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
