An ursane-type triterpenoid compound, preparation method and application thereof

Abstract

The present invention relates to the field of extraction of natural compounds, and specifically to an ursane-type triterpenoid compound, its preparation method, and application. The ursane-type triterpenoid compound of the present invention can be extracted from the Euphorbia genus medicinal plant Euphorbia ulmoides. The ursane-type triterpenoid compound can significantly inhibit the survival of liver cancer cells. The structural formula of the ursane-type triterpenoid compound of the present invention is:

Description

Technical Field

[0001] The present invention relates to the field of extraction of natural compounds, and in particular to an ursane-type triterpenoid compound, a preparation method and application thereof. Background Art

[0002] Euphorbia ebracteolata Hayata, a traditional Chinese medicine documented over 2,000 years ago in the Shennong Bencao Jing (Shen Nong's Classic of Materia Medica), is the dried root of the Euphorbia genus (Euphorbia ebracteolata Hayata). This herb is mild in nature and pungent in flavor. It is widely used to treat solid tumors, inflammation, asthma, indigestion, skin ulcers, and lymphatic diseases. Its chemical constituents primarily include diterpenes, triterpenes, phloroglucinol, flavonoids, phenylpropanoids, steroids, and essential oils. The diterpenes and triterpenes exhibit a wide range of anti-inflammatory, anti-tumor, and antiviral activities. However, research on Euphorbia ebracteolata Hayata has been limited in recent years, and much of the literature is outdated. Therefore, a systematic and in-depth review of its chemical composition and pharmacological activities is warranted. Summary of the Invention

[0003] The purpose of the present invention is to provide an ursane-type triterpenoid compound, which can be extracted from the root of Euphorbia lunatum, helping to solve or improve the current problem of little research on Euphorbia lunatum, and is conducive to the further development of the medicinal value of Euphorbia lunatum.

[0004] The ursane-type triterpenoid compound of the present invention adopts the following technical solution: the structural formula of the ursane-type triterpenoid compound is:

[0005] .

[0006] The present invention provides a method for preparing an ursane-type triterpenoid compound, which adopts the following technical scheme: A method for preparing an ursane-type triterpenoid compound, comprising the following steps: (1) crushing the dried root of Euphorbia lunulata, extracting it with an ethanol solution at room temperature, combining the extracts, and concentrating under reduced pressure to obtain a crude extract; (2) diluting the crude extract with water, extracting it with petroleum ether and ethyl acetate in sequence to obtain an ethyl acetate extract, and concentrating it under reduced pressure to obtain a concentrate; (3) passing the concentrate through an ODS-C18 medium-pressure column and gradient eluting it with a methanol solution with a volume concentration of 30%-100%, and combining the fractions after color development by thin-layer chromatography to obtain four fractions: Frs. I-IV; (4) fraction Frs. III is first subjected to gel column chromatography on Sephadex LH-20 to remove the drag, and then subjected to a first forward silica gel column chromatography to further obtain the subfraction Frs. III-1-III-3; during the first forward silica gel column chromatography, an eluent with a volume ratio of dichloromethane / methanol = 35:1-10:1 is used for elution; (5) the subcomponent Frs. III-2 is subjected to a second forward silica gel column chromatography to obtain components Frs. III-2A-III-2D; during the second forward silica gel column chromatography, an eluent with a volume ratio of petroleum ether / ethyl acetate = 20:1-1:1 is used for elution; (6) component Frs. III-2C is concentrated and purified, and then separated by semi-preparative HPLC to obtain the ursane-type triterpenoid compound.

[0007] Preferably, in step (1), the dried roots of Euphorbia lunulata are crushed until they can pass through a 100-mesh sieve; the concentration of the ethanol solution is 95%, and the amount of ethanol solution used is 9-11 times the mass of the dried roots of Euphorbia lunulata; the time for each extraction is 36-48 hours, and the number of extractions is 2-3 times; the temperature of the reduced pressure concentration is 50-55°C.

[0008] Preferably, in step (2), warm water at 50-60°C is added for dilution, and after dilution, insoluble matter is removed by filtration to obtain a dilution liquid; during extraction, the dilution liquid is first extracted with petroleum ether, and then with ethyl acetate; the volume ratio of petroleum ether or ethyl acetate to the dilution liquid is 1:1, and the number of extractions with petroleum ether and ethyl acetate is 3-4 times, respectively; the temperature of the reduced pressure concentration in step (2) is 50-55°C.

[0009] Preferably, in step (3), the mass ratio of the medium-pressure column packing to the concentrate is 300:1-500:1; during gradient elution, the concentrations of the methanol solution are 30%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 100%, respectively.

[0010] Preferably, during the first forward silica gel column chromatography and / or the second forward silica gel column chromatography, the silica gel in the forward silica gel column is 200-300 mesh silica gel; when a dry sample is selected, the silica gel used for mixing the sample is 80-100 mesh silica gel.

[0011] Preferably, in step (4), during the first forward silica gel column chromatography, eluents having a volume ratio of dichloromethane to methanol of 35:1, 20:1 and 10:1 are used for elution, respectively.

[0012] Preferably, in step (5), during the second forward silica gel column chromatography, elution is performed using eluents having a volume ratio of petroleum ether / ethyl acetate = 20:1, 15:1, 8:1 and 1:1, respectively.

[0013] Preferably, in step (6), the chromatographic column for semi-preparative HPLC is Agilent SB-C18, 9.4×250 mm, 5 μm, the mobile phase flow rate is 3 mL / min, and the detection wavelengths are 203 nm and 254 nm.

[0014] The present invention also provides the use of the ursane-type triterpenoid compound as described above, which adopts the following technical solution: the use of the ursane-type triterpenoid compound as described above in the preparation of anti-tumor drugs; the tumor includes but is not limited to liver cancer.

[0015] The present invention has the following advantages:

[0016] The present invention provides an ursane-type triterpenoid compound extracted from the Euphorbia genus medicinal plant Euphorbia oleifera, as well as a preparation method and the use of the compound in inhibiting hepatocellular carcinoma. This triterpenoid compound belongs to the ursane type, with a methyl group attached at positions 19 and 20, a hydroxyl group at position 30, and a methylcarbonyl group at position 3. This ursane-type triterpenoid compound can significantly inhibit the survival of liver cancer cells.

[0017] The ursane-type triterpenoid compound of the present invention provides a material basis for the research and development of new anti-tumor drugs, and is conducive to the further development of the medicinal value of Euphorbia lunata. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a high performance liquid chromatogram of the ursane-type triterpenoid compound of the present invention;

[0019] Figure 2 is a structural diagram of the ursane-type triterpenoid compound of the present invention;

[0020] Figure 3 This is a high-resolution mass spectrum of the ursane-type triterpenoid compound of the present invention;

[0021] Figure 4is the H NMR spectrum of the ursane-type triterpenoid compound of the present invention;

[0022] Figure 5 The ursane type triterpenoid compound of the present invention 13 C and DEPT NMR spectra;

[0023] Figure 6 HSQC spectrum of the ursane-type triterpenoid compound of the present invention;

[0024] Figure 7 The ursane type triterpenoid compound of the present invention 1 H- 1 HCOSY and key HMBC correlation diagrams;

[0025] Figure 8 The ursane type triterpenoid compound of the present invention 1 H- 1 HCOSY spectrum;

[0026] Figure 9 is the HMBC spectrum of the ursane-type triterpenoid compound of the present invention;

[0027] Figure 10 is the key ROESY correlation diagram of the ursane-type triterpenoid compound of the present invention;

[0028] Figure 11 ROESY spectrum of the ursane-type triterpenoid compound of the present invention. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention, and all such modifications and substitutions fall within the scope of the claims of the present invention.

[0030] In view of the problem that there is little research on Euphorbia lunatum at present, the present invention further studies Euphorbia lunatum.

[0031] The present invention provides an ursane-type triterpenoid compound, the structural formula of which is:

[0032] .

[0033] The ursane-type triterpenoid compounds of the present invention can be obtained from Euphorbia ulinatum ( Euphorbiaebracteolata The molecular formula of the ursane-type triterpenoid compound of the present invention is: C32 H 52 O2.

[0034] It is worth noting that, based on a SciFinder database search and comparison with previously reported triterpenoid compounds, the ursane-type triterpenoid compound of the present invention is a novel compound. Its structural characteristic is that C-3 is substituted with a carbonylmethyl group. This is the first discovery and report of the chemical structure and biological activity of this compound.

[0035] The present invention also proposes a method for preparing the ursane-type triterpenoid compound. The method for preparing the ursane-type triterpenoid compound in the embodiment of the present invention comprises the following steps: (1) crushing the dried root of Euphorbia lunulata, extracting it with an ethanol solution at room temperature, combining the extracts, and concentrating under reduced pressure to obtain a crude extract; (2) diluting the crude extract with water, extracting it with petroleum ether and ethyl acetate in sequence to obtain an ethyl acetate extract, and concentrating it under reduced pressure to obtain a concentrate; (3) passing the concentrate through an ODS-C18 medium-pressure column and gradient eluting it with a methanol solution with a volume concentration of 30%-100%, and combining the fractions after color development by thin-layer chromatography to obtain four fractions: Frs. I-IV; (4) fraction Frs. III is first subjected to gel column chromatography on Sephadex LH-20 to remove the drag, and then subjected to a first forward silica gel column chromatography to further obtain the subfraction Frs. III-1-III-3; during the first forward silica gel column chromatography, an eluent with a volume ratio of dichloromethane / methanol = 35:1-10:1 was used for elution; (5) the subfraction Frs. III-2 was subjected to a second forward silica gel column chromatography to obtain fractions Frs. III-2A-III-2D; during the second forward silica gel column chromatography, an eluent with a volume ratio of petroleum ether / ethyl acetate = 20:1-1:1 was used for elution; (6) fraction Frs. III-2C was concentrated and purified, and then separated by semi-preparative HPLC to obtain ursane-type triterpenoid compounds. Among them, Frs. I-IV are fractions obtained after elution with methanol solutions of different concentrations, Frs. III is the fraction obtained after elution with 70%-85% methanol solution; Frs. III-1-III-3 are three components divided according to TLC color development results, Frs. III-2 is the component containing pink spots; Frs. III-2A-III-2D are fractions obtained after elution with petroleum ether / ethyl acetate of different volume ratios, and Frs. III-2C is the fraction obtained after elution with an eluent of petroleum ether / ethyl acetate = 8:1.

[0036] In a preferred embodiment of the method for preparing ursane-type triterpenoid compounds of the present invention, in step (1), the dried roots of Euphorbia lunulata are crushed until they can pass through a 100-mesh sieve; the concentration of the ethanol solution is 95%, and the amount of the ethanol solution used is 9-11 times the mass of the dried roots of Euphorbia lunulata; the time for each extraction is 36-48 h (for example, 36 h, 38 h, 40 h, 42 h, 44 h, 46 h or 48 h), and the number of extractions is 2-3 times; the temperature for reduced pressure concentration is 50-55 ° C (for example, 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C or 55 ° C).

[0037] In a preferred embodiment of the method for preparing ursane-type triterpenoid compounds of the present invention, in step (2), warm water at 50-60°C (for example, 50°C, 52°C, 54°C, 56°C, 58°C or 60°C) is added for dilution, and after dilution, insoluble matter is removed by filtration to obtain a dilution liquid; during extraction, the dilution liquid is first extracted with petroleum ether, and then the dilution liquid is extracted with ethyl acetate; the volume ratio of petroleum ether or ethyl acetate to the dilution liquid is 1:1, and the number of extractions with petroleum ether and ethyl acetate is 3-4 times, respectively; the temperature of the reduced pressure concentration in step (2) is 50-55°C (for example, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C).

[0038] In a preferred embodiment of the method for preparing ursane-type triterpenoid compounds of the present invention, in step (3), the mass ratio of the medium-pressure column filler to the concentrate is 300:1-500:1 (for example, 300:1, 350:1, 400:1, 450:1 or 500:1); during gradient elution, the concentrations of the methanol solution are 30%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 100%, respectively.

[0039] In a preferred embodiment of the method for preparing ursane-type triterpenoid compounds of the present invention, during the first forward silica gel column chromatography and / or the second forward silica gel column chromatography, the silica gel in the forward silica gel column is 200-300 mesh silica gel; and when a dry sample is selected, the sample mixing silica gel is 80-100 mesh silica gel. If the mesh size of the silica gel in the forward silica gel column is greater than 200-300 mesh, the separation effect between different compounds is weakened, and compounds with similar polarity may be separated; if the mesh size of the silica gel in the forward silica gel column is less than 200-300 mesh, the separation time is too long; if the sample mixing silica gel is less than 80-100 mesh, the sample adsorption is too weak; and if the sample mixing silica gel is greater than 80-100 mesh, it is easy to cause dead adsorption or the sample mixing silica gel leaks into the column silica gel, affecting the separation.

[0040] In a preferred embodiment of the method for preparing ursane-type triterpenoid compounds of the present invention, in step (4), during the first forward silica gel column chromatography, eluents having a volume ratio of dichloromethane / methanol = 35:1, 20:1 and 10:1 are used for elution, respectively.

[0041] In a preferred embodiment of the method for preparing ursane-type triterpenoid compounds of the present invention, in step (5), during the second forward silica gel column chromatography, eluents having a volume ratio of petroleum ether / ethyl acetate = 20:1, 15:1, 8:1 and 1:1 are used for elution, respectively.

[0042] In a preferred embodiment of the method for preparing ursane-type triterpenoid compounds of the present invention, in step (6), the chromatographic column for semi-preparative HPLC is Agilent SB-C18, 9.4×250 mm (diameter×length), 5 μm (particle size), the mobile phase flow rate is 3 mL / min, and the detection wavelengths are 203 nm and 254 nm.

[0043] The present invention also proposes the application of the above ursane-type triterpenoid compounds: the application of the above ursane-type triterpenoid compounds in the preparation of anti-tumor drugs; tumors include but are not limited to liver cancer.

[0044] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0045] The sources of the main instruments and materials used in the following examples are as follows (unless otherwise explicitly stated, the instruments and materials used in the present invention can be purchased commercially):

[0046] Thin layer chromatography (TLC) silica gel plate GF 254 (Qingdao Ocean Chemical), silica gel for column chromatography (200-300 mesh, Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), hydroxypropyl dextran gel (Sephadex LH-20, 20-100 mmoL, Pharmacia), octadecyl silica gel (ODS-C 18 , 75 mmoL, YMC Co., Ltd., Japan); chromogenic agent 5% H2SO4-EtOH solution (heated appropriately after spraying); chromatographically pure methanol and acetonitrile (Fisher); other reagents were of analytical grade (Chengdu Kelong Chemical Co., Ltd.); Bruker DRX-500 and Bruker AVIII-600 nuclear magnetic resonance spectrometers (Bruker, Fällanden, Switzerland); Agilent 1206 high performance liquid chromatograph (Agilent) equipped with ZORBAXSB-C 18 Reversed-phase column and diode array detector (Agilent).

[0047] The dried roots of Euphorbia lunulata used in the experiment were collected from Linyi, Shandong Province in November 2021 and identified by Qi Huimin of Shandong Second Medical University as Euphorbia lunulata (Euphorbiaceae). Euphorbiaebracteolate The dried root of Psoralea corylifolia (Hayashita). Voucher samples are stored in the Laboratory of Medicinal Chemistry, Shandong Second Medical University.

[0048] Example 1

[0049] The preparation method of the ursane-type triterpenoid compound of this embodiment comprises the following steps:

[0050] (1) The dried roots of Euphorbia lunata (20 kg) were crushed to a particle size of less than 3 mm, and extracted with 95% ethanol water at room temperature for three times, 300 L each time, for 48 hours each time; the ethanol extracts were combined and concentrated under reduced pressure to obtain a crude extract;

[0051] (2) Add 50°C warm water to the crude extract and stir, filter out insoluble impurities with gauze, then extract the aqueous solution three times with petroleum ether (using 25L of petroleum ether for each extraction), and then extract it three times with ethyl acetate (using 25L of ethyl acetate for each extraction); combine the three ethyl acetate extracts and concentrate under reduced pressure to obtain the ethyl acetate extract of Euphorbia lunatum (205g);

[0052] (3) The ethyl acetate extract was passed through an ODS-C18 medium-pressure column (column height 80 cm, inner diameter 25 cm) and gradient eluted with methanol / water (30%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 100%, v / v). 10 L of each gradient elution was used, and a fraction was concentrated by rotary evaporation every 2 L. The fractions were visualized by thin layer chromatography (TLC) (developing solvent: petroleum ether: ethyl acetate = 3:2 (v / v) and dichloromethane: methanol = 15:1 (v / v); color developer 5% H2SO4-EtOH solution, sprayed with color developer and then heated appropriately) and the fractions were combined (the same fractions after TLC were combined) to obtain 4 fractions: Frs.I-IV (Frs.I is the elution part of 30%-50% methanol solution, Frs.II is the elution part of 60% methanol solution, Frs.III is the elution part of 70%-85% methanol solution, Frs. IV is the 90%-100% methanol solution elution portion);

[0053] (4) Frs.III (26.7 g) was first subjected to gel Sephadex LH-20 (column height 160 cm, inner diameter 4 cm) column chromatography (eluted with methanol, a total of 3 L) to remove the drag, and a fraction was concentrated by rotary evaporation for every 250 mL. The fractions were developed by TLC (developing solvent was petroleum ether: acetone = 2:1 v / v; the color developer was 5% H2SO4-EtOH solution, and the color developer was sprayed and heated appropriately) to obtain the fraction after removing the drag (2.1 g); then the fraction was subjected to normal silica gel column chromatography (column height 70 cm, inner diameter 10 cm, 200-300 mesh silica gel packing) The product was purified by centrifugation with a column (80-100 mesh silica gel mixed with sample) and gradient elution with dichloromethane / methanol (35:1, 20:1, 10:1, v / v). Each gradient elution was 500 mL. A fraction was concentrated by rotary evaporation for each 50 mL. The fractions were visualized by TLC (developing solvent: petroleum ether:ethyl acetate = 3:2 (v / v) and dichloromethane:isopropanol = 15:1 (v / v); the developer was 5% H2SO4-EtOH solution). The fractions were combined to further obtain subfractions: Frs.III-1-III-3 (the same fractions were combined after TLC development and divided into 3 components);

[0054] (5) The subfraction Frs.III-2 (157.2 mg) (Frs.III-2 refers to the component containing pink spots after color development with 5% H2SO4-EtOH solution; the color of Frs.III-2 is significantly different from that of Frs.III-1 and Frs.III-3) was subjected to normal silica gel column chromatography (column height 50 cm, inner diameter 1.5 cm, 200-300 mesh silica gel column, 80-100 mesh silica gel sample mixing), and gradient elution with petroleum ether / ethyl acetate (20:1, 15:1, 8:1, 1:1, v / v), with each gradient as a fraction (the fractions obtained were Frs.III-2A-III-2D in sequence), and the fractions were developed by TLC (developing solvent: dichloromethane / isopropanol 20:1 (v / v); color developer: 5% H2SO4-EtOH solution);

[0055] (6) The petroleum ether / ethyl acetate 8:1 fraction (Frs. III-2C) was then concentrated under reduced pressure and further purified by semi-preparative HPLC (Agilent 1206 high pressure liquid phase, Agilent SB-C18 column, 9.4×250 mm, 5 μm, mobile phase flow rate of 3 mL / min, detection wavelength of 203 nm and 254 nm, eluted with 72% methanol / water). After 9.7 min, the pure ursane-type triterpenoid compound of this example was obtained (the HPLC spectrum of the ursane-type triterpenoid compound of this example is shown in FIG. Figure 1 shown).

[0056] Experimental example

[0057] 1. Structural identification:

[0058] The ursane-type triterpenoid compound of Example 1 (structure shown in FIG. Figure 2 As shown, white powder), by high resolution mass spectrometry HR-ESI-MS ( m / z 507.3783[M+Na] + , calculated as 507.3809) (e.g. Figure 3 As shown) the molecular formula is determined to be C 32 H 52 O3, unsaturation is 7.

[0059] 1 H-NMR spectrum (Table 1, Figure 4 ) shows 7 singlet peaks of methyl hydrogen signals [ δ H 0.84 (3H, s, Me-23), 0.85 (3H, s, Me-24), 0.87 (3H, s, Me-25), 1.04 (3H, s, Me-26), 0.95 (3H, s, Me-27), 0.76 (3H, s, Me-28) and 2.05 (3H, s, OAc)]; 1 doublet of methyl hydrogen signal [ δ H 1.0(3H,d, J =6.5Hz,Me-29)]; 2 oxidized methylene hydrogen signals [ δ H 4.02(1H,d, J =12.7Hz,Me-30);4.12(1H,d, J =12.7 Hz,Me-30)] and one oxidized methine hydrogen signal [ δ H 4.49(1H,dd, J =10.9,5.6Hz,H-3)] and one double bond methine hydrogen signal [ δ H 5.59(1H,d, J =6.4Hz,H-21)].

[0060] 13 C-NMR and DEPT spectra (Table 1, Figure 5 ), showing 32 carbon signals, including 1 carbonyl ( δ C 171.1); 2 intracyclic alkenes ( δ C 120.7,143.6); 1 methylene oxide ( δ C 65.5); 1 oxymethyl group ( δC 81.0); 8 methyl groups ( δ C 14.7, 16.0, 16.3, 16.5, 17.7, 21.4, 22.5, 27.9); another 9 sp3 methylene groups; 5 sp3 methine groups; 5 sp3 quaternary carbon groups.

[0061] The HSQC spectrum ( Figure 6 ) analysis assigned the corresponding proton and associated carbon signals. NMR data of the compound indicated that it is an ursane-type triterpenoid with a carbonylmethyl group at position 3, a double bond at position 20, and a hydroxyl group at position 30.

[0062] exist 1 H- 1 HCOSY spectrum ( Figure 7 and Figure 8 ), the spin coupling structure fragment of H-1 / H-2 / H-3 was observed; and in the HMBC spectrum ( Figure 7 and Figure 9 ), the signals associated with Me-2' and C-1', and H-3 and C-1', C-1', C-2, C-4, C-23, and C-24, indicate the presence of a methylcarbonyl group at position 3. The signals associated with C-20 and H-29, and H-21 and C-19 and C-20, confirm a double bond at position 20. Furthermore, the observed signals associated with H-30 and C-20 and C-21 confirm a hydroxyl group at position 30.

[0063] In the ROESY spectrum ( Figure 10 and Figure 11 ), a series of correlations were observed between H-3 and H-5, H-5 and H-9, H-18 and Me-27 and Me-29, Me-23 and Me-27, and Me-27 and H-9 and H-18, indicating that H-3, H-5, H-9, H-18, Me-23, Me-27, and Me-29 are a In addition, a series of correlations between H-13, Me-25, Me-26, H-13, Me-28, and Me-24 and Me-25 confirm that H-13, Me-24, Me-25, Me-26, and Me-28 are Therefore, the structure of the ursane-type triterpenoid compound of the present invention is completely determined.

[0064] Table 1. Ursane-type triterpenoid compounds of this embodiment 1 H-and 13 C-NMR data

[0065]

[0066] 2. Screening of the inhibitory activity of the ursane-type triterpenoid compounds of the present invention on HepG2 cells

[0067] HepG2 cells (purchased from Jiangsu KeyGen Biotechnology Co., Ltd.) were revived, cultured, and passaged. The cells were then cultured at 37°C in a 5% CO2 atmosphere in a culture medium consisting of DMEM, 1% HEPES (4-hydroxyethylpiperazineethanesulfonic acid) (BioFroxx, Hesse, Einhausen), 1% mixed penicillin (10,000 μg / mL), 1% streptomycin (10,000 μg / mL), and 10% fetal bovine serum. Briefly, HepG2 cells (1 × 10 5 Cells were seeded in 96-well microplates (μM / well) and allowed to adhere for 12 hours before drug addition. Cisplatin or doxorubicin served as positive controls. Each cell line was treated three times with different concentrations (50, 25, 12.5, 6.25, and 3.12 μM) and cultured for an additional 48 hours.

[0068] Cell viability was determined by MTT colorimetry. Optical density ( λ = 490 nm). The IC50 values ​​of the samples were calculated based on the average OD and concentration curves of the three drugs. SPSS 21.0 software was used for data evaluation.

[0069] Experimental results

[0070] The ursane triterpenoids prepared in Example 1 were tested for their growth inhibitory activity against a tumor-sensitive cell line (HeG2), with doxorubicin (Adr) used as a positive control. The experimental results showed that the ursane triterpenoids prepared in Example 1 had a good inhibitory effect on HepG2 cells (the IC values ​​of doxorubicin and the ursane triterpenoids prepared in Example 1 were 2.37 and 2.76, respectively). 50 The results show that the inhibitory effect of the ursane-type triterpenoid compound in Example 1 on HepG2 cells is basically equivalent to that of doxorubicin.

[0071] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing an ursane-type triterpenoid compound, characterized in that: The structural formula of the ursane-type triterpenoid compound is: ； The preparation method of the ursane-type triterpenoid compound comprises the following steps: (1) The dried roots of Euphorbia lunata were crushed, extracted with ethanol solution at room temperature, the extracts were combined, and concentrated under reduced pressure to obtain a crude extract; (2) diluting the crude extract with water, extracting with petroleum ether and ethyl acetate in sequence to obtain an ethyl acetate extract, and concentrating under reduced pressure to obtain a concentrate; (3) The concentrate was passed through an ODS-C18 medium-pressure column and gradient eluted with a methanol solution having a volume concentration of 30%-100%. The fractions were visualized by thin-layer chromatography and combined to obtain four fractions: Frs. I-IV; (4) The fraction Frs. III was first subjected to gel column chromatography on Sephadex LH-20 to remove drag, and then subjected to a first forward silica gel column chromatography to further obtain subfractions Frs. III-1-III-3; during the first forward silica gel column chromatography, an eluent with a volume ratio of dichloromethane / methanol = 35:1-10:1 was used for elution; (5) Subfraction Frs. III-2 was subjected to a second normal silica gel column chromatography to obtain fractions Frs. III-2A-III-2D; during the second normal silica gel column chromatography, an eluent having a volume ratio of petroleum ether / ethyl acetate = 20:1 to 1:1 was used for elution; (6) The component Frs. III-2C was concentrated and purified, and then separated by semi-preparative HPLC to obtain the ursane-type triterpenoid compound.

2. The method for preparing the ursane-type triterpenoid compound according to claim 1, wherein In step (1), the dried roots of Euphorbia lunata are crushed to pass through a 100-mesh sieve; The concentration of the ethanol solution is 95%, and the amount of the ethanol solution is 9-11 times the mass of the dry roots of Euphorbia lunulata; Each extraction takes 36-48 hours, and the number of extractions is 2-3 times; The temperature of the reduced pressure concentration is 50-55°C.

3. The method for preparing the ursane-type triterpenoid compound according to claim 1, wherein In step (2), warm water at 50-60° C. is added for dilution, and after dilution, insoluble matter is removed by filtration to obtain a dilution solution; During the extraction, the dilution liquid is first extracted with petroleum ether, and then extracted with ethyl acetate; the volume ratio of petroleum ether or ethyl acetate to the dilution liquid is 1:1, and the number of extractions with petroleum ether and ethyl acetate is 3-4 times respectively; The temperature of the reduced pressure concentration in step (2) is 50-55°C.

4. The method for preparing the ursane-type triterpenoid compound according to claim 1, wherein In step (3), the mass ratio of the medium-pressure column filler to the concentrate is 300:1-500:1; During gradient elution, the concentrations of the methanol solution are 30%, 50%, 60%, 70%, 75%, 80%, 85%, 90% and 100% in sequence.

5. The method for preparing the ursane-type triterpenoid compound according to claim 1, wherein: During the first forward silica gel column chromatography and / or the second forward silica gel column chromatography, the silica gel in the forward silica gel column is 200-300 mesh silica gel; Select dry sample and use 80-100 mesh silica gel for sample mixing.

6. The method for preparing the ursane-type triterpenoid compound according to claim 1, wherein: In step (4), during the first forward silica gel column chromatography, eluents with volume ratios of dichloromethane / methanol = 35:1, 20:1 and 10:1 are used for elution, respectively.

7. The method for preparing the ursane-type triterpenoid compound according to claim 1, wherein: In step (5), during the second forward silica gel column chromatography, elution is performed using eluents having a volume ratio of petroleum ether / ethyl acetate = 20:1, 15:1, 8:1 and 1:1, respectively.

8. The method for preparing the ursane-type triterpenoid compound according to claim 1, wherein: In step (6), the chromatographic column for semi-preparative HPLC was Agilent SB-C18, 9.4×250 mm, 5 μm, the mobile phase flow rate was 3 mL / min, and the detection wavelengths were 203 nm and 254 nm.

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