Chapter Notes: Cyanogenetic glycosides, glucosinolate compounds, cysteine derivatives and miscellaneous glycosides

Table of Contents
1. Cyanogenetic glycosides
2. Wild cherry bark and related drugs
3. Glucosinolate (mustard oil) compounds
4. Mustard seeds and preparations
5. Cysteine derivatives (Allium sulphoxides)
6. Miscellaneous glycosides
7. Further reading and reviews
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Cyanogenetic glycosides

In addition to the well known groups of glycosides, a number of other plant glycosides are of medicinal and toxicological interest. Cyanogenetic (cyanophoric) glycosides are a large group of plant constituents which yield hydrocyanic acid (HCN, prussic acid) when hydrolysed. They occur widely in the plant kingdom - more than 2 000 species in roughly 110 families contain such compounds. Historically important examples isolated in the 19th century include amygdalin (from bitter almonds), linamarin (from linseed and cassava-related plants) and phaseolunatin (from Phaseolus lunatus).

Chemical nature

Cyanogenetic glycosides are O-glycosides whose aglycone portion is a cyanohydrin derivative (an α-hydroxynitrile). Many are derived from mandelonitrile or related mandelonitrile-type structures. The sugar moiety may be a monosaccharide (for example glucose) or a disaccharide such as gentiobiose or vicianose. When a disaccharide is present, hydrolysis by plant enzymes often proceeds in two stages.

Hydrolysis (general reaction)

Hydrolysis of a typical cyanogenetic glycoside proceeds to give the sugar, the corresponding aldehyde or ketone and hydrogen cyanide. For example, hydrolysis of amygdalin proceeds in stages:

Amygdalin → (enzymatic hydrolysis) → Prunasin + Glucose

Prunasin → (enzymatic hydrolysis) → Benzaldehyde + HCN + Glucose

Qualitative and quantitative tests

Qualitative test (simple, rapid): Finely broken plant material is moistened in a small flask and a strip of filter paper suspended in the neck. The paper is impregnated with either:

• Sodium picrate (yellow) - exposure to HCN converts it to sodium isopurpurate (brick-red).
• Guaiacum resin dried on the paper and then treated with very dilute copper sulphate - the paper turns blue in the presence of HCN.

If endogenous enzymes in the plant material are active, hydrolysis occurs at ambient warm temperature within about an hour. Adding a little dilute sulphuric acid and gentle heating accelerates hydrolysis. The depth of colour on sodium picrate paper may be used for a semiquantitative estimate.

Quantitative determination (classical): Plant material is steamed or acidified with tartaric acid and steam-distilled to collect evolved HCN. The distillate is made up to volume and aliquots titrated with standard silver nitrate (AgNO₃) solution. Modern analytical methods include gas-liquid chromatography (GLC) of trimethylsilyl (TMS) derivatives and other chromatographic assays for direct determination of individual glycosides.

Representative cyanogenetic glycosides and sources

Biogenesis and biosynthesis

The aglycones of cyanogenetic glycosides are biosynthetically derived from amino-acid nitrogenous intermediates. Classic feeding experiments using isotopically labelled amino acids (for example phenyl[¹⁴C]alanine with specific labelling positions and doubly labelled amino acids with ¹⁴C and ¹⁵N) demonstrated that:

• the carbon atoms of the nitrile group derive from specific carbons of the amino-acid precursor;
• the nitrile nitrogen originates from the nitrogen atom of the amino acid;
• intermediates such as aldoximes and nitriles are involved in the conversion from amino acid to cyanogenic glycoside.

Examples: prunasin and linamarin biosynthesis proceeds via oxime and nitrile intermediates derived from phenylalanine (for aromatic cyanogens) or tyrosine/valine-derived precursors for other types (dhurrin from tyrosine, for instance). Reviews and research papers summarising biosynthesis, compartmentation and catabolism of cyanogenic glycosides include Conn (Planta Med., 1991) and reviews by Nahrstedt (Proc. Phytochem. Soc. Europe, 1992) and D. A. Jones (Phytochemistry, 1998).

Wild cherry bark and related drugs

Wild cherry bark (Prunus serotina)

Botanical and commercial notes. The drug is the dried bark of Prunus serotina. The best commercial bark is collected in autumn and should be thoroughly dried and stored in airtight containers.

Macroscopic characters. Curved or channelled fragments of bark are typical, with an outer corky surface bearing whitish lenticels. The inner surface is reddish-brown and shows a striated, reticulately furrowed appearance due to phloem and medullary rays. The bark has a short, granular fracture and, when slightly moist, an odour of benzaldehyde; taste is astringent and bitter.

Microscopy and constituents. Microscopic features include groups of sclereids, prismatic and cluster crystals of calcium oxalate, cork cells with brown contents and starch granules. The bark contains the cyanogenic glycoside prunasin and the enzyme prunase. On hydrolysis the bark yields glucose, benzaldehyde and about 0.07-0.16% hydrocyanic acid. Other constituents reported include benzoic acid, trimethylgallic acid, p-coumaric acid, tannins and a resin giving scopoletin on hydrolysis. Amygdalin has been detected in leaves of several Prunus species by modern analytical methods (Santamour, Phytochemistry, 1998).

Uses. Wild cherry bark is used in cough preparations (syrups, tinctures) for its mild sedative and demulcent properties; it was historically valued for irritable or persistent coughs.

Cherry-laurel leaves (Prunus laurocerasus)

Leaves of Prunus laurocerasus contain the cyanogenic glycoside prulaurasin. Entire leaves have little odour, but when crushed they emit a benzaldehyde odour; a positive cyanogenetic test is obtained. Young small leaves may contain up to about 5% cyanide equivalents, decreasing rapidly with leaf age to roughly 0.4-1.0% in larger leaves.

Glucosinolate (mustard oil) compounds

Mustard oil glycosides - historically isolated as crystalline substances such as sinigrin (black mustard) and sinalbin (white mustard) - are widespread in plants used as condiments (mustard, horseradish) and in several plant families. The anionic glycoside species is now generally termed a glucosinolate. Glucosinolates have the common feature of a β-D-glucopyranosyl moiety linked through sulphur to an O-sulphated oxime (generalised as R-C(=N-O-SO₃-)-S-glucose). On hydrolysis by the enzyme myrosinase (β-thioglucoside glucohydrolase) they yield a variety of hydrolysis products including isothiocyanates (mustard oils), nitriles and other compounds; the particular product depends on substrate and reaction conditions.

Chemotaxonomy and distribution

Glucosinolates are found almost exclusively in dicotyledons and are especially abundant in the families Cruciferae (Brassicaceae), Capparidaceae and Resedaceae, with sporadic occurrences in several other families. The myrosinase enzyme is widely distributed in these plants. Mustard-type glycosides act as defensive compounds, increasing a plant's nonspecific resistance to some microorganisms and herbivores. Several glucosinolates and their hydrolysis products have antithyroid activity and can cause goitre in man when consumed in large amounts.

Biosynthesis

The side chain of glucosinolates is biosynthesised from suitable amino acids (for example methionine, phenylalanine, tryptophan or tyrosine) via nitrogenous intermediates. Isotopic labelling (¹⁴C and ¹⁵N) demonstrates direct incorporation of amino-acid carbon and nitrogen into the glucosinolate skeleton. Aldoximes are important intermediates in the conversion of amino acids to glucosinolates. In the case of sinigrin (the allyl glucosinolate), studies indicate that the carbon chain originates from homomethionine (produced by chain-lengthening of methionine) rather than directly from allylglycine. The sulphur atom present on the thioglucoside moiety can be introduced from organic sulphur sources such as cysteine or from inorganic sulphur sources, depending on the pathway and organism.

Mustard seeds and preparations

Black (brown) mustard

Black or brown mustard seeds are produced by Brassica nigra or Brassica juncea. The seeds are globular (about 1-1.6 mm diameter for black mustard) with a mucilaginous outer epidermis. The seeds contain the glucosinolate sinigrin and the enzyme myrosinase. On maceration with water, enzymatic hydrolysis produces volatile oil containing allyl isothiocyanate (the pungent oil). Black mustard seeds typically give 0.7-1.3% volatile oil, of which over 90% is allyl isothiocyanate. Seeds also contain fixed oil (~27%), proteins (~30%) and mucilage; sinapine hydrogen sulphate is present as a minor constituent.

White mustard

White mustard seeds (Sinapis alba) are larger, yellowish and contain the glucosinolate sinalbin and myrosinase. Hydrolysis yields an isothiocyanate with pungent taste but lower volatility than allyl isothiocyanate; sinapine hydrogen sulphate is also present. White mustard seeds contain approximately 30% fixed oil and about 25% proteins. Both black and white mustard are used as condiments and, traditionally, as rubefacients (mustard plasters) and counterirritants; in larger doses the preparations are emetic.

Cysteine derivatives (Allium sulphoxides)

Derivatives of the amino acid cysteine that occur as S-sulphoxides are characteristic of the genus Allium (onion, garlic, etc.). These sulphoxide precursors are responsible for the characteristic odour and many biological activities of Allium species.

Typical examples:

• Alliin (S-allyl-L-cysteine sulphoxide) - principal constituent in garlic bulbs.
• S-(trans-propen-1-yl)-cysteine sulphoxide - important in onions (lachrymatory factor precursor).

When the plant tissue is disrupted, the enzyme alliinase (stored in separate cells) comes into contact with alliin and converts it, under moist conditions, through allyl-sulphenic acid to allicin (diallyl thiosulfinate) and further to a complex mixture of volatile and non-volatile sulphur compounds such as diallyl disulphide and diallyl trisulphide. These products produce the characteristic garlic odour and are responsible for many pharmacological effects attributed to garlic.

Garlic (Allium sativum)

Garlic bulbs and cloves are used both as food and as a medicinal plant material. Powdered garlic is prepared by chopping and drying or freeze-drying at temperatures not exceeding about 65°C to retain active constituents. Commercial powders may vary depending on source and processing.

Constituents and assay. In the intact bulb the major precursor is alliin. When the bulb is damaged, alliinase converts alliin to allicin, which in turn yields various sulphur compounds during processing. The pharmacopoeial requirement (BP/EP) for dried garlic powder is a minimum allicin content of 0.45% (calculated on the dried drug); high-performance liquid chromatography with a suitable internal standard is used for assay. Volatile oil obtained by steam distillation contains diallyl sulphides and terpenes but not alliin or allicin (these are non-volatile or form under tissue disruption).

Biological activities. Garlic has been reported to possess numerous pharmacological activities; for detailed reviews see standard monographs and compilations (for example Barnes et al., Herbal Medicines, 3rd ed., 2007).

Miscellaneous glycosides

Several other glycoside classes of biological and pharmaceutical importance are summarised below.

Steroidal alkaloidal glycosides

Found particularly in Solanaceae and some Liliaceae, steroidal alkaloidal glycosides are nitrogen-containing steroidal aglycones glycosidically linked to one or more sugar units (typically attached at C-3). Like saponins, many possess haemolytic properties. Examples include:

• α-Solanine - potato (Solanum tuberosum).
• Soladulcin - Solanum dulcamara (bitter-sweet).
• Tomatin - tomato (Lycopersicon esculentum).
• Rubijervine - Veratrum species.

The sugar components (one to four sugars) may be glucose, galactose, rhamnose or xylose. Some steroidal alkaloids show spirosolane ring systems; stereochemistry about nitrogen and C-25 is important in their structural variation (for example solasodine versus 5-dehydrotomatidine as stereoisomeric spirosolanes).

Glycosidal resins

Complex resins in some Convolvulaceae drugs (e.g. jalap, scammony) are glycosidal in nature. Hydrolysis yields sugars such as glucose, rhamnose and fucose together with fatty acids and hydroxylated aglycone portions.

Glycosidal bitter principles

Certain bitter plant constituents are glycosides historically called "bitter principles." Representative examples include:

• Gentiopicroside (gentiopicrin) - gentian root.
• Picrocrocin (picrocroside) - saffron.
• Cucurbitacins - bitter principles of Cucurbitaceae (for example colocynth).

Betalain pigments

Betalains are nitrogen-containing plant pigments found mainly in the order Centrospermae (for example Beta vulgaris, red beet) and in certain Cactaceae and other taxa. They are classified as:

• Betacyanins - red-violet pigments (for example betanin, the glycoside found in beetroot).
• Betaxanthins - yellow pigments (for example indicaxanthin).

Betalains are not flavonoid derivatives; they are structurally distinct nitrogenous pigments. On hydrolysis, betanin yields the aglycone betanidin. Betalains are important as natural food colourants and are of chemotaxonomic significance. For recent advances in betalain research see Strach et al., Phytochemistry, 2003.

Antibiotic glycosides

Certain antibiotics are glycosides. Streptomycin is an example: the aglycone (streptidin, a nitrogen-containing cyclohexane derivative) is linked to the disaccharide streptobiosamine. Streptobiosamine is formed from the rare methylpentose streptose and N-methylglucosamine.

Nucleosides and nucleic acids

Nucleosides and nucleotides are biologically essential N-glycosides in which a sugar unit (ribose or 2-deoxyribose) is linked by an N-glycosidic bond to a purine or pyrimidine base (for example adenine, guanine, cytosine). Conjugation with phosphoric acid yields nucleotides; conjugation with proteins produces nucleoproteins. These N-glycosides differ fundamentally from the O-glycosides discussed elsewhere in this chapter.

Further reading and reviews

• E. E. Conn, "Biosynthesis, compartmentation and catabolism of cyanogenetic glycosides including amygdalin, linamarin and lotaustralin," Planta Med., 1991, 57 (Suppl. Issue No. 1), S1.
• Nahrstedt, Proc. Phytochem. Soc. Europe, 1992, 33, 249 (review on biology of cyanogenetic glycosides).
• D. A. Jones, "Why are so many plants cyanogenetic?", Phytochemistry, 1998, 47, 155 (review).
• L. Du and B. A. Halkier, Phytochemistry, 1998, 48, 1145 (biosynthesis in Brassica/sinapis systems).
• Strach D., Vigt T., Schliemann W., "Recent advances in betalain research," Phytochemistry, 2003, 62(3):247-269.