Application of isotoosendanin in preparation of medicine for preventing and treating colorectal cancer

Abstract

The isotoosendanin can effectively inhibit proliferation of colorectal cancer cells in vitro, induce cycle arrest of the colorectal cancer cells and induce apoptosis of the colorectal cancer cells. The tumor growth of the inflammation-related colorectal cancer can be inhibited in vivo, colon shortening caused by the inflammation-related colorectal cancer is inhibited, and liver and spleen organ enlargement caused by the inflammation-related colorectal cancer is recovered. The results show that the isotoosendanin has a good effect in preventing and / or treating the colorectal cancer, and can be used as a medicine with a development prospect for preventing and treating the colorectal cancer.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to use of isotoxin in preparing medicines for preventing and treating colorectal cancer. Background Art

[0002] Colorectal cancer is a common malignant tumor, and inflammation-related colorectal cancer (Colitis associated colorectal cancer, CAC) shows the development law of "inflammation-atypical hyperplasia-cancer". Its colon ulcers do not heal for a long time, and repeated stimulation of inflammatory infiltration eventually leads to cancer. How to improve the survival rate and quality of life of CAC patients has become a difficult problem that needs to be solved urgently in my country and even in the world.

[0003] Isotoosendanin (ITSN) is a tetracyclic triterpenoid compound extracted and separated from Toosendan Fructus Toosendans. It is the main active ingredient of Toosendan Fructus Toosendans, a traditional Chinese medicine, and is also widely found in many other Meliaceae plants. It is soluble in organic solvents such as methanol, ethanol, and DMSO. Currently, there are few reports on the pharmacological effects of isotoosendanin, and its pharmacological research reports are mainly on anti-inflammatory and analgesic effects, treatment of triple-negative breast cancer, and non-small cell lung cancer. So far, there have been no reports on the use of isotoosendanin as a monomer in the fight against colorectal cancer. Summary of the invention

[0004] To this end, the object of the present invention is to provide the use of isotomatolin in the preparation of drugs for preventing and / or treating colorectal cancer. The present invention finds that isotomatolin can effectively inhibit the proliferation of colorectal cancer cells in vitro, induce the cycle arrest of colorectal cancer cells, and induce the apoptosis of colorectal cancer cells, and can inhibit the tumor growth of inflammation-related colorectal cancer in vivo, inhibit the shortening of the colon caused by inflammation-related colorectal cancer, and restore the enlargement of the liver and spleen caused by inflammation-related colorectal cancer. Therefore, isotomatolin has a good effect in preventing and / or treating colorectal cancer.

[0005] The first aspect of the present invention provides the use of isotoxin in the preparation of drugs for preventing and / or treating colorectal cancer.

[0006] As used herein, the term "treat" refers to and includes improvement, alleviation and / or therapeutic use and outcome, or any combination thereof.

[0007] As used herein, the term "prevention" does not refer to or require absolute or complete prevention (i.e., 100% preventive or protective use or results). As used herein, "prevention" refers to the administration of a compound or composition to reduce or decrease the severity of a specific condition, symptom, or disorder described herein; to reduce or decrease the likelihood of experiencing a specific condition, symptom, or disorder described herein; or to delay the onset or recurrence of a specific condition, symptom, or disorder described herein; or any combination of uses and results of the above.

[0008] In some embodiments of the present invention, the colorectal cancer includes inflammation-related colorectal cancer.

[0009] In the present invention, "inflammation-related colorectal cancer" refers to a type of colorectal cancer that presents the development pattern of "inflammation-atypical hyperplasia-carcinogenesis". Due to the long-term non-healing of colon ulcers, repeated stimulation of inflammatory infiltration eventually causes cancer.

[0010] In some embodiments of the invention, the prevention and / or treatment comprises:

[0011] (i) inhibiting the proliferation of colorectal cancer cells;

[0012] (ii) induces cell cycle arrest in colorectal cancer cells;

[0013] (iii) induce apoptosis of colorectal cancer cells;

[0014] (iv) inhibiting tumor growth of inflammation-related colorectal cancer;

[0015] (v) inhibiting the shortening of the colon caused by inflammation-related colorectal cancer; and

[0016] (ⅵ) Restoration of liver and spleen organomegaly caused by inflammation-related colorectal cancer.

[0017] In some embodiments of the present invention, the colorectal cancer cells include HCT116, HT-29 and SW620 cells.

[0018] In some embodiments of the invention, the cycle arrest comprises G1 / G0 cycle arrest and S cycle arrest.

[0019] In some embodiments of the present invention, the medicament comprises isotoxin as an active ingredient and a pharmaceutically acceptable excipient.

[0020] In some embodiments of the present invention, the therapeutically effective amount of isotoxin in the medicine is 20 mg / kg-50 mg / kg.

[0021] As used herein, the term "therapeutically effective amount" refers to the amount of isotoxin for treating or preventing colorectal cancer. The dose is an amount sufficient to show a therapeutic or preventive or ameliorative effect. The effects include the treatment or prevention of colorectal cancer conditions listed herein. The exact effective amount for the subject will depend on the subject's weight and health, the nature and extent of the colorectal cancer condition, the treating physician's advice, and the therapy or combination of therapies selected for administration.

[0022] In some embodiments of the present invention, the medicament comprises isotoxin as the sole active ingredient and pharmaceutically acceptable excipients.

[0023] In some embodiments of the present invention, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0024] In some embodiments of the present invention, the oral dosage form is a tablet, a capsule, an oral liquid, a granule or a powder.

[0025] It is understandable that, for the convenience of medication, the active ingredient isoflavone can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch slurry and syrup, etc.), disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol and micropowder silica gel, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue and medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium pyrosulfite and sodium thiosulfate, etc.), antibacterial agents (such as phenol, benzyl alcohol and thimerosal, etc.), and isotonic regulators (such as sodium chloride and glucose, etc.).

[0026] It is to be understood that the oral tablet examples described in the embodiments of the present invention include but are not limited to enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratched tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, oral patches, etc.

[0027] It is to be understood that the oral capsules described in the embodiments of the present invention include but are not limited to hard capsules, soft capsules, enteric-coated capsules, sustained-release capsules, controlled-release capsules, and the like.

[0028] It is to be understood that the oral liquid preparations described in the embodiments of the present invention include but are not limited to oral suspensions, oral emulsions, syrups, oral liquids, oral emulsions, emulsions, colloidal solutions, mixtures, dews, drops, suspension drops, etc.

[0029] It is to be understood that the oral granules described in the embodiments of the present invention include but are not limited to enteric-coated granules, sustained-release granules, fine granules, teas, suspension granules, effervescent granules, and the like.

[0030] It is to be understood that the oral powders described in the embodiments of the present invention include but are not limited to medicinal powders, powders, dry suspensions, etc.

[0031] It can be understood that the injection dosage forms described in the embodiments of the present invention include but are not limited to injection solutions, injection solutions, injection solutions for intravenous drip, injection suspensions, sterile powders for injection, intravenous injections, water injections, injection emulsions, powder injections, injections, sterile powder injections, freeze-dried powder injections, etc.

[0032] The beneficial effects of the present invention are:

[0033] The present invention found that isotosenin can effectively inhibit the proliferation of colorectal cancer cells in vitro, induce the cycle arrest of colorectal cancer cells, and induce the apoptosis of colorectal cancer cells, and can inhibit the tumor growth of inflammation-related colorectal cancer in vivo, inhibit the shortening of the colon caused by inflammation-related colorectal cancer, and restore the enlargement of the liver and spleen caused by inflammation-related colorectal cancer. The above results show that isotosenin has a good effect in preventing and / or treating colorectal cancer, and can be used as a drug for preventing and treating colorectal cancer with development prospects.

[0034] The present invention also found that the toxicity of isoflavone to normal human intestinal epithelial cells is lower than that to colorectal cancer cells. Long-term administration of isoflavone was not found to have significant liver toxicity through in vivo experiments, indicating that isoflavone is safe to use as a drug for the treatment of colorectal cancer.

[0035] The above results indicate that isoflavone can be used as a safe and effective drug for the prevention and / or treatment of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0037] Figure 1 The IC values ​​of isotonin against various colorectal cancer cells were shown 50 Results; Figure 1 A is the IC of isotoxin on HCT116 cells 50 result, Figure 1 B is the IC of isotoxin on HT-29 cells 50 result, Figure 1 C is the IC of isotoxin on SW620 cells 50 result, Figure 1 D is the IC of isotoxin on normal intestinal epithelial cells NCM460 50 result.

[0038] Figure 2 A visual comparison chart showing the effect of isotropin on the formation of clone colonies of various colorectal cancer cells; Figure 2 A shows the effect of isotoxin on HCT116 cell colony formation at three concentrations: low (0.5 μM), medium (1.0 μM), and high (2.0 μM). Figure 2 B shows the effect of isotoxin on HT-29 cell clone colony formation at three concentrations: low (1.25 μM), medium (2.5 μM), and high (5.0 μM). Figure 2 C shows the effect of isotoxin on the clonal colony formation of SW620 cells at three concentrations: low (0.625 μM), medium (1.25 μM), and high (2.5 μM).

[0039] Figure 3 for Figure 2 Statistical results of Figure 3 A shows the statistical results of the effect of isotoxin on the colony formation of HCT116 cells. Figure 3 B shows the statistical results of the effect of isotoxin on the formation of HT-29 cell clones. Figure 3 C shows the statistical results of the effect of isotoxin on the clonal colony formation of SW620 cells.

[0040] Figure 4 The flow cytometry results show that isoflavone induces apoptosis in various colorectal cancer cells; Figure 4 A shows the flow cytometry of apoptosis induced by isoflavone (DMSO, 1 μM, 2 μM, 4 μM) in HCT116 cells. Figure 4 B shows the flow cytometry of apoptosis induced by isoflavone (DMSO, 2.5 μM, 5 μM, 10 μM) in HT-29 cells. Figure 4 C shows the flow cytometry graph of apoptosis induced by isoflurane (DMSO, 1.25 μM, 2.5 μM, 5 μM) in SW620 cells.

[0041] Figure 5 for Figure 4 Statistical results of Figure 5 A shows the statistical results of apoptosis induced by isoflavone in HCT116 cells. Figure 5 B shows the statistical results of apoptosis induced by isoflurane on HT-29 cells. Figure 5 C shows the statistical results of apoptosis induced by isoflurane in SW620 cells.

[0042] Figure 6 The results show that isoflavone induces cell cycle arrest in various colorectal cancer cells. Figure 6 A shows the fitting results of cell cycle arrest induced by isoflurane (DMSO, 1μM, 2μM, 4μM) in HCT116 cells. Figure 6 B shows the fitting results of cell cycle arrest induced by isoflurane (DMSO, 2.5μM, 5μM, 10μM) in HT-29 cells. Figure 6 C shows the fitting results of cell cycle arrest induced by isoflurane (DMSO, 1.25 μM, 2.5 μM, 5 μM) in SW620 cells.

[0043] Figure 7 for Figure 6 Statistical results of Figure 7 A shows the statistical results of cell cycle arrest induced by isoflurane in HCT116 cells. Figure 7 B shows the statistical results of cell cycle arrest induced by isoflavone in HT-29 cells. Figure 7 C shows the statistical results of cell cycle arrest induced by isoflurane in SW620 cells.

[0044] Figure 8 A graph showing the time-weight results of an in vivo experiment after administration of isoflavone to mice.

[0045] Fig. 9 A graph showing the time-disease activity index (DAI) results of an in vivo experiment after administration of isoflavone to mice.

[0046] Fig.10 The graph shows the comparison of the length of the mouse colon and the number of tumors in the in vivo experiment; Fig.10 A shows a direct comparison of the length of the mouse colon and the number of tumors. Fig.10 B shows the comparative statistical graph of mouse colon length. Fig.10 C shows the comparative statistical graph of the number of mouse colon tumors.

[0047] Fig.11 The graph shows the comparison results of the coefficients of the main organs of mice in the in vivo experiment; Fig.11 A-11F are intuitive group comparisons of the mouse heart, lung, kidney, liver, spleen and spleen, respectively. DETAILED DESCRIPTION

[0048] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0049] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In some embodiments, raw materials, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0050] The experimental materials and instrument information involved in the specific embodiments are as follows:

[0051] 1. Experimental Animals

[0052] SPF-grade C57BL / 6 male mice, 5 weeks old, were purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Guangdong) 2022-0063, animal quality certificate number: 44829700030730. The animals were raised in the Animal Experiment Center of the University Town Campus of Guangzhou University of Chinese Medicine, with experimental animal license number: SYXK (Guangdong) 2023-0342. Animal experiments were approved by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine (20240407005). They were given normal SPF-grade feed, bedding was changed regularly, and cages were disinfected. The experiments were conducted after 7 days of adaptive feeding.

[0053] 2. Main drugs and experimental materials

[0054] 2.1 Cell lines

[0055] HCT116 (human colorectal cancer cells), HT-29 (human colorectal cancer cells), and SW620 (human colorectal cancer cells) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences).

[0056] 2.2 Main drugs

[0057] Isotoosendanin (ITSN) was purchased from Med Chem Express, purity>99%, HY-N7694; azomethane AOM was purchased from Med Chem Express, purity>99%, HY-111375; dextran sulfate sodium DSS was purchased from Coolaber, MW: 36000-50000, CD33222105.

[0058] 2.3 Main reagents

[0059] Polyoxyl-15 hydroxystearate, MACKLIN, C16704849; DMEM, purchased from Gibco, 6124407; fetal bovine serum, purchased from PAN, ST181208; 1×PBS buffer, purchased from Biosharp, BL302A; penicillin and streptomycin, purchased from Gibco, 15140-122; cell-grade dimethyl sulfoxide (DMSO), purchased from MP Biomedicals LLC, 196055; CCK-8 kit, purchased from Biosharp, BS350B; crystal violet staining solution (2.5%, Gram), purchased from Solarbio, G1061; Annexin V-FITC / PI fluorescent double staining cell apoptosis detection kit, purchased from Precell, P-CA-201; PI staining cell cycle detection kit, purchased from Abbkine, KTA2020.

[0060] 2.4 Main instruments

[0061] Analytical balance, Ohaus Instruments (Shanghai) Co., Ltd., CP214; CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd., Q5200DB; CO2 incubator (universal type), ESCO Cel Mate, Singapore; biological safety cabinet, ESCO Class II BSC, Singapore; full-wavelength microplate reader, Thermo Multiskan GO, USA; biological microscope, OLYMPUSBX53, Japan; DxP Flow cytometer, Cytek, USA.

[0062] The statistical analysis method involved in the specific embodiment is as follows:

[0063] The experimental results were expressed as mean ± standard deviation (Mean ± SD). One-way anova analysis was used for statistical analysis of multiple groups. If the variance homogeneity was met, LSD analysis was used. If the variance homogeneity was not met, Dunnett T3 analysis was used. When the data were not normally distributed, the differences between the groups were analyzed by non-parametric Kruskal-Wallis test, and P < 0.05 was statistically significant. Graph Pad Prism 8.0.2 software was used for drawing.

[0064] In the in vitro experiments, * indicates P < 0.05 compared with the normal group, ** indicates P < 0.01 compared with the normal group, and *** indicates P < 0.001 compared with the normal group.

[0065] In the in vivo experiments, # indicates P<0.05 compared with the normal group, ## indicates P<0.01 compared with the normal group, and ### indicates P<0.001 compared with the normal group. * indicates P<0.05 compared with the model group, ** indicates P<0.01 compared with the model group, and *** indicates P<0.001 compared with the model group.

[0066] Example 1: CCK8 experiment

[0067] (1) HCT116 cells, HT-29 cells, SW620 cells and normal intestinal epithelial cells NCM460 in the logarithmic growth phase were digested and centrifuged and resuspended in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin (PS)), seeded into 96-well plates at a density of 6000 cells per well, and cultured in a 37°C, 5% CO2 incubator.

[0068] (2) After culturing for 24 h, the cells were treated with 0 μM, 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM of isothiocyanate for 48 h, respectively, with 6 replicate wells set up for each concentration.

[0069] (3) 48 hours after administration, a complete medium containing 10% CCK8 reagent was prepared at a volume ratio of 9:1, and 100 μL of the complete medium containing 10% CCK8 reagent was added to each well 2 hours before analysis. The absorbance was recorded at 450 nm using a full-wavelength microplate reader.

[0070] Results: By Figure 1 A-1C shows that isoflavone can significantly inhibit the growth of colorectal cancer cells HCT116, HT-29, and SW620. After fitting the survival curve using Graph Pad Prism 8.0.2, the IC value of isoflavone on HCT116 cells after 48 hours of administration was calculated. 50 The IC value for HT-29 cells is 4.088 μM. 50 The IC value for SW620 cells is 9.669 μM. 50 is 5.944μM. Figure 1 D It can be seen that the IC of isotoxin on normal intestinal epithelial cells NCM460 50It is 32.63μM. It can be seen that isotonin has the ability to inhibit the proliferation of colorectal cancer cells, and its toxicity to normal intestinal cells is significantly lower than that to colorectal cancer cells. 4μM, 2μM, and 1μM were selected as the dosing concentrations of HCT116 cells in subsequent experiments, 10μM, 5μM, and 2.5μM were selected as the dosing concentrations of HT-29 cells in subsequent experiments, and 5μM, 2.5μM, and 1.25μM were selected as the dosing concentrations of SW620 cells in subsequent experiments.

[0071] Example 2: Clonal colony formation experiment

[0072] HCT116 cells, HT-29 cells, and SW620 cells in the logarithmic growth phase were inoculated in 6-well plates (1000 cells per well), and HCT116 cells were treated with 2μM, 1μM, and 0.5μM isotoxin, HT-29 cells were treated with 5μM, 2.5μM, and 1.25μM isotoxin, and HT-29 cells were treated with 2.5μM, 1.25μM, and 0.625μM isotoxin. The cells were cultured for 12 days, and the culture medium was replaced every 3 days. After the cells in the blank group formed obvious colonies, the culture medium was discarded, and the cells were washed twice with PBS in each group, and then fixed with 4% paraformaldehyde for 30 minutes, and then stained with 0.1% crystal violet solution for 30 minutes, dried, and photographed. The number of clone colonies was quantified using Image J (version 1.51j8), and the experiment was repeated 3 times.

[0073] Results: By Figure 2 A-2C shows that isoflavone can significantly inhibit the growth of colorectal cancer cells. The results show that after long-term intervention, the number and size of clone colonies formed by the three cell lines decreased in a concentration-dependent manner. Figure 3 A, 3B, 3C show that under the condition of medium concentration administration, the clone formation rate of each cell line dropped to HCT116 (61.48±5.558%), HT-29 (3.307±0.4313%), and SW620 (13.40±10.52%), respectively, with extremely significant differences. Under the condition of high concentration administration, the clone formation ability of the three cell lines was almost lost, and almost no clone colony was formed.

[0074] Example 3: Flow cytometry detection of cell apoptosis by double staining of Annexin V-FITC / PI fluorescence

[0075] HCT116, HT-29, and SW620 cells in the logarithmic growth phase were inoculated in 6-well plates. A negative control group, FITC and PI single positive groups, and ITSN treatment groups of different concentrations (set according to the CCK8 experimental results) were set up according to the experimental needs. After 48 hours of administration, the cells were collected by digestion with EDTA-free trypsin, centrifuged at 300×g and 4°C for 5 minutes, the culture medium was discarded, and the cells were washed twice with PBS. The concentration was selected to be about 1×10 6 Cells with a concentration of 1 cell / mL were resuspended in 500 μL 1x Annexin V Binding Buffer. According to the different group settings, 5 μL Annexin V-FITC staining solution and 5 μL PI staining solution were added to the cell suspension, mixed, and incubated at 4°C in the dark for 20 minutes. Then, the cells were passed through a 40 μm cell sieve and flow cytometry was performed within 1 hour using the FITC channel and the Percp / Cy5.5 channel. After the detection, Flow jo v10.8.1 software was used to draw a two-dimensional graph and count the proportion of early, late, and total apoptotic cells.

[0076] Results: By Figure 4 A-4C shows that compared with the control group, the low, medium and high concentrations of each cell can induce HCT116, HT-29 and SW620 cells to undergo significant apoptosis to varying degrees. Figure 5 A, 5B, and 5C show that under high-concentration administration conditions, the total apoptosis rate of HCT116 reached 25.857±0.865%, the total apoptosis rate of HT-29 reached 39.967±2.454%, and the total apoptosis rate of SW620 reached 33.567±6.672%, indicating that ITSN can significantly induce an increase in the proportion of apoptotic cells in colorectal cancer cells in a concentration-dependent manner.

[0077] Example 4: PI staining cell cycle flow cytometry

[0078] HCT116, HT-29, and SW620 cells in the logarithmic growth phase were inoculated in 6-well plates. Negative control groups and ITSN treatment groups of different concentrations (set according to the CCK8 experimental results) were set up according to experimental needs. After 48 hours of administration, cells were collected by trypsin digestion, centrifuged at 600×g and 4°C for 5 minutes, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. Then the cells were slowly resuspended with pre-cooled 70% ethanol (PBS dilution preparation), and the cells were gently vortexed during the resuspension process to ensure that the cells were fixed in a single cell suspension, and placed at -20°C overnight for fixation. Centrifuge at 4°C and 1000×g for 5 minutes to remove ethanol, resuspend with 1mL PBS, and centrifuge at 4°C and 600g for 10 minutes to remove PBS. The above steps were repeated twice, and then the cells were resuspended in 0.5mL Staining Solution and incubated at 37°C in the dark for 30 minutes. The cells were washed twice with PBS, resuspended in 1 mL of pre-cooled PBS, passed through a 40 μm cell sieve, and flow cytometry was performed within 24 h using the PE channel. After the test, the cell cycle was fitted using Modfit4.0 software, and the proportion of cells in the G1 / G0, S, and G2 / M phases was counted.

[0079] Results: By Figure 6 A, 6B, and 6C show that compared with the control group, ITSN can significantly induce colorectal cancer cell cycle arrest. Figure 7 A, 7B, and 7C show that after 48 hours of ITSN administration and stimulation, the G1 / G0 phase of HCT116 cells increased from the normal group (68.900±0.891%) to the high-concentration group (80.237±1.468%), and the S phase decreased from the normal group (23.253±0.243%) to the high-concentration group (14.313±1.242%); the G1 / G0 phase of HT-29 cells increased from the normal group (56.137±1.936%) to the high-concentration group (84.317±1.085%), and the S phase decreased from the normal group (34.837±2.097%) to the high-concentration group (11.303±1.965%), indicating that HCT116 and HT-29 cells underwent significant G1 / G0 phase cycle arrest. The S phase of SW620 cells increased from the normal group (26.977±1.917%) to the high-concentration group (54.690±2.083%), and the G2 / M phase decreased from the normal group (21.223±1.066%) to the high-concentration group (5.697±1.181%), indicating that SW620 cells underwent significant S phase arrest.

[0080] Example 5: Isotropin inhibits the occurrence and development of inflammation-related colorectal cancer in vivo

[0081] 5.1 Drug administration and its preparation

[0082] Dissolve isoflurane in polyoxy-15-hydroxystearate, and then add 9 times the volume of normal saline to dilute it to prepare 0.2 mg / mL and 0.1 mg / mL solutions.

[0083] 5.2 Establishment of AOM / DSS inflammation-related colorectal cancer model in mice

[0084] 49 5-week-old SPF-grade C57BL / 6 male mice were purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd. They were randomly divided into 4 groups. They drank ordinary drinking water in the first week, and the mice were not treated in any way. After 7 days of adaptive feeding, they were randomly divided into normal control group (n=10), model group (n=15), low-dose administration group (1 mg / kg, n=12) and high-dose administration group (2 mg / kg, n=12).

[0085] After 7 days of oral gavage pre-intervention during the behavioral period, the AOM stock solution was diluted with normal saline on the first day of the first week and intraperitoneally injected into the model group, low-dose group, and high-dose group at a dose of 10 mg / kg.

[0086] One day after AOM injection, the low-dose group and the high-dose group were simultaneously given oral administration of isotonixol at the same concentration, and the administration was carried out every other day. The normal group and the model group were simultaneously given oral administration of the corresponding solvent (polyoxy-15 hydroxystearate: saline = 1:9 volume ratio)

[0087] One week later, the model group, low-dose group, and high-dose group were given drinking water containing 2.5% dextran sulfate DSS for one week, and then restored to normal drinking water for two weeks. This process was a three-week cycle, which was repeated three times. After the three cycles, normal drinking water was given until the end of the experiment. The normal group continued to drink normal saline until the end of the experiment.

[0088] The weight of mice was recorded every other day, and the activity status, feces, blood in stool, etc. of mice were closely observed to establish the disease activity index score (DAI) of mice to evaluate the disease activity. DAI is the average value of the scores of weight loss, stool characteristics and occult blood, that is, DAI = (weight loss index + stool characteristics index + blood in stool index) / 3. The cages of mice were changed regularly, and food and drinking water were added.

[0089] 5.3 Mouse tissue and organ collection

[0090] At the 20th week, the mice were killed by cervical dislocation, and the hearts, livers, spleens, lungs, and kidneys of the experimental mice were collected and weighed to calculate the relevant indexes according to the organ coefficient formula. The colon of the mice was collected, the colon length was measured, the number of colon tumors was counted, and photos were taken.

[0091] Results: By Figure 8 , 9 It can be seen that throughout the whole process, the mice in the normal control group maintained a high weight gain rate, their feces were grain-like, there was no loose stool, no blood in the stool, and their hair was shiny. However, the model group showed obvious weight loss, loose stools, and even blood in the stool during the DSS drinking cycle, and the overall recovery was slow during the inflammation recovery stage. During the entire modeling period, some mice died due to severe weight loss, severe blood in the stool, or intestinal obstruction. The weight loss and disease activity index scores of the mice in the drug-treated group were less than those in the model group, and decreased in a concentration-dependent manner. In the inflammation recovery stage, it can be seen that the mice in the drug-treated group recovered to normal levels faster than the model group.

[0092] Depend on Fig.10 A, 10B, and 10C show that the colon of mice in the model group was significantly shortened compared to the normal group, and multiple tumors were distributed on the colon, with more distributed in the distal part of the colon. In the drug-treated group, as the concentration increased, the colon length was significantly restored, and the number and size of colon tumors were significantly reduced. This proves that isoflavone can significantly restore the shortening of the colon caused by inflammation-related colorectal cancer and inhibit tumor growth.

[0093] Depend on Fig.11 A-11F shows that compared with the normal group, the heart, lungs, and kidneys of the mice in the model group and the drug-treated group did not show significant changes. However, the liver and spleen of the model group showed significant enlargement, and as the concentration increased, the liver and spleen organ coefficients of the mice in the drug-treated group were significantly restored, especially the spleen. This proves that isoflavone can significantly restore the enlargement of the liver and spleen caused by inflammation-related colorectal cancer.

[0094] Based on the above results, the model group mice had significantly shorter colons and more tumors than the normal group mice, and the liver and spleen organ coefficients increased significantly, proving that the inflammation-related colorectal cancer mouse model was successfully constructed. The mice in the drug-treated group had less weight loss during the entire modeling process than the mice in the model group, and the DAI scores and blood in the stool were improved. The colon length was significantly restored, the number and size of tumors were significantly reduced, and the liver and spleen organ coefficients were significantly restored. It is proved that isotonine can inhibit the occurrence and development of inflammation-related colorectal cancer in vivo in a concentration-dependent manner.

[0095] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of isotoxin in the preparation of a drug for preventing and / or treating colorectal cancer.

2. The use according to claim 1, characterized in that The colorectal cancer includes inflammation-related colorectal cancer.

3. The use according to claim 1 or 2, characterized in that The prevention and / or treatment includes: (i) inhibiting the proliferation of colorectal cancer cells; (ii) induces cell cycle arrest in colorectal cancer cells; (iii) induce apoptosis of colorectal cancer cells; (iv) inhibiting tumor growth of inflammation-related colorectal cancer; (v) inhibiting the shortening of the colon caused by inflammation-related colorectal cancer; and (ⅵ) Restoration of liver and spleen organomegaly caused by inflammation-related colorectal cancer.

4. The use according to claim 3, characterized in that: The colorectal cancer cells include HCT116, HT-29 and SW620 cells.

5. The use according to claim 3, characterized in that: The cycle arrest includes G1 / G0 phase cycle arrest and S phase cycle arrest.

6. The use according to claim 1, characterized in that: The isotoxin is used as the sole active ingredient for preparing medicine for preventing and / or treating colorectal cancer.

7. The use according to claim 1, characterized in that The medicine also includes pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that The auxiliary materials include diluents, absorbents, wetting agents, adhesives, disintegrants, lubricants, colorants, coating materials, solvents, acid-base regulators, antioxidants, antibacterial agents and isotonic regulators.

9. The use according to claim 7, characterized in that: The dosage form of the drug is an oral dosage form or an injection dosage form.

10. The use according to claim 9, characterized in that The oral dosage form is tablet, capsule, oral liquid, granule or powder.

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