Cycloartane saponins, extraction and purification method and application thereof

Abstract

This invention relates to the field of antitumor compound technology, providing a cyclic alphatin saponin compound, its extraction and purification method, and its applications. This invention is the first to extract, isolate, and identify a cyclic alphatin saponin compound (aspleniumside J) from the traditional Chinese medicine *Fernonia acutissima*. In vitro antitumor studies revealed that aspleniumside J can inhibit the proliferation of MDA-MB-231, HCC-1937, HeLa, and HepG2 cell lines, especially showing high activity against HepG2 liver cancer cells. Simultaneously, aspleniumside J can inhibit cell cycle arrest in the S phase of HepG2 liver cancer cells. In summary, the novel cyclic alphatin saponin compound provided by this invention exhibits high antitumor activity and has broad application prospects in the field of antitumor drugs, especially in the field of anti-liver cancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of antitumor compound technology, specifically relating to a cycloartin saponin compound, its extraction and purification method, and its application. Background Technology

[0002] Tumors are broadly classified into benign and malignant tumors. Malignant tumors, or cancer, are highly lethal due to their rapid growth, strong proliferative capacity, and high rate of metastasis. Currently, over 60% of anti-tumor drugs are derived from natural products, which have provided an important source for the discovery of chemotherapy drugs.

[0003] Ferns ( Asplenium ruprechtii Sa. Kurata, also known as Rejuvenating Grass, Madeng Grass, Madeng Grass, Guoqiao Grass, and Mati Grass, is the whole above-ground herb of Asplenium, a plant belonging to the genus Asplenium in the family Aspleniaceae (see Yan Zhongkai, Illustrated Catalogue of Medicinal Plants of Changbai Mountain, China, Beijing: People's Medical Publishing House, 1997: 96). It is used in folk medicine to treat thromboangiitis obliterans, hemiplegia, uterine bleeding, traumatic bleeding, neurodermatitis, and lower extremity ulcers. In Henan province, it is used to treat advanced liver cancer (see Yang Xinbao et al., Research Progress on Chemical Constituents and Pharmacological Activities of Asplenium, Modern Chinese Materia Medica, 2012, 14(5): 18). Modern pharmacological studies have shown that *Pteris vittata* has significant inhibitory activity against lung cancer (see Liu et al., Anti-lung cancer effect of total flavonoids from *Pteris vittata* and its inhibitory activity on lysine oxidase, Chinese Traditional and Herbal Drugs, 2014, 45(24):3573-3578). Its unique active components have been shown to have significant antitumor activity. For example, aspleniumside C, a cycloartin-type saponin-9,19-secreting-9,11-ene derivative extracted from *Pteris vittata*, can significantly inhibit the proliferation of liver cancer cells (see Fang Wang et al. Structural determination and...). in vitro tumorcytotoxicity evaluation of five new cycloartane glycosides from Asplenium ruprechtii Sa. Kurata, Bioorganic Chemistry, 2020, 102: 104085).

[0004] However, there are still relatively few compounds with antitumor activity extracted from *Dryopteris crassirhizoma*. Therefore, it is of great significance to search for novel compounds with excellent antitumor activity from *Dryopteris crassirhizoma*. Summary of the Invention

[0005] In view of this, the present invention provides a cyclic alpha-saccharide saponin compound, its extraction and purification method, and its application. The cyclic alpha-saccharide saponin compound provided by the present invention is a novel compound extracted from the traditional Chinese medicine *Fernonia acutissima*, and possesses excellent antitumor activity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A cycloartin saponin compound having the structure shown in Formula I:

[0007] Formula I This invention also provides a method for extracting and purifying the cyclic alpha saponins described in the above-mentioned scheme, comprising the following steps: The whole herb of *Dryopteris crassirhizoma* was extracted with ethanol, and the resulting extract was concentrated to obtain a paste. The extract was mixed with water to obtain an extract solution; the extract solution was extracted with ethyl acetate to obtain ethyl acetate and aqueous components. The aqueous phase was separated by macroporous adsorption resin chromatography to obtain four fractions, which were named A, B, C, and D in the order of elution. The D segment was separated by silica gel column chromatography to obtain 10 components, which were denoted as F1 to F10 in the order of elution. The F3 was separated by reversed-phase chromatography to obtain 18 components, which were named F3-1 to F3-18 in the order of elution. The F3-16 was purified by preparative high performance liquid chromatography to obtain cyclic alpha saponin compounds with the structure shown in Formula I.

[0008] Preferably, the volume fraction of ethanol is 50% to 95%, the extraction is reflux extraction, and the extraction is performed 3 to 5 times, with each extraction lasting 2 to 3 hours.

[0009] Preferably, the concentration method is vacuum concentration, and the vacuum concentration temperature is 40~55℃.

[0010] Preferably, the eluent used in the macroporous adsorption resin chromatography separation is ethanol and water; the elution method of the macroporous adsorption resin chromatography separation is gradient elution, in which the volume ratio of ethanol to water varies from 0:100 to 95:0.

[0011] Preferably, the eluent used in the silica gel column chromatography separation is 95% ethanol and ethyl acetate; the elution method of the silica gel column chromatography separation is gradient elution, in which the volume ratio of 95% ethanol and ethyl acetate changes from 1:0 to 0:1.

[0012] Preferably, the macroporous resin used in the macroporous adsorption resin chromatography separation is MCI macroporous adsorption resin; the silica gel used in the silica gel column chromatography separation has a mesh size of 100-200 mesh; and the chromatographic column used in the reversed-phase column chromatography separation is C14. 18 ODS medium pressure column.

[0013] Preferably, the eluent used in the reversed-phase column chromatography separation is an aqueous methanol solution; the elution method of the reversed-phase column chromatography separation is gradient elution, in which the volume fraction of methanol in the aqueous methanol solution changes from 15% to 100%.

[0014] Preferably, the mobile phase used in the preparative high-performance liquid chromatography purification is a methanol-water solution, wherein the volume ratio of methanol to water in the methanol-water solution is 50:50 to 90:10; and the flow rate of the mobile phase is 1 to 5 mL / min.

[0015] The present invention also provides the application of the cyclic alphatin saponins described in the above scheme or the cyclic alphatin saponins obtained by the extraction and purification method described in the above scheme in the preparation of antitumor drugs.

[0016] Preferably, the antitumor drug is an anti-liver cancer drug, an anti-breast cancer drug, or an anti-cervical cancer drug.

[0017] This invention provides a cyclic alpha saponin compound (denoted as aspleniumside J) having the structure shown in Formula I. This invention is the first to extract, isolate, and identify aspleniumside J from the traditional Chinese medicine *Fernonia acutissima*, which exhibits significant antitumor activity. In vitro antitumor studies revealed that aspleniumside J can inhibit the proliferation of MDA-MB-231, HCC-1937, HeLa, and HepG2 cell lines. Its inhibitory activity on HepG2 cell proliferation is superior to that of the positive control drug cisplatin, with an IC50 concentration of [missing value]. 50 The concentration was only 1.89 ± 0.03 μM; therefore, compound aspleniumside J has potential for development into a drug for treating breast cancer, liver cancer, and cervical cancer. Furthermore, this invention investigated the effects of aspleniumside J on apoptosis and cell cycle in HepG2 liver cancer cells. The results showed that aspleniumside J inhibited cell cycle arrest in the S phase, with the highest apoptosis rate at a concentration of 10 μmol / L. Therefore, the novel cyclic alpha-altin saponin compound provided by this invention exhibits high antitumor activity and has broad application prospects in the field of antitumor drugs, especially in anti-liver cancer drugs.

[0018] This invention also provides a method for extracting and purifying the cyclic alpha-altin saponins described in the above-mentioned scheme. The invention first extracts the whole herb of *Dryopteris crassirhizoma* with ethanol, then extracts with ethyl acetate to obtain ethyl acetate and water fractions. Subsequently, the cyclic alpha-altin saponins are obtained by macroporous adsorption resin chromatography, silica gel column chromatography, reversed-phase column chromatography, and preparative high-performance liquid chromatography. The extraction and purification method provided by this invention is simple and easy to operate. Attached Figure Description

[0019] Figure 1 HRESIMS spectra of the cycloartin saponins prepared in Example 1; Figure 2 The cyclic alpha saponins prepared in Example 1 1 1H NMR spectrum (600MHz, Pyridine- d 5); Figure 3 The cyclic alpha saponins prepared in Example 1 13 C10 NMR spectrum (150 MHz, Pyridine- d 5); Figure 4 DEPT spectrum (600 MHz, Pyridine-) of the cycloartin saponins prepared in Example 1 d 5); Figure 5 The cyclic alpha saponins prepared in Example 1 1 H- 1 H COSY spectrum (600MHz, Pyridine- d 5); Figure 6 HSQC spectra (600 MHz, Pyridine-) of the cycloartin saponins prepared in Example 1 d 5); Figure 7 HMBC spectrum (600 MHz, Pyridine-) of the cycloartin saponins prepared in Example 1 d 5); Figure 8 NOESY spectrum (600 MHz, Pyridine-) of the cycloartin saponins prepared in Example 1 d 5); Figure 9 This is a flow cytometry plot showing the effect of different concentrations of cycloaltin saponins on the HepG2 cell cycle in Example 3. Figure 10This is a flow cytometry diagram showing the effect of cyclic alphatin saponins at different concentrations on apoptosis in HepG2 cells in Example 4. Detailed Implementation

[0020] This invention provides a cyclic alpha saponin compound having the structure shown in Formula I:

[0021] Formula I.

[0022] This invention also provides a method for extracting and purifying the cyclic alpha saponins described in the above-mentioned scheme, comprising the following steps: The whole herb of *Dryopteris crassirhizoma* was extracted with ethanol, and the resulting extract was concentrated to obtain a paste. The extract was mixed with water to obtain an extract solution; the extract solution was extracted with ethyl acetate to obtain two fractions: ethyl acetate and an aqueous phase. The aqueous phase was separated by macroporous adsorption resin chromatography to obtain four fractions, which were named A, B, C, and D in the order of elution. The D segment was separated by silica gel column chromatography to obtain 10 components, which were denoted as F1 to F10 in the order of elution. The F3 was separated by reversed-phase chromatography to obtain 18 components, which were named F3-1 to F3-18 in the order of elution. The F3-16 was purified by preparative high performance liquid chromatography to obtain cyclic alpha saponin compounds with the structure shown in Formula I.

[0023] This invention uses ethanol to extract the whole herb of *Dryopteris crassirhizoma*, and concentrates the extract to obtain a paste. In this invention, the *Dryopteris crassirhizoma* is a plant of the family Acernaceae (family Acernaceae). Asplenium ruprechtii The aerial parts of Sa. Kurata; the volume fraction of ethanol is preferably 50-95%, and the extraction is preferably reflux extraction; the material-to-liquid ratio of Sa. Kurata to ethanol is preferably 1g:5 mL to 1g:20 mL; the number of extractions is preferably 3-5 times, and the extraction time for each extraction is preferably 2-3 hours.

[0024] In this invention, the concentration method is preferably vacuum concentration, and the vacuum concentration temperature is preferably 40~55℃; the vacuum concentration is preferably carried out using a rotary evaporator.

[0025] After obtaining the extract, the present invention mixes the extract with water to obtain an extract solution, and extracts the extract solution with ethyl acetate to obtain ethyl acetate and aqueous phase fractions.

[0026] After obtaining the aqueous phase fraction, the present invention performs macroporous adsorption resin chromatography separation on the aqueous phase fraction to obtain four fractions, which are designated as fractions A, B, C, and D in the order of elution. In the present invention, the eluent used in the macroporous adsorption resin chromatography separation is preferably ethanol and water; the elution method of the macroporous adsorption resin chromatography separation is gradient elution, in which the volume ratio of ethanol to water varies from 0:100 to 95:0, specifically 0:100, 3:7, 1:1, and 95:0; the macroporous adsorption resin used in the macroporous adsorption resin chromatography separation is preferably MCI macroporous adsorption resin.

[0027] After obtaining the distillation fraction, the present invention performs reversed-phase column chromatography and silica gel column chromatography to separate the D segment, obtaining 10 components, which are sequentially denoted as F1 to F10 according to the elution order of the components. In this invention, the eluent used in the silica gel column chromatography separation is preferably 95% ethanol and ethyl acetate; the elution method of the silica gel column chromatography separation is preferably gradient elution, in which the volume ratio of 95% ethanol and ethyl acetate in the gradient elution preferably varies from 1:0 to 0:1, specifically 1:0, 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1; the mesh size of the silica gel used in the silica gel column chromatography separation is preferably 100-200 mesh; in a specific embodiment of this invention, component D is preferably reconstituted with a mixed solvent of 95% ethanol and water, then mixed with silica gel, and then separated by silica gel column chromatography. The fractions in the separation process are identified by thin-layer chromatography (TLC), and the same fractions are combined; the developing solvent used in the thin-layer chromatography is preferably dichloromethane and methanol; the volume ratio of dichloromethane and methanol is preferably 12:1.

[0028] After obtaining the fraction, the F3 fraction is separated by reversed-phase chromatography to obtain 18 components, which are sequentially labeled F3-1 to F3-18 according to their elution order. In this invention, the chromatographic column used for the reversed-phase column separation is preferably a C14 column. 18 ODS medium-pressure column; the eluent used in the reversed-phase column chromatography separation is preferably an aqueous methanol solution; the elution method of the reversed-phase column chromatography separation is preferably gradient elution, wherein the volume fraction of methanol in the aqueous methanol solution during gradient elution preferably varies from 15% to 100%, specifically 15%, 30%, 50%, 70%, 90%, and 100%; in a specific embodiment of the present invention, the fractions obtained by reversed-phase column chromatography are preferably identified and segmented by thin-layer chromatography (TLC), and the same components are combined to finally obtain 18 components.

[0029] After obtaining component F3-16, the present invention purifies component F3-16 by preparative high-performance liquid chromatography (HPLC) to obtain cyclic alphatin saponins with the structure shown in Formula I. In the present invention, the mobile phase used for the preparative HPLC purification is preferably methanol-water solution, wherein the volume ratio of methanol to water solution is preferably 50:50 to 90:10; the flow rate of the mobile phase is preferably 1 to 5 mL / min; in a specific embodiment of the present invention, the retention time of the cyclic alphatin saponins with the structure shown in Formula I is 20.5 min.

[0030] This invention also provides the application of the cyclic alphatin saponins described in the above-described scheme or the cyclic alphatin saponins obtained by the extraction and purification methods described in the above-described scheme in the preparation of antitumor drugs; in this invention, the antitumor drugs are preferably anti-liver cancer drugs, anti-cervical cancer drugs, or anti-breast cancer drugs; the cyclic alphatin saponins provided by this invention have the activity of inhibiting liver cancer cells, cervical cancer cells, and breast cancer cells, especially with excellent anti-liver cancer activity, and have great development prospects in the development of anti-liver cancer drugs.

[0031] Example 1 The following embodiments will further illustrate the present invention, but do not limit the present invention.

[0032] (1) 17.4 kg of dried whole fern was extracted with 95% ethanol under reflux for 2 h each time. The extract was concentrated under reduced pressure at 50 °C using a rotary evaporator to obtain an extract (528.6 g). The extract was placed in 1.5 L of distilled water and extracted with ethyl acetate. The extraction was repeated 3 times to obtain ethyl acetate and aqueous fraction (97 g).

[0033] (2) The aqueous phase fraction was dissolved in 120 L of water and separated by macroporous resin column chromatography. The fractions were eluted with H2O (50 L), 30% EtOH (125 L), 50% EtOH (125 L) and 95% EtOH (100 L) to obtain four eluted fractions: A, B, C and D.

[0034] (3) Take 33.9 g of component D and redissolve it with 95% EtOH. Mix it with 100~200 mesh silica gel. After mixing, separate it by silica gel column chromatography (the eluent is 95% ethanol and ethyl acetate, and the volume ratio of 95% ethanol and ethyl acetate changes from 1:0 to 0:1, specifically 1:0, 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1). Identify it by thin layer chromatography (TLC) (the developing solvent is dichloromethane and methanol, and the volume ratio of dichloromethane and methanol is 12:1). Combine the same fractions to obtain 10 fraction segments, named F1~F10 segments.

[0035] (4) Take 7.6 g of F3 segment through C 18 ODS medium-pressure column (eluent is methanol aqueous solution, the volume fraction of methanol in the methanol aqueous solution varies from 15% to 100%, specifically 15%, 30%, 50%, 70%, 90%, 100%), TLC segmentation, obtained a total of 18 components from F3-1 to F3-18.

[0036] (5) The component F3-16 was purified by preparative high performance liquid chromatography. The mobile phase used for purification was methanol-water with a volume ratio of 65:35. Differential detection was performed at a flow rate of 2 mL / min. 29.8 mg of cycloaltin saponin compound (denoted as aspleniumside J) was obtained with a retention time of 20.5 min.

[0037] The structural identification of the cycloartin saponins obtained in this embodiment is as follows: Figure 1-8 As shown: aspleniumside J: white amorphous powder; high-resolution mass spectrometry (HRESIMS) yielded a quasi-molecular ion peak [MH]. - (m / z): 831.47644, and its molecular formula was determined to be C based on NMR data analysis. 42 H 73 O 16 .exist 1 In the H-NMR spectrum, two broad single peaks appear in the high-field region, located at... δ H 0.41, 0.19 (each 1H, br s), this should be attributed to the characteristic cyclopropane methylene proton (CH2-19, formula I). ​​In addition, there are 5 singlet methyl signals. δ H 1.76, 1.60, 1.58, 0.96 and 0.92, and in δ H 1.11 (d, J The presence of a bimodal methyl signal at 6.6 Hz suggests the presence of a cycloartane triterpenoid skeleton in the compound's structure (see Xu-YanWang et al. Cytotoxic 9,19-cycloartane type triterpenoid glycosides from the roots of Actaea dahurica , Phytochemistry, 2019, 160: 48-55). δ H The two double-split signals at 5.20 and 5.06 have coupling constants of... J = 7.8 Hz, and δ H The overlapping signal at 3.5–4.5 μm confirms the presence of two glycosyl groups. This information indicates that the compound is a cycloartin triterpenoid glycoside. 13 C-NMR and DEPT spectra show the carbon resonance signals corresponding to the above-mentioned units (Table 1). The NMR data of the compounds are compared with those of aspleniumside A~E previously isolated by our research group (see Fang Wang et al. Structural determination and in ex vitro tumor cytotoxicity evaluation of five new cycloartane glycosidesfrom Asplenium ruprechtii Sa. Kurata, Bioorganic Chemistry, 2020, 102: 104085), and similar to the artin triterpenoid glycosides isolated from the same plant species in Northeast my country (see Ning Li et al. New triterpene glycosides from Camptosorus sibiricus , Nat Prod Commun,2010, 5(10): 1557; Ning Li et al. New cycloartane glycosides from Camptosorus sibiricus Rupr., J Asian Nat Prod Res, 2008, 10(2): 119; Peng Zhang et al. Newcycloartane glycosides from Camptosorus sibiricus Rupr., J Asian Nat Prod Res,2008, 10(11): 1069; Ning Li et al. A new cycloartane glycoside from Camptosorus sibiricus Rupr., Nat Prod Commun, 2006, 20(12): 1041; Ning Li et al. A newcycloartane glycoside from Camptosorus sibiricus Rupr., J Asian Nat Prod Res, 2005, 7(2): 161). The compound's 13C-NMR data and known compounds 3,7,24,25,30-pentahydroxycycloartane-24- O - β Compared with -D-glucopyranoside, C-3, C-5-C-8, C-22, C-23, and C-30 were found to exist. δ The difference in size between C1 and C10 is in 1 H- 1 In H COSY, three spin-coupled systems CH2-CH2-CH(O); CH2-CH2-CH and CH2-CH2-CH-CH(CH3)-CH2-CH(O)-CH(O) indicate that the known compound contains an OH at position C-23 in addition to the OH at C-7. The final structure of the compound was determined using two-dimensional NMR. The hydrogen and carbon atoms of the compound were matched one-to-one using gHMQC experiments. In the HMBC spectrum, H-22a ( δ H 2.45) and C-23 ( δ C 67.7) is related, H3-26 / H3-27 is related to C-24 and C-25, confirming that the oxygen-bonded positions are at C-23, C-24 and C-25 of the branched chain. Additionally, H-3 ( δ H 3.78) and C-1′( δ C 106.2); and H-24 ( δ H 3.78) and C-1″ ( δ C The correlation with 107.0 indicates that one monosaccharide unit is linked at both C-3 and C-24. All glycosyl signals are similar to the glycosyl data of artin triterpenoid glycosides isolated from this plant reported in the literature, and are all... β -D-glucopyranosyl (see Fang Wang et al. Structural determination and in vitro tumorcytotoxicity evaluation of five new cycloartane glycosides from Asplenium ruprechtii Sa. Kurata, Bioorganic Chemistry, 2020, 102: 104085; Ning Li et al.New triterpene glycosides from Camptosorus sibiricus, Nat Prod Commun, 2010, 5(10): 1557; Ning Li et al. New cycloartane glycosides from Camptosorus sibiricus Rupr., J Asian Nat Prod Res, 2008, 10(2): 119; Peng Zhang et al. Newcycloartane glycosides from Camptosorus sibiricus Rupr., J Asian Nat Prod Res,2008, 10(11): 1069; Ning Li et al. A new cycloartane glycoside from Camptosorus sibiricus Rupr., Nat Prod Commun, 2006, 20(12): 1041; Ning Li et al. A newcycloartane glycoside from Camptosorus sibiricus Rupr., J Asian Nat Prod Res, 2005, 7(2): 161). This paper describes a co-TLC experiment using an aqueous phase obtained from acid hydrolysis of the compound and standard glucose [co-TLC, with chloroform-methanol 2:1 as the developing solvent, R]. f [The specific rotation value of the aqueous phase is 0.35, and the oxidative oxidation of the 5% sulfuric acid ethanol solution is black]. D 20 +50.3 (c 0.25, H2O) confirmed that all sugar groups in the compound are glucose groups. Therefore, the planar structure of the compound was determined to be 3- β -D-glucopyranosyl-24- O -β-D-glucopyranosyl-7,23,25,30-tetrahydroxyl-9,19-cycloartane. Since CH2-19 are both present in this type of structure... β Orientation was determined using 2D NOESY to establish the relative configuration of the compounds. H2-19 correlated with H2-30 and H-8, and H-8 correlated with H3-18, indicating that these protons are on the same plane as H2-19. β Orientation. H-3 correlates with H3-29 and H-5, H-5 correlates with H3-28; H3-28 correlates with H-17, indicating that these protons are on the other side of the plane. α Orientation. The cis configuration of H-23 and H-24 can be determined by the small coupling constant between them.J H-23 / H-24 = 3.0 Hz), therefore, the compound was identified as (23,24, cis )-3 β - O - β -D-glucopyranosyl-24- O - β -Dglucopyranosyl-7,23,25,30-tetrahydroxyl-9,19-cycloartane, commonly known as aspleniumside J.

[0038] Table 1 shows the obtained cyclic Altyn saponins. 1 H-NMR (600 MHz) and 13 C-NMR (150 MHz) in Pyridine- d NMR data in 5

[0039] a 1 H NMR data ( δ ) was measured at 600 MHz in pyridine- d 5. Protoncoupling constants ( J ) in Hz are given in parentheses. The assignments werebased on DEPT, 1 H- 1 H COSY, HSQC, and HMBC experiments; b the glycosyl grouplocated at C-3; c the glycosyl group located at C-24, respectively. Example 2 Antitumor activity experiment The cells used in this example were MDA-MB-231, HCC-1937, HeLa, and HepG2 cells. The compound to be tested was aspleniumside J, a cyclic alpha saponin compound prepared in Example 1. The experimental steps are as follows: Tumor cells in the logarithmic growth phase were selected, the old culture medium was discarded, and the cells were washed twice with 1 mL of PBS buffer. After digesting the cell clusters into single cells with 1 mL of trypsin cell digestion solution, 3 mL of cell culture medium was added to stop the digestion. After centrifugation for 5 min, the supernatant was discarded, and 1 mL of cell culture medium was added again to resuspend the precipitate into a single cell suspension and diluted to an appropriate cell concentration.

[0040] Using 96-well plates, 3000 log-phase cells (100 μL) were added to each well in the control and experimental groups, while only 100 μL of fresh cell culture medium was added to the blank group. Three replicates were set up for each of the control, experimental, and blank groups. After overnight cell growth, 100 μL of fresh cell culture medium was added to each well in the control group, 100 μL of the test compound diluted to final concentrations of 1 μmol / L, 5 μmol / L, 12.5 μmol / L, 25 μmol / L, and 50 μmol / L in each of the experimental groups, and only 100 μL of fresh culture medium was added to the blank group. After incubation at 37 °C with 5% CO2 for 24, 48, and 72 h, 20 μL of MTT (5 mg / mL) working solution was added to each group. The 96-well plates were incubated for 4 h, the supernatant was removed, and 200 μL of DMSO was added to dissolve the precipitate. The plates were then shaken at low speed at 37 °C for 10 minutes. The absorbance at 492 nm was measured using an ELISA reader.

[0041] Cell viability = (OD value of experimental group - OD value of blank group / OD value of control group - OD value of blank group) × 100%.

[0042] Table 2 shows the effect of aspleniumside J on tumor cell viability.

[0043] The results are averages of three independent experiments, and the data are expressed as means ± SD. As shown in Table 2, the cyclic alpha saponin compound aspleniumside J provided by this invention exhibits superior inhibitory activity against HepG2 cells compared to the positive control drug cisplatin, with an IC50 value of [missing value]. 50 The value was only 1.89±0.03 μM, indicating that it has significant inhibitory activity against the proliferation of liver cancer cells.

[0044] Example 3: Effects of different concentrations of cycloartin saponins on the HepG2 cell cycle HepG2 cells in logarithmic growth phase were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and 1 mL of cell culture medium was added and gently pipetted to form a homogeneous cell suspension. After cell counting under a microscope, the cells were divided into groups of 1×10⁻⁶ cells. 5 Cells were uniformly added to 6-well plates at a density of 2 mL / mL, and incubated overnight. The original culture medium was discarded, and the cells were washed twice with 1 mL PBS buffer. For the control group, 2 mL of cell culture medium was added, and for the drug groups, 2 mL of the drug solution diluted to final concentrations of 5 μmol / L and 10 μmol / L in cell culture medium was added. The plates were incubated at 37°C with 5% CO2 for 24 h. After incubation, the culture medium was discarded, and the cells were washed twice with 1 mL PBS buffer. Cells were then digested in each well with 500 μL of EDTA-free trypsin, and 1 mL of cell culture medium was added. The cells were gently pipetted and the cell suspension was transferred to 2 mL centrifuge tubes and centrifuged at 2000 rpm for 5 min at 4°C. Cells were washed twice with pre-chilled 1 mL PBS, the supernatant was discarded, and the cells were resuspended in 1 mL of 70% absolute ethanol and incubated overnight at -20°C.

[0045] Before testing, centrifuge at 2000 rpm for 5 min at 4℃ to remove fixative. Add 500 μL of PI / RNase A staining working solution to each sample (prepare the PI:RNase A working solution at a volume ratio of 9:1, derived from a cell cycle kit, before use). Incubate at room temperature in the dark for 15 min. Filter the cell suspension through a 200-mesh filter and analyze the samples using flow cytometry.

[0046] Test results are as follows Figure 9 As shown, Figure 9 In the diagram: G0 / G1 represents cells in the G0 / G1 phase, S represents cells in the S phase, and G2 / M represents cells in the G2 / M phase. According to... Figure 9 It was observed that in the control group, the majority of cells were in the G0 / G1 phase (65.7%), while fewer cells were in the S phase (16.7%) and G2 / M phase (17.6%). Figure 9 B). Incubation of HepG2 cells with the compound aspleniumside J at a treatment concentration of 5 μmol / L slightly increased the proportion of cells in S phase and G2 / M phase, from 16.7% to 18.2% and from 17.6% to 23.5%, respectively. Figure 9 B); At a treatment concentration of 10 μmol / L, the proportion of cells in S phase significantly increased from 16.7% to 30.5% (B). Figure 9 B). The novel cyclic alphatin saponin compound aspleniumside J of this invention can induce cell cycle arrest in HepG2 cells during the S phase.

[0047] Example 4: Effects of cyclic alpha-altin saponins at different concentrations on apoptosis in HepG2 cells HepG2 cells in logarithmic growth phase were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and 1 mL of cell culture medium was added and gently pipetted to form a homogeneous cell suspension. After cell counting under a microscope, the cells were divided into groups of 1×10⁻⁶ cells. 5 Cells were uniformly added to 6-well plates at a density of 2 mL / mL, and incubated overnight. The original culture medium was discarded, and the cells were washed twice with 1 mL PBS buffer. For the control group, 2 mL of cell culture medium was added, and for the drug group, 2 mL of diluted 5 μmol / L and 10 μmol / L of the test compound solution were added. The plates were incubated at 37°C with 5% CO2 for 24 h. After incubation, the culture medium was discarded, and the cells were washed twice with 1 mL PBS buffer. Cells were then digested in each well with 500 μL of EDTA-free trypsin, and 1 mL of cell culture medium was added. The cells were gently pipetted and transferred to 2 mL centrifuge tubes, which were then centrifuged at 2000 rpm for 5 min at 4°C. Each tube was washed twice with pre-chilled 1 mL PBS, and 1-5 × 10⁶ cells were collected. 5 Add 500 μL of Binding Buffer and gently mix to form a single-cell suspension; add 5 μL of Annexin V-FITC and 5 μL of Propidium Iodide, mix gently, and incubate at room temperature in the dark for 5–10 min. Analyze by flow cytometry within 1 h.

[0048] Flow cytometry was used to detect the green fluorescence of Annexin V-FITC (Ex=488 nm, Em=530 nm) via the FITC channel (FL1); and the red fluorescence of PI (Ex=488 nm, Em≥630 nm) via the PE channel (FL2 or FL3).

[0049] The proportion of apoptosis after treatment ( Figure 10 The results showed that aspleniumside J treatment significantly increased cell apoptosis, with the apoptosis rate reaching 29.65% at a concentration of 10 μmol / L (p<0.001). Figure 10B). Regarding different stages of apoptosis, most cells in the control group survived (quadrant 4 Q4: 89.9%), with very few cells in early apoptosis (quadrant 3 Q3: 3.48%) and late apoptosis (quadrant 2 Q2: 3.87%). However, after treatment with compound 1 at a concentration of 10 μmol / L, the number of late apoptotic cells significantly increased, while the number of surviving cells decreased accordingly (Q3: 1.71%, Q2: 26.3%, Q4: 69.3%). Figure 10 A). Therefore, the compound aspleniumside J can induce apoptosis, and the higher the concentration, the greater the proportion of cells undergoing late apoptosis.

[0050] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A cycloartane saponin compound, characterized in that, It has the structure shown in Equation I: Formula I.

2. The method for extracting and purifying the cycloartane saponins according to claim 1, characterized in that, Includes the following steps: The whole herb of *Dryopteris crassirhizoma* was extracted with ethanol, and the resulting extract was concentrated to obtain a paste. The extract was mixed with water to obtain an extract solution; the extract solution was extracted with ethyl acetate to obtain ethyl acetate and aqueous components. The aqueous phase was separated by macroporous adsorption resin chromatography to obtain four fractions, which were named A, B, C, and D in the order of elution. The D segment was separated by silica gel column chromatography to obtain 10 components, which were denoted as F1 to F10 in the order of elution. The F3 was separated by reversed-phase chromatography to obtain 18 components, which were named F3-1 to F3-18 in the order of elution. The F3-16 was purified by preparative high-performance liquid chromatography to obtain cyclic alpha saponin compounds with the structure shown in Formula I.

3. The extraction and purification method according to claim 2, characterized in that, The volume fraction of ethanol is 50-95%, and the extraction is reflux extraction; the extraction is performed 3-5 times, and the extraction time for each extraction is 2-3 hours. The concentration method is vacuum concentration, and the vacuum concentration temperature is 40~55℃.

4. The extraction purification method according to claim 2, characterized in that, The eluent used in the macroporous adsorption resin chromatography separation is ethanol and water; the elution method of the macroporous adsorption resin chromatography separation is gradient elution, in which the volume ratio of ethanol to water varies from 0:100 to 95:

0.

5. The method of claim 2, wherein the extraction purification method is characterized by, The eluent used in the silica gel column chromatography separation is 95% ethanol and ethyl acetate; the elution method of the silica gel column chromatography separation is gradient elution, in which the volume ratio of 95% ethanol and ethyl acetate changes from 1:0 to 0:

1.

6. The extraction and purification method according to claim 2 or 5, characterized in that, The macroporous adsorption resin chromatography separation uses MCI macroporous adsorption resin; the silica gel column chromatography separation uses silica gel with a mesh size of 100-200 meshes; the reverse phase column chromatography separation uses a C 18 ODS medium pressure column.

7. The extraction and purification method according to claim 2, characterized in that, The reversed-phase column chromatography uses methanol-water solution as the eluent; the elution method of the reversed-phase column chromatography is gradient elution, in which the volume fraction of methanol in the methanol-water solution changes from 15% to 100%.

8. The extraction and purification method according to claim 2, characterized in that, The mobile phase used in the preparative high-performance liquid chromatography purification is a methanol-water solution, wherein the volume ratio of methanol to water in the methanol-water solution is 50:50 to 90:10; for differential detection, the flow rate of the mobile phase is 1 to 5 mL / min.

9. The use of the cycloaltin saponin compound of claim 1 or the cycloaltin saponin compound obtained by the extraction and purification method of any one of claims 2 to 9 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The anti-tumor drugs mentioned are anti-liver cancer drugs, anti-breast cancer drugs, or anti-cervical cancer drugs.

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