A tenuissime extracted glycowithanolide and its preparation method

Abstract

The present application relates to a kind of Euphorbia kansui extracted glycowithanolide and its preparation method, the glycowithanolide has one of the structures of Munropene E, Munropene F.The glycowithanolide extracted from Euphorbia kansui Munropene E, F shows specific inhibitory effect to colon cancer cell, significantly better than the inhibitory effect to other types of tumor cells, surface glycowithanolide extracted from Euphorbia kansui has the hope to develop into new for treating colon cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of natural medicine, in particular to a glycowithanolide compound extracted from Munronia pinnata and a preparation method thereof. BACKGROUND

[0002] There are about 15 species of Meliastrum in the family Malvaceae distributed in Sri Lanka, India, Indochina, Indonesia, Philippines and other regions, of which 8 species and 1 variety are distributed in China. Previous studies on the chemical constituents of this plant have successfully isolated a series of structurally diverse compounds, including limonoids, triterpenoids, flavonoids, lignans, sterols, sesquiterpenes and diterpenes. Some of these compounds have significant biological activities, such as anti-inflammatory, anti-tumor, anti-tobacco mosaic virus and antifeedant activities.

[0003] Munronia pinnata (Wall.) W. Theob (synonyms: M. henryi Harms, M. pumila Wight, and M. sinica Diels) is a perennial herb growing in the temperate regions of southern China. The research group has previously isolated many new limonoids from Munronia pinnata.

[0004] Currently, there is no report on the glycowithanolide compound components in Munronia pinnata, and there is no record of the anti-cancer activity of glycowithanolide compounds in Munronia pinnata. SUMMARY

[0005] To solve the above technical problems, the present application provides a glycowithanolide compound extracted from Munronia pinnata and a preparation method thereof.

[0006] The present application provides a glycowithanolide compound extracted from Munronia pinnata, which has one of the structures of Munropene E and Munropene F, wherein the Munropene E is as shown in formula (I), and the Munropene F is as shown in formula (II):

[0007]

[0008] Formula (II).

[0009] A method for preparing a glycowithanolide compound extracted from Munronia pinnata, wherein the glycowithanolide compound extracted from Munronia pinnata is Munropene E or Munropene F, and the preparation method of Munropene E or Munropene F comprises the following steps:

[0010] S1: Take dry Munroa microphylla powder, add 95% ethanol according to a material-liquid ratio of 1 kg: 10-15 L, and soak and extract at room temperature for 2-4 times, once every 3 days, filter the filtrate, concentrate, and obtain an extract, then sequentially extract the extract with petroleum ether, ethyl acetate, and water in equal volumes, respectively, and then chromatograph the water layer on a macroporous resin column with 45-55 times the weight of the water layer, sequentially elute with 20%-80% ethanol, 2.0-3.0 L each time, collect the 80% ethanol elution part, and then chromatograph it on a gel column with 100 times the weight of the 80% ethanol elution part, and continue to elute with 100% methanol, 1.0-2.0 L, to obtain Fr2;

[0011] S2: Take Fr2 and chromatograph it on a C18 column with 100 times the weight of Fr2, and perform gradient elution with methanol-water in a volume ratio of 30:70-50:50, 1.0-1.5 L each time, collect one test tube every 10-15 ml, and observe each test tube by TLC spotting, and then combine the eluent containing the same components to obtain Fr2.1-Fr2.7, a total of 7 fractions;

[0012] S3: Take Fr2.4 and perform high-speed counter-current chromatography with 2.0-2.5 L of dichloromethane-methanol-water in a volume ratio of 2:2:1 as the mobile phase, collect one tube every 20-25 ml of the mobile phase, and combine the same components by TLC spotting to obtain Fr2.4.1-Fr2.4.6, a total of 6 components;

[0013] S4: Take Fr2.4.4 and then perform preparative high-performance liquid chromatography with a C18 preparative column, and elute with acetonitrile-water-formic acid in a volume ratio of 23:77:0.1, and sequentially obtain Munropene E and Munropene F at retention times of 10-16 min.

[0014] The gan seng alkane type triterpenoid compounds of the present application have the following characteristics and advantages:

[0015] 1. The gan seng alkane type triterpenoid compounds Munropene E and F extracted from Munroa microphylla have a specific inhibitory effect on colon cancer cells, which is significantly better than the inhibitory effect on other types of tumor cells, and the gan seng alkane type triterpenoid compounds extracted from Munroa microphylla have the potential to be developed into a new treatment for colon cancer.

[0016] 2. The preparation method of the present application is convenient to operate, easy to mass-produce, and stable in quality; the raw materials of the present application are abundant in China and suitable in price, so that the large-scale production of the present application is not limited by high costs. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 2D NMR correlation and planar structure diagrams of Munropene E and F.

[0018] Figure 2 Figure 6 is a ROESY spectrum stereogram of Munropene E.

[0019] Figure 3 Figure 7 is a ROESY spectrum stereogram of Munropene F. DETAILED DESCRIPTION

[0020] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0021] Example 1 Preparation of Munropene A-F, a triterpenoid compound from Euphorbia nivaeapunctata

[0022] Euphorbia nivaeapunctata plants were collected from Jingxi City, Guangxi in July 2021, and identified by Professor Li Dianpeng as Munronia pinnata (Wall.) W. Theob. of the family Meliaceae. The sample specimen is preserved in the Plant Specimen Room of the Natural Product Chemistry Research Center of the Guangxi Institute of Botany, Chinese Academy of Sciences (21-GX-001).

[0023] S1: Take dry Euphorbia nivaeapunctata powder (25 kg) and add 95% ethanol and soak for extraction at room temperature for 3 times, 250 L each time, once every 3 days. The filtrate is combined, filtered, and concentrated to obtain an extract, and then the extract (1.71 kg) is sequentially extracted with equal volumes of petroleum ether, ethyl acetate, and water, respectively. The water layer (560 g) is subjected to macroporous resin column chromatography (D-101) with 50 times the weight of the water layer, and eluted with 20%, 40%, and 80% ethanol, 2.5 L each. The 40% ethanol elution fraction is collected to obtain Fr3 (34 g). The 80% ethanol elution fraction (86 g) is subjected to LH-20 gel column chromatography with 100 times the weight of the 80% ethanol elution fraction, and eluted with 100% methanol, 1.5 L, to obtain Fr2 (15 g).

[0024] S2: Take Fr2 through its weight 100 times the amount of YMC 50 μm C18 column chromatography, using methanol-water with volume ratio of 30:70, 40:60, 50:50 each 1 L gradient elution, 1 test tube collection every 10 ml, each test tube by TLC point plate observation combined with the same components eluent, get Fr2.1 ~ Fr2.7 total 7 fractions (according to the order of the components flowing from the first, second, third …, and so on, the same below), wherein Fr2.1 is 1.46 g, Fr2.3 is 1.27 g, Fr2.4 is 1.39 g, Fr2.7 is 0.9 g.

[0025] S3: Take Fr2.1 (1.46 g) through its weight 100 times the amount of silica gel H column chromatography, using dichloromethane-methanol with volume ratio of 4:1 elution 900 mL, 1 test tube collection every 10 ml, each test tube by TLC point plate observation combined with the same components eluent, obtain Fr2.1.1 ~ Fr2.1.5 etc. 5 components. Fr2.1.2 (163 mg) by high-speed counter-current chromatography, using 2 L of dichloromethane-methanol-water with volume ratio of 2:2:1 as the mobile phase counter-preparation, 1 test tube collection every 20 ml of mobile phase, by TLC point plate combined with the same components, obtain Fr2.1.2.1 ~ Fr2.1.2.6 etc. 6 components, wherein component Fr2.1.2.3 is a monomer compound, namely Munropene B (2) (68 mg).

[0026] S4: Take Fr2.3 (1.27 g) by high-speed counter-current chromatography, using 600 mL of dichloromethane-methanol-water with volume ratio of 2:2:1 as the mobile phase, 1 test tube collection every 10 ml, each test tube by TLC point plate observation combined with the same components eluent, get Fr2.3.1 ~ Fr2.3.3 total 3 fractions. Take Fr2.3.3 by preparative high performance liquid chromatography, using Agilent ZORBAX SB-C18 preparative column, using acetonitrile-water-formic acid with volume ratio of 23:77:0.1 as the mobile phase, preparation and purification to obtain Munropene C (3) (26 mg, tR = 10.1 min).

[0027] S5: Take Fr2.4 (1.39 g) and elute by high-speed counter-current chromatography with 2 L of dichloromethane-methanol-water (2:2:1, by volume) as the mobile phase. Collect 1 tube every 20 ml of the mobile phase, and combine the fractions with the same components by TLC. Obtain Fr2.4.1-Fr2.4.6, and then take Fr2.4.4 (131 mg) and purify by preparative high-performance liquid chromatography with an Agilent ZORBAX SB-C18 preparative column and acetonitrile-water-formic acid (23:77:0.1, by volume) as the mobile phase. Obtain Munropene E (5) (10 mg, tR= 12.2 min) and Munropene F (6) (15 mg, tR= 14.7 min).

[0028] S6: Take Fr2.7 (0.9 g) and elute by high-speed counter-current chromatography with 1.5 L of dichloromethane-methanol-water (2:2:1, by volume) as the mobile phase. Collect 1 tube every 20 ml of the mobile phase, and combine the fractions with the same components by TLC. Obtain Fr2.7.1-Fr2.7.5, and then take Fr2.7.3 (184 mg) and purify by preparative high-performance liquid chromatography with an Agilent ZORBAX SB-C18 preparative column and acetonitrile-water-formic acid (23:77:0.1, by volume) as the mobile phase. Obtain Munropene D (4) (184 mg).

[0029] S7: Take Fr3 (30 g) and elute by column chromatography on silica gel H with 100 times the weight of the sample. Elute with dichloromethane-methanol (80:20, 90:10, and 100:0, by volume) as the mobile phase. Collect 1 tube every 10 ml, and combine the fractions with the same components by TLC. Obtain Fr3.1-Fr3.20, and then take Fr3.3 (1.49 g) and purify by preparative high-performance liquid chromatography with a ChromCore 120-C18 column and acetonitrile-water-formic acid (18:82:0.1, by volume) as the mobile phase. Obtain Munropene A (1) (321 mg, tR= 21.3 min).

[0030] Identification of the glywruic acid triterpenoids Munropene A-F

[0031] Optical rotations were measured on a Jasco P-1020 polarimeter. Infrared spectra were recorded on a TENSOR 27 Fourier transform infrared spectrometer (Therom Fisher, USA) with KBr pellets. CD spectra were recorded on a J-810 CD spectrometer. Mass spectra were measured on a LC / MS-IT-TOF mass spectrometer. NMR spectra were recorded on a Bruker AVANCE III-HD 500 spectrometer with TMS as internal standard. CCC was performed on a TBE-300C system (Tauto biotechnology, Shanghai, China). HPLC analysis was performed on an Agilent 1260 Infinity II LC (Agilent Technologies, USA) using Agilent Poroshell 120 cb-C18 (4 mm, 4.6 mm x 150 mm, Agilent, USA), ChromCore 120-C18 (5 mm, 10 mm x 250 mm, NanoChrom, China) and Agilent ZORBAX cb-C18 (5 mm, 9.4 mm x 250 mm, Agilent, USA). Column chromatography was performed on silica gel (200-300 mesh, Qingdao Haigong Factory, China) and MCI gel (Mitsubishi Chemical, Japan). TLC analysis was performed on pre-coated silica gel 60 F254 plates (Merck Millipore, Germany, silica gel plates were sprayed with 10% H2SO4 in ethanol, heated and observed for spots.

[0032] Munropene E: optically active colorless amorphous solid; molecular formula C 36 H 58 O 12 ; [a] D 20.0 = -24.04 (c 0.10, MeOH); HRESIMS: m / z 727.3839 [M+HCOO] - , Delta -7.1 mmu; 1H NMR spectra are shown in Table 3. 13C NMR spectra are shown in Table 3. 2D NMR correlations and planar structure diagram are shown in Figure 1 Figure 2

[0033] Munropene F: optically active colorless amorphous solid; molecular formula C 36 H 58 O 12 ; [a] D 20.0 = -6.80 (c 0.10, MeOH); HRESIMS: m / z 727.3863 [M+HCOO] - ​​Δ-4.7 mmu; the proton NMR spectrum is shown in Table 3. The carbon NMR spectrum is shown in Table 3. 2D NMR correlation and planar structure diagram are shown in... Figure 1 As shown, the ROESY spectrum is as follows Figure 2 As shown.

[0034] Munropen D-F acid hydrolysis and sugar derivatization of euphorbia triterpenoid compounds

[0035] The absolute configurations of sugars in Munropen D–F were determined according to the method of Tanaka et al. [Tanaka et al., 2007]. After hydrolysis, the compounds were reacted with 2.0 mL of 0.5 mol / L hydrochloric acid in a 90°C water bath for 2 h. The mixture was then adjusted to neutral using an IRA400 anion exchange resin, filtered, concentrated under reduced pressure to remove the solvent, and 0.2 mL of a pyridine solution containing 1 mg / L cysteine ​​formate hydrochloride was added. The reaction was carried out at 60°C for 1 h. Finally, 0.2 mL of a pyridine solution containing 1 mg of o-toluene isothiocyanate was added to the reactants, and the reaction was carried out at 60°C for 1 h.

[0036] The reaction mixture was analyzed by reversed-phase HPLC (Agilent Poroshell 120SB-C18 (4 mm, 4.6 mm x 150 mm, Agilent, USA) using acetonitrile-water (containing 0.1% formic acid) at a volume ratio of 25-75 as the mobile phase and 250 nm as the detection wavelength. The absolute configuration of the sugar was determined by comparing the retention time with that of D-glucose (tR = 9.55 min).

[0037] Cytotoxicity assays of Munropene A–F, a triterpenoid compound of Euphorbia kansui

[0038] The cytotoxicity of compounds Munropene A–F against human non-small cell lung cancer cell line A549, human colon cancer cell line HCT116, mouse mononuclear macrophage leukemia cell line RAW264.7, liver cancer cell line HepG2, triple-negative breast cancer cell line MCF7, and human breast ductal carcinoma cell line MDAMB was detected using the CCK-8 assay. 100 μL of cell suspension (2 × 10⁻⁶ cells / mL) was used as the initial cell suspension. ^5 Cells were seeded at 1000 cells / mL in 96-well microplates and cultured for 24 h. Then, different concentrations of Munropene A–F (5, 10, 20, 40, 80, 160 μM) were added to culture dishes, while an equal volume of DMSO was added to the control group. Each group was repeated in triplicate. After 24 h of culture, 10 μL of CCK-8 reagent was added to each well. One h later, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0039] Table 1. Proton NMR spectra ( 1H NMR data for munropenes A-D in CD3OD)

[0040]

[0041]

[0042]

[0043] Table 2 Nuclear magnetic resonance carbon spectra 13 C NMR data for munropenes A-D in CD3OD)

[0044]

[0045]

[0046] Table 3 Nuclear magnetic resonance carbon and hydrogen spectra 1 H and 13 C NMR data for munropenesE-F in CD3OD.

[0047]

[0048]

[0049] Table 4 Cytotoxic effects (IC 50 (μM))

[0050] Compounds HCT116 A549 HepG2 MCF7 MDAMB Munropene A 19.13 >160 >160 >160 >160 Munropene B 40.90 >160 >160 >160 >160 Munropene C >160 >160 >160 >160 >160 Munropene D 17.66 >160 >160 >160 >160 Munropene E 57.90 >160 >160 >160 >160 Munropene F 32.62 >160 >160 >160 >160

[0051] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application, and are not intended to limit the same; even though the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent replacements for some of the technical features therein; and such modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a euphorbia-type triterpenoid compound, characterized in that, The euphorbia-type triterpenoid compound has one of the structures of Munropene E and Munropene F, wherein Munropene E is as shown in formula (I) and Munropene F is as shown in formula (II): The preparation method of Munropene E or Munropene F includes the following steps: S1: Take dried Munropene powder, add 95% ethanol at a material-to-liquid ratio of 1kg:10-15L, and soak and extract at room temperature 2-4 times, once every 3 days. Combine the filtrates, filter, concentrate, and obtain an extract. Then, extract the extract sequentially by equal volumes of petroleum ether, ethyl acetate, and water. The aqueous layer is subjected to macroporous resin column chromatography with 45-55 times its weight, and eluted sequentially with 2.0-3.0L of 20%-80% ethanol. Collect the 80% ethanol eluent and perform gel column chromatography again with 100 times its weight, and continue eluting with 1.0-2.0L of 100% methanol to obtain Fr2. S2: Take Fr2 and perform C18 column chromatography with 100 times its weight. Use methanol-water gradient elution at a volume ratio of 30:70 to 50:50, 1.0 to 1.5 L each. Collect 10 to 15 ml in each test tube. Observe each test tube by TLC and combine the eluents containing the same components to obtain 7 fractions from Fr2.1 to Fr2.

7. S3: Fr2.4 was eluted by high-speed countercurrent chromatography using 2.0-2.5 L of dichloromethane-methanol-water at a volume ratio of 2:2:

1. Each tube containing 20-25 ml of mobile phase was collected and combined with the same components by TLC to obtain 6 components, namely Fr2.4.1 to Fr2.4.

6. S4: Fr2.4.4 was then subjected to preparative high performance liquid chromatography using a C18 column and eluted with acetonitrile-water-formic acid at a volume ratio of 23:77:0.

1. Munropene E and Munropene F were obtained sequentially at retention times of 10–16 min.

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