History

Drugs believed to have been derived from species of Colchicum have long been known under the names of ‘colchicum’, ‘hermodactyl’, ‘surinjan’ and ‘ephemeron’, and some have been identified as C. autumnale. Dioskurides was aware of the poisonous nature of a Colchicum which may or may not have been the species now used in medicine. The genus derives its name from Colchis on the Black Sea, one of the places where this plant is found. The drug was recommended in Arabian writings for use in gout, but it was little employed in either classical or mediaeval times, owing to the wholesome fear inspired by its poisonous properties. Colchicum corm appeared in the London Pharmacopoeias of 1618, 1627, 1632 and 1639. It was then deleted but reappeared in the edition of 1788. The uncertain action of the corm led Dr W. H. Williams, of Ipswich, to introduce the use of the seeds about 1820, and these were admitted to the Pharmacopoeia of 1824. Colchicine was isolated by Pelletier and Caventou in 1820.

Life cycle

The corm consists of an enlarged underground stem bearing foliage leaves, sheathing leaves and fibrous roots. If the plants are examined in the latter part of the summer, it will be found that a new corm is developing in the axil of a scale leaf near the base of the old corm, the new plant occupying an infolding in the side of the parent corm. In September the parent corm bears the remains of recently withered leaves and is very much larger than the daughter corm. For medicinal purposes the corm would have been collected shortly after the withering of the leaves (‘early summer’) and before the enlargement of its axial bud. The corms are surrounded by a dark, membranous coat. The young corm develops fibrous roots at its base, and in August or September two to six flowers emerge from it, but its foliage leaves do not appear above ground until the following spring. The flowers are 10–12 cm long. Each has six stamens and a perianth consisting of six lilac or pale-purple segments which fuse into an exceptionally long perianth tube, at the base of which lies the superior ovary. More than half the length of the flower is below ground, and the fruit lies protected throughout the winter by the surrounding corm and earth. The fruit is a three-lobed, three-celled, septicidal capsule, which is carried above ground in the spring by the expanding leaves. The fully grown leaves are radical, linear-lanceolate and about 12 cm long. During these changes the daughter corm grows at the expense of the parent, which now gradually perishes. Before doing so, however, it may produce in its second spring one or more small corms by means of which the number of plants may be increased.

Characters of seed

The seeds are collected when ripe, usually in July or August, and dried. They are ovoid or globular in shape and 2–3 mm in diameter. They are extremely hard and have a reddish-brown, minutely pitted testa. During drying the seeds darken in colour and become covered with a sugary exudation. The seed, as in most Liliaceae, develops from an amphitropous ovule. From a slight projection at the hilum there extends for about one-quarter of the circumference a well-marked strophiole. The small embryo lies embedded in horny endosperm.

Microscopical examination shows that the testa consists of somewhat thick-walled reddish-brown parenchyma; that the endosperm cells have pitted walls and contain fixed oil and aleurone grains up to 5 mm in diameter; and that the strophiole contains starch.

Colchicum seeds contain 0.6–1.2% of colchicine, a number of other colchicine-type alkaloids, a resin, fixed oil and reducing sugars.

Characters of corm

The corms are collected about July, cut into transverse slices and dried at a temperature not exceeding 65°C. The outer membranes are rejected. The whole corms are 2–3 cm diameter, but the dried drug consists of somewhat reniform, transverse slices and occasional more ovate longitudinal slices, about 2–5 mm thick. The epidermal surface is cinnamon-brown and slightly wrinkled. The interior is white and starchy and, if carefully smoothed, shows scattered fibrovascular bundles. The drug breaks with a short mealy fracture. The odour is much less marked than in the fresh drug. Taste, bitter.

Microscopical examination shows numerous starch grains contained in parenchyma, some simple but the majority consisting of two to seven components. Individual grains are from 6 to 30 μm diameter, and show a triangular or star-shaped hilum. Their shape varies from spherical or ovoid to polygonal. Vessels with a spiral or annular thickening and portions of brownish epidermis with very occasional circular stomata may also be seen.

On treating the drug with 60–70% sulphuric acid or with concentrated hydrochloric acid, a yellow colour, due to colchicine, is produced. The corms contain up to about 0.6% colchicine, other related alkaloids and starch.

Uses

Colchicum preparations are used to relieve gout, but must be employed with caution. Colchicine is frequently prescribed in tablet form and transdermal preparations containing colchicine are the subject of a Japanese patent (1991). The alkaloid is also used in biological experiments to produce polyploidy or multiplication of the chromosomes in a cell nucleus (see Chapter 14).

History

There is considerable doubt as to whether ergot and ergotism were known to the ancients, and it is impossible to say whether the ‘ignis sacer’ of the Romans referred to ergotism. The outbreaks of ‘ignis St Antonii’, or St Antony’s fire, which occurred during the Middle Ages, do, however, appear to have been of ergot origin. Outbreaks of ergotism occurred in Germany in 1581, 1587 and 1596 and at intervals in Europe until recent years. Ergotism was never common in England, probably owing to the fact that rye is little grown, and the only serious outbreak recorded, which took place in 1762, was caused by wheat.

World-wide, sporadic reports of ergot poisoning still appear in the literature and in 1992 an analysis of rye flour sold in Canada showed that low-level contamination by the fungus still exists; of 128 samples tested 118 proved positive for ergot alkaloids at concentrations of 70–414 ng g⁻¹ whereas with wheat flour the incidence and levels were much lower.

The obstetric use of ergot was known in the sixteenth century, but the drug was not widely employed until the nineteenth century. It was first introduced into the London Pharmacopoeia of 1836. The fungoid origin of ergot was recognized by Münchausen in 1764, while the life history of the fungus was worked out and the name Claviceps purpurea given to it by Tulasne in 1853.

Life history and collection

The fungus C. purpurea and other species such as C. microcephala Wallr., C. nigricans Tul. and C. paspali produce ergots on many members of the Gramineae (including the genera Triticum, Avena, Festuca, Poa, Lolium, Molinia and Nardus) and Cyperaceae (including the genera Scirpus and Ampelodesma). Many of these ergots appear to be extremely toxic and to produce typical ergotism.

For the life-cycle and illustrations of the fungus, see earlier editions.

Macroscopical characters

The drug consists almost entirely of sclerotia, the amount of other organic matter being generally limited to not more than 1%. Each sclerotium is about 1.0–4 cm long and 2–7 mm broad; fusiform in shape and usually slightly curved. The outer surface, which is of a dark, violet-black colour, is often longitudinally furrowed and may bear small transverse cracks. Ergot breaks with a short fracture and shows within the thin, dark outer layer a whitish or pinkish-white central zone of pseudoparenchyma in which darker lines radiating from the centre may be visible. Ergot has a characteristic odour and an unpleasant taste.

Powdered ergot when treated with sodium hydroxide solution develops a strong odour of trimethylamine. In filtered ultraviolet light it has a strong reddish colour by means of which its presence in flour may be detected.

Microscopical characters

Ergot shows an outer zone of purplish-brown, rectangular cells, which are often more or less obliterated. The pseudoparenchyma consists of oval or rounded cells containing fixed oil and protein, and possessing highly refractive walls which give a reaction for chitin. Cellulose and lignin are absent.

Constituents

The ergot alkaloids (ergolines) can be divided into two classes: (1) the clavine-type alkaloids, which are derivatives of 6,8-dimethylergoline and have been extensively studied in cultures of the mycelium of the ergot fungus; and (2) the lysergic acid derivatives, which are peptide alkaloids. It is the latter class that contains the pharmacologically active alkaloids that characterize the ergot sclerotium (ergot). Each active alkaloid occurs with an inactive isomer involving isolysergic acid; the inactive isomers are not formed initially in the sclerotium but tend to accumulate as a result of unsuitable processing and poor or long storage. These alkaloids have been studied over many years and were not easy to characterize. Thus ‘ergotoxine’, which since its isolation in 1906 (by Barger and Carr and independently by Kraft) had been accepted as a pure substance, and in the form of ergotoxine ethanosulphonate was formerly used as a standard, was shown to be a mixture of the three alkaloids ergocristine, ergocornine and ergocryptine.

Six pairs of alkaloids predominate in the sclerotium and fall into either the water-soluble ergometrine (or ergonovine) group or the water-insoluble ergotamine and ‘ergotoxine’ groups. Table 26.6 gives the morephysiologically active member of each pair first. Alkaloids of groups II and III are polypeptides in which lysergic acid or isolysergic acid is linked to other amino acids. In the ergometrine alkaloids lysergic acid or its isomer is linked to an amino alcohol. Ergometrine was synthesized by Stoll and Hofmann in 1943. Other, new, peptide alkaloids have been isolated from submerged cultures of C. purpurea and from the field-growing fungus (L. Cvak et al., Phytochemistry, 1996, 42, 231; 1997, 44, 365).

Table 26.6 Alkaloids of ergot.

Among the less important constituents of ergot may be mentioned histamine, tyramine and other amines and amino acids; acetylcholine; colouring matters; sterols (ergosterol and fungisterol); and about 30% fat. The cell walls are chitinous.

Variation in alkaloid constituents. Not only are chemical races very evident in C. purpurea with respect to alkaloid production but also the host plant is not without influence. Thus a new commercial strain of ergot adapted from a wild grass (Anthraxon lancifolius) to rye gave sclerotia containing 0.5% total alkaloids involving ergometrine (33%), ergotamine (17.6%), ergocornine (18.7%) and ergocryptine (22.7%). However, sclerotia produced on the grass as a result of natural infection did not contain ergometrine (K. K. Janardhanan et al., Planta Med., 1982, 44, 166). The application of specific amino acids to maturing sclerotia can also be used to influence the type of alkaloids produced (a technique also used with saprophytic cultures).

For recent studies and references on the investigation of the alkaloid gene cluster in C. purpurea see T. Haarmann et al., Phytochemistry, 2005, 66, 1312.

Alkaloid production in artificial cultures

The artificial culture of the ergot fungus has received considerable attention, and, obviously, large-scale submerged fermentation with selected strains to give alkaloids of choice has commercial possibilities. Abe’s initial work in Japan showed that submerged cultures did not produce the typical alkaloids associated with the sclerotium but, rather, a series of new non-peptide bases (clavines) which unfortunately possessed no significant pharmacological action. Attempts were made by many workers to influence alkaloid production by modification of the culture medium and the fungus strain. As a result of successful experiments in 1960, the commercial manufacture of simple lysergic acid derivatives by fermentative growth of a strain of Claviceps paspali became feasible. The alkaloids produced are converted to lysergic acid which is used for the part-synthesis of ergometrine and related alkaloids. Other strains are now available which produce the peptide alkaloids in culture; not only can different chemical races of the fungus be used to produce specific groups of alkaloids but synthesis can also be directed by the addition of certain amino acids or their analogues to the fermentation liquid. In this way new unnatural alkaloids can be produced.

Flieger et al. (J. Nat. Prod., 1989, 52, 1003) found that with submerged cultures in the postproduction stage both the alkaloid concentration and the composition of the alkaloid mixtures underwent dramatic changes including the production of two new alkaloids, 8-hydroxyergine and 8-hydroxyerginine. (For papers pertaining to the isolation of new and unnatural alkaloids from submerged cultures of C. purpurea see N. C. Perellino et al., J. Nat. Prod., 1992, 55, 424; 1993, 56, 489.)

Biosynthesis

The majority of biosynthetic studies were at first directed to the clavine alkaloids, which could be easily produced in cultures but, until recently, their biological relationship to the lysergic acid derivatives remained obscure. The ergoline nucleus is derived from tryptophan and mevalonate, and current work has involved elucidating the biosynthetic relationship between the various clavine alkaloids, determining which of these intermediates is the true natural precursor of lysergic acid, and studying the initial hydrogen elimination from the C-4 of mevalonate to yield the stereo-configuration of chanoclavine-I. Possible biosynthetic routes for lysergic acid involving two isomeric intermediates are given in Fig. 26.29. A major problem has been not so much in discovering which reactions the fungus can effect when supplied with a given substrate as which route is actually involved in its normal metabolism. As a result of work with cell-free systems, Abe assigned a primary role to 4-isopentenyltryptophan and lysergene rather than to the dimethylallyl compound, agroclavine or chanoclavine. Later work by Gröger et al. (Planta Med., 1980, 40, 109) appeared to favour a scheme involving the latter compounds and this has now been generally substantiated with the intermediates involved with the ring closure between dimethylallyltryptophan and chanoclavine having been investigated (A. P. Kozikowski et al., J. Amer. Chem. Soc., 1993, 115, 2482).

Fig. 26.29 Possible biogenesis of lysergic acid.

Work on the origin of the nitrogen of the peptide portions of the ergot alkaloids indicates that appropriate amino acids are specifically incorporated. Abe’s scheme is shown in Fig. 26.30 and he obtained intact incorporation of the units shown, in certain strains, but workers in Germany were unable to confirm these results. As has been mentioned earlier, unnatural amino acids can also be incorporated into the alkaloids.

Fig. 26.30 Biogenesis of ergot peptide alkaloids (after Abe).

Varieties

Commercial ergot varies considerably in activity from batch to batch, and the differences cannot be fully explained by differences in storage; further, it is often found that inferior-looking ergot is highly active. Such variations are apparently due to the fact that there are a number of different chemical races of C. purpurea and in cultivating ergot by modern methods it is obviously important to prepare the spore-cultures used for infecting the rye from a race of the fungus known to develop ergots having a high content of the requiredalkaloids. Cultivated ergot may contain up to 0.5% of total alkaloids, and 0.15% is a minimum commercial value. In addition, there may be a minimal requirement for water-soluble alkaloids. The alkaloids can be determined by colorimetry, as they give a blue colour with a solution of p-dimethylaminobenzaldehyde.

Substitutes

Ergot of wheat is now imported into Britain and has been used medicinally in France. The sclerotia are shorter and thicker than those of rye. Instances of ergot on barley and rye in Britain, and on wheat and rye in the USA and Canada have been reported.

Ergot of oats has been used medicinally in Algiers. The sclerotia are black in colour, 10–12 mm long and 3–4 mm diameter.

Ergot of diss, which is produced on the Algerian reed Ampelodesma tenax, has appeared in commerce and is said to be highly active. The sclerotia may attain as much as 9 cm in length and are spirally twisted.

Storage

Ergot is particularly liable to attack by insects, moulds and bacteria. After collection it should be thoroughly dried, kept entire, and stored in a cool, dry place. If powdered and not immediately defatted, the activity decreases, but if defatted and carefully stored in an air-tight container, it will remain active for a long period. However, as indicated above, under certain conditions, loss of activity arises by the conversion of the pharmacologically important alkaloids to inactive isomers. Any sample of ergot which shows worm holes or a considerable amount of insect debris will almost certainly deteriorate further on storage.

Uses

Although whole ergot preparations were traditionally used in labour to assist delivery and to reduce post-partum haemorrhage, ergot itself has been largely replaced in the pharmacopoeias by the isolated alkaloids. Only ergometrine produces an oxytocic (literally ‘quick delivery’) effect, ergotoxine and ergotamine having quite a different action. Ergometrine is soluble in water or in dilute alcohol. It is often known, particularly in the USA, as ergonovine. Ergotamine and the semisynthetic dihydroergotamine salts are employed as specific analgesics for the treatment of migraine. Lysergic acid diethylamide (LSD-25), prepared by partial synthesis from lysergic acid, is a potent specific psychotomimetic.

History

The seeds were formerly used by the west African tribes as an ‘ordeal poison’. They were first known in England in 1840. The myotic effect of the drug was noted in 1862 by Fraser, and physostigmine was isolated in 1864 by Jobst and Hesse.

Characters

Calabar beans have a somewhat flattened, reniform shape. They are 15–30 mm long, 10–15 mm wide and up to 15 mm thick. The seeds are extremely hard. The dark brown testa is smooth, except in the neighbourhood of the grooved hilum, which runs the whole length of the convex side and round one end, where it is somewhat wrinkled. On either side of the groove is a well-marked ridge and in the groove itself are the greyish, papery remains of the funiculus. A transverse section shows a large central cavity and two, very hard, concavo-convex cotyledons.

Constituents

The seeds contain the alkaloids physostigmine or eserine, eseramine, isophysostigmine, physovenine, geneserine, N-8- norphysostigmine, calabatine and calabacine. The structure of geneserine, long regarded as an N-oxide, has been revised to include the oxygen in a ring system. The chief alkaloid, physostigmine, is present to the extent of about 0.15%. It is derived from tryptophan (see Fig. 26.26). On exposure to air it oxidizes into a red compound, rubreserine, and should therefore be protected from air and light. Both physostigmine salicylate and sulphate are included in the BP/EP. The former of the two is more stable and non-deliquescent. For both salts there is a colorimetic test for the elimination of eseridine and a non-aqueous titration assay.

Uses

Physostigmine salicylate is used for contracting the pupil of the eye, often to combat the effect of mydriatics. It has also been investigated as an intravenous injection for reversing the effects of a number of sedatives. With Alzheimer’s disease it has shown some evidence of inducing a slight improvement in intellectual and cognitive performance (Pharm. J., 1992, 249, 376) but galanthamine (q.v.) may prove superior. Physovenine has the same order of activity but that of eseramine is much lower.

History

Nux vomica was known in Europe in the sixteenth century and was sold in England in the time of Parkinson (1640), mainly for poisoning animals. Strychnine was isolated in 1817 and brucine in 1819.

Collection and preparation

The fruit is a berry about the size of a small orange. When ripe it has a rather hard orange-yellow epicarp and a white, pulpy, interior in which 1–5 seeds are embedded. The seeds are washed free from pulp and dried. They are exported in small sacks, known as ‘pockets’, holding about 18–25 kg.

Macroscopical characters

Nux vomica seeds are extremely hard and should be boiled in water for at least an hour in order to soften them sufficiently for dissection. The seeds are greenish-grey, disc-shaped, 10–30 mm diameter and 4–6 mm thick. Most of the seeds are nearly flat and regular in shape, but a few are irregularly bent and somewhat oval in outline. The edge is rounded or acute. The testa is covered with silky, closely appressed, radiating hairs. In the centre of one of the flattened sides is a distinct hilum, and a small prominence on the circumference marks the position of the micropyle, which is joined to the hilum by a radial ridge. To examine further, a boiled seed should be cut transversely and another one opened like an oyster by inserting the blade of a small knife or scalpel at a point on the circumference opposite the micropyle. The small embryo with two cordate cotyledons and a cylindrical radicle, the latter directed towards the micropyle, will be seen embedded in a grey, horny endosperm (Fig. 26.31). In the centre of the seed is a slit-like cavity. The seeds are odourless when dry; but if soaked in water and left for a day or two, they develop a very unpleasant odour. They have a very bitter taste.

Fig. 26.31 A, Strychnos nux-vomica seed; B, S. nux-blanda seed. Surface and lateral views of entire seed and inner surface of horizontally split seeds. All ×0.8. m, Micropyle; r, ridge; h, hilum; b, lateral ridge; ep, epidermis; f, area of fusion of two endosperm halves; c, area of central cavity; em, embryo.

(Drawn by Dr. T. D. Turner. For further details, see J. Pharm. Pharmacol., 1963, 15, 594.)

Microscopical characters

A radial section shows a very thin testa consisting of collapsed parenchyma and an epidermal layer of very characteristic lignified hairs (Fig. 41.7M). The latter have a very large, thick-walled base with slit-like pits. Surface irregularities in the bases of the hairs cause them to interlock with one another. The upper portions of the hairs are set at almost a right angle to the bases and all radiate out towards the margin of the seed, giving the testa its characteristic silky appearance. On the ridge connecting hilum and micropyle, however, the hairs are irregularly arranged. The upper part of the wallof the hair is composed of about 10 longitudinal ridge-like thickenings united by a thin wall so that the lignified ribs readily separate from one another on powdering. The lumen is circular in the upper part, but in the base has branches corresponding with the oblique pits in the wall. Fragments of testa, removed from a soaked seed, may be disintegrated by treatment with 50% nitric acid and a little potassium chlorate; the hairs can then be separated.

The endosperm consists of large, thick-walled cells, which are nonlignified and yield galactose and mannose on hydrolysis. When mounted in solution of iodine, they show well-marked protoplasmic threads (plasmodesma) passing through the walls (see Fig. 42.1G) and an oily plasma containing a few aleurone grains and the alkaloids strychnine and brucine. Strychnine is most abundant in the inner part of the endosperm and brucine in the outer layers. The presence of strychnine is shown by mounting a section in a solution of ammonium vanadate in sulphuric acid, when a violet colour is produced; of brucine, by mounting in nitric acid, when a crimson colour is observed.

The length of lignified ribs of the hairs per milligram of nux vomica seed has been used for the determination of the content of seeds in veterinary medicines, see ‘Quantitative Microscopy’.

Constituents

Nux vomica usually contains about 1.8–5.3% of the indole alkaloids strychnine and brucine. Strychnine (formula Fig. 26.26) is physiologically much more active than brucine and the seeds are therefore assayed for strychnine and not for total alkaloids. They usually contain about 1.23% of strychnine and about 1.55% of brucine. Minor related alkaloids include α-colubrine, β-colubrine, icajine, 3-methoxyicajine, protostrychnine, vomicine, novacine, N-oxystrychnine, pseudo-strychnine and isostrychnine.

For a review (188 refs) of recent studies concerning the synthesis of strychnine, see M. Shibasaki and T. Ohshima, The Alkaloids, 2006, 64, 103.

Iridoids of the seeds include the glycoside loganin (Fig. 26.27), loganic acid and 7-O-acetyl loganic acid together with three new iridoids, 6′-O-acetyl loganic acid, 4′-O-acetyl loganic acid and 3′-O- acetyl loganic acid (X. Zhang et al., Phytochemistry, 2003, 64, 1341).

The seeds also contain chlorogenic acid (see Fig. 19.5) and about 3% of fixed oil.

Loganin, although present only in small amounts in the seed, occurs to the extent of about 5% in the fruit pulp together with secologanin; these compounds are intermediates in the biogenesis of the strychnine-type alkaloids (Fig. 26.27).

Seasonal variations in alkaloid content of S. nux-vomica have been studied (K. H. C. Baser and N. G. Bisset, Phytochemistry, 1982, 21, 1423).

Uses

The action of the whole drug closely resembles that of strychnine. The alkaloid was formerly used as a circulatory stimulant in such cases as surgical shock, but its use is now more limited to that of a respiratory stimulant in certain cases of poisoning. Like other bitters, strychnine improves the appetite and digestion, but it has been considerably misused as a ‘general tonic’. Nux vomica is used in Chinese medicine for much the same purposes as in Western medicine and the seeds are usually processed to reduce their toxicity. Heat-treatment of the seeds reduces the normal levels of the principal alkaloids and the amounts of isostrychnine, isobrucine, strychnine N-oxide and brucine N-oxide are increased (B.-C. Cai et al., Chem. Pharm. Bull., 1990, 38, 1295).

Allied drugs

The genus Strychnos continues to attract considerable attention. (For extensive reviews on the taxonomy, chemistry and ethnobotany of the American, African and Asian species see N. G. Bisset et al., Lloydia, 33, 201; 34, 1; 35, 95, 193; 36, 179; 37, 62; 39, 263; also Bisset’s review on alkaloids of the Loganiaceae in Indole and Biogenetically Related Alkaloids, 1980 (eds J. D. Phillipson and M. K. Zenk) p. 27, London: Academic Press; and J. Quetin-Leclercq et al., J. Ethnopharm., 1990, 28, 1, review with c. 150 refs.)

Ignatius beans are the seeds of Strychnos ignatii, a plant occurring in the Philippines, Vietnam and elsewhere. The fruits are larger than those of nux vomica and may contain as many as 30 seeds. These are about 25 mm long, dark grey in colour and irregularly ovoid in shape. The structure closely resembles that of nux vomica, but the testa, which bears irregularly arranged greyish hairs, is easily rubbed off and is almost entirely absent in the commercial drug. The seeds contain about 2.5–3.0% of total alkaloids, of which about 46–62% is strychnine. They are mainly used for the preparation of strychnine and brucine. The seeds of S. ignatii from Java (S. tieute) contain 1.5% strychnine and no brucine and from Hainen (S. hainanensis) mainly brucine with little strychnine.

In addition to the seeds, other parts of the plants of Strychnos spp. may contain alkaloids including strychnine (B. De Datta and N. G. Bisset, Planta Med., 1990, 56, 133; G. Massiot et al., Phytochemistry, 1992, 31, 2873).

S. potatorum, from India, and S. nux-blanda, from Burma, have been substituted for nux vomica; although they contain no strychnine or brucine, seeds of the former have been reported to contain the alkaloid diaboline and its acetyl derivative, triterpenes and sterols. They are best distinguished by means of the ammonium vanadate reagent. The seeds of S. potatorum are used in India for clearing water, whence the specific name. They will also flocculate heavy metal contaminants in water and are capable of mopping up radioactive isotopes from nuclear waste. The protein responsible for this property has now been isolated (Pharm. J., 1994, 252, 238). The tannins present in the seeds are suggested as the possible active constituents associated with the folklore treatment of chronic diarrhoea (S. Biswas et al., Fitoterapia, 2002, 73, 43).

History

Although used in India from time immemorial, it was not until 1942 that favourable reports were published of the use of the drug in powdered form. Since then research workers have studied the pharmacognosy, chemistry, pharmacology and clinical uses of many species of Rauwolfia and of the alkaloids obtained from them.

Collection and preparation

The drug is collected mainly from wild plants, but cultivation of the drug will probably increase as wild plants become more scarce; in parts of India collectors are required to leave some root from each plant in the ground for future growth. Nevertheless, and coupled with the low seed viability, the plant is regarded as an endangered species in India. Consequently, the potential for the regeneration of plants from cell cultures and the possible utilization of nodal culture has received some attention (see C. M. Ruyter et al., Planta Med., 1991, 57, 328; N. Sharma and K. P. S. Chandel, Plant Cell Rep., 1992, 11, 200).

As other species of Rauwolfia are found in India, care is needed to identify the correct plant. When first imported, many commercial samples were found to be adulterated; this was due in many cases to lack of knowledge, and substitution of, or adulteration with, other species has become much rarer in recent years. After collection the drug is cut transversely into convenient-sized pieces and dried.

Macroscopical characters

The first detailed description of the drug was made by Wallis and Rohatgi in 1949. It usually occurs in cylindrical or slightly tapering, tortuous pieces about 2–10 cm long and 5–22 mm in diameter (Fig. 26.32A). The roots are rarely branched and rootlets, 0.5–1 mm in diameter, are rare. Pieces of rhizome closely resemble the root but may be identified by a small central pith; they occasionally have attached to them small pieces of aerial stem.

Fig. 26.32 Rauwolfia serpentina and R. vomitoria roots. A, Root of R. serpentina (×1); a, transverse section (TS) of same (×1); B, root of R. vomitoria, ×1; b, TS of same (×1); C, diagrammatic TS of R. serpentina root (×15); D, diagrammatic TS of R. vomitoria root (×15); E, TS of cork of R. serpentina; F, TS of the secondary wood of R. serpentina; G, fibres and vessel of R. serpentina, isolated by maceration; H, TS of cork of R. vomitoria; I, TS of the secondary wood of R. vomitoria; E, F, G, H and I, (all ×150). ck, cork; f, fibre; g.r, growth ring; m.r, medullary ray; pd, phelloderm; ph, phloem; r, resinous material; s, starch; sc, group of sclereids; v, wood vessel; xy, xylem (J. D. Kulkarni, partly after T. E. Wallis and S. Rohatgi (R. serpentina) and W. C. Evans (R. vomitoria))

The outer surface is greyish-yellow, light brown or brown with slight wrinkles (young pieces) or longitudinal ridges (older pieces); occasional circular scars of rootlets. In this species the bark exfoliates readily, particularly in the older pieces, and may leave patches of exposed wood. The drug breaks readily with a short fracture. The smoothed transverse surface shows a narrow, yellowish-brown bark and a dense pale yellow wood, which occupies about three-quarters of the diameter. Both bark and wood contain abundant starch. Some commercial samples show mould. The recently dried drug has a slight odour which seems to decrease with age. Taste, bitter.

Microscopical characters

The cork is stratified into about two to eight zones (Fig. 26.32E), which consist of smaller and radially narrower suberized but unlignified cells alternating with larger radially broader cells which are lignified. In many pieces much of the cork is exfoliated, and for section cutting it may be best to select pieces with little exfoliation, separate these from the wood and cut sections of the bark and wood separately. Most of the cells of the secondary cortex are parenchymatous and contain starch; isolated latex cells may occur in this region, particularly in the Dehra Dun variety. The phloem is narrow and consists mainly of parenchyma with scattered sieve tissue. Sclerenchyma is absent (distinction from many other species such as R. tetraphylla (R. canescens), R. micrantha, R. densiflora, R. perakensis and R. vomitoria; see Fig. 26.32 C, D). Most of the parenchymatous cells of the bark contain starch grains, and others prisms or conglomerate crystals of calcium oxalate.

The xylem is entirely lignified and usually shows three to six annual rings. The medullary rays, which are one to five cells wide, contain starch and alternate with the rays of the secondary xylem, which consist of vessels, fibres and xylem parenchyma. Compared with many other species of Rauwolfia, the vessels of R. serpentina are small (up to 57 μm) and are less numerous than in most of the likely adulterants. The starch grains are larger in the wood than in the bark and measure from 5–8 to 12–20 μm.

Constituents

Rauwolfia contains at least 40 alkaloids, which total some 0.7–2.4%. Other substances present include phytosterols, fatty acids, unsaturated alcohols and sugars.

In 1931 Siddiqui and Siddiqui isolated ajmaline (rauwolfine), ajmalinine, ajmalicine, serpentine and serpentinine. The chief therapeutically important alkaloids are reserpine (isolated in 1952; formula Fig. 26.26) and rescinnamine (isolated in 1954). These are esters derived from methyl reserpate and trimethoxybenzoic acid in the case of reserpine and trimethoxycinnamic acid in the case of rescinnamine. New alkaloids continue to be isolated; recently, five anhydronium bases (e.g. 3,4,5,6-tetradehydroyohimbine) for the first time (O. Wachsmuth and R. Matusch, Phytochemistry, 2002, 61, 705) and five new indole alkaloids together with a new iridoid glycoside, 7-epiloganin, a new sucrose derivative and 20 known compounds (A. Itoh et al., J. Nat. Prod., 2005, 68, 848).

Cell and root cultures

R. serpentina cell suspension cultures have proved an important tool in the elucidation of monoterpenoid indole alkaloid biogenesis and in this connection the significance, and isolation, of strictosidine synthase has been considered. By the use of cell cultures Stöckigt in 1988 was able to clarify a 10-step biosynthetic pathway from strictosidine to the typical rauwolfia alkaloid, ajmaline. His group has also shown that the principal alkaloid of cell suspension cultures of R. serpentina is raucaffricine occurring in amounts of up to 1–6 g l⁻¹ in the nutrient medium, representing 2.3% of the dried cells, a value some 67 times higher than found for the roots. Ajmalicine, an antiarrhythmic drug, is also produced to the extent of 0.6% (cell dry wt.) together with over 30 different monoterpenoid alkaloids in trace amounts including five glucoalkaloids. Addition of high levels of ajmaline to the cell culture medium promoted the formation of a new group of alkaloids, the raumaclines, not found in the roots (for further details see S. Endress et al., Phytochemistry, 1993, 32, 725 and references cited therein).

Hairy root cultures of R. serpentina, produced by Agrobacterium rhizogenes transformation, synthesized ajmaline (0.045% dry wt.) and serpentine (0.007% dry wt.) (B. D. Benjamin et al., Phytochemistry, 1994, 35, 381); minor alkaloids have also been recorded (H. Falkenhagen et al., Can. J. Chem., 1993, 71, 2201). R. verticillata hairy roots have been shown to produce reserpine and aimaline. Working on hairy root cultures, Stockigt’s group has reported on the isolation of three new monoterpenoid indole alkaloids of the sarpagine group along with 16 known compounds together with the first natural occurrence (cf. cell cultures above), of the rare raumacline type alkaloids (Y. Sheludko et al., J. Nat. Prod., 2002, 65, 1006; Planta Medica 2002, 68, 435).

Two monoterpenoid indole alkaloids and four β-carbolines have been isolated from cultured hybrid cells of Rauwolfia serpentina and Rhazya stricta, not all of the compounds being found in the parent plants (N. Aimi et al., Chem. Pharm. Bull., 1996, 44, 1637).

Standardization

An assay for total alkaloids is not a true measure of therapeutic activity, since only some of the alkaloids have the desired pharmacological action. The BPC 1988 and USP/NF 1995 determine the reserpine-like alkaloids by utilizing the colour reaction between an acid solution of reserpine (and rescinnamine) and sodium nitrite solution.

Allied drugs

The 110 Rauwolfia species classified by Pichon in 1947 were reduced by Woodson in 1957 to 86. Some of these occur in more than one geographic area but their approximate geographic areas are as follows: Central and South America 34, Africa 20, Far East 24, India and Burma 7, Hawaii, New Guinea and New Caledonia 6. A large number of these species have been examined for reserpine and related alkaloids.

In the identification of the roots of species of Rauwolfia useful characters to be seen in transverse sections are: cork (whether stratified or lignified); cortex and phloem (presence or absence of sclereids or fibres); wood (relative number, distribution and size of vessels). As the species vary from herbs to large trees, the roots vary considerably in size. Some samples of drug contain aerial stems, which usually contain less reserpine than the roots and have unlignified pericyclic fibres.

R. tetraphylla (R. canescens, R. hirsuta) is a species of wide distribution—tropical South America, the Caribbean, India, Australia (Queensland). The root was at one time occasionally substituted for R. serpentina and could be recognized by its non-stratified cork, and sclereid groups in the phloem. It has served as a commercial source of reserpine and the alkaloid deserpidine, possibly particularly important as R. serpentina is now classed as an endangered species. Micropropagation protocols have been described for in vitro mass multiplication of the plant (D. Sarma et al., Planta Medica, 1999, 65, 277).

R. nitida is a West Indian species from the root-bark of which 33 indole alkaloids have been isolated.

Uses

Rauwolfia preparations and reserpine are used in the management of essential hypertension and in certain neuropsychiatric disorders. Ajmaline, which has pharmacological properties similar to those of quinidine, is marketed in Japan for the treatment of cardiac arrhythmias.

An estimated 3500 kg of ajmalicine is isolated annually from either Rauwolfia or Catharanthus spp. by pharmaceutical industries for the treatment of circulatory diseases.

Conflicting reports on the possible involvement of the rauwolfia alkaloids in breast cancer engendered a natural hesitation in their use. A report in the Lancet (1976) suggested that the alkaloids do not initiate the carcinogenic process but that they promote breast cancer from previously initiated cells.

Collection and preparation

As the tree may attain a height of 10 m, the roots are larger than those of R. serpentina. Before drying they are cut transversely, but are rarely sliced longitudinally. Roots up to 5 cm or more in diameter are sometimes found but the commercial drug usually consists of much smaller pieces. Occasional shipments have been made consisting of the bark only.

Macroscopical characters

The first detailed description of the drug was published by Evans in 1956. It occurs in cylindrical or flattened pieces, usually 0.15–1.5 cm diameter and up to 30 cm long. The roots taper slightly and are occasionally branched. The outer surface is greyish-brown, longitudinally furrowed or rubbed smooth, since the outer cork easily flakes off. Pieces do not break easily, but the fracture is short in the bark and splintery in the wood. The smoothed transverse surface shows a narrow brown bark and a buff or yellowish, finely radiate wood. Odourless; taste, bitter. Pieces of rootstock with attached stem-bases are sometimes found in the drug.

Microscopical characters

The drug is easily distinguished from R. serpentina by the groups of sclereids in the bark arranged in up to five discontinuous bands and by the large vessels of the wood which are up to 180 mu;m in diameter (Fig. 26.32 D and I).

Constituents

African rauwolfia contains reserpine and rescin-namine and alkaloids of the same type such as reserpoxidine and seredine. Many other alkaloids such as ajmaline, alstonine and yohimbine are also present. Court’s group (see Planta Med., 1982, 45, 105) isolated 42 indole alkaloids from the stem-bark and identified 39. The major alkaloids were heteroyohimbines (especially reserpiline) and N_a-demethyldihydroindoles. The interrelationship of these alkaloids with those in the root of the plant is discussed in the same paper. New indole alkaloids from R. vomitoria extracts have continued to be reported.

Allied drugs

Other African rauwolfias containing reserpine are R. caffra (R. natalensis), R. mombasiana, R. oreogiton, R. obscura, R. cumminsii, R. volkensii and R. rosea. Court and coworkers have made a systematic study of the microscopy and chemistry of African species (for a report see W. E. Court, Planta Med., 1983, 48, 228). The roots of R. caffra closely resemble those of R. vomitoria but the cork has not the same tendency to flake off. In sections the main difference is that in R. vomitoria there are alternating lignified and unlignified cork cells, while in R. caffra all the cork cells are lignified. R. caffra contains the alkaloid raucaffricine—one of relatively few examples of monoterpenoid indole glucoalkaloids within the group. R. mombasiana differs from R. vomitoria in the structure of the wood of the root; R. rosea, R. volkensii and R. obscura lack sclereid development.

History

The natives of South America do not appear to have been acquainted with the medicinal properties of cinchona bark, the bitter taste of which inspired them with fear. Although Peru was discovered in 1513, the bark was first used for the cure of fevers about 1630. The name ‘Cinchona’ is said to be derived from a Countess of Chinchon, wife of a viceroy of Peru who it was long believed was cured in 1638 from a fever by the use of the bark. According to recent study of the Count’s diary, it appears that the Countess never suffered from malaria or other fever during her stay in Peru, and although the Count himself did so, there is no record of his having been treated with cinchona bark. The remedy, which became known as ‘Pulvo de la Condesa’, acquired a considerable reputation and was known in Spain in 1639. The further distribution of the bark was largely due to the Jesuit priests, and the drug became known as Jesuit’s Powder or Peruvian Powder. It first appeared in the London Pharmacopoeia in 1677 under the name of ‘Cortex Peruanus’.

The bark was originally obtained by felling the wild trees, which were exterminated in many districts. Ruiz (1792) and Royle (1839) suggested the cultivation of cinchonas in other parts of the world. Weddell germinated seeds in Paris in 1848, and the plants were introduced into Algiers in the following year but without much success. A further attempt by the Dutch was made in 1854, seeds and plants being obtained from Peru by Hasskarl and introduced into Java. An English expedition under Markham in 1860 led to the introduction of C. succirubra (the most hardy species), C. calisaya, and C. micrantha into India. Seeds of C. ledgeriana were obtained in Bolivia by Charles Ledger in 1865 and were bought by the Dutch for their Javanese plantations. A fascinating book covering Ledger’s exploits is The Life of Charles Ledger (1818–1905) by G. Grammicia, Macmillan Press, London, 1988. World War II and subsequent fighting in Malaya and Vietnam increased the demand for cinchona and stimulated cultivation in Africa and Central and South America.

Cultivation, collection and preparation

The production of cinchona bark is a highly specialized section of tropical agriculture. An acid soil, rainfall and altitude are all important factors in cinchona production. Selection of high-yielding strains is of paramount importance, and grafting techniques with C. succirubra as stock may be employed. Seedlings need careful treatment and propagation to avoid disease attack, etc. Since the mid-1970s a disease of the cinchona tree, known as stripe canker, has posed a threat to the plantations of Central Africa. The disease, also known in Central America, is caused by the phytopathogenic fungus Phytophthora cinnamomi, which causes sunken necrotic stripes in the bark and kills thousands of trees a year.

Special characters

In view of the number of hybrids which are cultivated, the distinction of the various commercial cinchona barks is a matter of some difficulty. In Table 26.7 the notes on four important species have been made as concise as possible to facilitate comparison.

Table 26.7 Comparison of Cinchona species.

Microscopical characters

Cinchona barks have the general microscopical structure shown in Fig. 26.33. The cork is composed of several layers of thin-walled cork cells, arranged in regular radial rows and appearing polygonal in surface view. Their cell contents are dark reddish in colour. Within the cork cambium is a phelloderm of several layers of regular cells with dark walls. The cortex is composed of tangentially elongated, thin-walled cells containing amorphous reddish-brown matter or small starch grains 6–10 µm diameter. Scattered in the cortex are idioblasts containing microcrystals of calcium oxalate and secretion cells. The phloem consists of narrow sieve-tubes showing transverse sieve plates, phloem parenchyma resembling that of the cortex and large characteristic spindle-shaped phloem fibres with thick conspicuously striated walls traversed by funnel-shaped pits. The phloem fibres occur isolated or in irregular radial rows. The distribution and size of the phloem fibres differ in the various species(those of C. succirubra are 350–600–700–1400 mu;m long and 30–40–70–100 mu;m in diameter; for other species see 10th edition). The medullary rays are two or three cells wide, the cells being thin-walled and somewhat radially elongated.

Fig. 26.33 Cinchona bark. A, specimen of Cinchona succirubra (× 0.5); B, transverse section of bark (×25); C, isolated phloem fibres (×50); D, portion of phloem fibre with surrounding parenchyma; E, cork cells in surface view; F, idioblast with calcium oxalate; G, starch (all × 200). ck, Cork; ct, cortex; f, fibres protruding from fracture; id, idioblast; I, lichen patches; l.f, longitudinal fissure; m.r, medullary ray; pd, phelloderm; pg, phellogen; p.f, phloem fibres; s.c, secretory cell; t.f, transverse fissure.

Constituents

Cinchona bark contains quinoline alkaloids (see Fig. 26.34). The principal alkaloids are the stereoisomers quinine and quinidine and their respective 6′-demethoxy derivatives, cinchonidine and cinchonine. The quinine series has the configuration 8S, 9R and the quinidine 8R, 9S (Fig. 26.34); other alkaloids of lesser importance have been isolated. Some of these (e.g. quinicine and cinchonicine) are amorphous. The amount of alkaloids present and the ratios between them vary considerably in the different species and hybrids, also according to the environment of the tree and the age and method of collection of the bark.

Fig. 26.34 Outline of possible biogenetic pathway for Cinchona alkaloids. Marked atoms illustrate the structural changes.

The alkaloids appear to be present in the parenchymatous tissues of the bark in combination with quinic acid and cinchotannic acid. Quinicacid (see Fig. 19.5) is present to the extent of 5–8%. Cinchotannic acid is a phlobatannin and a considerable amount of its decomposition product, ‘cinchona red’, is also found in the bark. Other constituents are quinovin (up to 2%), which is a glycoside yielding on hydrolysis quinovaic acid and quinovose (isorhodeose).

Anthraquinones, which as a group of compounds are associated with the family Rubiaceae (see Table 21.3), are not normally found in quantity in the bark of cinchona as indicated by the isolation of norsolorinic acid, a tetrahydroxyanthraquinone, in 0.0008% yield from the bark of C. ledgeriana. However, they are produced in cell cultures of the plant and by infection of the bark with Phytophthora cinnamomi. The latter case may be associated with a phytoalexin defence mechanism; in infected material, alkaloid production is lowered. In connection with the production of anthraquinones in cell cultures, enzymes associated with the later stages of glycoside formation have been isolated. This work involved glucosidases in C. succirubra cell cultures and theisolation of five distinct glucosyltransferases (EC 2,4,1,-) which catalyse the transfer of the glucosyl moiety from UDP-glucose to the hydroxyl groups of those anthraquinones found in cinchona cultures (e.g. emodin, anthrapurpurin, quinizarin, etc.).

Biosynthesis of alkaloids

Grafting experiments and organ culture suggest that the alkaloids are formed principally in the aerial parts of the plant. Although the quinoline alkaloids have structures which by inspection might suggest anthranilic acid as a biological precursor, they are, in fact, as originally suggested by Janot et al. in 1950, derived from indolic precursors. This has been demonstrated by the specific incorporation of tryptophan (indole moiety), loganin and geraniol (terpenoid moiety) into the quinine of Cinchona spp. The pathway, largely established by Battersby’s and Leete’s groups, involves alkaloids of the serpentine type as illustrated in Fig. 26.34.

The proposed biosynthetic route has been supported and elaborated by enzyme studies. The important role of strictosidine synthase in the initial stages of the biogenesis of some tryptophan-derived alkaloids and its isolation from C. robusta was mentioned at the beginning of this section. The enzyme tryptophan decarboxylase (EC 4.1.1.28), which provides tryptamine, is also involved in these early reactions. An enzyme (cinchoninone: NADPH oxidoreductase) associated with the pathway has been isolated from cells of a suspension culture of Cinchona ledgeriana; it catalyses the reduction of cinchoninone to an unequal mixture of cinchonine and cinchonidine. The enzyme can be resolved (by ion exchange) into two isoenzymic forms both of which have an absolute requirement for NADPH and catalysed reversible reactions. Isoenzyme I acts specifically on cinchoninone in the forward direction of the pathway and on cinchonidine and cinchonine in the reverse direction. Isoenzyme II has a broad specificity acting on all the quinoline alkaloids of cinchona tested.

Allied drugs

The barks of certain species of Remijia (Rubiaceae) contain alkaloids. That of R. pedunculata is quoted (USP) as a source of quinidine. It also contains cupreine, an alkaloid which responds to the thalleioquin test and by methylation forms quinine. False cuprea bark (R. purdiena) contains no quinine but an alkaloid cusconidine and small proportions of cinchonine and cinchonamine.

Cinchona leaf alkaloids

Alkaloids of the indole type (e.g. cinchophylline) are generally considered to typify the leaves, so that it is of interest that Phillipson et al. (J. Pharm. Pharmacol., 1981, 33, 15P) isolated quinine from leaves of C. succirubra grown in Thailand. Thirteen alkaloids have been separated by HPLC.

Uses

Galenicals of cinchona have long been used as bitter tonics and stomachics. On account of the astringent action, a decoction and acid infusion are sometimes used as gargles. Before World War II, quinine was the drug of choice for the treatment of malaria but became largely superseded by the advent of synthetic antimalarials developed during that period. It has, however, remained of importance in Third World countries and has re-emerged as suitable for the treatment of Plasmodium falciparum infections (falciparum malaria) in the many areas where the organism is now resistant to chloroquine and other antimalarials.

Quinidine is employed for the prophylaxis of cardiac arrhythmias and for the treatment of atrial fibrillation; it also has antimalarial properties and like quinine is effective against chloroquine-resistant organisms.

Constituents

Maranham leaves contain about 0.7–0.8% of the alkaloids, pilocarpine, isopilocarpine, pilosine and isopilosine and about 0.5% of volatile oil. An examination of the volatile oil composition of a number of species indicated a total of 22 components occurring throughout the samples. These included monoterpenes (e.g. limonene, sabiene, α-pinene) sesquiterpenes (e.g. caryophyllene) but not in P. jaborandi, and 2-undecanone or 2-tridecanone.

Pilocarpine, the lactone of pilocarpic acid, contains a glyoxaline nucleus and with heat or alkalis is converted into its isomer isopilocarpine. Isopilocarpine occurs in small quantity in the leaf but more is formed during the extraction process. The dried leaves soon lose their activity on storage.

Uses

Salts of pilocarpine (e.g. Pilocarpine Hydrochloride BP/EP and Nitrate BP/EP) are used in ophthalmic practice, as they cause contraction of the pupil of the eye, their action being antagonistic to that of atropine. In early glaucoma treatment they serve to increase the irrigation of the eye and relieve pressure. A study in the USA involving 207 patients suffering from dry mouth resulting from radiation treatment for head or neck cancer indicated that oral pilocarpine can possibly offer relief (Pharm. J., 1993, 251, 215). In 1994 its use was approved for this purpose by the US Food and Drug Administration.

Biogenesis

The ring formation of the purine alkaloids appears to follow the classical scheme for the biosynthesis of purine nucleotides with C₁-moieties arising from such compounds as formates and formaldehyde. Methylamine is also effectively incorporated into the ring system; studies by Suzuki et al. indicated that methylamine is oxidized to formaldehyde and then metabolized as a C₁ compound. For caffeine, the purine bases such as hypoxanthine, adenine and guanine, and the nucleosides can also be incorporated by the plant into the molecule.

In both tea and coffee plants and in suspension cultures of Coffea arabica it has been clearly demonstrated that theobromine is methylated to caffeine. In 1979, as a result of work involving N-methyltransferases, Roberts and Waller suggested the pathway 7-methylxanthosine → 7-methylxanthine (heteroxanthin) → 3,7-dimethylxanthine (theobromine) → 1,3,7-trimethylxanthine (caffeine) which has been substantiated by later work. S-Adenosylmethionine is utilized as a donor of the methyl groups. Attempts to isolate the individual N-methyltransferase enzymes do not yet appear to have been successful due partly to their extreme lability. However progress has been made on their biochemical characterization and their time course during leaf development of Coffea arabica (S. S. Mösli Waldhauser et al., Phytochemistry, 1997, 44, 853). The origin of the caffeine molecule is shown in Fig. 26.36.

Fig. 26.36 Biogenetic origin of the caffeine molecule (SAM = S-adenosylmethionine).

(For a review on this aspect see T. Suzuki et al., Phytochemistry, 1992, 31, 2575.)

History

Cocoa has long been used in Mexico and was known to Columbus and Cortez. Cocoa butter was prepared as early as 1695.

Collection and preparation

Cocoa fruits are 15–25 cm long and are borne on the trunk as well as on the branches. Cocoa plantations are very vulnerable to pest attack and recently modern pheromone technology has been used to control the cocoa pod borer, also known as the cocoa moth (Conopomorpha cramerella), the most serious pest of the crop in S.E. Asia. Collection continues throughout the year, but the largest quantities are obtained in the spring and autumn. The fruits have a thick, coriaceous rind and whitish pulp in which 40–50 seeds are embedded. In different countries the seeds are prepared in different ways, but the following may be taken as typical: the fruits are opened and the seeds, embedded in the whole pulp or roughly separated from it, are allowed to ferment. Fermentation occurs in tubs, boxes or cavities in the earth; the process lasts 3–9 days, and the temperature is not allowed to rise above 60°C. In Jamaica fermentation is allowed to proceed for 3 days at a temperature of 30–43°C. During this process a liquid drains from the seeds, which change in colour from white or red to purple, and also acquire a different odour and taste. After fermentation the seeds may or may not be washed. They are then roasted at 100–140°C, when they lose water and acetic acid and acquire their characteristic odour and taste. Roasting facilitates removal of the testa. The seeds are cooled as rapidly as possible and the testa removed by a ‘nibbling’ machine. The nibs or kernels are separated from the husk by winnowing. Sometimes the seeds are simply dried in the sun but these are not as highly regarded owing to their astringent and bitter taste.

Plain or bitter chocolate is a mixture of ground cocoa nibs with sucrose, cocoa butter and flavouring. Milk chocolate contains in addition milk powder.

Macroscopical characters

Cocoa seeds are flattened ovoid in shape, 2–3 cm long and 1.5 cm wide. The thin testa is easily removed from prepared cocoa beans, but is difficult to remove from those that have not been fermented and roasted. The embryo is surrounded by a thin membrane of endosperm. The cotyledons form the greater part of the kernel and are planoconvex and irregularly folded. Each shows on its plane face three large furrows, which account for the readiness with which the kernel breaks into angular fragments. Both testa and kernel are of a reddish-brown colour, which varies, however, in different commercial varieties and depends on the formation of ‘cacao-red’ during processing.

Constituents

Cocoa kernels contain 0.9–3.0% of theobromine and the husks contain 0.19–2.98% of this alkaloid. The seeds also contain 0.05–0.36% caffeine, cocoa fat or butter (nibs 45–53%, husk 4–8%). During the fermentation and roasting, much of the theobromine originally present in the kernel passes into the husk. The constituents other than fat and theobromine are extremely complex and have been intensely studied in recent years. The fresh seeds contain about 5–10% of water-soluble polyphenols (epicatechol, leucoanthocyanins and anthocyanins) which are largely decomposed during processing, forming the coloured complex formerly known as ‘cocoa-red’. Condensed tannins are also present, and some 84 different volatile compounds, including glucosinolates, are responsible for the aroma (see M. S. Gill et al., Phytochemistry, 1984, 23, 1937).

Theobromine is produced on the commercial scale from cocoa husks. The process consists of decocting the husks with water, filtering, precipitating ‘tannin’ with lead acetate, filtering, removing excess of lead and evaporating to dryness. Theobromine is extracted from the residue by means of alcohol and purified by recrystallization from water.

Theobromine is 3,7-dimethylxanthine (see p. 409), the lower homologue of caffeine (trimethylxanthine). It is isomeric with theophylline (1,3-dimethylxanthine), which occurs in small quantities in tea. Theobromine crystallizes in white rhombic needles. It gives the murexide reaction (see p. 356), and may be distinguished from caffeine by the fact that it is precipitated from a dilute nitric acid solution by silver nitrate. Theobromine sublimes at 220°C, caffeine at 178–180°C.

Callus and suspension cultures of cocoa both produce caffeine, theobromine and theophylline and are considered useful for studying secondary metabolism in vitro.

Uses

Cocoa has nutritive, stimulant and diuretic properties. Theobromine is used as a diuretic. It has less action on the central nervous system than caffeine but is more diuretic. With its isomer, theophylline, the diuretic effect is even more marked. Oil of theobroma (q.v.) is used in pharmacy chiefly as a suppository base.

Allied products

Guarana (Pasta Guarana or Brazilian cocoa) is a dried paste prepared mainly from the seeds of Paullinia cupana (Sapindaceae). The seeds are collected from wild or cultivated plants in the upper Amazon basin by members of the Guaranis tribe. The kernels are roughly separated from the shell, broken and made into a paste with water, starch and other substances being frequently added. The paste is then made into suitable shapes and dried in the sun or over fires.

The drug usually occurs in cylindrical rolls 10–30 cm long and 2.5–4 cm diameter. Portions of broken seed project from the dark chocolate-brown outer surface. When broken, similar fragments project from the fractured surface. The drug has no marked odour but an astringent bitter taste.

Guarana contains 2.5–7.0% of caffeine, other xanthine derivatives, tannins about 12% (‘guarana red’) and other constituents resembling, as far as is known, those of cola and cocoa. Guarana resembles tea and coffee in its action and the powder grated from the masses is used in South America with water to make a drink. In the West it is now a popular remedy for combating fatigue, for slimming, and for the treatment of diarrhoea. The fat content of the drug is stated to effect a slow but steady release of the alkaloids (for a short article on guarana see P. Houghton, Pharm. J., 1995, 254, 435).

Coffee consists of the seeds of Coffea arabica and other species of Coffea (Rubiaceae). It contains caffeine (1–2%), tannin and chlorogenic (caffeotannic) acid (see Fig. 19.5), fat, sugars and pentosans.

Prepared coffee is the kernel of the dried ripe seeds of various species, including C. arabica (Arabica coffee), C. liberica and C. canephora (Robusta coffee) (Rubiaceae), deprived of most of the seed coat and roasted. The kernels are dark brown, hard and brittle, elliptical or planoconvex and about 1.0 cm long. Coffee has a characteristic odour and taste. A decoction is used as a flavouring agent in Caffeine Iodide Elixir BPC (1979). Prepared coffee contains about 1–2% of caffeine, probably combined with chlorogenic acid and potassium. Other constituents include nicotinic acid, fixed oil and carbohydrates caramelized during roasting.

C. arabica, both as whole plants and as cell suspension cultures, has been considerably employed to study purine alkaloid variations and biosynthesis (q.v.).

Tea consists of the prepared leaves of Camellia sinensis (Thea sinensis) (Theaceae), a shrub cultivated in India, Sri Lanka, East Africa, Mauritius, China and Japan. The leaves contain thease, an enzymic mixture containing an oxidase, which partly converts the phlobatannin into phlobaphene. This oxidase may be destroyed by steaming for 30 s. Tea contains 1–5% of caffeine and 10–24% of tannin; also small quantities of theobromine, theophylline and volatile oil. The alkaloid content of the leaves is very much dependent on age and season.

C. sinensis is known locally as the Chinese tea plant, the flower buds and seeds of which contain acylated oleane-type triterpenes (theasaponins) with antiallergic activities (M. Yashikawa et al., Chem. Pharm. Bull., 2007, 55, 57; 55, 598). The flower buds of C. japonica yield noroleane and oleane-type triterpenoids having gastroprotective and platelet aggregation activities (M. Yashikawa et al., Chem. Pharm. Bull., 2007, 55, 606).

Callus and root suspension cultures of C. sinensis have been shown to accumulate caffeine and theobromine (A. Shervington et al., Phytochemistry, 1998, 47, 1535).

The possible beneficial effects of drinking black or green tea have received considerable coverage in the medical and national press. An infusion of tea contains in addition to caffeine a mixture of polyphenols including epigallocatechin-3-gallate possessing strong antioxidant and free-radical scavenging properties. Possible beneficial effects are: inhibition of angiogenesis, a process involving the growth of blood vessels necessary for tumour growth and metastasis; the treatment of genetic haemochromatosis by the inhibition of absorption of iron by tannates and other ligands; treatment of blindness caused by diabetes (an angiogenic related condition); and a lowering of the risk of ischemic heart disease in older men (a finding not substantiated with tea with milk added) see M. G. L. Hertog et al., Amer. J. Clin. Nutr., 1997, 65, 1489; J. P. Kaltwasser, E. Werner, K. Schalk et al., Gut, 1998, 43, 649; Y. Cao and R. Cao, Nature, 1999, 398, 381.

History

Areca was known in China under the name pinlang (probably a corruption of the Malay name for the tree pinang) from at least 100 bc. Immense quantities have been consumed in the East from very early times in the form of a masticatory known as betel, which consists of a mixture of areca nuts, the leaves of Piper betle, and lime. The value of areca as a taenicide was also known in the East.

Collection and preparation

The fruits, of which about 100 are annually borne on each tree, are detached by means of bamboo poles and the seeds extracted. The latter, before exportation, are usually boiled in water containing lime, and dried.

Characters

The areca nut is about 2.5 cm long and rounded conical in shape. Patches of a silvery coat, the inner layer of the pericarp, occasionally adhere to the testa. The deep brown testa is marked with a network of depressed, fawn-coloured lines. The seed is very hard, has a faint odour when broken and an astringent, somewhat acrid taste. Sections of the seed show dark-brown, wavy lines (folds of testa and perisperm) extending into the lighter-coloured interior (ruminate endosperm). At the flattened end of the seed is a small embryo.

Constituents

Areca contains alkaloids which are reduced pyridine derivatives. Of these, arecoline (methyl ester of arecaine) (Fig. 26.38), arecaine (N-methylguvacine) and guvacine (tetrahydronicotinic acid) may be mentioned. Only arecoline, which is present to the extent of 0.1–0.5%, is medicinally important. Ether extraction yields about 14% of fat, consisting mainly of the glycerides of lauric, myristic and oleic acids; subsequent extraction with alcohol yields about 15% of amorphous red tannin matter (areca red) of phlobaphene nature.

Macroscopical characters

Aconite differs in appearance according to the season of collection. The aconite formerly cultivated in England was harvested in the autumn and consisted of both parent and daughter roots. Both are obconical in shape, dark-brown in colour, 4–10 cm long and 1–3 cm diameter at the crown. Most Continental aconite is collected from plants at the flowering stage and therefore consists mainly of parent roots. The parent roots bear the remains of aerial stems and are more shrivelled than the daughter roots, which bear large, apical buds. Rootlets may be present but these are usually broken off. The odour is usually slight but samples vary in this respect. Taste at first slightly sweet, followed by tingling and numbness (taste with care; long chewing may be painful).

Transverse sections cut about one-third of the length from the crown show a stellate cambium with five to eight angles. The amount of lignified tissues is small, the greater part of the root consisting of starch-containing parenchyma of the pith and secondary phloem.

Constituents

Aconite contains terpene ester alkaloids, of which the most important is aconitine.

Aconite also contains other alkaloids such as mesaconitine, hypaconitine, neopelline, napelline and neoline. Hikino et al. (J. Nat. Prod., 1984, 47, 190) isolated eight alkaloids from roots of Swiss origin, five being new to the species.

From ssp. vulgare Arlandini et al., (J. Nat. Prod., 1987, 50, 937) isolated N-deethylaconitine, and Chen et al., (J. Nat. Prod., 1999, 62, 701) obtained twelve diterpenoid alkaloids, characterized by NMR and MS, from the herb and flowers.

The percentage of total alkaloid in the drug is about 0.3–1.2%. About 30% of the total is ether-soluble aconitine. In view of the different groups of alkaloids reported by workers over the years, and the large variation in aconitine contents of roots, it seems that in all probability there is considerable chemical variation between varieties of A. napellus. Aconite also contains aconitic acid (see p. 177) and abundant starch.

Other Aconitum species may contain aconitine or similar alkaloids of very varied toxicity and the hydrolysis products of those given in Table 26.8 may be compared with aconitine. According to Zhu et al. (Phytochemistry, 1993, 32, 767) more than 96 spp. of Aconitum have been studied chemically, resulting in reports regarding over 250 C₁₉-diterpene alkaloids and a number of C₂₀-diterpene alkaloids. For a recent report on the alkaloids of the aerial parts of A. variegatum from the Carpathians and Pyrenees, see J. G. Diaz et al., Phytochemistry, 2005, 66, 837.

Table 26.8 Hydrolysis products of Aconitum alkaloids.

The employment of Aconitum spp. as arrow poisons in China, India and other parts of Asia has been reviewed by Bisset in a series of publications (J. Ethnopharmacology, 1981, 4, 247; 1984, 12, 1; 1989, 25, 1; 1991, 32, 71).

Uses

Aconite is a very potent and quick-acting poison which is now rarely used internally in the UK, except in homeopathic doses. The drug was included in the BPC (1973) and was formerly used for the preparation of an antineuralgic liniment.

History

The North American Indians were aware of the therapeutic activity of American hellebore and it was employed by the early European settlers. Its use spread to England about 1862. In Europe the closely allied drug obtained from V. album had long been used. Until about 1950 veratrums, except as insecticides, were being little used. Since then they have been the subject of much research and are now employed in the treatment of hypertension.

Collection and preparation

The rhizome is dug up in the autumn, often sliced longitudinally into halves or quarters to facilitate drying, and sometimes deprived of many of the roots.

Macroscopical characters

The rhizome, if entire, is more or less conical and 3–8 cm long and 2–3.5 cm wide; externally brownish-grey. The roots, if present, are numerous and almost completely cover the rhizome. Entire roots are up to 8 cm long and 4 mm diameter, light brown to light orange, and usually much wrinkled (for transverse section, see Fig. 41.8H). Commercial American veratrum is more frequently sliced than is the drug from V. album, and more of the roots remain attached to the rhizome. Odourless, but sternutatory; taste, bitter and acrid.

Microscopical characters

The various species of Veratrum resemble one another very closely in microscopical structure. The rhizomes of V. viride and V. album are virtually identical microscopically but minor differences occur in the roots. Microscopical distinction of the powders is nevertheless difficult.

Allied drugs

Youngken (1952) reported on the following substitutes for V. viride, which have been offered commercially: V. album, V. eschscholtzii, V. woodii, V. californicum and what is believed to be a variety of V. viride from Montana. In addition to these, V. fimbriatum has been the subject of chemical investigation.

Constituents

Numerous steroidal alkaloids are present in both V. album and V. viride; over 100 have been recorded in the former and new alkaloids of both groups (see below) continue to be isolated (Atta-ur-Rahman et al., J. Nat. Prod., 1992, 55, 565; K. A. El Sayed et al., Int. J. Pharmacognosy, 1996, 34, 111). V. nigrum L. var. ussuriense is used for the preparation of the Chinese drug ‘Li-lu’, together with other species (W. Zhao et al., Chem. Pharm. Bull., 1991, 39, 549). Both drugs have long been used as insecticides, but their more recent importance results from those alkaloids that have hypotensive properties. Alkaloids present in some other species, e.g. V. californicum, can cause serious damage to livestock grazing in locations where the plant occurs as they have teratogenic properties (see ‘Teratogens of Higher Plants’, Chapter 39).

There are two distinct chemical groups of veratrum steroidal alkaloids and these are now referred to as the jerveratrum and ceveratrum groups.

Jerveratrum alkaloids contain only 1–3 oxygen atoms and occur in the plant as free alkamines and also combined, as glucosides, with one molecule of D-glucose. Examples are pseudojervine derived from jervine and veratrosine derived from veratramine.

Ceveratrum alkaloids are highly hyroxylated compounds with 7–9 oxygen atoms. They usually occur in the plant esterified with two or more various acids (acetic, α-methylbutyric, α-methyl-α-hydroxybutyric, α-methyl-α,β-dihydroxybutyric), but are also found unconjugated. It is these ester alkaloids that are responsible for the hypotensive activity of veratrum; examples are the esters of germine, protoverine and veracevine.

Uses

American veratrum is used for the preparation of Veriloid, a mixture of the hypotensive alkaloids. European veratrum is used for the preparation of the protoveratrines. Both drugs, and the closely related cevadilla seeds (Schoenocaulon officinale), are used as insecticides.