Application of pittosporum glabra leaf volatile oil in preparation of medicine for treating non-small cell lung cancer

Abstract

The invention discloses an application of pittosporum glabra leaf volatile oil in preparation of a medicine for treating non-small cell lung cancer. The pittosporum glabra leaf volatile oil for treating the non-small cell lung cancer is extracted from pittosporum glabra leaves through steam distillation, and the volatile oil inhibits proliferation of non-small cell lung cancer cells in a G1 phase by blocking a cell cycle, induces apoptosis through a mitochondrial pathway and inhibits migration and invasion of the cells; therefore, the compound has the prospect of research and development of new anti-tumor drugs for treating non-small cell lung cancer and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of Pittosporum glabratum Lindl. leaf volatile oil in preparation of a medicine for treating non-small cell lung cancer. BACKGROUND

[0002] According to the global cancer statistics in 2020, lung cancer is the most common cancer, accounting for 11.4% of the total incidence of cancer and 18.0% of the total number of cancer deaths (CA Cancer J Clin, 2021, 71: 209-249). At present, chemotherapy combined with surgical operation and radiotherapy has been used for lung cancer treatment, but its side effects are large, such as nephrotoxicity and neurotoxicity, which are not conducive to the survival of patients (Mol Cell Biochem, 2021, 476(1): 57-68). Therefore, it is urgent to develop effective drugs with smaller toxic side effects. Natural products are an important source of antitumor drugs. Elemene derived from volatile oil of Curcuma zedoary has been developed into an antitumor drug: elemene emulsion injection and oral emulsion, in which the elemene emulsion injection has been used for treating lung cancer.

[0003] Pittosporum glabratum Lindl. is commonly known as Changguo Mantianxi, Yidu Yun and Guang Haitong, is a Pittosporaceae evergreen shrub of Pittosporum, and is distributed in Guangdong (Hainan Island), Guangxi, Guizhou and Hunan (Flora of China[M]. Beijing: Beijing Science Press, 1979, Vol. 35(2), pp: 11-12). In Guizhou, Pittosporum glabratum Lindl. is a Miao medicine, is commonly known as Shanzhicha in Miao language, grows in a mountain belly, a shrub or a streamside forest at an altitude of 500-1700 m; the root: tonifying deficiency and reducing blood pressure, tranquilizing and soothing the nerves, is used for treating neurasthenia, hypertension, dizziness and insomnia; the seed: clearing heat and relieving the throat, stopping diarrhea, is used for treating sore throat, thirst, hot and restless heart and the like; and Annaoshu Capsule and Jiulongdan Capsule both contain Pittosporum glabratum Lindl. (Chinese Herbal Medicine · Miao Medicine Volume[M]. Guiyang: Guizhou Science and Technology Press, 2005, pp: 260-261). Pittosporum glabratum Lindl. has great development value in medicine, but at present, the research on the chemical composition and pharmacological activity of Pittosporum glabratum Lindl. is less, and there is no relevant report on the anticancer effect of the volatile oil thereof. SUMMARY

[0004] The purpose of the application is to provide application of Pittosporum glabratum Lindl. leaf volatile oil in preparation of a medicine for treating non-small cell lung cancer, to develop a new use of Pittosporum glabratum Lindl. leaf volatile oil, and to provide a new choice for the medicine for treating non-small cell lung cancer.

[0005] The application also finds that the volatile oil of Pittosporum glabratum Merr. inhibits the proliferation of non-small cell lung cancer through G1 phase arrest, induces apoptosis through a mitochondrial pathway, and inhibits the metastasis of non-small cell lung cancer cells, and the chemical components of the volatile oil of Pittosporum glabratum Merr. are identified.

[0006] The technical scheme adopted by the application is as follows:

[0007] The volatile oil of Pittosporum glabratum Merr. is prepared by the following method:

[0008] Fresh Pittosporum glabratum Merr. leaves are crushed, the crushed raw materials are mixed with distilled water at a material-to-liquid ratio of 1:3 to 1:8, g / mL, the mixture is extracted by steam distillation in a volatile oil extractor for 3 to 5 hours, the volatile oil is collected, anhydrous sulfuric acid is used for water removal, and the volatile oil of Pittosporum glabratum Merr. is obtained.

[0009] The application determines the chemical components of the volatile oil of Pittosporum glabratum Merr. by GC-MS (see Table 1 in detail), wherein the main chemical components are alpha-caryophyllene (15.8%), (E)-beta-farnesene (11.9%), germacrene D (10.8%), caryophyllene (7.5%), n-hexanol (7.2%), delta-cadinene (3.9%), and bicyclic germacrene (3.6%). Figure 1

[0010] The application finds through experimental research that the cytotoxic activity of the volatile oil of Pittosporum glabratum Merr. on human non-small cell lung cancer cells is equivalent to that of the positive control elemene emulsion injection, and the cytotoxicity on normal cells is relatively low, which shows low toxicity and high antitumor activity; the volatile oil of Pittosporum glabratum Merr. has an obvious inhibitory effect on the proliferation of human non-small cell lung cancer cells through G1 phase arrest; the volatile oil of Pittosporum glabratum Merr. promotes the apoptosis of human non-small cell lung cancer cells through a mitochondrial apoptosis pathway; and the volatile oil of Pittosporum glabratum Merr. has a significant inhibitory effect on the metastasis of human non-small cell lung cancer.

[0011] By adopting the above technical scheme, the application first finds the new use of the volatile oil of Pittosporum glabratum Merr. in treating non-small cell lung cancer diseases, 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

[0012] Figure 1 The figure is a GC-MS chromatogram of the volatile oil of Pittosporum glabratum Merr.;

[0013] Figure 2 The figure is a cytotoxicity comparison of the volatile oil of Pittosporum glabratum Merr. on non-small cell lung cancer cells (A549), human prostate cancer cells (PC-3), colon cancer cell lines (HT-29), and normal human embryonic lung fibroblasts (MRC-5);

[0014] Figure 3 ​Effects of volatile oil from Pittosporum illicioides leaves on the colony formation of A549 cells

[0015] Figure 4 Effects of volatile oil from Pittosporum illicioides leaves on the cell cycle of A549 cells. (A) Flow cytometry detection results of A549 cell cycle after treatment with volatile oil from Pittosporum illicioides leaves, (B) Proportion statistics of A549 cells in different periods (G1, S, G2);

[0016] Figure 5 Effects of volatile oil from Pittosporum illicioides leaves on the expression levels of cell cycle-related proteins in A549 cells. (A) Western blot results of cell cycle-related proteins after treatment with volatile oil from Pittosporum illicioides leaves, (B) Quantitative statistics results of gray values of Western blot bands;

[0017] Figure 6 Morphological observation of A549 cells treated with volatile oil from Pittosporum illicioides leaves. (A) Morphological effects of volatile oil from Pittosporum illicioides leaves on A549 cells, (B) AO / EB staining analysis of apoptosis of A549 cells induced by volatile oil from Pittosporum illicioides leaves, (C) Hoechst 33258 staining analysis of apoptosis of A549 cells induced by volatile oil from Pittosporum illicioides leaves;

[0018] Figure 7 Flow cytometry analysis of apoptosis of A549 cells induced by volatile oil from Pittosporum illicioides leaves. (A) Analysis by flow cytometry after staining with Annexin V-FITC and PI of A549 cells treated with volatile oil from Pittosporum illicioides leaves at different concentrations for 24 h, (B) Statistics results of proportion of total apoptotic cells and living cells;

[0019] Figure 8 JC-1 staining analysis of effects of volatile oil from Pittosporum illicioides leaves on mitochondrial membrane potential of A549 cells;

[0020] Figure 9 Effects of volatile oil from Pittosporum illicioides leaves on mitochondrial apoptosis pathway-related proteins in A549 cells. (A, C) Expression levels of total Cyt c, mitochondrial Cyt c, Bax, Bcl-2, pro-caspase 9, cleaved-caspase 9, pro-caspase 3, cleaved-caspase 3, and cleaved-PARP protein levels after treatment with volatile oil from Pittosporum illicioides leaves for 48 h, (B, D, E) Quantitative statistics results of gray values of total Cyt c, mitochondrial Cyt c, Bax, Bcl-2, pro-caspase 9, cleaved-caspase 9, pro-caspase 3, cleaved-caspase 3, and cleaved-PARP bands;

[0021] Figure 10 Effects of volatile oil of Pittosporum illicioides on migration and invasion of A549 cells and expression of related proteins. (A, B) The migration ability of A549 cells treated with volatile oil of Pittosporum illicioides was detected by wound healing test, and the migration ability was quantitatively analyzed by migration rate (%); (C, D) The invasion ability (x100) was determined by transwell invasion test, and the invasion ability was quantitatively analyzed; (E, F) WB was used to detect the expression of transfer-related proteins (MMP-2, MMP-9 and N-cadherin) in A549 cells treated with volatile oil of Pittosporum illicioides, and the gray value of MMP-2, MMP-9 and N-cadherin bands was quantitatively analyzed. DETAILED DESCRIPTION

[0022] Embodiment of the application: Fresh Pittosporum illicioides leaf volatile oil (collection site: Guiyang, Guizhou; identified by Professor Hu Guoxiong of Guizhou University) was crushed, the crushed raw material was mixed with distilled water at a solid-liquid ratio of 1:4, the mixture was extracted in a volatile oil extractor for 4 h by steam distillation, the volatile oil was collected, anhydrous sulfuric acid was used for water removal, and Pittosporum illicioides leaf volatile oil was obtained, which was sealed and stored in a 4°C refrigerator

[0023] Chemical composition of Pittosporum illicioides leaf volatile oil in this embodiment: The chemical composition of Pittosporum illicioides leaf volatile oil was identified by GC-MS. GC-MS analysis conditions: sample amount 2 μL, chromatographic column HP-5MS (60 m x 0.25 mm x 0.25 μm) elastic quartz capillary column, initial temperature 70°C (retention 2 min), temperature rise 2°C / min to 180°C, then temperature rise 10°C / min to 310°C (retention 14 min), running time: 84 min; vaporization chamber temperature 250°C; carrier gas high-purity He (99.999%); pre-column pressure 18.53 psi, carrier gas flow rate 1.0 mL / min, split, split ratio 10:1, solvent delay time: 6.0 min; ion source EI source; ion source temperature 230°C; quadrupole rod temperature 150°C; electron energy 70 eV; emission current 34.6 μA; multiplier voltage 1847 V; interface temperature 280°C; mass range 29-500 amu. Each peak in the total ion chromatogram was searched and checked by the mass spectrometry computer data system against Nist2020 and Wiley275 standard mass spectra to determine the chemical composition, and the relative mass fraction of each chemical composition was determined by peak area normalization method. The chemical composition of Pittosporum illicioides leaf volatile oil is shown in Table 1. Figure 1 and Table 1, a total of 39 chemical compositions were identified, accounting for 99.1% of the total peak area, among which the main chemical compositions were a-caryophyllene (15.8%), (E)-β-farnesene (11.9%), germacrene D (10.8%), caryophyllene (7.5%), n-hexanol (7.2%), δ-cadinene (3.9%), bicyclic germacrene (3.6%).

[0024] Table 1 Chemical components of volatile oil from Pittosporum tobira leaves

[0025]

[0026]

[0027]

[0028] Note: a represents the compounds listed in the order of elution from the HP-5MS column; b represents the retention time; c represents the retention index in the NIST2020 database; d represents the retention index of n-alkanes (C6–C 20 ) The retention index was calculated. Pharmacological Example 1: Cytotoxicity of the essential oil from the leaves of Pittosporum tobira against human non-small cell lung cancer cells (A549), human prostate cancer cells (PC-3), colon cancer cell lines (HT-29) and normal human embryonic lung fibroblasts (MRC-5)

[0029] All experiments were performed using complete culture medium (RPMI-1640 medium, containing 10% fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin). Cells in the logarithmic phase were selected, trypsinized, counted using a hemocytometer, and diluted to the desired cell concentration using RPMI-1640 complete culture medium. 80 μL of the culture medium was seeded into a 96-well plate (5 × 10 cells per well). 3 Cells were cultured in a CO2 incubator for 24 hours. After the cells were completely stable or adhered, 20 μL of a Tween-80 solution of Pittosporum tobira leaf essential oil diluted in RPMI-1640 medium was added to each well. An equal volume of medium was added to the negative control group. Five parallel wells were set up in each group and cultured in a 37°C, 5% CO2 incubator for 48 hours. After co-culture, 10 μL of MTT (5 mg / mL) was added to each well and the cells were incubated in a 37°C, 5% CO2 incubator for another 4 hours. The 96-well plate was removed and the supernatant was aspirated. 150 μL of DMSO was added and the cells were shaken for 10 minutes to completely dissolve the MTT reaction product. The OD values ​​were read at a wavelength of 490 nm using a microplate reader. The results were repeated three times and the average value was calculated.

[0030] The calculation formula of the inhibition rate is as follows: cell line inhibition rate = [1-(OD value of the sample group-OD value of the zero-adjustment well) / (OD value of the control group-OD value of the zero-adjustment well)] × 100%.

[0031] SPSS 25.0 analysis software was used to calculate the concentration of the sample required to inhibit half of the cell proliferation (IC 50 ), using IC 50The cytotoxicity of the samples was evaluated. The results are shown in Table 2. The volatile oil from Pittosporum tobira leaves had an inhibitory effect on human non-small cell lung cancer cells A549 (IC 50 =35.32±2.89μg / mL) had the best cytotoxic activity, which was more effective than the positive control elemene emulsion injection (IC 50 =63.90±2.29μg / mL) and MRC-5 (IC 50 =152.20±7.73μg / mL) showed low cytotoxicity. In addition, the cytotoxicity to human tumor cells PC-3 (IC 50 =58.33±4.33μg / mL) and colon cancer cell line HT-29 (IC 50 =57.09±1.19μg / mL) showed good cytotoxicity. The inhibitory effect of Pittosporum tobira leaf volatile oil on A549, PC-3, HT-29 and MRC-5 was compared (such as Figure 2 As shown in the results, the volatile oil from the leaves of Pittosporum tobira had a better effect on A549 cells than the positive control drug elemene emulsion injection, and had lower toxicity to normal cells, indicating that the volatile oil from the leaves of Pittosporum tobira had good selectivity for cancer cells and was characterized by low toxicity and high anti-tumor activity.

[0032] Table 2 Cytotoxic activity of Pittosporum tobira leaf volatile oil against tumor cells and normal cells

[0033]

[0034] Pharmacological Example 2: Effect of Pittosporum tobira leaf volatile oil on A549 cell proliferation (plate clone formation assay)

[0035] A549 cells in the logarithmic phase were digested with 0.25% trypsin and resuspended in RPMI-1640 medium (containing 10% fetal bovine serum). After counting using a hemocytometer, 200 cells were seeded per well in a 6-well plate and dispersed by pipetting. The cells were then incubated in a CO2 incubator for 24 hours. The medium was discarded, and the blank group and the Pittosporum tobira leaf volatile oil-treated group were treated with 0, 30, 35, 40, 45, and 50 μg / mL of the drug, respectively. Three replicate wells were set up in each group, and routine culture was continued. After 3 days, fresh medium was replaced and cultured for approximately one week. Cell proliferation and colony formation were monitored under an inverted microscope until visible colonies formed. The medium was aspirated, the cells were rinsed twice with PBS, and 800 μL of fixative was added to each well. The cells were allowed to stand at room temperature for 30 minutes, after which the fixative was discarded and 800 μL of 0.1% crystal violet stain was added for staining. After being placed away from light for 15 minutes, the staining solution was discarded and washed off with distilled water. The plate was then allowed to stand at room temperature to evaporate the distilled water. The 6-well plate was placed under a white background cloth, and the cells were photographed and counted to calculate the monoclonal rate.

[0036]

[0037] The effect of the volatile oil of P. glabra leaves on the colony formation of A549 cells was investigated by a plate colony formation experiment, and the inhibitory effect of the volatile oil on the proliferation of A549 cells was explored, and the results are shown in Figure 3 Compared with the blank group without treatment of the volatile oil of P. glabra leaves, the volatile oil of P. glabra leaves significantly reduced the number and size of A549 cell colonies Figure 3 A). As shown in Figure 3 B, compared with the blank group (clone formation rate: 32.75 ± 0.53%), after the A549 cells were treated with the volatile oil of P. glabra leaves at concentrations of 30, 35, 40, 45 and 50 μg / mL, the clone formation rates of the A549 cells were 20.50 ± 0.71%, 16.00 ± 0.35%, 9.75 ± 0.53%, 4.75 ± 0.18% and 0.75 ± 0.18%, respectively, indicating that the volatile oil of P. glabra leaves significantly reduced the clone formation rate of A549 cells in a dose-dependent manner (p < 0.001). In summary, the volatile oil of P. glabra leaves significantly reduced the number and size of colonies, and significantly reduced the clone formation rate of A549 cells in a dose-dependent manner, and had a significant inhibitory effect on the proliferation of A549 cells.

[0038] Pharmacological Example 3: Effect of the volatile oil of P. glabra leaves on the cell cycle of A549 cells

[0039] The A549 cells in the logarithmic phase were counted by a hemocytometer, and then 3 × 10 5 cells / well were inoculated into a 6-well plate and cultured in a CO2 incubator for 24 h; the culture medium was discarded, and the cells were treated according to the blank group and the volatile oil of P. glabra leaves treatment group, and 0, 20, 40, 80, 100 and 160 μg / mL of the volatile oil of P. glabra leaves were added, respectively, and three replicate wells were set in each group, and the cells were cultured for another 24 h; the cells were collected in an EP tube by trypsinization, centrifuged at 800 r / min for 10 min, the culture medium was discarded, and the cells were washed with PBS, centrifuged to discard the supernatant, and then the cells were resuspended with PBS, counted and the cell concentration was adjusted to 1 × 10 6 cells / mL; 1 mL of the cell suspension was taken into an EP tube, centrifuged to discard the supernatant, and then washed with PBS and centrifuged to discard the supernatant; Permeabilization solution (10 μL) and DNA Staining solution (1 mL) were added, vortexed for 5-10 seconds, and mixed well, and then incubated at room temperature for 30 min in the dark; the red fluorescence at an excitation wavelength of 488 nm was detected on a flow cytometer, and then the cell cycle phases were analyzed.

[0040] The effect of the volatile oil of P. glabra leaves on the cell cycle of A549 cells was detected by a flow cytometer, and the results are shown in Figure 4The proportion of G1 phase cells was significantly increased by the leaf volatile oil of P. glabra, while the proportion of S phase and G2 phase cells was significantly decreased, leading to G1 phase arrest. Compared with the blank group (G1 phase: 45.41 ± 0.48%), the proportion of G1 phase cells in A549 cells was increased to 59.22 ± 0.46%, 68.89 ± 0.93%, 78.79 ± 0.96%, 84.53 ± 0.74% and 87.19 ± 1.72% after 20, 40, 80, 100 and 160 μg / mL of the leaf volatile oil of P. glabra was applied to A549 cells for 24 h, respectively, indicating that the leaf volatile oil of P. glabra significantly increased the proportion of G1 phase cells in a dose-dependent manner (p < 0.001). Therefore, the leaf volatile oil of P. glabra significantly increased the proportion of G1 phase cells, leading to G1 phase arrest in A549 cells.

[0041] The effect of the leaf volatile oil of P. glabra on the expression of cell cycle-related proteins in A549 cells was studied by Western blot, and the results are shown in Figure 5 Compared with the blank group, the expression of p21 was significantly up-regulated (p < 0.05 or p < 0.001) in a concentration-dependent manner after A549 cells were treated with the leaf volatile oil of P. glabra at concentrations of 40, 60 and 80 μg / mL. The expression of CDK4 and Cyclin D3 associated with CDK4 in A549 cells was inhibited (p < 0.05 or p < 0.01) after the cells were treated with the leaf volatile oil of P. glabra at concentrations of 60 and 80 μg / mL. The results showed that the expression of p21 was significantly up-regulated with the increase of the concentration of the leaf volatile oil of P. glabra, the expression of CDK4 and Cyclin D3 was inhibited at high concentrations, which inhibited the activity of the complex formed by them, leading to the obstruction of G1 phase progression and resulting in G1 phase arrest. Compared with the blank group, the expression level of CDK2 protein was significantly decreased (p < 0.05 or p < 0.001) with the increase of the concentration of the leaf volatile oil of P. glabra, and the expression of Cyclin E1 was also significantly down-regulated (p < 0.01 or p < 0.001). The leaf volatile oil of P. glabra significantly reduced the level of Cyclin E2 at concentrations of 60 and 80 μg / mL (p < 0.01). The results showed that the leaf volatile oil of P. glabra down-regulated the expression of CDK2 and Cyclin E1, and the expression of Cyclin E2 was down-regulated at high concentrations, thereby inhibiting the activity of Cyclin E1 / E2-CDK2 complex, obstructing the G1 / S transition, and leading to cell cycle arrest at the G1 phase.

[0042] Pharmacological Example 4: Effect of the leaf volatile oil of P. glabra on apoptosis of A549 cells

[0043] (1) Drug treatment: Take A549 cells in logarithmic phase, after digestion with 0.25% trypsin, resuspend with RPMI-1640 (containing 10% fetal bovine serum) culture solution; after counting with a hemocytometer, inoculate 5x10 5 individuals per well in a 6-well plate, and place in a CO2 incubator for 24 h; discard the culture medium, add 2 mL of drug-containing culture solution (0, 20, 40, 80, 100 and 160 μg / mL), and continue to culture for 48 h.

[0044] (2) After drug treatment, observe the morphological changes of A549 cells under an inverted microscope.

[0045] (3) After drug treatment as in (1), perform AO / EB staining. Mix AO staining solution and EB staining solution at a ratio of 1:1, vortex to fully dissolve; discard the culture medium, wash once gently with pre-cooled PBS buffer, add 800 μL of AO / EB staining solution per well, and incubate in the dark for 5 min; remove the staining solution, observe under an inverted fluorescence microscope, take photos and analyze.

[0046] (4) After drug treatment as in (1), perform Hoechst 33258 staining. Discard the culture solution, add 0.5 mL of fixing solution per well, fix for 10 min, discard the fixing solution, wash twice with pre-cooled PBS, shake back and forth several times, and remove the liquid; add 0.5 mL of Hoechst 33258 staining solution per well, stain for 5 min, shake several times during the period, discard the staining solution, wash twice with pre-cooled PBS, shake back and forth several times, and remove the liquid; observe under an inverted fluorescence microscope, take photos and analyze.

[0047] As shown in Figure 6 , after treatment of A549 cells with different concentrations of volatile oil of P. glabra leaves for 48 h, the number of shriveled cells significantly increased with increasing concentration of the volatile oil; as shown in Figure 6 , after treatment with different concentrations of volatile oil of P. glabra leaves, the proportion of green fluorescence gradually decreased, while the proportion of orange-red fluorescence gradually increased, indicating that the number of apoptotic cells gradually increased; as shown in Figure 6 , after treatment with different concentrations of volatile oil of P. glabra leaves, the proportion of cells showing bright blue fluorescence with dense nuclei gradually increased, which possessed the characteristics of cell apoptosis. The above morphological observations all indicated that the volatile oil of P. glabra leaves could effectively induce apoptosis of A549 cells.

[0048] Annexin V-FITC / PI staining combined with flow cytometry was used to further quantitatively detect the apoptosis-inducing effect of the volatile oil of P. glabra leaves on A549 cells, and the results are shown in Figure 7The proportion of A549 cell apoptosis was increased to 17.64±1.14%, 26.56±0.89%, 43.74±2.45%, 50.32±3.57%, 68.31±0.31% after 24h treatment of P. glabra leaf volatile oil at the concentration of 20μg / mL, 40μg / mL, 80μg / mL, 100μg / mL and 160μg / mL, respectively, compared with the blank group (apoptosis rate: 9.81±0.09%). It indicated that P. glabra leaf volatile oil significantly increased the proportion of apoptosis in a dose-dependent manner (p<0.001), and P. glabra leaf volatile oil significantly reduced the proportion of living cells. Therefore, P. glabra leaf volatile oil significantly induced 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 Figure 8 After 48h treatment of A549 cells with different concentrations of P. glabra leaf volatile oil, the proportion of red fluorescent cells gradually decreased, and the proportion of green fluorescent cells gradually increased. The results showed that P. glabra leaf volatile oil could effectively lead to down-regulation of mitochondrial membrane potential (ΔΨm) of A549 cells, thereby leading to apoptosis.

[0050] WB experiments were performed to study the expression of mitochondrial apoptosis pathway-related proteins. As shown in Figure 9 A and B. Compared with the untreated blank group, the expression of Cyt c in total protein was significantly up-regulated (p<0.01 or p<0.001) under the treatment of P. glabra leaf volatile oil at the concentration of 60μg / mL and 80μg / mL, and the expression of mitochondrial Cyt c was significantly down-regulated (p<0.01 or p<0.001) at the concentration of 40μg / mL, 60μg / mL and 80μg / mL. Therefore, P. glabra leaf volatile oil could up-regulate the expression of total Cyt c and down-regulate the expression of mitochondrial Cyt c, indicating the release of Cyt c from mitochondria.

[0051] As shown in Figure 9 As shown in C and D, P. glabra leaf volatile oil up-regulated the expression of Bax and inhibited the expression of Bcl-2, and the Bax / Bcl-2 ratio was significantly up-regulated in a dose-dependent manner (p<0.01 or p<0.001). At the concentration of 40μg / mL, 60μg / mL and 80μg / mL, the Bax / Bcl-2 ratio was significantly higher than that of the untreated blank group (p<0.01 or p<0.001). Figure 9In C and E, the volatile oil of P. glabra leaves significantly decreased the expression of Pro-Caspase 9 (p<0.05 or p<0.001) and increased the expression of cleaved-Caspase 9 (p<0.01 or p<0.001), indicating that Caspase 9 was activated. Meanwhile, the expression of Pro-Caspase 3 was significantly decreased (p<0.01 or p<0.001) and the expression of cleaved-Caspase 3 was increased (p<0.01 or p<0.001), indicating that Caspase 3 was activated. At the same time, the volatile oil of P. glabra leaves significantly up-regulated the expression of cleaved-PARP (p<0.001). Therefore, the volatile oil of P. glabra leaves activated the cascade reaction of Caspase 9 and Caspase 3 by increasing the expression of Bax and decreasing the expression of Bcl-2, resulting in the cleavage of PARP (up-regulation of cleaved-PARP expression), indicating that the volatile oil of P. glabra leaves can induce A549 cell apoptosis through the mitochondrial apoptosis pathway.

[0052] Pharmacological Example 5: Effect of the volatile oil of P. glabra leaves on the migration of A549 cells

[0053] Scratch: A549 cell suspension was collected and diluted to 2 x 10 5 cells / mL, and then 4 x 10 5 cells were added to a six-well plate. 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 the volatile oil of P. glabra leaves (prepared in serum-free medium) for 48 h of treatment. The scratch area was photographed at 0 h and 48 h using a Leica microscope. The cell migration rate was calculated as follows:

[0054]

[0055] Invasion: 750 μL of drug solution at different concentrations (15% FBS) was added to the lower chamber. 250 μL of A549 cell suspension (1 x 10 5 cells / well) was inoculated in the upper chamber and 250 μL of drug solution at different concentrations (containing 5% FBS) was added. After 48 h of incubation, fix with 10% paraformaldehyde for 2 min; incubate with anhydrous methanol for 20 min; stain with 0.1% crystal violet for 15 min; wash twice with PBS and then 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] The wound healing test and the invasion test were used to study the effect of the volatile oil of P. glabra leaves on the migration and invasion ability of A549 cells. As shown inFigure 10 A and B, the migration rate of A549 cells treated with different concentrations (10, 20, 30 and 40 μg / mL) of P. glabra leaf volatile oil was significantly lower than that of the control group (78.06 ± 0.93%), which was 57.92 ± 0.39%, 44.36 ± 1.31%, 33.63 ± 1.97% and 15.12 ± 0.43%, respectively. Therefore, the inhibition of P. glabra leaf volatile oil on the migration ability of A549 cells had obvious concentration dependence. The results of invasion test Figure 10 C and D) showed that P. glabra leaf volatile oil could significantly reduce the number of A549 cell invasion compared with the control group, and was related to the dose, which indicated that P. glabra leaf volatile oil could inhibit the invasion ability of A549 cells.

[0057] The effect of P. glabra leaf volatile oil on the migration-related proteins was detected by WB. As shown in Figure 10 E and F, P. glabra leaf volatile oil significantly down-regulated the expression of MMP-2, MMP-9 and N-cadherin in a concentration-dependent manner. The above results showed that P. glabra leaf volatile oil inhibited the migration and invasion ability of A549 cells by down-regulating the levels of MMP-2, MMP-9 and N-cadherin.

[0058] In summary, through morphological observation, AO / EB staining, Hoechst 33258 staining and flow cytometry apoptosis detection, the results showed that P. glabra leaf volatile oil induced A549 cell apoptosis; JC-1 staining results showed that P. glabra leaf volatile oil led to down-regulation of mitochondrial membrane potential (ΔΨm); combined with the results of Western Blot related to the mitochondrial apoptosis pathway, the mechanism of P. glabra leaf volatile oil inducing A549 apoptosis was as follows: P. glabra leaf volatile oil up-regulated the ratio of Bax / Bcl-2, thereby leading to increased mitochondrial membrane permeability, down-regulation of mitochondrial membrane potential (ΔΨm), release of Cyt C from mitochondria to cytoplasm (down-regulation of Mitochondria Cyt C and up-regulation of Total Cyt C), activation of Caspase-9 (down-regulation of Pro-Caspase 9 and up-regulation of Cleaved-Caspase 9), followed by cleavage and activation of Caspase-3 (down-regulation of Pro-Caspase 3 and up-regulation of Cleaved-Caspase 3), and then cleavage and inactivation of PARP (up-regulation of Cleaved-PARP), leading to apoptosis of A549; combined with the results of P. glabra leaf volatile oil on migration-related proteins, it was found that P. glabra leaf volatile oil inhibited the migration and invasion of A549 cells by down-regulating MMP-2, MMP-9 and N-cadherin.

[0059] In summary, the volatile oil of P. glomerata leaves could arrest the cell cycle of A549 cells at G1 phase and inhibit proliferation. The molecular mechanism of inducing apoptosis through mitochondrial-mediated pathway was as follows: Bax / Bcl-2↑→ΔΨm↓→mitochondria release Cyt C→activate Caspase-9→Caspase-3 activation→PARP inactivation→induce apoptosis. The inhibition of migration and invasion of A549 cells was achieved by MMP-2↓, MMP-9↓ and N-cadherin↓.

Claims

1. Application of the volatile oil of Pittosporum illicioides leaves in the preparation of a drug for treating non-small cell lung cancer.

2. The volatile oil of Pittosporum illicioides leaves according to claim 1 is prepared by the following method: Fresh Pittosporum illicioides leaves are crushed, the crushed raw material is mixed with distilled water at a ratio of 1:3-1:8, g / mL, the mixture is extracted by water vapor distillation in a volatile oil extractor for 3-5 hours, the volatile oil is collected, anhydrous sulfuric acid is used to remove water, and the volatile oil of Pittosporum illicioides leaves is obtained.

3. The use according to claim 1, wherein The drug for treating non-small cell lung cancer is a drug that causes G1 phase arrest, thereby inhibiting the proliferation of non-small cell lung cancer cells.

4. The use according to claim 1, wherein The drug for treating non-small cell lung cancer is a drug that induces apoptosis of non-small cell lung cancer cells through the mitochondrial apoptosis pathway.

5. The use according to claim 1, wherein The drug for treating non-small cell lung cancer is a drug that inhibits the metastasis of non-small cell lung cancer cells.

6. The use of the volatile oil of Pittosporum illicioides leaves according to claim 1 in the preparation of a medicament for treating non-small cell lung cancer, characterized in that, The volatile oil of Pittosporum illicioides leaves and a pharmaceutically acceptable carrier are prepared into a tablet, a capsule, a fat emulsion, a suppository, a dripping pill, or an ointment dosage form of a drug.

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