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 connection with searches for plant-derived substances with topical anti-irritant action. The main question to be answered was whether CAM-assay screening of plant extracts could provide a useful route to identifying promising anti-irritant extracts for follow-up clinical testing. To be useful, the method would have to flag materials with strong anti-irritant properties, and would have to avoid registering false negatives. The tests conducted provided positive indications. We measured the delays in onset of three manifestations of membrane irritation-vascular hemorrhaging, membrane lysis and membrane coagulation-observed with test substances relative to positive controls. Aqueous 15% lactic acid, a commonly used irritant in direct tests on human skin, was employed as the test irritant in this study. The ratio [irritation onset times after test substance pre-treatment]:[onset times without test substance pretreatment] was used to measure the anti-irritant power of test substances. A scoring notation was devised for this which treats the delay parameters as independent effects. Most tested plant extracts showed no significant irritant or anti-irritant effects. Among the apparently anti-irritant plant extracts (approx. 10% of all those tested), most showed their greatest effect against hemorrhaging. Lesser but still readily measurable effects against membrane lysis and coagulation were also observed in nearly all the apparently anti-irritant extracts. Two of the tested extracts proved to be membrane irritants. Some key CAM assay results were compared with results obtained in direct tests on human skin using the same test irritant (15% lactic acid). In these comparative tests on skin, an essentially similar pattern of efficacy was obtained, with the plant extract deemed best in the CAM screenings, outperforming the benchmark anti-irritant hydrocortisone. From these initial results it appears that physiological CAM assays may prove useful in screening natural materials for anti-irritant properties, as alternatives to mechanism-dependent biochemical assays, or expensive direct 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.