Use of ecklonia kurome and active ingredients thereof in preparation of drug for treating high fructose-promoted colitis associated colorectal cancer

Abstract

Traditional Chinese medicine Ecklonia kurome and ingredients thereof such as fucoidin and brown algae polyphenol can ameliorate high fructose-induced colitis associated colon cancer, which is mainly demonstrated by significant reduction in tumor load after drug administration. Thus, a medication regimen is provided for patients with clinical colitis associated colorectal cancer and for the prevention of potential hazards caused by a high-fructose diet.

Description

Application of kelp and its active ingredients in the preparation of medicines for treating high fructose-promoted colitis-related colorectal cancer Technical Field

[0001] The present invention relates to the use of kelp and its active ingredients fucoidan and brown algae polyphenol in the preparation of colitis-cancer transformation, particularly high-fructose-promoted colitis-related colorectal cancer drugs. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignancies and can be clinically divided into colitis-associated colorectal cancer (CAC) and sporadic colorectal cancer (SCRC). Compared with other subtypes, CAC has a more rapid disease progression and a higher mortality rate. The progression of CAC progresses through a staged progression of abnormal crypt lesions, polyps, adenomas, and carcinomas. Chronic intestinal inflammation induces mutations in oncogenes and tumor suppressor genes and genomic instability through mechanisms such as the production of cytokines, growth factors, reactive oxygen species, and nitrogen intermediates by immune cells. Persistent inflammation can activate the excessive proliferation and anti-apoptotic properties of precancerous cells and affect the permeability of epithelial cells, leading to epigenetic changes, inactivation of DNA repair mechanisms, and altered anti-tumor immune responses, promoting tumor formation, progression, and metastasis. Therefore, unlike sporadic colorectal cancer, colitis-associated colorectal cancer often occurs in patients with inflammatory bowel disease. Due to long-term exposure to chronic inflammation, the disease progression is more susceptible to factors such as dietary intake. Current chemotherapy regimens for colorectal cancer include single-drug and multi-drug therapies. However, chemotherapy has high toxicity and low response rates, making the improvement of existing colorectal cancer chemotherapy regimens a necessity.

[0003] Fructose, a common flavor enhancer, has been implicated in the development and progression of diseases such as diabetes, non-alcoholic fatty liver disease, cardiovascular disease, and colorectal cancer. Safe, effective, and long-term preventive and treatment options are still lacking to address the phenomenon that long-term daily fructose intake can worsen intestinal diseases.

[0004] Konbu (kelp) is the dried fronds of Laminaria japonica Aresch. (Laminaria japonica Aresch.) or Ecklonia kurome Okam. (Pterocarpus elegans). As a food and medicine substance, kelp possesses a wide range of pharmacological activities. Traditional medicine believes that kelp has the effects of softening and dispersing lumps, promoting diuresis and relieving heat. The Northern Song Dynasty's "Jiayou Materia Medica" records that kelp can eliminate phlegm, soften lumps, promote diuresis and reduce swelling. It has long been used to treat goiter, beriberi edema, and testicular swelling and pain. With the advancement of modern medicine, researchers have conducted more detailed studies on kelp and its components. However, there is currently no information on the use of kelp in the prevention or treatment of colon cancer related to high-fructose colitis.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an application of kelp in the preparation of a drug for treating high-fructose-promoted colitis-related colorectal cancer.

[0007] The present inventors determined the effect of high fructose on the inflammatory and adenoma stages of colitis-associated colon cancer using AOM / DSS and 30% high fructose-promoted AOM / DSS mouse models.

[0008] The present inventors used single-cell transcriptome sequencing to identify the key receptor ligand pairs that promote high fructose-induced colitis-associated colorectal cancer.

[0009] The inventors screened through the UNIQ system and found that kelp improves high-fructose-promoted colitis-related colorectal cancer.

[0010] The high-fructose-promoted colitis-associated colorectal cancer model used by the present inventors is an AOM / DSS mouse model induced by 30% high-fructose drinking water.

[0011] The present invention provides the use of fucoidan and phlorotannin, active ingredients of kelp, in the preparation of a drug for treating colitis-related colorectal cancer promoted by high fructose.

[0012] The kelp of the present invention improves the number and size of intestinal tumors in high-fructose-promoted colitis-related colorectal cancer.

[0013] The dosage of the kelp of the present invention for treating high-fructose-promoted colitis-related colorectal cancer is 400 mg / kg / day.

[0014] The dosage of the fucoidan and brown algae polyphenols of the present invention for treating high fructose-promoted colitis-related colorectal cancer is 200 mg / kg / day. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows the cellular interaction mechanism related to inflammatory-cancer transformation of colitis-associated colorectal cancer promoted by high fructose.

[0016] Figures 2a to 2d are four effector gene sets for high fructose-promoted colitis-associated colorectal cancer.

[0017] Figure 3 shows the ranking of various Chinese medicines with medicinal and edible properties in the four effector genes of high-fructose-promoted colitis-related colorectal cancer based on network pharmacology prediction.

[0018] Figure 4 shows the scores of various compound components in kelp in four effect gene sets based on network pharmacology prediction.

[0019] Figure 5 shows the pharmacological experiment of kelp, fucoidan and brown algae polyphenols on high fructose-promoted colitis-related colon cancer, and the changes in body weight.

[0020] Figure 6 shows the pharmacological experiment of kelp, fucoidan and brown algae polyphenols on high fructose-promoted colitis-related colon cancer, showing the changes in the appearance of the mouse colon.

[0021] Figure 7 shows the results of pharmacological experimental analysis of kelp, fucoidan, and brown algae polyphenols on high-fructose-promoted colitis-related colon cancer, and the statistics of the number and size of intestinal adenomas in mice.

[0022] Figure 8 shows the results of pharmacological experimental analysis of kelp, fucoidan, and brown algae polyphenols on high-fructose-promoted colitis-related colon cancer, and the results of HE staining of mouse intestine. DETAILED DESCRIPTION

[0023] 1. Network pharmacology screening of Kelp

[0024] In the present invention, C57BL / 6 mice were randomly divided into a normal group, an inflammation group (AOM / DSS 30 days), an adenoma group (AOM / DSS 75 days), a fructose inflammation group (Fructose+AOM / DSS 30 days), and a fructose adenoma group (Fructose+AOM / DSS 75 days). On day 0, each mouse except the normal group was intraperitoneally injected with 7.5 mg / kg of AOM solution. The mice in the fructose inflammation group and fructose adenoma group drank 30% fructose drinking water throughout the modeling period. On day 5, each mouse except the normal group was given 2.5% DSS drinking water for 5 days, and on day 11, it was changed to normal drinking water and 30% fructose drinking water for two weeks. The same cycle was repeated twice. On day 30, it was defined as the inflammation stage (tumor initiation stage), and on day 75, it was defined as the adenoma stage. Mouse tissues were taken for subsequent single-cell sequencing studies. In this study, single-cell transcriptome sequencing was used to identify key receptor-ligand pairs that promote high-fructose-induced colitis-associated colon cancer. Colon tissue was isolated and cleaned with sterile saline, minced, and digested to obtain a single-cell suspension. Single-cell sequencing was performed using Chromium Controller Single Cell 3' v3.1, specifically as follows:

[0025] (1) Gel bead generation and labeling: Add reagents and samples to the Chromium Next GEM Chip G as required, run the Chromium instrument, generate a gel bead GEM suspension through a microfluidic "double cross", and then recover and purify the GEM;

[0026] (2) cDNA amplification: GEM is mixed with relevant reagents for reverse transcription to form cDNA, which is then amplified by PCR, and the cDNA quality is tested and quantified using a Qubit instrument;

[0027] (3) Construction of gene expression library: cDNA was fragmented into 200-300 bp fragments, followed by end repair, adding adapters and indexes, and PCR was performed to obtain the target library, which was then quality-checked.

[0028] (4) Sequencing: Sequencing was completed using the Illumina sequencing platform. Cell Ranger (V3.1.0) software was used to process 10×Chromium single-cell gene expression data. Next, the R software Seurat package (V3.2.3) was used to normalize and standardize the data. The cell interactions in the single-cell transcriptome data were analyzed by the CellphoneDB method. By integrating the existing ligand information and interaction big data, the function of predicting intercellular communication was realized. In the present invention, high fructose significantly promoted the interaction between epithelial cells and stromal cells (Figure 1, each figure in Figure 1 represents the results of single-cell interactions in different groups, and the size and grayscale of the blocks in the figure are proportional to the intensity of the cell-to-cell interaction). The present invention compared the adenoma stage with the inflammation stage, and compared the high fructose group with the non-fructose group, and took the intersection to obtain the effector gene base A, which represents the ligand that changes alone during the transformation of colitis to colon cancer due to high fructose. Comparing the high-fructose inflammation group with the normal group, and the high-fructose adenoma group with the normal group, the intersection of the two yielded effector gene set B, representing the ligands that change during the transition from inflammation to tumorigenesis due to high fructose. Comparing the inflammation stage with the normal group, and the adenoma stage with the normal group, the intersection yielded effector gene set C, representing the ligand pairs that change during the transition from colitis to cancer. Finally, removing the ligand pairs from gene set C from gene set B yielded gene set D, highlighting the ligands affected by fructose during the transition from colitis to cancer. These four gene sets represent, in some ways, the effects of fructose on colitis-associated colon cancer (Figures 2a to 2d).

[0029] In the present invention, the inventors predicted the targets of the compounds contained in the Chinese medicine with medicinal and edible properties, and further predicted the targets of the Chinese medicine with medicinal and edible properties. The statistical models involved in the present invention include the Poisson binomial distribution model (Poisson binomial statistical model), the Pearson correlation coefficient statistical model (Pearson correlation statistical model) and the hypergeometric distribution statistical model (hypergeometric distribution statistical model). The statistical method is mainly the Fisher's exact test (Fisher's exact test), and the statistical correction method is the BH correction (Benjamini-Hochberg adjustment). The drug target calculation prediction method DrugCIPHER based on the relationship inference principle in the UNIQ (Using Network target for Intelligent and Quantitative) system was used to calculate the genome-wide target prediction scores of 4199 chemical components contained in the Chinese medicine with medicinal and edible properties, and further predicted the targets of the Chinese medicine with medicinal and edible properties. The targets of each Chinese medicine were enriched with the gene set. The higher the enrichment level, the more likely the Chinese medicine is to act on the gene set. The present invention screened a database of medicinal and edible herbs for potential intervention in this critical link. It was found that kelp (Kunbu) scored significantly higher than other medicinal and edible herbs in gene set A and ranked highly in gene sets B, C, and D. Based on this, the present invention selected kelp as a potential candidate for ameliorating high-fructose-promoted colitis-associated colon cancer (Figure 3).

[0030] The high ranking of Konbu in gene set C indicates the effect of Konbu on the cancer transformation of colitis. The high ranking of Konbu in gene sets A, B and D indicates the effect of Konbu on the cancer transformation of colitis induced by high fructose.

[0031] 2. Network pharmacological analysis of monomers in Kelp

[0032] Of the 48 components listed in the Kelp database, 27 have a druggability QED greater than or equal to 0.3. In addition to Kelp itself, the present invention also focuses on the main components of Kelp, brown algae polyphenols and fucoidan, which have high scores on gene sets A, B, C, and D (Figure 4).

[0033] 3. Pharmacological Experiments on Konbu

[0034] Fifty C57BL / 6 male mice were randomly divided into a normal group (Normal group), a high fructose group (Fru group), a kelp-treated group (Fru+Kunbu group), a fucoidan-treated group (Fru+Fuc group), and a fucoidan-treated group (Fru+Phl group). On day 0, each mouse except the normal group was intraperitoneally injected with 7.5 mg / kg of AOM solution. The mice in the normal group drank normal drinking water throughout the entire process. On day 5, the mice in the other groups were given 2.5% DSS drinking water for 5 days, and on day 11, they were changed to 30% fructose drinking water for two weeks. The kelp-treated group, fucoidan-treated group, and fucoidan-treated group were gavaged with 400 mg / kg / day, 200 mg / kg / day, and 200 mg / kg / day of kelp, fucoidan, and fucoidan solutions, respectively. The normal group and the high fructose group were administered with the same volume of solvent control. The same cycle was repeated twice, during which the weight, stool morphology and anal status of the mice were observed (Figure 5). At 75 days of the model, the mice were euthanized and the colon tissue of the mice was taken for preservation (Figure 6). The removed colon tissue was placed in a tissue fixative for 24 hours, and then the tissue was placed in 15% and 30% sucrose solutions overnight to allow the tissue to sink to the bottom. The tissue was placed in an embedding box, and after the OCT embedding agent submerged the tissue, it was quickly placed in a -80 degree refrigerator for storage or placed in a constant temperature box microtome for frozen sections.

[0035] 4. Analysis of the pharmacological experimental results of Kelp

[0036] 1) The effects of kelp on a high-fructose-induced colitis-associated colon cancer model in mice are shown in Figure 5. Only fucoidan significantly improved mouse body weight during the first two cycles of the colitis-associated colon cancer model, around day 30. However, with continued modeling and disease progression, kelp and fucoidan showed some improvement in mouse body weight, while fucoidan showed a lesser effect.

[0037] 2) As shown in Figures 5 to 8, the high-fructose group had more numerous and larger adenomas in the colon, most of which were concentrated near the anus. However, administration of kelp, fucoidan, and brown algae polyphenols significantly reduced tumor size and, to a certain extent, the number of adenomas.

[0038] 3) As shown in Figures 7 and 8, the high-fructose group showed significant destruction of the intestinal crypt structure, intestinal villus structure, and mucus layer, with significant inflammatory cell infiltration and tumor structures. However, administration of kelp, fucoidan, and brown algae polyphenols preserved the intestinal mucus layer and crypt structure to a certain extent, and exhibited fewer adenomatous structures.

[0039] 5. Advantages and positive effects

[0040] This study, using pharmacological experiments, demonstrates the beneficial effects of kelp and its monomeric components, fucoidan and brown algae polyphenols, on the improvement of colorectal cancer. Compared to existing treatments for colitis-related colorectal cancer, kelp offers enhanced safety, long-term suitability, and pharmacological activity, providing a safe and effective preventive and therapeutic option for patients with colitis-related colorectal cancer who have a high-fructose diet.

Claims

1. Use of kelp in the preparation of a drug for treating high fructose-induced colitis-related colorectal cancer.

2. Use of fucoidan in the preparation of a drug for treating high fructose-induced colitis-related colorectal cancer.

3. The application according to claim 2, characterized in that Fucoidan is an active ingredient of kelp.

4. Use of phlorotannins in the preparation of a drug for treating high fructose-induced colitis-related colorectal cancer.

5. The application according to claim 4, characterized in that Phlorotannins are active ingredients of kelp.

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