Identification of essential oils in Myrica rubra leaves and their inhibitory effect on the proliferation of lung cancer A549 cell

Abstract:【Objective】The secondary metabolites of essential oils in Myrica rubra leaves (MEO) have antioxidant, antibacterial, anticancer and other biological activities. In this paper, the chemical compositions and anti-cancer activity of MEO extracted from the leaves of four bayberry cultivars, ‘Ding'ao'‘Dongkui'‘Wandao'and‘Biqi', were analyzed. This study could provide a theoretical basis for the development of natural anti-cancer drugs.【Methods】The essential oils were extracted with steam distillation from the leaves of the four cultivars, ‘Ding'ao' (grown in Wenzhou, Zhejiang) , ‘Dongkui' (grown in Xianju, Zhejiang) , ‘Wandao' (grown in Zhoushan, Zhejiang) and‘Biqi' (grown in Yuyao, Zhejiang) . GC-MS was used to analyze the components and contents of essential oils. The chromatographic analysis column was a HP-5 type (30 m×0.25 mm×0.25 μm) . The initial was 50 ℃, which was kept for 1 min, rose to 250 degrees at 8 min and then maintained for 5 min. Carrier gas was high purity helium with a flow rate of 1 mL·min^-1. The temperature of the vaporizing chamber was 220 ℃. The in-jection volume was 1 µL. The split ratio was 20:1. Mass spectrometry conditions included an EI source with an ion source temperature of 200 ℃, a solvent delay time of 2 min, and a mass range of 50-500 u.Components were indentified based on the NIST'08 standard mass spectrometry database and related literatures. The relative GC contents of volatile oils were calculated by area normalization method. And then the compositions and contents of essential oils were comparatively analyzed. Human lung cancer A549 cells were used as the material to examine the inhibitory activities of four MEOs on the proliferation of the lung cancer A549 cells using 3- (4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide assay (MTT assay) . The concentrations of MEOs used were 50, 75, 100, 125, 150, 175 and 200 μ g· m L-1.The action durations were 24, 48, 72, 96 and 120 h, and the proliferation inhibition effect on human lung cancer A549 cells was measured at these treatment time points.【Results】After analysis with GCMS, 34, 33, 41 and 33 components of MEOs were identified from‘Ding'ao'‘Dongkui'‘Wandao'and‘Biqi', respectively, and the contents of the identified components accounted for 99.35%, 91.23%, 98.34% and 97% of total essential oils, respectively. MEOs included the three major components, such as terpenes, terpenols and terpene ethers, and trace components, such as fatty acids, pyrrolidone, and phenols, and so on. The contents of terpenes accounted for 63.58%, 74.13%, 72.12% and 52.42% of total essential oils in‘Ding'ao'‘Dongkui'‘Wandao'and‘Biqi', respectively. The terpene compounds of the essential oils in‘Dongkui'and‘Wandao'were significantly higher than those in‘Dingao'and‘Biqi'. There were 11 components of the essential oils found in the four varieties of Myrica rubra. Furthermore, 7, 6, 9 and 5 endemic components were found in the four kinds of MEOs from Myrica rubra, ‘Ding'ao'‘Dongkui'‘Wandao'and‘Biqi', respectively. The main components in the MEOs included 12.85%-23.87% of beta caryophyllene, 19.27%-24.26% of alpha olefin, 1.15%-23.47% of humulus nerolidol, 3.82%-7.51% of aromadendrene oxide, 2.93%-5.26% of caryophyllene oxide and 0.8%-5.2%of d-cadinene. However, there were significant differences in the contents and components of MEOs from different Myrica rubra varieties. The results displayed that the inhibitory effects of MEOs on lung cancer A549 cells were dependent on both the action time and dose. After the action of 24 h, the lethal dose IC50 of MEOs from‘Ding'ao'‘Dongkui'‘Wandao'and‘Biqi'leaves were 127.24, 127.14, 118.03 and 123.62 μg·mL^-1, respectively. The antiproliferative activity of MEO from‘Wandao'was slightly better than those from the other three varieties.【Conclusion】There were significant differences in the essential oil compositions among the four cultivars. And all the MEOs had a remarkable inhibitory activity on lung cancer A546 cells. However, the inhibitory effect of essential oils on the proliferation of A546 cells did not differ significantly (p > 0.05) among different Myrica rubra varieties.