Metabolomic Profiling of Liquid Echinacea Medicinal Products with In Vitro Inhibitory Effects on Cytochrome P450 3A4 (CYP3A4)

Abstract

Echinacea is a popular and widely used herbal medicinal product and consequently, studies of its interactions with conventional drugs are of particular importance. We have shown that Echinacea preparations and some common alkylamides weakly inhibit several cytochrome P450 (CYP) isoforms, with considerable variation in potency. We now report a detailed analysis of six commercial Echinacea liquid preparations, with emphasis on the metabolomic characterisation of the Echinacea compounds responsible for inhibiting CYP3A4. We separated each preparation into its ethanol- and water-soluble components, and then used ¹H‐NMR together with multivariate data analysis and partial least square regression analysis to investigate the nature of the compounds responsible for CYP3A4 inhibition. The results implicated alkylamides in the CYP3A4 inhibitory activity of Echinacea. One of the commercial preparations (Echinaforce®) was further fractionated using solid phase extraction. Analysis by ¹H‐NMR and mass spectroscopy (LC/MS, tandem MS, accurate mass) identified dodeca-2E,4E,8Z,10E/Z-tetraenoic acid (alkylamide 1) and a new compound (putative molecular formula C₁₈H₃₆ NO⁺) as major components of the inhibitory fractions. In addition, the alkylamide content of all six preparations was determined by reverse phase HPLC. Levels of alkylamides 1 and 3 (undeca-2E,4E/Z-diene-8,10-diynoic acid isobutylamide), correlated well with CYP3A4 inhibition. The acetylene tetradeca-8Z-ene-11,13-diyn-2-one was shown to be present in the E. purpurea as well as the E. pallida extracts. E. purpurea unlike E. pallida was thought to not contain significant amounts of acetylenes. Our results directly confirm the role of alkylamides in the inhibition of CYP3A4 by Echinacea and uncovered a new compound which may also be involved. Extensive differences in the composition of the commercially available preparations were found. This will inevitably impact on the product efficacy, safety and pharmacological effects, especially since the differences involve alkylamides, an important class of Echinacea’s active constituents. The metabolomic approach presented here may prove valuable as a screening or quality control tool.

Echinacea - Asteraceae (Compositae) - metabolomics - alkylamides - cytochrome P450 enzymes - herb‐drug interactions

References

• 1 Modarai M, Gertsch J, Suter A, Heinrich M, Kortenkamp A. Cytochrome P450 inhibitory action of Echinacea preparations differs widely and co-varies with alkylamide content.  J Pharm Pharmacol. 2007;  59 567-573
• 2 Coxeter P D, McLachlan A J, Duke C C, Roufogalis B D. Herb-drug interactions: an evidence based approach.  Curr Med Chem. 2004;  11 1513-1525
• 3 BfArM (Bundesinstitut für Arzneimittel und Medizinprodukte Germany) .www.phytotherapy.org/Interaktionen.pdf Accessed September 28, 2009
• 4 Guengerich F P. Role of cytochrome P450 enzymes in drug-drug interactions.  Adv Pharmacol. 1997;  43 7-35
• 5 Fiehn O. Metabolomics – the link between genotypes and phenotypes.  Plant Mol Biol. 2002;  48 155-171
• 6 Kim H K, Choi Y H, Verpoorte R. Metabolomic analysis of Catharanthus roseus using NMR and principal component analysis. Saito K, Dixon RA, Willmitzer L Biotechnology in agriculture and forestry 57: plant metabolomics. Berlin, Heidelberg; Springer-Verlag 2006
• 7 Bailey N J, Sampson J, Hylands P J, Nicholson J K, Holmes E. Multi-component metabolic classification of commercial feverfew preparations via high-field ¹H-NMR spectroscopy and chemometrics.  Planta Med. 2002;  68 734-738
• 8 Rasmussen B, Cloarec O, Tang H, Staerk D, Jaroszewski J W. Multivariate analysis of integrated and full-resolution ¹H-NMR spectral data from complex pharmaceutical preparations: St. John's Wort.  Planta Med. 2006;  72 556-563
• 9 Wang Z, Gorski J C, Hamman M A, Huang S M, Lesko L J, Hall S D. The effects of St. John's wort (Hypericum perforatum) on human cytochrome P450 activity.  Clin Pharmacol Ther. 2001;  70 317-326
• 10 Kim H K, Choi Y H, Erkelens C, Lefeber A W, Verpoorte R. Metabolic fingerprinting of Ephedra species using 1H-NMR spectroscopy and principal component analysis.  Chem Pharm Bull (Tokyo). 2005;  53 105-109
• 11 Cech N B, Eleazer M S, Shoffner L T, Crosswhite M R, Davis A C, Mortenson A M. High performance liquid chromatography/electrospray ionization mass spectrometry for simultaneous analysis of alkamides and caffeic acid derivatives from Echinacea purpurea extracts.  J Chromatogr A. 2006;  1103 219-228
• 12 Bauer R, Reminger P, Wagner H. Alkamides from the roots of Echinacea purpurea.  Phytochemistry. 1988;  27 2339-2342
• 13 Cravatt B F, Lerner R A, Boger D L. Structure determination of an endogenous sleep-inducing lipid, cis-9-octadecenamide (oleamide): a synthetic approach to the chemical analysis of trace quantities of a natural product.  J Am Chem Soc. 1996;  118 580-590
• 14 Miller S C, Yu H C. Echinacea: the genus Echinacea. London; CRC Press 2004
• 15 Heinrich M, Modarai M, Kortenkamp A. Herbal extracts used for upper respiratory tract infections: are there clinically relevant interactions with the cytochrome P450 enzyme system?.  Planta Med. 2008;  74 657-660