Application of oil tea flower bud ethanol extract as EGFR inhibitor in preparation of medicine for treating non-small cell lung cancer

Abstract

The application discloses application of an ethanol extract of Camellia oleifera flower buds as an EGFR inhibitor in preparation of a drug for treating non-small cell lung cancer. In the application, the ethanol extract of Camellia oleifera flower buds for treating non-small cell lung cancer is obtained by extracting Camellia oleifera flower buds with a solvent of 70% ethanol. The ethanol extract can inhibit EGFR kinase activity, can be used as an EGFR inhibitor, can inhibit proliferation of non-small cell lung cancer A549 cells by inducing G1 phase arrest, can induce apoptosis and inhibit migration and invasion through a mitochondria-mediated pathway, and can further play an antitumor role, and has a prospect of new drug research and development for treating non-small cell lung cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of an ethanol extract of Camellia oleifera flower buds in preparation of a medicament for treating non-small cell lung cancer. BACKGROUND

[0002] Cancer is the second leading cause of death, of which lung cancer accounts for 18% of all cancer deaths (CA Cancer J Clin, 2021, 71(3): 209-249). The cell types of lung cancer are divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), of which NSCLC accounts for 85% of all lung cancer cases (Pharmaceutics, 2022, 15(1): 139). Natural products are an important source of anticancer drugs (J Nat Prod, 2012, 75(3): 311-335). Many anticancer drugs used in clinical practice are derived from natural products, such as vinblastine, vincristine, paclitaxel, etc. (Molecules, 2022, 27(23): 8367; J Nat Prod, 2012, 75(3): 311-335; Curr Med Chem, 2004, 11(5): 607-628). Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) can significantly improve the survival period and quality of life of patients with EGFR-positive mutant non-small cell lung cancer, and the commonly used EGFR-targeted drugs in clinical practice are gefitinib, afatinib, osimertinib, etc. At present, chemotherapy combined with surgical operation and radiotherapy has been used for the treatment of lung cancer symptoms, but it has great side effects, such as nephrotoxicity and neurotoxicity, which are not conducive to the survival of patients (Mol Cell Biochem, 2021, 476(1): 57-68). Therefore, there is an urgent need to develop effective drugs with less toxic side effects.

[0003] Camellia oleifera Abel. is a kind of edible and medicinal plant of Camellia in Theaceae, widely distributed in the Yangtze River Basin and southern China, and is an important woody oil crop for preparing tea oil (Planta Med, 2014, 80 (07): 590-598; Chinese Flora [M]. Beijing: Beijing Science Press, 2007, Vol. 12: 366-478). As a traditional Chinese medicine, C. oleifera flower bud has the effect of cooling blood and stopping bleeding, and is used for treating hemoptysis, hematochezia and scalding (Chinese Herbal [M], 1999, Vol. 3: 561-564; Chem Pharm Bull, 2009, 57 (3): 269-275). C. oleifera seed and seed cake are used in traditional Chinese medicine for treating diarrhea, abdominal pain, constipation, itching, eczema and scalding. As a traditional Chinese medicine, C. oleifera root is used for treating stomach pain, pharyngitis, toothache, sprains and burns (Chinese Herbal [M], 1999, Vol. 3: 561-564). The flower bud of C. oleifera is used as a traditional Chinese medicine, but its chemical composition and pharmacological activity have not been deeply studied, and there is no related report on the anti-cancer effect of the ethanol extract of the flower bud of C. oleifera. SUMMARY

[0004] The purpose of the present application is to provide the application of the ethanol extract of the flower bud of C. oleifera as an EGFR inhibitor in the preparation of a drug for treating non-small cell lung cancer, to open up new uses of the ethanol extract of the flower bud of C. oleifera, and to provide a new choice for drugs for treating non-small cell lung cancer.

[0005] The present application also finds that the ethanol extract of the flower bud of C. oleifera inhibits the activity of EGFR, has an inhibitory effect on the proliferation of human non-small cell lung cancer cells, induces apoptosis and inhibits metastasis, and identifies the chemical composition of the ethanol extract of the flower bud of C. oleifera.

[0006] The technical solutions adopted by the present application are as follows:

[0007] The application of the ethanol extract of the flower bud of C. oleifera as an EGFR inhibitor in the preparation of a drug for treating non-small cell lung cancer.

[0008] The aforementioned ethanol extract of the flower bud of C. oleifera is prepared by the following method:

[0009] Fresh C. oleifera flower buds are crushed, the crushed raw materials are mixed with 70% ethanol at a ratio of 1 kg:2L-1 kg:4L, the mixture is refluxed and extracted for 2-5h, the extraction is repeated twice, then the filtrate is combined, concentrated by rotary evaporation, and freeze-dried to obtain the ethanol extract of the flower bud of C. oleifera.

[0010] The aforementioned drug for treating non-small cell lung cancer is a drug for targeting EGFR inhibition.

[0011] The aforementioned drug for treating non-small cell lung cancer is a drug for inhibiting proliferation of non-small cell lung cancer cells.

[0012] The aforementioned drug for treating non-small cell lung cancer is a drug for inducing apoptosis of non-small cell lung cancer cells.

[0013] The aforementioned drug for treating non-small cell lung cancer is a drug for inhibiting metastasis of non-small cell lung cancer cells.

[0014] The aforementioned oil-tea camellia bract ethanol extract and the pharmaceutically acceptable carrier are prepared into a tablet, a capsule, a fat emulsion, a suppository, a dripping pill, or a ointment.

[0015] The present application has the following beneficial effects:

[0016] The present application determines the chemical components of the oil-tea camellia bract ethanol extract by LC-MS; the present application finds through experiments that the oil-tea camellia bract ethanol extract has low cytotoxicity to normal cells and high cytotoxicity to A549 cells; inhibits EGFR activity and can be used as an EGFR inhibitor; inhibits proliferation of A549 cells by inducing G1 phase arrest; induces apoptosis of A549 cells through a mitochondrial-mediated pathway; and has an inhibitory effect on metastasis of A549 cells.

[0017] By adopting the above technical solution, the present application first finds a new use of the oil-tea camellia bract ethanol extract as an EGFR inhibitor in treating non-small cell lung cancer, provides a new choice for drugs for treating human non-small cell lung cancer, and has important application value in the pharmaceutical industry. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 A is cytotoxicity of the oil-tea camellia bract ethanol extract; B is cytotoxicity of cisplatin;

[0019] Figure 2 Effect of the oil-tea camellia bract ethanol extract on EGFR kinase activity;

[0020] Figure 3 Effect of the oil-tea camellia bract ethanol extract on A549 cell cloning;

[0021] Figure 4 Effect of the oil-tea camellia bract ethanol extract on A549 cell cycle (A is flow cytometry detection result of A549 cell cycle after treatment with the oil-tea camellia bract ethanol extract; B is proportion statistics of A549 cells in different periods (G1, S, G2));

[0022] Figure 5A549 cell morphological observation of Camellia oleifera flower bud ethanol extract (A: Camellia oleifera flower bud ethanol extract on A549 cell morphological influence; B: AO / EB staining analysis of Camellia oleifera flower bud ethanol extract induced A549 cell apoptosis; C: Hoechst 33258 staining analysis of Camellia oleifera flower bud ethanol extract induced A549 cell apoptosis);

[0023] Figure 6 Flow cytometry analysis of Camellia oleifera flower bud ethanol extract induced A549 cell apoptosis (A: A549 cells were treated with different concentrations of Camellia oleifera flower bud ethanol extract for 24h, then stained with Annexin V-PE and 7-AAD, and analyzed by flow cytometry; B: The statistical results of the proportion of total apoptotic cells and viable cells);

[0024] Figure 7 JC-1 staining analysis of Camellia oleifera flower bud ethanol extract induced A549 cell apoptosis through mitochondrial-mediated apoptosis pathway (A: Camellia oleifera flower bud ethanol extract on A549 cell mitochondrial apoptosis pathway related protein; B: After Camellia oleifera flower bud ethanol extract treatment for 48h, the expression levels of apoptosis related proteins Bax, cleaved-caspase 9, pro-caspase 3 and cleaved-PARP protein levels; C: Quantitative statistical results of Bax, cleaved-caspase 9, pro-caspase 3 and cleaved-PARP band gray value);

[0025] Figure 8 Effect of Camellia oleifera flower bud ethanol extract on A549 cell migration and invasion (A: Wound healing test was used to detect the migration ability of Camellia oleifera flower bud ethanol extract on A549 cells, B: Migration rate (%) was used to quantitatively analyze the migration ability, C: Transwell invasion test was used to determine the invasion ability, D: Quantitative analysis of invasion ability). DETAILED DESCRIPTION

[0026] Embodiment of the application: Fresh Camellia oleifera flower bud (collection site: Tongren, Guizhou; identified by Professor Hu Guoxiong of Guizhou University) was crushed, and the crushed raw material was mixed with 70% solvent at a solid-liquid ratio of 1:4, reflux extracted for 2h, repeated extraction twice, then filtered, combined the filtrate, concentrated under reduced pressure in a rotary evaporator to remove the solvent, then freeze-dried to obtain Camellia oleifera flower bud ethanol extract, which was stored in a 4℃ refrigerator.

[0027] Chemical composition of Camellia oleifera bud ethanol extract: UHPLC-Q-Orbitrap-MS (ultra-high performance liquid chromatography-quadrupole orbitrap mass spectrometry) was used to analyze the phytochemicals in Camellia oleifera bud ethanol extract. Liquid chromatography system: Dionex Ultimate 3000 RSLC (HPG). Mobile phase: acetonitrile (containing 0.1% formic acid) and 0.1% formic acid water; injection volume 5 μL; flow rate 0.3 mL / min; column temperature: 40 °C; chromatographic column: ACE Ultracore 2.5 Super C18 column (100 mm x 2.1 mm, 1.9 μm). Gradient elution program: 0-2 min, 5% A; 2-42 min, 5%→95% A; 42-47 min, 95% A; 47-47.1 min, 95%→5% A; 47.1-50 min, 5% A. Mass spectrometry system: Thermo Scientific Q Exactive Focus, HESI-II thermal spray ion source. Ion source parameters: spray voltage 3.0 kV (+) / 2.5 kV (-); sheath gas 35 arb; capillary temperature 320 °C; auxiliary gas 10 arb; block cone gas 0 arb; probe heater temperature 350 °C. Mass spectrometry scanning parameters: scanning mode is Full MS-ddMS2; resolution is 70000 (Full MS) 17500 (MS / MS); scanning range is 100-1500 m / z; maximum residence time is 100 ms (Full MS) and 50 ms (MS / MS); AGC target is 1e 6 (Full MS) and 2e 5 (MS / MS); minimum AGC target is 8e 3 . Mass spectrometry data was processed using Xcalibur 4.1 (Thermo Fisher Scientific, USA) and compared and identified by mzVault and mzCloud databases, allowing the relative mass deviation to be set to 10×10 -6 .

[0028]

[0029]

[0030]

[0031] Pharmacological Example 1: Cytotoxicity of Camellia oleifera bud ethanol extract (C. oleifera bud EE) on human non-small cell lung cancer cells (A549, NCI-H1299) and normal cells (L929 and MRC-5)

[0032] The experimental process used complete medium, i.e. RPMI-1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin. The cells in the logarithmic phase were trypsinized, counted by a hemocytometer, and diluted with RPMI-1640 complete medium to the desired cell concentration; 80 μL was inoculated in a 96-well plate (5 x 10 3 After the cells were completely stable or adherent, 20 μL of the Camellia oleifera flower bud ethanol extract diluted with RPMI-1640 medium was added to each well, and the same volume of medium was added to the negative control group, with 5 parallel holes in each group. The plates were incubated at 37°C in a 5% CO2 incubator for 48 h. After co-culture, 10 μL of MTT (5 mg / mL) was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator for 4 h. The 96-well plates were removed, and the supernatant was aspirated. DMSO 150 μL was added, and the plates were shaken for 10 min to completely dissolve the MTT reaction product. The OD value at 490 nm was read using a microplate reader, and the results were repeated three times and averaged.

[0033] The inhibition rate was calculated as follows: cell line inhibition rate = [1 - (sample group OD value - zero hole OD value) / (control group OD value - zero hole OD value)] x 100%.

[0034] The SPSS 25.0 analysis software was used to calculate the concentration of the sample required to inhibit half of the cell proliferation (IC 50 ), and IC 50 was used to evaluate the cytotoxicity of the sample. The results are shown in Table 2. The Camellia oleifera flower bud ethanol extract had the best cytotoxic activity on human non-small cell lung cancer cells (A549) (IC 50 = 57.53 ± 1.54 μg / mL), and the cytotoxicity on MRC-5 (IC 50 > 320 μg / mL) was lower. By comparing the inhibitory effect of the Camellia oleifera flower bud ethanol extract on A549 and MRC-5 cells, the Camellia oleifera flower bud ethanol extract had lower toxicity on normal cells, indicating that the Camellia oleifera flower bud ethanol extract had good selectivity for cancer cells and possessed the characteristics of low toxicity and high antitumor activity.

[0035] Pharmacological Example 2: Determination of the effect of the Camellia oleifera flower bud ethanol extract on EGFR enzyme activity based on enzyme activity detection

[0036] The Camellia oleifera flower bud ethanol extract was diluted to 50 μg / mL as the initial concentration, and 8 concentrations (2.5, 5, 10, 20, 40, 80, 160 and 320 μg / mL) were set by 2-fold dilution. The Camellia oleifera flower bud ethanol extract was diluted to 100 times of the final concentration in 100% DMSO solution in a 384-well plate. Then 250 μL was transferred to a 384-reaction plate by Echo550 for standby. 250 μL of 100% DMSO was added to the negative control and positive control wells, respectively. Meanwhile, 1×kinase buffer was prepared, and the kinase solution was prepared to 2.5 times of the final concentration. 10 μL of 2.5 times of the final concentration of the kinase was added to the test wells and positive control wells, respectively; 10 μL of 1×kinase buffer was added to the negative control wells. Centrifugation was performed at 1000 r / min for 30 s, and then the mixture was shaken and incubated at room temperature for 10 min. A mixed solution of 25 / 15 times of the final concentration of ATP and kinase substrate 22 was prepared by using 1×kinase buffer, and 15 μL of the mixed solution was added to the 384-well plate, which was centrifuged at 1000 r / min for 30 s, shaken and mixed, and then incubated at room temperature for 20 min. The kinase reaction was stopped by adding 30 μL of the termination detection solution, centrifuging at 1000 r / min for 30 s, shaking and mixing, and then reading the conversion rate by Caliper EZ Reader, and fitting the dose-effect curve.

[0037] EGFR, as a tyrosine kinase, can further activate multiple downstream signaling pathways after being stimulated by other ligands, thereby promoting the occurrence and development of tumors. Therefore, inhibiting the enzyme activity of EGFR can play an anti-tumor role. The Camellia oleifera flower bud ethanol extract has a strong inhibitory effect on the activity of EGFR kinase, and can inhibit the activity of EGFR kinase in a concentration-dependent manner Figure 2 ). The IC 50 of the Camellia oleifera flower bud ethanol extract is 21.56 μg / mL. The enzymatic detection results show that the Camellia oleifera flower bud ethanol extract can play an anti-non-small cell lung cancer role by inhibiting the function of EGFR, and can be used as an EGFR inhibitor.

[0038] Pharmacological Example 3: Evaluation of the proliferation ability of A549 cells by colony formation experiment.

[0039] Cells were seeded in six-well plates at a density of 200 cells per well, incubated for 24 h, and treated with different concentrations of C. camphora flower bud ethanol extract (0, 10, 20, 30, and 40 μg / mL) for 24 h. Then, we removed the culture medium, washed each well, and added fresh culture medium. After 7 d of incubation, the cells were washed twice with PBS, fixed with a formaldehyde solution (10%, 700 μL) for 30 min, permeabilized with anhydrous methanol (700 μL) for 20 min, and stained with crystal violet (0.1%, 700 μL) for 15 min. The six-well plates were washed with water, dried at room temperature, placed under a white background cloth, and photographed for counting and calculation of the monoclonal rate.

[0040]

[0041] The anti-proliferative activity of C. camphora flower bud ethanol extract was evaluated by a cell colony formation experiment. C. camphora flower bud ethanol extract significantly reduced the size and number of A549 cell colonies Figure 3 A). As shown in Figure 3 B, the colony formation rate of A549 cells treated with different doses of C. camphora flower bud ethanol extract (10, 20, 30, and 40 μg / mL) was significantly reduced to 21.25 ± 1.06%, 18.25 ± 0.35%, 12.50 ± 0.71%, and 8.50 ± 1.41%, respectively, compared with the control group (clony formation rate: 28.75 ± 2.48%). The above data indicate that C. camphora flower bud ethanol extract inhibits the proliferation of A549 cells in a concentration-dependent manner.

[0042] Pharmacological Example 4: Effect of C. camphora flower bud ethanol extract on the cell cycle of A549 cells

[0043] A549 cells were plated in 6-well plates (4 x 10 5 cells per well), incubated for 24 h, and then administered 0, 10, 20, 40, 80, and 160 μg / mL, with three replicate wells in each group, and incubated for another 24 h. The cells were trypsinized, collected in EP tubes, and centrifuged to discard the supernatant. They were washed once with PBS and centrifuged to discard the supernatant. Then, 1 mL of DNA Staining solution and 10 μL of Permeabilization solution were added, and the mixture was incubated at room temperature in the dark for 30 min. The red fluorescence at an excitation wavelength of 488 nm was detected on a flow cytometer, and the cell cycle phases were analyzed. The effect of C. camphora flower bud ethanol extract on the cell cycle of A549 cells was detected by flow cytometry.

[0044] To determine whether the anti-proliferative effect of C. camphora flower bud ethanol extract is caused by cell cycle arrest, we measured its effect on the cell cycle. The results are shown in Figure 3The proportion of G1 phase cells increased from 41.70 ± 0.61% in the control to 44.16 ± 1.62%, 49.22 ± 0.02%, 49.97 ± 1.19%, 51.18 ± 0.96% and 53.33 ± 0.08% after treatment with the Camellia oleifera flower bud ethanol extract at concentrations of 10, 20, 40, 80 and 160 μg / mL. The above results show that the Camellia oleifera flower bud ethanol extract inhibits the proliferation of A549 cells by arresting the cell cycle at the G1 phase.

[0045] Pharmacological Example 5: Effect of the Camellia oleifera flower bud ethanol extract on apoptosis of A549 cells

[0046] (1) Drug treatment: A549 cells in the logarithmic phase were taken, digested with 0.25% trypsin, resuspended with RPMI-1640 (containing 10% fetal bovine serum), counted with a hemocytometer, inoculated into a 6-well plate at 4 x 10 5 cells / well, and cultured in a CO2 incubator for 24 h. The culture medium was discarded, 2 mL of culture medium containing the drug (0, 20, 40, 80, 160 μg / mL) was added, and the culture was continued for 48 h. After drug treatment, the morphological changes of A549 cells were observed under an inverted microscope. After drug treatment, AO / EB staining was performed. AO staining solution was mixed with EB staining solution at a ratio of 1:1, vortexed to dissolve thoroughly, the culture medium was discarded, the cells were washed twice with pre-cooled PBS buffer, 1 mL of AO / EB staining solution was added to each well, and incubation was performed in the dark for 5 min. The staining solution was aspirated, and observation, photography and analysis were performed under an inverted fluorescence microscope. After drug treatment, Hoechst 33258 staining was performed. The culture medium was discarded, 0.5 mL of 4% paraformaldehyde solution was added to each well as a fixing solution, and the cells were fixed for 10 min. The fixing solution was discarded, the cells were washed twice with pre-cooled PBS, and the liquid was aspirated. 0.5 mL of Hoechst 33258 staining solution was added to each well, and the cells were stained for 5 min. The staining solution was discarded, the cells were washed twice with pre-cooled PBS, and the liquid was aspirated. Observation, photography and analysis were performed under an inverted fluorescence microscope.

[0047] As shown in Figure 5 A, the proportion of shrunken cells increased significantly after treatment of A549 cells with the Camellia oleifera flower bud ethanol extract at different concentrations for 48 h; as shown in Figure 5 B, the proportion of green fluorescence decreased gradually and the proportion of orange-red fluorescence increased gradually after treatment with the Camellia oleifera flower bud ethanol extract at different concentrations, indicating that the proportion of apoptotic cells increased gradually; as shown in Figure 5 C, the proportion of cells showing bright blue fluorescence of the dense nucleus increased gradually after treatment with the Camellia oleifera flower bud ethanol extract at different concentrations, which is a characteristic of cell apoptosis. The above morphological observations show that the Camellia oleifera flower bud ethanol extract can effectively induce apoptosis of A549 cells.

[0048] Compared with the blank group (apoptosis rate: 7.28 ± 0.08%), the proportion of A549 cell apoptosis was increased to 18.04 ± 0.98%, 22.09 ± 0.16%, 36.18 ± 0.80%, 42.92 ± 3.51% and 61.31 ± 4.43% respectively after the treatment of the Camellia reticulata Lindl. flower bud ethanol extract at the concentration of 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL and 160 μg / mL for 24 h, which indicated that the Camellia reticulata Lindl. flower bud ethanol extract significantly increased the proportion of apoptosis in a dose-dependent manner, and the Camellia reticulata Lindl. flower bud ethanol extract significantly reduced the proportion of living cells. Therefore, the Camellia reticulata Lindl. flower bud ethanol extract significantly induced the apoptosis of A549 cells in a dose-dependent manner.

[0049] Down-regulation of mitochondrial membrane potential is a hallmark event of apoptosis. JC-1 is a fluorescent probe for detecting mitochondrial membrane potential (ΔΨm). When the mitochondrial membrane potential is high, JC-1 forms a polymer in the mitochondrial matrix, producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot form a polymer in the mitochondrial matrix, and JC-1 is a monomer, producing green fluorescence. The ΔΨm value of mitochondria is measured by red and green fluorescence. The results of JC-1 staining are shown in FIG. 2A. After the treatment of A549 cells with different concentrations of the Camellia reticulata Lindl. flower bud ethanol extract for 48 h, the proportion of red fluorescent cells gradually decreased, and the proportion of green fluorescent cells gradually increased. The results indicated that the Camellia reticulata Lindl. flower bud ethanol extract could effectively lead to the down-regulation of mitochondrial membrane potential (ΔΨm) of A549 cells, thereby leading to apoptosis. Figure 7

[0050] The key proteins in the mitochondrial-mediated apoptosis pathway were detected by WB. As shown in FIG. 2B, the Camellia reticulata Lindl. flower bud ethanol extract up-regulated the expression of Bax, down-regulated the level of pro-caspase 3, up-regulated the level of cleaved-caspase 9 and cleaved-PARP, which indicated that the caspase cascade reaction was activated, leading to the cleavage of PARP. Therefore, the Camellia reticulata Lindl. flower bud ethanol extract could lead to the apoptosis of A549 cells through the mitochondrial apoptosis pathway. Figure 7

[0051] ​​In summary, by morphological observation, AO / EB staining, Hoechst 33258 staining and flow cytometry apoptosis detection, the results showed that the Camellia flower bud ethanol extract induced A549 cell apoptosis; JC-1 staining results showed that the Camellia flower bud ethanol extract caused the down-regulation of mitochondrial membrane potential (ΔΨm); combined with the results of Western Blot related to the mitochondrial apoptosis pathway, it was shown that the mechanism of the Camellia flower bud ethanol extract inducing A549 apoptosis was as follows: the Camellia flower bud ethanol extract up-regulated the expression level of Bax, thereby causing the increase of mitochondrial membrane permeability, the down-regulation of mitochondrial membrane potential (ΔΨm), the cleavage and activation of Caspase-9 (up-regulation of Cleaved-Caspase 9), followed by the cleavage and activation of Caspase-3 (down-regulation of Pro-Caspase 3), and then the cleavage and inactivation of PARP (up-regulation of Cleaved-PARP), leading to the apoptosis of A549.

[0052] Pharmacological Example 6: Effect of Camellia flower bud ethanol extract on A549 cell metastasis

[0053] (1) Scratch test: A549 cell suspension was added to a six-well plate at 3 x 10 5 cells per well. Incubate overnight until the cells cover the bottom of the six-well plate to form a single layer of confluent cells. Then use the tip of a 200 μL pipette to draw a straight line on the monolayer cells in each well. Next, wash the cells that float up due to the scratch with PBS, and then add different concentrations of Camellia flower bud ethanol extract (prepared in serum-free medium) for 24 h. Take pictures of the scratch area at 0 h and 24 h using a Leica microscope. The cell migration rate is calculated as follows:

[0054]

[0055] (2) Invasion test: 750 μL of different concentrations of drug solution (15% FBS) was added to the lower chamber. 250 μL of A549 cell suspension (4 x 10 5 cells per well) was inoculated in the upper chamber and 250 μL of different concentrations of drug solution (containing 5% FBS) was added. After 48 h of incubation, fix with 4% paraformaldehyde for 2 min; incubate with anhydrous methanol for 20 min; 0.1% crystal violet staining for 15 min; wash twice with PBS and take pictures using a Leica DMi8 microscope. Use ImageJ software to quantify the number of invasive cells in each field and perform statistical analysis.

[0056] Wound healing test and invasion test were used to study the effect of Camellia flower bud ethanol extract on the migration and invasion ability of A549 cells. As Figure 8A and B, the migration rate of A549 cells treated with different concentrations (10, 20, 30 and 40 μg / mL) of Camellia oleifera flower bud ethanol extract was significantly lower than that of the control group (96.04 ± 2.46%), which was 71.83 ± 4.25%, 53.60 ± 1.23%, 20.01 ± 3.94% and 6.20 ± 0.59%, respectively. Therefore, the inhibition of A549 cell migration ability by Camellia oleifera flower bud ethanol extract was obviously concentration-dependent. The results of the invasion test Figure 8 C and D) showed that Camellia oleifera flower bud ethanol extract could significantly reduce the number of A549 cell invasions compared with the control group, and was related to the dose, which indicated that Camellia oleifera flower bud ethanol extract could inhibit the invasion ability of A549 cells.

[0057] In summary of the above pharmacological examples, Camellia oleifera flower bud ethanol extract showed selective cytotoxicity to A549 cells, with lower cytotoxicity to normal cells; inhibited EGFR activity and could be used as an EGFR inhibitor; inhibited the proliferation of A549 cells by inducing G1 phase arrest; induced apoptosis of A549 cells through the mitochondrial-mediated pathway; and inhibited the metastasis of A549 cells.

Claims

1. The application of the ethanol extract of Camellia oleifera flower buds as an EGFR inhibitor in the preparation of drugs for treating non-small cell lung cancer, characterized in that: The ethanol extract of Camellia oleifera flower buds was prepared by the following method: Fresh camellia buds were crushed, and the crushed raw material was mixed with 70% ethanol at a material-liquid ratio of 1kg:2L~1kg:4L. The mixture was refluxed and extracted for 2~5 hours. The extraction was repeated twice, and then filtered. The filtrates were combined, concentrated by rotary evaporation, and freeze-dried to obtain the camellia bud ethanol extract.

2. The application according to claim 1, characterized in that: The drug used to treat non-small cell lung cancer is a drug that targets and inhibits EGFR.

3. The application according to claim 1, characterized in that: The drug used to treat non-small cell lung cancer is a drug that inhibits the proliferation of non-small cell lung cancer cells.

4. The application according to claim 1, characterized in that: The drug mentioned for treating non-small cell lung cancer is a drug that induces apoptosis in non-small cell lung cancer cells.

5. The application according to claim 1, characterized in that: The drug mentioned for treating non-small cell lung cancer is a drug that inhibits the metastasis of non-small cell lung cancer cells.

6. The application according to claim 1, characterized in that: The ethanol extract of the Camellia oleifera flower buds and a pharmaceutically acceptable carrier are used to formulate pharmaceutical dosage forms such as tablets, capsules, fat emulsions, suppositories, drops, and ointments.

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