Application of boniaea dickinsii in preparation of medicine for treating colorectal cancer

Abstract

The application discloses application of a white rot fungus in preparation of a medicine for treating colorectal cancer and belongs to the field of biological medicine.The application firstly analyzes and determines effective components in fruiting bodies of the white rot fungus, clearly defines the components of the white rot fungus, and proves through animal experiments that the white rot fungus can obviously improve the intestinal state of inflammation-related colorectal cancer mice, effectively reduces the cancerous tumor tissue area of the intestines, and can inhibit the production of PGE2 and the promotion effect of PGE2 on colorectal cancer by changing the structure of intestinal microbiota and regulating the metabolism, thereby relieving the occurrence and development of colorectal cancer.The application provides theoretical support for developing the medicine for treating or relieving colorectal cancer by taking the white rot fungus as an active component.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of *Porphyromonas Dickensburgii* in the preparation of medicaments for the treatment of colorectal cancer. Background Technology

[0002] Colorectal cancer is a common malignant tumor of the gastrointestinal tract, with extremely high incidence and mortality rates. Many people are asymptomatic in the early stages of the disease and are often diagnosed only in later stages when treatment options are limited. Common symptoms of colorectal cancer include changes in bowel habits, rectal bleeding (bright red or dark tarry stools), persistent abdominal cramps, pain or bloating, unexplained sudden weight loss without attempting to lose weight, frequent fatigue and lack of energy even with adequate rest, and iron deficiency anemia due to chronic bleeding, leading to fatigue, weakness, and paleness.

[0003] Current treatments for colorectal cancer include surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy. While early surgical resection can potentially cure colorectal cancer, its asymptomatic nature often causes patients to miss the optimal treatment window, making timely and proper care difficult. Furthermore, approximately 50% of colorectal cancer patients develop metastases, posing a greater threat to their lives. Currently, most treatments for colorectal cancer are accompanied by significant side effects and are not ideal. In this context, natural compounds, due to their low toxicity and easy absorption, have gained wider acceptance. Some traditional Chinese medicines or their extracts, or certain microorganisms, have been found to have therapeutic effects on colorectal cancer. Patent application CN117018153A discloses a traditional Chinese medicine composition for treating colorectal cancer and its application. The traditional Chinese medicine composition is composed of herbs such as Hedyotis diffusa, Paris polyphylla, Sophora flavescens, Taraxacum mongolicum, Curcuma longa, Myristica fragrans, Magnolia officinalis, Poria cocos, Curcuma longa, Lithospermum erythrorhizon, Atractylodes macrocephala, Paeonia lactiflora, Vitex trifolia, Platycodon grandiflorus, Polygonum cuspidatum, Cnidium monnieri, Forsythia suspensa, Cimicifuga foetida, Evodia rutaecarpa, Cinnamomum cassia, and fresh ginger. It can inhibit the proliferation of HCT116 colorectal cancer cells, promote cell apoptosis, and thus inhibit the occurrence of colorectal cancer in vivo. Patent CN114847483B discloses the application of Bifidobacterium longum BL21 and its containing agents in the preparation of products for the prevention, relief or treatment of colorectal cancer. Bifidobacterium longum BL21 can induce apoptosis of colorectal tumor cells in mice by regulating the expression of apoptosis-related genes in mouse colorectal tumor cells, thereby reducing the number of colorectal tumors in mice. It can also significantly alleviate the inflammatory response in the colon of colorectal cancer model mice, reduce oxidative stress damage in colorectal cancer model mice, improve the microecological environment of intestinal flora, and maintain intestinal health.

[0004] In traditional Chinese medicine, medicinal fungi have been studied for thousands of years. In recent years, with the continuous development of science and technology and research methods, research on medicinal fungi has also progressed rapidly. Numerous effective components, including polysaccharides, polypeptides, terpenes, polyhydrocarbons, glycosides, organic acids, and alkaloids, with various effects such as anti-tumor, immunomodulatory, antibacterial, and hypoglycemic properties, have been discovered in fungi, demonstrating their immense potential in drug development. *Bondarzewia dickinsii*, a novel edible and medicinal fungal resource that is being developed and utilized, has unclear components and pharmacological activities. Therefore, in-depth research into the components and pharmacological activities of *Bondarzewia dickinsii* is of great significance for developing new drugs for the treatment of colorectal cancer. Summary of the Invention

[0005] The purpose of this invention is to provide the application of *Fomitopsis Dickinsonii* in the preparation of drugs for treating colorectal cancer, thereby addressing the problems existing in the prior art. This invention clarifies the components of *Fomitopsis Dickinsonii* and confirms its beneficial effects on the intestinal flora, providing theoretical support for developing drugs for treating or alleviating colorectal cancer using *Fomitopsis Dickinsonii* as the active ingredient.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of *Bondarzewia dickinsii* in the preparation of medicaments for treating colorectal cancer.

[0008] Furthermore, the colorectal cancer is inflammation-associated colorectal cancer.

[0009] Furthermore, the *Phellinus Dickensbonni* is the fruiting body of *Phellinus Dickensbonni*.

[0010] Furthermore, the *Polyporus d'Dickens* strain achieves its therapeutic effect on colorectal cancer by regulating the metabolism of the gut microbiota.

[0011] Furthermore, the regulation of gut microbiota metabolism includes altering the gut microbiota structure and regulating gut microbiota metabolites.

[0012] Furthermore, the *Porphyromonas Dickensii* strain alleviates the occurrence and development of colorectal cancer by inhibiting the production of PGE2.

[0013] Furthermore, the drug includes pharmaceutically acceptable carrier substances and / or excipients.

[0014] The present invention discloses the following technical effects:

[0015] This invention is the first to analyze and determine the effective components in the fruiting body of *Pheromus d'Antinomyces*, and established a CAC mouse model through animal experiments to further verify the effects of *Pheromus d'Antinomyces* on mice with inflammation-associated colorectal cancer. The results demonstrate that *Pheromus d'Antinomyces* can significantly improve the intestinal condition of mice with inflammation-associated colorectal cancer, effectively reduce the area of ​​intestinal cancerous tumor tissue, and increase the number of intestinal goblet cells. Further research shows that *Pheromus d'Antinomyces* can alter the structure of the intestinal microbiota and, by regulating its metabolism, inhibit the production of PGE2 and suppress the promoting effect of PGE2 on colorectal cancer, thereby alleviating the occurrence and development of colorectal cancer.

[0016] This invention clarifies the composition of *Fomitopsis Dickensburgii* and confirms its beneficial effects on the gut microbiota, providing theoretical support for the development of drugs to treat or alleviate colorectal cancer using *Fomitopsis Dickensburgii* as an active ingredient. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Morphological illustration of wild *Porphyromonas d'Anse*.

[0019] Figure 2 Image showing the sequencing alignment results of wild-type F. Dickensburgia.

[0020] Figure 3 A schematic diagram of AOM / DSS modeling and drug delivery method;

[0021] Figure 4 Images showing the morphology of the colorectal region in mice under different treatment groups after dissection;

[0022] Figure 5 Effects of *Porphyromonas d'Eau* on colonic and rectal length (A), tumor size (B), colonic coefficient (C), and colonic index (D) in CAC model mice; ###P<0.001 compared with the Ctrl group; ***P<0.001 compared with the Model group;

[0023] Figure 6 H&E staining images of colorectal tissues from mice in different treatment groups, where the 40× scale bar is 500μm and the 200× scale bar is 100μm;

[0024] Figure 7 Venn diagrams (A) and α-diversity analysis results of fecal intestinal microbiota in mice from different treatment groups (B);

[0025] Figure 8 Principal coordinate analysis (PCoA) plots of fecal microbiota in the gut of mice in different treatment groups;

[0026] Figure 9 A graph showing the effect size (LEfSe) results of the linear discriminant analysis;

[0027] Figure 10 Heatmap of fecal intestinal microbiota in mice from different treatment groups;

[0028] Figure 11 Results of OPLS-DA analysis of serum metabolites in mice from different treatment groups;

[0029] Figure 12 Venn diagram of serum metabolites from mice in different treatment groups;

[0030] Figure 13 Heatmap of differential metabolites in the serum of mice in different treatment groups;

[0031] Figure 14 Correlation analysis of differential metabolism in serum of mice in different treatment groups;

[0032] Figure 15 A heatmap showing the correlation between metabolomics and the microbiome;

[0033] Figure 16 The levels of PGE2(A), COX(B), and IL-6(C) in the colorectal tissue of mice in the same treatment group were compared. ###P<0.001 with the Ctrl group; ***P<0.001 with the Model group. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1

[0040] The *Bondarzewia dickinsii* used in this invention is a strain that was artificially cultivated and domesticated after tissue isolation from the wild strain Bd-001. The wild strain Bd-001 was collected by the Qingyuan County Edible Fungus Industry Center in Baishanzu National Park, Qingyuan County, and was identified as *Bondarzewia dickinsii* through morphological and molecular biological identification.

[0041] The morphological identification results of wild strain Bd-001 are as follows: Figure 1 This strain is an annual basidiomycete, cap-shaped, with a central or lateral stalk, imbricate in shape. When fresh, it is corky, odorless and tasteless, becoming hard corky when dry. The cap is nearly semi-circular to fan-shaped, 6.8-9.5 cm in diameter and 0.5-0.8 cm thick. When fresh, the surface is light brown to yellowish-brown, with a lighter and sharper edge. The tubes are decurrent, with an irregular to polygonal, cream-colored pore surface when fresh, and are hard corky. The flesh is cream-colored to light yellow, corky, up to 1.4 cm thick, becoming thick, hard, and brittle when dry. The stipe is 8.4-10.6 cm long and 1.8-3.4 cm in diameter, cylindrical, and light yellow to light yellowish-brown.

[0042] Molecular biological identification results: The ITS sequence of wild-type strain Bd-001 was amplified and submitted to GenBank for BLAST homology search (www.ncbi.nlm.nih.gov). Similarity analysis with sequences of various strains in the database showed that its identity with the corresponding sequence of *Bondarzewia* in the database reached 93-97%. Figure 2 ITS sequences of *Bondarzewia* and *Heterobasidion annosum* were downloaded from the GenBank database, and an ML phylogenetic tree was constructed using PhyloSuite software with default parameters and a Botstrap test of 1000. The phylogenetic tree showed that the wild-type strain Bd-001 had a high degree of similarity to the *Bondarzewia dickinsii* species in the GenBank database, with a homology similarity of 98%.

[0043] Based on BLAST alignment and phylogenetic tree results, combined with morphological findings, wild-type strain Bd-001 was identified as *Bondarzewia dickinsii*. This strain was deposited on October 19, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40356.

[0044] Example 2: Extraction and content determination of effective components from the fruiting bodies of *Porphyra yezoensis*.

[0045] I. Experimental Methods

[0046] 1. Extraction and Detection of Conventional Nutrients in the Fruiting Bodies of *Porphyra yezoensis* (Dickens's Porphyra)

[0047] 1.1 Determination of total sugar content

[0048] Weigh 1 g of *Porphyromonas d'Anseltongue* fruiting body powder sample, mix with 30 mL of concentrated hydrochloric acid and 100 mL of deionized water, and hydrolyze under reflux in a boiling water bath for 3 h. Wash the filter residue with deionized water, combine the two filtrates, and dilute to the mark of 500 mL with deionized water. Mix well to obtain the sample solution to be tested.

[0049] Accurately measure 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose reference solution, respectively, and bring the volume to 1 mL with deionized water. Then, pipette 200 μL of each solution into a 1.5 mL EP tube, add 100 μL of freshly prepared 5% phenol solution, and then quickly add concentrated sulfuric acid to a final volume of 800 μL. Mix well and let stand for 15 min. Measure the absorbance at 490 nm and plot a standard curve, with glucose concentration on the x-axis and absorbance on the y-axis.

[0050] Take 200 μL of the sample solution and add it to a 1.5 mL EP tube. Measure the absorbance value and calculate the total sugar content according to the standard curve procedure in the previous step.

[0051] 1.2 Determination of reducing sugar content

[0052] Accurately weigh 2g of *Porphyromonas d'Anchis* fruiting body powder, place it in an Erlenmeyer flask, add 60mL of water, and incubate at 80℃ for 4 hours. After the water bath, cool and centrifuge to collect the supernatant, which is the test solution. Store at 4℃ for later use.

[0053] Preheat glucose to constant weight and prepare a 0.1 mg / mL glucose standard solution. Take 20, 40, 60, 80, and 100 μL of this glucose standard solution into 1.5 mL EP tubes, respectively, and add deionized water to 100 μL. Determine the reducing sugar content using the DNS method and plot a standard curve.

[0054] Accurately pipette 100 μL of the test solution into a 1.5 mL EP tube. Follow the same steps as the standard curve procedure. After measuring the absorbance value, calculate the total reducing sugar content in the fruiting body according to the standard curve equation.

[0055] 1.3 Determination of total protein content

[0056] Weigh 0.1g of methyl red and bromocresol green reagents respectively, dissolve them in 95% ethanol and make up to 100mL. Mix them well to obtain methyl red and bromocresol green ethanol solutions for later use.

[0057] Weigh 2g of *Porphyromonas d'Hippophae* fruiting body powder sample, add copper sulfate, potassium sulfate and sulfuric acid for digestion. After digestion, cool, add deionized water and distill using an automatic Kjeldahl nitrogen analyzer for 7 minutes. After distillation, add 1-2 drops of indicator, then add a certain amount of boric acid solution. Collect the distillate to 200mL and titrate with 0.1mol / L hydrochloric acid standard solution. After titration, calculate the protein content based on the data.

[0058] 1.4 Determination of crude fiber content

[0059] Weigh 2g of *Porphyra yezoensis* fruiting body powder sample into a 500mL Erlenmeyer flask, add 100mL of 1.25% sulfuric acid, and react thoroughly for 30min, keeping the sulfuric acid at a gentle boil throughout the reaction. After the reaction is complete, filter the residue and wash with boiling water until neutral. Add 100mL of 1.25% potassium hydroxide to the residue in the Erlenmeyer flask and continue to boil gently for 30min. After washing three times with boiling water, place the sample in a crucible with constant weight and record the weight, and wash once each with hot water, acetic acid, and ether. After washing, dry the crucible and its contents to constant weight, and weigh to calculate the crude fiber content.

[0060] 1.5 Determination of crude fat content

[0061] Accurately weigh 2g of *Porphyromonas d'Anseltongue* fruiting body powder sample, wrap it tightly with filter paper, and perform three replicates, taking the average value. Place the filter-wrapped sample into a Soxhlet extractor, assemble the receiving flask and condenser, and add petroleum ether into the Soxhlet extractor from the top of the condenser until the receiving flask is 2 / 3 full. Heat the receiving flask in a 70°C water bath to reflux for 7 hours. After reflux, recover the petroleum ether, evaporate the remaining filtrate to dryness in a water bath, dry it to constant weight, weigh it, and calculate the crude fat content.

[0062] 1.6 Total Ash Content Detection

[0063] Total ash content was determined by high-temperature ignition. A muffle furnace was used for ignition. The crucible was ignited at 550℃ for 30 min, cooled, and weighed. 2 g of *Pheromoneus Dickensii* was placed in the cooled crucible and ignited over a low flame until the sample was completely carbonized. Then, it was ignited at 550℃ for 4 h, with the ignition temperature fluctuation controlled within 25℃. After ignition and complete cooling, the sample was observed. Complete ash formation was considered achieved when no carbon particles were present, and the sample was then weighed. The ignition process was repeated two or more times until the weight difference was less than 0.5 mg. The total ash content in *Pheromoneus Dickensii* was the ratio of the weight of the total ash after ignition to the sample mass.

[0064] 1.7 Determination of total flavonoid content

[0065] Accurately weigh 1g of *Porphyromonas d'Hippophae* fruiting body powder sample, add 50mL of 60% ethanol solution, boil in a water bath for 20min, then filter. Add another 40mL of 60% ethanol, boil in a water bath for 20min again, and filter. Combine the filtrates, wash the filter residue with 10mL of hot 60% ethanol, and combine the filtrates to a final volume of 100mL, which is the test solution.

[0066] Accurately weigh 10 mg of rutin standard and dilute to 100 mL with deionized water to obtain the rutin standard solution. Pipette 0 μL, 100 μL, 200 μL, 300 μL, 400 μL, and 500 μL into 1.5 mL EP tubes, respectively, and perform colorimetric reactions using sodium nitrite solution, aluminum nitrate solution, and potassium hydroxide solution, respectively. Measure the absorbance at 516 nm and plot a standard curve.

[0067] Pipette 200 μL of the sample solution to be tested into a 1.5 mL EP tube. The remaining steps are the same as those for preparing the standard curve. After standing for 15 min, measure the absorbance at a wavelength of 516 nm and calculate the total flavonoid content.

[0068] 1.8 Determination of total phenol content

[0069] Weigh 2 g of *Porphyromonas dichotoma* fruiting body powder sample, add 5 mL of 70% ethanol, mix well, and extract in a 70℃ water bath for 10 min. After removing from the water bath and cooling to room temperature, centrifuge (8000 rpm, 8 min), and transfer the supernatant to a 10 mL volumetric flask. Extract the remaining residue once more with 5 mL of 70% ethanol using the same parameters, combine the extracts, and bring the volume to 10 mL. Mix well and filter through a 0.45 μm filter membrane to obtain the test solution.

[0070] Prepare a 0.1 mg / mL gallic acid standard solution. Add 0 μL, 10 μL, 20 μL, 40 μL, 50 μL, and 100 μL of this standard solution to 1.5 mL EP tubes, respectively. Perform a colorimetric reaction with Folin-Ciocalteu and sodium carbonate, and measure the absorbance value at a wavelength of 760 nm. Plot a standard curve.

[0071] Accurately pipette 100 μL of the test solution into a 1.5 mL EP tube. The remaining steps are the same as those for preparing the standard curve. Measure the absorbance and substitute it into the standard curve equation to obtain the total phenol concentration in the test solution. Then calculate the total phenol content in the sample.

[0072] 1.9 Determination of total sterol content

[0073] Accurately weigh 1g of *Porphyromonas dichotoma* fruiting body powder sample, add 25mL of chloroform to a 100mL Erlenmeyer flask, weigh the entire flask, and then sonicate for 30min. After sonication, cool to room temperature, then add a certain amount of chloroform to the original weight, mix thoroughly, and filter. Accurately measure 10mL of the filtrate and concentrate to dryness. Add 25mL of 10% sodium hydroxide-ethanol solution to the residue and reflux at 95℃ for 2h. After cooling, add 45mL of water, transfer to a separatory funnel, and extract three times with n-hexane, adding 20mL of n-hexane each time. Combine the three n-hexane layers, wash twice with water, adding 45mL of deionized water each time, discard the aqueous layer, combine the n-hexane layers, and add n-hexane to make up to 50mL. Mix thoroughly to obtain the test solution.

[0074] Prepare a 0.1 mg / mL β-sitosterol standard solution by drying the standard to constant weight. Accurately add 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the β-sitosterol standard solution to 5 mL EP tubes, dilute to 1 mL with deionized water, evaporate to dryness in a water bath, and perform a colorimetric reaction with vanillin-acetic acid solution, perchloric acid, and acetic acid. Then, plot a standard curve based on the absorbance at 543 nm.

[0075] Accurately pipette 1 mL of the test solution into a 5 mL EP tube. The remaining steps are the same as those for preparing the standard curve. Detect the absorbance at a wavelength of 543 nm. Calculate the total sterol concentration and sterol content according to the standard curve equation.

[0076] 1.10 Determination of total triterpenoid content

[0077] Take 1g of the fruiting body powder of *Porphyromonas d'Hippophae* and mix it with 50mL of ethanol. Sonicate the mixture for 45min, filter it, and wash the filter and filter residue with an appropriate amount of ethanol. Combine the filtrate and washing liquid and bring the volume to 100mL. Mix well to obtain the solution to be tested.

[0078] Prepare a 0.2 mg / mL oleanolic acid standard solution. Weigh 0 μL, 100 μL, 200 μL, 300 μL, 400 μL, and 500 μL of the standard solution into 15 mL EP tubes, evaporate to dryness in a water bath, and perform colorimetric reactions with vanillin-acetic acid solution, perchloric acid, and ethyl acetate. Measure the absorbance at 546 nm and plot a standard curve.

[0079] Accurately take 200 μL of the test solution and put it into a 15 mL test tube. Follow the same steps as the standard curve preparation method. Calculate the amount of oleanolic acid in the test solution based on the standard curve to obtain the total triterpenoid content.

[0080] 1.11 Determination of Mannitol Content

[0081] Dissolve 1 g of *Porphyromonas d'Hippophae* fruiting body powder in 25 mL of ethanol, weigh the sample, reflux in an 80 °C water bath for 2 h, then make up the weight and filter to obtain the supernatant. Take 20 mL of the supernatant, evaporate to dryness in a water bath, and then make up to 10 mL with acetonitrile-water. Filter through a 0.45 μm filter membrane for analysis. Detection is performed using liquid chromatography.

[0082] 2. Analysis of fatty acid content in the fruiting bodies of *Porphyra yezoensis*.

[0083] Weigh 0.05 g of *Porphyromonas Dickensii* fruiting body powder, dissolve it in chloroform-methanol (2:1) working solution, and evaporate to dryness; cool to room temperature and react with 4% potassium hydroxide-methanol solution at 50℃ for 10 min; after the reaction is complete, add 1 mL of 20% boron trifluoride-acetonitrile solution and continue the reaction for 15 min; finally, mix the reaction solution with n-hexane in a separatory funnel, let it stand, retain the n-hexane layer, wash the n-hexane layer with deionized water until neutral, filter it through a membrane, and then perform instrumental analysis.

[0084] The fatty acid composition and content of *Porphyromonas dichotoma* fruiting bodies were determined by gas chromatography. The program was as follows: initial column temperature 130℃, held for 5 min, then increased to 220℃ at a rate of 2℃ / min, held for 15 min. The experiment was repeated twice, and the average value was used to obtain the content of each fatty acid.

[0085] 3. Determination of amino acid content in the fruiting bodies of *Porphyra yezoensis* (Dickens's fungus)

[0086] (1) Preparation of mobile phase

[0087] B1: Weigh 6.19g of sodium citrate dihydrate and dissolve it in 700mL of distilled water. Then add 6mL of 1M sodium hydroxide solution, 5.66g of sodium chloride, 19.8g of citric acid and 135mL of ethanol solution in sequence. Mix well and then dilute to 1L with distilled water.

[0088] B2: Weigh 7.74g of sodium citrate dihydrate and dissolve it in 700mL of distilled water. Then add 20mL of 1mL sodium hydroxide solution, 7.07g of sodium chloride, 22g of citric acid and 25mL of ethanol solution in sequence. Mix well and then dilute to 1L with distilled water.

[0089] B3: Weigh 13.31g of sodium citrate dihydrate and dissolve it in 700mL of distilled water. Then add 3.74g of sodium chloride, 12.8g of citric acid and 9mL of ethanol solution in sequence. Mix well and then dilute to 1L with distilled water.

[0090] B4: Weigh 26.67g of sodium citrate dihydrate and dissolve it in 700mL of distilled water. Then add 54.35g of sodium chloride and 6.1g of citric acid in sequence, mix well, and then dilute to 1L with distilled water.

[0091] B5: Weigh 8g of sodium hydroxide and dissolve it in 700mL of distilled water, then add 100mL of ethanol solution, mix well, and then dilute to 1L with distilled water.

[0092] R1: Dissolve 39g of ninhydrin in 979mL of propylene glycol monomethyl ether, bubble with N2 for 5min, add 82mg of sodium borohydride, and bubble with N2 again for 30min.

[0093] R2: Mix 204g of anhydrous sodium acetate with 401mL of propylene glycol monomethyl ether, heat to 60℃ to dissolve the solid, and add 123mL of glacial acetic acid solution and 336mL of distilled water in sequence. After mixing evenly, add distilled water to make up to 1L, and bubble with N2 for 30min.

[0094] R3: Measure 50 mL of ethanol solution and add it to 950 mL of distilled water, then mix well.

[0095] (2) Experimental instruments and methods

[0096] Instrument: Automated Amino Acid Analyzer;

[0097] Ion exchange chromatography column: sulfonic acid type cation exchange resin;

[0098] Injection volume: 20 μL;

[0099] Reaction temperature: 135℃;

[0100] Detection wavelengths: 570nm, 440nm;

[0101] The flow rate of mobile phase B is 0.4 mL / min;

[0102] The flow rate of mobile phase R is 0.35 mL / min;

[0103] The mobile phase elution procedure is shown in Table 1:

[0104] Table 1 Mobile phase elution procedure

[0105]

[0106]

[0107] (3) Sample processing and instrument testing

[0108] Add 5 mg of *Porphyromonas d'Dickens* fruiting body powder and 12 mL of 6 M hydrochloric acid solution to a hydrolysis tube. After mixing, transfer the hydrolysis tube to a refrigerant and freeze for 5 min, during which time N2 is introduced. Then, transfer the hydrolysis tube to a constant temperature oven at 110 °C and react for 22 h. After the reaction is complete, immediately transfer it to room temperature for cooling. Then, filter the hydrolyzed liquid in the hydrolysis tube, discard the filter residue, take the filtrate, and add distilled water to make up to 50 mL. After the liquid is thoroughly mixed, take 1 mL of the liquid and evaporate the solvent at 40 °C. Redissolve the evaporated solid with 1 mL of sodium citrate buffer solution (pH = 2.2) to obtain the sample to be tested.

[0109] After the mixed amino acid standards and the sample to be tested are filtered through a 0.22 μm filter membrane, they are used for detection in an automatic amino acid analyzer. The amino acid components and contents contained in Fomitopsis pinicola can be determined based on the difference in peak time and peak area of ​​the standards.

[0110] 4. Determination of mineral and heavy metal elements in the fruiting bodies of *Porphyra yezoensis*.

[0111] 1.5 g of *Pheromus d'Anchis Dickens* fruiting body powder was mixed with 5 mL of nitric acid solution and digested to prepare the *Pheromus d'Anchis Dickens* sample. The elemental standard solution and the digested sample solution were loaded into an inductively coupled plasma mass spectrometer to detect the composition and content of minerals and heavy metals in the *Pheromus d'Anchis Dickens* fruiting body.

[0112] II. Experimental Results

[0113] 1. Analysis of major components in *Porphyromonas dichotoma*

[0114] This embodiment analyzed the content of 10 nutrients in *Porphyromonas dichotoma*, as shown in Table 2. The content of nutrients from highest to lowest is as follows: total sugar (37.29%) > total protein (24.90%) > mannitol (15.84%) > crude fiber (11.12%) > total ash (5.67%) > total flavonoids (3.47%) > crude fat (1.64%) > total triterpenes (0.60%) > total sterols (0.33%) > total phenols (0.08%). The top three nutrients are all important regulators of life activities. For example, polysaccharides have activities including anti-oxidation, anti-tumor, blood sugar and lipid regulation, anti-osteoporosis, alcohol detoxification and liver protection, and immune enhancement; proteins are important physiologically active substances that can participate in multiple biological processes such as immune regulation, oxidative metabolism, and body repair. Therefore, the results in this part indicate that *Porphyromonas Dickensii* is a nutrient-rich fungus that is both medicinal and edible and can be applied to pharmacological activity research.

[0115] Table 2. Analysis of the content of common nutrients in the fruiting bodies of *Porphyra yezoensis*.

[0116]

[0117]

[0118] 2. Amino acid composition analysis of the fruiting bodies of *Porphyromonas bubblytus*.

[0119] Arginine can inhibit the growth of tumor cells and reduce the risk of tumor metastasis. Supplementing cancer patients with arginine can help increase their survival time. *Pheromoneus dichotoma* is rich in protein; this example further examines the composition and content of its functional components—amino acids.

[0120] The amino acid composition and content percentage of each component in *Porphyromonas dichotoma* are shown in Table 3.

[0121] Table 3. Analysis of amino acid composition in the fruiting bodies of *Fomitopsis pinnatifida*.

[0122]

[0123] Table 3 shows that *Fomitopsis pinnatifida* contains 17 amino acids, including 8 essential amino acids (lysine, valine, isoleucine, methionine, threonine, leucine, alanine, and phenylalanine) and 9 non-essential amino acids (glutamic acid, aspartic acid, arginine, glycine, serine, tyrosine, proline, histidine, and arginine). The high amino acid content suggests that its consumption may promote protein synthesis in the body. Among the detected amino acids, the top three in abundance were glutamic acid (1.38%), leucine (1.10%), and aspartic acid (1.05%). Glutamic acid has been reported to regulate metabolism, and leucine has antioxidant properties. These results indicate that *Fomitopsis pinnatifida* contains a rich amino acid composition and has potential medicinal value.

[0124] 3. Fatty acid composition analysis of the fruiting bodies of *Porphyra yezoensis*.

[0125] The fatty acid composition and content of *Porphyromonas Dickensii* were analyzed by gas chromatography, and the results are shown in Table 4.

[0126] Table 4. Analysis of fatty acid composition in the fruiting bodies of *Porphyra yezoensis*.

[0127]

[0128]

[0129] Table 4 shows that only eight fatty acid components were detected in *Porphyromonas Dickensii*, namely C15:0 (pentadecanoic acid, 0.01%), C16:0 (palmitic acid, 0.24%), C17:0 (heptadecanoic acid, 0.01%), C18:0 (stearic acid, 0.38%), C18:1n9c (oleic acid, 0.36%), C18:2n6c (linoleic acid, 1.09%), C18:3n3 (linolenic acid, 0.01%), and C24:0 (lignotaric acid, 0.0089%), which together accounted for 2.1089% of the dry weight of *Porphyromonas Dickensii*. The composition of saturated fatty acids (including C15:0, C16:0, C17:0, C18:0, and C24:0) was 0.6489%, while unsaturated fatty acids (including C18:1n9c, C18:2n6c, and C18:3n3) accounted for 1.46%. These results indicate that the main fatty acid type in *Pheromus d'Antinomyces* is unsaturated fatty acids, with C18:2n6c being the most abundant. It has been reported that unsaturated fatty acids generally possess strong antibacterial effects, primarily by influencing the conformational changes of mycelial membrane proteins, leading to cytoplasmic disintegration. Studies have shown that C18:2n6c can affect the occurrence and development of cardiovascular and cerebrovascular diseases by influencing the expression of cholesterol in peripheral blood. Therefore, the results of this embodiment confirm that *Pheromus d'Antinomyces* contains a variety of fatty acids, providing insights for the development of *Pheromus d'Antinomyces* and its functional components.

[0130] 4. Mineral and heavy metal composition analysis of the fruiting bodies of *Porphyra yezoensis*.

[0131] Minerals cannot be produced by the body itself and often need to be obtained from external sources to maintain the body's required nutrition and energy. The mineral composition of *Pheromus d'Antinomyces* is shown in Table 5.

[0132] Table 5. Mineral and heavy metal composition analysis of the fruiting bodies of *Fomitopsis pinnatifida*.

[0133]

[0134] As shown in Table 5, the most abundant mineral element is potassium (2.24 × 10⁻⁶). 4 The content of iron (mg / kg) helps maintain the osmotic pressure and electrolyte balance of cells in organisms. Other mineral elements, in descending order of content, are: iron (132 mg / kg) > calcium (340 mg / kg) > sodium (59.2 mg / kg) > zinc (55.6 mg / kg) > copper (21.9 mg / kg) > manganese (9.84 mg / kg) > selenium (0.0639 mg / kg).

[0135] Example 3: Effects of *Porphyromonas Dickensii* on mice with inflammation-associated colorectal cancer

[0136] I. Experimental Methods

[0137] 1. Animal model and dosing regimen design

[0138] C57BL / 6J mice were randomly divided into the Ctrl group, the Model group, the 100 mg / mL *Pheromone mongolicum* administration group, and the 300 mg / mL *Pheromone mongolicum* administration group. All mice were kept in an SPF environment for one week to acclimatize.

[0139] Ctrl group: 15 C57BL / 6J mice, fed with standard diet;

[0140] Model group: 15 C57BL / 6J mice, fed with normal diet and gavage with physiological saline;

[0141] 100 mg / mL F. Dickensbond: 15 C57BL / 6J mice were fed with normal diet and administered F. Dickensbond fruiting body powder solution (BD) by gavage (100 mg / Kg);

[0142] 300 mg / mL F. Dickensbond: 15 C57BL / 6J mice were fed with normal feed and administered F. Dickensbond fruiting body powder solution by gavage (300 mg / kg).

[0143] On day 1, the Model group, the 100 mg / mL *Pheromone mica* administration group, and the 300 mg / mL *Pheromone mica* administration group were administered azomethane (AOM) via intraperitoneal injection, followed by normal feeding with saline. Mouse weight and activity levels were observed and recorded daily.

[0144] Starting in the second week, all groups except the Ctrl group had their daily drinking water replaced with a 2% dextran sulfate (DSS) solution for one week. In the third week, the 2% DSS solution was replaced with normal drinking water for two weeks. This cycle lasted three weeks. From the fifth week, starting in the second cycle, mice were administered medication daily via gavage. The Ctrl and Model groups were administered physiological saline, while the 100 mg / mL and 300 mg / mL *Pheromone mongolicum* groups were administered the corresponding dose of *Pheromone mongolicum* fruiting body powder solution via gavage. Mouse weight and activity were observed and recorded daily. AOM / DSS modeling and administration methods are as follows: Figure 3 .

[0145] 2. Specimen Collection

[0146] During the experimental feeding of mice, it is necessary to observe and record the weight, behavior, bloody stool, food intake, and mortality of each mouse.

[0147] Blood samples: After fasting for 12 hours (with free access to water), blood was collected from the heart of each group of animals, with a minimum of 0.8 mL of blood collected. After the blood samples were allowed to stand for 30 minutes, the upper serum layer was separated by centrifugation twice at 3000 r / min, 10 min each time. The serum samples were then stored at -80℃ for later use.

[0148] Tissue sample preparation: All mouse tissues and organs were divided into two groups. One group was cryopreserved, and the other group was fixed in 4% formaldehyde fixative. The contents of each organ, including the colon, rectum, heart, liver, kidneys, spleen, thymus, and intestines, were collected. Each organ was rinsed with physiological phosphate-buffered saline (PBS). After rinsing the colon and rectum thoroughly, photographs were taken, and the tissues and organs were dried and their weights recorded. The overall weight and the weight of major organs relative to the individual's body weight (organ coefficient, %) were then calculated using the following formula:

[0149] Organ Index % = Organ Mass (g) / Mouse Body Weight (g) × 100

[0150] 3. Pathological experimental analysis

[0151] 3.1 Observation of colorectal morphology

[0152] The collected colorectal specimens were rinsed clean, arranged neatly, and photographed. The effects of *Porphyromonas Dickensii* on CAC (colorectal cancer) mice were preliminarily assessed by observing colorectal length, tumor number, weight, and calculating the weight-to-length ratio.

[0153] 3.2 H&E staining

[0154] H&E is an acidic dye used to visualize cell nuclei, cytoplasm, and certain organelles, and is a fundamental method for histological and pathological examination. After sampling, the colon, rectum, and various organs are immersed in a neutral formalin solution to denature and coagulate proteins, preserving the original tissue morphology and structure. The tissue is then washed with PBS, followed by dehydration treatment with 75% ethanol, 80% ethanol, 95% ethanol, and anhydrous ethanol for 5 minutes each, and then cleared twice with xylene for 5 minutes each time. After this, the tissue is embedded in paraffin using a tissue embedding machine. It is then cut into 4-6 μm thick sections. The sections are flattened with hot water, mounted on glass slides, and dried at 45°C.

[0155] Paraffin sections were dewaxed to anhydrous, stained with hematoxylin for nuclear staining, differentiated, and then stained with eosin for cytoplasmic staining. After dehydration, the sections were allowed to air dry slightly before mounting. Subsequent microscopic observation and image acquisition and analysis were then performed.

[0156] 3.3 Gut microbiota analysis

[0157] DNA was extracted from collected intestinal contents. DNA purity was analyzed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and the quality of the extracted DNA was analyzed using 1.2% agarose gel electrophoresis. Polymerase chain reaction (PCR) amplification was performed on the V3-V4 region of the bacterial 16S rRNA gene. The amplification products were quantitatively analyzed, mixed according to sample requirements, and paired-end sequencing was performed on the cecal contents DNA. An OTU abundance table was obtained for subsequent analysis. Based on the OTU sequences, alpha diversity, and beta diversity indices of different samples, the diversity of the species within and between habitats was analyzed.

[0158] 3.4 Serum non-targeted metabolomics detection and analysis

[0159] After thawing, serum was mixed with pre-cooled methanol / acetonitrile / water solution, allowed to stand, centrifuged, and the supernatant was collected. Acetonitrile-water solution was added, vortexed, and centrifuged again; the supernatant was the extracted sample. The sample was separated using ultra-high performance liquid chromatography (UHPLC), and primary and secondary spectra were acquired using mass spectrometry (MS). Secondary mass spectrometry was performed in high-sensitivity information-dependent acquisition (IDA) mode. The collected raw data were format-converted and extracted using XCMS. After metabolite identification and preprocessing, the extracted data underwent quality assessment and analysis, followed by final data analysis.

[0160] 3.5 Cytokine Detection

[0161] The levels of PGE2, COX2, and IL-6 proteins in the colorectal region were detected according to the instructions of the ELISA kit.

[0162] II. Experimental Results

[0163] 1. Effects of *Porphyromonas Dickensii* on intestinal status in CAC mice

[0164] After the AOM / DSS-induced CAC model in mice and 42 days of continuous oral administration of *Porphyromonas Dickensii*, the colon and rectum of each group of mice were collected by euthanasia and dissection. Figure 4 As shown, the AOM / DSS-induced CAC model in mice showed a significant reduction in colon and rectum length. After fecal cleansing of all mice colon and rectum with saline, the intestines of mice in the Ctrl group were smooth with no obvious lesions, while mice in the Model group and the Diggins porphyria administration group showed different numbers and sizes of solid tumors, mostly occurring in the middle of the colon and rectum to the anus. The red arrows indicate the location of tumor occurrence in mice, indicating that the CAC model was successfully established.

[0165] Mice treated with *Porphyromonas Dickensii* showed significant changes in their intestinal condition, such as... Figure 5 As shown, treatment with *Pheromone d'Antiori* significantly reduced the number of tumors and increased the length of the colon and rectum in CAC mice. Furthermore, both the colon-rectal coefficient (colon-rectal weight / colon-rectal length) and the colon-rectal index (colon-rectal weight / mouse body weight) reflected the severity of CAC in mice. In the AOM / DSS-induced CAC mouse model, both the colon-rectal index and colon-rectal coefficient were significantly increased compared to the Ctrl group, while *Pheromone d'Antiori* significantly reduced both the colon-rectal coefficient and colon-rectal index, indicating that *Pheromone d'Antiori* can significantly reduce the severity of CAC in mice.

[0166] 2. The effects of *Porphyromonas Dickensii* on the pathology of the colorectal region.

[0167] In the development of the AOM / DSS-induced CAC model in mice, inflammatory infiltration plays a crucial role. Hematoxylin and eosin (H&E) staining allows for precise and detailed observation of the morphological structure of mouse colorectal tissue, revealing pathological features at the stained sites, such as... Figure 6 As shown in the figure, the cross-section of the colon and rectum of the Ctrl group mice shows that the structure of each layer of the tissue is relatively easy to distinguish and observe. The cells are arranged neatly and tightly, the tissue is relatively intact, the intestinal glands are normal in morphology, and there are no lesions or infiltration of inflammatory cells in the intestinal mucosal epithelium. In the colorectal tissue of the Model group mice, a large area of ​​cancerous tissue can be observed, and the cells are arranged in a disordered manner with a reduction in goblet cells. After administration of *Porphyromonas Dickensburgii*, the area of ​​intestinal cancerous tumor tissue decreased and the number of goblet cells increased significantly, indicating that *Porphyromonas Dickensburgii* can inhibit the development of CAC.

[0168] 3. Effects of *Porphyromonas Dickensii* on gut microbiota in CAC model mice

[0169] Gut microbiota imbalance can contribute to the development of various local and systemic diseases, including gastrointestinal cancers. The gut microbiota may directly or indirectly influence cancer through the secretion of metabolites, tissue invasion, and modulation of the host immune response. A Venn diagram was used to represent the number of fecal microbiome-assessable ASVs in the Ctrl, Model, and *Pheromoneus Dickensii* administration groups. Of the 4830 ASVs detected across all groups, 387 (8.01%) were common in each group. The number of specific ASVs was 1870 (38.72%) in the Ctrl group, 1234 (25.55%) in the Model group, and 997 (20.64%) in the *Pheromoneus Dickensii* administration group, indicating significant differences in microbiome composition among the groups. Figure 7 (A). α-diversity indicates that the combined index of species richness, diversity, and evenness does not vary significantly in locally homogeneous habitats. Figure 7(B). The β diversity index focuses on comparing the diversity of gut microbiota across different habitats. Principal coordinate analysis (PCoA) showed that the confidence intervals of the Ctrl group and the Model group were completely separated ( Figure 8 Treatment with *Pheromone d. Dickensburgii* significantly altered the gut microbiota structure influenced by AOM / DSS. The results showed that AOM / DSS treatment significantly affected the composition and distribution of the mouse gut microbiota, while *Pheromone d. Dickensburgii* treatment, to some extent, modified the effects of AOM / DSS treatment on the gut microbiota. Linear discriminant analysis (LDA) was used to identify differentially expressed microbiota among the Ctrl, Model, and BD groups. When the LDA threshold was set to 2.0, a total of 39 biological groups were significantly enriched across the three groups. Figure 9 Treatment with *Porphyromonas dichotoma* increased the levels of several major probiotics, including *Prevotella*, *Bacteroides*, *Parabacteroides*, and *Butyricimonas*, as well as *AKKermansia*, *Coprococcus*, *Lactobacillus*, and *Desulfovibrio*. Figure 10 ).

[0170] 4. Effects of *Porphyromonas Dickensii* on serum metabolites in CAC model mice

[0171] *Pheromone d'Anchionemae* affects both metabolites and metabolic pathways in a mouse CAC model. Changes in cellular metabolism are a common feature of tumors; tumor development and progression disrupt various metabolic processes, including amino acid metabolism and lipid metabolism. Through metabolomics analysis of the Model group, Ctrl group, and *Pheromone d'Anchionemae* administration group, this invention searches for differentially expressed metabolites and maps them to different metabolic pathways. Using orthogonal partial least squares discriminant analysis (OPLS-DA), significant differences in metabolite levels were found among the three groups. Figure 11 This indicates that AOM / DSS induction and administration of *Porphyromonas Dickensii* can alter the levels of metabolites in mouse serum. Venn diagram results show that there were 40 significantly different metabolites in the serum of mice in the Ctrl group and the Model group, and 7 significantly different metabolites in the serum of mice in the Model group and the *Porphyromonas Dickensii* administration group. Figure 12 By summarizing and statistically analyzing the differentially expressed metabolites in the three groups, and using row-normalized data to create a heatmap, the levels of these differentially expressed metabolites in the serum of the three groups of mice were displayed. Figure 13As shown, 37 differentially expressed metabolites were significantly altered, including arachidonic acid, linoleic acid, and vendolene, which were significantly increased after AOM / DSS treatment, while *Pheromone d. Dickensburgii* administration reduced the levels of these metabolites. Metabolites including L-asparagine, 5-aminovaleric acid, L-tryptophan, and acetylcarnitine were significantly decreased in the serum of Model group mice, and these decreases were reversed after oral administration of *Pheromone d. Dickensburgii*. Among the significantly altered metabolites, we noted that arachidonic acid and linoleic acid both decreased after *Pheromone d. Dickensburgii* administration. Arachidonic acid is a downstream product of linoleic acid; correlation analysis revealed a significant positive correlation between arachidonic acid and linoleic acid, and a negative correlation with L-asparagine. Figure 14 Furthermore, previous studies have shown that arachidonic acid can promote the development and progression of colorectal cancer. The administration of *Pheromoneus Dickensii* to treat CAC significantly affects various gut microbiota and metabolites, suggesting potential regulatory interactions. Correlation analysis can help measure the degree of metabolomics-microbiota interaction between significantly different metabolites and different gut microbiota. A heatmap of metabolomics-microbiota interactions was established, such as... Figure 15 As shown in the figure. Notably, arachidonic acid is negatively correlated with butyric acid carboxylates and positively correlated with turicibacter. This supports the claim that *Porphyromonas Dickensii* administration can achieve its anti-CAC effect by regulating gut microbiota metabolism.

[0172] 5. Regulation of cytokines and levels in CAC model mice by *Porphyromonas d'Antiquity*

[0173] Studies have confirmed that PGE2 is the main product of arachidonic acid, COX2 is the key synthase of PGE2, and IL-6 is a downstream protein of PGE2. Therefore, this invention determined the content of these three proteins in intestinal tissue, and the results are as follows: Figure 16 As shown, PGE2, COX2, and IL-6 were all elevated in the model group, but significantly decreased after administration of *Porphyromonas Dickensii*. This confirms that *Porphyromonas Dickensii* can inhibit the production of PGE2 and suppress the promoting effect of PGE2 on colorectal cancer, thereby alleviating the occurrence and development of colorectal cancer.

[0174] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Conophytum buergerianum ( Bondarzewia dickinsii The application of fruiting bodies in the preparation of drugs for treating colorectal cancer.

2. The application according to claim 1, characterized in that, The colorectal cancer mentioned is inflammation-associated colorectal cancer.

3. The application according to claim 1, characterized in that, The *Porphyromonas Dickensii* strain achieves therapeutic effects on colorectal cancer by regulating the metabolism of the gut microbiota.

4. The application according to claim 3, characterized in that, The regulation of gut microbiota metabolism includes altering gut microbiota structure and regulating gut microbiota metabolites.

5. The application according to claim 1, characterized in that, The *Porphyromonas Dickensii* strain described above alleviates the occurrence and development of colorectal cancer by inhibiting the production of PGE2.

6. The application according to claim 1, characterized in that, The drug includes pharmaceutically acceptable carrier substances.

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