Extraction of Artemisinin, an Active Antimalarial Phytopharmaceutical from Dried Leaves of Artemisia annua L., Using Microwaves and a Validated HPTLC-Visible Method for Its Quantitative Determination
A simple, rapid, precise, and accurate high-performance thin-layer chromatographic method coupled with visible densitometric detection of artemisinin is developed and validated. Samples of the dried Artemisia annua leaves were extracted via microwaves using different solvents. This method shows the advantage of shorter extraction time of artemisinin from leaves under the influence of electromagnetic radiations. Results obtained from microwave-assisted extraction (MAE) were compared with hot soxhlet extraction. Chromatographic separation of artemisinin from plant extract was performed over silica gel 60 F254 HPTLC plate using n-hexane？:？ethyl acetate as mobile phase in the ratio of 75？:？25, v/v. The plate was developed at room temperature 25？±？2.0°C. Artemisinin separation over thin-layer plate was visualized after postchromatographic derivatization with anisaldehyde-sulphuric acid reagent. HPTLC plate was scanned in a CAMAG’s TLC scanner 3 at 540？nm. Artemisinin responses were found to be linear over a range of 400–2800？ng spot？1 with a correlation coefficient 0.99754. Limits of detection and quantification were 40 and 80？ng spot？1, respectively. The HPTLC method was validated in terms of system suitability, precision, accuracy, sensitivity (LOD and LOQ), and robustness. Additionally, calculation of plate efficiency and flow constant were included as components of validation. Extracts prepared from different parts of the plant (leaves, branches, main stem, and roots) were analyzed for artemisinin content, in which, artemisinin content was found higher in the leaf extract with respect to branches and main stem extracts; however, no artemisinin was detected in root extract. The developed HPTLC-visible method of artemisinin determination will be very useful for pharmaceutical industries, which are involved in monitoring of artemisinin content during different growth stages (in vitro and in vivo) of A. annua for qualitative and quantitative assessment of final produce prior to commercial-scale processing for assessment of cost-benefit ratio. 1. Introduction Malaria is a vector-borne infectious disease, which affects approximately 400 million people every year, especially in Africa. The parasite responsible for fatal malarial infections is Plasmodium falciparum. The first effective antimalarial drug to treat this dangerous infection was quinine; since then, malaria has been treated with quinoline-based drugs like chloroquine, mefloquine, and pyrimethamine, but malaria parasite developed resistance to these drugs [1–3]. Artemisinin (Figure 1) is a natural
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