Volatile Oil Of Daucus Carota Linn. Induces Apoptosis Or Necrosis In Hela

BACKGROUND:Daucus carota Linn., belonging to Daucus carrot in Umbelliferae. The volatile oil of Daucus carota Linn. is the volatile and water-insoluble mixture extracted from Daucus carota Linn. Relevant researches indicate that the oil have been shown the effects of antifertility, insecticidal, antibacterial, antitumor. But the anticancer activities has not been reported. The objective of this study was to analyze constituents, determine the anticancer activity of the volatile oil of Daucus carota Linn., and then to study the influence and mechanisms of apoptosis and necrosis in Hela cells. Our research can provide experimental basis to the development and utilization of Daucus carota Linn..METHODS:Chemical compositions were identified by GC/MS-mass; MTT assay was used to evaluate the anticancer activitives of the oil; The antibacterial activitives was assayed with nephelometry method. Inverted microscope, electron microscope and AO/EB double staining were used to observe morphologic changes of Hela cells; DNA injury and break were detected by single cell gel electrophoresis and DNA Ladder; Flow cytometry was adopted to analyzed cell cycle and DNA content changes; To investigate the change of PARP, we adopted the Western Blot analysis.RESULTS:31compositions were identified form the volatile oil of Daucus carota Linn. and the major compounds were terpenoids such as β-Bisabolene accounting for63.06%of the total oil. The IC50of volatile oil of Daucus carota Linn. on NCI-H446for6h,12h,24h were244.82,113.13,61.76mg·L-1, the IC50on Huh7Cells were122.99,132.25,46.20mg·L-1, the IC50on Hep3B were55.74.31.82、50.88mg·L-1, the IC50on Hela were183.88,108.22,99.58mg·L-1, the IC50on A549were147.19,76.91,57.13mg·L-1, the IC50on PANC-28were47.86,26.20,34.32mg·L-1, the IC50on HUVEC were89.23,52.53,52.30mg·L-1. The inhibition rate of the oil against Acinetobacter Baumanii for24h was33.12%at high concentration of30g·L-1. Low dose group cells exhibited the typical morphological changes of early apoptosis such as cell shrinkage, existence of membrane bubble and nuclear condensation. In contrast, the cells in high dose group had fell off the bottom of cell plate and showed the necrotic morphology changes including nuclei pyknosis and appearance of small fragments. SCEG(single cell gel electrophoresis) showed that the injury of Hela cells started from the nuclear DNA. Treated with the volatile oil of Daucus carota Linn, for6h, significant induction of DNA migration was detected. Typical apoptosis DNA ladder by DNA agarose gel electrophoresis was observed and the stripe in100mg·L-1group was the brightest. The cell cycle was blocked in the Go/G1period at low dose of the oil, while it was blocked in the G2/M period at high dose. PARP cleavage was observe and it existed in the form of89kDa and116kDa.CONCLUSIONS:Above all, the main components of volatile oil of Daucus carota Linn. were terpenes such as β-Bisabolene and the oil exhibited antibacterial and anticancer activities. The oil could significantly inhibit the growth of Hela cells in a dose and time-dependent manner. Low dose groups cells exhibited the typical morphological changes of apoptosis, while the high dose group cells showed the necrotic morphology changes. The nuclear DNA of Hela was damaged by the oil. The cell cycle of low dose groups cells was blocked in the Go/G1period. But the high dose group was blocked in the G2/M period. The results can also suggest that the oil evokes Hela cells apoptosis is correlation with the change of PARP.