Artemisinin Of Extraction Process Optimization、content Determination And Cell Culture From Artemisia Annua L.

Artemisia annua L., one of the indigenous plants used in traditional Chinese remedies, is an annual herb which come from Artemisia, Compositae family. Artemisinin is an active ingredient isolated from the aerial parts of A. annua, which is recommend as "the world only effective antimalarial drug" by the World Health Organization in the field of modern medicine. In this work, A. annua was used as a material to investigate the extraction process of artemisinin and determine the content of artemisinin in wild and cultivated A. annua come from the southwest district (Sichuan provinces, Guizhou provinces and Chongqing). Meanwhile, tissue culture system of A. annua was established by biotechnology in order to get higher artemisinin and fast growing cell. The paper purpose was to provide the scientific basic for solving the chronic shortage of artemisinin. The main research results are as follows:1. The ultrasonic assisted extraction with petroleum ether was used as an extraction method, which investigated respectively the effect five process conditions on the extraction rate of artemisinin. The result was given that those factors (including extraction time, ultrasonic temperature and ratio of solvent to raw material) were beneficial role in promoting the extraction rate of artemisinin within a certain extent. But ultrasonic power has significant inhibition on the extraction of artemisinin. Extraction times had not significant effect with increasing in the number. The ratio of solvent to raw material (X1), ultrasonic temperature (X2), and ultrasonic power (X3) were further considered as independent variables, and the extraction rate of artemisinin (Y) was selecetd as a response variable. This experiment was performed in Box-Behnken factorial design. The extraction rate of artemisinin by UAE ranged from0.4234%to0.7353%in Box-Behnken Design (BBD) scheme. A second-order polynomial regression model was established by fitting the independent variable and response variable. Analysis of variance confirmed that the model was stable and credible, and it could be employed to predict the extraction rate of artemisinin. Ultimately, the largest response value was obtained by response surface methodology (RSM) as follow:ratio of solvent to raw material1:42.71, ultrasonic temperature41.86℃, and ultrasonic power120.00W. Under these optimal conditions, predicted extraction rate of artemisinin was0.7848%, and experimental value was0.7826%, in which the relative deviation was only-0.28%. The results undoubtedly demonstrated that RSM could be used to explore the optimum conditions of artemisinin extraction. Furthermore, ultrasonication is a simple complementary technique for extracting artemisinin efficiently from A. annua with advantage of shorter extraction time and easy operation.2. Eleven A. annua was determined and the result showed that stem, inflorescence and leaves of plant contain artemisinin, while it was a significant different in that three parts. The content of artemisinin was as follow:inflorescence> leaves> stem, and content of inflorescence was the range of12.7-75.6times compared to the stems. Even the content of artemisinin in the stems was the lowest, and mostly no more than0.1%. Artemisinin content of four materials in Chongqing area (except the materimal from Qianjiang district) was higher than those of Sichuan and Guizhou. Particularly, wild A. annua collected from Youyang country and Xiushan country (Chongqing province) were regarded as representative areas with higher artemisinin, in where the contents of artemisinin was higher than0.8%, mostly. The artemisinin content of A. annua collected from Sichuan and Guizhou province was less than0.6%, and did not reach the value of industrial development.3. In cell culture process, the results revealed that the stem of A. annua was induced easily to form callus with vigorous growth and loose texture in B5medium. Thus, take the B5medium as a basic medium, the different combinations of plant hormones was opt using central composite design in cell subculture growth process of A. annua. The optimal result was obtained by response surface analysis as follow:KT1.59mg/L+NAA1.83mg/L+6-BA0.89mg/L. At that condition, the actual callus weight of Artemisia annual could be up to259.60g/L, while artemisinin was not synthesized in this medium. The presence of artemisinin was only detected in a combination media of1.05mg/L KT+2.50mg/L NAA+1.60mg/L6-BA, and further confirmed that this media had a promoted role to synthetise artemisinin in callus. But available the content of artemisinin was103.9μ/g and compared with wild A. annua (0.702mg/g), it was still at a lower level.