Application of traditional Chinese medicine preparation in preparation of medicine for treating tumor diseases

Abstract

The invention relates to application of a traditional Chinese medicine preparation in preparation of a medicine for treating tumor diseases. The traditional Chinese medicine preparation comprises the following raw material medicines in parts by weight: 90-110 parts of sophora flower and 15-25 parts of honeysuckle. The traditional Chinese medicine preparation disclosed by the invention has an obvious inhibition effect on melanoma, can also be used in combination with tumor drugs such as temozolomide, enhances the inhibition effect on melanoma, also can reduce the toxic effect of the drugs on cells, and enhances the immune efficacy of the drugs.

Description

Technical Field

[0001] The invention discloses use of a traditional Chinese medicine preparation in preparing medicine for treating tumor diseases, belonging to the field of new clinical uses of medicines. Background Art

[0002] Melanoma is the deadliest type of skin cancer, and its incidence has been steadily increasing in the past, exceeding that of all other malignant tumors. Currently, common treatment options for melanoma include surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy. Early-stage carcinoma in situ can be cured by surgical resection. Middle- and late-stage melanoma is prone to metastasis, and chemotherapy, targeted therapy, and immunotherapy are the main treatments. Targeted therapy and immunotherapy are often costly and prone to drug resistance. Chemotherapy is a means of treatment based on chemical drugs and is one of the important methods for treating tumors. Among them, the more effective drugs include dacarbazine (DTIC) and tamiflu (TMZ). TMZ is an alkylating agent and has been shown to have comparable efficacy to DTIC, with similar side effects. The advantage of TMZ is that it can cross the blood-brain barrier and can treat brain metastases of melanoma. Its oral bioavailability is close to 100%, and compared with intravenous administration, oral administration is convenient. As a new alkylating agent, TMZ is increasingly widely used in clinical tumor chemotherapy and has been approved for first-line treatment of melanoma in Europe, the United States and other countries. Chemotherapy drugs have a certain therapeutic effect on malignant melanoma, but it is not ideal. To enhance its efficacy, TMZ can be combined with other drugs. However, compared with TMZ alone, multi-drug combination regimens based on TMZ, such as CVT (cisplatin + vincristine + TMZ), have no improvement in overall results. The combination of TMZ and thalidomide may cause thromboembolism. In addition, biochemotherapeutic combination therapy combines interferon (INF) with TMZ, but the combined treatment has no difference in overall survival rate and produces greater blood toxicity, and has not been widely accepted. Therefore, there is an urgent need to find effective chemical adjuvant drugs.

[0003] The applicant has systematically combed through the existing technical journals and patent documents and found that the reports that traditional Chinese medicine can enhance the efficacy and reduce toxicity as a chemotherapy adjuvant have attracted more and more attention from scholars. Sun Zhengyang, Liu Nannan, Fan Xuefei, et al., Resveratrol inhibits autophagy and promotes apoptosis of uveal melanoma cells through the miR-512-3P / DUSP1 axis, Chinese Pharmacological Bulletin, 2024-01-30, the article publicly recorded: Resveratrol inhibits DUSP1 by promoting the expression of miR-512-3P, thereby inhibiting autophagy and promoting apoptosis of uveal melanoma cells. Wang Zhining, Zhu Yinan, et al., Inhibitory effect of Ganoderma lucidum fruiting body extract on lung metastasis of melanoma in C57BL / 6 mice, Fungal Research, 2023-09-14, published: Ganoderma lucidum alcohol extract inhibits the occurrence of lung metastasis of mouse melanoma by upregulating the expression of MMP-2 and MMP-9 and downregulating the expression of TIMP-2, while Ganoderma lucidum polysaccharide can inhibit the lung metastasis of mouse melanoma by upregulating Bcl-2 and downregulating the expression of Bax and Caspase-3. Zhang Fen, Dai Gengwu, The effect and mechanism of cardamom on melanoma cells, Chinese Journal of Clinical Pharmacology, 2023-11-28, the article records: cardamomum may inhibit the proliferation, migration and invasion of melanoma B16-F10 cells by inhibiting the PI3K / Akt / mTOR signaling pathway and promote its apoptosis.

[0004] CN118160921A discloses a lung protection composition and a lung protection drink, which includes 2.1 parts of lung clearing components, 7.4 parts of lung moistening components, 0.7 parts of lung solidifying components, and 2.3 parts of immune function enhancing components by mass fraction; wherein the lung clearing component includes 1.5 parts of loquat leaf concentrated juice and 0.6 parts of honeysuckle powder, the lung moistening component includes 0.8 parts of Tremella polysaccharide powder, 0.7 parts of platycodon powder, 0.6 parts of tangerine peel powder, 1.5 parts of Momordica grosvenori concentrated juice, 3 parts of snow pear concentrated juice and 0.8 parts of mint powder, the lung solidifying component is 0.7 parts of Cordyceps militaris extract, and the immune function enhancing component includes 1.5 parts of Camellia chrysantha powder and 0.8 parts of Sophora japonica extract powder. The lung protection composition can remove harmful substances such as heat evil and phlegm turbidity in the lungs, nourish the lungs, strengthen the lungs and enhance the ability of the lungs to resist external invasion, and then have a significant protective effect on the lungs. CN117562155A discloses a health tea with liver protection effect, which includes 10-30 parts of radix scutellariae, 10-30 parts of bitter buckwheat buds, 5-20 parts of total flavonoids of flos inulae, 10-30 parts of polysaccharides of Pleurotus eryngii, 10-30 parts of wheat seedlings, 10-30 parts of Sophora japonica, 5-20 parts of licorice root, 5-20 parts of honeysuckle, 5-20 parts of scutellaria baicalensis, 5-20 parts of Artemisia capillaris, 1-10 parts of gardenia, and 5-15 jujubes. The recipe has the effects of protecting liver, lowering blood lipids, and resisting blood lipids.

[0005] CN106173083A discloses a kind of Sophora japonica tea, which is composed of the following raw materials in parts by weight: 10-20 parts of Sophora japonica, 5-7 parts of Scutellaria baicalensis, 3-5 parts of Prunella vulgaris, 1-2 parts of Patchouli, 5-7 parts of Honeysuckle, 2-4 parts of Rhubarb, and 3-5 parts of Tangerine peel. The recipe has the effects of lowering blood pressure and lipid, strengthening the spleen, resolving phlegm, and softening blood vessels. The recipe of the present invention contains Sophora japonica and Honeysuckle medicinal materials. The clinical uses of the above Chinese medicine are mainly concentrated in: lung diseases (clearing lungs and detoxifying, lung heat), eye diseases (presbyopia, myopia, dry eyes, sore eyes, blurred eyes and dark circles), oral ulcers, lowering blood pressure, lowering blood lipids, lowering blood sugar, etc., but no relevant reports on melanoma diseases have been found.

[0006] At present, the chemotherapy drug TMZ for melanoma has unsatisfactory therapeutic effects and has toxic and side effects. The existing technology combines it with other treatments, but has not achieved a breakthrough in efficacy. As the clinical efficacy of traditional Chinese medicine is recognized by a large number of patients, it has broad prospects as a way to make up for the shortcomings of existing drug treatment methods and effects. Therefore, it is particularly important to develop a drug with better efficacy for melanoma and less toxic and side effects. Summary of the invention

[0007] The purpose of the present invention is to provide a use of a traditional Chinese medicine preparation in the preparation of a drug for treating tumor diseases, specifically melanoma, and can also assist in the combined use of TMZ drugs to achieve the purpose of enhancing efficacy and reducing toxicity.

[0008] The technical solution of the patent application of this invention is as follows:

[0009] A use of a traditional Chinese medicine preparation in preparing a drug for treating tumor diseases, characterized in that the raw material composition of the traditional Chinese medicine preparation and the weight proportions thereof are: 90-110 parts of Sophora japonica flowers and 15-25 parts of honeysuckle flowers.

[0010] Preferably, the raw material composition and weight proportions of the traditional Chinese medicine preparation are: 100 parts of Sophora japonica flowers and 20 parts of honeysuckle flowers.

[0011] Formula explanation: Sophora japonica of the present invention has the effect of clearing heat and cooling blood, and honeysuckle has the effect of clearing heat and detoxifying. This side is an ancient prescription for treating skin diseases based on the law of clearing heat and detoxifying, and it can treat carbuncle, back ulcer and all sores, regardless of whether it has formed or not but is painful.

[0012] Preferably, the preparation method of the traditional Chinese medicine preparation is: take Sophora japonica flower and honeysuckle flower, mix them in proportion, add 30% alcohol, decoct and extract, and filter to obtain the traditional Chinese medicine preparation.

[0013] Preferably, the dosage form of the traditional Chinese medicine preparation is different oral preparations such as oral liquid, ointment, granule, capsule, and pill.

[0014] Oral liquid: add the extracts of Sophora japonica and honeysuckle of the traditional Chinese medicine preparation of the present invention, medicinal preservatives (ethyl paraben, sodium benzoate, sorbic acid, etc.), solubilizers (Tween-80 or β-cyclodextrin, etc.), and flavoring agents (sucrose, osmanthus essence, mannitol, lemon essence, aspartame or aspartame), mix and stir evenly to prepare the oral liquid.

[0015] Ointment: The extracts of Sophora japonica and honeysuckle of the Chinese medicine preparation of the present invention are mixed and stirred evenly with auxiliary materials such as honey or maltose to prepare a Chinese medicine ointment.

[0016] Granules: The extracts of Sophora japonica and honeysuckle in the Chinese medicine preparation of the present invention are mixed with appropriate amounts of dextrin, calcium sulfate, sucrose, microcrystalline cellulose and hydroxypropyl cellulose, wet granulated, and vacuum dried under reduced pressure to prepare granules.

[0017] Capsules: Add the extracts of Sophora japonica and honeysuckle in the traditional Chinese medicine preparation of the present invention, starch slurry, calcium dihydrogen phosphate, talcum powder and magnesium stearate, granulate and fill into capsules to prepare capsules.

[0018] Dropping pills: add the extracts of Sophora japonica and honeysuckle of the traditional Chinese medicine preparation of the present invention to a matrix (such as PEG4000, PEG6000, sodium stearate, etc.), heat-melt, stir evenly, and drip to make dropping pills.

[0019] Preferably, the tumor disease being treated is melanoma.

[0020] The Chinese medicine preparation of the present invention can also be used to improve the body's immunity.

[0021] Preferably, the tumor disease to be treated is glioblastoma multiforme or anaplastic astrocytoma.

[0022] A use of a traditional Chinese medicine preparation in the preparation of an auxiliary TMZ drug for attenuation of toxicity.

[0023] Preferably, the Chinese medicine preparation is used to alleviate weight loss and liver damage in mice caused by TMZ drug.

[0024] Beneficial effects of the Chinese medicine preparation of the present invention:

[0025] ⑴. In the early stage of the efficacy experiment, it was surprisingly found that the Chinese medicine preparation of the present invention has a significant inhibitory effect on melanoma, especially it can be used in combination with TMZ and other tumor drugs to enhance its melanoma inhibitory effect and reduce the toxic effect of drugs on cells.

[0026] ⑵, the inventive concept of the Chinese medicine preparation formula of the present invention is: the bitter and slightly cold Sophora japonica is selected as the main medicine in the prescription, which belongs to the liver meridian and the large intestine meridian, and has the effects of cooling blood and stopping bleeding, clearing the liver and purging fire. Its extract contains effective components such as rutin, quercetin, tannin, Sophora japonica diol, vitamin A, etc. Assisted by the cold and sweet honeysuckle as the minister, the medicine has the effects of clearing heat and detoxifying, cooling and dispersing wind and heat. The honeysuckle extract contains chemical components such as chlorogenic acid, isochlorogenic acid, ginkgo biloba, luteolin, volatile oil, terpenes, phenolic acid, etc. The two prescriptions match each other and play the effects of clearing heat and detoxifying, eliminating tumors and enhancing immunity. This prescription is an ancient prescription for treating skin diseases with the prescription of clearing heat and detoxifying, treating carbuncle, back hair and all sores, regardless of whether it has been formed or not but is painful. The prescription is simple and has only two medicines. The efficacy is good and has a broad market application prospect.

[0027] ⑶. The pharmacological experimental results of the Chinese medicine preparation of the present invention show that: ①. The experimental results of inhibiting the growth of A375 cells show that the Chinese medicine preparation of the present invention (Sophora japonica and honeysuckle extracts) SLE / TMZ can significantly inhibit the cell proliferation of A375 cells in vitro in a dose-dependent manner and induce cell apoptosis. The effect of the combination of the two is significantly different from that of the single use.

[0028] ②. The tumor volume and tumor weight of the Chinese medicine preparation (Sophora japonica and honeysuckle extract) SLE 1.2g / kg, TMZ 25mg / kg, TMZ 50mg / kg, SLE 1.2g / kg+TMZ 25mg / kg, and SLE 1.2g / kg+TMZ 50mg / kg of the present invention were significantly reduced compared with the control group. Compared with the SLE and TMZ single-use groups, the combination of the Chinese medicine preparation SLE of the present invention and TMZ can significantly reduce tumor weight (P<0.05). This shows that the two drugs have a significant synergistic effect in reducing tumor weight. We found that the Chinese medicine preparation SLE / TMZ temozole of the present invention has a synergistic anti-melanoma effect, and the Chinese medicine preparation SLE of the present invention can alleviate the weight loss and liver damage of mice caused by TMZ to a certain extent.

[0029] ③. Compared with Control, the Chinese medicine preparations SLE, SLE / TMZ 25, and SLE / TMZ 50 of the present invention increased the percentages of Th, Tc, and NK cells in the spleen of mice, and reduced the percentage of MDSC cells. TMZ 25 and TMZ 0 increased the percentage of MDSC cells and had no significant effect on the percentages of Th (helper T cells), Tc, and NK cells. Compared with SLE, TMZ 25, and TMZ50, SLE / TMZ 25 and SLE / TMZ 50 significantly increased the percentages of Th, Tc, and NK cells in the spleen of mice, and reduced the percentage of MDSC cells. These results show that SLE / TMZ 25 and SLE / TMZ 50 increased the proportion of Th and Tc cells in the spleen of mice, and reduced the proportion of MDSC cells. The above animal experimental results suggest that the Chinese medicine preparations (sophora japonica and honeysuckle extracts) of the present invention SLE combined with TMZ enhance the immune function of melanoma mice.

[0030] The above experimental data show that the Chinese herbal preparation (Sophora japonica and honeysuckle extracts) of the present invention combined with TMZ for SLE has a synergistic anti-melanoma effect both in vitro and in vivo, and can also enhance the immune function of melanoma mice, which further illustrates that the Chinese herbal preparation of the present invention can also be used to improve the body's immunity.

[0031] In order to more fully understand the implementation of the present invention, the present invention is further described below through typical examples. The following and through pharmacodynamics to prove the beneficial therapeutic effects of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Among them, SLE represents the Chinese medicine preparation prepared from Sophora japonica Flos and Honeysuckle Flos of the present invention, and TMZ represents temozolomide.

[0033] Figure 1 :SLE / TMZ inhibits the growth of A375 melanoma cells, (A) Survival rate of A375 cells. Cells were treated with different doses of SLE, TMZ and SLE / TMZ for 24 h, and cell survival rate was assessed using the CCK8 kit, (B–C) Proliferation rate of A375 cells. Cells were treated with different doses of SLE, TMZ and SLE / TMZ for 24 h. Cell growth was observed by crystal violet staining. Quantification of inhibition rate and representative images of living cells are shown. Scale bar is 200 μm. *p<0.05, **p<0.01 relative to the control group. #CDI<1, &CDI<0.7;

[0034] Figure 2:SLE / TMZ induced apoptosis in A375 melanoma cells. The left side is a representative scatter plot, and apoptotic cells were identified in the right quadrant of FITC-positive cells. The right side is the quantitative results. The experimental results are expressed as mean ± standard deviation. *p<0.05, **p<0.01 relative to the control group. #CDI<1, &CDI<0.7;

[0035] Figure 3 :SLE / TMZ shows synergistic anti-melanoma effect in the B16F10 melanoma mouse model. (A) The B16F10 melanoma mouse model includes the following treatment groups: control group (PBS), SLE (1.2g / kg), TMZ 25 (25mg / kg), TMZ 50 (50mg / kg), SLE / TMZ 25 (1.2g / kg SLE+25mg / kg TMZ), and SLE / TMZ 50 (1.2g / kgSLE+50mg / kg TMZ). Photos of dissected mouse tumors are shown (N=8). (B) Tumor weight of each group. (C) Tumor volume of each group. The experimental results are expressed as mean ± standard deviation. *p<0.05, **p<0.01 relative to the control group. #CDI<1, &CDI<0.7;

[0036] Figure 4 :SLE may have a reduced toxicity to TMZ in the B16F10 melanoma mouse model. (A) Body weight of mice in each group. (B) Organ index of mice in each group, including heart, liver, spleen, lung, and kidney. (C) H&E staining of the liver of mice in different treatment groups showed that the site of lymphocyte infiltration was indicated by the circled area. The experimental results are expressed as mean ± standard deviation. *p<0.05, **p<0.01 relative to the control group;

[0037] Figure 5 :SLE / TMZ increased the proportion of Th and Tc cells in the spleen of tumor-bearing mice. The spleens of the control group (PBS), SLE (1.2g / kg), TMZ 25 (25mg / kg), TMZ 50 (50mg / kg), SLE / TMZ 25 (1.2g / kg SLE + 25mg / kg TMZ) and SLE / TMZ 50 (1.2g / kg SLE + 50mg / kg TMZ) mice were removed and prepared into single cell suspensions. Flow cytometry was used to determine the proportion of Th and Tc cells in the spleen. CD3 / CD4 positive cells were Th cells, and CD3 / CD8 positive cells were Tc cells;

[0038] Figure 6:SLE / TMZ increased the proportion of NK cells in the spleen of tumor-bearing mice. The spleens of the control group (PBS), SLE (1.2g / kg), TMZ 25 (25mg / kg), TMZ 50 (50mg / kg), SLE / TMZ 25 (1.2g / kg SLE + 25mg / kg TMZ) and SLE / TMZ50 (1.2g / kg SLE + 50mg / kg TMZ) mice were removed and prepared into single cell suspensions. The proportion of NK cells in the spleen was determined by flow cytometry, and CD3 / NK1.1 positive was NK cells;

[0039] Figure 7 :SLE / TMZ reduced the proportion of MDSC cells in the spleen of tumor-bearing mice. The spleens of control group (PBS), SLE (1.2g / kg), TMZ 25 (25mg / kg), TMZ 50 (50mg / kg), SLE / TMZ 25 (1.2g / kg SLE + 25mg / kg TMZ) and SLE / TMZ 50 (1.2g / kg SLE + 50mg / kg TMZ) mice were removed and prepared into single cell suspensions. Flow cytometry was used to determine the proportion of MDSC cells in the spleen. Gr-1 / CD11b positive was MDSC cells. DETAILED DESCRIPTION

[0040] In order to better understand the present invention, several experimental examples are listed below to illustrate its formula ratio, preparation method, detection method, new use, etc. in traditional Chinese medicine. And the term of the Chinese medicine preparation of the present invention appearing in the specification generally refers to the Chinese medicine prepared according to the formula ratio and preparation method of Sophora japonica and honeysuckle in Example 1. The following experiments are intended to illustrate the present invention but not to limit the present invention.

[0041] Example 1 Chinese medicine preparation of the present invention

[0042] 1.1 Preparation of the Chinese medicine preparation of the present invention

[0043] Take 100g of Sophora japonica and 20g of honeysuckle, mix them in proportion, add 30% alcohol (according to the mass volume ratio of 1:10, the added concentration is 1200ml of 30% alcohol), decoct and extract (soak for 30min, condense and reflux for 3 times at 100°C, and extract for 1h each time), filter, and obtain. Combine all the extracts, concentrate by rotary evaporation, and freeze-dry in a freeze dryer to obtain the Chinese medicine preparation of the present invention (extracts of Sophora japonica and honeysuckle). Then add appropriate amounts of sodium benzoate and sucrose, mix, and stir to prepare an oral liquid preparation.

[0044] 1.2 Detection method of the Chinese medicine preparation of the present invention

[0045] Chromatographic conditions: the chromatographic column is a C18 column (2.1mm×100mm ID, 2.6μm); the mobile phase is water (A) and methanol (B) containing 0.1% formic acid, and the gradient elution program is: 0-5min, 95-70%A, 5-30min, 70%A, 30-50min, 70%-68%A; mobile phase flow rate: 0.2mL / min; column temperature: 35°C; injection volume: 5μL.

[0046] Preparation of reference solution: Determine according to the method of Chinese Pharmacopoeia: accurately weigh 2.0 mg of rutin and chlorogenic acid reference substances, add 50% methanol aqueous solution to dissolve and prepare 1 mg / mL reference solution. Then prepare the two reference solutions into a mixed reference solution and dilute the series of concentrations (rutin: 6.25-200.0 μg / mL, chlorogenic acid: 1.5625-50.0 μg / mL), and establish their own standard curves for calculation of content determination.

[0047] Preparation of test solution: Weigh 5 mg of Chinese medicine extract, accurately weigh it, dissolve it in 5 mL of 50% methanol, and filter it with a 0.22 μm microporous filter membrane to obtain a 1 mg / mL test solution. The main components of the extract are flavonoids, such as rutin, quercetin, luteolin, and ginsenoside.

[0048] Table 1 The active ingredients of the original Chinese medicine of the present invention are as follows:

[0049]

[0050]

[0051]

[0052]

[0053] 1.3 Determination of active ingredients in the Chinese medicine preparation of the present invention

[0054] 1.3.1 Instruments and analytical conditions

[0055] Liquid chromatography analysis was performed using an Agilent 1290UPLC system, which includes a 1290 binary pump solvent management system, a 1290TCC, and a 1290 autosampler. A C18 column (2.1 mm × 100 mm inner diameter, 1.7 μm) was used for sample separation, and the column temperature was maintained at 40°C. The mobile phase consisted of A (0.1% formic acid) and B (0.1% formic acid in acetonitrile), and a gradient elution was used, with B increasing from 12% to 25% from 0 to 6 minutes, from 25% to 45% from 6 to 8 minutes, from 45% to 80% from 8 to 11 minutes, and from 80% to 100% from 11 to 11.1 minutes, and then returned to the initial conditions, with a flow rate set to 0.40 mL / min. The autosampler temperature was set to 4°C, and the injection volume was 3 μL. Mass spectrometry detection was performed using an Agilent 6460 triple quadrupole MS equipped with an Agilent Jet Stream electrospray ionization (ESI) source. The ESI source was set in negative ion mode. The source parameters were set as follows: capillary voltage 3.5 KV; source temperature 150 °C; drying gas temperature 350 °C; drying gas flow rate 10 L / min; nebulizer 45 psi; sheath gas temperature 350 °C; sheath gas flow rate 8 L / min. Analyte detection was performed by multiple reaction monitoring (MRM).

[0056] 1.3.2 Sample preparation

[0057] Prepare methanol standard solution containing Jinshengcaosu (48.8-12,500.0ng / mL) for UPLC-MS / MS analysis. Record the peak area to make a standard curve. Accurately weigh the dried Chinese medicine extract and dissolve it in methanol (5mg / mL, w / v), then filter it using a 0.2μm filter membrane. Inject the supernatant. UPLC-MS / MS was used to determine the content of Jinshengcaosu in Chinese medicine preparations, and the results are as follows:

[0058] Table 2 The content of Jinshengcaosu in the Chinese medicine preparation of the present invention

[0059]

[0060] Embodiment 2 Pharmacological experiment of Chinese medicine preparation of the present invention

[0061] 1) SLE / TMZ inhibits the growth of A375 cells and induces cell apoptosis

[0062] The CCK-8 detection kit was used to evaluate the effects of SLE, TMZ and SLE / TMZ on the proliferation of A375 cells. The results showed that after 24 hours of treatment with SLE (200, 400μg / ml), TMZ (100, 200μM) or SLE / TMZ (200μgm / mlSLE+100μM TMZ, 200μg / mlSLE+200μM TMZ, 400μg / mlSLE+100μM TMZ, 400μg / mlSLE+200μM TMZ), the viability of A375 cells decreased in a dose-dependent manner (see the attached manual for details). Figure 1 A). The CDI values ​​were calculated to evaluate the synergistic effect of SLE / TMZ. After 24 hours, the CDI values ​​of 200 μg / ml SLE+100 μM TMZ, 200 μg / ml SLE+200 μM TMZ, and 400 μg / ml SLE+100 μM TMZ were all less than 0.7, and the CDI value of 400 μg / ml SLE+200 μM TMZ was less than 1.

[0063] The results showed that in the 24-hour treatment test, 400μg / ml SLE+200μM TMZ had a synergistic effect (CDI<1), and 200μg / ml SLE+100μM TMZ, 200μg / ml SLE+200μM TMZ, and 400μg / ml SLE+100μM TMZ had a significant synergistic effect on the proliferation of A375 cells (CDI<0.7). The crystal violet staining kit was used to evaluate the effects of SLE, TMZ, and SLE / TMZ on the proliferation of A375 cells (see the attached manual). Figure 1 B).

[0064] The results showed that after 24 h of treatment with SLE (200, 400 μg / ml), TMZ (100, 200 μM) or SLE / TMZ (200 μg / ml SLE + 100 μM TMZ, 200 μg / ml SLE + 200 μM TMZ), the number of A375 cells decreased in a dose-dependent manner (see Appendix 1 for details). Figure 1 C). The CDI value was calculated to evaluate the synergistic effect of SLE / TMZ. After 24 hours, the CDI of 200 μgm / ml SLE+100 μM TMZ and 200 μgm / ml SLE+200 μM TMZ were both less than 0.7, indicating that they had a significant synergistic effect on the proliferation of A375 cells (CDI<0.7).

[0065] Annexin V-FITC / PI double staining was used to detect the apoptosis-promoting effects of SLE, TMZ and SLE / TMZ on A375 cells. The results showed that the Chinese medicine preparations SLE, TMZ and SLE / TMZ of the present invention induced cell apoptosis in a dose-dependent manner (see the attached manual for details). Figure 2 ). Quantitative results showed that the apoptosis rates of A375 cells in Control, 200μg / ml SLE, 100μM TMZ, 200μg / ml SLE+100μM TMZ, and 200μg / ml SLE+200μM TMZ were 4.6% (P<0.05), 4.9% (P<0.01), 6.8% (P<0.01), 8.2% (P<0.01), and 9.6% (P<0.01), respectively. Further analysis of these data revealed that combined treatment had a stronger pro-apoptotic effect than single treatment. In summary, SLE / TMZ can significantly inhibit A375 cell proliferation and induce cell apoptosis in vitro in a dose-dependent manner.

[0066] 2) SLE / TMZ has a synergistic anti-melanoma effect in the B16F10 melanoma mouse model

[0067] To investigate whether SLE has a synergistic anti-melanoma effect with TMZ in vivo, B16F10 melanoma cells were injected subcutaneously into the shoulder of C57BL / 6 mice to construct a tumor-bearing mouse model. Figure 2 The dosage unit is 1.2 g / kg. The tumor volume and tumor weight of the Chinese medicine preparation SLE, 25 mg / kg TMZ, 50 mg / kg TMZ, 1.2 g / kg SLE + 25 mg / kg TMZ, and 1.2 g / kg SLE + 50 mg / kg TMZ of the present invention are significantly reduced compared with the control group (see the attached manual). Figure 3 A, B, C). Compared with the SLE and TMZ temozole groups, the Chinese medicine preparation SLE / TMZ of the present invention significantly reduced tumor weight (P<0.05). The CDI of the combination of the two drugs was less than 0.7, indicating that the two drugs had a significant synergistic effect in reducing tumor weight (see the attached manual). Figure 3 A, B).

[0068] Importantly, TMZ alone reduced the body weight of tumor-bearing mice, while SLE and SLE / TMZ 25 had no significant effect on the body weight of mice (see Appendix Figure 4 A). In addition, no abnormality was found in organ indexes (see Appendix Figure 4B), and then H&E staining was performed on the main organs of mice treated with PBS, the Chinese medicine preparation of the present invention, SLE, TMZ, and SLE / TMZ. It was found that the livers of the TMZ 25 and TMZ 50 groups had lymphocyte infiltration, indicating inflammation and damage, while the SLE, SLE / TMZ 25, and SLE / TMZ 50 groups had no abnormalities (see the appendix of the instruction manual). Figure 4 C). In summary, we found that SLE / TMZ has a synergistic anti-melanoma effect, and the Chinese medicine preparation SLE of the present invention can alleviate the weight loss and liver damage of mice caused by TMZ to a certain extent.

[0069] 3) SLE / TMZ increased the percentage of Th (helper T cells), Tc (cytotoxic T cells), and NK (natural killer cells) cells in the spleen of B16F10 melanoma mice, and decreased the percentage of MDSC cells

[0070] The onset and progression of melanoma triggers the immune response of the body's defense system, recruiting immune cells to the tumor site. These four cells are briefly introduced. In order to determine the effects of the Chinese medicine preparations SLE, TMZ temozole and SLE / TMZ temozole of the present invention on the number of these tumor-infiltrating immune cells, flow cytometry analysis was used. Compared with Control, SLE, SLE / TMZ 25, and SLE / TMZ 50 increased the percentages of Th, Tc, and NK cells in the spleen of mice, and reduced the percentage of MDSC cells. However, TMZ25 and TMZ 50 increased the percentage of MDSC cells and had no significant effect on the percentages of Th, Tc, and NK cells. Compared with SLE, TMZ 25, and TMZ 50, SLE / TMZ 25 and SLE / TMZ 50 significantly increased the percentages of Th and Tc in the spleen of mice (see the attached figure of the instruction manual). Figure 5 ) and NK (see the instructions for Figure 6 ) cells, reducing the percentage of MDSC (see the appendix of the instruction manual Figure 7 ) cells. These results show that SLE / TMZ 25 and SLE / TMZ 50 increased the proportion of Th and Tc cells in the spleen of mice and weakened the proportion of MDSC cells. The above animal experimental results suggest that the Chinese medicine preparation (Sophora japonica and honeysuckle extracts) SLE of the present invention can assist TMZ in enhancing the immune function of melanoma mice.

[0071] The above experimental data show that the Chinese medicine preparation (sophora japonica and honeysuckle extract) SLE of the present invention combined with TMZ has a synergistic anti-melanoma effect both in vitro and in vivo. These studies provide preclinical pharmacological support for developing the Chinese medicine preparation (sophora japonica and honeysuckle extract) SLE of the present invention into an effective Chinese medicine compound preparation and using it as an adjuvant drug for TMZ in the treatment of melanoma.

Claims

1. Use of a traditional Chinese medicine preparation in preparing a drug for treating tumor diseases, characterized in that: The raw material composition and weight proportions of the traditional Chinese medicine preparation are: 90-110 parts of Sophora japonica flower and 15-25 parts of honeysuckle flower.

2. The use according to claim 1, characterized in that The raw material composition and weight proportions of the traditional Chinese medicine preparation are: 100 parts of Sophora japonica flowers and 20 parts of honeysuckle flowers.

3. The use according to claim 1 or 2, characterized in that The preparation method of the traditional Chinese medicine preparation is as follows: taking Sophora japonica flower and honeysuckle flower, mixing them in proportion, adding 30% alcohol, decocting and extracting, filtering, and obtaining the traditional Chinese medicine preparation.

4. The use according to claim 1 or 2, characterized in that The dosage forms of the Chinese medicine preparations are oral liquids, pastes, granules, capsules, pills and other different oral preparations.

5. The use according to claim 1 or 2, characterized in that The tumor disease to be treated is melanoma.

6. The use according to claim 1 or 2, characterized in that The drug can also be used to improve the body's immunity.

7. The use according to claim 1 or 2, characterized in that The tumor disease to be treated is glioblastoma multiforme or anaplastic astrocytoma.

8. Use of a traditional Chinese medicine preparation in the preparation of auxiliary temozolomide drug attenuation.

9. The use according to claim 7, characterized in that The traditional Chinese medicine preparation is used in alleviating weight loss and liver damage caused by temozolomide.

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