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The ciliate Ichthyophthirius multifiliis is one of the most pathogenic parasites of fish maintained in captivity. In this study, effects of crude extracts, fractions, and compounds from the leaves of Macleaya cordata against I. multifiliis were investigated under in vitro conditions by bioactivity-guided isolation method. The dried ethanol extract of M. cordata was extracted successively in a separating funnel with petroleum ether, ethyl acetate, chloroform and n-butanol. Among them, only the chloroform extract showed promising activity and therefore, was subjected to further separation and purification using various chromatographic techniques. Four compounds were isolated from chloroform extract, but only one compound showed potent activity. The structure of the active compound was elucidated as sanguinarine by hydrogen, carbon-13 nuclear magnetic resonance spectrum and electron ionization mass spectrometry. In vitro antiparasitic efficacy tests exhibited that sanguinarine was 100% effective against I. multifiliis at a concentration of 0.7 mg l, with LC and LC values of 0.437 and 0.853 mg l after 4 h of exposure. In vivo antiparasitic efficacy tests showed that the number of I. multifiliis on the gills in the treatment group (in 0.9 mg l sanguinarine) was reduced by 96.8%, in comparison to untreated group at 25°C for 48 h. Mortality of fish did not occur in the treatment group during the trail, although 40% of untreated fish died. Our results indicate that the studied plant extracts, as well as sanguinarine might be potential sources of new antiparasitic drug for the control of I. multifiliis. [ABSTRACT FROM AUTHOR]