Antihelmintic effects of nutmeg (Myristica fragans) on Anisakis simplex L3 larvae obtained from Micromesistius potassou Víctor López *, Javier Gerique, Elisa Langa, César Berzosa, Marta Sofía Valero, Carlota Gómez-Rincón Facultad de Ciencias de la Salud, Universidad San Jorge, Autovía A-23 Zaragoza-Huesca km. 299, Villanueva de Gállego (Zaragoza), Spain ARTICLE INFO Article history: Received 26 December 2014 Accepted 29 March 2015 Keywords: Anisakis simplex Nutmeg Myristica fragans Myristicin Antihelmintic activity Natural products A B ST R AC T Anisakis simplex is a foodborne pathogen that can produce human infections and allergic reactions due to the high consumption of raw fish. The seeds of Myristica fragans (Myristicaceae), popularly known as nutmeg, are worldwide used as a culinary spice due to its flavour and properties in food preservation. A nutmeg extract was prepared, analyzed, screened for cytotoxicity and tested against Anisakis simplex L3 larvae. In order to detect the biologically active constituents of the extract, myristicin was tested on the larvae. An acetylcholinesterase inhibition bioassay was also carried out to investigate the antihelmintic mechanism of action. Our results demonstrate that nutmeg exerts antihelmintic effects on Anisakis simplex, being myristicin one of the active compounds. The extract induced a high rate of dead anisakis at con- centrations between 0.5 and 0.7 mg/ml without being considered cytotoxic; however, an inhibition of acetylcholinesterase was discarded as the molecular mechanism involved in the activity. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Bacteria, fungi and parasites are leading causes of deteriora- tion of food quality and human risks (Magnusson et al., 2012). Due to the increase and the worldwide acceptance of “sushi” and other traditional raw foods, the human parasitic infection of the gastro- intestinal tract caused by Anisakis simplex, known as anisakiasis, has become a big issue in the areas of food safety and public health (Madrid et al., 2012). Anisakis simplex is a parasitic nematode that hosts marine mammals like dolphins and whales but also small fishes. Humans usually eat L3 larvae hidden in raw and undercook fish and cephalopod (Chai et al., 2005; Hochberg and Hamer, 2010). Although anisakiasis is sometimes misdiagnosed with other gas- trointestinal diseases this parasite causes a high number of allergic reactions with urticaria and anaphylaxis as clinical symptoms (Audicana and Kennedy, 2008). The best protection against anisakiasis may be educating people about the risks of eating raw fish and rec- ommending cooking at temperatures over 60 °C. In the European Union, the legislation establishes that certain fishery products must be frozen at a temperature of not more than -20 °C in all parts of the product for not less than 24 hours in order to avoid parasites (EU, 2004). However, many other countries lack of regulation on food hygiene. In the attempt to reduce the use of chemicals due to the concern about human health and environmental toxicology, new and safer food control approaches such as the use of natural products are now- adays being developed (Abushelaibi et al., 2012; Chen et al., 2012; Davidson et al., 2013; Juneja et al., 2012; Negi, 2012). Myristica frangans (Myristicaceae) is a tree native to Indonesia, being nowadays cultivated in other Asian countries and in the Caribbean, whose seeds are popularly known as nutmeg and world- wide used as a spice in different types of cuisine (Vangils and Cox, 1994). Although the main value of nutmeg may be as an ingredi- ent in foods due to its flavour and preservative properties, this species is used in traditional medicine for the treatment of intestinal dis- eases like colitis in some Asian countries (Jazayeri et al., 2014; Kim et al., 2013; Touwaide and Appetiti, 2013). There are several papers reporting the chemical constituents and the biological effects of nutmeg. Nutmeg contains fatty acids, lignans, terpenes and phenylpropane derivatives such as myristicin, elemicin and safrol (Choo et al., 1999; Kapoor et al., 2013; Moreira Valente et al., 2011; Piaru et al., 2012; Simpson and Jackson, 2002). Common activities attributed to nutmeg extracts are antioxi- dant, antimicrobial, cytotoxic and anti-inflammatory effects (Jin et al., 2012; Olajide et al., 1999; Phuong Thien et al., 2014; Piaru et al., 2012; Shafiei et al., 2012; Sulaiman and Ooi, 2012). Antiparasitic proper- ties have also been reported (Pillai et al., 2012) and the effects of nutmeg essential oil in Anisakis larvae have been studied in an early work (Oishi et al., 1974). In this study, we have prepared, analyzed, screened for cyto- toxicity and tested against Anisakis L3 larvae a nutmeg ethanolic extract. Authors have also investigated a possible mechanism of * Corresponding author. Department of Pharmacy, Faculty of Health Sciences, San Jorge University, 50830Villanueva de Gállego (Zaragoza), Spain. Tel.: +34 626604895; fax: +34 +976 077584. E-mail address: ilopez@usj.es (V. López). http://dx.doi.org/10.1016/j.rvsc.2015.03.033 0034-5288/© 2015 Elsevier Ltd. All rights reserved. Research in Veterinary Science ■■ (2015) ■■–■■ ARTICLE IN PRESS Please cite this article in press as: Víctor López, et al., Antihelmintic effects of nutmeg (Myristica fragans) on Anisakis simplex L3 larvae obtained from Micromesistius potassou, Research in Veterinary Science (2015), doi: 10.1016/j.rvsc.2015.03.033 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc