Traditional Chinese medicine composition for treating colorectal cancer as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating colorectal cancer and a preparation method and application thereof. The traditional Chinese medicine composition comprises 20-30 parts of astragalus membranaceus, 8-12 parts of codonopsis pilosula, 10-20 parts of bighead atractylodes rhizome, 10-20 parts of poria cocos, 8-12 parts of liquorice, 20-30 parts of oldenlandia diffusa, 20-30 parts of sculellaria barbata and 10-15 parts of moutan bark. The traditional Chinese medicine composition provided by the invention can effectively inhibit cancer proliferation and enhance tumor cell apoptosis by down-regulating a PI3K / AKT signal pathway. A valuable insight is provided for the effect of the traditional Chinese medicine composition serving as a promising candidate medicine for treating colorectal cancer, and an important thought is provided for treating colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating colorectal cancer, and a preparation method and application thereof. Background Art

[0002] Colorectal cancer (CRC), a highly common malignancy worldwide, presents a dire situation. Global cancer statistics show that its incidence ranks third among all cancers and it is the second leading cause of cancer-related deaths. This situation urgently calls for more effective treatment strategies. Currently, clinical treatments for CRC primarily include surgical resection, chemotherapy, and targeted therapies. However, these approaches face numerous challenges in practical application. A significant number of patients experience recurrence or metastasis after treatment, and the side effects of conventional chemotherapy severely impact patients' daily lives, leading to a significant decline in their quality of life. Furthermore, CRC is highly heterogeneous, and different patients respond differently to existing treatments. Not all patients benefit from these therapies, increasing the uncertainty of treatment outcomes.

[0003] In recent years, numerous clinical studies and practices have been devoted to exploring the effectiveness of traditional Chinese medicine (TCM) combinations in the treatment of CRC. Some studies have shown that these combinations have a modest effect in improving patient symptoms, enhancing quality of life, and prolonging survival. Furthermore, TCM is generally considered to have relatively minimal side effects. However, certain ingredients in these combinations may pose potential safety concerns, such as liver and kidney damage and allergic reactions.

[0004] In view of this, it is necessary to propose a scientific, rigorous and comprehensive set of Chinese medicine compositions for the treatment of CRC, and to conduct in-depth research on the mechanism of action of the Chinese medicine compositions, clarify their effective ingredients and targets, so as to ensure the safe and effective application of the Chinese medicine compositions in the treatment of CRC and bring more treatment options and hope to CRC patients. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a traditional Chinese medicine composition for treating colorectal cancer, and its preparation method and application, which has solved the safety problems of traditional Chinese medicine compositions in the prior art for treating colorectal cancer, such as potential liver and kidney damage.

[0006] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions.

[0007] The present invention provides a traditional Chinese medicine composition for treating colorectal cancer. The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 20-30 parts of astragalus, 8-12 parts of codonopsis, 10-20 parts of atractylodes, 10-20 parts of poria, 8-12 parts of liquorice, 20-30 parts of oldenlandia diffusa, 20-30 parts of scutellaria barbata, and 10-15 parts of moutan bark.

[0008] In some embodiments of the present invention, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 25-30 parts of Astragalus, 9-10 parts of Codonopsis, 12-16 parts of Atractylodes, 12-16 parts of Poria, 8-10 parts of Licorice, 25-30 parts of Hedyotis diffusa, 25-30 parts of Scutellaria barbata, and 12-15 parts of Paeonia suffruticosa.

[0009] The invention also discloses a preparation method of the traditional Chinese medicine composition, comprising: weighing raw materials astragalus, codonopsis, atractylodes, poria, liquorice, oldenlandia diffusa, scutellaria barbata and moutan bark according to a proportion and extracting with water to obtain the composition.

[0010] In some embodiments of the present invention, the amount of water is 5 to 8 times the amount of the raw material drug.

[0011] In some embodiments of the present invention, the extraction method is selected from any one of a decoction method, a water heating reflux method, or an ultrasonic extraction method.

[0012] In some embodiments of the present invention, the decoction method includes: weighing the raw materials astragalus, codonopsis, atractylodes, poria, licorice, oldenlandia diffusa, scutellaria bark, and moutan bark according to the ratio, adding them to water, boiling over high heat for 40 to 80 minutes, filtering, and collecting the filtrate to obtain the traditional Chinese medicine composition.

[0013] In some embodiments of the present invention, the decoction method includes: boiling over high heat for a first time for 40 to 80 minutes, filtering, collecting the filtrate and filter residue separately, then adding the filter residue to water, boiling over high heat for a second time, after 40 to 80 minutes, collecting the filtrate, and combining the two filtrates to obtain the traditional Chinese medicine composition.

[0014] In some embodiments of the present invention, the decoction method includes: weighing the raw materials astragalus, codonopsis, atractylodes, poria, licorice, oldenlandia diffusa, scutellaria bark, and peony bark according to the ratio, adding them to 1L of water and boiling them twice over high heat, collecting the filtrates twice to obtain the traditional Chinese medicine composition.

[0015] The present invention also discloses a medicine for treating colorectal cancer, wherein the medicine uses the traditional Chinese medicine composition as an active ingredient.

[0016] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0017] In some embodiments of the present invention, the dosage form of the drug is a decoction, granules, pills, capsules, tablets, powders or oral liquids.

[0018] In some embodiments of the present invention, the method for preparing the granules comprises: preparing the Chinese medicine composition provided by the present invention by a decoction method, then precooling at -80°C for 4 hours, and then freeze-drying to obtain the granules.

[0019] In some embodiments of the present invention, the freeze-drying parameters are: ambient temperature: 20°C, vacuum: 0.01 mbar, freezing temperature: -20°C, and freeze-drying time: 72 hours.

[0020] The invention also discloses the use of the traditional Chinese medicine composition in preparing medicine for treating colorectal cancer.

[0021] The Chinese medicine composition of the present invention is composed of eight Chinese herbs: Astragalus, Codonopsis, Atractylodes, Poria, Licorice, Hedyotis diffusa, Scutellaria barbata, and Paeonia suffruticosa. Raw Astragalus and Codonopsis are used as the main herbs to tonify Qi; Scutellaria barbata and Hedyotis diffusa dissipate stasis and toxins as the auxiliary herbs; Atractylodes, Poria, and Licorice, inspired by the Four Gentlemen Decoction, invigorate Qi and strengthen the spleen; Paeonia suffruticosa promotes blood circulation and dissipates stasis, while Atractylodes and Licorice relieve pain and relieve pain, while Hedyotis diffusa also detoxifies and eliminates dampness, serving as adjuvants. Finally, Licorice harmonizes the herbs and serves as the guiding herb. The combination of these herbs works to tonify Qi and nourish Yin, strengthen the spleen and kidney, and activate blood circulation, dispel stasis, and detoxify, achieving the goal of strengthening the body and inhibiting tumors.

[0022] Based on the technical solution of the present invention, compared with the prior art, the present invention has the following beneficial effects:

[0023] The Traditional Chinese Medicine composition (FZYL) provided by this invention for the treatment of colorectal cancer effectively inhibits tumor proliferation and enhances tumor apoptosis by downregulating the PI3K / AKT signaling pathway. Comprehensive analyses, including tumor measurement, biochemical testing, molecular docking, and protein expression assessment, confirmed the therapeutic mechanism of FZYL. This invention provides valuable insights into the role of FZYL as a promising drug candidate for colorectal cancer treatment, highlighting its potential to mitigate disease progression while elucidating the underlying biological mechanisms. Overall, FZYL represents a significant advance in colorectal cancer treatment approaches.

[0024] It has been verified that the traditional Chinese medicine composition for treating colorectal cancer provided by the present invention inhibits tumor growth and colon inflammation in colorectal cancer mice, and can reduce organ damage and liver metastasis in colorectal cancer mice.

[0025] Through research, it was found that the Chinese medicine composition provided by the present invention can reduce the number of bone metastases, prolong survival, median survival and tumor progression time, and improve the quality of life in the treatment of prostate cancer. The preliminary study of the present invention showed that the modified Fuzheng Yiliu Decoction can effectively relieve diarrhea, vomiting, hand-foot syndrome and pain caused by chemotherapy after intestinal cancer surgery, improve the completion rate of chemotherapy, and improve the short-term and long-term quality of life and disease-free survival rate. At the same time, the preliminary cell experiment also found that the serum containing Fuzheng Yiliu Decoction has an inhibitory effect on the proliferation of colon cancer HT-29 cell line (P<0.05), suggesting that its mechanism may be related to downregulating Bcl-2 and upregulating the expression of p53, thereby inhibiting cell proliferation. In the Chinese medicine composition provided by the present invention, the number of medicinal materials is streamlined, all herbal medicines are selected, the price is low, and mass spectrometry detection has been carried out, and the ingredients are clear. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure 2 is the measurement result of tumor volume of mice in different treatment groups; Figure 1 A in the figure represents the phenotype diagram of tumor volume measurement results in colorectal tissues of mice in different treatment groups; Figure 1 B in the figure shows the statistical results of tumor volume measurement in colorectal tissues of mice in different treatment groups.

[0027] Figure 2 Figure 2 is a graph showing the colorectal measurement results and tumor number statistics in the colorectal tissues of mice in different treatment groups; Figure 2 A in the figure represents the colorectal measurement results in the colorectal tissues of mice in different treatment groups; Figure 2 Table B shows the statistical results of the number of tumors in mice in different treatment groups.

[0028] Figure 3 The results of H&E staining of tumors and colons of mice in different treatment groups are shown in Figure 2. Figure 3 A in the figure shows the staining results of tumors in mice in different treatment groups; Figure 3 B in FIG. 1 is a diagram showing the staining results of the colon of mice in different treatment groups. FIG.

[0029] Figure 4 The figure is a line graph showing the changes in body weight of mice in different treatment groups on different treatment days.

[0030] Figure 5 Statistical results of organ indexes in different treatment groups at different hours; Figure 5 A in the figure represents the cardiac index statistical result graph; Figure 5 B in the figure represents the statistical result of liver index; Figure 5 C in the figure represents the statistical result of spleen index; Figure 5 The D in the figure indicates that the lung is just a statistical result diagram; Figure 5 E in the figure represents the statistical results of the kidney index.

[0031] Figure 6 The staining results of liver and kidney in different treatment groups are shown in Figure 2. Figure 6 A in the figure represents the staining result of the liver; Figure 6 B in the figure shows the staining result of the kidney.

[0032] Figure 7 Figure 2 is the result of serum biochemical analysis of mice in different treatment groups; Figure 7 A in the figure represents the statistical result graph of ALT concentration in serum; Figure 7 B in the figure represents the statistical result of AST concentration in serum; Figure 7 C in the figure represents the statistical result of CK concentration in serum; Figure 7D in the figure represents the statistical result graph of CREA concentration in serum; Figure 7 E in the figure represents the statistical result graph of UREA concentration in serum.

[0033] Figure 8 This is a graph showing the chemical analysis results of the Chinese medicine composition for treating colorectal cancer provided by the present invention; wherein, Figure 8 A in the figure represents the identification result diagram under positive ion mode; Figure 8 B in the figure represents the identification result diagram under negative ion mode.

[0034] Figure 9 This is a target analysis result diagram of the traditional Chinese medicine composition for treating colorectal cancer provided by the present invention through network pharmacology; wherein, Figure 9 A in the figure represents the Venn diagram of FZYL and cancer cross-targets; Figure 9 B in the figure represents the PPI (protein-protein interaction) network diagram.

[0035] Figure 10 This is a molecular structure diagram of the key active compound of the traditional Chinese medicine composition for treating colorectal cancer provided by the present invention.

[0036] Figure 11 This is a KEGG bubble diagram of the traditional Chinese medicine composition for treating colorectal cancer provided by the present invention for treating colorectal cancer through network pharmacology.

[0037] Figure 12 The statistical results of the expression levels of tumor proliferation-related proteins and immunohistochemical staining results of FZYL in cancer mice are shown in Figure 2. Figure 12 A represents the results of immunohistochemical staining of tumor sections in mice in different treatment groups; Figure 12 B represents the statistical results of the expression levels of Ki67 protein in mice in different treatment groups; Figure 12 C represents the statistical results of the expression levels of PCNA protein in mice under different treatment groups.

[0038] Figure 13 The results of immunohistochemical staining of tumor apoptosis-related proteins and expression levels of apoptosis-related genes in mice with cancer inhibited by FZYL are shown in Figure 2. Figure 13 A in the figure shows the immunohistochemical staining results of apoptosis-related proteins in mice of different treatment groups; Figure 13 B in the figure shows the statistical results of Caspasse3 protein expression levels in mice under different treatment groups; Figure 13 C in the figure represents the statistical results of Bcl-2 protein expression levels in different treatment groups; Figure 13 D in the figure shows the statistical results of Bax protein expression levels in different treatment groups.

[0039] Figure 14 This is the CCK-8 assay result of the effect of FZYL on the proliferation activity of SW620 cells.

[0040] Figure 15 To observe the effect of different concentrations of FZYL on the proliferation activity of SW620 cells, the colony formation test bar graph is used as the statistical result. Figure 15 A in the figure represents the statistical results of RT-qPCR results showing the effects of FZYL on SW620 cell proliferation and related gene expression levels; Figure 15 B in the figure represents the statistical results of Ki67 expression level; Figure 15 C in the figure represents the statistical results of the PCNA expression level.

[0041] Figure 16 Flow cytometric analysis showed the effects of different concentrations of FZYL on the apoptosis of SW620 cells.

[0042] Figure 17 This is the statistical result of relative apoptosis level of SW620 cells in different treatment groups.

[0043] Figure 18 The results of the effect of FZYL on the protein levels of apoptosis-related markers in SW620 cells are shown in Figure 2. Figure 18 A in the figure represents the electrophoresis result of FZYL on the protein levels of apoptosis-related markers in SW620 cells; Figure 18 B in the figure represents the bar graph of Caspasse3 / β-actin protein levels; Figure 18 C in the figure represents the bar graph of statistical results of Bcl-2 / Bax protein levels.

[0044] Figure 19 This is the docking model diagram of the first 7 compounds and PI3K.

[0045] Figure 20 The results of the effect of FZYL on the protein level of PI3K / AKT pathway are shown in FIG. Figure 20 A in the figure represents the electrophoresis results of Western blot analysis showing the protein levels of the PI3K / AKT pathway; Figure 20 B in the figure represents the bar graph of statistical results of protein levels of p-PI3K / PI3K; Figure 20 C in the figure represents the bar graph of statistical results of p-AKT / AKT protein levels. DETAILED DESCRIPTION

[0046] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0047] The following describes the details in conjunction with specific embodiments.

[0048] 1. Experimental Materials

[0049] The Chinese medicinal raw materials used in the present invention, astragalus, codonopsis, atractylodes, tuckahoe, liquorice, oldenlandia diffusa, scutellaria barbata, and moutan bark were all purchased from Beijing Tong Ren Tang.

[0050] Example 1: A Chinese medicine composition for treating colorectal cancer and its preparation method

[0051] A traditional Chinese medicine composition for treating colorectal cancer comprises the following raw materials in parts by weight: 30g of astragalus, 10g of codonopsis pilosula, 15g of atractylodes macrocephala, 15g of poria, 10g of liquorice, 30g of oldenlandia diffusa, 30g of scutellaria barbata, and 15g of moutan bark.

[0052] A method for preparing a traditional Chinese medicine composition for treating colorectal cancer: the method adopts a decoction method.

[0053] The above-mentioned raw materials were weighed by weight, boiled in 1L of distilled water over high heat for 1 hour, and then filtered, retaining the filter residue and filtrate; the filter residue was added to 1L of distilled water and boiled again for 1 hour, and then filtered, and the two filtrates were combined to obtain the traditional Chinese medicine composition, which was named Fuzheng Yiliu Decoction.

[0054] Example 2: A Chinese medicine composition for treating colorectal cancer and its preparation method

[0055] A traditional Chinese medicine composition for treating colorectal cancer comprises the following raw materials in parts by weight: 20g of astragalus, 8g of codonopsis, 10g of atractylodes, 10g of poria, 8g of liquorice, 20g of oldenlandia diffusa, 20g of scutellaria barbata, and 10g of moutan bark.

[0056] A method for preparing a traditional Chinese medicine composition for treating colorectal cancer: the method adopts a decoction method.

[0057] The above-mentioned raw materials were weighed by weight, boiled in 1L of distilled water over high heat for 1 hour, and then filtered, retaining the filter residue and filtrate; the filter residue was added to 1L of distilled water and boiled again for 1 hour, and then filtered, and the two filtrates were combined to obtain the traditional Chinese medicine composition, which was named Fuzheng Yiliu Decoction.

[0058] Example 3: A Chinese medicine composition for treating colorectal cancer and its preparation method

[0059] A traditional Chinese medicine composition for treating colorectal cancer comprises the following raw materials in parts by weight: 25g of astragalus, 9g of codonopsis pilosula, 12g of atractylodes macrocephala, 12g of poria, 9g of liquorice, 25g of oldenlandia diffusa, 25g of scutellaria barbata, and 12g of moutan bark.

[0060] A method for preparing a traditional Chinese medicine composition for treating colorectal cancer: the method adopts a decoction method.

[0061] The above-mentioned raw materials were weighed by weight, boiled in 1L of distilled water over high heat for 1 hour, and then filtered, retaining the filter residue and filtrate; the filter residue was added to 1L of distilled water and boiled again for 1 hour, and then filtered, and the two filtrates were combined to obtain the traditional Chinese medicine composition, which was named Fuzheng Yiliu Decoction.

[0062] Example 4: A Chinese medicine composition granule preparation for treating colorectal cancer

[0063] Any of the Chinese medicine compositions provided in Examples 1 to 4 can be prepared into granule preparations. In this example, the Chinese medicine composition prepared in Example 1 is used as an example for illustration.

[0064] Preparation of a granular Chinese herbal composition for treating colorectal cancer: The Chinese herbal composition for treating colorectal cancer provided in Example 1 was pre-frozen at -80°C for 4 hours and then freeze-dried to obtain a powder, which is the granular Chinese herbal composition of this example. The final yield was 28 g of FZYL granules.

[0065] The freeze-drying parameters were as follows: ambient temperature: 20°C, vacuum: 0.01 mbar, freezing temperature: -20°C, and freeze-drying time: 72 hours.

[0066] Verification of the efficacy of a traditional Chinese medicine composition for treating colorectal cancer

[0067] Since the Chinese medicine composition provided in Example 1 of the present invention was used, a granular preparation was prepared according to the method shown in Example 4 for efficacy verification. Specifically, in order to study the therapeutic effect of the Chinese medicine composition provided by the present invention on colorectal cancer, a mouse colorectal tumor model was established and treated with the Chinese medicine composition, followed by efficacy evaluation and pharmacological analysis.

[0068] 1. Experimental Animals

[0069] Twenty 4- to 5-week-old BALB / c nu mice (SCXK[Yue]218-0051, SYXK[Yue]2022-0182) were obtained from Zhuhai Baishitong Co., Ltd. and housed in pairs in plastic cages in a temperature-controlled (25 ± 2°C) colony room under a 12 h / 12 h light / dark cycle with free access to food and water. All experimental protocols were approved by the Animal Center of Guangzhou University of Chinese Medicine.

[0070] 2. Grouping

[0071] All mice were fed distilled water for one week of adaptive feeding. Six mice served as a normal control group without any other intervention (N). The remaining mice were orthotopically inoculated into the cecum to establish a colorectal tumor model and were randomly divided into the model group (M), the low-dose FZYL group (FZYL-L), and the high-dose FZYL group (BZYL-H).

[0072] 3. Establishing a Colorectal Tumor Model

[0073] The method for establishing the colorectal tumor model is as follows:

[0074] ① Take BALB / c nude mice and inoculate SW620 cells in the logarithmic growth phase at 1.5×10 7 The cells were implanted subcutaneously to form subcutaneous tumors. After the tumors grew to a certain size, they were surgically removed and cut into uniform three-dimensional blocks. After being rinsed with PBS three times, they were placed in 1640 culture medium.

[0075] ② In situ modeling of Balb / c nude mice: 5-week-old nude mice were anesthetized with isoflurane gas, and the tumor block was inoculated into the mouse cecum and sutured with absorbable sutures. After closing the abdominal cavity, the abdominal incision was sutured and disinfected. The tumor block was fixed and did not move, indicating that the modeling was successful.

[0076] 4. Administer medication

[0077] Colorectal tumor model mice were randomly divided into a model group (M) and a drug administration group, with 5 mice in each group. The drug administration group was treated with the traditional Chinese medicine composition granules (FZYL) prepared in Example 2 for 21 days.

[0078] Specifically, the dosing groups were divided into a low-dose FZYL group (FZYL-L) and a high-dose FZYL group (FZYL-H). The FZYL-L group was orally administered with FZYL at 20 mg / kg / 0.2 mL / day per mouse; the FZYL-H group was orally administered with FZYL at 40 mg / kg / 0.2 mL / day per mouse; and the M group was orally administered with PBS at 0.2 mL / day per mouse.

[0079] The oral dose obtained by several conventional dose calculation methods is greater than 1g / kg. The final determination of the oral dose depends on the formula (EC50×bioavailability) / M mouse × 8% = IC50. The IC50 of FZYL for SW620 cells is 1 mg / mL. The oral bioavailability in nude mice is 20%. 8% is the percentage of blood to body weight. mouse ) with a body weight of 20 g and an EC50 of 40 mg / kg. Through preliminary experiments, the final dosages of FZYL-L (20 mg / kg / 0.2 mL / day / per mouse) and FZYL-H (40 mg / kg / 0.2 mL / day / per mouse) were determined.

[0080] At the end of the experiment, body and organ weights, tumor size and weight, colon length, and tumor number were measured. Histopathological and immunohistochemical analyses of the tumor, liver, kidney, and colon structures of the nude mice were performed. Blood samples were collected from the mice for serum biochemical analysis.

[0081] 5. Analysis of FZYL Extract

[0082] FZYL extracts were analyzed using a Vanquish UHPLC system (Thermo Scientific) at 35°C using an HSS-T3 column (100 x 2.1 mm, 1.8 μm, Waters). Mobile phases A consisted of H2O + 0.1% formic acid, and mobile phase B consisted of acetonitrile + 0.1% formic acid (LC-MS grade, Fisher Chemical).

[0083] The sample was separated using a gradient at 0.3 mL / min: 1 minute at 5% B, rising to 98% B over 16 minutes, returning to 5% B over 0.5 minutes, and 2.5 minutes at 5% B. A Q-Exactive HFX mass spectrometer (Thermo Fisher Scientific) was interfaced with a UHPLC system. Data were acquired in ESI positive and negative modes over a mass range of m / z 90–1300 using data-dependent acquisition (DDA). The top 10 most intense MS1 ions were selected for MS / MS analysis, with HCD collision energies of 20, 40, and 60 units, respectively. The capillary temperature was 320°C, and the probe heater temperature was 350°C.

[0084] Compound identification was accomplished by comparing accurate mass, isotope distribution, and MS / MS spectra with reference data from an in-house traditional Chinese medicine database (Shanghai Zhongke New Life Biotechnology Co., Ltd.), GNPS, ReSpectr, and Massbank. We commissioned Shanghai Zhongke New Life Biotechnology Co., Ltd. (Shanghai, China) to perform the analysis.

[0085] 6. Online pharmacological analysis of FZYL and CRC.

[0086] (1) Collection and arrangement of FZYL and colorectal cancer post-cancer biological targets

[0087] The chemical constituents of each herbal medicine presented in Example 1 were collected by integrating data from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ) and the HERB database (http: / / herb.ac.cn / ). Potential active ingredients of each herbal medicine were screened using the ADMET Lab 3.0 drug property prediction platform (https: / / admetlab3.scbdd.com / ). Selection criteria were based on Lipinski's five rules and a plasma protein binding rate (PPB) of less than 90%. The composition of FZYL was confirmed by UPLC-HRMS analysis. The Swiss Target Prediction Platform (http: / / www.swisstargetprediction.ch) and the SEA platform (https: / / sea.docking.org / ) were used to predict the targets of the herbal constituents in FZYL. For further network analysis, disease targets associated with colorectal cancer were obtained from the Genecards database (https: / / www.genecards.org / ) (with a correlation score >50) and the OMIM database (https: / / omim.org / ). The intersections of FZYL components and colorectal cancer-related targets were analyzed using Origin 2023 academic software to identify potential targets of FZYL for the treatment of colorectal cancer.

[0088] (2) Venn diagram analysis and target network construction of “cross-target diseases of effective ingredients of traditional Chinese medicine”

[0089] The Venny 2.10 platform was used to analyze drug targets and colorectal cancer targets, generating statistical intersections and forming a Venn diagram. The collected data were compiled and imported into Cytoscape 3.91 software to construct a target network diagram of "TCM active ingredients-cross-targets-diseases".

[0090] (3) Construction of PPI protein interaction network

[0091] Protein-protein interaction (PPI) network analysis of cross-targets was performed using the STRING database. The organism was set to Homo sapiens, and the minimum interaction score was set to 0.700 to generate a PPI network file. Cytoscape 3.9.0 software was used to construct the PPI network diagram and the "FZYL-targeted-colorectal cancer signaling pathway" network diagram. Core targets were identified based on their degree values within the PPI network.

[0092] (4) KEGG analysis

[0093] KEGG enrichment analysis of cross-targets between herbal medicines and diseases was performed using R 3.6.3 software and the MicroBioCloud online mapping platform. KEGG signaling pathways were obtained, and bubble plots of KEGG categories were generated based on the number of enriched genes.

[0094] (5) Molecular docking

[0095] Receptor protein and compound collection and preprocessing: The three-dimensional (3D) structure of the receptor protein was obtained by combining UniProt (https: / / www.uniprot.org / ) and the PDB (https: / / www1.rcsb.org / ) database. The protein structure was then processed using PyMOL version 2.3.0 to remove crystal water, irrelevant protein chains, and native ligands. The 3D structure of the compound was retrieved from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ) and optimized using the MMFF94 force field in OpenBabel 3.1.1 to achieve the lowest energy conformation.

[0096] Molecular Docking: Hydrogen atoms were added to the receptor protein and the compound using the AutoDock tool 1.5.6. Rotatable bonds in the compound were identified, and the structure was saved in pdbqt format. Docking grid parameters were set using the Grid module. The docking protocol was set to semi-flexible docking, and the Lamarckian genetic algorithm was used for docking with an exhaustive value of 25. Molecular docking was performed using AutoDock Vina 1.2.0 to generate binding free energies and docking result files.

[0097] (6) RNA isolation and quantitative analysis (RT-qPCR)

[0098] RNA was extracted from tumor tissue using RNAiso Plus (Vazyme, Product No. R401) according to the instructions. Then, cDNA was obtained using the ImProm II™ Reverse Transcription System (Promega), and RT-qPCR (95°C) 1 minute, 40 cycles of denaturation (95°C 15 seconds) and extension (60°C 1 minute) were performed in a total volume of 20 μL using the SweScript RT I FirstStrand cDNA Synthesis Kit (purchased from Servicebio, Product No. G3330) and 2X Universal Blue SYBR Green qPCR Master Mix (2X Universal Blue SYBR Green qPCR Master Mix, purchased from Servicebio, Product No. G3326). The experiment was performed in triplicate. After amplification, dissociation curve analysis was performed to confirm the amplicon specificity of each PCR run. The relative levels of gene expression in mouse colon tissue were normalized to mouse β-actin. Using 2 ( −ΔΔCT ) method for relative expression analysis. The primer sequences are shown in Table 1

[0099] Table 1 Primer sequences

[0100]

[0101] (7) Western blotting (WB)

[0102] Global colon tissue was dissected from treated mice, and proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat dry milk in Tris-buffered saline (20 mM Tris-HCl, 500 mM NaCl, pH 7.4) containing 0.2% Tween-20, the membranes were probed with antibodies overnight at 4°C and then incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibodies (1:2000) (Servicebio, G2210-2-A). Band intensity was quantified using ImageJ software (NIH).

[0103] Total proteins were washed with stripping buffer and incubated with corresponding phosphorylated proteins.

[0104] (8) Cell culture

[0105] SW620 cells were purchased from Wuhan Punosai Life Science Co., Ltd. Cells were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution (Corning, Cat. No. 30002304). All cells were grown in a humidified incubator at 37°C and 5% CO2.

[0106] (9) CCK-8 assay

[0107] Prepare a suspension of 6,000 SW620 cells per well in a 96-well plate. Allow the cells to attach. Add culture medium containing varying concentrations of FZYL (0 to 4.5 mg / mL) to the corresponding wells at each 0.5 mg / mL increase. The volume per well is 100 μL. Replace the culture medium with fresh cell culture medium and add 10 μL of CCK-8 solution to each well at 24, 48, and 72 hours.

[0108] (10) Clone formation experiment

[0109] Prepare a suspension of 2,000 SW620 cells per well in a 6-well plate. Allow cells to attach. Add culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution) containing 0 mg / mL FZYL, 0.5 mg / mL FZYL, and 1 mg / mL FZYL, 15 μM 740-YP, and 15 μM 740-Y-P plus 2 mg / mL FZYL to the corresponding wells, respectively. The volume per well is 2 mL. Culture for 15 days, replacing the corresponding culture medium with fresh medium every three days. On day 15, observe the size of the cell colonies under a microscope and wash with PBS. Fix with 4% paraformaldehyde for 30 minutes. Rinse three times with PBS. Add 1% crystal violet solution for 20 minutes. Rinse with PBS until cell colonies are clear. Take a photo of each well (wells in the microplate) using a mobile phone.

[0110] (11) Flow cytometry was performed using Annexin V / Propidium Iodide (Annexin V / PI, purchased from Melunbio, catalog number MA0229).

[0111] A suspension of 300,000 SW620 cells was prepared per well in a 6-well plate. After 12 hours, cells were allowed to adhere. Culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution) containing 0 mg / mL FZYL, 0.5 mg / mL FZYL, and 1 mg / mL FZYL, 15 μM 740-YP, and 15 μM 740-Y-P plus 2 mg / mL FZYL were added to the corresponding wells, respectively. The volume per well was 2 mL. After 48 hours, all wells were washed with PBS. 500 μL of trypsin was added to each well. After 2 minutes, 1 mL of culture medium was added to each well. The cells were centrifuged at 2000 rpm for 5 minutes. The culture medium was discarded. For each sample, 100 μL of 1X binding buffer, 5 μL of FITC-annexin V, and 5 μL of PI were added. After 15 minutes, apoptosis was assessed by flow cytometry.

[0112] 5. Experimental results

[0113] (1) FZYL inhibits colorectal tumor growth and intestinal inflammation in nude mice with cecal transplantation

[0114] After a one-week acclimation period, BALB / c nude mice were orthotopically implanted with cecal tumors to establish a colorectal tumor model. The tumors were immobilized, indicating successful modeling. The mice were then fed a diet for another three days, followed by daily gavage with FZYL. Various mouse indicators were measured after 22 days.

[0115] The results are as follows Figure 1 As shown in Figures A and B, FZYL treatment was found to reduce tumor volume and tumor weight in colorectal tissues of nude mice. The FZYL-H group showed statistically significant results. Figure 2 As shown in Figures A and B, FZYL treatment reduced the number of colorectal tumors and partially increased colorectal length, especially in the FZYL-H group. Figure 3 As shown in Figures A and B, the tumors in group M showed a more vascular distribution, uneven cell size, and the presence of giant cells and large nuclei. In contrast, the tumors in groups FZYL-L and FZYL-H showed almost no blood vessels, more uniform nuclei, more normal cell morphology, and fewer giant cells.

[0116] (2) FZYL is safe and can reduce multi-organ damage and liver metastasis in cancer nude mice.

[0117] After one week of adaptation, the body weight of nude mice was recorded. Statistical analysis showed that tumor development led to a decrease in body weight, while both FZYL-L and FZYL-H treatments mitigated this effect, maintaining body weight at normal levels (e.g., Figure 4 shown).

[0118] Subsequently, the organ indexes of nude mice were measured, and the results were as follows: Figure 5 As shown in Figures A to E, the liver index of the FZYL-L and FZYL-H groups was significantly lower than that of the M group. To exclude liver toxicity, H&E staining was performed. Figure 6 As shown in Figure A, the liver of the M group showed light staining, indicating hepatocyte edema and unclear hepatic cords or sinusoidal structures. In addition, cell clumps were observed in the central veins, which are tumor thrombi. In contrast, the liver tissue of the N, FZYL-L, and FZYL-H groups stained more red, with intact and clear cells and hepatic cord structures, and the central veins were filled with red blood cells and some immune cells. At the same time, the kidneys were analyzed, and the results are shown in Figure 4. Figure 6 As shown in Figure B, the kidneys of group M showed lighter staining, some glomeruli were atrophied and had unclear structures, and some renal tubular epithelial cells were arranged in disorder. Compared with group M, the staining of group FZYL-L was redder, the glomeruli were normal in morphology and clear in structure, and some renal tubules showed disordered epithelial cells. Groups FZYL-H and N showed redder staining, and the glomeruli and renal tubules were clear and normal in structure. Finally, the biochemical analysis of the serum of nude mice was performed, and the results were shown in Figure 3. Figure 7 As shown in Figures A to E, compared with group N, ALT, AST, CK, and CREA levels were increased in group M, whereas these levels were decreased in the FZYL-L and FZYL-H groups, with the decrease being more significant in FZYL-H. There were no significant differences in serum urea levels among the four groups.

[0119] (3) Chemical composition of FZYL

[0120] like Figure 8 As shown in Figures A and B, a total of 2,660 compounds (1,659 positive ion mode compounds and 1,099 negative ion mode compounds) were identified in FZYL, and 55 peaks were annotated. The compounds were annotated according to the NPClassifier classification method. The main pathways ranked by proportion included shikimic acid and phenylpropanoids (33%), terpenes (20%), alkaloids (19%), fatty acids (9%), amino acids and peptides (8%), polyketides (6%), and carbohydrates (4%). The most abundant compound classes were flavonoids (290), small peptides (175), and triterpenes (130). The distribution of annotated compounds in the peaks showed that shikimic acid and phenylpropanoids-flavonoids-flavonoids were the main groups.

[0121] (4) Network pharmacology study of the target of FZYL in the treatment of colorectal cancer

[0122] In the FZYL formula, 54 chemical components were identified from Hedyotis diffusa, 127 from Atractylodes macrocephala, 163 from Scutellaria barbata, 114 from Codonopsis pilosula, 185 from Poria cocos, 436 from Glycyrrhiza uralensis, 168 from Astragalus membranaceus, and 59 from Paeonia suffruticosa. UPLC-HRMS results combined with ADMET Lab 3.0 screening identified 26 potential bioactive components from Hedyotis diffusa, 41 from Atractylodes macrocephala, 63 from Scutellaria barbata, 54 from Codonopsis pilosula, 73 from Poria cocos, 77 from Glycyrrhiza uralensis, 68 from Astragalus membranaceus, and 33 from Paeonia suffruticosa. Swiss Target Prediction and the SEA database were used to predict targets for potential bioactive components in FZYL, resulting in 1,260 relevant targets. A total of 881 colorectal cancer-related targets were identified using gene cards and the OMIM database. This intersection generated 196 common targets between traditional Chinese medicine and the disease (e.g. Figure 9 (as shown in A in the figure).

[0123] The PPI protein interaction network and the “FZYL-targeting-colorectal cancer signaling pathway” network were constructed using the STRING platform and Cytoscape software (e.g. Figure 9 Finally, 10 core targets for FZYL in cancer treatment were identified: TP53, EGFR, CTNNB1, SRC, STAT3, AKT1, IL6, HSP90AA1, TNF, and BCL2. The degree, closeness, and betweenness centrality values of these targets are shown in Table 2. The "FZYL-targeted colorectal cancer signaling pathway" network revealed 12 key components for FZYL in colorectal cancer treatment, with their degree, closeness, and betweenness centrality values listed in Table 3. The molecular structures of these components are shown in Table 3. Figure 10 shown.

[0124] Table 2 Target information involved

[0125] Main objectives Degree value Closeness centrality value Betweenness centrality value TP53 96 0.658451 0.132778 EGFR 81 0.625418 0.069347 CTNNB1 79 0.613115 0.060739 STAT3 78 0.605178 0.039291 SRC 78 0.611111 0.057797 AKT1 77 0.607143 0.036176 IL6 68 0.578947 0.031328 HSP90AA1 67 0.582555 0.030563 TNF 66 0.568389 0.038188 BCL2 61 0.570122 0.020992

[0126] Table 3 Key ingredient information

[0127] Key Ingredients PubChem CID Degree value Closeness centrality value Betweenness centrality value GC11 195396 46 0.420026 0.018678 BZL63 122186903 44 0.366213 0.004171 FL60 139583733 41 0.38 0.004888 BHSSC20 / MDP26 12309055 40 0.380448 0.006764 GC17 2724360 39 0.392944 0.010241 HQ3 73067 39 0.373843 0.005533 BZL51 91895318 38 0.359288 0.002802 FL73 156582569 38 0.412516 0.014308 BZL49 46183384 37 0.379108 0.004167 BZL62 122186902 37 0.355727 0.002413 GC18 2724361 37 0.382249 0.009746 HQ10 336327 36 0.371692 0.004687

[0128] (5) Studying the pathway of FZYL in treating colorectal cancer through network pharmacology

[0129] like Figure 11As shown, KEGG analysis identified a total of 182 signaling pathways associated with intersection targets. The top 30 pathways were selected based on -logP values and the number of enriched genes and are presented in a KEGG bubble plot. The results showed that the most core signaling pathway in FZYL cancer treatment is the PI3K / AKT pathway, followed by the MAPK pathway.

[0130] (6) FZYL inhibits the proliferation of tumor cells in nude mice and increases cell apoptosis.

[0131] According to the previous results, the FZYL-H group showed good therapeutic effects. Subsequently, further experiments were conducted using tumor tissue sections from M and FZYL-H nude mice. Figure 12 A~C and Figure 13 As shown in Figures A to D, FZYL treatment was found to reduce tumor Ki67 protein (purchased from Servicebio, Cat. No. GB121141-50) and PCNA protein (purchased from Servicebio, Cat. No. GB12010-50), as well as their gene expression levels. Furthermore, FZYL treatment increased Caspase-3 protein levels in tumors and upregulated the gene expression of Caspase-3 and Bax. However, it downregulated the protein and gene expression levels of Bcl-2.

[0132] (7) FZYL inhibits SW620 cell proliferation and increases cell apoptosis.

[0133] In vitro experiments showed that FZYL effectively inhibited the growth of SW620 colorectal cancer cells, with IC50 values of 4.5 mg / mL, 2.5 mg / mL and 1 mg / mL at 24, 48 and 72 hours, respectively (e.g. Figure 14 Based on these results, we selected 72 hours as the experimental time point and applied a gradient concentration of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL. In addition, we also included the PI3K / AKT pathway activator 740-YP and 740-Y-P + 2 mg / mL FZYL groups for pathway validation. The results showed that FZYL inhibited the growth of SW620 cells, and the efficacy of the drug increased with increasing concentration. The activator 740-YP significantly accelerated the growth of SW620 cells, while FZYL slowed down their growth rate (as shown in Figure 2). Figure 15 In addition, we observed that FZYL accelerated the apoptosis of SW620 cells, and the apoptosis rate increased with increasing drug concentration. 740-YP significantly alleviated the apoptosis of SW620 cells, but FZYL reversed this effect (as shown in Figure 2A). Figure 16 and Figure 17In the present invention, the expression of proliferation-related genes Ki67 and PCNA was also evaluated, and it was found that FZYL reduced their expression levels. The 2 mg / mL FZYL group showed statistically significant changes; however, Ki67 did not show a concentration-dependent gradient, while PCNA showed a gradient change (as shown in Figure 2). Figure 15 Finally, we evaluated several apoptosis-related proteins and found that FZYL increased the protein level of Cleaved-Caspase-3, decreased the level of Bcl-2, and the Bcl-2 / Bax ratio showed an upward trend. However, this change did not reach statistical significance (as shown in Figure 2B and C). Figure 18 (as shown in A to C in the figure).

[0134] (8) FZYL can treat cancer through the PI3K / AKT pathway

[0135] Molecular docking of the main small molecules of FZYL with PI3K was performed, and the binding energies are shown in Table 4. It was found that the binding energies of seven compounds were greater than |-8| (e.g. Figure 19 Finally, we conducted in vitro experiments on the PI3K / AKT pathway and found that FZYL effectively inhibited the expression levels of p-PI3K and p-AKT in SW620 cells (as shown in Figure 20 shown).

[0136] Table 4 Binding energy of major FZYL small molecule compounds to PI3K protein

[0137]

[0138] In this study, using a BALB / c nu mouse model of colorectal cancer, the subjects were carefully assessed for tumor size, weight, and overall health using various biochemical analyses. The results demonstrated that FZYL significantly reduced tumor growth and enhanced apoptosis in CRC cells, suggesting its potential role in inhibiting tumor proliferation. Furthermore, integration of network pharmacology revealed key targets and pathways affected by FZYL, particularly the PI3K / AKT signaling pathway. These findings underscore the importance of FZYL as a promising candidate for CRC treatment and highlight its potential to modulate key molecular pathways involved in cancer progression. This study not only contributes to the existing body of knowledge on CRC therapy but also paves the way for future studies investigating the clinical application of FZYL in oncology.

[0139] Exploration of the relationship between the traditional Chinese medicine composition (FZYL) provided by this invention for the treatment of colorectal cancer and specific molecular targets highlights the compound's potential for the treatment of cancer (CRC). The results showed that FZYL significantly altered the expression of key proteins associated with tumor proliferation and apoptosis. Specifically, while FZYL administration resulted in a significant decrease in Ki67 and PCNA levels, indicators of cell proliferation, there was a concurrent increase in the expression of apoptotic markers such as Caspase-3 and Bax, and a decrease in BCL-2 levels. These changes in protein expression suggest that FZYL not only affects tumor biology but also actively regulates molecular pathways that determine cell fate. Furthermore, the involvement of the phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) pathway as a primary target of FZYL is noteworthy. Inhibition of proliferative signaling via PI3K / AKT may explain the observed phenotypic changes in tumor growth, weight, and metastatic potential. As demonstrated by molecular docking results, FZYL's ability to effectively bind to PIK3CA further solidifies its role as a regulator of this key pathway. Collectively, these insights reveal a strong interaction between FZYL and the PI3K / AKT axis, highlighting the therapeutic potential of this agent to control CRC progression through targeted molecular intervention.

[0140] In summary, the traditional Chinese medicine composition provided by this invention for the treatment of colorectal cancer can effectively inhibit cancer proliferation and enhance cell apoptosis by downregulating the PI3K / AKT signaling pathway. Comprehensive analyses, including tumor measurement, biochemical testing, molecular docking, and protein expression assessment, confirmed the therapeutic mechanism of FZYL. This invention provides valuable insights into the role of FZYL as a promising drug candidate for colorectal cancer treatment, highlighting its potential to mitigate disease progression while elucidating the underlying biological mechanisms. Overall, FZYL represents a significant advance in colorectal cancer treatment approaches.

[0141] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A Chinese medicine composition for treating colorectal cancer, characterized in that: The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 20-30 parts of astragalus, 8-12 parts of codonopsis, 10-20 parts of atractylodes, 10-20 parts of poria, 8-12 parts of liquorice, 20-30 parts of oldenlandia diffusa, 20-30 parts of scutellaria barbata, and 10-15 parts of moutan bark.

2. The Chinese medicine composition according to claim 1, wherein The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 25-30 parts of astragalus, 9-10 parts of codonopsis, 12-16 parts of atractylodes, 12-16 parts of poria, 8-10 parts of liquorice, 25-30 parts of oldenlandia diffusa, 25-30 parts of bark lotus, and 12-15 parts of moutan bark.

3. The method for preparing the Chinese medicine composition according to claim 1 or 2, wherein: include: The raw materials astragalus, codonopsis, atractylodes, tuckahoe, liquorice, hedyotis diffusa, scutellaria barbata and moutan bark are weighed according to the proportion and extracted with water to obtain the product.

4. The preparation method according to claim 3, wherein The water is 5 to 8 times the weight of the raw material.

5. The preparation method according to claim 4, wherein The extraction method is selected from any one of a decoction method, a water heating reflux method or an ultrasonic extraction method.

6. A drug for treating colorectal cancer, characterized in that: The medicine uses the traditional Chinese medicine composition according to any one of claims 1 to 2 as an active ingredient.

7. The drug according to claim 6, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. The drug according to claim 7, wherein The pharmaceutically acceptable excipients include at least one of a binder, a filler, a diluent, a lubricant, a disintegrant, a colorant, a flavoring agent, a wetting agent, a suspending agent, an emulsifier, and a preservative.

9. The drug according to claim 7, wherein The dosage form of the medicine is decoction, granules, pills, capsules, tablets, powders or oral liquid.

10. Use of the traditional Chinese medicine composition according to any one of claims 1 to 2 in the preparation of a medicament for treating colorectal cancer.

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