Abstract
Previously we have shown that commercially available echinacea extracts vary widely in their inhibitory activity on CYP3A4 (IC
50
values: 12.71µg/ml -1812µg/ml) in the supersome assay
1
. Modern NMR-spectroscopy and principal component analysis allows evaluation of differences between complex mixtures
2
. To correlate such differences to CYP3A4 inhibition we examined various Echinacea extracts. Six commercially available Echinacea extracts were chosen. Each extract was separated into ethanolic and water fractions, which were assayed for CYP3A4 inhibition. In tandem, 400MHz
1
H- NMR spectra were obtained in deuterated ethanol or deuterium oxide with internal standards. Principal component analysis was conducted on the data.
The inhibitory activity of the extracts resided mostly in the ethanolic fraction (with IC
50
values ten folds lower than original extract e.g. Echinaforce IC
50
: 22µg/ml, vs. ethanolic fraction 2µg/ml).
1
H NMR spectra of the ethanolic fractions showed a clear difference between the most and least active extracts. Greater inhibition was associated with the presence of peaks at 1–3 ppm and 6–8 ppm (visual inspection).
Principal component analysis revealed good correlation between differences in
1
H spectra and IC
50
values. Key contributors were identified in the regions: 0.875 ppm, 0.925 ppm, 1.275 ppm, 1.325 ppm. The ethanolic fraction of Echinaforce was further fractionated by SPE (C-18, water: ethanol 10% step gradient). Two potent fractions were identified (IC
50
: 0.43–0.58µg/ml).
1
H NMR analysis also revealed additional peaks at ˜7ppm, which were unique to these fractions. Amongst other compounds, we suspect that the ethanol soluble alkylamides, (potent CYP3A4 inhibitors) may be found in these fractions.
Acknowledgements:
Bioforce for funding this project.
References:
[1] Modarai, M. et al. (2007) Journal of Pharmacy and Pharmacology 59: 567–573 [2] Holmes, E. et al. (2006) Planta Med. 72: 771–785.