Glycolipid derivative, preparation method and application of glycolipid derivative in preparation of nerve injury resisting medicine

Abstract

The invention belongs to the technical field of medicines, and discloses a glycolipid derivative, a preparation method and application of the glycolipid derivative in preparation of nerve injury resisting medicines. The glycolipid derivative with a novel chemical structure is obtained by taking the sedge tricolor seeds as a separation target and comprehensively applying various separation technologies and means. An in-vitro nerve injury resisting experiment proves that the glycolipid derivative provided by the invention has remarkable nerve oxidative injury resisting activity, can be used as an active ingredient of a nerve injury resisting medicine, and has wide application.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a glycolipid derivative, a preparation method and application thereof in the preparation of an anti-nerve injury drug. Background Art

[0002] Convolvulus tricolor L. is a herbaceous plant of the genus Convolvulus in the family Convolvulaceae. Convolvulus tricolor is native to the Mediterranean coast, North Africa and other regions. Due to its rich flower colors and high ornamental value, it is cultivated in various parts of my country. At present, there are few studies on the chemical composition and biological activity of this plant. Only a small amount of alkaloids, coumarins, flavonoids and galactomannan compounds have been isolated from flowers and seeds. For example, Kooiman et al. isolated galactomannan compounds from the mature seeds of Convolvulus tricolor; Kacem et al. isolated caffeoylquinic acid glycoside compounds from the seed shells of Convolvulus tricolor.

[0003] Resin glycosides are characteristic glycolipid derivatives of Convolvulaceae plants. Pharmacological studies have shown that many resin glycoside compounds have significant biological activities, such as cytotoxicity, antiviral, anti-epileptic, sedative, vasodilation, and α-glucosidase inhibition, providing a variety of template molecules for drug development. Therefore, these novel structures are increasingly attracting the attention of drug researchers.

[0004] Therefore, it is necessary to study the active ingredients of some herbaceous plants of the genus Convolvulaceae and develop active resin glycoside compounds with novel structures. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a glycolipid derivative, a preparation method and use thereof in the preparation of an anti-nerve injury drug.

[0006] To solve the above problems, the present invention provides the following technical solutions:

[0007] In the first aspect of the present invention, the present invention provides a glycolipid derivative, wherein the glycolipid derivative is a compound having a structure represented by any of the following structural formulas:

[0008]

[0009] In the second aspect of the present invention, the present invention provides a method for preparing the above-mentioned glycolipid derivatives, specifically: isolating the glycolipid derivatives from the seeds of Convolvulus tricolor L.

[0010] In some embodiments of the present invention, the invention also provides a method for preparing the glycolipid derivatives, wherein the seeds of Convolvulus tricolor are selected, reflux extracted with a 95% ethanol aqueous solution, and then various separation techniques and means are used to obtain glycolipid derivatives of structures shown in Formula I to Formula V, which have significant anti-neural oxidative damage activity and can be used as an active ingredient for preparing anti-neural damage drugs, and have a wide range of uses.

[0011] In some embodiments of the present invention, the method for preparing the glycolipid derivative comprises the following steps:

[0012] 1) extracting the seeds of Convolvulus tricolor with an ethanol aqueous solution, and then concentrating them in vacuum to obtain an extract;

[0013] 2) separating the extract by silica gel column chromatography, using a dichloromethane-methanol two-phase system for gradient elution to obtain a resin glycoside enriched portion;

[0014] 3) repeatedly purifying the enriched part of the resin glycoside by reverse phase ODS column chromatography and amino silica gel column chromatography, collecting and combining the components to obtain the target component;

[0015] 4) Purifying the target component by reverse phase high performance liquid chromatography to obtain the glycolipid derivative.

[0016] In some embodiments of the present invention, in step 1) of the preparation method, the volume fraction of the ethanol aqueous solution is 90%-95%.

[0017] In some embodiments of the present invention, in step 2) of the preparation method, the dichloromethane-methanol two-phase system gradient elution is specifically: elution is carried out using dichloromethane-methanol mixed solvents with volume ratios of 1:0, 10:1, 5:1, 2:1, 1:1 and 0:1 in sequence.

[0018] In some embodiments of the present invention, in step 3) of the preparation method, the reversed-phase ODS column chromatography is followed by gradient elution using a methanol-water solution two-phase system.

[0019] In some embodiments of the present invention, in step 3) of the preparation method, the amino silica gel column chromatography is followed by gradient elution using a dichloromethane-methanol two-phase system.

[0020] In some embodiments of the present invention, in step 4) of the preparation method, the conditions for the reversed-phase high performance liquid chromatography purification are: a YMC RP-C18 chromatographic column with a specification of 10×200 mm, a methanol-water solution as the mobile phase, and a flow rate of 2.0 mL / min.

[0021] The third aspect of the present invention provides a use of the above-mentioned glycolipid derivatives or pharmaceutically acceptable salts thereof as active ingredients in the preparation of anti-nerve injury drugs.

[0022] The fourth aspect of the present invention provides a pharmaceutical composition, which comprises the glycolipid derivative according to claim 1 or a pharmaceutically acceptable salt thereof.

[0023] Compared with the prior art, the present invention selects the seeds of Convolvulus tricolor as the object, performs reflux extraction with ethanol, and then obtains the above-mentioned glycolipid derivatives of formula I to formula V through various separation techniques and means, which have novel oligosaccharide chain structures, wherein formulas I and II are rare glycolipid derivatives containing eight sugar residues. Such novel glycolipid derivatives have significant anti-nerve oxidative damage activity, can be used as active ingredients for preparing anti-nerve damage drugs, and have a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a hydrogen spectrum data diagram of compound I obtained in Example 1 of the present invention;

[0025] Figure 2 This is a hydrogen spectrum data diagram of compound II obtained in Example 1 of the present invention;

[0026] Figure 3 This is a hydrogen spectrum data diagram of compound III obtained in Example 1 of the present invention;

[0027] Figure 4 This is a hydrogen spectrum data diagram of compound IV obtained in Example 1 of the present invention;

[0028] Figure 5 This is a hydrogen spectrum data diagram of compound V obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The operating methods in the following examples where no specific conditions are indicated are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0030] Example 1: Preparation of compounds

[0031] The invention uses seeds of Convolvulus tricolor to perform reflux extraction with 95% ethanol and prepares the compound of the invention through various separation techniques and means. The seeds of Convolvulus tricolor are purchased in Beijing.

[0032] 1) Extraction

[0033] Tricolor bindweed seeds (5 kg) were crushed, and 5 times the volume of 95% ethanol aqueous solution was added, and hot reflux extraction was performed 3 times, each time for 3 hours. After the extracts were combined, they were concentrated under reduced pressure to obtain an extract (500 g).

[0034] 2) Silica gel chromatography separation

[0035] The extract was subjected to silica gel column chromatography and gradient elution was performed using a dichloromethane / methanol mixed solvent (1:0, 10:1, 5:1, 2:1, 1:1, 0:1, v / v) to obtain components 1 to 8, respectively.

[0036] 3) Reverse phase ODS column chromatography

[0037] Component 2 and component 4 were respectively chromatographed by reverse phase ODS column with methanol-water solution (50:50, 70:30, 80:20, 85:15, 90:10, 95:5, 100:0, v / v) as eluent to obtain components 2.1-2.3 and components 4.1-4.3, respectively.

[0038] 4) Amino silica gel column chromatography

[0039] Component 2.2 and component 4.2 were respectively chromatographed on an amino silica gel column with dichloromethane-methanol (5:1, 2:1, 1:1, 0:1, v / v) as eluent to obtain components 2.2.1 to 2.2.4 and components 4.2.1 to 4.2.4, respectively.

[0040] 5) Reverse phase ODS column chromatography

[0041] Component 2.2.2 and component 4.2.2 were subjected to reverse phase ODS column chromatography using methanol-water solution (60:40, 70:30, 80:20, 85:15, 90:10, 95:5, 100:0, v / v) and (70:30, 75:25, 80:20, 85:15, 90:10, 95:5, 100:0, v / v) as eluents to obtain components 2.2.2.1 to 2.2.2.8 and components 4.2.2.1 to 4.2.2.8, respectively.

[0042] 6) Amino silica gel column chromatography

[0043] Component 2.2.2.6 was subjected to amino silica gel column chromatography with dichloromethane-methanol (5:1, 2:1, 1:1, 0:1, v / v) as eluent to obtain components 4.2.2.6.1 to 4.2.2.6.7.

[0044] 7) Reverse-phase HPLC purification

[0045] Component 4.2.2.6.3 was purified by reverse phase high performance liquid chromatography. The preparation conditions were preparative chromatographic column YMC RP-C18 (10×200mm), methanol-water (72:28, v / v), flow rate 2.0mL / min, detector Shimadzu RID 10A, and compound formula I was prepared. Component 2.2.2.5 was purified by reverse phase high performance liquid chromatography. The preparation conditions were preparative chromatographic column YMC RP-C18 (10×200mm), methanol-water (79:21, V / V), flow rate 2.0mL / min, detector Shimadzu RID 10A, and compound formula II was prepared. Component 2.2.2.4 was purified by reverse phase high performance liquid chromatography. The preparation conditions are: preparative chromatographic column YMCRP-C18 (10×200 mm), methanol-water (74:26, v / v), flow rate 2.0 mL / min, detector Shimadzu RID 10A, and compounds of formula III to formula V are prepared.

[0046] The hydrogen spectrum data of compound IV is shown in the figure Figure 1-5 As shown, through Figure 1-Figure 5 The mass spectrum and spectroscopy data of compound IV are shown below.

[0047] Compound I: controlin III; main absorption peak of infrared spectrum (KBr) ν max :3420, 2930, 1735, 1650, 1075cm -1 ; [α] 25 D -45.4(c 0.10,MeOH); HRESIMS m / z 890.3856[M+2Na] 2+ (C 75 H 130 Na2O 44 , calculated value 890.3860); the H NMR and C NMR data are shown in Table 1.

[0048] Compound II: controlin VII; main absorption peak of infrared spectrum (KBr) ν max :3421,2933,1733,1650,1073 -1 ; [α] 25 D -21.7(c 0.12,MeOH); UV(MeOH)λmax(logε)218(4.37)nm; HRESIMS m / z981.4325[M+2Na] 2+ (C 85 H 144 Na2O 47, calculated value 981.4331); the H NMR and C NMR data are shown in Table 1.

[0049] Compound III: Controlin XIV; main absorption peak of infrared spectrum (KBr) ν max :3418,2931,1734,1075 -1 ; [α] 25 D -33.7(c 0.09,MeOH); HRESIMS m / z 809.3601[M+2Na] 2+ (C 69 H 120 Na2O 39 , calculated value 809.3596); the H NMR and C NMR data are shown in Table 1.

[0050] Compound IV: Controlin XV; main absorption peak of infrared spectrum (KBr) ν max :3416,2929,1636,1076 -1 ; [α] 25 D -36.4(c 0.05,MeOH); HRESIMS m / z 1249.5669[M+Na] + (C 53 H 94 NaO 31 , calculated value 1249.5671); the H NMR and C NMR data are shown in Table 2.

[0051] Compound V: Controlin XVI; main absorption peak of infrared spectrum (KBr) ν max :3417,2927,1616,1076 -1 ; [α] 25 D -27.5(c 0.04,MeOH); HRESIMS m / z 1249.5677[M+Na] + (C 53 H 94 NaO 31 , calculated value 1249.5671); the H NMR and C NMR data are shown in Table 2.

[0052] Table 1. H NMR and C NMR data of compounds I-III

[0053]

[0054]

[0055] a Overlapped signals are reported without designating multiplicity; b Abbreviations: Glc=glucopyranosyl, Rha=rhamnopyranosyl, Fuc=fucopyranosyl, Qui=quinovopyranosyl, Ag=12-hydroxyhexadecanoyl aglycone, Nla=3-hydroxy-2-methylbutanoyl, Tga=2-methyl-2-butenoyl.

[0056] Table 3. H NMR and C NMR data of compounds IV and V

[0057]

[0058]

[0059] a Overlapped signals are reported without designating multiplicity; b Abbreviations: Glc=glucopyranosyl, Rha=rhamnopyranosyl, Fuc=fucopyranosyl, Ag=12-hydroxyhexadecanoyl aglycone.

[0060] The above results show that the structural formula of the obtained compound IV is respectively chemical formula IV.

[0061] Example 2: Anti-neurooxidative damage activity of compounds I to V

[0062] 1) Experimental Materials

[0063] Instruments and reagents: CO2 incubator (Thermo Fisher Scientific, USA); DMEM culture medium (Gibco, Thermo Fisher Scientific, USA); MSO, MTT and trypsin (Sigma, USA); fetal bovine serum (Hangzhou Sijiqing Biomaterials Research Institute); other commonly used inorganic salts and other reagents were of analytical grade.

[0064] Test cell line: rat adrenal pheochromocytoma PC12 cell line.

[0065] Test samples: resin glycoside derivatives of formula I to formula V obtained from seeds of Convolvulus tricolor; donepezil hydrochloride was selected as a positive control drug, and each compound was dissolved in DMSO and then diluted.

[0066] 2) Experimental methods

[0067] The MTT method was used to determine the effects of each test compound on the activity of SH-SY5Y cells damaged by H2O2: the cells were counted after trypsin digestion, and the cell density of the cell suspension was adjusted to 1×10 5 / mL, add 200μL to each well in a 96-well culture plate, and culture overnight in a 5% CO2, 37℃ constant temperature incubator. The cells were divided into a control group, a model group, and a sample test group. Among them, blank culture medium was added to the control group and the model group, and samples of different concentrations were added to the sample test group. After incubation for 2h, blank culture medium was added to the control group, and H2O2 (500μM) was added to the model group and the sample test group. The final volume of each well was 200μL, and 3 parallels were set for each concentration. After 24h of drug treatment, 20μL of MTT solution (5mg / mL) was added to each well. After 4h, the culture medium was discarded, 150μLDMSO was added to each well, and the crystals were fully shaken and shaken. The absorbance of each well was measured at 570nm as the detection wavelength in the microplate reader, and the cell survival rate was calculated: cell survival rate = sample test group OD value / control group OD value.

[0068] 3) Experimental results

[0069] According to the MTT test results, the results of calculating the survival rate of PC12 cells damaged by H2O2 by compounds I-V of the present invention are shown in Table 3. ### p<0.001, compared with the control group; *p<0.05, **p<0.01, ***p<0.001, compared with the model group) Table 3. Results of the test samples on the survival rate of PC12 cells damaged by H2O2

[0070]

[0071] The results show that compounds I to V of the present invention can improve the PC12 nerve cell damage caused by H2O2 and can be used as active ingredients of drugs for treating nerve damage.

[0072] Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A glycolipid derivative, characterized in that: The glycolipid derivative is a compound represented by any of the following structural formulas: as well as 2. A method for preparing a glycolipid derivative according to claim 1, characterized in that: The preparation method comprises the following steps: separating and obtaining the glycolipid derivative from seeds of Convolvulus tricolor.

3. The preparation method according to claim 2, characterized in that: The steps include: 1) extracting the seeds of Convolvulus tricolor with an ethanol aqueous solution, and then concentrating them in vacuum to obtain an extract; 2) separating the extract by silica gel column chromatography and using a dichloromethane-methanol two-phase system gradient elution to obtain a resin glycoside enriched portion; 3) repeatedly purifying the enriched part of the resin glycoside by reverse phase ODS column chromatography and amino silica gel column chromatography, collecting and combining the components to obtain the target component; 4) Purifying the target component by reverse phase high performance liquid chromatography to obtain the glycolipid derivative.

4. The preparation method according to claim 3, characterized in that: In step 1), the volume fraction of the ethanol aqueous solution is 90%-95%.

5. The preparation method according to claim 3, characterized in that: In step 2), the dichloromethane-methanol two-phase system gradient elution is specifically: dichloromethane-methanol mixed solvents with volume ratios of 1:0, 10:1, 5:1, 2:1, 1:1 and 0:1 are used for elution.

6. The preparation method according to claim 3, characterized in that: In step 3), the reverse phase ODS column chromatography is followed by gradient elution using a methanol-water solution two-phase system.

7. The preparation method according to claim 3, characterized in that: In step 3), the amino silica gel column chromatography is followed by gradient elution using a dichloromethane-methanol two-phase system.

8. The preparation method according to claim 3, characterized in that: In step 4), the conditions for the reversed-phase high performance liquid chromatography purification are: a YMC RP-C18 chromatographic column with a specification of 10×200 mm, a methanol-water solution as the mobile phase, and a flow rate of 2.0 mL / min.

9. Use of the glycolipid derivative or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient in the preparation of an anti-nerve injury drug.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the glycolipid derivative according to claim 1 or a pharmaceutically acceptable salt thereof.

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