INTRODUCTION

The production of small ruminants in the world is limited by agro-meteorological, economic and sanitary factors, associated with the presence of microorganisms that cause infectious diseases. Parasitic diseases are a health problem, which mainly affects extensive small ruminant production systems; causing direct economic losses such as animal death, and indirect losses due to the decrease in productive and reproductive parameters (^{Pérez-Pérez et al., 2014}).

Hemonchosis is a frequent parasitic disease in small ruminants, the etiological agent is HC. HC is classified as a hematophagous nematode with tropism for the abomasum, where it attaches to feed. This parasite is associated with a picture of anemia and gastritis, which depending on the number of parasites and severity of clinical manifestations can cause death of adult and neonatal sheep (Kumarasingha et al., 2016).

The control of parasitosis is based on the administration of anthelmintics of synthetic chemical origin, including benzimidazole derivatives, macrocyclic lactones, aminocetonitrile monepantel derivatives and imidazothiazoles (^{Geary et al., 2015}). In this regard, the use of commercial anthelmintics once had promising results for livestock; however, irrational and inappropriate use has generated the development of nematode populations with resistance to anthelmintics; in addition, their ecological impact has been reported, by affecting non-target populations and in terms of food safety, due to the presence of residues of these molecules in products and by-products of animal origin (^{Zajíčková et al., 2020}).

Due to the aforementioned problems, several strategies have been implemented in Integrated Parasite Control (IPC); in this way, the aim is to reduce drug use of chemical origin to mitigate the negative impact on the environment, in addition to offering producers control alternatives that allow them to reduce production costs (^{Pérez-Pérez et al., 2014}).

With this approach, the use of plant extracts and their metabolites have been proposed as one of the most promising alternative methods to prevent and control diseases associated with the presence of some nematodes in ruminants, given the different biological activities that have been reported in plant extracts, including anthelmintic activity (^{Kotze & Prichard, 2016}).

The anthelmintic potential of plants has been associated with their content of secondary metabolites, such as terpenes (25,000 types), alkaloids (12,000 types) and phenolic compounds (8,000 types); of which diverse and possible mechanisms of action have been described according to their chemical structure and the bonds that could be formed with the structural molecules of the parasite egg or larva (^{Zajíčková et al., 2020}).

The mechanism of action of these secondary metabolites is unclear; however it has been hypothesized that they could cause from paralysis and/or larval mortality, interfere with larval development, inhibit egg hatching and interfere with female fertilization (^{Heeger et al., 2017}).

Several results have been obtained with plant extracts evidencing the anthelmintic potential that secondary metabolites possess against some nematodes of importance in ruminants, both in vitro and in vivo models. Under this premise, studies have been conducted with hydroalcoholic extracts obtained from the fruit and leaves of Caesalpinia coriaria, which inhibit the hatching of H. contortus and H. placei at 25 mg/mL as reported by (^{Rojo-Rubio et al., 2019}).

On the other hand, there is evidence of the evaluation performed by (^{Castillo-Mitre et al., 2017}) who evaluated the hydroalcoholic extract of Acacia cochliacantha leaves and reported the inhibition of 98 to 100 % the hatching of Haemonchus contortus eggs at concentrations of 0.62 to 1.56 mg/mL respectively; activity that was associated by the authors to the presence of caffeic and coumaric acid derivatives present in the extract.

In the same sense, (^{Von Son-de Fernex et al., 2015}) determined the ovicidal effect of the extract, obtained from Leucaena leucocephala leaves, on Cooperia spp. and identified quercetin and caffeic acid as the compounds responsible for this effect. With this, they suggest the possibility of a similar effect on other species of nematodes such as Haemonchus contortus.

In this regard, (^{Hernandez et al., 2014}) conducted an in vivo trial in growing lambs, which were administered 30 mL of Leucaena leucocephala extract for 63 days, observing a decrease in the parasite load of 54%; however, authors did not determine the mechanism of action of this effect.

Accordingly, the present study aimed to determine the effect of the hydroalcoholic extract of Leucaena leucocephala leaves on the hatching of Haemonchus contortus in vitro.

MATERIAL AND METHODS

RESULTS AND DISCUSSION

Qualitative phytochemical characterization indicated the presence of unsaturations (reduction to manganese dioxide), which are associated with compounds with ethylene bonds or compounds with two hydroxyl groups on adjacent carbons. A positive result was observed for phenolic oxydryls, which indicate the presence of hormones, pigments or essential oils. The presence of coumarins from the cycloaddition of siccaemic acid was also determined, as well as lactones, product of the union of an alcohol with a carboxylic acid in the same molecule. The positive reaction in the Salkowski test, indicative of the presence of sterols and triterpenes. The presence of flavonols and flavonones was also determined (^{Azcón-Bieto & Talón, 2003}).

The results concerning the tests for chalcones, quinones, Shinoda, sesquiterpenlactones, agitation and bicarbonate, Lieberman-Buchard, tannins and steroids, were negative. The absence of these compounds can be associated with the plant's own characteristics, environmental conditions, such as temperature, soil type, solar radiation and humidity; as well as factors of the extraction process, such as the polarity of the solvent with which they are extracted, among others (^{Zajíčková et al., 2020}).

The characterization of Leucaena leucocephala extract by gas chromatography-mass spectrometry was reported by (^{Salem et al., 2011}), in which they indicated that this forage plant has forty-four different secondary metabolites; of which the following chemical compounds stand out: 2(H)-benzofuranone-a-tetrahydro-4,4,7a-trimethyl (a volatile terpene: 23.1%) and pentadecanoic acid-14-methyl-methyl ester (a branched monomethyl acid: 8.2%), among others.

In this same study, it was reported that the alcoholic extract of Leucaena leucocephala is mainly constituted by oxygenated hydrocarbons of compounds C10 to C60, and contains predominantly phenolic hydrocarbons.

In the same context, (^{Von Son-de Fernex et al., 2015}) identified three phytochemicals in a biodirected fractionation of the hydroalcoholic extract of Leucaena leucocephala, these compounds were quercetin (82.21%), caffeic acid (13.42%) and scopolamine (4.37%).

Although the extract of L. leucocephala is rich in phenolic hydrocarbons, which as mentioned above can eventually generate the loss of egg viability, the participation of other types of metabolites, such as saponins and flavonoids, is not ruled out, generating a synergistic effect, since in plants and metabolites work under a joint dynamic, similar to the immune system, by conferring protection against bacteria, viruses, parasites and even predators. This is due to the formation of molecules that allow their survival, or simply by converging at some point in the same metabolic pathway (^{Azcón-Bieto & Talón, 2003}).

In most plants, the presence of secondary metabolites is distributed according to the parts that make up a plant (leaves, flower or fruit, stem and root). Accordingly, the presence of metabolites may present slight variations, since each of the parts that conform it is exposed to different stress factors, and therefore generate the necessary metabolites that confer protection and thus biological activities (^{Olguín-Rojas et al., 2019}).

Different concentrations of the hydroalcoholic extract of Leucaena leucocephala were evaluated on the inhibition of HC egg hatching. As can be seen in Table 1, there are significant statistical differences between treatments (p<0.05). The hydroalcoholic extract of Leucaena leucocephala leaves presented its best percentage of egg hatching inhibition at 100 mg/mL (71%), the concentrations of 90, 80 and 70 mg/mL did not show significant statistical differences among them (p>0.05), with 67, 65 and 62% inhibition of egg hatching respectively. The lowest percentages were obtained at concentrations of 60 and 50 mg/mL, which shows a dose-dependent behavior, where the lower the concentration of the extract, the lower the percentage of hatching inhibition.

Some research has reported favorable effects of a wide variety of extracts; however, they have not been able to determine the mechanisms by which the loss of egg viability occurs in nematodes.

In this regard, (^{Hernandez et al., 2014}) evaluated the hydroalcoholic extract of Leucaena leucocephala and observed a reduction in parasite load of 54% in growing lambs; however, the mechanism of action on the parasites in question was not determined. It has been reported that some compounds present in plant extracts possess metabolites such as saponins and tannins that interfere on fecundity in females, thus decreasing the parasite load in egg count (^{Zajíčková et al., 2020}). Given that background, results obtained in the present investigation could suggest that the decrease in parasite load in the above-mentioned study could be associated with the inhibition of egg hatching, induced by the hydroalcoholic extract of Leucaena leucocephala (71% at 100 mg/mL), and reported in the present investigation.

As mentioned, there are direct effects that suggest the ability of tannins to bind to intestinal mucosal proteins and cuticle glycoproteins, or structural proteins of eggs, interfering with development and growth and inhibiting hatching by loss of viability (^{Hoste et al., 2005}). From the ethnobotanical approach, the use of control alternatives based on plant extracts involve a variety of pharmacologically active compounds, especially tannins, flavonoids and phenylpropanoids, which are attributed biological activities such as anti-inflammatory, antioxidant and anthelmintic (^{Mravčáková et al., 2019}). Regarding the applications with anthelmintic activity of plant extracts, the proposed mechanisms are mentioned that may present a variation according to the content of the metabolites present or isolated, depending on the type and polarity of solvents used (^{Zajíčková et al., 2020}).

The ability of tannins to form complexes with glycoproteins can generate the inactivation of enzymes important for egg development, as well as to initiate with the hatching process. In this sense, (^{Rojo-Rubio et al., 2019}) determined that at a concentration of 25 mg/mL of hydroalcoholic extract of Caesalpinia coriaria leaves inhibits 95% of HC egg hatching, activity that was associated with the tannin content in Caesalpinia coriaria leaves (200 g/kg dry matter). According to the above, it has been documented that tannins can negatively affect the hatching of eggs of this nematode by binding to the egg cuticle, which is rich in glycoproteins, altering the development of the embryo and eventually the hatching process.

The evaluation of different extracts of Leucaena leucocephala, such as extracts of pod and aerial parts of the plant, has been reported, as reported by (^{Rivero-Perez et al., 2019}) who determined that the hydroalcoholic extract of Leucaena leucocephala pod inhibits the hatching of nematode eggs by 20% at a concentration of 50 mg/mL, a lower percentage than that obtained in the present investigation at the same concentration of 50 mg/mL (48%). In such a way, that it is inferred that the variation of pharmacologically active metabolites is a function of plant parts (^{Hernandez-Alvarado et al., 2018}).

In the study of (^{Von Son-de Fernex et al., 2015}), the effect of the hydroalcoholic extract of Leucaena leucocephala leaves on the inhibition of Cooperia spp. egg hatching was evaluated and an inhibition 90.49 ± 2.8% was observed; these were analyzed by scanning electron microscopy, observing irregular external damage with small projections and ruptures in the lateral walls of the egg membrane, noting direct damage of the extract fraction against the structural components of the Cooperia spp. egg, suggesting a similar behavior for the hydroalcoholic extract of Leucaena leucocephala evaluated in the present study.

Regarding effective concentrations (^{Carvalho et al., 2012}) found that ethyl acetate extract, ethanol and essential oils of Piper tuberculatum, Lippia sidoides, Mentha piperita, Hura crepitans and Capara guianensis presented an EC₅₀ on HC hatching inhibition at 0.031, 0.04, 0.037, 2.16 and 2.03 mg/mL respectively. The results of the present investigation indicate an EC₅₀ of 52.22 mg/mL and an EC₉₀ of 302.8 mg/mL, in comparison, the concentrations are higher; however, this may be associated to the use of different plants and solvents in the extraction process of secondary metabolites, see Figure 1.

Figura 1 Effective concentrations EC₅₀ and EC₉₀ of the hydroalcoholic extract of Leucaena leucocephala in the hatching test of Haemonchus contortus eggs  

CONCLUSION

The hydroalcoholic extract of Leucaena leucocephala leaves contains groups of secondary metabolites that inhibit HC egg hatching in vitro with a dose-dependent effect; however, it is necessary to identify the specific metabolite(s) that confer the activity, determine their toxicity and perform in vivo assays.

CONFLICT OF INTEREST

All authors declare that they have no conflicts of interest.