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Studies on the constituents of Helleborus purpurascens: analysis and biological activity of the aqueous and organic extracts

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Abstract

In Southeast Europe, the ethnomedicinal use of Helleborus species has a very long tradition. Cardiac steroids (Hellebrin), cysteine-rich proteins (Hellethionins) and several steroidal saponins have been identified in these plants. Aim of the present work was to investigate the amino acid composition of native extracts from the root and rootstock of Helleborus purpurascens. The amino acids have been identified by the GC–MS technique on the previously derivatised (Phenomenex Faast Kit) extract samples by comparison with the mass spectra and retention-time of the standards. A remarkable finding was a relatively intensive peak attributed to the non-proteinogenic Pipecolic acid (Pic). A cyclisation of the derivatised glutamine was observed during the GC measurement and a mechanistic pathway is described. Samples of the extract and of some isolated fractions have also been tested on; altogether 12 cancer cell lines aimed to identify further potentially cytostatic components which should be less toxic than Hellebrin. The finding of one Hellebrin-free fraction (IC50 = 0.007 mg/L) with higher cytotoxicity than Hellebrin (IC50 = 0.008 mg/L) is remarkable.

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Notes

  1. The term “hydroalcoholic” is not precisely but commonly used. Therefore, the term is also used by the authors for the extract which was generated with a mixture of water and ethanol.

  2. A congruence of retention time and fragmentation pattern between an unknown substance and an authentic material proves the identity of an unknown substance.

  3. In the publication of Segneanu et al. (2015) was announced an hydroalcoholic extract. This extract was dried and re-suspended in water. Furthermore, this suspension was extracted successively with hexane, Dichloromethane, Tetrachloromethane and n-butanol. Then, in the analysis part appeared an ethanolic extract. Nowhere in the publication could be found a description where this extract was generated. We supposed that the authors meant the hydroalcoholic extract which is more precisely an aqueous ethanolic extract. But this is just speculation. From the authors aren´t given any information.

  4. The mixture contained alanine, asparagine, glutamic acid, phenylalanine, histidine, isoleucine, threonine, proline, valine, Lysine, Glycine, Tyrosine, Serine and glutamine.

  5. Water-saturated n-butanol contains 20% water at 25 °C; see Hefter GT. Alcohols with water. In: Barton AFM, editor. Solubility Data Series. Vol. 15, Oxford: Pergamon Press; 1984: 32–93.

  6. The C.A.T. GmbH is specialised in the analysis of amino acids. They are certified and work under GMP conditions. For this work, they used a method for the trace analysis which they use for quality control of amino acids.

  7. The limit of detection was 1 mg/g.

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Acknowledgements

The authors want to thank Ing. Geta Serbanescu and Ing. Draga Todorov (both S.C. Exhelios S.R.L.) for fruitful discussions and for the support of this work with rhizome and roots of Helleborus purpurascens and an already prepared hydroalcoholic extract thereof. The authors want to thank also Dr. Raluca O. Pop, Dr. Carmen Lazau, Dr. Cornelia Bandas, Dr. Corina Orha, Dr. Macarie A. Corina, and Dr. Ionel Balcu (all Institutul National de Cercetare Dezvoltare pentru Electrochimie si Materie Condensata, Timisoara, Romania) for their contributions for this publication. In addition, this work was supported by the Romanian National Authority for Scientific Research through the (EXPLORATORY RESEARCH PROGRAM IDEI-PCE-PROJECT NR. 341-/05.10.2011—Immunomodulatory Fluoroglycopeptide Molecular Architectures (I. Neda).

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  1. Institutul National de Cercetare Dezvoltare Pentru Electrochimie si Materie Condensata, Str. Dr. A. Paunescu Podeanu, Nr. 144, 300569, Timisoara, Romania

    M. Heiko Franz, Roxana Birzoi, Catalin-Vasile Maftei, Elena Maftei & Ion Neda

  2. InnoChemTech GmbH, Hagenring 30, 38106, Brunswick, Germany

    M. Heiko Franz

  3. Institut für Anorganische und Analytische Chemie, Technische Universität Braunschweig, Hagenring 30, 38106, Brunswick, Germany

    Catalin-Vasile Maftei, Elena Maftei & Ion Neda

  4. Oncotest GmbH, Am Flughafen 12-14, 79108, Freiburg, Germany

    Gerhard Kelter & Heinz-Herbert Fiebig

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  1. M. Heiko Franz
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Correspondence to Ion Neda.

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Franz, M.H., Birzoi, R., Maftei, CV. et al. Studies on the constituents of Helleborus purpurascens: analysis and biological activity of the aqueous and organic extracts. Amino Acids 50, 163–188 (2018). https://doi.org/10.1007/s00726-017-2502-6

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