Antischistosomal Activity of the Terpene Nerolidol

Schistosomiasis is a neglected tropical disease that affects hundreds of millions of people worldwide. Since the treatment of this disease currently relies on a single drug, praziquantel, new and safe schistosomicidal agents are urgently required. Nerolidol, a sesquiterpene present in the essential oils of several plants, is found in many foods and was approved by the U.S. Food and Drug Administration. In this study we analysed the in vitro antiparasitic effect of nerolidol on Schistosoma mansoni adult worms. Nerolidol at concentrations of 31.2 and 62.5 μM reduced the worm motor activity and caused the death of all male and female schistosomes, respectively. In addition, confocal laser scanning microscopy revealed morphological alterations on the tegument of worms such as disintegration, sloughing and erosion of the surface, and a correlation between viability and tegumental damage was observed. In conclusion, nerolidol may be a promising lead compound for the development of antischistosomal natural agents.


Results and Discussion
Medicinal plants have been used for hundreds of years as therapeutics worldwide and the interest in natural products as new sources of antischistosomal drugs is rising. In this study, 49-day-old adult S. mansoni were cultured in RPMI 1640 medium in the presence of nerolidol, a sequisterpene present in essential oils of several plants.

Nerolidol Affected the Viability of Schistosomes
The results of the in vitro studies with schistosomes exposed to nerolidol at concentrations of 15.6, 31.2, 62.5, 125 and 250 μM and control groups are summarised in Figure 2 and Table 1. In the negative control group (RPMI 1640 medium containing 0.5% DMSO), schistosomes showed normal motor activity and had no observed mortality. In contrast, 3 µM praziquantel resulted in complete loss of motor activity and caused the death of all parasites. These observations in the negative and positive control groups are similar to that described in the literature [27][28][29].

Nerolidol Caused Tegumental Damage in Schistosomes
The schistosome worm is covered by a syncytial cytoplasmic layer, the tegument, which is of crucial importance for parasite survival [33]. Thus, the worm tegument is considered an important drug target in schistosomiasis [26]. We used confocal laser scanning microscopy as a tool to evaluate whether the exposure to nerolidol could affect the tegument of S. mansoni adult worms. The morphological features of adult S. mansoni in the control groups were in agreement with previous reports [27,34,35]. As can be observed in Figure 3, no abnormality was noticed in S. mansoni adult worms in the negative control group and, thus, the dorsal tegumental surface of male worms showed intact tubercles ( Figure 3A). In contrast, nerolidol at 62.5 to 250 µM caused morphological alterations in the tegument of parasites. For example, slight tegumental damage was observed in the schistosomes treated with nerolidol at 62.5 µM ( Figure 3B), whereas extensive tegumental damages were observed at 125 and 250 µM ( Figure 3C,D). The principal alterations in S. mansoni male worms exposed to nerolidol were disintegrated tubercles as well as sloughing and erosion of the surface. Additionally, we performed a quantitative analysis to observe tegumental damage on S. mansoni male worm. In this case, areas of 20,000 µm 2 of the tegument of schistosomes were assessed, and the number of tubercles was counted. Nerolidol caused changes in the tubercles of schistosomes in a concentration-dependent manner. As can be observed in Figure 4, the number of normal tubercles on schistosomes of the control group was 43 (± 4), while in the parasites treated with 62.5 µM of nerolidol, the number was 15 (± 5). In addition, when the concentration of nerolidol was increased to 250 μM, no intact tubercles were seen.
A recent study reported that nerolidol (10 to 100 μM) did not show any antischistosomal activity against S. mansoni adult worms [36]. However, in our hands, nerolidol exhibited antischistosomal properties at lower concentrations (31.2 and 62.5 µM). It has been demonstrated that different Schistosoma strains exhibit different drug sensitivity patterns [37] and thus we attribute the differences between our results and previous data to the use of different S. mansoni strains. Moreover, the researchers used trans-nerolidol [36], whereas we used a racemic mixture; therefore, it is possible that trans-nerolidol is the far less active isomer. Indeed, the results in the present study showed that the nerolidol has antischistosomal properties as well as revealed that nerolidol induced severe tegumental damage in adult schistosomes. Additionally, quantitative analysis showed that nerolidol caused alterations on the tubercles of male parasites in a concentration-dependent manner. Morphological changes on the tegument of S. mansoni male worms after treatment with nerolidol. Quantitative analysis, measured in a 20,000 µm 2 of area in a dorsal region of male parasite, was performed using three-dimensional images obtained from confocal microscope (see Figure 3). A minimum of three tegument areas of each parasite were assessed. Values are means ± SD (bars) of ten male adult worms. *** p < 0.001 compared with untreated groups.
The mechanism by which nerolidol exerts its in vitro schistosomicidal effect is not clear. However, a correlation between viability and tegumental damage was observed (Table 1, Figures 2-4). Due to its inherent lipophilicity, nerolidol will easily cross plasmatic membranes [19] and, consequently, may also interact with intracellular molecules of parasites. Comparable results were obtained by previous works using other antischistosomal natural compounds, such as epiisopiloturine [38], (+)-limonene epoxide [39], dermaseptin [40] and phytol [41].

Drugs
Nerolidol (a mixture of cis-and trans-nerolidol) (Figure 1) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and praziquantel was purchased from Merck (São Paulo, SP, Brazil). Stock solutions (8 mM nerolidol and 4 mM praziquantel) were prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and were used for in vitro experiments.

Parasite
Schistosoma mansoni (BH strain) was used in this study. Schistosomes were obtained from experimentally infected Mesocricetus auratus hamsters as described previously [26,34]. Animals were subcutaneously infected with approximately 150 cercariae following standard procedures of our laboratory [26]. Seven weeks post-infection, adult S. mansoni were removed from the hepatic portal system and mesenteric veins and cultured in RPMI 1640 culture medium supplemented with 200 IU/mL penicillin and 200 µg/mL streptomycin (Invitrogen, São Paulo, SP, Brazil) 10% and foetal bovine serum at 37 °C in an atmosphere of 5% CO 2 until use.

In Vitro Antischistosomal Assay
For the in vitro test with S. mansoni, parasites were incubated in a 24-well culture plate (TPP, St. Louis, MO, USA), placing one coupled worm pair in each well, containing the RPMI 1640 medium at 37 °C in a 5% CO 2 atmosphere [27,35]. Nerolidol was used at concentrations of 15.6 to 250 μM (15.6, 31.2, 62.5, 125 and 250 μM) in culture plates with a final volume of 2 mL. The parasites were kept for 120 h and monitored every 24 h using an inverted microscope. The effect of the drug was assessed with emphasis on changes in worm motor activity and alteration in the tegument as previously described [41]. Death was defined as no movement observed for at least 1 to 2 min of examination [26,42]. In addition, worms were prepared for confocal laser scanning microscopy examination whose details are described below. The control worms were assayed in RPMI 1640 medium as a negative control group and 3 μM praziquantel as a positive control group.

Microscopy Studies
To observe morphological alterations on the tegument of schistosomes after in vitro assays, male worms were monitored using a confocal laser scanning microscope as described elsewhere [26]. The parasites were fixed in a formalin-acetic acid-alcohol solution (FAA) and analysed under a confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Standorf Göttingen, Vertrieb, Germany). Autofluorescence was excited with a 488 nm line from an Argon laser, and emitted light was collected at 505 nm [43].
To assess the damage in the tegument of S. mansoni as a quantitative method, areas (20,000 µm 2 ) of the dorsal surface of male worms were assessed, and the numbers of tubercles were counted using three-dimensional images obtained from confocal microscopy according to standard procedures [26]. The area was calculated using LSM Image Browser software (Zeiss).

Conclusions
The results of the present study show that a racemic mixture of E-and Z-nerolidol possesses in vitro antischistosomal activity against Schistosoma mansoni adult worms. This terpene decreased the motor activity and caused the death of worms; additionally, nerolidol was able to cause morphological alterations in the tegument of adult schistosomes. In general, our results are important as there is an urgent need to develop new agents against schistosomiasis. Since nerolidol is generally recognized as safe, this compound may have antiparasitic applications. Further studies are necessary to elucidate mechanisms of action of nerolidol as well as to examine the in vivo effects of this natural compound in S. mansoni-infected animals.