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26Cyanogeneticglycosides,glucosinolatecompounds andmiscellaneousglycosides

In addition to the important groups of glycosides discussed in previouschapters, there are a number of other groups of some medicinal inter-est. Two of these, the cyanogenetic glycosides and the glucosinolatecompounds, are characteristic of certain groups of plants and havesimilarities 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 importantfoodstuff, having first found methods to remove its poison. In 1830 thecyanogenetic glycoside manihotoxin was isolated from it, and in thesame year amygdalin was obtained from bitter almonds, linamarinfrom linseed and phaseolunatin from a bean, Phaseolus lunatus. Theseyield prussic acid on hydrolysis and were the first discovered cyano-genetic or cyanophoric glycosides. Over 2000 plant species involvingabout 110 families are estimated to be cyanogenetic. Professor Lindley,a teacher of pharmaceutical students in London, realized as early as1830 that the presence or absence of HCN was of taxonomic import-ance and used it as a character for separating the subfamilies of theRosaceae. At the species level the presence or absence of prussic acidmay denote varieties or different chemical races of the same species(e.g. Prunus amygdalus yields both bitter and sweet almonds). Interestin cyanogenetic principles as chemotaxonomic characters continues toreceive much attention, as does the general biochemistry of cyanide inplants and microorganisms.

Many of these glucosides, but not all, are derived from the nitrile ofmandelic acid. Although they contain nitrogen their structure is that ofO-and not N-glycosides. The sugar portion of the molecule may be amonosaccharide or a disaccharide such as gentiobiose or vicianose. If adisaccharide, enzymes present in the plant may bring about hydrolysisin two stages, as in the case of amygdalin (amygdaloside).

Table 26.1 gives some well-known cyanogenetic glycosides isolatedfrom various sources between 1830 and 1907.

TestsTo test for a cyanogenetic glycoside qualitatively the material is wellbroken and placed in a small flask with sufficient water to moisten. Inthe neck of the flask a suitably impregnated strip of filter-paper is sus-pended by means of a cork. The paper may be treated in either of thefollowing ways to give a colour reaction with free hydrocyanic acid.Either sodium picrate (yellow), which is converted to sodium iso-purpurate (brick-red), or a freshly prepared solution of guaiacum resinin absolute alcohol which is allowed to dry on the paper and treatedwith very dilute copper sulphate solution. The latter test-paper turnsblue with prussic acid. If the enzymes usually present in the materialhave not been destroyed or inactivated, the hydrolysis takes place with-in about an hour when the flask is kept in a warm place. More rapidhydrolysis will result if a little dilute sulphuric acid is added and theflask gently heated. The depth of colour produced with sodium picratepaper can be used for semiquantitative evaluations.

For materials containing a fairly high percentage of cyanogeneticglycosides (e.g. bitter almonds) the amount may be determined quanti-tatively by placing the plant in a flask with water and tartaric acid andpassing steam through until all the hydrocyanic acid has distilled into areceiver. The distillate is then adjusted to a definite volume andaliquots titrated with standard silver nitrate solution. More sensitivemethods including the direct determination of individual glycosides byGLC of their TMS derivatives are now available.

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MISCELLANEOUS GLYCOSIDES 330

BiogenesisThe aglycones of cyanogenetic glycosides are derived solely from nitro-gen intermediates. The biosynthesis of prulaurasin (DL-mandelonitrileglucoside) has been studied in the leaves of Prunus laurocerasus.Phenyl[3-14C]alanine, phenyl[2-14C]alanine and phenyl[1-14C]alaninewere fed to the leaves and the hydrolytic products of the isolated glyco-sides were examined. The three labelled precursors gave, respectively,active benzaldehyde and inactive hydrocyanic acid; inactive benzalde-hyde and active hydrocyanic acid; and inactive benzaldehyde and activehydrocyanic acid; and inactive hydrolytic products consistent with thefollowing incorporation:

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

determine the nature of the intermediates involved in the above con-versions and, for prunasin and linamarin, the participation of oximesand nitriles has been demonstrated (Fig. 26.1).

For a report of a lecture on the biosynthesis, compartmentation andcatabolism of cyanogenetic glycosides including amygdalin, linamarinand lotaustralin see E. E. Conn, Planta Med., 1991, 57 (Suppl. Issue No1), SI. Nahrstedt (Proc. Phytochem. Soc. Europe, 1992, 33, 249)reviewed (84 refs) progress concerning the biology of cyanogeneticglycosides.

A review (107 refs) asking ‘Why are so many plants cyanogenetic?’(D. A. Jones, Phytochemistry, 1998, 47, 155) illustrates the continuinginterest in these plants, an interest which is, however, largely non-pharmaceutical.

Wild cherry barkWild cherry bark (Wild Black Cherry or Virginia Prune Bark; PrunusSerotina) is the dried bark of Prunus serotina (Rosaceae). The plantis a shrub or tree widely distributed in Canada and the USA, extend-ing from Ontario to Florida and westward to Dakota and Texas.Commercial supplies are obtained from Virginia, North Carolina andTennessee. The most esteemed bark is collected in the autumn, at whichtime it is most active. After careful drying it should be kept in airtightcontainers.

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Glycoside Source Family Constitution

Amygdalin Prunus amygdalus Rosaceae D(−)-Mandelonitrile-gentiobiosideLinamarin Linum usitatissimum Linaceae Acetone-cyanohydrin-glucosidePrulaurasin Prunus laurocerasus Rosaceae DL-Mandelonitrile-D-glucosideManihotoxin Manihot utilissima Euphorbiaceae Identical with linamarin (q.v.)Dhurrin Sorghum vulgare Gramineae β-Glucoside of p-hydroxymandelonitrileSambunigrin Sambucwas nigra Caprifoliaceae L(+)-Mandelonitrile-D-glucosideVicianin Vicia angustifolia Leguminosae Mandelonitrile-vicianosidePhaseolunatin Phaseolus lunatus Leguminosae Identical with linamarin (q.v.)Prunasin Prunus serotina Rosaceae D(−)-Mandelonitrile-D-glucoside

Table 26.1 Some cyanogenetic glycosides and their sources.

P. laurocerasusC6H5 *C

OH

+ CN

H*CH2 +

Fig..61.1NBiosynthetic pathway for

History. The drug was introduced into American medicine about1787 and appeared in the USP in 1820. It first attracted notice inBritain about 1863.

Macroscopical characters. The drug usually occurs in curved or chan-nelled pieces up to 10 cm long, 5 cm wide and 0.3–4.0 mm thick (Fig.26.2). Much larger pieces of trunk bark, up to 8 mm thick, may be foundbut the BP (1980) maximum thickness is 4.0 mm and is known commer-cially as ‘Thin Natural Wild Cherry Bark’. The branch bark, if unrossed, iscovered with a thin, glossy, easily exfoliating, reddish-brown to brownish-black cork, which bears very conspicuous whitish lenticels. In the rossedbark pale buff-coloured lenticel scars are seen and the outer surface issomewhat rough, some of the cortex having been removed and the phloemexposed. The inner surface is reddish-brown and has a striated and reticu-lately furrowed appearance, which is caused by the distribution of thephloem and medullary rays. Patches of wood sometimes adhere to theinner surface. The drug breaks with a short, granular fracture. When slight-ly moist it has an odour of benzaldehyde. Taste is astringent and bitter.

Features of the microscopy (Fig. 26.2) are numerous groups ofsclereids, prismatic and cluster crystals of calcium oxalate, cork cellswith brown contents, and starch granules.

Constituents. The bark contains prunasin (see above) and theenzyme prunase. Samples on hydrolysis yield glucose, benzaldehydeand about 0.07–0.16% of hydrocyanic acid, Also present are benzoicacid, trimethylgallic acid, p-coumaric acid, some tannin and a resinwhich gives scopoletin on hydrolysis. Modern methods of analysishave allowed detection, for the first time, of amygdalin in the leaves ofseveral 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 mainlyused in cough preparations, to which it gives mild sedative propertiesand a pleasant taste. It was regarded as particularly useful for irritableand persistent coughs.

Cherry-laurel leavesCherry-laurel leaves are obtained from Prunus laurocerasus(Rosaceae), an evergreen shrub common in Europe. They were former-ly official in the fresh state.

The leaves have little odour when entire, but when crushed an odourof benzaldehyde is soon apparent and a positive test for cyanogeneticglycoside is obtained. The cyanide content of small young leaves isreported as 5%, rapidly dropping to about 0.4–1.0% as leaf-sizeincreases. For the structure and hydrolysis of the glucoside prulaurasin,see Table 26.1.

GLUCOSINOLATE COMPOUNDS

Over a century ago sinigrin and sinalbin were isolated in crystallineform from black and white mustards. These and similar glycosideshave since been isolated from many plants, particularly those used ascondiments (e.g. horseradish) or in folk medicine; they have the gener-al structure:

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Fig. 26.2Wild cherry bark. A, Outer surface of bark; B, inner surface (both × 0.5); C, distribution of tissues in TS (× 25). D–J, 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 offibre 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.

In the above formulae, R represents CH2=CHCH2 in sinigrin andp-HOC6H4CH2 in sinalbin; in sinigrin the X represents an atom of potas-sium but can take the form of a more complex cation—for example,sinapine (C16H25O6N), in sinalbin. A suggestion made in 1961 to ration-alize the nomenclature of this enlarging group appears to have foundacceptance. This is that the anion of the formula be designated a glucosi-nolate; thus, sinalbin becomes sinapine, 4-hydroxybenzylglucosinolate.Many such glycosides, with a variety of side-chains, including indolyl,are now known; all contain the β-D-1-glucopyranosyl residue. They havebeen found only in dicotyledonous plants and are particularly abundant inthe families Cruciferae, Capparidaceae and Resedaceae with sporadicoccurrences in the Euphorbiaceae, Tovariaceae, Moringaceae,Tropaeolaceae and Caricaceae. The enzyme myrosinase has a similarwide distribution. With the Cruciferae it has been shown that the mustardoil glycosides significantly increase the non-specific resistance of theplants to microorganisms which disrupt plant cells; they do not appear toaffect the resistance of cruciferous plants to club root infections. Manyglucosinolates have an antithyroid and goitre-inducing effect in man.

BiosynthesisBiosynthesis of the glucosinolates of the relevant Cruciferae takes placeprincipally in the fruit wall with subsequent translocation to the seed.However it has been shown for oilseed rape (Sinapis alba) that the neces-sary enzymes for the biosynthesis of p-hydroxybenzylglucosinolate(derived from tryosine) are present in the seed where a limited synthesisdoes occur (L. Du and B. A. Halkier, Phytochemistry, 1998, 48, 1145).

The earlier feeding experiments with those members of theCruciferae which produce mustard oil glucosides showed that suitableamino acids are converted to thioglucosides by the plant. Doublylabelled (14C, 15N) amino acids afforded glucosides with 14C:15Nratios consistent with direct incorporation:

This means that all intermediates in this conversion are nitrogenouscompounds giving a similar situation to that found in the biosynthesisof cyanogenetic glycosides (see above). Following the work on cyano-genetic compounds, it was then demonstrated (1967) by differentgroups of workers that appropriate aldoximes were effective precursorsof these compounds in flax (linamarin), Cochlearia officinalis (gluco-putranjivan), Lepidium sativum (benzylglucosinolate) and Tropaeolummajus (benzylglucosinolate).

With sinigrin, the thioglucoside found in horseradish leaves and inblack mustard seeds, the most effective precursor of the carbon chainappears to be homomethionine rather than allylglycine which inspec-tion of the sinigrin structure might suggest. Homomethionine arises bychain lengthening of methionine with acetate by a mechanismanalogous to the formation of leucine from valine (see Fig. 19.16).Although the sulphur atom on the thioglucoside moiety may be intro-duced by feeding with methionine, Matsuo (1968) showed the sulphurof DL-[35S]cysteine to be a more efficient precursor. The sulphur of the

bisulphite portion of the molecule is more readily introduced frominorganic sources. Some incorporations consistent with the envisagedpathway for sinigrin are illustrated in Fig. 26.3.

Mustard seedBlack or brown mustard (Sinapsis) is the dried ripe seed of Brassicanigra or of B. juncea (Cruciferae) and their varieties. The formerspecies is cultivated in Europe and the USA, while B. juncea is grownin India and the former USSR.

Characters. The seeds are globular and 1–1.6 mm diameter. The testais dark reddish-brown to yellow and minutely pitted. The cells of theouter epidermis of the testa contain mucilage. The embryo is oily andgreenish-yellow or yellow in colour; it consists of two cotyledons fold-ed along their midribs to enclose the radicle. Powdered mustardacquires a much brighter yellow colour on treatment with alkali.

Constituents. Black mustard seeds contain sinigrin and myrosin andyield after maceration with water 0.7–1.3% of volatile oil. The lattercontains over 90% of allylisothiocyanate. The seeds also contain about27% of fixed oil, 30% of proteins, mucilage and traces of sinapinehydrogen sulphate (cf. white mustard).

Allied drug. White mustard, the seeds of Sinapsis alba, are globularand 1.5–2.5 mm diameter. The testa is yellowish and almost smooth,and contains mucilage in its outer epidermal cells. The kernel is oilyand the cotyledons are folded as in black mustard. On treatment withwater the powder develops a pungent taste but the pungent odour of theblack variety is absent. With alkali the powder acquires a bright yellowcolour.

White mustard seeds contain the glucoside sinalbin and myrosin. Inthe presence of moisture decomposition takes place with the formationof isothiocyanate, sinapine hydrogen sulphate and glucose. The iso-thiocyanate is an oily liquid with a pungent taste and rubefacient prop-erties but, owing to its slight volatility, it lacks the pungent odour ofallylisothiocyanate. Sinapine hydrogen sulphate, which is also found inblack mustard, is the salt of an unstable alkaloid. The seeds also containabout 30% of fixed oil, 25% of proteins and mucilage.

Uses. The mustards have been traditionally used, particularly in theform of plasters, as rubefacients and counterirritants. In large dosesthey have an emetic action. Both varieties are used as condiments.

MISCELLANEOUS GLYCOSIDES

Attention is drawn to the following types of glycoside, some of whichhave been mentioned under other headings.

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Steroidal alkaloidal glycosidesThese are particularly abundant in the families Solanaceae andLiliaceae. Like saponins, they have haemolytic properties. Examplesare α-solanin (potato, Solanum tuberosum), soladulcin (bitter-sweet, S.dulcamara), tomatin (tomato, Lycopersicon esculentum) and rubi-jervine (Veratrum spp.). The sugar components, one to four in number,are attached in the 3-position and may be glucose, galactose, rhamnoseor xylose. The formulae (as shown below), in which part of thesteroidal structure is omitted, illustrate three variations in the E and Fring systems of the aglycones. Solasodine and 5-dehydrotomatidineare stereoisomeric spirosolanes and the configuration of the nitrogenatom is apparently always linked to that at C-25. Thus, solasodine, thenitrogen analogue of diosgenin (q.v.), is ∆5,22β,25α-spirosolen-3β-oland 5-dehydrotomatidine is ∆5,22α,25β-spirosolen-3β-ol (see also‘Chemical Races’, Chapter 12 and ‘Saponins’, Chapter 24).

Glycosidal resinsThe complex resins of the Convolvulaceae such as those found in jalapand scammony (q.v.) are glycosidal; they yield on hydrolysis sugarssuch as glucose, rhamnose and fucose together with normal fatty acidsand the hydroxyl derivatives.

Glycosidal bitter principlesWhile many glycosides have a bitter taste, certain of them weredescribed as ‘bitter principles’ long before their chemical nature waselucidated. These compounds include gentiopicrin or gentiopicrosideof gentian root (q.v.); picrocrocin or picrocroside of saffron (q.v.); andcucurbitacins of the Cucurbitaceae (e.g. colocynth, q.v.).

BetalainsFor many years a group of plant pigments, associated with the orderCentrospermae and containing nitrogen, had been known. These com-pounds were termed ‘nitrogenous anthocyanins’. Following the initialisolation in crystalline form of one such compound in 1957, the struc-tures of two groups of pigments have now been determined; these arethe betacyanins and betaxanthins, the former being red-violet in colourand the latter yellow. These names were derived from a combination ofBeta vulgaris (the red beet) and the anthocyanin and anthoxanthin pig-ments to which they were thought to be related. That these new com-pounds contained nitrogen was confirmed, but they are not flavonoidderivatives (see structures below). Betanin, on hydrolysis, gives theaglycone betanidin; indicaxanthin, although not a glycoside, is includedhere for completeness. Betalains are also responsible for the bright col-orations of the flowers and fruits of the Cactaceae. In this case the sugarmoiety of betanin may be substituted at C-2 and C-6 by malonyl, apio-syl and feruloyl groups. For a report on betalains from Christmas cactussee N. Kobayashi et al., Phytochemistry, 2000, 54, 419. Musca-aurinand muscapurpurin are betalain pigments of the fly agaric, Amanita

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Fig. 26.3Biosynthesis of sinigrin.

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

Antibiotic glycosidesCertain antibiotics are of glycosidal nature. Streptomycin, for example,is formed from the genin streptidin (a nitrogen-containing cyclohexanederivative) to which is attached the disaccharide streptobiosamine. Thelatter is constituted from one molecule of the rare methylpentose strep-tose and one molecule of N-methylglucosamine.

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

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