Scopolamine: a journey from the field to clinics
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- Ullrich, S.F., Hagels, H. & Kayser, O. Phytochem Rev (2017) 16: 333. doi:10.1007/s11101-016-9477-x
Tropane alkaloids are present in many different plants of the Solanaceae family and widely known for their anticholinergic properties. Among them, most valued and increasingly demanded is scopolamine, also known under the name of hyoscine, which is used as pharmaceutical active substance in the treatment of postoperative nausea and vomiting, motion sickness and gastrointestinal, renal and biliary spasms for instance. It naturally occurs in various plant genera, e.g. Anisodus, Anthocercis, Atropa, Brugmansia, Datura, Duboisia, Hyoscyamus, Mandragora and Scopolia and the purified substance has a long history of use dating back to the nineteenth century. Until today, the supply in scopolamine is mainly covered by large scale field plant cultivation of hybrids between Duboisia myoporoides and Duboisia leichhardtii. Biotechnological approaches optimising the alkaloid biosynthesis, for example the use of callus cultures or genetically transformed hairy root cultures, are not competitive by now. The aim of this review is to give a comprehensive overview regarding the current knowledge on botanical origin, pharmacology, biosynthesis as well as agricultural and biotechnological production of scopolamine.
KeywordsAnticholinergics Hyoscine Solanaceae Tropane alkaloids
Central nervous system
Cytochrome P450 80F1
- E. coli
Hyoscyamine 6β-hydroxylase (enzyme/gene)
Muscarinic acetylcholine receptors
Putrescine N-methyltransferase (enzyme/gene)
- S. cerevisiae
Tropinone-reductase I (enzyme/gene)
Tropinone-reductase I (enzyme/gene)
Tropane alkaloids have been medicinally used for centuries due to their anticholinergic properties and are of great significance until today. They are chemically classified by their bicyclic tropane ring (N-methyl-8-azabicyclo [3.2.1] octane), which is characteristic for a group of approximately 200 compounds naturally occurring in mostly solanaceous plants, including scopolamine, hyoscyamine or cocaine (Grynkiewicz and Gadzikowska 2008). One widely used substances among them is scopolamine, also known under the name of hyoscine, which acts as competitive antagonist at muscarinic acetylcholine receptors and thereby exhibits a parasympatholytic effect (Palazón et al. 2008). Its commercial demand is assessed about tenfold higher than for hyoscyamine due to its fewer adverse effects and higher physiological activity (Palazón et al. 2003; Yun et al. 1992; Zhang et al. 2004). It is a more powerful mydriatic and suppressant of salivation than hyoscyamine, shows weaker spasmolytic effects and possesses central depressing effects already at low therapeutic doses, which can be used for example in the treatment of motion sickness (EFSA 2013; Finkel et al. 2009). Moreover, it is used as substrate for semisynthetic drugs like tiotropium bromide or scopolamine-N-butyl bromide. That is why there is a longstanding interest to increase the scopolamine production using large scale plant cultivation or different biotechnological approaches. Until today, its demand is continuing, as scopolamine and its derivatives like N-butyl scopolamine are pharmaceutically applied in many different therapeutic areas, e.g. as antiemetics or spasmolytics, and are still expanded to further applications, e.g. Clozapine-induced hypersalivation (Takeuchi et al. 2015). Moreover, other classes of compounds that can be substituted for these plant derived drugs are lacking (Oksman-Caldentey 2007). In order to further optimize the scopolamine production, an extensive knowledge on its botanical sources, biosynthetic pathway as well as agricultural and biotechnological production is required. This review gives a comprehensive overview on botanical origin, biosynthesis and medicinal applications focusing especially on the production of scopolamine.
Within the subfamily Solanoideae, it has been identified in Solandra (Evans et al. 1972), in Latua, (Munoz and Casale 2003) and in Mandragora (Jackson and Berry 1979). Referring to the tribe Datureae, it has been reported to be present in Datura (Evans and Wellendorf 1959) and Brugmansia (Griffin and Lin 2000). It is also occurring in Iochroma, tribe Iochrominae (Berger 2011) as well as in Lycium, tribe Lycieae (Rätsch 2012). Furthermore, plant genera belonging to the tribe Hyoscyameae, namely Scopolia (Samoryadov and Minina 1971), Hyoscyamus, Physochlaina, Przewalskia (Griffin and Lin 2000), Atropa (Phillipson and Handa 1976), Atrophante (Ripperger 1995) and Anisodus (Kai et al. 2007) are all known to contain scopolamine. Besides, Duboisia (Hills et al. 1953), Anthocercis, Cyphanthera, Crenidium (El Imam and Evan 1984), Anthotroche (Evans and Ramsey 1981), Symonanthus and Grammosolen (Evans and Ramsey 1983) of the tribe Anthocercideae all belong to the scopolamine-containing solanaceous plants. Apart from the family of Solanaceae, scopolamine has been identified in Benthamia alyxifolia within the Loranthaceae living as a parasite on Duboisia myoporoides and thereby accumulating scopolamine in its leaves (Rätsch 2012).
Until the nineteenth century, scopolamine was not separated as pure substance, but applied in extracts and tinctures containing a mixture of the plant-derived anticholinergics. The first record on the medicinal use of scopolamine and its derivatives can be traced back to the ancient Egyptians in 1500 BC. The use of one of its parent plants, namely Hyoscyamus, is described in the Ebers Papyrus, one of the oldest preserved medical documents. Therein, it is recommended in the treatment of “magic in the belly”, probably signifying colic and abdominal pain (Gyermek 1997; Thearle and Pearn 1982). Hereinafter, herbal preparations, which are nowadays known to contain Thearle and Pearn 1982scopolamine, were listed in several herbal books, e.g. Hyoscyamus in “De Materia Medica” of Dioscorides or Gerarde’s “The Herball or Generall Historie of Plantes” (Gerarde 1597; Osbaldeston 2000). Hyoscyamus, also known as Henbane and widespread in different regions all over the world, was also used as a potent ingredient in the various midnight brews and ointments prepared by witches in the Middle Ages (Carter 2003).
Datura species are largely distributed in warm climates all over the world (Griffin and Lin 2000) and have been used for their hallucinogenic properties for a long time. Their geographical origin is not fully elucidated yet (Rätsch 2012). It is assumed that Datura plants, also known under the name of thornapple, devil’s trumpet or Jimpson weed, were brought to Europe around 1500 AD, where they were utilized as potions by medieval witches at that time (Perger 1864; Thearle and Pearn 1982). They have also been applied by the Jivaroan people of Brazil (Thearle and Pearn 1982) and can be assigned to the Hindu god Shiva to whom the thornapples are provided as small offerings until today (Soni et al. 2012).
Different plant parts of Atropa, indigenous to Europe, North Africa and Western Asia, have been traditionally applied for ages for diverse purposes, e.g. the treatment of arthritis and muscle spasms as well as acute infections, asthma, colitis and many more (Godara et al. 2014; Nisar et al. 2013). Moreover, extracts of Atropa belladonna, commonly called belladonna or deadly nightshade, have also been used as poison already during the Roman empire (Thearle and Pearn 1982).
Scopolia, distributed in Europe as well as in East Asia, was used as mydriatic in the nineteenth century by applying an alkaloid-mixture called “Scopolein” extracted from its roots (Schmidt and Henschke 1888). Brugmansia has been cultivated by indigenous people in Central and South America until today due to its hallucinogenic, narcotic and medicinal properties. It is applied externally for broken bones, swellings and also in case of stomach or arthritic pain. Moreover, it is taken orally in case of pain of childbirth, untreatable illness or severe accidents, probably due to its narcotic properties (Shepard 2009). Solandra, endemic to South America, is associated with the Huichole god of wind and sorcery Kieri Tewiari and has been used by the Huichol of Mexico as a hallucinogenic agent to date (Knab 1977). Latua, native to Chile, is still relatively unknown compared to other plant genera and is mainly used by shamans and sorcerers on-site because of its ability to produce delirium, hallucinations and trance-like states (Plowman et al. 1971). Mandragora plants have been traditionally applied for medicinal purposes for a long time and are related to various myths due to their hallucinogenic effects. For instance Joan of Arc, burned as a heretic in 1431, was accused of the witches’ crime to own Mandrake among other things (Ungricht et al. 1998). Duboisia, endemic to Australia and New Caledonia, has been historically used by the Aborigines as a fish poison added to a billabong and, similar to Scopolia, its extract was used by ophthalmologists due to its mydriatic properties already in the 19th century (Foley 2006). All exemplary listed types of application can be traced back to the pharmacological properties of scopolamine and related tropane alkaloids.
Contemporary therapeutical use
Mechanism of action
Scopolamine is inhibiting the activity of the muscarinic acetylcholine receptors (mAChR). The mAChRs have widespread and diverse functions in the peripheral and in the central nervous system, e.g. the regulation of heart rate, smooth muscle contraction and glandular secretion (Bymaster et al. 2003). Five subtypes of the mAChRs exist, namely M1-M5 (Staskin and Zoltan 2007). M1 receptors are located in the CNS, in autonomic ganglia and glands. M2 receptors are widely expressed in the CNS as well as in heart, smooth muscle and autonomic nerve terminals. M3 receptors are also to be found in the CNS and present in smooth muscle and heart. M4 receptors are predominantly located in the forebrain, M5 receptors at low levels in the CNS and the periphery (EFSA 2013). Scopolamine acts at all five muscarinic receptor subtypes with relatively equal potency (Witkin et al. 2014). M1–M5 are coupled to heterotrimeric G-proteins and grouped based on the intracellular signalling pathway activated by ligand binding (Strang et al. 2010). The signaling pathways of M1, M3 and M5 are associated with the Gq/11 protein family and consist of an activation of the phospholipase C. M2 and M4 are linked with the Gi/o protein family and inhibit the adenylyl cyclase activity (Caulfield and Birdsall 1998; Volpicelli and Levey 2004). By blocking the parasympathetic system, many different physiological effects are observed, which can be pharmaceutically used. The main anti-muscarinergic effects consist of a decreased production of salivary, bronchial and glandular secretions, mydriasis, cycloplegia, increased heart rate, inhibited micturition, reduced gastrointestinal tone and gastric acid secretion.
Doses in (-)-scopolamine vary according to the indication with a maximum oral daily dose of 1.2 mg (Corallo et al. 2009). The pharmacokinetic parameters are largely dependent on the dosage form. In case of oral administration, the bioavailability is limited ranging between 3 and 27 %. Maximum plasma levels are to be found 23.5 ± 8.2 min after oral intake. Regarding ocular administration, scopolamine is rapidly, efficiently and systemically absorbed. In case of transdermal application, the systemic availability is comparable to an intravenous infusion suggesting a high transdermal bioavailability. Data concerning metabolism and renal excretion are limited, glucuronide or sulfate conjugation are supposed to be involved in biotransformation (Renner et al. 2005). An urinary excretion of ca. 30 % parent compound in addition to Phase II conjugates is observed within 24 h after oral administration (EFSA 2013).
Toxic doses of (-)-scopolamine cause restlessness, disorientation, hallucinations and delirium due to CNS stimulation. In the event of an overdose, CNS stimulation is followed by central depression and death through respiratory paralysis. The lethal dose is estimated to be about 100 mg (-)-scopolamine (EFSA 2013). Intoxications with scopolamine mainly occur in the following categories: unintended ingestions, intended ingestions of plant material and poisoning due to its abuse (Beyer et al. 2009). Unintended ingestions are mostly due to contamination of food, e.g. buckwheat or corn, and via intake of plant parts of Brugmansia, Datura or of Atropa, as for example the fruits of Atropa belladonna are sometimes confused with blackberries and the seeds of Datura stramonium are mixed up with those of poppy (Adamse and van Egmond 2010; Koleva et al. 2012). In Germany, Brugmansia belongs to the four plant genera leading to the most severe poisonings by botanicals in children (Pietsch et al. 2009).
In different regions all over the world, the seeds or flowers of Datura are intentionally taken by adolescents and young adults as a hallucinogenic agent leading to severe intoxications (Hall et al. 1977; Kintz et al. 2006). The same applies to Brugmansia, native to South America and spread over different regions worldwide, which is also frequently abused by adolescent recreational drug users (Kim et al. 2014).
Abuse as “truth serum”
Early in the twentieth century, medical doctors began to employ scopolamine, along with morphine and chloroform, as an agent for easing the pain of childbirth, inducing the so-called “twilight sleep” (Foley 2006). This sleep was characterized not only by a state of anaesthesia, but also by complete amnesia, in which the expectant mother lost all memories of the birth process. Moreover, doctors remarked that women in twilight sleep became talkative and were able to answer their questions accurately without remembering it later (Bimmerle 1993). In 1922, Robert House, an American obstetrician experienced with the usage of scopolamine in child birth, suggested a similar technique to be employed in the interrogation of suspected criminals. He also started to use scopolamine in the questioning of prisoners to show its potential in criminal investigations, published his results and thereby became famous as the “father of truth serum” (Geis 1959). But only a few cases in which scopolamine was used for police interrogation came to public notice (Bimmerle 1993). However, it is known that scopolamine was abused as “chemical straightjacket” in the former German Democratic Republic until the 1980s (Rätsch 2012).
These days, scopolamine derived from Brugmansia, commonly labelled Devil’s breath in this context, is widely abused by criminals in South America in order to render their victims unconscious and rob them. In Columbia for instance, unofficial data estimate scopolamine incidents to be at approximately 50,000 per year (OSAC 2014).
Plant biosynthetic pathway
In order to further elucidate the tropane alkaloid pathway, new approaches are currently under way, e.g. the use of isotope ratio monitoring by 13C NMR spectrometry. Romek et al. successfully showed that N-methylpyrrolinium, a precuroser of scopolamine as well as of nicotine, introduces similar isotope distribution patterns in the two target compounds, whereas the other atoms of both alkaloids, being of different origins, reflect their specific metabolic origin (Romek et al. 2016). Prospectively, those measured 13C distribution patterns can be targeted used in order to clarify aspects of enzymatic reactions still to be identified, as position-specific observations allow deductions as to the putative reaction mechanism involved.
Structure elucidation and full chemical synthesis
Scopolamine was first isolated from and named after Scopolia, native in Europe and Asia, by Ernst Schmidt and Hermann Henschke in 1888 (Schmidt 1892; Schmidt and Henschke 1888). A few years later, Willstätter was the first scientist who completely elucidated the structure of the tropane alkaloid ring and developed a chemical synthesis of tropine providing the possibility to produce hyoscyamine (Willstätter 1901). In 1917, Robinson published a short method for the synthesis of tropinone by using succinaldehyde, methylamine and an acetonedicarboxylic acid calcium salt, a proposal, which is of great significance to date (Humphrey and O’Hagan 2001; Robinson 1917). Based on Robinson’s synthesis of tropinone, it was supposed that the in vivo reactions could be similar to the chemical synthesis, which led to further progress in the elucidation of the tropane alkaloid pathway, exemplified in the following with ornithine and arginine. Leete showed the non-proteinogenic amino acid ornithine, as an equivalent to succinaldehyde, to be involved in the biosynthesis of the tropane skeleton by feeding Datura stramonium plants with radioactive labelled [2-14C] ornithine (Leete et al. 1954). As the metabolism of arginine is closely related to ornithine, it has also been proved to take part in the tropane alkaloid metabolism as a precursor (Walton et al. 1990). Robins demonstrated that arginine mainly contributes to the production of tropane alkaloids and that the responsible enzymes for the conversion of arginine and ornithine, namely the OrnDC and ArgDC, interact with each other (Robins et al. 1991).
The correct structure of scopolamine (Fig. 1) was first postulated by Gadamer and Hammer in 1921, but could not be reliably verified at that time (Gadamer and Hammer 1921). In 1923, Willstätter and Berner suggested four potential projection formulas of scopolamine (Willstätter and Berner 1923). Thirty years later, in 1953, Fodor and Kovács suggested the piperidine ring in the chair form, in analogy to Meinwald (Fodor and Kovács 1953; Meinwald 1953). In 1959, the first total synthesis of scopolamine was published by Dobó et al. starting from tropane-3α,6β-diol via 3α-Acetoxytrop-6-ene to 3α-acetoxy-6β,7β-epoxytropane, the latter acylated with O-acetyltropoyl chloride and hydrolysed to scopolamine (Dobo et al. 1959). Recently, a second approach to synthetically produce scopolamine was published by Nocquet and Opatz in a nine step reaction with 6,7-dehydrotropine as key intermediate including two different approaches to form the tropane skeleton with similar yields. The last reaction step, the chemoselective epoxidation of the 6,7-double bond, proved to be the limiting step of the full synthesis with only 16 % yield. Nevertheless, since higher yield was reported for related substrates, various possibilities for the optimisation of the last reaction step exist and need to be further investigated (Nocquet and Opatz 2016). Until today, the chemical synthesis does not seem to be competitive to direct extraction of plant material, as the synthetic routes are still expensive, low yielding and include too many reaction steps (Kai et al. 2007).
Overseas plantations of Duboisia species have also been started in other regions like India or South America (Mangathayaru 2013; Williams 2013). Related plant genera like Atropa spp. or Datura spp. are not used for commercial production due to their lower content in scopolamine (0.2-0.8 % compared to 2-4 % of total alkaloids in Duboisia spp.) (Grynkiewicz and Gadzikowska 2008). Main global producers providing plant material are Boehringer Ingelheim Pty Limited as well as Alkaloids of Australia Pty Limited, both located in Kingaroy, Australia, and Alkaloids Corporation in Calcutta, India. Also involved in the further processing to produce pure scopolamine are the Fine Chemicals Corperation in Cape Town, South Africa, and Phytex Australia Pty Ltd in Sydney, Australia.
Rosenblum already stated in 1954 that in the field various environmental factors, e.g. the daily exposure to sunlight and the soil composition, are able to mask genetic characteristics (Rosenblum 1954). This makes it difficult to get satisfactory results in field trials, especially by comparing plants grown in different geographical regions. However, in the past decades some field trials were carried out on Duboisia and related plants. In Queensland, Australia, Luanratana and Griffin observed the alkaloid content to decrease in autumn and winter in field grown Duboisia plants and assumed the low temperatures to be responsible for the decreased amount in tropane alkaloids (Luanratana and Griffin 1980b). As seasonal variations have also been observed in Duboisia hybrids grown in Japan (Ikenaga et al. 1985), plants were cultivated under controlled temperature in greenhouses in Thailand and showed stable scopolamine contents (Luanratana et al. 1990). Until now, no systematic assessment of the impact of individual climate factors as temperature, humidity, light intensity and duration on the alkaloid biosynthesis and biomass production has been published.
Not only the climate, but also the soil composition and fertilization have an influence on the alkaloid biosynthesis and plant development in Duboisia and related plant genera. In outdoor grown Duboisia plants in Queensland, Australia, Luanratana and Griffin observed no correlation between alkaloid yield and fertilization with nitrogen, potassium and sulfur (Luanratana and Griffin 1980b). Field trials in Saudi Arabia with Datura innoxia using Sangral® compound fertiliser showed increased scopolamine and hyoscyamine levels up to a concentration of 600 kg/ha, decreasing again at higher amounts (Al-Humaid 2004). By harvesting five Hyoscyamus species originating from different geographical origins in Iran including soil analysis, Nejadhabibvash et al. demonstrated positive correlations between N, P, K, Ca2+ and yield in scopolamine and hyoscyamine (Nejadhabibvash et al. 2012).
In contrast to field trials, experimental set-ups using hydroponic culture under glasshouse conditions allow a better differentiation regarding the specific effects of individual nutrients on alkaloid and biomass production, which will be specified in the following section. Referring to nitrogen, contradictory results have been published (Alaghemand et al. 2013; Luanratana and Griffin 1980a). Luanratana and Griffin observed a decrease in total alkaloids at higher nitrogen levels in case of Duboisia plants in contrast to Alaghemand measuring the highest content in scopolamine and hysocyamine with increased nitrogen concentrations using Hyoscyamus plants. Smolenski described a reduced biomass production and an elevated yield of total nitrogen and alkaloids, as the ratio of calcium to potassium was increased in Atropa plants (Smolenski et al. 1967). Moreover, Luanratana observed higher potassium levels leading to a significant increase in the percentage of scopolamine in hydroponically cultivated Duboisia plants (Luanratana and Griffin 1980a).
The previously described experiments show that there is still a lot of potential in order to improve the large scale cultivation of scopolamine producing plants. In a first step, systematic trials under controlled, reproducible conditions should be conducted in order to eliminate environmental influences that cannot be clearly allocated. Secondly, the knowledge gained by those trials could be transferred to field-grown plants and tested within specific field trials.
Callus and cell cultures
In 1957, West and Mika were the first scientists using callus cultures of Atropa belladonna instead of intact plants in order to verify the site of tropane alkaloid biosynthesis and thereby detecting atropine in root callus (West and Mika 1957). From now on, many different approaches were applied in order to use callus or suspension cultures for the targeted production of tropane alkaloids. The first publications showed the alkaloids in undifferentiated cultures to be low concentrated or hardly present compared to intact plants (Staba and Jindra 1968; Stohs 1969; Tabata et al. 1972). Endo and Yamada successfully demonstrated the production of tropane alkaloids in non-transformed roots derived from callus cultures of Duboisia. Interestingly, no alkaloids could be detected in the callus cultures used for differentiation indicating that the alkaloid production in Duboisia species is associated with organogenesis of roots (Endo and Yasuyuku 1985). This is most likely due to the specific location of key enzymes, e.g. the pericycle-specific location of the PMT and H6H (Hashimoto et al. 1991; Suzuki et al. 1999). Since the roots are supposed to be the main site of tropane alkaloid biosynthesis, from then on the focus was placed on in vitro root cultures (Parr 1989).
Hairy root cultures
During the eighties, Agrobacterium rhizogenes- transformed root cultures of many different plant genera (Atropa, Datura, Duboisia, Hyoscyamus, Scopolia) were used in order to further improve the production of hyoscyamine and/or scopolamine (Jaziri et al. 1988; Kamada et al. 1986; Mano et al. 1986, 1989). A. rhizogenes induces hairy root disease marked by extensive proliferation of roots originating from an infected plant wound (White and Nester 1980). The hairy root phenotype is generally characterized by fast growth and genetic stability in contrast to conventional root cultures. Furthermore, the secondary metabolites synthesized by hairy roots are comparable to those of in intact parent roots and found in similar or even higher amounts (Sevón and Oksman-Caldentey 2002). By using hyoscyamine-producing hairy root cultures of Datura stramonium, Payne et al. were also able to show that those cultures preserve the biosynthetic capacity of their mother plant and that formation of the desired products is less susceptible to manipulation than in callus or cell suspension cultures (Payne et al. 1987).
Many different cultivation parameters were optimized including culture media, pH-value, carbon source, nutrient concentrations and the use of biotic and abiotic elicitors, such as methyl jasmonate, chitosan, salicylic acid or silver nitrate improving the scopolamine production (Dupraz et al. 1994; Palazón et al. 2008; Pitta-Alavarez and Giulietti 1995; Pitta-Alvarez and Giulietti 1999; Pitta–Alvarez et al. 2000). The use of the wound stress hormone methyl jasmonate is still of current interest, as its stimulation of the tropane alkaloid biosynthesis is not fully understood and seems to be species dependent (Ryan et al. 2015). Previous studies reported a lack of jasmonate stimulation of tropane alkaloid biosynthesis in Atropa belladonna or Hyoscyamus muticus (Biondi et al. 2000; Suzuki et al. 1999), whereas methyl jasmonate-induction was successful in cultured roots of Datura stramonium (Zabetakis et al. 1999). Furthermore, systematic and repeated selection of promising lines for further culturing was applied and the influence of somaclonal variation was evaluated systematically (Maldonado-Mendoza et al. 1993; Sevón et al. 1998). Subroto et al. also tested the co-culture of Atropa belladonna shooty teratomas and hairy roots using hormone-free medium, thereby mimicking the whole plant by providing the possibility for localized metabolite synthesis as well as for transportation of compounds between organs. This led to a 3-11 times increased accumulation in scopolamine compared to the average levels found in leaves of intact plants (Subroto et al. 1996). Nevertheless, no breakthrough in the biotechnological production of tropane alkaloids was achieved by using hairy root cultures, which is shown using the following examples. In Duboisia myoporoides hairy root cultures the average content in scopolamine was significantly increased compared to the parent lines after several selections (3.2 % per dry weight compared to 0.15 %/dw), but at the same time the growth rate was decreased (Yukimune et al. 1994). Decreased growth combined with high alkaloid production and vice versa was also observed by Sauerwein and Shimomura using hairy roots of Hyoscyamus albus for testing different media and sucrose concentrations (Sauerwein and Shimomura 1991). And even though the alkaloid content in Datura candida hairy root cultures was 1.6- and 2.6-fold higher (up to 0.68 %/dw) compared to the aerial parts and roots of the parent plants, this is still not competitive with the commercially grown Duboisia hybrids containing up to 4 % of tropane alkaloids in their dried leaves (Christen et al. 1989; Grynkiewicz and Gadzikowska 2008). Moreover, one major restriction for the commercial production of scopolamine by hairy root cultures remains the scale up to an industrial level. Different bioreactor systems have been tested so far, including modified airlift and stirred tanks for Datura metel and Brugmansia candida, connective flow reactors for Hyoscyamus muticus as well as bubble-column and spray bioreactors for Hyoscyamus niger and Scopolia parviflora, and efforts to synthesize scopolamine in bioreactor systems appropriate for large scale production are still ongoing (Cardillo et al. 2010; Carvalho and Curtis 1998; Cusido et al. 1999; Jaremicz et al. 2014; Min et al. 2007).
Overexpression of the pmt gene
In 2001, Sato et al. showed transgenic hairy root clones of Atropa belladonna with a fivefold increased pmt transcript level to have quantitatively as well as qualitatively similar alkaloid profiles compared to the wild type (Sato et al. 2001). This indicates that the overexpression of the PMT enzyme might be not sufficient to boost the tropane alkaloid synthesis later in the biosynthetic pathway. In 2002, Moyano et al. introduced the pmt gene in Duboisia hybrids and found the N-methylputrescine levels of the resulting hairy roots to be 2–fourfold higher compared to wild type roots, but again no significant increase in tropane alkaloids (Moyano et al. 2002). Only shortly thereafter, Moyano et al. genetically engineered hairy root cultures of Datura metel and Hyoscyamus muticus by overexpressing the pmt gene and observed a more rapid ageing, growth reduction as well as an accumulation of higher amounts of tropane alkaloids than in control hairy roots. Thereby, hyoscyamine and scopolamine production were both increased in hairy root cultures of Datura, whereas in Hyoscyamus only hyoscyamine was found in higher amounts compared to controls (Moyano et al. 2003). In 2005, the overexpression of the pmt gene in Scopolia parviflora improved its production of hyoscyamine and scopolamine. However, the yields were still only comparable to or even lower than those synthesized by other species in the absence of such transformation (Lee et al. 2005). Furthermore, the pmt gene has been introduced in Hyoscyamus niger, thereby showing a significant increase in PMT activity and more than fivefold higher contents of N-methylputrescine compared to wild type hairy roots, but no increase in tropane alkaloids. Zhang et al. also demonstrated that the exposure of the roots to the elicitor methyl jasmonate positively affects both polyamine and tropane biosynthetic pathways in Hyoscyamus (Zhang et al. 2007). Those data already show that the same biosynthetic pathway in related plant species might be differently regulated. Moreover, the transgene allows bypassing of the endogenous control of metabolic flux to the alkaloids including metabolic changes not directly related to the transgene presence. Furthermore, most pathways do not have a single rate-limiting step, but the flux is controlled by multiple enzymes and feedback inhibition by the end-product (Kholodenko et al. 1998). This makes it difficult to predict the effect of the overexpression of single genes.
Overexpression of the tr-1 gene
The tropinone reductases TR-1 and TR-2 are another branch point within the biosynthesis of scopolamine. The TR-1 is thereby responsible for the biosynthesis of hyoscyamine and scopolamine via tropine, whereas the TR-2 catalyses the formation of calystegines via pseudotropine. Richter et al. showed that overexpression of the tr-1 or tr-2 gene in Atropa belladonna led to a higher enzyme activity and an increase in the respective enzyme products pseudotropine or tropine. Moreover, high pseudotropine levels resulted in an accumulation of calystegines in the roots and a high expression of the tr-1 increased hyoscyamine by a factor of three and scopolamine by a factor of five compared to controls (Richter et al. 2005).
Overexpression of the h6h gene
The hyoscyamine 6β-hydroxylase (H6H) is the last enzyme of the tropane alkaloid pathway which is needed in order to convert hyoscyamine into scopolamine and has been main target of transgenic approaches in order to improve the scopolamine biosynthesis in hairy root cultures and regenerated plants. In 1992, Yun et al. used hyoscyamine-rich Atropa belladonna plants for the overexpression of the h6h gene from Hyoscyamus niger. The single primary transformed plant and its subsequent generations almost exclusively stored scopolamine in the aerial plant parts indicating an nearby complete conversion of hyoscyamine to scopolamine (Yun et al. 1992). Shortly afterwards, Hashimoto et al. successfully engineered Atropa belladonna hairy roots using the h6h gene of Hyoscyamus niger. Thereby, increased amounts and high enzyme activities of the H6H were detected in the engineered hairy roots, which contained up to fivefold higher levels in scopolamine and elevated amounts of its direct precursor 6β-hydroxy-hyoscyamine compared to the wild type (Hashimoto et al. 1993b). Hyoscyamus muticus hairy roots overexpressing the same gene contained up to a 100-fold increased amounts in scopolamine compared to controls, but hyoscyamine still remained the main alkaloid indicating an incomplete conversion to the target substance scopolamine. Moreover, a large variation in the tropane alkaloid pattern was observed among the 43 positive clones with only 22 of these clones showing elevated levels of scopolamine, probably depending on the h6h expression level (Jouhikainen et al. 1999). The h6h gene was also introduced into the genome of a Duboisia hybrid rich in scopolamine. The resulting engineered hairy root lines contained increased amounts in scopolamine up to a factor of three with regard to the wild type hairy roots, but there was no significant enhancement in scopolamine production in the engineered regenerated plants compared to controls (Palazón et al. 2003). Rahman et al. used the Duboisia leichhardtii for the overexpression of the h6h, which led to high variations among the h6h gene positive clones. In case of clone 117 the conversion of hyoscyamine to scopolamine was almost complete with a rate of more than 95 %, whereas only half as much scopolamine than hyoscyamine was produced by clone 16 (Rahman et al. 2006). Transgenic hairy roots of Atropa baetica overexpressing the h6h showed hyoscyamine to be entirely converted into scopolamine. In the best clone, scopolamine accumulated in ninefold higher amounts compared to wild type plants (Zárate et al. 2006). Moreover, Moyano et al. demonstrated that plant cells, which do not naturally produce secondary compounds of interest, are able to do so after overexpression of the responsible gene and feeding with a suitable precursor. They used dedifferentiated root cultures of Nicotiana tabacum carrying the 35S-h6h gene from Hyoscyamus niger and successfully showed these cell cultures to be able to bioconvert 18 % of exogenous hyoscyamine into scopolamine using a 5 l turbine stirred tank bioreactor (Moyano et al. 2007).
However, apart from the high variations in alkaloid production and conversion rate of hyoscyamine to scopolamine which is to be found within the previous examples, also morphological anomalies were detected, both making it difficult to achieve a stable production of scopolamine. And even though hyoscyamine-rich plants, such as Hyoscyamus or Atropa, were actually converted into a potential source of scopolamine by significantly increasing its production, the amounts in scopolamine obtained so far are still too low for commercial application.
Engineering two steps
Besides the expression of single genes, the approach of multiple gene expression was pursued in order to further enhance the scopolamine production. This is illustrated within the examples given in the following. In 2004, the pmt and h6h gene were introduced and overexpressed in transgenic Hyoscyamus niger hairy root cultures. Transgenic hairy root lines overexpressing both genes accumulated significantly higher amounts of scopolamine (up to 411 mg/l) compared to the wild type and single gene transgenic lines overexpressing the pmt or the h6h gene (Zhang et al. 2004). This indicates that transgenic hairy roots overexpressing both pmt and h6h gene may have an enhanced flux in the tropane alkaloid biosynthetic pathway that increases the yield in scopolamine, thereby being more effective than plants hosting only one of the two genes. This was also shown by overexpressing pmt and tr-1 gene in Anisodus acutangulus producing significantly higher levels of tropane alkaloids compared to the wild type and single gene transformed lines. But as the H6H responsible for the conversion of hyoscyamine to scopolamine was not overexpressed, hyoscyamine remained the major alkaloid being only transformed to scopolamine on a small scale (Kai et al. 2011b). A simultaneous expression of the tr-1 and h6h gene in Anisodus acutangulus again led to a significant increase in tropane alkaloids compared to controls and single gene transformed lines. The levels in hyoscyamine, 6β–hydroxy–hyoscyamine (anisodamine) and α-hydroxyscopolamine (anisodine) could be significantly increased as well as those of scopolamine; however, the latter was still low concentrated compared to its precursors (Kai et al. 2012). As the examples show, the effect of overexpressing two enzymes at one time is difficult to predict and also undesired reactions might be favoured by that, for example the conversion from scopolamine to α-hydroxyscopolamine, a reaction probably catalysed by an unknown hydroxylase. Still, a co-overexpression of multiple biosynthetic enzymes might be a promising strategy, including the PMT responsible for the first committed step in the biosynthesis, the TR-1 as branch-controlling enzyme and the metabolically downstream enzyme H6H to the target substance scopolamine.
Another approach was the use of bacteria cultures, such as Escherichia coli, for the production of scopolamine. Hashimoto et al. already used E. coli in 1993 in order to characterize the H6H of Hyoscyamus niger by using a fusion protein with maltose-binding protein. Even though the expression level of the fusion protein was low, they were able to show that in case of a sufficient expression of the h6h gene, scopolamine was synthesized without substantial accumulation of its precursor 6β–hydroxy–hyoscyamine. In contrast to that, a limiting amount of h6h in relation to the supply of hyoscyamine led to an increase in 6β–hydroxy–hyoscyamine (Hashimoto et al. 1993a). The gene encoding H6H in Anisodus acutangulus was cloned by Kai et al. and expressed in E. coli by using a His-tag or GST-tag fusion protein. Hereby, the biofunctional assay with His-AaH6H and GST-AaH6H led to a conversion of hyoscyamine (at a concentration level of 40 mg/l) into scopolamine at 32 and 31 mg/l and 6β–hydroxy–hyoscyamine at 3.4 and 3.1 mg/l, respectively (Kai et al. 2011a). Only recently, random mutagenesis and site-directed saturation mutagenesis were applied for the enhancement of the hydroxylation activity of H6H from Anisodus acutangulus using E. coli. One double mutant, namely AaH6HM1(S14P/K97A), possessed a 3.4 times improved hydroxylation activity and an 2.3-fold enhanced in vivo epoxidation activity in comparison to the wild type enzyme. The total yield in scopolamine was 97 % (1.068 g) by using this mutant in a 5 l bioreactor (working volume: 3 l) with a space time yield of 251 mg/l/d (Cao et al. 2015). This is a considerable improvement compared to Hashimoto et al. and Kai et al. where scopolamine was produced from hyoscyamine with space time yields of 4.1 mg/l/d and 16 mg/l/d by cultivating recombinant E. coli cells in shake flasks, and shows that protein engineering in E. coli might be promising for further scale up. Not only Escherichia coli, but also Saccharomyces cerevisiae was applied as production organism for scopolamine. In this case, the h6h isolated from Brugmansia candida was used for functional expression, untagged and with a His-tag. The tagged enzyme converted hyoscyamine into 6β-hydroxy-hyoscyamine (35.7 % after 15 h of incubation), whereas the untagged protein was able to produce scopolamine as well as 6β-hydroxy-hyoscyamine (7.6 and 83.3 % respectively) (Cardillo et al. 2008). Even though S. cerevisiae has a long history of application in industry and combines the advantages of unicellular organisms with the ability of protein processing together with the lack of endotoxins, the yields in scopolamine are too low for commercial exploitation due to the incomplete conversion of hyoscyamine. Furthermore, the histidine tag fused to the protein and the epitope seems to reduce the capability of the enzyme to synthesize 6β-hydroxy-hyoscyamine and particularly scopolamine. If a large scale production of high amounts in scopolamine by microorganisms competitive to extraction from plant material will be possible in the future, remains questionable.
Doubled haploidy and polyploidization
The use of double haploid plants might also be one strategy towards homozygous lines, thereby improving breeding by the selection of lines showing enhanced biomass production and yielding high levels of the desired medicinal compounds. Microspore-derived embryos and haploid plants have already been successfully generated in case of Atropa belladonna and Hyoscyamus muticus (Chand and Basu 1998; Zenkteler 1971). However, compared to crops like wheat or barley, only little effort has been undertaken with regard to medicinal plants so far (Ferrie 2009). In order to successfully use this technique in breeding and cultivation, more work in this field of research has to be done.
Another promising strategy towards an enhanced scopolamine production will be the use of polyploid plants, which may differ in their cytological, biochemical, genetic and physiological character providing unique tolerances and developmental patterns. Within previous research, autopolyploids of many medicinal plants showed increased production of tropane alkaloids (Levin 1983). Lavania and Srivastava observed up to 22.5 % enhanced production of tropane alkaloids as well as higher fertility in artificial autotetraploids of Hyoscyamus niger (Lavania and Srivastava 1991). Only recently, Belabbassi et al. studied the effects of polyploidization in hairy roots of Datura stramonium revealing the content of hyoscyamine to be increased up to 276 % in tetraploid lines using salicylic acid as elicitor (Belabbassi et al. 2016). Dehghan et al. reported tetraploid plants of Hyoscyamus muticus to increase scopolamine production up to 200 % compared to their diploid counterparts, however, this did not apply to the corresponding induced hairy root cultures. Moreover, manipulation of the ploidy level and adaption of the culture conditions successfully shifted the scopolamine/hyoscyamine ratio towards scopolamine, even though hyoscyamine is known to be the main alkaloid Hyoscyamus (Dehghan et al. 2012).
These examples illustrate that it is worth to continue working with polyploid plants and to apply this knowledge also in order to optimise breeding in Duboisia hybrids towards improved production plants.
Conclusions and outlook
Solanaceous plants containing scopolamine have been medically used for centuries and scientific research on the active compounds including scopolamine started more than 100 years ago. Still, the global demand in scopolamine and its semisynthetic derivatives is increasing due to their various therapeutic applications, e.g. motion sickness or gastrointestinal spasms. Worldwide, many different botanical sources of scopolamine have been identified and characterized. From those plants, extracts of the aerial parts of field-grown hybrids of Duboisia are mainly used for its commercial production by today.
Alternatives to the agricultural production of scopolamine are being continued unabated. But the full chemical synthesis, being very complex and therefore quite expensive, is economically not feasible by now. Moreover, efforts using biotechnological approaches (genetically modified bacteria, plant cell cultures and transgenic plants) in order to optimize the output in scopolamine are ongoing. They are complicated by the complex interactions and various factors influencing the tropane alkaloid biosynthesis including biosynthetic steps which are not fully elucidated yet.
The metabolic engineering results achieved so far reveal that a thorough knowledge of all steps of the biosynthetic pathway is necessary including the sequences of the responsible enzymes and their regulation in order to optimize the biosynthesis in transgenic plants as well as in cell cultures towards the desired medicinal product scopolamine. Due to the high occurrence of multiple rate-limiting steps and the interaction of metabolic pathways and their metabolites involved, it is difficult to predict the results of overexpressing a single or multiple genes involved in the targeted pathway. Even though more and more knowledge is gained regarding the enzymes involved, their localisation and catalytic activities, there are still some bottlenecks to be found within the tropane alkaloid pathway. In order to fully elucidate the complete pathway, systematic research combining proteomics, transcriptomics and metabolomics and the use of metabolite correlation networks will be helpful. Besides, the selection of the most suitable host is crucial, as not only in cell cultures of the botanical sources of scopolamine like Duboisia or Hyoscyamus, but also in Echerichia coli or Saccharomyces cerevisiae cultures as well as plants like Nicotiana tabacum, a biotechnological production has been shown to be realizable. In addition, a stable and reproducible production system is required, which also needs to bring along the necessary characteristics for a scale-up to industrial levels for commercialisation. Until now, these requirements are not fully met and further studies are ongoing.
The research from the DISCO project leading to these results has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement 613513. This work is furthermore supported financially by Boehringer Ingelheim Pharma GmbH & Co. KG.. The authors are grateful to Andreas Rothauer for literature supply and proof reading of the manuscript.
|Funder Name||Grant Number||Funding Note|
|European Unions Seventh Framework Programme for research, technological development and demonstration|