Secondary Metabolites of Medicinal Plants. Bharat Singh

2.18.2 Culture Conditions

The synthesis of camptothecin and 10-hydroxycamptothecin was monitored with treatment of heavy metal ions, UV-B, methyl jasmonate (MeJA), abscisic acid, salicylic acid, and hydrogen peroxide in callus cultures of C. acuminata. During the course of treatments, it was found that UV-B light induced the synthesis of camptothecin by 10-fold higher than control. Similarly, the salicylic acid also stimulated the accumulation of 10-hydroxycamptothecin up to 25-fold higher than nontreated callus cultures (van Hengel et al. 1992; Pi et al. 2010). High-performance liquid chromatography (HPLC) analysis revealed that overexpression of ORCA3 gene in transgenic hairy roots enhanced the production of camptothecin (1.5-fold higher) than the control. The observed results show that ORCA3 can be considered as an effective regulatory gene for stimulating the metabolic flux in biosynthetic pathway of camptothecin (Ni et al. 2011).

      Camptothecin, a potent antitumor alkaloid endemic in North China, was isolated from C. acuminata (Wall et al. 1966). It is highly active against Walker 256 rat carcinosarcoma and mouse leukemia, p388 and L1210. The clinical trials in patients with gastrointestinal cancer were at first very promising, but subsequent trials showed toxicity. Callus induction in C. acuminata was achieved on MS medium containing dichlorophenoxyacetic acid (2,4-D) and kinetin and developed suspension cultures in the presence of gibberellins, L-tryptophan, and conditioned medium, which yielded camptothecin at about 0.0025% on a dry weight basis (Sakato and Misawa 1974); similarly, when the cultures were cultured on MS medium containing NAA, production of camptothecin reached its maximum peak (Van Hengal et al. 1992).

      The Chinese tree C. acuminata produces the anticancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25- to 30-day-old seedlings, presoaked for 48 hours in three different liquid media containing BA, kinetin, or thidiazuron, and subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the BA presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing BA. Individual shoots were removed from clusters and rooted on B5 supplemented with IBA (Jain and Nessler 1996).

      Camptothecin is an anticancer and antiviral monoterpene-derived indole alkaloid, which can be induced by plant hormone, jasmonates. To improve the production of the pharmaceuticals, the jasmonate biosynthesis-related gene allene oxide cyclase from C. acuminata was transferred back into C. acuminata by Agrobacterium-mediated genetic transformation and overexpressed. The camptothecin content of transgenic callus was higher than that of nontransgenic callus. However, camptothecin content in both transgenic and nontransgenic calli significantly decreased after further extrinsic MeJA's induction, whereas the content of camptothecin in transgenic callus was still higher than in nontransgenic one. All the results indicate that endogenic jasmonate's accumulation may be promoted after allene oxide cyclase gene was transformed into C. acuminata and overexpressed (Pi et al. 2012).

Hairy roots derived from the wild-type strains of Agrobacterium rhizogenes have also been developed in C. acuminata; the yields of the camptothecin in the two species were of approximately 1 mg/g dry weight (DW). The highest camptothecin content of the transgenic callus reached 3.9310 mg/g DW in this present research, which was higher than the one reported. The highest growth rate of callus was observed on MS medium with NAA, kinetin, and sucrose. All tissues and organs developed in vitro contain camptothecin and 10-hydroxycamptothecin. The presence of 10-hydroxycamptothecin in shoots and callus of C. acuminata Decne is reported for the first time (Wiedenfeld et al. 1997). The effects of I⁻, , , Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, and Zn²⁺ on the biosynthesis of camptothecin and the growth of suspension cultures of C. acuminata were studied. The increase of microelements in MS medium resulted in twofold higher camptothecin yield than that in standard MS medium, respectively (Pan et al. 2004).

      Camptothecin, a well-known monoterpenoid indole alkaloid originally identified in the extracts of the Chinese tree C. acuminata, exhibits antitumor activity due to its ability to kill cancer cells via topoisomerase I poisoning. Other plant species have since been shown to produce camptothecin and related compounds (Yamazaki et al. 2003). The complementary DNAs for strictosidine synthase, tryptophan decarboxylase (TDC), and cytochrome P₄₅₀ reductase were cloned from Ophiorrhiza pumila and evaluated for involvement in production of camptothecin in this species. RNA interference-mediated knockdown of gene expression indicated that the production of camptothecin, strictosidine, and camptothecin-related alkaloids was suppressed in a TDC expression-dependent manner in RNA interference hairy roots (Liu et al. 1998; Lorence and Nessler 2004; Yamazaki et al. 2013).

      The young flower buds had the highest alkaloid concentrations (camptothecin and 10-hydroxycamptothecin). Callus showed lower concentrations, but it should also be considered as a potential source of these pharmaceuticals (Lorence et al. 2004). The growth rate of C. acuminata cells in culture did not correlate with the contents of camptothecin and 10-hydroxycamptothecin (Lopez-Mayer et al. 1997; Zhang et al. 2007; Pi et al. 2010).

      Camptothecin production in regenerated plants was compared with its production in calli and the original seedlings. Dark incubation and seed coat removal led to a higher germination rate and a higher survival rate after germination. The best shoot induction medium was found to be Gamborg's medium + benzyl adenine. The leaf petiole calli had a higher shoot regeneration rate and a higher shoot number than did leaf disc calli on the best shoot induction medium. It took four to six weeks to regenerate shoots after transfer into shoot induction media. Camptothecin concentration in the regenerated plants was significantly higher than that in the calli and similar to that in the original seedlings (Li and Liu 2005).

      Concentrations of the DNA topoisomerase I inhibitor camptothecin were determined in different tissues of C. acuminata. Despite the presence of the toxic compound camptothecin, nine endophytic fungi were isolated from healthy C. acuminata plants and were tested for their camptothecin sensitivity. Even at a concentration of 100 μg/ml, the inhibition was moderate (Liu and Reinscheid 2004). Similarly, (Wiedenfeld et al. 1997) in callus cultures of C. acuminata and in shoot cultures has reported a low amount of camptothecin (Roja 2008).

The