A biflavonoid compound, its extraction method and application

Abstract

The application belongs to the field of medicine and chemical industry, and relates to a biflavonoid compound and an extraction method and application thereof. A structural formula of the biflavonoid compound is as shown in formula 1: The biflavonoid compound is prepared through a series of steps such as crushing, extraction, concentration, extraction, column chromatography, HPLC and drying, and has high purity. The anti-HCC activity of the C-O-C bonded biflavonoid compound is better than that of reported C-C bonded amentoflavone and C-O-C bonded taxifolin, and therefore the biflavonoid compound has high potential for preparing hepatocellular carcinoma drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemical industry, and relates to a biflavonoid compound, its extraction method and application. Background Technology

[0002] Hepatocellular carcinoma (HCC) accounts for 75-85% of all primary liver cancer cases. Early surgical resection or liver transplantation is considered the most effective treatment strategy. However, in addition to local invasion and multicentric growth within the liver, HCC is characterized by peripheral vascular invasion and distant multi-organ metastasis, leading to a poor prognosis. The postoperative recurrence rate is as high as 80%, and the 5-year survival rate is only 11%. Current treatment strategies focus on using multi-target tyrosine kinase inhibitors (such as sorafenib and cinvatinib), anti-angiogenic drugs (such as bevacizumab and ramoxirumab), and immune checkpoint inhibitors (such as atezolizumab and pembrolizumab) to delay tumor growth and metastasis. Unfortunately, because HCC is a multifocal disease with intratumoral and intertumoral heterogeneity, the tumor microenvironment and genome, as well as its transcriptional, translational, and post-modification levels, vary significantly across different sites. This partially explains the resistance to existing chemotherapeutic and biologic drugs.

[0003] Natural products, due to their multi-target regulatory capabilities, have become an important source of lead compounds and proprietary structures in drug development. The traditional Chinese medicine *Selaginella tamariscina* (P. Beauv.) Spring or *Selaginella pulvinata* (Hook. & Grev.) Maxim., belonging to the Selaginellaceae family, is the dried whole herb. It has a pungent taste, neutral properties, and enters the liver and heart meridians. When used raw, it treats amenorrhea, abdominal masses, traumatic injuries, abdominal pain, and asthma; when charred, it treats hematemesis, hematochezia, hematuria, and rectal prolapse. Multiple studies in China and abroad have shown that total extracts and different fractions of Selaginella tamariscina possess broad anticancer activities, involving lung cancer, prostate cancer, breast cancer, and colorectal cancer. For example, the 50% ethanol extract of Selaginella tamariscina can inhibit the invasion of human lung cancer cells A549 and LLC by downregulating tumor migration-related proteins MMP-2 and MMP-3, and it also exhibits in vivo activity. High-concentration dark green Selaginella tamariscina extract has a good inhibitory effect on human nasopharyngeal carcinoma cells TW03 by regulating the ratio of apoptosis-related proteins Bcl-2 and Bax. The benzene elution fraction of S. bryopteris water extract after silica gel column chromatography can inhibit the occurrence of 3,4-benzo[a]pyrene-induced lung adenomas and significantly reduce the proportion and number of skin cancers induced by methylphenylpropane and croton oil in mice. However, to date, research on the anticancer properties of biflavonoids identified in the whole herb of Selaginella has focused only on its major components, such as C-C bonded sotetsuflavone (SF), amentoflavone (AMF), and heveaflavone (HEF), as well as COC bonded hinokiflavone (HF), isocryptomerin (ISO), and cryptomerin. Moreover, research and application of biflavonoids from Selaginella plants in the field of HCC treatment are very limited. Only one COC bonded hinokiflavone has been shown to exert anti-HCC effects in mice by activating the mitochondrial ROS / JNK / cysteine ​​pathway and inhibiting the NF / κB pathway.

[0004] Existing research on COC-type biflavonoids from Selaginella tamariscina is extremely limited. For example, the Ming Dynasty patent CN105566271A relates to a biflavonoid compound extracted from Selaginella tamariscina using COC-linked A-ring and B-ring, which exhibits anti-non-small cell lung cancer activity.

[0005]

[0006] And invention patent CN108553476A discloses a chemical synthesis method for a COC-bonded type of cypress biflavonoid derivative, which has an anti-breast cancer effect:

[0007] Summary of the Invention

[0008] The purpose of this invention is to provide a novel COC-bonded cushion-like Selaginella biflavonoid compound and its extraction method, and to provide its use in the preparation of drugs for treating hepatocellular carcinoma.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0010] A biflavonoid compound, the structural formula of which is shown in Formula 1:

[0011]

[0012] The name of the biflavonoid compound is Selaginella tamariscina biflavonoid.

[0013] This invention also provides a method for extracting biflavonoid compounds, comprising the following steps:

[0014] S1. After crushing the cushion-shaped Selaginella, add solvent, heat and reflux to extract, and let stand to obtain the precipitate phase and the solution phase;

[0015] S2. The solution phase is filtered, concentrated under reduced pressure, extracted, and then concentrated under reduced pressure again to obtain crude total flavonoids.

[0016] S3. The crude total flavonoids were eluted by polyamide adsorption column chromatography, and the eluents were combined and concentrated under reduced pressure to obtain a solid.

[0017] S4. The solid was further purified by semi-preparative liquid chromatography to obtain the cushion-like Selaginella biflavonoid compound.

[0018] In one preferred embodiment, in step S1, the cushion-shaped Selaginella is crushed and then sieved.

[0019] In one preferred embodiment, in step S1, the mesh size of the sieve is 10-30 mesh.

[0020] In one preferred embodiment, in step S1, the solvent is an aqueous ethanol solution with a volume concentration of 85% or higher.

[0021] If the concentration of the ethanol aqueous solution is too low, the yield of the prepared product will be too low.

[0022] In one preferred embodiment, in step S1, the mass-to-volume ratio of the pad-shaped Selaginella to the solvent is 1:6-1:15.

[0023] In one preferred embodiment, in step S1, the heating reflux extraction is performed by heating to boiling and then adjusting the temperature to a stable value of 120-150°C for 2-4 hours; the extraction is performed twice or more.

[0024] If the temperature is too low, the extraction rate will be low.

[0025] In one preferred embodiment, the settling time in step S1 is 2-5 days.

[0026] In one preferred embodiment, in step S2, the extraction is performed by: extracting with petroleum ether 3-8 times, combining the extracts, and then extracting with dichloromethane 3-8 times.

[0027] The extraction solvent has a significant impact on the extracted substances and yield; other extraction solvents or extraction with only petroleum ether or dichloromethane will have an effect.

[0028] In one preferred embodiment, in step S3, the filler of the polyamide adsorption column is 300-500 mesh, and the filler is any one of silica gel, reversed C18 or gel.

[0029] In one preferred embodiment, in step S3, the crude total flavonoids are mixed with the packing material at a mass ratio of 1:3 to 1:5 and then added to the chromatographic column.

[0030] In one preferred embodiment, in step S3, elution is performed using an ethanol-water gradient elution system, with the gradient being: pure water, 15%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 95%, monitored by TLC.

[0031] In one preferred embodiment, in step S3, the eluent obtained by eluting with 55%-95% ethanol solution is concentrated under reduced pressure.

[0032] Low concentrations of ethanol cannot elute the target substance, meaning that what is eluted at low concentrations are impurities that do not contain the target compound.

[0033] In one preferred embodiment, in step S4, the eluent used in the semi-preparative liquid phase purification is an ethanol-water gradient solution, with the elution gradient being: 45%, 50%, 55%, and 95% ethanol solution, monitored by TLC.

[0034] In one preferred embodiment, in step S4, the chromatographic column used for the semi-preparative liquid chromatography is: YMC-pack-ODS-A, 250×30mm, 15μm; the injection volume is 80-120μL.

[0035] This invention also provides the application of biflavonoid compounds in the preparation of drugs for treating hepatocellular carcinoma.

[0036] In one preferred embodiment, the dosage form of the drug is: tablet, pill, capsule, granule, powder, powder, solution, suspension, ointment, liposome, granule, oral preparation, injection, injection, targeted drug delivery injection, suppository, patch, spray, lotion, drop, or liniment.

[0037] This invention utilizes a scientifically feasible method to prepare high-purity COC-bonded biflavonoid compounds through a series of steps including pulverization, extraction, concentration, extraction, column chromatography, HPLC, and drying. The preparation process is highly feasible and reproducible.

[0038] The biflavonoid compounds of this invention exhibit superior anti-HCC activity compared to previously reported C-bonded paclitaxel biflavonoids and COC-bonded cypress biflavonoids, thus ensuring their potential for use in the preparation of hepatocellular carcinoma drugs. Attached Figure Description

[0039] Figure 1 Infrared results for COC-bonded biflavonoids;

[0040] Figure 2 Mass spectrometry results for COC-bonded biflavonoids;

[0041] Figure 3 For COC-bonded biflavonoids 1 H-NMR results;

[0042] Figure 4 For COC-bonded biflavonoids 13 C-NMR results;

[0043] Figure 5 NMR signal assignment analysis for COC-bonded biflavonoids;

[0044] Figure 6 The IC50 statistical graph of COC-bonded biflavonoid PUF against human hepatocellular carcinoma lines HepG2 and Huh-7;

[0045] Figure 7 Image showing the proliferative activity of COC-bonded biflavonoid PUF on human hepatocellular carcinoma cell lines HepG2 (left) and Huh-7 (right);

[0046] Figure 8 The effect of COC-bonded biflavonoid PUF on the migration activity of the human hepatocellular carcinoma line HepG2 is shown in the figure.

[0047] Figure 9 Statistical graph of apoptosis induced by COC-bonded biflavonoid PUF in the human hepatocellular carcinoma line HepG2;

[0048] Figure 10 This is a first in vivo tumor inhibition experiment, showing tumor tissue images of mice 21 days after administration of the COC-bonded biflavonoid compound (PUF, 60 mg / kg), total flavonoid extract (60 mg / kg), CC-bonded paclitaxel biflavonoid (AMF, 30 mg / kg and 60 mg / kg) and solvent. Figure 10 A) and tumor tissue quality statistics ( Figure 10 B);

[0049] Figure 11 For the second in vivo tumor suppression experiment, tumor tissue images of mice 21 days after administration of the COC-bonded biflavonoids (PUF, 20 mg / kg and 50 mg / kg), 5-fluorouracil (5-Fu, 20 mg / kg), and solvent are shown. Figure 11 A); The tumor volume growth trend chart after 21 days of administration of the COC-bonded biflavonoids (PUF, 20 mg / kg and 50 mg / kg) and 5-fluorouracil (5-Fu, 20 mg / kg). Figure 11 B); Statistical chart of tumor tissue quality 21 days after drug administration ( Figure 11 C); (Note: n = 6, * indicates P < 0.05, indicating a significant difference; ns indicates P > 0.05, indicating no significant difference). Detailed Implementation

[0050] The present invention will be described in detail below with reference to embodiments. It should be noted that the selected embodiments are only used to further illustrate the essence and beneficial effects of the present invention in conjunction with the technical features of the claims. The present invention is not limited to the experimental materials and methods used in the following specific embodiments, and specific implementation methods can be determined according to the foregoing technical solutions and actual conditions. Unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0051] Example 1

[0052] Extraction and separation

[0053] Extraction Method 1: Extraction was performed using an 85% ethanol-water system.

[0054] The whole herb of Selaginella materia var. ... Add 15L of 85% ethanol-water solution, heat to boiling, reflux for 2 hours, and allow to settle for 2 days to obtain two phases: precipitate and solution. Filter the precipitate through gauze, dry it, and collect it for storage. Centrifuge the filtrate at 4000 rpm for 20 minutes until the solution is completely clear, then filter it through gauze. Concentrate the filtrate under reduced pressure and extract with petroleum ether (4L × 4 times) to wash away lipophilic impurities. Each time, after adding petroleum ether, shake thoroughly and allow to stand for 2 hours. Combine the extracts and extract with dichloromethane (4L) 4 times. Each time, after adding dichloromethane, shake thoroughly and allow to stand for 2 hours. Concentrate the extract under reduced pressure until completely dry to obtain 183g of crude total flavonoids.

[0055] Extraction Method 2: Extraction with anhydrous ethanol

[0056] The whole herb of Selaginella materia var. materia (1.5 kg) was crushed and the roots were retained. The mixture was filtered through a sieve (pore size 1.0 / wire diameter 0.35 / mesh 18). Ten times the volume of anhydrous ethanol was added to the filtrate and soaked for 24 h. The mixture was heated to boiling using an electric heating mantle and then the temperature was adjusted to stabilize at 150 °C. The mixture was refluxed for 2 h and the extract was allowed to cool to room temperature. After adding 15L of anhydrous ethanol, the mixture was heated to boiling and refluxed for 2 hours. After cooling to room temperature, another 15L of anhydrous ethanol was added, and the mixture was heated and kept at boiling point for 4 hours. After settling for 2 days, a precipitate and a solution phase were obtained. The solution was filtered through gauze and centrifuged at 4000 rpm for 20 minutes. The filtrates from the three previous extractions were concentrated under reduced pressure until completely dry to obtain a crude extract. Petroleum ether was used for extraction (4L × 4 times) to wash away lipophilic impurities. Each time, after adding petroleum ether, the mixture was shaken thoroughly and allowed to stand for 2 hours. The extracts were combined and extracted 4 times with dichloromethane (4L). Each time, after adding dichloromethane, the mixture was shaken thoroughly and allowed to stand for 2 hours. The extracts were concentrated under reduced pressure until completely dry to obtain 157g of crude total flavonoids.

[0057] Extraction Method 3: The extraction method described in invention patent CN104250240A.

[0058] The whole herb of Selaginella materia var. materia (1 kg) was crushed, with the roots retained. After filtration through a sieve (pore size 1.0 / wire diameter 0.35 / mesh 18), 15 kg of 95% ethanol-water solution was added. The mixture was heated with a heating mantle and stabilized at 70°C, and extracted twice by reflux for 2 hours each time. After settling for 2 days, two phases of precipitate and solution were obtained. The filtrate was filtered through gauze and centrifuged at 5000 rpm for 15 minutes until the solution was completely clear. The solution was then concentrated under reduced pressure to 2.5 kg. Subsequently, an equal volume of petroleum ether and water was added for extraction four times. Each extraction was thoroughly shaken and allowed to stand for 2 hours. The lower layer was collected and concentrated under reduced pressure until completely dry to obtain 81 g of crude total flavonoids.

[0059] Extraction Method 4: The extraction method described in invention patent CN105566271A.

[0060] The whole herb of *Selaginella materia var. materia* (1 kg) was crushed, with the roots retained. After filtration through a sieve (pore size 1.0 / wire diameter 0.35 / mesh 18), 15 kg of 70% ethanol-water solution was added. The mixture was heated using a heating mantle and stabilized at 40-50℃ for three low-temperature extractions, each lasting 2 hours. After settling for 2 days, two phases of precipitate and solution were obtained. The filtrate was filtered through gauze and centrifuged at 4000 rpm for 25 minutes until the solution was completely clear. The solution was then concentrated under reduced pressure to prepare 2 kg of ethanol extract. Subsequently, 20 kg of ultrapure water was added, and the mixture was stirred evenly. Equal volumes of petroleum ether, dichloromethane, and ethyl acetate were used for extraction in sequence. Each extraction was thoroughly shaken and allowed to stand for 2 hours. The ethyl acetate extract was concentrated under reduced pressure until completely dry to obtain 107 g of crude extract.

[0061] The above four crude extracts or crude flavonoids were separated and purified as follows:

[0062] The crude total flavonoids obtained in 1.1 were dissolved in methanol and mixed with 320g of 100-mesh polyamide packing material. The mixture was then packed into a column with 1500g of 300-mesh polyamide, pre-soaked in anhydrous ethanol overnight, and washed with ultrapure water the next day. The ethanol-water system was used as the mobile phase, with the following elution gradients: pure water, 15%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 95% ethanol solution, eluting 4 to 6 column volumes for each gradient. TLC was used to monitor the fractions by iodine fumigation and heating with 10% sulfuric acid-ethanol. The TLC development conditions were DCM:MeOH = 10:0.7 (v / v). The fraction eluted from the 55% ethanol solution was concentrated under reduced pressure and further purified using semi-preparative liquid chromatography. The chromatographic column used was a YMC-pack-ODS-A, 250×30mm, 15μm column, at room temperature. The injection volume was 100μL. The ethanol-water system was eluted sequentially with gradients of 45%, 50%, 55%, and 95%. Extraction method 1 yielded 621 mg of COC-bonded cushion-type Selaginella biflavonoids, with a yield of 0.03105%; extraction method 2 yielded 531 mg of COC-bonded cushion-type Selaginella biflavonoids, with a yield of 0.0354%; extraction method 3 yielded 97 mg of COC-bonded cushion-type Selaginella biflavonoids, with a yield of 0.0097%; and extraction method 4 yielded 108 mg of COC-bonded cushion-type Selaginella biflavonoids, with a yield of 0.0108%. Therefore, the extraction method has a significant impact on the type and yield of the obtained product. The obtained COC-bonded cushion-like Selaginella biflavonoid compound has the structural formula shown in Formula 1:

[0063]

[0064] Structural characterization of COC-bonded cushion-like Selaginella biflavonoids was performed, and the infrared results are as follows: Figure 1 As shown, the result is: 3399(-OH), 2973, 2917(CH3), 1659(conjugated C=O), 1604, 1497, 1444, 1358, 834. Mass spectrometry results are as follows: Figure 2 As shown, the result is: ESI-MS m / z: 565 (M + -1), 566(M) + ). 1 H-NMR results are as follows Figure 3 As shown, 13 C-NMR results are as follows Figure 4 As shown. The result is:

[0065] 1¹H-NMR (TMS, C D3OD, 400MHz) δ 3.90 (s, 3H), δ 3.87 (s, 3H) substituted by 7-OCH3 and / or 7″-OCH3. δ 6.38 (d, J = 2.0Hz, 1H, 6-H), δ 6.75 (d, J = 2.0Hz, 1H, 8-H), δ 7.12 (s, 1H, 8″-H), δ 6.92 (s, 1H, 3-H), δ 6.93 (s, 1H, 3″-H), δ 7.05 (d, J = 8″-H). 8Hz, 2H, 3′, 5′-H), δ6.96 (d, J = 8.8Hz, 2H, 3″′, 5″′-H), δ8.02 (t, J = 8.8, 8.8Hz, 4, 2′, 6′, 2″′, 6″′-H), δ10.40 (s, 1H, 4″′-OH), δ12.87 (s, 1H), δ13.10 (s, 1H) should be 5-OH and / or 5″-OH. δ10.40 (s, 1H, 4″′-OH).

[0066] 13 C-NMR (DMSO-d6, 100MHz) δ: 182.1 (C-4″), 181.9 (C-4), 165.2 (C-7), 163.3 (C-2), 164.4 (C-2″), 158.0 (C-7″), 161 .4(C-4″′), 161.1(C-5), 152.3(C-5″), 160.6(C-4′), 157.2(C-9), 154.0(C-9″), 128.4(C-2′), 128.6(C-6″′, C-2″ ′, C-6′), 124.2(C-1′), 115.1(C-3′), 120.9(C-1″′), 115.9(C-3″′, C-5″′), 115.1(C-5′), 92.0(C-8″), 104.7(C-1 0), 105.2(C-10″), 104.1(C-3), 102.8(C-3″), 97.9(C-6), 124.8(C-6″), 92.7(C-8), 56.0(7-OMe), 56.7(7″-OMe).

[0067] The specific data is shown in Table 1.

[0068] Table 1. NMR data of C-OC bonded cushion-type Selaginella biflavonoids

[0069]

[0070]

[0071] 1 H-NMR indicates that compound 4 has 22 protons. 13C10-NMR showed 32 carbon signals, with four carbon signals overlapping. Based on this, the molecular formula of the compound is inferred to be C10. 32 H 22 O 10 NMR signal attribution is as follows: Figure 5 As shown.

[0072] Example 2

[0073] CCK-8 assay for viability of HepG2 and Huh-7 cells

[0074] Experimental methods:

[0075] The cell viability assay kit was purchased from Dojindo Molecular Technologies (CK04). 6 × 10⁶ cells were used. 3 HepG2 and Huh-7 cells were seeded into 96-well plates and incubated for 48 hours with 0.5% DMSO (negative control), different concentrations of 5-fluorouracil (positive control), or different concentrations of crude total flavonoids from *Selaginella tamariscina* (TBF), CC-bonded paclitaxel flavonoids (AMF), Robesta flavonoids (ROF), COC-bonded cypress flavonoids (HF), and the *Selaginella tamariscina* flavonoids described in this invention (PUF) (experimental group). Subsequently, CCK-8 working solution was added to a final concentration of 10% (v / v), and the plates were incubated at 37°C and 5% CO2 for 2 hours. The absorbance was measured at 450 nm.

[0076] Data processing:

[0077] The cell proliferation inhibition ratio (IR) of each compound was calculated based on the measured absorbance values, using the following formula:

[0078] IR%=[AC-AD] / [AC-AB]×100%

[0079] AB: Absorbance of wells containing 0.5% DMSO, CCK-8, and cell-free culture medium.

[0080] AD: Absorbance of wells in the experimental group containing 0.5% DMSO, CCK-8, drug, and cell-containing culture medium.

[0081] AC: Negative control group, absorbance of wells containing 0.5% DMSO, CCK-8, and cell-containing culture medium.

[0082] Data were analyzed and plotted using GraphPad Prism 9 software, and the half-maximal inhibitory concentration (IC50) of each compound at 48 hours was calculated. 50 ).

[0083] Experimental results: The effects of each monomeric compound in the experimental group on the IC50 of HCC cells 50 (μM) are shown in Table 2 and Figure 6 .

[0084] Table 2. Inhibitory effects of the compounds of this invention on HepG2 and Huh-7 cells (IC50) 50 )

[0085]

[0086] Experimental results show that the compound (PUF) described in this invention has a good inhibitory effect on the proliferation of various HCC cells in vitro, and its effect is significantly better than that of the chemotherapy drugs 5-fluorouracil (5-Fu), crude total flavonoids (TBF), CC-bonded paclitaxel flavonoids (AMF), and COC-bonded cypress flavonoids (HF).

[0087] Example 3

[0088] Plate cloning assay to detect the proliferation capacity of HepG2 and Huh-7.

[0089] Experimental methods:

[0090] HepG2 and Huh-7 cells in logarithmic growth phase were seeded at 1×10³ cells / well into 12-well plates. After incubation at 37°C and 5% CO₂ for 24 hours, the cells were treated with low, medium, and high concentrations of fructan biflavonoids (HF) and purslane biflavonoids (PUF) as described in this invention for 5-10 days (control group: 0.5% DMSO). After culture, the culture medium was removed, the cells were washed once with PBS, and 1 mL of 4% paraformaldehyde was added to each well for fixation for 15 min. The cells were then washed 2-3 times with PBS, and finally stained with 0.5% crystal violet for 5 min. After washing three times with PBS, the number of cell colonies was counted.

[0091] Experimental results are as follows Figure 7 As shown:

[0092] In the plate cloning experiment, HepG2 and Huh-7 cells were seeded at the same density in 6-well plates and cultured adherently for 24 h. Afterward, they were co-incubated for 5 days with either fructan flavonoids (HF) or the cushion-shaped selaginella flavonoids (PUF) described in this invention at concentration gradients. The results showed that, compared to the untreated control group, PUF at a concentration of 4 μM effectively inhibited Huh-7 cell colony formation, while at a concentration of 8 μM, PUF completely inhibited HepG2 cell colony formation. At both concentrations, HF showed significantly weaker inhibitory activity against the proliferation of both HepG2 and Huh-7 cells than PUF.

[0093] Example 3

[0094] Transwell assays were used to assess the migration ability of HepG2.

[0095] Experimental methods:

[0096] HepG2 cells in logarithmic growth phase were digested with 1‰ trypsin and resuspended in serum-free DMEM medium. 8 × 10⁴ cells were added to 200 μL of serum-free medium, along with 2.5 μM and 5 μM of the described *Selaginella tamariscina* biflavonoid compound and 20 μg / mL of crude total flavonoids. The mixture was added to the upper chamber, with 0.5% DMSO as a control. Then, 500 μL of DMEM medium containing 10% fetal bovine serum was added to the lower chamber, and the cells were incubated at 37°C and 5% CO₂ for 20 hours. The old medium was discarded, and 200 μL of 4% paraformaldehyde was added to the lower chamber for fixation for 5 min. The cells were then stained with 0.1% crystal violet for 5 min, washed with PBS to remove excess stain, and photographed under a light microscope at 40× field of view. The crystal violet staining area was counted using ImageJ software.

[0097] Experimental results are as follows Figure 8 As shown:

[0098] Compared to the control group, the PUF compound of Selaginella tamariscina described in this invention can inhibit the migration activity of HepG2 cells in a dose-dependent manner.

[0099] Example 4

[0100] Inducing apoptosis in HepG2 cells

[0101] Experimental methods:

[0102] HepG2 cells in logarithmic growth phase were digested with 1‰ trypsin and seeded at 2×10⁵ cells / well in 6-well plates. The cells were cultured at 37°C and 5% CO₂ for 24 h. Then, 5 μM and 20 μM of the described *Selaginella tamariscina* biflavonoid compound and 20 μg / mL of crude total flavonoids were added to each well, with 0.5% DMSO as a control. After co-incubation for 24 h, the old culture medium was discarded, the cells were washed once with PBS, digested with 1‰ trypsin, and the cell suspension was collected and centrifuged at 1000 rpm for 3 min. The supernatant was removed, the cells were washed twice with PBS, and resuspended in 100 μL of working staining solution (Elabscience, E-CK-A217) containing 5 μL Annexin V-APC and 5 μL PI. The cells were stained at 37°C in the dark for 30 min. Finally, 300 μL of binding buffer was added, and the cells were analyzed by flow cytometry.

[0103] Experimental results are as follows Figure 9 As shown:

[0104] Compared to the control group, intervention with the pufferfish biflavonoid compound (PUF) described in this invention increased the proportion of cells in the early apoptotic phase by 1.3% and the proportion of cells in the late apoptotic phase by 4.3%. Intervention with crude total flavonoids (TBF) resulted in corresponding increases of 4.3% and 1.9%, respectively.

[0105] Example 5

[0106] Inhibits the growth of HepG2 cell subcutaneous xenografts

[0107] Cell lines and laboratory animals:

[0108] Human HCC cell line HepG2 was purchased from ATCC; 4-week-old male BALB / c nude mice, weighing 18±2g, were purchased from Hunan Silek Jingda Laboratory Animal Co., Ltd. This animal experiment complied with the ethical requirements of the Chinese Association for Laboratory Animal Science, and the experimental protocol was approved by the Animal Ethics Committee of the Medical School of Hunan Normal University. The mice were housed in an SPF-grade environment throughout the experiment, with 6 mice per cage, at 25℃ and 50±10% humidity, with light and dark cycles every 12 hours. Mice were given a one-week acclimatization period before the experiment, fed a standard diet.

[0109] Methods for establishing subcutaneous xenograft tumor models:

[0110] HepG2 cells in the logarithmic growth phase were digested and prepared into a concentration of 1×10⁻⁶. 7 Cell suspension at 1 / mL was aseptically extracted using a 1mL syringe (5# needle) and subcutaneously injected into nude mice 0.2mL below the right upper limb, 0.5cm below the limb. The injection continued until the tumor reached 100mm in size. 3 Then they were grouped.

[0111] The in vivo tumor inhibition experiment was conducted independently twice to compare the in vivo activity of pad-shaped Selaginella biflavonoids PUF prepared by different extraction processes (extraction method 1 and extraction method 2) and their combination with crude total flavonoids TBF and CC-bonded biflavonoid compound AMF.

[0112] The first time (extraction method 1), 5 groups are set:

[0113] ① Total flavonoids crude product TBF group (60mg / kg);

[0114] ②C-C bonded paclitaxel flavonoid low-dose group (30mg / kg);

[0115] ③ High-dose group of C-C bonded paclitaxel flavonoids (60 mg / kg);

[0116] ④ The COC-bonded cushion-like Selaginella biflavonoid compound PUF group (60 mg / kg);

[0117] ⑤ Negative control group (physiological saline containing 1% DMSO and 20% PEG400, pH=7).

[0118] The second extraction method (method 2) sets up 4 groups:

[0119] ① Positive control group (5-fluorouracil, 20 mg / kg);

[0120] ②The COC-bonded cushion-like Selaginella biflavonoid compound PUF low-dose group (20 mg / kg);

[0121] ③ The COC-bonded cushion-like Selaginella biflavonoid compound PUF high-dose group (50 mg / kg);

[0122] ④ Negative control group (physiological saline containing 1% DMSO and 20% PEG400, pH=7).

[0123] Except for the positive control group, which received the drug every other day, all other groups received the drug continuously for 21 days. The nude mice were observed daily, and their weight and tumor volume were measured. The major diameter L (mm) and minor diameter W (mm) of the solid tumor nodule were measured using calipers, and the tumor volume (mm³) was also measured. 3 )=1 / 2×L(mm)×W 2 (mm 2 24 hours after the last administration, the tumor was dissected and weighed. The tumor inhibition rate was calculated as follows: Tumor inhibition rate (%) = (1 - tumor weight in experimental group / tumor weight in negative control group) × 100%. GraphPad Prism9 software was used to analyze and plot the tumor volume, tumor weight, and body weight data.

[0124] Experimental results are as follows Figure 10 and Figure 11 As shown:

[0125] Compared to the negative control group, both batches of PUF prepared using different extraction processes significantly inhibited tumor growth in the first in vivo tumor suppression experiment. Figure 10 In experiments A and 10B), the tumor inhibition rate was 47.38% in the COC-bonded Selaginella cushionii flavonoid compound PUF (60 mg / kg) group, 17.80% in the crude total flavonoid TBF (60 mg / kg) group, and 4.63% in the high-dose (60 mg / kg) CC-bonded Taxus wallichiana flavonoid group. In the second in vivo tumor inhibition experiment (… Figure 11In (A), (B), and (C), the tumor inhibition rates of the COC-bonded Selaginella cushion-like biflavonoid compound PUF in the low-dose (20 mg / kg) and high-dose (50 mg / kg) groups were 53.49% and 59.05%, respectively; the tumor inhibition rate in the positive control group (5-fluorouracil, 20 mg / kg) was 37.86%. This indicates that the COC-bonded Selaginella cushion-like biflavonoid compound PUF described in this invention has good in vivo anti-HCC activity.

[0126] The above specific embodiments have shown and described the extraction and separation method of the COC-bonded biflavonoid compound PUF and its anti-HCC activity. It should be emphasized that the listed embodiments are exemplary, and those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of a biflavonoid compound in the manufacture of a medicament for the treatment of hepatocellular carcinoma, characterized in that, The structural formula of the biflavonoid compound is shown as formula 1: Formula 1 The liver cancer cells are HepG2 or Huh-7 cells.

2. Use according to claim 1, characterized in that, The extraction method of the biflavonoid compound comprises the following steps: S1, crushing the cushion-like Selaginella, adding a solvent and heating to reflux extraction, and standing to obtain a precipitate phase and a solution phase; S2, filtering the solution phase, reducing pressure concentration, extraction, and secondary reducing pressure concentration to obtain total flavone crude product; S3, using polyamide adsorption column chromatography to elute the total flavone crude product, and combining the eluent to reduce pressure concentration to obtain a solid; S4, using semi-preparative liquid phase to further purify the solid to obtain the cushion-like Selaginella biflavonoid compound; In the step S1, the solvent is an ethanol aqueous solution, and the volume concentration of the ethanol aqueous solution is 85% or above; In the step S1, the mass-volume ratio of the cushion-like Selaginella and the solvent is 1:6-1:15; In the step S1, the heating reflux extraction is: heating to boiling, adjusting the temperature to be stable at 120-150 DEG C, and extracting for 2-4 hours; the extraction times are 2 times or above; In the step S2, the extraction is: using petroleum ether to extract 3-8 times, combining the extraction liquid, and then using dichloromethane to extract 3-8 times; In the step S3, the filler of the polyamide adsorption column is 300-500 mesh, and the filler is any one of silica gel, reversed phase C18 or gel; the total flavone crude product is mixed with the filler at a mass ratio of 1:3-1:5, and then added to the chromatographic column; In the step S3, the elution is gradient elution with an ethanol-water system, and the gradient is in turn: pure water, 15% ethanol solution, 25% ethanol solution, 30% ethanol solution, 45% ethanol solution, 50% ethanol solution, 55% ethanol solution, 60% ethanol solution, 65% ethanol solution, 70% ethanol solution, 95% ethanol solution, and TLC monitoring; In the step S3, the eluent of the elution of the 55%-95% ethanol solution is reduced pressure concentrated.

3. Use according to claim 2, characterized in that, In the step S4, the eluent used in the semi-preparative liquid phase purification is an ethanol-water system gradient solution, and the elution gradient is in turn: 45% ethanol solution, 50% ethanol solution, 55% ethanol solution, and 95% ethanol solution, and TLC monitoring; the chromatographic column used in the semi-preparative liquid phase is YMC-pack-ODS-A, 250*30mm, 15μm; and the sample injection amount is 80-120μL.

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