[ II ]

Trans. Br. "!Y'0l. Soc. 49 (I), 11-17 (1966)Printed in Great Britain

INVESTIGATION OF PIGMENTS FROMRUSSULA SPP. BY THIN LAYER

CHROMATOGRAPHY

By PAULINE WATSON

Department ofBotany, University ofDurham

(With Plate I)

Pigments extracted from twenty-six 'representative' Russula spp. with 50%aqueous picoline were compared by thin layer chromatography on silica gel,using water-saturated phenol as developing solvent. The presence of at leastthree red, seven yellow and three blue substances was demonstrated and thespecies studied could be placed in four groups according to the major pigmentspresent. Chromatograms of pigments from some but not all species studied wereapparently characteristic. Further studies might well be useful taxonomically.

The notoriously difficult genus Russula includes many brightly colouredspecies and the cap colour is one of the major characters used in distin­guishing them. In spite of this importance the pigments appear to havebeen little studied, and although some monographs of the genus note thatthe red ones are easily washed out in wet weather and that the cap colouris a variable character, many do not refer to the pigments at all.

Phipson (1882) seems to have been one of the first workers to beinterested in the pigments of a Russula, when he studied the red extractfrom caps of Agaricus ruber (Russula sanguinea?). He referred to the redsubstance as 'ruberine' and also noted that the extract was fluorescent.Zellner (1907), summarizing this and other work, gave an account of thesolubility of Russula pigments in different solvents and other observations,including the absorption spectra of the solutions. Red, yellow and bluepigments were recognized, but few attempts seem to have been made toseparate them and there seem to be no references in the literature to anyone following Zellner's suggestion that capillary analysis of the pigmentsmight yield interesting results. After Zellner, there seem to be few referencesto the pigments of Russula spp. and they are not mentioned in Pastac'smonograph of fungal pigments (1942). More recently, Balenovic, Cerar,Pucar & Skaric (1955) studied the pigments of R. emetica and isolated themajor red component, which they called 'russularhodin'. They alsocompared extracts of ten species using paper chromatographic and electro­phoretic methods and showed that some fluorescent compounds werecommon to all the species tested but others were restricted to particularspecies. Deysson (1958) compared the fluorescence colours seen in differentspecies of Russula when they are examined under ultraviolet light andsuggested that they might be helpful taxonomically, but did not extractthe compounds responsible.

12 Transactions British Mycological SocietySome preliminary studies of pigments from various Russula spp., using

paper chromatography, were done by the author (Watson, 1956). How­ever, this method did not yield clear evidence on a number of points,although it did suggest that the colours of many species were due tovarying proportions of a relatively small number of pigments. Use of thinlayer chromatography has since made it possible to investigate the pig­ments more satisfactorily and the present paper gives an account of theinvestigation, which, although the complexity of the problem made it farfrom exhaustive, shows that further work of this kind could help to clarifythe taxonomy of the genus.

MATERIAL

Owing to the size of the genus and the difficulty of identifying many ofthe species, only twenty-six' good' species of Russula were chosen for study.These species were selected because they were relatively easily recognizedand identified, and represented different groups of species recognized byvarious authors within the genus. The nomenclature of the species studied(Table 2) follows that given in the New Check List of Agarics and Boleti(Dennis, Orton & Hora, 1960). Each collection studied normally con­sisted of a single typical fruit body and in almost all cases the collectionscame from different localities.

Table I. Characteristics of the major pigments extracted fromRussula spp.

Approx.Colour 'Type'species R p

R I Scarlet R. ernetica 0"3R 2 Crimson R. atropurpurea 0"35R 3 Pink R. vesca 0"25I Indigo R. cyanoxantha 0"3Y I Pale yellow R. ernetica 0"3*Y 2 Lemon yellow R. ochroleuca 0'35Y 3 Lemon yellow R. ochroleuca 0"3Y 4 Golden yellow R. velenovskyi 0"27Y 5 Golden yellow R. velenovskyi 0"2Y 6 Golden yellow R. velenovskyi 0"1

Y 7 Golden yellow R. arnoena 0"7B Blue 0"2-0'3t

* Paper chromatograms developed with 60 % aqueous acetone were prepared to confirm thepresence or absence of Y I, as it has a very distinctive R p (approx. 0'5) and shows a brilliantgreen fluorescence under these conditions.

t Three blue pigments were detected in extracts from many species, and although they werethought to be the same in different species, the Rp values were very variable, apparently due tointerference from other substances present, and they were not compared in detail.

METHOD

It was found that in all species studied, with the exception of those inthe IP group, the pigments were apparently unaltered by drying, so mostof the work was done on dried material, although at least one freshcollection of most species was included. The dried material consisted ofthe whole fruit bodies or only the coloured peel, dried rapidly in a warmroom and stored in the dark.

Russula pigments. Pauline Watson 13When pigments were required for investigation, material was placed in a

small amount of 50 %aqueous picoline. This was found after tests of manypossible extraction solvents to be the most effective, as it was the only onewhich readily removed all the colouring matter from the peel of all species(except those in the 0 group).

Silica gel (Merck Kieselgel G) was found to be the best adsorbant ofthose tested, which included kieselguhr, alumina and cellulose. Thin layerplates were prepared in the usual way, using a Shandon spreader to makelayers 2501" thick, and activated at goO C. for I hr. Both before and afterspotting, the activated plates were stored in a desiccator as poorer resultswere obtained otherwise.

The activated plates were spotted with the picoline extracts by meansof a micropipette and larger spots, approximately I em. diam., were madethan is usual for thin-layer chromatography. They were allowed to dryovernight in a desiccator, without heating. When developed, the pigmentsseparated as narrow bands, although the original spots were circular, andit was found that ifadjacent spots were just allowed to touch, the differenceor similarity between the bands from different spots could readily be seenin most cases.

A number ofdeveloping solvents were tested in preliminary experiments,but water-saturated phenol gave results so superior to the others that itwas the only one used in later work. It was found necessary to develop thechromatograms in a constant temperature room (230 C.) because of theextreme sensitivity of phenol to changes of temperature. As only one gooddeveloping solvent was known, only one-dimensional chromatograms wereprepared in most cases, but occasionally two dimensional chromatograms,prepared by developing with the same solvent in both directions, werefound to be helpful in detecting pigments present in small amounts. Afterdevelopment, the phenol was dried off in a current of air at room tempera­ture. As the pigments were unstable on silica gel, especially in strong light,the plates were kept in the dark as much as possible.

The chromatograms were examined by transmitted light on a KodakColdlight Illuminator. Examination under ultraviolet light did not add tothe observations which could be made using visible light, as the pigments donot fluoresce strongly on silica gel as they do on paper chromatograms.The RF values of the pigments werer ecorded, but, owing to the variabilityoften found on thin layer chromatograms, the patterns of bands andparticularly the interactions between bands from adjacent spots were foundto be more helpful in establishing whether pigments from different specieswere probably identical or not. Owing to their instability on silica gel, allattempts to elute the pigments were unsuccessful, and the conclusions as tothe identity or otherwise of pigments in different species are based on thecomparisons of adjacent spots, together with the data obtained in theprevious paper chromatographic study.

In the first stage of the investigation, the different collections of eachspecies were compared, to check on the constancy of the pigmentationwithin the species. In almost all those investigated, the extracts fromdifferent collections appeared to contain the same pigments, in similar pro­portions, so in later work only one collection of each of these species was

14 Transactions British Mycological Societyused. As noted in the results, in a few characteristically variable species,more than one collection was studied in detail. A standard 'type' specieswas chosen for each major pigment, as given in the results, and this wascompared with other species thought to contain the same substance, bydeveloping adjacent spots ofthem on several occasions. A series ofdilutionsof extracts from some of the more strongly coloured species was alsocompared, as anyone concentration of such extracts was not usuallyoptimum for detection of all the pigments present.

RESULTS

A much larger number ofpigments were detected on thin-layer chromato­grams than on the paper chromatograms previously studied, but many ofthem were only present in small amounts. Only the major ones wereinvestigated closely in this study (Table I).

Table 2 gives details of the pigments detected in the species studied. Thecolumns of the Table representing the pigments are arranged so thepigments most easy to detect, such as Y I, R I and R 2, are placed at theleft-hand side, and the most difficult ones at the extreme right, with theintermediates in between. The signs signifying that a particular pigmentwas not detected are of two kinds -'0' showing that the pigment wasalmost certainly not present and ' - ' showing that although the pigmentwas not detected it is possible that it was masked by others present, orthat it appeared to be relatively unstable or often present only in smallamounts. The species are arranged according to the major pigmentspresent and disregarding all other characters. When arranged in this way,they appear to fall into four groups, as shown in the Table, although thereappear to be some intermediate species. This grouping will be furtherdiscussed later.

NATURE AND PROPERTIES OF THE PIGMENTS

The work done in the present study consisted almost entirely of a purelyempirical comparison ofpigments from different species arid little is knownabout the nature of the substances concerned. However, a little is knownabout their solubility in various solvents and similar properties. Allreferences to the pigments note that the red and yellow ones are solubleonly in aqueous solvents and Zellner (1907) also notes that the bluepigments are insoluble in water or aqueous alcohol but can be extractedwith dilute acid. The present work has shown that they are also soluble inaqueous picoline or pyridine. Pigments of species in the 0 group, whichwere not extracted by any of the solvents tested, are presumably quitedifferent in nature from those of the other species.

All workers with the pigments have also noticed the characteristicfluorescence ofextracts containing the pigments, which is lost if the extractis acidified but regained on neutralization. Most of the observations onfluorescence, and also the records of absorbtion spectra made by variousworkers, were made on extracts which can be shown to contain a numberof fluorescent substances, both coloured and colourless, so that the resultsare due to the combined effects of mixtures of compounds. It is interesting

Table 2. Pigments extracted from Russula spp.No. of Pigments detectedcollec- t

. ,tiona YI RI R2 B Y2 Ys Y4 Ys Y6 I RS Y7 Others·

R. erne/ica S + ++ + 0 0 0 - - - - - - 2RR. mairei 5 + ++ + 0 0 0 - - - - - - 2RR. betularum 5 ++ + + (+) 0 0 - - - - - - 2RR. sardonia 5 + ++ + + 0 0 - - - - - - IR

~R·fragilis 5 + ++ + + 0 0 - - - - - - -R. erythropus 2 + ++ + + 0 0 - - - - - - rR ~

R. xerampelina (purple) 2 + ++ - + 0 0 - - - - - - - ~

RB ( d bl) ~ R. xerampelina (brown) r ++ + - + 0 0 - - - - - - - ~re - ue group R. caerulea 5 + ++ + + 0 0 + + + - - - -R. versicolor 5 + ++ - + 0 0 + + + - - - - ~.R. velenovskyi 4 + ++ + + 0 0 + + + - - - IRR. rosea 5 + ++ + (+) 0 0 + + + - - - - §R.lepida S + ++ + (+ ) 0 0 + + + - - - IRR. atropurpurea 5 + + ++ + 0 0 - - - - - - 2R ~{lR. lutea (apricot) 3 0 - + 0 ++ + - - - - - - -

Y ( II ) R. lutea (yellow) 2 0 - (+) 0 ++ + - - - - - - - ~ye ow group R. claroflava 2 0 0 0 0 ++ + - - - - - - -~R. ochroleuca 5 0 0 0 0 ++ + - - - ? ? - - ~

R. vesca 5 0 0 0 0 0 0 - - - + + ? I I, I Y ~.R. ryanoxantha 5 0 0 0 0 0 0 - - - + + ? I I, I Y

IP (indigo-pinki R. grisea S 0 0 0 0 0 0 - - - + ? ? -~group) R. heterophylla 5 0 0 0 0 0 0 - - - + ? ? I I, I R

{ R. amoena (yellow cap) 2 0 - (+) 0 0 0 - - - - - ++ - 1:::'R. amoena (purple stalk) 2 0 (+ ) + 0 0 0 - - - - - - - <::l

{ R·ftll" 5 0 0 0 0 0 0 0 0 0;:s

R·foetms 3 0 0 0 0 0 0 0 0 0

o (nil) group R. sororia 5 0 0 0 0 0 0 0 0 0R. nigricans 4 0 0 0 0 0 0 0 0 0R. virescens 2 0 0 0 0 0 0 0 0 0

+ +, large amounts of pigment present; +, small amounts present; (+), traces present; -, not detected; 0, almost certainly not present; ?, pigment of appropriatecolour and approx. R.... present but identity not certain.

• R, red; Y, yellow; I, indigo.

1-4V\

16 Transactions British Mycological Societythat Balenovic et al. showed that russularhodin (presumably = R I) wasonly fluorescent when adsorbed on to filter paper and did not fluorescein solution.

Apart from the demonstration by these workers that the pigments donot yield sugars or amino acids on hydrolysis, nothing is known of theirchemical composition, but they appear to be quite different from pigmentsfound in any other organisms, with one possible exception which will bementioned later.

The occurrence of some of the pigments, notably the blue and some ofthe yellow ones, in groups showing similar colours and apparently alwaysfound together, is interesting as it suggests that they represent series ofrelated compounds. It is, however, possible that the series do not in factoccur in the fruit bodies, but have been produced from single originalcompounds during the chromatographic process. It is known that sugarschromatographed on silica gel may be converted into various derivativesproducing multiple spots (Randerath, 1963) and it is possible that asimilar process may be occurring in the present case. However, as separa­tions are only good enough to separate the compounds on silica gel withphenol as developing solvent, it is difficult to devise methods of distin­guishing between the two possible explanations. All the observations madeso far suggest that the series are not artifacts produced by the method ofinvestigation, but do in fact exist in the fruit bodies, and it is well knownthat series of related compounds may be isolated from many organisms.

As the pigments were not successfully eluted it was not possible toinvestigate the effects of pH on the colour or fluorescence of individualpigments in solution. Observations of chromatograms kept in atmospheresof ammonia or acid fumes did not suggest that any of the pigments hadindicator properties.

DISCUSSION

The results obtained are interesting in several respects. As pigments fromsmall quantities of dried material can be investigated, it would be possibleto study pigments from herbarium material, and this could be usefultaxonomically. Some species, such as R. ochroleuca or R. amoena seem to givedistinctive patterns of pigments, and in many others the pattern, thoughnot apparently unique, is sufficiently characteristic and constant to beuseful. However, classification of species into groups based on the occur­rence of pigments in them does not in a number of cases correlate withclassifications now in use. An arrangement based on pigments might bemodified to take in other characters commonly used, such as taste andchemical reactions, but it is difficult to see how much account of sporecolour could be taken.

The species so far studied seem to fall into several groups, according tothe major pigments present. No colouring matter could be extracted fromspecies in the' nil' (0) group, whereas all the pigments could be extractedfrom the other species, and this seems to be a fundamental difference,suggesting that the pigments in the 0 group are very different chemicallyand perhaps resemble more the pigments of Lactarius spp. The speciescontaining extractable pigments could be placed in three groups-the

Trans. Br. myeol. Soc. Vol. 49. Plate I

(Facmg p. 17)

Russula pigments. Pauline Watson 17'red-blue' (RB), 'yellow' (Y) and 'indigo-pink' (IP) groups. Inter­mediate species can be demonstrated, however, as R. amoena appears to beintermediate between species in the RB and IP groups, R. ochroleucabetween those in the Y and IP groups, and R. lutca between those in theY and RB groups. It is possible that, if a larger number of species wasstudied, the species containing soluble pigments would still appear to formgroups, with a few intermediate species. Alternatively, the investigation ofmore species might show that the pigments really occur in more or lessrandom associations, and that the choice of species for the present studyaccidentally produced an impression of discontinuous distribution.

It would also be interesting to know more of the chemical nature of thesubstances concerned, as they appear to be highly characteristic of thisgenus of agarics. It has been suggested (Heim, 1942) that Elasmomycesrussuloides contains' rubeine' (presumably rubeine = ruberine = russula­rhodin = R 1) as found in Russula spp. and an investigation of this wouldbe of great interest, especially as other evidence, such as the spore orna­mentation, suggests that this gasteromycete may be related to the genusRussula.

Thanks are due to Mrs J. B. Scott for technical assistance, to Mr J. S.Redhead for taking the photograph, and to the Royal Society and theUniversity of Durham Research Fund for grants towards the cost of thecolour plate.

REFERENCES

BALENOVIC, K., CERAR, D., PUCAR, Z. & SKARIC, V. (1955). The chemistry of higherfungi. III. Contribution to the chemistry of the genus Russula. Arh. kem. 21, 15-19.

DENNIS, R. W. G., ORTON, P. D. & HORA, F. B. (1960). New check list of agarics andboleti. Trans. Br. mycol. Soc. 13, supp!., 225 pp.

DEYSSON, G. (1958). Sur l'examen des champignons en lumiere de Wood. Son interetpour l'etude du genre Russula. Bull. Soc. mycol. Fr. 74, 207-215.

HElM, R. (1942). Les pigments des champignons dans leurs rapports avec la systema­tique. Bull. Soc. Chim. biol. 29, 48-79.

PASTAC, I. A. (1942). Les matieres colorantes des champignons. Revue Mycol. MemoireHors-serie 2.

PHIPSON, T. L. (1882). On the colouring matter (Ruberine) and the alkaloid (agaryth­rine) contained in Agaricus ruber. Chem. News, Lond., 46, 435.

RANDERATH, K. (1963). Thin-layer chromatography. New York and London: AcademicPress.

WATSON, P. (1956). Some studies of a mould isolated by tissue culture from species ofRussula. Ph.D. Thesis, University of London.

ZELLNER, J. (19°7). Chemie der Hoheren Pilze. Leipzig: W. Engelmann.

EXPLANATION OF PLATE 1

Chromatogram of pigments from Russula spp. on silica gel, developed with water-saturatedphenol; lower edge ofchromatogramjust shows at bottom of plate and solvent front is just beyondextreme upper edge. The pigments were extracted from the following species, from left to righton the plate: R. caerulea, R. jragilis, R. cyanoxantha, R. emetica, R. ochroleuca, R. velenovskyi,R. erythropus, R. atropurpurea and R. caerulea. x approx. -.:r,;.

(Accepted for publication 30 April 1965)2 MYC.49