Ticks attaching to ear canals of humans and animals are the cause of otoacariasis, common in rural areas of Nepal. The plant Clerodendrum viscosum is used in multiple indigenous systems of medicine by ethnic communities in the Indo-Nepali-Malaysian region. Visiting the Chitwan National Park, we learned that in indigenous medicine, flower extract of C. viscosum is utilized to treat digestive disorders and extracts from leaves as tick repellent to prevent ticks from invading or to remove them from the ear canal. The objective of our study was to provide support to indigenous medicine by characterizing the in vivo effect of leave extracts on ticks under laboratory conditions and its phytochemical composition. We collected plant parts of C. viscosum (leaves and flowers) and mango (Mangifera indica) leaves at the Chitwan National Park, previously associated with repellent activity to characterize their effect on Ixodes ricinus ticks by in vivo bioassays. A Q-ToF high-resolution analysis (HPLC-ESI-QToF) was conducted to elucidate phenolic compounds with potential repellent activity. Clerodendrum viscosum and M. indica leaf extracts had the highest tick repellent efficacy (%E = 80-100%) with significant differences when compared to C. viscosum flowers extracts (%E = 20-60%) and phosphate-buffered saline. Phytochemicals with tick repellent function as caffeic acid, fumaric acid and p-coumaric acid glucoside were identified in C. viscosum leaf extracts by HPLC-ESI-QToF, but not in non-repellent flower extracts. These results support the Nepali indigenous medicine application of C. viscosum leaf extracts to repel ticks. Additional research is needed for the development of natural and green repellent formulations to reduce the risks associated with ticks resistant to acaricides.

7 Manantiales 2 Villanueva de los Infantes 13320 Spain

Center for Molecular Dynamics Nepal Thapathali Road 11 Kathmandu 44600 Nepal

Department of Molecular Biotechnology and Microbiology Faculty of Chemistry Gdańsk University of Technology Gdańsk 80 233 Poland

Department of Veterinary Pathobiology Center for Veterinary Health Sciences Oklahoma State University Stillwater OK 74078 USA

Institute of Parasitology Biology Centre Academy of Sciences of the Czech Republic Branišovská 1160 31 České Budějovice 37005 Czech Republic

Instituto de Investigación en Recursos Cinegéticos IREC CSIC UCLM JCCM Ronda de Toledo s n Ciudad Real 13005 Spain

Instituto Regional de Investigación Científica Aplicada Universidad de Castilla La Mancha Ciudad Real 13005 Spain

KGJ Collection Adelfa 75 Ciudad Real 13005 Spain

National Livestock Resources Research Institute Wakiso District P O Box 5704 Wakiso Uganda

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