Planta Med 2009; 75 - PD13
DOI: 10.1055/s-0029-1234492

A protocol for HPLC based activity profiling of plant and fungal extracts against tropical parasites

M Adams 1, S Zimmermann 1, M Kaiser 1, R Brun 2, M Hamburger 1
  • 1Institute of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
  • 2Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland

Of the more than two hundred thousand secondary metabolites known so far, many have become rewarding leads in the development of new therapeutics. In the area of tropical parasitic diseases, important natural product leads have been quinine, artemisinin, and the avermectins [1,2]. HPLC based activity profiling is an effective strategy to speed up the discovery of new leads. It conveniently combines the superior separation power of HPLC micro scale compound separation with efficient high-throughput biological screening methods. Active substances can be structurally characterized by HPLC hyphenated methods (HPLC-PDA, -MSn, -HR-MS, -NMR etc.), and if so desired, be effectively targeted. The need of handling large numbers of fractions is minimized [3].

We established a library of 640 plant and fungal extracts in 96-well format which was screened for antimalarial, antileishmanial and anti-trypanosomal activity. Active extracts were subjected to a newly developed medium throughput HPLC based activity profiling protocol, to identify the active compounds. 350µg of an extract were injected onto an analytical column (SunFire RP-18, 3.5 um, 3 * 150mm, Waters), and thirty five one- minute fractions were collected into 96 deep well microtiter plates. After parallel evaporation of the microfractions, suitable dilution schemes permitted parallel activity profiling for antimalarial, antileishmanial and anti-trypanosomal activity. The protocol was validated with extracts and positive controls such as Artemisia annua which were treated likewise. The activity was confined to one or few of the micro fractions, which could be chemically characterized online by HPLC-hyphenated methods. Examples of the use of this protocol for the identification of active constituents in a complex extract are shown.

References: [1] Cragg, G.M., Newman, D.J. (2005) Pure Appl Chem 77:7–24.

[2] Wilcox, M.L. et al. (2001) Trends Parasitol. 17:58–60.

[3] Potterat, O. et al. (2006) Curr. Org. Chem. 10:899–920.