Biflavone compound as well as extraction method and application thereof

Abstract

The invention belongs to the field of pharmaceutical chemicals, and relates to a biflavone compound as well as an extraction method and application thereof. The structural formula of the biflavone compound is as shown in formula 1: # imgabs0. The high-purity C-O-C bonding type biflavone compound is prepared through a series of steps of crushing, extraction, concentration, extraction, column chromatography, HPLC (High Performance Liquid Chromatography), drying and the like, and the anti-HCC activity of the compound is superior to that of reported C-C bond type amentotaxus biflavone and C-O-C type hinokiflavone; therefore, the compound has higher potential for preparing the hepatocellular carcinoma medicine.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemical industry and relates to a biflavonoid compound and an extraction method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) accounts for 75-85% of all primary liver cancer cases. Surgical resection or liver transplantation in the early stage of the disease is considered the most effective treatment strategy. However, in addition to local infiltration and multi-center growth in the liver, HCC also has the characteristics of peripheral vascular invasion and distant multi-organ metastasis, resulting in poor prognosis, with a postoperative recurrence rate of up to 80% and a 5-year survival rate of only 11%. Current treatment strategies focus on the use of multi-target tyrosine kinase inhibitors (such as sorafenib and canvatinib), anti-angiogenic drugs (such as bevacizumab and ramucirumab) and immune checkpoint inhibitors (such as atezolizumab and pembrolizumab) to delay tumor growth and metastasis. Unfortunately, since HCC is a multifocal disease with intra- and inter-tumor heterogeneity, the tumor microenvironment and genome and its transcription, translation and post-modification levels vary significantly from place to place, which partly explains the resistance of existing chemical and biological drugs.

[0003] Natural products have become an important source of lead compounds and privileged structures in drug development due to their multi-target regulation capabilities. The traditional Chinese medicine Selaginella is the dried whole herb of Selaginella Tamariscina (P.Beauv.) Spring or Selaginella pulvinata (Hook.&Grev.) Maxim. of the Selaginaceae family. It is pungent in taste and neutral in nature. It enters the liver and heart meridians. It is used raw to treat amenorrhea, lumps, traumatic injuries, abdominal pain, and asthma; and roasted charcoal is used to treat vomiting blood, blood in stool, hematuria, and rectal prolapse. Many studies at home and abroad have shown that the total extracts and different extraction fractions of Selaginella plants have a wide range of anti-cancer activities, involving lung cancer, prostate cancer, breast cancer, colorectal cancer, etc. For example, a 50% ethanol extract of Selaginella can inhibit the invasion of human lung cancer cells A549 and LLC by downregulating tumor migration-related proteins MMP-2 and MMP-3, and has in vivo activity; a high-concentration dark green Selaginella extract has a good inhibitory effect on human nasopharyngeal carcinoma cell TW03 by regulating the ratio of apoptosis-related proteins Bcl-2 and Bax; the benzene elution site of S. bryopteris water extract after silica gel column can inhibit the occurrence of 3,4-benzopyrene-induced lung adenomas and significantly reduce the proportion of mice with skin cancer induced by methylbenzanthracene and croton oil, as well as the number of tumors. However, to date, the anti-cancer research on the biflavonoid compounds identified in the whole plant of Selaginella has only focused on its major components, such as CC-bonded sotetsuflavone (SF), amentoflavone (AMF), heveaflavone (HEF), and COC-bonded hinokiflavone (HF), isocryptomerin (ISO) and cryptomerin. Moreover, the research and application of biflavonoid compounds derived from Selaginella plants in the field of HCC treatment are very limited. Only one COC-bonded cypress biflavonoid has been shown to exert anti-HCC effects in mice by activating the mitochondrial ROS / JNK / caspase pathway and inhibiting the NF / κB pathway.

[0004] There is little research on COC-type biflavonoids from Selaginella plants in the prior art. For example, patent CN105566271A relates to a biflavonoid compound extracted from Selaginella sphaerocephala, which is bonded by ring A and ring B with COC, and has anti-non-small cell lung cancer activity:

[0005]

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

[0007] Summary of the invention

[0008] The present invention aims to provide a novel COC-bonded Selaginella pulvinata biflavonoid compound and an extraction method thereof, and provide the use of the compound in preparing a drug for treating hepatocellular carcinoma.

[0009] In order to solve the above 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 biflavonoid compound is named Selaginella pulvinata biflavonoids.

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

[0014] S1. After crushing Selaginella pulverulosa, adding a solvent, heating under reflux for extraction, and standing to obtain a precipitate phase and a solution phase;

[0015] S2, filtering the solution phase, concentrating under reduced pressure, extracting and concentrating under reduced pressure twice to obtain a crude product of total flavonoids;

[0016] S3, eluting the crude total flavonoids by polyamide adsorption column chromatography, combining the eluates and concentrating under reduced pressure to obtain a solid;

[0017] S4. The solid is further purified by semi-preparative liquid phase to obtain the Selaginella pulvinata biflavonoid compound.

[0018] In a preferred embodiment, in the step S1, the Selaginella pulverulenta is crushed and then sieved.

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

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

[0021] The concentration of the ethanol aqueous solution is too low, and the yield of the prepared product is too low.

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

[0023] In one of the preferred embodiments, in step S1, the heating reflux extraction is: heating to boiling and then adjusting the temperature to stabilize at 120-150° C., extracting for 2-4 hours; the number of extractions is 2 times or more.

[0024] The temperature is too low and the extraction rate is low.

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

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

[0027] The extraction solvent has a great influence on the extracted substances and yield. Other extraction solvents or extraction with only petroleum ether or dichloromethane will have an impact.

[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, reverse phase C18 or gel.

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

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

[0031] In one preferred embodiment, in step S3, the eluates eluted with 55%-95% ethanol solution are combined and concentrated under reduced pressure.

[0032] Low concentrations of ethanol cannot elute the target substance, which means 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 system gradient solution, and the elution gradient is: 45%, 50%, 55%, 95% ethanol solution, and TLC monitoring.

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

[0035] The present invention also provides the use of the biflavonoid compounds in preparing drugs for resisting hepatocellular carcinoma.

[0036] In one preferred embodiment, the dosage form of the drug is: tablets, pills, capsules, granules, powders, powders, solutions, suspensions, ointments, liposomes, granules, oral preparations, injections, injections, targeted drug delivery injections, suppositories, patches, sprays, lotions, drops or ointments.

[0037] The present invention uses a scientifically feasible method to prepare the COC bonded biflavonoid compound with high purity through a series of steps such as crushing, extraction, concentration, extraction, column chromatography, HPLC, drying, etc. The feasibility and repeatability of the preparation process are high.

[0038] The anti-HCC activity of the biflavonoid compound of the present invention is superior to that of the reported CC-bond type aconitum biflavonoid and COC-bond type cypress biflavonoid, and thus the potential for use in the preparation of hepatocellular carcinoma drugs is guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the infrared result of COC bonded biflavonoid compounds;

[0040] Figure 2 The mass spectrometry results of COC bonded biflavonoids are shown;

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

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

[0043] Figure 5 It is the NMR signal attribution analysis of COC bonded biflavonoid compounds;

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

[0045] Figure 7 The images show the proliferation activity of COC-bonded biflavonoid compound PUF on human hepatocellular carcinoma cell lines HepG2 (left) and Huh-7 (right);

[0046] Figure 8 This is a graph showing the effect of COC-bonded biflavonoid compound PUF on the migration activity of human hepatocellular carcinoma cell line HepG2;

[0047] Fig. 9 This is a statistical diagram of apoptosis induced by COC-bonded biflavonoid compound PUF in human hepatocellular carcinoma cell line HepG2;

[0048] Fig.10 Figure 2 shows the tumor tissue of mice 21 days after administration of the COC-bonded biflavonoid compound (PUF, 60 mg / kg), total flavonoid extract (60 mg / kg), CC-bonded aconitum biflavonoid (AMF, 30 mg / kg and 60 mg / kg) and solvent in the first in vivo tumor inhibition experiment. Fig.10 A) and tumor tissue mass statistics ( Fig.10 B);

[0049] Fig.11 Figure 2 shows the tumor tissue of mice 21 days after administration of the COC-bonded biflavonoid compound (PUF, 20 mg / kg and 50 mg / kg), 5-fluorouracil (5-Fu, 20 mg / kg) and the vehicle in the second in vivo tumor inhibition experiment ( Fig.11 A); the tumor volume growth trend chart of the COC-bonded biflavonoid compound (PUF, 20 mg / kg and 50 mg / kg) and 5-fluorouracil (5-Fu, 20 mg / kg) after 21 days of administration ( Fig.11 B); Statistical graph of tumor tissue mass 21 days after administration ( Fig.11 C); (Note: n = 6, * indicates P < 0.05, with significant difference; ns indicates P > 0.05, with no significant difference). DETAILED DESCRIPTION

[0050] The present invention will be described in detail below in conjunction with the embodiments. It should be noted that the selected embodiments are only used to further illustrate the essential content and beneficial effects of the present invention in conjunction with the technical features of the claims of the present invention. The present invention is not limited by the experimental materials and methods used in the following specific embodiments, and the specific implementation method can be determined according to the above technical scheme and actual conditions. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0051] Example 1

[0052] Extraction and separation

[0053] Extraction method 1: Extraction with 85% ethanol-water system

[0054] The whole plant of Selaginella cuspidatum (2 kg) was crushed and the roots were retained, filtered through a sieve (pore size 1.0 / wire diameter 0.35 / mesh size 18), 10 times the volume of 85% volume concentration ethanol-water solution was added to the filtrate and soaked for 24 hours, heated to boiling with an electric heating mantle, then adjusted the temperature to a stable 120°C, refluxed for 2 hours, and the extract was cooled to room temperature. Add 15L of 85% ethanol-water solution and heat to boiling, reflux extraction for 2h, let it stand for 2 days to obtain two phases of precipitate and solution, filter with gauze, dry the precipitate and recover it for storage, take the filtrate and centrifuge at 4000rpm for 20min, filter it with gauze until the solution is completely clarified, and then concentrate the filtrate under reduced pressure. Extract with petroleum ether (4L×4 times) to wash away lipophilic impurities. Each time the extraction is performed, shake it thoroughly after adding petroleum ether and let it stand for 2h. Combine the extracts and extract with dichloromethane (4L) 4 times. Each time the extraction is performed, shake it thoroughly after adding dichloromethane and let it stand for 2h. Concentrate the extract under reduced pressure to complete dryness to obtain 183g of crude total flavonoids.

[0055] Extraction method 2: Extraction with anhydrous ethanol

[0056] The whole plant of Selaginella pulverulenta (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 size 18). 10 times the volume of anhydrous ethanol was added to the filtrate and soaked for 24 hours. The mixture was heated to boiling using an electric heating mantle and then the temperature was adjusted to a stable 150°C. The mixture was refluxed for 2 hours and the extract was cooled to room temperature. After adding 15L of anhydrous ethanol, heat to boiling, reflux extraction for 2h, let stand and cool to room temperature, add 15L of anhydrous ethanol again, heat and maintain boiling state, reflux extraction for 4h, let stand and settle for 2 days, obtain precipitate and solution phases, filter with gauze and take the solution, centrifuge at 4000rpm, 20min, reduce pressure and concentrate the filtrate of the above three extractions to complete dryness to obtain a crude extract, use petroleum ether extraction (4L×4 times) to wash and remove lipophilic impurities, each time after adding petroleum ether, shake thoroughly and let stand for 2h, combine the extracts, extract 4 times with dichloromethane (4L), each time after adding dichloromethane, shake thoroughly and let stand for 2h, reduce pressure and concentrate the extract to complete dryness to obtain 157g of crude total flavonoids.

[0057] Extraction method 3: Extraction method using invention patent CN104250240A

[0058] The whole herb of Selaginella cuspidatum (1kg) was crushed and the root was retained. After filtering with a sieve (pore size 1.0 / wire diameter 0.35 / mesh 18), 15kg of 95% ethanol-water solution was added, heated with an electric heating mantle and stabilized at 70°C, refluxed and extracted twice, each time for 2h, and allowed to stand for 2 days to obtain two phases of precipitate and solution. After filtering with gauze, the filtrate was centrifuged at 5000rpm for 15min until the solution was completely clarified and concentrated under reduced pressure to 2.5kg. Subsequently, equal volumes of petroleum ether and water were added for extraction 4 times. Each extraction was fully shaken and allowed to stand for 2h. The lower layer was removed and concentrated under reduced pressure to complete dryness to obtain 81g of crude total flavonoids.

[0059] Extraction method 4: Extraction method using invention patent CN105566271A

[0060] The whole plant of Selaginella cuspidatum (1kg) was crushed and the root was retained. After filtering with a sieve (pore size 1.0 / wire diameter 0.35 / mesh 18), 15kg of 70% ethanol-water solution was added, heated with an electric heating mantle and stabilized at 40-50°C, and low-temperature extraction was performed 3 times, each time for 2h. The mixture was allowed to stand and settle for 2 days to obtain two phases of precipitate and solution. After filtering with gauze, the filtrate was centrifuged at 4000rpm for 25min until the solution was completely clarified, and then concentrated under reduced pressure to prepare 2kg of ethanol extract. Subsequently, 20kg of ultrapure water was added, and after stirring evenly, equal volumes of petroleum ether, dichloromethane and ethyl acetate were used for extraction in turn. Each extraction was fully shaken and allowed to stand for 2h. The ethyl acetate extract was concentrated under reduced pressure to complete dryness to obtain 107g of crude extract.

[0061] The above four crude extracts or crude flavonoids are 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 filler, and 1500g of 300-mesh polyamide was loaded into the column, which was pre-soaked in anhydrous ethanol overnight and washed with ultrapure water the next day. The ethanol-water system was used as the mobile phase, and the elution gradient was: pure water, ethanol solution 15%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 95%, each gradient elution was 4 to 6 column volumes, and the TLC combined with iodine fumigation and 10% sulfuric acid-ethanol heating color development was used to monitor the flow fraction, and the TLC development condition was DCM: MeOH = 10: 0.7 (v / v). The fraction eluted after taking 55% ethanol solution was concentrated under reduced pressure and further purified by semi-preparative liquid phase. The chromatographic column used was: YMC-pack-ODS-A, 250×30mm, 15μm, room temperature, injection volume was 100μL, ethanol-water system was eluted with 45%, 50%, 55%, 95% gradient, and the COC-bonded Selaginella biflavonoid compound obtained by the method of extraction method 1 was 621mg, and the yield was 0.03105%; the COC-bonded Selaginella biflavonoid compound obtained by the method of extraction method 2 was 531mg, and the yield was 0.0354%; the COC-bonded Selaginella biflavonoid compound obtained by the method of extraction method 3 was 97mg, and the yield was 0.0097%; the COC-bonded Selaginella biflavonoid compound obtained by the method of extraction method 4 was 108mg, and the yield was 0.0108%. Therefore, the extraction method has a great influence on the type and yield of the obtained product. The obtained COC bonded Selaginella pulvinata biflavonoid compound has a structural formula as shown in Formula 1:

[0063]

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

[0065] 1H-NMR (TMS, CD3OD, 400MHz) δ3.90 (s, 3H), δ3.87 (s, 3H) for 7-OCH3 and / or 7″-OCH3 substitution. δ6.38 (d, J = 2.0 Hz, 1H, 6-H), δ6.75 (d, J = 2.0 Hz, 1H, 8-H), δ7.12 (s, 1H, 8″-H), δ6.92 (s, 1H, 3-H), δ6.93 (s, 1H, 3″-H), δ7.05 (d, J = 8. 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 are shown in Table 1.

[0068] Table 1 NMR data of C-OC bonded Selaginella pulvinata biflavonoids

[0069]

[0070]

[0071] 1 H-NMR suggests that compound 4 has 22 protons. 13C-NMR showed 32 carbon signals, four of which overlapped. Based on this, it was inferred that the molecular formula of the compound was C 32 H 22 O 10 The NMR signals are assigned as follows: Figure 5 shown.

[0072] Example 2

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

[0074] Experimental methods:

[0075] The cell viability assay kit was purchased from Dojindo Molecular Technologies (CK04). 3 HepG2 and Huh-7 cells were inoculated into 96-well plates and incubated for 48 hours with 0.5% DMSO (negative control group), different concentrations of 5-fluorouracil (positive control group) or different concentrations of crude total flavonoids of Selaginella pallida (TBF), CC-bonded aconitum biflavonoids (AMF), Robusta biflavonoids (ROF), COC-bonded cypress biflavonoids (HF) and Selaginella pallida biflavonoids (PUF) of the present invention (experimental group). Subsequently, CCK-8 working solution with a final concentration of 10% (v / v) was added, incubated at 37°C, 5% CO2 for 2 hours, and the absorbance value was detected at a wavelength of 450nm.

[0076] Data processing:

[0077] The cell proliferation inhibition ratio (IR) of each compound was calculated according to the measured absorbance value. The calculation formula is as follows:

[0078] IR%=[AC-AD] / [AC-AB]×100%

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

[0080] AD: Experimental group, absorbance of wells containing 0.5% DMSO, CCK-8, drugs, and culture medium with cells

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

[0082] GraphPad Prism 9 software was used to analyze the data and plot the graphs, and the half inhibitory concentration (IC) of each compound at 48 hours was calculated. 50 ).

[0083] Experimental results: IC values ​​of each monomer compound in the experimental group on HCC cells 50 (μM) See Table 2 and Figure 6 .

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

[0085]

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

[0087] Example 3

[0088] Plate cloning assay to detect proliferation ability of HepG2 and Huh-7

[0089] Experimental methods:

[0090] HepG2 and Huh-7 cells in the logarithmic growth phase were inoculated into 12-well plates at 1×103 / well. After culturing in a 37°C 5% CO2 incubator for 24 hours, 3 concentration gradients of low, medium and high concentrations of HF and PUF of the present invention were given according to IC50 for 5-10 days (the control group was 0.5% DMSO). After culturing, the culture medium was removed, washed once with PBS, 1 mL of 4% paraformaldehyde was added to each well for 15 minutes, then washed 2-3 times with PBS, and finally stained with 0.5% crystal violet stain for 5 minutes, washed 3 times with PBS, and the number of cell colonies was counted.

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

[0092] In the plate cloning experiment, HepG2 and Huh-7 cells were seeded in 6-well plates at the same density, and after 24 hours of adherent culture, they were given concentration gradients of cypress biflavonoids (HF) or selaginella biflavonoids (PUF) of the present invention and incubated for 5 days. The experimental results showed that compared with the blank control group without drug administration, PUF could effectively inhibit the formation of Huh-7 cell colonies at a concentration of 4 μM, and completely inhibited the formation of HepG2 cell colonies at a concentration of 8 μM. At these two concentrations, HF's inhibitory activity on the proliferation of HepG2 and Huh-7 cells was significantly weaker than that of PUF.

[0093] Example 3

[0094] Transwell assay to detect HepG2 migration ability

[0095] Experimental methods:

[0096] HepG2 cells in the logarithmic growth phase were digested with 1‰ trypsin and resuspended in serum-free DMEM medium. 8×104 cells were taken in 200μL serum-free medium, and 2.5μM and 5μM of the Selaginella pulvinata biflavonoid compound and 20μg / mL total flavonoid crude product were added respectively. After mixing, they were added to the upper chamber, with 0.5% DMSO as the control, and then 500μL of DMEM medium containing 10% fetal bovine serum was added to the lower chamber, and they were incubated together at 37°C and 5% CO2 for 20 hours. The old culture medium was discarded, 200μL of 4% paraformaldehyde was added to the lower chamber, fixed for 5min, and then stained with 0.1% crystal violet stain for 5min, PBS was washed to remove the floating color, and the photos were taken under a light microscope at a 40× field of view, and the crystal violet staining area was counted using ImageJ software.

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

[0098] Compared with the control group, the Selaginella pulvinata biflavonoid compound PUF of the present 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 the logarithmic growth phase were digested with 1‰ trypsin and inoculated into 6-well plates at 2×105 / well, and cultured at 37°C, 5% CO2 for 24h. Then, 5μM and 20μM of the selaginella biflavonoid compound and 20μg / mL crude total flavonoids were added to each well, respectively, and 0.5% DMSO was used as a control. After incubation for 24h, the old culture medium was discarded, washed once with PBS, digested with 1‰ trypsin and the cell suspension was collected, and centrifuged at 1000rpm for 3min. The supernatant was removed, washed twice with PBS, and 100μL Working staining solution (Elabscience, E-CK-A217) containing 5μL AnnexinV-APC and 5μL PI was added and resuspended, and stained for 30 minutes at 37°C in the dark. Finally, 300μL binding buffer was added and detected by flow cytometry.

[0103] The experimental results are as follows Fig. 9 As shown:

[0104] Compared with the control group, after intervention with the Selaginella pulvinata biflavonoid compound (PUF) described in the present invention, the proportion of cells in the early apoptotic stage increased by 1.3%, and the proportion of cells in the late apoptotic stage increased by 4.3%. After intervention with crude total flavonoids (TBF), the corresponding increases were 4.3% and 1.9%, respectively.

[0105] Example 5

[0106] Inhibits the growth of HepG2 cell subcutaneous transplanted tumors

[0107] Cell lines and experimental animals:

[0108] Human HCC cell line HepG2 was purchased from ATCC; 4-week-old BALB / c nude mice, weighing 18±2g (male), were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd. This animal experiment complies 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 School of Medicine of Hunan Normal University. The mice were kept in an SPF environment, 6 per cage, 25°C, 50±10% humidity, and light and dark cycles every 12h. They were adaptively fed for one week before the start of the experiment and given a standard diet.

[0109] Method for establishing subcutaneous transplant tumor model:

[0110] HepG2 cells in the logarithmic growth phase were digested and prepared to a concentration of 1×10 7 100mm / mL cell suspension was prepared and the cell suspension was aspirated with a 1mL (5# needle) syringe under sterile conditions and inoculated subcutaneously into the right upper limb of nude mice at a position 0.5cm below the mouse. Each mouse was injected with 0.2mL of cell suspension. 3 Then group them.

[0111] The in vivo tumor inhibition experiment was conducted twice independently to compare the in vivo activities of Selaginella pulvinata biflavonoids PUF prepared by different extraction processes (extraction method 1 and extraction method 2) and its comparison with total flavonoid crude TBF and CC-bonded biflavonoid compound AMF.

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

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

[0114] ② Low-dose group of C-C bonded aconitum biflavonoids (30 mg / kg);

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

[0116] ④ the COC-bonded Selaginella pulvinata 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 time (extraction method 2) set up 4 groups:

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

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

[0121] ③ The COC-bonded Selaginella pulvinata 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 was given the drug every other day, the other groups were given the drug continuously for 21 days. The nude mice were observed daily, and their body weight and tumor volume were measured. The long diameter L (mm) and short diameter W (mm) of the solid tumor nodules were measured with a vernier caliper, and the tumor volume (mm 3 )=1 / 2×L(mm)×W 2 (mm 2 ). 24 hours after the last administration, the tumor mass was removed and the tumor weight was weighed. The tumor inhibition rate was calculated as follows: tumor inhibition rate (%) = (1-tumor weight of experimental group / tumor weight of negative control group) × 100%. GraphPad Prism9 software was used to analyze the tumor volume, tumor weight and body weight data and plot them.

[0124] The experimental results are as follows Fig.10 and Fig.11 As shown:

[0125] Compared with the negative control group, the two batches of PUF prepared by different extraction processes could significantly inhibit tumor growth. Fig.10 A and 10B), the tumor inhibition rate of the COC-bonded Selaginella pulvinata biflavonoid compound PUF (60 mg / kg) group was 47.38%, the tumor inhibition rate of the total flavonoid crude product TBF (60 mg / kg) group was 17.80%, and the tumor inhibition rate of the CC-bonded Selaginella pulvinata biflavonoid high-dose (60 mg / kg) group was 4.63%. In the second in vivo tumor inhibition experiment ( Fig.11In the experiment, the COC-bonded Selaginella pulvinata biflavonoid compound PUF had a tumor inhibition rate of 53.49% and 59.05% in the low-dose (20 mg / kg) and high-dose (50 mg / kg) groups, respectively; the tumor inhibition rate of the positive control group 5-fluorouracil (20 mg / kg) group was 37.86%, indicating that the COC-bonded Selaginella pulvinata biflavonoid compound PUF of the present invention has a good anti-HCC effect in vivo.

[0126] The above specific embodiments have shown and described the extraction and separation method of the COC-bonded biflavonoid compound PUF and the anti-HCC activity of the present invention. It should be emphasized that the listed embodiments are exemplary, and ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A biflavonoid compound, characterized in that: Its structural formula is shown in Formula 1:

2. The method for extracting biflavonoid compounds according to claim 1, characterized in that: The following steps are involved: S1. After crushing Selaginella pulverulosa, adding a solvent, heating under reflux for extraction, and standing to obtain a precipitate phase and a solution phase; S2, filtering the solution phase, concentrating under reduced pressure, extracting and concentrating under reduced pressure twice to obtain a crude product of total flavonoids; S3, eluting the crude total flavonoids by polyamide adsorption column chromatography, combining the eluates and concentrating under reduced pressure to obtain a solid; S4. The solid is further purified by semi-preparative liquid phase to obtain the Selaginella pulvinata biflavonoid compound.

3. The extraction method according to claim 2, characterized in that In the step S1, the solvent is an ethanol aqueous solution, and the volume concentration of the ethanol aqueous solution is 85% or more; preferably, in the step S1, The mass volume ratio of Selaginella pulvinata to the solvent is 1:6-1:

15.

4. The extraction method according to claim 2, characterized in that In the step S1, the heating reflux extraction is: heating to boiling and then adjusting the temperature to stabilize at 120-150° C., extracting for 2-4 hours; the number of extractions is 2 times or more.

5. The extraction method according to claim 2, characterized in that In the step S2, the extraction is: The mixture was extracted with petroleum ether for 3-8 times, the extracts were combined, and then extracted with dichloromethane for 3-8 times.

6. The extraction method according to claim 2, characterized in that In the step S3, the filler of the polyamide adsorption column is 300-500 mesh, and the filler is any one of silica gel, reverse phase C18 or gel; the crude total flavonoids and the filler are mixed in a mass ratio of 1:3-1:5 and then added to the chromatographic column.

7. The extraction method according to claim 2, characterized in that In step S3, the elution is carried out by gradient elution in an ethanol-water system, and the gradient is: pure water, ethanol solution 15%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 95%, and monitored by TLC.

8. The extraction method according to claim 7, characterized in that In the step S3, the eluates eluted with the 55%-95% ethanol solution are combined and concentrated under reduced pressure.

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

10. Use of the biflavonoid compound according to claim 1 or the biflavonoid compound prepared by the preparation method according to any one of claims 2 to 9 in preparing drugs against hepatocellular carcinoma.

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