A marine galacto-oligosaccharide lipid and its preparation method and application

Abstract

The present invention discloses a marine galacto-oligosaccharide lipid and its preparation method and application, which belongs to the field of marine biological resource utilization and drug development. The present invention combines the marine red algae polysaccharide degradation technology with the high efficiency of reductive amination fatty chain modification for the first time, and realizes the modification of saturated fatty chains of different lengths of marine galacto-oligosaccharides with different polymerization degrees. And through cell-level pharmacodynamic evaluation, it is proved that marine galacto-oligosaccharide lipid can significantly inhibit the release of nitric oxide and the expression of inflammatory factors such as interleukin-6 and tumor necrosis factor α in microglia, and has the activity of inhibiting neuroinflammatory; at the same time, it can significantly inhibit the proliferation of tumor cells U87MG and GL261, has certain anti-tumor potential, and can be used to prepare drugs for preventing and / or treating neuroinflammation and anti-tumor related diseases. Therefore, the marine galacto-oligosaccharide lipid prepared by the present invention has high clinical application value and development prospects.

Description

Technical Field

[0001] The present invention belongs to the field of marine biological resource utilization and drug development, and specifically relates to the preparation of marine galacto-oligosaccharide lipids and their application in anti-neuroinflammatory and anti-tumor drugs. Background Art

[0002] The ocean covers over 70% of the world's total surface area, and this vast marine environment has spawned a vast marine ecosystem. Polysaccharides are one of the most abundant components of organic carbon in the marine environment. However, the large molecular weight and complex structure of marine polysaccharides result in low solubility and high solution viscosity, limiting their application. Marine oligosaccharides, which can be obtained by degrading marine polysaccharides, have excellent solubility and are easily absorbed and utilized by the body. They have been widely used in anti-tumor, immunomodulatory, and anti-inflammatory fields, and are a hot topic in marine drug research.

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

[0004] The purpose of the present invention is to provide a marine galacto-oligosaccharide lipid, a preparation method of the marine galacto-oligosaccharide lipid, and a specific application of the marine galacto-oligosaccharide lipid, so as to make up for the deficiencies of the prior art.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] A marine galacto-oligosaccharide lipid, the chemical formula structure of which is shown in Formula I or Formula II:

[0007] Formula I;

[0008] Formula II;

[0009] Wherein, in formula I, n=0-4, m=4-16; in formula II, n=0-4, m=4-16.

[0010] A method for preparing marine galacto-oligosaccharide lipids, using agar as raw material, and controllably degrading it through chemical and bioenzymatic methods to prepare galacto-oligosaccharides with different polymerization degrees, and then modifying the galacto-oligosaccharides with fatty chains of different lengths to obtain marine galacto-oligosaccharide lipids; the method specifically comprises the following steps:

[0011] (1) The β-agarase-mediated site-specific enzymatic hydrolysis process is synergistically combined with a controlled acid degradation process to achieve the selective preparation of target oligosaccharide chain length by precisely controlling the reaction pH, temperature, enzyme / substrate ratio, and hydrolysis time;

[0012] (2) Based on graphitized carbon column chromatography separation technology, high-purity galacto-oligosaccharide monomer components were purified by ethanol gradient elution, providing a highly uniform substrate for subsequent fatty chain modification studies.

[0013] (3) Agar-derived galacto-oligosaccharide with a degree of polymerization of 2-10 was used as a substrate for the fatty chain modification reaction. The reaction system was based on methanol. Galacto-oligosaccharide (1.0 equivalent), amino-modified saturated fatty chain (1.0 to 5.0 equivalent), and NaBH3CN (1.0 to 5.0 equivalent) were dissolved in the methanol system. The pH value of the reaction system was adjusted to 4.0 to 7.0, the reaction temperature was 10 to 60 °C, the rotation speed was 10-200 r / min, and the reaction was carried out under nitrogen protection for 10 to 24 h. The reaction progress was tracked by thin-layer chromatography, and a developing solvent of a mixture of n-butanol: formic acid: water = 4:6:1 by volume was used for detection.

[0014] (4) After obtaining the galacto-oligosaccharide lipid by the above-mentioned 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; finally, the target product is separated and purified using a silica gel column and a C18 column to obtain a marine galacto-oligosaccharide lipid. The mobile phase for silica gel column purification is dichloromethane:methanol = 8:1-1:1, and each gradient elution is 2-5 column volumes to collect the fraction containing the target product. Further, the target product is finely separated using a C18 column, and the mobile phase for C18 column purification is 20%-100% methanol, and each gradient elution is 3 column volumes. The product separation is monitored by TLC to achieve accurate preparation of the target product. The use of the marine galacto-oligosaccharide lipid in the preparation of anti-neuroinflammatory products; the product is a medicine or health product.

[0015] Application of the marine galacto-oligosaccharide lipid in the preparation of anti-tumor drugs.

[0016] Among them, anti-neuroinflammatory products include: neuroinflammatory inhibitors, products for treating and / or preventing nervous system diseases caused by neuroinflammation.

[0017] The nervous system diseases include: Parkinson's disease, Alzheimer's disease, multiple sclerosis and Huntington's disease, vascular dementia, stroke, depression, schizophrenia, meningitis, etc.; the tumors include: human brain glioma, liver cancer, lung cancer, skin cancer, prostate cancer, gastric cancer, breast cancer, colorectal cancer, melanoma, etc.

[0018] The drugs of the present invention containing the compounds represented by formula I-II as active ingredients can be introduced into the body, such as into muscle, intradermal, subcutaneous, intravenous, or mucosal tissues, by injection, spraying, nasal drops, eye drops, penetration, absorption, or physical or chemically mediated methods; or can be introduced into the body after being mixed or encapsulated with other substances.

[0019] When necessary, one or more pharmaceutically acceptable carriers may be added to the above-mentioned drugs. The carriers include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0020] The above-mentioned drugs can be prepared into various forms such as injection, tablet, powder, granule, capsule, oral solution, ointment, cream, etc. The above-mentioned drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0022] This invention combines, for the first time, marine red algae polysaccharide degradation technology with the high efficiency of reductive amination fatty chain modification, achieving saturated fatty chain modification of varying lengths in marine galacto-oligosaccharides with varying degrees of polymerization. Cell-based pharmacodynamic evaluations demonstrated that these marine galacto-oligosaccharide lipids significantly inhibited 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, demonstrating neuroinflammatory activity. Furthermore, they significantly inhibited the proliferation of tumor cells U87MG and GL261, demonstrating potential anti-tumor activity. These lipids could be used to prepare drugs for the prevention and / or treatment of neuroinflammatory and anti-tumor-related diseases. Therefore, they possess high clinical application value and promising development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The marine galacto-oligosaccharide lipid NMR in Example 1 1 H spectrum.

[0024] Figure 2 The marine galacto-oligosaccharide lipid NMR in Example 1 13 C spectrum.

[0025] Figure 3 : The lipid spectra of marine galacto-oligosaccharide in Example 1, wherein 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.

[0026] Figure 4 The effect of marine galacto-oligosaccharide lipids on LPS-induced cytotoxicity (A) and expression of inflammatory mediators such as NO (B), TNF-α (C), and IL-6 (D) in BV2 microglia cells in Example 2; 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).

[0027] Figure 5 The effect of different concentrations of marine galacto-oligosaccharide lipids on tumor cell viability in Example 3; A is a microscopic observation of U87MG cell morphology after treatment with compound 3, and B is the effect of compound 3 on U87MG cell viability; * indicates the difference between the experimental group and the control group (* P < 0.05, ** P < 0.01,*** P < 0.001, **** P < 0.0001).

[0028] Figure 6 The effect of different concentrations of marine galacto-oligosaccharide lipids on tumor cell viability in Example 3; A is a microscopic observation of GL261 cell morphology after treatment with compound 3, and B is the effect of compound 3 on GL261 cell viability; * indicates the difference between the experimental group and the control group (* P < 0.05, ** P < 0.01,*** P < 0.001, **** P < 0.0001). DETAILED DESCRIPTION

[0029] Next, the technical solution of the present invention will be further described with reference to specific embodiments, but the present invention is not limited thereto.

[0030] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0031] Example 1: Preparation and characterization of marine galacto-oligosaccharide lipids

[0032] The molecular reaction formula for preparing marine galacto-oligosaccharide lipids is:

[0033] .

[0034] Preparation Method: Agar oligosaccharides with a degree of polymerization of 2-10 were used as substrates for the fatty chain modification reaction. In a methanol-based reaction system, galacto-oligosaccharide (1.0 equivalent), amino-modified saturated fatty chains (1.0-5.0 equivalents), and NaBH3CN (1.0-5.0 equivalents) were dissolved in methanol. The pH of the reaction system was adjusted to 4.0-7.0. The reaction temperature was 10-60°C, and the rotation speed was 10-200 rpm under nitrogen for 10-24 hours. Reaction progress was monitored by thin-layer chromatography using a 4:6:1 volume ratio of n-butanol:formic acid:water as the developing solvent. After obtaining the galacto-oligosaccharide lipid via the above synthetic method, dilute hydrochloric acid (HCl) was added dropwise to the reaction solution to adjust the pH to 3-4 (operated under a fume hood), and the solvent was evaporated to dryness. The target product was isolated and purified using silica gel and C18 columns. The silica gel column purification process involved a mobile phase of dichloromethane to methanol (8:1 to 1:1) with a gradient elution of 2-5 column volumes. Fractions containing the target product were collected. Furthermore, the target product was finely separated using a C18 column with a mobile phase of 20% to 100% methanol with a gradient elution of 3 column volumes. Product separation was monitored by TLC, enabling precise preparation of the target product.

[0035] Compounds 1-4 mentioned in Table 1 correspond to the compounds described in Formula I, wherein, in Formula I, n=0-4, m=4-16; in Formula II, n=0-4, m=4-16.

[0036] Table 1 Structures of marine galacto-oligosaccharides

[0037]

[0038] This example uses compound 1-4 as an example 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 the glycolipid derivatives generated by using agar oligosaccharides with a degree of polymerization of 2-10 as reaction substrates are all within the scope of protection of the patent of this invention. Among them, the reducing end of agar oligosaccharides is a galactose unit, and having this structural feature can achieve an efficient reductive amination reaction. Therefore, using agar oligosaccharides with a degree of polymerization of 2-10 as reaction substrates and amino-modified saturated fatty chains as donors, after a reductive amination reaction, the compounds shown in formula I-II can be obtained. Compound 1-4 1 H NMR Figure 1As shown, characteristic peaks of the lipid chains appeared in the high-field region. The peak at δ 0.90 represents the terminal methyl group, and the peaks at δ 1.33–1.53 represent the methylene groups on the octylamine or stearylamine lipid chains, confirming successful modification of the lipid chains. In the low-field region, the anomeric proton signals of the neoagaron oligosaccharide appeared, along with the anomeric proton signals of the neoagaronobiose 3,6-ether galactose at δ 4.93 and the anomeric proton signals of the neoagaronatetraose 3,6-ether galactose at δ 5.13 and δ 4.96. Furthermore, the α-hydrogen signal of the anomeric position of the galactose reducing end of the oligosaccharide at δ 5.25 disappeared, indicating ring opening at the reducing end.

[0039] 13 C NMR results are as follows Figure 2 As shown, the carbon signal on the fatty chain appears in the high field area, and the anomeric carbon signal on the oligosaccharide appears in the low field area.

[0040] Combined with HRMS data to confirm that the molecular weight matches the theoretical value, e.g. Figure 3 As shown, compounds 1-4 respectively showed [M+H] + The signal peaks are m / z=438.27, 744.37, 578.43, and 884.53.

[0041] The above results indicate that marine galacto-oligosaccharide lipids were successfully prepared.

[0042] Example 2: Marine galacto-oligosaccharide lipids inhibit the release of inflammatory mediators in BV2 microglia induced by bacterial lipopolysaccharide (LPS)

[0043] CCK8 experimental steps:

[0044] Mouse microglial BV2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and a 1% penicillin-streptomycin mixture at 37°C in a 5% CO2 incubator. BV2 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 5,000–10,000 cells per well. Each well typically contained 200 μL of culture medium, and the edge wells were filled with sterile PBS to prevent edge effects. For the control group, 200 μL of culture medium was added to each well. For the experimental group, an equal volume of culture medium containing 50 μmol / L of drug was added to each well. For the blank group, an equal volume of culture medium alone was added without cells. Five replicates were set up for each group. The 96-well plates were returned to the incubator and removed after 24 hours. 10 μL of CCK-8 solution was added to each well and returned to the incubator. Two hours later, the absorbance (OD) of each well was measured at 450 nm using a microplate reader. Cell survival rate (%) was calculated for each concentration group: {(OD)}(% ) = ... 样品 -OD 空白 ) / (OD 对照 -OD 空白 )}×100%.

[0045] like Figure 4 As shown in Figure A, at a concentration of 50 μmol / L, compounds 1-2 (octylamine-glycolipid derivatives) had no significant effect on BV2 cell proliferation, demonstrating that octylamine-glycolipid derivatives have good biosafety and are non-cytotoxic. In contrast, compounds 3-4 (stearylamine-glycolipid derivatives) inhibited BV2 cell proliferation. These results indicate that saturated fatty acid chain length influences bioactivity.

[0046] Next, compound 1-2 is used as an example to further illustrate the anti-neuroinflammatory activity of marine galacto-oligosaccharide lipids.

[0047] Inflammatory mediator release detection steps:

[0048] BV2 mouse microglia in the logarithmic growth phase were cultured at a rate of 1×10 4 Cells were seeded at a density of 100 cells / well in a 96-well culture plate. After 24 hours of cell attachment, they were pre-incubated with different marine galacto-oligosaccharides at a final concentration of 50 μM for 2 hours and then incubated with or without LPS (final concentration of 1 μg / mL) for 24 hours. The supernatant was collected and centrifuged at 8000 rpm and 4°C for 10 minutes. The supernatant was taken and the levels of NO, TNF-α, and IL-6 were measured using a commercially available kit (Shanghai Beyotime Biotechnology Co., Ltd.) according to the manufacturer's instructions. Figure 4 As shown in Figure B, compared with the normal control group, the NO content in the supernatant of BV2 cells in the LPS-treated group was significantly increased (P < 0.0001), indicating that the neuroinflammation model was successfully established. When compound 1-2 was added for intervention, NO release showed a significant inhibitory trend. The expression levels of inflammatory factors are shown in Figure 4. Figure 4 As shown in C and D, compared with the normal control group, the levels of TNF-α and IL-6 in the supernatant of BV2 cells in the LPS stimulation group were significantly increased ( P < 0.05), and a final concentration of 50 μM marine galacto-oligosaccharide lipids (compounds 1-2) significantly inhibited the production of inflammatory factors in LPS-activated microglia, with a particularly significant inhibitory effect on IL-6. These results suggest that marine galacto-oligosaccharide lipids possess significant anti-neuroinflammatory activity.

[0049] The above experiments demonstrate that compounds 1-2 are able to 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 structural similarity of the compounds of Formula I-II is extremely high, differing only at the non-reducing end. The core pharmacological characteristic of this series of compounds is amino-fatty chain modification, and the structural differences of the compounds of Formula I-II are located away from this core pharmacophore. Therefore, based on the similarity of molecular structures and the general principles of medicinal chemistry, the present invention reasonably anticipates that all compounds encompassed by Formula I-II have anti-neuroinflammatory activity equivalent to or similar to that of the specific compounds 1 and 2.

[0050] Example 3: Marine galacto-oligosaccharide lipids inhibit tumor cell proliferation

[0051] In this example, the CCK-8 method was used to detect the growth inhibitory effect of compound 3 on brain tumor cells U87MG and GL261 to preliminarily explore its anti-tumor activity.

[0052] The experimental steps are as follows:

[0053] Mouse glioma cells GL261 were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and a 1% penicillin-streptomycin mixture. Human astrocytoblastoma cells U87MG were cultured in MEM supplemented with 10% fetal bovine serum and a 1% penicillin-streptomycin mixture. 5,000 U87MG and GL261 cells were seeded per well of a 96-well plate. After 24 hours of culture, the supernatant was aspirated. A control group (200 μL of culture medium) was added to each well. An equal volume of culture medium containing compound 3 at varying concentrations of 1.25, 2.5, 5, 10, 25, 50, and 100 μg / mL was added to each well. A blank control group was set up, in which only culture medium was added without cells. Three replicates were set up for each group. After 24 hours of culture, tumor cell growth was observed using an inverted microscope, and the inhibitory effect of the compound on tumor cell growth was determined using a CCK-8 assay.

[0054] Furthermore, the results are Figure 5 and Figure 6As shown, while the blank group showed normal growth of U87MG and GL261 cells, the compound 3 treatment group showed inhibition of cell growth with increasing concentration, with cells shrinking and becoming rounded, and significant cell fragmentation appeared at high concentrations. Furthermore, at concentrations of 50 and 100 μg / mL, the cell viability of tumor cells was less than 20%, indicating that compound 3 significantly inhibited tumor cell proliferation and growth, demonstrating its potential as an anti-tumor agent. Furthermore, it was shown that long-chain fatty acid modification can render oligosaccharides cytotoxic and enhance their anti-tumor activity.

[0055] The above experiments demonstrate that compound 3 significantly inhibits the proliferation of U87MG and GL261 tumor cells, demonstrating its potential as an anti-tumor agent. This anti-tumor activity stems from its specific core pharmacodynamic characteristic: amino-aliphatic chain modification, which significantly enhances biological activity. Based on this core structure and known principles of medicinal chemistry, the present invention reasonably anticipates that all compounds encompassed by Formulas I-II exhibit comparable or similar anti-tumor activity to compound 3.

[0056] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

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

2. The method for preparing the marine galacto-oligosaccharide lipid according to claim 1, characterized in that: Using agar as raw material, galacto-oligosaccharides with different polymerization degrees are prepared through controllable degradation by chemical and bioenzymatic methods. The galacto-oligosaccharides are then modified with fatty chains of different lengths to obtain marine galacto-oligosaccharide lipids.

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

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

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

6. The use according to claim 4, characterized in that Anti-neuroinflammatory products include products that treat and / or prevent neurological diseases caused by neuroinflammation.

7. The use according to claim 6, characterized in that The nervous system diseases include: Parkinson's disease, Alzheimer's disease, multiple sclerosis and Huntington's disease, vascular dementia, stroke, depression, schizophrenia, and meningitis.

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