The real nature of the indole alkaloids in Cortinarius infractus: Evaluation of artifact formation through solvent extraction method development

doi:10.1016/j.chroma.2007.02.074

Journal of Chromatography A

Volume 1148, Issue 1, 27 April 2007, Pages 1-7

https://doi.org/10.1016/j.chroma.2007.02.074 Get rights and content

Abstract

This study was triggered by the absence of infractine in EtOH extracts of Cortinarius infractus and the presence of the β-carboline-1-propionic acid [I. Brondz, K. Høiland, D.S. Bell, A.R. Annino, TheReporter, Supelco 24 (3) (2006) 6]. In previous studies it was determined that C. infractus contained indole alkaloid substances of the infractine group [W. Steglich, L. Kopanski, M. Wolf, M. Moser, G. Tegtmeyer, Indolalkaloide aus dem Blaetterpilz Cortinarius infractus (Agaricales). Tetrahedron Lett. 25 (1984) 2341]. It is quite possible that some of these compounds do not exist in any other mushroom species and therefore they could prove to be valuable chemotaxonomic markers. Proper classification of this species requires that the chemical markers are identified correctly. In this study, the presence of several compounds previously found in C. infractus (Pers.: Fr.) Fr. is questioned. In this investigation, it was shown by HPLC-MS that infractine and 6-hydroxyinfractine, previously described as constituents of C. infractus, were artifacts produced during the extraction process. Also, the molecular species that participate in the artefact formation were identified. Infractine and 6-hydroxyinfractine, reported earlier in C. infractus, originate from the precursor (pre-infractine) β-carboline-1-propionic acid during extraction with methanol.

Introduction

In the vast genus Cortinarius, which is comprised of brown spored, ectomycorrhizal mushrooms, C. infractus (Pers.: Fr.) occupies an isolated position. C. infractus is rather common in broadleaved deciduous forests with oak, beech, hazel, hornbeam, lime, or chestnut from Mediterranean to boreal or mountainous areas in Europe, especially on rich mull soil. On calcareous bedrock it may even be found in coniferous forests or under birch.

Basidiocarps of C. infractus have an unpleasant, fishy odour and a strong bitter taste. Odour and taste may partially be explained by the presence of the indole alkaloids infractine, 6-hydroxyinfractine and infractopicrine [1]. In 1984 it was claimed that indole alkaloids of the infractine series were present in higher plants and that infractine was a new β-carboline alkaloid under the name kumujanrine [2].

Among the mushrooms, infractine-like alkaloids may exclusively occur in C. infractus these compounds consequently may have profound value as chemotaxonomic markers in Cortinarius. For this reason, we previously investigated the possible content of infractine-like alkaloids in C. infractus and C. subtortus. In that study, however, we did not find infractine present in C. infractus or C. subtortus [3], [4].

Infractine is a methyl ester of β-carboline-1-propionic acid, and 6-hydroxyinfractine is a methyl ester of 6-hydroxy-β-carboline-1-propionic acid. Consequently, they should belong to the family of fatty acids which have an alkaloid moiety covalently bonded to the principal chain. Therefore, they ought to be treated as fatty acids for purposes of extraction solvent choice. This idea is firmly supported by Brondz [4], [5], [6]. The author strongly warns against the use of methanol as an extraction solvent for these compounds, because MeOH may cause artificial methyl esters of fatty acids in certain lipids in the presence of some organic or inorganic materials.

In the present work, we used HPLC–MS equipped with a Discovery ZR-PS column to verify the extraction procedures previously used and to establish the true nature of the substances present in C. infractus. The experimental design is important not only for this particular case but also for other works in the field of natural product analysis. These methods may prove invaluable in the determination of the origin of compounds found in plant materials and may allow researchers to differentiate between true plant constituents and fatty acid derivatives created by improper extraction procedures. This experimental design may also be useful in cases where commercial standards are unavailable and difficult to synthesize, thus making verification crucial.

Section snippets

Biological material and preparation of samples for analysis

Basidiocarps of C. infractus were sampled in a broadleaved deciduous forest dominated by lime and hazel on Ordovician calcareous clay schist; at about 10 m a.s.l. Samples were collected in the vicinity of Oslo, Southeast Norway. A voucher specimen is located in the Mycological Herbarium at the Botanical Museum of the University of Oslo, Oslo, Norway. An unpublished phylogeny based on DNA coding for the ribosomal RNA (viz. SSU, ITS1, 5.8S, ITS2, LSU) (Høiland pers. comm.) shows that C. infractus

Results and discussion

The present study was initiated by the absence of infractine noted in the previously described study of C. infractus [3] in which the presence of β-carboline-1-propionic acid was recorded. MeOH, when used as an extraction liquid for substances from natural sources, may produce methyl esters from fatty acids or other molecules having a carboxylic functionality [4], [5], [6]. Infractine and 6-hydroxyinfractine are aliphatic methyl esters that have one of hydrogen in principal chain substituted

Conclusion

The previously claimed [1] presence of infractine and 6-hydroxyinfractine in C. infractus was challenged. Infractine and 6-hydroxyinfractine in C. infractus were found to be artifacts produced during the extraction procedure.

Schemas of MS fragmentation for (pre-infractine) β-carboline-1-propionic acid, infractine, 6-hydroxyinfractine, ethyl analogues of infractine and ethyl analogues of 6-hydroxyinfractine were developed. In this study, the presence of (pre-infractine) β-carboline-1-propionic

Acknowledgements

The authors express their gratitude to company the Jupiter AS Norway for financial and technical support and to engineer John Vedde, Department of Chemistry, Laboratory of Mass-Spectrometry and engineer Jon Reierstad at the Technical Department, Photo and Graphic Laboratory, University of Oslo, Norway for technical support.

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The real nature of the indole alkaloids in Cortinarius infractus: Evaluation of artifact formation through solvent extraction method development

Abstract

Introduction

Section snippets

Biological material and preparation of samples for analysis

Results and discussion

Conclusion

Acknowledgements

Tetrahedron. Lett.

Anal. Chim. Acta

Benn. Acta Chim. Sinica

TheReporter, Supelco