Research On The Mechanism Of Apoptosis Induced By The Essential Oil Of Litsea Cubeba In Human Carcinoma Of Mouth Floor KB Cells

GC-MS was used to explore the composition of essential oil obtained from Litsea cubeba from different areas of China.The aim of this study was to identify the antiproliferation activity of KB cells,induction of apoptosis,cell cycle arrest and effect on mechanism of mitochondria pathway as a result of treatment with essential oil extracted from Litsea cubeba and Citral.The findings of this study showed that:（1）The chemical components of the essential oils obtained by hydrodistillation of Litsea cubeba,from different areas of Hubei,Guizhou and Chongqing were detected by GC-MS.Monoterpenes,Oxygenated monoterpenes and sesquiterpene hydrocarbons were found to be the dominant components in the essential oil of Litsea cubeba.The major proportion found in it was of citral,followed by D-limonene,β-Myrcene,α-Pinene andβ-Pinene.The essential oil of Litsea cubeba from Guizhou region has the highest citral content,followed by Hubei and Chongqing.D-sylvestrene,linalool andβ-elemene were only detected in LCEO（the essential oil of Litsea cubeba）of Hubei region,and Borneol was only detected in LCEO of Chongqing region.（2）LCEO and citral were found to inhibit KB cells proliferation by giving specific time-dose dependent treatment determined by MTT assay and RTCA assay.The IC₅₀and citral of LCEO from different areas had no significant difference（P>0.05）.（3）Both LCEO and citral induced time dependent strong and early apoptosis as measured by Annexin V-FITC/PI after staining for 3,6 and 12 h.There was a significant difference（P<0.05）between the blank control group and the positive drug suppression group.PI staining showed that LCEO and citral caused cell cycle arrest at G₁to S phase,indicated by the increased percentage of G₁cells with the decrease of S phase cells.（4）Mitochondrial membrane potential after treatment with LCEO and citral was measured by using the flow cytometry with JC-1 stain which indicated that with the increase of dose,these caused the loss of mitochondrial membrane potential.This effect shows that LCEO and citral not only induces an imbalance between the mitochondrial Bax and Bcl-2 levels but also increases mitochondrial cytochrome c,caspase-9 and caspase-3 expression,which is closely related to its ability to induce apoptosis based on mitochondria pathway.