Inhibition test of heme detoxification (ITHD) as an approach for detecting antimalarial agents in medicinal plants

Abstract

<strong>Background and objectives</strong>: There are several methods to assess the <em>in vitro</em> capability of heme inhibitory activity of antimalarial compounds; most of them require some specific equipment or toxic substances and sometimes the needed materials are not accessible. Regarding the necessity and importance of optimizing and standardizing experimental conditions, the present study has intended to improve the <em>in</em><em> vitro </em>assessment conditions of the β-hematin formation inhibitory activity for screening herbal samples. <strong>Methods:</strong> Hemin, tween 20, and samples (9:9:2) were incubated in different conditions including: hemin concentration (30, 60, and 120 µg/mL), duration (4, 24, 48, and 72 h), pH of buffer (3.6, 4, 4.4, 4.8, and 5), and temperature (37 and 60 °C) in 96-well plates. Also, a total of 165 plant extracts and fractions were tested in the most suitable conditions. <strong>Results</strong>: The reaction time and the incubation temperature were determined as the critical factors. The effective conditions for β-hematin formation were found to be 60 °C after 24 h incubation. In this method, proper correlations with respect to negative (69%) and positive (67%) predictive values were obtained in comparison with the anti-plasmodial assay. Antimalarial activities of <em>Pistacia atlantica</em>, <em>Myrtus communis</em>, <em>Pterocarya fraxinifolia</em>, and <em>Satureja mutica </em>were found to correlate significantly with inhibition of the heme detoxification assay. <strong>Conclusion:</strong> These results support a rapid, simple and reliable approach for selecting and identifying a number of herbs for further related antimalaria investigations.<br /> <strong> </strong>