Affordable and rapid HPTLC method for the

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(Liu et al., 2008), LC-MS/MS (Van Nieuwerburgh et al., 2006), qNMR. (Liu et al., 2010), Enzyme Linked Immunosorbent Assay (ELISA) (He et al., 2009) and ...
Research article Received: 18 August 2014,

Revised: 11 January 2015,

Accepted: 23 February 2015

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.3465

Affordable and rapid HPTLC method for the simultaneous analysis of artemisinin and its metabolite artemisinic acid in Artemisia annua L. Shazia Khan, Athar Ali, Shahzad Ahmad and Malik Zainul Abdin* ABSTRACT: Artemisinin (AN) and artemisinic acid (AA), valuable phyto-pharmaceutical molecules, are well known anti-malarials, but their activities against diseases like cancer, schistosomiasis, HIV, hepatitis-B and leishmaniasis are also being reported. For the simultaneous estimation of AN and AA in the callus and leaf extracts of A. annua L. plants, we embarked upon a simple, rapid, selective, reliable and fairly economical high performance thin layer chromatography (HPTLC) method. Experimental conditions such as band size, chamber saturation time, migration of solvent front and slit width were critically studied and the optimum conditions were selected. The separations were achieved using toluene–ethyl acetate, 9:1 (v/v) as mobile phase on pre-coated silica gel plates, G 60F254. Good resolution was achieved with Rf values of 0.35 ± 0.02 and 0.26 ± 0.02 at 536 nm for AN and 626 nm for AA, respectively, in absorption–reflectance mode. The method displayed a linear relationship with r2 value 0.992 and 0.994 for AN and AA, respectively, in the concentration range of 300–1500 ng for AN and 200–1000 ng for AA. The method was validated for specificity by obtaining in-situ UV overlay spectra and sensitivity by estimating limit of detection (30 ng for AN and 15 ng for AA) and limit of quantitation (80 ng for AN and 45 ng for AA) values. The accuracy was checked by the recovery studies conducted at three different levels with the known concentrations and the average percentage recovery was 101.99% for AN and 103.84% for AA. The precision was analyzed by interday and intraday precision and was 1.09 and 1.00% RSD for AN and 1.22 and 6.05% RSD for AA. The analysis of statistical data substantiates that this HPTLC method can be used for the simultaneous estimation of AN and AA in biological samples. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: artemisinin; artemisinic acid; HPTLC; Artemisia annua L.; malaria and artemisinin based combination therapy (ACTs)

Introduction The annual herbaceous plant, Artemisia annua L. (Chinese sweet wormwood) belongs to the family Asteraceae. It is a traditional medicinal plant native to China (Newton and White, 1999). Despite its origin, it has become naturalized and cultivated in many other regions as well, like equatorial Africa, Argentina, Bulgaria, France, Romania, Spain, Italy and India (Abdin et al., 2003). Its use is reported in ancient Chinese medicine for the treatment of fever caused by malaria. A. annua L. is now a well-known medicinal herb for anti-malarial phytochemical drug, artemisinin (Qinghaosu). Artemisinin (AN) is a highly oxygenated sesquiterpene lactone. It contains a unique 1,2,4- trioxane ring structure with an endoperoxide bridge, which is essential for the activity of AN and its derivatives (Fig. 1). The endoperoxide bridge is cleaved in the presence of ferrous ion (Fe2+) that comes from degradation of the heam group and forms highly reactive free radicals that chemically modify and inhibit a variety of parasite molecules, resulting in parasite death (Shandilya et al., 2013). AN is a safe and potent therapeutic agent against both drug-resistant and cerebral malaria-causing strains of Plasmodium sp. (Newton and White, 1999). In addition to its commercial use as antimalarial drug, it is also effective in other infectious diseases such as schistosomiasis, HIV, hepatitis-B and leishmaniasis. It also possesses herbicidal activity and effectiveness against a variety of cancer cell lines (Borrmann et al., 2001; Chen and Zhang, 1987; Crespo-Ortiz and Wei, 2012; Duke et al., 1987; Efferth et al., 2001; Jung and Schinazi, 1994; Klayman, 1985; Romero et al., 2005; Sen et al., 2007; Singh and Lai, 2001). Very recently artemisinic acid (AA), an intermediate precursor of AN has been shown to be effective against hepatitis-B,

Biomed. Chromatogr. 2015

schistosomiasis, several blood parasitic protozoans and a variety of cancer cell lines (Chaturvedi et al., 2010). Artemisinin was first isolated from the aerial parts ( glandular trichomes) of A. annua L. plants by scientists in China (Klayman, 1985; Olofsson et al., 2011). The World Health Organization has endorsed the use of artemisinin derivatives in combination with other effective antimalarial drugs in the treatment known as artemisinin-based combination therapies (ACTs) for the first-line treatment of malaria (Roll Back Malaria, World Health Organization and UNICEF, 2005). The number of ACT treatment courses delivered in 2005 was 11.2 million. It was increased to 76 million in 2006 and to 331 million in 2012 (World Malaria Report, 2013). However, in 2007–2008