WO2025138017A1 - Use of ecklonia kurome and active ingredients thereof in preparation of drug for treating high fructose-promoted colitis associated colorectal cancer - Google Patents

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 medicine for treating high fructose-promoted colitis-related colorectal cancer Technical Field

The invention relates to the use of kelp and its active ingredients fucoidan and brown algae polyphenol in the preparation of colitis-cancer transformation, especially high-fructose-promoted colitis-related colorectal cancer drugs.

Background Art

Colorectal cancer (CRC) is one of the most common malignant tumors, which can be clinically divided into colitis-associated colorectal cancer (CAC) and sporadic colorectal cancer (SCRC). Compared with other subtypes, CAC has a faster disease progression and a higher mortality rate. The development of CAC goes through the staged formation of abnormal crypt lesions, polyps, adenomas and cancers. Chronic intestinal inflammation induces mutations of 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, causing epigenetic changes, inactivation of DNA repair mechanisms, and changes in anti-tumor immune responses to promote the formation, development and metastasis of tumors. Therefore, unlike sporadic colorectal cancer, colitis-related colorectal cancer mostly occurs in patients with inflammatory bowel disease. Due to long-term exposure to chronic inflammation, the disease process is more susceptible to factors such as dietary intake. Current chemotherapy regimens for colorectal cancer include single-drug therapy and multi-drug therapy. However, chemotherapy has high toxicity and low response rate, making it inevitable to improve existing colorectal cancer chemotherapy regimens.

As a common flavor enhancer, fructose is involved in the occurrence and development of diseases such as diabetes, non-alcoholic fatty liver disease, cardiovascular disease, and colorectal cancer. There is still a lack of safe, effective, and long-term preventive and treatment measures and treatment plans for the phenomenon that long-term daily intake of fructose leads to the progression and aggravation of intestinal diseases.

Konbu is the dried thallus of Laminaria japonica Aresch. of the Laminariaceae family or Ecklonia kurome Okam. of the Pterygium family. As a substance with medicinal and edible properties, kelp has 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 "Jiayou Materia Medica" of the Northern Song Dynasty records that kelp can eliminate phlegm, soften hard masses, promote diuresis and reduce swelling. It has long been used to treat goiter, beriberi edema, and testicular swelling and pain. With the development of modern medicine, researchers have conducted more detailed studies on kelp and its components. However, there is currently no relevant content on the application of kelp in the prevention or treatment of high-fructose-promoted colitis-related colon cancer.

Summary of the invention

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.

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

The present inventors searched for and identified the key receptor pairs for high fructose-promoted colitis-associated colorectal cancer through single-cell transcriptome sequencing.

The inventors screened the colitis-related syndromes promoted by high fructose using the UNIQ system. Rectal cancer.

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.

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

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

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

The dosage of the fucoidan and brown algae polyphenols in the present invention for treating high fructose-promoted colitis-related colorectal cancer is 200 mg/kg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the cellular interaction mechanism related to inflammatory-cancer transformation of colitis-related colorectal cancer promoted by high fructose.

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

FIG3 shows the ranking of various Chinese medicines with medicinal and edible properties in the four effector gene sets of high-fructose-promoted colitis-related colorectal cancer based on network pharmacology prediction.

FIG4 shows the scores of various compound components in kelp in four effector gene sets predicted based on network pharmacology.

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.

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

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.

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 mouse intestinal HE staining.

DETAILED DESCRIPTION

1. Network pharmacology screening of Kelp

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 the 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 the 30th day, it was defined as the inflammatory stage (tumor initiation stage), and on the 75th day, it was defined as the adenoma stage. Mouse tissues were taken for subsequent single-cell sequencing studies. In the present invention, single-cell transcriptome sequencing was used to find the key receptor ligand pairs of high-fructose-promoted colitis-related colon cancer. After the colon tissue was removed from the body, sterile saline was used to clean the tissue, and the tissue was digested after cutting to obtain a single-cell suspension. Single-cell sequencing operations were performed according to Chromium Controller Single Cell 3'v3.1, specifically:

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

(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 the Qubit instrument;

(3) Construction of gene expression library: cDNA was fragmented into 200-300 bp fragments, followed by end repair, adding adapters and indexes, PCR was performed to obtain the target library, and the library quality was tested;

(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 CellphoneDB method was used to analyze the cell interactions in the single-cell transcriptome data. By integrating the existing receptor 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 cell-to-cell interaction). The present invention compares the adenoma stage with the inflammatory stage, and the high fructose group with the non-fructose group, and takes the intersection to obtain the effector gene base A, which represents the receptor that changes alone during the transformation of colitis to colon cancer due to high fructose. The high fructose inflammation group was compared with the normal group, and the high fructose adenoma group was compared with the normal group. The intersection of the two obtained the effect gene base B, which represents the ligands that change during the transformation of high fructose from inflammation to tumor. The inflammation stage was compared with the normal group, and the adenoma stage was compared with the normal group, and the intersection was obtained to obtain the effect gene base C, which represents the ligand pairs that change during the transformation of colitis to cancer. Finally, the ligand pairs in gene set B were removed from the ligand pairs in gene set C to obtain gene set D, highlighting the ligands affected by fructose in the process of transformation of colitis to cancer. These four gene sets represent the effects of fructose in colitis-related colon cancer in some aspects (Figures 2a to 2d).

In the present invention, the inventors predicted the targets of the compounds contained in the Chinese medicines with both medicinal and edible properties, and further predicted the targets of the Chinese medicines with both medicinal and edible properties. The statistical models involved in the present invention include Poisson binomial statistical model, Pearson correlation statistical model and hypergeometric distribution statistical model. The statistical method is mainly Fisher's exact test, and the statistical correction method is BH correction (Benjamini-Hochberg adjustment). The drug target computational 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 medicines with both medicinal and edible properties, and further predicted the targets of the Chinese medicines with both medicinal and edible properties. The targets of each Chinese medicine were enriched with the gene set. The higher the enrichment level, Then the Chinese medicine is more likely to act on this gene set. The present invention screened out Chinese medicines with medicinal and edible properties that have the potential to intervene in this key link from the medicinal and edible properties database. It was found that kelp scored much higher than other medicinal and edible properties in gene set A, and ranked high in gene sets B, C, and D. Based on this, the present invention selected kelp as a potential candidate drug for improving high-fructose-promoted colitis-related colon cancer (Figure 3).

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.

2. Network pharmacological analysis of monomers in kelp

Among the 48 components recorded in the kelp database, 27 components have a drugability 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).

3. Pharmacological Experiments on Kelp

Fifty C57BL/6 male mice were randomly divided into a normal group (Normal group), a high fructose group (Fru group), a kelp treatment group (Fru+Kunbu group), a fucoidan treatment group (Fru+Fuc group) and a brown algae polyphenol treatment 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 whole process. On the 5th day, the mice in the other groups were given 2.5% DSS drinking water for 5 days, and on the 11th day, it was changed to 30% fructose drinking water for two weeks. The kelp treatment group, the fucoidan treatment group and the brown algae polyphenol treatment group were gavaged with 400 mg/kg/day, 200 mg/kg/day, and 200 mg/kg/day of kelp, brown algae polyphenol, and fucoidan solutions, respectively, and 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 slicer for frozen sections.

4. Analysis of the results of the pharmacological experiments on kelp

1) The effect of kelp on the high fructose-promoted colitis-associated colon cancer model in mice is shown in Figure 5. Only brown algae polyphenols had a more obvious improvement on the weight of mice in the first two cycles of the colitis-associated colon cancer model, i.e., around 30 days. With the continuous modeling and the progression of the disease, kelp and brown algae polyphenols achieved a certain effect in improving the weight of mice, while fucoidan had a weaker effect on improving weight.

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

3) As shown in Figures 7 and 8, the intestinal crypt structure, intestinal villus structure and mucus layer in the high fructose group were largely destroyed, and a large number of inflammatory cell infiltration and tumor structures were present. However, the administration of kelp, fucoidan and brown algae polyphenols preserved the intestinal mucus layer and crypt structure to a certain extent, and fewer adenoma structures appeared.

5. Advantages and positive effects

The present invention verifies the good improvement effect of kelp and its monomer components fucoidan and brown algae polyphenols through pharmacological experiments. Compared with the existing colitis-related colon cancer treatment scheme, kelp as a therapeutic drug It is highly safe, suitable for long-term use, and has good pharmacological activity, providing a safe and effective prevention and treatment option for patients with colitis-related colorectal cancer who have a high-fructose dietary habit.

Claims (5)

Application of kelp in the preparation of medicine for treating high fructose-promoted colitis-related colorectal cancer. Use of fucoidan in the preparation of drugs for treating high fructose-promoted colitis-associated colorectal cancer. The use according to claim 2 is characterized in that fucoidan is the active ingredient of kelp. Application of brown algae polyphenols in the preparation of drugs for treating high fructose-promoted colitis-related colorectal cancer. The use according to claim 4, characterized in that brown algae polyphenols are the active ingredients of kelp.