Study On Screening And Inhibition On Human Cervical Cancer HeLa Cells Of Active Components From Walnut Pericarp

In this paper, both the effective parts and active components from the different solvent fractions which were petroleum ether part, ethyl acetate part, chloroform part, butanol part and water part in walnut pericarp were selected by MTT assay against human cervical cancer HeLa cells. The active ingredients were separated and purified by column chromatography, and identified by thin layer chromatography. The structure of the active ingredient was identified through LC-MS and NMR. The morphology of HeLa cells apoptosis was observed by Hoechst 33342 staining and data about HeLa cells apoptosis analyzed from flow cytometry. The safety performance was evaluated by MTD test in mice. In the same time, the method for examination of active components by RP-HPLC was studied.The results showed that the IC50 of petroleum ether part, ethyl acetate part, chloroform part, butanol 1 part and water part against HeLa cells was 158.81 mg/L,40.43 mg/L,130.49 mg/L,137.67 mg/L,5.10E+07 mg/L, respectively. These findings provided evidence that petroleum ether, ethyl acetate, chloroform, and butanol had considerable inhibition and showed the dose-effect manner to human cervical cancer HeLa cells. The chloroform extraction showed the strongest activity. The chloroform extract was isolated and purified by silica gel column chromatography and TLC. The eluent was chloroform:ethyl acetate (3:7, V/V). The structure of the active component was identified by LC-MS and NMR. The result indicated it was oleanolic acid. The inhibition rate of oleanolic acid to HeLa cells was 40.22±3.24% by MMT assay when the concentration of oleanolic acid was 10-4 mol/L; The apoptotic bodies of HeLa cells were observed under a fluorescence microscope after treatment of 24h by 40 mg/L oleanolic acid and further Hoechst 33342 staining. The early apoptosis rate of HeLa cells was 3.93% by flow cytometry after treatment of 24h by 40 mg/L oleanolic acid and further Annexin V-FITC/PI staining. The maximum tolerated dose in mice test to oleanolic acid was carried out and the mode of drug delivery with lavage. The result pointed out that mice's major organ coefficient also presented normally after 7 days of drug delivery; the maximum tolerated dose of mice to oleanolic acid was greater than 22.2 mg/kg which was equivalent to 200 times of 200 mg daily dosage of adult in 60 kg body weight. The method of RP-HPLC for determination of oleanolic acid in walnut pericarp was established. The optimal conditions were:the chromatographic column was Intersil ODS (150 mm×4.6 mm,5μm); the mobile phase was methanol:water:acetic acid (265:35:0.1, V/V), velocity of flow was 0.8 mL/min, the injection volume was 20μL, detection wavelength was 210 nm, and temperature of column was 30℃. The result showed that good linear relationship was presented when the injection volume of oleanolic acid was among 0.728～7.28μg (r=0.9997), the average recovery of oleanolic acid (n=9) was 101.1%, RSD was 2.44%. The content of oleanolic acid in walnut pericarp was 2.1 mg/100g. The method is simple, rapid, reliable and accurate, reproducible and stable.