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26
Cyanogenetic glycosides, glucosinolate compounds and miscellaneous glycosides

In addition to the important groups of glycosides discussed in previous chapters, there are a number of other groups of some medicinal interest. Two of these, the cyanogenetic glycosides and the glucosinolate compounds, are characteristic of certain groups of plants and have similarities in their biosynthetic origins.

CYANOGENETIC GLYCOSIDES
The poisonous properties of the roots of Manihot utilissima (cassava) have long been known to primitive tribes; they use it as an important foodstuff, having first found methods to remove its poison. In 1830 the cyanogenetic glycoside manihotoxin was isolated from it, and in the same year amygdalin was obtained from bitter almonds, linamarin from linseed and phaseolunatin from a bean, Phaseolus lunatus. These yield prussic acid on hydrolysis and were the first discovered cyanogenetic or cyanophoric glycosides. Over 2000 plant species involving about 110 families are estimated to be cyanogenetic. Professor Lindley, a teacher of pharmaceutical students in London, realized as early as 1830 that the presence or absence of HCN was of taxonomic importance and used it as a character for separating the subfamilies of the Rosaceae. At the species level the presence or absence of prussic acid may denote varieties or different chemical races of the same species (e.g. Prunus amygdalus yields both bitter and sweet almonds). Interest in cyanogenetic principles as chemotaxonomic characters continues to receive much attention, as does the general biochemistry of cyanide in plants and microorganisms. Many of these glucosides, but not all, are derived from the nitrile of mandelic acid. Although they contain nitrogen their structure is that of O-and not N-glycosides. The sugar portion of the molecule may be a monosaccharide or a disaccharide such as gentiobiose or vicianose. If a disaccharide, enzymes present in the plant may bring about hydrolysis in two stages, as in the case of amygdalin (amygdaloside). Table 26.1 gives some well-known cyanogenetic glycosides isolated from various sources between 1830 and 1907. Tests To test for a cyanogenetic glycoside qualitatively the material is well broken and placed in a small flask with sufficient water to moisten. In the neck of the flask a suitably impregnated strip of filter-paper is suspended by means of a cork. The paper may be treated in either of the following ways to give a colour reaction with free hydrocyanic acid. Either sodium picrate (yellow), which is converted to sodium isopurpurate (brick-red), or a freshly prepared solution of guaiacum resin in absolute alcohol which is allowed to dry on the paper and treated with very dilute copper sulphate solution. The latter test-paper turns blue with prussic acid. If the enzymes usually present in the material have not been destroyed or inactivated, the hydrolysis takes place within about an hour when the flask is kept in a warm place. More rapid hydrolysis will result if a little dilute sulphuric acid is added and the flask gently heated. The depth of colour produced with sodium picrate paper can be used for semiquantitative evaluations. For materials containing a fairly high percentage of cyanogenetic glycosides (e.g. bitter almonds) the amount may be determined quantitatively by placing the plant in a flask with water and tartaric acid and passing steam through until all the hydrocyanic acid has distilled into a receiver. The distillate is then adjusted to a definite volume and aliquots titrated with standard silver nitrate solution. More sensitive methods including the direct determination of individual glycosides by GLC of their TMS derivatives are now available.

CYANOGENETIC GLYCOSIDES GLUCOSINOLATE COMPOUNDS MISCELLANEOUS GLYCOSIDES

327 329 330

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Table 26.1 Glycoside Amygdalin Linamarin Prulaurasin Manihotoxin Dhurrin Sambunigrin Vicianin Phaseolunatin Prunasin Some cyanogenetic glycosides and their sources. Source Prunus amygdalus Linum usitatissimum Prunus laurocerasus Manihot utilissima Sorghum vulgare Sambucwas nigra Vicia angustifolia Phaseolus lunatus Prunus serotina Family Rosaceae Linaceae Rosaceae Euphorbiaceae Gramineae Caprifoliaceae Leguminosae Leguminosae Rosaceae Constitution
D()-Mandelonitrile-gentiobioside Acetone-cyanohydrin-glucoside DL-Mandelonitrile-D-glucoside Identical with linamarin (q.v.) -Glucoside of p-hydroxymandelonitrile L(+)-Mandelonitrile-D-glucoside Mandelonitrile-vicianoside Identical with linamarin (q.v.) D()-Mandelonitrile-D-glucoside

Biogenesis The aglycones of cyanogenetic glycosides are derived solely from nitrogen intermediates. The biosynthesis of prulaurasin (DL-mandelonitrile glucoside) has been studied in the leaves of Prunus laurocerasus. Phenyl[3-14C]alanine, phenyl[2-14C]alanine and phenyl[1-14C]alanine were fed to the leaves and the hydrolytic products of the isolated glycosides were examined. The three labelled precursors gave, respectively, active benzaldehyde and inactive hydrocyanic acid; inactive benzaldehyde and active hydrocyanic acid; and inactive benzaldehyde and active hydrocyanic acid; and inactive hydrolytic products consistent with the following incorporation:
+ CN *CH2

determine the nature of the intermediates involved in the above conversions and, for prunasin and linamarin, the participation of oximes and nitriles has been demonstrated (Fig. 26.1). For a report of a lecture on the biosynthesis, compartmentation and catabolism of cyanogenetic glycosides including amygdalin, linamarin and lotaustralin see E. E. Conn, Planta Med., 1991, 57 (Suppl. Issue No 1), SI. Nahrstedt (Proc. Phytochem. Soc. Europe, 1992, 33, 249) reviewed (84 refs) progress concerning the biology of cyanogenetic glycosides. A review (107 refs) asking Why are so many plants cyanogenetic? (D. A. Jones, Phytochemistry, 1998, 47, 155) illustrates the continuing interest in these plants, an interest which is, however, largely nonpharmaceutical. Wild cherry bark Wild cherry bark (Wild Black Cherry or Virginia Prune Bark; Prunus Serotina) is the dried bark of Prunus serotina (Rosaceae). The plant is a shrub or tree widely distributed in Canada and the USA, extending from Ontario to Florida and westward to Dakota and Texas. Commercial supplies are obtained from Virginia, North Carolina and Tennessee. The most esteemed bark is collected in the autumn, at which time it is most active. After careful drying it should be kept in airtight containers.

P. laurocerasus + N7.09 319.74 Tm.19 117 332.7*CH

C6H5

*C OH

Similarly, phenyl[2-14C]alanine fed to P. amygdalus gives amygdalin with most activity in the carbon atom of the nitrile. Experiments with doubly labelled amino acids have shown that the nitrile nitrogen of the cyanogen is derived from the nitrogen atom of the amino acid. Similar results have been obtained with dhurrin isolated from sorghum seedlings fed with labelled tyrosine. More recent work has sought to

cyanogenetic glycosides. Fig..61.1NBiosynthetic pathway for

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History. The drug was introduced into American medicine about 1787 and appeared in the USP in 1820. It first attracted notice in Britain about 1863. Macroscopical characters. The drug usually occurs in curved or channelled pieces up to 10 cm long, 5 cm wide and 0.34.0 mm thick (Fig. 26.2). Much larger pieces of trunk bark, up to 8 mm thick, may be found but the BP (1980) maximum thickness is 4.0 mm and is known commercially as Thin Natural Wild Cherry Bark. The branch bark, if unrossed, is covered with a thin, glossy, easily exfoliating, reddish-brown to brownishblack cork, which bears very conspicuous whitish lenticels. In the rossed bark pale buff-coloured lenticel scars are seen and the outer surface is somewhat rough, some of the cortex having been removed and the phloem exposed. The inner surface is reddish-brown and has a striated and reticulately furrowed appearance, which is caused by the distribution of the phloem and medullary rays. Patches of wood sometimes adhere to the inner surface. The drug breaks with a short, granular fracture. When slightly moist it has an odour of benzaldehyde. Taste is astringent and bitter. Features of the microscopy (Fig. 26.2) are numerous groups of sclereids, prismatic and cluster crystals of calcium oxalate, cork cells with brown contents, and starch granules. Constituents. The bark contains prunasin (see above) and the enzyme prunase. Samples on hydrolysis yield glucose, benzaldehyde and about 0.070.16% of hydrocyanic acid, Also present are benzoic acid, trimethylgallic acid, p-coumaric acid, some tannin and a resin which gives scopoletin on hydrolysis. Modern methods of analysis have allowed detection, for the first time, of amygdalin in the leaves of several Prunus spp. including P. serotina and a cultivar of P. virginiana (F. S. Santamour, Phytochemistry, 1998, 47, 1537).

Uses. Wild cherry bark in the form of a syrup or tincture is mainly used in cough preparations, to which it gives mild sedative properties and a pleasant taste. It was regarded as particularly useful for irritable and persistent coughs. Cherry-laurel leaves Cherry-laurel leaves are obtained from Prunus laurocerasus (Rosaceae), an evergreen shrub common in Europe. They were formerly official in the fresh state. The leaves have little odour when entire, but when crushed an odour of benzaldehyde is soon apparent and a positive test for cyanogenetic glycoside is obtained. The cyanide content of small young leaves is reported as 5%, rapidly dropping to about 0.41.0% as leaf-size increases. For the structure and hydrolysis of the glucoside prulaurasin, see Table 26.1.

26

GLUCOSINOLATE COMPOUNDS
Over a century ago sinigrin and sinalbin were isolated in crystalline form from black and white mustards. These and similar glycosides have since been isolated from many plants, particularly those used as condiments (e.g. horseradish) or in folk medicine; they have the general structure:

Fig. 26.2 Wild cherry bark. A, Outer surface of bark; B, inner surface (both 0.5); C, distribution of tissues in TS ( 25). DJ, fragments of powder (all 200): D, cork cells in surface view with associated fungal hypha; E, medullary ray in TLS; F, medullary ray in RLS with associated parenchyma; G, portion of fibre of the pericycle; H, sclereids; I, starch; J, prismatic crystals of calcium oxalate. a, Obliquely-cut edge of bark; ck, cork; e, exfoliating cork; g.c, greenish cortex; g.f, granular fracture; l, lenticel; m.r, medullary ray; ox1, ox2, prismatic and cluster crystals respectively of calcium oxalate; r.s, reticulately-marked inner surface; sc.c, sclereid groups of cortex; sc.ph, sclereid groups of secondary phloem; w, adhering wood.

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26

In the above formulae, R represents CH2=CHCH2 in sinigrin and p-HOC6H4CH2 in sinalbin; in sinigrin the X represents an atom of potassium but can take the form of a more complex cationfor example, sinapine (C16H25O6N), in sinalbin. A suggestion made in 1961 to rationalize the nomenclature of this enlarging group appears to have found acceptance. This is that the anion of the formula be designated a glucosinolate; thus, sinalbin becomes sinapine, 4-hydroxybenzylglucosinolate. Many such glycosides, with a variety of side-chains, including indolyl, are now known; all contain the -D-1-glucopyranosyl residue. They have been found only in dicotyledonous plants and are particularly abundant in the families Cruciferae, Capparidaceae and Resedaceae with sporadic occurrences in the Euphorbiaceae, Tovariaceae, Moringaceae, Tropaeolaceae and Caricaceae. The enzyme myrosinase has a similar wide distribution. With the Cruciferae it has been shown that the mustard oil glycosides significantly increase the non-specific resistance of the plants to microorganisms which disrupt plant cells; they do not appear to affect the resistance of cruciferous plants to club root infections. Many glucosinolates have an antithyroid and goitre-inducing effect in man. Biosynthesis Biosynthesis of the glucosinolates of the relevant Cruciferae takes place principally in the fruit wall with subsequent translocation to the seed. However it has been shown for oilseed rape (Sinapis alba) that the necessary enzymes for the biosynthesis of p-hydroxybenzylglucosinolate (derived from tryosine) are present in the seed where a limited synthesis does occur (L. Du and B. A. Halkier, Phytochemistry, 1998, 48, 1145). The earlier feeding experiments with those members of the Cruciferae which produce mustard oil glucosides showed that suitable amino acids are converted to thioglucosides by the plant. Doubly labelled (14C, 15N) amino acids afforded glucosides with 14C:15N ratios consistent with direct incorporation:

bisulphite portion of the molecule is more readily introduced from inorganic sources. Some incorporations consistent with the envisaged pathway for sinigrin are illustrated in Fig. 26.3. Mustard seed Black or brown mustard (Sinapsis) is the dried ripe seed of Brassica nigra or of B. juncea (Cruciferae) and their varieties. The former species is cultivated in Europe and the USA, while B. juncea is grown in India and the former USSR. Characters. The seeds are globular and 11.6 mm diameter. The testa is dark reddish-brown to yellow and minutely pitted. The cells of the outer epidermis of the testa contain mucilage. The embryo is oily and greenish-yellow or yellow in colour; it consists of two cotyledons folded along their midribs to enclose the radicle. Powdered mustard acquires a much brighter yellow colour on treatment with alkali. Constituents. Black mustard seeds contain sinigrin and myrosin and yield after maceration with water 0.71.3% of volatile oil. The latter contains over 90% of allylisothiocyanate. The seeds also contain about 27% of fixed oil, 30% of proteins, mucilage and traces of sinapine hydrogen sulphate (cf. white mustard). Allied drug. White mustard, the seeds of Sinapsis alba, are globular and 1.52.5 mm diameter. The testa is yellowish and almost smooth, and contains mucilage in its outer epidermal cells. The kernel is oily and the cotyledons are folded as in black mustard. On treatment with water the powder develops a pungent taste but the pungent odour of the black variety is absent. With alkali the powder acquires a bright yellow colour.

This means that all intermediates in this conversion are nitrogenous compounds giving a similar situation to that found in the biosynthesis of cyanogenetic glycosides (see above). Following the work on cyanogenetic compounds, it was then demonstrated (1967) by different groups of workers that appropriate aldoximes were effective precursors of these compounds in flax (linamarin), Cochlearia officinalis (glucoputranjivan), Lepidium sativum (benzylglucosinolate) and Tropaeolum majus (benzylglucosinolate). With sinigrin, the thioglucoside found in horseradish leaves and in black mustard seeds, the most effective precursor of the carbon chain appears to be homomethionine rather than allylglycine which inspection of the sinigrin structure might suggest. Homomethionine arises by chain lengthening of methionine with acetate by a mechanism analogous to the formation of leucine from valine (see Fig. 19.16). Although the sulphur atom on the thioglucoside moiety may be introduced by feeding with methionine, Matsuo (1968) showed the sulphur of DL-[35S]cysteine to be a more efficient precursor. The sulphur of the

White mustard seeds contain the glucoside sinalbin and myrosin. In the presence of moisture decomposition takes place with the formation of isothiocyanate, sinapine hydrogen sulphate and glucose. The isothiocyanate is an oily liquid with a pungent taste and rubefacient properties but, owing to its slight volatility, it lacks the pungent odour of allylisothiocyanate. Sinapine hydrogen sulphate, which is also found in black mustard, is the salt of an unstable alkaloid. The seeds also contain about 30% of fixed oil, 25% of proteins and mucilage. Uses. The mustards have been traditionally used, particularly in the form of plasters, as rubefacients and counterirritants. In large doses they have an emetic action. Both varieties are used as condiments.

MISCELLANEOUS GLYCOSIDES
Attention is drawn to the following types of glycoside, some of which have been mentioned under other headings.

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26

Fig. 26.3 Biosynthesis of sinigrin.

Steroidal alkaloidal glycosides These are particularly abundant in the families Solanaceae and Liliaceae. Like saponins, they have haemolytic properties. Examples are -solanin (potato, Solanum tuberosum), soladulcin (bitter-sweet, S. dulcamara), tomatin (tomato, Lycopersicon esculentum) and rubijervine (Veratrum spp.). The sugar components, one to four in number, are attached in the 3-position and may be glucose, galactose, rhamnose or xylose. The formulae (as shown below), in which part of the steroidal structure is omitted, illustrate three variations in the E and F ring systems of the aglycones. Solasodine and 5-dehydrotomatidine are stereoisomeric spirosolanes and the configuration of the nitrogen atom is apparently always linked to that at C-25. Thus, solasodine, the nitrogen analogue of diosgenin (q.v.), is 5,22,25-spirosolen-3-ol and 5-dehydrotomatidine is 5,22,25-spirosolen-3-ol (see also Chemical Races, Chapter 12 and Saponins, Chapter 24).

Glycosidal resins The complex resins of the Convolvulaceae such as those found in jalap and scammony (q.v.) are glycosidal; they yield on hydrolysis sugars such as glucose, rhamnose and fucose together with normal fatty acids and the hydroxyl derivatives. Glycosidal bitter principles While many glycosides have a bitter taste, certain of them were described as bitter principles long before their chemical nature was elucidated. These compounds include gentiopicrin or gentiopicroside of gentian root (q.v.); picrocrocin or picrocroside of saffron (q.v.); and cucurbitacins of the Cucurbitaceae (e.g. colocynth, q.v.). Betalains For many years a group of plant pigments, associated with the order Centrospermae and containing nitrogen, had been known. These compounds were termed nitrogenous anthocyanins. Following the initial isolation in crystalline form of one such compound in 1957, the structures of two groups of pigments have now been determined; these are the betacyanins and betaxanthins, the former being red-violet in colour and the latter yellow. These names were derived from a combination of Beta vulgaris (the red beet) and the anthocyanin and anthoxanthin pigments to which they were thought to be related. That these new compounds contained nitrogen was confirmed, but they are not flavonoid derivatives (see structures below). Betanin, on hydrolysis, gives the aglycone betanidin; indicaxanthin, although not a glycoside, is included here for completeness. Betalains are also responsible for the bright colorations of the flowers and fruits of the Cactaceae. In this case the sugar moiety of betanin may be substituted at C-2 and C-6 by malonyl, apiosyl and feruloyl groups. For a report on betalains from Christmas cactus see N. Kobayashi et al., Phytochemistry, 2000, 54, 419. Musca-aurin and muscapurpurin are betalain pigments of the fly agaric, Amanita

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Antibiotic glycosides Certain antibiotics are of glycosidal nature. Streptomycin, for example, is formed from the genin streptidin (a nitrogen-containing cyclohexane derivative) to which is attached the disaccharide streptobiosamine. The latter is constituted from one molecule of the rare methylpentose streptose and one molecule of N-methylglucosamine.

26
muscaria. Chemotaxonomically these compounds are of considerable interest and are of importance as food colourants (Chapter 33).

Nucleosides or nucleic acids These substances, which are of the highest biological importance, have three components: a sugar unit (either ribose or 2-desoxyribose), a purine or pyrimidine base or bases (e.g. adenine, guanine and cytosine) and phosphoric acid. These are N-glycosides. When conjugated with proteins (q.v.) they form nucleoproteins.