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Analysis of the Toxins of Amanitin-Containing ported in 1966 by Tyler et al. 5 by chromatographic Mushrooms and pharmacologic evaluation. The results are summarized in the table. For most Heinz Faulstich, Dionysos Georgopoulos, of the samples of A. phalloides the sum of the Maria Bloching, and Theodor Wieland toxins is about 0.7% of the dry weight, 0.1% being Max-Planck-Institut for Medical Research, Department a-amanitin. Sample (III) shows, however, that in of Chemistry, Heidelberg spite of a normal concentration of phallotoxins, the (Z. Naturforsch. 29 c, 86 — 88 [1974] ; received amount of a-amanitin may be only one twentieth October 25, 1973) of the value normally found. In all samples from A. phalloides the acidic phallotoxins are present in Amanitins, Phallotoxins, Toxic Mushrooms, Chromatography larger amount than the neutral ones. Hydroxylation of the leucine side chain, which is We have recently developed a method for the the cause of the large variety of phallotoxins in A. analysis of toxins in single specimens of the mush phalloides, is unusually high in the mushrooms of room Amanita phalloides (Fr.) Seer.1. Separation sample (I), in which high amounts of trihydroxy- of the different toxins was achieved by column chro lated toxins (phallisin and phallisacin) have been matography, on Sephadex LH20 in water, of a formed at the expense of dihydroxylated toxins methanolic extract of fresh or dried mushrooms, (phalloidin and phallacidin). However, hydroxyla followed by further purification on thinlayers of tion was less extensive in sample (III), where, be silica gel, spectrophotometric evaluation, and, final sides a high amount of the monohydroxylated phal- ly, identification of the toxins by amino acid analy loin, we detected traces of its acidic analog phalla- ses of their y-hydroxylated leucines or isoleucines cin, which was recently discovered by Walch6. In as lactones. This procedure afforded the quantitative addition to the well known series of phallotoxins determination of up to 10 toxins in 2 samples of and amatoxins, sample (I) contained 3 acidic A. phalloides from different sources. amanitins, none of which was identical with £-amani- In the present study the treatment outlined above tin 7 and which were not found in other A. phal was extended successfully to all the amanitin- loides mushrooms. containing species of mushrooms known in Europe so far. We examined two more samples of A. phal The content of toxins was, as a whole, lower in loides collected near Hirschhorn (Neckar, W.-Ger A. verna than in A. phalloides. This white mush many) in 1971 (sample III) and 1972 (sample IV), room had only 70% of the acidic phallotoxins, 50% so that the analytical results from the 4 different of the neutral phallotoxins and only 30% of the samples of this species should shed light on the amanitins in comparison with A. phalloides. Besides question as to what extent the toxic composition may the known peptides, however, 0.2 /mmoles each of differ, depending on location and season. A single two neutral phallotoxins were detected, which could small sample of the white species Amanita verna, of not be characterized because of their low concen only 1.47 g of dry weight, was found near Würz trations. burg (W.-Germany) in 19712. Two samples of The two samples of A. virosa, though of quite withe Amanita virosa were collected near Trento, different origin, corresponded well to each other in Italy, 1972 (sample I) 3 and in Hägnan, Femsjö the composition and concentration of toxins. (Smoland, Sweden) in 19724 (sample II). We a-Amanitin was 120 — 130%, and phalloidin up to also investigated a sample of Galerina marginata 200% of the values for A. phalloides. These mush (Fr.) Kühn, collected near Einsiedeln (Switzerland) rooms, however, were free from ß- and y-amanitin, in 19724; these small, brown mushrooms, which and, generally lacked the variety of toxins produced grow on coniferous trunks, are, together with the by hydroxylation of leucine and isoleucine side stirpes G. automnalis and G. venenata, the only chains in A. phalloides. In sample (I) trace amounts amanitin-containing mushrooms outside the Amanita of trihydroxylated phallotoxins were detected, the group known to date. This had already been re- acidic one in the form of its sulfoxide 8.
Requests for reprints should be sent to Dr. H. Faulstich. (OH) asp, D -ß {erythro) hydroxyaspartic acid; gly, glycine; Max-Planck-Institut für Medizinische Forschung, Abtei (OH) leu, y-hydroxyleucine; (O H )2leu, y,8-(erythro) dihy- lung Chemie, D-6900 Heidelberg 1, Jahnstr. 29. droxyleucine; (OH3)leu, y,<5,<5'-trihydroxyleucine; (OH)pro, Abbreviations 9: D- (OH) -abu, D -ß (threo) hydroxy-a-amino- y-hydroxyproline; a-(OH) pro, allo-y-hydroxyproline; ser, butyric acid ( = D-threonine) ; ala. alanine; asn. Aspara serine; thr, threonine; val, valine; cy(S)trp, /?-alanyl-2- gine; asp, aspartic acid; ile, isoleucine; (OH)ile, y-hy- tryptophylsulfide; cy(SO)trp (OH), /?-alanyl-2(6-hydroxy) droxyisoleucine; (O H )2ile, y,(5-dihydroxyisoleucine; D- tryptophylsulfoxide; R (S 0 2)trp, R-2-tryptophylsulfone. Table I. mg-Amounts of toxins found in various amanitin-containing mushrooms, related on 2.0 g dry weight each.
Amino acid composition A. phall. A. pliall. A. phall. A. phall. A. verna A. virosa A. virosa Galerina Toxin variable constant I II I ll IV I II marginata [1] [1] (OH) 3 leu Phallisacin val 6.0 2.6 1.7 2.3 <0.1 0.1 * D- (OH) asp (OH) 2 leu ala Phallacidin val 2.4 3.7 3.4 3.4 3.8 3.0 1.4 — cn D- (OH) asp a-(OH) pro G (OH) leu X Phallacin val CY (S) trp —— <0.1 — ———— O D- (OH) asp O (OH) 3 leu Phallisin ala 0.8 0.2 0.2 0.3 <0.1 —— CÖ D- (OH) abu 1.0 Oh (OH)., leu Phalloidin ala 0.9 2.0 2.2 2.2 4.1 4.2 D- (OH) abu (OH) leu Phalloin ala <0.1 0.3 0.3 D- (OH) abu ~ " " Notizen
(OH) 2 ile Amanin asp <0.1 0.6 cy (SO) trp (OH) 2 ile gly2 (/} /?-Amanitin asp 2.3 1.2 <0.1 1.3 0.4 —— <0.1 ** c cy (SO) trp (OH) ile X (OH) 2 ile o a-Ai«anitin asn (OH) pro 2.0 1.8 0.1 2.2 0.5 2.3 2.7 0.8 cd cy (SO) trp (OH) £ (OH) ile y-Amanitin asn 0.2 0.2 0.4 <0.1 cy (SO) trp (OH) ile Amanullin asn <0.1 0.2 cy (SO) trp (OH)
thr, ala, val, ser, Viroidin - - --- 2.2 /«moles 2.0 //moles - (OH) 2 leu, R (S02) trp Virosa- toxins
* As sulfoxide. 3 species 2 neutral 0.4 //moles 0.5 «moles * * Not identified by amino of acidic phallotoxins of a sulfone, closely « s i acid analysis. amatoxins 0.2 //moles related to viroidin r * - 3 §* o '3 each 2 phallo- toxines 88 Notizen
A. virosa contained two toxins not previously except for dihydroxylated leucine, which remains found in A. phalloides. Their UV absorption spectra uncertain, because of the small amount of material. indicated that they were a-tryptophyl sulfones8. In addition to these sulfones, sample (I) contained Their toxicity was 2.0 mg/kg on intraperitonal in 2 phallotoxins not further characterized. jection in white mice; the dose and progress of Finally, the sample of Galerina marginata was intoxication were very similar to that of phalloidin. devoid of any phallotoxins. It contained about 40% The major compound, about 2 /
1 H. Faulstich, D. Georgopoulos. and M. Bloching. J. Chro- 6 St. Walch, diploma work, University of Heidelberg 1973. matogr. [Amsterdam] 79,257 [1973]. 7 Th. Wieland and A. Buku, Liebigs Ann. Chem. 717, 215 2 It was a gift of Paul Matheis, Würzburg, to whom we wish [1968]. to express our gratitude. 8 H. Faulstich, Th. Wieland, and C. Jochum, Liebigs Ann. 3 We gratefully acknowledge this gift of Carlo Alberto Bauer, Chem. 713, 186 [1968]. Trento (Italy). 9 For formulas see the review of: Th. Wieland and 0. 4 A gift from Prof. M. Moser, Innsbruck, whom we wish to Wieland, Microbial Toxins, Vol. 8 (S. J. Ajl, S. Kadis, thank sincerely, also for the sample of Galerina marginata. and A. Giegler, ed.). Academic Press, New York, Lon 5 V. E. Tyler, L. R. Brady, R. G. Benedict, J. M. Khanna. don 1972. and M. H. Malone, Lloydia 26,154 [1963].