In vitro and in vivo efficacy of carvacrol against Echinococcus granulosus

Highlights

• The in vitro efficacy of carvacrol on E. granulosus metacestodes was demonstrated.

• Carvacrol induces apoptosis in the cells of protoscoleces after short incubation times.

• The in vivo treatment with carvacrol reduces the weight of cysts of E. granulosus.

Abstract

Currently, benzimidazoles are used as chemotherapeutic agents and as a complement to surgery and PAIR in the treatment of cystic echinococcosis (CE). They are generally applied at high doses causing side effects and, 50% of cases do not respond favorably to such chemotherapy. The use of essential oils obtained by distillation from aromatic plants would be an effective alternative or complementary to the synthetic compounds, because would not bring the appearance of side effects. Carvacrol and his isomer thymol are the main phenolic components from essential oils of Origanum vulgare (oregano) and Thymus vulgaris (thyme). The aim of the present work was to evaluate the in vitro and in vivo efficacy of carvacrol against Echinococcus granulosus metacestodes. For the in vitro assay, protoscoleces and cysts of E. granulosus were incubated with carvacrol at the following final concentrations: 10, 5 and 1 μg/ml of carvacrol. The maximum protoscolicidal effect was found with 10 μg/ml of carvacrol. Results of viability tests were consistent with the structural and ultrastructural damage observed in protoscoleces. Ultrastructural studies revealed that the germinal layer of cysts treated with carvacrol lost the multicellular structure feature. In the clinical efficacy study, a reduction in cyst weight was observed after the administration of 40 mg/kg of carvacrol during 20 days in mice with cysts developed during 4 months, compared to that of those collected from control mice. Given that the in vivo effect of carvacrol was comparable with the treatment of reference with ABZ and the fact that is a safe compound, we postulated that carvacrol may be an alternative option for treatment of human CE.

Introduction

The larval stage (metacestode) of Echinococcus granulosus causes cystic echinococcosis (CE) in humans and livestock. This cestode parasite has a worldwide distribution, particularly affecting pastoral and poor rural communities where people raise livestock in close contact with dogs (Alvarez Rojas et al., 2014). CE is characterized by cystic lesions, most commonly in the liver and lungs (Budke et al., 2013).

The WHO-Informal Working Group on Echinococcosis (WHO-IWGE) proposes four different treatment modalities for CE: surgery, percutaneous treatment (PAIR: puncture, aspiration, injection, re-aspiration), drug treatment and observation (watch and wait). Treatment indications are based on cyst characteristics, available medical/surgical expertise and equipment, and presence/absence of complications (Brunetti et al., 2010). Currently, chemotherapy is used as an adjuvant to surgery either preoperatively or postoperatively or both. This produces the decrease in the viability of cysts and reduces its tension, making surgery easier. It is also used complementarily with PAIR treatment and, in both cases; it prevents the recurrence of the disease in the form of secondary hydatidosis (Arif et al., 2008).

In human patients, benzimidazoles have to be applied at high doses for extended periods of time and adverse side effects are frequently observed, such as abnormalities in liver function, leucopenia and alopecia (Walker et al., 2004). Mebendazole, albendazole (ABZ) and its major metabolite albendazole sulfoxide, are the only anthelmintics approved by the FDA (Food and Drug Administration, USA), but its effectiveness is about 50% (Pawlowski et al., 2001).

Because of the serious difficulties in achieving treatment success and toxicity of the compounds used in chemotherapy, the proposal of an effective drug against hydatid disease would be a great alternative treatment to surgery. Many efforts have been made to discover new compounds from various kinds of sources such as plants, animals and microorganisms. Currently, herbal medicines are being increasingly used to treat many diseases including several infections (Khan et al., 2010). For this reason, the use of essential oils obtained by distillation from plants would be an effective alternative or complementary to the synthetic compounds, because they would not bring the appearance of side effects (Pessoa et al., 2002, Carson and Riley, 2003).

Essential oils are very complex natural mixtures, synthesized by all organs of the aromatic plants as secondary metabolites. They contain 20–60 components at very different concentrations, but they are characterized by two or three major components at fairly high concentrations (20–70%) compared to others components present in trace amounts. These major components determine the biological properties of the essential oils (Bakkali et al., 2008). Because of its hydrophobic character, the essential oils and their components present a great potential for pharmacological applications as antimicrobial agents (Edris, 2007, Santoro et al., 2007, Tavares et al., 2008, Arana-Sánchez et al., 2010, Machado et al., 2010).

Some essential oils have demonstrated activity against E. granulosus as Rosmarinus officinalis (rosemary), Pistacia khinjuk (pistachio), Mentha spp, Trachyspermum ammi (ajowan), Thymus vulgaris (thyme) and Origanum vulgare (oregano) (Albanese et al., 2009, Taran et al., 2009, Maggiore et al., 2012, Moazeni et al., 2012, Pensel et al., 2014).

Carvacrol and its isomer thymol are the main phenolic components from essential oils of thyme and oregano (Santoro et al., 2007). The in vitro effect of thymol was shown against protoscoleces, microcysts and cyst of E. granulosus, as well also its in vivo effect in a murine model (Elissondo et al., 2008, Elissondo et al., 2013, Maggiore et al., 2015). Nevertheless, the effect of carvacrol against E. granulosus it has not been studied yet. The aim of the present work was to determine the in vitro and in vivo efficacy of carvacrol against E. granulosus.