Use of ethyl extract of curcuma zedoary in preparation of medicine for treating colorectal cancer

Abstract

This invention belongs to the field of novel pharmaceutical uses, and more specifically, relates to the application of ethyl acetate extract of turmeric in the preparation of drugs for treating colorectal cancer. The preparation method of the extract is as follows: turmeric slices are soaked overnight in 15 times their weight of a 70% (v / v) ethanol solution, refluxed at 85°C three times, 2 hours each time. The extracts are combined and concentrated until no alcohol odor remains. The extract is then dried to constant weight to obtain the total extract. The total extract is reconstituted with water and extracted three times with ethyl acetate at an equal volume ratio. The extracts are combined and dried to constant weight to obtain the final product. Experimental results show that this extract is particularly effective in improving disease activity index, restoring colon length, reducing tumor number, and alleviating histopathological damage, and has no significant toxicity to major organs. This extract can be formulated into oral or injectable preparations, providing a new natural drug candidate for the treatment of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of new uses of pharmaceuticals, and more specifically, relates to the application of ethyl acetate extract of turmeric in the preparation of drugs for treating colorectal cancer. Background Technology

[0002] Natural medicines are pharmacologically active substances obtained from natural resources. Their sources are diverse, including plants, animals, minerals, and microorganisms. Furthermore, natural medicines contain abundant chemical components that exhibit anti-inflammatory, anti-tumor, antioxidant, antibacterial, and antiviral biological activities. Their rich chemical structures and biological activities can provide new ideas for developing novel drugs and improve clinical treatment levels.

[0003] As a representative herbal medicine, turmeric contains abundant natural chemical products, including terpenes, curcuminoids, and alkaloids, which have pharmacological effects such as anti-tumor, anti-inflammatory, and antioxidant properties. However, its complex chemical composition, unclear active ingredients, and unknown regulatory mechanisms severely restrict the clinical application and further in-depth research of turmeric and its active components. Summary of the Invention

[0004] The purpose of this invention is to provide the application of ethyl acetate extract of turmeric in the preparation of drugs for treating colorectal cancer.

[0005] This invention provides the application of ethyl acetate extract of turmeric in the preparation of drugs for treating colorectal cancer. The preparation method of the turmeric extract is as follows: turmeric is chopped and soaked overnight in 15 times its mass of a 70% ethanol solution. It is then refluxed at 85°C for 2 hours each time. The extracts are combined and concentrated until no alcohol odor remains. The extract is dried to constant weight to obtain the total extract. The total extract is reconstituted with water and extracted 3 times with ethyl acetate in an equal volume ratio. The ethyl acetate layers from the three extractions are combined and dried to constant weight to obtain the ethyl acetate extract of turmeric.

[0006] This invention stems from the potential value of natural medicines in the field of anti-tumor therapy, particularly turmeric, which, while known to possess anti-inflammatory and antioxidant activities, has had its specific pharmacological components and mechanisms of action against colon cancer remaining unclear. The study first obtained a total extract and different polarity components of turmeric through systematic extraction and fractional separation. UPLC-MS / MS was used for component identification, revealing its rich content of terpenes, alkaloids, and other active substances. Subsequently, an AOM / DSS-induced mouse colorectal cancer model was established to comprehensively evaluate the intervention effects of each component on colon cancer. The experimental design included multi-dimensional analysis of disease activity index, colon morphology, tumor burden, histopathology, and key protein expression, ultimately identifying the ethyl acetate component as the core effective fraction. Further Western blot analysis preliminarily revealed that it may exert its therapeutic effect by regulating the JAK2 / STAT3 pathway and ferroptosis-related proteins. This invention, progressing step-by-step from component analysis to efficacy screening and preliminary mechanism exploration, provides experimental evidence and material basis for the anti-colon cancer application of turmeric.

[0007] Furthermore, the drug is used to relieve inflammatory infiltration of colonic tissue and inhibit colonic shortening caused by colorectal cancer.

[0008] Furthermore, the drug is used to reduce the number of colorectal cancer tumors.

[0009] Furthermore, the drug is used to inhibit the phosphorylation of STAT3 and JAK, downregulate the expression levels of HO-1, Nrf2, FTH1 and GPX4, and upregulate the protein expression of TFR1, thereby inducing ferroptosis in tumor tissue.

[0010] Furthermore, the content of ethyl acetate extract of turmeric in the drug is 0.1 wt% to 99 wt%.

[0011] Furthermore, the drug is made from the ethyl acetate extract of turmeric tablets and pharmaceutically acceptable excipients.

[0012] Furthermore, the excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

[0013] Furthermore, the diluent is either water or physiological saline.

[0014] The beneficial effects of this invention are as follows: First, the ethyl acetate component of turmeric extract was identified as the key active site for anti-colon cancer treatment, providing a clear material basis for subsequent drug development. Second, in vivo experiments confirmed that EA significantly improved the disease activity index, inhibited tumor growth, and alleviated colonic shortening and histopathological damage in a colon cancer model, with effects superior to the positive control drug sulfasalazine. Third, safety assessments showed that turmeric extract and its components did not cause significant pathological changes in major organs such as the heart, liver, spleen, lungs, and kidneys at experimental doses, demonstrating good biocompatibility. Fourth, preliminary mechanistic studies indicate that EA may exert its therapeutic effect by inhibiting the JAK2 / STAT3 signaling pathway and regulating the expression of ferroptosis-related proteins such as GPX4, FTH1, and TFR1, providing a molecular-level explanation of its mechanism of action. In summary, this invention not only expands the medicinal uses of turmeric but also provides a promising candidate formulation for the natural drug treatment of colon cancer, possessing good prospects for research and translation. Attached Figure Description

[0015] Figure 1 This is the mass spectrum of the TE component.

[0016] Figure 2 This is the mass spectrum of the PE component.

[0017] Figure 3 This is the mass spectrum of the EA component.

[0018] Figure 4 This is the mass spectrum of the BU component.

[0019] Figure 5 This is the mass spectrum of the AF component.

[0020] Figure 6 The effects of turmeric extract and its components on DAI and body weight in CRC mice are shown in Figure A, where DAI score is plotted and body weight is plotted in Figure B.

[0021] Figure 7 The effects of turmeric extract and its components on colorectal length and tumor number in CRC mice are shown in Figure A, where A represents the appearance of the colon and B represents the appearance of colon tumors. Figure 8 The graphs are statistical representations of the colon, where A is a statistical graph of colon length and B is a statistical graph of tumor number.

[0022] Figure 9 H&E staining images of mouse CRC colorectal tissue containing turmeric extract and its components.

[0023] Figure 10 The figure shows the effects of the total extract of turmeric and its components on the heart of mice. In the figure, A is the H&E staining of the heart tissue of mice in each group, and B is the statistical graph of the heart-to-body ratio of mice in each group.

[0024] Figure 11 The graph shows the effects of the total extract of turmeric and its components on mouse liver. In the graph, A is the H&E staining of liver tissue of mice in each group, and B is the statistical graph of the liver-to-body ratio of mice in each group.

[0025] Figure 12 The figure shows the effects of the total extract of turmeric and its components on the spleen of mice. In the figure, A is the H&E staining of the spleen tissue of each group of mice, and B is the statistical graph of the spleen-to-body ratio of each group of mice.

[0026] Figure 13 The figure shows the effects of the total extract of turmeric and its components on mouse lungs. In the figure, A is the H&E staining of lung tissue of mice in each group, and B is the statistical graph of lung-to-body ratio of mice in each group.

[0027] Figure 14 The figure shows the effects of the total extract of turmeric and its components on mouse kidneys. In the figure, A is the H&E staining of mouse kidney tissue in each group, and B is the statistical graph of the kidney-to-body ratio in each group.

[0028] Figure 15 The graph shows the effects of the total extract of turmeric and its components on the expression of STAT3 and p-STAT3, which are iron death-related proteins, in the colon of CRC mice. In the graph, A is the Western blot map of the protein, B is the statistical graph of the expression level of STAT3 protein, and C is the statistical graph of the expression level of p-STAT3 protein.

[0029] Figure 16 The graph shows the effects of the total extract of turmeric and its components on the expression of iron death-related proteins JAK, p-JAK and HO-1 in the colon of CRC mice. In the graph, A is the Western blot map, B is the statistical graph of JAK protein expression, C is the statistical graph of p-JAK protein expression, and D is the statistical graph of HO-1 protein expression.

[0030] Figure 17 The graph shows the effects of the total extract of turmeric and its components on the expression of Nrf2 and FTH1, iron death-related proteins, in the colon of CRC mice. In the graph, A is the Western blot map, B is the statistical graph of Nrf2 protein expression, and C is the statistical graph of FTH1 protein expression.

[0031] Figure 18 The graph shows the effects of total extract of turmeric and its components on the expression of ferromorxin-associated proteins TFR1 and GPX4 in the colon of CRC mice. A represents the Western blot image, B represents the TFR1 protein expression level, and C represents the GPX4 protein expression level. * indicates a significant difference between the treatment group and the model group (P < 0.05, ** indicates P < 0.01, *** indicates the most significant difference). # indicates a significant difference between the model group and the control group (P < 0.001). Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0033] A table of Chinese and English abbreviations is shown in Table 1.

[0034] Table 1: Comparison of Chinese and English Abbreviations Example 1: Preparation of turmeric extract.

[0035] 1. Experimental Materials The turmeric slices were purchased in December 2023 from the Wenyujin Traditional Chinese Medicine GAP Base in Ruian City, Wenzhou City, Zhejiang Province. The turmeric slices have a yellow outer skin, a rough surface covered with wrinkles, and a yellowish-white to brownish-yellow cut surface with obvious ring patterns and numerous small veins. They vary in shape, mostly being oblong or irregular slices, with a size of 6-9 cm. The slices are firm and brittle, with a strong aroma and a slightly bitter taste.

[0036] 2. Extraction and separation of turmeric slices and their components Weigh 2 kg of turmeric slices, chop them, and place them in a round-bottom flask. Add 15 times the weight of the turmeric slices of a 70% ethanol solution to submerge the slices and soak overnight. Extract by reflux at 85℃ three times, 2 hours each time. Combine the filtrates, concentrate the filtrate until no alcohol odor remains, and dry to constant weight to obtain a total extract of 309.66 g. Redissolve the extract and extract three times each with petroleum ether, ethyl acetate, and n-butanol reagents at a volume ratio of 1:1. Combine the filtrates and dry to constant weight to obtain a petroleum ether fraction of 109.4 g, an ethyl acetate fraction of 31.05 g, and a n-butanol fraction of 35.22 g. Finally, dry the aqueous phase to obtain an aqueous fraction of 112.62 g.

[0037] 3. Identification of the total extract of turmeric and its components (1) Sample preparation: First, the five components of turmeric slices were freeze-dried in a freeze dryer for 63 hours under vacuum, and then ground into powder using a grinder. Next, 30 mg of sample powder was weighed using an electronic balance and 1500 μL of pre-cooled 70% methanol-water internal standard extraction solution was added. Then, the sample was vortexed every 30 min for 30 s each time, for a total of 6 vortexes. Finally, after centrifugation at 12000 rpm for 3 minutes, the supernatant was collected, the sample was filtered through a 0.22 μm microporous membrane, and stored in a sample vial for UPLC-MS / MS analysis.

[0038] (2) Chromatographic conditions: Column: Agilent SB-C18 1.8µm, 2.1mm*100mm; Mobile phase: Phase A is ultrapure water containing 0.1% formic acid, and Phase B is acetonitrile containing 0.1% formic acid; Elution gradient: The proportion of Phase B is 5% at 0.00min, and the proportion of Phase B increases linearly to 95% within 9min and is maintained for 1min. From 10.00 to 11.10min, the proportion of Phase B decreases to 5% and is equilibrated to 5% at 14min; Flow rate: 0.35mL / min; Column temperature: 40℃; Injection volume: 2μL.

[0039] (3) Mass spectrometry conditions: Electrospray ionization source temperature 500℃; positive ion mode ion spray voltage 5500V / negative ion mode -4500V; ion source gas I, gas II, and curtain gas were set to 50, 60, and 25 psi, respectively. QQQ scans used MRM mode with collision gas N2 set to medium. The declustering voltage and collision energy of each MRM ion pair were optimized through further optimization. A specific set of MRM ion pairs was monitored in each period based on the metabolites eluted in each period.

[0040] (4) Data processing: Based on a self-built database, the substances were qualitatively identified according to the secondary spectral information. Isotope signals were removed during the analysis, including those containing K. + Ions, Na + Ions, NH 4+ Repeated signals of ions, as well as repeated signals of fragment ions that are themselves other substances with larger molecular weights. Data filling and screening method for all sample metabolites: 1 / 5 of the minimum value of each metabolite in each row is used to fill missing values, and then the CV value of the QC sample is calculated. Substances with CV values ​​< 0.5 are retained.

[0041] Qualitative analysis of the chemical components in TE, PE, EA, BU, and AF was performed. The retention times of the compounds were compared with those of each component using ion chromatograms, and primary and secondary mass spectrometry information was also analyzed. The experimental results are as follows: Figures 1-5 As shown, the five components were well separated under the experimental conditions, indicating that the extraction and separation design of this experiment was reasonable and provided a solid foundation for subsequent evaluation of the efficacy of different components.

[0042] Example 2: In vivo effects of turmeric extract on colorectal cancer in mice.

[0043] I. Experimental Materials and Design.

[0044] 1. Laboratory animals Six-week-old male C57BL / 6J mice, SPF grade, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and raised in the Animal Experiment Center of Wenzhou Medical University. The animal implementation license number is SYXK (Zhe) 2021-0020. They were kept in an SPF-class clean animal room with the temperature maintained at 25±2°C and the relative humidity at 40% - 70%, and a 12h light / dark cycle was satisfied. All experiments strictly adhered to the relevant regulations of the "Regulations on the Administration of Laboratory Animals". The ethical approval number for animal experiments is xmsq2024-0437.

[0045] 2. Experimental design One hundred and thirty male C57BL / 6J mice after adaptive feeding were randomly divided into groups of 10 each, namely: the blank control group was denoted as Ctrl, the DSS model group was denoted as M, the sulfasalazine enteric-coated tablet group was denoted as P with a dosing dose of 125 mg / kg. The low-dose groups of TE, PE, EA, BU, and AF at 150 mg / kg were denoted as TEL, PEL, EAL, BUL, and AFL respectively, and the high-dose groups of TE, PE, EA, BU, and AF at 300 mg / kg were denoted as TEH, PEH, EAH, BUH, and AFH respectively.

[0046] The drugs were prepared with a 1% (v / v) aqueous solution of Tween 80 as the solvent and were freshly prepared before use. During the experiment, the mice in each group were weighed every 24h, and the body weight data of the mice were recorded. On the 0th day of the experiment, except for the control group, the remaining mice were intraperitoneally injected with 12.5 mg / kg of AOM. Subsequently, the health status of the mice was closely observed within 5 days. On the 5th day, after the mice freely ingested a 2.5% (v / v) aqueous solution of DSS for 5 days, the 2.5% (v / v) aqueous solution of DSS for the mice was replaced with normal drinking water and continuously consumed for 14 days. This process took 19 days and was recorded as one cycle. A total of 3 cycle periods were used to construct the AOM / DSS mouse colorectal cancer model. On the 5th - 10th day, the 24th - 29th day, and the 43rd - 48th day, the dosing groups were given a 2.5% (v / v) aqueous solution of DSS, and normal drinking water was given during the remaining time periods. The sulfasalazine enteric-coated tablet group, the low-dose dosing group, and the high-dose dosing group were administered by gavage once a day throughout the process.

[0047] One day before the end of the experiment, the mice were fasted for 24h. On the 63rd day, blood was collected from the eyes of the mice. Under the conditions of 12000 rpm and 4°C, it was centrifuged for 20 min, and the upper serum was taken and stored at -20°C for later use. The colorectal tissue from the ileocecal valve to the anus of the mice was separated, and 1 cm of the cecum was intercepted and placed in 7 mL of tissue fixative. It was longitudinally dissected along the mesenteric margin, and the colonic contents were rinsed clean with physiological saline and immediately placed in dry ice and stored at -80°C for later use. At the same time, the heart, liver, spleen, lungs, and kidneys were collected, the weights of the mouse organs were measured, and the organ indices were calculated.

[0048] II. Basic physiological signs of mice.

[0049] 1. Experimental Methods: During the experiment, the mental and limb status, fur condition, and water intake of mice in each group were closely observed daily. The daily weight changes, fecal characteristics, and fecal blood occurrences of the mice were recorded in detail. DAI = (weight loss score + fecal characteristics score + occult blood score) / 3. DAI was used as the scoring standard, and the DAI scoring standard is shown in Table 3.

[0050] Table 3: Disease Activity Index Scoring Criteria Note: — indicates no data.

[0051] (1) The weight loss rate is based on the previous day's weight, according to Weight loss (%) = (W (n+1)d -W nd The weight loss is calculated using the formula 100% × 100%, and the degree of weight loss is scored according to the DAI scoring table.

[0052] (2) Stool viscosity: The stool characteristics are scored according to the characteristics of the stool, which mainly include normal formed stool, soft stool, and watery stool. The scores are based on Table 3.

[0053] (3) Fecal occult blood test: Fecal occult blood was detected in mouse feces using a fecal occult blood qualitative test kit. Take an appropriate amount of feces, add equal amounts of o-toluidine solution and oxidant, and score according to the color change of the feces within 20 seconds. If blood is visible around the anus or there is obvious blood in the stool, the kit test is not required. Refer to Table 3 for DAI score.

[0054] 2. Experimental Results: Compared with the Ctrl group, where the DAI score remained at a consistently low level, the DAI score in the M group showed a significant upward trend with the extension of the modeling time, increasing from the initial stage and maintaining a high level in the middle and late stages, indicating a continuous increase in disease activity. The CRC model was successfully established. Figure 6 As shown in A.

[0055] The changes in DAI scores across the groups revealed that groups P, AFH, AFL, and BUL alleviated the increase in DAI scores during the modeling process. In contrast, groups TEH and EAH showed more significant alleviating effects. The DAI scores of other groups did not differ significantly from those of group M. Based on the combined results of the DAI scores, it can be concluded that component EA is a key component in turmeric tablets that improves CRC in mice.

[0056] Mouse body weight change rate is a key indicator in disease-associated acute injury (DAI), closely related to disease severity and providing important information for subsequent research. During the modeling period, changes in mouse body weight are helpful in assessing the therapeutic effect of drugs and screening for effective treatments.

[0057] according to Figure 6 Data from group B shows that the body weight of mice in the Ctrl group generally maintained an increasing trend. The body weight of mice in the other groups fluctuated with the administration of the modeling drug; that is, their weight decreased during the modeling period and then increased after the modeling was stopped. Among these groups, the EA group experienced the smallest decrease in body weight, indicating that EA has a good effect on slowing down the weight loss in mice.

[0058] In conclusion, the EA component may be a key component in improving colorectal cancer in mice, and it may have potential application value in the treatment of colorectal cancer. Future research can further explore the mechanism of action of the EA component, as well as its efficacy and safety in CRC treatment, in order to provide new treatment options for CRC patients.

[0059] III. Analysis of colorectal length and tumor number in mice.

[0060] 1. Experimental Methods: Colorectal tissue from the ileocecal valve to the anus of mice was isolated. Changes in colon length are an important indicator for evaluating the progression and pathological damage of colorectal cancer.

[0061] 2. Experimental Results: Changes in colon length are an important indicator for evaluating the progression and pathological damage of colorectal cancer. For example... Figure 7 and Figure 8 As shown in Figure A, AOM / DSS-induced CRC mice generally exhibited colonic shortening, and the degree of shortening was positively correlated with tumor burden. EAH treatment significantly restored colonic length, indicating that EAH components can significantly improve the abnormal colonic morphology induced by colorectal cancer modeling and alleviate the pathological damage process of colonic tissue.

[0062] Tumor number is a core quantitative indicator in colorectal cancer experimental research, directly reflecting the tumor burden level and tumor development process in model animals. It is also a key observational basis for evaluating the tumor-suppressing efficacy of various interventions. Experimental results are shown below. Figure 7 and Figure 8 Compared with the Ctrl group, the number of tumors in the M group mice was significantly increased, while the P, TEH, EAH and EAL groups all significantly improved this situation, with the EAH group showing the most significant treatment effect.

[0063] IV. H&E staining of mouse colorectal intestine.

[0064] 1. Experimental Methods: First, the contents of the cecal colon (1 cm postcecal) tissue from each component were thoroughly cleaned with pre-cooled PBS solution. The cecum was then longitudinally cut and placed in 4% (v / v) neutral paraformaldehyde tissue solution for fixation for 24 h. After removal, the tissue was eluted using a gradient ethanol system and then cleared twice with xylene solution, 20 min each time. First, xylene I solution was used to remove ethanol from the tissue, initially clearing it. Then, xylene II solution was used to ensure complete clarity. The tissue was then transferred to an embedding cassette, filled with paraffin wax, and allowed to cool into a block. The block was then cut to an appropriate thickness and mounted on a glass slide in preparation for subsequent staining. After dewaxing and hydration, the paraffin sections were stained with hematoxylin for 5 min, followed by rinsing with running water for 1 min to thoroughly remove excess staining agent. Next, the sections were differentiated using 1% (v / v) hydrochloric acid ethanol, with a 5-second waiting period to further enhance staining contrast. Subsequently, the slides were placed in a 0.2% ammonia solution (v / v), and a bluing reaction occurred after approximately 30 seconds, enhancing the staining effect on cell nuclei. Finally, the slides were thoroughly rinsed with running water. The slides were then immersed in eosin staining solution for approximately 30 seconds, followed by rinsing off excess stain with running water. The stained slides were dehydrated and cleared again, and finally mounted with neutral resin. A coverslip was gently pressed to remove air bubbles and avoid interfering with the experimental results. The pathological morphology of mouse colorectal tissue was evaluated under an optical microscope. Images were collected using a slide scanner and viewed and analyzed using CaseViewer software.

[0065] 2. Experimental Results: From Figure 9 It was found that the colon and rectum of mice in the Ctrl group had normal morphology, intact colonic mucosa, regular arrangement of crypt structures, abundant goblet cells, and no inflammatory cell infiltration or pathological damage. Compared with the Ctrl group, the colon tissue of the M group showed obvious pathological changes, including disordered crypt structure, glandular atrophy and deformation, accompanied by severe inflammatory cell infiltration, and a significant reduction in the number of goblet cells.

[0066] The microscopic findings of the TEL, PE, BU and AF groups were similar, with increased and irregularly shaped crypt branches, local necrosis and sloughing, and no significant relief of inflammatory infiltration, indicating that this group failed to effectively reverse the pathological process of the CRC model.

[0067] The colonic tissue structure of groups P, THE, EAH, and EAL was significantly better than that of other treatment groups, with EAH showing the most ideal effect. The crypt structure became more regular, glands were more neatly arranged, goblet cell count was significantly restored, inflammatory infiltration was mild, and mucosal integrity was well maintained, suggesting that the EA component can significantly improve colorectal tissue damage, especially at high doses. In summary, the EAH group exhibited the best therapeutic activity in the CRC experimental model, with its histological characteristics most similar to the Ctrl group, and its efficacy was higher than that of groups P and TEH. The other components showed no significant efficacy. The effects of different turmeric components on colorectal cancer are significantly different, requiring further screening for effective active ingredients.

[0068] V. Changes in the heart, liver, lungs, kidneys, and spleen of mice.

[0069] 1. Experimental methods: Collect mouse heart, liver, spleen, lungs and kidneys, weigh the mouse organs and perform H&E staining.

[0070] 2. Experimental Results: The H&E staining results of mouse hearts are shown in the figure. Figure 10 Figure A shows the pathological changes in the mouse heart tissue structure after drug intervention: the cardiomyocytes in the Ctrl group had normal structure, with oval-shaped and neatly arranged nuclei; compared with the Ctrl group, there was no significant difference in cardiomyocyte morphology between the M group and the drug-treated group, and the nuclei and cell arrangement remained normal. The statistical results of their heart-to-body ratio are shown in [Figure A]. Figure 10 B also indicates that there is no significant difference between the groups.

[0071] H&E staining results of mouse liver are shown in the figure. Figure 11 As shown in Figure A, the hepatocytes in the Ctrl group were morphologically intact, with large, round nuclei and clear lobular structures; the hepatocyte structures in the M group and the drug-treated group were similar to those in the Ctrl group, with no obvious pathological changes. The statistical results of their liver-to-body ratio are shown in [Figure A]. Figure 11 B also indicates that there are no significant differences between the groups.

[0072] H&E staining results of mouse spleen are shown in the figure. Figure 12 As shown in Figure A, the spleen tissue structure of the Ctrl group was normal, with regular cell arrangement and a clear boundary between the red and white medulla. The tissue structure of the M group and the drug-treated group was similar to that of the Ctrl group, with no obvious pathological changes. The spleen-to-body ratio statistical results are shown below. Figure 12 B also indicates that there are no significant differences between the groups.

[0073] H&E staining results of mouse lungs are shown in the figure. Figure 13 As shown in Figure A, the alveolar structure of the Ctrl group was normal, with tightly packed and regularly arranged epithelial cells; the lung tissue structure of the M group and the drug-treated group was similar to that of the Ctrl group, with no obvious pathological changes. The lung-to-body ratio statistical results are shown below. Figure 13 B also indicates that there are no significant differences between the groups.

[0074] H&E staining results of mouse kidneys are shown in the figure. Figure 14 As shown in Figure A, the renal cell nuclei in the Ctrl group were small, regular, and compactly arranged; the renal tissue structure of the M group and the drug-treated group was similar to that of the Ctrl group, with no obvious pathological changes. The statistical results of their kidney-to-body ratio are shown in [Figure A]. Figure 14 B also indicates that there are no significant differences between the groups.

[0075] These results indicate that, at the tested doses, the total extract of turmeric and its components did not produce significant toxic effects on the major organs of mice, demonstrating good safety.

[0076] VI. Protein blotting.

[0077] 1. Experimental Methods: Protein sample preparation was performed on colonic tissue from CRC mice. First, 30 mg of mouse colonic and rectal tissue was weighed and placed in a 2 mL centrifuge tube. Then, lysis buffer was added to each centrifuge tube, and the tissue was thoroughly homogenized using a multi-sample tissue homogenizer for 30 min. After complete lysis, the tissue was centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was collected and transferred to pre-chilled EP tubes. Subsequently, BCA protein quantification was performed by adding an appropriate amount of protein supernatant and incubating at 37°C for 30 min. The absorbance of the protein sample was measured using a microplate reader, and the values ​​were calculated based on the results. The remaining sample was added to 4× protein loading buffer and heated in a metal bath at 100°C for 10 min to completely denature the protein sample. The sample was then stored at -20°C.

[0078] The target protein was separated using SDS-PAGE protein gel electrophoresis. PVDF membranes were pre-activated with methanol and transferred using a wet transfer method. After transfer, the membrane was blocked by immersing it in 5% (v / v) skim milk powder for 1 hour, followed by washing three times with PBST for 10 minutes each time. The transferred PVDF membrane was then completely immersed in primary antibody solution and incubated overnight at 4°C, followed by washing three times with PBST for 10 minutes each time. It was then incubated with secondary antibody at room temperature for 1 hour, followed by washing three times with PBST for 10 minutes each time. Finally, the bands were placed flat in a chemiluminescence gel imaging system, and ECL ultrasensitive luminescent solution was evenly added above the bands for exposure and development to obtain clear images. The saved files were imported into ImageJ for grayscale analysis, using β-actin as an internal control protein to calculate the relative expression level of the target protein.

[0079] 2. Experimental results: JAK kinase activation can phosphorylate STAT3, promote the formation of STAT3 homodimers and translocation into the nucleus, thereby regulating cell proliferation, survival and transcription of inflammation-related genes.

[0080] FTH1 is the heavy chain subunit of ferritin, an intracellular iron storage protein that binds and stores iron ions, preventing oxidative stress damage caused by iron overload.

[0081] Nrf2 is a transcription factor that plays a crucial role in cellular antioxidant stress responses. Under normal conditions, Nrf2 is inhibited by the Keap1 protein, but under oxidative stress, Nrf2 is activated and translocated to the cell nucleus, initiating the expression of a series of antioxidant stress genes, such as glutathione S-transferase, superoxide dismutase, and glutathione peroxidase.

[0082] HO-1 is a stress-response enzyme that converts heme into biliverdin, iron, and carbon monoxide. HO-1 plays an important role in anti-oxidative stress, anti-inflammation, and cell protection.

[0083] TRF1 is transferrin receptor 1, which is involved in cellular iron uptake. TRF1 transports iron ions into the cell by binding to transferrin, and plays a role in maintaining cellular iron homeostasis and regulating iron metabolism.

[0084] GPX4 is a key enzyme regulated by Nrf2, which can specifically clear lipid peroxides and acts as a gatekeeper protein that inhibits ferroptosis.

[0085] These proteins constitute the "iron metabolism-antioxidant" axis that regulates ferroptosis. Nrf2 is located upstream and achieves a protective response through bidirectional regulation: on the one hand, it activates HO-1 and GPX4 to enhance antioxidant capacity, and on the other hand, it inhibits ferroptosis; FTH1 and TRF1 play roles at both ends of iron metabolism—FTH1 reduces free iron, and TRF1 promotes iron absorption. An imbalance between the two leads to iron overload, which drives the Fenton reaction to produce ROS and induce ferroptosis.

[0086] like Figures 15-18 As shown, compared with the Ctrl group, the M group showed significantly increased protein expression levels of p-STAT3, p-JAK, HO-1, Nrf2, FTH1, and GPX4, and significantly decreased TFR1 protein expression. This indicates that there is abnormal activation of the JAK2 / STAT3 pathway and enhanced ferroptosis mechanism in the tumor tissue of CRC model mice. Notably, among the total extract of turmeric and each component intervention group, the EAH group showed the most significant regulatory effect on key proteins, specifically: EA significantly inhibited the phosphorylation of STAT3 and JAK, downregulated the expression levels of HO-1, Nrf2, FTH1, and GPX4, and upregulated the protein expression of TFR1. In this experiment, it was found that the EAH group can induce significant ferroptosis by inhibiting the JAK2 / STAT3 / GPX4 axis in tumor tissue, which is quite significant in the treatment of CRC in mice and has potential clinical application value.

[0087] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. The application of ethyl acetate extract of turmeric in the preparation of drugs for treating colorectal cancer, characterized in that, The preparation method of the turmeric extract is as follows: After turmeric is chopped, it is soaked overnight in 15 times its mass of 70% ethanol solution, refluxed at 85°C for 3 times, 2 hours each time, the extracts are combined and concentrated until there is no alcohol odor, and dried to constant weight to obtain the total extract. The total extract is reconstituted with water and extracted 3 times with ethyl acetate at an equal volume ratio. The extracts are combined and dried to constant weight to obtain the ethyl acetate extract of turmeric.

2. The application according to claim 1, characterized in that, The drug is used to relieve inflammatory infiltration of colonic tissue and inhibit colonic shortening caused by colorectal cancer.

3. The application according to claim 1, characterized in that, The drug is used to reduce the number of colorectal cancer tumors.

4. The application according to claim 1, characterized in that, The drug is used to inhibit the phosphorylation of STAT3 and JAK, downregulate the expression levels of HO-1, Nrf2, FTH1 and GPX4, and upregulate the protein expression of TFR1, thereby inducing ferroptosis in tumor tissue.

5. The application according to claim 4, characterized in that, The content of ethyl acetate extract of turmeric in the drug is 0.1wt% to 99wt%.

6. The application according to claim 4, characterized in that, The drug is made from the ethyl acetate extract of turmeric tablets and pharmaceutically acceptable excipients.

7. The application according to claim 4, characterized in that, The excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

8. The application according to claim 4, characterized in that, The diluent is either water or physiological saline.

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