Application of glycyrrhiza flavone in preparation of drugs for preventing and / or treating colon cancer

Abstract

The present application relates to the application of glycyrrhizin flavone in the preparation of drugs for preventing and / or treating colon cancer, and belongs to the technical field of antitumor biological medicine. The glycyrrhizin flavone includes ganomacerone, isolongifolen-9-one, nor-dihydroguaiaretic acid and glabrone. The present application extracts the main active compound glycyrrhizin flavone from traditional Chinese medicine glycyrrhiza, confirms the treatment effect of glycyrrhizin flavone on colon cancer through various in-vivo and in-vitro models, and can inhibit the diffusion of tumor cells to the whole body. The present application confirms four active monomer compounds ganomacerone, isolongifolen-9-one, nor-dihydroguaiaretic acid and glabrone from glycyrrhizin flavone, and finds through in-vivo and in-vitro experiments that they can inhibit the proliferation of colon cancer cells DLD-1 and HCT-116 cells, delay the diffusion speed of tumor cells, have no obvious effect on normal colon epithelial cells NCM460, reduce the toxicity of glabrone to normal cells, and have a synergistic effect.

Description

Technical Field

[0001] This invention relates to the field of antitumor biomedicine technology, and in particular to the application of glycyrrhizin in the preparation of drugs for the prevention and / or treatment of colon cancer. Background Technology

[0002] Colorectal cancer (CRC) is one of the three most common cancers worldwide, with a high incidence rate in developed countries, accounting for approximately 10% of all cancer cases and being the second leading cause of cancer-related deaths globally. It is caused by numerous risk factors, including diet, lack of exercise, obesity, smoking, and alcohol consumption, while family history or certain genetic syndromes can also increase susceptibility. Current treatments for colorectal cancer include endoscopic local resection, palliative chemotherapy, targeted therapy, and immunotherapy.

[0003] While these treatments have prolonged patient survival, their effectiveness is not satisfactory due to the high recurrence and metastasis rates of colorectal cancer. Surgical resection has drawbacks such as high recurrence rates and high costs; chemotherapy, while killing tumor cells, may damage normal cells and immune cells, making it a double-edged sword; targeted therapy and immunotherapy have relatively fewer side effects, but they are not suitable for all patients and vary greatly from person to person. Therefore, finding safe and effective treatment options to alleviate the burden of this disease is crucial.

[0004] Traditional Chinese medicine (TCM) is a mainstream form of complementary and alternative medicine. However, some TCM herbs have limited therapeutic effects and certain adverse reactions. Therefore, it is necessary to conduct component analysis on relevant TCM herbs to improve therapeutic efficacy and safety. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the use of glycyrrhizin in the preparation of drugs for the prevention and / or treatment of colon cancer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides the use of glycyrrhizin flavonoids in the preparation of drugs for the prevention and / or treatment of colon cancer, wherein the glycyrrhizin flavonoids include Ganoderma lucidum flavonoids, isophyllene-9-one, nordihydroguaiacol, and glycyrrhizin.

[0008] The combination of Ganoderma lucidum equine, isolongen-9-one, nordihydroguaiac acid and glycyrrhizin can further enhance the anti-colorectal cancer effect, reduce the adverse reactions of glycyrrhizin to normal cells, and improve safety.

[0009] Furthermore, the drug is a drug that inhibits the proliferation of colorectal cancer. Furthermore, the drug is a drug that inhibits the metastasis of colorectal cancer.

[0010] Furthermore, the drug is a drug for repairing the intestinal barrier.

[0011] In a specific embodiment of the present invention, the drug inhibits the proliferation and / or metastasis of colorectal cancer HCT-116 cells and / or DLD-1 cells.

[0012] Furthermore, the concentration of Ganoderma lucidum equine, isolongfelene-9-one, nordihydroguaiacol, or glycyrrhizin is 1~128 μM.

[0013] In a preferred embodiment, the concentration of Ganoderma lucidum equine, isophylline-9-one, nordihydroguaiacol, or glycyrrhizin is 32-128 μM; more preferably, the concentration is 64-128 μM; and even more preferably, the concentration is 128 μM. At this concentration, the combination of Ganoderma lucidum equine, isophylline-9-one, nordihydroguaiacol, and glycyrrhizin has the best inhibitory effect on the proliferation of colorectal cancer, and exhibits low toxicity to normal colonic epithelial cells (NCM460 cells).

[0014] Furthermore, the drug is a pharmaceutical preparation made with glycyrrhizin as the active ingredient and pharmaceutically acceptable excipients.

[0015] Furthermore, the drug or drug formulation includes tablets, granules, or liquid formulations.

[0016] Furthermore, the method for preparing the glycyrrhizin flavonoids includes the following steps:

[0017] S11: Mix the licorice sample with water, vortex and grind; the ratio of licorice sample to water is licorice sample:water = (10~250)mg:(0.1~2.5)mL.

[0018] S12: The product ground in step S1 is subjected to ice bath ultrasonic extraction for 30-60 min.

[0019] S13: Centrifuge the product extracted in step S2 at a speed of 5000~15000 rpm for 5~15 min.

[0020] In a specific embodiment of the present invention, in step S11, the ratio of licorice sample to water is 100 mg to 1 mL.

[0021] In a specific embodiment of the present invention, the extraction time in step S12 is 60 min.

[0022] In a specific embodiment of the present invention, in step S13, the centrifugation speed is 14000 rpm and the centrifugation time is 10 min.

[0023] Furthermore, the quality control method for the licorice flavonoids includes using liquid chromatography combined with mass spectrometry to detect Ganoderma lucidum quinone, isolongifene-9-one, nordihydroguaiacol, and glycyrrhizin in the licorice flavonoids.

[0024] Furthermore, the mobile phase in the liquid chromatography-mass spectrometry detection and analysis includes mobile phase A and mobile phase B. Mobile phase A is a 0.1%~0.5% w / v formic acid aqueous solution, and mobile phase B is acetonitrile. The chromatographic column is ACQUITY UPLCHSS T3, with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm.

[0025] In a specific embodiment of the present invention, a 0.1% w / v formic acid aqueous solution is used as mobile phase A for detection.

[0026] Furthermore, the liquid chromatography-mass spectrometry detection and analysis is performed using gradient elution.

[0027] From 0 to 2 min, the volume ratio of mobile phase A to mobile phase B is mobile phase A: mobile phase B = 95: 5;

[0028] In 2-4 minutes, the volume ratio of mobile phase A to mobile phase B changed from mobile phase A: mobile phase B = 95: 5 to mobile phase A: mobile phase B = 70: 30.

[0029] Over 4-8 minutes, the volume ratio of mobile phase A to mobile phase B changed from 70:30 to 50:50.

[0030] In 8-10 minutes, the volume ratio of mobile phase A to mobile phase B changed from 50:50 to 20:80.

[0031] In 10-14 minutes, the volume ratio of mobile phase A to mobile phase B changed from mobile phase A: mobile phase B = 20: 80 to mobile phase A: mobile phase B = 0: 100.

[0032] At 14-15 min, the volume ratio of mobile phase A to mobile phase B is mobile phase A: mobile phase B = 0:100.

[0033] After 15-16 minutes, the volume ratio of mobile phase A to mobile phase B changed from mobile phase A: mobile phase B = 95: 5.

[0034] The column temperature is 40~50℃; the flow rate is 0.2~0.5 mL / min; and the diode array detector has a scanning range of 210~400 nm.

[0035] In a specific embodiment of the present invention, the column temperature is 45°C and the flow rate is 0.35 mL / min.

[0036] Furthermore, the mass spectrometry conditions were as follows: positive ion spray voltage of 3800 V, negative ion spray voltage of -3000 V; sheath gas flow rate of 35 Arb; S-lens RF level of 50; normalized fragmentation energies of 10, 20, and 40; secondary mass spectrometry resolution of 15000; mass range of 100–1500 m / z; full resolution of 60000; capillary temperature of 32 °C; auxiliary gas heater temperature of 350 °C; and auxiliary gas flow rate of 8 Arb.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] This invention extracts the main active compound, glycyrrhizin, from the traditional Chinese medicine licorice. The therapeutic effect of glycyrrhizin on colon cancer was confirmed through multiple in vivo and in vitro models. It can inhibit the spread of tumor cells throughout the body, solve the problem of difficult treatment caused by rapid tumor spread in clinical practice, prolong the survival time of patients, and confirm its safety through toxicity experiments, thus solving the problem of large side effects of clinical colon cancer treatment drugs.

[0039] This invention identified four active monomeric compounds from glycyrrhiza flavonoids: Ganoderma lucidum quinone, isolongifene-9-one, nordihydroguaiacol, and glycyrrhizin. In vitro and in vivo experiments verified that these compounds can inhibit the proliferation of DLD-1 and HCT-116 colon cancer cells and slow the spread of tumor cells. Furthermore, the combination of these compounds had no significant effect on normal colonic epithelial cells (NCM460 cells), reducing the toxicity of glycyrrhizin to NCM460 cells and exhibiting a synergistic effect. This provides a reference for the safe development of small molecule targeted drugs in clinical practice. Attached Figure Description

[0040] Figure 1 The image shows the EIC chromatogram of Ganoderma lucidum equinox and its MS / MS chromatogram compared with the standard library LuMet-TCM.

[0041] Figure 2 EIC plot of isophyllen-9-one and its MS / MS plot compared with the public library HerbDB.

[0042] Figure 3 EIC plot of nordihydroguaiaric acid and its MS / MS plot compared with the public library HerbDB.

[0043] Figure 4 EIC chromatogram of glycyrrhizin and its MS / MS chromatogram compared with the standard library LuMet-TCM.

[0044] Figure 5The effect of glycyrrhizin on survival time in mice with AOM / DSS colon cancer.

[0045] Figure 6 The effect of glycyrrhizin flavonoids on the intestines of AOM / DSS colon cancer mice. Where A represents intestinal length and B represents the number of tumors in the colon.

[0046] Figure 7 The effect of glycyrrhizin on the intestinal barrier in AOM / DSS colon cancer mice.

[0047] Figure 8 The effect of glycyrrhizin on the proliferation of colon cancer cells in a zebrafish xenograft model.

[0048] Figure 9 The effect of glycyrrhizin on the metastasis of colon cancer cells in a zebrafish xenograft model.

[0049] Figure 10 The effects of glycyrrhizin flavonoids on HCT-116 and DLD-1 colon cancer cells are shown. A represents HCT-116 cells; B represents DLD-1 cells.

[0050] Figure 11 The effect of glycyrrhizin on the clonogenic ability of HCT-116 and DLD-1 colon cancer cells.

[0051] Figure 12 This study describes the Edu staining experiment of licorice flavonoids on HCT-116 and DLD-1 colon cancer cells.

[0052] Figure 13 The effects of licorice flavonoids on mouse organs.

[0053] Figure 14 The effect of glycyrrhizin flavonoids on liver function in mice. A represents aspartate aminotransferase (AST) / gOT; B represents alanine aminotransferase (ALT) / gPT.

[0054] Figure 15 The effect of glycyrrhizin on the proliferation of NCM460 cells.

[0055] Figure 16 The effect of glycyrrhizin on the cell colony formation ability of NCM460 cells.

[0056] Figure 17 The effects of Ganoderma lucidum equine and isophylline-9-one on the proliferation of HCT-116 and DLD-1 colon cancer cells were investigated. A represents the effect of Ganoderma lucidum equine on HCT-116 cells; B represents the effect of Ganoderma lucidum equine on DLD-1 cells; C represents the effect of isophylline-9-one on HCT-116 cells; and D represents the effect of isophylline-9-one on DLD-1 cells.

[0057] Figure 18 The effects of Ganoderma lucidum equine and isophylline-9-one on the proliferation of NCM460 cells were investigated. A represents Ganoderma lucidum equine; B represents isophylline-9-one.

[0058] Figure 19 The effects of Ganoderma lucidum equine and isophylline-9-one on the proliferation of colon cancer cells in a zebrafish xenograft model.

[0059] Figure 20 The effects of Ganoderma lucidum equine and isophylline-9-one on the metastasis of colon cancer cells in a zebrafish xenograft model.

[0060] Figure 21 The effect of nordihydroguaiacol on the proliferation of HCT-116, DLD-1, and NCM460 cells was investigated. A represents HCT-116 cells; B represents DLD-1 cells; and C represents NCM460 cells.

[0061] Figure 22 The effect of glycyrrhizin on the proliferation of HCT-116, DLD-1, and NCM460 cells is shown. A represents HCT-116 cells; B represents DLD-1 cells; and C represents NCM460 cells.

[0062] Figure 23 The effects of a combination of glycyrrhizin, ganoderic acid, isophylline-9-one, and nordihydroguaiacol on the proliferation of NCM460 cells.

[0063] Figure 24 The effects of combinations of glycyrrhizin, ganoderic acid, isophylline-9-one, and nordihydroguaiacol on the proliferation of HCT-116 and DLD-1 cells were investigated. In this study, A represents HCT-116 cells, and B represents DLD-1 cells. Detailed Implementation

[0064] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0065] Example 1: Isolation and identification of Ganoderma lucidum equine, isophyllene-9-one, glycyrrhizin and nordihydroguaiaric acid

[0066] I. Experimental Methods

[0067] 1. Sample processing

[0068] The licorice sample was taken out and ground evenly with liquid nitrogen. Approximately 100 mg of the sample was weighed into a 1.5 mL centrifuge tube. 1 mL of water was added, and the mixture was vortexed for 1 min. Steel balls were added, and the mixture was pre-cooled in a -40℃ refrigerator for 2 min. The mixture was then ground in a grinder (60 Hz, 2 min), and extracted by sonication in an ice-water bath for 60 min. After centrifugation for 10 min (14000 rpm, 4℃), the mixture was diluted 5 times with water to obtain the diluted supernatant, which is licorice flavonoid. 200 μL of the diluted supernatant was placed into an LC-MS vial with an inner liner for analysis.

[0069] 2. Liquid Chromatography-Mass Spectrometry Conditions

[0070] (1) The analytical instrument is a liquid chromatography-mass spectrometry system consisting of an ACQUITY UPLC I-Class HF ultra-high performance liquid chromatography-tandem QE high resolution mass spectrometer.

[0071] (2) Chromatographic conditions

[0072] Column: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); Column temperature: 45℃; Mobile phase: Mobile phase A was 0.1% (w / v) formic acid aqueous solution, and mobile phase B was acetonitrile; Flow rate: 0.35 mL / min; Gradient elution information is shown in Table 1; Injection volume: 5 μL; PDA (diode array detector) scan range: 210~400 nm.

[0073] (3) Mass spectrometry conditions

[0074] Ion source: HESI (Heated Elektrospray Ionization source).

[0075] The mass spectrometry signals of the samples were acquired using both positive and negative ion scanning modes.

[0076] Data acquisition mode: DDA (Data Dependency Acquisition Mode).

[0077] Scanning method: Secondary ion scanning mode Full MS / dd-MS2 (TOP 8).

[0078] The parameters for positive and negative ions are shown in Table 2.

[0079] Table 1 Elution gradient information

[0080]

[0081] Table 2 Positive and Negative Ion Parameters

[0082]

[0083] 3. Data Analysis

[0084] Before pattern recognition, the raw data underwent preprocessing using Progenesis QI v3.0 software (Nonlinear Dynamics, Newcastle, UK) for baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization. Compound identification was based on precise mass number, secondary fragments, and isotopic distribution, using the TCM database for qualitative analysis. The TCM database is a dedicated database of traditional Chinese medicine components for plant samples, containing information on over 5000 TCM component standards (purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd., etc.). For each identified component, an EIC plot and an MS2 mirror image with secondary fragment structure annotations were generated. Chemical composition was determined by comparing MS / MS plots with the LuMet-TCM database of TCM metabolites (a standard library) and the HerbDB public database.

[0085] II. Experimental Results

[0086] 1. Identification of Ganoderma lucidum equine

[0087] like Figure 1 The image shows the EIC chromatogram of Ganodermanontriol and its MS / MS chromatogram compared with the standard library LuMet-TCM. The MS / MS chromatogram compared with the standard library LuMet-TCM shows a retention time of 11.40 min, which largely overlaps with the standard chromatogram. Therefore, this compound can be considered as Ganodermanontriol (CAS: 106518-63-2).

[0088] 2. Identification of isophyllene-9-one

[0089] like Figure 2 The image shows the EIC spectrum of isolongifolen-9-one and its MS / MS spectrum compared with the HerbDB library. The EIC spectrum of this compound largely overlaps with the MS / MS spectrum compared with the HerbDB library, confirming that this compound is isolongifolen-9-one (CAS: 23747-14-0).

[0090] 3. Identification of nordihydroguaiac acid

[0091] like Figure 3The image shows the EIC chromatogram of nordihydroguaiaretica acid and its MS / MS chromatogram compared with the HerbDB public library. Based on a retention time of 6.86 min and qualitative analysis using RT+MS1 (comparison with a standard library) or predicted RT+MS1 (comparison with a public library), this compound can be identified as nordihydroguaiaretica acid (CAS: 500-38-9).

[0092] 4. Identification of glycyrrhizin

[0093] like Figure 4 The image shows the EIC chromatogram of glabrone and its MS / MS chromatogram compared with the standard library LuMet-TCM. The MS / MS chromatogram compared with the standard library LuMet-TCM shows a retention time of 10.09 min, which largely overlaps with the standard chromatogram. Therefore, this compound can be identified as glabrone (CAS: 60008-02-8).

[0094] Example 2: Inhibitory effect of glycyrrhizin on AOM / DSS-induced inflammation-related colon cancer in mice.

[0095] I. Experimental Methods

[0096] 1. Animal models

[0097] SPF-grade C57BL / 6 male mice were acclimatized for one week before model initiation. Model initiation was divided into three phases: Phase 1: Except for the blank control group (which received intraperitoneal injection of saline), the other groups of mice received intraperitoneal injection of the carcinogen azomethane (AOM) (10 mg / kg), followed by free access to 2% (w / v) sodium dextran sulfate (DSS) in drinking water for 7 days, then switched to regular drinking water for 14 days; Phase 2: Free access to 2% (w / v) DSS in drinking water for 10 days, then switched to regular drinking water for 14 days; Phase 3: Free access to 2% (w / v) DSS in drinking water for 10 days, then switched to regular drinking water for 11 days. The blank control group received regular drinking water throughout the entire phase. Modeling was considered successful when mice exhibited lethargy, bloody stools, or mucus-bloody stools, indicating the presence of colon cancer mice. Mouse weight and food intake were recorded daily.

[0098] 2. Drug intervention

[0099] Following AOM injection, mice were orally administered glycyrrhizin at doses of 208 mg / kg and 416 mg / kg daily until the endpoint. The 208 mg / kg dose was calculated based on the human clinical prescription dose (1.6 g / 70 kg = 0.0228 g / kg) and the body surface area between humans and animals (0.0228 g / kg × 9.1 = 208 mg / kg). Regorafenib (Selleck, USA) at 20 mg / kg served as a positive control.

[0100] 3. Colon length analysis

[0101] After the experiment, the colon of the mice was removed, from above the anus to below the cecum, and the changes in colon length in each group of mice were measured.

[0102] 4. Tumor Count Statistics

[0103] After the experiment, the mouse colons were cut open, cleaned in saline, and observed on a white light plate to assess the tumors in the colon. The findings were then statistically analyzed.

[0104] 5. Hematoxylin-eosin staining (HE staining)

[0105] At the end of the experiment, mouse colon tissue was collected, fixed with 4% (w / v) paraformaldehyde for 48 h, treated with 80% (v / v) ethanol for 5 h, 90% (v / v) ethanol for 5 h, and left overnight with 95% (v / v) ethanol. Anhydrous ethanol I, II and III were treated for 30 min each, xylene I, II and III were treated for 30 min each, paraffin I and II were treated for 30 min each, and paraffin III was treated for 1 h. After dehydration, paraffin infiltration, embedding and sectioning were completed to obtain paraffin sections.

[0106] Paraffin sections were stained with hematoxylin and eosin (HE) according to the steps in Table 3 below, and then mounted with neutral resin. All percentages in Table 3 are volume concentrations.

[0107] Table 3 HE staining steps

[0108]

[0109] 6. Periodic acid Schiff staining (PAS staining)

[0110] Obtain paraffin sections according to step 5.

[0111] Dewaxing paraffin sections: The paraffin sections were placed in xylene for dewaxing twice, 15 minutes each time, to obtain dewaxed sections.

[0112] Hydration: The dewaxed sections were hydrated by sequentially passing them through a gradient of alcohols. The specific steps were: 5 min of anhydrous ethanol, 3 min of 90% ethanol, 3 min of 80% ethanol, 3 min of 70% ethanol; 3 min of distilled water, repeated 3 times; and 3 min of PBS buffer, repeated 3 times, to obtain hydrated sections.

[0113] Oxidation: The hydrated sections were placed in a 1% (w / v) periodic acid solution and oxidized for 6-20 min to obtain oxidized sections.

[0114] Washing: Rinse the oxidized sections thoroughly with distilled water to remove excess periodic acid solution for 10 min to obtain washed oxidized sections.

[0115] Staining: Place the washed oxidized sections into fuchsin-sulfurite reagent (Schiff reagent) and stain for 10-20 minutes. The staining time can be adjusted according to the room temperature. In summer, when the room temperature is high, the staining time can be reduced, while in winter, when the room temperature is low, the staining time can be extended to 20 minutes to obtain Schiff-stained sections.

[0116] Rinsing: Rinse the Schiff stained sections with running water until the water color changes from red to colorless for 10 minutes to obtain the rinsed Schiff stained sections.

[0117] Counterstaining: The rinsed Schiff stained sections are counterstained with hematoxylin to make the cell nuclei appear blue. This usually takes 2 to 4 minutes to obtain counterstained sections.

[0118] Differentiation: If the cell nuclei are stained too darkly during the counterstaining process, hydrochloric acid alcohol can be used to differentiate the counterstained sections for about 2 to 5 seconds to obtain differentiated sections.

[0119] Blueing: Use Scott's Bluing Reagent to blue the counterstained or differentiated sections for 1-2 minutes, then rinse with running water for 3 minutes to obtain the blue-rebounded sections.

[0120] Dehydration: The blue-returned sections were sequentially dehydrated by gradient alcohol treatment. The specific steps were: 70% (v / v) ethanol for 1 min, 80% (v / v) ethanol for 1 min, 95% (v / v) ethanol for 2 min, and 100% (v / v) ethanol for 4 min to obtain dehydrated sections.

[0121] Clearing: The dehydrated sections are cleared with xylene, usually twice, for 15 minutes each time, to obtain clear sections.

[0122] Mounting: Dry the transparent slide, add a drop of neutral resin to mount the slide, and cover with a coverslip.

[0123] 7. Detection of cell proliferation antigen (Ki67)

[0124] Following step 6, hydrated sections are obtained. These sections are then immersed in a 3% (v / v) hydrogen peroxide solution for 10 minutes to block endogenous peroxidase activity. After rinsing with tap water, hydrogen peroxide-treated sections are obtained.

[0125] Hydrogen peroxide-treated sections were placed in citrate buffer (pH 6.0) for heat-induced antigen retrieval, and then microwaved for 20 min to obtain retrieval sections.

[0126] The repaired sections were allowed to cool naturally to room temperature, washed with PBS buffer, covered with bovine serum albumin (BSA), and incubated at room temperature for 10 min to reduce non-specific binding, resulting in BSA-treated sections.

[0127] Add 3 drops of Ki67 antibody reagent to each BSA-treated slide (preferably enough to completely cover the slide tissue), incubate at room temperature for 30 min, wash with PBS buffer for 5 min each time, repeat 3 times to obtain Ki67-treated slides.

[0128] Secondary antibody incubation: Add approximately 100 μL of enzyme-labeled goat anti-mouse / rabbit IgG polymer to each Ki67-treated slide (enough to completely cover the slide tissue), incubate at room temperature for 30 min, wash with PBS buffer for 5 min each time, repeat 3 times to obtain secondary antibody-incubated slides.

[0129] The secondary antibody-incubated sections were stained with freshly prepared DAB (diaminobenzidine) chromogenic solution. Approximately 100 μL of DAB chromogenic solution was added to each secondary antibody-incubated section, and the chromogenic time was 1–5 min (staining was controlled under a microscope). The chromogenic reaction was terminated by rinsing with tap water to obtain DAB-treated sections.

[0130] Follow the method in step 6 to counterstain, dehydrate, clear, and mount the DAB-treated sections.

[0131] II. Experimental Results

[0132] 1. Licorice flavonoids prolong the survival time of mice with AOM / DSS colon cancer.

[0133] like Figure 5 As shown, glycyrrhizin prolongs the survival time of mice in a dose-dependent manner. In the control group, the number of mice dying increased continuously with the extension of the experimental time. The number of surviving mice in the low-dose glycyrrhizin group tended to stabilize in the middle and late stages of treatment, while the number of mice in the high-dose glycyrrhizin group remained stable throughout the entire treatment process.

[0134] 2. Licorice flavonoids reduce the number of tumors in the colon of mice.

[0135] like Figure 6 As shown in Figure A, the colon length of mice in the glycyrrhizin-treated group was significantly longer than that in the control group, indicating that glycyrrhizin treatment can significantly reduce colonic inflammation in mice. Figure 6 As shown in Figure B, compared with the control group, the number of tumors in the colon of mice treated with licorice flavonoids was significantly reduced, indicating that licorice flavonoids have a significant inhibitory effect on colon cancer.

[0136] 3. Licorice flavonoids repair the colonic mucosa of mice, improve intestinal damage, and inhibit the malignant proliferation of tumor cells.

[0137] like Figure 7 As shown in the results of HE staining, PAS staining, and Ki67 staining of mouse intestines, the intestinal barrier of control mice was significantly damaged, with increased intercellular spaces, reduced goblet cells, and a high degree of malignant proliferation of tumors in the colon. Treatment with different doses of glycyrrhizin repaired the intestinal barrier, increased goblet cells, and reduced the degree of malignant tumor proliferation. This indicates that glycyrrhizin can repair the colonic mucosa of mice, improve intestinal damage, inhibit the malignant proliferation of tumor cells, and has a therapeutic effect on colon cancer.

[0138] Example 3: Glycyrrhizin inhibits the development of colon cancer in zebrafish xenografts.

[0139] I. Experimental Methods

[0140] 1. Establish a model

[0141] AB-type zebrafish were used as a model organism, and the fish were reared according to standard procedures. Embryos were obtained from naturally spawning fish and reared in a 28.5°C, 12:12 light-dark (LD) cycle in water containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.3 mM MgSO4; pH=7. All rearing and experimental procedures complied with Italian and European animal protection regulations for scientific purposes (Directive 2010 / 63 / EU). For all experimental data, embryonic age is expressed in hours post-fertilization (hpf), hours post-injection (hpi), and days post-fertilization (dpf).

[0142] 2. Cell Culture and Labeling

[0143] Human colon cancer cells HCT-116 were screened based on dedifferentiation phenotype, invasion, metastatic potential, and clinical significance. They were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640) medium supplemented with 10% (v / v) fetal bovine serum (FBS) at 37°C in a humidified air incubator with 5% CO2. For xenografting, HCT-116 cells were washed, digested, and cultured in a Dil Vybrant solution.TM Resuspend the cells in RPMI 1640 medium containing red fluorescent dye (brand: Thermo Fisher Scientific, USA; catalog number: V22885) for 20 min. Centrifuge the cells at 1500 rpm for 5 min, discard the supernatant, and wash twice with PBS to obtain dil-labeled HCT-116 cells. Resuspend the dil-labeled HCT-116 cells in PBS (phosphate-buffered saline) and inject them into zebrafish yolk.

[0144] 3. Xenotransplantation

[0145] AB-type zebrafish embryos at 48 hpf were fixed on 2% (w / v) agarose pads with 0.04% tricaine (Merck, Germany; catalog number: E10521). 200 dil labeled HCT-116 cells were injected into zebrafish yolk sacs to observe tumor volume and its interactions with the host and therapeutic agents. Tumor volume changes at 3 dpf were observed using fluorescence microscopy. The fluorescence diffusion of tumor cells at 5 dpf 19 was observed to investigate the metastatic potential of the tumor.

[0146] 4. Drug intervention

[0147] Glycyrrhizin flavonoids were dissolved in DMSO (dimethyl sulfoxide), and zebrafish were exposed to three dose groups at concentrations of 5, 10, and 20 µg / mL, selected based on in vivo toxicology studies in zebrafish. The control group of zebrafish was exposed to water containing the same concentration of DMSO. The final concentration of DMSO in each group was controlled to be below 1% (v / v).

[0148] Quantitative analysis of tumor volume and metastatic potential: Zebrafish successfully injected with HCT-116 cells into their yolk sacs were exposed to drugs (glycyrrhizin or DMSO) for 24 h and 72 h, respectively, to observe the effects of the drugs on tumor volume and tumor spread. Fluorescence stereomicroscopy imaging was used, and ImageJ analysis was performed to analyze the fluorescence area and intensity, characterizing the effects of the drugs on tumor volume and tumor dissemination.

[0149] II. Experimental Results

[0150] 1. Licorice flavonoids inhibit the proliferation of colon cancer cells in a zebrafish xenograft model.

[0151] like Figure 8 As shown, compared with the control group, the fluorescence area of ​​yolk sac tumor cells in the licorice flavonoid-treated group was significantly reduced, and the effect was concentration-dependent, with the effect becoming more pronounced as the concentration increased.

[0152] 2. Licorice flavonoids inhibit tumor cell metastasis.

[0153] like Figure 9As shown, the extent of tumor cell spread throughout the body was significantly reduced in the glycyrrhizin-treated group, and the effect was better with increasing concentration, indicating that glycyrrhizin can inhibit tumor cell metastasis.

[0154] Example 4: Licorice flavonoids inhibit the proliferation of colon cancer cells in vitro.

[0155] I. Experimental Methods

[0156] 1. DLD-1 cells and HCT-116 cells

[0157] Human colon cancer cells HCT-116 and DLD-1 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium supplemented with 10% (v / v) FBS and 1% penicillin-streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin) in an incubator with humidified air at 37°C and 5% CO2.

[0158] 2. CCK-8 assay for cell proliferation

[0159] CCK-8 assay for cell viability: Cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 Each experiment was repeated 4 times. After incubation at 37°C for 24 h, different final concentrations of glycyrrhizin (0, 1, 2, 4, 8, 16, 32, 64 and 128 μg / mL) were added and cultured for 24 h. 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 h. The optical density (OD) value was measured at 450 nm using a microplate reader.

[0160] 3. Cell clone formation experiment

[0161] The colony formation assay was performed as follows: 3000 cells / well were inoculated into a 6-well plate, and different final concentrations of glycyrrhizin (0, 2, 4, 8, 16 and 32 μg / mL) were added. After culturing for 1 week, the colonies were clearly visible. The colonies were fixed with 4% (w / v) paraformaldehyde and stained with crystal violet. The colonies were dissolved with 1% (w / v) SDS (sodium dodecyl sulfate), and the absorbance was measured at 570 nm for quantitative analysis.

[0162] 4. Edu (5-ethynyl-2'-deoxyuridine) staining experiment

[0163] Edu assay for cell proliferation: Cells were cultured on slides and incubated for 24 h with different final concentrations of glycyrrhizin (0, 2, 4, 8, 16, and 32 μg / mL). Edu was added to each slide, and the cells were incubated for 1.5 h in a 37°C incubator with 5% CO2 humidified air. Cells were then fixed with 4% (w / v) paraformaldehyde and analyzed using the E-clik EDU cell proliferation Imaging Assay Kit (Green, Elab Fluor® 488). Edu-positive cells were recorded using confocal microscopy, and quantitative fluorescence analysis was performed using ImageJ.

[0164] II. Experimental Results

[0165] 1. Licorice flavonoids have a killing effect on colon cancer cells.

[0166] like Figure 10 A and Figure 10 As shown in Figure B, with increasing glycyrrhizin concentration, the number of dead cells increased, and the IC50 of glycyrrhizin in HCT-116 and DLD-1 was higher. 50 The values ​​were 20.92 μg / mL and 27.93 μg / mL, respectively.

[0167] 2. Licorice flavonoids inhibit the clonal formation of colon cancer cells.

[0168] like Figure 11 As shown, the colony-forming ability of HCT-116 and DLD-1 cells was significantly weakened after treatment with glycyrrhizin, and the number of cell colonies decreased significantly with increasing concentration, indicating that glycyrrhizin can weaken the proliferation ability of colon cancer cells in vitro.

[0169] 3. Licorice flavonoids inhibit tumor cell proliferation.

[0170] like Figure 12 As shown, Edu staining revealed that as the concentration of glycyrrhizin increased, the number of Edu-positive cells gradually decreased. DLD-1 and HCT-116 cells showed a similar trend, further indicating that glycyrrhizin can inhibit the proliferation of colon cancer cells.

[0171] Example 5: In vivo safety evaluation of glycyrrhizin flavonoids

[0172] I. Experimental Methods

[0173] 1. HE staining

[0174] According to Example 2, mouse heart, liver, spleen, lung and kidney sections were fixed, hydrated and stained with hematoxylin to reveal the fine structure of the cell nucleus. Then, the cytoplasm was contrast stained with eosin. After a series of gradient alcohol dehydration and clearing treatments, the sections were mounted and images were obtained using a digital slide scanner (KFBIO, China).

[0175] 2. Assay of ALT and AST activities

[0176] According to Example 2, peripheral blood was collected from each group of mice, and serum was obtained by centrifugation at 3000 rpm and 4℃ for 10 min. The liver function of the mice was detected using an aspartate aminotransferase (AST / GOT) test kit (microplate method); manufacturer: Nanjing Jiancheng Biotechnology Research Institute; catalog number: C010-2-1) and an alanine aminotransferase (ALT / GPT) test kit (microplate method); manufacturer: Nanjing Jiancheng Biotechnology Research Institute; catalog number: C009-1-1.

[0177] II. Experimental Results

[0178] 1. Licorice flavonoids have no obvious toxicity to mouse organs.

[0179] like Figure 13 As shown, compared with the control group, the mice in the glycyrrhizin-treated group showed no obvious damage or inflammatory infiltration in the heart, liver, spleen, lungs and kidneys, indicating that glycyrrhizin is safe for in vivo administration.

[0180] 2. Licorice flavonoids have no significant effect on liver function in mice.

[0181] like Figure 14 A and Figure 14 As shown in Figure B, compared with the control group, the activities of aspartate aminotransferase (AST / GOT) and alanine aminotransferase (ALT / GPT) in the serum of mice were not significantly reduced after administration of glycyrrhizin, indicating that glycyrrhizin has no effect on liver function in mice and also demonstrates its safety.

[0182] Example 6: In vitro safety evaluation of glycyrrhizin flavonoids

[0183] I. Experimental Methods

[0184] The effect of licorice flavonoids on the proliferation and cell colony formation of normal human colonic epithelial cells NCM460 was detected using the method in Example 4.

[0185] II. Experimental Results

[0186] 1. Licorice flavonoids have little effect on the proliferation of NCM460 cells.

[0187] like Figure 15As shown, after 24 h of administration of glycyrrhizin to NCM460 cells, the cells still maintained a viability of over 50% until the glycyrrhizin concentration reached its maximum of 128 µg / mL, indicating the relative safety of in vitro administration of glycyrrhizin.

[0188] 2. Licorice flavonoids do not affect NCM460 cell clone formation.

[0189] like Figure 16 As shown, glycyrrhizin has no significant effect on the number of NCM460 cell clones and no significant inhibitory effect on the size of individual clones, indicating that glycyrrhizin has little effect on the proliferation of normal human colonic epithelial cells, thus demonstrating its safety.

[0190] Example 7: In vitro inhibition of colon cancer cell proliferation by Ganoderma lucidum equine and isophylline-9-one.

[0191] I. Experimental Methods

[0192] 1. Following the method in Example 4, the effects of Ganoderma lucidum equine and isophylline-9-one on the proliferation of human colon cancer cells DLD-1 and HCT-116, as well as normal human colonic epithelial cells NCM460, were detected by the CCK-8 assay. Cells were cultured with different concentrations of Ganoderma lucidum equine and isophylline-9-one for 24 h and 48 h, respectively.

[0193] II. Experimental Results

[0194] 1. Ganoderma lucidum equine and isophylline-9-one inhibit the proliferation of colon cancer cells.

[0195] like Figure 17 As shown in A-D and Table 4, both Ganoderma lucidum equine and isophylline-9-one significantly inhibited cell proliferation in the two colon cancer cell lines, indicating that Ganoderma lucidum equine and isophylline-9-one have an in vitro intervention effect on colon cancer.

[0196] Table 4. Effects of Ganoderma lucidum malone and isophorone-9-one on the proliferation of HCT-116 and DLD-1 colon cancer cells.

[0197]

[0198] 2. Ganoderma lucidum equine and isophylline-9-one do not affect the proliferation of NCM460 cells.

[0199] like Figure 18 A, Figure 18 As shown in B and Table 5, treatment of NCM460 cells with Ganoderma lucidum equine and isophylline-9-one for 24 h and 48 h did not show significant inhibition of cell proliferation, indicating that Ganoderma lucidum equine and isophylline-9-one have certain safety.

[0200] Table 5. Effects of Ganoderma lucidum malone and isophylline-9-one on NCM460 cell proliferation.

[0201]

[0202] Example 8: In vivo intervention of Ganoderma lucidum equine and isophylline-9-one in the development and progression of colon cancer.

[0203] I. Experimental Methods

[0204] Using the method in Example 3, the effects of Ganoderma lucidum equine and isophylline-9-one on colon cancer cells in a zebrafish xenograft model were evaluated.

[0205] II. Experimental Results

[0206] 1. Ganoderma lucidum equine and isophylline-9-one inhibit tumor cell proliferation.

[0207] like Figure 19 As shown, the fluorescence area of ​​zebrafish yolk sac tumor cells was significantly reduced after treatment with Ganoderma lucidum equine and isophylline-9-one, and the effect was concentration-dependent, with the effect becoming more pronounced as the concentration increased, indicating that both Ganoderma lucidum equine and isophylline-9-one can inhibit tumor proliferation.

[0208] 2. Ganoderma lucidum equine and isophylline-9-one delay the spread of tumor cells.

[0209] like Figure 20 As shown, treatment with Ganoderma lucidum equine and isophylline-9-one significantly reduced the spread of tumor cells throughout the zebrafish, and the effect was better with increasing concentration, indicating that Ganoderma lucidum equine and isophylline-9-one delayed the spread of tumor cells.

[0210] Example 9: Inhibitory effect of nordihydroguaiaric acid on colon cancer

[0211] I. Experimental Methods

[0212] Following the method in Example 4, the effects of nordihydroguaiaric acid on the proliferation of human colon cancer cells DLD-1 and HCT-116, as well as normal human colonic epithelial cells NCM460, were detected by the CCK-8 assay. Cells were cultured with different concentrations of nordihydroguaiaric acid for 24 h and 48 h.

[0213] II. Experimental Results

[0214] 1. Nordihydroguaiac acid inhibits the proliferation of colon cancer cells.

[0215] like Figure 21As shown in A~C and Table 6, nordihydroguaiaric acid has a certain inhibitory effect on the proliferation of HCT-116 and DLD-1 colon cancer cells, and this effect is time-dependent, while it has no significant inhibitory effect on NCM460 cells.

[0216] Table 6. Effects of nordihydroguaiac acid on the proliferation of HCT-116, DLD-1, and NCM460 cells.

[0217]

[0218] Example 10: Inhibitory effect of glycyrrhizin on colon cancer

[0219] I. Experimental Methods

[0220] Following the method in Example 4, the effects of glycyrrhizin on the proliferation of human colon cancer cells DLD-1 and HCT-116, as well as normal human colonic epithelial cells NCM460, were detected using the CCK-8 assay. Cells were cultured with different concentrations of glycyrrhizin for 24 h and 48 h.

[0221] II. Experimental Results

[0222] like Figure 22 As shown in A-C and Table 7, glycyrrhizin strongly inhibits the proliferation of HCT-116 and DLD-1 colon cancer cells in a time-dependent manner. However, glycyrrhizin also strongly inhibits the proliferation of NCM460 cells, exhibiting significant toxicity.

[0223] Table 7 Effects of glycyrrhizin on the proliferation of HCT-116, DLD-1, and NCM460 cells.

[0224]

[0225] Example 11: Inhibitory effect of the combination of glycyrrhizin, ganoderic acid, isophylline-9-one, and nordihydroguaiaric acid on colon cancer.

[0226] I. Experimental Methods

[0227] Following the method in Example 4, the effects of the combination of glycyrrhizin, ganoderic acid, isophylline-9-one, and nordihydroguaiaric acid on the proliferation of HCT-116 and DLD-1 colon cancer cells and NCM460 normal human colon epithelial cells were detected by CCK-8 assay. Cells were cultured at different concentrations of the combination for 24 h and 48 h.

[0228] Nine gradient concentrations were set up for the combination of glycyrrhizin, Ganoderma equine, isophyllene-9-one, and nordihydroguaiaric acid, namely 0, 1, 2, 4, 8, 16, 32, 64 and 128 μM. Each gradient concentration represents the concentration of each component, and the concentration of each component is the same.

[0229] II. Experimental Results

[0230] like Figure 23 As shown in Table 8, when glycyrrhizin was administered in combination with Ganoderma lucidum equine, isophyllene-9-one, and nordihydroguaiaric acid, it was found that it could significantly reduce the toxicity of glycyrrhizin to normal human colonic epithelial cells NCM460, indicating that these four components have a synergistic effect and reduce toxicity.

[0231] like Figure 24 As shown in Table 8, co-administration of glycyrrhizin with Ganoderma lucidum equine, isophyllene-9-one, and nordihydroguaiaric acid significantly inhibited the proliferation of HCT-116 and DLD-1 colon cancer cells, with effects stronger than those of any single component administered alone. This indicates a synergistic effect among the four components.

[0232] Table 8. Effects of combinations of glycyrrhizin, ganoderic acid, isoflavone-9-one, and nordihydroguaiac acid on the proliferation of HCT-116, DLD-1, and NCM460 cells.

[0233]

[0234] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of glycyrrhizin in the preparation of drugs for treating colon cancer, characterized in that, The licorice flavonoids are Ganoderma lucidum quinone, isolongifene-9-one, nordihydroguaiac acid and glycyrrhizin; The concentrations of Ganoderma lucidum equine, isolongifene-9-one, nordihydroguaiacol, or glycyrrhizin are 32–128 μM.

2. The application as described in claim 1, characterized in that, The drug in question is a drug that inhibits the proliferation of colorectal cancer.

3. The application as described in claim 1, characterized in that, The drug in question is a drug that inhibits the metastasis of colorectal cancer.

4. The application as described in claim 1, characterized in that, The drug in question is a drug for repairing the intestinal barrier.

5. The application as described in claim 2 or 3, characterized in that, The drug inhibits the proliferation and / or metastasis of colorectal cancer HCT-116 cells and / or DLD-1 cells.

6. The application as described in claim 1, characterized in that, The drug is a pharmaceutical preparation made by adding pharmaceutically acceptable excipients to glycyrrhizin as the active ingredient.

7. The application as described in claim 6, characterized in that, The dosage form of the drug includes tablets, granules, or liquid preparations.

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