0022] In the following examples, the experimental methods are all conventional methods unless otherwise specified; the reagents and biological materials can be obtained from commercial channels unless otherwise specified.

[0023] Example 1: Preparation and characterization of marine galactooligosaccharide lipids The molecular reaction formula for preparing marine galactooligosaccharide lipids is: .

[0024] Preparation method: Agar oligosaccharides with a degree of polymerization of 2 - 10 are used as the substrate for the fatty chain modification reaction. The reaction system is based on methanol. Galacto-oligosaccharides (1.0 equivalent), amino-modified saturated fatty chains (1.0 - 5.0 equivalents), and NaBH3CN (1.0 - 5.0 equivalents) are dissolved in the methanol system. The pH value of the reaction system is adjusted to 4.0 - 7.0, the reaction temperature is 10 - 60 °C, the rotation speed is 10 - 200 r / min, and the reaction is carried out for 10 - 24 h under nitrogen protection. The progress of the reaction is tracked by thin-layer chromatography, and the developing agent prepared by mixing n-butanol:formic acid:water = 4:6:1 by volume is used for detection. After obtaining galacto-oligosaccharide lipids through the above synthesis method, dilute hydrochloric acid (HCl) is added dropwise to the reaction solution to adjust the pH to 3 - 4 (operating in a fume hood), and the solvent is evaporated to dryness. The target product is separated and purified using a silica gel column and a C18 column. Among them, the mobile phase for silica gel column purification is dichloromethane:methanol = 8:1 - 1:1, and each gradient is eluted for 2 - 5 column volumes, and the fractions containing the target product are collected. Further, the C18 column is used for fine separation of the target product, and the mobile phase for C18 column purification is 20% - 100% methanol, and each gradient is eluted for 3 column volumes. The separation of the product is monitored by TLC to achieve the precise preparation of the target product.

[0025] Compounds 1 - 4 mentioned in Table 1 respectively correspond to the compounds described by Formula I, where, in Formula I, n = 0 - 4, m = 4 - 16; in Formula II, n = 0 - 4, m = 4 - 16.

[0026] Table 1 Structures of Marine Galacto-oligosaccharide Lipids

[0027] In this example, compounds 1 - 4 are taken as examples to illustrate the results of the structural identification of marine galacto-oligosaccharide lipids, but the marine galacto-oligosaccharide lipids involved in the present invention are not limited thereto. And all glycolipid derivatives generated with agar oligosaccharides with a degree of polymerization of 2 - 10 as the reaction substrate are within the protection scope of this invention patent. Among them, the reducing end of agar oligosaccharides is a galactose unit, and the high-efficiency reductive amination reaction can be achieved with this structural feature. Therefore, using agar oligosaccharides with a degree of polymerization of 2 - 10 as the reaction substrate and amino-modified saturated fatty chains as donors, the compounds shown in Formulas I - II can be obtained through reductive amination reaction. The 1 1H NMR is as Figure 1As shown, characteristic peaks of the fatty chain appeared in the high-field region. The terminal methyl group was at δ 0.90, and the methylene groups on the fatty chain of octylamine or stearylamine were at δ 1.33 - 1.53, confirming the successful modification of the fatty chain. In the low-field region, anomeric proton signals of neoagaro-oligosaccharides appeared. The anomeric proton signal of 3,6-anhydrogalactose in neoagaro-disaccharide was at δ 4.93, and the anomeric proton signals of 3,6-anhydrogalactose in neoagaro-tetrasaccharide were at δ 5.13 and δ 4.96. In addition, the α-hydrogen signal of the anomeric position of the galactose at the reducing end of the oligosaccharide disappeared at δ 5.25, indicating that ring opening occurred at the reducing end.

[0028] 13 The results of 13C NMR were as Figure 2 shown. Carbon signals of the fatty chain appeared in the high-field region, and anomeric carbon signals of the oligosaccharide appeared in the low-field region.

[0029] Combined with the HRMS data, it was confirmed that the molecular weight matched the theoretical value. As Figure 3 shown, signal peaks of [M + H]+ for compounds 1 - 4 appeared at m / z = 438.27, 744.37, 578.43, and 884.53 respectively. +

[0030] All of the above results indicated the successful preparation of marine galacto-oligosaccharide lipids.

[0031] Example 2: Inhibition of the release of inflammatory mediators in BV2 microglial cells induced by bacterial lipopolysaccharide (LPS) by marine galacto-oligosaccharide lipids CCK8 experimental procedure: Mouse microglial BV2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture in a 37 °C, 5% CO2 incubator. BV2 cells in the logarithmic growth phase were taken and seeded into 96-well plates at 5000 - 10000 cells per well, with a volume of 200 μL per well generally. The edge wells were filled with sterile PBS to prevent edge effects. 200 μL of medium was added to each well in the control group, 200 μL of medium containing 50 μmol / L drug was added to each well in the experimental group, and only 200 μL of medium without cell seeding was added to the blank group. Each group was set with 5 replicates. The 96-well plates were returned to the incubator, taken out after 24 h, 10 μL of CCK-8 solution was added to each well, then returned to the incubator, and the absorbance (OD value) of each well was measured at a wavelength of 450 nm with an enzyme-linked immunosorbent assay reader after 2 h. Calculate the cell viability of each concentration group. Viability (%) = { (OD 样品 - OD 空白 ) / (OD 对照 - OD 空白 )} × 100%.

[0032] As Figure 4As shown in A, at a concentration of 50 μmol / L, compound 1-2 (octylamine-glycolipid derivative) had no obvious effect on the proliferation of BV2 cells, indicating that the octylamine-glycolipid derivative has good biosafety and no cytotoxicity. However, compound 3-4 (stearylamine-glycolipid derivative) inhibited the proliferation of BV2 cells. The above results show that the length of the saturated fatty acid chain affects its biological activity.

[0033] Next, taking compound 1-2 as an example, the anti-neuroinflammatory activity of marine galactooligosaccharide lipids will be further described.

[0034] Detection steps for inflammatory mediator release: BV2 mouse microglial cells in the logarithmic growth phase were seeded in 96-well culture plates at a density of 1×10 4 cells / well. After the cells adhered for 24 h, they were pre-incubated with different marine galactooligosaccharide lipids at a final concentration of 50 μM for 2 h, and then incubated with or without LPS (final concentration 1 μg / mL) for 24 h. The supernatant was collected, centrifuged at 8000 r / min at 4 °C for 10 minutes, and the supernatant was taken. According to the manufacturer's instructions, the levels of NO, TNF-α, and IL-6 were measured using a commercially available kit (Shanghai Beyotime Biotechnology Co., Ltd.). As Figure 4 shown in B, compared with the normal control group, the content of NO in the supernatant of BV2 cells in the LPS treatment group increased significantly (P < 0.0001), indicating that a neuroinflammation model was successfully constructed. When compound 1-2 was added for intervention, the release of NO showed an obvious inhibitory trend. The expression levels of inflammatory factors are as Figure 4 shown in C and D. Compared with the normal control group, the contents of TNF-α and IL-6 in the supernatant of BV2 cells in the LPS-stimulated group increased significantly ( P < 0.05), and the marine galactooligosaccharide lipid (compound 1-2) at a final concentration of 50 μM could significantly inhibit the generation of inflammatory factors in LPS-activated microglial cells, and the inhibitory effect on IL-6 was more significant. The results show that marine galactooligosaccharide lipids have significant anti-neuroinflammatory activity.

[0035] The above experiments prove that both Compounds 1 and 2 can significantly inhibit the release of nitric oxide (NO) and the expression of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in microglia induced by bacterial lipopolysaccharide (LPS). The structures of the compounds in Formula I-II series are highly similar, with differences only at the non-reducing end position, and the core pharmacodynamic feature of this series of compounds is amino fatty chain modification. The position where the structural differences of the compounds in Formula I-II are located is far from the core pharmacophore. Therefore, based on the similarity of molecular structures and the general principles of medicinal chemistry, it can be reasonably expected in the present invention that all compounds covered by Formula I-II have anti-neuroinflammatory activities equivalent or similar to those of specific Compounds 1 and 2.

[0036] Example 3: Inhibition of tumor cell proliferation by marine galactooligosaccharide lipids In this example, the growth inhibitory effects of Compound 3 on brain tumor cells U87MG and GL261 cells were detected by the CCK-8 method to preliminarily explore its anti-tumor activity.

[0037] The experimental steps are as follows: Mouse glioma cells GL261 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture. Human astroglioblastoma cells U87MG were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture. 5000 tumor cells U87MG and GL261 were seeded in each well of a 96-well plate. After culturing for 24 h, the supernatant was aspirated. In the control group, 200 μL of medium was added to each well, and medium with equal volume and containing different mass concentrations of Compound 3 was added to each well. The concentrations were 1.25, 2.5, 5, 10, 25, 50, 100 μg / mL. A blank group was set up, and only medium was added to the blank group without inoculating cells. Each group was set with 3 replicates. After culturing for 24 h, the growth status of tumor cells was observed by an inverted microscope, and the inhibitory effect of the compound on tumor cell growth was detected by a CCK-8 kit.

[0038] Furthermore, the results are as Figure 5 and Figure 6 shown. The growth status of U87MG and GL261 cells in the blank group was normal. In the Compound 3 intervention group, as the administration concentration increased, cell growth was inhibited, the cells shrank and became round, and obvious cell debris appeared at high concentrations. And at the mass concentrations of 50 and 100 μg / mL, the cell viability of tumor cells was lower than 20%, indicating that Compound 3 can significantly inhibit the proliferation of tumor cells, play an obvious inhibitory role in the growth of tumor cells, and has certain anti-tumor potential. And it further shows that long-chain fatty acid modification can make oligosaccharides exhibit cytotoxicity and improve their anti-tumor activity.

[0039] The above experiments prove that compound 3 can significantly inhibit the proliferation of tumor cells U87MG and GL261, and has certain anti-tumor potential. The anti-tumor activity stems from its specific core pharmacodynamic feature of amino aliphatic chain modification, and the aliphatic chain modification can significantly enhance the biological activity. Based on this core structure and the general principles of known medicinal chemistry, it can be reasonably expected in the present invention that all compounds covered by Formula I-II have anti-tumor activity equivalent to or similar to that of compound 3.

[0040] Although the above embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. Other embodiments can be obtained according to these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A marine galactooligosaccharide lipid, characterized in that, The chemical formula structure of the marine galactooligosaccharide lipid is shown in Formula I or Formula II: Formula I; Formula II; Among them, in Formula I, n = 0 - 4, m = 4 - 16; in Formula II, n = 0 - 4, m = 4 - 16.

2. The preparation method of the marine galactooligosaccharide lipid according to claim 1, characterized in that, Using agar as a raw material, it is subjected to controllable degradation by chemical methods and bioenzymatic methods to prepare galactooligosaccharides with different degrees of polymerization, and then the galactooligosaccharides are modified with fatty acid chains of different lengths to obtain marine galactooligosaccharide lipids.

3. The preparation method according to claim 2, characterized in that, This method includes the following steps: (1) Adopting the synergistic effect of a β-agarase-mediated site-specific enzymatic hydrolysis process and a controllable acid degradation process, by regulating the reaction pH, temperature, enzyme / substrate ratio, and hydrolysis time, the selective preparation of the target oligosaccharide chain length is achieved; (2) Based on the graphitized carbon column chromatography separation technology, high-purity galactooligosaccharide monomer components are purified by ethanol gradient elution to prepare a highly homogeneous substrate; (3) Using the agar-derived galactooligosaccharides with a degree of polymerization of 2 - 10 as the substrate for the fatty acid chain modification reaction, the reaction system is based on methanol. 1.0 equivalent of galactooligosaccharides, 1.0 - 5.0 equivalents of amino-modified saturated fatty acid chains, and 1.0 - 5.0 equivalents of NaBH3CN are dissolved in the methanol system. The pH value of the reaction system is adjusted to 4.0 - 7.0, the reaction temperature is 10 - 60 °C, and the reaction is carried out for 10 - 24 h; the progress of the reaction is tracked by thin-layer chromatography, and the developing agent prepared by mixing n-butanol: formic acid: water = 4:6:1 by volume is used for detection; (4) Then, dilute hydrochloric acid is added dropwise to the reaction solution to adjust the pH to 3.0 - 4.0, and the solvent is evaporated to dryness; finally, the target product is separated and purified using a silica gel column and a C18 column to obtain the marine galactooligosaccharide lipid.

4. Use of the marine galactooligosaccharide lipid according to claim 1 in the preparation of anti-neuroinflammatory products.

5. Use of the marine galactooligosaccharide lipid according to claim 1 in the preparation of anti-tumor drugs.

6. The application according to claim 4, wherein The anti-neuroinflammatory products include: neuroinflammatory inhibitors, products for treating and / or preventing nervous system diseases caused by neuroinflammation.

7. The application according to claim 4, characterized in that, The nervous system diseases include: Parkinson's disease, Alzheimer's disease, multiple sclerosis, Huntington's disease, vascular dementia, stroke, depression, schizophrenia, meningitis.

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