Tropane alkaloids constitute one of the distinctive groups of secondary metabolites of the Solanaceae and many plants containing them have long been utilized for their medicinal, hallucinogenic, and poisonous properties. Hyoscyamus plants are a natural source for the isolation of hyoscyamine (atropine) and scopolamine, 6-7 epoxide of hyoscyamine. Both alkaloids are of medicinal importance because of their suppressive activity on the parasympathetic nervous system. In addition, scopolamine is also applied to suppress the central nervous system, whereas hyoscyamine excites it. Ratios of hyoscyamine content to scopolamine content vary markedly between plant species. These differences result in a higher commercial demand for scopolamine than for hyoscyamine (and its racemic form atropine). Both appear in the USA in the list of the ten most used compounds of plant origin. Because many tropane alkaloid-producing species accumulate hyoscyamine as the major alkaloid and scopolamine in minor quantities, it is of commercial importance to increase scopolamine content in these species. Moreover, these plants also synthesize the calystegines, a pseudotropine-derived group of alkaloids, found in considerable amounts in Atropa and Hyoscyamus species, exerting a strong inhibitory activity on β-glycosidases, which can be of great importance for cell recognition. The latter can possibly result in treatment of some types of cancers.
This chapter mainly deals with H. reticulatus and describes the optimization of culture conditions for growth and productivity of tropane alkaloids from transformed H. reticulatus roots. Methodologies to enhance secondary metabolite levels are also discussed, together with a new technique of permeabilization of H. reticulatus-hairy roots for the release of stored tropane alkaloids. These recent developments are largely based on our published and unpublished experimental results.
Distribution and Importance of the Plant
The four genera of subtribe Hyoscyaminae (Scopolia, Physochlaina, Prezewalskia, Hyoscyamus) produce hyoscyamine as the usual major alkaloid with some hyoscine, but some Hyoscyamus species may contain hyoscine as the principal alkaloid.
Hyoscyamus plants are herbaceous, differing essentially from other solanaceous genera by having a fruit composed of a thornless seed capsule with a cover. The genus consists of 14 species: H. albus L. (including var. desertorum, var. canariensis), H. arenarius Dun., H. aureus L., H. ceratophyllus Fisch., H. eminens Kunce, H. falezlez Coss., H. grandiflorus Franch., H. muticus L., H. niger L. (including var. agrestis, var. bohemicus, var. pallidus), H. physaloides L., H. pseudophysaloides Roth., H. pussilus L., H. reticulatus L. and H. senecionis Willd.
Hyoscyamus reticulatus, henbane, is an annual or biennial plant, native to arid and semi-arid regions of Egypt, south-west Asia, Iran, and Turkey. The plant consists of an erect, simple ore branched stem, covered with basal and cauline leaves petiolate, upper leaves sessile, pinnatipartite, lobate or entire. Calyx is 10-20 mm, fruit 20-30 mm, not constricted at middle, teeth cuspidate, pungent, patent to deflexed. The corolla is pale yellowish, soon becoming purplish-violet with darker reticulate veins, 20-30 mm, almost campanulate with yellow anthers. It is found on cereal fields, waste places, roadsides, vineyards from 10 to 1700 m.
Hyoscyamus reticulatus as a Source of Tropane Alkaloids
Most pharmacological effects of Hyoscyamus drugs can be attributed to tropane alkaloids – hyoscyamine and scopolamine. They have a wide range of pharmaceutical applications, including uses as mydriatica, spasmolytica, analgetica, sedativa, antiperspirants, antidotes to intoxications, or for their beneficial effects in cases of asthma, Parkinson’s disease, ulcers, and motion sickness. These species also contain various other tropane alkaloids: apohyoscyamine, norhyoscyamine, littorine, tropine, cuscohygrine, tigloidine, tigloyloxytropane. Recently, the importance of the henbane drug has decreased, being often replaced by the pure tropane alkaloids, which allow a more controlled and specific application. The Hyoscyamus genera produce hyoscyamine as the major alkaloid. Scopolamine is more active than hyoscyamine, and is formed from hyoscyamine via 6-hydroxyhyo-scyamine. The extent to which this conversion occurs often depends on the age of the plant, particular variety or species, and geographical source, and is an important factor in determining the ultimate use and pharmacological properties of the plant. Other secondary metabolites detected in Hyoscyamus plants are: flavonoids (rutin), chlorogenic acid, coumarins, tannins.
Pelt et al. reported that the main tropane alkaloid of the H. reticulatus plant is hyoscyamine in the range from 0.033 to 0.056% dry weight (DW), followed by less scopolamine from 0.011 to 0.015% DW, depending on the strain and the age of the plant and growth conditions. In our investigation with the H. reticulatus plant, we have determined the content of hyoscyamine and scopolamine during different growth periods – the plant with ten leaves, plant before flowering and plant during seed formation. The content of both alkaloids in the leaves and roots is maximal before the flowering stage, and maximal flower scopolamine content is before flowering. The hyoscyamine and scopolamine contents in the leaves are 0.631 and 0.320% DW, respectively (plant before flowering), whereas the production in the intact roots at the same time is even four times less. The changes in the content of both main alkaloids in H. reticulatus were greater in the leaves than in the roots during different growth periods. In contrast to the data of Pelt et al., the maximum hyoscyamine and scopolamine yields (11 and 20 times higher, respectively) were found in the leaves before flowering. In addition, apoatropine and littorine were detected. The roots produced trace amounts of hygrine, tropine and cuscohygrine.
Hyoscyamus reticulatus L.: 3Conclusion
We discussed the approaches applied to establish high producing lines of H. reticulatus-transformed roots that might meet the productivity requirements for a commercial process. In our studies with H. reticulatus, it was shown that in tropane alkaloid production, the Agrobacterium-mediated transformation may be favorable for the full expression of potential biosynthetic capacity. In a screening program, a number of cell lines are set up and the production of the compounds of interest was determined. The best producing lines were selected. Transformation with different Agrobacterium strains might have an effect, not only on total alkaloid content, but also on the ratio of the alkaloids. The effects of two natural agents (colchicine and glycyrrhizin) in the regulation of tropane alkaloid biosynthesis were demonstrated. The influence of saponins on the release of tropane alkaloids in culture medium maybe applicable to a continuous culture system for alkaloid production. Lines capable of exporting large amounts of alkaloids into the medium offer the best operational system for large-scale production.
Selections from the book: “Medicinal and Aromatic Plants XII” (2002).