Study On Essential Oils Extracted From The Leaves Of Artemisia Argyi Against Hepatocellular Carcinoma And Preparation Of Its ?-Cyclodextrin Polymer Microsphere

Artemisia argyi is a famous species with a long history in traditional Chinese medicine,which is the dry leaf of Artemisia argyi Lévl.et Vant..Essential oils extracted from Artemisia argyi leaves?AAEO?by the common way of hydrodistillation method.The main chemical composition of AAEO were eucalyptol,camphor and borneol.Our previous study showed that AAEO significantly inhibits the SMMC-7721cell proliferation.The research about AAEO and the AAEO-CDP?AAEO-?-cyclodextrinpolymer?microsphere will merit further investigation.The aim of this study can be divided into four parts.1.AAEO inhibits HCC cell proliferation in vitro.The inhibitory effect of AAEO on human HCC cell was evaluated by MTT assay.The IC_50s0s were 353.83,330.88 and 295.50?g/mL for 24,48 and 72 h.The inhibition rate of SMMC-7721 cells by AAEO is the highest for 72 h.The cells were stained with Annexin V-FITC and PI and analysed by flow cytometry.The results showed AAEO led to cell apoptosis in a dose-dependent manner.The cells were treated with AAEO induced a significant apoptosis rate about80.84%.To visualize the morphological alterations of apoptotic nuclei,fluorescent staining of AAEO treated cells was performed:with the dose of AAEO increase,cells showed early stage apoptotic cells,late stage apoptotic cells,the formation of apoptotic bodies,chromatin condensation and necrotic cells gradually.This process illustration that cell shape has experienced a lot of changes.The cells were stained with PI and analysed by flow cytometry:the result illustrated that AAEO can arrest cell cycle at G₂/M phase.The induction of apoptosis by AAEO was confirmed by arresting G₂/M phase in whole cell cycle process.2.Assessment of AAEO anti-HCC in nude miceThe nude mice bearing hepatoma model was used to investigate the antitumor activity of AAEO.The mice were divided into five groups:control group,positive group,210,105,and 52.5 mg/kg of AAEO.The significant antitumor rates?68.42%and 44.74%?were observed in mice administered with 5-FU and AAEO at the dose of 210mg/kg.A considerable reduction in mice body weight?from 19.23 g to 14.33 g?was observed in mice treated with 5-FU.It resulted in serious damages in tumor-bearing mice.The tests showed the reduction in mice body weight were from19.33 g to 17.93 g of AAEO.AAEO might be an ideal candidate agent for treating HCC with low toxicity on tissues.3.Preparation and quality evaluation of AAEO-CDP microsphere.To establish a method for the deterring the contents determination of AAEO,and methodology was examined.Selecting the optimum prescription and process to get the optimum encapsulation rate and productive rate by the BOX response-surface method.After software analysis and experimental verification,the optimum preparation process was as follows:mixing time was 3 h,mixing temperature was 45?and?-CDP:AAEO was 7:1.?-CDP microsphere was shape spherical,size equality,regular in morphology,surface smooth,with the mean diameter of 57.67±5.64?m and the span of 1.46±0.03.The swelling ratio and?-CD content of?-CDP microsphere were?33.7±6.5?%and?36.3±7.7?%.IR and DSC show the prepared microsphere reserved the cavity structure of?-CD and with a high content of it.The?-CDP microsphere has a cross-linking three dimensions,reticular internal structure.The encapsulation rate of eucalyptol,camphor and borneol were 82.49%,77.48%and 75.62%.AAEO-CDP microsphere preparation release characteristics was slowly in vitro.The stability of the AAEO-CDP microsphere was detected by putting them under the condition of illumination,high temperature and high humidity.The results showed that AAEO-CDP microsphere has good stability within 90 days.And the microsphere should protected from light in a dry place under 4?.4.Study on pharmacokinetics of AAEO and AAEO-CDP microsphere.The mice were ig administration of AAEO-CDP microsphere and AAEO.Pharmacokinetics parameters were obtained by the concentration-time curves of AAEO and?-CDP microsphere and software analysis.It turned out that the time to peak of AAEO-CDP microsphere was 2 h.And the plasma dose was the 2.03times to AAEO.In summary,this paper investigated the antitumor activity of AAEO on hepatocellular carcinoma in vitro and in vivo.AAEO-CDP microsphere can increase the solubility and stability of AAEO.The drug in plasma of AAEO-CDP microsphere was higher than AAEO.These findings might be important to explore new therapeutic drug for treatment of liver cancer and merit further investigation.Main Innovative Points:This research is the first time to investigate the anti-hepatoma activity which deep into cell apoptosis and cell cycle of AAEO in vitro.To solve the problems about AAEO,we use?-CDP microsphere as carrier to prepare AAEO-CDP microsphere firstly.And establish a method for the deterring the contents determination of AAEO.At the same time,it is the first time to investigate the pharmacokinetics of AAEO-CDP microsphere in vivo.