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Revista mexicana de ciencias pecuarias
On-line version ISSN 2448-6698Print version ISSN 2007-1124
Rev. mex. de cienc. pecuarias vol.14 n.4 Mérida Oct./Dec. 2023 Epub Nov 17, 2023
https://doi.org/10.22319/rmcp.v14i4.6339
Articles
Anthelmintic evaluation of four fodder tree extracts against the nematode Haemonchus contortus under in vitro conditions
a Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán. Cuautitlán Izcalli, Estado de México, México.
b Universidad Autónoma Chapingo. Posgrado en Producción Animal. Chapingo, Estado de México, México.
c Universidad Autónoma Chapingo. Unidad Regional Universitaria Sur Sureste. Teapa, Tabasco, México.
d Instituto Nacional de Investigaciones Agrícolas, Forestales y Pecuarias. Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad. Carr. Fed. Cuernavaca-Cuautla 8534, Jiutepec 62574, Morelos, México.
e Universidad Autónoma de Guerrero. Facultad de Ciencias Agropecuarias y Ambientales. Iguala, Guerrero, México.
f Islamic Azad University. Faculty of Veterinary Medicine, Department of Parasitology. Tabriz, Iran.
The objective was to evaluate the nematocidal effect of four hydroalcoholic extracts (HAE) of Brosimum alicastrum (HAE-Ba), Guazuma ulmifolia (HAE-Gu), Erythrina americana (HAE-Ea) and Leucaena leucocephala (HAE-Ll) against Haemonchus contortus. The tests of egg hatching inhibition (EHI) and larval (infective larvae) mortality were used. The treatments were HAEs at concentrations of 6.25-50 mg/mL for EHI and 25-100 mg/mL for larval mortality, ivermectin (5 mg/mL, positive control) and distilled water (negative control). Data were analyzed using an ANOVA and treatments with a concentration-dependent effect were subjected to a regression analysis to determine lethal concentrations (LC50 and LC90). In addition, a phytochemical analysis was performed on the extracts to identify the presence of the main secondary metabolites. The best ovicidal and larvicidal activity was observed in HAE-Gu with 96.78 % EHI at 6.25 mg/mL and 57.2 % larval mortality at 75 mg/mL. Followed by HAE-Ba showing 90 % EHI at 6.25 mg/mL and 58.0 % larval mortality at 75 mg/mL. The LC50 and LC90 of HAE-Gu on EHI were 2.7 and 4.4 mg/mL, respectively. While the LCs of this same extract on larvae were LC50= 64 and LC90= 125 mg/mL. The phytochemical analysis indicates that all extracts contain tannins, coumarins, flavonoids and terpenes. The fodder species G. ulmifolia and E. americana could be candidate plants for the control of H. contortus.
Keywords Fodder trees; Secondary metabolites; Haemonchus contortus; Larval mortality; Egg hatching inhibition
El objetivo del presente estudio fue evaluar el efecto nematicida de cuatro extractos hidroalcohólicos (EHA) de Brosimum alicastrum (EHA-Ba), Guazuma ulmifolia (EHA-Gu), Erythrina americana (EHA-Ea) y Leucaena leucocephala (EHA-Ll) contra Haemonchus contortus. Se usaron las pruebas de inhibición de la eclosión de huevos (IEH) y mortalidad larval (larvas infectantes). Los tratamientos fueron los EHA´s a concentraciones de 6.25-50 mg/ml para IEH y de 25-100 mg/ml para mortalidad larval, ivermectina (5 mg/ml, control positivo) y agua destilada (control negativo). Los datos se analizaron mediante un ANOVA y los tratamientos con efecto dependiente a la concentración se sometieron a un análisis de regresión para determinar las concentraciones letales (CL50 y CL90). Además, se realizó un análisis fitoquímico a los extractos para identificar la presencia de los principales metabolitos secundarios. La mejor actividad ovicida y larvicida fue observada en EHA-Gu con un 96.78 % de IEH a 6.25 mg/ml y 57.2 % de mortalidad larval a 75 mg/ml. Seguido de EHA-Ba mostrando 90 % IEH a 6.25 mg/ml y un 58.0 % de mortalidad larval a 75 mg/ml. Las CL50 y CL90 del EHA-Gu sobre la IEH fueron 2.7 y 4.4 mg/ml, respectivamente. Mientras que las CL´s de este mismo extracto sobre larvas fue de CL50=64 y CL90=125 mg/ml. El análisis fitoquímico indica que todos los extractos contienen taninos, cumarinas, flavonoides y terpenos. Las especies forrajeras G. ulmifolia y E. americana podrían ser plantas candidatas para el control de H. contortus.
Palabras clave Arboles forrajeros; Metabolitos secundarios; Haemonchus contortus Mortalidad de larvas; Inhibición de la eclosión de huevos
Introduction
In tropical regions, gastrointestinal nematodes (GIN) represent a serious problem in small ruminants; and to reduce the impact that these organisms have on animals, it is necessary to perform some type of treatment1. Haemonchus contortus is a hematophagous nematode with the highest prevalence worldwide in sheep and goats, which affects their health2,3. This parasite causes different alterations in its host, including reduced growth rate, anemias and can cause sudden death3. The main method for the control of GIN in small ruminants, including H. contortus, is through the use of broad-spectrum anthelmintics such as benzimidazoles, macrocyclic lactones, imidazothiazoles and more recently the amino-acetonitrile derivative. The inappropriate and excessive use of these antiparasitics has triggered a problem of multiple anthelmintic resistance worldwide4.
In small ruminant production systems under grazing conditions, the use of tree species with fodder potential represents a viable option for their feeding, because they contain a rich source of energy and protein5. It has been determined that these tree species contain secondary metabolites, so they could have anthelmintic activity6. Among the best-known tree species are: Brosimum alicastrum, which contains 14 to 17 % crude protein (CP)7,8; Guazuma ulmifolia, which contains 17 % CP9,10; Erythrina americana, which provides 14 to 18.9 % CP11,12; and Leucaena leucocephala, which provides 23.4 to 33.2 % of CP, depending on the age of regrowth and the season of the year13,14. Some secondary metabolites have been identified in these tree species; for example, in the foliage of B. alicastrum, phenols such as gallic acid are reported15, G. ulmifolia presents phenols such as caffeic acid, chlorogenic acid and flavonoids such as catechin, quercetin and luteolin16. Erythrina americana contains alkaloids (erysotrine) in seeds, flowers and foliage17 and phenols such as hydrolysable tannins12. For its part, L. leucocephala contains flavonoids such as quercetin, kaempferol, luteolin, among others18.
Due to the presence of these secondary metabolites in the foliage of these plants and their availability in tropical regions, it is interesting to know their effect on the GINs of small ruminants; however, there is limited information on some of these plants. In sheep fed G. ulmifolia for 30 days, a highly significant decreasing trend (P<0.001) was found in the count of eggs per gram of feces19, while the methanolic extract of the seed of E. americana exerts a nematocidal and insecticidal effect on Panagrellus redivivus and Anopheles sp., respectively20-22. On the other hand, aqueous extracts of L. leucocephala and G. ulmifolia showed inhibitory effect of egg hatching of 50 % at 1.25 mg/ml on GINs from sheep23. In order to know the effect of B. alicastrum, G. ulmifolia, E. americana and L. leucocephala, on the nematode H. contortus, hydroalcoholic extracts were evaluated on eggs and infective larvae of the parasite H. contortus under in vitro conditions.
Material and methods
Fodder samples
The collection of plant material was carried out in the Huasteca Potosina region, located in the state of San Luis Potosí. This region has a subhumid climate with rains in summer24. Leaves and stems of mature trees aged 3, 12, 20 and 30 yr for L. leucocephala, G. ulmifolia, B. alicastrum and E. americana, respectively, were collected. It should be noted that the material collected were non-senescent leaves and stems. The collection was carried out during the months of June to October 2017. The material was then dried in a forced air oven and ground to a particle size of 0.5 cm.
Hydroalcoholic extract
Each tree species was macerated with a hydroalcoholic solution, placing 300 g of the dried and ground plant material in a solution of 70 % water and 30 % methanol and it was left to macerate for 24 h. Each extract was then filtered to remove the plant material. After obtaining the liquid part, the solvents were removed by distillation under reduced pressure using an R-300 rotary evaporator (BUCHI, Switzerland) until semisolid extracts were obtained. Then each extract was frozen at -80 ° C for 24 h and finally they were brought to total dryness by lyophilization processes and stored at -40 ° C until further use.
Qualitative analysis of secondary compounds of extracts
The chemical profile of the hydroalcoholic extracts was determined by following different phytochemical procedures using reference compounds25. The identification of alkaloids was performed using the technique by Dragendorff, Mayer and Wagner25. The presence of coumarins was determined with the Bornträger test, while the flavonoid content was determined with the Mg2+ and HCl test26,27. The ferric chloride, saline and gelatin test was used to identify tannins28,29. The identification of terpenes was determined using the Liebermann-Burchard and Salkowski tests and foam formation was the indicator used to identify the presence of saponins27.
Biological material
Eggs and larvae of Haemonchus contortus were obtained from a donor sheep free of gastrointestinal nematodes, of three and a half months of age and 22 kg of live weight, previously artificially infected with a monospecific strain of the parasite under study (strain INIFAP-HcIVMr-SAI). The sheep was housed in an elevated individual cage provided with alfalfa, commercial feed and freely accessible water. The lamb was cared for following health and welfare care according to the standard NOM-062-ZOO-1999.
Collection of H. contortus eggs
Feces were collected directly from the rectum of the infected animal. Subsequently, they were washed with clean water through sieves of different diameters (240, 150, 120 and 30 μm) and the suspension of the last sieve was collected in 15 mL Falcon tubes containing the parasites. Then the tubes were centrifuged at 3,500 rpm for 5 min (three times) in order to obtain eggs free of fecal residues. Finally, they were quantified by aliquots to verify a concentration of 100 ± 15 eggs in an aqueous suspension of 50 μL30.
Obtaining of infective larvae (L3) of H. contortus
The L3 were obtained by stool cultures of the donor animal. The feces collected from the animal were kept moist at room temperature for 7 d. After the required time, the larvae were recovered using the Baermann technique31. The L3 obtained were stored in culture dishes at 4 °C. Prior to performing the bioassays, the L3 were suspended in hypochlorite (187 μL chlorine and 4,813 mL of distilled water) for 5 min so that they unsheathed. Then the L3 were washed with distilled water three times by centrifugation (3,500 rpm for 5 min). Subsequently, different dilutions were made until obtaining 100 ± 15 L3 contained in 50 μL of an aqueous suspension.
Egg hatching inhibition (EHI)
Bioassays were performed on 96-well microtiter plates. Each extract was evaluated individually in triplicate considering four repetitions per replication (n= 12). The HA-Es of the four tree species were evaluated at concentrations of 50, 25, 12.5 and 6.25 mg/mL. In addition, each bioassay included distilled water was as a negative control and ivermectin (5 mg/mL) as a positive control. Fifty microliters of an aqueous suspension containing 100 ± 15 eggs were added to each well and then 50 μL of extract at the required concentration or controls were added as appropriate. The plates were incubated in a wet chamber at 25-30 °C for 48 h. After this time, the number of eggs and larvae in each well was counted (Motic® 10x microscope). The percentage of egg hatching inhibition (%EHI) was determined by the following formula:
Larval mortality
Bioassays were performed on 96-well microtiter plates (n=12). Each extract was evaluated individually in triplicate considering four repetitions per replication (n=12). The treatments were the extracts at different concentrations (100, 75, 50 and 25 mg/mL). Ivermectin (5 mg/mL) and distilled water were used as positive and negative controls, respectively. An aqueous suspension of 50 μL containing 100 ± 15 L3 was added to each well and then 50 μL of the treatments was added as appropriate. The plates were incubated in a wet chamber at 25-30 °C for 48 h. Subsequently, the live and dead larvae contained in each well were quantified based on the criteria described by Olmedo-Juárez et al32. The percentage of larval mortality (LM) was determined by the following equation:
Statistical analysis
The percentages of EHI and LM were previously normalized using the square root and analyzed by ANOVA under a completely randomized design with the general linear model (PROC GLM) of the SAS statistical package version 9.033. The comparison of means was performed using the Tukey test at a significance level of 0.05. Treatments with concentration-dependent effect were subjected to a regression analysis to determine lethal concentrations 50 and 90 (LC50 and LC90) using the PROC PROBIT system of the SAS statistical package33.
Results
Egg hatching inhibition and larval mortality
Table 1 shows the results of the ovicidal and larvicidal activity of the HAE of B. alicastrum on the nematode H. contortus. This activity was different (P<0.05) in each concentration evaluated, obtaining the greatest inhibitory effect of egg hatching at 50 mg/mL. On the other hand, in the larval mortality test, only a mortality percentage of 29 % was achieved at 100 mg/mL.
Table 1 Percentage of egg hatching inhibition (%EHI) and mortality of infective larvae (L3) of Haemonchus contortus caused by a hydroalcoholic extract of Brosimum alicastrum
| Treatments | Average eggs and larvae | %EHI ± SD | Average live and dead larvae | % Mortality ± SD | ||
|---|---|---|---|---|---|---|
| Eggs | Larvae | Dead | Live | |||
| Distilled water | 2.9 | 138.2 | 2.07 ± 1.0f | 2.8 | 74.5 | 4.6 ± 4.3c |
| Ivermectin (5 mg/ml) | 127.2 | 0.8 | 99.9 ± 0.2a | 139.3 | 0 | 100a |
| HAE-Ba (mg/ml) | ||||||
| 100.0 | --- | --- | --- | 34.8 | 87.7 | 29.0 ± 11.1b |
| 75.0 | --- | --- | --- | 32.3 | 94.1 | 26.1 ± 7.6b |
| 50.0 | 92.8 | 21.1 | 81.4 ± 3.5b | 14.3 | 103.6 | 14.4± 14.4b |
| 25.0 | 82.0 | 36.6 | 69.1 ± 5.1c | 11.3 | 121.6 | 8.5 ± 5.2c |
| 12.5 | 75.5 | 43 | 63.8±2.4d | --- | --- | --- |
| 6.25 | 70.0 | 56.1 | 55.5 ± 1.7e | --- | --- | --- |
| Coefficient of variation | 0.62 | 23.1 | ||||
| R2 | 0.99 | 0.95 | ||||
| Standard error of the mean (SEM) | 0.04 | 0.15 | ||||
| P value | <0.001 | <0.0001 | ||||
HAE-Ba= Hydroalcoholic extract of Brosimum alicastrum. ---= not evaluated. SD= standard deviation.
a-f Means with different literal within the same column indicate a difference (P<0.05).
The HAE of G. ulmifolia exhibited an ovicidal effect close to 100 % from the concentration 6.25 mg/mL, being statistically equal to that obtained with ivermectin up to the concentration of 12.5 mg/mL (Table 2). A similar effect was observed using the HAE of E. americana at concentrations of 50, 25, and 12.5 mg/mL (Table 3).
Table 2 Percentage of egg hatching inhibition and mortality of infective larvae (L3) of Haemonchus contortus caused by a hydroalcoholic extract of Guazuma ulmifolia
| Treatments | Average eggs and larvae | %EHI ± SD | Average live and dead larvae | % Mortality ± SD | ||
|---|---|---|---|---|---|---|
| Eggs | Larvae | Dead | Live | |||
| Distilled water | 5.5 | 135.3 | 2.07 ± 1.0f | 1.9 | 153.8 | 1.1 ±1.8e |
| Ivermectin (5 mg/ml) | 127.3 | 0 | 99.9 ± 0.2a | 158.1 | 0 | 100a |
| HAE-Gu (mg/ml) | ||||||
| 100.0 | --- | --- | --- | 109.7 | 18.5 | 85.9 ± 7.4b |
| 75.0 | --- | --- | --- | 85.3 | 60.5 | 57.2 ± 15.5c |
| 50.0 | 118.8 | 0.4 | 99.5 ± 0.7ab | 43.5 | 103.8 | 26.8± 15.0d |
| 25.0 | 112.5 | 2.3 | 97.8 ± 2.8ab | 11.3 | 138 | 7.7 ± 4.9e |
| 12.5 | 120.8 | 0.75 | 99.4± 0.9ab | --- | --- | --- |
| 6.25 | 113.4 | 3.9 | 96.78± 5.3b | --- | --- | --- |
| Coefficient of variation | 3.12 | 21.3 | ||||
| R2 | 0.99 | 0.95 | ||||
| Standard error of the mean (SEM) | 0.03 | 0.18 | ||||
| P value | <0.001 | <0.0001 | ||||
SD= standard deviation; HAE-Ba= Hydroalcoholic extract of Guazuma ulmifolia. ---= not evaluated.
a-f Means with different literal within the same column indicate a difference (P<0.05).
Table 3 Percentage of egg hatching inhibition and mortality of infective larvae (L3) of Haemonchus contortus caused by a hydroalcoholic extract of Erythrina americana
| Treatments | Average eggs and larvae | %EHI ± SD | Average live and dead larvae | % Mortality ± SD | ||
|---|---|---|---|---|---|---|
| Eggs | Larvae | Dead | Live | |||
| Distilled water | 5.9 | 134.0 | 4.1 ± 2.0c | 3.0 | 96.3 | 3.6 ±2.9d |
| Ivermectin (5 mg/mL) | 111.5 | 0.2 | 99.7± 0.6a | 145.4 | 0 | 100a |
| HAE-Ea (mg/mL) | ||||||
| 100.0 | --- | --- | --- | 93.4 | 49.1 | 60.0± 13.5b |
| 75.0 | --- | --- | --- | 102.7 | 50.1 | 58.0 ± 24.8b |
| 50.0 | 111.4 | 2.6 | 97.0 ± 7.5ab | 86.8 | 49.2 | 62.6± 10.2b |
| 25.0 | 86.4 | 0.5 | 99.5 ± 0.7a | 50.3 | 93.3 | 35.8± 7.3c |
| 12.5 | 91.3 | 2.0 | 97.7 ± 2.6a | --- | --- | --- |
| 6.25 | 94.0 | 9.3 | 88.8 ± 19.0ab | --- | --- | --- |
| Coefficient of variation | 10.7 | 21.4 | ||||
| R2 | 0.94 | 0.89 | ||||
| Standard error of the mean (SEM) | 0.12 | 0.16 | ||||
| P value | <0.0001 | <0.0001 | ||||
HAE-Ba= Hydroalcoholic extract of Erythrina americana. ---= not evaluated. SD= standard deviation.
a-d Means with different literal within the same column indicate a difference (P<0.05).
The highest larvicidal activity (85 % LM) of the extract of G. ulmifolia was achieved using the highest concentration (100 mg/ml). While the HAE of E. americana only caused 60 % mortality at the same concentration. On the other hand, the results obtained with the HAE from L. leucocephala showed the highest percentage of EHI (83.2 %) when the concentration of 50 mg/mL was used. And for LM, only 63 % was achieved using 100 mg/ml of the HAE (Table 4).
Table 4 Percentage of egg hatching inhibition and mortality of infective larvae (L3) of Haemonchus contortus caused by a hydroalcoholic extract of Leucaena leucocephala
| Treatments | Average eggs and larvae | %EHI ± SD | Average live and dead larvae | % Mortality ± SD | ||
|---|---|---|---|---|---|---|
| Eggs | Larvae | Dead | Live | |||
| Distilled water | 7.7 | 131.3 | 5.6 ± 3.5c | 4.7 | 122.0 | 5.2 ±2.9d |
| Ivermectin (5 mg/mL) | 112.5 | 0.1 | 99.9 ± 0.2a | 145.4 | 0 | 100a |
| HAE-Ll (mg/mL) | ||||||
| 100.0 | --- | --- | --- | 75.7 | 40.0 | 63.0± 22.9b |
| 75.0 | --- | --- | --- | 27.6 | 99.5 | 21.7 ± 8.4c |
| 50.0 | 97.0 | 20.4 | 83.2 ± 12.4a | 13.0 | 95.2 | 12.0± 2.1cd |
| 25.0 | 53.9 | 66.8 | 48.9 ± 31.7b | 7.5 | 114.2 | 6.2± 2.9d |
| 12.5 | 50.5 | 65.9 | 48.4 ± 35.3b | --- | --- | --- |
| 6.25 | 44.4 | 65.9 | 45.9 ± 38.6b | --- | --- | --- |
| Coefficient of variation | 46.1 | 29.2 | ||||
| R2 | 0.59 | 0.93 | ||||
| Standard error of the mean (SEM) | 0.35 | 0.21 | ||||
| P value | <0.0001 | <0.0001 | ||||
HAE-Ba= Hydroalcoholic extract of Leucaena leucocephala. ---= not evaluated. SD= standard deviation.
a-d Means with different literal within the same column indicate a difference (P<0.05).
Lethal concentrations (LCs)
The Cs 50 and 90 required to cause EHI and larval mortality are shown in Table 5. The regression analysis indicated that the extracts with the best inhibitory effect on egg hatching were HAE-Ea (LC50=0.16 mg/mL and LC90=4.41 mg/mL) and HAE-Gu (LC50=2.7 mg/mL and LC90=4.4 mg/mL). Regarding larval mortality, the best treatment was observed in HAE-Gu with LC50 and LC90 of 64.0 and 125.2 mg/mL, respectively.
Table 5 Lethal concentrations (LC50 and LC90) of hydroalcoholic extracts of four fodder tree species required to inhibit egg hatching and kill infective larvae (L3) of Haemonchus contortus at 48 hours
| % Egg hatching inhibition | % Mortality of infective larvae (L3) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Plant | LC50 | CI 95% limits (lower-upper) | LC90 | CI 95% limits (lower-upper) | LC50 | CI 95% limits (lower-upper) | LC90 | CI 95% limits (lower-upper) | |
| HAE-Ba | 4.8 | (3.88-5.70) | 197 | (145.6-293.1) | 187.8 | (156.67-2.70.6) | 608.7 | (376.7- ..) | |
| HAE-Gu | 2.7 | (2.6-2.8) | 4.4 | (2.62-2.80) | 64.0 | (62.45-65.66) | 125.2 | (119.6-132.0) | |
| HAE-EA | 0.16 | (0.04-0.38) | 4.1 | (2.8-5.4) | NA | --- | NA | --- | |
| HAE-LL | 17.9 | (16.8-19.1) | 201.9 | (167.6-251.0) | 93.12 | (91.61-94.71) | 124.5 | (119.6-131.36) | |
CI= confidence interval. NA= not active. HAE-Ba= Brosimum alicastrum, HAE-Gu= Guazuma ulmifolia, HAE-Ea= Erythrina americana.
Identification of secondary metabolites
The phytochemical analysis showed the presence of secondary metabolites in the four plant extracts, such as tannins, coumarins, saponins, alkaloids and flavonoids (Table 6).
Table 6 Results of the qualitative phytochemical analysis of the hydroalcoholic extracts
| Metabolite | Colorimetric reaction | Hydroalcoholic extract (HA-E) | |||
|---|---|---|---|---|---|
| Brosimum alicastrum | Guazuma ulmifolia | Erythrina americana | Leucaena leucocephala | ||
| Alkaloids | Dragendorff | - | - | - | + |
| Mayer | - | - | - | + | |
| Wagner | - | - | - | ++ | |
| Coumarins | Borntraeguen | - | + | + | + |
| Flavonoids | Mg2+ and HCL | - | - | + | + |
| Tannins | Ferric chloride | +++ | +++ | +++ | +++ |
| Gelatin solution | - | - | - | - | |
| Gelatin and saline | - | - | - | - | |
| Saline | +++ | +++ | +++ | +++ | |
| Triterpenes/ Steroids | Liebermann-Burchard | - | + | - | + |
| Salkowski | + | + | + | + | |
| Saponins | Foam formation | + | - | + | ++ |
(-) Not detected (+) positive light reaction (++) positive reaction (+++) strong positive reaction.
Discussion
Natural products obtained from plants rich in secondary metabolites have been evaluated for different medicinal purposes, such as antioxidants, antimicrobials and antiparasitics34-36. The four hydroalcoholic extracts evaluated in the present study exhibit nematocidal activity against Haemonchus contortus, a hematophagous parasite of greater prevalence in sheep and goats, which affects their health. There are few studies on the use of Brosimum alicastrum as an anthelmintic, although it is an abundant resource in tropical regions; the extract of acetone:water (70:30) on H. contortus larvae has been observed to inhibit 95 % of the ability to unsheathe at a concentration of 1.2 mg/mL37. While in the present study, using extract based on methanol:water, 187.8 mg/mL was required to cause 50 % mortality. On the other hand, an acetonic extract of G. ulmifolia has been shown to exhibit ovicidal activity on Cooperia punctata, another parasitic nematode of cattle, inhibiting up to 70 % of hatching at a concentration of 9.6 mg/mL38. Likewise, an ethanolic extract (100 mg/mL) of this plant species has shown nematocidal effect on Pheritima posthuma39. In a recent study, a hydroalcoholic extract of G. ulmifolia has been shown to exhibit significant ovicidal effect (90 % EHI) at a concentration of 0.50 mg/mL40. The ovicidal activity reported in the present study with the hydroalcoholic extract of G. ulmifolia indicates that a higher concentration (LC50=4.4 mg/mL) than reported by the previous work is required. This could be explained by the fact that a plant species collected in a different region was used and probably the content of bioactive compounds could be different between both plant species. Although in the present work it has been reported that G. ulmifolia contains some secondary compounds such as tannins, flavonoids, coumarins and terpenes, it is very important to know the content of each of these compounds to relate them to anthelmintic activity. On the other hand, in vivo studies have also been conducted in kids artificially infected with infective larvae of H. contortus, which were fed with 10 % of G. ulmifolia foliage and no differences were obtained in the count of eggs per gram of feces (EPG) compared to the control group41. The same results were observed in Pelibuey ewes fed with 30 % of G. ulmifolia, however, a highly significant trend (P<0.001) towards the decrease of EPG was observed in these ewes19.
It is known that species of the genus Erythrina have a wide variety of alkaloids that have been identified and are attributed a neuromuscular blocking effect20, in addition, the use of methanolic extract on Daphnia magna turned out to be highly toxic21, so the nematocidal effect found in the present study could be attributed to those compounds. A methanolic extract of E. variegate has been evaluated against crustaceans of the genus Artemia, as well as earthworms (Eisenia foetida) and parasitic helminths of birds such as Ascardi galli and Raillietina spiralis and mortality was reported in these biological models using concentrations of 10 mg/mL42,43. On the other hand, in a study conducted in Pelibuey sheep fed with E. americana foliage, no changes in egg count were observed during the experimental phase12.
The LCs 50 and 90 for B. alicastrum in gastrointestinal nematode larvae reported in another study were 291.6 and 666.6 mg/mL, respectively44, which were similar to those reported in the present study (187.8 and 608.7 mg/mL). Regarding G. ulmifolia, the results of the present study indicate that, to inhibit 50 % of the hatching of H. contortus eggs, 2.2 mg/mL of the hydroalcoholic extract is required, while in another study with an extract of acetone:water (70:30) of G. ulmifolia against C. punctata, it was 8.84 mg/mL38. In the same study, the authors report a LC50 of 11.77 mg/mL of the extract of acetone:water 70:30 of L. leucocephala38. In the present research work, the LCs calculated for the HAE of the leaves of this tree species were higher (LC50=52.8 and LC90=308 mg/mL) respectively (Table 5)45. The LC of E. americana on H. contortus has not been previously reported, however, for the species E. variegata, on crustaceans of the genus Artemia, the LC50 was 3.99 mg/mL43, a value higher than that of the present study (0.19 mg/ml).
Some secondary metabolites such as tannins, saponins and coumarins have been identified in the bark and leaves of B. alicastrum46,47. In the present study, the chemical profile in the extract of B. alicastrum indicated the presence of tannins and saponins. On the other hand, saponins, cyanogenic glycosides, phenols and steroids, which were also found in the present study, have been reported qualitatively in G. ulmifolia48. In other species of the genus Erythrina, they have been reported to contain secondary metabolites similar to those found in the hydroalcoholic extract of E. americana. E. variegate has been reported to contain alkaloids, saponins and flavonoids43. In another study in E. americana from Tabasco, Mexico, high levels of tannins have been identified12. The secondary metabolites reported in L. leucocephala depend on the type of extract; for instance, saponins, phenols, tannins, terpenes, among others, have been identified in aqueous and ethanolic extracts, similar to the profile found in this study45,49-51.
Conclusions and implications
It is concluded that the hydroalcoholic extract of the four trees studied may be an option for the control of Haemonchus contortus in small ruminants, especially G. ulmifolia and E. americana. It is recommended to continue with their study to identify the active compounds in each case.
Acknowledgements
The authors thank the National Council of Humanities, Sciences and Technologies for the financing during the period of Doctoral Studies of the main author (grant number: 429558).
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Received: October 13, 2022; Accepted: June 12, 2023
Este es un artículo publicado en acceso abierto bajo una licencia Creative Commons
