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Introduction
Parasitism is one of the main problems that affect small ruminants, and in this group, gastrointestinal nematodes (GIN) are the most important cause of mortality in sheep and goats in the tropics of Mexico (Delgado, A. et al., 2016; Zapata et al., 2016; Canul-Ku et al., 2012). H. contortus is a gastroenteric nematode that parasitizes the abomasum of ruminants (sheep, goats and cattle) and is epidemiologically the most important with 70% prevalence in tropical regions (Olivares et al., 2012; Ehsan et al., 2020) This parasite is the most pathogenic (Baltrusis et al., 2020), it feed on blood, injury the abomasal epithelium and clinically causes epithelium inflammation, emaciation, anemia and hypoproteinemia with losses of up to 500 mL of blood/day, submandibular edema, drooping of the productive parameters (production of wool, milk and meat), and in many cases death of infected animals (Eshan et al., 2020). The frequent use of anthelmintic for the parasites control has been one of the causes that has led to the resistance development of these microorganisms (Laca-Megyesi et al., 2020). The parasites are a problem from a biological and economic point of view, especially when the inadequate use of chemical dewormers is abused, this has promoted the development of the anthelminthic resistance (Muñiz-Lagunes et al., 2015). Kaplan & Vidyashankar (2012) reported resistance of H. contortus to ivermectin, moxidectin, levamizole and albendazole in 76 %, 24 %, 98 % and 54 % of the studied goat and sheep herds; to tetrahydropyrimidines and macrocyclic lactones (Baltrusis et al., 2020).
The impact of parasitism by GIN has motivated the development of investigation in alternative medicine, such as the use of biological agents (fungi and hematophagous mites) (Pérez-Pérez et al., 2014; Von de - Fernex et al., 2015; Olmedo et al., 2014; García-Ortiz et al., 2015) and medicinal plants or extracts of tree leaves with nematicidal properties of use in ruminants (León-Castro et al., 2015; Olivares et al., 2012; Carvalho et al., 2012). Manuel-Pablo et al. (2020) reported that the supply of a diet with 4.5 % tannins from the C. coriaria fruits in goats; they had a daily consumption of 45 g of the secondary compounds without negative effects on health, weight gain and feed conversion of the animals. The arboreal legume C. coriaria Jacq. Willd, is commonly known as "Cascalote", is widespread in the Tierra Caliente region of Guerrero and contains a high variety of secondary metabolites such as tannins, gallic acid, and flavonoids (Sánchez-Carranza et al., 2017), gallotannins (methyl gallate) and their derivatives were also identified (De Jesús-Martínez et al., 2018). The objective of the study was to evaluate the acetonic and ethanolic extracts from C. coriaria fruits against H. contortus eggs.
Material and Methods
Vegetative material corresponded to C. coriaria dried fruit (5000 g) were collected in march in the Tierra Caliente region of Guerrero, Mexico, located at 18° 20´ 30" NL and 100 39`18" WL, which were brought to total dryness through of a forced-air heater at 40 °C. Subsequently they were subjected to a milling with a Mini Wiley Mill, to obtain a particle size of 1 mm.
Preparation of acetonic and ethanolic extracts
Each acetone and ethanol extracts were elaborated separately preparing a solution of 300 g of dried fruits of C. coriaria suspended in 2000 mL of the solvents, during 72 h, at room temperature, to extract polar secondary compounds and intermediate polarity. Then the liquid solutions of the extracts were filtered with different filters separately in the following order, first gauze, then cotton and finally filter paper, the residual solvents were removed by distillation under reduced pressure with the help of a rotary evaporator (Buchi R-114) at 60 °C, and finally, they were dried by Lyophilization processes (Labconco FreeZone -105 °C) to obtain the semi-solid extracts. The solvent-free dry extracts (36 g) were stored at -40 °C until their use in the in vitro bioassays.
Biological material
Obtaining Haemonchus contortus eggs
The H. contortus eggs were used obtained of the feces from an ovine experimentally infected with infective larvae (L3) of the parasite (strain INIFAP, 350 L3/kg of BW of the animal). The eggs were concentrated through the passage in different sieves (200, 100, 75 and 37 μm in diameter) and by density gradients in 40 % sucrose solution.
Eggs eclosion inhibition (% EEI)
Eight 96-well microtiter plates were used. The treatments were acetonic and ethanolic extracts at different concentrations (20, 10, 5, 2.5, 1.2 and 0.6 mg / mL) and (6.15, 3.12, 1.56 and 0.78 mg / mL), 4 % methanol as a negative control and ivermectin injectable solution (5 mg / mL; Ivomec® Pour ON Boehringer Ingelheim laboratory) as a positive control. Fifty µL of an aqueous suspension containing 100 ± 150 H. contortus eggs were placed in each well. Subsequently, aliquots of 50 μL of the extracts and controls were added, having a final volume of 100 μL per well. The plates were incubated by 48 hours at a temperature of 28 ° C, with 100 % humidity (at an incubator Ecoshel model Cl-80). The egg eclosion process was stopped by adding 10 μL of 5 % lugol solution. Finally, a total count of eggs or larvae of each well was performed and the EEI percentage was determined by the following formula: % EEI = [(number of eggs) / (number of larvae + number of eggs)] * 100.
Statistical analysis
The data were analyzed under a completely randomized design, with the following statistical model: Y ij = μ + T i + ξ ij ; where: Y ij = eclosion inhibition; μ = general mean; T i = effect of the extracts and controls ξ i = the random error of the treatment. The difference between means was compared with the Tukey test (p < 0.05). In addition, minimum (LC50) and maximum (LC90) lethal concentrations were determined using the PROBIT procedure of the SAS statistical package (SAS, 2002).
Results and Discussion
The results obtained in the study showed evidence that the extracts of the fruits of this arboreal legume have inhibitory effects on the eclosion of H. contortus eggs. In the percentages of EEI, the effects were close to 100 % at the concentration doses of 2.5 and 3.12 mg L of the acetonic (Figure 1) and ethanolic (Figure 2) extracts respectively, this means that both extracts used effectively inhibited the eclosion of eggs to the interrupt their development. Figure 3 shows the images with the readings of the eggs after their incubation with the different treatments, the images 3A, 3B and 3C showed the high eclosion of eggs to L1 larvae that were observed when they were incubated with the negative controls (methanol at 4 % and H2O). The 3D, 3E and 3F images showed the remaining eggs that were inhibited by exposure to the C. coriaria fruit extracts. Poné et al. (2011) reported that the active compounds in the extracts penetrate the envelope (cuticle) of the egg and prevent its development and/or paralyze the larvae of the first embryonic stage. Delgado-Nuñez et al. (2020) reported an interruption of embryonic development and a 30 % reduction in the cell mass and shell of the egg. In addition, some eggs showed irregular edges and deformations that produced a wrinkled surface appearance.
Figure 1. Eggs eclosion inhibition of H. contortus exposed to acetonic extract elaborated with C. coriaria fruits (abc different literals eclosion inhibition differed between treatments)
Figure 2 Eggs eclosion inhibition of H. contortus exposed to ethanolic extract elaborated with C. coriaria fruits (abc different literals eclosion inhibition differed between treatments)
Figure 3 Microscopic images that represented the readings of the H. contortus eggs: (A, B and C) showed the high eggs eclosion to L1 larvae in the negative controls, (D, E and F) showed the remaining eggs inhibited by exposure to C. coriaria fruits extracts
Ademola et al. (2011), Zabré et al. (2017) when using an acetonic solvent observed that tannins extracted from Cassia alata and Acacia raddiana, respectively, were responsible of eggs eclosion inhibition and adult stage H. contortus mortality inducing, but damages in the enveloped of the eggs was not they described. Similar results were obtained by Carvalho et al. (2012), Akkari et al. (2014), Cabardo & Portugaliza (2017) when they used ethanol solvent observed that tannins extracted polarly from several plants rich in condensed tannins, showed in studies in vitro activity to inhibit the eclosion of eggs, infective stage larvae (L3) and caused paralysis and / or death in adult parasites. These antecedents indicated that the use of acetonic and ethanolic solvents extracted secondary polar compounds that showed activity against H. contortus eggs. Veloz-Garcia et al. (2004) reported as main phenolic compounds to gallic and tannic acids in the cascalote pods, in another study De Jesús-Martínez et al. (2018) reported to the gallotannins (methyl gallate) and their derivatives as primordial secondary compounds, for this reason, the effects observed against the parasite eggs in this study, could be attributed to the action of these compounds.
The lethal concentrations (LC50 and LC90) of the extracts used are shown in figure 4A and 4B. In the acetonic extract, an LC50 of 0.23 mg/mL and LC90 of 1.04 mg/mL are observed, and for the ethanolic extract an LC50 of 0.014 mg/mL and CL90 of 0.14 mg/mL, are observed. The results showed a concentration-dependent effect in the different extracts, however, comparatively between the two extracts it can be seen that the lethal concentrations of the ethanolic extract were lower compared to acetonic extract, which may indicate that the polar compounds extracted of the C. coriaria fruits with the ethanolic solvent, turned out to be more lethal against the eggs of the parasite. Al - Rawahi et al. (2013) reported that the solubility of polyphenols is affected by the type of solvents used and their polarity, resulting in extracts with different properties despite being elaborated from the same plant. Dai & Mumper (2010), Al-Farsi et al. (2007) observed that 100% acetone extracted flavonoid and phenolic compounds of low polarity. Ringuelet & Viña (2013) mentioned that ethanol is a solvent of excellent solubility to extract polar compounds. The same phenomenon was observed by Castillo-Mitre et al. (2017) who reported the different effect of extracts elaborated with Acacia cochliacantha leaves using different solvents, against eggs of H. contortus and attributed it to polar and non-polar compounds present in the different fractions. Sánchez-Carranza et al. (2017) reported that fruits and leaves of C. coriaria are a rich source in condensed tannins, such as gallic acid, ethyl gallate and tannic acid, as a basis of hydrolyzable tannins, so the biological activity reported in this study could be related to these metabolites. In addition, the concentration doses used in the study were less than 9% (90 g/kg dry matter) of tannins to cause mortality in the animal (Nawab et al., 2020). Frutos et al. (2004) fed finishing lambs daily with diets added with 20.8 g/kg of dry matter, and they did not observe toxic effects or decrease in the production of the animals. Manuel-Pablo et al. (2020) provided daily 45 g of secondary compounds in the fruits of C. coriaria without negative effects on health, weight gain and feed conversion of the goats. Pérez, V. et al. (2011) reported symptoms such as methemoglobinemia, kidney failure, anorexia, depression and diarrhea in ruminants when they consumed tannins in the diet to higher quantities at 4400 mg/kg of body weight.
Conclusions
It is concluded that the extract elaborated withC. coriariafruits in acetonic and ethanol solvents eclosion inhibitsH. contortuseggs, so it could be an option in the treatment of nematodes in small ruminants, however, it requires more research as antiparasitic for direct and reliable use in animals. The effect on the eggs eclosion inhibition of the parasite was different between the extracts and was attributed to the solvent used because it was the same arboreal fruit. The findings in this study require additional investigations for the identification of the compounds responsible for the EEI, by means of studies of identification of bioactive compounds by high-performance liquid chromatography (HPLC).
