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2.15 Atropa Species
2.15.1 Ethnopharmacological Properties and Phytochemistry
Atropa belladonna L. (Fam. – Solanaceae) is a perennial plant endemic to Central and Southern Europe and India and is being cultivated worldwide. It contains tropane alkaloids and possesses anticholinergic and spasmolytic properties; it is also used in the treatment of headache, menstrual symptoms, peptic ulcer, inflammation and motion sickness, bronchial spasms and whooping cough, Parkinson's disease, antidote for snake bite, and gastric agent (Tyler et al. 1988; Paul and Datta 2011). A. belladonna, known as perennial herb, as well as toxic drug (Greek word Atropa – Atropos, means to cut the fate of life; belladonna – beautiful women [in Italian]). Since ancient times, the extract of aerial parts has been used by women to dilate pupils and also applied to the cheeks to give a pinkish-red glow to the skin. In European civilization, the plant species was used in the treatment of various diseases (Bousta et al. 2001). The A. belladonna is used as antidote, anodyne, analgesic, hallucinogenic, mydriatic, narcotic and sedative and against parkinsonism, encephalitis, carcinoma, and spastic dysmenorrhea; its pharmacologically active ingredients include atropine, scopolamine, hyoscyamine, and all tropane alkaloids (Chopra et al. 1986). The plant species is considered as extremely toxic due to the presence of alkaloids (atropine, scopolamine, and hyoscyamine). The toxic Atropa alkaloids cause delirium, hallucination, tachycardia, mydriasis, dry mouth, flushed skin, urinary retention, vomiting, and anhidrosis in humans (Rajput 2013). The roots, leaves, fruits, and stems of Atropa species contain tropane alkaloids (e.g. atropine, hyoscyamine, and scopolamine). By mixing activated charcoal, it helps in decontamination of gastrointestinal tract (Joshi et al. 2003; Berdai et al. 2012). No elevated creatine kinase was observed in one case with acute subdural hematoma and hyperamylasemia (Cikla et al. 2011). Besides A. belladonna, four other species of Atropa are found in different regions of the world including Atropa acuminata in Asia, Atropa baetica in Spain and Morocco, and Atropa caucasica and Atropa komarovii in Russia (Chadha 1985; Harborne and Khan 1993; Maqbool et al. 2014). The yield of scopolamine was higher (1.8-fold) in seeds than in leaves and roots (Ashtiania and Sefidkonb 2011).
The main root, lateral roots, and leaves of A. baetica were investigated for tropane alkaloids by GC–MS analysis. The main root and leaves showed the presence of the maximum number of alkaloids (hygrine, tropinone, tropine, pseudotropine, N-methylpyrrolidinyl-hygrine A, N-methylpyrrolidinyl-hygrine B, cuscohygrine, 3α-phenylacetoxytropane, 6-hydroxyapoatropine, and 6-hydroxyhyoscyamine). Only five alkaloids were present in the stem region (3α-isobutyryloxytropane, apoatropine, atropine, aposcopolamine, and scopolamine) of this plant species (Zárate et al. 1997; Gadzikowska and Grynkiewicz 2002). The main source of scopolamine and hyoscyamine, the drugs used commercially, is A. belladonna. For the commercial synthesis, the nine structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, and TRII) were involved in tropane alkaloid biosynthesis. The digital expression patterns suggested that four genes, ODC, ADC, AIH, and CPA, were involved in upstream pathway of tropane alkaloids. The maximum contents of hyoscyamine were obtained in tender stems, leaves, and roots, while maximum synthesis of scopolamine occurred in fruit sepals. The older parts of stems and roots contained the lowest levels of hyoscyamine and scopolamine (Qiang et al. 2014).
2.15.2 Culture Conditions
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
