## 2 Results and Discussion  
### Effects of DIMEB on Intracellular and Extracellular Carotenoids and Quinones  
In a previous study, chemically modified \(\beta\)-CDs (2,6-dimethyl-\(\beta\)-cyclodextrintrins, DIMEB) were effectively used to significantly increase the production and release into the culture medium of artemisinin in _Artemisia annua_ suspension cell cultures [6]. To study the effects of DIMEB on the production of other important isoprenoid metabolites, carotenoids and quinones were analyzed in actively growing _A. annua_ suspension cell cultures treated with DIMEB. The concentration of 50 mM for two different time intervals, three and seven days, was chosen on the basis of previous results obtained using different concentrations of DIMEB and various time intervals as for the production of the terpenoid artemisinin and according to recent literature data [6,12,13]. The effects of DIMEB on _A. annua_ cell growth were previously reported showing that DIMEB had no negative effects on the growth of these cultures [6]. The viability assay, carried out using the fluorescein diacetate staining method [14], confirmed that DIMEB treatments did not affect the viability of the cultures (data not shown). This indicates that the addition of CDs to the culture medium does not affect the growth ability of plant cell cultures as also reported by other authors in _Vitis vinifera_ cell cultures [15]. When carotenoids were analyzed in cell extracts of _A. annua_ suspension cultures, cells treated for three days with 50 mM DIMEB showed slight increases of violaxanthin, zeaxanthin and \(\beta\)-carotene and a highly significant increase of lutein (more than 10-fold) compared to untreated cells, thus showing the ability of DIMEB to enhance carotenoid production (Figure 1A). After a seven-day treatment the intracellular levels of carotenoids resulted similar compared to the untreated cell cultures (Figure 1C). Intracellular quinone levels in treated and untreated _A. annua_ suspension cultures were also measured. In comparison with the untreated cells, after three days of 50 mM DIMEB treatment, significant increases of intracellular levels of Q9 (about 3-fold) and Q10 (2.5-fold) were observed (Figure 2A). After seven days the intracellular Q9 level decreased by 20% compared to the content of the control cells while the Q10 level was very similar to the level of the untreated cells (Figure 2C). These results clearly indicate that DIMEB were able to induce an early increase of intracellular carotenoid and quinone content, which could be observed to a higher extent for lutein, Q9 and Q10. It has been shown that DIMEB can act as enhancer of secondary metabolite biosynthesis in different plant cell cultures alone or in combination with methyl jasmonate [5,6,12,13]. This activity was explained by the similarity of CD chemical structure to pectic oligosaccharides released by the cell wall after a fungal infection Thus, the DIMEB induction of carotenoid and quinone metabolism here detected, to our knowledge for the first time, could resemble a defense response of stressed plant cell cultures. Since _A. annuua_ cell cultures had already been shown to produce extracellular amounts of the terpenoid artemisinin [11], the culture medium of 50 mM DIMEB treated cultures was analyzed for carotenoid and quinone contents in comparison with the medium of untreated cultures. The results showed a considerable increase of extracellular levels induced by the DIMEB treatment that could be detected for most carotenoids after three and seven days, while hardly detectable levels of carotenoids with the exception of lutein, were observed in the medium samples of untreated cultures. Moreover, in the medium of DIMEB treated cells a time-related increase of carotenoid levels was observed by analyzing three- and seven-day-treated samples (Figure 1B,D). When the medium of _A.