A gansuline-type triterpenoid compound with anti-tumor effect and its preparation method

Abstract

The present invention relates to a gansuane-type triterpenoid compound with anti-tumor effect and a preparation method thereof. The gansuane-type triterpenoid compound has a structure of one of Munropene A, Munropene B, and Munropene C. The gansuane-type triterpenoid compounds Munropene A to Munropene C show specific inhibitory effects on colon cancer cells, which are significantly better than the inhibitory effects on other types of tumor cells. This indicates that the gansuane-type triterpenoid compound extracted from Dalbergia odorifera has the potential to be developed into a new treatment for colon cancer.

Description

Technical Field

[0001] The present invention relates to the field of natural medicines, and in particular to a gansuline-type triterpenoid compound with anti-tumor effect and a preparation method thereof. Background Art

[0002] Approximately 15 species of Rehmanniae (Gleditsia chinensis) from the Meliaceae family are found in nature, distributed throughout Sri Lanka, India, Indochina, Indonesia, the Philippines, and other regions. Eight of these species and one variant are found in China. Previous studies on the chemical composition of this plant have successfully isolated a diverse array of compounds, including limonoids, triterpenes, flavonoids, lignins, sterols, sesquiterpenes, and diterpenes. Some of these compounds exhibit significant biological activities, including anti-inflammatory, anti-tumor, anti-tobacco mosaic virus, and antifeedant properties.

[0003] Munronia pinnata (Wall.) W. Theob (synonyms: M. henryi Harms, M. pumila Wight, and M. sinica Diels), a member of the Meliaceae family, is a perennial herb found in temperate regions of southern China. The research team has previously isolated numerous new limonoid compounds from Munronia pinnata.

[0004] At present, there are no reports on the research on the presence of gansuline-type triterpenoid ring compounds in dwarf ... Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a gansuline-type triterpenoid compound with anti-tumor effect and a preparation method thereof.

[0006] The present invention provides a gansuiane-type triterpenoid compound with anti-tumor effect. The gansuiane-type triterpenoid compound has a structure of one of Munropene A, Munropene B, and Munropene C. The Munropene A is represented by formula (I), the Munropene B is represented by formula (II), and the Munropene C is represented by formula (III):

[0007]

[0008] A method for preparing a gansuline-type triterpenoid compound with anti-tumor effect, wherein the gansuline-type triterpenoid compound with anti-tumor effect is Munropene A. The preparation method of Munropene A comprises the following steps:

[0009] S1: Take dry dwarf powder and add 95% ethanol at a material-liquid ratio of 1kg:10-15L and soak and extract at room temperature for 2-4 times, once every 3 days. Combine the filtrates, filter, and concentrate to obtain an extract. Then, extract the extract with equal volumes of petroleum ether, ethyl acetate, and water, respectively. The aqueous layer is chromatographed on a macroporous resin column with 45-55 times its weight, and eluted with 2.0-3.0L of 20%-80% ethanol, respectively. Collect the 40% ethanol eluate to obtain Fr3;

[0010] S2: Fr3 was subjected to silica gel column chromatography (100 times its weight) using a mobile phase of dichloromethane-methanol (1.0-2.0 L each) at a volume ratio of 80:20 to 100:0 for gradient elution. 10-15 ml of the mobile phase was collected into a test tube. The eluates from each test tube containing the same components were combined by TLC spot plate observation to obtain 20 fractions, Fr3.1 to Fr3.20.

[0011] S3: Fr3.3 was purified by preparative high performance liquid chromatography using a C18 high performance liquid chromatography column with acetonitrile-water-formic acid in a volume ratio of 18:82:0.1 as the mobile phase to obtain Munropene A at a retention time of 20 to 25 minutes.

[0012] A method for preparing a gansuline-type triterpenoid compound with anti-tumor effect, wherein the gansuline-type triterpenoid compound with anti-tumor effect is Munropene B. The preparation method of Munropene B comprises the following steps:

[0013] S1: Take dry dwarf powder and add 95% ethanol at a material-liquid ratio of 1kg:10-15L and soak and extract at room temperature for 2-4 times, once every 3 days. Combine the filtrates, filter, and concentrate to obtain an extract. Then, extract the extract with equal volumes of petroleum ether, ethyl acetate, and water, respectively. The aqueous layer is chromatographed on a macroporous resin column in an amount of 45-55 times its weight, and eluted with 2.0-3.0L of 20%-80% ethanol in sequence. The 80% ethanol elution fraction is collected and chromatographed on a gel column in an amount of 100 times its weight, and further eluted with 100% methanol for 1.0-2.0L to obtain Fr2;

[0014] S2: Fr2 was subjected to C18 column chromatography (100 times its weight) using a gradient elution of 1.0 to 1.5 L of methanol and water at a volume ratio of 30:70 to 50:50. Each 10 to 15 mL fraction was collected into a test tube. The eluates from each test tube containing the same components were combined by TLC spot plate observation to obtain 7 fractions, Fr2.1 to Fr2.7.

[0015] S3: Fr2.1 was subjected to silica gel column chromatography (100 times its weight). 850-950 mL was eluted with dichloromethane-methanol at a volume ratio of 4:1. 10-15 mL was collected into a test tube. The eluates from each test tube containing the same components were combined by TLC spot plate observation to obtain five components, namely Fr2.1.1-Fr2.1.5.

[0016] S4: Fr2.1.2 was prepared by high-speed countercurrent chromatography using 1.5-2.5 L of dichloromethane-methanol-water with a volume ratio of 2:2:1 as the mobile phase. Every 20-25 ml of mobile phase was collected into one test tube. The same components were combined by TLC spot plate to obtain 6 components, including Fr2.1.2.1 to Fr2.1.2.6, among which component Fr2.1.2.3 was a monomer compound, namely Munropene B.

[0017] A method for preparing a gansuline-type triterpenoid compound with anti-tumor effect, wherein the gansuline-type triterpenoid compound with anti-tumor effect is Munropene C. The preparation method of Munropene C comprises the following steps:

[0018] S1: Take dry dwarf powder and add 95% ethanol at a material-liquid ratio of 1kg:10-15L and soak and extract at room temperature for 2-4 times, once every 3 days. Combine the filtrates, filter, and concentrate to obtain an extract. Then, extract the extract with equal volumes of petroleum ether, ethyl acetate, and water, respectively. The aqueous layer is chromatographed on a macroporous resin column in an amount of 45-55 times its weight, and eluted with 2.0-3.0L of 20%-80% ethanol in sequence. The 80% ethanol elution fraction is collected and chromatographed on a gel column in an amount of 100 times its weight, and further eluted with 100% methanol for 1.0-2.0L to obtain Fr2;

[0019] S2: Fr2 was subjected to C18 column chromatography (100 times its weight) using a gradient elution of 1.0 to 1.5 L of methanol and water at a volume ratio of 30:70 to 50:50. Each 10 to 15 mL fraction was collected into a test tube. The eluates from each test tube containing the same components were combined by TLC spot plate observation to obtain 7 fractions, Fr2.1 to Fr2.7.

[0020] S3: Fr2.3 was subjected to high-speed countercurrent chromatography using 550-650 mL of dichloromethane-methanol-water in a volume ratio of 2:2:1 as the mobile phase. One test tube was collected for every 10-15 mL. The eluents containing the same components in each test tube were combined after TLC spot plate observation to obtain three fractions, Fr2.3.1 to Fr2.3.3. Fr2.3.3 was subjected to preparative high-performance liquid chromatography using a C18 preparative column and acetonitrile-water-formic acid in a volume ratio of 23:77:0.1 as the mobile phase. Munropene C was prepared and purified at a retention time of 10-15 min.

[0021] The gansuline-type triterpenoid compounds of the present invention have the following characteristics and advantages:

[0022] 1. The munropene A-C triterpenoids extracted from the dwarf ...

[0023] 2. The preparation method of the present invention is easy to operate, easy to mass produce, and has stable quality; the raw materials of the present invention are sufficient in China and are reasonably priced, so that its large-scale production does not have too high cost restrictions. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 2D NMR correlation and planar structure diagram of Munropene A and Munropene B.

[0025] Figure 2 2D NMR correlation and planar structure diagram of Munropene C.

[0026] Figure 3 This is the X-ray crystal structure of Munropene A.

[0027] Figure 4 This is the ROESY spectrum stereo structure diagram of Munropene A.

[0028] Figure 5 This is the ROESY spectrum stereo structure diagram of Munropene B.

[0029] Figure 6 This is the ROESY spectrum stereo structure diagram of Munropene C.

[0030] Figure 7 Schematic diagram of the skeleton rotation model of Munropene A, where a) is C-17-C-20, b) is C-20-C-22, c) is C-22-C-23, and d) is C-23-C-24. DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0032] Example 1 Preparation of Munropene A to F

[0033] The dwarf dwarf plant was collected from Jingxi, Guangxi, in July 2021 and identified by Professor Li Dianpeng as Munronia pinnata (Wall.) W. Theob. The specimen is stored in the herbarium of the Center for Natural Product Chemistry, Guangxi Institute of Botany, Chinese Academy of Sciences (21-GX-001).

[0034] S1: Take dry dwarf dwarf powder (25kg), add 95% ethanol and soak and extract 3 times at room temperature, 250L each time, once every 3 days, combine the filtrates, filter and concentrate to obtain an extract, then extract (1.71kg) is sequentially extracted with equal volumes of petroleum ether, ethyl acetate and water, the aqueous layer (560g) is chromatographed on a macroporous resin column (D-101) 50 times its weight, eluted with 20%, 40%, and 80% ethanol 2.5L each, and the 40% ethanol eluate is collected to obtain Fr3 (34g). The 80% ethanol elution portion (86g) is again chromatographed on a LH-20 gel column 100 times its weight, and further eluted with 100% methanol for 1.5L to obtain Fr2 (15g).

[0035] S2: Fr2 was chromatographed on a YMC 50μmC18 column (100 times its weight) using a gradient elution method using methanol-water at a volume ratio of 30:70, 40:60, and 50:50 (1 L each). Every 10 ml of the fraction was collected in one test tube. The eluates from each test tube containing the same components were combined after TLC spot plate observation to obtain seven fractions, Fr2.1 to Fr2.7 (numbered 1, 2, 3, etc. according to the order of the eluted components, the same below). Among them, Fr2.1 was 1.46 g, Fr2.3 was 1.27 g, Fr2.4 was 1.39 g, and Fr2.7 was 0.9 g.

[0036] S3: Fr2.1 (1.46 g) was subjected to silica gel H column chromatography (100 times its weight) using dichloromethane-methanol at a volume ratio of 4:1 to elute 900 mL. Each 10 mL fraction was collected into a test tube. The eluates containing the same components in each test tube were combined by TLC spot plate observation to obtain 5 components, including Fr2.1.1 to Fr2.1.5. Fr2.1.2 (163 mg) was prepared by high-speed countercurrent chromatography using 2 L of dichloromethane-methanol-water at a volume ratio of 2:2:1 as the mobile phase. Each 20 mL mobile phase was collected into a test tube. The same components were combined by TLC spot plate to obtain 6 components, including Fr2.1.2.1 to Fr2.1.2.6. Among them, component Fr2.1.2.3 was a monomer compound, namely Munropene B (2) (68 mg).

[0037] S4: Fr2.3 (1.27 g) was subjected to high-speed countercurrent chromatography using 600 mL of dichloromethane-methanol-water in a volume ratio of 2:2:1 as the mobile phase. One test tube was collected for every 10 mL. The eluents from each test tube were combined after TLC spot plate observation to obtain three fractions, Fr2.3.1 to Fr2.3.3. Fr2.3.3 was subjected to preparative high-performance liquid chromatography using an Agilent ZORBAXSB-C18 preparative column and acetonitrile-water-formic acid in a volume ratio of 23:77:0.1 as the mobile phase to obtain Munropene C (3) (26 mg, tR = 10.1 min).

[0038] S5: Fr2.4 (1.39 g) was subjected to high-speed countercurrent chromatography using 2 L of dichloromethane-methanol-water with a volume ratio of 2:2:1 as the mobile phase for elution. Each 20 ml of mobile phase was collected into one tube, and the same components were combined on a TLC plate to obtain 6 components, including Fr2.4.1 to Fr2.4.6; Fr2.4.4 (131 mg) was then subjected to preparative high-performance liquid chromatography using an Agilent ZORBAXSB-C18 preparative column and acetonitrile-water-formic acid with a volume ratio of 23:77:0.1 as the mobile phase for elution to obtain Munropene E (5) (10 mg, tR = 12.2 min) and Munropene F (6) (15 mg, tR = 14.7 min), respectively.

[0039] S6: Fr2.7 (0.9 g) was subjected to high-speed countercurrent chromatography using 1.5 L of dichloromethane-methanol-water with a volume ratio of 2:2:1 as the mobile phase for elution. Each 20 ml of mobile phase was collected into one tube and the same components were combined by TLC spot plate to obtain five components, namely Fr2.7.1 to Fr2.7.5, among which component Fr2.7.3 was a monomer compound, namely Munropene D(4) (184 mg).

[0040] S7: Fr3 (30 g) was subjected to chromatography on a silica gel H column in an amount 100 times its weight, and 1.5 L of dichloromethane-methanol was used as the mobile phase in a volume ratio of 80:20, 90:10, and 100:0 for gradient elution. One test tube was collected for every 10 ml, and the eluents containing the same components in each test tube were combined after TLC spot plate observation. There were 20 fractions in total, Fr3.1 to Fr3.20; Fr3.3 (1.49 g) was subjected to preparative high performance liquid chromatography using a ChromCore 120-C18 high performance liquid chromatography column and purified with acetonitrile-water-formic acid in a volume ratio of 18:82:0.1 as the mobile phase to obtain Munropene A (1) (321 mg, tR = 21.3 min).

[0041] Identification of Munropene A~F

[0042] Optical rotation was measured using a Jasco P-1020 polarimeter. Infrared spectroscopy was performed on KBr pellets using a TENSOR 27 Fourier transform infrared spectrometer (TheromFisher, USA). CD spectra were recorded on a J-810 CD spectrometer. Mass spectra were determined using an LC / MS-IT-TOF mass spectrometer. Nuclear magnetic resonance spectra were recorded using a Bruker AVANCE III-HD 500 spectrometer with TMS as the internal standard. CCC was performed on a TBE-300C system (Tauto biotechnology, Shanghai, China). HPLC analysis was performed using an Agilent 1260 Infinity II LC (Agilent Technologies, USA) using an Agilent Poroshell 120cb-C18 (4 mm, 4.6 mm x 150 mm, Agilent, USA), a ChromCore 120-C18 (5 mm, 10 mm x 250 mm, NanoChrom, China), and an Agilent ZORBAX cb-C18 (5 mm, 9.4 mm x 250 mm, Agilent, USA). Column chromatography was performed using silica gel (200-300 mesh, Qingdao Ocean Chemical Plant, China) and MCI gel (Mitsubishi Chemical Corporation, Japan). TLC analysis was performed using pre-coated silica gel 60f254 plates (Merck Millipore, Germany). The silica gel plates were sprayed with 10% H2SO4 in ethanol and heated for observation of spots.

[0043] Munropene A: a colorless, amorphous solid with optical activity; its molecular formula is C 32 H 50 O 12 ; [α] D 20.1 +4.98(c0.10,MeOH); HRESIMS:m / z 608.2496([M+Na] + ,Δ+2.4mmu; IR: 1748 and 1689cm-1, carbonyl functional group absorption peaks. The H NMR spectrum is shown in Table 1. The C NMR spectrum is shown in Table 2. The 2D NMR correlation and planar structure diagram are shown in Figure 1 As shown, the X-ray crystal structure is shown in Figure 3 As shown, the ROESY spectrum is Figure 4 As shown, the skeleton rotation model diagram is as follows Figure 7 shown.

[0044] Munropene B: a colorless, amorphous solid with optical activity; its molecular formula is C32 H 50 O 12 ; [α] D 20.1 -5.00(c0.10,MeOH); HRESIMS: m / z 608.2493([M+Na]+,Δ+2.1mmu; IR: 1748and 1689cm -1 , carbonyl functional group absorption peak. The H NMR spectrum is shown in Table 1. The C NMR spectrum is shown in Table 2. The 2D NMR correlation and planar structure diagram are shown in Figure 1 As shown, the ROESY spectrum is Figure 5 shown.

[0045] Munropene C: a colorless, optically active amorphous solid; its molecular formula is C 32 H 52 O 13 ; [α] D 20.0 =-50.51 (c 0.10, MeOH); HRESIMS: m / z 643.3216 ([MH]-, Δ-11.9 mmu); H NMR spectrum is shown in Table 1. C NMR spectrum is shown in Table 2. 2D NMR correlation and planar structure diagram are shown in Figure 2 As shown, the ROESY spectrum is Figure 6 shown.

[0046] Cytotoxicity experiments of munropene A~F triterpenoids

[0047] The CCK-8 assay was used to detect the cytotoxicity of compounds Munropene A to 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 cancer cell line MDAMB. 100 μL cell suspension (2 × 10 ^5 Cells were seeded in 96-well microtiter plates (100 cells / mL) and cultured for 24 hours before compound addition. Different concentrations of compounds Munropene A–F (5, 10, 20, 40, 80, and 160 μM) were added to the culture dish. An equal volume of DMSO was added to the blank control group. Three replicates were added to each well for 24 hours. Then, 10 μL of CCK-8 reagent was added to each well. After 1 hour, absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0048] Table 1 H NMR spectrum ( 1 H NMR data for munropenes AD in CD3OD)

[0049]

[0050]

[0051] Table 2 C NMR spectrum ( 13 C NMR data for munropenes AD in CD3OD)

[0052]

[0053]

[0054] Table 3 C NMR spectrum and H NMR spectrum (C NMR spectrum and H NMR spectrum ( 1 H and 13 C NMRdata formunropenesE-F in CD3OD)

[0055]

[0056]

[0057] Table 4 Cytotoxicity (IC 50 (μM)

[0058] 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

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A gansuline-type triterpenoid compound having anti-tumor effect, characterized in that: The gansuiane-type triterpenoid compound has a structure of either Munropene A or Munropene B, wherein Munropene A is represented by formula (I), and Munropene B is represented by formula (II): Formula (I); Formula (II).

2. A method for preparing the gansuline-type triterpenoid compound with anti-tumor effect according to claim 1, characterized in that: The preparation method of Munropene A comprises the following steps: S1: taking dry dwarf dwarf powder, adding 95% ethanol at a material-liquid ratio of 1 kg:10-15 L, and soaking and extracting at room temperature for 2-4 times, once every 3 days, combining the filtrates, filtering, and concentrating to obtain an extract, then extracting the extract with equal volumes of petroleum ether, ethyl acetate, and water, respectively, subjecting the aqueous layer to macroporous resin column chromatography in an amount 45-55 times its weight, eluting with 2.0-3.0 L of 20%-80% ethanol, and collecting the 40% ethanol eluate to obtain Fr3; S2: Fr3 was subjected to silica gel column chromatography (100 times its weight) using a mobile phase of dichloromethane-methanol (volume ratio 80:20 to 100:0) with a gradient elution of 1.0-2.0 L each. 10-15 ml of fractions were collected in one test tube. The eluates from each test tube containing the same components were combined after TLC spot plate observation to obtain 20 fractions, Fr3.1 to Fr3.

20. S3: Fr3.3 was purified by preparative high performance liquid chromatography using a C18 high performance liquid chromatography column with acetonitrile-water-formic acid in a volume ratio of 18:82:0.1 as the mobile phase to obtain Munropene A at a retention time of 20-25 min.

3. A method for preparing the gansuline-type triterpenoid compound with anti-tumor effect according to claim 1, characterized in that: The preparation method of Munropene B comprises the following steps: S1: taking dry dwarf dwarf powder, adding 95% ethanol at a material-liquid ratio of 1kg:10-15L, and soaking and extracting at room temperature for 2-4 times, once every 3 days, combining the filtrates, filtering, and concentrating to obtain an extract, then extracting the extract with equal volumes of petroleum ether, ethyl acetate, and water, respectively, subjecting the aqueous layer to macroporous resin column chromatography in an amount 45-55 times its weight, eluting with 2.0-3.0L of 20%-80% ethanol, collecting the 80% ethanol elution portion, subjecting it to gel column chromatography in an amount 100 times its weight, and further eluting with 100% methanol for 1.0-2.0L to obtain Fr2; S2: Fr2 was subjected to C18 column chromatography (100 times its weight) using a gradient elution of 1.0 to 1.5 L of methanol-water at a volume ratio of 30:70 to 50:

50. Each 10-15 ml fraction was collected into a test tube. The eluates from each test tube containing the same components were combined by TLC spot plate observation to obtain 7 fractions, Fr2.1 to Fr2.

7. S3: Fr2.1 was subjected to silica gel column chromatography (100 times its weight). 850-950 mL was eluted with dichloromethane-methanol at a volume ratio of 4:

1. 10-15 mL was collected into a test tube. The eluates from each test tube containing the same components were combined by TLC spot plate observation to obtain five components, namely Fr2.1.1 to Fr2.1.

5. S4: Fr2.1.2 was prepared by high-speed countercurrent chromatography using 1.5-2.5 L of dichloromethane-methanol-water with a volume ratio of 2:2:1 as the mobile phase. Every 20-25 ml of mobile phase was collected into one test tube. The same components were combined by TLC spot plate to obtain 6 components, namely Fr2.1.2.1 to Fr2.1.2.6, among which component Fr2.1.2.3 was a monomer compound, namely Munropene B.

4. Use of the gansuline-type triterpenoid compound with anti-tumor effect according to claim 1 in the preparation of a drug for treating colon cancer.

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