A traditional Chinese medicine composition and application thereof in preparation of an anti-tumor drug

Abstract

This invention provides a traditional Chinese medicine composition and its application in the preparation of antitumor drugs, belonging to the field of traditional Chinese medicine technology. The traditional Chinese medicine composition includes *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa*, with each component accounting for 20% to 45% by weight, and the weight difference between *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa* not exceeding 15%. This traditional Chinese medicine composition exhibits significant direct antitumor activity and can be used as an effective technical means in tumor treatment, providing a new and practical option for tumor therapy.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine composition and its application in the preparation of anti-tumor drugs. Background Technology

[0002] Malignant tumors are a major disease that seriously endangers human health. Their high invasiveness, easy metastasis, and significant heterogeneity are the core reasons for low clinical treatment response rates and poor prognosis. Despite continuous advancements in modern medical techniques such as surgery, radiotherapy, and chemotherapy, we still face severe challenges in dealing with recurrence, metastasis, and drug resistance in mid-to-late-stage tumors. Many patients develop treatment resistance due to low drug sensitivity, poor tolerance to toxic side effects, or post-treatment drug resistance, resulting in limited efficacy and a significant decline in quality of life. Against this backdrop, the holistic regulation and multi-target intervention characteristics of Traditional Chinese Medicine (TCM) demonstrate its unique value in the field of tumor treatment. TCM compound formulas are composed of multiple active ingredients, which, through synergistic effects via multiple pathways and mechanisms, can simultaneously intervene in multiple key aspects of tumor development and progression. This "multi-target, holistic" mode of action holds the promise of overcoming the limitations of insufficient response to single-target drugs, providing a new synergistic treatment strategy for improving treatment response and controlling disease progression.

[0003] *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa* are all traditional Chinese medicinal herbs. Their decoction usage, meridian tropism, and applications are recorded in ancient books and are widely used in modern clinical practice. However, no reports have been found in domestic or international literature or patent documents regarding compound decoctions using *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa* as active ingredients for the preparation of anti-tumor drugs, or their use in enhancing the efficacy of tumor treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a traditional Chinese medicine composition, which is formed by combining three leaves of *Trifolium repens*, *Panax quinquefolium*, and *Hedyotis diffusa* in a specific ratio, and has anti-tumor effects.

[0005] This invention provides a traditional Chinese medicine composition comprising the following traditional Chinese medicine raw materials: *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa*; wherein the mass ratio of *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa* is 20~45:20~45:20~45.

[0006] Preferably, the weight percentages of the three-leaf green, cat ginseng, and white flower tongue grass do not differ by more than 15%.

[0007] Preferably, the mass ratio of the three-leaf green, cat ginseng and white flower tongue grass is 1:1:1.

[0008] Preferably, the traditional Chinese medicine composition further includes coix seed or a combination of coix seed and astragalus, codonopsis, wolfberry, privet fruit, poria, polyporus, and raw licorice; The amount of Job's tears added is twice that of Clematis armandii; The mass ratio of Astragalus membranaceus, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Poria cocos, Polyporus umbellatus, Codonopsis pilosula, Hedyotis diffusa, Trifolium repens, Coix lacryma-jobi, and raw Glycyrrhiza uralensis is 3:2:3:2:3:3:3:3:3:6:1.

[0009] This invention provides a method for preparing the aforementioned traditional Chinese medicine composition, comprising the following steps: The raw materials of traditional Chinese medicine are mixed and then heated under reflux for extraction. The extract is collected to obtain a traditional Chinese medicine composition.

[0010] Preferably, the solvent used for heating and reflux extraction includes water or an aqueous solution of ethanol with a volume concentration of 77% to 95%; The temperature for the heating reflux extraction is 98~102℃ or 78~90℃; The number of heating and reflux extractions is 1 to 5 times.

[0011] Preferably, the Chinese herbal raw materials are pulverized before mixing, and the particle size of the pulverized Chinese herbal raw materials is less than 20 mesh.

[0012] This invention provides the application of the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method in the preparation of anti-tumor drugs.

[0013] Preferably, the tumor includes at least one of the following: colorectal cancer, lung cancer, gastric cancer, liver cancer, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, esophageal cancer, glioma, melanoma, nasopharyngeal carcinoma, thyroid cancer, osteosarcoma, gallbladder cancer, and soft tissue sarcoma. Preferably, the intestinal cancer includes colon cancer.

[0014] This invention provides an antitumor drug, the active ingredient of which includes the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method.

[0015] This invention provides a traditional Chinese medicine composition comprising the following raw materials: *Trifolium repens*, *Panax ginseng*, and *Hedyotis diffusa*; the mass ratio of *Trifolium repens*, *Panax ginseng*, and *Hedyotis diffusa* is 20-45:20-45:20-45. This invention provides a simplified composition with significant anti-tumor effects. The core drug group was refined from traditional tonifying and eliminating pathogenic factors formulas (such as F1), and a series of formula screening studies were conducted. Experimental results show that the simplified formula (F4) composed of *Panax ginseng*, *Hedyotis diffusa*, and *Trifolium repens* has a better anti-tumor effect than the original formula containing tonifying drugs such as *Astragalus membranaceus* and *Codonopsis pilosula* (F1) and other control groups (F2, F3, F5). This discovery breaks through the conventional thinking that "anti-tumor formulas must be combined with a large number of traditional tonifying drugs," revealing that the specific combination of these three drugs is the core contributing unit to the anti-tumor effect of this scheme, and produces a unique synergistic effect. The dosage of the composition of this invention is determined based on the commonly used effective dosage range of each drug in clinical tumor treatment, ensuring the safety and practicality of the prescription. Its formulation principle is based on the multi-target synergistic effect of modern pharmacology: *Tripterygium wilfordii*, as a direct anti-tumor component, primarily inhibits tumor cell proliferation and metastasis; *Gynostemma pentaphyllum*, as an immunomodulatory component, focuses on improving the tumor immune microenvironment and also has the effect of supporting the body's vital energy; *Hedyotis diffusa*, as a heat-clearing and anti-inflammatory component, exerts anti-inflammatory, detoxifying, and tumor angiogenesis-inhibiting effects. The three herbs combined construct a highly effective and streamlined anti-tumor combination that prioritizes eliminating pathogenic factors while simultaneously tonifying and attacking the tumor. Attached Figure Description

[0016] Figure 1 A bar chart showing the final tumor volume of the blank group and the five candidate traditional Chinese medicine composition groups (subcutaneous xenograft tumors in the MC38 black mouse disease model). Figure 2 Curves showing the growth of colorectal cancer tumors in the control group and the traditional Chinese medicine combination group (subcutaneous xenografts in HCT116 nude mice, a disease model). Figure 3 The graphs show the growth of colorectal cancer tumors in the blank group and the group with traditional Chinese medicine composition (subcutaneous xenograft tumors in MC38 black mice, a disease model). Detailed Implementation

[0017] This invention provides a traditional Chinese medicine composition comprising the following traditional Chinese medicine raw materials: *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa*; wherein the mass ratio of *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa* is 20~45:20~45:20~45.

[0018] In this invention, the weight ratio of *Trifolium repens*, *Gynostemma pentaphyllum*, and *Hedyotis diffusa* does not differ by more than 15%, preferably 30-35:30-35:30-35. The preferred mass ratio of *Trifolium repens*, *Gynostemma pentaphyllum*, and *Hedyotis diffusa* is 1:1:1. This traditional Chinese medicine composition can effectively inhibit the proliferation of colon cancer cells. In this composition, *Trifolium repens* is the principal herb, playing a core role in clearing heat and detoxifying, directly inhibiting tumor proliferation, and inducing apoptosis in cancer cells; *Hedyotis diffusa* is the assistant herb, assisting the principal herb in enhancing its heat-clearing and detoxifying effects, and providing an outlet for pathogenic factors through diuresis and urination; *Gynostemma pentaphyllum* is the adjuvant herb, assisting the principal and assistant herbs in softening and dispersing nodules to eliminate tumors, and also counteracting the cold nature of the entire formula, thus strengthening the body's resistance. The three herbs work synergistically to form an organic whole based on the principle of "clearing heat and toxins, dispersing nodules and reducing swelling," achieving excellent anti-tumor, immune-regulating, and toxicity-reducing effects through multi-target and multi-pathway synergistic action. The three need to form a "synergistic and complementary" ratio.

[0019] In this invention, the traditional Chinese medicine composition further includes coix seed or a combination of coix seed and astragalus, codonopsis, wolfberry, privet fruit, poria, polyporus, and licorice. The amount of coix seed added is twice that of *Trifolium repens*. The mass ratio of astragalus, codonopsis, wolfberry, privet fruit, poria, polyporus, ginseng, *Hedyotis diffusa*, *Trifolium repens*, coix seed, and licorice is 3:2:3:2:3:3:3:3:3:6:1. This invention does not impose special restrictions on the source of the above-mentioned traditional Chinese medicine raw materials; any traditional Chinese medicine raw materials well known in the art can be used.

[0020] This invention provides a method for preparing the aforementioned traditional Chinese medicine composition, comprising the following steps: The raw materials of traditional Chinese medicine are mixed and then heated under reflux for extraction. The extract is collected to obtain a traditional Chinese medicine composition.

[0021] In this invention, the herbal raw materials are preferably pulverized before mixing to maximize the extraction of active ingredients. This invention does not impose any particular limitation on the pulverization method; any pulverization method well-known in the art, such as chopping, can be used. The particle size of the pulverized herbal raw materials is preferably less than 20 mesh, and more preferably 40-100 mesh.

[0022] In this invention, the solvent for reflux extraction includes water or an aqueous ethanol solution with a volume concentration of 77% to 95%. The volume concentration of the aqueous ethanol solution can also be 85% to 90%. When water is used as the solvent, the reflux extraction temperature is preferably 98 to 102°C, but can be 100°C. When an aqueous ethanol solution is used as the solvent, the reflux extraction temperature is preferably 78 to 90°C, but can be 85°C. The number of reflux extractions is 1 to 5 times, 2 to 4 times, or 3 times. The extraction time for each extraction is 0.5 to 3 hours, 1 to 2.5 hours, or 1.5 to 2 hours. During each extraction, the volume of the solvent is 5 to 15 times the total mass of the herbal raw materials, 7 to 12 times, or 10 times. After multiple reflux extractions, the extracts are combined. The extraction process preferably includes concentration and drying. Concentration removes the extraction solvent, and drying removes moisture and solvent.

[0023] In this invention, the herbal raw materials can also be extracted by decoction, preferably 2-3 times. The amount of water used for each decoction is 5-10 times, or 6-8 times, the total weight of the herbs. Each decoction lasts 1-2 hours. The decoction liquids from each decoction are combined and concentrated to obtain the herbal composition.

[0024] This invention provides the application of the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method in the preparation of anti-tumor drugs.

[0025] In this invention, the tumor preferably includes at least one of the following: colorectal cancer, lung cancer, gastric cancer, liver cancer, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, esophageal cancer, glioma, melanoma, nasopharyngeal carcinoma, thyroid cancer, osteosarcoma, gallbladder cancer, and soft tissue sarcoma; the colorectal cancer preferably includes colon cancer. The traditional Chinese medicine composition has the effect of inhibiting tumor cell proliferation.

[0026] This invention provides an antitumor drug, the active ingredient of which includes the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method.

[0027] In this invention, the antitumor drug preferably further includes pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients include, but are not limited to, one or more of fillers, binders, disintegrants, flavoring agents, lubricants, suspending agents, or solvents. The pharmaceutical formulation preferably includes at least one of the following dosage forms: decoction, tablets, pills, capsules, granules, suspension, ointment, oral liquid, and injection, preferably tablets, capsules, or oral liquids. The therapeutic scope of the antitumor drug is the same as the types of tumors described above, and will not be repeated here.

[0028] In this embodiment of the invention, in vivo pharmacodynamic studies of colon cancer (e.g., MC38, HCT116 mouse xenograft models) confirmed that the composition of *Trifolium repens*, *Panax ginseng*, and *Hedyotis diffusa* has a significant therapeutic effect on malignant tumors conforming to the TCM syndrome of "spleen deficiency, damp-heat, blood stasis, and toxin accumulation." Based on the core TCM theory of "treating different diseases with the same method," which states that different diseases exhibiting the same core pathogenesis during their development can be treated with the same approach, this "spleen deficiency, damp-heat, blood stasis, and toxin accumulation" pathogenesis is not unique to colon cancer but is a key common pathological link in the occurrence and development of various solid tumors (especially digestive system tumors, and some respiratory and reproductive system tumors). For example, gastric cancer often belongs to "spleen deficiency, phlegm, and blood stasis," and liver cancer is often diagnosed as "liver stagnation, spleen deficiency, damp-heat, blood stasis, and toxin accumulation," the core pathogenesis of which is closely related to "spleen deficiency" and "damp-heat, blood stasis, and toxin accumulation." Although lung cancer often manifests as "phlegm-heat and blood stasis," the formation of its pathological products of "phlegm," "heat," and "blood stasis" is also often based on the internal foundation of "spleen deficiency" and impaired digestion. Therefore, the aforementioned tumors share a high degree of overlap or similarity with the pathogenesis targeted by this invention in terms of key pathogenic mechanisms. Thus, based on its proven efficacy against colon cancer, and combined with traditional Chinese medicine theory, it is anticipated that the herbal composition of this invention also has therapeutic potential for other solid tumors that conform to the core pathogenesis of "spleen deficiency, damp-heat, blood stasis, and toxin accumulation," such as gastric cancer, liver cancer, lung cancer, and ovarian cancer. This provides sufficient theoretical and practical basis for summarizing the scope of protection of this invention.

[0029] The following detailed description, in conjunction with embodiments, illustrates a traditional Chinese medicine composition provided by the present invention and its application in the preparation of antitumor drugs. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 Preparation method of traditional Chinese medicine composition Formula F1: Astragalus membranaceus 30g, Codonopsis pilosula 20g, Lycium barbarum 30g, Ligustrum lucidum 20g, Poria cocos 30g, Polyporus umbellatus 30g, Codonopsis pilosula 30g, Hedyotis diffusa 30g, Clematis armandii 30g, Coix lacryma-jobi 60g, Glycyrrhiza uralensis 10g.

[0031] Formula F2: Astragalus membranaceus 30g, Codonopsis pilosula 20g, Lycium barbarum 30g, Ligustrum lucidum 20g, Poria cocos 30g, Polyporus umbellatus 30g.

[0032] Formula F3: Astragalus membranaceus 30g, Codonopsis pilosula 20g, Lycium barbarum 30g, Ligustrum lucidum 20g, Poria cocos 30g, Polyporus umbellatus 30g, Coix lacryma-jobi 60g.

[0033] Formula F4: 30g of cat ginseng, 30g of white flower tongue grass, and 30g of three-leaf green.

[0034] Formula F5: 30g of cat ginseng, 30g of white flower tongue grass, 30g of three-leaf green, and 60g of coix seed.

[0035] After mixing the ingredients of formulations F1 to F5 respectively, the mixture was extracted three times by heating and reflux with water. The amount of water added each time was 42.4 kg, 42.4 kg, and 31.8 kg respectively. The extraction time for each extraction was 1.5 hours, 1.5 hours, and 1 hour respectively. The extracts from the three extractions were combined, concentrated, and dried to obtain five groups of traditional Chinese medicine compositions.

[0036] Example 2 Validation of the antitumor function of different traditional Chinese medicine combinations 1. Experimental Materials 1.1 Reagents The five groups of traditional Chinese medicine compositions prepared in Example 1 were mixed with water to prepare medicinal solutions containing the corresponding amounts of raw herbs, wherein F1 was 4.38 g / mL, F2 was 2.19 g / mL, F3 was 3.01 g / mL, F4 was 1.23 g / mL, and F5 was 2.05 g / mL.

[0037] 1.2 Cell lines The mouse colon cancer cell line MC38 and the human colon cancer cell line HCT116 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0038] 1.3 Experimental Animals The specific information of the experimental animals used in this invention is as follows: 5-week-old female C57BL / 6J black mice, weighing 20 ± 2g, and 5-week-old female BALB / c-nude mice, weighing 20 ± 2g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All animal handling and research procedures were completed at the Animal Experiment Center of Hangzhou Normal University, and the entire process complied with the ethical review standards established by the institution's Animal Ethics Committee. The experimental animals were housed in SPF (Specific Pathogen Free) grade animal experimental facilities, and the housing environment met relevant regulatory requirements; during the experiment, the animals had free access to standard feed and sterile water.

[0039] 2. Establishment of animal models 2.1 Culture of MC38 colon cancer cells MC38 cells were seeded in DMEM medium containing 10% fetal bovine serum, 100 U / ml sodium penicillin, and 100 U / ml streptomycin, and cultured in a 37°C incubator containing 5% CO2. When the cells reached approximately 80% confluence, they were digested with 0.25% trypsin containing 0.02% EDTA for 2 minutes, followed by the addition of culture medium containing 10% fetal bovine serum to terminate the digestion. After the reaction was terminated, the cells were centrifuged and passaged at a 1:3 ratio twice weekly. MC38 cells in the logarithmic growth phase were selected for the experiment to obtain the MC38 colon cancer cells.

[0040] 2.2 Establishment of a mouse model of colorectal cancer Forty-five female C57BL / 6J mice (18-22 g) were used, and each mouse was subcutaneously inoculated with 5 × 10⁵ lbs of 500 mg / kg solution on the right ventral side. 5 One MC38 colon cancer cell was inoculated. Tumor formation was confirmed on day 4 post-inoculation. From the successfully modeled tumor-bearing mice, 36 individuals with uniform tumor size were selected and randomly assigned to 6 experimental groups (n=6) to ensure consistent initial tumor burden among the groups.

[0041] 2.3 Grouping and Sampling of Experimental Animals Animal Experiment 1 on the Anti-cancer Effects of Traditional Chinese Medicine Compositions: Black mice with intestinal cancer were randomly divided into 6 groups: a control group, a group receiving traditional Chinese medicine composition F1, F2, F3, F4, and F5. Treatment began on day 5 after tumor formation was observed. The control group received physiological saline, while the groups receiving different compound traditional Chinese medicine compositions were administered via gavage. Each group received the medication once daily for continuous administration. Two hours after the last administration, the mice were sacrificed, and tumor tissue was collected, photographed, and recorded. A portion of the tumor tissue from each group was flash-frozen in liquid nitrogen and then stored at -80°C for later use. Another portion of the tumor tissue was fixed in formalin for later use.

[0042] 3. Experimental Methods 3.1 Tumor volume measurement Starting from the first day of drug administration, the long and short diameters of the tumor were measured for the first time using vernier calipers, and the average size of the tumor was calculated and recorded. Thereafter, the above measurements (measuring the long and short diameters of the tumor and calculating the average size) and recording were repeated every 2 days until the end of the experimental period.

[0043] 3.2 Statistical Analysis All experimental data are expressed as mean ± SEM (mean ± standard error of mean). P A value <0.05 was used as the criterion for determining statistical significance, and all data were statistically analyzed using GraphPad Prism 10.1.2 software.

[0044] 4. Experimental Results This invention used MC38 colon cancer cells to construct a subcutaneous mouse tumor-bearing model of colon cancer. Through this series of experiments, the anti-tumor activity of a traditional Chinese medicine composition with *Trifolium repens*, *Panax ginseng*, and *Hedyotis diffusa* as its core active ingredients was systematically evaluated. Detailed experimental results can be found in [link to relevant documentation]. Figure 1 .like Figure 1The results showed that, compared with the blank group, the traditional Chinese medicine composition (i.e., F4) with Trifoliate orange, ginseng and oldenlandia diffusa as active ingredients, and the traditional Chinese medicine compositions (i.e., F1 and F5) containing the above active ingredients could significantly inhibit the tumor proliferation process of subcutaneous tumor-bearing black mice with colorectal cancer. The proliferation inhibition rate was higher than any other compound traditional Chinese medicine composition, and the inhibitory effect was significant.

[0045] Example 3 A method for preparing a traditional Chinese medicine composition Weigh out 30g of cat ginseng, 30g of white flower tongue grass, and 30g of three-leaf green, mix them, and extract them three times by heating and refluxing with water. The amount of water added each time was 42.4kg, 42.4kg, and 31.8kg respectively, and the extraction time was 1.5 hours, 1.5 hours, and 1 hour respectively. Combine the three extracts, concentrate them, and dry them to obtain the traditional Chinese medicine composition 1.

[0046] Example 4 A method for preparing a traditional Chinese medicine composition Weigh out 30g of cat ginseng, 30g of white flower tongue grass, and 30g of three leaves of green, mix them, and extract them twice with 95 vol% ethanol aqueous solution under reflux. The solvent used for each extraction is 42.4 kg, and the extraction time is 2 hours each time. Combine the two extracts, concentrate and dry them to obtain Chinese medicine composition 2.

[0047] Example 5 A method for preparing a traditional Chinese medicine composition Weigh out 30g of cat ginseng, 30g of white flower tongue grass, and 30g of three-leaf green, pulverize them separately until the particle size is less than 20 mesh, mix them evenly, and obtain the Chinese herbal composition 3.

[0048] Example 6 Validation of anti-tumor drugs 1. Verification reagent instructions The traditional Chinese medicine composition prepared in Example 3 was mixed with water to prepare medicinal solutions with raw drug concentrations of 0.62 g / ml, 1.23 g / ml, and 2.46 g / ml, respectively, which were denoted as low concentration, medium concentration, and high concentration.

[0049] 2. Culture of HCT116 colorectal cancer cells HCT116 cells were seeded in 5A medium containing 10% fetal bovine serum, 100 U / ml sodium penicillin, and 100 U / ml streptomycin, and cultured at 37°C in a 5% CO2 incubator. When the cells reached approximately 80% confluence, they were digested with 0.25% trypsin containing 0.02% EDTA for 2 minutes, followed by the addition of medium containing 10% serum to terminate the digestion. After the reaction was terminated, the cells were centrifuged and passaged at a 1:4 ratio twice weekly. HCT116 cells in the logarithmic growth phase were selected for the experiment to obtain the prepared HCT116 colon cancer cells.

[0050] 3. Establishment of nude mouse and black mouse models of colorectal cancer Twenty-five female BALB / c nude mice were used, and 5 × 10⁵ cells were subcutaneously injected into the right ventral side of each mouse. 6 HCT116 colon cancer cells were inoculated. Tumor formation was confirmed on day 7 post-inoculation. From the successfully modeled tumor-bearing mice, 20 individuals with uniform tumor size were selected and randomly assigned to 4 experimental groups (n=5) to ensure consistent initial tumor burden among the groups.

[0051] Twenty-five female C57BL / 6J mice (18-22 g) were used, and each mouse was subcutaneously inoculated with 5 × 10⁵ cells on the right ventral side. 5 One MC38 colon cancer cell was inoculated. Tumor formation was confirmed on day 4 post-inoculation. From the successfully modeled tumor-bearing mice, 20 individuals with uniform tumor size were selected and randomly assigned to 4 experimental groups (n=5) to ensure consistent initial tumor burden among the groups.

[0052] 4. Animal experiments on the anticancer effects of traditional Chinese medicine compositions Nude or black mice with colorectal cancer, as established above, were randomly divided into four groups: a control group, a low-concentration group of the traditional Chinese medicine (TCM) composition, a medium-concentration group of the TCM composition, and a high-concentration group of the TCM composition. Drug administration began on day 7 or 5 after tumor formation was observed. The control group received physiological saline, while the TCM composition groups received different concentrations of the TCM composition. Each group received the drug via gavage once daily for continuous administration. Two hours after the last administration, the nude or black mice with colorectal cancer in each group were sacrificed, and tumor tissue was collected, photographed, and recorded. A portion of the tumor tissue from each group was then flash-frozen in liquid nitrogen and stored at -80°C for later use. Another portion of the tumor tissue from each group was fixed in formalin for later use.

[0053] Figure 2 The results showed that, compared with the control group, the traditional Chinese medicine composition of the present invention, with Trifolium repens, Codonopsis pilosula, and Hedyotis diffusa as the core active ingredients, could significantly inhibit the tumor proliferation process in nude mice with subcutaneous tumors of colorectal cancer during the administration period, and the inhibitory effect was significant.

[0054] at the same time Figure 3The results showed that when the traditional Chinese medicine composition was applied to the subcutaneous MC38 tumor-bearing black mouse model of colorectal cancer, it could effectively inhibit tumor proliferation during the administration period, further verifying its inhibitory activity against colorectal cancer.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, The ingredients include the following Chinese medicinal materials: *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa*; the mass ratio of *Trifolium repens*, *Codonopsis pilosula*, and *Hedyotis diffusa* is 20~45:20~45:20~45.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The weight percentages of the three-leaf green, cat ginseng, and white flower tongue grass do not differ by more than 15%.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The mass ratio of the three-leaf green, cat ginseng and white flower tongue grass is 1:1:

1.

4. The traditional Chinese medicine composition according to any one of claims 1 to 3, characterized in that, The traditional Chinese medicine composition also includes coix seed or a combination of coix seed and astragalus, codonopsis, wolfberry, privet fruit, poria, polyporus, and raw licorice; The amount of Job's tears added is twice that of Clematis armandii; The mass ratio of Astragalus membranaceus, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Poria cocos, Polyporus umbellatus, Codonopsis pilosula, Hedyotis diffusa, Trifolium repens, Coix lacryma-jobi, and raw Glycyrrhiza uralensis is 3:2:3:2:3:3:3:3:3:6:

1.

5. A method for preparing the traditional Chinese medicine composition according to any one of claims 1 to 4, characterized in that, Includes the following steps: The raw materials of traditional Chinese medicine are mixed and then heated under reflux for extraction. The extract is collected to obtain a traditional Chinese medicine composition.

6. The preparation method according to claim 5, characterized in that, Solvents used for heating and reflux extraction include water or an aqueous solution of ethanol with a volume concentration of 77% to 95%; The temperature for the heating reflux extraction is 98~102℃ or 78~90℃; The number of heating and reflux extractions is 1 to 5 times.

7. The preparation method according to claim 5, characterized in that, The raw materials of traditional Chinese medicine are pulverized before mixing, and the particle size of the pulverized raw materials is less than 20 mesh.

8. The use of the traditional Chinese medicine composition according to any one of claims 1 to 4 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 5 to 7 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The tumors include at least one of the following: colorectal cancer, lung cancer, stomach cancer, liver cancer, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, esophageal cancer, glioma, melanoma, nasopharyngeal carcinoma, thyroid cancer, osteosarcoma, gallbladder cancer, and soft tissue sarcoma. Preferably, the intestinal cancer includes colon cancer.

10. An antitumor drug, characterized in that, The active ingredients include the traditional Chinese medicine composition according to any one of claims 1 to 4 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 5 to 7.

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