Читать книгу Secondary Metabolites of Medicinal Plants - Bharat Singh - Страница 50

The hyoscyamine and scopolamine are found in A. belladonna as well as other members of Solanaceae family. The tropine, tropananone, belladonnine, norhyoscyamine, apoatropine, hyoscyamine, 6β-hydroxyhyoscyamine, and scopolamine have been simultaneously identified from A. belladonna L (Arráez-Román et al. 2008). The enzymatic activity of tropine-forming reductase increased the synthesis of hyoscyamine and scopolamine in transformed roots (Richter et al. 2005). Cell culture studies were established in Atropa plant species by using various explants of A. belladonna for the enhancement of yield of the tropane alkaloids. The callus cultures were established by using as root and stem explants. For the induction of callus, the Wood and Braun's culture medium was found to be more suitable for the production of tropane alkaloids (Simola and Nieminen 1988). The induction of hairy root culture of A. belladonna was achieved by using Agrobacterium rhizogenes to increase the accumulation of tropane alkaloids. Earlier reported studies revealed that the production of the tropane alkaloids in the cell cultures of A. belladonna was similar or even higher than of intact plants. It has also been proved that hyoscine was produced in higher concentration in transgenic A. belladonna L. in comparison to nontransformed A. belladonna plant (Kamada et al. 1986; Yang et al. 2011; Asha Rani and Prasad 2014). High-performance liquid chromatography (HPLC) results suggest that transgenic hairy roots produced maximum content of scopolamine as compared with nontransgenic hairy roots. The gene expression profile indicated that both putrescine N-methyltransferase and hyoscyamine 6β-hydroxylase were expressed at different levels in different transgenic hairy roots, which would be helpful for biosynthesis of scopolamine (Zheng 2005; Long et al. 2013).

The Nicotiana tabacum pmt gene was engineered in cauliflower mosaic virus 35S promoter and then incorporated into the genome of A. belladonna. In the regenerated transgenic plants, it was observed that accumulation of tropane alkaloids was found unchanged or somewhat the production reduced in comparison to nontransgenic plants. Similarly, the production of calystegines was enhanced by treating the hairy roots with 5% sucrose. When Murashige and Skoog (1962) medium was supplemented with 1.0 mM of auxin, the concentration reduced the accumulation of calystegine, but the production of tropane alkaloids remain unchanged in transgenic hairy roots (Rothe et al. 2003). On estimation it was observed that the in vitro hyoscyamine alkaloid production in differentiated leaves was more in yield in comparison to the original plant (Al-Ashaal et al. 2013; Khater et al. 2013).

Scopolamine and hyoscyamine like alkaloids, considered as anesthetic and antispasmodic drugs, are produced commercially by applying hairy root culture technology in A. belladonna. The well-regenerated hairy roots accumulate more scopolamine, hyoscyamine, and/or nicotine in A. belladonna (Endo and Yamada 1985; Yang et al. 2011). By HPLC determination, it was concluded that the accumulation of tropane alkaloids was higher in in vitro regenerated leaves but lower in the roots and shoots. Similarly, the atropine accumulation was higher, but scopolamine accumulated in lower concentration in the leaves of A. acuminata (Banerjee et al. 2008; Ashtiania and Sefidkonb 2011). The transgenic cells (containing H6H gene/s) of A. baetica were elicited with salicylic acid, acetyl salicylic acid, and methyl jasmonate for the induction of synthesis of tropane alkaloids. The synthesis of scopolamine was increased by the acetyl salicylic acid and methyl jasmonate, while salicylic acid did not affect the synthesis. This happened due to the expression of engineered h6h genes and other genes involved in the biosynthesis of alkaloids. The synthesis of scopolamine was increased by 25-fold higher than in control (Jaber-Vazdekis et al. 2008).

The biotransformation of betuligenol into an oxidized product betuloside was performed in the hairy roots of A. belladonna. The yield of biotransformed products increased twofold higher than the control root mass. On the 5th and 10th days of incubation, the highest biotransformation of betuligenol into raspberry ketone and betuligenol into betuloside was reported in hairy roots. The application of hairy roots for biotransformation of raspberry ketone and betuloside opens up new opportunities in the production of medicinally significant secondary metabolites (Srivastava et al. 2013).

The pharmaceutically important tropane alkaloids were isolated from the A. belladonna. The putrescine N-methyltransferase from N. tabacum and hyoscyamine 6β-hydroxylase from Hyoscyamus niger were overexpressed in transgenic A. belladonna for investigation of biosynthesis of tropane alkaloids in stem roots and fruits. The expression of putrescine N-methyltransferase and hyoscyamine 6β-hydroxylase was not reported in wild plant species but were found transgenic plants. The maximum conversion of hyoscyamine was observed in aerial parts, not in underground parts. In aerial parts, the hyoscyamine was fully transformed into scopolamine. The synthesis of hyoscyamine, anisodamine, and scopolamine was reported to have higher yield in underground parts than in aerial parts (Xia et al. 2016).

The effects of KCr(SO₄)₂ on the accumulation of tropane alkaloids as well as the expression of hyoscyamine 6β-hydroxylase gene was investigated in in vitro regenerated plantlets of A. belladonna. The chromium treatment to plantlets decreased the weights, lengths of the plantlets, and chlorophyll contents but enhanced the levels of proline contents. The chromium treatment also increased the concentration of hyoscyamine and scopolamine. The levels of scopolamine can be correlated with the expression levels of h6h gene with several concentrations of chromium (Jaber-Vazdekis et al. 2009; Vakili et al. 2012).