Aporphine-protoberberine alkaloid dimer as well as preparation method and application thereof

Abstract

The invention relates to a preparation method and application of aporphine-protoberberine alkaloid dimers, and belongs to the field of natural medicines and biological medicines. The aporphine-protoberberine alkaloid dimer disclosed by the invention has a structural general formula shown in the specification, in the formula, R1, R2, R3, R5, R6, R7, R8 and R9 are independently selected from hydrogen, methoxyl, hydroxyl or two adjacent substituent groups are methylenedioxy, so that a five-membered ring is formed; r4 is selected from hydrogen, methoxyl or hydroxyl. Six novel aporphine-protoberberine alkaloid dimers are extracted and separated from thalictrum omeiensis roots, and tests show that the aporphine-protoberberine alkaloid dimers have obvious growth inhibition effects on human acute promyelocytic leukemia cells HL-60 and human prostate cancer cells PC-3, can cause HL-60 cell cycle arrest in an S phase, induce HL-60 cell apoptosis, and can be used for preparing the aporphine-protoberberine alkaloid dimers. The compound has a prospect of preparing medicines for preventing and treating tumors.

Description

Technical Field

[0001] This invention relates to the preparation method and application of apophyte-proberberine alkaloid dimers, specifically to apophyte-proberberine alkaloids and their derivatives and pharmaceutical compositions with antitumor activity extracted and isolated from *Thalictrum emeiense*, their preparation methods, and their application in the preparation of drugs for treating and / or preventing tumors, belonging to the fields of natural medicines and biomedicine. Background Technology

[0002] According to global cancer epidemiology reports, in recent years, my country has ranked first in the world in both new cancer cases and cancer deaths, posing a significant challenge to cancer prevention and control. Developing effective prevention strategies and novel therapeutic drugs is a crucial direction for addressing this challenge. Searching for lead compounds with anti-tumor activity in natural products is one of the important pathways in anti-cancer drug development. *Thalictrum emeiense* (Emei Mountain Thrips) Thalictrum omeiense WTWang et *Thalictrum spp.* is a plant belonging to the genus *Thalictrum* in the family Ranunculaceae. Modern pharmacological studies have shown that various extracts and compounds from *Thalictrum* species exhibit antitumor potential in in vitro and in vivo models, especially alkaloids, which have attracted considerable attention due to their antitumor activity. *Thalictrum spp.* is traditionally used in folk medicine to treat damp-heat, abdominal pain, malaria, and other ailments. However, research on the chemical composition of *Thalictrum spp.* is relatively limited, and reports on its antitumor active components, particularly novel and highly active alkaloid compounds, are extremely scarce. In-depth exploration of the active components in *Thalictrum spp.*, especially the novel alkaloids, is of positive significance for the discovery of new anticancer drug candidates. Summary of the Invention

[0003] This invention provides an apophene-proberberine alkaloid dimer, its preparation method, and its applications. Six novel apophene-proberberine alkaloid dimers were extracted and isolated from the roots of *Thalictrum emeiense*. Testing showed that these dimers significantly inhibited the growth of human acute promyelocytic leukemia cells (HL-60) and human prostate cancer cells (PC-3), induced cell cycle arrest in the S phase of HL-60 cells, and induced apoptosis in HL-60 cells.

[0004] Apophene-proberberine alkaloid dimer or a pharmaceutically acceptable salt thereof, wherein the general structural formula of the apophene-proberberine alkaloid dimer is shown below:

[0005] in, R1, R2, R3, R5, R6, R7, R8, and R9 are each independently selected from hydrogen, methoxy, hydroxyl, or the two adjacent substituents are methylenedioxy, thus forming a five-membered ring; R4 is selected from hydrogen, methoxy, or hydroxyl.

[0006] In this invention, R1, R2, R3, R5, R6, R7, R8, and R9 are each independently selected from two adjacent substituents that are methylenedioxy groups to form a five-membered ring. This means that when two adjacent substituents on the benzene ring (such as R1 and R2, R2 and R3, etc.) form a methylenedioxy group (-O-CH2-O-), they will form a five-membered ring structure together with the carbon atom on the benzene ring.

[0007] Preferably, the specific structure of the apophyll-proberberine alkaloid dimer is selected from the following compounds: .

[0008] Preferably, the apophyll-proberberine alkaloid dimer is extracted and isolated from the roots of *Thalictrum emeiense*.

[0009] Another object of the present invention is to provide a method for preparing the aforementioned apophyll-proberberine alkaloid dimer or a pharmaceutically acceptable salt thereof, comprising the following steps: S1: The roots of *Thalictrum emeiense* were extracted with an ethanol-water solution, the extracts were combined, and the crude extract was obtained by vacuum concentration. S2: Disperse the crude extract obtained in S1 with water, adjust the pH to 2-3, and extract with dichloromethane or trichloromethane. Adjust the pH of the aqueous solution after extraction to 9-10, and then extract with dichloromethane or trichloromethane to obtain the dichloromethane or trichloromethane extract, i.e., total alkaloids. S3: The total alkaloids obtained in S2 were separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol with a volume ratio of 100:0 to 100:50. The fractions with a volume ratio of 100:3 to 100:5 were collected and designated as Fr.2; the fractions with a volume ratio of 100:5 to 100:7 were collected and designated as Fr.3. S4: Fraction Fr.2 obtained in S3 was separated and purified by ODS column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative high performance liquid chromatography and preparative TLC to obtain compounds 2, 3, 5 and 6; Fraction Fr.3 obtained in S3 was separated and purified by ODS column chromatography, Sephadex LH-20 gel column chromatography and semi-preparative high performance liquid chromatography to obtain compounds 1 and 4.

[0010] 5. The preparation method according to claim 4, characterized in that: the specific separation and purification process of fraction Fr.2 and fraction Fr.3 in step S4 is as follows: The fraction Fr.2 was separated by ODS column chromatography, and gradient elution was performed using a methanol-water solvent system with a volume ratio of 10% to 100%. The fraction with a volume ratio of 50% to 60% was collected and denoted as Fr.2D; the fraction with a volume ratio of 60% to 80% was collected and denoted as Fr.2E. The fraction Fr.2D was separated by ODS column chromatography and eluted with a gradient of 50% to 100% methanol-water solution. The fraction with a volume ratio of 60% to 70% was collected and denoted as Fr.2D2. The fraction Fr.2D2 was separated by semi-preparative high-performance liquid chromatography, using a mobile phase of methanol-water containing 0.5‰ diethylamine at a volume ratio of 80:20 to elute compound 3. t R = 49.5 min); The fraction Fr.2E was separated by ODS column chromatography, and gradient elution was performed using a methanol-water solvent system with a volume ratio of 50% to 100%. The fraction with a volume ratio of 60% to 70% was collected and designated as Fr.2E1; the fraction with a volume ratio of 70% to 80% was collected and designated as Fr.2E2. The fraction Fr.2E1 was separated by Sephadex LH-20 column chromatography with methanol as the eluent to obtain fractions Fr.2E1-1 and Fr.2E1-2. Fraction Fr.2E1-1 was separated by preparative TLC chromatography, using a dichloromethane-methanol solvent system with a volume ratio of 10:1 as the developing solvent to obtain compounds 5 and 6; The fraction Fr.2E2 was separated by Sephadex LH-20 column chromatography using methanol as the eluent, followed by preparative high-performance liquid chromatography (HPLC) purification. Elution was performed using a mobile phase of methanol-water or acetonitrile-water containing 0.3‰–0.5‰ diethylamine (v / v) at a ratio of 70:30 to obtain compound 2. t R = 72.3 min); The fraction Fr.3 was separated by ODS column chromatography and eluted with a methanol-water solvent system with a volume ratio of 10% to 100%. The fraction with a volume ratio of 60% to 80% was collected and denoted as Fr.3A. The fraction Fr.3A was separated by ODS column chromatography and eluted with a 10%–100% methanol-water solvent system. The fraction with a volume ratio of 60%–70% was collected and designated as Fr.3A1. The fraction Fr.3A1 was separated by Sephadex LH-20 column chromatography with methanol as the eluent, followed by preparative high-performance liquid chromatography (HPLC) purification. Elution was performed using a mobile phase of methanol-water or acetonitrile-water containing 0.3‰–0.5‰ diethylamine at a volume ratio of 68:32 to obtain compound 1 (… tR = 75.5 min) and 4 ( t R = 82.5 min).

[0011] Furthermore, in step S1, the root of *Thalictrum emeiense* is extracted three times by reflux with 50%–95% ethanol aqueous solution or methanol aqueous solution, each time for two to three hours; the mass-to-volume ratio of the root of *Thalictrum emeiense* to the ethanol aqueous solution or methanol aqueous solution is 1:5–10 kg / L.

[0012] Further, in step S2, the pH value is adjusted to 2-3 using a 2-6% hydrochloric acid aqueous solution, and then extracted with dichloromethane or trichloromethane. The extracted aqueous solution is then adjusted to pH 9-10 using a 1-3 mol / L sodium hydroxide aqueous solution, and then extracted again with an equal volume of dichloromethane or trichloromethane to obtain a dichloromethane or trichloromethane extract.

[0013] Another object of the present invention is to provide a pharmaceutical composition comprising one or more of the following: apophyll-proberberine alkaloid dimer, a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer, a solvate or a salt of a solvate, and a pharmaceutically acceptable excipient.

[0014] Furthermore, the dosage form of the pharmaceutical composition is an oral dosage form or a non-oral dosage form, wherein the oral dosage form is a tablet, capsule, powder or granule, and the non-oral dosage form includes suppositories or injections.

[0015] Another object of the present invention is to provide the use of the aforementioned apophyll-proberberine alkaloid dimer or a pharmaceutically acceptable salt thereof or the aforementioned pharmaceutical composition in the preparation of drugs for the prevention and / or treatment of tumors.

[0016] This invention revealed that compounds 1-6 all exhibited strong anti-proliferation activity against HL-60 and PC-3 cells. Mechanistic studies of compound 1 showed that flow cytometry induced apoptosis in HL-60 cells and arrested them in the S phase. Hoechst 33258 staining experiments showed that compound 1 caused morphological changes in HL-60 cells, inducing apoptosis in a concentration-dependent manner. Mitochondrial membrane potential assays showed that compound 1 dose-dependently reduced the mitochondrial membrane potential of HL-60 cells, indicating that it triggers programmed cell death by activating the mitochondrial apoptosis pathway.

[0017] The beneficial effects of the present invention: The compounds 1-6 obtained by the present invention have novel structures and have significant growth inhibitory effects on human acute promyelocytic leukemia cells HL-60 and human prostate cancer cells PC-3. Among them, compound 1 causes HL-60 cells to arrest in the S phase and induces HL-60 cell apoptosis, which has the prospect of preparing drugs for the prevention and treatment of tumors. Attached Figure Description

[0018] Figure 1 The image shows the HR-ESI-MS spectrum of compound 1 obtained in Example 1.

[0019] Figure 2 The image shows the IR spectrum of compound 1 obtained in Example 1.

[0020] Figure 3 The UV spectrum of compound 1 obtained in Example 1 (solvent CH3OH).

[0021] Figure 4 The ECD spectrum of compound 1 obtained in Example 1 (solvent CH3OH).

[0022] Figure 5 Compound 1 obtained in Example 1 1 1H NMR spectrum (solvent CDCl3).

[0023] Figure 6 Compound 1 obtained in Example 1 13 C10 NMR spectrum (solvent CDCl3).

[0024] Figure 7 The HSQC spectrum of compound 1 obtained in Example 1 (solvent CDCl3).

[0025] Figure 8 The HMBC spectrum of compound 1 obtained in Example 1 (solvent CDCl3).

[0026] Figure 9 The NOESY spectrum of compound 1 obtained in Example 1 (solvent CDCl3).

[0027] Figure 10 The figure shows the results of flow cytometry analysis of the effect of compound 1 on apoptosis of HL-60 cells. It can be seen that compound 1 induces apoptosis of HL-60 cells in a concentration-dependent manner.

[0028] Figure 11 The figure shows the results of flow cytometry analysis of the effect of compound 1 on the cell cycle of HL-60 cells. It can be seen that compound 1 caused HL-60 cells to be arrested in the S phase.

[0029] Figure 12The figure shows the effect of different concentrations of compound 1 on the morphology of HL-60 cells. It can be seen that compound 1 causes changes in the morphology of HL-60 cells and induces apoptosis.

[0030] Figure 13 The figure shows the effect of compound 1 on the mitochondrial membrane potential of HL-60 cells. It can be seen that compound 1 can significantly reduce the mitochondrial membrane potential of HL-60 cells, increase mitochondrial membrane permeability, and thus promote cell apoptosis. Detailed Implementation

[0031] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0032] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0033] Example 1 The preparation method of apophyll-proberberine alkaloid dimers 1-6 includes the following steps: Take 30 kg of *Thalictrum emeiense* root and extract three times using 300 L of 95% ethanol aqueous solution under reflux. The first two extractions are for three hours each, and the third for two hours. Combine the extracts and concentrate to obtain a crude extract. Suspend the crude extract in 5 L of water and adjust the pH to 2-3 using 5% hydrochloric acid aqueous solution. Extract this acidic aqueous solution with dichloromethane. The resulting aqueous solution is then treated with 3... The pH was adjusted to 9-10 with a mol / L sodium hydroxide aqueous solution, and then extracted with an equal volume of dichloromethane to obtain the dichloromethane extract, i.e., the total alkaloids. The obtained dichloromethane extract was separated by silica gel column chromatography with a 200-300 mesh, using a gradient elution system of dichloromethane-methanol solvent with a volume ratio of 100:0 to 0:100, specifically dichloromethane:methanol = 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:20, and 100:50, to obtain 7 fractions (Fr.1-Fr.7). The fraction with a volume ratio of 100:3 to 100:5 was collected and designated as Fr.2; the fraction with a volume ratio of 100:5 to 100:7 was collected and designated as Fr.3.

[0034] The fraction Fr.2 was separated by ODS column chromatography and eluted with a 10%–100% methanol-water solvent system to obtain 5 fractions (Fr.2A–Fr.2E). The fraction with a volume ratio of 50%–60% was collected and denoted as Fr.2D; the fraction with a volume ratio of 60%–80% was collected and denoted as Fr.2E.

[0035] The fraction Fr.2D was separated by ODS column chromatography with gradient elution using 50%–100% methanol-water solution to obtain two fractions. The fraction with a volume ratio of 60%–70% was collected and designated as Fr.2D2. This fraction was then separated by semi-preparative high-performance liquid chromatography (using a methanol-water mixture containing 0.5‰ diethylamine at a volume ratio of 80:20) to obtain compound 3. t R = 49.5 min).

[0036] The fraction Fr.2E was separated by ODS column chromatography and eluted with a gradient of 50%–100% methanol-water solution to obtain two fractions. The fraction with a volume ratio of 60%–70% was collected and designated as Fr.2E1; the fraction with a volume ratio of 70%–80% was collected and designated as Fr.2E2.

[0037] Fraction Fr.2E1 was separated by Sephadex LH-20 column chromatography (eluent: methanol) to obtain fractions Fr.2E1-1 and Fr.2E1-2; fraction Fr.2E1-1 was separated by preparative TLC chromatography (developing solvent: dichloromethane-methanol, 10:1) to obtain compounds 5 and 6.

[0038] Fraction Fr.2E2 was purified by Sephadex LH-20 column chromatography (eluent: methanol) and further separated by preparative HPLC (using methanol-water containing 0.5‰ diethylamine at a volume ratio of 70:30 as the mobile phase) to obtain compound 2 ( t R =72.3 min).

[0039] Fraction Fr.3 was separated by ODS column chromatography with gradient elution using a 10%–100% methanol-water solvent system to obtain three fractions. The fraction with a volume ratio of 60%–80% was collected and designated as Fr.3A. Fraction Fr.3A1 was also separated by ODS column chromatography with gradient elution using a 10%–100% methanol-water solvent system and collected with a volume ratio of 60%–70%. After purification by Sephadex LH-20 column chromatography (using methanol as the eluent), it was further separated by preparative HPLC (using a methanol-water mixture containing 0.5‰ diethylamine at a volume ratio of 68:32 as the mobile phase) to obtain compound 1. t R = 75.5 min) and 4 ( t R = 82.5 min).

[0040] The structural identification results of compounds 1-6 obtained in Example 1 are as follows: Compound 1 was named Thalibearine B: a pale yellow amorphous powder; -115.7 ( c 2.24, CH3OH); HR-ESI-MS m / z 711.3276 [M+H] + (cacld for C 41 H 47 N₂O₉ (711.3276); IR (KBr) υ max :2935, 2838, 1610, 1509, 1460, 1394, 1340, 1269, 1119, 1020 cm -1 UV (CH3OH) λ max (log ε ): 282 (4.35), 297 (4.25), 302 (4.22) sh, 310 (4.19) nm; ECD (CH3OH) λ max (Δ ε ): 195 (-30.82), 233 (+27.96), 271 (-5.39), 316 (-0.08) nm; 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (100 MHz, CDCl3) data are shown in Tables 1 and 2. High-resolution mass spectrometry data are shown in Tables 1 and 2. Figure 1 IR spectrum see Figure 2 See UV spectrum Figure 3 ECD spectrum can be found Figure 4 , 1 H-NMR spectrum see Figure 5 , 13 C-NMR spectrum see Figure 6 HSQC spectrum Figure 7 HMBC spectrum Figure 8 See NOESY spectrum Figure 9 .

[0041] Compound 2 was named Thalibearine C: a pale yellow amorphous powder; -130.3 ( c 0.39, CH3OH); HR-ESI-MS m / z 711.3276 [M+H] + (cacld for C 41 H 47 N₂O₉ (711.3276); IR (KBr) υ max:2923, 2853, 1632, 1512, 1463, 1384, 1268, 1116 cm -1 UV (CH3OH) λ max (log ε ): 282(4.37), 300 (4.24), 302 (4.23) sh, 315 (4.19) nm; ECD (CH3OH) λ max (Δ ε ): 195 (-8.39), 232 (+7.93), 267 (-1.78), 315 (-0.10) nm; 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (100 MHz, CDCl3) data are shown in Tables 1 and 2.

[0042] Compound 3 was named Thalibearine D: a pale yellow amorphous powder; -110.7 ( c 1.55, CH3OH; HR-ESI-MS m / z 741.3389 [M+H] + (cacld for C 42 H 49 N2O 10 , 741.3382); IR (KBr) υ max :2935, 2838, 1611, 1504, 1461, 1394, 1340, 1252, 1118, 1016 cm -1 UV (CH3OH) λ max (log ε ): 281 (4.33), 301 (4.27), 303 (4.20) sh, 317 (4.17) nm; ECD (CH3OH) λ max (Δ ε ): 197 (-19.90), 232 (+21.38), 269 (-4.71), 316 (-0.01) nm; 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (100 MHz, CDCl3) data are shown in Tables 1 and 2.

[0043] Compound 4 was named Thalibearine G: a pale yellow amorphous powder; -118.7 ( c 0.10, CH3OH); HR-ESI-MS m / z : 727.3233 [M+H] + (cacld for C 41 H 47 N2O 10 , 727.3225); ECD(CH3OH) λ max (Δ ε ): 214 (-49.06), 242 (+75.72), 277 (-16.55), 301 (-11.30) nm; 1 H-NMR (400 MHz, CDCl3) and 13 C-NMR (100 MHz, CDCl3) data are shown in Tables 1 and 2.

[0044] Compound 5 was named Thalibearine E: a pale yellow amorphous powder; -77.1 ( c 0.50, CH3OH); HR-ESI-MS m / z 739.3213 [M+H] + (cacld for C 42 H 47 N2O 10 , 739.3225); IR (KBr) υ max :2932, 2850, 1614, 1502, 1460, 1392, 1337, 1249, 1197, 1124, 1087, 1010cm -1 UV (CH3OH) λ max (log ε ): 261 (4.55), 271 (4.58) nm; ECD (CH3OH) λ max (Δ ε ): 194 (-4.43), 202 (-6.49), 211 (-4.83), 217 (-5.44), 237 (+1.85), 264 (-0.23), 304(-0.56), 321 (+0.45) nm; 1H-NMR (600 MHz, CDCl3) and 13 C-NMR (100 MHz, CDCl3) data are shown in Tables 1 and 2.

[0045] Compound 6 was named Thalibealine F: a pale yellow amorphous powder; -86.5 ( c 0.56, CH3OH); HR-ESI-MS m / z 723.3299 [M+H] + (cacld for C 42 H 47 N₂O₉ (723.3276); IR (KBr) υ max :2923, 1561, 1499, 1458, 1384, 1116 cm -1 UV (CH3OH) λ max (log ε ): 261 (4.57), 272 (4.60) nm; ECD (CH3OH) λ max (Δ ε ): 195 (-3.82), 203 (-11.35), 212 (-5.82), 221 (-9.54), 247 (+4.45), 278 (-0.62), 304 (-0.22), 327 (+0.35) nm; 1 H-NMR (600 MHz, CDCl3) and 13 C-NMR (100 MHz, CDCl3) data are shown in Tables 1 and 2.

[0046] Table 1. Compounds 1 Chemical shift values ​​of 1H NMR (solvent: CDCl3)

[0047] Table 2. Compounds 13 Chemical shift values ​​of 10⁻⁶ C NMR (solvent: CDCl₃)

[0048] a Indicates signal absence Example 2 In vitro antitumor cell proliferation activity assay of compounds 1-6 obtained in Example 1 (1) Experimental methods The antiproliferative activity of compounds 1–6 against PC-3 and HL-60 cells was evaluated using the MTT assay and trypan blue staining. Logarithmically growing PC-3 cells were seeded in 96-well plates and incubated overnight at 37°C with 5% CO2. Different concentrations of the test compounds were added, and the cells were cultured for another 48 hours. 20 μL of the test compounds was added to each well. μ After adding 5 mg / mL MTT solution, continue incubation for 4 hours. Discard the solution in each well and add 200 mL of MTT solution to each well. μ L DMSO was used, and the absorbance (OD) value was measured at 490 nm using a microplate reader. The growth inhibition rate at different concentrations was calculated using the following formula:

[0049] Add 2 mL of HL-60 cell suspension (5.0 × 10⁻⁶) 4 Cells / mL were added to 24-well plates, and pre-prepared compound solutions of different concentrations were added to the corresponding wells, with control wells included. After culturing for 72 hours, trypan blue staining was performed, and cells were observed and counted under a light microscope. The anti-cell proliferation activity of the compound was measured using IC50. 50 Indicated. The IC is calculated using SPSS software. 50 Values. Experimental data are expressed as mean ± standard error. 5-Fu was used as a positive control, and 0.1% DMSO was used as a negative control.

[0050] (2) Experimental results The antiproliferative activities of compounds 1-6 against human leukemia cells HL-60 and human prostate cancer cells PC-3 were tested. The results are shown in Table 3. It can be seen that these compounds all exhibited strong activity against HL-60 and PC-3 cells, with IC50 values ​​of [missing data]. 50 The ranges are 2.01 to 5.23. μ M and 5.71~15.14 μ M, positive control drug 5-fluorouracil IC 50 They are 2.80 and 22.04 respectively. μ M. These compounds exhibited activity against cancer cell proliferation and show promise for the development of antitumor drugs.

[0051] Table 3. Antiproliferative effects of compounds 1-6 on HL-60 and PC-3 cancer cells (IC50, 100%) 50 ± SD in μ M)

[0052] Example 3 Apoptosis and cell cycle experiments of compound 1 obtained in Example 1 (1) Experimental methods Apoptosis detection: HL-60 cell suspension was added to 6-well plates, and pre-prepared concentrations of 1, 2, and 3 were added. μ Compound M solution was added to the corresponding wells, and control wells were also set up. After incubation for 24 hours, the cells were centrifuged, the supernatant was discarded, and the cells were washed with pre-cooled PBS. The cells were then collected and resuspended in 500 mL of water. μ Add 1× Binding Buffer to L. Add Annexin V-APC and 7-AAD, and incubate the mixture at room temperature in the dark for 15 minutes. Centrifuge to remove the supernatant, resuspend the cells in pre-chilled PBS, and immediately detect apoptosis using flow cytometry.

[0053] Cell cycle detection: Cells were seeded and incubated using the same method as described above. After centrifugation, the culture supernatant was discarded. Cells were washed once with pre-chilled PBS, centrifuged again, and the supernatant was discarded. Pre-chilled 70% ethanol solution was added for fixation overnight at 4°C. Before staining, the fixative was washed away with PBS, and the cells were then resuspended at 100°C. μ Add 400 ml of L RNase A solution. μ The cells were incubated with PI staining solution at 4°C in the dark for 30 minutes, and the cell cycle was detected by flow cytometry.

[0054] (2) Experimental results Flow cytometry results showed that, compared with the control group, compound 1 was significantly reduced in cells 1, 2, and 3. Figure 10 At M concentration, dose-dependent apoptosis was induced in HL-60 cells ( Figure 11 ), and cause cell cycle arrest in the S phase ( μ ).

[0055] Example 4 Hoechst 33258 staining experiment of compound 1 obtained in Example 1: (1) Experimental methods HL-60 cells were seeded in 6-well plates, compound 1 was added, and the cells were incubated for 24 hours. The cells were collected and placed on glass slides, and fixed with 4% paraformaldehyde for 30 minutes. After washing the cells three times with PBS, they were stained with Hoechst 33258 working solution for 10 minutes in the dark, then washed again with PBS and observed under a fluorescence microscope.

[0056] (2) Experimental results Control group cells showed uniform staining and no morphological abnormalities. HL-60 cells were treated with compounds 1 (1, 2, and 3). Figure 12 M) morphological changes occurred 24 hours after treatment ( μ The presence of apoptotic features indicates that compound 1 can induce apoptosis in HL-60 cells in a dose-dependent manner.

[0057] Example 5 Detection of mitochondrial membrane potential of compound 1 obtained in Example 1: (1) Experimental methods HL-60 cells were treated with compound 1 or cultured in medium for 24 hours, then washed with PBS and stained with JC-1 dye. The medium was discarded, and the cells were washed with PBS, then incubated with JC-1 staining working solution in the dark for 20 minutes. After centrifugation and discarding the supernatant, the cells were washed twice with JC-1 buffer dilution, resuspended in JC-1 staining working solution, and analyzed by flow cytometry. The percentage of mitochondrial membrane depolarization was detected by flow cytometry.

[0058] (2) Experimental results Flow cytometry analysis showed that compounds 1 (1, 2 and 3) were involved in the detection of the virus. Figure 13 After 24 hours of treatment with M, the proportion of mitochondrial depolarized cells increased in a dose-dependent manner. ​ The results showed that compound 1 could reduce mitochondrial membrane potential in a dose-dependent manner and induce apoptosis.

Claims

1. Apophyll-proberberine alkaloid dimer or a pharmaceutically acceptable salt thereof, characterized in that: The general structural formula of the apofel-proberberine alkaloid dimer is shown below: in, R1, R2, R3, R5, R6, R7, R8, and R9 are each independently selected from hydrogen, methoxy, hydroxyl, or the two adjacent substituents are methylenedioxy, thus forming a five-membered ring; R4 is selected from hydrogen, methoxy, or hydroxyl.

2. The apophyll-proberberine alkaloid dimer or its pharmaceutically acceptable salt according to claim 1, characterized in that: The apofel-proberberine alkaloid dimer is one of the following compounds: 。 3. The apophyll-proberberine alkaloid dimer or its pharmaceutically acceptable salt according to claim 1 or 2, characterized in that: The apophyte-proberberine alkaloid dimer was extracted and isolated from the roots of *Thalictrum emeiense*.

4. The method for preparing the apophyll-proberberine alkaloid dimer according to claim 2, characterized in that: Includes the following steps: S1: The roots of *Thalictrum emeiense* were extracted with an ethanol-water solution, the extracts were combined, and the crude extract was obtained by vacuum concentration. S2: Disperse the crude extract obtained in S1 with water, adjust the pH to 2-3, and extract with dichloromethane or trichloromethane. Adjust the pH of the aqueous solution after extraction to 9-10, and then extract with dichloromethane or trichloromethane to obtain the dichloromethane or trichloromethane extract, i.e., total alkaloids. S3: The total alkaloids obtained in S2 were separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol with a volume ratio of 100:0 to 100:

50. The fractions with a volume ratio of 100:3 to 100:5 were collected and designated as Fr.2; the fractions with a volume ratio of 100:5 to 100:7 were collected and designated as Fr.

3. S4: Fraction Fr.2 obtained in S3 was separated and purified by ODS column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative high performance liquid chromatography and preparative TLC to obtain compounds 2, 3, 5 and 6; Fraction Fr.3 obtained in S3 was separated and purified by ODS column chromatography, Sephadex LH-20 gel column chromatography and semi-preparative high performance liquid chromatography to obtain compounds 1 and 4.

5. The preparation method according to claim 4, characterized in that: The specific separation and purification process of fractions Fr.2 and Fr.3 in S4 is as follows: The fraction Fr.2 was separated by ODS column chromatography, and gradient elution was performed using a methanol-water solvent system with a volume ratio of 10% to 100%. The fraction with a volume ratio of 50% to 60% was collected and denoted as Fr.2D; the fraction with a volume ratio of 60% to 80% was collected and denoted as Fr.2E. The fraction Fr.2D was separated by ODS column chromatography and eluted with a gradient of 50-100% methanol-water solution. The fraction with a volume ratio of 60%-70% was collected and denoted as Fr.2D2. The fraction Fr.2D2 was separated by semi-preparative high-performance liquid chromatography, using a mobile phase of methanol-water containing 0.5‰ diethylamine at a volume ratio of 80:20 to elute compound 3. t R = 49.5 min); The fraction Fr.2E was separated by ODS column chromatography, and gradient elution was performed using a methanol-water solvent system with a volume ratio of 50% to 100%. The fraction with a volume ratio of 60% to 70% was collected and designated as Fr.2E1; the fraction with a volume ratio of 70% to 80% was collected and designated as Fr.2E2. The fraction Fr.2E1 was separated by Sephadex LH-20 column chromatography with methanol as the eluent to obtain fractions Fr.2E1-1 and Fr.2E1-2. Fraction Fr.2E1-1 was separated by preparative TLC chromatography, using a dichloromethane-methanol solvent system with a volume ratio of 10:1 as the developing solvent to obtain compounds 5 and 6; The fraction Fr.2E2 was separated by Sephadex LH-20 column chromatography using methanol as the eluent, followed by preparative high-performance liquid chromatography (HPLC) purification. Elution was performed using a mobile phase of methanol-water or acetonitrile-water containing 0.3‰–0.5‰ diethylamine (v / v) at a ratio of 70:30 to obtain compound 2. t R = 72.3 min); The fraction Fr.3 was separated by ODS column chromatography and eluted with a methanol-water solvent system with a volume ratio of 10% to 100%. The fraction with a volume ratio of 60% to 80% was collected and denoted as Fr.3A. The fraction Fr.3A was separated by ODS column chromatography and eluted with a 10%–100% methanol-water solvent system. The fraction with a volume ratio of 60%–70% was collected and designated as Fr.3A1. The fraction Fr.3A1 was separated by Sephadex LH-20 column chromatography with methanol as the eluent, followed by preparative high-performance liquid chromatography (HPLC) purification. Elution was performed using a mobile phase of methanol-water or acetonitrile-water containing 0.3‰–0.5‰ diethylamine at a volume ratio of 68:32 to obtain compound 1 (… t R = 75.5 min) and 4 ( t R = 82.5 min).

6. The preparation method according to claim 4, characterized in that: In step S1, the root of *Thalictrum emeiense* is extracted three times by reflux with 70%–95% ethanol aqueous solution or methanol aqueous solution for two to three hours each time; the mass-to-volume ratio of the root of *Thalictrum emeiense* to the ethanol aqueous solution or methanol aqueous solution is 1:5–10 kg / L.

7. The preparation method according to claim 4, characterized in that: In step S2, the pH value is adjusted to 2-3 using a 2-6% hydrochloric acid aqueous solution, and then extracted with dichloromethane or trichloromethane. The pH value of the extracted aqueous solution is adjusted to 9-10 using a 1-3 mol / L sodium hydroxide or saturated sodium carbonate aqueous solution, and then extracted again with an equal volume of dichloromethane or trichloromethane to obtain a dichloromethane or trichloromethane extract.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises one or more of the following: apophyll-proberberine alkaloid dimer as described in claim 1 or 2, a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer, a solvate or a salt of a solvate, and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, characterized in that: The dosage form of the pharmaceutical composition is an oral dosage form or a non-oral dosage form, wherein the oral dosage form is a tablet, capsule, powder or granule, and the non-oral dosage form includes suppositories or injections.

10. The use of the apophyll-proberberine alkaloid dimer of claim 1 or 2 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 8, in the preparation of a drug for the prevention and / or treatment of tumors.

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