A modified HET-CAM assay approach to the assessment of anti-irritant properties of plant extracts

Hen's egg-chorioallantoic membranes were used to screen for and assess anti -irritant properties among aqueous extracts of plants (HET-CAM tests), in c onnection with searches for plant-derived substances with topical anti-irri tant action. The main question to be answered was whether CAM-assay screeni ng of plant extracts could provide a useful route to identifying promising anti-irritant extracts for follow-up clinical testing. To be useful, the me thod would have to Rag materials with strong anti-irritant properties, and would have to avoid registering false negatives. The tests conducted provid ed positive indications. We measured the delays in onset of three manifesta tions of membrane irritation-vascular hemorrhaging, membrane lysis and memb rane coagulation-observed with test substances relative to positive control s, Aqueous 15% lactic acid, a commonly used irritant in direct tests on hum an skin, was employed as the test irritant in this study. The ratio [irrita tion onset times after test substance pre-treatment]:[onset times without t est substance pretreatment] was used to measure the anti-irritant power of test substances. A scoring notation was devised for this which treats the d elay parameters as independent effects. Most tested plant extracts showed n o significant irritant or anti-irritant effects. Among the apparently anti- irritant plant extracts (approx. 10% of all those tested), most showed thei r greatest effect against hemorrhaging. Lesser but still readily measurable effects against membrane lysis and coagulation were also observed in nearl y all the apparently anti-irritant extracts. Two of the tested extracts pro ,ed to be membrane irritants. Some key CAM assay results were compared with results obtained in direct tests on human skin using the same test irritan t (15% lactic acid). In these comparative tests on skin, an essentially sim ilar pattern of efficacy was obtained, with the plant extract deemed best i n the CAM screenings, outperforming the benchmark anti-irritant hydrocortis one. From these initial results it appears that physiological CAM assays ma y pro,ve useful in screening natural materials for anti-irritant properties , as alternatives to mechanism-dependent biochemical assays, or expensive d irect screening tests on human subjects. Further work remains to extend the CAM screening approach to irritants other than lactic acid, and to assess its quantitative powers of prediction of topical anti-irritancy. (C) 2000 E lsevier Science Ltd. All rights reserved.