A phenylpropanoid compound and its extraction and purification method and application

Abstract

The present invention relates to the technical field of anti-tumor compounds, and provides a phenylpropanoid compound and an extraction and purification method and application thereof. The present invention extracts, separates and identifies a phenylpropanoid compound (euphebrarin A) from the Chinese medicine wolfsbane (Euphorbia chamaejasme) for the first time. Through in vitro anti-tumor effect studies, it is found that the compound euphebrarin A can inhibit the proliferation of HL-60, SMMC-7721 and SW480 cell lines, especially the activity of inhibiting SMMC-7721 liver cancer cells is high; at the same time, the compound euphebrarin A can inhibit the cell cycle arrest of SMMC-7721 liver cancer cells in the G2 / M phase, and is concentration-dependent. In summary, the novel phenylpropanoid compound provided by the present invention has high anti-tumor activity and has broad application prospects in the field of anti-tumor drugs, especially in the field of anti-liver cancer drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor compounds, and in particular to a phenylpropanoid compound and an extraction and purification method and application thereof. Background Art

[0002] Tumors are divided into two categories: benign tumors and malignant tumors. Malignant tumors, also known as cancer, have a high mortality rate due to their rapid growth, strong proliferation ability, and easy metastasis. Among the new anticancer drugs discovered in recent years, natural products are an important source for the development of new anticancer drugs.

[0003] Radix Euphorbiae Ebracteolatae is a perennial herbaceous plant of the Euphorbiaceae family. As one of the origins of the Chinese medicine wolfsbane, it is mainly used clinically to treat lymph node tuberculosis, psoriasis, etc. Modern pharmacological studies have shown that Radix Euphorbiae Ebracteolatae has activities such as inhibiting leukemia, anti-gout and anti-cancer. Its special active ingredients have been shown to have significant anti-tumor activity. For example, Ebracteolatain A is an acetylphloroglucinol compound extracted from Radix Euphorbiae Ebracteolatae, which can significantly inhibit the proliferation of breast cancer cells.

[0004] However, there are still few compounds with anti-tumor effects extracted from Euphorbia lunatum. It is of great significance to find new compounds with excellent anti-tumor activity from Euphorbia lunatum. Summary of the invention

[0005] In view of this, the present invention provides a phenylpropanoid compound and an extraction and purification method and application thereof. The phenylpropanoid compound provided by the present invention is a new compound extracted from the traditional Chinese medicine Euphorbia lunatum and has excellent anti-tumor activity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A phenylpropanoid compound having a structure shown in Formula I:

[0008] Formula I.

[0009] The present invention also provides a method for extracting and purifying the phenylpropanoid compounds described in the above scheme, comprising the following steps:

[0010] Using ethanol to extract the Euphorbia lunulata medicinal material powder, concentrating the obtained extract to obtain an extract;

[0011] The extract is mixed with water to obtain an extract solution; the extract solution is extracted with ethyl acetate to obtain an ethyl acetate portion;

[0012] The ethyl acetate portion is subjected to silica gel column chromatography to obtain 10 fractions, which are sequentially recorded as A, B, C, D, E, F, G, H, I, and J according to the order of the fractions flowing out;

[0013] The F segment is separated by a reverse phase chromatography column to obtain 18 components, which are denoted as F1 to F18 in the order of their elution;

[0014] The F16 is purified by preparative high performance liquid chromatography to obtain a phenylpropanoid compound with a structure shown in Formula I.

[0015] Preferably, the volume fraction of the ethanol is 80-90%, and the extraction is reflux extraction; the number of extractions is 3-5 times, and the time of each extraction is 2-3 h.

[0016] Preferably, the concentration method is reduced pressure concentration, and the temperature of the reduced pressure concentration is 47-50°C.

[0017] Preferably, the eluents used in the silica gel column chromatography separation are dichloromethane and methanol; the elution method of the silica gel column chromatography separation is gradient elution, and the volume ratio of dichloromethane and methanol in the gradient elution changes from 1:0 to 0:1.

[0018] Preferably, the mesh size of the silica gel used in the silica gel column chromatography separation is 100-200 mesh; the chromatographic column used in the reverse phase column chromatography separation is a C18ODS medium pressure column.

[0019] Preferably, the eluent used in the reverse phase column chromatography separation is a methanol-water solution; the elution method of the reverse phase column chromatography separation is gradient elution, and the volume fraction of methanol in the methanol-water solution changes from 15% to 100% during the gradient elution.

[0020] Preferably, the mobile phase used for the preparative high performance liquid chromatography purification is acetonitrile-formic acid aqueous solution, the volume ratio of acetonitrile to formic acid aqueous solution in the acetonitrile-formic acid aqueous solution is 85:15~90:10; the volume fraction of formic acid in the formic acid aqueous solution is 0.1~0.5%; and the flow rate of the mobile phase is 8~10 mL / min.

[0021] The present invention also provides the use of the phenylpropanoid compounds described in the above scheme or the phenylpropanoid compounds obtained by the extraction and purification method described in the above scheme in the preparation of anti-tumor drugs.

[0022] Preferably, the anti-tumor drug is an anti-liver cancer drug, an anti-colon cancer drug or an anti-leukemia drug.

[0023] The present invention provides a phenylpropanoid compound (denoted as euphebrarin A) having a structure shown in formula I. The present invention extracts, separates and identifies the compound euphebrarin A from the Chinese medicine wolfsbane (Euphorbia chamaejasma) for the first time, and the compound has significant anti-tumor activity. In vitro anti-tumor effect studies have shown that the compound euphebrarin A can inhibit the proliferation of HL-60, SMMC-7721 and SW480 cell lines, wherein the activity of inhibiting the proliferation of SMMC-7721 and SW480 cells is better than that of the positive drug cisplatin, especially the IC of inhibiting SMMC-7721 liver cancer cells. 50 The value is only 1.878±0.072 μM; therefore, the compound euphebrarin A has certain potential in developing drugs for the treatment of leukemia, liver cancer and colon cancer; at the same time, the present invention further detected the effect of compound euphebrarin A on apoptosis and cell cycle of SMMC-7721 liver cancer cells, and the results showed that compound euphebrarin A can inhibit the cell cycle arrest in the G2 / M phase, and is concentration-dependent, with the most apoptosis at a concentration of 0.3 μM. Therefore, the novel phenylpropanoid compound provided by the present invention has high antitumor activity and has broad application prospects in the field of antitumor drugs, especially in anti-liver cancer drugs.

[0024] The present invention also provides a method for extracting and purifying the phenylpropanoid compounds described in the above scheme. The present invention first uses ethanol to extract the Euphorbia lunatum powder, then extracts it with ethyl acetate to obtain the ethyl acetate part, and then separates it by silica gel column chromatography, reverse phase chromatography column separation and preparative high performance liquid chromatography purification to obtain the phenylpropanoid compounds of the present invention; the extraction and purification method provided by the present invention has simple steps and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The phenylpropanoid compound prepared in Example 1 1 H-NMR spectrum;

[0026] Figure 2 The phenylpropanoid compound prepared in Example 1 13 C-NMR spectrum;

[0027] Figure 3 LC-IT-TOF spectrum of the phenylpropanoid compound prepared in Example 1;

[0028] Figure 4 The flow cytometry staging diagram of the effect of phenylpropanoid compounds at different concentrations on the cell cycle of SMMC-7721 cells in Example 3;

[0029] Figure 5The flow cytometric graphs of the effects of phenylpropanoid compounds at different concentrations on apoptosis of SMMC-7721 cells in Example 4;

[0030] Figure 6 These are the WB experimental results of PTEN and EGFR proteins in Example 5. DETAILED DESCRIPTION

[0031] The present invention provides a phenylpropanoid compound having a structure shown in Formula I:

[0032] Formula I.

[0033] The present invention also provides a method for extracting and purifying the phenylpropanoid compounds described in the above scheme, comprising the following steps:

[0034] Using ethanol to extract the Euphorbia lunulata medicinal material powder, concentrating the obtained extract to obtain an extract;

[0035] The extract is mixed with water to obtain an extract solution; the extract solution is extracted with ethyl acetate to obtain an ethyl acetate portion;

[0036] The ethyl acetate portion is subjected to silica gel column chromatography to obtain 10 fractions, which are sequentially recorded as A, B, C, D, E, F, G, H, I, and J according to the order of the fractions flowing out;

[0037] The F segment is separated by a reverse phase chromatography column to obtain 18 components, which are denoted as F1 to F18 in the order of their elution;

[0038] The F16 is purified by preparative high performance liquid chromatography to obtain a phenylpropanoid compound with a structure shown in Formula I.

[0039] The present invention uses ethanol to extract the Euphorbia lunulata medicinal material powder, and concentrates the obtained extract to obtain an extract. In the present invention, the Euphorbia lunulata medicinal material powder is preferably obtained by crushing the dried Euphorbia lunulata medicinal material; the Euphorbia lunulata medicinal material is specifically the root of Euphorbia lunulata; the volume fraction of the ethanol is preferably 80-90%, and the extraction is preferably reflux extraction; the solid-liquid ratio of the Euphorbia lunulata and ethanol is preferably 1g:10mL-1g:15mL; the number of extractions is preferably 3-5 times, and the time of each extraction is preferably 2-3h.

[0040] In the present invention, the concentration method is preferably reduced pressure concentration, and the temperature of the reduced pressure concentration is preferably 47-50° C.; the reduced pressure concentration is preferably performed using a rotary evaporator.

[0041] After obtaining the extract, the present invention mixes the extract with water to obtain an extract liquid, and extracts the extract liquid with ethyl acetate to obtain an ethyl acetate portion. In the present invention, the amount ratio of the extract to water is preferably 1.4 kg: 1.5 L; the water is preferably distilled water; the volume ratio of the extract liquid to ethyl acetate is preferably 1:1 to 1:2; the number of extractions with ethyl acetate is preferably 3 times, and the ethyl acetate phases obtained by the 3 extractions are combined to obtain the ethyl acetate portion.

[0042] After obtaining the ethyl acetate portion, the present invention performs silica gel column chromatography on the ethyl acetate portion to obtain 10 fraction segments, which are sequentially recorded as A, B, C, D, E, F, G, H, I, and J segments according to the elution order of the fractions. In the present invention, the eluents used for the silica gel column chromatography separation are preferably dichloromethane and methanol; the elution mode of the silica gel column chromatography separation is preferably gradient elution, and the volume ratio of dichloromethane and methanol in the gradient elution is preferably changed from 1:0 to 0:1, specifically 1:0, 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1; the mesh number of the silica gel used for the silica gel column chromatography separation is preferably 100-200 mesh; in a specific embodiment of the present invention, the ethyl acetate portion is preferably redissolved with ethyl acetate, and then the sample is mixed with silica gel, and then silica gel column chromatography is performed, and the fractions in the separation process are detected by thin layer chromatography (TLC), and the same fractions are combined; the developing solvents used for the thin layer chromatography are preferably dichloromethane and methanol; the volume ratio of dichloromethane and methanol is preferably 12:1.

[0043] After obtaining the fraction segments, the present invention performs reverse phase chromatography column separation on the F segment to obtain 18 components, which are recorded as F1 to F18 in the order of component outflow. In the present invention, the chromatographic column used for the reverse phase column chromatography separation is preferably a C18ODS medium pressure column; the eluent used for the reverse phase column chromatography separation is preferably a methanol aqueous solution; the elution method of the reverse phase column chromatography separation is preferably gradient elution, and the volume fraction of methanol in the methanol aqueous solution during the gradient elution is preferably changed from 15% to 100%, specifically 15%, 30%, 50%, 70%, 90%, 100%; in a specific embodiment of the present invention, the fraction obtained by reverse phase column chromatography separation is preferably segmented by thin layer chromatography (TLC), and the same components are combined to finally obtain 18 components.

[0044] After obtaining component F16, the present invention performs preparative high performance liquid chromatography purification on the component F16 to obtain a phenylpropanoid compound of the structure shown in formula I. In the present invention, the mobile phase used in the preparative high performance liquid chromatography purification is preferably an acetonitrile-formic acid aqueous solution, and the volume ratio of acetonitrile and formic acid aqueous solution in the acetonitrile-formic acid aqueous solution is preferably 85:15~90:10; the volume fraction of formic acid in the formic acid aqueous solution is preferably 0.1~0.5%; the flow rate of the mobile phase is preferably 8~10mL / min; in a specific embodiment of the present invention, the retention time of the phenylpropanoid compound of the structure shown in formula I is 23.8min.

[0045] The present invention also provides the use of the phenylpropanoid compounds described in the above scheme or the phenylpropanoid compounds obtained by the extraction and purification method described in the above scheme in the preparation of anti-tumor drugs; in the present invention, the anti-tumor drug is preferably an anti-liver cancer drug, an anti-colon cancer drug or an anti-leukemia drug; the phenylpropanoid compounds provided by the present invention have the activity of inhibiting liver cancer cells, colon cancer cells and leukemia cells, especially excellent anti-liver cancer activity, and have great development prospects in the development of anti-liver cancer drugs.

[0046] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only 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 creative work are within the scope of protection of the present invention.

[0047] Example 1

[0048] (1) Take 20 kg of dried Euphorbia lunulata medicinal materials and crush them. Use 80% ethanol to reflux and extract for 3 times, 2 h each time. Use a rotary evaporator to reduce the pressure and concentrate at 47°C to obtain an extract. Take 1.4 kg of the extract and put it in 1.5 L of distilled water. Use ethyl acetate to extract it. Repeat 3 times. Combine the ethyl acetate phases obtained from the 3 extractions to obtain the ethyl acetate part.

[0049] (2) Take 322 g of the ethyl acetate portion and dissolve it in ethyl acetate. Mix the sample with 100-200 mesh silica gel. After mixing, separate the sample by silica gel column chromatography (the eluent is dichloromethane and methanol, and the volume ratio of dichloromethane and methanol varies from 1:0 to 0:1, specifically 1:0, 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1), and identify it by thin layer chromatography (TLC) (the developing solvent is dichloromethane and methanol, and the volume ratio of dichloromethane and methanol is 12:1). Combine the same fractions to obtain 10 fraction segments, which are named A, B, C, D, E, F, G, H, I, and J.

[0050] (3) Take 32.5 g of segment F and pass it through a C18ODS medium-pressure column (the eluent is methanol-water solution, and the volume fraction of methanol in the methanol-water solution varies from 15% to 100%, specifically 15%, 30%, 50%, 70%, 90%, and 100%). Then, identify the segments by TLC and obtain a total of 18 components, F1-F18.

[0051] (4) Component F16 was purified by preparative HPLC with a mobile phase of acetonitrile-0.1% formic acid water in a volume ratio of 85:15 at a flow rate of 8 mL / min to obtain 6.9 mg of euphebrarin A with a retention time of 23.8 min.

[0052] The structural identification data of the phenylpropanoid compounds obtained in this example are as follows:

[0053] Structural formula:

[0054]

[0055] Molecular formula: C 28 H 44 O8.

[0056] LC-IT-TOF m / z: [MH] - 507.2964.

[0057] State: Yellow amorphous powder.

[0058] Specific rotation: [α] 20 D = -73.00 (c 0.8, CH3OH).

[0059] Figure 1 The phenylpropanoid compounds obtained in this example 1 H-NMR spectrum, Figure 2 The phenylpropanoid compounds obtained in this example 13 C-NMR spectrum, 1 H-NMR and 13 C-NMR data are shown in Table 1.

[0060] Table 1 1 H-NMR, 13 C-NMR data

[0061]

[0062] Figure 3 LC-IT-TOF spectrum of the phenylpropanoid compounds obtained in this example.

[0063] According to the above structural identification data, the phenylpropanoid compound of the present invention has the structure shown in Formula I.

[0064] Example 2 Antitumor Activity Experiment

[0065] The cells used in this example are HL-60, SMMC-7721 and SW480 cells, and the compound to be tested is the phenylpropanoid compound euphebrarin A prepared in Example 1. The experimental steps are as follows:

[0066] Inoculation of cells: Prepare a single cell suspension with culture medium containing 10% fetal bovine serum, inoculate 5000-15000 cells per well into a 96-well plate, with a volume of 100 μL per well. Cells should be inoculated and cultured 24 h in advance.

[0067] Add the solution of the compound to be tested: the compound was dissolved in DMSO, and the compound was screened at concentrations of 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0.064 μM. The final volume of each well was 200 μL, and 3 replicate wells were set for each treatment.

[0068] Color development: After culturing at 37°C for 48 h, discard the culture medium in the wells, add 20 μL of MTS solution and 100 μL of culture medium to each well; discard 100 μL of culture supernatant of suspended cells, add 20 μL of MTS solution to each well; set up 3 blank replicate wells (a mixture of 20 μL MTS solution and 100 μL culture medium), continue incubation for 4 h, and measure the absorbance value after the reaction is fully carried out.

[0069] Colorimetry: Select 492nm wavelength, use a multifunctional microplate reader to read the light absorption value of each well, record the results, and after data processing, draw a cell growth curve with concentration as the horizontal axis and cell survival rate as the vertical axis. Use the two-point method to calculate the IC value of the compound. 50 value.

[0070] Positive control drug: The experimental steps are the same as above. Cisplatin (DDP) is used as the positive control drug in each experiment. The cell growth curve is drawn with concentration as the horizontal axis and cell survival rate as the vertical axis. The two-point method (Reed and Muench method) is used to calculate the IC of the positive drug. 50 value.

[0071] The experimental results show that the IC values ​​of euphebrarin A, a phenylpropanoid compound provided by the present invention, on HL-60, SMMC-7721 and SW480 cells are 50 The values ​​were 13.18±0.39, 1.878±0.072, and 17.48±0.35μM, respectively; the IC values ​​of the positive control drug cisplatin against the above three tumor cell lines were 50The values ​​were 2.968±0.140, 9.599±0.667, and 35.63±1.16 μM, respectively. According to the above results, it can be seen that the phenylpropanoid compounds of the present invention have inhibitory activity against HL-60, SMMC-7721, and SW480 cells, and the inhibitory activity against SMMC-7721 and SW480 cells is better than that of the positive drug cisplatin, especially the IC of SMMC-7721 liver cancer cells. 50 The value was only 1.878±0.072 μM, indicating that it has significant activity in inhibiting the proliferation of liver cancer cells.

[0072] Example 3 Effects of different concentrations of phenylpropanoid compounds on the cell cycle of SMMC-7721 cells

[0073] This example tests the effect of phenylpropanoid compounds at different concentrations on the cell cycle of SMMC-7721 cells. The compound to be tested is euphebrarin A prepared in Example 1. The experimental steps are as follows:

[0074] Cell inoculation: Prepare a single cell suspension in a culture medium containing 10% fetal bovine serum, with 10 cells per well. 6 The cells were inoculated into 6 cm small dishes with white transparent bottom, 4 mL / dish, and 3 replicates were made. The cell plates were placed in a 37°C, 5% CO2 incubator for incubation.

[0075] Starving cells: After cells adhered to the wall, discard the culture medium in the wells and add 4 mL of serum-free culture medium to each well for 24 h.

[0076] Add the solution of the compound to be tested: After starvation is completed, discard the liquid in the wells, add phenylpropanoid compounds at concentrations of 0, 0.1, 0.3, and 0.6 μM, and a positive control drug 0.1 μM doxorubicin (DOX), with 3 replicates in each group; place in a 37°C, 5% CO2 incubator and culture for 24 h.

[0077] Cell fixation: Add trypsin to digest the cells and collect them, centrifuge for 5 min, discard the supernatant, and use PBS to prepare a single cell suspension. 6 Resuspend the cells, centrifuge for 5 min, and discard the supernatant. Add 1 mL of ice-cold 70% ethanol, gently pipette to mix, fix at 4℃ for 4 h, centrifuge for 5 min, and discard the supernatant; add about 1 mL of ice-cold PBS, resuspend the cells, centrifuge again to precipitate the cells, and discard the supernatant.

[0078] Staining: Add 0.5 mL of propidium iodide (PI) staining solution to each tube of cell sample, slowly and fully resuspend the cell pellet, warm it at 37℃ in the dark for 30 min, and then store it in an ice bath in the dark.

[0079] Flow cytometric detection and analysis: Flow cytometry was used to detect red fluorescence at an excitation wavelength of 488 nm and light scattering at the same time. Flow cytometric analysis software was used to analyze cell DNA content and light scattering.

[0080] The test results are as follows Figure 4 As shown, Figure 4 Middle: G0 / G1 indicates cells in the G0 / G1 phase, S indicates cells in the S phase, and G2 / M indicates cells in the G2 / M phase. Figure 4 It can be found that the novel phenylpropanoid compound euphebrarin A of the present invention can induce cell cycle arrest in the G2 / M phase of SMMC-7721 cells in a concentration-dependent manner.

[0081] Example 4 Effects of phenylpropanoid compounds at different concentrations on apoptosis of SMMC-7721 cells

[0082] This example tests the effect of phenylpropanoid compounds at different concentrations on apoptosis of SMMC-7721 cells, and the compound to be tested is euphebrarin A prepared in Example 1. The experimental steps are as follows:

[0083] The experimental steps of inoculating cells, starving cells and adding the test compound solution are the same as in Example 3.

[0084] Flow cytometry apoptosis detection: add trypsin to digest the cells and collect them, centrifuge for 5 min, discard the supernatant, and use PBS to prepare a single cell suspension. 6 Resuspend the cells, centrifuge for 5 min, discard the supernatant; add 195 μL Annexin V-FITC binding solution and gently resuspend the cells; add 5 μL Annexin V-FITC and mix gently; add 10 μL PI staining solution and mix gently. Incubate at room temperature in the dark for 20 min, then place in an ice bath, and observe Annexin V-FITC as green fluorescence and PI as red fluorescence using a flow cytometer.

[0085] The test results are as follows Figure 5 As shown. Figure 5 The results show that after the SMMC-7721 cells were treated with the novel phenylpropanoid compound euphebrarin A of the present invention, the apoptotic cells and necrotic cells increased compared with the control group; and the concentration of 0.3 μM had the greatest effect on the cells, with the most apoptosis, and then the effect weakened.

[0086] Example 5 Effects of phenylpropanoid compounds on proteins in SMMC-7721 cells

[0087] This example tests the effects of phenylpropanoid compounds on proteins (PTEN, EGFR) in SMMC-7721 cells. The compound to be tested is euphebrarin A prepared in Example 1.

[0088] The experimental equipment and main experimental reagents used in this example are shown in Table 2 and Table 3.

[0089] Table 2 Experimental equipment

[0090]

[0091] Table 3 Main experimental reagents

[0092]

[0093] The experimental steps are as follows:

[0094] Cell culture: Prepare a single cell suspension in culture medium containing 10% fetal bovine serum, and add 10 6 The cells were inoculated into a 6 cm small dish with a white transparent bottom cell plate and incubated at 37°C, 5% CO2 incubator. After the cells were stable, the phenylpropanoid compounds of the present invention and 1 positive control drug 0.1 μM DOX were added at concentrations of 0 and 0.3 μM, with 3 replicate dishes in each group; the plates were placed in a 37°C, 5% CO2 incubator and cultured for 24 h.

[0095] Total protein extraction: After 24 h of drug treatment, wash once with PBS, add 500 μL of lysis buffer (RIPA:PMSF:phosphatase inhibitor = 100:1:1) to each 6 cm dish, place on ice for 30 min, centrifuge at 4°C, 12,000 rpm for 15 min, and take the supernatant into a new centrifuge tube.

[0096] Protein concentration determination: Prepare BCA working solution (A:B=50:1) according to the sample volume and place on ice. Take 10 μL BSA standard and dilute it to 100 μL with PBS to a final concentration of 0.5 mg / mL. Add 0, 2, 4, 6, 8, 12, 16, and 20 μL of the standard to the protein standard wells of the 96-well plate and add PBS to make up to 20 μL. Dilute the sample appropriately and add 20 μL to the sample well. Add 200 μL BCA working solution to each well and place at 37°C for 30 min. Measure A562 nm with an enzyme reader and calculate the protein concentration based on the standard curve.

[0097] SDS-PAGE gel electrophoresis: constant voltage 80 V, 90 min, until bromophenol blue reaches the bottom.

[0098] Transfer (wet transfer): Assemble the transfer sandwich in the buffer, place a sponge on top and then three layers of filter paper, cover the glue on the filter paper, and cover the membrane on the glue. Cover the membrane with three layers of filter paper, and finally cover it with a layer of sponge. Place the transfer sandwich in the transfer tank, black side facing black side, and add transfer buffer. Place the transfer tank in ice, insert the electrode, and constant current 300 mA for 1 hour.

[0099] Blocking: The PVDF membrane was washed in TBST for 5 min and incubated with an appropriate amount of protein-free rapid blocking solution for 1 h.

[0100] Primary antibody incubation: Dilute 5 mL of antibody diluent (TBST as diluent) according to the primary antibody product instructions and incubate on a roller at 4°C overnight. Wash the membrane with TBST three times after incubation, 5 min each time.

[0101] Secondary antibody incubation: Select HRP secondary antibody according to the selected primary antibody. Dilute HRP secondary antibody (TBST as diluent). Incubate on roller at room temperature for 1 hour. Wash the membrane with TBST 3 times after incubation, 5 minutes each time.

[0102] Color development: Use ECL color development and chemiluminescence instrument to take pictures.

[0103] Figure 6 These are the WB experimental results of PTEN and EGFR proteins.

[0104] according to Figure 6 It can be seen that after adding different concentrations of compounds to SMMC-7721 cells, the expression of PTEN and EGFR proteins is related to the concentration of euphebrarin A. The higher the concentration of euphebrarin A, the lower the expression of PTEN and EGFR proteins.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A phenylpropanoid compound, characterized in that It has the structure shown in formula I: Formula I.

2. The method for extracting and purifying phenylpropanoid compounds according to claim 1, characterized in that: The following steps are involved: Using ethanol to extract the Euphorbia lunulata medicinal material powder, concentrating the obtained extract to obtain an extract; The extract is mixed with water to obtain an extract solution; the extract solution is extracted with ethyl acetate to obtain an ethyl acetate portion; The ethyl acetate portion is subjected to silica gel column chromatography to obtain 10 fractions, which are sequentially recorded as A, B, C, D, E, F, G, H, I, and J according to the order of the fractions flowing out; The F segment is separated by a reverse phase chromatography column to obtain 18 components, which are denoted as F1 to F18 in the order of their elution; The F16 is purified by preparative high performance liquid chromatography to obtain a phenylpropanoid compound with a structure shown in Formula I.

3. The extraction and purification method according to claim 2, characterized in that: The volume fraction of the ethanol is 80-90%, and the extraction is reflux extraction; the number of extractions is 3-5 times, and the time of each extraction is 2-3 hours.

4. The extraction and purification method according to claim 2, characterized in that: The concentration method is reduced pressure concentration, and the temperature of the reduced pressure concentration is 47-50°C.

5. The extraction and purification method according to claim 2, characterized in that: The eluents used in the silica gel column chromatography separation are dichloromethane and methanol; the elution method of the silica gel column chromatography separation is gradient elution, and the volume ratio of dichloromethane and methanol in the gradient elution changes from 1:0 to 0:

1.

6. The extraction and purification method according to claim 2 or 5, characterized in that: The mesh number of the silica gel used in the silica gel column chromatography separation is 100-200 mesh; the chromatographic column used in the reverse phase column chromatography separation is a C18ODS medium pressure column.

7. The extraction and purification method according to claim 2, characterized in that: The eluent used in the reverse phase column chromatography separation is methanol aqueous solution; the elution method of the reverse phase column chromatography separation is gradient elution, and the volume fraction of methanol in the methanol aqueous solution changes from 15% to 100% during the gradient elution.

8. The extraction and purification method according to claim 2, characterized in that: The mobile phase used in the preparative high performance liquid chromatography purification is acetonitrile-formic acid aqueous solution, the volume ratio of acetonitrile to formic acid aqueous solution in the acetonitrile-formic acid aqueous solution is 85:15-90:10; the volume fraction of formic acid in the formic acid aqueous solution is 0.1-0.5%; and the flow rate of the mobile phase is 8-10 mL / min.

9. Use of the phenylpropanoid compound according to claim 1 or the phenylpropanoid compound obtained by the extraction and purification method according to any one of claims 2 to 8 in the preparation of anti-tumor drugs; the anti-tumor drug is an anti-liver cancer drug, an anti-colon cancer drug or an anti-leukemia drug.

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