Planta Med 2009; 75 - PJ71
DOI: 10.1055/s-0029-1234876

Influence of chlorophyll and tannins in plant extracts on cell-based luciferase reporter gene assays

S Vogl 1, P Picker 1, N Fakhrudin 1, A Atanasov 1, E Heiß 1, G Reznicek 1, J Saukel 1, C Wawrosch 1, VM Dirsch 1, B Kopp 1
  • 1Department of Pharmacognosy, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria

Various studies indicate that several ubiquitous plant compounds, like chlorophyll and tannins, possibly interfere with biological in vitro assays. Chlorophyll might interact with fatty acids, whereas tannins can form tight complexes with metal ions, proteins and polysaccharides [1]. The aim of this study was, to examine whether the chlorophyll and/or tannin content of plant extracts leads to false positive or false negative results in several luciferase gene assays.

Therefore, two model plants, Sambucus nigra and Urtica dioica, were extracted with dichloromethane (DCM) and methanol (MeOH) using the Accelerated Solvent Extractor (Dionex ASE200). From the DCM extract chlorophyll was removed, whereas tannins were separated from the MeOH extract. The chlorophyll separation method was based on a liquid-liquid-repartition between DCM and MeOH:H2O (1:1). For the separation of tannins a liquid-liquid-solvent partition in CHCl3 and 1% NaCl [2] was used. In addition HPLC-MS fingerprints were generated. Samples, with and without the possible interfering substances, were examined for their potential to activate the peroxisome proliferator-activated receptors (PPAR)-α and -γ as well as to inhibit the transcription factor NF-κB. Assays were performed in HEK293 cells transfected with luciferase-reporter constructs for PPAR α/γ or pNF-κB and with green fluorescent protein as internal normalization control. Further on, luciferase activity and fluorescence intensity were quantified.

The results indicated that in all three assay systems the purified extracts were more active. Thus, the pure substances chlorophyll a and b, tannic acid, and epicatechin gallate were tested in the different assay formats too. Since, neither the pure chlorophylls nor the pure tannins had any influence in our assay systems, the higher activity of the purified extracts might be due to enrichment of the active compounds in those.

Acknowledgements: This work is funded by the Austrian Science Fund, FWF: S10704-B037.

References: [1] Potterat, O. and Hamburger, M. (2006) Curr. Org. Chem. 10:899–920.

[2] Wall, M.E. et al. (1996) Phytomedicine 3:281–285.