methods from A. annua (Khan et al. 1991). The high-performance liquid chromatography (HPLC) determination of methanolic extract of Artemisia capillaris revealed the presence of chlorogenic acid, 3,5-dicaffeoylquinic acid, and 3,4-dicaffeoylquinic acid, and their chemical structures were elucidated by spectral analysis (Seo et al. 2003). Artemisinin showed antiprotozoal activity against Trypanosoma cruzi and Trypanosoma brucei rhodesiense at several concentrations. Artemisinin inhibits calcium-dependent ATPase activity in T. cruzi membranes, proposing a mechanism of action through membrane pumps (Mishina et al. 2007).
2.13.2 Culture Conditions
Due to the epidemic prevalence of malaria and resistance acquired by Plasmodium falciparum, a search for plant-based molecule was imminent. The artemisinin from A. annua is widely known as antimalarial agent. For cell manipulations in A. annua, the leaf of plant was used as explant for the regeneration of callus. The explants were inoculated onto MS culture medium, supplemented with naphthaleneacetic acid (NAA), BAP, and sugar. The chloroform extract of callus showed larvicidal activity against Anopheles stephensi (Bilia et al. 2006; Bartarya et al. 2009). The callus browning is a major problem in tissue culture system and occurred due to excessive accumulation of phenolic compounds in the callus. As for preventive measures of this problem, the culture medium was incorporated with 2-aminoindane-2-phosphonic acid. This compound stopped the browning of callus tissue by decreasing the accumulation of phenolic compounds. The microscopic analysis of the cells revealed that the accumulation of phenolic compounds is more prevalent in brown cells. The cell wall of these cells was broken so the phenolic compounds were released into culture medium. The 2-aminoindane-2-phosphonic acid inhibited the phenylpropanoid pathway by which the browning of callus tissue is stopped (Nair et al. 1986; Jones and Saxena 2013).
To evaluate the effect of dichlorophenoxyacetic acid (2,4-D) on callogenesis and total contents of chlorophylls, carotenoids, phenolics, flavonoids, and proteins, callus cultures of A. annua were established. The maximum callus growth was obtained in MS culture medium with supplementation of 2,4-D (Geldre et al. 1997). By increasing the concentration of 2,4-D, the production of chlorophylls, carotenoids, and flavonoids was enhanced but it did not increase the accumulation of phenols and proteins. The 2,4-D also increased the polymorphism in cell cultures (Rai et al. 2014). Seeds of A. annua were obtained from different locations of the United States, Europe, and India. The maximum level of artemisinin was obtained from Indian cultivar. The explants of American, European, and Indian cultivars were inoculated onto MS culture medium. The culture medium was supplemented with various concentrations of BA + IAA, NAA + kinetin. Maximum artemisinin accumulation was reported in European cultivars with BA + IAA supplementations of MS culture medium (Fulzele et al. 1991). As per other strategy, the accumulation of artemisinin was induced in hormone free culture medium. In the second set of experiment, the culture medium was supplemented with casein hydrolysate and gibberellins. Precursor feeding technology was also implemented for the enhancement of artemisinin production. Along with precursor feeding, interference in biosynthetic pathway of sterols was also done by feeding of miconazole, terbinafine, or naftifine. The maximum production of artemisinin was reported in presence of gibberellic acid, casein hydrolysate, and naftifine in cell cultures of A. annua (Woerdenbag et al. 1993; Jaziri et al. 1995). Other combinations of growth hormones were also tested for the enhancement of artemisinin production. In the first set of experiment, only auxins were supplemented in MS culture medium. In other experiment, only cytokinins were mixed into culture medium. In the third set, the combination of auxin (NAA) and cytokinin (BA) was added to the culture medium (Mannan et al. 2008). The maximum accumulation of artemisinin was observed in the combination of 2,4-D and NAA. The artemisinin was not synthesized in suspension cultures, but trace amount of artemisinin was obtained in adventitious shoots (Brown 1994; Paniego and Giulietti 1994; Zia et al. 2007a). Both 6-benzylaminopurine and 2-isopentenyladenine inhibited root growth; however, only 2-isopentenyladenine stimulated artemisinin production, more than twice that of the B5 controls, and more than any other hormone studied. These results will prove useful in increasing hairy root growth and artemisinin production (Bhakuni et al. 2001; Kim et al. 2003; Weathers et al. 2005).
However, artemisinin production from callus and cell suspension cultures of A. annua was reported to have extremely very low yields (Vishweshwar Rao and Lakshmi Narasu 1998a; Vishweshwar Rao et al. 1998b; Dhingra et al. 1999). The artemisinin production in shoot cultures of such species as Artemisia pontica, Artemisia judaica, Artemisia vulgaris, A. annua, and Artemisia scoparia was investigated by a number of researchers (Gulati et al. 1996; Liu et al. 2004; Sujatha and Rajnitha Kumari 2007; Mannan et al. 2010; Singh and Sarin 2010).
Artemisinin is considered as an important antiplasmodial drug and used in Chinese traditional system of medicine. For the enhancement of production of artemisinin, hairy root cultures were established. The effect of gibberellic acid on growth of hairy roots and contents of artemisinin was evaluated. Maximum growth of hairy roots and total artemisinin content was observed in the presence of gibberellic acid. The gibberellic acid-treated hairy roots attained stationary phase of growth rapidly compared with nontreated hairy roots (Smith et al. 1997). The regenerated hairy roots were tested for scaled-up production of artemisinin (Xie et al. 2000). From the hairy roots of A. annua, artemisinin, artemistene, artemisinic acid, and arteannuin B were isolated, and it was concluded that this technology might be considered as feasible and more economical for the production of artemisinin (Weathers et al. 1994). Rapid growth of hairy roots and maximum accumulation of artemisinin was obtained in the presence of sucrose. Low concentration of NAA increased the growth of the roots but inhibited the production of artemisinin (Weathers et al. 1997, 2004). The growth and artemisinin production in hairy root cultures were greatly promoted by the addition of gibberellin to the medium (Cai et al. 1995).
The complete biosynthesis of artemisinin is tedious and quite expensive (Schmid and Hofheinz 1983); therefore an alternative semi-synthesis mode of artemisinin or via artemisinic acid (immediate
