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.

History

Belladonna was probably known to the ancients but it is not clearly recorded until the beginning of the sixteenth century. The leaves were introduced into the London Pharmacopoeia of 1809, but the root was not used in Britain until a liniment prepared from it was introduced by Squire in 1860.

Cultivation, collection and preparation

Belladonna is grown from seed. The leaves are said to be richest in alkaloid at the end of June or in July, and a sunny position is said to give more active leaves than a shady one. Plants about 3 years old are sufficiently large to give a good yield of leaves and, if the roots are being collected, it would seem to be best to replant about every third year (see also ‘Belladonna Root’). Two or more crops of leaves may be collected annually. Leaves left in an imperfectly dry state deteriorate and give off ammonia. They should therefore be dried immediately after collection and be carefully stored. Leaves of a good colour may be obtained by drying in thin layers starting with a moderate heat which is gradually increased to about 60°C and then gradually decreased. Sometimes the leaves are badly attacked by insects and the roots by a fungus.

Macroscopical characters

The drug consists of leaves and the smaller stems, the latter seldom exceeding 5 mm diameter, together with flowers and fruits as described above. If the drug is little broken, the arrangement of the leaves in unequal pairs may be seen. The leaves are dull green or yellowish-green in colour, the upper side being somewhat darker than the lower. Each has a petiole about 0.5–4 cm long and a broadly ovate, slightly decurrent lamina about 5–25 cm long and 2.5–12 cm wide. The margin is entire and the apex acuminate. A few flowers and fruits may be found. If the leaves are broken, the most useful diagnostic characters are the venation and roughness of the surface. The latter is due to the presence of calcium oxalate in certain of the mesophyll cells which causes minute points on the surface of the leaf as the other cells contract more on drying.

Microscopical characters

A transverse section of the leaf of A. belladonna is shown in Fig. 26.8A. It has a bifacial structure. The epidermal cells have wavy walls and a striated cuticle (Fig. 26.8E). Stomata of the characteristic anisocytic type and also some of the anomocytic type are present on both surfaces but are most common on the lower. Hairs are most numerous on young leaves. Some of the hairs are uniseriate, two- to four-celled clothing hairs; others resemble these but have a unicellular glandular head; while a third kind has a short pediceland a multicellular glandular head (Fig. 26.8F). Certain of the cells of the spongy mesophyll are filled with microsphenoidal (‘sandy’) crystals of calcium oxalate (Fig. 26.8B, C). The midrib is convex above and shows the usual bicollateral vascular bundle. A zone of collenchyma underlies both epidermi in the region of the midrib.

Fig. 26.8 Atropa belladonna leaf. A, Transverse section of midrib (×40); B, transverse section of portion of lamina (×200); C, distribution of idioblasts, surface view of leaf cleared in chloral (×50); D, upper epidermis; E, lower epidermis; F, trichomes (all ×200); G and H, Scanning electron micrographs (G) of glandular trichome and epidermal cells with striated cuticle and (H) stoma and striated cuticle. a, Striations of cuticle; c, collenchyma; e, endodermis; ep, epidermis; id, idioblast containing crystals of calcium oxalate; i.ph, intraxylary phloem; m, mesophyll; ox, calcium oxalate crystals; p, palisade layer; ph, phloem; st, stoma; vt, veinlet; xy, xylem.

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

Constituents

The drug from A. belladonna contains 0.3–0.60% of alkaloids, the chief of which is hyoscyamine. Small quantities of volatile bases, such as pyridine and N-methylpyrroline, are present, and if not removed during the assay of the drug by heating, increase the titration and appear in the result as hyoscyamine. The leaves also contain a fluorescent substance, β-methylaesculetin (scopoletin), and calcium oxalate. They yield about 14% of ash and not more than 4% of acid-insoluble ash.

Prepared Belladonna Herb is the finely powdered drug adjusted to contain 0.28–0.32% of total alkaloids. Note the ‘loss on drying’ requirement.

Allied drugs

Indian belladonna from A. acuminata Royle ex Lindley differs from that derived from A. belladonna in that its flowers are yellowish-brown and its leaves brownish-green, oblong-elliptical and tapering towards both base and apex. It grows wild in the Himalayan regions of northern India (1800–3400 m) and is cultivated in the Kashmir valley.

Atropa baetica Willk. is a species native to southern Spain and northern Morocco; it produces yellow flowers and black berries and is regarded as an endangered species. R. Zárate et al. have described a rapid in vitro propagation method for the plant, and from hairy root cultures have isolated tigloylpseudotropine—alkaloid not found in the mature plant (Plant Cell Rep., 1999, 18, 418).

Adulterants

Of the numerous recorded adulterants of belladonna leaves, those of Phytolacca decandra (Phytolaccaceae) and Ailanthus glandulosa (Simaroubaceae) are perhaps the most important. In Phytolacca the lamina is denser and less decurrent than in belladonna; the epidermal cells have straight walls, the stomata are of the anomocytic type and some of the mesophyll cells contain bundles of needle-shaped crystals of calcium oxalate. Ailanthus leaves are triangular-ovate, have straight-walled epidermal cells showing a strongly striated cuticle, cluster crystals of calcium oxalate, and on both surfaces white, unicellular clothing hairs which are lignified (Fig. 42.3F).

Uses

Belladonna leaves are mainly used for internal preparations which are used as sedatives and to check secretion. Preparations of the root are mainly used externally.

Collection and preparation

Much of the A. belladonna drug is of small size and poor quality. The first-year roots are not profitable to collect from the commercial point of view, although they contain a high proportion of alkaloids. The autumn of the third year would seem to be a suitable time for collection. The roots are dug up, washed, sliced and dried.

Macroscopical characters

Atropa belladonna rapidly develops a large branching root. The aerial stems die back each year and new ones arise independently from the large crown. Dried roots of 3-year-old plants are about 3 cm diameter and roots over 4 cm diameter are exceptional. Most commercial drug is about half this thickness.

The drug is usually cut into short lengths, which are sometimes split longitudinally. The outer surface is a pale greyish-brown. The root breaks with a short fracture and then shows a whitish or, if overheated during drying, brownish interior. A yellowish-green colour in the region of the cambium is often seen.

A transverse section of the bark is non-fibrous and the wood does not show a radiate appearance. The wood consists of scattered groups of vessels, tracheids and fibres which are most abundant near the cambium; there is a central mass of primary xylem (Fig. 41.8G). The extensive parenchyma of bark and wood contains sandy crystals of calcium oxalate and abundant simple and compound starch grains.

The structure gradually changes as the roots pass into rhizome, the wood becoming denser and exhibiting a distinctly radiate structure; the rhizome also shows a distinct pith and internal phloem. The aerial stems found on the upper surface of the crown are hollow.

Constituents

Atropa belladonna root contains about 0.4–0.8% of alkaloids calculated as hyoscyamine.

Samples of belladonna root examined by Kuhn and Schäfer showed 0.3–1.0% of alkaloids, of which 82.8–97.3% was hyoscyamine, 2.7–15.2% atropine, and 0.0–2.6% scopolamine. Capillary GLC–mass spectrometry data revealed the presence of hygrine, hygroline, cuscohygrine, tropinone, tropine, pseudotropine and nine tropanol esters (F. Oprach et al., Planta Med., 1986, 513). Other constituents previously reported include belladonnine together with β-methylaesculetin, calcium oxalate and starch.

A pseudotropine-forming, tropinone reductase (see biogenesis of tropane alkaloids), not entirely similar in chemical and catalytic properties to other samples of the enzyme previously described, has been isolated from transformed belladonna root cultures. The pseudotropine could have implications for the formation of calystegines (q.v). Littorine has been detected in both non-transformed and hairy root-cultures (F. Nakanishi et al., Plant Cell Rep., 1998, 18, 249).

Allied drug

Indian belladonna root from Atropa acuminata (see under ‘Belladonna Leaf’) consists of brownish-grey roots, stolons, rootstock and stem bases. It has been described in detail by Melville. The roots are cylindrical, longitudinally wrinkled, occasionally branched, and 0.5–3 cm diameter. Young roots resemble those of A. belladonna but older ones show concentric zonation of the secondary xylem. The rootstock is 3–9 cm diameter at the top and bears the bases of 4–12 aerial stems. The rootstock, stem bases and stolons all possess a pith which becomes hollow in the stem bases. The constituents are similar to those of European belladonna.

Adulterant

The root of Phytolacca decandra (Phytolaccaceae) is sometimes sliced and mixed with samples of belladonna. It bears little resemblance to belladonna root, but a casual and inexperienced observer might perhaps mistake it for pieces of an old belladonna crown. The transverse section shows a number of concentric cambia, each producing a ring of wood bundles. The parenchyma contains abundant acicular crystals of calcium oxalate.

History

Coca leaves have been used in South America as a masticatory from very early times. They were formerly reserved for the sole use of the native chiefs and Incas. Coca was introduced into Europe about 1688 and cocaine was isolated in 1860. By employing the alkaloid in ophthalmic surgery in 1884 Carl Koller was the first to introduce it into clinical practice so heralding the era of modern anaesthetics. For reviews covering both the historical and other aspects of coca see the special issue of The Journal of Ethnopharmacology (1981), Vol. 3: Coca and Cocaine.

Cultivation and collection

These differ depending on geographical source. For Andean coca, plants are raised from seed and cultivated at an altitude of 500–2000 m. Pruning limits the height to about 2 m and traditionally three harvests are collected annually, the first from the pruned twigs, the second in June and the third in November. The leaves are artificially or sun dried and packed into bags. On the other hand, in the Amazon, plants are raised from cuttings, often in jungle clearings and interplanted between other staple crops.

Varieties and characters:

Microscopical characters

A transverse section of a coca leaf shows upper epidermis, palisade parenchyma containing prisms of calcium oxalate, spongy parenchyma and a very characteristic lower papillose epidermis with numerous stomata. The midrib is partly surrounded by an arc of pericyclic fibres, above and below which is a considerable amount of collenchyma. A surface preparation of the lower epidermis shows the papillae as well-marked circles, and numerous stomata (see Fig. 42.2J), each with four subsidiary cells, two of which have their long axes parallel to the pore.

DNA analysis

For a report on the differentiation of the cocaine-producing species and varieties of Erythroxylum using AFLP DNA analysis, see E. L. Johnson et al., Phytochemistry, 2003, 64, 187; 132 Erythroxylum samples were examined.

Constituents

Coca leaves contain about 0.7–1.5% of total alkaloids, of which cocaine, cinnamylcocaine and α-truxilline are the most important (Fig. 26.9). They occur in different proportions in different commercial varieties. Javanese leaves are usually richest in total alkaloids, of which the chief is cinnamylcocaine, while the Bolivian and Peruvian leaves contain less total alkaloid but a higher proportion of cocaine. Other substances isolated from various varieties of the leaves are hygrine, hygroline, cuscohygrine, dihydrocuscohygrine, tropacocaine (3β-benzoyloxytropane), crystalline glycosides and cocatannic acid. 1-Hydroxytropacocaine (free hydroxyl situated at a bridgehead carbon) has been isolated as a major alkaloid of greenhouse-cultivated E. novogranatense var. novogranatense; much lower amounts were detected in var. truxillense and in field cultivated coca from Colombia and Bolivia (J. M. Moore et al., Phytochemistry, 1994, 36, 357).

Fig. 26.9 Constituents of coca.

The leaves also contain essential oil and as early as 1894 Van Romburgh identified methyl salicylate as a component; this was confirmed (13.6%) in a recent study, together with N-methylpyrrole (3.7%) and possibly N,N-dimethylbenzylamine (0.5%) and two dihydrobenzaldehydes (38.9%). The grassy odour of the leaves is explained to a large extent by the presence in the oil of trans-2-hexenal (10.4%) and cis-3-hexen-1-ol (16.1%); no mono- or sesquiterpenes were detected (M. Novák et al., Planta Med., 1987, 53, 113).

Although it had been generally assumed that ecgonine, the basic moiety of the cocaines, was ornithine-derived (Fig. 26.2), the practical demonstration of the incorporation of the usual precursors proved difficult. Then Leete (J. Am. Chem. Soc., 1982, 104, 1403) obtained a significant level of radioactivity in cocaine isolated from Erythroxylum coca, the leaves of which were painted with an aqueous solution of DL-[5-¹⁴C]ornithine HCl. The pathway to ecgonine appears to be similar to that for tropine except that the carboxyl is retained and the different stereospecificities need to be accommodated. The benzoyl moiety of cocaine is derived from phenylalanine. For work on the incorporation of labelled 1-methyl-Δ¹-pyrrolinium chloride into cuscohygrine, indicating the alkaloid to be a mixture of its meso and optically active diastereomers, see E. Leete et al., Phytochemistry, 1988, 27, 401.

Fig. 26.10 Formation and liver metabolism of pyrrolizidine-ester alkaloids.

Manufacture of cocaine

The crude alkaloids may be extracted with dilute sulphuric acid or by treatment with lime and petroleum or other organic solvents. Non-alkaloidal matter is roughly separated bytransferring the alkaloids from one solvent to another. The crude alkaloids are obtained in solid form either as free bases by precipitation with alkali, or as hydrochlorides by concentrating an acidified solution.

Pure cocaine is prepared from the leaves, the crude bases or the crude hydrochlorides. The process depends on the fact that cocaine, cinnamylcocaine and α-truxilline are closely related derivatives of ecgonine (Fig. 26.2), which is produced by hydrolysing them with boiling dilute hydrochloric acid.

The ecgonine hydrochloride is purified and converted into the free base. This is benzoylated by interaction with benzoic anhydride and the benzoylecgonine purified. The benzoylecgonine is methylated with methyl iodide and sodium methoxide in methyl alcohol solution, to give methylbenzoylecgonine or cocaine. The latter is converted into the hydrochloride and purified by recrystallization.

Much illicit cocaine is extracted locally in South America and despite the unsophisticated methods employed a high degree of purity can be attained.

In view of the importance of quantitatively determining cocaine and its metabolite, benzoylecgonine, in body fluids, etc., many assays are available for these alkaloids.

Allied species

There are over 200 species of Erythroxylum found throughout the tropical and pantropical regions of the world. Few of the non-cocaine-producing species have been systematically examined but the majority of those that have contain a range of tropane alkaloids (W. C. Evans, J. Ethnopharmacol., 1981, 3, 265 and for Pt. 12 of a further series of papers see P. Christen et al., Phytochemistry, 1995, 38, 1053). Subsequently, other workers have isolated a number of the same alkaloids from other species, together with the characterization of new tropane alkaloids, some named pervilleines and others catuabines. Nortropanols (calystegins, see below) are present in some species. Trimethoxybenzoic acid and trimethoxycinnamic acid commonly occur as esterifying acids. D. Bieri et al. (J. Ethnopharmacology, 2006, 103, 439) have studied the cocaine distribution in 51 species of Erythroxylum from S. America, 28 of which had received no previous phytochemical examination; cocaine was reported for the first time in 14 species, with E. laetevirens having the highest cocaine content of the wild species. It is of interest to note that in this study, the time between collection and analysis of the samples varied from 20 to 25 years.

Uses

Cocaine and its salts were the earliest of the modern local anaesthetics but, because of their toxic and addictive properties, their use is now almost entirely confined to ophthalmic, ear, nose and throat surgery.

History

Lobelia has long been used by the North American Indians. It was recommended for use in asthma by Cutler in 1813 and was introduced to the English medical profession by Reece in 1829.

Cultivation and collection

Lobelia is grown from seed which is sown either in the autumn or in March and April. The plant produces an aerial stem about 50 cm high. It bears alternate leaves 3–8 cm long and pale blue, bilabiate flowers. The inferior ovary develops into an inflated capsule. The plants are cut in August or September, when they bear numerous capsules. After drying, the drug is exported in bales or compressed packets. The seeds are sometimes separated by thrashing.

Macroscopical characters

Up to about 60% (BP, 1988, upper limit) of the drug consists of stems. These are green or purplish, winged and very hairy in the upper part but becoming more rounded and channelled and less hairy below. The pale green leaves are usually more or less broken and are covered with bristly hair. Entire leaves are ovate to ovate-lanceolate in shape. The margin is irregularly serrate-dentate and the teeth bear water-pores. The flowers are rarely seen in the drug. The fruits are 5–8 mm long, ribbed and crowned by the calyx teeth. Each is bilocular and contains numerous oval-oblong, brown reticulated seeds about 0.5–0.7 mm long. The drug has a slightly irritating odour and an acrid taste.

Microscopical characters

The epidermis of the stem is composed of rectangular cells, covered with a striated cuticle and with anticlinal walls clearly pitted, giving a characteristic beaded appearance. The epidermis bears stomata with the pore parallel to the stem axis and large, conical, warty-walled, unicellular hairs up to 600 μm long (Fig. 42.3). The cortex is composed of rounded, thin-walled, chlorophyll-containing parenchyma except in the wings, where the cells are collenchymatous. The endodermis is well-differentiated, the cells clearly showing the Casparian strip. A pericycle composed of small groups of fibres is distinguishable in the lower part of the stem. The phloem is composed of small groups of delicate sieve-tube tissue enclosing the anastomosing latex vessels, readily seen after staining with iodine. The xylem is composed of elongated, thick-walled xylem fibres and spiral and scalariform vessels. The pith is composed of pitted lignified parenchyma.

With the leaf the upper epidermis is composed of straightwalled, papillose cells with the anticlinal walls showing the beaded appearance (Fig. 42.2). The lower epidermal cells have wavy walls, and numerous stomata, without special subsidiary cells, are present. Unicellular covering hairs, like those present on the stem, are borne on both epidermal surfaces. The mesophyll is differentiated into a single-layered palisade tissue and a spongy mesophyll. The mesophyll cells contain small fat crystals. The palisade tissue is interrupted in the midrib and groups of collenchyma occur above and below the midrib bundle. The phloem contains the characteristic latex vessels. Numerous water-pores occur on the upper surface of the marginal teeth.

The surface of the seed is characteristically reticulate. The pollen grains are roughly spherical, 20–30 μm diameter, and show three pores.

Constituents

Lobelia contains about 0.24–0.4% of alkaloids (BP 1988, not less than 0.25% as determined by a standard Stas-Otto procedure) the most important of which is lobeline. This and many related alkaloids including lobelidine, lobelanine, lobelanidine and isolobelanine, have a piperidine nucleus (Fig. 26.12). Others are piperideines (i.e. the ring system is unsaturated).

Fig. 26.12 Alkaloids of lobelia, pomegranate, broom and pepper.

By analogy with the biosynthesis of coniine (Fig. 26.38), the above alkaloids could be formed from a polyketo acid involving two benzoyl units and acetate. However, the demonstration that phenylalanine can be incorporated by the plant into the lobelia alkaloid lobinaline and into the related sedamine (Sedum acre, Crassulaceae), and the incorporation of lysine into the piperidine ring of lobeline, does not support this hypothesis. A number of Brazilian species contain similar constituents.

Root cultures of L. inflata transformed by Agrobacterium rhizogenes have been shown to produce lobeline at the same concentration as normal pot-grown plants (H. Yonemitsu et al., Plant Cell Rep. 1990, 9, 307).

Uses

Lobelia is used in spasmodic asthma and chronic bronchitis; it is included in some antismoking preparations. In its pharmacological action, lobeline resembles nicotine in having both central and peripheral effects. In toxic doses the drug has a paralytic effect and its continuous use should be avoided.

Production

The vines are grown on poles or trees. The inflorescence is a spike of about 20–30 sessile flowers, which develop into sessile fruits (see earlier editions for diagrams). The latter are picked when the lower fruits of the spike turn red. They are then removed from the axis and dried, either in the open air or by artificial heat. The fire-dried spice is most esteemed but the ground spice is usually a blend of different varieties.

White pepper, which is largely used in the East, is also obtained from P. nigrum, but the fruits are allowed to become more completely ripe. After storing them for some days or soaking them in water, the outer part of the pericarp is removed by rubbing and washing and the fruits are dried.

Black pepper fruits are almost globular and 3.5–6 mm diameter. The surface is dark brown or greyish-black and strongly reticulated. The apex shows the remains of the sessile stigmas and a basal scar indicates the point of attachment to the axis. Pepper has an aromatic odour and pungent taste.

Bacterial and fungal contamination of the stored peppers can be reduced by washing and redrying with subsequent maintenance of the moisture content at less than 11%.

In white pepper, owing to the removal of the outer part of the pericarp, the vascular bundles, about 16 in number, run on the outside of the fruit from base to apex.

Constituents

Pepper contains 1–2.5% of volatile oil, 5–9% of the crystalline alkaloids piperine and piperettine, and a resin. The aroma of the spice is due to the volatile oil, which consists largely of terpenes, while the pungency is ascribed to piperine and the resin. Piperine, first isolated in 1819, is also found in the long pepper (1–2%) and in Ashanti pepper, the fruits of Piper guineense. Analyses for the volatile oil are given as β-caryophyllene (21.59–27.70%), limonene (21.06–22.17%), sabinene (8.5–17.6%), β-pinene (9.16–11.08%), α-pinene (5.07–6.18%), myrcene (2.2–2.3%), p-cymeme (0.0–0.18%) and oxygenated constituents (3.39–5.68%); 40 compounds were identified in oil from Sao Tome e Principe (A. P. Martíns et al., Phytochemistry, 1998, 49, 2019).

Pepper was once employed in the treatment of gonorrhoea and chronic bronchitis. Large quantities are used as a condiment.

Long pepper is the dried unripe fruit of Piper retrofractum (P. officinarum) and P. longum, grown in Indonesia, India and the Philippines. The spice consists of whole spikes of small fruits forming a structure about 4 cm long and 6 mm in diameter. Individual fruits show a similar structure to black pepper. For a report on the component alkamides and other constituents see B. Das et al., Planta Med., 1996, 62, 582. Guineesine, a recently isolated alkaloid, is an acyl-CoA:cholesterol acyltransferase inhibitor and has been proposed as an attractive target for the prevention and treatment of hypercholesterolemia and atherosclerosis (S. W. Lee et al., Planta Medica, 2004, 70, 678).

Cubebs or tailed pepper are the dried, full-grown fruits of Piper cubeba, a native of Indonesia, Borneo and Sumatra. The fruits are collected while green and dried in the sun. They were used in Europe as a spice as early as the eleventh century. The spikes of cubebs bear more fruits than those of pepper and become falsely stalked as they mature, owing to an abnormal development of the base of the pericarp. The upper part of the cubeb fruit is globular, 3–6 mm diameter and covered with a greyish-brown, reticulated pericarp, which is prolonged at the base into a straight stalk. Cubebs yield 10–18% of volatile oil containing terpenes and sesquiterpenes, a crystalline inodorous substance (−)-cubebin (formula Table 21.7) and a number of other lignans, a white amorphous substance cubebic acid (1%), and amorphous resin (3%).

The above gives only a token appreciation of the genus as a whole—there are some 700 species of Piper globally of which only about 12% have been studied phytochemically. Nevertheless the literature is extensive and in a review (341 refs) covering the secondary metabolites (V. S. Parmar et al., see ‘Further reading’) nearly 600 constituents are listed. Classes of metabolites considered are alkaloids/amides, propenylphenols, lignans, neolignans, terpenes, steroids, kawa pyrones, piperolides, chalcones, dihydrochalcones, flavones, flavanones and miscellaneous compounds.

Collection

The drug is collected in the autumn, this being important, as the amount of alkaloid present shows considerable variation at different seasons. The drug is imported in bales. It consists of slender, more or less broken aerial stems which are woody and usually branch only at the base.

Characters

The stems of the ephedras bear numerous, fine, longitudinal ridges. The leaves are small, connate at the base, and usually in whorls of two (less commonly, whorls of three or four) and decussate.

Constituents

The ephedras contain about 0.5–2.0% of alkaloids. Of the total, ephedrine (and its isomers) forms from 30 to 90%, according to the species. Pseudoephedrine is also present (see Fig. 26.14). The roots also contain a number of macrocyclic alkaloids (ephedradines) and feruloylhistamine which have hypotensive properties.

Biosynthesis of ephedrine and related alkaloids

These alkaloids are formed by a union of a C₆–C₁ unit and a C₂ unit. For many years it has been known that phenylalanine is the originator of the C₆–C₁ moiety, being converted first to benzaldehyde or benzoic acid. GrueSørensen and Spenser (J. Am. Chem. Soc., 1993, 115, 2052 and references quoted therein) using ¹³C- and ²H-labelled precursors in feeding experiments with E. gerardiana have shown that benzoic acid combines with the intact CH₃CO group of pyruvic acid to form ephedrine and related alkaloids with 1-phenylpropan-1,2-dione and (S)-(−)-2-amino-1-phenylpropan-1-one (cathinone) serving as intermediates. The route is illustrated in Fig. 26.14. 1-Phenylpropan-1,2-dione and cathinone are known constituents of Catha edulis (see below) but the previous non-isolation of cathinone from ephedra is attributed to its high efficiency of incorporation into the nor-alkaloids.

Fig. 26.14 Biogenesis of ephedrine and related alkaloids.

Uses

Ephedrine is used for the relief of asthma and hay fever. Its action is more prolonged than that of adrenaline, and it has the further advantage that it need not be given by injection but may be administered by mouth. In oriental medicine the ephedras are also used as anti-inflammatory drugs and this action is ascribed to an oxazolidone related to ephedrine. In contrast to the herb, which has a sudorific action, the root has been used clinically in China for its antisudorific effect.

History

Opium was well known to the ancients. Dioskurides, about AD 77, distinguishes between the latex of the capsules, opos, and an extract of the whole plant, mekonion. The use of opium spread from Asia Minor to Persia, where opium eating became popular, and from there to India and China. However, it was not until the second half of the eighteenth century that opium smoking began to be extensively practised in China and the Far East.

Asia Minor has from very early times been an important source of opium production. In Macedonia cultivation was started as recently as 1865. Persian opium was imported into England from about 1870 to 1955. Opium was cultivated in India during the Middle Ages, and the monopoly of the Mogul Government was taken over first by the East India Company and then by the British Government. Formerly, Indian opiums, being prepared mainly for smoking, were little esteemed for pharmaceutical purposes. However, that now imported is of good quality and constitutes the principal British source for the manufacture of alkaloids.

Production

The plant cultivated in India under licence is P. somniferum var. album. Sowing takes place in November and collection from April to June. The incisions are made in the afternoon with an instrument known as a ‘nushtur’. This bears narrow iron spikes which are drawn down the capsule to produce several longitudinal cuts. The incision must not penetrate into the interior of the capsule or latex will be lost. The latex, which is at first white, rapidly coagulates and turns brown. Early in the morning of the day following the making of the incisions the partly dried latex is scraped off with a trowel-like ‘seetooar’. Each capsule is cut several times at intervals of 2 or 3 days. After collection the latex is placed in a tilted vessel so that the dark fluid (pussewah) which is not required may drain off. By exposure to air the opium acquires a suitable consistency for packing.

Indian opium is exported in 5 kg blocks, packed 12 to a lightweight wooden case to facilitate air transport. Each block is wrapped in grease-proof paper, tied with tape and placed in a polythene bag. The drug has a soft consistency and so arrives as rounded, somewhat flattened, cakes. It contains about 9–12% of morphine. Being difficult to dry and powder because of its plastic nature, Indian opium is less suitable than some other types for the preparation of powdered opium.

Former varieties of legal opium were obtained from Turkey (Turkish Government Monoply Opium) and the former Yugoslavia, and opium is also produced in former Kirghiz SSR and China under government control for national use. Iran and Egypt, former producers, no longer cultivate the plant. Such opiums were very characteristic in appearance and packaging, and illustrations of some different varieties together with tools for production will be found in the 10th edition (1972) of this book. Turkish government opium was until relatively recently much used and consisted of cubical stamped blocks each weighing 2 kg; it was much easier to powder than the Indian drug.

In addition to the above countries, opium has been produced, often only on an experimental scale, in most European countries, the USA, the East Indies and parts of Africa. Experiments have shown that a hot climate is not essential—opium of excellent quality has been produced in Scotland and Norway.

Microscopy

Opium examined under the microscope shows agglomerated latex granules in irregular masses. Other smaller amounts of characteristic material which arise as a result of the preparation process are best seen by examining the residue left after water-extraction of the opium. These particles include occasional spherical pollen grains, fragments of vessels and portions of the epicarp of the capsule the latter showing in surface view polygonal thick-walled cells with a stellate lumen. Pointed trichomes and a few starch grains may be present.

Constituents

Opium contains some 30 alkaloids, which are largely combined with the organic acid meconic acid; the drug also contains sugars, salts (e.g. sulphates), albuminous substances, colouring matters and water. Six principal alkaloids are listed in Table 26.4.

Table 26.4 Historic isolations of principal opium alkaloids.

The first group (e.g. morphine) consists of alkaloids which have a phenanthrene nucleus whereas those of the papaverine group have a benzylisoquinoline structure. Some of the less important opium alkaloids (e.g. protopine and hydrocotarnine) are of different structuraltypes. The morphine molecule has both a phenolic and an alcoholic hydroxyl group, and when acetylated forms diacetyl morphine or heroin. Codeine is an ether of morphine (methylmorphine), and other morphine ethers which are used medicinally are ethylmorphine and pholcodine.

New alkaloids continue to be isolated from P. somniferum—a number were recognized during investigations on the biogenesis of morphine and Repasi et al. (Planta Med., 1993, 59, 477) obtained 5′-O-demethylnarcotine during the purification of narcotine from poppy straw; it was also detected in a sample of Indian opium.

Meconic acid, a dibasic acid, is easily detected either in the free state or as a meconate by the formation of a deep red colour on the addition of a solution of ferric chloride. As it is invariably found in opium, its presence has long been used to indicate opium. However, research has shown that some species of Papaver which produce no morphine but other morphinanes may also contain this acid; it may serve as a chemotaxonomic marker for the Papaveraceae. It is notable that the related acid, chelidonic acid, is found in some other members of the Papaveraceae such as in the root of Greater Celandine (q.v).

Role of reticuline in alkaloid biosynthesis

The alkaloids involved in Fig. 26.15 are derived from (−)-reticuline, and the enzymic racemization of reticuline, which is essential for the biosynthesis of the principal opium alkaloids, is very substrate specific. Thus the N-ethyl, 6-ethoxy and 4′-ethoxy analogues of reticuline are completely resistant to racemization. (+)-Reticuline also gives rise to anumber of bases: narcotine (noscopine) and narceine of opium; canadine, berberine and hydrastine of Hydrastis (Berberidaceae); and sinomenine (the enantiomer of the opium alkaloid salutaridine, Fig. 26.15, of Sinomenium acutum, Menispermaceae). With the exception of sinomenine, these alkaloids are termed ‘berberine bridged’ alkaloids and they arise from norlaudanosoline and a one-carbon unit, which is derived from the N-methyl group of (+)-reticuline. The methylenedioxy group of these alkaloids is formed by oxidative cyclization of an o-methoxyphenol function. A scheme indicating the origin of some of these alkaloids is given in Fig. 26.18. For berberine, 13 enzymes are involved in its biosynthesis from two molecules of tyrosine. The enzyme associated with the final reaction, the formation of the methylenedioxy bridge, has now been detected in microsomal preparations from different Ranunculaceae and Berberidaceae cell cultures (M. Rueffer and M. H. Zenk, Phytochemistry, 1994, 36, 1219).

Fig. 26.18 Some alkaloids derived from (+)-reticuline.

Enzyme studies

As indicated in Chapter 13 cell cultures of P. somniferum do not produce morphine-type alkaloids but accumulate large amounts of sanguinarine and other alkaloids. However, some of the enzymes of the morphinan pathway are present in such cell cultures as shown by the reduction of codeinone to codeine by immobilized cells of P. somniferum and by the isolation from cell cultures of the enzyme which reduces salutaridine to (7S)-salutaridinol. This enzyme has been fully characterized (R. Gerardy and M. H. Zenk, Phytochemistry, 1993, 34, 125) as has 1,2-dehydroreticuline reductase isolated from poppy seedlings. The same group has shown (R. Wilhelm and M. H. Zenk, Phytochemistry, 1997, 46, 701) that the enzyme necessary for the enol cleavage in the conversion of thebaine to neopinone (Fig. 26.15) is present in cell cultures, as evidenced by the formation of a new alkaloid, thebainone, as a result of feeding labelled thebaine to low-thebaine producing P. somniferum cultures. For work on genes encoding (S)-N-methylcoclaurine-5′-hydroxylase and codeinone reductase see F.-C. Huang and T. M. Kutchan, Phytochemistry, 2000, 53, 555.

Tests for opium alkaloids

Tests for morphine and other alkaloids are given in the pharmacopoeias. The solubility of morphine in sodium hydroxide solution is explained by its phenolic nature. Conversely, codeine is precipitated by sodium hydroxide.

Storage

Opium requires careful storage if the morphine content is to be maintained. In the past, Indian opium appears to have suffered more morphine loss during preparation and storage than the other varieties, but when it is dried at 100°C and stored out of contact with air, the loss of morphine is small. Abraham and Rae (1926) ascribe the lossof morphine to a peroxidase which they called opiase. More recently a phenoloxidase which acts on morphine has been isolated from poppy capsules.

Adulteration

Opium has been adulterated with sugary fruits, gum, powdered poppy capsules and other substances too numerous to mention. As far as legitimate commerce is concerned, such adulteration is now pointless because the product is analysed and the price paid is governed by the content of morphine and other alkaloids.

Uses

The alkaloids present in opium in greatest proportion decrease in narcotic properties in the order morphine, codeine, noscopine. Opium and morphine are widely used to relieve pain and are particularly valuable as hypnotics, as, unlike many other hypnotics, they act mainly on the sensory nerve cells of the cerebrum. Codeine is a milder sedative than morphine and is useful for allaying coughing. Both morphine and codeine decrease metabolism, and the latter, particularly before the introduction of insulin, was used for the treatment of diabetes. Opium, while closely resembling morphine, exerts its action more slowly and is therefore preferable in many cases (e.g. in the treatment of diarrhoea). Opium is also used as a diaphoretic. The habitual use of codeine may, in some individuals, produce constipation.

Manufacture of opium alkaloids

The majority of legal opium is used for the isolation of its constituent alkaloids, and in Britain some 90% of the morphine produced is converted into other bases such as codeine, ethylmorphine and pholcodine. In recent years attempts have been made to reduce the illicit traffic in opium either by banning the cultivation of the opium poppy or by cultivation under strict licences. However, two methods by which the opium stage is eliminated are by extraction of the whole poppy capsule and by the use of other species (e.g. P. bracteatum) which do not contain morphine.

Extraction of poppy capsules and straw

The feasibility of extracting poppy straw has long been known and utilized in Europe and recently there has been a world-wide trend towards the extraction of the dried poppy capsules (e.g. in Hungary, former USSR, Tasmania). In a study of poppy capsule drying and storage under commercial conditions in Tasmania it has been shown that kiln-drying of immature capsules (42 days old) at various temperatures (40–100°C) resulted in a loss of morphine content of up to about 11% without effect on codeine and thebaine. However, this morphine loss was significantly less than with the field-dried material. To avoid deterioration of the dried product the moisture content should not exceed 16%. Processes have also been developed in France and in the UK for the harvesting and processing of the green capsule, one technical difficulty being the separation of the seed from the fruit at this stage of development.

Use of morphine-free species of Papaver

The increasing abuse of opiates has stimulated the search for raw materials other than Papaver somniferum which would meet the requirements of the pharmaceutical industry. Thus, plants containing non-addictive thebaine as principal alkaloid could be used for the manufacture of codeine, naloxone (a narcotic antagonist prescribed for babies of heroin addicts) and etorphine (a ‘Bentley’ compound used for sedating large wild animals).

In this respect attention focused on three closely related perennial species of Papaver of the section Oxytona Bernh. of the family, namely, P. bracteatum, P. orientale and P. pseudo-orientale. These are indigenous to the mountainous districts of Iran, eastern Turkey and the Transcaucasian former USSR. Confusion concerning the identity of the characteristic alkaloids for each species had arisen from various factors, including incorrect identification, variations of chromosome number within a species, the existence of chemical races and geographical influences.

Characters

The drug consists of almost cylindrical rhizomes about 1–5 cm long and 2–10 mm diameter. The rhizomes grow more or less obliquely and bear numerous, short branches, which terminate in cup-shaped scars and bear encircling cataphyllary leaves. Similar scale leaves are found on the rhizome, the outer surface of which is yellowish-brown or greyish-brown. The roots, which originate on the ventral and lateral surfaces, are long and wiry, and in the commercial drug are often broken at a distance of a centimetre or so from the rhizome. The drug breaks with a short, waxy fracture. It has a slight but distinctive odour and bitter taste.

A transverse section of the rhizome shows a fairly thick, yellow or yellowish-brown bark; 12–20 radially elongated, bright yellow wood bundles, separated by wide medullary rays; a large pith.

Constituents

Hydrastis contains the alkaloids hydrastine, berberine, canadine and other minor ones. Commercial samples yield 1.5–4% of hydrastine and 0.5–6.0% of berberine. The latter, as a constituent of an extract of the root, is responsible for activity against multiple drug resistant Mycobacterium tuberculosis (see E. J. Gentry et al., J. Nat. Prod., 1998, 61, 1187)

The BP/EP requires a minimum content of 2–5% (dried drug) for hydrastine and a minimum of 3.0% for berberine. These are determined by liquid chromatography with absorbance measurements at 235 nm using a reference solution of the above alkaloids for comparison. These alkaloids are also identified in the TLC test for the drug using visualization with UV light at 365 nm.

Uses

The use of hydrastis to check uterine haemorrhage, as a bitter stomachic and locally in the treatment of catarrhal conditions of the genito-urinary tract is largely based on empirical observations. Hydrastine hydrochloride and hydrastinine hydrochloride have been used in various forms to control uterine haemorrhage.

Constituents

A range of isoquinoline-type alkaloids includes protoberberines, spirobenzylisoquinolines, benzophenanthridines and indenobenzazepines. Protopine (Fig. 26.19) is the principal alkaloid, the BP/EP requiring a minimum total alkaloid content of 0.40% calculated as this alkaloid. A titrimetric assay is employed, sonication being used for dissolving the plant extract produced in the alkaloid purification procedure.

Other constituents include flavonoids, principally glycosides of quercetin, and acids, including chlorogenic and caffeic acids.

Uses

Fumitory is employed for its choleretic action, see Chapter 29.

Constituents

Greater celandine contains up to 4% of alkaloids including α- and β-allocryptopine, berberine (Fig. 28.1), chelerythrine (Fig. 26.19), chelidonine (Fig. 26.18), chelirubine, choline, coptisine, hydroxychelidonine, hydroxysanguinarine, protopine (Fig. 26.19), sanguinarine (Fig. 26.19), sparteine (Fig. 26.12), and others.

Fig. 26.19 Selected alkaloids of drugs described in the accompanying text.

The BP/EP TLC test for identity produces a number of unidentified separated components; the assay for total alkaloids is performed on an extract of the drug treated in acid solution with the BP chromotropic, sodium salt reagent and absorbance measurements at 570 nm.

Uses

The drug has many traditional uses based on its reputed anodyne, antispasmodic, caustic, diaphoretic, diuretic, hydragogue, narcotic and purgative properties, Some of these activities have support from pharmacological tests involving particular alkaloids. C. majus is also used in homoeopathic practice and in Chinese medicine.

Collection

Attempts to cultivate the drug in various areas do not appear to have been successful and collection is from the wild. The plant possesses a somewhat slender rhizome from which numerous large fusiform roots arise. The older reports state that these are dug up during dry weather (March), the rhizomes are rejected and the roots cut into transverse or oblique slices and dried in the shade. The imported ‘natural calumba’ is frequently washed, brushed and graded, the product being known as ‘washed calumba’.

Macroscopical characters

Calumba occurs in circular or oblique slices. These are usually 2–8 cm diameter and 3–12 mm thick.

The cork is thin, greyish-brown or reddish-brown in colour and longitudinally wrinkled. Within it lies a broad, greenish-yellow zone which extends to the cambium and contains in its outer part isolated sclerenchymatous cells within which are dark-grey, sinuous strands of sieve tissue. The greyish wood, which is separated from the bark by a dark cambium line, shows numerous narrow, radiating lines of yellow vessels separated by abundant parenchyma. The vessels are close together in the region near the cambium and again in the extreme centre of the root, but they are less numerous in the intermediate zone, which therefore shrinks considerably and becomes depressed on drying. Some pieces show two or more concentric zones of wood. The fracture is short and starchy; odour, slight and somewhat musty; taste, bitter.

Calumba frequently contains occasional slices of calumba rhizome. These average about 2–3 cm diameter. The structure is markedly radiate and, owing to its greater woodiness in that region, is not depressed in the centre.

Microscopical characters

The drug is characterized by the sclereids which have unevenly thickened, yellow walls and contain a number of prisms of calcium oxalate, by abundant parenchymatous cells containing starch grains, each grain measuring about 20–85 μm long and having an eccentric, very distinct radiate or cleft hilum, and by the yellow reticulate vessels. The walls of both the sclerenchymatous cells and vessels on treatment with 66% v/v sulphuric acid change colour from yellow to green.

Constituents

Calumba contains about 2–3% of isoquinoline alkaloids, palmatine, jatrorrhizine and columbamine. Bisjatrorrhizine is a quaternary dimeric alkaloid formed by ortho-oxidative coupling of the phenolic group of jatrorrhizine. Other constituents are the non-alkaloidal furanoditerpenes columbin, isocolumbin, palmarin, chasmanthin, jateorin and isojateorin. Some of these occur as glucosides and have been named palmatosides A to G.

Uses

Calumba is used as a bitter tonic and, as it contains no tannin, may be prescribed with iron salts. In the BHP it is specifically indicated for anorexia and flatulent dyspepsia.

Macroscopical characters

The drug has a yellowish colour when fresh, becoming brown on keeping. It consists of small rhizomes bearing the remains of subaerial stems and numerous wiry roots. The rhizomes are about 1–2 cm long and 2–3 mm diameter, while the roots are about 10 cm long and 0.2–1.2 mm diameter. The drug contains up to 10% of subaerial stems. Odour, camphoraceous; taste, camphoraceous and bitter.

A transverse section of the rhizomes shows a starchy, eccentric pith (nearer the upper surface of the rhizome than the lower), wedge-shaped, yellowish vascular bundles separated by wide medullary rays, and a narrow bark.

Allied drugs

Virginian snake-root, from Aristolochia serpentaria, was formerly official but its regular importation has now ceased. It closely resembles the Texan drug, but has smaller rhizome and more wiry roots. Indian aristolochia or Indian birthwort consists of the roots and rhizome of Aristolochia indica; it contains aristolochic acid together with other phenanthrene derivatives, an N-glycoside and steroids. A. heterophylla is used in China, and A. constricta, recently investigated (L. Pastrelli et al., J. Nat. Prod., 1997, 60, 1065), is widely employed in folk medicine in S. America. A. clematis (birthwort) is European and has been used both internally and externally. It contains similar constituents to other species.

Constituents

Many species of Aristolochia including A. reticulata contain aristolochic acid and the tumour-inhibiting properties of this compound are of interest. However, in experimental animals it can cause tumour formation and has been associated with cases of renal failure. Aristolochic acid is not alkaloidal, belonging to a small group of naturally occurring nitro-compounds, but is included here because of its direct derivation from isothebaine derivatives in the plant.

(For a review on the structure of the aristolochic acids and their corresponding lactams (aristolactams) see D. B. Mix et al., J. Nat. Prod., 1982, 45, 657 and for the isolation of aristolochic acids I–IV and aristolactams I–III from A. auricularis see P. J. Houghton and M. Ogutveren, Phytochemistry 1991, 30, 253.)

Uses and dangers

Snakeroot has been traditionally employed as an aromatic bitter but other Aristolochia spp. have also been employed in Chinese herbal medicines for various treatments. As from 28 July 1999 an emergency ban on the import, sale and supply of medicinal products containing Aristolochia spp. came into force for the UK. This arose in part from two UK cases of end-stage renal failure in patients using Chinese medicines containing Aristolochia and from many cases of renal failure in Belgium when Aristolochia was substituted for Stephania in a herbal preparation. Apparently in Chinese herbal preparations it is liable to be substituted for other innocuous components such as Stephania, Akebia or Clematis (MCA/CSM, UK, Current Problems in Pharmacovigilance, 1996, 22, 10; see also the report, Pharm. J., 2000, 265, 10).

History

There is a close botanical relationship between Pareira Brava Radix of the seventeenth- and eighteenth-century pharmacopoeias and the menispermaceous plants yielding curare. In both cases the botanical source of the drugs has long been in doubt. It is now known that Pereira Brava is obtained from two species of Chondrodendron, namely, C. microphyllum, which contains (+)- bebeerine, and C. platyphyllum, which contains (−)-bebeerine. A similar case is the so-called C. tomentosum, which sometimes yields

(+)-tubocurarine and sometimes the less active (−)-tubocurarine. This led King (1948) to write: ‘It seems very probable therefore that two species are involved under the name C. tomentosum’.

Boehm (1895) distinguished three kinds of curare differentiated first by their containers, and second by their different chemical characteristics.

Curare in tins

Much curare has been imported in tins containing about 1 kg of a viscous dark-brown or blackish extract. It has little odour but a very bitter taste.

This drug is similar to the specimen examined by King (1948), who received it from Asher Kates y Cia S.A. of Lima, Peru, together with the leaves of the plant from which it was prepared. These leaves were indistinguishable from those of C. tomentosum and its menispermaceous nature, and similarity to the old tube-curare, was confirmed by the isolation of (+)-tubocurarine chloride and four other alkaloids.

Constituents

(1) Menispermaceous tube-curare and the form now imported in tins contain tubocurarine. From the tin-curare King (1948) isolated (+)-tubocurarine chloride and four non-quaternary bases (isochondrodendrine dimethyl ether, (−)-curine (bebeerine), (+)-chondrocurine and (+)-isochondrodendrine). (2) Loganiaceous calabash-curare derives its activity largely from Strychnos species, particularly S. toxifera. King (1949) has shown that the bark of this species contains 12 crystalline quaternary alkaloids, the toxiferines I–XII, of which two had previously been isolated by Wieland et al. (1937–41). These latter authors examined calabash-curare, probably from Venezuela, and isolated several alkaloids known as C-curarines (C signifies calabash). Toxiferines I and II have been found in calabash-curare.

More recently calabash-curares and Strychnos spp. have been examined by Karrer and his colleagues with the isolation of a large number of new alkaloids. (+)-Tubocurarine is a bisbenzylisoquinoline alkaloid and as such is derived from dopamine, whereas the Loganiaceous curares are indolic and contain C₄₀ compounds of the dimeric strychnine type.

Uses

Curare is now little used except as a source of alkaloids. Tubocurarine chloride, official in the BP/EP, is used to secure muscular relaxation in surgical operations and in certain neurological conditions.

History

What appears to have been ipecacuanha was mentioned, under the name of Igpecaya, by a Portuguese friar around 1600. It was introduced into Europe in 1672.

Collection and preparation

In the Matto Grosso district of Brazil the drug is collected from wild plants. The collector, using a pointed stick, levers the plant from the ground and, having removed most of the roots, replaces it in the ground, where it usually lives to produce further crops. The roots are dried in the sun or by fires and transported down river to ports such as Rio de Janeiro, Bahia and Pernambuco from which they are exported in bales. Other South American ipecacuanhas are collected in a similar way. The supply of S. American ipecacuanha has been erratic for many years as a result of habitat destruction, overcollection and the uprooting of plants. The high price of the drug has stimulated both cultivation in other areas (see above) and the promotion of cell and root cultures for alkaloid production (see below).

Macroscopical characters

The underground portion consists of thin, horizontal rhizomes from the lower surface of which roots are given off. Some of the latter remain thin, while others develop an abnormally thick bark and become annulated.

The Matto Grosso drug occurs in tortuous pieces up to 15 cm long and 6 mm diameter, but it is usually smaller. The colour of the outer surface varies from a deep brick-red to a very dark brown, the colour being very largely dependent on the type of soil in which the plant has been grown. Most of the roots are more or less annulated externally, and some have a portion of the rhizome attached (Fig. 26.20A), while separate portions of rhizome and non-annulated roots are also found. Generally, the drug of present-day commerce is less markedly annulated than was formerly the case, a fact that points to earlier collection. The ridges are rounded and completely encircle the root; here and there the bark has completely separated from the wood.

Fig. 26.20 Ipecacuanha. A, Cephaëlis ipecacuanha roots with rhizome; B, C. acuminata roots with rhizome (both ×1); C, transverse section of root; D, transverse section of rhizome (both ×4); E, cork cells in surface view; F, starch granules (mounted in cold lactophenol); G, idioblast containing calcium oxalate crystals; H, elements from Schultze maceration of wood (all ×200). a₁, Complete annulation of C. ipecacuanha; a₂, incomplete annulation of C. acuminata; ck, cork; e, endodermis; f, fibrous cell; id, idioblast containing calcium oxalate; p, pith; pd, phelloderm; ph, phloem; rh, rhizome; tr.v, tracheid vessel; xy, xylem; xy.p, xylem parenchyma.

The root breaks with a short fracture and shows a thick greyish bark and a small, dense wood, but no pith. The rhizomes, on the other hand, have a much thinner bark and a definite pith (Fig. 26.20C, D). The drug has little odour, but is irritating and sternutatory when in fine powder, and has a bitter taste.

The Costa Rica drug (Fig. 26.20B) is exported from Cartagena and Savanilla. The main differences between the Rio and Cartagena drugs are listed in Table 26.5.

Table 26.5 Comparison of ipecacuanha (Cephaëlis) roots.

Ipecacuanha stems, although containing the same alkaloids as the roots, usually contain them in smaller proportion. An excessive amount of stem must, therefore, be regarded as an adulteration.

Microscopical characters

A transverse section of the root (Fig. 26.20C) shows a thin, brown cork, the cells of which contain brown, granular material. Within this is a wide secondary cortex (phelloderm), the cells of which are parenchymatous and contain starch, usually in compound grains with from two to eight components, or raphides of calcium oxalate. The individual starch grains are muller-shaped and up to 15 or 20 μm diameter. The phloem is entirely parenchymatous, containing no sclerenchymatous cells or fibres. The compact central mass of xylem is composed of small tracheidal vessels, tracheids, substitutefibres, xylem fibres and xylem parenchyma. Starch is present in the xylem parenchyma and substitute fibres contain starch (Fig. 26.20E–H).

The transverse section of ipecacuanha rhizome (Fig. 26.20D) shows the presence of a ring of xylem and a large pith. The pericycle contains characteristic sclerenchymatous cells. Spiral vessels occur in the protoxylem. The pith is composed of pitted parenchyma which shows some lignification.

Adulterants

At one time other ‘ipecacuanhas’ were regularly imported, the name being applied in South America to a number of different roots which were reputed to have emetic properties. Most of these, briefly described in the 10th edition, are very easily distinguished from the genuine drug and are now rarely imported.

Constituents

Ipecacuanha contains the alkaloids emetine (Pelletier and Magendie, 1817), cephaëline (Paul and Cownley, 1894), psychotrine, psychotrine methylether and emetamine. These are isoquinoline derivatives of a group only known with certainty to occur in the families Alangiaceae, Icacinaceae and Rubiaceae. However, emetine-type alkaloids are not necessarily a characteristic of the genus Cephaëlis as a whole as Solis et al. (Phytochemistry, 1993, 33, 1117) recorded a new indole alkaloid and four other known indole alkaloids from the aerial parts of C. dichroa from Western Panama.

In a review (411 refs) of the ipecacuanha and related bases (T. Fujii and M. Ohba, The Alkaloids, 1998, 51, 271) 39 new alkaloids from ipecacuanha and Alangium are recorded for the 14 years to 1997. For further new alkaloids of A. longiflorum, see for example N. Sakurai et al., Phytochemistry, 2006, 67, 894.

Other constituents of the official drug are monoterpenoid isoquinoline glucosides including ipecoside (Fig. 26.22), alangiside and, reported in a series of papers (1989–), a number of other novel glycosides closely related to the known ipecacuanha alkaloids (A. Itoh et al., Phytochemistry, 2002, 59, 91 and the refs cited therein). The iridoid glucosides sweroside and 7-dehydrologanin together with starch and calcium oxalate are also found in the root. Ipecacuanhin and ipecacuanhic acid, originally designated glycosidal tannins, are now thought to have been impure mixtures of ipecoside and sucrose.

As may be seen from Fig. 26.21, the principal alkaloids are closely related to one another; emetine and psychotrine methylether are nonphenolic, whereas cephaëline and psychotrine are phenolic.

Fig. 26.21 Relationship between the principal alkaloids of ipecacuanha.

Thus, emetine, which is the alkaloid usually required in medicine, may be prepared by methylating the cephaëline originally present in the drug. These alkaloids may be regarded as being formed in the plant from two phenylethylamine units and a C₉ terpenoid precursor. The latter is provided in the plant by secologanin and is incorporated via desacetylisoipecoside into the emetine alkaloids (Fig. 26.22). The glucosidic compound ipecoside is formed from the epimer desacetylipecoside (cf. Fig. 26.22, which illustrates the involvement of secologanin in the formation of some indole alkaloids; in this case, however, only the α-epimer, strictosodine, is formed, but it can serve as a precursor for alkaloids with β-configuration). The isolation by Itoh et al., (Chem. Pharm. Bull., 1994, 17, 1460) of tetrahydroisoquinoline-monoterpenoid glucosides, from Alangium lamarckii fruits, with the same stereo-configuration as desacetylisoipecosides supported the role of the latter as an intermediate in the formation of emetine-type alkaloids. Furthermore cell-free extracts of A. lamarckii have been shown to contain two enzyme activities promoting the condensation of dopamine and secologanin to give the (S)- and (R)-products respectively (W. De-Eknamkul et al., Phytochemistry, 1997, 45, 477).

Fig. 26.22 Biosynthetic sequence for the biosynthesis of cephaeline, emetine and the alkaloidal glucoside ipecoside.

Various varieties of ipecacuanha contain different proportions of the principal alkaloids. Thus, the Rio drug, which is now difficult to obtain commercially and is the most esteemed, contains 2–2.4% alkaloids, of which 60–75% is emetine. Those varieties derived from C. acuminata yield 2–3.5% alkaloids, of which emetine may constitute 30–50%.

Cell and root cultures

Production of the ipecacuanha alkaloids by artificial culture would be commercially highly desirable and some research in this area has been reported. The composition of the culture medium and the nature of the added hormones greatly influences alkaloid production but generally root cultures appear to be more satisfactory than callus or suspension cultures. Increased growth of roots is obtained by the induction of hairy roots. In contrast to whole roots, cell cultures produce more cephaëline than emetine, and immobilized cell systems give higher amounts of cephaëline compared with static cell cultures. (For original research papers see K. Yoshimatsu et al., Phytochemistry, 1991, 30, 507; S. Jha et al., ibid., 30, 3999; C. Veeresham et al., ibid., 1994, 35, 947.) The roots of C. ipecacuanha transformed with Agrobacterium rhizogenes grow well in a gamboge medium yielding 112 mg/l of cephaeline and 14 mg/l of emetine after eight weeks of culture (K. Yoshimatsu et al., Planta Med., 2003, 69, 1018).

A simple one-step method for the production of ipecacuanha plants from root cultures has been described (K. Yoshimatsu and K. Shimomura, Plant Cell Rep., 1994, 14, 98).

Test for emetine

Mix 0.5 g of the powdered drug with 20 ml of hydrochloric acid and 5 ml of water; filter, and to 2 ml of the filtrate add 0.01 g of potassium chlorate; if emetine is present, a yellow colour appears, which, on standing for about 1 h, gradually changes to red.

Prepared Ipecacuanha is the drug reduced to a fine powder and adjusted to contain 1.90–2.10% of total alkaloids. It is assayed (BP/EP) by extraction of the alkaloids followed by back-titration of the standarized acid solution with sodium hydroxide.

Uses

Ipecacuanha is used as an expectorant and emetic and in the treatment of amoebic dysentery (Chapter 28). Emetine has a more expectorant and less emetic action than cephaëline, a fact that accounts for the preference shown for the Brazilian drug. In the treatment of amoebic dysentery emetine hydrochloride is frequently given by injection, and emetine and bismuth iodide by mouth. Psychotrine and its O-methyl ether are selective inhibitors of human immunodeficiency virus and their study could lead to the development of therapeutically useful agents (G. J. Tan et al., J. Biol. Chem., 1991, 266, 23529).

Macroscopical characters

The commercial bark which has a slight aromatic odour, occurs in fairly large flattish or curved pieces, up to 60 cm long and 5–20 mm thick. Externally, the cork may be quite extensive and fissured, but the outer layers are missing in some areas and covered by lichen patches in others. The inner surface shows longitudinal striations and is lighter in colour than the grey-brown or orange brown outer tissues.

Microscopical characters

The lignified cork cells occur in bands alternating with layers of lignified parenchyma and sclereids. In the phloem the narrow medullary rays, some cells of which are sclerenchymatous, run between numerous fibre groups, each containing aprism sheath. Many cells contain pigmented contents and a little starch is present. For illustrations showing the macroscopical and microscopical features of the drug, see previous editions.

Constituents

There appears to be no recent work which delineates the active constituents of the drug and it is placed in this position because of its association with ipecacuanha. Arising from work carried out at the end of the nineteenth century, the bark is described as containing 2.3% resins, 2.5% fixed oil, tannin, a small quantity of alkaloid and possibly a glycoside. A study carried out in 1966, which duplicated the earlier methods of extraction and fractionation, gave different results and added credence to the widespread belief that present commercial supplies may not be identical with the earlier ones.

Uses

A liquid extract, with other ingredients, is used in the form of a linctus as an expectorant giving an alternative to ipecacuanha in the treatment of coughs.