105Supplementary references
4.5 SULPHUR COMPOUNDS .1 Chemistry and distribution
(b) Procedure
Sulphur compounds 179
(1) Three medium-sized fresh carrots are cut into small pieces, covered with ether and stored in the refrigerator for at least 24h.
(2) The ether is then decanted, the extract dried over MgS04and evapo- rated to dryness at below 35°C. The residue is taken up in a small volume of n-hexane and applied as a streak to a narrow (5 X 20 em) and full size (20 x 20cm) silica gel plate.
(3) The plates are developed in benzene-ehloroform (10:1) for 1 h. The smaller plate is then sprayed with 0.4% isatin in conc.H2S04 and heated at 110°C for 5 min. The most intense brown spot is due to fa1carinol and the position of the major band on the unsprayed plate can be determined by comparison with the marker plate.
(4) The band is then scraped off, eluted with ether (3m1) and the UV spectrum measured. Several intense peaks should be obtained, corre- sponding in position to those of fa1carinol (Table 4.4). Extra peaks may also be present due to contamination with related acetylenes. Ifthis is so, fa1carinol can be purified by re-running it on silica gel in pentane-ether (9:1).
(c) Other sources of polyacetylenes
Another plant which can be used as a convenient source of acetylenics is the water dropwort, Oenanthe crocata, which grows abundantly near ditches and streams in temperate regions of the Northern hemisphere.
The roots are poisonous and the plant has been described as the 'five finger death' because of their shape. Fresh roots can be collected, and after careful washing, extracted in the same way as in the carrot experiment above. A TLC plate of a concentrated extract developed in pure chloro- form will show the presence of several acetylenes after spraying with isatin in cone.H2S04• One of the major toxins, a ketone, has spectral maxima at 210, 249, 266, 291, 314 and 336nm.
4.5 SULPHUR COMPOUNDS
180 Organic acids, lipids and related compounds
disulphides of Allium, since the flavours of mustard, radish, onion and garlic are due to these substances. A third group are the acetylenic thiophenes found in roots and leaves of members of the Compositae.
There are also a few miscellaneous sulphur derivatives, such as the sul- phur alkaloids of Nuphar, Nympheaceae, which will not be considered further here.
The glucosinolates are thein vivo precursors of the mustard oils them- selves and only yield acrid volatile flavours after enzymic hydrolysis. This occurs whenever fresh crucifer tissue is crushed, since the glucosinolates are always accompanied by the appropriate hydrolytic enzyme, a thioglucosidase known as myrosinase. The products of enzymic release are the isothiocyanates, formed by hydrolysis and rearrangement of the parent thioglucosides according to the scheme shown in Fig. 4.9. Thus, when screening plants for mustard oil glycosides, it does not matter whether detection is via the glucosinolate, the isothiocyanate formed on hydrolysis or the enzyme myrosinase.
About seventy glucosinolates are known, the majority being aliphatic derivatives (e.g. glucocapparin, sinigrin) with the remainder having ben- zyl substituents (e.g. sinapine) of one sort or another. The basic structure is the same (see. Fig. 4.9) and the sulphur-linked sugar is always glucose.
Glucosinolates are biosynthesized from amino acids, the pathway being
Glucosinolates
Sulphides
S-Glc
R - ! Myrosinase.. R-N ==C==S+Glc+HS04"
~N-O-S03
-
R=CH3-, glucocapparin R=CH2==CH-CH2-,sinigrin R=MeS(CH2>:J-,glucoibervirin R=PhCH2-,glucotropaeolin R=p-HO-C6H4-,sinapine
Me-S-S-Me dimethyldisulphide n-Pr-S-S-Pr-n di-n-propyldisulphide
CH2==CH-CHrS-S-CHrCH=C~diallyldisulphide CH2=CH-CHrS-S-CH3 methlallyldisulphide CH2=CH-S-CH=CH2diallysulphide
Thiophenes
0-0-f[J
S S S
Fig. 4.9 Sulphur compounds of plants.
Sulphur compounds 181 related to that utilized for cyanogenic glucoside sYnthesis (see p. 214).
Their function in plants is still being elucidated; they are known to have antibacterial properties and some are feeding attractants to caterpillars and aphids feeding on crucifers.
The glucosinolates are universally distributed in the Cruciferae and also occur in related families in the same plant order, the Rhoeadales, namely the Capparaceae, Resedaceae, Tovariaceae and Moringaceae. The absence of mustard oil glucosides from the Papaveraceae, a family often closely associated with the Cruciferae, is good chemotaxonomic evidence for the separation of the poppy family into another plant order (Kjaer, 1963;
1966). There are a number of miscellaneous occurrences in unrelated plant groups, notably of glucotropaeolin (giving benzyl isothiocyanate) in seeds ofCarica papaya,Caricaceae, the classic source of the proteolytic enzyme, papain.
Sulphides, which are widely distributed in the species of Allium (Saghir et al., 1968; Bernhard, 1970) are immediately recognizable by their pun- gent smells and lachrymatory properties. They probably occur in intact tissue bound as sulphur amino acids; during isolation, they can give rise by decomposition to alkyl thiols (e.g. methylmercaptan, MeSH), sub- stances with even more repellent odours. The sulphides of Allium are mainly alkyl mono- or disulphides; the structures of some typical mem- bers are illustrated in Fig 4.9.
Finally, there are the thiophenes, a remarkably diverse group of natural products (Ettlinger and Kjaer, 1968) occurring almost entirely in one fam- ily, the Compositae, and then in association with polyacetylenes (see p.
174). The first to be discovered a-terthienyl, found in marigold, Tagetes petal, is a simple trimer of thiophene itself, but most of the 100 or so thiophenes have side-chains containing acetylenic substitutions (e.g.
methylphenyltriacetylene from Coreopsis). They are normally isolated along with the polyacetylenes and are purified and identified by very similar procedures.
4.5.2 Recommended techniques (a) Glucosinolates
These are extracted with boiling alcohol from fresh tissue; it is important to avoid enzYmic hydrolysis during this procedure. They are purified on columns of anion exchange resin (Amberlite IR-400) or of acid washed 'anionotropic' alumina (Kjaer, 1960). They can then be separated and isolated by PC in n-butanol-ethanol-water (4: 1: 4) or n-butanol- pYridine-water (6: 4: 3), detection being by means of ammoniacal AgN03•Alternatively, TLC on silica gel can be used, with solvents such as chloroform-methanol (17: 3) and ethyl acetate-ethanol-water (9: 1: 2),
182 Organic acids, lipids and related compounds
when they can be detected as yellow spots with iodine vapour (Matsuo, 1970).
The HPLC of aromatic and indolic glucosinolates can be conducted on Nuc1eosil 5 C]8 columns using an ion-pairing reagent, tetraheptyl ammo- nium bromide, in phosphate buffer and aqueous MeOH with UV detection at 235 nm.
For quantifying ~ucoSinolatesin plant extracts two indirect methods are recommended.,.The first provides the concentration of total glucosinolate and involves hydrolysis of the purified glucosinolate frac- tion with myrosinase and measuring the glucose released with a standard glucose assay kit (see Chapter 6). The second provides the concentrations of individual glucosinolates that may be present and requires hydrolysis of the purified glucosinolate fraction with sulphatase for 30 min and sub- sequent HPLC separation of the desulphoglucosinolates produced. These are separated on a Spherisorb 0052 column, with gradient elution with 0.5% and 20% MeCN in H20 at 30°C and UV detection at 230nm (Heaney and Fenwick, 1993).
(b) Isothiocyanates
These are obtained by steam-distillation of crushed crucifer tissue, or by enzymic or acid hydrolysis of isolated glucosinolates. They have a charac- teristic pungent taste and smell. The non-volatile isothiocyanates can be separated on paper in similar solvents as for glucosinolates, detection being with ammoniacal AgN03• TLC is carried out on silica gel G in carbon tetrachloride-methanol-water (20: 10:1). The volatile iso- thiocyanates are either separated by GLC or, more frequently, by PC as their thiourea derivatives. Thioureas are prepared by warming 0.3 g of the crude isothiocyanate oil with 1 ml 95% ethanol and 1 ml 25% NH40H. An exothermic reaction takes place and on cooling, the thiourea derivative(s) should crystallize out. The derivatives are then separated either on paper in water-saturated chloroform or n-butanol-ethanol-water (4: 1: 4) or on silica gel G plates in ethyl acetate-ehloroform-water (3:3:4) (Wagneret al., 1965). Thioureas give characteristic blue colours on treatment with Grote's reagent. This is prepared by adding two drops of bromine to a mixture of sodium nitroprusside (0.5 g), water OOm1), hydroxylamine hydrochloride (0.5 g) and NaHC030 g); after aeration and filtration, the solution is made up to 25ml and is stable for up to 2 weeks (Grote, 1931).
(c) Sulphides
These have been routinely separated in the volatile fraction of Allium bulbs or leaves, by GLC on 5% Carbowax 20m on a Firebrick 000-120 mesh) support using a 3mX3.2mm column at 90°C. In order to improve
General references 183
the sensitivity, it is advantageous to use dual channel GLC, with both hydrogen flame and electron capture detectors (Saghir et al., 1964;Bernhard, 1970). This procedure gives good separation of the various alkyl sulphides present.
A protocol for the separation and identification of the sulphides and other volatile sulphur compounds in garlic using GC-MS has been de- scribed by Vernin and Metzger(1991).
(d) Thiophenes
These are isolated and purified by similar procedures as for polyacetylenes (see p.176).TLC is carried out on silica gel or Al203plates in pentane-ether (9:1)or benzene-ehloroform(10:1). Plates are sprayed with0.4%isatin in cone.H2S04and then heated. Thiophenes give intense purple, red, blue or blue-green colours and can be distinguished from simple polyacetylenes, which give brown or green colours. The presence of a thiophene in a plant extract has to be confirmed by isolation of the pure compound, followed by spectral studies. 2,5-Disubstituted thiophenes, for example, have a characteristic band at 838 em-1 inthe IR spectrum (measured in KBr) and also several distinctive signals in the NMR spectrum.
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