Essential oil extracted from leaf of Phoebe bournei (Hemsl.) Yang: Chemical constituents, antitumor , antibacterial, Hypoglycemic activities

: The essential oil were extracted from the leaf of Phoebe bournei (Hemsl.) Yang by a hydrothermal method and then analyzed by gas chromatography–mass spectrometry. The leaf oil mainly included α-copaene(5.44%), α-muurolene(7.32%), δ-cadinene(11.44%), 1s-calamenene(5.18 % ). Phoebe bournei (Hemsl.) Yang leaf essential oil had significant inhibitory activity against Epidermophyton floccosum and Microsporum gypseum , the potential antitumor activity towards leukemia, breast, and colon cancer cell lines was good. Phoebe bournei (Hemsl.) Yang leaf essential oil had weaker activity on the four tested bacteria, it exhibited a certain role in promoting glucose uptake by adipocytes.


Material
The leaf of P. bournei are from Gongdong Township, Rongshui County, Liuzhou City, Guangxi Province. The sample was obtained on January 23, 2018 by the team consisting of researchers from Sichuan Agricultural University and the special committee of China Nanmu Association. The leaf, the bark and tree core was taken.
Some of the tree core samples were sliced and observed for microscopic structure, which was consistent with the microstructure of P. bournei wood (Chen et al. 2015).
The samples of fresh bark and leaves were identified, which were consistent with the shape characteristics of P. bournei (Delecti Flora Reipublicae Popularis Sinicae Agendae Academiae Sinicae. 1982). In summary, the sample species was regarded to be P. bournei.
At present, the tree core, bark, leaf samples and tree core wood chips (three slices) are kept in the Wood Engineering Laboratory of Sichuan Agricultural University, and their numbers are 20180123-MNP, 20180123-MNY, 20180123-MNM and 20180123-MNM (01 -03). The moisture content of leaf was 12.1%。

Extraction of Essential Oils
First, a crushed sample of P. bournei leaf weighing 200±0.01g was accurately measured using an analytical balance and added to a 2000mL round-bottomed flask.
Next, distilled water (1200 mL) was introduced, and several glass beads were added to prevent bumping. The extraction apparatus was assembled and heated for 5 h (until the volume of the essential oil remained constant). The volume of the extract was then recorded after settling for 0.5h.
Gas Chromatography Analysis.
Chromatographic column: Agilent HP-5 column (30m× 0.25mm×0.50μm); sample injection volume: 1μL; split ratio: 50:1; Detector temperature:250°C; carrier gas: ultra-pure helium; oven temperature program: 120°C for 3 min, to 140°C at 5°C/min for 5 min, to 160°C at 2°C/min for 10 min; flow rate: 1 mL/min. equipped with flame ionization detector. The components in the essential oils were identified by retention indices that were determined with C7-C20 alkane standards as the reference and that were confirmed by GC-MS.
Gas Chromatography-Mass Spectrometry Analysis.
Gas chromatography conditions. Chromatographic column: Agilent HP-5ms column (30m×0.25mm×0.25μm); sample injection volume: 1μL; split ratio: 50:1; injection port: 250°C; carrier gas: ultra-pure helium; oven temperature program: 120°C for 3 min, to 140°C at 5°C/min for 5 min, to 160°C at 2°C/min for 10 min; flow rate: 1 mL/min. MS conditions. Ion source: electron ionization; ionization energy: 70eV; auxiliary heating zone: 280°C; ion source: 230°C; quadrupole: 150°C; data acquisition mode: full scan; mass scanning range: m/z 50-550; solvent delay: 3 min. The GC-MS spectra were analyzed via a computer-based automatic search, the automated and manual analyses of a mass spectral library (NIST), and a literature search to identify each component. Finally, the relative contents of each constituent were calculated using the peak area normalization method.

Experimental Method
Procedure. First, the samples were diluted to specific concentrations and added to the individual wells of a 96-well microplate. Next, the bacterial solutions were added, diluted to obtain a final concentration of 5 × 10 5 CFU/mL, and then cultured for 24 hat 37°C. A microplate photometer was used to measure the optical density(OD) below625 nm, and each treatment was the same as the three controls, and the mean value was taken. Control cultures, blank media, bacterial cultures, and penicillin-streptomycin, and penicillin G sodium-positive controls were prepared.
Inhibition rates were calculated based on the following formula. Inhibition rate=(1-OD value of sample/OD value of control well)×100% Figure  Cisplatin(DDP) and Taxol were also provided by the institute. All reagents were of analytical grade.

Experimental Method
Dulbecco's modified Eagle medium(DMEM) culture medium containing 10% fetal bovine serum was used to formulate a single-cell suspension culture.
Approximately 3000-15000 cells per well were seeded into the 96-well plates, with a volume of 100 µL in each well. Adherent cells were cultured 12-24 h in advance.
Next, 20 µL of the supernatant from the samples was removed and dissolved in dimethyl sulfoxide. The essential oil samples were rescreened using five