Application of licoflavone in preparation of medicine for preventing and / or treating colon cancer

Abstract

The invention relates to application of licoflavone in preparation of a medicine for preventing and / or treating colon cancer, and belongs to the technical field of anti-tumor biological medicines. The licoflavone is prepared from ganoderma lucidum mascone, isolongifolen-9 ketone, nordihydroguaiacol and glabridin. The main active compound licoflavone is extracted from the traditional Chinese medicine liquorice, the treatment effect of the licoflavone on colon cancer is confirmed through multiple in-vivo and in-vitro models, and the licoflavone can inhibit tumor cells from spreading to the whole body. According to the application disclosed by the invention, four active monomer compounds, namely ganoderma lucidum mascone, isolongifolenone, nordihydroguaiacol and glabridin, are confirmed from licoflavone, and in-vivo and in-vitro experiments prove that the licoflavone can inhibit proliferation of colon cancer cells DLD-1 and HCT-116 and can delay the diffusion speed of tumor cells at the same time; in addition, the combination of the glabridin has no obvious influence on normal colonic epithelial cells NCM460, the toxicity of the glabridin to normal cells is reduced, and a synergistic effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor biomedicine, and in particular to the application of glycyrrhizin in the preparation of drugs for preventing and / or treating colon cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the three most common cancers in the world. Its incidence is higher in developed countries, accounting for about 10% of all cancer cases and the second leading cause of cancer-related deaths worldwide. It is caused by many risk factors, including diet, lack of exercise, obesity, smoking and drinking, while family history or certain genetic syndromes can also increase susceptibility. Currently, the treatment of colorectal cancer includes endoscopic local resection, palliative chemotherapy, targeted therapy and immunotherapy.

[0003] Although these treatments prolong the survival of patients, their effects are not satisfactory due to the high recurrence and metastasis rates of colorectal cancer. Surgical resection has disadvantages such as high recurrence and high cost; chemotherapy may damage normal cells and immune cells while killing tumor cells, which is a "double-edged sword"; targeted therapy and immunotherapy have relatively small side effects, but are not suitable for all patients and have high individual differences. Therefore, it is crucial to find safe and effective treatment options to reduce the burden of the disease.

[0004] Traditional Chinese medicine is the mainstream form of complementary and alternative medicine. However, some Chinese medicines are not very effective and have certain adverse reactions. Therefore, it is necessary to analyze the ingredients of relevant Chinese medicines to improve their therapeutic effects and safety. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the use of glycyrrhiza flavonoids in the preparation of drugs for preventing and / or treating colon cancer.

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

[0007] The invention provides the use of licorice flavonoids in preparing a drug for preventing and / or treating colon cancer. The licorice flavonoids include ganoderma lucidum ketone, isolongifolia 9-ketone, nordihydroguaiaretic acid and glabrizone.

[0008] The combination of ganoderma lucidum ketone, isothiocyanate 9-one, nordihydroguaiaretic acid and licorice root can further enhance the anti-colorectal cancer effect, reduce the adverse reactions of licorice root to normal cells and improve safety.

[0009] Furthermore, the drug is a drug for inhibiting the proliferation of colorectal cancer.

[0010] Furthermore, the drug is a drug for inhibiting colorectal cancer metastasis.

[0011] Furthermore, the drug is a drug for repairing the intestinal barrier.

[0012] 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.

[0013] Furthermore, the concentration of the ganoderma lucidum ketone, isothiocyanate 9-one, nordihydroguaiaretic acid or glycyrrhizin is 1 to 128 μM.

[0014] As a preferred embodiment, the concentration of the ganoderma lucidum ketone, isolongifolia 9-ketone, nordihydroguaiaretic acid or glabridinone is 32-128 μM; more preferably, the concentration is 64-128 μM; further preferably, the concentration is 128 μM. At this concentration, the combination of the ganoderma lucidum ketone, isolongifolia 9-ketone, nordihydroguaiaretic acid and glabridinone has the best effect in inhibiting the proliferation of colorectal cancer, and has less toxicity to normal colon epithelial cells NCM460.

[0015] Furthermore, the medicine is a pharmaceutical preparation made of the glycyrrhizin as an active ingredient and pharmaceutically acceptable excipients.

[0016] Furthermore, the medicine or pharmaceutical preparation includes tablets, granules or liquid preparations.

[0017] Furthermore, the preparation method of the licorice flavonoids comprises the following steps:

[0018] S11: Mix the licorice sample with water, vortex and shake to mix, and grind; the ratio of the licorice sample to water is licorice sample: water = (10-250) mg: (0.1-2.5) mL.

[0019] S12: subjecting the product ground in step S1 to ice bath ultrasonic extraction for 30 to 60 minutes.

[0020] S13: centrifuging the extracted product in step S2 at a speed of 5000 to 15000 rpm for 5 to 15 min.

[0021] In a specific embodiment of the present invention, in step S11, licorice sample: water = 100 mg: 1 mL.

[0022] In a specific embodiment of the present invention, in step S12, the extraction time is 60 minutes.

[0023] In a specific embodiment of the present invention, in step S13, the centrifugal speed is 14000 rpm and the centrifugal time is 10 min.

[0024] Furthermore, the quality inspection method of the licorice flavonoids includes using liquid chromatography combined with mass spectrometry to detect ganoderma lucidum ketone, isolongifolia 9-ketone, nordihydroguaiaretic acid and glabrizone in the licorice flavonoids.

[0025] Furthermore, the mobile phase in the liquid chromatography combined with mass spectrometry detection analysis includes mobile phase A and mobile phase B, the mobile phase A is 0.1% to 0.5% w / v formic acid aqueous solution, and the mobile phase B is acetonitrile; the chromatographic column is ACQUITY UPLCHSS T3, the column length of the chromatographic column is 100 mm, the inner diameter is 2.1 mm, and the particle size is 1.8 μm.

[0026] In a specific embodiment of the present invention, 0.1% w / v formic acid aqueous solution is used as mobile phase A for detection.

[0027] Furthermore, the liquid chromatography combined with mass spectrometry detection and analysis is gradient elution,

[0028] 0-2min, the volume ratio of mobile phase A to mobile phase B is mobile phase A: mobile phase B = 95:5;

[0029] 2-4 min, 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;

[0030] 4-8 min, the volume ratio of mobile phase A to mobile phase B changed from mobile phase A:mobile phase B=70:30 to mobile phase A:mobile phase B=50:50;

[0031] 8-10 min, the volume ratio of mobile phase A to mobile phase B changed from mobile phase A:mobile phase B=50:50 to mobile phase A:mobile phase B=20:80;

[0032] 10-14 min, 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;

[0033] 14-15 min, the volume ratio of mobile phase A to mobile phase B is mobile phase A: mobile phase B = 0:100;

[0034] 15-16 min, the volume ratio of mobile phase A to mobile phase B changed from mobile phase A:mobile phase B= to mobile phase A:mobile phase B=95:5;

[0035] The column temperature is 40-50°C; the flow rate is 0.2-0.5 mL / min; and the scanning range of the diode array detector is 210-400 nm.

[0036] In a specific embodiment of the present invention, the column temperature is 45° C. and the flow rate is 0.35 mL / min.

[0037] Furthermore, the mass spectrometry conditions were as follows: positive ion spray voltage was 3800 V, negative ion spray voltage was -3000 V; sheath gas flow rate was 35 Arb, S-lens RF level was 50, normalized fragmentation energy was 10, 20, 40, secondary mass spectrometry resolution was 15000, mass range was 100-1500 m / z, full resolution was 60000, capillary temperature was 32°C, auxiliary gas heater temperature was 350°C, and auxiliary gas flow rate was 8 Arb.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention extracts the main active compound - glycyrrhizin - from the traditional Chinese medicine liquorice, and confirms the therapeutic effect of glycyrrhizin on colon cancer 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 spread of tumors in clinical practice, prolong the survival time of patients, and confirm its safety through toxicity experiments, solving the problem of large side effects of clinical colon cancer treatment drugs.

[0040] The present invention identifies four active monomer compounds, namely, ganoderma lucidum flavonoids, isolongifolia 9-one, nordihydroguaiaretic acid and glabridin, and verifies them through in vivo and in vitro experiments. It is found that the compounds can inhibit the proliferation of colon cancer cells DLD-1 and HCT-116 and also slow down the diffusion rate of tumor cells. Moreover, the combination of the compounds has no significant effect on normal colon epithelial cells NCM460, reduces the toxicity of glabridin to normal colon epithelial cells NCM460, has a synergistic effect, and provides a reference for the safe development of small molecule targeted drugs in clinic. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The EIC graph of Ganoderma lucidum ketone and its MS / MS graph compared with the standard library LuMet-TCM.

[0042] Figure 2 This is the EIC graph of isothiocyanate 9-one and its MS / MS graph compared with the public library HerbDB.

[0043] Figure 3 The EIC graph of nordihydroguaiaretic acid and its MS / MS graph compared with the public library HerbDB.

[0044] Figure 4 The EIC graph of glabridinone and its MS / MS graph compared with the standard library LuMet-TCM.

[0045] Figure 5To investigate the effect of licorice flavonoids on the survival time of AOM / DSS colon cancer mice.

[0046] Figure 6 The effect of licorice flavonoids on the intestine of AOM / DSS colon cancer mice. A is the length of the intestine; B is the number of tumors in the colon.

[0047] Figure 7 To investigate the effects of licorice flavonoids on the intestinal barrier of AOM / DSS colon cancer mice.

[0048] Figure 8 Effects of licorice flavonoids on the proliferation of colon cancer cells in a zebrafish xenograft model.

[0049] Fig. 9 Effects of licorice flavonoids on metastasis of colon cancer cells in a zebrafish xenograft model.

[0050] Fig.10 The effect of licorice flavonoids on colon cancer cells HCT-116 and DLD-1. A is HCT-116 cells; B is DLD-1 cells.

[0051] Fig.11 The effect of licorice flavonoids on the cell clone-forming ability of colon cancer cells HCT-116 and DLD-1 cells.

[0052] Fig.12 This is an experiment on Edu staining of licorice flavonoids on colon cancer cells HCT-116 and DLD-1 cells.

[0053] Fig.13 The effect of licorice flavonoids on mouse organs.

[0054] Fig.14 The effect of licorice flavonoids on the liver function of mice. A is aspartate aminotransferase / AST / GOT; B is alanine aminotransferase / ALT / GPT.

[0055] Fig.15 The effect of licorice flavonoids on the proliferation of NCM460 cells.

[0056] Fig.16 This is the effect of glycyrrhizin flavonoids on the cell clone-forming ability of NCM460 cells.

[0057] Fig.17 The effects of ganoderma lucidum ketone and isolongifolia 9-ketone on the proliferation of colon cancer cells HCT-116 and DLD-1. A is the effect of ganoderma lucidum ketone on HCT-116 cells; B is the effect of ganoderma lucidum ketone on DLD-1 cells; C is the effect of isolongifolia 9-ketone on HCT-116 cells; D is the effect of isolongifolia 9-ketone on DLD-1 cells.

[0058] Fig.18 The figure shows the effects of ganoderma lucidum ketone and isolongifolia 9-ketone on the proliferation of NCM460 cells. A is ganoderma lucidum ketone; B is isolongifolia 9-ketone.

[0059] Fig.19 Effects of ganoderma lucidum ketone and isolongifolia ketone on the proliferation of colon cancer cells in a zebrafish xenograft model.

[0060] Fig. 20 Effects of ganoderma lucidum ketone and isothiocyanate 9-ketone on the metastasis of colon cancer cells in a zebrafish xenograft model.

[0061] Fig.21 The effect of nordihydroguaiaretic acid on the proliferation of HCT-116, DLD-1 and NCM460 cells. A is HCT-116 cells; B is DLD-1 cells; C is NCM460 cells.

[0062] Fig. 22 The figure shows the effect of licorice on the proliferation of HCT-116, DLD-1 and NCM460 cells. A is HCT-116 cells; B is DLD-1 cells; C is NCM460 cells.

[0063] Fig.23 The effect of the combination of licorice root, ganoderma lucidum ketone, isothiocyanate and nordihydroguaiaretic acid on the proliferation of NCM460 cells.

[0064] Fig.24 The figure shows the effect of the combination of licorice root, ganoderma lucidum, isothiocyanate and nordihydroguaiaretic acid on the proliferation of HCT-116 and DLD-1 cells. A is HCT-116 cells; B is DLD-1 cells. DETAILED DESCRIPTION

[0065] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0066] Example 1 Isolation and Identification of Ganoderma Lucidum ketone, Ganoderma lucidum ketone, Licorice ketone and Nordihydroguaiaretic acid

[0067] 1. Experimental Methods

[0068] 1. Sample processing

[0069] The licorice sample was taken out, ground evenly with liquid nitrogen, and about 100 mg of the sample was weighed into a 1.5 mL centrifuge tube; 1 mL of water was added, vortexed for 1 min, steel balls were added, precooled in a -40°C refrigerator for 2 min, and then ground in a grinder (60HZ, 2 min), ultrasonically extracted in an ice-water bath for 60 min, centrifuged for 10 min (14000 rpm, 4°C), diluted 5 times with water to obtain the diluted supernatant, i.e., licorice brass, and 200 μL of the diluted supernatant was placed in an LC-MS injection vial with an inner liner for analysis.

[0070] 2. Liquid chromatography-mass spectrometry conditions

[0071] (1) The analytical instrument is a liquid chromatography-mass spectrometry system consisting of an ACQUITY UPLC I-Class HF ultra-high performance liquid phase tandem QE high-resolution mass spectrometer.

[0072] (2) Chromatographic conditions

[0073] Chromatographic column: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); column temperature: 45 ° C; mobile phase: mobile phase A is 0.1% (w / v) formic acid aqueous solution, mobile phase B is acetonitrile; flow rate: 0.35 mL / min; gradient elution information is shown in Table 1; injection volume: 5 μ L; PDA (diode array detector) scanning range: 210 ~ 400 nm.

[0074] (3) Mass spectrometry conditions

[0075] Ion source: HESI (Heated Elektrospray Ionization source).

[0076] The sample mass spectrometry signals were collected in positive and negative ion scanning modes, respectively.

[0077] Data acquisition mode: DDA (data dependent acquisition mode).

[0078] Scanning mode: Secondary product ion scanning mode Full MS / dd-MS2 (TOP 8).

[0079] The positive and negative ion parameters are shown in Table 2.

[0080] Table 1 Elution gradient information

[0081] Time (min) Mobile phase A (%, v / v) Mobile phase B (%, v / v) 0 95 5 2 95 5 4 70 30 8 50 50 10 20 80 14 0 100 15 0 100 15.1 95 5 16 95 5

[0082] Table 2 Positive and negative ion parameters

[0083]

[0084]

[0085] 3. Data Analysis

[0086] Preprocessing Before pattern recognition, the raw data were subjected to baseline filtering, peak identification, integration, retention time correction, peak alignment and normalization by the metabolomics processing software Progenesis QI v3.0 (Nonlinear Dynamics, Newcastle, UK). The identification of compounds was based on accurate mass, secondary fragments and isotope distribution, and the TCM database was used for qualitative analysis. The TCM database is a database of traditional Chinese medicine components established for plant samples. The database contains information on 5000+ traditional Chinese medicine component standards (standard products were purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd., etc.). For each identified component, an EIC diagram and an MS2 mirror comparison diagram with secondary fragment structure annotations were drawn. The chemical components were determined by comparing the MS / MS diagram with the standard library traditional Chinese medicine metabolite database LuMet-TCM and the public library HerbDB.

[0087] 2. Experimental Results

[0088] 1. Identification of Ganoderma lucidum ketone

[0089] like Figure 1 The following is the EIC graph of Ganodermanontriol and its MS / MS graph compared with the standard library LuMet-TCM. The retention time of the MS / MS graph compared with the standard library LuMet-TCM is 11.40min, which is mostly overlapped with the standard spectrum. It can be considered that the compound is Ganodermanontriol (CAS: 106518-63-2).

[0090] 2. Identification of isothiophenone

[0091] like Figure 2 As shown, the EIC graph of isolongifolen-9-one and its MS / MS graph compared with the public library HerbDB. The ECI graph of the compound is basically overlapped with the MS / MS graph of the public library HerbDB, and the compound can be considered to be isolongifolen-9-one (Isolongifolen-9-one, CAS: 1135-66-6).

[0092] 3. Identification of nordihydroguaiaretic acid

[0093] like Figure 3The EIC graph of nordihydroguaiaretic acid and its MS / MS graph compared with the public library HerbDB are shown. The retention time is 6.86 min, and the qualitative analysis is performed by RT+MS1 in comparison with the standard library or by predicted RT+MS1 in comparison with the public library. It can be considered that the compound is confirmed to be nordihydroguaiaretic acid (CAS: 500-38-9).

[0094] 4. Identification of Licorice

[0095] like Figure 4 The following is the EIC graph of glabrone and its MS / MS graph compared with the standard library LuMet-TCM. The retention time of the MS / MS graph compared with the standard library LuMet-TCM is 10.09min, which is mostly overlapped with the standard spectrum. It can be considered that the compound is confirmed to be glabrone (CAS: 60008-02-8).

[0096] Example 2 Inhibitory effect of licorice flavonoids on AOM / DSS-induced inflammation-related colon cancer in mice

[0097] 1. Experimental Methods

[0098] 1. Animal Model

[0099] SPF-grade C57BL / 6 male mice were adaptively fed for 1 week before modeling. The modeling was divided into three stages: in the first stage, except for the blank control group (intraperitoneal injection of normal saline), the other groups of mice were intraperitoneally injected with the carcinogen azoxymethane AOM (10 mg / kg), and were given 2% (w / v) dextran sulfate sodium DSS free drinking water the next day for 7 days, and then replaced with regular drinking water for 14 days; in the second stage, 2% (w / v) DSS was given free drinking water for 10 days, and then replaced with regular drinking water for 14 days; in the third stage, 2% (w / v) DSS was given free drinking water for 10 days, and then replaced with regular drinking water for 11 days. The blank control group was given regular drinking water throughout the whole process. When the mice were unresponsive, had bloody stools or mucous bloody stools, the modeling was successful, and colon cancer mice were obtained. The weight and food intake of the mice were recorded every day.

[0100] 2. Drug intervention

[0101] After AOM injection, mice were orally administered 208 mg / kg and 416 mg / kg of licorice flavonoids every day until the end point. The 208 mg / kg dose was calculated based on the human clinical prescription dose (1.6 g / 70 kg = 0.0228 g / kg), accounting for the body surface area between humans and animals (0.0228 g / kg × 9.1 = 208 mg / kg). Regorafenib (Selleck, USA) 20 mg / kg was used as a positive control.

[0102] 3. Colon length analysis

[0103] After the experiment, the colon part of the mice, that is, from above the anus to below the cecum, was removed and the changes in colon length of each group of mice were measured.

[0104] 4. Tumor number statistics

[0105] After the experiment, the mouse colon was cut open and cleaned in saline. The tumor condition in the mouse colon was observed on a white light board and statistically analyzed.

[0106] 5. Hematoxylin-eosin staining (HE staining)

[0107] At the end of the experiment, the mouse colon tissue was taken, 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, 95% (v / v) ethanol overnight, anhydrous ethanol I, II and III for 30 min each, xylene I, II and III for 30 min each, paraffin I and II for 30 min each, paraffin III for 1 h, dehydration, wax immersion, embedding and slicing were completed to obtain paraffin sections.

[0108] Paraffin sections were stained with HE according to the steps in Table 3 below and sealed with neutral gum. The percentages in Table 3 are all volume concentrations.

[0109] Table 3 HE staining steps

[0110]

[0111] 6. Periodic acid Schiff staining (PAS staining)

[0112] Follow step 5 to obtain paraffin sections.

[0113] Dewax the paraffin sections: Dewax the paraffin sections in xylene twice, each time for 15 min, to obtain dewaxed sections.

[0114] Hydration: The dewaxed sections were hydrated with gradient alcohols in sequence. The specific steps were as follows: anhydrous ethanol treatment for 5 min, 90% ethanol treatment for 3 min, 80% ethanol treatment for 3 min, and 70% ethanol treatment for 3 min; distilled water treatment for 3 min, repeated 3 times; PBS buffer treatment for 3 min, repeated 3 times to obtain hydrated sections.

[0115] Oxidation: Place the hydrated sections in a 1% (w / v) periodic acid solution and oxidize for 6 to 20 minutes to obtain oxidized sections.

[0116] Water washing: Rinse the oxidized sections thoroughly with distilled water, remove excess periodic acid solution for 10 minutes, and obtain water-washed oxidized sections.

[0117] Staining: Place the washed oxidized sections in a fuchsin sulfite reagent (Schiff reagent) and stain for 10 to 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. In winter, when the room temperature is low, the staining time can be extended to 20 minutes to obtain Schiff-stained sections.

[0118] Rinse: Rinse the Schiff-stained sections with running water until the color of the running water changes from red to colorless for 10 minutes to obtain the rinsed Schiff-stained sections.

[0119] Counterstaining: Counterstain the washed Schiff-stained sections with hematoxylin to make the cell nuclei appear blue. It usually takes 2 to 4 minutes to obtain counterstained sections.

[0120] Differentiation: If the cell nucleus is 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.

[0121] Bluing: Use Scott's Bluing Reagent to blue the counterstained sections or differentiated sections for 1 to 2 minutes, and rinse with running water for 3 minutes to obtain blue-returned sections.

[0122] Dehydration: The blue-returned slices were dehydrated in gradient alcohol in sequence, with the specific steps as follows: 70% (v / v) ethanol treatment for 1 min, 80% (v / v) ethanol treatment for 1 min, 95% (v / v) ethanol treatment for 2 min, and 100% (v / v) ethanol treatment for 4 min to obtain dehydrated slices.

[0123] Transparency: Use xylene to make the dehydrated sections transparent, which usually takes two times, each time for 15 minutes, to obtain transparent sections.

[0124] Sealing: Dry the transparent slices, add neutral gum to seal the slices, and cover with a coverslip.

[0125] 7. Cell proliferation antigen (Ki67) detection

[0126] According to step 6, hydrated sections were obtained, and the hydrated sections were placed in a 3% (v / v) hydrogen peroxide solution and soaked for 10 minutes to block endogenous peroxidase activity, and washed with tap water to obtain hydrogen peroxide-treated sections.

[0127] The hydrogen peroxide-treated sections were placed in a citric acid buffer (pH 6.0) for heat-induced antigen retrieval and treated in a microwave oven for 20 min to obtain repaired sections.

[0128] The repaired slices were naturally cooled to room temperature, washed with PBS buffer, covered with bovine serum albumin (BSA), and incubated at room temperature for 10 min to reduce nonspecific binding, thereby obtaining BSA-treated slices.

[0129] Three drops of Ki67 antibody reagent were added to each BSA-treated slice (to completely cover the slice tissue), incubated at room temperature for 30 min, and washed with PBS buffer for 5 min each time, repeated 3 times to obtain Ki67-treated slices.

[0130] Perform secondary antibody incubation: add about 100 μL of enzyme-labeled goat anti-mouse / rabbit IgG polymer to each Ki67-treated section (to completely cover the section tissue), incubate at room temperature for 30 min, wash with PBS buffer for 5 min each time, repeat 3 times, and obtain secondary antibody incubation sections.

[0131] Use freshly prepared DAB (diaminobenzidine) colorimetric solution to develop the secondary antibody incubated sections. Add about 100 μL of DAB colorimetric solution to each secondary antibody incubated section. The color development time is 1 to 5 min (microscope examination to control staining). Rinse with tap water to terminate the color development reaction and obtain DAB-treated sections.

[0132] Counterstain, dehydrate, clear and mount the DAB-treated sections according to step 6.

[0133] 2. Experimental Results

[0134] 1. Licorice flavonoids prolong the survival time of AOM / DSS colon cancer mice

[0135] like Figure 5 As shown in the figure, licorice flavonoids prolonged the survival time of mice in a dose-dependent manner. The number of mice in the control group increased as the experimental time prolonged. The number of mice surviving in the low-dose licorice flavonoids group tended to stabilize in the middle and late stages of treatment, while the mice in the high-dose licorice flavonoids group maintained stable survival throughout the treatment process.

[0136] 2. Licorice flavonoids reduce the number of tumors in the colon of mice

[0137] like Figure 6 As shown in A, the length of the colon of mice in the licorice flavonoids group was significantly longer than that in the control group, indicating that licorice flavonoids treatment can significantly reduce colon inflammation in mice. Figure 6 As shown in B, compared with the control group, the number of tumors in the colon of mice in the glycyrrhizin-treated group was significantly reduced, indicating that glycyrrhizin has a significant inhibitory effect on colon cancer.

[0138] 3. Licorice flavonoids repaired the intestinal mucosa of mice, improved intestinal damage, and inhibited the malignant proliferation of tumor cells

[0139] like Figure 7As shown in the results of HE staining, PAS staining and Ki67 staining of the mouse intestine, the intestinal barrier of the mice in the control group was significantly damaged, the intercellular space increased, the goblet cells decreased, and the malignant proliferation of tumors in the colon was high. After treatment with different doses of licorice flavonoids, the intestinal barrier was repaired, the goblet cells increased, and the malignant proliferation of tumors decreased. This shows that licorice flavonoids can repair the colon mucosa of mice, improve intestinal damage, inhibit the malignant proliferation of tumor cells, and have a therapeutic effect on colon cancer.

[0140] Example 3 Licorice flavonoids inhibit the development of zebrafish xenograft colon cancer

[0141] 1. Experimental Methods

[0142] 1. Build the model

[0143] AB zebrafish were used as model subjects, and the fish were maintained according to standard procedures. Embryos were obtained from naturally spawned fish and maintained at 28.5°C, 12:12 light-dark (LD) cycle, in rearing water (rearing water supplemented with 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.3 mM MgSO4; pH = 7). All rearing and experimental procedures were in accordance with Italian and European regulations on the protection of animals for scientific purposes (Directive 2010 / 63 / EU). For all experimental data, the age of the embryos is expressed in hours post fertilization (hpf), hours post injection (hpi), and days post fertilization (dpf).

[0144] 2. Cell culture and labeling

[0145] Human colon cancer cells HCT-116 were screened for dedifferentiation phenotype, invasion, metastasis potential and clinical significance and were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). They were cultured in RPMI 1640 (RoswellPark Memorial Institute 1640) medium supplemented with 10% (v / v) fetal bovine serum (FBS) at 37°C in an incubator with humidified air and 5% CO2. For xenografting, HCT-116 cells were washed, digested and cultured in Dil Vybrant TM The cells were resuspended in RPMI 1640 medium containing DIL Red fluorescent dye (brand: Thermo Fisher Scientific, USA; catalog number; V22885) for 20 min, the cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with PBS to obtain DIL-labeled HCT-116 cells. The DIL-labeled HCT-116 cells were resuspended in PBS (phosphate buffered saline) and injected into zebrafish yolk.

[0146] 3. Xenotransplantation

[0147] AB zebrafish embryos at 48 hpf were fixed on 2% (w / v) agarose pads with 0.04% tricaine (Brand: Merck, Germany; Cat. No.: E10521). 200 dil-labeled HCT-116 cells were injected into zebrafish yolks to observe tumor volume and its interaction with the host and therapeutic drugs. Fluorescence microscopy was used to observe changes in tumor volume at 3 dpf. The degree of fluorescence diffusion of tumor cells at 5 dpf19 was observed to study the metastatic potential of the tumor.

[0148] 4. Drug intervention

[0149] Licorice flavonoids were dissolved in DMSO (dimethyl sulfoxide) and divided into three dose groups for zebrafish exposure experiments. The concentrations of licorice flavonoids were 5, 10 and 20 μg / mL, respectively. The concentrations were selected based on the in vivo toxicology experiment of zebrafish. The zebrafish in the control group were exposed to water containing the same concentration of DMSO. The final concentration of DMSO in each group was controlled within 1% (v / v).

[0150] Tumor volume and metastatic potential quantification: Zebrafish successfully injected with HCT-116 cells in the yolk were exposed to drugs (licorice flavonoids or DMSO) for 24h and 72h, and the effects of drugs on tumor volume and tumor spread were observed. Fluorescence stereomicroscope imaging and Image J analysis of fluorescence area and intensity were used to characterize the effects of drugs on tumor volume and tumor spread.

[0151] 2. Experimental Results

[0152] 1. Licorice flavonoids inhibit the proliferation of colon cancer cells in zebrafish xenograft models

[0153] like Figure 8 As shown, compared with the control group, the fluorescence area of ​​yolk sac tumor cells in the licorice flavonoids administration group was significantly reduced, and it was concentration-dependent, and the effect became more obvious with the increase of concentration.

[0154] 2. Licorice flavonoids inhibit tumor cell metastasis

[0155] like Fig. 9 As shown, the extent of tumor cell spread throughout the body in the glycyrrhizin-treated group was significantly reduced, and the effect became better with increasing concentration, indicating that glycyrrhizin can inhibit tumor cell metastasis.

[0156] Example 4 Licorice flavonoids inhibit colon cancer cell proliferation in vitro

[0157] 1. Experimental Methods

[0158] 1. DLD-1 cells and HCT-116 cells

[0159] 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) at 37°C in an incubator with humidified air containing 5% CO2.

[0160] 2. CCK-8 detection of cell proliferation

[0161] CCK-8 assay for cell viability: Cells were seeded in 96-well plates (5 × 10 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.

[0162] 3. Cell clone formation experiment

[0163] The steps of colony formation experiment are as follows: 3000 cells / well were inoculated in 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, fixed with 4% (w / v) paraformaldehyde, stained with crystal violet, dissolved with 1% (w / v) SDS (sodium dodecyl sulfate), and the absorbance was measured at 570 nm for quantitative analysis.

[0164] 4. Edu (5-ethynyl-2'-deoxyuridine) staining experiment

[0165] Edu assay for cell proliferation: cells were cultured on glass slides and incubated with different final concentrations of glycyrrhizin (0, 2, 4, 8, 16, and 32 μg / mL) for 24 h. Edu was added to each glass slide and incubated for 1.5 h in an incubator at 37°C and 5% CO2 in humidified air. The cells were fixed with 4% (w / v) paraformaldehyde and analyzed using the E-clik EDU cell proliferation Imaging Assay Kit (Green, Elab Edu-positive cells were recorded using confocal microscopy and fluorescence quantitative analysis was performed using ImageJ.

[0166] 2. Experimental Results

[0167] 1. Licorice flavonoids have a killing effect on colon cancer cells

[0168] like Fig.10 A and Fig.10As shown in B, with the increase of licorice flavonoids concentration, the number of cell deaths increased, and the IC 50 The values ​​were 20.92 μg / mL and 27.93 μg / mL respectively.

[0169] 2. Licorice flavonoids inhibit colon cancer cell cloning

[0170] like Fig.11 As shown in the figure, the colony-forming ability of HCT-116 and DLD-1 cells was significantly weakened after treatment with glycyrrhizin. The number of cell clones decreased significantly with increasing concentration, indicating that glycyrrhizin can weaken the proliferation ability of colon cancer cells in vitro.

[0171] 3. Licorice flavonoids inhibit tumor cell proliferation

[0172] like Fig.12 As shown, from Edu staining, it can be found that with the increase of glycyrrhizic flavonoids concentration, Edu-positive cells gradually decreased, and DLD-1 and HCT-116 cells showed a similar trend, further indicating that glycyrrhizic flavonoids can inhibit the proliferation of colon cancer cells.

[0173] Example 5 In vivo safety evaluation of licorice flavonoids

[0174] 1. Experimental Methods

[0175] 1. HE staining

[0176] According to Example 2, mouse heart, liver, spleen, lung and kidney sections were fixed, hydrated, and stained with hematoxylin to show the fine structure of the cell nucleus, and then the cytoplasm was contrast stained with eosin, followed by a series of gradient alcohol dehydration and clearing treatments, sealing, and images were obtained using a digital slide scanner (KFBIO, China).

[0177] 2. Determination of ALT and AST activity

[0178] According to Example 2, peripheral blood was collected from each group of mice and centrifuged at 3000 rpm and 4°C for 10 min to obtain serum. The liver function of mice was detected using an aspartate aminotransferase (AST / GOT) test kit (microplate method; manufacturer: Nanjing Jiancheng Bioengineering Institute; item number: C010-2-1) and an alanine aminotransferase (ALT / GPT) test kit (microplate method; Nanjing Jiancheng Bioengineering Institute; item number: C009-1-1).

[0179] 2. Experimental Results

[0180] 1. Licorice flavonoids have no obvious toxicity to mouse organs

[0181] like Fig.13As shown, compared with the control group, there was no obvious injury reaction and obvious inflammatory infiltration in the heart, liver, spleen, lung and kidney of mice in the glycyrrhizin treatment group, indicating that glycyrrhizin is safe for in vivo administration.

[0182] 2. Licorice flavonoids have no significant effect on mouse liver function

[0183] like Fig.14 A and Fig.14 As shown in B, compared with the control group, after the administration of glycyrrhizin, the activities of aspartate aminotransferase / AST / GOT and alanine aminotransferase / ALT / GPT in the mouse serum did not decrease significantly, indicating that glycyrrhizin had no effect on the liver function of mice and also demonstrated its safety.

[0184] Example 6 In vitro safety evaluation of licorice flavonoids

[0185] 1. Experimental Methods

[0186] The method of Example 4 was used to detect the effects of licorice flavonoids on the proliferation and cell clone formation of normal human colon epithelial cells NCM460.

[0187] 2. Experimental Results

[0188] 1. Licorice flavonoids have little effect on the proliferation of NCM460 cells

[0189] like Fig.15 As shown, 24 hours after glycyrrhizin was administered to NCM460 cells, until glycyrrhizin reached the highest concentration of 128 μg / mL, the cell survival rate still remained above 50%, indicating the relative safety of glycyrrhizin administration in vitro.

[0190] 2. Licorice flavonoids do not affect the colony formation of NCM460 cells

[0191] like Fig.16 As shown, licorice flavonoids had no significant effect on the number of NCM460 cell clones, and had no significant inhibitory effect on the size of a single clone, indicating that licorice flavonoids had little effect on the proliferation of normal human colon epithelial cells, indicating its safety.

[0192] Example 7 Ganoderma lucidum ketone and isothiocyanate 9-ketone inhibit the proliferation of colon cancer cells in vitro

[0193] 1. Experimental Methods

[0194] 1. According to the method of Example 4, the effects of ganoderma lucidum ketone and isolongifolia ketone on the proliferation of human colon cancer cells DLD-1 cells and HCT-116 cells and human normal colon epithelial cells NCM460 were detected by CCK-8 method. The cells were cultured with different concentrations of ganoderma lucidum ketone and isolongifolia ketone for 24 hours and 48 hours respectively.

[0195] 2. Experimental Results

[0196] 1. Ganoderma lucidum ketone and isothiocyanate 9-ketone inhibit the proliferation of colon cancer cells

[0197] like Fig.17 As shown in A to D and Table 4, after the two colon cancer cells were treated with ganoderma lucidum ketone and isolongifolia 9-one, they had a significant inhibitory effect on cell proliferation, indicating that ganoderma lucidum ketone and isolongifolia 9-one have an intervention effect on colon cancer in vitro.

[0198] Table 4 Effects of Ganoderma lucidum ketone and Isophyllotoxin 9 on the proliferation of colon cancer cells HCT-116 and DLD-1

[0199]

[0200]

[0201] 2. Ganoderma lucidum ketone and isothiocyanate 9-ketone do not affect the proliferation of NCM460 cells

[0202] like Fig.18 A. Fig.18 As shown in B and Table 5, the treatment of NCM460 cells with ganoderma lucidum ketone and isolongifolia 9-one for 24 h and 48 h did not show obvious proliferation inhibition, indicating that ganoderma lucidum ketone and isolongifolia 9-one have certain safety.

[0203] Table 5 Effects of Ganoderma lucidum ketone and Isophyllotoxin 9 on the proliferation of NCM460 cells

[0204]

[0205]

[0206] Example 8: Ganoderma lucidum ketone and isothiocyanate 9-ketone intervene in the occurrence and development of colon cancer in vivo

[0207] 1. Experimental Methods

[0208] The method of Example 3 was used to evaluate the effects of ganoderma lucidum ketone and isothiocyanate 9-ketone on colon cancer cells in a zebrafish xenograft model.

[0209] 2. Experimental Results

[0210] 1. Ganoderma lucidum ketone and isothiocyanate 9-ketone inhibit tumor cell proliferation

[0211] like Fig.19 As shown in the figure, the fluorescence area of ​​zebrafish yolk sac tumor cells was significantly reduced after treatment with ganoderma lucidum ketone and isolongifolia 9-one, and it was concentration-dependent. The effect became more obvious with the increase of concentration, indicating that ganoderma lucidum ketone and isolongifolia 9-one can inhibit tumor proliferation.

[0212] 2. Ganoderma lucidum ketone and isothiocyanate 9-ketone delay the spread of tumor cells

[0213] like Fig. 20 As shown, after treatment with Ganoderma lucidum ketone and Isophyllotoxin 9-ketone, it was found that the degree of tumor cell spread throughout the zebrafish was significantly reduced, and the effect became better with increasing concentrations, indicating that Ganoderma lucidum ketone and Isophyllotoxin 9-ketone delay the spread of tumor cells.

[0214] Example 9 Inhibitory Effect of Nordihydroguaiaretic Acid on Colon Cancer

[0215] 1. Experimental Methods

[0216] The effect of nordihydroguaiaretic acid on the proliferation of human colon cancer cells DLD-1 and HCT-116 cells and human normal colon epithelial cells NCM460 was detected by CCK-8 method according to the method of Example 4. The cells were cultured with different concentrations of nordihydroguaiaretic acid for 24 h and 48 h.

[0217] 2. Experimental Results

[0218] 1. Nordihydroguaiaretic acid inhibits the proliferation of colon cancer cells

[0219] like Fig.21 As shown in A to C and Table 6, nordihydroguaiaretic acid has a certain proliferation inhibitory effect on colon cancer cell HCT-116 and DLD-1 cells, and has a certain time dependence, while it has no obvious inhibitory effect on NCM460 cells.

[0220] Table 6 Effect of nordihydroguaiaretic acid on proliferation of HCT-116, DLD-1 and NCM460 cells

[0221]

[0222]

[0223] Example 10 Inhibitory effect of licorice root on colon cancer

[0224] 1. Experimental Methods

[0225] The effect of glabridin on the proliferation of human colon cancer cells DLD-1 and HCT-116 cells and human normal colon epithelial cells NCM460 was detected by CCK-8 method according to the method of Example 4. The cells were cultured with different concentrations of glabridin for 24 h and 48 h.

[0226] 2. Experimental Results

[0227] like Fig. 22As shown in A to C and Table 7, glabridin has a strong inhibitory effect on the proliferation of colon cancer cells HCT-116 and DLD-1 cells, and has a certain time dependence. However, glabridin also strongly inhibits the proliferation of NCM460 cells and has a relatively large toxicity.

[0228] Table 7 Effects of licorice on proliferation of HCT-116, DLD-1 and NCM460 cells

[0229]

[0230]

[0231] Example 11 Inhibitory effect of a combination of licorice root, ganoderma lucidum, isothiocyanate and nordihydroguaiaretic acid on colon cancer

[0232] 1. Experimental Methods

[0233] According to the method of Example 4, the effect of the combination of glabridin, ganoderma lucidum, isothiocyanate and nordihydroguaiaretic acid on the proliferation of colon cancer cells HCT-116, DLD-1 and human normal colon epithelial cells NCM460 was detected by CCK-8 method. The cells were cultured with different concentrations of the combination for 24h and 48h.

[0234] The combination of licorice root, ganoderma lucidum, isothiocyanate and nordihydroguaiaretic acid was set to have 9 gradient concentrations, namely 0, 1, 2, 4, 8, 16, 32, 64 and 128 μM, each gradient concentration represents the concentration of each component therein, and the concentrations of each component are the same.

[0235] 2. Experimental Results

[0236] like Fig.23 As shown in Table 8, the co-administration of licorice root with ganoderma lucidum ketone, isolongifolia 9-one and nordihydroguaiaretic acid was found to significantly reduce the toxic effect of licorice root on normal human colon epithelial cells NCM460, indicating that the four components have a synergistic effect and reduce toxicity.

[0237] like Fig.24 As shown in Table 8, the combination of licorice root, ganoderma lucidum ketone, isolongifolia 9-ketone and nordihydroguaiaretic acid was found to have a significant inhibitory effect on the proliferation of colon cancer cells HCT-116 and DLD-1, and the effect was stronger than that of a single component alone. This shows that the four components can synergize. Table 8 Effect of the combination of licorice root, ganoderma lucidum ketone, isolongifolia 9-ketone and nordihydroguaiaretic acid on the proliferation of HCT-116, DLD-1 and NCM460 cells

[0238]

[0239]

[0240] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. The use of licorice flavonoids in the preparation of a drug for preventing and / or treating colon cancer, characterized in that: The licorice flavonoids include ganoderma lucidum ketone, isolongifolia ketone, nordihydroguaiaretic acid and glabridinone.

2. The use according to claim 1, characterized in that The drug is a drug for inhibiting the proliferation of colorectal cancer.

3. The use according to claim 1, characterized in that The drug is a drug for inhibiting colorectal cancer metastasis.

4. The use according to claim 1, characterized in that The drug is a drug for repairing the intestinal barrier.

5. The use according to 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 use according to claim 1, characterized in that The concentration of the ganoderma lucidum ketone, isolongifolia 9-ketone, nordihydroguaiaretic acid or glycyrrhizinone is 1-128 μM.

7. The use according to claim 1, characterized in that The medicine is a pharmaceutical preparation prepared by taking the glycyrrhiza flavonoids as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

8. The use according to claim 7, characterized in that The medicine or pharmaceutical preparation includes tablets, granules or liquid preparations.

9. The use according to claim 1, characterized in that The preparation method of the licorice flavonoids comprises the following steps: S11: Mix the licorice sample with water, vortex and shake to mix, and grind; the ratio of the licorice sample to water is licorice sample: water = 10-250 mg: 0.1-2.5 mL. S12: subjecting the product ground in step S1 to ice bath ultrasonic extraction for 30 to 60 minutes. S13: centrifuging the extracted product in step S2 at a speed of 5000 to 15000 rpm for 5 to 15 min.

10. The use according to claim 1, characterized in that The quality inspection method of the licorice flavonoids comprises the steps of using liquid chromatography combined with mass spectrometry to detect ganoderma lucidum ketone, isolongifolia 9-ketone, nordihydroguaiaretic acid and glabridinone in the licorice flavonoids.

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