Glycolipid derivatives, preparation methods and their application in the preparation of drugs for treating nerve damage

Abstract

This invention belongs to the field of pharmaceutical technology and discloses a glycolipid derivative, its preparation method, and its application in the preparation of drugs for treating neuropathic damage. This invention uses Convolvulella tricolor seeds as the isolation target and comprehensively utilizes multiple separation techniques and methods to obtain a glycolipid derivative with a novel chemical structure. In vitro anti-neuropathic damage experiments have confirmed that the glycolipid derivative provided by this invention has significant anti-neurooxidative damage activity and can be used as an active ingredient in drugs for treating neuropathic damage, thus having a wide range of applications.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a glycolipid derivative, its preparation method, and its application in the preparation of drugs for preventing nerve damage. Background Technology

[0002] *Convolvulus tricolor* L. is a herbaceous plant belonging to the genus *Convolvulus* in the family Convolvulaceae. Native to the Mediterranean coast and North Africa, it is widely cultivated in my country due to its rich flower colors and high ornamental value. Currently, research on the chemical composition and biological activities of this plant is limited; only small amounts of alkaloids, coumarins, flavonoids, and galactomannans have been isolated from its flowers and seeds. For example, Kooiman et al. isolated galactomannan compounds from the mature seeds of *Convolvulus tricolor*; Kacem et al. isolated caffeoylquinic acid glycosides from the seed coat of *Convolvulus tricolor*.

[0003] Resin glycosides are characteristic glycolipid derivatives of plants in the Convolvulaceae family. Pharmacological studies have shown that many resin glycoside compounds possess significant biological activities, such as cytotoxicity, antiviral activity, antiepileptic activity, sedation, vasodilatory activity, and α-glucosidase inhibition, providing diverse 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 components of some Convolvulaceae family and Convolvulium genus herbaceous plants and develop novel active resin glycoside compounds. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a glycolipid derivative, a preparation method thereof, and its application in the preparation of drugs for preventing nerve damage.

[0006] To address the above problems, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a glycolipid derivative, said glycolipid derivative being a compound with any of the following structural formulas:

[0008]

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned glycolipid derivative, specifically: the glycolipid derivative is isolated 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, which involves selecting seeds of Convolvulia tricolor, extracting them by reflux with a 95% ethanol aqueous solution, and then obtaining glycolipid derivatives with structures shown in Formulas I-V through various separation techniques and methods. These derivatives exhibit significant anti-neurooxidative damage activity and can be used as active ingredients in the preparation of anti-neurodamage drugs, thus having a wide range of applications.

[0011] In some embodiments of the present invention, the preparation method of the glycolipid derivative includes the following steps:

[0012] 1) Extract the seeds of Convolvulus tricolor using an aqueous ethanol solution, and then concentrate them under vacuum to obtain an extract;

[0013] 2) The extract was separated by silica gel column chromatography and gradient elution was performed using a dichloromethane-methanol two-phase system to obtain the enriched fraction of resin glycosides;

[0014] 3) The enriched fractions of the resin glycosides were repeatedly purified by reversed-phase ODS column chromatography and amino silica gel column chromatography, and the fractions were collected and combined to obtain the target fraction;

[0015] 4) The target component is purified by reversed-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 gradient elution using a dichloromethane-methanol two-phase system specifically involves eluting with a dichloromethane-methanol mixed solvent in 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, gradient elution is performed using a dichloromethane-methanol two-phase system after the amino silica gel column chromatography.

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

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

[0022] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the glycolipid derivative of claim 1 or a pharmaceutically acceptable salt thereof.

[0023] Compared with existing technologies, this invention selects the seeds of *Convolvulus tricolor* as the target, extracts them using ethanol under reflux, and then obtains the aforementioned glycolipid derivatives of formulas I-V through various separation techniques and methods. These derivatives possess novel oligosaccharide chain structures, with formulas I and II being rare glycolipid derivatives containing eight sugar residues. These novel glycolipid derivatives exhibit significant anti-neurooxidative damage activity and can be used as active ingredients in the preparation of anti-neurodamage drugs, possessing a wide range of applications. Attached Figure Description

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

[0025] Figure 2 This is the proton NMR spectrum of compound II obtained in Example 1 of the present invention;

[0026] Figure 3 This is the proton NMR spectrum of compound III obtained in Example 1 of the present invention;

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

[0028] Figure 5 This is a hydrogen spectrum of compound V obtained in Example 1 of the present invention. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0030] Example 1: Preparation of the compound

[0031] This invention utilizes the seeds of *Convolvulus tricolor* (tricolor bindweed) for reflux extraction with 95% ethanol, and prepares the compound of this invention through various separation techniques and methods. The *Convolvulus tricolor* seeds were purchased in Beijing.

[0032] 1) Extraction

[0033] Five kilograms of Tricolor Convolvulia seeds were crushed and then refluxed three times with a 95% ethanol aqueous solution (5 times the volume of the extract) for three hours each time. The extracts were combined and concentrated under reduced pressure to obtain an extract (500g).

[0034] 2) Separation by silica gel chromatography

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

[0036] 3) Reverse-phase ODS column chromatography

[0037] Components 2 and 4 were subjected to reversed-phase ODS column chromatography 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 4.1–4.3, respectively.

[0038] 4) Amino silica gel column chromatography

[0039] Components 2.2 and 4.2 were subjected to amino silica gel column chromatography with dichloromethane-methanol (5:1, 2:1, 1:1, 0:1, v / v) as the eluent to obtain components 2.2.1–2.2.4 and 4.2.1–4.2.4, respectively.

[0040] 5) Reversed-phase ODS column chromatography

[0041] Components 2.2.2 and 4.2.2 were subjected to reversed-phase ODS column chromatography, using methanol-water solutions (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, respectively, to obtain components 2.2.2.1–2.2.2.8 and components 4.2.2.1–4.2.2.8.

[0042] 6) Amino silica gel column chromatography

[0043] Fraction 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 the eluent to obtain fractions 4.2.2.6.1 to 4.2.2.6.7.

[0044] 7) Reversed-phase high-performance liquid chromatography purification

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

[0046] The proton NMR spectrum of compound IV is shown below. Figure 1-5 As shown, by analyzing Figures 1-5 The analysis was performed, and the mass spectrometry and wavelength spectrometry data of compound IV are shown below.

[0047] Compound I: controlin III; major absorption peak in 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 proton and carbon NMR spectra are shown in Table 1.

[0048] Compound II: controlin VII; major absorption peak in 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 proton and carbon NMR spectra are shown in Table 1.

[0049] Compound III: Controlin XIV; Major absorption peak in 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 proton and carbon NMR spectra are shown in Table 1.

[0050] Compound IV: Controlin XV; Major absorption peak in 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 proton and carbon NMR spectra are shown in Table 2.

[0051] Compound V: Controlin XVI; Major absorption peak in 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 proton and carbon NMR spectra are shown in Table 2.

[0052] Table 1. 1H and 1C NMR spectra 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. 1H and 1C NMR spectra 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 indicate that the structural formula of the obtained compound IV is chemical formula IV.

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

[0062] 1) Experimental materials

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

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

[0065] Test samples: Resin glycoside derivatives of formulas I to V obtained from seeds of Convolvulia tricolor; donepezil hydrochloride was selected as a positive control drug. All compounds were dissolved in DMSO and then diluted.

[0066] 2) Experimental methods

[0067] The effect of each test compound on the viability of H2O2-damaged SH-SY5Y cells was determined using the MTT assay: cells were digested with trypsin and counted, and the cell density of the cell suspension was adjusted to 1×10⁻⁶. 5 Cells were cultured at a concentration of 100 μL / mL in 96-well plates, 200 μL per well, and incubated overnight in a 5% CO2 incubator at 37°C. Cells were divided into control, model, and sample test groups. Blank culture medium was added to the control and model groups, while different concentrations of sample were added to the sample test groups. After 2 hours of incubation, blank culture medium was added to the control group, and H2O2 (500 μM) was added to the model and sample test groups. The final volume of each well was 200 μL, with three replicates for each concentration. After 24 hours of drug treatment, 20 μL of MTT solution (5 mg / mL) was added to each well. After 4 hours, the culture medium was discarded, and 150 μL of LDMSO was added to each well. The crystals were thoroughly shaken and mixed, and the absorbance of each well was measured at 570 nm using a microplate reader. Cell viability was calculated as: Cell viability = OD value of sample test group / OD value of control group.

[0068] 3) Experimental Results

[0069] Based on the MTT assay results, the survival rates of PC12 cells damaged by H2O2 induced by compounds I–V of this invention were calculated and 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 PC12 cell survival rate under H2O2 damage by test samples

[0070]

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

[0072] Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A glycolipid derivative, characterized in that, The glycolipid derivative is a compound with any of the following structural formulas:

2. A method for preparing a glycolipid derivative as described in claim 1, characterized in that, Includes the following steps: 1) Extract the seeds of Convolvulus tricolor using an ethanol-water solution, and then concentrate them under vacuum to obtain an extract; 2) The extract was separated by silica gel column chromatography and eluted with a gradient of dichloromethane-methanol two-phase system to obtain the enriched fraction of resin glycosides; 3) The enriched fractions of the resin glycosides were repeatedly purified by reversed-phase ODS column chromatography and amino silica gel column chromatography, and the fractions were collected and combined to obtain the target fraction; 4) The target component is purified by reversed-phase high-performance liquid chromatography to obtain the glycolipid derivative.

3. The preparation method according to claim 2, characterized in that, In step 1), the volume fraction of the ethanol aqueous solution is 90%-95%.

4. The preparation method according to claim 2, characterized in that, In step 2), the gradient elution using a dichloromethane-methanol two-phase system specifically involves eluting with a dichloromethane-methanol mixed solvent in volume ratios of 1:0, 10:1, 5:1, 2:1, 1:1, and 0:

1.

5. The preparation method according to claim 2, characterized in that, In step 3), the reversed-phase ODS column chromatography is followed by gradient elution using a methanol-water solution two-phase system.

6. The preparation method according to claim 2, characterized in that, In step 3), the amino silica gel column chromatography is followed by gradient elution using a dichloromethane-methanol two-phase system.

7. The preparation method according to claim 2, characterized in that, In step 4), the conditions for the reversed-phase high-performance liquid chromatography purification are: a YMC RP-C18 column with a specification of 10×200 mm, a mobile phase of methanol-water solution, and a flow rate of 2.0 mL / min.

8. The use of a glycolipid derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of an anti-neural injury drug.

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

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