Purification Of Essential Oil From Ginger By High-speed Counter-current Chromatography And Its Anti-tumor Activity

In the study,three bioactive components(6-gingerol,zingerone and sesquiterpenes)of essential oil from ginger extracted by supercritical CO2 fluid were purificated by high-speed counter-current chromatography(HSCCC).The purities,chemical compositions and anti-proliferation activities of the three compounds were determined to prove that the essential oil from ginger could achieve high-purity separation and the bioactive substances with anti-tumor potential could be obtained by HSCCC.The main contents were as follows:1.The essential oil from ginger extracted by supercritical CO2 fluid extraction was separated and purified by high-speed counter-current chromatography.According to the partition coefficients(k)of three bioactive components from the essential oil from ginger in the selected solvent systems,it was determined that the solvent system n-hexane-ethyl acetate-methanol-water(7:3:5:5)was used to separate the compound 1,the n-hexane-methanol-water(3:2:1)was used to isolate the compound 2 and the n-hexane-chloroform-acetonitrile(6:2:5)was used to separate the compound 3.The other factors were also optimized:the flow rate of the mobile phase was 2 mL/min,the injection volume was 600 μL,the revolution rate was set to 800 rpm and the separation temperature was 25℃.2.The purities and chemical compositions of three different compounds separated from HSCCC were analyzed by high performance liquid chromatography(HPLC)and gas chromatography-mass spectrometry(GC-MS).The HPLC analysis manifested that the liquid chromatograms of the three compounds were singel peaks without heteropolar peaks.The purities of compounds 1-3 evaluated by HPLC were 98.6%,99.4%and 99.2%.The GC-MS results were as follows:compound 1 was 6-gingerol,compound 2 was zingerone,and compound 3 was sesquiterpenes.And their relative contents reached to 84.07%,94.67%and 95.17%,respectively.3.MTT assay was performed to preliminarily evaluate the proliferation inhibition activities of the three compounds to three cancer cells:the human non-small lung cancer A549 cells,HepG2 cells and MDA-MB-231 breast cancer cells.The results showed that all three compounds significantly inhibited the proliferation of cancer cells in a concentration-dependent manner,and A549 had the most significant loss of cell activity.By comparing the 50%inhibitory concentration(IC50),it was found that 6-gingerol had the strongest inhibitory effect on A549 cells.4.After confirming that 6-gingerol had the strongest antitumor activity,the effect of 6-gingerol on inducing apoptosis of A549 cells was further investigated.Inverted light microscope observation and Hoechst 33342/PI double staining results showed that 6-gingerol could induce A549 cells to exhibit some typical features of apoptosis,such as cell shrinkage,volume contraction,chromatin condensation,cell membrane blebbing,etc.;The results of Annexin V-FITC/PI double staining showed that the apoptosis rate of A549 cells increased from 2.19%to 83.56%after the action of 6-gingerol at different concentrations,indicating that the cells gradually transformed from early apoptosis to late apoptosis.Cell cycle assay showed that 6-gingerol disrupted the continuity of cell cycle and triggered S-G2 phase arrest in a dose-dependent manner.