## 2 Results and Discussion
### Effects of DIMEB on Intracellular and Extracellular Carotenoids and Quinones
annuua_ cell cultures was analyzed for quinones, a significant Q9 increase in the medium of three-day-treated cells was observed compared to the untreated samples, while no detectable Q10 levels were observed both in the medium of untreated and treated samples (Figure 2B). After 7 days, extracellular Q9 and Q10 levels significantly increased in the medium of treated cells compared to the control (Figure 2D). Figure 1: Intracellular and extracellular carotenoid levels in _A. annuua_ cell cultures untreated (CTRL) or treated (DIMEB) with 50 mM DIMEB. Data are expressed as mean \(\pm\) standard deviation from three independent experiments, **_p_\(<\) 0.01; ***_p_\(<\) 0.001 in comparison with control assessed by Anova-one-way _post hoc_ Holm-Sidak test. Intracellular (**A**) and extracellular (**B**) carotenoid levels after three days of treatment; intracellular (**C**) and extracellular (**D**) carotenoid levels after seven days of treatment. These results indicate that the addition of DIMEB to _A. annua_ suspension cultures not only induced an early increase of intracellular carotenoid and quinone content, but also an enhanced release of carotenoids and quinones into the culture medium. It is known that CDs are able to form inclusion complexes with non-polar compounds, favoring their accumulation in aqueous solution. In recent years, the complexation of hydrophobic antioxidant compounds, including carotenoids and quinones, in aqueous medium with molecules such as CDs has been reported in order to increase their stability, bioavailability and solubility (for a review [16]). Therefore this ability to form inclusion complexes with hydrophobic molecules, such as isoprenoids, could explain the increase of carotenoids and quinones released into the culture medium by _A. annua_ cell cultures treated with 50 mM DIMEB, possibly as an effect of enhanced membrane permeability [17] or improved stability of the isoprenoid molecules in the medium. The addition of \(\beta\)-CDs to plant cell cultures to improve the production and the extracellular accumulation of various secondary metabolites such as resveratrol and phytosterols has been described in suspension cell cultures of _Vitis vinifera_ and _Daucus carota_, respectively [5, 6, 7, 8, 9, 10, 11, 12]. In a previous work, we showed the ability of \(\beta\)-CDs to improve the release of the sesquiterpene lactone artemisinin from _Artemisia annua_ cell cultures [6]. The results of this work suggest that \(\beta\)-CDs could  
Figure 2: Intracellular and extracellular quinone levels in _A. annua_ cell cultures untreated (CTRL) or treated (DIMEB) with 50 mM DIMEB. Data are expressed as mean \(\pm\) standard deviation from three independent experiments, * \(p\) \(<\) 0.05; ** \(p\) \(<\) 0.01; *** \(p\) \(<\) 0.001 in comparison with control assessed by Anova-one-way _post hoc_ Holm-Sidak test. Intracellular (**A**) and extracellular (**B**) quinone levels after three days of treatment; intracellular (**C**) and extracellular (**D**) quinone levels after seven days of treatment. also be successfully used for enhancing the production and release of carotenoids and quinones in _A. annua_ suspension cell cultures. Although plant _in vitro_ cultures have received a lot of attention as an effective technology for the production of valuable secondary metabolites, however, in most cases, the desired products are stored, often as low amounts, within the cells and this prevents an efficient and continuous production of useful metabolites increasing the complexity and the cost of the whole process [1]. Finding out strategies to overcome these problems, by enhancing product yields and inducing the release or exudation of useful products into the culture medium can be actually helpful. The secretion of isoprenoids induced by DIMEB in the culture medium of _A. annua_ cell cultures could be promising in view of making easier the isolation of these molecules to be exploited for the production of nutraceuticalals, food additives or ingredients for cosmetics.