Diterpenoid compound as well as preparation method and application thereof

Abstract

The invention provides a diterpenoid compound and a preparation method and application thereof.The structural formula of the diterpenoid compound is shown in the formula (1), and the diterpenoid compound is separated from isodon amethystoides, has good biological activity, can be used for preparing anti-tumor drugs and is expected to be applied to the fields of medicine, health care products, cosmetics and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a diterpenoid compound and a preparation method and application thereof. Background Art

[0002] Isodon ternifolia (D.Don) Hara is a plant of Isodon (Schrader ex Bentham) Spac, Lamiaceae. It is also known as Wormtooth Grass in the Buyi area. It is mainly distributed in Zhenning, Ceheng, Xingyi, Luodian, Dushan and other areas in Yunnan. It is a Yunnan specialty medicinal plant. Isodon ternifolia tastes bitter and cold in nature. The whole plant is used as medicine. It has the effects of dispelling wind and cold, resolving phlegm and dampness, relieving pain, stopping bleeding, and promoting tissue regeneration. It is mainly used to treat colds, coughs, icteric hepatitis, dysentery, enteritis, pharyngitis, tonsillitis, urinary tract infection, toothache, rheumatic swelling and pain, and knife wound bleeding; it is used for external washing to treat various poisons, eczema, etc.

[0003] Diterpenoids are one of the most typical and abundant structural types in the genus Ampelopsis. At present, more than 1,000 diterpenoids have been reported from Ampelopsis plants, the main type of which is kaurene-type diterpenes. Among them, kaurene-type diterpenes with α,β-unsaturated ketone units are the main material basis for the efficacy of Ampelopsis plants. Current studies have shown that diterpenoids have antioxidant, anti-inflammatory, and anti-tumor pharmacological activities. The discovery of diterpenoids with novel structures and strong pharmacological effects from plants is of great significance for a deep understanding of their biological functions and the development of new drugs and health products. Summary of the invention

[0004] In view of the above technical problems, the present invention discloses a diterpenoid compound and a preparation method and application thereof, wherein the compound has an anti-tumor effect.

[0005] To this end, the technical solution adopted by the present invention is:

[0006] A diterpene compound, the structural formula of which is shown in the following formula (1):

[0007]

[0008] After identification, the diterpenoid compound is 6α,7β,14β-trihydroxy-7α,20epoxy-ent-kaur-16-en-15-one. This type of compound provides a chemical basis and material reference for the comprehensive development of Camellia chrysantha medicinal materials and the research on the pharmacological activity of natural plants.

[0009] The present invention also discloses a method for preparing the diterpenoid compound as described above, comprising the following steps:

[0010] Step S1, preparing a three-leaf fragrant tea extract;

[0011] Step S2, adding water to the extract of the three-leaf fragrant tea and stirring until a suspension is formed;

[0012] Step S3, extracting the suspension obtained in step S2 with petroleum ether, dichloromethane and ethyl acetate, filtering, and concentrating under reduced pressure to obtain a petroleum ether fraction (IT-PE), a dichloromethane fraction (IT-DCM), an ethyl acetate fraction (IT-EA), and a remaining water layer fraction;

[0013] Step S4, using silica gel to gradiently elute the dichloromethane fraction (IT-DCM) obtained in step S3 with petroleum ether (PE) / ethyl acetate (EtOAc) solution to obtain a fraction containing diterpenoid compounds of Ipomoea batatas;

[0014] Step S5, subjecting the fraction obtained in step S4 to silica gel column chromatography, and performing gradient elution with a PE / ACT solution to obtain a secondary fraction containing diterpenoid compounds;

[0015] Step S6, subjecting the secondary fraction of step S5 to gradient elution with a CH2Cl2 / MeOH solution to obtain a sub-fraction containing diterpenoid compounds;

[0016] Step S7, separating and purifying the subfractions from step S6 by semi-preparative high performance liquid chromatography to obtain the diterpenoid compounds.

[0017] As a further improvement of the present invention, step S1 includes taking the medicinal material of Tripterygium wilfordii, drying it, refluxing and extracting it three times with an ethanol solution, combining the extracts, and concentrating under reduced pressure to remove ethanol to obtain a Tripterygium wilfordii extract.

[0018] As a further improvement of the present invention, the volume percentage concentration of the ethanol solution is 95%.

[0019] As a further improvement of the present invention, in step S4, the silica gel is 200-300 mesh; the volume ratios of petroleum ether (PE) / ethyl acetate (EtOAc) solution used for gradient elution are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:1, MeOH (i.e., pure methanol is used for elution in the last step).

[0020] As a further improvement of the present invention, in step S5, the volume ratios of the PE / ACT solutions for gradient elution are 20:1, 15:1, 10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:100, respectively.

[0021] As a further improvement of the present invention, in step S6, the volume ratios of CH2Cl2 / MeOH solution for gradient elution are 100:0, 100:1, 80:1, 50:1, 30:1, 10:1 and MeOH, respectively.

[0022] As a further improvement of the present invention, in step S7, the conditions of the semi-preparative high performance liquid chromatography are: the chromatographic column is selected as a Welch Ultimate XB-C18 semi-preparative column, the mobile phase is a mixture of methanol and water, the volume ratio of acetonitrile to water is 75:25; the detection wavelength is 210 and 254 nm; or the chromatographic column is selected as a Welch Ultimate XB-C18 semi-preparative column, the mobile phase is a mixture of methanol and water, the volume ratio of acetonitrile to water is 70:30; the detection wavelength is 210 and 254 nm.

[0023] The present invention also discloses the use of the above-mentioned diterpenoid compound in the preparation of anti-tumor drugs. Furthermore, the tumor is triple-negative breast cancer. In vitro pharmacological activity experiments show that the diterpenoid compound 6α,7β,14βtrihydroxy-7α,20-epoxy-ent-kaur-16-en-15-one of the technical solution of the present invention has a significant inhibitory effect on BT549 (triple-negative breast cancer cells) and has good prospects in the field of tumor treatment.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] By adopting the technical scheme of the present invention, diterpenoid compounds separated from the three-leaf balsam pear have good biological activity, can be used to prepare anti-tumor drugs, and are expected to be applied to the fields of medicine, health care products and cosmetics; the method for separating diterpenoid compounds is practical and feasible for industrial production, and provides a new way for the comprehensive development and utilization of the three-leaf balsam pear resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 1H NMR spectrum of the diterpenoid compound of the present invention.

[0027] Figure 2 It is the 13C NMR spectrum of the diterpenoid compound of the present invention.

[0028] Figure 3 1H 1H COSY spectrum of the diterpenoid compound of the present invention.

[0029] Figure 4 It is the HMBC spectrum diagram of the diterpenoid compound of the present invention.

[0030] Figure 5Figure 1 is a graph showing the ability of the diterpenoid compounds of the present invention to inhibit the survival of BT549 cells; wherein (a) is the survival rate of BT549 cells at different diterpenoid concentrations and different treatment times, and (b) is the IC 50 Calculation curve of .

[0031] Figure 6 The results of the ability of diterpenoid compounds of different concentrations to inhibit the proliferation of BT549 cells in the examples of the present invention; wherein, (a) to (e) are blank, 0.25 μM, 0.5 μM, 0.75 μM, and 1 μM, respectively, and (f) is a comparison chart of the number of clones of BT549 cells to which diterpenoid compounds of different concentrations were added.

[0032] Figure 7 The results show that the diterpenoid compounds of the present invention inhibit the migration ability of BT549 cells; wherein (a) is a microscope observation diagram of different diterpenoid compounds at different treatment times, and (b) is a comparison diagram of scratch healing rates of different diterpenoid compounds at different treatment times.

[0033] Figure 8 The figures are graphs of ROS content in BT549 cells after intervention with different concentrations of diterpenoid compounds according to the examples of the present invention; wherein (a) and (b) are changes in intracellular ROS caused by drug treatment, (c) is a comparison of ROS content in BT549 cells after intervention with different concentrations of diterpenoid compounds, and (d) to (h) are staining images of BT549 cells after intervention with blank, 0.25 μM, 0.5 μM, 1 μM, and 1.5 μM concentrations of diterpenoid compounds, respectively.

[0034] Fig. 9 The results of different concentrations of diterpenoid compounds inducing apoptosis in BT549 cells in the examples of the present invention; (a) to (f) are blank, 0.25 μM, 0.5 μM, 0.75 μM, 1 μM, and 1.5 μM, respectively, and (g) is a comparison chart of apoptosis rates.

[0035] Fig.10 These are cell staining pictures of BT549 cells induced by diterpenoid compounds of different concentrations in the present invention to cause apoptosis; (a) to (f) are blank, 0.25 μM, 0.5 μM, 0.75 μM, 1 μM, and 1.5 μM, respectively. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention are described in detail and clearly below. It is obvious that the embodiments disclosed here are only part of the present invention, not all of them. All other embodiments based on the embodiments of the present invention and obtained by those skilled in the art without creative work are within the protection scope of the present invention.

[0037] Example 1

[0038] The preparation of diterpenoid compounds is extracted from the three-leaf fragrant tea, and specifically comprises the following steps:

[0039] (1) taking a medicinal material of three-leafed chrysanthemum, drying it, and extracting it three times with a 95% ethanol aqueous solution at 60° C. under hot reflux, combining the extracts, and concentrating under reduced pressure to remove ethanol to obtain an ethanol extract of three-leafed chrysanthemum;

[0040] (2) adding water to the alcohol extract of Ipomoea batatas obtained in step (1) and stirring until a suspension is formed;

[0041] (3) extracting the suspension of step (2) five times with equal volumes of petroleum ether, dichloromethane, and ethyl acetate, filtering the extracted suspension, and concentrating under reduced pressure to obtain a petroleum ether fraction (IT-PE), a dichloromethane fraction (IT-DCM), an ethyl acetate fraction (IT-EA), and a remaining aqueous fraction;

[0042] (4) using silica gel to gradiently elute the dichloromethane fraction (IT-DCM) of the trifoliate chrysanthemum in step (3) with petroleum ether (PE) / ethyl acetate (EtOAc) solutions of different volume fractions to obtain fractions Fr.1 to Fr.8 containing diterpenoid compounds of the trifoliate chrysanthemum;

[0043] (5) subjecting the fraction Fr.8 of step (4) to silica gel column chromatography and eluting with PE / ACT solutions of different volume fractions to obtain secondary fractions Fr.8.1 to Fr.8.29 containing diterpenoid compounds;

[0044] (6) eluting the secondary fraction Fr.8.1.17 of step (5) with CH2Cl2 / MeOH solution of different volume fractions to obtain sub-fractions Fr.8.17.1 to Fr.8.17.21 containing diterpenoid compounds;

[0045] (7) The subfraction Fr.8.17.15 of step (6) is separated and purified by semi-preparative high performance liquid chromatography to obtain the diterpenoid compound.

[0046] Furthermore, in step (4), the silica gel is (200-300 mesh); the petroleum ether (PE) / ethyl acetate (EtOAc) gradient elution system is 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:1, MeOH, v / v.

[0047] Furthermore, in step (5), the PE / ACT gradient elution system is 20:1, 15:1, 10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:100, v / v.

[0048] Furthermore, in step (6), the CH2Cl2 / MeOH gradient elution system is 100:0, 100:1, 80:1, 50:1, 30:1, 10:1 and MeOH, v / v.

[0049] Furthermore, in step (7), the conditions of the semi-preparative HPLC are as follows: the chromatographic column is a Welch Ultimate XB-C18 semi-preparative column, the mobile phase is a mixture of methanol and water, and the volume ratio of methanol to water is 75:25; the detection wavelengths are 210 and 254 nm.

[0050] The diterpenoid compound prepared in this example is colorless needle-shaped crystals in appearance. The 1H NMR spectrum obtained by identification is as follows: Figure 1 As shown, the 13C NMR spectrum is as follows Figure 2 As shown, the 1H 1H COSY spectrum is as follows Figure 3 As shown, the HMBC spectrum is as follows Figure 4 shown.

[0051] [α] 25 -147.8 (c 0.05, MeOH), HR-ESI-MS (positive) gave m / z 371.1825 [M+Na] + (The calculated value is 371.1834, C 20 H 28 O5Na), and the molecular formula of the compound is determined to be C 20 H 28 O5, its unsaturation degree is calculated to be 7. In addition, UV spectrum (MeOH) gives characteristic absorption of λmax:240nm. After thin layer development and color development, it is determined that the compound may be a terpene.

[0052] exist 1 In the H-NMR (500MHz, in CDCl3) spectrum, δ H 6.18 (1H, brs) and 5.56 (1H, brs) are hydrogen signals on a terminal double bond; δ H 4.81 (1H, s), 3.76 (1H, d, J = 7.2 Hz) are hydrogen signals on two oxygen-linked methine groups; δ H 4.13 (1H, d, J = 9.9 Hz) and 3.81 (1H, d, J = 9.9 Hz) are hydrogen signals on a group of oxymethylene groups; δ H 1.11 (3H, s) and 1.10 (3H, s) are two groups of methyl hydrogen signals.

[0053] 13The C-NMR (125MHz, in CDCl3) spectrum combined with the DEPT 135 spectrum showed a total of 20 carbon signals, including one carbonyl carbon signal δ C 207.4, 2 sp2 hybridized double bond carbon signals δ C 151.4,121.0, 1 sp3 hybridized oxymethylene carbon signal δ C 67.1, 3 sp3 hybridized oxygen-linked methylene carbon signals δ C 97.5, 74.5, 72.7, 5 sp3 hybridized methylene carbon signals δ C 41.4, 31.3, 29.8, 18.7, 16.8, three sp3 hybridized methine carbon signals δ C 59.2, 53.2, 42.9, three sp3 hybridized quaternary carbon signals δ C 62.3, 36.5, 33.8, and 2 methyl carbon signals δ C 33.5, 22.6. Based on 1H-NMR, 13C-NMR, molecular formula and unsaturation, the compound was determined to be a kauri-type diterpenoid compound.

[0054] 1 H- 1 In the H COSY spectrum, we can see δ H There is a correlation between 1.32 (H-2β) and 0.97 (H 1β), 1.46 (H-3β); δ H There is a correlation between 1.26 (H-5) and 3.76 (H-6); δ H There is a correlation between 1.68 (H-9) and 1.63 (H-11α); δ H There is a correlation between 2.47 (H-12α) and 1.63 (H-11α), 3.05 (H-13); δ H There is a correlation between 4.81 (H-14α) and 3.05 (H-13). Combined with the HSQC spectrum, the following fragments can be deduced: C1-C2-C3, C5-C6, C9-C 11- C 12 -C 13 -C 14 .

[0055] In the HMBC spectrum, H-18 (1.10) has a long-range correlation with C-3 (41.4), C-5 (59.2), and C-19 (22.6); H-19 (1.11) has a long-range correlation with C-3 (41.4), C-5 (59.2), and C-18 (33.5); H-6β (3.76) has a long-range correlation with C-7 (97.5); H-20β (4.13) has a long-range correlation with C- C-1 (31.3), C-5 (59.2), C-9 (53.2), C-10 (36.5) have long-range correlations, and the above correlations give fragment 1; H-14 (4.81) has long-range correlations with C-7 (97.5), C-8 (62.3), C-9 (53.2), C-15 (207.4), and H-17α (6.17) has long-range correlations with C-13 (42.9), C-15 (207.4), C 16 (151.4), and the above correlations give fragment 2. Fragments 1 and 2 share C-7 and C-9, and after connection, the planar structure of formula (1) is obtained.

[0056] By comparison, it was found that the diterpenoid compound of formula (1) has the same planar structure as longikaurin A, but different optical rotations. The optical rotation of formula (1) is [α] 25 -147.8 (c 0.05, MeOH), and the optical rotation of longikaurin A is [α] 25 +97.8 (c 0.04, MeOH). At the same time, the NOESY spectra of the two compounds are also different.

[0057] The relative configuration of compound formula (1) was determined by NOESY spectrum. In the NOESY spectrum, H-18 (1.10) was correlated with H-6 (3.76), and H-14 (4.81) was correlated with H-13 (3.05). Therefore, H-18 and H-6 were in the same plane and were β-oriented; H-14 and H-13 were in the same plane and were α-oriented.

[0058] In summary, the carbon and hydrogen signals of formula (1) were assigned by combining HSQC and HMBC spectrum data, and the results are shown in Table 1. After searching SciFinder scholar, no literature reports were found, indicating that compound formula (1) is a new kaur-type diterpenoid compound, named 6α,7β,14β-trihydroxy-7α,20-epoxy ent-kaur-16-en-15-one.

[0059] Table 1 Formula (1) 1 H(500MHz)and 13 C NMR (125 MHz) NMR data (deuterated reagent: CDCl3)

[0060]

[0061] Example 2

[0062] The in vitro activity evaluation of the diterpenoid compounds prepared in Example 1 in inhibiting BT549 triple-negative breast cancer cells was performed using the following experimental steps:

[0063] (1) Test on the inhibitory activity of diterpenoid compounds on the proliferation of BT549 triple-negative breast cancer cells

[0064] Weigh an appropriate amount of formula (1), add cell-grade dimethyl sulfoxide to dissolve, and dilute to 0.5, 1, 2, 4, 8, 10 μM test solution using RPMI-1640 culture medium.

[0065] BT549 (triple-negative breast cancer cells) in the logarithmic growth phase were cultured at a rate of approximately 2×10 4 Each well was inoculated with 100 μL of CCK8 solution at a ratio of 1:10 and incubated for 1 hour. The OD value of each well was measured at 450 nm. The inhibition rate of the compound on tumor cells was calculated according to formula 1, and the IC was calculated using prism software. 50 value.

[0066] The inhibition rate was calculated as follows:

[0067] Inhibition rate (%) = [1-(A2-A0) / (A1-A0)] × 100%, where: A2 is the test group added with the culture medium containing the test solution; A1 is the control group added with the culture medium without the test solution; A0 is the blank control group without any liquid and cells.

[0068] The experimental results are as follows Figure 5 As shown, within the investigated range, the cell survival rate of BT549 showed a downward trend with the increase of the concentration of formula (1), indicating that the inhibitory effect of formula (1) on BT549 cells was concentration-dependent within the investigated range.

[0069] like Figure X It can be seen that by calculation, the IC of the diterpenoid compound of formula (1) is 50 Value, visible IC 50 The values ​​were 1.488 μM respectively.

[0070] (2) Experiment on inhibition of BT549 colony formation by diterpenoid compounds

[0071] BT549 cells in the logarithmic growth phase were inoculated in a 6-well plate at a ratio of 1000 cells / well and incubated in a 37°C, 5% CO2 saturated humidity incubator. The culture medium was replaced every 3 days to maintain a suitable environment for the cells. After the single cells formed small clusters of 5 to 10 cells, the culture medium was replaced with the drug-added culture medium and cultured for about 1 week. When the cells formed small clusters visible to the naked eye, the culture medium was removed and gently rinsed with phosphate-buffered saline. Fixation was performed with 4% paraformaldehyde at room temperature for 30 minutes. After removing the 4% paraformaldehyde, 1 mL of crystal violet (Beyotime) staining solution was added to each well, left to stand at room temperature for 30 minutes, then rinsed with clean water, dried, and photographed and counted cell colonies.

[0072] Experimental results: The effect of compound IT1 (Formula (1)) on the proliferation of BT549 cells was determined by clone formation assay. Figure 6 As shown, different concentrations of IT1 were added to BT549 cells. Compared with the blank control group, the number of cell colonies was significantly reduced after IT1 treatment in a concentration-dependent manner, indicating that IT1 can reduce the clone-forming ability of BT549 cells and inhibit the proliferation of BT549 cells.

[0073] (3) Experiment on the inhibition of BT549 scratch damage by diterpenoid compounds

[0074] BT549 cells were seeded in 6-well plates at 1×10 6 Cells were placed in a 37°C, 5% CO2 saturated humidity incubator and incubated overnight to a density of 100%. A 200 μL pipette was used to vertically scratch the bottom of the well plate, and then gently rinsed with PBS to remove the cells remaining in the scratch area. Subsequently, serum-free medium containing drugs (DMSO as a blank control group) was added, and the cells were placed in a cell culture incubator for incubation. The healing of the scratches at different time points (0h, 24h, 48h) was observed under a microscope.

[0075] Experimental results:

[0076] In the scratch injury experiment, BT549 cells were treated with different concentrations of IT1 (Formula (1)). Figure 7 As shown, compared with the blank control group, the healing ability of BT549 cell scratches was significantly weakened after IT1 treatment for 48 hours, and the open scratch area (%) increased with the increase of IT1 concentration, indicating that IT1 inhibited the migration of BT549 cells in a time- and dose-dependent manner.

[0077] (4) Experiment on the inhibition of ROS by diterpenoids in BT549

[0078] BT549 cells were seeded in 6-well plates at 1×106 When the cells adhered to the wall, the culture medium was replaced with different concentrations of drug-added culture medium. After 48 hours of drug action, the drug-containing culture medium was aspirated and 1 mL of DCFH-DA was added. The cells were reacted at 37°C in the dark for 30 minutes. After the DCFH-DA solution was aspirated, the cells were rinsed with PBS three times, observed under a fluorescence microscope, and photographed for analysis.

[0079] Experimental results:

[0080] After treating BT549 cells with different concentrations of IT1, ROS detection was performed. The experimental results are shown in Figure 8 As shown, compared with the blank control group, 1.5 μM IT1 could significantly promote the production of hydrogen peroxide and superoxide anions in BT549 cells, indicating that IT1 may promote cell apoptosis by inducing the production of ROS in BT549 cells.

[0081] (5) Flow cytometry analysis of diterpenoids inhibiting BT549

[0082] BT549 cells were plated in 6-well plates at 1 × 10 6 cells. After 24 hours of drug action, the cells in the culture medium were collected by centrifugation. Subsequently, the attached cells were rinsed with PBS and the residual cells were removed with a cell scraper. 100 μL of 1×binding buffer was added to each sample and mixed thoroughly. At the same time, 5 μL of AnnexinⅤ-FITC and 10 μL of PI staining solution were added and mixed evenly. Incubate at 37°C for 15 minutes in a dark environment. After the incubation, 400 μL of 1×binding buffer was added and stored in a dark environment at 4°C. Detection was performed by flow cytometry within 1 hour after the staining was completed.

[0083] Experimental results:

[0084] like Fig. 9 As shown, the Annexin V-FITC / PI double staining experiment showed that after adding different concentrations of IT1 for 48 hours, there was no significant difference between the 0.25, 0.75, 1 and 1.25 μM drug groups and the blank control group, while after the action of 1.5 μM IT1, the cell apoptosis rate was significantly increased.

[0085] (6) Diterpenoids inhibit BT549 Hoechst staining experiment

[0086] 7×10 5 BT549 cells were evenly distributed in a 6-well plate, and the cells in the plate were stained with Hoechst for 15 minutes after 24 hours of drug intervention.

[0087] Experimental results:

[0088] After the BT549 cells were treated with compound IT1, Hoechst 33342 was used to stain the nuclei of live cells. Fig.10 As shown, compared with the blank control group, the nuclear staining of the 1 μM and 1.5 μM groups was significantly enhanced, the fluorescence was brighter, and it showed a round structure, indicating that the cell death mode treated with compound IT1 was apoptosis, and it was dose-dependent.

[0089] In summary, compound IT1 has a significant inhibitory effect on BT549 cells.

[0090] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A diterpenoid compound, characterized in that: Its structural formula is shown in the following formula (1):

2. The method for preparing diterpenoid compounds according to claim 1, characterized in that: The following steps are involved: Step S1, preparing a three-leaf fragrant tea extract; Step S2, adding water to the extract of the three-leaf fragrant tea and stirring until a suspension is formed; Step S3, extracting the suspension obtained in step S2 with petroleum ether, dichloromethane and ethyl acetate, filtering, and concentrating under reduced pressure to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, and a remaining water layer fraction; Step S4, using silica gel to gradiently elute the dichloromethane fraction obtained in step S3 with a petroleum ether / ethyl acetate solution to obtain a fraction containing diterpenoid compounds of the spatholobifolia; Step S5, subjecting the fraction obtained in step S4 to silica gel column chromatography, and performing gradient elution with a PE / ACT solution to obtain a secondary fraction containing diterpenoid compounds; Step S6, subjecting the secondary fraction of step S5 to gradient elution with a CH2Cl2 / MeOH solution to obtain a sub-fraction containing diterpenoid compounds; Step S7, separating and purifying the subfractions from step S6 by semi-preparative high performance liquid chromatography to obtain the diterpenoid compounds.

3. The method for preparing diterpenoids according to claim 2, characterized in that: Step S1 comprises taking the medicinal material of Tripterygium wilfordii, drying it, refluxing and extracting it three times with an ethanol solution, combining the extracts, and concentrating under reduced pressure to remove the ethanol, thereby obtaining a Tripterygium wilfordii extract.

4. The method for preparing diterpenoid compounds according to claim 3, characterized in that: The volume percentage concentration of the ethanol solution is 95%.

5. The method for preparing diterpenoids according to claim 3, characterized in that: In step S4, the silica gel is 200-300 mesh; the volume ratios of petroleum ether / ethyl acetate solution used for gradient elution are 1:0, 100:1, 50:1, 20:1, 10:1, 5:1, 3:1, 1:1, 0:1, MeOH.

6. The method for preparing diterpenoid compounds according to claim 5, characterized in that: In step S5, the volume ratios of the PE / ACT solutions for gradient elution are 20:1, 15:1, 10:1, 8:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:100, respectively.

7. The method for preparing diterpenoid compounds according to claim 6, characterized in that: In step S6, the volume ratios of CH2Cl2 / MeOH solution for gradient elution are 100:0, 100:1, 80:1, 50:1, 30:1, 10:1 and MeOH, respectively.

8. The method for preparing diterpenoid compounds according to claim 6, characterized in that: In step S7, the conditions of the semi-preparative high performance liquid chromatography are: the chromatographic column is selected as a Welch Ultimate XB-C18 semi-preparative column, the mobile phase is a mixture of methanol and water, the volume ratio of acetonitrile to water is 75:25; the detection wavelength is 210 and 254 nm; or the chromatographic column is selected as a Welch Ultimate XB-C18 semi-preparative column, the mobile phase is a mixture of methanol and water, the volume ratio of acetonitrile to water is 70:30; the detection wavelength is 210 and 254 nm.

9. Use of the diterpenoid compound as claimed in claim 1 in the preparation of anti-tumor drugs.

10. Use of the diterpenoid compound according to claim 9 in the preparation of an anti-tumor drug, wherein the tumor is triple-negative breast cancer.

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