Method for separating and extracting epicatechin, formononin and genistin from caulis spatholobi and application of formononin

Abstract

The invention discloses a method for separating and extracting epicatechin, formononin and genistin from caulis spatholobi and application of the formononin, belongs to the technical field of natural product extraction and medical application, and relates to an efficient and controllable extraction process. According to the method, the three active ingredients of epicatechin, ononin and genitin can be enriched and purified at the same time from the caulis spatholobi, the potential medicinal value of ononin in gastric cancer treatment is further revealed, and a scientific basis is provided for deep development and comprehensive utilization of the caulis spatholobi.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and pharmaceutical application technology, specifically relating to a method for separating and extracting epicatechin, gentiopicrin and genistein from chicken blood vine, and a new application of gentiopicrin in the pharmaceutical field. Background Technology

[0002] Chicken blood vine is a legume plant called *Dendrobium nobile* (also known as chicken blood vine). Spatholobus suberectus Chicken blood vine (Dunneria spp.), also known as blood vine, blood wind vine, and three-leaf chicken blood vine, is mainly distributed in Guangxi and Guizhou provinces. It has a bitter and sweet taste, is warm in nature, and enters the liver and kidney meridians; it has the effects of relaxing muscles and tendons, regulating menstruation and relieving pain, and nourishing and activating blood circulation. Chicken blood vine is rich in various chemical components such as flavonoids, phenolic acids, sterols, terpenes, polysaccharides, and anthraquinones. For example, flavonoids such as luteolin, glycyrrhizin, and isoliquiritin, as well as phenolic acids such as proanthocyanidins and epigallocatechin, all have various activities such as anti-tumor, antioxidant, and anti-inflammatory effects; terpenoids play an important role in regulating cell growth, immune regulation, and antiviral activity; steroidal components participate in the anti-inflammatory process; and polysaccharides have immunomodulatory effects. However, although the chemical components of chicken blood vine have been partially revealed, further in-depth and systematic extraction and separation studies will help to further clarify its pharmacological material basis and provide solid support for subsequent pharmacological research and drug development.

[0003] In recent years, an increasing number of studies have demonstrated that *Spatholobus suberectus* possesses significant pharmacological activity in antioxidation and antitumor activity. Its antioxidant effect can effectively scavenge excess reactive oxygen species in the body, reducing oxidative stress-induced cell damage, thereby preventing and treating diseases related to oxidative stress. In terms of antitumor activity, various chemical components of *Spatholobus suberectus* can exert their antitumor effects through multiple pathways, including inducing tumor cell apoptosis, inhibiting tumor cell proliferation, inhibiting tumor angiogenesis, and regulating the body's immune function. However, the specific components and molecular mechanisms by which *Spatholobus suberectus* exerts its antioxidant and antitumor effects are not yet fully elucidated. Therefore, in-depth research into its novel antitumor active ingredients and mechanisms of action has significant scientific and application value, providing crucial theoretical basis for the in-depth development of *Spatholobus suberectus* and the research and development of new antitumor drugs.

[0004] In the detection of active components of *Caulis Spatholobi*, the existing technology *Study on HPLC chromatographic fingerprint of anti-tumor active site SSCE of *Caulis Spatholobi** constructed fingerprints for 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, epicatechin, puerarin, daidzein, glycyrrhizin, verbascoside, genistein, gentianin, and chrysanthin. Regarding the anti-tumor application of active components of *Caulis Spatholobi*, Chinese patent CN102579425B reported an extraction method for *Caulis Spatholobi* extract containing isoglycyrrhizin (composed of isoglycyrrhizin, gallocatechin, epicatechin, and catechin) and its application in the preparation of drugs for the prevention or treatment of triple-negative breast cancer. Chinese patent CN108578464B also reports the application of *Spatholobus suberectus* extract (ethyl acetate extract or n-butanol extract) in the preparation of antitumor (colon cancer) hematogenous metastasis drugs.

[0005] Currently, no methods for the extraction and separation of gentianin from *Spatholobus suberectus* have been reported in the literature. Therefore, establishing a method for extracting gentianin and other active ingredients from *Spatholobus suberectus* and exploring new medicinal applications of gentianin has significant research value and application prospects. Summary of the Invention

[0006] This invention aims to provide an efficient and controllable extraction process that can simultaneously enrich and purify three active ingredients—epicatechin, ononin, and genistin—from *Spatholobus suberectus*, and further reveals the potential medicinal value of ononin in the treatment of gastric cancer, providing a scientific basis for the in-depth development and comprehensive utilization of *Spatholobus suberectus*.

[0007] This invention is achieved through the following technical solution: a method for separating and extracting epicatechin, gentiopicroside, and genistein from *Spatholobus suberectus*, comprising the following steps: S1. After crushing the chicken blood vine, soak it in ethanol and heat it under reflux to extract the ethanol extract. Then extract it with n-butanol to obtain n-butanol extract and aqueous extract. S2. The n-butanol extract and the aqueous extract were dissolved in methanol and then fed into an alumina chromatography column. The column was eluted with dichloromethane-methanol in a gradient ratio of (20:1) → (0:1) to obtain genistein and gentianin from the n-butanol extract and epicatechin from the aqueous extract.

[0008] After crushing the chicken blood vine, add 90% ethanol at a material-to-liquid ratio of 1:6, mix well, soak for 24 h, and then heat the soaking solution under reflux for 3 h to obtain the ethanol extract.

[0009] The ethanol extract was filtered, concentrated, and the ethanol was removed. It was then diluted with water by 2 times to prepare a suspension. The suspension was then extracted with n-butanol at a 1:1 extraction volume. After extraction, the suspension was concentrated and dried to obtain n-butanol extract and aqueous extract.

[0010] The n-butanol extract was dissolved in methanol and fed into an alumina chromatography column. The eluent during the dichloromethane phase was collected to obtain genistein, and the eluent during the dichloromethane phase was collected to obtain gentiopicroside. The aqueous extract was dissolved in methanol and fed into an alumina chromatography column. The eluent during the methanol phase was collected to obtain epicatechin.

[0011] The eluent was concentrated to dryness under reduced pressure at 60°C, dissolved in chloroform, and the pH was adjusted to 2.0. The resulting solid was then dissolved in methanol, crystallized, and dried. Finally, the corresponding compounds were determined by nuclear magnetic resonance spectroscopy and high-performance liquid chromatography.

[0012] The high-performance liquid chromatography (HPLC) conditions for determining genistein were as follows: a C18 column was used, with an acetonitrile-0.2% phosphoric acid aqueous solution (v / v) at a ratio of 18:82 as the mobile phase, and a flow rate of 0.4 mL / min. -1 The column temperature was 35 ℃, and the detection wavelength was UV-260 nm.

[0013] The high-performance liquid chromatography (HPLC) conditions for determining gentianin were as follows: a C18 column was used, with a mobile phase of ACN-0.1% H3PO4 solution (25:75 v / v) and a flow rate of 1.0 mL / min. -1 The column temperature was 35 ℃, and the detection wavelength was UV-260 nm.

[0014] The high-performance liquid chromatography (HPLC) conditions for determining epicatechin were as follows: a C18 column was used, with a mobile phase of methanol-0.1% phosphoric acid aqueous solution (v / v) at a ratio of 30:70, and a flow rate of 0.4 mL / min. -1 The column temperature was 35 ℃, and the detection wavelength was UV-278 nm. This invention also provides the application of gentianin in the preparation of a drug for treating gastric cancer, wherein the gentianin is obtained by the above-described method.

[0015] The gastric cancer treatment includes inhibiting the proliferation of gastric cancer cells, inhibiting tumor cell metastasis, and inducing tumor cell apoptosis.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention is the first to use alumina chromatography column technology to successfully separate and enrich genistein, gentiopicrin and epicatechin from the n-butanol and water fractions of chicken blood vine. This method is highly efficient and controllable, and can achieve high-purity extraction of target components, providing reliable technical support for in-depth research on the active components of chicken blood vine.

[0017] (2) This invention confirms that the isolated genistein, gentiopicrin, and epicatechin have significant antioxidant and antitumor activities. Among them, the antioxidant activity is mainly contributed by genistein and epicatechin; the antitumor activity is mainly derived from gentiopicrin, providing a scientific basis for the application of Spatholobus suberectus in the fields of antioxidant and antitumor.

[0018] (3) This invention first proposed that gentianin has multiple anti-gastric cancer effects, including: significantly inhibiting the proliferation of gastric cancer cells, effectively inhibiting tumor cell metastasis and inducing tumor cell apoptosis, providing a potential natural drug candidate molecule for the treatment of gastric cancer, and has important clinical application prospects. Attached Figure Description

[0019] Figure 1 This is the 1H-NMR spectrum of compound 1.

[0020] Figure 2 The image shows the 13C-NMR spectrum of compound 1.

[0021] Figure 3 This is the mass spectrum of compound 1.

[0022] Figure 4 This is the HPLC chromatogram of compound 1.

[0023] Figure 5 The image shows the 1H-NMR spectrum of compound 2.

[0024] Figure 6 The image shows the 13C-NMR spectrum of compound 2.

[0025] Figure 7 The mass spectrum of compound 2 is shown in (1).

[0026] Figure 8 Here is the mass spectrum of compound 2 (2).

[0027] Figure 9 This is the HPLC chromatogram of compound 2.

[0028] Figure 10 The image shows the 1H-NMR spectrum of compound 3.

[0029] Figure 11 The image shows the 13C-NMR spectrum of compound 3.

[0030] Figure 12This is the mass spectrum of compound 3.

[0031] Figure 13 This is the HPLC chromatogram of compound 3.

[0032] Figure 14 The scavenging rate of hydroxyl radicals by monomers of different concentrations is shown.

[0033] Figure 15 The scavenging rate of superoxide anion free radicals by monomers of different concentrations is shown.

[0034] Figure 16 The scavenging rates of DPPH free radicals by monomers of different concentrations are shown.

[0035] Figure 17 The scavenging rates of ABTS free radicals by monomers of different concentrations are shown.

[0036] Figure 18 Different concentrations of monomers and Fe 2+ Chelating ability.

[0037] Figure 19 For different concentrations of monomers on Fe 3+ Its restorative ability.

[0038] Figure 20 The cell proliferation inhibition rate of mangosteen glycoside on gastric cancer cells at different time points.

[0039] Figure 21 The healing rate of gastric cancer cell scratches by spatholobi glycoside.

[0040] Figure 22 This study investigated the apoptosis-promoting effect of spatholobus suberectus glycoside on gastric cancer cells.

[0041] Figure 23 Enrichment analysis of GO, a potential anti-gastric cancer target of spatholobi glycoside from chicken blood vine.

[0042] Figure 24 KEGG enrichment analysis of the potential anti-gastric cancer target of spatholobi glycoside from chicken blood vine.

[0043] Figure 25 Molecular docking of HSP90AA1 (a), BCL-2 (b), CASP3 (c) with gentianoside.

[0044] Figure 26 The expression levels of apoptosis-related genes in gastric cancer cells AGS under the influence of gentianin.

[0045] Figure 27 The expression levels of apoptosis-related proteins in gastric cancer cells AGS under the influence of gentianin. Detailed Implementation

[0046] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0047] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] Chicken blood vine, also known as "the holy medicine for blood," is believed in traditional Chinese medicine to have the effects of promoting blood circulation, replenishing blood, regulating menstruation, relieving pain, and relaxing muscles and tendons. Modern research shows that chicken blood vine possesses various pharmacological activities, including antioxidant and anti-tumor effects, demonstrating significant potential for medicinal development. However, current research on the active ingredients in chicken blood vine still has certain limitations, particularly regarding its pharmacodynamic material basis and mechanism of action, which have not yet formed a systematic and complete scientific framework. Therefore, further isolating and identifying new active ingredients from chicken blood vine and exploring their specific pharmacodynamic mechanisms is of great significance for promoting the modernization of its research and clinical application.

[0049] This invention innovatively isolates and extracts three high-purity active ingredients—genistein, gentiopicrin, and epicatechin—from the n-butanol and aqueous fractions of *Spatholobus suberectus*. After efficient purification, the purity of each ingredient reaches over 99.0%, providing high-quality standard compounds for subsequent pharmacological research. Furthermore, this invention systematically investigates the inhibitory effects of the three extracted active ingredients on various tumor cell types, including breast cancer cells (MCF-7, MDA-MB-231), gastric cancer cells (SGC-7901, AGS), and liver cancer cells (HepG2, BEL-7404). Experimental results show that all three components exhibit significant tumor-suppressive activity, with gentiopicrin showing particularly prominent inhibitory effects on gastric cancer cells. Therefore, based on the specific inhibitory effect of gentiopicrin on gastric cancer cells, this invention also provides a potential lead compound for gastric cancer treatment, offering an important material basis and research foundation for the development of novel anti-gastric cancer drugs.

[0050] The specific implementation of the present invention will be described below with reference to the embodiments. Of course, the scope of protection of the present invention is not limited to the following embodiments.

[0051] Example 1: Extraction, separation and purification of three active ingredients The purchased dried *Spatholobus suberectus* was pulverized, and 25 kg was weighed. 90% ethanol was added at a ratio of 1:6, mixed thoroughly, and soaked for 24 h. The soaking solution was then heated under reflux for 3 h. The ethanol extract obtained under reflux was filtered and concentrated in a rotary evaporator. The concentrated ethanol extract was collected and heated in a 55 ℃ water bath to evaporate excess ethanol solvent. The ethanol extract was diluted twice with pure water to prepare a suspension, which was then extracted with n-butanol at a volume ratio of 1:1 for 30 min each time, repeated 3 times. Extraction continued until the organic layer was colorless, and the remaining fraction was the aqueous portion. After evaporation, concentration, and drying, *Spatholobus suberectus* n-butanol and aqueous extracts were obtained and stored at 4 ℃ for later use.

[0052] The concentrated extract was dissolved in methanol and then placed in an alumina chromatography column. A gradient elution was then performed using dichloromethane-methanol as the eluent at a ratio of (20:1) → (0:1), and the eluents from the dichloromethane, dichloromethane, and methanol eluents were collected. The collected eluents were filtered, concentrated to dryness under reduced pressure at 60 °C, dissolved in chloroform, and then the pH was adjusted to 2.0 with 10% HCl, followed by filtration. The resulting solid was dissolved in methanol and crystallized. The crystallized solid was then subjected to drying at 50 °C under forced air and then under reduced pressure at 40 °C to finally obtain compound 1 (200 mg), compound 2 (228 mg), and compound 3 (250 mg).

[0053] Example 2: Identification and Purity Detection of Genistein Compound 1 (200 mg) obtained in Example 1 was subjected to 1H NMR spectroscopy (NMR spectroscopy). 1 H-NMR, carbon nuclear magnetic resonance (NMR) 13 C-NMR and mass spectrometry (ESI-MS), based on their results (see Table 1, Table 2, and...). Figures 1 to 3 Its structure was determined to be genistein, with the molecular formula C. 21 H 20 O 10 Its molecular weight is 432.38. The specific structure is shown in formula (2-1): (2-1) Table 1: Signal assignment of proton NMR spectra ( 1 H-NMR)

[0054] Table 2: Signal assignment of proton NMR spectra ( 13 C-NMR)

[0055] The percentage of the compound's area was determined using high-performance liquid chromatography (HPLC) area normalization method, and the purity of compound 1 (200 mg) was calculated to be greater than 99%. Specific methods are shown in Table 3 below. Figure 4 .

[0056] Table 3: Detection conditions and results of compound 1

[0057] Example 3: Identification and purity determination of gentianin Compound 2 (228 mg) obtained in Example 1 was subjected to 1H NMR spectroscopy (NMR spectroscopy). 1 H-NMR, carbon nuclear magnetic resonance (NMR) 13 C-NMR and mass spectrometry (ESI-MS), based on their results (see Tables 4 and 5) Figures 5 to 8 Its structure was determined to be gentianin, with the molecular formula C. 22 H 22 O9 has a molecular weight of 430.40. Its specific structure is shown in formula (3-1): (3-1) Table 4: Signal assignment of proton NMR spectra ( 1 H-NMR)

[0058] Table 5: Signal assignment of proton NMR spectra ( 13 C-NMR)

[0059] The percentage of the compound's area was determined by high-performance liquid chromatography (HPLC) area normalization method, and the purity of compound 2 (228 mg) was calculated to be greater than 99%. Specific methods are shown in Table 6 below. Figure 9 .

[0060] Table 6: Detection conditions and results of compound 2

[0061] Example 4: Identification and purity determination of epicatechin Compound 3 (250 mg) obtained in Example 1 was subjected to 1H NMR spectroscopy (NMR spectroscopy). 1 H-NMR, carbon nuclear magnetic resonance (NMR) 13 C-NMR and mass spectrometry (ESI-MS), based on their results (see Tables 7 and 8) Figures 10 to 12 Its structure was determined to be epicatechin, with the molecular formula C0. 15 H 14 O6 has a molecular weight of 290.26. Its specific structure is shown in formula (4-1): (4-1) Table 7: Signal assignment of proton NMR spectra ( 1 H-NMR)

[0062] Table 8: Signal assignment of proton NMR spectra ( 13 C-NMR)

[0063] The percentage of the compound's area was determined by high-performance liquid chromatography (HPLC) area normalization method, and the purity of compound 3 (250 mg) was calculated to be greater than 99%. Specific methods are shown in Table 9 below. Figure 13 .

[0064] Table 9: Detection conditions and results of compound 3

[0065] Example 5: Determination of the antioxidant activity of three active ingredients The antioxidant activities of the three active ingredients obtained in Example 1—genistein, gentiopicrin, and epicatechin—were determined, including their hydroxyl radical scavenging capacity, superoxide anion radical scavenging capacity, DPPH radical scavenging capacity, ABTS radical scavenging capacity, and their interaction with Fe... 2+ Chelating ability and Fe 3+ The specific methods and results for determining reducing power are as follows: (1) Determination of hydroxyl radical scavenging ability Prepare concentrations (µg·mL) separately -1 The solution contained monomers (mangosteenin, epicatechin, and genistein) at concentrations of 5, 10, 20, 30, and 40, and the positive control BHT solution; 1 mL of FeSO4 solution (9 mmol·L⁻¹) was added. -1 ), 1 mL H2O2 solution (8.8 mmol·L) -1 ) and 1 mL of salicylic acid-ethanol solution (9 mmol·L) -1 Mix thoroughly, then add 1 mL of distilled water and measure the absorbance at 510 nm. A 0); The absorbance was determined using the same method, replacing distilled water with 1 mL of monomer solutions of different concentrations ( A 1). Calculate the hydroxyl radical scavenging rate according to formula (5-1).

[0066] Hydroxyl radical scavenging rate = [1- ( A 0- A 1) / A 0]×100 %(5-1) The test results are shown below. Figure 14 As shown.

[0067] Depend on Figure 14 It can be seen that with increasing concentration, the scavenging rates of the three monomers and BHT on hydroxyl radicals gradually increased; at the same concentration, epicatechin and gentiopicroside had higher scavenging rates on hydroxyl radicals than BHT and genistein; when the concentration was 40 μg·mL －1 At that time, the order of the scavenging abilities of the three monomers and BHT against hydroxyl radicals was: epicatechin > gentiopicroside > BHT > genistein.

[0068] (2) Determination of superoxide anion free radical scavenging ability Add 4 mL of Tris-HCl buffer (pH 7.4, 1 mol·L⁻¹) －1 Add the solution to a colorimetric tube, let it stand at room temperature for 30 minutes, then add 1 mL of anhydrous ethanol and 0.4 mL of pyrogallol solution (2.5 mmol·L⁻¹). －1 React at room temperature for 5 min, then add 1 mL of HCl solution (8 mmol·L⁻¹). －1 The reaction was stopped, the zeroing point was adjusted with distilled water, and the absorbance at 320 nm was measured. A 0); The absorbance was determined using the same method, replacing anhydrous ethanol with 1 mL of monomer solutions of different concentrations ( A 1); Replace the pyrogallol solution with 0.4 mL of distilled water and determine its absorbance using the same method ( A 2). Calculate the superoxide anion radical scavenging rate according to formula (5-2).

[0069] Superoxide anion radical scavenging rate = [1- ( A 1- A 2) / A 0]×100 %(5-2) The test results are shown below. Figure 15 As shown.

[0070] Depend on Figure 15 It can be seen that when the concentration is 40 μg·mL －1 At that time, the scavenging rates of the three monomers and BHT on superoxide anion free radicals were similar, all between 23% and 30%. The order of their superoxide anion free radical scavenging ability was: epicatechin > BHT > genistein > gentiopicroside.

[0071] (3) Determination of DPPH free radical scavenging ability Take 2 mL each of anhydrous ethanol and DPPH solution (0.1 mmol·L⁻¹). －1 Mix well, let stand in the dark for 30 min, and measure the absorbance at 517 nm. A0); The absorbance was determined using the same method, replacing anhydrous ethanol with 2 mL of monomer solutions of different concentrations ( A 1); Replace the DPPH solution with 2 mL of monomer solutions of different concentrations, and determine its absorbance using the same method ( A 2). Calculate the scavenging rate of DPPH free radicals according to formula (5-3).

[0072] DPPH free radical scavenging rate = [1- ( A 1- A 2) / A 0]×100%(5-3) The test results are shown below. Figure 16 As shown.

[0073] Depend on Figure 16 It can be seen that all three monomers have a certain degree of scavenging ability against DPPH free radicals, and their scavenging rate against hydroxyl free radicals gradually increases with increasing concentration; when the concentration is 40 μg·mL －1 At that time, epicatechin showed a strong scavenging ability against DPPH free radicals, with a scavenging rate as high as 94.04%, while the scavenging rates of gentiopicrin and genistein were less than 30%. The order of the scavenging abilities of the three monomers and BHT against DPPH free radicals was: epicatechin > BHT > gentiopicrin > genistein.

[0074] (4) Determination of ABTS free radical scavenging ability From 7 mmo1·L －1 ABTS solution and 2.45 mmol·L －1 Mix potassium persulfate solution thoroughly at a volume ratio of 1:1, let stand in the dark for 16 h, and then use 0.2 mol·L⁻¹ potassium persulfate solution. －1 Dilute the absorbance at 734 nm with phosphate buffer (pH 7.4) to a range of (0.70 ± 0.02) to prepare the ABTS working solution. Take 2 mL of phosphate buffer (0.2 mol·L⁻¹) and dilute to a range of (0.70 ± 0.02) to prepare the ABTS working solution. －1 Mix the ABTS working solution and let stand at room temperature for 30 min. Measure the absorbance at 734 nm. A 0); The absorbance was measured using the same method, replacing phosphate buffer with 2 mL of monomer solutions of different concentrations. A 1); Replace the ABTS working solution with 2 mL of monomer solutions of different concentrations, and determine its absorbance using the same method ( A 2). Calculate the scavenging rate of ABTS free radicals according to formula (5-4).

[0075] ABTS radical scavenging rate = [1- ( A 1- A 2) / A 0]×100%(5-4) The test results are shown below. Figure 17 As shown.

[0076] Depend on Figure 17 It can be seen that when the concentration is 40 μg·mL －1 At that time, BHT showed the strongest scavenging ability against ABTS free radicals, with a scavenging rate of 98.58%, followed by epicatechin with a scavenging rate of 93.32%, a difference of only 5.26%. It can be inferred that among the three monomers, epicatechin has a relatively stronger antioxidant capacity. The order of the scavenging abilities of the three monomers and BHT against ABTS free radicals is: BHT > epicatechin > genistein > gentiopicroside.

[0077] (5) With Fe 2+ Determination of chelating capacity Add 3 mL of distilled water and 0.1 mL of ferrous chloride solution (2 mmol·L⁻¹). －1 0.2 mL phenanthridine solution (5 mmol·L) －1 Mix well, then add 2 mL of distilled water, let stand for 10 min, and measure the absorbance at 562 nm. A 0); The absorbance was determined using the same method, replacing distilled water with 2 mL of monomer solutions of different concentrations ( A 1). Calculate its relationship with Fe according to formula (5-5). 2+ Its chelating ability.

[0078] Fe 2+ Chelating ability = [( A 0- A 1) / A 0]×100%(5-5) The test results are shown below. Figure 18 As shown.

[0079] Depend on Figure 18 It can be known that Fe 2+ It exhibited different chelating effects on the three monomers, and the chelating ability increased with increasing concentration. Among them, BHT and Fe showed the best chelating effect. 2+ The chelating ability is relatively weak, and epicatechin and Fe 2+ It has strong chelating ability; at a concentration of 40 μg·mL －1 At that time, with Fe 2+ The order of chelating ability is: epicatechin > gentiopicroside > genistein > BHT.

[0080] (6) For Fe 3+ Measurement of reducing power Take 1 mL of monomer solutions of different concentrations and add 2 mL of phosphate buffer (pH 6.6, 0.2 mol·L⁻¹) to each solution. －1The reaction was carried out with 1% potassium ferricyanide solution and heated in a water bath at 50 °C for 25 min, followed by termination of the reaction with 10% trichloroacetic acid solution; 3000 r·min －1 Centrifuge for 10 min, take 2.5 mL of the supernatant, add 2.5 mL of distilled water and 0.5 mL of FeCl3 solution (1%), let stand for 10 min to allow the solution to react completely, and measure the absorbance at 700 nm. A 1); Replace the monomer solutions of different concentrations with 1 mL of distilled water and determine their absorbance using the same method. A 2). Calculate its effect on Fe according to formula (5-6). 3+ Restorative ability.

[0081] Fe 3+ Restoration ability = A 1- A 2 (5-6) The test results are shown below. Figure 19 As shown.

[0082] Depend on Figure 19 It can be seen that the absorbance gradually increases with increasing concentration, meaning that the absorbance of the three monomers and BHT on Fe... 3+ The reducing power of Fe gradually increases; within a certain concentration range, the reducing power of Fe... 3+ The order of reducing power is: gentiopicroside > BHT > genistein > epicatechin.

[0083] In summary, antioxidant activity assays showed that all three monomeric components of *Spatholobus suberectus* possess free radical scavenging ability, reducing ability, and metal ion (Fe) activity. 2+ ) chelating ability, among which gentianin has an effect on Fe 3+ Its restoration ability is the strongest.

[0084] Example 6: Inhibitory effect of three active ingredients on tumor cells The three active ingredients obtained in Example 1—genistein, gentiopicroside, and epicatechin—were compared to inhibit the proliferation of liver cancer cells (HepG2, BEL-7404), gastric cancer cells (SGC-7901, AGS), and breast cancer cells (MCF-7, MDA-MB-231). The specific process is as follows: Experimental materials: Liver cancer cells BEL-7404, gastric cancer cells SGC-7901, and breast cancer cells MCF-7 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; breast cancer cells MDA-MB-231, liver cancer cells HepG2, and gastric cancer cells AGS were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences.

[0085] Experimental method: Three monomeric compounds were accurately weighed and dissolved in DMSO to prepare stock solutions, which were then diluted with complete culture medium to prepare working solutions: epicatechin (0, 50, 100, 200, 400, 600, 800 μg·mL). －1 ), gentianin and genistein (0, 10, 20, 40, 60, 80, 100 μg·mL) －1 The working solution contained 0.1% DMSO, and a complete culture medium containing 0.1% DMSO was used as a control group. The solution was heated in an ultrasonic instrument (37 ℃) and sonicated for 1 h.

[0086] (1) Screening of antitumor activity of monomeric compounds of *Spatholobus suberectus* The antitumor activity of three monomeric compounds from *Spatholobus suberectus* was screened using the CCK-8 assay. Liver cancer cells (HepG2, BEL-7404), gastric cancer cells (SGC-7901, AGS), and breast cancer cells (MCF-7, MDA-MB-231) in logarithmic growth phase were respectively screened at 8 × 10⁸ wells. 3 Cells were seeded in 96-well plates and incubated for 24 h at 37 ℃ and 5% CO2. 100 μL of different concentrations of monomeric compounds were added to the experimental groups. A control group (complete medium containing 0.1% DMSO) and a blank group (no cells added) were also included. After another 24 h of incubation, culture medium containing 10% CCK-8 was added, and incubation continued for 1 h. The optical density at 450 nm was then measured. OD First calculate the cell inhibition rate using formula (6-1), then calculate the IC50. 50 .

[0087] Cell inhibition rate = 1 - [( OD experiment- OD blank) / ( OD Comparison - OD [Blank)]×100% (6-1) The experimental results are shown in Table 10 below.

[0088] Table 10: IC50 of single compounds from *Spatholobus suberectus* against tumor cells 50 Value (unit: μg·mL) －1 )

[0089] Table 10 shows that the three monomers of *Spatholobus suberectus* all have inhibitory effects on liver cancer cells (HepG2, BEL-7404), gastric cancer cells (SGC-7901, AGS), and breast cancer cells (MCF-7, MDA-MB-231). Among them, gentianin showed the highest IC50 value after acting on gastric cancer cells. 50 The smallest value was 78.21 μg·mL. －1and 59.32 μg·mL －1 .

[0090] (2) Effect of Spatholobus suberectus glycoside on the proliferation of gastric cancer cells Gastric cancer cells (SGC-7901, AGS) were cultured at 5 × 10⁻⁶ cells per well. 3 Inoculated into 96-well plates and cultured for 24 h, 100 μL of high, medium, and low concentration monomer compound solutions (SGC-7901: 60, 80, and 100 μg·mL⁻¹) were added to the experimental groups, respectively. －1 AGS: 40, 60, 80 μg·mL －1 A control group (complete culture medium containing 0.1% DMSO) and a blank group (without cells) were set up and cultured for 12, 24, 36 and 48 h respectively before termination. The absorbance was measured and the cell inhibition rate was calculated according to the above method.

[0091] The experimental results are shown in Figure 20 As shown (Note: compared with 12 h, ns indicates no significant difference; * indicates...), P <0.05; ** indicates P <0.01; *** indicates P <0.001).

[0092] Depend on Figure 20 It was found that when gastric cancer cells were treated with the same concentration of gentianin, the inhibition rate increased in a time-dependent manner with prolonged treatment time. Within the same time frame, the inhibitory effect on gastric cancer cells also increased with increasing concentration of gentianin.

[0093] (3) Effect of Spatholobus suberectus glycoside on scratch healing of gastric cancer cells Five equidistant lines were drawn on the bottom of a 6-well plate to mark gastric cancer cells (SGC-7901, AGS) in logarithmic growth phase, with each well containing 5 × 10⁻⁶ cells. 5 Cells were seeded in 6-well plates. When the adherent cell density reached over 90%, a 10 μL pipette tip was used to streak vertically along the marked line. The cells were washed 2-3 times with PBS. 2 mL of different concentrations of gentianin were added to the experimental groups, while 2 mL of complete culture medium containing 2% fetal bovine serum was added to the control group. A photograph was taken under an inverted microscope, and this was recorded as 0 h. The cells were incubated for another 48 h, and a photograph was taken again, recording this as 48 h. The streaked area was delineated and its area measured using ImageJ software. The wound healing rate was calculated using formula (6-2).

[0094] Wound healing rate = [(scratch area at 0 h - scratch area at 48 h) / scratch area at 0 h] × 100% (6-2) Scratch assays can reflect the metastatic ability of tumor cells to some extent. When gastric cancer cells uniformly cover the bottom of a 6-well plate, artificial scratches (circled in red) are created and used as the 0-hour scratches, and their areas are measured. Subsequently, cells are treated with different concentrations of gentianin for 48 hours, and the scratches (circled in red represent areas where cells have not yet migrated and filled the cell line) are recorded again, and their areas are measured. By comparing the scratch area before and after drug treatment, the anti-metastatic effect of gentianin can be evaluated.

[0095] Experimental results are as follows Figure 21 As shown (compared to the control group, * indicates...), P <0.05; ** indicates P <0.01; *** indicates P <0.001).

[0096] Depend on Figure 21 It can be seen that the scratches in the blank control group of the two gastric cancer cells have been significantly reduced, while the scratch healing area in the drug-treated group has decreased significantly with increasing concentration, with high concentration showing the most significant inhibitory effect. P <0.001).

[0097] (4) Effect of Spatholobus suberectus glycoside on apoptosis of gastric cancer cells The apoptosis rate was determined according to the method provided in the Annexin V-FITC / PI Apoptosis Kit. Old cell culture medium was collected, and 300 μL of EDTA-free trypsin was added to each well for digestion. The digested cells were also collected in the old culture medium. The cells were centrifuged at 1000 g for 5 min, and the supernatant was discarded. The cells were washed twice with pre-chilled PBS, centrifuged again with the same parameters, and the PBS was discarded. The cells were resuspended in 1× Binding Buffer, and the cell concentration was adjusted to 1×10⁻⁶. 6 For each cell / mL sample, transfer 100 μL of cell suspension to a new tube, add 5 μL of Annexin V-FITC, mix well, and incubate at room temperature in the dark for 5 min. Then add 10 μL of propidium iodide (PI), mix well, and incubate at room temperature in the dark for 15 min. Finally, add 400 μL of 1× Binding Buffer, mix gently, and then perform the analysis.

[0098] The proportion of gastric cancer cells that underwent apoptosis 24 h after treatment with gentianin was detected by flow cytometry using Annexin V-FITC / PI staining. The results are shown below. Figure 22 As shown (Note: a, c, and d represent the apoptosis rate, total apoptosis rate, and early apoptosis rate of SGC-7901 cells treated with gentianin, respectively; b, e, and f represent the apoptosis rate, total apoptosis rate, and early apoptosis rate of AGC cells treated with gentianin, respectively; compared with the control group, *** indicates...).P <0.001).

[0099] Depend on Figure 22 It can be seen that, compared with the control group, the total proportion of apoptosis in both gastric cancer cell lines significantly increased with the increase of the concentration of gentianin intervention. p The total apoptosis rates of SGC-7901 gastric cancer cells were 7.94 ± 0.13, 8.03 ± 0.42, and 13.14 ± 0.55, respectively, while those of AGS gastric cancer cells were 14.54 ± 0.37, 15.50 ± 0.67, and 16.69 ± 0.35, respectively. Further analysis revealed a significant difference in the proportion of cells undergoing early apoptosis between the two gastric cancer cell lines. (<0.001)

[0100] In summary, CCK-8 assay showed that the three monomers of *Spatholobus suberectus* (chicken blood vine) inhibited liver cancer cells (HepG2, BEL-7404), gastric cancer cells (SGC-7901, AGS), and breast cancer cells (MCF-7, MDA-MB-231). This indicates that the three active components of *Spatholobus suberectus*—epicatechin, gentiopicrin, and genistein—all possess antitumor efficacy. Among them, gentiopicrin showed the strongest inhibitory effect on gastric cancer cells, with an IC50 concentration of [missing value]. 50 The values ​​were 78.21 μg·mL. －1 and 59.32 μg·mL －1 Further experiments demonstrated that gentianin has the effects of inhibiting the proliferation, metastasis, and inducing apoptosis of gastric cancer cells (SGC-7901 and AGS).

[0101] Example 7: Mechanism of action of gentianin on gastric cancer cells This embodiment uses the gentianin obtained in Example 1 as the object, and detects the mRNA expression of gastric cancer-related target genes by qPCR, and verifies the changes in protein levels by Western blot (WB), further confirming that gentianin acts on key targets of gastric cancer.

[0102] Using the CAS number and SMILES number of styracin, and utilizing the Swiss Target Prediction database (http: / / swisstargetprediction.ch) and the SEA database (…), http: / / sea.bkslab.org ), TCMSP database ( http: / / old.tcmsp-e.com / tcmsp.phpThe target sites were predicted using the TargetNet database (http: / / targetnet.scbdd.com) and the CTD database (http: / / ctdbase.org). The SEA screening criterion was: Max TC ≥ 0.3. After summarizing the target sites predicted by the five databases and removing duplicates, 54 potential targets for *Spatholobus suberectus* glycosides were obtained.

[0103] Using "Gastric Cancer" as the keyword, in the GeneCarde database ( http: / / www.genecards.org), TTD database( http: / / db.idrblab.net / ttd Disease targets were retrieved from the OMIM database (http: / / www.omim.org), and the results were filtered by median and deduplication, resulting in 1748 disease targets. A Venn diagram was drawn using the online tool MicroBio, and it was found that there were 47 intersection targets between gentianin and gastric cancer.

[0104] Forty-seven overlapping target sites were imported into the STRING database with a confidence level of 0.7. Discrete points were removed, and a protein-protein interaction network was constructed. The data exported from STRING was processed in Cytoscape 3.8.0 software, and a plugin was used to calculate and screen out the core targets, which were 10 in total: BC > 54.10, CC > 0.01, and DC > 6.44. These targets were then sorted according to their DC values ​​as follows: IL-6, BCL-2, SRC, HIF1A, HSP90AA1, PTGS2, TNF, CASP3, ALB, and MAPK14.

[0105] Enrichment analysis was performed on the intersection targets of mangosteen and gastric cancer in the DAVID database. Within the GO enrichment set, the top 10 items with the highest enrichment levels were selected for analysis across three dimensions: cell composition (CC), molecular function (MF), and biological process (BP). Figure 23 It is known that the biological processes involved by gentianin mainly focus on the regulation of apoptosis, responses to exogenous stimuli, and intrinsic apoptosis signaling pathways mediated by DNA damage. Its molecular function is primarily binding, including enzyme binding, protease binding, and binding to similar proteins. The cellular components involved mainly include mitochondria, cytoplasm, and BCL-2 family protein complexes. In KEGG enrichment analysis, the top 20 enriched items were selected for further analysis. Figure 24 It is known that the signaling pathways involved in gentianin mainly include cancer pathways, VEGF signaling pathways, and MAPK signaling pathways.

[0106] Molecular docking was performed on 10 core target sites with gentianin, and the binding energies obtained showed that all sites had good binding ability, as shown in Table 11 below.

[0107] Table 11: Molecular docking between spatholobin and corresponding target sites of *Spatholobus suberectus*

[0108] GO enrichment analysis revealed that the biological functions of the potential targets of gentianin are mainly related to apoptosis regulation. Based on Example 6, it was hypothesized that the anti-gastric cancer mechanism of gentianin is related to apoptosis. Therefore, from 10 core targets, CASP3 and BCL-2, which are directly related to apoptosis, and HSP90AA1, which is indirectly related, were selected. Molecular docking visualization analysis was performed with gentianin, and the results are shown in […]. Figure 25 As shown.

[0109] Depend on Figure 25 It is known that gentianin binds to threonine (Thr)-195, glutamic acid (Glu)-196, and arginine (Arg)-201 residues of HSP90AA1 via hydrogen bonds; to glutamic acid (Glu)-25, arginine (Arg)-26, aspartic acid (Asp)-102, and serine (Ser)-105 residues of BCL-2; and to lysine (Lys)-137, glycine (Gly)-125, and tyrosine (Tyr)-197 residues of CASP3.

[0110] The expression levels of relevant genes and proteins were detected using qPCR and WB techniques, respectively.

[0111] (1) qPCR detection Gastric cancer cells in the logarithmic growth phase, AGS, were divided into groups of 5 × 10⁻⁶ cells per well. 5 One cell was seeded into a 6-well plate. After the cells adhered, 2 mL of gentianin (60 μg·mL⁻¹) was added. －1 The control group was cultured in 2 mL of medium containing an equal volume of DMSO for 24 h. Cells were then treated according to the instructions of the total RNA extraction kit to extract total RNA. During extraction, it was important to maintain low temperature and avoid enzyme contamination. The extracted mRNA was reverse transcribed into cDNA, diluted 3-fold, and amplified using a PCR program. The relative expression level is calculated using (-ΔΔCt), as shown in Table 12 below.

[0112] Table 12: Primer sequence numbers

[0113] qPCR test results are shown below Figure 26 shown (Note: HSP90 / GAPDH (a) BCL-2 / GAPDH (b) CASP3 / GAPDH (c) Compared with the control group, *** indicates P <0.001).

[0114] (2) WB detection Protein extraction: After pretreatment of cells according to method 5.2.6, discard the old culture medium, wash once with pre-chilled PBS, add 1 mL of pre-chilled PBS, scrape off cells with a spatula, collect in centrifuge tubes, and centrifuge at 1500 rpm for 5 min. Simultaneously, prepare lysis buffer: add 10 μL of phenylmethanesulfonylfluoride (PMSF), 10 μL of phosphorylated protease inhibitor, and 20 μL of 50×Cocktail protease inhibitor to each 1 mL of RIPA lysis buffer, mix well, and place on ice. After centrifugation, discard the supernatant, add 500 μL of the prepared lysis buffer to each tube, mix by pipetting or shaking until the lysis buffer is no longer viscous, and lyse on ice for 30 min, shaking every 10 min. After complete lysis, centrifuge at 12000 g for 5 min, collect the supernatant, which is the total protein solution.

[0115] Protein concentration determination: Using the BCA protein quantification kit, a standard curve was first plotted according to the instructions to generate the calculation equation. The protein solution was then diluted 10-fold, and the protein concentration was determined according to the instructions. Based on the equation, the protein concentration and the required loading volume for electrophoresis were calculated.

[0116] Electrophoresis: Prepare stacking and separating gels according to the instructions of the gel preparation kit. The concentration of the PAGE separating gel is determined based on the molecular weight of the protein. A 10% PAGE separating gel is used to separate proteins of 20-100 kDa, and a 12% PAGE separating gel is used to separate proteins of 10-60 kDa. Install the electrophoresis tank, add electrophoresis buffer until the liquid level covers the upper edge of the glass plate, remove the gel comb, and use a 10 μL pipette tip to rinse away any residual gel in the sample wells. Then add the boiled protein sample and marker protein separately, and add the samples vertically. Electrophoresis conditions: When separating HSP90AA1 (90 kDa), BCL-2 (26 kDa), and GAPDH (36 kDa) proteins, maintain a constant voltage of 80 V. After approximately 30 minutes, when the bromophenol blue in each lane is compressed into a straight line and located at the boundary between the stacking gel and the separating gel, switch to a constant voltage of 120 V for electrophoresis. Refer to the marker position, and electrophoresis ends when the target protein is effectively separated. When separating Cle-CASP3 (17 kDa) protein, maintain a constant voltage of 220 V throughout the electrophoresis process. After approximately 30 minutes, electrophoresis ends when the target protein is effectively separated.

[0117] Transfer: Place the membrane in the following order: sponge, filter paper, gel, PVDF membrane, filter paper, sponge, taking care to avoid air bubbles. Adjust to a constant current of 400 mA. The transfer time depends on the molecular weight.

[0118] Immunological reaction: After the membrane is transferred, wash it twice with TBST, then add rapid blocking buffer, place it on a shaker, block it at room temperature for 10 min, discard the blocking buffer, add diluted primary antibody, and incubate it on a shaker at 4 ℃ for 12-16 h; recover the primary antibody, wash it three times with TBST, add diluted secondary antibody, and incubate it on a shaker at room temperature for 2 h; recover the secondary antibody, and wash it three times with TBST.

[0119] Chemiluminescence: Take out the eluted PVDF membrane and place it in the 1:1 mixed ECL luminescent solution. After reacting for 1 minute, take out the membrane and place it on the chemiluminescence instrument tray. Perform chemiluminescence according to the program and save the image.

[0120] The WB test results are shown below. Figure 27 As shown (Note: HSP90AA1 / GAPDH (b), BCL-2 / GAPDH (c), CASP3 / GAPDH (d), compared with the control group, * indicates...), P <0.05; ** indicates P <0.01).

[0121] Depend on Figure 26 and Figure 27 It can be seen that, compared with the control group, the expression levels of HSP90AA1 and the anti-apoptotic protein BCL-2 were significantly decreased after treatment of gastric cancer cells AGS with gentianin. P <0.01, P <0.05), while the expression level of the pro-apoptotic protein Cle-CASP3 splice was significantly increased ( P <0.05). This result suggests that gentianin may exert its anti-gastric cancer effect by inducing apoptosis in gastric cancer cells through its action on HSP90AA1, BCL-2, and CASP3 targets.

[0122] In summary, using qPCR and Western blotting, when gentianin was applied to AGS gastric cancer cells, the expression levels of HSP90AA1 and BCL-2 proteins significantly decreased, while the expression level of Cle-CASP3 protein significantly increased. Based on these experimental results, the anti-gastric cancer mechanism of gentianin may be achieved by acting on HSP90AA1 protein, thereby activating the mitochondrial apoptosis pathway.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for separating and extracting epicatechin, gentiopicroside, and genistein from *Spatholobus suberectus*, characterized in that: Includes the following steps: S1. After crushing the chicken blood vine, soak it in ethanol and heat it under reflux to extract the ethanol extract. Then extract it with n-butanol to obtain n-butanol extract and aqueous extract. S2. The n-butanol extract and the aqueous extract were dissolved in methanol and then fed into an alumina chromatography column. The column was eluted with dichloromethane-methanol in a gradient ratio of (20:1) → (0:1) to obtain genistein and gentianin from the n-butanol extract and epicatechin from the aqueous extract.

2. The method according to claim 1, characterized in that: After crushing the chicken blood vine, add 90% ethanol at a material-to-liquid ratio of 1:6, mix well, soak for 24 h, and then heat the soaking solution under reflux for 3 h to obtain the ethanol extract.

3. The method according to claim 2, characterized in that: The ethanol extract was filtered, concentrated, and the ethanol was removed. It was then diluted with water by 2 times to prepare a suspension. The suspension was then extracted with n-butanol at a 1:1 extraction volume. After extraction, the suspension was concentrated and dried to obtain n-butanol extract and aqueous extract.

4. The method according to claim 1, characterized in that: The n-butanol extract was dissolved in methanol and fed into an alumina chromatography column. The eluent during the dichloromethane phase was collected to obtain genistein, and the eluent during the dichloromethane phase was collected to obtain gentiopicroside. The aqueous extract was dissolved in methanol and fed into an alumina chromatography column. The eluent during the methanol phase was collected to obtain epicatechin.

5. The method according to claim 4, characterized in that: The eluent was concentrated to dryness under reduced pressure at 60°C, dissolved in chloroform, and the pH was adjusted to 2.

0. The resulting solid was then dissolved in methanol, crystallized, and dried. Finally, the corresponding compounds were determined by nuclear magnetic resonance spectroscopy and high-performance liquid chromatography.

6. The method according to claim 5, characterized in that: The high-performance liquid chromatography (HPLC) conditions for determining genistein were as follows: a C18 column was used, with an acetonitrile-0.2% phosphoric acid aqueous solution (v / v) at a ratio of 18:82 as the mobile phase, and a flow rate of 0.4 mL / min. -1 The column temperature was 35 ℃, and the detection wavelength was UV-260 nm.

7. The method according to claim 5, characterized in that: The high-performance liquid chromatography (HPLC) conditions for determining gentianin were as follows: a C18 column was used, with a mobile phase of ACN-0.1% H3PO4 solution (v / v) at a ratio of 25:75, and a flow rate of 1.0 mL / min. -1 The column temperature was 35 ℃, and the detection wavelength was UV-260 nm.

8. The method according to claim 5, characterized in that: The high-performance liquid chromatography (HPLC) conditions for determining epicatechin were as follows: a C18 column was used, with a mobile phase of methanol-0.1% phosphoric acid aqueous solution (v / v) at a ratio of 30:70, and a flow rate of 0.4 mL / min. -1 The column temperature was 35 ℃, and the detection wavelength was UV-278 nm.

9. The application of gentianin in the preparation of drugs for treating gastric cancer, characterized in that: The gentianin was obtained by separation and extraction using the method described in any one of claims 1 to 8.

10. The application according to claim 9, characterized in that: The gastric cancer treatment includes inhibiting the proliferation of gastric cancer cells, inhibiting tumor cell metastasis, and inducing tumor cell apoptosis.

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