Marsdenia tenacissima extract as well as preparation method and application thereof

Abstract

The invention discloses a marsdenia tenacissima extract as well as a preparation method and application thereof, and belongs to the technical field of medicines. The marsdenia tenacissima extract is Marsdenside A or Marsdenside C, and the structural formulas of the marsdenside A and the Marsdenside C are respectively shown as a formula I and a formula II. Marsdenside C can induce ferroptosis and inhibit liver cancer, and the liver cancer is liver cancer caused by HepG2 or Huh7 cells; the Marsdenside A induces pyroptosis to play a role in inhibiting the gastric cancer, and the gastric cancer is caused by HGC-27 or AGS cells, so that the Marsdenside C can be used for preparing the medicine for resisting the liver cancer, and the Marsdenside A can be used for preparing the medicine for resisting the gastric cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and specifically relates to an extract of Marsdenia tenacissima, a preparation method thereof, and applications thereof. Background Art

[0002] Marsdenia tenacissima Marsdenia tenacissima (Roxb.) Moon Wight et Arn. is a plant of the Asclepiadaceae family, and its dried vine stems are widely used in the traditional medicine of the Yi and Dai ethnic groups. According to "Compendium of Materia Medica of the Yunnan Region", Marsdenia tenacissima is bitter, astringent, and cold in nature, enters the lung, stomach, and bladder meridians, and has the effects of clearing heat and detoxifying, relieving cough and asthma, etc. In the field of cancer treatment and prevention, the single-drug therapy of Marsdenia tenacissima has been proven to have significant efficacy. Its anti-cancer preparation, Xiaoaiping, is widely used clinically to treat various malignant tumors including lung cancer and esophageal cancer. When used in combination with targeted therapy, this therapy can enhance the therapeutic effect and reduce toxicity, showing excellent safety and effectiveness, with very few adverse reactions, and can effectively enhance the immune response of patients and prolong the survival period. At present, the research on the pharmacological effects and mechanisms of action of individual chemical components in Marsdenia tenacissima is still relatively limited and superficial, and the bioactive substances that play a key role in specific curative effects have not been verified, and the existing screening methods and mechanisms of action for the compounds contained in Marsdenia tenacissima also need to be further explored.

[0003] Primary liver cancer is one of the most common malignant tumors globally. Given the high incidence, high mortality, and poor prognosis of hepatocellular carcinoma (HCC), exploring its pathogenesis is crucial for developing new treatment regimens. Ferroptosis, as a new type of programmed cell death mechanism, is an Fe 2+ -dependent non-apoptotic death pathway, which is significantly different from other cell death mechanisms. At present, targeted therapy for ferroptosis has been regarded as a potential therapeutic intervention for various diseases including cancer. Gastric carcinoma (GC), as the second most common lethal malignant tumor globally, has attracted much attention due to its huge disease burden and epidemiological characteristics. As a highly prevalent malignant tumor, the treatment of gastric cancer faces severe challenges. Pyroptosis, as a form of programmed cell death, is characterized by cell membrane perforation, cell swelling and lysis, releasing cytoplasmic contents and inflammatory mediators, including IL-1β, IL-18, ATP, and HMGB1. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides an extract of Marsdenia tenacissima, a preparation method thereof, and applications thereof, isolates the active substances in Marsdenia tenacissima, and uses them to prepare anti-tumor drugs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is to provide the application of the extract of *Tylophora indicum* in the preparation of antitumor drugs, wherein the extract of *Tylophora indicum* is Marsdenoside A or / and Marsdenoside C, and the structural formulas are shown in Formula I and Formula II, respectively. .

[0006] Based on the above technical solution, the present invention can be further improved as follows.

[0007] Furthermore, the tumor is liver cancer and / or stomach cancer.

[0008] Furthermore, liver cancer is caused by HepG2 or Huh7 cells, and gastric cancer is caused by HGC-27 or AGS cells.

[0009] Furthermore, the application of Marsdenoside C in the preparation of drugs for treating liver cancer, and the application of Marsdenoside A in the preparation of drugs for treating gastric cancer.

[0010] Furthermore, drugs also include pharmaceutically acceptable salts or aglycones.

[0011] Furthermore, the dosage form of the drug is a lyophilized powder injection, an injectable preparation, or an oral preparation.

[0012] Furthermore, the oral preparations are tablets, capsules, pellets, or soft capsules.

[0013] Furthermore, the extract of *Tongguanteng* was prepared by the following steps: (1) Crush the *Thunb.* vine and soak and reflux extract it with 70% ethanol; (2) The extract obtained in step (1) is dispersed in water and then extracted sequentially with petroleum ether, ethyl acetate and n-butanol; (3) The n-butanol phase product obtained in step (2) is purified by silica gel column chromatography, Diaion HP-20, ODS, MCI and Sephadex LH-20 column chromatography techniques, combined with thin layer chromatography and rapid preparative liquid chromatography.

[0014] Furthermore, in step (1), the *Tongguanteng* was crushed and soaked in 70% ethanol overnight, and then refluxed for extraction for 1.5 h each time, for a total of 3 extractions; in step (2), each solvent was used for repeated extraction 8 to 10 times.

[0015] Furthermore, in step (3), the sample was separated using a Diaion HP-20 macroporous adsorption resin column and eluted sequentially with distilled water, 10%, 20%, 30%, 50%, 70%, and 95% ethanol.

[0016] The beneficial effects of this invention are as follows: This invention analyzes the components of a 70% ethanol extract of *Tongguanteng* (a type of orchid), employing silica gel column chromatography, Diaion HP-20, ODS, MCI, and Sephadex LH-20 column chromatography techniques, combined with thin-layer chromatography and rapid preparative liquid chromatography for purification. Finally, the structures of the separated compounds are identified by spectral analysis and mass spectrometry. The isolated Marsdenoside C and Marsdenoside A exhibit significant inhibitory effects on liver cancer and gastric cancer, respectively. Marsdenoside C inhibits liver cancer by inducing ferroptosis, while Marsdenoside A inhibits gastric cancer by inducing pyroptosis. Both can be used to prepare antitumor drugs, providing a new therapeutic approach and method for the treatment of liver and gastric cancer. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the extraction and purification process of chemical components from the vine. Figure 2 The effects of compounds Marsdenoside C and Marsdenoside A on the proliferation of hepatocellular carcinoma cells and gastric cancer cells were investigated. Specifically, A represents the half-maximal inhibitory concentration (WMC) of Marsdenoside C on hepatocellular carcinoma cells and Marsdenoside A on gastric cancer cells; B represents the survival rate of hepatocellular carcinoma cells after 24 h of treatment with Marsdenoside C; C represents the survival rate of gastric cancer cells after 48 h of treatment with Marsdenoside A; D represents the effect of Marsdenoside C on the proliferation of normal hepatocytes; and E represents the effect of Marsdenoside A on the proliferation of normal gastric cells. Figure 3 The effects of Marsdenoside A on the clonogenic ability of gastric cancer cells (AC) and Marsdenoside C on hepatocellular carcinoma cells (DF); Figure 4 The effects of Marsdenoside C on the lateral migration ability of hepatocellular carcinoma cells (A) and Marsdenoside A on gastric carcinoma cells (B); Figure 5 The effects of Marsdenoside C on the migration (A) and invasion (B) abilities of liver cancer cells, and the effects of Marsdenoside A on the migration (C) and invasion (D) abilities of gastric cancer cells; Figure 6 The effects of Marsdenoside C on apoptosis (A and B) and cell cycle (C and D) in liver cancer cells; Figure 7The effect of Marsdenoside C on the proliferation of HepG2 (A) and Huh7 (B) liver cancer cells; Figure 8 To observe the effects of Marsdenoside C on the ultrastructure of HepG2 and Huh7 liver cancer cells using transmission electron microscopy; Figure 9 The effect of Marsdenoside C on ferroptosis-related indicators; where AD represents the effect of Marsdenoside C on Fe in HepG2 and Huh7 liver cancer cells, respectively. 2+ The impact of MDA, GHS and ROS levels; Figure 10 Western blot results for the proteins that Marsdenoside C inhibits tumor growth and induces ferroptosis and apoptosis in HepG2 cells (A), BJ shows the statistical results of each protein, *P<0.05, **P<0.01; Figure 11 Western blot results for the protein immunoblotting of Marsdenoside C in inhibiting tumor growth and inducing ferroptosis and apoptosis in Huh7 cells (A), BJ are the statistical results of each protein, *P<0.05, **P<0.01; Figure 12 The images show the pyroptosis induced by Marsdenoside A in gastric cancer cells HGC-27 and AGS. A represents cell morphology under an optical microscope, B represents the ultrastructure of the cell membrane and mitochondria under a transmission electron microscope (scale bar = 1 μm), C represents the release of lactate dehydrogenase from HGC-27 and AGS cells, and D represents the cell viability of HGC-27 and AGS cells. Figure 13 The results show the pyroptosis induced by Marsdenoside A in gastric cancer cells HGC-27 and AGS. Among them, A and B are the detection results of HGC-27 cells after different concentrations of Marsdenoside A, C and D are the detection results of AGS cells after different concentrations of Marsdenoside A, and EG are the detection results of HGC-27 and AGS cells after different treatment times with Marsdenoside A. Figure 14 The results show the intracellular reactive oxygen species content of HGC-27 and AGS cells after treatment with Marsdenoside A or 5-FU. Figure 15 The effects of Marsdenoside A on apoptosis (A) and cell cycle (B) in HGC-27 and AGS cells; Figure 16The results of Western blot analysis of the proteins by which Marsdenoside A inhibits tumor growth and induces pyroptosis in gastric cancer cells HGC-27 are shown in Figure (A). Figures B and J represent the statistical results of each protein. *P<0.05, **P<0.01; Figure 17 The results of Western blot analysis of the proteins by which Marsdenoside A inhibits tumor growth and induces pyroptosis in gastric cancer cells AGS are shown in Figure A. BJ represents the statistical results of each protein. *P<0.05, **P<0.01; Figure 18 A schematic diagram illustrating the mechanism by which Marsdenoside C induces ferroptosis in liver cancer cells. Detailed Implementation

[0018] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0019] Example 1. Materials (1) Chemicals and reagents: Methanol and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). HPLC-grade formic acid (Shanghai, China) was supplied by Shanghai Aladdin Biochemical Technology Co., Ltd. Other analytical grade reagents were supplied by Tongguang Fine Chemical Co., Ltd. (Beijing, China). Ultrapure water was supplied by Wahaha Co., Ltd. (Hangzhou, China). Cisplatin for injection (lyophilized) was supplied by Qilu Pharmaceutical Co., Ltd. (Shandong, China). (2-hydroxypropyl)-β-cyclodextrin was supplied by Maijin Biotechnology Co., Ltd. (Shanghai, China). Cell culture reagents such as high glucose medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, trypsin / EDTA solution, and phosphate-buffered saline (PBS) were supplied by Beyotime (China) Co., Ltd. (Shanghai, China). CCK-8 kit, biuret kit, MDA detection kit, and ROS detection kit were supplied by Beyotime Biotechnology Research Institute (Shanghai, China). Ferrous ion kit and GSH detection kit were supplied by Solarbio Science & Technology Co., Ltd. (Beijing, China). Nrf2 antibody, GPX4 antibody, S100A4 antibody, xCT antibody, Ferritin antibody, GAPDH antibody, etc. are provided by Abdominal Mart (Beijing, China).

[0020] (2) Plant material: The stem of *Thunb. guanyinensis* was collected in September 2021 in Tanglishan, Yongde County, Lincang City, Yunnan Province, and verified by Kunming Caizhi Biotechnology Co., Ltd. (Kunming, China). The sample (No.: MT-202109) is now preserved at the School of Pharmacy, Minzu University of China (Beijing, China).

[0021] 2. Experimental Methods 2.1 Extraction and separation of active substances from *Thunb. tung* (1) Weigh an appropriate amount of *Tongguanteng* (a type of vine), crush it, and soak it in 70% ethanol overnight. After the sample is completely soaked, reflux extraction is performed at a mass ratio of 10:1 between the sample and 70% ethanol for 1.5 h each time, for a total of 3 extractions. The 3 extracts are combined, concentrated under reduced pressure, and freeze-dried to obtain a 70% ethanol extract. (2) The obtained 70% ethanol extract is subjected to four-stage extraction. An appropriate amount of 70% ethanol extract is dispersed in a small amount of water and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol. Each solvent is used for repeated extraction 8-10 times. After each extract is concentrated under reduced pressure, petroleum ether phase, ethyl acetate phase, n-butanol phase, and aqueous phase are obtained, respectively. (3) n-Butanol phase is separated by macroporous adsorption resin: n-Butanol phase is dissolved in water, centrifuged and filtered, and then concentrated through a Diaion HP-20 macroporous adsorption resin column. It is eluted sequentially with distilled water, 10%, 20%, 30%, 50%, 70%, and 95% ethanol. Each eluent is concentrated under reduced pressure to obtain each eluent component. (4) Each eluted component was rapidly separated and purified using a combination of various column chromatography methods and rapid liquid chromatography (FLSH), and finally purified by high-pressure preparative liquid chromatography to obtain the monomeric compound. The extraction and separation steps of the active substances in *Tinospora sinensis* are as follows: Figure 1 As shown in the figure, the substances named starting with TGT are the numbers of different active substances isolated from *Thunb. tung*.

[0022] 2.2 UPLC-MS Analysis Component analysis of *Tinospora sinensis* was performed using an ExionLC14 ionization system connected to a SCIEX Triple TOF 5600 mass spectrometer; the mobile phase was acetonitrile (A) and an aqueous solution containing 0.1% formic acid (B). Chromatographic analysis was performed using an Acquity HSS T3 reversed-phase column (1.8 μm, 100 mm × 2.1 mm). The gradient elution conditions were as follows: 0 min 5% B; 2 min 5% B; 14 min 98% B; 17 min 98% B; 17.1 min 5% B; 20 min 5% B, with a flow rate of 0.3 mL / min. The column temperature was 30℃; the injection volume was 1.0 μL; mass spectrometry was performed using an ESI source; the sample was detected in both positive and negative ion modes, and the MS1-MS2 mass spectrometry scan range was 100–1200 m / z. The ion source temperature was 100℃, and the desorption temperature was 500℃.

[0023] 2.3 Cell lines HepG2 cells were provided by the Chinese Academy of Sciences and are currently stored at the School of Pharmacy, Minzu University of China; Huh-7 cells were purchased from the Institute of Pharmacy, Peking Union Medical College; HL-7702, AGS, and GES-1 cells were purchased from EallBio (Beijing, China); and HGC-27 cells were purchased from Lebaiha (Shanghai, China). Cells were cultured in RPMI Medium 1640 Medium (Gibco), DMEM (Gibco), or Ham's F-12K Medium (EallBio) containing 10% fetal bovine serum (FBS) and 1% bispecific antibody, respectively. Cells were cultured at 37°C and 5% CO2, with the medium changed periodically until cell confluence reached 80-90%.

[0024] 2.4 Cell viability assay Liver cancer cells and gastric cancer cells were respectively treated at 5 × 10⁻⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. Hepatocellular carcinoma cells were treated with different concentrations of Marsdenoside C, and gastric carcinoma cells were treated with different concentrations of Marsdenoside A. The original culture medium was discarded after 24, 48, and 72 h of treatment. Cisplatin or 5-fluorouracil (5-FU) were used as positive controls, and untreated culture medium served as a negative control. Cell counts were determined using a CCK-8 assay kit, and the half-maximal inhibitory concentration (WMC) was determined using nonlinear regression analysis.

[0025] 2.5 Clonogenesis Assay Liver cancer cells and gastric cancer cells were respectively treated at a density of 1×10⁻⁶ per well. 3 Cells were seeded at a density of [number] cells per well in 12-well plates and cultured overnight. Hepatocellular carcinoma cells were treated with different concentrations of Marsdenoside C, and gastric carcinoma cells were treated with different concentrations of Marsdenoside A. After treatment, cells were incubated at 37°C in a 5% CO2 incubator for 7–14 days, with the complete culture medium changed every three days. After colony formation, cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet solution, and observed under a microscope.

[0026] 2.6 Scratch Healing Test HepG2 and Huh7 liver cancer cells and HGC-27 and AGS gastric cancer cells were seeded into 6-well plates. When the cell density reached 90%, the cells were scratched with a pipette tip and washed twice with PBS buffer. Then, culture medium containing different concentrations of Marsdenoside C or Marsdenoside A was added to the liver and gastric cancer cells, respectively, and the plates were incubated at 37°C in a 5% CO2 incubator. Random images were acquired using a microscope at 0, 24, and 48 h to assess lateral cell migration.

[0027] 2.7 Transwell Chamber Experiment HepG2 and Huh7 liver cancer cells and HGC-27 and AGS gastric cancer cells were respectively 1×10⁻⁶ per well. 5 Cells were seeded at a density of [number] cells in the upper chamber. After 24 h of culture, the culture medium for liver cancer cells and gastric cancer cells was replaced with medium containing different concentrations of Marsdenoside C or Marsdenoside A, respectively. Complete medium containing 10% fetal bovine serum was added to the lower chamber, and after 48 h of culture, residual cells on the basement membrane surface were carefully removed with cotton swabs. Cells were fixed with 4% paraformaldehyde at room temperature for 20 min, followed by staining with 0.1% crystal violet solution at room temperature for 20 min. Cell migration ability was recorded using microscopy.

[0028] The same steps were repeated on a matrix pre-coated with Matrigel gelatin, and cell invasion ability was observed under a microscope.

[0029] 2.8 Cell cycle and apoptosis detection HepG2 and Huh7 liver cancer cells were treated with different concentrations of Marsdenoside C for 24 h. Gastric cancer cells HGC-27 and AGS were treated with different concentrations of Marsdenoside A for 48 h. After treatment, the samples were stored in a refrigerator and fixed with 70% ethanol for 2 h (4℃). Then, 0.5 mL of PI staining solution was added, and the cells were incubated in a dark room at 37℃ for 30 min. Cell cycle progression was detected by flow cytometry, and the data were analyzed and a flow chart was generated using FlowJo 10.8.1 software.

[0030] To detect apoptosis, cells were first washed with PBS buffer, then resuspended in 195 μL Annexin V-FITC solution (Beyotime Biotechnology Co., Ltd.), followed by the addition of 10 μL PI staining solution. The stained cells were then placed on ice, shielded with aluminum foil, and stained for 30 min. During incubation, cells were resuspended to improve staining efficiency. Finally, flow cytometry was performed, and the data were analyzed and a flowchart generated using FlowJo 10.8.1 software.

[0031] 2.9 Microscopic Imaging HepG2 and Huh7 liver cancer cells and HGC-27 and AGS gastric cancer cells were seeded into 6-well plates. When the cells reached approximately 70% confluence, HepG2 and Huh7 liver cancer cells were treated with Marsdenoside C and Erastin, respectively, while HGC-27 and AGS gastric cancer cells were treated with Marsdenoside A and 5-FU. After 24 h of treatment, bright-field cell images were captured using a microscope, and the cells were observed using transmission electron microscopy.

[0032] 2.10 Cell proliferation assay (Edu assay) This invention uses the Edu experiment to detect the effect of Marsdenoside C on the proliferation ability of liver cancer cells, and uses the ferroptosis activator Erastin as a positive control.

[0033] 2.11 Intracellular Fe 2+ Measurement HepG2 and Huh-7 cells were seeded into 10 cm culture dishes, 5 × 10⁶ cells per dish. 6 Cells were treated with different concentrations of Marsdenoside C, with 10 μM Erastin as a positive control, and cultured at 37°C for 24 h. The absorbance was measured at 593 nm using a microplate reader according to the iron ion content detection kit instructions, and the data were calculated as per the kit instructions.

[0034] 2.12 Determination of ROS generation and glutathione (GSH) biosynthesis Intracellular reactive oxygen species (ROS) generation was detected by fluorescence microscopy using the fluorescent dye 2',7'-dichlorofluorescein diacetate (DCFH-DA, Beyotime Biotechnology Research Institute). Gastric cancer cells HGC-27 and AGS were seeded into 6-well plates and incubated for 24 h. After treatment with Marsdenoside A and 5-FU for a specified time, DCFH-DA (10 mmol / L) was incubated at 37°C in the dark for 20 min. Observations were performed using fluorescence microscopy, and images were processed using ImageJ.

[0035] According to the kit instructions, intracellular GSH levels were monitored using monobromoxylene, and changes in intracellular GSH content were calculated based on the kit.

[0036] 2.13 Measurement of intracellular MDA HepG2 and Huh-7 cells were seeded into 10 cm culture dishes, 5 × 10⁶ cells per dish. 6Cells were treated with different concentrations of Marsdenoside C, with 10 μM Erastin as a positive control, and incubated at 37°C for 24 h. Lipid oxidation (MDA) assays were performed according to the kit instructions, and the relative MDA levels between groups were calculated using a standard curve.

[0037] 2.14 Hoechst 33342 / PI staining Gastric cancer cells HGC-27 and AGS were seeded into 96-well plates, and drugs were added and their concentrations adjusted at specified time points. Hoechst 33342 (5 mL) and PI dye (5 mL) were then added, and the mixture was incubated at 4°C in the dark for 20 min. Finally, the cells were observed using a fluorescence inverted microscope.

[0038] 2.15 LDH Release Experiment Gastric cancer cells HGC-27 and AGS were treated with different concentrations of Marsdenoside A and 5-FU, respectively. Cell culture supernatants were collected after treatment, and lactate dehydrogenase activity was detected using a lactate dehydrogenase assay kit (Beyotime Biotechnology Research Institute). The necrosis inhibitor Necrostatin-1 was also used for observation to differentiate between necrosis and apoptosis.

[0039] 2.16 Western blot for protein immunoblotting Proteins were extracted from hepatocellular carcinoma cells HepG2 and Huh7, and gastric carcinoma cells HGC-27 and AGS. Nucleoproteins were isolated using a nucleoprotein extraction kit (according to the manufacturer's instructions), and their concentrations were determined using a BCA protein quantification kit. After separation by SDS-PAGE, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk powder and then co-incubated with primary and secondary antibodies sequentially. Finally, the membranes were imaged and analyzed using an ECL imaging system.

[0040] 2.17 Statistical Analysis SPSS 27 software was used to perform normality analysis and homogeneity of variance tests on the data for each group. A p-value > 0.05 indicated homogeneity of variance, and the LSD test was used for within-group comparisons. If p < 0.05, Dunnett's T3 test was used. One-way ANOVA was used for between-group comparisons, and independent samples t-tests were used for comparisons between two independent samples. Statistical data analysis and image visualization were performed using GraphPad Prism 9.5 software. A p-value less than 0.05 was considered statistically significant.

[0041] 3. Experimental Results 3.1 Structural identification of Marsdenoside C and Marsdenoside A The planar structure of the extract of *Tricholoma matsutake* was analyzed using nuclear magnetic resonance (NMR) spectroscopy, and the relative configuration of the compounds was determined. The molecular weight and chemical formula were confirmed by high-resolution mass spectrometry, the structural characteristics were verified by ultraviolet / infrared chromatography, and circular dichroism spectroscopy was used for absolute configuration determination. After processing the n-butanol phase of *Tricholoma matsutake*, two monomeric compounds—Marsdenoside C (TGT-111) and Marsdenoside A (TGT-110)—were separated, with chemical structures shown in Formula I and Formula II, respectively.

[0042]

[0043] The nuclear magnetic resonance (NMR) spectra of the compound Marsdenoside C are shown below: HR-ESI-MS m / z: 857.46 [M+H] + . 1 H-NMR (CDCl3, 600MHz) δ H : 0.53 (3H, t, J =6.0 Hz, 11α- O -Bu-H-4'),0.82 (3H, d, J =8.0 Hz, 11α- O -Bu-H-5'),1.07 (3H, s, H-18), 1.14 (3H, s, H-19), 1.24 (3H, d, J = 6.5 Hz, Ole-H-6 ), 1.35 (3H, d, J =6.0Hz, Allo-H-6"''),2.26 (3H, s, H-21), 2.97 (1H, d, J = 6.5 Hz, H-17), 3.34 (3H, s, Ole-OCH3), 3.64 (3H, s, Allo-OCH3), 4.57 (1H, d, J = 8.0 Hz, Ole-H-1 ), 4.78 (1H, d, J =8.0 Hz, Allo-H-1"''), 5.22 (1H, d, J =12.0 Hz, H-12), 5.52 (1H, t, J= 10.0 Hz,H-11), 7.40 (t, 2H, J =9.0Hz, 12 β - O -Bz-H-4'',6''), 7.53 (t, 1H, J = 6.0 Hz,12 β - O -Bz-H-5''), 7.94 (d, 2H, J =6.0 Hz, 12 β - O -Bz-H-3'',7''); 13 C NMR (CDCl3, 150 MHz) δC: 37.8 (C-1), 31.9 ( C-2), 76.3 (C-3), 39.2(C-4), 44.0 (C-5), 26.9 (C-6), 29.1 (C-7), 66.9 (C-8), 51.2 (C-9), 34.8 (C-10), 68.6 (C-11), 75.5 (C-12) , 46.2 (C-13) ,71.6 (C-14), 26.7 (C-15), 25.8(C-16), 60.1 (C-17), 12.9 (C-18), 16.9 (C-19), 210.9 (C-20), 30.0 (C-21),175.8 (11α- O -Bu-C-1'), 41.3 (11α- O -Bu-C-2'), 25.1 (11α- O -Bu-C-3'), 11.5(11α- O -Bu-C-4'), 15.2 (11α- O -Bu-C-5'), 166.2 (12 β-O -Bz-C-1''), 129.5 (12 β - O -Bz-C-2''), 129.9 (12 β - O -Bz-C-3'',7''),128.6 (12 β-O -Bz-C-4'',6''),133.4 (12 β-O - Bz-C-5''), 97.0 (Ole-C-1 ), 36.2 (Ole-C-2 ), 78.9 (Ole-C-3 ),79.3 (Ole-C-4 ), 71.4 (Ole-C-5 ), 18.7 (Ole-C-6 ), 55.8 (Ole-3 -OCH,), 99.3(Allo-C-1"''), 71.8 (Allo- C-2"''), 81.1(Allo-C-3"''), 72.9 (Allo-C-4"'') ,71.4 (Allo-C-5"''), 18.0 (Allo-C-6"''), 62.0 (Allo-3"''-OCH).

[0044] The nuclear magnetic resonance (NMR) spectra of compound Marsdenoside A are shown below: HR-ESI-MS m / z: 835.48 [M+H] + . 1 H-NMR (CDCl3, 600MHz) δH: 0.75 (3H, t, J =6.0 Hz, 11α- O -Bu-H-4'),0.92 (3H, d, J =8.0 Hz, 11α- O -Bu-H-5'),1.01 (3H, s, H-18), 1.04 (3H, s, H-19), 1.21 (3H, d, J = 6.5 Hz, Ole-H-6), 1.34 (3H, d, J =6.0Hz, Allo-H-6), 1.75(3H, d, J =5.5 Hz, 12 β-O -Tig-H-4''), 1.76 (3H,s, 12 β-O -Tig-H-5''), 2.17 (3H,s, H-21), 2.87 (1H, d, J = 6.5 Hz, H-17), 3.34 (3H, s, Ole-OCH3), 3.62 (3H, s, Allo-OCH3), 4.54 (1H, d, J = 8.0 Hz, Ole-H-1 ), 4.76 (1H, d, J= 8.0 Hz, Allo-H-1"''), 5.01 (1H, d, J =12.0 Hz, H-12), 5.36 (1H, t, J = 10.0 Hz, H-11), 6.75(1H, s, 12 β-O -Tig-H-3''); 13 C NMR (CDCl3, 150 MHz) δC: 37.7 (C-1), 31.8 ( C-2), 76.2 (C-3), 39.1(C-4), 44.0 (C-5), 29.0 (C-6), 30.6 (C-7), 71.3 (C-8), 51.1 (C-9), 39.1 (C-10), 68.6 (C-11), 65.6 (C-12) , 46.1 (C-13) ,71.6 (C-14), 26.8 (C-15), 26.6(C-16), 60.1 (C-17), 12.8 (C-18), 16.8 (C-19), 210.8 (C-20), 30.0 (C-21),175.6 (11α- O -Bu -C-1'), 41.4 (11α- O -Bu -C-2'), 26.0 (11α- O -Bu -C-3'), 11.7(11α- O -Bu-C-4'), 15.2 (11α- O -Bu-C-5'), 167.4 (12 β-O -Tig-C-1''), 128.1 (12 β-O -Tig-C-2''), 138.6 (12 β-O -Tig-C-3''),12.0 (12 β-O -Tig-C-4''), 14.5 (12 β- O -Tig-C-5''), 96.9 (Ole-C-1 ), 36.2 (Ole-C-2 ), 78.8 (Ole-C-3 ), 79.3 (Ole-C-4 ), 71.4 (Ole-C-5 ), 18.7 (Ole-C-6 ), 55.7 (Ole-3 -OCH,), 99.2 (Allo-C-1"''), 71.8 (Allo- C-2"''), 81.1(Allo-C-3"''), 72.9 (Allo-C-4"'') , 71.4(Allo-C-5"''), 18.0 (Allo-C-6"''), 62.0 (Allo-3"''-OCH).

[0045] 3.2 Effects of Marsdenoside C and Marsdenoside A on Cancer Cell Viability The results of the CCK-8 experiment showed that ( Figure 2 Marsdenoside C treatment for 24 h resulted in dose- and time-dependent growth inhibition of HepG2 and Huh7 liver cancer cells, with half-inhibitory concentrations (IC50) of 16.54 μM and 17.69 μM for HepG2 and Huh7 cells, respectively. Gastric cancer cells HGC-27 and AGS, after treatment with different concentrations of Marsdenoside A for 48 h, also showed dose- and time-dependent growth inhibition, with half-inhibitory concentrations of 41.02 μM and 46.93 μM, respectively. Figure 2 As shown in D and 2E, cell viability was assessed by the CCK-8 assay. Marsdenoside C showed no significant toxicity to normal human hepatocytes HL-7702, and Marsdenoside A showed no significant toxicity to normal human gastric epithelial cells GES-1.

[0046] 3.3 Effects of Marsdenoside C and Marsdenoside A on cancer cell colony formation In the results of cell colony formation experiments ( Figure 3 The number of cloned cells in the experimental group was significantly less than that in the control group. Different concentrations of Marsdenoside C showed significant inhibitory effects on liver cancer cells, and different doses of Marsdenoside A showed significant inhibitory effects on gastric cancer cells, indicating that Marsdenoside C and Marsdenoside A have dose-dependent inhibitory effects on the proliferation of liver cancer cells and gastric cancer cells, respectively.

[0047] 3.4 Effects of Marsdenoside C and Marsdenoside A on cancer cell migration and invasion In the scratch healing experiment ( Figure 4Untreated hepatocellular carcinoma cells HepG2 and Huh7, as well as gastric carcinoma cells HGC-27 and AGS, all exhibited normal migration ability. Cells treated with Marsdenoside C and Marsdenoside A, respectively, showed significantly impaired lateral migration ability compared to the control group. Transwell assays showed that Marsdenoside C dose-dependently inhibited the invasion and vertical migration abilities of HepG2 and Huh7 cells, while Marsdenoside A similarly dose-dependently inhibited these abilities in HGC-27 and AGS cells. Figure 5 ).

[0048] 3.5 Effect of Marsdenoside C on apoptosis in HepG2 and Huh7 liver cancer cells Annexin V and PI staining experiments revealed that Marsdenoside C treatment significantly increased the proportion of apoptotic cells. Figure 6 AB), while the number of cells in the G0 / G1 phase significantly increased. This compound effectively inhibited the proliferation, migration, and invasion of HepG2 and Huh7 liver cancer cells. This was achieved by inducing G0 / G1 checkpoint-mediated cell cycle arrest (AB). Figure 6 Marsdenoside C (CD) ultimately triggers apoptosis.

[0049] 3.6 Marsdenoside C inhibits the proliferation of HepG2 and Huh7 liver cancer cells. The effects of different concentrations of Marsdenoside C on HepG2 and Huh7 liver cancer cells were evaluated using the Edu assay. After 24 h of treatment, the drug effectively inhibited DNA synthesis in the S phase of the cell cycle in HepG2 and Huh7 cells, and its therapeutic effect was comparable to that of Erastin. Figure 7 Preliminary analysis indicates that Marsdenoside C exerts its effects on liver cancer cells through a mechanism that induces ferroptosis.

[0050] 3.7 Marsdenoside C-induced ferroptosis in liver cancer cells is associated with mitochondrial damage. HepG2 and Huh7 cells were treated with different concentrations of Marsdenoside C and Erastin (10 μM) for 24 h. Observation of mitochondrial ultrastructure revealed that, compared with the control group, Marsdenoside C treatment resulted in reduced mitochondrial volume, increased double membrane density, decreased cristae structure, and outer membrane rupture in both HepG2 and Huh7 cells. Figure 8This indicates that Marsdenoside C mediates ferroptosis in liver cancer cells by inducing mitochondrial damage.

[0051] 3.8 Marsdenoside C induces ferroptosis in HCC cells Ferroptosis is an iron-dependent cell death mechanism closely related to excessive lipid peroxidation, ultimately leading to cell membrane rupture. Ferrous ion detection kits can effectively monitor intracellular Fe in living cells. 2+ Changes in levels. The DCFH-DA kit is commonly used to detect specific reactive oxygen species such as superoxide anions and hydroxyl radicals, while glutathione (GSH) and malondialdehyde (MDA) assay kits are used to quantitatively detect the degree of intracellular lipid peroxidation and the content of reduced glutathione, respectively. For example... Figure 9 As shown, these detection methods together constitute a comprehensive system for assessing intracellular lipid peroxidation; compared with the control group, Marsdenoside C and Erastin treatments increased intracellular Fe... 2+ Increased levels of MDA and decreased levels of GSH, all in a concentration-dependent manner, led to an increase in MDA content and a decrease in GSH content. Figure 9 AC); when stained with the DCFH-DA probe, the control group showed weak fluorescence, while the fluorescence intensity of cells treated with Marsdenoside C and Erastin was significantly enhanced. Figure 9 (D) proves that the ROS level is significantly increased.

[0052] 3.9 Marsdenoside C-induced ferroptosis in HCC cells is mediated via the Nrf2 / HO-1 / GPX4 axis. Oxidative stress is a key factor in ferroptosis. As an antioxidant transcription factor, Nrf2 protects cells from oxidative stress damage by regulating downstream antioxidant genes such as HO-1 and NQO1. Activation of Nrf2 can promote iron storage, inhibit reactive oxygen species (ROS) generation, and prevent ferroptosis. To explore this mechanism, the expression levels of Nrf2 and ferroptosis-related proteins GPX4, SLC7A11, FSP1, and FTH1 were detected using Western blotting. Figure 10 and Figure 11As shown, compared with the control group, Marsdenoside C induced a decreasing trend in the expression levels of Nrf2, HO-1, NQO1, GPX4, SLC7A11, FSP1, and FTH1 in HepG2 and Huh7 hepatocellular carcinoma cells, with effects similar to those of ferroptosis inducers. This indicates that Marsdenoside C can induce ferroptosis in HepG2 and Huh7 hepatocellular carcinoma cells through the Nrf2 / HO-1 / GPX4 pathway. Furthermore, the expression levels of apoptosis-regulating proteins Bax and Bcl-2, as well as Caspase9, in gastric cancer cells were also examined. The results showed that Marsdenoside C increased the expression of Bax protein in HepG2 and Huh7 cells while decreasing the expression of Bcl-2 and Caspase9 proteins.

[0053] 3.10 Marsdenoside A altered the morphology and plasma membrane permeability of gastric cancer cells HGC-27 and AGS. HGC-27 and AGS cells treated with Marsdenoside A or 5-FU showed numerous gas bubbles in the cell membrane and cell swelling under a light microscope, changes that more closely resembled the characteristic morphology of pyroptosis cells. Figure 12 A, the arrow indicates that pyroptosis cells exhibit swelling characteristics. Transmission electron microscopy (TEM) observation showed that the cell membranes of control group cells were intact, and organelles such as mitochondria and endoplasmic reticulum were in normal condition; in the Marsdenoside A treatment group, cell membranes showed damage or pore-like structures, and organelles such as mitochondria and endoplasmic reticulum were highly swollen and round compared to the normal group. Figure 12 B, arrow indicates typical damage or swelling features). HGC-27 and AGS cells treated with Marsdenoside A release lactate dehydrogenase (LDH) in a dose-dependent manner. Figure 12 C), further indicating cell membrane rupture and leakage. The necrosis inhibitor Necrostatin-1 was used to differentiate pyroptosis from necrosis; experimental results showed that Necrostatin-1 had no significant effect on Marsdenoside A-induced cell death (C). Figure 12 D).

[0054] In pyroptosis cells, pores form on the cell membrane, allowing impermeable staining agents such as PI to enter the cell and stain the nucleus. Microscopic observation showed that Marsdenoside A treatment increased the proportion of PI-positive cells in HGC-27 or AGS cells in a dose- and time-dependent manner. Figure 13 The increased activity of lactate dehydrogenase and the increase in PI-positive cells indicate that Marsdenoside A triggers cell death by inducing changes in cell membrane permeability.

[0055] ROS not only participate in the apoptosis process but are also key upstream factors regulating the apoptosis pathway. After treating HGC-27 and AGS cells with different concentrations of Marsdenoside A, fluorescence images were captured under a microscope, and the fluorescence intensity was calculated using FijiImageJ software to detect intracellular ROS levels. Compared with the control group, ROS levels in HGC-27 and AGS cells treated with Marsdenoside A showed a significant concentration-dependent increase. Figure 14 ).

[0056] 3.11 Effect of Marsdenoside A on apoptosis in gastric cancer cells HGC-27 and AGS Annexin-V and PI staining showed that treatment with Marsdenoside A significantly increased the proportion of apoptotic cells. Figure 15 A), and the proportion of cells in the G0 / G1 phase also increased significantly ( Figure 15 B). This indicates that Marsdenoside A can significantly inhibit the proliferation, migration, and invasion of HGC-27 and AGS cells, and at the same time induce cell cycle arrest at the G0 / G1 checkpoint, ultimately leading to apoptosis.

[0057] 3.12 Marsdenoside A regulates the pyroptosis pathway in gastric cancer cells HGC-27 and AGS. The caspase 1 / GSDMD pathway is a classic activation pathway for pyroptosis. This experiment examined the expression levels of caspase-1, NLRP3, GSDMD, and GSDMD-N proteins in gastric cancer cells. Figure 16 and Figure 17 As shown, compared with the control group, Marsdenoside A significantly increased the protein expression levels of NLRP3, caspase-1, and GSDMD-N in HGC-27 and AGS cells, while the expression level of GSDMD-N showed a decreasing trend, indicating that GSDMD protein was cleaved. This suggests that Marsdenoside A can induce pyroptosis in HGC-27 and AGS gastric cancer cells, and may mediate this process through the caspase 1 / GSDMD pathway. The regulatory effects of Marsdenoside A on the apoptosis proteins Bax, Bcl-2, and PARP1 were also investigated. The results showed that Marsdenoside A significantly increased the expression level of Bax protein in HGC-27 and AGS gastric cancer cells, while decreasing the expression levels of Bcl-2 and PARP1.

[0058] 4. Conclusion This study discovered that Marsdenoside C exerts its anti-hepatocellular carcinoma effect through the ferroptosis pathway and elucidated its molecular mechanism. Experimental results showed that Marsdenoside C significantly increased intracellular iron (Fe2+) levels. 2+ Marsdenoside C reduced the accumulation of ferroptosis-related marker GSH, while increasing intracellular lipid reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and downregulated the expression of xCT and GPX4, ultimately inducing ferroptosis in both HCC cell lines; the efficacy of high-dose Marsdenoside C in inducing ferroptosis was comparable to that of Erastin. These findings reveal for the first time a novel mechanism by which Marsdenoside C induces ferroptosis in HCC cells. Figure 18 Marsdenoside C induces ferroptosis by inhibiting the activation of the Nrf2 / HO-1 / GPX4 signaling pathway, accompanied by a reduction in mitochondrial volume and an increase in double membrane density, thereby disrupting mitochondrial structure and ultimately inhibiting liver cancer cells.

[0059] Marsdenoside A was studied to investigate its ability to induce pyroptosis in gastric cancer cells. Marsdenoside A effectively inhibited the proliferation of gastric cancer cells and induced typical pyroptosis features in the treated cells: balloon-like vesicle structures, organelle swelling, lactate dehydrogenase (LDH) release, increased reactive oxygen species (ROS) levels, and GSDME cleavage.

[0060] In summary, Marsdenoside A exhibits significant anti-tumor effects against gastric cancer, and its mechanism of action has been preliminarily elucidated. Treatment with Marsdenoside A induces pyroptosis, providing a novel therapeutic approach for gastric cancer. Marsdenoside C effectively inhibits the proliferation of liver cancer cells through mechanisms such as blocking cell cycle progression, inducing apoptosis, and inhibiting cell migration and invasion. Marsdenoside C inhibits liver cancer by inducing ferroptosis. This invention provides new insights into tumor treatment and is of great significance for the development of anti-tumor drugs.

[0061] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. The use of extracts of the plant Tylophora benthamii in the preparation of anti-tumour medicaments characterised in that: The Marsdenoside A or / and Marsdenoside C has a structural formula as shown in formula I and formula II respectively. 。 2. Use according to claim 1, characterized in that: The tumor is liver cancer or / and gastric cancer.

3. Use according to claim 2, characterized in that: The liver cancer is caused by HepG2 or Huh7 cells, and the gastric cancer is caused by HGC-27 or AGS cells.

4. Use according to claim 2, characterized in that: The Marsdenoside C is used for preparing a medicine for resisting liver cancer, and the Marsdenoside A is used for preparing a medicine for resisting gastric cancer.

5. The use according to claim 1, characterized in that: The medicine further comprises a pharmaceutically acceptable salt or aglycone.

6. Use according to claim 5, characterized in that: The medicine is in a dosage form of a freeze-dried powder injection, an injection preparation or an oral preparation.

7. Use according to claim 6, characterized in that: The oral preparation is a tablet, a capsule, a dripping pill or a soft capsule.

8. The use according to claim 1, characterized in that, The Marsdenoside A or / and Marsdenoside C is prepared by the following steps: (1) crushing Marsdenia tenacissima and soaking and refluxing extraction with 70% ethanol; (2) dispersing the extract obtained in step (1) with water, and then extracting with petroleum ether, ethyl acetate and n-butanol in sequence; (3) purifying the n-butanol phase product obtained in step (2) by silica gel column chromatography, Diaion HP-20, ODS, MCI and Sephadex LH-20 column chromatography technologies, combining thin layer chromatography and fast preparation liquid chromatography, to obtain the product.

9. Use according to claim 8, characterized in that: In step (1), the crushed Marsdenia tenacissima is soaked in 70% ethanol overnight, and then refluxed and extracted for 1.5 h each time, totally for 3 times; in step (2), each solvent is repeatedly extracted for 8-10 times.

10. Use according to claim 8, characterized in that: In step (3), the sample is separated by Diaion HP-20 macroporous adsorption resin column, and eluted with distilled water, 10%, 20%, 30%, 50%, 70% and 95% ethanol in sequence.

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