Skip to main content

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


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.

This is a preview of subscription content, access via your institution.

Fig. 1
Scheme 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Scheme 2
Fig. 6
Fig. 7
Scheme 3
Scheme 4
Scheme 5
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13
Fig. 14
Fig. 15
Fig. 16
Fig. 17
Fig. 18
Fig. 19
Fig. 20
Fig. 21
Fig. 22
Fig. 23
Fig. 24
Fig. 25
Fig. 26
Fig. 27


  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.


  • Belitz HD, Grosch W, Schieberle P (2008) Lehrbuch der Lebensmittelchemie, 6th edn. Springer, Berlin, Heidelberg

    Google Scholar 

  • Bouché N, Lacombe B, Fromm H (2003) GABA signaling: a conserved and ubiquitous mechanism. Trends Cell Biol 13:607–610

    Article  PubMed  Google Scholar 

  • Byun SM, Jeong SW, Cho DH, Kim YH (2015) Optimized conversion of l-lysine to l-pipecolic acid using recombinant lysine cyclodeaminase from Streptomyces pristinaespiralis. Biotechnol Bioprocess Eng 20:73–78

    CAS  Article  Google Scholar 

  • Choi SS, Song MJ, Kim OB, Kim Y (2013) Fragmentation patterns of protonated amino acids formed by atmospheric pressure chemical ionization. Rapid Commun Mass Spectrom 27:143–151

    CAS  Article  PubMed  Google Scholar 

  • Dengler WA, Schulte J, Berger DP, Mertelsmann R, Fiebig HH (1995) Development of a propidium Iodide fluorescence assay for proliferation and cytotoxicity assays. Anticancer Drugs 6:522–532

    CAS  Article  PubMed  Google Scholar 

  • Dirsch V, Lacaille-Dubois MA, Wagner H (1994) Dracoside, a new steroidal saponin from Helleborus purpurascens. Nat Prod Lett 4(1):29–33. doi:10.1080/10575639408043888

    CAS  Article  Google Scholar 

  • Dunn MS, Ross FJ (1938) Quantitative investigations of amino acids and peptides: IV. The solubility of amino acids in water–ethyl alcohol mixtures. J Biol Chem 125:309–332

    CAS  Google Scholar 

  • Fendler JH, Nome F, Nagyvary J (1975) Compartmentalization of amino acids in surfactant aggregates. J Mol Evol 6:215–232

    CAS  Article  PubMed  Google Scholar 

  • Fiebig HH, Berger DP, Dengler WA, Wallbrecher E, Winterhalter BR (1992) Combined in vitro/in vivo test procedure with human tumor xenografts. In: Fiebig HH, Berger DP (eds) Contributions to oncology: immunodeficient mice in oncology, vol 42. Karger, Basel, pp 321–351

    Google Scholar 

  • Fiebig HH, Dengler WA, Roth T (1999) Human tumor xenografts: predictivity, characterization, and discovery of new anticancer agents. In: Fiebig HH, Burger AM (eds) Contribution to oncology: relevance of tumor models for anticancer drug development, vol 54. Karger, Basel, pp 29–50

    Google Scholar 

  • Fringuelli R, Utrilla Navarro MP, Milanese L, Bruscoli S, Schiaffella F, Riccardi C, De Simone C (2004) Synthesis and evaluation of anti-apoptotic activity of l-carnitine cyclic analogues and amino acid derivatives. Il Farmaco 59:271–277

    CAS  Article  PubMed  Google Scholar 

  • Fujioka S, Sakurai A, Yamaguchi I, Murofushi N, Takahashi N, Kaihara S, Takimoto A (1987) Isolation and identification of l-pipecolic acid and nicotinamide as flower-inducing substances. Lemna Plant Cell Physiol 28:995–1003

    CAS  Google Scholar 

  • Germann UA, Shlyakhter D, Mason VS, Zelle RE, Duffy JP, Galullo V et al (1997) Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of P-glycoprotein-mediated multidrug resistance in vitro. Anticancer Drugs 8:125–140

    CAS  Article  PubMed  Google Scholar 

  • Gopalakrishnan R, Kozany C, Gaali S, Kress C, Hoogeland B, Bracher A, Hausch F (2012) Evaluation of synthetic FK506 analogues as ligands for the FK506-binding proteins 51 and 52. J Med Chem 55:4114–4122

    CAS  Article  PubMed  Google Scholar 

  • Harper HA, Martin DW, Mayes PA, Rodwell VW (1986) Medizinische biochemie. Springer, Berlin, Heidelberg

    Book  Google Scholar 

  • Hill AE, Malisoff WM (1926) The mutual solubility of liquids. III. The mutual solubility of phenol and water. IV. The mutual solubility of normal Butyl alcohol and water. J Am Chem Soc 48:918–927

    CAS  Article  Google Scholar 

  • Karrer W (1943) Über Hellebrin, ein krystallisiertes glykosid aus radix Hellebori nigri. Helv Chim Acta 26:1353–1367

    CAS  Article  Google Scholar 

  • Kerek F (2000) The structure of the digitalislike and natriuretic factors identified as macrocyclic derivatives of the inorganic carbon suboxide. Hypertens Res 23:S33–S38

    CAS  Article  PubMed  Google Scholar 

  • Kerek F (2004) Peptides having a high cysteine content. WO Patent 2004/058813

  • Kerek F, Szegli G, Cremer L, Lupu AR, Durbaca S, Calugaru A, Herold A, Radu DL (2008) The novel arthritis-drug substance MCS-18 attenuates the antibody production in vivo. Acta Microbiol Immunol Hung 55:15–31

    CAS  Article  PubMed  Google Scholar 

  • Kißmer B, Wichtl M (1987) Ecdysone aus wurzeln und samen von helleborus-arten. Arch Pharm 320:541–546

    Article  Google Scholar 

  • Maftei CV, Fodor E, Jones PG, Freytag M, Franz MH, Kelter G, Fiebig HH, Tamm M, Neda I (2015) N-heterocyclic carbenes (NHC) with 1,2,4-oxadiazole-substituents related to natural products: synthesis, structure and potential antitumor activity of some corresponding gold(I) and silver(I) complexes. Eur J Med Chem 101:431–441

    CAS  Article  PubMed  Google Scholar 

  • Maftei CV, Fodor E, Jones PG, Freytag M, Franz MH, Kelter G, Fiebig HH, Tamm M, Neda I (2016a) Novel 1,2,4-oxadiazoles and trifluoromethylpyridines related to natural products: synthesis, structural analysis and investigation of their antitumor activity. Tetrahedron 72:1185–1199

    CAS  Article  Google Scholar 

  • Maftei E, Maftei CV, Jones PG, Freytag M, Franz MH, Kelter G, Fiebig HH, Tamm M, Neda I (2016b) Trifluoromethylpyridine-substituted N-heterocyclic carbenes related to natural products: synthesis, structure and potential antitumor activity of some corresponding gold(I), rhodium(I) and iridium(I) complexes. Helv Chim Acta 99:469–481

    CAS  Article  Google Scholar 

  • McPhee DJ, Ferreira ICFR (2016) Expression of Concern: Segneanu et al. Helleborus purpurascens—Amino acid and Peptide Analysis Linked to the Chemical and Antiproliferative Properties of the Extracted Compounds. Molecules 2015 20:22170–22187

    Google Scholar 

  • Miehl H (1957) Über freie aminosäuren im gifte der gelbbauchunke Bombina variegata L. Monatsh Chem 88:701–702

    Article  Google Scholar 

  • Milbradt AG, Kerek F, Moroder L, Renner C (2003) Structural characterization of hellethionins from Helleborus purpurascens. Biochemistry 42:2404–2411

    CAS  Article  PubMed  Google Scholar 

  • Morrison RI (1953) The isolation of l-pipecolinic acid from Trifolium repens. Biochem J 53:474–478

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Muhr P, Kerek F, Dreveny D, Likussar W, Schubert-Zsilavecz M (1995) The structure of Hellebrin. Eur J Org Chem 1995(2):443–444

    Google Scholar 

  • Nagyvary J, Fendler JH (1974) Origin of the genetic code: a physical–chemical model of primitive codon assignments. Orig Life 5:357–362

    CAS  Article  PubMed  Google Scholar 

  • Návarová H, Bernsdorff F, Döring AC, Zeier J (2012) Pipecolic acid, an endogenous mediator of defence amplification and priming, is a critical regulator of inducible plant immunity. Plant Cell 24:5123–5141

    Article  PubMed  PubMed Central  Google Scholar 

  • Neacsu C, Ciobanu C, Barbu I, Toader O, Szegli G, Kerek F, Babes A (2010) Substance MCS-18 isolated from Helleborus purpurascens is a potent antagonist of the capsaicin receptor, TRPV1, in rat cultured sensory neurons. Physiol Res 59:289–298

    CAS  PubMed  Google Scholar 

  • Nernst W (1891) Verteilung eines Stoffes zwischen zwei Lösungsmitteln und zwischen Lösungsmittel und Dampfraum. Z Phys Chem 8:110–139

    Article  Google Scholar 

  • Nozaki Y, Tanford C (1971) The solubility of amino acids and two glycine peptides in aqueous ethanol and dioxane solutions establishment of a hydrophobicity scale. J Biol Chem 246:2211–2217

    CAS  PubMed  Google Scholar 

  • Otsuka H (2005) Purification by solvent extraction using partition coefficient. Nat Prod Isol Methods Biotech 20:269–273

    CAS  Google Scholar 

  • Ro KE, Keener CS, McPheron BA (1997) Molecular phylogenetic study of the Ranunculaceae; utility of the nuclear 26S ribosomal DNA in inferring intrafamilial relationships. Mol Phylogenet Evol 8:117–127

    CAS  Article  PubMed  Google Scholar 

  • Roth T, Burger AM, Dengler W, Willmann H, Fiebig HH (1999) Human tumor cell lines demonstrating the characteristics of the patient tumors as useful models for anticancer drug screening. In: Fiebig HH, Burger AM (eds) Contributions to oncology: relevance of tumor models for anticancer drug development, vol 54. Karger, Basel, pp 145–156

    Google Scholar 

  • Schmutz J (1947) Glycosides and aglycones. XXII. Hellebrin. Pharm Acta Hel 22:373–380

    CAS  Google Scholar 

  • Schmutz J (1949) Die konstitution des hellebrigenins: glykoside und aglykone. Helv Chim Acta 32:1442–1452

    CAS  Article  Google Scholar 

  • Segneanu AE, Grozescu I, Cziple F, Berki Damian D, Niculite CM, Florea A, Leabu M (2015) Helleborus purpurascens-amino acid and peptide analysis linked to the chemical and antiproliferative properties of the extracted compounds. Molecules 20:22170–22187

    CAS  Article  PubMed  Google Scholar 

  • Wissner W, Kating H (1974) Botanical and phytochemical investigations of species of the genus Helleborus growing in Europe and Asian Minor—III. The quantitative contents of hellebrin in plants of the natural biotops and in culture. Planta Med 26:364–374

    CAS  Article  PubMed  Google Scholar 

  • Zacharius RM, Thompson JF, Steward FC (1954) The detection, isolation and identification of l(−)-pipecolic acid in the non-protein fraction of beans (Phaseolus vulgaris). J Am Chem Soc 76:2908–2912

    CAS  Article  Google Scholar 

  • Zeier J (2013) New insights into the regulation of plant immunity by amino acid metabolic pathways. Plant, Cell Environ 36:2085–2103

    CAS  Article  Google Scholar 

Download references


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).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Ion Neda.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Additional information

Handling Editor: M. S. Palma.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 2058 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Helleborus purpurascens
  • Pipecolic acid
  • Biological activity
  • Hellebrin
  • Glutamine fragmentation