Opculinic acid macrocyclic resin glycoside compound as well as preparation method and application thereof
By isolating and preparing the Operculinic acid macrocyclic resin glycoside compounds from the whole grass of the heart calyx, the problem of lack of structurally novel active resin glycoside compounds in the prior art was solved, and the preparation of compounds with significant anti-tumor activity was achieved, and its application prospects in anti-tumor drugs were opened.
Patent Information
- Application Number
- CN202411990381.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-15
AI Technical Summary
There is a lack of research and development of novel structures in the prior art, especially the Operculinic acid macrocyclic resin glycoside compounds, and their application in anti-tumor drugs has not been fully developed.
By isolating and preparing the Operculinic acid macrocyclic resin glycoside compounds from the calyxagrass, the Operculinic acid D and Operculinic acid A compounds with significant biological activity were obtained by using polar solvent extraction, ethyl acetate extraction, silica gel column chromatography separation, ODS column chromatography separation, gel column chromatography separation and reverse phase high performance liquid chromatography purification.
The unreported macrocyclic resin glycoside compound of Operaculinic acid has been successfully prepared, which has significant anti-tumor activity and can be used as an active ingredient of anti-tumor drugs and has a wide range of uses.
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Figure CN119823199A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medicines and relates to an Operculinic acid macrocyclic resin glycoside compound and a preparation method and application thereof. Background Art
[0002] Resin glycosides are characteristic components of Convolvulaceae plants, and their structure is usually composed of three parts: oligosaccharide chain, aglycone and modified fatty acyl group. Oligosaccharide chain is generally composed of 3 to 7 monosaccharide residues, and aglycone is usually a long-chain hydroxy fatty acid, and is often connected to the hydroxyl group on the oligosaccharide chain through an ester bond to form a 15-27-membered macrolide structure. Due to the changes in the type of oligosaccharide chain, the position of the lactone bond, the esterification position of different substituents, and the formation of dimers, the structure of resin glycoside compounds is complex and changeable. Pharmacological studies have shown that many resin glycoside compounds have significant biological activities, such as cytotoxicity, anti-tumor cell migration, antiviral, anti-epileptic, vasodilation, α-glucosidase inhibition, and reversal of tumor and bacterial multidrug resistance, etc., providing a variety of template molecules for drug development for a variety of diseases.
[0003] Therefore, it is necessary to study the active ingredients of some plants and develop new active resin glycoside compounds with novel structures. Summary of the invention
[0004] In view of this, the object of the present invention is to provide an Operculinic acid macrocyclic resin glycoside compound and a preparation method and application thereof.
[0005] To solve the above problems, the present invention provides the following technical solutions:
[0006] In the first aspect of the present invention, an Operculinic acid macrocyclic resin glycoside compound is provided, wherein the Operculinic acid macrocyclic resin glycoside compound is an Operculinic acid D macrocyclic resin glycoside compound having any of the following structural formulas:
[0007]
[0008] Operculinic acid D resin glycoside is a pentasaccharide macrocyclic resin glycoside compound. Its oligosaccharide chain consists of one D-xylose (Xyl), three L-rhamnose (Rha), and one D-glucose (Glc), and the aglycone is 11S-hydroxyhexadecanoic acid. So far, there has been no report on macrocyclic resin glycoside compounds with Operculinic acid D as the parent nucleus.
[0009] In a second aspect of the present invention, there is provided an Operculinic acid macrocyclic resin glycoside compound, wherein the Operculinic acid macrocyclic resin glycoside compound is an Operculinic acid A macrocyclic resin glycoside compound having any of the following structural formulas:
[0010]
[0011] Operculinic acid A resin glycoside is a pentasaccharide macrocyclic resin glycoside compound. Its oligosaccharide chain is composed of one D-fucose (Fuc), three L-rhamnoses (Rha), and one D-glucose (Glc), and the aglycone is composed of 11S-hydroxyhexadecanoic acid. Its molecular structure is similar to that of Operculinic acid D, with the main difference being that the first sugar in the oligosaccharide chain is D-fucose instead of D-xylose. The carboxyl end of the aglycone is usually connected to the 2nd or 3rd position of the second sugar residue on the oligosaccharide chain to form an 18- or 19-membered macrocyclic ring. Due to the differences in the macrolide connection site and the modified fatty acyl group, this type of resin glycoside has a wide range of significant biological activities.
[0012] Ipomoea biflora (L.) Pers. is a plant of the family Convolvulaceae. It grows in areas with an altitude of 150 to 1,800 meters, mostly in valleys, hillsides, roadsides and under forests. In Guangxi, the stems and leaves are used to treat infantile malnutrition, and the seeds are used to treat bruises and snake bites. However, there is no research on its active ingredients.
[0013] The third aspect of the present invention provides a method for preparing the above-mentioned Operculinic acid macrocyclic resin glycoside compound, wherein the preparation method comprises: isolating the Operculinic acid macrocyclic resin glycoside compound from the whole herb of Dioscorea sylvestris.
[0014] In some embodiments, the preparation method comprises the following steps:
[0015] S1. extracting the whole plant of the sweet potato with a polar solvent to obtain an extract, and then extracting the extract with ethyl acetate after vacuum concentration to obtain an ethyl acetate extract;
[0016] S2. The ethyl acetate extract was separated by silica gel column chromatography, using a dichloromethane-methanol two-phase system gradient elution to obtain a resin glycoside enrichment;
[0017] S3. The enriched product is sequentially separated by ODS column chromatography, using a methanol-water two-phase system gradient elution to obtain a resin glycoside component;
[0018] S4. separating the resin glycoside component by gel column chromatography and eluting with methanol to obtain the target component;
[0019] S5. Purify the target component by reverse phase high performance liquid chromatography to obtain the Operculinic acid macrocyclic resin glycoside compound.
[0020] In some embodiments, in step S1 of the above preparation method, the polar solvent is an ethanol aqueous solution, and the volume fraction of the ethanol aqueous solution is 90%-95%.
[0021] In some embodiments, in step S2 of the preparation method, the dichloromethane-methanol two-phase system is used for gradient elution, specifically, dichloromethane / methanol mixed solvents with volume ratios of 1:0, 10:1, 5:1, 2:1 and 1:1 are used for elution.
[0022] In some embodiments, in step S3 of the above preparation method, the elution is carried out using a methanol-water two-phase system, specifically, eluting using a methanol / water mixed solvent with a volume ratio of 50:50, 70:30, 80:20, 85:15, 90:10, 95:5, and 100:0 in sequence.
[0023] In some embodiments, in step S5 of the above preparation method, the reverse phase high performance liquid chromatography uses a 10×200 mm YMC RP-C18 chromatographic column, and the mobile phase is a methanol-water solution.
[0024] The fourth aspect of the present invention provides a use of the above-mentioned Operculinic acid macrocyclic resin glycoside compound as an active ingredient in the preparation of an anti-tumor drug.
[0025] In some embodiments, the tumor is one of colon cancer, lung cancer, and liver cancer.
[0026] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises the Operculinic acid macrocyclic resin glycoside compound or a pharmaceutically acceptable salt thereof according to claim 1.
[0027] Compared with the prior art, the present invention selects the whole herb of Dioscorea paniculata, uses ethanol for reflux extraction, extracts with ethyl acetate, and then uses various separation techniques and means to obtain the Operculinic acid macrocyclic resin glycoside compounds, which are all new compounds that have never been reported. Among them, compound formula I-II are the first discovered macrocyclic resin glycoside compounds with Operculinic acid D as the parent nucleus. Compound formula III-V are Operculinic acid A macrocyclic resin glycoside compounds, and the aglycone of compound formula III is connected to the 2-hydroxyl group of the third sugar residue of the pentasaccharide chain to form a 23-membered ring, which is the first discovered Operculinic acid A resin glycoside compound with a 23-membered ring. In addition, this type of novel resin glycoside compound has significant anti-tumor activity, can be used as an active ingredient of anti-tumor drugs, and has a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a hydrogen spectrum data diagram of compound I obtained in Example 1 of the present invention;
[0029] Figure 2 This is a carbon spectrum data diagram of compound I obtained in Example 1 of the present invention;
[0030] Figure 3 This is a hydrogen spectrum data diagram of compound II obtained in Example 1 of the present invention;
[0031] Figure 4 This is the carbon spectrum data diagram of compound II obtained in Example 1 of the present invention;
[0032] Figure 5 This is a hydrogen spectrum data diagram of compound III obtained in Example 1 of the present invention;
[0033] Figure 6 This is the carbon spectrum data diagram of compound III obtained in Example 1 of the present invention;
[0034] Figure 7 This is a hydrogen spectrum data diagram of compound IV obtained in Example 1 of the present invention;
[0035] Figure 8 This is the carbon spectrum data diagram of compound IV obtained in Example 1 of the present invention;
[0036] Fig. 9 This is a hydrogen spectrum data diagram of compound V obtained in Example 1 of the present invention;
[0037] Fig.10 This is the carbon spectrum data of compound V obtained in Example 1 of the present invention. DETAILED DESCRIPTION
[0038] To further illustrate the present invention, the preparation method of the Operculinicacid resin glycoside compound in O. calyx provided by the present invention and its application in the preparation of anti-tumor drugs are described in detail below in conjunction with examples.
[0039] Example 1: Preparation of compounds
[0040] The present invention adopts the whole herb of Dioscorea calyx to carry out reflux extraction with 95% ethanol, and prepares the compound of the present invention through the steps of extraction, enrichment, separation and the like. The whole herb of Dioscorea calyx is collected from Nanning, Guangxi.
[0041] 1) Extraction, enrichment and initial separation
[0042] The dried whole herb of Dioscorea calyx was crushed (8.0 kg), and 50 L of 95% ethanol aqueous solution was added, and reflux extraction was performed three times, each time for 3 hours. The extracts were combined and concentrated in vacuo to obtain the total extract.
[0043] The total extract was resuspended in water and extracted three times with ethyl acetate to obtain ethyl acetate extracts.
[0044] The ethyl acetate extract (600 g) was separated by silica gel column chromatography and gradient elution was performed using a dichloromethane / methanol mixed solvent. The elution solvents were dichloromethane / methanol in a volume ratio of 1:0, 10:1, 5:1, 2:1, and 1:1, respectively, to obtain 8 components AH.
[0045] 2) ODS-C18 column chromatography separation
[0046] Component D was separated by ODS-C18 column chromatography, using methanol / water with a volume ratio of 50:50, methanol / water with a volume ratio of 70:30, methanol / water with a volume ratio of 80:20, methanol / water with a volume ratio of 85:15, methanol / water with a volume ratio of 90:10, methanol / water with a volume ratio of 95:5, and methanol / water with a volume ratio of 100:0 for elution, and three resin glycoside components DA, DB, and DC were obtained in sequence.
[0047] 3) Gel column chromatography separation
[0048] The components DA-DC were separated by gel column chromatography, and pure methanol was used as the elution solvent to obtain components DAA, DBA and DCA respectively.
[0049] 4) Reverse-phase HPLC purification
[0050] The component DAA was purified by reverse phase high performance liquid chromatography. The preparation conditions were: chromatographic column YMC RP-C18 (10×200 mm), methanol-water (85:15, V / V), flow rate 2.0 mL / min, detector Shimadzu RID 10A, and compounds I and IV were prepared. The structures of compounds I and IV were identified as follows: Figure 1 , Figure 2 , Figure 7 , Figure 8 As shown, Figure 1 is the hydrogen spectrum data diagram of compound I, Figure 2 is the carbon spectrum data diagram of compound I, Figure 7 is the hydrogen spectrum data of compound IV, Figure 8 This is the carbon spectrum data of compound IV.
[0051] The component DCA was purified by reverse phase high performance liquid chromatography. The preparation conditions were: chromatographic column YMC RP-C18 (10×200 mm), methanol-water (90:10, V / V), flow rate 2.0 mL / min, detector Shimadzu RID 10A, and compounds II and V were prepared. The structures of compounds II and V were identified as follows: Figure 3 , Figure 4 , Fig. 9 , Fig.10 As shown, Figure 3 is the hydrogen spectrum data diagram of compound II, Figure 4 is the carbon spectrum data of compound II, Fig. 9 is the hydrogen spectrum data of compound V, Fig.10 This is the carbon spectrum data of compound V.
[0052] The component DBA was purified by reverse phase high performance liquid chromatography. The preparation conditions were: chromatographic column YMC RP-C18 (10×200 mm), methanol-water (86:14, V / V), flow rate 2.0 mL / min, detector Shimadzu RID 10A, and compound III was prepared. The structure of compound III was identified as follows: Figure 5 , Figure 6 As shown, Figure 5 is the hydrogen spectrum data diagram of compound III, Figure 6 This is the carbon spectrum data of compound III.
[0053] Through Figure 1-Figure 10 The mass spectrum and spectroscopy data of compound IV are shown below.
[0054] Compound I: ipomofin I; optical rotation (c 0.10, MeOH); UV absorption (MeOH)λ max(logε)218(4.49),280(4.97)nm, the main absorption peak of infrared spectrum (KBr)ν max 3435,2929,2856,1734,1636,1457,1040cm -1 ; High resolution mass spectrum m / z is 1199.5846 [MH] - (calcd for C 59 H 91 O 25 1199.5849), and the H NMR and C NMR data are shown in Table 1.
[0055] Compound II: ipomofin II; optical rotation (c 0.10, MeOH); UV absorption (MeOH)λ max (logε)219(4.31),280(4.54)nm, the main absorption peak of infrared spectrum (KBr)ν max 3434,2929,1736,1636,1458,1380,1049cm -1 ; High resolution mass spectrum m / z is 1223.5815 [M+Na] + (calcd for C 59 H 92 O 25 Na1223.5825), the H NMR and C NMR data are shown in Table 1.
[0056] Compound III: ipomofin IV; optical rotation (c 0.26, MeOH); main absorption peak of infrared spectrum (KBr) ν max 3436,2929,2856,1726,1460,1383,1137,1069cm -1 ; High resolution mass spectrum m / z is 1199.5846 [MH] - 1085.5748[M+H] + (calcd for C 51 H 89 O 24 1085.5744), and the H NMR and C NMR data are shown in Table 2.
[0057] Compound IV: ipomofinV; optical rotation (c 0.36, MeOH); UV absorption (MeOH)λ max (logε)218(4.45),279(4.66)nm, the main absorption peak of infrared spectrum (KBr)ν max3436,2934,1725,1638,1457,1138,1069cm -1 ; High resolution mass spectrum m / z is 1237.5988 [M+Na] + (calcd for C 60 H 94 O 25 Na1237.5982), the H NMR and C NMR data are shown in Table 2.
[0058] Compound V: ipomofin VI; optical rotation (c 0.13, MeOH); UV absorption (MeOH)λ max (logε)218(4.46),280(4.71)nm, the main absorption peak of infrared spectrum (KBr)ν max 3436,2932,2858,1722,1455,1265,1139,1071cm -1 ; High resolution mass spectrum m / z is 1213.6010 [MH] - (calcd for C 60 H 93 O 25 1213.6006), and the H NMR and C NMR data are shown in Table 2.
[0059] Table 1. H NMR and C NMR data of compounds of formula I-II
[0060]
[0061]
[0062] a Overlapped signals are reported without designating multiplicity; b Abbreviations:
[0063] Table 2. H NMR and C NMR data of compounds of formula III to formula V
[0064]
[0065]
[0066] a Overlapped signals are reported without designating multiplicity; b Abbreviations:Xyl=
[0067] xylopyranosyl, Rha=rhamnopyranosyl, Glc=glucopyranosyl, Jal=11S-hydroxyhexadecanoyl, Mba=2S-methylbutanoyl, Cna=trans-cinnamoyl.
[0068] The above results show that the structural formula of the obtained compound IV is respectively chemical formula IV.
[0069] Example 2: Antitumor activity test of compound IV
[0070] 1) Experimental Materials
[0071] Instruments and reagents: CO2 incubator (Thermo Fisher Scientific, USA); DMEM / RPMI 1640 culture medium (Gibco, Thermo Fisher Scientific, USA); DMSO, MTT, trypsin and cisplatin (Sigma, USA); fetal bovine serum (Hangzhou Sijiqing Biomaterials Research Institute); other commonly used inorganic salts and other reagents were of analytical grade.
[0072] Tumor cell lines used for testing: human colon cancer HCT-15, SW480 and HT-29 cell lines, human lung cancer A549 cell line, human liver cancer HepG2 cell line.
[0073] Test samples: Operculinic acid macrocyclic resin glycosides of formula I to formula V obtained from the whole plant of Dioscorea calyx; cisplatin was selected as the positive control drug, and each compound was dissolved in DMSO and then diluted.
[0074] 2) Experimental methods
[0075] The MTT method was used to determine the half inhibitory concentration (IC) of the compounds on tumor cell lines. 50 Value: Tumor cells in the logarithmic growth phase were inoculated into 96-well culture plates, with 90 μL per well and a cell density of 5×10 3 / well. After overnight culture, add different concentrations of samples or positive drugs, 10μL per well, and add the same amount of culture medium to the negative control group, and set up 3 parallel wells for each concentration. Continue to culture for 24h, add 20μL of MTT (5mg / mL) to each well, discard the culture medium after 4h, add 150μL DMSO to each well, shake the crystals thoroughly, place them in a microplate reader with 570nm as the detection wavelength to measure the absorbance of each well, and calculate the inhibition rate [inhibition rate = (1-experimental group A value / negative control group A value) × 100%]. Repeat the experiment 3 times. Calculate the half inhibitory concentration (IC50) of each test sample on tumor cells for 24h 50 ).
[0076] 3) Experimental results
[0077] According to the MTT test results, calculate the IC of the test sample for the above cells 50 The results are shown in Table 2.
[0078] Table 2. In vitro cytotoxic activity screening results of test samples
[0079]
[0080] a IC 50 Values are presented as means (95% confidence intervals)
[0081] The results showed that the IC values of compound IV of the present invention on human colon cancer HCT-15, SW480 and HT-29 cell lines were 50 The values are in the range of 5.35 μM to 30.29 μM. In addition, the IC values of compound IV of the present invention on human lung cancer A549 cell line and human liver cancer HepG2 cell line are 50 The values were 23.63 μM and 49.48 μM, respectively. The above results show that compound IV of the present invention can be used as an active ingredient of anti-tumor drugs.
[0082] 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. An Operculinic acid macrocyclic resin glycoside compound, characterized in that: The Operculinic acid macrocyclic resin glycoside compound is an Operculinic acid A macrocyclic resin glycoside compound having any of the following structural formulas:
2. A method for preparing the Operculinic acid macrocyclic resin glycoside compound as claimed in claim 1, characterized in that: The preparation method comprises the following steps: isolating the Operculinic acid macrocyclic resin glycoside compound from the whole herb of Dioscorea calyx.
3. The preparation method according to claim 2, characterized in that: The steps include: S1. extracting the whole plant of the sweet potato with a polar solvent to obtain an extract, and then extracting the extract with ethyl acetate after vacuum concentration to obtain an ethyl acetate extract; S2. The ethyl acetate extract is separated by silica gel column chromatography, using a dichloromethane-methanol two-phase system gradient elution to obtain a resin glycoside enrichment; S3. The enriched product is sequentially separated by ODS column chromatography, using a methanol-water two-phase system gradient elution to obtain a resin glycoside component; S4. separating the resin glycoside component by gel column chromatography and eluting with methanol to obtain the target component; S5. Purify the target component by reverse phase high performance liquid chromatography to obtain the Operculinic acid macrocyclic resin glycoside compound.
4. The preparation method according to claim 3, characterized in that: In step S1, the polar solvent is an ethanol aqueous solution, and the volume fraction of the ethanol aqueous solution is 90%-95%.
5. The preparation method according to claim 3, characterized in that: In step S2, the dichloromethane-methanol two-phase system is used for gradient elution, specifically, dichloromethane / methanol mixed solvents with volume ratios of 1:0, 10:1, 5:1, 2:1 and 1:1 are used for elution in sequence.
6. The preparation method according to claim 3, characterized in that: In step S3, the elution is carried out using a methanol-water two-phase system, specifically, eluting using a methanol / water mixed solvent with a volume ratio of 50:50, 70:30, 80:20, 85:15, 90:10, 95:5, and 100:0 in sequence.
7. The preparation method according to claim 3, characterized in that: In step S5, the reverse phase high performance liquid chromatography uses a 10×200 mm YMC RP-C18 chromatographic column, and the mobile phase is a methanol-water solution.
8. Use of the operculinic acid macrocyclic resin glycoside compound according to claim 1 as an active ingredient in the preparation of anti-tumor drugs.
9. The use according to claim 8, characterized in that: The tumor is one of colon cancer, lung cancer and liver cancer.
10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Operculinicacid macrocyclic resin glycoside compound or a pharmaceutically acceptable salt thereof according to claim 1.