Experimental Research On Extraction Technology And Antitumor Activities Of Volatile Oil From V. Jatamansi Jones Of Genuine Medicinal Materials In Guizhou

Objective:To study the extraction technology and anti-tumor effect of volatile oil from V.jatamansi Jones,explore its antitumor mechanism,provide the experimental basis for the development of V.jatamansi Jones which is the genuine medicinal materials in Guizhou.Methods:?1?The volatile oil of V.jatamansi Jones was extracted by SFE-CO₂.With the extraction rate of oil as index,orthogonal experiment was used to selecte the best extraction process.Its chemical composition were analyzed and identified by GC-MS.?2?The method was used to research the acute toxicity which is intragastricly administered with volatile oil to the mice and the rats.The acute toxicity test and stimulation test were carried out on rats by vaginal administration.?3?Detected the affect of the proliferation on vitro of the volatile oil on human cervical carcinoma Hela cells by MTT and LDH assay,the rate of apoptosis was determined by flow cytometer,the expression of apoptosis related proteins which are Bax?Bcl-2?Caspase-3 were detected by Western Blot.?4?The model of human cervical cancer Hela cells in nude mice subcutaneous transplantation tumor was established,set up the volatile oil to high,middle and low dose group,the positive control group and the model control group,hypodermic injection 1 times in the daily,continuous dosing 15 days,nude mice were euthanized at the next day after the treatment,recorded the weight of nude mice and the volume of transplanted tumor,calculated the inhibitory rate,observed tumor tissue of the morphological changes by HE stain,detected the express of HIF-1,VEGF and VEGFR by immunohistochemical methods,signed the vascular endothelial cell with CD34 to count MVD.Results:?1?The optimum process parameters which can be used to extract the volatile oil from V.jatamansi Jones for:extraction pressure 25 MPa,extraction temperature 45?,extraction time 1.5 h,the flow rate of CO₂ is 25 L/h,parsing temperature 50?,parsing pressure 4.2 MPa,the extraction rate is 6.52%.There are 80 kinds of compositions in volatile oil of V.jatamansi Jones,among which67 kinds of compositions were indentified.Its ingredients include acids?37.07%?,valepotrates?16.93%?and terpenoids?3.4%?.The content of 6 kinds of ingredients of the highest were isovaleric acid?19.04%?,valeric aldehyde?15.13%?,Pentanoic acid,oct-4-yl ester?10.4%?,linoleic acid?7.05%?,oleic acid?5.53%?and palmitic acid?3.7%?.?2?LD₅₀ of the volatile oil ig in mice is 21.989g?kg^-1,95%confidence limits of 16.304²9.655g?kg^-1.MTD of the volatile oil ig in rats is21.726g?kg^-1.The volatile oil has no acute toxicity and stimulation of the rat vagina.?3?The volatile oil could inhibit the proliferation of Hela cells.IC₅₀ are35.48?g?mL^-1?24h?and 22.39?g?mL^-1?48h?.As the dose increased,apoptosis rate of Hela cells rises,increase the expression of Caspase-3 and Bax,reduce the expression of Bcl-2.?4?The inhibition rates of the volatile oil to high,middle,low dose and the positive control group in nude mice model were 72.33%,58.89%,48.62%and 65.61%.HE staining showed that the tumor cells in each group appear different degree of degeneration and necrosis.Each treatment group compared with the model control group,the expression of HIF-1,VEGF,VEGFR decreased obviously,angiogenesis of tumor interstitial tissue decreased observably?P<0.01?.Conclusion:The volatile oil of V.jatamansi Jones could inhibit the proliferation of human cervical cancer Hela cells significant,It induce cell apoptosis to display antitumor activity in vitro by means of the mechanism of action;to inhibit the formation of tumor blood vessels by cutting the expression of VEGF,VEGFR and HIF-1,in the end,it can inhibit the growth of cervical cancer in nude mice transplantation tumor.