Chinese and western medicine composition as well as preparation method and application thereof

Abstract

The invention discloses a Chinese and western medicine composition and a preparation method and application thereof, and belongs to the technical field of anti-lung cancer targeted drugs, the traditional Chinese medicine composition and EGFR-TKI cooperate to treat EGFR mutation advanced non-small cell lung cancer, the lifetime of a patient is prolonged, the treatment effective rate is increased, the life quality is improved, and the side effect of the targeted drugs is relieved. Compared with EGFR-TKI single medicine treatment, the traditional Chinese medicine composition is combined with EGFR-TKI, proliferation of HCC827 lung cancer cells is remarkably enhanced and inhibited, cell apoptosis is promoted, cycle division of cells is interfered, the traditional Chinese medicine and western medicine composition provided by the invention has the advantages that by regulating and controlling tumor cell energy metabolism and regulating and controlling expression of metabolism-related proteins LDHA, HK2, GLUT1 and HIF-1alpha, the effect of inhibiting HCC827 lung cancer cells is achieved, and the curative effect of the HCC827 lung cancer cells is improved. Therefore, effective control of the advanced non-small cell lung cancer is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-lung cancer targeted drugs, and particularly relates to a traditional Chinese and western medicine composition, a preparation method thereof and an application thereof. Background Art

[0002] Non-small cell lung cancer (NSCLC) is the most common pathological type in lung cancer, accounting for about 85% of lung cancer, and is a disease with high specificity in terms of histology, pathophysiology, genomics, etc. Epidermal growth factor receptor (EGFR) gene mutation is the most common driver gene mutation in non-small cell lung cancer (NSCLC), and is also called a classical mutation. Targeted drugs represented by epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have replaced classical platinum-based doublet chemotherapy and are recommended as the first-line treatment for advanced NSCLC with classical EGFR mutations.

[0003] EGFR-TKI is a small molecule inhibitor, which mainly competitively binds to the ATP site in the tyrosine kinase region of EGFR through an endogenous ligand, inhibits the autophosphorylation of the receptor, thereby blocking the EGFR signaling pathway, inhibiting the proliferation and metastasis of tumor cells, promoting their apoptosis, and achieving the effect of inhibiting tumor development. First-generation / second-generation EGFR-TKIs are used for the first-line treatment of patients with advanced NSCLC with classical EGFR mutations (EGFR exon 19 deletion mutation or exon 21 L858R point mutation), which can increase the median PFS of patients to 9.2 - 14.7 months compared with chemotherapy, but disease progression caused by drug resistance is inevitable. Third-generation EGFR-TKIs represented by osimertinib can not only overcome the drug resistance problems of the first- and second-generation EGFR-TKIs caused by T790M, but also can be applied to the first-line treatment of advanced NSCLC with classical EGFR mutations, effectively improving the progression-free survival and overall survival of patients. However, unfortunately, over time, patients will still develop secondary drug resistance, resulting in poor treatment effects and shortened survival of patients. Summary of the Invention

[0004] The present invention provides a traditional Chinese and Western medicine composition, its preparation method and application, effectively solving the technical problems that when using first-generation EGFR-TKIs to treat advanced non-small cell lung cancer, the progression-free survival and overall survival of patients cannot be prolonged, and adverse reactions such as rash and diarrhea occur in patients. The traditional Chinese medicine composition composed of Astragalus membranaceus, Pseudostellaria heterophylla, Curcuma aromatica Salisb., Curcuma longa L., Concha Ostreae, Prunella vulgaris L., Hedyotis diffusa Willd. and Nidus Vespae synergistically enhances the inhibition of the proliferation of HCC827 lung cancer cells, promotes cell apoptosis, and interferes with cell cycle division by epidermal growth factor receptor tyrosine kinase inhibitors, effectively prolonging the progression-free survival and overall survival of patients, and alleviating the adverse reactions of rash and diarrhea caused by EGFR-TKI drugs.

[0005] The first object of the present invention is to provide a traditional Chinese and Western medicine composition, which is composed of a traditional Chinese medicine composition and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0006] The traditional Chinese medicine composition is made from the following raw materials in parts by mass: 25-60 parts of Astragalus membranaceus, 10-18 parts of Pseudostellaria heterophylla, 8-15 parts of Curcuma aromatica Salisb., 8-15 parts of Curcuma longa L., 15-45 parts of Concha Ostreae, 10-20 parts of Prunella vulgaris L., 12-30 parts of Hedyotis diffusa Willd., 10-20 parts of Nidus Vespae and 0-12 parts of Artemisia anomala S. Moore.

[0007] In the traditional Chinese and Western medicine composition, the dosage ratio of the traditional Chinese medicine composition to the epidermal growth factor receptor tyrosine kinase inhibitor is 4g-20g: 3nmol-10nmol.

[0008] As a preferred embodiment, the traditional Chinese medicine composition is made from the following raw materials in parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., 15 parts of Nidus Vespae and 10 parts of Artemisia anomala S. Moore.

[0009] As a preferred embodiment, the dosage ratio of the traditional Chinese medicine composition to the epidermal growth factor receptor tyrosine kinase inhibitor is 1g: 1nmol.

[0010] As a preferred embodiment, the epidermal growth factor receptor tyrosine kinase inhibitor is erlotinib hydrochloride, gefitinib or icotinib hydrochloride.

[0011] The second object of the present invention is to provide a preparation method of the above-mentioned traditional Chinese and Western medicine composition, including the following steps:

[0012] Weigh according to the weight parts of each raw material: 25-60 parts of Astragalus membranaceus, 10-18 parts of Pseudostellaria heterophylla, 8-15 parts of Curcuma aromatica Salisb., 8-15 parts of Curcuma longa L., 15-45 parts of Concha Ostreae, 10-20 parts of Prunella vulgaris L., 12-30 parts of Hedyotis diffusa Willd., 10-20 parts of Nidus Vespae and 0-12 parts of Artemisia anomala S. Moore.

[0013] Mix the above raw materials, soak them in water, decoct, filter to obtain the original medicinal liquid, freeze-dry to obtain the freeze-dried powder, dissolve the freeze-dried powder in water, perform ultrasonic centrifugation to obtain the supernatant, and filter and sterilize to obtain the traditional Chinese medicine composition solution.

[0014] Dissolve and dilute the epidermal growth factor receptor tyrosine kinase inhibitor with sodium sulfobutyl ether β-cyclodextrin to obtain the epidermal growth factor receptor tyrosine kinase inhibitor solution.

[0015] Mix the traditional Chinese medicine composition solution with the epidermal growth factor receptor tyrosine kinase inhibitor solution to obtain the Chinese and Western medicine composition.

[0016] As a preferred embodiment, in the Chinese and Western medicine composition, the dosage ratio of the traditional Chinese medicine composition to the epidermal growth factor receptor tyrosine kinase inhibitor is 4 g - 20 g: 3 nmol - 10 nmol.

[0017] As a preferred embodiment, during decoction, the raw materials soaked in water are decocted once to obtain the first decoction liquid and the medicinal residues, the medicinal residues are decocted a second time to obtain the second decoction liquid, and the first decoction liquid and the second decoction liquid are combined to obtain the original medicinal liquid.

[0018] The third object of the present invention is to provide the application of the above Chinese and Western medicine composition in the preparation of drugs for treating non-small cell lung cancer.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention provides a Chinese and Western medicine composition, which is prepared by compounding a traditional Chinese medicine composition composed of astragalus membranaceus, pseudostellaria heterophylla, curcuma aromatica, curcuma longa, raw oyster, prunella vulgaris, hedyotis diffusa, nidus vespae and artemisia anomala and an epidermal growth factor receptor tyrosine kinase inhibitor to obtain the Chinese and Western medicine composition. The present invention uses the traditional Chinese medicine composition to synergistically treat EGFR-mutated advanced non-small cell lung cancer with EGFR-TKI, prolonging the survival period of patients, improving the treatment efficiency, improving the quality of life, and reducing the side effects of targeted drugs. Compared with single-agent EGFR-TKI treatment, the present invention combines the traditional Chinese medicine composition with EGFR-TKI, significantly enhancing the inhibition of the proliferation of HCC827 lung cancer cells, promoting cell apoptosis, and interfering with cell cycle division. The Chinese and Western medicine composition provided by the present invention controls advanced non-small cell lung cancer effectively by regulating the energy metabolism of tumor cells and regulating the expression of metabolism-related proteins LDHA, HK2, GLUT1 and HIF-1α. Description of the Drawings

[0021] Figure 1 It is the survival analysis function for the comparison of PFS of two groups in the present invention.

[0022] Figure 2 This is the survival analysis function for comparing two groups of OS in the present invention.

[0023] Figure 3 This is for the present invention to detect the cell viability of BEAS-2B cells and HCC827 cells after 24-hour exposure to different concentrations of Erlotinib using CCK8. Among them, Figure A is for HCC827 cells and Figure B is for BEAS-2B cells.

[0024] Figure 4 This is for the present invention to detect the cell viability of BEAS-2B cells and HCC827 cells after 24-hour exposure to different concentrations of traditional Chinese medicine compositions in Comparative Examples 2 to 7 and the blank control group of Comparative Example 16 using CCK8. Among them, Figure A is for BEAS-2B cells, Figure B is for HCC827 cells, and Figure C is for the cell viability of BEAS-2B cells and HCC827 cells after 24-hour exposure to the traditional Chinese medicine compositions of Comparative Examples 7 and 8.

[0025] Figure 5 This is for the present invention to detect the proliferation of HCC827 cells after 24-hour treatment with different concentrations of Erlotinib (5, 10, 15 nM) and different concentrations of traditional Chinese medicine compositions (1, 2, 5, 10, 20 mg / mL) using EdU. Among them, Figure A is for different concentrations of Erlotinib, Figure B is for different concentrations of traditional Chinese medicine compositions (nucleus in blue, EdU-positive cells in red, scale bar is 500 μm), and Figure C is the quantitative data of EdU proliferation detection (* indicates P < 0.05 compared with the Cont group, i.e., the blank control group).

[0026] Figure 6 This is a representative image for the present invention to detect the proliferation of HCC827 cells after 24-hour treatment with different concentrations of Erlotinib (5, 10 nM) combined with a traditional Chinese medicine composition (5 mg / mL) using EdU (nucleus in blue, EdU-positive cells in red, scale bar is 500 μm).

[0027] Figure 7 This is the quantitative data of each group after EdU proliferation detection for the treatment with Erlotinib combined with a traditional Chinese medicine composition (* indicates P < 0.05 compared with the Cont group).

[0028] Figure 8 This is for the present invention to detect the apoptosis of HCC827 cells after 24-hour treatment with different concentrations of traditional Chinese medicine compositions (Cont, 1, 2, 5, 10 mg / mL) by flow cytometry. Among them, Figures A to E are representative images of each group, and Figure F is the quantitative data of each group (* indicates P < 0.05 compared with the Cont group).

[0029] Figure 9This invention uses flow cytometry to detect the apoptosis of HCC827 cells after being treated with Erlotinib combined with a traditional Chinese medicine composition for 24 hours. Among them, Figures A - D are representative images of each group, and Figure E is the quantitative data of each group (* indicates P < 0.05 compared with the Cont group).

[0030] Figure 10 This invention uses flow cytometry to detect the cell cycle of HCC827 cells after being treated with the control group, traditional Chinese medicine composition, Erlotinib, and the group of Erlotinib combined with traditional Chinese medicine composition for 24 hours. Among them, Figure A is the representative image of each group, and Figure B is the quantitative data of each group (* indicates P < 0.05 compared with the Cont group).

[0031] Figure 11 This invention uses Western blot to analyze the protein expression map of HCC827 cells after being treated with different concentrations of traditional Chinese medicine composition (0, 1, 2, 5, 10 mg / mL) for 24 hours. Among them, (a) shows Caspase8, Caspase 3, BAX, Bcl - 2; (b) shows CyclinA2, Cyclin B1, CDK4, CDK6; (c) shows the protein expression of LDHA, HK2, GLUT1, and HIF - 1α.

[0032] Figure 12 This invention shows the tumor volume and tumor weight of nude mice in each group after 14 days of drug administration. Among them, Figure A shows the tumor volume, Figure B shows the tumor weight, and Figure C shows the quantitative data of each group (* indicates P < 0.05 compared with the Cont group). Detailed implementation method

[0033] To enable those skilled in the art to better understand and implement the technical solution of this invention, the following further explains this invention in combination with specific examples and experimental data, but the examples cited do not limit this invention.

[0034] Aiming at the technical problems that when using first - generation EGFR - TKI to treat advanced non - small cell lung cancer, it cannot extend the progression - free survival and overall survival of patients, and it causes adverse reactions such as rash and diarrhea in patients, this invention provides a traditional Chinese and Western medicine composition, its preparation method, and application.

[0035] The following details the technical content of this invention.

[0036] Experimental part

[0037] The first - generation EGFR - TKI representative drug erlotinib hydrochloride (Erlotinib) and the traditional Chinese medicine composition (XYT) are used as research objects.

[0038] 1. In vitro experiments were conducted to expose BEAS-2B cells (normal lung epithelial cells) and HCC827 cells (lung cancer cells with EGFR mutations) to Erlotinib solutions at different concentrations, XYT freeze-dried powder solutions, and the combination of Erlotinib and XYT. The CCK8 assay was used to detect cell viability and screen for appropriate exposure concentrations.

[0039] 2. The effects of the control group, XYT group, Erlotinib group, and the combination of Erlotinib and XYT on the proliferation, apoptosis, and cell cycle of HCC827 cells were observed by EdU and flow cytometry. Furthermore, to explore the mechanism of action of XYT from the perspective of cellular energy metabolism, the effects of XYT on the metabolism of ATP, lactate, and glucose in HCC827 cells were detected.

[0040] 3. To further clarify the mechanism of action of XYT, Western blot was used to detect the expression of related effector proteins involved in apoptosis, namely Cleaved Caspase-8, Cleaved Caspase-3, BAX, and Bcl-2, as well as the expression of key effector proteins in the cell cycle, namely CyclinA2, Cyclin B1, CDK4, and CDK6, and the expression changes of key effector protein molecules related to metabolism, namely LDHA, HK2, GLUT1, and HIF-α.

[0041] 4. In vivo experiments were carried out by establishing a nude mouse xenograft model of HCC827 cells. The tumor volume and tumor weight were compared among groups, the tumor inhibition rate was calculated, and the expression of Ki67 and Caspase-8 was detected by immunohistochemistry for further verification.

[0042] The present invention prepared the following 5 groups of traditional Chinese and Western medicine compositions with different concentrations and the comparative drugs of each comparative ratio, and in vitro experiments were respectively carried out using the traditional Chinese and Western medicine compositions and comparative drugs of each group, and the results are as follows.

[0043] Example 1

[0044] A traditional Chinese and Western medicine composition is prepared from a traditional Chinese medicine composition and a representative epidermal growth factor receptor tyrosine kinase inhibitor.

[0045] The traditional Chinese medicine composition is made from the following raw materials in parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., and 15 parts of Nidus Vespae. The representative epidermal growth factor receptor tyrosine kinase inhibitor is Erlotinib hydrochloride.

[0046] The preparation method of the above traditional Chinese and Western medicine composition includes the following steps:

[0047] S1. Preparation of traditional Chinese medicine composition solution: Weigh and mix 1400 g of raw materials according to the following parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., and 15 parts of Nidus Vespae. Soak the mixed raw materials in water, decoct twice, combine the decoctions obtained twice, concentrate by evaporation and filter through a sieve. Place the filtered decoction in a sterile tray for freeze-drying to obtain a freeze-dried powder. Dissolve the freeze-dried powder with water, shake well and use ultrasonic assistance for dissolution. Put it into a centrifuge and centrifuge at 4000 rpm for 20 min. Take the supernatant, filter and sterilize it with a 0.22 μm filter membrane, and prepare a stock solution of 100 mg / mL. Seal and store it in an environment of -20 °C to obtain the traditional Chinese medicine composition solution.

[0048] S2. Dissolve and dilute the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride with sodium sulfobutyl ether β-cyclodextrin to obtain an epidermal growth factor receptor tyrosine kinase inhibitor solution.

[0049] S3. Dilute the traditional Chinese medicine composition solution and mix it with the epidermal growth factor receptor tyrosine kinase inhibitor solution to obtain a traditional Chinese and Western medicine composition. In the traditional Chinese and Western medicine composition, the concentration of the traditional Chinese medicine composition is 5 mg / mL, and the concentration of the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride is 5 nM.

[0050] Example 2

[0051] A traditional Chinese and Western medicine composition is prepared from a traditional Chinese medicine composition and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0052] The traditional Chinese medicine composition is made from the following raw materials by parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., and 15 parts of Nidus Vespae. The epidermal growth factor receptor tyrosine kinase inhibitor is erlotinib hydrochloride.

[0053] The preparation method of the above traditional Chinese and Western medicine composition includes the following steps:

[0054] S1. Preparation of traditional Chinese medicine composition solution: Weigh and mix 1400 g of raw materials according to the following parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., and 15 parts of Nidus Vespae. Soak the mixed raw materials in water, decoct twice, combine the decoctions obtained twice, concentrate by evaporation and filter through a sieve. Place the filtered decoction in a sterile tray for freeze-drying to obtain a freeze-dried powder. Dissolve the freeze-dried powder with water, shake well and use ultrasonic wave to assist dissolution. Put it into a centrifuge and centrifuge at 4000 rpm for 20 min. Take the supernatant, filter and sterilize it with a 0.22 μm filter membrane, and prepare a mother liquor with a concentration of 100 mg / mL. Seal and store it in an environment of -20 °C to obtain the traditional Chinese medicine composition solution.

[0055] S2. Dissolve and dilute the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride with sulfobutyl beta-cyclodextrin sodium to obtain an epidermal growth factor receptor tyrosine kinase inhibitor solution.

[0056] S3. Dilute the traditional Chinese medicine composition solution and mix it with the epidermal growth factor receptor tyrosine kinase inhibitor solution to obtain a traditional Chinese and Western medicine composition. In the traditional Chinese and Western medicine composition, the concentration of the traditional Chinese medicine composition is 5 mg / mL, and the concentration of the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride is 10 nM.

[0057] Example 3

[0058] A traditional Chinese and Western medicine composition is prepared from a traditional Chinese medicine composition and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0059] The traditional Chinese medicine composition is made from the following raw materials by parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., and 15 parts of Nidus Vespae. The epidermal growth factor receptor tyrosine kinase inhibitor is erlotinib hydrochloride.

[0060] The preparation method of the above traditional Chinese and Western medicine composition includes the following steps:

[0061] S1. Preparation of traditional Chinese medicine composition solution: Weigh and mix 1400 g of raw materials according to the following parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., and 15 parts of Nidus Vespae. Soak the mixed raw materials in water, decoct twice, combine the decoctions obtained twice, concentrate by evaporation and filter through a sieve. Place the filtered decoction in a sterile tray for freeze-drying to obtain a freeze-dried powder. Dissolve the freeze-dried powder with water, shake well and use ultrasonic assistance for dissolution. Put it into a centrifuge and centrifuge at 4000 rpm for 20 min. Take the supernatant, filter and sterilize it with a 0.22 μm filter membrane, and prepare a mother liquor with a concentration of 100 mg / mL. Seal and store it in an environment of -20 °C to obtain the traditional Chinese medicine composition solution.

[0062] S2. Dissolve and dilute the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride with sulfobutyl ether β-cyclodextrin sodium to obtain an epidermal growth factor receptor tyrosine kinase inhibitor solution.

[0063] S3. Dilute the traditional Chinese medicine composition solution and mix it with the epidermal growth factor receptor tyrosine kinase inhibitor solution to obtain a traditional Chinese and Western medicine composition. In the traditional Chinese and Western medicine composition, the concentration of the traditional Chinese medicine composition is 10 mg / mL, and the concentration of the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride is 5 nM.

[0064] Example 4

[0065] A traditional Chinese and Western medicine composition is prepared from a traditional Chinese medicine composition and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0066] The traditional Chinese medicine composition is made from the following raw materials by parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., and 15 parts of Nidus Vespae. The epidermal growth factor receptor tyrosine kinase inhibitor is erlotinib hydrochloride.

[0067] The preparation method of the above traditional Chinese and Western medicine composition includes the following steps:

[0068] S1. Preparation of traditional Chinese medicine composition solution: Weigh and mix 1400 g of raw materials according to the following parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., and 15 parts of Nidus Vespae. Soak the mixed raw materials in water, decoct twice, combine the decoctions obtained twice, concentrate by evaporation and filter through a sieve. Place the filtered aqueous decoction in a sterile tray for freeze-drying to obtain a freeze-dried powder. Dissolve the freeze-dried powder with water, shake well and use ultrasonic assistance for dissolution. Put it into a centrifuge and centrifuge at 4000 rpm for 20 min. Take the supernatant, filter and sterilize it with a 0.22 μm filter membrane, and prepare a stock solution of 100 mg / mL, which is sealed and stored in an environment of -20 °C to obtain the traditional Chinese medicine composition solution.

[0069] S2. Dissolve and dilute the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride with sulfobutyl ether β-cyclodextrin sodium to obtain an epidermal growth factor receptor tyrosine kinase inhibitor solution.

[0070] S3. Dilute the traditional Chinese medicine composition solution and mix it with the epidermal growth factor receptor tyrosine kinase inhibitor solution to obtain a traditional Chinese and Western medicine composition. In the traditional Chinese and Western medicine composition, the concentration of the traditional Chinese medicine composition is 10 mg / mL, and the concentration of the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride is 10 nM.

[0071] Example 5

[0072] A traditional Chinese and Western medicine composition is prepared from a traditional Chinese medicine composition and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0073] The traditional Chinese medicine composition is made from the following raw materials by parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of Concha Ostreae, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., and 15 parts of Nidus Vespae. The epidermal growth factor receptor tyrosine kinase inhibitor is erlotinib hydrochloride.

[0074] The preparation method of the above traditional Chinese and Western medicine composition includes the following steps:

[0075] S1. Preparation of traditional Chinese medicine composition solution: Weigh and mix 1400 g of raw materials according to the following parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of raw oyster, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., 15 parts of Nidus Vespae. Soak the mixed raw materials in water, decoct twice, combine the decoctions obtained twice, concentrate by evaporation and filter through a sieve. Place the obtained water decoction in a sterile tray for freeze-drying to obtain a freeze-dried powder. Dissolve the freeze-dried powder with water, shake well and use ultrasonic assistance for dissolution. Put it into a centrifuge and centrifuge at 4000 rpm for 20 min. Take the supernatant, filter and sterilize it with a 0.22 μm filter membrane, and prepare a mother liquor with a concentration of 100 mg / mL, and store it sealed in an environment of -20 °C to obtain the traditional Chinese medicine composition solution.

[0076] S2. Dissolve and dilute the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride with sodium sulfobutyl ether β-cyclodextrin to obtain an epidermal growth factor receptor tyrosine kinase inhibitor solution.

[0077] S3. Dilute the traditional Chinese medicine composition solution and mix it with the epidermal growth factor receptor tyrosine kinase inhibitor solution to obtain a traditional Chinese and Western medicine composition. In the traditional Chinese and Western medicine composition, the concentration of the traditional Chinese medicine composition is 20 mg / mL, and the concentration of the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib hydrochloride is 10 nM.

[0078] Example 6

[0079] A traditional Chinese and Western medicine composition is prepared from a traditional Chinese medicine composition and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0080] The traditional Chinese medicine composition is made from the following raw materials in parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of raw oyster, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., 15 parts of Nidus Vespae, 10 parts of Artemisia anomala S. Moore. The epidermal growth factor receptor tyrosine kinase inhibitor is erlotinib hydrochloride.

[0081] The preparation method of the above traditional Chinese and Western medicine composition includes the following steps:

[0082] S1. Preparation of the traditional Chinese medicine composition solution: Weigh and mix 1400 g of raw materials according to the following parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of raw oyster, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., 15 parts of Nidus Vespae, and 10 parts of Herba Artemisiae Anomalae. Soak the mixed raw materials in water, decoct twice, combine the decoctions obtained twice, concentrate by evaporation and filter through a sieve. Place the filtered decoction in a sterile tray for freeze-drying to obtain a freeze-dried powder. Dissolve the freeze-dried powder with water, shake well and use ultrasonic assistance for dissolution. Put it into a centrifuge and centrifuge at 4000 rpm for 20 min. Take the supernatant, filter and sterilize it with a 0.22 μm filter membrane, and prepare a mother liquor with a concentration of 100 mg / mL. Seal and store it in an environment of -20 °C to obtain the traditional Chinese medicine composition solution.

[0083] S2. Dissolve and dilute erlotinib hydrochloride, a representative epidermal growth factor receptor tyrosine kinase inhibitor, with sodium sulfobutyl ether β-cyclodextrin to obtain a solution of a representative epidermal growth factor receptor tyrosine kinase inhibitor.

[0084] S3. Dilute the traditional Chinese medicine composition solution and mix it with the solution of a representative epidermal growth factor receptor tyrosine kinase inhibitor to obtain a traditional Chinese and Western medicine composition. In the traditional Chinese and Western medicine composition, the concentration of the traditional Chinese medicine composition is 5 mg / mL, and the concentration of erlotinib hydrochloride, a representative epidermal growth factor receptor tyrosine kinase inhibitor, is 5 nM.

[0085] To further illustrate the effects of the present invention, the present invention also sets up comparative examples as follows:

[0086] Comparative Example 1

[0087] Compared with Example 1, only the traditional Chinese medicine composition is used, with a concentration of 1 mg / mL.

[0088] Comparative Example 2

[0089] Compared with Example 1, only the traditional Chinese medicine composition is used, with a concentration of 2 mg / mL.

[0090] Comparative Example 3

[0091] Compared with Example 1, only the traditional Chinese medicine composition is used, with a concentration of 5 mg / mL.

[0092] Comparative Example 4

[0093] Compared with Example 1, only the traditional Chinese medicine composition is used, with a concentration of 10 mg / mL.

[0094] Comparative Example 5

[0095] Compared with Example 1, only the traditional Chinese medicine composition is used, with a concentration of 20 mg / mL.

[0096] Comparative Example 6

[0097] Compared with Example 1, only the traditional Chinese medicine composition was used, with a concentration of 50 mg / mL.

[0098] Comparative Example 7

[0099] Compared with Example 1, only the traditional Chinese medicine composition was used, with a concentration of 100 mg / mL.

[0100] Comparative Example 8

[0101] Compared with Example 6, only the traditional Chinese medicine composition was used, with a concentration of 100 mg / mL.

[0102] The above Comparative Examples 1 to 8 are denoted as the XYT group.

[0103] Comparative Example 9

[0104] Compared with Example 1, only the first-generation EGFR-TKI representative drug erlotinib hydrochloride was used, with a concentration of 1 nM.

[0105] Comparative Example 10

[0106] Compared with Example 1, only the first-generation EGFR-TKI representative drug erlotinib hydrochloride was used, with a concentration of 5 nM, denoted as E5.

[0107] Comparative Example 11

[0108] Compared with Example 1, only the first-generation EGFR-TKI representative drug erlotinib hydrochloride was used, with a concentration of 10 nM, denoted as E10.

[0109] Comparative Example 12

[0110] Compared with Example 1, only the first-generation EGFR-TKI representative drug erlotinib hydrochloride was used, with a concentration of 15 nM, denoted as E15.

[0111] Comparative Example 13

[0112] Compared with Example 1, only the first-generation EGFR-TKI representative drug erlotinib hydrochloride was used, with a concentration of 50 nM.

[0113] Comparative Example 14

[0114] Compared with Example 1, only the first-generation EGFR-TKI representative drug erlotinib hydrochloride was used, with a concentration of 100 nM.

[0115] Comparative Example 15

[0116] Compared with Example 1, only the first-generation EGFR-TKI representative drug erlotinib hydrochloride was used, with a concentration of 500 nM.

[0117] The above Comparative Examples 9 to 15 are denoted as the Erlotinib group.

[0118] Comparative Example 16

[0119] Blank control group

[0120] The technical effects of the Chinese and Western medicine compositions prepared in Examples 1 to 5 of the present invention and the drugs in Comparative Examples 1 to 8 were detected, and the results are as follows

[0121] In terms of clinical observation, compared with the targeted therapy using the EGFR-TKI single drug (control group) in Comparative Example 8, the Chinese and Western medicine composition of Example 1 of the present invention, that is, the combination of the Chinese medicine composition and the first-generation EGFR-TKI (observation group), can extend the progression-free survival period of patients with advanced NSCLC with EGFR mutation by 2.7 months and the overall survival period by 7.1 months, and can significantly relieve the rash and diarrhea caused by the targeted drug, such as Figure 1 and Figure 2 , which is worthy of clinical promotion and application

[0122] Both the Chinese medicine composition and the first-generation EGFR-TKI erlotinib hydrochloride of the present invention can inhibit the viability of lung cancer cells HCC827 and BEAS-2B, and have no cytotoxicity to normal lung epithelial cells under conventional dosing doses, such as Figure 3 and Figure 4 shown, 10 parts by weight of Artemisia anomala was added to the Chinese medicine composition of Comparative Example 7 (30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica, 10 parts of Curcuma longa, 30 parts of raw oyster, 15 parts of Prunella vulgaris, 15 parts of Hedyotis diffusa, 15 parts of Nidus Vespae) to obtain a new Chinese medicine composition in Comparative Example 8. It can be seen from Figure C in Figure 4 that compared with the Chinese medicine composition of Comparative Example 7, the new Chinese medicine composition has a stronger inhibitory effect on the cell viability of lung cancer cells HCC827 and BEAS-2B at the same exposure concentration

[0123] In terms of cell proliferation and apoptosis, both the Chinese medicine composition and erlotinib can effectively inhibit cell proliferation and promote cell apoptosis, but the combined exposure of the Chinese medicine composition and erlotinib can play a synergistic role, such as Figures 5 to 9 shown. The EdU labeling method was used to detect the effects of different concentrations of Erlotinib (0, 5, 10, 15 nM) and XYT (0, 1, 2, 5, 10, 20 mg / mL) on the proliferation of HCC827 cells, and the results are as Figure 5As shown in Figures A, B, and C, in Figures A and B, blue represents the cell nucleus, and red represents proliferating EdU-positive cells. The results in Figure C show that with the increase in concentration, both Erlotinib and XYT can effectively inhibit the proliferation of HCC827 cells, causing a significant decrease in EdU-positive cells. When the concentration of Erlotinib reaches 5 nM, there is a statistically significant difference compared with the control group (Comparative Example 9) (P < 0.05). When the concentration of XYT reaches 5 mg / mL, there is a statistically significant difference compared with the control group (P < 0.05). Subsequently, the EdU labeling method was used to detect the proliferation of HCC827 cells treated with Erlotinib and XYT in combination. The results are as Figure 6 and Figure 7 shown. When the Chinese and Western medicine composition of Example 1, that is, Erlotinib (5 nM) and XYT (5 mg / mL), was used to treat HCC827 cells in combination, it could significantly inhibit EdU-positive cells, and there was a statistically significant difference compared with the treatment with a single drug (P < 0.05). In addition, 10 parts by weight of Artemisia anomala was added to the Chinese medicine composition of Example 1 (30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica, 10 parts of Curcuma longa, 30 parts of raw oyster, 15 parts of Prunella vulgaris, 15 parts of Hedyotis diffusa, 15 parts of Nidus Vespae) to obtain the Chinese medicine composition of Example 6. When the Chinese medicine composition of Example 6 (5 mg / mL) and Erlotinib (5 nM) were used to treat HCC827 cells in combination, it can be seen from Figure 7 Figure C that compared with the inhibitory effect of the Chinese and Western medicine composition of Example 1 on HCC827 cells, when the Chinese medicine composition of Example 6 was used in combination with Erlotinib (5 nM) to treat HCC827 cells, the inhibitory effect on EdU-positive cells was greatly improved. Thus, it can be seen that the combination of Artemisia anomala with Astragalus membranaceus, Pseudostellaria heterophylla, Curcuma aromatica, Curcuma longa, raw oyster, Prunella vulgaris, Hedyotis diffusa, and Nidus Vespae produced a better inhibitory effect on lung cancer cells, whether using a single Chinese medicine composition or a Chinese and Western medicine composition composed of a Chinese medicine composition and a representative epidermal growth factor receptor tyrosine kinase inhibitor. Compared with the inhibitory effect of the Chinese and Western medicine composition of Example 2 on EdU-positive cells, the inhibitory effect of the Chinese and Western medicine composition of Example 6 of the present invention on EdU-positive cells is slightly weaker. However, it should be noted that the concentration of Erlotinib (5 nM) in the Chinese and Western medicine composition of Example 6 is significantly lower than the concentration of Erlotinib (10 nM) in Example 2. Thus, it can be seen that the combination of the Chinese medicine composition of Example 6 (5 mg / mL) and Erlotinib (5 nM) in the present invention greatly reduces the dosage of Erlotinib, thereby gradually weakening the drug resistance consequences caused by the long-term use of a representative epidermal growth factor receptor tyrosine kinase inhibitor by patients.

[0124] The apoptosis rates of HCC827 cells treated with different concentrations of XYT (0, 1, 2, 5, 10 mg / mL) and erlotinib combined with XYT for 24 h were detected by flow cytometry. The results showed that Figure 8 as can be seen, with the increase of XYT concentration, the apoptosis rate of HCC827 cells could be gradually increased, and the apoptosis rates were 6.67%, 6.95%, 7.99%, 9.56%, 10.61% respectively. When the XYT concentration reached 2 mg / mL, there was a statistically significant difference compared with the control group (P < 0.05); furthermore, the apoptosis rates of the control group, XYT group, erlotinib group and XYT + erlotinib were detected by flow cytometry as 5.66%, 9.72%, 24.21% and 33.48% respectively. The application of XYT combined with erlotinib could significantly induce the apoptosis of HCC827 cells compared with single drug exposure, and there was a statistically significant difference between groups (P < 0.05), as Figure 9 shown.

[0125] Compared with the single drug exposure in Comparative Examples 1 to 8, the Chinese medicine composition combined with erlotinib could effectively reduce the division of HCC827 cells and inhibit the cell proliferation ability as Figure 10 shown. The cell cycle status of HCC827 cells treated with the control group, XYT, erlotinib, and XYT + erlotinib groups for 24 h was detected by flow cytometry. Among them, the G2 phase, S phase, and M phase reflected the proliferation activity of cells, especially the G2 phase and S phase (because the M phase was shorter). Therefore, G2 / M and S reflected the cell proliferation ability. The results are shown in the figure. The results showed that the average proportions of G0 / G1 phase, S phase, and G2 / M phase cells in the control group were 54.99%, 31.59%, and 13.41% respectively; the average proportions of G0 / G1 phase, S phase, and G2 / M phase cells in the XYT group were 64.43%, 24.52%, and 10.96% respectively; the average proportions of G0 / G1 phase, S phase, and G2 / M phase cells in the erlotinib group were 65.71%, 23.49%, and 10.77% respectively; the average proportions of G0 / G1 phase, S phase, and G2 / M phase cells in the erlotinib combined with XYT group were 71.83%, 20.01%, and 8.11% respectively. Compared with the single drug exposure, XYT combined with erlotinib could effectively reduce the division of HCC827 cells and inhibit the cell proliferation ability, and there was a statistically significant difference between groups (P < 0.05).

[0126] The traditional Chinese medicine composition adopted by the present invention can promote the apoptosis of lung tumor cells HCC827 by up-regulating the expression levels of Cleaved Caspase-8, Cleaved Caspase-3, and BAX, and inhibiting the expression level of Bcl-2; it can inhibit cell division by reducing the expression levels of cell cycle checkpoint proteins CyclinA2, Cyclin B1, CDK4, and CDK6; at the same time, by detecting the expression levels of key effector molecules related to energy metabolism, such as LDHA, HK2, GLUT1, and HIF-1α, it is found that the traditional Chinese medicine composition can regulate the key effector proteins affecting cell energy metabolism, thereby inhibiting the production of ATP, as Figure 11 shown.

[0127] The present invention further confirms through in vivo experiments that the conclusion of the in vitro experiment that the traditional Chinese medicine composition combined with erlotinib can significantly inhibit the proliferation of tumor cells and promote apoptosis, as Figure 12 shown. After 14 days, the tumor volumes of nude mice in the XYT group, Erlotinib group, and XYT+Erlotinib group were all smaller than those in the control group. Among them, compared with the tumor volume of the control group, the difference in the XYT group was not statistically significant, while the differences in the Erlotinib group and the XYT+Erlotinib group were statistically significant (P<0.05). In addition, the tumor volume of the XYT+Erlotinib group was smaller than that of the single-drug administration groups, and the difference was statistically significant (P<0.05); in terms of tumor weight, the tumor weights of each group were smaller than those of the control group. Among them, compared with the tumor weight of the control group, the difference in the XYT group was not statistically significant, while the differences in the Erlotinib group and the XYT+Erlotinib group were statistically significant (P<0.05). In addition, the tumor weight of the XYT+Erlotinib group was smaller than that of the single-drug administration groups, and the difference was statistically significant (P<0.05). The tumor inhibition rates of the XYT group, Erlotinib group, and combined group were 28.73%, 37.29%, and 74.65% respectively.

[0128] In summary, the present invention combines a traditional Chinese medicine composition with erlotinib, a representative drug of the first-generation EGFR-TKI, which significantly enhances the inhibition of the proliferation of HCC827 lung cancer cells, promotes apoptosis, and interferes with cell cycle division. The Chinese and Western medicine composition provided by the present invention realizes the effective control of advanced non-small cell lung cancer by regulating the energy metabolism of tumor cells and regulating the expression of metabolism-related proteins LDHA, HK2, GLUT1, and HIF-1α.

[0129] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. A traditional Chinese and Western medicine composition, characterized in that, The traditional Chinese and Western medicine composition is composed of a traditional Chinese medicine composition and an epidermal growth factor receptor tyrosine kinase inhibitor; The traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 25-60 parts of Astragalus membranaceus, 10-18 parts of Pseudostellaria heterophylla, 8-15 parts of Curcuma aromatica Salisb., 8-15 parts of Curcuma longa L., 15-45 parts of raw oyster, 10-20 parts of Prunella vulgaris L., 12-30 parts of Hedyotis diffusa Willd., 10-20 parts of Nidus Vespae, and 0-12 parts of Artemisia anomala S. Moore; In the traditional Chinese and Western medicine composition, the dosage ratio of the traditional Chinese medicine composition to the epidermal growth factor receptor tyrosine kinase inhibitor is 4 g-20 g: 3 nmol-10 nmol.

2. The Chinese and Western medicine composition according to claim 1, wherein The traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 30 parts of Astragalus membranaceus, 15 parts of Pseudostellaria heterophylla, 10 parts of Curcuma aromatica Salisb., 10 parts of Curcuma longa L., 30 parts of raw oyster, 15 parts of Prunella vulgaris L., 15 parts of Hedyotis diffusa Willd., 15 parts of Nidus Vespae, and 10 parts of Artemisia anomala S. Moore.

3. The Chinese and Western medicine composition according to claim 1, wherein, The dosage ratio of the traditional Chinese medicine composition to the epidermal growth factor receptor tyrosine kinase inhibitor is 1 g: 1 nmol.

4. The Chinese and Western medicine composition according to claim 1, characterized in that The epidermal growth factor receptor tyrosine kinase inhibitor is erlotinib hydrochloride, gefitinib or icotinib hydrochloride.

5. A preparation method of the traditional Chinese and western medicine composition according to claim 1, characterized in that, It includes the following steps: Weigh according to the parts by weight of each raw material: 25-60 parts of Astragalus membranaceus, 10-18 parts of Pseudostellaria heterophylla, 8-15 parts of Curcuma aromatica Salisb., 8-15 parts of Curcuma longa L., 15-45 parts of raw oyster, 10-20 parts of Prunella vulgaris L., 12-30 parts of Hedyotis diffusa Willd., 10-20 parts of Nidus Vespae, and 0-12 parts of Artemisia anomala S. Moore; Mix the above raw materials, soak them in water, decoct them, filter them to obtain the original medicinal liquid, freeze-dry it to obtain the freeze-dried powder, dissolve the freeze-dried powder in water, perform ultrasonic centrifugation to obtain the supernatant, and filter and sterilize it to obtain the traditional Chinese medicine composition solution; Dissolve and dilute the epidermal growth factor receptor tyrosine kinase inhibitor with sodium sulfobutyl ether β-cyclodextrin to obtain the epidermal growth factor receptor tyrosine kinase inhibitor solution; Mix the traditional Chinese medicine composition solution with the epidermal growth factor receptor tyrosine kinase inhibitor solution to obtain the traditional Chinese and Western medicine composition.

6. The preparation method according to claim 5, characterized in that, In the traditional Chinese and Western medicine composition, the dosage ratio of the traditional Chinese medicine composition to the epidermal growth factor receptor tyrosine kinase inhibitor is 4 g-20 g: 3 nmol-10 nmol.

7. The preparation method according to claim 5, wherein The number of decoction times is two, and the medicinal liquids after two decoctions are combined to obtain the original medicinal liquid.

8. Use of the traditional Chinese and Western medicine composition according to any one of claims 1-4 in the preparation of a drug for treating advanced non-small cell lung cancer.

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