Preparation method of natural flavanone compound and application of natural flavanone compound in preparation of anti-hepatoma drugs

Abstract

The invention belongs to the technical field of extraction of active ingredients, and provides a preparation method of a natural flavanone compound and application of the natural flavanone compound in preparation of anti-liver cancer drugs, and the application is that a sophora alopecuroide rhizome extraction mixture and a monomer are applied to preparation of anti-liver cancer drugs. According to the method, the HSCCC linear elution technology is utilized, a large number of high-purity fractions of active flavanone compound components are rapidly, efficiently and simultaneously separated from a large number of sophora alopecuroide rhizome crude extracts, and the effect of a mixture and a monomer in the sophora alopecuroide rhizome extracts combined with sorafenib on liver cancer cells MHCC97H is evaluated; the result shows that the mixture of the two flavanone compounds Alopecurone A and Alopecurone B of which the total purity is greater than 90% can be obtained by the preparation method disclosed by the invention, and the mixture and the monomer substances thereof have a growth inhibition effect on hepatoma carcinoma cells MHCC97H.

Description

Technical Field

[0001] This invention belongs to the field of active ingredient extraction technology, and relates to natural dihydroflavonoids, specifically to the preparation method of natural dihydroflavonoids and their application in the manufacture of anti-liver cancer drugs. Background Technology

[0002] *Sophora alopecuroides* L., a perennial herb belonging to the genus *Sophora* in the legume family, is also known as bitter bean grass. In my country, it is mainly distributed in Xinjiang, Gansu, Ningxia, and Inner Mongolia. Its roots, stems, whole plant, and seeds are all used medicinally. *Sophora alopecuroides* has a bitter taste and cold properties, possessing effects such as dispelling wind and dampness, clearing heat and detoxifying, relieving pain, and killing parasites. The chemical composition of *Sophora alopecuroides* is rich and complex, mainly including alkaloids, flavonoids, flavonoid glycosides, polysaccharides, volatile oils, and organic acids. Pharmacological studies have shown that various types of compounds isolated from *Sophora alopecuroides* have anti-tumor, immunomodulatory, antitoxin, and hepatitis B treatment pharmacological effects, some of which have been developed into clinical drugs. The diverse pharmacological activities of *Sophora alopecuroides* stem from its various structural types; flavonoids, including dihydroflavonoids, are one such class of components. These diverse pharmacological activities make *Sophora alopecuroides* a widely used medicinal plant. However, the different types of pharmacological effects of the various compounds in bitter bean seeds lead to uncertainty regarding their efficacy when used as a medicinal material. Bitter bean seed tablets, once used clinically, ceased production in the late 1990s for this reason. Therefore, employing specific methods to separate the components from bitter bean seeds is of great significance for the effective use of this medicinal material.

[0003] High-speed counter-current chromatography (HSCCC) is a highly efficient and continuous liquid-liquid partition chromatography technique. It utilizes the high-speed planetary motion of two immiscible solvent phases in a spiral tube, causing multiple partitions of the separated substances between the two phases, thus achieving rapid and efficient separation and preparation of target components. HSCCC technology avoids irreversible adsorption or contamination during separation, operates under mild conditions, and is rapid, exhibiting characteristics such as minimal component denaturation and no loss. Under appropriate separation conditions, HSCCC can efficiently, rapidly, and in large quantities separate and prepare phytochemicals. HSCCC technology has been widely applied to the separation and purification of natural products such as flavonoids, alkaloids, anthraquinones, saponins, polyphenols, and coumarins. With technological advancements, early HSCCC techniques have become increasingly inadequate for separating complex natural products, leading to technological innovations in solvent systems and elution methods. Existing literature reports the separation of several flavonoids from the mangrove plant *Pongamia pinnata* using a three-phase solvent system (Hao et al., 2013). Regarding elution methods, conventional linear elution is time-consuming and has poor separation efficiency for structurally similar compounds. Gradient elution, internal circulation elution, online storage technology, reverse elution, and two-step elution can significantly improve separation efficiency (Sun ZL et al, 2017; Wang M et al, 2017; Shaheen N et al, 2017). For bitter bean, conventional HSCCC technology is difficult to rapidly separate high-purity, structurally complex, and similar monomeric flavonoids.

[0004] Based on the current state of existing technologies, this invention provides a two-step elution method for the rapid and efficient separation and preparation of large quantities of natural dihydroflavonoids using high-speed countercurrent chromatography (HSCCC). This method utilizes HSCCC linear elution to rapidly separate and purify a mixed fraction of two dihydroflavonoid epimers with anti-hepatocellular carcinoma activity from Sophora flavescens plant extract. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing natural dihydroflavonoids and their application in the manufacture of anti-liver cancer drugs. By applying dihydroflavonoids to the preparation of drugs for treating liver cancer, a new approach and means for liver cancer treatment is provided.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a method for separating dihydroflavonoids from the rhizomes of Sophora flavescens using high-speed countercurrent chromatography, comprising the following steps:

[0008] (1) Sample preparation: Take the root and rhizome of bitter bean, crush it, and extract it with 95% ethanol at least 3 times. Combine the extracts and concentrate them under reduced pressure and evaporate to dryness to obtain bitter bean root extract.

[0009] (1) Initial high-speed countercurrent chromatography separation: Petroleum ether, ethyl acetate, methanol and water were thoroughly mixed in a volume ratio of 2~3:2~3:3:2 to form a two-phase solvent system 1; The above-mentioned Sophora flavescens root extract sample was dissolved in system 1 with a mass-volume fraction of 15% g / mL, and the stationary phase G and each crude fraction AF were obtained by initial high-speed countercurrent chromatography separation. The crude fraction AF was evaporated to dryness under reduced pressure and then injected again to separate the compounds.

[0010] (2) Secondary high-speed countercurrent chromatography separation: Petroleum ether, ethyl acetate, methanol and water are thoroughly mixed in a volume ratio of 6~8:6~8:4:2 to form a two-phase solvent system 2; the above crude fraction 1 is dissolved in system 2 with a mass-volume fraction of 26.67% g / mL, and the fraction containing a pair of dihydroflavonoid epimers is obtained by secondary high-speed countercurrent chromatography separation.

[0011] This invention also provides a method for separating dihydroflavonoid monomers from the rhizomes of Sophora flavescens using high-speed countercurrent chromatography. Specifically, the method includes: thoroughly mixing petroleum ether, ethyl acetate, methanol, and water in a volume ratio of 6-8:6-8:4:2 to form a two-phase solvent system 3; fractionally dissolving the mixture containing a large amount of dihydroflavonoid compounds into system 3 at a mass-volume fraction of 2.25% g / mL; and separating the monomers by high-speed countercurrent chromatography to obtain two diastereomers, Alopecurone A and Alopecurone B.

[0012] Preferably, the settings parameters of the high-speed countercurrent chromatography include: a stationary phase pumping flow rate of 500~1000 mL / min, a mobile phase pumping flow rate of 30~100 mL / min, a pumping volume of mobile phase:stationary phase = 1:1, an HSCCC main unit rotation speed of 500~700 rpm, a temperature of 15~25℃, and a dual-wavelength detection range of 254~365 nm.

[0013] Preferably, the settings parameters of the high-speed countercurrent chromatography include: a stationary phase pumping flow rate of 500 mL / min, a mobile phase pumping flow rate of 40~60 mL / min, a main unit rotation speed of 600 rpm, a temperature of 20 ℃, and a dual-wavelength detection range of 254 nm and 365 nm.

[0014] Preferably, the volume ratio of petroleum ether, ethyl acetate, methanol and water in the two-phase solvent system 1 is 2:3:3:2.

[0015] Preferably, the volume ratio of petroleum ether, ethyl acetate, methanol and water in the two-phase solvent system 2 is 7:7:4:2.

[0016] Preferably, the volume ratio of petroleum ether, ethyl acetate, methanol and water in the two-phase solvent system 3 is 7:7:4:2.

[0017] The present invention also provides a mixture containing a pair of dihydroflavonoid epimers prepared by the above preparation method.

[0018] This invention also provides two dihydroflavonoid monomer compounds prepared by the above-described method, wherein the dihydroflavonoid monomer compounds are Alopecurone A and Alopecurone B, and the structural formulas of Alopecurone A and Alopecurone B are as follows:

[0019] .

[0020] The present invention also provides the use of the above-described mixture or the two dihydroflavonoid monomer compounds described above in the preparation of a drug for treating liver cancer.

[0021] The beneficial effects of this invention are:

[0022] This invention utilizes HSCCC linear elution technology to rapidly and efficiently separate and purify a large quantity of highly pure active dihydroflavonoid compounds from crude extracts of *Sophora flavescens* rhizomes. These fractions exhibit inhibitory effects on the growth of hepatocellular carcinoma cells MHCC97H. The method of this invention features short operation time, large separation volume, simple and rapid operation, and minimal loss of target components due to material adsorption during the separation process. Attached Figure Description

[0023] Figure 1 This is the first high-speed countercurrent chromatogram in this invention;

[0024] Figure 2 This is the chromatogram of the secondary high-speed countercurrent flow in this invention;

[0025] Figure 3 This is a high-speed countercurrent chromatogram of the three phases in this invention;

[0026] Figure 4 This is the high-performance liquid chromatography chromatogram of this invention;

[0027] Figure 5 This is a high-performance liquid chromatogram of the monomeric dihydroflavonoid in this invention (A is compound 1; B is compound 2).

[0028] Figure 6These are the results of single-drug cell activity and model evaluation in this invention (A is the effect of crude product, petroleum ether extract PE, ethyl acetate extract EA, and methanol blank control MeOH on cell activity; B is the effect of SAR02-2, SAR02-3, SAR02-5, SAR02-8, and SAR02-10 on cell activity).

[0029] Figure 7 The results of the synergistic drug anti-liver cancer cell activity and model evaluation in this invention are as follows: (A is the effect of SAR02-2, SAR02-3, SAR02-6-8, SAR02-8, and SAR02-10 on cell activity; B is the effect of SG, SAR02-6-2, SAR02-6-6, SAR02-21, SAR02-23, SAR02-5, and SAR02-6-7 on cell activity; C is the synergistic index heatmap). Detailed Implementation

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] Example 1: High-speed countercurrent chromatography separation of a mixture of epimeric strychnine.

[0034] This invention utilizes a SuperChrome-5L high-speed countercurrent chromatograph (manufactured by Jiangsu Jiangyin Countercurrent Technology Co., Ltd.) for sample separation and purification, an Agilent 1200 high-performance liquid chromatograph (manufactured by Agilent Technologies, Inc.) for sample purity analysis, an Agilent 1260 Infinity II-LC-MSD for molecular weight determination, and a Mercury Plus 400MHz nuclear magnetic resonance spectrometer (manufactured by Bruker Technologies, Inc.) for NMR data acquisition. The *Sophora flavescens* rhizomes used in this invention were collected from the western suburbs of Shihezi City, Xinjiang.

[0035] 1. Experimental Methods

[0036] (1) Set up a high-speed countercurrent chromatograph

[0037] The HSCCC has a total of 6 outlet ports and 1 inlet port. The outlet ports can be connected to the inlet port via external pipelines to switch between normal elution and cyclic elution modes.

[0038] (2) Preparation of extract samples

[0039] The dried bitter bean rhizomes were crushed and extracted three times with 95% ethanol under cold soaking. The extracts were combined, concentrated under reduced pressure, and evaporated to dryness (crude extract).

[0040] (3) Preparation of two-phase solvent system: Petroleum ether (4 L, AR), ethyl acetate (6 L, AR), methanol (6 L, AR) and water (4 L, AR) are thoroughly mixed in a ratio of 2:3:3:2 (system 1, v:v) and allowed to stand for separation; the supernatant (upper phase) is taken as the mobile phase and the lower clear liquid (lower phase) is taken as the stationary phase. The mobile phase and the stationary phase are two-phase solvent system 1.

[0041] (4) Preparation of sample solution

[0042] Dissolve 12 g of the crude extract of Sophora flavescens root and stem obtained in (2) in 80 mL of the above solvent system 1 (40 mL of each phase).

[0043] (5) High-speed countercurrent chromatography separation

[0044] The stationary phase of System 1 was pumped into the chromatographic column at a flow rate of 500 mL / min. After the stationary phase filled the entire column, the main unit was started and the rotation speed was set to 600 rpm. Once the set rotation speed was reached, the mobile phase of System 1 was pumped into the column at a flow rate of 60 mL / min. After the two phases reached dynamic equilibrium in the column, 80 mL of sample solution was injected into the column. The fractions containing each compound (AF) and the pumped stationary phase (G) were collected separately through the sample outlet. The fraction containing the target compound was evaporated under reduced pressure to obtain the crude fractions of each compound. The crude fractions were then injected again to separate the compounds. During the separation process, the fractions were detected using a UV detector with dual wavelengths of 254 nm and 365 nm. Data acquisition and processing were performed using a chromatography workstation.

[0045] (6) Preparation of two-phase solvent system

[0046] Petroleum ether (7 L, AR), ethyl acetate (7 L, AR), methanol (4 L, AR) and water (2 L, AR) were thoroughly mixed in a ratio of 7:7:4:2 (system 2, v:v) and allowed to stand for separation. The supernatant (upper phase) was taken as the mobile phase and the lower clear liquid (lower phase) was taken as the stationary phase. The mobile phase and the stationary phase constituted a two-phase solvent system 2.

[0047] (7) Preparation of crude sample solution

[0048] Take 8 g of the above coarse fraction G (stationary phase G) and dissolve it in 30 mL of the above solvent system 2 (15 mL each of the upper and lower phases).

[0049] (8) High-speed countercurrent chromatography separation

[0050] The stationary phase of System 2 was pumped into the column at a flow rate of 500 mL / min. After the stationary phase filled the entire column, the main unit was started and the rotation speed was set to 600 rpm. Once the set rotation speed was reached, the mobile phase of System 2 was pumped into the column at a flow rate of 40 mL / min. After the two phases reached dynamic equilibrium in the column, 50 mL of sample solution was injected into the column. The fraction was collected at the sample outlet based on the UV absorption signal. During the separation process, the fractions were detected using a UV detector at wavelengths of 254 nm and 365 nm, and the data were acquired and processed using a chromatography workstation.

[0051] 2. Experimental Results

[0052] The *Sophora flavescens* rhizome extract obtained in (2) was injected and separated by high-speed countercurrent chromatography (HSCLC), then evaporated under reduced pressure to obtain 7 crude fractions of AF. The results of the AF fractions separated by the HSCLC are as follows: Figure 1 As shown, the pumped phases are, in order: SAR02-1 (71.0-78.8 min, 540 mL), SAR02-2 (78.8-82.8 min, 240 mL), SAR02-3 (82.8-87.1 min, 258 mL), SAR02-4 (87.1-97.5 min, 627 mL), SAR02-5 (97.5-115.7 min, 1093 mL), SAR02-6 (115.5-129.2 min, 805 mL), and the pumped stationary phase G (SAR02-10, 5 L).

[0053] The fractions containing the above compounds were evaporated under reduced pressure to obtain crude fractions of each compound. The crude fractions of each compound were then re-injected. Fraction D was separated to obtain compound SAR02-6-2 (97.8-119.0 min, 1273 mL), fraction E was separated to obtain compound SG (124.3-241.1 min, 7005 mL), and fraction F was separated to obtain compound SAR02-6-6 (51.3-81.8 min, 1818 mL), as well as fractions SAR02-6-7 (81.8-125.9 min, 2646 mL) and SAR02-6-8 (125.9-178.5 min, 3159 mL).

[0054] Flow fraction G was separated by a high-speed countercurrent chromatography system. Flow fractions SAR02-7 (124.0–137.5 min, 540 mL) and SAR02-8 (137.5–176.8 min, 1572 mL) were collected by controlling the sample outlet based on the UV absorption signal. Flow fraction SAR02-8 contained a pair of dihydroflavonoid epimers. The high-speed countercurrent chromatogram is shown below. Figure 2 As shown, efficient separation of dihydroflavonoid compounds is achieved.

[0055] Example 2: High-speed countercurrent chromatography separation of sophorone monomers A and B

[0056] 1 Experimental Methods

[0057] (1) Structural verification of dihydroflavonoid epimers: A two-phase solvent system 3 was selected: petroleum ether (0.7 L, AR): ethyl acetate (0.7 L, AR): methanol (0.4 L, AR): water (0.2 L, AR) (v:v:v:v = 7:7:4:2). The supernatant (upper phase) was used as the mobile phase, and the lower clear liquid (lower phase) was used as the stationary phase. The mobile phase and the stationary phase constituted the two-phase solvent system 3. The epimer mixture of the obtained fraction SAR02-8 was separated at a flow rate of 10 mL / min and a rotation speed of 1200 rpm / min. 225.0 mg of fraction SAR02-8 was dissolved in 10 mL of the mobile phase of the above solvent system 3 and loaded onto the sample. The sample was received based on the ultraviolet chromatographic peaks observed by the high-speed countercurrent chromatogram.

[0058] (2) Purity analysis: The purity of the collected fractions was determined by high performance liquid chromatography. The chromatographic separation conditions were as follows: Agilent Eclipse XDB-C18 column (5 μm, 4.6 mm × 250 mm), column temperature 40℃, mobile phase acetonitrile / water system (40-100%, 0-20 min), flow rate 1 mL / min, and detection wavelength 254 nm.

[0059] (3) Mass spectrometry and nuclear magnetic resonance

[0060] Each fraction (5 mg) was dissolved in 500 μL of methanol, filtered through a 0.22 μm filter membrane, and then subjected to UV absorption and mass spectrometry data acquisition using LC-MS (Agilent 1260 Infinity II-LC-MSD). The chromatographic separation conditions were as follows: Agilent Poroshell EC-C18 column (2.7 μm, 3.0 mm × 50 mm), column temperature 40℃, mobile phase acetonitrile / water system (40-90%, 0-5 min), flow rate 0.8 mL / min, and detection at all wavelengths.

[0061] Take 20 mg of each fraction, dissolve it in 0.6 mL of deuterated acetone (Acetone-d6), and place it in an NMR tube. Collect the compounds using a nuclear magnetic resonance spectrometer (Brucker Mercury Plus 400 MHz). 1 H and 13 C NMR data.

[0062] 2. Experimental Results

[0063] The sample is received based on the UV chromatographic peaks of high-speed countercurrent chromatography (countercurrent chromatogram as shown). Figure 3 (As shown). Finally, two main component monomer compounds, 1 (163.6–205.3 min, 41.7 mL) and 2 (207.0–247.1 min, 40.1 mL), were obtained and named SAR02-21 and SAR02-23, respectively. The structural analysis results are as follows:

[0064] Compound SAR02-21 (80.0 mg) had a purity of 96.0% as determined by HPLC. Figure 4 The yield was 35.0%. Compound SAR02-21 was a yellow solid; ESI-MS m / z: 651.2 [M+H] + The compound has a molecular weight of 650 and a molecular formula of C1. 39 H 38 O9. The NMR data of this compound (Table 1, 400 MHz, Acetone-d6) are consistent with the data of compound Alopecurone A reported in the literature, confirming that compound SAR02-21 is Alopecurone A.

[0065] Compound SAR02-23 (48.2 mg) had a purity of 96.2% as determined by HPLC. Figure 4 The yield was 21.0%. Compound SAR02-23 was a pale yellow solid; ESI-MS m / z: 651.2 [M+H] + The compound's molecular weight is indicated to be 650, and its calculated molecular formula is C1. 39 H 38 O9. The NMR data of this compound are consistent with those of compound Alopecurone B reported in the literature, confirming that compound SAR02-23 is Alopecurone B.

[0066] The structures of the two compounds are as follows Figure 5As shown, compounds SAR02-21 and SAR02-23 are dihydroflavonoid epimers. In the structural formula, the hydroxyphenyl groups on the dihydrofuran ring are β and α, respectively; in the resulting compounds, the two phenyl groups on the dihydrofuran ring have opposite and same configurations, respectively.

[0067] Table 1. Nuclear magnetic resonance (NMR) spectra of compounds SAR02-21 (1) and SAR02-23 (2) (Acetone-d6)

[0068]

[0069] Application example: The synergistic effect of bitter bean root and rhizome extract with sorafenib in the anti-hepatocellular carcinoma activity

[0070] Using hepatocellular carcinoma cells MHCC97H as the research object, the individual and synergistic anti-hepatocellular carcinoma activities of substances isolated from the root and stem extract of Sophora flavescens were determined.

[0071] 1 Experimental Methods

[0072] (1) MTT assay for screening drug anti-hepatocellular carcinoma activity

[0073] The MTT assay uses 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide, a reducing agent for live cell metabolites, to detect cell viability and growth. MTT is a yellow compound and a hydrogen-accepting dye that acts on the respiratory chain in the mitochondria of live cells. Under the action of succinate dehydrogenase and cytochrome C, the tetrazolium ring cleaves, generating blue formazan crystals that deposit in the cells. The amount of formazan crystals generated is directly proportional to the number of live cells (dead cells lose this function). The generated formazan crystals can be dissolved with DMSO. The absorbance (OD) value at 490 nm is measured using an enzyme-linked immunosorbent assay (ELISA) monitor to determine the number of live cells. This method can be used to detect the cytotoxic activity of drugs on tumor cells; compared with the control group, the smaller the OD value, the stronger the cytotoxic activity of the drug.

[0074] Drug concentration settings and grouping: The final concentration of sorafenib was set at 5 μg / mL. Concentration gradients of 5, 10, and 20 μg / mL were set for 16 samples (crude product, petroleum ether extract PE, ethyl acetate extract EA, methanol blank control MeOH; 5 monomeric compounds SG, SAR02-6-2, SAR02-6-6, SAR02-21, SAR02-23 and 7 fractions SAR02-2, SAR02-3, SAR02-5, SAR02-8, SAR02-10, SAR02-6-8, SAR02-6-7). Based on preliminary experiments, crude extract concentrations with almost no cytotoxicity or inhibition rates less than 50% were selected as fixed values ​​to match different concentrations of sorafenib, and the drug was administered simultaneously.

[0075] Plating: When the cells reach approximately 70-80% confluence, add trypsin for digestion and then count the cells, adjusting the cell concentration to 5 x 10^6 cells / year. 4 Add 100 μL of cell culture to each well of a 96-well plate. Incubate overnight in a cell culture incubator, and administer the drug the next day.

[0076] Drug administration (monoclonal antibody): The monotherapy efficacy of *Sophora flavescens* extract samples (crude product, petroleum ether extract PE, ethyl acetate extract EA, methanol blank control MeOH, SAR02-2, SAR02-3, SAR02-5, SAR02-8, SAR02-10) was assessed. 100 μL of pre-prepared samples at different concentrations were added to 96-well plates, with final concentration gradients of 0, 1, 3, 11, 33, and 100 μg / mL. The final volume was 200 μL. Drug intervention was based on the final concentration. Each experiment had three replicates. After 48 h of incubation, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were cultured for another 4 h. The supernatant was aspirated with a 1 mL disposable syringe, and 150 μL of DMSO was added to dissolve the supernatant. The absorbance at 490 nm was then measured.

[0077] Synergistic Effect of Drug Addition: The synergistic effect of sorafenib (SF) on the extract samples (SAR02-2, SAR02-3, SAR02-5, SAR02-8, SAR02-10, SG, SAR02-6-2, SAR02-6-6, SAR02-21, SAR02-23, SAR02-6-8, SAR02-6-7) was detected by adding sorafenib (SF). 50 μL of different concentrations of single and dual-drug formulations were added to 96-well plates, with a blank complete culture medium (containing DMSO) added to the 0-concentration group, resulting in a final volume of 200 μL. The final concentration was used as the drug concentration for all drug interventions. Two replicates were set up for each experiment. After incubation for 48 h, 20 μL of 5 mg / mL MTT solution was added to each well and the cells were cultured for another 4 h. The supernatant was aspirated with a 1 mL disposable syringe, and 150 μL of DMSO was added to dissolve the supernatant. The absorbance at 490 nm was then measured.

[0078] Cell Viability (%) = (OD-treated group - OD-blank group) / (OD-control group - OD-blank group) * 100%

[0079] (2) Evaluation of drug combination effects

[0080] Four mathematical models in the Synergy Finder software were used to evaluate the synergistic index of combined drug use. Among them, the ZIP model was used to analyze the synergy score of each concentration combination. Orange represents the synergistic effect of the drug combination, and blue represents the additive / antagonistic effect of the drug combination.

[0081] To investigate the antitumor effect of the chemical components of Sophora flavescens extract combined with sorafenib, a synergistic experimental design was employed. For most crude extracts, the effective drug concentrations were set within the IC50 range, and proximal concentrations were used to observe the synergistic antitumor effect of crude extracts with sorafenib at different concentrations.

[0082] 2. Experimental Results

[0083] The cytotoxic activity assay results of the SAR fraction showed that ( Figure 6 The crude product, MeOH, SAR02-2, SAR02-3, and SAR02-10 showed no significant antitumor inhibitory effects, while other fragments effectively inhibited the growth of 97-h cell lines, and within a certain range, a dose-response relationship was observed with increasing drug concentration.

[0084] The effect of SAR samples combined with sorafenib on cell proliferation was investigated using MHCC97H hepatocellular carcinoma cells. Synergy Finder software was used for analysis, and four mathematical models—ZIP, Loewe, HSA, and Bliss—were used to score the synergistic index of the compound and drug. A Synergy Score > 5 indicated a certain synergistic effect at this dosage combination, with higher scores indicating stronger synergistic activity. Simultaneously, based on the MTT assay, Synergy Finder software calculated the corresponding drug sensitivity scores according to the IC50 values ​​of the compound and drug; higher scores indicated greater sensitivity of tumor cells to the compound.

[0085] MTT test results show ( Figure 7 At a concentration of 5 μg / mL, most samples showed no synergistic effect or an additive effect when used in combination with sorafenib (Synergy Score < 5). At a concentration of 10 μg / mL, only SAR02-21 (sobacterium ketone A) and SG showed significant synergistic effects when used in combination with sorafenib (Synergy Score > 5).

[0086] In summary, the HSCCC linear elution technique can rapidly and efficiently separate and purify a large number of high-purity dihydroflavonoid compounds from crude extracts of Sophora flavescens rhizomes, yielding a mixture of two dihydroflavonoid compounds, Alopecurone A and Alopecurone B. The monomeric dihydroflavonoids can then be further separated. Both the mixture and its monomeric components exhibit inhibitory effects on the growth of hepatocellular carcinoma cells MHCC97H.

[0087] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for separating dihydroflavonoids from the rhizomes of Sophora flavescens based on high-speed countercurrent chromatography, characterized in that, Includes the following steps: (1) Sample preparation: Take the root and rhizome of bitter bean, crush it, and extract it with 95% ethanol at least 3 times. Combine the extracts and concentrate them under reduced pressure and evaporate to dryness to obtain bitter bean root extract. (2) Initial high-speed countercurrent chromatography separation: Petroleum ether, ethyl acetate, methanol and water were thoroughly mixed in a volume ratio of 2~3:2~3:3:2 to form a two-phase solvent system 1; The above-mentioned Sophora flavescens root extract sample was dissolved in system 1 with a mass-volume fraction of 15% g / mL, and the stationary phase G and each crude fraction AF were obtained by initial high-speed countercurrent chromatography separation. The crude fraction AF was evaporated to dryness under reduced pressure and then injected again to separate the compounds. (3) Secondary high-speed countercurrent chromatography separation: Petroleum ether, ethyl acetate, methanol and water are thoroughly mixed in a volume ratio of 6~8:6~8:4:2 to form a two-phase solvent system 2; the above stationary phase G is dissolved in system 2 with a mass-volume fraction of 26.67% g / mL, and a mixture fraction containing a large amount of dihydroflavonoid compounds is obtained by high-speed countercurrent chromatography separation.

2. A method for separating dihydroflavonoid monomers from the rhizomes of Sophora flavescens based on high-speed countercurrent chromatography, characterized in that, Specifically, it includes: Petroleum ether, ethyl acetate, methanol, and water were thoroughly mixed in a volume ratio of 6-8:6-8:4:2 to form a two-phase solvent system 3. The mixture containing a large amount of dihydroflavonoids from claim 1 was fractionally dissolved in system 3 at a mass-volume fraction of 2.25% g / mL. Two epimeric monomers, Alopecurone A and Alopecurone B, were obtained by high-speed countercurrent chromatography.

3. The preparation method according to claim 1, characterized in that, The settings parameters for the high-speed countercurrent chromatography include: a stationary phase pumping flow rate of 500~1000 mL / min, a mobile phase pumping flow rate of 30~100 mL / min, a mobile phase:stationary phase ratio of 1:1, a main unit rotation speed of 500~700 rpm, a temperature of 15~25℃, and a dual-wavelength detection range of 254~365 nm.

4. The preparation method according to claim 3, characterized in that, The settings parameters for the high-speed countercurrent chromatography include: a stationary phase inlet flow rate of 500 mL / min, a mobile phase inlet flow rate of 40~60 mL / min, an HSCCC main unit rotation speed of 600 rpm, a temperature of 20 ℃, and dual wavelengths of 254 nm and 365 nm.

5. The preparation method according to claim 1, characterized in that, In the two-phase solvent system 1, the volume ratio of petroleum ether, ethyl acetate, methanol, and water is 2:3:3:

2.

6. The preparation method according to claim 1, characterized in that, In the two-phase solvent system 2, the volume ratio of petroleum ether, ethyl acetate, methanol, and water is 7:7:4:

2.

7. The preparation method according to claim 2, characterized in that, In the two-phase solvent system 3, the volume ratio of petroleum ether, ethyl acetate, methanol, and water is 7:7:4:

2.

8. A mixture containing a pair of dihydroflavonoid epimers prepared by the preparation method according to claim 1.

9. The two dihydroflavonoid monomer compounds prepared by the preparation method according to claim 2, characterized in that, The dihydroflavonoid monomer compounds are Alopecurone A and Alopecurone B, and the structural formulas of Alopecurone A and Alopecurone B are as follows: 。 10. The use of the mixture of claim 8 or the two dihydroflavonoid monomer compounds of claim 9 in the preparation of a drug for treating liver cancer.

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