Phenolic lactones as chemotaxonomic indicators in the genera Leucadendron and Leucospermum (Proteaceae)

Analysis by paper chromatography has shown that 58 out of 59 Leucadendron spp., so far studied, contain the phenolic lactone leucodrin as a leaf metabolite; similarly, out of 30 Leucospermum spp. studied, about half contain conocarpin, a diastereoisomer of leucodrin, while a further number contain leucodrin itself. These results have a bearing on the taxonomy of these two genera of the family Proteaceae and indicate the possibility of chemotaxonomic assistance in the grouping of the species of these genera. S. Afr. J. Bot. 1984, 3: 103-107

A paper-chromatographic analysis was subsequently developed to allow screening of a large number of spp. for these leaf constituents; the optimal conditions follow hereunder. Thin layer chromatography on silica gel was more rapid, but afforded less information, as leucodrin and conocarpin showed the same RF value in various solvent mixtures.
An air-dried sample (200 mg) was continuously extracted (Soxhlet) with methanol for 16 h, the extract evaporated to dryness and the residue shaken at room temperature with five times (volume in cm 3 ) its mass (in g) of ethanol-watel (1 + 1 v/v). This solution was filtered (cotton wool) to afford a clear filtrate for analysis.
A Whatman No. 2 filter paper sheet (46 x 16 em) was drawn through a solution of glycerol (10%) in methanol and allowed to dry in air for 30 min. Spots of 1, 2 and 5 mm 3 of the extract solution were applied at 2 em spacing together with a reference spot of Ieucodrin (20 f.J,g) on a base line 6 em    from the narrow edge of the paper. The prepared sheet was equilibrated for 10 h in a tank over water saturated with n-butanol and toluene, and then eluted by descending chromatography with n-butanol-toluene (1:1 v/v, saturated with water) for 6 h when the solvent front was at some 30 em beyond the base line. The sheet was air-dried and sprayed with Pauly's reagent, i.e. diazotized sulphanilic acid (50 mg) in 10% sodium carbonate solution in water (10 cm 3 ). [The same spots could be revealed with Fast Blue Salt B but the colours were then practically all of the same blue tint and hence less informative.] The colour of each spot was assessed after the sprayed chromatogram had dried in air for 30 min. The hRL ( = 100 x RL) values for individual spots were calculated relative to the distance travelled by the standard spot of pure leucodrin.
The hRL values were rounded off in decades (Tables 1 & 2) and were found to be replicable within a few per cent. With this system the leucodrin (reference) spot appeared at RF 0,65.

Discussion
Of the 59 Leucadendron spp. analysed (Table 1) all but one show red spots at hRL 100 for leucodrin and 29 of them show spots at hRL 50-60 for leudrin. The single exception is Leucadendron platyspermum which shows spots only above hRL 100 and below hRL 80. Spots above hRL 100 occur at various positions with various colours for various spp. and are frequently not well defined. Spots below hRL 50 are also often streaky, except for well-defined plum-coloured spots at hRL 10 in the subsection A lata, identified as corresponding to arbutin (hydroquinone /3-D-glucoside) by running the pure compound as a reference.
Of the 30 Leucospermum spp. studied (Table 2), 14 spp. show the co-occurrence of conocarpin and (as expected) S.-Afr. Tydskr. Plantk., 1984, 3(2) reflexin as red spots at hRL 90 and 80 respectively. In the case of 12 spp. (in Groups 3, 7, 8, and 9) chromatograms show the pattern, found for Leucadendron spp., of red and mauve spots at hRL 100 and 60 for leucodrin and leudrin respectively. Four out of the six representatives of Group 4 of the genus Leucospermum show still different behaviour in that only brown and yellow, but no red spots are shown, i.e. neither leucodrin nor conocarpin is present in these instances. Of the remaining two representatives of this Group, Leucospermum cordatum shows the presence of conocarpin and Table2 Paper chromatograms of leaf extracts of Leucospermum in groups Colours of spots are entered as: B =brown; M =mauve; 0 =orange; P =plum; R =red; Y =yellow.
reftexin , while Leucospermum patersonii shows the presence of leucodrin and leudrin. There are 81 known (Williams 1972) spp. of the genus Leucadendron and in 58 out of 59 spp . examined so far , leucodrin (and frequently leudrin) appears as a leaf constituent. There are 47 established (Rourke 1972) members of the genus Leucospermum ; conocarpin and reftexin appear as leaf constituents of about half the 30 spp . studied, while leucodrin and leudrin occur in a somewhat smaller number of these spp. The atypical behaviour of four out of the six representatives of Group 4 of the genus Leucospermum, in that neither of the two series of phenolic lactones occurs, is still being studied.
These findings have a direct bearing on the proposed mechanism of the biogenesis of these phenolic lactones (Diamand & Rogers 1964;Glennie & Perold 1980), viz. that the two diastereoisomeric series of compounds arise via enzyme-mediated coupling between p-coumaric acid and L-galactono-y-lactone (or their biochemical equivalents) in either of two mirror-image relationships in different subdivisions of these two genera. Where the stereo-specificity of enzyme systems is genetically controlled, these findings may have a bearing on the possibility of an evolutionary relationship between the genera Leucadendron and Leucospermum (Rourke 1975, pers. comm .).