Diterpenoid compound extracted from euphorbia fischeriana as well as preparation method and application of diterpenoid compound

Abstract

The invention provides a diterpenoid compound extracted from euphorbia fischeriana as well as a preparation method and application of the diterpenoid compound, and belongs to the technical field of plant medicines. The diterpenoid compound (A3710) in the medicinal material euphorbia fischeriana is found for the first time, the diterpenoid compound (A3710) has a remarkable anti-lung cancer effect, and the cytotoxicity IC50 value of the diterpenoid compound (A3710) to lung cancer cells reaches the nanomole level; in a mouse subcutaneous transplantation tumor model of cell line-derived allograft tumor and patient-derived tumor xenotransplantation, the diterpenoid compound A3710 can effectively inhibit the growth of lung cancer cells, and shows a clear anti-lung cancer drug effect. The invention further provides a method for preparing the diterpenoid compound A3710 from the medicinal material euphorbia fischeriana, the extraction rate of the diterpenoid compound A3710 prepared according to the method can reach 0.05% or above, and operation is convenient and fast.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant medicine, and particularly relates to a diterpenoid compound extracted from Euphorbia fischeriana Steud., a preparation method and application thereof. BACKGROUND

[0002] Lung cancer is derived from uncontrolled growth of lung tissue cells. Globally, lung cancer ranks second in new cancer cases and is the leading cause of cancer death. Epidemiological surveys have shown that smoking and passive smoking are the primary factors that induce lung cancer. In addition, air pollution, occupational exposure, and genetic factors also play an important role that cannot be ignored. At the same time, age is an independent factor affecting the survival rate of lung cancer.

[0003] The dry roots of Euphorbia fischeriana Steud. can be used as medicine, i.e. Euphorbia fischeriana Steud. as a traditional Chinese medicine. It has a bitter and pungent taste, and a flat nature, and has the effects of purging water, resolving nodules, breaking accumulation, and killing insects. Modern pharmacological studies have also shown that the extract of Euphorbia fischeriana Steud. has anti-inflammatory, analgesic, anti-tumor, and treatment of pulmonary tuberculosis effects.

[0004] At present, a number of studies have been carried out on diterpenoid compounds in Euphorbia fischeriana Steud. Some patent documents disclose specific types of diterpenoid compounds isolated from Euphorbia fischeriana Steud. For example, Chinese Patent No. CN 106279081 A discloses the abietane-type diterpenoid compounds Fischeriolide G and Fischeriolide H with strong inhibitory activity against Mycobacterium tuberculosis, providing new candidate substances for the research and development of anti-tuberculosis drugs. For another example, Chinese Patent No. CN 117582426 A reports a preparation method and application of a diterpenoid compound, further enriching the preparation technology system of diterpenoid compounds in Euphorbia fischeriana Steud. In addition, Kaicheng Du et al. isolated 8 unreported glycosides, including 2 diterpene glycosides, from the polar extract of the roots of Euphorbia fischeriana Steud. through phytochemical research. The two diterpene glycosides have cytotoxicity to Hep-G2, Hep-3B, A549, NCI-H460, and AGS cells, with IC 50 values between 5.4 and 16.2 μM. Chen Hu et al. identified 5 diterpenoid compounds and 2 triterpenoid compounds, including 7-ketodehydroabietic acid, from the 80% acetone extract of the roots of Euphorbia fischeriana Steud. using various column chromatography separation methods. Liu Wenji et al. showed that the diterpene lactone compounds 16-hydroxypseudojolkinolide B and jolkinolide B isolated from Euphorbia fischeriana Steud. have obvious dose-dependent inhibitory effects on human erythroleukemia K562 cells and human nasopharyngeal carcinoma CNE 2 cells.

[0005] Although the prior art has proved that some diterpenoids in Euphorbia fischeriana have cytotoxic or antitumor activity, these compounds are mostly targeted at hepatoma cells, leukemia cells, etc., and diterpenoids with significant inhibitory activity specifically targeting lung cancer are still rare. From the chemical structure, the Euphorbia fischeriana components with cytotoxic or antitumor activity in the prior art are mostly diterpenoids of abietane type, and there is no report on the activity of diterpenoids of crotonate type in Euphorbia fischeriana. Moreover, the inhibitory efficiency (such as IC 50 value) of the diterpenoids with anti-lung cancer activity in the prior art is micromolar, which still has a large room for improvement and is difficult to meet the demand for high-efficiency drugs in clinical treatment. In addition, the prior art mostly uses mouse animal experiments based on immortalized cell lines to evaluate drug efficacy, and lacks tumor tissues from clinical patients for drug efficacy evaluation. Furthermore, the prior art lacks evaluation of the toxic effects of diterpenoids with antitumor effect in Euphorbia fischeriana on normal cells and organs. Therefore, further in-depth exploration of new diterpenoids with excellent anti-lung cancer activity in Euphorbia fischeriana, establishment of an efficient preparation method, and further testing in samples from clinical patients have important theoretical and application values for promoting the research and development of anti-lung cancer drugs. SUMMARY

[0006] To solve the above technical problems, the present application provides a diterpenoid extracted from Euphorbia fischeriana, and a preparation method and application thereof.

[0007] To achieve the above object, the present application provides the following technical solutions.

[0008] The present application provides a diterpenoid (diterpenoid A3710) extracted from Euphorbia fischeriana, and the structural formula of the diterpenoid is as follows:

[0009] .

[0010] The present application also provides a preparation method of the above-mentioned diterpenoid extracted from Euphorbia fischeriana, comprising the following steps:

[0011] (1) crushing the dried branches of Euphorbia fischeriana, and extracting with ethanol under reflux;

[0012] (2) concentrating the extract obtained in step (1) under reduced pressure to obtain total extract;

[0013] (3) separating the total extract by reduced pressure silica gel column, using a mixture of dichloromethane and methanol, and gradient eluting according to the concentration of CH2Cl2 in the mixture from high to low, to obtain four crude fractions in turn;

[0014] (4) the first crude fraction obtained by gradient elution in step (3) is separated by a polyamide resin, gradient elution is performed using a mixture of ethanol and water, the concentration of ethanol in the mixture is gradually increased from low to high, and two fractions are obtained in sequence;

[0015] (5) the first fraction obtained by gradient elution in step (4) is separated by reverse-phase ODS column chromatography, gradient elution is performed, and the diterpenoid compound is prepared.

[0016] Further, in step (1), the concentration of ethanol is 80-95 wt.%, and the extraction is performed 2-3 times by reflux extraction.

[0017] Further, in step (2), the temperature for vacuum concentration is 55-65°C.

[0018] Further, in step (3), the gradient elution conditions for the vacuum silica gel column chromatography are elution using CH2Cl2 (dichloromethane) and MeOH (methanol) in a volume ratio of 100:1-0:1; specifically, the volume ratio of CH2Cl2 (dichloromethane) and MeOH (methanol) is 100:1, 50:1, 10:1, or 0:1.

[0019] Further, in step (4), the gradient elution conditions for the polyamide resin are elution using EtOH (ethanol) and H2O (water) in a volume ratio of 30:70-90:10; specifically, the volume ratio of EtOH and H2O is 30:70, 60:40, or 90:10.

[0020] Further, in step (5), the gradient elution conditions for the reverse-phase ODS column chromatography are elution using MeOH and H2O in a volume ratio of 20:80-90:10; specifically, the volume ratio of MeOH and H2O is 20:80, 40:60, 60:40, 80:20, or 90:10.

[0021] Further, the preparation method of the diterpenoid compound A3710 specifically comprises the following steps:

[0022] (1) dry branches of Euphorbia fischeriana are crushed, and extracted 2-3 times by reflux extraction using ethanol with a concentration of 80-95 wt.%;

[0023] (2) the extract is concentrated under reduced pressure at 55-65°C to obtain total extract;

[0024] (3) the total extract is separated by a vacuum silica gel column, gradient elution is performed using CH2Cl2 and MeOH in a volume ratio of 100:1-0:1, and four crude fractions Fr.1, Fr.2, Fr.3, and Fr.4 are obtained;

[0025] (4) The crude fraction Fr.1 was separated by polyamide resin and gradient elution was performed using EtOH and H2O with a volume ratio of 30:70-90:10 to obtain products Fr.1-1 and Fr.1-2.

[0026] (5) The product Fr.1-2 was separated by reversed-phase ODS column chromatography and eluted with a gradient of MeOH and H2O in a volume ratio of 20:80-90:10 to prepare the diterpenoid compound A3710.

[0027] The present invention also proposes a pharmaceutical composition comprising the above-mentioned diterpenoid compound and a pharmaceutically acceptable carrier or excipient.

[0028] The present invention also proposes the use of the above-mentioned diterpenoid compound or the above-mentioned pharmaceutical composition in the preparation of a drug for treating lung cancer.

[0029] Compared with the prior art, the present invention has the following advantages and technical effects:

[0030] This invention marks the first discovery that the diterpenoid compound A3710 from the medicinal herb *Euphorbia fischeriana* possesses significant anti-lung cancer activity. In a mouse subcutaneous xenograft model, diterpenoid compound A3710 effectively inhibited lung cancer cell growth, demonstrating a clear anti-lung cancer efficacy. This invention also provides a method for preparing diterpenoid compound A3710 from *Euphorbia fischeriana*. The dried roots of *Euphorbia fischeriana* are extracted with ethanol under reflux, concentrated under reduced pressure to obtain a total extract, and rapidly separated into four crude fractions by reduced-pressure silica gel column chromatography. Then, the fractions are thoroughly and systematically separated using silica gel, polyamide resin, reversed-phase ODS, and cation exchange resin to obtain a novel diterpenoid compound A3710. The extraction rate of diterpenoid compound A3710 prepared by this method can reach over 0.05%, featuring convenient operation and high extraction rate. Attached Figure Description

[0031] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0032] Figure 1 Here is a high-resolution mass spectrum of the diterpenoid compound prepared in Example 1;

[0033] Figure 2 The diterpenoid compound A3710 prepared in Example 1 1 H-NMR spectrum;

[0034] Figure 3 The diterpenoid compound A3710 prepared in Example 1 13 C-NMR spectrum;

[0035] Figure 4 HSQC spectrum of diterpenoid compound A3710 prepared in Example 1;

[0036] Figure 5 HMBC spectrum of diterpenoid compound A3710 prepared in Example 1;

[0037] Figure 6 NOESY spectrum of diterpenoid compound A3710 prepared in Example 1;

[0038] Figure 7 Results of the effect of different concentrations of A3710 on the survival rate of lung cancer cells A549;

[0039] Figure 8 Results of the effect of A3710 on the survival rate of normal human lung epithelial cells BEAS-2B;

[0040] Figure 9 Results of tumor volume test of control group and administration (A3710, same below) group mice;

[0041] Figure 10 Actual tumor size of control group and administration group mice;

[0042] Figure 11 Results of tumor weight test of control group and administration group mice;

[0043] Figure 12 Body weight change of control group and administration group mice;

[0044] Figure 13 Liver index change of control group and administration group mice;

[0045] Figure 14 Spleen index change of control group and administration group mice;

[0046] Figure 15 Lung index change of control group and administration group mice;

[0047] Figure 16 Kidney index change of control group and administration group mice;

[0048] Figure 17 Results of tumor volume test of control group and administration group mice in PDX model;

[0049] Figure 18 Actual tumor size of control group and administration group mice in PDX model;

[0050] Figure 19The tumor weight test results of the control group and the administration group mice in the PDX model;

[0051] Figure 20 The body weight change of the control group and the administration group mice in the PDX model. DETAILED DESCRIPTION

[0052] The detailed description set forth below of various illustrative embodiments explains the principles of the application and the best mode presently contemplated by the inventors for carrying out the application. It will be understood that the detailed description is merely illustrative of certain aspects of the application, and the scope of the application should not be limited to the specifically enumerated embodiments set forth in the description below.

[0053] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, where particular ranges of values are given, it is intended to include every intervening value between the upper and lower limit of that range. If the upper and lower limits of a range are identical, the range is intended to include only that single value. Any smaller range that falls within the broader range is also intended to be included. These smaller ranges are not by way of limitation.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present application, the preferred methods and materials are described herein. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to the extent allowed by law. In the case of conflict between the description herein and the incorporated material, the description herein shall control.

[0055] In the description of the application specific embodiments, numerous specific details are set forth in order to provide a thorough understanding of the application. However, those of ordinary skill in the art will recognize that the application can be practiced without the specific details set forth herein. In other instances, well-known methods, procedures and components have not been described in detail so as not to unnecessarily obscure aspects of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from a review of the description of the application and practice of the application.

[0056] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0057] The embodiment of the present application provides a diterpenoid compound (denoted as diterpenoid compound A3710), which is extracted from Euphorbia fischeriana and has a structural formula as follows:

[0058] .

[0059] The embodiment of the present application further provides a preparation method of the diterpenoid compound, comprising the following steps:

[0060] (1) Dry branches of Euphorbia fischeriana Steud. are cut into pieces and extracted by refluxing with ethanol;

[0061] (2) The extract obtained in step (1) is concentrated under reduced pressure to obtain total extract;

[0062] (3) The total extract is separated by silica gel column under reduced pressure, and gradient elution is performed with a mixture of dichloromethane and methanol, and the concentration of CH2Cl2 in the mixture is gradually reduced from high to low, and four crude fractions are obtained in sequence;

[0063] (4) The first crude fraction obtained in step (3) is separated by polyamide resin, and gradient elution is performed with a mixture of ethanol and water, and the concentration of ethanol in the mixture is gradually increased from low to high, and two fractions are obtained in sequence;

[0064] (5) The first fraction obtained in step (4) is separated by reverse-phase ODS column chromatography, and gradient elution is performed to prepare a diterpenoid compound.

[0065] In step (1) of the preferred embodiment of the present application, the concentration of ethanol is 80-95 wt.%, and the extraction is performed 2-3 times by refluxing.

[0066] In step (2) of the preferred embodiment of the present application, the temperature for concentration under reduced pressure is 55-65℃.

[0067] In step (3) of the preferred embodiment of the present application, the gradient elution conditions for silica gel column chromatography under reduced pressure are elution with CH2Cl2 (dichloromethane) and MeOH (methanol) in a volume ratio of 100:1-0:1; specifically, the volume ratio of CH2Cl2 and MeOH is 100:1, 50:1, 10:1, and 0:1.

[0068] In step (4) of the preferred embodiment of the present application, the gradient elution conditions for polyamide resin are elution with EtOH (ethanol) and H2O (water) in a volume ratio of 30:70-90:10; specifically, the volume ratio of EtOH and H2O is 30:70, 60:40, and 90:10.

[0069] In step (5) of the preferred embodiment of the present application, the gradient elution conditions for reverse-phase ODS column chromatography are elution with MeOH and H2O in a volume ratio of 20:80-90:10; specifically, the volume ratio of MeOH and H2O is 20:80, 40:60, 60:40, 80:20, and 90:10.

[0070] The present application also provides a pharmaceutical composition comprising the diterpenoid compound described above and a pharmaceutically acceptable carrier or excipient.

[0071] The application further provides application of the diterpenoid compound or the pharmaceutical composition in preparation of a drug for treating lung cancer.

[0072] The technical solutions of the application are further described below through examples.

[0073] Example 1

[0074] A preparation method of a diterpenoid compound specifically comprises the following steps:

[0075] (1) 5 kg of dried branches of Euphorbia fischeriana are crushed, and are extracted three times by reflux extraction with ethanol with a concentration of 95 wt. %;

[0076] (2) The extraction liquid is concentrated under reduced pressure at 60 ℃ to obtain 100 g of total extract;

[0077] (3) The total extract is separated by a reduced-pressure silica gel column, and is subjected to four-stage gradient elution by using CH2Cl2 and MeOH with a volume ratio of 100:1, 50:1, 10:1 and 0:1, and the elution volume of each solvent ratio is 8 L, to obtain four crude fractions Fr.1, Fr.2, Fr.3 and Fr.4;

[0078] (4) 40 g of the crude fraction Fr.1 is separated by a polyamide resin, and is subjected to gradient elution by using EtOH and H2O with a volume ratio of 30:70, 60:40 and 90:10, and the elution volume of each elution is 4 L, to obtain fractions Fr.1-1 and Fr.1-2;

[0079] (5) 20 g of the fraction Fr.1-1 is separated by a reversed-phase ODS column chromatography, and is subjected to gradient elution by using MeOH and H2O with a volume ratio of 20:80, 40:60, 60:40, 80:20 and 90:10, to prepare 2.5 g of the diterpenoid compound A3710 (the extraction rate of the diterpenoid compound A3710 in Euphorbia fischeriana can be more than 0.05 %).

[0080] The high-resolution mass spectrum (HRESIMS) figure, hydrogen spectrum (H-NMR), carbon spectrum (C-NMR), HSQC spectrum, HMBC spectrum and NOESY spectrum of the diterpenoid compound A3710 prepared in Example 1 are shown in Figures 1 to 6 respectively. 1 H-NMR 13 C-NMR Figures 1-6 .

[0081] According to HRESIMS m / z 637.4442 [M + Na] + (C 38 H 62 O6Na + , the calculated value is 637.4439), the molecular formula is C 38 H62 O6. 1 H-NMR (600 MHz, CDCl3): δ H 7.55 (1H, t, J = 1.9 Hz), δ H 5.68 (1H, dd, J = 5.7, 2.1 Hz) is the hydrogen signal on the two olefin carbons, δ H 4. 04 (1H, d, J = 2.8 Hz), δ H 3.98 (1H, d, J = 2.8 Hz) is the hydrogen signal on the oxygen-bound carbon. δ H 1.61 (3H, m), δ H 1.19 (3H, s), δ H 1.07 (3H, s), δ H 0.88 (3H, m) represents the signal of 4 methyl groups. 13 C-NMR (150 MHz, CDCl3) showed 38 carbon signals, including 2 carbonyl groups (δ). C 209.2, 176.0), 4 olefinic carbons (δ C 161.3, 139.8, 132.8, 130.3), 4 oxygen-bound carbons (δ C 76.0, 73.7, 68.3, 63.3). δ H 1.26 (m) and δ C NMR data at 22.7-31.9 nm indicate that compound A3710 possesses long-chain fatty acid groups. The NMR data for compound A3710 are listed in Table 1. From the above, it can be seen that the chemical structure of compound A3710 is (1aR,4aR,7aS,7bR,8R,9aS)-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[1,2-e]azulen-9a-yl stearate, which is a newly discovered diterpenoid compound.

[0082] Table 1. Compound A3710 1 H (600 MHz, CDCl3) and 13 C (150 MHz, CDCl3) data (δ inppm, J in Hz)

[0083]

[0084] Performance test

[0085] Test of the inhibitory effect of diterpenoid compound A3710 prepared in Example 1 on the proliferation of lung cancer cells

[0086] 1. Experimental materials

[0087] 1.1 Test product: diterpenoid compound A3710

[0088] 1.2 Instruments: enzyme label instrument (INFINITE E PLEX, Switzerland, Tecan), vernier caliper (Shenzhen Qiji Duoyou Precision Instrument Co., Ltd.), ML104 electronic analytical balance (Mettler Toledo, NY, USA), animal weight balance (Shanghai Yuyan Scientific Instrument Co., Ltd.).

[0089] 1.3 Reagents: CCK-8 kit (Bi Yun Tian Biotechnology Co., Ltd., Shanghai), F12-K cell culture medium (GIBCO), DMEM cell culture medium (GIBCO), PBS-phosphate buffer (Dalian Milab Biotechnology Co., Ltd.), physiological saline (Shandong Chenxin Pharmaceutical Co., Ltd.), DMSO-dimethyl sulfoxide (Shanghai Macklin Biochemical Technology Co., Ltd.).

[0090] 1.4 Cells: human lung cancer A549 cells were purchased from Wuhan Punuo Life Science and Technology Co., Ltd.; human normal lung epithelial BEAS-2B cells were purchased from Shanghai Zhijiao Xin Zhou Biological Technology Co., Ltd.

[0091] 1.5 Patient-derived xenograft (PDX)

[0092] The PDX model PJ-KS-KY-2024-382 of the application was provided by the First Affiliated Hospital of Dalian Medical University, and the P1 generation was used for the experiment.

[0093] 1.6 Animals: The cells used to construct the animal tumor transplantation model were human lung cancer A549 cells. The experimental animals were SPF NYG mice, 12 male, 6 weeks old, weighing 20-30 g, purchased from Liaoning Changsheng Biological Technology Co., Ltd.

[0094] 2. Evaluation experiment of the activity of compound A3710 on A549 cells

[0095] 2.1 Preparation method of A3710 test solution

[0096] Take 1 mg of diterpenoid compound A3710 powder, prepare a 10 μM stock solution with DMSO, and then dilute the dosing test solution with blank F12-K medium to concentrations of 150, 75, 37.5, 18.75, 9.375, 4.6875 nM, and store at 4°C for later use.

[0097] 2.2 Human lung cancer A549 cell plating

[0098] A549 cells in the logarithmic growth phase were trypsinized, centrifuged, and counted. The cells were plated in a 96-well plate at a density of 6000 cells per well, with 100 μL of cell suspension added to each well. The edge wells of the plate were filled with PBS to avoid edge effects. The cells were incubated in a 37°C, 5% CO2 incubator for 24 h, and then drug treatment was performed after the cells adhered.

[0099] 2.3 Grouping and drug administration

[0100] The experiment was divided into three groups: the experimental group was added with 50 μL of A3710 solution at different concentrations, the control group was added with an equal amount of blank F12-K medium, and the blank group only contained blank F12-K medium (without cells, used for background correction). After drug administration, the final concentrations of the drugs in each group were 50, 25, 12.5, 6.25, 3.125, and 1.5625 nM, respectively. After 48 h of drug action, subsequent detection was performed.

[0101] 2.4 CCK-8 method for detecting the inhibition of A3710 on A549

[0102] 10% CCK-8 working solution was prepared using blank F12-K medium. The original liquid in the 96-well plate was discarded, and 100 μL of CCK-8 working solution was added to each well. After incubation at 37°C in the dark for 1 h, the OD value of each well was measured using a microplate reader at a wavelength of 450 nm, and the cell survival rate was calculated. Cell survival rate (%) = (experimental group OD value - blank group OD value) / (control group OD value - blank group OD value) x 100%.

[0103] 2.5 Data processing and IC 50 value calculation

[0104] The cell survival rate data corresponding to different concentrations of A3710 were imported into GraphPad Prism 9.5 software for curve fitting, and the IC 50 value was 3.7 ± 0.9 nM. The results of the effect of different concentrations of A3710 on cell survival rate are shown in Figure 7 .

[0105] 3. Evaluation of the activity of compound A3710 on normal human lung epithelial cells BEAS-2B

[0106] 3.1 A3710 solution preparation method

[0107] 1 mg of diterpenoid compound A3710 powder was prepared into a 10 μM stock solution with DMSO, and then the drug solution was diluted with blank DMEM medium to a concentration of 300 nM, and stored at 4°C for use.

[0108] 3.2 BEAS-2B cell plating

[0109] BEAS-2B cells in logarithmic growth phase were trypsinized, centrifuged and counted, and then inoculated into 96-well plates at a density of 6000 cells per well with the addition of 100 μL of cell suspension per well. The edge wells of the plate were filled with PBS to avoid edge effects. The cells were incubated in a 37°C, 5% CO2 incubator for 24 h, and then dosed after the cells adhered.

[0110] 3.3 Grouping and dosing

[0111] The experiment was divided into three groups: the experimental group was added with 50 μL of A3710 test solution with a concentration of 300 nM, the control group was added with an equal amount of blank DMEM medium, and the blank group only contained blank DMEM medium (no cells, for background correction). The final concentration of the drug was 100 nM after dosing. The drug was used for 48 h before subsequent detection.

[0112] 3.4 CCK-8 method for detecting the inhibition of A3710 on BEAS-2B

[0113] 10% CCK-8 working solution was prepared with blank DMEM medium. The original liquid in the 96-well plate was discarded, and 100 μL of CCK-8 working solution was added to each well. After incubation at 37°C in the dark for 1 h, the OD value of each well was measured at 450 nm wavelength using a microplate reader, and the cell survival rate was calculated. Cell survival rate (%) = (experimental group OD value - blank group OD value) / (control group OD value - blank group OD value) x 100%.

[0114] 3.5 Data processing and IC 50 value calculation

[0115] The cell survival rate data of A3710 at a concentration of 100 nM were imported into GraphPad Prism 9.5 software for plotting, and the results of the effect of A3710 on the survival rate of BEAS-2B cells are shown in Figure 8 .

[0116] 4. Evaluation of the activity of compound A3710 in mouse subcutaneous tumor formation experiment of A549 cells

[0117] 4.1 Preparation method of test solution

[0118] According to the body weight of the mice and the given drug dose (0.5 mg / kg), the total amount and volume of the drug required for each administration were calculated. 1 mg of A3710 powder was prepared into a 10 mg / mL stock solution with DMSO, and then diluted with normal saline to prepare a 0.05 mg / mL administration test solution. The control group was prepared in the same way without drug, and the same volume as the drug group was used for intragastric administration.

[0119] 4.2 Establishment of human lung cancer A549 tumor-bearing mouse model

[0120] Mice were raised in SPF (specific pathogen free) environment, and acclimated for 5 days to adapt to laboratory conditions. A549 cells were trypsinized, centrifuged, resuspended with PBS buffer and adjusted to a cell concentration of 2.5 x 10 7 / mL; using a syringe, the mice were subcutaneously inoculated in the right axillary, each mouse injected with 0.2 mL of cell suspension (actual amount of cells inoculated was 5 x 10 6 On day 7, round or oval tumor masses with moderate texture and clear boundaries were observed at the subcutaneous inoculation site of the mice, indicating that the tumor-bearing mouse model was successfully constructed.

[0121] 4.3 Randomization and administration

[0122] On day 15 after inoculation, the volume of the transplanted tumor in the mice reached 100 mm 3 around, with clear boundaries from the surrounding tissues, and the tumor-bearing mice were randomly divided into 2 groups: control group and administration group. The administration group was given A3710 solution by gavage every day, and the control group was given the same volume of normal saline. After 14 days of continuous administration, the experiment was terminated.

[0123] 4.4 Observation of subcutaneously transplanted tumors and determination of various indicators

[0124] The mice were observed and recorded every day: including water and food intake, stool characteristics, response to external stimuli, mental state, etc. Every three days, the length (L) and width (W) of the tumor in the mice were measured and recorded using a vernier caliper, and the formula for calculating the volume of the tumor was V = 0.5 x L x W 2 , and a curve of tumor volume versus time was plotted, and the body weight of the mice was also measured and recorded.

[0125] 4.5 Mouse sacrifice and sample collection

[0126] After termination of drug intervention, the mice were sacrificed by cervical dislocation, and the subcutaneously transplanted tumors were carefully and completely peeled off, and the liver, spleen, lung, and kidney of each mouse were removed and weighed using an electronic analytical balance.

[0127] 4.6 Experimental results

[0128] The results of the tumor volume test in the control and administration groups of mice are shown in Figure 9 , the actual tumor size is shown in Figure 10 , and the results of the tumor weight test are shown in Figure 11 It can be seen that, compared with the control group, the tumor volume of the mice in the administration group was significantly reduced and had a statistically significant difference (P < 0.05). At the same time, the tumor weight in the A3710 group also decreased significantly, with a statistically significant difference. Figure 12The weight change of mice in the control group and the administration group was observed. Compared with the control group, the weight of mice in the administration group did not change significantly, which indirectly indicated that the administration dose of A3710 had no obvious toxicity. The liver, spleen, lung and kidney indexes of mice in the two groups also had no significant change. Figures 13-16 The above results show that A3710 has a significant anti-lung cancer effect and does not show obvious toxic side effects on major organs.

[0129] 5. Evaluation of the activity of compound A3710 on patient-derived xenograft (PDX) models

[0130] 5.1 Preparation method of test solution

[0131] The administration dose and the preparation method are the same as those in 4.1.

[0132] 5.2 Establishment of patient-derived xenograft models

[0133] The mice were raised in a SPF (specific pathogen free) environment and adaptively fed for 5 days to adapt to the laboratory conditions. Under sterile conditions, the patient tumor was cut into small pieces of 1 × 1 mm in a culture dish, and the tissue pieces were implanted into the subcutaneous tissue layer of the right axillary of the mice through a sterile cannula needle, with one piece of tissue (about 1 mm 3 ) implanted in each mouse. After implantation, the skin at the implantation site was gently pressed to ensure that the tissue pieces were tightly attached to the subcutaneous tissue and did not fall off.

[0134] 5.3 Random grouping and administration

[0135] When the PDX tumor grew to about 80 to 120 mm 3 in size and had a clear boundary with the surrounding tissue, the tumor-bearing mice were randomly divided into two groups: a control group and an administration group. The administration group was given A3710 solution by gavage at a dose of 0.5 mg / kg per day, and the control group was given an equal volume of normal saline. After 14 days of continuous administration, the experiment was terminated.

[0136] 5.4 Observation of subcutaneously implanted tumors and determination of various indexes

[0137] After implantation, the mental state, diet, and presence or absence of redness, infection, and shedding of the implantation site were observed weekly, and the tumor volume was measured using a vernier caliper (volume calculation formula: V = 0.5 × L × W 2 ), a tumor growth curve was drawn, and the body weight of the mice was recorded.

[0138] 5.5 Sacrifice of mice and sample collection

[0139] After termination of drug intervention, the mice were sacrificed by cervical dislocation, and the subcutaneously implanted tumors were carefully and completely peeled off and weighed using an electronic analytical balance.

[0140] 5.6 Experimental results

[0141] The tumor volume test results of the control group and the administration group mice are shown in Table 1 Figure 17 , the actual tumor size is shown in Table 2 Figure 18 , and the tumor weight test results are shown in Table 3 Figure 19 It can be seen that, compared with the control group, the tumor volume of the administration group mice is significantly reduced and has a statistical difference (P<0.05). The tumor weight of the A3710 group is also significantly decreased, has a significant statistical difference, and the body weight of the A3710 group mice is not significantly changed Figure 20

[0142] In summary, the diterpenoid compound A3710 prepared from the medicinal material Euphorbia fischeriana Steud has a significant anti-lung cancer effect. In a mouse subcutaneous transplantation tumor model, the diterpenoid compound A3710 can effectively inhibit lung cancer cell growth, showing a clear anti-lung cancer efficacy, and the extraction rate of the diterpenoid compound A3710 prepared according to the method can reach 0.05% or more, having the characteristics of convenient operation and high extraction rate.

[0143] The above merely describes the preferred embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.​

Claims

1. A diterpenoid compound extracted from Euphorbia fischeriana, characterized in that, The structural formula is: 。 2. A method for preparing the diterpenoid compound extracted from Euphorbia fischeriana according to claim 1, characterized in that, The method comprises the following steps: (1) crushing dry branches of Euphorbia fischeriana Steud, and extracting with ethanol by refluxing; (2) concentrating the extract obtained in step (1) under reduced pressure to obtain total extract; (3) separating the total extract by a silica gel column under reduced pressure, and gradient eluting with a mixture of dichloromethane and methanol, wherein the concentration of CH2Cl2 in the mixture is from high to low, and four crude fractions are obtained in sequence; (4) separating the first crude fraction obtained in step (3) by a polyamide resin, and gradient eluting with a mixture of ethanol and water, wherein the concentration of ethanol in the mixture is from low to high, and two fractions are obtained in sequence; (5) separating the first fraction obtained in step (4) by a reversed-phase ODS column chromatography, and gradient eluting to prepare the diterpenoid compound.

3. The method of claim 2, wherein the diterpenoid compound is extracted from Euphorbia macoantha. In step (1), the concentration of ethanol is 80-95 wt.%, and the extraction is performed 2-3 times.

4. The method of claim 2, wherein the diterpenoid compound is extracted from Euphorbia macoantha. In step (2), the temperature for concentration under reduced pressure is 55-65℃.

5. The method of claim 2, wherein the diterpenoid compound is extracted from Euphorbia macoantha. In step (3), the silica gel column is gradient eluted with a mixture of dichloromethane and methanol in a volume ratio of 100:1-0:

1.

6. The method of claim 2, wherein the diterpenoid compound is extracted from Euphorbia macoantha. In step (4), the polyamide resin is gradient eluted with a mixture of ethanol and water in a volume ratio of 30:70-90:

10.

7. The method of claim 2, wherein the diterpenoid compound is extracted from Euphorbia macoantha. In step (5), the reversed-phase ODS column chromatography is gradient eluted with a mixture of methanol and water in a volume ratio of 20:80-90:

10.

8. A pharmaceutical composition, characterized by, A pharmaceutical composition comprising the diterpenoid compound extracted from Euphorbia fischeriana Steud according to claim 1 and a pharmaceutically acceptable carrier or excipient.

9. Use of the diterpenoid compound extracted from Euphorbia fischeriana Steud according to claim 1 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating lung cancer.

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