Ruta chalepensis full extract and organic phases exhibit nematocidal activity against Haemonchus contortus eggs and infective larvae (L₃)

This study was performed at the Laboratory of the Research Unit in Helminthology from Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, (CENID-SAI), belonging to the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), situated in Progreso, Jiutepec Municipality, Morelos State, Mexico. The plant extraction and the chromatographic techniques for partitions were performed at Centro de Investigación Biomédica del Sur (CIBIS-IMSS) in Xochitepec Municipality, Morelos State, Mexico.

Five kg of the plant R. chalepensis (in fresh) was collected from a particular garden at La Milpa, locality, Villa García Municipality, Zacatecas State, Mexico. The plant material was deposited at the ethnobotanical garden of Cuernavaca city, State of Morelos, Mexico and the taxonomic identification of the plant, was performed by the Biologist Margarita Avilés, from the Autonomous University of the State of Morelos, Mexico.

The plant material (R. chalepensis aerial parts) was washed with tap water and was dried into a conventional stove at 45 °C for 3 days. Dried plant material (1.1 Kg) was ground to obtain small particles (1 mm) and transferred to a 4 L Erlenmeyer flask and distilled water was added to afore up to 4 L. The material remained under water maceration for 24 h. After this period, material was filtered through a Whatman paper (Nº 4). The extract was concentrated into a rotary evaporator (Heidolph Laborota 4000, Germany) at 50 °C with 90 rpm and at continuous vacuum. The whole solvent was eliminated until obtain a dense mixture that was frozen at -80 °C. Later on, the extract was lyophilized using a Heto spywinner lyophilizer to obtain a brown powder (308 g) which was finally maintained under darkness at -20 °C until use.

The R. chalepensis full aqueous extract (FE, 100 g) was subjected to a liquid-liquid bipartition with Ethyl acetate (EtOAc), what is a water immiscible solvent (used in 1:1 proportion). The liquid separation of the whole extract was performed as follows: One hundred grams of the extract were eventually resuspended into 1000 ml of distilled water. The liquid extract was processed for a bi-partition with Ethyl Acetate to obtain one aqueous phase and an EtOAc phase. The mixture was put into a crystal separation funnel (Pixer^® 2000 mL N° 6400, pear-shape) and remained for 5 min to allow the phases separation. Then, the funnel key was opened to recover the individual phases. The denser phase was the aqueous phase (Aq-Ph) and this was the first collected phase and the less dense phase (EtOAc-Ph) was the last recovered phase. This method was repeated several times until the whole aqueous extract was processed. Both phases were concentrated using the previously described rotary evaporator. The aqueous phase was concentrated at 50 °C with 90-rpm rotations and at continuous vacuum. The solvent was eliminated until obtaining a dense mixture that it was frozen at -80 °C. The phase EtOAc-Ph was concentrated at 46 ^oC with 90-rpm rotations and also at a continuous vacuum until eliminating the whole solvent. Both phases were eventually lyophilized using the previously described lyophilizer and were maintained at -20 °C under dark conditions until use (García, 2015).

A phytochemical screening of the EtOAc phase of the R. chalepensis aqueous extract was performed in silica gel 60 F254 pre-coated plates. Samples were seeded using capillary tubes. Plates were developed into a crystal chamber with a low polarity system (95:5 dichloromethane:methanol) to allow the visualization of the highest amount of compounds present in the analysed sample. After developing, plates were dried though a soft agitation. Once the plates were dried these were observed under ultraviolet light at 245 nm and 365 nm wavelengths searching for compounds. Plates were developed using reagents specific for flavonoids, 2-aminoethyl diphenylborinate, Cerium (IV) sulphate solution and 4-dimethylamino benzaldehyde. After developing plates were dried on a hot stirrer until, visualize the coloured bands indicating the presence of compounds Delgado-Núñez et al. (2020). The bands corresponding to compounds of interest were identified by comparison with pure compounds either isolated or commercial that showed both a similar colorimetric pattern and similar development (von Son-de Fernex et al., 2015).

The identification of molecules using Gas chromatography/mass spectrometry analysis was performed based on the protocol described by Pineda-Alegría et al. (2017). The chemical composition of EtOAc phase was analysed on a Gas Chromatograph-Mass Spectrometry (GC-MS) equipped with a quadruple mass detector in electron impact mode at 70 eV. Volatile compounds were separated onto a HP 5MS capillary column (25 m long, 0.2 mm i.d., with 0.3-μm film thickness). Oven temperature was set at 40 °C for 2 min, then programmed from 40 – 260 °C at 10 °C/min and maintained for 20 min at 260 °C. Mass detector conditions were as follows: interphase temperature 200 °C, and mass acquisition range, 20 – 550. Injector and detector temperatures were set at 250 and 280 °C, respectively. Splitless injection mode was carried out with 1 μL of each fraction (3 mg/mL solution). The carrier gas was helium at a flow rate of 1 mL/min. Identification of the compounds was performed with the comparison of mass spectra with those of the National Institute of Standards and Technology (NIST, 1.7 Library) and with data from the literature, which identified eighteen compounds (1 – 18).

One 10 months, Pelibuey male sheep previously infected with 350 H. contortus infective larvae (L3) per kg of body weight (BW) (per os) was used as an egg donor of the parasite. Faeces from this animal were processed to recover the eggs (in fresh) following the technique descried by Coles et al. (1992). The faeces were suspended in water and were passed through different sized meshes to separate detritus and residues of large size and eggs were eventually retained in a 37-μm-size mesh. Eggs were recovered into a precipitate glass and then the egg suspension was centrifuged at 300 rpm for 1 min with a separation gradient using saturated saline solution. After spinning down eggs floated up forming a white ring in the upper part of the centrifuge tube. Eggs were recovered using a crystal Pasteur pipette and re-suspended in water. Then, several washes with water were necessary to obtain perfectly clean egg suspension (Jasso-Díaz et al., 2014). Likewise, faeces put into a plastic bowl for the elaboration of coprocultures. Faeces were crushed using a wooden spoon and tap water and small particles of rubber foam were added and mixed to create an adequate water and oxygen micro-environment for promoting the best nematode egg hatching (MAFF, 1986). Coprocultures were maintained under room temperature (18 – 25 ºC) for 5 days to allow the highest larval rate recovering. Infective larvae were obtained through the Baermann funnel technique for 12 h (Mesquita et al., 2018).

The egg hatching inhibition assay was performed on 96-well micro-titration plates. Five treatments were settle down as follows:

1) the whole extract, 2) aqueous (Aq-Ph) and 3) ethyl acetate (EtOAc-Ph) phases at 1.25, 2.5, 5, 10 and 20 mg/mL. 4) distilled water (a negative control, water was used to dissolve the whole extract and Aq-Ph) and 4) 4 % methanol (another negative control, methanol was used to dissolve the EtOAc-Ph). Fifty microliters of an aqueous suspension containing 100±10 H. contortus eggs were deposited in every well (n=4) and additionally 50 μL of the corresponding to extract, phases and controls were added in each well obtaining a 100 μL final volume. The plates were maintained at 28 °C for 48 h into a humidity chamber. After incubation 5-μL of lugol were deposited on each well in order to stop the egg hatching. Both first stage larvae (L1) and eggs of the parasite into each well were counted. The results were considered using an egg hatching inhibition rate; where the number of recovered larvae was considered as 100 % hatching eggs. The results were eventually expressed as egg hatching inhibition percentage. This experiment was performed by triplicate.

This assay was also performed in 96-well micro-titration plates considering four well per treatment (n=4). It is important to mention that in preliminary results the same doses used in the ovicidal assay did not show any important larvicidal effect. So, we decided to increase the concentrations to identify some potential important larvicidal effect. The treatments were the serial concentrations of whole extract (at 30, 60, 90, 120, 150 and 200 mg/mL). Likewise, distilled water was used as control. Ten microliters of an aqueous suspension containing 200±15 H. contortus infective larvae (L3) were deposited in each well (n=4 wells). Additionally 90 μL of the corresponding extract at different concentrations were added to each well, giving 100 μL as a final volume. The plates were maintained at 28 °C for 72 h into a humidity chamber. After the confrontation period, live and dead larvae were counted in ten 5-μL aliquots and the means of dead or live larvae per treatment were recorded. This assay was performed by triplicate. Mortality rate was expressed as a percentage mortality and was calculated according to the following formula:

Mortality= [(dead larvae mean) / (live larvae mean + dead larvae mean)] × 100

Data were arcsin√Xij transformed; since the distribution was not normal. Transformed data were analysed with a fix effect model completely random with six treatments with the PROC GLM program using the statistical package SAS and a Tukey mean comparison prove (SAS, 1999). The lethal concentrations (LC 50) for egg hatching inhibition and larvae mortality were estimated by the Probit analysis.