Traditional Chinese medicine preparation for treating esophageal cancer and preparation method thereof

Abstract

The application provides a traditional Chinese medicine preparation for treating esophageal cancer and a preparation method thereof, and relates to the technical field of traditional Chinese medicine pharmacy. The traditional Chinese medicine preparation is composed of Wilingxian, Shijianchuan, Kelp, Caenotus, Zhongjie wind, Lotus leaf peduncle, Chutoucao, Shashen, Jiuxiangchong, Shiqiti, Huangyaozi, Jiluo, and Luhongfang. The application verifies the anti-esophageal cancer effect and safety of the traditional Chinese medicine preparation through in-vitro and in-vivo experiments. The in-vitro experiment shows that the traditional Chinese medicine preparation can significantly reduce the TNF-alpha, IL-1beta and IL-8 levels in the supernatant of esophageal cancer cells Eca-109, and plays an anti-tumor role by regulating the inflammatory microenvironment. The in-vivo experiment shows that the traditional Chinese medicine preparation can inhibit the growth of esophageal cancer EC9706 cell transplanted tumors in nude mice, remodel the immune balance of the organism by bidirectional regulation of immune cell subgroups, and enhance the anti-tumor immunity. Meanwhile, the traditional Chinese medicine preparation does not cause liver and kidney damage to the nude mice at an effective dose, and provides a new scheme with safety and effectiveness for the clinical treatment of esophageal cancer.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine pharmaceutical technology, specifically to a traditional Chinese medicine preparation for treating esophageal cancer and its preparation method. Background Technology

[0002] Esophageal cancer is a malignant tumor originating from the esophageal mucosal epithelium. Its occurrence is related to various factors such as smoking, alcohol consumption, long-term consumption of excessively hot food, and genetics. Typical symptoms in the middle and late stages include progressive dysphagia, reflux, and persistent pain, severely impacting patients' quality of life and survival. Currently, clinical treatment for esophageal cancer is primarily based on Western medicine, including surgery, radiotherapy, chemotherapy, and targeted therapy. However, existing treatments have significant limitations: low early diagnosis rates (only about 20% of patients are diagnosed at an early stage), and a recurrence rate as high as 50%-70% after surgical resection; first-line chemotherapy drugs, such as cisplatin, are associated with severe liver and kidney damage, bone marrow suppression, and gastrointestinal adverse reactions; radiotherapy easily causes complications such as radiation esophagitis and tissue fibrosis; while targeted drugs such as PD-1 inhibitors are effective in some patients, the overall response rate is less than 25%, and drug resistance is easily developed. In summary, Western medicine treatment faces challenges such as "limited efficacy, significant toxic side effects, and large individual differences," urgently requiring new treatment strategies with low toxicity and high efficacy.

[0003] Traditional Chinese medicine (TCM) categorizes esophageal cancer under the category of "dysphagia," believing its pathogenesis is closely related to emotional distress, improper diet, and stagnation of qi, phlegm, blood stasis, and toxin accumulation due to old age and physical weakness. TCM treatment emphasizes a holistic approach and syndrome differentiation, often using compound formulas such as Qi Ge San and Sha Shen Mai Dong Tang, which exert synergistic effects through multiple components, including anti-tumor activity, immune regulation, and toxicity reduction. Modern research confirms that TCM has multi-target intervention characteristics, including inhibiting tumor cell proliferation and inducing apoptosis, inhibiting angiogenesis, and regulating the body's immune function. Furthermore, the combined use of TCM and chemotherapy can reduce the toxic side effects of drugs such as cisplatin, thereby improving patients' quality of life.

[0004] Although traditional Chinese medicine (TCM) has demonstrated unique advantages in the treatment of esophageal cancer, its clinical application still faces certain limitations. On the one hand, TCM has a relatively slow onset of action and limited direct tumor-suppressing ability, limiting its ability to rapidly control advanced tumors. On the other hand, its mechanisms of action are not fully understood; most TCM formulas rely on traditional empirical formulations, and while some active ingredients have been shown to induce apoptosis or inhibit angiogenesis, the overall regulatory network still lacks systematic and in-depth validation at the modern molecular biology level. Therefore, developing a TCM preparation that can inhibit tumor growth and regulate immune function through a clearly defined mechanism, while also possessing good safety profile, is of significant practical importance and application value for addressing current deficiencies in esophageal cancer treatment and improving the overall level of treatment. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] In view of the shortcomings of the prior art, the present invention provides a traditional Chinese medicine preparation for the treatment of esophageal cancer and its preparation method.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a traditional Chinese medicine preparation for treating esophageal cancer, which is composed of the following raw materials in parts by weight: Clematis chinensis 5-25 parts, Hedyotis diffusa 5-25 parts, Laminaria japonica 3-21 parts, Actinidia chinensis root 3-21 parts, Rhizoma Sinapis alba 2-20 parts, Nelumbo nucifera leaf 2-22 parts, Rhizoma Scutellariae var. chinensis 2-22 parts, Radix Adenophorae 1-20 parts, Cortex Eupolyphagae 1-20 parts, Persimmon calyx 1-18 parts, Radix Dioscoreae 1-18 parts, Citrus reticulata peel 2-20 parts, and Vespa nidus 2-20 parts.

[0010] Specifically, the traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 10-20 parts, Hedyotis diffusa 10-20 parts, Laminaria japonica 8-17 parts, Actinidia chinensis root 8-17 parts, Rhizoma Sinapis alba 6-15 parts, Nelumbo nucifera leaf stalk 6-17 parts, Chlorella vulgaris 6-17 parts, Adenophora stricta 5-15 parts, Eupolyphaga sinensis 5-15 parts, Persimmon calyx 3-12 parts, Dioscorea bulbifera 3-12 parts, Citrus reticulata peel 6-15 parts, and Vespa nidus 6-15 parts.

[0011] Specifically, the traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 15 parts, Hedyotis diffusa 15 parts, Laminaria japonica 13 parts, Actinidia chinensis root 13 parts, Rhizoma Sinapis alba 11 parts, Nelumbo nucifera leaf calyx 12 parts, Cortex Prunella vulgaris 12 parts, Radix Adenophorae 10 parts, Cortex Eupolyphaga sinensis 10 parts, Persimmon calyx 8 parts, Dioscorea bulbifera 8 parts, Citrus reticulata peel 12 parts, and Vespa nidus 12 parts.

[0012] This invention provides a method for preparing the traditional Chinese medicine preparation, wherein the traditional Chinese medicine preparation is prepared into an oral dosage form, including pills, powders, granules, oral liquids, decoctions, and tablets, by conventional pharmaceutical methods and with medically acceptable excipients.

[0013] Specifically, taking the decoction as an example, the steps include:

[0014] (1) Weigh each medicinal material according to the above weight proportions, wash them, and dry them in an oven at 60℃;

[0015] (2) Place Clematis chinensis, kelp, Hedyotis diffusa, Actinidia chinensis root, Adenophora stricta, and Dioscorea bulbifera in a clay pot, add an appropriate amount of distilled water and soak for 30 minutes. Bring to a boil over high heat and then simmer over low heat for 20 minutes.

[0016] (3) Then wrap the rice bran and honeycomb in sterile gauze, add them together with the rhizome of the swollen joint, lotus leaf stalk, orange peel, persimmon stalk, and nine-fragrant insect, continue to decoct for 10 minutes, filter, and collect the filtrate.

[0017] (4) Add warm water back to the dregs, bring to a boil over high heat, then simmer over low heat for 20 minutes, filter, and combine the two filtrates to obtain the decoction of the Chinese medicine preparation of the present invention.

[0018] This invention provides the application of the aforementioned traditional Chinese medicine preparation in the preparation of drugs for the prevention and / or treatment of esophageal cancer.

[0019] The pharmacological effects of each medicinal material in the traditional Chinese medicine preparation described in this invention are as follows:

[0020] Clematis chinensis: It has a pungent and salty taste, is warm in nature, and enters the bladder meridian; it has the effects of dispelling wind and dampness and unblocking the meridians; it is used for rheumatic pain, numbness of limbs, muscle contracture, and difficulty in flexion and extension.

[0021] Rock-leaved herb: bitter and pungent in taste, neutral in nature, and enters the liver and spleen meridians; it has the effects of clearing heat and detoxifying, promoting blood circulation and relieving pain; it is used for jaundice-type hepatitis, cancer, nephritis, leukorrhea, dysmenorrhea, lymph node tuberculosis, and elephantiasis; externally it is used to treat facial nerve paralysis, mastitis, and boils.

[0022] Kelp: Salty in taste, cold in nature, and enters the liver, stomach, and kidney meridians; it has the effects of resolving phlegm, softening hard masses, dispersing nodules, promoting diuresis, and reducing swelling; it is used for goiter, scrofula, testicular swelling and pain, and phlegm-dampness edema.

[0023] The root of *Actinidia chinensis* is sweet and astringent in taste, cool in nature, and enters the heart, kidney, liver, and spleen meridians. It has the effects of clearing heat and detoxifying, dispelling wind and dampness, promoting blood circulation and reducing swelling. It is used for hepatitis, dysentery, indigestion, urinary tract infections, leukorrhea, rheumatic joint pain, edema, traumatic injuries, boils, scrofula, gastrointestinal tumors, and breast cancer.

[0024] Swollen Joint Wind: It tastes bitter and pungent, is neutral in nature, and enters the heart and liver meridians; it has the effects of clearing heat and cooling blood, promoting blood circulation and eliminating spots, and dispelling wind and unblocking collaterals; it is used for blood heat causing spots and rashes, rheumatic pain, and traumatic injuries.

[0025] Lotus leaf stalk: bitter and astringent in taste, neutral in nature, and enters the liver, spleen, and large intestine meridians; it has the effects of relieving summer heat and dampness, removing blood stasis and stopping bleeding, and calming the fetus; it is used for summer heat and dampness diarrhea, bloody dysentery, metrorrhagia, and threatened abortion.

[0026] Pestove chaff: It tastes sweet and pungent, is warm in nature, and enters the stomach and large intestine meridians; it has the effects of stimulating appetite and relieving bloating; it is used for esophageal obstruction, nausea, and beriberi.

[0027] Adenophora stricta: It has a sweet taste and slightly cold nature, and enters the lung and stomach meridians; it has the effects of nourishing yin and clearing the lungs, benefiting the stomach and promoting body fluids, resolving phlegm, and benefiting qi; it is used for dry cough due to lung heat, cough due to yin deficiency, dry cough with sticky phlegm, stomach yin deficiency, poor appetite and vomiting, qi and yin deficiency, and irritability and dry mouth.

[0028] Nine-fragrant insect: salty in taste, warm in nature, and enters the liver, spleen, and kidney meridians; it has the effects of regulating qi and relieving pain, warming the middle and assisting yang; it is used for stomach cold and bloating pain, liver and stomach qi pain, kidney deficiency and impotence, and soreness and pain in the waist and knees.

[0029] Persimmon calyx: bitter and astringent in taste, neutral in nature, and enters the stomach meridian; it has the effect of relieving nausea and hiccups; it is used for hiccups.

[0030] Dioscorea bulbifera: It tastes bitter and pungent, and is cool in nature. It enters the lung and liver meridians. It has the effects of detoxifying and reducing swelling, resolving phlegm and dissipating nodules, cooling blood and stopping bleeding. It is used for goiter, lymph node tuberculosis, sore throat, hematemesis, hemoptysis, pertussis, and cancer. It is also used externally to treat boils and carbuncles.

[0031] Orange pith: It tastes sweet and bitter, is neutral in nature, and enters the liver and spleen meridians; it has the effects of clearing the meridians, regulating qi, and resolving phlegm; it is used for qi stagnation in the meridians, chronic cough with chest pain, blood in sputum, and thirst due to alcohol consumption.

[0032] Honeycomb: Sweet in taste, neutral in nature, and enters the liver, stomach, and kidney meridians; it has the effects of dispelling wind and relieving pain, detoxifying and reducing swelling, killing insects and relieving itching; it is mainly used for rheumatic pain, toothache caused by wind and insects, carbuncles and malignant sores, scrofula, sore throat and tongue, hemorrhoids, urticaria and itching, and stubborn skin diseases.

[0033] (III) Beneficial Effects

[0034] This invention provides a traditional Chinese medicine preparation for treating esophageal cancer and its preparation method. The preparation is made from Clematis chinensis, Hedyotis diffusa, Laminaria japonica, Actinidia chinensis root, Rhizoma Sinapis alba, Nelumbo nucifera leaf stalk, Rhizoma Scutellariae, Radix Adenophorae, Inula japonica, Persimmon calyx, Dioscoreae scabra, Citrus reticulata peel, and Vespa nidus. The formula uses Clematis chinensis, Hedyotis diffusa, and Laminaria japonica as the principal herbs: Clematis chinensis is good at penetrating and dispersing, and its salty nature can soften hardness. It is especially good at penetrating the bone to dispel wind, soften hardness and disperse nodules, and is used for esophageal stenosis and dysphagia caused by phlegm and turbidity in esophageal cancer; Hedyotis diffusa is bitter and pungent, drains blood stasis and toxins, clears heat and invigorates blood, and is used for the core of phlegm and toxin obstruction in esophageal cancer, enhancing the power of expelling pathogens; Laminaria japonica is salty and cold, moistens and descends, and is good at eliminating phlegm, softening hardness, promoting diuresis and reducing swelling, and is used for the symptoms of phlegm and turbidity congealing into lumps and obstructing the esophagus in esophageal cancer; The three herbs combined work together to resolve phlegm and disperse nodules, detoxify and eliminate masses, directly targeting the root cause of phlegm and blood stasis in esophageal cancer. The formula uses *Actinidia chinensis* root, *Hedyotis diffusa*, lotus leaf stalk, and *Paeonia lactiflora* husk as assistant herbs: *Actinidia chinensis* root clears heat and detoxifies, promotes diuresis and dissipates nodules, and is good at clearing damp-heat in the stomach and intestines; *Hedyotis diffusa* clears heat and detoxifies, invigorates blood and relieves pain, and helps the principal herb to enhance its blood-activating power while relieving pain; lotus leaf stalk ascends clear and descends turbid, harmonizes the stomach and stops vomiting, and is effective for common symptoms of esophageal cancer such as reflux, hiccups, and upward reversal of stomach qi; *Paeonia lactiflora* husk opens the stomach and lowers qi, eliminates stagnation and promotes the spleen and stomach to transform and transport, and prevents the internal generation of phlegm and turbidity; the four herbs work together to enhance the principal herb's ability to dissipate nodules and detoxify, while also protecting the qi mechanism of the middle jiao and preventing the expelling herbs from damaging the spleen and stomach. The formula uses Adenophora stricta, worm, persimmon calyx, and Dioscorea bulbifera as adjuvant herbs: Adenophora stricta nourishes yin and generates fluids, benefits the stomach and moistens the lungs, replenishing stomach yin to moisten the esophagus, and also restraining the dryness and heat of the principal herb from damaging fluids; worm is warm in nature and regulates qi, while persimmon calyx is neutral in nature and stops hiccups. The two, one warm and one neutral, one regulating qi and one descending rebellious qi, are aimed at the chest and diaphragm distending pain and intractable hiccups commonly seen in esophageal cancer; Dioscorea bulbifera softens hard masses, disperses nodules, clears heat and detoxifies, and helps the principal herb enhance its tumor-reducing power; the combination of these four herbs not only compensates for the insufficient power of the principal and adjuvant herbs in eliminating pathogens, but also restrains their biases, achieving a balance of "attacking pathogens without harming the body's vital energy". Citrus reticulata peel and bee honeycomb are used as guiding herbs: Citrus reticulata peel unblocks the meridians and resolves phlegm, taking advantage of its "leaf and reticulata unblocking the meridians" property to guide the herbs directly to the affected area; bee honeycomb contains melitin, which can enhance anti-tumor activity, while also harmonizing the cold and hot properties of the entire formula; the two herbs together serve as guiding herbs, ensuring that "the medicinal power is focused but not too strong, and the medicinal properties are mild and not biased".

[0035] This invention systematically verified the anti-esophageal cancer efficacy and safety of the traditional Chinese medicine preparation through in vitro and in vivo experiments, with the following results:

[0036] (1) In vitro experiments show that the traditional Chinese medicine preparation of the present invention can significantly reduce the levels of inflammatory factors such as TNF-α, IL-1β and IL-8 in the culture supernatant of esophageal cancer cells Eca-109 under the premise of ensuring biosafety, and exert its potential anti-tumor effect by regulating the inflammatory microenvironment;

[0037] (2) In vivo experiments showed that the traditional Chinese medicine preparation of the present invention can inhibit the growth of esophageal cancer EC9706 cell xenografts in nude mice in a dose-dependent manner; it can also reshape the body's immune balance and enhance anti-tumor immunity by bidirectionally regulating immune cell subsets; and at the same time, it did not cause liver or kidney damage in nude mice at effective doses.

[0038] In summary, the traditional Chinese medicine preparation of this invention provides a new approach for the clinical treatment of esophageal cancer that combines safety and effectiveness. Attached Figure Description

[0039] Figure 1 The growth curves of EC9706 esophageal cancer xenografts in nude mice are shown for each group; Note: Compared with the model control group. * P<0.05, ** P<0.01.

[0040] Figure 2 The serum levels of ALT, AST, Cr, and BUN in nude mice with EC9706 esophageal cancer xenografts were measured in each group. Note: Compared with the blank control group, * P<0.05, ** P<0.01. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1

[0043] A traditional Chinese medicine preparation for treating esophageal cancer, the preparation comprising the following raw materials in parts by weight: Clematis chinensis 15 parts, Hedyotis diffusa 15 parts, Laminaria japonica 13 parts, Actinidia chinensis root 13 parts, Rhizoma Sinapis alba 11 parts, Nelumbo nucifera leaf calyx 12 parts, Rhizoma Scutellariae var. chinensis 12 parts, Radix Adenophorae 10 parts, Cortex Eupolyphagae 10 parts, Persimmon calyx 8 parts, Radix Dioscoreae 8 parts, Citrus reticulata peel 12 parts, and Vespa nidus 12 parts.

[0044] Example 2

[0045] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 5 parts, Hedyotis diffusa 5 parts, Laminaria japonica 3 parts, Actinidia chinensis root 3 parts, Rhizoma Sinapis alba 2 parts, Nelumbo nucifera leaf 2 parts, Cortex Prunella vulgaris 2 parts, Radix Adenophorae 1 part, Inula japonica 1 part, Persimmon calyx 1 part, Dioscorea bulbifera 1 part, Citrus reticulata peel 2 parts, and Vespa nidus 2 parts.

[0046] Example 3

[0047] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 23 parts, Hedyotis diffusa 23 parts, Laminaria japonica 20 parts, Actinidia chinensis root 19 parts, Rhizoma Sinapis alba 18 parts, Nelumbo nucifera leaf calyx 20 parts, Cortex Prunella vulgaris 20 parts, Radix Adenophorae 17 parts, Inula japonica 15 parts, Persimmon calyx 15 parts, Dioscorea bulbifera 15 parts, Citrus reticulata peel 18 parts, and Vespa nidus 18 parts.

[0048] Example 4

[0049] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 25 parts, Hedyotis diffusa 25 parts, Laminaria japonica 21 parts, Actinidia chinensis root 21 parts, Rhizoma Sinapis alba 20 parts, Nelumbo nucifera leaf 22 parts, Cortex Prunella vulgaris 22 parts, Radix Adenophorae 20 parts, Cortex Eupolyphaga sinensis 20 parts, Persimmon calyx 18 parts, Dioscorea bulbifera 18 parts, Citrus reticulata peel 20 parts, and Vespa nidus 20 parts.

[0050] Example 5

[0051] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: 10 parts Clematis chinensis, 10 parts Hedyotis diffusa, 7 parts Laminaria japonica, 7 parts Actinidia chinensis root, 7 parts Rhizoma Sinapis alba, 5 parts Nelumbo nucifera leaf calyx, 5 parts Chlorella vulgaris, 5 parts Adenophora stricta, 5 parts Eupolyphaga sinensis, 4 parts Persimmon calyx, 5 parts Dioscorea bulbifera, 7 parts Citrus reticulata peel, and 6 parts Vespa nidus.

[0052] Example 6

[0053] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 7 parts, Hedyotis diffusa 7 parts, Laminaria japonica 5 parts, Actinidia chinensis root 5 parts, Rhizoma Sinapis alba 4 parts, Nelumbo nucifera leaf 4 parts, Rhizoma Scutellariae 4 parts, Radix Adenophorae 3 parts, Inula japonica 3 parts, Persimmon calyx 3 parts, Dioscorea bulbifera 3 parts, Citrus reticulata peel 5 parts, and Vespa nidus 5 parts.

[0054] Example 7

[0055] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 20 parts, Hedyotis diffusa 20 parts, Laminaria japonica 18 parts, Actinidia chinensis root 17 parts, Rhizoma Sinapis alba 17 parts, Nelumbo nucifera leaf stalk 18 parts, Cortex Prunella vulgaris 15 parts, Radix Adenophorae 14 parts, Inula japonica 12 parts, Persimmon calyx 15 parts, Dioscorea bulbifera 12 parts, Citrus reticulata peel 15 parts, and Vespa nidus 15 parts.

[0056] Example 8

[0057] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 12 parts, Hedyotis diffusa 12 parts, Laminaria japonica 12 parts, Actinidia chinensis root 9 parts, Rhizoma Sinapis alba 9 parts, Nelumbo nucifera leaf stalk 9 parts, Cortex Prunella vulgaris 7 parts, Radix Adenophorae 7 parts, Inula japonica 7 parts, Persimmon stalk 7 parts, Dioscorea bulbifera 9 parts, Citrus reticulata peel 9 parts, and Vespa nidus 2-20 parts.

[0058] Experimental Example 1

[0059] 1. Materials and Methods

[0060] 1.1 Experimental Drugs

[0061] Test substance 1: Clematis chinensis 15 g, Hedyotis diffusa 15 g, Laminaria japonica 13 g, Actinidia chinensis root 13 g, Rhizoma Sinapis alba 11 g, Nelumbo nucifera leaf stalk 12 g, Chaenomeles speciosa 12 g, Adenophora stricta 10 g, Insect 10 g, Persimmon calyx 8 g, Dioscorea bulbifera 8 g, Citrus reticulata peel 12 g, Vespa nidus 12 g.

[0062] Test substance 2: 15 g of *Hemiberlesia lingua*, 13 g of kelp, 13 g of *Actinidia chinensis* root, 11 g of *Euphorbia humifusa*, 12 g of lotus leaf stalk, 12 g of *Centipeda minima* husk, 8 g of persimmon calyx, 8 g of *Dioscorea bulbifera*, 12 g of orange peel, and 12 g of bee honeycomb.

[0063] Test substance 3: Clematis chinensis 15 g, Actinidia chinensis root 13 g, Rhizoma Sinapis alba 11 g, Nelumbo nucifera leaf stalk 12 g, Cortex Prunella vulgaris 12 g, Radix Adenophorae 10 g, Insect 10 g, Persimmon calyx 8 g, Citrus reticulata peel 12 g, Vespa nidus 12 g.

[0064] Test substance 4: Clematis chinensis 15 g, Hedyotis diffusa 15 g, Laminaria japonica 13 g, Nelumbo nucifera leaf stalk 12 g, Adenophora stricta 10 g, Eupolyphaga sinensis 10 g, Persimmon calyx 8 g, Dioscorea bulbifera 8 g, Citrus reticulata peel 12 g, Vespa nidus 12 g.

[0065] Test substance 5: Clematis chinensis 15 g, Hedyotis diffusa 15 g, Laminaria japonica 13 g, Actinidia chinensis root 13 g, Rhizoma Sinapis alba 11 g, Rhizoma Scutellariae var. chinensis 12 g, Radix Adenophorae 10 g, Inula japonica 10 g, Dioscoreae scutellariae 8 g, Vespa nidus 12 g.

[0066] 1.2 Drug Preparation

[0067] Weigh each herb according to the above weight ratio, wash them, and dry them at 60℃. Place Clematis chinensis, kelp, Hedyotis diffusa, Actinidia chinensis root, Adenophora stricta, and Dioscorea bulbifera in a clay pot, add an appropriate amount of distilled water and soak for 30 minutes. Bring to a boil over high heat, then simmer over low heat for 20 minutes. Subsequently, wrap the rice bran and bee honeycomb in sterile gauze and add them together with Rhizoma Sinapis alba, Nelumbo nucifera leaf calyx, Citrus reticulata peel, Persimmon calyx, and Eupolyphaga sinensis. Continue to decoct for 10 minutes, filter, and collect the filtrate. Add warm water to the dregs again and decoct a second time using the same method (bring to a boil over high heat, then simmer over low heat for 20 minutes). Filter. Combine the two filtrates and concentrate under vacuum at 45℃ to a relative concentration of 1.13. Store at 4℃ for later use.

[0068] 1.3 Cell Culture

[0069] Esophageal cancer cell line Eca-109 was cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin as a complete medium. The cells were placed in a constant temperature incubator at 37°C and 5% CO2. When the cell confluence reached 70%-80%, the cells were passaged by digestion with 0.25% trypsin-EDTA.

[0070] 1.4 Detection Indicators

[0071] 1.4.1 CCK-8 assay for cell viability

[0072] Eca-109 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 3 Cells were seeded at a concentration of 1 cell / well in 96-well plates and cultured for 24 h. A blank control group and test substance groups 1-5 were set up, with 3 replicates in each group. The blank control group was given 200 µL of culture medium, and test substance groups 1-5 were given 200 µL of complete culture medium containing the corresponding test substance (the final concentration of each test substance was 0.2 mg / mL). After 24 h of culture, the culture medium was changed, and 180 µL of culture medium and 20 µL of CCK-8 were added to each well. After incubation for 1-4 h, the absorbance was measured at 450 nm using a microplate reader. A blank group (only 200 µL of complete culture medium was added, and no cells were seeded) was set up to remove background interference. Cell viability was calculated according to formula (1).

[0073] Cell viability = (OD) 药物组 -OD 空白组 ) / (OD 空白对照组 -OD 空白组 Formula (1) × 100%

[0074] 1.4.2 ELISA detection of TNF-α, IL-1β, and IL-8 levels

[0075] Eca-109 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates and cultured for 24 h. A blank control group and test substance groups 1-5 were included, with three replicates per group. The blank control group was given 200 µL of culture medium, while test substance groups 1-5 were given 200 µL of complete culture medium containing the corresponding test substance (final concentration of each test substance was 0.2 mg / mL). Culture was continued for another 24 h. Cell culture supernatants were collected from each well, and the levels of TNF-α, IL-1β, and IL-8 in the supernatant were measured strictly according to the ELISA kit instructions.

[0076] 1.5 Statistical Methods

[0077] The data were analyzed using Graphpad Prism 8 statistical software. Experimental data are expressed as mean ± standard deviation. The data indicates that one-way ANOVA was used for comparisons between groups, and a p-value < 0.05 was considered statistically significant.

[0078] 2 Results

[0079] 2.1 Effects on cell viability

[0080] As shown in Table 1, there was no significant difference in cell viability between the test group 1-5 and the blank control group (P>0.05), indicating that the traditional Chinese medicine preparation of the present invention did not have a significant effect on esophageal cancer cells Eca-109 and has good biosafety.

[0081] 2.2 Effects on the levels of inflammatory factors

[0082] As shown in Table 1, compared with the blank control group, the levels of TNF-α, IL-1β, and IL-8 in test groups 1-5 were significantly reduced. Specifically, test group 1 showed the most significant reduction in the levels of the three inflammatory factors (P<0.01), and its inhibitory effect on the inflammatory response was the best among all test groups. In contrast, test groups 2-5, due to the absence of some key active ingredients in the formulation, had significantly inhibited their ability to downregulate inflammatory factors, and the reduction was less than that of test group 1.

[0083] Table 1 Comparison of cell viability and inflammatory factor levels in each group

[0084] Grouping Cell viability (%) TNF-α (pg / mL) IL-1β (pg / mL) IL-8 (pg / mL) Blank control group 100 281.80±18.33 72.40±7.75 106.45±7.77 Test substance 1 101.46±4.57 <![CDATA[123.42±17.64 ** ]]> <![CDATA[27.18±6.91 ** ]]> <![CDATA[41.16±7.12 ** ]]> Test substance 2 95.28±3.62 266.28±11.75 <![CDATA[54.34±5.93 * ]]> 91.13±8.14 Test substance 3 99.79±6.10 <![CDATA[160.11±16.29 ** ]]> <![CDATA[39.80±6.57 ** ]]> <![CDATA[83.38±7.11 * ]]> Test substance 4 100.96±4.55 <![CDATA[242.83±160 * ]]> 63.65±4.24 <![CDATA[63.19±6.89 ** ]]> Test substance 5 103.11±3.88 <![CDATA[183.15±14.60 ** ]]> <![CDATA[33.18±6.16 ** ]]> <![CDATA[58.71±8.13 ** ]]>

[0085] Note: Compared with the control group, * P<0.05, ** P<0.01.

[0086] 3. Conclusion

[0087] This experiment systematically evaluated the effects of the traditional Chinese medicine preparation of this invention on esophageal cancer cells Eca-109 from two dimensions: biosafety and anti-inflammatory activity, through cell viability detection and inflammatory factor level measurement. The specific analysis is as follows: From a biosafety perspective, all test compounds 1-5 exhibited good safety. From an anti-inflammatory activity perspective, the anti-inflammatory effects of test compounds 1-5 showed clear differences, with test compound 1 (complete components) showing the most significant downregulation of inflammatory factors, indicating that the complete formula can exert its optimal effect through the synergistic effect of multiple components. In summary, the traditional Chinese medicine preparation of this invention exerts its potential anti-tumor effect by regulating the inflammatory microenvironment while ensuring biosafety.

[0088] Experimental Example 2

[0089] 1. Materials and Methods

[0090] 1.1 Animals and Cells

[0091] Forty BALB / c-nu nude mice, 5-6 weeks old, weighing 18-20 g, were housed in an environment with a temperature of 24-26℃ and a humidity of 65%-70%, with free access to food and water.

[0092] Human esophageal squamous cell carcinoma cell line EC9706 was routinely cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0093] 1.2 Experimental Drugs

[0094] Clematis chinensis 15 g, Hedyotis diffusa 15 g, Laminaria japonica 13 g, Actinidia chinensis root 13 g, Rhizoma Sinapis alba 11 g, Nelumbo nucifera leaf 12 g, Rhizoma Scutellariae 12 g, Radix Adenophorae 10 g, Cortex Eupolyphaga sinensis 10 g, Persimmon calyx 8 g, Dioscoreae sibiricum 8 g, Citrus reticulata peel 12 g, Vespa nidus 12 g.

[0095] Weigh each ingredient according to the above weight ratio, wash them, and dry them at 60℃. Place Clematis chinensis, kelp, Hedyotis diffusa, Actinidia chinensis root, Adenophora stricta, and Dioscorea bulbifera in a clay pot, add an appropriate amount of distilled water and soak for 30 minutes. Bring to a boil over high heat, then simmer over low heat for 20 minutes. Subsequently, wrap the chaff and bee honeycomb in sterile gauze and add them together with Rhizoma Sinapis alba, Nelumbo nucifera leaf calyx, Citrus reticulata peel, Persimmon calyx, and Eupolyphaga sinensis. Continue to decoct for 10 minutes, filter, and collect the filtrate. Add warm water to the dregs again and decoct a second time using the same method (bring to a boil over high heat, then simmer over low heat for 20 minutes). Filter. Combine the two filtrates and concentrate under vacuum at 45℃ to a relative concentration of 1.13. Store at 4℃ for later use.

[0096] 1.3 Model construction, grouping, and drug administration

[0097] EC9706 cells were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum and incubated at 37°C with 5% CO2. When the cell density reached 80%, the cells were digested with 0.25% trypsin-EDTA, collected, centrifuged, washed twice with serum-free RPMI 1640 medium, counted using a hemocytometer, resuspended, and diluted to 2×10⁶ cells / mL. 7 Prepare a cell suspension of cells / mL for later use.

[0098] Eight BALB / c-nu nude mice were randomly selected as a blank control group after one week of acclimatization, while the remaining nude mice were used to establish a tumor model. 0.1 mL of cell suspension was subcutaneously injected into the axilla of the right forelimb of each nude mouse. The growth of the tumor implanted in the axilla was observed daily until the tumor volume reached 100-200 mm. 3The model was considered successful when the tumor was established. The tumor-bearing nude mice were randomly divided into a model control group (gavaged with an equal volume of physiological saline), a low-dose group, a medium-dose group, a high-dose group (50, 100, 200 mg / kg / day, administered by gavage), and a cisplatin group (3 mg / kg, 0.2 mL / mouse, once a week) according to the tumor volume. Each group consisted of 8 mice, and the mice were administered the medication for 4 consecutive weeks.

[0099] 1.4 Detection Indicators

[0100] 1.4.1 Tumor volume and weight

[0101] Every week, the long diameter (a) and short diameter (b) of the tumor were measured with calipers. The tumor volume was calculated according to formula (2), and the tumor growth curve was plotted. After the last administration, the nude mice were euthanized, the tumor tissue was dissected, weighed, and the tumor inhibition rate was calculated according to formula (3).

[0102] Tumor volume (V) = ab 2 / 2 formula (2)

[0103] Tumor inhibition rate (%) = (1 - average tumor weight in the treatment group / average tumor weight in the model group) × 100% Equation (3)

[0104] 1.4.2 Immune function tests

[0105] Peripheral blood was collected from nude mice using the ocular blood sampling method after the last administration. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation at 4°C and 3000 rpm for 15 min. CD4+ levels in the PBMCs were measured by flow cytometry. + T cells, CD4 + / CD25 + Tr cell levels.

[0106] 1.4.3 Safety Evaluation

[0107] After the last administration, three nude mice were randomly selected from each group, and 3 mL of venous blood was collected by ocular blood collection. The blood was centrifuged at 4℃ and 3000 rpm for 15 min, and the supernatant was collected. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (Cr), and blood urea nitrogen (BUN) levels were detected by a fully automated biochemical analyzer.

[0108] 1.5 Statistical Methods

[0109] The data were analyzed using Graphpad Prism 8 statistical software. Experimental data are expressed as mean ± standard deviation. The data indicates that one-way ANOVA was used for comparisons between groups, and a p-value < 0.05 was considered statistically significant.

[0110] 2 Results

[0111] 2.1 Tumor volume and weight

[0112] Depend on Figure 1 The tumor growth curves and data in Table 2 show that, compared with the model group, the traditional Chinese medicine preparation of this invention significantly reduced the volume and weight of EC9706 cell xenografts in nude mice, and the difference was statistically significant (P<0.05, P<0.01). Further analysis showed that the antitumor effect of this preparation exhibited a clear dose-dependent effect. Calculated by tumor weight, the tumor inhibition rate reached 57.85% in the high-dose group, 36.68% in the medium-dose group, and 22.09% in the low-dose group. These results fully demonstrate that the traditional Chinese medicine preparation of this invention has a clear inhibitory effect on the growth of esophageal cancer EC9706 cell xenografts.

[0113] Table 2 Comparison of tumor inhibition rates among groups

[0114] Grouping Tumor inhibition rate (%) Blank control group — Model control group — low-dose group 22.09±0.06 medium dose group 36.68±0.01 High-dose group 57.85±0.04 cisplatin group 74.39±0.03

[0115] 2.2 Immune function

[0116] Table 3 shows that, compared with the blank control group, the serum CD4 levels in the model control group nude mice were significantly higher. + The level decreased significantly, CD4 + / CD25 + The levels were significantly elevated (P<0.01), suggesting that the tumor-bearing state led to a significant immunosuppressive microenvironment in nude mice; compared with the model control group, the low, medium, and high dose groups of the traditional Chinese medicine preparation of this invention showed a clear dose-dependent improvement effect: CD4 + Levels significantly increased, CD4 + / CD25 + The levels decreased significantly (P<0.05, P<0.01), indicating that the traditional Chinese medicine preparation of this invention can exert its effect by bidirectionally regulating the balance of immune cell subsets: on the one hand, increasing CD4 levels... + On the one hand, it increases the number of effector T cells to enhance the immune response, and on the other hand, it reduces the proportion of Tregs to alleviate excessive immunosuppression, thereby reshaping the body's immune balance and enhancing anti-tumor immunity.

[0117] Table 3 Comparison of serum immune indicators in nude mice of different groups

[0118] Grouping <![CDATA[CD4 + (%)]]> <![CDATA[CD4 + / CD25 + ]]> Blank control group 47.19±1.76 0.60±0.09 Model control group <![CDATA[32.78±1.85 ## ]]> <![CDATA[1.54±0.11 ## ]]> low-dose group <![CDATA[37.12±1.24 * ]]> <![CDATA[1.25±0.13 * ]]> medium dose group <![CDATA[39.59±2.03 ** ]]> <![CDATA[1.03±0.11 ** ]]> High-dose group <![CDATA[43.04±1.58 ** ]]> <![CDATA[0.84±0.10 ** ]]> cisplatin group <![CDATA[44.68±2.26 ** ]]> <![CDATA[0.70±0.08 ** ]]>

[0119] Note: Compared with the blank group, # P<0.05, ## P<0.01; compared with the control group, * P<0.05, ** P<0.01.

[0120] 2.3 Safety Evaluation

[0121] Depend on Figure 2 It can be seen that although the levels of ALT, AST, Cr, and BUN in each dosage group of the traditional Chinese medicine preparation of this invention were slightly higher than those in the blank control group, the differences were not statistically significant (P>0.05), and all indicators were within the normal reference range. This indicates that the traditional Chinese medicine preparation of this invention did not cause significant liver and kidney damage in nude mice at effective doses, further verifying its good biocompatibility. However, the serum ALT, AST, Cr, and BUN levels in the cisplatin group of nude mice were significantly higher than those in the blank control group (P<0.01), and some indicators were close to or exceeded the upper limit of the normal reference range, revealing that cisplatin has certain hepatotoxicity and nephrotoxicity.

[0122] 3. Conclusion

[0123] The traditional Chinese medicine preparation of this invention can inhibit the growth of esophageal cancer EC9706 cell xenografts in nude mice in a dose-dependent manner; it can also reshape immune balance by bidirectionally regulating immune cell subsets and enhancing the body's anti-tumor immunity; at effective doses, it did not cause liver or kidney damage in nude mice, and its safety is superior to cisplatin. These experimental results provide a new approach for the clinical treatment of esophageal cancer that combines safety and efficacy.

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine preparation for treating esophageal cancer, characterized in that, The traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 5-25 parts, Hedyotis diffusa 5-25 parts, Laminaria japonica 3-21 parts, Actinidia chinensis root 3-21 parts, Rhizoma Scrophulariae 2-20 parts, Nelumbo nucifera leaf stalk 2-22 parts, Glutinosa scabra 2-22 parts, Adenophora stricta 1-20 parts, Eupolyphaga sinensis 1-20 parts, Persimmon calyx 1-18 parts, Dioscorea bulbifera 1-18 parts, Citrus reticulata peel 2-20 parts, and Vespa nidus 2-20 parts; the traditional Chinese medicine preparation is prepared according to the following method: (1) Weigh each medicinal material according to the above weight proportions, wash them, and dry them in an oven at 60℃; (2) Place Clematis chinensis, kelp, Hedyotis diffusa, Actinidia chinensis root, Adenophora stricta, and Dioscorea bulbifera in a clay pot, add an appropriate amount of distilled water and soak for 30 minutes. Bring to a boil over high heat and then simmer over low heat for 20 minutes. (3) Then wrap the rice bran and honeycomb in sterile gauze, add them together with the rhizome of the swollen joint, lotus leaf stalk, orange peel, persimmon stalk, and nine-fragrant insect, continue to decoct for 10 minutes, filter, and collect the filtrate. (4) Add warm water to the dregs, bring to a boil over high heat, then simmer over low heat for 20 minutes. Filter and combine the two filtrates to obtain the final product.

2. A traditional Chinese medicine preparation for treating esophageal cancer according to claim 1, characterized in that, The traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 10-20 parts, Hedyotis diffusa 10-20 parts, Laminaria japonica 8-17 parts, Actinidia chinensis root 8-17 parts, Rhizoma Sinapis alba 6-15 parts, Nelumbo nucifera leaf stalk 6-17 parts, Cortex Prunella vulgaris 6-17 parts, Radix Adenophorae 5-15 parts, Inula japonica 5-15 parts, Persimmon calyx 3-12 parts, Dioscorea bulbifera 3-12 parts, Citrus reticulata peel 6-15 parts, and Vespa nidus 6-15 parts.

3. A traditional Chinese medicine preparation for treating esophageal cancer according to claim 1, characterized in that, The traditional Chinese medicine preparation is composed of the following raw materials in parts by weight: Clematis chinensis 15 parts, Hedyotis diffusa 15 parts, Laminaria japonica 13 parts, Actinidia chinensis root 13 parts, Rhizoma Sinapis alba 11 parts, Nelumbo nucifera leaf calyx 12 parts, Cortex Prunella vulgaris 12 parts, Radix Adenophorae 10 parts, Cortex Eupolyphaga sinensis 10 parts, Persimmon calyx 8 parts, Dioscoreae scutellariae 8 parts, Citrus reticulata peel 12 parts, and Vespa nidus 12 parts.

4. A method for preparing a traditional Chinese medicine preparation for treating esophageal cancer as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Weigh each medicinal material according to the above weight proportions, wash them, and dry them in an oven at 60℃; (2) Place Clematis chinensis, kelp, Hedyotis diffusa, Actinidia chinensis root, Adenophora stricta, and Dioscorea bulbifera in a clay pot, add an appropriate amount of distilled water and soak for 30 minutes. Bring to a boil over high heat and then simmer over low heat for 20 minutes. (3) Then wrap the rice bran and honeycomb in sterile gauze, add them together with the rhizome of the swollen joint, lotus leaf stalk, orange peel, persimmon stalk, and nine-fragrant insect, continue to decoct for 10 minutes, filter, and collect the filtrate. (4) Add warm water back to the dregs, bring to a boil over high heat, then simmer over low heat for 20 minutes, filter, and combine the two filtrates to obtain the decoction of the Chinese medicine preparation of the present invention.

5. The use of the traditional Chinese medicine preparation according to any one of claims 1 to 3 in the preparation of a medicament for treating esophageal cancer.

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