Introduction
Infections by gastrointestinal nematodes (GIN) influence sheep and goat health and their welfare status dramatically, limiting productivity by removing nutrients from the ingesta and producing indirect effects on the immune response (Sargison, Reference Sargison2012). Anthelmintic drugs in association with other strategies, such as grazing management, genetic selection and nutritional manipulation, are essential for the control of GIN infestations (Sargison, Reference Sargison2008; Sutherland & Scott, Reference Sutherland and Scott2010; Hoste & Torres-Acosta, Reference Hoste and Torres-Acosta2011). In particular, strategies of grazing management aim to decrease the timing and extent of parasite risk by dispersion of larval density and by accelerating larval mortality (Torres-Acosta & Hoste, Reference Torres-Acosta and Hoste2008). In recent years, interest in the use of forages and plants with anthelmintic properties in the diets of small ruminants has increased (Hoste & Torres-Acosta, Reference Hoste and Torres-Acosta2011). Plants are considered a valid alternative to conventional anthelmintic drugs (Hammond et al., Reference Hammond, Fielding and Bishop1997, Torres-Acosta & Hoste, Reference Torres-Acosta and Hoste2008, Hoste & Torres-Acosta, Reference Hoste and Torres-Acosta2011). In particular, studies have been conducted to evaluate the anthelmintic activity of the Meliaceae family, such as Melia azedarach L. Szewczuk et al. (Reference Szewczuk, Mongelli and Pomilio2006) reported that the drupe extracts of this plant showed anthelmintic activity against Bunostomum trigonocephalum and Oesophagostomum columbianum. Moreover, the seed ethanol extract of M. azedarach revealed activity against Haemonchus contortus eggs, while in a larval development test the leaf ethanol extract showed an LC50 = 9.18 mg/ml (Maciel et al., Reference Maciel, Morais, Bevilaqua, Camurça-Vasconcelos, Costa and Castro2006). Currently, the condensed tannins contained in tannin-rich fodders are the most studied compounds with anthelmintic activity in small ruminants infected by GIN (Hoste & Torres-Acosta, Reference Hoste and Torres-Acosta2011). Molan et al. (Reference Molan, Meagher, Spencer and Sivakumaran2003) studied the in vitro anthelmintic effects of monomer units of condensed tannins called 3-flavan-3-ols and their galloyl derivatives on Trichostrongylus colubriformis, finding that epigallocatechin gallate was more active than the other 3-flavan-3-ols in an egg hatch assay.
Ntalli et al. (Reference Ntalli, Manconi, Leonti, Maxia and Caboni2011) reported the nematicidal activity of the essential oil and the methanol extract of Ruta chalepensis L. on root knot nematodes. Ruta chalepensis essential oil was more active if compared with the methanol extract, and 2-undecanone was considered to be the main component responsible for the nematicidal effect. In addition, essential oils of fruits and leaves showed inhibition effects on H. contortus egg hatching (Hussien et al., Reference Hussien, Urgessa, Regassa, Jemal, Abajebel and Hussien2011) and adult worm motility (Akkari et al., Reference Akkari, Ezzine, Dhahri, B'chir, Rekik, Hajaji, Darghouth, Jamâa and Gharbi2015).
The aim of the present study was to evaluate the in vitro anthelminthic activity of R. chalepensis methanol extract (RME), the essential oil (REO) and its metabolites 2-nonanone, 2-undecanone and 2-decanone. In addition, their anthelmintic activity was compared with that of other phytochemicals, such us furfural, salicylaldehyde, (E,E)-2-4-decadienal and epigallocatechin gallate.
Materials and methods
Chemicals and plant materials
Standards of 2-undecanone (UND), 2-nonanone (NON), 2-decanone (DEC), (E,E)-2-4-decadienal (DDI), furfural (FUR), salicylaldehyde (SAL), epigallocatechin gallate (ECG), gallic acid (GAA) and levamisole hydrochloride (LEV) of purity greater than 98%, as well as Tween 20, sodium phosphate dibasic, sodium phosphate monobasic and methanol were purchased from Sigma-Aldrich (Milano, Italy). The methanol, ethanol and water used were high-performance liquid chromatography (HPLC) grade.
Aerial parts of R. chalepensis were collected in February 2014 at Cagliari, Italy and were dried in the absence of light at room temperature in paper bags for 30 days. Botanical identity of the plants was verified and they were deposited in the Department of Life and Environmental Sciences, University of Cagliari, Italy. Dried aerial plant parts (100 g) were ground and extracted with methanol (1:10 w/v). After 20 h samples were homogenized with a sonicator for 15 min, filtered through a Whatman 40 filter and centrifuged for 15 min at 13,000 rpm, according to Ntalli et al. (Reference Ntalli, Manconi, Leonti, Maxia and Caboni2011). Aerial parts of R. chalepensis were subjected to hydro-distillation for 4 h. Essential oil was extracted in hexane and was dried over anhydrous magnesium sulphate. After filtration, the hexane was evaporated at low temperature under low pressure. The essential oil obtained and the methanol extract were kept in dark glass vials at −20°C until use. Yields of the essential oil and the extract were determined on average over four replicates.
GC–MS analysis
The essential oil was diluted (1:200 v/v) with hexane and injected for gas chromatography–mass spectrometry (GC–MS) analysis according to Ntalli et al., (Reference Ntalli, Manconi, Leonti, Maxia and Caboni2011) and Caboni et al. (Reference Caboni, Ntalli, Aissani, Cavoski and Angioni2012) with slight modifications. The chromatographic separation and identification of the main components were performed on a Trace GC ultra gas chromatograph (Thermo Finnigan, San Jose, California, USA) coupled with a Trace DSQ mass spectrometry detector, a split-splitless injector and an Xcalibur MS platform. The column used was polar fused silica capillary DB-5MS (5% phenylmethylpolysyloxane, 30 m × 0.25 mm; film thickness, J&W 0.25 μm; Scientific Fisons, Folsom, California, USA). The injector and transfer line were at 250°C, while the oven operating conditions were as follows: from 50°C (held for 5 min) to 280°C at 9°C/min heating rate, and kept at this temperature for 8 min. The carrier gas was helium with a constant flow rate of 1 ml/min; the sample (0.20 μl) was injected in splitless mode (60 s). Mass spectrometry acquisition was carried out using the following conditions: ionization voltage, 70 eV; scan rate 1.6 scan/s; mass range, 50–550 amu. Essential oil components were identified by comparison of their retention times and mass fragmentation with those of authentic standards and computer matching against a NIST98 commercial library, as well as Kovats retention indices, for alkanes C9–C24 compared with those reported by Adams (Reference Adams2007). Moreover, concentrations of essential oil components were expressed as area percent.
In vitro assays
Faecal samples were collected from the rectum of Sarda dairy ewes and kept at 4°C until use. Third-stage larvae (L3) of GIN were obtained by coproculture: briefly, the faeces were broken up and placed into a glass jar, which was closed and kept at a temperature of about 27°C for 14 days. After that, larvae were separated following the Baermann technique (Euzeby, Reference Euzeby1981) and identified using the morphological keys by MAFF (1986). Larvae were identified as belonging to the following GIN species: Teladorsagia spp. (52%), H. contortus (25%) and Trichostrongylus spp. (23%). A preliminary experiment was conducted to evaluate the lowest concentration giving 100% paralysis.
Ruta chalepensis methanol extract and its essential oil were tested on L3 GIN at a dose range of 0.02–0.20 and 0.40–6.30 mg/ml, respectively, and EC50 values were calculated. Pure compounds 2-nonanone, 2-undecanone and 2,4-decadienal, constituents of R. chalepensis extract, were tested individually at a concentration range of 0.03–1.60, 0.40–3.90 and 0.02–0.65 mg/ml, respectively. Moreover, (E,E)-2-4-decadienal, furfural, salicylaldehyde and epigallocatechin gallate were tested on L3 at dosages ranging from 0.30 to 2.30, 0.10 to 3.00, 0.35 to 6.00, and from 0.70 to 5.45 mg/ml, respectively. A stock solution of methanol extract was diluted with distilled water containing the polysorbate surfactant Tween 20, while the stock solution of essential oil was prepared in ethanol and successively diluted with water containing Tween 20. Stock solutions of test compounds were prepared using distilled water containing Tween 20 and, to overcome insolubility, methanol was used. For the same reason, solutions of SAL and ECG were prepared using aqueous 0.1 m phosphate-buffered saline solution (PBS). Therefore, the solvents were used as carriers in treatment wells and as controls for death/paralysis correction in untreated wells. Final concentrations of solvent and Tween 20 in treatment wells never exceeded 1 and 0.3%, respectively. Aqueous solutions of PBS and methanol were used as negative controls, while levamisole was the positive control. Stock solutions of LEV were prepared at 1 mg/ml using water. Larval bioassays were performed in Cellstar 96-well cell-culture plates (Greiner Bio-One, Kremsmunster, Austria) and each treatment consisted of 25 L3 per well (Martin & Le Jambre, Reference Martin and Le Jambre1979). The larval paralysis test was replicated six times per experiment and was carried out in the dark (Gill et al., Reference Gill, Redwin, van Wyk and Lacey1991) and at room temperature (Szewezuk et al., Reference Szewczuk, Mongelli and Pomilio2006). L3 were analysed at 1, 24 and 48 h for pure compounds and 96 h for the extracts, with the aid of an inverted microscope (Euromex, The Netherlands) at 40 × magnification. Nematodes were ranked into two categories: motile and immotile/paralysed. The classification of the larvae into immotile/paralysed was ascertained by absence of motility during an observation period of 6–8 s.
Data analysis
The percentages of L3 paralysed during the bioassay tests were corrected by elimination of the natural paralysis in the negative control according to the Schneider Orelli formula (Puntener, Reference Puntener1981):
$$\eqalign{{\rm corrected}\,\% = &\; \displaystyle{{{\rm mortality}\,\,\% \,{\rm in}\,{\rm treatment}\, - \,{\rm mortality}\, \% \,{\rm in}\,{\rm control}} \over {100 - {\rm mortality}\,\% \,{\rm in}\,{\rm control}}}\cr & \times 100}.$$
The corrected percentages of treated L3 were analysed by Probit analysis for the determination of EC50 values using Minitab 16 software (Minitab Inc., State College, Pennsylvania, USA) and a probability of 0.05 was used. EC50 values indicate the concentration of the compound required for 50% paralysis after correction of percentages for natural paralysis. The results were expressed as EC50 ± SD mg/ml. Moreover, linear regression was used to evaluate dose–response relationships.
Results
Mass spectrometry coupled to gas chromatography was used for the chemical characterization of the essential oil. According to GC–MS analysis, the essential oil comprised sabinene, α-pinene, limonene, 2-nonanone, 2-nonanol, geijerene isomer, geijerene, 2-decanone, octyl acetate, 2-undecanone, 2-dodecanone and 2-tridecanone (fig. 1). Mass chromatographic characteristics along with the percentage composition of the oil are reported in table 1. Levels of 2-nonanone and 2-undecanone were 25.3 and 24.0%, while limonene and octyl acetate were 12.8 and 10.4%, respectively. The oil yield was 1.09 ± 0.05% w/w expressed on a dry basis. Overall GC–MS analysis allowed the identification of 12 compounds, comprising 89.1% of the total oil composition; other unknown substances were present at low concentrations (table 1).
GC–MS chromatogram of R. chalepensis L. essential oil to show peaks of: (1) sabinene, (2) α-pinene, (3) limonene, (4) 2-nonanone, (5) 2-nonanol, (6) geijerene isomer, (7) geijerene, (8) 2-decanone, (9) octyl acetate, (10) 2-undecanone, (11) 2-dodecanone and (12) 2-tridecanone. The remaining peaks are unknown.
GC–MS analysis of Ruta chalepensis L. essential oil (REO). For each compound the molecular weight (MW) and two characteristic fragment ions with different mass to charge (m/z) ratios are shown.
When REO and RME were tested for anthelmintic activity, a clear dose-dependent effect was observed. EC50 values after 1 day of exposure in test solution to R. chalepensis methanol extract and its essential oil were 0.10 ± 0.08 and 1.29 ± 1.10 mg/ml, respectively. This result was confirmed 96 h after treatment, with an EC50 of 0.10 ± 0.06 mg/ml for RME and 1.91 ± 1.61 mg/ml for REO. Furthermore, a linear relationship between the mean values of paralysis percentage and the log concentration of the tested solution was observed, with coefficient of determination (R 2) values at 96 h of 0.981 and 0.984 for REO and RME, respectively. Levamisole was used as the positive control, with EC50 values at 24 and 48 h after treatment of 0.63 ± 1.15 and 0.66 ± 0.65 mg/l, respectively (table 2). 2-Decanone, 2-nonanone and 2-undecanone showed anthelmintic activity with EC50 values at 24 h after treatment of 0.07 ± 0.06, 0.25 ± 0.29 and 0.88 ± 0.73 mg/ml, respectively (table 2); these activities were confirmed at 48 h with EC50 values of 0.11 ± 0.08, 0.25 ± 0.22 and 1.06 ± 1.05 mg/ml, respectively. A linear relationship between the mean values of paralysis percentage and the log concentration of solution tested was observed, with R 2 values at 48 h of 0.994 for UND, 0.978 for NON and 0.959 for DEC, respectively (table 2). For comparison, we tested the anthelmintic activity of four phytochemicals – 2,4-decadienal, furfural salicylaldehyde and epigallocatechin gallate. These compounds exhibited the highest activity at 24 h with EC50 values of 1.03 ± 0.79, 1.83 ± 1.50, 2.12 ± 2.09 and 2.10 ± 2.14 mg/ml, respectively, while ECG at 48 h had an EC50 value of 1.79 ± 1.72 mg/ml (table 2). The four compounds studied showed a linear relationship between the mean values of paralysis percentage and the log concentration of solution tested, with R 2 values of 0.933, 0.901 and 0.970 at 24 h for DDI, FUR and SAL, and 0.887 and 0.847 at 48 h for ECG and LEV, respectively (table 2).
EC50 (mg/ml) ± SD and coefficient of determination (R 2) values of tested compounds on larval nematodes 1, 24 and 48 h after treatment.
Discussion
According to other authors (Günaydin & Savci, Reference Günaydin and Savci2005; Dob et al., Reference Dob, Dahmane, Gauriat-Desrdy and Daligault2008; Ntalli et al., Reference Ntalli, Manconi, Leonti, Maxia and Caboni2011; Ali et al., Reference Ali, Demirci, Kiyan, Bernier, Tsikolia, Wedge, Khan, Baser and Tabanca2013; Haddouchi et al., Reference Haddouchi, Chaouche, Zaouali, Ksouri, Attou and Benmansour2013; Caboni & Ntalli, Reference Caboni, Ntalli, Gross, Coats, Duke and Seiber2014; Akkari et al., Reference Akkari, Ezzine, Dhahri, B'chir, Rekik, Hajaji, Darghouth, Jamâa and Gharbi2015), 2-undecanone and 2-nonanone were the major constituents of the essential oil. Also Mejri et al. (Reference Mejri, Abderrabba and Mejri2010) and Tounsi et al. (Reference Tounsi, Wannes, Ouerghemmi, Msaada, Smaoui and Marzouk2011) reported 2-undecanone as the predominant compound, but followed by 2-decanone (Mejri et al., Reference Mejri, Abderrabba and Mejri2010) or 2-nonanol (Tounsi et al., Reference Tounsi, Wannes, Ouerghemmi, Msaada, Smaoui and Marzouk2011). The oil yield was 1.09 ± 0.05% w/w expressed on a dry basis. Ntalli et al. (Reference Ntalli, Manconi, Leonti, Maxia and Caboni2011) reported a yield of REO of 0.36 ± 0.10%, Tounsi et al. (Reference Tounsi, Wannes, Ouerghemmi, Msaada, Smaoui and Marzouk2011) described yields in the range of 0.39–2.46% for leaves, flowers, stems and fruits, whereas Günaydin & Savci (Reference Günaydin and Savci2005) found a yield of 0.70%. Results are in accordance with the variability of different plant organs, the altitude, the climate, the soil where plants grew, the harvest period and the extraction technique used (Ntalli et al., Reference Ntalli, Manconi, Leonti, Maxia and Caboni2011; Tounsi et al., Reference Tounsi, Wannes, Ouerghemmi, Msaada, Smaoui and Marzouk2011; Bouzidi et al., Reference Bouzidi, Latrèche, Attaoui, Benabderrahmane, Mehdadi and Benyahia2012). Furthermore, the chemical profile of the methanol extract of R. chalepensis obtained by time of flight mass spectrometry coupled to liquid chromatography was in accordance with the profile of the extracts studied by Ntalli et al. (Reference Ntalli, Manconi, Leonti, Maxia and Caboni2011), where rutin, angelicin and 8-methoxypsoralen were the main constituents, and by Günaydin & Savci (Reference Günaydin and Savci2005), where psoralen, chalepin and chalepensis were identified (data not shown). Nematode paralysis was evaluated by counting moving larvae with the use of an inverted microscope, as suggested by Gill et al. (Reference Gill, Redwin, van Wyk and Lacey1991). In accordance with Martin & Le Jambre (Reference Martin and Le Jambre1979), larvae were considered immotile or paralysed when there were no observable movements during 6–8 s. Considering the high variability of percentage content of nematode species in real samples, R. chalepensis methanol extract showed highest anthelmintic activity if compared with its essential oil. At 48 h R. chalepensis secondary metabolites 2-decanone, 2-nonanone and 2-undecanone showed an anthelmintic activity of 0.11 ± 0.08, 0.25 ± 0.22 and 1.06 ± 1.05 mg/ml, respectively (table 2). A light micrograph of nematodes treated with DEC is shown in fig. 2. For comparison (E,E)-2,4-decadienal, an anthelmintic component of Ailanthus altissima (De Martino & De Feo, Reference De Martino and De Feo2008), showed an EC50 of 1.09 ± 0.88 mg/ml. In addition, the in vitro anthelmintic activity of the secondary metabolites furfural, salicylaldehyde and epigallocatechin gallate on sheep GIN was studied. FUR was also found to be a volatile nematicidal component of A. altissima (Ntalli et al., Reference Ntalli, Vargiu, Menkissogluspiroudi and Caboni2010) while SAL was reported to be active against Meloidogyne incognita (Caboni et al., Reference Caboni, Aissani, Cabras, Falqui, Marotta, Liori, Ntalli, Sarais, Sasanelli and Tocco2013). These phytochemicals showed a linear relationship between the mean values of paralysis percentage and the log concentration of solution tested (EC50 values at 48 h of 2.18 ± 1.19 and 2.89 ± 2.50 mg/ml for FUR and SAL, respectively). On the other hand, when we tested ECG, we recorded an anthelmintic activity of 1.79 ± 1.72 mg/ml at 48 h. Molan et al. (Reference Molan, Meagher, Spencer and Sivakumaran2003) reported that ECG possesses ovicidal and larvicidal activity against T. colubriformis in vitro. Interestingly, gallic acid did not show anthelmintic activity at the concentrations tested (table 2). Moreover, levamisole anthelmintic activity (EC50 value of 0.63 ± 1.15 mg/l at 24 h) can be compared with data reported by Martin & Le Jambre (Reference Martin and Le Jambre1979), who obtained an LC50 against Ostertagia spp., after 24 h of treatment, between 1.3 and 9.0 μg/ml. Lower EC50 values can be explained hypothetically by the different species tested.
Third-stage strongyloid larvae: (a) untreated; and treated with (b) 2-decanone at 0.20 mg/ml and (c) levamisole at 1.2 mg/l, both after 24 h.
The present investigation clearly indicated that the R. chalepensis plant extracts RME and REO showed good anthelmintic activity against GIN if compared with other botanical extracts (Ademola et al., Reference Ademola, Fagbemia and Idowub2004; Maciel et al., Reference Maciel, Morais, Bevilaqua, Camurça-Vasconcelos, Costa and Castro2006; Kamaraj et al. Reference Kamaraj, Rahuman, Elango, Bagavan and Zahir2011, Elandalousi et al., Reference Elandalousi, Akkari, B'chir, Gharbi, Mhadhbi, Awadi and Darghouth2013), while they were less active if compared with the conventional anthelmintic, levamisole. Moreover, their constituents 2-undecanone, 2-nonanone, 2-decanone, together with (E,E)-2,4-decadienal, were the most active tested compounds. A lower anthelmintic activity was recorded for furfural, salicylaldehyde and epigallocatechin gallate. Taking these results together, the use of phytochemicals is an opportunity for the development of valid alternatives for control of strongyle infestations in small ruminant herds. In conclusion, these compounds may find an application in the future as anthelmintics in veterinary practices. On the other hand, more studies are needed to evaluate the mode of action of these active compounds and to optimize the anthelmintic activity through studies of structure–activity relationships.
Financial support
This research was supported by a PhD grant from the University of Sassari, Department of Agriculture (Scuola di Dottorato di Ricerca in Scienze e Biotecnologie dei Sistemi Agrari e Forestali e delle Produzioni Alimentari), and by the Regione Autonoma Sardegna, L.R. 7/2007–2012, Research Project ‘Nuovi approcci alla gestione eco-compatibile di parassiti degli allevamenti zootecnici’, University of Sassari, Italy.
Conflict of interest
None.
