Correlation of the Expression Level of Amorpha Diene Yynthase Gene (ADs) with the Artemisinin Level in Artemisia annua L. plant

Artemisinin is an important therapeutic drug that, along with its derivatives, is a proven treatment for a number of diseases besides malaria (Dhingra et al., 2000), other parasites like schistosomiasis (Utzinger et al., 2001; Borrmann et al., 2001), and more recently cancer and hepatitis B (Romero et al., 2005). Artemisinin has also been shown to be effective against a variety of cancer cell lines including breast cancer, human leukemia, colon, and small-cell lung carcinomas (Efferth et al., 2001; Singh and Lai, 2001). Furthermore, artemisinin may be especially effective in treating drug resistant cancers (Sadava et al., 2002; Efferth et al., 2002). However, the drug is in short supply as its complex structure still requires extraction from plants. Although other researchers are working on a synthetic trioxolane (Vennerstrom et al., 2004) and bacterial produced artemisinin precursors (Martin et al., 2003) that may replace artemisinin as an inexpensive therapeutic. A. annua plants are still the only current source of the drug. In spite of the therapeutic importance of artemisinin, its biosynthetic pathway post-FDP production is not yet completely elucidated. It is clear that the first dedicated step in the biosynthesis of artemisinin is the cyclization of farnesyl diphosphate (FDP) by amorphadiene synthase (ADS) to amorpha-4,11-diene (Fig. 1; Bouwmeester et al., 1999). Several authors have demonstrated that artemisinic acid and/or dihydroartemisinic acid are further intermediates in the formation of artemisinin (Fig. 1; Wallaart et al., 1999a; Abdin et al., 2003). Little is known about the enzymes involved in the conversion of amorpha-4,11-diene to dihydroartemisinc acid, and until recently (Bertea et al., 2005), none of the putative intermediate products had been identified. Modification of the amorpha-4,11-diene carbon skeleton to produce artemisinic acid was thought to involve cytochrome P450 enzyme leading to the production of artemisinic alcohol which could then be oxidized twice by either cytochrome P450 enzymes or dehydrogenases to yield artemisinic acid (Fig. 1; Bouwmeester et al., 1999). The C11– C13 double bond of artemisinic acid is thought to be reduced to yield dihydroartemisinic acid, which was shown by others to be converted nonenzymatically to artemisinin (Wallaart et al., 1999a,b; Abdin et al., 2003). The first committed step of artemisinin biosynthesis is the cyclization of farnesyl diphosphate (FDP) by a ses-quiterpene synthase (cyclase) to produce the characteristic 15 carbon ring system, Amorpha-4,11-diene. Although the complete biosynthetic pathway for artemisinin has not yet been established, artemisinic acid is now considered to be the biogenic precursor of artemisinin (Wallaart et al.,1999a,b; Brown et al., 2004). Artemisinic acid and amorpha-4,11-diene are structurally closely related, which made the latter the more likely candidate for the cyclization product. Detection and partial purification of amorpha-4,11-diene synthase (ADS) from the plant was first reported by Bouwmeester et al. (1999). The low level of the volatile amorpha-4,11-diene in the plant and the high amorphadiene synthase activity were considered to be strong evidence that amorpha-4,11-diene is an intermediate in the biosynthesis of artemisinin. Several analytical methods to assay artemisinin have been described in the literatures, including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography coupled with mass spectrometry (GC-MS), and immunoassay. In addition, analytical procedures for biosynthetic precursors of artemisinin as well as metabolites in biological fluids have been developed. In this study we used Q260 HPLC method for estimation of artemisinin. Artemisinin has no chromophoric group and only absorbs at the low end of the UV spectrum, below 220 nm. In order to obtain a product with more specific and sensitive spectrometric charcteristics, Zhao and Zeng (1985) converted artemisinin into a product named Q260. First, artemisinin is treated with alkalie and converted into Q292, possessing a UV absorbtion at 292. Upon acidification, Q292 yields Q260, which has a strong UV absorption at 260 nm and is more stable than Q292. The reaction scheme is shown in Fig. (2). Q260 can be separated by means of HPLC on a reversed phase C18 column, with phosphate buffer-methanol as the mobile phase.