Use of glychalon A and glychalon A in combination with glycyrrhizine and glycyrrhizine derivatives for the preparation of a medicament for the treatment of colorectal cancer

Abstract

The application provides a kind of licorice chalcone A and the application of the composition of light glycyrrhizin and licorice chalcone A in preparing the drug for treating colorectal cancer.Belongs to the technical field of antitumor biological medicine.The inhibitory activity of LCA and CMC on human colon cancer cells is verified by CCK-8 method, and the survival rate is significantly reduced.The influence of high-dose and medium-dose LCA and CMC on the apoptosis of human colon cancer cells is detected by flow cytometry technology, and the apoptosis rate is increased.The influence of LCA and CMC on the migration of human colon cancer cells is studied by cell scratch test, and the migration ability of cells can be significantly inhibited.The influence of LCA and CMC on the invasion ability of human colon cancer cells is studied by Transwell experiment, and the number of invasive cells is significantly reduced.LCA and CMC can inhibit the proliferation of human colon cancer cells in vitro, and induce tumor cell apoptosis, and the mechanism may be related to the inhibition of PI3K / AKT / mTOR signal pathway.

Description

Technical Field

[0001] This invention belongs to the field of anti-tumor biomedical technology, and specifically relates to the application of a composition of glycyrrhizin and glycyrrhizin in the preparation of a drug for treating colorectal cancer. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors of the digestive tract, and its incidence is gradually increasing. Currently, treatment primarily involves a combination of surgery, radiotherapy, chemotherapy, and targeted therapy, but it suffers from short survival and poor prognosis. Traditional Chinese medicine (TCM), as an adjunct to cancer treatment, can improve patient prognosis and quality of life, and has gained widespread clinical recognition. Licorice is a commonly used qi-tonifying herb in TCM. Liang Jun's analysis of ancient TCM prescriptions for treating colon cancer revealed that licorice was extensively used as a tonifying herb, ranking fourth in frequency among all herbs, and serving as a core ingredient in many colon cancer treatment formulas.

[0003] Although literature reports that crude extracts of licorice, effective components of flavonoids or saponins, and monomeric compounds have varying degrees of inhibitory effects on tumors such as breast cancer, prostate cancer, liver cancer, gastric cancer, bladder cancer, and lung cancer, the mechanism of action of traditional Chinese medicine itself, characterized by "multiple components, multiple pathways, and multiple targets," needs further in-depth research. Modern scientific and technological methods are needed to reveal the pharmacodynamic material basis and mechanism of action of traditional Chinese medicine, thereby providing a basis for the classic clinical application of ancient prescriptions.

[0004] Licochalcone A (CAS No.: 58749-22-7) is a natural phenolic compound, as shown in Formula 1. It can be isolated from licorice and has shown in vitro antimalarial, anticancer, antibacterial, and antiviral properties (especially against influenza virus neuraminidase).

[0005]

[0006] In the prior art, for example, Chinese invention patent No. 201210403351.8 discloses the application of glycyrrhizin A in the preparation of anticancer drugs or health products, and specifically discloses that glycyrrhizin A can significantly inhibit the proliferation and colony formation of 4T1 mouse breast cancer cells, MDA-MB-231 human breast cancer cells, A375 human melanoma cells, AGs human gastric cancer cells, human pancreatic cancer PANC-1, human liver cancer cell lines HepG2, SMMC-7721, and human lung cancer cell line H2126. However, on the one hand, the mechanism of action of glycyrrhizin A in inhibiting cancer cell proliferation is unclear, and on the other hand, the effect of glycyrrhizin A on colon cancer is rarely reported. Summary of the Invention

[0007] Based on this, the present invention provides the application of glycyrrhizin A in the preparation of a drug for treating colorectal cancer.

[0008] The present invention also provides the use of a composition of glycyrrhizin and glycyrrhizin chalcone A in the preparation of a medicament for treating colorectal cancer.

[0009] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0010] The application of glycyrrhizin A in the preparation of a drug for treating colorectal cancer.

[0011] Preferably, glycyrrhizin A is used as the sole active ingredient in the preparation of a drug for treating colorectal cancer.

[0012] Preferably, the action of glycyrrhizin A significantly increases the expression levels of Cleaved-caspase3, Cleaved-caspase9, and Bax proteins, and significantly decreases the expression levels of Bcl-2 and MMP9 proteins.

[0013] Preferably, in the prepared drug for treating colorectal cancer, glycyrrhizin A exerts its effect by regulating the PI3K / Akt / mTOR signaling pathway.

[0014] Preferably, in the prepared drug for treating colorectal cancer, the concentration of glycyrrhizin A is 5 μg / mL to 40 μg / mL.

[0015] The use of a composition of glycyrrhizin and glycyrrhizin chalcone A in the preparation of a medicament for treating colorectal cancer.

[0016] Preferably, a combination of glycyrrhizin and glycyrrhizin chalcone A is used as the sole active ingredient in the preparation of a drug for treating colorectal cancer.

[0017] Preferably, the combination of glycyrrhizin and glycyrrhizin chalcone A significantly increases the expression levels of Cleaved-caspase3, Cleaved-caspase9, and Bax proteins, while significantly decreasing the expression levels of Bcl-2, MMP2, and MMP9 proteins.

[0018] Preferably, in the prepared medicament for treating colorectal cancer, the combination of glycyrrhizin and glycyrrhizin chalcone A exerts its effect by regulating the PI3K / Akt / mTOR signaling pathway.

[0019] Preferably, in the prepared medicament for treating colorectal cancer, the concentration of the combination of glycyrrhizin and glycyrrhizin chalcone A is 5 μg / mL to 40 μg / mL.

[0020] Compared with the prior art, the present invention has at least the following advantages:

[0021] Glycyrrhizin chalcone A was used to prepare a drug for treating colorectal cancer. The inhibitory activity of glycyrrhizin chalcone A (LCA) on human colon cancer cells SW480 and SW620 was verified by the CCK-8 assay. The results showed that after treatment with 5 μg / mL, 20 μg / mL, and 40 μg / mL LCA for 24 h, 48 h, and 72 h, the survival rates of SW480 and SW620 cells were significantly reduced in all groups. Flow cytometry was used to detect the effect of high- and medium-dose LCA groups (40 μg / mL and 20 μg / mL) on apoptosis of human colon cancer cells SW480 and SW620. The results showed that the apoptosis rates of SW480 and SW620 cells treated with 20 μg / mL and 40 μg / mL LCA were significantly different, with increased apoptosis rates (P < 0.001). The effect of 40 μg / mL LCA on the migration of human colon cancer cells SW480 and SW620 was investigated using a cell scratch assay. The migration rate of SW480 cells treated with LCA was (44.93±0.49)%, which was not statistically significant compared to the control group. However, the migration rate of SW620 cells treated with LCA was (30.33±0.91)%, which was statistically significant (P<0.001), indicating a significant inhibition of cell migration. The effect of 40 μg / mL LCA on the invasive ability of SW480 and SW620 cells was investigated using a Transwell assay. The number of invasive cells in SW480 cells treated with LCA was (63.60±8.91), which was not statistically significant compared to the control group. However, the number of invasive cells in SW620 cells treated with LCA was (53.6±6.47), which was statistically significant (P<0.01), indicating a significant reduction in the number of invasive cells. The effects of LCA active ingredient intervention on the expression levels of metastasis-invasion-related proteins in human colon cancer SW480 and SW620 cells were detected by Western blotting. After LCA drug intervention, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased in SW480 cells (P<0.05, P<0.001, P<0.001), while the expression levels of Bcl-2, MMP2, and MMP9 proteins were significantly decreased (P<0.01). In SW620 cells, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased (P<0.05, P<0.001, P<0.001), while the expression levels of Bcl-2 and MMP9 proteins were significantly decreased (P<0.05). LCA can inhibit the proliferation of human colon cancer SW480 and SW620 cells in vitro and induce tumor cell apoptosis. Its mechanism of action may be related to the inhibition of the PI3K / AKT / mTOR signaling pathway.

[0022] A combination of glycyrrhizin and glycyrrhizin chalcone A (CMC) was used to prepare a drug for treating colorectal cancer. The inhibitory activity of CMC on human colon cancer cells SW480 and SW620 was verified by the CCK-8 assay. The results showed that after treatment with 5 μg / mL, 20 μg / mL, and 40 μg / mL CMC for 24 h, 48 h, and 72 h, the survival rates of SW480 and SW620 cells were significantly reduced in all groups. Flow cytometry was used to detect the effect of high- and medium-dose CMC groups (40 μg / mL and 20 μg / mL) on apoptosis of human colon cancer cells SW480 and SW620. The results showed that the apoptosis rates of SW480 and SW620 cells treated with 20 μg / mL and 40 μg / mL CMC were significantly different, with increased apoptosis rates (P < 0.001). The effect of 40 μg / mL CMC on the migration of human colon cancer cells SW480 and SW620 was investigated using a cell scratch assay. The migration rate of SW480 cells treated with CMC was (33.62±1.86)%, and that of SW620 cells treated with CMC was (22.23±0.77)%, both statistically significant (P<0.001), indicating a significant inhibition of cell migration. The effect of 40 μg / mL CMC on the invasive ability of human colon cancer cells SW480 and SW620 was investigated using a Transwell assay. The number of invasive cells in SW480 cells treated with CMC was (37.80±4.66), and the number of invasive cells in SW620 cells treated with CMC was (32.80±3.27), both statistically significant (P<0.01), indicating a significant reduction in the number of invasive cells. The effects of CMC intervention on the expression levels of metastasis-invasion-related proteins in human colon cancer SW480 and SW620 cells were detected by Western blotting. After CMC intervention, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased in SW480 cells (P<0.05, P<0.001, P<0.001), while the expression levels of Bcl-2, MMP2, and MMP9 proteins were significantly decreased (P<0.01). In SW620 cells, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased (P<0.05, P<0.001, P<0.001), while the expression levels of Bcl-2, MMP2, and MMP9 proteins were significantly decreased (P<0.05). CMC can inhibit the proliferation of human colon cancer SW480 and SW620 cells in vitro and induce tumor cell apoptosis. Its mechanism of action may be related to the inhibition of the PI3K / AKT / mTOR signaling pathway. Attached Figure Description

[0023] Figure 1The effects of TFGR, LCA, GBN, and CMC on the proliferation of SW480 cells were detected by CCK-8 assay (A: LCA; B: TFGR; C: GBN; D: CMC; Compared with the control group, *P<0.05, **P<0.01, n=3).

[0024] Figure 2 The effects of TFGR, LCA, GBN, and CMC on the proliferation of SW620 cells were detected by CCK-8 assay (A: LCA; B: TFGR; C: GBN; D: CMC; Compared with the control group, *P<0.05, **P<0.01, n=3).

[0025] Figure 3 The effects of different experimental groups of licorice active ingredients on apoptosis in human colon cancer SW480 and SW620 cells were investigated.

[0026] Figure 4 The effect of different experimental groups of licorice active ingredients on the nuclear morphology of human colon cancer SW480 and SW620 cells (400×, arrows in the figure indicate condensed chromatin or fragmented nuclei).

[0027] Figure 5 The effects of different experimental groups of licorice active ingredients on the migration of human colon cancer SW480 and SW620 cells (100×, compared with the control group, *p<0.05, **p<0.01, ***p<0.001).

[0028] Figure 6 The effects of TFGR, LCA, CMC, and GBN on the invasive ability of human colon cancer SW480 and SW620 cells (x±s, n=3).

[0029] Figure 7 The effects of TFGR, LCA, and GBN on the expression of apoptosis-related protein in human colon cancer SW480 and SW620 cells (x ± s, n = 3).

[0030] Figure 8 The effect of CMC on the expression of apoptosis-related protein in human colon cancer SW480 and SW620 cells (x±s, n=3).

[0031] Figure 9 The effects of TFGR, LCA, CMC, and GBN on the expression of apoptosis-related gene-related factors in SW480 and SW620 cells (x±s, n=4).

[0032] Figure 10 The effect of CMC on the expression levels of PI3K-Akt-mTOR pathway-related proteins in human colon cancer cells (*P<0.05, ***P<0.01, vs. model group). Detailed Implementation

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other. The technical solutions of the present invention will be further described below with reference to the accompanying drawings of the embodiments of the present invention. The present invention is not limited to the following specific embodiments.

[0034] 1.1 Experimental Materials

[0035] 1.1.1 Cell lines

[0036] Human colon cancer cell lines SW480 and SW620 were purchased from Shanghai Luyu Biotechnology Co., Ltd. All cells were cultured in L15 medium containing 10% fetal bovine serum (FBS) with added antibiotics (100 μg / mL streptomycin and 100 units / mL penicillin), and incubated at 37°C in a 5% CO2 incubator. Cells were passaged every two days at a 1:3 ratio, and only cells in the logarithmic growth phase were used in the experiments.

[0037] 1.1.2 Laboratory Animals

[0038] Sixty male SPF-grade BALB / c nude mice, 3-4 weeks old, weighing 18.0±2.0g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., Certificate No.: NO110322200101294471. The animal experiments were approved by the Ethics Committee of the Animal Experiment Center of Ningxia Medical University.

[0039] 1.1.3 Experimental reagents

[0040] TFGR (UVHPLC ≥98%, batch number: 20141015) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0041] GBN standard (batch number: G006171216) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0042] LCA standard (batch number: P21O8F46473) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0043] 5-Fluorouracil (fluorouracil injection, 5-FU), produced by Tianjin Jinyao Pharmaceutical Co., Ltd., batch number 2004201, specification 10mL: 0.25g;

[0044] 10% chloral hydrate was purchased from the Second Hospital of Lanzhou University.

[0045] 1.1.4 Experimental Reagents

[0046] All experimental reagents were selected from commercially available reagents that meet the standard requirements.

[0047] 1.1.5 Instruments and Equipment

[0048]

[0049] 1.2 Experimental Methods

[0050] 1.2.1 Establishment of a mouse model bearing human colon cancer cells SW480

[0051] SPF-grade male BALB / c nude mice, 3-4 weeks old, weighing 18.0±2.0g, were acclimatized for 5 days at the Experimental Animal Center of Ningxia Medical University after passing quarantine, in preparation for subsequent human colon cancer SW480 and SW620 cell line xenograft tumor modeling.

[0052] Human colon cancer SW480 and SW620 cell lines were cultured and passaged using standard cell culture methods. SW480 cells in good growth phase were collected, and the cell suspension concentration was adjusted to 2 × 10⁻⁶. 6 Cells / mL, dispensed into syringes, with 0.1 mL of cell suspension drawn from each syringe for later use.

[0053] After disinfecting the skin at the injection site with povidone-iodine, the prepared cell suspension was injected subcutaneously into the right axilla of BALB / c nude mice. Following inoculation, the mice were fed as usual. When the transplanted tumor nodules reached approximately 4-5 mm in diameter, they were randomly assigned to groups based on their weight: a blank control group (0.05% sodium carboxymethyl cellulose) 5 mL / kg. -1 Positive control group (5-FU) 20 mg / kg -1 ; TFGR high, medium and low dose groups were administered at 400 mg / kg -1 200mg·kg -1 100mg·kg -1 Glycyrrhizin chalcone A (LCA) high, medium and low dose groups were administered at 40 mg / kg. -1 20 mg·kg -1 5mg·kg -1 ; Glycyrrhizin (GBN) high, medium and low dose groups were administered at 40 mg / kg -1 20 mg·kg -1 5mg·kg -1 The combination of glycyrrhizin chalcone A and glycyrrhizin (CMC) was administered at high, medium, and low doses of 40 mg / kg. -1 20 mg·kg -1 5mg·kg -1 In CMC, the molar ratio of glycyrrhizin A to glycyrrhizin is 1:1.

[0054] Administration method: The positive control group was administered via intraperitoneal injection every other day; the blank control group and the high, medium, and low dose groups of TFGR, LCA, GBN, and CMC were administered via gavage once daily at regular intervals, with the dosage calculated based on body weight, for 14 consecutive days. The behavior and tumor growth of the nude mice were observed daily after administration. The diameter of the subcutaneous tumor was measured every 3-4 days using calipers, and the long axis (A) and short axis (B) of the tumor were measured. The transplanted tumor volume V = A × B was calculated. 2 / 2, plot the tumor volume curve. Mice in each group were anesthetized with 10% chloral hydrate and euthanized by cervical dislocation the day after the last administration. The transplanted tumor tissue was dissected and weighed, and the tumor inhibition rate was calculated for each group: Tumor inhibition rate (%) = [Average tumor mass of model group (g) - Average tumor mass of administered group (g)] / Average tumor mass of model group (g) × 100%. Tissue collection should be rapid to ensure freshness. A portion of the extracted tumor tissue was fixed in 4% paraformaldehyde for 24 hours before subsequent pathological experiments. A portion of the tissue was also preserved in liquid nitrogen for detecting the expression of relevant proteins and mRNAs in the tumor tissue.

[0055] 1.2.2 Preparation of paraffin sections

[0056] After the fixed tumor tissue blocks were trimmed, they were placed in the embedding cassette and marked. Then, they were placed in a fully automatic dehydrator for dehydration and paraffin infiltration. After paraffin infiltration, they were embedded in the cassette. After the paraffin blocks were completely solidified, they were sectioned to a thickness of 5 μm, spread, baked, and stored at 4°C.

[0057] 1.2.3 Pathological observation of tumor tissue from tumor-bearing mice after HE staining

[0058] For HE staining, first dewax the sections sequentially with xylene I and xylene II for 10 min each, then dewax using descending alcohol (in the order of anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and water, 5 min each). Immerse the sections in hematoxylin staining solution for 5 min, quickly wash away excess stain with water, differentiate with hydrochloric acid alcohol for a few seconds, rinse with running water for 15 min to achieve blue reversal. Next, immerse the sections in eosin staining solution for 5 min, quickly wash away excess stain with water. Dehydrate using ascending alcohol (in the order of 70% ethanol, 80% ethanol, and 90% ethanol, 5 min each in 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II). After dehydration, immerse the sections in xylene I and xylene II for 5 min each to achieve transparency. Remove the sections from the xylene and allow them to dry slightly (avoid drying the sections completely). Add neutral resin, cover with a coverslip, and place the sealed slide in a fume hood to dry. Finally, the slides were placed on the microscope stage for image acquisition, observation, and analysis.

[0059] 1.2.4 Immunohistochemistry was used to detect protein expression in transplanted tumor tissue.

[0060] The sections were dewaxed sequentially with xylene I and xylene II for 10 min each, followed by dewaxing with descending alcohol (in the order of anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol, 5 min each). The sections treated with xylene and the graded alcohols were then hydrated in water for several minutes. The hydrated sections were then placed in a retrieval box containing an appropriate amount of citric acid (pH 6.0) retrieval solution and microwaved (sealed with tape to prevent boiling over). The microwave was heated on medium for 8 min, then turned off and kept warm for 8 min, followed by heating on medium-low for 7 min. After retrieval, the sections were allowed to cool naturally and washed with PBS for 5 min (3 times). They were then incubated with 3% hydrogen peroxide for 25 min and washed with PBS for 5 min (3 times). A circular incubation pen was used to prevent reagent runoff, and 10% normal goat serum was added to evenly cover the tissue and blocked at room temperature for 30 min. The serum was discarded, and the primary antibody diluted with PBS was added to evenly cover the tissue. The tissue was then placed in a 4°C refrigerator overnight. The next day, the sections were removed from the refrigerator and warmed to 30 min before being microwaved with PBS. Wash with PBS for 5 min (3 times), add diluted secondary antibody and incubate at room temperature for 50 min, then wash with PBS for 5 min (3 times). Add freshly prepared DAB chromogenic solution to evenly cover the tissue, control the chromogenic process under a microscope, and rinse with pure water to stop the chromogenic process. Counterstain the sections with hematoxylin for 2 min, wash with tap water, differentiate with hydrochloric acid alcohol for a few seconds, and then rinse with tap water to turn blue. Dehydrate with ascending alcohol (in the order of 70% ethanol, 80% ethanol, and 90% ethanol, each for a few seconds, and then in 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II for 5 min each), clear with xylene for 5 min, mount with neutral resin, and air dry in a fume hood. Finally, place the sections on the microscope stage for image acquisition.

[0061] 1.2.5 Western blotting was used to detect the expression levels of tumor-related proteins in xenograft tissue.

[0062] Tissue samples from each group of transplanted tumors were minced into approximately 3mm × 3mm pieces and placed in separate 2mL pre-chilled centrifuge tubes. 0.5-1mL of prepared pre-chilled lysis buffer was added to every 100mg of tumor tissue, along with two small steel balls. The tubes were then placed in an automated cryogenic homogenizer and homogenized at 60Hz for 90s. The lysed tissue was centrifuged at 12000rpm / min at 4℃ for 5min. The supernatant was transferred to newly labeled, pre-chilled centrifuge tubes to obtain the total protein extracts from the tumor tissues of each treatment group. The total protein content of the total protein extracts from each group of tumor tissues was measured using a BCA protein quantification kit. Loading buffer was added to the total protein extracts from each group of tumor tissues at a ratio of 4:1. The mixture was boiled for 5min to denature the proteins before aliquoting. The prepared electrophoresis plates were placed in an electrophoresis tank, electrophoresis buffer was added, and the protein samples from each group were added. Electrophoresis was performed at a constant voltage of 120V for separation. Under 200 mA conditions, transfer the target protein bands from the gel to a PVDF membrane (activated with methanol), wash three times with PBST for 10 min each time. Then, place the PVDF membrane in an antibody incubation chamber, add 5% skim milk powder (prepared with PBST), and block on a shaker at room temperature for 1 h. Then wash the bands once with PBST. Prepare the antibody concentrations according to the table below using primary antibody dilution buffer. Incubate the prepared primary antibody on the corresponding bands and freeze overnight at 4°C. The next day, recover the primary antibody. Wash the bands three times with PBST for 10 min each time. According to the number of bands, prepare secondary antibody with 5% skim milk powder according to Table 5 and incubate at room temperature for 1 h. After incubation, wash three times with PBST for 10 min each time. Incubate the chemiluminescence solution on the bands, expose using a fully automated chemiluminescence analyzer, and store the band images.

[0063] 1.2.6 The expression level of tumor-related mRNAs in xenograft tissues was detected by q-PCR.

[0064] ①Total RNA extraction

[0065] Place the frozen tissue in a pre-chilled RNase-free EP tube. Add 300 μL of lysis buffer RL to every 10-20 mg of tissue, homogenize using a cryo-mortar, and centrifuge at 12000 rpm (~13400 × g) for 5 min. Collect the supernatant and slowly add 0.5 times the volume of anhydrous ethanol, mix well. Transfer the resulting solution and precipitate to the adsorption column CR3 (place the adsorption column in the collection tube), centrifuge at 12000 rpm (~13400 × g) for 30 s, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Add 500 μL of protein removal buffer RD, centrifuge at 12000 rpm (~13400 × g) for 30 s, remove the waste liquid, and return the adsorption column to the collection tube. Add 500 μL of washing buffer RW to the center of the adsorption column CR3, let stand for 2 min, and incubate at 4°C, 12000 rpm (~13400 × g) for 5 min. Centrifuge at 12000 rpm (~13400×g) for 30 seconds, discard the waste liquid, and repeat this step once; place the adsorption column back into the collection tube; add 500 μL of wash buffer RW (containing ethanol) to the adsorption column CR3, let stand at room temperature for 2 min, centrifuge at 12000 rpm (~13400×g) for 1 min, discard the waste liquid, and place the adsorption column back into the collection tube. Then repeat the washing process by adding wash buffer once more, centrifuging at 12000 rpm (~13400×g) for 2 min, discard the waste liquid, and place the adsorption column at room temperature for several minutes to dry the residual wash liquid in the material. Transfer the adsorption column CR3 into a new RNase-free EP tube, add 100 μL of RNase-free ddH2O dropwise to the middle of the adsorption membrane, let stand at room temperature for 2 min, centrifuge at 12000 rpm (~13400×g) for 2 min to obtain the total RNA extract, and determine its purity.

[0066] ②Reverse transcription to synthesize cDNA

[0067] Take an EP tube containing total RNA extract (<5 μg) and place it on ice. Add 1 μL of Oligo(dT) to the tube, then add nuclease-free water to a total volume of 12 μL. Briefly centrifuge and incubate at 65°C for 5 min in a PCR instrument. After the reaction, cool the PCR product on ice and briefly centrifuge. Add the following components to an enzyme-free PCR tube in the specified order: 5× Reaction Buffer (4 μL), Ribolock RNase Inhibitor (20 U / μL) (1 μL), 10 mm dNTP Mix (2 μL), and RevertAid M-MuLV RT (200 U / μL) (1 μL). The total reaction volume is 20 μL. Gently mix, briefly centrifuge, and then incubate in a PCR instrument for cDNA synthesis. The conditions are: 42°C for 60 min, 70°C for 5 min, and then terminate the reaction. Place the reaction product in an ice box and briefly centrifuge to obtain reverse-transcribed cDNA, which can be used directly for experiments or stored at -80°C.

[0068] ③ RT-qPCR detection of expression levels of relevant mRNAs in transplanted tumor tissues

[0069] The internal reference gene was selected as β-actin, and the mRNA primers for the target gene are shown in the table below:

[0070] RT-qPCR primer sequences for each group

[0071]

[0072] Add 90-100 μL of RNase-Free ddH2O to the reverse transcribed cDNA samples of each treatment group to dilute the samples to an appropriate concentration. Place each group of samples in a dedicated eight-tube set and prepare the reaction system according to the table below: 2*AceQ UniversalSYBR qPCR Master Mix (10 μL), ROX Passive Reference Dye (50×) (0.5 μL), cDNA sample of each group (2 μL), PCR Forward Primer 0.4 μL (0.5 μL), PCR Reverse Primer 0.4 μL (0.5 μL), RNase-Free ddH2O (0.5 μL).

[0073] Place the above reaction system into an ABI StepOne Plus Real-Time PCR instrument and set the reaction conditions as follows:

[0074]

[0075] After the reaction, the relative expression level of the target mRNA was obtained, and then analyzed using 2... -△△Ct The expression level of the target gene relative to the internal reference is calculated to obtain the relative quantitative detection of the gene to be measured, and the expression level is expressed as a multiple.

[0076] 1.2.7 Cell Culture

[0077] ①Cell resuscitation

[0078] Remove the cell lines frozen in liquid nitrogen and thaw them rapidly. Then, use a pipette to transfer them to a pre-prepared sterile 10mL centrifuge tube and centrifuge (1000rpm, 5min). After centrifugation, discard the supernatant. Next, add 1mL of cell culture medium containing 10% fetal bovine serum and repeatedly pipette until a single-cell suspension is formed. Transfer the suspension to a cell culture flask, add 5-6mL of complete culture medium, and place the flask in a 5% CO2 incubator. Adjust the temperature of the incubator to 37℃ for cell culture.

[0079] ② Cell passage

[0080] When the cell density reaches approximately 85% proliferation under a microscope, the cells are ready for passage. Discard the used culture medium from the cell culture flask, wash with 3 mL of PBS, and repeat three times. Then add 1 mL of 0.25% trypsin digestion solution to the flask, gently shake to distribute the digestion solution evenly, and place the flask in an incubator for several minutes. After removing it, observe under a microscope until the cells become rounded, then immediately add 3 mL of complete culture medium to stop digestion. Gently pipette the cells to the flask wall until most of the adherent cells detach. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells in 1 mL of complete culture medium and pipette to form a single-cell suspension. Count the cells. Depending on the specific experimental conditions, the cell suspension can be divided into 2-3 culture flasks, with 5-7 mL of complete culture medium added to each flask. Incubate at 37°C in a 5% CO2 incubator.

[0081] ③ Cell cryopreservation

[0082] Collect cells at a density of 60-70% in the logarithmic growth phase, digest them with 0.25% trypsin solution according to the conditions for cell passage, and pipette into a single-cell suspension. Transfer the suspension to a 10mL centrifuge tube, centrifuge at 1000rpm for 5min, discard the culture medium, add 1mL of serum-free cryopreservation solution, mix well, and transfer again to a cell cryopreservation tube, labeling it carefully. Place the cryopreservation tubes in a cell cryopreservation box and store at -80℃ overnight. After overnight storage, transfer to a liquid nitrogen tank for long-term preservation.

[0083] 1.2.8 Preparation of Drug Storage Solution

[0084] TFGR was weighed and prepared to a concentration of 100 mg / mL, and then serially diluted to 50, 25, and 12.5 mg / mL. LCA and GBN were accurately weighed and dissolved separately in DMSO solution to prepare stock solutions. These stock solutions were filtered through a 0.22 μm microporous membrane and stored at 4°C for later use. Before each cell administration, each drug group was diluted with complete culture medium to a final concentration of 100, 50, 25, and 12.5 μg / mL.

[0085] Drug preparation for subsequent experiments: LCA, GBN, and CMC were prepared according to the above method, with final concentrations of 40, 20, 10, and 5 μg / mL, respectively, and the final concentrations of TFGR extract were 100, 75, 50, and 25 μg / mL.

[0086] 1.2.9 Flow cytometry analysis of the effects of TFGR, LCA, GBN, and CMC on apoptosis in human colon cancer cells

[0087] Cells were seeded in 6-well plates and cultured overnight before drug administration. Cells were treated with different concentrations of LCA (20 μg / mL, 40 μg / mL), GBN (20 μg / mL, 40 μg / mL), CMC (20 μg / mL, 40 μg / mL), and total flavonoids (75 μg / mL, 100 μg / mL). After culturing for 24 h in a cell culture incubator at 37°C and saturated humidity, cells were collected by trypsin digestion without EDTA, centrifuged at 2000 rpm for 5 min at room temperature, washed twice with cold PBS, and resuspended in 400 μL of 1× Binding Buffer (approximately 1×10⁻⁶ cells / mL). 6 Cells / mL; Add 5 μL Annexin V-FITC to the cell suspension, mix gently, and incubate at 2-8℃ in the dark for 15 min. Add 10 μL PI, mix gently, and incubate at 2-8℃ in the dark for 5 min. Analyze the total apoptosis rate using a flow cytometer within 1 h.

[0088] 1.2.10 Hoechst 33258 fluorescence staining method to observe the effects of TFGR, LCA, GBN, and CMC on the nuclear morphology of human colon cancer cells.

[0089] Soak clean coverslips in 70% ethanol for 5 minutes or longer, wash three times with PBS (cell culture medium), and then wash once with cell culture medium. Place the coverslips in a 6-well plate, seed cells, and culture overnight at a cell density of approximately 50%-80%. Treat cells with the respective drugs (40 μg / mL LCA, 40 μg / mL GBN, 40 μg / mL CMC, 100 μg / mL TFGR), aspirate the culture medium, add 0.5 mL of fixative, and fix for 10 minutes or overnight at 4°C. Remove the fixative, wash twice with PBS for 3 minutes each time, discard the liquid, add 0.5 mL of Hoechst 33258 staining solution, stain for 5 minutes, and gently shake to ensure thorough contact. Wash twice with PBS for 3 minutes each time, then mount with anti-quenching mounting medium. Observe the staining using a fluorescence microscope and acquire images.

[0090] 1.2.11 Scratch assay to detect the effects of TFGR, LCA, GBN, and CMC on the migration ability of human colon cancer cells.

[0091] Select cells in the logarithmic growth phase, culture each group of cells to a density of 80%, and then add them at a rate of 1×10⁻⁶. 5 Cells were seeded into six-well plates and incubated in an incubator. After adhesion, the cells were observed and marked on the back of the plate. Using a 10 μL sterile pipette tip, scratches were made perpendicularly to the marked areas of the six-well plate. The original culture medium was discarded, and the cells were treated with drug-containing culture media (40 μg / mL LCA, 40 μg / mL GBN, 40 μg / mL CMC, 100 μg / mL TFGR). The cells were incubated at 37°C for 0 h and 24 h, and photographs were taken to record the results. The scratch healing distance was measured. Each experiment was repeated three times.

[0092] 1.2.12 Transwell assay to detect the effects of TFGR, LCA, GBN, and CMC on the invasive ability of human colon cancer cells.

[0093] First, thaw the Matrigel gel at 4°C overnight. Place the Matrigel gel on ice in a clean bench and dilute it 1:3 with serum-free culture medium. Coat the upper part of the chamber with 40 μL of the pre-diluted Matrigel gel. Remove the Transwell chamber and place it in a 24-well plate. Incubate for 2 hours to allow the gel to solidify and the basement membrane to form.

[0094] Logarithmic growth phase cells were seeded in 60 mm cells. 3 In a culture dish, when the cell density reaches 80%, the original culture medium is discarded. The cells are washed twice with PBS, digested with trypsin, centrifuged at 1000 rpm for 5 min, and the supernatant is discarded. Serum-free culture medium is added to the tube to prepare a cell suspension, and the cells are counted. 200 μL of the cell suspension is added to the upper chamber of a Transwell chamber, with a cell count of 3 × 10⁶ cells / mL.4 Cells were placed in 24-well plates and allowed to adhere to the walls. The culture medium was then aspirated. For each treatment group, drug-containing medium (40 μg / mL LCA, 40 μg / mL GBN, 40 μg / mL CMC, 100 μg / mL TFGR) without FBS was added to the upper chamber for 24 h. 500 μL of 20% FBS medium was added to each well of the lower chamber. The plates were then incubated in a cell culture incubator. The culture medium was aspirated, and the Transwell chambers were washed twice with PBS. They were then fixed with 4% paraformaldehyde at room temperature for 30 min (200 μL for the upper chamber and 500 μL for the lower chamber). The fixative was discarded, and the cells were washed twice with PBS. Staining with 0.1% crystal violet for 15 min was performed, and the staining solution was discarded. The cells were rinsed twice with PBS. Cells invading the lower layer of the microporous membrane were counted under a microscope. Five fields of view were selected for each sample to count the cells, and the data were processed.

[0095] 1.2.13 Western blotting analysis of the effects of TFGR, LCA, GBN, and CMC on the expression of apoptosis-related proteins in human colon cancer cells.

[0096] After aspirating the culture medium from adherent tumor cells, wash twice with 10 mL / 150 mm cold PBS, shaking several times each time to remove as much culture medium as possible. Scrape the adherent cells off the plate with a cell scraper, transfer the cells and culture medium to centrifuge tubes, centrifuge at 800 × g for 10 min, discard the supernatant, and wash twice more with 10 mL / 150 mm cold PBS, centrifuging at 800 × g for 5 min. Add 10 μL of phosphatase inhibitor, 1 μL of protease inhibitor, and 5 μL of 100 mM PMSF to each 1 mL of cold Lysis Buffer, mix well, and store on ice for several minutes until needed. After washing the cells, transfer them to new pre-chilled centrifuge tubes, add 1 mL of the prepared cold Lysis Buffer, place on a 4°C shaker, shake vigorously for 30 s, place on ice for 4 min, and repeat 5 times. Then centrifuge at 12000 rpm, 4°C for 5 min, and collect the supernatant as the total protein extract. The total protein content of the total protein extract from each group of tumor tissues was detected using a BCA protein quantification kit. Proteins from each treatment group were tested according to method 1.2.5.

[0097] 1.2.14 Effects of qRT-PCR on the levels of apoptosis-related mRNAs in human colon cancer cells

[0098] Adherent cells were aspirated from culture medium, washed with PBS, the PBS was removed, and the cells were digested normally. The cell solution was transferred to an RNase-Free centrifuge tube and centrifuged at 300×g for 5 min. The cell pellet was collected, and the supernatant was removed. 350 μL of lysis buffer RL was added to the tube, and all the solution was transferred to a CS filter column and centrifuged at 12000 rpm (~13400×g) for 2 min. The filtrate was collected. Add 350 μL of 70% ethanol to the filtrate, mix well, and transfer the resulting solution and precipitate to CR3. Centrifuge at 12000 rpm (~13400 × g) for 1 min, discard the waste liquid in the collection tube, and return the adsorption column CR3 to the collection tube. Add 350 μL of protein removal solution RW1 to the adsorption column, centrifuge at 12000 rpm (~13400 × g) for 1 min, discard the waste liquid, and return the adsorption column to the collection tube. Add 80 μL of DNase I working solution (70 μL of RDD solution added to 10 μL of DNase I stock solution, mixed well) to the center of the adsorption column CR3, incubate at room temperature for 15 min, add 350 μL of protein removal solution RW1 to the adsorption column, and centrifuge at 12000 rpm (~13400 × g) for 1 min. Centrifuge at 12000 rpm (~13400×g) for 1 min, discard the waste liquid, and return the adsorption column to the collection tube. Add 500 μL of wash buffer RW (containing ethanol) to the adsorption column CR3, let stand at room temperature for 2 min, centrifuge at 12000 rpm (~13400×g) for 1 min, return the adsorption column to the collection tube, repeat the washing with wash buffer once, centrifuge at 12000 rpm (~13400×g) for 2 min, discard the waste liquid, let the adsorption column stand at room temperature for several minutes to dry, transfer the adsorption column CR3 to a new RNase-free EP tube, add 100 μL of RNase-free ddH2O dropwise to the middle of the adsorption membrane, let stand at room temperature for 2 min, centrifuge at 12000 rpm (~13400×g) for 2 min to obtain the total RNA extract, and determine its purity. Reverse transcription cDNA and PCR experiments are performed according to method 1.2.6.

[0099] 1.2.15 Using reverse virtual screening technology to explore the potential targets and signaling pathways of GBN and LCA in the fight against colorectal cancer.

[0100] Using the whole target library of the reverse virtual screening platform, the crystal structures of targets corresponding to disease genes were fully resolved, including 2119 active sites for 140 targets; the partial target library, containing partial structures of targets corresponding to disease genes, was resolved, including 5487 active sites for 506 targets. Structural files of LCA and GBN were obtained by searching the DrugBank database and submitted to the reverse virtual screening platform for molecular virtual docking. The binding energies of the compounds to proteins were scored, and the top 200 results were returned. A higher absolute score indicates more stable drug binding to the target. The results were compared with the ProteinBank and KEGG databases for target prediction and pathway analysis.

[0101] 1.2.16 Proteomics study of LCA, GBN, and CMC in vitro against colon cancer

[0102] The experiment consisted of eight groups: four groups of human colon cancer SW480 cells and four groups of human colon cancer SW620 cells. SW480 cells were treated with the following methods: blank control, 40 μg / mL LCA, 40 μg / mL GBN, 40 μg / mL CMC, and 100 μg / mL TFGR, with each treatment lasting 24 h. SW620 cells were treated in the same manner as SW480 cells. Cell culture methods are described in section 1.2.7. Protein extraction and quantification were performed as previously described.

[0103] Enzymatic digestion and labeling:

[0104] Reductive alkylation and enzymatic digestion: Take 100 μg of protein sample and make up to 90 μL with lysis buffer. Add 10 mmol / L TCEP reducing agent and react at 37 °C for 60 min. Add 40 mmol / L iodoacetamide and react at room temperature in the dark for 40 min. Add pre-cooled acetone (acetone:sample volume ratio = 6:1) to each tube, precipitate at -20 °C for 4 h, centrifuge at 10000×g for 20 min, and collect the precipitate. Dissolve the sample thoroughly with 50 mmol / L TEAB, add Trypsin at a mass ratio of 1:50 (enzyme:protein), and digest overnight at 37 °C. TMT labeling and mixing: Remove TMT reagent (Thermofisher) at -20 °C and allow to return to room temperature, add acetonitrile, vortex centrifuge, add one tube of TMT reagent for every 100 μg of peptide, and label the samples as shown in the table below. Incubate at room temperature for 2 hours; add hydroxylamine and react at room temperature for 15 minutes. Mix equal amounts of the labeled product in one tube and dry using a vacuum concentrator.

[0105] List of sample labeling order for each group

[0106]

[0107] One-dimensional RPLC separation: Peptide samples were reconstituted with UPLC loading buffer and separated using a reverse-phase C18 column (ACQUITY UPLC BEHC18 Column 1.7μm, 2.1mm × 150mm, Waters, USA) at high pH. Phase A: 2% acetonitrile (adjusted to pH 10 with ammonia); Phase B: 80% acetonitrile (adjusted to pH 10 with ammonia). Chromatography intervals were as follows: 0–1.9 min, 100% A; 2–17 min, 0–5% B; 17–18 min, 5%–10% B; 18–35.5 min, 10%–30% B; 35.5–38 min, 30%–36% B; 38–39 min, 36%–42% B; 39–40 min, 42%–100% B; 40–44 min, 100% B; 44–45 min, 100%–0% B (see table below). The UV detection wavelength was 214 nm, the flow rate was 200 μL / min, and the elution time was 48 min. Twenty fractions were collected based on peak shape and time, combined into ten fractions, and concentrated by vacuum centrifugation.

[0108] UPLC loading and elution gradient

[0109]

[0110]

[0111] Protein identification was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS): The second dimension was analyzed using nano-level LC-MS / MS (Easy-nLC 1200 combined with a Q_Exactive HF-X mass spectrometer). Peptides were dissolved in mass spectrometry loading buffer, loaded, and then separated on a C18 column (75 μm × 25 cm, Thermo, USA) for 120 min at a flow rate of 300 μL / min. EASY-nLC gradient elution was used: phase A with 2% acetonitrile (plus 0.1% formic acid) and phase B with 80% acetonitrile (plus 0.1% formic acid), as shown in the table below. Automatic switching between MS and MS / MS acquisition was used, with mass spectrometry resolutions of 70 K and 35 K, respectively. MS was performed as a full scan (m / z 350-1500) in DDA mode with a cycle time of 2 s; the top 20 precursor ions were selected for secondary fragmentation, with a primary mass spectrometry resolution of 120,000. TurboTMT (Intelligent Acquisition Improves Report Ion Isotope Resolution); Maximum Injection Time: 20ms, Fragmentation Mode: HCD; Secondary Resolution: 45000, Maximum Injection Time: 86ms, Fixed First Mass: 110m / z; Minimum AGC Target: 8e3, Intensity Threshold: 8.3e4, Dynamic Exclusion Time: 40s.

[0112] EASY-nLC liquid phase gradient

[0113]

[0114] Protein search library:

[0115] The raw mass spectrometry data were analyzed using ProteomeDiscoverer™ Software 2.4, with NCBInr as the database for the search. During the search, the false discovery rate (FDR) for peptide identification was set to ≤0.01, the precursor mass tolerance to 20 ppm, and the fragment mass tolerance to 0.02 Da, ensuring at least one specific peptide was present. For the proteins detected in this study, the p-value for statistical significance between samples was calculated using the t.test function in R, and the fold change (FC) between groups was also calculated. Proteins with a significance p-value <0.05 and a fold change greater than 1.2 were considered differentially expressed proteins.

[0116] Bioinformatics analysis: GO (gene ontology, http: / / geneontology.org / ) was used to perform functional annotation analysis on all differentially expressed proteins from three aspects: biological processes, cellular components, and molecular functions. The KEGG (Kyoto encyclopedia of gene and genomes, http: / / www.genome.jp / kegg / / ) pathway database was used to analyze the metabolic and signaling pathways involved in the differentially expressed proteins.

[0117] 1.2.17 Statistical Methods

[0118] Data processing was performed using SPSS 26.0 statistical software. All experimental data and measurement data are expressed as mean ± standard deviation. A t-test was used to compare the means of two groups (if σ1...). 2 ≠σ2 2 If σ1 < σ', then the t' test is used. For comparisons of means among multiple groups, a one-way ANOVA test is used (if σ1 < σ'). 2 ≠σ2 2 The Dunnett-t test is then used, with P < 0.05 considered statistically significant.

[0119] 1.3 Experimental Results

[0120] 1.3.1 The inhibitory effects of TFGR, LCA, CMC, and GBN on the proliferation of human colon cancer SW480 and SW620 cells were verified by CCK-8 cytotoxicity assay.

[0121] The experimental results are shown in Figure 1 and Figure 2 The inhibitory activities of TFGR, LCA, GBN, and CMC on human colon cancer cells SW480 and SW620 were verified using the CCK-8 assay. The effects of different concentrations of each drug group on tumor cell proliferation at different time points were measured, and the inhibition results are shown below. Figure 1 and Figure 2 As shown. Compared with the control group, all dose groups of total licorice flavonoids showed statistical significance (P<0.01), and all inhibited the proliferation of SW480 and SW620 cells to varying degrees. The inhibitory effect gradually increased with time and dosage, showing a certain dose- and time-dependent effect. Since the 24-hour treatment group already showed significant inhibitory effects on proliferation, the high-dose groups of each 24-hour treatment group were selected for subsequent experiments. The proliferation inhibition rates of the four drug groups at different doses at 24 hours were different, as shown when acting on SW480 cells... Figure 1 As shown, the inhibition rate of 20 μg / mL LCA was (91.16±0.69)%; the inhibition rate of 20 μg / mL GBN was (34.85±10.04)%; the inhibition rate of 20 μg / mL CMC was (79.05±3.50)%; and the inhibition rate of 50 μg / mL TFGR was (81.76±1.46)%. Among these, the inhibition rate of the monomeric compound LCA at the same concentration was higher than that of GBN, and the differences were statistically significant compared with the control group (p<0.05, p<0.01). When acting on SW620 cells, the inhibition rate of 20 μg / mL LCA was (84.62±0.87)%; the inhibition rate of 20 μg / mL GBN was (92.34±0.24)%; the inhibition rate of 20 μg / mL CMC was (93.87±0.54)%; and the inhibition rate of 50 μg / mL TFGR was (81.74±1.76)% (see...). Figure 2 At the same concentration, both monomeric compounds LCA and GBN showed inhibitory effects. SW620 cells were more sensitive to LCA, GBN, and CMC than SW480 cells, exhibiting a higher cell proliferation inhibition rate. Under the same drug concentration, SW620 cell proliferation was significantly inhibited, while the inhibition rate of SW480 cells was lower than that of SW620. Subsequent mechanism of action and proteomics experiments used 20 μg / mL and 40 μg / mL of each monomeric compound and its combination, as well as 75 μg / mL and 100 μg / mL of total licorice flavonoids, for apoptosis experiments, with an action time of 24 h.

[0122] 1.3.2 Effects of TFGR, LCA, CMC and GBN on apoptosis in human colon cancer cells

[0123] Flow cytometry was used to detect LCA, GB), and CMC in high and medium dose groups (40 μg / mL). -1 and 20 μg·mL -1 )High- and medium-dose groups of TFGR (100 μg·mL -1 and 75 μg·mL -1 The effect of ) on apoptosis of human colon cancer cells SW480 and SW620 is shown in Figure 3 As shown, the apoptosis rate of SW480 cells in the control group was only (1.13±0.11)%, and the apoptosis rate of SW620 cells in the control group was only (1.79±0.15)%.

[0124] 20 μg·mL -1 The apoptosis rate of LCA-treated SW480 cells was only (2.39 ± 0.17)%, showing no significant difference compared to the control group SW480 cells; however, the apoptosis rate of LCA-treated SW620 cells was (4.56 ± 0.26)%, which was statistically significant compared to the control group SW620 cells, indicating an increased apoptosis rate (p < 0.01). 40 μg·mL -1 The apoptosis rate of SW480 cells treated with LCA was only (5.59±0.66)%, while the apoptosis rate of SW620 cells treated with LCA was (11.56±1.44)%, both showing statistically significant differences compared to the apoptosis rates of the control groups (p<0.001). The apoptosis rate increased to varying degrees after drug treatment. (20 μg / mL) -1 The apoptosis rate of GBN-treated SW480 cells was only (7.77±0.28)%, which was statistically significant compared to the control group SW480 cells, showing an increased apoptosis rate (p<0.01); the apoptosis rate of GBN-treated SW620 cells was (6.35±0.53)%, which was not statistically significant compared to the control group SW620 cells. (40 μg·mL) -1 The apoptosis rate of GBN-treated SW480 cells was only (16.35±0.88)%, while that of SW620 cells was (16.38±1.17)%, both showing statistically significant differences compared to the apoptosis rates of the control groups (p<0.001). The apoptosis rate increased to varying degrees after treatment. (20 μg / mL) -1 The apoptosis rate of CMC-treated SW480 cells was only (10.50±0.77)%, while that of SW620 cells was (12.55±0.13)%, which was statistically significant compared to the control groups (p<0.05, p<0.01). 40 μg·mL -1The apoptosis rate of CMC-treated SW480 cells was only (18.54±1.16)%, while the apoptosis rate of CMC-treated SW620 cells was (27.90±2.06)%, which was statistically significant compared to the control groups, indicating a significantly increased apoptosis rate (p<0.001). 75 μg·mL -1 The apoptosis rate of TFGR-treated SW480 cells was only (17.29±0.94)%, with no significant difference compared to the control group SW480 cells; while the apoptosis rate of TFGR-treated SW620 cells was (15.84±0.71)%, at 100 μg·mL⁻¹. -1 The apoptosis rate of TFGR in SW480 cells was only (33.78±2.50)%, while that in SW620 cells was (32.97±0.51)%. All dose groups showed statistically significant differences compared to the control groups, with a significantly increased apoptosis rate (p<0.001). The experimental results indicate that medium-dose groups of GBN, CMC, and TFGR can all induce apoptosis in human colon cancer cells SW480; medium-dose groups of LCA, CMC, and TFGR can all induce apoptosis in human colon cancer cells SW620; and high-dose groups of LCA, GBN, and CMC (40 μg·mL⁻¹) can induce apoptosis. -1 ) and the high-dose group of TFGR (100 μg·mL) -1 Both drugs significantly induced apoptosis in human colon cancer SW480 and SW620 cells. All subsequent experiments used the high-dose group.

[0125] 1.3.3 Effects of TFGR, LCA, CMC and GBN on the nuclear morphology of human colon cancer cells

[0126] After staining with Hoechst 33258 fluorescent dye, the effect of the drug on cell nuclear morphology was investigated. The results are shown in [Figure number missing]. Figure 4 As shown. Microscopic observation revealed that the nuclei of both SW480 and SW-620 colon cancer cells in the control group exhibited weak blue fluorescence, and the nuclear structure remained intact. The high-dose groups of LCA, GBN, and CMC (40 μg / mL) -1 ) and the high-dose group of TFGR (100 μg·mL) -1 In all groups, the cell nuclei showed dense staining and bright blue fluorescence, exhibiting varying degrees of apoptotic changes: nuclear condensation and shrinkage, nuclear fragmentation, and incomplete cell disruption. Apoptotic bodies were observed in some groups (specific nuclear fragmentation and condensation are indicated by arrows in the diagram). From a cellular morphological perspective, LCA, GBN, CMC, and TFGR all induced varying degrees of apoptosis in human colon cancer SW480 and SW620 cells.

[0127] 1.3.4 Effects of TFGR, LCA, CMC and GBN on the migration of human colon cancer cells

[0128] The high-dose group (40 μg / mL) of LCA, GBN, and CMC was studied using a cell scratch assay. -1 ) and the high-dose group of TFGR (100 μg·mL) -1 The effects of ) on the migration of human colon cancer cells SW480 and SW-620 were as follows: Figure 5 As can be seen, the migration rate of SW480 cells in the control group 24 h after scratching was (45.14±1.92)%, indicating faster scratch healing; the migration rates of human colon cancer cells SW480 treated with LCA, GBN, CMC, and TFGR were (44.93±0.49)%, (34.10±2.34)%, (33.62±1.86)%, and (24.09±3.68)%, respectively. The migration rate of SW620 cells in the control group 24 h after scratching was (39.01±3.95)%, indicating faster scratch healing; the migration rates of human colon cancer cells SW620 treated with LCA, GBN, CMC, and TFGR were (30.33±0.91)%, (25.11±0.73)%, (22.23±0.77)%, and (19.01±1.35)%, respectively. LCA showed no statistically significant difference in migration rate compared to the SW480 control group, but a statistically significant difference compared to the SW620 control group (p<0.05), thus reducing the migration ability of SW620 cells in scratch healing. The GBN group showed statistically significant differences in migration rate compared to the control groups of both cell lines (p<0.01, p<0.001), significantly inhibiting cell migration. The CMC group showed statistically significant differences in migration rate compared to the control groups of both cell lines (p<0.01, p<0.001), significantly inhibiting cell migration. The TFGR group showed statistically significant differences in migration rate compared to the control groups of both cell lines (p<0.001), significantly inhibiting the migration ability of SW480 and SW620 cells. The experimental results indicate that GBN, CMC, and TFGR can significantly inhibit the migration of human colon cancer cells SW480 and SW620, slowing down scratch healing; LCA only inhibited the migration of human colon cancer cells SW620.

[0129] 1.3.5 Effects of TFGR, LCA, CMC and GBN on the invasive ability of human colon cancer cells

[0130] The Transwell assay was used to investigate the high-dose group (40 μg / mL) of LCA, GBN, and CMC. -1 ) and the high-dose group of TFGR (100 μg·mL) -1 The effects of ) on the invasive ability of human colon cancer cells SW480 and SW-620 were as follows: Figure 6As can be seen, the number of invasive cells in the human colon cancer SW480 cell control group was (71.00±8.72), the number of invasive cells in the LCA group treated with SW480 was (63.60±8.91), the number of invasive cells in the GBN group was (51.00±6.48), the number of invasive cells in the CMC group was (37.80±4.66), and the number of invasive cells in the TFGR group was (33.8±4.82); the number of invasive cells in the human colon cancer SW620 cell control group was (65.80±8.44), the number of invasive cells in the LCA group treated with SW620 was (53.6±6.47), the number of invasive cells in the GBN group was (51.00±6.56), the number of invasive cells in the CMC group was (32.80±3.27), and the number of invasive cells in the TFGR group was (30.0±4.18). There was no statistically significant difference between the LCA treatment group and the SW480 control group, but a statistically significant difference compared with the SW620 control group (p<0.001), indicating a decrease in the number of invasive cells after treatment. The GBN treatment group showed statistically significant differences compared with both the SW480 and SW620 control groups (p<0.01, p<0.001), with a significant decrease in the number of invasive cells. The CMC and TFGR treatment groups showed statistically significant differences compared with the SW480 and SW620 control groups (p<0.001), with a significant decrease in the number of invasive cells. The experimental results indicate that GBN, CMC, and TFGR can significantly inhibit the invasive ability of human colon cancer cells SW480 and SW620, significantly reducing the number of invasive cells; LCA only significantly inhibited the invasive ability of human colon cancer cells SW620.

[0131] 1.3.6 Effects of TFGR, LCA, CMC and GBN on the expression levels of proteins related to apoptosis, metastasis and invasion in human colon cancer cells

[0132] The effects of licorice active ingredient intervention on the expression levels of metastasis and invasion-related proteins in human colon cancer SW480 and SW620 cells were detected by Western blotting. Results are shown below. Figure 7 Compared with the control group, after LCA drug intervention, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased in SW480 cells (P<0.01, P<0.001, P<0.001), while the expression levels of Bcl-2, MMP2, and MMP9 proteins were significantly decreased (P<0.001, P<0.01, P<0.001); in SW620 cells, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased (P<0.01, P<0.001, P<0.001), while the expression levels of Bcl-2 and MMP9 proteins were significantly decreased (P<0.001, P<0.01). (See...) Figure 7 (As shown in A).

[0133] Compared with the control group, after GBN drug intervention, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased in SW480 cells (P<0.05, P<0.001, P<0.001), while the expression levels of Bcl-2 and MMP9 proteins were significantly decreased (P<0.01); in SW620 cells, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased (P<0.001), while the expression levels of Bcl-2, MMP2, and MMP9 proteins were significantly decreased (P<0.05) (see...). Figure 7 (As shown in B).

[0134] Compared with the control group, after TFGR drug intervention, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased in SW480 cells (P<0.001), while the expression levels of Bcl-2, MMP2, and MMP9 proteins were significantly decreased (P<0.001, P<0.01, P<0.001); in SW620 cells, the expression levels of Bax, Cleaved-caspase3, Cleaved-caspase9, and Bcl-2 proteins were significantly increased (P<0.001), while the expression levels of MMP2 and MMP9 proteins were significantly decreased (P<0.001, P<0.01, P<0.01) (see...). Figure 7 (As shown in C).

[0135] Compared with the control group, after CMC drug intervention, the expression levels of Bax, Cleaved-caspase3, and Cleaved-caspase9 proteins were significantly increased in SW480 cells (P<0.001), while the expression levels of Bcl-2, MMP2, and MMP9 proteins were significantly decreased (P<0.001, P<0.01, P<0.001); in SW620 cells, the expression levels of Bax, Cleaved-caspase3, Cleaved-caspase9, and Bcl-2 proteins were significantly increased (P<0.001), while the expression levels of MMP2 and MMP9 proteins were significantly decreased (P<0.001, P<0.01, P<0.01) (see...). Figure 8 (As shown).

[0136] The above results indicate that GBN, LCA, CMC, and TFGR can all promote apoptosis in human colon cancer SW480 and SW620 cells through the Caspase apoptosis pathway. GBN and TFGR may also inhibit the migration and invasion of human colon cancer SW480 and SW620 cells.

[0137] 1.3.7 Effects of TFGR, LCA, CMC, and GBN on the expression levels of mRNAs related to apoptosis, metastasis, and invasion in human colon cancer cells. qRT-PCR results showed that, compared with the control group, after LCA intervention, the gene levels of Caspase3, Caspase9, and Bax were significantly increased in SW480 cells (P<0.001, P<0.01, P<0.05), while the gene level of MMP2 was significantly decreased (P<0.05); in SW620 cells, the gene levels of Caspase3 and Caspase9 were significantly increased (P<0.001), while the gene levels of Bcl-2 and MMP2 were significantly decreased (P<0.001, P<0.001) (see...). Figure 9 (As shown in A).

[0138] Compared with the control group, GBN drug intervention significantly increased the gene levels of Caspase3, Caspase9, and Bax in SW480 cells (P<0.001, P<0.001, P<0.05) and significantly decreased the gene level of MMP2 (P<0.01); in SW620 cells, the gene levels of Caspase3, Caspase9, and Bax were significantly increased (P<0.001), while the gene levels of Bcl-2 and MMP2 were significantly decreased (P<0.001) (see...). Figure 9 (As shown in B).

[0139] Compared with the control group, after TFGR drug intervention, the levels of Caspase3, Caspase9, and Bax genes were significantly increased in SW480 cells (P<0.01, P<0.001, P<0.001), while the levels of Bcl-2 and MMP2 genes were significantly decreased (P<0.01, P<0.001); in SW620 cells, the levels of Caspase3, Caspase9, and Bax genes were significantly increased (P<0.05, P<0.001, P<0.001), while the levels of Bcl-2 and MMP2 genes were significantly decreased (P<0.01, P<0.05). (See...) Figure 9 (As shown in C).

[0140] Compared with the control group, after CMC drug intervention, the levels of Caspase3, Caspase9, and Bax genes were significantly increased in SW480 cells (P<0.01, P<0.001, P<0.001), while the levels of Bcl-2 and MMP2 genes were significantly decreased (P<0.01, P<0.001); in SW620 cells, the levels of Caspase3, Caspase9, and Bax genes were significantly increased (P<0.05, P<0.001, P<0.001), while the levels of Bcl-2 and MMP2 genes were significantly decreased (P<0.01, P<0.05). (See...) Figure 9 (As shown in D).

[0141] The above results indicate that GBN, LCA, CMC, and TFGR affect the expression levels of mRNAs related to apoptosis, metastasis, and invasion in human colon cancer cells.

[0142] Furthermore, Western blotting was used to observe the effects of CMC drugs on the expression of PI3K-Akt-mTOR pathway-related proteins in SW480 and SW620 cells.

[0143] Compared with the control group, CMC drug intervention significantly decreased the expression levels of PI3K, AKT, mTOR, p-PI3K, p-AKT, and p-mTOR proteins in SW480 and SW620 cells (P<0.05); see Figure 9 Western blotting results suggest that CMC drugs can promote apoptosis in SW480 and SW620 cells via PI3K-AKT-mTOR.

[0144] 1.3.8 The reverse virtual screening technique was used to explore the potential targets and signaling pathways of glycyrrhizin (GBN) and glycyrrhizin chalcone A (LCA) in the anti-colon cancer activity. The results showed that:

[0145] ① Among the candidate proteins that are both upregulated in colorectal cancer cells and act on signaling pathways, 13 candidate targets were screened using glycyrrhizin, and 16 candidate protein targets were identified for LCA. Ultimately, 8 protein targets were predicted to potentially act on both LCA and GBN: BRAF, TGFBR1, CSNK2A1, PPARD, CDK2, HSP90AB1, RXRA, and MAPK14. These targets are primarily concentrated in the PI3K / AKT, mTOR, MAPK, and Wnt signaling pathways.

[0146] ② Gene information was compared with that in the Cancer Cell Line Encyclopedia (CCLE). Targets screened by GBN and LCA were compared with SW480 cell gene information, resulting in 7 target genes; and with SW620 cell gene information, resulting in 5 target genes. These target genes were then compared with candidate target genes among those screened by GBN and LCA that are both upregulated proteins in colorectal cancer cells and act on signaling pathways. Three target genes were found to be comparable: PPARD, TGFBR1, and HSP90AB1. This target gene is involved in the Wnt, TGF-β, MAPK, and PI3K / AKT signaling pathways.

[0147] 1.3.9 Results of in vitro proteomics study on the anti-colon cancer effects of TFGR, GBN, LCA, and CMC

[0148] Proteomics was used to detect the differential expression of proteins in human colon cancer cells after the action of glycyrrhiza flavonoids. Bioinformatics analysis was then performed on the differentially expressed proteins to infer the signaling pathways involved, thereby exploring the potential mechanism of action of glycyrrhiza flavonoids against colon cancer. Results showed that:

[0149] ① A total of 57,965 peptides and 6,750 proteins were identified. Human colon cancer SW620 cells were compared with the treatment groups (LCA, GBN, and TFGR). Among the LCA groups, 183 proteins were differentially expressed, with 31 upregulated and 152 downregulated in the treatment groups; among the GBN groups, 150 proteins were differentially expressed, with 43 upregulated and 107 downregulated in the treatment groups; and among the TFGR groups, 180 proteins were differentially expressed, with 62 upregulated and 118 downregulated in the treatment groups. Compared with the human colon cancer SW480 cell control group, the drug-treated groups (LCA, GBN, and TFGR) showed 165 differentially expressed proteins among the LCA groups, including 29 upregulated proteins and 136 downregulated proteins; the GBN groups showed 55 differentially expressed proteins, including 23 upregulated proteins and 32 downregulated proteins; and the TFGR groups showed 200 differentially expressed proteins, including 66 upregulated proteins and 134 downregulated proteins.

[0150] ②GO functional annotation analysis showed that, compared with the SW620 cell control group, GO annotation analysis of differentially expressed proteins in the LCA group indicated involvement in 21 biological processes, 3 cellular components, and 11 molecular functions; in the GBN group, it indicated involvement in 19 biological processes, 2 cellular components, and 8 molecular functions; and in the TFGR group, it indicated involvement in 15 biological processes, 2 cellular components, and 11 molecular functions. Compared with the SW480 control group, GO annotation analysis of differentially expressed proteins in the LCA group indicated involvement in 18 biological processes, 3 cellular components, and 14 molecular functions; in the GBN group, it indicated involvement in 16 biological processes, 2 cellular components, and 7 molecular functions; and in the TFGR group, it indicated involvement in 18 biological processes, 2 cellular components, and 12 molecular functions.

[0151] ③ KEGG functional analysis results showed that after LCA, GBN, and TFGR acted on SW620 cells, KEGG annotation analysis of differentially expressed proteins between groups showed that a total of 193 differentially expressed proteins were involved in the KEGG pathway.

[0152] ④ A total of 382 differentially expressed proteins were found in the control group of human colon cancer SW620 cells across all treatment groups, involving 260 KEGG metabolic pathways. Further KEGG pathway analysis of 29 differentially expressed proteins in the main signal transduction pathway revealed that these proteins were most involved in the cancer, mTOR, and PI3K-Akt signaling pathways. In the drug-treated groups, except for GTP, heat shock stress proteins, and intercellular adhesion molecules, the expression of other signal transduction-related proteins showed varying degrees of downregulation. A total of 348 differentially expressed proteins were found in the control group of human colon cancer SW480 cells across all treatment groups, involving 225 KEGG metabolic pathways. Further KEGG pathway analysis of 27 differentially expressed proteins in the signal transduction pathway revealed that these proteins were most involved in the cancer and PI3K-Akt signaling pathways. Except for heme oxidase-1 and ARF-6 proteins, the expression of other signal transduction-related proteins showed varying degrees of downregulation, indicating that drug treatment had a good restorative effect on signaling pathways. Among these proteins, the regulation of some important proteins is related to apoptosis, metastasis, and invasion in colon cancer.

[0153] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of a composition of glycyrrhizin and glycyrrhizin chalcone A in the preparation of a medicament for treating colorectal cancer, characterized in that, The molar ratio of glycyrrhizin A to glycyrrhizin is 1:

1.

2. The use of the composition of glycyrrhizin and glycyrrhizin chalcone A as described in claim 1 in the preparation of a medicament for treating colorectal cancer, characterized in that, A combination of glycyrrhizin and glycyrrhizin chalcone A was used as the sole active ingredient in the preparation of a drug for treating colorectal cancer.

3. The use of the composition of glycyrrhizin and glycyrrhizin chalcone A as described in claim 1 or 2 in the preparation of a medicament for treating colorectal cancer, characterized in that, The combination of glycyrrhizin and glycyrrhizin chalcone A significantly increased the expression levels of Cleaved-caspase 3, Cleaved-caspase 9, and Bax proteins, while significantly decreasing the expression levels of Bcl-2, MMP2, and MMP9 proteins.

4. The use of the composition of glycyrrhizin and glycyrrhizin chalcone A as described in claim 3 in the preparation of a medicament for treating colorectal cancer, characterized in that... In the prepared drug for treating colorectal cancer, the combination of glycyrrhizin and glycyrrhizin chalcone A exerts its effect by regulating the PI3K / Akt / mTOR signaling pathway.

5. The use of the composition of glycyrrhizin and glycyrrhizin chalcone A as described in claim 3 in the preparation of a medicament for treating colorectal cancer, characterized in that... In the prepared drug for treating colorectal cancer, the combination of glycyrrhizin and glycyrrhizin chalcone A is used at a concentration of 5 μg / mL to 40 μg / mL.

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