, Volume 10, Issue 4, pp 574–588 | Cite as

Metabolite profiling and fingerprinting of Hypericum species: a comparison of MS and NMR metabolomics

  • Andrea Porzel
  • Mohamed A. Farag
  • Julia Mülbradt
  • Ludger A. Wessjohann
Original Article


Hypericum perforatum, commonly known as St. John’s wort, is a popular herbal supplement used for the treatment of mild to moderate depression. The major secondary metabolites of St. John’s wort extracts include phenylpropanoids, flavonoids, xanthones, phloroglucinols, and naphthodianthrones. There are over 400 species in the genus Hypericum world-wide, most of which are little or not characterized in terms of phytochemical or pharmacological properties. Metabolomics techniques were used to investigate the natural product diversity within the genus Hypericum (Hypericaceae) and its correlation to bioactivity, exemplified by cytotoxic properties. Utilizing nuclear magnetic resonance (NMR) fingerprinting and mass spectrometry (MS) metabolic profiling techniques, MS and NMR spectra of extracts from H. perforatum, H. polyphyllum, H. tetrapterum, H. androsaemum, H. inodorum, H. undulatum and H. kouytchense were evaluated and submitted to statistical multivariate analyses. Although comparable score plots in principal component analysis were derived from both MS and NMR datasets, loading plots reveal, that different set of metabolites contribute for species segregation in each dataset. Major peaks in 1H NMR and MS spectra contributing to species discrimination were assigned as those of hyperforins, lipids, chlorogenic and shikimic acid. Shikimic acid and its downstream phenylpropanoids were more enriched in H. perforatum, H. androsaemum, H. kouytchense and H. inodorum extracts; whereas a novel hyperforin was found exclusively in H. polyphyllum. Next to H. perforatum, H. polyphyllum and H. tetrapterum show the highest levels of hypericins, and H. perforatum and H. polyphyllum are highest in phloroglucinols, suggesting that the latter species might be used as an alternative to St. John’s wort. However, the major hyperforin-type compound in H. polyphyllum possesses a novel constitution of yet unknown bioactivity. Anti-cancer in vitro assays to evaluate the ability of extracts from Hypericum species in inhibiting prostate and colon cancer growth suggest that such bioactivity might be predicted by gross metabolic profiling.


H. perforatum H. polyphyllum 1H NMR-based metabolomics LC–MS Hyperforin Anticancer activity prediction 



Electrospray ionisation


Liquid chromatography


Mass spectrometry


Tandem mass spectrometry


Nuclear magnetic resonance


Photodiode array detection


Principal component analysis


Hierarchical cluster analysis



Mohamed A. Farag thanks the Alexander von Humboldt-Foundation, Germany for financial support. We also thank Christoph Böttcher for assistance with the UPLC–MS. We are grateful to Steffen Neumann and Tilo Lübken for providing R scripts for NMR and MS data analysis.

Supplementary material

11306_2013_609_MOESM1_ESM.pdf (542 kb)
Supplementary material 1 (PDF 543 kb)


  1. Adam, P., Arigoni, D., Bacher, A., & Eisenreich, W. (2002). Biosynthesis of hyperforin in Hypericum perforatum. Journal of Medicinal Chemistry, 45(21), 4786–4793.CrossRefPubMedGoogle Scholar
  2. Agnolet, S., Jaroszewski, J. W., Verpoorte, R., & Staerk, D. (2010). H-1 NMR-based metabolomics combined with HPLC–PDA–MS–SPE–NMR for investigation of standardized Ginkgo biloba preparations. Metabolomics, 6(2), 292–302.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Alali, F. Q., & Tawaha, K. (2009). Dereplication of bioactive constituents of the genus Hypericum using LC-(+/−)–ESI–MS and LC-PDA techniques: Hypericum triquetrifolium as a case study. Saudi Pharmaceutical Journal, 17(4), 269–274.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Alali, F. Q., Tawaha, K., & Ghareibeh, M. (2009). LC–MS and LC–PDA analysis of Hypericum empetrifolium and Hypericum sinaicum. Zeitschrift fur Naturforschung, Section C: Journal of Biosciences, 64(7/8), 476–482.Google Scholar
  5. Bilia, A. R., Bergonzi, M. C., Mazzi, G., & Vincieri, F. F. (2001). Analysis of plant complex matrices by use of nuclear magnetic resonance spectroscopy: St. John’s wort extract. Journal of Agricultural and Food Chemistry, 49(5), 2115–2124.CrossRefPubMedGoogle Scholar
  6. Bonkanka, C. X., Smelcerovic, A., Zuehlke, S., Rabanal, R. M., Spiteller, M., & Sanchez-Mateo, C. D. (2008). HPLC–MS analysis and anti-oedematogenic activity of Hypericum grandifolium Choisy (Hypericaceae). Planta Medica, 74(7), 719–725.CrossRefPubMedGoogle Scholar
  7. Brolis, M., Gabetta, B., Fuzzati, N., Pace, R., Panzeri, F., & Peterlongo, F. (1998). Identification by high-performance liquid chromatography diode array detection mass spectrometry and quantification by high-performance liquid chromatography UV absorbance detection of active constituents of Hypericum perforatum. Journal of Chromatography A, 825(1), 9–16.CrossRefGoogle Scholar
  8. Brunarska, Z. O. F. I. (1962). A study of some hypericin species of the genus Hypericum 1. II. Hypericum polyphyllum Boiss et Bal. Dissertationes Pharmaceuticae, 14(1), 89–97.Google Scholar
  9. Butterweck, V., Wall, A., Lieflaender-Wulf, U., Winterhoff, H., & Nahrstedt, A. (1997). Effects of the total extract and fractions of Hypericum perforatum in animal assays for antidepressant activity. Pharmacopsychiatry, 30, 117–124.CrossRefPubMedGoogle Scholar
  10. Charchoglyan, A., Abrahamyan, A., Fujii, I., Boubakir, Z., Gulder, T. A. M., Kutchan, T. M., et al. (2007). Differential accumulation of hyperforin and secohyperforin in Hypericum perforatum tissue cultures. Phytochemistry, 68, 2670–2677.CrossRefPubMedGoogle Scholar
  11. de Rijke, E., Zappey, H., Ariese, F., Gooijer, C., & Brinkman, U. A. T. (2003). Liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry of flavonoids with triple-quadrupole and ion-trap instruments. Journal of Chromatography A, 984(1), 45–58.CrossRefPubMedGoogle Scholar
  12. Farag, M. A., Porzel, A., Schmidt, J., & Wessjohann, L. A. (2012a). Metabolite profiling and fingerprinting of commercial cultivars of Humulus lupulus L. (hop): A comparison of MS and NMR methods in metabolomics. Metabolomics, 8(3), 492–507.CrossRefGoogle Scholar
  13. Farag, M. A., Porzel, A., & Wessjohann, L. A. (2012b). Comparative metabolite profiling and fingerprinting of medicinal licorice roots using a multiplex approach of GC–MS, LC–MS and 1D NMR techniques. Phytochemistry, 76, 60–72.CrossRefPubMedGoogle Scholar
  14. Farag, M. A., & Wessjohann, L. A. (2012). Metabolome classification of commercial Hypericum perforatum (St. John’s wort) preparations via UPLC–qTOF-MS and chemometrics. Planta Medica, 78(5), 488–496.CrossRefPubMedGoogle Scholar
  15. Fornal, C. A., Metzler, C. W., Mirescu, C., Stein, S. K., & Jacobs, B. L. (2001). Effects of standardized extracts of St. John’s wort on the single-unit activity of serotonergic dorsal raphe neurons in awake cats: Comparisons with fluoxetine and sertraline. Neuropsychopharmacology, 25(6), 858–870.CrossRefPubMedGoogle Scholar
  16. Giese, J. (1999). Tests for nutraceutical products. Food Technology, 53(10), 93.Google Scholar
  17. Gioti, E. M., Fiamegos, Y. C., Skalkos, D. C., & Stalikas, C. D. (2009). Antioxidant activity and bioactive components of the aerial parts of Hypericum perforatum L. from Epirus, Greece. Food Chemistry, 117(3), 398–404.CrossRefGoogle Scholar
  18. Gobbi, M., & Mennini, T. (2001). Is St John’s wort a ‘Prozac-like’ herbal antidepressant? Trends in Pharmacological Sciences, 22(11), 557–559.CrossRefPubMedGoogle Scholar
  19. Goodacre, R., Shann, B., Gilbert, R. J., Timmins, E. M., McGovern, A. C., Alsberg, B. K., et al. (2000). Detection of the dipicolinic acid biomarker in Bacillus spores using Curie-point pyrolysis mass spectrometry and Fourier transform infrared spectroscopy. Analytical Chemistry, 72(1), 119–127.CrossRefPubMedGoogle Scholar
  20. Heinke, R., Arnold, N., Wessjohann, L., & Schmidt, J. (2013). Negative ion tandem mass spectrometry of prenylated fungal metabolites and their derivatives. Analytical and Bioanalytical Chemistry, 405(1), 177–189.CrossRefPubMedGoogle Scholar
  21. Hillwig, M. L., Hammer, K. D., Birt, D. F., & Wurtele, E. S. (2008). Characterizing the metabolic fingerprint and anti-inflammatory activity of Hypericum gentianoides. Journal of Agricultural and Food Chemistry, 56(12), 4359–4366.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hoelscher, D., Shroff, R., Knop, K., Gottschaldt, M., Crecelius, A., Schneider, B., et al. (2009). Matrix-free UV-laser desorption/ionization (LDI) mass spectrometric imaging at the single-cell level: Distribution of secondary metabolites of Arabidopsis thaliana and Hypericum species. Plant Journal, 60(5), 907–918.CrossRefGoogle Scholar
  23. Jiang, H. L., Xie, Z. Z., Koo, H. J., McLaughlin, S. P., Timmermann, B. N., & Gang, D. R. (2006). Metabolic profiling and phylogenetic analysis of medicinal Zingiber species: Tools for authentication of ginger (Zingiber officinale Rosc.). Phytochemistry, 67(15), 1673–1685.CrossRefPubMedGoogle Scholar
  24. Keller, J. H., Karas, M., Muller, W. E., Volmer, D. A., Eckert, G. P., Tawab, M. A., et al. (2003). Determination of hyperforin in mouse brain by high-performance liquid chromatography/tandem mass spectrometry. Analytical Chemistry, 75(22), 6084–6088.CrossRefPubMedGoogle Scholar
  25. Kitanov, G. M. (2001). Hypericin and pseudohypericin in some Hypericum species. Biochemical Systematics and Ecology, 29(2), 171–178.CrossRefPubMedGoogle Scholar
  26. Kusari, S., Zuhlke, S., Borsch, T., & Spiteller, M. (2009). Positive correlations between hypericin and putative precursors detected in the quantitative secondary metabolite spectrum of Hypericum. Phytochemistry, 70(10), 1222–1232.CrossRefPubMedGoogle Scholar
  27. Laakmann, G., Dienel, A., & Kieser, M. (1998a). Clinical significance of hyperforin for the efficacy of Hypericum extracts on depressive disorders of different severities. Phytomedicine, 5(6), 435–442.CrossRefPubMedGoogle Scholar
  28. Laakmann, G., Schule, C., Baghai, T., & Kieser, M. (1998b). St. John’s Wort in mild to moderate depression: The relevance of hyperforin for the clinical efficacy. Pharmacopsychiatry, 31, 54–59.CrossRefPubMedGoogle Scholar
  29. Lee, J., Duke, R. K., Tran, V. H., Hook, J. M., & Duke, C. C. (2006). Hyperforin and its analogues inhibit CYP3A4 enzyme activity. Phytochemistry, 67(23), 2550–2560.CrossRefPubMedGoogle Scholar
  30. Liu, F., Pan, C., Drumm, P., & Ang, C. Y. W. (2005). Liquid chromatography–mass spectrometry studies of St. John’s wort methanol extraction: Active constituents and their transformation. Journal of Pharmaceutical and Biomedical Analysis, 37(2), 303–312.CrossRefPubMedGoogle Scholar
  31. Meruelo, D., Lavie, G., & Lavie, D. (1988). Therapeutic agents with dramatic antiretroviral activity and little toxicity at effective doses—Aromatic polycyclic diones hypericin and pseudohypericin. Proceedings of the National Academy of Sciences of the United States of America, 85(14), 5230–5234.CrossRefPubMedPubMedCentralGoogle Scholar
  32. Nahrstedt, A., & Butterweck, V. (1997). Biologically active and other chemical constituents of the herb of Hypericum perforatum L. Pharmacopsychiatry, 30, 129–134.CrossRefPubMedGoogle Scholar
  33. Osinska, E. (2003). Morphological, developmental and chemical analysis of 7 species of genus Hypericum. Annals of Warsaw Agricultural University, Horticulture (Landscape Architecture), 24, 23–26.Google Scholar
  34. Payne, R., & Edmonds, M. (2005). Isolation of shikimic acid from star aniseed. Journal of Chemical Education, 82(4), 599–600.CrossRefGoogle Scholar
  35. Politi, M., Zloh, M., Pintado, M. E., Castro, P. M. L., Heinrich, M., & Prieto, J. M. (2009). Direct metabolic fingerprinting of commercial herbal tinctures by nuclear magnetic resonance spectroscopy and mass spectrometry. Phytochemical Analysis, 20(4), 328–334.CrossRefPubMedGoogle Scholar
  36. Pongsuwan, W., Fukusaki, E., Bamba, T., Yonetani, T., Yamahara, T., & Kobayashi, A. (2007). Prediction of Japanese green tea ranking by gas chromatography/mass spectrometry-based hydrophilic metabolite fingerprinting. Journal of Agricultural and Food Chemistry, 55(2), 231–236.CrossRefPubMedGoogle Scholar
  37. Poutaraud, A., Lobstein, A., Girardin, P., & Weniger, B. (2001). Improved procedure for the quality control of Hypericum perforatum L. Phytochemical Analysis, 12(6), 355–362.CrossRefPubMedGoogle Scholar
  38. Rasmussen, B., Cloarec, O., Tang, H. R., Staerk, D., & Jaroszewski, J. W. (2006). Multivariate analysis of integrated and full-resolution H-1-NMR spectral data from complex pharmaceutical preparations: St. John’s wort. Planta Medica, 72(6), 556–563.CrossRefPubMedGoogle Scholar
  39. Sagratini, G., Ricciutelli, A., Vittori, S., Öztürk, N., Öztürk, Y., & Magg, E. (2008). Phytochemical and antioxidant analysis of eight Hypericum taxa from Central Italy. Fitoterapia, 79(3), 210–213.CrossRefPubMedGoogle Scholar
  40. Schempp, C. M., Kirkin, V., Simon-Haarhaus, B., Kersten, A., Kiss, J., Termeer, C. C., et al. (2002a). Inhibition of tumour cell growth by hyperforin, a novel anticancer drug from St. John’s wort that acts by induction of apoptosis. Oncogene, 21(8), 1242–1250.CrossRefPubMedGoogle Scholar
  41. Schempp, C. M., Müller, K. A., Winghofer, B., Schöpf, E., & Simon, J. C. (2002b). Saint John’s wort in dermatology. Der Hautarzt, 53(5), 316–321.CrossRefGoogle Scholar
  42. Schmidt, B., Jaroszewski, J. W., Bro, R., Witt, M., & Staerk, D. (2008). Combining PARAFAC analysis of HPLC-PDA profiles and structural characterization using HPLC-PDA-SPE-NMR-MS experiments: Commercial preparations of St. John’s wort. Analytical Chemistry, 80(6), 1978–1987.CrossRefPubMedGoogle Scholar
  43. Smelcerovic, A., & Spiteller, M. (2006). Phytochemical analysis of nine Hypericum L. species from Serbia and the FYR Macedonia. Pharmazie, 61(3), 251–252.PubMedGoogle Scholar
  44. Smelcerovic, A., Spiteller, M., & Zuehlke, S. (2006a). Comparison of methods for the exhaustive extraction of hypericins, flavonoids, and hyperforin from Hypericum perforatum L. Journal of Agricultural and Food Chemistry, 54(7), 2750–2753.CrossRefPubMedGoogle Scholar
  45. Smelcerovic, A., Verma, V., Spiteller, M., Ahmad, S. M., Puri, S. C., & Qazi, G. N. (2006b). Phytochemical analysis and genetic characterization of six Hypericum species from Serbia. Phytochemistry, 67(2), 171–177.CrossRefPubMedGoogle Scholar
  46. Smith, C. A., Want, E. J., O’Maille, G., Abagyan, R., & Siuzdak, G. (2006). XCMS: Processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Analytical Chemistry, 78(3), 779–787.CrossRefPubMedGoogle Scholar
  47. Tatsis, E. C., Boeren, S., Exarchou, V., Troganis, A. N., Vervoort, J., & Gerothanassis, I. P. (2007). Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS. Phytochemistry, 68(3), 383–393.CrossRefPubMedGoogle Scholar
  48. Umek, A., Kreft, S., Kartnig, T., & Heydel, B. (1999). Quantitative phytochemical analyses of six Hypericum species growing in Slovenia. Planta Medica, 65(4), 388–390.CrossRefPubMedGoogle Scholar
  49. van den Berg, R. A., Hoefsloot, H. C. J., Westerhuis, J. A., Smilde, A. K., & van der Werf, M. J. (2006). Centering, scaling, and transformations: Improving the biological information content of metabolomics data. BMC Genomics, 7, 142.CrossRefPubMedPubMedCentralGoogle Scholar
  50. Xie, Z. Z., Ma, X. Q., & Gang, D. R. (2009). Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism. Journal of Experimental Botany, 60(1), 87–97.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Andrea Porzel
    • 1
  • Mohamed A. Farag
    • 1
    • 2
  • Julia Mülbradt
    • 1
    • 3
  • Ludger A. Wessjohann
    • 1
  1. 1.Department of Bioorganic ChemistryLeibniz Institute of Plant BiochemistryHalle (Saale)Germany
  2. 2.Pharmacognosy Department, College of PharmacyCairo UniversityCairoEgypt
  3. 3.FB Biologie/ChemieUniversität BremenBremenGermany

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