The Essential Oils from Pittosporum tobira Flowers Display Antiproliferative Properties and Inducing Apoptosis on Lung Carcinoma Cells

Background: Pittosporum tobira (Thunb.) Aiton is an aromatic medicinal plant widely cultivated in the world. However, the essential oils (EOs) from P. tobira flowers for anti-cancer potential is still not systematically studied. The present aim to elucidate the phytochemical composition of the EOs and to explore mechanism of anticancer action. Methods: The EOs were extracted and analyzed by headspace solid-phase microextraction (HS-SPME) with gas chromatography-mass spectrometry (GC-MS). Volatile components were identified according to Kovats retention index (KI) and NIST database. The anti-neoplasm mechanisms of the EOs was comprehensively investigated in lung carcinoma A549 and H460 cells. Results: A total of 47 secondary metabolites representing 94.18% of the EOs were successfully identified: monoterpenes and sesquiterpenes were the dominant terpenoids. The EOs exhibited antiproliferative activity on A549 and H460 lung carcinoma cells. Hoechst 33324 fluorescent staining indicated the typical characteristics of apoptosis and induced cycle phase arrest. AnnexinV/PI staining revealed that the number of apoptotic cells was increased. Furthermore, the EOs also induced the caspase-mediated mitochondrial apoptosis pathway. Conclusions: Findings suggest that the full-scale chemical composition and first characterization of anticancer activities of the EOs, it could be used for integrative natural anti-cancer agents in health care should be pay more attention.


Introduction
Pittosporum tobira (Thunb.)Aiton belongs to the Pittosporaceae family that originates from East Asia, but is presently being cultivated as ornamental plant in many parts of the world [1] because it is resistant to pollution and environmental stress [2,3].The inflorescence is a cluster of fragrant flowers sitting at the end of branches.The flowers have five pure white petals which gradually turn yellow [4].The significant feature of this evergreen tree with thick leathery leaves is its prominent scent of flower; also named "Bάi lḹ xίang" in Chinese [5].The volatile oils from P. tobira flowers has been many studied before [6], main components are α-pinene, n-nonane, nerolidol and β-ocimene [7].However, the composition varies strongly between origins [8].Differences in chemical compositions can affect the biological activities of volatile oils, which include anti-inflammation [9], anti-fungal [10], antioxidant [11] and anticancer activities [12].The present study mainly focused on the phytochemical analysis of the EOs from P. tobira flowers cultivated in China.Furthermore, we firstly studied the antiproliferative potentials of the EO against the human non-small cell lung cancer cell lines A549 and H460.

Phytochemical composition of the EOs
In the present work, the total of 47 components representing 94.18% of the EOs were identified according to KI value and NIST11 database (Figure a, Table 1).The main components were mono-       1 KI, Relative retention index was calculated against n-alkanes (C8-C20) on DB-5 column.

Effect of the EOs on apoptosis and cell cycle distribution
To quantify the extent of apoptosis, A549 and H460 cells treated with the EOs were stained with Annexin V-FITC/PI followed by flow cytometry analysis.After treatment with the EOs for 24 h, the percentages of both early and late apoptotic cells gradually increased in a dose dependent fashion.

Effect of the EOs on cell cycle distribution
A549 and H460 cells were stained with PI.The percentages of cells in the G0/G1 phase with a hypodiploid DNA histogram decreased from roughly 43.94% (negative control) to 13.85% for A549 and from 46.94% to 12.67% for H460 cells, respectively (Figure 4a).Subsequently, the number of cells in the G2/M phase also correspondingly elevated from 6.63% to 49.09% for A549 and from 9.81% to 40.12% for H460 cells, respectively (Figure 4b).The EOs decreased the percentages of cells in the G0/G1 phase in a dose-dependent manner and significantly increased the percentages of cells in the

Discussion
In our present phytochemical investigation on the EOs from P. tobira flowers growing in China, we found that natural terpenoids dominated the oil with 56.7% consisting to 24.1% monoterpenes (C10H16) and 32.7% sesquiterpenes (C15H24).Especially monoterpenes and sesquiterpenes appear to be responsible for the anticancer activity in several medicinal plants.The main compounds of EOs such as α-humulene, β-caryophyllene, myrcene and β-pinene exhibited popularbioactivities against fungal infectious diseases, viruses and tumors [13,14].Camphene isolated from Piper cernuum EO exerted antitumor activity against melanoma cells [15].Limonene and β-elemene can be regarded as broad-spectrum antitumor agents [16][17][18].These volatile compounds could contribute to inhibit lung tumor cell proliferation; however, data from animals experiments are needed to validate this assumption.
In addition, EOs induced A549 and H460 cells apoptosis and mediated the mitochondrial apoptosis pathway by up-regulation the level of Bax and down-regulation of Bcl-2.It is well known that mitochondrial pathway is the best known intrinsic apoptosis pathway.The mitochondrion is the main site of action for members of the apoptosis-regulating protein family, the balance of Bax and Bcl-2 plays a critical role in mitochondrial apoptosis process [19].Human non-small cell lung cancer cell lines A549 and H460 were obtained from the Chinese Academy of Sciences, Shanghai Institutes for Cell Resource Center.The cell lines were maintained on Dulbecco´s modified Eagle high glucose medium (Gibco, USA), supplemented with 10% fetal bovine serum (Hyclone, USA), 100 U/mL penicillin and 100 U/mL streptomycin.The cells were cultured in an incubator with 5% CO2 and 95% humidity; the experiments were performed with cells in the logarithmic growth phase.

Extraction of EOs by headspace solid-phase microextraction (HS-SPME)
The

GC-MS analysis
Agilent-Technologies 6890 N/MS ： 5973I N Network GC system equipped with a flame ionization detector and DB-5 column(30 m×0.25 mm×0.25 µm)were employed for the EOs analysis.
Helium was the carrier gas with a flow rate of 1.0 mL/min.The column temperature was initially kept at 50 °C for 5 min, gradually increased to 210 °C at 3 °C /min rate, held to 10 min and finally raised to 230 °C at 15 °C /min.For GC-MS detection, an ionization system, with ion source temperature of 230 °C and ionization energy of 70 eV were used.A scan rate of 0.6 s was applied covering a mass range from 35-400 amu.The standard mass spectral library NIST11 was used to identify individual components of the EOs.Mixed alkanes (C8-C20, Sigma) were analyzed to determine Kovats retention indexes.

Cytotoxicity assay
MTT assay was employed to evaluate the effect of the EOs on the viability of human lung carcinoma A549 and H460 cells.1.0×10 4 cells were plated in 96-well plates and treated with different concentrations of the EOs for 24 h.5.0 mg/mL MTT (20 µL) was added to each well and cells were incubated for another 4 h at 37 °C.The reaction was terminated by addition of 150 µL DMSO and optical density at 570 nm was determined on a microplate reader (BioTek).50% inhibitory concentration (IC50) was calculated from growth-inhibitory curves of cells by SigmaPlot 12.5 software.

Cell nucleus morphology observed by fluorescence staining
A549 and H460 (2.5×10 5 cells/well) were seeded into 6-well plates and incubated to 90% confluence.Subsequently, the cells were treated with different concentrations (0, 15.0, 30.0, 60.0 µL/mL) of the EOs for 24 h, and then indirectly incubated with Hoechst 33342 (20 µg/mL) for 30 min in the dark.The dyed fluorescent cells were observed under a fluorescence microscope (Olympus Co., Japan).

Cell protein extraction and immunoblotting
Immunoblotting was performed as described previously [20].After A549 and H460 cells were treated with the EOs for 48 h.They were then resuspended in cold PBS after removal of the nutrient solution, and then centrifuged at 1500 rpm for 5 min and lysed in RIPA lysis buffer with freshly added 100 mM PMSF by ice bath for about 30 min; afterwards, the protein supernatant was centrifuged at 12000 rpm for 10 min at 4 °C.The protein supernatant was quantified using a BSA standard curve.The proteins were denatured with loading buffer at 100 °C.40 µg protein was separated by using 10-12% SDS-PAGE and transferred to PVDF membranes (Millipore Corporation, USA).The membranes were blocked with 5% skim milk (BD Biosciences, Franklin), which dissolved in pH-adjusted TBST at room temperature for 2 h.The membranes were incubated with diluted primary antibodies including Caspase-3, Caspase-8, Caspase-9, PARP, Bcl-2, Bax and β-actin diluted by 1:1000, followed by washing and incubation with HRP-linked anti-rabbit and anti-mouse IgG secondary antibodies diluted by 1:10000 in TBST.Protein bands were visualized with UltraECL (BI, Beit Haemek, Isreal) by super sensitive chemiluminescence imaging (Tanon Co., Shanghai, China).

Statistical analysis
Quantitative data are presented as mean ± SEM from at least three independent experiments.Sigmaplot 12.5 software (Systat Software, Inc., Germany) was employed for graph construction.
Student's t-test was used to evaluate the significance of differences in multiple comparisons, *P<0.05 and **P<0.001were considered as the statistical significance.

Conclusions
In summary, we provide a thorough phytochemical characterization of the flower EOs from Chinese P. tobira.In agreement with other studies, the EOs exhibited remarkable antiproliferative properties and induced apoptosis in cancer cells.However, in ecology, pharmacy and medicine, more data are needed to elucidate the role of individual compositions of the EOs or combinations of them for the observed biological activities.

Figure 2 .
Figure 2. (a) Antiproliferative effects of the EOs on A549 and H460 cells by MTT assay.A549 and H460 cells were treated with various concentrations of the EOs for 24 h.The results represented the average of three independent experiments ± S.D.(b) Morphological observation by inverted fluorescence microscopy of A549 and H460 cells stained with Hoechst 33342.Photomicrographs of fluorescence staining were randomly examined with a magnification of 40.Apoptotic cells containing fragmented nuclear chromatin are indicated by red arrows.

Figure 3 .
Figure 3. EO induced apoptosis in A549 (a) and H460 cells (b) were detected from labeled Annexin V-FITC/PI by flow cytometric histogram analyses.Columns show mean values of three experiments expressed as mean ± S. D. (n=3).Statistical analyses were performed by Student′s t-test (*P﹤0.05,**P﹤ 0.01) compared with the negative control group.

Figure 4 .Figure 5 .
Figure 4. Effect of the EOs on cell cycle in A549 (a) and H460 cells (b) stained with PI and analyzed on FL2-A channel of flow cytometer equipped with 488 nm laser.The X-axis represents the DNA content and the Y-axis represents the cell counts.The graph depicts the percentage of apoptotic cells.Columns show mean values of three independent experiments expressed as mean ± S. D. (n=3).Statistical analyses were performed by Student′s t-test (*P﹤0.05,**P﹤0.01)compared to untreated control.
Flowers of P. tobira were collected from the campus of Zhejiang Sci-Tech University during the flowering season in May, 2015-2016, and identified by Prof. Hongfei Lu of Botany Taxonomy.Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 17 May 2017 doi:10.20944/preprints201705.0132.v1Voucher specimens(No.PP2016050649)were deposited at the Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, China.
solid-phase microextraction holder consistent match a replaceable extraction fibre, coated with 100 µm polydimethylsiloxane (CAR/PDMS; Supelco) and extra block heater, headspace vials (7 mL) and accessories.The solid phase microextraction device is the trace sampler to insert a quartz fiber coated with extraction phase, when the extraction equilibrium, enter the amount of analyte extraction phase [23].The freshly harvested flowers (1 g) were ground in liquid nitrogen and immediately transferred into headspace vials.Samples were heated at 70 °C for 40 min, and then, volatile substances were automatically collected by HS-SPME with simultaneous heating and agitation.The fibre was conditioned at 270 °C for 3 min to stop any contamination.

) Molecular formula Molecular weight KI
. Phytochemical composition of the EO from P. tobira flowers via HS-SPME/GC-MS.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 May 2017 doi:10.20944/preprints201705.0132.v1
Apoptosis was monitored using the common Annexin V-FITC/PI detection kit (BD, Biosciences Pharmingen) as described by the manufacture′s instruction.A549 and H460 cells in 6-well plates were treated with different concentrations of the EOs for 24 h, collected and washed twice with PBS, gently resuspended in AnnexinV-binding buffer, and incubated with Annexin V-FITC and PI in