Silybum marianum ethanolic extract: in vitro effects on protoscolices of Echinococcus granulosus G1 strain with emphasis on other Iranian medicinal plants

Abstract

Background: Cystic echinococcosis (CE), is a parasitic zoonosis caused by Echinococcus granulosus (E. granulosus) larvae in liver and lungs of both humans and animals. Surgical intervention is the mainstay for CE treatment, using scolicidal agents that inactivate live protoscolices. This study evaluated the scolicidal effects of Silybum marianum ethanolic extract and its combination with albendazole in vitro for the first time. Moreover, in a literature review, we investigated the effects of a wide range of Iranian medicinal plants on protoscolices of E. granulosus.

Methods: S. marianum ethanolic extract was prepared and high-performance liquid chromatography (HPLC) analysis was used to establish the proportions of its component compounds in the extract. Cytotoxicity was evaluated in mouse macrophage cells (J774A.1 cell line) using MTT method. Next, the scolicidal activity of the extract alone and combined with albendazole was tested as triplicate at various concentrations incubated for 5, 10, 20, 30, and 60 min. Finally, protoscolex viability was determined using 0.1% eosin as a vital stain. PCR-RFLP and DNA sequencing techniques were used to characterize the genotype of E. granulosus.

Results: HPLC analysis showed that S. marianum ethanolic extract contained mostly silydianin (14.41%), isosilybin A (10.50%), and silychristin (10.46%). The greatest scolicidal effects were obtained with the combination of S. marianum with albendazole (79%), S. marianum ethanolic extract alone (77%) and albendazole (69%), at a concentration of 500 μg/ml for 60 min, respectively (P < 0.05). Molecular analysis showed that all the cysts used were G1 genotype.

Conclusion: The data suggest that S. marianum ethanolic extract is a potential scolicide in vitro; however, further investigations are required to determine its efficacy in vivo.

Keywords: Cystic echinococcosis; In vitro; Scolicide; Silybum marianum.

Fig. 1

Electrophoresis of PCR amplification (462 bp) provided from liver of sheep sample (lane C− negative control, C +  positive control, lane 1 and 2 positive samples, L ladder 100 bp)

Fig. 2

Agarose gel electrophoresis of ITS1-PCR products of E. granulosus isolates from sheep after digestion with the restriction enzyme Bsh1236I. (lane C− negative control, C +  positive control, lane 1 and 2 positive samples, L ladder 50 bp)

Fig. 3

HPLC of silymarin constituents of the Silybum marianum ethanolic extract

Fig. 4

Scolicidal effects of ethanolic extract of S. marianum after exposure at different concentrations for 60 min. Each point represents the mean percentage of fatality rate

Fig. 5

Scolicidal effects of albendazole after exposure at different concentrations for 60 min. Each point represents the mean percentage of fatality rate

Fig. 6

Scolicidal effects of ethanolic extract of S. marianum with albendazole after exposure at different concentrations for 60 min. Each point represents the mean percentage of fatality rate