Application of berberine combined with formononetin in preparation of anti-tumor drugs

Abstract

The application discloses application of berberine combined with formononetin in preparation of an antitumor drug, and particularly relates to treatment of nasopharyngeal carcinoma. After the two drugs are combined, an unexpected synergistic effect exists, and no toxic side effect is caused, so that the application has great application value in treatment of nasopharyngeal carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to berberine and cypermethrin as drugs for treating tumors and their applications, and more specifically to drugs for treating nasopharyngeal carcinoma. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is an epithelial malignant tumor, accounting for approximately 80% of all NPC cases in my country, making it the leading cause of head and neck cancer in the country. Its pathogenesis is not fully understood, but it is currently believed to be closely related to genetic factors, microbial infections (such as EBV), environment, diet, and social factors. Diagnosis of NPC mainly relies on routine imaging and pathological examinations, lacking reliable early diagnostic indicators and methods. Therefore, when patients seek medical attention, 70%-80% already have cervical lymph node metastasis, and 75% are diagnosed at stage III or IV.

[0003] Due to the deep and narrow location of the nasopharynx, and the presence of numerous important blood vessels, nerves, and lymphatic tissues in its vicinity, surgical treatment is extremely difficult. Therefore, radiotherapy remains the primary treatment for nasopharyngeal carcinoma. However, radiotherapy only targets the local lesion and cannot prevent distant metastasis. Furthermore, over 95% of nasopharyngeal carcinomas are poorly differentiated or undifferentiated, exhibiting high malignancy, rapid growth, and a high likelihood of local recurrence, lymphatic or hematogenous metastasis. This is a major reason for radiotherapy failure, resulting in a 5-year survival rate of only 40%-60%. Distant metastasis is a prominent factor leading to further deterioration of the disease. Statistics show that the 5-year cumulative recurrence rate for nasopharyngeal carcinoma patients is 19.8%-36.0%, and the distant metastasis rate is 21.2%-41.8%. Finding effective drugs to treat nasopharyngeal carcinoma remains a pressing issue in the field of nasopharyngeal carcinoma treatment.

[0004] Currently, common chemotherapy drugs for nasopharyngeal carcinoma include cisplatin, 5-fluorouracil, and nedaplatin. These drugs all have certain cytotoxicity, and monotherapy easily leads to drug resistance. Furthermore, the toxic side effects of radiotherapy combined with chemotherapy are significant, substantially reducing the patient's quality of life. Nasopharyngeal carcinoma requires induction chemotherapy, concurrent chemoradiotherapy, or adjuvant chemotherapy depending on the stage. Generally, taxane-based chemotherapy combined with platinum-based drugs is used, while some patients choose oral tegafur or capecitabine. Side effects of radiotherapy manifest as systemic and local reactions. Systemic reactions include a series of functional disorders and imbalances, such as lethargy, decreased appetite, weakness, fatigue, nausea, vomiting, and postprandial fullness. Local reactions include dry skin manifesting as itching, pigmentation, and peeling, which can produce permanent light brown spots; and moist skin manifesting as eczema and blisters at the irradiation site, which can lead to erosion and ulceration in severe cases. There are also oral mucosal reactions. If the oral and pharyngeal mucosa is located within the radiotherapy area, acute oral and pharyngeal mucosal reactions are common, usually manifesting as redness, swelling, ulceration, altered taste, pain, and difficulty swallowing. The side effects of radiotherapy are often unbearable for patients in poor physical condition. Chemotherapy side effects include anemia, leukopenia, thrombocytopenia, and weakened immunity; neurotoxic reactions such as numbness in the hands and feet; gastrointestinal reactions, with most patients experiencing nausea, vomiting, and loss of appetite; a tendency to develop oral ulcers; potential hair loss; and the possibility of allergic reactions to chemotherapy drugs.

[0005] Traditional Chinese medicine (TCM) treatment is characterized by its multi-stage, multi-target, and multi-pathway approach. Its clinical application can effectively control tumors, not only reducing the toxicity and enhancing the efficacy of radiotherapy and chemotherapy, but also playing a vital role in tumor rehabilitation. Given the characteristics of TCM treatment, identifying effective Chinese herbal medicines, parts, or individual components for treating nasopharyngeal carcinoma is crucial for improving efficacy and prognosis. Therefore, the search for low-toxicity, highly effective, and natural novel anticancer drugs and the development of combination therapy regimens are current hot topics in nasopharyngeal carcinoma research.

[0006] Berberine is a major component of the traditional Chinese medicine Coptis chinensis, and it can inhibit the growth of various tumor cells, including ovarian cancer, cervical cancer, breast cancer, lung cancer, liver cancer, colorectal cancer, gastric cancer, kidney cancer, bladder cancer, and prostate cancer. Astragalin is one of the main active components of the traditional Chinese medicine Astragalus membranaceus, and it can inhibit the growth of breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, liver cancer, gastric cancer, lung cancer, osteosarcoma, and melanoma cells. Their effects are mainly related to inhibiting cell proliferation, interfering with the cell cycle, inducing apoptosis, and increasing the sensitivity to chemotherapy drugs. Regarding nasopharyngeal carcinoma, berberine can improve radiosensitivity and inhibit the proliferation, tumorigenesis, and migration of nasopharyngeal carcinoma cells. Astragalin can inhibit the proliferation of nasopharyngeal carcinoma cells and induce apoptosis; however, there are also reports that astragalin promotes the proliferation of nasopharyngeal carcinoma cells. Whether it is anti- or pro-nasopharyngeal carcinoma-specific remains controversial. There are currently no reports of a synergistic anti-tumor effect between berberine and astragalin. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing tumor treatment technologies by providing an application of berberine combined with cypermethrin in the preparation of antitumor drugs.

[0008] To achieve the above objectives, the present invention provides an antitumor drug composition, characterized in that its active ingredients are berberine and cypermethrin.

[0009] The anti-tumor effect is achieved by inhibiting tumor cell proliferation, inducing tumor cell apoptosis, and inhibiting tumor cell migration.

[0010] The drug also includes one or more pharmaceutically acceptable excipients.

[0011] The antitumor drugs are available in capsule, tablet, oral, microcapsule, or injection formulations. The administration methods include injection, oral, parenteral, inhalation spray, or transdermal delivery.

[0012] The tumor targeted by this invention is a solid tumor, more preferably a primary or secondary solid tumor, and more specifically nasopharyngeal carcinoma.

[0013] In one embodiment, the corresponding drug is prepared according to the following dosages: berberine dosage is 5 mg / kg-20 mg / kg, cypermethrin dosage is 5 mg / kg-30 mg / kg, and the dosages of the two drugs meet the ratio of 1:0.5 to 1:2.

[0014] On the other hand, a medicine box for treating nasopharyngeal carcinoma is characterized by comprising individually packaged berberine and gentianin preparations. Preferably, the effective amounts of berberine and gentianin in the medicine box are in a ratio of 1:0.5 to 1:2.

[0015] Furthermore, this invention also provides the use of berberine and cypermethrin as active ingredients in the preparation of drugs for treating nasopharyngeal carcinoma.

[0016] The technical solution adopted in this invention is the application of berberine combined with cypermethrin in the preparation of antitumor drugs. The optimal combination of the two is: berberine at a dose of 5 mg / kg-20 mg / kg and cypermethrin at a dose of 5 mg / kg-30 mg / kg, used in a ratio of 1:1, 1:2, or 2:1. Preferably, the effective therapeutic dose of berberine and cypermethrin is 10 mg / kg, which can be used in a 1:1, 1:2, or 2:1 ratio, with the dosage appropriately increased by 2-3 times. In clinical applications, the dosage can be calculated using the animal-to-human dosage conversion formula.

[0017] This invention is based on research findings demonstrating the significant synergistic effect of berberine and cytosolic flavonoids. The synergistic effect, achieved at the optimal ratio, can reach a CI value as low as 0.0022, significantly exceeding expectations. Therefore, at the same dosage, the combined use of the two drugs exhibits a significantly better therapeutic effect than berberine or cytosolic flavonoids alone. Furthermore, the side effects of each drug were observed in the experiments, revealing that the combined use of the two drugs resulted in virtually no adverse reactions. The inventors further demonstrated through experiments that both berberine and cytosolic flavonoids, when used alone, can induce apoptosis in nasopharyngeal carcinoma CNE2 cells via the mitochondrial-cytochrome C and endoplasmic reticulum pathways. The combination of the two drugs induces apoptosis in nasopharyngeal carcinoma CNE2 cells via these pathways, and simultaneous use results in a stronger apoptotic effect. If the same effect as using either drug alone is desired, the concentrations of both drugs can be reduced. Furthermore, the two drugs have different mechanisms of action. For example, berberine, in addition to its direct anti-tumor effect, can also enhance the body's innate immune function, inhibit spleen cell apoptosis and increase its transformation rate, thereby exerting an indirect anti-tumor effect. Additionally, berberine can exert anti-fatigue effects by reducing free radical generation, enhancing antioxidant enzyme activity, and regulating energy metabolism. Berberine, besides its direct anti-tumor effect, can also exert immunomodulatory effects through mechanisms such as inhibiting Th1 and Th17 cell differentiation, regulating the Th1 / Th2 balance, inhibiting T cell proliferation, inducing apoptosis of dendritic cells and macrophages, inhibiting the production of pro-inflammatory factors and antibodies, and protecting epithelial barrier function, thus producing an indirect anti-tumor effect. The two drugs exert their effects from different aspects through different molecular mechanisms, ultimately synergistically producing a strong anti-tumor effect, which is stronger than expected. Attached Figure Description

[0018] Figure 1 The induction effect of berberine combined with gentianin on apoptosis in nasopharyngeal carcinoma cells and the combination index;

[0019] Figure 2 The effect of berberine combined with gentianin on the nuclei of nasopharyngeal carcinoma cells;

[0020] Among them, A.Control; B.Ber5μM; C.Ber10μM; D.Form5μM; E.Form10μM; F.Ber10μM+Form10μM; G.Ber10μM+Form5μM; H.Ber5μM+Form10μM; -1 ;

[0021] Figure 3 The effect of berberine combined with gentianin on mitochondrial membrane potential in nasopharyngeal carcinoma cells;

[0022] Among them, compared with the control group, **P<0.01;

[0023] Figure 4 Inhibitory effect of berberine combined with gentianin on the migration of nasopharyngeal carcinoma cells (scratch assay);

[0024] Figure 5 Inhibitory effect of berberine combined with gentianin on the migration of nasopharyngeal carcinoma cells (Transwell assay);

[0025] Among them, A: Control; B: Ber 5μM; C: Ber 10μM; D: Form 5μM; E: Form 10μM; F: Ber 5μM+Form 5μM; G: Ber 5μM+Form 10μM; H: Ber 10μM+Form 5μM; 10μM; J:5-Fu2μg·mL -1 ;

[0026] Figure 6 Berberine combined with human cypermethrin inhibited the growth of nasopharyngeal carcinoma xenografts in nude mice (n=5);

[0027] Wherein, Con: physiological saline of the same volume as the experimental group; Ber: 10 mg / kg berberine; Form: 10 mg / kg cypermethrin; Ber+Form: 10 mg / kg berberine + 10 mg / kg cypermethrin; Cis: 3 mg / kg cisplatin;

[0028] Compared with the Control group, *P<0.05; compared with the Ber+Form group, #P<0.05.

[0029] Figure 7 Tunel method for detecting cell apoptosis in transplanted tumor tissue (n=5);

[0030] Wherein, Con: physiological saline of the same volume as the experimental group; Ber: 10 mg / kg berberine; Form: 10 mg / kg cypermethrin; Ber+Form: 10 mg / kg berberine + 10 mg / kg cypermethrin; Cis: 3 mg / kg cisplatin; compared with the Control group, *P<0.05; compared with the Ber+Form group, #P<0.05;

[0031] Figure 8 Effects of berberine combined with gentianin on body weight in nude mice;

[0032] Wherein, Con: physiological saline of the same volume as the experimental group; Ber: 10 mg / kg berberine; Form: 10 mg / kg cypermethrin; Ber+Form: 10 mg / kg berberine + 10 mg / kg cypermethrin; Cis: 3 mg / kg cisplatin. Figure 9 Effects of berberine combined with ginsenoside Rg3 on liver and kidney coefficients in nude mice;

[0033] Figure 9 Effects of berberine combined with gentianin on liver and kidney coefficients in nude mice;

[0034] Among them, (A) liver coefficient, compared with the control group, *P<0.05; compared with the Cis group, #P<0.05, n=5. (B) kidney coefficient, compared with the control group, *P<0.05; compared with the Cis group, #P<0.05, n=5;

[0035] Con: Physiological saline of the same volume as the experimental group; Ber: 10 mg / kg berberine; Form: 10 mg / kg cypermethrin; Ber+Form: 10 mg / kg berberine + 10 mg / kg cypermethrin; Cis: 3 mg / kg cisplatin;

[0036] Figure 10 The synergistic effects of berberine combined with cypermethrin on the survival, apoptosis, and expression of apoptosis-related proteins in nasopharyngeal carcinoma CNE2 cells;

[0037] Compared with the solvent control group, **P<0.01; compared with the single-use group, #P<0.05, ##P<0.01;

[0038] Figure 11 The effect of adding inhibitors on the expression of proteins related to the mitochondrial and endoplasmic reticulum pathways in nasopharyngeal carcinoma CNE2 cells was **P<0.01 compared with the solvent control group; the drug plus inhibitor group was #P<0.05, ##P<0.01 compared with the combination group.

[0039] Figure 12The effect of adding inhibitors on the apoptosis rate of nasopharyngeal carcinoma CNE2 cells;

[0040] Among them, compared with the solvent control group, **P<0.01; compared with the combination group, ##P<0.01. Detailed Implementation

[0041] The present invention will be explained more clearly through a detailed description of specific embodiments, but this does not constitute a limitation thereof.

[0042] Example 1: Synergistic effect experiment of berberine combined with gentianin to inhibit the proliferation of nasopharyngeal carcinoma cells.

[0043] 1. Inhibitory effect of berberine alone on nasopharyngeal carcinoma cells

[0044] Treatment of CNE2 nasopharyngeal carcinoma cells with berberine alone significantly inhibited the proliferation of nasopharyngeal carcinoma cells at 24, 36, 48 and 72 hours, with inhibition rates shown in Table 1.

[0045] Table 1. Inhibitory effect of berberine on the proliferation of nasopharyngeal carcinoma cells ( %, n=3)

[0046]

[0047] 2. The inhibitory effect of gentianin alone on nasopharyngeal carcinoma cells.

[0048] Treatment of CNE2 nasopharyngeal carcinoma cells with form alone significantly inhibited the proliferation of nasopharyngeal carcinoma cells at 24, 36, 48 and 72 hours, with inhibition rates shown in Table 2.

[0049] Table 2. Inhibitory effect of basidiostanoic acid on the proliferation of nasopharyngeal carcinoma cells ( %, n=3)

[0050]

[0051] 3. The effect of berberine combined with gentianin on inhibiting the proliferation of nasopharyngeal carcinoma cells.

[0052] The effects of different concentrations of berberine and different concentrations of gentianin on the proliferation of nasopharyngeal carcinoma cells were observed.

[0053] Berberine (Ber) and form were used in combination at different doses. The combination index (CI) was calculated to reflect the effect of the two drugs. CI > 1 indicates an antagonistic effect, CI = 1 indicates an additive effect, and CI < 1 indicates a synergistic effect. For CI less than 1, the smaller the CI value, the stronger the synergistic effect, meaning the better the synergistic effect of the two drugs used together. The results are shown in Table 3.

[0054] At 24 hours of drug exposure, the CI values ​​for the (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 5), (Ber 10+Form 10), (Ber 10+Form 20), and (Ber 20+Form 20) groups were 0.18, 0.02, 0.0022, 0.067, 0.011, and 0.0088, respectively. At 36 hours of drug exposure, the CI values ​​for the (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 5), (Ber 10+Form 10), (Ber 10+Form 20), and (Ber 20+Form 20) groups were 0.26, 0.26, 0.0598, 0.41, 0.21, and 0.20, respectively. At 48 hours of drug exposure, the CI values ​​for the (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 10), (Ber 10+Form 20), and (Ber 20+Form 20) groups were 0.18, 0.02, 0.0022, 0.067, 0.011, and 0.0088, respectively. The CI values ​​for groups 10), (Ber 10+Form 5), (Ber 10+Form 10), (Ber 10+Form 20), and (Ber 20+Form 20) were 0.32, 0.16, 0.0196, 0.29, 0.12, and 0.09, respectively. After 72 hours of drug treatment, the CI values ​​for groups (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 5), (Ber 10+Form 10), (Ber 10+Form 20), and (Ber 20+Form 20) were... The CI values ​​for groups 20 were 0.759, 0.469, 0.07577, 0.53308, 0.47703, and 0.179, respectively. All of these groups had CI values ​​less than 1, indicating a synergistic effect, and the synergistic effect was very strong. In particular, after 24 hours of combined drug action, the CI values ​​were all much less than 1, indicating a very strong synergistic effect. The (Ber 10 + Form 5) group showed a very strong synergistic effect at all time points.

[0055] Table 3. Inhibitory effect of berberine combined with gentianin on the proliferation of nasopharyngeal carcinoma cells and the combination index ( %, n=3)

[0056]

[0057] Example 2: Berberine combined with gentianin has a synergistic effect in inducing apoptosis in nasopharyngeal carcinoma cells.

[0058] Based on the results of Example 1, a factorial design was used to select berberine (Ber) at different concentrations of 0, 5 μM, and 10 μM, combined with berberine (Form) at different concentrations of 0, 5 μM, and 10 μM, and divided into 9 groups: 0 (control, solvent control group), Ber 5, Ber 10, Form 5, Form 10, (Ber 5 + Form 5), (Ber 5 + Form 10), (Ber 10 + Form 5), and (Ber 10 + Form 10). A positive control cisplatin group (concentration 4.0 μg / mL) was also set up. -1 To observe the effect of the combined use of the two drugs on apoptosis of nasopharyngeal carcinoma cells.

[0059] 1. Flow cytometry detection of the effect of berberine combined with gentianin on induced apoptosis in nasopharyngeal carcinoma cells.

[0060] Flow cytometry was used to further investigate the effect of drugs on apoptosis of nasopharyngeal carcinoma cells. Berberine (0, 5 μM, and 10 μM) was combined with 0, 5 μM, and 10 μM berberine, respectively, to observe their effects on apoptosis of nasopharyngeal carcinoma cells, and the combination drug index was calculated. According to the method for analyzing the combined drug effect, a combination index CI > 1 indicates an antagonistic effect, CI = 1 indicates an additive effect, and CI < 1 indicates a synergistic effect. After CI value less than 1, a smaller CI value indicates a stronger synergistic effect, meaning a better effect from the combined use of the two drugs. Results were obtained after 36 hours of drug treatment. Figure 1 The CI values ​​for the (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 5), and (Ber 10+Form 10) groups were 0.06087, 0.03191, 0.12777, and 0.05164, respectively. Since the CI values ​​were much less than 1, the combined use of the two drugs had a very strong synergistic effect.

[0061] 2. Hoechst 33342 staining observation of the effect of berberine combined with gentianin on the morphology of nasopharyngeal carcinoma cells.

[0062] The effect of Hoechst 33342 staining on the morphology of CNE2 nasopharyngeal carcinoma cells after 36 hours of drug treatment was observed. Hoechst 33342 staining was performed after 36 hours of drug treatment. Figure 2 The results showed that the nuclei of cells in the untreated group were uniformly pale blue (A), while the cells in the drug-treated groups exhibited obvious apoptotic characteristics, namely, some nuclei were granular and bright blue, accompanied by nucleus marginalization, fragmentation, and shrinkage. Furthermore, the fluorescence intensity of the combined groups (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 5), and (Ber 10+Form 10) was stronger than that of the single-drug groups (BH). This suggests that the combined drug treatment has a strong apoptosis-inducing effect.

[0063] 3. Effects of berberine combined with gentianin on mitochondrial membrane potential in nasopharyngeal carcinoma cells

[0064] The mitochondrial membrane potential of nasopharyngeal carcinoma cells after drug treatment was detected using a mitochondrial membrane potential assay kit (JC-10). Data and fluorescence images showed that berberine and gentianin, alone or in combination, significantly affected the mitochondrial membrane potential of nasopharyngeal carcinoma cells, disrupting mitochondrial membrane integrity and indirectly reflecting drug-induced mitochondrial apoptosis. (See [link to kit]). Figure 3 Red represents normal cells, and green represents apoptotic cells.

[0065] Example 3: Berberine combined with gentianin has a synergistic effect in inhibiting the migration of nasopharyngeal carcinoma cells.

[0066] 1. Observation of the effect of berberine combined with gentianin on inhibiting the migration of nasopharyngeal carcinoma cells using scratch assay.

[0067] Berberine (0, 5 μM, and 10 μM) was combined with berberine (0, 5 μM, and 10 μM) to observe its effect on nasopharyngeal carcinoma cell migration using a scratch assay, and the combination drug index was calculated. According to the method for analyzing the combined drug effect, a combination index (CI) > 1 indicates an antagonistic effect, CI = 1 indicates an additive effect, and CI < 1 indicates a synergistic effect. For CI values ​​less than 1, a smaller CI value indicates a stronger synergistic effect, meaning a better effect from the combined use of the two drugs. Results are shown below. Figure 4 See Table 4. Based on the migration inhibition rate, all drug groups significantly inhibited nasopharyngeal carcinoma cell migration. The migration inhibition rates of the (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 5), and (Ber 10+Form 10) groups were significantly higher than those of the single-drug groups. The combination indices for the (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 5), and (Ber 10+Form 10) groups were 0.44, 0.02, 0.72, and 0.40, respectively, all significantly less than 1, indicating a strong synergistic inhibitory effect from the combination of the two drugs.

[0068] Table 4. Inhibitory effect of berberine combined with gentianin on nasopharyngeal carcinoma cell migration and the combination index (scratch assay).

[0069]

[0070] 2. Transwell assay to observe the effect of berberine combined with gentianin on inhibiting the migration of nasopharyngeal carcinoma cells.

[0071] Berberine (0, 5 μM, and 10 μM) was combined with berberine (0, 5 μM, and 10 μM) to observe its effects on nasopharyngeal carcinoma cell migration using Transwell assays, and the combination drug index was calculated. According to the analytical method for drug combination effects, a combination index (CI) > 1 indicates an antagonistic effect, CI = 1 indicates an additive effect, and CI < 1 indicates a synergistic effect. For CI values ​​less than 1, a smaller CI value indicates a stronger synergistic effect, meaning a better effect from the combined use of the two drugs. Results are shown below. Figure 5 According to Table 5, based on the migration inhibition rate, all drug groups significantly inhibited the migration of nasopharyngeal carcinoma cells. The migration inhibition rates of the (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 5), and (Ber 10+Form 10) groups were significantly higher than those of the single-drug groups. The combination indices of the (Ber 5+Form 5), (Ber 5+Form 10), (Ber 10+Form 5), and (Ber 10+Form 10) groups were 0.71, 0.71, 0.98, and 0.87, respectively, all less than 1, indicating that the combination of the two drugs had a synergistic inhibitory effect.

[0072] Table 5. Inhibitory effect of berberine combined with gentianin on nasopharyngeal carcinoma cell migration and the combination index (Transwell assay).

[0073]

[0074] Example 4: Berberine combined with cypermethrin synergistically inhibits the growth of nasopharyngeal carcinoma xenografts.

[0075] A nasopharyngeal carcinoma xenograft model was established in nude mice. Nude mice with xenograft tumors were treated intraperitoneally with 10 mg / kg berberine combined with 10 mg / kg berberine. After 18 days of treatment, the weight of the xenograft tumors was measured, and the ratio of tumor weight in each treatment group to that in the solvent control group (i.e., the untreated group) was calculated to obtain the relative weight. The average tumor weights in the berberine monotherapy group, berberine monotherapy group, and berberine combined with berberine group were 63.8%, 75.2%, and 29.4% of those in the solvent control group, respectively. The average tumor weights in the berberine monotherapy group and the berberine group were 2.17 times and 2.56 times the average tumor weights in the combined treatment group, respectively. (See...) Figure 6 The average tumor weight in the combination therapy group was significantly lower than that in the single-drug group (P<0.05), and statistical analysis showed that there was an interaction between the drugs (P<0.05), indicating a synergistic effect in inhibiting the growth of nasopharyngeal carcinoma xenografts.

[0076] Example 5: Berberine combined with gentianin induces apoptosis in nasopharyngeal carcinoma xenograft cells

[0077] A nasopharyngeal carcinoma xenograft model was established in nude mice. Cisplatin (3 mg / kg) was used as a positive control. Berberine (10 mg / kg) combined with 10 mg / kg berberine was administered intraperitoneally to the xenograft mice. After 18 days of treatment, tumor cell apoptosis was detected using the TUNEL assay. Results are shown below. Figure 7 The average apoptosis rates of the xenografts in the solvent control group, berberine alone group, berberine alone group, and berberine combined with berberine were 13.2%, 38.5%, 29.1%, and 79.5%, respectively. The average apoptosis rates of the berberine alone group and the berberine alone group were 48.4% and 36.6% of the combined treatment group, respectively. The average apoptosis rate of the combined treatment group was significantly higher than that of the single-drug group (P<0.05). Statistical analysis showed an interaction between the drugs (P<0.05), indicating that berberine combined with berberine has a synergistic effect in inducing apoptosis in nasopharyngeal carcinoma xenograft cells.

[0078] Example 6: Study on the toxic side effects of berberine combined with cypermethrin on nude mice

[0079] A nude mouse xenograft model of nasopharyngeal carcinoma was established. Berberine (10 mg / kg) combined with 10 mg / kg cypermethrin was administered intraperitoneally to the xenograft mice. After 18 days of drug treatment, the mice's condition and weight were observed. HE staining was used to detect pathological changes in tumor tissue, liver, and kidneys after drug treatment. The effects of berberine combined with cypermethrin on the liver and kidneys of nude mice were analyzed.

[0080] During the experiment, nude mice in the cisplatin group experienced a decrease in body weight in the later stages of drug administration, while the nude mice in the solvent control group and other drug administration groups showed no significant changes in body weight, food intake, defecation, activity, or mental state. Figure 8 After drug administration, liver and kidney tissues were harvested from nude mice, weighed, and the liver / kidney ratio was calculated for each group. The results showed that the liver ratio increased and the kidney ratio decreased in the cisplatin group, while there were no significant differences in the liver and kidney ratios among the other groups. Figure 9 HE staining revealed that the hepatic cord cells in the cisplatin group were not arranged very neatly, while the hepatic lobule structure was present in all other groups. Hepatocytes showed no obvious swelling or necrosis, and there was no fibrosis in the portal areas. The renal cortex and medulla structure were clear in all groups, and glomerular cells and renal tubular epithelial cells showed no obvious swelling, degeneration, or necrosis. These results indicate that berberine and gentianin have no significant toxicity to nude mice. This demonstrates that using an additional drug did not increase toxicity. Furthermore, compared with the positive control group (cisplatin), the toxicity was low.

[0081] Example 7: Study on the molecular mechanism of antitumor activity of berberine combined with cypermethrin

[0082] Examples 2 and 5 only illustrate part of the drug's mechanism of action, namely, that the drug exerts its anti-nasopharyngeal carcinoma effect by synergistically inducing apoptosis. This part further explains the molecular mechanism of the drug's action, namely, the mechanism by which berberine and cypermethrin synergistically induce apoptosis in nasopharyngeal carcinoma cells.

[0083] 1. The synergistic effect of berberine combined with gentianin on the survival, apoptosis, and expression of apoptosis-related proteins in nasopharyngeal carcinoma CNE2 cells.

[0084] Westorn blot results showed that after 36 hours of drug treatment, the expression levels of relevant proteins changed compared to the Control group. Specifically, the expression of cell survival protein Survivin and anti-apoptotic protein Bcl-2 showed a decreasing trend, while the expression of pro-apoptotic protein Bax increased; and the trend was more pronounced in the combination group than in the single-drug group. The expression levels of proteins related to each apoptosis pathway also changed compared to the Control group. Specifically, the expression levels of cleaved caspase-3 and cleaved caspase-7 increased, as did the expression levels of cleaved caspase-9 (related to the mitochondrial-cytochrome C pathway) and cleaved caspase-12 (related to the endoplasmic reticulum pathway). There was no significant difference in the expression level of cleaved caspase-8 (related to the death receptor pathway) among the groups (P > 0.05). Figure 10 Therefore, it is preliminarily determined that berberine combined with gentianin induces apoptosis in nasopharyngeal carcinoma CNE2 cells through the mitochondrial-cytochrome C pathway and the endoplasmic reticulum pathway.

[0085] 2. The roles of mitochondrial and endoplasmic reticulum apoptosis pathways in the synergistic promotion of apoptosis in nasopharyngeal carcinoma CNE2 cells by berberine combined with gentianin.

[0086] To further verify the roles of mitochondrial and endoplasmic reticulum apoptosis pathways in promoting CNE2 cell apoptosis, caspase-9 inhibitor (Z-LEHD-FMK) and caspase-12 inhibitor (Z-ATAD-FMK) were added alone or in combination. The expression levels of related apoptotic proteins and the apoptosis rate in cells were then measured, as shown in the table below. Figure 11 and Figure 12 Western blot results ( Figure 11The results showed that, compared with the combination therapy group, the expression levels of cleaved caspase-3 and cleaved caspase-7 were significantly reduced in the three groups treated with caspase-9 inhibitor (Z-LEHD-FMK) alone or in combination with caspase-12 inhibitor (Z-ATAD-FMK) (all P < 0.01). The reduction in cleaved caspase-3 and cleaved caspase-7 expression was more significant in the group treated with both inhibitors, showing a statistically significant difference compared with the group treated with only one inhibitor (all P < 0.01). Furthermore, there was no significant difference in expression levels between the group treated with both inhibitors and the control group (P > 0.05). The expression levels of cleaved caspase-9 and cleaved caspase-12 were significantly reduced in their respective inhibitor groups, but there was no statistically significant difference compared with the control group (P > 0.05). This indicates that the inhibition of cleaved caspase-9 and cleaved caspase-12 activity led to a decrease in protein expression, demonstrating that the inhibitors exerted their corresponding effects. Fluorescence double staining flow cytometry results showed that the apoptosis rate in the Control group was 1.805±0.064%, and the Ber 5 μmol·L -1 The apoptosis rate in the group was 24.5 ± 1.813%, Form 5 μmol·L -1 The apoptosis rate in the group was 21.797±1.951%, Ber 5 μmol·L -1 +Form5μmol·L -1 The apoptosis rate in the group was 28.437±1.274%, Ber 5μmol·L -1 +Form5μmol·L -1 The apoptosis rate in the +Z-LEHD-FMK group was 12.447±0.451%, Ber 5μmol·L -1 +Form5μmol·L -1 The apoptosis rate in the +Z-ATAD-FMK group was 11.387±0.473%, Ber 5μmol·L -1 +Form5μmol·L -1 The apoptosis rate in the Z-LEHD-FMK+Z-ATAD-FMK group was 2.983±0.075%. The data showed that the addition of Z-LEHD-FMK or Z-ATAD-FMK respectively reduced the drug-induced apoptosis rate (both P<0.01), but still had an apoptosis-inducing effect, and the difference compared with the Control group was still statistically significant (P<0.01). Simultaneous treatment with both inhibitors significantly reduced the drug-induced apoptosis rate (both P<0.01), and there was no significant difference between the two groups and the Control group (P>0.05). Figure 12 The changes in apoptosis rate were consistent with protein changes, namely, both caspase-9 inhibitors (Z-LEHD-FMK) and caspase-12 inhibitors (Z-ATAD-FMK) weakened the apoptosis-inducing effect of berberine combined with gentianite, and the combined effect of the inhibitors was stronger. This further proves that berberine combined with gentianite induces apoptosis in nasopharyngeal carcinoma CNE2 cells through the mitochondrial-cytochrome C pathway and the endoplasmic reticulum pathway.

Claims

1. An antitumor pharmaceutical composition, characterized in that, Its active ingredients are berberine and cypermethrin, with the effective drug amounts of the two meeting a ratio of 1:1 to 1:2; the tumor is nasopharyngeal carcinoma.

2. The pharmaceutical composition according to claim 1, characterized in that, The anti-tumor effect is achieved by inhibiting tumor cell proliferation, inducing tumor cell apoptosis, or inhibiting tumor cell migration.

3. The pharmaceutical composition according to claim 1, characterized in that, It also includes one or more pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 1, characterized in that, The antitumor drug dosage form is a capsule, tablet, microcapsule preparation or injection.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The corresponding drugs are prepared according to the following dosages: berberine dosage is 5mg / kg-20mg / kg, and cypermethrin dosage is 5mg / kg-30mg / kg, with the two dosages meeting a ratio of 1:1 to 1:

2.

6. A medicine box for treating nasopharyngeal carcinoma, characterized in that, It includes individually packaged berberine and cypermethrin preparations, with the effective amounts of berberine and cypermethrin in a ratio of 1:1 to 1:

2.

7. Berberine and cypermethrin are used as active ingredients in the preparation of drugs for treating nasopharyngeal carcinoma, wherein the effective amounts of berberine and cypermethrin meet the ratio of 1:1 to 1:2.

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