Alkaloid N-oxides

N-oxidation products of alkaloids, particularly the N-oxides of tertiary alkaloids, are well-known laboratory products, easily prepared from the original base. As early as the 1920s quite extensive pharmacological and toxicological comparisons had been made of common alkaloids such as morphine, strychnine and hyoscyamine and their corresponding N-oxides. Some enthusiasm for the clinical use of N-oxides was engendered by their purported delayed-release properties, low toxicities and low addictive properties compared with the corresponding tertiary alkaloids.

Although the formation of N-oxides and other N-oxidation products of alkaloids in animal systems is well-known, forming part of the wider scheme for the metabolism of amines, the occurrence of such compounds in plants has, until relatively recently, received little attention. This was possibly due to a belief that such compounds represented artefacts arising during the extraction and work-up of tertiary alkaloids. Secondly, because of the high polarity, and water-solubility of alkaloid N-oxides, they were discarded by the normal alkaloid extraction procedures.

One group of alkaloids known to occur extensively as the natural N-oxides comprises the quinolizidines of the Boraginaceae, Compositae and Papilionaceae; these are alkaloids, including those of Senecio spp., which cause extensive liver damage in animals using plants containing them as fodder. A number of N-oxide alkaloids of the indole series have been isolated from plant materials, and among those of pharmaceutical significance are the simple hallucinogenic indole derivatives of Amanita spp., reserpine, strychnine, and some Mitragyna alkaloids. Fresh Atropa, Datura, Hyoscyamus, Scopolia and Mandragora each contain the two isomeric N-oxides of hyoscyamine.

One of the two possible N-oxides of hyoscine has been isolated from species of the first four genera above. Morphine and codeine N-oxides are natural constituents of the opium poppy latex, and Nicotiana spp. contain two isomeric nicotine N-oxides based on the pyrrolidine nitrogen. Some N-oxides—for example, aspergillic acid and iodinin (1,6-dihydroxyphenazine dioxide)—isolated from microorganisms, possess antibacterial activity.

As with the tertiary alkaloids themselves, there is little evidence to suggest what role the N-oxides may play in the plant’s metabolism. Ontogenetic studies of hyoscyamine N-oxide production in belladonna indicate a dynamic role for the N-oxide with a maximum build-up in the developing fruits. Oxidation–reduction involving N-oxides and tertiary bases is a probability. It has been suggested that N-oxides may be involved in demethylations and their participation in the biosynthesis of benzylisoquinoline alkaloids has also been proposed. The solubility properties of N-oxides could influence the transport of alkaloids both throughout the plant and also within the cell itself.

Process A

The powdered material is moistened with water and mixed with lime which combines with acids, tannins and other phenolic substances and sets free the alkaloids (if they exist in the plant as salts). Extraction is then carried out with organic solvents such as ether or petroleum spirit. The concentrated organic liquid is then shaken with aqueous acid and allowed to separate. Alkaloid salts are now in the aqueous liquid, while many impurities remain behind in the organic liquid.

Process B

The powdered material is extracted with water or aqueous alcohol containing dilute acid. Pigments and other unwanted materials are removed by shaking with chloroform or other organic solvents. The free alkaloids are then precipitated by the addition of excess sodium bicarbonate or ammonia and separated by filtration or by extraction with organic solvents.

Large-scale extractions based on the above principles are sometimes done in the field and the crude mixtures of alkaloids afterwards sent to a factory for separation and purification. This has been done for both cinchona and coca alkaloids in South America and Indonesia, the crude alkaloids then being sent to Europe, USA or Japan for purification. The separation and final purification of a mixture of alkaloids may sometimes be done by fractional precipitation or by fractional crystallization of salts such as oxalates, tartrates or picrates. Chromatographic methods are particularly suitable if the mixture is a complex one and if small quantities of alkaloids will suffice. Supercritical fluid extraction (Chapter 17), although not yet applied to many alkaloids, will probably become of increasing importance for these compounds.

Volatile liquid alkaloids such as nicotine and coniine are most conveniently isolated by distillation. An aqueous extract is made alkaline with caustic soda or sodium carbonate and the alkaloid distilled off in steam. Nicotine is an important insecticide, and large quantities of it are prepared from those parts of the tobacco plant which cannot be used for tobacco manufacture.

Tropane moiety

The available evidence suggests that the formation of the tropane ring system is generally similar for all Solanaceae studied but there are still apparent variations between species, particularly in the stereospecific incorporation of some precursors.

Early work with isotopes indicated that ornithine and acetate were precursors of the tropane nucleus; later, the incorporation of ornithine was shown to be stereospecific. Hygrine can also serve as a precursor of the tropane ring but is not now considered to lie on the principal pathway. The N-methyl group of the tropane system can be supplied by methionine and can be incorporated at a very early stage of biosynthesis, as demonstrated by the intact incorporation of N-methylornithine into hyoscine and hyoscyamine of Datura metel and D. stramonium. Early involvement of the N-methyl group was reinforced by the isolation in 1981 of naturally occurring δ-N-methylornithine from belladonna plants. Also supporting the stereospecificity of the ornithine incorporation was the work of McGaw and Woolley (Phytochemistry, 1982, 21, 2653) which showed that for D. meteloides the C-2 of hygrine was specifically incorporated into the C-3 of the tropine moiety of the isolated alkaloid. Putrescine (the symmetrical diamine formed by the decarboxylation of ornithine) and its N-methyl derivative also serve as precursors, which, taken in conjunction with the stereospecific incorporation of ornithine, makes it difficult to construct a single pathway for tropane ring formation. A scheme for the biogenesis of the tropane moiety, consistent with the above findings, is shown in Fig. 26.3.

Fig. 26.3 Possible biogenetic routes for tropine and pseudotropine (see text for additional comments).

Studies on the enzyme putrescine N-methyltransferase in cultured roots of Hyoscyamus albus support the role of this enzyme as the first committed enzyme specific to the biosynthesis of tropane alkaloids. (N. Hibi et al., Plant Physiol., 1992, 100, 826.)

It will be observed from Fig. 26.3 that the reduction of tropinone yields both tropine (3α-hydroxytropane) and pseudotropine (3β-hydroxytropane). These reductions are brought about by two independent tropinone reductases (EC 1.1.1.236), often referred to as TR-I and TR-II, which accept NADPH as coenzyme. After considerable research involving principally D. stramonium root cultures both enzymes were separately purified and characterized. Furthermore, cDNA clones coding for the two separate enzymes TR-I and TR-II have been isolated and shown to involve polypeptides containing 272 and 260 amino acids respectively. These clones were expressed in Escherichia coli and the same reductive specificity demonstrated as for the natural TRs isolated from plant material.

As indicated in Table 26.2 for solanaceous alkaloids, hydroxyls and ester groups are also common at C-6 and C-7 (R² and R³) of the tropane ring system. Current evidence suggests that hydroxylation of these carbons probably occurs after the C-3 hydroxyl has been esterified.

Esterification

The next stage in the biosynthesis of hyoscyamine, the esterification of tropine and tropic acid, has been demonstrated by feeding experiments and isolated enzymes. It was some 40 years ago that Kaçzkowski first recorded the presence of a hyoscyamine esterase in D. stramonium; later, Robins et al. (FEBS Lett., 1991, 292, 293) demonstrated the involvement of two acetyl-CoA-dependent acyltransferases in the respective formation of 3α- and 3β-acetoxytropanes in D. stramonium-transformed root cultures.

Acid moiety

The tropic acid fragment of hyoscine and hyoscyamine is derived from phenylalanine, as is the α-hydroxy-β-phenylpropionic acid (phenyllactic acid) of the tropane alkaloid littorine. The specific incorporations obtained with phenylalanine are given in Fig. 26.4. The sequence involved in the rearrangement of the side-chain in the conversion of phenylalanine to tropic acid has been the subject of longstanding debate. Ansarin and Woolley (Phytochemistry, 1994, 35, 935), by feeding phenyl [1,3¹³C₂]lactic acid to D. stramonium and examining the ¹³C-NMR spectra of the subsequently isolated hyoscine and hyoscyamine, have substantiated the hypothesis that tropic acid is formed by an intramolecular rearrangement of phenyllactate. Furthermore, it has been demonstrated that hyoscyamine is biosynthesized from littorine by a process involving the intramolecular rearrangement of the phenyllactate moiety of the alkaloid. In concordance with this, transformed roots of Datura stramonium will convert exogenously added littorine to hyoscyamine (35% metabolism recorded) but, in contrast, exogenously added hyoscyamine is not metabolized to littorine.

Fig. 26.4 Demonstrated incorporations of phenylalanine into the tropic acid and α-hydroxy-β-phenylpropionic acid (phenyllactic acid) moieties of hyoscyamine and littorine, respectively.

Isoleucine serves as a precursor of the tigloyl and 2-methylbutanoyl moieties of various mono- and di-esters of the hydroxytropanes.

Biogenesis of hyoscine (scopolamine)

Work initiated by Romeike in 1962 showed that hyoscine appeared to be formed in the leaves of D. ferox from hyoscyamine via 6-hydroxyhyoscyamine and 6,7-dehydrohyoscyamine. The former intermediate has been well substantiated, as indicated below, and occurs in quantity in some other genera (Scopolia, Physochlaina, Przewalskia) but the latter, although incorporated into hyoscine when fed as a precursor to D. ferox, has never been isolated from normal plants. Some 25 years later Hashimoto’s group, using Hyoscyamus niger cultured roots, isolated and partially purified the enzyme responsible for the conversion of hyoscyamine to 6-hydroxyhyoscyamine. They used this enzyme to prepare [6-¹⁸O]-hydroxyhyoscyamine from hyoscyamine and showed that when the labelled compound, fed to Duboisia myoporoides, was converted to hyoscine the ¹⁸O was retained, thus eliminating 6,7-dehydrohyoscyamine from the pathway (Fig. 26.5). For this reaction to proceed the epoxidase enzyme requires 2-oxo-glutarate, ferrous ions and ascorbate as cofactors, together with molecular oxygen.

Fig. 26.5 Route for the formation of hyoscine from hyoscyamine (partial formulae).

The elucidation of the above pathway, which has spanned many years, aptly illustrates the value of biotechnology and enzymology in contributing to the resolution of some uncertainties resulting from traditional labelled-precursor experiments.

Ontogenesis

In some plants of the Solanaceae (e.g. belladonna and scopolia) hyoscyamine is the dominant alkaloid throughout the life cycle of the plant. In D. stramonium hyoscyamine is the principal alkaloid at the time of flowering and after, whereas young plants contain principally hyoscine; in many other species of Datura (e.g. D. ferox) hyoscine is the principal alkaloid of the leaves at all times. The relative proportions of hyoscine and hyoscyamine in a particular species not only vary with age of the plant, but also are susceptible to other factors, including day length, light intensity, general climatic conditions, chemical sprays, hormones, debudding and chemical races. Isolated organ cultures of belladonna, stramonium and hyoscyamus indicate that the root is the principal site of alkaloid synthesis; however, secondary modifications of the alkaloids may occur in the aerial parts, for example, the epoxidation of hyoscyamine to give hyoscine, and the formation of meteloidine from the corresponding 3,6-ditigloyl ester.

Plant

D. Stramonium is a bushy annual attaining a height of about 1.5 m and having a whitish root and numerous rootlets. The erect aerial stem shows dichasial branching with leaf adnation. The stem and branches are round, smooth and green. The flowers are solitary, axillary and short-stalked. They have a sweet scent. Each has a tubular, five-toothed calyx about 4.5 cm long, a white, funnel-shaped corolla about 8 cm long, five stamens and a bicarpellary ovary. The plant flowers in the summer and early autumn. The fruit is originally bilocular but as it matures a false septum arises, except near the apex, so that the mature fruit is almost completely four-celled. The ripe fruit is a thorny capsule about 3–4 cm long. Stramonium seeds (see Fig. 41.6) are dark brown or blackish in colour, reniform in outline and about 3 mm long. The testa is reticulated and finely pitted. A coiled embryo is embedded in an oily endosperm.

D. stramonium var. tatula closely resembles the above; its stems are reddish and the leaves have purplish veins, as also have the lavender-coloured corollas. Varieties of both the above forms occur with spineless capsules.

History

Stramonium was grown in England by Gerarde towards the end of the sixteenth century from seeds obtained from Constantinople. The generic name, Datura, is derived from the name of the poison, dhât, which is prepared from Indian species and was used by the Thugs.

Macroscopical characters

Fresh stramonium leaves or herbarium specimens should first be examined, since the commercial leaves are much shrunken and twisted, and their shape can only be ascertained by careful manipulation after soaking them in water.

The dried leaves are greyish-green in colour, thin, brittle, twisted and often broken. Whole leaves are 8–25 cm long and 7–15 cm wide; they are shortly petiolate, ovate or triangular-ovate in shape, are acuminate at the apex and have a sinuate-dentate margin. They are distinguished from the leaves of the Indian species, D. innoxia, D. metel and D. fastuosa, by the margin, which possesses teeth dividing the sinuses, and by the lateral veins which run into the marginal teeth.

The commercial drug contains occasional flowers and young capsules, which have been described above. The stems are often flattened, longitudinally wrinkled, somewhat hairy and vary in colour from light olive brown (D. stramonium) to purplish-brown (var. tatula). Stramonium has a slight but unpleasant odour, and a bitter taste.

Microscopical characters

A transverse section of a leaf (Fig. 26.6) shows that it has a bifacial structure. Both surfaces are covered with a smooth cuticle and possess both stomata and hairs. Cluster crystals of calcium oxalate are abundant in the mesophyll (Fig. 26.6F, G), and microsphenoidal and prismatic crystals are also found. The stomata are of the anisocytic and anomocytic types. The epidermal cells have wavy walls, particularly those of the lower epidermis. The uniseriate clothing hairs are three- to five-celled, slightly curved, and have thin, warty walls (Fig. 26.6E). The basal cell is usually more than 50 μm long (distinction from D. metel). Small glandular hairs with a one- or two-celled pedicel and others with a two-celled pedicel and an oval head of two to seven cells are also found. If portions of the leaf are cleared with chloral hydrate solution, the abundance of the cluster crystals of calcium oxalate and their distribution with regard to the veins may be noted.

Fig. 26.6 Datura stramonium leaf. A, Transverse sections of midrib (×15); B, transverse section portion of lamina; C, lower epidermis with stomata and glandular trichome; D, glandular trichome over vein; E, clothing trichomes (all ×200); F, arrangement of calcium oxalate crystals in crystal layer, surface view (×50); G, calcium oxalate crystals in cells; H, upper epidermis showing cicatrix and stomata (G and H, ×200). I, J, Scanning electron micrographs of (I) clothing trichome and (J) glandular trichome. c, Collenchyma; cic, cicatrix; c.l, crystal layer; e, endodermis; id, idioblast containing micro-crystals; i.ph, intraxylary phloem; I.ep, lower epidermis with stoma; m, mesophyll; ox, calcium oxalate crystal; p, palisade layer; ph, phloem; u.ep, upper epidermis with stoma; vt, veinlet; xy, xylem.

(Photographs: L. Seed and R. Worsley.)

The midrib shows a bicollateral structure and characteristic subepidermal masses of collenchyma on both surfaces. The xylem forms a strongly curved arc. Sclerenchyma is absent.

Stems are present, but few of these should exceed 5 mm diameter. They possess epidermal hairs up to 800 μm long and have perimedullary phloem. The stem parenchyma contains calcium oxalate similar to that found in the leaf.

Constituents

Stramonium usually contains 0.2–0.45% of alkaloids, the chief of which are hyoscyamine and hyoscine, but a little atropine may be formed from the hyoscyamine by racemization. At the time of collection these alkaloids are usually present in the proportion of about two parts of hyoscyamine to one part of hyoscine, but in young plants hyoscine is the predominant alkaloid. The TLC test for identity given in the BP/EP enables other Datura species containing different proportions of alkaloids to be detected. The larger stems contain little alkaloid and the official drug should contain not more than 3% stem with a diameter exceeding 5 mm. Stramonium seeds contain about 0.2% of mydriatic alkaloids and about 15–30% of fixed oil. The roots contain, in addition to hyoscine and hyoscyamine, ditigloyl esters of 3,6-dihydroxytropane and 3,6,7-trihydroxytropane, respectively, and a higher proportion of alkamines than the aerial portions. For a recent report on the distribution of alkaloids in different organs and in three varieties of D. stramonium, see S. Berkov et al., Fitoterapia, 2006, 77, 179. D. stramonium cell and root cultures have been considerably utilized in biogenetic studies.

Prepared Stramonium BP/EP is the finely powdered drug adjusted to an alkaloid content of 0.23–0.27%.

Allied species

All Datura species examined to date contain those alkaloids found in stramonium, but frequently hyoscine, rather than hyoscyamine, is the principal alkaloid.

Commercial ‘datura leaf’ consists of the dried leaves and flowering tops of D. innoxia and D. metel; it is obtained principally from India. Like those of stramonium, the dried leaves are curled and twisted, but are usually somewhat browner in colour, with entire margins and with differences in venation and trichomes. The leaves contain about 0.5% of alkaloids. Variations in hyoscine and atropine contents in different organs of D. metel during development have been studied (S. Afsharypuor et al., Planta Med., 1995, 61, 383). Over 30 alkaloids have been characterized from D. innoxia by capillary GLC–mass spectrometry. For studies on the anatomy of the leaf of D. metel, see V. C. Anozie, Int. J. Crude Drug Res., 1986, 24, 206; and for the isolation of 3α-anisoyloxytropane see S. Siddiqui et al., J. Nat. Prod., 1986, 49, 511. ‘Datura seeds’ are derived from D. metel and possibly other species. Each seed is light brown in colour and ear-shaped. They are larger and more flattened than stramonium seeds but resemble the latter in internal structure. The alkaloid content, hyoscine with traces of hyoscyamine and atropine, is about 0.2%. D. ferox, a species having very large spines on its capsules, contains as its major alkaloids hyoscine and meteloidine.

The ‘tree-daturas’ constitute Section Brugmansia of the genus; these arboraceous, perennial species are indigenous to South America and are widely cultivated as ornamentals. They produce large, white or coloured trumpet-shaped flowers and pendant unarmed fruits. Some species constitute a potential source of hyoscine (W. C. Evans, Pharm. J., 1990, 244, 651) and D. sanguinea, in particular, has proved a most interesting plant with respect to its wide range of tropane alkaloids and has been cultivated commercially in Ecuador. It yields about 0.8% hyoscine. Plantations have an economically useful life of about 10 years. Chemical races of D. sanguinea are evident, particularly one producing relatively large amounts of 6β-acetoxy-3α-tigloyloxytropane. Various tree datura hybrids developed at Nottingham University, UK, have been used by a number of workers for alkaloid studies involving hairy root and root cultures; as an example see P. Nussbaumer et al., Plant Cell Rep., 1998, 17, 405.

The South American Indians have long cultivated various races of these plants for medicinal and psychotropic use (for a comparison of native assessment of their potency with alkaloid content, see Bristol et al., Lloydia, 1969, 32, 123).

Withanolides (q.v.) have also been recorded in a number of species of the genus; these include various hydroxywithanolides.

Adulteration

Adulterants cited are the leaves of species of Xanthium (Compositae), Carthamus (Compositae) and Chenopodium (Chenopodiaceae), which are, however, easily distinguished from the genuine drug.

Uses

Atropine has a stimulant action on the central nervous system and depresses the nerve endings to the secretory glands and plain muscle. Hyoscine lacks the central stimulant action of atropine; its sedative properties enable it to be used in the control of motion sickness. Hyoscine hydrobromide is employed in preoperative medication, usually with papaveretum, some 30–60 min before the induction of anaesthesia. Atropine and hyoscine are used to a large extent in ophthalmic practice to dilate the pupil of the eye.

Plant

Henbane is a biennial (var. α-biennis) or annual (var. β-annua) plant. It is found wild, chiefly near old buildings, both in the UK and in the rest of Europe, and is widely cultivated. Before examining commercial henbane leaves it is advisable to study growing plants or herbarium specimens. The differences tabulated in Table 26.3 should be noted.

Table 26.3 Comparison of commercial varieties of hyoscyamus.

Henbane flowers have the formula K(5), C(5), A5, G(2). The hairy, five-lobed calyx is persistent. The fruit is a small, two-celled pyxis (see Fig. 41.6B), which contains numerous seeds.

Henbane seeds are dark grey in colour, somewhat reniform in shape and about 1.5 mm long. They have a minutely reticulated testa and an internal structure closely resembling that of stramonium seeds. Henbane seeds contain about 0.06–0.10% of alkaloids (hyoscyamine with a little hyoscine and atropine) together with calystegines (nortropane alkaloids). A number of non-alkaloidal components include various lignanamides (C.-Y. Ma et al., J. Nat. Prod., 2002, 65, 206).

History

Henbane, probably the Continental H. albus, was known to Dioskurides and was used by the ancients. Henbane was used inEngland during the Middle Ages. After a period of disuse in the eighteenth century the drug was restored to the London Pharmacopoeia of 1809, largely owing to the work of Störck.

Collection and preparation

Biennial henbane was the variety traditionally grown in England, but much of the current drug is now of the annual variety or is derived from the allied species H. albus. The germination of henbane seeds is slow and often erratic and may often be assisted by special treatments (e.g. concentrated sulphuric acid, gibberellic acid or splitting of the testa). The plant may be attacked by the potato beetle, and spraying with derris or pyrethrum may be necessary.

The annual plant usually flowers in July or August and the biennial in May or June. The leaves should be dried rapidly, preferably by artificial heat at a temperature of about 40–50°C.

Macroscopical characters

Commercial henbane consists of the leaves and flowering tops described above. The leaves are more or less broken but are characterized by their greyish-green colour, very broad midrib and great hairiness. If not perfectly dry, they are clammy to the touch, owing to the secretion produced by the glandular hairs. The stems are mostly less than 5 mm diameter and are also very hairy. The flowers are compressed or broken but their yellowish corollas with purple veins are often seen in the drug. Henbane has a characteristic, heavy odour and a bitter, slightly acrid taste.

Microscopical characters

A transverse section of a henbane leaf shows a bifacial structure (Fig. 26.7A). Both surfaces have a smooth cuticle, epidermal cells with wavy walls, stomata of both anisocytic and anomocytic types, and a large number of hairs, which are particularly abundant on the midrib and veins. The hairs are up to 500 μm long; some are uniseriate and two to six cells long, while others have a uniseriate stalk and a large, ovoid, glandular head, the cuticle of which is often raised by the secretion (Fig. 26.7E). Similar hairs are found on the stems. The spongy mesophyll contains calcium oxalate, mainly in the form of single and twin prisms, but clusters and microsphenoidal crystals are also present (Fig. 26.7B,D). The broad midrib contains a vascular bundle, distinctly broader than that of stramonium, showing the usual bicollateral arrangement, which is also to be seen in the stems. The mesophyll of the midrib is made up of two thin zones of collenchyma immediately within the epidermi and a ground mass of colourless parenchyma showing large, intercellular air spaces and containing prisms or, occasionally, microsphenoidal crystals of calcium oxalate.

Fig. 26.7 Hyoscyamus niger. A, Transverse section of midrib of leaf (×40); B, transverse section of portion of leaf lamina; C, portion of leaf upper epidermis, surface view; D, calcium oxalate crystals; E, trichomes; F, pollen grains; G, portion of epidermis of corolla with attached glandular trichome (all ×200). b, Base of trichome; c, collenchyma; cic, cicatrix; c.I, crystal layer; e, endodermis; g.t, glandular trichome or portion of; id, idioblast; i.ph, intraxylary phloem; I.ep, lower epidermis; m, mesophyll; p, palisade layer; ph, phloem; st, stoma; tr₁, tr₂, whole and broken clothing trichomes, respectively; u.ep, upper epidermis; vt, veinlet; xy, xylem.

The calyx possesses trichomes and stomata, as in the leaf. The corolla is glabrous on the inner surface but exhibits trichomes on the outer surface, particularly over the veins (Fig. 26.7G). Those cells of the corolla which contain bluish anthocyanins turn red with chloral hydrate solution. Numerous pollen grains are present, about 50 μm diameter, tricolpate with three wide pores and an irregularly, finely pitted exine (Fig. 26.7F). The testa of the seeds has an epidermis with lignified and wavy anticlinal walls, and sclereids are present in the pericarp.

Constituents

Henbane leaves contain about 0.045–0.14% of alkaloids and yield about 8–12% of acid-insoluble ash (BP/EP 2001 not more than 12%). Hyoscyamine and hyoscine are the principal alkaloids. The petiole appears to contain more alkaloid than the lamina or stem.

Prepared Hyoscyamus BP/EP 2001 is the drug in fine powder adjusted to contain 0.05–0.07% of total alkaloids. It has a ‘loss on drying’ requirement of not more than 5.0%.

Allied drugs

Hyoscyamus albus is grown on the Continent, particularly in France, and in the Indian subcontinent. It has petiolate stem-leaves and the flowers have pale yellow, non-veined corollas. Unlike H. niger, the fruits are barely swollen at the base. Quantitatively and qualitatively its alkaloids appear similar to those of H. niger. It has been used in biogenetic studies (q.v.) and the hairy roots (transformed with Agrobacterium rhizogenes) have been analysed for 7β- and 6β-hydroxyhyoscyamine, littorine, hyoscine and hyoscyamine (M. Sauerwein and K. Shimomura, Phytochemistry, 1991, 30, 3277). In traditional medicine of the Tuscan archipelago the seeds are pressed into the cavities of decayed teeth to obtain pain relief (R. E. Uncini Manganelli and P. E. Tomei, J. Ethnopharmacology, 1999, 65, 181).

Hyoscyamus muticus is indigenous to India and Upper Egypt; it has been introduced into Algiers. For further details, see below.

Indian henbane. Under this name considerable quantities of drug were imported into Britain during World War II. Although H. niger is grown in India and Pakistan, much of the drug came from a closely related plant, H. reticulatus. This contains hyoscine and hyoscyamine and microscopically it is almost identical to H. niger.

Hyoscyamus aureus and H. pusillus are two species which produced hyoscine as the principal alkaloid.

Uses

Henbane resembles belladonna and stramonium in action but is somewhat weaker. The higher relative proportion of hyoscine in the alkaloid mixture makes it less likely to give rise to cerebral excitement than does belladonna. It is often used to relieve spasm of the urinary tract and with strong purgatives to prevent griping.

Hyoscyamus for Homoeopathic Preparations

This is described in the BP/EP Vol. III and consists of the whole, fresh, flowering plant of Hyoscyamus niger L. There is a limit for foreign matter (max. 5%) and a minimal loss on drying at 100–105°C for 2 h of 50%. An assay is described for the Mother Tincture.

Macroscopical characters

The drug consists of leaves, stems, flowers and fruits. The leaves are usually matted and form a lower proportion of the drug than in the case of European henbane. The leaves are pubescent, pale green to yellowish, rhomboidal or broadly elliptical and up to about 15 cm long. Midrib broad, venation pinnate, margin entire or with about five large teeth on each side. Petiole almost absent or up to 9 cm long. The stems are greyish-yellow, striated, slightly hairy and hollow. The flowers are shortly stalked, with large hairy bracts, a tubular five-toothed calyx and a yellowish-brown corolla which in the dry drug may show deep purple patches. The fruit is a cylindrical pyxidium surrounded by a persistent calyx and containing numerous yellowish-grey to brown seeds. Odour, slightly foetid; taste, bitter and acrid.

Microscopical characters

Egyptian henbane is easily distinguished from H. niger by the numerous branched and unbranched glandular trichomes, which have a one- to four-celled stalk and unicellular heads. Additional characters are the striated cuticle, the prisms of oxalate 45–110 μm, twin prisms and occasional clusters and microsphenoids.

Constituents

Ahmed and Fahmy found about 1.7% of alkaloids in the leaves, 0.5% in the stems and 2.0% in the flowers. The chief alkaloid is hyoscyamine for the isolation of which (as atropine) the plant is principally used. The alkaloidal mixture of plants grown in Afghanistan had the following composition: hyoscyamine 75%, apoatropine 15%, hyoscine 5%, with smaller quantities of noratropine and norhyoscine. A number of non-alkaloidal ketones, an acid and sitosterol have been characterized from plants raised in Lucknow, India. The formation of alkaloids in suspension cell cultures has been widely investigated with variable results; with callus cultures the addition of phenylalanine to the medium produced maximum alkaloid production (3.97%) whereas isoleucine gave the greatest growth (M. K. El-Bahr et al., Fitoterapia, 1997, 68, 423).

Plant

The deadly nightshade, A. belladonna, is a perennial herb which attains a height of about 1.5 m. Owing to adnation, the leaves on the upper branches are in pairs, a large leaf and a smaller one.

The flowers appear about the beginning of June. They are solitary, shortly stalked, drooping and about 2.5 cm long. The corolla is campanulate, five-lobed and of a dull purplish colour. The five-lobed calyx is persistent, remaining attached to the purplish-black berry. The latter is bilocular, contains numerous seeds and is about the size of a cherry (see Fig. 41.6C). In the USA the plant is often known as the ‘Poison Black Cherry’, while the German name is ‘Tollkirschen’ (i.e. Mad Cherry). A yellow variety of the plant lacks the anthocyanin pigmentation; the leaves and stems are a yellowish-green and the flowers and berries yellow.