Extraction And Purification Of Artemisinin From Artemisia Annua L.

Artemisinin, which is isolated from artemisia annua L., is considered to be the most promising drug for treatment of malaria. It was developed in China and has a large demand. Conventional methods based on solvent extraction of artemisinin are time-consuming, energy-consuming and of low extraction yield since they require multiple extraction steps. Supercritical CO₂ extraction is considered as a new and green process for value-added natural products. The main objective of this dissertation is to study the process of extracting artemisinin with supercritical CO₂ and purifying the extracts. The new separation process of artemisinin, which includes supercritical CO₂ extraction, column chromatographic separation and crystallization, is expected to be applied commercially.The method for detection and quantification of artemisinin by reversed-phase HPLC has been developed. This quantitative method is accurate, sensitive, and reproducible, which is suitable to the analysis of artemisinin in crude plant extracts. The relative standard deviation is less than 1.1%. The recovery is 98.9%.A flow-type apparatus has been built for solubility determination of solid in supercritical CO₂. The solubility of artemisinin in supercritical CO₂ was measured in a pressure range of 10 MPa to 27 MPa and a temperature range of 37℃ to 65 ℃. The range of solubility is from 9.8×10^-5 to 2.7×10^-3 mol/mol. The solubility increases with pressure or temperature at pressure range between 18 and 27 MPa. However, the solubility increases with pressure but decreases with temperature at pressure range between 10 and 18 MPa. Based on the molecular association assumption, a four-parameter molecular association model for solid solubility in supercritical CO₂ has been deduced, wherein the phase equilibrium was considered. This model is in good agreement with the experimental results, with the average relative deviation is 4.28 %.The supercritical CO₂ extraction of artemisinin was studied on the ground of the solubility measurement. The operating conditions: particle size between 40 and 100 mesh, moisture of artemisia annua L. between 2.5% and 11.3%, extraction pressure between 15 and 30 MPa, extraction temperature between 40 and 60℃, flow rate of CO₂ between 0.55 and 6.24 kg/(hkg), and extraction time, etc., have been optimized in terms of the yield and the extraction selectivity of the artemisinin. The extraction yield increase with pressure, temperature, extraction time, but decrease with particle size in the range of observed operating conditions. The effects of above operating conditions on extraction selectivity were just reversed, however. The moisture content of approximately 11.3% was appropriate in terms of extraction yield and selectivity. At the optimal operating conditions: extraction pressure 20 MPa, temperature 50℃,CO₂ flow rate 1.00 kg/(h·kg), particle size between 60 and 80 mesh and extraction time 4 h, artemisinin content in the extract was about 15% with 95% yield of artemisinin. Two collection methods of pressure-reduction and supercritical adsorption were compared. The results showed that pressure-reduction collection method was superior to supercritical adsorption method due to the low adsorption capacity of artemisinin on silica gel. Moreover, the second supercritical CO₂ extraction was conducted. The results showed that there was no use to increase the content of artemisinin by the second supercritical CO₂ extraction. The process flow chart of artemisinin was designed according to the experimental results.The hot-ball model and the mass-balance model were applied to analyze the experimental results. The mass-balance model successfully fitted the kinetic extraction of the artemisinin. The relative deviation was between 1.2% and 14.8%.In column chromatography process, silica gel and hexane/ether (80/20, v/v) were used as adsorbent and the elution solvent, respectively. The adsorption capacity of artemisinin on silica gel was 35.2 mg/g at 30℃. The R_f value of artemisinin was 0.16. The operating conditions of column chromatography have been optimized in terms of the recovery and the purity of the artemisinin. In Φ2.0 cm ×50 cm column, the optimal operating conditions were as follows: flow rate 1.5 ml/min, temperature 25℃, load 15.0 mg/(g silica). As a result, the content of artemisinin reached 70% from 12.25% with 88.5% recovery of artemisinin. The silica gel could be regenerated with hexane/ chloroform (60/40, v/v). The purity of artemisinin could be further improved to 98.9% by crystallization in 70% alcohol (about 17 ml/g) with 85.2% recovery of artemisinin.The total recovery of artemisinin in the new separation process was more than 71%.