Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: Effects on growth, viability, adherence and ultrastructure M. Machado a,b , A.M. Dinis c , L. Salgueiro a , José B.A. Custódio d , C. Cavaleiro a , M.C. Sousa a,⇑ a Faculdade de Farmácia/CEF, Universidade de Coimbra, Azinhaga de Santa Comba, 3030-548 Coimbra, Portugal b Departamento Farmácia, Escola Superior de Saúde do Vale do Ave/Centro de Investigação em Tecnologias da Saúde IPSN-CESPU, 4760 Vila Nova de Famalicão, Portugal c Laboratório de Microscopia Electrónica, Departamento das Ciências da Vida, Faculdade de Ciências e Tecnologia da Universidade de Coimbra, Portugal d Centro de Neurociências da Universidade de Coimbra, 3000 Coimbra, Portugal article info Article history: Received 22 October 2010 Received in revised form 26 November 2010 Accepted 19 January 2011 Available online 25 January 2011 Keywords: Giardia lamblia Essential oils In vitro activity Drug action Infectious diseases (ID) Ultrastructure Protozoa Drug development Medicinal plants abstract The present work evaluates the anti-Giardia activity of Syzygium aromaticum and its major compound eugenol. The effects were evaluated on parasite growth, adherence, viability and ultrastructure. S. aromaticum essential oil (IC 50 = 134 lg/ml) and eugenol (IC 50 = 101 lg/ml) inhibited the growth of G. lamblia. The essential oil inhibited trophozoites adherence since the first hour of incubation and was able to kill almost 50% of the parasites population in a time dependent manner. The eugenol inhibited G. lamblia trophozoites adherence since the third hour and not induce cell lyses. The main morphological alterations were modifications on the cell shape, presence of precipitates in the cytoplasm, autophagic vesicles, internalization of flagella and ventral disc, membrane blebs, and intracellular and nuclear clear- ing. Taken together, our findings lead us to propose that eugenol was responsible for the anti-giardial activity of the S. aromaticum essential oil and both have potential for use as therapeutic agents against giardiasis. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction The flagellated protozoan Giardia lamblia (syn. Giardia intestinal- is, Giardia duodenalis) is a worldwide cause of human and animal diseases. It is responsible for intestinal infection and diarrhea that may lead to nutritional deficiencies and significant morbidity and mortality, especially among children, the elderly and travelers in developing countries and patients who have immune defects (Faubert, 2000; Huang and White, 2006). In developed nations, a rising incidence of giardiasis has been noted leading to the designation of giardiasis as a ‘‘re-emerging’’ infectious disease (Thompson, 2000). Its life cycle consists of two stages, cysts and trophozoites, which are adapted to survive in very diverse and hostile environments. Transmission to mammals takes place mainly through contaminated water and foods, and often exhibit zoonotic transmission (Gillin et al., 1996). In children, infections may result in severe diarrhea, malabsorp- tion, and failure to thrive. In contrast, approximately one-half of infected people, children and adults, remain asymptomatic (Farthing, 1997). Giardiasis is generally treated with nitroimidazoles, nitrofurans, benzimidazoles and pyrantel pamoate. However, in areas where it is endemic the emergence of resistant strains has led to a high rate of clinical failures and to an urgent need for more effective drugs (Katelaris et al., 1994; Adam, 2001). The identification of novel active plant derived compounds could increase the number of available chemotherapeutic agents, thereby reducing the frequency of resistance phenomena and pro- viding alternative drugs with greater acceptance. Essential oils have a long history of use as natural antimicrobial agents with many industrial applications as they effectively inhibit the growth of a wide range of microorganisms and they cause fewer side effects. Despite the widespread use of essential oils by humans, little is known about the exact mechanism of their antimicrobial action (Edris, 2007; Bakkali et al., 2008). Clove oils have been widely investigated due to their popularity, availability, and high essential oil content. In fact, the clove oil of Syzygium aromaticum (L.) Merr. & Perry (Syn. Eugenia caryophyllata L. Merr. & Perry, Eugenia aromatic L., Caryophyllus aromaticus L.), belonging to the family Myrtaceae, has long been considered to have medicinal properties such as a stimulant against digestive disorders and diarrhea (Chaieb et al., 2007). 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.01.011 ⇑ Corresponding author. Address: Faculdade de Farmácia da, Universidade de Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa, Comba, 3030-548 Coimbra, Portugal. E-mail address: mcsousa@ci.uc.pt (M.C. Sousa). Experimental Parasitology 127 (2011) 732–739 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr