Rapid determination of nonsteroidal anti-inflammatory properties of alpine medicinal plants (NAIP)

The determination in liquid medium of the in vitro nonsteroidal anti-inflammatory activity of all kinds of matrices has been the subject of quite a number of studies. Such measurements avoid but do not replace, in vivo experiments on animals. Nevertheless they are very useful in systematic studies. They enable the quantification of the inhibition of certain enzymes that are responsible for the biochemical signal of inflammations, e.g. phospholipase A₂, 5- and 12-lipoxygenase and cyclooxygenase. Commercially available standard microcuvette colorimetric tests work well with pure dissolved substances. However, the usefulness of these tests is limited when the active substance is present in a complex matrix, such as raw and refined plant extracts. Recently it has been shown for acetylcholinesterase [1], that it is possible to apply an enzymatic reaction to a thin layer, after separation of the matrix, in order to characterize by colorimetry the inhibitory effect of each individual fraction. As the characterization of the nonsteroidal anti-inflammatory activity without previous separation of a plant extract is questionable, transferring commercial microcuvette tests to thin layers allows a more reliable direct comparison of raw plant extracts when carrying out a series of comparative measurements. Applying such enzymatic tests requires partial separation of the constituents by TLC, and colorimetric detection in situ by spraying well-defined quantities per surface unit of a solution of the enzyme, the substrate (lipoxygenase inhibition) or the developer (phospholipase A2). A second treatment is sometimes needed after fixation of the reagents (cyclooxygenase). The global in vitro nonsteroidal anti-inflammatory activity is presented, based on the IC₅₀ values measured for each specific enzymatic reaction. The results obtained for microcuvettes and for thin layers will be compared and presented for screening tests of raw extracts of a dozen alpine plants. The results obtained confirm the advantages of the newly developed method.

Reference: [1] A. Marston et al. (2002) Phytochem. Anal. 13: 51