, Volume 8, Issue 2, pp 335–346 | Cite as

1H NMR-based metabolic profiling and target analysis: a combined approach for the quality control of Thymus vulgaris

  • Valerio Pieri
  • Sonja Sturm
  • Christoph Seger
  • Chlodwig Franz
  • Hermann Stuppner
Original Article


The characterization of T. vulgaris plant material for quality control purposes was performed by NMR-based methods. Direct extraction of 141 T. vulgaris samples with DMSO-d 6 enabled the obtainment of crude extracts with a representative composition in terms of both volatile and non-volatile constituents. The acquisition of 600 MHz 1H NMR spectra resulted in a dataset which was analyzed by a combination of metabolic profiling and target analysis approaches. Preliminary analysis of the 1H NMR spectra was performed by principal component analysis, which revealed sample discrimination on a chemotype basis (thymol, carvacrol and linalool chemotypes). Further minor discriminative constituents were identified as p-cymene, γ-terpinene, rosmarinic acid, and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl. Metabolite identification was accomplished by 1D and 2D NMR techniques and supported by spiking experiments. Fast dereplication of constituents not available as reference compounds was performed by HPLC–SPE–NMR experiments. A targeted approach based on qHNMR was validated for quantification of the identified secondary metabolites. Validation was performed in terms of precision (intra-day RSD ≤ 4.51%, inter-day RSD ≤ 4.18%), repeatability (RSD ≤ 2.30%), accuracy (recovery rates within 93.4 and 103.4%), linearity (correlation coefficients ≥ 0.9990), robustness, and stability. The amount of the dominant monoterpene in thymol, carvacrol, and linalool chemotypes was respectively found to be within 0.4–2.6, 0.7–2.3, and 1.1–3.6% (w/w). Variable amounts of the precursors p-cymene and γ-terpinene were found in thymol and carvacrol chemotypes. The highest amount of rosmarinic acid and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl in the analyzed samples was respectively 4.6 and 0.4% (w/w). Since quantification is performed on a weight basis, the essential oil content can be estimated based on the sum of the quantified monoterpenes. The NMR-based analysis of T. vulgaris represents a more comprehensive approach in comparison to traditional chromatographic methods such as GC and LC, respectively employed for the analysis of volatile and non-volatile constituents. Further advantages lie in the simple sample preparation, rapidity and reproducibility of the NMR analysis.


Thymus vulgaris 1H NMR Metabolic profiling Target analysis Principal component analysis qHNMR 



This work was financially supported by Bionorica Research GmbH, 6020 Innsbruck, Austria.

Supplementary material

11306_2011_317_MOESM1_ESM.docx (4.8 mb)
Supplementary material 1 (DOCX 4888 kb)


  1. Abdel-Farid, I. B., Kim, H. K., Choi, Y. H., & Verpoorte, R. (2007). Metabolic characterization of Brassica rapa leaves by NMR spectroscopy. Journal of Agricultural and Food Chemistry, 55, 7936–7943.PubMedCrossRefGoogle Scholar
  2. Agnolet, S., Jaroszewski, J. W., Verpoorte, R., & Staerk, D. (2010). 1H NMR-based metabolomics combined with HPLC–PDA–MS–SPE–NMR for investigation of standardized Ginkgo biloba preparations. Metabolomics, 6, 292–302.PubMedCrossRefGoogle Scholar
  3. Chizzola, R., Michitsch, H., & Franz, C. (2008). Antioxidative properties of Thymus vulgaris leaves: Comparison of different extracts and essential oil chemotypes. Journal of Agricultural and Food Chemistry, 56, 6897–6904.PubMedCrossRefGoogle Scholar
  4. Choi, Y. H., Choi, H.-K., Hazekamp, A., Bermejo, P., Schilder, Y., Erkelens, C., et al. (2003). Quantitative analysis of bilobalide and ginkgolides from Ginkgo biloba leaves and Ginkgo products using 1H-NMR. Chemical and Pharmaceutical Bulletin, 51, 158–161.CrossRefGoogle Scholar
  5. Council of Europe. (2008). European pharmacopoeia (6th ed.). Strasbourg Cedex: Council of Europe.Google Scholar
  6. Dastmalchi, K., Ollilainen, V., Lackman, P., Boije af Gennaes, G., Dorman, H. J. D., Jaervinen, P. P., et al. (2009). Acetylcholinesterase inhibitory guided fractionation of Melissa officinalis L. Bioorganic & Medicinal Chemistry, 17, 867–871.CrossRefGoogle Scholar
  7. Deans, S. G., & Ritchie, G. (1987). Antibacterial properties of plant essential oils. International Journal of Food Microbiology, 5, 165–180.CrossRefGoogle Scholar
  8. Frederich, M., Choi, Y. H., & Verpoorte, R. (2003). Quantitative analysis of strychnine and brucine in Strychnos nux-vomica using 1H-NMR. Planta Medica, 69, 1169–1171.PubMedCrossRefGoogle Scholar
  9. Gottlieb, H. E., Kotlyar, V., & Nudelman, A. (1997). NMR chemical shifts of common laboratory solvents as trace impurities. Journal of Organic Chemistry, 62, 7512–7515.PubMedCrossRefGoogle Scholar
  10. Granger, R., & Passet, J. (1973). Thymus vulgaris spontane de France: Races chimiques et chemotaxonomie. Phytochemistry, 12, 1683–1691.CrossRefGoogle Scholar
  11. Haraguchi, H., Saito, T., Ishikawa, H., Date, H., Katoka, S., Tamura, Y., et al. (1996). Antiperoxidative components in Thymus vulgaris. Planta Medica, 62, 217–221.PubMedCrossRefGoogle Scholar
  12. Helander, I. M., Alakomi, H.-L., Latva-Kala, K., Mattila-Sandholm, T., Pol, I., Smid, E. J., et al. (1998). Characterization of the action of selected essential oil components on Gram-negative bacteria. Journal of Agricultural and Food Chemistry, 46, 3590–3595.CrossRefGoogle Scholar
  13. Holmes, E., Tang, H., Wang, Y., & Seger, C. (2006). The assessment of plant metabolite profiles by NMR-based methodologies. Planta Medica, 72, 771–785.PubMedCrossRefGoogle Scholar
  14. Lecomte, J., Giraldo, L. J. L., Laguerre, M., Barea, B., & Villeneuve, P. (2010). Synthesis, characterization and free radical scavenging properties of rosmarinic acid fatty esters. Journal of the American Oil Chemists’ Society, 87, 615–620.CrossRefGoogle Scholar
  15. Mallakin, A., McConkey, B. J., Miao, G., McKibben, B., Snieckus, V., Dixon, D. G., et al. (1999). Impacts of structural photomodification on the toxicity of environmental contaminants: anthracene photooxidation products. Ecotoxicology and Environmental Safety, 43, 204–212.PubMedCrossRefGoogle Scholar
  16. Maniara, G., Rajamoorthi, K., Rajan, S., & Stockton, G. W. (1998). Method performance and validation for quantitative analysis by 1H and 31P NMR spectroscopy. Applications to analytical standards and agricultural chemicals. Analytical Chemistry, 70, 4921–4928.PubMedCrossRefGoogle Scholar
  17. Miura, K., Inagaki, T., & Nakatani, N. (1989). Structure and activity of new deodorant biphenyl compounds from thyme (Thymus vulgaris L.). Chemical and Pharmaceutical Bulletin, 37, 1816–1819.CrossRefGoogle Scholar
  18. Pauli, G. F., Jaki, B. U., & Lankin, D. C. (2005). Quantitative 1H NMR: Development and potential of a method for natural products analysis. Journal of Natural Products, 68, 133–149.PubMedCrossRefGoogle Scholar
  19. Rasmussen, B., Cloarec, O., Tang, H., Staerk, D., & Jaroszewski, J. W. (2006). Multivariate analysis of integrated and full-resolution 1H-NMR spectral data from complex pharmaceutical preparations: St: John’s Wort. Planta Medica, 72, 556–563.PubMedCrossRefGoogle Scholar
  20. Reiter, M., & Brandt, W. (1985). Relaxant effects on tracheal and ileal smooth muscles of the guinea pig. Arzneimittel-Forschung, 35, 408–414.PubMedGoogle Scholar
  21. Smith-Palmer, A., Stewart, J., & Fyfe, L. (1998). Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Letters in Applied Microbiology, 26, 118–122.PubMedCrossRefGoogle Scholar
  22. Tarachiwin, L., Ute, K., Kobayashi, A., & Fukusaki, E. (2007). 1H NMR based metabolic profiling in the evaluation of Japanese green tea quality. Journal of Agricultural and Food Chemistry, 55, 9330–9336.PubMedCrossRefGoogle Scholar
  23. Ternes, W., Gronemeyer, M., & Schwarz, K. (1995). Determination of p-cymene-2,3-diol, thymol, and carvacrol in different foodstuffs. Zeitschrift fuer Lebensmittel-Untersuchung und -Forschung, 201, 544–547.CrossRefGoogle Scholar
  24. Thompson, J. D., Chalchat, J.-C., Michet, A., Linhart, Y. B., & Ehlers, B. (2003). Qualitative and quantitative variation in monoterpene co-occurrence and composition in the essential oil of Thymus vulgaris chemotypes. Journal of Chemical Ecology, 29, 859–880.PubMedCrossRefGoogle Scholar
  25. 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.Google Scholar
  26. Van den Broucke, C. O., & Lemli, J. A. (1981). Pharmacological and chemical investigation of thyme liquid extracts. Planta Medica, 41, 129–135.PubMedCrossRefGoogle Scholar
  27. Van den Broucke, C. O., & Lemli, J. A. (1982). Antispasmodic activity of Origanum compactum. Part 2. Antagonistic effect of thymol and carvacrol. Planta Medica, 45, 188–190.PubMedCrossRefGoogle Scholar
  28. Van Den Broucke, C. O., & Lemli, J. A. (1983). Spasmolytic activity of the flavonoids from Thymus vulgaris. Pharmaceutisch weekblad. Scientific Edition, 5, 9–14.CrossRefGoogle Scholar
  29. Venskutonis, P. R. (2002). Harvesting and post-harvest handling in the genus Thymus. In E. Stahl-Biskup & F. Saez (Eds.), Thyme: The genus Thymus (pp. 197–223). London: Taylor & Francis.Google Scholar
  30. Wang, Y., Tang, H., Nicholson, J. K., Hylands, P. J., Sampson, J., Whitcombe, I., et al. (2004). Metabolomic strategy for the classification and quality control of phytomedicine: a case study of chamomile flower (Matricaria recutita L.). Planta Medica, 70, 250–255.PubMedCrossRefGoogle Scholar
  31. Ward, J. L., Baker, J. M., Miller, S. J., Deborde, C., Maucourt, M., Biais, B., et al. (2010). An inter-laboratory comparison demonstrates that [1H]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection. Metabolomics, 6, 263–273.PubMedCrossRefGoogle Scholar
  32. Yang, S. Y., Kim, H. K., Lefeber, A. W. M., Erkelens, C., Angelova, N., Choi, Y. H., et al. (2006). Application of two-dimensional nuclear magnetic resonance spectroscopy to quality control of ginseng commercial products. Planta Medica, 72, 364–369.PubMedCrossRefGoogle Scholar
  33. Zarzuelo, A., & Crespo, E. (2002). The medicinal and non-medicinal uses of thyme. In E. Stahl-Biskup & F. Saez (Eds.), Thyme: The genus Thymus (pp. 263–292). London: Taylor & Francis.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Valerio Pieri
    • 1
  • Sonja Sturm
    • 1
  • Christoph Seger
    • 1
  • Chlodwig Franz
    • 2
  • Hermann Stuppner
    • 1
  1. 1.Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences InnsbruckUniversity of InnsbruckInnsbruckAustria
  2. 2.Institute for Applied Botany and PharmacognosyUniversity of Veterinary Medicine ViennaViennaAustria

Personalised recommendations