as well as H2O2-induced oxidative damage in β-TC6 cells (Tiong et al. 2013; Vuong 2017). The high performance liquid chromatography (HPLC) determination of flowers and leaves of C. roseus showed the presence of rutin, quercetin, and gallic acid (Nisar et al. 2017). Phenolic acids such as p-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, vanillic acid, and gallic acid were also isolated from C. roseus (Mustafa and Verpoorte 2007), as well as α-amyrin, β-amyrin, and lupeol to ursolic acid, oleanolic acid, and betulinic acid (Huang et al. 2012).
2.23.2 Culture Conditions
Metabolic engineering may be a potent tool for increasing the production of terpenoid indole alkaloids in C. roseus cell platforms. Usually, enzymes of the biosynthetic pathway are selected as targets for gene cloning and then manipulated by genetic engineering, e.g. to increase metabolic flow rate toward the compound of interest and hence enhance its levels. This approach has a limited value since a more predictable control of metabolic flux could be achieved using transcription factors because they can regulate one or more catalytic steps from terpenoid indole alkaloid biosynthesis (Capell and Christou 2004; Giri and Narasu 2000; Goyal et al. 2008). In addition, the complex interactions among biosynthetic pathways and sophisticated regulatory mechanisms further complicate engineering efforts. Therefore, among the strategies used to enhance terpenoid indole alkaloid biosynthesis in C. roseus in vitro cultures, the overexpression of transcription factors and genes of their biosynthetic pathways could be highlighted as well as the overexpression of genes from the terpenoid indole alkaloid biosynthesis in other organisms (Wilson and Roberts 2014).
These compounds were extracted commercially from large quantities of C. roseus. Since the intact plant contains very low concentrations, plant cell cultures have been employed as an alternative to produce large quantities of these alkaloids. Since vindoline is more abundant than catharanthine in intact plants, it is less expensive. An economically feasible process consisting of catharanthine production by plant cell fermentation and a simple chemical coupling was also established (Misawa et al. 1988; Jung et al. 1995). Maximum callus induction was achieved in V. rosea by culturing several types of explants on different combinations of growth hormones. Estimation studies reveal that metabolites accumulated in higher yield in callus cultures than in vivo plants. The effects of various compounds, like vanadyl sulfate, abscisic acid, and sodium chloride, on catharanthine production have been established (Smith et al. 1987).
The accumulation of vindoline in C. roseus intact plant was 0.2%, a level much higher than that of catharanthine, while the cost of vindoline is less expensive compared to catharanthine and vinblastine. The observed results showed that the MS medium (Murashige and Skoog 1962) was the most favorable for optimization of catharanthine production in different cell lines of C. roseus. Addition of various biotic and abiotic precursors to the medium as “inducers” was found to induce the production of Vinca alkaloids. When abscisic acid was used as an elicitor in cell cultures, the accumulation of catharanthine was raised maximum on the seventh day of cultivation. Circular dichroism confirmed that α-coupling exists between the two monomeric units of both vinblastine and vincristine produced enzymatically (Whitmer et al. 2000). This is an efficient and novel method to produce vinblastine and is likely to be used for the commercialization of vinblastine. The production of anhydrovinblastine was enhanced by a two-enzyme system (horse radish peroxidase and glucose oxidase) for catalyzation of the anhydrovinblastine to catharanthine and vindoline (Bede and DiCosmo 1992; Kumar et al. 2013).
MS medium supplemented with kinetin and 6-benzylaminopurine (BAP) each with 2,4-D and indole-3-acetic acid (IAA) combinations showed good callus production. Similarly MS + kinetin and BAP 2 mg/l each and MS + 2,4-D and IAA combinations showed green and resin-secreting callus. When the leaf explants were cultured in MS + BAP and 1-naphthaleneacetic acid (NAA), they showed large number of root formation. When combination of MS + kinetin and 2,4-D and MS + BAP and IAA were used, a quick callus induction was seen (Negi 2011; Koul et al. 2013). The production of vinblastine via chemical coupling was enhanced in the presence of ferric chloride, oxalate, maleate, stemmadenine, and sodium borohydride (El-Sayed et al. 2004). Effects of various parameters like stress, addition of bioregulators, elicitors, and synthetic precursors on indole alkaloids production were also studied (Zhao et al. 2001a,b). Metabolic rate limitations through precursor feeding and the effects of elicitor dosage on biosynthesis of indole alkaloids in hairy root cultures of C. roseus have also been studied (Morgan and Shanks 2000; Rijhwani and Shanks 1998; Almagro et al. 2014).
Alkaloids were produced from callus, roots, and petiole of C. roseus in the presence of kinetin and NAA. MS with NAA + kinetin had the highest vindoline, catharanthine, and vincristine. But the level of these alkaloids and ajmalicine were very low compared to that in the petiole of intact plant, and the level of serpentine was similar. The largest amount of alkaloids was produced in new roots and callus. The indole alkaloid levels of new roots in new media were higher than in petioles of intact plants (El-Sayed and Verpoorte 2007; Courdavault et al. 2014). The most interesting result was the presentation of two important anticancer dimeric alkaloids, 20-fold for vinblastine and sixfold for vincristine, compared with that in the petioles of intact plants (Ataei-Azimi et al. 2008).
The complex development in environment, organs, and cell-specific controls involved in the expression of monoterpenoid indole alkaloids (MIA) pathways is attributed to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the surface of Catharanthus leaves, as well as its insect toxicity, provides an additional biological
